The Tumor Microenvironment (Cancer Drug Discovery and Development)

Cancer Drug Discovery and Development Series Editor: Beverly A. Teicher For further volumes: http://www.springer.com/s...

Author: Rebecca G. Bagley

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Cancer Drug Discovery and Development Series Editor: Beverly A. Teicher

For further volumes: http://www.springer.com/series/7625

Rebecca G. Bagley Editor

The Tumor Microenvironment

Editor Rebecca G. Bagley Genzyme Corporation Framingham MA, USA [email protected]

ISBN 978-1-4419-6614-8 e-ISBN 978-1-4419-6615-5 DOI 10.1007/978-1-4419-6615-5 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2010934382 © Springer Science+Business Media, LLC 2010 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer soft-ware, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

The fact that tumors are composed of both tumor cells and host cells has long been known. These tumor-associated cells include vascular endothelial cells and pericytes, as well as inflammatory cells such as neutrophils, monocytes, macrophages, mast cells and eosinophils, and lymphocytes. The tumor cells also interact with stromal cells and with elements of the tissue extracellular matrix. What has been less appreciated is the role that these cells could have in modulating the growth, invasion, and metastasis of the tumor. Early on, the elements of what we now call the tumor microenvironment were considered to be more or less innocent bystanders to the role of the tumor cells as they grew and invaded local sites. Today, there is an increased understanding of the critical role of the tumor microenvironment as dramatically influencing the course of tumor development and dissemination. This volume represents a superb compilation of the latest thoughts and data regarding the role of each essential component of the tumor microenvironment in cancer development and progression. Perhaps, the earliest recognition of the role of nonmalignant cells as cancer regulators was the recognition that lymphocytes can participate in what was termed “immune surveillance” in the 1960s. Our understanding of tumor immunity has improved markedly since then, and there are now successful clinical studies showing the potential use of immune-based therapies in cancer treatment. The role of tumor infiltrating lymphocytes is nicely discussed here in the chapter by Fu and Yu, with a special emphasis on the potential prognostic implications of the presence of these cells at a tumor site. Natural killer cells, first shown to be involved in tumor surveillance by Herlyn in the 1980s, also have multiple effects on tumors as discussed in the chapter by Arai. Additional immune and inflammatory cells are also discussed. These include macrophages, which have effects on both tumor cells and blood vessels, and mast cells, which have a potent and somewhat underappreciated effect on tumor growth that is clarified in the chapter by Angelidou and Zhang. A picture emerges of the tumor as a city filled with diverse cellular inhabitants who operate in the role of a “zoning board” in telling the tumor how fast it can expand and into what spaces it can grow. An extremely important regulator of tumor growth and metastasis is the tumor vasculature. Again, tumors were known to be filled with functional blood vessels for many decades before Judah Folkman explained how they might influence tumor v

vi

Preface

growth. His theory of tumor angiogenesis predicted that anti-angiogenic agents might be developed to combat cancer and this prediction has been borne out. In many cases, the predominant signal for angiogenesis is tissue hypoxia and a very strong section of this book deals with the effects of hypoxia on the tumor and on the vascular components of the tumor. The potential translational aspects of this field is just emerging and a compelling discussion of the novel therapies that target the hypoxia induced transcription factor, HIF-1-alpha can be found in the book’s opening chapter by Rapisarda and Melillo. Along with the immune cells, inflammatory cells, and vascular cells, the tumor surrounds stromal cells including fibroblasts and other mesenchymal cells. The interplay between stromal cells and tumor cells is an extremely vibrant area of study and is nicely discussed in the chapter by Orimo. Of course, a principal function of these cells is to produce and remodel the elements of the extracellular matrix as described in the contribution by DeClerck. These proteins can present a barrier to tumor invasion and metastasis. Excellent and comprehensive discussions of the role of collagens and of fibronectin as well as their binding partners the integrins and tetraspanins can be found in the section on the extracellular matrix. The modulation of the matrix by cancer cells is discussed in detail in the chapter on matrix metalloproteases and cancer cell invasion by Zucker and Cao. The interplay of tumor cells and the microenvironment is mirrored in the complex interactions of tumor cells, fibroblasts, matrix elements, and the various cells that produce either matrix metalloproteases or their natural inhibitors. No contemporary discussion of the role of the tumor microenvironment could be complete without an in-depth examination of the existence and potential role of stem cells in the establishment of human tumors and in their metastatic dissemination. This is a relatively new field and comes with the attendant controversies that often characterize new areas of investigation. This can make for very exciting reading, as it does in the chapters on cancer stem cells by Giordano, on endothelial cell precursors by Shaked and on mesenchymal stem cells by Banerjee. It is safe to say that the exact contribution of all of these progenitor, precursor and stem cell populations to cancer is an emerging and truly important field of inquiry. These chapters beautifully capture the scientific foment that characterizes the study of these cells and their role in cancer. Perhaps, the most exciting aspect of the increased attention paid to the tumor microenvironment is the promise that new approaches to cancer therapy can be derived from our paying attention to these non-tumor elements and to their interactions with the tumor cells. This is beautifully captured throughout this book. One senses an emerging optimism that the tumor microenvironment is not only important, but that it represents a treasure trove of potential therapeutic opportunities such as the ones described in the chapter on secreted growth factors as therapeutic targets by Teicher. Whether one starts out as a skeptic, or as a devotee, this excellent and comprehensive compilation will educate the reader and stimulate new ideas about tumors as an integrated combination of cellular, secreted, stromal, and matrix elements that each represents a potential therapeutic target. Boston, MA

Bruce Zetter

Contents

Part I: Physiological Parameters 1 Combination Strategies Targeting Hypoxia Inducible Factor 1 (HIF-1) for Cancer Therapy.................................................... Annamaria Rapisarda and Giovanni Melillo

3

2 The Tumor Microenvironment: New Insights into Regulation of Tumor pH by Carbonic Anhydrases.............................. Pawel Swietach, Adrian L. Harris, and Richard D. Vaughan-Jones

23

3 Hypoxia, Gene Expression, and Metastasis........................................... Olga V. Razorenova and Amato J. Giaccia 4 Molecular Mechanisms Regulating Expression and Function of Cancer-Associated Carbonic Anhydrase IX..................... Jaromir Pastorek and Silvia Pastorekova 5 Glycolytic Pathway as a Target for Tumor Inhibition.......................... Weiqin Lu and Peng Huang

43

59 91

Part II: Malignant Cells 6 Aberrant DNA Methylation in Cancer Cells......................................... Toshikazu Ushijima

121

7 DNA Repair and Redox Signaling.......................................................... Mark R. Kelley, Millie M. Georgiadis, and Melissa L. Fishel

133

8 Cancer Stem Cells and Microenvironment............................................ Mario Federico and Antonio Giordano

169

9 Epithelial–Mesenchymal Transition in Development and Diseases...................................................................... Yadi Wu and Binhua P. Zhou

187 vii

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Contents

10 Invasion and Metastasis......................................................................... Douglas M. Noonan, Giuseppina Pennesi, and Adriana Albini 11 Dormancy of Disseminated Tumor Cells: Reciprocal Crosstalk with the Microenvironment.................................................. Paloma Bragado, Aparna C. Ranganathan, and Julio A. Aguirre-Ghiso

213

229

Part III: Vasculature and Stroma 12 Impact of Endothelial Progenitor Cells on Tumor Angiogenesis and Outcome of Antiangiogenic Therapy: New Perspectives on an Ongoing Controversy.................................... Robert S. Kerbel, Francesco Bertolini, and Yuval Shaked 13 Bone Marrow Derived Mesenchymal Stem/Stromal Cells and Tumor Growth....................................................................... Pravin J. Mishra and Debabrata Banerjee 14 Integrin Signaling in Lymphangiogenesis............................................ Barbara Garmy-Susini 15 Role of Pericytes in Resistance to Antiangiogenic Therapy................................................................................................... Koji Matsuo, Chunhua Lu, Mian M.K. Shazad, Robert L. Coleman, and Anil K. Sood 16 Tumour-Promoting Stromal Myofibroblasts in Human Carcinomas........................................................................... Urszula M. Polanska, Kieran T. Mellody, and Akira Orimo

257

275 289

311

325

Part IV: Immune-Mediated Cells 17 Mast Cells and Tumor Microenvironment.......................................... Theoharis C. Theoharides, Konstantinos-Dionysios Alysandratos, Asimenia Angelidou, and Bodi Zhang

353

18 Macrophages in the Tumor Microenvironment.................................. Monica Escorcio-Correia and Thorsten Hagemann

371

19 The Prognostic Significance of Tumor-Infiltrating Lymphocytes........................................................................................... Ping Yu and Yang-Xin Fu

385

Contents

20 The Pro-inflammatory Milieu and Its Role in Malignant Epithelial Initiation................................................................................ Adam Yagui-Beltrán, Qizhi Tang, and David M. Jablons 21 Natural Killer Cells for Adoptive Immunotherapy............................ Jonathan E. Benjamin and Sally Arai

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409 431

Part V: Extracellular Matrix 22 Fibronectin.............................................................................................. Andreas Menrad

457

23 Collagen in Cancer................................................................................. Janelle L. Lauer and Gregg B. Fields

477

24 Integrins and Cancer............................................................................. Laurie G. Hudson and M. Sharon Stack

509

25 Matrix Metalloproteinases and Cancer Cell Invasion/Metastasis................................................................................ Stanley Zucker and Jian Cao 26 Tetraspanins and Cancer Metastasis.................................................... Margot Zöller

531 555

Part VI: Secreted Proteins 27 Chemokines and Metastasis.................................................................. Kalyan C. Nannuru, Seema Singh, and Rakesh K. Singh

601

28 Transforming Growth Factor-b in Lung Cancer, Carcinogenesis, and Metastasis............................................................ Sonia B. Jakowlew

633

29 Cooperative Interactions Between Integrins and Growth Factor Signaling in Pathological Angiogenesis.................................... Jennifer Roth, Eric Tweedie, and Peter C. Brooks

673

30 The Extracellular Matrix and the Growth and Survival of Tumors......................................................................... Yves A. DeClerck

695

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Contents

31 Secreted Growth Factors as Therapeutic Targets............................... Beverly A. Teicher

711

32 Adrenomedullin...................................................................................... Rebecca G. Bagley

733

Index................................................................................................................

749

Contributors

Julio A. Aguirre-Ghiso Division of Hematology and Oncology, Departments of Medicine and Department of Otolaryngology, Tisch Cancer Institute, Mount Sinai School of Medicine, One Gustave L Levy Place, Box 1079, New York, NY 10029, USA Adriana Albini IRCCS Multimedica, Via Fantoli 16/15, 20138 Milano, Italy Konstantinos-Dionysios Alysandratos, MD Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111, USA Asimenia Angelidou, MD Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111, USA Sally Arai, MD Division of Blood and Marrow Transplantation, Stanford School of Medicine, 300 Pasteur Drive, H3249 MC 5623, Stanford, CA 94305, USA, Rebecca G. Bagley Genzyme Corporation, 49 New York Ave, Framingham, MA 01701, USA Debabrata Banerjee Department of Medicine and Pharmacology, Robert Wood Johnson Medical School, The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08901, USA Jonathan E. Benjamin Division of Blood and Marrow Transplantation, Stanford University Medical Center, Stanford, CA, USA

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Contributors

Francesco Bertolini, MD/PhD Department of Hematology-Oncology, European Institute of Oncology, Milan, Italy Paloma Bragado, PhD Division of Hematology and Oncology, Department of Medicine and Department of Otolaryngology, Mount Sinai School of Medicine – NYU, New York, NY 10029, USA Peter C. Brooks Maine Medical Center Research Institute, Center for Molecular Medicine, 81 Research Drive, Scarborough, ME, USA Jian Cao, PhD Stony Brook University, Room 004, Life Sciences Building, Stony Brook, NY 11794-5200, USA Robert L. Coleman, MD Department of Gynecologic Oncology, M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA; Center for RNA Interference and Non-Coding RNA, University of Texas, Houston, TX, USA Yves A. DeClerck Departments of Pediatrics and Biochemistry and Molecular Biology, University of Southern California and The Saban Research Institute of Childrens Hospital Los Angeles, Los Angeles, CA 20027, USA Monica Escorcio-Correia, PhD Barts and The London School of Medicine and Dentistry, Institute of Cancer, Charterhouse Square, London, UK EC1M 6BQ Mario Federico, MD Sbarro Health Research Organization, Temple University, BioLife Science Building, Suite 333, 1900 N. 12th Street, Philadelphia, PA 19122, USA; Section of Clinical Oncology, University of Palermo, Palermo, Italy Gregg B. Fields Department of Biochemistry, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA Melissa L. Fishel, PhD Herman B. Wells Center for Pediatric Research, Indiana University, 980 W. Walnut Street, Indianapolis, IN 46202, USA

Contributors

Yang-Xin Fu, MD/PhD Department of Pathology and Section of Dermatology/ Department of Medicine, University of Chicago, 5841 S. Maryland Ave, MC 3083, Chicago, IL 60637, USA Barbara Garmy-Susini Unité mixte Inserm U858, Institut de Médecine Moléculaire de Rangueil, IFR 150, 1, Avenue Jean Poulhès, BP 84225 31432, Toulouse Cedex 4, France Millie M. Georgiadis, PhD Herman B. Wells Center for Pediatric Research, Indiana University, 980 W. Walnut Street, Indianapolis, IN 46202, USA Amato J. Giaccia Division of Radiation and Cancer Biology, Stanford University, Stanford, CA 94305, USA Antonio Giordano Biology Department, Sbarro Health Research Organization, Temple University, Philadelphia, PA, USA Thorsten Hagemann Barts and The London School of Medicine and Dentistry, Institute of Cancer, Charterhouse Square, London, EC1M 6BQ, UK Adrian L. Harris Weatherall Institute of Molecular Medicine, Oxford University, Oxford OX3 9DS, UK Peng Huang Department of Molecular Pathology, The University of Texas MD, Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA Laurie G. Hudson, PhD University of New Mexico College of Pharmacy, Albuquerque, NM, USA David Jablons, MD Department of Surgery, University of California San Francisco, San Francisco, CA 94143, USA Sonia B. Jakowlew National Cancer Institute, Center for Cancer Training, Cancer Training Branch, Bethesda, MD 20892, USA

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Contributors

Mark R. Kelley Department of Pediatrics, Section of Hematology/Oncology, Herman B Wells Center for Pediatric Research, Indiana University, Indianapolis, IN 46202, USA Robert S. Kerbel, PhD Department of Cellular and Molecular Biology, Sunnybrook Health Sciences Centre, University of Toronto, Toronto, ON, Canada Janelle L. Lauer, PhD The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA Chunhua Lu, MD/PhD Department of Gynecologic Oncology, M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA Weiqin Lu, PhD MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA Koji Matsuo, MD Department of Gynecologic Oncology, M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA Giovanni Melillo SAIC-Frederick, Inc., DTP-Tumor Hypoxia Laboratory, Developmental Therapeutics Program, National Cancer Institute – Frederick, Building 432, Room 218, Frederick, MD 21702, USA Kieran T. Mellody, BSc./MPhil Paterson Institute for Cancer Research, The University of Manchester, Wilmslow Road, Manchester, England M20 4BX Andreas Menrad Antibody Therapeutics Genzyme Europe Research, 310 Cambridge Science Park, Milton Road, Cambridge, CB4 OWG, UK Pravin J. Mishra, MS Department of Medicine, Robert Wood Johnson Medical School, The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey, 195 Little Albany St, New Brunswick, NJ 08901, USA Kalyan C. Nannuru, PhD Department of Pathology and Microbiology, University of Nebraska Medical Center, 985900 Nebraska Medical Center, Omaha, NE 68198-5900, USA

Contributors

xv

Douglas M. Noonan, PhD Universita degli Studi dell’Insubria, Varese, Italy Akira Orimo CR-UK Stromal-Tumour Interaction Group, Paterson Institute for Cancer Research, The University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK Jaromir Pastorek Centre of Molecular Medicine, Slovak Academy of Sciences, Dubravska cesta 9, 845 05 Bratislava, Slovak Republic Silvia Pastorekova Centre of Molecular Medicine, Slovak Academy of Sciences, Dubravask cesta 9, Bratislava 845 05, Slovak Republic Giuseppina Pennesi, MD IRCCS Multimedica, Via Fantoli 16/15, 20138 Milano, Italy Urszula M. Polanska, Msc/PhD Paterson Institute for Cancer Research, The University of Manchester, Wilmslow Road, Manchester, England M20 4BX Aparna C. Ranganathan, PhD Division of Hematology and Oncology, Department of Medicine and Department of Otolaryngology, Mount Sinai School of Medicine – NYU, New York, NY 10029, USA Annamaria Rapisarda, PhD SAIC-Frederick, Inc., DTP-Tumor Hypoxia Laboratory, Developmental Therapeutics Program, National Cancer Institute – Frederick, Building 432, Room 218, Frederick, MD 21702, USA Olga V. Razorenova, PhD Division of Radiation and Cancer Biology, Stanford University School of Medicine, 269 Campus Drive, Stanford, CA 94305, USA Jennifer M. Roth, MS Center for Molecular Medicine, Maine Medical Center Research Institute, 81 Research Drive, Scarborough, ME 04074, USA Yuval Shaked Department of Molecular Pharmacology, Rappaport Faculty of Medicine, Technion – Israel Institute of Technology, Haifa, Israel

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Contributors

M. Sharon Stack University of Missouri School of Medicine, M214C Medical Sciences Building, 1 Hospital Drive, Columbia, MO 65212, USA Mian M. K. Shazad, MS/MD Department of Gynecologic Oncology, M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA; Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, TX, USA Rakesh K. Singh Department of Pathology and Microbiology, University of Nebraska Medical Center, 985900 Nebraska Medical Center, Omaha, NE 68198-5900, USA Seema Singh, PhD Department of Pathology and Microbiology, University of Nebraska Medical Center, 985900 Nebraska Medical Center, Omaha, NE 68198-5900, USA Anil K. Sood Department of Gynecologic Oncology, Cancer Biology, M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA Center for RNA Interference and Non-Coding RNA, University of Texas, Houston, TX, USA Pawel Swietach, DPhil Deparment of Physiology, Anatomy & Genetics, Oxford University, Oxford OX1 3PT, UK Qizi Tang, PhD Department of Surgery, University of California San Francisco, San Francisco, CA 94143, USA Beverly A. Teicher Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701-9322, USA [email protected] Theoharis C. Theoharides Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111, USA Eric Tweedie, BS Center for Molecular Medicine, Maine Medical Center Research Institute, 81 Research Drive, Scarborough, ME 04074, USA

Contributors

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Toshikazu Ushijima National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, Japan Richard D. Vaughan-Jones, PhD Deparment of Physiology, Anatomy & Genetics, Oxford University, Oxford OX1 3PT, UK Yadi Wu, PhD Department of Molecular and Biomedical Pharmacology, Markey Cancer Center and University of Kentucky School of Medicine, BBSRB room B336, 741 South Limestone, Lexington, KY 40506-0509, USA Adam Yagui-Beltrán Department of Surgery, Division of Adult Thoracic Surgery, The Helen Diller Family Comprehensive Cancer Center Thoracic Surgery, University of California San Francisco, 1600 Divisadero, Room A-743, San Francisco, CA 94143-1724, USA Ping Yu The Committee on Immunology and Department of Pathology and Section of Dermatology/Department of Medicine, University of Chicago, 5841 S. Maryland Ave, Chicago, IL 60637, USA Bruce Zettler, PhD Childrens’s Hospital, Boston Harvarel Medical School, 300 Lougwood Ave., Boston, MA 02115, USA Bodi Zhang, MD/MPH Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111, USA Binhua P. Zhou Markey Cancer Center, University of Kentucky School of Medicine, Lexington, KY, USA and Department of Molecular and Cellular Biochemistry, University of Kentucky School of Medicine, Lexington, KY, USA

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Margot Zöller Department of Tumor Cell Biology, University Hospital of Surgery, Im Neuenheimer Feld 365, D-69120, Heidelberg, Germany and Department of Tumor Progression and Immune Defense, German Cancer Research Center, Heidelberg, Germany and Department of Applied Genetics, University of Karlsruhe, Karlsruhe, Germany Stanley Zucker Veterans Affairs Medical Center, Northport, NY 11768, USA and Stony Brook University, Stony Brook, NY 11794, USA

Contributors

Part I

Physiological Parameters

Chapter 1

Combination Strategies Targeting Hypoxia Inducible Factor 1 (HIF-1) for Cancer Therapy Annamaria Rapisarda and Giovanni Melillo

Abstract Solid tumors often present regions of decreased oxygen levels (hypoxia) due to an imbalance between increased oxygen consumption and insufficient oxygen delivery from the aberrant tumor vasculature. Intratumor hypoxia is associated with altered cellular metabolism, an invasive and metastatic phenotype, as well as resistance to radiation and chemotherapy. The discovery of Hypoxia Inducible Factor-1 (HIF-1), a transcription factor critically involved in cellular responses to hypoxia and tumor progression, has provided evidence of a potential molecular target of intratumor hypoxia that could be exploited for the development of novel cancer therapeutics. A growing number of small molecule inhibitors of HIF-1, which act by distinct molecular mechanisms, have been described so far. However, HIF-1 expression in human cancers is focal and heterogeneous, consistent with the possibility that single agent HIF-1 inhibitors may have limited clinical activity. It is then plausible that combination strategies aimed at maximizing the clinical potential of HIF-1 inhibition may be more effective. We will discuss current approaches used for targeting HIF-1, emphasizing in particular opportunities for rationally designed combination strategies aimed at exploiting vulnerable features of the tumor microenvironment.

Introduction Whereas each tumor type reflects a heterogeneous collection of genetic and epigenetic alterations, hypoxia represents a unifying characteristic of human tumors, being a widely represented feature of the tumor microenvironment. Intratumor hypoxia not only affects cells that are located far from a functional blood vessel but also originates from an imbalance between increased oxygen consumption of rapidly G. Melillo (*) DTP-Tumor Hypoxia Laboratory, Bldg. 432, Room 218, SAIC – Frederick, Inc., NCI at Frederick, Frederick, MD 21702, USA e-mail: [email protected] R.G. Bagley (ed.), The Tumor Microenvironment, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-6615-5_1, © Springer Science+Business Media, LLC 2010

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A. Rapisarda and G. Melillo

growing tumor cells and inadequate oxygen supply from a structurally and functionally abnormal tumor vasculature. The fluctuation of oxygen levels in the tumor microenvironment results in the induction of an array of responses in tumor cells, such as inactivation of apoptotic pathways and activation of pro-survival pathways, induction of a more invasive and metastatic phenotype, switch to a glycolytic metabolism and induction of angiogenesis, responses aimed at providing adaptation to, or escape from, the hypoxic microenvironment. Clinically, hypoxia is associated with resistance to standard treatment, including chemotherapy and radiation therapy, and is predictive of metastasis and poor outcome in a variety of tumor types (Hockel and Vaupel 2001; Brown and Giaccia 1998; Brizel et al. 1996; Semenza 2007). Hypoxia controls the expression of hundreds of genes, many of which are regulated by Hypoxia Inducible Factor-1 (HIF-1), a master regulator of the transcriptional response to oxygen deprivation (Semenza 2007; Manalo et al. 2005). HIF-1 is a heterodimeric protein comprised of a constitutively expressed HIF-1b (also known as aryl hydrocarbon receptor nuclear translocator, ARNT) subunit and a HIF-a subunit, which is regulated by oxygen concentrations (Wang et al. 1995; Wang and Semenza 1995). Under normoxic onditions, HIF-a is rapidly hydroxylated on two proline residues by oxygen-dependent prolyl hydroxylases (PHDs) and targeted for ubiquitylation and proteasomal degradation in a VHL-dependent fashion. In addition, HIF-1a can be hydroxylated on an asparagine residue (Asn803) by Factor Inhibiting HIF-1 (FIH), hence inhibiting the recruitment of the co-activator p300/CBP and transcriptional activity. In contrast, under hypoxic conditions the HIF-a subunit is stabilized and translocates to the nucleus where it dimerizes with HIF-1b and, by binding to hypoxia responsive elements (HREs), activates transcription of hundreds of target genes involved in key steps of tumorigenesis, including angiogenesis, metabolism, proliferation, metastasis, and differentiation (Semenza 2008) (Fig. 1.1). Overwhelming evidence indicates that HIF-a (HIF-1a and/or HIF2a) is indeed over-expressed in the majority of human cancers (Zhong et al. 1999; Talks et al. 2000; Bos et al. 2001), where it is associated with patient mortality and poor response to treatment (Table 1.1) (Aebersold et al. 2001; Birner et al. 2000, 2001b; Bos et al. 2003; Koukourakis et al. 2006). Notably, increased HIF-1a expression is associated with a variety of genetic alterations frequently detected in human cancers, including loss of function of tumor suppressor genes, such as VHL, PTEN, and CDKN2A (which encodes ARF) or oncogenic gain of function, such as RAS, SRC, and BCR-ABL (Semenza 2003). In addition, the involvement of receptor tyrosine kinase-dependent signaling pathways, including EGFR, HER2/Neu, and the PI3K/AKT/mTOR and MAPK pathways, in the induction of HIF-1a expression also suggests that many of these pathways converge on or implicate HIF-1 in mediating cell survival and growth (Pouyssegur et al. 2006; Majumder et al. 2004; Pore et al. 2006; Hudson et al. 2002). Therefore, HIF-1 has become an attractive target for the development of novel cancer therapeutics and despite the challenges associated with the discovery and development of small molecule inhibitors of transcription factors, many HIF-1 inhibitors which may potentially be useful for cancer therapy have been described so far (Onnis et al. 2009; Melillo 2007; Welsh et al. 2006; Melillo 2006).

1 Combination Strategies Targeting Hypoxia Inducible Factor 1

5

Angiogenic factors

(VEGF, FGF2, PDGF, PIGF, Tie2)

VHL binding Proteosomal degradation

Inhibition of transcriptional activity

P402 OH

Drug resistance

(Telomerase, Oct4)

(MDR1/ABCB-1, ABCG-2)

Glycolitic metabolism, pH regulation

HIF- α P564 OH HIF- α N803OH PHDs

RTKs/mTOR

(GLUT1, PDK1, PGK1, HK2, LDH-A, CA9, MCT4)

FIH

Survival, Proliferation

HIF- α

HIF-1 translation

mRNA transcription

O2 concentration

Immortalization and stem cells

β

α

Dimerization Cofactors recruitment DNA binding

(Cyclin D1, VEGF, IGF2, TGF-α)

Migration (CXCR4, SDF-1 α)

Invasion Metastasis (UPAR, Met, LOX)

Fig. 1.1 Regulation of Hypoxia Inducible factor-a (HIF-a) and HIF-1 dependent gene expression. Under normal oxygen conditions, HIF-a is continuously translated and rapidly degraded through the VHL-proteasome pathway following hydroxylation of proline residues by PHDs. Factor inhibiting HIF-1 mediates the hydroxylation of an asparagine residue, thus inhibiting the recruitment of the co-activator p300/CBP and HIF-1 transcriptional activity. All these processes are inhibited when oxygen concentration decreases, hence the HIF-a subunit is stabilized, translocates to the nucleus where it dimerizes with HIF-1b and induces the transcription of a large number of genes involved in crucial aspects of tumorigenesis. In addition, receptor tyrosine kinase-dependent signaling pathways can increase the rate of HIF-1a translation, at least in part, through the activation of mTOR

Small Molecule Inhibitors of HIF-1 The approach most widely used so far to identify HIF-1 inhibitors has been cell-based high throughput screens (HTS) using reporter gene assays (Rapisarda et al. 2002). The main advantage of cell-based HTS is the potential for the identification of unrecognized pathways associated with the activation of the target being investigated. However, determining the mechanism of action of active “hits” may be time consuming and chances to identify highly specific inhibitors is very low. Indeed, the majority of HIF-1 inhibitors described so far lack specificity, which complicates the pharmacological validation of HIF-1 inhibitors for cancer therapy, and either target signaling pathways that are involved in HIF-1 regulation or inhibit HIF-1a protein accumulation and/or transcriptional activity. Examples of agents that target signaling pathways implicated in the HIF-1a expression include

6


Table 1.1 Human cancers in which HIF-1a over-expression has been associated with patient mortality Cancer type

Reference

Astrocytoma (diffuse) Bladder (superficial urothelial)a Bladder (transitional cells) Breast Breast (c-Erb2 positive) Breast (positive linfonode) Breast (negative linfonode) Cervix (early stage) Cervix (radiation therapy) Cervix (grade IB-IIIB, radiation therapy) Endometrial Gastric Gastrointestinal stromal tumor (stomach) Lung (nonsmall cell lung carcinoma) Melanoma (malignant)b Oligodendrioglioma Oropharynx-squamous cell carcinoma Ovariana Pancreas

Korkolopoulou et al. (2004) Theodoropoulos et al. (2004) Theodoropoulos et al. (2005) Vleugel et al. (2005) and Dales et al. (2005) Giatromanolaki et al. (2007) Schindl et al. (2002) Bos et al. (2003) Birner et al. (2000) Burri et al. (2003) Bachtiary et al. (2003) Sivridis et al. (2002) Griffiths et al. (2007) Takahashi et al. (2003) Swinson et al. (2004) Giatromanolaki et al. (2003) Birner et al. (2001a) Aebersold et al. (2001) Birner et al. (2001b) Sun et al. (2007)

Combination of HIF-1a over-expression and mutant p53 HIF-2a over-expression

a

b

inhibitors of mTOR (Majumder et al. 2004; Hudson et al. 2002), AKT (Tan et al. 2005), Her2/Neu (Laughner et al. 2001), EGFR (Pore et al. 2006; Koukourakis et al. 2003; Zhong et al. 2000), and Bcr-Abl (Mayerhofer et al. 2002) (Fig. 1.2). Since HIF-1a accumulation is controlled primarily at the level of protein translation or protein degradation, many of the HIF-1 inhibitors identified thus far do indeed decrease HIF-1a protein levels by a number of distinct molecular mechanisms. HIF-1 inhibitors that affect protein translation, although by mechanisms not always clearly elucidated, include topoisomerase I (topotecan and EZN-2208, a pegylated form of SN38) (Rapisarda et al. 2004a, b; Sapra et al. 2008) and topoisomerase II inhibitors (NSC644221) (Creighton-Gutteridge et al. 2007), microtubule stabilizing (taxanes and epothilones) and destabilizing agents (Mabjeesh et al. 2003; Escuin et al. 2005), cardiac glycosides (Zhang et al. 2008), PX-478, which also increases HIF-1a degradation (Koh et al. 2008) and farnesyltransferase inhibitors (Han et al. 2005). Agents that appear to affect primarily HIF-1a degradation include inhibitors of the chaperon protein Hsp90 (17-AAG, 17-DMAG) (Isaacs et al. 2002; Mabjeesh et al. 2002), and HDAC inhibitors (vorinostat, LAQ824, FK228) (Ellis et al. 2009), which may also block HIF-1a transcriptional activity (Kong et al. 2006), and YC-1, which activates soluble guanylate cyclase (sGC) (Kim et al. 2006a). Another mechanism by which HIF-1 may be inhibited is at the level of transactivation. In addition to chetomin (Kung et al. 2004), which


HIF-1 β

α

Co-factors Recruitment Transcriptional activity DNA binding

NSC50352

7 Chetomin Bortezomib Vorinostat Amphotericin

HIF-1-dependent gene expression

Echinomycin Polyamides Doxorubicin

Dimerization

HIF-1 β 17AAG,17DMAG YC-1, SCH66336 NS398, ibuprofen celecoxib

HIF-1 α

RTKs/mTOR

Translation Degradation

Temsirolimus Everolimus Gefitinib Erlotinib Cetuximab Herceptin

TPT,SN38 NSC644221, Digoxin Candidaspongiolide PX478, 2ME2

Proteasome HIF-1 α mRNA

HIF-1 α antisense RNA Aminoflavone

Fig. 1.2 Signaling pathways targeted by small molecule inhibitors of HIF-1

was originally identified as a small molecule that interferes with the interaction between HIF-1a C-TAD and p300/CBP, inhibition of the proteasome (bortezomib, Velcade®) has also been associated with blockade of HIF-1 transcriptional activity, despite the paradoxical increase of HIF-1a protein, although the exact mechanism of action remains to be fully elucidated (Shin et al. 2008; Kaluz et al. 2008). Finally, modulation of HIF-1a mRNA levels using antisense oligonucleotides (EZN-2968) (Greenberger et al. 2008) or the AhR ligand aminoflavone (Terzuoli et al. submitted) also leads to inhibition of HIF-1 expression. However, in order to identify HIF-1 small molecule inhibitors with a higher degree of selectivity, efforts are ongoing to target protein–protein interaction, a challenging yet potentially highly rewarding strategy, in an attempt to interfere with HIF-a/HIF-1b dimerization (Yang et al. 2005; Park et al. 2006; Scheuermann et al. 2009). In addition, examples of HIF-1 inhibitors that affect HIF-1 DNA binding to the HRE have been provided, including synthetic polyamides (Olenyuk et al. 2004), echinomycin (Kong et al. 2005), and anthracyclines (Lee et al. 2009), which represent a conceptually attractive strategy to inhibit HIF-1. Many of the HIF-1 inhibitors described above are either FDA approved or are in early clinical development (Table 1.2). Results of ongoing clinical trials of HIF-1 inhibitors in patients with cancer will provide information regarding both inhibition of HIF-1 and potential clinical activity of this emerging therapeutic approach.

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Table 1.2 HIF-1 inhibitors FDA approved or in clinical development with potential mechanism of action Agent

Target

Mechanism

Reference

EZN-2968 Aminoflavone

HIF-1a mRNA AhR

Greenberger et al. (2008) Terzuoli, submitted

Topotecan EZN-2208 PX-478 Erlotinib, gefitinib, cetuximab Trastuzumab Temsirolimus, everolimus 17AAG, 17DMAG

Topoisomerase 1 Topoisomerase 1 unknown EGFR

Degradation HIF-1a mRNA processing Translation Translation Translation Signaling

Her2/Neu mTOR

Translation Translation

HSP90

Degradation

Doxorubicin Bortezomib

DNA Proteasome

Vorinostat, LAQ824, FK228

HDAC

DNA binding Transcriptional activity Degradation, transcriptional activity

Rapisarda et al. (2004b) Sapra et al. (2008) Welsh et al. (2004) Pore et al. (2006) and Luwor et al. (2005) Koukourakis et al. (2003) Del et al. (2006) and Wan et al. (2006) Isaacs et al. (2002) and Mabjeesh et al. (2002) Lee et al. (2009) Kaluz et al. (2006) Kong et al. (2006), Mie et al. (2003), and Qian et al. (2006)

Targeting HIF-1: Single Agent or Combination? The potential therapeutic benefit of HIF-1 inhibition has been extensively documented in preclinical models using both genetic and pharmacological tools, and consistent inhibition of tumor growth and angiogenesis has been demonstrated in different tumor types (Semenza 2003; Melillo 2006). However, the lack of selective HIF-1 inhibitors and reliable biomarkers associated with HIF-1 inhibition in tumor tissue, associated with the hardly predictive nature of results obtained in xenograft models, have significantly hampered the validation of HIF-1 as a target in human cancers. For instance, it is still largely unclear whether tumor regression should be an expected, although desirable, outcome of single agent HIF-1 inhibition. Evidence has been provided that HIF-1 inhibition might be most effective when used in early, rather than late, stages of tumor progression (Li et al. 2005), a situation rarely encountered in clinical oncology practice, where the majority of patients present with metastatic disease. In addition, even conceptually, inhibition of HIF-1 expressing cells, as a single agent strategy, may not be particularly effective for a number of reasons, including the focal and heterogeneous expression of HIF-1a in solid tumors, the reliance of cancer cells on HIF-1-independent pathways in oxygenated areas of the tumor, and the overall well-established redundancy of oncogenic signaling pathways that may be hardly affected by single agent strategies. In addition, the broad involvement of HIF-1 in a number of biological pathways that are


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relevant for tumorigenesis provides unique opportunities for combination therapies that may take advantage of the different approaches available in the current therapeutic armamentarium. A better understanding of the role that HIF-1 plays in the biology of human cancers and in the context of available therapeutic strategies will provide valuable information for the design of rational combinations that may target vulnerable aspects of the tumor microenvironment.

Molecularly Targeted Agents and HIF-1 Inhibition Combination strategies aimed at targeting multiple and redundant signaling pathways that contribute to oncogenesis are a mainstay of current therapeutic efforts in oncology for the potential to both prevent the emergence of resistant clones and translate in a better therapeutic outcome. Indeed, combinations of molecularly targeted agents, with or without chemotherapy, are currently being tested in virtually any tumor type with the goal of assessing safety of administration and potential therapeutic efficacy. As mentioned above, several novel targeted agents currently in clinical development or FDA approved also inhibit HIF-1, raising the possibility that combination strategies incorporating these agents may already provide examples in which inhibition of HIF-1 may potentially contribute to the therapeutic efficacy. Experimental evidence in support of this conclusion has been provided by combination of the mTOR inhibitor rapamycin with the HDAC inhibitor LBH589, which showed increased antitumor activity in prostate and renal cell cancer xenografts models (Verheul et al. 2008). Interestingly, the concomitant inhibition of HDACs and mTOR resulted in a greater inhibition of HIF-1a protein levels relative to what was achieved by each agent alone, possibly due to the activity of these inhibitors at different levels of HIF-1a regulation including translation, protein stability, and transcriptional activity. In addition, experiments conducted in colon cancer xenograft models using low doses of rapamycin in combination with the camptothecin analog irinotecan (a topoisomerase I inhibitor) showed a marked increase in antitumor activity associated with a profound inhibition of HIF-1a protein accumulation that was not observed with either agent alone (Pencreach et al. 2009). Interestingly, previous studies in a human glioma xenograft model had suggested that daily, but not intermittent, administration of single agent topotecan, a topoisomerase I HIF-1 inhibitor, downregulated HIF-1a expression in tumor tissue and caused inhibition of tumor growth and angiogenesis (Rapisarda et al. 2004c). These examples emphasize how HIF-1 inhibition may be an underappreciated therapeutic consequence of molecularly targeted agents currently in clinical development. A better understanding of the potential contribution of HIF-1 inhibition to therapeutic activity of these combinations may help in selecting patients more likely to respond and in designing optimal schedules of administration to maximize inhibition of HIF-1-dependent responses.

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Hypoxic Cells Are More Resistant to Chemotherapy and Radiation Therapy It has long been recognized that hypoxic cells are more resistant to conventional therapeutic approaches, including chemotherapy and radiation therapy. Likewise, it is well appreciated that resistant hypoxic cells may contribute to treatment failure and poor prognosis of patients whose cancers are more hypoxic. Resistance to radiotherapy and chemotherapy in hypoxic tumors has been attributed to a multitude of mechanisms, including direct effects (decreased generation of oxygendependent radical formation or decreased drug accessibility to the more distant hypoxic cells) and indirect effects (clonal selection of cells adapted to hypoxia) (Brown and Giaccia 1998; Vaupel et al. 2001). Hypoxia exerts selective pressure on cells for loss of p53, regulates the transcription of several genes pivotal for the selection of a resistant phenotype, such as multidrug resistance gene 1 (MDR-1) and ABCG-2 and ABCB-1 (ABC transporters), induces expression of VEGF and other pro-angiogenic factors (important for the survival of endothelial cells following radiation), downregulates genes involved in DNA mismatch repair (MSH2, MSH6, NSB1, and BRCA1) leading to increased genomic instability (Huang 2008), and finally induces genes important for survival of cancer cells (Bertout et al. 2008; Huang 2008). Many of the effects described above are mediated by HIF-1; therefore, it is conceivable that inhibition of HIF-1 may contribute in a number of ways to combination therapies by counteracting mechanisms that are activated in the tumor microenvironment to generate resistance and by sensitizing cancer cells to available therapeutic strategies.

Combination of HIF-1 Inhibitors with Chemotherapy The potential contribution of HIF-1a to cellular resistance to chemotherapy has been suggested by evidence in both experimental models and the clinical setting. Initial clinical observations indicated that high levels of HIF-1a expression were associated with incomplete responses to radiation and chemotherapy in patients with head and neck squamous cancers (Aebersold et al. 2001; Koukourakis et al. 2003). Formal evidence that HIF-1a may be implicated in mediating resistance to conventional chemotherapeutic agents was provided by studies showing that MDR-1, one of the most widely expressed efflux pumps that mediate resistance to chemotherapy, is a direct HIF-1 target gene (Comerford et al. 2002). In addition, it was shown that HIF-1a deficient mouse embryonic fibroblasts were more sensitive to carboplatin, etoposide (a topoisomerase II inhibitor), and irradiation (agents that cause double-strand DNA breaks) but not to the topoisomerase 1 inhibitor SN38 (that generates single-strand DNA breaks) (Unruh et al. 2003), suggesting that HIF-1-mediated resistance to chemotherapy cannot simply be explained by upregulation of MDR-1. Resistance to etoposide, a


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topoisomerase II inhibitor, also appears to correlate with HIF-1-dependent induction of anti-apoptotic genes and downregulation of topoisomerase IIa (Sermeus et al. 2008; Sullivan and Graham 2009). The potential synergistic activity of HIF-1 inhibition in combination with chemotherapy has been formally tested in a glioma xenograft model in which HIF-1a expression was downregulated by an inducible shRNA system (Li et al. 2006). Earlier evidence had indicated that HIF-1a downregulation by shRNA effectively inhibited tumor growth, although the effect was more pronounced in early rather than late stages of tumor progression (Li et al. 2005). The combination of the cytotoxic drug temozolomide with HIF-1a knockdown exhibited a super-additive therapeutic effect compared with either approach alone, demonstrating that HIF-1 inhibition impacted on sensitivity to chemotherapy (Li et al. 2006). However, the same authors showed no benefit of a combination between HIF-1 inhibition and the angiogenesis inhibitor ABT-869 (a multitargeted receptor tyrosine kinase inhibitor). It is interesting to note that following treatment with ABT-869, large tumors that are less likely to be responsive to HIF-1a inhibition became sensitive to HIF-1a knockdown, suggesting that anti-angiogenic treatment might partially alleviate the “resistance” of large tumors to HIF-1a knockdown and sensitize tumors to HIF-1 inhibition. Further studies in preclinical models, ultimately with validation in clinical trials, are required to fully explore the potential of HIF-1 inhibition to modulate cellular sensitivity to chemotherapeutic agents and to block stress pathways induced by chemotherapy in the tumor microenvironment. Combination of HIF-1 Inhibitors with Radiation Therapy Hypoxia is an important factor contributing to tumor radioresistance, a phenomenon initially explained by the reduced generation of oxygen radicals in hypoxic tissues and consequent decrease in radiation-induced DNA damage (Moeller et al. 2007). In reality, more complex biological pathways are implicated in the effects of radiation therapy on hypoxic tumors. Clinical studies have suggested that high levels of HIF-1a expression correlate with poor local control in patients with oropharyngeal cancers (Aebersold et al. 2001) and evidence from preclinical studies has indicated that HIF-1 deficient tumors are more radiosensitive than their wild-type counterpart (Zhang et al. 2004; Moeller et al. 2005; Williams et al. 2005). Moreover, it has been shown that radiation increases HIF-1a levels and induces the expression of HIF-1-dependent genes in tumors (Moeller et al. 2004), consistent with the possibility that HIF-1 activation may be part of compensatory pathways that might mediate resistance to radiation therapy. However, further studies have shown that HIF-1 may have divergent effects on radiosensitivity. On the one hand, HIF-1 might be involved in the induction of apoptosis by increasing p53 phosphorylation following radiation treatment, thus sensitizing cancer cells to radiation (Moeller et al. 2005). On the other side, it is well appreciated that the tumor vasculature is an important target of radiation therapy and a major determinant of response

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(Garcia-Barros et al. 2003), thus the induction of angiogenic factors, including VEGF, by HIF-1 may actually protect endothelial cells from radiation-induced apoptosis (Gupta et al. 2002). Indeed, combination of the HIF-1 inhibitor YC-1 with radiation blocked radiation induced HIF-1 upregulation and resulted in significant vascular destruction, suggesting that combining HIF-1 blockade with radiation may increase antitumor efficacy (Moeller et al. 2004). In addition, the HIF-1 inhibitor PX-478 increased therapeutic efficacy of radiation by blocking HIF-1-dependent effects on the tumor microenvironment in addition to a direct effect on tumor cell survival (Schwartz et al. 2009). Finally, recent evidence also suggests that HIF-2a inhibition sensitizes renal cancer cells to radiation therapy by increasing p53 activity and inducing apoptosis (Bertout et al. 2009). Given the complexity of the role that HIF signaling may play in the response of tumor tissue to radiation therapy, careful consideration should be given to the identification of tumor types and features of the tumor microenvironment that may predict for or be associated with a positive interaction between radiation therapy and HIF-1 inhibition.

Intratumor Hypoxia as a Potential Mechanism of Resistance to Anti-angiogenic Therapies The excitement associated with the introduction of anti-angiogenic agents for cancer therapy has been partially blunted by the limited single agent activity of this therapeutic strategy and by the development of resistance, a phenomenon originally unanticipated for an approach targeted to “normal,” genetically stable endothelial cells. While anti-angiogenic therapies have shown clinical benefit in combination with chemotherapy in metastatic colorectal cancer, advanced nonsmall cell lung cancer, and metastatic breast cancer (Ferrara 2005; Shojaei and Ferrara 2007; Ellis and Hicklin 2008), the single agent activity has been well below expectations and has been demonstrated only in few tumor types, including in renal cell carcinoma and more recently gliomas. More importantly, clinical benefit has only been transient and most tumors eventually become resistant and relapse. In addition to “intrinsic” resistance in patients who do not respond to anti-angiogenic agents, several mechanisms of “acquired” resistance have been proposed, including upregulation of alternative pro-angiogenic signals, production of pro-angiogenic factors by stromal cells, recruitment of bone marrow derived pro-angiogenic cells, increased pericyte coverage of the vasculature, and activation of invasive phenotypes (Rapisarda and Mellilo 2009). Interestingly, many of the responses implicated in resistance to anti-angiogenic therapy may be mediated by the products of genes induced by HIF-1. The consequences of effective anti-angiogenic therapy on the tumor microenvironment are still poorly understood and partly controversial, which limits our ability to develop rational combination therapies. On the one hand, “normalization” of tumor vasculature has been proposed, with consequent decrease in interstitial pressure and


13

better delivery of chemotherapy (Jain 2005); on the other hand, vascular “regression” has been demonstrated in several experimental models and appears to be a logical consequence of effective inhibition of tumor vasculature (Kerbel and Folkman 2002). The latter scenario is associated with an increase in intratumor hypoxia, which not only might be mechanistically involved in mediating resistance to anti-angiogenic therapies but also might provide unique opportunities for combination strategies (Paez-Ribes et al. 2009; Loges et al. 2009). Indeed, while expression of HIF-1a in solid tumors is focal and heterogeneous, detected predominantly in perinecrotic areas, “therapy-induced” hypoxia, which may be associated with the administration of antiangiogenic therapies, will lead to a more persistent and protracted expression of HIF-1a. Higher levels of HIF-1 in the tumor microenvironment may not only contribute to drug resistance in general, but also provide a mechanism of “escape” for tumor cells overcoming the potential therapeutic effects of anti-angiogenic agents. Thus, it is conceivable that combination of anti-angiogenic therapy with HIF-1 inhibition may have synergistic therapeutic activity by targeting HIF-1 in the context of a “hypoxic stressed” tumor microenvironment. Combination of Anti-angiogenic Therapies and HIF-1 Inhibitors To test the hypothesis that anti-angiogenic therapies may be more efficacious in combination with HIF-1 inhibition, we combined topotecan, a topoisomerase I inhibitor that downregulates HIF-1a protein in vitro and in vivo (Rapisarda et al. 2004b, c), with the anti-VEGF antibody bevacizumab in a glioma xenograft model. Indeed, we found that inhibition of HIF-1a by topotecan in a hypoxic stressed tumor microenvironment resulted in a more pronounced antitumor effect, relative to either agent alone. The effects on tumor growth were associated with significantly decreased HIF-1 transcriptional activity and reduced tumor cell proliferation (Rapisarda et al. 2009), consistent with the hypothesis that targeting HIF-1a activity may abrogate compensatory pathways required for cancer cell survival. Moreover, recent studies showed that genetic disruption of both HIF-1a and HIF-2a expression in colon cancer xenografts improves tumor response to sunitinib (a multitargeted receptor tyrosine kinase inhibitor) (Burkitt et al. 2009). This effect was mediated by a marked decrease in tumor angiogenesis and perfusion, through inhibition of multiple pro-angiogenic factors, as well as a marked decrease in tumor cells proliferation. To further support the role that HIF-1 inhibition might play in combination with anti-angiogenic therapies, it is interesting to point out that the combination of bevacizumab and irinotecan (another topoisomerase I inhibitor that also inhibits HIF-1) has shown clinical benefit in glioblastoma patients with a six-month overall survival of 62–77% (Vredenburgh et al. 2007; Chen et al. 2007). Further studies are required to better define the patient population that may benefit from these combination approaches, in particular by better understanding when and to what extent anti-angiogenic therapy is indeed associated with increased intratumor hypoxia and HIF-1a expression.

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Cancer Cell Metabolism and the Hypoxic Tumor Microenvironment More than 80 years ago, Otto Warburg observed that cancer cells avidly take up glucose and produce lactic acid under aerobic conditions, a process subsequently referred to as the Warburg effect or aerobic glycolysis. The exact molecular mechanism(s) regulating cancer cells dependence on glycolysis remain unclear, but it is likely that a combination of effects due to oncogenic alterations and the hypoxic tumor microenvironment play a role. Many genetic alterations associated with the cancer phenotype promote glycolysis and, at the same time, suppress oxidative phosphorylation. For instance, p53 plays an important role in regulating both “aerobic” and “anaerobic” respiration, while c-Myc expression activates the transcription of several glycolytic enzymes (Kroemer and Pouyssegur 2008). The largest functional group of genes consistently upregulated by HIF-1 in a number of cell types is associated with glucose metabolism. HIF-1 increases the expression of the transporters necessary for the entry of glucose into the cell, the genes involved in the enzymatic breakdown of glucose to pyruvate and the enzymes involved in the metabolism of pyruvate. In hypoxic cells, pyruvate is converted by lactate dehydrogenase (LDH) to lactate, which is then released into the extracellular space. HIF-1 also controls cellular oxygen consumption by (a) inducing the expression of PDH kinase 1 (PDK1), which inhibits pyruvate dehydrogenase (PDH) and the conversion of pyruvate to acetyl-CoA, ultimately decreasing mitochondrial respiration (Papandreou et al. 2006; Kim et al. 2006b) and (b) reducing mitochondrial mass, by counteracting the stimulatory action of c-Myc on mitochondrial biogenesis (Zhang et al. 2007). It is conceivable then that these metabolic changes utilized by cancer cells to survive in a hypoxic, nutrient deprived tumor microenvironment might also offer therapeutic opportunities to selectively target hypoxic cells and spare normal tissues.

HIF-1 Inhibitors in Combination Strategies Targeting Tumor Metabolism Cancer cells, but not normal cells, are thought to be “addicted” to glycolysis and attempts are being pursued to explore the possibility to target glycolysis for cancer therapy. However, the activity of 2-deoxyglucose (2-DG, a competitive inhibitor of glucose), as single agent, has been limited by evidence of toxicities in clinical trials (Denko 2008). Interestingly, genetic knockdown of HIF-1a, as well as treatment with CCI-779 (an inhibitor of mTOR that decreases HIF-1 activity), sensitizes cancer cells in vitro to 2-DG (Maher et al. 2007; Wangpaichitr et al. 2008), suggesting that HIF-1 inhibitors might be used to increase the therapeutic efficacy of 2-DG. An original approach has been proposed to increase oxygen consumption in cancer cells by reversing the inhibition of mitochondrial function using either the HIF-1 inhibitor echinomycin or the PDK-1 inhibitor dichloroacetate (DCA) (Cairns et al. 2007). The pharmacological induced increase in oxygen consumption, associated


15

with the limited oxygen supply due to the dysfunctional tumor vasculature, made the tumors more hypoxic and sensitized cancer cells to the hypoxic cytotoxin tirapazamine. These data suggest that HIF-1 inhibitors might have a potential role in combination strategies aimed at targeting tumor metabolism or oxygen consumption.

Conclusion In the complex biology of human cancers, HIF-1 is yet another attractive target for the development of cancer therapeutics. The role of HIF-1 in human cancers has been extensively investigated and accumulating evidence implicates HIF-1 in a number of biological processes associated with resistance to therapy and tumor progression. Hence, HIF-1 inhibition appears as a logical therapeutic strategy and many small molecule inhibitors of HIF-1 have been described, several of which are validated in preclinical models. However, we have still limited understanding of when and to what extent inhibition of HIF-1 in cancer patients may be effective. Single agent studies are required to have a better appreciation of the biological consequence associated with HIF-1 inhibition in human cancers, yet the lack of selective pharmacological inhibitors of HIF-1 significantly hinders this task. On the other hand, we have learned over the last few years that targeting multiple signaling pathways deregulated in cancer cells may be a more effective therapeutic strategy. The broad involvement of HIF-1 in many biological processes associated with and required for tumor progression provides unique opportunities for the development of combination therapies. A better understanding of the role played by HIF-1 in pathways associated with the administration of chemotherapy, radiation and anti-angiogenic therapies may unveil vulnerable aspects of the tumor microenvironment that could be exploited for the rational design of combination strategies. Acknowledgments The authors would like to thank members of the Tumor Hypoxia Laboratory and Dr. R. H. Shoemaker for helpful discussion. This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. N01-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government. This research was supported in part by the Developmental Therapeutics Program, DCTD, of the National Cancer Institute, NIH.

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Chapter 2

The Tumor Microenvironment: New Insights into Regulation of Tumor pH by Carbonic Anhydrases Pawel Swietach, Adrian L. Harris, and Richard D. Vaughan-Jones

Abstract Intracellular pH (pHi) is a powerful modulator of cell function. There is a narrow range of pHi, usually between 7.0 and 7.5, over which cell growth and development is optimal. Metabolism, particularly cellular respiration, tends to disturb this favorable steady state. The survival of cells undergoing such an acid challenge relies on efficient mechanisms for regulating pHi. In solid tumors, pHi regulation is challenged by their heavy demand for energy and poor blood supply. Inadequately perfused cancer cells deprive the milieu of oxygen, forcing a switchover to the less energy efficient, anaerobic mode of respiration. To meet energy demands, cells upregulate respiration and excrete more acid which lowers extracellular pH (pHe). The tumor extracellular milieu is believed to exert Darwinian selection in favor of cancer cells and against normal cells, based on the cell’s capacity to protect pHi under hypoxia and low pHe. In this chapter, we describe the pathways operating in tumors for removing acid and regulating pHi, with particular emphasis on membrane transporters and carbonic anhydrase (CA) enzymes. We discuss these pathways in terms of survival adaptations and possible targets for anticancer therapy.

Biological Importance of pH The hydrogen atom is very common in the human body, and despite being the lightest of elements, it comprises about a tenth of total body mass. Virtually all hydrogen atoms are bonded by means of electron transfer or sharing with other elements. In many such compounds, the hydrogen nucleus can dissociate reversibly from the remainder of the molecule. In the case of the most common isotope 1 H, the hydrogen nucleus is solely a proton, and therefore the H+ dissociation and

A.L. Harris (*) Weatherall Institute of Molecular Medicine, Oxford University, Oxford OX3 9DS, UK e-mail: [email protected] R.G. Bagley (ed.), The Tumor Microenvironment, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-6615-5_2, © Springer Science+Business Media, LLC 2010

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association reactions are dubbed deprotonation and protonation, respectively. Water, the biological solvent that contributes towards 60% of the body’s mass, also undergoes reversible deprotonation, and in pure form equilibrates at a “neutral” H+ ion concentration ([H+]) of 1027 M. Acids and bases are defined as molecules that increase and decrease [H+], respectively. To avoid the use of exponential notation, the acidity/basicity of a solution is commonly expressed as the negative logarithm of molar [H+], and given the symbol pH (Boron 2004). Many biologically relevant molecules are weak acids and weak bases. Proteins, for instance, are chains of weak acids/bases. Weak acids/bases exist in dynamic equilibrium between their protonated and unprotonated forms, the concentration ratio of which is very sensitive to pH. Since the H+ ion has very high charge density, pH will have a major impact on the physico-chemical properties of weak acids/ bases. This is particularly relevant to proteins, where charge distribution is linked to structure and function (Boron 2004; Swietach et al. 2007). Indeed, intracellular pH (pHi) is a potent and universal regulator of cellular physiology (Boron 2004; Swietach et al. 2007; Roos and Boron 1981; Vaughan-Jones et al. 2008; Isfort et al. 1993; McConkey and Orrenius 1996). Displacements of pHi by only a fraction of a unit can lead to patho-physiological responses and apoptotic death (Park et al. 1999). Cells must defend their optimal pHi by corrective (homeostatic) mechanisms. A major challenge to these housekeeping mechanisms is metabolic acid production, a process that takes place in all respiring cells throughout their life. To counteract metabolic acid loading, excess intracellular H+ ions must be removed across the cell membrane into the extracellular space and out of the body at the lungs and kidneys. Despite its tiny radius, the highly-polarizing H+ ion is poorly permeant across the lipid bilayer of cell membranes. To allow transmembrane H+ - traffic, cells have evolved transport proteins for H+ ions (e.g. Na+/H+ exchange), or other means of facilitated H+ ion diffusion (e.g. its reaction with HCO3– to form membrane-permeant CO2). The performance of these mechanisms can be compromised by the accumulation of extracellular acid, either as a result of allosteric H+ - inhibition of transport proteins (Vaughan-Jones and Wu 1990; Aronson 1985) or by the collapse diffusion gradients that drive acid-efflux (Swietach et al. 2008, 2009). Through these mechanisms, extracellular pH (pHe) can feedback onto pHi homeostasis. In turn, pHe depends on the diffusive coupling across the extracellular space and with blood. Efficient pHi regulation is central to the survival of metabolically-active tissues, particularly those with relatively poor blood perfusion. Solid tumors are characterized by a high acid production rate (Gatenby and Gillies 2004; Gillies et al. 2008) and inadequate blood supply (Swietach et al. 2007; Vaupel et al. 1989; Kallinowski et al. 1989; Stubbs et al. 2000). Although cancer cells, by definition, harbor a number of mutated gene products, the bulk of cellular protein is no different from normal cells and bears a similar pHi sensitivity, carving a narrow pHi range for survival. The growth of tumors may depend critically on the ability of cancer cells to remove intracellular acid more efficiently than normal cells. The conditions that are characteristic of tumors, notably hypoxia and low pHe (Griffiths et al. 2001; Gatenby et al. 2006), may be tolerated by cancer cells, but not by normal cells, thereby selecting against the latter (Fang et al. 2008). In this chapter, we describe

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the mechanisms for controlling pHi in solid tumors, with particular emphasis on the role of CO2/HCO3– buffer and its enzyme catalyst, carbonic anhydrase (CA).

Sources of Cellular Acid Cellular Respiration A large number of biochemical reactions produce or consume H+ ions, but production of lactic acid by anaerobic respiration and carbon dioxide (CO2) by aerobic respiration are quantitatively the most important acid fluxes that can alter pHi. Glucose, the major respiratory substrate in tumors, produces 2 × H+ plus 2 × lactate (i.e. ionized lactic acid) by the cytosolic O2-independent glycolytic pathway, or 6 × CO2 plus 6 × H2O through O2-dependent mitochondrial pathways. In addition, during periods of nucleic acid synthesis, glucose can yield 1 × CO2 through an additional cytosolic pathway called the pentose phosphate shunt (Helmlinger et al. 2002). Upon hydration, CO2 yields H+ and HCO3−. Assuming equilibrium at pH 7, the molar H+ – yield per aerobically and anaerobically respired glucose is 5.3 and 2.0, respectively (aciddissociation constants for CO2 and lactic acid are 1026.2 and 1024 M, respectively). Tumors demand a particularly high respiratory rate to energize their substantial growth and development. Glucose yields almost 20 times more energy (ATP molecules) when it is respired to CO2 rather than lactic acid. The carbon atoms of lactic acid are less oxidized than those in CO2, and still have considerable energy trapped in their bonds. Indeed, lactic acid can be respired further to CO2. Based on energy efficiency alone, it would be sensible for tumors to prefer aerobic respiration. But to benefit from this higher energy yield, tumors need functional mitochondria and adequate O2 supply. As solid tumors develop, they grow away from their basement membrane and vasculature. This increases the diffusion distance for O2 delivery. Furthermore, the elevated O2 demand in cancer cells that are closer to blood vessels greatly reduces O2 availability in more distal cells, and shortens the distance over which cells are adequately oxygenated. This distance, known as the Krogh radius (Krogh 1919), can be as little as a few cell layers thick in tumors (Dewhirst et al. 1994). Beyond the Krogh radius, tissue becomes hypoxic. In addition to inadequate and highly disorganized vasculature (Thomlinson and Gray 1955), the extent of hypoxia within tumors is increased by variable and discontinuous blood flow (Vaupel et al. 1989; Helmlinger et al. 1997). The size of the hypoxic region can be visualized by pimonidazole staining (Fig. 2.1a). Low O2 tension (50% of patients) (Klein 2008; Wikman et al. 2008) than the rate of metastasis in the BM.

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Therefore, it has been proposed that the bone marrow microenvironment may be able to induce dormancy. BM stores a variety of growth factors including IGFs, TGFb, PDGF, and BMP (Mundy 1997; Mundy 2002; Roodman 2004). In addition, there is experimental evidence showing that TGFb is released from the bone matrix by bone osteoclasts (Guise and Chirgwin 2003). Moreover, majority of breast tumors, including their metastases, are positive for nuclear phosphorylated Smad2, which suggests they have an actively signaling TGFb pathway (Kang et al. 2005; Xie et al. 2002). In addition, Salm et al. (2005) have showed that TGFb is necessary to maintain dormancy of normal prostatic stem cells in the proximal region of ducts. Thus, TGFb is another potential signaling pathways that might participate in the induction of dormancy of DTCs in the BM. TGFb has been shown to exert its anti-proliferative activities by regulating epithelial cell and fibroblasts, which synthesize and secrete a variety of cytokines, growth factors, and extracellular matrix (ECM) proteins that mediate tissue homeostasis and inhibit cancer cell proliferation (Alexandrow and Moses 1995; Huang and Huang 2005). For example, studies show that TGFb stimulation of epithelial cells induces a G1 cell cycle arrest via the transcriptional upregulation of the cyclindependent kinase (CDK) inhibitors p21Cip1/WAF1 and p15Ink4b (Florenes et al. 1996). In addition, TGFb also causes transcriptional repression of transcription factors that regulate proliferation such as c-Myc and the inhibitors of differentiation Id1, Id2, and Id3 (Wu et al. 2009). Therefore, it is possible that TGFb induces quiescence of DTC through any of these mechanisms (Fig. 11.2). TGFb growth response can be inhibited by changes in active signaling networks or by the availability of transcriptional co-repressors or co-activators that regulate the Smad pathway. For instance, c-Ski is an important corepressor of TGF-b signaling that binds to and represses the activity of the Smad proteins. In breast and lung cancer cells, TGF-b induces Ski degradation through the ubiquitin-dependent proteasome and suppresses its ability to inhibit tumor metastasis (Deheuninck and Luo 2009; Reed et al. 2001). It has also been shown that in melanoma cells Id2 upregulation suppresses TGFb induction of p15, and this way circumvent TGFb-mediated inhibition of proliferation (Schlegel et al. 2009). The discovery that TGF-b-induced growth inhibition requires p53 activity suggests that mutational inactivation of p53 may contribute to the development of resistance to TGF-b antiproliferative effects by tumor cells (Atfi and Baron 2008; Cordenonsi et al. 2003). Recently, Adorno et al. (2009) showed that TGFb-dependent metastasis are empowered by mutantp53 and opposed by p63. They showed that TGFb collaborates with Ras and mutant-p53 to induce the assembly of a mutant-p53/p63 protein complex and inhibit p63 functions. They also have identified two candidate metastasis suppressor genes regulated by TGFb and associated with metastasis risk in a large cohort of breast cancer patients. Thus, it is possible that some of the above described regulatory mechanisms could be operational in the BM microenvironment and convert dormant DTC into proliferative active DTCs that may be even able to migrate to other tissues or grow in the bone microenvironment like in breast and prostate cancer. One of the metastasis suppressor genes regulated by TGFb described by Adorno et al. is BHLHB3, which is a transcriptional repressor involved in differentiation

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and a regulator of the circadian rhythm (Azmi et al. 2004; Fujimoto et al. 2007). In addition, in breast cancer, high BHLHB3 expression in primary tumors from patients correlates with good prognosis at 5-year survival (van de Vijver et al. 2002), suggesting that it influences behavior of residual disease. Work from our laboratory has recently showed that BHLHB3 is required for D-HEp3 cell quiescence in vivo and that its expression is repressed by ERK and induced by p38 (Adam et al. 2009). These results suggest that BHLHB3 is a novel negative regulator of tumor growth functionally linked to p38 signaling. Based on these results it is tempting to speculate that regulation of BHLHB3 through TGFb might limit expansion of DTCs into micrometastasis via cellular dormancy (i.e., quiescence) (Fig. 11.2). A study by Hoek et al. (2008) illustrates the dual role of TGFb. They found two different transcriptional signatures – one corresponding to a proliferative melanoma that was sensitive to TGFb inhibition and another corresponding to an invasive melanoma that was resistant to TGFb inhibition. They showed that upon transplantation in nude mice, the proliferative TGFb-sensitive cells produced growing tumors faster than the invasive variant. The authors concluded that there was a switch between both transcription signatures in vivo, probably regulated by environmental conditions (Hoek et al. 2008). Therefore, from all these studies we can conclude that there are two possible scenarios for TGFb regulation of DTCs fate. At early stages of progression, DTCs that lodge in tissues rich in TGFb like the BM, might be sensitive to its antiproliferative effect, and thus, TGFb may induce quiescence of DTC, that can be in that state for months, or even years. It is even possible that TGFB might be produced in an autocrine fashion to maintain dormancy. At a given point, these cells will become resistant to TGFb growth inhibitory effect, either because of changes in the microenvironment or DTCs newly acquired mutations, which will favor expansion.

Models to Study Dormancy Clinical dormancy has long been reported in breast, prostate, head and neck cancer, and melanoma (Fehm et al. 2008; Kell et al. 2000; Klein 2008; Morgan et al. 2007; Ossowski and Aguirre-Ghiso 2010). These dormant DTCs can be detected in the blood and bone marrow of these patients, months, years, and even decades after remission. Detection of these DTCs usually correlates with poor patient outcome (Allan et al. 2006; Braun et al. 2009; Ignatiadis et al. 2008; Janni et al. 2000). DTCs are usually found in the BM, therefore most of the studies regarding the biology of DTC are done in the BM, mainly because it is easier to obtain BM samples. However, there are other tissues where dormant DTC can also be found, for instance, studies in mice injected intravenously with melanoma and breast cancer cells, show a proportion of dormant cells in lungs and liver (Cameron et al. 2000; Logan et al. 2008). Most of the mouse models of cancer reproduce primary tumor development. Much less effort has been dedicated to model the progression of spontaneous MRD

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and metastasis development despite the abundance of mouse transgenic models. Dormancy is a hidden state and thus is difficult to observe and study directly. In addition, characterization of disseminated tumor cells is very complicated because of their low abundance (one disseminated tumor cell per 106 bone marrow cells) (Braun et al. 2009). As we have discussed before, DTCs and primary tumor cells appear to be two different entities. Therefore, the models that we have classically used to study primary tumor growth cannot be used to extrapolate information about the DTCs. Because, metastasis is still the leading cause of mortality in patients and it arises from DTCs, we urgently need new models that better recapitulate MRD and DTC biology so that we can gather more information about the mechanisms that regulate their behavior. This will allow us to design new therapies specifically designed either to eradicate DTCs or to maintain them in a state of dormancy forever. The ideal model of solitary dormant tumor cells would consist of a population of cells which become dormant upon arrival to a secondary site, stay dormant for a period of time that is indicative of a prolonged inactive state (weeks to months), and then escape dormancy to grow. Importantly, it would be ideal that this occurs to all cells that disseminate. If only a subpopulation is dormant and other resume aggressive growth concomitantly in the same organ like in some of the above studies using i.v. injections, this argues that clonal heterogeneity is driving these differences. These may be informative, but if all cells that disseminate enter dormancy in a specific site it suggests more powerful microenvironmental mechanisms driving this phenotypic change independently of the genetics of those tumor cells. In order to study minimal residual disease, we need to have models that better recapitulate what happens in patients. The most commonly used metastatic models generate very rapid development of metastasis or rely on intravenous or intracardial injection and also, in the case of transgenic mice, are accompanied by exaggeratedly large primary tumor masses that shed enormous amounts of cells. Therefore, including surgery of the primary tumors that are smaller than 500 mm3, and then allowing the animals to recover and follow the residual cells might help better model MRD and dormancy. Bone marrow is the most important site to detect DTCs from epithelial tumors (Fehm et al.2008; Pantel et al. 2009; Riethdorf and Pantel 2008). DTCs are present in BM aspirate from 20 to 40% of breast and prostrate patients (Msaouel et al. 2008; Zhang et al. 2009). Spontaneous bone metastasis in animals is uncommon. For instance, most spontaneous mammary carcinomas in mice do not metastasize to bone but readily do to lungs and lymph nodes. However, this does not mean that they do not disseminate to bone and stay dormant. In fact, Husemann et al. (2008) and Husemann and Klein (2009) used HER-2 and PyMT transgenic mice, transplanted with premalignant HER-2 transgenic glands, and showed that they displayed disseminated tumor cells and micrometastasis in bone marrow and lungs. MMTV-Neu cells disseminated to the bone marrow of mice fail to expand unless they are transplanted into recipient mice that were irradiated. This suggests that the bone microenvironment may delay cancer progression and thus prolong patient survival, but also that perturbations of this microenvironment might trigger growth.


