Signaling Pathways in Squamous Cancer

Signaling Pathways in Squamous Cancer

Adam B. Glick    Carter Van Waes ●

Editors

Signaling Pathways in Squamous Cancer

Editors Adam B. Glick, Ph.D. Associate Professor Center for Molecular Toxicology and Carcinogenesis Department of Veterinary and Biomedical Sciences The Pennsylvania State University and Department of Dermatology Penn State Milton S. Hershey Medical Center [email protected]

Carter Van Waes, M.D., Ph.D. Clinical Director and Chief, Head and Neck Surgery Branch, National Institute on Deafness and Other Communication Disorders, Senior Investigator, Radiation Oncology Branch, National Cancer Institute National Institutes of Health [email protected]

ISBN 978-1-4419-7202-6 e-ISBN 978-1-4419-7203-3 DOI 10.1007/978-1-4419-7203-3 Springer New York Dordrecht Heidelberg London  Springer Science+Business Media, LLC 2011 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 software, 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. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

Squamous epithelia form the lining surface of tissues in contact with the environment, including the skin, upper aerodigestive, respiratory and genital tracts, and several other specialized tissues. Cancers that form in squamous epithelia are among the most common human solid tumors due to increased exposure to environmental carcinogens such as ultraviolet light, tobacco smoke, and other genotoxic compounds, as well as infectious agents such as human papilloma virus. Late stage cancers of the upper aerodigestive tract, esophagus and cervix have high morbidity and there has been little improvement in survival. Thus there is compelling need to identify critical signaling pathways that regulate the development of squamous cancer and translate these findings into therapeutic targets to improve patient survival. In general, squamous epithelia are multilayered or stratified epithelia in which proliferation is confined to the basal layer in contact with the basement membrane, and squamous differentiation occurs as cells move away from the basal layer. As in any epithelium, proliferation and differentiation are tightly regulated by signaling pathways that respond to the external tissue and cellular microenvironment, and become dysregulated during progression to malignancy. This text addresses some of the most important signaling pathways that regulate normal growth and differentiation in squamous epithelia; how they are altered during progression to carcinoma; and their potential as therapeutic targets. The reviews include studies from human squamous cancers and cancer cell lines, as well as mouse two-stage skin carcinogenesis and genetically engineered mice, which provide meaningful animal models for the development of squamous cancers in multiple tissues. Because these different squamous tissues likely share similar regulatory networks, studies in one tissue or animal model are likely to have general significance for cancer development and therapy in other squamous epithelia. While each chapter focuses on a specific pathway and its role in squamous cancer, it is clear that these represent a network of interacting pathways that control many different aspects of normal keratinocyte homeostasis. Alterations in any one pathway during cancer progression are likely to impact several others. Interaction of epithelial cells with the extracellular matrix in the basement membrane through integrin receptors is critical for tissue integrity and control of epithelial cell proliferation. Chapters by Dr. Kramer, University of Pennsylvania and Dr. DiPersio, Albany Medical College and colleagues review recent studies on the alterations in v

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expression and adhesive interactions between integrins and their ECM ligands that drive local tissue invasion and progression squamous cell carcinoma. Equally important, extracellular signals that regulate cell growth come from both positive and negative growth factor signaling pathways. The epidermal growth factor receptor family and its ligands are critical regulators of both normal keratinocyte proliferation and differentiation, and aberrant expression and activation of this pathway is a consistent feature of squamous cancers. Chapters by Dr. Hansen, Creighton University and Dr. Grandis, University of Pittsburgh and their colleagues discuss recent data on the role of the epidermal growth factor receptor in mouse models of squamous cancer, human head and neck squamous cell carcinoma (HNSCC) and targeting this signaling pathway for therapy of HNSCC. The role of another important growth factor pathway, HGF/cMet, in the development of squamous cell cancer, and therapeutic targeting of this pathway is also discussed in a chapter by Dr. Zhong Chen, NIDCD, NIH. Transforming growth factor beta (TGFb1) is a critical negative regulator of keratinocyte proliferation but with important autocrine and paracrine roles in cancer pathogenesis that may both inhibit and enhance the malignant phenotype. The chapter by Drs. Reiss and Xie, UMDNJ-Robert Wood Johnson Medical School, examines the role of the TGFb1 signaling pathway and mutations in this pathway in HNSCC. Many of these growth factor pathways activate intracellular signaling molecules that are the center of important regulatory nodes controlling proliferation differentiation and inflammatory signaling in keratinocytes. Thus, this book contains several chapters which review studies in humans and mouse models that indicate an important role of Ras (Drs. Cataisson and Yuspa, NCI), Protein Kinase C (Dr. Denning, Loyola University), AKT and mTOR (Dr. Nathan et  al., LSU; Drs. Lin and Rocco, Harvard Medical School; Dr. Gukind et  al., NIDCR) and Cox-2 (Drs. Rundaug and Fischer, UT M.D. Anderson Cancer Center) in the development of squamous cancer, and the potential of these molecules as therapeutic targets. These signaling pathways converge on two transcription factor families AP-1 and NF-kB that play critical roles in gene expression that regulates keratinocyte proliferation, differentiation and inflammatory signaling. Drs. Bowden and Alberts, University of Arizona, and Hess and Angel, German Cancer Research Center review studies on ultraviolet activation of AP-1 signaling and potential therapeutic targets in this pathway and the role of AP-1 in mouse skin carcinogenesis, while Dr. Karin and colleagues, University of California, San Diego discuss recent studies on the role of NF-kB and IkB kinases in squamous cancer and the interaction with other signaling pathways. Other nuclear transcription factor families play critical roles in normal keratinocyte homeostasis and are frequently altered during progression to squamous cell carcinoma. Chapters on PPARs (Drs. Peters and Gonzalez, Pennsylvania State University and NCI), p. 63 (Drs. Roop and Koster, University of Colorado, Denver), retinoic acid receptors (Drs. Kadara and Lotan, UT M.D. Anderson Cancer Center) and vitamin D receptors review the important role of these transcription factor families in the regulation of epidermal proliferation and differentiation their role in squamous cancer. Finally the chapter by Drs. Zhou, Hu, and Wong, University of California at Los Angeles, describes

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recent advances in high throughput molecular profiling as a means to identify new genomic alterations and therapeutic targets for oral cancer. While this is not an exhaustive survey of all signaling pathways that regulate squamous cancer development we hope that pulling together this diverse research into one monograph will provide potential for cross-fertilization between researchers studying different aspects of squamous cancer, stimulate new research directions and highlight potential new targets for therapeutic intervention. The editors would like to thank their colleagues who contributed chapters to this book and to everyone in the research community that have made significant contributions to our understanding of this disease. Adam B. Glick Carter Van Waes

Acknowledgments

CVW supported by NIDCD Intramural Research Projects ZIA-DC-000016, 000073 and 000074.

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Contents

1 Cell Adhesion Molecules in Carcinoma Invasion and Metastasis......... Barry L. Ziober, Joseph O. Humtsoe, and Randall H. Kramer

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2 Roles of Integrins in the Development and Progression of Squamous Cell Carcinomas.................................................................. John Lamar and C. Michael DiPersio

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3 Alterations of Transforming Growth Factor-b Signaling in Squamous Cell Carcinomas.................................................................. Wen Xie and Michael Reiss

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4 Aberrant Activation of HGF/c-MET Signaling and Targeted Therapy in Squamous Cancer........................................... Zhong Chen

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5 The Epidermal Growth Factor Receptor in Normal and Neoplastic Epithelia............................................................................ 113 Susan K. Repertinger, Justin G. Madson, Kyle J. Bichsel, and Laura A. Hansen 6 Cyclooxygenase-2 Signaling in Squamous Cell Carcinomas................. 131 Joyce E. Rundhaug and Susan M. Fischer 7 Interacting Signaling Pathways in Mouse Skin Tumor Initiation and Progression............................................................ 149 Christophe Cataisson and Stuart H. Yuspa 8 Protein Kinase C and the Development of Squamous Cell Carcinoma........................................................................................... 165 Mitchell F. Denning

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  9 The Transcription Factor AP-1 in Squamous Cell Carcinogenesis: Lessons from Mouse Models of Skin Carcinogenesis............................................................................. 185 Jochen Hess and Peter Angel 10 NF-kB, IkB Kinase and Interacting Signal Networks in Squamous Cell Carcinomas................................................................ 201 Antonio Costanzo, Giulia Spallone, and Michael Karin 11 Regulation of Squamous Cell Carcinoma Carcinogenesis by Peroxisome Proliferator-Activated Receptors.................................. 223 Jeffrey M. Peters and Frank J. Gonzalez 12 p63 in Squamous Differentiation and Cancer....................................... 241 Dennis R. Roop and Maranke I. Koster 13 Effects of Natural and Synthetic Retinoids on the Differentiation and Growth of Squamous Cancers................... 261 Humam Kadara and Reuben Lotan 14 Regulation of Keratinocyte Differentiation by Vitamin D and Its Relationship to Squamous Cell Carcinoma.............................. 283 Arnaud Teichert and Daniel D. Bikle 15 Epidermal Growth Factor Receptor-Targeted Therapies.................... 305 Sun M. Ahn, Seungwon Kim, and Jennifer R. Grandis 16 Targeting UVB Mediated Signal Transduction Pathways for the Chemoprevention of Squamous Cell Carcinoma...................... 335 G. Tim Bowden and David S. Alberts 17 Molecular-Targeted Chemotherapy for Head and Neck Squamous Cell Carcinoma..................................................... 365 Harrison W. Lin and James W. Rocco 18 Effects and Therapeutic Potential of Targeting Dysregulated Signaling Axes in Squamous Cell Carcinoma: Another Kinase of Transcription and Mammalian Target of Rapamycin............................................................................... 383 Cheryl Clark, Oleksandr Ekshyyan, and Cherie-Ann O. Nathan 19 Head and Neck Cancer and the PI3K/Akt/mTOR Signaling Network: Novel Molecular Targeted Therapies................... 407 Panomwat Amornphimoltham, Vyomesh Patel, Alfredo Molinolo, and J. Silvio Gutkind

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20 High Throughput Molecular Profiling Approaches for the Identifications of Genomic Alterations and Therapeutic Targets in Oral Cancer............................................... 431 Xiaofeng Zhou, Shen Hu, and David T. Wong Index.................................................................................................................. 453

Contributors

Sun M. Ahn Department of Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA David S. Alberts Department of Medicine, College of Medicine, University of Arizona, Tucson, AZ, USA and Arizona Cancer Center, University of Arizona, Tucson, AZ, USA Panomwat Amornphimoltham Oral and Pharyngeal Cancer Branch, National Institute of Craniofacial and Dental Research, National Institutes of Health, Bethesda, MD, USA Peter Angel DKFZ-ZMBH Alliance, Division of Signal Transduction and Growth Control (A100), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany [email protected] Kyle J. Bichsel Biomedical Science, Creighton University School of Medicine, Omaha, NE, 68178, USA Daniel D. Bikle Endocrine Unit, University of California, San Francisco, CA, USA G. Tim Bowden Department of Cell Biology and Anatomy, Arizona Cancer Centre, University of Arizona, College of Medicine, University of Arizona, Tucson, AZ, USA and Arizona Cancer Center, University of Arizona, Tucson, AZ, USA [email protected] xv

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Christophe Cataisson Laboratory of Cancer Biology and Genetics, National Cancer Institute, NIH, Bethesda, MD, 20892, USA Zhong Chen Head and Neck Surgery Branch, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, 10 Center Drive, Building 10, 5D55, Bethesda, MD 20892, USA [email protected] Cheryl Clark Department of Otolaryngology-Head and Neck Surgery, Louisiana State University Health Sciences Center, Shreveport, LA, USA and Feist-Weiller Cancer Center, Shreveport, LA, USA Antonio Costanzo Department of Dermatology, University of Rome “Tor Vergata”, 00133, Rome, Italy Mitchell F. Denning Department of Pathology, Cardinal Bernardin Cancer Center, Loyola University Medical Center, 2160 S. First Avenue, Maywood, IL 60153, USA [email protected] C. Michael DiPersio Center for Cell Biology & Cancer Research, Albany Medical College, 47 New Scotland Avenue, MC 165, Albany, NY 12208, USA [email protected] Oleksandr Ekshyyan Department of Otolaryngology-Head and Neck Surgery, Louisiana State University Health Sciences Center, Shreveport, LA, USA and Feist-Weiller Cancer Center, Shreveport, LA, USA Susan M. Fischer The University of Texas M.D. Anderson Cancer Center, Science Park – Research Division, Smithville, TX 78957, USA [email protected] Frank J. Gonzalez Laboratory of Metabolism, National Cancer Institute, Bethesda, MD, 20892, USA

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Jennifer R. Grandis Department of Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA [email protected] J. Silvio Gutkind Oral and Pharyngeal Cancer Branch, National Institute of Craniofacial and Dental Research, National Institutes of Health, 30 Convent Drive, Building 30, Room 212, Bethesda, MD 20892-4340, USA [email protected] Laura A. Hansen Department of Biomedical Sciences, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA [email protected] Jochen Hess DKFZ-ZMBH Alliance, Division of Signal Transduction and Growth Control (A100), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany Shen Hu School of Dentistry, Dental Research Institute, University of California at Los Angeles, Los Angeles, CA, USA and Jonsson Comprehensive Cancer Center, University of California at Los Angeles, Los Angeles, CA, USA Joseph O. Humtsoe Department of Cell and Tissue Biology, University of California San Francisco, 521 Parnassus Avenue, Room C-640, San Francisco, CA, 94143-0640, USA Humam Kadara Department of Thoracic/Head and Neck Medical Oncology, University of Texas, M.D. Anderson Cancer Center, Houston, TX, USA Michael Karin Laboratory of Gene Regulation and Signal Transduction, School of Medicine, University of California, San Diego, 9500 Gilman Drive MC 0723, La Jolla, CA 92093-0723, USA [email protected] Seungwon Kim Department of Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

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Contributors

Maranke I. Koster Department of Dermatology, Charles C. Gates Center for Regenerative Medicine and Stem Cell Biology, University of Colorado Denver, Aurora, CO 80045, USA [email protected] Randall H. Kramer Department of Cell and Tissue Biology, University of California San Francisco, 521 Parnassus Avenue, Room C-640, San Francisco, CA 94143-0640, USA [email protected] John Lamar Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, E17-23002139, USA [email protected] Harrison W. Lin Department of Otolaryngology (H.W.L., J.W.R.), Massachusetts Eye and Ear Infirmary, Boston, MA, USA and Department of Otology and Laryngology (H.W.L., J.W.R), Harvard Medical School, Boston, MA, USA and Department of Surgery (J.W.R.), Massachusetts General Hospital, Boston, MA, USA Reuben Lotan Department of Thoracic/Head and Neck Medical Oncology, University of Texas, M.D. Anderson Cancer Center, Houston, TX, USA [email protected] Justin G. Madson Departments of Biomedical Science, Creighton University School of Medicine, Omaha, NE, 68178, USA and Department of Dermatology, The University of Oklahoma College of Medicine, Oklahoma City, OK, 73126, USA Alfredo Molinolo Oral and Pharyngeal Cancer Branch, National Institute of Craniofacial and Dental Research, National Institutes of Health, Bethesda, MD, USA

Contributors

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Cherie-Ann O. Nathan Department of Otolaryngology-Head and Neck Surgery, Louisiana State University Health Sciences Center, Shreveport, LA, USA and Feist-Weiller Cancer Center, Shreveport, LA, USA [email protected] Vyomesh Patel Oral and Pharyngeal Cancer Branch, National Institute of Craniofacial and Dental Research, National Institutes of Health, Bethesda, MD, USA Jeffrey M. Peters Department of Veterinary and Biomedical Sciences, The Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, University Park, PA 16802, USA [email protected] Michael Reiss Division of Medical Oncology, Department of Internal Medicine, The Cancer Institute of New Jersey, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ 08901, USA [email protected] Susan K. Repertinger Department of Pathology, Creighton University School of Medicine, Omaha, NE, USA James W. Rocco Department of Surgery, Massachusetts General Hospital, Jackson 904G, 55 Fruit Street, Boston, MA 02114, USA [email protected] Dennis R. Roop Department of Dermatology, Charles C. Gates Center for Regenerative Medicine and Stem Cell Biology, University of Colorado Denver, Aurora, CO, 80045, USA Joyce E. Rundhaug The University of Texas M.D. Anderson Cancer Center, Science Park – Research Division, Smithville, TX, 78957, USA Giulia Spallone Department of Dermatology, University of Rome “Tor Vergata”, 00133, Rome, Italy

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Contributors

Arnaud Teichert Endocrine Unit, University of California, San Francisco, CA, USA [email protected] David T. Wong School of Dentistry, Dental Research Institute, University of California at Los Angeles, Los Angeles, CA, USA and Jonsson Comprehensive Cancer Center, University of California at Los Angeles, Los Angeles, CA, USA Wen Xie Division of Medical Oncology, Department of Internal Medicine, The Cancer Institute of New Jersey, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ, 08901, USA Stuart H. Yuspa Laboratory of Cancer Biology and Genetics, National Cancer Institute, NIH, Bethesda, MD 20892, USA [email protected] Xiaofeng Zhou Center for Molecular Biology of Oral Diseases, College of Dentistry, University of Illinois at Chicago, Chicago, IL, USA [email protected] Barry L. Ziober Oncology, Centocor Ortho Biotech Inc, Radnor, PA, 19087, USA and Department of Otorhino-laryngology: Head and Neck Surgery, University of Pennsylvania, Philadelphia, PA, 19104, USA

Chapter 1

Cell Adhesion Molecules in Carcinoma Invasion and Metastasis Barry L. Ziober, Joseph O. Humtsoe, and Randall H. Kramer

Abstract  The primary reason for treatment failure in patients with head and neck squamous cell carcinoma (HNSCC) is local tumor cell invasion. HNSCC invasion is a necessary component of metastasis where tumor cells infiltrate into adjacent tissues, degrading basement membranes and extracellular matrix (ECM), and ­disrupting tissue architecture. These adhesive interactions of integrins with their ECM ligands are important not only in physically modulating HNSCC migration and invasion, but also in regulating the pathways required for survival and continued tumor expansion. During tumor progression, tumor cells must overcome a hostile microenvironment that can include hypoxia, growth factor deprivation, and loss of adhesion to the ECM. A second class of receptors expressed in HNSCC, the cadherins, form intercellular adhesions and are also relevant to the invasive process. These cell–cell adhesions are responsible for forming stratifying cell layers, but also influence the differentiated state of the tumor cells, and tend to restrain invasion. In these epithelial tumors, cadherin engagement can promote cell survival by a process termed “synoikis” that involves the receptor tyrosine kinase, EGFR. The complex signaling pathways transduced by integrin and cadherin receptors are poorly understood but are known to coordinately regulate such diverse cellular processes as apoptosis, proliferation, and the invasive phenotype.

