Signaling Pathways and Molecular Mediators in Metastasis

Signaling Pathways and Molecular Mediators in Metastasis Alessandro Fatatis Editor Signaling Pathways and Molecular ...

Author: Alessandro Fatatis

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Signaling Pathways and Molecular Mediators in Metastasis

Alessandro Fatatis Editor

Signaling Pathways and Molecular Mediators in Metastasis

Editor Alessandro Fatatis Drexel University College of Medicine 245 N. 15th Street Philadelphia, PA 19102 USA [email protected]

ISBN 978-94-007-2557-7 e-ISBN 978-94-007-2558-4 DOI 10.1007/978-94-007-2558-4 Springer Dordrecht Heidelberg London New York Library of Congress Control Number: 2011944018 © Springer Science+Business Media B.V. 2012 No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

To Mara, Enrico and Fabrizio, for introducing me to life. To Olimpia and our son Andrea, for making my life worthwhile.

Preface

Since the dawn of modern medicine, the spreading of a tumor has always been regarded as an unfavorable event. However, until recently patients would refer to this condition as a tumor that had ‘returned’ and simply prepare for additional rounds of treatment, as if the recurrence would be a replica of the original disease, just in a different site. Today, patients and physicians alike are fully aware that the detection of metastases is a negative prognostic factor, in most cases leading to serious repercussions on the quality of life and overall survival. Indeed, the treatment of metastatic patients with curative intent is frequently a daunting task, albeit not always bond to fail. The first chapter of this book provides an excellent overview of the problem. The gravity of metastatic disease is often due to the multiplicity of lesions to be treated, combined to locations not easily amenable of surgical excision or irradiation. Although cytotoxic, targeted and in selected cases hormone-deprivation systemic therapies can initially bypass some of these limitations, the onset of resistance mechanisms will eventually obligate physicians to change chemotherapy protocol, switch to different targeted drugs and eventually resolve to palliative measures. Furthermore, we now recognize that metastatic lesions might share very little with their tumors of origin as a myriad of events occurring either at primary or secondary sites can dramatically alter genotypic and phenotypic features of cancer cells during the progression of the disease. Thus, aiming to the successful treatment of metastatic lesions based on information gained from primary tumors should be considered a dangerous overlook. Several chapters of this book provide a compelling review of the current knowledge on the changes occurring in different organ microenvironments that permit malignant colonization and subsequent progression of clinically overt metastases. These changes affect not only epithelial cancer cells, but also the resident cells of the surrounding stroma, immune cells and bone marrow-derived cells that can be locally recruited to create a pre-metastatic niche. A significant percentage of solid tumors are currently diagnosed at their initial stages. However, the progress in screening and diagnostic procedures only marginally translates into major survival benefits for patients, as too many still succumb to vii

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Preface

metastatic disease often years after the initial diagnosis. A better understanding of the mechanisms and molecular mediators promoting local tumor progression and invasion into the circulatory and lymphatic systems will lead to appropriate therapeutic strategies aiming to limit the extent and duration of cancer spreading. A number of chapters in this book very effectively address these particular issues. This volume presents the work of scientists at the forefront of the metastasis research field and it is a testimony of the power of their intellect, dedication and efforts to improve the range of treatment options for cancer patients and effectively counteract the most lethal complication of their disease. I knew some of them personally prior to undertaking this project and learned more about the others because of it. It has been a true privilege for me to work on this text with such a group of brilliant co-authors. They all have been enthusiastic about this book from the very beginning and demonstrated a remarkable willingness to participate, despite the variety of academic and clinical commitments they had to attend and the demands of their research groups and medical teams. For this, and for their contribution of competent writing and effective illustrations, I am extremely grateful. I am also indebted to all the staff at Springer, especially Melania Ruiz (Publishing Editor), Ilse Hensen (Publishing Assistant) and Sunil Padman (Project Manager). Their enthusiasm and knowledgeable assistance during the different phases of this project have been very much appreciated. The ensuing of metastatic disease is too often responsible for the demise of patients that would be otherwise successfully treated for their primary tumors. The scientists involved in this project, along with many others in the field, intend to change this grim scenario forever. Philadelphia, PA

Alessandro Fatatis

Contents

1

Introduction ............................................................................................. Robert B. Den and Adam P. Dicker

Part I 2

1

Local Invasion

Reactivation of Epithelial-Mesenchymal Transition in Invasive and Metastatic Cancer ........................................................ Michael D. Amatangelo and Mark E. Stearns

13

3

Biomechanical ECM Switches and Tumor Metastasis ........................ Jacquelyn J. Ames, Calvin P.H. Vary, and Peter C. Brooks

71

4

Moving Aggressively: S100A4 and Tumor Invasion ............................ Reniqua P. House, Sarah C. Garrett, and Anne R. Bresnick

91

5

Regulation of TGF-b Signaling and Metastatic Progression by Tumor Microenvironments ............................................................... 115 Michael K. Wendt and William P. Schiemann

6

Rho GTPases and Their Activators, Guanine Nucleotide Exchange Factors (GEFs): Their Roles in Glioma Cell Invasion ............................................................................................ 143 Bo Hu, Marc Symons, Bodour Salhia, Shannon P. Fortin, Nhan L. Tran, James Rutka, and Shi-Yuan Cheng

7

Impact of Tumor Hypoxia, Src, and Met Signaling in the Dissemination of Tumor Cells ..................................................... 171 Yao Dai, Wenyin Shi, Nikolett Molnar, and Dietmar Siemann

Part II 8

Homing and Colonization at Distant Sites

Cellular and Molecular Biology of Cancer Cell Extravasation .......... 197 J. Matthew Barnes and Michael D. Henry

ix

x

Contents

9

Role of CXCL12 and CXCR4 in Tumor Biology and Metastasis ......................................................................................... 221 Ramesh K. Ganju, Yadwinder S. Deol, and Mohd W. Nasser

10

Transcription Factors Stat5a/b and Stat3 in Prostate Cancer Growth and Metastases ............................................................. 245 Tuomas K. Mirtti, Pooja Talati, and Marja T. Nevalainen

11

Survival and Growth of Prostate Cancer Cells in the Bone: Role of the Alpha-Receptor for Platelet-Derived Growth Factor in Supporting Early Metastatic Foci ......................................... 261 Qingxin Liu, Yun Zhang, Danielle Jernigan, and Alessandro Fatatis

12

Androgen Receptor Regulation of Prostate Cancer Progression and Metastasis .................................................................... 277 R.S. Schrecengost, M.A. Augello, and Karen E. Knudsen

13

Integrin Signaling in Angiogenesis and Metastatic Cancer Progression in the Brain............................................................ 311 Mihaela Lorger and Brunhilde Felding-Habermann

14

Tumor and Host Determinants of Pulmonary Metastasis in Bladder Cancer ................................................................ 331 Neveen Said and Dan Theodorescu

15

The Influence of Metastatic Breast Cancer on the Bone Microenvironment ............................................................. 347 Andrea M. Mastro, Donna M. Sosnoski, Venkatesh Krishnan, and Karen M. Bussard

Index ................................................................................................................. 369

Chapter 1

Introduction Robert B. Den, MD and Adam P. Dicker, MD, PhD

Abstract Local therapies have continued to evolve with advances in surgical and radiation therapy techniques. This has contributed to improvements in survivorships in early stage disease. However, survival rates for metastatic disease remain poor. There has been increasing evidence that local therapy directly influences metastatic growth and overall survival. Both clinical and laboratory evidence support this hypothesis and an increasing understanding of the role of circulating tumor cells has further galvanized the idea of crosstalk between distant sites and the local disease. The current clinical arena is ripe for new innovations and the scientific advancements described in the remainder of this text set the stage upon which such progress will occur.

Cancer is the second leading cause of death in the United States for both men and women [1]. Currently, there are 1.5 million new cases diagnosed per year with a declining cancer-specific mortality rate of 600,000 per year due in large part to early detection and improved therapeutics. Five-year survivorship is markedly decreased between localized and metastatic disease. Regardless of the initial tumor type, 5-year overall survival with stage IV malignancy ranges from 2% to 30% in comparison to

R.B. Den, MD Assistant Professor, Department of Radiation Oncology and Cancer Biology, Kimmel Cancer Center, Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA 19107, USA A.P. Dicker, MD, PhD () Professor and Chair of Radiation Oncology, Professor of Pharmacology and Experimental Therapeutics, Department of Radiation Oncology, Kimmel Cancer Center, Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA 19107, USA e-mail: [email protected]

A. Fatatis (ed.), Signaling Pathways and Molecular Mediators in Metastasis, DOI 10.1007/978-94-007-2558-4_1, © Springer Science+Business Media B.V. 2012

1

2

R.B. Den and A.P. Dicker 100

25

90 20

70 60

15

50 40

10

DM Rate

Local Control

80

30

20

5

10 0

0 90%) are carcinomas which arise from epithelial glands. Surgery to remove the primary tumors is an effective treatment for many cancers and patients treated prior to metastases are often cured. Unfortunately, treatment of advanced cancer is often complicated by metastatic disease where cancer cells have migrated to distant sites. Thus, the majority of cancer deaths are caused by the ability of cancer cells to become detached from the neoplastic epithelia and form metastases where surgery becomes impossible. It has become increasingly apparent that in order for cancer cells to accomplish this, an EMT-like program must be activated to prime nodular epithelial cells for the dissemination, movement and invasion required for metastatic spread [7]. The concept that EMT events are involved in the formation of metastatic cancer are primarily based on mechanistic studies done in vitro and in mouse models and observations that loss of epithelial characteristics and acquisition of mesenchymal markers within tumors is often associated with advanced disease. It must be noted, however, that EMT changes do not appear to occur in consistent patterns in cancers nor is the commitment to a mesenchymal phenotype always permanent and the reverse process, mesenchymalepithelial transition (MET), is also observed [8]. Although many similarities in the major concepts of developmental and pathological EMTs exists, Kalluri and Weinberg have classified EMT events into three separate categories to help clarify key differences in the functional consequences and regulation of these events [9]. Accordingly, EMT events associated with early

16

M.D. Amatangelo and M.E. Stearns

embryo and organ development are considered type-1 EMT. This type of EMT is tightly regulated, does not induce fibrosis or systemic, uncontrolled invasion and results from cells which have not fully matured. Type-2 EMT events are associated with wound healing, tissue regeneration and fibrosis. This type of EMT is less controlled and occurs in adult tissues in response to inflammation. Type-3 EMT occurs in carcinoma cells in which changes in oncogenes and tumor suppressor genes in conjunction with tumor associated inflammation utilize the EMT machinery to induce a migratory phenotype and invasion. While there are functional differences between tumor progression and normal embryonic development it also has become quite clear that many similarities also exist [10]. Thus, it is limiting to assert that type-1 and type-3 EMT events are distinctly different and it should be considered that a type-3 EMT event is a reactivation of developmental pathways observed in type-1 EMT resulting from the cellular and environmental changes in cancer. This reactivation in the context of cancer consequently enhances epithelial cell plasticity and promotes aberrant invasive and migratory activities of the cancer cell. Unfortunately, and unlike type-1 and type-2 EMT events, it has been difficult for pathologists to conclusively document type-3 EMT events associated with tumor progression and metastasis in humans [11]. EMT has, however, been directly observed at the leading edge of a spontaneously driven breast cancer model in vivo in mice utilizing stromal and epithelial cell specific cre-transgene markers [12]. Although this study definitively showed cells of epithelial origin becoming mesenchymal during tumor progression and being associated with increased invasion and metastasis, not all tumors that gave rise to metastases exhibited robust EMT. Therefore, it could be concluded that EMT is not the only mechanism by which tumors become invasive and metastatic. Invasive carcinomas may invade surrounding tissues as multicellular epithelial sheets which maintain cell-cell junctions and polarity in a process known as collective migration [13]. However, it has recently been demonstrated that cells restricted to collective invasion were only capable of lymphatic invasion and metastasis to adjacent organs, but not entry into blood vessels or dissemination to distant organ sites [14]. Furthermore, while in many tumors the presence or absence of local lymph node metastasis is a strong predictor of distant metastatic disease, in others, as many as 30% of patients free of lymph-node metastasis still develop disease at distant sites [15]. In agreement with these observations, cancers which display clear forms of collective migration, such as squamous cell carcinomas, rarely form distant metastases [16]. Collectively migrating cells also appear to always follow the tracks of a leading stromal cell but may also follow tracks or signals of migrating epithelial cells that have undergone EMT, suggesting that a cooperative interaction by cells with each other and the surrounding stroma is required for collective invasion [17]. The overall picture emerging from such observations is that collective migration may not be a sufficient for the formation of distant metastases. Regardless, it is clear that changes in the local extracellular matrix and adaptation of a motile phenotype are required for metastasis and such changes can occur as a result of EMT.

2 Reactivation of Epithelial-Mesenchymal Transition in Invasive and Metastatic Cancer

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We suggest that type-3 EMT events play a significant role in the formation of distant metastasis. These events may represent transient processes of EMT-MET conversions whereby invasive cells which undergo EMT regularly revert back to a normal morphology following specific interactions with the surrounding stroma or to a new environment. Cells which exhibit the plasticity to undergo EMT and MET conversions are likely to be highly adaptable, metastatic and capable of forming distant metastases. While proof that such events occur during metastasis is not conclusive, given the discrete nature in which EMT might occur, standard pathological examination is not conducive to observing EMT or associating EMT with advanced and metastatic cancer. This is primarily because type-3 EMTs occur to different extents and exhibit different phenotypes, with some cells retaining more epithelial traits then others. Interestingly, many cells undergoing EMT adopt a cancer stem cell-like phenotype and are able to produce metastases in mice at very low titer [18, 19]. These results suggest that cells undergoing EMT might not have to en mass in order to promote the formation of metastasis. Accordingly, such events might go unobserved during pathological observation of tumors and the frequency of cells displaying EMT markers might not correlate with metastatic spread. So, while it is apparent in animal and in vitro models that different type-3 EMT events are related to metastatic potential, it is unclear whether the extent which a cell loses epithelial characteristics or gains mesenchymal traits or whether the quantity of cells undergoing EMT has any correlation to metastasis in humans. Thus, evidence in support of EMT will require real-time Imaging since the dynamic nature of such EMTs are difficult to capture in fixed tissues where only a small number of tumor cells might exhibit EMT markers at any one time. Furthermore, standard histological examination of human tumors is observational and not mechanistic in nature and therefore limited in its ability to understand the contribution EMT might have to metastatic progression. Until experimental techniques are available which resolve these issues in humans, the importance of EMT to human cancer metastasis will remain controversial.

2.2

EMT Biomarkers and Their Functional Significance

In order to generate cells with specific functions cells during development must exhibit a level of plasticity that allows them to give rise to or morph into other phenotypes. As this process proceeds, cells must change the repertoire of proteins they express in order to function in their new role. Identification of such changes in protein expression can be used as biomarkers to identify cells associated with a specific purpose. A variety of biomarkers specific to cells of epithelial and mesenchymal origin have been identified to demonstrate the three subtypes of EMT. Indeed, the changes in these biomarkers, including changes in ‘cell-cell’ adhesion, cytoskeleton dynamics and matrix remodeling, have functional consequences and clinical significance as they all play important roles in metastatic progression.

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2.2.1


Disruption of Epithelial Cell-Cell Junctions

E-cadherin is an adherence junction protein that is expressed at ‘cell-cell’ junctions in nearly all epithelial cells but is absent in mesenchymal cells [20]. Furthermore, E-cadherin expression is critical for proper organization and differentiation of epithelial cells during development, as well as for maintaining the apical-basal polarity that is a hallmark of epithelial tissues [21, 22]. Decreases in E-cadherin expression are nearly always observed during EMT associated with development, tissue fibrosis, wound healing and cancer progression and thus its loss is the major biomarker for an EMT event [23]. The most convincing evidence for EMT being associated with invasive and metastatic disease is that loss of E-cadherin-based adherence junctions is consistently associated with progression to invasive carcinoma and poor prognosis in most human epithelial cancers, including carcinomas of the breast, colon, prostate, stomach, liver, esophagus, skin, bladder, kidney and lung [24]. The loss of E-cadherin at ‘cell-cell’ junctions promotes metastasis by enabling cells to detach in response to the shear forces found in lymphatic vessels, venules and arterioles, facilitating their dispersion from the tumor mass [25]. Impairment of E-cadherin-mediated cell adhesion during tumor progression has been shown to occur by deletion, mutation, chromatin rearrangement and hypermethylation. In addition, loss of E-cadherin promoter activity has been found to occur in many metastatic malignancies [24, 26]. In fact, knockdown of E-cadherin alone can induce wide-ranging transcriptional and functional changes which manifest in EMT and contributes to metastatic dissemination [27]. Conversely, several groups have demonstrated that forced re-expression of E-cadherin in malignant cells results in a reversion of the EMT phenotype and inhibits invasion and metastasis [28–31]. Additionally, in an in vivo model for spontaneous pancreatic cancer, maintenance of E-cadherin expression during beta-cell tumorigenesis inhibited invasion and arrested tumor development at the adenoma stage [30]. The implication being that loss of the E-cadherin adherence junction complex is one of the rate limiting step in EMT and progression to invasive cancer in vivo and thus serves as the gatekeeper of the epithelial phenotype [32]. Critical for maintaining E-cadherin based adherence junctions is its interaction with p120 CAS and alpha-, beta-, and gamma-catenin, which link E-cadherin to the actin cytoskeleton. Disruption of the intracellular E-cadherin–catenin complex alone is sufficient for a loss of ‘cell-cell’ adhesions and the tissue rigidity characteristics of epithelial tissues which occurs in tandem with tumor invasion [33]. In particular, beta-catenin is released from E-cadherin complexes into the cytoplasm when these ‘cell-cell’ junctions are disrupted and can act as a signaling molecule. The signaling activity of beta-catenin regulates cellular plasticity and type-1 EMT during heart cushion development [34]. Accordingly, beta-catenin localization has been used as a biomarker of EMT in both fibrosis and cancer [35, 36]. In normal epithelium, cytoplasmic beta-catenin is degraded by the ubiquitin-proteasome pathway through a multiprotein destruction complex containing APC and GSK-3beta. Upon activation of Wnt signaling pathways within the cell (which regulate


19

proliferation, morphology, motility, and fate during embryonic development) the activity of GSK-3beta is inhibited. This results in the accumulation of beta-catenin in the cytoplasm which can translocate into the nucleus where it can regulate gene expression. Thus, cytoplasmic or nuclear localization of beta-catenin is indicative of and a biomarker for EMT and an invasive phenotype [36]. For example, in advanced colorectal cancers the central region of the tumor exhibits membrane localized betacatenin associated with E-cadherin at cell junctions. In contrast, at the invasive front membrane staining is lost and beta-catenin is localized in the nucleus [36, 37]. Active Wnt/beta-catenin signaling and nuclear beta-catenin accumulation also correlates with EMT, invasion and a poor prognosis in breast cancers [38, 39]. During EMT loss of the E-cadherin cell junction complex is often accompanied by a concomitant up-regulation of mesenchymal cadherins, such as N-cadherin and cadherin-11 [40]. This process is described as “cadherin switching” and readily occurs during type-1 EMT when cells separate from the epiblast layer to ingress the primitive streak and when epithelial cardiomyocytes migrate toward the endocardium during heart morphogenesis [41, 42]. N-Cadherin is typically expressed by mesenchymal cells, fibroblasts and neuronal cells, however, aberrant expression of N-cadherin is also observed in invasive breast, prostate and melanoma cancer cells [43–45]. During type-3 EMT events, N-cadherin expression promotes loss of epithelial cell polarity and increased cell motility, invasion and metastasis, having the opposite effect as E-cadherin expression [46, 47]. Like N-cadherin, cadherin-11 is not expressed by normal epithelial cells but is induced during EMT events associated with development [48]. In addition, cadherin-11, is also expressed in aggressive melanoma, breast and prostate cancer cell lines and appears to coincide with greater cellular invasiveness and a poor clinical prognosis in patients [43, 49, 50]. Expression of these mesenchymal cadherins promotes tumor cell dissemination and invasion independent of E-cadherin down-regulation, highlighting their potent tumor promoting activities [51]. It appears that mesenchymal cadherins promote local invasion by allowing dynamic interactions with the endothelial and stromal components surrounding epithelial tumors [44]. In sum, loss of E-cadherin, nuclear beta-catenin localization and gain of mesenchymal cadherin expression are all currently recognized as key biomarkers for EMT and metastatic potential.

2.2.2

Changes in Cytoskeleton Dynamics

Studies of EMT have also revealed that the expression of vimentin and Fibroblast Specific Protein-1 (FSP1), also known as S100A4, are commonly associated with EMT during embryogenesis and highly invasive tumor cells. Vimentin is a mesenchymal intermediate filament which is known to play a role in maintaining cell integrity in response to mechanical stress [52]. Vimentin also plays a key functional role in the migration and contractibility of cells, as vimentin null mice have fibroblasts with a decreased ability to migrate and exhibit impaired wound healing [53]. During embryogenesis vimentin is expressed in mesodermal cells which exhibit a

20


highly motile and invasive phenotype [54, 55]. Specifically, vimentin is turned on at the onset of when cells first detach and migrate from the epithelium to form the mesoderm, and continues to be expressed in all mesenchymal cells making it an excellent biomarker for the mesenchymal phenotype [56]. Carcinoma progression to an invasive phenotype is also often accompanied by increased expression of vimentin in a wide range of cancers [57–61]. Conversely, knocking down the expression of vimentin in highly malignant colon, prostate and breast cancer cells inhibits their ability to migrate and become invasive [62, 63]. Vimentin still remains a controversial type-3 EMT marker, however, as pathologists do not always observe significant increases in vimentin expression in cancer and it is sometimes associated with benign tissue [64]. However, the limitations in histological examination of human tumor sections appear to be part of the problem. Advances in analysis of circulating tumor cells have recently associated vimentin expression with metastatic disease, thus vimentin positive circulating tumor cells might be indicative of tumor associated EMT [65, 66]. In addition to up-regulation of vimentin, rigid epithelial cytokeratins are also down-regulated during EMT and actin filaments are organized into stress fibers [67]. The consequence of this dramatic remodeling of the cytoskeleton is facilitating pseudopod formation at the leading edge of the cell to promote invasion and migration. Fibroblast Specific Protien-1 (FSP-1) appears to be another biomarker for EMT that is important for the transformation of epithelial cells to a metastatic phenotype. FSP-1 is a member of the S100 family of cytoplasmic proteins and is homologous to S100A4. Members of this family are calcium binding, low molecular weight proteins that function as both homo and heterodimers and are implicated in cytoskeletal-membrane interactions, calcium signal transduction, and cellular growth and differentiation [68]. FSP-1 expression was first observed in cells of mesenchymal origin during mesoderm formation and during inflammation induced fibrogenesis [69]. FSP-1 is absent in normal epithelial cells but is prevalent in mesenchymal cells including fibroblasts, monocytes, macrophages and lymphocytes, all of which exhibit a migratory phenotype [69–71]. Transfection studies have shown that FSP-1 is involved in the stimulation of cellular motility and it has been shown to colocalize with myosin IIA and actin filaments at the leading edge of migrating cells [72]. FSP-1 expression is commonly observed in cultured epithelial cells undergoing growth factor induced EMT as well as in EMT during renal fibrosis in transgenic mice [73, 74]. The FSP1 gene is a direct target of nuclear beta-catenin and is associated with promoting the progression of invasive and metastatic cancer [75]. Overexpression of FSP-1 in non-metastatic breast cancer and bladder cancer cells has been shown to promote local tumor invasion and metastasis to lungs and lymph nodes [76, 77]. Conversely, knock down of FSP-1 reduces cell motility, invasiveness and metastatic potential in vivo [78]. In addition, FSP-1 is expressed by invasive tumor cells undergoing EMT in PyV-mT induced mammary tumors in mice and mice over-expressing FSP1 crossed into tumorigenic backgrounds have offspring which exhibit increased frequency of metastasis even tough primary tumor incidence and size do not change [7, 79]. Finally, human patient studies have further revealed that in a panel of 349 breast cancer patients, FPS-1 expression was the


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most significant predictor of patient survival [80]. Likewise, FSP-1 expression by tumor cells is a significant negative prognostic indicator in several other cancers [81]. In sum, cells undergoing EMT appear to undergo a reorganization of their cytoskeleton to support the mesenchymal phenotype. The studies highlighted above clearly demonstrate the functional significance of FPS-1 and vimentin expression and their role as an important positive marker for EMT and metastatic potential.

2.2.3

Matrix Remodeling

The extracellular matrix (ECM) is a ‘mesh- like’ scaffolding of proteins localized on the cell surface which provides support and structure for cells and tissues [82]. Cells adhere to the extracellular matrix through integrin receptors which bind to specific matrix proteins, link the ECM to the cytoskeleton and induce intracellular signaling cascades. Epithelial tissues are normally enclosed by an ECM rich in laminin and type-IV collagen proteins called the basement membrane, which is able to impede invasion and the advancement of benign growths [83]. Loss of the basement membrane is one of the key steps in the transition to invasive cancer and the most reliable indicator of poor prognosis in most epithelial carcinomas [84]. Therefore, it appears that during EMT cells significantly alter the way in which they interact with the ECM and there is a sustained remodeling of the adjacent ECM to allow for increased cell migration and invasion. ECM remodeling during EMT appears to involve increased secretion and organization of fibronectin. Fibronectin is a large molecular-weight matrix glycoprotein that is secreted and organized by fibroblasts. Normally, fibronectin is not secreted by epithelial cells, however, it is the most prominent ECM protein produced by cells undergoing type-1 EMT during gastrulation [85]. As a result of EMT, these mesenchymal cells secrete and organize fibronectin to create and facilitate a migratory path for the neural crest [86]. Abundant increases in fibronectin expression and secretion are also readily observed and serve as a type-3 EMT biomarker in vitro [87]. Functional studies have further revealed that fibronectin expression by melanoma cells enhances their invasive ability and synthetic peptides which inhibit fibronectin-integrin interactions inhibit melanoma cell invasion [88, 89]. Likewise, fibronectin promotes the invasion of prostate, breast and gastric cancer cells [90–92]. Furthermore, fibronectin is consistently up-regulated in a variety of solid tumors and is the major constituent of the fibrillar extracellular matrix characteristic of tissue fibrosis and the desmoplastic tumor associated stroma [93]. Tumor modeling studies further show that dense, fibrotic, fibronectin rich matrices create a rigid tumor microenvironment that enhances metastatic tumor progression and is a predictor of poor clinical outcome with breast cancer [94–96]. Fibronectin specifically binds to the fibronetcin receptor on cells, the alpha5beta1 integrin. Thus, many cellular responses to, as well as proper secretion and organization of fibronectin are dependent on expression of alpha5beta1 integrin. Interestingly,

22


alpha5beta1 integrin is also associated with type-1 EMT events and in conjunction with fibronectin is critical for generating productive cellular protrusions and inducing motility [97]. In regards to tumor progression, alpha5beta1 expression positively correlates with and is required for melanoma metastasis [98, 99]. Thus, co-expression of fibronectin and alpha5beta1 integrin may be needed to promote invasion and both proteins may serve as biomarkers for cells undergoing EMT. Matrix degradation at the leading edge of cells is also apparent during EMT and can be accomplished by the expression of a family of proteases called matrix metalloproteinases (MMPs). MMPs play an important role in matrix remolding in which they activate growth factors and other MMPs, disrupt ‘cell-cell’ and ‘cell-ECM’ contacts, and cleave several ECM components facilitating invasion. Approximately 23 different MMPs have been reported and each contains a pro-peptide, a catalytic domain with a Zn2+ binding site and a c-terminal tail that determines substrate specificity. Mechanistically, most MMPs are secreted in a latent pro-form and the pro-peptide must be cleaved off outside the cell to activate the enzyme. For example, MMP-2 is most notably cleaved and activated by MT1-MMP, a membrane associated MMP, and MMP-9 is efficiently activated by both MMP-2 and MMP-3 [100–102]. Unique to MMP-9 is its ability to also form a disulfide linked homodimer which is a strong stimulator of cellular migration and appears to be induced by similar pathways that activate EMT [103, 104]. Increases in MMP expression are well documented during type-1 and type-2 EMT events. During EMT associated with neural crest migration cells express MMP-8 and induction of MMP-2 has been shown to be required for EMT and mesenchyme formation in embryos [105–107]. Furthermore, MT1-MMP and MMP-2 have been shown to be induced and play an important part in regulating EMT during heart organogenesis [108, 109]. Cells undergoing type-2 EMT and migration in wounded bronchial epithelial tissues consistently express a variety of MMPs including MMP-9, MMP-3 and MMP-11 [110–112]. Several MMPs which efficiently degrade basement membrane components, including MMP-2, MMP-9, and MT1MMP, have also been linked to progression of benign tumors to invasive cancers of the ovaries, stomach, colon, breast and prostate [113–117]. In particular, expression of MT1-MMP plays an important role in cancer cell invasion and is specifically implicated in prostate cancer metastasis to the bone [118, 119]. Likewise, expression of MMPs has been associated with cells undergoing EMT. In vitro, it has been shown that breast, prostate and bladder cancer cells undergoing EMT, as characterized by decreases in E-cadherin and increases in vimentin, also frequently up-regulate MMP-2, MMP-9 and MT-MMP1 which contributes to their invasive potential [120–122]. MMP-2, MMP-9 and MT-MMP1 are also up-regulated in human breast cancer cell lines that are more mesenchymal in phenotype, further suggesting that increases in MMP production are a relevant marker for EMT [123]. Interestingly, the matrix remodeling that occurs during EMT, including both ECM protein organization and MMP production, also seems to regulate the EMT phenotype, perhaps acting as a positive feedback loop to maintain the mesenchymal state. Functionally blocking the association between alpha5beta1 integrin and fibronectin with a peptide mimetic can inhibit EMT induction and invasion of cells


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[98]. Neutralizing antibodies to the beta1 integrin subunit and pharmacological inhibition of beta3 integrin subunit also inhibits induction of EMT [124, 125]. Furthermore, spontaneous breast tumors that develop in mice overexpressing MMP-3 are more metastatic and show mesenchymal characteristics and exposure of cultured mouse mammary epithelial cells to MMP-3 cleaves E-cadherin, dissociates cells and directly activates EMT [126, 127]. In addition, MT-MMP1 has also been shown to induce EMT in prostate cancer cells and MMP-9 has been shown to activate EMT in ovarian cancer cells [128, 129]. Therefore, it appears that the local extracellular changes which occur as a result of EMT also play important roles in regulating EMT. While it is generally thought that fibroblasts in the reactive stroma are responsible for tumor associated matrix remodeling, it is increasingly clear that such activities are also part of the EMT program. For example, MMP-2 expression by stromal cells is not associated with metastatic progression of prostate cancer, whereas MMP-2 expression by malignant epithelial cells is [130]. Thus, it can be concluded that tumor cell associated MMP production during EMT serves an important function in metastatic potential. Studies comparing the functional role of fibronectin expression by tumor associated fibroblasts versus cells undergoing EMT would also be helpful to understand how EMT related matrix remodeling effects metastatic potential. As the tumor microenvironment clearly influences many cell processes, it is most likely stromal cells and cells undergoing EMT work in concert to effect and regulate changes promoting dissemination, motility and invasion during tumor progression. Although the heterogeneity, genetic diversity and uniqueness of individual cancers make it difficult to draw general conclusions regarding EMT, the fact that many characteristics of EMT are associated with more advanced cancers and metastasis offers assurance that this process plays an important role in metastatic progression.

2.3

Cell Signaling Networks Regulating EMT

During development, type-1 EMT is tightly controlled by receptor kinases that are activated by different extracellular factors to induce signaling pathways in cells. Activation of these pathways directs gene transcription and the changes in protein expression that accompany EMT. Studies in cell lines and transgenic mouse models have confirmed that similar signaling pathways regulate type-2 EMT associated with fibrosis and type-3 EMT associated with carcinogenesis. It appears that factors involved in these pathways are often inappropriately expressed during tumor progression from tumor associated inflammation, tumor associated stroma or by the tumor cells. The spectrum of EMT inducers implicated in all three types of EMT is vast and most notably includes signaling pathways such as TGF-beta, EGF, Ras, HGF and Wnt/beta-catenin. Recently it has become clear that significant cross-talk between these pathways must occur to coordinate the elaborate transformation of

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phenotype characteristic of EMT. Importantly, aberrations in these pathways are associated with tumor progression and metastatic potential in many different cancers and are considered attractive drugable targets.

2.3.1

TGF-beta

Transforming Growth Factor-beta (TGF-beta) is a multifunctional cytokine that acts as the master regulator of EMT in normal, developmental and malignant tissues. The TGF-beta superfamily of genes encodes for over 30 signaling peptides including the TGF-beta family, the activin family, the Growth and Differentiation Factors (GDFs) family and the Bone Morphogenetic Proteins (BMPs) family. As part of the TGFbeta family, mammals express three different TGF-beta isoforms: TGF-beta1, TGFbeta2, and TGF-beta3. Each of these appear to be functionally interchangeable in vitro, and although they exhibit a differential expression pattern in the developing embryo, each is associated with regions of active morphogenesis involving epithelial-mesenchymal interactions [131]. TGF-beta ligands act as homo- and heterodimmers and bind to transmembrane TGF-beta type-II receptors. Binding results in autophosphorylation which triggers transphosphorylation of intercellular serines and threonines and activation of the transmembranre TGF-beta type-I receptor. In the canonical TGF-beta signaling pathway, the activated type-I receptor phosphorylates and activates latent Smad proteins. Smads 1, 5 and 8 are activated by the BMP family, while the activin and TGF-beta families activate Smads 2 and 3. When activated, each of these proteins heterodimerizes with Smad4 in the cytoplasm forming a transcription factor complex that enters the nucleus and regulates the expression of TGFbeta responsive genes. One of the TGF-beta responsive genes is Smad7, which functions as a negative feedback loop for the canonical pathway. Smad7 expression competitively inhibits further Smad interactions with the TGF-beta type-I receptor as well as recruits the Smurf family of ubiquitin ligases to the TGF-beta receptor complex to promote its degradation [132, 133]. TGF-beta has also been shown to activate a variety of other noncanonical pathways, including, the AKT, mitogen activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK) and NF-kappaB pathways [134–137]. However, direct activation of these pathways and the role this plays in TGF-beta induced EMT appears to be cell line and context dependent. Members of the TGF-beta superfamily play a critical role in all subtypes of EMT. The TGF-beta superfamily members, Nodal and Vg1, are required for and stimulate EMT in primitive streak cells during gastrulation [138, 139]. BMPs are essential to the induction of EMT during neural crest formation and expression of TGF-beta2 and 3 controls EMT during heart morphogenesis [140, 141]. Signaling through TGF-beta1, which is secreted by immune and epithelial cells, has also been implicated as the main culprit of EMT induced renal and lung fibrosis after injury [142, 143]. In cancer, TGF-beta and activin family members seem to play a more significant role in inducing type-3 EMT events and invasion whereas pathways of the BMP branch do not [144, 145]. TGF-beta1, -2 and -3 have each been shown to


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induce EMT in ovarian cancer cells [146]. TGF-beta1 also appears to alters cells at the invasive edge of cancer from cohesive migration to single cell motility reminiscent of EMT, which results in a higher propensity to create distant metastases [14]. Furthermore, increases in TGF-beta1 mRNA and protein are commonly detected in advanced invasive and metastatic tumors of the breast, prostate, colon, skin and correlate with poor prognosis [147–150]. Mechanistic studies have revealed that TGF-beta stimulation and TGF-beta receptor activity result in down-regulation of E-cadherin and tight junction proteins such as ZO-1 and up-regulation of MMPs, fibronectin, FSP-1 and vimentin [151]. For example, during adherence junction disassembly, TGF-beta1 appears to function independent of Smad signaling but in cooperation with the activated oncogene Raf via clathrin-mediated endocytosis and lysosomal degradation of E-cadherin [152]. Additionally, TGF-beta1 treatment in conjunction with phosphatidylinositol 3 kinase (PI3K) can increase tyrosine phosphorylation of alpha- and beta-catenin resulting in disruption of the E-cadherin/catenin complex from the actin cytoskeleton thus reducing ‘cell-cell’ adhesion and promoting EMT [153]. The TGF-beta type-II receptor also plays a direct role in the dismantling of tight junctions. Recently it has been shown that TGF beta type-I receptors are located in the tight junctions where they interact with occludins and the polarity protein Par6 [154]. TGF-beta type-II receptors in turn phosphorylate both TGF-beta type-I receptors and Par6 upon TGF-beta1 stimulation. This controls the interaction of Par6 with the E3 ubiquitin ligase Smurf1 which leads to degradation of the small GTPase RhoA and tight junction dissolution. Additionally, expression of dominant negative TGFbeta type-II receptors in highly malignant cells has been shown to inhibit EMT, invasion and metastasis in mice, further demonstrating the importance of TGF-beta receptor activity in cancer progression and EMT [155]. TGF-beta signaling is also a strong inducer of matrix remodeling. TGF-beta1 promotes fibronectin induction through activation of JNK in a Smad4 independent manner while simultaneously promoting expression of the fibronectin receptor, alpha5beta1, as cells undergo EMT [98, 156]. TGF-beta has also been shown to induce expression of fibrillar collagens and the basement membrane protein laminin-5 in a Smad4 dependent manner [157, 158]. While laminin-5 has been shown to induce migration and EMT in hepatocellular carcinoma cell lines, its role in tumor progression remains controversial as in other systems its expression appears to be lost during EMT and progression to invasive cancer in vivo [98, 159]. Several immunohistochemical studies have however shown a positive correlation between expression of both protein and RNA of the gamma-2 chain, but not the alpha-3 or beta-3 chain, of laminin-5 and the invasive front of colorectal and gastric cancers (in contrast cancer cells deeper in the tumor were negative) [160–163]. TGF-beta through p38 MAPK signaling has also been shown to induce expression of both MMP-2 and MT-MMP1, both of which are known to cleave laminin-5 gamma-2 chain and expose a cryptic promigratory site that triggers cell motility in cancer cells [164– 166]. Thus, while the exact role of laminin-5 during EMT and cancer cell invasion remains obscure, it is clear that TGF-beta mediated ECM remodeling promotes migration, invasion and an EMT phenotype.

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Tumor modeling studies have established that TGF-beta receptor singling through Smads 2 and 3 helps mediate the EMT response to TGF-beta family members and primes cells for metastasis [167]. Transgenic mouse models have revealed that Smad-specific knockouts or expression of mutant type-I TGF-beta receptors that specifically fail to activate Smad2 or Smad3 all block an EMT response in tumors [151]. Canonical TGF-beta signaling by Smad2 and Smad3 also depends on Smad4; likewise, knockout of Smad4 significantly reduces TGFbeta’s ability to down regulate E-cadherin expression and prevents EMT and cell migration in pancreatic tumors [168, 169]. Furthermore, Smad4 knockdown in a highly malignant breast cancer cell line was sufficient to inhibit EMT and bone metastasis in mice [170]. It should, however, be noted that Smads 2 and 3 seem to mediate distinct biological activities in response to TGF-beta and are not completely functionally redundant [171]. It appears that Smad2 activity inhibits EMT while Smad3 activity stimulates EMT and invasion. For example, Smad2 deletion has been shown to enhance the progression of squamous cell carcinomas by inducing EMT and in hepatocytes EMT induction depends on Smad3 but not Smad2 [172, 173]. Moreover, knockdown of Smad3 prevents TGF-beta induced EMT and invasion in ovarian cancer cells and nuclear Smad3 is as a marker for poor prognosis [146]. Smad3 deletion also prevents type-2 EMT during injury and type-1 EMT during gastrulation [174, 175]. Smad signaling is further complicated by its interactions with other signaling networks. Thus, despite the importance of Smad signaling as an inducer of EMT, it is becoming increasing clear that other signaling pathways must be activated in conjunction with Smad signaling to stimulate a robust EMT program.

2.3.2

EGF and Ras

While TGF-beta is the most widely studied cytokine which stimulates EMT, many cancer cell lines which exhibit proficient TGF-beta signal transduction, do not undergo TGF-beta mediated EMT [176]. It is therefore clear that other growth factors and their respective signaling pathways must play an important role in regulating EMT, such as the ErbB family of ligands and receptors. Notably, and unlike TGF-beta receptors, the ErbB family of receptors are frequently over-expressed in many solid human tumors, including lung, colon, breast, prostate, ovarian and bladder carcinomas [177, 178]. Over-expression of ErbB receptors increases their sensitivity to low concentrations of growth factors and thus amplifies the intracellular signaling networks activated by these receptors. Importantly, over-expression of ErbB receptors has been shown to correlate with poor clinical prognosis and promotes tumor cell motility, invasion and an EMT phenotype [179–181]. The ErbB family of receptors include 4 Receptor Tyrosine Kinases (RTKs) including the epidermal growth factor receptor (EGFR; ErbB1, HER1), ErbB2 (Neu, HER2), ErbB3 (HER3) and ErbB4 (HER4). ErbB ligands that activate these receptors are divided into three groups. The first group includes Epidermal Growth Factor


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(EGF) and Transforming Growth Factor-alpha (TGF-alpha) which bind specifically to EGFR. The second group includes Betacellulin, Heparin-Binding Growth Factor (HB-EGF) and Epiregulin (EPR), which bind both EGFR and ErbB4. The final group is composed of the Neuregulins (NRGs) which can bind ErbB3 and ErbB4. Ligand binding and receptor activation results in either homo- or heterodimerization and leads to phosphorylation of specific intracellular tyrosine residues which serve as docking sites for proteins containing Src Homology 2 (SH2) and Phosphotyrosine Binding (PTB) domains. In particular, this leads to initiation of the Ras-Raf-MAPK and PI3K-AKT cascades as well as activation of Phospholipase C-gamma (PLCgamma) and Src. Notably, none of the EGF family of ligands binds ErbB2. However, ErbB2 is the preferred heterodimerization partner of the other ErbB family members, is constitutively present in the active state and ErbB2-containing heterodimers have the strongest signaling output [182, 183]. The signaling pathways activated by the ErbB receptors have been consistently implicated in stimulating EMT events in embryogenesis and cancer. Specifically, ErbB receptor signaling induces loss of ‘cell-cell’ junctions, loss of epithelial cell polarity, actin stress fiber polymerization, and induction of migration and invasion [182]. Thus, EGF ligands and receptors have a significant role in inducing EMT and the metastatic phenotype. EGF family member signaling has key functions across vertebrate species in inducing type-1 EMT events and is essential for heart morphogenesis [184, 185]. Direct evidence of EGF family member involvement in type-2 EMT events is lacking. Studies have, however, demonstrated a positive effect of EGF, TGF-alpha and HB-EGF on wound repair and EGF immunoreactivity is associated with the advancing epithelial margin [186]. In type-3 EMT there is stronger evidence which links EGF and its family of receptors to EMT events during tumor progression. Signaling through EGFR has been shown to induce EMT, invasion, and metastasis in several different human prostate and breast cancer cells [187–189]. One important role of EGF signaling in regards to EMT is the dismantling of the cobblestone network and cell polarity characteristic of normal epithelial tissues. For example, persistent exposure to EGF leads to the disruption of cell adherence junctions by caveolin-induced endocytosis of E-cadherin [187]. Over- expression of ErbB2 also disrupts apical–basal polarity organization by interacting with and inhibiting the function of the Par6/aPKC polarity complex [190]. Furthermore, inhibiting EGFR signaling has been shown to restore E-cadherin expression and prevent the mesenchymal phenotype [189]. EGF signaling through the MAPK pathway also induces expression of MT-MMP1 (which activates MMP2) and MMP-9 through activation of the Ets-1 and Ets-2 transcription factors [191–193]. ErbB2 over-expression also induces the expression of MMP-2 through p38 MAPK and AKT signaling, thereby promoting EMT and invasion [194]. Taken together, this evidence suggests that pathways induced by EGF signaling play a significant role in regulating EMT and tumor invasion. Dissection of the EGF signaling pathways have also provided significant insights into how EGF induces tumor cell motility once epithelial cell adhesions are dismantled. Several elegant studies have revealed that EGF signaling activates Coflin at the plasma membrane by PLC-gamma mediated hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP2). The Arp2/3 complex and Cofilin then work together

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to reorganize actin filaments at the leading edge by nucleating F-actin branches to form sheet like protrusions in the cell to facilitate lamellipodia mediated migration [195]. The significance of this is demonstrated by the fact that EGFR expression enhances prostate cancer cell motility and invasion and inhibition of EGFR signaling to block phosphorylation of PLC-gamma prevents invasion and metastasis to lungs in mice without an effect on proliferation [196]. Likewise, activation of PI3K by EGF is also an important regulator of actin polymerization and motility in carcinoma cells [197]. PI3K, composed of regulatory (p85) and catalytic (p110) subunits, binds to phosphorylated ErbB receptor family members and phosphorylates PIP2 to from phosphatidylinositol (3,4,5) trisphosphate (PIP3). The presence of PIP3 at the plasma membrane then activates Rho GTPases (Rho, Rac and Cdc42) which initiate and regulate the formation of lamelipodia, filapodia and invadapodia, the cell protrusions that drive cell motility [198]. Specifically, Rho GTPases promote motility through the activation of WAVE and WASP nucleating factors which work in concert with the Arp2/3 complex to form actin projections. Studies in a variety of cell lines have confirmed that both of these pathways stimulated by EGF are required for full induction of migration and invasion by metastatic cells [199, 200]. Albeit, the role of these pathways (and the different types of protrusions they induce) in the enhanced migration seen during EMT is poorly understood. The most significant downstream effector of EGF signaling promoting EMT is the Ras oncogene. The Ras family of GTP-binding proteins are downstream effectors of many growth factor receptors and mutations leading to hyper-activation of Ras signaling pathways are frequent in cancers [201]. The most well characterized action of Ras activation is recruitment of Raf kinases to the plasma membrane where Raf in turn activates the MAP kinase signaling pathway through MEK (MAPKK). GTP-Ras can also activate PI3K leading to phosphorylation and activation of AKT. Activation of both of these pathways makes Ras a powerful oncogene and promoter of cell proliferation. Ras also cooperates with Smads 2 and 3 to drive EMT and metastatic phenotypes where it promotes nuclear accumulation of Smad proteins [202]. In this regard, pharmacological inhibition of MEK1/2 activation also prevents TGF-beta from stimulating EMT [203]. While non-canonical MAPK activation by TGF-beta appears to be required for TGF-beta induced EMT, it is also apparent that constitutive Ras activation along with TGF-beta stimulation can act cooperatively to induce EMT when TGF-beta alone cannot [204, 205]. Ras-Raf-MAPK signaling and TGF-beta also work synergistically to induce endocytosis and lysosomal degradation of E-cadherin as well as increases in MMP-2, MMP-12 and MMP-13 [152, 206]. Furthermore, co-stimulation of cells with TGF-beta and EGF induces robust EMT and ErbB2 over-expression induces migration and invasive behaviors in cooperation with TGF-beta dependent on sustained elevated levels of activate MAPK [207, 208]. Interestingly, it appears that Ras-MEK1/2 activation of Erk2, but not Erk1, is required for EMT [209]. Considering that MEK1 and MEK2 have differential effects on Erk2 localization and function, it is plausible that EGF and Ras’s ability to differentially effect Erk2 activation might serve as a mechanism by which Ras and TGF-beta signaling work synergistically to induce EMT [210, 211]. In agreement with this hypothesis, other studies have shown that while the ability of


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Ras to activate PI3K can induce cell scattering in TGF-beta treated cells, it is the ability of Ras to activate the Raf-MAPK pathway which promotes robust and sustainable EMT in response to TGF-beta treatment [212]. The studies highlighted in the above discussion suggest that TGF-beta must cooperate with other signaling pathways either from hyper-activation of oncogenes, such as Ras or ErB2, or exposure to other growth factors, such as EGF, to elicit EMT. The sources of multiple EMT inducing ligands might result from chronic inflammation or the reactive stroma associated with tumors, where macrophages and other immune and stromal cells secrete TGF-beta and EGF into the tumor microenvironment. In animal models of breast cancer, it is apparent that breast cancer cells often co-migrate with macrophages which secrete EGF and EGF is required for stimulation of carcinoma cell invasion and induces an EMT phenotype [213]. Consequently, this might be a mechanism by which early stage carcinoma cells that lack gross oncogenic abnormalities might be induced to undergo EMT and participate in the early dissemination model of tumor metastasis [214].

2.3.3

HGF and FGF

Besides TGF-beta and EGF, other growth factor receptor pathways also play a role in the induction of EMT and invasion; in particular fibroblast growth factor (FGF) and hepatocyte growth factor/scatter factor (HGF) seem to be an important. HGF is a multifunctional factor produced by many cell types, including normal fibroblasts, epithelial and endothelial cells. HGF binds to the RTK c-Met, which causes dimerization and autophosphorylation of tyrosines which create multisubstrate docking sites for the adaptor proteins Grb2 and Shc, and the p85 subunit of PI3K, resulting in activation of the Ras–MAPK, PI3K, PLC-gamma and Src kinase pathways. In addition, Gab1 is recruited to the receptor as well as the ubiquitin ligases c-Cbl and Cbl-b, which negatively regulate the c-Met signal. Gab1 is the major substrate for c-Met, and HGF binding promotes a prolonged Gab1 phosphorylation that is not produced upon activation of other RTKs, such as EGFR [215]. HGF was first identified as a fibroblast secreted factor that induced epithelial cell scattering and actin remodeling [216]. Since then, HGF has been found to induce EMT as well as increase cell motility and invasiveness [217]. c-Met over-expression is evident in many cancers including hepatocarcinomas and carcinomas of the colon, prostate, ovary and breast and correlates more invasive tumors and poor prognosis in each [218–222]. Likewise, HGF over-expression in the tumor microenvironment is also observed and is shown to directly induce localized invasion [223]. Mechanistically, HGF induces EMT and invasion by activating the PI3K pathway rather than Gab1, as specific inhibition of c-Met’s ability to activate PI3K prevents dismantling of ‘cell-cell’ contacts, cytoskeletal rearrangements and c-Met driven metastasis [224, 225]. This seems to rely on AKT-mTOR phosphorylation of p70 S6 kinase (p70S6K). Neither MAPK nor PLC-gamma activity are required for HGF to activate p70S6K. Additionally, HGF induction of p70S6K activity has been

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shown to inhibit E-cadherin expression and promote EMT and invasion in ovarian cancer [226, 227]. Thus, in contrast from EGF studies indicating Ras-MAPK pathway as a key signaling network required for EMT, in other contexts, such as in HGF signaling, AKT activation appears to be sufficient to induce EMT as characterized by down-regulation of E-cadherin and up-regulation of vimentin [228]. The FGF family of ligands includes at least 23 member which signal through four high-affinity RTKs (and their alternatively spliced isoforms), designated FGFR-1 to −4, which can promote increased cellular motility and invasiveness in specific contexts [229]. Much like EGF and HGF, ligand binding to FGFRs results in receptor dimerization and activation of the intrinsic tyrosine kinase activity which activates several downstream signaling pathways including Ras-MAPK, PI3K, PLC-gamma and Src kinase. However, functional consequence of FGF activation appear to be very cell type specific and studies from one cell line or tissue may not be generally applicable to all tumors. In most cellular contexts FGFs appear to induce proliferation and migration, however it can also induce differentiation and/ or cell cycle arrest [230]. Thus, the mechanisms by which deregulated FGF signaling may promote tumorigenesis are still poorly understood despite the fact that there is solid evidence that FGF signaling supports EMT. FGF is a critical ligand in development, promoting mesodermal formation, vimentin expression and mesenchymal cell migration through the primitive streak during gastrulation [231]. FGFs also play a role in EMT during wound healing where they stimulate tissue remodeling and migration and epithelial cells as well as inflammation induced fibrosis [232]. In adult tissues FGFs are secreted by fibroblasts often after TGF-beta stimulation, and are localized along epithelial-stromal boundaries [233]. In studies regarding type-3 EMT, FGF-1 stimulation of bladder cancer cells has been shown to enhance cell migration and induce EMT [234]. In contrast to TGF-beta, EGF and HGF, FGF stimulation does not cause a decrease in expression of adherence junction proteins. Rather, E-cadherin based adherence junctions become internalized, cells fail to make ‘cell-cell’ junctions and desmosomal components disappear [235]. In in vivo prostate cancer modeling studies, active FGFR-1 has been shown to promote EMT and metastasis to liver and lymph nodes further substantiating that FGFs can play a role in tumor associated EMT and metastasis [236]. In sum, FGF clearly plays an important role in type-1 and type-2 EMTs, however, less has been explored about its role in type-3 EMTs and little is known about the intercellular signaling cascades that govern FGF stimulated EMT during tumorigeneis. Thus, the specific mechanisms by which FGF signaling may promote metastasis and invasion are poorly understood.