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Apart from breast cancer, in prostate cancer, the main cause of death is bone metastasis and BM-DTC can be found in patients (Alix-Panabieres et al. 2005; Morgan et al. 2007). In fact, in a recent publication Morgan et al. (2009) have showed that finding DTC in prostate cancer patients, who have undergone radical prostatectomy and do not show evidence of disease, predicts biochemical recurrence. BM-DTCs were detected in 72% of patients prior to radical prostatectomy. Significant effort has been taken on trying to characterize BM-DTCs. Orthotopic injection of human cancer cells into the prostate usually results in a low incidence of bone metastasis from late-stage cancers. For instance, Vesella and collaborators (2003) has developed a prostate cancer xenograft tumor cell dormancy model. They have used LuCaP 23.8 and LuCaP 35, hormone-sensitive CaP xenografts that express PSA and the wild-type androgen receptor and grew them orthotopically in SCID mice. Once the tumor volumes reached 250–500 mg, primary tumors were removed to allow micrometastases to grow. Using this procedure they were able to generate macroscopic metastases (>20 mg) in 71% (LuCaP 23.8) and 100% (LuCaP 35) of animals, respectively. Visible metastases in pelvic lymph nodes were observed in nearly half of the animals and microscopic lung metastases (1–40 cells) were observed in 80% of the animals. However, even using the prostatectomy procedure with the orthotopic implants, the authors were unable to generate bone metastasis. Due to the fact that they found that some bone marrow samples were PSA RT-PCR positive, which suggests the presence of CaP micrometastases in the bone, they propose that the lack of bone metastasis may be due to the time frame, and that if the observation time would have been longer maybe dormant BM-DTC might have developed into visible metastases. But this information also suggests that the timing to develop overt lesions is in part controlled by the target tissue microenvironment. Thus, the bone marrow microenvironment delays growth and this model can be used to study BM-DTC dormancy of CaP xenografts. In addition to this Shachaf et al. has published that inactivation of the MYC oncogene is sufficient to induce sustained regression of invasive liver cancers. In liver cells, MYC inactivation uncovers stem cell properties and triggers differentiation, but some of the cells remain in a dormant state and can become cancerous again by MYC reactivation. They showed that in MYC-induced hepatocellular carcinoma, MYC inactivation initially induces differentiation and apoptosis of most of the tumor cells. However under this circumstances, reactivation of MYC causes these differentiated tumor cells to rapidly become tumorigenic again (Shachaf and Felsher 2005; Shachaf et al. 2004 2008). These studies suggest that cancer cells retain the capacity to become biologically normal behaving cells, and that this change can be controlled by oncogene induction. Depending on the context oncogene reactivation can inhibit or activate the malignant phenotype. Based on our discussions above it is essential to include DTC as therapeutic targets. Further, molecular and functional information about these cells and the molecular mechanisms driving their behavior are necessary to design and develop more efficient therapies. Screening of BM for DTC could provide information about the therapeutic efficiency of a tested drug against DTCs. Identifying the specific

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genetic and epigenetic changes, that DTC undergo during cancer progression, is essential to develop efficient therapies that target DTC, before they develop in overt metastasis. Acknowledgments This work was supported by grants from the Samuel Waxman Cancer Research Foundation Tumor Dormancy Program (to JAG), NIH/NCI (CA109182 to JAG), NIEHS (ES017146 to JAG), New York State Stem Cell Science – NYSTEM (to JAG) and NIDCR (DE020121 to AR).

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Part III

Vasculature And Stroma

Chapter 12

Impact of Endothelial Progenitor Cells on Tumor Angiogenesis and Outcome of Antiangiogenic Therapy: New Perspectives on an Ongoing Controversy Robert S. Kerbel, Francesco Bertolini, and Yuval Shaked Abstract Tumor angiogenesis is driven not only by proliferation of differentiated endothelial cells in sprouting capillaries from pre-existing mature vessels, but also by mobilization of bone marrow derived circulating endothelial progenitor cells (EPCs). The latter are thought to home to the tumor site and incorporate into the lumen of newly growing blood vessels. Over the past several years, a growing number of reports have challenged the hypothesis concerning the involvement of EPCs in tumor angiogenesis, and instead suggest that such cells only have a minor role, if any at all, in the formation of tumor-associated blood vessels. Consequently, these studies implicate EPCs as a minor or negligible target for cancer therapy. In this review, we discuss the arguments for and against a significant role of EPCs in tumor angiogenesis and growth, and as possible surrogate markers of angiogenesis as well as valuable therapeutic targets.

Introduction Over the last decade, various types of bone marrow derived cell (BMDCs) have emerged as key players in tumor angiogenesis, growth, and metastasis (Shaked and Voest 2009; Kaplan et al. 2007); most such cell types originate from the hematopoietic lineage (Coffelt et al. 2009; De Palma and Naldini 2006; Grunewald et al. 2006; Shojaei et al. 2007). They can contribute to angiogenesis by various mechanisms including paracrine secretion of different growth factors mediated by perivascular residing BMDCs, and by contributing to the stability of the vasculature. Research into the development of antiangiogenic drugs to treat cancer has revealed that some BMDCs may compensate for vessel-targeting antiangiogenic effects, thus allowing for continued tumor growth. These bone marrow derived Y. Shaked (*) Department of Molecular Pharmacology, Rappaport Faculty of Medicine, Technion – Israel Institute of Technology, Haifa, Israel e-mail: [email protected] R.G. Bagley (ed.), The Tumor Microenvironment, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-6615-5_12, © Springer Science+Business Media, LLC 2010

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tumor-associated stromal cells include various myeloid, macrophage, and monocytic cell populations. They all express the pan-hematopoietic surface marker, CD45, along with various endothelial cell/angiogenesis-related surface markers. However, a subset of BMDCs which are CD45-negative has been found to promote tumor angiogenesis by acting as an alternative source of endothelial cells; these cells are termed endothelial precursor or progenitor cells (EPCs). In contrast to all other proangiogenic BMDC types, EPCs are thought to merge with the wall of a growing blood vessel, where they differentiate into mature endothelial cells, and contribute to vessel growth. The various types of BMDC promoting angiogenesis are summarized in Fig. 12.1.

Fig. 12.1 Cellular players in tumor angiogenesis. Representation of various BMDC populations which have been shown to stimulate or amplify tumor angiogenesis. The various hematopoietic (CD45+) cell types appear to have a perivascular location with respect to the tumor neovasculature, whereas the CD45-negative endothelial progenitor cells can actually incorporate into the lumen of a growing vessel and differentiate into a mature endothelial cell. TEMs tie-2 expressing monocytes, HSC hematopoietic stem cell, ECs endothelial cell, TASC tumor associated stromal cell, HPC hematopoietic progenitor cell, VLC vascular leukocytes. Mature endothelial cells detach from the basal membrane following damage to tumor blood vessel, and in the blood flow become apoptotic circulating endothelial cells (CECs). Circulating endothelial progenitors (CEPs) that might derive from hemangioblast are mobilized from the bone marrow to incorporate in the vasculature as part of physiological tissue repair. In micro-metastatic lesions CEPs may play a significant role in the angiogenic switch, which promote metastatic growth. Reproduced from Martin-Padura and Bertolini (2008), with permission from the publisher

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There are a number of controversial issues regarding the origin, nature, and relevance of EPCs to angiogenesis, including tumor angiogenesis which are summarized below, along with some recent findings that may help resolve some of these controversies.

The Identification of EPCs In 1997, Isner’s group reported that post-natal vasculogenesis and angiogenesis can be driven by BMDCs circulating in peripheral blood which are home to sites of ongoing angiogenesis. The systemic process of de novo formation of capillaries and vessels from angioblasts of endothelial progenitor, previously shown to be involved in the formation of an embryonic vessel network, has been investigated in adults. Activated endothelial cells have been shown to express CD34 along with VEGFR1. In addition, CD34 is expressed by all hematopoietic stem cells but not mature hematopoietic cells. Using this distinguishing surface marker, Asahara et al. (1997) isolated CD34-negative and -positive cells and assessed their possible characteristics as endothelial cells, both morphologically and functionally. Injection of CD34+ cells labeled with the fluorescent dye Dil into ischemic hind-limb bearing mice resulted in massive homing of CD34+ but not CD34- cells to the mouse ischemic limb, and incorporation into the blood vessel wall. This study was the first to show that circulating CD34+ cells, obtained from human peripheral blood, can contribute to angiogenesis in adult tissues implicating the possibility that CD34+ cells are, in fact, EPCs. This discovery opened up new directions in angiogenesis research including tumor angiogenesis.

The Controversy Surrounding Functions of EPCs Following the first study by Asahara et al. (1997), implicating a role for EPCs in adult angiogenesis, many investigators reported results confirming the involvement of EPCs in tumor growth; various studies revealed highly contrasting results. Perhaps the most compelling evidence to support a role for EPCs in tumor angiogenesis emerged from studies using knockout mice bearing mutations in the Id family of transcriptional repressors. Id proteins are helix–loop–helix transcription factors that regulate various physiological processes including angiogenesis. These mice have been used to investigate the role of EPCs in tumor angiogenesis (Lyden et al. 1999). In vivo studies revealed that Id1-null mice exhibit decreased neovascularization as assessed by the Matrigel perfusion assay, compared to their wildtype counterparts, and this is regulated by the endogenous inhibitor of angiogenesis, thrombospondin 1 (TSP-1) (Volpert et al. 2002). It was shown that loss of Id1 in tumor endothelial cells resulted in downregulation of several proangiogenic genes, including those encoding a6 and b4 integrins, matrix metalloprotease-2, and fibroblast

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growth factor receptor-1 (Ruzinova et al. 2003). Benezra and colleagues showed tumors growing in mice with a defect in the Id1 and Id3 genes (Id1+/–Id3–/–) are defective in angiogenesis, and hence growth. However, if such mice are irradiated and then given a transplant of wild-type bone marrow derived cells, tumor angiogenesis and progressive growth are restored (Lyden et al. 2001). Thus this in vivo data seemingly confirm the existence of EPCs, and suggest that these cells can contribute to overall tumor growth. In contrast to the in vivo and ex vivo evidence regarding a significant role of EPCs in tumor growth, several recent studies have challenged the definition, function, and contribution of EPCs. Perhaps the major controversy is whether putative EPCs can truly incorporate into tumor blood vessels. Some investigators have raised doubts about this possibility, in part, due to the presence of other BMDCs that are found in close proximity to blood vessels, but which do not directly incorporate to the blood vessel wall. Such cells do not express VEGFR2 or CD31 surface markers like EPCs or mature endothelial cells, but express a variety of surface markers associated with angiogenesis such as Tie-2, CXCR4, and VEGFR1 (Grunewald et al. 2006; De Palma et al. 2005; Jin et al. 2006). These cells are found to colonize tumors, and contribute to their growth. Another factor which has led to this increasing controversy is the fact that some tumor models exhibit relatively high number of EPCs, whereas in others the number detected is minimal or even nonexistent: a range of 0–50% of tumor endothelium consisting of EPCs has been reported. Machein et al. (2003) reported that tumor type is one such factor as this may significantly alter vasculogenesis and relative colonization of EPCs. They reported that the majority of bone marrow cells expressing hematopoietic and/or microglia markers, did not contribute to glioma vasculature. They also showed that overexpression of VEGF in glioma cells produced highly vascularized tumors, but the number of EPCs incorporated into the tumor vasculature was not increased. In a more recent study, Machein and colleagues reported that EPCs do not contribute at all to the growth of Lewis Lung carcinomas. The study evaluated the colonization of BMDCs tagged with GFP using several imaging techniques (Wickersheim et al. 2009). Ziegelhoeffer et al. (2004) reported that in mice bearing methylcholanthrene-induced fibrosarcoma (BFS-1), there were little, if any, BMDCs found to be incorporated into the tumor vasculature. In addition to tumor type, tumor grade has also been shown to alter the percentage of EPCs. Li et al. (2004) found that Id1 and Id3 transcription factors are highly expressed in endothelial cells of poorly differentiated prostate adenocarcinomas but not in the vasculature of well-differentiated tumors. Overall, these studies suggest that neovascularization in certain tumor types and of different tumor grade may be mediated by division and migration of differentiated endothelial cells from pre-existing vessels and that EPCs may not play a significant role in these circumstances. A clinical study by Peters et al. (2005) evaluated the number of BMDCs incorporated into the tumor blood vessel wall. They studied tumors obtained from patients who had previously undergone bone marrow transplantation obtained from individuals of the opposite sex. By using fluorescent In Situ hybridization (FISH)


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staining techniques and confocal laser scan microscopy the authors counted the number of endothelial cells in tumors which expressed mismatched sex chromosomes, indicating that they were derived from the transplanted donor bone marrow cells. They found that approximately 5% of tumor blood vessels consisting of bone marrow origin. These results indicate that there is a low number of EPCs incorporating into tumor blood vessels, and thus the possibility that they may function as endothelial cells contributing to the formation of a tumor vasculature. In recent studies, we have offered another possibility to explain the differences in the number of EPCs contributing to tumor growth reported by different investigators. We have shown that the genetic background of the host may play a significant role in the extent of adult vasculogenesis (Shaked et al. 2005a). Using a number of mouse strains, we evaluated the levels of EPCs circulating in the blood, and found that there are some strains, e.g., BALB/c, which exhibit high levels of EPCs in peripheral blood whereas other strains, e.g., C57Bl showed a low number of EPCs in up to a tenfold difference. We also showed that these levels strikingly correlated with the angiogenic responsiveness of the mouse strains to angiogenic stimuli measured by the corneal neovascular micropocket assay (Rohan et al. 2000) as well as in cases where mice were genetically manipulated to induce or reduce angiogenesis factors (Shaked et al. 2005a). These results indicated that in addition to tumor grade and type, the genetic background of the host may play a significant role in tumor neoangiogenesis. Only in the past few years several investigators have refuted the existence of EPCs in tumors. Purhonen et al. (2008) studied the mobilization and tumor homing of EPCs. They induced angiogenesis by using melanomas, APCmin adenomas, adenoviral VEGF delivery, and Matrigel plugs in four different genetically tagged universal or endothelial cell-specific chimeric mouse models. They failed to detect VEGFR2 cells mobilized from the bone marrow, nor incorporation directly into blood vessels. They thus concluded that EPC contribution is an extremely rare event, and perhaps might not even exist. Similarly, as noted above, Machein et al. (2003) who studied BMDCs expressing GFP, found that local VEGF induces a massive tumor infiltration of BMDCs, but no evidence of vessel wall integration of these cells. Furthermore, Kim et al. (2009) investigated acute and chronic phases of angiogenesis in wound healing assays, gel foam angiogenesis, in a parabiosis mouse model, and found that BMDCs expressing CD31 homing to sites of angiogenesis are macrophages (or monocytes) as they also express F4/80, a specific pan-macrophage marker. This approach was undertaken to avoid radiation of mice followed by a bone marrow transplant as such radiation might have affected the outcome of some of the prior studies using GFP+ bone marrow cells. These observations constitute yet another challenge to the EPC concept and the role of such cells in tumor angiogenesis, by suggesting that CD31 is expressed on angiogenic monocytes which do not differentiate into endothelial cells (Horrevoets 2009). In summary, these particular studies suggest that during tumor development and progression, new capillary formation is dependent only on “sprouting angiogenesis”, i.e., by migration and division of differential endothelial cells from pre-existing mature vessels. They clearly contradict the hypothesis of an involvement of EPCs in angiogenesis and tumor growth, and suggest that such cells might even be an

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experimental artifact. It should be noted, however, that several criticisms have been made regarding the study and conclusions reported by Purhonen et al. (2008). For example, APCmin mice develop only obstructive adenomas, rather than adenocarcinomas; therefore, it is an inappropriate model to study EPC incorporation in tumors (Kerbel et al. 2008). Furthermore, the study of 6-month-old VEGF-Aloaded Matrigel plugs in mice is not possible because Matrigel plugs are degraded within 2 months, particularly when VEGF-A by itself is unlikely to induce neoangiogenesis in Matrigel plugs. A recent study by Madlambayan et al. (2009) attempted to resolve this ongoing controversy by comparing a variety of models of tumor angiogenesis in a single mouse background. These included the Lewis Lung carcinoma, B16 melanoma as well as a model of retinopathy. The authors evaluated a spectrum of BMDC types including Tie2 expressing monocytes (TEMs) (De Palma et al. 2005), tumor associated macrophages (Coffelt et al. 2009), EPCs, and hemangiocytes (Jin et al. 2006). They found that the contribution of systemic BMDCs to tumor angiogenesis is dependent primarily on the expression of SDF-1 in the model/site tested. Manipulation of SDF-1 expression in such sites revealed large differences in the number of BMDCs recruited. Since the majority of EPCs express the CXCR4 chemokine receptor (Pitchford et al. 2009), the results suggest that EPCs, among other bone marrow derived CXCR4+ cells, home to active sites of angiogenesis, and promote tumor growth. This study offers a resolution for the finding of different values of BMDCs, including EPCs contributing to tumor growth in different models, as reported by some investigators.

The Controversy About the Definition of EPCs In addition to the questions raised about the possible numbers of EPCs detected in tumor vessels and their functional importance, yet another controversy which has emerged concerns the precise definition and origin of such cells. It is likely that the difficulties associated with some of the techniques used to isolate, sort, enumerate, and quantitate EPCs have contributed to this situation. In peripheral blood, EPCs are measured by using a combination of antibodies recognizing various endothelial and hematopoietic surface markers, as detected by multiparametric flow cytometry techniques (Bertolini et al. 2006). The lack of a single definitive antigen to define and detect EPC populations has contributed to the debate regarding the evaluation of EPCs. Initial studies using both in vitro quantitative and flow cytometry characterization of EPCs revealed that VEGFR2 and CD34 are the “ultimate” surface markers of EPCs; however, it has been shown that AC133 (CD133), an orphan receptor that is expressed on some hemoatopoietic progenitor cells, and lost once they differentiate, is also considered as a marker of EPCs (Peichev et al. 2000; Rafii et al. 2002). Furthermore, CD146, for example, has been evaluated as a specific endothelial cell marker (Solovey et al. 2001), and is still used to detect EPCs circulating in peripheral blood. However, other studies have recently reported that mesenchymal cells


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(Elshal et al. 2005), and activated T lymphocytes (Duda et al. 2006) can also express CD146. Moreover, VE-cadherin (CD144), an endothelial-specific marker which was also thought to be expressed specifically by endothelial cells, has been reported to be expressed by some hematopoietic stem cells (Kim et al. 2005). Overall, currently, such results indicate that the most useful markers for identifying EPCs circulating in the blood are CD34, VEGFR2, and CD133, which appear to constitute a phenotypically distinct population of cells capable of differentiating into endothelial cells that can play a role in post-natal angiogenesis/vasculogenesis. To add complexity to the phenotypic characterization of EPCs (and thus to the confusion in the field) recent studies by Yoder, Ingram, and colleagues indicated that established commercially available kits used to repopulate and isolate EPCs from human peripheral blood by identifying methylcellulose endothelial cell colony forming units (CFU-ECs) do not represent truly putative EPCs (Case et al. 2007; Yoder et al. 2007). In a number of studies they identified another population of endothelial colony-forming cells (ECFCs), and tested the origin, proliferative potential, and differentiation capacity of ECFCs as well as CFU-ECs (obtained from human peripheral blood). They compared the function of CFU-ECs and ECFCs and determined that CFU-ECs are derived from the hematopoietic system, and possess myeloid progenitor cell activity, whereas ECFCs display robust proliferative potential, and form perfused vessels in vivo (Yoder et al. 2007). Overall, their results suggest that CFU-ECs, obtained from commercially available kits to isolate EPCs, are in fact not authentic EPCs, whereas ECFCs may actually represent a rare EPC population. These studies indicate that in order to improve quantitation and isolation of putative EPC populations, there is a clear need to discover a specific EPC marker which would constitute verification of EPCs in endothelial engraftment experiments in vivo. The development of monoclonal antibodies to detect endothelial cell-associated antigens has led to the extensive use of flow cytometry based assays to measure the number of EPCs circulating in peripheral blood. The use of multiparametric flow cytometry analysis on whole blood labeled simultaneously using a combination of conjugated antibodies has provided a useful tool to adequately measure rare populations in the blood. The combination of antibodies to detect EPCs in the blood usually consists of first excluding cells expressing the hematopoietic marker CD45, and then detecting cells expressing progenitor markers such as CD34, CD133, in addition to VEGFR2 as an endothelial cell marker. However, the lack of a standardized method to detect EPCs and mature endothelial cells circulating in the blood (CECs) has resulted in very different values of EPCs detected using flow cytometry techniques by different investigators, and this has caused increasing confusion about the nature of these cells, both clinically and preclinically. Since flow cytometry approaches require accurate sequential gating and are prone to operator and instrument-associated variability, a greater effort has been made recently to try and standardize the methodology for detecting EPCs and CECs in peripheral blood, especially for clinical studies (Bertolini et al. 2006; Mancuso et al. 2009). Thus, the definition and detection of EPCs requires further refinement and improvement in order to become widely accepted when used in different centers and by different operators/groups.

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EPCs as a Surrogate Biomarker for Antiangiogenic Therapy The identification of EPCs in peripheral blood has led to emerging data regarding the levels of EPCs in different pathologies including cardiovascular disease (Werner et al. 2005), diabetes (Cubbon et al. 2009), arthritis (Distler et al. 2009), eclampsia (Gammill et al. 2007), and cancer. With respect to cancer patients, some early studies evaluated the number of circulating endothelial cells (CECs) and their precursor subset (CEPs). It has been shown that levels of CECs, including viable CECs in cancer patients is higher than in normal healthy controls (Mancuso et al. 2001). This is thought to occur by virtue of the fact that the production of proangiogenic factors by the tumor may promote CEC and CEP mobilization. In some of our early studies, we hypothesized that certain antiangiogenic drugs may target CEPs or at least may decrease their mobilization into the bloodstream, despite the production of angiogenic stimuli by the tumor. To test this hypothesis, we took advantage of a previous study by Rohan et al. (2000) who reported that angiogenic responsiveness measured by the corneal neovascular micropocket assay using bFGF, is markedly heterogeneous in different strains of mice. We found a striking correlation between the angiogenic responsiveness and the levels of CECs and CEPs in the same mouse strain (Shaked et al. 2005a). We next asked whether CEPs are targeted by antiangiogenic drugs. We showed that mice treated with DC101, a monoclonal antibody directed to mouse VEGFR2 (Prewett et al. 1999) or ABT-510, a peptide mimetic of TSP-1 (an endogenous angiogenic inhibitor (Lawler 2002)) (Yap et al. 2005), caused a significant dose-dependent drop in levels of CEPs (defined by the phenotype CD45-,VEGFR2+,CD117+, and CD13+). We also found that the maximum decline (the “nadir”) in CEP levels correlated with the optimal biological dose (OBD) of the drug based on prior dose-response studies of anti-tumor activity (Shaked et al. 2005a). Similar results were obtained when we used another antiangiogenic treatment strategy, namely, metronomic low-dose chemotherapy (Klement et al. 2000; Hanahan et al. 2000; Shaked et al. 2005c), i.e., the administration of chemotherapy drugs using lower doses than the maximum tolerated dose (MTD), given at frequent, regular intervals with no long drug-free break periods (Klement et al. 2000). These results not only provided evidence that some antiangiogenic drugs and treatment strategies target putative CEPs, but also suggested that CEPs can be used as a surrogate pharmacodynamic biomarker to determine the OBD of antiangiogenic therapies (Schneider et al. 2005), an approach which has been further evaluated in clinical studies (Mancuso et al. 2006; Buckstein et al. 2006; Bender et al. 2008). Mancuso et al. (2006) have raised the possibility that apoptotic CECs may be used as a biomarker for antiangiogenic drug or treatment outcome. In their study of 121 breast cancer patients treated with metronomic cyclophosphamide and methrotraxate levels of CECs were evaluated. In contrast to the perception, at that time, that antiangiogenic drug therapy would reduce the levels of CECs and CEPs (Shaked et al. 2005a), the authors found that increases in CECs were associated with tumor response. When the authors distinguished between apoptotic and viable CECs, they


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found that in responding patients, the majority of CECs are in fact apoptotic (Mancuso et al. 2006). Supported by preclinical models of metronomic chemotherapy, the authors showed that the elevation in apoptotic CEC levels were likely tumor derived (i.e., derived from the tumor vasculature) and not host derived, since studies in non-tumor bearing mice indicated no detectable rise in apoptotic CECs levels. Comparable results were generated in additional clinical studies. For example, in a phase I clinical study of pediatric cancer patients undergoing bevacizumab treatment (Bender et al. 2008), it was reported that the ratio between the number of apoptotic CECs and total CECs was associated with the number of bevacizumab treatment cycles, indicating patients responding to therapy (Bender et al. 2008). In another clinical study, patients with advanced breast cancer received metronomic oral capecitabine and cyclophosphamide along with bevacizumab and levels of CECs were measured during the treatment; higher baseline CECs were found to be correlated with overall response, clinical benefit, and improved progression-free survival (Dellapasqua et al. 2008). Along with this analysis, the pharmacokinetics of CECs was also evaluated, and a pattern of decreased CECs and increased levels of various angiogenic growth factors have been reported in relapsed patients (Calleri et al. 2009). Overall, the aforementioned preclinical and clinical studies suggest that CECs and perhaps also CEPs may be used as surrogate biomarkers to determining the OBD of an antiangiogenic drug as well as predictive biomarkers of clinical outcome.

Therapy-Induced EPC Mobilization and Tumor Vessel Incorporation The lack of a specific antigen to define and detect EPCs, and the use of techniques to enumerate and quantitate EPCs in peripheral blood using at least four different surface markers have become extremely challenging technical issues for investigators in the field. However, in addition to studying CECs and CEPs as possible biomarkers for treatment outcome, our efforts have also been directed to studying the functional properties and effects of EPCs in different therapeutic circumstances. The results we have obtained provide an entirely new perspective on their impact and importance to angiogenesis and cancer therapy not only using antiangiogenic drugs, but also other therapies, including chemotherapy, both conventional and metronomic. We first investigated the possible impact of CEPs on tumor angiogenesis immediately after therapy with various cytotoxic agents. The rationale was based on our initial observations that MTD chemotherapy using cyclophosphamide rapidly increases the mobilization and viability of CEPs in NOD/SCID mice bearing human lymphomas whereas, in contrast, a low-dose metronomic regimen using the same chemotherapy drug, had the very opposite effect (Bertolini et al. 2003). These results suggested that MTD chemotherapy transiently induces angiogenesis (vasculogenesis) during the follow-up drug-free break periods which are usually instituted when administrating MTD cytotoxic therapy regimens, in order to allow recovery of

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patients (or animals) from the toxic side effects of the treatment, e.g., myelosuppression. In contrast, the chemotherapy may induce an antiangiogenic effect during the therapy, by targeting dividing endothelial cells (Kerbel 1991). Thus this sequence of events may be likened to an “action” (antiangiogenesis) followed by a “reaction” (pro-angiogenesis). As such, the “drug holidays” might promote, at least in part, tumor cell repopulation between successive courses of MTD chemotherapy. The surviving repopulating tumor cells are obviously one of the most important causes for treatment failure or limitations in the effects of such treatments (Kim and Tannock 2005). In this regard, we have recently found that shortly after treatment with any one of a number of different cytotoxic or cytotoxic-like drugs such as microtubule inhibiting vascular disrupting agents (VDAs), and certain chemotherapy drugs, e.g., paclitaxel, the levels of CEPs rise in peripheral blood within a matter of hours, after which they subsequently home to the tumor site and incorporate into or around the lumen of tumor blood vessels (Shaked et al. 2006b; Shaked et al. 2008). In the case of VDAs, these drugs cause rapid tumor vessel ischemia and occlusion followed by massive tumor hypoxia and necrosis. However, a viable and perfused rim tumor tissue usually remains from which tumor re-growth rapidly resumes (Tozer et al. 2005; Siemann et al. 2004), similar in some respects to rapid tumor cell repopulation observed after MTD chemotherapy-induced tumor shrinkage (Kim and Tannock 2005). Since the mechanisms which underline tumor cell repopulation after cytotoxic (or cytotoxic-like) drug therapy are poorly understood, we sought to investigate whether CEPs contribute to tumor re-growth from the viable tumor rim. We found that mice treated with VDAs, e.g., combretastatin A 4 phosphate (CA4-P) or OXi-4503, a second generation (more potent derivative) of CA4-P, exhibited substantial increases in CEPs within 4h. Subsequently, these cells home to the tumor site in large numbers and promote angiogenesis which accompanies the rapid tumor growth from the viable rim. An example of the massive homing and invasion of BMDCs, including EPCs (which incorporate into the lumen of the blood vessel wall), after therapy with a VDA is presented in Fig. 12.2. We also demonstrated that when an antiangiogenic treatment (i.e., DC101) which can block CEP mobilization (Shaked et al. 2005a; Shaked et al. 2006b) was coadministered with the VDA, or when tumor bearing mice that are deficient in CEP mobilization (i.e., Id-1+/-Id-3-/- mice) (Lyden et al. 2001) are used, a minimal residual viable tumor rim was observed after OXi-4503 treatment. Importantly, this phenomenon was not restricted to VDAs, as it was also observed following administration of other (but not all) cytotoxic chemotherapeutic drug therapies. Thus we found that certain chemotherapy drugs administered near or at the MTD, e.g., paclitaxel can induce a rapid spike in CEP levels but, again, this can be largely blocked by DC101 which enhanced treatment impact. However, other chemotherapy drugs, e.g., gemcitabine, which does not induce such a CEP spike, the addition of DC101, had no additional therapeutic benefit (Bertolini et al. 2006; Shaked et al. 2008). Thus, these observations indicate that in addition to the anti-tumor activity of chemotherapy, the subsequent tumor growth that occurs may be facilitated by a rapid influx of CEPs. The administration of an antiangiogenic drug (or other treatment strategies (Shaked et al. 2005b)) concomitantly with chemotherapy that can block


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Fig. 12.2 Colonization of GFP± bone marrow cells in LLC tumors grown in mice treated with OXi-4503, DC101, or the combination of the two drugs. Lewis Lung carcinoma grown in lethally irradiated mice that were previously transplanted with GFP+ bone marrow tagged cells (green) were treated with DC101, the VEGFR-2 blocking antibody, OXi-4503 or a combination of the two drugs. Three days later, tumors were removed and stained for blood vessels using the CD31 endothelial cell marker (red). Untreated mice revealed some GFP+ BMDCs that did not directly incorporated into the tumor blood vessels. In contrast, massive invasion of BMDCs to the viable tumor rim was observed in mice treated with OXi-4503, some of which were incorporated into the tumor blood vessel lumen. The combination of DC101 and OXi-4503 resulted in the absence of some BMDCs reside at the tumor viable rim. Bottom panels demonstrate the incorporation of BMDCs into the tumor vasculature (co-localization of CD31 and GFP), but also a number of other BMDCs which are adjacent to the tumor vessels and may support tumor angiogenesis in a paracrine manner. Scale bars: Upper 50 µm; bottom 20 µm. Reproduced from Shaked et al. (2006b), with permission from the publisher

tumor cell repopulation mediated by rebound angiogenesis can result in prolongation of the duration of tumor response. These results may also suggest how antiangiogenic drugs may act to enhance the efficacy of standard chemotherapy (Shaked and Kerbal 2007; Kerbal 2006). So how do these results provide an entirely new perspective regarding the ongoing controversy of the contribution of CEPs to tumor angiogenesis? The answer is that all previously published studies showing very low or non-existent levels of EPC incorporation in tumor vessels were performed on untreated tumors, which were removed at various stages of tumor growth. In contrast, there are circumstances, e.g., after acute cytotoxic therapy, where our results show that CEPs may play a significant role in tumor angiogenesis and tumor (re)growth; thus, targeting these cells at these critical time points may lead to significant therapeutic benefits. The robust and acute CEP host responses induced by drugs such as VDAs or paclitaxel may be a consequence, at least in part, of an attempt to repair damage to the tumor vasculature caused by such drugs. We should stress, however, that many other BMDC populations may also contribute to the overall host response and tumor vessel repair process following cytotoxic drug therapy, i.e., this phenomenon

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in all likelihood is not restricted to CEPs. It will be of great interest to assess the possibility that cytotoxic therapies, including radiation, induce the mobilization and tumor homing of other BMDC populations such as monocytes and macrophages (Brown et al. 2010). Another circumstance which implicates an important role of EPCs in tumor growth and angiogenesis even when they are present in very limited numbers comes from a study by Mittal and colleagues (Gao et al. 2008). Since the development of macroscopic metastases from micrometastases also requires angiogenesis (Woodhouse et al. 1997), Gao et al. (2008) recently studied whether EPCs are crucial regulators of the “angiogenic switch” mediating progression of metastasis. By using two models of pulmonary metastasis in mice, they demonstrated that the vessels of macrometastases contained approximately 11% EPCs. They found that this was regulated by the Id1 transcription factor (Shaked et al. 2006b), since the blockade of Id1 by short hairpin RNA (shRNA) resulted in impaired angiogenesis, and lack of formation of macrometastases. Importantly, this study not only demonstrated a role of EPCs in metastatic growth, but also indicated that even a relatively small percentage of EPC incorporation into metastatic blood vessels (~11%), can nevertheless induce a major biologic effect in tumor biology. As such, pharmacologically targeting these relatively rare cells may be therapeutically promising. The rapid and robust increase in CEP levels following cytotoxic drug therapy has been recently confirmed in clinical studies. With respect to VDAs, in a phase I clinical study patients treated with cisplatinum in combination with the tubulin binding VDA, AVE8062, exhibited increases in the number of CEPs within the first 3 days (Farace et al. 2007). In this study, CEPs were evaluated by flow cytometry and were defined by the surface markers CD45(dim), VEGFR2+, and CD34+. Similar results were reported based on a phase I clinical study of the VDA, ZD6126, conducted by Beerepoot et al. (2006). The authors measured levels of CECs (but not CEPs), and found that levels of such cells are significantly elevated within 2–8h after therapy. The authors suggested that the rise in CECs levels is probably a consequence of endothelial cell shedding from the damaged tumor vasculature in response to VDA therapy. Although the authors’ conclusion focused on the levels of CECs, no precursor marker for CEP subset was evaluated at that time, and it is plausible that some of the cells detected by flow cytometry are in fact CEPs (Shaked et al. 2006). Furthermore, an additional clinical study testing the combination of a VDA with an antiangiogenic drug has been conducted in a phase I trial involving combretastatin A 4 phosphate (CA4P) and bevacizumab (Nathan et al. 2008). Preliminary results have been presented by Nathan and colleagues (2008) who reported that the combination treatment revealed statistically significant reductions in tumor perfusion/vascular permeability which reversed after CA4P alone but which were sustained following bevacizumab treatment using DCE-MRI. In addition they observed increases in circulating CD34+ and CD133+ bone marrow progenitors following CA4P treatment as well as increases in VEGF and GCSF levels, in a similar manner to a recent preclinical study (Shaked et al. 2009). Nathan et al. (2008) concluded that CA4P in combination with bevacizumab appears safe and well tolerated with evidence of clinical activity. With respect to chemotherapy,


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we reported that cancer patients treated with paclitaxel based therapy but not gemcitabine- or doxorubicin-based therapies exhibited increases in CEP levels along with SDF-1 and other proangiogenic factors (Shaked et al. 2008; Shaked et al. 2009). In addition, Voest and colleagues have also reported that MTD paclitaxel exhibit an immediate increase in CEP levels 4h after treatment was initiated, but these immediate changes did not correlate with response to therapy. However, sustained and consistent high levels of CEPs measured on day 7 (regardless of the chemotherapy drug used) were associated with an increase in tumor growth and could predict progression free and/or overall survival (Roodhart et al. 2010). Overall, these results further reinforce the concept of rapid cytotoxic drug induced host CEP mobilization, and its blockade by an antiangiogenic drug which results in increased treatment efficacy.

Conclusions The role of EPCs in post-natal angiogenesis, and specifically during tumor growth remains controversial. Although many studies have shown vessel incorporation and a functional role of EPCs in tumor biology, other studies have failed to reproduce such results. We and others have shown that in certain circumstances EPCs may play a major role in tumor angiogenesis, mainly with respect to repair of therapy-induced tumor vascular damage induced by certain drugs, e.g., VDAs and MTD chemotherapy. In such circumstances we have found that EPCs rapidly home to the tumor site and promote an active angiogenesis process. Clinically, EPCs and CECs have been investigated as possible surrogate biomarkers to predict clinical outcome in patients undergoing antiangiogenic therapies. However, such measurements require further evaluation and validation using sophisticated flow cytometry technology. Overall, a consensus regarding the nature of EPCs and their relative functional importance in tumor angiogenesis and anti-cancer therapy remains to be determined.

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Chapter 13

Bone Marrow Derived Mesenchymal Stem/Stromal Cells and Tumor Growth Pravin J. Mishra and Debabrata Banerjee

Abstract Carcinoma associated fibroblasts (CAFs) play an important role in the growth of epithelial solid tumors. The origin of these tumor or CAFs has not been conclusively established. There is experimental evidence to suggest that part of the tumor or CAFs may arise from bone marrow derived mesenchymal stromal/stem cells or MSCs. It is well known that bone marrow derived MSCs can give rise to cells of different lineages: muscle, bone, fat, and cartilage. Based on recent work from our own laboratory and that of others, we now suggest that human BM-derived MSCs exposed to tumor-conditioned medium (TCM) over a prolonged period of time can give rise to cells that assume a CAF-like phenotype. Thus, MSCs may be a source of CAFs and can be used experimentally for modeling tumor-stroma interactions. Although the importance of the dialog between cancer cells and other components of the tumor milieu has been increasingly appreciated, it is as yet unclear whether the stromal cells themselves harbor cancer promoting mutations or changes. Activated stromal cells have been shown to promote tumor growth and metastasis in experimental models and we speculate on the possibility of increased activation of bone marrow derived MSCs by higher levels of chemokines under certain physiological situations and how this may impact tumor growth.

D. Banerjee (*) Department of Medicine, The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08901, USA and Department of Pharmacology, The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08901, USA e-mail: [email protected] R.G. Bagley (ed.), The Tumor Microenvironment, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-6615-5_13, © Springer Science+Business Media, LLC 2010

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Introduction Most solid tumors are made up of two discernable compartments, tumor or neoplastic cells and the stromal platform on which the tumor cells are scattered. Besides these, there are many other cell types present in the tumor milieu including tumor associated macrophages (TAMs), lymphocytes, endothelial cells, fibroblasts, and pericytes that interact with each other either directly or via paracrine mechanisms and influence tumor growth. The origin and function of tumor stroma has been the subject of intense investigation ever since Virchow first observed that cancers were infiltrated with inflammatory and immune cells (Balkwill and Mantovani 2001). The presence of fibroblast populations within human tumors often referred to as carcinoma associated fibroblasts (CAFs) has been associated with poor outcome and an increase in metastatic potential (Tsujino et al. 2007; Yazhou et al. 2004). Experimental evidence from tumor models as well as from clinical specimens suggest that CAFs may promote tumor growth and metastasis. In this chapter we will discuss the likely source of these CAFs as well as their characteristics and hope to convince the readers that bone marrow derived mesenchymal stem/stromal cells (MSCs) may be a likely source of CAFs. We will also discuss how this can be an important model system to study tumor-stroma interaction. Lastly, we will speculate on the possibility of increased activation of bone marrow derived MSCs by higher levels of chemokines in circulation under certain physiological situations and how this may impact tumor growth.

Characteristics of CAFs Over the years, characterization of CAFs has been largely based on immunohistochemical staining and more recently by specific gene expression signatures (Allinen et al. 2004; Hu and Polyak 2008; Chang et al. 2005). These have relied upon identification and or isolation of the CAFs from tumor specimens. We and others have developed in vitro methods to generate CAFs and study them in model systems to understand the role of these CAFs in tumor growth and metastasis as well as to better define paracrine factors that participate in the tumor stroma dialog (Mishra et al. 2008; Spaeth et al. 2009). Advantages of an in vitro system include better control over cell type, identification and characterization of CAFs and generation of large number of cells in culture to carry out studies in various model systems including xenograft studies. In order to generate CAFs (also referred to as tumor associated fibroblasts or TAFs) in vitro, long term exposure to tumor cell conditioned medium has been the method of choice. Spaeth et al. (2009) have defined the tumor or CAFs involved in ovarian cancer growth (as well as breast and prostate) by expression of four qualifying factors: (1) fibroblast markers fibroblast-specific protein (FSP)

13 Bone Marrow Derived Mesenchymal Stem/Stromal Cells and Tumor Growth

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and fibroblast activating protein (FAP); (2) genes associated with an increase of tumor aggression, including stromelysin-1 (SL-1), thrombospondin-1 (Tsp-1), and tenascin-C (Tn-C); (3) myofibroblast/provascularizing potential including desmin, a-smooth muscle actin (a-SMA), and vascular endothelial growth factor (VEGF); and lastly, (4) growth factors, transforming growth factor-beta (TGF-b), HGF/ scatter factor (SF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF). Bone marrow derived MSCs have been suggested to play a major role in tissue regeneration and repair based on their multilineage differentiation potential (Pittenger et al. 1999; Picinich et al. 2007) as well as the propensity of these cells to migrate to areas of injury, inflammation and tumor (Fox et al. 2007). Studeny et al. (2002) originally demonstrated the capacity of MSCs, also known as multipotent stromal cells, to home to tumors and participate in tumor stroma formation, suggesting MSCs as a potential source of stroma. The observed tendency of MSCs to migrate to sites of injury, inflammation and tumors is attributed to common chemoattractants or factors present in the wound and tumor milieu leading to the concept that tumors are unhealed wounds (Dvorak 1986). Our own work on role of MSCs in wound healing has revealed remarkable similarities in the process of wound healing and tumor growth with some subtle differences such as in production of cytokines by MSCs in the wound bed or in the presence of keratinocytes, the principal epithelial cells in skin (Mishra et al. unpublished observations). The ability of MSCs to migrate to tumor sites has been exploited to deliver therapeutic genes to tumor sites using MSCs as carriers. (Studeny et al. 2002, 2004; Nakamizo et al. 2005; Hung et al. 2005).

Bone Marrow Derived MSCs as Source of CAFs Accumulating evidence suggests that CAFs or TAFs play an important role in the growth of epithelial solid tumors. It has been demonstrated that a significant fraction of the stroma in some breast cancers consists of fibroblasts (Bissell and Radisky 2001). More recent studies have revealed that CAFs from breast cancer specimens promote tumor cell growth as compared to fibroblasts obtained from non-neoplastic locations. In addition to tumor growth, tumor stroma has also been shown to be involved in other important processes such as angiogenesis and metastasis. Orimo and colleagues (2005) defined several important characteristics of breast CAFs including promotion of breast carcinoma cell growth, promotion of angiogenesis, and expression of myofibroblast traits. Expression of the chemokine stromal-derived factor 1 (SDF-1) has also been shown to be of paramount importance in the interaction between tumor cells and stromal fibroblasts (Orimo et al. 2005). Given the importance of tumor stroma in promoting tumor growth, it becomes necessary to identify the likely source(s) of cells that make up the tumor stroma, particularly the carcinoma associated fibroblasts or CAFs.

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It has been suggested that the likely source (s) of tumor stroma include (1) the locally resident tissue stem cells that are recruited to the tumor site; (2) epithelial to mesenchymal transition or EMT; (3) fibroblast recruitment into the tumor stroma; and (4) recruitment of bone marrow-derived cells from the circulation (Udagawa et al. 2006; Koyama et al. 2008; Jodele et al. 2005). We and others have demonstrated that bone marrow derived MSCs, like other bone marrow-resident cells, have the capacity to differentiate within the tumor microenvironment into myofibroblastic-like cells commonly referred to as; myofibroblasts, tumor-associated (myo)fibroblasts (TAF), CAF, fibrocytes or pericytes (Ogawa et al. 2006; Mishra et al. 2008; Spaeth et al. 2009) which have been shown to play an important role in tumor formation, growth and metastasis. Although the cell type of origin of myofibroblasts has not been conclusively established it has been shown that they may be bone marrow derived (Direkze et al. 2004). In a recent study we demonstrated that human bone marrow-derived mesenchymal stem cells (hMSCs) when exposed to tumor-conditioned medium (TCM) over a prolonged period of time assume a CAF-like phenotype (Mishra et al. 2008) and this was confirmed by Spaeth et al. (2009). More importantly, these cells exhibit functional properties of CAFs including sustained expression of SDF-1 and the ability to promote tumor cell growth both in vitro and in an in vivo co-implantation model. These cells also express myofibroblast markers including a-SMA and FSP. Gene expression profiling revealed similarities between TCM exposed hMSCs and CAFs. This suggests that hMSCs are a source of CAFs and can be used in modeling tumor-stroma interactions (Mishra et al. 2008; Karnoub et al. 2007). Further evidence for a bone marrow source of CAFs comes from studies using a gastric cancer mouse model (Gan mice) in which prostaglandin E2 (PG E2) and Wnt signaling were simultaneously activated in the gastric mucosa (Guo et al. 2008). Since PGE2 and Wnt pathways both play a role in human gastric tumorigenesis, the Gan mouse model may display important aspects of the molecular etiology of human gastric cancer. Bone marrow transplantation (BMT) experiments revealed that subsets of gastric myofibroblasts were derived from bone marrow (Guo et al. 2008). In a recent report, Worthley et al. (2009) have shown for the first time that human gastrointestinal neoplasia associated myofibroblasts can develop from bone marrow derived cells following allogeneic BMT based on identification of SMA expressing, Y chromosome positive CD45 negative cells in the tumor milieu of female recipients of BMT who went on to develop solid tumors following allogeneic male bone marrow/stem cells transplants. This clinical study supports the previous in vitro as well as in vivo animal model studies that suggested bone marrow origin of CAFs (Ogawa et al. 2006). Although literature supports the bone marrow origin of CAFs, alternate sources including resident fibroblasts and adipose tissue cells are also sources of CAFs. In a recent report Zhang et al. (2009) demonstrate that white adipose tissue derived stromal cells are also recruited to tumor stroma and promote cancer progression in animal models of human cancers including Kaposi’s sarcoma, prostate and breast cancer models.


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Alterations in Tumor Associated Stromal Cells Although the importance of the dialog between cancer cells and other components of the tumor milieu has been increasingly appreciated, the question of whether the stromal cells themselves harbor cancer promoting mutations or changes is just beginning to be addressed. For example, Patocs et al. (2007) hypothesized that mutational inactivation of the tumor-suppressor gene TP53 and genomic alterations in stromal cells of a tumor’s microenvironment may be correlated to clinical outcome. The authors, in an analysis of somatic and stromal cell mutations, demonstrated that stroma-specific loss of heterozygosity or allelic imbalance was associated with somatic p53 mutations and regional lymph-node metastases in sporadic breast cancer but not in hereditary breast cancer. A previous study in head and neck squamous cell carcinoma (SCC) by the same investigators looked at whether the apparently “normal” stroma surrounding the tumor epithelium can acquire genomic alterations and contribute to cancer initiation and or progression. Tumor-associated stroma of head and neck SCC from smokers was found to have a high degree of genomic alterations. The results indicated that stroma-specific genetic alterations may have been associated with smoking-related head and neck SCC genesis (Weber et al. 2007). Whether the identified mutations correlate with stromal changes or whether this is a reflection of methodological artifacts remains unresolved and larger studies on tumor associated stromal cells from a variety of tumor specimens may be helpful in settling this contentious issue (Eng et al. 2009; Campbell et al. 2009). It has become clear that the initiation and progression of carcinomas depend not only on alterations in tumor epithelial cells, but also on changes in their microenvironment. To study changes in stromal cells within the tumor milieu Hasawi et al. (2008) undertook to characterize CAFs and their tumor counterpart fibroblasts (TCFs) at the cellular and molecular level in a small subset of breast cancer patients using normal breast fibroblasts (NBFs) from plastic surgery as a control. The results suggested that the p53/p21 response to gamma-radiation was attenuated in 70% of CAFs, whereas it was normal in all the TCF and NBF cells. These results indicate that alterations in the p53 pathway can occur in breast CAFs and their corresponding adjacent counterparts, further pointing to the important role that stroma may play in breast carcinogenesis and treatment. Alterations in the tumor suppressor p53 have been reported in tumor-associated stromal cells, however, the consequence of these alterations are not well understood. Dudley et al. (2008) have investigated p53 status and response to p53-activating drugs using tumor-associated stromal cells from A375 melanoma and PC3 prostate carcinoma xenografts, as well as from a spontaneous prostate tumor model (TRAMP). Unlike normal stromal cells, tumor associated stromal cells failed to arrest in G2 after etoposide treatment, failed to upregulate p53-inducible genes, and failed to undergo apoptosis after treatment with vincristine. Tumor-associated stromal cells were also found to be less sensitive to p53-activating drugs. Knockdown of p53 in normal stromal cells produced similar results strongly supporting the

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contention that there was loss of p53 response in tumor associated stromal cells. Investigations into the role of tumor associated stromal cells in supporting aerobic glycolysis (also called the Warburg effect) has revealed that activation of highly conserved mammalian uncoupling proteins through interaction with the tumor milieu may facilitate the Warburg effect in the absence of permanent respiratory impairment (Samudio et al. 2008). Additionally, tumor associated stromal cells have been reported to cooperate with tumor cells by taking up the lactate produced by tumor cells and secreted into the tumor microenvironment. The stromal cells take up the secreted lactate via monocarboxylate transporters (MCT1 and MCT2) and after conversion to pyruvate secrete it back into the extracellular milieu where it can be used by local cell constituents for oxidative phosphorylation (Samudio et al. 2008; Koukourakis et al. 2006). Our own observations indicate that exposure of bone marrow derived MSCs to 15 mM lactate for periods as short as 5 min results in upregulation of MCT1 protein as determined by western blotting (Rattigan and Banerjee 2009, unpublished data). Although little is known regarding how changes in stromal gene expression affect epithelial tumor progression, it is becoming clear that cancer is influenced by signals emanating from tumor stroma. Finak et al. (2008) studied gene expression profiles of tumor stroma from 53 primary breast tumors obtained by laser capture microdissection and were able to generate a novel stroma-derived prognostic predictor (SDPP) that correlated with disease outcome independently of standard clinical prognostic factors. The SDPP gene expression signature when applied to several previously published tumor-derived expression data sets was able to identify poor-outcome individuals from amongst multiple clinical subtypes, including lymph node-negative tumors. The SDDP signature appears to be an improved prognostic predictor compared to previously published methods, particularly for HER2-positive breast tumors. Genes showing strong prognostic tendencies included those associated with differential immune responses and angiogenic and hypoxic responses underscoring the importance of stromal biology in tumor progression (Kroemer and Pouyssegur 2008). The plasticity of both the epithelial tumor cells and bone marrow derived MSCs and its impact on tumor biology remain a subject of intense investigation. On the one hand tumor cells are known to undergo epithelial mesenchymal transition (Salomon and Thiery 2003; Radisky 2005) while on the other hand, MSCs are also capable of mesenchymal epithelial transition (Chaffer et al. 2006) thus adding to the complexity of cell types in the microenvironment.

Implications of MSCs as a Source of CAFs: A Model to Study Tumor Stroma Interactions In vitro generation of CAFs following exposure to TCM as well as the admixed injection of tumor cells and bone marrow derived MSCs hold great promise as systems where tumor stroma interaction can be manipulated and studied. Additionally, this may also provide a cell culture method for generating one of the important cell types of the tumor stroma, the activated myofibroblasts.


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A better understanding of the dialog between various bone marrow derived cell types and tumor cells within the tumor milieu will be important for improved tumor therapy that takes into account influence of the tumor milieu on survival and growth of tumor cells. We have initiated studies to model the tumor-stroma interaction in vitro by culturing TCM exposed BMDMSCs (representing CAFs), reporter gene marked tumor cells (to report growth of tumor cells in the cell mix) and other components of the tumor milieu. It is now becoming clearer that CAFs and other cells in the tumor milieu actively participate in altering the growth and drug response of tumors (Iwamoto et al. 2007; Mantovani et al. 2006). For example, MSCs, which have very high levels of asparaginase expression, can protect leukemic cells from asparaginese cytotoxicity by providing increased concentrations of asparagine in the vicinity of leukemic cells. This system can be exploited to study the contribution of freshly harvested TAFs from dissected tumors on growth of a similar type of tumor cell. By establishing a panel of reporter gene marked tumor cells representing a variety of tumor types and subtypes, it may be possible to determine influence of TAFs on tumor growth. Specific reporters can also be generated for pathways that may be activated in different tumors, for example one can study whether androgen independent prostate cancer cells are still influenced by the tumor stroma. The relative ease with which the reconstituted tumor in its microenvironment can be transplanted as a xenograft may also permit improved drug studies in vivo. An important aspect of the reconstituted tumor microenvironment is the ability to evaluate chemopreventive measures in vitro and in vivo. By targeted pretreatment of critical components of the reconstituted tumor microenvironment, it may be possible to rank chemopreventive agents by potency as well as cell type specificity. Further applications of the reconstituted tumor milieu system include study of gap junctions and other direct communication means between tumor cells and other cell types. This type of experimental system may provide a better simulation of an in vivo solid tumor and may be a more realistic model for investigation of tumor biology and experimental therapeutics.

Activation of BMD MSCs and Growth of Tumors Speculation on Role of Chemokines on Activation of Circulating MSCs and Effect on Tumor Growth in African American Individuals with Breast Cancer Caucasian American women have higher age-adjusted breast cancer incidence rates compared with African American women (143 per 100,000 versus 119 per 100,000; Ries et al. 2002). However, African American women have higher age-adjusted mortality rates from the disease. Breast cancer mortality rates among younger African American women are approximately twice that of younger Caucasian American women (Amend et al. 2006; Albain et al. 2009). African American breast

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cancer patients also more frequently develop basal like subset of breast cancer (triple negative; ER-, PR- and Her2 low; greater stromal involvement), which has a poorer prognosis (van de Vijver et al. 2002; Sorlie et al. 2001). Gene expression studies reveal that pathways related to tumor angiogenesis and chemotaxis may be upregulated in tumors from African American women as compared with women of Caucasian origin. Increased microvessel density and increased TAMs in tumors from African American patients have been reported (Martin et al. 2009) suggesting involvement of the tumor microenvironment. A majority of African Americans are negative (~68% as compared to 3.4% of Caucasian Americans) for Duffy antigen receptor for chemokines (DARC) and it has been suggested that absence of this non-signaling receptor can increase migration of several types of blood cells from circulation to tissues (Tournamille et al. 1995; Fukuma et al. 2003; Lentsch 2002; Pruenster et al. 2009) supporting the idea of increased migration of activated MSCs to the tumor milieu. A mutation in the erythroid specific promoter GATA-1 binding site in the promoter of the gene encoding DARC results in lack of expression of DARC on erythrocytes. The erythroid DARC clears ELR chemokines such as CXCL8 from circulation while the endothelial DARC may facilitate transport of chemokines from tissue to vascular lumen for presentation (Lee et al. 2003). Overexpression of DARC in breast cancer cells correlates with a tumor and metastasis suppressor phenotype underscoring the importance of the DARC-negative phenotype in tumor progression. Wang et al. (2006) have shown a strong negative correlation between DARC expression and lymph node metastasis, ER status and microvessel density suggesting that low DARC expression correlated with aggressive disease in an analysis of 75 breast tumors. We have re-analyzed data compiled previously from two separate gene expression profiling studies and discovered that lower relative expression of DARC correlates with basal subtypes (unpublished data; analysis carried out in collaboration with Dr. S. Ganesan at CINJ from data published by Alexe et al. 2007; Wang et al. 2005). Our analysis suggests that lower relative expression of DARC correlates with basal subtypes of breast cancer. The basal subtypes, basal A1 and basal A2 had the lowest expression as compared with the luminal type as well as normal breast tissue. African American breast cancer patients more frequently develop basal subtype of breast cancer. Our analysis did not separate the population by ethnicity supporting an independent association between DARC negativity and the basal subtype. Lack of DARC expression in African American individuals correlates with elevated levels of free chemokines, such as CXCL8, and leads to pathological conditions as shown below. Lack of DARC Expression, Circulating Chemokines and Pathological Conditions It has been shown that DARC negative individuals have higher levels of CXCL8 in supernatants of red cell concentrates than DARC positive individuals suggesting that plasma levels of CXCL8 are higher in the former (Wadhwa et al. 2000).


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Moreover, high levels of ELR chemokines such as CXCL8 are found in renal biopsies from patients with HIV associated renal neuropathies seen predominantly in African American children with HIV. It is postulated that individuals who lack the DARC sink for chemokines would have higher levels of circulating chemokines available for endothelial cells to bind and deliver to renal tissues and increase risk of renal injury (Liu et al. 1999). DARC Expression and Cancer in African American Men DARC has been found to act as a multispecific receptor for chemokines of both the C–C and C–X–C families. Using a transgenic model of prostate cancer with DARC-deficient mice, Shen et al. (2006) demonstrated that lack of DARC expression contributed to enhanced prostate tumor growth. Their studies showed that DARC functions to clear angiogenic CXC chemokines (ELR chemokines, the key one being CXCL8) from the prostate tumor microcirculation and that lack of DARC expression in 70% of African Americans may be a contributing factor to the increased mortality to prostate cancer in this population but not to tumor initiation. Although DARC and elevated chemokine levels were implicated, no mechanistic insight was provided by these studies. We have developed the idea further and have generated the novel hypothesis that increased circulating chemokines results in greater number of activated MSCs leading to aggressive tumor growth in DARC negative African American breast cancer patients. Recent data from Mayr et al. (2007) demonstrates that individuals of African origin have higher levels of CXCL8 than individuals of Caucasian origin. In the analysis from Mayr et al. (2007) 41 Caucasians were of European origin, whereas the remaining 31 individuals of sub-Saharan African origin. CXCL-8 levels averaged 5.7 pg/mL (CI 4.4–7.0) in Africans and 3.3 pg/mL (CI 2.7–3.8) in Caucasians (P< 0.0008*). These levels are consistent with previously determined values in Caucasians. This shows that the lack of erythroid DARC expression correlates with elevated CXCL8 level in plasma.

Activation of Bone Marrow-Derived MSCs and Metastasis Karnoub et al. (2007) demonstrated that bone-marrow-derived human MSCs increased metastasis potency of MDAMB231 (otherwise weakly metastatic human breast carcinoma cells), when this cell mixture was introduced subcutaneously in nude mice. The MSCs were stimulated by the breast cancer cells to secrete CCL5, which acted in a paracrine fashion on the cancer cells to enhance metastasis. Thus, MSCs as part of the tumor microenvironment promoted metastatic spread by eliciting reversible changes in the phenotype of cancer cells. The influence of MSCs on tumor growth and metastasis was recently described by Shinagawa et al. (2009) who examined the role of human MSCs in the

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Experimental model systems for Support Tumor growth studying tumor stroma interactions

Bone Marrow

CAFs Injected with cancer cell Therapeutic Potential /Regenerative medicine CAFs

TCM-Activated

MSCs

Differentiation

In DARC negative individuals Increased levels of circulating bioactive molecules such as CXCL8

Aggressive tumor growth ?

Fig. 13.1 Mesenchymal stem cells and tumor growth. TCM tumor conditioned medium, CAF carcinoma associated fibroblasts

tumor stroma using an orthotopic nude mice model of KM12SM colon cancer. Systemically injected MSCs migrated to the stroma of colon tumors, both orthotopically implanted as well as metastatic liver tumors. Orthotopically implanted KM12SM cells mixed with MSCs resulted in larger tumors than KM12SM cells alone suggesting that MSCs promoted tumor growth. Survival rate was significantly lower in the group of animals receiving admixed MSCs, and liver metastasis was seen only in this group. Tumors resulting from admixed cells had a significantly higher proliferating cell nuclear antigen labeling index, a significantly greater proportion of microvessels and a significantly lower apoptotic index. Splenic injection of KM12SM cells admixed with MSCs resulted in greater number of liver metastases than when KM12SM cells were injected alone. MSCs incorporated into the stroma of primary and metastatic tumors and expressed markers of carcinoma-associated fibroblasts (CAFs) such as a-SMA and platelet-derived growth factor receptor-b. In in vitro experiments, KM12SM cells attracted MSCs and MSCs stimulated migration and invasion of tumor cells. The results of studies carried out by Shinagawa et al. (2009) suggest that MSCs migrate and differentiate to CAFs in tumor stroma, and promote growth and metastasis of human colon cancer in a nude mouse model.


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Conclusion Solid tumors are often surrounded by a variety of cell types including myofibroblasts also known as tumor TAFs or CAFs, tumor associated macrophages (TAMs), lymphocytes, and pericytes. We have discussed how BMD MSCs can be activated in vitro to generate myofibroblasts that resemble CAFs and the advantages of this system to model tumor stroma interaction. Bone marrow derived MSCs are suggested to be a source of tumor or CAFs and influence tumor growth and metastasis. There is emerging clinical data to suggest that in some instances tumor associated myofibroblasts may indeed be of bone marrow origin. We have also taken the liberty to speculate on the possibility that activation of MSCs derived from bone marrow under certain physiological conditions may lead to aggressive tumor growth such as seen in African American individuals with breast and prostate cancers (Fig. 13.1).

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Chapter 14

Integrin Signaling in Lymphangiogenesis Barbara Garmy-Susini

Abstract The lymphatic vasculature is essential for the maintenance of normal fluid balance and immune response. The lymphatic vessels are also crucially involved in pathological processes such as lymphedema and tumor metastasis. In the adult, lymphangiogenesis is only involved in pathological conditions. Induction of tumoral lymphangiogenesis allows tumor cells to enter the lymphatics to induce metastasis to the lymph nodes and distant loci. Dynamic interactions between lymphatic endothelial cells (LEC) and components of their surrounding extracellular matrix are necessary for the invasion, migration, and survival of LEC during lymphangiogenesis. A major part of these interactions are mediated by integrins, a cell matrix receptors family that initiate intracellular signaling cascades in response to binding to specific extracellular matrix molecules. Understanding the molecular events that define this subset of invasive LEC will facilitate the development of new treatment strategies. Therefore, understanding the role of integrin in tumor lymphangiogenesis remains crucial. Here, we review the role of integrins as major regulator of lymphangiogenesis.

Introduction Lymphatic system collects the extravasated fluid to maintain normal tissue fluid balance. In addition, lymphatic vessels absorb and transport fat released by enterocytes and represent an important part of immune surveillance by carrying immune cells and antigens. Furthermore to these physiologic tasks, lymphatic system participates to pathological conditions such as lymphedema, inflammatory diseases, and tumor metastasis. Many studies have demonstrated the existence of proliferative peri- and intratumoral lymphatic vessels (Skobe et al. 2001; Alitalo et al. 2005). Additionally, tumoral lymphangiogenesis correlates with an increase of

B. Garmy-Susini (*) Unité mixte Inserm U858, Institut de Médecine Moléculaire de Rangueil, IFR 150, 1, Avenue Jean Poulhès, BP 84225 31432, Toulouse Cedex 4, France e-mail: [email protected] R.G. Bagley (ed.), The Tumor Microenvironment, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-6615-5_14, © Springer Science+Business Media, LLC 2010

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metastases (Williams et al. 2003; Mumprecht and Detmar 2009) and detection of lymphangiogenic growth factors is associated with poor prognosis in many human tumors (Stacker et al. 2004; Renyi-Vamos et al. 2005; Zeng et al. 2005). Development and remodeling of lymphatic system requires complex interactions between lymphatic endothelial cells (LECs) and extarcellular matrix (ECM). The molecular mechanisms leading to LEC–ECM interactions remain crucial for the understanding of lymphatic development by contributing to cell movement, proliferation, and survival. Integrins represent the most important family of receptors mediating cell adhesion to ECM. A large body of evidences has demonstrated that integrins are not only adhesion receptors, but influenc e the biological activity of several other molecular systems within the cell. Their role in angiogenesis has also been clearly established. Here, we reviewed emerging results highlighting new roles of integrins in lymphangiogenesis.

Lyphangiogenesis Lymphatic Vasculature Lymphangiogenesis, the outgrowth of novel lymphatic vessels, plays a central role in maintaining the interstitial fluid balance via transporting extravasated tissue fluid, macromolecules and cells back into the blood circulation. Lymphatic vessels also play a crucial role in promoting the immune functions by controlling cellular and antigen trafficking. Initial lymphatics combine to form larger vessels called precollectors and collectors, which in turn lead to four major groups of lymph nodes in the axillary and inguinal regions. Present in the skin and in most internal organs, the lymphatic vasculature is composed of vessels with distinct morphological features. Similar to blood capillaries, lymphatic capillaries consist of a single layer of thin-walled, nonfenestrated LECs, but they are not covered by pericytes or smooth muscle cells, and have an absent or poorly developed basement membrane. In addition, they lack tight junctions and adherens junctions, which allow easy access for fluid, macromolecules, and cells into the vessel lumen (Leak 1976). Endothelial cells of lymphatic capillaries are oak leaf shaped and are interconnected by specialized discontinuous button-like junctions, whereas collecting lymphatic vessels downstream have continuous zipperlike junctions found also in blood vessels (Baluk et al. 2007). Overlapping endothelial cell–cell contacts (also called primary valves) in initial lymphatic vessels prevent fluid escaping back into the interstitial space (Trzewik et al. 2001; Schmid-Schonbein 2003). The recent discovery of specific markers and growth factors for lymphatic endothelium and the establishment of genetic mouse models with impairment of lymphatic function have provided novel insights into the molecular control of the lymphatic system in physiology and in embryonic development. Based upon these findings, novel therapeutic strategies are currently being developed that aim at inhibiting or promoting the formation and function of lymphatic vessels in disease.

14 Integrin Signaling in Lymphangiogenesis

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Lymphatic Makers Research into the role of the lymphatic system in cancer metastasis has been hampered by the lack of specific markers that distinguish lymphatic vessels from blood vessels, and also by the lack of known lymphatic-specific growth factors (Alitalo et al. 2005). However, novel lymphatic-specific markers have been recently identified such as lymphatic vascular endothelial-cell hyaluronan receptor-1 (LYVE-1) (Prevo et al. 2001) a new homologue of the CD44 glycoprotein, which is a lymphspecific receptor for hyaluronan; Prox1, a homeobox transcription factor that induces lymphatic lineage-specific differentiation and is essential for the embryonic development of the lymphatic system (Wigle and Oliver 1999; Hong et al. 2002; Petrova et al. 2002) and Podoplanin, a transmembrane glycoprotein molecule (Wicki et al. 2006) expressed in the cardinal vein and in Prox1-positive lymphatic progenitor cells in the embryos and restricted to lymphatic endothelium during later development. VEGFR-3 (also known as FLT-4) has largely been described as an major marker of lymphatics (Karkkainen et al. 2000; Kilic et al. 2007) as its expression in the adult becomes restricted to the lymphatic endothelium (Makinen et al. 2001; Bridenbaugh 2005; Breslin et al. 2007). However, recent finding have shown that VEGFR-3 is also upregulated on vascular endothelial cells in angiogenic sprouts and is present on vessels in tumors and wounds (Petrova et al. 2008; Tammela et al. 2008).

Induction of Lymphangiogenesis The spread of cancer cells from the primary tumor to distant organs usually first occurs via the sentinel lymph node. Tumors can induce lymphangiogenesis in both primary tumor and lymph node via release of the lymphangiogenic growth factors vascular endothelial growth factor VEGF-C or VEGF-D, leading to increased rates of metastasis to the draining sentinel lymph nodes and beyond (Mandriota et al. 2001; Skobe et al. 2001; Stacker et al. 2001) (Fig. 14.1). Several mediators of lymphangiogenesis have been identified. Fibroblast growth factor FGF-2 promotes lymphatic vessel growth in the mouse cornea (Kubo et al. 2002; Chang et al. 2004) and also stimulates proliferation and migration of LECs by binding to the receptor FGFR-3, which is upregulated by the transcription factor Prox1 in lymphatic endothelium (Shin et al. 2006). Hepatocyte growth factor (HGF; also known as scatter factor) also induces proliferation, migration, and tube formation of LECs and increases lymphangiogenesis in vivo (Kajiya et al. 2005). Despite the growing number of novel potential lymphangiogenic factors, the VEGF family represent the most extensive lymphangiogenic stimuli in the majority of human and experimental cancers, in particular VEGF-C and VEGF-D. VEGF-C promotes lymphangiogenesis by activating VEGF receptor (VEGFR)-2 and

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Fig. 14.1 Mechanisms regulating tumor lymphangiogenesis. (1) Tumor cells near preexisting blood vessels secrete growth factors and chemokines such as VEGF-A and FGF-2 that stimulate quiescent vascular endothelium to enter the cell cycle. (2)Tumors also secrete factors such as VEGF-C, VEGF-D, VEGF-A, FGF-2 and HGF that stimulate the growth of new lymphatic vessels in the peritumoral space. (3) These growth factors activate or upregulate expression of integrins such as avb3, avb5, a4b1, and a5b1 on blood vessels and a1b1, a2b1, a4b1, and a9b1 on lymphatic vessels. (4) These integrins then promote lymphatic endothelial cell migration, proliferation and survival, resulting in the creation of new lymphatic vessels. (5) Tumor-derived VEGF-C also promotes new lymphatic vessel growth in sentinel lymph nodes. (6) Whereas the new blood vessels induce tumor growth providing oxygen and nutriments, new lymphatic vessels represent an easier way for tumor cells to metastasize due to their vascular wall more permeable. (7) Tumor cells form metastases to proximal draining lymph nodes and then to distant organs such as lung, liver or bone

VEGFR-3 on LECs (Makinen et al. 2001). VEGF-C-deficient mice fail to develop a functional lymphatic system (Karkkainen et al. 2004), and transgenic expression of soluble VEGFR-3 results in pronounced lymphedema (Makinen et al. 2001). Recently, VEGF-A was identified as a strong lymphangiogenic mediator. Adenoviral delivery of murine VEGF-A to the skin of mice strongly promotes lymphatic vessel growth, and transgenic mice that overexpress murine VEGF-A, specifically in the skin, show increased lymphangiogenesis during wound healing and inflammation (Nagy et al. 2002; Hong et al. 2004; Hirakawa et al. 2005). Moreover both VEGF-C- and VEGF-A-overexpressing primary tumors induce lymphangiogenesis in sentinel lymph nodes before metastasizing to these tissues (Hirakawa et al. 2007) indicates that primary tumors can prepare their future metastatic site in advance of their arrival, partly by producing lymphangiogenic factors that mediate their transport. Thus, tumors might actively modify their future distant loci to make it more suitable for their further metastatic spread. This hypothesis is further supported by recent findings that attraction of bone marrow-derived hematopoietic progenitor


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cells to premetastatic sites was related to tumor-specific upregulation of fibronectin in resident fibroblasts (Kaplan et al. 2005). It will be important to determine the exact mechanisms by which lymphangiogenesis induces tumor-cell migration, via the lymphatic vasculature, to the lymph nodes and other tissues. Systemic blockade of VEGFR-3 has been recently shown to block tumor metastasis not only to lymph nodes, but also to lungs in experimental models of breast cancer (Krishnan et al. 2003; Roberts et al. 2006). Treatment with a VEGF-C inhibitor could potentially prevent the systemic spread of an early or even advanced-stage malignancy, by inhibiting lymphangiogenesis in tumors and lymph nodes. Recent reports have shown that the overexpression of VEGF-C or VEGF-D induces tumor lymphangiogenesis and promotes lymphatic metastasis in mouse tumor models (Stacker and Achen 2008). However, few clinical studies have investigated the association between the expression of VEGF-C and VEGF-D, and lymphangiogenesis and lymphatic metastasis. VEGF-D increases lymphatic vessel growth and lymphatic metastasis (Stacker et al. 2001). Recent studies revealed that VEGF-C is more significantly correlated with lymph node metastasis than VEGF-D. These findings suggest that VEGF-D is less important in lymphatic metastasis than VEGF-C, but is still necessary for metastasis (Sugiura et al. 2009).

Lymphangiogenesis and Pathology In adult organisms, lymphangiogenesis takes place only in certain pathological conditions. Abnormal function of the lymphatics is implicated in the diseases, such as lymphedema, inflammation, immune diseases, and tumor metastasis. Lymphedema is a disorder of the lymphatic vascular system characterized by impaired lymphatic return and swelling of the extremities. When the lymphatic system has been damaged during surgery or radiation treatment, its capacity to absorb excess water and cells from the interstitial space is reduced. If the transport capacity of the lymphatic system is reduced enough so that it cannot handle this increase in lymphatic load, an insufficiency of the lymphatic system may occur. Lymphedema can be an unfortunate side effect of cancer treatment. It is a chronic condition that, if ignored, can lead to disfigurement, immobilization, and severe infections. Without treatment, the swelling may continue to increase. Inflammation is thought to contribute to the development and progression of various cancers, including lung (Ardies 2003), breast (Van der Auwera et al. 2004), gastrointestinal (Jaiswal et al. 2001; Biarc et al. 2004; Brower 2005), ovarian (Altinoz and Korkmaz 2004), prostate (Wang et al. 2004), skin (Hussein and Ahmed 2005), and liver cancers (Bartsch and Nair 2004). Inflammatory breast cancer exhibits increased angiogenesis and lymphangiogenesis and has a higher metastatic potential than noninflammatory breast cancer (Angelo and Kurzrock 2007). Blocking lymphangiogenesis in chronic inflammatory diseases may become an important means of ameliorating the severity of some of these pathologies.