1.1 Introduction Despite significant advancements in surgery, radiation, and chemotherapy, only about half of individuals diagnosed with head and neck squamous cell carcinoma (HNSCC) will survive for 5 years. HNSCC spreads by local and distant metastasis, and it is this aspect of the disease that is the most challenging for developing

R.H. Kramer (*) Department of Cell and Tissue Biology, University of California San Francisco, 521 Parnassus Avenue, Room C-640, San Francisco, CA 94143-0640, USA e-mail: [email protected] A.B. Glick and C. Van Waes (eds.), Signaling Pathways in Squamous Cancer, DOI 10.1007/978-1-4419-7203-3_1, © Springer Science+Business Media, LLC 2011

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s­ uccessful clinical approaches to therapy. The rich lymphatic drainage from the oral cavity and the highly invasive behavior of HNSCC facilitate the early spread to regional lymph nodes. Timely detection of malignant lesions is the most critical step at the present time to reduce the morbidity and mortality of HNSCC, but it is important that we gain a better understanding about the basic mechanisms of invasion that lead to recurrence and metastasis. The process of squamous cell carcinoma invasion and dissemination requires active cell migration through the extracellular matrix with the simultaneous remodeling of intercellular adhesions. These cellular processes are poorly understood and more effort is needed to identify signaling pathways regulating the invasive tumor phenotype, including how ­specific adhesion receptors are involved. Integrins are one class of adhesion receptors that mediate interactions with the surrounding extracellular matrix (ECM) and are clearly important in the invasive process. The intercellular adhesion receptors, predominately the cadherins, are another class of adhesion receptors that may restrain invasion and promote a more differentiated phenotype. In various carcinomas, cadherins can modulate cell locomotion by contact inhibition that can also lead to suppression of cell growth. Importantly, there is evidence for cross-talk and synergy between the two types of receptors whereby they act together to control various cellular functions.

1.2 Overview of Cell–ECM Adhesion Epithelial cell–matrix adhesion complexes mechanistically and functionally link cells to their underlying basement membrane. The underlying basement membrane is rich in ligands such as collagens, laminins, and fibronectin. It acts as an adhesive scaffolding to help maintain anchorage and organize cell layers and tissue architecture. The basement membrane also provides an important barrier between the ­epithelium and lamina propria. The integrins, a family of cell transmembrane receptors, are responsible for mediating the cell’s adhesive interactions with the ECM ligands (Patarroyo et al. 2002). Integrins provide a linkage between the cell cytoskeleton and the extracellular environment. Each integrin is a heterodimer composed of a noncovalently associated a and b subunit. In the case of head and neck squamous cell carcinoma (SCC), the major integrin receptors include the a2b1, a3b1, a5b1, a6b1/a6b4, and the av complexes (reviewed Ziober and Kramer 2003; Ziober et al. 2001). It is now well recognized that integrin interactions with the ECM can transduce signaling cascades that are not only important for adhesive and migratory functions, but are also important for cell growth, apoptosis, epithelial-mesenchymal transition, angiogenesis, protease production, differentiation, and gene expressions – all properties involved in malignant conversion and invasion (Aumailley et al. 2003; Goldfinger et al. 1998, 1999; Kosmehl et al. 1999; Niki et al. 2002; Shang et al. 2001). The interaction between integrins and laminins can regulate most, it not all, of these properties.

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1.3 Laminin-332 and SCC 1.3.1 Laminin-332 Expression in SCC Laminins are large, heterotrimeric extracellular glycoproteins composed of an a, b, and g subunit. To date, more than 12 different laminin heterotrimers have been identified (Colognato and Yurchenco 2000). The primary laminin expressed in stratified epithelium and derived carcinomas is laminin-5 (new nomenclature, laminin 332 Aumailley et al. 2005). Laminin-332 is composed of an a3, b3, and g2 subunit (Nguyen et  al. 2000; Rousselle et  al. 1991) and plays a significant role in SCC tumor biology. In SCC, laminin-332 is overexpressed primarily at the invasive front (Pyke et al. 1995). It was determined by immunohistochemistry and in situ hybridization, that in normal mucosa and lichen planus, laminin-332 was present as a thin, continuous line located in the basement membrane region. In epithelial dysplasia, this staining was discontinuous and more diffuse (Kainulainen et  al. 1997). However, there was a strikingly intense cytoplasmic staining of the carcinoma cells along the invasive border and in the individual infiltrating carcinoma cells in invasive carcinomas and lymph node metastasis (Kainulainen et  al. 1997). As tumor invasion is typically associated with hypoxic environments, recent evidence has determined that the hypoxia induced transcription factor HIF-1 contributes to cell motility and invasion by up-regulating laminins-5 expression and deposition (Fitsialos et al. 2008). These observations indicate that in SCC tumor progression, synthesis and secretion of laminin-332 are altered; the most intense expression occurs at the hypoxic invasive front and in the lymph node metastasis. Furthermore, it has been demonstrated that along with tumor cells, mesenchymal cells contribute to the synthesis and deposition of laminin-332 at the invasive front (Franz et al. 2007). Finally, it has been demonstrated that within distinct domains of laminin-332 reside sites for normal tumor adhesion as well as sites required for SCC tumorigenesis (Waterman et al. 2007). In addition to being expressed and contributing to SCC development and ­progression, laminin-332 has also been identified as a clinically unique tumor marker. Ono et  al. found a significant correlation between laminin-332-­ immunopositive SCC cells and increased infiltrative growth and poorer differentiation (Ono et  al. 1999). In addition, these same researchers, by analyzing patient survival data, found that increased laminin-332 expression was significantly associated with poorer patient outcome. Furthermore, several reports have indicated that the laminin-332 g2 chain is expressed in SCC of the skin, colon, esophagus, larynx, oral cavity, and recurrent lesions of these cancers (Ginos et  al. 2004; Patarroyo et al. 2002; Patel et al. 2002). Additionally, as described above, immunohistochemical staining indicates that the g2 chain of laminin-332 is dispersed in the invasive front and is a good indicator of a less favorable outcome (Kosmehl et al. 1999; Niki et  al. 2002; Pyke et  al. 1995). As a monomer, the g2 chain has been found frequently expressed in several cancers without the expression of the α3 or b3 chains (Koshikawa et al. 1999; Seftor et al. 2001). As such, the g2 chain of laminin-332

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has been detected circulating in patients’ serum with several cancer types and in particular invasive carcinomas (Katayama et al. 2005). Furthermore, oral mucosal lesions that express the laminin-332 g2 chain show an increased risk for tumor progression (Nordemar et  al. 2003). Recently, a monoclonal antibody to the g2 chain has been developed that can specifically detect the monomer g2 chain and not the laminin-332 ­heterotrimer (Koshikawa et al. 2008). This antibody should be a powerful diagnostic tool for detecting potential oral cancer lesions, g2-monmer expressing tumor cells and can also be a possible agent in cancer therapy. Overall, increased laminin-332 expression may provide a significant marker for SCC and tumor invasion, and be indicative of a poorer outcome for patients with SCC.

1.3.2 Role of Laminin-332 Processing in SCC The laminin-332 (Ln-5) heterotrimer is secreted as a 460-kDa precursor that undergoes specific proteolytic processing after secretion (Fig. 1.1). Ln-5 extracted from tissue is composed of a3 (165 or 145 kDa), b3 (145 kDa), and g2 (105 kDa) chains. In culture, keratinocytes synthesize a precursor form of a3 (190 kDa) and g2 (155 kDa) chains, which are processed to the tissue forms extracellularly (Ebihara et al. 2000; Koshikawa et al. 2000). Ln-5 is considered fully processed when the ­heterotrimer contains a 145-kDa a3 chain, a 145-kDa b3 chain, and a 105-kDa g2 chain. Recently

Cadherin Integrin

BM

Fig.  1.1  Adhesion receptors in head and neck squamous cell carcinoma cells. Integrins and c­ adherins mediate cell–matrix and cell–cell adhesions, respectively. Integrins are heterodimeric adhesion receptors and provide a linkage between the cell cytoskeleton and the extracellular environment. Cadherins form junctional adhesions between adjacent epithelial cells and involve the linker proteins, catenins, and the cytoskeleton. During tumor invasion, migrating cell penetrate the underlying laminin-rich basement membrane matrix (BM) and interact with the lamina propria interstitial extracellular matrix.

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it has been shown that the laminin-332 b3 chain is also proteolytically processed. The processing of all 3 chains of laminin-332 suggests another level of complexity in regards to the functions and activities of laminin-332. The function of Ln-5 processing is somewhat controversial. It appears that ­different proteolytically cleaved products of laminin-332 can yield diverse cellular responses when secreted and deposited. In normal tissues, the G4–G5 region of the laminin-332 a3 chain is proteolytically removed. However, in SCC, this a3 region is retained and plays a role in tumorigenesis by activating cell signaling pathways, metalloproteinase production, invasion, and tumorigenicity in  vivo (Tran et  al. 2008). In agreement, Bachy et  al. have shown that retention of the a3 precursor chain in laminin-332 provides a binding site for integrin a3b1 and syndecan-1 which when bound together triggers intracellular events required for keratinocyte migration (Bachy et al. 2008). The a3 chain of laminin-332 can also be cleaved by several enzymes including plasmin and astacins (bone morphogenic protein-1 and m-tolloid) (Amano et  al. 2000; Veitch et  al. 2003). Cleavage by these enzymes occurs between the globular modules 3 and 4 (LG3 and LG4) of the a3 chain favoring hemidesmosome assembly and stability. In support, the recombinant LG3 module of the a3 chain has been shown to support cell adhesion and migration in an integrin a3b1-dependent manner (Shang et al. 2001). Unlike the a3 chain, cleavage of the g2 chain by matrix metalloproteinase-2 (MMP-2), membrane type-1 matrix metalloproteinase (MT1-MMP) or astacins can promote binding of a3b1 to the a3 chain of laminin-332 and induction of cell migration (Giannelli et  al. 1997; Koshikawa et  al. 2000). Expression of both MMP-2 and MT1-MMP are up-regulated (Dumas et  al. 1999; Ondruschka et  al. 2002; Yoshizaki et al. 1997), while reports have indicated both a loss and a gain of the astacins in head and neck SCC (Veitch et al. 2003; Ziober et al. 2006). Recent work has demonstrated that only when g2 is completely processed is Ln-5 incorporated into the ECM, resulting in cells that are more resistant to enzymatic detachment (Gagnoux-Palacios et al. 2001). These results suggest that complete processing of Ln-5 is required for ECM development and the formation of stable adhesions. Conversely, unprocessed g2 subunits may support cell motility until processed. In support of this concept, we have recently shown in the oral cancer cell lines JHU022 and UM-SCC1 that the g2 chain is fully processed in JHU-022 while it is not processed in UM-SCC1 (Fig.  1.2) (Yuen et  al. 2005; Ziober in preparation). Furthermore, UM-SCC1 cells contain higher levels of GTP bound Rac1 and are more mobile and invasive through MatrigelTM than the JHU-022 cells (Yuen et al. 2005; Ziober in preparation; Fig.  1.2). Interestingly, UM-SCC1 invasion can be inhibited by infection with an adenoviral vector expressing a dominant negative Rac1. Together, these results suggest that failure to process Ln-5, in particular the g2 chain, may lead to activation of Rac1, cell motility, and tumor invasion. Finally and in contrast, it has been suggested that cell motility requires the processing of the g2 chain to the 105-kDa form (Veitch et al. 2003). It is believed that the g2 chain can negatively regulate the phosphorylation of laminin-332 binding integrin a6b4 and hemidesmosome formation (cell adhesion). This activity of the g2 chain involves syndecan-1 binding and its signaling which eventually leads to enhanced

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γ2

β3

Laminin 332

integrin

1 2

155 kDa 5 3 4

G domain

c

UM-SCC1

b

α3

JHU-022

a

Laminin-5 γ2 chain

105 kDa

d

Invasion through Matrigel

Rac1-GTP Total cellular Rac1

Fig. 1.2  Unprocessed Ln-5 correlates with increased Rac1 activity and head and neck squamous cell carcinoma (HNSCC) invasion. (a) Structure of Laminin-332 (Laminin-5). Laminin 5 is a heterotrimer consisting of the a3, b3, and g2 chains forming the typical laminin cross-shaped structure. Integrin receptors bind to the base region of the molecule. Both the a3 and g2 chains can be processed by proteolytic cleavage as indicated. (b) Conditioned medium from the poorly invasive JHU-022 and the more invasive UM-SCC1 HNSCC cell lines were immunoblotted with an anti-human laminin-332 g2 chain antibody. (c) Cell lysates from JHU-022 and UM-SCC1 HNSCC cell lines were assayed in the GTPase pulldown assay for Rac1 activity. GTP bound Rac1 (top) and total cellular Rac1 (bottom) were immunoblotted with an anti-human Rac1 antibody. (d)  SCC1 cells were infected with an adenoviral vector expressing green fluorescent protein (GFP) or dominant negative Rac1 (N17; DN-Rac1). Expression of DN-Rac1 in the UM-SCC1 cells inhibited cellular invasion in the Matrigel invasion assay as compared to both the control (uninfected) and GFP-infected SCC1 cells.

cell adhesion and inhibition of cell motility (Ogawa et al. 2007). Thus, it is possible that the binding of syndecan-1 to the g2 chain may be an important factor influencing cell migration or adhesion. More work is required to fully understand g2 processing and cell motility. Finally, the b3 chain of laminin-332 has recently been found to be cleaved as well. The processing of the b3 chain can occur by matrilysin (MMP-7) and hepsin, (Nakashima et al. 2005; Remy et al. 2006; Tripathi et al. 2008) and appears to be important for matrix assembly as well as cell adhesion and motility. More work is required to identify the regulatory factors responsible for laminin-332 cleavage and to understand how these cleavage products regulate the static and dynamic mechanisms of cell movement and tumor invasion.

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1.4 Laminin-332 Receptors and SCC Invasion and Motility 1.4.1 Signaling Pathways Activated by a3b1 Integrin SCC cells from the oral cavity express a number of integrin receptors for laminin, including a3b1, a6b1, and a6b4. The integrin a3b1 is now considered to be the major laminin-332 receptor (Baker et al. 1996; Mizushima et al. 1997) and has been reported to be responsible for mediating cell spreading and motility on laminin-332 substrates (Carter et al. 1991; DiPersio et al. 1997; Zhang and Kramer 1996). a3b1 is up-regulated in several SCC tumor cell lines and biopsies (Jones et  al. 1996). In particular, a3b1 is increased in the suprabasilar area during development of SCC (Van Waes 1995). In contrast, reduced expression of a3b1 has been reported to correlate with poor histological differentiation in SCC (Shang et al. 2001). Based on these studies, it is likely that a3b1 plays a significant role in SCC tumor development and tumor invasion; however, more thorough studies are required. Previously, it has been shown that laminin-332 promoted rapid cell scattering in HNSCC cells, whereas fibronectin and collagen-I did not (Kawano et al. 2001). The role of GTPases is probably important in such integrin-mediated responses to ­specific ECM ligands and may be important during tumor invasion. On laminin-332 substrate, a3b1 integrin preferentially inactivated RhoA and induced activation of Cdc42 and PAK1, and thereby promoted migration of oral SCC cells (Zhou and Kramer 2005). In contrast, on type I collagen, a3b1 integrin strongly activated RhoA, leading to enhanced focal contact formation, and thereby hindered cell migration. These results suggest that Rho signaling in SCC may be important in defining a cell’s invasive phenotype. Activation of RhoA/ROCK may be important in regulating invasion of HNSCC cells. For example, laminin-332 and a3b1 integrin have the ability to inactivate the RhoA pathway (Zhou and Kramer 2005) and enhance cell motility and invasion. Clearly, further studies are needed to establish whether inactivation of RhoA/ ROCK promotes invasion and metastasis in head and neck cancer. However, the RhoA/ROCK pathway plays an important role in metastasis in other malignancies including those of the bladder and breast cancer, and melanoma (Kamai et al. 2003; Nakajima et al. 2003; Nishimura et al. 2003). The influence of RhoA/ROCK in this process can be explained by work done by Sahai and Marshall (2003) where they identified two types of tumor cell motility in 3-dimensional matrices that involved different Rho signaling functionality and mode of invasion. Rho signaling through ROCK promoted a rounded bleb-associated mode of motility that does not require pericellular proteolysis. In contrast, elongated cell motility did not require Rho, ROCK, or ezrin function but was rather dependent on Rac for cell movement. Thus, it is apparent that the RhoA/ROCK pathway is a strong regulator of cell motility and invasion. a3b1 appears to be the major receptor for activating this pathway as well as other pathways required for SCC tumor development and progression. Adhesion of a3b1 to laminin-332 can stimulate a signaling pathway involving mitogen-activated protein kinase (MAPK), which appears to be important in

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r­ egulating cell growth and survival, thus presumably contributing to the development of SCC (Gonzales et al. 1999). In addition, a recent study has indicated that a3b1, when bound to laminin-332, can induce cross talk between the beta-catenin and Smad signaling pathways. When activated by laminin-332, these two pathways along with TGF-b1 can activate epithelial-mesenchymal transition during injury, tumor development, and tumor invasion (Giannelli et al. 2005; Kim et al. 2009). Similarly, a3b1 has also been shown to contribute to tumor invasion by up-regulating MMP-9 during p53 null and activated H-Ras epithelial cell transformation (Lamar et  al. 2008). Likewise, a3b1 when attached to laminin-332 regulates Src kinase signaling through FAK. This pathway eventually activates Rac1 and promotes lamellipodium extension which is indicative of migratory/invasive cells (Choma et al. 2004). Together, it appears that laminin-332 and a3b1 binding stimulates the phenotypic changes and pathways required for cell motility and invasion. However, work using mice that lack a3b1 specifically in the basal layer of the epidermis has indicated that a3b1 can also delay keratinocyte migration during wound healing (Margadant et al. 2009). Thus, it will take further investigations to decipher how a3b1 contributes to the migratory/invasive properties of SCC. For example, the migratory/invasive phenotype displayed by SCC tumor cells may be a result of signaling pathways activated by engagement of both a3b1 and the a6 integrin subunits to laminin-332.

1.4.2 Signaling Pathways Activated by a6b4 Integrin The a6 integrin is another important laminin-binding receptor in SCC cells (Zhang et al. 1996; Van Waes et al. 1991). a6 can pair with b1 or b4 in SCC cells, potentially giving rise to two laminin receptors. Because a6 preferentially combines with b4, this usually is the dominant complex found in SCC. In contrast to a3b1, a6b4 was originally believed to be involved in the static structures known as hemidesmosomes and to contribute little to cell migration. The extracellular laminin-332 anchoring molecules are directly linked with the keratin filament network within the cell by hemidesmosomes (Nguyen et  al. 2000). During wound healing, the dermal-epidermal adhesive structure provided by a6b4 is undesirable. Thus, for epithelial cells to migrate, they must first disassemble their hemidesmosomes (Goldfinger et  al. 1999). In fact, SCC tumor invasion may represent a normal wound repair process, involving laminin-332 and its two primary receptors a6b4 and a3b1 that are no longer properly controlled. a6 has been reported to show a higher expression in non-metastatic cells than in metastatic SCC cell lines (Jones et al. 1996). However, this subunit is upregulated in tumor biopsies from invasive and metastatic cases (Jones et al. 1996). Analysis of a6b4 levels in poorly differentiated SCCs revealed pronounced expression of this receptor along with laminin-332 at the invasive front (Mercurio et al. 2001). Furthermore, in tumors of patients with SCC, a6b4 levels have also been reported to be increased, localizing along the invasive border (Ilic and Damsky 2002).