2.3.4

Wnt, Notch and Hedgehog

The Wnt/beta-catenin, Notch and Hedgehog pathways all play a critical role during embryogenesis and regulate type-1 EMTs during development [237]. Wnt growth factors constitute a family of 19 closely related glycoproteins that are secreted and


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activate members of Frizzled family and LDL receptor-related protein 5/6 (LRP-5/6) receptors. Signaling through the Frizzled receptor causes release of the protein disheveled from the receptor complex which inhibits a multicomponent destruction complex composed of Axin, APC, Glycogen Synthase Kinase 3-beta(GSK3-beta) and Casein Kinase I (CKI). This complex regulates the stability of cytoplasmic beta-catenin by phosphorylation which targets it for degradation by the proteasome. As a consequence of activated Wnt signaling, beta-catenin is able to accumulate in the nucleus where it interacts with the TCF/LEF family of transcription factors which contain DNA binding sites and regulate gene expression. Wnt pathway activation is critical for induction of EMT in gastrulation and cardiac valve formation as stabilization of beta-catenin causes premature mesoderm formation and beta-catenin knockout mice fail to develop heart cushions during development [34, 238]. Activation of the Wnt pathway and beta-catenin nuclear accumulation also induces vimentin expression and EMT in several breast cancer cell lines as well as progressive loss of E-cadherin in colon cancer [37, 239, 240]. Furthermore, beta-catenin/TCF/LEF transcriptional activity induces invasion and metastasis through the expression of MT1-MMP, MMP-7 and MMP-26 [241–243]. Well characterized mutations in the Wnt/beta-catenin pathway have also been identified in the tumor suppressor gene APC which leads to inherited familial adenomatous polyposis, a spontaneous form of colon cancer that is associated with increased incidence of invasive colorectal cancers [244]. Aberrant Wnt signaling which causes nuclear accumulation of beta-catenin also contributes to EMT, invasion and increased metastatic potential in melanoma [245]. The Wnt/beta-catenin/LEF signaling pathway also exhibits extensive crosstalk with other receptor tyrosine kinases associated with EMT. Activation of c-Met by HGF results in tyrosine phosphorylation and cellular accumulation of beta-catenin, which amplifies TCF/LEF-mediated gene transcription [246]. beta-Catenin and TGF-beta/Smad signaling also cooperate via Smad and TCF/LEF interactions which can act as transcription factor complexes to synergistically activate target genes and EMT [247]. While most known receptor-ligand interactions governing intracellular signaling pathways involve diffusible proteins, Notch signaling involves protein ligands which remain bound to the cell surface thus inducing signaling via ‘cell-cell’ contacts. Activation of the Notch family of receptors, Notch-1, -2, -3 and -4, is mediated by the interaction with one of five different identified Notch ligands, Jagged-1/-2, and Delta-like-1/3/4 on a neighboring cell. Activation of Notch results in the cleavage of the Notch intracellular domain (NICD) inside the cell by gamma-secretase, which then translocates to the nucleus where it partners with CSL (C protein binding factor/Suppressor of hairless/Lag-1) to form an active transcriptional complex. Like Wnt-signaling, Notch is also critical for type-1 EMT associated with heart morphogenesis as well as neural crest migration during development [248, 249]. Like TGF-beta, Notch signaling also seems to act in both a tumor suppressive and tumor promoting role depending on cell context and cooperation with oncogenes. Activation of Notch has, however, been associated in vivo with promoting tumorigenesis and metastasis in the prostate cancer, promoting invasion in pancreatic cancers and over-expression of Notch1 and Jagged1 correlates with poor prognosis in

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breast cancer patients [250–252]. Hypoxia within rapidly growing tumors has also been known to induce EMT and is linked to increased metastatic potential [253]. Studies have shown that activation of the PI3K-AKT pathway is critical for this process [254]. In an array of cell lines from different cancers, Notch signaling also seems to be required to convert this hypoxic stimulus and Hypoxia induced factor 1-alpha (HIF1-alpha) signaling into EMT, increased motility, and invasiveness [255]. The NICD/CSL transcription complex resulting from notch activation also directs the expression of multiple genes including the transcription factors, hairy enhancer of split (HES) and hairy enhancer of split-related YPRW motif (HEY) [256]. Interestingly, expression of HEY1 and Jagged-1 is required in a variety of cells for TGF-beta induced EMT as well, where the proteins themselves are induced by TGF-beta treatment in a Smad3 dependent manner [257]. These studies suggest that while Notch signaling itself maybe not direct EMT, its integration into other oncogenic pathways can facilitate EMT and invasive phenotypes in tumor cells. Another major pathway regulated during development is the Hedgehog pathway which is essential for stem cell maintenance and directing embryonic tissue patterning of the various organ systems [258, 259]. In mammals, three secreted Hedgehog ligands have been identified: Sonic Hedgehog (SHH), Indian Hedgehog (IHH), and Desert Hedgehog (DHH). Hedgehog signaling is mediated by Patched receptors (PTCH1 or PTCH2), which can bind each Hedgehog ligand, and by its co-receptor, Smoothened (SMO). When PTCH is unbound to ligand, signaling by SMO is inhibited. Upon binding of Hedgehog, SMO is released and initiates a cascade of signaling events that activates a microtubule-associated complex containing the proteins Fused (Fu), Suppressor of Fused (SuFu), and Costal-2 (Cos-2). Activation of the Fu-SuFu-Cos-2 complex results in the activation and nuclear translocation of the Gli family of transcription factors. The Gli family consists of Gli-1 and −2 which function as transcriptional activators and Gli-3 which acts primarily as a repressor [260, 261]. In regards to EMT, Hedgehog signaling has been shown to be required for EMT during somite development and heart morphogenesis [262]. Gli proteins also participate in conjunction with FGF and Wnt signaling in EMT during neural and mesodermal development [263]. Hedgehog signaling also promotes type-2 EMT events and is a significant contributor to the evolution of biliary fibrosis during chronic cholestasis [264]. Elevated Hedgehog signaling is also associated with type-3 EMTs which causes increased metastatic potential in prostate cancer and basal cell carcinoma cells [265, 266]. In fact, exogenous expression of Gli-1 in non-metastatic cell lines results in E-cadherin repression and enhancement of migration, invasion and establishment of metastasis in vivo [267]. In addition, cyclopamine, a potent inhibitor of Hedgehog signaling, inhibits Gli-1 induction of EMT and invasion. Hedgehog signaling is also associated with induction of the EMT in conjunction with the Wnt pathway in skin cancer further demonstrating the intricate crosstalk between EMT signaling pathways [265]. Collectively, the role of highly conserved developmental pathways such as Wnt, Notch and Hedgehog signaling in type-3 EMT events implies that metastatic cancer cells reactivate latent developmental programs to enable the required cellular plasticity needed for such drastic changes in phenotype.


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Despite the established role of receptor signaling in EMT, the studies highlighted above clearly demonstrate that activation of a single pathway may not be sufficient to elicit robust EMT. While many oncogenic signals can result in abrogate epithelial polarity, tight and adherence junctions; acting alone they are insufficient to induce a mesenchymal, migratory and invasive phenotype [190]. These observations agree with the overwhelming evidence that EMT in various carcinomas involves an intricate interplay of multiple signaling pathways which crosstalk in a precise manner to elicit the drastic change in cellular phenotype seen during EMT. In addition, many of these pathways activate intracellular reactive oxygen species (ROS), which inhibit the activity of phosphatases thus amplifying signals [268]. To further complicate the dynamic signaling networks regulating EMT, various pathways can also regulate the secretion of other ligands and thus the activation of other pathways in an autocrine manner. For example, TGF-beta stimulation has been shown to induce PDGF secretion and PDGF receptor activation in hepatocellular carcinoma, and was required to induce nuclear beta-catenin and a migratory phenotype [269]. As the signaling networks regulating EMT become explored in greater depth the future challenge will be to identify the specific circumstances governing when and why certain pathways must be activated and when and why others are not necessary.

2.4

Transcriptional Regulators of EMT

Over the last 10 years much effort has been given to identifying and understanding several families of transcription factors which are up-regulated during EMT induced signaling. Members of the Snail (SNAI1 and SNAI2/Slug), Zeb (ZEB1 and ZEB2/SIP1), basic helix-loop-helix (Twist and Twist2), and homeodomain transcription factors are now considered to be master transcriptional regulators of EMT in both development and cancer related pathologies. In addition, chromatin associated proteins found to regulate EMT and plasticity during development have also been associated with the EMT phenotype and promote the invasive and metastatic potential of a variety of cancers. Given the obscurity of the vast array of signaling cascades which promote EMT, understanding the mechanisms of action of these transcription factors and their role in cancer metastases will likely be critical to development of therapeutic agents which prevent EMT, invasion and metastases.

2.4.1

Snail and Slug

SNAI1 (Snail) and SNAI2 (Slug) belong to the Snail superfamily and were the first such transcription factors shown to directly regulate EMT and invasive activities of cancer cells. Both Snail and Slug act as transcriptional repressors and bind to the promoter of E-cadherin and block its expression [270–272]. Snail family

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protein expression regulates developmental EMT and their expression is regulated by cross-talk between various signaling molecules, including the TGF-beta, EGF, FGF, HGF, Notch, hedgehog and Wnt pathways [248, 257, 273, 274]. In particular, Ras activation of the MAPK and PI3K signaling pathways cooperate with TGF-beta to induce strong activation of the Snail promoter [275]. Snail also appears to function as the main transcriptional regulator of Hedgehog and betacatenin induced EMT during tumorigenesis [276]. Studies have also shown that both Snail and Slug are induced by HIF-1alpha during hypoxia to repress E-cadherin expression and promote EMT and migration in ovarian cancer cells [277]. Analysis of Snail family promoters has identified several conserved elements including AP1 and AP4 sites, Smad-binding elements, LEF1 binding sites and two E-boxes which regulate their transcriptional activity [278, 279]. Recent studies have also shown that Snail proteins can bind and represses their own promoters in a negative feedback loop [280]. Snail family members are also regulated through post-transcriptional modifications. For example, p21-activated kinase1 (PAK1) appears to promote the nuclear localization and the transcriptional repressor activity of Snail on E-cadherin and occludins by phosphorylation of Ser 246 [281]. Conversely, GSK3-beta binds to and phosphorylates Snail at its nuclear export signal (NES) and destruction box, promoting its cytoplasmic export and proteasomal degradation [282]. The post translational regulation of Slug is less understood. However, Slug, like Snail, appears to be a very unstable protein and is efficiently targeted for proteasomal degradation by the F-box protein, Partner of Paired (Ppa) independent of GSK3beta activity [283]. Structurally, members of the Snail family are zinc-finger transcription factors that share a highly conserved C-terminal region, containing four to six zinc fingers which bind consensus E-box elements in the promoters of genes (CAGGTG) [284]. Their repressor activity is dependent on the SNAG domain at their N-terminus, which is involved in the direct recruitment of a chromatin modifying repressor complex formed by the co-repressors SIN3A, and HDAC1 and HDAC2 [285]. Studies beyond E-cadherin have implicated the Snail family as repressors of a variety of other common epithelial genes including claudins and occludins, which are required for tight junctions and epithelial cell polarity, as well as epithelial cytokeratins [286]. Snail has also recently been shown to interact with Smad3 and Smad4 to form a transcriptional repressor complex which targets the promoters of CAR, occludin, claudin-3 and E-cadherin during TGF-beta driven EMT [287]. Thus, as a transcription factor, Snail appears to play a key role in controlling genes that regulate tight junction stability, epithelial cell polarity and morphology. Developmental biologists have shown that the expression of Snail family proteins triggers EMT during embryonic development; triggering mesoderm formation, gastrulation, neural crest migration and heart morphogenesis [288]. Studies in carcinoma cell lines and transgenic mouse tumor models have also confirmed that Snail and Slug expression induce type-3 EMT associated with carcinogenesis [279]. In pre-malignant breast epithelial cells, Slug in cooperation with activated Ras induces vimentin expression and migration [289]. Furthermore, whereas little


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or no Snail expression is detected in well differentiated, non-invasive carcinomas, expression is high in a range of metastatic cell lines and invasive human cancers [290, 291]. In breast carcinomas, Snail and Slug expression are elevated and associated with repression of E-cadherin and increases in lymph node metastasis, tumor recurrence and poor prognosis [292, 293]. Studies in colorectal cancer have also indicated that both Snail and Slug expression are associated with the down regulation of E-cadherin, poor prognosis, and development of distal metastases [294, 295]. Likewise, Snail activates the MMP-2 promoter, up-regulates vimentin and induces invasion of squamous cell carcinomas, and has also been shown to induce MMP-7, MMP-9 and MT-MMP1 in a variety of cell lines to confer invasion and basement membrane degradation [296–299]. Snail over-expression is also observed in oesophageal squamous cell carcinomas and invasive edges of hepatocarcinomas, where it is associated with the down-regulation of E-cadherin and the development of distant metastasis [300, 301]. Similarly, highly metastatic prostate cancer cells require Slug to activate invasion and EMT [302]. Importantly, functional expression of Slug has also conclusively been shown to be essential for metastasis of aggressive melanoma cells in vivo, as its knock-out inhibited lung metastasis without effects on primary tumor growth [303]. Collectively, these studies implicate the Snail family of transcription factors in cancer associated EMT, invasion, poor clinical outcome, and development of metastases in a variety of cancer types.

2.4.2

Zeb1 and Zeb2

Zeb1 (delta-EF1) and Zeb2 (Sip1) are zinc finger transcription factors which have also emerged as direct transcriptional repressors of the E-cadherin gene and EMT, and are associated with increased invasive capacity in cells [304, 305]. Both Zeb1 and Zeb2 were identified as proteins capable of interacting with and modulating the activity of receptor activated Smads (Smads 1, 2 and 3) [306, 307]. Although the role of Zeb1 and Zeb2 in influencing Smad signaling is poorly understood, a conserved region downstream of the N-terminal zinc finger acts as a Smad interacting domain in both proteins, suggesting that Zeb proteins might play critical roles in integrating signals from TGF-beta and other signaling molecules [307]. Zeb proteins, like Snail family proteins, have also been shown to be post-transcriptionally modified by phosphorylation, however, the functional consequences of these events is unknown [308]. It is tempting to believe that such phosphorylation events might alter interactions with co-repressor and activators, regulating the transcriptional activity of Zeb factors and influence how they integrate signals from multiple pathways. Sumoylation also appears to be an important posttranslational modification on both Zeb1 and Zeb2, and is mediated by polycomb protein, Pc2, which has the functional consequence of decreasing Zeb mediated E-cadherin repression [309]. Zeb protein expression has been shown to be regulated by TGF-beta, NF-KappaB and hypoxia induced signaling [310, 311]. TGF-beta induction of Ets1 and

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activation of Smads has been shown to be an upstream transcriptional activator of both Zeb1 and Zeb2 [312]. Treatment with TNF-alpha or induction of constitutive NF-kappB signaling in breast epithelial cells results in a decrease in E-cadherin and increase in vimentin in a Zeb1/2 dependent manner [311]. NF-kappaB signaling also appears to be essential for maintenance of the EMT phenotype and metastasis in Ras transformed breast cancer cells, suggesting that regulation of Zeb proteins might play a key role in EMT induction and metastasis promoted by NF-kappaB [313]. Zeb proteins also contain hypoxia response elements in their promoters and appear to mediate HIF-1alpha-dependent E-cadherin down-regulation in renal clear cell carcinoma cells, indicative of a role for Zeb proteins in inducing hypoxic mediated cell migration [310, 314]. Structurally, Zeb proteins contain two separated clusters of zinc fingers, one N-terminal and one C-terminal, which bind independently to bipartite e-box elements composed of one CACCT and one CACCTG sequence as found in the E-cadherin promoter [315]. Studies have indicated that the transcriptional repression activity of Zeb family proteins may be attributed to its association with the co-repressor CtBP (C-terminal binding protein) which aids in the recruitment of HDAC1, HDAC2 and the histone methyltransferases G9a and EuHMT to the promoters of genes [316]. Indeed, CtBP expression and its association with Zeb factors is associated with the repression of many epithelial genes, including E-cadherin and promotes cancer cell migration in response to hypoxia [317, 318]. Zeb1 has also recently been shown to interact with the SWI/SNF chromatin-remodeling protein Brg1 to down-regulate E-cadherin and induce vimentin independently of CtBP [319]. Interestingly, colocalization of Zeb1 and Brg1 is found in conjunction with nuclear beta-catenin staining at the invasive edge of colorectal tumors. Beyond E-cadherin, Zeb2 also down-regulates several other tight junction, adherence junction, desmosome and gap junction proteins by repression of promoter activity contributing to the dissociation of epithelial cells from their tissues [320]. Furthermore, conditional expression of Zeb2 in cells has been shown to be sufficient to disrupt ‘cell-cell’ adhesions and induces invasion in vitro [304]. Zeb proteins can also act as transcriptional activators, which is dependent on their ability to associate with p300 and displace CtBP [321]. While developmental biologists have observed Zeb family protein expressed in distinct regions during development of the central nervous system, heart, and in other cells of mesenchymal origin, genetic studies have shown their activities might not be functionally redundant or additive [322]. For example, in embryos lacking Zeb2 neural crest cells fail to undergo EMT and migrate, and they exhibit a host of abnormalities which Zeb1 expression cannot rescue [323]. Congruent with a role in type-3 EMT, expression of Zeb proteins are also associated with more aggressive, invasive and metastatic tumors. Zeb2 expression is associated with more advanced ovarian cancers, increases in circulating tumor cells and is an indicator of poor survival [291]. Zeb2 is also linked with advanced pancreatic tumors which are highly aggressive, have an extremely poor prognosis and are associated with a robust EMT and fibrotic response [324]. Head and neck squamous cell carcinoma cells which express high levels of Zeb1 are also more mesenchymal in phenotype and are highly invasive and


37

migratory [325]. Zeb1 is also associated with advanced colorectal cancers and seems to be specifically expressed in those that lack Snail expression suggesting that some redundancy amongst EMT inducing transcription factors might exist [321]. However, the relationship between Zeb and Snail family transcription factors is complicated. The expression of Snail indirectly induces expression of Zeb1 and Zeb2 in some cell lines to promote EMT [326, 327]. Moreover, Zeb family members have been shown to be necessary, but not sufficient, for TGF-beta induced EMT in other cells [307]. This suggests that there is a coordinated transcriptional response needed to induce and maintain EMT that likely involves activation of more than one transcription factor. The complexities of these interactions and their relationship to EMT and metastatic potential remain to be unraveled.

2.4.3

Twist1 and Twist2

The Twist proteins are members of the basic-helix-loop-helix (bHLH) family of transcription factors and were originally shown to be central to embryonic development and to control cell fate at the early stages of embryogenesis. More recent studies have shown Twist1 and Twist2 to play many roles in the development and progression of tumors, including transcriptional control of genes associated with EMT. bHLH transcription factors are classified into three major classes: Class A factors are ubiquitously expressed and include E12/E47; Class B factors are tissue restricted and include Twist1 and Twist2; and the inhibitory HLH proteins, which lack DNA binding sites, include the Id proteins. We have largely restricted this review to discussion of class B factors as expression of these has been most commonly linked to metastatic cancer progression. Like the Snail and Zeb family of proteins, the mechanisms leading to the aberrant reactivation of Twist factors seem to result from signaling pathways that normally direct the expression of genes during embryonic development. Wnt has been shown to induce Twist1 expression and Twist1 promoters are responsive to betacatenin/TCF complexes as well as c-Jun, and Ets transcription factors [328]. Twist1 is also induced in the mesoderm during embryogenesis by a combination of FGF and SHH signaling, but not by individual application of FGF or SHH alone [329]. The relevance of this has not been studied in cancer associated EMT however. In addition, stress conditions seem to play an important role in inducing Twist activity. Both Twist1 and Twist2, are induced during inflammation by a cytokine signaling pathway that requires the p65 NF-kappaB family of transcription factors [330]. Furthermore, like the Snail and Zeb transcription factors, HIF-1alpha also regulates the expression of Twist during hypoxia by binding directly to the hypoxia-response element (HRE) in the Twist promoter [331]. Significantly, knockdown of either Twist or HIF-1alpha prevents hypoxia induced EMT. Post-translational regulation of bHLH factors is more complex than that of Snail and Zeb factors because the specific expression pattern of members and their affinities for each other regulate their capacity to form functional dimers. Twist

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transcription factors are unique in their ability to form functional homodimers and heterodimers with Class A bHLH proteins. The Id proteins, on the other hand, exhibit greater affinity for class A factors and actively alter dimerization pools by binding and sequestering class A factors [332]. It is therefore the composition of these partner pools that determines cellular response and thus the balance between different Twist homo and heterodimers directs the functional consequences of Twist expression [333, 334]. Both Twist1 and Twist2 genes have been found to be overexpressed and induce EMT and metastasis in a large set of human and murine tumors suggesting that the ability of Twist proteins to form homodimers and out compete Id proteins is sufficient for functionality [335, 336]. However, phosphorylation also influences bHLH activity by regulating their homo and heterodimerization affinity as well as DNA-binding capacity. For example, phosphorylation of the class A bHLH E47 on serine 140 increases its affinity to heterodimerize with other class A bHLH members [337]. Furthermore, Notch activation of Erk1 and Erk2 can phosphorylate class A bHLH factors and induce their proteasomal degradation [338]. Twist1 and Twist2 can also be phosphorylated on threonine 125 and serine 127, which is regulated by Protein Kinase A and Protein Kinase C and increases their DNA binding affinity in a dimerization partner dependent manner [339, 340]. Recently, AKT has also been linked to the phosphorylation of Twist1 at serine 42 which promotes cell survival and resistance to apoptosis [341]. AKT activation also appears to be required for Twist mediated EMT in response to hypoxia [254]. The basic structure of all bHLH family members involves two parallel amphipatic alpha-helices joined by a loop which is required for dimerization. bHLH proteins bind to DNA using a consensus E-box (CANNTG) site as homo- or heterodimers. bHLH protein complexes can also act as both transcriptional repressors and transcriptional activators. Transactivation activity seems to be dependent on the recruitment of histone acetyl transferase (HAT) proteins, such as p300 or the SPT–ADA–GCN5– acetyltransferase (SAGA) complex [342]. Twist proteins can also directly bind histone acetyltransferases, p300 and p300/CBP–associated factor (PCAF) and inhibit the ability of other transcription factors to promote transcription [343]. Both Twist1 and Twist2 also interact with and recruit HDAC complexes to exert transcriptional repression [344, 345]. Taken together, it might be implied that repression and activation functions of Twist proteins depend on dimer choice, however, such evidence in mammalian systems has not been consistent. Studies indicated that like Snail and Zeb family of transcription factors, Twist1 and Twist2 promote all three types of EMT events. In particular, Twist1 is a critical factor regulating TGF-beta’s ability to induce type-1 and type-3 EMT events as well as the TGF-beta induced fibrotic response during type-2 EMT [346–350]. Increased Twist expression is associated with invasive ductal carcinomas of the breast and positively correlates with EMT and the formation of distant solid metastasis in prostate cancer, oesophageal squamous cell carcinomas and hepatocarcinomas [351–354]. Others have shown that Twist expression is associated with circulating tumor cells in breast cancer and is essential for metastasis to the lung [335, 355]. Twist1 over-expression also correlates with nuclear beta-catenin and invasive cells in a mouse model of colorectal cancer [356]. Moreover, Twist


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proteins have been shown to directly up-regulate N-cadherin and down-regulate E-cadherin, placing them at the center of the cadherin switch seen during some EMT events [357]. Furthermore, ectopic Twist expression results in the activation of several other mesenchymal genes including vimentin, fibronectin MMP-2 and alpha-smooth muscle actin, correlating with the adoption of a motile and invasive phenotype [335, 355, 358]. Overall, the significance of the overlap in Twist functions with Snail and Zeb factors remains unresolved. While it is clear that each of these factors is important in promoting EMT and metastatic potential in different systems, considerable work is required to understand how each of these transcriptions factors work in a coordinate manner to control gene expression generally associated with EMT, invasion and metastasis.

2.4.4

Homeodomain Proteins

Homeobox proteins are a group of highly conserved transcription factors that bind homeobox DNA sequences found within genes. The homeobox family of transcription factors acts as critical regulators of growth, migration and differentiation during embryogenesis. In addition, they have recently gained attention as promoters of invasion and type-3 EMT. Homeobox proteins generally do not function alone but in tandem with various members of the homeobox family which determines functional consequences of expression thus making it difficult to discern the roles of distinct members. Specifically, however, expression of members of the sine oculis homeobox (Six) protein family, Six1, Six2 and Six4, have been implicated in EMT events during development and cancer [10]. Six family protein expression is regulated at the level of transcription, by proteasome-mediated degradation and in some cancers by gene amplification [359]. Notably, Six1 expression is regulated during the cell cycle, with its maximum expression during S and G2 phase, and its transcription is induced by E2F1 [360, 361]. Six1 is then degraded via the proteasomal pathway at the G2/M phase transition via the anaphase-promoting complex [362]. Structurally, the Six family of transcription factors are characterized by a helixturn-helix motif and the presence of the homeobox nucleic acid recognition domain. While Six1, Six2 and Six4 show similarity in the binding to MEF3 sites on DNA (TCAGGTTC) to induce transcription, Six1 also binds structural DNA features, like nonsequence-specific HMG protein recognition sites [363–365]. The Six family of proteins appear to regulate a wide variety of genes that are critical for the migration of myogneic cells as well as organogenesis of the eye and kidney during development [366, 367]. Specifically, Six1 and Six2 are associated with cells of mesenchymal origin and Six2 appears to be critical for maintaining the mesenchymal phenotype of renal progenitor cells [368]. Interestingly, Six1 overexpression activates TGF-beta signaling and is associated with nuclear Smad3 accumulation and increased expression of TGF-beta responsive genes [369]. Furthermore, Six1 induces features of EMT in vivo in tumors derived from mammary specific Six1 over-expression, including loss of E-cadherin, cytokeratin-18,

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nuclear accumulation of beta-catenin and expression of alpha-smooth muscle actin [370]. Over-expression of Six1 in mammary carcinoma cells in vitro also induces an EMT phenotype that is characterized by dissociation of ‘cell-cell’ contacts, decreases in cytokeratin-18 expression and increases in metastatic potential [371]. Consistent with other regulators of EMT, Six1 over-expression is seen in 90% of human metastatic breast cancer lesions and correlates with adverse outcomes in numerous other cancers including brain, cervical, prostate, colon, kidney, and liver [359, 369]. While the mechanism by which Six1 activates TGF-beta signaling and EMT remains unclear, it must be noted that inhibition of TGF-beta signaling is unable to completely reverse Six1-induced EMT, suggesting that Six1 activates additional pathways responsible for maintaining EMT [369]. Other homeobox transcription factors associated with development also appear to be inducers of EMT. Ladybird homeobox 1 (Lbx1) protein is a regulator of c-Met induced muscle precursor migration in the developing embryo [371]. Recently expression of Lbx1 was found to be associated with a robust EMT characterized by the down regulation of E-cadherin and occludins as well as the up-regulation of N-cadherin, vimentin and fibronectin [372]. Interestingly Lbx1 also induced Snail, Zeb1, Zeb2 and TGF-beta2 expression, although the contribution of TGF-beta2 induction toward this EMT response was not investigated. Significantly, inhibition of Lbx1 in metastatic cells also inhibits migration and increased Lbx1 expression is associated with more invasive subtypes of human breast cancers [372]. Msx2 is a member of muscle-segment homeobox genes and has also been implicated in developmental EMT events during organogenesis and neural tube migration [373, 374]. Msx2 is expressed at the sites of epithelial-mesenchymal interactions mediating the signaling between the two different tissues. Msx2 has also been shown to induce EMT in vitro in breast and pancreatic cancer cell lines as characterized by the loss of E-cadherin, nuclear accumulation of beta-catenin and up-regulation of N-cadherin, vimentin and Twist1 [375, 376]. Furthermore, Msx2 over-expression enhances the metastatic potential of cells and its expression is specifically observed in tumor cells that display an invasive phenotype in clinical samples. Interestingly, part of the mechanism by which Msx2 initiates EMT is by up-regulation of the signaling molecule Cripto [375]. Cripto-knockout mice lack of mesoderm formation and fail to initiate early body patterning, indicating Cripto plays a critical role in type-1 EMT events and inducing the mesenchymal phenotype. Likewise, Cripto expression increases the invasive and migratory properties of cells and induces down-regulation of E-cadherin and up-regulation of vimentin and N-cadherin [377, 378]. Cripto expression is, however, also associated with non-invasive tumor types in vivo, suggesting that induction of Cripto by Msx2 is necessary, but not sufficient to induce an EMT event that stimulates robust invasion and metastasis [10]. The Goosecoid (Gsc) homeobox transcription factors are also a well known activator of cell migration. During development, Gsc is expressed during primitive streak formation and its activity is required for embryonic cell migration and dorsoventral patterning of mesodermal tissues [379–381]. Gsc expression appears to be induced by TGF-beta signaling and down-regulates E-cadherin expression in cells possibly by inducing expression of Snail, Slug, Zeb2 and Twist1 [382, 383]. Studies


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have shown that Gsc is also over-expressed in a majority of human breast tumors and ectopic expression of Gsc in human breast cells induces EMT and enhances the ability of breast cancer cells to form pulmonary metastases in mice [382]. While the specific roles of the various homeobox transcription factors during tumorigeneis and EMT are just starting to become unraveled, it is clear that they do play a significant role in regulating type-3 EMT events and metastatic potential in cancer cells.

2.4.5

HMGA2 and Polycomb-Group Proteins

The role of chromatin associated proteins which alter DNA and chromatin structure to regulate the transcriptional activity of several genes associated with EMT is just beginning to be investigated. Of particular interest in regards to EMT and tumor progression is the high mobility group A2 protein (HMGA2, also known as HMGI-C). While HMGA2 is widely expressed during early embryogenesis, it is almost completely absent in differentiated cells and normal adult tissues [384]. During development HMGA2 is expressed in all mesenchymal cell populations and its expression is required for heart organogenesis, all suggesting a possible role for HMGA2 in regulating the mesenchymal cell phenotype [385, 386]. Pathological studies have shown that HMGA2 is often re-expressed in tumors and correlates with occurrence of metastasis and poor prognoses in several human cancers including breast, pancreatic and lung carcinomas and in squamous carcinomas of the oral cavity [387–390]. Ectopic expression of HMGA2 in adult cells induces a mesenchymal phenotype characterized by robust down-regulation of E-cadherin and induction of Snail, Slug, Zeb1 and Twist1 [391]. Mechanistically, HMGA2 induces Snail by binding to the proximal region of the Snail promoter and synergizes with Smad3/Smad4 to activate Snail transcription [391]. Furthermore, HMGA2 induction appears to be required for TGF-beta stimulated EMT [347]. The HMGA family of proteins binds fairly ubiquitously to the minor groove of AT-rich DNA sequences. The DNA-binding domain is located in their N-terminus which contains three separate AT-hooks characterized by eight to nine basic amino acid repeats [392]. HMGA proteins do not display direct transcriptional activity, rather they function by altering chromatin architecture and interacting with transcriptional machinery to form multiprotein complexes on promoter/enhancer regions of genes [393]. HMGA proteins also participate in protein–protein interactions with other DNA bound proteins to affect transcriptional activity. For example, expression of HMGA2 enhances the transcriptional activity of E2F1 target genes by displacing HDAC1 from the pRB–E2F1 complex and inducing acetylation of both E2F1 and DNA-associated histones [394]. HMGA2 also binds the p50/p65 NF-kappaB heterodimer and enhances its transcriptional activity at various promoters [395, 396]. HMGA2 expression is induced by binding of the transcription factors c-Jun to AP1 sites, c-Myc to E-boxes and by TGF-beta stimulated Smad 4 binding to sites in the HMGA2 promoter [347, 397, 398]. Interestingly, HMGA2 appears to be one of the more post-transcriptionally modified proteins in the cell, via phosphorylation,

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acetylation and sumolyation, however, the contribution of these events to HMGA2 function remain to be elucidated. While more work is clearly needed to fully understand HMGA2¢s role in EMT and its regulation of EMT associated transcription factors, the ability for it to interact with and enhance various signaling pathways, as well its promiscuous binding to DNA, might very well put it at the forefront as a key master regulator of EMT. Another family of chromatin associated proteins that have recently become of interest in regards to EMT are the Polycomb-group of Proteins (PcG) which are evolutionarily conserved chromatin modifying factors that repress transcription. These proteins regulate covalent modification of specific residues within aminoterminal tails of histones. There are two core PcG complexes which are made up of multiple protein subunits, the polycomb repressive complex 1 and 2 (PRC1 and PRC2 respectively). The PRC1 genes include Bmi-1, Mel-18, RING, Hpc and Hph, while PRC2 includes Eed, Ezh1/2, Suz12 and YH1. In general, PRC2 binds polycomb repressive elements (PREs) on DNA and through the action of the subunit enhancer of zeste homologue 2 (Ezh2) which trimethylates histone H3 at lysine 27 to induce transcriptionally inactive chromatin [399]. These changes in DNA conformation established by PRC2 are recognized by PRC1, which stabilizes the inactive state by ubiquitylating histone H2A to maintain transcriptional repression memory and impede RNA polymerase II initiation of transcription and elongation [400]. In addition to histone methylation, PcG complexes can also recruit proteins that facilitate other covalent modifications including the recruitment of sirtuin 1 (SIRT1) and HDAC2 to acetylate histones as well as DNA methyltransferases (DNMTs) which mediated direct methylation of DNA [401]. Notably, PRC2 proteins and their triMeK27-H3 marks reside at and transcriptionally repress many regulatory genes that control specific developmental lineages. Thus PcG proteins and their antagonist, the trithorax group of proteins, serve to fine tune gross changes in gene expression required for differentiation during embryonic organogenesis [402, 403]. Bmi-1, was the first PcG protein to be implicated in cancer and was initially identified as an oncogene that cooperates with Myc to induce proliferation and resistance to senescence [404]. Subsequently, Bmi-1 has been found to be over-expressed in aggressive and invasive breast cancer, prostate cancer, bladder cancer, melanoma, hepatocellular carcinomas and esophageal squamous cell carcinomas [405–410]. The combined over-expression of Bmi-1 and Ras in epithelial cells results in a mesenchymal-type change in cell morphology, increased invasive properties, and increased metastatic potential [411]. Conversely, knockdown of Bmi-1 in metastatic breast cancer cell lines reduces their invasive capacity. Together, these observations suggest that Bmi-1 may have a role beyond regulation of proliferation and senescence and may regulate EMT and invasion. Indeed, Ras and Bmi-1 expressed in combination can induce the expression of vimentin and fibronectin and decrease E-cadherin expression in normal breast epithelial cells [412]. Furthermore, Twist1 appears to be a direct regulator of Bmi-1 and Twist1 and Bmi-1 are mutually essential to promote EMT and act cooperatively to repress expression of E-cadherin [413]. Bmi-1 expression can also induce EMT and invasion in nasopharyngeal epithelial cells by the stabilization of Snail via modulation of GSK-3beta signaling [414].


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Ezh2, a member of the PRC2 complex, has been shown to play a role in controlling actin polymerization in fibroblast cells, suggesting Ezh2 expression could also contribute to invasion by regulation of actin-dependent cell adhesion and migration [415]. Ezh2 might directly impact EMT by mediating transcriptional silencing of E-cadherin by trimethylation of H3 lysine 27 [416]. Additionally, Ezh2 has been shown to silence DAB2IP, a Ras GAP, and induce Ras hyperactivity and prostate cancer metastasis [417]. Taken together, these studies suggest a significant role for PcG in the regulation of EMT related pathways during tumorigenesis and highlight how epigenetic changes in cells might impact their ability to undergo EMT. In sum, it is clear that aberrant reactivation of developmental transcription factors is a critical event for EMT to occur, and that this enhances tumor cell migration, invasion and metastatic potential. One important concern at this juncture surrounds understanding the relative contribution of these different transcription factors and chromatin associated proteins during EMT. Future studies should focus on the relative interdependence of these transcription factors to induce EMT, whether a particular hierarchy of expression between factors exists and the relative robustness of EMT induced by each. For example, differences in potency of gene regulation exist as Slug inhibition of E-cadherin appears to be less effective than that of Snail, correlates with weaker binding affinity to the E-cadherin promoter and is associated with a different phenotypes in vivo [418]. In addition, a hierarchical pattern might exist where a dedifferentiated cell state induced in part by expression of PcG proteins, homoeobox proteins and HMGA2 is required for Snail and Slug expression in response to TGF-beta, leading to the activation of Zeb and Twist family members [419]. These transcription factors then orchestrate a down-regulation of adherence and tight junctions and a remodeling of the cytoskeleton and ECM to induce tumor cell invasion and metastasis as part of an EMT program. Some reports also suggest that EMT and tumor cell dissemination may be an early event in tumor progression before gross genetic abnormalities arise [214, 420]. Therefore, understanding the regulation of EMT in primary cell lines from early stage cancers might be key to understanding critical interactions amongst these transcription factors to control for active background pathways which may be contributing to the EMT phenotype.

2.5

MicroRNAs and EMT

Since the discovery of microRNAs (miRNAs), over 900 miRNA genes been identified in the human genome [421]. In many cases, it also appears that each mRNA species can influence global patterns of gene expression. Thus, miRNAs constitute one of the most significant classes of gene-regulatory molecules in humans. As such, miRNAs have been shown to be key players in a broad range of developmental and physiological processes and their deregulation has been closely linked to human diseases including cancer. MicroRNA profiling studies in a variety cancers have revealed that their expression varies considerably between malignant and normal

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tissues [422–425]. While it appears that many miRNAs are down-regulated as tumors become less differentiated, consistent with a role for miRNAs in determining cellular differentiation states, miRNAs appear to act as critical components of both oncogenic and tumor suppressor pathways [426, 427]. Notably, as the genes and processes which miRNAs regulate expand, it has become increasing clear that miRNA expression indeed serves as an important regulator of cellular differentiation, cell fate, stemness and EMT. miRNAs are a diverse family of 18–24 nucleotide non-coding RNAs that posttranscriptionally regulate the stability and translational efficiency of target mRNAs. Generally, miRNAs function through imperfect complimentary base pairing with specific sequences in the 3¢ untranslated regions (UTRs) of target mRNAs. The key region of the miRNA that largely governs its target specificity is known as the seed sequence, and encompasses bases 2–7 from its 5¢ end [428]. In the nucleus, miRNAs are transcribed as a primary transcripts (pri-miRNAs), by RNA polymerase II or III, which can be several 1,000 nucleotides in length. These transcripts are 5¢-capped, spliced, and polyadenylated, and often contain more than one functional miRNA [429]. In the nucleus, pri-miRNAs are then cleaved into 70–80 nucleotide hairpin-loop structures called precursor miRNAs (pre-miRNA) by a multiprotein complex called the microprocessor which contains Drosha, an RNase III enzyme, and DGCR8/Pasha, a double-stranded RNA-binding protein [430]. Drosha cleavage generates a 2 nucleotide 3¢ overhang that is recognized by exportin 5 which transports the pre-miRNA into the cytoplasm in a RanGTP-dependent manner [431]. Once in the cytoplasm, pre-miRNAs are further processed by the RNase-III enzyme Dicer, which removes the stem loop and produces a 22 nucleotide RNA duplex. Of this duplex, one strand (the guide strand) stably associates with an Argonaute (Ago) protein within the RNA-induced silencing complex (RISC), while the other strand (passenger strand) is discarded [432]. This miRNA-RISC complex is then recruited to target mRNAs. There are multiple mechanisms by which miRNA expression can be changed during tumor development. To begin with, changes in miRNA expression between normal and cancer associated tissues can often be attributed to the location of miRNA genes in regions of chromosomal instability characteristic of certain cancers [433]. The expression and activation of specific transcription factors (often fueled by activation of signaling networks described above) also play an important role in regulating miRNA expression. Many miRNAs have been shown to be regulated by well-established tumor suppressor and oncogene pathways, such as p53, Myc and Ras in which miRNAs and their transcriptional regulators participate in complex feedback regulation loops which appear to drive tumorigenesis [434]. Interestingly, DICER is a haploinsufficient tumor suppressor and 27% of various tumors are found to have a hemizygous deletion [435]. Knockdown of Dicer1 also appears to increase the malignant and invasive potential of already transformed cell lines and to increase the rate of tumor formation in cancer mouse models [436, 437]. The phosphorylation of the Dicer cofactor TARBP2 by the MAPK pathway has also been shown to positively affect the processing of oncogenic miRNAs and negatively regulate the tumor suppressor let-7a [438]. Also, TGF-beta, through Smad signaling, promotes a rapid increase in expression of oncogenic miR-21 by promoting the


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recruitment of pri-miR-21 to the Drosha microprocessor complex [439]. Thus regulation of miRNA processing also appears to play an important role in miRNA expression and function during tumorigenesis. While multiple mechanisms contribute to aberrant miRNA transcription and function in cancer, it has become apparent that various RNA binding proteins also play an important role in regulating the processing and stability of miRNAs in normal and malignant tissues. Lin28A (Lin28), Lin28B and RPS2 (Ribosomal Protein S2) all have been shown to be over-expressed in advanced tumors and bind to the pre-let7 hairpin and inhibit expression of the mature form of this potent tumor suppressor [440–442]. Furthermore, while it appears that pre-miRNA complexing with these proteins interferes with functional incorporation into the RISC silencing complex, Lin28 has also been shown promote miRNA degradation by binding to the GGAG sequence in the terminal loop and mediating terminal uridylation of preLet-7, 107, 143, and 200c miRNAs and recruiting 3¢ terminal uridylyl transferases [443, 444]. Many of these very miRNAs seem to be critical for all three types of EMT events. Recently it has been shown that over-expression of Lin28B is sufficient to induce EMT and invasion in hepatocellular carcinoma cell lines [445]. Given that miRNAs play critical roles in regulating multiple pathways and global gene expression patterns, it is not surprising that they also play an important role in orchestrating the extensive changes that occur during EMT. Thus, regulation of miRNA expression and function appears to be one of the most important regulators EMT and cancer cell invasion and metastasis. The extent of which is just now beginning to be realized. miRNAs represent a critical downstream component of key developmental, oncogenic and tumor suppressor pathways, and as has been discussed in previous sections, EMT appears to be driven by developmental and oncogenic pathways [427]. Thus, aberrant activation of these pathways in tumor cells directly influences miRNA expression, which may consequently affect EMT and metastasis [437]. This is elegantly illustrated by the process of miRNA expression controlled by the oncogenic transcription factor c-Myc, one of the most commonly activated oncoproteins in human cancer [446]. Myc directly induces expression of the miR17–92 cluster, which subsequently promotes proliferation, inhibits apoptosis, and promotes tumor angiogenesis [447]. Moreover, Myc activation leads to widespread repression of many additional miRNA genes including several with known tumor suppressor activity such as members of the Let-7 family, miR-15a/16–1, the miR-29 family, and miR-34a [446]. It appears that the predominant consequence of activation of Myc is widespread repression of miRNA expression as a direct result of Myc binding to miRNA promoters. This supports a critical role for miRNA repression in the Myc oncogenic mechanism of action. Furthermore, Myc is negatively regulated by the developmental miRNA, Let-7, whose functionality must be reduced for Myc expression [448]. Thus, there is a complex interplay and delicate balance of developmental and oncogenic transcription factor expression and miRNAs whose disruption appears to lie at the heart of tumorigenesis and the metastatic cascade.

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2.5.1


The miRNA-200 Family

The best characterized miRNAs involved in regulating EMT are the miR-200 family. A number of reports have identified changes in their expression as a fundamental regulator of EMT [449–451]. Microarray analysis comparing mesenchymal cell lines to epithelial lines has revealed that a reduction in the levels of the miRNA-200 family and miRNA-205 are the most consistent markers associated with cells which lack E-cadherin and express vimentin [449]. miR-200 family members are also routinely found to be down-regulated during TGF-beta induced EMT; likewise, inhibition or ectopic expression of the miRNA-200 family members can induce EMT or epithelial cell characteristics respectively [451]. Accordingly, miR-200 family can reverse/inhibit EMT and are powerful inducers of epithelial differentiation. The miR-200 family consists of 5 highly homologous RNAs: miR-141, miR-429, miR-200a, miR-200b, and miR-200c. These miRNAs differ by only a single nucleotide in their seed sequence. miR-200b, miR-429, and miR-200c share the seed sequence 5¢-AAUACU-3¢ while miR-200a and miR-141 share the sequence 5¢-AACACU-3¢. Functionally, miR-200 family members act through the targeted silencing of the E-cadherin transcriptional repressors, Zeb1 and Zeb2, thus increasing the levels of E-cadherin and enforcing the epithelial phenotype [451]. miR-205 has also been shown to target both Zeb1 and Zeb2 [451]. Interestingly, Zeb1 and Zeb2 both suppress miR-200 family member expression, creating a double negative feedback loop that enables miR-200 family and Zeb proteins to respectively enforce the epithelial and mesenchymal cellular states [452]. The miR-200 family also inhibits the expression of other targets known to contribute to EMT, including TGFbeta2 by miR-141, beta-catenin by miR-200a, fibronectin by miR-200c and Bmi-1 by miR-200c [453–456]. Other EMT related targets of the miR-200 family may still be identified. In several cancer cells and models, expression of miR-200 family members inversely correlate with invasive and metastatic potential. For example, downregulation of the miR-200 family is part of a distinct miRNA signature of metastases from primary tumors in mice predisposed to develop pancreatic neuroendocrine tumors [457]. Metastasis and EMT, but not primary tumor growth rate, has also been shown to be entirely dependent on TGF-beta down-regulation of miR-200 family expression, where forced expression of the miR-200b cluster completely abrogates EMT and metastatic potential [458]. Additionally miR-200a expression inhibits the in vitro migration and invasion of highly metastatic and invasive nasopharyngeal carcinoma cells, miR-200b expression inhibits the invasive capacity of LNCaP prostate adenocarcinoma cells and MDA-MB-231 breast cancer cells and miR-200b inhibition supports invasion of non-invasive HT29 colon cancer cells [453, 459]. Interestingly, in cell lines isolated from a spontaneously arising mammary carcinoma which exhibit different metastatic potential, 4T1 cells, the most aggressively metastatic, express comparatively high levels of members of the miR-200 family [460]. These results seemingly contradict our current understanding of the tumor suppressor functions of the miR-200 family. However, this study highlights


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three issues regarding EMT and metastasis. First, cells might need to re-establish epithelial characteristics by inducing a mesenchymal to epithelial transition (MET) in order to successfully form macroscopic secondary tumors. Therefore, expression of miR-200 family members might support the ability for cells to proliferate at secondary sites. Second, type-3 EMT events which lead to metastasis might not need to be full EMTs in that they do not require all the characteristics of an EMT to effectively disseminate and metastasize. It is quite likely that cells only need to surpass a threshold of sufficient EMT characteristics to successfully orchestrate their metastatic dissemination. In this regard, it should be noted that 4T1 cells undergoing EMT express E-cadherin while also expressing vimentin and Twist1 [460]. Lastly, given the heterogeneity of tumors, it is possible that a subset of highly metastatic 4T1 cells transiently loses miR-200 expression and induces an EMT which is undetectable but sufficient for invasion and metastasis. Regardless, the miR-200 family remains one of the most significant regulators of EMT. Future work is however needed to understand the results from 4T1 cells to clarify the roles of EMT and miR-200 expression in metastasis.

2.5.2

The Let-7 Family

The Let-7/miR-98 family was one of the first mammalian miRNAs to be identified and is ubiquitously expressed in normal adult tissues [461, 462]. The Let-7 family has 12 members (Let-7-a1, a2, a3, b, c, d, e, f1, f2, g, i and miR-98) with nine distinct mature miRNA sequences all with identical seed sequences (5¢-GAGGUA-3¢). Let-7 is expressed late in mammalian embryonic development and plays an evolutionarily conserved role of promoting cell and tissue differentiation [463, 464]. With regards to cancer, a plethora of human malignancies, including lung, ovarian, gastric, prostate cancer and melanoma, appear to down regulate Let-7 family members during tumor development [422, 465–468]. Multiple loci that encode Let-7 miRNAs are also frequently deleted in cancer cells [433]. The human Let-7 family of miRNAs possesses potent tumor suppressor activity by targeting multiple oncogenes, including those involved in EMT. Specifically Let-7 was shown to directly target Ras, negatively correlate with Ras expression and inhibit growth of human lung cancer cells [469, 470]. At least nine members of the Let-7 family efficiently bind multiple complementary sites in the 3¢-UTRs of all three human Ras genes [470]. Given the key role of Ras signaling in inducing EMT, Let-7 down-regulation might help to induce EMT by amplifying Ras signals. Indeed over-expression of Let-7 in lung cancer cells represses genes regulated by Ras [466]. Let-7 miRNAs are also a strong negative regulator of HMGA2, binding to multiple target sites in the 3¢ UTR [471]. HMGA2 is known to be expressed in a variety of tumors which also express low levels of Let-7 and correlates with poor prognosis [389, 472, 473]. Most importantly, HMGA2 has been described as a critical factor necessary for TGF-beta induced EMT in mouse primary mammary epithelial cells and its expression in conjunction with oncogenic Ras signaling seems to be required

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for Snail expression and EMT in human pancreatic cancer cells [347, 474]. Taken together these studies suggest that Let-7 family members might be potent negative regulators of EMT. While detailed analysis of the direct role for Let-7 in EMT is lacking, interestingly, miRNA expression analysis of cancer cell lines suggests that Let-7 is a marker for less advanced cancers and negatively correlates with reactivation of embryonic mesenchymal genes [473]. Let-7a has also been shown to down-regulate expression of the proliferative transcription factor Myc and its target genes [448]. As eluded to above, Myc participates in a feedback loop, inhibiting Let-7 expression by transcriptional repression [446]. Myc also transcriptionally induces the Let-7 negative regulator, Lin28 [475]. Taken together with the observation that MAPK signaling through Erk2 can increase Myc activity, it appears that a signaling cascade involving Ras-MAPK-Myc-Lin28 down regulation of Let-7 might play a critical role in regulating metastasis and EMT [210, 475, 476]. Thus, disrupting the delicate balance of this loop is likely a potent facilitator of tumor progression, invasion and metastasis. Interestingly, recent findings have also connected Lin28 and Myc expression with stem cell maintenance and the pluripotent state, where expression of Myc and Lin28 appear to be interchangeable [477, 478]. Given that expression of Let-7 leads to differentiation, this data suggests that regulation of Let-7 expression by Myc and Lin28 might pose as a connection between EMT, pluripotency and cancer stem cells. For example, TGF-beta induction of EMT in human mammary epithelial cells not only involves up-regulation of EMT transcriptional regulators (Snail, Zeb2 and Twist), but also includes induction of classical stem cell markers [18, 479]. In mouse tumor modeling studies, these cells are able to form metastasis at very low titers and are thought to function as tumor initiating cells. Down-regulation of Let-7 expression appears to play a significant role in the activation of these tumor initiating cells. Tumor initiating cells isolated from breast cancers express very low levels of Let-7, and induction of Let-7 in these cells inhibits metastasis [480]. Ultimately it appears that the main function of Let-7 miRNAs are to suppress early embryonic genes which may have activities in the maintaining stem cell cell-like phenotype which is essential for EMT and induction of the metastatic phenotype. In future studies, development of therapeutics which prevents Lin28 or RPS2 binding to pre-Let-7 RNA may be highly beneficial in blocking EMT and metastasis.

2.5.3

Other miRNAs and EMT

In addition to the miR-200 family and let-7 family, several other miRNAs have been shown to alter the expression of EMT related genes, impacting cellular morphology, invasion and metastasis. Some miRNAs have been identified in TGF-beta induced EMT, and are directly regulated by TGF-beta stimulation. Levels of miR-155 have been shown to be increased during TGF-beta induced EMT in mammary epithelial cells via transcriptional activation by Smad4 [481]. While ectopic expression of


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miR-155 does not induce EMT, it does induce disruption of cell polarity and tight junctions and loss of miR-155 suppresses TGF-beta induced EMT. miR-29a and miR-21 levels also appear to increase during TGF-beta induced EMT and are expressed at higher levels in mesenchymal cells when compared to epithelial cells [449, 482]. At this juncture the direct role of miR-29a in metastatic progression is unclear, however, miR-21 is found to be over-expressed in lung, breast, stomach, prostate, colon, brain, and pancreas carcinomas and stimulates invasion and metastasis in colon and breast cancer cells [483–485]. miR-21 transcription appears to be regulated by TGF-beta activation of the AP-1 and Zeb1 transcription factors in a Smad4 independent manner [486]. Twist1, as part of its ability to regulate EMT, activates miR-10b, which contributes to the migration, invasion and metastasis of breast cancer cells and its expression in primary tumors correlates with clinical progression [487]. miR-9 is also up-regulated by Myc and directly targets E-cadherin mRNA resulting in increased cell motility, invasiveness and metastatic potential [488]. Furthermore, miR-9 is expressed at high levels specifically in primary breast tumors from patients who have developed metastases [488]. Conflicting with these results, miR-10a and miR-9 do not appear to be expressed at high levels in actual distant metastasis [489, 490]. This is understandable if we assume EMT at the invasive front of tumors only leads to transient activation of miR-9 and miR-10b to promote dissemination and invasion and upon metastatic seeding, expression of these miRNAs is lost as cells undergo an MET. In conclusion, the network of oncogenes and miRNAs that play a role in controlling genes associated with EMT is in its infancy. Undoubtedly, many more pathways which regulate EMT will emerge that will help our understanding of the mechanisms involved in metastatic dissemination. As global regulators of gene expression and differentiation, the field of miRNAs remains an extraordinarily exciting opportunity to identify key regulators of EMT that are not context dependent. Most importantly, the prospects of developing novel diagnostics for cancer or discovering therapeutics which target miRNA regulatory pathways present enormous opportunities to improve cancer patient outcomes. As cancers are dynamic and heterogeneous in nature, future drug discovery efforts need to consider which cancer cells pose the greatest threat to patient survival. It is clear from the literature that the cellular characteristics adopted during an EMT, such as dissolution of epithelial cell contacts, cytoskeletal changes and matrix remodeling, all promote progression to metastatic and deadly disease. Furthermore, the key signaling pathways which induce EMT, namely TGF-beta, Wnt, MAPK and AKT, also promote tumor progression and metastasis. Targeting these EMT pathways is a promising therapeutic strategy as knockdown of several transcription factors regulating EMT, such as those of the Snail, Twist and Zeb families, effectively inhibit metastasis in mouse tumor modeling studies. Moreover, EMT is increasingly associated with dedifferentiation and tumor initiating cells, thus targeting cells with stem cell-like characteristics or pharmacological induction of differentiation may also have therapeutic value. Finally, therapeutic delivery of endogenous miRNAs or miRNAs which specifically target EMT regulators may represent a potentially potent therapy to prevent metastasis and extend patient survival.

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Chapter 3

Biomechanical ECM Switches and Tumor Metastasis Jacquelyn J. Ames, Calvin P.H. Vary, and Peter C. Brooks

Abstract Data is rapidly emerging that physical or biomechanical changes in extracellular matrix molecules (ECM) may allow differential control of cell signaling pathways and therefore, these structural alterations may actively regulate diverse cell types that comprise the tumor stroma. These mechanical changes in the structure of ECM molecules may trigger the exposure of cryptic ECM elements that help create a local microenvironment that facilitates initiation and progression of inflammation, tumor growth and metastasis. To this end, we will highlight the accumulating experimental evidence for the existence of biomechanical ECM switches and discuss how the selective triggering of these switches may be used by diverse cell types to promote malignant tumor progression. Finally, we will discuss scientific and clinical advancements that have lead to the recent translation of these exciting biological concepts into human clinical trials for cancer imaging and therapy.

3.1

Introduction

Communication links between distinct populations of cells and between cells and the surrounding solid-state extracellular matrix (ECM) molecules are among the most fundamental requirements for essentially all biological processes. Implicit within this general concept is the importance of a metered flow of mechanical, biochemical and molecular information that can be precisely controlled to provide a selective means to impart specificity to such divergent biological processes as embryonic development and tumor progression. Bi-directional communication

J.J. Ames • C.P.H. Vary • P.C. Brooks (*) Maine Medical Center Research Institute, Center for Molecular Medicine, 81 Research Drive, Scarborough, ME 04074, USA e-mail: [email protected]


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Fig. 3.1 Triggering biomechanical ECM switches. Accumulating evidence suggest that the local stromal microenvironment of a tumor plays critical roles in regulating tumor growth and metastasis. Localized structural changes within individual solid-state ECM molecules may represent “Biomechanical Switches”. The selective triggering of these biomechanical switches may provide a mechanism to fine tune cellular responses to mechanical changes within the tumor microenvironment

circuits are known to exist between cells and the ECM and these circuits can be connected by cell surface receptors such as integrins [1–3]. Interestingly, integrin-ECM interactions have been shown to regulate a wide array of cellular and molecular processes ranging from mechanical adhesion, motility, proliferation, survival and differentiation, to the regulation of gene expression and biochemical signaling [1–3]. Given the broad functional importance of these communication links, it is not be surprising that dysregulation of these information highways might result in profound changes in homeostasis, which in turn might govern pathological events such as tumor growth and metastasis. In some respects, the flow of biological information between cells and the ECM might be viewed in a similar manner to that of the flow of electricity within a network of circuits regulated by switches. It is well-accepted that the interconnected network of ECM molecules that compose the non-cellular microenvironment of a particular tissue is dynamic, with transient alterations occurring in the molecular composition and structural integrity [4, 5]. The dynamic nature of the ECM may provide a tissue specific mode for cells to respond in appropriate ways to transient changes resulting from external stimuli. Viewed from this perspective, structural or mechanical alterations in the integrity of individual ECM molecules might function as biomechanical switches, with the capacity to initiate, stop or divert the flow of biological information in different pathways when a specific need arises (Fig. 3.1). However, if problems occur in specific switches within this network of circuits, the resulting aberrant information flow may result in unintended or detrimental consequences. Within this context, we will discuss the diverse roles by which structural alterations in ECM molecules may function as biomechanical switches and examine the potential of these switches to contribute to tumor growth and metastasis.

