Signal Transduction in Cancer Metastasis (Cancer Metastasis - Biology and Treatment, 15) 9048195217, 9789048195213

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Signal Transduction in Cancer Metastasis

Cancer Metastasis – Biology and Treatment VOLUME 15 Series Editors Richard J. Ablin, Ph.D., University of Arizona, College of Medicine and The Arizona Cancer Center, AZ, U.S.A. Wen G. Jiang, M.D., Wales College of Medicine, Cardiff University, Cardiff, U.K. Advisory Editorial Board Harold F. Dvorak, M.D. Phil Gold, M.D., Ph.D. Danny Welch, Ph.D. Hiroshi Kobayashi, M.D., Ph.D. Robert E. Mansel, M.S., FRCS. Klaus Pantel, Ph.D. Recent Volumes in this Series Volume 7: DNA Methylation, Epigenetics and Metastasis Editor: Manel Esteller ISBN 978-1-4020-3641-8 Volume 8: Cell Motility in Cancer Invasion and Metastasis Editor: Alan Wells ISBN 978-1-4020-4008-3 Volume 9: Cell Adhesion and Cytoskeletal Molecules in Metastasis Editors: Anne E. Cress and Raymond B. Nagle ISBN 978-1-4020-5128-X Volume 10: Metastasis of Prostate Cancer Editors: Richard J. Ablin and Malcolm D. Mason ISBN 978-1-4020-5846-2 Volume 11: Metastasis of Breast Cancer Editors: Robert E. Mansel, Oystein Fodstad and Wen G. Jiang ISBN 978-1-4020-5866-7 Volume 12: Bone Metastases: A Translational and Clinical Approach Editors: Dimitrios Kardamakis, Vassilios Vassiliou and Edward Chow ISBN 978-1-4020-9818-5 Volume 13: Lymphangiogenesis in Cancer Metastasis Editors: Steven A. Stacker and Marc G. Achen ISBN 978-90-481-2246-2 Volume 14: Metastasis of Colorectal Cancer Editors: Nicole Beauchemin and Jacques Huot ISBN 978-90-481-8832-1 Volume 15: Signal Transduction in Cancer Metastasis Editors: Wen-Sheng Wu and Chi-Tan Hu ISBN 978-90-481-9521-3

Signal Transduction in Cancer Metastasis Edited by

Wen-Sheng Wu Associated Professor, Institute of Medical Biotechnology, College of Medicine, Tzu-Chi University, Hualien, Taiwan and

Chi-Tan Hu Associated Professor, Research Center for Hepatology, Buddhist Tzu-Chi General Hospital, Hualien, Taiwan

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Editors Dr. Wen-Sheng Wu Institute of Medical Biotechnology College of Medicine Tzu-Chi University Chung-Yang Rd, Sec 3 701 970 Hualien Taiwan R.O.C. [email protected]

Dr. Chi-Tan Hu Research Center for Hepatology Department of Medicine Buddhist Tzu-Chi General Hospital Chung-Yang Rd, Sec 3 707 907 Hualien Taiwan R.O.C. [email protected]

ISSN 1568-2102 ISBN 978-90-481-9521-3 e-ISBN 978-90-481-9522-0 DOI 10.1007/978-90-481-9522-0 Springer Dordrecht Heidelberg London New York Library of Congress Control Number: 2010936099 © Springer Science+Business Media B.V. 2010 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)

Preface

Cancer remains to be one of the most devastating diseases world wide since long ago. The poor prognosis of cancer is largely due to metastasis. Metastasis is often depicted as a multistage process in which malignant cells spread from the primary locus to distant organs via circulation. Whereas genetic alterations were suggested to be essential for transformation of primary tumor cells into metastatic phenotype, epigenetic events are equally important, which may be triggered by metastatic factors wherever in the primary tumor locus, blood circulation and the secondary loci. Signal transductions initiated by the metastatic factors are responsible for mediating the molecular and cellular processes leading to metastasis. Blockade of the relevant molecular pathways is one of the most effective strategies for prevention of tumor metastasis. Clinical trials are underway with promising outcome. In this book, we take comprehensive review in regard with this exciting field of cancer research. Chapter 1 takes a brief overview of recently identified signal mechanisms for each step of tumor metastasis including the initiation stage, intravasation, anti-anoikis in blood circulation, homing, extravasation and final survival in the metastatic site. Chapter 2 makes a completed review for the molecular and cellular events involved in initiation of metastasis. Especially, the signaling mechanisms for mediating tumor progression induced by some important metastatic factors are described. In Chapters 3 and 4, the central roles of MAPK and its downstream effectors MAPKAPK play in each step of tumor metastasis are well delineated. Chapter 5 further describes detailedly about how Grb2 and other adaptor proteins, upstream of MAPK cascade, contribute to metastasis. In Chapter 6, the role of reactive oxygen species (ROS) in tumor progression are highlighted. Moreover, the potential contribution of ROS to cross talk between major signaling cascades that lead to sustained MAPK activation are proposed in Chapter 7. Chapter 8 takes an insight into the signaling mechanisms for dynamic trafficking and turnover of focal adhesion proteins in regulation of traction and retraction forces, which are needed for cell locomotion and invasion. Chapter 9 describes the involvement of Notch signaling pathway which is not only essential for embryonic development but also plays important role in tumor progression. Chapter 10 reviewed the recently identified cancer- and metastasis-initiating cells involved in tumor progression. Especially, signal pathways that are frequently deregulated in cancer stem/progenitor cells

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during cancer progression are highlighted. Chapter 11 describes the role of lipid rafts, a special component within membrane lipid domain, in signal transduction triggered by growth factor receptors leading to tumor metastasis. Finally, Chapters 12, Chapters 13, and Chapters 14 present the signaling pathways responsible for metastatic progression of specific tumors including ovarian cancer, uveal melanoma and hepatoma, respectively. We thank all the contributors of every Chapter in the book including Jia-Ru Wu, Chi-Tan Hu, Laure Voisin, Stéphanie Duhamel, Sylvain Meloche, Alexey Shiryaev, Marijke Van Ghelue, Ugo Moens, Alessio Giubellino, Praveen R. Arany, Moulay A. Alaoui-Jamali, Krikor Bijian, Panagiota Toliopoulos, Pingyu Zhang, Patrick A. Zweidler-McKay, Murielle Mimeault, Surinder K. Batra, Samir Kumar Patra, Lydia W. T. Cheung, Carman K. M. Ip, Alice S. T. Wong, Cécile Laurent, Jérôme Couturier, Xavier Sastre-Garau, Laurence Desjardins, Emmanuel Barillot, Sophie Piperno-Neumann, Simon Saule and Rajagopal N. Aravalli. We hope this book might stimulate more cancer biologists to emphasize this field which benefits devising more effective molecular targeting strategies for prevention of cancer metastasis. Hualien, Taiwan

Wen-Sheng Wu Chi-Tan Hu

Contents

1 Overview of Signal Transduction in Tumor Metastasis . . . . . . . Wen-Sheng Wu and Jia-Ru Wu

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2 Microenvironment Triggers EMT, Migration and Invasion of Primary Tumor via Multiple Signal Pathways . . . . . . . . . . Wen-Sheng Wu and Chi-Tan Hu

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3 The ERK1/2 MAP Kinase Signaling Pathway in Tumor Progression and Metastasis . . . . . . . . . . . . . . . . . . . . . . Laure Voisin, Stéphanie Duhamel, and Sylvain Meloche

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4 Mitogen-Activated Protein Kinase-Activated Protein Kinases and Metastasis . . . . . . . . . . . . . . . . . . . . . . . . . Alexey Shiryaev, Marijke Van Ghelue, and Ugo Moens

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5 Grb2 and Other Adaptor Proteins in Tumor Metastasis . . . . . . Alessio Giubellino and Praveen R. Arany

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6 The Role of ROS Signaling in Tumor Progression . . . . . . . . . . Wen-Sheng Wu and Jia-Ru Wu

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7 Signal Cross Talks for Sustained MAPK Activation and Cell Migration Mediated by Reactive Oxygen Species: The Involvement in Tumor Progression . . . . . . . . . . . . . . . . Chi-Tan Hu, Jia-Ru Wu, and Wen-Sheng Wu 8 Insights into the Dynamics of Focal Adhesion Protein Trafficking in Invasive Cancer Cells and Clinical Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Moulay A. Alaoui-Jamali, Krikor Bijian, and Panagiota Toliopoulos 9 Notch Signaling in Cancer Metastasis . . . . . . . . . . . . . . . . . Pingyu Zhang and Patrick A. Zweidler-McKay

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New Concepts on the Critical Functions of Cancerand Metastasis-Initiating Cells in Treatment Resistance and Disease Relapse: Molecular Mechanisms, Signaling Transduction Elements and Novel Targeting Therapies . . . . . . . Murielle Mimeault and Surinder K. Batra Involvement of Lipid Rafts in Growth Factor Receptors-Mediated Signaling for Cancer Metastasis . . . . . . . . Samir Kumar Patra

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Cadherin-Catenin Signaling in Ovarian Cancer Progression . . . . Lydia W.T. Cheung, Carman K.M. Ip, and Alice S.T. Wong

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PTP4A3, a Signal Molecule Deregulated in Uveal Melanoma Metastasis . . . . . . . . . . . . . . . . . . . . . . . . . Cécile Laurent, Jérôme Couturier, Xavier Sastre-Garau, Laurence Desjardins, Emmanuel Barillot, Sophie Piperno-Neumann, and Simon Saule

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Signal Transduction Pathways Involved in Hepatocarcinogenesis and Metastasis of Hepatoma . . . . . . . . Rajagopal N. Aravalli

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

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Contributors

Moulay A. Alaoui-Jamali Faculty of Medicine, Departments of Medicine and Oncology, Segal Cancer Center of the Jewish General Hospital, McGill University, Montreal, Canada, [email protected] Praveen R. Arany Harvard University, 49 Oxford Street, 415 ESL, Cambridge, MA 02138, USA, [email protected] Rajagopal N. Aravalli Department of Radiology, University of Minnesota Medical School, Minneapolis, MN 54455, USA, [email protected] Emmanuel Barillot Institut Curie, Paris F-75248, France; INSERM, U900, Paris F-75248, France; Ecoles des Mines ParisTech, Fontainebleau F-77300, France, [email protected] Surinder K. Batra Department of Biochemistry and Molecular Biology, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68198-5870, Nebraska, USA, [email protected] Krikor Bijian Faculty of Medicine, Departments of Medicine and Oncology, Segal Cancer Center of the Jewish General Hospital, McGill University, Montreal, Canada, [email protected] Lydia W.T. Cheung School of Biological Sciences, University of Hong Kong, Pokfulam Road, Hong Kong, [email protected] Jérôme Couturier Département Biologie des Tumeurs, Institut Curie, Hôpital, Paris F-75248, France, [email protected] Laurence Desjardins Département d’Ophtalmologie, Institut Curie, Hôpital, Paris F-75248, France, [email protected] Stéphanie Duhamel Institut de Recherche en Immunologie et Cancérologie, Université de Montréal, Montreal, Quebec H3C 3J7, Canada; Departments Molecular Biology, Université de Montréal, Montreal, Quebec H3C 3J7, Canada, [email protected]

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Xavier Sastre-Garau Département Biologie des Tumeurs, Institut Curie, Hôpital, Paris F-75248, France, [email protected] Marijke Van Ghelue Department of Medical Genetics, University Hospital of North Norway, N-9038 Tromsø, Norway, [email protected] Alessio Giubellino National Cancer Institute, National Institutes of Health, 10 Center Drive, Bldg. 10-CRC, Rm. 1W-5832, Bethesda, MD 20892, USA, [email protected] Chi-Tan Hu Research Center for Hepatology, Department of Medicine, Buddhist Tzu Chi General Hospital, School of Medicine and Graduate Institute of Clinical Medicine, Tzu Chi University, Hualien, Taiwan, [email protected]; [email protected] Carman K.M. Ip School of Biological Sciences, University of Hong Kong, Pokfulam Road, Hong Kong, [email protected] Cécile Laurent Institut Curie, Paris F-75248, France; CNRS, UMR3347, Orsay F-91405, France; INSERM, U1021, Orsay F-91405, France; Université Paris-Sud 11, Orsay F-91405, France; INSERM, U900, Paris F-75248, France; Ecoles des Mines ParisTech, Fontainebleau F-77300, France, [email protected] Sylvain Meloche Institut de Recherche en Immunologie et Cancérologie, Université de Montréal, Montreal, Quebec H3C 3J7, Canada; Departments of Pharmacology, Université de Montréal, Montreal, Quebec H3C 3J7, Canada; Departments Molecular Biology, Université de Montréal, Montreal, Quebec H3C 3J7, Canada, [email protected] Murielle Mimeault Department of Biochemistry and Molecular Biology, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68198-5870, Nebraska, USA, [email protected] Ugo Moens Medical Faculty, Department of Medical Biology, University of Tromsø, N-9037 Tromsø, Norway, [email protected] Sophie Piperno-Neumann Département D’oncologie Médicale, Institut Curie, Hôpital, Paris F-75248, France, [email protected] Samir Kumar Patra Department of Life Science, National Institute of Technology, Rourkela, Orissa, India, [email protected]; [email protected] Simon Saule Institut Curie, Paris F-75248, France; CNRS, UMR3347, Orsay F-91405, France; INSERM, U1021, Orsay F-91405, France, [email protected] Alexey Shiryaev Medical Faculty, Department of Medical Biology, University of Tromsø, N-9037 Tromsø, Norway, [email protected]