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Earlier studies indicated that in normal squamous epithelium, a6b4 expression is limited to the contact sites of the basement membrane. In aggressive SCC, recurrent tumors, and metastatic tumor cell lines, this normal basal polarity of a6b4 is lost and there is a more intense suprabasal staining of a6b4 (Juliano 2002; Wolf and Carey 1992). This suggests that cytoplasmic or membrane components, possibly CD151 and bullous pemphigoid antigens 1 and 2, which function in a6b4 polarity and hemidesmosome formation, may be defective or lacking during SCC tumor progression (St Croix et al. 1996; St Croix and Kerbel 1997). Recent work using mouse tumor-initiating cells has begun to answer some of the questions regarding a6b4’s role in tumor development and progression. In this study, it was shown that a6b4 by not binding to laminin-332 but rather by recruiting the cytoplasmic linker protein, plectin, to the plasma membrane can suppress tumor growth (Raymond et  al. 2007). In contrast, when mouse tumor-initiating cells were further transformed with Ras, a6b4 stimulated tumor growth. Thus, depending on the transformation context, a6b4 can either mediate adhesion-independent tumor suppression or act as a tumor promoter. An interesting question arises as to the regulation of a6b4 in normal epithelial architecture versus tumor invasion: if a6b4 is primarily involved in inhibiting cell migration through the formation of hemidesmosomes, why does an increased expression of this receptor not inhibit SCC tumor invasion? Studies directed at understanding the signaling pathways elicited by a6b4, including the signaling pathways that disrupt the hemidesmosome structure, the factors responsible for proper cell polarity, and the proteolytic regulation of the motility-inducing laminin-332 isoforms, are required to answer this question. For example, in keratinocytes phosphorylation of the cytoplasmic tail of a6b4 by protein kinase C-delta redistributes this integrin from the hemidesmosome to the cytosol (Alt et al. 2001). It should also be noted that the cytoplasmic interactions of a6b4 may also play important roles in whether this receptor functions in cell adhesion or motility. In this regard, BPAG1, which localizes to the inner surface of hemidesmosomal structures containing a6b4, positively influences cell migration when removed by homologous recombination (Day et al. 1999). In agreement with this work, it has recently been shown that BPAGe1 is required for efficient regulation of keratinocyte polarity and migration by activating Rac1 (Hamill et al. 2009). This body of work suggests that a6b4 is probably a more important regulator of SCC cell motility and tumor invasion than originally thought. a6b4, by signaling to Rac1 and the actin-severing protein, cofilin, regulates the assembly of laminin-332 tracks required for keratinocyte migration (Kligys et al. 2007). Furthermore, a6b4 via activation of Rac1, 14-3-3 proteins, and slingshot phosphatase family members (SSH) can regulate cell polarity and migration (Kligys et al. 2007). Interestingly, that cofilin is a major regulator and is involved in invadopodia, suggests that a6b4, laminin-332, and the Rac1/SSH pathway may be instrumental in regulating SCC tumor invasion. Finally, more recent studies have confirmed this work by showing that 14-3-3zeta/tau heterodimers regulate the activity of SSH and cytoskeleton remodeling during cell migration in keratinocytes (Kligys et al. 2009).

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Decreased expression of a6b4, in direct contrast to the reports above, has also been described in SCC. Normally, a6b4 expression shows a basally polarized ­distribution. Lack of a restricted basal polarity of a6b4, by absence of b4 expression, has been suggested to be an early marker of oral malignancy (Garzino-Demo et al. 1998). Examination of normal and epithelial SCC tissue sections for a6 expression demonstrated that the staining intensity of this subunit was significantly reduced in SCC compared to normal epithelium (Maragou et  al. 1999). Similarly, a6b4 was found to be down-regulated in oral SCC, and this reduction in a6b4 correlated with poor histological differentiation (Shang et  al. 2001). Thus, loss of a6b4 and the absence of hemidesmosome formation may result in a more motile and invasive SCC tumor. This notion is supported by the fact that blocking antibodies to b4 can result in stimulation of cell migration and an increase in MMP-2 activity (Daemi et  al. 2000). Finally, transfection of the b4 subunit into a neoplastic keratinocyte cell line failed to restore differentiation capacity or proliferation properties, suggesting that a6b4 is not required for these properties in SCC (Jones et al. 1996).

1.5 Intercellular Adhesion and Signaling In normal epithelial cells, survival, growth, and proliferation are dependent on the coordinated involvement of ECM and growth factors (Cabodi et al. 2004; Miranti and Brugge 2002). The loss of attachment to ECM can result to cellular stress that eventually may trigger programmed cell death or anoikis (Frisch and Screaton 2001; Gilmore 2005). However, during tumor progression, SCC cells have the ability to adapt to adverse microenvironmental conditions that include hypoxia as well as nutrient, growth factor, and anchorage deprivation. For tumor cells to continue to survive and grow, they may use adaptive mechanisms that favor survival through inter- and intracellular signaling pathways. The cellular processes controlling these signaling pathways remain complex and poorly understood. Understanding how SCC cells become insensitive to anoikis is important because such survival mechanism may not only permit continued tumor expansion, but may also favor tissue invasion and metastasis. One mechanism by which carcinoma cells may overcome these diverse biological stresses is through their ability to form intercellular adhesions (Alt-Holland et al. 2005; Bates et al. 2000).

1.5.1 E-Cadherin Mediated Signaling In the absence of proper cell attachment to ECM, acquisition of cell–cell adhesion may induce critical signaling pathways to promote survival and growth regulating responses (Bates et al. 2000; Santini et al. 2000). Typical SCCs are characterized as nests of 3-dimensional aggregates or tumor islands with an extensive network of intercellular adhesions. Many of the cells in the tumor nests lack a direct interaction

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with the surrounding extracellular matrix and these cells could depend on the ­survival signaling generated by cell–cell contacts. In some SCCs, cell aggregation induces adhesion-mediated signaling that is dependent on the engagement of ­cellular adhesion receptors, such as E-cadherin. For example, previous work in a 3-dimensional multicellular model showed that E-cadherin-dependent intercellular adhesion mediated the anchorage independent survival and growth of SCC cells (Kantak and Kramer 1998). Another study further demonstrated that cell–cell adhesion through E-cadherin could trigger a ligand-independent transactivation of EGFR to promote survival primarily through activation of the ERK signaling pathway (Shen and Kramer 2004). These studies have led to the use of the Greek term, synoikis, to describe the process of intercellular adhesion-dependent cell survival (Kramer et al. 2005; Shen and Kramer 2004). Other studies in Ewing tumor (Kang et al. 2007; Lawlor et al. 2002) and breast epithelial cells (Fournier et al. 2008) have shown that E-cadherin can mediate survival and growth in anchorage independent cultures. The E-cadherin mediated Ewing cell spheroids induce PI3-kinase through the ErbB4 tyrosine kinase receptor (Kang et  al. 2007). In different experimental settings where the cells in study were subjected to ECM-adherent condition, E-cadherin engagement has been shown to activate various signaling components that also affect cellular function. For example, E-cadherin was found to mediate the induction of MAPK (Pece and Gutkind 2000; Reddy et al. 2005), Rac (Betson et al. 2002; Kovacs et al. 2002), STAT3 (Onishi et al. 2008), and PI3-kinase (Pang et al. 2005) activity following cell–cell contact formation in monolayer adherent conditions. This intercellular adhesion-mediated signal transduction appears to involve several tyrosine and non-tyrosine protein kinases. The activation of MAPK and Rac appears to occur through EGFR signaling (Betson et al. 2002; Pece and Gutkind 2000; Reddy et  al. 2005), while PI3-kinase is controlled by c-Src kinases (McLachlan et al. 2007; Pang et al. 2005). The JAK and Src signaling pathway also seems to play a major role in regulating E-cadherin induced STAT3 activation (Onishi et  al. 2008). Using recombinant E-cadherin-Fc to ligate E-cadherin, Liu et al. (2006) have shown that direct E-cadherin engagement alone is sufficient to promote proliferation through Rac activation. Besides E-cadherin’s role in serving as a mechanical support between neighboring cells, it is strongly evident that E-cadherin can also directly influence and modulate distinct signaling pathways potentially dictating cellular functions in a context dependent manner. A recent study showed that intercellular adhesion is sufficient to induce STAT3 activation in HNSCC (Onishi et al. 2008). This observation is in support of other previous studies in monolayer adherent culture conditions where cell–cell contact induced enhanced STAT3 activation (Vultur et al. 2004). STAT3, a member of the STAT family proteins, is generally activated by multiple receptor and nonreceptor tyrosine kinases in response to various cytokines, hormones, and growth factors (Levy and Darnell 2002). Constitutive activation of STAT3 has been demonstrated in several human cancers, including HNSCC (Grandis et  al. 2000). It has been ­suggested that STAT3 signaling induced by E-cadherin-mediated cellular adhesions may also play a role in conferring resistance to anoikis in SCC cells (Onishi et al. 2008). Understanding how intercellular adhesions in SCC cells activate STAT3 is

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important since targeting STAT3 is being viewed as an effective therapeutic strategy in SCC of the head and neck (Boehm et al. 2008; Leeman et al. 2006). Recent studies add more to the growing evidence that suggests that intercellular adhesion mediated signaling may recruit different signaling components depending on the cell type. For example, aggregations of colonic epithelial cells mediate cell survival through Src and PI3-kinase signaling that involves beta-catenin (Hofmann et  al. 2007). Aggregates of malignant pleural mesothelioma cells have also been shown to exhibit the ability to resist anoikis (Daubriac et  al. 2009). This study showed that the inactivation of the SAPK/JNK signaling pathway due to loss of anchorage may be involved. Because of its role in survival and growth, further studies to elucidate the molecular mechanism of intercellular adhesion-mediated signaling is needed.

1.5.2 E-Cadherin Mediated EGFR Activation The molecular signaling networks and mechanisms promoting synoikis appear to be complex and remain to be understood. One of the prime signaling axes that appears to regulate this process in SCCs is through the E-cadherin-mediated ligandindependent activation of EGFR (Shen and Kramer 2004). Despite the existing evidence of a molecular crosstalk between E-cadherin and EGFR signaling, the mechanisms by which E-cadherin transactivates EGFR still remain unclear. Earlier work from Kemler’s group (Hoschuetzky et al. 1994) showed that the cytoplasmic E-cadherin/EGFR association is mediated through interaction with b-catenin. Subsequent reports from others provide evidence that the extracellular region of E-cadherin is necessary for its interaction with EGFR (Fedor-Chaiken et al. 2003; Qian et al. 2004). Several studies carried out in adherent or anchorage independent condition have demonstrated ligand-independent EGFR activation and the ability of EGFR to associate with E-cadherin (Fedor-Chaiken et al. 2003; Pece and Gutkind 2000; Shen and Kramer 2004). These studies point to the competence of EGFR to associate with E-cadherin as an essential event in the process of E-cadherin induced EGFR activation (Gavard and Gutkind 2008). In addition to this finding, our laboratory has analyzed the EGFR/E-cadherin interactions following chemical cross-linking and resolution in two-dimensional gel (Fig. 1.3). First, it is evident that for efficient physical interactions between the two receptors, a stable cell–cell contact is essential. Secondly, it reveals that EGFR can exist in multiple structural complexes. The ability of EGFR to form these ligand-independent higher order structures appears to be dependent on the extent of cell–cell contact formation. These structures may represent the different EGFR oligomers that are formed during adhesion-mediated receptor activation. Generally, EGFR undergoes homo- and heterodimerization in response to ligand binding, an initial event in the EGFR signaling pathway (Jorissen et  al. 2003; Schlessinger 2002). It is possible that upon initiation of stable cell–cell adhesions through E-cadherin, EGFR may dimerize or heterodimerize with other EGFR family ­members,

High density (ML) Ca++ Switch Assay

Low density (ML)

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EGFR

E-Cad

EGFR

EGTA

E-Cad EGFR

Ca++

E-Cad

MCA

EGFR E-Cad

Fig. 1.3  Two-dimensional gel analysis of EGFR structural complexes induced during cell–cell adhesions. The cell membrane impermeable protein crosslinker, DTSSP, was added to HSC-3 cells cultured at very low- and high-density or as multicellular aggregates (MCA) (Onishi et al. 2008). The high-density monolayer cell culture was subjected to a Ca++-switch assay prior to DTSSP treatment. Cell lysates were then prepared for immunoprecipitation with an EGFR antibody. The EGFR immunocomplex was then resolved in the first dimension (4% non-reduced tube gel), followed by incubation in b-mercaptoethanol containing buffer, and then resolved in the second dimension (7% reducing slab gel). Western blotting was then performed for EGFR and E-cadherin. Arrowheads indicate monomers, while the arrows indicate the slower mobile structures representing homo- and heteroligomerization.

or become physically associated with other cell surface molecules, including E-cadherin (Fig. 1.3). The formation of such hetero-oligomers could then lead to higher EGFR aggregation, and thus EGFR transactivation in a ligand-independent manner (Gavard and Gutkind 2008). Nevertheless, the presence of multiple EGFR forms reflects the complexity of the mechanism of EGFR signaling that is induced by E-cadherin mediated intercellular adhesions. In addition, the nature of E-cadherin dependent EGFR signaling needs to be further addressed. For example, such mode of EGFR signaling appears to be distinct from that of ligand-induced signaling events (Humtsoe and Kramer, submitted). While ligand treatment can stimulate efficient phosphorylation of EGFR tyrosine residues, intercellular adhesion alone produces inefficient tyrosine phosphorylation at residue EGFR-Y1086. Furthermore, in the multicellular spheroid model, intercellular adhesion-mediated EGFR

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a­ ctivation alone fails to transduce efficient PI3K/AKT signaling which eventually regulates cell proliferation (Humtsoe and Kramer, submitted). Perhaps, in tumor islands of cadherin-mediated compact cell aggregates, the nature and specificity of EGFR signaling may be determined by the extensiveness of cell–cell junctions and the microenvironment. Unraveling the signaling mechanism that drives the process of synoikis can be greatly challenging. Signals produced by intercellular adhesion-mediated activation maybe important for prolonged survival and growth of tumor cell aggregates that are commonly present in SCC lesions in vivo. The dissemination of metastatic cells occurs when malignant cells detach from their primary tumor site and infiltrate to surrounding tissues. The malignant cells develop mechanisms to suppress anoikis while transiting through the ECM-deficient lymphatic and vascular systems. Thus, in malignant cell aggregates, cell survival through synoikis may be advantageous in rendering a greater likelihood of implanting and forming metastatic lesions. Selective targeting of cell–cell adhesion-induced signaling pathways during tumor metastasis may represent an effective therapeutic approach. Interestingly, maintenance of cadherin-mediated cell–cell interactions not only promotes cell survival, but enhances resistance to chemotherapeutic agents (Kang et  al. 2007; Nakamura et  al. 2003). Future studies are needed to elucidate the complicated crosstalk of cell surface molecules in cell–cell and cell–ECM adhesion and in understanding how these interactions induce a complex array of important downstream signaling pathways.

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Kligys K, Claiborne JN, DeBiase PJ, Hopkinson SB, Wu Y, Mizuno K, Jones JC (2007) The slingshot family of phosphatases mediates Rac1 regulation of cofilin phosphorylation, laminin-332 organization, and motility behavior of keratinocytes. J Biol Chem 282:32520–32528 Kligys K, Yao J, Yu D, Jones JC (2009) 14-3-3zeta/tau heterodimers regulate Slingshot activity in migrating keratinocytes. Biochem Biophys Res Commun 383:450–454 Koshikawa N, Moriyama K, Takamura H, Mizushima H, Nagashima Y, Yanoma S, Miyazaki K (1999) Overexpression of laminin gamma2 chain monomer in invading gastric carcinoma cells. Cancer Res 59:5596–5601 Koshikawa N, Giannelli G, Cirulli V, Miyazaki K, Quaranta V (2000) Role of cell surface metalloprotease MT1-MMP in epithelial cell migration over laminin-5. J Cell Biol 148:615–624 Koshikawa N, Minegishi T, Nabeshima K, Seiki M (2008) Development of a new tracking tool for the human monomeric laminin-gamma 2 chain in vitro and in vivo. Cancer Res 68:530–536 Kosmehl H, Berndt A, Strassburger S, Borsi L, Rousselle P, Mandel U, Hyckel P, Zardi L, Katenkamp D (1999) Distribution of laminin and fibronectin isoforms in oral mucosa and oral squamous cell carcinoma. Br J Cancer 81:1071–1079 Kovacs EM, Ali RG, McCormack AJ, Yap AS (2002) E-cadherin homophilic ligation directly signals through Rac and phosphatidylinositol 3-kinase to regulate adhesive contacts. J Biol Chem 277:6708–6718 Kramer RH, Shen X, Zhou H (2005) Tumor cell invasion and survival in head and neck cancer. Cancer Metastasis Rev 24:35–45 Lamar JM, Iyer V, DiPersio CM (2008) Integrin alpha3beta1 potentiates TGFbeta-mediated induction of MMP-9 in immortalized keratinocytes. J Invest Dermatol 128:575–586 Lawlor ER, Scheel C, Irving J, Sorensen PH (2002) Anchorage-independent multi-cellular spheroids as an in vitro model of growth signaling in Ewing tumors. Oncogene 21:307–318 Leeman RJ, Lui VW, Grandis JR (2006) STAT3 as a therapeutic target in head and neck cancer. Expert Opin Biol Ther 6:231–241 Levy DE, Darnell JE Jr (2002) Stats: transcriptional control and biological impact. Nat Rev Mol Cell Biol 3:651–662 Liu WF, Nelson CM, Pirone DM, Chen CS (2006) E-cadherin engagement stimulates proliferation via Rac1. J Cell Biol 173:431–441 Maragou P, Bazopoulou-Kyrkanidou E, Panotopoulou E, Kakarantza-Angelopoulou E, Sklavounou-Andrikopoulou A, Kotaridis S (1999) Alteration of integrin expression in oral squamous cell carcinomas. Oral Dis 5:20–26 Margadant C, Raymond K, Kreft M, Sachs N, Janssen H, Sonnenberg A (2009) Integrin alpha3beta1 inhibits directional migration and wound re-epithelialization in the skin. J Cell Sci 122:278–288 McLachlan RW, Kraemer A, Helwani FM, Kovacs EM, Yap AS (2007) E-cadherin adhesion activates c-Src signaling at cell-cell contacts. Mol Biol Cell 18:3214–3223 Mercurio AM, Rabinovitz I, Shaw LM (2001) The alpha6beta4 integrin and epithelial cell migration. Curr Opin Cell Biol 13:541–545 Miranti CK, Brugge JS (2002) Sensing the environment: a historical perspective on integrin signal transduction. Nat Cell Biol 4:E83–E90 Mizushima H, Takamura H, Miyagi Y, Kikkawa Y, Yamanaka N, Yasumitsu H, Misugi K, Miyazaki K (1997) Identification of integrin-dependent and -independent cell adhesion domains in COOHterminal globular region of laminin-5 alpha 3 chain. Cell Growth Differ 8:979–987 Nakajima M, Hayashi K et al (2003) Effect of Wf-536, a novel ROCK inhibitor, against metastasis of B16 melanoma. Cancer Chemother Pharmacol 52:319–324 Nakamura T, Kato Y, Fuji H, Horiuchi T, Chiba Y, Tanaka K (2003) E-cadherin-dependent intercellular adhesion enhances chemoresistance. Int J Mol Med 12:693–700 Nakashima Y, Kariya Y, Yasuda C, Miyazaki K (2005) Regulation of cell adhesion and type VII collagen binding by the beta3 chain short arm of laminin-5: effect of its proteolytic cleavage. J Biochem 138:539–552 Nguyen BP, Ryan MC, Gil SG, Carter WG (2000) Deposition of laminin 5 in epidermal wounds regulates integrin signaling and adhesion. Curr Opin Cell Biol 12:554–562 Niki T, Kohno T et al (2002) Frequent co-localization of Cox-2 and laminin-5 gamma2 chain at the invasive front of early-stage lung adenocarcinomas. Am J Pathol 160:1129–1141