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Biomechanical ECM Switches and Tumor Metastasis

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Tumor Cell Metastasis

It has been appreciated for some time that metastasis or the spread of tumor cells from the primary tumor mass to distant sites is a major obstacle to the successful management of malignant tumors. In fact, metastasis has been implicated as a leading cause for the majority of deaths associated with diverse forms of malignant tumors. Metastasis has often been described as a highly inefficient process due in large part to the requirement of tumor cells to complete all the interconnected steps within the metastatic cascade to successfully establish secondary lesions [6, 7]. Due to the physical distances and numerous cellular and non-cellular components that tumor cells may encounter during their journey, a wide array of factors influence the capacity of tumor cells to successfully establish secondary metastatic foci. Given these well-established caveats, it is not surprising that effective therapeutic strategies to manage metastatic disease have been slow to emerge. In general, the metastatic cascade (Fig. 3.2) can be thought of as an interdependent set of steps that facilitates the capacity of tumor cells to spread from the primary tumor to multiple sites throughout the body [6, 7]. These complex cellular events include alterations in cell-cell interactions via changes in expression and activity of cell-cell adhesion molecules such as E-Cadherin [8]. This overall reduction in cellcell adhesion can be accompanied by a multitude of additional changes, including increased expression of matrix altering enzymes as well as alterations in the expression and activation of numerous cell surface receptors that mediate interactions with soluble factors, other cells, and with the ECM [9, 10]. These changes and many more can lead to localized structural remodeling within the interconnected network of ECM molecules. Evidence suggests that multiple cell types within the tumor microenvironment including stromal fibroblasts and inflammatory infiltrates such as mast cells, neutophils and macrophages secrete an array of ECM altering enzymes including members of the serine, cystiene, heparinase and matrix metalloproteinase (MMP) families [11]. Several of these proteases have been shown to localize to the cell surface of tumor and stromal cells via diverse mechanisms, some of which include interactions with cell surface receptors such as integrins [12, 13]. Localized ECM remodeling as a consequence of either secreted or cell associated enzymes, results in more than simply degradation of individual molecules to provide a less restrictive pathway for cell movement. For example, many serine proteases as well as MMPs have been shown to release bioactive ECM peptides, while enzymes such as lysyl oxidase (LOX) can modify ECM molecules resulting in altered cross-linking leading to changes in ECM biomechanical properties [14, 15]. Finally, a number of proteolytic enzymes have been shown to contribute to the exposure of functionally active cryptic or hidden structural ECM domains that exhibit unique functions [16–18]. Collectively, these changes may govern divergent processes ranging from growth factor localization and changes in mechanical stability and stiffness to modifying numerous cellular interactions which ultimately impact the ability of cells to move, proliferate, differentiate and survive within a particular tissue microenvironment.

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Fig. 3.2 Tumor cell metastasis. Tumor cell metastasis involves a complex interconnected series of cellular and molecular events. This complex process is governed by numerous distinct cell types that functionally interact with each other as well as with an array of diverse extracellular matrix (ECM) molecules. Localized changes in cell-cell and cell-ECM interactions along with structural alterations within ECM molecules facilitate cellular invasion and ultimately the formation of secondary metastatic foci at remote site from the primary tumor lesion

A growing body of evidence now shows that changes in tensional and contractile forces can generate increased stiffness and rigidity of the ECM and may actually promote malignant cellular behavior. For example, increased interstitial pressure from a rapidly growing tumor mass may enhance the physical forces on individual ECM molecules such a collagen and fibronectin [19, 20]. In this regard, it is

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known that many of the multi-domain ECM molecules that make up the tumor microenvironment can respond to these alterations in mechanical forces by changing their native conformation and altering other biomechanical properties including their contractility and rigidity [21, 22]. Changes in ECM rigidity have been shown to alter integrin function and activation resulting in changes in focal adhesion formation, re-organization of the cytoskeleton and enhanced activation of critical intracellular signaling pathways such as MAPK/Erk and Rho signaling, which act to control cellular migration and invasion [23, 24]. Therefore, it is not surprising that structural remodeling resulting from either mechanical forces or proteases, facilitates invasion and migration away from the primary tumor mass into the surrounding interstitial tissue. Recent evidence suggests that under some circumstances, initial tumor cell invasion during metastasis may be associated with epithelial to mesenchymal transition (EMT) like processes [25]. Studies have shown that molecular regulators of EMT including Snail and Twist may play roles in carcinoma cell invasion and metastasis. EMT within this context may be transient, occurring predominantly at the invasive front of tumors and governed, at least in part, by extrinsic factors from the surrounding tissue microenvironment [26]. The potential transient nature of carcinoma-associated EMT may explain in part, the conflicting data for EMT during metastases of tumors from distinct histological origins. As cellular invasion and migration continue, tumor cells may sense specific gradients of soluble factors that help direct them towards lymphatic and/or blood vessels. These soluble factors range from growth factors; cytokines and chemokines to small ECM derived peptides [27]. Experiments have suggested that protease generated fragments of collagen and elastin can function as cellular chemoattractants and some studies have demonstrated that certain PPG amino acid containing peptides bind chemokine receptors and stimulate directed cellular motility [28]. Interestingly, new work suggests that macrophages, which are well–known to respond to chemokines, may contribute to the re-orientation of tumor cells toward blood vessels to facilitate intravasation [29]. Supporting this notion, evidence suggests that macrophages are often present within the immediate vicinity of tumor cells invading the host circulation and moreover, that reduction in macrophages may correlate with reduced tumor intravasation [30, 31]. In order to gain entry into the circulation, tumor cells are thought to remodel the structural integrity of basement membranes. Malignant cells may use a combination of proteolytic enzymes such as MMPs, serine proteases and heparinases to cleave the major components of the basement membranes including collagen type-IV, laminin and proteolgycans such as perlecan [32]. These altered basement membranes, along with changes in the biomechanical properties of the tumor cells themselves, help facilitate cellular invasion. Once in the circulation, tumor cells must survive physical shear forces as well as evade host immune surveillance. It has been suggested that platelet-tumor cell interactions result in cellular aggregates that help protect and hide the circulating tumor cells from mechanical shear forces and immunological attack [33, 34]. Multiple mechanisms have been described by which tumor cells arrest within the vasculature

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and these may involve a combination of mechanical trapping and weak interactions between tumor cells and specific cell-cell adhesion molecules such as selectins that are expressed on the luminal side of the endothelium. In addition, it has been appreciated for some time that the endothelia of distinct organs may display a unique repertoire of surface molecules termed vascular addressins, which may contribute to the ability of tumor cells to preferentially arrest at tissue-specific sites [35, 36]. This concept is thought to contribute to organ-specific metastasis. Following initiation of these weak cellular interactions, more stable physical connections between integrins and the underlying ECM proteins have been suggested to occur. While the mechanisms by which tumor cells exit the vasculature, a process termed extravasation, are complex, new insight is rapidly adding to the overall understanding of this highly dynamic process. For example, while endothelial cell contraction resulting in exposure of basement membrane components is thought to occur at the site of extravasation, evidence also suggests that cells often exit the vasculature at unique sites that express low levels of matrix proteins called LERs (Low Expression Regions). These LER’s may allow more efficient proteolytic and biomechanical remodeling of the basement membrane to promote cellular extravasation [37, 38]. Moreover, leukocytes such as neutrophils may contribute to the structural modification at LERs and the formation of these vascular exit gates may allow easier egress routes for tumor cells during extravasation. Once the tumor cells have escaped the confines of the circulation and entered the microenvironment of a distant organ a new set of challenges emerge. Tumor cells must either migrate into a biocompatible area or establish their own local microenvironment that allows continued survival. In some circumstances, tumor cells have been shown to co-opt an existing blood vessel to provide a sufficient source of localized nutrients needed for growth and expansion, while in other cases tumor cells initiate structural and mechanical remodeling of ECM, releasing matrix-bound growth factors, generating pro-migratory ECM fragments and secreting soluble molecules that function to promote recruitment of endothelial cells and bone marrow progenitor. Several studies have suggested that prior to tumor cell arrival at sites distant from the primary lesion, subsets of bone marrow progenitor cells may be recruited to create a localized pre-metastatic niche [39, 40]. These pre-metastatic niches may be created in part by altering the composition and possibly the structural integrity of the ECM by mechanisms involving alterations in the levels of fibronectin and MMPs [39]. In addition, infiltration of immune cells such as mast cells and neutrophils may also help modify or condition the microenvironment to potentiate cellular proliferation and support angiogenesis or vasculogenesis [41, 42]. In this regard, heparin, a major component of mast cell granules, has been shown to bind fibronectin, facilitating a biomechanical change that allows exposure of normally cryptic vascular endothelial growth factor (VEGF) binding sites [43]. These intriguing observations provide an important example by which structural alterations of ECM molecules may act as biomechanical switches to help create a local tissue microenvironment that is conducive to angiogenesis and tumor cell survival. In addition, experimental evidence suggests that biomechanical contraction of tissues may also be employed to physically translocate existing blood vessels to areas of

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hypoxia [44]. Whether this mechanism occurs in the context of tumor development remains to be confirmed. Taken together, these examples help illustrate some of the ECM remodeling mechanisms that might be utilized to ensure cell survival at sites distant from the primary lesion.

3.3

Importance of the Dynamic Nature of the ECM Microenvironment in Tumor Metastasis

It is well established that many secreted factors such as matrix-modifying enzymes; growth factors, cytokines and chemokines all play distinct roles in regulating the metastatic cascade. However, in addition to these soluble components, it has also been known for decades that cellular interactions with solid-state ECM molecules such as collagen (Coll), laminin (LN) and fibronectin (FN) play crucial roles to governing numerous aspects of both normal and pathological cell behavior. Just as farmers often need to plow or mechanically disrupt the hard packed soil of their fields in order to prepare nutrient rich soil for their seeds to grow and survive, it is also likely that tumor cells need to mechanically remodel the ECM to create a nutrient rich microenvironment to ensure their growth and survival. In many respects, this general concept was proposed in the late 1800s by Dr. Paget and was termed the seed and soil hypothesis [45]. Once the ECM has been remodeled and the biological information within the ECM rendered readily accessible, tumor cells need a means to detect and assimilate this information for use inside the cell. To this end, integrin receptors provide a key component that allows cells the capacity to tap into and utilize the vast amount of biological information contained within remodeled ECM molecules. These heterodimeric transmembrane receptors may act as functional hubs by connecting the extracellular compartment with the cell’s interior, thereby helping to establish bidirectional circuits that distribute the complex biological information among numerous intracellular signaling pathways [1, 46]. These important information circuits physically connect the extracellular environment to the cell’s signaling machinery via integrin-associated cytosolic adaptor proteins (Fig. 3.3). Following integrin engagement of extracellular ligands, conformational changes are induced within integrins. Studies suggest that these conformational changes are associated with a physical separation of the a and b integrin chains that is propagated across the plasma membrane to their cytoplasmic tails [47]. The physical separation of the a and b integrin cytoplasmic tails is thought to facilitate the formation of scaffolds containing signaling molecules such as Fak, and Src [48]. Recent estimates have suggested that these protein-protein signaling complexes could be composed of different combinations of many molecules and can lead to distinct patterns of phosphorylation that help initiate as well as modify downstream signaling cascades such as PI3K/Akt and MAP/Erk pathways [49]. Importantly, sustained activation of both PI3K/Akt and MAP/Erk signaling pathways have been implicated in regulating tumor cell proliferation, invasion, migration and survival; all critical processes required to complete the metastatic cascade.

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Fig. 3.3 Integrins as sensors of the extracellular microenvironment. Integrins are a family of heterodimeric cell surface receptors with diverse activities. A major functional role for integrins during tumor growth and metastasis involves acting as bidirectional sensors of changes within the intra and extracellular environments. Integrin receptors can gather and interpret unique changes within these compartments and differentially distribute this molecular information into distinct biochemical signaling pathways that regulate cellular behavior

As can be appreciated from this brief discussion, metastasis involves a number of potential biomechanical processes that might contribute to the ability of tumor cells to metastasize. These events range from changes in mechanical properties of individual tumor cells to allow alterations in cell shape and deformability to modulation of the physiochemical properties of individual ECM molecules that may impart distinct functions. To illustrate this important concept, we will discuss a few examples of how changes in physiochemical and mechanical properties of widely expressed ECM proteins such as FN, may act as a biomechanical switches to facilitate unique functions. It is known that changes in ionic strength and pH can vary substantially within a tumor microenvironment and these physiochemical parameters can alter the folding properties of ECM molecules such as FN [50, 51]. In this regard, structural alterations of FN can uncover cryptic self-association sites that are crucial for higher-order fibrillar network formation [52]. These mechanical changes in structural integrity can have a pronounced impact on numerous cellular signaling pathways resulting in alterations in adhesion, migration and gene expression that might impact tumor cell metastasis. Changes in the elastic properties of FN for example, may alter cellular accessibility to a variety of cryptic sites within this

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molecule [53]. In fact, it has been suggested that the precise physical distance and orientation between the integrin binding PHSRN synergy site within the 9th type-III FN module and that of the well-known cell binding RGD site within the 10th type-III FN module can alter the ability of specific integrin receptors to bind FN [54]. Experimental modeling suggests that mechanical stretching and unfolding of FN can increase the distance between these receptor binding sites, thereby converting FN from an a5b1- and aIIbb3-binding ligand to one that can be recognized by avb3 [54]. In this regard, it is well known that signaling through integrins such as a5b1 and avb3 can regulate distinct signaling cascades such as MAP/Erk and PI3K/ Akt pathways leading to modulation of protease expression, cellular proliferation, invasion and survival. For example, a5b1-mediated interactions with FN are associated with high levels of activated RhoA following epithelial cell spreading, which lead to phosphorylation and inactivation of the actin-modifying protein cofilin [55]. In addition, focal adhesion structures were shown to be translocated to spike-like protrusions at the cell periphery, which was associated with random migration patterns [55]. In contrast, avb3-mediated adhesion to FN results in little phosphorylation of coflin, extensive reorganization of the actin cytoskeleton resulting in actin polarization and a more directed cellular migration [55]. Given these clearly distinct cellular responses it might not be surprising that mechanical changes in FN structure may impact a cell’s migratory and invasive behavior within the tumor microenvironment. Thus, mechanically induced switching of integrin binding can be associated with initiation of different molecular signaling cascades. Another interesting example shows that proteolytic cleavage may facilitate exposure of enzymatically active FN sub-domains that exhibit independent disulfide isomerase activity as well as unique collagen degrading activity [56, 57]. These intriguing studies demonstrate the possibility that an ECM protein primarily known for its ability to regulate adhesive functions may be converted into a protein with enzymatic activities. Importantly, this biomechanically-induced switching of integrin utilization or ECM function is not restricted solely to FN, as studies have shown that alterations in the triple-helical structure of collagen can expose cryptic RGD sites that can lead to a shift from predominantly b1 integrin-mediated signaling to that of both b1- and avb3-mediated signaling [58]. Biomechanical switching of integrin binding to collagen was shown to protect melanoma and endothelial cells from apoptosis by providing a survival advantage associated with changes in the ratio of Bcl-2 to Bax expression [58, 59]. These examples are just a few of the novel cryptic functions that may be activated following the triggering of biomechanical switches within solid-state ECM molecules.

3.4

Biomechanical ECM Switches in the Interstitial Matrix

The ECM can be thought of as being organized into at least two major compartments, which include a thin, sheet-like arrangement of proteins that comprise the basement membrane and the loosely organized interconnected network of molecules

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that make up the interstitial matrix. Given the tissue-specific variations in the molecular composition of these distinct matrix compartments, it would be well beyond the scope of this review to discuss in detail all of the molecules that comprise the ECM. Therefore, we will limit our discussion to a few well-characterized examples to illustrate the significance of biomechanical switches in regulating tumor growth and metastasis. Rapidly proliferating primary tumor cells can elicit a strong angiogenic response, resulting in tumor-associated blood vessels that are often leaky, which in turn can facilitate the deposition of provisional matrix proteins from the circulation such as vitronectin (VN), fibrinogen (FB) and fibronectin (FN). As with many ECM proteins, the functional impact of these provisional matrix molecules is governed in part by their structural conformation. For example, it has been suggested that the cell-binding RGD site within monomeric VN in its native configuration within the circulation is cryptic and therefore not readily accessible [60]. However, when VN is deposited into the ECM it may undergo multimerization, resulting in activation of a biomechanical switch that allows exposure of its cell binding RGD site [60]. Integrin-mediated interactions with cryptic RGD sites have been suggested to regulate tumor cell adhesion and invasion especially in association with glioblastoma [61]. In addition to mechanical force induced triggering of cryptic ECM switches, proteolytic activation of cryptic ECM switches can also occur. In this regard, a similar paradigm can be observed with other RGD-containing ECM proteins such as Collagen and Tenacin-C, both of which harbor cryptic RGD sites that can be proteolytically exposed [59, 62]. It is important to point out that additional peptide sequences outside of the classical cell binding RGD site may also represent cryptic regulatory elements. To this end, proteolytic exposure of EGF-like repeats within proteins such as Tenacin-C and LN have been shown to bind EGF receptors and thereby stimulating downstream signaling, which has been implicated in regulating the behavior of a number of distinct tumor cell types [63, 64]. Interestingly, subtractive screening of an antibody display library allowed the isolation of a novel antibody that selectively binds to a cryptic epitope within activated VN [65]. This cryptic VN site was preferentially localized within human renal cell, colon and breast carcinomas as compared to their corresponding normal tissue counterparts [65]. While it remains unclear whether this specific VN cryptic site contains an RGD epitope or whether it plays a functional role in regulating tumor progression, the selective distribution of this cryptic site is consistent with the notion that a subset of biomechanical ECM switches may be triggered within the tumor microenvironment. In addition to VN, another important provisional ECM protein often seen in association with malignant tumors is fibrinogen (FB). A number of studies have implicated cellular interactions with FB in the regulation of angiogenesis and tumor metastasis [66]. Fibrinogen has been shown to contain cryptic integrin binding sites that may be exposed by proteolytic mechanisms. An interesting example of a cryptic biomechanical ECM switch within FB involves the proteolytic exposure of a cryptic avb6 binding site. Studies suggest that MMP-9 may cleave FB, triggering

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the exposure of an avb6-binding element that facilitates cellular interactions that promote activation of MAPk/Erk signaling [67]. As we discussed above, one of the most thoroughly studied ECM proteins within the interstitial matrix in terms of its cryptic functions is FN. Fibronectin is known to harbor cryptic self-association sites that, when exposed following mechanical unfolding, promote fibrillogenesis and the formation of a more elaborate interconnected network that can provide mechanical support for tumor cells as well as providing a number of distinct binding sites for additional ECM molecules such as fibrin, tenascin and collagen [68]. Moreover, as detailed above, changes in FN conformation following heparin binding may expose unique cryptic sites that facilitate VEGF binding. The elevated abundance of angiogenic growth factors may provide a biological reservoir that can be utilized to stimulate tumor-associated angiogenesis, which in turn may facilitate tumor cell survival as well as provide readily accessible sites for tumor cells to gain access to the circulation. Beyond providing binding sites for multiple ECM molecules and a variety of growth factors, FN provides critical integrin binding sites that have been shown to regulate diverse molecular and cellular processes. For example, cellular interactions with FN mediated by a5b1 have been shown to promote cell survival by mechanisms involving differential regulation of Bcl-2 as well as differentially regulating the expression of proteolytic enzymes such as MMPs [69]. In addition to regulating the expression of matrix-modifying enzymes, proteolytic cleavage of FN may expose sub-domains that actually exhibit intrinsic protease activity by themselves. Thus, it would be interesting to speculate that localized exposure of these unique matrix-modulating fragments within the tumor microenvironment contributes to tumor cell metastasis. In addition, the a4b1 FN receptor has been shown to play a role in regulating myeloid cell recruitment to metastatic sites [39]. Finally, while studies have demonstrated that cryptic sites within FN promote cell adhesion, other studies have also provided evidence for a cryptic site within the 14th type-III FN module reduces cell adhesion [70]. It is interesting to point out that a reduction in cell adhesion is necessary to facilitate cellular invasion and migration. Taken together, the dynamic mechanical and elastic properties of these multi-domain ECM molecules within the interstitial tumor microenvironment appear well-suited to serve as cryptic biomechanical switches.

3.5

Biomechanical ECM Switches in the Basement Membrane

A second major ECM compartment within the tumor microenvironment that is known to regulate tumor metastasis is the basement membrane. The molecular composition of basement membranes can vary substantially between different organ systems but collectively serves to regulate a variety of tissue-specific cellular processes ranging from tissue compartmentalization to gene expression. To illustrate the functional importance of cryptic biomechanical switches within basement membrane, we will focus primarily on laminin and collagen type-IV, the two major

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structural molecules within this compartment. The functional trimmer of LN is formed from distinct combinations of three chains termed a, b and g. Various combinations of these chains can result in the generation of LN isoforms with tissuespecific distribution patterns [71]. Cells can interact with LN by several cell surface molecules including integrins and non-integrin molecules such as a-dystroglycan, the 37/67Kda laminin receptor and some galactoside-binding lectins [71, 72]. As with many other ECM molecules, LN contains a number of functional domains that may regulate tumor metastasis including growth factor binding sites and integrin interaction elements. An extensive analysis of LN-1 has identified multiple peptides that exhibit differential biological activities in angiogenesis and tumor metastasis. For example, a YIGRS-containing peptide from the LN b1 chain was shown to reduce angiogenesis and tumor metastasis in experimental models [73]. In contrast, a SIKVAV-containing peptide from the a1 chain of LN was shown to increase metastasis [74]. These studies as well as others are consistent with the notion that unique cryptic sites within LN may play functional roles in controlling specific parts of the metastatic cascade. A well-characterized example of this concept can be illustrated by work demonstrating that MMP-mediated cleavage of the g2 chain of LN-5 can expose a cryptic site that promotes tumor cell motility [17]. To identify and characterize potentially functional cryptic ECM biomechanical switches in LN, our laboratory used a subtractive screening approach to examine a peptide phage display library in order to generate synthetic peptides that selectively bound denatured and proteolyzed LN-1 [75]. These studies resulted in the generation of a synthetic peptide termed STQ that bound denatured and proteolyzed LN-1, while exhibiting little if any interactions with intact LN. This cryptic LN site was preferentially exposed within basement membranes associated with malignant melanomas and tumor-blood vessels but failed to show significant exposure within corresponding normal tissues [75]. The STQ-peptide selectively inhibited tumor and endothelial cell adhesion, migration and proliferation on denatured LN, but failed to significantly alter these cellular events on intact LN, suggesting that this novel cryptic element can selectively modulate cellular behavior. The ability of this cryptic LN epitope to regulate these cellular processes may involve modulation of the cyclin-dependent kinase inhibitor P27KIP1 as the STQ-peptide induced elevated levels of P27KIP1 in melanoma cells seeded on denatured LN. Importantly, targeting of this selectively triggered biomechanical switch inhibited both angiogenesis and melanoma tumor metastasis in vivo [75]. Taken together, these findings suggest that the cryptic LN site defined by STQ-peptide may represent a functionally relevant biomechanical ECM switch that is triggered within the tumor microenvironment to promote tumor metastasis. A second major glycoprotein component of the basement membrane is collagen type-IV. Collagen type-IV is a triple-helical molecule composed of different combinations of a-chains with a characteristic tri-peptide repeat, where glycine is often present at every third amino acid position. This tri-peptide motif along with hydroxylation of proline residues have been shown to be important for the triple-helical structure of collagen and the packaging of collagen into more complex fibrillar networks [76, 77]. Similar to many other ECM glycoproteins, collagen type-IV also has discrete functional domains that provide unique binding sites for cell surface

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receptors, matrix altering enzymes and possibly secreted growth factors. Similar to what was accomplished with LN, studies on collagen type-IV have uncovered a wide array of peptide fragments with potent biological activities. The majority of these functional peptides were shown to exist within the non-collagenous (NC) domains and were shown to inhibit angiogenesis and tumor growth by mechanisms involving alterations in integrin signaling [78, 79]. Using a subtractive immunization approach, we developed a panel of antibodies that preferentially bound to denatured or proteolyzed collagen [80]. A monoclonal antibody (Mab HUIV26) generated from this procedure was shown to bind a cryptic epitope restricted to basement membrane collagen type-IV. This novel cryptic epitope may function as a biomechanical ECM switch as exposure of this site can be triggered by thermal denaturation as well as MMP-mediated cleavage [16]. While exposure of the HUIV26 cryptic site could not be readily detected in basement membranes of most normal tissues examined, it was exposed within basement membranes of malignant tumors and angiogenic blood vessels [81, 82]. The functional relevance of the HUIV26 biomechanical switch in tumor progression and angiogenesis was shown as a Mab directed to this site selectively inhibited angiogenesis, tumor growth and metastasis in several animal models [16, 81–84]. Importantly, studies have indicated that cellular interactions with this cryptic site were mediated by integrin avb3 in a non-RGD dependent manner. These studies provide an additional example of a mechanism by which cells may sense and utilize biological information following triggering of a unique basement membrane biomechanical switch. Using a similar subtractive immunization approach, we generated a second antibody termed HU177 that binds denatured and proteolyzed forms of multiple types of collagen including collagen type-IV [80, 85]. Peptide-based mapping analysis revealed that Mab HU177 recognized, in part, a consensus sequence containing the amino acids GPPG and also demonstrated a requirement for proline hydroxylation [85, 86]. As was observed with the HUIV26 cryptic site, little exposure of the HU177 cryptic epitope was detected within the ECM of normal tissues, while the HU177 cryptic site was readily detectable within the ECM of malignant tumors and angiogenic blood vessels [85, 87]. Importantly, antibodies specifically targeting the HU177 cryptic element were shown to inhibit both angiogenesis and tumor growth in vivo [85, 88]. Taken together, these studies suggest that the selective exposure of these functional biomechanical switches within the tumor microenvironment may represent clinically useful therapeutic and imaging targets.

3.6

Biomechanical ECM Switches as Therapeutic and Imaging Targets

Given the multiple cellular functions governed by solid-state ECM molecules, the non-cellular tissue microenvironment might represent a relatively untapped source of therapeutic and imaging targets. However, due to the ubiquitous expression of these molecules a clear strategy is needed to allow selective targeting of functional

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ECM sites associated with pathological conditions to avoid interactions in normal tissues. In this regard, the concept that selective triggering of biomechanical ECM switches may occur within the non-cellular tumor compartment may facilitate the development of novel approaches to selectively exploit the unique mechanical changes within tissues during tumor growth and metastasis. Evidence is beginning to accumulate that conformational alterations of ECM molecules may be useful for selective imaging of pathological processes such as tumor growth. One example involves the alternatively spliced form of FN termed ED-B Fibronectin [89]. Insertion of the ED-B domain into fibronectin was shown to create a conformational change that resulted in exposure of a cryptic epitope [90]. Interestingly, ED-B FN has been shown to be selectively expressed within tumorassociated vasculature, but not within quiescent adult blood vessels [89]. Preclinical studies have demonstrated that targeting this unique form of FN can lead to a preferential accumulation in experimental tumors growing in vivo. Clinical testing using L19, a radiolabeled antibody that specifically interacts with ED-B FN, has been initiated to examine its use in detection of colorectal and lung tumors [91]. With the initiation of these early proof of concept clinical investigations, along with the generation of antibodies and peptides that selectively bind novel biomechanical ECM switches in matrix molecules such as collagen, laminin and vitronectin, the clinical use of these novel reagents in combination with imaging modalities such as MRI, and Ultrasound may significantly enhance the ability to detect tumor growth and metastasis at an early stage. Similarly, the potential of targeting selectively exposed biomechanical ECM switches for the treatment of malignant tumors are beginning to be investigated in the clinical setting. For example, a murine Mab directed to the HU177 cryptic collagen epitope has recently been humanized and optimized (TRC093) for clinical use [92]. In preclinical studies, humanized Mab TRC093 was shown to selectively localize to tumor-associated blood vessels in animal models [88]. Moreover, TRC093 has been shown to inhibit tumor-associated angiogenesis and effectively enhanced the anti-tumor activity of Paclitaxel in vivo [88]. These and other studies have lead to a Phase-I human clinical trial of TRC093. Results of this clinical trial indicated that TRCO93 was well-tolerated with no dose-limiting toxicities observed [92]. Importantly, evidence of clinical activity was detected as stable disease was observed in several subjects and a reduction in liver lesions was noted in a patient with ovarian carcinoma. While all patients eventually progressed, these studies provide some of the first evidence consistent with the notion that selective targeting of cryptic biomechanical ECM switches may represent a useful therapeutic strategy.

3.7

Conclusion

The growing appreciation for the functional importance of the stromal microenvironment in governing malignant tumor development and metastasis has stimulated many investigators to search for innovative new strategies to exploit the increasing

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molecular understanding of the stroma to help control tumor progression. In addition to the progress that has been made in our knowledge of the diverse cell populations that contribute to tumor growth and metastasis, new molecular insight is rapidly emerging into the importance of unique biomechanical changes within individual ECM molecules as they pertain to regulation of the malignant phenotype. While it has been known for some time that the mechanical properties of a given ECM molecule can regulate its function, the concept that selective triggering of specific conformational changes within solid-state ECM molecules during pathological tissue remodeling may serve as biomechanical switches to regulate information flow is rapidly gaining acceptance. In fact, exciting basic research over the last decade has now led to examining the potential of targeting these unique biomechanical switches for both imaging and therapy in human clinical trials. It is likely that the translational applications of this new biological insight will expand into other clinical areas in the coming years. Acknowledgements This work was supported in part from the National Institute of Health Grant CA91645 to PCB, The Maine Cancer Foundation (CPHV and PCB) and the National Institute of Health Center for Research Resources P20-RR-15555 (CPHV and PCB) and NIH Grant HL083151 (CPHV). Jacquelyn Ames is supported by the Integrative Graduate Education and Research Traineeship (NSF-IGERT DGE0221625) from UMaine. We would like to apologize to those investigators whose important work was not cited do to space limitations.

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81. Hangai M, Kitaya N, Xu J et al (2002) Matrix metalloproteinase-9-dependent exposure of a cryptic migratory control site in collagen is required before retinal angiogenesis. Am J Pathol 161(4):1429–1437 82. Odaka C, Tanioka M, Itoh T (2005) Matrix metalloproteinase-9 in macrophages induces thymic neovascularization following thymocyte apoptosis. J Immunol 174(2):846–853 83. Roth JM, Caunt M, Cretu A et al (2006) Inhibition of experimental metastasis by targeting the HUIV26 cryptic epitope in collagen. Am J Pathol 168(5):1576–1586 84. Jo N, Ju M, Nishijima K et al (2006) Inhibitory effect of an antibody to cryptic collagen type IV epitopes on choroidal neovascularization. Mol Vis 12:1243–1249 85. Cretu A, Roth JM, Caunt M et al (2007) Disruption of endothelial cell interactions with the novel HU177 cryptic collagen epitope inhibits angiogenesis. Clin Cancer Res 13(10): 3068–3078 86. Freimark B, Clark D, Pernasetti F et al (2007) Targeting of humanized antibody D93 to sites of angiogenesis and tumor growth by binding to multiple epitopes on denatured collagens. Mol Immunol 44:3741–3750 87. Brooks PC, Roth JM, Lymberis SC et al (2002) Ionizing radiation modulates the exposure of the HUIV26 cryptic epitope within collagen type IV during angiogenesis. Int J Radiat Oncol Biol Phys 54(4):1194–1201 88. Pernasetti F, Nickel J, Clark D et al (2006) Novel anti-denatured collagen humanized antibody D93 inhibits angiogenesis and tumor growth: An extracellular matrix-based therapeutic approach. Int J Oncol 29(6):1371–1379 89. Ebbinghaus C, Sheuermann J, Neri D et al (2004) Diagnostic and therapeutic applications of recombinant antibodies: targeting the extra-domain B of fibronectin, a marker of tumor angiogenesis. Curr Pharm Des 10(13):1537–1549 90. Ventura E, Sassi F, Parodi A et al (2010) Alternative splicing of the angiogenesis associated extra-domain B of fibronectin regulates the accessibility of the B-C loop of the type III repeat 8. PLoS One 5(2):e9145 91. Santimaria M, Moscatelli G, Viale GL et al (2003) Immunoscintigraphic detection of the ED-B domain of fibronectin, a marker of angiogenesis, in patients with cancer. Clin Cancer Res 9(2):571–579 92. Robert F, Gordon MS, Rosen LS et al (2010) Final results from a phase 1 study of TRC093 (humanized anti-cleaved collagen antibody) in patients with solid cancer. ASCO annual meeting abst # 3038

Chapter 4

Moving Aggressively: S100A4 and Tumor Invasion Reniqua P. House, Sarah C. Garrett, and Anne R. Bresnick

Abstract Tumor cell invasion and metastasis are complex multi-step cellular processes that involve the ability of malignant cells to escape the primary tumor, migrate to distant sites within the body and establish secondary tumors. S100A4, a member of the S100 family of Ca2+-binding proteins, is a prognostic marker in a number of human cancers and has a direct role in promoting metastatic disease. S100A4’s metastasis promoting properties are attributed to diverse biological functions including the regulation of apoptosis, cell motility, invasion and angiogenesis. In this chapter we discuss the contribution of tumor and stromal-derived S100A4 in supporting metastatic dissemination and examine the biochemical properties of S100A4 and the S100A4/target protein interactions that may contribute to its function as a metastasis factor.

4.1

S100 Protein Family

S100A4 is a member of the S100 family of Ca2+-binding proteins and is implicated in the modulation of tumor cell motility, invasion, and angiogenesis (Fig. 4.1). The term “S100” arose from the observation that these proteins are soluble in 100% saturated ammonium sulfate at neutral pH [82]. In humans there are 21 S100 family members, the majority of which cluster on the long arm of human chromosome 1, with the remaining members spread throughout the genome on chromosomes 4, 5, 7, 21 and the X chromosome [74, 98]. The S100 proteins are expressed in a cell and tissue specific manner in vertebrates [22]. Due to the large number of family members and the diversity of their biological targets, S100 proteins regulate multiple

R.P. House • S.C. Garrett • A.R. Bresnick (*) Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA e-mail: [email protected]


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Fig. 4.1 Proposed functions of S100A4-mediated metastatic dissemination. S100A4 (red circles) is expressed by diverse cells within the tumor microenvironment, including tumor cells, fibroblasts, macrophages and T cells. Intracellular S100A4 is associated with increased cell migration and invasion through interactions with cytoskeletal components such as myosin-IIA and via unidentified mechanisms. S100A4 is also released by tumor and stromal cells, and elicits a variety of cellular responses within the tumor microenvironment. Through interactions with annexin A2, S100A4 stimulates the conversion of plasminogen to plasmin on the endothelial cell surface to promote angiogenesis. Extracellular S100A4 also activates NF-kB in tumor cells; however, the receptors responsible for S100A4 signaling have not been identified

cellular processes and are involved in a number of human pathologies such as neurodegenerative and inflammatory disorders, fibrosis, cardiomyopathies and cancer [74, 98, 104]. The S100 family members share 25–65% sequence identity, and as expected exhibit overall similar three-dimensional structures. The majority of the S100 proteins exist as symmetric, anti-parallel dimers except for S100G, which is monomeric [2, 74, 98, 104]. For dimeric S100 proteins, each monomer consists of four helices and a small b-strand. Two helices from each subunit (1, 4, 1¢, and 4¢) form a tight X-type four-helix bundle that comprises the dimer interface [23, 95, 119] (Fig. 4.2). In addition, each S100 monomer contains two helix-loop-helix motifs, referred to as EF-hands, which bind calcium. The 12 residue canonical C-terminal EF-hand coordinates calcium using mostly side-chain carbonyl oxygens and exhibits a high affinity for calcium [23, 33, 73, 90, 132]. The N-terminal EF-hand, also known as the

4 Moving Aggressively: S100A4 and Tumor Invasion Fig. 4.2 Ribbon and surface diagrams of apo-S100A4 and Ca2+-S100A4 with one monomer shown in blue and one shown in green. Dark gray spheres denote calcium ions. Residues colored yellow exhibit significant chemical shift perturbations upon binding a myosin-IIA peptide comprised of residues 1908–1923

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“S100-specific” or pseudo EF-hand, differs from the canonical EF-hand found in Ca2+-binding proteins such as calmodulin and troponin C, and in the C-terminus of the S100 proteins [37, 73, 113]. The N-terminal pseudo EF-hand is comprised of 14 instead of 12 residues and coordinates calcium primarily via main-chain backbone carbonyl oxygens [23, 37, 73]. The reduced flexibility of the main-chain atoms may account for the 10 to 250-fold weaker affinity exhibited by the N-terminal EF-hand for calcium as compared to the C-terminal EF-hand [33, 73, 76]. Calcium has a pivotal role in S100 protein activation. Most S100 proteins, including S100A4, undergo a significant conformational rearrangement following Ca2+-binding to the C-terminal EF-hand [74, 98, 104]. In the apo state, helices 3 and 4, which flank the C-terminal EF-hand, have a roughly parallel orientation with respect to one another. Following Ca2+-binding, helix 3 in each S100A4 subunit rotates by 55° with respect to helix 4, exposing a hydrophobic cleft that serves as the binding pocket for target proteins (Fig. 4.2) [73, 119]. Of particular relevance to S100A4, NMR studies examining the binding of a 16 residue peptide from the target myosin-IIA demonstrated that residues in the hinge and helices 3 and 4 exhibited significant chemical shift perturbations following peptide addition [73]. These observations suggest that myosin-IIA binds in the Ca2+-induced hydrophobic cleft, which is consistent with other S100-target complexes [8, 63, 100, 103]. These structural studies highlight the dependency of S100 protein-target interactions on Ca2+activation and demonstrate that S100 proteins function as Ca2+-activated switches.

4.2

S100A4 as a Cancer Prognostic Marker

The first indication that S100A4 could serve as a prognostic marker came from the observations of Lukanidin and colleagues, who isolated S100A4 mRNA from metastatic CSML100 mouse mammary adenocarcinoma cells and noted that S100A4 mRNA was absent in nonmetastatic CSML0 cells [26]. Subsequently, S100A4 expression has been shown to be a predictive indicator of poor patient survival in several studies of breast cancer. In a study of 349 patients with stage I or II invasive breast cancer, the 19-year median survival rate was increased significantly in S100A4 negative patients (>228 months) compared to S100A4 positive patients (47 months) with 80% of the S100A4 negative patients and 11% of the S100A4 positive patients alive at the conclusion of the study [102]. The association between S100A4 expression and patient mortality was confirmed in two additional studies. In a cohort of 92 patients with stage I breast cancer, patients with S100A4 positive tumors had a significantly worse 10 year survival (29%) than those with S100A4 negative tumors (68.9%) [62]. Moreover, a 312 patient study of women with minimally invasive stage I or II breast cancer showed that the 18-year median for survival was >204 months for S100A4 negative patients as compared to 186 months for S100A4 positive patients [21]. Together these studies suggest that in early stage breast cancer S100A4 may be a prognostic indicator for tumor progression and patient mortality.

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In contrast to these findings, two additional studies found no correlation between S100A4 expression and patient outcome. In a study of 66 stage I-IV breast carcinomas, S100A4 expression did not correlate with patient survival; however, there was an inverse correlation between S100A4 expression and the loss of estrogen receptor expression [92]. Similarly in a study examining S100A4 mRNA levels from 295 stage I-III breast cancer patients, S100A4 mRNA expression correlated with the loss of estrogen receptor expression and was associated with basal-type cancers, but was not predictive with respect to patient outcome [79]. The discrepancy between these two studies and studies demonstrating that S100A4 expression can predict patient outcome may result from several factors, including (1) small sample size and a relatively short follow-up period [92]; (2) the evaluation of S100A4 mRNA instead of S100A4 protein levels [79]; and (3) the inclusion of patients with more advanced disease [79, 92]. While there is substantive data supporting S100A4 as a predictive marker for patient outcome, additional studies using larger patient populations are needed to confirm that S100A4 is indeed a prognostic factor for early stage breast cancers. In addition to breast cancer, positive S100A4 expression correlates with (1) blood vessel invasion and poor patient survival in lung adenocarcinoma [77]; (2) lymph node and peritoneal metastases in gastric cancers [128, 140]; (3) recurrence and/or metastatic dissemination of colorectal cancers [39, 60]; (4) poor differentiation of pancreatic ductal adenocarcinomas [101]; (5) metastatic relapse in bladder cancer [1, 19]; (6) poor prognosis in renal cell carcinoma [6]; and (7) lymph node metastases in papillary thyroid carcinomas [80]. The association of S100A4 expression with metastatic disease and poor patient survival in a number of cancers suggests that S100A4 may have widespread use as a marker for disease progression. Current methods rely on tumor size and histological grade to predict potential metastatic dissemination. The identification and development of molecular markers, such as S100A4, which are better predictors of metastatic disease, will be necessary to improve treatment strategies and patient outcomes.

4.3

S100A4 Promotes Tumor Metastasis

Tumor invasion and metastasis are complex multi-step cellular processes that account for the majority of cancer related deaths [109, 111]. Although S100A4 has been considered a marker of metastatic disease, there is significant evidence demonstrating that S100A4 has a causal role in the metastatic process. In an orthotopic model of breast cancer, overexpression of S100A4 in nonmetastatic rat mammary adenocarcinoma cells resulted in an approximately 60% increase in the number of tumor-bearing animals with metastatic lesions in the lungs as compared to untransfected cells or control transfectants, which did not produce metastases [18]. In addition, S100A4 expression in hormone dependent nonmetastatic MCF-7 breast cancer cells promoted hormone-independent tumor invasion of the surrounding muscle and adipose tissue, as well as metastasis to the lungs and lymph nodes [42 ] . Conversely the loss

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of S100A4 expression in highly metastatic cells is associated with a reduction in tumor metastasis. Transfection of metastatic osteosarcoma cells with either a S100A4 specific ribozyme or S100A4 shRNA resulted in a 50% reduction in the number of animals with metastatic lesions, and a 75–85% decrease in the number of metastatic lung nodules, respectively [31, 70]. Similarly, transfection or retroviral infection of Lewis lung carcinoma cells with a S100A4 antisense oligonucleotide caused a significant reduction in cell motility and invasion in vitro and a corresponding 50–90% decrease in the number of metastatic lung lesions in vivo [115]. S100A4 transgenic mice do not develop tumors, indicating that S100A4 itself is not tumorigenic [4, 20], but S100A4 overexpression will facilitate metastasis in an already tumorigenic background. The mammary epithelium of wild-type mice and mice overexpressing S100A4 under the control of the mouse mammary tumor promoter are phenotypically indistinguishable. However, S100A4 overexpression in the mammary epithelium of MMTV-neu mice, which express the HER2/neu oncogene, resulted in a fourfold increase in the number of lung metastases that was associated with a 66-fold increase in the affected areas of the lung as compared to MMTV-neu mice [20]. S100A4 overexpression also enhanced tumor metastasis in GRS/A mice, which normally have a high incidence of tumor formation, but a low rate of metastasis [120]. Crossing GRS/A and MMTV-S100A4 mice produced progeny that exhibited a tenfold increase in the number of animals that developed metastatic disease as compared to non-transgenic littermates [4]. Notably, S100A4 expression in tumor cells had only a modest affect on tumor latency [20]. Conversely, when transgenic mice expressing the PyMT antigen, which normally exhibit a high incidence of metastasis, are crossed with mice carrying null alleles for S100A4, they display a 60% reduction in the number of metastatic lesions [46, 136]. Altogether these animal studies provide strong evidence that in tumor cells, S100A4 functions as a factor that facilitates the progression of metastatic disease.

4.4

S100A4 and the Tumor Microenvironment

The tumor microenvironment consists of extracellular matrix and diverse cell types, including fibroblasts, myofibroblasts, endothelial cells, inflammatory and immune cells, and various bone marrow derived progenitor cells that support the growth, local invasion and metastatic dissemination of tumors [51, 52]. Although S100A4 expression in tumor cells clearly induces a metastatic phenotype, there is accumulating data that S100A4 expression in cells within the tumor microenvironment has an equally important role in promoting tumor progression. S100A4, which is also known as fibroblast-specific protein (FSP1), has been described as being expressed exclusively in fibroblasts [112], and is used frequently as a marker to identify cancer-associated fibroblasts [52, 141] and fibroblasts in tissues undergoing remodeling and EMT [50, 61, 142]. However, there is substantial data demonstrating S100A4 expression in a variety of stromal cell types, including macrophages


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[66, 89], myeloid dendritic cells [12], and lymphocytes [24]. Moreover, immunohistochemistry of human breast cancer biopsies showed that S100A4 is expressed in multiple cell types within the tumor stroma, with high S100A4 expression levels detected in fibroblasts, macrophages, and activated lymphocytes [15]. Studies examining the contribution of stromal S100A4 to tumor development showed that orthotopic injection of highly metastatic, S100A4-expressing mouse mammary adenocarcinoma cells (CSML100) into S100A4−/− mice resulted in reduced tumor uptake and decreased tumor incidences compared to S100A4+/+ mice [45]. Moreover, mice that did develop tumors in the S100A4 null background did not develop metastatic disease, demonstrating that S100A4 expression in host stroma contributes to tumor progression. Interestingly, co-injection of CSML100 cells and immortalized S100A4+/+ mouse embryonic fibroblasts (MEFs) into S100A4−/− mice reduced the number of tumor free animals and restored the ability of the CSML100 cells to metastasize in a low percentage of animals (~12%), whereas co-injection of S100A4−/− MEFs had no effect on tumor development or metastasis [45]. While these studies demonstrate a role for host-derived S100A4 in driving tumor progression, the inability of S100A4+/+ MEFs to rescue the full metastatic potential of CSML100 cells suggests that S100A4 expression in additional stromal cells within the tumor microenvironment contributes to tumor progression. Cancer-associated fibroblasts (CAFs) promote tumor progression through paracrine signaling with both tumor cells and other cells present in the tumor microenvironment. CAFs secrete vascular endothelial growth factor (VEGF), a chemoattractant for endothelial cells that promotes angiogenesis [9, 52], as well as chemotactic factors and matrix metalloproteinases that aid in tumor growth, invasion and metastasis [9, 52]. Although S100A4+/+ MEFs partially restore the metastatic capabilities of tumor cells in S100A4-deficient mice, the mechanisms by which fibroblast-derived S100A4 promotes tumor metastasis are unknown. S100A4 null MEFs exhibit impaired 2D and 3D motility, thus the metastasis-promoting effects of S100A4+/+ MEFS may be a consequence of the motile properties of these cells and their ability to more efficiently invade the extracellular matrix [45]. In addition, breast tumor cells have been reported to stimulate the release of S100A4 from fibroblasts, thus S100A4 may exert its prometastatic effects as an extracellular factor [30, 105]. The loss of S100A4 expression also modulates the recruitment of immune cells to the neoplastic stroma. Using the PyMT breast cancer model, recent studies demonstrated a significant reduction in the recruitment of CD45+ leukocytes and CD3+ T lymphocytes to adenoma/MIN tumors of S100A4−/− mice [46]. Moreover, increased S100A4 expression was observed in the tumor stroma during the transition from adenoma/MIN to early carcinoma, which was coincident with the infiltration of T-lymphocytes. In an orthotopic breast cancer model, co-injection of CSML100 cells and S100A4+/+ MEFs into S100A4−/− mice increased the recruitment of T lymphocytes to the tumor [46]. These observations suggest that stromal S100A4 may facilitate T lymphocyte recruitment to the site of tumor growth, possibly through the release of S100A4 by cancer-associated fibroblasts. T cells also express S100A4 [15] and it is unknown if the loss of S100A4 has a direct and adverse impact on T cell functions that could affect recruitment to mammary tumors.

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In human tumors, the population of infiltrating T-lymphocytes can fluctuate greatly with respect to CD4/CD8 expression and Th1/Th2 regulatory status and significantly impact disease progression and patient survival [11, 56, 71]. Further classification of the infiltrating T lymphocytes in S100A4−/− animals will be important for establishing the mechanism by which stromal S100A4 establishes a tumor microenvironment that favors metastatic progression. CD45 is a marker for multiple cell types in the tumor microenvironment, including tumor-associated macrophages, TIE2-expressing monocytes, B cells and T cells [51]. Therefore it will be of interest to determine if the reduced recruitment of CD45+ leukocytes to mammary tumors in S100A4−/− mice reflects the loss of other bone-marrow derived cells from the tumor microenvironment. In breast cancer, tumor-associated macrophages promote tumor angiogenesis, invasion and metastasis [40, 68, 133]. Based on immunohistochemistry with the macrophage marker F4/80, there was a trend towards reduced numbers of tumor-associated macrophages in S100A4−/− adenoma/MIN tumors [46]. This observation is intriguing as recent studies in a model of liver fibrosis demonstrated that S100A4 is expressed in a specific subpopulation of macrophages that are characterized by increased expression of proinflammatory cytokines, chemokines and osteopontin, which is highly expressed in tumor-associated macrophages [85, 89]. In addition, studies with S100A4−/− bone marrow-derived macrophages showed that S100A4 is required for the regulation of colony stimulating factor-1 signaling, and macrophage recruitment and chemotaxis in vivo [66]. These observations support S100A4 as a regulator of macrophage function and warrant a more thorough evaluation of the role of S100A4 expression in modulating the tumor-promoting functions of macrophages. As there is accumulating evidence that distinct subpopulations of macrophages support tumor-associated angiogenesis, modulate tumor cell migration, invasion and intravasation, and enhance the seeding and establishment of metastatic cells [96, 133, 135], it will be important to examine how S100A4 regulates the biological activities of these specialized subsets of macrophages.

4.5

S100A4-Target Interactions

At present 16 S100A4/target protein interactions have been reported (Table 4.1). The current list of S100A4 binding partners are involved in diverse biological processes and include cytoskeletal proteins, signaling molecules, transcription factors and extracellular proteins and growth factors. Although in vitro data support the interaction of S100A4 with each of these targets, for the majority of these proteins the biological relevance of the interaction with S100A4 has not been examined. A common feature of S100A4 expression in both cancer and non-transformed cells is the observation that S100A4 levels correlate strongly with cell motility. For instance, fibroblasts and epithelial tumor cells that overexpress S100A4 display increased migratory properties [110, 114]. Conversely, genetic deletion of S100A4 reduces the motility of fibroblasts and macrophages [45, 66], and


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Table 4.1 S100A4 targets Target Cellular function Reference Intracellular Myosin-IIA Cytoskeleton [29, 59, 67, 81] Tropomyosin Cytoskeleton [116] F-actin Cytoskeleton [67, 129] liprin b1 Scaffolding/cell adhesion [57] MetAP2 Post-translational processing [27] p53 Transcription [28, 43, 122] p63 Transcription [121] p73 Transcription [121] MDM2 Ubiquitination [123] Smads 2 and 3 Transcription [78] Extracellular CCN3 Matricellular signaling [64] Annexin II Signaling [108] Amphiregulin Growth factor [54] RAGE Cell surface receptor [138] Tag7 Innate immunity [24] MFAP 4 Cell adhesion [130] MetAP2 methionine aminopeptidase 2, MDM2 murine double minute oncogene, CCN3 cysteinerich 61-connective tissue growth factor-nephroblastoma overexpressed gene, RAGE receptor for advanced glycation end products, Tag7 peptidoglycan recognition protein, MFAP4 microfibrilassociated glycoprotein 4

reduction of S100A4 expression in tumor cells correlates with decreased cellular motility [10, 115]. Consistent with a role in regulating motility, immunofluorescence studies showed that S100A4 localizes to the leading edge of polarized breast cancer cells [53, 65], and proteomic studies demonstrated that S100A4 is enriched in the pseudopodia of migrating cells [127]. Moreover, using a fluorescent S100A4 biosensor that reports on the Ca2+-bound, activated form of S100A4 [33], we observed activated S100A4 in the leading edge and dorsal ruffles of breast cancer cells migrating into a wound (Fig. 4.3). Given the connection between S100A4 expression, the enhancement of cell motility and the ability of S100A4 to promote tumor invasion, we will focus our discussion on S100A4 targets involved in modulating cell migration.

4.6 4.6.1

Intracellular Targets Myosin-II

Three myosin-II heavy chain isoforms are expressed in nonmuscle cells, myosinIIA (MYH9), myosin-IIB (MYH10) and myosin-IIC (MYH14). Although the three myosin-II isoforms share 64–80% sequence identity [38], they exhibit distinct

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Fig. 4.3 S100A4 activation in human breast cancer cells crawling into a wound. (a and b) Ratio images of two representative MDA-MB-231 cells injected with a 1:2 ratio of Flu-S100A4:MeroS100A4. Purple to red signifies low to high S100A4 activation and the white dashed line denotes the location of the wound edge. (c–f) Higher magnification image of boxed regions in left panel showing phase contrast (c and d) and ratio images (e and f). Mero-S100A4 activation is observed in cellular regions extending towards the wound and in dorsal ruffles (arrowheads). Scale bar = 10 mm. More information on the S100A4 biosensor can be found in reference [33]

patterns of tissue and cell expression, have different enzymatic activities, interact with different proteins, and have unique functional roles in vivo (reviewed in [124]). More importantly, myosin-II regulates and integrates multiple steps during cell motility, including cell polarization and protrusion, and the assembly and turnover of adhesions [35, 125]. In animal models, myosin-II regulatory pathways are activated in invasive breast cancer cells [126], and myosin-II activity is required for matrix deformation and breast cancer cell motility through a three-dimensional matrix [93, 134]. These observations suggest that myosin-II-based contractility is a critical component of tumor cell invasion.


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In vitro characterization of the S100A4/myosin-II interaction showed that S100A4 binds to both myosin-IIA and myosin-IIB in a Ca2+-dependent manner; however, the affinity of S100A4 for myosin-IIB is at least tenfold weaker than that observed for myosin-IIA (myosin-IIB: Kd = 23.1 ± 2.7 mM; myosin-IIA: Kd = 2.7 ± 0.6 mM) [29, 67]. The S100A4 binding site maps to the C-terminal end of the coiled-coil of the myosin-IIA heavy chain (residues 1908–1923) and S100A4 binding promotes the monomeric, unassembled state of myosin-IIA [58, 67]. Although the S100A4 binding site overlaps a PKC phosphorylation site at Ser1916, S100A4 binding is unaffected by PKC phosphorylation; however, phosphorylation on Ser1943, which is located downstream of the S100A4 binding site, inhibits S100A4 binding [25]. Thus myosin-IIA phosphorylation on Ser1943 provides an additional regulatory mechanism for modulating the S100A4/myosin-IIA interaction. In addition, recent fluorescence cross-correlation spectroscopy studies showed that S100A4 specifically depolymerized myosin-IIA filaments in heteroassemblies comprised of myosin-IIA and myosin-IIB filaments, demonstrating that S100A4 selectively targets myosin-IIA filaments [81]. With respect to cell migration, S100A4 modulates cellular motility by effecting the localization and stability of cellular protrusions. Phenotypic analysis of mammary adenocarcinoma cells undergoing directional migration showed that S100A4 expression resulted in the formation of extensive forward protrusions and a suppression of side protrusions, resulting in enhanced cell polarization during chemotaxis [65]. Moreover, these effects were due to the specific interaction of S100A4 with myosin-IIA. Microinjection of an antibody that recognizes the S100A4 binding site on myosin-IIA and promotes myosin-IIA disassembly, into cells that do not express S100A4 produced a similar increase in forward protrusions [65]. Consistent with these observations, CSF-1 stimulated S100A4−/− macrophages form numerous, highly unstable forward and side protrusions and, as a consequence, turn more frequently and migrate with less persistence than wild-type macrophages [66]. In addition, the loss of S100A4 increased myosin-IIA filament assembly in vivo, which is in agreement with in vitro studies demonstrating that S100A4 promotes myosin-IIA filament depolymerization. Together these data support a role for S100A4 in maintaining cell polarization during chemotactic migration and suggest that S100A4 may modulate cell invasion via the regulation of myosin-IIA assembly.