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Panagiota Toliopoulos Faculty of Medicine, Departments of Medicine and Oncology, Segal Cancer Center of the Jewish General Hospital, McGill University, Montreal, Canada, [email protected] Laure Voisin Institut de Recherche en Immunologie et Cancérologie, Université de Montréal, Montreal, Quebec H3C 3J7, Canada, [email protected] Alice S.T. Wong School of Biological Sciences, University of Hong Kong, Pokfulam Road, Hong Kong, [email protected] Jia-Ru Wu Department of Medical Technology, Graduate Institute of Medicine, Tzu Chi University, Hualien, Taiwan, [email protected] Wen-Sheng Wu Department of Medical Technology, Graduate Institute of Medicine, Tzu Chi University, Hualien, Taiwan; Institute of Medical Biotechnology, College of Medicine, Tzu Chi University, Hualein, Taiwan, [email protected] Pingyu Zhang Department of Surgical Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA, [email protected] Patrick A. Zweidler-McKay Division of Pediatrics, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA; University of Texas Graduate School of Biomedical Sciences, Houston, Texas, USA, [email protected]

Chapter 1

Overview of Signal Transduction in Tumor Metastasis Wen-Sheng Wu and Jia-Ru Wu

Abstract The signal transductions mediating each stage of tumor metastasis are gradually unraveled. A lot of growth factors and cytokines secreted in the tumor environment may trigger epithelial–mesenchymal transition (EMT), migration and invasion of the primary tumor via integration of multiple pathways. Further, TGF-β1/Smad signaling may switch the tumor cell from cohesive to single cell movement that facilitates the invasive tumor to enter blood circulation (intravasation). In the circulation, tumor cell may survive in an anchorage independent manner by developing signals counteracting the apoptotic machinery responsible for anoikis. In the extravasation stage, tumor cells that express higher selectin ligands my bind to platelet via P-selectin which facilitates the formation of tumor-platelet-leukocyte emboli. The arrested emboli may promote the E-selectinmediated activation of ERK(MAPK) to trigger transendothelial passage. Moreover, various chemokine receptor/ligand pairs such as CCR4/CXCL12 can act to achieve organ preference in metastatic colonization. Distinct signaling pathways may also be responsible for determining the destination of tumor cells. The final change faced by the metastatic tumor cells is how to survive in the secondary loci. Recently, Srcmediated signaling was found to be responsible for metastatic breast cancer cells to survive in the bone marrow microenvironment. Blockade of the aforementioned molecular pathways for each stage of tumor progression could be one of the most effective strategies for prevention of metastasis. Keywords Tumor metastasis · Signal transductions · EMT · Migration · Invasion · Intravasation · Extravasation · MAPK · TGF-β · Src

W.-S. Wu (B) Department of Medical Technology, Tzu Chi-University, No. 701, Chung Yang Rd, Sec. 3, Hualien 970, Taiwan, Republic of China e-mail: [email protected]

W.-S. Wu, C.-T. Hu (eds.), Signal Transduction in Cancer Metastasis, Cancer Metastasis – Biology and Treatment 15, DOI 10.1007/978-90-481-9522-0_1,  C Springer Science+Business Media B.V. 2010

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Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . Signaling for the Initiation of Tumor Metastasis . . . . . . . . . . . Signaling for Intravasation . . . . . . . . . . . . . . . . . . . . Signaling for Tumor Progression in Circulation . . . . . . . . . . . 1.4.1 Anti-Anoikis of Circulating Tumor Cell . . . . . . . . . . . 1.4.2 Signaling for Extravasation . . . . . . . . . . . . . . . . . 1.5 Signaling for Homing and Survival of the Tumor in the Metastatic Loci 1.6 Conclusion and Perspective . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1.1 Introduction Metastasis is a complicated pathological processes consisting of several stages [1]. Initially, the primary tumor cell exhibit epithelial–mesenchymal transition (EMT) followed by migration and invasion to the surrounding tissues. Subsequently, the tumor cells may penetrate the endothelium to enter blood circulation (intravasation). The tumor cells that survive in the blood may move out of blood vessel (extravasation) and finally proliferate in the secondary loci. In addition, angiogenesis may support the growth of the tumor cells. During these processes, the tumor cells may be endowed with increasingly metastatic capability by a lot of environmental factors. The signal transductions mediating each stage of tumor metastasis are gradually unraveled and will be briefly overviewed in this Chapter.

1.2 Signaling for the Initiation of Tumor Metastasis The microenvironment in the primary tumor locus is rather complicated. A variety of the stromal cells, inflammatory cells, and tumor cell itself may interact with each other to constitute a microenvironment in favor of tumor progression. A lot growth factors and cytokines including hepatocyte growth factor (HGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), stromal cell-derived factor (SDF) and transforming growth factor-β (TGFβ), which can be secreted during tumor-stromal and/or tumor-macrophage interactions [2–4], can trigger EMT, migration and invasion of primary tumor cells. Various signal pathways such as Ras-Raf-MAPK [5], PKC [6], PI3K/AKT [7, 8] and integrinrelated pathway [9] were involved in these processes. A detailed review for tumor progression at this stage can be found in next Chapter.

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1.3 Signaling for Intravasation It is a general concept that most of the metastatic factors in the tumor environment can trigger the invasion of tumor cell not only toward the surrounding tissue but also across the endothelium into blood circulation. However, the detailed cellular and molecular events of intravasation are not clearly established yet. A recent study [10] suggested that the modes of cell movement determine whether tumor cells can penetrate blood vessel. During spreading of breast cancer cells, collective and single-celled movement can be observed. Interestingly, single cell motility is essential for tumor cells to enter blood circulation (intravasation) whereas cells restricted to collective movement were capable of lymphatic invasion. Moreover, TGF-β1, one of the most important metastatic factors, may switch tumor cells from cohesive to single cell motility through Smad 2/4-dependent pathway. This may lead to upregulation of various signal components such as EGFR, Nedd9, M-RIP, FARP and RhoC facilitating intravasation. The involvement of MAPK-dependent pathway in intravasation of breast tumor cell was implicated in a recent study for Raf kinase inhibitory protein (RKIP), a negative regulator of MAPK [11]. The Ras-Raf-1-MEK-ERK cascade initiated by Erb2 may relieve the negative regulation of a chromatin remodeling protein HMGA2 by let-7 microRNA, resulting in activation of pro-invasive and prometastatic genes, including Snail. The blockade of Ras-MAPK-Snail axis by RIPK suppressed intravasation of breast tumor cell.

1.4 Signaling for Tumor Progression in Circulation Following intravasation, tumor cells undertake a dangerous voyage in the hostile environment of the blood circulation. Importantly, anchorage-independent growth (anti-anoikis) is required for survival of tumor cell in circulation, mediated by specific signal pathways.

1.4.1 Anti-Anoikis of Circulating Tumor Cell 1.4.1.1 The Basic Concept for Anoikis Cell to matrix adhesion is a key factor for cellular homeostasis and disruption of such interaction has adverse effects on cell survival. It leads to a specific type of apoptosis known as ‘‘anoikis’’. The molecular mechanisms governing anoikis have been well reviewed [12]. Essentially, loss of the integrin signaling is the most critical cause for anoikis. Various type of integrins can sense mechanical forces arising from the matrix, converting these stimuli to diverse signals responsible for modulating a variety of cellular behaviors including adhesion, migration and survival

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[for review 13]. Without adhesion on ECM, the integrin-mediated survival signaling will be lost and the cells enter apoptosis. The BH3-only family proteins such as Bid and Bim are the most critical pro-apoptotic proteins executing anoikis. Bim is sequestered in the dynein complex until the loss of integrin engagement resulting in its release and translocation to mitochondria. Activation of Bid and Bim rapidly promote the assembly of channel protein Bax-Bak oligomers within the outer mitochondrial membrane followed by release of cytochrome C. Cytochrome C may trigger the caspase cascade resulting in activation of the effector caspase3. Caspase-3 then cleaves a lot of molecules including signaling molecules like focal adhesion-kinase (FAK), Cas, and paxillin, which are key components in integrin-mediated signal transduction for adhesive survival. 1.4.1.2 Signal Mechanism for Anti-Anoikis of Tumor Cell in Circulation In the circulation, tumor cell may escape anoikis by several ways. The basic strategy is to develop a constitutive survival signals counteracting the apoptotic machinery responsible for anoikis. Changing the Pattern of Integrin Expression One way for cancer cells to avoid anoikis is to change the pattern of integrin expression so that the incorrect survival signals may be received continually. Several integrins (α1β1, α2β1, α3β1, α5β1, α6β1, α6β4, and αvβ3) have profound impact on cell survival without engagement with ECM [14]. One recent report [15] demonstrated that overexpression of αvβ3 in tumor cell can substantially increase anchorage-independent cell survival in vitro and metastasis in vivo. These effects were mediated by the activation of the non-receptor tyrosine kinase Src and its down stream signal components including ERK and PI3K/Akt , which are capable of phosphorylating Bim, leading to its proteasome-dependent degradation [12]. Constituting Autocrine Growth Factor Loops Another way for the tumor cell to achieve anti-anoikis is constituting the autocrine growth factor loops which sustainedly activates the survival pathways. For example, overexpression of TrkB, a neurotrophic tyrosine kinase receptor, greatly activates the PI3K/Akt pathway and Twist-Snail transcriptional apparatus rendering the tumor cell anoikis-resistant [16, 17]. The Role of ROS Reactive oxygen species (ROS) are recently recognized to be critical mediators of signal transduction. The role of ROS in tumor migration and invasion is detailed in Chapter 6. Interestingly, Giannoni et al. [18] demonstrated that ROS, produced upon integrin engagement and activation of the small GTPase Rac-1, play essential role in transducing the pro-survival signal for protection from anoikis. In particular,

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ROS are responsible for the redox mediated activation of Src, leading to the ligand-independent trans-phosphorylation of EGFR. The redox-dependent EGFR activation switches both ERK and PKB/Akt pathways, resulting in degradation of Bim.

1.4.2 Signaling for Extravasation Extravasation of cancer cells, also called diapedesis, is the passage of circulating tumor cells out of blood vessel. Essentially, tumor cells ought to adhere to the endothelium followed by trans-endothelial migration and invasion. This process can be facilitated by interaction of the tumor cells with circulating platelets and leukocytes, resulting in the formation of emboli capable of binding to endothelial cells. Platelet is the most adhesive component in the blood. One of the previous reports demonstrated that activation of αvβ3 integrin promoted the interaction of metastatic human breast cancer cells with platelet and supported adhesion of cancer cell within the vasculature [19]. On the other hand, leukocytes are the most invasive blood cell, capable of infiltrating to inflammatory tissue loci when ever needed. It was proposed that extravasation of tumor cell follows that of infiltration of leukocytes [20, 21]. Moreover, selectin, which is the adhesion receptors on the platelet and endothelium are essential mediator of extravasation [for review 22]. Tumor cells express higher selectin ligands for binding to platelet via P-selectin (on the platelet) which facilitate the formation of tumor-platelet-leukocyte emboli. The arrested emboli may then induce signals that further promote the E-selectin (on the endothelium)mediated transendothelial passage including the opening of endothelial cell junctions [23]. The intracellular signal pathway mediating extravasation has not been fully established. Two important signal molecules, Rho GTPases [24] and MAPK [23] which are well known to be critical for regulation of cytoskeletal rearrangements and motility, were found to be involved. Tremblay et al. indicated that E-selectindependent paracellular extravasation requires the activation of ERK(MAPK) downstream of E-selectin [23]. In addition, the critical role of p38 (MAPK) in adhesion of tumor cell to endothelial cells [25] was demonstrated by Matsuo et al. They found platelets and lung endothelial cells of p38α(+/−) mice were poorly bound to tumor cells compared with those of WT mice suggesting that p38alpha plays an important role in the early step of extravasation.