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Nishimura Y, Itoh K, Yoshioka K, Tokuda K, Himeno M (2003) Overexpression of ROCK in human breast cancer cells: evidence that ROCK activity mediates intracellular membrane traffic of lysosomes. Pathol Oncol Res 9:83–95 Nordemar S, Hogmo A, Lindholm J, Auer G, Munck-Wikland E (2003) Laminin-5 gamma 2: a marker to identify oral mucosal lesions at risk for tumor development? Anticancer Res 23:4985–4989 Ogawa T, Tsubota Y, Hashimoto J, Kariya Y, Miyazaki K (2007) The short arm of laminin gamma2 chain of laminin-5 (laminin-332) binds syndecan-1 and regulates cellular adhesion and migration by suppressing phosphorylation of integrin beta4 chain. Mol Biol Cell 18:1621–1633 Ondruschka C, Buhtz P, Motsch C, Freigang B, Schneider-Stock R, Roessner A, Boltze C (2002) Prognostic value of MMP-2, -9 and TIMP-1,-2 immunoreactive protein at the invasive front in advanced head and neck squamous cell carcinomas. Pathol Res Pract 198:509–515 Onishi A, Chen Q, Humtsoe JO, Kramer RH (2008) STAT3 signaling is induced by intercellular adhesion in squamous cell carcinoma cells. Exp Cell Res 314:377–386 Ono Y, Nakanishi Y, Ino Y, Niki T, Yamada T, Yoshimura K, Saikawa M, Nakajima T, Hirohashi S (1999) Clinocopathologic significance of laminin-5 gamma2 chain expression in squamous cell carcinoma of the tongue: immunohistochemical analysis of 67 lesions. Cancer 85: 2315–2321 Pang JH, Kraemer A, Stehbens SJ, Frame MC, Yap AS (2005) Recruitment of phosphoinositide 3-kinase defines a positive contribution of tyrosine kinase signaling to E-cadherin function. J Biol Chem 280:3043–3050 Patarroyo M, Tryggvason K, Virtanen I (2002) Laminin isoforms in tumor invasion, angiogenesis and metastasis. Semin Cancer Biol 12:197–207 Patel V, Aldridge K, Ensley JF, Odell E, Boyd A, Jones J, Gutkind JS, Yeudall WA (2002) Laminingamma2 overexpression in head-and-neck squamous cell carcinoma. Int J Cancer 99:583–588 Pece S, Gutkind JS (2000) Signaling from E-cadherins to the MAPK pathway by the recruitment and activation of epidermal growth factor receptors upon cell-cell contact formation. J Biol Chem 275:41227–41233 Pyke C, Salo S, Ralfkiaer E, Romer J, Dano K, Tryggvason K (1995) Laminin-5 is a marker of invading cancer cells in some human carcinomas and is coexpressed with the receptor for urokinase plasminogen activator in budding cancer cells in colon adenocarcinomas. Cancer Res 55:4132–4139 Qian X, Karpova T, Sheppard AM, McNally J, Lowy DR (2004) E-cadherin-mediated adhesion inhibits ligand-dependent activation of diverse receptor tyrosine kinases. EMBO J 23:1739–1748 Raymond K, Kreft M, Song JY, Janssen H, Sonnenberg A (2007) Dual Role of alpha6beta4 integrin in epidermal tumor growth: tumor-suppressive versus tumor-promoting function. Mol Biol Cell 18:4210–4221 Reddy P, Liu L et al (2005) Formation of E-cadherin-mediated cell-cell adhesion activates AKT and mitogen activated protein kinase via phosphatidylinositol 3 kinase and ligand-independent activation of epidermal growth factor receptor in ovarian cancer cells. Mol Endocrinol 19:2564–2578 Remy L, Trespeuch C, Bachy S, Scoazec JY, Rousselle P (2006) Matrilysin 1 influences colon ­carcinoma cell migration by cleavage of the laminin-5 beta3 chain. Cancer Res 66:11228–11237 Rousselle P, Lunstrum GP, Keene DR, Burgeson RE (1991) Kalinin: an epithelium-specific basement membrane adhesion molecule that is a component of anchoring filaments. J Cell Biol 114:567–576 Sahai E, Marshall CJ (2003) Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis. Nat Cell Biol 5:711–719 Santini MT, Rainaldi G, Indovina PL (2000) Apoptosis, cell adhesion and the extracellular matrix in the three-dimensional growth of multicellular tumor spheroids. Crit Rev Oncol Hematol 36:75–87 Schlessinger J (2002) Ligand-induced, receptor-mediated dimerization and activation of EGF receptor. Cell 110:669–672 Seftor RE, Seftor EA, Koshikawa N, Meltzer PS, Gardner LM, Bilban M, Stetler-Stevenson WG, Quaranta V, Hendrix MJ (2001) Cooperative interactions of laminin 5 gamma2 chain, matrix metalloproteinase-2, and membrane type-1-matrix/metalloproteinase are required for mimicry of embryonic vasculogenesis by aggressive melanoma. Cancer Res 61:6322–6327

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Shang M, Koshikawa N, Schenk S, Quaranta V (2001) The LG3 module of laminin-5 harbors a binding site for integrin alpha3beta1 that promotes cell adhesion, spreading, and migration. J Biol Chem 276:33045–33053 Shen X, Kramer RH (2004) Adhesion-mediated squamous cell carcinoma survival through ligandindependent activation of epidermal growth factor receptor. Am J Pathol 165:1315–1329 St Croix B, Kerbel RS (1997) Cell adhesion and drug resistance in cancer. Curr Opin Oncol 9:549–556 St Croix B, Florenes VA, Rak JW, Flanagan M, Bhattacharya N, Slingerland JM, Kerbel RS (1996) Impact of the cyclin-dependent kinase inhibitor p27Kip1 on resistance of tumor cells to anticancer agents. Nat Med 2:1204–1210 Tran M, Rousselle P, Nokelainen P, Tallapragada S, Nguyen NT, Fincher EF, Marinkovich MP (2008) Targeting a tumor-specific laminin domain critical for human carcinogenesis. Cancer Res 68:2885–2894 Tripathi M, Nandana S, Yamashita H, Ganesan R, Kirchhofer D, Quaranta V (2008) Laminin-332 is a substrate for hepsin, a protease associated with prostate cancer progression. J Biol Chem 283:30576–30584 Van Waes C (1995) Cell adhesion and regulatory molecules involved in tumor formation, hemostasis, and wound healing. Head Neck 17:140–147 Van Waes C, Kozarsky KF, Warren AB, Kidd L, Paugh D, Liebert M, Carey TE (1991) The A9 antigen associated with aggressive human squamous carcinoma is structurally and functionally similar to the newly defined integrin alpha 6 beta 4. Cancer Res 51:2395–2402 Van Waes C, Surh DM et al (1995) Increase in suprabasilar integrin adhesion molecule expression in human epidermal neoplasms accompanies increased proliferation occurring with immortalization and tumor progression. Cancer Res 55:5434–5444 Veitch DP, Nokelainen P et al (2003) Mammalian tolloid metalloproteinase, and not matrix metalloprotease 2 or membrane type 1 metalloprotease, processes laminin-5 in keratinocytes and skin. J Biol Chem 278:15661–15668 Vultur A, Cao J, Arulanandam R, Turkson J, Jove R, Greer P, Craig A, Elliott B, Raptis L (2004) Cell-to-cell adhesion modulates Stat3 activity in normal and breast carcinoma cells. Oncogene 23:2600–2616 Waterman EA, Sakai N, Nguyen NT, Horst BA, Veitch DP, Dey CN, Ortiz-Urda S, Khavari PA, Marinkovich MP (2007) A laminin-collagen complex drives human epidermal carcinogenesis through phosphoinositol-3-kinase activation. Cancer Res 67:4264–4270 Wolf GT, Carey TE (1992) Tumor antigen phenotype, biologic staging, and prognosis in head and neck squamous carcinoma. J Natl Cancer Inst Monogr 13:67–74 Yoshizaki T, Sato H, Maruyama Y, Murono S, Furukawa M, Park CS, Seiki M (1997) Increased expression of membrane type 1-matrix metalloproteinase in head and neck carcinoma. Cancer 79:139–144 Yuen HW, Ziober AF, Gopal P, Nasrallah I, Falls EM, Meneguzzi G, Ang HQ, Ziober BL (2005) Suppression of laminin-5 expression leads to increased motility, tumorigenicity, and invasion. Exp Cell Res 309:198–210 Zhang K, Kramer RH (1996) Laminin 5 deposition promotes keratinocyte motility. Exp Cell Res 227:309–322 Zhang K, Kim JP, Woodley DT, Waleh NS, Chen YQ, Kramer RH (1996) Restricted expression and function of laminin 1-binding integrins in normal and malignant oral mucosal keratinocytes. Cell Adhes Commun 4:159–174 Zhou H, Kramer RH (2005) Integrin engagement differentially modulates epithelial cell motility by RhoA/ROCK and PAK1. J Biol Chem 280:10624–10635 Ziober BL, Kramer RH (2003) Adhesion receptors in oral cancer invasion. In: Ensley JF, Gutkind JS (eds) Head and neck cancer. Elsevier Science, New York, pp 65–79, Chap 6 Ziober BL, Silverman SS Jr, Kramer RH (2001) Adhesive mechanisms regulating invasion and metastasis in oral cancer. Crit Rev Oral Biol Med 12:499–510 Ziober AF, Falls EM, Ziober BL (2006) The extracellular matrix in oral squamous cell carcinoma: friend or foe? Head Neck 28:740–749

Chapter 2

Roles of Integrins in the Development and Progression of Squamous Cell Carcinomas John Lamar and C. Michael DiPersio

Abstract  The identification of therapeutic targets for inhibiting malignant p­ rogression and metastasis remains a critically important step in combating cancer mortality in the clinic. Integrins, the major cell surface receptors for cell adhesion to the extracellular matrix, are involved in all stages of carcinogenesis and are promising targets for anti-cancer therapies. Indeed, roles for integrins in cancer have been the focus of intense investigation since this family of cell adhesion receptors was first discovered in the early 1980s, and many studies during the past three decades have described critical functions for integrins expressed on carcinoma cells in controlling proliferation, survival, migration, and angiogenesis. In addition to mediating cell adhesion, integrins serve as conduits of signal transduction across the plasma membrane, thereby mediating information flow between the interior of the tumor cell and the extracellular microenvironment that promotes angiogenesis and drives malignant growth and metastasis. Although a number of integrin antagonists are currently in pre-clinical and clinical development, the repertoire of integrins that is expressed by tumor cells varies considerably among different types of cancer. Therefore, the most effective combination of integrins to target will vary among cancer types and must be determined in each case. In this chapter, we will provide an overview of current knowledge regarding­ tumor-promoting functions of integrins that are expressed in squamous cell ­carcinoma (SCC), and we will consider the prospect of exploiting these integrins as therapeutic­ targets for inhibiting SCC in the clinic.

C.M. DiPersio (*) Center for Cell Biology & Cancer Research, Albany Medical College, 47 New Scotland Avenue, MC 165, Albany, NY 12208, USA e-mail: [email protected] A.B. Glick and C. Van Waes (eds.), Signaling Pathways in Squamous Cancer, DOI 10.1007/978-1-4419-7203-3_2, © Springer Science+Business Media, LLC 2011

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2.1 Introduction Normal structure, function, and repair of stratified epithelial tissues are regulated by adhesive interactions of individual cells with both neighboring cells and the extracellular matrix (ECM), and abnormal changes in cell adhesion contribute to the development of squamous cell carcinoma (SCC) (Janes and Watt 2006). Integrins are the major receptors for cell adhesion to the ECM (Hynes 2002), while epithelial cell-cell interactions are controlled largely through cadherins, although there is considerable crosstalk between these two receptor families, as reviewed elsewhere (Chen and Gumbiner 2006; DiPersio 2008). While integrins are well known for their roles in cell adhesion, they can also modulate signal transduction pathways that control a variety of cell functions important in normal and pathological tissue remodeling, including proliferation, survival, motility, cytoskeletal dynamics, and gene expression (Hynes 2002; Giancotti and Ruoslahti 1999; Ridley et al. 2003). Indeed, integrins are important at every stage of carcinogenesis (Janes and Watt 2006; Kramer et al. 2005; Ziober et al. 2001), and they are potential therapeutic targets for inhibiting cancer progression (Rust et al., 2002; Stupp and Ruegg 2007). This chapter provides an overview of current knowledge regarding the roles that integrins play in the development and malignant progression of SCC. Initial sections offer a brief review of known integrin functions in normal stratified epithelia, which provides the foundation for subsequent sections that are focused on integrin functions in SCC. The latter sections are structured to emphasize key concepts regarding various mechanisms that are used by different integrins to regulate SCC cell functions, and they draw on specific examples to illustrate these concepts. As  space limitations preclude a complete discussion of the numerous published studies that have contributed to this field, the reader is directed to several excellent reviews for further coverage of relevant topics (Janes and Watt 2006; Kramer et al. 2005; Ziober et al. 2001).

2.2 Integrins as Potential Targets for Anticancer Therapies All members of the integrin family are heterodimeric, transmembrane glycoproteins consisting of an a and a b subunit (Hynes 2002). Eighteen a subunits and eight b subunits can dimerize in various combinations to form at least 24 different integrins with distinct, though often overlapping, ligand-binding specificities (Hynes 2002). As a group, integrins bind to a wide variety of extracellular ligands, many of which are associated with the ECM. Simultaneously, integrin cytoplasmic domains can interact with cytoskeletal proteins to mediate a transmembrane linkage of the ECM to the cytoskeleton, which is critical for controlling cell shape, polarization, and motility (Chen and Gumbiner 2006; Delon and Brown 2007; Litjens et al. 2006; Liu et al. 2000; Ridley et al. 2003). In addition, although integrins lack intrinsic enzymatic activity, they are conduits of bidirectional signal transduction across the plasma membrane (Hynes 2002; Schwartz and Ginsberg 2002). Indeed,

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through interactions of their cytoplasmic domains with a wide variety of signaling effectors inside the cell (Legate and Fassler 2009; Liu et al. 2000), integrins regulate intracellular pathways in response to extracellular cues (i.e., “outside-in” signal transduction). In addition, some cytoplasmic interactions can regulate the activation state of an integrin, thereby modulating its binding affinity for extracellular ligands (i.e., “inside-out” signal transduction) (Askari et al. 2009; Hynes 2002). Signaling functions of some integrins can be modulated by lateral interactions with other cell surface proteins, such as tetraspanins, urokinase receptor (uPAR), or caveolin, at sites of cell adhesion or from within specialized membrane microdomains (reviewed in Berditchevski 2001; Chapman et al. 1999; Del Pozo and Schwartz 2007; Hemler 2005; Porter and Hogg 1998). As discussed later in Sect.  2.4.3, some integrins signal through cooperative interactions with cell surface receptors for growth factors or cytokines (reviewed in Comoglio et  al. 2003; French-Constant and Colognato 2004; Giancotti and Ruoslahti 1999; Guo and Giancotti 2004). Integrins regulate many cell functions associated with epithelial-to-mesenchymal transition (EMT), and they have important roles in the development and malignant progression of SCC and other carcinomas (Brakebusch et al. 2002; Janes and Watt 2006; White et  al. 2004). In their capacity as bidirectional signaling receptors, integrins regulate both tumor cell-mediated changes to the microenvironment that promote cancer progression, and tumor cell responses to such changes, implicating them as potential targets for antagonistic agents in anti-cancer therapies (Mulgrew et  al. 2006; Rust et  al. 2002; Stupp and Ruegg 2007; Wu et  al. 1998). However, most integrin inhibitors in clinical development are thought to alter angiogenesis by targeting integrins on endothelial cells of the tumor vasculature (Alghisi and Ruegg 2006; Stupp and Ruegg 2007), and there remains a critical need to identify and validate specific integrin targets on tumor cells. As will be discussed, several integrins have been shown to regulate skin tumorigenesis, malignant progression, and metastasis, identifying them as possible therapeutic targets for SCC (Felding-Habermann 2003; Janes and Watt 2006; Ziober et al. 2001).

2.3 Roles of Integrins in Stratified Epithelia Expression patterns of individual integrins in normal epidermis and other epithelia are well documented, and several integrins are known to have critical roles in regulating epithelial growth, differentiation, and wound repair (Watt 2002). Importantly, some integrin functions that are involved in maintenance of the stem cell compartment, or that promote epithelial regeneration during wound healing, also contribute to SCC (Janes and Watt 2006; Ziober et al. 2001). Indeed, it has long been recognized that wound healing and carcinogenesis share intriguing similarities regarding epithelial cell behaviors and microenvironmental factors that drive each process (Dvorak 1986). We will briefly review the expression and functions of specific integrins in normal stratified epithelia, and how they change during wound healing, to provide a foundation for our discussions in later sections on integrin functions in SCC.