4.6.2

Cytoskeletal Elements

The actin-based cytoskeleton is involved in the maintenance of cell morphology and is essential for cell migration and invasion [94, 137]. S100A4 is reported to exhibit Ca2+-dependent binding to F-actin [67, 129] and to the nonmuscle tropomyosin isoform 2 (TM2) [116]. Multiple tropomyosin splice variants are expressed in nonmuscle cells, which exhibit different affinities for F-actin, modulate myosin motor activity to varying extents and regulate the activity of actin filament severing proteins such as gelsolin and ADF/cofilin [47, 88]. S100A4-mediated regulation of

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tropomyosin function would be expected to significantly affect actin dynamics as well as cell polarization and migration; however, future studies will be required to evaluate how S100A4 binding impacts TM2 function and the subsequent consequences on cell motility and invasion.

4.6.3

p53

Altered p53 expression either through mutation or down regulation is thought to occur in approximately one-half of human cancers [48, 49, 62]. Wild-type p53 is activated in response to cellular stresses such as hypoxia, temperature shock and DNA damage, and regulates the expression of genes involved in cell cycle arrest and apoptosis to maintain the integrity of the genome. The initial report of the S100A4/ p53 interaction mapped the S100A4 binding site to the p53 C-terminal negative regulatory domain (residues 364–393) [43]; however, recent reports indicate that S100A4 binds weakly to the negative regulatory domain (Kd = 700 ± 50 mM), and instead binds a peptide derived from the p53 tetramerization domain with moderate affinity (Kd = 17 ± 1 mM) [28]. Notably, subsequent analytical centrifugation and size exclusion chromatography studies showed that S100A4 did not form a stable complex with either the C-terminal 100 residues of p53, which encompasses the tetramerization domain, or with full-length p53 [122]. These observations bring into question the relevance of the putative S100A4/p53 interaction. The cellular effects of S100A4 on p53 function have been equally complex. An evaluation of p53-regulated pro-apoptotic and pro-survival genes in S100A4 transfected cells demonstrated that expression was up-regulated for some genes (e.g., Bax); down-regulated for other genes (e.g. p21/WAF, thrombospondin-1) and for some genes such as MDM-2 expression was down-regulated initially and then upregulated [43]. These studies are difficult to interpret as there is no clear correlation in the expression patterns of the pro-survival or pro-apoptotic genes. Moreover, the density of the cell cultures significantly affected the gene expression profiles, suggesting that S100A4-mediated regulation of p53 transcriptional activity may depend on environmental signals. Lastly, S100A4 was reported to induce apoptosis in tumor cells expressing wild-type p53, whereas apoptosis was not triggered in cells expressing mutated p53 [43]. These observations led to the proposal that the induction of apoptosis in tumor cells expressing wild-type p53 might be a mechanism in which S100A4 expression selects for tumor cells expressing mutated p53, thus allowing for the survival of tumor cells with greater invasive potential. However, studies in colon carcinoma and lung cancer lines demonstrated high S100A4 expression in cells expressing wild-type p53 and the loss of S100A4 expression in cells deficient for p53 or expressing mutated p53 [17, 87], thus there is no clear consensus on p53 mutational status and S100A4 expression in cancer cells. Although p53 is considered primarily a regulator of cell proliferation and apoptosis, there is growing evidence supporting the involvement of p53 in the suppression of cell motility and invasion. The loss of wild-type p53 or expression


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of mutant p53 enhances cell migration and invasion [83]. Normally p53 levels are maintained by the ubiquitin E3 ligase MDM2 [14]. Recent studies demonstrated that S100A4 binds with relatively high affinity to the N-terminal domain of MDM2 (Kd = 2.5 ± 0.6 mM) and inhibits MDM2-mediated ubquitination of p53 [123], which could provide a mechanism for stabilizing wild-type or mutant p53 proteins. Interestingly, the expression of mutant p53 protein in mouse embryonic fibroblasts, melanoma and pancreatic cancer cells enhanced RhoA-ROCK signaling and increased invasion into a 3D matrix [32, 118]. Thus S100A4 could indirectly modulate cell migration by inhibiting MDM2 function and preventing down-regulation of p53.

4.7

Extracellular S100A4

There is accumulating evidence that S100A4 is released by multiple cell types, including smooth muscle cells, fibroblasts, macrophages, tumor cells and chondrocytes [15, 30, 91, 105, 130, 139]. In addition, extracellular S100A4 is detected in the tumor interstitial fluid collected from pieces of freshly dissected invasive breast carcinomas [15] and in the synovial fluid collected from individuals with rheumatoid arthritis and osteoarthritis [55]. Notably, S100A4 does not contain any of the known signal peptide sequences required for export via the endoplasmic reticulum/ Golgi-dependent secretory pathway and therefore is thought to be released via an unconventional secretory process. Indeed, recent studies suggested that S100A4 release from fibroblasts occurs through a mechanism that involves the shedding of plasma membrane microvesicles [30]. The application of exogenous recombinant S100A4 has been reported to elicit a variety of cellular responses in multiple cell types. In neuronal cells, exogenous application of S100A4 promoted neurite outgrowth [84]. Extracellular S100A4 also stimulated angiogenesis in a corneal vascularization model and enhanced endothelial cell invasion into a 3D matrix [3, 106]. Further characterization showed that extracellular S100A4 stimulated the production of MMP-13 in endothelial cells, suggesting that S100A4 contributes to the angiogenic process by facilitating the degradation of the extracellular matrix [106]. Recent studies have indicated that exogenous S100A4 mediates mammary branching morphogenesis via the induction of MMP-3 expression [5], suggesting a role for extracellular S100A4 in normal mammary development. Despite these varied cellular responses, the receptors responsible for S100A4 binding and their associated signal transduction pathways remain largely unknown; although, in tumor cells extracellular S100A4 has been shown to activate the transcription factor NF-kB [13, 44, 106], which is a major contributor to the development and progression of cancer. A potential issue with all of these studies on the effects of extracellular S100A4 is the use of recombinant N-terminal hexahistidine-tagged S100A4, which forms dimers (2 S100A4 monomers), tetramers and large oligomers that range in size from 143 to 200 kDa (approximately 10–15 S100A4 monomers) [84]. Notably,

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these large S100A4 multimers are not observed with recombinant untagged S100A4 [36, 73]; however, many of the cellular responses described above are associated specifically with the oligomeric forms of S100A4 rather than S100A4 dimers or tetramers [3, 84, 106]. Size exclusion chromatography of human plasma and synovial fluid demonstrated the presence of dimeric S100A4 and also identified S100A4 in fractions corresponding to high molecular weight protein species [3, 55]. Although these high molecular weight species were interpreted to be S100A4 oligomers, S100A4 elution profiles were evaluated with S100A4-specific antibodies without consideration or assessment of additional proteins that may co-elute with S100A4. Thus these immunological detection approaches cannot distinguish between S100A4 oligomers or complexes comprised of S100A4 bound to other unidentified proteins. In addition, recent studies with S100B showed that imidazole, which is used in the purification of hexahistidine-tagged proteins, acts as a trigger to induce the formation of S100B tetramers and higher order oligomers that are stable following imidazole removal [117]. Despite these concerns, it is highly noteworthy that a few studies have shown that the application of untagged recombinant S100A4 can elicit cellular responses such as the stimulation of glioma cell motility [7], endothelial cell capillary tube formation [108] and activation of NF-kB [138]. As it is unknown if imidazole specifically induces S100A4 oligomerization or if the hexahistidine tag promotes S100A4 aggregation, the biological activities observed following the application of extracellular hexahistidine-tagged S100A4 need to be confirmed with untagged S100A4 or with S100A4 purified from conditioned medium derived from appropriate stromal or tumor cells.

4.7.1

Annexin A2

Annexins are cytoplasmic proteins that bind negatively charged phospholipids in a Ca2+-dependent manner; however, they are also found on the surface of activated endothelial cells and some tumor cells. In conjunction with tissue plasminogen activator (tPA), extracellular annexin A2 facilitates the conversion of plasminogen to plasmin, a serine protease that mediates tissue fibrinolysis [34]. S100A4 binds to the N-terminus of annexin A2 in a Ca2+-independent manner and increases the catalytic efficiency of tPA-mediated conversion of plasminogen to plasmin both in vitro and in cell-based assays with human cerebromicrovascular cells [108]. Plasmin mediates the degradation of the extracellular matrix and also activates latent matrix metalloproteinases and proangiogenic factors to promote angiogenesis. The release of S100A4 by tumor or stromal cells and subsequent binding to annexin A2 on the endothelial cell surface could allow for a local increase in tPA-mediated production of plasmin, thus providing a mechanism for facilitating angiogenesis. Consistent with a role for S100A4 in tumor angiogenesis, the mammary tumors from GRS/A mice that overexpress S100A4 in the mammary epithelium exhibited increased blood vessel densities compared to non-transgenic mice [3], whereas early and late carcinomas from S100A4−/− PyMT mice displayed reduced vascular densities [46].


4.8

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S100A4-Targeted Therapeutics

At present there is substantial data supporting the involvement of S100A4 in several aspects of tumor progression, including cell migration, invasion and angiogenesis. Moreover, the universality of S100A4 expression suggests that S100A4 contributes to metastasis and disease progression in a variety of cancers and highlights S100A4 as a potential target for therapeutic intervention. Recent efforts have identified inhibitors for several S100 family members, demonstrating that these small Ca2+binding proteins can be targeted with small molecules. A combination of computeraided drug design and high throughput fluorescence polarization assays identified small molecules that disrupt the interaction of S100B and p53 [75, 131], and structurebased virtual screening allowed for the discovery of compounds that inhibit the binding of S100A10 to annexin A2 [99]. For S100A4, a fluorescent biosensor (Mero-S100A4) that reports on the conformational rearrangements associated with Ca2+-binding and protein activation, was used to identify several phenothiazines that block S100A4-mediated depolymerization of myosin-IIA filaments [33]. Moreover, phenothiazine-mediation inhibition of S100A4 occurs by a novel mechanism involving the sequestration of S100A4 into small molecule-induced oligomers [72] (Fig. 4.4). The use of S100A4 as a therapeutic target has also attracted the attention of the pharmaceutical industry. SP-MET-X1, developed by Supratek Pharma, is a modified version of the anti-allergy drug Amlexanox, which has been shown to bind several S100 proteins [69, 86, 97]. Since Amlexanox has an excellent safety record and is used widely for the treatment of aphthous ulcers and bronchial asthma, it is anticipated that long-term treatment may be used to prevent metastatic disease. Although the S100 proteins exhibit significant sequence identity, there is some indication that selectivity can be achieved with respect to S100 inhibitors. For example, the S100B inhibitor pentamidine, which disrupts the binding of S100B to p53 [16, 75], has no effect on the S100A4/myosin-IIA interaction (A.R. Bresnick, unpublished observations). Given the contribution of S100 proteins to a multitude of biological processes, the issues and challenges of specificity and selectivity will continue to be an active area of investigation as new S100 inhibitors are discovered.

4.9

Conclusions

S100A4 is a proven mediator of metastatic dissemination; however, the molecular mechanisms by which S100A4 promotes tumor invasion are not understood. Deciphering the contribution of S100A4 to the metastatic process is complicated by the observations that tumor and stromal-derived, as well as intracellular and extracellular S100A4 all contribute to metastatic progression. Although a number of intracellular, cell surface and extracellular S100A4 protein targets have been described, the details of how S100A4 regulates the functions of these protein targets

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Fig. 4.4 Structures of S100 protein/inhibitor complexes. (a) Ribbon diagram of the Ca2+–S100B/ pentamidine complex. The two S100B monomers are shown in blue and green. Two pentamidine molecules (pink) are bound per S100B monomer. Calcium ions are shown as gray spheres. (b) Ribbon diagram of the Ca2+-S100A4/trifluoperazine oligomer. Trifluoperazine (pink) binding induces the assembly of five Ca2+-S100A4/trifluoperazine dimers into a pentameric ring. Calcium ions are shown as gray spheres

and how these interactions synergize and influence tumor and stromal cell activities has not been examined. Biochemical and biological analysis of the mechanisms through which S100A4 modulates its protein targets will be essential for understanding how S100A4 promotes disease progression not only in cancer, but in other


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pathological conditions as well [41, 107]. Moreover, this mechanistic information can be applied to the development of S100A4-targeted therapies. Most cancer therapeutics block tumor cell proliferation; however, the successful treatment of cancer by surgery and chemotherapy is limited by metastatic disease. As a consequence, there is a recognized need for cancer therapeutics that directly target metastatic disease by inhibiting the invasive capabilities of tumor cells. In particular, the signaling pathways and proteins that regulate cell motility and invasion, such as S100A4, are attractive candidates for drug development.

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Chapter 5

Regulation of TGF-b Signaling and Metastatic Progression by Tumor Microenvironments Michael K. Wendt and William P. Schiemann

Abstract The TGF-b signaling system comprises a complex and dynamic cascade of molecular interactions that invoke a variety of intracellular and extracellular reactions that coalesce to maintain tissue homeostasis. A rapidly accumulating body of scientific literature clearly demonstrates a conversion in TGF-b function from that of a powerful tumor suppressor in normal epithelium and early-stage carcinomas to that of a prometastatic molecule in their late-stage counterparts. Collectively, this malicious switch in TGF-b behavior is termed the “TGF-b Paradox.” Historically, cell autonomous changes that transpire during tumor development and progression have been studied extensively as a means to decipher the “TGF-b Paradox.” Although highly informative and intriguing, these findings have yet to unravel the molecular underpinnings of the “TGF-b Paradox,” thereby suggesting involvement of additional signaling components and players that originate beyond the confines of developing carcinomas. Indeed, recent studies have been directed at interrogating the microenvironments of developing carcinomas and how changes within this unique cellular niche manifest the “TGF-b Paradox.” For instance, tumor microenvironments house an array of essential cellular, structural, and humoral factors that include stromal cells and altered elastic moduli, integrins and their engagement of matrix proteins, hypoxic zones, and a host of cytokines, growth factors, and chemokines that collectively influence the response of carcinoma cells to TGF-b. Here we review recent findings demonstrating the importance of the tumor microenvironment to regulate TGF-b signaling and its stimulation of metastatic progression. In addition, we also highlight recent in vitro and in vivo scientific advances capable of recapitulating various aspects of the metastatic process and its regulation by TGF-b.

M.K. Wendt • W.P. Schiemann (*) Division of General Medical Sciences-Oncology, Case Comprehensive Cancer Center, Case Western Reserve University, Wolstein Research Building, 2103 Cornell Road, Cleveland, OH 44106, USA e-mail: [email protected]


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Indeed, incorporating and extending these novel systems to analyses of the “TGF-b Paradox” may offer new inroads in resolving this enigma and improving the overall survival of cancer patients.

Abbreviations 2D 3D CSF-1 Dab2 ECM EGF EMT ERK FAK Hgs IHC JNK LOX MAPK MEC MMP PTK RBM SARA TAK-1 TGF-b TbR-I TbR-II TbR-III VEGF

5.1

2-Dimensional 3-Dimensional Colony stimulating factor-1 Disabled-2 Extracellular matrix Epidermal growth factor Epithelial-mesenchymal transition Extracellular signal-regulated kinase Focal adhesion kinase Hepatocyte growth factor-regulated tyrosine kinase substrate Immunohistochemistry c-Jun N-terminal kinase Lysyl oxidase Mitogen-activated protein kinase Mammary epithelial cell Matrix metalloproteinase Protein tyrosine kinase Reconstituted basement membrane Smad anchor for receptor activation TGF-b-activated kinase 1 Transforming growth factor-b TGF-b type I receptor TGF-b type II receptor TGF-b type III receptor Vascular endothelial growth factor

Introduction

Transforming growth factor-b (TGF-b) is a ubiquitously expressed cytokine that regulates an assortment of biological activities in essentially all cell types and tissues. Besides its role in regulating cell development, differentiation, and survival, TGF-b also inhibits the proliferation of epithelial, endothelial, and hematopoietic cell lineages [1–4]. Interestingly, resistance to TGF-b-mediated cytostasis is a hallmark of neoplastic transformation, which ultimately transform the signals produced by this cytokine into oncogenic activities, particularly enhanced cancer cell invasion

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Regulation of TGF-β Signaling and Metastatic Progression…

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and metastasis [2, 5]. This conversion in TGF-b function is known as the “TGF-b Paradox” [5–7], which underlies the lethality of TGF-b in developing carcinomas, including their acquisition of EMT, metastatic, and chemoresistant phenotypes [5, 8, 9]. The molecular mechanisms whereby TGF-b mediates tumor suppression in normal and neoplastic cells remain to be fully elucidated. Likewise, how TGF-b promotes tumor progression in late-stage carcinomas is even more mysterious. Scientific investigations into TGF-b action have historically applied “cell-centric” approaches to interrogate the “TGF-b Paradox.” Indeed, these analyses have relied heavily on the differential expression of gene transcripts, proteins, and microRNAs between normal and malignant cells as a potential means to explain the dichotomous of TGF-b in responsive cells. Although these studies have yielded enormous volumes of data related to potential alterations in the coupling of TGF-b to its downstream effectors [10–12], they have failed to accurately recapitulate the “TGF-b Paradox” and unlock its ability to promote the development and progression of human tumors. In light of these limitations, recent studies are now actively investigating the impact that dynamic microenvironment alterations play in dictating the behaviors of TGF-b during metastatic progression. Here we review recent findings that elucidate novel mechanisms whereby tumor microenvironments manifest the “TGF-b Paradox.” Likewise, we also expound the virtues of novel 3-dimensional (3D)-organotypic culture techniques to recapitulate the changing microenvironments encountered by metastatic cancer cells as they disseminate throughout the body to distant locales.

5.2

The TGF-b Signaling System

Mammals express three genetically distinct TGF-b ligands (e.g., TGF-bs 1–3) whose mature and biologically active forms are ~97% identical and exhibit virtually indistinguishable actions in vitro [1, 13]. Interestingly, individual TGF-b ligands are expressed in a spatiotemporal manner during embryogenesis and tissue morphogenesis, which contributes to the array of diverse and nonredundant phenotypes displayed by mice lacking distinct TGF-b isoforms [14]. Transmembrane signaling by TGF-b is propagated by its binding to three high-affinity receptors, TGF-b type I (TbR-I), type II (TbR-II), and type III (TbR-III or betaglycan). When expressed, TbR-III is the most abundant TGF-b receptor present on the cell surface where it was originally believed to function solely as an accessory receptor that binds and modulates TGF-b function in responsive cells. However, recent findings implicate TbR-III as an essential mediator of the tumor suppressing activities of TGF-b. Indeed, loss of TbR-III expression associates with disease progression and poor overall survival in patients with cancers of the breast, ovary, prostate, lung, pancreas, kidney, and endometrium [15], suggesting that measures capable of elevating TbR-III expression or activity may circumvent the “TGF-b Paradox” and its initiation of oncogenic TGF-b signaling. Although TbR-III lacks intrinsic enzymatic activity, TbR-I and TbR-II both possess Ser/Thr protein kinases in their cytoplasmic

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domains that serve to initiate downstream signaling [2, 3, 16–18]. In fact, the binding of TGF-b to TbR-II enables this polypeptide to transphosphorylate and activate TbR-I, which subsequently binds, phosphorylates, and stimulates the latent transcription factors, Smad2 and Smad3 [2, 3, 19]. Once phosphorylated, Smads 2 and 3 rapidly form higher order complexes with the common Smad, Smad4, with the resulting heterotrimeric complexes that rapidly translocate and accumulate in the nucleus where they cooperate with an ever expanding list of transcriptional activators or repressors to govern gene expression in a gene- and cell-specific manner [2, 3, 16–18]. Changes in cell behavior regulated by the activation of Smad2/3 is referred to as “canonical TGF-b signaling” and is modulated in all subcellular compartments by numerous effector molecules. For example, the specific recruitment and phosphorylation of Smad2/3 by TbR-I is regulated by several adaptor proteins, including SARA (Smad Anchor for Receptor Activation; [20]), Hgs (Hepatocyte Growth factor-regulated tyrosine kinase Substrate; [21]), and Dab2 (Disabled-2; [22]). Incorporation of the inhibitory Smad, Smad7, into active TGF-b receptor complexes displaces Smad2/3 and prevents their phosphorylation and activation by TbR-I [23–25]. Elevated Smad7 expression also inhibits TGF-b signaling through its ability to recruit Smurf E3 ubiquitin ligases, which promote the ubiquitination and degradation of TbR-I [26, 27]. Smad2/3 signaling is also governed in the nucleus by their association with a variety of interacting proteins, including ATF-3, Sp-1, AP-1, Jun/Fos, Stats, and members of the Forkhead family of transcription factors [2, 3, 16–18]. Finally, Smad2/3 signaling is also subject to fine-tuning following their phosphorylation by cellular protein kinases, as well as to inactivation by their dephosphorylation and ubiquitination by phosphatases and E3 ubiquitin ligases, respectively [28]. Figure 5.1 depicts the TGF-b signaling system and the potential role of Smads 2 and 3 in regulating metastatic progression driven by TGF-b. Additional details linking Smad2/3 to tumor metastasis are discussed in the following sections. TGF-b also couples to a variety of noncanonical signaling systems (i.e., Smad2/3-independent), including ERK1/2, p38 MAPK, JNK, AKT, the small GTP-binding proteins Rho, Rac, and Cdc42 (see [5, 9, 17, 29]). The specific sequence of events and molecular mechanisms that couple TGF-b receptors to the activation of these pathways remain to be fully delineated. However, we recently defined a novel signaling axis comprised of avb3 integrin:Src:FAK:p130Cas:TbR-II:Grb2 that is critical for TGF-b stimulation of MAP kinases, EMT, and pulmonary metastasis of breast cancer cells [30–32]. Additional evidence suggests that the ability of TGF-b to activate MAPKs can also emanate from TbR-I and its physical interaction with the E3 ubiquitin ligase, TRAF6, which ubiquitinates itself and the MAPK kinase kinase TAK-1 (TGF-b-activated kinase 1) to facilitate JNK and p38 MAPK activation [33, 34]. TGF-b displays a dichotomous connection with NF-kB, whose transcriptional activity is inhibited by TGF-b in normal epithelial cells [35], but is robustly stimulated by this cytokine in their transformed counterparts [36–38]. The inverted coupling of TGF-b to NF-kB in normal and malignant cells may reflect a loss of TbR-III expression, which suppresses NF-kB activity through the formation of TbR-III:b-arrestin-2:NF-kB complexes [39]. Likewise, the coupling

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Fig. 5.1 The TGF-b signaling system during metastatic progression. TGF-b initiates transmembrane signaling by binding to its three cell surface receptors, TbR-I, TbR-II, and TbR-III. Once activated, TbR-I phosphorylates Smads 2 and 3, which form hetrotrimeric complexes with Smad4 that translocate to the nucleus to regulate gene expression. The transduction of messages via Smad2/3 constitutes the canonical arm of the TGF-b signaling system. Activated TGF-b receptors also stimulate a variety of non-Smad2/3-based messages that constitute the noncanonical arm of the TGF-b signaling system. Also shown are the influences and outcomes of TGF-b signaling within the primary tumor versus those at metastatic sites

of TGF-b to epithelial-mesenchymal transition (EMT; see below) is critically reliant on NF-kB activity [38]. We extended these findings to show that mammary tumorigenesis and EMT were both sufficient induce the formation of TbRI:xIAP:TAB1:TAK1:IKKb complexes operant in activating NF-kB [40–42], as well as its initiation of an autocrine Cox-2:PGE2:EP2 signaling axis that drives breast cancer metastasis [43]. Interestingly, IKKa also impacts canonical TGF-b signaling by interacting physically with Smad3 to enhance its binding at EMT-regulated

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promoters [44]. Finally, the ability of TGF-b to regulate cellular behaviors also transpires through its stimulation of a number of nonreceptor protein tyrosine kinases (PTKs), including focal adhesion kinase (FAK; [45–49]), c-Src [30–32, 50], and c-Abl [51–54]. Collectively, the preferential coupling of TGF-b to its noncanonical effectors appears inappropriately amplified in metastatic cancer cells, thereby generating a signaling imbalance that overrides and/or dampens the tumor suppressing messages transduced by Smad2/3 in human tumors ([46]; Fig. 5.1). Unfortunately, precisely how noncanonical effectors are activated by TGF-b in normal and malignant cells remains to be determined definitively, as does the manner in which these pathways become dysregulated and magnified during the acquisition of metastatic phenotypes by cancer cells. These relationships and their regulation by tumor microenvironments during metastatic progression driven by TGF-b are discussed below.

5.3

EMT

The impact of TGF-b on metastatic progression is strongly linked to EMT, which reflects the ability of immotile, polarized epithelial cells to acquire highly motile, apolar fibroblastoid-like phenotypes [8, 9, 55–57]. More specifically, epithelial cells undergoing EMT exhibit several distinct features, including (i) the loss of cell polarity due to downregulated expression of epithelial cell markers (e.g., E-cadherin, zona occluden-1, and b4 integrin); (ii) cytoskeletal architecture reorganization and intracellular organelle redistribution; (iii) upregulated expression of fibroblast markers (e.g., vimentin, N-cadherin, a-smooth muscle actin); and (iv) elevated invasion factors (e.g., MMP-9, fibronectin) [8, 9, 55–57]. In general, these steps underlie the pathophysiological reactions associated with EMT, which were recently categorized into three distinct subtypes: (i) Type 1 EMT, which reflects the epithelial plasticity associated with embryogenesis and tissue morphogenesis; (ii) Type 2 EMT, which reflects the epithelial plasticity of tissue regeneration and fibrotic reactions; and (iii) Type 3 EMT, which reflects the plasticity of carcinoma cells during metastatic progression [58]. Indeed, the initiation of Type 3 EMT confers carcinoma cells a selective invasive advantage to exit both the primary tumor [45] and the circulation at sites of dissemination [47, 59]. At first blush, this classification scheme acknowledges that the extent to which an EMT reaction transpires and/or resolves likely reflects the overall health and well-being of the epithelium and its immediate microenvironment. Unfortunately, the vast majority of EMT studies employ “cellcentric” approaches to assess the functional consequences of EMT in normal and malignant cells, and as such, the contributions of the microenvironment to regulating epithelial plasticity remains a critical and underexplored question. Despite these limitations and knowledge gaps, recent inroads into how microenvironments influence EMT have been gleaned through the use of novel 3D-organotypic culture systems capable of modeling distinct steps of the metastatic cascade. The impact of these cultures systems on the morphologies and phenotypes associated with Type 3

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EMT are discussed below. Readers desiring in-depth summaries pertaining to the molecular mechanisms whereby TGF-b promotes EMT are directed to several recent reviews [8, 9].

5.4

The “TGF-b Paradox” in Animal Models

TGF-b was originally defined and purified 30 years ago by its ability to promote the morphological transformation and anchorage-independent growth of normal rat kidney fibroblasts (NRK-49 cells; [60, 61]). However, ensuing examinations of TGF-b action in epithelial cells quickly established this cytokine as a potent inhibitor of cell cycle progression [62], thus providing the initial framework for the “TGF-b Paradox” in traditional culture systems. Extending these analyses to animal studies quickly confirmed the dichotomous nature of TGF-b in regulating tumorigenesis. Indeed, implantation of slow-release TGF-b pellets into the mammary gland significantly inhibited its growth and morphogenesis in a reversible manner [63]. In contrast, administering neutralizing TGF-b antibodies suppressed breast cancer development in mice in part by enhancing splenic natural killer (NK) cell activity [64]. Likewise, transgenic expression of dominant-negative TbR-II enhanced mammary tumorigenesis in carcinogen-treated mice [65], while transgenic expression [66] or systemic administration [67] of a soluble Fc:TbR-II fusion protein both inhibited metastatic progression in mice. In addition, canonical TGF-b signaling was shown to inhibit the tumorigenicity of normal, premalignant, and malignant breast cancer cells, while stimulating that of highly invasive and metastatic breast cancer cells [68–70]. These findings coalesce to support a model wherein TGF-b drives the metastatic progression of late-stage carcinomas, which can upregulate their secretion of TGF-b by as much as 40-fold as compared to their normal counterparts [71]. Accordingly, conditional expression of active TGF-b1 in established mammary tumors significantly enhanced their metastatic progression without affecting the proliferation or growth of the corresponding primary tumors [72]. Similarly, targeted expression of active TGF-b1 in mouse keratinocytes promoted their proliferation in response to carcinogens, as well as induced their progression to aggressive spindle cell carcinomas [73, 74]. Additional insights into the importance of TGF-b signaling during normal development were gleaned from genetically engineering mice to lack expression of individual TGF-b isoforms and their receptors. For example, homozygous deletion of TGF-b1 results in ~50% of the developing pups to die in utero at embryonic day E10.5 [75, 76], while those that survive to term typically succumb to massive inflammatory reactions that result in multifocal infiltration of lymphocytes and macrophages into the heart, lungs, and salivary glands [77]. Homozygous deletion of TGF-b2 elicited perinatal lethality due to multiple developmental defects indicative of aberrant Type I EMT reactions during organogenesis and tissue morphogenesis [78], while disruption of the TGF-b3 locus also produces perinatal lethality resulting from defective EMT that occurred during lung and palate development [79].

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Mice lacking TbR-II exhibit yolk sac hematopoiesis and vasculogenesis defects that phenocopy those observed in TGF-b1-deficient mice [80], while mice deficient in TbR-I expression die during midgestation due to placental and yolk sac vascular defects [81]. Collectively, these and numerous additional studies have helped to define the essential role of TGF-b signaling regulating organ development and immune surveillance and privilege [82–85]. Recent technological advancements have enabled the generation of mice that harbor alleles of the TGF-b signaling system that are flanked by LoxP sites (Floxed) as a means to circumvent the embryonic lethality associated with traditional gene targeting approaches. Indeed, conditional deletion of TbR-II in has provided new understanding of its role in regulating cardiac, skull, and palate development [86–88], and in mediating mammary gland involution and wound healing [89, 90]. It is important to note that the biology of TGF-b can largely be divided into two broad categories: regulation of cell cycling versus regulation of cell microenvironments (i.e., extracellular matrix (ECM) remodeling, angiogenesis, fibroblast activation, and immune cell infiltration). Thus, besides its ability to induce cytostasis in epithelial cells, TGF-b also governs the behaviors of adjacent fibroblasts and their synthesis and secretion of paracrine factors and ECM molecules that collectively suppress carcinoma development. Indeed, rendering fibroblasts deficient in TbR-II expression led to the formation of prostate intraepithelial neoplasia and invasive carcinoma of the forestomach [91]. Likewise, conditional deletion of TbR-II in mammary gland fibroblasts expanded their abundance and proliferative potential in a manner correlating with abnormal ductal development [92]. Moreover, these same TbR-II-deficient mammary fibroblasts greatly exaggerated the growth and invasion of breast cells simultaneously engrafted under the renal capsule in part through their upregulated expression of TGF-a, MSP (macrophage-stimulating protein), and HGF (hepatocyte growth factor) [91–94]. Even more remarkably, similar deletion of TbR-II in mammary carcinoma cells promoted the inappropriate activation of two distinct paracrine signaling axes – i.e., SDF-1:CXCR4 and CXCL5:CXCR2 – whose activation led to the recruitment of immature GR1+CD11b+ myeloid cells that drive breast cancer metastasis by inhibiting host tumor immunosurveillance, and by inducing MMP expression [95]. Interestingly, targeted deletion of Smad4 in T cells also elicited carcinoma formation within the gastrointestinal track (e.g., colon, rectum, intestine, and stomach) due to aberrant stromal expansion and signaling [96]. Finally, similar conditional deletion of TbR-I was targeted to the oral cavity of mice, which facilitated carcinogen-induced tumor formation in a manner correlated with constitutive PI3K/AKT activation [97]. Moreover, mice rendered heterozygous null for TbR-I displayed twice as many intestinal tumors as compared to their wild-type counterparts when crossed onto an Apc(Min/+) background [98]. Collectively, these findings highlight the importance of tissue homeostasis and cell microenvironments to facilitate the tumor suppressing activities of TGF-b, as well as establish the pathophysiological basis of the “TGF-b Paradox” in preclinical models of TGF-b-responsive carcinomas. In the succeeding sections, we present recent findings that frame our understanding of the “TGF-b Paradox” and its regulation by the diversity of microenvironments encountered by carcinoma cells during their acquisition of metastatic phenotypes.

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5.5.1

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TGF-b Signaling Within Primary Tumor Microenvironments Mechanotransduction

The use of traditional 2-dimensional (2D) tissue culture systems to compare the behaviors and activities of normal versus malignant cells have proven to be wholly insufficient in recapitulating the “TGF-b Paradox.” To circumvent this experimental deficiency, researchers have begun employing novel 3D-organotypic cultures to more accurately model and assess how changes within tumor microenvironments impact the functions of TGF-b. For example, culturing normal and malignant mammary epithelial cells (MECs) on top of or embedded into cushions of reconstituted basement membranes (RBMs) has provided numerous insights into how malignant transformation alters acinar development [99]. Although the precise conditions employed in these analyses can impart dramatic alterations in organoid development and behavior [100], it is nonetheless widely accepted that these 3D-organotypic systems more accurately recapitulate the signaling dynamics experienced by MECs and their microenvironments during mammary gland development [101]. It is important to note that tumor development is typically accompanied by intense desmoplastic and fibrotic reactions that enable primary tumors to be palpable in the context of surrounding normal tissue architectures. Moreover, tumor fibrosis results in the formation of mechanically rigid tumor microenvironments that (i) enhance the selection and expansion of developing neoplasms, particularly that of late-stage metastatic tumors, and (ii) predict for poor clinical outcomes in patients with cancers of the colon, ovary, and breast [102–105]. Interestingly, these aberrant cellular activities are highly reminiscent of those attributed to TGF-b [1, 2, 6, 106], whose upregulated expression dictates the composition tumor reactive stroma. In a reciprocal manner, tumor reactive stroma plays an important role during cancer initiation and progression by determining whether TGF-b suppresses or promotes tumor formation [5, 91–94]. In fact, it has been argued that “phenotypes dominate genotypes,” a statement referring to the ability of the ECM and cell microenvironments to either suppress or promote tumorigenesis in a manner independent of genotypic alternations in MECs [107]. Dramatic evidence supporting this idea was provided by the findings that normal mice readily developed from (i) blastocysts injected with stable teratoma cells [108], and (ii) enucleated oocytes injected with nuclei isolated from melanoma cells [109]. Unfortunately, a role for TGF-b to impact these events remains surprisingly unexplored. However, infecting chicken embryos with RSV in ovo was shown to elicit cellular transformation and tumor formation only in response to tissue wounding, a reaction mediated by TGF-b [110, 111]. Collectively, these findings provide compelling evidence to support the notion that “phenotypes dominate genotypes,” as well as implicate TGF-b as a principal player operant in overseeing this phenomenon. As such, inclusion of collagen and other matrices to RBM preparations represented a significant scientific achievement to initiate mechanotransduction in

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modeled tumor microenvironments [112, 113]. For instance, elevated collagen concentrations results in integrin clustering and increased activation of focal adhesion complexes, growth factor receptors, and MAP kinase pathways [102–105, 113]. Along these lines, aberrant lysyl oxidase (LOX) activity also associates with cancer progression and the development of desmoplasia [104]. Indeed, elevated expression of LOX family members, particularly LOX, LOXL (LOX-like), and LOXL2, correlates with increased malignancy and the acquisition of invasive/metastatic phenotypes, and with the induction of EMT and the formation of the premetastatic niche [103, 113–118]. In particular, LOX expression is essential for hypoxia-induced metastasis of human MDA-MB-231 breast cancer cells in mice [115, 116]. Moreover, elevated LOX expression in breast cancer is observed most frequently in poorly differentiated, high grade tumors and, consequently, predicts for increased disease recurrence and decreased patient survival [115, 116]. Likewise, LOX expression has recently been validated as a prognostic marker for metastasis development in patients with head and neck cancers [119]. Interestingly, we observed TGF-b to be a potent inducer of LOX expression and activity, an event coupled to the acquisition of oncogenic TGF-b signaling in cancers of the breast [120]. Finally, two-photon intravital imaging analyses demonstrated that cancer cells attempting to exit the primary tumor utilize a paracrine signaling axis comprised of carcinoma-derived colony stimulating factor-1 (CSF-1) and macrophage-derived epidermal growth factor (EGF) that facilitates tumor cell migration and invasion [121–124]. Importantly, TGF-b plays a critical role in establishing this paracrine signaling network through its ability to (i) upregulate CSF-1 receptor expression [121]; (ii) stabilize EGF receptor (EGFR) expression at the cell surface [47]; and (iii) facilitate tumor-infiltration of macrophages via a FAK-dependent pathway [45]. The ability of mechanotransduction to alter function and composition of TGF-b signaling modules in normal and malignant cells has recently been linked to signals originating from integrins. For example, we observed TbR-II to interact physically with b3 integrin following its activation by vitronectin or in response to EMT induced by TGF-b [30–32]. Furthermore, the formation of b3 integrin:TbR-II complexes is critically dependent upon the expression and activity of FAK, such that depleting cells of FAK expression not only disrupts this interaction, but also markedly reduces the ability of TGF-b to stimulate breast cancer invasion and metastasis [45]. Along these lines, b1 integrin also interacts physically with TbR-II [30] and is required for its stimulation of EMT and p38 MAPK [125]. Additionally, the FAK effector, p130Cas, can bind and sequester Smad3 to prevent its phosphorylation by TbR-I and subsequent transit to the nucleus [126]. In extending these findings, we found elevated p130Cas expression to mark metastatic progression in breast cancers, as well as to distort the balance of TGF-b signaling from canonical to noncanonical effectors in metastatic MECs [46]. Indeed, genetic depletion of p130Cas was sufficient in drastically reducing the ability of TGF-b to promote breast cancer development and progression [46], presumably by enhancing their sensitivity to apoptotic stimuli [127]. Taken together, these findings highlight the direct influence integrins and focal adhesion complexes possess in promoting tumor progression,

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particularly that driven by TGF-b. These studies also emphasize the power of 3D-organotypic models to faithfully recapitulate these events in sensitive and rapid cell-based assays capable of interrogating the “TGF-b Paradox.” The use of collagen to increase matrix rigidity strongly suggests that integrins, particularly those that ligate collagen, have the ability to influence TGF-b signaling. This notion is supported by our recent findings demonstrating that propagating malignant MECs in compliant 3D-organotypic cultures restored the cytostatic activities of TGF-b in highly metastatic 4T1 cells, which normally fail to undergo growth arrest in response to TGF-b in traditional 2D-cultures [32, 41, 51, 128]. Importantly, inclusion of type I collagen in these 3D-organotypic cultures to increase their rigidity/tension and to initiate mechanotransduction dose-dependently uncoupled TGF-b from the regulation of cell cycle progression in 4T1 cells [51]. Conversely, treating 4T1 organoids with the small molecule TbR-I inhibitor, TbR-I Inhibitor II stimulated the growth of 4T1 cells in compliant 3D-organotypic cultures wherein TGF-b functions as a tumor suppressor. However, administering this same pharmacological treatment regimen to tense 3D-organotypic cultures wherein TGF-b functions as a tumor promoter greatly inhibited the growth of the resultant 4T1 organoids [120], thereby validating the first in vitro system that recapitulates the “TGF-b Paradox” solely by modulating the tension sensed by malignant MECs. Interestingly, a recent study suggests that dramatic decreases in matrix compliance (i.e., elastic modulus) can serve as the driving force to alter cell signaling. Indeed, increasing matrix rigidity not only induced dramatic differences in cell proliferation, but also suppressed E-cadherin expression in a manner associated with the exaggerated development of EMT phenotypes [129]. Analogous alterations in tumor microenvironments also enabled E-cadherin-negative breast cancer cells to reinitiate expression of this junctional protein during metastatic outgrowth in the lungs of mice [130]. Although a direct participatory role for TGF-b during these events was not addressed, these analyses have nonetheless linked mechanotransduction to the activation of known TGF-b effectors. Moreover, increased matrix rigidity elicits increased TGF-b production [120], presumably via augmented integrin-mediated activation of latent TGF-b complexes [131]. Taken together, these studies stress the importance of integrins and matrix rigidity in regulating TGF-b signaling modules, and in enhancing the release of TGF-b from inactive matrix depots.

5.5.2

Hypoxia

It has long been appreciated that tumor development and progression significantly deprives these neoplasms access to sufficient nutrients and oxygen that facilitates their unabated growth, leading to the hypothesis that chemotherapeutic targeting of tumor angiogenesis would prove effective in starving tumors to death [132, 133]. Unfortunately, administering angiostatic agents to cancer patients has offered little improvement in overall patient survival, presumably reflecting the fact that hypoxic tumors house significantly elevated quantities of oncogenic factors that collectively

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drive carcinoma cell dissemination to more hospitable environments [134]. Indeed, hypoxic culture conditions are sufficient to produce hyperinvasive and EMT phenotypes in carcinoma cells [135]. Moreover, hypoxia and TGF-b act in concert to induce the expression of vascular endothelial growth factor (VEGF) and the chemokine receptor CXCR4, two molecules essential to the metastatic process [136]. As expected, these oncogenic TGF-b activities are mediated by p38 MAPK [136], and by Smad7, which somewhat counterintuitively actually promoted carcinoma invasion under hypoxic conditions [137]. Collectively, these findings support a paradigm in which the activities of canonical and noncanonical TGF-b signaling systems are balanced under normal homeostatic conditions [138], but suddenly become unbalanced and slanted towards noncanonical TGF-b signaling under hypoxic tumor conditions. Indeed, these findings highlight the intimate relationship between hypoxia and oncogenic TGF-b signaling and their ability to be interrogated mechanistically via employment of 3D-organotypic culture systems.

5.5.3

Reactive Tumor Stroma

The cellular and structural composition of tumor microenvironments has a profound impact on tumor progression and TGF-b signaling. As mentioned above, targeted deletion of TbR-II in mammary carcinoma cells facilitated exaggerated infiltration of immature myeloid cells that greatly enhanced tumor invasion and metastasis in part via upregulated production of matrix metalloproteinases (MMPs) and TGF-b1 [95]. Interestingly, targeted inactivation of the TGF-b signaling system in fibroblasts [91] or the Smad4 pathway in T cells [96] both elicited carcinoma development due to disruptions of tumor suppressing paracrine signaling networks. Attempts to model these complex microenvironmental events in vitro have recently been initiated. For instance, the influence of CD4+ T cells and their production of cytokines, particularly TGF-b, to drive breast cancer metastasis was determined to be dependent upon the development of M2-type macrophages. Indeed, co-culturing M2 macrophages with mammary carcinoma cells in 3D-organotypic cultures led to the production of invasive structures from resulting organoids in a manner indicative of increased carcinoma malignancy [139]. Although the synthesis of protruding branched structures is believed to reflect the extent of metastatic progression, it should be noted that the validity of this assumption remains to be rigorously examined. Thus, while certain metastatic cells do in fact grow as branched structures [140, 141], others readily assume a dense spherical morphology when propagated in 3D-organotypic systems. Indeed, we recently observed nonmetastatic cells to display dysmorphic branching structures that readily disappear during metastatic progression and in response to EMT induced by TGF-b (Fig. 5.2), which also promoted luminal filling of established acinar structures [47, 140]. Collectively, these findings highlight the potential of 3D-organotypic cultures to model distinct cell morphologies and behaviors in a manner that recapitulates defined aspects of tumor:stromal interactions. Extending these systems to include additional stromal

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Fig. 5.2 The influence of TGF-b signaling on normal and malignant MEC acinar morphology. Photomicrographs of MECs grown in 3D-organotypic cultures (left to right): (i) normal MECs grown in the absence (a, 400×) or presence (b, 100×) of EGF; (ii) nonmetastatic breast cancer cells (c, 100×); and (iii) metastatic breast cancer cells (d, 100×). Accompanying schematic depicts these morphologies and how TGF-b signaling drives the acquisition of metastatic phenotypes

mediators (e.g., fibroblasts, endothelial, and immune cells) propagated under varying oxygen tensions and elastic moduli will greatly enhance the ability of science and medicine to decipher and manipulate the molecular underpinnings of the “TGF-b Paradox.”

5.5.4

Visualizing Metastatic Progression Stimulated by TGF-b

Immunohistochemistry (IHC) is one of the most powerful tools available to pathologists attempting the grade and subtype tumor biopsies. Moreover, as the molecular basis of tumorigenesis continues to unfold, these IHC analyses will become more sophisticated in their ability to accurately diagnose and prognose patient outcomes, as well as to monitor the effectiveness of specific therapeutic regimens. However, employing IHC to gauge the extent of TGF-b function in human tissues is fraught with caveats and misinterpretations because this technique only captures a small snap-shot of the dynamics built into the TGF-b signaling system. Powerful developments in intravital imaging have greatly accelerated our understanding of (i) “how,” “when,” and “where” cancer cells systemically disseminate, and (ii) the role of TGF-b in mediating discrete steps of the metastatic cascade [45–47]. By employing dual bioluminescent imaging techniques, Korpal et al. [142] demonstrated the essential importance of canonical TGF-b

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Fig. 5.3 Smad2/3 Signaling is decreased dramatically during the outgrowth of pulmonary metastases. (a) Metastatic 4T1 breast cancer cells stably expressing CMV-renilla luciferase in conjunction with either SBE (4T1-SBE)- or p3TP (4T1-p3TP)-driven firefly luciferase were propagated (±) TGF-b in 2D- or 3D-cultures. Data are the fold-induction of luciferase activity stimulated by TGF-b. (b) 4T1-SBE cells were engrafted onto the mammary fat pad of Balb/C mice. Primary tumor growth and metastasis was assessed 4 weeks later using dual bioluminescent imaging to visualize these events (CMV-renilla), as well as the extent of TGF-b-specific Smad2/3 signaling (SBE-firefly). Data are the mean luciferase ratios of SBE-firefly:CMV-renilla (±SD; n = 4)

signaling to promote the establishment of osteolytic bone lesions by metastatic breast cells. Importantly, bisphosphonate administration was shown to be more effective in suppressing osteolytic bone lesions and canonical TGF-b signaling early in the metastatic process as opposed to after these lesions were fully established [142]. These findings suggest that canonical TGF-b signaling is differentially regulated during specific stages of the metastatic cascade. Accordingly, transient activation of Smad2/3 by TGF-b was sufficient in converting the migration of breast cancer cells from cohesive to single cell programs

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[143, 144]. Remarkably, the ability of breast cancer metastases to resume proliferative programs within the pulmonary microenvironment required these cells to first inactivate Smad2/3 signaling [143, 144], and as such, it is tempting to speculate that altered elastic moduli govern the coupling of TGF-b to Smad2/3. In support of this supposition, Fig. 5.3 shows that TGF-b becomes selectively uncoupled from the activation of Smad2/3 in (i) compliant 3D-organotypic cultures relative to rigid tissue culture plastic (Fig. 5.3a), and (ii) pulmonary metastases relative to their site of origin (Fig. 5.3b). Collectively, these intriguing findings demonstrate the plasticity present in the TGF-b signaling system as carcinoma cells undergo EMT and metastatic outgrowth, presumably reflecting a shift from canonical (i.e., Smad2/3-based) to noncanonical (i.e., non-Smad2/3-based) signaling that originates from altered mechanotransduction within the tumor microenvironment [9, 46, 47]. Clearly, extending these novel intravital imaging techniques holds great promise to delineate and define the mechanics of the TGF-b signaling during distinct stages of metastatic progression.

5.6

TGF-b Signaling Within the Circulation

After exiting the confines of the primary tumor, disseminated cancer cells are confronted with the challenges of traversing and surviving the nonadherent microenvironments provided by the lymphatic and circulatory systems. In fact, the lack of ECM support, coupled with shear forces encountered in circulation, make this process of the metastatic cascade one of the harshest and deadliest faced by metastatic cells. This notion is bolstered by “experimental metastasis” studies which demonstrated that 95% of inoculated cancer cells die prior to entering a metastatic niche [145, 146]. At present, a direct role for TGF-b in regulating the behaviors of circulating tumor cells remains largely unexplored. However, TGF-b does promote the generation of “mammospheres,” which are nonadherent structures enriched for stem-like progenitor cells [147]. These data suggest that TGF-b and its induction of EMT functions to promote the selection and expansion of cancer initiating cells that are ideally suited for exiting the primary tumor and surviving nonadherent conditions. Anoikis is a specialized form of apoptosis exhibited by adherent cells when they are deprived of their normal cell: ECM interactions [148]. Interestingly, anoikis also plays a critical role during acinar development by promoting “nonadherent” luminal cells to undergo apoptosis during organoid hollowing [149]. As mentioned previously, EMT induced by TGF-b prevents acinar hollowing [47, 140], suggesting that TGF-b confers resistance to anoikis in post-EMT cell populations. Along these lines, myofibroblasts can be protected from anoikis following TGF-b-mediated activation of FAK and AKT [48]. Although additional studies are clearly warranted to solidify the relationship between TGF-b and anoikis, these findings do coalesce to support a model wherein circulating tumor cells activated by TGF-b are afforded a selective survival advantage during their rapid and violent transit to distant metastatic sites.

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5.7 5.7.1


TGF-b Signaling Within Metastatic Sites Pulmonary Microenvironment

As mentioned previously, FAK mediates the interaction between integrins and TGF-b receptors [9, 46], as well as promotes the metastatic outgrowth of breast cancer cells in pulmonary tissues [150]. Interestingly, recent evidence demonstrates that the success or failure of breast cancer cells grow in pulmonary microenvironments can be modeled and recapitulated by the sparse plating of cells in 3D-organotypic cultures [150–152]. Moreover, culturing normal MECs in these 3D-organotypic cultures is sufficient to induce acinar formation and milk production [153], strongly suggesting that these culture systems represent suitable surrogates to model the differentiation and development of normal mammary glands [99], as well as metastatic dormancy in isolated low-grade carcinoma cells [151]. The dichotomous nature of these two examples (i.e., acinar development versus metastatic dormancy) raises two important questions for future analyses. First, what are the underlying mechanisms that enable a single microenvironment to elicit such varying responses in MECs? And second, what role does TGF-b play in directing these disparate functions of MECs? Although both questions remain unanswered, a recent study described a metastatic gene signature associated with the ability of breast cancers to metastasize to the lungs [154]. Central players to this response were IDs (Inhibitor of Differentiation) 1 and 3, which promoted breast cancer cells to extravasate and colonize the lung [154]. Along these lines, we found TGF-b to strongly suppress the expression of IDs 1 and 3 in normal MECs, but to dramatically induce their expression in late-stage breast cancer cells [106]. Thus, future studies need to gauge the extent to which dysregulated coupling of TGF-b to ID1/3 expression manifests the “TGF-b Paradox,” as well as dictates the response of normal and malignant MECs to microenvironmental signals. TGF-b stimulation of angiopoietin-like 4 expression enabled breast cancer cells to specifically extravasate and colonize the lungs, but not the bone [59], suggesting that TGF-b signaling is critical in determining metastatic tropism. Likewise, activation of Smad2/3 by TGF-b was shown to be critical in promoting the invasive exodus of breast cancer cells out of the primary tumor; however, reinitiation of proliferation programs necessary for metastatic colonization of the lungs required the activity of Smad2/3 to be silenced (Fig. 5.3; [143, 144]). Unfortunately, these analyses only employed a “Smad2/3-centric” approach, and in doing so, failed to address the relative contribution of noncanonical TGF-b signaling inputs to the latter steps of metastasis. Given the established and essential function of noncanonical TGF-b signaling to EMT and metastasis [8, 9], we propose a provocative explanation that the inherent plasticity of the TGF-b signaling system enables metastatic cells to actively induce or repress specific branches of this unique signaling system metastatic progression via a microenvironment-dependent manner. Future studies need to address this hypothesis using an intricate combination of 3D-organotypic cultures and in vivo metastasis models in mice.

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5.7.2

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Skeletal Microenvironment

Despite the fact that skeletal metastases pose a significant health concern for many cancers, the development of appropriate 3D-organotypic cultures to model the interactions of carcinoma cells with a bone microenvironment have yet to be fully exploited. However, a recent study did employ bone hydroxyapatite (HA) to mineralize polymeric scaffolds which supported the growth of breast cancer cells [155]. Importantly, mineralized microenvironments rendered breast cancer cells more competent to synthesize pro-osteoclastic interleukin-8, as well as more sensitive to bisphosphonate treatment [155]. Because TGF-b induces interleukin-8 expression [156], and bisphosphonates inhibit TGF-b signaling [142], it is tempting to speculate that mineralized microenvironments support oncogenic TGF-b. Accordingly, inhibiting TGF-b signaling via administration of the TbR-I inhibitor, SD-208 [157] or a soluble TbR-II molecule [158] both effectively reduced bone metastases. Moreover, Smad4-deficiency or expression of a dominant-negative TbR-II mutant both abrogated breast cancer metastasis to bone in part via diminished the expression of PTHrP, IL-11, and CTGF [10, 159, 160]. Collectively, these findings highlight the importance of TGF-b signaling in mediating bone metastases, and more importantly, the role of bone microenvironments to directly impact the pathophysiology of TGF-b. Indeed, the continued refinement of mineralized microenvironmental cultures clearly holds tremendous promise to elucidate the molecular mechanisms whereby skeletal-derived signals manifest the “TGF-b Paradox.”