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The extent of lymph node metastasis is a major determinant for the staging and the prognosis of most human malignancies. Although the clinical significance of lymph node involvement is well documented, molecular mechanisms that promote tumor spread into the lymphatic or blood vascular systems and widespread dissemination are not well understood. Recent studies have shown a large body of evidence that newly visualized lymphatics facilitates formation of metastases. High tumor interstitial fluid pressure is thought to promote tumor cell entry into lymphatic vessels that have lower fluid pressure (Jain 1994; Padera et al. 2002). Intratumoral lymphatic vessel growth often correlates with metastasis of human melanoma, breast, or head and neck cancers (Maula et al. 2003; Choi et al. 2005; Dadras et al. 2005), where tumor cells can be observed within lymphatic vessels, demonstrating that lymphatic vessel growth is important for tumor spread.

Integrins Many studies have implicated a number of endothelial cell integrins in the regulation of cell growth, survival and migration during angiogenesis; nevertheless, their role in lymphangiogenesis remains unclear. The role of cell adhesion molecules and extracellular matrix proteins (ECM) in various pathological processes including angiogenesis, thrombosis, apoptosis, cell migration, and proliferation are well documented. These processes can lead to both acute and chronic disease states such as ocular diseases, metastasis, myocardial infarction, stroke, osteoporosis, a wide range of inflammatory diseases, vascular remodeling, and neurodegenerative disorders. As several integrin-targeted therapeutic agents are in clinical trials for cancer therapy, future clinical studies will likely determine whether integrin inhibitors will be best used against select tumors, such as those in which tumor cells themselves express the targeted integrin. As integrins are clearly a family of critical and fundamental regulators of angiogenesis and lymphangiogenesis, the future of integrin antagonists in cancer therapy is promising.

Integrin Expression and Function Integrin induce several physiological processes including cell activation, migration, proliferation, differentiation, and many other processes require direct contact between cells or ECM proteins. Cell–cell and cell–matrix interactions are mediated through several different families of CAM including the selectins, the integrins, the cadherins, and the immunoglobulins. The integrin family is an extensive group of structurally related receptors for ECM proteins and immunoglobulin superfamily molecules. Integrins are heterodimeric membrane glycoproteins comprised of noncovalently associated a and b subunits that promote cell attachment and migration on the surrounding extracellular matrix. Eighteen a and eight b subunits can


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Fig. 14.2 Integrin family of adhesion receptors. Each integrin receptor heterodimer binds a specific set of endogenous ligands, which may include ligands in the ECM, soluble ligands, and ligands on other cell surfaces. Integrins are divalent cation-dependent heterodimeric membrane glycoproteins comprised of noncovalently associated a and b subunits. Each integrin subunit consists of an extracellular domain, a single transmembrane region, and a short (approximately 30–40 amino acids) cytoplasmic region. Upon ligand binding, a series of intracellular signaling events is initiated. These pathways are associated with enhanced cell proliferation, migration, and survival. The role of integrins lymphangiogenesis is at an early stage of understanding. Nevertheless, recent studies have shown an important role integrins a1b1, a2b1, a4b1, and a9b1 in promoting lymphatic endothelial cell migration and survival (green)

associate to form 24 unique integrin heterodimers (Fig. 14.2). Each integrin subunit consists of an extracellular domain, a single transmembrane region, and a short (approximately 30–40 amino acids) cytoplasmic region (Hynes 2002). The main ligands for integrins in the extracellular space are extracellular matrix proteins, such as laminin and collagen, as well as cellular counter-receptors. Integrins are linked to the cytoskeleton through their cytoplasmic domains. Integrins modulate the cytoskeleton via various submembrane adaptor proteins and kinases (Zamir and Geiger 2001). They transduce signals across the plasma membrane in both directions. Most of the time, they are expressed on the cell surface in an inactive state. The activation of integrins leads to conformational changes in the extracellular domain allowing ligand binding. Integrin binding to its ligands requires its activation by inside-out signals. Conversely, integrin ligation triggers outside–in signals that regulate different aspects of cell behavior, including cell survival, control of transcription, cell proliferation, cell motility, and cytoskeletal organization (Hynes 2002). Integrin bidirectional signals are thought to be transmitted via protein–protein interactions but how the integrin cytoplasmic tail orchestrates the transmission of bidirectional signals is not well understood. Specific binding of the cytoskeletal protein to integrin b subunit cytoplasmic tails leads to the conformational rearrangements of integrin extracellular domains that increase their affinity (Tadokoro et al. 2003). Studies support a model in which intracellular protein interactions with the integrin b

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subunit tail play key roles for both inside–out and outside–in signal transduction (Arias-Salgado et al. 2003; Tadokoro et al. 2003; Arias-Salgado et al. 2005). Outside–in signal: During migration, cells are constantly making and breaking integrin contacts, activating intra-cellular signaling pathways. These signals are associated with growth factors signals and regulate cell invasion into their microenvironment (Hood and Cheresh 2002). They are mediated by intracellular signaling molecules such as FAK, SHC, and RHO family of small GTPases. Inside–out signal: Integrins not only send signals to the cell in response to the extracellular environment, but also respond to intracellular stimuli to modify the way in which they interact with extracellular matrix proteins (Hood and Cheresh 2002). This process modulates the affinity and the avidity of integrins for their ligands.

Role of Integrins in Promoting Endothelial Cells Migration, Proliferation, and Survival During tumorigenesis, a switch of integrin expression can be observed, in as much as growth-promoting and growth-attenuating integrins are up- and downregulated, respectively. ECM-ligand binding to an integrin initiates signals, which eradiating from the integrins are transmitted via different interconnecting pathways and elicit various cell functions, such as morphological changes, adhesion, migration, and gene activation. Any of these functions takes part in the metastatic cascade of tumor progression, such as epithelial-to-mesenchymal transition of carcinoma cells, tumor cell contact with the basement membrane, invasion into neighboring tissues as well as production and activation of ECM-degrading MMPs. Besides their direct involvement in tumor progression as cell surface molecules on tumor cells, integrins on endothelial surrounding a tumor can also determine various cancer characteristics, such as tumor-induced neoangiogenesis, lymphangiogenesis, and immune resistance. Therefore, integrins are relevant pharmacological targets in tumor biology.

Ligand Specificity of Integrins Each unique a-chain combines with a b-chain to form heterodimers with unique selectivity for ECM, cell surface molecules, and plasma proteins (Loftus et al. 1994; Plow et al. 2000; van der Flier and Sonnenberg 2001). Integrins binds to their ligand to a divalent-cation dependent fashion (Smith et al. 1994; Mould et al. 1995). While some integrins selectively recognize primarily a single ECM protein ligand (e.g., integrin a5b1 recognizes primarily fibronectin), others can bind several ligands (e.g., integrin avb3 binds vitronectin, fibronectin, collagen, and other matrix proteins).


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Many integrins, including avb3, a5b1, aIIbb3, avb6, and a3b1, recognize the tripeptide Arg-Gly-Asp (RGD) in their ligands (Plow et al. 2000). Sequences flanking the RGD peptide are also important for integrin selectivity (Haas and Plow 1994). Other integrins recognize alternative short peptide sequences; for example, integrin a4b1 recognizes Glu-Ile-Leu-Asp-Val (EILDV) and Arg-Glu-Asp-Val (REDV) in the alternatively spliced fibronectin domain known as IIICS (Komoriya et al. 1991). Some integrins, such as a4b1, can also bind cell surface receptors, such as Vascular Cell Adhesion Molecule-1 (VCAM-1), to promote cell–cell adhesion (Jin and Varner 2004) in addition to alternatively spliced CS-1 fibronectin. Spurred by the recent success to generate pharmaceutical mimetics of RGDdependent integrins and by the integrin’s easy accessibility on the cell surface, the hope is rising that also RGD-independent integrins, such as the collagen- and laminin-binding integrins, can be pharmacologically manipulated to fight integrindependent functions of cancer cells, which are necessary and at least partially specific for their proliferation and progression.

Integrin Signaling Integrins are essential for cell migration and invasion. They mediate adhesion to extracellular matrix and regulate intracellular signaling pathways that control cytoskeletal organization. These pathways involve phosphorylation of focal adhesion kinase (FAK), recruitment of adaptator proteins, activation of small GTPases, and downstream effector molecules (Fig. 14.3). Integrin ligation promotes integrin clustering and subsequent integrin-mediated intracellular signal transduction. Unlike growth factor receptors, integrins have no intrinsic enzymatic or kinase activities, but activate complex signaling pathways by coclustering with kinases and adaptor proteins in focal adhesion complexes. A number of signaling pathways are activated by integrins and many of these are found within focal adhesion complexes. Focal adhesion complexes are comprised of integrins, protein kinases – such as FAK and Src – adaptor proteins such as Shc, signaling intermediates such as Rho family GTPases, actin-binding cytoskeletal proteins such as talin, a-actinin, paxillin, tensin, and vinculin and other signaling proteins. Integrin signaling promotes cell migration, proliferation, and survival. Loss of integrin ligation inhibits these events and unligated integrins can actively initiate apoptosis, even without loss of cell attachment. This form of death is stress response- and death-receptor-independent, but caspase 8-dependent, and has been called “integrin mediated death” (Stupack et al. 2006). Fak FAK is a cytoplasmic protein kinase that colocalizes with integrins at structures called focal adhesion. Integrins binding of ECM ligands induces integrin clustering

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Fig. 14.3 Each integrin subunit consists of an extracellular domain, a single transmembrane region, and a short (approximately 30–40 amino acids) cytoplasmic cytoplasmic tails and no intrinsic enzymatic or kinase activities. Upon ligand binding, a series of intracellular signaling events is initiated. These pathways are associated with enhanced cell proliferation, migration, and survival. To integrate signals and activate intracellular signaling pathways, integrins cocluster with serine, threonine and tyrosine kinases, phosphatases, and adaptor proteins in focal adhesions. Focal adhesion complexes are comprised of integrins, protein kinases such as focal adhesion kinase (FAK), Src and many other kinases, adaptor proteins such as Shc, signaling intermediates such as PI-3-kinase, Rho and Rac GTPases and actin-binding cytoskeletal proteins such as talin, a-actinin, paxillin, tensin, and vinculin. Integrin signaling promotes cell migration by providing traction along the extracellular matrix and by promoting actin remodeling through the Rho family small GTPases. This actin remodeling leads to cytoplasmic flow in the direction of cell migration and cell body retraction at the trailing end of the cells. Individual components of integrin-mediated signaling cascades, such as FAK, Shc and Raf, play essential roles in angiogenesis. For example, FAK is a mediator of signal transduction by integrins and growth factor receptors in endothelial cells. In addition, Shc, an important adaptor protein that potentiates MAP kinase pathway signaling is activated by both integrins and growth factor receptors and plays critical roles in early vascular development. Like Shc, Raf-1 is an integral component of the MAP kinase signaling pathway. This signaling intermediate is activated by integrins and is critical for vascular morphogenesis. Thus, integrin mediated signaling likely plays important roles in vascular development in the adulthood

and FAK activation (Schlaepfer et al. 1999). Numerous studies have linked FAKmediated signaling pathways to cancer and a variety of other biological and disease processes. Integrin-mediated cell adhesion is the major upstream activator of FAK, and increased activation and tyrosine phosphorylation of FAK have been observed in essentially all adherent cells analyzed so far. Once it is activated, FAK undergoes


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autophosphorylated at Y397, which creates a binding site for Src via the SH2 domain (Schaller and Parsons 1994; Xing et al. 1994; Cary et al. 1999). FAK also binds p85 subunit of PI3K through autophosphorylated Y397 (Reiske et al. 2000). Phospholipid production stimulated by FAK association and activation of PI3K can activate Akt kinase, which inhibits apoptosis by regulating various cell death machinery proteins (Luo et al. 2003; Hennessy et al. 2005). Several FAK signaling pathways have been shown to play an important role in cell proliferation. The formation of FAK/Src complex allows Src to phosphorylate Y925 on FAK to mediate its interaction with Grb2 leading to the activation of RAS-Erk signaling pathway (Schlaepfer et al. 1994). FAK interaction with another adaptor molecule SHC also contributes to the activation of this pathway (Schlaepfer and Hunter 1996). Numerous studies have shown that autophosphorylation of FAK at Y397 and its association with Src at the site is essential for FAK’s ability to promote cell migration, as mutation of Y397 to F both disrupted FAK association with Src and its stimulation of cell migration in many cell types including FAK-/- cells (Cary et al. 1996; Cary et al. 1998; Owen et al. 1999). Inhibition of cell spreading by blocking FAK functions with FRNK (C-terminal FAK fragment functioning as a dominant negative FAK) could be partially rescued by overexpression of Src (Richardson and Parsons 1996). Taken together, these studies demonstrate the crucial role of FAK in cell migration, an essential process during lymphangiogenesis. Shc The adapter protein Shc is a prototype adapter protein that has been quite useful in the understanding of the function of adapter proteins in cellular signaling. The importance of Shc in vivo has been demonstrated by the knockout of the shcA gene, which results in embryonic lethality at day 11.5 (Lai and Pawson 2000) due to defects in blood vessels formation and cardiovascular development (Lai and Pawson 2000). Although a role for Shc in activation of the Ras/MAPK pathway and its role in mitogenic signaling has been better described (Bonfini et al. 1996), what other roles it may play in signaling and why this protein is so often recruited/targeted for phosphorylation by many different types of receptors is not clear. SHC is recruited to activated tyrosine kinases in response to ligation of integrins a1b1, a6b4, a5b1, and avb3 (Wary et al. 1996, 1998). Studies from various laboratories in different model systems have shown an important role for Shc in leading to Ras activation. Shc is tyrosine-phosphorylated by receptor activation and it subsequently interacts with Grb2. Grb2, binds to Ras guanine nucleotide exchange factor, Sos. The Shc/Grb2/Sos complex gets localized to the membrane through the interaction of Shc with the phosphorylated receptor. In the case of the integrin family of receptors and G-protein-coupled receptors, the precise mechanism by which the Shc/Grb2/Sos complex is localized to the membrane is not clear. Sos has been found preferentially in complexes that also contain Shc (Pronk et al. 1994). Shc may also influence the extent of Ras activation. A number of studies using

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dominant negative Shc proteins and mice lacking Shc expression have definitively established a role for Shc in MAPK activation (Salcini et al. 1994; Gotoh et al. 1997; Pratt et al. 1999; Lai and Pawson 2000). Together, studies identify Shc as a critical regulator of integrin signaling pathways. Rho Family of Small GTPases Integrin engagement and subsequent clustering of these receptors in focal adhesions leads to the generation of intracellular macromolecular complexes (Miyamoto et al. 1995). Numerous proteins present at the cytoplasmic face of focal adhesions are considered to be structural components of focal adhesions, including cytoskeletal proteins such as vinculin and talin (Jockusch and Rudiger 1996). In addition, numerous “signaling” proteins with enzymatic activity (e.g., kinases and GTPases) are also components of focal adhesions. The Rho family of GTPases appears poised to contribute to these integrin-mediated signals, in particular, signals that control cytoskeletal organization involved in changes in cell morphology. Rho family members such as Cdc42, Rac1, and RhoA are part of the Ras superfamily of proteins that cycle between active and an active state. Activated RhoA is capable of stimulating microfilament bundling in serum-starved cells that are already adherent (Ridley and Hall 1992), similar to the response of cells to plating on an ECM-coated surface. Rho is also essential for the formation of focal complexes (Hotchin and Hall 1995). The Rho family member Rac controls growth factor-stimulated membrane ruffling and formation of lamellipodia (Ridley and Hall 1992). Finally, Cdc42 activation triggers the extension of filopodia (Kozma et al. 1995; Nobes and Hall 1995). Studies such as these have defined how soluble extracellular factors induce the assembly of focal adhesions and stress fibers in serum-starved adherent Swiss 3T3 fibroblasts through activation of the Rho family of GTPases (Ridley 1996; Tapon and Hall 1997). Rho family GTPases are therefore necessary to induce the signaling and cellular responses that are required to reorganize the actin cytoskeleton into an invasive and migratory phenotype. Talin Talin is a major cytoskeletal protein that colocalizes with and binds to integrins, and to actin and actin-binding proteins such as vinculin (Calderwood et al. 1999, 2002; Tadokoro et al. 2003; Rose et al. 2007). Talin interacts directly with the b-chain cytoplasmic domain of integrins. Knockdown of talin expression in CHO (Chinesehamster ovary) cells inhibits the activation of both b1 and b3 integrins without altering integrin expression, and this cannot be compensated for by the expression of activating molecules such as activated R-Ras or the CD98 heavy chain (Tadokoro et al. 2003). Talin colocalizes with activated integrins, and overexpression of talin’s N-terminus activates integrins (Calderwood et al. 1999). Talin represents therefore a potential key regulator of integrins signaling during lymphangiogenesis.


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Vinculin Vinculin is a cytoskeletal protein associated with the cytoplasmic part of matrix adherens-type junctions. Vinculin is one of several interacting proteins involved in anchoring actin to the membrane (Weller et al. 1990). Vinculin also plays a pivotal role in cell adhesion and migration by providing the link between the actin cytoskeleton and the transmembrane receptors, integrin and cadherin. Additionaly, modulation of b1-integrin association with vinculin in human coronary artery endothelial cells (HCAEC) alter endothelial wound closure under shear stress (Albuquerque and Flozak 2003) demonstrating its role in integrin-mediated endothelial migration.

Paxillin Paxillin connect the actin cytoskeleton to the extracellular matrix within focal adhesions. Studies have shown that mutant of paxillin formed focal adhesions and exhibited limited movement associated with cell migration and wound-healing assays (Huang et al. 2003). Paxillin has also been shown to bind specifically the cytoplasmic tail of alpha4 chain to induce cell migration (Liu et al. 2000; Liu and Schnellmann 2003). This a4 binding protein suggests a potential role of the integrin alpha chain in regulating activation and signaling pathways.

Intergrins and Lymphangiogenesis The role of integrins in angiogenesis has been previously indicated in tumor angiogenesis (Hynes et al. 2002), but their role in the remodeling of lymphatic vessels is at an early stage of understanding.

a 9b1 Recent studies have shown that integrin a9b1 is expressed on quiescent LEC. Integrin a9b1 binds tenascin and oxteopontin (Kanayama et al. 2009) and is required for development of the fully functional lymphatic system because mice deficient in a9b1 integrin die 6–12 days after birth due to chylothorax, an accumulation of lymph in the pleural cavity (Huang et al. 2000). Integrin a9b1 participates to the lymphatic valves morphogenesis as it has been recently demonstrated an upregulation of integrin a9b1 on LEC and deposition of its ligand fibronectin in the extracellular matrix during embryonic development (Bazigou et al. 2009). Integrin a9b1 plays a role in growth factor mediated lymphangiogenesis as Prox-1, a LEC selective transcription factor coordinately upregulates integrin a9b1 and VEGFR-3

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expression and endothelial cell motility in vivo (Mishima et al. 2007). Furthermore, studies have shown that this integrin promotes VEGF-C- and -D-stimulated cell migration by directly binding these growth factors (Vlahakis et al. 2005). Importantly, antagonism of a9b1 suppresses VEGF-C induced motility. Taken together, these studies indicate that a9b1 plays unique yet critical roles in lymphangiogenesis.

a 1b1 and a2b1 Other studies have shown that integrins a1b1 and a2b1 are expressed on lymphatic endothelium in healing wounds in response to VEGF-A. Inhibition of these integrins blocked lymphangiogenesis in these wounds (Hong et al. 2004). These integrins bind laminin and collagen and also play roles in regulating angiogenesis. Studies in cultured LECs revealed that VEGF-A induced expression of the alpha1 and alpha2 integrins, which promoted their in vitro tube formation and their haptotactic migration toward type I collagen. It has also been shown that systemic blockade of the alpha1 and alpha2 integrins inhibits VEGF-A-driven lymphangiogenesis in vivo (Hong et al. 2004).

a 5b1 Integrin a5b1 is receptor for fibronectin. The role of integrins a5b1 in tumor angiogenesis has been clearly demonstrated (Yang et al. 1993; Francis et al. 2002) its their role in tumor lymphangiogenesis remains unclear. Integrins a5b1 and av are expressed by a subpopulation of lymphatic vessels in the inflamed cornea and small molecule antagonists of this integrin inhibited inflammatory lymphangiogenesis (Dietrich et al. 2007). Based on these studies, the role of integrin a5b1 during lymphangiogenesis seems to be restricted to inflammatory diseases. Recently, integrin a5b1 has been shown to be upregulated on lymphatic sprouts in a mice model of airways inflammation (Okazaki et al. 2009) suggesting a role in lymphatic growth. The av integrins appear to play little or no role in lymphangiogenesis and integrin a5b1 appears to play no role in tumor lymphangiogenesis due to the lack of expression on lymphangiogenic LEC during tumor progression (Garmy-Susini et al. 2007; Avraamides et al. 2008; Garmy-Susini and Varner 2008).

a 4b1 Integrin a4b1 not only allows cells to interact with the ECM after binding a spliced variant of fibronectin (CS-1 fibronectin), but also participates to cell–cell adhesion after binding a cell-surface molecule VCAM-1, a member of the immunoglobulin


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superfamily. Integrin a4b1 promotes angiogenesis after binding its ligand VCAM-1 on mural cells during blood vessels formation (Garmy-Susini et al. 2005). Integrin a4b1 regulate embryonic development as loss of its gene causes embryonic lethality by E11.5-E12.5 due to vascular defects (Kwee et al. 1995; Yang et al. 1995; Sengbusch et al. 2002). Integrin a4b1 is also highly expressed on tumor lymphatic endothelium (Garmy-Susini et al. 2007) and antagonists of this integrin can block lymphangiogenesis and tumor metastasis.

Conclusion Most of blood and lymphatic vessels remain quiescent in the adulthood and are activated during pathological conditions such as wound healing, inflammatory disease, or tumor development. Several integrins appear to play important roles in regulating lymphangiogenesis. Their profile during lymphangiogenesis is distinct from those regulating angiogenesis. Integrin expressed during lymphangiogenesis promote LEC migration, proliferation and survival, providing new ways for tumor cells to escape and metastasize to distant loci. They represent potentially relevant targets for antilymphangiogenic therapy. Many studies exhibit the evidence that drug therapies have to be considered in connection with the cell microenvironment that controls lymphangiogenesis. Targeting integrins for antilymphangiogenic therapy remains crucial, as they are expressed on activated vessels but not on quiescent vessels and therefore accessible by drugs only during lymphangiogenesis. Antagonists of these integrins may be useful in preventing tumor metastasis by blocking lymphangiogenesis. Moreover, agents targeting integrins receptors are now in clinical development for treating solid tumors. Due to the lack of side effect associated with integrins inhibitors the challenge for the future will be to optimize their use in combination with other treatment to fully exploit their therapeutic potential in lymphangiogenesis inhibition. However, the molecular events underlying the regulatory function of integrin receptors are not well understood and targeting of integrin function and signaling may be an alternative strategy to extracellular integrin antagonists for the therapeutic inhibition of tumor lymphangiogenesis.

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Chapter 15

Role of Pericytes in Resistance to Antiangiogenic Therapy Koji Matsuo, Chunhua Lu, Mian M.K. Shazad, Robert L. Coleman, and Anil K. Sood

Abstract Angiogenesis plays an important role in the progressive growth of primary tumor and metastasis, and targeting tumor angiogenesis as a therapeutic strategy is showing promise. While these approaches have shown improved survival for some cancer patients, most eventually develop progressive disease due to resistance to antiangiogenic therapy. Recent evidence suggests a functional role for pericytes in acquired resistance to antiangiogenesis agents. Pericytes play an important role in stabilizing blood vessels in the microvasculature regulated by the PDGF ligand (PDGF-BB) and receptor (PDGFR-b) homeostasis, and serve as a local source of survival factors for endothelial cells. Therefore, dual targeting of pericytes (PDGF axis blockers) and endothelial cells (VEGF pathway blockers) may be more efficacious than targeting either cell type alone.

Introduction Oxygen is vitally important to the survival of most organisms. Due to limitations in oxygen diffusion, mammalian cells require blood supply within 100–200 µm of its cell lining (Carmeliet and Jain 2000). As a result, it has been estimated that for a tumor to grow beyond 1mm in size, angiogenesis is required (Naumov et al. 2006). Recruitment of new blood vessels can occur by many pathways including vasculogenesis and angiogenesis (Carmeliet and Jain 2000; Semenza 2003). Pericytes and endothelial cells are

A.K. Sood (*) Department of Gynecologic Oncology, University of Texas M. D. Anderson Cancer Center, Houston, TX, USA and Center for RNA Interference and Non-Coding RNA, University of Texas M. D. Anderson Cancer Center, Houston, TX, USA and Department of Cancer Biology, University of Texas M. D. Anderson Cancer Center, Houston, TX, USA e-mail: [email protected] R.G. Bagley (ed.), The Tumor Microenvironment, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-6615-5_15, © Springer Science+Business Media, LLC 2010

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two critical components of blood vessel formation, and the physical and chemical interactions between the two cells are indispensable for normal endothelial function (Allt and Lawrenson 2001; Cuevas et al. 1984; Gerhardt and Betsholtz 2003). For cancer, angiogenesis plays an important role in progressive growth of primary tumor and metastasis. Therefore, targeting tumor angiogenesis may provide an efficient strategy to block tumor growth. Previous studies targeting angiogenesis have widely focused on tumor endothelial cells and their progenitors. While these approaches have shown improved survival for some cancer patients, most eventually develop progressive disease due to resistance to antiangiogenic therapy (Kerbel 2008). Several mechanisms for this acquired antiangiogenic resistance have been hypothesized, one of which is the functional role of pericytes in the tumor microenvironment (Bergers and Hanahan 2008; Kerbel 2008). In this chapter, we will focus on physiology of pericytes in tumor vasculature, targeting pericytes as a therapeutic modality, role of pericytes in resistance to antiangiogenesis therapy, and strategies to overcome pericyte-associated antiangiogenic resistance.

Biology, Physiology, and Pathology of Pericytes Despite decades of research, the origin of pericytes is still not fully understood. It is currently believed that pericytes develop from various types of progenitors depending on their anatomic location in the body. For example, epicardial, mesenchymal, and neural crest cells are believed to be a source for pericytes in the cardiac coronary vasculature, dorsal aorta, and cardiac outflow tract, respectively (Bergwerff et al. 1998; Drake et al. 1998; Vrancken Peeters et al. 1999). Pericytes play an important role in stabilizing blood vessels in the microvasculature (Nehls and Drenckhahn 1993; Sims 1986). A hallmark of pericyte function is their ability to provide vascular stability by depositing matrix or releasing factors that can promote endothelial cell differentiation or quiescence (Armulik et al. 2005). For example, it was demonstrated that pericytes release angiopoietin-1 (Ang-1) that binds to epidermal growth factor homology domains-2 (Tie2) receptor (von Tell et al. 2006). Tie2 receptor expression recently has been identified in mesenchymal cells that are present in the stroma; implicating a repository for tumor vessel pericytes (De Palma et al. 2005). Pericyte homeostasis in normal biology is regulated in significant part by signaling through the PDGF ligand and receptor system (Fig. 15.1) (Lindahl et al. 1999; Pietras and Hanahan 2005). PDGF is a potent mitogen for pericytes and fibroblasts and is composed of A, B, C, and D polypeptide chains and forms homodimers PDGF-AA, BB, CC, and DD and heterodimer PDGF-AB (Andrae et al. 2008). Its biological activities are linked to two tyrosine kinase receptors, PDGFR-a and PDGFR-b (Heldin et al. 1998; Kelly et al. 1991). PDGFR-a binds to PDGF-AA, BB, AB, and CC, whereas PDGFR-b interacts with BB and DD (Betsholtz et al. 2001). Previous studies have shown that up to 90% reduction in pericyte coverage in mice is compatible with postnatal survival (Enge et al. 2002), whereas loss of >95% of pericytes is lethal (Enge et al. 2002; Lindahl et al. 1997), suggesting that a rather

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Fig. 15.1 PDGF pathways in tumor biology. PDGF ligands and receptor pathway is suggested to be involved in the recruitment of pericytes and stabilization of blood vessels

low threshold density of pericytes is required for basal function of microvasculature. Pericyte deficiency, seen in knockout mice lacking platelet-derived growth factor-B (PDGF-B) and its receptor, PDGF receptor-beta (PDGFR-b), results in various changes in microvasculature, including endothelial hyperplasia, vessel dilation, tortuosity, leakage, and rupture, leading to widespread and lethal microhemorrhage and edema in late gestation (Hellstrom et al. 1996b, 2001; Lindahl et al. 1997). Studies of implanted tumors have shown that pericytes initially accumulate at the interface of tumor and host tissue and later around new blood vessels exhibiting close contacts with endothelial cells. Maturation of the tumor-associated vasculature is accompanied quantitatively by a reduced pericyte volume and qualitatively by morphological changes wherein pericytes become flattened and elongated (Verhoeven and Buyssens 1988).

Pericytes and Tumor Angiogenesis Pericytes are tightly attached to the endothelial cells in normal vasculature; however, it is becoming apparent that pericyte coverage in tumor blood vessels is quite distinct. Studies with insulinoma, breast, and lung carcinoma models have demonstrated that pericytes were present on most blood vessels, but 30–50% of the endothelial surface had no pericyte coverage (Morikawa et al. 2002). The extent of pericyte coverage in different tumors ranges from expansive (Schlingemann et al. 1990; Wesseling et al. 1995) to little or none (Benjamin et al. 1999; Eberhard et al. 2000; Johnson and Bruce 1997). Some of these differences may be explained by

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Fig. 15.2 Pericyte coverage on the microvasculature. Dual immunofluorescence staining for CD31 (red) and desmin (green) was performed to visualize endothelial cells and pericytes, respectively. (a) In the normal vasculature, pericytes cover vessels extensively and tightly. (b) In the tumor vasculature, pericytes are morphologically abnormal and tortuous in shape, attach to endothelial cells loosely, and paradoxically extend cytoplasmic processes away from the vessel wall

differences in pericyte marker expression among tumors. Pericytes in tumor vasculature are morphologically abnormal and tortuous in shape, attach to endothelial cells loosely, and paradoxically extend cytoplasmic processes away from the vessel wall (Fig. 15.2) (Lu et al. 2008). Several markers, including a-SMA, NG2, and desmin, have been found to be useful for detecting pericytes (Schlingemann et al. 1990, 1991). Pericytes also play an important role in regulating local blood flow, phagocytosis, modulation of new blood vessel growth and protecting endothelial cells from apoptosis (Hirschi and D’Amore 1996; Reinmuth et al. 2001). It has been demonstrated that pericytes can serve as a local source of vascular endothelial growth factor (VEGF), which is a survival factor for endothelial cells (Brown et al. 2001; Fukumura et al. 1998; Lu et al. 2008; Reinmuth et al. 2001) (Fig. 15.2). Tumor vessels lacking pericytes appear to be more dependent on VEGF for their survival than are vessels invested by pericytes (Benjamin et al. 1999). It is possible that decreased pericyte coverage may make the vascular endothelium vulnerable to VEGF blockage. Tyrosine kinase inhibitors affecting multiple receptor tyrosine kinases may exert their antitumor activity in part by reducing pericyte density in tumor vessels, thereby sensitizing them to inhibition of endothelial receptor tyrosine kinases (Lindblom et al. 1999, 2003; Shaheen et al. 2001). Variable recruitment of pericytes into tumor-associated vasculature may be explained by differences in expression of the PDGF ligands. Recent studies suggest that PDGF-B expression stimulates pericyte recruitment in gliomas (Guo et al. 2003). Both PDGF-AA and BB ligands are expressed in most ovarian cancer samples (Apte et al. 2004). PDGF-B produced by tumor endothelial cells has been shown to be required for the recruitment of adequate number of pericytes as well as proper integration of pericytes in the vascular wall (Abramsson et al. 2003). This may be further enhanced by PDGF-B production directly from tumor cells (Abramsson et al. 2003). Genetic ablation of PDGF-B in endothelial cells leads to


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impaired recruitment of pericytes, resulting in glomerular, cardiac and placental abnormalities using Cre-lox techniques (Bjarnegard et al. 2004). The close interrelationship between these control mechanisms suggests a short-range paracrine system. Using tyrosine kinase inhibitors, a functional role for PDGFR-b has also been implicated in pericyte recruitment in mouse insulinomas (Bergers et al. 2003). Interestingly, the tyrosine kinase inhibitors had no effect on normal tissues with regard to pericyte detachment, but they disrupted the association of pericytes from endothelial cells in tumors (Bergers et al. 2003). These results suggest that there may be differential sensitivity of pericytes in developing immature blood vessels compared to the mature vasculature with regard to dependence on PDGF.

Pericytes and Resistance to Antiangiogenic Therapy One of the first lines of evidence regarding the role of pericytes in protecting the tumor endothelium came from observations following treatment with anti-VEGF therapy. It was noted that anti-VEGF treatment with receptor tyrosine kinase inhibitors in RIP-Tag mice resulted in significant reductions in tumor vasculature, however, the antiangiogenic therapy failed to affect the basement membrane and pericytes (Mancuso et al. 2006).

VEGF Pathway and Pericytes in Tumor Angiogenesis VEGF affects not only endothelial cell proliferation but also cell survival (Carmeliet et al. 1996; Senger et al. 1983). Among several strategies for platinum refractory ovarian cancer, it was concluded that antiangiogenesis approaches targeting VEGF pathway were among the most efficacious (Burger et al. 2007). However, it is possible that anti-VEGF therapy selectively “prunes” blood vessels that have little or no pericyte coverage (Bergers and Hanahan 2008). Upon stimulation of pericytes (e.g., PDGF-BB), local production of factors such as VEGF may serve as a survival factor for the remaining endothelial cells (Lu et al. 2008; Reinmuth et al. 2001). For example, antiangiogenesis therapy with a VEGF receptor inhibitor (AEE788) significantly reduced microvessel density in orthotopic mouse models of ovarian cancer, but the percentage of pericyte-coated vessels was significantly increased, suggesting that pericytes may protect these vessels (Lu et al. 2007).

Resistance to Antiangiogenic Therapy The mechanisms of resistance to antiangiogenic compounds are not well understood, but may overlap to some degree with those factors responsible for resistance to cytotoxic agents (Broxterman and Georgopapadakou 2007; Shahzad et al. 2009).

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For instance, similar to cytotoxic drugs, antiangiogenic agents may face resistance due to decreased bioavailability in the tumors or by upregulation of factors that are involved in apoptosis inhibition. Antiangiogenic agents that affect endothelial cells may not be effectively targeting other components of the tumor vasculature. There are currently many antiangiogenic agents in clinical development for ovarian cancer. While antiangiogenesis treatment strategies are showing promise in clinical trials for patients with ovarian and other cancers (prolonging progressionfree and/or overall survival), most patients eventually develop progression resulting in eventual death (Burger et al. 2007; Jain et al. 2006; Spannuth et al. 2008). Emerging evidence from preclinical and clinical data suggest at least two modes of resistance to antiangiogenic therapy (Bergers and Hanahan 2008). The first mode is adaptive (evasive) resistance and the second is intrinsic (preexisting) nonresponsiveness (Bergers and Hanahan 2008). Possible mechanisms associated with adaptive resistance to anti-VEGF therapy include: (1) activation and/or upregulation of alternative proangiogenic pathways in the tumor; (2) activation and enhancement of invasion and metastasis to provide access to normal tissue vasculature without neovascularization; (3) recruitment of bone marrow-derived proangiogenic cells that differentiate into pericytes; and (4) increased pericyte coverage of tumor microvasculature serving to support survival functions for tumor endothelial cells (Bergers and Hanahan 2008). Antiangiogenic approaches are highly effective in tumor vessels lacking adequate pericyte coverage (Benjamin et al. 1999). It was found that antiangiogenic therapy can effectively reduce the overall vessel density and increase endothelial cell apoptosis in pericyte-negative vessels, but the fraction of pericyte-positive vessels significantly increases after such therapy (McCarty et al. 2004). For example, a VEGFR inhibitor was effective against endothelial cells, affecting early stage angiogenic vasculature, but not against the more mature vasculature in larger tumors (Bergers et al. 2003). The vessels in the tumors intrinsically resistant to antiangiogenic therapy showed slim and tightly covered blood vessels with pericytes (Jain and Booth 2003; Mancuso et al. 2006). These blood vessels are quite distinct from the untreated tumor vasculature, which is often characterized by pericytes that are loosely attached to endothelial cells (Jain 2005; Jain and Booth 2003). On the basis of such data, it is thought that endothelial cells may induce pericyte recruitment to protect themselves from the effects of antiangiogenic therapy. While disruption of the connections between pericytes and endothelial cells may be an attractive approach to increase the antiangiogenic effects on endothelial cells, there is an emerging concern of increased risk of metastasis associated with facilitation of tumor cell entry into the circulation through the disrupted vasculature (Xian et al. 2006). Recruitment of pericytes for external coating and reinforcement of immature blood vessels is essential in the process of normal vascular development (Allt and Lawrenson 2001; Cuevas et al. 1984; Gerhardt and Betsholtz 2003). Antiangiogenic agents may inhibit other kinases as well, and whether their effects on pericytes are mediated by kinases other than PDGFR-b cannot be ruled out. In addition, PDGF-B and PDGFR-b have other roles beside pericyte recruitment,


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including autocrine growth stimulation, formation of tumor stroma, and control of interstitial tumor pressure that may also affect tumor growth (Forsberg et al. 1993; Heldin and Westermark 1999; Pietras et al. 2001; Westermark and Heldin 1991). Other growth factors that may affect pericyte function include endothelin-1 (produced by endothelial cells) (Egginton et al. 1996), interleukin-2 (may increase localization of pericytes to endothelial cell junctions) (Sims et al. 1994), and transforming growth factor-b (produced by pericytes and may inhibit endothelial cell proliferation) (Wakui et al. 1997). However, the precise mechanism underlying recruitment of pericytes to endothelial cells is not well understood. PDGF signaling is suggested as the key factor for pericyte recruitment due to its critical role in pericyte development and function (Hellstrom et al. 1999a, 2001; Lindahl et al. 1997). Tumor hypoxia due to the vascular regression following antiangiogenic treatment appears to induce recruitment of various bone marrow-derived cells to the tumor microenvironment (Du et al. 2008). In the presence of inducible HIF1-a, populations of CD45+ myeloid cells containing Tie2, VEGFR1, CD11b, and F4/80 subpopulations, as well as endothelial and pericyte progenitor cells are released into the circulation where they promote neovascularization in glioblastoma (Du et al. 2008). In absence of HIF1-a, fewer bone marrow-derived cells are recruited to the tumors, which severely impair tumor growth. These data suggest paradoxical induction of tumor angiogenesis via bone marrow-derived vessel progenitor cells after antiangiogenic therapy.

Targeting Pericytes for Antivascular Strategies Signaling through PDGF ligand and receptor system significantly regulates pericyte homeostasis (Lindblom et al. 2003; Pietras and Hanahan 2005). On the basis of the functional and biological roles of the PDGD-BB/PDGFR-b axis on pericyte function, inhibitors of this pathway may be effective in targeting pericytes. Several available clinical or preclinical approaches to inhibiting PDGF-B and PDGFR-b pathway include: (1) tyrosine kinase inhibitors of PDGF receptor, such as imatinib mesylate (Lu et al. 2007 2008), SU6668 (Erber et al. 2004); (2) PDGF-B aptamer, a modified DNA-based aptamer to PDGF-B chain that blocks binding of PDGF-B to its cell-surface receptor (Fredriksson et al. 2002); (3) PDGF Trap targeting PDGF-BB (Lu et al. 2008); and (4) RNA interference. Treatment of RIP-Tag tumors with AX102, an aptamer blocking PDGF-BB, showed loss of pericytes with subsequent loss of endothelial cells; thereby emphasizing the importance of pericytes in tumor associated vasculature (Sennino et al. 2007). However, not surprisingly, monotherapy with these agents has been ineffective and may actually accelerate tumor growth by making the tumor vasculature more immature (Jayson et al. 2005; Spannuth et al. 2008). In contrast, combination of PDGF-BB/ PDGFR-b inhibitors with anti-VEGF or cytotoxic agents may enhance efficacy of

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antiangiogenic therapy. For example, several studies with various tumor models recently have shown that dual targeting of endothelial cells with agents such as AEE788 or SU5416, and pericytes with STI571 or SU6668, is more efficacious than targeting either cell type alone, even in established or drug-resistant tumors (Bergers et al. 2003; Lu et al. 2007). Furthermore, triple combination therapy with a cytotoxic agent, AEE788, and STI571 was effective in regressing bulky tumors in orthotopic mouse models of ovarian cancer using in vivo bioluminescence imaging (Lu et al. 2007). Other dual targeting approaches that showed efficacy in inhibiting in vitro tumor growth include combination of VEGF Trap and PDGF Trap targeting PDGF-BB (Lu et al. 2008), and bevacizumab with AX102. We and others have shown that the PDGF-BB aptamer AX102 can significantly reduce pericyte coverage, and also enhance efficacy of anti-VEGF therapy. It is possible that the efficacy of some of the new tyrosine kinase inhibitors such as SU11248 and SU14813 may be related to targeting both endothelial cells (VEGFR) and pericytes (PDGFR) (Hartmann et al. 2009; Hopkins et al. 2008; Patyna et al. 2006). Similarly, dual targeting of endothelial cells and pericytes has been reported to cause regression of pancreatic tumors in RIP1 Tag 2 mice (Bergers et al. 2003). Thus, for bulky tumors, which are often seen in recurrent disease, these approaches may have clinical relevance. In addition to PDGF-B/PDGFR-b pathway, other pathways such as Ang1 and a receptor tyrosine kinase with immunoglobulin and Tie2 pathway, sphingosine 1-phosphate (S1P) and endothelial differentiation gene-1 (Edg-1), TGF-b1 and activin-like kinase receptor (Alk5) may also offer additional opportunities for therapeutic targeting of pericytes (Erber et al. 2004). Neuropilin is a receptor of semaphorin that was initially implicated in the development of nervous system and in axon guidance (Capparuccia and Tamagnone 2009). However, recent studies have found that neuropilin is expressed in pericytes associated with angiogenesis. It is also suggested that blocking neuropilin-1 function inhibits vascular remodeling, rendering vessels more susceptible to anti-VEGF therapy (Pan et al. 2007). Neuropilin-2 is a high-affinity kinase-deficient receptor for VEGF and semaphorin, and expressed in certain types cancer (Gray et al. 2008). Dual targeting of neuropilin in tumor and pericytes may offer novel approaches for antivascular therapy.

Conclusions In summary, pericytes appear to be emerging as an important target for antivascular approaches. Pericytes not only function to stabilize the vasculature, but may play an important role in conferring resistance to anti-VEGF therapy. Therefore, dual endothelial and pericyte targeting holds promise for more efficacious cancer treatment strategies.


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Fig. 15.3 Interactions between tumor, endothelial cells, and pericytes. Cancer and endothelial cells produce PDGF-BB. PDGF-BB promotes VEGF production by pericytes. VEGF provides local survival signals for mature vasculature (adapted and modified, Lu et al. 2008)

Acknowledgments The authors declare that there is no conflict of interest. KM is supported by the GCF/OCRF Ann Schreiber Ovarian Cancer Research grant and an award from the Meyer and Ida Gordon Foundation #2. MMS is supported by the GCF-Molly Cade ovarian cancer research grant and the NIH/NICHD Baylor WRHR scholarship grant (HD050128). Portions of this work were supported by NIH grants (CA 110793 and 109298), the Ovarian Cancer Research Fund, Inc. (Program Project Development Grant), U. T. M. D. Anderson Cancer Center SPORE (P50CA083639 and P50CA098258), the Marcus Foundation, the Entertainment Industry Foundation, the Blanton-Davis Ovarian Cancer Research Program, and the Betty Anne Asche Murray Distinguished Professorship. We thank Ms. Alison E. Schroeer for assistance with graphic design for Figs. 15.1 and 15.3.

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Chapter 16

Tumour-Promoting Stromal Myofibroblasts in Human Carcinomas Urszula M. Polanska, Kieran T. Mellody, and Akira Orimo

Abstract Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the “stroma.” The stroma consists of an extracellular matrix (ECM) and a variety of mesenchymal cells, notably including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes. During tumourigenesis, the tumour-associated stroma is continuously exposed to substantial paracrine signals released by nearby carcinoma cells, and is often populated by considerable numbers of myofibroblasts. These cells are a hallmark of “activated fibroblasts” that are commonly observed in injured and fibrotic tissue. Importantly, their presence in large numbers within the stroma of human carcinomas is related to high-grade malignancies and poor prognoses in patients. Tumour-derived stromal myofibroblast-rich cell populations propagated in vitro stably maintain their myofibroblastic state, as well as an ability to significantly promote tumour growth in xenograft models. Differentiation of stromal cells into myofibroblasts within the tumour corroborates the evolution of the normal stroma towards a tumour-promoting stroma. However, the selective pressures responsible for instigating the generation of an altered stroma, and the molecular alterations that stably maintain the unique tumour-promoting myofibroblastic phenotype remain unclear. This chapter highlights the biological role of the tumour-associated stroma, with a particular focus on myofibroblasts and their ability to promote tumour progression through their interactions with carcinoma cells.

Introduction The epithelium is the tissue composed of highly organised cells that line the cavities and surfaces of structures in organs. The epithelium in the mammary gland, for example, consists of an outer layer of myoepithelial cells and an inner layer of A. Orimo () CR-UK Stromal-Tumour Interaction Group, Paterson Institute for Cancer Research, The University of Manchester, Wilmslow Road, Manchester M20 4BX, UK e-mail: [email protected] R.G. Bagley (ed.), The Tumor Microenvironment, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-6615-5_16, © Springer Science+Business Media, LLC 2010

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Fig. 16.1 Histology shows differences between stroma of the normal mammary gland and of a breast carcinoma. Paraffin sections prepared from an invasive breast cancer region (c, f ) and a non-tumour region (b, e) dissected from the breast tissue of a patient, and the normal breast region (a, d) prepared from a healthy donor, were stained with hematoxylin and eosin (H&E) (a–c) or immunostained with an anti-a-smooth muscle actin (a-SMA) antibody (d–f). Large numbers of myofibroblasts (indicated by arrows) in the cancer region (f) and myoepithelial cells (indicated by an arrow in d and e) surrounding luminal epithelial cells are shown. Scale bar, 75 mm [from Orimo et al. (2005)]

luminal epithelial cells attached to a basement membrane (Fig. 16.1a–e). The epithelium is surrounded by an adjacent stroma that consists of mesenchymal cells and a specialised ECM that helps maintain tissue integrity and homeostasis (Fig. 16.1a, b). Not only is the stroma important for cell differentiation and proliferation during tissue development, but it also plays a major role in wound healing, fibrosis and tumourigenesis. The malignant transformation of epithelial cells depends on genetic and/or epigenetic alterations that accumulate within certain oncogenes and/or tumour suppressor genes [reviewed in Hanahan and Weinberg (2000) and Vogelstein and Kinzler (2004)]. These perturbations alone, however, are not sufficient for the carcinoma to fully develop into a high-grade malignancy [for reviews, see DePinho (2000), Bissell and Radisky (2001), Coussens and Werb (2002) and Mueller and Fusenig (2004)]. Malignant epithelial cells require additional support from the surrounding stroma that evolves alongside and promotes tumourigenic progression [reviewed in Rowley (1998), Cunha et al. (2003), Bhowmick et al. (2004b), Lorusso and Ruegg (2008) and Polyak et al. (2009)]. The presence of large numbers of stromal cells and a dense ECM is characteristic of the stroma found in many types of carcinomas, including breast cancer, and is often referred to as a “desmoplastic” or “reactive” stroma (Fig. 16.1c, f). The desmoplastic stroma is believed to play a major role in promoting the growth and progression of the carcinoma [reviewed in Ronnov-Jessen et al. (1996), Bissell and Radisky (2001), Elenbaas and Weinberg (2001), Desmouliere et al. (2004), Kalluri and Zeisberg (2006), De Wever et al. (2008) and Shimoda et al. (2009)].

16 Tumour-Promoting Stromal Myofibroblasts in Human Carcinomas

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Fibroblasts and myofibroblasts, collectively designated carcinoma-associated fibroblasts (CAFs), are one of the most significant cell types found within the desmoplastic stroma. CAFs interact with cancer cells and collaborate with other components of the stroma through their production and secretion of various growth factors, cytokines and chemokines. During tumourigenesis, signalling molecules secreted by these cells effectively mediate neoangiogenesis, carcinoma cell proliferation, survival, motility and invasion [reviewed in Ronnov-Jessen et al. (1996), De Wever and Mareel (2003), Kalluri and Zeisberg (2006), Orimo and Weinberg (2006) and Ostman and Augsten (2009)].

Myofibroblasts Involved in Tissue Fibrosis Share Characteristics with Tumour-Associated Myofibroblasts Myofibroblasts exhibit a highly contractile phenotype through their expression of a-smooth muscle actin (a-SMA), which assembles into a robust network of stress fibers. These cells share characteristics with smooth muscle cells and pericytes [Desmouliere et al. 1993; reviewed in Serini and Gabbiani (1999)]. Myofibroblasts are commonly found in injured tissues (Gabbiani et al. 1971), and play essential roles in regulating the healing response by facilitating contraction of the wound. They also secrete a number of soluble growth factors, pro-inflammatory cytokines, chemokines and matrix metalloproteinases (MMPs), which act together in concert to induce angiogenesis and further facilitate tissue repair [reviewed in Powell et al. (1999) and Ronnov-Jessen et al. (1996)]. Myofibroblasts are activated and proliferate in injured tissues to promote wound healing. These cells are cleared from the tissue once the healing process is complete. Myofibroblasts are also found in a number of pathological fibrotic diseases including rheumatoid arthritis, Dupuytren’s disease, idiopathic pulmonary fibrosis, scleroderma, pulmonary hypertension and arteriosclerosis [Kim et al. 2009; reviewed in Varga (2002), Leask (2006), Varga and Abraham (2007) and Ihn (2008)]. In contrast to the normal healing response, myofibroblasts in fibrotic tissues continue to persist in an activated state. Their presence perpetuates the inflammatory process resulting in an improper tissue repair response that disrupts organ and tissue function. Several studies have identified crucial roles for the pleiotropic cytokine TGF-b in promoting fibrosis [for review, see Varga and Abraham (2007)]. TGF-b promotes mesenchymal cell proliferation, migration, differentiation, adhesion, survival, cytokine induction and ECM protein synthesis [reviewed in Derynck et al. (2001), Bierie and Moses (2006) and Massague (2008)]. It also induces and activates many profibrotic gene signalling pathways in stromal cells such as PDGF signalling that promotes the proliferation and survival of myofibroblasts during fibrogenesis [reviewed in Bonner (2004)]. This cytokine also induces expression level of endothelin-1 (ET-1) which signals through the ETA and ETB receptors. Binding of ET1 to the ETA and ETB receptors activates the JNK pathway that increases further expression of endogenous ET-1 (Shi-Wen et al. 2006). The resulting autocrine

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signalling loop contributes to the persistence of the myofibroblastic phenotype in myofibroblasts derived from scleroderma (Shi-Wen et al. 2006). In addition, TGF-b induces Jagged1-Notch autocrine signalling that is involved in myofibroblast differentiation during fibrosis (Liu et al. 2009). Importantly, myofibroblasts extracted from pathological fibrotic tissues, frequently show the establishment of a TGF-b-Smad autocrine signalling loop that is stably maintained during propagation of these cells in vitro [Kim et al. 2009; reviewed in Varga (2002)]. Myofibroblasts, derived from fibrotic tissues, secrete high levels of TGF-b and show upregulated expression levels of TGF-b receptors. This results in a constitutive activation of the TGF-b-Smad signalling pathway in an autocrine fashion that helps to maintain the myofibroblastic phenotype. Inhibition of this pathway not only substantially abates the myofibroblastic phenotype, as demonstrated using both in vitro and in vivo models of fibrosis, but is also associated with decreased ECM deposition and suppression of the inflammatory cytokine response [reviewed in Varga and Abraham (2007) and Varga and Pasche (2009)]. However, the mechanism(s) by which TGF-b autocrine signalling is initially triggered and programmed in these cells remain unclear. One possibility is that sustained exposure of stromal cells to chronic inflammation, and other selective pressures, results in altered gene expression and epigenetic modifications that generate the myofibroblastic phenotype. The tissue remodelling processes involved in wound healing and fibrosis physiologically resemble those occurring within the tumour-associated stroma [reviewed in Dvorak (1986) and Schafer and Werner (2008)]. An important study highlighted the ability of activated fibroblasts, derived from injured tissue, to actively promote tumourigenesis (Hu et al. 2009). Such fibroblasts, extracted from an inflamed rheumatoid arthritis patient, were co-injected with carcinoma cells into a recipient mouse. These activated fibroblasts increased levels of cyclooxygenase-2 (COX-2) expression in the carcinoma cells, an enzyme that is involved in inflammationassociated tumourigenesis and which boosts tumour progression. The carcinoma exploits the host’s wound healing response in order to promote tumourigenesis [reviewed in Bissell and Radisky (2001), Furlow (2005), Kalluri and Zeisberg (2006) and Orimo and Weinberg (2006)]. However, it is not known if molecular signalling, such as TGF-b autocrine signalling, which plays a central role in myofibrogenesis during fibrosis, also mediates the tumour-promoting myofibroblastic phenotype observed in tumour-associated myofibroblasts.

Carcinoma-Associated Fibroblast Characterised as Tumour-Promoting Myofibroblasts The presence of myofibroblasts in large numbers in the stroma of human tumours (Fig. 16.1c, f) is often associated with high-grade malignancies and poor prognoses in patients (Cardone et al. 1997; Maeshima et al. 2002; Tsujino et al. 2007; Kellermann et al. 2008). In order to examine the functional role of tumour-associated myofibroblasts during tumourigenesis, CAFs have been isolated from various different


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types of human carcinomas including carcinomas of the breast (Allinen et al. 2004; Orimo et al. 2005; Lebret et al. 2007; Casey et al. 2008; Mercier et al. 2008), prostate (Olumi et al. 1999; Ao et al. 2007), ovary (Yang et al. 2006), pancreas (Hwang et al. 2008), skin (Sneddon et al. 2006) and esophagus (Zhang et al. 2009). Our own observations indicate that CAFs extracted from a human breast tumour mass exhibit a different morphology in culture compared to “counterpart fibroblasts” extracted from a noncancerous region of the breast in the same individual or “normal fibroblasts” isolated from a healthy donor (Fig. 16.2a). CAFs appear more slender and spindly and upon reaching confluency, they often lose their contact inhibition of cell growth, continuing to proliferate and stack on top of one another (Fig. 16.2a). These observations are consistent with the findings of an earlier pioneer study that characterised CAFs isolated from human prostate carcinomas (Olumi et al. 1999). Primary cultured fibroblasts, extracted from human normal breast tissues, are a heterogeneous cell population that contains a small number of cells exhibiting a myofibroblastic characteristic, as determined by a-SMA expression. However, CAFs contain a noticeably larger proportion of myofibroblasts compared to those present within counterpart and normal fibroblasts (Fig. 16.2b) (Ronnov-Jessen et al. 1992; Orimo et al. 2005). Moreover, CAFs in comparison with control fibroblasts, when embedded within a collagen gel, display a more contractile phenotype that is indicative of the activated fibroblastic trait in myofibroblasts (Fig. 16.2c). They are also able to retain the activated phenotype during propagation in vitro for at least ten population doublings (PDs), without further ongoing interaction with carcinoma cells (Fig. 16.2c). Taken together, these observations demonstrate that CAFs include increased proportions of myofibroblasts that stably maintain an activated fibroblast phenotype. Genetic analyses of CAFs have previously been performed to determine if these cells are themselves neoplastic. Karyotype analysis to date has not detected any chromosomal abnormalities in CAFs (Olumi et al. 1999; Orimo et al. 2005). Consistently, genome-wide genetic analyses have also failed to detect any significant loss of heterozygosity (LOH) in these cells (Allinen et al. 2004; Qiu et al. 2008; Walter et al. 2008); (discussed in “Carcinoma-Associated Fibroblast Characterised as Tumour-Promoting Myofibroblasts” section). CAFs exhibit no anchorage-independent growth ability in vitro and no tumourigenicity in vivo when injected alone, without carcinoma cells, into a recipient mouse (Fig. 16.3a–c). They also undergo cellular senescence in vitro after 15 PDs (Orimo et al. 2005). Collectively, these findings strongly suggest that CAFs are intrinsically non-tumourigenic. CAFs, however, can promote tumour growth through their interactions with carcinoma cells. When injected along with human breast carcinoma MCF-7-ras cells subcutaneously into an immunodeficient mouse, CAFs show an ability to more significantly increase the rate and size of tumour growth compared to control counterpart and normal fibroblasts (Orimo et al. 2005). Importantly, CAFs retain such a tumour-promoting phenotype until, at least, ten PDs in the absence of continuous interaction with carcinoma cells (Orimo et al. 2005). Several other independent studies have also confirmed the ability of CAFs extracted from a number of different human carcinomas to promote tumourigenesis

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Fig. 16.2 CAFs include a larger proportion of myofibroblasts. (a) Normal fibroblasts (i) were extracted from a reduction mammoplasty performed on a healthy donor. Counterpart fibroblasts (ii) were also extracted from a noncancerous region of the breast at least 2 cm from the outer tumour margin. In addition, CAFs (iii, iv) were extracted from an invasive ductal breast carcinoma region in the same individual. CAFs (iv) show a more spindle-like morphology when they reach near confluency compared with normal (i) and counterpart fibroblasts (ii) (scale bar 50 mm). (b) An increased proportion of a-SMA-positive myofibroblasts are observed in CAFs. Immunofluorescence of normal fibroblasts (i), counterpart fibroblasts (ii) or CAFs (iii) was performed using an antia-SMA antibody. CAFs (iv) were stained with control IgG as a negative control (scale bar: 50 mm); a–d from Orimo et al. (2005). (c) Equal numbers of various fibroblasts were incubated in a collagen gel for 72 h. CAFs exhibit a greater ability to contract collagen gels compared to normal or counterpart fibroblasts. The graph shows the rate of contraction of the collagen gels for each group of fibroblasts tested. Error bars indicate standard deviation of the mean [from Orimo et al. (2005)]

(Olumi et al. 1999; Yang et al. 2006; Hwang et al. 2008; Hu et al. 2009). Immunohistochemical analysis using a human-specific anti-vimentin antibody, which detects human mesenchymal cells but not MCF-7-ras carcinoma cells or the mouse host cells, shows that either the injected CAFs or control fibroblasts are present in considerable numbers within the advanced tumour xenografts (Fig. 16.3d) (Orimo et al. 2005). Taken together, these studies suggest that CAFs promote


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Fig. 16.3 CAFs themselves are not neoplastic but are capable of promoting tumour growth through their interactions with apposed carcinoma cells. (a) Various human mammary fibroblasts, including CAFs, show no anchorage-independent growth activity when propagated in vitro. Tissue sections from mice inoculated with CAFs alone without cancer cells were stained with hematoxylin and eosin (b), and with a human-specific anti-vimentin antibody and hematoxylin (c). (d) CAFs survive alongside carcinoma cells within the advanced tumour xenografts. Immunohistochemical analysis of a paraffin section prepared from a 62-day-old tumour with antibodies against human vimentin (brown) and GFP (pink), allowing specific detection of human vimentin-expressing CAFs and GFP-labelled MCF-7-ras carcinoma cells, respectively. Nuclear staining was performed using hemotoxylin (blue) [from Orimo et al. (2005)]. Scale bars: 100 mm (a) and 50 mm (b–d)

tumour growth via their ongoing interactions with the apposed carcinoma cells throughout tumour progression. Moreover, these fibroblasts are able to retain their stable tumour-promoting properties in a cell-autonomous fashion.

Somatic Genetic and Epigenetic Alterations in Tumour-Associated Stroma The CAFs are non-neoplastic cells capable of promoting tumourigenesis through their interactions with carcinoma cells. However, there is much debate as to whether tumour-associated stromal cells harbour somatic genetic alterations [reviewed in

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Weinberg (2008b), Haviv et al. (2009) and Polyak et al. (2009)]. Furthermore, the molecular mechanism(s) responsible for maintaining the stable tumour-promoting CAF phenotype also remain unclear. A number of studies support the presence of somatic genetic alterations, including LOH and mutations, in stromal regions microdissected from various human carcinomas, including those of the breast (Moinfar et al. 2000; Wernert et al. 2001; Kurose et al. 2002; Patocs et al. 2007), ovary (Tuhkanen et al. 2004), colon (Wernert et al. 2001), bladder (Paterson et al. 2003) and head and neck (Weber et al. 2007). Indeed, 25.6% in the hereditary group and 19.4% in the sporadic group of breast cancer patients show somatic p53 mutations in microdissected tumour-associated stromal regions (Patocs et al. 2007). In addition, there is a higher risk associated with regional lymph node metastases in these patients. In prostatic cell-specific oncogene-driven transgenic mice with a p53+/- background, the developing tumour puts selective pressure on the tumour-associated stroma. This results in frequent loss of the wild type p53 allele within the stroma (Hill et al. 2005). Carcinoma cell-derived paracrine signals may act upon the adjacent stroma to give rise to p53-negative highly proliferative mesenchymal cells. However, whilst alterations in the p53 gene may confer upon stromal cells their tumour-promoting properties, it is unclear whether such alterations do give rise to the myofibroblast-rich desmoplastic stroma often seen in human carcinomas. Furthermore, patients with Li-Fraumeni syndrome are prone to developing soft tissue and bone sarcomas due to inherent p53 alterations, as do p53 knockout transgenic mice. However, it is not known why breast cancer patients, who may lose p53 function within the tumourassociated stroma, very rarely develop tumours of mesenchymal origin (Bolton and Sieunarine 1990; Tokudome et al. 2005). Most of the studies that detected genetic alterations in the tumour stromal compartments were performed using paraffin-fixed tumour tissues. Conversely, genome-wide genetic analyses, including single nucleotide polymorphism and comparative genomic hybridisation arrays, performed using freshly frozen tissues derived from human breast carcinomas, fail to detect any significant genetic alterations in cancer-associated stroma (Qiu et al. 2008). This was highlighted in a recent study that reported only one chromosomal LOH event among 35 samples of cancer-associated stromal regions dissected from different breast and ovarian tumours [Qiu et al. 2008; reviewed in Haviv et al. (2009)]. Similar analyses performed using CAFs have also failed to detect any significant genetic alterations, as described earlier (Allinen et al. 2004; Qiu et al. 2008; Walter et al. 2008). Therefore, detection of genetic alterations in some studies, whilst not in others, may be in part due to the different methods used to acquire and prepare tissues for analysis, the use of paraffin-fixed tissues instead of frozen tissues or the different types of genomic analyses performed [commented in Weinberg (2008b) and reviewed in Haviv et al. (2009)]. Future independent analyses are therefore required to clarify these issues. The presence of altered profiles of epigenetic modifications, such as DNA methylation within the genome of CAFs, has also been reported (Hu et al. 2005; Fiegl et al. 2006; Hanson et al. 2006; Jiang et al. 2008). In addition, CAFs, unlike


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normal fibroblasts, are able to confer epigenetic modifications upon apposed human breast epithelial cells. This was demonstrated in an important study in which CAFs were co-cultured for three weeks with human breast epithelial cells in an assay that allowed cell-to-cell contacts to form (Lin et al. 2008). CAFs activated the AKT signalling pathway in the epithelial cells, which hypermethylated the promoter region for the tumour suppressor gene cystatin M. Future studies are required to determine if the CAF-induced epigenetic modifications are stably reprogrammed within the epithelial cells, and if this, in turn, promotes tumourigenesis in vivo. Understanding what epigenetic modifications in CAFs mediate their phenotype, and how these cells confer epigenetic alterations upon neighbouring epithelial cells, will further highlight the molecular mechanisms that underlie their tumour-promoting properties.

Heterogeneous Cellular Origins of Carcinoma-Associated Myofibroblasts Human dermal fibroblasts are heterogeneous in nature and often display locationspecific gene expression signatures (Chang et al. 2002). Similarly, tumour-associated stromal fibroblasts are also considered to be a heterogeneous cell population (Sugimoto et al. 2006; Anderberg and Pietras 2009). Although the expression of a-SMA defines the myofibroblastic phenotype, to date, no specific protein markers have been identified that determine the cellular origins of myofibroblasts found in the stroma of tumour. These cells are therefore characterised by expression of a number of mesenchymal protein markers that include tenascin-C, fibroblast specific protein-1, fibroblast activation protein, PDGF receptor-a/b, NG2 chondroitin sulfate proteoglycan, vimentin and prolyl 4-hydroxylase [Sugimoto et al. 2006; reviewed in Kalluri and Zeisberg (2006), De Wever et al. (2008) and Anderberg and Pietras (2009)]. Various cell types are thought to act as a source for the emergence of tumourpromoting CAF myofibroblasts (Fig. 16.4). Resident fibroblasts are likely a major source of myofibroblasts [reviewed in Powell et al. (1999) and Hinz (2007)], whilst other mesenchymal cell types including endothelial cells, pericytes, smooth muscle cells and preadipocytes are also capable of converting into myofibroblasts [Zeisberg et al. 2007; reviewed in Kalluri and Zeisberg (2006) and De Wever et al. (2008)]. Bone marrow-derived progenitors, such as fibrocytes and mesenchymal stem cells (MSCs), are also reported to differentiate into myofibroblasts within the tumour. It has been shown, for example, that bone marrow-derived cells contribute to the generation of nearly 40% of myofibroblasts present in the tumour-associated stroma of 28-day-old pancreatic tumour xenografts (Ishii et al. 2003), and of approximately 25% in spontaneous pancreatic tumours developing for 16–18 weeks in another transgenic mouse model (Direkze et al. 2004). It is also possible that a small number of myofibroblasts present within the normal fibroblast populations are clonally expanded in response to selective pressure imposed by the tumour

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Carcinoma in situ Invasive carcinoma Fig. 16.4 Schematic representation of cellular origins of CAF myofibroblasts during tumour progression; CAFs are proposed to be derived from various cell types, such as pre-existing fibroblasts, pericytes or endothelial cells. Bone marrow-derived progenitor cells (BMDCs) including MSCs and fibrocytes also differentiate into myofibroblasts. A small population of residual myofibroblasts or mesenchymal cells that carry genetic alterations may be clonally selected during tumour progression. The genetically altered mesenchymal cells may or may not differentiate into myofibroblasts

microenvironment. In addition, the stromal cells that have acquired epigenetic or genetic alterations may differentiate into myofibroblasts and these cells are then also likely to undergo clonal expansion. The tumour stroma continues to remodel itself during tumour progression and actively recruits various cell types into the tumour mass where they act as different sources for myofibroblasts. It is not known, however, if different cells of origins in the myofibroblast populations in the stroma of tumour exhibit different tumourpromoting properties. Once generated, myofibroblasts maintain their ability to substantially promote tumour growth and progression in many aspects via their interactions with carcinoma cells and other host stromal cells.


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Normal Stroma-Derived Tumour-Suppressive Signalling and Tumour Stroma-Derived Tumour-Promoting Signalling TGF-b signalling functions as a potent tumour suppressor in epithelial cells during the early stages of tumourigenesis. However, the role of this signalling pathway within the stroma is unclear. A study using a transgenic mouse model, in which TGF-b type II receptor (TbRII) gene is deleted specifically in stromal fibroblasts, has demonstrated the inherent tumour-suppressing effects of stromal TGF-b signalling on epithelial cells (Bhowmick et al. 2004a). The TbRII-deficient fibroblasts secrete high levels of hepatocyte growth factor (HGF) that causes spontaneous malignant transformation of c-Met receptor-expressing forestomach epithelial cells and tumourigenesis within the mice. More recently, the tumour-suppressive role of Notch signalling within the stroma has been demonstrated in a transgenic mouse model. The chimeric deletion of the Notch 1 gene, within the epidermis of a conditional Notch 1flox/flox mouse, causes a wound-like microenvironment, as characterised by the infiltration of myofibroblasts and leukocytes into the affected tissue (Demehri et al. 2009). These cells secrete elevated levels of TGF-b, keratinocyte growth factor and stromal cell-derived factor 1 (SDF-1, also called CXCL12). This altered microenvironment, rich in growth factors and cytokines, promotes chemical carcinogeninduced skin tumourigenesis of Notch 1-expressing keratinocytes in a non-cell autonomous manner. The inherent tumour-suppressive role of stromal Notch signalling is therefore important in maintaining tissue integrity and homeostasis within the skin. In addition to TGF-b and Notch signalling, phosphatase and tensin homologue (PTEN) signalling within the stroma plays an important function in inhibiting tumourigenesis. Myofibroblasts in lung tissues from idiopathic pulmonary fibrosis patients show decreased levels of PTEN expression (White et al. 2006). Inhibition of PTEN also increases numbers of myofibroblasts observed in bleomycin-induced fibrotic lung (White et al. 2006). Furthermore, suppression of PTEN expression and inhibition of its activity stimulate differentiation of cultured fibroblasts into myofibroblasts. Indeed, the loss of PTEN expression in mammary stromal fibroblasts within a transgenic mouse model accelerates the initiation and progression of ErbB2-driven breast epithelial cancers (Trimboli et al. 2009). The ErbB2 tumours grow more rigorously in the presence of PTEN-deficient stromal fibroblasts and show a highly desmoplastic stromal reaction, as determined by an increase in the numbers of infiltrating macrophages. However, it is not known if the PTENdeficient stromal fibroblasts per se are a source of tumour-associated myofibroblasts in the stroma. In addition, a gene expression profile obtained from PTEN-null mammary stromal fibroblasts indicates upregulation of genes predominantly involved in ECM remodelling, inflammation and fibrosis. These observations further highlight the inherent tumour-suppressive role of the normal stroma, and specifically stromal PTEN, TGF-b and Notch signalling pathways, involved in suppressing tumourigenesis.