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2.3.1 Integrin Functions in Normal Stratified Epithelia Stratified epithelia are continually renewed by stem cells that give rise to committed progenitor cells, or transit-amplifying cells, which in turn give rise to differentiated keratinocytes (Fuchs 2008; Owens and Watt 2003; Watt 2002). Proliferating keratinocytes, including stem cells and transit-amplifying cells, are normally restricted to the basal cell layer where they are adhered through integrins to the underlying basement membrane (BM), a specialized ECM that separates epithelial cell layers from adjacent connective tissue. In the epidermis, integrin expression is normally restricted to cells of the basal layer and outer root sheath of the hair follicle (Hertle et  al. 1991; Watt 2002). Differentiating keratinocytes down-regulate integrin expression as they detach from the BM and are displaced into the suprabasal layers (Watt 2002). Several integrins are expressed constitutively in normal, unwounded epidermis and oral squamous epithelium, including a3b1 and a6b4 (both laminin-332 receptors), a2b1 (a collagen receptor), a9b1 (a fibronectin and tenascin receptor) and avb5 (a vitronectin receptor) (Thomas et al. 2006; Watt 2002). Integrin a6b4 is an essential component of hemidesmosomes, which are adhesion structures on the basal surfaces of keratinocytes that anchor the epidermis to the dermis (Litjens et  al. 2006). Consistently, deletion of a6b4 (through null mutation of either the Itga6 or Itgb4 gene, encoding the a6 or b4 subunit, respectively) leads to extensive epidermal blistering (Dowling et al. 1996; Georges-Labouesse et al. 1996; van der Neut et al. 1996). Deletion of a3b1 (through null mutation of the Itga3 gene encoding the a3 subunit) causes minor perinatal blistering at the epidermal-dermal junction, but this is caused by rupture of the BM, which is disorganized in a3-null mice (DiPersio et al. 1997). Interestingly, mice that lack a3b1 and a6b4, either alone or in combination, show essentially normal epidermal stratification (DiPersio et  al. 1997; Dowling et al. 1996; Georges-Labouesse et al. 1996; van der Neut et al. 1996; DiPersio et  al., 2000a). Similarly, individual deletion of integrin a2b1, a9b1, or avb5 does not substantially alter epidermal differentiation (Grenache et al. 2007; Huang et al. 2000; Singh et al. 2009; Zweers et al. 2007). In contrast, ablation of all b1 integrins from epidermis (through null mutation of the Itgb1 gene encoding the b1 subunit) leads to proliferation defects, loss of hair follicles and sebaceous glands and, depending on the genetic model, a modest increase in terminally differentiating keratinocytes (Brakebusch et  al. 2000; Grose et  al. 2002; Raghavan et al. 2000). Thus, there appears to be overlap in the roles of different integrins in maintaining epidermal homeostasis. The level of integrin expression in keratinocytes is thought to control the balance between stem cell renewal and terminal differentiation, which is important for maintaining tissue homeostasis (Fuchs 2008; Jones et  al. 1995; Watt 2002; Zhu et al. 1999). Indeed, b1 integrins and a6b4 are expressed at relatively high levels in epidermal stem cells (Janes and Watt 2006; Jones and Watt 1993; Jones et al. 1995; Terunuma et  al. 2007), and some integrin signaling pathways have been linked to maintenance of the epidermal stem cell compartment, such as those

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involving mitogen-activated protein kinases (MAPKs), or the Rho family guanosine triphosphatase (GTPase), Rac1 (Benitah et al. 2005; Haase et al. 2001; Zhu et al. 1999). Presumably, it is these resident stem cells that accumulate mutations in oncogenes and tumor suppressor genes that lead to tumorigenesis, since differentiated keratinocytes are eventually shed from the outer layer of stratified epithelia (Owens and Watt 2003; Watt 2002). Therefore, changes in integrins that disrupt the balance between stem cell renewal and differentiation are likely to greatly influence SCC development and progression. It is important to point out, however, that altered integrin expression on differentiating cells of the suprabasal layers, as occurs in SCC and other hyperproliferative states such as wound healing and psoriasis, can also influence the stem cell compartment and, therefore, affect tumor growth and progression (reviewed in Janes and Watt 2006). Indeed, skin carcinogenesis studies performed in transgenic mice showed that forced integrin expression in suprabasal keratinocytes can influence both the clonal expansion of tumor progenitor cells and malignant progression of resulting tumors to SCC (Nguyen et  al. 2000; Owens and Watt 2001; Owens et  al. 2003, 2005). Interestingly, suprabasal expression of different integrins had distinct effects. For example, suprabasal a2b1 had no effect on SCC progression, while suprabasal a3b1 suppressed malignant conversion of papillomas (Owens and Watt 2001). In contrast, suprabasal a6b4 or a5b1 each increased tumor incidence and progression to SCC, although through distinct mechanisms (Owens and Watt 2003; Owens et al. 2005). Thus, new integrin signals that are turned on in suprabasal cells can influence nearby tumor progenitor/stem cells.

2.3.2 Integrin Functions in Wound Healing As mentioned above, there are compelling similarities between wound healing and SCC progression (Dvorak 1986), and integrins regulate a number of epithelial functions important in both processes, including migration, proliferation, and the production of proangiogenic factors. Consistently, expression patterns of integrins in SCC often mirror those that occur in wound healing (Thomas et  al. 2006; Watt 2002). During cutaneous wound healing, several integrins that are expressed in unwounded epidermis at high or moderate levels (i.e., a3b1, a9b1, a6b4) or low levels (i.e., a5b1) show sustained or increased expression, while avb5 is downregulated and replaced by avb6. As a group, these integrins can bind multiple ligands that are present in the provisional ECM of the wound, including fibronectin (a5b1, a9b1, avb6), vitronectin (avb6), and tenascin (a9b1, avb6), as well as laminin-332 (a3b1, a6b4) that is newly deposited by migrating keratinocytes (Nguyen et al. 2000; Thomas et al. 2006; Watt 2002). Furthermore, numerous studies in cultured cells have shown that these integrins regulate keratinocyte adhesion and migration on their respective ECM ligands [for example, (Grose et al. 2002; Carter et  al. 1990a, b; Choma et  al. 2004; Frank and Carter 2004; Pilcher et  al. 1997;

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Sehgal et  al. 2006)]. Therefore, it is perhaps not surprising that wound healing ­studies in integrin knockout mice have indicated considerable overlap in the ­abilities of different integrins to mediate epidermal migration. Indeed, while mice with epidermis-specific deletion of all b1 integrins showed impaired wound reepithelialization (Grose et al. 2002), mice that lack certain b1 integrins individually (a2b1, a3b1, or a9b1) did not show such a defect (Grenache et al. 2007; Zweers et al. 2007); Singh et al. 2009; (Margadant et al. 2009). Similarly, absence of integrin avb6 did not cause impaired wound healing in young adult mice (although it caused delayed wound healing in old mice) (AlDahlawi et al. 2006). On the other hand, epidermis-specific deletion of individual integrins has revealed important roles in regulating other aspects of wound healing. For example, deletion of a3b1 from epidermis was associated with reduced wound angiogenesis, indicating a3b1-dependent secretion of pro-angiogenic factors (Mitchell et  al. 2009). In addition, deletion of a9b1 from epidermis caused proliferation defects in wound keratinocytes (Singh et al. 2009). Thus, the repertoire of distinct integrins expressed in wounded epidermis is important for coordinating diverse keratinocyte functions (migration, proliferation, ECM remodeling, secretion of pro-angiogenic factors) that collectively ensure efficient wound repair and epidermal regeneration. Importantly, these same integrin-mediated cell functions are also likely to contribute to SCC progression.

2.4 Roles of Integrins in SCC Roles for integrins in promoting both early and late stages of SCC have been investigated extensively (reviewed in Janes and Watt 2006; Kramer et  al. 2005; Marinkovich 2007; Thomas et  al. 2006; Ziober et  al. 2001). Integrins regulate a number of tumor cell functions that facilitate initial tumor growth, including proliferation, survival, and secretion of pro-angiogenic factors. In addition, integrinmediated cell survival, migration, invasion, and ECM proteolysis are important for later stages of malignant tumor progression and metastasis (Brakebusch et al. 2002; Felding-Habermann 2003). Integrins can influence tumor cell behavior directly through their cell adhesion and signaling functions, or indirectly through effects on ECM remodeling. Indeed, there are many reports of integrins regulating expression or activities of extracellular proteases, such as matrix metalloproteinases (MMPs) or urokinase plasminogen activator (uPA), that can promote tumor angiogenesis and carcinoma progression [for example, (Brooks et  al. 1996; Ellerbroek et  al. 1999; Morini et al. 2000; Thomas et al. 2001a; Ghosh et al. 2000, 2006; Gu et al. 2002; Han et al. 2002; Iyer et al. 2005; Symowicz et al. 2007)]. In this section, we will review what is currently known about integrin expression and function in SCC. Although changes in relevant ECM ligands that occur in SCC will be mentioned where appropriate, the reader is directed to several excellent reviews for further details on this subject (Marinkovich 2007; Ziober et al. 2001). Because of space limitations, our discussion is concentrated on tumor cell-autonomous functions

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of integrins on SCC cells and their potential value as therapeutic targets. However, integrins expressed on stromal cells, such as endothelial cells, macrophages, and fibroblasts, also regulate the abilities of these cells to alter the tumor microenvironment and influence carcinoma progression. Therefore, the importance of integrins on these nontumor cells as therapeutic targets should not be overlooked (Hofmeister et al. 2008).

2.4.1 Integrin Expression in SCC There is evidence that the expression levels of certain integrins in SCC may serve as useful biomarkers for clinical outcome (Kurokawa et al. 2008). As already mentioned, altered integrin expression in SCC bears similarities to that which occurs during wound healing and includes sustained expression, increased expression, or loss of expression (Bagutti et al. 1998; Jones et al. 1993). For example, integrins a5b1 and avb6 are expressed at low or negligible levels in normal epidermis but are increased in SCC (Gomez and Cano 1995; Shinohara et al. 1999), while avb5 is downregulated (Janes and Watt 2004, 2006). On the other hand, expression of a3b1 and a6b4 often persists in SCC (Janes and Watt 2006), although reduced expression has also been reported in some cases (Bagutti et al. 1998; Maragou et al. 1999). Colocalization of a9b1 and its ligand, tenascin, has also been reported in SCC tumors, although inflamed areas often showed focal loss of both at the BM zone (Hakkinen et al. 1999). While some studies reported increased expression of a2b1 in metastatic SCC cell lines and tumor biopsies (Shinohara et  al. 1999), others reported that loss of a2b1 and its ­collagen ligands is correlated with SCC progression (reviewed in (Ziober et al. 2001)). Importantly, there can also be considerable variation in expression of an individual integrin either within a tumor or between different SCC tumors, possibly reflecting differential expression in distinct cellular compartments of the tumor and/or at distinct stages of tumor progression (Janes and Watt 2006; Watt 2002). However, while the expression pattern of an individual integrin might reflect its involvement in SCC, by itself it reveals no information about the functional role of the integrin at a particular stage of carcinogenesis. In fact, there is increasing evidence that some integrins that are already expressed on normal epithelial cells acquire new functions during SCC progression (see Sect.  4.5). As discussed in the following ­sections, numerous preclinical studies have identified key roles for specific integrins in SCC, and they also suggest that the malignant phenotype is influenced by the cumulative roles of several integrins, rather than by any particular integrin alone.

2.4.2 Integrin Signaling in SCC Integrins expressed on tumor cells can relay signals bidirectionally across the plasma membrane that control basic cell functions important for cancer progression­,

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invasion, and metastasis, as reviewed in detail elsewhere (Brakebusch et al. 2002; Felding-Habermann 2003; Giancotti and Ruoslahti 1999; Gilcrease 2007; Guo and Giancotti 2004). As mentioned above and discussed in several reviews (Berditchevski 2001; Chapman et al. 1999; Del Pozo and Schwartz 2007; Hemler 2005; Porter and Hogg 1998; Salanueva et al. 2007), integrin signaling functions can be modulated through lateral associations with other cell surface proteins, including growth factor receptors (see Sect. 2.4.3). This discussion is focused on integrin-mediated outsidein signaling; however, changes in integrin activation state that are regulated by inside-out signals also control cell functions that promote carcinoma progression (Legate and Fassler 2009; Schwartz and Ginsberg 2002). Focal adhesion kinase (FAK) has emerged in recent years as a particularly important effector of integrin-mediated signal transduction in tumor cells, and its regulation serves as a useful paradigm of outside-in integrin signaling that promotes malignant cell behavior (Brunton and Frame 2008; McLean et  al. 2005; Mitra and Schlaepfer 2006; Zhao and Guan 2009). FAK is a nonreceptor tyrosine kinase that associates with several integrins at focal adhesions or other cell-matrix contacts, and its activation by cell adhesion is the initial enzymatic step in several integrin-dependent signaling pathways. Integrin-mediated FAK activation can contribute to many different tumor cell functions, including proliferation, survival, motility, and invasiveness, and it has been linked to the stimulation of various pathways involving the MAPKs, extracellular signal-regulated kinase (ERK) and Jun N-terminal kinase (JNK), certain Rho family GTPases (CDC42, Rho, RAC1), and the serine/threonine kinase AKT (Felding-Habermann 2003). FAK can also be activated by growth factor receptors, identifying it as a potential integrator of growth factor and integrin signaling (Brunton and Frame 2008; Sieg et al. 2000). Importantly, FAK expression is enhanced in invasive SCCs (Kornberg 1998), where it appears to regulate both early and late stages of cancer progression (reviewed in (Ziober et al. 2001)). Indeed, deletion of FAK from the epidermis suppresses carcinogen-induced skin tumorigenesis, as well as malignant progression of benign papillomas to carcinomas (McLean et al. 2004). An early step in many integrin-FAK signaling pathways is the direct binding of a SRC-family kinase (SFK) to activated FAK at sites of cell adhesion (Schaller et al. 1999), as described in detail in several excellent reviews (Brunton and Frame 2008; Cary and Guan 1999; McLean et  al. 2005; Mitra and Schlaepfer 2006). Briefly, integrin binding to ECM ligands leads to FAK clustering and auto-phosphorylation of Y397, creating a high-affinity binding site for the Src-homology 2 (SH2) domain of SRC (or another SFK) and leading to formation of a FAK/SRC complex. Subsequent phosphorylation of other FAK tyrosines by SRC creates binding sites for a number of signaling intermediates, such as GRB2, p130CAS, and phosphatidylinositol 3¢-kinase (PI3-K). These intermediates link the FAK/SRC complex to different downstream effectors, including the RAS-to-ERK pathway, AKT, and JNK (Giancotti and Ruoslahti 1999; Grille et  al. 2003; Mitra and Schlaepfer 2006), thereby activating several pathways that promote EMT by enhancing proliferation, survival, migration, invasion, and expression of ECMdegrading proteases and pro-angiogenic factors. As mentioned above, FAK/SRC

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can also signal through Rho family GTPases (CDC42, Rho, and RAC1) to modulate cytoskeletal dynamics and cell migration (Felding-Habermann 2003). Furthermore, RAC1 plays important roles in keratinocyte proliferation and migration in  vivo (Tscharntke et  al. 2007), and it is critical for maintaining epidermal stem cell compartments (Benitah et al. 2005; Castilho et al. 2007; Chrostek et al. 2006), suggesting that enhanced FAK/SRC-to-RAC1 signaling may contribute to clonal expansion of tumor progenitor/stem cells. Consistently, mice that lack Tiam1, a guanine nucleotide-exchange ­factor (GEF) that activates RAC1, are resistant to RAS-induced skin tumors (Malliri et al. 2002). Given the importance of the FAK/SRC complex as a major signaling nexus that links integrin-mediated adhesion to pathways that promote several cancer cell functions, it is not surprising that small molecule inhibitors of both FAK and SRC have been the focus of recent clinical studies to inhibit tumor progression (reviewed in (Brunton and Frame 2008; McLean et  al. 2005; Mitra and Schlaepfer 2006)). However, integrins in epithelial cells can also signal through effectors other than FAK, such as integrin-linked kinase (ILK) and phospholipase C (PLC), as reviewed in detail elsewhere (Gilcrease 2007). Therefore, FAK-independent pathways should not be overlooked as potential therapeutic targets for SCC.

2.4.3 Functions of Individual Integrins in SCC In the following subsections we will discuss tumor cell-autonomous functions of individual integrins that are expressed on SCC cells. This discussion is focused on integrins avb6, a3b1, and a6b4, since their roles have been studied most extensively. However, several other integrins with less-defined roles should also be mentioned briefly. For example, increased expression of integrin a9b1 and two of its ECM ligands, fibronectin and tenascin, has been reported in some SCCs (Hakkinen et al. 1999; Ziober et al. 2001). However, functional roles for a9b1 in SCC are poorly defined, in part because this integrin is down-regulated in cultured keratinocytes and has been largely overlooked in studies of integrin-mediated keratinocyte function. Although high expression of a2b1 (a receptor for certain laminins and collagens) and a5b1 (a receptor for fibronectin) has been reported in some SCC cell lines and tumor biopsies (Shinohara et al. 1999), expression patterns are quite variable and roles for these integrins in SCC require further study (Hakkinen et al. 1999; Ziober et al. 2001). 2.4.3.1 Integrin avb6 Regulatory roles for integrin avb6 in SCC have been studied quite extensively, as reviewed in (Thomas et al. 2006). Although not expressed constitutively by normal epithelium, avb6 is upregulated during wound healing and in many carcinomas (Breuss et al. 1995; Hamidi et al. 2000; Jones et al. 1997), and it has been correlated

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with malignant progression of SCC (Hazelbag et al. 2007). Furthermore, numerous studies have demonstrated roles for this integrin in promoting SCC cell motility and invasion (for example, (Thomas et al. 2001a, b; Ramos et al. 2002)). avb6 binds to a tripeptide motif, arginine-glycine-aspartic acid (RGD), that occurs within several of its ECM ligands including fibronectin, tenascin, and vitronectin (Hynes 2002; Thomas et al. 2006). In addition to mediating cell migration on these ligands, avb6 may promote invasion by regulating expression of extracellular proteases that degrade or remodel ECM. For example, avb6 promotes invasion of oral keratinocytes through up-regulation of MMP-9 and, to a lesser extent, MMP-2 (Thomas et al. 2001a). Other invasion-promoting proteases that can be regulated by avb6 in carcinoma cells include MMP-3 (Ramos et al. 2002) and uPA (Ahmed et al. 2002). Signaling intermediates that have been implicated in avb6-mediated SCC cell invasion include cyclooxygenase-2 (COX-2) (Nystrom et al. 2006), RAC1 (Yap et al. 2009), and the SRC-family member FYN (Li et al. 2003). In addition to promoting an invasive phenotype, the de novo expression of avb6 has been shown to prevent oral SCC cells from undergoing differentiation, as well as protect them from anoikis (i.e., apoptosis caused by reduced or inappropriate cell adhesion) when they are deprived of normal attachments to BM (Janes and Watt 2004). The latter function involves avb6-­ mediated activation of an AKT survival pathway (Janes and Watt 2004). One of the most important functions of avb6 in SCC cells may be its ability to activate the ECM-associated pool of latent TGFb, thereby initiating TGFb signaling pathways that influence tumor progression (Sheppard 2005; Thomas et al. 2006). As discussed further in Sect. 2.4.4.1, avb6 binds to an RGD motif within the latent TGFb complex, thereby inducing a conformational change that activates TGFb (Munger et  al. 1999). Although this mechanism has been best characterized in colon cancer cells, it also occurs in keratinocytes and is likely to be important for regulating TGFb-mediated signaling in SCC progression (Munger et al. 1999). Finally, it is important to note that some studies have indicated that avb6 has tumor suppressing roles, rather than tumor promoting roles, in SCC. For example, mice that are doubly-deficient for avb6 and thrombospondin showed increased incidence of skin papillomas and SCCs, suggesting that avb6 suppresses tumor formation in this model (Ludlow et al. 2005). Similarly, genetic deletion of av integrins in epithelial cells of the eyelid skin and conjunctiva lead to increased SCC (McCarty et al. 2008). In another study, increased expression of avb6 suppressed the invasive phenotype of transformed oral keratinocytes (Mogi et al. 2005). The paradoxical findings regarding roles for avb6 in SCC may, to some extent, be reflective of the well known biphasic roles of TGFb, which suppresses early stages of skin tumorigenesis but promotes progression to malignancy at later stages (Wakefield and Roberts 2002; Wang 2001), such that effects of avb6-mediated TGFb activation on tumor cells are dependent on the stage of cancer development at which this activation occurs (Thomas et al. 2006). 2.4.3.2 Laminin-332-Binding Integrins, a6b4 and a3b1 In recent years, a prominent role in SCC growth and invasion has emerged for laminin-332 (previously known as laminin-5, kalinin, nicein, or epiligrin) [for a