1.5 Signaling for Homing and Survival of the Tumor in the Metastatic Loci The final stages of tumor metastasis include the homing of tumor cell to specific organs. The underlying mechanisms for this intriguing process are largely unexplored but are highlighted in several recent studies. One plausible mechanism

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for organ preference in metastatic colonization is the communication between the circulating tumor cells and the target host tissue via interaction of chemokine receptors and their ligands. This is reminiscent of the homing process of circulating leukocytes and stem cells to specific organs. Of the chemokine receptor/ligand pairs involved in the homing of metastatic tumors, CCR4 and CXCL12 are the most well studied [for review 26, 27]. The intracellular signaling for the organ preference in metastatic colonization has not been clearly delineated yet. One recent study [28] raised the possibility that distinct signaling pathways may determine the destination of metastatic tumor cells. They found that human prostate cancer cell DU145 can be triggered by oncogenic Ras for metastasis to multiple organs, including bone and brain. Interestingly, Raf/ERK, one of the effector pathways of Ras, determine colonization of the brain, whereas the other that mediated by RalGEF is responsible for metastasis to bone, the most common organ site for prostate cancer metastasis. After reaching the target tissue, the final challenge faced by the tumor cells is how to survive in the secondary loci, which is totally different from where they are derived. This may be revealed by variable latency in metastasis of different tumors. In some types of cancer, metastasis occurs soon after a primary tumor develops, whereas in others it emerges years after the primary tumor is removed. It has also been proposed that in order to achieve the final stage of metastasis, primary tumors must produce factors that induce the formation of a suitable and appropriate environment in the organ where metastatic cells will be seeded. Recently, the signal mechanisms that support the survival of disseminated breast cancer cells in bone marrow was investigated by Zhang et al. [29]. In a clinical approach, they found association between Src pathway and late-onset bone metastasis. In vitro studies further demonstrated that Src was required for the prosurvival pathway triggered by CXCL12 coupled with its G protein-coupled receptor CXCR4 and down stream effector AKT. On the other hand, c-Src signaling may confer resistance to the apoptotic effect of TNF-related apoptosis-inducing ligand (TRAIL). Both pathways support the survival of breast cancer cell in the bone marrow microenvironment.

1.6 Conclusion and Perspective Metastasis is a long process during which the tumor cell develops increasingly metastatic potential in order not only to migrate and invasion but also to face the subsequent challenges for surviving in unfavorable environments outside of their original loci. The signal transduction triggered by the metastatic factors in the primary locus, blood circulation and the secondary loci mediate each stage of tumor progression (Fig. 1.1). Blockade of the relevant molecular pathway could be one of the most effective strategies for prevention of tumor metastasis. Clinical trials are underway with promising outcomes as will be mentioned in the later Chapters of this book.

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Phenotypical Changes in Tumor Microenvironment HGF/cMet; TGFβ/Smad; Integrin and other signals

Invasion

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Altered integrin expression, autocrine growth factor loops integrin-Rac-ROS signaling

Survive in secondary loci Src-mediated signaling

Extravasation E-selectin-mediated activation of MAPK

Fig. 1.1 Signal pathway triggering tumor metastasis. In the tumor environment phenotypical changes including epithelial–mesenchymal transition (EMT), migration and invasion can be triggered by TGF-β, HGF-cMet, integrin-mediated signaling. In the intravasation stage, TGF-β1/Smad signaling may switch the tumor cell from cohesive to single cell movement facilitating the invasive tumor to enter blood circulation. In the circulation, tumor cell may survive in an anchorage independent growth fashion by developing several signaling strategy (as indicated) to counteract the apoptotic machinery responsible for anoikis. In the extravasation stage, tumor cells may develop E-selectin-mediated ERK (MAPK) signals that trigger the E-selectin-mediated transendothelial passage. Finally, Src-mediated signaling was responsible for metastatic cancer cells (such as breast cancer) to survive in the secondary loci (such as bone marrow microenvironment)

References 1. Gupta GP, Massague J. Cancer metastasis: building a framework. Cell 2006; 127: 679–695. 2. Nakamura T, Matsumoto K, Kiritoshi A, et al. Induction of hepatocyte growth factor in fibroblasts by tumor-derived factors affects invasive growth of tumor cells: in vitro analysis of tumor-stromal interactions. Cancer Res. 1997; 57: 3305–3313. 3. Kajita T, Ohta Y, Kimura K, et al. The expression of vascular endothelial growth factor C and its receptors in non-small cell lung cancer. Br. J. Cancer 2001; 85: 255–260. 4. Koshiba T, Hosotani R, Miyamoto Y, et al. Expression of stromal cell-derived factor 1 and CXCR4 ligand receptor system in pancreatic cancer: a possible role for tumor progression. Clin. Cancer Res. 2000; 6: 3530–3535. 5. Leicht DT, Balan V, Kaplun A, et al. Raf kinases: function, regulation and role in human cancer. Biochim. Biophys. Acta 2007; 1773: 1196–1212. 6. Oka M, Kikkawa U. Protein kinase C in melanoma. Cancer Metastasis Rev. 2005; 24: 287–300. Review.

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7. Brader S, Eccles SA. Phosphoinositide 3-kinase signalling pathways in tumor progression, invasion and angiogenesis. Tumor 2004; 90: 2–8. Review. 8. Sheng S, Qiao M, Pardee AB. Metastasis and AKT activation. J. Cell Physiol. 2009; 218: 451–454. Review. 9. Desgrosellier JS, Cheresh DA. Integrins in cancer: biological implications and therapeutic opportunities. Nat. Rev. Cancer 2010; 10: 9–22. Review. 10. Giampieri S, Manning C, Hooper S, et al. Localized and reversible TGFbeta signalling switches breast cancer cells from cohesive to single cell motility. Nat. Cell Biol. 2009; 11: 1287–1296. 11. Dangi-Garimella S, Yun J, Eves EM, et al. Raf kinase inhibitory protein suppresses a metastasis signalling cascade involving LIN28 and let-7. EMBO J. 2009; 28: 347–358. 12. Chiarugi P, Giannoni E. Anoikis: a necessary death program for anchorage-dependent cells. Biochem. Pharmacol. 2008; 76: 1352–1364. 13. Guo W, Giancotti FG. Integrin signalling during tumour progression. Nat. Rev. Mol. Cell Biol. 2004; 5: 816–826. 14. Alahari SK, Reddig PJ, Juliano RL. Biological aspects of signal transduction by cell adhesion receptors. Int. Rev. Cytol. 2002; 220: 145–184. 15. Desgrosellier JS, Barnes LA, Shields DJ, et al. An integrin αvα3–c-Src oncogenic unit promotes anchorage-independence and tumor progression. Nat. Med. 2009; 15: 1163–1170. 16. Lagadec C, Meignan S, Adriaenssens E, et al. TrkA overexpression enhances growth and metastasis of breast cancer cells. Oncogene 2009; 28: 1960–1970. 17. Smit MA, Geiger TR, Song JY, et al. A Twist-Snail axis critical for TrkB-induced epithelialmesenchymal transition-like transformation, anoikis resistance, and metastasis. Mol. Cell Biol. 2009; 29: 3722–3737. 18. Giannoni E, Buricchi F, Grimaldi G, et al. Redox regulation of anoikis: reactive oxygen species as essential mediators of cell survival. Cell Death Differ. 2008; 15: 867–878. 19. Felding-Habermann B, O’Toole TE, Smith JW, et al. Integrin activation controls metastasis in human breast cancer. PNAS 2001; 98: 1853–1858. 20. Petri B, Bixel MG. Molecular events during leukocyte diapedesis. FEBS J. 2006; 273: 4399–4407. 21. Johnston B, Butcher EC. Chemokines in rapid leukocyte adhesion triggering and migration. Semin. Immunol. 2002; 14: 83–92. 22. Witz IP. The selectin-selectin ligand axis in tumor progression. Cancer Metastasis 2008; 27: 19–30. 23. Tremblay PL, Huot J, Auger FA. Mechanisms by which E-selectin regulates diapedesis of colon cancer cells under flow conditions. Cancer Res. 2008; 68: 5167–5176. 24. Gout S, Tremblay PL, Huot J. Selectins and selectin ligands in extravasation of cancer cells and organ selectivity of metastasis. Clin. Exp. Metastasis 2008; 25: 335–344. 25. Matsuo Y, Amano S, Furuya M, et al. Involvement of p38alpha mitogen-activated protein kinase in lung metastasis of tumor cells. J. Biol. Chem. 2006; 281: 36767–36775. 26. Ben-Baruch A. Organ selectivity in metastasis: regulation by chemokines and their receptors. Clin. Exp. Metastasis 2008; 25: 345–356. Review. 27. Vandercappellen J, Van Damme J, Struyf S. The role of CXC chemokines and their receptors in cancer. Cancer Lett. 2008; 267: 226–244. 28. Yin J, Pollock C, Tracy K, et al. Activation of the RalGEF/Ral pathway promotes prostate cancer metastasis to bone. Mol. Cell Biol. 2007; 27: 7538–7550. 29. Zhang XHF, Wang Q, Gerald W, et al. Latent bone metastasis in breast cancer tied to Srcdependent survival signals. Cancer Cell 2009; 16: 67–78.

Chapter 2

Microenvironment Triggers EMT, Migration and Invasion of Primary Tumor via Multiple Signal Pathways Wen-Sheng Wu and Chi-Tan Hu

Abstract In the tumor microenvironment, the primary tumor cells may recruit and interact with stromal cells and inflammatory cells including macrophages, mast cells and neutrophiles. A great multitude of growth factors, cytokines, chemokines and growth/motility factors may be secreted by aforementioned cells in the microenvironment to trigger epithelial-mesenchymal transition (EMT), migration and invasion of tumor cells and facilitate neovasculation and modifications of the ECM. Multiple signal pathway induced by various metastatic factors may be integrated to establish an amplified and sustained driving force for tumor progression. The first part of this review focus on the cellular and molecular processes occurring in the microenvironment that are fundamental for triggering the initiation stage of tumor metastasis. In the second part, altered signal pathways induced by several critical metastatic factors such as HGF, TGFβ and that elicited by integrin-ECM engagement are delineated. In the future, more effective cancer therapy can be developed by targeting the critical signal molecules responsible for the initiation stage of tumor metastasis. Keywords Tumor metastasis · Signal transductions · EMT · Migration · Invasion · MAPK · HGF · TGF-β · Integrin

Contents 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Phenotypical Changes of Tumor Cells in the Initial Stage of Tumor Metastasis: EMT, Migration and Invasion of Primary Tumor . . . . . . . . . . 2.2.1 Epithelial–Mesenchymal Transition: the Initiation Step of Tumor Metastasis 2.2.2 Microenvironment in the Primary Tumor Locus . . . . . . . . . . . . .

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C.-T. Hu (B) Division of Gastroenterology, Department of Medicine, Buddhist Tzu Chi Hospital and University, Section 3, No.707, Chung-Yan Road, Hualien, Taiwan e-mail: [email protected]; [email protected]

W.-S. Wu, C.-T. Hu (eds.), Signal Transduction in Cancer Metastasis, Cancer Metastasis – Biology and Treatment 15, DOI 10.1007/978-90-481-9522-0_2,  C Springer Science+Business Media B.V. 2010

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2.3 Signal Transduction Triggered by Metastatic Factors Within Microenvironment 2.3.1 HGF/c-Met . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2 TGF-β . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.3 The Integrin-ECM Engagement . . . . . . . . . . . . . . . . . . . 2.4 Conclusion and Perspective . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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2.1 Introduction Cancer metastasis, or dissemination of the primary tumor to distant body sites, is one of the most complicated pathological processes [1]. It is also the major determinant of mortality in cancer patients. The critical steps in tumor metastasis include (a) detachment of the primary cancer cells followed by migration and invasion to adjacent tissues, (b) penetrating the extracellular matrix (ECM) and blood vessels (intravasation) and then (c) penetrating out of the vessels (extravasation) and finally proliferate in a secondary site. The microenvironment in the primary tumor locus is rather complicated [2]. Various types of cells including the primary tumor itself may secret a lot growth factors and cytokines to trigger phenotypical changes in tumor cells responsible for the initiation of tumor metastasis. This review focus on the molecular and cellular events occurred in this critical stage. Especially, the signal transduction pathways involved in tumor metastasis will be focused.

2.2 Phenotypical Changes of Tumor Cells in the Initial Stage of Tumor Metastasis: EMT, Migration and Invasion of Primary Tumor The phenotypical changes which eventually endow tumor cells with metastatic ability are epithelial-mesenchymal transition (EMT) followed by migratory and invasive properties.

2.2.1 Epithelial–Mesenchymal Transition: the Initiation Step of Tumor Metastasis The conversion of epithelial cells to mesenchymal cells is fundamental for embryonic development during which cell–cell adhesion and cell polarity are lost accompanied with the acquisition of fibroblastoid morphology with motile and invasive properties [3]. Transcriptional reprogramming during EMT resulted in combined loss of epithelial cell junction proteins (e.g., E-cadherin, catenins and mucin-1) and the appearance of mesenchymal markers (e.g., fibronectin, vitronectin, vimentin and smooth muscle actin).