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Through its interactions with carcinoma cells, the initially tumour-suppressing stroma, however, overtime acquires an ability to promote tumourigenesis. SDF-1 is an important proangiogenic chemokine that is known to actively regulate tumourigenesis [reviewed in Burger and Kipps (2006), Nagasawa (2006), Ratajczak et al. (2006), Kryczek et al. (2007) and Petit et al. (2007)]. Unlike fibroblasts found within the non-cancerous stroma, myofibroblasts present within the tumour stroma actively secrete SDF-1 (Allinen et al. 2004; Orimo et al. 2005; Ao et al. 2007; Tait et al. 2007). This chemokine, upon binding to its cognate receptor CXCR4 constitutively expressed on the cell surface of carcinoma cells, stimulates carcinoma cell proliferation (Hall and Korach 2003; Allinen et al. 2004; Orimo et al. 2005). In the later stages of tumour progression, TGF-b signalling in epithelial cancer cells also promotes tumourigenesis. A recent study showed that elevated secretion of TGF-b by CAFs (San Francisco et al. 2004; Rosenthal et al. 2004; Ao et al. 2007), upregulates the expression levels of CXCR4 in prostatic epithelial cells. This in turn boosts stromal SDF-1-stimulated tumourigenesis in vivo (Ao et al. 2007). TGF-b within the stroma therefore also plays a permissive role in promoting some aspects of tumourigenesis. The Hedgehog (Hh) signaling is a key regulator of embryonic development but has also been implicated in the development of many different type of cancers [Karhadkar et al. 2004; reviewed in Taipale and Beachy (2001)]. The activity of the transmembrane receptor Smoothened (Smo) is constitutively suppressed by the unbound Patched 1 (PTCH1) receptor, the cognate receptor for Hh ligands. However, subsequent binding of Hh ligands to PTCH1 reverses this effect, and thus activates the Smo signalling pathway. Recent studies have demonstrated that ligand-dependent activation of the Hh pathway in the tumour-associated stroma plays an important role in influencing tumourigenesis [reviewed in Theunissen and de Sauvage (2009)]. For example, the human pancreatic carcinoma cell-secreted Sonic hedgehog (Shh) ligand has been shown to induce a desmoplastic stromal reaction within the tumour (Bailey et al. 2008). Exposure of cultured pancreatic stellate cells to Shh also facilitated their differentiation into myofibroblasts (Bailey et al. 2008). Moreover, the expression levels of both PTCH1 and Gli1, a downstream target of the Smo signalling pathways, are elevated in the stromal region of human pancreatic carcinomas (Tian et al. 2009). Hh ligands, however, are expressed within the epithelial carcinoma compartment. Importantly, inhibition of Smo protein activity on the tumour-associated stromal cells using a potent, small molecular antagonist of the Hh pathway attenuated growth of human colon adenocarcinoma cells in a tumour xenograft model (Yauch et al. 2008). These findings demonstrate that the Hh ligands secreted by carcinoma cells instigate tumour-promoting Hh signalling in nearby stromal cells in a paracrine fashion, through binding of the PTCH1 receptor expressed by stromal cells. However, the contribution of soluble paracrine factors, released by the tumourpromoting stroma in response to the activation of Hh signalling, to tumourigenesis remains unknown. A recent study highlighted an ability of Hh signalling within the tumour-associated stroma to affect drug delivery in cancer (Olive et al. 2009). Systemic administration


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of a Smo inhibitor into a murine model of pancreatic cancer, which expresses endogenous mutant K-ras and p53 alleles, inhibits Hh signalling. This alters the composition of the desmoplastic stroma and attenuates proliferation of stromal myofibroblasts. The authors also claim that the associated increase of the tumour vascular density facilitates efficient delivery of the chemotherapeutic agent into the tumour. As another possible mechanism, the increase in drug delivery may be due to decreased interstitial fluid pressure that is normally instigated by the tumourassociated stroma with high Hh signalling. This may prevent transport of the drug from the vasculature into the tumour tissue [for reviews, see Jain (2001) and Pietras et al. (2003)]. Stromal fibroblasts interact with carcinoma epithelial cells within the tumour, yet this relationship between the fibroblasts and carcinoma cells is reciprocal. It is likely that carcinoma cells initially secrete cell signalling molecules (e.g., TGF-b), which can induce the conversion of fibroblasts and/or their progenitors into myofibroblasts. In vitro experiments have demonstrated that stromal fibroblasts exposed to medium conditioned by carcinoma cells can undergo differentiation into myofibroblasts (Guo et al. 2008; Kellermann et al. 2008; Noma et al. 2008). Importantly, the ability of stromal fibroblasts to promote tumourigenesis and tumour progression is substantially associated with myofibroblast differentiation [De Wever et al. 2004; Orimo et al. 2005; reviewed in Desmouliere et al. (2004) and De Wever et al. (2008)]. Carcinoma cells can induce myofibroblast differentiation of the apposed stromal cells and the resulting myofibroblasts can in turn reciprocate to promote growth and progression of carcinoma cells. The development of the malignant tumour and promotion towards advanced stages of carcinomas require the successful co-evolution of both cancer cells and stromal cells [reviewed in De Wever et al. (2008), Weinberg (2008b), Polyak et al. (2009) and Shimoda et al. (2009)].

Tumour-Associated Stroma Promotes Neoangiogenesis The CAF-promoted neoangiogenesis is mediated by a number of proangiogenic cytokines and chemokines including SDF-1, vascular endothelial growth factor-A (VEGF-A) and basic fibroblast growth factor 2 (FGF2) (Fig. 16.5a) (Orimo et al. 2005; Guo et al. 2008; Noma et al. 2008; Pietras et al. 2008). The elevated levels of VEGF promoter activity have been detected in stromal fibroblasts present in tumours developing within transgenic mice expressing VEGF promoter-driven GFP (Fukumura et al. 1998). Other studies have shown that inhibition of stroma cell-derived VEGF significantly decreases the growth rate of fibrosarcoma cells (Dong et al. 2004). In addition, TGF-b-primed myofibroblasts secrete increased levels of VEGF that stimulates vascular formation by endothelial cells co-cultured in a 3D assay (Noma et al. 2008). These studies further support the pivotal roles of stromal VEGF in stimulating neoangiogenesis. A recent study has reported that tumour-associated stromal fibroblasts not only stimulate tumour angiogenesis but also mediate resistance to anti-angiogenic therapy.

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Fibroblast-enriched cell fractions extracted from the mouse lymphomas, which were resistant to anti-VEGFA antibody treatment, upregulated PDGF-C expression by 200-fold compared to fibroblasts isolated from non-refractory tumours (Crawford et al. 2009). This increase in stromal PDGF-C expression in the therapy-resistant tumours was attributed to an increase in neoangiogenesis occurring during the course of anti-VEGF treatment. Similarly, in a cervical squamous cell carcinoma

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Fig. 16.5 Tumour-promoting effects exerted by CAFs. (a) Tumour-promoting paracrine signalling exerted by tumour-associated myofibroblasts: HGF (Tokunou et al. 2001; De Wever et al. 2004), SDF-1 (Orimo et al. 2005; Daly et al. 2008), IGF-2 (insulin-like growth factors 2) (Zhu et al. 2007), Gremlin-1 (Sneddon et al. 2006), SFRP1 (secreted frizzled-related protein) (Joesting et al. 2005), FGF2 (fibroblasts growth factor 2) (Pietras et al. 2008), VEGF-A (vascular endothelial growth factor A) (Fukumura et al. 1998), TGF-b (Weinberg 2007), MMPs, tenascin C (De Wever et al. 2004) and tenascin W (Degen et al. 2007). Carcinoma-derived signalling molecules causing myofibroblast differentiation and proliferation: TGF-b (Desmouliere et al. 1993; Ronnov-Jessen et al. 1996; Serini and Gabbiani 1999; Hinz et al. 2001), PDGF (Bostrom et al. 1996; Jester et al. 2002; Bonner 2004), Sonic hedgehog (Shh) (Bailey et al. 2008; Olive et al. 2009). (b) CAFs stimulate neoangiogenesis; Human breast MCF-7-ras tumours that developed in the presence of CAFs are highly angiogenic. Sections prepared from MCF-7-ras tumour xenografts containing various fibroblasts were stained with anti-CD31 antibody to identify endothelial cells (iv, v, vi) or by Masson’s trichrome (i, ii, iii), which stains collagen in blue, red blood cells in red and cancer cells in purple. Tumours, developed in the presence of CAFs, are rich in collagen (iii) and have a highly vascularised stroma (iii, vi). Scale bar: 100 mm; (c) Human breast MCF-7-ras tumours developed in the presence of CAFs contain increased numbers of Sca1+CD31+ endothelial progenitor cells (EPCs) compared to tumours derived from MCF-7-ras admixed with control fibroblasts. The advanced tumours were dissociated into a single cell suspension and stained with anti-Sca1 and anti-CD31 antibodies; p80% of metastases and in >85% of studies downregulation was associated with a poor prognosis (Zöller 2009).

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In agreement with these findings, CD82 expression is able to suppress metastasis formation in animal models (Phillips et al. 1998; Takeda et al. 2007a, b). An opposing result on CD82-supported hematogenous spread of human lung cancer in SCID mice may be related to the xenogeneic system (Shinohara et al. 2001). In vitro, CD82 overexpression inhibits migration and invasion (Jackson et al. 2005; Tonoli and Barrett 2005; Liu and Zhang 2006). Studies using human tumor lines have revealed several pathways through which CD82 achieves this. In general, CD82 likely suppresses tumor progression by inhibiting cell motility via regulating signal transduction through associated proteins (reviewed in Liu and Zhang 2006). First, the association of CD82 with the a6 integrin chain and the epidermal growth factor receptor (EGFR) is accompanied by impaired laminin adhesion and migration, which is due to cointernalization with CD82, and is abolished by mutating the CD82 sorting motif (He et al. 2005; Odintsova et al. 2000, 2003). Similarly, the Ly6 antigen associates with CD82 and CD63 in TEM and promotes internalization of these tetraspanins. Accordingly, downregulation of Ly6 is accompanied by increased levels of CD82 and CD63 surface expression and reduced motility (Lekishvili et al. 2008). Second, the association of CD82 with EWI-2 strengthens the motility inhibitory activity of EWI-2 on laminin and fibronectin (Zhang et al. 2003a, b) by preventing the activation of ERM proteins, which is required for the linkage with actin (Devaux and Morris 2004). Third, uPAR colocalizes with a5b1 in focal adhesions only in the presence of CD82. The stable association between uPAR and a5b1 prevents binding of uPA to its receptor and pericellular proteolysis, a necessary step in invasion, is strikingly reduced (Bass et al. 2005). In multiple myeloma CD82, CD81 overexpression affects motility and invasive potential accompanied by reduced MMP9 secretion. In addition, a decrease in Ki67 and an elevated intracellular glutathione level are accompanied by reduced survival (Tohami et al. 2007). Fourth, CD82 interferes with c-Met signaling such that hepatocyte growth factor (HGF, also known as scatter factor)-induced cell migration, but not proliferation is reduced. In a nonsmall cell lung cancer line overexpressing CD82, phosphorylation of c-Met by HFG stimulation was unimpaired, but CD82 interfered with binding of growth factor receptor-bound protein 2 (Grb2). Grb2 is a key molecule in intracellular signal transduction, linking activated cell surface receptors to downstream targets by binding to specific phosphotyrosine-containing and proline-rich sequence motifs. CD82 interfering with Grb2 binding was accompanied by inhibition of downstream signaling via phosphoinositide 3-kinase (PI3K) and the ras–raf–MAPK (mitogen-activated protein kinase) pathway with activation of rac and Cdc42 GTPases. Lamellipodia formation and cell migration were severely impaired (Takahashi et al. 2007). Fifth, CD82 can recruit negative inhibitors that affect c-Met and src activation. Src are cytoplasmic tyrosine kinases, which are controlled by multiple membrane receptors and signal to a variety of downstream effectors (reviewed in Fuss et al. 2008) In a prostate cancer line, where CD82 interferes with integrin-dependent, HGF-induced activation of c-Met, src activation as well as c-Met phosphorylation

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was impaired, although CD82 did not interact with c-Met. Instead, in the absence of CD82 c-Met activation was unimpaired and the formation of the focal adhesion kinase (FAK)–p130CAS–Crk complex downstream of src initiated the molecular switch for motility (Sridhar and Miranti 2006). p130CAS and FAK are downstream substrates of src. P130CAS is a multiadaptor and scaffold molecule that spatially and temporally controls signaling events through changes in phosphorylation and association with effector molecules like Crk, FAK, PTP-1B, 14-3-3, PI3K, and src family kinases (reviewed in Defilippi et al. 2006). Downregulation of the p130CAS– Crk complex by CD82/KAI1 has also consequences on integrin-mediated cell migration (Zhang et al. 2003a, b). Sixth, some activities of CD82 can only be explained by taking into account the organization of tetraspanin complexes in TEM and the contribution of gangliosides (Ono et al. 1999; Todeschini and Hakomori 2008; Hakomori 2010). For example, the impact of CD82 on EGFR activation varies depending on the presence of ganglioside GD1a, which is required for the relocalization of the CD82–EGFR complex in TEM. GD1a is important for the spatial organization of CD82-enriched microdomains, which can interfere with the capacity of CD82 to recruit molecules that negatively regulate EGFR activation, e.g., a tyrosine phosphatase (Odintsova et al. 2006). The CD82–a3–Met crosstalk also becomes regulated via formation of a complex of GM2/GM3 with CD82, which interferes with c-Met activation. Both Grb2 and Ras upstream of the MAPK pathway and Gab1 (Grb2-associated binder 1) upstream of PI3K do not become activated. This blockade impairs not only cell motility, but also proliferation (Todeschini et al. 2008). It is discussed that the CD82/GM2/GM3 complex-inhibited tumor cell proliferation proceeds similar to the PKCa-mediated inhibition of EGFR-induced proliferation, where GM3 together with CD82 account for translocation and phosphorylation of PKCa that induces EGFR phosphorylation and internalization (Wang et al. 2007a, b). Seventh, two pathways of CD82-mediated metastasis suppression are motility independent. KITENIN is a metastasis-supporting four-TM protein that does not belong to the tetraspanin family. It binds to the C-terminal tail of CD82. Over expression of KITENIN in a murine colon carcinoma line promotes adhesion to ECM elements, tumor cell migration, and metastasis formation (Rowe and Jackson 2006). This may, in part, be due to the expression of a CD82 splice variant, which does not bind KITENIN and, thus, does not interfere with its metastasis-promoting activity (Jackson et al. 2007). There exists some kind of a feedback loop, because KITENIN expression by itself supports expression of this CD82 splice variant (Cherukuri et al. 2004; Clark et al. 2004). Finally, CD82 interacts in trans with DARC (Duffy antigen receptor for chemokines) on vascular ECs. This induces tumor cell senescence via reduced expression of the senescence-related transcription factor TBX2 (T-box 2) gene and upregulation of the cyclin-dependent kinase inhibitor p21WAF1, which is repressed by TBX2 (Prince et al. 2004). Accordingly, the metastasis-suppressor activity of CD82 is significantly reduced in DARC-/- mice (Bandyopadhyay et al. 2006). Besides the latter two exceptions, CD82 is a convincing example that blocking one step of the metastatic cascade, migratory activity of primary tumor cells, is

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sufficient to prevent metastasis. CD82 inhibits migration and invasion by associating directly or via bridging integrins with a multitude of different molecules as well as by the recruitment of the partner molecules in TEM, where gangliosides support or counteract the metastasis-prohibiting activity of CD82. In this respect, it is particularly challenging that perturbation of TM interactions of CD82 significantly affect the molecule’s intrinsic activities (Bari et al. 2009). A therapeutic perturbation of TM interactions may open a new avenue to prevent cancer invasion, which could be far easier approached than a blockade of individual signaling pathways. Rescuing CD82 gene expression also should prevent tumor progression (see “Rescuing the Metastasis Suppressor Gene CD82” section).

CD9 Interferes with Distinct Steps of the Metastatic Cascade CD9, expressed by ECs, brain tissue, peripheral nerves, vascular smooth muscle cells, cardiac muscles, epithelial cells, some hematopoietic cells including platelets (Horejsí and Vlcek 1991; Maecker et al. 1997), is not a metastasis suppressor gene in the strict sense (Boucheix and Rubinstein 2001; Wright et al. 2004a, b), as CD9 expression can favor metastasis formation in some tumors like gastric cancer (Hori et al. 2004). However, a positive correlation between CD9 downregulation and tumor progression has been described in colon cancer (Mori et al. 1998; Ovalle et al. 2007), peritoneal dissemination of ovarian cancer (Houle et al. 2002; Drapkin et al. 2004; Furuya et al. 2005), endometrial cancer (Miyamoto et al. 2001), small cell lung carcinoma (Higashiyama et al. 1995, 1997; Adachi et al. 1998; Miyake et al. 1999; Funakoshi et al. 2003; Saito et al. 2006; Takeda et al. 2007a, b), breast cancer (Huang et al. 1998, 2005; Mimori et al. 2005), cervical carcinoma (Sauer et al. 2003a, b) and squamous cell carcinoma of head and neck (Kusukawa et al. 2001; Mhawech et al. 2004), bladder cancer (Mhawech et al. 2003), and brain tumors (Kawashima et al. 2002). For prostate cancer, different effects of CD9 expression were described (Bérubé et al. 1994; Zvieriev et al. 2005; Wang et al. 2007a). The opposing activities of CD9 may depend on the associating molecules in the tetraspanin web. CD9 homoclustering is promoted by a3b1, a6b4, and by palmitoylation of CD9 and b4. Instead EWI-F- and EWI-2-associated or unpalmitoylated CD9 forms heteroclusters, which particularly are seen on malignant epithelial tumors (Yang et al. 2006). Distinct to CD82, CD9 can interfere with tumor progression at several steps of the metastatic cascade. CD9 can have an impact on tumorigenicity. V-Jun transformation of chicken or mouse fibroblasts interferes by an unknown mechanism with GM3 synthase transcription, such that Jun-induced oncogenic transformation was accompanied by loss of CD9–GM3 association, leading to integrin activation, enhanced motility, and soft agar colony formation. Transfection with the GM3 synthetase gene reverted the oncogenic phenotype (Miura et al. 2004). Furthermore, ectopic expression in colon carcinoma cells has an impact on in vivo tumorigenicity in nude mice (Ovalle et al. 2007). Growth inhibition by CD9 can also rely on increased expression of tumor necrosis factor (TNF)a, where the inhibitory effect


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of CD9 is mediated by membrane-bound TNFa (Ovalle et al. 2007), whose production is delayed in CD9-/- mice (Yamane et al. 2005). A similar phenomenon has been described in hepatic carcinoma (Li et al. 2006). How CD9 influences TM TNFa activity has not been clarified, but affecting the molecules stability by inhibiting the activity of ADAM in cleaving TNFa could provide a possible explanation (Moss and Bartsch 2004). CD9 might also interfere with the EMT, the initiating step of the metastatic cascade. CD9 expression induces downregulation of several Wnt family genes, such as Wnt1, Wnt2b1, and Wnt5a and their targets including WISP-1, WISP-3, c-Myc, VEGF-A, and MMP26. Wnt proteins are a large family of secreted glycoproteins that activate signal transduction pathways to control a wide variety of cellular processes such as determination of cell fate, proliferation, migration, and polarity. Wnts are capable of signaling through several pathways, the best characterized being the canonical b-catenin/Tcf-mediated pathway (Coombs et al. 2008). Notably, CD9 expression had no impact on expression of genes involved in cell motility-associated signaling or cell cycle regulation. Instead a CD9-specific antibody inhibited downregulation of Wnt genes, which suggests that the CD9 signal is located upstream of the Wnt signaling pathway (Huang et al. 2004). Thereby, downregulation of Wnt signaling by CD9 could well result in suppression of transformation and epithelial– mesenchymal transition (Huang et al. 2004) as Wnt1 stimulates the canonical Wnt/b-catenin signaling pathway that leads to changes in cell fate and/or transformation (You et al. 2002). The impact of CD9 on Wnt signaling may also affect later stages in the metastatic cascade-like cell motility, where Wnt5a stimulates the Wnt/ Ca2+ signaling pathway that directly affects actin reorganization (Ishitani et al. 2003) and invasiveness, the downregulated target genes WISP-1 and MMP26 being associated with aggressive tumor growth (Yamamoto et al. 2004). CD9 can hamper metastasis formation by prohibiting integrin-mediated motility. In ovarian carcinoma cells, expression levels of CD9 and b1, a2, a3, a5, and a6 integrin chains correlate, and downregulation of CD9 is accompanied by weaker matrix adhesion and diffuse growth in vitro (Ikeyama et al. 1993; Furuya et al. 2005). In addition, CD9 can associate with gangliosides that modulates the activity of CD9. A noninvasive bladder cancer line expresses the GM3–CD9 complex at a high level. This correlates with a strong association with a3b1 and low cell motility. The reverse is true for an invasive bladder cancer line. When GM3 is expressed at a low level, it activates Src, whereas a high level GM3 causes Csk (C-terminal Src kinase, an endogenous inhibitor of the Src-family protein tyrosine kinases) translocation into TEM microdomains with subsequent inhibition of Src by its inhibitor phoshatase 2. By the recruitment of Csk, GM3 interferes with Src activity with the consequence of impaired Rac and PI3K/Akt activation (Mitsuzuka et al. 2005). CD9 also can hamper the migration of the isolated metastasizing cells. CD9 associates with the b1 integrin chain and the EGFR. CD9 crosslinking or EGF stimulation in cells overexpressing CD9 promotes EGFR internalization, which results in a reduction in EGFR autophosphorylation after stimulation and reduced SHC phosphorylation and recruitment of Grb2 (Murayama et al. 2008). An additional pathway, whereby CD9 could regulate ligand-induced activation of the

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EGFR relies on its association with TM transforming growth factor (TGF)a, which binds, like proteolytically cleaved soluble TGFa, to the EGFR and activates EGFRinduced signaling pathways. Metalloproteinase ectodomain cleavage of TGFa is important in many cellular signaling pathways (Fan and Derynck 1999; Herrlich et al. 2008). Soluble TGFa activates the EGFR and induces receptor downregulation. This also accounts for CD9-associated TGFa, which enhances EGFR activation and proliferation in juxtacrine assays, but decreases autocrine growth stimulation (Shi et al. 2000). This is a sequel of persisting EGFR stimulation by CD9-stabilized TGFa, which leads to pronounced EGFR internalization, reorganization of the cytoskeleton, an increase in cell adhesion, and a decrease in cell migration (Imhof et al. 2008). Finally, by a not yet defined mechanism, though not proceeding through the Wnt signaling pathway (Takenawa and Suetsugu 2007), CD9 expression is accompanied by transcriptional downregulation of WAVE2 (Huang et al. 2006). WAVE2, a member of the WASP (Wiskott–Aldrich syndrome proteins) family of proteins, serves as a scaffold that links upstream signals to the activation of the ARP2/3 (actin-related proteins 2 and 3) complex, which leads to a burst of actin polymerization, where WAVE2 is crucial for lamellipodium and filipodium formation. CD9 also can interfere with transendothelial migration of tumor cells, another essential step in the metastatic cascade. CD9, CD81, and CD151 colocalize at the tumor cell–endothelial cell contact area, where CD9 promotes strong adhesion via the b1 integrin chain, which hampers transendothelial migration of the tumor cell (Longo et al. 2001). On the other hand, although downregulated in metastases, high level CD9 expression at tumor cones can support transendothelial migration in cervical carcinoma and recovery of these cone-localized CD9 “hot spots” is a highly significant indicator of lymphangiogenesis (Sauer et al. 2003a, b). Strong CD9 expression was also observed on myeloma cells in close contact to bone marrow ECs (De Bruyne et al. 2006). The reason(s) for these opposing observations may rely on differences in the CD9 web of individual tumor cells. Within the blood stream, the tumor cell can profit again from CD9 downregulation. CD9 associates with the platelet aggregation-inducing factor podoplanin. Ectopic expression of CD9 in podoplanin-expressing tumor cells leads to reduced lung metastasis formation accompanied by impaired platelet aggregation (Nakazawa et al. 2008). Platelets bind via CLEC-2 (C-type lectin-like receptor-2) to podoplanin that induces platelet degranulation (Suzuki-Inoue et al. 2006). Because CLEC-2 does not recognize CD9-associated podoplanin (Nakazawa et al. 2008), platelet aggregation is impaired. Tumor cell platelet aggregates facilitate embolization of the microvasculature and metastasis formation. Platelet aggregates embedded tumor cells may also be protected from an immune attack (Sierko and Wojtukiewicz 2007). Depending on the prevailing conditions, CD9 can mitigate the aggressiveness of the metastasizing tumor cell and of metastatic growth. Ectopic CD9 expression can suppress motility and neurite-like outgrowth and promotes apoptotic cell death when grown in vitro under starvation. This correlates with impaired adhesion-dependent Akt phosphorylation and suppression of MMP2 secretion (Saito et al. 2006). In contrast, under physiological conditions, CD9 overexpression revealed increased


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MMP2 expression at the cone of invading cells that facilitates transendothelial migration (Hong et al. 2005). Inhibition of p38MAPK and JNK as well as siRNA transfection abrogated MMP2 expression, which points towards CD9-induced MMP2 expression to be mediated through an AP-1 site (Hong et al. 2005). Because metastasizing tumor cells less likely are exposed to starvation, CD9-induced MMP2 expression appears more likely to reflect the in vivo situation. Though partly opposing activities have been observed, which may be due to differences in the tetraspanin web, the potential of CD9 to suppress metastasis relies on inhibiting tumorigenicity and proliferation, EMT, motility, aggressiveness, and survival including the recruitment of a protective environment. CD81 and CD63 and Metastasis Suppression CD81 is widely expressed in human tissue with the exception of erythrocytes and platelets. Similar expression profiles were found in mouse tissue (Levy et al. 1998). CD81 downregulation has been observed in hepatocellular carcinoma (Inoue et al. 2001; Schöniger-Hekele et al. 2005), multiple myeloma (Drucker et al. 2006), melanoma (Xu and Hynes 2007), and glioblastoma (Staflin et al. 2009). In hepatocellular carcinoma, the interaction of CD81 with PI4KII suppresses cell motility by promoting the formation of CD81-enriched vesicles that sequestered actinin-4. It is discussed that the CD81 association-mediated redistribution of PI4KII to intracellular vesicles negatively affects actin-bundling activity of actinin (Fraley et al. 2003; Janmey and Lindberg 2004; Mazzocca et al. 2008). In melanoma, GPR56 forms a complex with Gaq and CD81. GPR56 binds tissue transglutaminase 2 (TG2), a major crosslinking enzyme in the ECM. The binding of the GPR56– Gaq–CD81 complex to TG2 may support adhesion, thereby interfering with tumor cell migration (Xu and Hynes 2007). CD81, but also CD82, have been described to cause multiple myeloma death. The antimyeloma effect of CD81 and CD82 involves downregulation of Akt, activation of FoxO (forkhead box, subgroup O) transcription, and a decrease in the active mechanistic target of rapamycin (mTOR). This is surprising because inhibitors of mTOR frequently activate Akt and vice versa (Lishner et al. 2008). The authors speculate that CD81/CD82 may act as a switch in signaling pathways rather than affect a single factor. Though the complexity of tetraspanin networks could allow for such switching, further studies are required before considering therapeutic application. Another pathway whereby CD81 is involved in metastasis inhibition proceeds via its association with EWI-2 in glioblastoma. EWI-2 expression causes diminished cell motility and invasion. EWI-2 affects the organization of CD9 and CD81 as well as of MMP2 and MT1-MMP. EWI-2 strengthens tetraspanin–tetraspanin associations and the association of CD81 with MMP-2 and MT1-MMP becomes weakened (Kolesnikova et al. 2009). CD63 is expressed rather ubiquitously. It was first described as a molecule expressed on activated platelets as platelet glycoprotein 40, and in early stage human melanoma cells as melanoma antigen 491. CD63 is present in TEM as well as in late endosomes and lysosomes. It is also enriched in exosomes. Endocytosis

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is supposed to be clathrin dependent, though not exclusively (reviewed in Pols and Klumperman 2009). CD63 interacts with CD9, CD81, CD82, CD151 (Radford et al. 1996; Cannon and Cresswell 2001; Berditchevski and Odintsova 2007), several integrins (Berditchevski et al. 1995; Radford et al. 1995; Rubinstein et al. 1996; Skubitz et al. 1996), cells surface receptors including the chemokine receptor CXCR4 (Kitani et al. 1991; Levy and Shoham 2005a, b; Yoshida et al. 2008), kinases, APs, L6, syntenin, tissue inhibitor of metalloproteinase (TIMP)1, H,K-ATPase, and MT1-MMP (Skubitz et al. 1996; Hirst et al. 1999; Duffield et al. 2003; Takino et al. 2003; Jung et al. 2006; Latysheva et al. 2006; Lekishvili et al. 2008). An inverse correlation between CD63 and tumor progression has been described for ovarian cancer (Zhijun et al. 2007), lung cancer (Kwon et al. 2007), breast and colon cancer (Sordat et al. 2002; Sauer et al. 2003a, b), and melanoma (Hotta et al. 1988; Radford et al. 1995; Jang and Lee 2003). However, for melanoma the opposite finding has also been reported (Lewis et al. 2005). As recently reviewed (Pols and Klumperman 2009), potential pathways, whereby CD63 prohibits metastasis formation, may rely on integrin endocytosis, lysosomal degradation of MT1-MMP (Takino et al. 2003), and recruitment of TIMP-1 (Jung et al. 2006). Another pathway, whereby CD63 becomes involved in tumor progression relies on its association with syntenin-1. Syntenin-1 regulates cell migration and promotes metastasis (Koo et al. 2002; Boukerche et al. 2005). Syntenin-1 directly interacts with CD63 and overexpression of syntenin-1 decreases the rate of CD63 internalization (Latysheva et al. 2006). Besides a report on Tspan13 (TM4SF13) to suppress breast carcinoma invasion in vitro (Huang et al. 2007), to my knowledge nothing is known about the involvement of additional tetraspanins in metastasis suppression.

Tetraspanins and Tumor Progression CD151 and Tumor Cell Motility CD151/PETA-3 has a broad tissue distribution. It is expressed in epithelial cells and high in ECs, platelets, megakaryocytes, and monocytes (Horejsí and Vlcek 1991; Hasegawa et al. 1997; Maecker et al. 1997; Sincock et al. 1997). First evidence of CD151 as a metastasis-promoting molecule was derived from a blinded study. An antibody for an unknown target inhibited metastasis formation of a human epidermoid carcinoma line. The antibody was found to recognize CD151. The antibody inhibited cell migration without having any effect on cell adhesion or cell growth (Testa et al. 1999). Meanwhile, overexpression of CD151 has been described in many tumor types like nonsmall cell lung cancer (Tokuhara et al. 2001), where the overall survival rate was much lower for patients with CD151+ as compared to patients with CD151- tumors. Overexpression of CD151 in squamous cell carcinoma and prostate cancer correlates with a poor prognosis, CD151 expression being a better predictor than histological grading (Ang et al. 2004).


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In brain, mammary, pancreatic and colorectal cancer (Hashida et al. 2003; Bredel et al. 2005; Gesierich et al. 2005; Yang et al. 2008), too, high CD151 expression mostly is associated with a poor prognosis (Zöller 2006; Lazo 2007; Le Naour and Zöller 2008). CD151 also has been shown to inhibit tumor progression in several animal models (Testa et al. 1999; Kohno et al. 2002; Zijlstra et al. 2008). Several lines of evidence point towards a link between MMPs and CD151. CD151 contributes to pericellular activation of MMPs by associating with proMMP7, which results in activation of MMP7; this can be prevented by antiCD151 antibodies. Capture and activation of MMP7 at the cell membrane allows for focal proteolysis of the surrounding ECM (Shiomi et al. 2005). In addition, CD151 influences MMP9 expression, which was inhibited in the presence of FAK, Src p38, and JNK inhibitors and was mediated by c-Jun binding to AP1 sites in the MMP9 promoter. Signaling was initiated via CD151-associated a3b1 or a6b1 and was stimulated by CD151 homodimerization (Fitter et al. 1995; Hong et al. 2006; Yang et al. 2008). Reduced expression of MMP2, MMP7, and MMP9 in a CD151knockdown carcinoma line confirmed the involvement of CD151 in MMP expression, complex formation, and colocalization at the leading edge of lamellipodia (Shiomi et al 2005; Hasegawa et al. 2007). Transfection of FAK competent and deficient fibroblasts with CD151 cDNA provided evidence that FAK is needed for CD151-mediated increased migration, matrigel invasion, and metastasis formation (Kohno et al. 2002). Further studies confirmed that CD151 has no effect on steady state a3 and a6 integrin chain expression, but is important for proper localization of laminin5-binding integrins during tumor cell–stromal cell interactions, where under stimulatory conditions migration and invasion are strengthened by a3b1 cointernalization with CD151 (Hasegawa et al. 2007). Accordingly, CD151-knockdown cells display impaired motility, anomalously persistent adhesive contacts, and impaired a3b1 internalization (Winterwood et al. 2006). Notably, too, CD151 regulates glycosylation of a3b1 with reduced a3b1 glycosylation in CD151-knockdown cells showing strongly impaired migration towards laminin (Baldwin et al. 2008). Confirming the importance of CD151 for integrin traffic, mutating the CD151 sorting motif markedly attenuated a3b1, a5b1, and a6b1 endocytosis and accumulation in intracellular vesicular compartments (Liu et al. 2007). Thus, CD151 plays a critical role in integrin recycling as a mechanism to regulate tumor cell migration. CD151 is also an important regulator of collective tumor cell migration. Monolayers of CD151-knockdown cells display strikingly increased remodeling rates and junctional instability, which is caused by excessive RhoA activation and loss of actin organization at cell–cell junctions. There is evidence that CD151 regulates the stability of tumor cell–cell interaction through its association with a3b1 (Johnson et al. 2009). The importance of CD151 for metastasis formation has been confirmed in vivo (Zijlstra et al. 2008). A CD151 blocking antibody inhibits matrix-mediated migration, but has no impact on extravasation. Migration inhibition is due to a failure to detach at the rear end. As migration of CD151-knockout cells is unimpaired, the authors suggest that – if present – CD151 might recruit partner molecules that

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control deadhesion, but are hampered in their activity in the presence of the CD151 blocking antibody (Zijlstra et al. 2008). Taking into account that rear end adhesion was not restricted to selective components of the ECM, one could hypothesize that recruitment of MMPs may only be hampered by a CD151-specific antibody if CD151-associated integrins are involved in the adhesion process, which might be a consequence of a direct interaction with CD151, but not with other tetraspanins. The contribution of CD151 to cancer metastasis provides another convincing example of the facilitator role of tetraspanins. CD151 regulates cell migration, mostly through its association with a3b1, a6b4, and MMPs. The TEM location, which facilitates the recruitment of integrins, additional transmembrane and cytosolic proteins like E-cadherin, b-catenin, kinases, and phosphatases, in multimolecular complexes, contributes to this dominating theme (Hemler 2005). Tspan8 and Metastasis Expression of Tspan8, formerly CO-29 in human (Sela et al. 1989; Szala et al. 1990) and D6.1A in the rat (Matzku et al. 1989), has not been systemically evaluated in humans. It is relatively broad in rats, but it is not expressed in the epidermis, lymphocytes, and platelets (Claas et al. 1996). Human and rat Tspan8 associates with CD9, CD81, CD151, and several integrins including a3b1 and a6b4, but both a3b1 and a6b4 associations are weaker than described for CD151. Nonintegrin partners are EWI-F, EpCAM, CD13, CD44, PKC, and PI4KII (Claas et al 1998, 2005; Herlevsen et al. 2003; Schmidt et al. 2004; Gesierich et al. 2005; Jung et al. 2009). The Yxxf sorting motif of Tspan8 is located close to the TM region, which could hamper binding of the µ subunit of the AP-2 adaptor complex (Nakatsu and Ohno 2003; Berditchevski and Odintsova 2007). Overexpression of Tspan8 (CO-029) in colorectal cancer was described in 1989 (Sela et al. 1989) and was subsequently confirmed for colorectal, pancreatic, hepatocellular carcinoma, and esophageal cancer (Kanetaka et al. 2001; Gesierich et al. 2005; Kuhn et al. 2007; Zhou et al. 2008; reviewed in Zöller 2006, 2009). Tspan8 overexpression correlates with poor differentiation and intrahepatic spread of hepatoma rather than with the primary tumor growth. Only a Tspan8overexpressing hepatoma clone develops intrahepatic metastases (Kanetaka et al. 2003). Profiling the tetraspanin web of human colon cancer revealed Tspan8 expression in two metastatic, but not a nonmetastatic subline (Le Naour et al. 2006). Increased CO-029 expression in a metastasis versus the primary tumorderived colon carcinoma line supports a role in tumor progression (Huerta et al. 2003). First evidence that Tspan8 acts via associating molecules is derived from the observation that expression of an EpCAM–claudin-7 complex in human colorectal cancer promotes tumor progression only when Tspan8 is associated in TEM (Kuhn et al. 2007). Tspan8 can exert four tumor growth-promoting features, support of tumor cell proliferation, apoptosis protection, angiogenesis induction, and strengthening tumor cell motility. Only the latter is directly related to the metastatic process.


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Increased Tspan8 expression in a dedifferentiated rat hepatoma cell line promoted cell proliferation (Tanaka et al. 2002). Furthermore, an interaction with platelets and leukocytes was suggested to provide tumor cells with a survival advantage in the hostile environment encountered during metastatic spread (Kanetaka et al. 2001, 2003). Several groups observed high Tspan8 expression to be associated with increased apoptosis resistance (Huerta et al. 2003; Kuhn et al. 2007), likely via a Tspan8-associated EpCAM–claudin-7 complex. This has been deduced from the striking decrease in drug resistance upon a knockdown of EpCAM or claudin-7 in human and rat cancer lines, which is accompanied by reduced PI3K activation and loss of Akt and downstream anti-apoptotic protein phosphorylation. Signals are initiated by the recruitment of the EpCAM-claudin-7 complex into TEM, which is accompanied by claudin-7 phosphorylation, possibly via Tspan8-associated PKC (Ladwein et al. 2005; Kuhn et al. 2007; Nübel et al. 2009). Tspan8-promoted tumor cell motility and liver metastasis formation may proceed via its association with a6b4, as it is seen in a Tspan8 and a6b4 overexpressing tumor line, but not a Tspan8 or a6b4, only, expressing line (Herlevsen et al. 2003; Gesierich et al. 2005). Tspan8 associates with a6b4 only after stimulation and disassembly of hemidesmosomes, which is accompanied by transient internalization of the Tspan8–a6b4 complex and increased motility (Huerta et al. 2003; Herlevsen et al. 2003). This kind of continuing internalization to the endosomal compartment and rapid recycling back to the cell surface via a short loop recycling machinery under the control of rab4 has been described for several integrins (Caswell and Norman 2008). An additional feature of pro-metastatic activity of Tspan8, angiogenesis induction, will be detailed in the following section. However, it should be mentioned that the Tspan8-mediated support of tumor cell migration or angiogenesis strikingly depends on the tumor cell’s integrin profile. Angiogenesis induction dominates in the absence of a6b4 and may be actively suppressed by a6b4 (Gesierich 2006). These comparably few studies point towards metastasis-promoting activities of Tspan8. In line with the general feature of tetraspanins, the TEM localization of Tspan8 is decisive and functional activity is determined by complex formation with associating molecules.

Tetraspanins, Premetastatic Niche, Angiogenesis, Thrombosis, and Exosomes Tetraspanins and Exosomes The MVB derive from vesicles sorted from the trans-Golgi network or from internalized membranes. MVB fuse with lysosomes for protein degradation (Stahl and Barbieri 2002) or their intraluminal vesicles fuse with the plasma membrane and are released as exosomes (Denzer et al. 2000; Stoorvogel et al. 2002; Lakkaraju and Rodriguez-Boulan 2008; Simpson et al. 2009). The latter process, first described for

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the release of the transferrin receptor during reticulocyte maturation, was considered as a mode to eliminate obsolete proteins (Pan et al. 1985). Exosome research became highly stimulated when it was noted that antigen presenting cells release exosomes derived from the MVB of the MHC class II compartment, which function similar to antigen presenting cells and stimulate T cells in vitro and in vivo (Denzer et al. 2000; André et al. 2002; Admyre et al. 2006; Iero et al. 2008; Schorey and Bhatnagar 2008). Exosomes are abundantly released by tumor cells (de Gassart et al. 2004; Keller et al. 2006; Wieckowski and Whiteside 2006; Lakkaraju and Rodriguez-Boulan 2008; Schorey and Bhatnagar 2008; van Niel et al. 2006). The molecular composition of exosomes reflects their origin from intraluminal vesicles. Besides a common set of membrane and cytosolic molecules, which includes several tetraspanins, like CD9, CD37, CD53, CD63, CD81, CD82, CD151, and Tspan8, exosomes harbor subsets of proteins that are linked to cell type-specific functions (Schorey and Bhatnagar 2008; Mathivanan et al. 2010) (Fig. 26.2). Exosomal proteins maintain their functional activity. Thus, microglial-derived exosomal CD13, a zink-dependent metalloprotease (Zhang and Xu 2008) is active in cleaving neuropeptides (Potolicchio et al. 2005); ADAM10 (a disintegrin and metalloprotease), a member of these multidomain membrane proteins, which share a disintegrin and a zinc metalloprotease domain (Edwards et al. 2008), cleaves proteins, like CD44 and L1/CD171 within exosomes (Stoeck et al. 2006). Another notable feature of exosomes is expression of phosphatidylserine at the outer membrane leaflet, which appears to be essential for exosome budding in the late endosomes (Fomina et al. 2003) and also can trigger exosome uptake by phosphatidylserinebinding proteins like scavenger receptors, integrins, and complement receptors (Zakharova et al. 2007). Most importantly, exosomes contain mRNA and microRNA, so-called shuttle RNA that is transferred to the target cell. The horizontal transfer of exosomal genetic material between cells can induce exogenous gene expression and mediate RNA silencing (Ratajczak et al. 2006; Deregibus et al. 2007; Valadi et al. 2007; Burghoff et al. 2008; Lakkaraju and Rodriguez-Boulan 2008). Exosomemediated horizontal gene transfer is specific with respect to the target cell, such that it is transcribed in one, but not another type of target cells (Valadi et al. 2007; Simons and Raposo 2009). The relative abundance of proteins, mRNA, and miRNAs differs between exosomes and donor cells, which implies active sorting into MVBs (Lakkaraju and Rodriguez-Boulan 2008). The mechanisms underlying selective sorting of mRNA and miRNA into exosomes are unknown (Subra et al. 2007). Instead, it is known that sorting of proteins into exosomes can rely on monoubiquitination (Gruenberg and Stenmark 2004; Hurley and Emr 2006; Smalheiser 2007), localization in cholesterol-rich membrane microdomains, or higher order oligomerization (Fang et al. 2007; Smalheiser 2007). Though many questions remain to be answered, it is well appreciated that exosomes may be one of the most important delivery systems (Johnstone 2006; Belting and Wittrup 2008; Simpson et al. 2008; Pap et al. 2009; Seow and Wood 2009; Xiao et al. 2009). Tetraspanins are a constitutive component of exosomes (Escola et al. 1998). Though targeting into exosomes apparently differs for individual tetraspanins and their associated molecules (Abache et al. 2007), tetraspanin enrichment in


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exosomes is well in line with the preferential internalization of oligomeric proteins and/or proteins localized in cholesterol-rich membrane domains and may be supported by the internalization motif of some tetraspanins and/or their associated proteins. As exosomes could well contribute to premetastatic niche preparation and have been demonstrated to be most important in angiogenesis as well as in coagulation and homeostasis (Al-Nedawi et al. 2009), defining the function of tetraspanins in exosomes becomes demanding. Though not yet proven, tetraspanins have been implicated in the adhesion of exosomes to target cells (Pols and Klumperman 2009; Zöller 2009). Tetraspanins and the Premetastatic Niche Settlement of metastasizing tumor cells is facilitated by the establishment of special niches in (pre)metastatic organs (Bissell and Labarge 2005). Niche preparation involves stimulation of local fibroblasts by tumor-derived factors and chemokines that attract tumor cells and hematopoietic progenitors (Kaplan et al. 2005; Kaplan et al. 2006), lysyl oxidase being important for marrow cell recruitment (Erler et al. 2009). Nonetheless, information on long-distance communication between a tumor and host organs is still limited. We suggest that tumor cells avail special delivery systems and hypothesize that a concerted activity between tumor-derived factors and exosomes is required (Fevrier and Raposo 2004). An involvement of exosomes in metastasis was first described for platelet-derived exosomes. These transferred the aIIb integrin chain to lung cancer cells, stimulated the MAPK pathway and MT1-MMP expression, increased cyclinD2 expression, stimulated angiogenic factor expression as well as adhesion to fibrinogen and human umbilical vein ECs (Janowska-Wieczorek et al. 2005). A direct transfer of metastatic capacity by exosomes was demonstrated for B16 melanoma cells. Exosomes derived from a highly metastatic variant transferred metastatic capacity to low metastatic B16F1 cells. Lung metastasis formation by B16F1 was accompanied by protein uptake from exosomes of the metastasizing subclone (Hao et al. 2006). Our own findings suggest that Tspan8 and/or CD151-containing exosomes, depending on the exosomal tetraspanin web, also contribute to premetastatic niche formation (Jung et al. 2009). As the metastasizing capacity of a rat pancreatic adenocarcinoma, which metastasizes via the lymphatic system, but does not grow locally, is strikingly reduced by a knockdown of CD44v4-v7, this model allowed to define tumor cellderived components required for (pre)metastatic niche formation. Conditioned medium of the metastasizing subline strongly supports (pre)metastatic niche preparation, where fractionation of the conditioned medium revealed that the soluble matrix of the metastasizing subline can also cooperate with exosomes of the nonmetastatic subline. This implies that exosomes are the final actors, but require a soluble matrix, whose assembly depends on CD44v. A dominating component of the exosomes is the tetraspanins CD151 and Tspan8, which coimmunoprecipitate, besides others, with a6b4 (Jung et al. 2009). These exosomes preferentially bind to and are taken up by lymph node stroma cells and lung fibroblasts and binding is inhibited by anti-Tspan8 and even more efficiently by anti-CD151 and anti-a6b4 (unpublished finding).

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According to published evidence and our own findings, we suppose that exosomal tetraspanins and their associated molecules are most important for selective targeting and delivery of exosomes. Our work on the role of Tspan8 in angiogenesis strengthens this hypothesis. Tetraspanins and Angiogenesis CD151 has repeatedly been reported to be important in angiogenesis induction. This has been suggested to be due to CD151 being expressed abundantly on EC and particularly on EC–cell junctions (Fitter et al. 1995; Maecker et al. 1997). However, patients with mutations of the CD151 gene show skin and kidney disorders, but no obvious defects in vasculogenesis (Karamatic Crew et al. 2004) and in CD151knockout mice vasculogenesis is unimpaired. Instead, defects are seen in angiogenesis (Takeda et al. 2007a, b; Wright et al. 2004a, b; Sachs et al. 2006). Thus, CD151 expression of the tumor-bearing host facilitates tumor growth due to angiogenesis induction (Takeda et al. 2007a, b). CD151 supports EC invasiveness, migration, cable formation, matrigel contraction, tube formation and sprouting, which activities are all impaired in CD151-knockout mice (Takeda et al. 2007a, b). Selective defects in adhesion-dependent signaling activation of PKB/c-Akt, e-NOS, Rac, and Cdc42 on laminin substrates contribute to impaired angiogenesis induction (Takeda et al. 2007a, b; Zheng and Liu 2007). Also, overexpression of CD151 promotes neovascularization and improves blood perfusion in an ischemia model (Lan et al. 2005). Taken together, like in tumor cells, CD151 supports functional activity of ECs via the association with integrins, particularly laminin-binding integrins, that organization is distorted in CD151-/- mice. Instead, EC proliferation, ERK, p38 MAPK, FAK, src, and Raf activation are unimpaired in CD151-/- ECs (Takeda et al. 2007a, b). Besides the contribution of EC CD151 in angiogenesis, expression of tetraspanins in tumor cells and tumor-derived exosomes can be most important in angiogenesis. Rats receiving a Tspan8 overexpressing tumor line develop disseminated intravascular coagulation, which could be prevented by a Tspan8-specific antibody (Claas et al. 1998). A prothrombotic state that can culminate in disseminated intravascular coagulation is frequent in cancer patients. Though multifactorial, tumor-initiated angiogenesis and the leakiness of tumor vessels are considered to be important (De Cicco 2004). In fact, Tspan8 is a strong angiogenesis inducer that contributes to a systemic angiogenic switch by delivery of Tspan8 in tumor cell-derived exosomes (Gesierich et al. 2006). Based on these observations, we explored how Tspan8-containing, tumorderived exosomes interact with EC. Tspan8 contributes to a selective recruitment of proteins and mRNA into exosomes. Furthermore, possibly among others, Tspan8-associated CD49d is essential in exosome–EC binding. Transient recovery of selective Tspan8-exosomes enriched mRNA in EC revealed that Tspan8–CD49d complex-containing exosomes are internalized by EC. Exosome uptake induced VEGF-independent regulation of several angiogenesis-related genes, which was accompanied by enhanced proliferation, migration, and sprouting of EC.


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Importantly, Tspan8 exosomes also bind to EC progenitors and suffice for EC progenitor maturation (Nazarenko et al. 2010). Both CD151 and Tspan32 (TSSC6) also are important for normal platelet functions. Though CD151-knockout mice also revealed only mild deficiencies (Karamatic Crew et al. 2004; Wright et al. 2004a, b; Sachs et al. 2006), CD151and Tspan32-knockout mice show defective platelet aggregation, impaired spreading on fibrinogen, and delayed clot retraction (Lau et al. 2004; Goschnick et al. 2006). Both tetraspanins associate with aIIbb3 and modulate outside-in signaling. Tspan32 additionally is required for stable platelet thrombus formation. CD63 may regulate platelet spreading by its association with aIIbb3 (Israels and McMillan-Ward 2005). Platelet-derived exosomes constitute about 70–90% of circulating exosomes in the plasma (Berckmans et al. 2001) with a life span of about 30 min (Flaumenhaft 2006). The procoagulant activity of platelet-derived exosomes is well known. It is suggested that exosomes provide negatively charged phospholipids, which are required for factor IXa and Xa activation (Kessels et al. 1994; Shet et al. 2003). Though still controversial, the therapeutic efficacy of antiglycoprotein IIb/IIIa is suggested to be a consequence of altered platelet exosome formation (Craft and Marsh 2003; Morel et al. 2004; Razmara et al. 2007). Taking into account the abundance of platelet-derived exosomes and their functional activity in coagulation, it will be important to analyze the contribution of exosomal CD151, Tspan32 and CD63. Taken together, by their localization in TEM supported by an internalization motif of some tetraspanins, tetraspanins are prone for internalization. Independent of the donor cell, tetraspanins are enriched in exosomes and the tetraspanin web is mostly maintained (Abache et al. 2007). It remains to be clarified, why some endosomes are delivered to the degradation machinery of the cell, whereas others are preferentially released as exosomes. It is also unanswered whether tetraspanins are involved in this vesicle sorting process (Gibbings et al. 2009; Simons and Raposo 2009). Instead, our data provide convincing evidence that tetraspanins are important in target cell selection, where in the two systems, which we analyzed, tetraspanin-associated integrins account for stable binding. Additional tetraspanin partners have not been excluded. It is also likely that other tetraspanin partners might account for tetraspanin uptake by different cells. Tetraspanins are involved in membrane fusion events (Hemler 2005; Levy and Shoham 2005a, b), which could facilitate the exosome uptake by target cells. However, further elaboration of the fusogenic activity of tetraspanins is required. So far, there is no evidence that the fate of the target cell and its modulation by the transferred proteins and/or the exosomal mRNA and/or microRNA is influenced by the tetraspanins contained in the exosome. Yet, it remains to be explored, whether tetraspanins contribute not only to the protein composition of exosomes, but also to the incorporation of mRNA and miRNA. In view of the exciting power of exosomes and our preliminary evidence for the engagement of tetraspanins in target cell selection, it is demanding to further elaborate the function of tetraspanins in exososmes as a possible tool to interfere with pathological angiogenesis and metastasis formation, two serious handicaps in cancer therapy, that rely on long-distance communication.

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Tetraspanins and Cancer Therapy Taking into account the importance of some tetraspanins in tumor progression and angiogenesis, it is demanding to consider these molecules as therapeutic targets. Due to their mode of activity as molecular facilitators this will not be an easy task. Indeed, the author is not aware of any standard therapy based on selectively attacking tetraspanins. As some tetraspanins function as metastasis suppressors, while others promote metastasis, the author will discuss these two aspects of tetraspanin-based therapies.

Rescuing the Metastasis Suppressor Gene CD82 Rescuing the metastasis suppressor gene CD82 offers itself for therapeutic interference, which requires an awareness of the regulation of CD82 gene transcription as well as of the mechanisms that downregulate CD82 expression in tumor cells (Tonoli and Barrett 2005; Liu and Zhang 2006). Regulation of CD82 transcription and silencing are complex processes (Gao et al. 2003; Tonoli and Barrett 2005; Liu and Zhang 2006). There is no evidence for gene mutation or loss of heterozygosity (Tagawa et al. 1999; Liu et al. 2000) and hypermethylation of CpG islands in the CD82 gene has only been seen in patients with multiple myeloma, where combined demethylation and deacetylation induced increased expression of CD82 mRNA (Jackson et al. 2000; Drucker et al. 2006). Histone deacetylase has also been described to target CD82 in glioma (Gensert et al. 2007). CD82 downregulation has also been related to the p53 status. Binding motifs for the transcription factor AP2 in the CD82 promoter function synergistically with p53 and junB such that the absence of wild-type p53 and/or loss of junB and AP2 protein expression correlate with CD82 mRNA downregulation (Marreiros et al. 2003, 2005). There have been some controversial results on the involvement of NFkB in CD82 transcription, which likely can be explained by the nature of the recruited cofactors. In nonmetastatic cells, IL-1b supports the recruitment of a Tip60 (HIV-1 TAT-interactive protein 60)/Fe65-Pontin complex, which acts as a coactivator together with NFkB p50 and accounts for the dismissal of the corepressor N-Cor/TAB2 (TAK1-binding AP)/HDAC3 (histone deacetylase 3) complex from NFkB p50, which would turn off CD82 transcription. In metastasizing tumor cells, Tip60 is downregulated and a b-catenin–reptin complex replaces the Tip60– Pontin complex and represses NFkB activity (Kim et al. 2005; Telese et al. 2005). An additional mode of CD82 downregulation could rely on alternative splicing. Expression of a splice variant lacking exon 7, frequently seen in metastatic tissue, confers increased metastatic potential (Lee et al. 2003). The potential therapeutic efficacy of CD82 was already demonstrated. CD82-transfected Lewis lung carcinoma cells loose the capacity to form lymph node metastasis (Takeda et al. 2007a, b). Furthermore, nerve growth factor has been shown to rescue CD82 expression and to abrogate tumorigenicity (Sigala et al. 1999). Besides reviving CD82 expression at the transcriptional level, CD82 expression may also become rescued by proteasome inhibitors or by targeting specific


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components of the ubiquitin system, as ubiquitin ligase gp78 regulates CD82 expression (Tsai et al. 2007).

Interfering with Metastasis-Promoting Activities of Tetraspanins Therapeutic approaches concerned about interference with metastasis-promoting activities of tetraspanins are mostly based on antibodies, recombinant soluble ECL2, or posttranscriptional gene silencing via siRNA (Hemler 2008). Tetraspanin-specific antibodies, though not yet commercially available, have been shown in several instances to be of potential clinical relevance. Intratumoral application of anti-CD9 inhibited colon carcinoma growth (Ovalle et al. 2007), anti-CD37 improved the survival of B-CLL xenografted mice (Levy et al. 1998), and anti-CD151 interfered with metastasis formation (Testa et al. 1999; Kohno et al. 2002; Zijlstra et al. 2008). Though the underlying mechanisms have not been fully elucidated, it has been suggested that antibodies may interfere with the lateral associations of tetraspanins or promote clustering of tetraspanins and tetraspanin-associated molecules in TEM and thereby interfere with the activity not only of the targeted tetraspanin, but also of associated molecules including cytoplasmic partners. In line with this suggestion, tetraspanin antibodies have in some instances been shown to exert stronger effects than the knockout of an individual tetraspanin, e.g., anti-CD81 (Oren et al. 1990; Boismenu et al. 1996; Miyazaki et al. 1997; Tsitsikov et al. 1997; Levy et al. 1998). Taking this into account, one has to be aware that particularly the activity of tetraspanin-specific antibodies may vary depending on the recognized epitope (Serru et al. 1999; Yauch et al. 2000; Geary et al. 2001), which may enhance or block the effect of a tetraspanin as demonstrated for anti-CD151-promoting adhesion (Zijlstra et al. 2008) and for antiCD9 that can amplify the tumor suppressor function (Ovalle et al. 2007). Besides there blocking or enhancing activity, tetraspanin-specific antibodies, like therapeutic antibodies recognizing other molecules, have been described to induce apoptosis (Murayama et al. 2004) and to support complement and antibodydependent cellular cytotoxicity (Zhao et al. 2007). Finally, antibodies can be used as drug transporter as reported for 131I-labeled anti-CD37 (Press et al. 1989) or for transporting nanoparticles with siRNA (Peer et al. 2008). The latter has not yet been explored for tetraspanins. However, several approaches have been undertaken to silence tetraspanins via siRNA. CD9 silencing resulted in pronounced ovarian cancer dissemination (Furuya et al. 2005) and CD151 silencing interfered with integrin-dependent adhesion and migration (Winterwood et al. 2006). Though not metastasis-related, lentiviral CD81 shRNA delivery into the nucleus accumbens or the ventral tegmental resulted in a significant decrease in locomotor activity (Bahi et al. 2005). So far, the soluble form of the large extracellular domain (ECL2) of tetraspanins as a competitor has mainly been tested with respect to leukocyte–EC interaction via CD9 and CD151 (Barreiro et al. 2005), egg–sperm fusion (Zhu et al. 2002) and infectivity, where the ECL2 by exerting direct and indirect effects may be superior to antibodies (Molina et al. 2008).

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Other therapeutic setting based on tetraspanins is currently being discussed. These include modulation of AA important for TM folding (Tarasova et al. 1999), where perturbation of TM interactions of CD82 significantly affect the molecules intrinsic activities (Bari et al. 2009). The authors argue that a therapeutic perturbation of TM interactions may open a new avenue to prevent cancer invasion, which could be far easier approached than a blockade of individual signaling pathways. Modulation of the PDZ domain (Dev 2004; Latysheva et al. 2006), of key interaction sites in the ECL2 (Yauch et al. 2000; Seigneuret 2006), of palmitoylation sites (Berditchevski et al. 2002; Charrin et al. 2002; Yang et al. 2002, 2004; Kovalenko et al. 2005) including targeting of the responsible acyltransferase (Sharma et al. 2008) are additional therapeutic approaches currently being discussed. Last, not least, taking into account the increasingly appreciated role of exosomes as intercellular communicators and the strong presence of tetraspanins in exosome membranes, it is tempting to speculate that tetraspanins could be used as delivery system. This requires further elaboration of the engagement of tetraspanins and the associated molecule that together selectively bind to and become internalized by selective targets (Nazarenko et al. 2010). Knowledge on the exosomal binding to and uptake of tetraspanin complexes by selective target cells could well allow to generate competitive exosomes carrying desired siRNA or other drugs that interfere with exosome-initiated premetastatic niche preparation, angiogenesis, and thrombosis.

Conclusion Tetraspanins function as molecular facilitators that assemble a web including many distinct families of TM proteins in specialized membrane microdomains that serve as a scaffold for signal transducing and cytoskeletal proteins and are prone for internalization. The reversibility of TEM and their composition, which depends on the cell’s activation state, the abundance of associating molecules and their ligands adds a major constraint in defining tetraspanin functions. Nonetheless, modulation of cell motility, cell fusion and, as defined more recently, intercellular communication via exosomes may well cover the essential activities of tetraspanins. Taking a simplified view (exemplified in Fig. 26.3), the involvement of tetraspanins in these

Fig. 26.3 Modes of tetraspanin activity. (a) Tetraspanins may act as a receptor for defined ligands: CD82 binds DARC on endothelial cells (ECs). Thereby TBX2 becomes downregulated, ARF and p21 are released from repression and force tumor cell senescence. (b) Tetraspanins can directly influence adhesion, signal transduction, or gene transcription via associated molecules: CD151 can associate with proMMP7. This facilitates matrix degradation and strengthens invasiveness. (c) Tetraspanins only indirectly initiate activities via the recruitment of different molecules into TEM, a process frequently involving gangliosides. High level of a GM3–CD9 complex is accompanied by strong a3b1 ECM adhesion. High-level GM3 expression also initiates recruitment of Csk into TEM, where it inhibits src activation with the consequence of reduced rac and PI3K/Akt activation, which results in pronounced adhesion and increased apoptosis susceptibility. (d) Tetraspanins initiate internalization and relocation in distinct


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Fig. 26.3 (continued) membrane regions of associated molecules: CD151 recruits the major laminin-binding integrins a3b1 and a6b4; via the CD151 Yxxf motif, the CD151–integrin complex becomes internalized. The integrin redistribution with re-expression in the leading lamella is associated with reduced adhesion and increased motility. (e) Tetraspanins initiate recruitment into MVB and release of TEMs in exosomes: Transfection of a nonmetastasizing tumor line with Tspan8 is accompanied by systemic angiogenesis and thrombosis as well as by upregulation of a4b1 and MMP9. Both a4b1 and MMP9 are enriched in exosomes and exosomes suffice for angiogenesis induction, where exosomes bind via Tspan8-associated a4b1 to endothelial VCAM as the initial step of exosome uptake and modulation of gene transcription in ECs. Thus, exosomal tetraspanins and tetraspanin-associated molecules are of major importance in target cell selection and target cell modulation

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actions can follow five routes: (a) tetraspanins may act as a receptor for defined ligands, this is rather exceptionally the case; (b) tetraspanins directly influence adhesion, signal transduction, or gene transcription via associated molecules; (c) tetraspanins only indirectly initiate activities via the recruitment of different molecules into TEM, a process that frequently involves gangliosides; (d) tetraspanins initiate internalization and relocation in distinct membrane regions of associated molecules; and (e) tetraspanins initiate recruitment into MVB and release of TEMs in exosomes. Exosomal tetraspanin-associated molecules may be of major importance in target cell selection and exosomal tetraspanins in exosome fusion with the target cell. All these activities have been demonstrated to contribute to tetraspaninmediated metastasis inhibition and promotion, to premetastatic niche formation, to angiogenesis and the tumor-associated prothrombotic state. Nonetheless, one of the key questions, why some tetraspanins rather consistently suppress (CD82, CD81, CD9, CD63) or promote (CD151, Tspan8) tumor progression remains unanswered. Neither the structure of the tetraspanins (CD82 and Tspan8 belong to the same subgroup) (Seigneuret et al. 2001) nor the assembly of the associating TM and signal transduction molecules in TEM differs fundamentally between CD9, CD63, CD81, CD82, CD151, and Tspan8 (Hemler 2005; Levy and Shoham 2005a, b). A well-positioned sorting motif is present in CD82 and CD151 (Berditchevski and Odintsova 2007) and, thus, cannot account for the distinct activity. Taking into account that the most consistent distinction between metastasis-suppressing and -promoting tetraspanins relates to the strong adhesion initiated by CD82 or CD9 versus CD151- and Tspan8-initiated transient internalization and motility, it is tempting to speculate that distinct vesicular journeys of internalized tetraspanins may be decisive for metastasis inhibition versus promotion. This hypothesis urgently requires to become answered, which may provide a solid ground for therapeutic interference with tetraspanin activities in tumor progression. Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (grant ZO 40/12-1), the Deutsche Krebshilfe, and the Tumorzentrum Heidelberg/Mannheim.

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Yang XH, Richardson AL, Torres-Arzayus MI et al (2008) CD151 accelerates breast cancer by regulating alpha 6 integrin function, signaling, and molecular organization. Cancer Res 68: 3204–3213 Yates AJ, Rampersaud A (1998) Sphingolipids as receptor modulators. An overview. Ann N Y Acad Sci 845:57–71 Yauch RL, Hemler ME (2000) Specific interactions among transmembrane 4 superfamily (TM4SF) proteins and phosphoinositide 4-kinase. Biochem J 351:629–637 Yauch RL, Berditchevski F, Harler MB et al (1998) Highly stoichiometric, stable, and specific association of integrin alpha3beta1 with CD151 provides a major link to phosphatidylinositol 4-kinase, and may regulate cell migration. Mol Biol Cell 9:2751–2765 Yauch RL, Kazarov AR, Desai B et al (2000) Direct extracellular contact between integrin alpha(3)beta(1) and TM4SF protein CD151. J Biol Chem 275:9230–9238 Yoshida T, Kawano Y, Sato K et al (2008) A CD63 mutant inhibits T-cell tropic human immunodeficiency virus type 1 entry by disrupting CXCR4 trafficking to the plasma membrane. Traffic 9:540–558 You Z, Saims D, Chen S et al (2002) Wnt signaling promotes oncogenic transformation by inhibiting c-Myc-induced apoptosis. J Cell Biol 157:429–440 Yunta M, Lazo PA (2003) Tetraspanin proteins as organisers of membrane microdomains and signalling complexes. Cell Signal 15:559–564 Zakharova L, Svetlova M, Fomina AF (2007) T cell exosomes induce cholesterol accumulation in human monocytes via phosphatidylserine receptor. J Cell Physiol 212:174–181 Zhang X, Xu W (2008) Aminopeptidase N (APN/CD13) as a target for anti-cancer agent design. Curr Med Chem 15:2850–2865 Zhang XA, Bontrager AL, Hemler ME (2001) Transmembrane-4 superfamily proteins associate with activated protein kinase C (PKC) and link PKC to specific beta(1) integrins. J Biol Chem 276:25005–25013 Zhang XA, Kazarov AR, Yang X et al (2002) Function of the tetraspanin CD151-alpha6beta1 integrin complex during cellular morphogenesis. Mol Biol Cell 13:1–11 Zhang XA, He B, Zhou B et al (2003a) Requirement of the p130CAS-Crk coupling for metastasis suppressor KAI1/CD82-mediated inhibition of cell migration. J Biol Chem 278:27319–27328 Zhang XA, Lane WS, Charrin S et al (2003b) EWI2/PGRL associates with the metastasis suppressor KAI1/CD82 and inhibits the migration of prostate cancer cells. Cancer Res 63:2665–2674 Zhao X, Lapalombella R, Joshi T et al (2007) Targeting CD37-positive lymphoid malignancies with a novel engineered small modular immunopharmaceutical. Blood 110:2569–2577 Zheng ZZ, Liu ZX (2007) Activation of the phosphatidylinositol 3-kinase/protein kinase Akt pathway mediates CD151-induced endothelial cell proliferation and cell migration. Int J Biochem Cell Biol 39:340–348 Zhijun X, Shulan Z, Zhuo Z (2007) Expression and significance of the protein and mRNA of metastasis suppressor gene ME491/CD63 and integrin alpha5 in ovarian cancer tissues. Eur J Gynaecol Oncol 28:179–183 Zhou B, Liu L, Reddivari M et al (2004) The palmitoylation of metastasis suppressor KAI1/CD82 is important for its motility- and invasiveness-inhibitory activity. Cancer Res 64:7455–7463 Zhou Z, Ran YL, Hu H et al (2008) TM4SF3 promotes esophageal carcinoma metastasis via upregulating ADAM12m expression. Clin Exp Metastasis 25:537–548 Zhu GZ, Miller BJ, Boucheix C et al (2002) Residues SFQ (173–175) in the large extracellular loop of CD9 are required for gamete fusion. Development 129:1995–2002 Zijlstra A, Lewis J, Degryse B et al (2008) The inhibition of tumor cell intravasation and subsequent metastasis via regulation of in vivo tumor cell motility by the tetraspanin CD151. Cancer Cell 13:221–234 Zöller M (2006) Gastrointestinal tumors: metastasis and tetraspanins. Z Gastroenterol 44:573–586 Zöller M (2009) Tetraspanins: push and pull in suppressing and promoting metastasis. Nat Rev Cancer 9:40–55 Zvieriev V, Wang JC, Chevrette M (2005) Over-expression of CD9 does not affect in vivo tumorigenic or metastatic properties of human prostate cancer cells. Biochem Biophys Res Commun 337:498–504

Part VI

Secreted Proteins

Chapter 27

Chemokines and Metastasis Kalyan C. Nannuru, Seema Singh, and Rakesh K. Singh

Abstract Chemokines are a large group of low-molecular-weight cytokines that are known to direct migration of leukocytes in response to inflammation and pathologic stimuli. Although the primary function of chemokines is well recognized as leukocyte attractants, recent evidences indicate that chemokines and their receptors influence tumor development, growth, angiogenesis, invasion, and metastasis. Chemokines activate cells through cell surface seven trans-membrane, G-protein-coupled receptors (GPCR), resulting in cell invasion, motility, and survival. The role played by chemokines and their receptors in tumor pathophysiology is complex as some chemokines favor tumor growth and metastasis, whereas others may enhance anti-tumor immunity. These diverse functions of chemokines establish them as key mediators between the tumor cells and their microenvironment and play a critical role during tumor progression and metastasis. Manipulating chemokine–chemokine receptor network is an emerging novel targeted therapeutic strategy for various malignancies. In this chapter, we will review recent advances in chemokine research with special emphasis on their role in host–tumor interaction during tumor progression, angiogenesis, and metastasis.

Introduction Despite advances in the use of aggressive adjuvant chemotherapy and radiotherapy, which in combination with surgery, are often successful in the eradication of the primary tumor, most deaths in cancer patients result from metastasis. The process of cancer progression and metastasis comprises a series of sequential interrelated steps, each of which can be rate-limiting (Fidler 2003). The major steps in the formation of a metastasis are as follows: (1) following initial transforming event,

R.K. Singh (*) Department of Pathology and Microbiology, University of Nebraska Medical Center, 985900 Nebraska Medical Center, Omaha, NE 68198-5900, USA e-mail: [email protected] R.G. Bagley (ed.), The Tumor Microenvironment, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-6615-5_27, © Springer Science+Business Media, LLC 2010

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growth of neoplastic cells must be progressive which requires extensive vascularization if a tumor mass is to exceed 2-mm in diameter; (2) local invasion of the host stroma by some tumor cells occurs as thin-walled venules, like lymphatic channels, offer very little resistance to penetration by tumor cells and provide the most common pathway for tumor-cell entry into the circulation; (3) detachment and embolization of tumor-cell aggregates occurs with subsequent arrest in the capillary beds of organs; (4) extravasation to secondary organ; and (5) proliferation and neovascularization within the distant organ parenchyma to produce detectable metastatic lesions. Intrinsic properties of the tumor cells, as well as their surrounding microenvironment, are crucial in defining the progression of cancer and the fate of metastases. Several factors have been identified that facilitate the interplay between tumor cells and their microenvironment. Proliferation, neovascularization, invasion, and migration of malignant cells to distinct organs are crucial steps for tumor progression and metastasis that can be regulated by chemokines (Fig. 27.1). Over the last 25 years, it has been increasingly recognized that chemokines play an important part in regulation of the metastatic cascade, as they are known to be expressed by tumor cells, as well as by host cells in their proximity and at metastatic Proliferation/Vascularization Migration

Inflammation

Circulation Metastasis Chemokine receptor

Chemokines

Intravasation

Extravasation Chemokines

Primary Tumor Chemokine Receptors

Leukocytes

Chemokines

Angiogenesis

Invasion and motility

Cell growth/survival Immunosuppression

Tumor Progression and Metastasis Fig. 27.1 The multifaceted role of chemokines in tumor growth, invasion, and metastasis. Chemokines produced by tumor cells attract infiltrating leukocyte and/or promote proliferation and can also affect the microenvironment by promoting vascularization. Chemokines can stimulate their specific receptors that alter the adhesive capacity of tumor cells and their migration/invasion into circulation, and extravasation toward distant organs

27 Chemokines and Metastasis

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sites (Singh et al. 2007; Balkwill 2004). The interactions between chemokine receptors and their ligands coordinate cellular trafficking and responses within different microenvironments. Recent evidences demonstrate the multifunctional role of chemokines and their receptors in regulating cellular growth, survival, angiogenesis, motility and metastasis, and controlling leukocyte infiltration to affect anti-tumor immunity (Ruffini et al. 2007; Balkwill 2004; Vandercappellen et al. 2008; Singh et al. 2007; Ransohoff 2009). A clear understanding of emerging roles of chemokines and the mechanisms of their actions in the processes of malignant progression and metastasis will open new doors for therapeutic interventions. This chapter highlights the functional role of chemokines and their receptors, with focus on angiogenesis, tumor-stromal interaction, and distant metastasis.