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review, see (Marinkovich 2007)]. Laminin-332 expression is enhanced at the ­invasive fronts of SCCs (Pyke et al. 1995) and is correlated with poor prognosis in SCC patients (Ono et al. 1999). Laminin-332 also disrupts cell-cell adhesions and induces scattering in SCC and other carcinoma cells (Kawano et al. 2001; Miyazaki et al. 1993), suggesting that it can act as a pro-invasive autocrine factor (Marinkovich 2007). Laminin-332 can be cleaved by MMPs or other proteases (Ziober et  al. 2001), and specific proteolytic events have been linked to carcinoma cell migration and invasion [for example, see (Gianelli et al. 1997; Goldfinger et al. 1998; Schenk et al. 2003)]. The effects of laminin-332 on behaviors of both normal keratinocytes and carcinoma cells are mediated largely through its main integrin receptors, a3b1 and a6b4 (Carter et  al. 1991; Nguyen et  al. 2000; Giannelli et  al. 2002a; Dajee et al. 2003), although other receptors such as syndecan-1 also contribute (Okamoto et al. 2003). For simplicity, functions of a3b1 or a6b4 are discussed individually below, although as receptors for a common ECM ligand these two integrins are likely to function coordinately to regulate some aspects of SCC growth and invasion (Marinkovich 2007). There is also substantial evidence that some signaling functions of a3b1 or a6b4 occur independently of binding to laminin-332, and instead involve lateral interactions with other cell surface proteins (see below). Integrin a6b4 Expression of integrin a6b4 is often high in SCCs and has been correlated with malignant conversion and poor prognosis (Jones et  al. 1993; Rabinovitz and Mercurio 1996; Tennenbaum et al. 1993; Van Waes et al. 1991, 1995). Therefore, it is not surprising that roles for a6b4 in carcinogenesis have been studied extensively by several groups. These studies have revealed that a6b4 is critical for SCC formation (Dajee et al. 2003), and that it can promote survival and invasion of SCC and other carcinoma cells (Lipscomb and Mercurio 2005; Marinkovich 2007). a6b4 can also control organization of laminin-332 in the ECM, which is an important regulator of keratinocyte migration (Sehgal et al. 2006). However, a6b4 functions in carcinoma cells appear to be quite complex, and multiple mechanisms have been proposed for its effects on tumor cell behavior, as described below. As already mentioned, a6b4 in normal keratinocytes mediates stable adhesion through its association with the intermediate filaments in hemidesmosomes (Carter et al. 1990a; Litjens et al. 2006). In contrast, a6b4 in invasive carcinoma cells is mobilized out of hemidesmosomes and associates instead with the actin cytoskeleton in membrane protrusions (Mercurio and Rabinovitz 2001; Mercurio et  al. 2001), where it facilitates migration and invasion rather than stable adhesion. Signaling pathways through which a6b4 regulates cell behavior are complex and have been shown to involve several effectors, including PI3-K, RAC and Rho GTPases, and Shc/RAS-to-MAPK pathways (Lipscomb and Mercurio 2005; Mainiero et al. 1997; O’Connor et al. 2000; Russell et al. 2003). Adding further to this complexity, a6b4 often signals in collaboration with receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR) (Mariotti et  al. 2001), the MET receptor for hepatocyte growth factor (HGF) (Trusolino et al. 2001), and the

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Ron receptor for macrophage stimulating protein (MSP) (Santoro et al. 2003) (see Sect. 2.4.3). Integrin a6b4 has also been implicated in the regulation of early stages of SCC progression, where it was shown to cooperate with RAS and IkBa to induce transformation of primary keratinocytes (Dajee et  al. 2003). In this study, blocking antibodies against either a6b4 or laminin-332 blocked the formation of SCCs, and keratinocytes that lacked b4 were unable to form SCC after transformation with Ras. There is also evidence that a6b4 can regulate carcinoma cell survival (Chung et al. 2002; Zahir et al. 2003). While a6b4-mediated signaling pathways mentioned above are likely to contribute to this regulation, a6b4-mediated induction of VEGF protein translation in carcinoma cells can also regulate autocrine cell survival (Chung et al. 2002). The fact that a6b4 impacts SCC development and progression at multiple steps, and through multiple mechanisms, identifies this integrin as an attractive target for anticancer therapies, especially since at least some of these mechanisms are acquired by carcinoma cells (see Sect. 2.4.5.2). Integrin a3b1 Integrin a3b1 has also been implicated in invasion and metastasis of many cancer cell types (Morini et al. 2000; Tawil et al. 1996; Tsuji et al. 2002; Wang et al. 2004). In vivo and in vitro studies have identified roles for a3b1 in regulating several functions in epithelial cells that contribute to both wound healing and carcinogenesis, including ECM deposition and organization (deHart et  al. 2003; Hamelers et  al. 2005), cell polarization and migration (Choma et al. 2004; Frank and Carter 2004), adhesion-dependent survival (Manohar et al. 2004), proliferation (Gonzales et al. 1999), and secretion of ECM proteases and pro-angiogenic factors (Marinkovich 2007; Mitchell et al. 2009; Sugiura and Berditchevski 1999; DiPersio et al. 2000b). There is also solid evidence that some of these a3b1-mediated functions involve regulation of TGFb signaling (see Sect. 2.4.2). Integrin a3b1 in immortalized/transformed keratinocytes regulates the expression of MMP-9 (Iyer et al. 2005; Lamar et al. 2008a), a known promoter of both tumor angiogenesis and SCC invasion (Bergers et  al. 2000; McCawley and Matrisian 2001). Using oncogenically-transformed keratinocytes generated from mice that either express or lack a3b1, we showed that a3b1 is required for MMP-9 gene expression, and also regulates tumor growth in vivo and cell invasion in vitro (Iyer et  al. 2005; Lamar et  al., 2008a). Although promigratory effects of a3b1laminin binding probably contribute to invasion, RNAi and overexpression studies indicated that MMP-9 was largely responsible for a3b1-dependent invasion (Lamar et al. 2008a). a3b1 may also be involved in later stages of metastasis, as it was able to promote arrest of circulating carcinoma cells in the lungs (Wang et al. 2004). Many functions of a3b1 are clearly dependent on its interactions with its laminin ligands in the ECM (Kreidberg 2000). However, some a3b1 functions have been reported to be mediated, or modulated, by direct or indirect interactions with other cell surface proteins, including tetraspanins (Berditchevski et al. 1996; Sugiura and

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Berditchevski 1999), uPAR (Wei et al. 2001), and components of adherens junctions (Chattopadhyay et al. 2003; Kim et al. 2009). Thus, roles of a3b1 in tumor cells appear complex and may involve both ECM-dependent and ECM-independent signaling. In particular, a3b1 binds directly and robustly to the tetraspanin CD151 (Yauch et  al. 2000), and this interaction has been shown to regulate both a3b1mediated signaling (Yauch et al. 1998) and motility of epidermal carcinoma cells (Winterwood et  al. 2006). Interestingly, recent studies have identified important roles for CD151-integrin interactions in several aspects of carcinoma progression and metastasis in  vivo (Sadej et  al. 2009; Yang et  al. 2008; Zijlstra et  al. 2008). Although the full extent to which CD151 modulates a3b1-dependent functions in tumor cells is still unclear, it seems likely that this interaction will prove to be important for SCC progression and metastasis.

2.4.4 Cooperative Functions of Integrins and Growth Factors in SCC Growth factors influence cancer growth and progression through both autocrine and paracrine effects on tumor cells and stromal cells. Multiple studies in both normal and cancer cells have revealed significant signaling crosstalk and complex formation between integrins and growth factor receptors (French-Constant and Colognato 2004; Gilcrease 2007; Guo and Giancotti 2004). In the following ­sections, we will discuss several examples that serve to illustrate different mechanisms whereby integrins can collaborate with growth factors to promote tumor growth and progression, including forming complexes with growth factor receptors, regulating the expression of growth factors or their receptors, and activating ECMbound growth factors. As described below, some of these mechanisms are best illustrated by roles that have been demonstrated for certain integrins in regulating cellular responses to TGFb. 2.4.4.1 Integrin-Dependent Activation of Latent Growth Factors In what is perhaps the best characterized example of direct activation of a latent growth factor by an integrin, avb6 plays a critical role in the activation of the latent TGFb complex (Sheppard 2005). Each of the three mammalian TGFb isoforms (TGFb1, 2, and 3) is secreted as an inactive complex consisting of the latencyassociated protein (LAP) and the latent TGFb binding protein (LTBP). This latent complex is covalently linked through LTBP to certain ECM proteins (e.g., fibronectin) (Sheppard 2005; Taipale et al. 1994), and it must be activated either through proteolytic release of TGFb from the LAP [for example, mediated by MMP-9 or plasmin (Lyons et al. 1990; Sato et al. 1990; Yu and Stamenkovic 2000)], or through a ­conformational change in the complex [for example, induced by thrombospondin-1 (Crawford et  al. 1998; Schultz-Cherry et al. 1995)]. As already mentioned, avb6 can activate latent

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TGFb1 or TGFb3 (but not TGFb2) by binding to an RGD motif within the LAP and inducing a conformational change in the complex (Annes et al. 2004; Munger et al. 1999; Sheppard 2005). It is well known that TGFb acts as a tumor suppressor at early stages of tumorigenesis, but switches to a promoter of EMT at later stages of progression (for several excellent reviews, see Derynck et  al. 2001; He et  al. 2001; Wakefield and Roberts 2002; Wang 2001; Zavadil et  al. 2001). Given that avb6 is upregulated in SCC, its ability to activate latent TGFb could play a role in these biphasic effects of TGFb on cancer progression. In addition to direct growth factor activation, there is evidence that some integrins­ can activate latent or ECM-sequestered growth factors through less direct mechanisms. For example, certain integrins, such as avb6 and a3b1, can induce the expression of MMP-9, uPA, or other extracellular proteases (Thomas et al. 2001a; Ghosh et al. 2000, 2006; Iyer et al. 2005), which can then degrade ECM and release reservoirs of ECM-associated growth factors (i.e., VEGF) that can promote tumor proliferation or angiogenesis (Bergers et al. 2000; McCawley and Matrisian 2001). 2.4.4.2 Integrin-Dependent Enhancement of Growth Factor Signaling Once activated, TGFb interacts with its type I and type II serine/threonine kinase receptors to initiate signaling pathways that modulate transcriptional or post-­ transcriptional gene regulation. Cellular responses to TGFb can be mediated by the Smad family of transcription factors, or by Smad-independent pathways of TGFb signaling, such as MAPK pathways (Derynck and Zhang 2003). Many studies have identified interactions between integrins and TGFb signaling pathways that regulate motility or invasiveness of keratinocytes or carcinoma cells (Gailit et al. 1994; Zambruno et  al. 1995; Giannelli et  al. 2002b; Decline et  al. 2003; Galliher and Schiemann 2007; Jeong and Kim 2004; Reynolds et al. 2008). We recently discovered that integrin a3b1 potentiates the ability of TGFb to induce MMP-9 gene expression in immortalized keratinocytes through a mechanism that is independent of changes in the levels of TGFb or its receptors (Lamar et al. 2008b). However, a3b1 did not enhance all TGFb signaling pathways, since TGFb-mediated Smad phosphorylation remained intact in a3b1-deficient (Itga3-/-) keratinocytes, suggesting that this integrin is a selective modulator of a subset of TGFb signaling pathways (Lamar et al., 2008b). These findings raise the intriguing possibility that a3b1 is involved in the above-mentioned switch in TGFb function from tumor suppressor to tumor promoter during SCC progression. Although the mechanism whereby a3b1 enhances TGFb signaling is not yet clear, it is unlikely that this integrin activates latent TGFb as described above for avb6 (Sect. 2.4.1), since a3b1 does not bind RGD ligands efficiently, and such a mechanism was not indicated for b1 integrins (Munger et al. 1999). Rather, the ability of a3b1 to potentiate MMP-9 induction in response to exogenous pre-­activated TGFb, and the dependence of this regulation on SRC (Lamar et al. 2008b), suggests similarities to a previously described mechanism used by integrin avb3 to modulate

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a subset of TGFb signaling pathways in breast cancer cells (Galliher and Schiemann 2007). In the latter study, avb3 was shown to activate the TGFb type II receptor in a SRC-dependent manner, which was required for TGFb-mediated activation of p38 MAPK, but not for TGFb-mediated stimulation of Smad2/3 (Galliher and Schiemann 2007). Further studies are required to determine if MMP-9 induction requires the formation of a a3b1/TGFb receptor signaling complex, or results from distinct pathways that are initiated independently by TGFb or a3b1 and ­converge on a common intermediate. In any case, it appears that different integrins may modulate TGFb activation and signaling through different mechanisms, thereby cooperating to determine the overall response of the tumor cell to TGFb. Although TGFb exerts its effects on tumor cells in large part through signaling pathways that ultimately regulate the transcription of target genes (Derynck et  al. 2001; Derynck and Zhang 2003), it is well known that TGFb can also regulate the expression of EMT-associated genes through post-transcriptional mRNA stability (Dibrov et al. 2006). Integrin a3b1 promotes Mmp9 mRNA stability in immortalized mouse keratinocytes (Iyer et al. 2005), raising the intriguing possibility that TGFb and a3b1 cooperate to stabilize mRNA transcripts of EMT genes. The MAPK p38 is a potential effector for this regulation, since it has been implicated in both EMTpromoting effects of TGFb (Bakin et  al. 2002; Zavadil and Bottinger 2005) and TGFb-mediated mRNA stability (Dibrov et al. 2006), and it can be activated through cooperative interactions between b1 integrins and TGFb (Bhowmick et al. 2001). The MAPK ERK is another potential intermediate in this regulation. Indeed, TGFb activates ERK pathways in cell culture models of EMT (Zavadil and Bottinger 2005), and ERK is both activated by a3b1, and required for induction of Mmp9 mRNA in immortalized keratinocytes (Iyer et al. 2005; Manohar et al. 2004). In addition, TGFb signaling through the type I receptor, ALK5, leads to MEK/ERK-dependent induction of MMP9 mRNA in human breast cancer cells (Safina et al. 2007). 2.4.4.3 Formation of Integrin-Growth Factor Receptor Signaling Complexes Association of integrin a6b4 with the MET receptor provides a compelling example of an integrin-growth factor receptor interaction that regulates signal transduction in carcinoma cells, most likely in a manner that is independent of a6b4 binding to laminin-332 (Trusolino et  al. 2001). Indeed, in a complex formed with activated MET, a6b4 acts as an essential adapter protein that facilitates HGF-mediated cell invasion through a signaling mechanism that involves Shc and PI3-K (Trusolino et al. 2001). a6b4 can also form a complex with the activated Ron receptor, dependent on 14-3-3 binding, which displaces a6b4 from hemidesmosomes and activates new signaling pathways that promote keratinocyte migration (Santoro et al. 2003). Other studies in carcinoma cells have revealed crosstalk between a6b4 and EGFR that leads to Rho activation (Gilcrease et al. 2009), as well as complex formation between a6b4 and ERBB2 (a binding partner of EGFR) that enhances activation of the transcription factors STAT3 and c-Jun (Guo et  al. 2006). In a less direct

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mechanism of enhanced growth factor signaling, a6b4 can also regulate translation­ of ERBB2 (Yoon et al. 2006) and VEGF (Chung et al. 2002).

2.4.5 Integrin Switches That Promote SCC As described in the following section, SCC cells can acquire new adhesion properties and signaling pathways either through changes in the expression of particular integrins, or through alterations in the signaling functions of integrins that were already expressed in normal epithelial cells. Elucidating the mechanisms that control these integrin switches may identify novel targets for therapeutic agents that inhibit cancer cell-specific integrin functions with minimal off-target effects on normal cells. Figures 2.1, 2.2, and 2.3 illustrate three different ways in which SCC cells can acquire new integrin functions: (1) expression of new integrins (Fig. 2.1), (2) changes in functions of pre-existing integrins (Fig. 2.2), and (3) integrin mutations (Fig. 2.3). The following sections will focus on examples of each mechanism from studies performed in keratinocytes or SCC cells. There are several points to keep in mind when considering these examples. First, as discussed below, there is evidence that some integrin switches may be linked to specific stages of carcinogenesis, and perhaps associated with specific oncogene or tumor suppressor mutations. Second, new integrin expression that is acquired during the clonal expansion of tumor progenitor cells could contribute to both heterogeneous expression ­patterns within a tumor, and variations in expression between different SCC samples (Janes and Watt 2006; Jones et al. 1993). Third, it is still not clear whether a “new” integrin function observed in SCC cells arises as a result of de  novo activation of the function, or reflects the clonal expansion of a stem cell population within which the function pre-existed.

Fig. 2.1  Integrin switches that promote SCC: expression of new integrins.

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Fig. 2.2  Integrin switches that promote SCC: altered function of pre-existing integrins.

Fig. 2.3  Integrin switches that promote SCC: integrin mutations.

2.4.5.1 av Integrin Switch Upregulation of avb6 in SCC occurs at the expense of avb5 expression, probably due to higher affinity of the av subunit for the b6 subunit (Janes and Watt 2004). Consequently, there is a switch from avb5 to avb6 as keratinocytes undergo malignant transformation, similar to that which has been described in wound healing (Clark et  al. 1996). This switch in av integrin expression from avb5 to avb6 ­provides a

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clear example of new integrin expression that facilitates SCC progression­(Fig. 2.1). Recent studies indicate that the switch from avb5 to avb6 protects SCC cells from undergoing differentiation or anoikis when they are deprived of normal attachments to BM, due to the very different effects that these two integrins have on both cell survival pathways and cell death pathways (Janes and Watt 2004). For example, protection of SCC cells from anoikis results in part from the ability of newly expressed avb6 to activate AKT survival pathways in cells that have lost adhesion to the BM and would otherwise undergo anoikis (Janes and Watt 2004). Loss of avb5 may also enhance cell survival, since this integrin had pro-apoptotic effects in its unligated form (Janes and Watt 2004). The switch from avb5 to avb6 may also provide temporal control of TGFb activation by avb6 (see Sect. 2.4.1), since avb5 binds to the latent TGFb complex with significantly lower avidity and probably does not support efficient TGFb activation (Sheppard 2005). Although the mechanism that triggers the switch in av integrins is not yet clear, possible effectors of avb6 induction include TNFa (Scott et al. 2004) and, interestingly, TGFb itself (Zambruno et al. 1995).

2.4.5.2 Functional Switch in a6b4 In addition to de novo integrin expression described above, there is increasing evidence that some pre-existing integrins undergo functional changes during carcinoma progression, leading to the activation of new signaling pathways that promote tumorigenesis and invasion. Integrin a6b4 provides a paradigm for this sort of regulation, as during malignant conversion it switches from a predominantly adhesive receptor to a proinvasive signaling protein that can form complexes with growth factor receptors (Lipscomb and Mercurio 2005; Mercurio et al. 2001) (Fig. 2.2). As already mentioned, a6b4 is associated with the intermediate filaments in hemidesmosomes of normal, undamaged epidermis (Litjens et al. 2006), but in invasive carcinoma cells this integrin relocates to actin-associated filopodia and lamelipodia through a mechanism that involves PKCa-mediated phosphorylation of the b4 cytoplasmic domain (Mercurio et  al. 2001; Rabinovitz et  al. 1999). There is also evidence that EGFR signaling through FYN kinase can disrupt a6b4 function in hemidesmosomes (Mariotti et al. 2001). This relocalization of a6b4 not only relieves tumor cells of stable adhesion that would presumably suppress invasive growth, but it also frees a6b4 to form new signaling complexes with growth factor receptors, such as those described above in Sect. 2.4.4.3, that promote tumor growth and malignant progression. Interestingly, the SCC-associated switch in a6b4 function can be influenced by mutations in key oncogenes and tumor suppressor genes. Indeed, a recent study showed that a6b4 acts as either a tumor suppressor or tumor promoter depending on specific mutations that are acquired by keratinocytes (Raymond et al. 2007). Specifically, a6b4 inhibited tumor growth in tumorigenic keratinocytes that harbor loss-of-function mutations in the genes that encode p53 (Trp53) and Smad4 (Smad4), but it promoted tumor growth when these same cells were transformed by oncogenic RAS, indicating that RAS-mediated transformation induces a switch in a6b4 function (Raymond et  al.