5.8

Summary

Attempts to solve the “TGF-b Paradox” have yet to be actualized since the discovery of this phenomenon nearly 30 years ago. Indeed, the inability of science and medicine to solve the “TGF-b Paradox” reflects their collective failure to develop a sensitive and rapid cell-based assay that fully recapitulates this phenomenon in vitro. Here we discussed recent advances in our understanding of the “TGF-b Paradox” and its role in promoting metastatic progression, and in doing so, highlighted the importance of novel 3D-organotypic culture systems to deconstruct the metastatic cascade to better investigate the molecular connections between tumor microenvironments and the TGF-b signaling system. Collectively, these findings coalesce to support the model in Fig. 5.4 that depicts the potential role of ECM and microenvironmental rigidity to alter the response of carcinoma cells to TGF-b. Indeed, we propose that early-stage carcinomas evolve in compliant microenvironments that favor canonical Smad2/3 signaling stimulated by TGF-b. The continued growth of the developing neoplasm enhances ECM rigidity by upregulating TGF-b production, which further enhances the demsoplastic process and its inappropriate formation of receptor complexes comprised of integrins, growth factor receptors, and TGF-b receptors. Once formed, these hyperactivated signaling modules greatly

Fig. 5.4 Schematic depicting the role of microenvironmental and ECM rigidity in mediating oncogenic TGF-b signaling. Tumor-initiated carcinomas evolve in compliant microenvironments that favor canonical signaling by TGF-b. Neoplastic growth enhances microenvironmental rigidity, leading to the inappropriate formation of integrin:TbR-II and TGF-b receptor:EGFR receptor complexes and the amplification of noncanonical signaling TGF-b. These unbecoming events culminate in maximal noncanonical TGF-b signaling, leading to the acquisition of EMT, stem-like, and metastatic phenotypes. Deposition of metastatic cells at compliant micrometastatic niches partially restores the cytostatic function of TGF-b, resulting in tumor dormancy. Over time, this microenvironment cycle is repeated, leading to disease recurrence and poor clinical outcomes in patients harboring metastatic disease

132 M.K. Wendt and W.P. Schiemann

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amplify the activation of noncanonical effectors by TGF-b, which culminate in the acquisition of EMT, stem-like, and metastatic phenotypes. Arrival of disseminated carcinoma cells to metastatic niches once again places these cells in compliant microenvironments, which partially reinstates the cytostatic activities of TGF-b and initiates micrometastatic dormancy. Over time, this vicious microenvironment cycle is repeated, leading to disease recurrence and poor clinical outcomes in carcinoma patients harboring metastatic disease. The basic tenets of this model clearly are supported by the findings presented herein, and as such, this model should serve as a launching point for future studies aimed at identifying the individual effectors operant in regulating ECM tension and TGF-b function in distinct carcinoma subtypes. The use of advanced 3D-organotypic cultures, together with powerful intravital imaging techniques will undoubtedly delineate the complex interactions whereby tumor microenvironments govern oncogenic TGF-b signaling and its stimulation of metastatic progression. In doing so, we believe that it will one day be possible to manipulate the “TGF-b Paradox” and prevent its pathophysiological manifestations, thereby improving the prognosis and overall survival of patients with metastatic disease. Acknowledgements We thank members of the Schiemann Laboratory for critical comments and reading of the manuscript. W.P.S. was supported by grants from the National Institutes of Health (CA129359), the Komen Foundation (BCTR0706967), the Department of Defense (BC084651); and the Case Comprehensive Cancer Center and the University Hospitals Seidman Cancer Center. M.K.W. was supported by the American Cancer Society (PF-09-120-01-CS).

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Chapter 6

Rho GTPases and Their Activators, Guanine Nucleotide Exchange Factors (GEFs): Their Roles in Glioma Cell Invasion Bo Hu, Marc Symons, Bodour Salhia, Shannon P. Fortin, Nhan L. Tran, James Rutka, and Shi-Yuan Cheng

Abstract Accumulated studies showed that constitutively activated Rho GTPases such as Rac1 in malignant human glioblastomas are responsible for the highly invasive phenotype observed in these aggressive brain cancers. Notably, no activating mutations of Rac1 have been reported in human glioblastomas or other types of cancers. Moreover, Rac1 and several GEFs have been implicated in cell invasion and metastasis of glioblastomas and other types of human cancers. Mechanistically, the functions of the Rho GTPases are regulated by three distinct classes of molecules. Among them, guanine nucleotide exchange factors (GEFs) are the activators for Rho GTPases. Here, we review the role of Rho GTPases, particularly Rac1 and

B. Hu Cancer Institute, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15213-1862, USA Department of Medicine, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15213-1862, USA M. Symons Center for Oncology and Cell Biology, The Feinstein Institute for Medical Research at North Shore-LIJ, 350 Community Drive, Manhasset, NY 11030, USA B. Salhia • S.P. Fortin • N.L. Tran Translational Genomics Research Institute, 445 N. Fifth Street, Phoenix, AZ 85004, USA J. Rutka The Arthur and Sonia Labatt Brain Tumor Research Center, Hospital for Sick Children, Toronto, ON M5G 1X8, Canada S.-Y. Cheng (*) Cancer Institute, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15213-1862, USA Department of Pathology, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15213-1862, USA e-mail: [email protected]


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several GEFs including Trio, SWAP-70, Ect2, Vav3 and Dock180-ELMO1 in glioblastoma cell invasion. Since studies of Rho GTPases and their GEFs in glioblastomas are just emerging, we place specific emphasis on the current knowledge of their roles in cell motility and cancer cell invasion as well as potential response to extracellular stimuli that promotes glioma cell invasion. Additionally, we include studies of two membrane receptor proteins, Fn14 and TROY that promote glioma cell invasion through activation of Rho GTPases. Lastly, we discuss future directions for understanding the functions of Rac1 and GEFs in glioma cell invasion and implications to establish these key modulators as potential targets to inhibit diffusely invasive glioblastomas in the brain. The insight provided by this review will help to develop new therapeutic approaches to treat patients with malignant gliomas.

Abbreviations BAI-1 Crk Dbl DD DH domain DHR domain Dock Ect2 EGFR ELMO1 Fn14 GAP GBM GDI GEF p130Cas PDGFR PH domain PtdIns Pyk RNAi ROCK SH2 or 3 SWAP TNF TNFRSF TRAF Trio

Brain-specific angiogenesis inhibitor-1 v-crk sarcoma virus CT10 oncogene homolog Diffuse B-cell lypmphoma Death domain Dbl homology (DH) domain Dock-homology region Dedicator of cytokinesis Epithelial cell transforming sequence 2 Epithelial growth factor receptor Engulfment and cell motility-1 Fibroblast growth factor inducible 14 GTPase-activating protein Glioblastoma multiforme Guanine nucleotide dissociation inhibitor Guanine nucleotide exchange factor (GDP–GTP exchange factor) crk-associate substrate 130 kDa protein Platelet-derived growth factor receptor Pleckstrin homology domain Phosphatidylinositol proline-rich tyrosine kinase RNA interference Rho kinase src homologous domain 2 or 3 switching B-cell complex 70 kDa subunit Tumor necrosis factor Tumor necrosis factor receptor superfamily Tumor necrosis factor receptor associated factor Triple function domains (PTPRF interacting)

6 Rac1 and its GEFs in Glioma Cell Invasion

TROY/Tnfrsf19 TSC TWEAK Vav3 VEGFR

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Tumor necrosis factor receptor superfamily member 19 Triple sex combs Tumor necrosis factor-like weak inducer of apoptosis vav 3 guanine nucleotide exchange factor Vascular endothelial growth factor receptor

Introduction

Human glioblastoma multiforme (GBM) is the most common malignant brain cancer in adults and portends a dismal prognosis [1, 2]. A hallmark of human GBM is the intrinsic ability of single tumor cells to infiltrate throughout the brain, rendering these tumors virtually incurable by all existing therapies and also leading to their great propensity for recurrence [1, 2]. Genetic and clinical studies demonstrated that a number of signaling pathways are constitutively activated in actively infiltrating glioma cells [3–5]. A key group of molecules that are involved in glioma cell invasion are the Rho GTPases. Rho GTPases are molecular switches that are essential in controlling cell movement, morphology, and survival [6, 7]. As with other members of the Ras family of GTPases, most Rho GTPase cycle between an active GTPbound state and an inactive GDP-bound state. In their GTP-bound state, GTPases recognize downstream targets to elicit intracellular responses including the regulation of actin cytoskeleton dynamics [7, 8]. Biochemical and genetic studies have led to the identification of three classes of regulatory proteins that control the nucleotide exchange or degradation, and therefore activation state of Rho GTPases. These are the guanine nucleotide exchange factors (GDP–GTP exchange factors or GEFs), the GTPase-activating proteins (GAPs) and the guanine nucleotide dissociation inhibitors (GDIs) [8–10]. Rho GTPases are activated by GEFs that promote the exchange of GDP to GTP. Subsequently, Rho GTPases are inactivated by GTPase activating proteins (GAPs), which stimulate the intrinsic GTP hydrolyzing activity of the GTPases [11]. Rho GTPases are also controlled by Rho GDIs. Rho GDIs can regulate GTPases in both a negative fashion, by preventing the interaction of GTPases with GEFs for example, and a positive fashion, by regulating proper intracellular targeting of GTPases [12, 13]. Importantly, the activity of all three classes of regulatory proteins is controlled by signal transduction events [12–15]. Rho GTPases are ubiquitously expressed and 22 members have been described in humans [16]. Among them, Rac1 is the most studied and has been shown to play a central role in promoting cancer cell invasion [17, 18]. Rac1 causes lamellipodia formation thus participating in actin-driven cell migration [17, 19]. Rac1 relays signals from a wide variety of growth factors, cytokines and adhesion proteins to a large array of effector proteins [20, 21]. GEFs activate Rho GTPases such as Rac1 by catalyzing the exchange of GDP for GTP in response to various extracellular signals. Many GEFs were identified as oncogenes after transfection of immortalized fibroblast cell lines with cDNA expression libraries [8–10]. Moreover, only a few GEFs such as Vav and Tiam1 have been

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implicated in tumor cell migration and invasion [22–25]. Until recently, very little was known regarding the expression or function of GEFs in GBM. However, during the last several years, with the knowledge that Rac1 is a potent inducer of glioma cell migration and invasion [18, 26, 27], a number of investigators who study glioma biology and tumor invasion have used various approaches to study the role of Rho GEFs, especially those that can activate Rac1, in stimulating glioma cell invasion. In this review, we describe our current knowledge of the Rho GTPases including Rac1 and several Rac1 GEFs in cell motility and the role of these molecules in the modulation of human glioma cell invasion. We also present our studies of two members of the TNF receptor superfamily (TNFRSF), Fn14 and TROY in glioma cell invasion. Lastly, we will discuss futuristic implications of the function and regulation of Rac1 GEFs in glioma cell invasion.

6.2

6.2.1

The Role of Rho GTPases and Rac1 in Glioma Cell Invasion Rho GTPases in Glioma Cell Invasion

Rho GTPase family proteins are thought to regulate cell invasion in a large part via controlling cell adhesion, cell polarity and the organization of the actin cytoskeleton [6, 7, 20, 28]. In human cancers, tumor cells can employ different modes of invasive behavior, termed mesenchymal and amoeboid [29, 30]. Mesenchymal invasion relies on Rac-mediated signaling and extension of lamellar structures which attach and pull the cell forwards, whereas amoeboid invasion is mediated by the Rho-ROCK signaling axis and involves blebbing protrusions and cortical contractility to squeeze the cell through pores in the extracellular matrix [29, 31, 32]. Notably, during the process of invasion and metastasis, tumor cells can switch between the mesenchymal and amoeboid modes of invasion. The mechanisms that control this plasticity are under intense investigation [33, 34]. An important consequence of this dual migratory behavior is that it may allow tumor cells to escape therapeutics that specifically target either mode of migration and therefore combinations of drugs targeting the respective modes of invasion may be needed to achieve clinical benefit. There is mounting evidence that Rho GTPases are deregulated in human tumors [16, 35, 36]. Unlike Ras itself where activating mutations are commonplace, with the exception of RhoH [37], which is frequently rearranged in lymphomas, thus far no activating mutations of Rho proteins have been identified in tumors [38, 39]. Rac1, Cdc42, RhoA and RhoC, however, have been found to be frequently overexpressed at the protein level in a wide range of tumors and in some cases, expression levels have been correlated with tumor grade [16, 40]. Notably, in astrocytomas, both Rac1 and RhoA proteins have been found to be overexpressed in high-grade versus low-grade tumors [27, 41]. In addition, the marked plasma membrane


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localization of Rac observed in a significant subset of GBM tumors, but not in low-grade astrocytomas or non-neoplastic brain, indicates that this GTPase is hyperactivated in GBMs [27].

6.2.2

Rac1 in Glioma Cell Invasion

A role for the Rac1 GTPase in the invasive behavior of GBM cells has been established in several studies, both using an in vitro Matrigel invasion assay and an ex vivo organotypic brain slice invasion assay [18, 26, 42, 43]. In gliomas, Rac1 mediates glioma cell invasion promoted by fibroblast growth factor-inducible 14 [43], Ephrin-B3 [44] or inhibition of ROCK [26]. One potential mechanism that mediates the role of Rac1 in invasion is the activation of NF-kB [43]. Another mechanism is that Rac1, via its effector synaptojanin 2 [18], controls the formation of invadopodia, which are specialized domains of the plasma membrane at which extracellular matrix degradation takes place [45]. Interestingly, siRNA-mediated depletion of Rac3, a GTPase that is highly homologous with Rac1, also potently inhibits GBM cell invasion through Matrigel in vitro [18]. This finding suggests non-redundant roles for Rac1 and Rac3 in glioma cell invasion. The observation that inhibition of ROCK, an effector of both RhoA and RhoC, stimulates the invasion of GBM cells through Matrigel, both in two-dimensional [26] and three-dimensional settings (Symons M et al, unpublished observations), indicates that in these conditions, GBM cells employ a mesenchymal mode of invasion. In line with this, siRNA-mediated depletion of Rac1 [18], or inhibition of matrix-degrading proteases (Symons M et al, unpublished observations) also inhibits glioblastoma cell invasion through Matrigel. Interestingly however, inhibition of ROCK strongly inhibits the invasive behavior of glioblastoma cells in organotypic brain slices [46]. Thus, these observations support the notion that GBM cells can adapt their invasive behavior to their environmental conditions. The roles of RhoA and RhoC, as well as other Rho GTPase family members, in GBM invasiveness remain to be explored.

6.3

6.3.1

The Role of Rho GEFs, Trio, SWAP-70, Vav3 and Ect2 and in Glioma Cell Invasion Guanine Nucleotide Exchange Factors (GEFs)

The first mammalian Rho GEF was identified as a transforming gene in diffuse B-cell lymphoma cells, and was therefore designated Dbl [9]. Since then 80 Rho GEFs were identified in humans and at least 27 GEFs stimulate Rac1 activity [8]. According to their protein structures Rho GEFs are divided into two distinct families.

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The classical GEF family contains the Dbl homology (DH) domain and an adjacent Pleckstrin homology (PH) domain [8–10]. DH domains are responsible for catalyzing the exchange of GDP for GTP within Rho GTPases by transiently promoting GTPase intermediates that are devoid of nucleotide and Mg2+ [8]. Cellular GTP is present in approximately tenfold molar excess relative to GDP, and binding of GTP stimulates the release of the protein from the GEF. DH-associated PH domains, by binding phosphoinositides, have been proposed to localize Dbl proteins to plasma membranes and to regulate their GEF activity through allosteric mechanisms [8]. This in turn renders the GTPase active and allows it to initiate a signaling complex with one of several effectors. Outside the DH-PH domains, Dbl-family proteins show significant divergence and typically contain other protein domains that underlie the unique cellular functions of the different family members [8]. Since the identification of Dbl, 69 distinct mammalian members of this Dbl family have been identified in humans [8, 47]. More recently, another smaller family of Rho GEFs has been identified related to the founding member Dock1 (dedicator of cytokinesis 1, also called Dock180) [48]. These are unrelated in primary sequence to the Dbl family proteins and instead are characterized by two regions of high sequence conservation that are designated Dock-homology region -1 and -2 (DHR-1 and DHR-2). The Dock family members are considered unconventional or atypical GEFs. Within the Dock family, 11 are found in humans [8, 14, 49]. In the case of Dock180, the prototypical Dock in this family, the DHR-1 domain was shown to mediate a specific interaction with phophatidylinositol (PtdIns)(3, 5)-biphosphate and PtdIns(3,4,5)P3 signals in vitro and in cells [50]. The DHR-1 domain was shown to have a critical role in Dock-Rac signaling by positioning the Dock GEF downstream of PI3K which produces PtdIns(3,4,5)P3 in response to various extracellular stimuli and Rac activation [51]. Similar to DH-PH modules in Dbl GEFs, the DHR-2 domain mediates interactions with the nucleotide-free form of the Rho GTPases that they catalytically target, leading to the exchange of GDP to GTP on Rho GTPases [52]. Several DHR-2 domains have been shown to be both necessary and sufficient to promote specific guanidine nucleotide exchange on various GTPases in vitro and in vivo [49, 51, 53]. Although several Dbl GEFs such as Vavs and Tiams are regulated by receptor tyrosine kinases (RTKs) through protein phosphorylation and direct interaction with the RTKs [54], little is known about how GEFs are regulated. Our understanding of the function and regulation of these GEFs will be central to determining the mechanisms that underlie the spatial, as well as the temporal, activation of GTPases during glioma cell invasion in response to outside influences. With the knowledge that Rac1 is a potent inducer of glioma cell migration and invasion [18, 26, 27], a gene expression array database was examined comparing human gliomas to non-neoplastic tissue to identify GEFs that act on Rac proteins and may be important for glioma invasion [27]. Indeed, targeting Rho GEFs is likely to generate less generalized toxicity than direct targeting of Rac. Based on increased expression when compared with non-neoplastic brain; association with higher tumor grade and poor patient outcome, we selected Trio, SWAP-70, Ect2 and Vav3 for further study [27, 55, 56]. Expression levels of these Rac GEFs were validated


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using quantitative real-time polymerase chain reaction (PCR) and protein levels assessed using immunohistochemistry in independent glioma specimens [18, 26, 27]. Depletion of the respective GEFs using RNA interference significantly inhibited the migratory and invasive behavior of glioblastoma cells using radial cell and organotypic brain slice models [27, 55, 56].

6.3.2

Trio and SWAP-70

Most of the Rac GEF family members are expressed preferentially in specific tissues and at different developmental stages, suggesting involvement in specific events during embryogenesis [8]. The multidomain protein Trio is the founding member of an intriguing family of Rho-GEFs that contains two GEF domains, a kinase domain and numerous accessory domains [57, 58]. The first DH–PH domain module of Trio is N-terminally situated (TrioN or GEFD1) and activates the Rac pathway directly [59] or indirectly through RhoG [60]. The second GEF domain of Trio situated in the C-terminus (TrioC or GEFD2) acts on RhoA [57], suggesting that full-length Trio could activate several GTPases in vivo. The members of the Trio family play an important role in neurogenesis, phagocytosis, and myogenesis [61–63] and their structural organization is very well conserved through evolution [64]. Trio is a modulator of the cytoskeleton through the Rho and/or Rac pathways and is vital in axon guidance, neuronal cell migration [58], in regulation of focal adhesion dynamics [65] as well as in anchorage-independent growth [66]. In the adult mammalian CNS, Trio expression in the nervous system is limited to neurons, and neither mRNA nor protein levels were detected in blood vessel walls, the ependymal layer of the third ventricle or in glial cells [67, 68]. In addition to Trio being upregulated grade-dependently at both the mRNA and protein levels in tumor cells of human GBMs [27], high expression levels of Trio were also seen in breast tumors, especially in those with poor prognosis [69]. Tgat, an alternative Trio isoform derived from TrioC, was identified as a new oncogene in adult T-leukemia [70]. Accordingly, glial, mammary and T-cell transformation is somehow associated with increased expression of Trio. Trio is also involved in the invasive behavior of glioblastoma and breast cancers [27, 69]. In GBM, Trio is expressed at higher levels in the tumor rim versus the core lending further support to the importance of Trio during the invasive process (unpublished data). This suggests that aberrant regulation and activity of Trio may have an important prognostic value in breast cancer and leukemia as well as gliomas. Although considered to be promising therapeutic targets, the only GEF inhibitors identified so far act against Trio [10, 71–73]. To date, two types of Trio inhibitors have been characterized: Tripa and derived peptides, which associate with and inhibit TrioC [10, 72], and NPPD and ITX3, which are compounds biochemically active on TrioN. NPPD and ITX3 were identified from a screen of 3,500 chemical compounds using a GEF activity assay in a permeant yeast strain [71, 73]. NPPD

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is toxic in mammalian cells and blocked TrioN-mediated activity of RhoG and Rac1 thus disrupting various physiological processes [71]. ITX3 is a nontoxic inhibitor and in transfected mammalian cells, ITX3 blocks TrioN-mediated dorsal membrane ruffling and Rac1 activation while having no effect on other GEFmediated RhoA or Rac1 activation. Thus unlike NSC23766 which targets a Rac region involved in binding TrioN- and Tiam1 [74], ITX3 has no effect on Tiam1. ITX3 specifically inhibited endogenous TrioN activity, as evidenced by its ability to inhibit neurite outgrowth in nerve growth factor (NGF)–stimulated PC12 cells or C2C12 differentiation into myotubes [73]. The emergence of GEF compounds like ITX3 validate the notion that GEFs can specifically be targeted in cancer cells with minimal toxicity. SWAP-70 is a multifunctional signaling protein involved in membrane ruffling that works cooperatively with activated Rac [56, 75]. It has been suggested that SWAP-70 acts as an effector or adaptor in response to PI3-kinase activity and is important in regulating the actin cytoskeleton and cell motility by promoting PtdIns(3,4,5)P3–dependent activation of Rac [75, 76]. SWAP-70 is expressed in various tissues, such as heart, kidney, spleen, and liver [76] but not expressed to any significant degree in the brain [56]. Src-transformed mouse embryonic fibroblasts lacking SWAP-70 were found to be significantly less invasive than their parental counterparts, and expression of both SWAP-70 and Src induced constant membrane ruffling but not found with Src alone [77].

6.3.3

Ect2

Epithelial cell transforming sequence 2 (Ect2) was originally identified as a proto-oncogene capable of transforming NIH3T3 cells and causing tumors in nude mice when an N-terminally truncated Ect2 is expressed [78–80], suggesting a role of Ect2 overexpression in tumorigenesis. Paradoxically, full-length Ect2 is overexpressed in several human tumor types, including brain, lung, esophageal, pancreatic bladder and ovarian tumors [27, 55, 81, 82]. Moreover, Saito et al. demonstrated that both the removal of the negative regulatory domain and alteration of subcellular localization are required to induce the oncogenic activity of Ect2 [80]. Since the oncogenic activity of Ect2 is strongly inhibited by dominant negative Rho GTPases, Ect2 sequestered in the nucleus during interphase is thought to be inactive because nucleus-Ect2 is unable to activate Rho GTPases situated in the cytoplasm [80]. A study of GBMs using comparative genomic hybridization and a BAC DNA microarray of the human genome identified a chromosomal gain of 3q26.3, the ECT2 locus, in 3/10 GBMs [83]. Previous studies have also shown an important role for Ect2 in the regulation of cytokinesis [84]. A dominant-negative C-terminally truncated Ect2, or microinjection of anti-Ect2 antibody prevents contractile ring formation during cytokinesis and leads to the formation of multinucleated cells [84, 85]. This finding was validated in a study showing that inhibition of Ect2 by siRNA leads to formation of large multinucleated cells in human glioma SNB19 and U87MG cells [27].


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Interestingly, whereas low-grade astrocytomas show predominantly nuclear Ect2 staining, GBMs display prominent staining of Ect2 in both the cytoplasm and nucleus [27]. It is highly probable that the sub-cellular localization of Ect2 in human astrocytomas dictates the functional significance and state of activation. This knowledge could be utilized in future studies for pre-screening patients, for example, for eligibility with anti-Ect2 therapy.

6.3.4

Vav3

In mammals, there are three Vav protein family members; Vav1 is specifically expressed in the hematopoietic system, whereas Vav2 and Vav3 are more ubiquitously expressed [86]. Vav proteins are evolutionarily conserved from nematodes to mammals and play a pivotal role in many aspects of cellular signaling, coupling cell surface receptors to various effector functions [86]. Vav proteins have several protein-protein interaction domains including a Src homology 2 (SH2) and two Src homology 3 (SH3) domains that can, respectively, interact with a large variety of tyrosine kinase receptors or adaptor proteins [87]. Vav proteins are regulated by tyrosine phosphorylation [86]. The participation of the Vav proteins in several processes that require cytoskeletal reorganization, such as the formation of the immunological synapse, phagocytosis, platelet aggregation, spreading, and transformation have been well documented [86]. Several lines of evidence show that Vav proteins are crucial for developmental, mitogenic, and pathological processes. Vav3 is expressed at low levels in nonneoplastic brain and low-grade gliomas [27] but expressed at high levels by immunohistochemistry in both the tumor cells and tumor-associated endothelial cells in all GBMs examined [27]. A number of growth factor receptors that can recruit Vav proteins, including EGFR, PDGFR, IGF1R, and TrkA are abundantly expressed in malignant glioblastomas, providing a potential mechanism for the role of Vav3 in glioblastoma cell migration and invasion [27]. Increased expression of Vav3 has also been reported in prostate cancer, whereby through the PI3KAkt pathway, Vav3 inappropriately activates androgen receptor signaling and stimulates growth in prostate cancer cells [88]. Vav3 is overexpressed in over 80% of human breast cancer specimens, particularly in poorly differentiated lesions [89]. Vav3 complexes with and activates estrogen receptor alpha partially via PI3K-Akt signaling leading to stimulated growth of breast cancer cells [89]. In anaplastic large cell lymphomas (ALCL), aberrant expression of the oncogenic fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is present in 75% of cases. Vav3 is phosphorylated in NPM-ALK positive biopsies from patients suffering from ALCL, demonstrating the pathological relevance of this observation [90]. The use of Vav3-specific shRNA and a dominant negative Rac1 mutant inhibited the migration and invasion of NPM-ALK-expressing cells [90]. Hunter et al. demonstrated that overexpression of Vav2 or Vav3 in primary microvascular endothelial cells promotes Rac1 activation, cell migration and assembly

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in response to ephrin-A1 stimulation [91]. These findings collectively suggest that Vav3 overexpression may aberrantly enhance development and/or progression in various types of tumors including gliomas.

6.4

Dock180, a Rac1 GEF That Promotes Glioma Cell Invasion

6.4.1

The Dock Family Members

Based on the homologies in their protein sequences [48], the Dock family can be divided into four classes, DockA, which include Dock1, Dock2 and Dock5; DockB, which has Dock3 (modifier of cell adhesion, MOCA) and Dock4; DockC, which includes Dock6, Dock7 and Dock8 (Zir1, Zir2, and Zir3) and DockD, which has Dock9, Dock 10 and Dock11 (Zizimin1, Zizimin2 and Zizimin3) [49]. In addition to the well-studied Dock1, the roles of Dock2, Dock4, Dock7 and Dock9 in cell motility are better understood than other Docks. For the cellular functions of other Docks, please see more comprehensive reviews [47, 49, 51, 92].

6.4.1.1

Dock2

Dock2 was identified as a haematopoietic cell specific Dock essential for lymphocyte migration, interstitial motility and sphingosine-1-phosphate-mediated egress [93–95]. Dock2 associates with engulfment and cell motility 1 (ELMO1) through its Src-homology 3 (SH3) domain and CrkII, and regulates Rac1 activation of lymphocyte adhesion, motility and cytoskeletal re-organization [96–101]. Dock2 also regulates Rac1 and Rac2 stimulation of neutrophil chemotaxis [102, 103]. Moreover, intracellular Dock2 dynamics are sequentially regulated by distinct phospholipids to localize Rac activation during neutrophil chemotaxis [104]. Additionally, Dock2 is involved in Rac, Erk and other signals in activation of dendritic cells, T cells and cancer cells [105–107].

6.4.1.2

Dock4

Dock4 was initially cloned as a tumor suppressor gene that is homozygously deleted in a mouse NF2 and TP53 osteosarcoma model [108]. Dock4 mutations are present in a subset of human cancer cell lines and myelogenous leukemia, ovarian cancers, autism and dysplaxia [109–114]. Dock4 rescues deficiency caused by loss of a Dock1 homolog in C. elegans and suppresses tumor growth and invasion in Dock4deleted tumors [108]. Dock 4 mediates RhoG activation of Rap and Rac1 GTPases and promotes cell motility through interaction with ELMO1 or ELMO2 in cancer


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cells and during dendritic development [115–118]. Additionally, Dock4 also enhances Wnt/beta-catenin activation of Rac1 and increases cell growth and transformation [119].

6.4.1.3

Dock7

Dock7 was identified as an activator of Rac GTPases through a yeast two-hybrid screening in the fetal brain. Dock7–Rac activation leads to phosphorylation and inactivation of the microtubule destabilizing proteins stathmin/Op18 thus regulating neuronal polarity during axonal development [120, 121]. In Schwann cells, stimulation of ErbB2 by neuregulin-1 that phosphorylates Dock7 at a tyrosine residue at its C-terminus, resulting in activation of Dock7-Rac1/Cdc42-JNK signaling and enhancement of cell migration [122]. Additionally, proteomic studies identified Dock7 as a binding partner for the TSC1 complex, suggesting its role in TSC1regulated tumorigenesis [123–125].

6.4.1.4

Dock9

A biochemical approach identified Dock9 (Zizimin1) as a specific activator for Cdc42 through direct interaction with a Cdc42-binding domain (CZH2). Dock9 activates Cdc42 and promotes cell motility ad dendrite growth in hippocampal neurons [49, 53, 126–128]. Dock9 also activates Rac2 through interacting with a specific region of Rac2 [129]. Recently, structural analysis identified a nucleotide sensor within the DHR2 domain that contributes to release of GDP and then to discharge of the activated GTP-bound Cdc42, thus providing the most complete picture of a GDP-GTP exchange cycle of modulation of Rho GTPase activation to date [130, 131].

6.4.2

Dock180-ELMO1 is a Bipartite GEF That Mediates Rac1-Promoted Cell Motility

Dock1 or Dock180 is the prototypical member of the Dock family [8, 47, 49, 51, 92]. Dock180 was first identified as a CrkII-binding protein that regulates NIH 3T3 cell morphology [132]. Dock180 interacts with Crk and p130Cas, leading to activation of Rac1 and stimulation of cell migration [133–136]. Genetic approaches identified a Dock180 homolog in C. elegans (CED-5) and Drosophila (myoblast city) that modulates cell body engulfment, cell migration, myoblast fusion, dorsal closure and cytoskeletal organization through interaction with CED-2/CrkII and CED-10/Rac [137–147]. Dock180 binds to PtdIns(3,4,5)P3 through its unconventional Docker DHR-1 domain, resulting in its membrane translocation [50, 148]. Dock180

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facilitates nucleotide exchange on Rac1 activation through its DHR-2 domain [48, 52, 145, 149]. Importantly, Dock180 forms a bipartite complex with engulfment and cell motility 1 (ELMO1) that is required for GDP/GTP exchange and stimulation of Rac1 activity and cell motility [52]. In C. elegans, Drosophila and mammalian cells, ELMO1 and its homolog, CED-12 enhances phagocytosis and cell migration by forming a bipartite complex with CED-5/Dock180 [150]. This bipartite GEF synergistically functions upstream of CED-10/Rac1 promoting Rac-dependent cell migration and cell corpse engulfment [151–153]. The Dock180-ELMO1 complex interacts with CrkII that in turn regulates the stability and function of this bipartite GEF [154]. Dock180-ELMO1 further forms a complex including paxillin-Crk-p130Cas and Rac1 that mediates early flow-induced polarization of actin-based protrusion of the lamellipodium in wounded monolayers and single cells [155]. Moreover, other evidence showed that Dock180-ELMO1 and CrkII might act independently for efficient removal of apoptotic cells [156]. During bacterial invasion into epithelial cells, IpgB1, an effector of Shigellar driver mimic the role of RhoG by interacting with the ELMO1-Dock180 complex, thus stimulating Rac1 and promoting membrane ruffle formation [157]. However, RhoG is not required for integrin-mediated ELMO1-Dock180-Rac1 signaling and cell migration [158]. In Dictyoselium, DdELMO1 associates with DockD but not DockA and activates both Rac1A and RacC, stimulating cell migration [159]. Interestingly, Dock180 itself is subjected to constant ubiquitin-mediated protein degradation and ELMO1 acts as an inhibitor of ubiquitination of Dock180 thus preventing Dock180 protein from degradation [160]. This also explains a constant association of ELMO1 with Dock180 in unstimulated cells [150].

6.4.3

Dock180-ELMO1 Couples Receptor Signaling to Rac

Similar to classical GEFs, the bipartite GEF Dock180-ELMO1 couples receptor signaling to its target Rho GTPase, Rac1, thus promoting receptor-induced cell motility, engulfment, myoblast fusion, dorsal closure and cytoskeletal organization [54]. In Drosophila, PDGFR and EGFR are essential for guidance of cell migration of embryonic precursors of blood cells and border cell clusters [161–166]. Significantly, Dock180 was found directly downstream of PDGFR/VEGFR- and EGFR- signaling during Drosophila development. Genetic evidence showed that myoblast city/Dock180 mediates two separate steps of PDGFR/VEGFR- and EGFR-induced blood cell precursor migration [163–166]. In mammalian cells, integrin receptors recruit the CrkII-Dock180-Rac1 complex to the membrane for phagocytosis of apoptotic cells and cell adhesion to laminin10/11 and fibronectin [136, 167–169]. Dock180-ELMO1 also mediates urokinase plasminogen activator receptor (uPAR)-induced cell migration [170]. Additionally, the p130Cas-CrkII-Dock180 complex was found to be involved in lysyl oxidase regulation of actin filament formation and Fcg receptor-mediated phagocytosis [171, 172].


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In mice, Dock180 is essential during embryonic development and deletion of Dock180 impaired myoblast fusion during muscle development leading to postnatal death of pups [173], corroborating what had been observed for the role of myoblast city/Dock in Drosophila [161–164] and zebrafish [146]. The role of ELMO1 during development has recently been investigated. In zebrafish, ELMO1 is expressed in the vascular and neuronal system. Morpholino-knockdown of ELMO1 severely impaired the formation of vasculature and Netrin-1 and its receptor, Unc5B were identified as the upstream activator of the Dock180ELMO1 complex leading to activation of Rac1 in endothelial cells and vessel formation [174]. During engulfment of apoptotic cells, phosphatidylserine acts as an “eat-me” signal that binds to its receptor, brain-specific angiogenesis inhibitor (BAI). BAI cooperates with Dock180-ELMO1-Rac1 complex to promote maximal engulfment of apoptotic cells [175]. Moreover, ELMO1-deficient mice were unexpectedly viable and grossly normal. However, in the testis of ELMOnull mice, disrupted seminiferous epithelium, multinucleated giant cells, uncleaned apoptotic germ cells and decreased sperm production were found, indicating a selective requirement for ELMO1 in clearance of apoptotic germ cells in the testis [176, 177]

6.4.4

ELMO1-Dock180 Plays an Important Role in Promoting Glioma Cell Invasion

Although it has been established that the atypical bipartite Rac1 GEF Dock180ELMO1 plays critical roles in the activation of Rac1 signaling and stimulation of cell motility, studies of the role of Dock180-ELMO1 in human cancer invasion and metastasis are limited [8, 47, 49, 51, 92]. Increased expression or mutation of Dock180 has not been reported in human cancers and other diseases. However, genetic variations of ELMO1 were found to associate with type 1 and type 2 diabetes [178, 179–181]. In human gliomas, expression of ELMO1 is elevated in a subtype of clinical glioblastomas [182]. In Src-transformed fibroblasts, the p130Cas-Dock180 complex was found to mediate Src-FAK stimulation of cell motility and invasion [183]. In human ovarian cancer cells knockdown of Dock180 decreased expression of ELMO1 and Rac1 activity, changed cell morphology and reduced cell proliferation and migration [184]. Some of us have reported that the bipartite GEF Dock180-ELMO1 plays a critical role in glioma cell invasion [185]. Immunohistochemical analysis of primary human glioma specimens showed that Dock180 and ELOM1 are highly expressed in actively infiltrating glioma cells within the invasive regions along blood vessels, neuronal structures, and corpus callosum, but not in the central regions of these tumors. Elevated expression of ELMO1 and Dock180 was also found in various human glioma cell lines compared to normal human astrocytes. Furthermore, suppression of endogenous ELMO1 and Dock180 expression by each type of siRNA significantly impeded glioma cell migration and invasion

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in vitro, and in brain tissue slices with a concomitant reduction in Rac1 activation. Conversely, exogenous expression of ELMO1 and Dock180 in glioma cells with low levels of endogenous expression enhances their migratory and invasive capacities in vitro and in brain tissue. These data suggest that the bipartite GEF, ELMO1 and Dock180 play an important role in promoting cancer cell invasion and could be potential therapeutic targets for the treatment of diffuse malignant gliomas [185].

6.5

6.5.1

The Role of the TNFRSF Members, Fn14 and TROY in Glioma Cell Invasion Fn14

Cytokines are membrane bound or soluble proteins released from cells that act mechanistically via autocrine, paracrine, or endocrine signaling. These signals regulate important biological activities including reproduction, growth, development, homeostasis, injury response and repair, and inflammation [186]. The tumor necrosis factor (TNF) superfamily of ligands is a subgroup of cytokines heavily researched for their role in many disease states, including human cancers and glioblastomas. The TNF cytokines are type II proteins that can exist in either a membrane embedded or cleaved soluble form. The ligands can be active in either form, via assembly as a non-covalent homotrimer [187]. TNF receptor superfamily (TNFRSF) members are known to have a scaffold of disulfide bridges that gives rise to the cysteine rich domains required to be a family member. TNF receptors signal via protein-protein interactions to promote either cell death or cell survival [188]. Cytoplasmic domains may contain TNF receptor associated factors (TRAFs) and death domains (DDs); the subset of TNFRSF members which contain a DD are referred to as death receptors [187], and the DD signals for cell death via caspase dependent signaling and activation of JUN kinase pathways [188, 189]. TRAF mediated survival pathways have been associated with the activation of NFkB [189]. Of the TNF receptors characterized for their role in cancer progression, TNFRSF12a, also known as Fibroblast Growth Factor-Inducible 14 or Fn14, is one member of the TNF receptor superfamily that has been implicated in tumor progression [190, 191]. Fn14 was first identified in murine cells as an immediate-early response gene that encodes a cell surface localized type Ia transmembrane protein [192]. Fn14 is the smallest known TNF receptor family member and contains only one cysteine rich domain in the extracellular region. The Fn14 cytoplasmic domain lacks a death domain but contains TRAF binding sites specific for TRAFs 1, 2, 3, and 5 [190]. Fn14 serves as a cell surface receptor for the multifunctional cytokine


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tumor necrosis factor-like weak inducer of apoptosis (TWEAK) [193]. In humans, Fn14 mRNA is expressed at the highest relative level in heart, placenta, and kidney, at an intermediate level in lung, skeletal muscle and pancreas, and is relatively low in brain and liver tissue. Expression of Fn14 was found to associate with liver regeneration and hepatocarcinogenesis [194], and endothelial growth and migration [195]. Fn14 expression is induced during nerve regeneration and tissue injury, and in several types of human cancers including brain, breast, cervical, esophageal, hepatocellular, and testicular carcinoma in situ [191]. In brain tumors, Fn14 expression level is elevated in advanced glial tumors and is also up-regulated in migrating glioma cells in vitro [196] and in invading GBM cells in vivo [43]. Some of us have reported that activation of Fn14 by TWEAK promotes glioma cell migration, invasion and survival [43, 196, 197]. Inhibition of TWEAK-Fn14 binding by the Fn14 decoy receptor abrogates TWEAK-induced glioma cell migration and invasion [43, 196], suggesting that TWEAK-stimulated glioma cell motility via activation of the Fn14 receptor. In addition, several groups, including ours, have reported that Fn14 may be able to signal independent of the ligand when expressed ectopically in vitro. Specifically, ectopic expression of Fn14 has been shown to induce the NFkB and Rac1 signaling pathways [43, 190, 198] in various cellular systems including PC12 cell neurite extension and outgrowth, migration of rat aortic smooth muscle cells [199], pro-apoptotic stimulation of glioma cells [197], glioma cell migration and invasion [43], and invasion of oesophageal adenocarcinoma cells [200]. To date, it is unknown whether TWEAK-independent Fn14 signaling occurs in vivo. In gliomas, Fn14 mRNA is highly expressed, whereas TWEAK mRNA expression is low [196], which may favor a TWEAK-independent Fn14 signaling event. Together, these findings stipulate that activation of Fn14 results from either TWEAK binding the receptor or under circumstances of Fn14 overexpression independent of TWEAK. Dysregulation of the actin cytoskeleton contributes to the migratory phenotype of cancer cells, and promotes the invasion so descriptive of GBM. Rho GTPases are a family of molecular switches that regulate the actin cytoskeleton, cell polarity, and microtubule dynamics [17]. The TWEAK-Fn14 signaling axis mediates glioma migration and invasion via the Rac1 GTPase, and fosters a self-promoting feedback loop whereby Rac1 mediated TWEAK-Fn14 signaling induces Fn14 gene expression via the NFkB pathway [43]. TWEAK stimulated glioma cells were found to induce rapid phosphorylation of IKKb and IkBa coincident with total IkBa protein degradation. Use of either an Fn14-Fc decoy receptor or transduction of glioma cells with a dominant negative Rac1N17 expressing adenovirus abrogates these events. Furthermore, the Fn14 promoter region contains an NF-kB consensus sequence that has been shown to be occupied by the NF-kB transcription factor upon TWEAK stimulation. siRNA-mediated depletion of Rac1 expression strongly suppresses TWEAK-induced glioma invasion and Fn14 mRNA transcript levels [43], suggesting that the TWEAK-Fn14 signaling axis may be one of the key inputs to Rac hyperactivation in GBM and consequently the malignant invasive behavior of glial tumors.

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TROY

Similar to Fn14, TROY (TNFRSF19) has been shown to play an important role in GBM invasion. TNFRSF19/TROY gene encodes a type I cell surface receptor that is expressed on migrating or proliferating progenitor cells of the hippocampus, thalamus, and cerebral cortex [201–206]. TROY is an orphan member of the TNF receptor superfamily that lacks a cytoplasmic death domain but contains a single TRAF binding site [201]. TROY mRNA expression directly correlates with increasing glial tumor grade, and among malignant gliomas TROY expression correlates inversely with overall patient survival. During embryonic development, TROY is widely expressed, but its expression in the postnatal organism is restricted [201– 203, 205, 206]. The strict control of TROY expression indicates that aberrant expression may be unfavorable. The aberrant re-expression of TROY in GBM may play an important role in GBM progression, specifically cell invasion. Some of us also recently reported that TROY is able to promote glioma cell migration and invasion; TROY signals via Rac1 to direct glial cell motility, and TROY is found localized to the leading edge of migrating cells, notably enriched in lamellipodia, when overexpressed in glioma cells [207]. siRNA mediated depletion of Rac1 results in decreased migration of glioma cells overexpressing TROY. Importantly, the non-receptor tyrosine kinase Pyk2 was shown to be a cytoplasmic binding partner of TROY via mass spectrometry analysis of proteins that immunoprecipitated with the TROY receptor [207]. Pyk2 is highly expressed in the nervous system is uniquely localized within the cells to transduce signal from the extracellular matrix and soluble mediators through cell surface proteins including integrins, receptor tyrosine kinases, and G-protein coupled receptors into activated intracellular signaling pathways that regulates cell growth, survival and migration [208, 209]. Up-regulation of Pyk2 expression has been noted in several human tumors including hepatocellular carcinoma [210, 211], NSCLC [82] and breast cancer [212] and correlates with increased metastatic potential and reduced survival. Pyk2 expression is elevated in glioma and expression is increased with advancing WHO grade [213] and is seen up-regulated in the invasive tumor rim cells versus tumor core of glioblastoma samples [214, 215]. Knockdown of Pyk2 expression via shRNA or inhibition of Pyk2 activity by kinase-deficient Pyk2 suppresses TROY-induced glioma cell migration and Rac1 activation, thus implicating Pyk2 as a proximal signaling effector for TROY [207]. Taken together, these data suggest the important role of TROY-Pyk2-Rac1 signaling axis in glioma progression. To date, it is unclear how Fn14 and TROY stimulate Rac1 activation to drive glioma migration and invasion. It is likely that Fn14 and TROY modulates cytoskeleton dynamics by influencing one or more of Rac GEF. Since several Rac GEFs, including Ect2, Vav3, Trio, and Dock180, have been shown to be overexpressed in GBM specimens and modulate Rac1-dependent glioma cell migration and invasion [27, 185], it is possible that Fn14 and TROY signal through one or more of these GEFs to drive the hyperactivation of Rac1 to enhance glioma cell migration and invasion.


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6.6 Concluding Statements: Targeting GEFs as an Anti-invasive Strategy for Glioblastoma Treatments The stimulatory role of Rac1 and its cognate GEFs in promoting glioma cell invasion has been firmly established. Targeted therapy to block functions of Rac1 and its GEFs in gliomas is predicted to abrogate the highly invasive nature of glioma cells. Much work is still needed, however, to fully understand the activity of this large family of GEF proteins as it pertains to glioma biology. One of the most challenging tasks is to decipher the relative contribution of each GEF towards the activation of one GTPase versus another since GEFs commonly have exchange factor activity for more than one GTPase. This is true for Trio, Ect2, Vav3 and perhaps other GEFs that will be identified in regulating glioblastoma cell invasion. Such a phenomenon will factor strongly into the issue of molecular redundancy – that is with a relatively large number of GEFs that perform similar functions understanding the context of vulnerability will be an important component for targeting strategies. Work in this realm is currently ongoing to determine the signaling pathways that mediate the activation of different Rho GTPases and their downstream effectors. This will ultimately require the establishment of assays, such as the G-LISA (Cytoskeleton, Inc) assay that should allow us to do this, designed to measure the activity of Rac and other GTPases in human specimens. Also of importance is the heterogeneous nature of malignant glioblastomas, i.e. the tumor-to-tumor variability of GEF expression in clinical gliomas, whereby some of the tumors express GEFs at low levels and others express the same GEFs at high levels. In fact, decreased expression of SWAP-70 in clinical glioma samples was found to correlate with favorable outcome of patients with glioblastomas [56]. In the future, it is hoped that several targets will become available for halting the migration of GBM cells which will represent strategic inhibitory points along well characterized signaling pathways. Discovery and optimization of existing small molecule inhibitors (such as NS237600, an inhibitor that interferes with the interaction of Rac1 with a number of different GEFs, including Trio and Tiam1) against target GEFs are also among the next important steps to this emerging field. These and future studies hold great promise in identifying novel therapeutic approaches against the intrinsically invasive phenotype of diffuse gliomas based on inhibiting GEF function.

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Chapter 7

Impact of Tumor Hypoxia, Src, and Met Signaling in the Dissemination of Tumor Cells Yao Dai, Wenyin Shi, Nikolett Molnar, and Dietmar Siemann

Abstract Metastasis is the major cause of therapeutic failure and high mortality in cancer patients. The metastatic cascade consists of a series of cellular processes including migration, invasion and survival. Hypoxia has been identified as a key contributor to critical functions associated with tumor cell dissemination. Hypoxiainduced alterations are primarily mediated by the transcription factor hypoxiainducible factor 1 (HIF-1), a factor that induces the expression of an array of genes associated with cell aggressiveness. In addition, altered intracellular signaling pathways, such as c-Src and c-Met, aid tumor cells to progress towards a metastatic phenotype and strongly correlates with poor prognosis. Given the pivotal role of hypoxia/HIF-1, c-Src and c-Met in the metastatic process, the potential for developing therapeutic strategies targeting these pathways is significant. Although the effects of HIF-1 inhibitors require further investigation, small molecule kinase inhibitors targeting Src or Met already have been shown to have considerable efficacy in metastatic diseases. Overall, the fact that cancer cell dissemination can be driven by both external (hypoxia) and internal features (c-Src, c-Met) suggests that the combined targeting of tumor microenvironment and intracellular signaling pathways may have particular utility in impeding cancer metastasis.

Y. Dai • D. Siemann (*) Department of Radiation Oncology, Shands Cancer Center, University of Florida, Gainesville, FL 32610, USA e-mail: [email protected] W. Shi Department of Radiation Oncology, Thomas Jefferson University, Philadelphia, PA 19107, USA N. Molnar Department of Radiation Oncology, Shands Cancer Center, University of Florida, Gainesville, FL 32610, USA Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL 32610, USA


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Introduction

Despite improvement in early detection, more refined diagnostic modalities, and a better understanding of the natural history of the disease, metastatic cancers remain largely incurable. The presence of metastases results in a substantial reduction of the quality of life and the drastic worsening of the prognosis. For example, though highly curable if locally confined, nearly 90% of prostate cancer patients with advanced diseases develop skeletal metastases, a result which lowers the 5-year survival rate to ~31% [1]. In colorectal cancer, the 5-year survival rate is less than 10% in patients with liver metastases [2], compared to nearly 90% in patients with early stages of the disease. The tumor microenvironment has long been considered a critical player facilitating cancer progression and dissemination [3]. Hypoxia, a consequence of inadequate vascular supply, is a common feature of most solid tumors detected in 50–60% of all human tumors [4]. From a therapeutic perspective, the existence of low oxygen tensions in tumors has been associated with increased resistance to cytotoxic therapies and elevated metastatic capacity [5, 6]. Acquisition of the metastatic phenotype is also the result of abnormal intracellular signaling pathways that enhance multiple functions associated with cell motility and invasiveness. Key among complex signaling networks, c-Src and HGF/c-Met trigger a variety of signaling cascades and facilitate the ability of tumor cells to metastasize. Indeed, their dysregulated expression has been strongly associated with cancer progression and metastasis in a variety of human cancers.

7.2

Hypoxia Is an Extrinsic Driving Force That Promotes Tumor Cell Dissemination

As tumors grow the associated blood vessel network often is inadequate to provide nutritional support to the rapidly expanding neoplastic cell population. As a consequence, regions within solid tumors experience oxygen deprivation and become hypoxic. Tumor cells acquire the ability to adapt to hypoxic environments by lowering oxygen consumption, switching to anaerobic glycolysis, co-opting blood vessel formation, and migrating toward blood vessels [7]. Using a variety of measurement techniques including microelectrodes, hypoxic cell markers, and hypoxia-associated molecules, hypoxia has been demonstrated to be a common feature in most solid human cancers and associated with poor prognoses [8, 9]. Importantly, cancer cells existing in low oxygen microenvironments not only contribute to therapeutic resistance [5] but also express a more metastatic phenotype [6].

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Impact of Tumor Hypoxia, Src, and Met Signaling in the Dissemination…

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Fig. 7.1 HIF-1 activation of selected genes involved in metastasis

7.2.1

Impact of Hypoxia on Metastasis

Increasingly laboratory [11] and clinical [10] evidence supports a role for hypoxia in the development of metastatic disease. In both pre-clinical models and patients, hypoxic tumors are generally characterized to have a higher tendency to metastasize Hypoxia regulates a myriad of genes predominantly via hypoxia-inducible factor 1 (HIF-1), a transcriptional factor that mediates hypoxia-induced alterations [9, 12]. These HIF-1-induced genes are widely involved in metastasis-associated phenotypes, such as epithelial-to-mesenchymal transition (EMT), migration, invasion, survival and angiogenesis (Fig. 7.1) [6, 9, 13]. For example, hypoxic exposure enhances the transition from epithelial characteristics to a dispersed morphology, a typical aspect of EMT, due to loss of E-cadherin and increased expression of EMT-associated genes [14]. Hypoxia also increases cancer cell invasiveness by activating several proteolytic enzymes responsible for extracellular matrix degradation, such as matrix metalloproteinase-2 (MMP-2) and urokinase plasminogen activator receptor (uPAR) and uPA; although it should be noted that the expression of some of these genes is independent of HIF-1 [13]. Vascular endothelial growth factor (VEGF) is another hypoxia mediated critical HIF-1 target gene. VEGF is known to stimulate endothelial cell proliferation and migration, as well as the breakdown of the endothelial cell barrier through disruption of adhesion junctions which increases blood vessel permeability [15]. Thus hypoxic exposure affects many of the key aspects of the metastatic cascade including angiogenesis, intravasation, extravasation, and secondary growth [13].

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Acute Hypoxia and Chronic Hypoxia

Tumor hypoxia is generally classified into two types: chronic and acute hypoxia [16]. Tumor cells residing at the limits of oxygen diffusion from functional blood vessels may experience chronic or “diffusion-limited” hypoxia. Such hypoxic conditions typically last for relatively long periods of hours or days [16]. In contrast, some tumor cells may be exposed to short-term, transient hypoxia as a result of intermittent blood flow due to tumor vasculature abnormalities [17]. Such acute or “perfusion-limited” hypoxia is characterized by rapid reoxygenation and hypoxicoxic cycles [18] having periodicities of minutes to hours. However, it should be recognized that such temporal distinctions are somewhat artificial and it is most likely that there is no discrete temporal boundary between these two states in the tumor [7]. Although both types of hypoxia coexist in human tumors, their relative impacts on the metastatic dissemination of cancer cells may vary [19]. While some studies appear to favor chronic hypoxia as the key factor [20, 21], others suggest that acute hypoxia may be equally or more critical in mediating a cancer cell’s metastatic behavior [19, 22, 23]. For example, when tumor-bearing mice were exposed to repeated short-term intervals of hypoxia, the frequency of lung metastases was markedly increased [22, 24]. This complexity is further illustrated by reported conflicting findings often in the same tumor type. For example, in prostate cancer models, while one study proposed that chronic hypoxia may achieve a more aggressive phenotype than acute hypoxia [21], others reported that 48 h chronic hypoxic exposure resulted in decreased invasion [25] and increased apoptosis [26].

7.2.3

HIF-1 Signaling and Metastasis

HIF-1 over-expression has been implicated as a potential prognostic and predictive factor in human cancers. In the presence of ambient oxygen, the HIF-1a subunit is degraded by the ubiquitin-proteasome system via binding to the von Hippel-Lindau (VHL) protein. Under hypoxic conditions, HIF-1a is rapidly stabilized and functionally activated [27]. In some cancers, HIF-1 can be constitutively activated in an O2-independent manner under aerobic conditions as a consequence of dysregulated signaling pathways that involve over-activation of oncogenes (EGFR, Akt, Src) or inactivation of tumor suppressor genes (PTEN, VHL) [27]. The finding that HIF-1 can be activated through either oncogene gain of function or tumor suppressor loss of function suggests that increased HIF-1 signaling activity represents a final convergent pathway in cancer cell dissemination regardless of oxygen status. Since HIF-1 is believed to mediate a pleiotropic role under both aerobic and anaerobic conditions, it may serve as a surrogate marker of tumor oxygenation and response. Multiple experimental studies have linked HIF-1 activity with cancer progression. For example, immuno-histochemical analysis of human cancer biopsies displayed increased levels of HIF-1a protein in the majority of primary tumors and

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their metastases [28]. In breast cancer, HIF-1 is suggested to contribute to lung [29] and bone [30] metastases. Furthermore, studies from early stage of breast, cervical, and endometrial cancers suggest that a subset of patients whose tumors exhibit high HIF-1a expression have a significantly increased mortality, indicating the important role of HIF-1 in metastasis irrespective of primary tumor status [31].

7.3

Src and Met Kinases Are Intrinsic Contributors in Facilitating Cancer Metastasis

Deregulated activation of oncogenic proteins has been widely accepted as a cause of cancer onset, progression and metastasis. Src non-receptor tyrosine kinase and Met receptor tyrosine kinase are two crucial signaling kinase families that orchestrate a plethora of cellular functions involved in the metastasis process. In human cancers, Src hyperactivation occurs frequently and aberrant Met activation, by both liganddependent and -independent mechanisms, is a typical event.