Chemokines and Their Receptors The chemokine superfamily includes a large number of low-molecular-weight chemotactic proteins that regulate the trafficking of leukocytes to inflammatory sites (Locati et al. 2002; Murphy et al. 2000; Zlotnik and Yoshie 2000). Chemokines are generally 8–15kDa in size and contain four conserved cysteine amino acid residues linked by disulfide bonds. Structurally, chemokines are classified into four (CXC, CC, C, and CX3C) subfamilies (Murphy et al. 2000; Zlotnik and Yoshie 2000). There are more than 50 chemokines, the majority of which belong to the major CC and CXC chemokine subfamilies (Tables 27.1 and 27.2). According to a new classification, chemokine ligands/receptors are named ‘L’ or ‘R’, respectively (Zlotnik and Yoshie 2000). Receptors are also grouped into four subfamilies, as each receptor binds to one of the four chemokine subfamilies (Tables 27.1 and 27.2). To avoid confusion, we have used the new designations and have listed their old and new names along with their respective receptors in Tables 27.1 and 27.2. Members of the CXC (or a-chemokine) subfamily contain one non-conserved amino acid (X) between the first and second cysteine residues. On the basis of the presence or absence of a Glu-Leu-Arg (ELR) motif, the CXC chemokines can be further subdivided into two groups (ELR+and ELR¯) (Murphy et al. 2000; Murphy 2002; Zlotnik and Yoshie 2000). The ELR motif is located at the N-terminus immediately before the first cysteine amino acid residue (Baggiolini et al. 1997). Extensive investigations regarding the functions of the CXC subfamily have revealed that the presence/absence of the ELR motif determines whether the chemokine is angiogenic or angiostatic (Strieter et al. 1995b, 2006). The ELR+chemokines are primarily chemotactic for endothelial cells and neutrophils. These chemokines are potent promoters of angiogenesis, as the recruited neutrophils are known to synthesize and store angiogenic molecules like vascular endothelial growth factors (VEGF)-A (Scapini et al. 2004; Belperio et al. 2000; Luster 1998; Strieter et al. 1995b, 2006). On the other hand, the main targets for ELR¯ members are T cells and B cells and are potent inhibitors of angiogenesis (Strieter et al. 1995a). The angiogenic CXC chemokine family members include CXCL1-3 and CXCL5-8

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Table 27.1 Human CXC, C, and CX3 C chemokines in cancer Chemokines Alternative name Known receptor(s) CXC (a-chemokines) CXCL1 GRO-a /SCYB-1/MGSA/ CXCR1, CXCR2 GRO-1/NAP-3

CXCL2

CXCR2

CXCL9

GRO-b/SCYB-2/GRO-2/ MIp-2a GRO-g/SCYB-3/GRO-3/ MIp-2b PF-4/SCYB-4 ENA-78/SCYB-5 GCP-2/SCYB-6 NAP-2/(SCYB-7/PBP/ CTAP-III/b-TG SCYB-8/GCP-1/NAP-1/ MDNCF MIG/SCYB-9

CXCL10

IP-10/SCYB-10

CXCL11

I-TAC/SCYB-11/b-R1/ H174/IP-9 SDF-1/SCYB-12/PBSF BCA-1/SCYB-13

CXCR3, KSHVGPCR CXCR3

CXCL3 CXCL4 CXCL5 CXCL6 CXCL7 CXCL8

CXCL12 CXCL13

CXCL14 CXCL16

BRAK/SCYB-14/ Bolekine Small inducible cytokine B6

C (g-chemokines) XCL1 Lymphotactin/ SCYC1/SCM-1a/ Lymphotactin a XCL2 SCM-1b/SCYC2/ Lymphotactin b CX3C (DELTA chemokines) Fractalkine/SCYD1 CX3CL1

CXCR2 Unknown CXCR2 CXCR1, CXCR2 CXCR1, CXCR2 CXCR1, CXCR2 CXCR3

CXCR4 CXCR5

Unknown CXCR6

Tumor type Melanoma, breast cancer, myeloma, colon cancer, ovarian cancer Melanoma, breast cancer Melanoma, colon cancer Lung cancer Lung cancer Lung cancer Myelodysplastic syndrome Various malignancies Breast cancer, melanoma Colon cancer, breast cancer Colon cancer, breast cancer Various malignancies Prostate cancer, melanoma, breast cancer Breast cancer, oral carcinoma Prostate cancer, pancreatic cancer

XCR1

Anti-tumor activity

XCR1

Anti-tumor activity

CX3CR1

Various malignancies

Cancer cells express different chemokine and chemokine receptors and modulate tumor growth angiogenesis and metastasis

(Strieter et al. 1995b), whereas angiostatic CXC chemokine members include CXCL4 (Maione et al. 1990) and CXCL9-11 (Maione et al. 1990; Nagpal et al. 2004; Strieter et al. 1995a, 2006). Although CXCL12, an ELR¯chemokine, was originally described as a pre-B-cell growth-stimulating factor (Nagasawa et al. 1996), it has been shown to exhibit angiogenic activity (Mirshahi et al. 2000; Orimo et al. 2005; Salcedo et al. 1999; Tachibana et al. 1998). Furthermore,


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Table 27.2 Human CC chemokines in cancer Chemokines Alternative name Known receptor(s) CC (b-chemokines) CCL1 I-309 /SCYA1 CCR8 CCL2 MCP-1/SCYA2/ CCR2, CCR5,CCR10 MCAF/HC11

Tumor types

MIP-1a/SCYA 3/ LD78a/SIS- a MIP-1b/SCYA4/ACT2/G-26/HC21/ LAG-1/SIS-g RANTES/SCYA5/ SIS-d

CCR1,CCR5

Colon cancer Prostate cancer, melanoma, colon cancer, breast cancer Lung cancer

CCR5, CCR10

Lung cancer

CCR1, CCR3, CCR5, CCR10

CCL7

MCP-3/SCYA7

CCL8

MCP-2/SCYA8/HC14

CCL11

Eotaxin/SCYA11

CCR1, CCR2, CCR3 CCR5 CCR2, CCR3, CCR5 CCR1 CCR3

Prostate cancer, melanoma, colon cancer, breast cancer, renal cell carcinoma Anti-tumor activity

CCL13

MCP-4/SCYA13/Ck b10/NCC-1 HCC-1/SCYA14/Ck b1/MCIF/NCC-2/ CC-1 MIP-1 d/SCYA15/ Lkn-1/HCC-2/ MIP-5/NCC-3/CC-2 HCC-4/SCYA16/Ck b12/LEC/LCC-1/ NCC-4/ILINCK/LMC/ Mtn-1 TARC/SCYA17

CCR1, CCR2, CCR3, CCR5 CCR1

PARC/SCYA18/Ckb7/ DC-CK1/ AMAC-1/MIP-4/ DCtactin MIP-3b/SCYA19/ Ckb11/ELC/ Exodus-3 MIP-3a/SCYA20/ LARC/Exodus-1 6Ckine/SCYA21/ Ckb9/SLC/ Exodus-2 MDC/SCYA22

Unknown

CCL3 CCL4

CCL5

CCL14

CCL15

CCL16

CCL17 CCL18

CCL19

CCL20 CCL21

CCL22

Anti-tumor activity Renal cell carcinoma, Colon cancer Unknown Ovarian cancer

CCR1, CCR3

Melanoma

CCR1

Anti-tumor activity

CCR4

Non-Hodgkin’s lymphoma Melanoma

CCR7

Breast cancer, colon cancer

CCR6

Colon cancer

CCR7

Breast cancer, colon cancer, prostate cancer Breast cancer

CCR4

(continued)

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Table 27.2 (continued) Chemokines Alternative name

Known receptor(s)

Tumor types

CCL23

MPIF/SCYA23/Ckb8/ Ckb8-1/MIP-3/ MPIP-1 Eotaxin-2/SCYA24/ Ckb6/MPIF-2 TECK/SCYA25/ Ckb15

CCR1

Unknown

CCR3

Colon cancer

CCR9

Eotaxin-3/SCYA26/ MIP-4a/TSC-1/ IMA CTACK/SCYA27/ ESkine/Skinkine CCL28/SCYA28/MEC

CCR3

Prostate cancer, melanoma, colon cancer, breast cancer Unknown

CCR3, CCR2, CCR10

Melanoma

CCR10, CCR3

Unknown

CCL24 CCL25

CCL26

CCL27 CCL28

Cancer cells express different chemokine and chemokine receptors and modulate tumor growth angiogenesis and metastasis

ELR¯CXC chemokines have also been shown to inhibit neovascularization induced by classical angiogenic factors, such as basic fibroblast growth factor (FGF-2) and vascular endothelial cell growth factor (VEGF) (Strieter et al. 1995a). The CC chemokines (or b-chemokines) represent the largest family of chemokines and have adjacent cysteine residues. The known members of this family are CCL1-5, CCL7-8, CCL11, and CCL13-28, which are listed in Table 27.2. Members of this family exhibit the most diverse range of target cell specificities. They generally attract leukocytes, including monocytes, macrophages, T cells, B cells, basophils, eosinophils, dendritic cells, mast cells, and natural killer cells (Baggiolini et al. 1994; Bischoff et al. 1993; Dahinden et al. 1994; Garcia-Zepeda et al. 1996; Imai et al. 1996; Jose et al. 1994; Kameyoshi et al. 1992; Ponath et al. 1996; Rot et al. 1992). So far, neutrophils have not been shown to respond to chemotactic stimuli from any of the CC chemokines. Chemokines of the C subfamily (g-chemokines) have only one of the cysteine residues, and XCL1 (lymphotactin-a) and XCL2 (lymphotactin-b; Kelner et al. 1994) are the two members of this subfamily, with a molecular size of 16kDa. These chemokines seem to be lymphocyte specific (Kelner et al. 1994). The CX3C chemokine (d-chemokines) has three non-conserved amino acids between the first two cysteines (Bazan et al. 1997). This family also has only one known member, CX3CL1 (Fractalkine), which has been shown to induce both the migration and the adhesion of leukocytes (Segerer et al. 2002; Umehara and Imai 2001). In general, chemokines are secretory proteins, and CX3CL1 is the only exception being a membrane-bound chemokine (Pan et al. 1997). All chemokines exert their biological function by binding to G-protein-coupled receptors (GPCR; Murphy et al. 2000; Murphy 2002; Thelen 2001). Chemokine receptors have an N-terminus outside the cell, three extracellular and three intracellular


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loops, and a C-terminus containing serine and threonine phosphorylation sites in the cytoplasm. In addition, they also possess seven hydrophobic trans-membrane domains. Currently, 6 CXC receptors, 11 CC receptors, and 1 receptor each for C and CX3C have been identified (Tables 27.1and 27.2). Of the six CXC receptors, CXCR1 binds both CXCL8 and CXCL6 with high affinity (Lee et al. 1992; Wuyts et al. 1998). CXCR2, another receptor for CXCL8, has 78% identity with CXCR1 at the amino acid level. It has also been reported that CXCR2 can bind to all of the ELR+CXC chemokines with high affinity (Baggiolini and Loetscher 2000; Locati and Murphy 1999; Wuyts et al. 1998). The third receptor, CXCR3, has been shown to bind the ELRˉ CXC chemokines (CXCL9-11; Cole et al. 1998; Farber 1997; Loetscher et al. 1998a). CXCR4 has been shown to be a cofactor for human immunodeficiency virus (HIV) infection of T lymphocytes (Feng et al. 1996). For CXCR4, the only known ligand is CXCL12, and this ELR¯ CXC chemokine can inhibit HIV infection by competing with lymphotropic HIV virus for binding of CXCR4 (Oberlin et al. 1996). CXCR5, identified as a receptor on B lymphocytes, is responsible for B cell chemotaxis mediated by CXCL13 (Legler et al. 1998). CXCR6 is a receptor for CXCL16, and was described previously as a fusion cofactor for HIV-1 and simian immunodeficiency virus (SIV) (Deng et al. 1997; Matloubian et al. 2000). Among the CC chemokine receptors, CCR1 is a receptor for CCL3, CCL5, CCL7-8, CCL13-16, and CCL23 (Berkhout et al.2000; Gong et al.1996; Hwang et al.2005; Neote et al.1993; Tsou et al.1998).CCR2 exists as two splice variants, CCR2a and CCR2b, among them CCR2b is the most studied and appears to bind at least CCL2, CCL7-8, CCL13, and CCL27 (Charo et al. 1994; Moore et al. 1997; Stellato et al. 1997). CCR3 binds to CCL5, along with CCL7-8, CCL11, CCL13, NNY-CCL14 (CCL14 analogue), CCL15, CCL24, and CCL26-28 (Forssmann et al. 1997, 2004; Ponath et al. 1996; Uguccioni et al. 1997; Youn et al. 1997). CCR4 is a receptor for CCL17 and CCL22 (Imai et al. 1997, 1998), whereas CCR5 is a major co-receptor for macrophage (M)-tropic HIV-1, HIV-2, and SIV strains (Littman 1998). CCR5 can bind to other CC chemokines (CCL2, CCL7-8, and CCL13) with decreased affinity (Blanpain et al. 1999). CCR6 has been shown to be a receptor for CCL20 (Baba et al. 1997). CCL19 and CCL21 bind to CCR7 (Yoshida et al. 1997a), while CCR8 appears to bind CCL1 specifically (Tiffany et al. 1997). CCR9 is a receptor for CCL25 (Zaballos et al. 1999). Functional studies conducted on CCR10 indicate that it has high binding affinity for CCL2, CCL4, CCL27, and CCL28 (Bonini and Steiner 1997; Wang et al. 2000). So far, one receptor each for C (XCR1) and CX3C (CX3CR1) chemokines have been identified (Table 27.1). One of the remarkable features of the chemokine receptor superfamily is their promiscuity in ligand binding (Mantovani 1999). This suggests that the regulation of chemokine activities and the response in target cells are complex events. The intricate functional and regulatory natures of chemokine activities give rise to diverse responses in normal homeostasis and pathological conditions, including tumor growth, invasion, angiogenesis, and metastasis.

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Chemokines on Leukocyte Recruitment and Activation in Malignant Tumors Chemokines and their receptor expression, which is dependent on intrinsic properties of the individual cells and the microenvironment, regulate their recruitment and activation within specific microenvironment. The pattern of chemokine receptors’ and their ligands’ expression in a tissue generally correlates with the frequency and type of leukocyte infiltrates. The chemokine gradient that regulates the recruitment can be created by different cell population in response to pathological and inflammatory stimuli. The leukocyte infiltration within tumor microenvironment is regulated by a complex network of chemokines, which influences frequency and phenotype of the immune cells, critical for modulating tumor growth, progression, and metastasis. Most malignant tumors contain tumor cells and stromal cells (macrophages, T and B lymphocytes, eosinophils, granulocytes, natural killer cells, endothelial cells, and fibroblasts; Brigati et al. 2002; Coussens and Werb 2002; Balkwill 2004; Pollard 2009). The number and type of leukocyte infiltration depend on the type of chemokine present in the microenvironment and the specific receptors expressed on the infiltrating cells. The presence of CC chemokines is an important determinant for macrophage and lymphocytic infiltration in a variety of human malignancies (Balkwill and Mantovani 2001; Zlotnik 2006). The extent of macrophage and lymphocyte infiltration into tumors of the same histological origin can vary widely. However, the percentage of tumor-associated macrophages (TAMs) and lymphocytes (TILs) for each tumor is usually maintained as a relatively stable property of a particular tumor during progression (Talmadge et al. 1981; Whiteside et al. 1986). The functional role that these macrophages play in tumor growth is controversial. These cells are located predominantly at the tumor and host cellular interface and represent a potential target for therapy based on immune manipulation. Infiltrating lymphocytes are multifunctional and capable of producing cytokines, enzymes, and different growth inhibitory/stimulatory factors to regulate the initiation, maintenance, and termination of tumor (Perussia 1992; Mantovani et al. 2008). The mechanism of recruitment and the significance of macrophages and lymphocytes in the growth and metastasis of breast cancer have not been studied. In addition, a better understanding of “cross-talk” between these cells and the malignant cell population is essential before the potential of macrophages and lymphocytes as antitumor effector cells can be realized and successful therapeutic strategies implemented. Accumulation of macrophages and lymphocytes at tumor sites has been shown to be mediated by tumor-derived CC-chemokines (Balkwill and Mantovani 2001; Zlotnik 2006). By virtue of receptors on the target cells, CC-chemokines have the potential to preferentially recruit macrophages and T lymphocytes, NK cells, and dendritic cells into the tumors (Balkwill 2004; Ruffini et al. 2007). However, the relationship between CC-chemokines and macrophage- and lymphocyte-mediated immune response in different malignancies remains poorly understood.


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CCL2 was initially considered a monocyte-specific chemoattractant, until in vitro studies showed that CCL2 was the prime agent in phytohemagglutinin (PHA)-stimulated leukocyte culture responsible for T cell chemotaxis (Carr et al. 1994). Importantly, flow cytometric analysis of responding population indicated that only T cells with memory phenotype (CD45RA+, CD45RO+, CD29+, L-selectin+) were involved, because naïve T cells (CD45RA+, CD45RO+CD29 weak, L-selectin+) were unresponsive. In addition, although monocytes began to migrate in response to CCL2 within 1h, a significant T cell response to CCL2 was not evident until 4h. These data were of great significance because although CCL2 had been previously demonstrated within inflammatory sites characterized by monocytes and lymphocytes infiltration, there was no concept of a casual link. This theme of CCL2 involvement in T cell recruitment was elaborated in subsequent studies that confirmed a T cell chemotactic response to CCL2 and extended the data by showing analogous response to CCL2 and CCL7 (Loetscher et al. 1998b, 2000; Loetscher and Clark-Lewis 2001; Youngs et al. 1997). Recent reports demonstrate that differential expression of chemokine receptors and their responsiveness may dictate, to a large extent, the migration and homing of T helper cell type 1 (Th1) and Th2s (Negus et al. 1995, 1998; Milliken et al. 2002; Gu et al. 2000). A two way interaction (negative, positive, or both) occurs between tumor cells and infiltrating leukocytes at certain stage of tumor progression. The role of infiltrating macrophages and lymphocytes in regulating tumor growth can best be viewed as a balance between stimulatory and inhibitory activities. The host response against tumors can be exerted in situ by interaction of different subsets of leukocytes, which results into either positive (by production of immunosuppressive factors, tumor growth, or angiogenic factors) or negative (potent cellular immune response) effects on tumor growth (Locati et al. 2002; Sica et al. 2002). Functional data demonstrate that tumor-infiltrating macrophages and lymphocytes are usually unable to react against the tumors and display a depressed function. There is evidence that macrophage and lymphocyte recruitment and activation is systemically impaired in some cancer patients (Dinapoli et al. 1996; Young and Wright 1992; Varney et al. 2005a, b). If this is a cytokine defect, and not an inherent macrophage and/or lymphocyte defect, manipulation of CC-chemokines may enhance antitumor responses (Huang et al. 1994, 1995). Aberrant levels of chemokines in tumor microenvironment can influence immunosuppressive type 2 macrophage (M2), which release immunosuppressive cytokines interleukin 10 and transforming growth factor (TGF)b (Sica et al. 2000). M2 macrophages also produce higher amounts of CCL2, which can contribute to T-helper 2 (Th2) polarized immunity (Gu et al. 2000). In addition to being immunosuppressive, infiltrating leukocytes contribute to tumor progression by producing matrix metalloproteinases (MMPs), growth, and angiogenic factors (Varney et al. 2005a, b; Lu et al. 2007a, b; Kuroda et al. 2005; Nakashima et al. 1995). Moreover, chronic expression of CCL2 in tumor microenvironment directly or indirectly enhance tumorigenecity and metastasis (Lu et al. 2007a, b; Kuroda et al. 2005; Nakashima et al. 1995; Sica et al. 2002; Varney et al. 2009).

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The relationship between patient prognosis, the extent of lymphoreticular infiltration and chemokine, and their receptor expression remains poorly understood. The mechanism(s) of leukocytic recruitment and activation and the significance of this process in tumor growth and metastasis are intensely investigated. A detailed model is required to determine the interrelations (whether positive, negative, or both) between leukocytic infiltration, chemokine and their receptor expression, prognosis, and therapeutic effectiveness, which may offer strategies for the development of novel adjuvant therapies.

Chemokines in Tumor Angiogenesis Angiogenesis is a biological process of new blood vessel formation from preexisting blood vessels and hence is also referred to as neovascularization. It is fundamental to many physiological as well as pathological processes in living organisms (Folkman and Cotran 1976; Folkman 1985; Folkman and Klagsbrun 1987; Leibovich and Wiseman 1988). The process of angiogenesis is regulated by many angiogenic growth factors, enzymes, lipids, and carbohydrates, including the members of the chemokine superfamily (Strieter et al. 2004, 2006). Specific members of the chemokine superfamily can act as pro-angiogenic molecules and support the formation of new blood vessels, while others can antagonize these activities and therefore are angiostatic (Belperio et al. 2000; Strieter et al. 2004). Among all the chemokines, CXCL8 is extensively studied as a potent mediator of angiogenesis. The pro-angiogenic activity of CXCL8 in vivo was confirmed through the use of the rat mesenteric window assay, the rat and rabbit corneal assay, and a subcutaneous sponge model (Hu et al. 1993; Koch et al. 1992; Norrby 1996; Strieter et al. 1992). Human recombinant CXCL8 was shown to be angiogenic when implanted in the rat cornea and induced proliferation and chemotaxis of human umbilical vein endothelial cells (HUVEC) (Koch et al. 1992). In addition, the angiogenic properties of conditioned media from activated monocytes and macrophages were attenuated by CXCL8 anti-sense oligonucleotides (Koch et al. 1992). Furthermore, it was shown that CXCL8 can act directly on vascular endothelial cells by promoting their survival (Yoshida et al. 1997b). Studies from our lab and other groups suggest that CXCL8 stimulates both endothelial cell proliferation and capillary tube formation in vitro in a dose-dependent manner, and both of these effects can be blocked by monoclonal antibodies to CXCL8 (Li et al. 2003; Shono et al. 1996). In addition, CXCL8 was shown to inhibit apoptosis of endothelial cells (Li et al. 2005). CXCL8 exerts its angiogenic activity by up-regulating MMP-2 and MMP-9 in tumor and endothelial cells (Inoue et al. 2000; Li et al. 2005; Luca et al. 1997). MMP-mediated degradation of the extracellular matrix is required for endothelial cell migration, organization, and hence, angiogenesis (McCawley and Matrisian 2000). It has been demonstrated by our group that CXCL8 directly enhances endothelial cell proliferation, survival, and MMP expression in


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CXCR1- and CXCR2-expressing endothelial cells, thus may be an important player in the process of angiogenesis (Li et al. 2003). Several investigators have suggested the angiogenic effect of CXCL8 is independent of its chemotactic and pro-inflammatory effects, since CXCL8 promotes angiogenesis in the absence of inflammatory cells (Hu et al. 1993; Strieter et al. 1992). In addition, it has been reported that there is a direct correlation between high levels of CXCL8 and tumor angiogenesis, progression, and metastasis in nude xenograft models of human cancer cells (Luca et al. 1997; Xie 2001). In an experimental model of ovarian cancer, the expression of CXCL8 was directly correlated with neovascularization and poor survival (Yoneda et al. 1998). CXCL8 may also play an important role in angiogenesis in prostate and breast cancers as elevated serum levels of CXCL8 in the patients with these cancers correlate with disease stage (Benoy et al. 2004; Veltri et al. 1999; Aalinkeel et al. 2004; Lehrer et al. 2004; Uehara et al. 2005). The ability of CXCL8 to elicit angiogenic activity depends on the expression of its receptor by endothelial cells. CXCL8 and its receptors, CXCR1 and CXCR2, have been observed on endothelial cells and have been shown to play a role in endothelial cell proliferation (Koch et al. 1992; Murdoch et al. 1999; Salcedo et al. 2000b). Recent studies indicate that CXCR1 is highly and CXCR2 is moderately expressed on human microvascular endothelial cells (HMEC), whereas HUVEC show low levels of CXCR1 and CXCR2 expressions (Salcedo et al. 2000b). Neutralizing antibodies to CXCR1 and CXCR2 abrogated CXCL8-induced migration of endothelial cells, indicating that these two receptors are critical for the CXCL8 angiogenic response (Li et al. 2005; Salcedo et al. 2000b). Of these two high affinity receptors for CXCL8, the importance of CXCR2 in mediating chemokine-induced angiogenesis was demonstrated to be fundamental to CXCL8-induced neovascularization (Addison et al. 2000; Strieter et al. 2004). The role of CXCR2 in promoting tumor-associated angiogenesis has been confirmed in other tumor systems (Keane et al. 2004; Mestas et al. 2005). In addition to CXCL8, other members of the chemokine family have been shown to play important roles in angiogenesis. Elevated levels of CXCL5 and CXCL8 correlated with the vascularity of non-small cell lung cancer (NSCLC) (Numasaki et al. 2005; Strieter et al. 2004). In a severe, combined immune-deficient (SCID) mouse model system, depletion of CXCL5 resulted in the attenuation of tumor growth, angiogenesis, and spontaneous metastasis (Arenberg et al. 1998). In renal cell carcinoma, elevated levels of CXCL1, CXCL3, CXCL5, and CXCL8 were found to be expressed in the tumor tissue and detected in the plasma, and CXCR2 was found to be expressed on endothelial cells within the tumor biopsies (Mestas et al. 2005). Thus, multiple studies mentioned above demonstrate the important role of ELR+CXC chemokines in tumor angiogenesis. Despite of being a non-ELR, CXC chemokine, CXCL12 is of major interest in tumor angiogenesis. Several lines of evidence indicate that CXCL12 induces endothelial cell migration, proliferation, and tube formation, and increases VEGF release by endothelial cells (Kanda et al. 2003; Neuhaus et al. 2003; Salcedo and Oppenheim 2003). Blockade of CXCL12/CXCR4 signaling results in decreased

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tumor growth in vivo due to inhibition of angiogenesis in a VEGF-independent manner (Guleng et al. 2005). The source of CXCL12 that drives angiogenesis is likely to be derived from specialized stromal cells and tumor cells (Barbero et al. 2003). In a recent study, it has been suggested that CXCL12 is partly responsible for the ability of breast carcinoma-associated fibroblasts to promote angiogenesis (Orimo et al. 2005). However, the effects of CXCL12 on angiogenesis cannot be generalized to all tumor systems, as inhibition of metastasis in a model of NSCLC by CXCL12 neutralization did not show reduction of tumor angiogenesis (Phillips et al. 2003). These observations suggest that the action of CXCL12 may be tumor specific and/or may act in cooperation with other angiogenic proteins. As mentioned above, the ELRˉ CXC chemokines include angiostatic members that are known to inhibit neovascularization (Belperio et al. 2000; Salcedo and Oppenheim 2003; Strieter et al. 2004). The angiostatic role of these chemokines has been demonstrated in several studies. For example, CXCL10 potently inhibited CXCL8- and FGF-2-induced angiogenesis (Strieter et al. 1995a). Delivery of CXCL9 or CXCL10 into tumors by injection or by genetic manipulation has been shown to suppress tumor angiogenesis (Arenberg et al. 1996b; Sgadari et al. 1996, 1997). In murine cancer models, intratumoral delivery of immunotherapeutic agents correlates with increased expression of CXCL9 and/or CXCL10 (Dorsey et al. 2002; Ruehlmann et al. 2001). Clinical studies of renal carcinoma patients have also indicated that the intratumoral expression of CXCL9 and CXCL10 results in a decrease in the tumor size (Kondo et al. 2004). It has been shown that CXCR3 is expressed on endothelial cells in a cell cycle-dependent manner, and its expression mediates the angiostatic activity of CXCL9-11 (Romagnani et al. 2001). Recently, it has been suggested that overexpression of CXCL10 in human prostate LNCaP cells activates CXCR3 expression and inhibits cell proliferation (Nagpal et al. 2006). These findings provide definitive evidence of CXCR3-mediated angiostatic activity by angiostatic ELRˉ CXC chemokines. The presence of angiogenic and angiostatic regulators in the CXC chemokine family suggests that tumor angiogenesis may also be affected by the relative expression/activities of the different chemokines in the tumor microenvironment. Recent reports demonstrate that CC chemokines can also participate in angiogenic activity in addition to members of CXC chemokine (Salcedo et al. 2000a). CCL2 has been added to the growing list of angiogenic modulators (Hong et al. 2005; Salcedo et al. 2000a). Previous studies suggest that CCL2 indirectly stimulates angiogenesis (Goede et al. 1999; Leung et al. 1997); however, it has recently been shown that CCL2 may also mediate angiogenic effects by acting directly on endothelial cells and increasing vascularity (Hong et al. 2005). Fractalkine (FKN, CX3CL1), a member of the CX3C chemokine family, also belongs to the list of angiogenesis regulators (Blaschke et al. 2003; Nanki et al. 2004; Volin et al. 2001). Recent studies have suggested that the interaction of FKN and CX3CR1 contributes to the pathogenesis of atherosclerosis (Combadiere et al. 2003; Eriksson 2004) and kidney diseases through the firm adhesion of leukocytes to endothelial cells (Segerer et al. 2002; Umehara et al. 2001). FKN has also been shown to participate in the pathogenesis of rheumatoid arthritis, probably by


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increasing the angiogenic process through endothelial cell activation (Blaschke et al. 2003; Nanki et al. 2004; Volin et al. 2001). The in vivo effect of FKN on angiogenesis has clearly shown that FKN plays a significant role in facilitating inflammatory angiogenesis by activating the GPCR (Lee et al. 2006). Several lines of evidence suggest that a biological imbalance in the production of angiogenic and angiostatic factors, such as chemokines, contributes to the pathogenesis of several angiogenesis-dependent disorders, including cancer, rheumatoid arthritis, and psoriasis (Singh and Fidler 1996; Folkman 1995; Keane et al. 1997; Arenberg et al. 1997; Strieter et al. 1995a). The relative levels of these chemokines and their role in regulating tumor angiogenesis are not clear. More studies are needed to provide evidence, if any, that the imbalance in the expression of angiogenic or angiostatic chemokines regulates tumor angiogenesis. On the basis of the published reports, one can predict that a shift in the balance of expression of these angiogenic and angiostatic chemokines dictates whether the tumor grows and develops metastasis or regresses. If this is correct, it will provide an opportunity to shift this imbalance in favor of angiostasis by modulating the expression of the specific chemokine by pharmacological intervention, which will inhibit tumor growth and metastasis.

Chemokines in Tumor Growth and Metastasis Cancer metastasis consists of multiple, complex interacting and interdependent steps (Fidler 1995; Fidler and Ellis 1994; Nicolson 1991; Singh and Fidler 1996; Fidler et al. 2000) where tumor cells exploit the host responses, which are a part of normal, physiological processes, in order to grow and metastasize, and chemokines play important roles in tumor–host interaction. To begin with, chemokines can provide chemo-attractive signaling that can be critical for cellular trafficking to distant organ sites. Infiltrating leukocytes are not the only cells that express chemokine receptors and respond to chemokine gradient in cancer, many cancer cells themselves express chemokine, chemokine receptors and respond to chemokine gradient (Singh et al. 2007; Balkwill 2003; Muller et al. 2001; Murphy 2001). It has been shown that the site of metastasis depends on the characteristics of neoplastic cells and the specific microenvironment of the secondary organ (Fidler 2002). Similar to the process of leukocyte trafficking, constitutive expression of chemokines by the secondary organ of metastasis can provide signaling cues for malignant cell homing. The role of chemokines in organ-specific metastasis was initially suggested in breast cancer. It was demonstrated that CCR7 and CXCR4 that are highly expressed in breast cancer cells determine the invasion and organ specificity of breast cancer metastasis (Muller et al. 2001). The ligands CCL21 and CXCL12 for these receptors are highly expressed in the organs (lung, liver, and bone) that are preferred sites for breast cancer metastasis (Muller et al. 2001). Recently, it has been suggested that osteoclasts may promote metastasis of lung cancer cells expressing CCR4 to the bone marrow by producing its ligand CCL22 (Nakamura et al. 2006).

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Stimulation of osteoclast-like cells with CCR4 results in up-regulation of its ligand, CCL22. In addition, it has been demonstrated that a human lung cancer cell line which expresses CCR4 metastasizes to bone when injected intravenously into NK cell-depleted SCID mice, (Nakamura et al. 2006). Together, the above data supports the idea that the expression of chemokine ligands or their receptors in the organ environment or by malignant cells plays a major role in organ-specific metastasis. CXCR4 appears to be the major chemokine receptor expressed on cancer cells. The expression of CXCR4 has been reported in malignant cells from more than 23 different types of cancers. The accumulating evidence suggests that CXCR4 is an important regulator of breast cancer metastasis and can be predictive of lymph node metastasis (Cabioglu et al. ; Hao et al. 2007; Smith et al. 2004). In experimental studies, treatment of CXCR4-expressing breast cancer cells with neutralizing anti-CXCR4 antibody reduced metastasis to lung in a mouse model (Muller et al. 2001). Gene array analysis of human breast cancer myoepithelial cells and myofibroblasts demonstrated up-regulation of both CXCR4 and CXCL12 which serve to enhance migration and invasion (Allinen et al. 2004). CXCR4 expression also mediates organ-specific metastasis of pancreatic cancer cells and a strong association of CXCR4 with advanced pancreatic cancer has also been suggested (Saur et al. 2005; Wehler et al. 2006). In vitro stimulation of CXCR4-positive cancer cells with CXCL12 resulted in directed migration/ invasion of ovarian cancer cells (Scotton et al. 2002). Stimulation with CXCL12 also up regulated integrin expression and facilitated adhesion in lung cancer cells (Burger et al. 2003). Furthermore, it has been shown that the expression of CXCR4 on human renal cell carcinoma (RCC) correlates with their metastatic ability in both heterotopic and orthotopic SCID mouse models (Pan et al. 2006). In an orthotopic SCID mouse model of RCC, treatment with specific antiCXCL12 antibodies markedly abrogated metastasis of RCC to target organs expressing high levels of CXCL12, without significant changes in tumor cell proliferation, apoptosis, or tumor-associated angiogenesis (Pan et al. 2006). The above findings support the notion that the CXCL12/CXCR4 axis plays a critical role in regulating tumor growth and metastasis. In addition, the clinical relevance of CXCR4 expression has been demonstrated in various types of cancers. In esophageal cancer and melanoma, CXCR4 expression is associated with poor clinical outcome (Kaifi et al. 2005; Scala et al. 2005). In breast cancer, CXCR4 expression predicted lymph node metastasis (Cabioglu et al. 2005). Patients with high CXCR4 expression in NSCLC, colorectal or prostate cancer was more prone to metastasis (Arya et al. 2004; Schimanski et al. 2005; Su et al. 2005). The expression of CXCR4 in nasopharyngeal carcinoma and osteosarcoma is also associated with metastasis (Hu et al. 2005; Laverdiere et al. 2005). In colorectal cancer, CXCR4 expression in primary tumors demonstrated significant association with recurrence, survival, and liver metastasis (Kim et al. 2005). In contrast, a study on neuroblastoma patients found that, although neuroblastoma cells expressed CXCR4, but was not functional (Airoldi et al. 2006), and a study on approximately 300 breast cancer patients found that expression of CXCR4 was not


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associated with metastasis (Weigelt et al. 2005). Above data strongly suggest the role of CXCR4 in regulating the progression of tumor cells to metastasize; however, additional investigations are needed to resolve the ambiguities in different studies. CXCL8, a potent chemoattractant, has been demonstrated to contribute to human cancer progression through its potential function as a mitogenic and angiogenic factor. Elevated levels of CXCL8 have been detected in variety of tumors, such as ovarian carcinoma (Ivarsson et al. 2000), NSCLC (Arenberg et al. 1996a), metastatic melanoma (Varney et al. 2006), and colon carcinoma (Grimm et al. 1996). Studies from our laboratory and others suggest that the expression of CXCL8 correlates positively with disease progression (Nurnberg et al. 1999; Scheibenbogen et al. 1995; Singh et al. 1994; Ugurel et al. 2001). A concomitant up-regulation of one of the two putative CXCL8 receptors has been reported in human melanoma specimens. Analysis of CXCR1 in human melanoma specimens from different Clark levels demonstrated that it is expressed ubiquitously in all Clark levels. In contrast, CXCR2 is expressed predominantly by higher grade melanoma tumors and metastases, suggesting an association between expression of CXCL8 and CXCR2 with vessel density in advanced lesions and metastasis (Varney et al. 2006). More specifically, the effect of CXCL8 can be mediated by CXCR1 and CXCR2, with CXCR1 being a selective receptor for CXCL8 (Addison et al. 2000). Together, these data implicate that CXCL8 can directly modulate growth and the metastatic phenotype of cancer cells. Antibodies to chemokines have shown some promise as a therapeutic modality for treatment of malignant melanoma. Earlier studies have also demonstrated that neutralizing antibodies to CXCR1 and CXCR2 inhibits melanoma cell proliferation and their invasive potential (Varney et al. 2003). Humanized antibodies to CXCL8 have also been shown to inhibit melanoma growth, angiogenesis, and metastasis (Villares et al. 2008; Melnikova and Bar-Eli 2006). Neutralizing antibodies against other chemokines also shows similar results as shown by antibodies against CXCL8 suggesting that melanoma may utilize more than one chemokine ligand pathway to support its growth (Fujisawa et al. 1999). All these evidences emphasize on targeting CXCL8 receptors rather than CXCL8 alone. Also, it has been reported that 17beta-estradiol, progesterone, and dihydrotestosterone suppress the growth of melanoma by inhibiting CXCL8 production in a receptordependent manner (Bendrik and Dabrosin 2009). Antagonist for CXCL8 receptors is also under consideration for melanoma therapy. Small molecule inhibitors with affinity for CXCR1 such as repertaxin or affinity for CXCR2 such as SB-225002 or SB-332235 have been used against inflammatory diseases (Bertini et al. 2004; Thatcher et al. 2005). A recent study has shown the potential of the CXCR2/1specific inhibitors, SCH-479833 and SCH-527123, in inhibiting human melanoma growth by decreasing tumor cell proliferation, survival, and invasion (Singh et al. 2009a). Treatment of melanoma cells with SCH-479833 or SCH-527123 also inhibited tumor growth. Histological and histochemical analyses showed significant (p50%) the growth of the primary tumor. However, its effects on metastasis were more striking, with more than 90% reduction of metastases in lungs and no metastasis in the liver. BALB/c mice or severe combined immunodeficient-beige mice were treated with the immunosuppressive drug, tacrolimus, also known as Fujimycin, and the effect of treatment on mouse renal cancer cell pulmonary metastasis was investigated. Treatment with tacrolimus resulted in a dose-dependent increase in the number of pulmonary metastases in the BALB/c mice and in the severe combined immunodeficient-beige mice (Maluccio et al. 2003). Tacrolimusinduced TGF-b1 overexpression may be a pathogenetic mechanism in tumor progression. Bryostatin 1 and phorbol-12-myristate-13-acetate (PMA) have been used to reduce the intracellular melanin level in high metastatic overexpressing nPKCdelta BL6 (BL6T) cells, thereby inducing white experimental metastasis in syngeneic mice. The possible differences between white and black metastases induced by both treatments on the proliferative and metastatic potential as well as on the expression of some cytokines involved in the metastatic process such as TGF-b, interleukin-10 (IL-10), and interferon (IFN)-gamma were evaluated after the injection of bryostatin 1- or PMA-treated cells into the tail vein of syngenic mice. The results showed only one significant difference between bryostatin I and phorbol ester, namely the cells obtained from white bryostatin 1-treated cells return to a black phenotype after a few passages in culture (La Porta and Comolli 2000). In vivo white metastases showed higher levels of TGF-b and IFN-gamma and when re-injected into syngeneic mice, gave big black metastases. Therefore, in murine melanoma cells, treatment with bryostatin 1 induces the appearance of a white population expressing different levels of TGF-b and IFN-gamma. The effect of TGF-b and PMA alone and in combination was examined in human NSCLC and normal human bronchial epithelial (NHBE) cells and shown to increase expression of TGF-b1 mRNA 24h after their addition to both cell lines (Jakowlew et al. 1997a, b). TGF-b1 and PMA both caused a persistent increase in expression of the mRNAs for both PAI-1 and PA up to 24h in most NSCLC cells, with the increase in PAI-1 mRNA beginning several hours before that of PA mRNA. In contrast, while

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TGF-b1 also increased expression of PAI-1 mRNA in NHBE cells, the expression of PA mRNA decreased simultaneously. The effect of PMA on PAI-1 and PA mRNAs was opposite of TGF-b1 in these cells, with expression of PAI-1 mRNA decreasing and PA mRNA increasing after addition of PMA. The responses of the mRNAs and proteins of TGF-b1, PAI-1 and PA to TGF-b1 and PMA were inhibited by the serine/threonine kinase inhibitor H7 in NSCLC cells. These studies show that there is parallel regulation of the genes for TGF-b1, PAI-1 and PA by TGF-b1 and PMA in NSCLC, but differential regulation of the genes for PAI-1 and PA by these agents in NHBE cells and because the effects of TGF-b and PMA on the different TGF-b isoforms, PA, and PAI, as well as on and fibronectin in NHBE and NSCLC cells are complex, these studies suggest that there are distinct mechanisms for controlling the different TGF-b isoforms, PA, PAI and extracellular matrix proteins in normal lung and lung cancer cells. Both PMA and TGF-b1 can also induce early growth response gene-1 (Egr) mRNA expression in NSCLC cells and NHBE cells with PMA inducing Egr-1 mRNA similarly in both cell types, while TGF-b1 induces Egr-1 mRNA expression more rapidly and more transiently in NSCLC cells than in NHBE cells (You and Jakowlew 1997). 13-cis-Retinoic acid can mediate differentiation of transformed cells and slow the proliferation of malignant cells, suggesting its use as a potential intervention tool. Specific cDNA probes for retinoic acid receptors demonstrated the expression of mRNAs for the different retinoic acid receptor isoforms in SCLC cell lines (Avis et al. 1995). Addition of 13-cis-retinoic acid to SCLC cells resulted in an increase in the level of the retinoic acid receptor-beta mRNAs and in a significant dosedependent, growth-inhibitory effect using serum-free conditions that decreased when cells were cultured in medium containing serum or serum components. Preincubating serum with triglycerides restored the inhibitory effects of 13-cisretinoic acid demonstrated in serum-free systems and suggests that 13-cis-retinoic acid preferentially binds to serum albumin, restricting its inhibitory effects on epithelial cell receptors. Blocking retinoic acid–albumin interactions with a fatty acid source may improve the bioavailability of 13-cis-retinoic acid and significantly enhance the inhibitory effect in vivo. In addition, TGF-b2 transcripts increased while TGF-b3 transcripts decreased in multiple NSCLC cells upon treatment with 13-cis-retinoic acid (Jakowlew et al. 2000). Rapamycin is an effective inhibitor of human renal cancer metastasis. A human renal cell cancer pulmonary metastasis model was developed using human RCC 786-O as the tumor challenge and the severe combined immunodeficient (SCID) beige mouse as the host. Rapamycin reduced the number of pulmonary metastases, whereas interestingly, cyclosporine increased pulmonary metastases (Luan et al. 2003). Rapamycin was effective in cyclosporine-treated mice, and rapamycin or rapamycin plus cyclosporine prolonged survival. The modifying effects of a Kunitz trypsin inhibitor (KTI) and a Bowman-Birk trypsin inhibitor (BBI), purified from soybean trypsin inhibitor, as dietary supplements on experimental and spontaneous pulmonary metastasis of murine Lewis lung carcinoma 3LL cells as well as peritoneal disseminated metastasis model in human ovarian cancer HRA cells were also investigated in mouse models. It was shown in an in vivo spontaneous metastasis

28 Transforming Growth Factor-b in Lung Cancer, Carcinogenesis, and Metastasis

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assay that diet supplementation with KTI, but not with BBI, for 28days immediately after tumor cell inoculation significantly inhibited the formation of lung metastasis in C57BL/6 mice in a dose-dependent manner (Kobayashi et al. 2004). The inhibition of lung metastasis was not due to direct antitumor effects of KTI. In an in vivo experimental metastasis assay, the diet supplementation with KTI or BBI for 21days after tumor cell inoculation did not reduce the number of lung tumor colonies. In addition, KTI treatment in a peritoneal disseminated metastasis model of HRA cells resulted in a 40% reduction in total tumor burden when compared with control animals. These results suggest that dietary supplementation of KTI more efficiently regulates the mechanism involved in the entry into vascular circulation of tumor cells in intravasation than in extravasation during the metastatic process. KTI treatment may also be beneficial for ovarian cancer patients with or at risk for peritoneal disseminated metastasis; it greatly reduces tumor burden in part by inhibiting phosphorylation of MAP kinase and PI3 kinase, leading to suppression of uPA expression. Calcium homeostasis is another area of drug study. Calcium homeostasis is a tightly regulated process that involves the co-ordinated efforts of the skeleton, kidney, parathyroid glands, and intestine. Neoplasms can alter this homeostasis indirectly through the production of endocrine factors resulting in humoral hypercalcemia of malignancy. Relatively common with breast and lung cancer, this paraneoplastic condition is most often due to tumor production of parathyroid hormone-related protein (PTHrP) and ensuing increased osteoclastic bone resorption. The metastasis of tumor cells to bone represents another skeletal complication of malignancy (Clines and Guise 2005). As explained in the “seed and soil” hypothesis (Fidler 2003), bone represents a fertile ground for cancer cells to flourish. In the case of osteolytic bone disease, tumor-produced PTHrP stimulates osteoclasts that in turn secrete tumor-activating TGF-b that further stimulates local cancer cells. This vicious cycle of bone metastases represents reciprocal bone/cancer cellular signals that likely modulate osteoblastic bone metastatic lesions as well. The development of targeted therapies to block initial cancer cell chemotaxis, invasion and adhesion or to break the vicious cycle is dependent on a more complete understanding of bone metastases. Although bisphosphonates delay progression of skeletal metastases, it is clear that more effective therapies are needed. Cancer-associated bone morbidity remains a major public health problem, and to improve therapy and prevention it is important to understand the pathophysiology of the effects of cancer on bone. Decorin is a major extracellular matrix protein which has become the focus of various cancer studies. The potential of decorin as a novel biological target for the treatment of osteosarcoma has been studied, with lung metastases being the most crucial event affecting the therapeutic outcome of osteosarcoma. In one study, the LM8 murine osteosarcoma cell line with high metastatic potential to the lung was used and two cell lines were established, including LM8-DCN which stably expressed human decorin and LM8-mock control. When the LM8-DCN cell line was subcutaneously injected into the backs of mice, significantly fewer pulmonary metastases were observed in mice with LM8-DCN compared to mice inoculated

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with LM8 and LM8-mock (Shintani et al. 2008). In addition, the mice in the LM8DCN inoculated group survived significantly longer than those in the LM8 and LM8-mock inoculated group. There was no difference in the morphology and growth rates, but the motility and invasion of LM8 were inhibited by decorin. These results suggest that decorin has the therapeutic potential to prevent lung metastasis in osteosarcoma. Doxorubicin is a DNA-damaging drug, commonly used for treatment of cancer patients. Doxorubicin causes not only cytotoxic and cytostatic effects, but also inhibits metastasis formation. The influence of doxorubicin on TGF-b signaling in tumor cells was examined and demonstrated that Doxorubicin inhibited TGF-b signaling in human lung adenocarcinoma A549 cells; namely, it blocked TGF-b1induced activation of Smad3-responsive CAGA(12)-Luc reporter, but did not affect c-myc-Luc reporter (Filyak et al. 2008). That effect was observed as early as after 1–3h of treating these cells with doxorubicin, while the other drugs cisplatin or methotrexate did not alter activation of CAGA(12)-Luc reporter under the same conditions. Also, after 1h, doxorubicin abrogated TGF-b-induced translocation of Smad3-protein from the cytoplasm to the nucleus. Down-regulation of expression of Smad2, Smad3, and Smad4 proteins, and up-regulation of inhibitory Smad7 protein upon doxorubicin treatment, were found after 12–24h of doxorubicin treatment. Phosphorylation of Smad2/3 proteins was also affected by doxorubicin.

Genomics Lung cancer often metastasizes to bone in patients with advanced disease. The bone is a rich source of many chemokines and growth factors, including: insulin-like growth factor (IGF) I and II, TGF-b, interleukins, and tumor necrosis factor-alpha (TNF-a). Identification of other factors involved in the interactions between lung cancer cells and bone will improve the prevention and treatment of bone metastases. Changes in metastasis-related gene expression of human HARA lung squamous carcinoma cells co-cultured with neonatal mouse calvariae using a pathwayspecific microarray analysis. Nine genes were up-regulated and two genes downregulated in HARA cells co-cultured with mouse calvariae (Deng et al. 2007). Five of the nine up-regulated genes, including caveolin 1, CD44, ephrin-B2 (EphB2), ezrin, and PTHrP, and one down-regulated gene, secretory leukocyte protreinase inhibitor I (SLPI), were further confirmed by RT-PCR amplification. A mouse model was subsequently used to study the role of PTHrP and ezrin in bone metastasis in vivo. PTHrP (all three isoforms) and ezrin were up-regulated in HARA cells at sites of bone metastasis as detected by RT-PCR amplification and immunohistochemical analysis. The PTHrP-141 mRNA isoform was increased by the greatest extent in bone metastases compared to PTHrP-139 and PTHrP-173 mRNA. A HARA cell line was generated in which PTHrP expression was inducibly silenced by RNA interference. Silencing of PTHrP expression caused significant reduction of submembranous F-actin and decreased HARA cell invasion. Ezrin up-regulation


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was confirmed by western blots on HARA cells co-cultured with adult mouse long bones. Further, TGF-b was identified as one of the factors in the bone microenvironment that was responsible for the up-regulation of ezrin. The identification of PTHrP and ezrin as important regulators of lung cancer bone metastasis offers new mechanistic insights into the metastasis of lung cancer and provides potential targets for the prevention and treatment of lung cancer metastasis. It has also been proposed that exposure of breast cancer cells to the bone microenvironment results in alterations in gene expression that favor the growth and proliferation of tumor cells in the bone. To investigate this, MDA-MB-231 breast carcinoma cells were exposed to bone-derived conditioned media generated by culturing fetal rat calvaria for 24h under serum free conditions. Using cDNA microarray technology, the insulin-like growth factor family of binding proteins (IGFBPs) was identified as genes whose expression profiles are consistently and significantly altered with exposure to this simulated bone environment in vitro, when compared with untreated controls (Giles and Singh 2003). These findings suggest that the up-regulation of IGFBP-3 seen with exposure to the bone microenvironment is directly linked to an increase in TGF-b-mediated cell proliferation and appears to be functioning through an IGF-independent mechanism. Gene expression profiling of metastatic brain tumors from primary lung adenocarcinoma, using a 17K-expression array, also revealed that 1561 genes were consistently altered (Zohrabian et al. 2007). Further functional classification placed the genes into seven categories: cell cycle and DNA damage repair, apoptosis, signal transduction molecules, transcription factors, invasion and metastasis, adhesion, and angiogenesis. Genes involved in apoptosis, such as caspase 2 (CASP2), transforming growth factor-b-inducible early gene (TIEG), and neuroprotective heat shock protein 70 (Hsp70) were underexpressed in metastatic brain tumors. Alterations in Rho GTPases (ARHGAP26, ARHGAP1), as well as down-regulation of the metastasis suppressor gene kisspeptin-1 (KiSS-1) were noted, which may contribute to tumor aggression. Overexpression of the invasion-related gene neurofibromatosis 1 (NF1), and angiogenesis-related genes VEGF-b and placental growth factor (PGF) was also observed, while brain-specific angiogenesis inhibitors 1 and 3 (BAI1 and BAI3) were underexpressed as well. Examination of cell-adhesion and migration-related genes revealed an increased expression of integrins and extracellular matrices collagen and laminin. The study also showed alterations in p53 protein-associated genes, among these increased gene expression of p53, up-regulation of Reprimo or candidate mediator of the p53-dependent G2-arrest, down-regulation of p53-regulated apoptosis-inducing protein 1 (p53 AIP1), p53 decreased expression of tumor protein inducible nuclear protein 1 (p53 DINP1), and down-regulation of transformed 3T3 cell double minute 4 (Mdm4). The results demonstrated that genes involved in adhesion, motility, and angiogenesis were consistently up-regulated in metastatic brain tumors, while genes involved in apoptosis, neuroprotection, and suppression of angiogenesis were markedly down-regulated, collectively making these cancer cells prone to metastasis. To identify and functionally characterize genes involved in the mechanisms of osseous metastasis, a murine lung cancer model was developed. Comparative transcriptomic

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analysis identified genes encoding signaling molecules (such as transcription factor 4 [TCF4] and protein kinase D [PRKD3]) and cell anchorage-related proteins (melanoma cell adhesion molecule [MCAM] and sushi domain-containing protein 5 precursor [SUSD5]), some of which were basally modulated by TGF-b in tumor cells and in conditions mimicking tumor–stromal interactions (Vicent et al. 2008). Triple gene combinations induced not only high osteoclastogenic activity but also a marked enhancement of global metalloproteolytic activities in vitro. These effects were strongly associated with robust bone colonization in vivo, whereas this gene subset was ineffective in promoting local tumor growth and cell homing activity to bone. Interestingly, global inhibition of metalloproteolytic activities and simultaneous TGF-b blockade in vivo led to increased survival and a remarkable attenuation of bone tumor burden and osteolytic metastasis. Thus, this metastatic gene signature mediates bone matrix degradation by a dual mechanism of induction of TGFb-dependent osteoclastogenic bone resorption and enhancement of stroma-dependent metalloproteolytic activities. These findings suggest the cooperative contribution of host-derived and cell autonomous effects directed by a small subset of genes in mediating aggressive osseous colonization.

Animal Models Various mouse animal model systems have been used to examine the role of TGF-b in lung cancer metastasis. The roles of TGF-b1, TGF-b2, and TGF-b3 were determined in the in-bred A/J mouse model challenged with the carcinogen ethyl carbamate to induce lung adenomas. Immunostaining for the TGF-b ligands and receptors was detected in the epithelia of the bronchioles of untreated and treated A/J mice at similar levels and in adenomas by two months (Jakowlew et al. 1998a, b). While immunostaining for TGF-b1, TGF-b2, and TGF-b3 and TGF-b RI in adenomas was detected at levels comparable to those in bronchioles, immunostaining for TGF-b RII was less intense in adenomas than in bronchioles, and decreased immunostaining for TGF-b RII in adenomas persisted for at least 8 months after exposure to ethyl carbamate. In situ hybridization studies conducted with TGF-b receptor riboprobes showed a corresponding reduction in expression of TGF-b RII mRNA, but not of TGF-b RI mRNA in adenomas compared with expression in normal bronchioles. Expression of TGF-b RII mRNA was also examined in nontumorigenic and tumorigenic mouse lung cells and found to be lower in tumorigenic cells derived from ethyl carbamate-induced lung tumors. These findings suggest that a decrease in expression of TGF-b RII may contribute to autonomous cell growth and may play an important role in mouse lung carcinogenesis induced by ethyl carbamate. Whereas expression of the proteins and mRNAs for TGF-b1 and TGF-b RI was found to be comparable in lung adenomas and bronchioles of in-bred A/J mice treated with benzo(a)pyrene in chemically-induced mouse lung tumorigenesis, decreased immunostaining and hybridization for TGF-b RII protein and mRNA was detected in 50% of lung adenomas in these mice (Kang et al. 2000)


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along with increased levels of surfactant protein D (Zhang et al. 2003). The non-steroidal anti-inflammatory drug indomethacine was shown to be able to reduce lung tumor number in these mice (Moody et al. 2001). Interestingly, expression of TGF-b1 and the TGF-b receptor proteins in A/J mice was similar to that of bronchioles in C57B1/6 mice and their littermates heterozygous for deletion of the TGF-b1 gene treated with diethylnitrosamine (Tang et al. 1998). Such findings show that reduced levels of expression of TGF-b RII occur in some, but not all, human and mouse lung tumors. This suggests that different mechanisms of action, some of which may involve the TGF-b signaling pathway, may contribute to the progression of mouse lung tumorigenesis. To elucidate the role of TGF-b1 and TGF-b RII as tumor-suppressor genes in lung carcinogenesis, C57BL/6 mice heterozygous (HT) for deletion of the TGF-b1 gene were mated with A/J mice to produce AJBL6 TGF-b1 HT progeny and their wild-type (WT) littermates. Immunohistochemical staining, in situ hybridization, and northern blot analyses showed decreased staining and hybridization for TGF-b1 protein and mRNA, respectively, in the lungs of normal HT mice versus WT mice and competitive reverse transcription polymerase chain reaction (CRT-PCR) amplification showed the level of TGF-b1 mRNA in the lungs of HT mice to be fourfold lower than the level in WT lung (Kang et al. 2000). When challenged with ethyl carbamate, lung adenomas were detected in 55% of HT mice by 4 months but only in 25% of WT littermates at this time. Whereas all HT mice had adenomas by 6 months, it was not until 10 months before all WT mice had adenomas. After 12 months, the average number of adenomas was fivefold higher in HT lungs than in WT lungs. Most dramatic was the appearance of lung carcinomas in HT mice 8months before they were visible in WT mice. Increased susceptibility of HT mice to chemically-induced lung tumorigenesis was also shown to occur independently of K-ras (McKenna et al. 2001) and to result in reduced levels of expression of Nkx2 homeobox 1 transcription factor (Nkx2.1), also known as thyroid transcription factor 1 (TTF-1) (Kang et al. 2004). In addition, weak immunostaining for TGF-b RII was detected in 67% of HT carcinomas at 12 months, whereas only 22% of WT carcinomas showed weak staining for this protein. Individual lung carcinomas showing reduced TGF-b RII expression and adjacent normal bronchioles were excised from HT lungs using laser capture microdissection, and CRT-PCR amplification of the extracted RNA showed 12-fold less TGF-b RII mRNA in these carcinomas compared with bronchioles. Decreasing TGF-b RII mRNA levels occurred with increasing tumorigenesis in lung hyperplasias, adenomas, and carcinomas, with carcinomas having four- and sevenfold lower levels of TGF-b RII mRNA than adenomas and hyperplasias, respectively. These findings show enhanced ethyl carbamate-induced lung tumorigenesis in AJBL6 HT mice compared with WT mice, suggesting that both TGF-b1 alleles are necessary for tumor-suppressor activity. Reduction of TGF-b RII mRNA expression in progressive stages of lung tumorigenesis in HT mice suggests that loss of TGF-b RII may play an important role in the promotion of lung carcinogenesis in mice with reduced TGF-b1 gene dosage when challenged with carcinogen. Multiple components of the cyclin D/cyclin-dependent kinase-4 (CDK-4)/p16(ink4a)pRb signaling pathway were

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demonstrated to be frequently altered early in lung tumors of HT mice induced by ethyl carbamate (Kang et al. 2002). Heterozygous TGF-b1 mice were also mated with latent activatable (LA) mutated K-ras mice to generate TGF-b1(+/+), K-ras LA (wild-type (WT)/LA) and TGF-b1(+/–), K-ras LA (HT/LA) mice. While both HT/LA and WT/LA mice developed spontaneous lung tumors, HT/LA mice progressed to adenocarcinomas significantly earlier compared with WT/LA mice and HT/LA adenocarcinomas had significantly lower angiogenic activity compared with WT/LA adenocarcinomas (Pandey et al. 2007). Thus, while oncogenic K-ras mutation and insensitivity to the growth regulatory effects of TGF-b1 is essential for initiation and progression of mouse lung tumors to adenocarcinoma, a full gene dosage of TGF-b1 is required for tumor-induced angiogenesis and invasive potential. The influence of TGF-b signaling on Neu-induced mammary tumorigenesis and metastasis was examined with transgenic mouse models using mice expressing an activated TGF-b RI or dominant negative TGF-b RII under control of the mouse mammary tumor virus promoter. When crossed with mice expressing activated forms of the Neu receptor tyrosine kinase that selectively couple to the growth factor receptor-bound protein 2 (Grb2) or Src homology 2 domain-containing protein (Shc) signaling pathways, the activated TGF-b increased the latency of mammary tumor formation and also enhanced the frequency of extravascular lung metastasis (Siegel et al. 2003). Conversely, the expression of dominant-negative TGF-b RII decreased the latency of Neu-induced mammary tumor formation, while significantly reducing the incidence of extravascular lung metastases. These observations argue that TGF-b can promote the formation of lung metastases while impairing Neu-induced tumor growth and suggest that extravasation of breast cancer cells from pulmonary vessels is a point of action of TGF-b in the metastatic process. To address the role of TGF-b in the progression of established tumors while avoiding the confounding inhibitory effects of TGF-b on early transformation, doxycyclineinducible triple transgenic mice were generated in which active TGF-b1 expression could be conditionally regulated in mouse mammary tumor cells transformed by the polyomavirus middle T antigen. Doxycycline-mediated induction of TGF-b1 for as little as 2weeks increased lung metastases more than ten-fold without a detectable effect on primary tumor cell proliferation or tumor size (Muraoka-Cook et al. 2004). Doxycycline-induced active TGF-b1 protein and nuclear Smad2 were restricted to cancer cells, suggesting a causal association between autocrine TGF-b and increased metastases. Antisense-mediated inhibition of TGF-b1 in polyomavirus middle T antigen-expressing tumor cells also reduced basal cell motility, survival, anchorage-independent growth, tumorigenicity, and metastases. Thus, induction and/or activation of TGF-b in hosts with established TGF-b-responsive cancers can rapidly accelerate metastatic progression. Using the MMTV/PyVmT transgenic model of metastatic breast cancer, it was shown that administration of ionizing radiation or doxorubicin caused increased circulating levels of TGF-b1 as well as increased circulating tumor cells and lung metastases (Biswas et al. 2007). Circulating polyomavirus middle T antigen-expressing tumor cells did not grow ex vivo in the presence of the


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TGF-b antibody, suggesting autocrine TGF-b is a survival signal in these cells. Radiation failed to enhance lung metastases in mice bearing tumors that lack TGF-b RII, suggesting that the increase in metastases was due, at least in part, to a direct effect of TGF-b on the cancer cells. Such findings implicate TGF-b induced by anticancer therapy as a pro-metastatic signal in tumor cells and provide a rationale for the simultaneous use of these therapies in combination with TGF-b inhibitors. Use of small interfering RNA has been perfected in the past few years to demonstrate functional roles of gene products. Inhibition of TGF-b1 expression with small interfering RNA constructs targeting TGF-b1 in metastatic breast cancer MDA-MB-435 cells showed a 35% decrease in migration and a 55% decrease in invasion in vitro, with a 50% increase in proliferation and no effect on apoptosis (Moore et al. 2008). While in vivo analysis indicated a 90% decrease in the number of mice bearing macroscopic lung metastases, primary tumors did not show any difference in the growth kinetics when compared with the parental MDA-MB-435 cells. Interestingly, analysis of TGF-b signaling pathways in the clonal derivatives showed a decrease in Smad2 activation and an increase in Akt/protein kinase B and ERK activation and decreased TGF-b RI and TGF-b RII expression in TGF-b1 silenced cells. These findings suggest that inhibition of TGF-b1 ligand may act as a negative feedback loop to disrupt the function of all TGF-b isoforms. Furthermore, therapies targeting the TGF-b signaling pathway may be more effective in latestage disease to prevent organ metastasis but not primary tumor formation and may be combined with other tumor-targeted therapies normally limited by increased circulating TGF-b levels. Clonal populations of 4NQO-induced rat malignant oral keratinocytes were also examined for metastatic capacity following orthotopic transplantation into athymic mice for effects on TGF-b. Polygonal and spindle cells formed well-differentiated squamous cell carcinomas and undifferentiated spindle cell tumors in almost 100% of animals at the site of inoculation (Davies et al. 1999). Transplantation of cells of either cell type at high cell density resulted in approximately 50% of animals forming pulmonary metastases, while inoculation of low density differentiated polygonal cells resulted in the formation of significantly fewer pulmonary metastases than the undifferentiated spindle cells. A single well-differentiated clone of polygonal cells and three of four of the undifferentiated spindle cell lines produced comparable levels of TGF-b1. One undifferentiated spindle cell line expressed significantly more TGF-b1 and following transplantation orthotopically, fewer animals formed pulmonary metastases despite the formation of primary tumors in almost all grafted animals, suggesting that TGF-b1 can act as a tumor suppressor in this cell type. The clones of polygonal cells were markedly inhibited and the spindle cells were only partially inhibited by exogenous TGF-b1. Both cell types expressed high-affinity TGF-b cell surface receptors. The results suggest that differentiated rat malignant oral keratinocytes are less aggressive and have a decreased potential to metastasise than their undifferentiated spindle cell counterparts and may be attributable, in part, to a change in TGF-b receptor profile leading to the partial loss of response to exogenous TGF-b1.

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The effects of TGF-b1 on the proliferation and experimental pulmonary metastasis of MCS-1 cells, undifferentiated type cloned tumor cells established from a mesenchymal chondrosarcoma which spontaneously occurred in the soft tissue of a female Chinese hamster were also examined. Treatment of MCS-1 cells with TGF-b1 was tested for in vitro growth using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) method, in vivo growth by subcutaneous inoculation into athymic nude mice and experimental pulmonary metastasis by injection into the tail vein of athymic nude mice. TGF-b1 significantly inhibited in vitro growth of MCS-1, depending on concentration, and also experimental metastasis (Fujisawa et al. 1999). TGF-b1, however, was ineffective for in vivo subcutaneous growth of MCS-1 cells. These results indicate that TGF-b1 might be an inhibitor of metastasis of mesenchymal chondrosarcomas including other types of non-epithelial cartilage or bone formation tumors.

Conclusions The acquisition of metastatic characteristics that are associated with lung cancer often predicts significant cancer-associated mortality and morbidity as lung tumorigenesis progresses. Although drug treatments and therapies are being developed that target primary lung tumors, additional new drugs and therapies are required to combat the challenges of metastatic tumors that occur in the lung as well as in other organs. Because there is ample clinical and experimental evidence that shows that TGF-b and its downstream signaling components have an important role in the metastatic process, TGF-b has become an attractive candidate for use in anti-metastasis treatments and therapies. Several small molecule inhibitors targeting the TGF-b receptor kinases have been developed recently along with specific neutralizing antibodies and nucleic acid-based therapies to inhibit various components of the TGF-b signaling pathway. Some of these drug treatments and therapies have shown promise in mouse model and cell culture systems that include lung. Developing these drug therapies has not been easy, partially because of the complex role of TGF-b in tumor progression to metastasis. This is because TGF-b can act as a tumor suppressor or a tumor promoter depending on the tumor type and the stage of tumor progression. Although many investigators have already devoted several years to understanding the role of TGF-b in tumorigenesis, there is still a lot of research that needs to be performed in order to develop new strategies that can utilize the complex nature of TGF-b to make significant inroads into the prevention and elimination of lung cancer, carcinogenesis, and metastasis. Acknowledgments It is not possible to include every important contribution that has been made to our understanding of TGF-b in lung cancer and metastasis. Apologies are extended to those investigators whose contributions could not be included.


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Chapter 29

Cooperative Interactions Between Integrins and Growth Factor Signaling in Pathological Angiogenesis Jennifer Roth, Eric Tweedie, and Peter C. Brooks

Abstract As with most complex biological processes, angiogenesis requires the integration of a number of molecular signaling networks to coordinate multiple cues from both the extracellular tissue microenvironment as well as the cell’s interior. Thus an important area of angiogenesis investigation involves understanding the mechanisms that facilitate cooperation between multiple receptor–ligand signaling pathways. Two crucial networks that play active role in angiogenesis include growth factor/growth factor receptors and extracellular matrix/integrin receptor signaling systems. Emerging evidence suggests that these two important signaling systems depend in large part on each other, and function cooperatively to control new blood vessel development. Given the tissue-specific variations in the expression of components within each of these systems, significant challenges exist in order to exploit these signaling pathways for clinical intervention. A more detailed understanding of how the molecular components of these two signaling systems communicate with each other to direct and coordinate downstream effector functions may lead to optimized anti-angiogenic strategies to control malignant tumor progression. In this regard, we will discuss the multiple ways by which growth factor and integrin signaling pathways function cooperatively to regulate pathological angiogenesis within the context of the tissue microenvironment.

Introduction Both physiological and pathological blood vessel formation occurs within a complex tissue microenvironment composed of interconnected networks of extracellular matrix (ECM) molecules and a variety of different cell types. It has been appreciated for decades that cellular behavior is regulated by a highly orchestrated P.C. Brooks (*) Maine Medical Center Research Institute, Center for Molecular Medicine, 81 Research Drive, Scarborough, ME, USA e-mail: [email protected] R.G. Bagley (ed.), The Tumor Microenvironment, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-6615-5_29, © Springer Science+Business Media, LLC 2010

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system of communication links between different cell types, secreted growth factors, cytokines, chemokines, and a variety of insoluble solid-state ECM molecules that constitute the tissue microenvironment (Contois et al. 2009; Rapisarda and Melillo 2009). However, how cells are able to assimilate and interpret this vast amount of information in a meaningful way to regulate processes such as new blood vessel development or angiogenesis remains in many respects unknown. Given the diverse interwoven signaling networks that are required to complete the angiogenic cascade, it is critical that we begin to understand in more detail the impact that disrupting one communication pathway may have on another, especially as it relates to development of anti-angiogenic strategies for controlling tumor progression. This concept is of particular importance given the highly integrated nature of feed back systems that exist between signaling pathways such as PI3K/Akt and MAPK/Erk cascades which have been implicated in angiogenesis (Huang and Fraenkel 2009; Wagner and Nebreda 2009; Engelman 2009). Viewed from an integrated systems perspective, disruption of one signaling node or hub may have unexpected consequences on a second distinct pathway. Thus, studying angiogenesis from a more global network systems approach may offer unique molecular insight into the coordinated events necessary for new vessel formation. This molecular insight may facilitate the development of more effective therapeutic strategies to control pathological blood vessel growth. As the complexities of angiogenesis continue to be unraveled, new studies are rapidly adding to the growing list of cell types that play active roles in controlling neovascularization. In the past, the majority of studies on angiogenic mechanisms focused largely on signaling within endothelial cells. Now, evidences indicates that pericytes, smooth muscle cells, fibroblasts, inflammatory infiltrates including monocytes, macrophages, mast cells, as well as platelets, bone marrow-derived progenitor cells, and malignant tumor cells all contribute to new blood vessel formation (Murdoch et al. 2008; Colmone and Sipkins 2008; Greenberg et al. 2008; Klement et al. 2008). These distinct cell types have been suggested to contribute to angiogenesis by mechanisms as diverse as physical incorporation into the vascular structure, to modulating the local concentrations of secreted pro- and anti-angiogenic factors, and to mechanically altering the local insoluble matrix surrounding vessels (Murdoch et al. 2008; Colmone and Sipkins 2008; Greenberg et al. 2008; Klement et al. 2008). Intriguing new data now suggests that cell-mediated biomechanical tissue contraction may contribute to the movement of pre-existing vessels into areas of hypoxia, thereby enhancing local tissue perfusion (Kilarski et al. 2009). Given the increasing numbers of distinct cell types and mechanisms thought to regulate angiogenesis, coupled with the diversity of soluble factors, understanding how a specific tissue microenvironment modulates a cells capacity to produce as well as response to both pro- and anti-angiogenic molecules will likely lead to more effective therapeutic strategies to control abnormal vessel growth. It is well accepted that the ability of a cell to respond to external cues such as pro- and anti-angiogenic regulatory molecules depends on bi-directional communication with secreted factors, insoluble ECM elements, and between different cell types. Integrins represent a major family of cell surface receptors known to play

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important roles in mediating cell-matrix communication. This multifunctional family of receptors not only binds to insoluble ECM, but also bind a variety of soluble molecules (Contois et al. 2009; Avraamides et al. 2008). Given their capacity to sense and transmit signals derived from biochemical and mechanical alteration originating from inside and outside the cell, integrins are well suited to function as microenvironmental sensors, which integrate diverse signaling cascades. Thus, integrins may serve as “functional hubs” in the interconnected network of angiogenesis signaling (Contois et al.2009). Many growth factors and cytokines are known to regulate angiogenesis. Thus, distinct molecular mechanisms have evolved to allow cells to sense and utilize the diversity of soluble regulatory factors in specific ways. While it would be well beyond the scope of this review to discuss all the growth factors and cytokines thought to regulate angiogenesis, we will highlight a number of examples to illustrate the complexity by which these molecules contribute to new vessel growth. Given the diversity of soluble factors that impact vessel formation, it is becoming increasingly clear that in order to gain more complete understanding of how growth factor signaling impacts angiogenesis, a greater appreciation of the roles that integrin-mediated sensing of the surrounding microenvironment has on modulating angiogenic growth factor signaling is needed. Therefore, we will focus our discussion on the multitude of ways by which integrins, growth factors, and their receptors function cooperatively to regulate angiogenesis (Fig. 29.1).