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As described by a recent review [4], the significance of EMT during cancer progression and even its relevance in human cancer tissues have remained a matter of debate until very recently. EMT is now proved to be a fundamental step in tumor progression and metastasis in a lot of studies. For example, in colon carcinoma, EMT occurs at the invasive front and produces migratory cells that lose E-cadherin expression [5]. Moreover, direct in vivo imaging has also yielded evidence of EMT in cancer progression [6]. 2.2.1.1 Tumor Migration: The Essential Driving Force for Metastasis After EMT, tumor cells acquire higher migratory ability to move to the surrounding tissue. Cell movement is a complex and tempo-spatially controlled process involving the coordinated activity of hundreds of different proteins [7]. The cytoskeletal network consisting of actin, myosin and many proteins associated with them is essentially responsible for the typical morphological changes observed in cell migration. In the initial stage of cell migration, the leading edge of a cell is pushed forward by the extension of the internal cytoskeleton leading to the formation of a large, flat structure that is often termed the lamellipodium. The leading edge is then attached to the surface by adhesion molecules such as integrins. This allows the rest of the cell body to be pulled forward. Integrins, the cytoskeleton-associated cell surface receptor can bind to the ECM including laminins, fibronectin and proteoglycans, which is responsible for adhesion and de-adhesion during cell migration. Moreover, engagement of integrins with ECM may stimulate intracellular signal transduction mediating a lot of cellular effects including migration. The integrinmediated signaling may integrate with those from other metastatic factors in the tumor microenvironment, endowing tumor cells with sustained migratory capability which is the essential driving force for tumor metastasis. 2.2.1.2 Tumor Invasion in Metastasis Invasion is another essential step for tumor progression during which tumor cells follow a chemoattractive path across the ECM and basement membrane of tissue boundary, which are the structural barriers against tumor invasion. Degradations of the basement membrane and ECM are fundamental for tumor cell invasion [8]. A chorioallantoic membrane invasion assay has been used to demonstrate proteolysis in the invasive process [9]. Tumor cells and most of the cells in the tumor environment may overexpress and secret one or more of the matrix degradation enzymes (proteinases) which can be divided into four groups: (1) the matrix metalloproteinases (MMPs), a family of secreted and membrane-anchored proteinases, (2) the adamalysin-related membrane proteinases, (3) the bone morphogenetic protein-1-type metalloproteinases, and (4) tissue serine proteinases, including tissue plasminogen activator, urokinase, thrombin and plasmin [10]. The activities of these enzymes are tightly regulated by a series of activation steps and specific inhibitors. One of the principle matrix degradation enzymes is matrix metalloproteases (MMPs). MMPs are a family of neutral metalloenzymes secreted as latent

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proenzymes, requiring activation through proteolytic cleavage of the amino-terminal domain in the presence of zinc and calcium [11, 12]. Five MMP subclasses were grouped according to substrate specificity: interstitial collagenases, gelatinases, stromelysins, membrane type-MMPs (MT-MMPs), and elastases [13]. Increased MMP activity has been detected and shown to correlate with invasion and metastatic potential in a wide range of cancers, including ovary, lung, prostate, breast, and pancreas cancers [11, 14, 15].

2.2.2 Microenvironment in the Primary Tumor Locus The formation of a clinically progressing tumor requires interactions with the surrounding normal stroma. The tumor-stroma compartment as a whole constitutes the tumor microenvironment. Recent evidence indicates that the microenvironment not only maintains the survival of cancer stem cells/cancer initiating cells but also promotes cancer cells metastasis. Furthermore, inflammatory cells and immunomodulatory mediators present in the tumor microenvironment polarize host immune response toward specific phenotypes favoring tumor progression. A lot of studies demonstrate a positive correlation between poor prognosis with angiogenesis, carcinoma-associated fibroblasts, and the effect of inflammation on tumor progression. Thus, the dynamic interactions between tumor cells and cells of the tumor microenvironment are critical to tumor metastasis [2]. 2.2.2.1 The Constitution of Tumor Microenvironment The concept of tumor microenvironment as an integrated and essential part of the cancer tissue has been proposed decades ago. However, not until recent years, the cellular and molecular components of tumor microenvironment were identified, and the mechanisms for how they affect tumor progression were delineated. The constitution of tumor microenvironment (Fig. 2.1) comprises the primary tumor itself which may recruit and interact with stroma through production and secretion of stimulatory growth factors and cytokines [16, 17]. A variety of cell types populate the stromal compartment, including smooth muscle cells, myofibroblasts, carcinoma associated fibroblasts, vascular cells (such as lymph-endothelial, vascular-endothelial cells and pericytes) and immune cells including lymphocytes, tumor associated monocytes/macrophages and dendritic cells [16, 17]. The interactions between tumor cells and stroma cells lead to secretion of growth factors and cytokines for neovasculation and modifications of the ECM, which greatly support growth and survival of tumor cells [16, 17] (Fig. 2.1). 2.2.2.2 The Effect of Inflammation on Tumor Progression It has been proposed since 1863 by Rudolf Virchow that cancer originates at sites of chronic inflammation. This notion was further strengthened by recent studies demonstrating that chronic inflammation is critical for cancer progression [18].

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Angiogenesis

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Fig. 2.1 The constitution of tumor microenvironment. Transition to invasive carcinoma is preceded by the interaction/cross-talk among the premalignant cells (PMC) and stromal cells such as fibroblasts (Fi), smooth muscle cells (SMC), endothelial cells (EC), or immune cells (IC) (dashed lined zones). Cancer cells further modify directly or indirectly (tumor or tissue hypoxia/necrosis) the microenvironment by activating other stromal cells, such as tumor associated monocytes/macrophages (TAM), dendritic cells (DC) or carcinoma associated fibroblasts (CAFi) (solid blue arrows; dashed blue arrows represent differentiation). In turn, growth factors, cytokines and proteinases released from cancer cells, immune cells and the activated stromal cells (dashed red arrows) promote tumor growth, epithelial-mesenchymal transition (EMT), migration, invasion (by degradating the ECM and basement membrane) of primary cancer cells, and angiogenesis (solid red arrows). MSC mesenchymal stem cell, MyoFi myofibroblast

Inflammatory responses within the microenvironment can be triggered by tumor hypoxia and necrosis secondary to excessive tumor cell proliferation. The inflammatory cytokines, including colony stimulating factor (CSF)-1, granulocyte–monocyte (GM)-CSF, transforming growth factor (TGF)-β, and chemokines (e.g., CCL2, CCL7, CCL3, CCL4) [19] are chemoattractive for monocytes and macrophages. The recruited macrophages in turn secrete growth factors, cytokines, chemokines and growth/motility factors that affect tumor cell behavior (e.g., induction of motility and invasiveness). These include vascular endothelial growth factor (VEGF)-A and -C, basic fibroblast growth factor (bFGF), tumor necrosis factor (TNF), hepatocyte growth factor (HGF), epidermal growth factor (EGF) family members, platelet-derived growth factor (PDGF), and chemokines such as CXCL12 and

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interleukin (IL)-8. These factors may also promote the recruitment of additional inflammatory cells, such as mast cells and neutrophils which also secret tumorpromoting cytokines, growth factors and proteases, thus amplifying the initial inflammatory reaction [20] and facilitating tumor progression including EMT, migration, invasion of the tumor cells coupled with angiogenesis for supporting the growth of cancer tissue (Fig. 2.1).

2.3 Signal Transduction Triggered by Metastatic Factors Within Microenvironment As described above, a variety of growth factor/cytokine and extracellular matrix may be secreted by tumor cells, stromal cells and inflammatory cells. These factors have great impacts on the initiation of tumor progression and may be collectively called as the “metastatic factors”. Of these, TGF-β [21], HGF [22], EGF [23], and insulin-like growth factor (IGF) [24] have been demonstrated to be capable of inducing EMT and migration of tumor cells. Also, HGF, EGF and TGF-β released from the degraded matrix may guide the path in front of tumor cells. The signal transductions responsible for tumor progression induced by some of these factors are well established as described in the following sections.

2.3.1 HGF/c-Met Hepatocyte growth factor (HGF), or so called scatter factor (SF) was capable of inducing scatter (i.e., dissociation) of epithelial cells and stimulating proliferation of hepatocytes. SF/HGF is synthesized by mesenchymal cells as a paracrine effector of epithelial cells. The HGF-induced cell scatter may endow the cell with increasing motility and invasiveness, effects that are physiologically required for embryonic development [for review 25, 26] and also pathologically involved in tumor metastasis [27, 28]. 2.3.1.1 The HGF-cMet Signaling The cellular receptor of HGF is the tyrosine kinase receptor c-Met. C-Met receptor consists of a disulphide-linked heterodimeric complex containing an extracellular portion for ligand binding, a membrane spanning segment, a juxtamembrane domain, a catalytic domain, and a C-terminal docking site [29]. Binding of HGF to c-Met triggers autophosphorylation of its cytoplasmic domain. The phosphorylation at two tyrosine residues in the catalytic domain, Y1234 and Y1235, is crucial for kinase activity, while phosphorylation at Y1349 and Y1356 in the C-terminal docking site is essential for recruiting adaptor proteins. Many adaptor proteins such as Shc [30], Src, Grb2, the p85 regulatory subunit of PI3K [29] and Gab1 [31] may bind directly or indirectly with the c-Met receptor. Most of them contain a Src

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homologous2 (SH2) domain interacting with c-Met receptor and a Src homologous3 (SH3) domain that binds to downstream signal molecules. Several down stream signal pathways were well known to be triggered by HGF/ c-Met, including Raf-ERK (MAPK) [32, 33], p38 (MAPK) [34, 35], and PI3KAkt/PKB [33, 36] pathways. HGF/c-Met can also crosstalk with integrin-initiated signal cascade leading to activation of FAK-Src-paxillin, Ras-Rac1/Cdc42-PAK and Gab1-Crk-C3G-Rap1 cascades. In addition, by activation of the PKC isozyme ε, c-Met may recruit ERK1/2 to focal adhesion complex to phosphorylate and activate the migration-related signal components [for review 37]. 2.3.1.2 The Role of HGF/SF-Met Signaling in Tumor Progression All of the aforementioned HGF/c-Met signalings are known to be provital for cytoskeletal rearrangement, cell scattering, migration, invasion and angiogenesis. Thus, elevated activation of HGF-cMET signaling may lead to eventual metastasis. In the animal studies, cells which over-express either c-MET or HGF are extremely metastatic when implanted into nude mice [38]. Consistently, transgenic mice for either MET or HGF develop metastatic tumors [39]. A lot of patho-physiological evidences demonstrated that c-MET can be constitutively activated by establishment of ligand-receptor autocrine loops, or activating point mutations [for review 40]. Moreover, altered HGF secretion has been reported in both solid and hematologic malignancies. In the past decades, mounting studies have demonstrated the critical role of HGF/c-Met in malignant progression of a lot of tumors [for review 41, 42, 43, 44]. These include small cell lung cancer [45], melanoma cells [35], ovarian cancer [46], colorectal carcinoma [47], human stomach cancer [32] prostate adenocarcinoma cells [48], breast cancer [36, 49], pancreatic cancer [34], head and neck squamous cell carcinoma [33], oral squamous cell carcinoma [50] and renal carcinoma cells [51]. 2.3.1.3 Blockade of HGF/c-Met for Prevention of Tumor Metastasis The use of HGF as a target for prevention of metastasis has been intensively studied [for review 52]. Previously, NK4, a competitive antagonist of HGF/c-Met was employed to block HGF-induced invasion and metastasis of hepatocellular carcinoma. NK4 is an internal fragment of HGF and able to bind to but not activate the c-Met receptor, thereby competitively suppressing the biological activities of HGF [for review 53]. Recently, a single-agent and combination therapeutic strategies to inhibit HGF/c-Met signaling for prevention of cancer progression was proposed [54].

2.3.2 TGF-β The transforming growth factor-β (TGF-β) superfamily of growth factors consist of TGF-βs, activins and bone morphogenetic proteins (BMPs). In physiological

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context, TGF-β is involved in the proliferation and differentiation of cells, embryonic development, angiogenesis, and wound healing. Previously, TGF-β was known to be a regulatory molecule that signals cell cycle arrest [55]. However, later studies demonstrated that members of the TGF-β family produce different cellular effects, either growth promoting or antiproliferative, depending on the types and states of cells [for review 56].