7.3.1

Src Signaling and Metastasis

Src tyrosine kinase family consists of nine members that share a unique N-terminal region, two Src homology domains, a highly conserved kinase domain, and a C-terminal tail containing a negative regulatory tyrosine residue [32]. c-Src is the prototypical member that plays a pleiotropic role in controlling multiple functions including proliferation, adhesion, migration, invasion, and angiogenesis. In cancer, Src family kinases generally lack mutation or gene amplification but their enzymatic activities frequently increase in primary tumors with further enhancement in metastatic lesions [33–36]. Aberrant expression and activation of c-Src have been correlated with aggressiveness and adverse outcome.

7.3.1.1

Role of Src in Metastasis-Associated Functions

Src signaling participates in multiple steps of tumor cell dissemination including angiogenesis, motility, invasion, and distant colonization [37–40]. Constitutively activated c-Src induces cadherin-catenin complex loss-of-function, thereby promoting cell differentiation and invasiveness [41]. Moreover, c-Src has long been known to regulate the dynamics of the cytoskeleton, resulting in increased cell motility and deceased cell-matrix adhesion [42]. In addition, c-Src is a pivotal contributor in the invasion process by up-regulating matrix proteases that lead to degradation of the basement membrane [43]. The function of c-Src in endothelial cells also has been extensively studied. c-Src mediates VEGF/VEGFR signaling

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Fig. 7.2 c-Src promotes bone metastasis. Nude mice were intracardially injected with an MDA-MB-231-derived highly metastatic subline that was transfected with a control vector (Control), a c-Src shRNA vector (Src RNAi), or c-Src shRNA and shRNA-resistant c-Src expression vectors (Rescued). Bioluminescent, radiographic and H&E analysis of bone lesions from representative mice in each group were shown at the indicated times after inoculation. In the X-ray images, areas of bone lysis are indicated by dotted lines. In the H&E staining, asterisks indicate tumor (Data are modified from Zhang et al. [158], with permission)

and results in endothelial cell migration, invasion and tube formation, aspects of angiogenesis, a critical process that aids the dissemination of tumor cells [40]. Src can also phosphorylate VE-Cadherin, a cell adhesion molecule that mediates vascular cell-cell junction, leading to increased vascular permeability and facilitating entry of tumor cells into blood vessels [44]. c-Src is also a key mediator of bone resorption due to its essential role in osteoclast function [45]. Disruption of Src signaling in osteoclasts results in decreased cell motility and prevents the formation of ruffled membranes, a key step during bone resorption [46]. Mice injected with human breast cancer cells with a constitutively active Src developed increased osteolytic bone metastases [47]. In a highly metastatic breast cancer subline c-Src knockdown significantly decreased outgrowth of the bone lesions; this could be rescued by c-Src protein reintroduction (Fig. 7.2). Taken together, the role of c-Src in bone microenvironment makes it an attractive target for anti-metastatic therapy in cancers with osteolytic lesions such as breast, lung, prostate, and kidney.

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Src Signaling Pathways

Src participates in the activation of various downstream pathways through molecular interactions with a host of proteins including trans-membrane receptors, such as growth factor receptors and G protein-coupled receptors, and other non-receptor tyrosine kinases including focal adhesion kinase (FAK) and signal transducer and activator of transcription proteins (STATs) [37, 48]. As a biological consequence of interactions with Src, each of these effectors functions in unique signaling pathways. FAK, a key protein in modulating the formation and turnover of focal adhesions, is perhaps the best known partner of c-Src. FAK and c-Src have been shown to form a transient, active complex following integrin engagement by extracellular matrix proteins. As a consequence both kinases are activated on at least four different tyrosine residues, leading to amplification of FAK kinase activity and triggering of other signaling molecules [49]. Upon association, the FAK/c-Src complex activates a cascade of downstream cytoskeletal components including paxillin and p130Cas, recruits ERK, Jun kinase (JNK) and Rho GTPase pathways, and triggers actin stress fiber rearrangement, focal adhesion and cell migration. Recent studies have also documented that both c-Src and FAK catalytic activities are important in promoting VEGF-associated angiogenesis and protease-associated invasion, suggesting that c-Src cooperating with FAK generates signals leading to tumor growth and metastasis [50]. In addition to VEGFR, c-Src has also been shown to be downstream of a number of other growth factor receptors, including epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) [37]. Thus upon activation, c-Src can amplify a great number of critical signaling pathways and effector molecules that are responsible for migration, invasion, survival, and angiogenesis [37, 38, 51].

7.3.2

Met Signaling and Metastasis

Receptor tyrosine kinase c-Met is encoded by MET proto-oncogene. Oncogenic c-Met signaling is widely implicated in various human malignancies and the expression of both the hepatocyte growth factor (HGF) ligand and the c-Met receptor has been correlated with tumor progression and poor prognosis in patients. Although c-Met is normally activated by HGF under physiological conditions, abnormal c-Met activation is typically observed in human cancers [52, 53] and the HGF/c-Met axis has been linked to malignant transformation and metastasis [54]. In lung cancer for example, expression levels of both HGF and c-Met have been associated with advanced tumor stage and worse clinical outcome [55]. In prostate cancer, increased serum levels of HGF have served as an independent prognosticator in advanced disease [56]; also, Met is expressed more frequently in metastatic lesions than in primary tumors [57], with 100% of prostate cancer bone metastases being Met-positive [58]. These findings support the notion that elevated HGF and c-Met may be used as predictive markers for metastatic formation in cancer patients.

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Table 7.1 Abnormal MET status and metastasis in human cancers MET status Cancer types Metastatic phenotypes HGF-dependent Paracrine loop SCC Scattering, lung metastasis [70] Thyroid cancer Morphology, invasion, adhesion [71] HCC Morphology, motility [72] Prostate cancer Scattering, motility, invasion [73] Autocrine loop NSCLC Spontaneous metastasis [74] Melanoma Liver metastasis [75] HGF-independent Gene amplification Activating mutation

Crosstalk with other proteins Overexpresison

CRC Multiple cancers HNSCC HNSCC NSCLC Breast cancer CRC Melanoma

Liver metastasis [65, 76] Migration, invasion, lymph node and pulmonary metastasis [77] Lymph node metastasis [63] Distant metastasis [78] Morphology, motility [79] Migration [80] Liver metastasis [81] Motility, invasion, lung metastasis [82]

HNSCC head and neck squamous cell carcinoma, HCC hepatocellular carcinoma, NSCLC nonsmall cell lung carcinoma, CRC colorectal cancer

7.3.2.1

c-Met Is Aberrantly Activated in Human Cancers

c-Met is one of the most common abnormally activated signaling molecules in human cancers [59]. Alterations in MET gene amplification as well as activating mutations and crosstalk between the c-Met receptor and other signaling molecules have been reported to result in constitutive activation of the c-Met kinase independent of the HGF ligand [60, 61]. These abnormalities have been found in papillary renal cell carcinoma [62], head and neck cancer [63], gastric cancer [64] and metastatic colorectal cancer [65]. In some cases, tumor cells express both HGF and c-Met, thus establishing an autocrine loop in which tumor cell secreted HGF binds to c-Met and causes its activation. Such HGF-dependent autocrine c-Met activation is considered to be a self-supportive mechanism involved in cancer cell transformation, proliferation and survival. It has been detected in a variety of human primary and metastatic tumors, including breast cancers [66], gliomas [67] and osteosarcomas [68]. Although aberrant MET activation occurs in only some cancer types, in a majority of human malignancies wild-type c-Met over-expression, due to MET mRNA up-regulation, is a predominant event [69]. In this circumstance, the c-Met receptor still depends on the HGF ligand for activation, but increased expression of c-Met on the cell surface would favor HGF-independent activation through spontaneous receptor dimerization [61]. The contribution of the abnormal MET status on tumor progression and response to therapy has been widely studied. In addition, there exists a growing body of preclinical and clinical evidence that

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supports the notion that a tumor’s metastatic potential can be affected by HGF/c-Met axis abnormalities. Selected examples are shown in Table 7.1.

7.3.2.2

c-Met, Invasive Growth and Metastasis

Functionally, c-Met orchestrates invasive growth by activating multiple cell functions including proliferation, scattering, migration, invasion, branching morphogenesis, and angiogenesis [83, 84]. Under normal conditions the HGF/c-Met axis is essential for embryogenesis and tissue homeostasis, while in cancer, both the ligand (HGF) and the receptor (c-Met) have been closely linked to the activation of invasive growth and metastatic spread [83]. Activating somatic mutations of MET and associated with increased aggressiveness and extensive metastases [77]. Similarly, the induction of HGF/c-Met autocrine loops in cell lines or in transgenic animals can lead to the generation of invasive tumors [75]. HGF-binding enhances the potential of invasive growth that contributes to tumor cell dissemination in cancer cells with highly expressed c-Met [85]. For example, in PC-3 prostate cancer cells that over-express c-Met [73, 86], human HGF induces cell scatter, migration, invasion (Fig. 7.3a) and clonogenic growth (Fig. 7.3b). The process of cell scattering can be divided into three phases, namely cell-cell dissociation, cell migration, and cell spreading [87]. The scattered phenotype is typically induced by HGF (hence its original designation as scatter factor) [88]. Mechanistically, HGF/c-Met acts as a stimulator to increase cell migration and invasion (Table 7.1) by promoting cytoskeleton rearrangement and stimulating the production of proteases responsible for basement membrane degradation [87]. HGF also functions as a potent pro-angiogenic molecule that directly stimulates endothelial cell migration, invasion and tube formation [89]. Furthermore, HGF can promote the production of other angiogenic factors in tumor cells [90], thus amplifying tumor angiogenesis. MET activation may also lead to cell survival advantages as suggested by the finding of MET-induced protection from anoikis [91], an anchorage-independent cell death mode that negatively affects tumor cell dissemination [92].

7.3.2.3

HGF/c-Met Signaling Pathways

The c-Met protein is synthesized as a 170 kDa precursor which is glycosylated and proteolytically cleaved into a 50 kDa a-chain and a 140 kDa b-chain thus forming a heterodimeric molecule. Typically, c-Met is autophosphorylated by HGF at tyrosines 1234 and 1235 in the activation loop of the kinase domain in order to achieve full enzyme activation [51]. Phosphorylated c-Met further triggers activation of two main downstream signaling pathways, Ras-mitogenactivated protein kinase (MAPK) and phosphoinositide 3-kinases (PI3K)-Akt (Fig. 7.3c), via the adaptor proteins Gab-1 and Grb2 [85]. Both ERK and PI3K pathways have been found to be involved in cell spreading; HGF-induced cancer cell scattering is dependent on the activity of Rho family GTPases mediated by

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Fig. 7.3 Effects of human HGF (25 ng/ml) on cellular functions and c-Met signaling pathways in PC-3 cells. (a) Cells were treated with HGF for 24 h. Cell scattering, motility and invasion were detected using a scatter assay, a wound-healing assay, and a Matrigel-based chamber assay, respectively. Magnification, ×5. (b) Cells (700 cells/well) were seeded into a 24-well plate and incubated with HGF for 10 days. Cell proliferation was assessed using an MTT assay. Columns, mean; bars, SD (n = 6). ***P < 0.001 (t-test). (c) Serum-starved cells were exposed to HGF for 10 min. c-Met signaling molecules were shown by Western blot analysis. C = control (Data are adapted from Dai and Siemann [73])

the PI3K pathway [93]. Met also activates the migration-associated components Ras, Rac1 and p21-activated kinase (PAK) and controls cytoskeletal rearrangement, cell adhesion and cell motility [94]. PI3K activation of AKT also promotes cell survival. c-Src is another downstream target of c-Met [95]. In addition, other signaling molecules, such as Jun amino-terminal kinase (JNK), nuclear factor-kB (NF-kB) and b-catenin, can participate in the HGF/c-Met pathway [52]. Overall, Met signaling is highly complex and involves distinct but interacting cascades [53]. In addition to ligand activation, c-Met can also be transactivated by other transmembrane receptors. For example, EGFR can physically interact with c-Met in tumor cells [96] such that c-Met is phosphorylated by an EGFR ligand in the absence of HGF [96]. Reciprocally, HGF/c-Met signaling may induce expression of EGFR ligands thus leading to EGFR signaling activation [97], suggesting a crosstalk

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between these two receptor tyrosine kinases. In addition to EGFR, other receptors including HER2 [98] and Ron [99], another Met family member, have been shown to interact with c-Met to enhance the malignant phenotype.

7.4

Interplay Between HIF-1, c-Src and c-Met Signaling Pathways

The crosstalk between HIF-1 and other signaling pathways has been extensively studied [12]. Under hypoxic conditions HIF-1 up-regulates pro-survival and proinvasive signaling pathways such as Akt [100] and c-Met [101] while under aerobic conditions HIF-1 is regulated by a variety of genetic factors including SRC [12].

7.4.1

Hypoxia, HIF-1 and c-Src Signaling

Although data concerning the role of c-Src signaling under hypoxic conditions are somewhat limited, it has been suggested that Src activation may promote survival of hypoxic cells [102]. A recent study in pancreatic and cervical carcinoma xenografts also indicated that the expression levels of total Src protein were higher in the chronically hypoxic regions of the tumors [103]. Several other studies have reported that c-Src is required for increased expression of VEGF. In the presence of oxygen, consistent with the finding that constitutively activated c-Src leads to elevated HIF-1a expression [104], Jiang et al. reported that increased HIF-1a synthesis and VEGF gene activation are caused by SRC gene activation [105]. In contrast, oxygen deprivation can increase the kinase activity of c-Src leading to up-regulation of VEGF mRNA [106]. Furthermore, Src-dependent hypoxia-induced VEGF expression appears to be regulated by STAT3 activation and HIF-1a accumulation [107]. Clearly, definitive insights into the relationship between HIF-1 and c-Src signaling are lacking and continuing exploration of possible interactions of these pathways is needed.

7.4.2

Hypoxia, HIF-1 and HGF/c-Met Signaling

Hypoxia directly promotes motility and invasion by inducing MET transcription [101, 108, 109] and sensitizing cells to HGF stimulation [101]. This invasive phenotype can be reversed by MET [101] or HIF-1a [108] knockdown. Since the HGF/c-Met pathway induces both the MET gene and HIF-1 activity [13], amplification of HGF-induced c-Met signaling by hypoxia provides a molecular mechanism by which low oxygen tensions such as can occur in solid tumors could cause malignancies to become more aggressive, invasive and metastatic. Although this possibility is intriguing, it should be noted that the MET gene is not always found to be up-regulated by oxygen deprivation [110–112].

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With regard to an association between HIF-1 and c-Met, immuno-histochemical studies in breast cancer patients showed HIF-1a and MET to be positively correlated, independent predictors for distant metastasis, and associated with poor survival [113]. These findings strengthen the possibility that tumor hypoxia promotes metastasis through MET induction. Similar relationships have been reported for pancreatic adenocarcinoma [114] and papillary thyroid carcinoma cells [115]. However, MET transcription and cell invasiveness can also be induced by the genetic inactivation of VHL, which leads to HIF-1a stabilization in the absence of hypoxia and suggests an aerobic regulation of c-Met by alterations of hypoxia-associated proteins [116]. Collectively, the current uncertainties surrounding the effects of hypoxia on MET expression may reflect the complexities of the crosstalk between c-Met receptor and HIF-1 signaling.

7.4.3

Interplay Between c-Src and the HGF/c-Met Axis

c-Src and c-Met are two critical signaling molecules that trigger multiple similar cell functions including migration, invasion, survival, and angiogenesis. Not surprisingly increased activation of both c-Src and c-Met have been associated with enhanced metastatic potential in cancer cells. Although a direct physical interaction between these two functional related proteins has not been reported, the molecules do display close relationships at least in a certain cancer types. In breast cancer, activation of c-Src results in elevated HGF expression and activation of autocrine HGF/c-Met signaling [117]. c-Src also is activated in response to HGF stimulation in both breast carcinoma cells and breast epithelial cells; corresponding to a coassociation between c-Met and c-Src [118]. In prostate cancer cells, c-Src activation of MAPKs plays a key role in HGF-mediated cell protection against chemotherapeutic agents [119]. In colorectal cancer, c-Met activation of c-Src was observed to be critical in mediating tumorigenesis and liver metastasis [120]. In addition, a majority of colorectal cell lines found to express high Src family kinase activity also demonstrated activated c-Met [121]. Moreover, inhibition of Src reduced c-Met phosphorylation in most cases, indicating a reversed signal flow from Src to c-Met [121]. All these findings indicate that c-Src can function both as an upstream initiator and a downstream effector of c-Met, clearly highlighting the potential association between these two key signaling molecules.

7.5

Therapeutic Interventions Targeting Hypoxia/HIF-1, Src, or Met Signaling to Inhibit Metastasis

The development of novel therapeutic strategies that impair the metastatic process is critical to improving cancer patient survival. Two approaches to interfere with the dissemination of cancer cells are to target the regulation of signaling pathways associated with invasiveness and angiogenesis, essential components of the

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metastatic cascade. Given their pleiotropic roles in microenvironmental responsive signaling cascades, targeting HIF-1, c-Src and c-Met would be expected to have therapeutic efficacy in constraining tumor cell dissemination.

7.5.1

Targeting Hypoxia and HIF-1

Given its central role in tumor cell response to oxygen deprivation, HIF-1 has become an attractive target for drug development [27]. HIF-1 inhibitors can tentatively be divided into several categories that target different processes during HIF1a activation, including mRNA expression, transcriptional activity, protein translation, DNA binding, and protein degradation [146]. Interestingly, most of these inhibitors were not developed as HIF-1 inhibitors per se; belonging instead to a wide variety of molecules including signaling pathway inhibitors, chaperone inhibitors, chemotherapeutic agents and natural compounds [147]. Many of the targeted compounds have been found to have antiangiogenic effects likely due to the elimination of HIF-1 activity in tumor cells and/or endothelial cells. Such agents include gefitinib (EGFR inhibitor), trastuzumab (HER2 inhibitor), and rapamycin (mTOR inhibitor). Although the primary focus of such agents has been the assessment of their effects on primary tumor growth and angiogenesis, studies evaluating the impact of HIF-1 inhibitors on metastasis have begun (Table 7.2).

7.5.2

Targeting Src Kinase

The pivotal role of Src in neoplastic cell progression has made it a relevant target in cancer management. Currently, the three lead small molecule ATP competitors, dasatinib, saracatinib and bosutinib, are being evaluated in clinical trials for many different types of solid tumors [40]. Preclinical studies are promising and indicate that Src kinase inhibitors can affect not only primary tumor growth [148], but also the development of secondary metastases [127, 128, 149–151]. For example, in vitro, dasatinib inhibits PC-3 cell adhesion (Fig. 7.4a), migration (Fig. 7.4b) and invasion (Fig. 7.4c) as well as significantly reducing prostate cancer cell induced angiogenesis (Fig. 7.4d). Saracatinib and bosutinib also have been shown to effectively suppress the metastatic phenotype in vitro and impair the formation of metastases in vivo in a variety of cancer models (Table 7.2), further supporting the potential of Src inhibitors as anti-metastatic agents.

7.5.3

Targeting Met Kinase

Dysregulation of c-Met signaling is strongly associated with metastasis and poor prognosis in multiple cancers including carcinomas, sarcomas, and gliomas, making MET a potentially important prognostic indicator for cancer progression [87].

ATP-competitor

ATP-competitor

ATP-competitor

ATP-competitor

ATP-competitor ATP-competitor ATP-competitor ATP non-competitor

Src targeting Dasatinib

Saracatinib

Bosutinib

Met targeting SU11274

BMS-777607 PHA-665752 PF-2341066 ARQ197

Melanoma; Multiple cancers; Ovarian cancer Prostate cancer Multiple cancers Lung carcinoma Lung carcinoma; Colon carcinoma

Prostate cancer [135]; Breast cancer [136, 137]; Colon cancer and lung cancer [138]

Bladder cancer [129]; Sarcoma [130]; Prostate cancer [131, 132]; HSCC [133]; Breast cancer [134]

Pancreatic cancer [125]; Melanoma [126]; Prostate cancer [127, 128];

Migration, liver metastasis [139]; Scatter, invasion, tubulogenesis [140]; Motility, invasion [141] Scatter, motility, invasion [73] Morphology, motility, invasion [142] Invasion [143] Invasion [144]; Liver metastasis [145]

Migration [125, 128]; Invasion [125, 128]; Lung metastasis [126]; Secondary growth [127]; Lymph node metastasis [128] Migration [129, 130, 132, 134]; Invasion [129–131, 133, 134]; Scatter [129]; Adhesion [130]; Lymph node metastasis [129, 133]; Lung metastasis [130]; Bone metastasis [131] Migration [136]; Invasion [135, 136]; Extravasation [138]; Skeletonal metastasis [135]; Spontaneous metastasis [137]

Table 7.2 Selective small molecule inhibitors targeting HIF-1, Src or Met for the inhibition of metastasis in preclinical studies Compounds Targets Cancer models Disrupted functions HIF-1 targeting 2-methoxyestradiol HIF-1a translation Gastric adenocarcinoma Migration, invasion, adhesion, systemic tumor inhibitor spread [122] YC-1 Multiple mechanisms Hepatoma Migration, invasion, lung and liver metastasis [123] Vitexin Multiple mechanisms Multiple cancer cell lines Migration, invasion, angiogenesis [124]

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Fig. 7.4 Effects of dasatinib on PC-3 cell functions. (a) Sub-confluent cells were treated with dasatinib for 24 h and floating cells were counted using a hemocytometer. (b) A confluent cell monolayer was scratched and exposed to dasatinib (10 nM) for 24 h. Magnification, ×5. (c) Cells were seeded into Matrigel-based chambers (104/well) using serum as a chemo-attractant and incubated with dasatinib for 24 h. Invaded cells were counted under a microscope. (d) Dasatinibpretreated (24 h) cells were injected (105 cells/site) intradermally in four locations on the chests of nude mice. Three days later the skin flaps were removed and the blood vessels grown to the tumor sites were counted using a dissecting microscope. (a and c): Columns, mean; bars, SD (n = 3); (d): Whiskers, 90th (above) and 10th (below) percentiles (n = 12). *P < 0.05; **P < 0.01 [compared to control, t-test for (a and c), Wilcoxon rank sum test for (d)]

The prevalence of HGF/c-Met pathway activation in human malignancies has led to rapid and extensive growth in the development of agents targeting this signaling pathway. Many intervention strategies have been developed including antagonistic compounds (competitors), monoclonal antibodies (mAbs) and small molecule kinase inhibitors [152]. Therapeutic mAbs specifically blocking c-Met activation are being evaluated in patients with advanced malignancies. These include mAbs

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targeting both HGF (AMG102) and c-Met (MetMAb) [59]. Although neutralizing antibodies have shown promise when inhibiting HGF-stimulated functions, Met kinase inhibitors may have a broader range of application, having found to be effective in cancers driven by HGF [153], Met [154], or both [74, 155, 156]. To date, the primary clinical evaluations of these compounds have focused on their ability to inhibit primary tumor growth. However, preclinical studies clearly indicate that Met targeting agents may have considerable utility in interfering with the metastatic process (Table 7.2).

7.5.4

Combining Agents to Target Cancer Cell Dissemination

Metastasis is a complex process. Although targeted therapies have shown some promise in treating metastatic disease, successful therapeutic interventions are likely to require a combinatorial agent treatment approach. Given the heterogeneity of malignant cells [3] and the complexity and redundancy of molecular events involved in the metastatic process, eliminating one aspect of the metastatic cascade may still allow another to proceed uninterrupted [157]. Thus the simultaneous targeting of two or more signaling pathways involved in the metastatic process should provide superior outcomes than either intervention alone. For example, targeting both HIF-1 and c-Src may provide complimentary treatment strategies for controlling cancer cell spread from primary tumors containing hypoxic microenvironments. In cancers in which c-Src and c-Met are active, the combined targeting of both these two kinases should result in superior therapeutic efficacy. Ultimately a better understanding of the tumor microenvironment and molecular signatures in involved in cancer cell dissemination will offer opportunities to optimize the application of novel targeting agents alone or in combination to improve treatment outcomes.

7.6

Concluding Remarks

Hypoxia, a common feature of human cancers, is known to accelerate tumor cell dissemination. It is generally believed that in most cases HIF-1links the hypoxic cell response to metastatic progression. Indeed, positive correlations between tumor hypoxia and increased metastatic capacity have been suggested by both clinical and preclinical investigations. Altered intracellular signaling pathways such as c-Src and HGF/c-Met axis also have been identified as key contributors to the metastatic phenotype. Evidence exists to support the notion that such signaling cascades may be amplified by oxygen deprivation. Furthermore, the interrelated pathways of signaling molecules, HIF-1, c-Src and c-Met may cooperate to promote cell functions that lead to an elevated metastatic potential. The metastatic process of cancer cells may therefore be independently augmented by both extrinsic environmental factors (hypoxia) and intrinsic cellular features (dysregulated signaling pathways) (Fig. 7.5).

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Fig. 7.5 Scheme of hypoxia/ HIF-1, c-Src kinase and the HGF/c-Met axis in the metastatic process

Given the pivotal role of hypoxia/HIF-1, c-Src and c-Met in tumor cell dissemination, small molecule therapeutic agents targeting these pathways have significant potential to interfere with the metastatic phenotype. While the impact of HIF-1 inhibitors remains to be investigated, small molecules targeting Src and Met kinases have been efficacious in impeding metastatic disease. Clearly it is of interest to explore the strategy of combining therapeutic interventions targeting both the tumor microenvironment and the intracellular signaling pathways associated with the spreading of cancer cells. Establishment of such combinatorial treatment modalities holds great promise to successfully interrupt cancer metastasis.

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Part II

Homing and Colonization at Distant Sites

Chapter 8

Cellular and Molecular Biology of Cancer Cell Extravasation J. Matthew Barnes and Michael D. Henry

Abstract Hematogenous metastasis involves the entry of cancer cells into the circulation at a primary tumor site and the extravasation of those cells at a secondary organ which may ultimately support the growth of a metastatic tumor. Although extravasation is likely an obligate step in metastasis, it is relatively poorly understood in part due the difficulty studying this process in appropriate experimental models in vivo. Thus, there remain open questions about how cancer cells interact with the vascular wall during this process including the extent to which non-selective mechanisms such as size restriction versus specific adhesive interactions determine the behavior of extravasating cancer cells; how cancer cells cross the endothelium; and the degree to which extravasation limits the overall efficiency of metastasis. The answers to these questions are influenced by specific properties of both the cancer cells and the nature of the vascular bed involved. In this Chapter, we review our current understanding of the cellular and molecular biology of cancer cell extravasation and discuss how this knowledge impacts clinical issues related to the biology of circulating tumor cells and cancer therapy.

Abbreviations CTC DTC EMT

Circulating tumor cell Disseminated tumor cell Epithelial-to-mesenchymal transition

J.M. Barnes • M.D. Henry (*) Departments of Molecular Physiology and Biophysics and Pathology, Holden Comprehensive Cancer Center, University of Iowa Carver College of Medicine, Iowa City, IA, USA e-mail: [email protected]


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EpCAM MET SCID TEM

8.1

Epithelial cell adhesion molecule Mesenchymal-to-epithelial transition Severe and combined immune deficient Trans-endothelial migration

Introduction

Metastasis, or the spread of cancer cells from a primary tumor to a distant tissue, is associated with the lethal consequences of cancer progression. Metastasis is comprised of a series of events, beginning with local tissue invasion, followed by entrance into the bloodstream or lymphatic system, survival within circulation, extravasation (exit from the vasculature), and finally colonization of and proliferation at a secondary organ site. The focus of this chapter is on cancer cell extravasation, a topic which remains poorly understood in part because tracking the fate of individual cancer cells from the circulation into the vascular beds of various organs remains a technically daunting task. Nonetheless both in vivo and ex vivo analyses have provided insight into the cellular and molecular mechanism of cancer cell extravasation. Although leukocyte diapedesis has provided an instructive paradigm for understanding cancer cell extravasation, the latter exhibits numerous differences and it is likely that the mechanism of cancer cell extravasation is both cell-type and organ-specific. Another aspect of this process which remains a matter of some debate is the extent to which metastatic efficiency is limited by cancer cell extravasation. Here, we discuss evidence from both sides of this issue. Finally, we discuss the relationship between epithelial-to-mesenchymal transition (EMT) and cancer cell extravasation. A growing body of evidence indicates that EMT is one mechanism driving the invasive phenotype of various cancers, but the role of EMT in extravasation has been unclear. Recent data suggests that EMT phenotype augments the ability of prostate cancer cells to extravasate, in part through modulating interactions between cancer cells and endothelial cells. A better understanding of the connection between EMT and extravasation capability may have an impact on the analysis of circulating tumor cells, as well as provide potential therapeutic targets to forestall metastatic disease.

8.2

Mechanisms of Cancer Cell Extravasation

In order for circulating tumor cells (CTCs) to form metastatic tumors, they must exit the bloodstream and enter the parenchyma of a secondary tissue. Extravasation involves passage of cells across the endothelium, termed transendothelial migration (TEM), as well as transit through the subendothelial basal lamina. Several mechanisms of cancer cell extravasation have been reported, and this variation may be due in part to differences in experimental models. Results from many groups support a model by which cancer cells adopt certain leukocyte-like properties allowing them

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to adhere to endothelial cells by specific and dynamic cell surface interactions, thereby facilitating passage between or through the endothelial cells and through the underlying basement membrane. Others report static, nonselective mechanisms of cancer cell adhesion followed by either TEM, often involving retraction of endothelial cells, or proliferation within the lumen of the vessel.

8.2.1

Leukocyte Versus Cancer Cell Extravasation

Leukocyte extravasation is a highly regulated process which allows immune cells to home to lymphatic and other tissues throughout the body. The cellular and molecular biology of leukocyte extravasation is generally well understood and has been reviewed recently [1–3]. Each step of leukocyte extravasation is mediated by specific combinatorial interactions between a receptor and a ligand. Temporal and spatial regulation of some of these molecules dictates the selectivity of a leukocyte for a given tissue. Selectins are surface glycoproteins expressed on the endothelium which initiate rolling of leukocytes by binding to various selectin ligands on the surface of these cells. While rolling, leukocytes encounter a gradient of chemokines presented by endothelial cells. Subsequent activation of chemokine receptor signal transduction facilitates changes in the affinity and avidity of leukocyte integrins for their endothelial ligands, the intercellular adhesion molecules (ICAMs). This results in integrin-mediated firm adhesion of leukocytes which is followed by diapedesis. Cancer cells of various histological origins have been shown to express cell surface proteins known to be important in leukocyte extravasation, including cell adhesion and chemokine receptors which interact with corresponding ligands at the luminal surface of the endothelium, and this has been implicated in the organ tropism of metastasis as recently reviewed [4, 5]. As an example, bone marrow endothelial cells constitutively express the chemokine SDF-1 and the adhesion molecules VCAM-1 and E-selectin, which are used to facilitate adhesion of circulating leukocytes [6]. Numerous reports indicate that a variety of cancers express the SDF-1 receptor, CXCR4 [7]. Antibody neutralization of CXCR4 has been shown to greatly reduce metastatic colonization of breast cancer cells in mice [8]. In a panel of prostate cancer cells, CXCR4 expression levels were shown to correlate with metastatic aggressiveness, and targeting this receptor with a small molecule, T140, reduced chemotactic invasion of PC-3 cells towards SDF-1 [9]. Extravasation of PC-3 cells has also been shown to be facilitated by expression of monocyte chemoattractant protein-1 on bone marrow endothelial cells [10]. In vitro-derived bonetropic clones of the MDA.MB.231 cell line have been shown to overexpress CXCR4 as compared to the heterogeneous parent [11]. Recently Jamieson et al. detected overexpression of the monocyte chemokine receptor, CX3CR1, in human prostate cancer tissue samples and show that its ligand, fractalkine, is expressed on the plasma membrane of osteoblasts, mesenchymal, and other stromal cells of the bone marrow [12]. Furthermore, dihydrotestosterone induces cleavage of stromal, but not endothelial plasma membrane-associated fractalkine; thus establishing a soluble chemotactic gradient promoting prostate cancer cell homing to bone and ultimately

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adhesion to the bone marrow endothelium [12]. Forced expression of the E-selectin ligand, Sialyl Lewis X, in PC-3 prostate cancer cells has been shown to induce cell rolling and adhesion to IL-1b-stimulated bone marrow endothelial cells in vitro and this interaction is blocked with anti-E-selectin antibody [13]. These and other studies support a role for leukocyte extravasation molecules and activated endothelial cells in the bone tropism of cancers such as breast and prostate. However, chemokine signaling may play roles relevant to metastasis beyond extravasation [14, 15]. On the other hand, N-cadherin is an example of an adhesion molecule implicated in cancer cell extravasation, which is not known to be involved in leukocyte diapedesis. N-cadherin is well positioned to be involved in the association between cancer cells and endothelial cells as it is present on the luminal surface of endothelia and not enriched at cell junctions [16]. Melanoma cells have been shown to adhere to endothelial cells via homophilic N-cadherin binding which leads to retraction of the VE-cadherin and PECAM junctions between neighboring endothelial cells [17]. Engagement of melanoma cell N-cadherin leads to transactivation of Src kinase and nuclear localization of b-catenin, which facilitate the formation of pseudopodia into the disrupted endothelial cell junction and subsequent TEM to the basal side of the endothelium [18, 19]. Similarly, siRNA-mediated knockdown of N-cadherin in PC-3 prostate cancer cells was shown to reduce TEM [20]. Although the signaling consequences of N-cadherin binding were not addressed here, PC-3 TEM has been shown to involve Rac1-mediated invadapodia formation [10, 21]. Most of the in vitro studies of extravasation discussed above are performed by seeding cancer cells onto a monolayer of endothelial cells growing on a semipermeable transwell insert or on a layer of extracellular matrix proteins. While this provides a convenient model for extravasation, tumor cells in vivo are in the presence of flowing blood cells. To more accurately mimic physiological conditions, dynamic in vitro models of extravasation have been used in which mixtures of cancer cells and leukocytes are subjected to flow through chambers lined with endothelial monolayers. Using this approach, evidence has been provided for cooperation between leukocytes and cancer cells in extravasation. Strell et al. showed that breast cancer cells, which lack b2-integrin but express its ligand ICAM-1, bind to b2-expressing neutrophils and this hetero-cellular complex then binds to the endothelial cells via ICAM-1 [22]. These results are in agreement with other reports of neutrophilassisted extravasation of breast cancer cells [23] and melanoma cells [24]. Platelets have also been shown to bind to cancer cells of various types, often through selectin-mediated interactions, leading to formation of emboli which exhibit enhanced adhesion to the endothelium [25–28]. It may be the case that certain cancer cells are actually dependent upon leukocyte and/or platelet interactions in order to form metastatic tumors, which is interesting considering the growing understanding of the role of inflammation in cancer progression and metastasis [29]. Although leukocytes provide an attractive mechanistic paradigm for understanding cancer cell extravasation, the selective pressures which would drive epithelial cells to acquire detailed leukocyte gene expression patterns and cellular behavior are not obvious. As discussed in the following sections, cancer cell extravasation does not follow a strict paradigm and has been reported to occur by a number of mechanisms,


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which appear to be both cell type- and organ-specific. One feature of cancer cell extravasation, observed primarily in vitro, is that cancer cells induce the retraction of endothelial cells, unlike leukocytes [30]. There are even alternatives to classical extravasation, such as expansive intraluminal proliferation, which have been proposed as a means for metastatic colonization.

8.2.2

Cancer Cell Adhesion to the Endothelium in Extravasation

Many studies on extravasation have focused on the mechanisms of tumor cell adhesion to the endothelium, as this is presumed to be the initiating event in this process. Here, intravital videomicroscopy and detailed accounting of cancer cell fate following systemic injection have provided important insights into the process of extravasation in vivo. These studies are summarized in Table 8.1. Two general mechanisms are thought to be involved: (1) non-selective and passive lodgment of cancer cells in the microvasculature by size restriction and (2) selective adhesion between cancer cells and the vasculature of particular organ sites, including larger diameter vessels. Although these mechanisms are often regarded to be in conflict, both size restriction and selective adhesion could influence the overall process of extravasation. Determining the relative contribution of these mechanisms will be important in potential therapeutic contexts.

8.2.2.1

Mechanical Arrest of Circulating Tumor Cells in the Microvasculature

Ewing first proposed that cancer cells become entrapped in the first capillary beds they encounter; thus contributing to organ-site preference for metastasis [31]. Intuitively, this mechanism derives from the fact that epithelial-derived cancer cells are typically larger than the largest of the white blood cells (human monocytes range from about 12–20 mm in diameter [32]). Although CTCs are deformable and may thus be able to negotiate this restrictive barrier to some degree, they may not be as successful as leukocytes [33, 34]. CTCs would thus seem susceptible to size restriction when encountering the microvasculature (human capillaries are as small as 7 mm in diameter [35]). Indeed, it has long been appreciated in experimental mouse models of metastasis that cancer cells injected into the tail vein accumulate predominantly in the lung [36]. Here, this is demonstrated utilizing bioluminescence imaging (Figs. 8.1 and 8.2). Early efforts aimed at observing the behavior of CTCs in the microvasculature of the rabbit ear described the association of cancer cells with the endothelium and subsequent extravasation, but did not conclude that size restriction was the sole means of cancer cell arrest at this site [37]. However, the pioneering work of Ann Chambers, Alan Groom, and colleagues utilizing in vivo video microscopy have provided abundant support for the concept of size restriction in the lung and other organs [38–43].

FVB/N mouse

Nude mouse

Nude and SCID mouse SD and Nude rat

C57BL/6 mouse

SD rat

C57BL/6 mouse

Human breast

Human lung and melanoma

Human breast

Mouse melanoma

Human and rat colon

Mouse mammary

Human colon

Host FVB/N mouse

Cell type Human breast

Mesenteric vein

Intra-arterial

Tail vein

Intra-arterial

Tail vein

Carotid artery

Spleen

Injection route Tail vein

Liver

Liver

Lung

Liver

Lung

Brain

Liver

Organ site Lung

Table 8.1 Selected references reporting cancer cell extravasation in vivo Observation technique and timeline 2PM –resected and perfused tissue At 2 h, 2% of cells had extravasated At 24 h, 22% of cells had extravasated 2PM – resected and perfused tissue At 2 h, 4% of cells had extravasated At 24 h, 56% of cells had extravasated MPLSM – living animals Extravasation occurred between days 1–9 >36% lung cancer cells extravasated >45% melanoma cells extravasated 2PM – resected and perfused tissue Extensive extravasation at 48 h IVVM – living animals 5–30 min (peak at 20 min) ESCM – fixed and embedded tissue At 5 h, nearly all cells had extravasated IVVM Most cells had extravasated at 30 min IVVM – living animals At day 3, >80% of cells had extravasated Size restriction

Specific adhesion

Not directly addressed

Specific adhesion

Not directly addressed

Size restriction

Specific adhesion

Primary method of CTC arrest Specific adhesion

Luzzi [41]

Haier [70]

Voura [17]

Schluter [51]

Gupta [78]

Kienast [44]

Martin [69]

Reference Martin [69]

202 J.M. Barnes and M.D. Henry

Chick embryo CAM

Nude mouse

Chick embryo CAM

Mouse melanoma

Mouse mammary

Mouse melanoma

CAM vein

Mesenteric vein

CAM vein

Injection route

CAM

Liver

CAM

Organ site IVVM Extravasation started at 2 h At 24 h, >80% of cells had extravasated IVVM – living animals At day 1, 51–63% of cells had extravasated At day 21, 97–100% of cells had extravasated IVVM After 8 h, 83% of cells had extravasated

Observation technique and timeline

Size restriction

Size restriction

Size restriction

Primary method of CTC arrest

Chambers [43]

Morris [39]

Koop [40]

Reference

MPLSM multi-photon laser scanning microscopy, 2PM two-photon-microscopy, IVVM in vivo videomicroscopy, ESCM emission scanning confocal microscopy, FM fluorescence microscopy, REF rat embryonic fibroblast, SCID severe and combined immune deficient, SD Sprague-Dawley, CAM chorioallantoic membrane

Host

Cell type

8 Cellular and Molecular Biology of Cancer Cell Extravasation 203

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Fig. 8.1 Long-term bioluminescence imaging of tail vein injected cancer cells. A luciferase expressing metastatic derivative of PC-3 prostate cancer cells was injected systemically into SCID mice via the tail vein route. In an image taken minutes after injection, these cells are observed arresting in the lung (a). At 2 months post-injection, colonization of various tissues is observed (b, dorsal image on top, ventral image on bottom). Note that not all mice develop tumors in the region of the lungs, despite their original arrest in this organ


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Fig. 8.2 Short-term serial bioluminescence imaging of tail vein injected cancer cells. Luciferase expressing B16.F10 melanoma cancer cells were injected systemically into SCID mice via the tail vein route. At the indicated time points after injection, bioluminescence images were obtained. Note that by 48 h, nearly all of the signal seen at 4 h is gone, indicating clearance of cells from the lung vasculature. Between 120 and 168 h, bioluminescence signal steadily increases, indicating outgrowth of lung-colonized cancer cells. Graphical quantification of the bioluminescence images is shown at the bottom of the figure

Recently, monitoring circulating lung carcinoma and melanoma cells in the brains of living mice via multi-photon microscopy over an extended time course, Kienast et al. have stunningly visualized detailed, individual steps of extravasation [44]. Minutes after injection, all adherent cells were reported to be arrested by sizerestriction in vessels of similar diameter than the cancer cells or in vascular branch points. Some cells were observed dislodging from their original point of arrest only to arrest in another microvascular branch point for periods of up to 48 h. Both lung carcinoma and melanoma cells were observed extravasating into brain parenchyma at days 1–9 after injection. The process of TEM involved extension of invadopodia through the endothelium as described in the PC-3 and melanoma cell lines in

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Sect. 8.2.1. Intriguingly, intraluminal proliferation of lung carcinoma cells was only observed when host mice were treated with anti-VEGF therapy, indicating a critical role for neoangiogenesis in the metastatic colonization by this cell line. This finding is consistent with reports that VEGF production by cancer cells facilitates extravasation by activating Src-family kinases in endothelial cells, leading to disruption of their VE-cadherin junctions [45]. Although intraluminal proliferation was observed, colonies of cells were never witnessed rupturing through the vascular wall and often would regress [44]. It is possible that the architecture of the brain vasculature is nonpermissive to this type of metastatic colonization (differences in organ vasculature are the focus of Sect. 8.3).

8.2.2.2

Specific Cancer-Endothelial Cell Interactions

Although it can be experimentally demonstrated that the majority of cancer cells injected into the tail vein are retained in the lung following injection, extrapulmonary tumors do develop in some tumor models, as demonstrated in Fig. 8.1. Glinskii et al. show adherent DU-145 prostate cancer cells in the microvasculature of the lung, vertebrae, sternum, kidney, and thyroid gland after tail vein injection. Surprisingly, nearly equal numbers of cells were quantified in the lung and vertebrae [46]. Furthermore, treating Du-145 cells with blocking antibodies recognizing either Thomas Friedenreich (TF) antigen (expressed on DU-145 cells) or its receptor, galectin-3 (expressed on the surface of endothelial cells), greatly reduced CTC arrest in all organs. Treatment with E, P, or L-Selectin blocking antibodies, on the other hand, did not significantly affect tumor cell adhesion [46]. b3 integrins have been implicated in cancer cell transendothelial migration, although these are likely to engage ligands in the subendothelial matrix [47, 48]. Wang et al. found that blocking a3 and b1 integrin subunits on human fibrosarcoma cells leads to significant reduction of CTC arrest in the lung vasculature following renal injection in mice [49]. Similar results were obtained when infusing blocking antibodies to laminin-332 into mice prior to cancer cell injection. It was concluded that there are small, naturally occurring foci of exposed basement membrane throughout the lung vasculature, exposing laminin-332 to integrins on CTCs, facilitating adhesion [49]. However laminin-332 is not normally a component of the subendothelial basement membrane [50], so details of this mechanism remain to be explained. To determine if metastatic potential correlates with the ability of cancer cells to adhere to and extravasate through the endothelium, colon cancer cell lines of low, medium, and high aggressiveness were monitored via intravital fluorescence microscopy following systemic injection into Sprague-Dawley rats [51]. Regardless of metastatic potential, each cell line bound efficiently to the endothelium of the two most common tissues of colon cancer metastasis, the liver and lung. In less common sites of colon cancer metastasis, including kidney, intestine, skin, muscle, and spleen these cell lines all adhered poorly. Metastatic potential did, however, correlate with the frequency of extravasation and tissue invasion in the lung and liver. Surprisingly, mechanical lodging of cancer cells in the microvasculature was rare, and most adherent cells did not block blood flow through the lumen of the vessel. Schluter


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et al. did not observe rolling of cancer cells on the endothelium prior to adhesion in vivo, although colon cancer cells have been observed rolling on endothelial cells in vitro under laminar flow [52]. These data suggest that adhesion to the endothelium occurs through cell- and organ-specific interactions and further molecular characterization may help explain the tropism of metastatic colon cancer to the liver and lung. Importantly, these data support the argument that metastatic tumor formation reflects the efficiency of extravasation [51]. Ex vivo fluorescence imaging of isolated perfused lung preparations from mice injected systemically with human and rat fibrosarcoma cells revealed attachment of cancer cells to the endothelial walls of the pre-capillary arterioles, which are much larger in diameter than the cancer cells themselves. Furthermore, these arterioles remained perfusable, showing that the cancer cells had not occluded the lumens of the microvasculature. These data provide support for a model in which specific interactions between cancer and endothelial cells facilitate adhesion [53]. An unexpected finding of this work was that adherent fibrosarcoma cells rarely extravasated and that most viable cells began to proliferate within the lumen of the blood vessel, leading to colony formation which caused enlargement of the capillaries. Additional support for intravascular proliferation was obtained using this same imaging approach with lungs from mice with subcutaneous breast cancer tumors [54]. These experimental data support a longstanding hypothesis that intraluminal proliferation and subsequent colony growth through the vessel wall can lead to metastatic colonization in humans [55–57].

8.3

Cancer Cell Extravasation and Vascular Architecture of Common Sites of Metastasis

The tendency of primary cancers to metastasize to certain organs was first observed by Paget in the late 1800s [58]. The ability of CTCs to colonize some tissues and not others has since been referred to as the “seed and soil” hypothesis, describing the metastatic compatibility of a cancer cell with a given tissue. Because it is assumed that CTCs must first extravasate in order to colonize a tissue, observed differences in tumor tropism may be due to the ability of these cells to extravasate. One factor which may contribute to site-variable tumor cell extravasation efficiency is differences in organ vascular structure. Here, we focus on four organs which are common target tissues of metastatic carcinoma: bone, brain, liver, and lung. This subject has recently been reviewed in brief [59].

8.3.1

Bone Marrow Microvasculature

The bone marrow consists of two compartments, the hematopoietic compartment, which produces blood cells, and the stroma which contains the vasculature and its supporting fibroblasts, as well as adipocytes and nerves. The bone marrow

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microvascular endothelial cells are fenestrated and arranged in discontinuous sinusoids with little to no basement membrane and are thus surrounded only by hematopoietic tissue [6]. Functionally, this allows easy passage of blood cells into and out of the bone marrow sinusoids. While it is possible that CTCs which are poor at TEM may favor colonization of bone due its vascular structure, there is molecular evidence for bone-specific adhesion and extravasation (discussed above) which may help explain why bone is a common site of metastasis for several cancers. In prostate cancer, the lumbar spine is a common site of metastasis which reflects the fact that this site encounters high numbers of cancer cells draining through the Batson’s venous plexus structure in the pelvic floor [60]. On a clinical note, the detection of disseminated prostate, breast, and colon tumor cells in bone marrow may have important prognostic and predictive utility [61].

8.3.2

Brain Microvasculature

At the microvascular level, the most unique architectural feature of the brain vasculature is the blood-brain-barrier (BBB). The BBB is formed as a consequence of specialized tight junctions between brain endothelial cells; the result is markedly low passive diffusion of polar solutes and macromolecules, making the BBB highly selective to gasses, nutrients, and drugs [62]. This vascular structure would certainly seem to pose a challenge to CTC extravasation. Indeed, it is known that leukocyte extravasation into brain parenchyma is reduced compared to other tissues [4, 62]. Furthermore, it has been shown that when monocytes traverse the brain vasculature, they do so by a specialized trans-cellular route (through the endothelial cell as opposed to common monocyte diapedesis between endothelial cells), thus leaving the BBB tight junctions intact [63]. The fidelity of the BBB thus remains high except for conditions of trauma, ischemia, and various disease states which can cause leakiness or breakdown of the BBB, resulting in increased transport of macromolecules and leukocytes into brain parenchyma. In light of this information, it is surprising that the brain is a common site for metastatic colonization by several cancers. Breast cancer metastasis to brain is common and has been reviewed [64]. It is possible that cancer cells utilize unique mechanisms which facilitate brain extravasation. In vitro TEM assays using BBB-forming brain microvascular endothelial cells (BMECs) have shown that MDA.MB.231 breast cancer cells are able to disrupt BBB adherens junctions and transmigrate through the monolayer. This process was shown to be enhanced by exogenous addition of VEGF, which led to increased cytoskeletal rearrangement and permeability of BMECs [65]. In support of these findings, brain metastatic derivatives of MDA. MB.231 cells were shown to overexpress VEGF-A as well as the angiogenic interleukin IL-8 [66]. Finally, oral administration of a VEGF receptor tyrosine kinase inhibitor, PTK787, led to a large reduction of brain tumor formation in mice [66]. These studies, along with those outlined in Sect. 8.2.2.1, highlight an important role for angiogenesis and VEGF signaling in brain metastasis. Many conventional therapeutic strategies to combat brain metastasis are excluded by the BBB. It is tempting


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to speculate that VEGF signaling has a direct impact on the extravasation of CTCs in the brain. VEGF targeting may prevent extravasation of CTCs that have potential to form brain metastases and may improve patient survival.

8.3.3

Liver Microvasculature

The liver is unique in that it has both an arterial blood supply from the aorta as well as a portal venous supply, carrying blood from the gastrointestinal tract. These blood sources both drain into specialized liver sinusoids which deliver blood to hepatocytes and eventually to the large hepatic vein. These sinusoids are composed of extensively fenestrated endothelial cells and phagocytic Kupffer cells with no basal lamina [67]. Functionally, the sinusoids act as a sieve, allowing free passage of large macromolecules and blood cells. The fenestrations in hepatic circulation are both maintained and induced by VEGF signaling [67], which has been shown to augment extravasation and metastatic colonization of both the liver and of the lung, an organ which lacks fenestrations [45]. The liver undergoes constitutive leukocyte trafficking, and extravasation appears to occur in the sinusoids more often than in post-capillary venules [68]. It is thus believed by many that the discontinuous, fenestrated hepatic microcirculation is more permissive to tumor colonization than the vasculature of other organs [4]. In support of this hypothesis, Martin et al. showed that the same breast cancer cell line extravasates at a higher rate and frequency in the liver than in the lung [69]. The liver is the most common site of colon and pancreatic cancer metastasis. Here the liver is the first organ encountered by cancer cells via portal circulation. As discussed in Sect. 8.2.2.2 data from several labs have demonstrated adhesion of CTCs at pre-sinusoidal venules and in sinusoids of larger diameter than cancer cells, suggesting that colon cancer cell extravasation within the liver is initiated by specific interactions [51, 70]. Similarly, B16F1 melanoma cells injected into the portal veins of IL-1a-treated mice exhibited reduced velocity and increased retention in the liver vasculature compared to control mice, suggestive of cell rolling and adhesion to the activated endothelium [71]. Consistent with these findings are various studies showing that E-selectin ligands, expressed on colorectal cancer and melanoma cells, play an important role in CTC arrest and metastatic extravasation [52, 72]. Additionally, Rosenow et al. showed that extracellular matrix collagens and laminins, exposed in the numerous liver endothelial fenestrations, interact with integrin-a2b1 on colorectal cancer cells facilitating tumor cell adhesion and extravasation. Furthermore, blockage of this interaction with lebein-1 and rhodocetin reduced tumor formation [73].

8.3.4

Lung Microvasculature

The lung is a common metastatic site for various cancers, including breast, colon, and melanoma, and is one of the most studied organs in experimental metastasis. The microvasculature within the lung is smaller than many organs, with diameters as small

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as 7 mm [35], similar to the diameter of a red blood cell. The pulmonary capillaries are arranged in a dense, sheet-like network which covers the surface of alveoli, facilitating efficient gas exchange. These capillaries are composed of continuous, non-fenestrated endothelial cells attached firmly to a thin basement membrane [68]. The lung is an active site of leukocyte trafficking and extravasation, and the concentration of leukocytes in pulmonary blood is as much as 100-fold greater than in systemic blood [4, 35]. Leukocyte adhesion and extravasation in the pulmonary vasculature is thought to occur most frequently at the level of the capillaries, whereas in most systemic tissues this occurs in post-capillary venules [68, 74]. Furthermore, leukocyte adhesion in pulmonary capillaries appears to occur by size restriction and is not preceded by a rolling step as occurs in other tissues [75]. Once adherent, specific molecular interactions involving CD11/18, ICAM-1, and VCAM have been reported to be important in maintenance of adhesion and in leukocyte infiltration of lung parenchyma [76]. Evidence exists for specific molecular interactions between CTCs and the lung endothelium, which contribute to the observed lung tropism of some cancers. Using a phage-display library, Brown and Ruoslahti identified a surface protein, metadherin, which targets phage specifically to lung microvasculature [77]. After producing an antibody to metadherin, it was shown that this protein is upregulated in breast cancer samples when compared to healthy tissue. Both metadherin-neutralizing antibody and metadherin-siRNA treatment of metastatic 4T1 mammary carcinoma cells significantly reduced lung metastases in syngeneic hosts [77]. Minn et al. isolated a lung-tropic subpopulation of MDA.MB.231 breast carcinoma, named LM2, by serial tail vein passage in mice. RNA from this cell line and the parent were subjected to comparative microarray analysis, which revealed a lung-metastatic gene signature of 54 transcripts [11]. A follow-up to this work showed that four of the genes within this signature, including epiregulin, matrix metalloprotease 1 and 2, and COX2, contribute to vascular remodeling in the primary tumor, which promotes both tumor growth and dissemination of LM2 cells [78]. When these genes were knocked down simultaneously in LM2 cells, pulmonary extravasation and colonization were greatly reduced. Furthermore, small molecule inhibition of these four gene products led to reduced primary tumor growth as well as reduced pulmonary extravasation and tumor formation. These findings support a genetic basis for lung tropism and suggest that mutations acquired during tumorigenesis are co-opted for primary tumor growth as well as invasion, extravasation, and metastatic tumor formation [78] In support of this hypothesis, TGFb signaling at the site of a primary breast cancer tumor has been shown to induce production of angiopoietin-like 4 in invading cells, which facilitates retention of CTCs in the lungs of mice, but not bone, and enhances TEM in vitro [79].