Blood Vessel Formation While the formation of functional blood vessels can occur by several processes that share many common mechanisms, it is important to note that distinct differences also exist, especially between normal and pathological vessel formation (St Croix et al. 2000; Alavi et al. 2007; Hida and Klagsbrun 2005; Ghosh et al. 2008). These differences between normal vascular development and pathological angiogenesis may be due in part to the unique tissue microenvironments within which vessels form. For example, the local variability in stromal cell types that express a distinct repertoire of pro- and anti-angiogenic factors, combined with the composition and integrity of ECM molecules associated with blood vessels is likely to be quite different between normal and tumor vessels. Examples of the major ways by which blood vessel formation occurs include embryonic vasculogenesis, which involves the de novo formation of vessels from precursor cells, arteriogenesis, a process by which functional vessels form by a mechanism of activation, dilation and remodeling of small pre-existing nonfunctional vessels and finally angiogenesis, a process by which blood vessels form from pre-existing vessels (Hodivala-Dilke et al. 2003; Fischer et al. 2006). New evidence now suggests that enhanced vascularization of hypoxic microenvironments may also occur by a mechanism involving cell-mediated tissue contraction that physically translocates existing functional vessels into a hypoxic area where

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Cooperation Between Receptor / Ligand Networks Regulate New Blood Vessel Formation

GF

ECM

CRD CRD

FN FN FN FN III III JM

JM

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Fibroblasts/Stromal Cells Inflammatory Cells Bone Marrow Progenitor Cells Tumor Cells Vascular Cells

Pathological Angiogenesis Fig. 29.1 Cooperation between receptor/ligand networks regulate new blood vessel formation. A wide variety of cell types within the tissue microenvironment are thought to contribute to the formation of new blood vessels. Two of the major receptor/ligand signaling networks that function cooperatively to control angiogenesis include the growth factor (GF)/growth factor receptor system and extracellular matrix (ECM)/integrin receptor system

few vessels previously existed (Kilarski et al. 2009). Given the diversity by which blood vessels form, we will limit the remainder of our discussion largely to angiogenesis and in particular pathological angiogenesis.

Pathological Angiogenesis Angiogenesis can be sub-categorized as to whether it occurs by intussuception or sprouting (Burri et al. 2004; Hlushchuk et al. 2008). Intussuception involves the splitting of pre-existing vessels by the formation of translumenal tissue bridges that


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result in the formation of a diversity of branching patterns (Burri et al. 2004; Hlushchuk et al. 2008). This form of angiogenesis is thought to occur more rapidly than sprouting angiogenesis and may involve minimal if any cellular proliferation and extracellular matrix (ECM) remodeling (Burri et al. 2004; Hlushchuk et al. 2008). Sprouting angiogenesis on the other hand can be organized temporally into three general steps, including an initiation phase, an invasive phase and a maturation phase, and both integrins and growth factor signaling play vital roles in each of these steps. The sprouting form of angiogenesis involves growth factor-stimulated proliferation and invasion of tip cells from an existing vessel. Endothelial cells line up and organize into solid cords, which is followed by the process of canulization, which ultimately results in the formation of a functional lumen (Folkman 2007). Understanding the particular type of vascular development occurring may help direct the most appropriate therapeutic approach to control aberrant angiogenesis. For example, targeting a specific angiogenic growth factor to inhibit pathological blood vessel formation that is occurring predominately by intussuception or by biomechanical tissue contraction may result in only limited therapeutic benefit. In fact, new studies are emerging suggesting that blocking one growth factor signaling pathway such as VEGF may initiate an anti-angiogenic rescue program that allows alternative growth factor signaling cascades to substitute or compensate in part for the disrupted pathway (Ebos et al. 2009; Fernado et al. 2008; Puigvert et al. 2009). Thus, more molecular insight into these regulatory feed back loops will likely lead to more efficacious therapeutic intervention.

Integrins and Their ECM Ligands in Angiogenesis It is known that understanding both cell surface receptors and their cognate ligands in growth factor signaling systems is critical to appreciate their biological functions, and this same principal applies to integrins and their ligands. Integrins have been shown to bind a host of different regulatory molecules including: ECM protein, proteolytic enzymes, protease receptors, other cell adhesion molecules, and both growth factors and growth factor receptors (Contois et al. 2009; Avraamides et al. 2008). Given this diversity of binding partners, it is not surprising that a growing number of studies have demonstrated functional roles for integrins in modulating growth factor signaling. Integrins are transmembrane heterodimers composed of a and b chains. Distinct combinations of these separate gene products give rise to at least 24 different integrins with specific and sometimes overlapping ligandbinding capacity (Avraamides et al. 2008). While integrins lack catalytic domains traditionally associated with signaling receptors, they retain the capacity to promote signaling in a bi-directional manner by binding to a wide array of cytosolic adaptor molecules (Wegener and Campbell 2008). These transmembrane heterodimers associate with many cytoplasmic molecules to form large multicomponent signaling complexes that facilitate communication between the extracellular tissue microenvironment and the cell’s interior. Some recent estimates have suggested

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that integrin cytoplasmic domains may have the capacity to associate with over 100 different proteins, which facilitate their ability to integrate and define specific responses to numerous signaling networks (Wegener and Campbell 2008; Zaidel-Bar et al. 2007). The functional integration of integrins within growth factor signaling cascades may explain in part the rapidly expanding list of different integrins, which have been shown to regulate angiogenesis. For example, integrin such as a1b1, a2b1, a3b1, a4b1, a5b1, a6b1 as well as a6b4 have been implicated in the control of angiogenesis (Avraamides et al. 2008; Akalu et al. 2005). Experimental evidence also indicates members of the av family of integrins such as avb3, avb5, and avb8 in blood vessel formation (Avraamides et al. 2008; Akalu et al. 2005). Interestingly, early studies have linked the functional impact of integrins on angiogenesis to their interactions with insoluble ECM, resulting in enhanced endothelial cell adhesion, migration, and proliferation. However, new studies have now uncovered novel mechanisms by which integrins regulate angiogenesis by processes that are dependent on integrin binding to soluble pro- and anti-angiogenic molecules (Contois et al. 2009). Given the growing list of integrins thought to contribute to angiogenesis, it is clear that the ability of vascular cells to sense their immediate microenvironment plays important roles in new vessel development. Equally important in the contribution of integrins to angiogenesis are their respective ligands. For example, studies have shown that cellular interactions with distinct types of ECM components can alter the expression of integrins by mechanisms such as regulating mRNA stability (Feng et al. 1999; Retta et al. 2001; Delcommenne and Streuli 1995). Moreover, cellular interactions with distinct ECM components can differentially alter a cells response to a particular growth factor (Madri et al. 1988; Walker et al. 2005). In addition, certain ECM molecules have specific binding sites that allow secreted growth factors to become localized within the matrix (Schultz and Wysocki 2009; Hynes 2009). Collectively, these studies imply that different microenvironments composed of distinct ECM molecules may differentially regulate integrin expression and growth factor signaling. The composition and integrity of ECM ligands can vary widely between distinct tissue microenvironments such as those associated with embryonic development and those in the adult. Studies have indicated that embryonic vessels as well as angiogenic tumor vessels express alternatively spliced forms of fibronectin containing EIIIA and EIIIB domains, which are normally lacking in adult vessels (Astrof and Hynes 2009). Moreover, the major type of laminin within basement membranes during murine embryonic development is largely laminin-8 which contains the a4 laminin chain, while in postnatal mice, laminin-10 expression is predominate which contains the a5 laminin chain and a functionally exposed RGD avb3 binding site (Sasaki and Timpl 2001). Thus, developmentally regulated expression of ECM ligands may alter the functional significance of a given signaling pathway triggered by the particular integrin. As an example, if avb3-mediated interactions with laminin-10 contribute to the regulation of vessel development, it would not be surprising to observe only minimal impact from the lack of avb3 on embryonic vascular development since laminin-10 is not highly expressed. Conversely, in adult pathological


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angiogenesis when laminin-10 is strongly expressed along with other RGD containing provisional matrix proteins such as fibrinogen and vitronectin, blocking avb3 disrupts pathological angiogenesis (Brooks et al. 1994). It is interesting to note that during angiogenesis associated with tumors and wound healing, provisional ECM components such as fibrin and denatured collagen are often found and studies suggest that endothelial cell interactions with these ECM proteins can increase the expression of integrin avb3 by a mechanisms associated with enhanced b3 integrin mRNA stability (Feng et al. 1999; Retta et al. 2001).

Modulation of Growth Factor/Growth Factor Receptor Systems within Different Tissue Microenvironments As mentioned above, when examining receptor-mediated signaling systems it is important to have an appreciation of the mechanisms that impact expression, biodistribution, and activity of both the receptors and their ligands. Given the diversity of growth factors and cytokines thought to play roles in regulating tumor angiogenesis, it is likely that tissue-specific processes exist within a particular microenvironment that may alter the mechanisms by which growth factors are presented to cells since many growth factors can be physically bound and stored within the ECM (Clark 2008; Hutchings et al. 2003). Thus, the relative ratio and bioavailability of different growth factors alter the functional impact of these angiogenic molecules during blood vessel development. The ability of secreted growth factors to become deposited and stored within the ECM of a particular tissue microenvironment contributes to their function during angiogenesis and tumor progression (Clark 2008; Hutchings et al. 2003). In fact, studies have suggested that matrix immobilized growth factors as well as proteolytically liberated growth factors can impact angiogenic signaling pathways. Thus, a more detailed understanding of control mechanisms regulating tissue-specific expression and localization of angiogenic growth factors will likely provide important molecular insight into how these receptor/ ligand systems function during new blood vessels formation.

Integrin–ECM Interactions Regulate Growth Factor Expression and Bio-distribution As discussed previously, integrin-mediated reciprocal communication links between cells and ECM can play roles in determining the expression, bio-distribution, and activity of specific components within growth factor signaling systems (Somanath et al. 2008; Streuli and Akhtar 2009; Ivaska and Heino 2010). For example, retinalpigmented epithelial cell interaction with the matricellular protein thrombospondin-1 has been shown to increase expression of VEGF and FGF2 as compared to interactions with collagen and laminin (Mousa et al. 1999). These studies illustrate

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the differential impact that ECM composition may have on expression of angiogenic growth factors. In other studies, monocyte adhesion to fibronectin resulted in elevated levels of PDGF and IGF-1 as compared to non-adherent cells (Shaw et al. 1990; Jendraschak et al. 1998). These findings are consistent with a potential role for monocyte/macrophage infiltration into sites of hypoxia serving to modulate the relative ratios of growth factors that promote angiogenesis. In this regard, a number of studies have shown a correlation between elevated migration and invasion of macrophages into the tumor microenvironment and enhanced angiogenesis and tumor progression (Toge et al. 2009; Chung et al. 2009). In studies with carcinoma cells, experiments have suggested that the laminin receptor a6b4 may play a role in translation of VEGF by a mechanism involving regulation of e4F1 (Chang et al. 2002). These studies, as well as many others, suggest that the cellular interactions with specific ECM molecules can alter the local growth factor profile within a give tissue microenvironment. Thus, understanding the ECM composition of a particular tissue microenvironment may help direct the most efficacious therapeutic approaches to control pathological angiogenesis occurring at a given site. While it is known that organ-specific differences exist in the composition of ECM components, how these variations in the insoluble microenvironment impacts organ-specific vasculature is not completely understood. Given the striking differences in ECM composition between brain tissue and other organs, the unique brain microenvironment provides multiple examples to help illustrate how angiogenic signaling cascades might be altered by the composition of the local microenvironment. Studies suggest that glioblastoma-derived tumor endothelial cells exhibit enhanced resistance to chemotherapeutic drugs as compared to normal brain endothelial cells and this resistance may be partly due to elevated levels of survivin, which can be regulated by b1-integrin-mediated interactions with ECM (Virrey et al. 2008; Fornaro et al. 2003) In addition, targeting avb3 has been shown to enhance the anti-tumor activity of radiation in brain tumors (Mikkelsen et al. 2009). In this regard, it is interesting to note that while normal vascular development did occur in avb3-integrin null mice defects were noted in brain vasculature (Bader et al. 1998). Collectively, these studies and others provide additional evidence that the specific composition of a given tissue microenvironment may alter the response of vascular cells to angiogenesis regulatory factors. Interestingly, elevated levels of the VEGF121 have been detected in association with angiogenic glioma tumors growing intracranially, while similar findings were not observed within glioma tumors growing within subcutaneous microenvironments (Guo et al. 2001). Recent studies have also identified brain-specific growth factors, some of which have been shown to bind integrins and activated MAP kinase signaling (Staniszewska et al. 2008). In an intriguing new study, evidence was provided that activated avb3 expressed within metastatic breast tumor cells growing intracranially were associated with elevated levels of VEGF (Lorger et al. 2009). This avb3-mediated upregulation of VEGF expression was dependent on phosphorylation-mediated inactivation of 4EBP1, which can suppress translation of VEGF (Lorger et al. 2009). Remarkably, this avb3-dependent upregulation of VEGF was only observed in metastatic tumors growing intracranially and was not


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observed in similar tumors growing in the mammary fat pad, suggesting a specific microenvironmental influence. These important findings along with recent evidence, that the growth of brain tumors may depend in large part on the unique repertoire of ECM, may explain in part the enhanced efficacy for avb3 antagonists for brain tumors in animal models and in human clinical trials (Carbonell et al. 2009; MacDonald et al. 2008).

Modulation of Growth Factor Signaling by Integrins Just as cell–ECM signaling may alter growth factor expression in a tissue-specific way, so can growth factors stimulation in turn, modulate integrin and ECM expression, thereby altering the molecular composition of a particular microenvironment. While it is known that growth factors such as FGF2, IGF-1, VEGF, and TGF-b can differentially regulate expression of integrins and modulate a variety of important cellular parameters critical to angiogenesis and tumor progression, the molecular mechanisms by which they accomplish this may be quite different and depend on the particular tissue microenvironment (Somanath et al. 2008; Streuli and Akhtar 2009; Ivaska and Heino 2010). Therefore, understanding the microenvironmental context within which these signaling pathways operate may provide unique insight to optimize the development of specific drugs and therapeutic strategies. Given the ability of both integrin and ECM components within defined tissue microenvironment to modulate expression and downstream signaling activity of growth factors, it is not surprising that evidence is emerging that distinct integrin-mediated communication links modulate different angiogenic signaling pathways. One of the better-characterized examples of this concept includes the differences between VEGF and FGF in their ability to regulate endothelial cell survival during angiogenesis. Experimental observation suggested that antagonists of integrin avb3 inhibit FGF-induced angiogenesis, while exhibiting minimal impact on VEGFinduced angiogenesis (Friedlander et al. 1995). Conversely, antagonists of integrin avb5 inhibited VEGF-induced angiogenesis while showing limited impact on FGF stimulated angiogenesis. In other studies, investigators have shown distinct differences in the ability of FGF and VEGF to regulate cellular responses in endothelial cells derived from distinct microenvironments. For example, FGF2 but not VEGF stimulated reorganization of the actin cytoskeleton in bovine microvascular endothelial cells (BMEs), but similar results were not observed in bovine aortic endothelial cells (BAEs) (Cavallaro et al. 2001). Moreover, while FGF2 stimulation of BME cells resulted in a reduction in expression of the angiogenesis inhibitor TSP-1, neither VEGF nor FGF2 altered expression of TSP-1 in BAEs (Cavallaro et al. 2001). These studies, along with experiments demonstrating differential chemoresistance in brain tumor endothelial cells as compared to normal endothelial cells, provide further evidence that growth factor-stimulated responses within endothelial cells derived from different tissue microenvironments may be quite different.

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Since the initial observations concerning the differential integrin dependency for growth factor-stimulated responses, a number of studies have begun elucidating molecular mechanisms to account for these findings. Evidence suggests that c-Src plays a role in VEGF-stimulated angiogenesis in both the chick CAM and mouse models while playing minimal roles in FGF2-induced angiogenesis (Eliceiri et al. 1999). Moreover, VEGF but not FGF stimulation of endothelial cells resulted in Src-mediated phosphorylation of Fak at tyrosine 861 which facilitated FAK association with avb5 (Eliceiri et al. 2002; Ricono et al. 2009). Providing further insight into the molecular mechanisms by which distinct integrins modulate growth factor responses in endothelial cells, comes from studies showing that FGF2 stimulated, p21 activated protein kinase-1 (PAK-1)-dependent phosphorylation of Raf-1 at tyrosine residues 338 and 339, while VEGF stimulated Src-dependent phosphorylation of Raf-1 at tyrosine 340 and 341 (Hood et al. 2003; Alavi et al. 2003). This FGF-mediated phosphorylation of Raf-1 resulted in its translocation to the mitochondria independent from MEK/Erk activation and promoting resistance to apoptosis mediated by activation of the intrinsic pathway. In contrast, VEGF stimulation resulted in Src-mediated suppression of apoptosis mediated by the extrinsic pathway that depended on MEK/Erk activation (Hood et al. 2003; Alavi et al. 2003). In more recent studies, evidence suggested that c-abl may play a functional role in mediating FGF2- and avb3-dependent angiogenesis, while exhibiting little impact on VEGF/avb5-dependent angiogenesis (Yan et al. 2008). Further complicating our understanding of the biological relevance of these two integrin-dependent pathways have on angiogenesis are studies indicating that VEGF stimulation results in association of b3 integrin with the VEGFR2, thereby enhancing VEGF-mediated signaling (Mahabeleshwar et al. 2007; Borges et al. 2000). Surprisingly, in mice lacking b3 integrin, enhanced angiogenesis was observed and this enhanced angiogenesis was associated with elevated VEGF signaling (Reynolds et al. 2002, 2004). In contrast, pathological angiogenesis was significantly inhibited in b3 integrin knockin mice harboring a signaling deficient avb3 receptor (Mahabeleshwar et al. 2006). Thus, given the known complexity and compositional differences that exist between normal and tumor microenvironments, along with signaling feedback mechanisms that exist between distinct growth factor signaling cascades, the cooperative interactions between integrins and growth factors signaling pathways may provide new molecular understanding into these contrasting observations.

Integrin/Growth Factor Cooperation in Angiogenesis As discussed above, pathological angiogenesis can be organized into three general stages including an initiation phase, an invasive phase, and a maturation phase. The cooperative interactions between integrins and growth factor signaling play crucial roles in each of these overlapping steps. A growing body of evidence indicates that integrins may not only influence growth factor signaling indirectly by


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Integrin Associations with Growth Factors VEGF FGF

TGF-β α β

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Ang -1 Cyr61

Fig. 29.2 Integrin associations with growth factors. Integrin receptors are known to associate with a number of distinct types of molecules and secreted growth factors are emerging as an important class of molecules that may form functional associations with integrins. Representative examples of growth factor/integrin associations. VEGF vascular endothelial growth factor, TGF-b transforming growth factor-beta, CTGF connective tissue growth factor, Ang-1 angiopoietin-1, Cyr61 cysteine-rich protein 61, NGF nerve growth factor, PTN pleiotrophin, FGF fibroblast growth factor

modulating growth factor expression and bio-distribution, but integrins may also form functional associations with growth factors (Fig. 29.2), thereby modulating downstream signaling pathways (Mori et al. 2008; Vlahakis et al. 2007; Munger et al. 1998; Mikelis et al. 2009; Chen et al. 2001; Aidoudi et al. 2008). When considering the potential impact of cooperative interactions between integrins and growth factor signaling, it is important to have an appreciation of the particular tissue microenvironment within which these signaling cascades are occurring. During tumor angiogenesis, the local ECM may be enriched with provisional ECM proteins such as denatured collagens, fibronectin, fibrin, and vitronectin. These ECM proteins have been shown to contain functional binding sites for a number of angiogenic growth factors, thus facilitating immobilization of these pro-angiogenic molecules within the ECM (Clark 2008; Rahman et al. 2005). This ECM-mediated storage of secreted growth factors may either enhance or inhibit their activity by altering receptor recognition. Integrins have been shown to have the capacity to bind some immobilized pro-angiogenic growth factors. Integrins such as avb3 and a3b1 have been shown to bind immobilized VEGF-A, which may facilitate endothelial cell adhesion, migration, and survival within a particular microenvironment (Hutchings et al. 2003). Interestingly, antibodies directed to a9b1 were shown to inhibit VEGF but not FGF2-induced angiogenesis suggesting that the repertoire of integrins expressed within a particular tissue may differentially regulate growth factor utilization and signaling (Mori et al. 2008). In addition to VEGF, a number of other angiogenic factors have also been shown to bind integrins including FGFs, angiopoietins (Ang1), connective tissue growth

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factor (CTGF), cysteine-rich angiogenic protein 61 (Cyr61), semaphorin 7A, and nerve growth factor (NGF) (Mori et al. 2008; Vlahakis et al. 2007; Munger et al. 1998; Mikelis et al. 2009; Aidoudi et al. 2008; Rahman et al. 2005; Hutchings et al. 2003). Studies have shown that avb3 may directly bind immobilized FGF1 and enhanced cell adhesion, migration, and proliferation (Mori et al. 2008). Studies have also demonstrated that a5b1 may associate with angiogenesis regulatory factor Ang-1 and that this interaction promotes signaling leading to vascular cell migration that was not dependent on the Ang-1 receptor Tie-2 (Shim et al. 2007). Experiments continue to provide additional examples by which integrins may associate with angiogenic growth factors and influence there downstream signaling. For example, interesting new work suggests that the heparin-binding angiogenic growth factor pleiotrophin (PTN), which was shown to stimulate endothelial cell migration, may interact with integrin avb3 in a non-RGD-dependent fashion (Mikelis et al. 2009). Moreover, function-blocking antibodies directed to avb3, but not a5b1 blocked PTN-induced endothelial cell migration mediated through the PTN receptor protein tyrosine phosphatase (Mikelis et al. 2009). Interestingly, PTN treatment of glioblastoma cells that lacked avb3 resulted in decreased migration, suggesting the possibility that avb3 may differentially regulate the utilization and activity of growth factors such as PTN in distinct cell types. Finally, a well-studied group of growth factors known to play multiple roles in regulating cellular behavior, angiogenesis, and tumor growth belong to the TGF-b family. Many studies have provided convincing evidence that multiple integrins including members of the av integrin subfamily (avb1, avb3, avb5, avb6, and avb8) interact with the latent form of TGF-b1 by binding to the latency-associated peptide (LAP) within its small latency complex (SLC) (Wipff and Hinz 2008). These TGF-b complexes can associate with TGF-b binding proteins (LTBPs) to form large latent complexes that bind ECM and become integrated within insoluble tissue matrix (Wipff and Hinz 2008). Importantly, some members of this av integrin family are thought to facilitate activation of TGF-b1 by at least two general but incompletely understood mechanisms including protease-dependent and -independent pathways (Wipff and Hinz 2008). Integrin-associated activation of TGF-b is known to promote diverse cellular signaling leading to control of cellular proliferating and survival (Wipff and Hinz 2008). Interestingly, studies have indicated that avb3 can modulate and enhance the TGF-b signaling in fibroblasts and avb6dependent activation of TGF-b was shown to be associated with regulation of pulmonary inflammation and fibrosis (Scaffidi et al. 2004). Finally, a number of anti-angiogenic factors are also thought to bind integrins thereby regulating the balance of pro- and anti-angiogenic signaling activity (Petitclerc et al. 2000; Wickström et al. 2004; Woodal et al. 2008). Recent evidence suggests that the anti-angiogenic chemokine CXCL4 may bind avb3 in endothelial cells and inhibit adhesion and migration (Aidoudi et al. 2008). Furthermore, angiogenesis inhibitors derived from the ECM such as Canstatin and Tumstatin may bind b1 and b3 integrins leading to inhibition of endothelial cell proliferation and survival by altering expression of cell cycle inhibitors and apoptosis regulatory proteins (Magnon et al. 2005; Sudhakar and Boosani 2008). Taken together, evidence supports the notion that integrin


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receptors play important tissue-specific roles in integrating and coordinating the response of many cell types to angiogenic growth factors.

Integrin/Growth Factor Receptor Cooperation in Angiogenesis As can be appreciated from the discussion above, when one considers the molecular and biological impact of a given ligand/receptor signaling system, it is crucial to have an understanding of both the ligand and its cognate receptor. Thus, just as important as growth factor–integrin cooperation is in modulating angiogenesis, it is also crucial that we have a molecular understanding of the consequences of growth factor receptor interactions with integrins in controlling new blood vessel development. A steadily increasing number of studies demonstrate interactions between growth factor receptors and integrins (Fig. 29.3) (Borges et al. 2000; Mikelis et al. 2009; Scaffidi et al. 2004; Elsegood et al. 2006; Cascone et al. 2005; Lazova et al. 2009; Sahni et al. 2005; Kim et al. 2009). In recent years, a considerable amount of attention has been paid to the association of VEGFR2 with avb3. Studies have revealed that following stimulation of endothelial cells with VEGF-A, VEGFR2

Integrin Associations with Growth Factor Receptors VEGFR Tie-2

TGF-βR

α β HGFR

EGFR

FGFR

cFms

IGF-1R

Fig. 29.3 Integrin associations with growth factor receptors. In addition to the ability of integrin receptors to associate with secreted growth factors, integrins also can form functional associations with growth factor receptors. Representative examples of growth factor receptor/integrin associations. VEGFR vascular endothelial growth factor receptor, TGF-bR transforming growth factorbeta receptor, FGFR fibroblast growth factor receptor, IGF-1R insulin-like growth factor receptor-1, cFms c-MCF receptor, EGFR epidermal growth factor receptors, HGFR hepatocyte growth factor receptor, Tie-2 angiopoietin receptor

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recruits Src, which promotes the phosphorylation of the cytoplasmic tail of b3 integrin (Soldi et al. 1999). This phosphorylation event has been suggested to facilitate the formation of a complex between avb3 and VEGFR2. In turn, this functional complex may enhance downstream signaling promoting endothelial cell proliferation and survival, thereby facilitating angiogenesis (Soldi et al. 1999). While significant attention has been focused on the ability of avb3 to associate with VEGFR2, avb3 is well known to bind and associate with multiple molecules. Thus it is not surprising that avb3 has also been shown to interact with an array of other angiogenic growth factor receptors such as FGFRs, IGF-1Rs, PDGFbRs, and HGFRs (Toledo et al. 2005; Clemmons and Maile 2005; Schneller et al. 1997; Rahman et al. 2005). In a recent study, endothelial cell expression of the angiogenesis regulator nitric oxide (NO) following IL-1b stimulation was significantly enhanced in the presence of the avb3 ligand fibrinogen (Sahni et al. 2005). This elevated expression of NO was dependent on avb3, as anti-avb3 antibodies blocked this effect (Sahni et al. 2005). To this end, a stable association between avb3 and the IL-R was detected and further work suggested that the avb3/IL-1R association may play a role in regulating NO production in endothelial cells (Sahni et al. 2005). While some of these avb3-growth factor receptors interactions have been document in cell types other than endothelial cells, given the well-established contribution of tumor cells, inflammatory cells and stromal cells to blood vessel formation, it is likely that the formation of these interesting complexes in non-endothelial cells may also play tissue-specific roles in pathological angiogenesis. For example, previous studies have suggested that macrophage colony-stimulating factor (M-CSF) plays a role in regulating tumor angiogenesis in part by modulating expression of VEGF as well as recruitment of endothelial progenitor-like cells into the tumor microenvironment (Kubota et al. 2009). Moreover, recent studies have provided evidence that inhibition of M-CSF may selectively inhibit pathological angiogenesis (Kubota et al. 2009). Interestingly, recent studies suggest that M-CSF stimulation in osteoclasts resulted in a stable interaction between avb3 and the M-CSF receptor cFms (Elsegood et al. 2006). Even though the molecular mechanisms governing these associations are not fully understood, these avb3–growth factor receptor complexes likely enhance the activation of numerous signaling cascades such as the MAP/Erk and PI3k/Akt pathways, which are well known to play roles in new blood vessel formation. Finally, additional examples of growth factor receptor association with avb3 include interactions between avb3 and the pleiotrophin (PTN) receptor phosphatase as well as avb3 interactions with TGF-bRII (Mikelis et al. 2009; Scaffidi et al. 2004). Interestingly, a common occurrence in many of these growth factor receptor/integrin associations is the apparent requirement for growth factor stimulation prior to complex formation. For example, in human lung fibroblast, TGF-b1 stimulation was required to induce a functional association between avb3 and TGF-bRII (Scaffidi et al. 2004). Furthermore, VEGF stimulation was necessary to induce VEGFR2/avb3 association in endothelial cells, PTN stimulation was needed to induce association of the PTN receptor phosphatase with avb3 and finally IL-1b stimulation was required to induce an association between IL-1R and avb3 (Mikelis et al. 2009; Sahni et al. 2005; Soldi et al. 1999).


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The biological implication of avb3 association with TGF-b receptors is complex and may be cell- and or tissue-type specific. Studies have shown that TGF-b1induced association of avb3 with the TGF-bIIR within human lung fibroblasts was associated with an amplification of TGF-b1-stimulated proliferation and migration and this cellular response could be inhibited by blocking avb3 (Scaffidi et al. 2004). These findings are surprising given that many reports implicate TGF-b signaling in inhibiting cellular proliferation and promoting differentiation in normal cell types (Wipff and Hinz 2008). In contrast, several studies have suggested that TGF-b may enhance proliferation, migration, invasion, and metastasis in transformed cells (Wipff and Hinz 2008). In this regard, it is well known that the repertoire and activation state of integrins can be significantly modified in tumor cells as compared to that observed in normal cells and thus it would be interesting to speculate that the particular ECM microenvironment and integrin repertoire may contribute to the differential response of cells to TGF-b stimulation. Interestingly, recent studies have suggested that mice deficient in b3 integrin exhibited elevated levels of TGF-b1, TGF-bIR, and TGF-bIIRs (Reynolds et al. 2005). Moreover, b3 integrin knockout fibroblasts derived from these mice exhibited elevated levels of TGF-b1 signaling, enhanced SMAD2 phosphorylation, but interestingly, reduced phosphorylation of SMAD3 and reduced localization of SMAD3 to the nucleus (Reynolds et al. 2005). Importantly, wound re-epithelialization within these b3-deficient mice was significantly enhanced (Reynolds et al. 2005). This enhanced re-epithelialization response was associated with elevated levels of fibronectin and vitronectin within the wound. Finally, increased fibroblast infiltration into the wound as compared to wildtype mice was also observed. These interesting findings suggest functional role for b3 containing integrins in regulating the response of cells to TGF-b in vitro and in vivo. Other studies also suggest that b1-containing integrins including a3b1 may associate with TGF-b1 receptor in a complex with E-cadherin expressed in epithelial cells and this complex may alter the response of epithelial cells to TGF-b1 by a unique b-catenin/SMAD signaling pathway (Kim et al. 2009). Taken together, with previous work demonstrating a role for integrins in activating TGF-b, along with other studies are consistent with the notion that the particular tissue microenvironment and integrin expression profile of cells may contribute to the differential response of cells to TGF-b. In addition to avb3, a number of b1 containing integrins have also been shown to physically associate with growth factor receptors. The fibronectin receptor a5b1 has been shown to interact with the Tie-2 receptor thereby altering Ang-1 signaling (Cascone et al. 2005). These studies indicate the Tie-2/a5b1 association may play a role in reducing the levels of Ang-1 needed to stimulate Tie-2 phosphorylation thereby modulating endothelial cell response to this angiogenic growth factor (Cascone et al. 2005). Moreover, studies have also shown that members of the b1integrin family may associate with the EGFR and promote PI3 kinase signaling, which is thought to play an important role in regulating endothelial cell survival and angiogenesis (Falcioni et al. 1997). Interestingly, the roles of b1 integrins in controlling angiogenesis may depend in part on the particular integrin heterodimer and the ECM ligands present within

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the specific tissue microenvironment. For example, while the a1b1 and a2b1 integrins have been implicated in regulating angiogenesis and antibodies directed to these collagen binding integrins inhibit VEGF-induced angiogenesis, studies have suggested that collagen binding mediated specifically through a1b1 can limit EGF-stimulated proliferation by activating the protein tyrosine phosphates TCPTP, which in turn lead to inhibition of EGF-induced EGFR signaling by de-phosphorylation (Mattila et al. 2004). In the case of endothelial cells, studies have indicated that collagen-I-mediated endothelial cell adhesion caused a reduction of VEGFR signaling as a result of recruitment of SHP2 and de-phosphorylation of VEGFR (Mittola et al. 2006). Moreover, recent studies have also shown that a1b1 binding to intact collagen, but not denatured collagen can also activate TCPTP resulting in reduction of VEGFR signaling, inhibition of endothelial cell proliferation, migration, and endothelial cell sprouting (Mattila et al. 2008). These studies provide convincing evidence that integrins can have either a positive or negative impact on growth factor receptor signaling and that these changes can further depend on the particular integrin and ECM ligands expressed within the specific tissue microenvironment.

Conclusions A growing body of evidence indicates that pathological angiogenesis involves highly complex sets of cellular, biochemical, molecular, and mechanical events that are controlled by an interconnected network of signaling systems that depend in large part on the particular tissue microenvironment within which angiogenesis is occurring. Thus, it is becoming increasingly clear that the development of more effective strategies to control pathological angiogenesis and tumor progression will depend on a more in-depth molecular understanding of how a particular tissue microenvironment modulates the integrated network of angiogenic signaling pathways. As can be appreciated from the discussion above, growth factor signaling networks present within a particular tissue microenvironment need to be sensed, integrated, and responded to in a meaningful way in order for functional blood vessels to form. To this end, integrin receptors are emerging as a central group of molecules that play critical roles in modifying and integrating these diverse signaling inputs from a variety of angiogenic growth factors. Thus, from a network systems perspective, integrins may serve as network hubs, by allowing tissue-specific detection, assimilation, and distribution of these signaling cues within the local microenvironment to allow angiogenesis to proceed in an effective manner. With the appreciation of the ability of integrins to modulate angiogenic growth factor signaling at multiple levels, a crucial challenge in the coming years will be to decipher how to exploit this new molecular understanding of the cooperative interactions of integrins and growth factor signaling networks to improve the efficacy of anti-angiogenic strategies for the treatment of human diseases.


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Acknowledgments This work was supported in part by grant 2ROICA91645 to PCB and grant P20RR15555 to Robert Friesel and subproject to PCB. We would like to apologize to all those investigators whose important work was not discussed due to space limitations.

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Chapter 30

The Extracellular Matrix and the Growth and Survival of Tumors Yves A. DeClerck

Abstract The role of the extracellular matrix (ECM) in the tumor microenvironment extends well beyond the formation of a barrier against tumor invasion. Through a combination of physical forces and chemical signals generated upon contact between cells and ECM proteins, the ECM exerts a control on the proliferation and survival of cancer cells, which were previously considered to be solely affected by genetic alterations. The interaction between tumor cells and the ECM is also a dynamic one that changes upon modification of the ECM by proteases produced by tumor cells and by normal cells in the tumor microenvironment. These proteases modify the ECM generating cryptic epitopes, producing proteolytic fragments that are biologically active and releasing growth factors and cytokines that are trapped in the ECM. Our knowledge of the mechanisms by which the ECM controls cancer cells has significantly improved over the last 10 years and is now leading toward clinical trials testing agents disrupting tumor cell–ECM interaction.

Introduction Although able to proliferate and survive in the absence of exogenous signals, cancer cells remain deeply influenced by extracellular stimuli. The proliferation and survival of tumor cells is influenced by a large variety of external factors of a physical (interstitial pressure, mechanical forces) and chemical (acidosis, hypoxia) nature, as well as by cell–cell contact, soluble factors like growth factors, chemokines and cytokines, and by contact with the extracellular matrix (ECM). The ECM is an integral component of the tumor microenvironment that can affect cancer at multiple stages of progression, from initiation to metastasis. The importance of the interaction Y.A. DeClerck (*) Departments of Pediatrics and Biochemistry and Molecular Biology, University of Southern California and The Saban Research Institute of Childrens Hospital Los Angeles, Los Angeles, CA 20027, USA e-mail: [email protected] R.G. Bagley (ed.), The Tumor Microenvironment, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-6615-5_30, © Springer Science+Business Media, LLC 2010

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between the ECM and tumor cells became clear in the late 1990s when the laboratory of M. Bissell made the seminal observation that when grown in three-dimensional (3D) laminin-rich basement membrane matrices, malignant breast epithelial cells reverse to a normal polarized epithelial phenotype in the presence of integrin blocking antibodies (Weaver et al. 1997). The mechanisms by which the ECM affects cancer progression are highly complex and involve physical forces and biochemical signals, based on protein–protein interactions. Contact between the ECM and tumor cells can either inhibit or promote cancer progression. Importantly, this contact is a dynamic one that substantially changes during cancer progression. As cancer cells invade surrounding tissues and enter in contact with different ECM proteins, they modify the ECM by the action of a large variety of proteases that they produce or whose expression they stimulate in non-malignant stromal and inflammatory cells. This proteolytic modification of the ECM releases proteins that are trapped in the ECM and generates proteolytic fragments of ECM proteins that are biologically active and exert diverse functions on cancer progression and angiogenesis. In this chapter, we will focus on reviewing the mechanisms by which contact between tumor cells and ECM proteins and proteolytic modification of the ECM affect the proliferation and survival of tumor cells and influence angiogenesis.

Mechanical Forces and the ECM in Cancer Progression The stiffness of a tumor has long been recognized by clinicians as a sign of malignancy, and the presence of a desmoplastic stroma in a tumor is typically an indicator of a more aggressive behavior (Paszek and Weaver 2004). In breast cancer, a stiffening of the tissue surrounding the tumor is caused by a response of normal fibroblasts to the presence of invasive malignant epithelial cells and is known as the desmoplastic response. Fibroblasts not only increase their production and deposition of ECM proteins like fibronectin, tenascin, and collagen types I, III and IV but also increase collagen cross-linking by overproducing the extracellular enzyme lysyl oxidase (LOX) that catalyses covalent intermolecular cross-linking between collagen molecules and with elastin (Payne et al. 2007). This increases collagen insolubility and ECM stiffness (Payne et al. 2005). A rigid stroma generates three types of physical stress on cells: compressive stress applied by forces perpendicular to the cell surface and resulting in cell compaction, tensile stress applied by perpendicular forces exerting a traction on cells, and shear stress due to forces that are applied parallel to the cell surface. These forces are all sensed by cells via mechanoreceptors (Butcher et al. 2009). On the basis of Newton’s third law of action and reaction, cells respond to these external forces by adjusting intracellular tensions through the network of cytoskeletal proteins, a process called “mechanoreciprocity” (Lopez et al. 2008). Under normal conditions, this mechanoreciprocity maintains a tensional homeostasis in the cells and tissue that prevents an increase in internal forces. However in cancer cells this mechanoreciprocity is often lost, and consequently integrins become activated and cluster. This clustering causes activation of the focal adhesion kinase (FAK)-125, a-actinin tyrosine phosphorylation and interaction

30 The Extracellular Matrix and the Growth and Survival of Tumors

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with FAK and Src, and stimulation of Rho-GTPases (Craig et al. 2007). Consistent with this concept, it has been demonstrated that when normal epithelial cells are maintained in the presence of a rigid matrix, there is an increase in integrin activation, enhancement of Erk activation, and ROCK-dependent contractility that promote malignant transformation (Paszek and Weaver 2004). A rigid matrix can thus promote malignant transformation. Cell stretching contributes to this phenomenon as it is an important stimulator of cell proliferation. For example, when forced to spread by changing the space between multiple points of focal adhesions, cells proliferate and escape apoptosis regardless of the type of ECM they are in contact with (Chen et al. 1997). For the same level of matrix rigidity, tumor cells stretch much more than normal cells and thus activate integrin-mediated Rho signaling much more effectively. The consequences of Rho-GTPase activation are an increased contractility in tumor cells associated with increased migratory behavior and proliferation via activation of Erk1/2. At the same time, Rho-GTPase activation increases matrix stiffness, providing a positive feedback signal that further activates integrin-mediated signaling (Huang et al. 1998). Forces generated when cells are in contact with the ECM also affect gene expression. For example, the expression of integrin is much higher in fibroblasts and epithelial cells that are in contact with a rigid matrix and signaling pathways like Erk and JNK become hyperactivated. Consequently, transcription factors like AP-1, STATs, MYC, CEBP and NFkB translocate to the nucleus, and their transcriptional activity is increased (Avvisato et al. 2007). A rigid ECM can also affect cancer cell proliferation by having a direct mechanical effect on the cell nucleus. This nuclear mechanotransduction acts like an epigenetic determinant by distorting the shape of the nucleus, altering the structure of the chromatin and affecting gene expression (Dahl et al. 2008). The exact mechanism linking deformation of the nuclear cytoskeleton and gene expression is still poorly understood, but it involves cadherin and integrin and cytoskeletal protein like actin (Gieni and Hendzel 2008). Proteases are also upregulated by stress forces that apply to cells. As a result of their overproduction, the ECM is degraded and its stiffness negatively affected. By decreasing the stiffness of the ECM, ECM degrading proteases may therefore have a negative effect on cancer cell proliferation and provide a negative feedback loop that limits the effect of tissue stiffness on tumor cells. These fundamental concepts on the role of mechanical forces in tumor cell growth have recently reached clinical application. It has long been well recognized that the presence of increased tissue densities in mammographic images of the breast is associated with a significant increase in the risk of developing a breast tumor (Wolfe 1976), an observation that is consistent with the concept that increased tissue stiffness promotes malignant transformation and progression (Tlsty and Coussens 2006). Loss of elastic properties in a tissue can now be accurately measured by elastography, using non-invasive ultrasound and magnetic resonance-based imaging techniques. Preliminary studies aimed at testing the use of such techniques to differentiate a benign tumor from a malignant tumor in prostate and breast cancer have been encouraging but studies on large cohorts will be needed to fully evaluate their potential (Garra 2007; Linden and Halpern 2007; Siegmann et al. 2009; Tse et al. 2009).

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Contact Between ECM and Tumor Cells Regulates Proliferation and Survival Although the ability to proliferate in the absence of exogenous stimulatory signals is a well recognized hallmark of cancer cells, cancer cells remain sensitive to stimuli from ECM proteins. Not every ECM protein however exerts a function on cell proliferation and survival. To distinguish between ECM proteins that have an effect on these cell functions from those that do not, the term matricellular proteins has been proposed (Bornstein and Sage 2002). These matricellular proteins can stimulate or inhibit cell proliferation and can have a pro-survival as well as a proapoptotic effect on cancer cells (Table 30.1) (Reddig and Juliano 2005).

Regulation of Tumor Cell Proliferation by ECM Proteins Most ECM proteins have a growth stimulatory activity on cells, including tumor cells. This growth stimulatory activity is typically mediated by integrins present at the cell surface. The contact between tumor cells and ECM proteins promotes the clustering and activation of integrins at points of focal adhesion. As a result, p130CAS is activated and phosphorylates FAK at Tyr397. Activated FAK recruits the Src family of kinases and activates phosphoinositol-3 kinase (PI3-K). Src also activates p130CAS, which in turn activates Rac, which can also be directly activated by FAK. This ultimately results in activation of paxillin and extracellular Table 30.1 Matricellular proteins Protein Domain/structure Fibronectin

Function Increases survival and proliferation

Mechanism b1 integrin medicated, stimulates Erk1/2, ROCK and PI3-K and Akt

Laminin

Domain 10/11

Increases survival and proliferation

PI3-K and Akt

Collagen type I

Non-fibrillar

Stimulates proliferation

a1 or a3b1 integrin, FAK and Erk1/2

Fibrillar

Inhibits proliferation

DDR-2, increases p27KIP-1, p21CIP-1 and p15INK4B

CCN1

Stimulates apoptosis in EC

a6b1 integrin and syndecan, activates Bax and caspase-3 and -9

TSP-1

Stimulates apoptosis in EC

Binds to CD 36, activates p59fyn and p38MAPK

SPARC

Stimulates apoptosis

Unknown

Decorin

Stimulates apoptosis

Caspase-3 activation

EMILIN2

Stimulates apoptosis in tumor cells

DR4 and DR5 activation and caspase-8 activation


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signal-related kinase (MEK) and Erk that upregulates cell cycle-dependent kinases (CDK) and downregulates inhibitors of CDKs like p21CIP-1and p27KIP-1(Aplin et al. 1999; Aplin and Juliano 1999). This is the pathway by which fibronectin exerts its well known growth stimulatory effect on cancer cells. Binding of epithelial cells to fibronectin via a5b1 integrin inhibits the expression of p21CIP-1and PTEN, activates Erk1/2, Rho kinase and PI3-K-protein kinase B (PkB/Akt) and increases the expression of c-MYC and cyclin D1 (Han and Roman 2006). Another ECM protein with a well known growth stimulatory activity is laminin, a major component of the basement membrane (Kim et al. 1999). Contact between tumor cells and collagen type I can also stimulate proliferation, however the effect is a function of the native state of the collagen. Collagen type I is typically present in tissues in an insoluble multimeric fibrillar form. In this form it inhibits the proliferation of normal and malignant cells (Henriet et al. 2000; Koyama et al. 1996). This growth inhibitory effect is associated with an upregulation of the expression of p27KIP-1, p21CIP-1, and p15INK-4. p21CIP-1is responsible for blocking of cells in the G1/G0 and p15INK-4for blocking cells at the G2 phase of the cell cycle (Wall et al. 2007). In contrast to growth stimulation, which is mediated by integrin, growth inhibition is mediated by the discoidin domain receptor-2 (DDR-2), a tyrosine kinase cell surface receptor that binds to fibrillar collagen and not to denatured collagen (Wall et al. 2005). When partially denatured (gelatin), monofibrillar collagen, like fibronectin, exerts a growth stimulatory activity by promoting cell spreading, integrin clustering and activation, formation of focal adhesions, actin cytoskeleton organization, and FAK phosphorylation (Hotary et al. 2003). The effect that collagen type I exerts on cell proliferation is therefore complex and highly dependent of its native state. When highly cross-linked with other collagen molecules and elastin though the action of LOX, it exerts a tensile stress on cells that stimulates proliferation. When present in a fibrillar multimeric structure, it inhibits proliferation and when partially denatured it again promotes cell growth. A second mechanism by which integrin activation promotes proliferation involves a crosstalk with growth factor receptors tyrosine kinase (RTKs) (Juliano 1996). Binding of integrin to the ECM enhances the activity of soluble growth factors and their affinity for RTKs (Guo and Giancotti 2004; Larsen et al. 2006), which provides an amplification system where insoluble ECM proteins and soluble growth factors interacting with their corresponding receptors (integrins and RTKs) cooperate to stimulate cell proliferation.

Regulation of Apoptosis by ECM Proteins In the absence of contact with the ECM, most normal cells undergo apoptosis (anoikis). A hallmark of malignant cells is their ability to escape apoptosis, which is commonly achieved by genetic alterations that change the balance between survival and death signals in favor of survival signals such as the overexpression of Bcl-2 associated with 8q24:14q32 translocation in Burkitt’s lymphoma, loss of p53 function

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associated with a majority of human cancers or loss of PTEN function in glioma (Hanahan and Weinberg 2000). However, tumor cells remain sensitive to mechanisms of survival that are triggered by contact with the ECM. Central to this mechanism is PI3-K, which is activated by integrins and integrin-linked kinase (ILK), and generates 3¢ phosphorylated inositides that promote the recruitment of PkB/Akt to the cell membrane and its activation by phosphorylation at Thr 308 by phosphoinositide-dependent kinase 1 (PDK1). As a result, a series of survival pathways become activated that inhibit caspase-9, decrease Bad, and increase the expression of survival proteins like survivin (Reddig and Juliano 2005). A second pathway is via FAK, activated by integrin clustering upon contact with ECM proteins. FAK activates p130CAS, an inhibitor of Bax and interacts with the receptor-interacting protein (RIP), a death domain containing serine/threonine kinase that is a major component of the death receptor complex (Kurenova et al. 2004). Through the adaptor proteins Grb2 and son of sevenless (SOS), FAK also activates the Ras/ Raf-1/MEK1/Erk1/2 pathway that not only stimulates proliferation but also negatively regulates Bim, by promoting its degradation after phosphorylation, thus preventing Bim from antagonizing Bcl-2 (Reginato et al. 2003). Integrin-mediated cell adhesion also protects cells from the extrinsic apoptotic pathway and caspase-8 activation by inhibiting the production of the death-associated protein-3 (DAP-3), that binds to Fas-associated death domain (FADD) and promotes caspase-8 activation (Miyazaki et al. 2004). Integrin-mediated contact between tumor cells and ECM proteins like fibronectin, laminin and vitronectin, protects tumor cells from stress-induced apoptosis (Gu et al. 2002; Trikha et al. 2002), including drug-induced apoptosis, providing therefore a reversible mechanism of acquired chemoresistance (Zutter 2007). There are however a few noticeable exceptions that are discussed in the next section.

Stimulation of Apoptosis by ECM Proteins A few ECM proteins are known to stimulate rather than inhibit apoptosis. Although their function has been primarily studied in normal cells, there is evidence that they could also induce apoptosis in malignant cells (Marastoni et al. 2008). Among these proteins is a secreted ECM-associated heparin-binding protein CCN1. The pro-apoptotic function of this protein is mediated by a6b1 and syndecan-4, an ECM heparan-sulfate proteoglycan. It culminates with activation of Bax which induces cytochrome C release from the mitochondria and activation of caspase-9 and -3. In prostate cancer cells it has a dual function, promoting proliferation and enhancing TRAIL-mediated apoptosis (Franzen et al. 2009). Thrombospondin-1 (TSP-1) is a secreted glycoprotein that interacts with its receptor CD36 present on endothelial cells. This results in association of CD36 with the Src-family tyrosine kinase protein p59fyn and activation of p38MAPK. In endothelial cells, this mechanism is responsible for the antiangiogenic activity of TSP-1 (Armstrong and Bornstein 2003). The effect of


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TSP-1 on tumor cells remains controversial with stimulatory and inhibitory effects that can be in part explained by conformational changes induced by proteases (Sid et al. 2004). The secreted protein acidic and rich in cysteine (SPARC) stimulates apoptosis in pancreatic tumor cells and inhibits growth (Puolakkainen et al. 2004). Decorin has a similar effect on other cancer cells (Goldoni and Iozzo 2008). A different mechanism of apoptosis is triggered by EMILIN-2, a member of the EMILIN family of ECM proteins. This protein activates the extrinsic pathway of apoptosis through direct binding to the death receptor (DR) 4 and 5 (although to a lower extent) present at the cell surface. This results in receptor clustering, death-induced signal complex (DISC) and caspase-8 activation (Mongiat et al. 2007). This ECM protein thus mimics the activity of a soluble death receptor ligand. Interestingly, in the particular case of EMILIN2, non-malignant cells like fibroblasts do not seem to be sensitive to this pathway of ECM-induced apoptosis, whereas it is active in several cancer cell lines.

Proteolytic Modification of the ECM and Tumor Growth and Survival Proteolytic Modification of the ECM and Cancer Progression During the different stages of cancer progression, the ECM is actively modified by proteases either produced by cancer cells or secreted in the tumor microenvironment by stromal and inflammatory cells. Matrix metalloproteinases (MMPs), serine proteases, cysteine proteases and aspartic proteases, as well as heparanase (an enzyme degrading proteoglycans) all have been shown to contribute to the proteolytic modification and degradation of the ECM (Lopez-Otin and Matrisian 2007). It was initially thought that the primary role of these proteases was to permit the invasion of tumor cells through the basement membrane and underlying connective tissues, a key hallmark of malignancy and a critical step in metastasis. In fact, whether the degradation of the ECM is always a necessary step for invasion and metastasis of cancer cells has been recently questioned as it became clear that cancer cells can adopt amoeboid movements that do not require proteases and ECM degradation to migrate through the ECM (Wolf et al. 2003). It is now clear that ECM degrading proteases do much more than allow tumor cells to invade. The proteolytic cleavage of ECM proteins has multiple effects on the tumor microenvironment. By altering the structure of proteins, these enzymes can make apparent new functional domains that were cryptic. They release cleaved peptides that are biologically active and finally, by dissolving the ECM, they liberate growth factors and cytokines in soluble forms increasing their biological activity. It is therefore more correct to refer to proteolytic modification rather than degradation of the ECM by proteases.

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Proteolytic Modification of the ECM Reveals Cryptic Domains in ECM Proteins (Matricryptins) ECM degrading proteases cleave ECM proteins at specific cleavage sites. In the majority of the cases, this initial proteolytic cleavage denatures the ECM protein and promotes its degradation by other less specific proteases. However in some cases, the cleavage generates a partially denatured protein in which a cryptic domain becomes exposed. This domain can exert a new function. Proteins having such cryptic domains that become active upon proteolytic cleavage are designated matricryptins, a term that underlines their biological activity on cell proliferation, migration, and differentiation. They are recognized by specific integrins expressed by cells. Among those, the integrin avb3, an integrin expressed by angiogenic endothelial cells (EC) and melanoma cells, has been well described for its ability to recognize, in addition to many other proteins, matricryptins that are unfolded upon denaturation or degradation of collagen type IV (Xu et al. 2001). Other matricryptins present in collagen, fibronectin or laminin, and exposed upon partial proteolysis of these ECM proteins interact with growth factor receptors and stimulate cell proliferation (Tran et al. 2005; Watanabe et al. 2000).

Proteolytic Modification of the ECM Releases Soluble Active Peptides (Matrikines) Among peptides that are generated from the proteolytic cleavage of precursor ECM proteins and have a biological activity is a family of anti-angiogenic peptides (Table 30.2). Most of these inhibitors have only an indirect effect on tumor cell growth by their anti-angiogenesis activity. The concept that proteolytic degradation of proteins by proteases like MMPs can generate biologically active peptides came from an original observation made in J. Folkman’s laboratory in the early 1990s. His laboratory had observed that in Lewis Lung carcinoma-bearing mice, the development of lung metastasis was dramatically accelerated upon surgical excision of the primary tumor. In an attempt to understand this process, the laboratory identified in the serum of these mice a factor produced by the primary tumor, that inhibited angiogenesis. This factor was later purified and identified as a 38-kDa protein called angiostatin (O’Reilly et al. 1994). It was then determined that angiostatin was generated by the cleavage of plasminogen by MMPs like MMP-2, and was a potent inhibitor of EC proliferation (Cao et al. 1996; O’Reilly et al. 1999). Since this initial discovery, many other peptides with a similar activity have been identified, all fragments of ECM precursor molecules, mainly collagen (Mundel and Kalluri 2007). Whereas the proteases generating some of these peptides are known, others are still unknown. Endostatin is a 20-kDa C-terminal fragment of collagen type XVIII that enhances apoptosis in EC by suppressing apoptosis inhibitors like Bcl-2. It is generated by cleavage by elastase (Wen et al. 1999). It binds to the integrin a5 and av

30 The Extracellular Matrix and the Growth and Survival of Tumors Table 30.2 Anti-angiogenic matrikines Name Mr (kDa) ECM source Angiostatin 38 Plasminogen Endostatin

20

Neostatin 7

28

Neostatin 14

23

Arresten

26

Canstatin

24

Tumstatin

28

TSP1 and 2 Neostatin

110 and 36 50

Collagen type XVIII NC domain Collagen type XVIII NC domain Collagen type XVIII NC domain Collagen type IV a1 NC domain Collagen type IV a2 NC domain Collagen type IV a3 NC domain Thrombospondin 1 & 2 Fibulin 1

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Cleavage MMP-2, MMP-9 MMP-12 Elastase MMP-7 MMP-14 Not reported Not reported Not reported ADAMS-TS Cathepsin D

on the surface of human EC (Rehn et al. 2001). Neostatin 7 (28 kDa) and 14 (23 kDa) are two other anti-angiogenic factors derived from the cleavage of collagen type XVIII by MMP-7 and MMP-14, respectively (Chang et al. 2005; Kojima et al. 2008). They inhibit EC proliferation but their mechanism of action has not been fully investigated. Arresten is a 26 kDa proteolytic fragment of the non-collagenous (NC1) N-terminal domain of a1 collagen type IV by MMPs (Colorado et al. 2000). It inhibits the proliferation of basic fibroblast growth factor (bFGF)-stimulated endothelial cells and induces apoptosis (Nyberg et al. 2008). It also inhibits EC migration and tube formation in Matrigel. In vivo it inhibits the growth of xenotransplanted tumor cells and their metastasis. Its activity is mediated by binding to b1 integrin and inhibition of Erk signaling. It has not been shown to have a direct inhibitory effect on tumor cells. Canstatin is a 24-kDa proteolytic fragment of the NC1 domain of a2 chain of collagen type IV that binds avb3 and avb5 integrins and induces apoptosis in EC by inhibiting the phosphorylation of PkB/Akt and the activation of FAK (Magnon et al. 2005). It also induces FasLmediated apoptosis and activation of caspase-8 and -9 cleavage. It does not directly inhibit tumor cell growth (He et al. 2003; Kamphaus et al. 2000). Tumstatin is a 28-kDa proteolytic fragment of the a3 chain of collagen type IV that binds to integrin avb3 and a6b1 and inhibits activation of FAK, PI3-K, PkB/Akt, and a mammalian target of rapamycin (mTOR), in an EC-specific manner (Maeshima et al. 2002). Whereas arresten and canstatin have no direct effect on tumor cells, tumstatin has a direct inhibitory effect on tumor cells and inhibits their growth in a PTEN and PkB/Akt-mediated mechanism (Kawaguchi et al. 2006). The fact that several of these angiogenesis inhibitors are generated by the activity of MMPs on ECM proteins in part explains the failure of MMP inhibitors in clinical trials. In some of these trials, a paradoxical increase in tumor growth in patients

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treated with MMP inhibitors was reported. It later became apparent that these inhibitors have an undesirable and unanticipated effect by decreasing the production of MMP-generated anti-angiogenic peptides from their precursor molecules (Coussens et al. 2002; Houghton et al. 2006). Experiments in mice confirmed that this was in fact the case and investigators demonstrated that inhibition of MMP-9 in tumor-bearing animals increased tumor growth and vascularization and decreased the levels of angiostatin in the serum (Pozzi et al. 2002). The production of antiangiogenic peptides from precursor molecules is however not limited to the activity of MMPs. Cathepsin D digestion of fibulin-1 produces a fragment (also designated Neostatin) with nearly the same molecular weight as fibulin-5 that inhibits EC proliferation (Xie et al. 2008). TSP1 and 2 are also substrates for ADAMTS1. The cleavage of TSP by ADAMTS1 releases polypeptides from the trimeric structure of both TSP1 and 2, generating a pool of anti-angiogenic fragments from matrixbound thrombospondin (Iruela-Arispe et al. 1999; Lee et al. 2006). It is clear that the list of peptides released by the proteolytic remodeling of ECM proteins will continue to increase. Whereas many of those peptides have an anti-angiogenic activity by directly binding to surface receptors on EC (mainly integrins), some have a direct inhibitory activity on tumor cells.

Proteolytic Degradation of the ECM Releases Soluble Growth Factors Many growth factors share in their structure the presence of heparin binding domains and other motifs that mediate their binding to components of the ECM. This property explains that the ECM is an important reservoir of growth factors and cytokines. By doing so, the ECM increases their stability and limits their bioavailability. Upon proteolytic degradation of the ECM, these factors are released in soluble forms and can exert their biological function much more efficiently. This process plays an important role, particularly in bone metastasis and angiogenesis. When arrested in the bone marrow, tumor cells secrete a series of hormones and growth factors that activate pathways that alter the homeostatic balance between the formation of new bone by osteoblasts and the degradation of the bone matrix by osteoclasts (Guise 2000; Roodman 2004). This is mediated by the production of a large variety of stimulatory molecules like parathormone related peptide (PTHrP) that stimulates the production of the receptor activator of NFkB ligand (RANKL) by osteoblasts. By binding to RANK present on the surface of osteoclast precursor cells, RANKL activates their maturation and osteolytic activity. Alternatively, tumor cells secrete osteoclast activating factors like interleukin-1 and 6, granulocytic monocyte colony stimulating factor (GMCSF), and macrophage inflammatory protein (MIP)-1a. Activated osteoclasts demineralize the bone and degrade collagen type I through the combined activities of proteases like MMP-7, 9 and cathepsin K. The degradation of the bone matrix releases growth factors including insulin-like growth factor 1 (IGF-1) and transforming growth factor-b (TGF-b) that


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directly stimulate the proliferation of tumor cells and further increase the production of RANKL by osteoblasts and the production of PTHrP by tumor cells (Guise and Chirgwin 2003; Yin et al. 1999). This process further fuels bone degradation and is known as the vicious circle of bone metastasis. Degradation of the ECM by proteases like MMP-9 has also been shown to release vascular endothelial growth factor (VEGF) and thus to stimulate angiogenesis. This mechanism explains the critical role of MMP-9 in initiating the angiogenic switch in transgenic mouse models of pancreatic cancers and squamous cell carcinoma (Bergers et al. 2000; Coussens et al. 2000). The degradation of glycosaminoglycans by heparanase is another mechanism releasing many growth factors like VEGF in the microenvironment that is responsible for the pro-tumorigenic and pro-angiogenic activities of heparanase (Vlodavsky et al. 2007).

Clinical Implications As we increasingly appreciate the importance of the interactions between tumor cells and the ECM and how they regulate the proliferation and survival of cancer cells, the possibility to target these interactions in the treatment of cancer has been considered and some agents are currently tested in clinical trials.

Broad Inhibitors of ECM Degradation Blocking the degradation of the bone matrix by tumor-activated osteoclasts has been among the first attempts to affect the interaction between tumor cells and the ECM. It can be effectively achieved by bisphosphonates, a group of phosphoric acid-based molecules that tightly bind to the bone matrix and are potent stimulators of apoptosis in osteoclasts and tumor cells residing in the bone. Several of these bisphosphonates, like ibandronate and zoledronic acid, have been successfully tested in prostate and breast cancer metastatic to the bone. Their use has been a powerful strategy to interrupt the vicious circle of osteolysis in patients with cancer metastasis to the bone (Clezardin 2002; Coleman 2004; Lipton 2003) and they have been successfully tested in myeloma and breast and prostate cancer metastasis (Michaelson and Smith 2005).

Integrin Inhibitors Because integrin plays such a central role in mediating the interactions between tumor cells and the ECM, and in particular by protecting tumor cells from apoptosis, inhibitors of integrin have been first considered as potential anti-cancer agents.

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A small RGD peptide inhibitor of avb3 and avb5 integrin labeled Cilengitide has been developed, and is currently tested in patients, including children, with brain tumors (MacDonald et al. 2008; Reardon et al. 2008). It has shown little toxicity but the results of the clinical trials have not been entirely conclusive. One reason is that, at low concentration, this inhibitor is pro-angiogenic and stimulates tumor cell proliferation (Reynolds et al. 2009). A monoclonal antibody against a5b1 integrin (Volociximab) has been developed as an anti-angiogenic agent and has been shown to be well tolerated in patients with solid tumors (Ricart et al. 2008). It is presently too early to determine the potential value of these inhibitors in the clinic and more testing will be needed.

Protease Inhibitors In the late 1990s, several small molecule inhibitors of MMPs were tested in clinical trials in patients with a variety of cancers. The rationale behind their use was based on their anti-invasive and anti-metastatic activity, as well as their anti-angiogenic and growth inhibitory activities well demonstrated in pre-clinical models. The results of these trials were however disappointing, to the point that by 2001 all trials were abandoned (Coussens et al. 2002). One of the reasons for this has already been discussed and is related to the fact that inhibition of MMP activity also inhibits the release of anti-angiogenic matrikines like endostatin or angiostatin. There are however other reasons for these disappointing results. Most of the inhibitors tested had a broad spectrum of activity and were thus inhibiting most MMPs. Clinical studies were done without a complete understanding of the complex role of MMPs and of their many substrates, and a lack of realization that some MMPs have an anti-tumor activity. Studies did not include the monitoring of their effect on proteolytic activity and unanticipated side effects in the form of an arthritis-like syndrome requiring interruption of the treatment were observed in almost 30% of the cases. As we understand better the complex action of MMPs and the large number of proteins they cleave, the use of MMP inhibitors in cancer clinical trials may be reconsidered (Overall and Kleifeld 2006).

Angiogenesis Inhibitors Several native angiogenic inhibitors like endostatin and angiostatin made as recombinant proteins are currently in clinical phase I trials. Recombinant human angiostatin in combination with paclitaxel and carboplatin has been tested in patients with advanced non-small cell lung cancer and an overall response rate of 39% was reported (Kurup et al. 2006). Recombinant human endostatin (Endosteal) was also tested with or without interferon-a2 in metastatic melanoma but failed to show any activity (Moschos et al. 2007). Although well tolerated, these inhibitors have however not yet shown clinical efficacy (Kulke et al. 2006).


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Conclusion It is now clear that the ECM is not an innocent bystander in the tumor microenvironment, and that its role is not limited to providing a barrier against the expansion of tumor cells. Through a combination of physical and chemical signals, the ECM controls essential cellular functions like growth, survival and angiogenesis, and therefore controls and can even reverse the malignant behavior of tumor cells. Our understanding of the mechanism by which the ECM controls tumor cell proliferation and apoptosis has significantly improved over the last decade, to a point that such knowledge is now leading to the identification of molecules interfering with tumor cell–ECM interaction or ECM-derived peptides that could have a therapeutic value.

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Chapter 31

Secreted Growth Factors as Therapeutic Targets Beverly A. Teicher

Abstract Secreted growth factors directed from malignant cell to malignant cell, malignant cell to stromal cells, vascular cells and immune system cells or from stromal cells, vascular cells and immune system cells to malignant cells are essential for the growth and progression of malignant disease. Proangiogenic factors including members of the vascular endothelial growth factor (VEGF)-A family, placental growth factor, members of the fibroblast growth factor family, semaphorins, ephrins, angiopoietins, stromal-cell derived factor-1, EG-VEGF, Bv8, transforming growth factor-bs (TGFbs), and others act on endothelial cells, endothelial precursor cells and endothelial progenitor cells from the bone marrow to promote tumor growth. Platelet-derived growth factors act on pericytes. The VEGF-C and VEGF-D family members stimulate lymphangiogenesis. TGFb secreted by malignant cells and stromal cells acts on the tumor stromal cells and on immune system cells to promote growth of the malignancy. Urokinase plasminogen activator and tissue plasminogen activator stimulate malignant cell and vascular cell migration as do numerous secreted matrix metalloproteinases. Secreted protein acidic and rich in cysteine (SPARC) stimulates the growth of malignant cells and influences malignant cell invasion and metastasis. In the bone, RANK ligand secretion can be stimulated by malignant disease to stimulate osteoclast-mediated bone resorption and allow metastasis growth. The interleukin family of secreted proteins mediates immune system activity. Immune system damping interleukins are involved in tumor immune evasion as are the chemokines monocyte chemoattractant protein 1 (MCP1) and RANTES. The large epidermal growth factor family stimulates the proliferation of epithelial malignant cells. Similarly, insulin-like growth factors and hepatocyte growth factor increase the growth of malignant tumors. Recently, the Wnt family of secreted growth factors has been identified as deregulated in multiple epithelial tumors. Many secreted growth factors are potential therapeutic targets for neutralizing antibodies or soluble receptor constructs.