2.3.2.1 TGF-β/Smad Signaling The signal pathway for the action of TGF-β was well established [for review 57]. The membrane receptors of TGF-β consist groups of serine/threonine kinase family of which the type I and type II TGF-β receptors may form heteromeric complexes responsible for intracellular signaling. Ligand binding of TGF-β receptors may activate its cytoplasmic mediators, the Smads. The mammalian Smad family consists of 8 members, which can be divided into 3 groups according to their functions: receptor-activated Smads (R-Smads), common-mediator Smads (Co-Smads) and inhibitory Smads (I-Smads) [58]. After receptor activation, Smad2 and Smad3, which belong to the group of R-Smads, can be phosphorylated in their C-terminus and associated with Smad 4, one of the Co-Smad. This Smad complex translocates into the nucleus and associate with transcriptional coactivators or corepressors to regulate the transcriptional activation of various TGF-β responsive genes. Several reports demonstrated that TGF-β/Smad can crosstalk with MAPK cascade for regulation of growth, survival and motility of cells [59]. The MAPK family proteins including ERK1 and ERK2; JNK1 and JNK2; and the four p38 isoforms, p38α, p38β, p38γ and p38δ can be activated by TGF-β and have been implicated as upstream regulators of Smad. For example, all three MAPK can phosphorylate Smad3 in the linker region and facilitate both activation and nuclear accumulation of Smad3 [60]. On the other hand, Smads can act upstream of MAPKs and mediate their activation [59]. These studies suggested that a sustained signal circuit may be established by crosstalk of both TGF-β/Smad and MAPK pathway.

2.3.2.2 The Role of TGF-β in Tumor Progression The potential involvement of TGF-β in cancer has been noted by cancer biologists decades ago. Previously, it was suggested that TGF-β play as a tumor suppressor and cancer cells may acquire genetic alterations involved in the TGF-β signaling pathway to bypass the antiproliferative action of TGF-β. Moreover, the role of TGF-β1 itself as a potent inducer of tumor progression was highlighted in recent studies. TGF-β is among the prevalent growth factors overproduced in tumor microenvironment exerting autocrine or paracrine action on the tumor cells. TGF-β may induce EMT, migration and invasion of tumor cell in cultured condition [21, 61, 62]. TGF-β can also enhances proteinase expression by tumor cells which in turn leads to degradation of the ECM with a consequent release of stored TGF-β. Moreover, excess TGF-β production is associated with poor prognosis of a lot of cancer including breast tumor, lung cancer, hepatocarcinoma and prostate carcinoma [63].

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2.3.2.3 TGF-β/Smad Signaling for Tumor Progression Both Smad and MAPK signaling has been implicated in mediating the tumor progressive effects of TGF-β on cancer cells. For example, overexpression of Smad3 had prometastatic effects on breast cancer cell lines [64], and Smad signaling is required for TGF-β-induced bone metastasis of breast cancer cells [65]. Also, Smad signaling mediates the invasive phenotype of head and neck squamous carcinoma cells by regulating their collagenase production [66]. In addition, TGF-β-stimulated invasion of head and neck SCC cells is mediated via p38 (MAPK) of which p38α and p38 δ isotypes are the most important [66]. 2.3.2.4 Blockade of TGF-β Signal for Prevention of Tumor Metastasis Due to its important role in tumor progression, the TGF-β signaling were regard as promising targets for anti-metastasis. Large molecular inhibitors, such as TGF-β antibodies and antisense oligonucleotides, have been used quite widely in preclinical and clinical studies [67, 68]. Also, small molecular inhibitors were developed to target TGF-β receptor kinases. However, because of the multifunctional activities of TGF-β, complete loss of TGF-β signaling would eventually be detrimental, such as promotion of inflammation, development of autoimmune diseases and even enhancement of tumor growth in situations where cancer cells are still responsive to the antiproliferative effects of TGF-β.

2.3.3 The Integrin-ECM Engagement In addition to aforementioned growth factors and cytokines, the extracellular matrix (ECM) is another potent metastatic factors in the tumor microenvironment. The ECM serve as the scaffold for the organization of cells in tissues exerting an extraordinary control on cell growth, survive migration and invasion. The interaction between tumor cells and stromal cells may lead to altered secretion and/or modifications of extracellular matrix which greatly facilitate tumor progression. The effects of the ECM on cells are mainly mediated by integrins, a large family of cell-surface receptors that bind and mediate adhesion to ECM components. Each integrin consists of two type-I transmembrane subunits: α and β. In mammals, 18α and 8β subunits associate in various combinations to form 24 integrins that can bind to distinct, although partially overlapping, subsets of ECM ligands. Engagement of integrin with ECM lead to clustering of integrins at the focal adhesion site which impart polarity to the cell and remodel its cytoskeleton. Moreover, the focal adhesion site serves as a platform for signal transduction mediating a lot of cellular effects including proliferation, adhesion, migration and eventually metastasis [69, 70, 71, 72]. 2.3.3.1 The Integrin-Mediated Signaling A lot of signal cascades can be activated by integrin engagement, of which the mitogen activated protein kinase (MAPK) is the most relevant to tumor

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progression. Integrins can activate extracellular signal-regulated kinase (ERK)/(MAPK) through Shc or FAK. Whereas FAK signaling is necessary for directional cell movement, Shc promotes random cell motility [73]. The Shc-dependent pathway may trigger the conventional Ras-Raf-MEK cascade for ERK activation. In the FAK-dependent pathway, FAK may autophosphorylate at Y397 for binding to SH2 domain of Src followed by phosphorylation on multiple residues resulting in elevation of its kinase activity. Activated FAK/Src may phosphorylate the adaptor protein P130CAS and scaffold protein paxillin. Paxillin may be phosphorylated at tyrosine residue 31 and 118 between its N-terminal LD1 and LD2 motif generating docking site for P130CAS. This may facilitate the recruitment of another SH2/SH3 adaptor CRK and trigger activation of GTPases Rac via guanidine exchange factor (GEF) DOCK180 leading to JNK(MAPK) activation. The effects of integrin-triggered ERK and JNK (MAPK) activation in cell migration has two-fold. One is the phosphorylation of cytoskeletal components, the other is the regulation of gene expression. ERK(MAPK) may phosphorylate and activate the myosin light chain (MLC) kinase (MLCK), which induces the contraction of actomyosin fibres through phosphorylation of MLC [74]. On the other hand, ERK and JNK (MAPK) also control cell migration through induction of activator protein-1(AP-1)-dependent gene expression. Interestingly, after integrin engagement, MAPK are not only activated but also recruited to focal adhesion complex to induce phosphorylation of paxillin. Paxillin phosphorylation might further promote the turnover of focal adhesions facilitating cell migration [75]. Another important down stream component of integrin signal cascade is Rho family protein. The RhoGTPases mediate many of the integrin-dependent modifications of the actin cytoskeleton that are necessary for cell migration [76]. Whereas Cdc42 and Rac promote actin polymerization at the leading edge, and thereby the formation of filopodia and lamellipodia, Rho induces the assembly and contraction of the actomyosin fibres, which contributes to pulling the trailing edge forwards. 2.3.3.2 Altered Integrin Signaling Promotes Tumor Progression There are several mechanisms for integrin signaling to contribute tumor metastasis. One of them is altered expression of integrin, the other is integration of integrin signaling with that triggered by other metastatic factors. Pathological studies of human cancer have provided evidence that tumor cells switch expression of their integrins. These changes are complex and depend on the tissue origin of the tumor, its histological type, and the stage of progression of the disease [77, 78, 79].One example is that elevated levels of integrins α6β4 can be observed in metastatic breast tumor cell. Consistently, introduction of β4 to β4-negative breast carcinoma cells activates signaling from phosphatidylinositol 3-kinase (PI3K) to Rac and increases the invasive ability of these cells [79]. In addition, one recent report [80] demonstrated that overexpression of αvβ3 in tumor cell can substantially increases anchorage-independent cell survival in vitro and metastasis in vivo.

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2.3.3.3 Crosstalk of Integrin Signaling With Other Pathway Since many growth factors and ECM are located in the tumor microenvironment as described in the above section, it is tempting to speculate that integrin signaling might crosstalk with others to enhance tumor progression. Indeed, a lot of evidences have indicated that deregulated joint integrin–RTK signaling contributes to tumor metastasis. For example, αvβ3 integrin combines with several RTKs, including the receptors for EGF, PDGF, insulin and vascular endothelial growth factor (VEGF), and cooperates with them to promote cell migration [81, 82]. In addition, αvβ6 and αvβ8 might promote EMT by contributing to the activation of TGF-β [83]. The mechanisms underlying the crosstalk of integrin with RTK or other signal cascade have been intensively studied. Guo et al. indicated that both the EGF receptor and c-Met induce phosphorylation of β4 and enhance Shc activity by disrupting hemidesmosomes. These suggested that RTKs may decrease the ability of α6β4 to mediate stable adhesion while increase its function in signaling [70]. On the other hand, one of the studies demonstrated that the integrin β4 tail may serve as an adaptor for amplification of pro-invasive signals elicited by HGF/c-met [84]. Recently, Wu et al. suggested that integrin may crosstalk with RTK and PKC by establishing a signal circuit in the focal adhesion complex mediated by reactive oxygen species (ROS), resulting in sustained ERK activation and cell migration [85]. The detailed mechanisms are described in Chapter 6. One recently highlighted mechanisms for sustained integrin and RTK signaling involved the endocytic-exocytic cycling of both receptors [86, 87]. Interestingly, Caswell et al. reported that α5β1 and EGFR recycle to the plasma membrane in complex with one another, resulting in enhanced cell migration and invasion. This is associated with enhanced signaling from EGFR to the proinvasive kinase, AKT [86]. Moreover, coordination of α5β1 and EGFR trafficking depend on their association with the Rab11 effector Rab-coupling protein (RCP, Rab11-FIP1) [86]. The role of Rab-GTPases and its down stream effector as central regulators of integrin traffics and tumor progression are well reviewed in Chapter 8 of this book. 2.3.3.4 Targeting Integrin Signaling for Prevention of Tumor Progression Since the role of integrin signaling in migration and invasion of cell are so provital, it become one of the most promising targets for prevention of tumor metastasis. One recent review [87] indicated that the use of integrin antagonists for cancer therapy have shown encouraging outcome. One of the drug cilengitide, which is an inhibitor of αvβ3 and αvβ5, is currently being tested in a Phase III trial for patients with glioblastoma.

2.4 Conclusion and Perspective Growth factors and cytokines secreted by tumor cells, stromal cells and inflammatory cells in the complicated tumor microenvironment can induce signal transductions for eliciting phenotypical changes required for tumor progression (Fig. 2.1).

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Moreover, there is significant overlap between the pathways induced by each metastatic factors resulting in crosstalk of signal transduction. In the future, more effective cancer therapy can be developed by targeting the critical signal molecules responsible for the initiation stage of tumor metastasis.

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Chapter 3

The ERK1/2 MAP Kinase Signaling Pathway in Tumor Progression and Metastasis Laure Voisin∗ , Stéphanie Duhamel∗ , and Sylvain Meloche

Abstract The extracellular signal-regulated kinase (ERK) 1/2 mitogen-activated protein (MAP) kinase module is a conserved signaling pathway that plays a major role in the control of cell proliferation, survival and differentiation. This pathway is typically turned on by engagement of growth factor receptors, which leads to the activation of the small GTPase Ras and to sequential phosphorylation and activation of Raf, MEK1/MEK2 and ERK1/ERK2 protein kinases. ERK1 and ERK2 are multifunctional Ser/Thr kinases that phosphorylate a panoply of substrates involved in multiple cellular processes. Hyperactivation of the ERK1/2 pathway is frequently observed in human malignancies as a result of aberrant activation of receptor tyrosine kinases or by gain-of-function mutations in RAS or RAF genes. This dysregulation is believed to provide a proliferation and survival advantage to cancer cells. Accumulating evidence suggest that the ERK1/2 signaling pathway also contributes to the increased motility, invasiveness and dissemination of tumor cells. In this chapter, we review the role of ERK1/2 MAP kinase signaling in the pathogenesis of tumor metastasis. Keywords Adhesion · Angiogenesis · Anoikis · Apoptosis · Cancer · Cell survival · E-cadherin · Epithelial-mesenchymal transition (EMT) · Extracellular matrix (ECM) · Extracellular signal-regulated kinase 1/2 (ERK1/2) · Extravasation · Immune-system evasion · Integrins · Invasion · Invasiveness · Intravasation · Matrix metalloproteinases (MMPs) · Mitogen-activated protein kinase (MAPK) · Metastasis · Micrometastases · Motility · Polarity · Proteases · Ras/ Raf/MEK/ERK1/2 pathway · Rho GTPases · Signaling pathway · Snail · Tumorigenesis · Tumor · Urokinase-type plasminogen-activated receptor (uPAR)

S. Meloche (B) Institut de Recherche en Immunologie et Cancérologie, Université de Montréal, 2950, Chemin de Polytechnique, Montreal, Quebec, Canada H3C 3J7 e-mail: [email protected] ∗ These

authors contributed equally to this work

W.-S. Wu, C.-T. Hu (eds.), Signal Transduction in Cancer Metastasis, Cancer Metastasis – Biology and Treatment 15, DOI 10.1007/978-90-481-9522-0_3,  C Springer Science+Business Media B.V. 2010

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Contents 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Hyperactivation of the Ras-ERK1/2 MAP Kinase Pathway in Cancer . 3.3 The ERK1/2 MAP Kinase Pathway in Cancer Metastasis . . . . . . 3.3.1 Loss of Cellular Contacts, Detachment From the Primary Tumor and Local Invasion . . . . . . . . . . . . . . . . . . . . . 3.3.2 Intravasation and Dissemination to Distant Organ Sites . . . . 3.3.3 Extravasation, Formation of Micrometastases and Outgrowth of Secondary Tumors . . . . . . . . . . . . 3.4 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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3.1 Introduction Mitogen-activated protein (MAP) kinase pathways are evolutionarily conserved protein kinase cascades that play a key role in signal transduction [58]. These pathways process information from a wide variety of extracellular stimuli and cellular perturbations to control multiple physiological processes required to maintain normal cellular and tissue homeostasis [16, 47, 58]. Furthermore, deregulated activity of MAP kinase pathway components has been linked to many diseases, including cancer, inflammatory disorders, diabetes and cardio-facio-cutaneous syndromes [49]. These enzymes are therefore viewed as attractive targets for novel targeted therapies. Among the best-characterized MAP kinase pathways is the Ras-dependent extracellular signal-regulated kinase (ERK) 1/2 pathway, which conveys signals from mitogenic factors, cytokines and differentiation cues. Typically, ligand binding to growth factor receptors leads to the activation of the small GTPase Ras, which allows the recruitment of Raf to the membrane and mediates the sequential phosphorylation and activation of Raf, MEK1/MEK2 and ERK1/ERK2 protein kinases [67]. Once activated, the multifunctional kinases ERK1/ERK2 phosphorylate a vast array of substrates, including membrane receptors, cytoskeletal proteins, transcriptional regulators and signaling enzymes, to impact on cell growth, cell cycle progression, survival, adhesion and motility [99]. Not surprisingly, the ERK1/2 MAP kinase pathway is found commonly deregulated in cancer.