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2007). Consistently, a6b4 was also shown to be essential for SCC formation caused by oncogenic RAS and the inhibitor of kBa (IkBa) (Dajee et al. 2003). 2.4.5.3 Functional Switch in a3b1 Integrin a3b1 provides another example of an integrin that can acquire novel signaling functions as keratinocytes accumulate cancer-promoting mutations (Fig. 2.2). We showed that a3b1-dependent Mmp9 gene expression (discussed above in Sect.  2.3.2) was acquired in mouse keratinocytes as a result of immortalization caused by loss of p53, and was retained in RASV12-transformed versions of these cells where it promoted cell invasion (Lamar et  al. 2008a). Importantly, a3b1dependent MMP-9 expression was also observed in immortalized human keratinocytes and human SCC cell lines that harbor p53 mutations (Lamar et  al. 2008a). Cancer-cell specific pathways whereby a3b1 induces expression of the MMP9 gene or other EMT-promoting genes, would be attractive therapeutic targets. However, it is not yet known whether the acquisition of a3b1-dependent MMP-9 expression by immortalized cultures of keratinocytes represents a switch in a3b1 function that occurred de novo within tumor progenitor cells that have lost p53, or reflects the a3b1-dependent outgrowth of stem/tumor-progenitor cells that already possess this pathway. That a3b1 may promote the expansion of a tumor progenitor/ stem cell compartment is an intriguing possibility, especially since a3b1 has been reported to be expressed at higher levels in epidermal stem cells (Jones et al. 1995). Consistent with such a role, a3b1 in keratinocytes is a known regulator of RAC1 signaling pathways (Choma et al. 2004), and RAC1 is essential for maintenance of the stem cell compartment in the epidermis (Benitah et al. 2005). 2.4.5.4 Gain-of-Function Mutations in Integrins Another potential mechanism whereby altered integrin function may contribute to SCC progression is through rare gain-of-function mutations in integrin genes that predispose keratinocytes to the transforming effects of oncogenes. To date, the best example of such a mutation is T188Ib1, which was first identified as a heterozygous mutation in the b1 gene (ITGB1) of a human cell line derived from a poorly differentiated SCC of the tongue (Evans et al. 2003). This mutation, which occurs in a region of the b1 I-like domain that determines ligand-binding specificity, leads to constitutive activation of all ab1 integrin heterodimers and causes enhanced cell spreading, sustained ERK signaling, and reduced differentiation (Evans et al. 2003; Ferreira et al. 2009). Nevertheless, transgenic expression of T188Ib1 did not alter normal architecture or homeostasis of the epidermis (Ferreira et al. 2009), consistent with the notion that this mutation is a genetic polymorphism that has no deleterious effects on epidermal development or function (Evans et al. 2003). However, following chemical carcinogenesis to induce skin tumors, mice expressing T188Ib1 in the epidermis showed an increase in the frequency and rate of papilloma conversion to

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SCCs, and also formed more poorly differentiated SCCs, compared with mice expressing only wild type b1 (Ferreira et al. 2009). These intriguing findings suggest that the T188Ib1 mutation both predisposes benign tumors to malignant conversion and promotes formation of less differentiated tumors, providing an example of an integrin mutation that may influence both susceptibility to SCC and disease progression. Although other polymorphisms in the genes that encode b integrin subunits have been reported to occur in SCCs or other human cancers (Evans et  al. 2003, 2004), their effects on carcinogenesis are not yet known. It also remains to be determined whether activating mutations in other domains of the b1 subunit, or in other b or a integrin subunits, can similarly predispose epidermis to SCC.

2.5 Exploiting Integrins in the Clinic As described in the preceding sections, preclinical studies using cell culture and in vivo models of SCC have identified critical roles for integrins in the regulation of tumor growth, invasion, and metastasis. These studies provide a solid foundation for the development of therapeutic strategies to inhibit SCC using agents that target integrins, particularly since the location of integrins on the cell surface make them readily accessible to therapeutic compounds. Indeed, several types of integrin antagonists are currently in preclinical and clinical development, including humanized monoclonal antibodies (i.e., volociximab against a5b1, and Vitaxin against avb3), RGDcontaining peptides (i.e., cilengitide), and non-peptide antagonists (Mulgrew et  al. 2006; Rust et al. 2002; Stupp and Ruegg 2007; Thomas et al. 2006; Van Waes et al. 2000; Wu et al. 1998). The majority of these compounds are intended for use as angiogenesis inhibitors with a demonstrated ability to target integrins expressed on endothelial cells in the tumor vasculature (reviewed in (Stupp and Ruegg 2007; Tucker 2006)). However, preclinical studies in mice suggest that these and other compounds also effectively target integrins on tumor cells to reduce tumor cell growth, survival and metastasis (Chen et al. 2008; Gramoun et al. 2007; Harms et al. 2004; Landen et al. 2008; Park et  al. 2006; Stoeltzing et  al. 2003). Importantly, investigators have also exploited the increased or newly acquired expression of integrins on tumor cells, or tumor vessels, to deliver chemotherapies specifically to tumors (Abraham et al. 2007; Arap et al. 1998; Hallahan et al. 2003). Integrin-targeted micelles or liposomes have also been utilized to specifically deliver genes and antisense oligonucleotides to tumors (Bachmann et al. 1998; Cemazar et al. 2002; Oba et al. 2007). Despite the success of some integrin antagonists in preclinical studies, these compounds have had only minimal success in early clinical trials (reviewed in (Stupp and Ruegg 2007; Tucker 2006)). In addition, recent evidence suggests that at least some integrin antagonists may enhance, rather than inhibit, tumor growth and angiogenesis under certain circumstances. For example, the avb3/avb5-specific RGD-mimetic, cilengitide, was shown to have antitumor activity in preclinical and clinical studies of certain forms of glioblastoma (Reardon et al. 2008), but it was recently reported to stimulate tumor growth and angiogenesis when administered at low doses in a murine model of tumor growth (Reynolds et  al. 2009). These

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discordant­ findings could be due to dose-dependent effects of cilengitide, or they may reflect differences between cancer types in the roles that av integrins play on tumor cells and endothelial cells (Weller et al. 2009). In any case, they highlight the importance of understanding the roles of the intended integrin targets within distinct cellular compartments of the tumor type being tested, in order to predict the overall effect of a particular integrin antagonist. Future preclinical studies using inducible and cell-specific transgenic/knockout models, in which candidate integrins and signaling proteins can be manipulated with temporal and spatial precision, should reveal roles of individual integrins within different cellular compartments of the tumor, and help develop effective strategies to inhibit SCC by targeting integrins. Another potential challenge that must be overcome before integrins can be fully exploited as therapeutic targets is that most integrins expressed on SCC cells also perform essential functions in normal cells; therefore systemic delivery of integrin inhibitors may cause adverse side effects. This problem might be avoided by targeting integrins that are expressed at high levels on SCC cells but at low or negligible levels on normal cells, such as avb6 (Fig. 2.1). In addition, identifying mechanisms that trigger new functions of pre-existing integrins, such as a3b1 and a6b4 (Fig. 2.2), or determining how gain-of-function mutations in integrins predispose keratinocytes to SCC (Fig.  2.3), may reveal intermediate molecules or pathways that are activated specifically in cancer cells and can be targeted with minimal effects on normal cell function. Another challenge is that the malignant phenotype is probably influenced by the cumulative functions of the various integrins expressed on SCC cells, such that combinatorial targeting of more than one integrin may be necessary to effectively inhibit disease progression. Integrin antagonists may prove to be most useful in combination with other types of therapeutics. For example, preclinical studies have already demonstrated that cilengitide can synergize with either chemotherapy or radiotherapy (Albert et al. 2006; Burke et al. 2002; Tentori et al. 2008), and other compounds targeting integrins a5b1 and aVb3 were also demonstrated to enhance chemosensitivity (Menendez et  al. 2005; Stoeltzing et  al. 2003) and augment the effects of radiotherapy (Abdollahi et al. 2005). Whether administered alone or in combination with other therapies, the efficacy and specificity of integrin antagonists or inhibitors of integrin signaling pathways could be improved by exploiting newly developed strategies for targeted delivery of compounds to tumors and tumor stroma. It may also be advantageous to target integrin function in tumor cells by combining ­targeted delivery systems with antisense oligonucleotides or RNAi to inhibit specific integrin expression. In support of this approach, antisense oligonucleotides that target avb3 were found to be effective against hepatocellular and mammary carcinoma in preclinical studies (Li et al. 2007; Townsend et al. 2000). In summary, integrins represent promising targets for therapeutic strategies to inhibit SCC development and progression. Although most studies to test integrin antagonists have focused on cancers other than SCC, one preclinical study demonstrated that a non-peptide antagonist of integrin av, SM256, significantly inhibited the in vivo growth of a murine SCC (Van Waes et al. 2000), suggesting that integrin av antagonists are likely to be effective inhibitors of SCC in the clinic, as well. Future clinical and preclinical studies in SCC model systems, using antagonists or RNAi

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strategies developed against other integrins discussed in this chapter, should ­determine if these integrins will also serve as effective therapeutic targets to treat SCC. Acknowledgments  The authors are grateful to the members of the DiPersio laboratory, and c­ olleagues at Albany Medical College, for valuable discussions and insights. Studies conducted by the authors were supported by grants from the NIH/NCI to C.M. DiPersio (R01CA84238, R01CA129637). In addition, J. Lamar was supported by a predoctoral training grant from the National Heart, Lung, and Blood Institute (NIH-T32-HL-07194) and a predoctoral fellowship from the National Cancer Center (06118). We offer our apologies to the many researchers whose valuable contributions to the field could not be cited due to space constraints.

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Chapter 3

Alterations of Transforming Growth Factor-b Signaling in Squamous Cell Carcinomas

Wen Xie and Michael Reiss

Abstract  Genetic mouse models have clearly demonstrated that either activation or attenuation of the transforming growth factor-(TGF-)b and the TGF-b signaling pathway can have a major impact on either the genesis and/or the progression of squamous cell carcinomas (SCC) in the epidermis as well as in the head-and-neck region. In general, inactivation of the TGF-b signaling pathway in stratified squamous epithelium promotes the de novo emergence of benign papillomas that have the potential to progress to invasive SCC. On the other hand, activation of TGF-b signaling in established SCC clearly favors their progression to highly invasive and metastatic SCC. Furthermore, a large number of reports of structural and functional alterations in TGF-b pathway components in human SCC cell lines as well as tumor specimens strongly support the idea that this pathway in general, and TGF-b receptors in particular, play an important role in human SCC as well. Attenuation of either Type I TGF-b receptor (TbR)-I or -II signaling promotes SCC development in mice, and mutation and/or loss of expression of TbR-I or -II receptors are commonly seen in human SCC. Thus, approximately 10–15% of head and neck squamous cell carcinoma (HNSCC) display evidence of functional inactivation of TbR receptor signaling, as defined by the absence of pSmad2 and -3 or the presence of an inactivating TGFBR gene mutation. Patients with this tumor type appear to have a particularly favorable clinical outcome. On the other hand, in approximately 40–60% of human SCC TbR expression is reduced but not eliminated. In this context, exposure of the tumor cells to bioactive TGF-b will activate a proinvasive and -metastatic gene expression program, thereby conferring a worse clinical outcome. Therefore, we would like to propose that a structural and functional analysis of the TbR receptors potentially represents a powerful prognostic tool for the management of patients with SCC.

M. Reiss (*) Division of Medical Oncology, Department of Internal Medicine, The Cancer Institute of New Jersey, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ 08901, USA e-mail: [email protected] A.B. Glick and C. Van Waes (eds.), Signaling Pathways in Squamous Cancer, DOI 10.1007/978-1-4419-7203-3_3, © Springer Science+Business Media, LLC 2011

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Abbreviations DMBA ECM EMT HNSCC IHC MMP SCC SSCP TGFBR1, Tgfbr-1 TGFBR2, Tgfbr-2 TGF-b TPA TbR-I TbR-II

7,12-Dimethylbenz(a)anthracene Extracellular matrix Epithelial-to-mesenchymal transition Human head-and-neck squamous cell carcinoma(s) Immunohistochemistry Matrix metalloproteinase Squamous cell carcinoma(s) Single-strand conformation polymorphism Type I TGF-b receptor gene Type II TGF-b receptor gene Transforming growth factor-b 12-tetradecanoyl-phorbol-13-acetate Type I TGF-b receptor Type II TGF-b receptor

3.1 Transforming Growth Factor-b Signaling in Nonneoplastic Keratinocytes The transforming growth factor-(TGF)-b family of polypeptides comprises a group of highly conserved proteins with a molecular weight of about 25 kDa (Roberts and Sporn 1993). Following activation of latent TGF-b, the ligand binds to the type II TGF-b receptor (TbR-II). The type I receptor (TbR-I) is then recruited into the ligand/TbR-II complex and phosphorylated and activated by the TbR-II kinase. The activated TbR-I receptor then phosphorylates the receptor-associated Smads, Smad2, and Smad3. These, in turn, form complexes with the common Smad, Smad4, and accumulate in the nucleus. Along with coactivators and cell-specific DNA-binding factors, these nuclear activated Smad complexes regulate gene expression and cellular responses. It is important to realize that, besides this classical pathway, the TbR-II receptor is capable of partnering with other members of the type I receptor family, including Alk-1, Alk-2 and, possibly, Alk-3 (Bharathy et al. 2008; Daly et al. 2008; Konig et al. 2005; Lebrin et al. 2005; Liu et al. 2009). In these cases, TGF-b signals can also activate the BMP Smads 1, 5, and 8. This alternate pathway appears to activate a distinct genetic program (Bharathy et al. 2008; Daly et al. 2008; Konig et al. 2005; Lebrin et al. 2005; Liu et al. 2009). Furthermore, in the context of cancer, this second pathway can become constitutively activated and drive epithelial-to-mesenchymal transitions (EMT), cell motility and invasiveness (Bharathy et al. 2008). In keratinocytes of the skin and other stratified epithelia, TGF-b appears to exert two major functions, tissue homeostasis and the response to tissue injury.

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3.1.1 Homeostatic Functions of TGF-b In the epidermis, TGF-b plays a key role in maintaining the balance between cellular self renewal and differentiation and loss. This function is probably mediated by a low level of active TGF-b signaling. Several lines of evidence support this idea. First, using a transgenic mouse model, Cui et al. (1995) showed that constitutive expression of TGF-b1 in suprabasal epidermal keratinocytes protects against 12-tetradecanoyl-phorbol-13-acetate (TPA)-induced hyperplasia preceded by a strong induction of TbR-II expression. Thus, TGF-b1 and its type II receptor are components of the endogenous homeostatic regulatory machinery in the mouse epidermis. In addition, low levels of endogenous phosphorylated Smad2 (pSmad2) can usually be detected in human keratinocytes in culture (Kareddula et al. 2008). Furthermore, when these cells are treated with selective TbR-I kinase inhibitors, pSmad2 becomes dephosphorylated and cell growth is stimulated (Kareddula et al. 2008). In aggregate, these observations indicate that these cells are subject to a low level of endogenous TGF-b signaling and some degree of growth inhibition even in the absence of exogenous TGF-b. Consistent with these in vitro findings, pSmad2 is normally detectable in murine as well as human epidermis (Xie et al. 2003). This suggests that even though most of the TGF-b secreted into the extracellular matrix (ECM) remains latent, a small amount is activated at the cell surface, presumably to control normal cell proliferation and differentiation. Finally, it is likely through this homeostatic function that TGF-b suppresses tumor development. This is clearly illustrated by mice that are homozygous for a hypomorphic allele of the latent TGF-b binding protein, LTBP-4. These animals fail to express pSmad2 precisely in those epithelial tissues that normally express this particular LTBP isoform, such as colon and lung (Sterner-Kock et al. 2002). Furthermore, the mice are prone to developing colon cancer, supporting the idea of a tissue-specific failure of TGFb’s homeostatic function. Finally, we have found that in vivo most human head and neck squamous cell carcinomas (HNSCC) continue to express pSmad2 (Xie et al. submitted; Xie et al. 2003). As these tumors are actively growing, they have presumably escaped from TGF-b-mediated growth arrest. Besides its role in cell cycle control, TGF-b also maintains tissue homeostasis by regulating apoptosis and perhaps others forms of cell death. For example, TGFb-3 plays a key role in mediating the massive apoptosis of mammary glandular epithelium during postlactational involution, an effect that appears to be mediated by Smad3 (D’Cruz et al. 2002; Faure et al. 2000; Yang et al. 2002). A third important homeostatic function of TGF-b is to protect keratinocytes against DNA damage (Glick et  al. 1996, 1999). For example, keratinocytes from Tgfb1 null animals displayed a higher frequency of N-phosphonoacetyl-l-aspartate (PALA)-induced gene amplification than those from wild type animals (Glick et al. 1996, 1999). Moreover, when these cells were transduced with a Ha-Ras oncogene, the frequency of aneuploidy and chromosomal abnormalities was higher than in keratinocytes from TGF-b1 wild-type littermates. Furthermore, exogenous TGF-b1 suppressed gene amplification, aneuploidy, and chromosome breaks in TGF-b1 null

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keratinocytes at lower concentrations than those required for cell cycle arrest, suggesting that the TGF-b signaling pathway directly mediates genomic stability (Glick et al. 1999). It has been demonstrated by more recent studies that genotoxic stress and DNA damage induced by ionizing radiation or cytotoxic chemotherapy are associated with dramatic activation of TGF-b (Biswas et al. 2007; Kirshner et al. 2006). In addition, TGF-b is required for activation of the ATM kinase, which, in turn, coordinates the cellular program of damage control to ionizing radiationinduced DNA damage (Ewan et al. 2002; Kirshner et al. 2006). Thus, in addition to its roles in homeostatic control of cell cycle and -survival, TGF-b1 plays an important role in regulating responses to genotoxic stress, the failure of which may well contribute to cancer development and progression (Andarawewa et al. 2007).