8.4

Metastatic Inefficiency and Extravasation

As mentioned above, early studies on the fate of circulating tumor cells led to the observation that after systemic injection, cells became arrested at the first capillary bed encountered [36, 80]. For example, cells injected via the tail vein would arrest in the


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lung, and those injected into the hepatic portal vein would arrest in the liver. A sharp drop in cell viability in these experiments occurred immediately after injection, with as many as 99% of cells dying within 24 h (see Fig. 8.2). It was concluded that the majority of these cells died due to hemodynamic shear stress and by mechanical rupture following size restriction in the microvasculature [81]. Of the small percentage of surviving cells, fewer yet gave rise to metastatic lesions. Thus, great emphasis was placed on the ability of CTCs to extravasate and subsequently proliferate in order to successfully colonize a tissue. These observations introduced the concept of metastatic inefficiency, for which there is direct clinical evidence [82]. To date, debate continues over which steps of metastasis contribute most to the inefficiency of this process. Koop et al. took a quantitative approach to measure the rate limiting steps of metastasis by adding inert microspheres to suspensions of B16 melanoma cells and injecting these systemically into chick embryos. The cell:sphere ratio was monitored over time to determine the percentage of viable cells at each step of metastasis. Twelve to 24 h post injection 85% of the melanoma cells survived circulation and had extravasated [40]. Strikingly similar results were recapitulated by using the same method of cell accounting in a mouse model following injection into the mesenteric vein [41]. Interestingly, this latter study showed that although the majority of injected cells extravasated, only 0.07% led to micrometastases and 0.02% gave rise to tumors. Because B16F10 cells are a highly aggressive cell line, the question of whether less metastatic cell lines could extravasaste was addressed. Using the chick chorioallantoic membrane model, ras-transformed or wild type NIH/3T3 cells, as well as wild type mouse embryonic fibrobalasts, were compared for their extravasation potential. No differences were found between the three cell lines, as all were able to successfully extravasate, but only the transformed cells were capable of tumor formation [42]. These are among the seminal reports arguing that although extravasation is necessary for metastatic tumor formation, it is an efficient step and not related to the overall aggressiveness of the cancer cell. These findings supported the idea that the ability to survive and proliferate in the parenchyma of a secondary tissue ultimately dictates the efficiency of metastasis. Recent support for the hypothesis that late steps of metastasis are the predominant contributors to metastatic inefficiency was provided by Podsypanina et al. Mice were injected systemically with primary mouse mammary cells containing a doxycycline-dependent promoter driving the expression of the oncogenes MYC and KrasD12. At 1.5, 8, and 17 weeks post-injection, mice were placed on either a control or doxycycline diet and in all cases, pulmonary tumor growth was observed in the doxycyline-fed animals using bioluminescence imaging. Intriguingly, although no tumors formed in the control-diet mice, histological analysis of lung tissue showed that in the absence of oncogene expression, these control cells had colonized the lung tissue, were viable, and mitotically active. These findings suggest that nontumorigenic cells in circulation are capable of extravasation and colonization but secondary tumor growth requires oncogenic transformation [83]. In contrast, recent studies demonstrate that extravasation of a particular breast cancer cell type occurs at different rates in lung versus liver. Six hours after injection, two-photon microscopy revealed that most cells were arrested in the lumen of the microvasculature in both organs. However, at 24 h post-injection, over half of the

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cancer cells had extravasated into liver parenchyma, whereas less than one quarter had in the lung [69]. Thus, experimental determination of the extent to which extravasation contributes to metastatic inefficiency may depend on: (1) the cancer cell type involved, as melanoma cells appear to be an overall more aggressive lung-metastatic cell than many carcinomas, (2) the organs in which extravasation is being studied, as structural differences at the vascular level likely have a large impact on the permissiveness of tumor cell adhesion and extravasation, and (3) the imaging technique being used to qualify extravasation, as even with high resolution fluorescence imaging, delineation of intravascular versus extravascular location of cancer cells can be very challenging, especially in capillary-dense tissues such as the lung.

8.5

Epithelial-to-Mesenchymal Transition and Cancer Cell Extravasation

EMT is a process by which a polarized epithelial cell undergoes morphological changes resulting in a mesenchymal phenotype. EMT is characterized by loss of functional epithelial markers, such as E-cadherin and cytokeratin, and gain of mesenchymal markers such as N-cadherin and vimentin. EMT is often accompanied with large changes in the production of extracellular matrix and basement membrane proteins, and increases in protease secretion. These changes result in loss of epithelial polarity and the acquisition of migratory and invasive characteristics. This process is highly regulated both spatially and temporally during embryogenesis [84] and also plays a role in the processes of inflammation and wound healing [85]. Recent evidence has shown that EMT contributes to the progression of solid tumors by permitting detachment of cells from their primary site and by inducing a migratory phenotype, allowing the cell to invade local tissue and enter the lymphatic system or bloodstream [86]. Despite a large number of publications supporting a role for EMT in tumor progression and metastasis, information linking EMT and cancer cell extravasation is lacking. To identify novel genes involved in cancer cell extravasation, Drake et al. serially passaged PC-3 cells through a monolayer of human lung endothelial cells seeded onto transwell inserts and isolated a subpopulation named TEM4–18. Compared to the PC-3 input, these cells were enhanced in TEM and were shown to colonize tissues at a significantly higher rate in SCID mice. Furthermore, TEM4–18 cells exhibited an EMT-like morphology and near complete loss of E-cadherin mRNA and protein. Microarray analysis showed upregulation of a zinc-finger homeodomain transcription factor, Zeb1, in TEM4–18 cells [20]. Zeb1 had previously been implicated in EMT in development and pathological contexts [ 87, 88]. shRNA-mediated knockdown of Zeb1 in TEM4–18 cells resulted in a partial reversal of EMT, including restoration of E-cadherin and epithelial morphology, and reduction of TEM [20]. These data provide evidence that repressors of an epithelial phenotype, such as Zeb1, facilitate extravasation of prostate cancer cells.


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However, other studies suggest that cells bearing epithelial characteristics are capable of metastasis. To study the role of EMT in individual steps of metastasis in vivo, Tsuji et al. compared the invasive and metastatic properties of normal epithelial and EMT (p12CDK2-AP1-driven) hamster cheek pouch carcinoma cells. Both cell lines formed subcutaneous tumors at a similar rate, but only the EMT cells invaded local tissue and could be detected in the blood, consistent with previous reports of EMT-driven invasion [89]. When these cell lines were injected intravenously into mice, only the wild-type epithelial cells gave rise to lung tumors, suggesting that the EMT cells are incapable of metastatic colonization. When the two cell lines were differentially labeled, mixed, and injected subcutaneously, lung tumors formed which only consisted of wild type cells and, although EMT cell DNA was readily detectable in blood, none was detected in lung tumors. These data suggest that EMT cells are responsible for early events in metastasis, namely invasion and intravasation, and allow neighboring epithelial tumor cells to enter the circulation. During later steps in this model, it appears that only the epithelial cells are able to extravasate and proliferate at a secondary tissue, although extravasation, per se was not evaluated. These studies are in agreement with reports that epithelial-like MDA.MB.231 cells are enhanced in metastasis from a subcutaneous site when mixed with mesenchymal stem cells than on their own but that the resulting metastatic tumors do not contain detectable mesenchymal cells [90]. As described in Sect. 8.4, the work by Podsypanina et al. provides evidence that epithelial cells are capable of lung colonization [83], although it is difficult to ascertain whether these cells had, in fact, extravasated in the lung microvasculature. It is possible that epithelial cells have a propensity for intravascular colonization and proliferation, while the EMT phenotype facilitates trans-endothelial migration.

8.6

Clinical Aspects of Cancer Cell Extravasation

A more thorough understanding of cancer cell extravasation is likely to impact efforts to better predict the likelihood of occult metastasis and perhaps to inhibit metastasis. One area relates to the biology of CTCs. By definition, CTCs represent the precursor to extravasating cancer cells, and as the forgoing discussion illuminates, their biology is linked. The isolation and quantification of CTCs provides promise as a prognostic and predictive tool in the clinic. Early CTC studies successfully showed correlation of CTC number with poor prognosis in breast cancer patients [91, 92]. Longitudinal monitoring of CTCs during therapy of metastatic breast cancer patients was also shown to be prognostic of progression-free and overall survival [92]. These findings encourage clinicians that CTC quantification may be able to replace testing of disseminated tumor cells (DTCs). DTCs have been identified in the bone marrow of patients with breast, colon, and prostate cancer and have been shown to correlate with poor prognosis and cancer progression [93, 94], however isolation of bone marrow is painful and complicated

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compared to drawing a blood sample. Most current methods of CTC isolation rely on the physical selection of epithelial cells by exploiting their surface expression of epithelial cellular adhesion molecule (EpCAM) [95]. However, with growing support for EMT in tissue invasion and intravasation, it is possible that some CTCs have undergone at least a partial EMT. Such cells will have reduced expression or possible loss of EpCAM, thus potentially important CTCs will be missed entirely by standard isolation approaches. Isolation of CTCs by targeting mesenchymal markers, such as N-cadherin, alongside EpCAM may provide a more powerful prognostic outcome than standard approaches. Furthermore, molecular analysis of CTCs may provide information on the ability of these cells to extravasate and form metastatic tumors; such information would help tailor patient-specific therapies. Finally, one may question whether there is any therapeutic utility in blocking cancer cell extravasation. From this chapter it is apparent that a number of cell adhesion and intracellular signaling molecules may be viable targets for inhibiting cancer cell extravasation. To the extent that these targets overlap with those involved in leukocyte trafficking, one might anticipate side effects due to leukocyte dysfunction. In this regard, one particularly interesting target is N-cadherin which may not be involved in leukocyte trafficking and for which antibody and inhibitory peptides already exist [96, 97]. However, since metastatic dissemination may be an early event, possibly occurring prior to initial diagnosis, whether an opportunity exists to intervene in this step of metastasis is unclear. If secondary metastasis (metastasis not from the primary tumor) plays an important role clinically, blockade of extravasation may benefit patients who already have disseminated disease. Alternatively, there may be situations such as in the peri-operative state in which large numbers of cancer cells are liberated into the bloodstream for which temporary inhibition of cancer cell extravasation may be useful.

8.7

Conclusions

In order for a circulating tumor cell to form a metastatic tumor, at some point it must exit the vasculature and gain access to the parenchyma of a metastatic organ site. This is most often thought to occur by the process of extravasation. We have discussed many reported mechanisms of cancer cell extravasation, which have been derived from various cancer cell lines, in vitro models, and organs within several animal hosts. Although many differences in the cellular and molecular interactions between cancer cells and the endothelium have been observed, two general arguments prevail: (1) cancer cell extravasation is facilitated by specific molecular interactions and the ability to extravasate correlates with metastatic potential, and in some cases, organ tropism and, (2) extravasation of circulating tumor cells is non-selective, efficient, and unrelated to metastatic potential. As we have discussed throughout this chapter, these possibilities are not necessarily mutually exclusive. While size restriction is most certain to occur in some


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organs, productive extravasation may yet depend on specific cell adhesion mechanisms. It is also clear that at least some cancer cells can transit the first restrictive vascular bed they encounter to gain access to other distal organs that may be more conducive to metastatic colonization. The mechanism by which a cancer cell extravasates likely depends on the tissue-derivation of the cancer cell, the vascular bed in which it encounters, and the expression of various proteins on both the cancer and endothelial cells. Many knowledge gaps in cancer cell extravasation are beginning to be filled, thanks to the persistent interest in this important aspect of tumor biology. As highlighted above, there is recent evidence for a role of EMT in cancer cell extravasation, and this has clinical implications on both the isolation and analysis of circulating tumor cells. Ongoing and future work will hope to clarify the genetic, molecular, and cellular basis for cancer cell extravasation and determine the feasibility of targeting this step of metastasis therapeutically. Acknowledgements We thank Jones Nauseef for critical reading of the manuscript. JMB was supported by a Department of Defense pre-doctoral fellowship, PC094754. Work on cancer cell extravasation in the Henry lab has been supported by a Grant-in-Aid from the American Heart Association.

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Chapter 9

Role of CXCL12 and CXCR4 in Tumor Biology and Metastasis Ramesh K. Ganju, Yadwinder S. Deol, and Mohd W. Nasser

Abstract Chemokine receptor CXCR4 and its cognate ligand CXCL12, also known as stromal derived factor-1 (SDF-1), have been shown to play an important role in growth and metastasis of various tumors. CXCR4 is the most common chemokine receptor that has been demonstrated to be overexpressed in several cancers. Its overexpression is also correlated with poor clinical outcomes and survival in various cancers, including breast, prostate, and lung. CXCR4/CXCL12 signaling axis plays an important role in organ selective metastasis since CXCR4 overexpressing cancer cells have been shown to metastasize to organs such as bone, lymph nodes, liver, lung, and brain, which produce high amounts of CXCL12 and thus provide a favorable microenvironment. CXCR4/CXCL12 axis has been shown to enhance tumor growth and metastasis by maintaining cancer stem cells and modulating tumor stroma through activation of cancer-associated fibroblasts and recruitment of CXCR4+ endothelial precursor cells, thereby enhancing angiogenesis. CXCR4/CXCL12 axis has been shown to mediate both pro-tumorigenesis and metastasis by activating multiple signaling pathways, including protein tyrosine pathways through activation of Pyk2. Several CXCR4/CXCL12 antagonists/agonists have been shown to have potential therapeutic effects as they significantly inhibit tumor growth and metastasis of various cancers in both in vitro and in vivo mouse models. These studies suggest that the CXCR4/CXCL12 signaling axis is an important target for developing innovative therapies against various cancers.

R.K. Ganju (*) • Y.S. Deol • M.W. Nasser Department of Pathology, The Ohio State University Medical Center, Columbus OH, USA e-mail: [email protected]


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CXCR4/CXCL12 Signaling Pathways in Cancer

CXCR4/CXCL12 axis has been shown to mediate pro-tumorigenic effects and metastasis by activating multiple signaling pathways [1–3]. CXCR4 is a seventransmembrane receptor that belongs to the G-protein coupled receptor (GPCR) family. Therefore, CXCR4 has been shown to partly mediate its functional effects through GPCR signaling pathways [4]. GPCRs have been shown to bind to heterotrimeric G-protein composed of Ga, Gb and Gg subunits [5–7]. CXCR4-Gprotein complex in basal state is bound to GDP. However, upon binding its ligand CXCL12, GDP is replaced by GTP which disrupts the G-protein into a bg dimer and the a-monomer. Based on the sequence similarity, the Ga subunit is divided into four families: Gas, Gai, Gaq, and Ga12. However, CXCR4 has been shown to mainly mediate its signaling through Gai. Gai modulates the downstream signaling by inhibiting adenyl cyclase [8, 9]. Gai signaling has also been associated with CXCR4 signaling through activation of Src related kinases (Src), Erk 1/2 and NF-kB [4, 9–12]. ERK can phosphorylate and activate other cellular proteins as well as translocate into the nucleus and phosphorylate and activate transcription factors, leading to changes in gene expression and cell cycle progression. CXCL12-mediated activation of MAPK extracellular signal regulated kinases (MEK) has been shown to inactivate BCL-2 which is pro-apoptotic and thus can inhibit apoptosis of cells [10, 13]. Therefore, CXCR4/CXCL12-axis may promote cell survival by posttranslational inactivation of the cell death machinery as well as by increased transcription of cell survival-related genes. CXCR4/CXCL12-mediated chemotaxis and proliferation is also mediated by PI3 Kinase which can be activated both by Gbg and Ga subunits [10, 14]. PI3K then can activate the serine-threonine kinase AKT that plays a key role in tumor cell survival, proliferation, and chemotaxis [15]. In addition to GPCR signaling pathways, CXCR4 also has been shown to activate protein kinase pathways, especially focal adhesion tyrosine kinases [16, 17]. We have shown that focal adhesion kinase (FAK) and related adhesion focal tyrosine kinase (RAFTK), also known as Pyk2, plays an important role in CXCR4-mediated signaling in breast cancer, Kaposi sarcoma, and T-cells [18, 19]. CXCR4-mediated signaling was also shown to enhance motility and invasion in breast cancer cells [18]. In addition, CXCR4 has also been shown to activate components of focal adhesion complexes such as Crc and paxilin [18] (Fig. 9.1). CXCR4 also mediates its signaling through tyrosine phosphatases SHP2 and an adaptor-ubiquitin ligase like protein Cbl [4, 18, 20, 21]. Cbl protein was tyrosine-phosphorylated upon CXCL12 treatment in breast cancer cells [18]. Cbl, a 120-kDa protein that contains a tyrosine-kinase-binding (TKB) domain, can function as an adaptor molecule by binding to various proteins [22, 23]. It also contains a RING finger domain and negatively regulates signaling by directing the ubiquitination and degradation of activated receptor tyrosine kinases [24, 25]. The C-terminal region of Cbl contains a proline-rich domain that binds to the SH3-domain containing proteins. It also contains a cluster of tyrosine residues that bind to SH2-domain containing proteins [26–28]. Some of the domain-SH3 containing proteins that bind to Cbl may also

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Role of CXCL12 and CXCR4 in Tumor Biology and Metastasis

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Fig. 9.1 Schematic representation of CXCL12-induced and CXCR4-mediated signaling mechanisms that regulate chemotaxis and proliferation in tumor cells

bind to components of lipid rafts, which play an important role in chemotaxis [29–31]. The Cbl protein has been shown to regulate cell spreading in response to integrin engagement and is involved in the functional organization of the actin cytoskeleton [32, 33]. CXCL12 treatment also activated PI3 kinase increasing its association with Cbl and SHP2. CXCL12-induced chemotaxis and chemoinvasion was significantly inhibited by PI3K, RAFTK/Pyk2 and tyrosine phosphatase inhibitors [4, 18, 20]. It has been proposed that CXCL12-induced chemotaxis may be mediated through the activation and formation of multimeric-signaling complex comprised of Pyk2, SHP2 and PI3 kinase [4, 18, 20]. The complex then results in cytoskeletal changes and activation of MAP kinases and transcription factors. CXCL12 treatment of PC-3 cells led to MEK, IKK and IkBa phosphorylation leading to nuclear localization of NF-kB [34]. These transcription factors may in turn enhance expression of metallo-proteinases and other proteins. In this regard, CXCL12 has also been shown to activate MMP2 and MMP9 in breast and prostrate cancer cells [35]. Further, CXCR4 dependent signaling has been shown to decrease TIMP2 expression which in combination with enhanced matrix metalloproteinase secretion may increase the invasiveness of the prostate cancer cells [36]. Serratì et al. showed that CXCL12 produced by endothelial as well as epithelial cells induces the expression of urokinase-type plasminogen activator receptor (uPAR) in the CXCR4-expressing breast cancer cells [37]. Expression of uPAR has been linked to transition from dormant to metastatic status of tumor cells in vivo [38, 39]. In addition, CXCL12 can up-regulate the expression of adhesion molecules like VLA-4 which are involved

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in tumor cell invasion [40, 41]. CXCL12/CXCR4 axis has also been shown to up-regulate the expression of b3 integrin leading to the activation of avb3 receptors. Activated avb3 integrins have been shown to mediate adhesion of prostrate cancer cells to bone marrow epithelium [42, 43]. These processes may regulate CXCL12-mediated chemoinvasion and chemotaxis that may lead to the development of metastasis. CXCR4 has been shown to contain a short C-terminal domain (CTD) that contains tyrosine residues, which are phosphorylated upon ligand activation. CTD plays a major role in regulating CXCR4 receptor desensitization and down-regulation [44, 45]. CXCR4 CTD has been shown to play an important role in the process of epithelial to mesenchymal transition (EMT). Aberrant CXCR4 chemokine receptor function due to c-terminal truncation mutation has been shown to be present in various diseases, including warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis syndromes. In the case of breast cancer cells, it has been shown that overexpression of CXCR4 receptor containing c-terminal truncated cytoplasmic tails results in altered morphology as indicated by EMT and higher growth rates [44]. Breast cancer MCF-7 cells expressing truncated CXCR4 (CXCR4-ΔCTD) exhibited higher growth rate and EMT transition compared to wild type CXCR4 expressing cells. Furthermore, cells expressing truncated CXCR4 showed decreased E-cadherin expression and increased ERK activation [44]. These studies indicate that CTD region of CXCR4 is important for its regulation, expression and recycling. Various factors have been shown to regulate CXCR4 including tumor suppressor protein p53. p53 negatively regulates the expression of CXCR4 in breast cancer cells as downregulation of wild type p53 was shown to increase endogenous CXCR4 expression in breast cancer cells [46]. PRIMA-1 and CP-31398, which have been shown to rescue p53, were shown to reduce expression of CXCR4, at both mRNA and cell-surface levels [46]. These studies indicate that p53 targeting drugs may also reduce CXCL12/CXCR4-mediated cell proliferation and metastasis. In addition, p53 has also been shown to inhibit CXCL12 expression in fibroblasts, thereby modulating adjacent cancer cell migration and invasion [47]. Recently, it was shown that wt p53 reduces expression of CXCL12 in stromal fibroblasts [48]. Furthermore, p53 mutant expressing stromal fibroblasts were shown to express high amounts of CXCL12, thereby augmenting the tumor growth in prostrate cancer. CXCR4 has also been shown to cross-talk with other receptors such as epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and transforming growth factor beta (TGF-b), thereby regulating tumor growth and metastasis. In ovarian cancer cells, CXCR4 activation was shown to induce EGFR trans-activation by enhancing EGFR phosphorylation [49, 50]. Furthermore, it was shown that CXCL12 induced c-Src activation that may enhance EGFR phosphorylation and trans-activation [49]. CXCL12 has also been shown to trans-activate HER2/neu through Src kinase activation in breast cancer cells [11]. Furthermore, HER2 has been shown to enhance CXCR4 expression, thereby promoting growth and metastasis in lung and breast cancer cells [11, 51, 52]. EGFR has also been shown to enhance CXCR4 expression in non-small lung cancer cells through

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increased expression and activation of HIF-1 alpha [53]. Breast cancer patients showing poor overall survival rate has been shown to co-express CXCR4 and EGFR/ HER2 [51]. In breast cancer cells, CXCR4 was also shown to form a complex with insulin like growth factor-1 receptor (IGF-1R) [54]. Furthermore, IGF was shown to enhance migration and chemotaxis in these cells by activating CXCR4-mediated signaling pathways [54]. In addition, cross-talk between CXCL12/CXCR4 and TGF-b1 signaling was shown to initiate and maintain the differentiation of fibroblasts into myofibroblasts, thereby promoting tumor growth and metastasis in breast cancer [55]. Recently, there have been several reports suggesting cross-talk between estrogen receptor and CXCR4 signaling. It was shown that enhanced CXCR4 signaling drives estrogen receptor positive breast cancer to endocrine therapy resistant phenotype [56]. Furthermore, CXCR4 over-expression in ER positive MCF-7 cells was shown to enhance hormone independence [57].

9.2

CXCR4/CXCL12 in Breast Cancer Progression and Metastasis

CXCR4 is expressed in a wide variety of tissues including bone marrow, blood, spleen, thymus, lymph nodes, pituitary and adrenal glands [3, 58]. However, CXCR4 was recently shown to be one of the most common chemokine receptors to be overexpressed in several human cancers such as breast, ovarian, melanoma, prostrate, etc. [59]. Figure 9.2 shows the CXCR4 expression in a variety of human and neoplastic tissues as detected by immunohistochemical staining using anti-CXCR4 antibody UMB-2 from Dr. Schulz’s laboratory [59]. It has been demonstrated that CXCR4 levels measured by immunohistochemistry are extremely low or absent in normal breast epithelium, while >90% of specimens with atypical ductal hyperplasia showed positive staining for CXCR4 [60, 61]. CXCR4 is also present in ductal carcinoma in situ (DCIS). Moreover, CXCR4-expression is observed in about 75% of biopsy specimens of invasive ductal carcinoma and high expression of CXCR4 is correlated with decreased overall survival of patients in breast cancer [61, 62]. Similarly, increased expression levels of CXCR4 have been linked to cancer progression from atypical hyperplasia to invasive cancer [63]. Recently, it was shown that poor clinical outcome in triple negative breast cancer patients was shown to be co-related with high CXCR4 expression [64]. The CXCR4/CXCL12 pathway is involved in several aspects of breast tumor progression, including metastasis [65–67]. Metastatic cells characteristically lose growth inhibitory responses, undergo alterations in adhesiveness, and migrate to other distant organs by invading the blood and lymphatic vessels [66, 67]. This migration leads to secondary tumor formation that represents the most devastating feature of breast and other cancers. One of the important steps in the intricate process leading to the formation of metastases is tumor cell motility [68, 69]. It has been observed that more motile tumor cells may have increased metastatic potential [70]. We have shown that CXCL12 induces chemotaxis, chemoinvasion and

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Fig. 9.2 CXCR4 immunohistochemical staining in human formalin-fixed and paraffin-embedded tissues. (a–k) CXCR4 immunohistochemical staining of a variety of human normal and neoplastic tissues using anti-CXCR4 antibody UMB-2. Sections were dewaxed, microwaved in citric acid and incubated with UMB-2 at a dilution of 110. Sections were sequentially treated with biotinylated anti-rabbit IgG and AB solution. Sections were then developed in diaminobenzidine and lightly counterstained with hematoxylin. Note that UMB-2 detected CXCR4 receptors at the plasma membrane of a subset of tumor cells in a variety of human tumors including mammary carcinoma (a), endometrial carcinoma (b), cervical carcinoma (c), ovarian carcinoma (d), gastric carcinoma (e), pancreatic carcinoma (f), colonic carcinoma (g) and malignant pheochromocytoma (j). In nearly all of these cases UMB-2 immunoreactive tumor cells exhibited a heterogeneous distribution throughout the tumor. UMB-2 revealed predominant cytoplasmic staining in prostate carcinoma (h). In glioblastoma, UMB-2 detected CXCR4 receptors predominantly on the plasma membrane of endothelial cell of tumoral blood vessels (i). UMB-2 also detected CXCR4 receptors in the zona fasciculata of the adrenal cortex (k). Inset in (c), peptide adsorption control. Arrowheads in (i), tumoral blood vessels. C capsule; Z.g. zona glomerulosa, Z.f. zona fasciculata, Z.r. zona reticularis. Scale bars, A=B=C=D=E=F=G=H=I=J=K=50 mm, K=100 mm (Reprinted by permission of Dr. Schulz [59])

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adhesion of CXCR4 positive breast cancer cells in vitro. Molecules, which enhance the motility of tumor cells, have been implicated to play a role in the development and progression of tumors to the metastatic step [68, 70–73]. Metastasis is a frequent cause of death in patients with malignant neoplasms. Increased expression of CXCR4 has also been correlated with lymph node metastasis in breast cancer [74]. It is known that during metastasis, tumor cells employ several mechanisms to regulate the trafficking of tumor cells [66, 67]. It is now evident that CXCL12/CXCR4 plays a significant role in organ-selective cancer metastasis in breast cancer [75]. In particular, breast cancer cells were shown to express the chemokine receptors, CXCR4, whereas organs such as lymph nodes, bones and lungs that represent important sites of breast cancer metastasis were shown to express ligands of these receptors [72, 76] (Fig. 9.3). Furthermore, in vivo and in vitro neutralization of the CXCL12/CXCR4 interaction leads to a marked inhibition of metastasis [72, 77, 78]. CXCR4 was recently shown to be abundantly expressed by a subpopulation of breast cancer cells that have enhanced metastatic activity to bone or the adrenal medulla [79]. Moreover, breast cancer cells isolated from mammary fat pad xenografts were observed to have high surface expression of CXCR4 and enhanced CXCL12-induced chemotaxis [12]. Also, cancer cells that metastasize to lungs were found to have increased expression of CXCR4 and migration towards CXCL12 [12]. These studies suggest that the CXCR4 receptor and its cognate ligand CXCL12 are involved in regulating the trafficking and metastasis of these tumor cells to various tissues. Muller et al. reported that CXCR4 and CXCL12 are central players in regulating metastasis [72, 76]. Furthermore, using a mouse model of breast cancer, they have shown that neutralizing antibodies to CXCR4 significantly limited metastases to lymph nodes and lung [72]. These data were the first to identify a key function for CXCL12/CXCR4 in metastatic breast cancer. We have also shown that CXCL12-induced and CXCR4-mediated signaling plays an important role in breast cancer migration in vitro [18, 20]. These results were supported by Liang et al., who showed that blocking CXCR4 expression at the mRNA level by siRNAs decreased breast cancer cell invasion in in vitro assays and inhibited metastasis in an animal model [80]. CXCR4 has been shown to be one of the few genes that are enriched in a subpopulation of metastatic breast cancer cells [81]. Over-expression of CXCR4 alone has been shown to significantly increase the bone metastases in vivo. Li et al. demonstrated that HER2/neu enhances the expression and function of CXCR4 by inhibiting CXCR4 degradation [52]. Potentially, co-expression of CXCR4 with EGFR/HER2-neu may help to select for a metastatic clone with a particular propensity to metastases to the bone marrow. Further studies have demonstrated that elevated levels of pAkt and CXCR4 are induced by factors that are both dependent and independent of HER-2, and the activation of HER-2/ CXCR4/Akt signaling pathway in primary breast tumors may contribute to the formation of bone metastasis in breast cancer [82]. CXCL12/CXCR4 axis is also implicated in the bone metastasis of prostrate cancers [83]. CXCR4/CXCL12 signaling has been shown to stimulate production of matrix metalloproteinases and increase integrin activity [35, 43, 84]. Further, metastasis triggering mechanisms like hypoxia that enhance expression of hypoxia-inducible factor-1 (HIF-1) have

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Fig. 9.3 Model of chemokine regulation of breast-cancer metastasis. Metastasis is an orderly, multistep process involving the movement of cancer cells from the primary tumor to specific organs under the guidance of specific chemokines. First, cancerous mammary epithelial cells undergo clonal proliferation, invade local tissue, induce angiogenesis, and express CXC chemokine receptor 4 (CXCR4) on their surface. Then, cancer cells detach from the primary tumor, migrate across lymphatic and vascular walls in the tumor, and enter the systemic circulation. Cancer cells are arrested in vascular beds in organs that produce high levels of the CXCR4 ligand (CXCL12), which is expressed on the surface of vascular endothelial cells. Binding of CXCL12 to CXCR4 induces the migration of cancer cells into normal tissue, where the cells proliferate, induce angiogenesis, and form metastatic tumors. Breast-cancer cells do not usually metastasize to organs that produce low levels of CXCL12, such as the kidney (Reprinted by permission of Dr. Murphy [76])

also been shown to induce CXCR4 expression [85]. It has been shown that inactivating mutation of von Hippel Lindau (VHL) tumor suppressive gene, which normally targets HIF-1 for degradation, increases CXCR4 expression in adrenal cell carcinomas [86]. VEGF has also been shown to enhance CXCR4 expression during breast cancer progression and metastasis [12, 87]. Oncoproteins, such as RET/PTC,

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have been shown to enhance the transforming ability of breast cancer cells by enhancing CXCR4 expression [88]. In addition, the Pax3-FKHR has been shown to increase CXCR4 expression of rhabdomyosarcoma that leads to enhanced migration and adhesion of these cells [89].

9.3

CXCR4/CXCL12 in Lung Cancer Growth and Metastasis

There are some reports which suggest a correlation between CXCR4 and clinical outcomes in non-small cell lung cancer (NSCLC). It has been suggested that CXCR4 expression may be increased in patients with metastatic disease [90]. CXCR4 expression has also been shown to be in both cytoplasm and nucleus; however, there are conflicting reports regarding the clinical relevance of such differential expression [91, 92]. It has also been shown that nuclear expression of CXCR4 in NSCLC tumor cells significantly correlates to lymph node metastasis [91]. In contrast, Wagner et al. showed nuclear staining to be significantly associated with improved disease free survival whereas cell-membrane staining was associated with distant metastasis and decreased disease-free survival in lung adenocarcinoma patients [92]. Interestingly, it has also been shown previously that differential expression of CXCR4 correlated with metastatic potential in vitro and in vivo [93]. This suggests that the movement of NSCLC from primary sites to metastatic nodes might be dependent on the level of CXCR4 [93]. It has been shown that high expression levels of CXCR4 are correlated with metastasis to the brain in NSCLC that have shorter survival rates [94]. Further, since CXCR4 expression is inducible by hypoxia, Liu et al. showed that when HIF-1a and HIF-2a were knocked down using RNAi approach in NSCLC cells, it reduced the invasion, adhesion and migration in response to CXCL12 [95]. Burger et al. showed high levels of functional CXCR4 receptors in small cell lung cancer (SCLC) cells [96, 97]. They further showed that CXCR4 activation induces migratory and invasive responses and adhesion to marrow stromal cells in a CXCR4 and integrin-dependent fashion [96, 97]. CXCR4/CXCL12 axis is an important mediator for the adherence of SCLC cells to extra cellular matrix (ECM) proteins like fibronectin, collagen I and VCAM-1 [98]. CXCR4-induced adhesion of SCLC cells to marrow stromal cells protected them against etoposide-induced apoptosis [98]. The CXCR4 antagonist T140 and its derivatives have been shown to block the CXCR4-mediated adhesion to ECM components and thereby affect SCLC chemo-sensitivity [98]. Collectively, these studies indicate that expression of CXCR4 by SCLC cells facilitates the activation of integrins that regulate the adhesion of tumor cells to the bone marrow microenvironment, which in turn confers drug resistance and tumor cell growth. Moreover, hypoxia is able to induce a significant increase in the expression level of CXCR4 on NSCLC cells through the VHLHIF-1a pathway, supporting the notion that HIF-1a-mediated up-regulation of CXCR4 may be a common response of tumor cells to hypoxia [53, 99]. Recent studies also implicate that activation of EGFR by EGF increases expression of CXCR4 and chemotactic potential of lung cancer cell lines. It has been suggested that EGFR

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within the primary tumors of NSCLC provides the microenvironment and signals necessary to up-regulate CXCR4 expression and promote metastasis [100]. A series of targets and therapeutic strategies for the treatment of lung cancer are currently being investigated [53]. However, all patients ultimately develop resistance against these agents, including chemotherapy, possibly due to abnormal signal transduction and high EGFR expression level. CXCL12 has been shown to modulate EGFRmediated signaling pathways [100].

9.4

CXCL12/CXCR4 and Other Cancers

Several other neoplasias are known to express CXCR4 chemokine receptors. Most notably, CXCR4 activation by CXCL12 induces migration and/or survival of neuronal and glial tumors, neuroblastoma cells, colorectal cancer, esophageal cancer, melanoma, renal cell cancer, hepatocellular carcinoma, ovarian cancer and rhabdomyosarcoma [101]. In other cancers, such as prostate and pancreatic, the CXCL12/ CXCR4 axis was shown to promote tumor cell trans-endothelial chemotaxis [102, 103]. In patients with colorectal cancer and melanoma, CXCR4 expression of primary tumor cells correlates with recurrence, metastasis, and survival [104–106]. Interestingly, in these cancer patients, rate of bone metastasis is not as high as that of prostate and breast cancers indicating that CXCL12/CXCR4 may not be the only mechanism required for the establishment and localization of tumors to the bone marrow. Expression of CXCR4 in hepatocellular carcinoma has been shown to decrease 3-year survival rates and correlates to local tumor progression, lymphatic and distant metastasis in these patients [107]. In addition, increased levels of nuclear expression of CXCR4 and VEGF-C was shown to be correlated with lymph node metastasis and worse prognosis in these patients [107]. This finding has also been observed in colorectal cancer patients where nuclear expression of CXCR4 is related to poor outcome [108]. In renal cell carcinomas, CXCR4 expression has been found to localize in cytoplasm of metastatic renal cell carcinomas whereas in the case of primary non-metastatic carcinomas, its expression was observed in the nucleus [109–111]. In melanoma cancer cells, increased metastasis of CD133+ cells was shown to be mediated through CXCR4/CXCL12 axis [106]. Direct co-relation has been observed between grade and CXCR4 expression in prostate cancer [112]. CXCR4 neutralizing antibody and a blocking peptide have been shown to decrease the metastasis of prostrate cancer to bone [113]. A positive co-relation has been observed between tissue levels of CXCL12 and metastatic lesions [114]. Signaling through CXCR4 could alter the ability of cancer cell lines to adhere to endothelium and invade through extracellular matrix components in the bone marrow [42, 115]. CXCR4/CXCL12 has also been shown to play an important role in the development of hematopoietic malignancies such as acute leukemias, multiple myeloma, B-cell chronic lymphocytic leukemia, and acute myelogenous leukemias [116]. CXCL12 produced by bone marrow stromal cells has been implicated in providing growth and survival signals through activation of CXCR4 receptors present on various hematopoietic malignant cells (91).

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Role of CXCR4/CXCL12 Axis in Enhancing Growth Through Modulation of Tumor Stroma

CXCL12/CXCR4 axis has also been shown to regulate growth and metastasis by enhancing development of carcinoma-associated fibroblasts (CAFs), thereby modulating tumor stroma, an active element of tumor microenvironment [117, 118]. In tumors, cancer cells coexist with other cell stypes such as fibroblast, endothelial, immune cells, and extracellular matrix (ECM) that form the tumor stroma [2, 118, 119]. Higher incidence of tumor development has been observed in tissues with chronically inflamed stroma in which resting fibroblasts get activated [120, 121]. Such activated subsets of fibroblasts are CAFs or myofibroblasts and have been observed in the stroma of invasive human breast cancers [118, 119]. Myofibroblasts possess increased contractile ability, promote angiogenesis and stimulate epithelial cell growth through production of ECM and secretion of growth factors, cytokines, and chemokines [122, 123]. Recently, it was shown that CAFs derived from breast tumors secrete high levels of CXCL12 which enhance tumor growth through paracrine mechanism by activating CXCR4 receptor present on tumor cells [117]. In addition, CXCL12 has also been shown to increase angiogenesis through endocrine mechanisms by recruiting CXCR4+ endothelial pre-cursor cells to the tumor stroma [5, 117, 118] (Fig. 9.4). Similarly, CXCL12, secreted by CAFs, stimulates the in vivo growth of benign prostate hyperplasia (BPH) that expresses CXCR4 [124, 125]. In addition, TGF-b and CXCL12 autocrine signaling mechanisms have been shown to enhance tumor progression by development of tumor-promoting CAF myofibroblasts from preexisting stromal fibroblasts [55]. It was shown that TGF-b released by carcinoma cells can elicit enhanced endogenous TGF-b and CXCL12 production via TGF-b receptor-Smad signaling pathway [55]. Furthermore, TGF-b was also shown to induce CXCR4 expression in stromal fibroblasts and tumor cells, thereby facilitating the generation of two autocrine signaling loops, mediated by TGF-b and CXCL12, acting in a positive feedback manner [55]. Such autocrine signaling loops self stimulate and cross-communicate with each other to maintain the myofibroblastic phenotype. CAFs have also been shown to promote the proliferation of breast cancer CD44+CD24− stem cells through their ability to secrete CXCL12. Furthermore, CXCL12/CXCR4 has been shown to play an important role in breast and lung cancer stem cells survival and drug resistance [117, 118, 126]. CXCL12/CXCR4 axis may also stimulate tumor growth by enhancing recruitment of Gr1+CD11b+ myeloid derived suppressor cells (MDSCs) to the tumor stroma [127]. It was shown recently that in Wnt signaling-induced mammary gland tumorigenesis, CXCL12 produced by myoepithelial cells and stromal fibroblasts supports tumorigenesis by involving Gr1+CD11b+ MDSCs [128]. MDSCs have been shown to enhance tumor growth by inhibiting tumor immune response [127, 128]. Recently, Hiratsuka et al., using CXCR4 conditional knockout mice, showed that inhibition of CXCR4 in myeloid cells suppress tumor growth, angiogenesis and metastasis by decreasing recruitment of Gr-1+CD11b+ bone marrow derived myeloid cells (BMDCs) to the primary tumors [129]. Thus, targeting CXCL12/CXCR4

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Fig. 9.4 Schematic representation of tumor-promoting effects provoked by CXCL12 released by stomal fibroblasts in invasive human mammary carcinomas. CXCL12 released by stomal fibroblasts facilitates tumorigenesis by two different mechanisms. Stromal fibroblast-derived CXCL12 enhances tumor growth by stimulating angiogenesis through recruiting circulating EPCs into the tumor mass (endocrine effect) and by direct paracrine stimulation of tumor cells through CXCR4 expressed on carcinoma cells (paracrine effect) (Reprinted by permission of Dr. Weinberg and the Cell Press, as published in Cell. 2005;121(3):335–48)

signaling axis will prevent the development and maintenance of tumor promoting myofibroblast and therefore, may prove to be a useful antitumor therapeutic strategy in the future.

9.6

CXCR4/CXCL12 and Angiogenesis

CXCL12/CXCR4 axis has been shown to play an important role in vasculature and angiogenesis. CXCR4 or CXCL12 knockout mice are embryonically lethal due to disruptions in the development of hematopoietic, vasculature and neuronal network in many organs [105, 130]. During tumor development, CXCL12 produced by stromal cells recruits CXCR4 positive endothelial precursor cells to tumors, thereby enhancing endothelial cell growth and vasculature [117, 118]. The ability to recruit and sustain an active vasculature is critical for the survival of tumors. Recently, prostate cancer tumor-associated blood vessels and basal cell hyperplasia were shown to express CXCL12 [131]. Tumors derived from CXCR4 over-expressing

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PC3 cells show significantly higher blood vessel density, functionality and invasiveness [132]. CXCL12/CXCR4 has been shown to enhance angiogenesis at metastatic sites by down-regulating glycolytic enzyme phospho-glycerate kinase (PGK) [133]. PGK has angiostatic properties as it cleaves extracellular plasminogen to produce angiostatin that releases the angiostatic clamp [133]. This angiogenetic switch has been shown to play an important role for enhancing growth of metastasized colonies and thereby enhance metastasis. Vascular endothelial growth factor (VEGF), which plays an important role in angiogenesis and the survival of metastatic breast carcinoma cells, was recently shown to induce expression of the CXCR4 receptor in carcinoma cells, which promotes their chemotaxis [87]. Tumor angiogenesis can effect the tumor growth and neo-vascularization by several ways [117, 134]. New blood vessels arise from pre-existing vessels through the dissociation, migration, and division of differentiated endothelial cells. Further, these blood vessels can also be synthesized de novo from bone marrow-derived cells (BMDCs) [135]. BMDCs, such as endothelial progenitor cells, tumor-associated macrophages, Tie2-expressing monocytes, and myeloid progenitor cells, have also been shown to enhance tumor angiogenesis, thereby facilitating tumor growth [135–139]. Some BMDCs express high levels of CXCR4 receptor and can be mobilized from the bone marrow to sites producing the chemokine ligand, CXCL12 [117, 118, 129]. The importance of the stromal derived factor-1 (SDF-1)-CXCR4 axis in angiogenesis is apparent from the lack of gastrointestinal blood vessels in CXCR4−/− mice [140].

9.7

Inhibitors of CXCR4/CXCL12 Signaling Axis

CXCR4/CXCL12 has been shown to be a potential therapeutic target because targeting CXCR4/CXCL12 axis in cancer will block migration and dissemination of tumor cell metastasis, paracrine growth and survival signals and pro-angiogenesis effects. Currently, several types of CXCR4/CXCL12 antagonist/agonists are being used to inhibit this axis, such as bicyclams (AMD3100), T22, TN14003, CTCE9908, and ALX40–4C, which are analogs and peptides based on the amino-terminal region of the chemokine, CXCL12 [141]. AMD3100 is a specific antagonist of CXCL12 binding to CXCR4, inhibiting CXCL12-mediated calcium mobilization, chemotaxis and GTP binding, and does not cross-react with other chemokine receptors [142, 143]. AMD3100 antagonists are macrocyclic polyamines which were shown to inhibit X4 strain of HIV binding to CXCR4 which acts as co-receptor for HIV [142, 143]. Functional studies have shown that a strong and direct interaction occurs between CXCR4 and bicyclams without receptor internalization [144]. AMD3100 (recently re-named as Plerixafor or Mozobil) was shown to inhibit CXCR4/CXCL12 mediated tumor growth and metastasis both in vitro and in vivo in various types of cancers [144]. Recently, radiolabeled AMD3100 was developed to selectively target and monitor CXCR4 positive tumor growth and metastasis [145]. CXCR4 antagonists, such as Plerixafor (AMD3100), have been shown to sensitize

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SCLC cells to cytotoxic drugs, such as etoposide [146], suggesting the CXCR4/ CXCL12 signaling axis as a novel target in drug-resistant SCLC therapy. Combining CXCR4 antagonist with cytotoxic chemotherapy may be an attractive strategy to overcome chemo-resistance and relapse in lung cancer. Another CXCR4 inhibitor, TN14003, derived from 14-mer peptide antagonists of CXCR4 (T140) has been used in various preclinical studies, including in vivo models for breast cancer [147]. These studies show that in addition to limiting metastasis of breast cancer via inhibiting migration, TN14003 may also be useful as a diagnostic tool to identify CXCR4 receptor positive tumor cells in culture and tumor samples [148]. TN14003 has also been shown to be effective in other cancers including small cell lung cancer (SCLC) [146], malignant melanoma and pancreatic cancer [141]. Peptide analogs of CXCL12 (CTCE-9908 and CTCE-0214), which have shown tumor and metastatic inhibiting activity, recently received orphan drug status by the Food and Drug Administration for the treatment of osteogenic sarcoma [149, 150]. CTCE-9908 has shown both inhibition of the primary tumor and anti-metastatic effects in animal models of melanoma, osteosarcoma, breast, and prostate tumors [149, 151–153]. In prostrate cancer, CTCE-9908 delivered intraperitoneally was also associated with inhibition of VEGF and angiogenesis and reduced recruitment of myeloid host cells [152]. Recently, using a transgenic model for breast cancers, the administration of CTCE-9908 resulted in a 30–40% decrease in lung metastasis [154]. Chemotherapy drugs or an anti-angiogenic agent in combination with CTCE-9908 have been shown to possess enhanced anti-tumorigenic and metastatic effects [154]. Currently, CTCE-9908 is undergoing Phase 1 and Phase II clinical trials in hepatocellular carcinoma [149]. Other molecules, such as CD26/dipeptidyl IV (DPPIV), MMPs and serine proteases, have been shown to regulate CXCL12/CXCR4 pathway by cleaving CXCL12 rendering it inactive [155]. Recent studies from our group suggest that synthetic non-psycho active cannabinoids inhibit breast or lung cancer growth and metastasis. Synthetic cannabinoids CP55,940 and WIN55,212–2, which bind to cannabinoid receptor CB1 and cannabinoid receptor CB2, as well as the CB2-selective agonist JWH-015, inhibited CXCL12-induced and CXCR4-mediated chemotaxis and invasion of Jurkat T cells [156]. These synthetic cannabinoids have also been shown to inhibit tumor growth in transgenic breast cancer tumor model systems [157, 158]. We have also recently shown that Slit-2, which is hyper methylated and inactivated in breast and lung cancer cells, inhibits CXCL12/CXCR4-induced breast cancer cell chemotaxis, chemoinvasion, and adhesion, the fundamental components that promote metastasis [20, 159, 160]. Slit, which has been reported to play an important role in axonal movement [161–165], was recently shown to modulate CXCR4-induced leukocyte trafficking [166]. Slit is a ~200 kDa secretory protein that consists of a family of three genes: Slit 1, Slit 2 and Slit 3. Slit, which mediates its function by binding to the Roundabout (Robo) was shown to inhibit CXCL12induced tyrosine phosphorylation of focal adhesion components such as RAFTK/ Pyk2 at residues 580 and 881, focal adhesion kinase at residue 576, and paxillin [20]. Recent studies from Hinck’s group found that loss of Slits in murine mammary gland or human breast carcinoma cells result in up-regulation of the CXCL12 and

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CXCR4 signaling axis, specifically within mammary epithelium [167]. In addition, inverse correlation between Slit and CXCR4 expression is observed in human breast tumor tissues [167]. Furthermore, Slit over-expression in breast cancer cell line MDA-MB-231, was shown to significantly suppress tumor growth in in vivo mouse models [167]. Furthermore, they have shown that Slit2 may inhibit tumor growth by down-modulating CXCR4 expression [167]. We have also shown that Slit2 inhibits tumor growth in MCF-7 cells by modulating b-catenin/TCF pathway [159]. Our group also showed that Slit2 inhibited CXCL12/CXCR4-mediated chemotaxis, chemoinvasion and adhesion by decreasing the activation of RAFTK/Pyk2, FAK and paxillin [160]. These findings suggest that Slits may act as a negative regulator of CXCL12/CXCR4 axis in tumor development.

9.8

Conclusive Remarks

Chemokine receptor CXCR4 and its ligand CXCL12 have been shown to be crucial for tumor proliferation, migration, adhesion, angiogenesis, and metastasis. CXCR4/CXCL12 axis has been shown to possess a multi-faceted role in diverse processes of tumor progression and metastasis through regulation of cancer stem cells, modulation of tumor stroma, enhancing angiogenesis, formation of premetastatic niche and organ selective metastasis. Therefore, it is of utmost importance to understand the mechanisms by which CXCL12/CXCR4 modulate microenvironment to enhance growth and help in homing and progression of cancer cells to different organs. This will help to identify novel targets for development of therapies to inhibit both growth and metastasis against various types of cancers, including breast, liver, prostate, pancreas, and leukemia. This is especially imperative considering the resistance of various cancers to chemotherapy and the poor prognosis of CXCR4 positive cancer patients. Furthermore, identification of CXCR4 as an early novel biomarker and therapeutic target may lead to the improvement of the overall survival of cancer patients.

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Chapter 10

Transcription Factors Stat5a/b and Stat3 in Prostate Cancer Growth and Metastases Tuomas K. Mirtti, Pooja Talati, and Marja T. Nevalainen

Abstract The majority of prostate cancer fatalities are caused by development of castration-resistant growth and metastatic spread of the primary tumor. There are currently no effective treatments for advanced prostate cancer and therefore it is imperative to develop more effective therapies for prostate cancer. Transcription factors Stat5a/b and Stat3 both represent potential therapeutic target proteins for advanced prostate cancer. Stat5 is the major signal transducer downstream of Prolactin (Prl) receptors in human prostate cancer cells, while Stat3 is known as the key mediator of the IL-6 effects. Stat5a/b and Stat3 both are constitutively active in clinical prostate cancers of high histological grades and in distant prostate cancer metastases. In addition, Stat5a/b and Stat3 both functionally interact with the androgen receptor in human prostate cancer cells. While both Stat5a/b and Stat3 bind to the same DNA consensus sequence, Stat5a/b regulates largely different genes from those that are regulated by Stat3 in human prostate cancer cells. Importantly, Stat5a/b

Tuomas K. Mirtti and Pooja Talati contributed equally to this chapter. T.K. Mirtti Department of Pathology, Haartman Institute, University of Helsinki, Helsinki 00014, Finland P. Talati Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA M.T. Nevalainen (*) Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA Department of Medical Oncology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA Department of Urology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA e-mail: [email protected]; [email protected]


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has a preferential role over Stat3 in the promotion of prostate cancer cell viability and tumor growth. At the same time, both Stat5 and Stat3 stimulate metastatic behavior of human prostate cancer cells in vitro and in vivo. The Janus kinase family proteins (Jaks) are primary activators of Stat proteins. Inhibitors of Jak2 would target both transcription factors and theoretically provide control of prostate tumor growth as well as metastatic potential.

10.1

Introduction

Prostate cancer is the most common non-skin malignancy in the US and the second leading cause of cancer related deaths in males in the western countries [1]. The majority of prostate cancer fatalities are caused by metastatic spread of the primary tumor and development of castration resistant growth. The complex molecular mechanisms underlying progression of prostate cancer from organ-confined cancer to metastatic disease are largely unknown and, currently, there are no effective therapies for disseminated castration-resistant prostate cancer. Identification of the molecular changes associated with the progression of prostate cancer will provide a rational basis for the development of improved therapies for advanced disease. Currently, there is a lack of reliable markers that would identify those prostate cancers that are most likely to progress to aggressive disease and would benefit from extensive treatment. Here, we review the recent published data on transcription factors Stat3 and Stat5a/b in the regulation of prostate cancer growth and metastasis formation, as well as their interaction with the androgen receptor. The clinical importance of these transcription factors, as prognostic markers and therapeutic targets, will be discussed.

10.2

Structure and Function of STAT3 and STAT5a/b

Signal Transducer and Activator of Transcription (Stat)-3 and Stat5 belong to the seven member family of transcription factors (Stat1, Stat2, Stat3, Stat4, Stat5a, Stat5b and Stat6)[2]. The homology between the protein structures suggests that all Stats have a common genetic origin and have later diverged through duplications into three separate genetic loci: Stat1 and Stat4 in chromosome 2, Stat2 and Stat6 in chromosome 12 and Stat3, and Stat5a and Stat5b in chromosome 17 [3–7]. All the Stat proteins are coded by separate genes, even the highly homologous isoforms Stat5a and Stat5b [2]. The common genetic locus of Stat3 and Stat5a/b is chromosome 17 (bands q11–1 to q22), homologous to mouse chromosome 11[7–9]. The major structural features of all Stat proteins are similar. The N-terminal domain is involved in stabilizing interactions between two Stat dimers to form tetramers, thus facilitating the transcriptional activity of weak promoters. The coiled-coil domain facilitates protein-protein interactions by chaperone interactions

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[10] and co-activator recruitment [11]. The DNA-binding domain interacts with the Gamma-interferon Activation Sequence (GAS) motifs in the target gene regulatory elements [12]. The linker domain stabilizes DNA-binding [13]. The SH2 (Src homology 2) domain mediates Stat dimerization and receptor-specific recruitment. During dimerization, a phosphorylated tyrosine residue of one Stat subunit binds to the SH2 domain of another Stat molecule [14]. In the C-terminal part of Stat proteins there is a transcriptional activation (TA) domain which interacts with critical co-activators and is directly involved in the initiation of transcription [15, 16]. Additionally, Stat5 proteins are post-translationally glycosylated on threonine 92 near the N-terminus, which allows interaction with the CREB-binding protein [17]. The highly homologous isoforms Stat5a and Stat5b differ in their C-terminal amino acid sequence. Stat5a (94-kDa) has 20 unique C-terminal amino acids while Stat5b (92 kDa) has eight distinct C-terminal amino acids in its sequence. Stat3 shares sequence homology with Stat5a/b, but the main differences are in the length and sequence of the TA domains [15]. Stat3 is activated by phosphorylation of tyrosine residue Y705 whereas Stat5a and Stat5b are activated at tyrosines Y694 and Y699, respectively [7, 18, 19]. Serine 727 phosphorylation supplements the activation of Stat3, whereas phosphorylation of serine 725 and serine 730 may negatively control the transcriptional activity of Stat5a and Stat5b, respectively [18, 20]. While the nuclear translocation of Stat dimers is an energy-dependent process [21, 22], there is recent evidence that non-phosphorylated Stat3 could regulate gene expression also [23, 24]. Ligands activating different Stat proteins vary considerably. Stat5a is the predominant signal transmitter of prolactin receptor activation, while Stat5b mediates growth hormone response [16]. Stat3 mediates primarily the action of interleukin-6 (IL-6) family proteins [25]. As the cytokine receptors that activate Stats usually lack tyrosine kinase activity, this activity has to be provided by other cytoplasmic kinases. The predominant kinases in Stat signaling are the Janus kinase family proteins (Jaks), Jak2 being the primary activator of both Stat5a/b and Stat3 [26, 27]. The cytokine Prl is locally expressed at high levels in high-grade prostate cancers [28], distant prostate cancer metastases, and castration-resistant prostate cancers [29]. In the canonical Prl-Jak2-Stat5a/b signaling pathway (Fig. 10.1), ligand binding-induced conformational changes in the prolactin receptor (PrlR) activate Jak2 molecules, which then phosphorylate tyrosine motifs in PrlR. Stat5a/b SH2 domains recognize these phosphorylated motifs, come close to their docking sites, and are subsequently phosphorylated by Jak2[2]. The IL-6 receptor (IL-6R) has two subunits: gp80 or IL-6Ra, which is cytokinespecific; and gp130, which is shared by IL-6 and related cytokines [30]. IL-6 binds first to IL-6Ra [31], which subsequently recruits the signal-transducing subunit gp130 (Fig. 10.1). The association of gp130 with IL-6 and IL-6Ra leads to the formation of the high-affinity IL-6R complex [32, 33] and phosphorylation of Jak2 preassociated with the cytoplasmic domain of gp130[34]. Jak2 phosphorylates Stat3 at Tyr705[25, 35], and phosphorylated Stat3 dimers translocate to nucleus where they bind to GAS elements of target genes. In addition to Stat3, IL-6 also can activate Ras and induce its translocation to the cell membrane, where Ras activates Raf,

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Fig. 10.1 The Janus kinase family proteins (Jaks) are primary activators of Stat proteins. In the canonical Prl-Jak2-Stat5a/b signaling pathway ligand binding induces conformational changes in the prolactin receptor (PrlR) and activates Jak2 molecules. Jak2 molecules then phosphorylate tyrosine motifs in PrlR, which are recognized by the SH2 domain on Stat5a/b and allow subsequent recruitment and phosphorylation of Stat5a/b by Jak2 [2]. Phosphorylated Stat5a/b dimerizes, undergoes nuclear translocation and binds to GAS elements of target genes. The IL-6 receptor (IL6R) has two subunits: gp80, or IL-6Ra, and gp130. IL-6 binds first to IL-6Ra [31], which subsequently recruits the signal-transducing subunit gp130. The association of gp130 with IL-6 and IL-6Ra leads to phosphorylation of Jak2 preassociated with the cytoplasmic domain of gp130 [34]. Jak2 phosphorylates and activates Stat3. Stat3, similar to Stat5, dimerizes, nuclear translocates and binds to GAS elements on the DNA. In addition to Stat3, IL-6 also activates Ras which activates Raf, leading to activation of mitogen-activated protein kinase kinase (MEK) and MAPK (Erk1/2) [36]. The third signaling pathway activated by IL-6 through a Jak2-mediated pathway is activation of the phosphoinositol 3 kinase (PI3K)-protein kinase B –Akt pathway [37, 38]

leading to activation of mitogen-activated protein kinase kinase (MEK) and MAPK (Erk1/2)[36]. The third signaling pathway activated by IL-6 via Jak2 is phosphoinositol 3 kinase (PI3K)-protein kinase B –Akt pathway [37, 38] (Fig. 10.1).