3.2 Hyperactivation of the Ras-ERK1/2 MAP Kinase Pathway in Cancer Hyperactivation of the Ras/Raf/MEK/ERK1/2 pathway by aberrant activation of receptor tyrosine kinases or by gain-of-function mutations in RAS or RAF genes is a highly prevalent event in human cancer. Gene amplification and mutational activation of members of the HER (human EGF receptor) family of receptors are

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frequently observed in human epithelial tumors and gliomas [37]. Activating mutations in RAS genes, most frequently in KRAS, occur in ∼30% of cancers and are often acquired early in the tumorigenic process [26, 76]. Although Ras is known to regulate multiple effector pathways, the discovery that BRAF is mutated in ∼20% of all cancers and in 44% of melanomas has provided genetic evidence for the involvement of the Raf/MEK/ERK1/2 effector branch in oncogenic signaling [19, 26]. Raf relays its oncogenic signals mainly via the MAP kinase kinases MEK1 and MEK2. Several studies have shown that expression of activated alleles of MEK1 or MEK2 is sufficient to deregulate the proliferation and transform immortalized fibroblast and epithelial cell lines [17, 52, 55, 63, 90]. In vivo, orthotopic transplantation of mammary or intestinal epithelial cells expressing activated MEK1/MEK2 into mice induces the formation of metastatic tumors [63, 90]. Transgenic expression of activated MEK1 in mouse skin induces hyperproliferative and inflammatory lesions and inhibits epidermal differentiation, mimicking features of squamous cell carcinomas [35, 74]. Although mutations in MEK1/MEK2 genes are rare in cancer [53], numerous studies have documented the hyperactivation of MEK1/MEK2 and ERK1/ERK2 in solid tumor and leukemia clinical specimens [36, 79]. Consistent with its physiological involvement in the regulation of cell proliferation and survival, aberrant activation of the ERK1/2 pathway is expected to relax the growth factor dependency and enhance the survival of cancer cells. In addition, accumulating evidence suggest that this signaling pathway also contributes to the increased motility and invasiveness of tumor cells, another hallmark of cancer [33]. In this chapter, we discuss the evidence for a role of ERK1/2 MAP kinase signaling in the pathogenesis of cancer metastasis.

3.3 The ERK1/2 MAP Kinase Pathway in Cancer Metastasis Metastasis is a complex process by which tumor cells spread from a primary site to form new tumors in distant organs [14, 28]. The metastatic process consists of a series of sequential and rate limiting steps that can be defined as follows: (1) detachment from the primary tumor and local invasion; (2) intravasation into the lymphatic or blood circulation; (3) transport to distant anatomical sites and arrest in microvessels; (4) extravasation into the surrounding tissue; (5) formation of micrometastases; and (6) colonization and formation of large clinically detectable metastases. The outcome of the metastatic process is dependent on both the intrinsic properties of the cancer cells and the host response.

3.3.1 Loss of Cellular Contacts, Detachment From the Primary Tumor and Local Invasion 3.3.1.1 The Epithelial-Mesenchymal Transition (EMT) One of the first steps in the metastasis process involves changes in the intercellular adhesion properties of cancer cells. Carcinoma cells undergo a drastic modification

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known as epithelial-mesenchymal transition (EMT), whereby epithelial cells switch to a fibroblast-like mesenchymal phenotype, enabling their detachment from epithelial sheets within the primary tumor and favoring their motility and invasiveness [32, 42]. EMT is characterized by morphological changes, loss of apical-basal polarity and alterations in gene expression profiles. A major determinant of the reduced intercellular adhesion observed during EMT is the loss of the epithelial adherens junction protein E-cadherin [61]. Various mechanisms can lead to silencing of E-cadherin expression during tumor progression, but transcriptional repression has emerged as a fundamental mechanism. Several transcriptional repressors, including Snail, ZEB and bHLH factors (E47/E2A, Twist), have been found to inhibit the expression of the E-cadherin gene and to induce EMT [60]. Multiple signaling pathways can induce EMT, including transforming growth factor-β (TGF-β), Wnt, NF-kB, Notch, phosphoinositide 3-kinase and ERK1/2 MAP kinase [60, 83]. Early studies have shown that expression of activated MEK1 in MDCK cells results in morphological transformation and conversion to invasive mesenchymal-like cells [34, 55, 75]. Other studies also documented the essential role of the ERK1/2 pathway in hepatocyte growth factor (HGF)/scatter factorinduced scattering of epithelial cells [34, 65, 82]. The role of Ras effector pathways in epithelial plasticity was analyzed in EpH4 mammary epithelial cells using Ras effector mutants and signaling inhibitors. These experiments revealed that hyperactivation of the ERK1/2 pathway cooperated with TGF-β to induce a full EMT in these cells [38]. Treatment with the MEK1/2 inhibitor PD98059 completely prevented and reversed EMT. Similarly, in TGF-β-responsive pancreatic cancer cells bearing activating KRAS mutations, TGF-β1 treatment caused an EMT that was reduced or abolished by pretreatment with the MEK1/2 inhibitor [23]. More recently, we have reported that expression of activated forms of MEK1 or MEK2 induces a partial EMT in IEC-6 intestinal epithelial cells, characterized by loss of epithelial polarity, adoption of a fibroblastoid morphology and downregulation of E-cadherin expression [90]. No induction of the mesenchymal proteins vimentin and smooth muscle α-actin was observed in IEC-6 cells. These results suggest that constitutive ERK1/2 signaling, while disrupting normal epithelial morphology and polarization, is not sufficient to induce a full EMT. In agreement with this idea, conditional activation of Raf was shown to induce an EMT in MDCK cells, but this was dependent on the operation of an autocrine loop producing TGF-β [51]. Activation of the ERK1/2 pathway can also contribute to the induction of TGF-β1 by TGF-β3 in epithelial cells [100]. One mechanism by which ERK1/2 signaling contributes to EMT execution is through induction of the Snail family of transcriptional repressors. In MDCK cells, treatment with PD98059 was found to decrease basal Snail promoter activity and to abolish Snail induction in response to TGF-β or activated Ras [59]. HGF-induced Snail expression and cell scattering is also dependent on ERK1/2 activation in hepatocytes [31]. Notably, the upregulation of Snail by HGF requires the activity of the zinc finger transcription factor Egr-1, a known target of ERK1/2 MAP kinases. Activation of the ERK1/2 pathway by receptor tyrosine kinases also leads to the induction of Slug and to repression of E-cadherin transcription in colon carcinoma

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cells [15]. Another mechanism by which oncogenic Ras-mediated ERK1/2 activation decreases intercellular adhesion in lung epithelial cells is through induction of metalloproteinase (MMP)-9 and proteolytic cleavage of E-cadherin, resulting in partial disruption of adherens junctions [92]. Interestingly, a recent study has identified RSK1 and RSK2 as key effectors of the ERK1/2 pathway for eliciting cell scattering and mesenchymal gene expression in epithelial cell lines of diverse origins [21]. RSK activity is also important for Raf-induced secretion of TGF-β in MDCK cells.

3.3.1.2 Extracellular Proteases and Invasiveness The most characteristic trait of malignant cells is their ability to invade normal adjacent tissues. Dissemination of cancer cells involves the breakdown and remodeling of extracellular matrix (ECM) components by extracellular proteases to allow movement of individual or collective mass of cells into new passage ways. Several classes of proteases contribute to ECM remodeling, but the most important are probably the MMPs, a family of zinc-dependent proteinases that can degrade all components of the ECM [20, 40]. The role of MMPs is not limited to tumor cell invasion, as these enzymes can proteolytically process a large number of growth factors, cytokines, adhesion molecules and precursor of biologically active proteins to impact on tumor initiation, angiogenesis and immune surveillance [22]. Cancer cells frequently upregulate MMP expression, but the bulk of these proteases are secreted by host-derived cells like fibroblasts, endothelial cells or inflammatory cells that contribute to the tumor microenvironment. It should be noted that individual tumor cells can switch to an amoeboid mode of movement and invade local tissue in the absence of extracellular proteolysis [73]. The ERK1/2 MAP kinase pathway plays a major role in inducing proteolytic enzymes and promoting tumor cell invasiveness. Numerous studies have reported that pharmacological inhibition of MEK1/2 markedly decreases the invasiveness of various carcinoma cells in vitro [39, 68, 78, 80]. Importantly, constitutive activation of MEK1 or MEK2 is sufficient to upregulate the expression of ECM proteases and to increase the invasive capacity of mammary and intestinal epithelial cells [63, 90]. Biochemical studies and transcriptional profiling experiments have revealed that the ERK1/2 pathway regulates the expression of several MMP genes, such as MMP-1, MMP-3, MMP-7, MMP-9, MMP-10, MMP-11, MMP-13, MMP-14, MMP-19 and MMP-25 [21, 45, 68, 89, 90]. ERK1/2 signaling also coordinately enhances the expression of α2-integrin and CD44, which act as receptors for MMP-1 and MMP-9, respectively [21]. Compelling evidence that these MMPs regulate tumor progression and metastasis was provided by genetic studies employing engineered mouse models [20]. Another protease that plays a crucial role in tumor cell invasion and metastasis is the serine protease urokinase-type plasminogen activator (uPA) [9, 48]. Activated uPA bound to its cell surface receptor uPAR on tumor cells converts the zymogen plasminogen into the active protease plasmin, which in turn cleaves pro-MMPs into MMPs and latent TGF-β1 into its active form. Hyperactivation of ERK1/2 MAP

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kinases was shown to upregulate the expression of uPA and of its receptor uPAR in a variety of stromal and tumor cell types [1, 21, 48, 63, 90]. 3.3.1.3 Tumor Cell Motility The acquisition of cell motility is essential for tumor cells to invade surrounding tissues and enter into the blood or lymphatic circulation. Cancer cells can move by at least three distinct mechanisms: mesenchymal motility, amoeboid motility and collective motility [73]. Studies with cultured cells and gene expression profiling have provided strong evidence for the involvement of the ERK1/2 signaling pathway in mesenchymal motility of epithelial cells [21, 88]. The cell motility cycle consists of the following steps: formation of protrusions at the leading edge, integrin-mediated adhesion to the underlying substrate, cell contraction and disassembly of adhesions at the rear [70]. The Rho GTPases Cdc42 and Rac, which are key regulators of actin dynamics, control the formation of protrusions (filopodia and lamellipodia) at the front of the moving cell [66]. Rho, on the other hand, regulates the assembly of stress fibers and focal adhesions assembly and is required for cell body contraction. One of the mechanisms by which the ERK1/2 pathway promotes cell motility is by regulating the activity of Rho family GTPases. In colon carcinoma cells, ERK1/2 signaling was shown to coordinately modulate the activity of Rho and Rac via transcriptional upregulation of the Fra-1 transcription factor and uPAR [87]. Induction of Fra-1 inactivates β1-integrin signaling by an unknown mechanism, resulting in the repression of Rho-GTP levels and reduced activation of the Rho effector ROCK. This prevents the formation of excessive stress fibers and focal adhesions, allowing Rac-induced membrane ruffles to be extended into protrusions. Elevated expression of uPAR drives the activation of Rac and membrane ruffling by a Fra1-independent mechanism. Active ERK1/ERK2 MAP kinases localize to cell adhesions [25] and several studies have shown that local ERK1/2 activity is required for focal adhesion disassembly and migration [43, 94, 97]. Various molecular mechanisms have been proposed to explain how ERK1/2 signaling affects adhesion, including stimulation of calpain 2 activity and proteolysis of FAK, downregulation of ROCKI/II expression or activity, and induction of Rnd3 expression [12, 30, 43, 57, 72]. A more recent study reported that oncogenic Ras-mediated ERK1/2 activation induces tyrosine dephosphorylation and inhibition of FAK activity, resulting in decreased adhesion and enhanced migration and metastasis [101]. Another target of ERK1/2 MAP kinases involved in the regulation of cell motility is myosin light chain kinase (MLCK). Together with ROCK, MLCK regulates actinmyosin contractility by phosphorylating the regulatory light chain of myosin II. A study showed that ERK1/2 directly phosphorylate MLCK leading to increased myosin light chain phosphorylation and enhanced cell migration [44]. It has been suggested that ERK1/2-mediated activation of MLCK is required to generate the traction force necessary for pseudopodial protrusion formation at the side of the cell facing the chemotactic gradient [11]. Rho activity, on the other hand, facilitates pseudopodia retraction. It should also be emphasized that mesenchymal motility

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is dependent upon proteolytic degradation of the ECM, which is regulated by the ERK1/2 pathway as discussed above.