3.1.2 Role of TGF-b in Epidermal Wound Repair In addition to controlling homeostasis, the second major function of TGF-b is to orchestrate and mediate the local response to tissue injury. Wounding results in brisk local activation of TGF-b, which induces epithelial cells to detach from each other, assume a fibroblastoid and motile phenotype (EMT), and to secrete ECM proteins that become incorporated into scar tissue (Roberts et  al. 2001). Phenotypically, EMT is characterized by realignment of the actin cytoskeleton from its submembranous location to a cytoplasmic stress-fiber network connected to focal adhesions, down-regulation of epithelial adhesion molecules such as E-cadherin and zonula occludens 1 (ZO-1), and up-regulation of mesenchymal markers such as fibronectin, Fsp1, a-smooth muscle actin and vimentin (Xu et al. 2009). Normally, this process is self-limited in space and time, so that epithelial cells eventually revert back to their cohesive epitheloid phenotype (Barcellos-Hoff 1998). TGF-b is a potent inducer of EMT in HaCaT human immortalized keratinocytes in vitro (Xu et al. 2009; Zavadil et al. 2001). Moreover, treatment of keratinocytes with TbR kinase inhibitors blocked TGF-b-induced EMT in keratinocytes, and enhanced the epitheloid phenotype (Ge et al. 2004; Peng et al. 2005). Consistent with this, mouse skin keratinocytes stably transfected with a dominant-negative Tgfbr-2 gene were unable to undergo the EMT switch in vivo, indicating that EMT was mediated directly by TGF-b signaling in  vivo as well (Portella et  al. 1998). Furthermore, in TGF-b1/dominant-negative Tgfbr-2 compound transgenic mice, loss of the TbR-II receptor was associated with decreased EMT (Han et al. 2005). In vitro and in vivo studies using dominant-negative Smad mutants, Smad RNAi, tissue-specific Smad knock-out or Tgfbr-1 mutants that cannot bind and activate R-Smads have clearly established the role of Smad signaling in TGF-b-induced EMT (Xu et  al. 2009). For example, skin keratinocytes derived from Smad3 null mice have a reduced migration response to TGF-b in vitro (Ashcroft et al. 1999). In contrast, in a mouse model with a targeted deletion of Smad2 in skin keratinocytes, the absence of Smad2 promoted EMT and accelerated skin tumor formation, implying that Smad2 may function to maintain an epithelial phenotype (Hoot et al. 2008).

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The results suggest the possibility that the Smad3 to Smad2 ratio may be the main determinant of EMT. In addition to receptor-associated Smads, Smad4 is also indispensable for EMT. Knockdown expression of SMAD4 by RNAi or expression of a dominant negative mutant of SMAD4 resulted in preserving E-cadherin expression and in suppressing the profibrotic type I collagen in human epidermal keratinocytes in vitro (Valcourt et al. 2005). The Ras-Erk MAP kinase pathway, activated by either EGF or an oncogenic Ras gene, appears to cooperate with TGF-b in inducing EMT in keratinocytes. In cells expressing wild type Ras, TGF-b activates ERK and p38 MAPK, and levels of activation are increased further by treatment with EGF. Activation of MEK/Erk/ MAP kinase signaling enhances TGF-b induced transcription responses, leading to downregulation of E-cadherin, upregulation of N-cadherin and increased matrix metalloproteinase (MMP) expression (Janda et  al. 2002; Lehmann et  al. 2000). Conversely, inhibition of the MAPK pathway blocked the induction of EMT by TGF-b (Davies et al. 2005). Bae et al. (2009) recently examined the interactions between ras and TGF-b signaling in primary wild type and Smad3 null mouse epidermal keratinocytes. Oncogenic ras and hyperactivation of the ERK1/2 pathway did not affect Smad2 phosphorylation, nuclear translocation or regulation of Smad3 dependent homeostatic gene responses. In contrast, the induction of many extracellular matrix TGF-b/Smad3 target genes was attenuated by v-rasHa. Thus, ERK1/2 activation has distinct effects on the TGF-b transcriptome, especially favoring EMT and matrix remodeling. Besides the MAPK pathway, Notch signaling pathway has also been implicated in the regulation of EMT by TGF-b. In response to TGF-b, the immediate early induction of Hey1, a transcriptional target of Notch, followed by induction of the Notch-ligand, Jagged1 (Jag1), contribute to EMT by disassembly of E-cadherin adherens junctions, resulting in cell–cell separation, and increased cell motility of epidermal keratinocytes (Zavadil et al. 2004). Silencing of Jag1 or Hey1 expression using siRNA or chemical inactivation of Notch signaling block TGF-b-induced EMT (Zavadil et al. 2004). Activation of Hey1 and delayed expression of Jag1 by TGF-b is Smad3-dependent, as it does not occur in Smad3-deficient cells (Zavadil et  al. 2004). In addition, Jag1 and Hey were activated in chemically induced squamous cell carcinomas (SCC) in Tgfb-1 transgenic mice, but not in Tgfb-1/ dominant-negative Tgfbr-2 bigenic mice in which the epidermal cells fail to undergo EMT in vivo (Han et al. 2005). In chronic inflammatory conditions, persistent activation of TGF-b causes epithelial cells to be permanently converted to myofibroblasts, eventually resulting in the loss of epithelial structures and progressive tissue fibrosis (Border and Noble 1994; Branton and Kopp 1999; Roberts et al. 2006). Similarly, persistent activation of TGF-b signaling in the context of cancer appears to drive invasion and metastasis via constitutive induction of EMT (Massague 2008). For example, in mouse models of skin carcinogenesis, the highly invasive and metastatic mesenchymal phenotype that is characteristic of late stage SCC appears to be associated with a persistent autocrine or intrinsic activation of TGF-b signaling (Oft  et  al. 2002; Portella et  al. 1998). Thus, in concert with a Ha-ras oncogene,

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activated Smad2 drives the progression of well-differentiated skin SCC to highly invasive undifferentiated SCC by breaking down cell cohesion, increasing cell motility and inducing invasion (Oft et al. 2002). Furthermore, these spindle cells carcinoma with elevated levels of activated Smad2 acquire the capability to metastasize (Oft et al. 2002). Thus, in this case, constitutive activation of TGF-b’s tissue repair function represents an oncogenic event. Loss of E-cadherin expression is a key feature of EMT, particularly in tumor cells (Xu et  al. 2009). Consistent with the genetic mouse models, E-cadherin expression is inversely correlated both with tumor grade and with the presence of lymph node metastases in human HNSCCs, suggesting that the loss of the cell adhesion molecule E-cadherin plays an important role in the progression of human HNSCC in  vivo (Schipper et  al. 1991). Using a dominant-negative form of E-cadherin to repress endogenous E-cadherin expression, Andl et al. (2006) demonstrated this to result in decreased cell adhesion, enhanced migration and invasion of human primary esophageal keratinocytes. Interestingly, overexpression of wildtype E-cadherin was associated with elevated TbR-II mRNA and protein levels. Moreover, the extracellular domains of E-cadherin and TbR-II appear to physically interact, suggesting a coordinated role for E-cadherin and TbR-II loss in epithelial carcinogenesis (Andl et al. 2006).

3.1.3 TGF-b Signaling and Angiogenesis In addition to its effects on normal and malignant keratinocytes, TGF-b exerts a wide range of effects on the normal host microenvironment that indirectly play an important role in the development and progression of SCC and other epithelial carcinomas. For example, both in  vitro studies and mouse models have demonstrated that TGF-b1 is a potent inducer of angiogenesis associated with wound repair and tumor development (reviewed in Pardali and ten Dijke (2009)). Increased angiogenesis was observed in preneoplastic head and neck lesions in Tgfb-1 transgenic mice (Lu et al. 2004). Similarly, in xenografts of human HNSCC tumor cells, TGF-b1 appears to attract tumor associated macrophages into the tumor microenvironment, and to induce these cells to secrete angiogenic factors, such as VEGF and interleukin (IL)-8. Moreover, this process can be blocked by treatment with anti-TGF-b1 antibody, suggesting the possibility that such an antibody could be used as an antiangiogenic agent (Liss et al. 2001).

3.1.4 TGF-b Signaling and Immune Suppression Secretion and activation of TGF-b1 by tumor cells also stimulate tumor development and progression indirectly via immune evasion. TGF-b inhibits the proliferation and functional differentiation of T lymphocytes, lymphokine-activated killer cells,

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natural killer cells (NK cells), neutrophils, macrophages as well as B cells (Li and Flavell 2008; Wan and Flavell 2008; Wrzesinski et al. 2007). Consistent with TGFb1’s immune-suppressive but potent chemotactic effects on macrophages and neutrophils, HNSCC arising in Tgfbr-2 null mice are associated with increased expression of endogenous TGF-b and increased macrophage and neutrophil infiltration (Lu et al. 2006). Furthermore, TGF-b expression by tumor cells promotes tumorigenicity by locally repressing immune functions. Thus, Dasgupta et  al. (2005, 2006) examined the effects of vaccination with a recombinant vaccinia virus expressing IL-2 (rvv-IL-2) on NK cell-mediated anti-tumor immunity in an orthotopic murine model of HNSCC (SCC VII/SF). SCC VII/SF tumors expressed high levels of TGFb-1, which were down modulated by vaccination with rvv-IL-2. Incubation of NK cells with tumor homogenate or cultured supernatant of SCC VII/ SF cells reduced the expression of NKG2D and CD16. This inhibition appeared to be mediated by TGFb-1, as it could be blocked by treatment with a TGF-bneutralizing antibody (Dasgupta et al. 2005, 2006).

3.1.5 TGF-b Signal Strength Determines Response Type in Human Keratinocytes As summarized above, TGF-b maintains keratinocyte homeostasis on the one hand, and orchestrates EMT and the response to tissue injury on the other. However, surprisingly little is known about how these two fundamentally different responses to TGF-b are regulated. We recently examined the TGF-b-regulated gene expression programs and cellular responses in human keratinocytes as a function of TbR-I kinase activity and TGF-b level (Kareddula et  al. 2008). The TGF-b-mediated homeostatic gene response program and cellular growth arrest were extremely sensitive to a reduction in receptor kinase activity, while much stronger inhibition of TGF-b receptor activity was required to block the tissue injury response gene expression program and EMT. Both endogenous TGF-b and high exogenous levels of TGF-b controlled homeostasis, while higher levels of TGF-b were required to induce EMT. The results suggest the working model that two major changes in TGF-b signaling might occur during SCC development: First, a global reduction in receptor signaling and results in loss of homeostatic control and of TGF-b’s tumor suppressive activity. At a later stage of tumor progression, overproduction of bioactive TGF-b might result in activation of a proinvasive, -angiogenic, and -metastatic TGF-b-regulated gene expression program. On the other hand, complete inactivation of TGF-b signaling by, for example, inactivating mutations or deletion of one of the TGFBR genes, would result not only in loss of TGF-b-dependent homeostatic control, but also eliminate TGF-b’s proinvasive and -metastatic actions. Therefore, one might predict that tumors with somatic mutation or deletion of a TGFBR gene might be less aggressive than those in which TGF-b signaling is partly retained. Finally, this model predicts that TGF-b pathway antagonists that target the intracellular signaling machinery (for example, chemical receptor kinase

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inhibitors) may alter the cellular gene expression profile and phenotype in ways that are quite distinct from agents that trap excess ligand (for example, neutralizing TGF-b antibodies). While the former may mimic the effects of lowering receptor expression or receptor mutation, the latter may be more selective in blocking the metastasis-associated gene expression profile. These are important issues to consider in the design of clinical trials of these agents.

3.2 Alterations of TGF-b Signaling and Squamous Cell Cancer In humans, SCCs arise primarily in the skin, the aerodigestive tract (which includes the head and neck region, esophagus, and bronchi), and the uterine cervix. Based on the experimental evidence summarized in the earlier sections, one might predict that loss of TGF-b signaling, both by defects of TGF-b receptors or of Smads, would result in uncontrolled proliferation of squamous epithelial cells and promote the development of SCC. In addition, constitutive activation of TGF-b signaling in advanced stage SCC cell may play a role in SCC progression. In the following sections, we will review the changes in genomic sequence, expression and function of TGF-b, it’s receptors, and the Smads that have been found in human SCC and how these might contribute to the development of human SCC.

3.2.1 TGF-b Ligands 3.2.1.1 TGF-b in Mouse Models of Squamous Carcinogenesis Several studies have shown that TGF-b1 exerts potent effects on the malignant transformation of keratinocytes (Glick et al. 1993, 1994). For example, TGF-b1 null (TGF-b1−/−) primary mouse keratinocytes undergo spontaneous transformation at significantly higher frequency than wild type cells. Furthermore, v-Ha-Ras transformed Tgfb-1−/− keratinocytes transplanted onto the skin of athymic mice gave rise to papillomas with dysplasia that rapidly progressed to multifocal SCC, irrespective of the dermal fibroblast genotype (Tgfb-1 wild-type or null), while grafts from v-Ha-Ras transformed keratinocytes with wild-type Tgfb-1 did not progress beyond well-differentiated papillomas (Glick et al. 1993, 1994). Similarly, when TGF-b1 was conditionally overexpressed in skin keratinocytes and the mice were exposed to the 7,12-dimethylbenz(a)anthracene (DMBA)-TPA two-stage chemical carcinogenesis protocol, TGF-b1 suppressed skin papillomas formation (Cui et al. 1996). Conversely, ectopic expression of constitutively active Tgfb-1 in keratinocytes was associated with increased resistance to TPA-induced benign skin tumor formation (Cui et al. 1995, 1996). These studies clearly demonstrated

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that TGF-b1 acts as a potent tumor suppressor of skin carcinogenesis (Cui et al. 1995, 1996). In a genetic mouse model of HNSCC, in which TGF-b1 expression levels was selectively induceable in squamous epithelia in a dose-dependent manner by the synthetic progesterone inhibitor RU486, increased levels of TGF-b1 were associated with severe inflammation and angiogenesis (Lu et al. 2004). Moreover, Tgfb-1 transgenic epithelia exhibited a phenotype of hyperproliferation in the buccal mucosa, tongue and esophagus. This result appears to contradict previous in vivo studies that had suggested that induction of TGF-b1 inhibits proliferation of keratinocytes (Cui et al. 1996; Wang et al. 1999). One possible explanation is that the increased proliferation of keratinocytes was caused indirectly by inflammation and angiogenesis, which may override the antiproliferative effect of TGF-b. Therefore, some of the growth promoting effects associated with the overexpression of TGF-b at early stages of SCC might be attributed to a tumor-promoting microenvironment induced by TGF-b. In contrast to its ability to suppress initial papilloma and SCC development, TGF-b appears to promote progression of SCC once they have formed. Thus, when TGF-b1 is conditionally overexpressed in skin keratinocytes and the mice are exposed to the two-stage chemical carcinogenesis protocol, TGF-b1 increases the invasiveness and metastatic potential of the SCC once they arise (Cui et al. 1996). Under the influence of Tgfb-1 transgene expression, benign skin tumors underwent EMT, forming invasive spindle carcinoma cells in vivo, which expressed high levels of TGF-b3. Metastatic SN161 cells, derived from a chemically induced mouse skin carcinoma, underwent a reversible conversion to a fibroblastoid phenotype in vitro following treatment with TGF-b1. Furthermore, these SCC cells spontaneously converted to a fibroblastoid phenotype after subcutaneous inoculation in nude mice. Conversely, SN161 clones that were stably transfected with a dominant-negative TGFBR2 gene failed to develop into spindle cell carcinomas in vivo, demonstrating that the EMT was mediated directly by the TGF-b signaling pathway, and was sufficient to enhance tumorigenicity and invasive characteristics of the tumor in vivo (Portella et al. 1998). To address whether the two disparate effects of TGF-b, i.e., inhibition of papilloma formation and enhancement of tumor metastasis, are both dependent on TGF-b receptor signaling, Han et al. (2005) used a mouse skin cancer model that allows stage-specific overexpression of Tgfb-1 in the context of keratinocyte-­ specific overexpression of a dominant-negative Tgfbr-2 gene. These investigators found that, in a wild type Tgfbr-2 background, induction of TGF-b1 early during carcinogenesis suppressed tumor formation, while, at later stages, TGF-b1 not only failed to inhibit tumor growth but also induced EMT and metastasis. Conversely, in a dominant-negative Tgfbr-2 background, induction of TGF-b1 early in carcinogenesis failed to suppress benign tumor growth. However, even in the presence of TGF-b1, metastases failed to develop, indicating that TbR-II also mediates TGFb’s ability to promote metastasis in a cell autonomous manner (Han et al. 2005). Thus, the effects of TGF-b1 overexpression on tumor development and progression depend, in large part, on the tumor cells’ ability to respond to the ligand.

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3.2.1.2 TGF-b Expression in Human SCCs Cancer-associated increases in TGF-b expression either at the level of protein or mRNA expression have been reported for a number of different tumor types (reviewed in Gold, 1999). Several studies have addressed the question whether development and progression of human SCC are associated with changes in TGF-b expression (Table 3.1). In general, TGF-b expression tends to be increased in SCC compared to adjacent normal tissues (Eisma et al. 1996; Fukai et al. 2003; Hagedorn et al. 2001; Lu et al. 2004). On the other hand, several studies have suggested that SCC-associated TGF-b expression might decrease as a function of tumor grade (El-Sherif et  al. 2000; Mincione et  al. 2008; Natsugoe et  al. 2002; Torng et  al. 2003). Thus, these studies of TGF-b expression in SCC have yielded conflicting results and no clear pattern has emerged. This is likely a result of, at least in part, differences in the types of tissue specimens examined, in the specific comparisons reported, the different methods used to assess TGF-b expression levels, and specific TGF-b isoforms examined. However, the differences across studies could also be reconciled if one assumes that SCC can be classified into two major subgroups, one in which TGF-b signaling is completely inactivated, and one in which the signaling pathway is attenuated or altered. The signaling pathway appears to be fundamentally altered in tumor cells in such a way that the tumor cells interpret incoming signals as proinvasive, while they are no longer growth inhibited. Thus, a microenvironment rich in bioactive TGF-b would provide a selective pressure that favors growth and invasion of tumor cells with this particular phenotype.

3.2.2 TGF-b Type I Receptor (TbR-I) 3.2.2.1 TbR-I in Mouse Models of Squamous Carcinogenesis Bian et al. (2009) recently developed a genetic mouse model that allows the conditional deletion of the Tgfbr-1 gene in epithelia of the head-and-neck region. This was accomplished by crossing Tgfbr-1 floxed mice with K14-CreER(tam) mice in which a Cre recombinase is fused to a human estrogen receptor, which can be activated by treatment with tamoxifen in vivo. This fusion protein is driven by a keratin 14 (K14) promoter, which specifically targets gene expression to the basal layer of stratified epithelia. Applying tamoxifen to the oral cavity to induce Cre expression resulted in conditionally deleting Tgfbr-1 in the mouse head and neck epithelia. Four weeks following initiation with DMBA and tamoxifen treatment, basal epithelial cells of Tgfbr-1 knock-out mice displayed enhanced proliferation and loss of apoptosis, which were associated with a decrease in pSmad2 and -3 levels as well as activation of the phosphoinositide 3-kinase/Akt pathway. Moreover, almost half the Tgfbr-1 conditional knockout mice began developing SCC in the head and neck area at 16 weeks, while no tumors were observed in control littermates. These results confirmed the critical role of TGF-b signaling in general and of the TbR-I receptor in particular in suppressing head and neck carcinogenesis.

52

15

13

38

258

48

C

C

HN

HN

HN

C

N/A

148 (57)

N/A

0 (0)

N/A

N/A

Decrease

Decrease

No detectable change Increase

Decrease

Decrease

IHC

IHC

IHC

IHC

IHC, qRT-PCR

IHC

Table 3.1  Alterations in TGF-b signaling in human SCC cell lines and tumors Number with TGF-b/Smad signaling defect (%) Alteration type Detection method SCC type n TGF-b (in vivo) Expression HN 47 0 (0) No detectable IHC change HN 17 N/A Increase ELISA

Trend for increased TGF-b1, 2, 3 expression compared to adjacent stromal cells Only TGF-b1 examined. Decrease defined as £ 10% of cells positive TGF-b1 expression decreased in CIN 1, 2, 3 compared to normal (p