B.A. Teicher (*) Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701-9322, USA e-mail: [email protected] R.G. Bagley (ed.), The Tumor Microenvironment, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-6615-5_31, © Springer Science+Business Media, LLC 2010

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Pro-angiogenic and Lymphangiogenic Factors Secreted growth factors are ideal targets for antibody therapeutics which can neutralize the target. One of the most successful anticancer drugs available is a humanized antibody, bevacizumab, which neutralizes the pro-angiogenic secreted growth factor vascular endothelial growth factor (VEGF)-A. VEGF signaling is a critical potentially rate-limiting step in physiological angiogenesis. The VEGF family includes seven secreted glycoproteins which have been designated VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor (PlGF) and VEGF-F. These proteins stimulate angiogenesis through a signaling process initiated by binding to their receptors which have been designated VEGF receptor (VEGFR)-1, VEGFR-2, VEGFR-3 and neuropilins (NP-1 and NP-2) (Otrock et al. 2007). VEGFs are potent, multifunctional cytokines that with important, possibly independent actions on blood and lymph vascular endothelium. VEGF-A was discovered as a tumor-secreted protein which caused large and small veins to be hyperpermeable to circulating macromolecules. VEGF-B promotes angiogenesis; however, its role has not been elucidated. VEGF-C drives in lymphangiogenesis during embryogenesis and functions in the maintenance of differentiated lymphatic endothelium in adults. VEGF-D stimulates the growth of vascular and lymphatic endothelial cells. Although initially characterized in the placenta, PlGF is expressed by a variety of cells, tissues, and organs. PlGF is involved in angiogenesis, wound healing, and the inflammatory response (Roskoski 2008). In cell culture VEGF induces tube formation, proliferation, transient accumulation of calcium, shape change, cell division, migration, and invasion of mature and precursor endothelial cells. In addition, exposure to VEGF alters endothelial cell gene expression and in vivo induces angiogenesis (Dvorak et al. 1995; Folkman 1995). VEGF neutralizing therapies were hypothesized to inhibit new blood vessel growth and inducing hypoxia and “starving” the tumor of oxygen and nutrients. It is becoming clear that the efficacy of VEGF neutralizing agents arises from multiple effects on varied cell types (Ellis and Hicklin 2008). The majority of anti-angiogenic agents approved and currently in clinical trial are directed toward neutralizing VEGFA or blocking VEGFR intracellular signaling. However, although many patients benefit from, for example, bevacizumab, many patients derive little or no benefit from treatment. Those patients who initially benefit through disease stabilization and prolonged progression-free and overall survival after treatment with VEGF-directed agents, their tumors eventually become non-responsive to continued anti-VEGF treatment so that the increased survival duration is months rather than years. Various mechanisms may contribute to initial lack of response or decreased in responsiveness after initial response to VEGF-directed therapy including dependence upon or up-regulation of other pro-angiogenic factors (Fischer et al. 2008). One pro-angiogenic factor which is emerging as a potentially important therapeutic target is PlGF. PlGF is a pleiotropic cytokine that stimulates endothelial cell growth, migration, and survival. PlGF is a chemoattractant for angiocompetent

31 Secreted Growth Factors as Therapeutic Targets

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macrophages and bone marrow (BM)-derived endothelial progenitor cells. PlGF signals through binding to VEGFR-1 and the co-receptors neuropilin-1 and -2. VEGFR-1 is expressed not only by endothelail cells but also by macrophages, bone marrow-derived endothelial progenitor cells, and some tumor cells (Fischer et al. 2007). While VEGF signal primarily through VEGFR-2, PlGF binds selectively to VEGFR-1 and thus produces a specific signal distinct from VEGF. Upon binding to VEGFR-1, PlGF amplifies VEGF-induced signaling through VEGFR-2 by intrareceptor crosstalk, resulting in potentiation of the pro-angiogenic effect of VEGF. PlGF is a key player in resistance mechanisms, used by tumors to escape anti-VEGF therapies. PlGF was upregulated when pancreatic tumors, initially responsive to blockade of VEGFR-2, became resistant and broke through the treatment. PlGF homozygous knockout mice grow up normally and give birth to healthy litters. However, when challenged with cancer, ischemia, inflammation, or wounds, PlGF homozygous knockout mice demonstrate defective angiogenesis and plasma extravasation. A monoclonal anti-PlGF has been tested as an anticancer agent in several syngeneic mouse tumor models. The anti-PlGF was an effective treatment at a dose of 50 mg/kg. The anti-PlGF inhibited neo-angiogenesis and induced regression of existing tumor vessels, inhibited lymphangiogenesis, inhibited recruitment of angiocompetent macrophages, and had a direct cytostatic effect on certain tumor cells (Loges et al. 2008). PlGF seems to be highly inducible in the clinic especially during treatment of patients with VEGF neutralizing agents or inhibitors of VEGFR kinase activity (Kopetz et al. 2009; Motzer et al. 2006; Rosen et al. 2007). Semaphorins are a large family of secreted, transmembrane and GPI-linked proteins initially characterized in the development of the nervous system and axonal guidance. Semaphorins are expressed in many tissues where they regulate normal development, organ morphogenesis, immunity, and angiogenesis. They affect the cytoskeleton, actin filament organization, microtubules, and cell adhesion. Several semaphorins and their receptors (neuropilins and plexins) participate in vascular development, angiogenesis, and cancer. Neuropilins, which are high-affinity receptors for class-3 semaphorins, are also co-receptors for VEGF and other growth factors, and their expression is often abnormal in cancer. Class-3 semaphorin signaling is transduced by neuropilin receptors. In cancer, semaphorins have both tumor suppressor and tumor promoting functions. In tumor-bearing mice, treatment with anti-neuropilin antibodies inhibited tumor angiogenesis and was additive when combined with an anti-VEGF antibody (Potiron et al. 2009). The large family of fibroblast growth factors (FGF) regulates a wide range of developmental processes, including brain patterning, branching morphogenesis and limb development. The functions of individual members of the FGF family differ in nearly every aspect including cell types affected and cellular functions affected. The angiogenic properties of FGF2, also known as basic FGF (bFGF) are well known. Exogenous FGF2 stimulates migration and proliferation of endothelial cells in vivo, and enhances survival and proliferation of smooth muscle cells and fibroblasts, which induces the development of large. Mice engineered to over-express of FGF2 have normal vasculature, possibly due to balancing by negative regulators of

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vascular growth. Interferon-a (IFNa) and IFNb downregulate FGF2 in human kidney, bladder and prostate tumor cell lines. Exposure of bladder carcinoma to IFNa or b inhibits FGF2 expression and cellular proliferation. There is evidence that inhibition of FGF2 signaling can slow tumor growth by inhibiting angiogenesis. However, FGF2 levels do not correlate with tumor microvessel density indicating that the anti-tumor effects of interferons mediated through FGF2 blockade may not be due only to anti-angiogenesis (Beenken and Mohammadi 2009). In a fastgrowing malignant tissue, tumor blood vessels are exposed to multiple growth factors and cytokines. Although the roles of individual factors and their signaling pathways in regulation of tumor angiogenesis and neovascularization are well-established, understanding the interactions amongst these factors in stimulating tumor angiogenesis and metastasis remains an area of intense investigation. Quiescent vascular endothelial cells remain non-responsive to platelet-derived growth factor (PDGF)-BB stimulation alone; however, exposure to the combination of PDGF-BB and FGF2 switches on the PDGF receptor and activates endothelial cells. PDGF-BB transduces positive feedback signals to the FGF2 signaling system by amplifying FGF receptor expression in vascular pericytes. These reciprocal interactions in the tumor microenvironment lead to the formation of the tumor vasculature; thus, providing a rationale for development of anti-angiogenic agent combinations for the treatment of cancer (Beenken and Mohammadi 2009; Cao et al. 2008). Growth factors that stimulate lymphangiogenesis have been identified. The growth of lymphatic vessels is controlled by proteins related to VEGF, namely VEGF-C and VEGF-D. Increased expression of VEGF-C and VEGF-D is associated with several human tumor types. The experimental inhibition of VEGF-C and VEGF-D signaling in animal models suggests that lymphangiogenic growth factors facilitate the metastatic spread of tumor cells via lymphatics. Anti-lymphangiogenesis has not yet been tested as a therapeutic approach. Lymphangiogenesis inhibitors may block the metastatic spread of cancers and may be very useful when cancer is detected earlier in the course of the disease (Stacker and Achen 2008). It is well-established that the metastatic spread of tumor cells frequently occurs via lymphatic vasculature. Experimental tumor data and human clinicopathologic data indicate that growth of lymphatic vessels near a tumor mass is often associated with lymph node metastasis. Changes in the adhesive properties of lymphatic endothelium near tumors facilitate metastatic spread. Lymphangiogenic growth factors that promote formation of tumor lymphatics and metastatic spread of tumor cells to lymph nodes are known. These include VEGF-C and VEGF-D which act through binding to receptor tyrosine kinase VEGFR-3 located on lymphatic endothelial cells. Several other signaling molecules promote lymphangiogenesis and/or lymphatic metastases in cancer include VEGFA, PDGF-BB, hepatocyte growth factor (HGF), and certain chemokines. Recently, it was shown that lymphangiogenic growth factors secreted by a primary tumor can induce lymphangiogenesis in nearby lymph nodes, prior to migration by tumor cells, thus further facilitating metastasis (Achen and Stacker 2008). The invasion of lymphatic vessels by colorectal cancer and subsequent spread of the tumor to draining lymph nodes is a key determinant of prognosis in colon cancer. Although lymphangiogenesis most likely contributes to this process a simple


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correlation between lymphatic vessel density and colorectal cancer metastasis has been difficult to prove. Attempts to correlate expression of VEGF-C and VEGF-D with the lymphatic metastasis in colon cancer have provided contradictory results. Evidence from models of tumor metastasis suggests that interactions between the tumor microvasculature and varied cell types within the tumor microenvironment are involved in the spread of metastasizing tumors. Strategies similar to those which have been instrumental to increasing understanding of vascular angiogenesis are being applied to tumor lymphangiogenesis and suggest tumor lymphatics have gene expression profiles distinct from those of normal lymphatic vessels and that promote metastasis (Royston and Jackson 2009). As with all anticancer therapies, most tumors development resistance to treatment with anti-angiogenic agents, while a fraction of tumors are non-responsive to these therapies from the outset. These two forms of resistance have been termed acquired resistance and intrinsic resistance. Both forms of resistance have been associated with the genetic instability of malignant disease. Similarly, two modes of resistance have been described for tumors treated with anti-angiogenic therapies: evasive resistance, an adaptation to circumvent the specific angiogenic blockade; and intrinsic or pre-existing indifference. Multiple mechanisms can be invoked that manifest in evasive or intrinsic resistance. The two modes of resistance in response to anti-angiogenic therapy imply adaptive evasion and intrinsic non-responsiveness of tumors. Adaptive or evasive resistance refers to the ability of a tumor, after an initial response phase, to evade the therapeutic blockade by altering the secreted proangiogenic factors or by reducing dependence on such growth of new blood vessels such growth along host vessels. Intrinsic non-responsiveness is defined by the absence of benefit from anti-angiogenic therapy. Acquired/adaptive resistance may occur by activation and/or upregulation of non-VEGF pro-angiogenic signaling pathways such as PlGF, FGF, ephrin, and angiopoietins. Recruitment of bone marrow-derived endothelial precursor cells (EPC) through secretion of certain chemokines such as stromal cell-derived factor 1 can restore neovascularization (Bergers and Hanahan 2008). Tumor stromal and vascular cells can be derived from bone marrow-derived progenitor cells including mesenchymal stem cells (MSC), EPC, pericyte progenitor cells, VEGFR-1+ progenitor cells, and Tie2-expressing monocytes, which are mobilized into circulation and incorporate into tumor microenvironment. In the tumor microenvironment, MSC may promote tumor growth by secretion of growth factors, participation in vessel formation and possibly development of tumor stem cell niches. It is clear that bone marrow-derived cells can functionally contribute to tumor growth. Precursor and progenitor cells mobilized from the bone marrow into circulation migrate to tumor sites and incorporate into the tumor microenvironment. The contribution of these cells to tumor growth seems to be highly variable and whether blockade of recruitment of cells from the bone marrow to the tumor can be of therapeutic value (Roorda et al. 2009). Bone marrow-derived cells participate in tumor angiogenesis. The secreted growth factors, EG-VEGF and Bv8 proteins (also called prokineticin1 (Prok1) and prokineticin 2 (Prok2)), promote both tissue-specific angiogenesis and hematopoietic cell mobilization. Bv8 and EG-VEGF bind two highly related G-protein coupled

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receptors (GPCRs), EG-VEGFR/PKR1 and EG-VEGFR/PKR2. Both EG-VEGF and Bv8 are mitogens selective for endothelial cell types. Bv8 and EG-VEGF can induce hematopoietic cell mobilization in vivo and stimulate production of granulocytic and monocytic colonies in vitro. Bv8 is expressed in the bone marrow and granulocyte colony-stimulating factor up-regulates Bv8 expression. Anti-Bv8 antibodies reduced CD11b+Gr1+ myeloid cell mobilization elicited by granulocyte colony-stimulating factor. Anti-Bv8 inhibited growth of several tumors in mice and suppressed tumor angiogenesis. Anti-Bv8 treatment also reduced the number of CD11b+Gr1+ myeloid cells in circulation and in tumors. Bv8 may be interesting candidate for a mediator of inflammatory-cell-dependent angiogenesis (Shojaei et al. 2007). Pro-angiogenic bone marrow cells include subsets of hematopoietic cells that provide vascular support and EPC, which under certain conditions can differentiate into functional vascular cells. The chemokine stromal-cell derived factor-1 (SDF-1; CXCL12) recruits and retains CXCR4+ bone marrow cells to angiogenic niches supporting ischemic tissue revascularization and tumor growth. The mechanism by which activation of CXCR4 modulates angiogenesis is not clear. SDF-1 promotes revascularization by binding CXCR4 expressed on vascular cells and supports mobilization of pro-angiogenic CXCR4+VEGFR1+ hematopoietic cells, thus stimulating ischemic site revascularization. SDF-1–CXCR4 signaling pathway has multiple functions in the regulation of vascularization during acute ischemia and tumor growth. By modulating plasma SDF-1 levels, the short-lived CXCR4 antagonist AMD3100 acutely promotes, while chronic AMD3100 treatment inhibits, mobilization of pro-angiogenic cells (Petit et al. 2007). In malignant disease, SDF-1+ or CXCR4+ various lineages cells are found in tumor tissues. Blockade of the SDF-1/CXCR4 axis decreases growth of preclinical gastrointestinal tumors through an anti-angiogenic effect. CXCR4 Neutralization can suppress the growth of mouse Colon38 and PancO2 tumors. The suppression of tumor growth was independent of CXCR4 expression by the malignant cells. CD31+ intratumoral vessels were decreased 55% and intratumor blood flow was decreased 35% by CXCR4 blockade. Intratumoral VEGF was not altered by CXCR4 neutralization. CXCR4+ endothelial cells were detected in the tumors suggesting that the anti-angiogenic effects of CXCR4 blockade lead to decreased tumor vessels in a VEGF independent process (Guleng et al. 2005). In patients with multiple myeloma, elevated SDF-1 levels in peripheral blood plasma are associated with osteolysis and with tumor angiogenesis. High SDF-1 levels produced by multiple myeloma plasma cells promote osteolysis and angiogenesis (Martin et al. 2006). Cellular niches are key in regulating normal stem cell differentiation and regeneration and appear to support malignant metastasis. Using dynamic in vivo confocal imaging, it has been shown that murine bone marrow contains unique anatomic regions defined by specialized endothelium which express the adhesion molecule E-selectin and the chemoattractant SDF-1 in discrete, discontinuous areas. Circulating leukemic cells can engraft around these regions suggesting that these may be a microenvironment for metastatic tumor spread in bone marrow. Purified hematopoietic stem/progenitor cells and lymphocytes localize to the same microdomains. Disruption of the


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interaction between SDF-1 and CXCR4 blocked migration of Nalm-6 acute lymphoblastic leukemia cells to these regions (Sipkins et al. 2005). Multiple myeloma plasma cells produce significant levels of SDF-1. Multiple myeloma patients have elevated SDF-1 plasma which positively correlates with the presence of multiple radiological bone lesions, suggesting a potential role for SDF-1 in osteoclast precursor recruitment and activation. There is increased osteoclast motility and activation in the presence of SDF-1 as well as an increase in osteoclast activation-related gene expression including receptor activator of nuclear factor-kB ligand (RANKL), RANK, TRAP, matrix metalloproteinases (MMP)-9, CA-II, and Cathepsin K. A small molecule CXCR4 inhibitor effectively blocked osteoclast formation stimulated by RPMI-8226 myeloma cells. In multiple myeloma patients, high circulating SDF-1 may recruit osteoclast precursors to local sites within the bone marrow and enhance bone resorbing activity (Zannettino et al. 2005). A large array of small molecule VEGF receptor tyrosine kinase inhibitors and a few biologic therapeutic primarily directed toward VEGF are in clinical trial. Drugs which target VEGF and PDGF pathways have revolutionized the treatment of patients with metastatic renal cell cancer. Approximately 75% of patients with clear cell renal cell carcinoma have mutations or silencing of the von Hippel Lindau gene producing an accumulation of HIF 1 alpha. These tumors produce high levels of proangiogenic factors such as VEGF and PDGF leading to angiogenesis and endothelial stabilization. Two small molecules and one biological therapeutic targeting these pathways have reached FDA approval. Both sunitinib and sorafenib target VEGF and PDGF receptor tyrosine kinases and bevacizumab, a VEGFtargeting monoclonal antibody. These three agents have superior progression free survival in patients with metastatic renal cell carcinoma compared with interferon or placebo. Several additional receptor tyrosine kinase inhibitors such as axitinib, pazopanib, and cediranib are under investigation and may provide further treatment options (Heng and Bukowski 2008). Several clinical studies have investigated treatment with bevacizumab for patients with recurrent malignant glioma. Treatment with bevacizumab is commonly combined with cytotoxic chemotherapy. These combination regimens often produce marked responses radiographically, prolongation of progression-free survival, and decreased corticosteroids use. Small molecule VEGFR receptor tyrosine kinase inhibitors, such as cediranib can achieve similar results. Anti-angiogenic treatment is generally well tolerated but common adverse effects include hypertension and proteinuria, whereas the potentially more serious adverse effects, such as thromboembolic disease and haemorrhage, occur infrequently. Approximately 50% of patients fail to respond to anti-angiogenic treatment and the response duration is variable. Resistance to anti-VEGF therapy implicates alternative pro-angiogenic factors such as bFGF, SDF-1, the angiopoietin receptor Tie2, and PlGF in the angiogenic process (Norden et al. 2008). Several agents in development for the treatment of non-small cell lung cancer (NSCLC) target the VEGF pathway. Most clinical trial data generated to date are with either bevacizumab or small-molecule inhibitors of VEGF receptor tyrosine kinase activity (sunitinib, sorafenib, and ZD6474). VEGF Trap, an engineered soluble receptor made from extracellular domains of VEGFR1 and VEGFR2, binds to

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VEGF and to PlGF. VEGF Trap binds to VEGFA with higher affinity than does bevacizumab. The toxicities in phase I trials with subcutaneous and intravenous administration of VEGF Trap, hypertension and proteinuria, are similar to those seen with other molecules that target the VEGF pathway. Ongoing phase I trials are evaluating combinations of VEGF Trap with platinum-based doublets and single agent docetaxel. The activity of single agent VEGF Trap in NSCLC is being assessed in a multicenter phase II trial (Riely and Miller 2007). VEGF Trap is in clinical trial in a wide variety of malignant diseases.

Pro-stromal Factors Transforming growth factor-b (TGFb) is a key player in malignant disease through its actions on host tissues and cells. Malignant cells often secrete large amounts of TGFb that act on non-transformed cells present in the tumor mass as well as distal cells in the host to suppress antitumor immune responses creating an environment of immune tolerance, augmenting angiogenesis, invasion and metastasis, and increasing tumor extracellular matrix (ECM) deposition (Pinkas and Teicher 2006; Teicher 2007). The tumor stroma actively contributes to tumorigenesis; it is more than a bystander. The involvement of stroma may occur early during epithelial cell transformation. Stroma consists of many cell types including fibroblasts, immune cells, endothelial cells, and pericytes lining blood and lymphatic vessels, which are embedded in ECM and produce soluble factors. The percentage and composition of tumor stroma varies amongst malignant diseases but is different from normal stroma. Malignant cells produce growth factors which induce a “reactive” stroma supporting tumor cell proliferation, migration and invasion, and angiogenesis (Mueller and Fusenig 2004). “Reactive stroma” is also called desmoplasia. Tumor stroma resembles the stroma usually found in injured tissues (Dvorak et al. 1995). Communication between malignant cells and tumor stroma components is facilitated by a network of growth factors including TGFb (Naber et al. 2008). Fibroblasts are the predominant stromal cells in carcinomas suggesting that fibroblasts may support or induce tumor progression. Progression to invasive carcinoma involves differentiation of fibroblasts to myofibroblasts or so-called cancer-associated fibroblasts (CAF) (Zeisberg et al. 2007). CAFs secrete growth factors including insulinlike growth factor (IGF), VEGF, epithelial growth factor (EGF), HGF, and TGFb, as well as MMP and ECM components. TGFb is critical to communication between epithelial malignant cells and stromal cells. TGFb stimulates production of ECM components such as collagens, fibronectin, tenascin and basement membrane components such as laminin. TGF regulates expression of MMPs and induces expression of protease inhibitors such as tissue inhibitors of metalloproteases (TIMPs) and plasminogen activator inhibitor-1 (PAI-1). TGFb is important in regulation of immune system response to malignancy. Tumor associated macrophages (TAM) are a major source of pro-angiogenic factors and are involved in tumor invasion. TGFb stimulates chemotaxis of monocytes, macrophage precursors (Naber et al. 2008). In malignant disease, the host immune


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system can promote tumor progression, invasion, and dissemination to distant sites. TGFb secreted by the tumor cells and stromal cells recruits leukocytes to secrete chemokines, growth factors, cytokines, and proteases which support the tumor and neutralizes the immune response to the tumor. As a potent immunosuppressant, TGFb, both directly and through the generation of regulatory T cells supports tumor growth (Moutsopoulos et al. 2008). Cells of the innate immune system contribute to the high concentrations of TGFb found in tumor masses. In addition, dendritic cell subpopulations secreting TGFb contribute to the generation of regulatory T cells that actively inhibit the activity of other T cells. Elevated levels of plasma TGFb are associated with advanced stage disease and may separate patients into prognostically high-risk populations. Anti-TGFb therapy could reverse the immunosuppressive effects of this cytokine on the host as well as decrease ECM formation, decrease angiogenesis, decrease osteolytic activity, and increase the sensitivity of the malignant cells to cytotoxic therapies and immunotherapies. Clinical trials of an inhibitor of TGFb receptor type I kinase activity and a TGFb neutralizing antibody are underway (Teicher 2007). In normal tissues, fibrosis is a failure of tissue remodeling that is the result of an excessive inflammatory response, representing an imbalance between enhanced production and deposition and impaired degradation of ECM components. Urokinase-type plasminogen activator (uPA) and TGFb1 are critical in ECM deposition and degradation (Philippou et al. 2008). The plasminogen activator system controls intravascular fibrin deposition and a variety of other physiologic and pathologic processes. In cancer, components of the plasminogen activator system are involved in tumor growth, invasion and metastasis through effects on angiogenesis and cell migration. Both plasminogen activators, tPA and uPA, are expressed by tumor cells. uPA with its receptor (uPAR) are involved in cellular functions, while tPA with its receptor annexin II on the endothelial cell surface regulates intravascular fibrin deposition. Among the inhibitors of fibrinolysis, PAI-1 has a major role in the pathogenesis of many vascular diseases as well as in cancer. Therapeutic intervention, either using plasminogen activators or agents directed against PAI-1, have shown encouraging results in experimental tumors (McMahon and Kwaan 2007). The human MMP family includes 23 enzymes which degrade the ECM and are associated with cancer invasion and metastasis. In recent studies, some MMPs such as collagenase 2 (MMP8), macrophage metalloelastase (MMP12) and matrilysin 2 (MMP26), showed a protective effect in different stages of cancer progression. Stromelysin1 (MMP3), gelatinase B (MMP9), stromelysin 3 (MMP11), and MMP19 may also be protective enzymes in some specific situations (Lopez-Otin and Matrisian 2007). MMPs are produced in latent forms are prototypically modified to be active. MMPs are classified into groups designated matrilysins, collagenases, stromelysins, gelatinases, membrane-type metalloproteinases, and others. Matrilysins (MMP-7, MMP-26) have the simplest structure, consisting of a signal peptide, a prodomain, and a catalytic domain with a zinc-binding site and are expressed in physiological and pathological cells in the endometrium, small intestine, breast, pancreas, liver, and prostate. Collagenases (MMP-1, MMP-8, MMP-13) and stromelysins have a hemopexin-like domain and regulate fetal bone

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development and fetal wound repair by rapidly remodeling ECM. MMP-13 is expressed in malignant melanoma and breast, head and neck, and bladder carcinoma. The gelatinases (MMP-2 and MMP-9) have three-fibronectin type II repeats within their catalytic domains and catalyze the degradation of fibral collagens after initial cleavage by collagenases. Gelatinases are key proteases in malignant tumor metastasis. The membrane-type metalloproteinases (MMP-14, MMP-15, MMP-16, MMP-17, MMP-24, MMP-25) have a glycosylphosphtidylinositol anchor that provides cell surface binding. MMP-2 is important in gastric, pancreatic, prostate, and breast cancer cell migration. MMP-2 expression is dependent on extracellular matrix metalloproteinase inducer (EMMPRIN). The detection of active MMP-2 alone or the rate of pro-MMP-2 and active MMP-2 has been used as an indicator of cancer metastasis. Modulation of MMP-2 expression and activation through specific inhibitors and activators may thus provide a new mechanism for breast cancer treatment. Degradation of the cellular network established by adhesion molecules such as E-cadherin or ALCAM/CD166 causes tumor tissue relaxation, increases metastasis, and correlates with shortened survival in patients with primary breast carcinoma (Jezierska and Motyl 2009). Secreted protein acidic and rich in cysteine (SPARC) is a prototypic matricellular protein. Matricellular proteins modulate cell–cell and cell–matrix interactions and are expressed during morphogenesis, development, tissue injury, and tissue remodeling. The SPARC-related protein sub-family share three domains: (1) an N-terminal acidic and low-affinity calcium-binding domain; (2) a disulfide-bonded, copper-binding follistatin domain (homologous to TGFb inhibitors activin and inhibin); and (3) a C-terminal extracellular calcium-binding domain. SPARC is counter-adhesive that is, SPARC impairs cell attachment to the ECM in a concentration-dependent manner. Upon exposure to SPARC, primary cultured cells lose focal adhesions and exhibit decreased cell spreading (Ledda et al. 1997). SPARC may affect the tumor–host interaction in the metastatic niche, where metastatic tumor cells migrate through the ECM. Understanding SPARC influence on tumor invasion and metastasis requires continued investigation (Clark and Sage 2008). In a clinical study, increased SPARC expression in pancreatic tumors resulted in improved response to albumin-bound paclitaxel (Smith et al. 2008). RANKL is the primary mediator of osteoclast formation, function, and survival. RANK and RANKL are expressed by cells involved in bone remodeling, by cells of the immune system, and by cells in other tissues. Bone loss is mediated by osteoclasts, cells whose formation, function and survival depend on the RANKL. RANKL binds RANK on pre- and mature osteoclasts and activates and maintains osteoclast-mediated bone resorption. In preclinical inflammatory disease models, inhibition of RANKL prevents bone loss and has no detectable effect on immune mediators or inflammation. A phase 2 clinical study of the fully human RANKLneutralizing antibody, denosumab, in postmenopausal women with low bone mineral density using showed increased bone mineral density similar adverse event frequency as placebo and open-label alendronate. A subset of patients in the trial tested for immunological markers had no differences in T, B, or NK cell numbers or in immunoglobulin levels across dose or treatment groups (Stolina et al. 2007).


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In another clinical trail, women with non-metastatic breast cancer and low bone mineral density who were receiving adjuvant aromatase inhibitor therapy, twiceyearly administration of denosumab led to significant increases in bone mineral density over 24 months at trabecular and cortical bone, with overall adverse event rate similar to those of placebo (Ellis et al. 2008). Denosumab has shown benefit in other breast cancer clinical trials (Fizazi et al. 2009; Lipton et al. 2008). Blockade of the RANKL–RANK pathway may be of benefit in additional cancers (Schwartz 2008; Sung et al. 2009; Roodman 2009).

Immune System Modulators Solid tumors are infiltrated by leukocytes. Interaction between malignant and blood borne cells have profound effects on tumor progression. Leukocytes account for up to 50% of the tumor cellular component; these are mainly lymphocytes and macrophages. The presence of immunocompetent cells in tumors is believed to indicate a host immunological response to the malignancy. In some tumor types (colon, ovarian, melanoma), high T lymphocyte density can be an indication of a better clinical outcome. In most tumors, a high macrophage density is an indication of increased angiogenesis, tumor invasion, and poor prognosis (Sica et al. 2008). Activated immune T cells can kill cancer cells. Cancer vaccines function by increasing the activated immune T cells in the tumor. Immunotherapeutic agents that can increase the number of activated tumor-directed T cells include: (1) dendritic cell activators and growth factors, (2) vaccine adjuvants, (3) T-cell stimulators and growth factors, (4) immune checkpoint inhibitors, and (5) agents to neutralize or inhibit suppressive cells, cytokines, and enzymes (Cheever 2008). A few immune system stimulators are secreted factors. Interleukin-15 (IL-15) is a T-cell growth factor, similar to interleukin-2 (IL-2). Preclinical data suggest IL-15 could improve the therapeutic effect of cancer vaccine and adoptive T-cell regimens. IL-15 inhibits antigen-induced T cells death, whereas IL-2 promotes antigen-induced cell death. IL-15 is secreted by dendritic cells, macrophages, and stromal cells. IL-15 acts on CD8 T cells, CD4 T cells, natural killer (NK) cells, and mast cells. IL-15 is required to maintain CD8 memory cells and NK cell development. It stimulates development of long-lived and highavidity CD8 T cells that kill tumor cells effectively (Cheever 2008). Interleukin-7 (IL-7) is a T-cell growth factor responsible for homeostatic expansion of naive cells. In mice and primates, IL-7 treatment induces dramatic expansion in peripheral T-cell numbers without obvious toxicity. In preclinical bone marrow transplant studies, IL-7 increases the rate and degree of immune reconstitution. In humans, IL-15 can reverse T-cell anergy. A phase I clinical trial was completed in patients with cancer (Rosenberg et al. 2006). Treatment with human recombinant IL-7 increased in total CD4 and CD8 T cells with modest increases in NK cells and with no change in mature B cells or increase in T regulatory cells (Tregs). IL-12 is a

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potent immune adjuvant. IL-12 promotes interferon-g release by IL-12R-expressing T and NK cells and induces T-helper 1 cell (Th1) polarization and proliferation of interferon-g-expressing T cells. IL-12 plays a central role in resistance to mycobacterial and intracellular pathogens. Phase I and II clinical trials IL-12 showed very modest efficacy alone with a handful of melanoma and renal cell cancer responses and proved challenging to administer safely (Younces et al. 2004). Flt3 ligand (Flt3L) is a hematopoietic growth factor that stimulates proliferation and differentiation of dendritic cell progenitors, especially interferon-producing killer and plasmacytoid dendritic cells. Administration of Flt3L increases dendritic cells in circulating blood, secondary lymphoid tissues and tumors. A Phase I study examined Flt3L alone, with peptide vaccines, as dendritic cell stimulators, and after bone marrow transplant and increases in circulating dendritic cells occur; however, studies were too small and variable to determine clinical efficacy (Fong et al. 2001). When activated CD4+ T cells develop into varied T helper cell subsets with specific cytokine profiles and effector functions. IL-17-producing effector T helper cells, Th17 cells, produce IL-17, IL-17F, IL-21, and IL-22, resulting in massive tissue reaction because IL-17 and IL-22 receptors are widely expressed. Because TGF-b is involved in the differentiation of Th17 cells, similar to CD4+CD25+Foxp3+ regulatory T cells (Tregs) (Korn et al. 2009). Chemokines are key molecules involved in the migration and homeostasis of immune cells (Krieg and Boyman 2009). The predominant tumor infiltrating cells are macrophages and CD8+ T lymphocytes (Sica et al. 2008; Balkwill 2004). Specifically, monocyte chemoattractant protein 1 (MCP1; CCL2) localizes to tumor epithelial areas. MCP1 levels correlate with the numbers of lymphocytes and macrophages that localize in the same area. RANTES (CCL5) localizes with tumor-infiltrating leukocytes and the CCL5 concentrations reflect the extent of CD8+ T-lymphocyte infiltration. High numbers of tumor-infiltrating leukocytes and production of chemokines that attract leukocytes is a poor prognostic sign in human breast cancer, where CCL5 and CCL2 correlate with tumor progression, macrophage infiltrates, lymph-node metastasis, and clinical aggressiveness. In esophageal squamous-cell carcinoma, CCL2 expression was associated with macrophage infiltration, tumor cell invasion and tumor vascularity.

Malignant Cell Growth Factors Epidermal growth factor receptor (EGFR, HER1), HER2/neu (ErbB2), HER3 (ErbB3) and HER4 (ErbB4), the EGFR family of receptor tyrosine kinases and their secreted protein ligands comprise an important growth factor pathway in cancer. A subset of breast cancers are driven by overactive EGFR or HER2 tyrosine kinases and continued activity of these pathways stimulates cancer progression (Sergina et al. 2007). In humans, there are more than 30 ligands and the EGFR family of four receptors initiates a complex, multi-layered signal-transduction network. The varied activated receptor–ligand complexes produce cellular responses differing strength and type. Multiple processes can modulate EGFR


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signal transduction including receptor heterodimerization and endocytosis. The multiple ligands and receptors allow the EGFR signaling system with greater specificity and a large repertoire of responses. The four receptors can potentially form ten distinct homo- and heterodimers that are activated by different ligands (Fig. 31.1) (Yarden 2001). Epidermal growth factor (EGF) and transforming growth factor-a (TGFa) are two predominant EGFR ligands. The binding of these ligands to the EGFR extracellular domain results in receptor monomer dimerization forming either EGFR homodimers or heterodimers with other HER proteins. Under normal conditions, ligand binding results in EGFR tyrosine kinase activation. In tumor cells a variety of other events can initiate EGFR tyrosine kinase activity, for example, certain mutations in the EGFRgene such as the EGFRvIII mutation, result in constitutive activation of EGFR kinase (Riely et al. 2006). The activated EGFR pathway supports solid tumor malignant progression. TGFa and EGF induce angiogenesis by up-regulating the expression of VEGF in tumor cells. Increased microvessel density is found in tumors with an activated EGFR pathway. An activated EGFR pathway also alters cell–cell adhesion, in a manner which favors the up-regulation or activation of MMP and tumor cell motility and invasion of adjacent and distal tissues (Herbst and Bunn 2003; Ciardiello et al. 2001). Although the functioning of signaling pathways requires the presence both ligand and receptor, many preclinical and clinical studies test only for the presence of the receptor. Measurements of EGFR expression do not reliably predict therapeutic responses to EGFR inhibitors (Gusterson and Hunter 2009). In one study, the ligand TGFa was detected in only 2.7% of tumors and the low rate of expression of TGFa was associated with the low rate of response to EGFR tyrosine kinase inhibitors. It is likely that the patients most likely to benefit from treatment with EGFR tyrosine kinase inhibitors are those whose tumors express both the receptor(s) and the ligands (Onn et al. 2004). Interestingly, a connection between response to EGFR inhibitors and KRAS mutational status has been made. Now, all patients with metastatic colorectal carcinoma who are candidates for anti-EGFR antibody therapy must have their tumor tested for KRAS mutations. If KRAS mutation in codon 12 or 13 is detected, then patients with metastatic colorectal carcinoma should not receive anti-EGFR antibody therapy as part of their treatment (Allegra et al. 2009). The neuregulins are the largest subclass of EGFR ligands. Neuregulins were identified by searching for HER2 receptor activators, thus, neuregulins are also called heregulins (Montero et al. 2008). The neuregulins can up-regulate the number of acetylcholine receptors in neuromuscular junctions and therefore these factors are also called acetylcholine receptor-inducing activity (ARIA). Other names for neuregulins include Neu differentiation factor or glial growth factors due to their breast and glial cells activities. The neuregulins are synthesized as membrane-bound, biologically active growth factors that bind to the HER/ErbB receptors. Preclinically, increased expression and function of neuregulins can induce cancer. They are potent mitogens for cell expressing HER receptors. In mice overexpression of neuregulins in the mammary tissue can generate adenocarcinomas and stimulate breast cancer metastatic spread. Neuregulin expression is detected in several neoplasias and tends to correlate with response to HER receptor-targeting treatments such as trastuzumab. The growth and progression of many ovarian cancers are regulated by the HER/EGFR

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Fig. 31.1 The epidermal growth factor pathway includes of many ligands and a system of receptors which can homo-dimerize and hetero-dimerize. These ligand– receptor complexes can trigger multiple intracellular signal transduction routes to reach activation of nuclear transcription factors that usually result in cell growth (adapted from Yarden (2001))

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family of receptor tyrosine kinase pathway. The receptors are activated by the ligands especially TGFa (activating HER1) and heregulin (activating HER3 and HER4) (Fig. 31.1). HER2 (HER2/neu) has no direct ligand and is activated via either homodimerization or heterodimerization with HER1, HER3 or HER4. Through heterodimerization with other members of the HER family, TGFa and heregulin (HRGh1) stimulate the growth of ovarian cancer cells (Mullen et al. 2007). HER2/neu overexpression is associated with increased tumor progression and metastasis. HER2/neu receptors are important in heregulin-induced angiogenesis. Overexpression of the HER2/neu receptor alone results in increased secretion of VEGF and exposure to heregulin enhances VEGF secretion in breast cancer cells (Konecny et al. 2004). The neuregulins are implicated in resistance to anti-HER therapies, so targeting neuregulins therapeutically useful in neoplastic diseases in which these ligands contribute to tumor progression (Montero et al. 2008). The hypothalamic decapeptide gonadotropin-releasing hormone (GnRH) controls pituitary gonadotropin secretion. GnRH hormone and receptor are also expressed in extra-pituitary tissues and tumor cells including epithelial ovarian cancers. Outside of the CNS, the GnRH pathway may function as an autocrine regulatory system. GnRH and several synthetic analogs have a direct antiproliferative effect on ovarian cancer cell lines. This effect is attributed to multiple steps in the GnRH signaling cascade such as cell cycle arrest at G0/G1. In ovarian cancer cells, GnRH receptors appear to couple to the pertussis toxin-sensitive protein Gai activating a protein phosphatase, which blocks growth factor-induced mitogenic signals. Recently, a regulatory role of GnRH analogs in ovarian cancer cell migration and invasion emerged. GnRH actions in ovarian cancer indicate that clinical application of GnRH analogs in ovarian cancer patients may be useful (So et al. 2008). Activation of the HGF/MET signaling pathway often has a role in oncogenesis, cancer metastasis, and drug resistance. The MET gene encodes a high-affinity receptor for HGF (also called scatter factor). The transforming properties of MET were identified first in a human osteosarcoma cell line following chemically induced mutagenesis. HGF is secreted by mesenchymal cells and MET is widely expressed by epithelial cancer cells. HGF binding to MET induces receptor homodimerization, phosphorylation of the tyrosine kinase domain and activation of MET-mediated signaling. HGF/MET signaling is essential during embryogenesis and is important in normal adult cells including hepatocytes, renal tubule cells, and myoblasts. Deregulation of the MET pathway occurs in many human malignancies. Sustained MET activation has been well characterized in preclinical models. Activation of HGF/MET signaling promotes cell invasiveness and triggers metastases through direct involvement of angiogenic pathways. HGF can stimulate endothelial cell proliferation and migration through induction of VEGF expression and down-regulation of thrombospondin-1 (You and McDonald 2008). Altered HGF secretion occurs in both solid and hematologic malignancies. Both malignant and mesenchymal cells can secrete increased HGF leading to paracrine and/or autocrine mechanisms of receptor activation. Inhibition of HGF/MET signaling represents a promising cancer treatment either alone or in combination therapy regimens. Clinical trials with agents targeting HGF/MET signaling are underway. A key issue

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in the clinical studies is appropriate patient selection strategies. Methods for assessment of HGF/MET overexpression and MET gene amplification are under development. Understanding the other key activated signaling pathways that occur concurrently with HGF/MET activation may allow rational combination therapeutic strategies (Toschi and Janne 2008). Insulin and IGFs differ from many other regulatory peptides that are relevant to cancer because they regulate whole organism cellular functions. Insulin and IGFs are tissue growth factors and hormones regulating whole organism growth and energy metabolism. Insulin is produced by pancreatic b-cells and is distributed to tumors through the circulation. Circulating insulin-like growth factor 1 (IGF1) and insulin-like growth factor 2 (IGF2) are produced in the liver as well as by tumor cells and stromal fibroblasts. IGF1 and 2 function through autocrine, paracrine, or endocrine mechanisms (Pollack 2008). The IGF1 receptor (IGF1R) is activated by IGF1 and IGF2. In cells that express both IGF1R and insulin receptor heterodimers form by association of insulin half-receptor with IGF half-receptor. These heterodimers are similarly activated by IGF1 and IGF2 (Chitnis et al. 2008). Insulin and IGF are relevant to cancer. Several therapeutic candidates that target IGF1 signaling have antineoplastic activity in preclinical tumor models as single agents and in combination regimens. Many tumors have altered levels of IGF1R, IGF2 and/or IGF binding proteins. Tumors can also express insulin receptor and heterodimers. Changes in IGF may be initiating events in tumorigenesis. Individuals with highnormal circulating IGF1 are at increased risk of later development of common solid tumors, possibly because IGFs stimulate neoplastic progression of occult lesions. Tumor growth can be stimulated by increasing circulating IGFl in experimental models. Rigorous prospective studies provided evidence for a relationship between circulating IGF1 and risk of developing prostate, breast, colorectal or other cancers, such that individuals at the high end of the normal range of serum IGF1 had more than double the risk of a cancer diagnosis than those at the low end of the normal range (Shukla et al. 2008; Kleinberg et al. 2009; Kawada et al. 2006; Rowlands et al. 2009). Several investigational antibody therapeutics directed toward IGFR are in early clinical trial including AmG479 (Amgen), AvE1642 (Sanofi-Aventis), A12 (Lilly), mK0646 (Merck) and R1507 (Roche). A compensatory increase in the circulating concentrations of growth hormone and IGF1 occurs on administration of IGF1R-specific antibodies (Pollack 2008; Rodon et al. 2008). Netrin-1 is a member of a large family of conserved 60–80 kDa proteins with sequence homology to laminins. Secreted netrins, netrin-1, -3, and -4 are expressed in mammals. The functions of netrin-1 are mediated by two families of receptors: the UNC5 family (human UNC5H1-H4 or UNC5A-D) and DCC, including the paralog neogenin, which are members of the immunoglobulin superfamily. Netrin-1 and the receptors DCC and UNC5 are involved in normal development in the brain– gut axis by regulating axonal guidance and survival, pancreatic, and mammary gland morphogenesis and angiogenesis. Netrin receptors are expressed by vascular endothelial cells and are implicated in the control of vascular endothelial and smooth muscle cell morphogenesis. Tumor development is associated with the loss of netrin-1 receptors and increased production of netrin-1, thus escaping the control


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of homeostasis mediated by the balance between receptor and ligand providing a selective advantage for tumor development. Overexpression of netrin-1 in the gastrointestinal tract of transgenic mice is associated with tumor initiation and progression. In human colorectal tumors netrin-1 is only rarely overexpressed (7%); however, loss of DCC/UNC5 is more frequent. Bernet et al. showed that 60% of breast cancers with distant metastases at diagnosis and 37% of tumors with axillary node involvement show increased netrin-1 mRNA expression and immunohistochemistry (Bernet and Fitamant 2008). Netrin-1 may be a useful therapeutic target in cancer. S100A4 (mts1, pEL-98, 18A2, p9Ka, CAPL, calvasculin, and FSPl) belongs to the S100 family of calcium binding proteins. S100A4 expression in tumor cells is correlated with an aggressive metastatic phenotype. Up-regulation of S100A4 in tumor and stroma cells is associated with poor prognosis and survival of patients with cancer. Immune cells including macrophages, neutrophils, certain lymphocytes, dendritic and mast cells, and human endothelial cells express and release S100A4 into the extracellular space (Grigorian et al. 2008). S100A4 does not have a signal sequence and is released from the cell through an atypical pathway. Extracellular S100A4 can affect tumor progression by stimulation of angiogenesis, cell motility, upregulation of MMPs, modulation of tumor-related transcription factors, and stromal factors. It is not known whether S100A4 has a cell surface receptor. However, the short exposure time (1–2 min) needed to generate the response of neurons to S100A4 indicate receptor-mediated signaling is likely (Helfman et al. 2005). The Wnt family of secreted growth factors has key roles in directing cell patterning both during development and in adult tissues (Musgrove 2004). There are 19 Wnt proteins with a highly regulated pattern of expression and distinct roles in development and tissue homoeostasis. Wnts activate b-catenin signaling and consequently modulate the expression of specific target genes that regulate cell proliferation, apoptosis, and cell fate. Wnts are involved in oncogenesis. Early, Wnt-1 was found to be overexpressed in mammary epithelial adenocarcinomas. Since then, deregulated Wnt expression and Wnt signaling has been found in multiple epithelial cancers (Giles et al. 2003). The Wnt signaling pathways are activated through binding of a Wnt to one of nine seven-pass transmembrane receptors called the Frizzled receptor proteins (Fz) and LRP5/6 coreceptors. Following receptor binding, Wnt signals are transmitted by Wnt receptors and Dishevelled (Dvl) which triggers the disruption of the complex containing APC, Axin, GSK-3, and b-catenin, preventing phosphorylation-dependent b-catenin degradation (Jin et al. 2008). The Wnts are divided into two classes by signaling through the “canonical” or the “non-canonical” signaling pathway. Canonical Wnts are thought to activate a signal-transduction pathway that induces nuclear accumulation and transcriptional activation of b-catenin (Giles et al. 2003). The non-canonical signaling pathway describes all Wnt-activated cellular signaling pathways that do not promote b-catenin-mediated transcription. Emerging data suggest that these pathways are not as autonomous (Hopfner et al. 2008; Sirica 2008; McDonald and Silver 2009). For example, Wnts activate erbB signaling in addition to stimulating Wnt and b-catenin signaling, elucidating another level of regulation by Wnt family members and providing

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increased interest in these and other developmental pathways in breast cancer (Musgrove 2004).

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Chapter 32

Adrenomedullin Rebecca G. Bagley

Abstract Adrenomedullin is a multifunctional regulatory and vasoactive peptide that was identified in the early 1990s. Since then, research into this secreted hormone related to malignant disease has been emerging. Adrenomedullin is present in some normal tissues and is upregulated in a multitude of cancers. Adrenomedullin overexpression has been detected in human adrenal, breast, endometrial, lung, ovarian, pancreatic, prostate, and renal cancers. In certain indications, adreno-medullin expression correlated to disease progression and therefore adrenomedullin could possibly serve as a prognostic biomarker. Adrenomedullin promoted tumor development in preclinical models that can be attributed in part to an increase in angiogenesis. Both cancer cell lines and endothelial cells exposed to exogenous adrenomedullin displayed enhanced proliferative, migratory, and invasive properties. Efforts to target adrenomedullin under certain conditions have generated results which suggest that neutralizing adrenomedullin activity or interfering with receptor activity may have potential as a novel strategy to treat cancer.

Structure and Function Adrenomedullin (AM) is a secreted peptide hormone that was originally isolated from human pheochromocytoma, a rare tumor of the adrenal gland, and AM is also abundant in the normal adrenal medulla (Kitamura et al. 1993). AM has vasodilatory properties that were discovered by monitoring elevated platelet cAMP. Adrenomedullin consists of 52 amino acids with one intramolecular disulfide bond resulting from the cleavage of a 185 amino acid protein (pre-proadrenomedullin), which also produces proadrenomedullin N-terminal 20 peptide (PAMP).

R.G. Bagley (*) Genzyme Corporation, 49 New York Ave, Framingham, MA 01701, USA e-mail: [email protected] R.G. Bagley (ed.), The Tumor Microenvironment, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-6615-5_32, © Springer Science+Business Media, LLC 2010

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Adrenomedullin belongs to the calcitonin gene peptide superfamily due to the shared homology with the calcitonin gene-related peptide (CGRP) and amylin (Poyner et al. 2002). Several receptors for adrenomedullin have been reported. The first putative receptor described as L1 was isolated from rat lung. In COS-7 cells transfected with L1, there was an increase in cAMP upon exposure to AM (Kapas et al. 1995). However, these findings were subsequently thrown into question when the results could not be duplicated by other researchers (Kennedy et al. 1998). A second putative AM receptor, RDC-1, was recognized from a dog thyroid library and was shown to be able to bind both AM and CGRP (Kapas and Clark 1995). Additional research indicated that RDC-1 and AM were upregulated under hypoxic conditions in rat astrocytes in the blood–brain barrier (Ladoux and Frelin 2000). Subsequently, the calcitonin receptor-like receptor (CRLR) was shown to bind to AM (Njuki et al. 1993). CRLR is a seven-transmembrane, G-protein coupled receptor that forms heterodimers with receptor activity modifying protein (RAMP)-2 and -3 (Chakravarty et al. 2000; McLatchie et al. 1998). The vasodilatory properties of adrenomedullin were elucidated upon the discovery of AM in human pheochromocytoma tissue (Kitamura et al. 1993). Soon thereafter, the multifunctional characteristics of adrenomedullin emerged. The physiological and pathological roles of adrenomedullin were studied in genetically engineered mice that either lacked or overexpressed AM (Caron and Smithies 2001; Imai et al. 2001; Shindo et al. 2001). In homozygous knockout mice, the lack of AM proved to be embryonic lethal. However, the heterozygotes were viable and had a hypertensive phenotype compared with the wild-type mice. In transgenic mice that overexpressed AM, blood pressure was significantly lower than in the wild-type mice. The AM-transgenic mice were resistant to vascular injuries and lipopolysaccharide-induced septic shock. These results implied a significant role for AM in circulatory homeostasis and also indicated the importance of AM in vascular development during gestation. The role of AM in angiogenesis is further described below. In addition to maintaining physiological vascular homeostatsis and to supporting embryogenesis during pregnancy, adrenomedullin can regulate insulin secretion and blood glucose metabolism. A survey of pancreatic tissues from multiple species identified adrenomedullin protein in the pancreas of the rat, hamster, dog, and guinea pig (Martinez et al. 1996). mRNA transcripts for AM and its receptor were also present in multiple human adrenal and pancreatic cell lines that produce insulin. In the rat pancreas, in situ hybridization confirmed the expression of the AM receptor in the islets of Langherans. Using isolated rat islets, Martinez et al. demonstrated that AM can inhibit insulin secretion in a concentration-dependent manner and that exposure to an AM-neutralizing monoclonal antibody can result in an increase in insulin secretion. The co-expression of three receptors (L1, RDC-1, and CRLR) for AM in pancreatic b-cells is further evidence linking AM to the regulation of insulin release (Martinez et al. 2000). Adrenomedullin is a ubiquitous peptide with numerous biological functions that can also influence the CNS and renal function, and exert effects on the

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endocrine system. Several reports describe in detail the synthesis and secretion of AM, AM receptors and intracellular signal transduction pathways triggered by AM binding to its receptor, and AM-mediated activity in various organ systems (Hinson et al. 2000; Martinez and Cuttitta 1998). The remainder of this chapter will focus on the properties of AM related to angiogenesis, expression in tumors and in the tumor microenvironment, and the potential value of targeting AM with novel therapeutics.

Angiogenesis The ability of adrenomedullin to induce and regulate angiogenesis is well-documented in multiple preclinical models and in clinical specimens (Nikitenko et al. 2006). Transgenic mice were generated that lacked the AM gene or were heterozygous for AM to further elucidate significant functions of AM. In knockout mice, the absence of adrenomedullin gene expression was embryonic lethal (Caron and Smithies 2001). The mice died at mid-gestation with extreme hydrops fetalis and cardiovascular abnormalities. These abnormalities included overdeveloped ventricular trabeculae and underdeveloped arterial walls. In a separate study, investigators obtained similar results, noting a high mortality rate of embryos and abnormal vasculature causing hemorrhage and high blood pressure in the surviving embryos (Shindo et al. 2001). Detailed examination of the vessels revealed detached endothelial cells and incomplete basement membranes. The CRLR mediates AM signaling when the receptor is associated with RAMP-2 (Fernandez-Sauze et al. 2004). Additional evidence supporting the observation that adrenomedullin signaling is necessary for embryogenesis was revealed in knockout mice lacking CRLR or RAMP-2 genes (Fritz-Six et al. 2008). These mice also died mid-gestation after interstitial lymphedema occurred. In the knockout mice that were deficient in CRLR expression, the lack of AM signaling resulted in reduced lymphatic endothelial cell proliferation that caused abnormal jugular lymphatic vessels. Thus, these results establish a role for AM in the development of lymphatic vasculature. The placenta is a highly angiogenic tissue. Placental expression of adrenomedullin during pregnancy was investigated at the mRNA and protein levels (Moriyama et al. 2001). AM mRNA was detected in human placental trophoblastic tissues obtained during the first, second, and third trimesters. AM peptide was identified by immunohistochemical methods in the cytotrophoblasts but not in the syncytiotrophoblasts or endothelial cells of fetal stroma. AM peptide expression in the cytotrophoblasts was the highest during the first trimester and then decreased throughout the course of the pregnancy. In rare instances, choriocarcinomas, a trophoblastic cancer of the placenta, can develop during pregnancy. A choriocarcinoma cell line, JAr, was included in this study for analysis. The JAr cells expressed AM mRNA, secreted AM in culture, and also expressed a receptor for AM as demonstrated by the specific binding of radiolabeled rat AM to the cells.

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The effects of endothelial cell exposure in culture to adrenomedullin were investigated in several studies. The addition of AM to human umbilical vein endothelial cells (HUVEC) increased migration and tube formation in culture (FernandezSauze et al. 2004). Experiments with HUVEC and AM revealed that AM signaled through the Akt, ERK, and focal adhesion kinase pathways (Kim et al. 2003). Inhibitors to those pathways were able to suppress partially AM-induced HUVEC tube formation. In rat endothelial cells, AM functioned via autocrine and paracrine mechanisms as an apoptosis survival factor (Kato et al. 1997). In addition to adrenomedullin, the 20 amino acid peptide (PAMP) that results from the cleavage of pre-proadrenomedullin also possesses angiogenic properties. In a series of angiogenesis assays, PAMP was able to enhance the sprouting of new vessels in the chick embryo aortic ring assay and increase migration and cord formation in human microvascular endothelial cells (HMVEC) that expressed PAMP receptors (Martinez et al. 2004). Exposure of HMVEC to PAMP also increased molecular levels of other potent angiogenic factors such as VEGF, bFGF, and PDGF-C. In vivo, the angiogenic potential of adrenomedullin was demonstrated in a rabbit hindlimb ischemia model via gene delivery with a plasmid DNA (Nagaya et al. 2005). Transgenic expression of adrenomedullin resulted in an increase in capillary density, calf muscle blood pressure ratio, and blood flow measured by laser Doppler methodology. Similarly, recombinant human AM protein enhanced blood flow to the ischemic limb in the mouse hindlimb ischemia model and upregulated VEGF production in the affected tissue (Iimuro et al. 2004). These results suggest that AM could offer therapeutic value in ischemic disease. In mice, adrenomedullin stimulated angiogenesis in “angioreactors”, silicone tubes filled with matrix proteins, that were implanted subcutaneously in nude mice (Martinez et al. 2004). AM stimulated neo-vascularization in a traditional Matrigel plug assay and increased blood flow that was characterized by immunohistochemistry (IHC), hemoglobin assays, and laser Doppler perfusion image analysis (Kim et al. 2003; Miyashita et al. 2003). Analysis of the cellular components of the newly formed vessels in Matrigel plugs whereby angiogenesis was induced by exogenous AM revealed the recruitment of not only endothelial cells and pericytes but also myeloid precursor cells and macrophages (Kaafarani et al. 2009). This influx of cells that contribute to the tumor microenvironment was inhibited by an antibody directed toward the CLRL receptor. Adrenomedullin is involved in angiogenesis not only during embryogenesis but also during tumor development in a wide variety of human cancers. In renal cell carcinoma (RCC) cell lines and human biopsies, a positive correlation existed between AM and VEGF mRNA levels. Both AM and VEGF mRNA correlated with microvessel density (MVD) (Fujita et al. 2002; Deville et al. 2009). Under hypoxic conditions, the increase in AM mRNA was significantly greater than the increase in VEGF mRNA levels (10.6- to 26.7-fold increase vs. 1.5- to 1.9-fold) in several RCC cell lines. Similarly, hypoxia induced AM expression in hepatocellular carcinoma (HCC) (Park et al. 2008). AM expression in the HCC study also correlated with vascular invasion and N-cadherin expression by IHC, factors that may be associated with a poor prognosis.

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Adrenomedullin in Cancer Expression of adrenomedullin in normal and malignant tissues is not limited to pheochromocytomas, the adrenal glands, or the pancreas (Kitamura et al. 1993; Martinez et al. 1996). Adrenomedullin mRNA transcripts were detected by RT-PCR in 18 of 20 major organs including the brain, heart, and lung (Miller et al. 1996). AM transcripts were not found in normal thyroid or thymus tissues. In the same survey, 55 of 58 (95%) of human cancer cell lines were positive for AM mRNA. The few tumor cell lines that were negative included the H187 small cell lung carcinoma (SCLC), H23 lung adenocarcinoma, and H460 lung large cell carcinoma. The human cancer cell lines where AM was detected were derived from malignancies of the bone marrow, breast, cartilage, colon, lung, nervous system, ovary, and prostate. Based upon these findings, the study of AM expression in malignant tissues was expanded to the examination of multiple samples from cancer patients and preclinical studies in tumor models.

Breast Cancer There have not been many reports focused on the role of adrenomedullin in breast cancer. One study investigated the effects of AM overexpression on a subline of the human breast cancer cell line T47D that was stably transfected to overexpress AM (Martinez et al. 2002). The morphology of T47D cells that overexpressed AM changed by becoming more pleiotropic and the cells also demonstrated an increased angiogenic potential in vitro and in vivo. In vitro, the transfected T47D breast cancer cells served as a feeder layer in the chick aortic ring assay and promoted more sprouting of new vessels compared to the control cells. In vivo silicone tubes were filled with Matrigel-containing transfected T47D cells, or conditioned medium collected in culture was used to further evaluate the angiogenic potential of AM. After intravenous injection of FITC-dextran, there was an increase in fluorescence in the implants containing the cells that overexpressed AM as well as in those exposed to conditioned medium compared to that in the controls. Under serum deprivation, the T47D cells that overexpressed AM were more resistant to apoptosis and had lower levels of pro-apoptotic proteins such as Bax, Bid, and caspase 8 compared to T47D cells transfected with an empty vector. The T47D cells that overexpressed AM also had higher levels of proteins associated with oncogenic signal transduction pathways such as Ras, Raf, PKC, and MAPKp49, and were more tumorigenic when injected subcutaneously in nude mice. These data indicate that AM can confer a survival advantage in breast cancer cells, a finding that was also observed with human prostate cancer cells (Abasolo et al. 2006). The preclinical data on the role of adrenomedullin in breast cancer are supported by clinical data that examined tissue and plasma expression of AM in patients with breast cancer, some of whom had metastatic disease (Oehler et al. 2003). Immunohistochemically detected AM peptide expression was present with

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moderate to strong staining intensity that was homogeneous and cytoplasmic in 82% of the breast tumor specimens (27 of 33). Importantly, AM expression significantly correlated with axillary lymph node metastasis. When plasma concentrations of AM were compared between breast cancer patients and healthy controls, no meaningful differences were found, although there was a significant positive correlation between tumor size and AM plasma levels. In addition, elevated circulating plasma levels of AM in patients with breast cancer correlated with the presence of lymph node metastasis. These findings suggested a possible role for AM in metastatic disease, although AM offered little value as a diagnostic biomarker for breast cancer since there was no significant difference between plasma levels between healthy volunteers and breast cancer patients in this study.

Central Nervous System Adrenomedullin expression has been studied in glioblastoma, one of the more aggressive malignancies (Ouafik et al. 2002). AM mRNA transcripts were detected by RT-PCR and Northern blot in multiple human glioblastoma cell lines. Western blot confirmed protein expression in the conditioned media of U87, SW1088, and U373 glioblastoma cultures. RT-PCR methods also detected mRNA transcripts for CLRL/RAMP-2 and -3 in glioblastoma cell lines and human glioma tissues. A survey of AM mRNA in human brain tumor biopsies revealed that AM expression correlated with tumor type and grade. While high expression was present in glioblastomas, lower AM mRNA levels were found in anaplastic astrocytomas. In low-grade astrocytomas and oligodendrogliomas, levels of AM mRNA were negligible. The signal transduction pathway of adrenomedullin was further investigated in human glioblastoma cells (Ouafik et al. 2009). In the study, AM was found to promote cell transit with a concomitant increase in cyclin D1 protein level and proliferation through the activation of the cJun/JNK pathway. Adrenomedullin peptide was also detected by radioimmunoassay in human ganglioneuroblastomas and neuroblastomas (Satoh et al. 1995). The same study revealed high levels of adrenomedullin in every region of the human brain, with the highest concentrations present in the thalamus and hypothalamus. These data suggest that adrenomedullin could function as a neurotransmitter, neuromodulator, or a neurohormone.

Endometrial Cancer Endometrial cancer is an indication where AM has been shown to correlate with more aggressive disease both in preclinical tumor models and from the study of clinical samples. In mouse models, adrenomedullin promoted the growth of endometrial xenograft tumors. Two human endometrial carcinoma cell lines were

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transfected with a cDNA encoding AM and then implanted subcutaneously into mice (Oehler et al. 2002). AM expression enhanced in vitro proliferation of RL95.2 cells and in vivo tumor growth compared to controls. IHC of RL95.2 tumors with an antibody against CD31 revealed greater MVD when AM was overexpressed. Transfected Ishikawa endometrial tumor cells did not exhibit a faster growth rate in vitro but tumor growth in vivo was enhanced. These cells were also resistant to apoptosis under hypoxic conditions (Oehler et al. 2001). The oncoprotein Bcl-2 was upregulated in Ishikawa endometrial tumor cells overexpressing AM, and therefore AM may confer resistance to hypoxia-induced cell death. The potential contribution of AM to endometrial cancer growth was investigated in a collection of human tumor biopsies (Nunobiki et al. 2009). A total of 180 clinical endometrial specimens were surveyed for AM expression and MVD by an immunoperoxidase method. The panel of endometrial cancers included 30 proliferative phase specimens, 30 simple hyperplasias, 30 atypical simple hyperplasias, 30 complex hyperplasias, 30 atypical complex hyperplasias, and 30 grade 1 adenocarcinoma specimens. Significant differences existed between AM expression between the normal proliferation samples and those representing hyperplasia without atypia. The levels of expression of AM, the area of the venules, and the MVD increased in a stepwise manner from normal, simple, or complex hyperpasia with or without atypia to specimens of grade 1 adenocarcinomas. These results not only suggest a link between AM expression and stage of the disease but also illustrate the role of AM as an angiogenic protein.

Lung Cancer Little research has been carried out in lung cancer in associated with adrenomedullin but a retrospective study was conducted using archived lung tumor specimens. In a survey of 22 squamous cell carcinomas, 15 adenocarcinomas, and 13 small cell carcinomas, AM expression was more prevalent in the non-small cell carcinomas (Buyukberber et al. 2007). Of the biopsies examined, 91% of squamous cell carcinomas and 87% of adenocarcinomas had moderate to strong levels of AM compared to non-neoplastic lung tissue. By comparison, there was immunoreactivity in only 23% of small cell carcinomas where AM was weakly expressed. These results indicate that adrenomedullin is overexpressed in lung cancer; however, in this study, there was no correlation between AM expression and tumor differentiation, cancer stage, or overall survival.

Mast Cells and the Tumor Microenvironment In human tumors, mast cells can secreted and responded to adrenomedullin (Zudaire et al. 2006). Under hypoxic conditions in culture, differentiated human mast cells increased production of AM mRNA and AM protein expression. AM was also

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chemotactic for human mast cells and stimulated the production of VEGF, monocyte chemoattractant protein-1, and basic fibroblast growth factor at the molecular level. Mast cells also released histamine or b-hexosaminidase upon exposure to AM. Mast cells expressing AM were identified by immunohistochemical methods in human breast and lung carcinoma tumors.

Ovarian Cancer Adrenomedullin expression has been studied in ovarian cancer both in preclinical experiments and from clinical specimens. One report described incorporating the human ECV ovarian tumor cell line into in vitro experiments, the results of which demonstrated that the addition of exogenous AM to ECV cells promoted in an increase in motility (Martinez et al. 2002). Data generated in preclinical studies with CAOV3 ovarian epithelial cancer cells demonstrated that CAOV3 cells express both AM mRNA and AM protein in vivo (Zhang et al. 2009). The study also indicated that bFGF can induce increased AM expression through the JNK-AP-1 pathway. Ovarian cancer is an indication where adrenomedullin expression was significantly associated with poor prognosis. Survival data and RT-PCR data on AM levels were generated from 60 fresh surgical specimens of epithelial ovarian cancer (29 serous, 14 mucinous, 13 endometrioid, 3 clear cell, and 1 undifferentiated carcinoma) (Hata et al. 2000). In addition to survival, a distinct association existed between the histological grade of the tumors and AM gene expression. IHC methods applied to the ovarian cancer biopsies detected AM protein in the cytoplasm or outer cell membrane of the malignant cells, and in the endothelial cells of the tumor vasculature. In a separate investigation, adrenomedullin expression was evaluated in ovarian granulosa cell tumors and in fibrothecomas, stromal tumors of the ovary that tend to be rich in lipids (Liu et al. 2009). The levels of AM mRNA transcripts as analyzed by Northern blots were higher in the granulosa cell tumors than that in the fibrothecomas and normal ovaries. Within normal ovaries, AM protein expression was localized in both granulosa and theca cells, and in the oocyte of a preovulatory follicle. However, any immunoreactivity of the anti-adrenomedullin antibody with AM protein in the granulosa cell tumors or fibrothecomas was typically considered to be weak. The conclusions from this report are that any correlation between AM levels and the prognosis of ovarian cancer patients may depend upon the histology of the tumor. The findings from these studies that incorporated human ovarian cancer specimens are intriguing, and further research is warranted into the role of AM in ovarian cancer.

Pancreatic Cancer AM and its receptors were identified in normal pancreatic tissue, where it can regulate insulin secretion and blood glucose metabolism (Martinez et al. 1996, 2000).

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To determine if AM may be involved in pancreatic cancer, a panel of pancreatic cancer cell lines were surveyed for AM mRNA expression by RT-PCR, and numerous human pancreatic tumor specimens were analyzed by IHC for AM protein expression (Ramachandran et al. 2007). All eight pancreatic cancer cell lines expressed AM mRNA, and the IHC results revealed AM protein in the epithelium of 90% (43 of 48) of human pancreatic ductal adenocarcinomas. In culture, all five pancreatic cancer cell lines secreted AM that was detected by ELISA. When several of those cell lines (Panc-1, BxPC3, and MPanc96) were exposed to exogenous AM, cellular proliferation, invasion, and nuclear factor kB activity increased. In vivo, silencing AM expression with shRNA in MPanc96 pancreatic cancer cells that expressed relatively high levels of AM resulted in slower growth of orthotopic tumors and metastasis to the lung and liver. Human Panc-1 pancreatic cancer cells that expressed relatively low levels of AM and which were genetically engineered to overexpress AM resulted in larger orthotopic tumors. These observations imply that AM overexpression can promote more aggressive disease in patients with pancreatic cancer, which overall is associated with a poor outcome. The expression levels of the AM receptor L1 and the calcitonin receptor-like receptor (CRLR) were further investigated in pancreatic cancer cells and the cells that contribute to the tumor microenvironment (Ramachandran et al. 2007, 2009). Human pancreatic cancer cells were found to express the AM receptor but not CRLR by RT-PCR and Western blotting methods. Human pancreatic stellate cells (HPSC) derived from pancreatic adenocarcinoma samples, HUVEC, and mouse lung endothelial cells (MLEC) expressed both the receptors as determined by the same techniques. Similar to the human pancreatic cancer cells, HPSC, HUVEC, and MLEC also secreted AM in culture that could be quantified by ELISA. These cells responded to exogenous AM with an increase in proliferation and in vitro tube formation on extracellular matrix proteins. Using shRNA to silence the AM receptor by lentiviral infection in several pancreatic cancer cell lines resulted in a reduction in growth of BxPC3, MPanc96, and Panc-1 cells and invasion of MPanc96 compared to controls when exposed to exogenous AM. Knockdown of the AM receptor or CRLR by siRNA in HPSC, HUVEC, and MLEC indicated that the silencing of the AM receptor but not CRLR reduced the growth of the cells in vitro and polygon formation by endothelial cells. The results generated from experiments with human pancreatic cancers cells and cells of the tumor microenvironment indicated that the autocrine effects of AM are mediated by the AM receptor but not by CRLR. Three receptors associated with AM were co-expressed in pancreatic b-cells: L1, RDC1, and CRLR (Martinez et al. 2000). Overexpression of adrenomedullin was also associated with pancreatic cancer, and the expression of adrenomedullin in pancreatic cancer was observed in clinical samples. In a survey that quantified mRNA levels by QRT-PCR in pancreatic adenocarcinoma vs. normal pancreatic tissue, AM transcripts were 1.5- to 2.4-fold higher in the malignant samples, particularly when lymph node metastasis was present (Keleg et al. 2007). Serum levels of AM were significantly increased in pancreatic cancer patients compared to that in healthy controls or those with chronic pancreatitis. Hypoxia induced AM expression

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in multiple human pancreatic cell lines and although exogenous recombinant human AM inhibited proliferation slightly, the invasiveness of the cells increased in vitro.

Prostate Cancer Adrenomedullin is expressed in both the normal and malignant prostate tissues. In the healthy human prostate tissue, AM protein was detected by immunocytochemistry primarily in the basal cells of the glandular epithelium and the utriculus (Jimenez et al. 1999). The same study found that PAMP, the other peptide resulting from the cleavage of pre-proadrenomedullin, was expressed in neuroendocrine (NE) cells throughout the epithelium of the prostate glands. QRT-PCR methods applied to human prostate cancer specimens generated results indicating that AM mRNA levels were 3-fold higher in prostate adenocarcinomas with a high Gleason’s score compared to those in benign prostate hyperplasia (Rocchi et al. 2001). The AM protein detected in the prostate tumor tissues was localized in the carcinomatous epithelial compartment. Adrenomedullin expression was specifically associated with androgen-independent prostate cancer. The production of adrenomedullin by prostate cancer cells was investigated in the hormone-dependent LNCaP and hormone-independent PC3 and DU145 cell lines (Rocchi et al. 2001). AM protein was detected in both the cell extracts and conditioned medium of androgen-nonresponsive PC3 and DU145 cells derived from therapeutic hormone-derived prostate cancer but not the androgenresponsive LNCaP cells. AM mRNA was present in xenograft tumors generated from the subcutaneous implantation of PC3 and DU145 cells but not the LNCaP cells. Exogenous AM added to the three prostate cancer cells in culture resulted in enhanced proliferation of the DU145 cells only. In contrast, overexpression of AM by PC3 cells inhibited proliferation through a G0/G1 cell cycle arrest (Abasolo et al. 2004). The transfected PC3 cells also resulted in slower tumor growth in vivo compared to that in the mock transfected cells, suggesting that in certain prostate cancers, AM may inhibit the growth of prostate cancer cells. PC3, DU145, and LNCaP cells expressed the CLRL/RAMP-2 receptor complex, indicating that AM functions in an autocrine manner in prostate cancer cells. Additional experiments conducted with human prostate cancer cells in culture indicated that adrenomedullin can prevent apoptosis (Abasolo et al. 2006). PC3, DU145, and LNCaP human prostate cancer cell lines were transfected with an expression vector to overexpress AM. The overexpression of AM conferred a survival advantage under conditions when the parental cells, cultured with exogenous AM, or the three genetically engineered prostate cancer cell lines were serum deprived or exposed to the topoisomerase II inhibitor etoposide. AM exposure prevented apoptosis in the DU145 and PC3 prostate cancer cells after serum removal and prevented apoptosis in the PC3 and LNCaP cells exposed to etoposide. Western blotting methods applied to the parental PC3 cell line after treatment with etoposide

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indicated that protein levels of phosphorylated ERK1/2, kinases that regulate cell cycle growth and progression, increased. However, the PC3 cells that overexpressed AM had lower phosphorylated ERK1/2 basal levels that did not increase upon etoposide exposure. Similarly, control PC3 cells treated with etoposide resulted in a significant increase in PARP, a protein involved in programmed cell death, but the PC3 clones that overexpressed AM only showed a modest increase in fragmented PARP under the same conditions. The findings from these experiments imply that AM may have a protective role against drug-induced apoptosis in prostate cancer cells. NE differentiation in prostate cancer is of interest as it may be an early marker associated with androgen independence and more aggressive disease (Abrahamsson 1999). Adrenomedullin has recently been implicated in NE differentiation under conditions of androgen withdrawal in models of prostate cancer. The earlier investigations with the LNCaP prostate cancer cell line did not find significant levels of AM produced in vitro or in vivo (Rocchi et al. 2001). However, a more recent study revealed that levels of AM increased 4- to 7-fold in androgen-sensitive LNCaP cells after androgen withdrawal in vitro and in xenograft tumors following castration (Berenguer et al. 2008). The expression of the AM receptors did not change under these conditions. In culture, LNCaP cells that were exposed to AM adopted a NE phenotype including extension of neuritic processes and expression of the neuronspecific enolase (NSE). The administration of AM also increased NSE levels in the serum of noncastrated mice bearing LNCaP xenograft tumors, although there were no changes in tumor growth. However, when exogenous AM was delivered to castrated animals, a significant increase in LNCaP tumor volume was observed 36 days after treatment. These data demonstrate that AM mediates the NE phenotype and can promote tumor regrowth in a hormone-independent manner. These findings have potentially significant implications for the clinic and warrant further investigation.