3.3.2 Intravasation and Dissemination to Distant Organ Sites 3.3.2.1 Intravasation Metastasising cells that detach from the primary tumor must enter into the bloodstream and survive in the circulation until they can arrest at a secondary site. Both active and passive mechanisms have been proposed to explain how tumor cells enter the vasculature [10]. Interestingly, a recent study reported that expression of the Raf/MEK/ERK1/2 pathway inhibitor RKIP in the metastatic breast cancer cell line MDA-MB-231 inhibits cell intravasation in a murine orthotopic model [18]. 3.3.2.2 Surviving Anoikis Following intravasation, tumor cells undertake a dangerous voyage in the hostile environment of the blood circulation, where they are devoided of contact with the ECM and are threatened by high shear stress. Epithelial cells, as well as other cell types, that lose appropriate contact with the matrix or neighboring cells undergo a form of apoptosis known as anoikis [29]. Surviving anoikis, therefore, is a prerequisite for cancer progression and establishment of metastases. An early study revealed that transformation of MDCK epithelial cells with oncogenic Ras confers resistance to anoikis, although the downstream effectors mediating this response were not characterized at that time [27]. Subsequently, several studies reported that activation of the ERK1/2 MAP kinase pathway is both necessary and sufficient to protect against cell detachment-induced apoptosis in fibroblast, endothelial and epithelial cell models [5, 41, 50, 71, 77, 95]. One of the main mechanisms by which ERK1/2 signal anoikis survival is repressing the expression or activity of pro-apoptotic Bcl-2 family proteins such as Bim, and inducing the expression of pro-survival members like Bcl-2 and Mcl-1 [7]. In a pioneer study, Reginato et al. [69] have shown that expression of H-RasV12 , Raf-CAAX or MEK2DD in the mammary epithelial cell line MCF-10A completely inhibits the upregulation of Bim expression in suspension and prevents anoikis. Other mechanisms such as upregulation of c-FLIP expression [5] or autocrine production of EGF receptor ligands [77] may also contribute to the survival effect of the ERK1/2 pathway. 3.3.2.3 Escape From Immune Response In addition to surviving anoikis, tumor cells in the circulation must also escape recognition and destruction by the immune system. Although the role of the ERK1/2 MAP kinase pathway in immune-mediated antitumor responses has not been studied extensively, recent reports suggest that ERK1/2 may also control the escape of tumor cells from immune surveillance. The expression of major histocompatibility complex (MHC) class II genes is necessary for presentation of antigens to CD4+ lymphocytes and induction of an effective immune response. Both the

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constitutive and inducible expression of MHC class II proteins is regulated by the transcriptional activator class II transactivator (CIITA) [81]. Studies have shown that ERK1/2 signaling negatively regulates the expression and function of CIITA in dendritic cells and macrophages [91, 98]. Interestingly, a recent study identified the Ig superfamily membrane glycoprotein CD200 as a transcriptional target of the Raf/MEK/ERK1/2 pathway in melanoma cells [62]. Expression of CD200 mRNA is higher in melanoma than in other tumors and correlates with progression from nevi to melanoma. Importantly, the authors showed that melanoma cell lines expressing high CD200 repressed primary T cell activation by dendritic cells, an effect abrogated by RNAi silencing of CD200 expression. These findings suggest that hyperactivation of ERK1/2 in metastatic melanomas attenuates the host antitumor immune response. Another study reported that stimulation of human keratinocytes with EGF or expression of oncogenic Ras activates ERK1/2 and markedly decreases expression of the skin-associated chemokine CCL27, which regulates the recruitment of T cells to the skin during immune surveillance [64]. Treatment of keratinocytes with the MEK1/2 inhibitor U0126 was found to abrogate the suppression of CCL27 expression. This suggests that skin tumors may evade T cell-mediated antitumor response by downregulating CCL27 expression through activation of the Ras-ERK1/2 pathway.

3.3.3 Extravasation, Formation of Micrometastases and Outgrowth of Secondary Tumors 3.3.3.1 Extravasation Once at the secondary site, tumor cells must adhere to the endothelium and transmigrate across endothelial cells by a process known as diapedesis. Expression of the adhesion molecule E-selectin, which is induced by proinflammatory cytokines, appears to play an important role in the homing of cancer cells [96]. Early studies have shown that metastatic Ras-transformed NIH 3T3 fibroblasts and parental nontransformed cells extravasate with the same kinetics in the chick chorioallantoic membrane model or after injection in the mouse mesenteric vein [46, 86]. However, a recent study has suggested that the ERK1/2 MAP kinase pathway may play an active role in diapedesis [85]. Indeed, it has been reported that activation of E-selectin by the binding of circulating colon cancer cells triggers the activation of ERK1/2, and that inhibition of ERK1/2 signaling with PD98059 or by adenoviral expression of a dominant-negative ERK1/2 mutant inhibits E-selectin-mediated transendothelial migration. 3.3.3.2 Establishment and Outgrowth of Micrometastases Into Macrometastases The outgrowth of single extravasated cells into micrometastases and eventually macrometastases is a very inefficient step of the metastatic cascade [14, 28]. Tumor cells must adapt to their new microenvironment, resist to immune attack, survive

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apoptosis, and start proliferating. Early observations have shown that cancer cells do not colonize tissues randomly but have a predilection to form macrometastases in specific organs. This compatibility between disseminated tumor cells and host tissues has led to the formulation of the “seed and soil” hypothesis [24]. Several mechanisms have been proposed to explain the tropism of metastasizing cells. Many evidence point to a role of the ERK1/2 MAP kinase pathway in the outgrowth of micrometastases. Webb et al. [93] compared the ability of Ras effector domain mutants expressed in NIH 3T3 fibroblasts to mediate tumorigenic and metastatic phenotypes in nude mice. While all Ras effector mutants displayed comparable tumorigenic properties, only the V12S35 mutant that activates the Raf/MEK/ERK1/2 pathway was able to induce the formation of lung metastases when injected intravenously. To confirm this finding, the authors further showed that expression of the Mos oncogene, a strong activator of MEK1/MEK2, or activated MEK1 also confer metastatic properties to the cells. These results suggest that constitutive activation of the ERK1/2 pathway is necessary and sufficient to promote the last steps of the metastatic cascade. Another study showed that Ras does not enhance the ability of NIH 3T3 cells to extravasate from liver sinusoids or to initiate proliferation of cells to form micrometastases in a liver metastasis model [86]. However, only micrometastases formed by Ras-transformed cells persisted to form macrometastases by day 14, whereas most NIH 3T3 micrometastases disappeared from liver. Ras signaling was able to shift the balance between apoptosis and cell division in favor of proliferation in this model. Interestingly, an elegant series of in vivo studies using the chick chorioallantoic membrane model and nude mice has highlighted the importance of the ERK1/2 and p38 MAP kinase pathways in regulating metastasis dormancy [2–4]. These studies showed that the ratio of ERK1/2-to-p38 activity dictates cell proliferation or growth arrest and dormancy in various cancer cell lines. High expression of uPAR, by interacting with and activating α5β1 integrin, initiates a signaling cascade that leads to sustained activation of ERK1/2 and cell proliferation. High ERK1/2 activity is maintained by a positive feedback loop that induces uPA and uPAR expression (see above). Downregulation of uPAR expression decreases ERK1/2 activity and increases p38 activity, inducing dormancy of carcinoma cells. ERK1/2 signaling may contribute to metastasis outgrowth via several molecular mechanisms. The central role of ERK1/2 in cell cycle progression and cellular survival is well documented and has been the subject of recent reviews [6, 7, 13, 54, 84]. In addition, growing metastases like primary tumors are critically dependent on the recruitment of new blood vessels (angiogenesis) or on co-option of existing vessels to provide the cells with the nutrients and oxygen necessary to reach a clinically detectable size [8]. This angiogenic switch is controlled by a balance between pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), and anti-angiogenic factors like thrombospondin. Activation of the ERK1/2 pathway can stimulate angiogenesis by upregulating VEGF expression through the regulation of Sp1 and HIF-1 transcription factors [56]. Finally, ERK1/2 signaling may also contribute to metastasis expansion by favoring immune-system evasion.

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3.4 Concluding Remarks The ERK1/2 MAP kinase signaling pathway has gained notoriety for its role in cell cycle progression and cell proliferation. However, later work has provided convincing evidence for the involvement of this pathway in other cellular processes that are essential hallmarks of cancer. In vitro studies of cancer cell lines and in vivo findings from animal models have highlighted the role of ERK1/2 signaling in cell survival, motility, invasiveness and angiogenesis. Through its various effects, the ERK1/2 pathway may contribute to every step of the metastatic process, from the early escape of cancer cells from the primary tumor loci to the late step of metastatic colonization (Fig. 3.1). Indeed, expression of activated forms of MEK1 or MEK2 in intestinal epithelial cells is sufficient to induce the formation of intestinal tumors and to promote tumor progression and metastasis in a mouse orthotopic transplantation model [90]. These pre-clinical studies suggest that blockade of the ERK1/2 pathway with small-molecule inhibitors of Raf or MEK1/2 should prove effective not only in shrinking the primary tumor but also in restraining metastatic dissemination, which is the major cause of cancer-associated mortality.

Fig. 3.1 The ERK1/2 MAP kinase signaling pathway in cancer metastasis. Schematic diagram illustrating how ERK1/2 signaling may contribute to every step of the metastatic process, from the early escape of cancer cells from the primary tumor to the late steps of metastatic colonization. See text for details

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Acknowledgments Work in the author’s laboratory was supported by grants from the National Cancer Institute of Canada and the Cancer Research Society. S. Duhamel is recipient of a studentship from the Canadian Institutes for Health Research. S. Meloche holds the Canada Research Chair in Cellular Signaling.