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10.3

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Regulators of Stat Signaling

The physiological actions of Stats are regulated by interactions with different cellular proteins and dephosphorylation [15]. In cancer, however, the various mechanisms that lead to persistent activity of Stat5a/b and Stat3 are under extensive research. Although Stat3 and Stat5a/b both recognize the same GAS consensus sequence TTCN2–4GAA, the gene expression regulatory functions are largely different for Stat5a/b versus Stat3, probably because of different co-activator, co-repressor, and chaperone expression profiles and interactions [39]. In prostate cancer, the positive regulatory mechanisms related to Stat activation may involve autocrine prolactin production activating the Jak2-Stat5a/b signaling [28, 29], gain-of-function mutations of Jak2, or somatic changes of Stat5a/b genes. Other tyrosine kinases, such as Src and Fer, have also been suggested to activate Stat5a/b in prostate cancer cells [40, 41]. In addition, receptor tyrosine kinases of EGFR family have been shown to activate Stat3 signaling [42]. Caspase-3, an apoptosis inhibitor, was found to be the probable effector of EGF/Stat3 action in DU145 prostate cancer cells [42]. In addition, there is evidence of somatic missense mutations in EGFR of prostate cancer patients, which leads to phosphorylation of Stat3. However, these effects were ligand-independent and phosphorylation of Stat3 disappeared upon addition of EGF [43]. Signaling pathways that negatively regulate Stat3 and Stat5a/b function may affect prostate cancer growth. The most studied of these regulatory mechanisms are Protein Inhibitors of Activated Stat proteins (PIAS), cytoplasmic and nuclear Protein Tyrosine Phosphatases (PTP), and Suppressors of Cytokine Signaling (SOCS) proteins. PIAS family of inhibitors includes PIAS1, PIAS3, PIASx, and PIASy, and alternative splicing variants of PIASx [44]. These proteins are located in the nucleus and repress Stat3 and Stat5a/b activity by a direct interaction. PIAS3 negatively regulates Stat3 DNA binding and represses the PRL-induced transcriptional activity of Stat5a/b [45, 46]. PTPs regulate the intranuclear dephosphorylation of the Jak2/ Stat5 signaling components and one of the PTP member, SHP-2, has been shown to interact directly with Stats [47]. Recently, another member of PTP family, SHP-1, has been shown to suppress IL-6-induced, Stat3-mediated prostate cancer cell proliferation in hormone-dependent LNCaP cancer cell model [48]. The third established down-regulatory mechanism of Stat signaling encompasses the suppressors of cytokine signaling -protein family. This family has eight members, SOCS1–7 and CIS (cytokine-inducible SH2 domain protein), and they block the upstream Jak activators by direct inhibition and/or by competitive binding to receptors [49]. Alternatively, SOCS proteins may bind to phosphorylated receptors competing for Stat binding [50, 51]. The SOCS1, SOCS3, SOCS5 and CIS protein levels are lower in hormone-independent cell lines DU145 and PC-3, but higher in wild type, hormone-dependent LNCaP cells as compared to normal epithelial cells [52]. Others have recently demonstrated that inhibition of SOCS-3 leads to reactivation of Stat1 and Stat3, but causes activation of extrinsic and intrinsic apoptosis pathways [53]. In castration-refractory clinical cancers, SOCS-3 protein levels are elevated [53].

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Stat5a/b and Stat3 Expression Patterns in Clinical Prostate Cancer and as Biomarkers of the Disease Progression

The findings on the constitutively active autocrine Prl-Jak2-Stat5a/b and IL-6Jak2-Stat3 pathways in prostate tissue [54–56] have led to studies focused on the role of Stat3 and Stat5a/b in prostate cancer progression. Li et al. (2004) showed that nuclear Stat5a/b expression was associated with high Gleason grade in clinical prostate cancer specimens [28]. In addition to the association with the loss of prostate cancer differentiation, nuclear Stat5a/b and prolactin expression, both were elevated in distant prostate cancer metastases and castration-resistant prostate cancers [29, 57]. Li et al. (2005) further showed that activated nuclear Stat5a/b predicted early prostate cancer recurrence in a univariate analysis [58]. Furthermore, multivariate analysis using Cox proportional hazard regression showed that Stat5a/b activation remained an independent prognostic factor (HR 1.63, 95% CI 0.99–2.69) of short progression-free survival of prostate cancer. However, the patient cohort in this study was heterogenous in terms of treatment modalities and therefore the significance of active Stat5a/b in predicting the clinical outcome should be addressed in a more homogenous group of patients with disease-specific mortality as an additional end-point. It has also been shown that Stat5a/b is constitutively active in over 60% of clinical prostate cancer metastasis [59], and another study indicated a higher frequency of Stat5a/b activation in castration-resistant prostate cancers treated by an androgen blockade vs. hormone naïve prostate cancers [57]. IL-6 is an autocrine cytokine in prostate cancer [55], and Stat3 is constitutively activated in human prostate cancer tissue [56]. Activation of Stat3 has been associated with high Gleason score in immunohistochemical and proteomic studies [56, 60–62]. The study by Abdulghani et al. (2008) showed that Stat3 is activated in the majority of clinical prostate cancer metastases [63]. In the study by Horinaga et al. (2005), including 92 patients, the increased phospho-Stat3 immunoreactivity was associated with shorter time to biochemical recurrence after radical prostatectomy [64]. In contrast, the nuclear expression of Stat3 correlated inversely with distant metastases formation in another study, and low Stat3 expression was an independent predictor of metastasis in patients treated with radiotherapy or combined radio- and chemotherapy [65]. In a large scale prognostic analysis by Li et al. (2005) including 357 primary prostate cancer patients treated with radical prostatectomy, active Stat3 did not predict prostate cancer recurrence (unpublished data), while active Stat5 was a marker of poor clinical outcome in the same cohort [58]. A recent study reported that Stat3associated regulatory protein SHP-1 inversely correlated with disease progression and biochemical recurrence after radical prostatectomy [66]. Considering the abundance of Stat3 in later stages of prostate cancer, i.e., metastasis and castration resistance, the prognostic significance of Stat3 should be evaluated with disease-specific mortality as the end-point.

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Stat5a/b and Stat3 in Growth Promotion of Prostate Cancer

Both Stat5a/b and Stat3 have been shown to be involved in the regulation of viability and proliferation of human prostate cancer cells. The crucial role of Stat5a/b in promotion of prostate cancer cell viability and tumor growth has been demonstrated in several different studies. Inhibition of Stat5a/b transcriptional activity by adenoviral delivery of a dominant negative (DN) mutant of Stat5a/b (lacking the TA domain) results in apoptotic death of a battery of human prostate cancer cell lines in culture [41, 67, 68]. In addition, inhibition of Stat5 markedly decreased the growth of human prostate xenograft tumors in nude mice [41, 67, 68], whereas normal prostate epithelial cells did not respond to Stat5a/b inhibition [41]. Blocking the protein expression of Stat5a/b by RNA interference or antisense oligonucleotides resulted in a rapid cell death of human prostate cancer cells [41, 68]. Moreover, Stat5b siRNAs, but few Stat5a siRNAs, decreased the viability of DU145 prostate cancer cells, suggesting that Stat5b might have the predominant role over Stat5a in regulating prostate cancer cell growth [41]. Inhibition of Stat5a/b also blocked the progression of prostate cancer in the TRAMP mouse prostate cancer model [69]. Mediators of Stat5a/b effects on prostate cancer cell viability include Cyclin D1 and BclXL proteins [68], and additional targets were recently identified by gene expression profiling [41, 59]. The role of Stat3 in prostate cancer growth is more controversial. Induction of transcription factor Stat3 expression has been reported to induce a change of normal prostate epithelial cells to a malignant phenotype [70]. In addition, blocking Stat3 function has been shown to induce apoptosis of DU145 prostate cancer cells in vitro [60, 71]. There is evidence that active Stat3 binds to DNA in benign tissue adjacent to prostate cancer in whole tissue sections suggesting that Stat3 activation may occur before detectable histological alteration of the prostate [56]. On the contrary, others have shown that LNCaP cells that express a dominant-negative form of Stat3 exhibit a proliferative response to IL-6 stimulation [72], indicating that Stat3, in fact, mediates growth inhibition of human prostate cancer cells. In addition, IL-6 produced by prostate stromal cells derived from benign hyperplasia had a growth inhibitory effect on LNCaP cells [73]. When Stat5a/b and Stat3 were compared side-by-side for their significance on prostate cancer cell viability and survival, it became evident that Stat5a/b has the predominant role in growth promotion of prostate cancer over Stat3. Specifically, siRNA inhibition of Stat5a/b resulted in 70% decrease in the viability of DU145 human prostate cancer cells, while Stat3-inhibition reduced the DU145 cell viability only by 20%[41]. Similarly, inhibition of transcriptional activity of Stat3 by adenoviral delivery of a DN form of Stat3 decreased the number of viable DU145 cancer cells by 30% as compared to 87% decrease induced by DNStat5a/b [41]. Both the DNStat5a/b and DNStat3 constructs lack the C-terminal transactivation domains, resulting in Stat5 and Stat3 proteins able to bind to DNA but unable to initiate transcription. In the same study, the inhibition of Stat3 activity by DNStat3

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did not have any significant effect on the growth of orthotopic or subcutaneous prostate tumors in nude mice, whereas DNStat5a/b reduced both the growth rates and the volumes of the tumors robustly [41].

10.6

Stat5a/b and Stat3 in Promotion of Metastatic Behavior of Prostate Cancer

Both Stat5a/b and Stat3 induce metastatic behavior of human prostate cancer cells in vitro and in vivo [41, 63]. Adenoviral expression, of wild-type (WT) Stat3 in DU145 cells led to 33–50-fold increase in metastases formation in the lungs of nude mice after tail-vein injections of the cells in comparison to the control DU145 cells. Stat3 increased migration of prostate cancer cells, in addition to reorganization of actin cytoskeleton and the microtubule network [63]. Furthermore, Abdulghani et al. (2008) showed that Jak2 inhibition by a dominant-negative mutant of Jak2 (DNJak2) blocked tyrosine phosphorylation of Stat3 and led to similar morphological changes as Stat3 inhibition, whereas overexpression of WTJak2 increased Stat3 activity and cell migration [63]. Similarly to Stat3, Stat5a/b is activated in the majority of distant human prostate cancer metastases [41]. Introduction of an active form of Stat5 promoted DU145 prostate cancer cell migration and invasion in vitro, and increased the in vivo lung metastasis formation by 11-fold in nude mice after tail-vein injections [41, 59]. Surprisingly, when wild-type Stat5, not the active form of Stat5, was introduced to DU145 prostate cancer cells, it increased metastases formation only by four to fivefold [59]. These data indicate that the autocrine Stat5-activating signaling pathway is not fully functional in DU145 cells, since wild-type Stat5 was not able affect the metastatic potential of the cells to the same extent as the active form of Stat5. Disrupted Prl-Jak2-Stat5 signaling pathway in DU145 cells may be due to low level expression of Prl-receptors in these cells [74]. Metastases-related mechanisms associated with active Stat5a/b include increased migration, decreased cellsurface E-cadherin levels, and increased heterotypic adhesion of prostate cancer cells to endothelial cells [59]. Gene expression profiling indicated that the majority of Stat5a/b regulated genes were metastases-related [41, 59].

10.7

Molecular Mechanisms Underlying Stat5/Stat3 -Induced Metastatic Behavior of Human Prostate Cancer Cells

Prostate cancer metastases formation is a multi-step process. First, prostate cancer cells have to move from the primary tumor mass into the bloodstream where they must endure the hemodynamic stresses, evade the natural host immune response, and arrest at the secondary site. Once at the secondary site, the metastatic prostate

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cancer cells need to adapt to the local microenvironment, proliferate, and induce angiogenesis to form successful secondary colonies. Failure to complete any step in this cascade prevents formation of distant metastases. Both Stat5a/b and Stat3 have been shown to promote metastasis formation of blood-borne prostate tumor cells in vivo after tail-vein injections [41, 59]. Hematogenous metastasis is believed to be a relatively inefficient process. This is due to the destruction of cells in the bloodstream by shear stress, the immune system, and a slow rate of extravasation. In addition, one key contributor for metastatic potential of cancer cells is the capability for anchorage-independent growth and survival in the bloodstream and at the distant site. While both Stat5a/b and Stat3 promoted metastases formation in the in vivo experimental metastasis assay, only Stat5a/b, not Stat3, had a major role in induction of prostate cancer cell survival [41, 68]. In addition, Stat5a/b regulated a largely different set of genes from those of Stat3 in human prostate cancer cells [41]. This suggests that the two transcription factors likely affect different steps of the metastases process (i.e., survival, arrest at the secondary site, extravasation, invasion, migration, colonization) and have different mechanisms of action to induce colonization of the circulating tumor cells. Both Stat5a/b and Stat3 induce migration of human prostate cancer cells [41, 59, 63]. Acquisition of a migratory phenotype is concurrent with the epithelial-mesenchymal transition (EMT), whereby epithelial cells lose polarity and homotypic adhesion and undergo dramatic remodeling of the cytoskeleton. Both Stat5a/b and Stat3 were shown to affect the cellular organization of the microtubule network while only Stat3 induced polarization of the actin cytoskeleton [41, 63]. Ng et al. has shown that Stat3 interacts with stathmin, a small cytosolic phosphoprotein that binds a/b-tubulin heterodimers, and facilitates cell migration [75]. The interactions of Stat5a/b or Stat3 with stathmin in human prostate cancer cells remain to be investigated. In general, hallmarks of EMT in prostate cancer cells include loss of epithelial markers, such as E-cadherin, concomitant with up-regulation of mesenchymal markers, such as N-cadherin, also known as “cadherin switching”[76, 77]. E-cadherin, the major component of epithelial adherens junctions, is a single-span transmembrane glycoprotein with a cytosolic domain that binds to a, b -catenin and p120 proteins, which interact with the cytoskeleton and facilitate cell-cell adhesion [78–81]. Importantly, Stat5 activation induced down-regulation of cell surface E-cadherin in human prostate cancer cells in vitro [59], and low cell surface E-cadherin expression correlated with high nuclear Stat5 levels in clinical prostate cancers [58]. Decreased homotypic adhesion of prostate cancer cells was induced by constitutively active Stat3 shown in another study [82]. In functional E-cadherin complexes, which are located in adherens junctions, the cytosolic domains of E-cadherins bind to a and b- catenin which interact with the microtubule network, thus the loss of homotypic adhesion may initiate depolymerization of microtubules, promoting prostate cancer cell motility. Recently, decreased b-catenin expression was shown to predict early prostate cancer-specific death [83]. However, b-catenin expression alone is not able to induce EMT of cancer cells [84]. Also, decreased p120 expression, which stabilizes cadherins, has been associated with a shorter, recurrence-free survival of prostate cancer [83]. In summary, the individual and

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detailed roles of Stat5a/b and Stat3 in EMT of human prostate cancer cells need to be determined. Loss of E-cadherin can be achieved by mutation, DNA methylation, or silencing of the promoter region. Genes that suppress E-cadherin expression are zinc finger proteins that bind to the E-cadherin gene (CDH1) promoter and include Snail, Slug, SIP1, and TWIST [85–89]. TWIST, a basic helix-loop-helix transcription factor, is a crucial modulator of a prostate-specific metastatic phenotype. This phenotype is achieved through TWIST-induced loss of E-cadherin-mediated cell-cell adhesion and expression of mesenchymal markers [84, 87, 90–92]. In prostate cancer, TWIST expression is correlated with high Gleason grade and TWIST is expressed at high levels in metastatic human prostate cancer cell lines, DU145 and PC-3[90, 91]. Two groups have independently demonstrated that active Stat3 binds to the TWIST promoter and directly regulates the transcriptional activity of TWIST [93, 94]. Regulation of TWIST expression by Stat5a/b and Stat3 in human prostate cancer has yet to be determined. In order to escape into the extravascular tissue to establish metastases, circulating tumor cells arrested in capillary beds of different organs must invade the endothelial cell lining of blood vessels and degrade its underlying basement membrane. Tumor cell metastasis is facilitated by formation of “premetastatic niches” in the destination organs [95]. At premetastatic sites, unidentified tumor-secreted factors are thought to induce elevated fibronectin expression [95] which may serve as an anchor to arrest circulating tumor cells. The key integrin receptor subunit binding to fibronectin is beta1. Integrins are transmembrane heterodimer receptors consisting of an alpha and beta subunit [96]. By binding to extracellular matrix (ECM) components, such as laminin, collagen, and fibronectin, they convey heterotypic signals and interactions. There are at least 24 members in the integrin family, 18 alpha and 8 beta subunits. Beta 1, 3 and 6 are upregulated in human prostate cancer – they are known to localize in focal contacts and to mediate spreading and cytoskeletal rearrangements in normal cells [97, 98]. There are 5 different cytoplasmic variants of the human beta1 subunit: beta 1A, beta 1B, beta 1C, beta1C-2 and betaD, of which only b1A and b1C are expressed in prostate epithelium [99, 100]. b1A is upregulated in prostate cancer, stimulates proliferation, and allows for anchorageindependent growth [100]. The splice variant b1C mRNA and protein levels are reduced in prostate cancer, compared to levels in benign prostate epithelial cells [101, 102], and integrin b1C is an inhibitor of prostate cancer cell proliferation. In prostate cancer, active Stat5a has been shown to promote heterotypic adhesion of prostate cancer cells to endothelial cells [59]. Constitutively active Stat3 induces transcriptional up-regulation of b6 bound to fibronectin in human prostate cancer cell lines [82]. There is no information in the literature on Stat5 regulation of integrins in human prostate cancer cells or xenograft tumors. Finally, prostate cancer migration and invasion may result from secretion of proteases by the cancer cells. Matrix metalloproteases (MMPs), a multigene family of zinc-dependent endopeptidases, degrade ECM components and have been implicated in EMT by cleavage of E-cadherin [103–105]. MMP-9 and MMP-2 expression positively correlate with Gleason score and promote development of metastases

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of prostate cancer cells [106–108]. Stat3, in turn, has been shown to regulate MMP-2 and MMP-1 expression by direct promoter binding [109, 110]. In summary, both Stat5a/b and Stat3 are involved in the development of disseminated prostate cancer. Little is known about the molecular mechanisms mediating the metastases process of Stat5a/b vs. Stat3, which are likely to differ and therefore require further studies. Both Stat5a/b and Stat3 may provide effective therapeutic targets for metastatic prostate cancer. Acknowledgments We would like to thank Ms Elyse Amico for her editorial contributions to the chapter.

References 1. Jemal A, Siegel R, Xu J, Ward E (2010) Cancer statistics, 2010. CA Cancer J Clin 60(5):277–300 2. Ihle JN (2001) The Stat family in cytokine signaling. Curr Opin Cell Biol 13(2):211–217 3. Haddad B, Pabon-Pena CR, Young H, Sun WH (1998) Assignment1 of STAT1 to human chromosome 2q32 by FISH and radiation hybrids. Cytogenet Cell Genet 83(1–2):58–59 4. Yamamoto K, Kobayashi H, Arai A, Miura O, Hirosawa S, Miyasaka N (1997) cDNA cloning, expression and chromosome mapping of the human STAT4 gene: both STAT4 and STAT1 genes are mapped to 2q32.2– shaft

injected mice (Fig. 15.6). MIG levels the femurs of mice inoculated with human breast cancer cells were lower at day 19 compared to femurs of mice inoculated with PBS. However, by day 27, the metaphyses of femurs from mice inoculated with human breast cancer cells expressed larger amounts of MIG than the femurs of mice inoculated with PBS. It was generally seen that cytokine concentration in the femurs of mice inoculated with MDA-MB-231BRMS1 cells were expressed in similar quantities to the femurs from mice inoculated with MDA-MB-231 cells.

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Fig. 15.5 Comparison of cytokines detected in the metaphyses of femurs of mice following intracardiac inoculation of metastatic and metastasis suppressed breast cancer cells. Femurs were removed from mice 3 weeks following inoculation of MDA-MB-231GFP cells, MDA-MB231BRMS1GFP cells or PBS into the left ventricle of the heart. The femurs were treated as described in the legend to Table 15.2. Femurs from eight mice (16 femurs) were assayed. Shown is the mean log of the cytokine concentration. The results were analyzed by a statistical model in which femurs from the same mouse were considered repeated measurements and the mice were grouped according to treatments with PBS, MDA-MB-231 (231) or MDA-MB-231BRMS1 (BRMS). *P < 0.05, **P < 0.01, ***P < 0.001

IL-6 increased earlier; by day 11 it was higher in the cancer- injected mice. Eotaxin showed no significant differences among groups until day 27. For the most part the cytokines from the MDA-MB-231BRMS1 GFP inoculated mice followed the same expression pattern as the MDA-MB-231GFP cells. The fact that the metastatic and non-metastatic cell lines elicit the same cytokine responses in the bone implies that the local cytokine environment is not responsible for the inability of the BRMS1 cells to proliferate in bone.

15.4

A Three-Dimensional Model of the Bone Microenvironment

Due to the sheer expanse of the human skeleton as well as difficulty in accessing the bone marrow cavity in the clinic, tumors and osteolytic lesions are usually not detected until late in the metastatic process. Lack of easy access makes it very difficult to study cellular and molecular mechanisms involved in cancer colonization of bone and hampers rapid development of therapeutic interventions. Although excised tissue captures end stages of metastases in bone, the critical initial stages of disease remain substantially inaccessible. Bioengineers have been attempting to develop

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Fig. 15.6 Changes in MCP-1 and MIG concentrations over time in the metaphyses of femurs following intracardiac inoculation of MDA-MB-231GFP cells, MDA-MB-231BRMS1GFP cells or PBS into the left ventricle of the heart. The femurs were treated as described in the legend to Table 15.2. Shown are the median log transformed values ± standard deviations for MCP-1 (left) and MIG (right). The numbers indicate that the values on a particular day are statistically different (P < 0.5) from the days listed above the box

cell culture systems that mimic the in vivo system [32–34]. These culture constructs, often referred to as bioreactors, attempt to bridge the complexity from tissue culture polystyrene to an animal model. Ideally, a bioreactor allows a rapid and orderly development of tissue starting from single cells. A novel, compartmentalized bioreactor based on the principle of simultaneous growth and dialysis [35] has been developed which allows this process to occur. A dialysis membrane separates a cell growth space from a larger volume medium reservoir. Cells are inoculated into the growth chamber in complete medium including serum. The reservoir is filled only with basal medium. During culture, cells are bathed in pH-equilibrated and oxygenated medium continuously dialyzing from the reservoir. At the same time, metabolic waste products such as lactic acid dialyze out of the growth compartment, maintaining low pericellular concentrations. Serum constituents or macromolecules synthesized by cells with molecular weights in excess of the dialysis membrane cutoff (typically 6–8KD) are retained and concentrated within the growth compartment. The pericellular environment is unperturbed by refreshing only the media reservoir (not cell compartment). Thus, the culture environment is very stable; various chemical gradients that guide

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Fig. 15.7 Morphological progression of MC3T3-E1 cells over 10 months of bioreactor culture. Cultures were monitored by confocal microscopy of Alexa Fluor 568 phalloidin stained cells (a–d). Osteoblasts progress from rapidly proliferating cells (a and b) to ‘cobble stone’ shaped cells (c) to stellate shaped osteocyte-like cells (d). Metastatic cancer cells co-cultured with 2 month old, osteoblasts (red, stained with Cell Tracker Orange) for a period of 7 days (e–h). MDA-MB-231 cells (e and f) adhere, proliferate and form colonies and invade the multi-layered osteoblast tissue (f). The MDA-MB-231 cells form single files of cells (arrows f), and also cause a change in osteoblast morphology from cuboidal (c) to spindle shaped. In contrast, MDA-MB-231-BRMS1 cells (g), a metastasis suppressed variant of MDA-MB-231, adhere lightly to and proliferates on the osteoblast tissue. By day 7 of co-culture, the BRMS1 cells show signs of apoptosis (arrows g). Multiple myeloma cells (h) appear to aggregate in groups that undergo rapid proliferation and eventually cover over the osteoblast tissue

osteoblast maturation can develop. As a consequence, a thick and highly mineralized osteoblastic tissue can be grown and maintained for long terms [36, 37]. The bioreactor has allowed direct observation of osteoblast development; i.e. proliferation of pre-osteoblasts and differentiation into mineralizing osteoblasts that become engulfed in a thick, cell-secreted mineralized matrix that completely surrounds the cells (Fig. 15.7) [38]. Development and maturation of cell-derived matrix is important because it has been shown that interaction of cancer cells with reconstituted collagen layers is fundamentally different than interaction with authentic cell-produced matrix [39]. Phenotypic maturation of osteoblastic tissue was measured as a function of time in the bioreactor (Fig. 15.7a–d). In some cultures, after 5 months, contiguous, macroscopic mineral deposits were formed that proved consistent with bone hydroxyapatite by X-ray scattering and IR spectroscopy [38]. In addition, the gene expression profiles of characteristic osteoblastic proteins such as type 1 collagen, alkaline phosphatase, osteonectin, osteopontin and osteocalcin, mirrored that observed in vivo. After several months, cell morphology strongly resembled osteocytes (Fig. 15.7d) and cultures expressed molecules indicative of osteocytes;

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i.e. E11, DMP1, and sclerostin (Fig. 15.7a–d). The compartmentalized bioreactor represents a valid in vitro model for studies of bone biology and pathology. The introduction of MDA-MB-231GFP human metastatic breast cancer cells (known to invade the murine skeleton) [40] onto MC3T3-E1 osteoblast tissue grown in this bioreactor to various stages of phenotypic maturity allowed for observation of early stages of cancer cell colonization in real time by confocal microscopy (Fig. 15.7e, f) [41]. These stages included breast cancer cell to osteoblast tissue adhesion, cancer cell proliferation, tissue penetration, formation of non-vascularized colonies of tumor cells, and degradation of osteoblast derived extracellular matrix (ECM). Cancer cells proliferated and organized into single cell files (Fig. 15.7f, arrows) as described for infiltrating lobular or metaplastic breast carcinomas [42]. Migration of cancer cells along tracks of remodeled ECM produced by preceding invading cell(s) is likely responsible for the characteristic cell alignment patterns [43]. Invasion by chains of tumor cells linked together by cell-cell contacts is considered to be an effective penetration mechanism, [44] conferring high metastatic capacity and commensurately poor prognosis when observed in patients [42, 43]. Observation of single-cell filing in the osteoblast tissue model indicated a considerable degree of physiological relevance. Single-cell filing is common in soft tissue but also found in bone metastasis [45]. Also, the osteoblasts increased their production of inflammatory cytokines, i.e. IL-6, KC, MIP-2, MCP-1, VEGF. Concomitantly, production of collagen, osteocalcin and other differentiation proteins responsible for bone accretion was reduced. To clarify the effect of metastatic cancer cells on the osteoblast tissue, metastasis-suppressed MDA-MB-231-BRMS1 cells were cocultured with the mature osteoblast tissue (Fig. 15.7g). MDA-MB-231-BRMS1 cells interacted with the tissue in a very different manner than the metastatic MDA-MB-231 cells. BRMS1 cells formed small colonies on osteoblasts but did not penetrate the tissue, or form micro-tumors. Furthermore, the metastasis suppressed cells did not cause changes in osteoblast morphology. However, they showed marked signs of apoptosis (Fig. 15.7g, arrow). Thus we have demonstrated that 3D, multiple-cell-layer osteoblast tissue grown in the simultaneous-growth-and-dialysis bioreactor is a clinically relevant in vitro model in which to study osteoblastic aspects of metastatic breast cancer in bone. This bioreactor tissue model is an ideal tool to study various other cancer types, such as multiple myeloma, and prostate cancer that preferentially metastasize to bone but behave differently than breast cancer [46, 47]. This model allows one to control the bone microenvironment while varying types of cancer. Multiple myeloma arises from a hematopoietic lineage. The cells are found in close association with sites of active bone resorption; their ability to stimulate osteoclast formation and activity has been well characterized [48]. In the early stages of multiple myeloma, bone formation is actually increased. This increase is thought to reflect a compensation for the increase in osteoclastic resorption [49]. However, as the disease progresses, bone formation is rapidly reduced and as a consequence, the osteolytic aspect of multiple myeloma increases, resulting in the release of a number of pro-tumorogenic factors that promote cancer cell growth and survival. The bioreactor was employed to co-culture multiple myeloma cells with osteoblast tissue

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Fig. 15.8 Three dimensional reconstruction of confocal Z-stack images of multiple-myeloma cells on 2 month old osteoblast tissue after 7 days of co-culture. Multiple-myeloma cells adhere to the top layer of osteoblast tissue and proliferate to from a confluent layer (~11–12 mm thick optical sections), but fail to penetrate through to the bottom layer of osteoblasts

(Figs. 15.7h and 15.8). Myeloma cells were observed to adhere to the osteoblasts and eventually formed a confluent monolayer. Unlike breast cancer cells that quickly penetrated the osteoblastic tissue, the myeloma cells remained in a layer over the osteoblasts. It has long been appreciated that the extracellular matrix (ECM) is a primary determinant of cell phenotype [50]. In recent years, the role of ECM in controlling malignancy and the remodeling of ECM that occurs during cancer cell colonization of tissue has become a focus of research [51]. A pantheon of important adhesins and proteases have been identified [52], and the overall structure of the ECM has been shown to control cancer cell migration [53]. For example, the interaction of cancer cells with reconstituted collagen layers is fundamentally different than interaction with authentic cell-produced matrix [39]. In an effort to investigate changes induced in the osteoblast ECM by the MDA-MB-231 cells, osteoblasts were exposed to conditioned medium from the cancer cells. After 4 h of incubation with MDA-MB-231 breast cancer conditioned medium, osteoblast RNA was extracted and analyzed

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Table 15.3 Gene expression analysis of 3 month old osteoblasts treated with breast cancer cell conditioned media (CM) in bioreactor culture system Fold change when treated with CM Up regulated genes Procollagen, type IV, alpha3 (Col4a3) 4.0 Elastin microfibril interfacer 1 (Emilin1) 4.0 Ectonucleoside triphosphate 5.0 diphhosphohydrolase 1 (Entpd1) Integrin alpha 2 (Itga2) 3.0 Integrin alpha 5 (Itga5) 3.6 Integrin beta 3 (Itga3) 4.0 Matrix metallopeptidase 1a (MMP1a) 4.5 Matrix metallopeptidase 3 (MMP3) 3.0 Down regulated genes Fibulin 1 (Fbln1) Hemolytic Complement (Hc) Synaptotagmin 1 (Syt1) A disintegrin-like and metallopeptidase with thrombospondin type1 motif, 5 (Adamts5) Cadherin 1 (Cdh1)

−7.0 −7.2 −4.0 −3.1 −4.2

MC3T3-E1 osteoblasts grown in the bioreactor for 3 months were treated with 50% MDA-MB-231 conditioned medium for 4 h. RNA was collected and profiled for expression of 84 genes involved in cell-cell and cell-matrix interaction using an SABiosciences gene expression array. Genes that exhibited a threefold or greater change are listed

with a gene array specific for expression of ECM related genes. Of the 84 genes on the array, three to fivefold increases were detected in several integrins, MMPs, and procollagen type IV (Table 15.3). Furthermore, several proteases and adhesion molecules were decreased. These preliminary findings along with observed morphology changes in direct co-cultures suggest that the osteoblast matrix was remodeled as a result of molecules secreted by the osteoblasts and the cancer cells and by cell-cell contact. The matrix that forms in the 3D culture is critical for an understanding of the cancer cell colonization of the host tissue. In order to increase the biological complexity of the system and to begin to model the vicious cycle, osteoclasts have also been added to the system. The aim is to create a tri-culture system containing the key cells that define the vicious cycle (osteoblasts, osteoclasts and metastatic cancer cells) and monitor their interaction. This model could be an invaluable tool in targeted therapeutic studies. In summary, the 3D in vitro culture system enables both tumor and non-tumor cells to grow and organize in a more biologically relevant manner, enhancing our ability to observe and to manipulate cancer progression. Three dimensional model systems that more accurately mimic in vivo phenotypes, such as the cell perfusion culture system in microfluidic channels, are being designed to advance drug toxicity, metabolism, and stem cell differentiation studies [54]. Most importantly, cell-cell interactions can be closely monitored in a 3D culture system, without specialized microscopy or sacrifice of animals.

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Conclusions

A variety of systems described in this chapter and ranging from standard cell culture to three-dimensional bioreactors to mouse models have been used to study the interaction of breast cancer cells with the bone microenvironment. These observations, taken together, support the hypothesis that normal osteoblast function (i.e. deposition of matrix) is impaired in the presence of breast cancer cells. Furthermore, osteoblasts undergo a stress response and secrete increased levels of inflammatory cytokines that create an environment conducive to cancer cell propagation and to osteoclast activation. Thus the metastatic bone environment results from the synergy among the resident bone marrow cells and the invading cancer cells. While current therapies are directed towards activated osteoclasts and are effective in slowing lesion progression, new therapeutic strategies should focus beyond inhibition of osteoclasts and include enhancement or restoration of osteoblast function. Further understanding of the cellular and molecular basis for breast cancer interactions with bone requires a complete and in-depth understanding of the bone microenvironment including cell-cell interaction, cell-matrix interaction and cytokine crosstalk as they occur over the time of cancer colonization. Models that increase the biological complexity by inclusion of other cells of the metastatic bone environment could reveal unknown interactions and possible new treatment options. Acknowledgements This work was supported through grants from the U.S. Army Medical and Materiel Command Breast Cancer Research Program (WX81XWH-06-1-0432 and WX81XWH-0801-0448), The Susan G. Koman Breast Cancer Foundation (BCTR 0601044), with help from The National Foundation for Cancer Research, and The Penn State Hershey Cancer Institute. We would like to thank Erwin Vogler, Carol Gay, Ravi Dhurjati, and Laurie Shuman for their work with various aspects of the projects.

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Index

A Androgen depletion therapy (ADT), 280–281 Androgen receptor (AR), oncogenes, and metastasis c-MYC (MYC) CTCs, 295–296 Ezrin expression, 295 Lo-Myc model, 294 proto-oncogene, 294 transgenic mice, 294–295 c-SRC, 296–297 Androgen response elements (AREs), 279

B Benign prostate hyperplasia (BPH), 231 Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE), 7 Biomechanical extracellular matrix switches basement membrane, 81–83 biological information, 72 embryonic development and tumor progression, 71 integrins, 72 interstitial matrix, 79–81 physiochemical and mechanical properties, 78 therapeutic and imaging targets, 83–84 tumor metastasis (see Tumor metastasis) Bladder cancer CD24, 338–339 endothelin axis, 335–337 lung microenvironment ECs, 333 premetastatic niche, 334

primary tumor and shed tumor cells, 332–333 TAMs, 334 vascular endothelium, 332 metastasis suppressor RhoGDI2, 335 Ral GTPases, 338 UC, 331–332 versican, 337–338 Blood-brain-barrier (BBB), 208 Bone marrow derived myeloid cells (BMDCs), 231, 233 Bone marrow microvasculature, 208 Bone microenvironment bioreactor, 361–362 BRMS1 cells, 363 confocal microscopy, 363 confocal Z-stack images, 364 3D culture system, 365 ECM, 364 gene expression, 365 myeloma cells, 364 single-cell filing, 363 Brain cancer cancer cell migration and invasion b1-containing integrins, 316 avb3 and avb5 integrins, 317 ECM components, 314–315 experimental models and clinical data, 313–314 glioma cancer stem cell function, 323–324 glioma treatment a5b1 integrin antagonists, 323 radiation and chemotherapy, 321–322 avb3/avb5 integrin antagonists, 322–323

A. Fatatis (ed.), Signaling Pathways and Molecular Mediators in Metastasis, DOI 10.1007/978-94-007-2558-4, © Springer Science+Business Media B.V. 2012

369

370 Brain cancer (cont.) integrins a and b subunits, 311–312 cell types, 313 ECM binding, 313 metastatic colonization, 315 regulating growth and angiogenesis animal models, 319, 320 glioma cells, 320–321 TGF-b activation, 321 avb3 ligands, 318–319 VEGF expression, 321 avb3 expression, 314 Brain microvascular endothelial cells (BMECs), 208

C Cancer androgen suppression therapy, 4 biochemical progression, 4 bladder (see Bladder cancer) brain (see Brain cancer) breast cancer, 5 chemotherapy and combination therapy, 7 cytoreductive nephrectomy, 5 DMFS, 6–7 malignancy ranges, 3 monoclonal antibody Cetuximab, 5 oligometastatic disease, 7 post treatment prostate biopsy, 4 primary tumor treatment, 5–6 radiation dose escalation, 4 radiation therapy, 3–4 stereotactic body radiation therapy, 7 tumor self seeding hypothesis, 7 Cancer cell extravasation cellular and molecular interactions, 214 clinical aspects, 213–214 EMT and, 212–213 mechanisms endothelium, 201–207 leukocyte vs. cancer cell extravasation, 199–201 metastatic inefficiency and extravasation, 210–212 metastatic organ, 214 vascular architecture bone marrow microvasculature, 208 brain microvasculature, 208–209 liver microvasculature, 209 lung microvasculature, 209–210 Cancer cell migration and invasion, 315–317 Cancer stem cells (CSCs), 323

Index Carbohydrate recognition domain (CRD), 337 Carcinoma-associated fibroblasts (CAFs), 231 Castration resistant prostate cancers (CRPCs), 279 Chondroitin sulfate (CS), 337 Circulating tumor cells (CTC), 198, 262, 295 Colony stimulating factor-1 (CSF-1), 124 Complement binding protein (CBP), 337 C-terminal domain (CTD), 224 CXCL12 and CXCR4 angiogenesis, 232–233 breast cancer progression and metastasis chemokine regulation, 227, 228 EGFR/HER2, 227 immunohistochemistry, 225, 226 lymph node metastasis, 227 hepatocellular carcinoma, 230 inhibitors AMD3100, 233 CTCE-9908, 234 Slit, 234–235 TN14003, 234 lung cancer growth and metastasis, 229–230 renal cell carcinomas, 230 signaling pathways breast cancer cells, 224, 225 Cbl protein, 222–223 EGFR phosphorylation, 224–225 GPCR, 222 p53, 224 prostate cancer cells, 223–224 tumor stroma, 231–232 Cyclooxygenase 2 (COX-2), 350

D Death domains (DDs), 156 5a-Dihydrotestosterone (5a-DHT), 279 Disseminated tumor cells (DTCs), 213, 262 Distant metastases free survival (DMFS), 6–7 DNA methyltransferases (DNMTs), 42 Ductal carcinoma in situ (DCIS), 225

E Early Breast Cancer Trialists’ Collaborative Group (EBCTCG), 5 E-cadherin, 18–19 ECM. See Extracellular matrix (ECM) EMT. See Epithelial-mesenchymal transition (EMT) Endothelial cells (ECs), 333

Index Endothelium circulating tumor cells, 201, 204–206 extravasation in vivo, 201–203 specific cancer-endothelial cell interactions, 206–207 Epidermal growth factor (EGF) and Ras cofilin, 27 ErbB family, 26 human prostate and breast cancer cells, 27 plasma membrane, 28 Raf-MAPK pathway, 29 signaling pathways, 26–27 Epidermal growth factor receptor (EGFR), 224 Epithelial cell transforming sequence 2 oncogene (Ect2), 150–151 Epithelial cellular adhesion molecule (EpCAM), 214 Epithelial-mesenchymal transition (EMT) basement membrane, 14 biomarkers and functional significance cytoskeleton dynamics changes, 19–21 ECM remodeling (see Extracellular matrix (ECM)) epithelial cell-cell junctions disruption, 18–19 cell signaling networks EGF and Ras, 26–29 HGF and FGF, 29–30 receptor kinases, 23 TGF-beta, 24–26 Wnt, Notch and Hedgehog, 30–33 cellular phenotype, 14 cobblestone network and apical-basal polarity, 15 CXCR4 CTD, 224 epithelial cells, 14–15 metastatic cancer, 15 metastatic cells, 13 MET conversions, 17 migratory phenotype and invasion, 16 miRNAs (see MicroRNAs (miRNAs)) N-cadherin and vimentin, 212 polarized epithelial cell, 212 prostate cancer cells, 253 solid human tumors, 15 squamous cell carcinomas, 16 transcriptional regulators HMGA2 and PcG (see High mobility group A2 protein (HMGA2) and Polycomb-Group Proteins (PcG)) homeodomain proteins, 39–41 Snail and Slug, 33–35 Twist1 and Twist2, 37–39 Zeb1 and Zeb2, 35–37

371 tumor cell invasion, 75 tumor metastasis, 13 type-3, 16 type-1 and type-2, 16 Erythroblast transformation specific (ETS), 288 Express green fluorescent protein (eGFP), 265 Extracellular matrix (ECM), 363 alpha5beta1 integrin, 21–22 CXCL12/CXCR4, 231 epithelial tissues, 21 fibronectin, 21 integrins bind, 311 laminin, collagen, and fibronectin, 254 lung microenvironment, 332 metastatic progression, 23 MMPs, 22–23 type-1 and type-2 EMT, 22

F Fibroblast growth factor (FGF), 29–30 Fibroblast growth factor inducible 14 (Fn14), 156–157 Fibroblast Specific Protein-1 (FSP1), 19–21 Fibronectin (FN), 78–79 Focal adhesion kinase (FAK), 177, 222, 313

G Gamma-interferon Activation Sequence (GAS), 247 Glioblastoma multiforme (GBM), 145 Glioma cancer stem cell function, 323–324 Glioma cell invasion Dock180-ELMO1 bipartite Rac1 GEF, 155 cancer cell invasion, 156 couples receptor signaling-rac, 154–155 human gliomas, 155 immunohistochemical analysis, 155 Rac1-promoted cell motility, 153–154 Dock family members, 152–153 Ect2, 150–151 Fn14, 156–157 GEFs, 147–148 glioblastoma treatments, 159 Rac1, 147 Rho GTPases, 145–147 Trio and SWAP-70, 149–150 TROY, 158 Vav3, 151–152 Glycosaminoglycan (GAG), 337 Gonadotropinreleasing hormone (GnRH), 280

372 G-protein coupled receptor (GPCR), 222 Green fluorescent protein (GFP), 349 GTPase activating protein (GAP), 268 Guanine nucleotide exchange factors (GEFs), 147–149

H Heat shock proteins (HSPs), 279, 280 Hepatocyte growth factor (HGF), 29–30, 177 High mobility group A2 protein (HMGA2) and Polycomb-Group Proteins (PcG) Bmi-1, 42 DNA and chromatin structure, 41 DNMTs, 42 E-cadherin, 43 ectopic expression, 41 Ezh2, 43 human cancers, 41 mesenchymal cell phenotype, 41 PRC1 genes, 42 protein–protein interactions, 41 Histone acetyltransferases (HATs), 294 Human epidermal growth factor receptor 2 (HER2), 224 Hypoxia acute and chronic, 174 HIF-1 signaling and metastasis, 174–175 metastasis impacts, 173 Hypoxia-inducible factor 1 (HIF-1), 173, 227

I Immunohistochemistry (IHC), 292 Insulin like growth factor-1 receptor (IGF-1R), 225 Integrin linked kinase (ILK), 313 Intercellular adhesion molecule (ICAM)-1, 199, 333

K Krüppel-like-factor 5 (KLF5), 288

L Leukocyte vs. cancer cell extravasation, 199–201 Lewis Lung Carcinoma (LLC) cells, 96 Ligand binding domain (LBD), 279 Liver microvasculature, 209 Low expression regions (LER), 76

Index Lung microvasculature, 209–210 Luteinizing hormone (LH), 280 Lysyl oxidase (LOX), 124

M Mammary epithelial cells (MECs), 123 Matrix metalloproteinases (MMPs), 22–23, 254, 289 Mesenchymal-epithelial transition (MET), 15–16 Metastatic breast cancer bone remodeling, 349–350 osteoblast apoptosis, 352 osteoblast cytokine responses bone marrow microenvironment, 357–360 cells interact, 354 MCP-1, 355 murine VEGF, 355–356 osteoblast inflammatory response, 353–354 skeleton, 349 three-dimensional model bioreactor, 361–362 BRMS1 cells, 363 confocal microscopy, 363 confocal Z-stack images, 364 3D culture system, 365 ECM, 364 gene expression, 365 myeloma cells, 364 single-cell filing, 363 vicious cycle, 351 Methylguanine methyltransferase (MGMT), 322–323 MicroRNAs (miRNAs) aberrant activation, 45 AP-1 and Zeb1 transcription factors, 49 DICER, 44 Drosha microprocessor complex, 45 gene-regulatory molecules, 43 human genome, 43 Let-7/miR-98 family, 47–48 Lin28A, Lin28B and RPS2, 45 mammary epithelial cells via transcriptional activation, 48 miRNA-200 family, 46–47 miR-21 transcription, 49 Myc expression, 45 oncogenic and tumor suppressor pathways, 44 precursor miRNAs, 44 primary transcripts, 44

Index RISC, 44 TGF-beta stimulation, 48 tumorigenesis, 45 untranslated regions, 44 Monocyte chemotactic protein 1 (MCP-1), 334, 349 Myeloid derived suppressor cells (MDSCs), 231

N Non-small cell lung cancer (NSCLC), 229 Nuclear Co-Repressor 1 (NCoR1), 284 Nuclear factor-kB (NF-kB), 334

O Osteoblast cytokine responses bone marrow microenvironment breast cancer cells, 357 MDA-MB231BRMS1 GFP, 358 mice femurs, 359–360 MIG levels, 359 mouse cytokines, 357 cells interact, 354 MCP-1, 355 murine VEGF, 355–356 Osteoprotegerin (OPG), 263, 350

P Parathyroid hormone related peptide (PTHrP), 263, 350 Phosphatidylinositol 3’-kinase (PI3K), 268 Phospho-glycerate kinase (PGK), 233 Phospholipase C (PLC), 268 Platelet derived growth factor (PDGF), 353 Polycomb-group (PcG), 293 Polycomb repressive complex 1 and 2 (PRC1 and PRC2), 42 Primary tumor microenvironments hypoxia, 125 mechanotransduction CSF-1, 124 3D-organotypic systems, 123 FAK expression, 124 LOX, 124 MECs, 123 phenotypes dominate genotypes, 123 TGF-b Paradox, 125 tumor fibrosis, 123 metastatic progression stimulation, 127–129 reactive tumor stroma, 126–127

373 Prolactin receptor (PrlR), 247 Prostate cancer cells bone marrow microenvironment, 263 bone-metastatic cancer, 269 early stages, 266–267 metastatic process and bone-tropism, 262–263 osteoclast bone-resorption activity bisphosphonates, 264 bone metastases, 265 OPG, 263 RANK activation, 263–264 skeletal foci, 265 ZA, 264 PDGFR expression, 267–268 PDGFR structure and signaling, 268 skeletal metastasis, 270 Prostate cancer progression and metastasis AKR1C3 and SRD5A1/2, 284 androgen-regulated miRNAS, 292 AR, 279–280 AR and chemokine receptors, 286–288 AR-dependent chromosomal translocations clinical implications, 291–292 TMPRSS2, 289–291 AR, oncogenes, and metastasis c-MYC (MYC), 294–296 c-SRC, 296–297 characteristics, 285 CRPC, 281–282 cyclin D1, 284 LBD mutations, 283 metastatic disease, 301–302 therapeutic intervention direct AR targeting, 299–301 indirect AR targeting, 299 treatment, 280–281 Prostate specific antigen, (PSA), 280 Protein Inhibitors of Activated Stat proteins (PIAS), 249 Protein tyrosine phosphatases (PTP), 249

R Reactive oxygen species (ROS), 333 Receptor Activator of NF-kB ligand (RANKL), 263 Receptor tyrosine kinases (RTK), 33, 312 Related adhesion focal tyrosine kinase (RAFTK), 222 Rho family GDPD issociation inhibitor 2 (RhoGDI2), 335 RNA-induced silencing complex (RISC), 44

374 S S100A4 and tumor invasion cancer prognostic marker, 94–95 extracellular annexin A2, 104 corneal vascularization model, 103 human plasma and synovial fluid, 104 oligomerization, 104 tumor interstitial fluid, 103 intracellular targets cytoskeletal elements, 101–102 myosin-II, 99–101 p53 expression, 102–103 LLC, 96 mammary epithelium, 96 metastatic process, 95 molecule-induced oligomers, 105–106 pharmaceutical industry, 105 S100 protein family Ca 2+-binding proteins, 91 calcium, 94 calmodulin and troponin C, 94 human chromosome, 91 human pathologies, 92 monomer, 92–93 pseudo EF-hand, 92, 94 tumor cell motility, invasion, and angiogenesis, 91–92 target protein interactions, 98–99 therapeutic intervention, 105 transgenic mice, 96 tumor cell proliferation, 107 tumor microenvironment, 96–98 Skeletal related events (SREs), 264 Small cell lung cancer (SCLC), 229, 234 Src family kinases (SFK), 296, 313 Stat5a/b and Stat3, prostate cancer cadherin switching, 253 castration resistant growth, 246 disease progression, 250 E-cadherin, 254 growth promotion, 251–252 metastatic behavior, 252 MMPs, 254–255 promote metastasis formation, 261–253 stat signaling regulators, 249 structure and function, 246–248 transcription factors, 246 TWIST expression, 254 Steroid receptor coactivator (SRC), 283 Stromal derived factor-1 (SDF-1), 233 Suppressors of Cytokine Signaling (SOCS), 249

Index T TGF-b. See Transforming Growth Factor-b (TGF-b) Thomas Friedenreich (TF) antigen, 206 Thyroid receptor a (TRa ), 318 Transcriptional activation (TA), 247 Transendothelial migration (TEM), 198 Transforming growth factor-b (TGF-b) animal models, 121–122 canonical TGF-b signaling, 118 carcinoma cells, 131–132 3D-organotypic models, 123, 125 E-cadherin and tight junction proteins, 25 EMT, 120–121 human tumors, 117 lymphatic and circulatory systems, 129 mammospheres, 129 matrix-bound factors, 351 matrix remodeling, 25 metastatic, EMT and chemoresistant phenotypes, 117 metastatic progression, 118–120 multifunctional cytokin, 24 neoplastic transformation, 116 noncanonical signaling systems, 118 ovarian cancer cells, 25 oxygen tensions and elastic moduli, 127 primary tumor microenvironments (see Primary tumor microenvironments) pulmonary microenvironment, 130 skeletal microenvironment, 131 Smad proteins, 24 Smad2/3 signaling, 118 Smurf E3 ubiquitin ligases, 118 TbR-III expression, 117 TGF-b ligands, 117 transgenic mouse models, 26 transmembrane signaling, 117 tumor modeling, 26 Transgenic adenocarcinoma of the mouse prostate (TRAMP) model, 296 Tumor cells cell-cell adhesion molecules, 76 cellular and non-cellular components, 73 collagen and fibronectin, 74 E-cadherin, 73 EMT, 75 FN, 78–79 heterodimeric transmembrane receptors, 77 HIF-1, c-Src and c-Met signaling pathways, 181–182 hypoxia acute hypoxia and chronic hypoxia, 174

Index HIF-1 signaling and metastasis, 174–175 metastasis impacts, 173–174 LER, 76 malignant tumors, 73 matrix-modifying enzymes, 77 melanoma and endothelial cells, 79 metastatic cascade, 73–74 metastatic phenotype, 171 metastatic process, 186–187 met signaling and metastasis c-Met, invasive growth and metastasis, 179 HGF/c-Met signaling pathways, 179–181 human cancers, 178 receptor tyrosine kinase c-Met, 177 pre-metastatic niches, 76 proteolytic enzymes, 73 signaling cascades, 79 Src signaling and metastasis metastasis-associated functions, 175–176 Src signaling pathways, 177 stromal fibroblasts and inflammatory infiltrates, 73 therapeutic interventions cancer cell dissemination, 186 hypoxia and HIF-1, 183–184

375 Met Kinase, 183–186 Src Kinase, 183–185 tumor microenvironment, 75, 171 Tumor necrosis factor (TNF), 156 Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), 157 Tumor necrosis factor receptor associated factor (TRAF), 156 Tumor necrosis factor receptor superfamily (TNFRSF) members, 156 Tyrosine-kinase-binding (TKB), 222

U urokinase-type plasminogen activator receptor (uPAR), 223 Urothelial cancer (UC), 331

V Vascular cell adhesion molecule (VCAM)-1, 333 Vascular endothelial growth factor (VEGF), 233 Vertebral-cancer of the prostate (VCaP), 289 von Hippel Lindau (VHL), 174, 228

Z Zoledronic acid (ZA), 264

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