Renal Cancer The role of adrenomedullin was investigated in human renal cancer cell lines and in clinical specimens (Deville et al. 2009). In culture, the BIZ and 786-O clear-cell renal carcinoma (cRCC) cell lines secreted AM into the medium. Addition of exogenous AM to the cultures stimulated cellular proliferation, migration, and invasion. In human tumor specimens, levels of AM mRNA transcripts were higher in advanced cRCC and in chromophobe renal cell carcinomas (chRCC) compared to that in normal renal tissue. The molecular expression of AM in these samples correlated with levels of VEGF-A mRNA transcripts. Immunohistochemical analyses showed that in RCC tumors, the AM receptors CRLR/RAMP-2 were expressed by the malignant carcinoma cells, whereas RAMP-3 was expressed by infiltrating inflammatory cells, suggesting cross-talk within the tumor microenvironment. Clinically, AM demonstrated a prognostic value as high AM mRNA levels were

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associated with an increased risk of relapse after curative nephrectomy for cRCC. The results generated from this study in renal cancer implicate AM in the metastatic process and support the potential value of AM as a prognostic biomarker in RCC.

Adrenomedullin as a Therapeutic Target The overexpression of AM in human cancer cells increased angiogenic activity in vitro and in vivo. In preclinical tumor models, studies have demonstrated that AM can promote tumor growth. In clinical specimens, high AM levels correlated with disease grade in some malignancies. These findings suggest that AM should be evaluated as a novel therapeutic target. Preclinically, several strategies have been employed to interfere with AM function. In vitro, an antibody that neutralizes adrenomedullin was effective at inhibiting MCF-7 breast cancer cell growth (Miller et al. 1996). In vivo, intratumoral injections of an anti-AM antibody inhibited the growth of well-established subcutaneous tumors in the U87 glioblastoma xenograft model compared to a vehicle or an irrelevant antibody (Ouafik et al. 2002). Additional studies in several preclinical models using an antibody against the AM receptors (CLRL, RAMP-2, and RAMP-3) either alone or in combination further demonstrated the therapeutic potential of targeting the AM pathway. The antibodies were effective when delivered by intraperitoneal injection to mice bearing subcutaneous U87 glioblastoma, HT29 colon carcinoma, or A549 lung carcinoma xenograft tumors (Kaafarani et al. 2009). In the U87 tumors, immunofluorescent methods employing anti-CD31 and anti-SMA antibodies revealed depletions in endothelial cell and pericyte coverage. Immunohistochemical analysis utilizing anti-vWF antibody confirmed a reduction in mean vessel area; furthermore, an increase in apoptosis was quantified in the U87 model following treatment with the antibodies by the detection of single-stranded DNA. Pancreatic cancer is an indication where targeting adrenomedullin may offer clinical benefits. An AM antagonist (AM 22–52) demonstrated that AM promotes pancreatic cancer by both enhancing angiogenesis and stimulating the malignant cells (Ishikawa et al. 2003). When delivered in vivo to mice bearing PCI-43 xenograft tumors, the AM antagonist delayed tumor formation compared to control. Angiogenesis was reduced with the resulting blood vessels being smaller in diameter. In addition, the tumor proliferation index was lower. In an orthotopic model of pancreatic cancer, sublines of BxPC3 and MPanc 96 pancreatic carcinoma cells stably bearing shControl or shADMR vectors were developed. Sublines of both tumor lines transfected with shADMR generated tumors that were reduced by ~90% in tumor volume compared to tumors grown following the implantation of shControl-transfected cells (Ramachandran et al. 2009). The formation of lung and liver metastasis was also inhibited in the shADMR-transfected models. Nanoliposomes were used to deliver siADMR to mice with orthotopic MPanc96 pancreatic tumors. Both human and mouse siRNAs to ADMR were delivered to target both the host stroma and human cancer cells.

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Bioluminescence imaging indicated that the siRNAs resulted in decreased tumor burden. Subsequent staining of the tumors with an anti-CD31 antibody and IHC revealed collapsed blood vessels in the siADMR-treated tumors compared to the vasculature in the control tumors that had well-developed lumen formation. Targeting pro-adrenomedullin (PAMP) or the adrenomedullin pathway has resulted in anti-angiogenic effects in other preclinical models as well. A peptide fragment of PAMP that inhibited PAMP signaling slowed tumor growth in the A549 xenograft tumor model and decreased angiogenesis in “angioreactors” implanted subcutaneously into nude mice (Martinez et al. 2004). In HUVEC cells, AM antagonists and inhibitors for PKA or PI3K were effective at inhibiting the neo-vascularization induced by AM (Miyashita et al. 2003). In the Matrigel plug assay, angiogenesis was inhibited by an antibody against the AM receptor (Kaafarani et al. 2009). In a xenograft model, AM promoted the growth of subcutaneous mouse sarcoma S180 tumors that could then be inhibited with an antiangiogenic competitive inhibitor of AM (Iimuro et al. 2004). The increases in capillary density and weight of the tumors in mice that were treated with AM were comparable in mice injected with VEGF. S180 tumors in the heterozygous AM+/mice were smaller and had a lower capillary density compared to tumors grown in wild-type mice.

Conclusion Adrenomedullin is a multifunctional peptide that is important for embryogenesis and vascular homeostasis, and for regulating endocrine functions as well as exerting effects on the renal and central nervous systems. AM is upregulated in many cancers from patients in the clinic and in preclinical tumor models. The data from many investigations have demonstrated that AM is an angiogenic peptide and that it conferred a survival advantage in malignant cells. Several strategies that target AM or the AM receptors have been tested in human tumor xenograft models with the resulting data suggesting that neutralizing AM signaling may be a valuable therapeutic approach in cancer patients.

References Abasolo I, Wang Z, Montuenga LM, Calvo A (2004) Adrenomedullin inhibits prostate cancer cell proliferation through a cAMP-independent mechanism. Biochem Biophys Res Commun 322:878–886 Abasolo I, Montuenga LM, Calvo A (2006) Adrenomedullin prevents apoptosis in prostate cancer cells. Regul Pept 133(1–3):115–122 Abrahamsson PA (1999) Neuroendocrine differentiation in prostatic carcinoma. Prostate 39:135–148 Berenguer C, Foudouresque F, Dussert C, Daniel L, Muracciole X, Grino M, Rossi D, Mabrouk K, Figarella-Branger D, Martin P-M Ouafik L’H (2008) Adrenomedullin, an auocrine/paracrine

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Keleg S, Kayed H, Jiang X, Penzel R, Giese T, Buchler MW, Friess H, Kleeff J (2007). Adrenomedullin is induced by hypoxia and enhances pancreatic cancer cell invasion. Int J Cancer 121:21–32 Kennedy SP, Sun D, Oleynek JJ, Hoth CF, Kong J, Hill RJ (1998) Expression of the rat adrenomedullin receptor or a putative human adrenomedullin receptor does not correlate with adrenomedullin binding or functional response. Biochem Biophys Res Commun 244(3):832–837 Kim W, Moon S-O, Sung MJ, Kim SH, Lee S, So J-N, Park SK (2003) Angiogenic role of adrenomedullin through activation of Akt, mitogen-activated protein kinase, and focal adhesion kinase in endothelial cells. FASEB J 17:1937–1939 Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H, Eto T (1993) Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun 192(2):553–560 Ladoux A, Frelin C (2000) Coordinated up-regulation by hypoxia of adrenomedullin and one of its putative receptors (RDC-1) in cells of the rat blood-brain barrier. J Biol Chem 275(51):39914–39919 Liu J, Butzow R, Hyden-Granskog C, Voutilainen R (2009) Expression of adrenomedullin in human ovaries, ovarian sex cord-stromal tumors and cultured granulosa-luteal cells. Gynecol Endocrinol 25(2):96–103 Martinez A, Cuttitta F (1998) Adrenomedullin. IOS Press, Netherlands Martinez A, Weaver C, Lopez J, Bhathena SJ, Elsasser TH, Miller M-J, Moody TW, Unsworth EJ, Cuttitta F (1996) Regulation of insulin secretion and blood glucose metabolism by adrenomedullin. Endocrinology 137(6):2626–2632 Martinez A, Kapas S, Miller M-J, Ward Y, Cuttitta F (2000) Coexpression of receptors for adrenomedullin, calcitonin gene-related peptide, and amylin in pancreatic b-cells. Endocrinology 141(1):406–411 Martinez A, Vos M, Guedez L, Kaur G, Chen Z, Garayoa M, Pio R, Moody T, Stetler-Stevenson WG, Kleinman HK, Cuttitta F (2002) The effects of adrenomedullin overexpression in breast tumor cells. J Nat Cancer Inst 94(16):1226–1237 Martinez A, Zudaire E, Portal-Nunez S, Guedez L, Libutti SK, Stetler-Stevenson WG, Cuttitta F (2004) Proadrenomedullin NH2-terminal 20 peptide is a potent angiogenic factor, and its inhibition results in reduction in tumor growth. Cancer Res 64:6489–6494 McLatchie LM, Fraser NJ, Main MJ, Wise A, Brown J, Thompson N, Solari R, Lee MG, Foord SM (1998) RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature 393:333–339 Miller MJ, Martinez A, Unsworth EJ, Thiele CJ, Moody TW, Elsasser T, Cuttitta F (1996) Adrenomedullin expression in human tumor cell lines. J. Biol. Chem. 271(38):23345–23351 Miyashita K, Itoh H, Sawada N, Fukunaga Y, Sone M, Yamahara K, Yurugi-Kobayashi T, Park K, Nakao K (2003) Adrenomedullin provokes endothelial Akt activation and promotes vascular regeneration both in vitro and in vivo. FEBS Lett 544:86–92 Moriyama T, Otani T, Maruo T (2001) Expression of adrenomedullin by human placental cytotrophoblasts and choriocarcinoma JAr cells. J Clin Endocrinol Metab 86(8):3958–3961 Nagaya N, Mori H, Murakami S, Kangawa K, Kitamura S (2005) Adrenomedullin: angiogenesis and gene therapy. Am J Physiol Regul Integr Comp Physiol 288:R1432–R1437 Nikitenko LL, Fox SB, Kehoe S, Rees MCP, Bicknell R (2006) Adrenomedullin and tumour angiogenesis. Br J Cancer 94(1):1–7 Njuki F, Nicholl CG, Howard A, Mak JC, Barnes PJ, Girgis SI, Legon S (1993) A new calcitonin receptor-like sequence in rat pulmonary blood vessels. Clin Sci 85:385–388 Nunobiki O, Nakamura M, Taniguchi E, Utsunomiya H, Mori I, Tsubota Y, Mabuchi Y, Kakudo K (2009) Adrenomedullin, Bcl-2 and microvessel density in normal, hyperplastic and neoplastic endometrium. Pathol Int 59:530–536 Oehler MK, Norbury C, Hague S, Rees MCP, Bicknell R (2001) Adrenomedullin inhibits hypoxic cell death by upregulation of Bcl-2 in endometrial cancer cells: a possible promotion mechanism for tumour growth. Oncogene 20:2937–2945

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Oehler MK, Hague S, Rees MCP, Bicknell R (2002) Adrenomedullin promotes formation of xenografted endometrial tumors by stimulation of autocrine growth and angiogenesis. Oncogene 21:2815–2821 Oehler MK, Fischer DC, Orlowska-Volk M, Herrle F, Kieback DG, Rees MCP, Bicknell R (2003) Tissue and plasma expression of the angiogenic peptide adrenomedullin in breast cancer. Br J Cancer 89:1927–1933 Ouafik L’H, Sauze S, Boudouresque F, Chinot O, Delfino C, Fina F, Vuaroqueaux V, Dussert C, Palmari J, Dufour H, Grisoli F, Casellas P, Brunner N, Martin P-M (2002) Neutralization of adrenomedullin inhibits the growth of human glioblastoma cell lines in vitro and suppresses tumor xenograft growth in vivo. Am J Pathol 106(4):1279–1292 Ouafik L’H, Berenguer-Daize C, Berthois Y (2009) Adrenomedullin promotes cell cycle transit and up-regulates cyclin D1 protein level in human glioblastoma cells through the activation of c-Jun/JNK/AP-1 signal transduction pathway. Cell Signal 21(4):597–608 Park SC, Yoon JH, Lee JH, Yu SJ, Myung SJ, Kim W, Gwak GY, Lee SH, Lee SM, Jang JJ, Suh KS, Lee HS (2008) Hypoxia-inducible adrenomedullin accelerates hepatocellular carcinoma cell growth. Cancer Lett 271(2):314–322 Poyner DR, Sexton PM, Marshall I, Smith DM, Quirion R, Born W, Muff R, Fischer JA, Foord SM (2002) The mammalian calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors. Pharmacol Rev 54:233–246 Ramachandran V, Arumugam T, Hwang RF, Greenson JK, Simeone DM, Logsdon CD (2007) Adrenomedullin is expressed in pancreatic cancer and stimulates cell proliferation and invasion in an autocrine manner via the adrenomedullin receptor, ADMR. Cancer Res 67(6):2666–2675 Ramachandran V, Arumugam T, Langley R, Hwang RF, Vivas-Mejia P, Sood AK, Lopez-Berestein G, Logsdon CD (2009) The ADMR Receptor mediates the effects of adrenomedullin on pancreatic cancer cells and on cells of the tumor microenvironment. PLoS One 4(10):e7502 Rocchi P, Bodouresque F, Zamora AJ, Muracciole X, Lechevallier E, Martin P-M, Ouafik L’H (2001) Expression of adrenomedullin and peptide amidation activity in human prostate cancer and in human prostate cancer cell lines. Cancer Res 61:1196–1206 Satoh F, Takahashi K, Murakami O, Totsune K, Sone M, Ohneda M, Abe K, Miura Y, Hayashi Y, Sasano H (1995) Adrenomedullin in human brain, adrenal glands, and tumor tissues of pheochromocytoma, ganglioneuroblastoma and neuroblastoma. J Clin Endocrinol Metab 80(5):1750–1752 Shindo T, Kurihara Y, Nishimatsu H, Moriyama N, Kakoki M, Wang Y, Imai Y, Ebihara A et al (2001) Vascular abnormalities and elevated blood pressure in mice lacking adrenomedullin gene. Circ Res 104:1964–1971 Zhang Y, Zhang S, Shang H, Pang X, Zhao Y (2009) Basic fibroblast growth factor upregulates adrenomedullin expression in ovarian epithelial carcinoma cells via JNK-AP-1 pathway. Regul Pept 157(1–3):44–50 Zudaire E, Martinez A, Garayoa M, Pio R, Kaur G, Woolhiser MR, Metcalfe DD, Hook WA, Siraganian RP, Guise TA, Chirgwin JM, Cuttitta F (2006) Adrenomedullin is a cross-talk molecule that regulates tumor and mast cell function during human carcinogenesis. Am J Pathol 168(1):280–291

Index

A Aberrant DNA methylation altered methylation involvement cancer stromal cells, 129–130 EMT, 128–129 field cancerization, 128 somatic mutations, 129 unique natures, 127–128 characteristics DNA methylation status maintenance, 122–123 gene transcription regulation, 124–125 maintenance and de novo DNA methylases, 125 epigenetic changes, 122 epilogue, 130 methylation alterations CpG islands, 126–127 driver methylation and passenger methylation, 127 genome-overall hypomethylation, 125–126 Acetylcholine receptor-inducing activity (ARIA), 723 Acid-extruding mechanisms, 29 Acid production, 27 Acid transport H+ ions buffering, 30 extrusion, 30–31 nonrespiratory sources, 31 intracellular and extracellular pH, 28 metabolic acid efflux, 29–30 pH regulation, 28–29 Activator protein-1 (AP-1), 152–153 Adhesion receptors, family, 295

Adipokine angiopoietin-like 4 (ANGPTL4), 647–648 Adrenomedullin (AM) angiogenesis, 735–736 breast cancer, 737–738 central nervous system, 738 endometrial cancer, 738–739 lung cancer, 739 mast cells, 357, 361, 739–740 ovarian cancer, 740 pancreatic cancer, 740–742 prostate cancer, 742–743 renal cancer, 743–744 structure and function blood glucose metabolism, 734 definition of AM, 733 insulin secretion, 734 intracellular signal transduction, 735 PAMP, 733 vasodilatory properties, 733, 734 therapeutic target, 744–745 Adult stem cells (ASCs), 170 Aerobic ATP generation, 99 Aerobic glycolysis. See Warburg effect Alkaline pHe restoration, 35 AM. See Adrenomedullin Anatomic distribution, NK cell, 433 Angiogenic CXC chemokines, 283 Angioreactor angiogenesis, 736 therapeutic target, 745 Anti-a5b1 integrin function-blocking antibody, 469 Antiangiogenic drug therapy, 264–265

R.G. Bagley (ed.), The Tumor Microenvironment, Cancer Drug Discovery and Development, DOI 10.1007/978-1-4419-6615-5, © Springer Science+Business Media, LLC 2010

749

750 Apurinic/apyrmidinic endonuclease 1 (APE1) AP-1, 152–153 APE1-regulated transcription factors, 148–149 BER, 139–140 GRX/GSH system, 146 HIF-1a, 153–154 p53 APE1, role, 150, 151 BER pathway, 151 DNA-binding domain, 149 DR pathway, 152 glycosylases, 151 HR and NHEJ pathways, 152 MMR pathway, 152 polymerase b (Pol b), 151 proteins interaction, 149 redox functions, 148 Arg-Gly-Asp (RGD), 297 Autophagy, 216 B Baculovirus system, 62–63 Base excision repair (BER), 139–140 Bioluminescence imaging, 318 Blood-brain-barrier (BBB), 358 BMDMSCs. See Bone marrow derived mesenchymal stem/stromal cells Bmi-1, 178–179 Bone marrow derived cell (BMDCs), 257 Bone marrow derived mesenchymal stem/stromal cells (BMDMSCs) activation and metastasis, 283–284 activation and tumor growth breast cancer, 281–282 DARC expression, 282–283 as source, 277–278 tumor stroma interactions, 280–281 Bovine aortic endothelial cells (BAEs), 681 Bovine microvascular endothelial cells (BMEs), 681 Bowman-Birk trypsin inhibitor (BBI), 656–657 C CA. See Carbonic anhydrase CAFs. See Carcinoma associated fibroblasts CA9 gene expression, 66–68 CA IX. See Carbonic anhydrase IX

Index Calcitonin gene-related peptide (CGRP), 734 Calcitonin receptor-like receptor (CRLR), 734, 735, 741 Cancer progression CA IX, 72–74 disseminated tumor cells cellular dormancy, 236–237 chromosomal aberrations, 230–231 dissemination theory, 230 gene expression analysis, 230 immunocytochemical techniques, 231 metastatic cascade, 230 micrometastasis (See Micrometastasis) mechanical forces, 696–697 proteolytic modification, 701 Cancer stem cells (CSC) ASCs, 170 Bmi-1, 178–179 CAF, 180 carcinogenesis, 178 clonal selection, 169 durotaxis, 178 dynamic equilibrium, 180 ESCs cell cycle regulatory mechanisms, 174 characteristics, 172 ChIP-on-chip analysis, 173 epigenetic control, 173 mitogen signaling, MAPK pathway, 176 molecular cues, 177 pluripotent cell characteristics, 174, 175 POU5FI, 172–173 Rb/p105, 174 R point, 174–176 telomerase, 172 transcriptional regulators, 172–173 hypothesis, 170, 171 mechanobiology, 177–178 self-renewal process deregulation, 170 stem cell niche, 170, 177 tumor hypoxia, 179 tumorigenesis, 170 tumor progenitor, 179–180 Cancer stromal cells, 129–130 Carbonic anhydrase (CA) activity, 32 cytosolic isoforms, 61 dominant role, 35 facilitated CO2 diffusion, 32–34

Index facilitated H+ diffusion, 34 human CA isoforms, 60 intracellular and extracellular anhydrase isoforms, 32 metabolons, 61 N-terminal proteoglycan-like region, 61 transport metabolon, 34 zinc-binding enzymes, 59 Carbonic anhydrase IX (CA IX) clinical value anticancer treatment, 72 biopsies, 75 cancer progression, prognosis, and treatment outcome, 72–74 hypoxia marker, 72 pre-surgical diagnosis, 75 RCC, 72, 75 in vivo imaging, 75 molecular features baculovirus system, 62–63 biochemical and crystallographic analysis, 63–64 catalytic domain, 62–63 MN antigen structure, 62 molecular weight, CA IX, 63 regulation CA9 gene expression, 66–68 CA IX ECD shedding, 68 extracellular acidosis, 67 HIF, 65, 66 HRE, genomic position, 66–67 normoxic conditions, 67 posttranslational level, 68 transcription, 65 role in cancer acidosis, 69 catalytic domain, 69 cell adhesion, 71 cell migration, 71 ectopic expression, 70–71 extracellular acidification, 71 oncogenic metabolism, 69 pH regulation, 68–70 targeting strategies cancer-related CA, 76–80 FITC-CAI binding, 79 pH control, 79 pre-clinical and clinical experiment, 76 SK-RC-52 (RCC) xenograft model, 80 sulfonamide inhibitors, 77–79 therapeutic target stems, 75 tissue distribution, 64–65

751 Carcinoma associated fibroblasts (CAFs) BMDMSCs activation and metastasis, 283–284 activation and tumor growth, 281–283 as source, 277–278 tumor stroma interactions, 280–281 cell-autonomous fashion, 331 characteristics, 276–277 counterpart and normal fibroblasts, 329, 330 CSC, 180 genetic and karyotype analysis, 329 heterogeneous cellular origins, 333–334 high-grade malignancy and poor prognosis, 328 immunohistochemical analysis, 330, 331 a-SMA expression, 329 TGF-b, 647 tumor associated stromal cells, 279–280 tumor stroma, 276 tumour-promoting stromal myofibroblasts, 327 CD4+CD25+ regulatory T cells, 396–397 CD34 cell, 259 CD9 interferes EGFR, 567–568 homoclustering, 566 integrin-mediated motility, 567 MMP2 expression, 569 positive correlation, 566 transendothelial migration, 569 tumorigenicity, 566, 569 WAVE2, 568 Wnt proteins, 567, 568 CD16 receptor, 436–437 CD44s cell surface-docking molecule, 537 CSPG, 485, 486 epidermal melanocytes, 484 galactosylation, 486 melanoma motility, 485 MMPs, 486–487 proteolytic removal, 485 shedding, 487 CD8+ T cells CTLs cell memory response, 391–392 MHC class I and MHC class II, 387–388 murine model, 388 TGF-b, 653 Cell culture method, 280 Cellular fibronectin (cFN), 459

752 Chemokines biological and molecular mechanisms, 19 intrinsic properties, tumor cells, 602 leukocyte recruitment CCL2, 609 immunosuppressive type 2 macrophage, 609 inflammatory and pathological stimuli, 608 lymphoreticular infiltration, 610 macrophages, 608, 609 primary tumor eradication, 601 receptors CC chemokines, 605–607 C chemokines, 606 classification, 603 CXC chemokines, 603, 604, 606 CX3C chemokines, 604, 606–607 leukocyte trafficking, 603 promiscuity, 607 targeting and chemotherapy, 617 tumor angiogenesis angiostasis, 613 CXCL5, 611 CXCL8, 610–611 CXCL10, 612 CXCL12, 611–612 CXCR3, 612 definition, 610 fractalkine, 612–613 renal cell carcinoma, 611 tumor growth and metastasis CCR4, 613–614 CCR7, 616 CXCL8, 615 CXCL9 and CXCL10, 616, 617 CXCR3, 616–617 CXCR4, 614–615 CXCR1 and CXCR2, 615 multifaceted role, 602, 603 SCH-479833 and SCH-527123, 615–616 tumor-host interaction, 613 Chemokine stromal-derived factor 1, 277 Chondroitin sulfate proteoglycan (CSPG), 485, 486 Chromatin, 173 Chronic inflammation, 124 vs. acute inflammation leukocyte infiltration, 411 pro-inflammatory response phase, 412–413

Index resolution phase, 414 vasculitis and pre-neoplastic syndromes, 412 dynamic renaissance, 410 Helicobacter pylori colonization, 410, 411 neoplastic transformation, 410 soluble mediator, immune response, 417 tumor immune evasion and progression, 415–416 Circulating endothelial cells (CECs), 264–265 Circulating endothelial progenitors (CEPs), 264–265 c-kit positive mast cells, 360 Collagen adhesion receptor binding CD44s, 484–487 DDRs, 487–488 a2b1 integrin, 483–484 RTKs, 487 THP, 483, 484, 488 angiogenesis, 482 cell homeostasis, 481 ECM cell proliferation, 479 b1 integrin and dystroglycan-dependent process, 481 intermolecular self-assembly, 481 pericytes, 480 fibronectin fragments, 496–497 fragments biological activities, 493, 494 cryptic sites, 493, 496 endostatin, 495 homotypic multimerization, 496 integrins, 493, 495 pentastatin-1, 495 synthetic RGD-containing peptides, 495 matrix metalloproteinases collagenolytic MMPs, 492 heterotrimeric type I and II collagen, 492 LOX, 482 metastatic melanoma, 491 tumor progression, 492 VEGF, 492, 493 mechanotransduction and oncogenic signaling pathways, 482 protein–collagen interactions decorin, 490–491 FN, 488–490 LN, 490

Index proteins C-terminal propeptides, 478–479 glycosylation, 478 homotrimeric and heterotrimeric collagens, 477 left-handed polyPro II helix, 477 NC1 domain, 479 nonfibrillar type IV collagen, 479 Connective tissue mast cells (CTMC), 356 Cortocotropin-releasing hormone (CRH), 356–358, 363 CO2 transmembrane efflux, 28 CpG site, 122–123 Cre-lox techniques, 315 CRH. See Cortocotropin-releasing hormone CRLR. See Calcitonin receptor-like receptor Crystallographic analysis, 63–64 CSC. See Cancer stem cells CSPG. See Chondroitin sulfate proteoglycan C-terminal Cys, 145 CTLs. See Cytotoxic T lymphocytes CXCL8, 282–283 CXCL12, 416. See also Stromal cell-derived factor 1 CXCR4, 416 Cytokine IL-17, 386 Cytotoxic agent, 318 Cytotoxic T lymphocytes (CTLs) CD8+ T cell cell memory response, 391–392 MHC class I and MHC class II, 387–388 murine model, 388 CD4+ T cells induction, 389–390 maintenance, 391 T helper 1 vs. T helper 2 responses, 392–393 D DDRs. See Discoidin domain receptors Decathlon, 214–215 2-Deoxyglucose (2-DG), 14 Desmoplasia, 461, 718 Diethylnitrosamine (DEN), 376 Discoidin domain receptors (DDRs), 242, 487–488 Disseminated tumor cells cancer progression cellular dormancy, 236–237 chromosomal aberrations, 230–231 dissemination theory, 230

753 gene expression analysis, 230 immunocytochemical techniques, 231 metastatic cascade, 230 micrometastasis (See Micrometastasis) dormancy models bone marrow, 246 cancer xenograft tumor cell model, 247 mouse models, 245–246 MYC oncogene, 247 phenotypic change, 246 solitary dormant tumor cells, 246 stroma-associated factors collagen matrix signaling, 241–242 hormone depletion dormancy, 242–243 TGFb signaling, 243–245 tumor microenvironment cellular dormancy, 240 deregulated ligand-dependent signaling, 240–241 ERK signaling, 241 genetic ablation, b1 integrin, 238 mitogenic signaling, 240 p38a activation, 241 paracrine tumor-stroma interactions, 238 DNA repair APE1, redox activity (See Apurinic/ apyrmidinic endonuclease 1) BER, 139–140 cancer therapeutic approach, 159–161 DR, 137–139 global influences, APE1 angiogenesis, 156–157 cell survival, 155–156 inflammation, 157–158 HR, 143–144 MMR, 140–141 NER, 141–142 NHEJ, 143 redox signaling GRX/GSH system, 146–147 Trx system, 145 schematics, 137, 138 tumor microenvironment, 158–159 Driver methylation, 127 Drosophila melanogaster, 190 Duffy antigen receptor for chemokines (DARC) expression cancer, 283 circulating chemokines and pathological conditions, 282–283 lymph node metastasis, 282 Durotaxis, 178

754 E ECM. See Extracellular matrix EDA-FN. See Extra Domains A-fibronectin EDB-FN. See Extra Domains B-fibronectin EGFR. See Epidermal growth factor receptor Embryonic stem cells (ESCs) cell cycle regulatory mechanisms, 174 characteristics, 172 ChIP-on-chip analysis, 173 epigenetic control, 173 mitogen signaling, MAPK pathway, 176 pluripotent cell characteristics, 174, 175 POU5FI, 172–173 Rb/p105, 174 R point, 174–176 telomerase, 172 transcriptional regulators, 172–173 EMT. See Epithelial–mesenchymal transition Endothelial cell colony forming units (CFU-ECs), 263 Endothelial colony-forming cells (ECFCs), 263 Endothelial progenitor cells (EPCs) BMDCs, 257 cellular players, 258 definition ECFCs, 263 hematopoietic marker, 263 multiparametric flow cytometry techniques, 262 VE-cadherin (CD144), 263 functions angiogenesis, 259 confocal laser scan microscopy, 261 FISH staining techniques, 260–261 Id proteins, 259 SDF-1 expression, 262 sprouting angiogenesis, 261 tumor vasculature, 260 VEGFR2 cells, 261 identification, 259 neoangiogenesis, 340 surrogate biomarker, 264–265 therapy-induced cell mobilization and tumor vessel incorporation antiangiogenic drug, 266 CEPs impact, 265–266 colonization, 266, 267 cytotoxic therapy, 267 macroscopic metastases, 268 MTD chemotherapy, 265–266 surface markers, 268–269 VDAs, 266

Index Epidermal growth factor receptor (EGFR) CD9 interferes, 567–568 CD82 interferes, 564, 565 malignant cell growth factors, 722–723 M-CSF receptor, 378 Epilogue, 130 Epithelial–mesenchymal transition (EMT) biomarkers, 188, 189 characteristics and classifications, 188, 517 definition, 649 differentiated epithelial cells, 517 DNA methylation, 128–129 E-cadherin cell–cell adhesion molecule, 188–189 gene expression and protein, 518–520 Rb down-regulation, 651 FBLN5, 649 FOXC2, 650 HMW-tropomyosins, 650 human hepatocellular carcinoma, 652 MECs, 649 metastatic process, 49–50 MMPs, 519 N-cadherin expression, 518–519 perspective, 204–205 phenotypic conversion, 187–188 signaling molecules, 518 Smad gene expression, 651 tetraspanins, 562, 567 tumour-promoting stromal myofibroblasts, 341 type 1 blastula stage, 189–190 mesoderm formation, 190–191 MET, 191 neural crest formation, 191 type 2 fibrosis, 191–192 re-epithelialization, wounded skin, 192 type 3 CSC generation, 202 cytokines, 199–201 EMT stimuli, 193–195 genetic and epigenetic control, 202–203 hypoxia, 201 micro RNA, 203–204 molecular regulation, EMT, 196 signaling pathways, 197–199 vimentin, 189 vimentin expression, 650–651 Epithelial ovarian cancer (EOC) metastasis model, 515, 516

Index ESCs. See Embryonic stem cells Extracellular acidosis, 67 Extracellular carbonic anhydrase, 32, 33 Extracellular matrix (ECM) adipocytes, 542 angiogenesis inhibitors, 706 apoptosis regulation, 699–700 stimulation, 700–701 broad inhibitors, 705 cell proliferation, 479 cellular regulation, 481 collagen fragments, 493, 496 collagen-mediated tumor cell behavior, 488 components, 538 3D cell migration, 545 decorin, 491 definition, 695 degradation, 542, 548 fibrillar collagen, 538 fibrillar proteins, 543 FN polymerization and incorporation, 489 b1 integrin and dystroglycan-dependent process, 481 integrin inhibitors, 705–706 intermolecular self-assembly, 481 invasive tumor and transformed cell growth, 544 LOX, 547 matrix component hydrolysis, 482 mechanical forces, 696–697 MMPs, 490, 491 pericytes, 480 pro-stromal factor, 718–720 protease inhibitors, 706 protein-protein interactions, 696 protein vitronectin, 497 proteolytic modification cancer progression, 701 cryptic domains, 702 soluble active peptides, 702–704 soluble growth factors, 704–705 remodeling, 536 sequestered TGFb, 539 tumor cell proliferation regulation, 698–699 type IV collagen, 538 type VII collagen, 490 VEGF, 543 Extra Domains A-fibronectin (EDA-FN) gene structure and FN-knock out, 458 tumor growth and angiogenesis, 463–464

755 Extra Domains B-fibronectin (EDB-FN) gene structure and FN-knock out, 458 potential function, 463 tumor growth and angiogenesis angiogenic switch, 463 cryptic site exposure, 464–465 EDA/EDB-double null mutants, 464 extensive immunohistochemical data, 462 F FAK. See Focal adhesion kinase Fibroblast growth factors (FGF), 713–714 Fibronectin (FN) fibrils, 467 knock out gene structure, 458–459 mice and phenotype, 460–461 molecular structure, 458 MSF, 465–466 potential function, EDB-domain, 463 synthesis and matrix assembly, 459, 460 therapeutic interventions anti-a5b1 integrin function-blocking antibody, 469 endogenous inhibitors, angiogenesis, 469–470 isoforms, 468–469 therapeutic mode, 467, 468 tumor angiogenesis definition, 462 EDA-FN, 463–464 EDB-FN, 462–463 tumor dormancy, 466–467 tumor growth autocrine/paracrine mitogenic factor, 461 desmoplasia, 461 EDA-FN, 463–464 EDB-FN, 462–465 TAMs, 461 tumor invasion and metastasis, 465 tumor stroma, 457 Fibulin-5 (FBLN5), 649 FKN. See Fractalkine Flt3 ligand (Flt3L), 722 Fluorescein-conjugated carbonic anhydrase inhibitor thioureidohomosulfanilamide (FITC-CAI), 79 Fluorescent in situ hybridization (FISH), 260–261

756 Fluorodeoxyglucose positron emission tomography, 27 FN. See Fibronectin Focal adhesion kinase (FAK) cell cycle progression, 513 cytoplasmic tails, 510, 512 downstream signaling pathways, 512 expression, 522 integrin signaling, 297–299 intracellular signals, 510 nuclear localization, 513 Src activation, 512 Forkhead box C2 (FOXC2), 650 Foxp3+ Tregs, 373 Fractalkine (FKN), 612–613 18

G Gastrulation, 190 Gene promoter region, 124 Glucose-6-phosphate (G6P), 92 Glucose transporters (GLUT1), 95–96 Glutaredoxin/glutathione (GRX/GSH) system, 146–147 Glycolysis ATP generation, 93 cancer AMP-activated protein kinase (AMPK), 104 cell growth and proliferation, 94 c-Myc, 104–105 GLUT1, 95–96 HIF-1, 100–103 HKII, 96 LDH-A, 97–98 mitochondrial dysfunction, 99–100 PFK1, 96–97 PI3K/Akt Pathway, 105–106 PPP, 98 p53 regulation, mitochondrial respiration, 103–104 putative mechanisms, 95 pyruvate kinase (PKM2), 97 Ras, 105 chemotherapy, 93 FDG-PET scan, 94 G6P, 92 metabolic alterations, 94 NAD+, 93 overview, 92 tumor inhibition 3-bromopyruvate, 107–108 2-deoxyglucose, 107

Index dichloroacetate, 109 lonidamine, 108 metabolic modulators, 110 metabolic regulatory mechanisms, 102, 107 oxythiamine and 6-aminonicotinamide, 109 Glycosylases, 151 Gonadotropin-releasing hormone (GnRH), 725 Graft-versus-host disease (GVHD), 438 Growth factor cytokines, 675, 679 expression and bio-distribution avb3-dependent upregulation, 681–682 ECM composition, 680 integrin-mediated reciprocal communication, 679 monocyte/macrophage infiltration, 680 VEGF, 680, 681 integrin cooperation ECM proteins, 683 functional associations, 683 initiation, invasive and maturation phase, 682 integrin receptors, 684–685 PTN receptor, 684 pulmonary inflammation and fibrosis regulation, 684 relative ratio and bioavailability, 679 signaling, 681–682 H Hedgehog (Hh) signaling, 336–337 Helicobacter pylori, 410, 411, 418 Helix-loop-helix transcription factor, 50 Hepatocarcinogenesis, 421 Hepatocellular carcinoma (HCC), 736 Hepatocyte growth factor (HGF), 714, 718, 725–726 HER, 722–723, 725 Hexokinases (HKII), 96 HGF. See Hepatocyte growth factor HIF-1. See Hypoxia inducible factor-1 Histidine decarboxylase (HDC), 362 HMC-1. See Human leukemic mast cells Homologous recombination (HR), 143–144 Human leukemic mast cells (HMC-1), 357, 358, 363

Index Human Leukocyte Antigen (HLA), 435–436 Human microvascular endothelial cells (HMVEC), 736 Human umbilical vein endothelial cells (HUVEC), 736, 741, 745 HUVEC. See Human umbilical vein endothelial cells Hybrid resistance model, 440 Hyperproliferating tumor cells, 213 Hypoxia, gene expression, and metastasis causes and consequences, 45–46 CSC CD133 expression, 52–53 iPS cell state, 51 leukocyte trafficking control, 53 PTEN tumor, 52 resistance, 51 striking correlation, CSC and cancer cell, 51 HIF regulation by genetic alterations of upstream regulators, 47–49 regulation by oxygen, 46–47 target genes, 49–50 metastatic process, 44, 45 Hypoxia inducible factor-1 (HIF-1) cancer cell metabolism and tumor microenvironment aerobic glycolysis, 14 HIF-1 inhibitors, 14–15 chemotherapy, 10–11 glycolysis, 100–101 heterodimeric protein, 4 human cancers, 4, 6 intratumor hypoxia anti-angiogenic therapies and HIF-1 inhibitors, 13 tumor vasculature normalization, 12–13 MDR-1, 10 mitochondria, 101–103 molecularly targeted agents, 9 oncogenic signaling pathways, 8 radiation therapy, 11–12 regulation, 4, 5 small molecule inhibitors cell-based HTS, 5 mechanism of action, 7, 8 protein–protein interaction, 7 signaling pathways, 6, 7 xenograft models, 8 Hypoxia inducible factor (HIF) CA IX, 65, 66 degradation, 25

757 HIF-a molecules, 46 regulation genetic alterations of upstream regulators, 47–49 oxygen, 46–47 signaling, complexity, 12 target genes, 49–50 Hypoxia-response element (HRE), 66 I IkB protein, 375 IKKb, 375–377 IL-15 cytokines, 434 Imatinib, 110 Induced pluripotent stem (iPS) cell state, 51 Inducible nitric oxide synthase (iNOS), 372 Infinite replication paradigm, 216 Insulin-like growth factor family of binding proteins (IGFBPs), 659 b3 Integrin, 682 Integrins EMT characteristics and classifications, 517 differentiated epithelial cells, 517 E-cadherin protein, 518–520 MMPs, 519 N-cadherin expression, 518–519 signaling molecules, 518 endothelial cells migration, proliferation, and survival, 296 expression and function, 294–296 growth factor receptor cooperation a1b1 and a2b1 integrins, 688 ligand/receptor signaling system, 685 nitric oxide production, 686 SMAD2 phosphorylation, 687 TCPTP, 688 TGF-bIIR, 686, 687 Tie-2/a5b1 association, 687 avb3, 685–687 VEGFR2, 685, 686 ligand specificity, 296–297 lymphangiogenesis (see Lymphangiogenesis) mechanotransduction, 520–521 motility, and invasion actin-rich protrusion, 514 ACTN4 and CTGF, 517 EOC metastasis model, 515, 516 filopodia and lamellipodia, 514 gene expression, 516–517 MT1-MMP, 515–516

758 Integrins (cont.) provisional tumor stroma, 514 receptor occupancy vs. aggregation, 515 pathological angiogenesis ECM ligands, 678 functional hubs, 675 functional integration, 678 growth factor (see Growth factor) growth factor receptor cooperation, 685–688 laminin-10 expression, 678–679 transmembrane heterodimers, 677 avb3, 678, 679 signaling antiadhesion strategy, 522 antitumor efficacy, 521 biomimetic scaffolds, 523 FAK, 297–299, 522 ILK expression, 522–523 LECs and ECM, interactions, 290 paxillin, 301 Rho family, GTPases, 300 SHC, 299–300 talin, 300 vinculin, 301 structure and function cell–matrix contact, 510 domain structure and conformational alteration, 510, 511 FAK, 510, 512–513 integrin-linked kinase, 513–514 monomeric ligand, 510 Interferon-a (IFN-a), 362 Interferon g, 434 Interferon-g (IFN-g), 362 Interleukin-7 (IL-7), 721 Interleukin-12 (IL-12), 721–722 Interleukin-15 (IL-15), 721 Interleukin-17 (IL-17), 722 Interleukin-like EMT inducer (ILEI), 652 Interwoven signaling network, 674 Intracellular oxidative stress, 146 Isocitrate dehydrogenase 1 (IDH1), 48 K Killer cell immunotherapy adoptive therapy adjunctive strategies, 444–445 adoptively transferred NK cells, 444 alternative NK cell sources, 443 haploidentical NK cells, 441–442 host factors, 445

Index NK cell lines, 443–444 in vitro NK cell expansion, 442–443 LAK cells, 441 Killer immunoglobulin-like receptors (KIR) gene, 435–436 KM12SM cells, 284 Krogh radius, 25 KTI. See Kunitz trypsin inhibitor Kunitz trypsin inhibitor (KTI), 656–657 L Lactate dehydrogenase (LDH-A), 97–98 Lewis lung carcinoma (LLC) cell line, 379 Loss of heterozygosity (LOH), 48 LOX. See Lysyl oxidase Lymphangiogenesis a4b1, 302–303 a5b1, 302 a9b1, 301–302 a1b1 and a2b1, 302 angiogenesis, 220 induction, 291–293 lymphatic makers, 291 lymphatic vasculature, 290 pathology, 293–294 Lymphatic vessels, 290 Lymphedema, 293 Lymphocytes, 433 Lymphoid progenitors, 432–433 Lymphokine activated killer (LAK) cells, 441 Lysyl oxidase (LOX) ECM, 547 HIF target genes, 50 hypoxia, 201 MMPs, 482 Lytic granule exocytosis, 435 M Macromolecules biosynthesis, 98 Macrophage-colony stimulating factor (M-CSF), 372, 378, 686 Macrophages angiogenesis, 376–377 anti-tumor potential/therapeutic implications, 379–380 chemoattractants, 372 chemokines, 372, 374, 378 immunosuppressive phenotype, 373–374 inflammation, 375–376 iNOS, 372

Index metastasis definition, 377 LLC cell line, 379 M-CSF and EGF receptor, 378 MMP-9, 379 MMTV-PyMT mice, 378 TAMs, 378–379 monocytes, 371 Maladaptive response, tissue or organ, 214 Mammary carcinogenesis, 359 Mammary epithelial cells (MECs), 649 Mast cells benefits chemoattractants, 357 histamine, 357, 358 immunosuppression, 358, 359 metalloproteinases, 357, 358 tumor angiogenesis, 357 biology, 356–357 breast cancer, 359–360 cancer cell proliferation, 355 degranulation nitric oxide (NO), 355 pro-tumor molecules, 361 SCF, 357 tumor-derived blockers, 362 heparan sulfate proteoglycans, 362 IFN-a and IFN-g, 362 lung cancer, 361–362 melanoma and basal cell carcinoma, 360 metastasis, 354 pancreatic cancer, 360–361 TRAIL, 362–363 tumor enhancing effect, 356 W/Wv mast cell deficient mice, 356 Matrikines angiostatin, 702, 704 anti-angiogenic peptides, 702, 703 arresten and canstatin, 703 endostatin, 702–703 fibulin-1 and fibulin-5, 704 MMP inhibitors, 703–704 TSP1 and 2, 704 tumstatin, 703 Matrix metalloproteinases (MMPs) anticancer effects, 540–541 biology, 532–533 cathepsin K, 704 chemistry, 533–534 chemokines, 538 collagenolytic MMPs, 492 fibrillar collagen, 538 function regulation

759 CD44, 537 epigenetic mechanism, 536 furin-like recognition domain, 536 gene expression, 535–536 hypothetical model, proMMP-2 activation, 536, 537 MT1-MMP, 536–537 in vivo activity, 535 gene expression signatures, 539–540 heterotrimeric type I and II collagen, 492 HMGA1, 539 inflammation and cancer, 547 inhibitors, 703–704 integrins, 519 invasive/metastatic cancer phenotype cancer cell invasion, three-dimensional matrix, 543–545 epithelial-to-mesenchymal transition, 545–546 premetastatic niche, 546–547 protease-independent cell invasion, 545 LOX, 482 malignant transformation, 531 metastatic melanoma, 491 migration, invasion, and metastasis, 217–219 natural inhibitors, 535 PAR-1, 539 physiologic vs. neoplastic invasion, 539 pro-stromal factors, 719–720 protease classification, 532 stromal cell production fibroblasts, 541, 542 immunohistochemical examinations, 541 squamous carcinoma cells, 542 tumor angiogenesis, 542–543 therapeutic targets catalytic Zn2+, 548 exocyte binding and alosteric inhibitors, 549 impressive tumor regression, 548 MT1-MMP, 548–549 RNAi technology, 549–550 type IV basement membrane collagen, 547 tumor progression, 492 VEGF anti-VEGF antibody, 549 ECM and glycosaminoglycan degradation, 705 primary tumors, 547 tumor angiogenesis, 542–543 type IV collagen, 492, 493

760 Maximum tolerated dose (MTD), 264 M-CSF. See Macrophage-colony stimulating factor Mechanism regulating tumor, 291, 292 Mechanoreciprocity, 696 Mechanosensitivity, 520 MEK/Erk activation, 682 Membrane transporters, role, 29 Membrane type 1 matrix metalloproteinase (MT1-MMP), 515–516 Mesenchymal–epithelial transition (MET), 188 Metastasis cascades decathlon, 214–215 infinite replication paradigm, tumor cells, 216 potential mechanisms, 215 CD81 and CD63, 569–570 CD9 interferes EGFR, 567–568 homoclustering, 566 integrin-mediated motility, 567 MMP2 expression, 569 positive correlation, 566 transendothelial migration, 569 tumorigenicity, 566, 569 WAVE2, 568 Wnt proteins, 567, 568 chemokines CCR4, 613–614 CCR7, 616 CXCL8, 615 CXCL9 and CXCL10, 616, 617 CXCR3, 616–617 CXCR4, 614–615 CXCR1 and CXCR2, 615 multifaceted role, 602, 603 SCH-479833 and SCH-527123, 615–616 tumor-host interaction, 613 disease, 214 EMT, 562 hypoxia, 44–45 macrophages definition, 377 LLC cell line, 379 M-CSF and EGF receptor, 378 MMP-9, 379 MMTV-PyMT mice, 378 TAMs, 378–379 migration and invasion angiogenesis process, 219–220 chemotaxis, 216

Index classic metastatic cascade, 217–218 clinical trials, MMP, 218 emboli formation, 217–218 heparin-binding factors, 217 lymphangiogenesis, 220 mechanistic similarities, 219, 220 MMP, blockage, 217 tumor-cell-centric view, 218 promoting activities, 579–580 rethinking metastasis angioprevention, 223–224 born metastatic hypothesis, 221–222 metastatic decathlon, 221 metronomic therapy, 224 pre-metastatic niche, 223 stem/cancer-initiating cells, 222 transcriptome profiles, 221 tumor cell colonization, 223 suppressor gene CD82/KAI1 c-Met signaling, 564, 565 cysteines, 563 EGFR, 564, 565 gangliosides contribution, 565 integrins and cadherins, 563 KITENIN, 565 rescue, 578–579 TM interactions, 566 uPAR and a5b1, 564 xenogeneic system, 564 Tspan8, 572–573 tumors as tissues, 213–214 Metastatic promoter, 243 Metformin, 110 Micrometastasis angiogenic dormancy, 231–234 immunity-driven dormancy apoptosis resistance mechanism, 236 cytotoxic role, 235 immunesurveillance, 234 non-Hodgkin’s lymphoma, 234 tumor dormancy vs. immune-mediated control, 234–235 Migration-stimulating factor (MSF), 465–466 Mismatch repair (MMR), 140–141 Mitochondrial DNA (mtDNA), 99 Mitochondrial fumarate hydratase, 100 Mitochondrial genomic integrity, 99 MMPs. See Matrix metalloproteinases MN antigen, 62. See also Carbonic anhydrase IX MSF. See Migration-stimulating factor MT1-MMP. See Membrane type 1 matrix metalloproteinase

Index Mucosal mast cells (MMC), 356 Multidrug resistance gene 1 (MDR-1), 10 Multivesicular bodies (MVB), 558, 573–574 MVB. See Multivesicular bodies Myeloid-derived suppressor cells (MDSCs) EMT stimuli, 195 mast cells, 359 myeloid cell recruitment, 423 N NADPH, 98 Na+/H+ exchangers (NHE), 30–31 Natural killer cells (NKC) activation, 434 anti-tumor response, 437 cytokine secretion, 434 cytotoxicity, 435 haploidentical transplantation allogeneic HCT, 438 GVHD risk, 438–439 non-myeloablative transplantation, 440 potential benefits, NK cell, 439 umbilical cord transplantation, 440 immunophenotype adhesion glycoprotein CD56, 432 ontogeny, 432–433 killer cell immunotherapy adoptive therapy, 441–445 LAK cells, 441 licensing, 437 localization and trafficking, 433 receptors activation, 436–437 signals activation and inhibitory, 435–436 tumor infiltrating lymphocytes, 438 Neoangiogenesis angiogenesis APE1, 156 metastasis, 376, 377 EPCs controversy surrounding functions, 261 Matrigel plugs, 262 integrins, 296 MMPs, 542 tumour-associated stroma anti-angiogenic therapy, 337 human breast MCF-7-ras tumours, 338–339 PDGF, 338, 340

761 proangiogenic cytokines and chemokines, 337, 338 therapy-resistant tumour, 338 VEGF, 337–338 tumourigenesis, 327 Neovascularization, 610 Neuropilin, 318 NF-kB. See Nuclear factor-kappa B NKC. See Natural killer cells NKG2D receptor, 437 Non-homologous end joining (NHEJ), 143 Non-small cell lung cancer (NSCLC), 362, 717, 718 Non-steroidal anti-inflammatory drugs (NSAIDs), 375 Notch signal, 191 Nuclear factor-kappa B (NF-kB) pro-inflammatory milieu, 419 proteins, 375–376 Nucleosome-free region (NFR), 124 Nucleotide excision repair (NER), 141–142 O Oncogenes activation c-Myc, 104–105 PI3K/Akt pathway, 105–106 Ras, 105 Oncogenesis, 9 Osseous metastasis, 659 Oxygen-dependent HIF regulation, 46, 47 P PAMP. See Proadrenomedullin N-terminal 20 peptide Pancreatic ductal adenocarcinoma (PDAC), 360 Parathormone related peptide (PTHrP), 704, 705 Passenger methylation, 127 Pasteur effect, 26 Patched 1 (PTCH1) receptor, 336 Pathological angiogenesis blood vessel formation arteriogenesis, 675 ECM molecules, 673, 674 embryonic vasculogenesis, 675 interconnected networks, 673 intussuception/sprouting, 676, 677 normal and pathological vessel formation, 675

762 Pathological angiogenesis (cont.) integrins ECM ligands, 678 functional hubs, 675 functional integration, 678 growth factor (see Growth factor) growth factor receptor cooperation, 685–688 laminin-10 expression, 678–679 transmembrane heterodimers, 677 avb3, 678, 679 neovascularization control, 674 receptor/ligand networks, 675, 676 Paxillin, 301 PDGF pathways, 312, 313 Pentose phosphate pathway (PPP), 98 Perforin, 435 Pericytes biology, physiology, and pathology, 312–313 resistance-antiangiogenic therapy adaptive resistance, 316 antivascular strategies, 317–318 bioavailability, tumor, 316 HIF1-a, 317 metastasis risk, 316 PDGF signaling, 316–317 VEGF pathway, 315 tumor angiogenesis, 313–315 Pharmacokinetics, CECs, 265 Phosphatase and tensin homologue (PTEN) signalling, 335 Phosphatidylinositol 3-kinase (PI3K), 105–106 Phosphofructokinase (PFK1), 96–97 Phosphoinositide 3 kinase (PI-3K) pathway, 48–49 Plasmacytoid and myeloid dendritic cells, 434 Platelet-derived growth factor (PDGF)-BB stimulation, 714 Pleiotrophin (PTN) receptor, 684, 686 Pluripotency, 172 31 P nuclear magnetic resonance (NMR), 28 POU5FI, 172–173 Proadrenomedullin N-terminal 20 peptide (PAMP) angiogenesis, 736 prostate cancer, 742 structure and function, 733 therapeutic target, 745 Progenitors, 312

Index Pro-inflammatory milieu bone marrow-derived cells, 424 chronic inflammation vs. acute inflammation, 411–414 dynamic renaissance, 410 Helicobacter pylori colonization, 410, 411 neoplastic transformation, 410 soluble mediator, immune response, 417 tumor immune evasion and progression, 415–416 malignant epithelial initiation, 414–415 matrix remodeling proteases, 418 myeloid cell recruitment chemokines, 422, 423 immune cell subpopulation, 422 MDSCs, 423 M2 polarization, 423, 424 TAM (See Tumor associated macrophages) oxidative stress species, 417–418 transcription factors and primary inflammatory cytokines IL-1, 421–422 IL-6, 420–421 NF-kB, 419 TGF-b, 420 TNF, 422 tumor progression, metastatic potential, and inflammation, 416–417 Proly hydroxylase, 101 Protease-activated receptor-1 (PAR-1), 539 Prox1, 291 PTHrP. See Parathormone related peptide Pyruvate kinase (PKM2), 97 R RANKL. See Receptor activator of nuclear factor-kB ligand RCC. See Renal cell carcinomas Receptor activator of nuclear factor-kB ligand (RANKL), 704, 705, 720–721 Receptor activity modifying protein (RAMP)2, 734, 735, 744 Receptor tyrosine kinases (RTKs), 487 Redox signaling DNA repair APE1, redox activity (See Apurinic/ apyrmidinic endonuclease 1) BER, 139–140 cancer therapeutic approach, 159–161 DR, 137–139

Index global influences, APE1, 154–158 HR, 143–144 MMR, 140–141 NER, 141–142 NHEJ, 143 schematics, 137, 138 tumor microenvironment, 158–159 GRX/GSH system, 146–147 Trx system, 145 Re-epithelialization, 192 Regional hypermethylation, 126–127 Regulatory CD4+ cells CD4+CD25+ regulatory T cells, 396–397 markers, 394–395 suppression, 397 tumor-induced CD4+ regulatory T cells anti-inflammatory mechanisms, 395 immune response, 396 origin, 398 types, 394 Renal cell carcinomas (RCC) adrenomedullin cell lines and human biopsies, 736 immunohistochemical analysis, 743 prognostic biomarker, 744 CA IX, 72, 75 CXCL1, CXCL3, CXCL5, and CXCL8, 611 CXCR4, 614 RENCAREX®, 76 Rho family, GTPases, 300 Rho-GTPase activation, 697 RNA interference (RNAi) technology, 549–550 S SDF-1. See Stromal cell-derived factor 1 Secreted growth factors immune system modulators, 721–722 malignant cell growth factors EGFR, 722–723 GnRH, 725 heregulin, 725 HGF/MET signaling, 725–726 homo- and heterodimers, 723, 724 insulin and IGFs, 726 KRAS mutation, 723 netrin-1, 726–727 neuregulins, 723 S100A4 protein, 727 Wnt family, 727–728

763 pro-angiogenic and lymphangiogenic factors adaptive/evasive resistance, 715 bevacizumab, 712, 717, 718 CXCR4, 716–717 EG-VEGF and Bv8 proteins, 715–716 FGF, 713–714 IFNa and IFNb, 714 lymphangiogenesis, 714, 745 metastatic renal cell cancer, 717 multiple myeloma plasma cells, 716 NSCLC, 717, 718 PDGF-BB stimulation, 714 PlGF, 712–713 receptor tyrosine kinase inhibitors, 9–10 signaling process, 712 tumor stromal and vascular cells, 715 pro-stromal factors MMP, 719–720 RANKL, 720–721 reactive stroma, 718 SPARC, 720 TGFb, 718–719 uPA, 719 Secreted protein acidic and rich in cysteine (SPARC), 720 SHC, 299–300 Small cell lung carcinomas (SCLC), 362 Squamous cell carcinoma (SCC), 279, 616 STAT3, 190–191 Stem cell factor (SCF), 357 Stroma-derived prognostic predictor (SDPP), 280 Stroma fibroblasts, 213 Stromal cell-derived factor 1 (SDF-1), 335 Stromal matrix metalloproteinases, 359 S180 tumor, 745 Succinate dehydrogenase (SDH), 100 Suppressor gene CD82/KAI1 c-Met signaling, 564, 565 cysteines, 563 EGFR, 564, 565 gangliosides contribution, 565 integrins and cadherins, 563 KITENIN, 565 rescue, 578–579 TM interactions, 566 uPAR and a5b1, 564 xenogeneic system, 564 Surrogate biomarker, 264–265 Synthesis of cytochrome c oxidase 2 (SCO2), 103–104

764 T Talin, 300 TAMs. See Tumor associated macrophages ab T cells, 386–387 Telomerase, 172 Tetraspanins angiogenesis CD151, 576, 577 mRNA and miRNA, 577 plasma protein extravasation, 562 platelet-derived exosomes, 577 Tspan8, 576–577 Tspan32, 577 CD151 and tumor cell motility adhesion process, 572 FAK competent and deficient fibroblasts, 571 human epidermoid carcinoma line, 570 knockdown cells, 571 TEM location, 572 egg–sperm fusion, 561, 579 exosomes coagulation and homeostasis, 575 mRNA and microRNA, 574 MVB, 573–574 proteins, 574 exosome–target cell interaction, 561 major functional activities cellular penetration, invasion, and fusion, 560 exosomal tetraspanins, 561 functional divergence, 562 MMP transcription and secretion, 561 molecule trafficking and biosynthesis, 560 metastasis CD81 and CD63, 569–570 CD9 interferes, 566–569 EMT, 562 promoting activities, 579–580 suppressor gene CD82/KAI1, 563–566, 578–579 Tspan8, 572–573 premetastatic niche, 575–576 structure, 556, 557 web CD82, 560 CD63 and CD151, 559 cytosolic signal transduction molecules, 557 exosome composition, 558, 559 MVB, 558 PKC, PI4KII and PLCg, 557–558 protein-protein interactions, 558

Index TEM, 558 transmembrane and cytosolic proteins, 556 TGF-b. See Transforming growth factor-b TGFb1, 192 TGF-b-response signature (TBRS), 647 Thioredoxin (Trx) system, 145 THP. See Triple-helical peptide Thrombospondin-1 (TSP-1), 700–701, 704 TILs. See Tumor-infiltrating lymphocytes Tissue fibroblasts generation, 188 Toll-like-receptors (TLRs), 412, 419 Transcription-coupled repair (TCR), 12 Transforming growth factor-b (TGF-b) adenocarcinoma, 634 ANGPTL4, 647–648 animal models CRT-PCR amplification, 661 doxycycline-mediated induction, 662 HT and WT mice, 661–662 immunostaining, 660, 661 MCS-1 cells, 664 MDA-MB-435 cells, 663 MMTV/PyVmT transgenic model, 662 Neu-induced mammary tumorigenesis and metastasis, 662 4NQO-induced rat malignant oral keratinocytes, 663 bronchioloalveolar carcinoma (BAC), 634 CAFs, 647 CD8+ T cells, 653 drugs, treatments, and therapies BBI and KTI, 656–657 bryostatin 1 and phorbol-12-myristate13-acetate, 655 calcium homeostasis, 657 13-cis-retinoic acid, 656 cyclooxygenase-2 metabolism, 654 decorin, 657–658 doxorubicin, 658 Fujimycin, 655 LM8-DCN cell, 657–658 NHBE and NSCLC cells, 655–656 rapamycin, 656 retinoid metabolism, 654 tranilast (N-[3,4dimethoxycinnamonyl]-anthranilic acid), 655 EMT, 649–652 ER-negative breast tumor, 647 genomics bone metastasis, 658, 659 gene expression, 659 HARA cells, 658–659

Index host-derived and cell autonomous effects, 660 p53 protein-associated genes, 659 PTHrP and ezrin identification, 658 immune system, 653–654 intratumoral histologic heterogeneity, 634 isoforms A549 cells, 639 immunohistochemical staining, 638 MTLn3 cells, 640 PC3 cells, 640 R3327-MATLyLu cells, 639 TGF-b1, 639–640 TGF-b3, 638–639 metastasis, 633–634 molecular cloning and functional analysis, 635 polypeptide growth factor, 646–647 primary carcinomas, 646 pro-stromal factors, 718–719 receptors Cre/LoxP technology, 644 Fc:TGF-b RII fusion protein, 644 integrins, 642 MDA-MB-435-F-L cells, 641–642 microarray analysis, 642 overexpressing cells, 643 potent immunosuppressive cytokine, 643 Smad2/3 signaling, 640–641 TGF-b RI gene mutation, 641 TGF-b RIIDN, 643–644 TGF-b RII expression, 642–643 TGF-b sRIII, 645 seed and soil theory, 636 signaling antiproliferative effects, 243 BHLHB3, 244–245 cancer cell invasion and metastasis, 341 cysteine knot, 636 growth response, 244 mechanisms, cellular dormancy, 239, 244 normal stroma-derived tumoursuppressive signalling, 335–336 role, DTC dormancy, 243–244 Smad family protein, 637–638 transcriptional signatures, 245 type I and type II receptors, 636–637 Smads, 645–646 TBRS, 647 tumor-associated host cells, 636 uPAR, 648 Transketolase and transaldolase, 98 Tregs. See T regulatory cells

765 T regulatory cells (Tregs), 373–374, 378 Triple combination therapy, 318 Triple-helical peptide (THP), 483, 484, 488 TSP-1. See Thrombospondin-1 Tumor associated fibroblast (TAFs), 277 Tumor associated macrophages (TAMs) EMT stimuli, 193–194 fibronectin, 461, 462, 465 macrophages anti-tumor potential/therapeutic implication, 379–380 immunosuppressive phenotype, 373–374 inflammation, 375, 376 iNOS and arginase, 372 metastasis, 378–379 myeloid cell recruitment functional and phenotypic properties, 423 IL-23/IL-17 axis, 424 leukocytes, 422 MDSCs, 423 M2 polarization, 423–424 pro-angiogenic factor, 718 squamous cell carcinoma invasion, 355 tumor angiogenesis and growth, 354 Tumor-conditioned medium (TCM), 278 Tumor hypoxia, 25, 26 Tumor-infiltrating lymphocytes (TILs) antitumor immunity, 387, 392–393 CD4+ T cells adaptive immunity, 388 CTLs (see Cytotoxic T lymphocytes) hallmark phenotype, 388 predominant effector mechanism, 389 T helper 1 vs. T helper 2 response, 392–393 CD8+ T cells cell memory response, 391–392 MHC class I and MHC class II, 387–388 murine models, 388 cytokine IL-17, 386 malignant tumors, 385 myeloid cell recruitment, 386 prognosis, 399–401 regulatory CD4+ cells CD4+CD25+ regulatory T cells, 396–397 markers, 394–395 suppression, 397 tumor-induced CD4+ regulatory T cells, 395–396, 398 types, 394 targeted tumor tissues, 401–402 ab T cells, 386–387 Th17 cells, 398–399

766 Tumor milieu, 281 Tumor pH acid transport H+ ion buffering, 30 H+ ions extrusion, 30–31 H+ ions, nonrespiratory sources, 31 intracellular and extracellular pH, 28 metabolic acid efflux, 29–30 pH regulation, 28–29 CA dominant role, 35 facilitated CO2 diffusion, 32–34 facilitated H+ diffusion, 34 intracellular and extracellular anhydrase isoforms, 32 transport metabolon, 34 cellular acid sources cellular respiration, 25–26 Warburg effect, 26–27 pH biological importance hydrogen nucleus, 23 pHi displacements, 24 pHi regulation, 24–25 weak acids/bases, 24 Tumor radioresistance, 11 Tumor suppressor p53, 279–280 Tumour-promoting stromal myofibroblasts CAFs, 327 cell-autonomous fashion, 331 counterpart and normal fibroblasts, 329, 330 genetic and karyotype analysis, 329 heterogeneous cellular origins, 333–334 high-grade malignancy and poor prognosis, 328 immunohistochemical analysis, 330, 331 a-SMA expression, 329 cancer cell invasion and metastasis EMT, 341 metastatic carcinoma cells, 340 pre-metastatic niche, 342 TGF-b-Smad signalling, 341 tumour cell-derived paracrine soluble factors, 342 desmoplastic/reactive stroma, 326 drug resistance, 343 epithelium, 325 neoangiogenesis anti-angiogenic therapy, 337 EPCs, 340 human breast MCF-7-ras tumours, 338–339

Index PDGF, 338, 340 proangiogenic cytokines and chemokines, 337, 338 therapy-resistant tumour, 338 VEGF, 337–338 normal stroma-derived tumour-suppressive signalling carcinoma cells, 337 Hh and Smo signaling pathways, 336–337 Notch and PTEN signalling, 335 SDF-1, 335 TGF-b signalling, 335–336 somatic genetic and epigenetic alterations, 331–333 tissue fibrosis share characteristics, 327–328 Twist, 50 Tyrosine kinase inhibitors, 314 U Umbilical cord blood (UCB) transplants, 440 uPA receptor (uPAR), 648 Urokinase-type plasminogen activator (uPA), 719 V Vascular disrupting agents (VDAs), 266 Vascular endothelial growth factor (VEGF) MMPs anti-VEGF antibody, 549 ECM and glycosaminoglycan degradation, 705 primary tumors, 547 tumor angiogenesis, 542–543 type IV collagen, 492, 493 neoangiogenesis, 337–338 pro-angiogenic and lymphangiogenic factors anti-VEGF antibody, 713 cell culture, 712 EG-VEGF, 715–716 glycoproteins, 712 lymphatic vessel growth, 714–715 PDGF, 717 PlGF, 712–713 Trap, 717–718 type IV collagen, 492, 493 VEGFR-3, 291 VEGF receptor (VEGFR) bone marrow-derived progenitor cells, 715 CXCR4+VEGFR1+ hematopoietic cells, 716

Index kinase activity, 713 PlGF signal, 713 receptor tyrosine kinase, 714, 717 signaling process, 712 Vimentin, 189 Vinculin, 301

767 Volociximab. See Anti-a5b1 integrin functionblocking antibody Von Hippel-Lindau syndrome, 48 W Warburg effect, 14, 26–27, 100, 280

The Tumor Microenvironment (Cancer Drug Discovery and Development)

Tumor Models in Cancer Research (Cancer Drug Discovery and Development)

Cancer Genome and Tumor Microenvironment

Cancer Genome and Tumor Microenvironment (Cancer Genetics)

Tumor Microenvironment

Antiangiogenic Agents in Cancer Therapy (Cancer Drug Discovery and Development) (Cancer Drug Discovery and Development)

Cancer Drug Resistance (Cancer Drug Discovery and Development)

Anticancer Drug Development Guide (Cancer Drug Discovery and Development)

Principles of Anticancer Drug Development (Cancer Drug Discovery and Development)

Genomics in Cancer Drug Discovery and Development

DNA Topoisomerases and Cancer (Cancer Drug Discovery and Development)

Apoptosis, Senescence and Cancer (Cancer Drug Discovery and Development)

Tumor Suppressor Genes in Human Cancer (Cancer Drug Discovery and Development)

Stem Cells and Cancer (Cancer Drug Discovery and Development)

Tumor Suppressor Genes in Human Cancer (Cancer Drug Discovery and Development)

Tumor Models in Cancer Research, Second Edition (Cancer Drug Discovery and Development)

DNA Repair in Cancer Therapy (Cancer Drug Discovery and Development)

Targets for Cancer Chemotherapy (Cancer Drug Discovery and Development)

Checkpoint Responses in Cancer Therapy (Cancer Drug Discovery and Development)

Chemokine Receptors in Cancer (Cancer Drug Discovery and Development)

Fluoropyrimidines in Cancer Therapy (Cancer Drug Discovery and Development)

Camptothecins in Cancer Therapy (Cancer Drug Discovery and Development)

Deoxynucleoside Analogs in Cancer Therapy (Cancer Drug Discovery and Development)

Farnesyltransferase Inhibitors in Cancer Therapy (Cancer Drug Discovery and Development)

Farnesyltransferase Inhibitors in Cancer Therapy (Cancer Drug Discovery and Development)

Antiangiogenic Agents in Cancer Therapy (Cancer Drug Discovery and Development)

Regional Cancer Therapy (Cancer Drug Discovery and Development)

Chemoradiation in Cancer Therapy (Cancer Drug Discovery and Development)

Gene Therapy for Cancer (Cancer Drug Discovery and Development)

Cancer Drug Design and Discovery

The Tumor Microenvironment (Cancer Drug Discovery and Development)

Tumor Models in Cancer Research (Cancer Drug Discovery and Development)

Cancer Genome and Tumor Microenvironment

Cancer Genome and Tumor Microenvironment (Cancer Genetics)

Tumor Microenvironment

Antiangiogenic Agents in Cancer Therapy (Cancer Drug Discovery and Development) (Cancer Drug Discovery and Development)

Cancer Drug Resistance (Cancer Drug Discovery and Development)

Anticancer Drug Development Guide (Cancer Drug Discovery and Development)

Principles of Anticancer Drug Development (Cancer Drug Discovery and Development)

Genomics in Cancer Drug Discovery and Development

DNA Topoisomerases and Cancer (Cancer Drug Discovery and Development)

Apoptosis, Senescence and Cancer (Cancer Drug Discovery and Development)

Tumor Suppressor Genes in Human Cancer (Cancer Drug Discovery and Development)

Stem Cells and Cancer (Cancer Drug Discovery and Development)

Tumor Suppressor Genes in Human Cancer (Cancer Drug Discovery and Development)

Tumor Models in Cancer Research, Second Edition (Cancer Drug Discovery and Development)

DNA Repair in Cancer Therapy (Cancer Drug Discovery and Development)

Targets for Cancer Chemotherapy (Cancer Drug Discovery and Development)

Checkpoint Responses in Cancer Therapy (Cancer Drug Discovery and Development)

Chemokine Receptors in Cancer (Cancer Drug Discovery and Development)

Fluoropyrimidines in Cancer Therapy (Cancer Drug Discovery and Development)

Camptothecins in Cancer Therapy (Cancer Drug Discovery and Development)

Deoxynucleoside Analogs in Cancer Therapy (Cancer Drug Discovery and Development)

Farnesyltransferase Inhibitors in Cancer Therapy (Cancer Drug Discovery and Development)

Farnesyltransferase Inhibitors in Cancer Therapy (Cancer Drug Discovery and Development)

Antiangiogenic Agents in Cancer Therapy (Cancer Drug Discovery and Development)

Regional Cancer Therapy (Cancer Drug Discovery and Development)

Chemoradiation in Cancer Therapy (Cancer Drug Discovery and Development)

Gene Therapy for Cancer (Cancer Drug Discovery and Development)

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