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76. Schubbert S, Shannon K, Bollag G. Hyperactive Ras in developmental disorders and cancer. Nat. Rev. Cancer 2007; 7: 295–308. 77. Schulze A, Lehmann K, Jefferies HB, McMahon M, Downward J. Analysis of the transcriptional program induced by Raf in epithelial cells. Genes Dev. 2001; 15: 981–994. 78. Sebolt-Leopold JS, Dudley DT, Herrera R, Van Becelaere K, Wiland A, Gowan RC, Tecle H, Barrett SD, Bridges A, Przybranowski S, Leopold WR, Saltiel AR. Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo. Nat. Med. 1999; 5: 810–816. 79. Sebolt-Leopold JS, Herrera R. Targeting the mitogen-activated protein kinase cascade to treat cancer. Nat. Rev. Cancer 2004; 4: 937–947. 80. Simon C, Juarez J, Nicolson GL, Boyd D. Effect of PD 098059, a specific inhibitor of mitogen-activated protein kinase kinase, on urokinase expression and in vitro invasion. Cancer Res. 1996; 56: 5369–5374. 81. Steimle V, Otten LA, Zufferey M, Mach B. Complementation cloning of an MHC class II transactivator mutated in hereditary MHC class II deficiency (or bare lymphocyte syndrome). Cell 1993; 75: 135–146. 82. Tanimura S, Chatani Y, Hoshino R, Sato M, Watanabe S, Kataoka T, Nakamura T, Kohno M. Activation of the 41/43 kDa mitogen-activated protein kinase signaling pathway is required for hepatocyte growth factor-induced cell scattering. Oncogene 1998; 17: 57–65. 83. Thiery JP. Epithelial-mesenchymal transitions in tumour progression. Nat. Rev. Cancer 2002; 2: 442–454. 84. Torii S, Yamamoto T, Tsuchiya Y, Nishida E. ERK MAP kinase in G cell cycle progression and cancer. Cancer Sci. 2006; 97: 697–702. 85. Tremblay PL, Huot J, Auger FA. Mechanisms by which E-selectin regulates diapedesis of colon cancer cells under flow conditions. Cancer Res. 2008; 68: 5167–5176. 86. Varghese HJ, Davidson MT, MacDonald IC, Wilson SM, Nadkarni KV, Groom AC, Chambers AF. Activated ras regulates the proliferation/apoptosis balance and early survival of developing micrometastases. Cancer Res. 2002; 62: 887–891. 87. Vial E, Sahai E, Marshall CJ. ERK-MAPK signaling coordinately regulates activity of Rac1 and RhoA for tumor cell motility. Cancer Cell 2003; 4: 67–79. 88. Viala E, Pouyssegur J. Regulation of tumor cell motility by ERK mitogen-activated protein kinases. Ann. N. Y. Acad. Sci. 2004; 1030: 208–218. 89. Vincenti MP, Brinckerhoff CE. Signal transduction and cell-type specific regulation of matrix metalloproteinase gene expression: can MMPs be good for you? J. Cell Physiol. 2007; 213: 355–364. 90. Voisin L, Julien C, Duhamel S, Gopalbhai K, Claveau I, Saba-El-Leil MK, Rodrigue-Gervais IG, Gaboury L, Lamarre D, Basik M, Meloche S. Activation of MEK1 or MEK2 isoform is sufficient to fully transform intestinal epithelial cells and induce the formation of metastatic tumors. BMC Cancer 2008; 8: 337. 91. Voong LN, Slater AR, Kratovac S, Cressman DE. Mitogen-activated protein kinase ERK1/2 regulates the class II transactivator. J. Biol. Chem. 2008; 283: 9031–9039. 92. Wang XQ, Li H, Van Putten V, Winn RA, Heasley LE, Nemenoff RA. Oncogenic K-Ras regulates proliferation and cell junctions in lung epithelial cells through induction of cyclooxygenase-2 and activation of metalloproteinase-9. Mol. Biol. Cell 2009; 20: 791–800. 93. Webb CP, Van Aelst L, Wigler MH, Woude GF. Signaling pathways in Ras-mediated tumorigenicity and metastasis. Proc. Natl. Acad. Sci. USA 1998; 95: 8773–8778. 94. Webb DJ, Donais K, Whitmore LA, Thomas SM, Turner CE, Parsons JT, Horwitz AF. FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly. Nat. Cell Biol. 2004; 6: 154–161. 95. Welch DR, Sakamaki T, Pioquinto R, Leonard TO, Goldberg SF, Hon Q, Erikson RL, Rieber M, Rieber MS, Hicks DJ, Bonventre JV, Alessandrini A. Transfection of constitutively active mitogen-activated protein/extracellular signal-regulated kinase kinase confers tumorigenic and metastatic potentials to NIH3T3 cells. Cancer Res. 2000; 60: 1552–1556.

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96. Witz IP. The selectin-selectin ligand axis in tumor progression. Cancer Metastasis Rev. 2008; 27: 19–30. 97. Xie H, Pallero MA, Gupta K, Chang P, Ware MF, Witke W, Kwiatkowski DJ, Lauffenburger DA, Murphy-Ullrich JE, Wells A. EGF receptor regulation of cell motility: EGF induces disassembly of focal adhesions independently of the motility-associated PLCgamma signaling pathway. J. Cell Sci. 1998; 111(Pt 5): 615–624. 98. Yao Y, Xu Q, Kwon MJ, Matta R, Liu Y, Hong SC, Chang CH. ERK and p38 MAPK signaling pathways negatively regulate CIITA gene expression in dendritic cells and macrophages. J. Immunol. 2006; 177: 70–76. 99. Yoon S, Seger R. The extracellular signal-regulated kinase: multiple substrates regulate diverse cellular functions. Growth Factors 2006; 24: 21–44. 100. Yue J, Mulder KM. Requirement of Ras/MAPK pathway activation by transforming growth factor beta for transforming growth factor beta 1 production in a Smad-dependent pathway. J. Biol. Chem. 2000; 275: 30765–30773. 101. Zheng Y, Xia Y, Hawke D, Halle M, Tremblay ML, Gao X, Zhou XZ, Aldape K, Cobb MH, Xie K, He J, Lu Z. FAK phosphorylation by ERK primes ras-induced tyrosine dephosphorylation of FAK mediated by PIN1 and PTP-PEST. Mol. Cell 2009; 35: 11–25.

Chapter 4

Mitogen-Activated Protein Kinase-Activated Protein Kinases and Metastasis Alexey Shiryaev, Marijke Van Ghelue, and Ugo Moens

Abstract Cancer is characterized by cells that disobey the stringent control mechanisms of cellular processes such as division and growth, survival, homeostasis, motility, and tissue invasion. Signalling pathways, including the mitogenactivated protein kinase (MAPK) signalling pathways, regulate these cellular processes. Not unexpectedly, cancer cells display defects in signalling pathways due to mutations in genes encoding signal transduction proteins. The typical MAPK pathways transmit, amplify and translate signals through consecutive phosphorylation events engaging a MAPK kinase kinase, a MAPK kinase, and a MAPK, which finally phosphorylates substrates. These substrates can be non-protein kinases or protein kinases. The latter are referred to as MAPK-activating protein kinases. Escalating evidence exists that the MAPK signal transduction pathways can be implicated in metastasis. This review focuses on the specific roles of MAPKactivating protein kinases in metastasis and summarizes potential small inhibitors against MAPK-activating protein kinases that may find their way in cancer therapy. Keywords MAPK-activating protein kinases · Inhibitors · Metastasis · Cytoskeleton · Gene expression Abbreviations AMPK BRSK2 CaMKK CHK2 CK DYRK EPCG ERK8

AMP-activated protein kinase brain-specific kinase 2 calmodulin-dependent kinase checkpoint kinase 2 casein kinase dual-specificity tyrosine-(Y)-phosphorylation regulated kinase Epigallocatechin gallate extracellular-signal-regulated kinase 8

U. Moens (B) Medical Faculty, Department of Medical Biology, University of Tromsø, N-9037 Tromsø, Norway e-mail: [email protected]

W.-S. Wu, C.-T. Hu (eds.), Signal Transduction in Cancer Metastasis, Cancer Metastasis – Biology and Treatment 15, DOI 10.1007/978-90-481-9522-0_4,  C Springer Science+Business Media B.V. 2010

41

42

GSK3β HIPK IKK LCK MARK3 MELK MST2 PDK1 PHK PIM PKA PKBα PKCα PRK2 ROCK-II SGK1 Src SRPK1 S6K1

A. Shiryaev et al.

glycogen synthase kinase 3β homeodomain-interacting protein kinase 2 IkappaB kinase lymphocyte cell-specific protein tyrosine kinase microtubule-affinity-regulating kinase maternal embryonic leucine-kinase mammalian homologue Ste20-like kinase 2 3-phosphoinositide-dependent protein kinase phosphorylase kinase provirus integration site for Moloney murine leukaemia virus protein kinase A/cAMP-dependent protein kinase protein kinase B isoform α protein kinase C isoform α protein kinase C-related kinase 2 Rho-dependent kinase II serum- and glucocorticoid-induced kinase 1 sarcoma kinase serine-arginine protein kinase 1 p70 ribosomal protein S6 kinase 1

Contents 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 MAPKAPK and Cancer . . . . . . . . . . . . . . . . . . . . . 4.2.1 Mutations in the MAPKAPK-encoding Genes in Cancer Tissue 4.2.2 MAPKAPK Expression Levels in Cancers . . . . . . . . . . 4.2.3 MAPKAPK and Cell Cycle . . . . . . . . . . . . . . . . . 4.2.4 MAPKAPK and Cell Survival . . . . . . . . . . . . . . . 4.3 MAPKAPK and Metastasis . . . . . . . . . . . . . . . . . . . . 4.3.1 MAPKAPK and Regulation of the Cytoskeleton . . . . . . . 4.3.2 MAPKAPK and Gene Regulation . . . . . . . . . . . . . . 4.4 Role of MAPKAPK in Tumour Angiogenesis Triggered by VEGF . . 4.5 MAPKAPK Inhibitors . . . . . . . . . . . . . . . . . . . . . . 4.6 Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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42 45 45 47 47 50 50 51 56 62 63 63 64

4.1 Introduction Many cellular processes such as proliferation, differentiation, apoptosis, development, gene expression, and cell motility are governed by signal transduction pathways. Such pathways often consist of protein kinases that transmit and convert signals through a relay of protein phosphorylation events [131, 156]. One of

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43

the major signalling pathways in mammalian cells is the mitogen-activated protein kinase (MAPK) pathway. Seven distinct mammalian MAPK pathways have been identified that mediate cellular responses to a wide variety of extracellular signalling molecules (Fig. 4.1). The typical MAPK pathways, represented by the ERK1/2, ERK5, JNK, and p38 modules consist of a cascade of three consecutive phosphorylation events exerted by a MAPK kinase kinase (MAPKKK or MAP3K), a MAPK kinase (MAPKK or MAP2K), and a MAPK. The atypical MAPKs ERK3, ERK4, ERK7, and ERK8 comprise the less characterized MAPK pathways [36, 37, 89, 94, 112, 167, 180, 218, 235]. MAPKs not only target substrates without kinase activity, but also phosphorylate other protein kinases designated as MAPK-activated protein kinases (MAPKAPK). The MAPKAPK include the ribosomal-S6-kinases (RSK1–4 or MAPKAPK1a-d), the MAPK-interacting kinases (MNK1 and 2), the mitogenand stress-activated kinases (MSK1 and 2), and the MAPKAPK (MK2, 3 and 5) subfamilies [4, 5, 18, 59, 157, 177]. The first MAPKAPK to be isolated was ribosomal S6 kinase (RSK), a 90 kDa protein kinase that phosphorylated the 40S ribosomal subunit protein S6 in Xenopus laevis oocytes [53]. This family of serine/threonine kinase consists of four human isoforms referred to as RSK1–4 (p90Rsk1−4 ) or MAPKAPK1a-d that share 65–73% amino acid identity [4, 23]. RSKs regulate cell growth, cell proliferation, cell survival, transcriptional and translational regulation, and cell motility [4, 23].

MEKK1-4 MEKK1-4 MLK2/3 MLK2/3 ASK1 TAO1/2 TAK1 TAK1 ASK1/2

MAPKKK

RAF1/A/B C-MOS

???

MEKK 2/3

???

MAPKK

MEK 1/2

ERK3K

MEK5

???

MEK 4/7

MEK 3/6

MAPK

ERK 1/2

ERK 3/4

ERK5

ERK 7/8

JNK 1/2/3

p38 α/β/γ/δ

MAPKAPKs (MKs)

RSKs

MNKs

MSKs

MK5

MK2/3

Fig. 4.1 Schematic presentation of the mammalian MAPK pathways. The classical MAPK pathways, represented by the MEK/ERK, JNK, p38 MAPK, and MEK5/ERK5 pathways, consist of a module of three kinases that subsequently phosphorylate and activate each other. The MAPK kinase kinase (MAPKKK or MAP3K) phosphorylates MAPK kinase (MAPKK or MAP2K), which in turn phosphorylates MAPK. Downstream of MAPK are substrates including other protein kinases referred to as MAPK-activated protein kinases (MAPKAPK or MK). The atypical pathways include ERK3, ERK4, ERK7, and ERK8. MAPKs can converge to different MAPKAPK as shown in this figure. ERK7/8 and the JNKs 1–3 do not seem to activate MAPKAPK

44

A. Shiryaev et al.

Studies with knockout mice revealed that Rsk1−/− and Rsk3−/− were viable with no obvious phenotype, while Rsk2−/− mice had impaired learning and cognitive functions, poor coordination, suffered of osteopaenia, and had a specific loss of white adipose tissue. Mutations in the human rsk2 gene are associated with the X-linked mental retardation Coffin-Lowry syndrome [203]. Remarkably, the triple Rsk1−/− /Rsk2−/− /Rsk3−/− knockout mice were viable, while reports on Rsk4−/− mice are lacking so far [4]. The MAPK-interacting or MAPK signal-integrating kinases 1 and 2 (MNK1 and MNK2) were originally identified by screening ERK substrates, but later it was shown that they are activated in vivo by both ERK and p38 MAPK. Two C-terminal different human MNK1 (MNK1a and MNK1b) and two human MNK2 (MNK2a and MNK2b) isoforms, have been identified in human and mouse [18]. The exact in vivo functions of MNK1 and MNK2 are not known because mice deficient in MNK1, MNK2, or both appear normal (reviewed in Gerits et al. [63]). A recent study reported that transgenic mice expressing a constitutive active MNK1 mutant developed tumours rapidly compared to control mice, suggesting that MNK1 can act as an oncogene [216]. MNK1 and MNK2 seem to be involved in anti-apoptotic signalling in response to serum withdrawal as MNK1−/− /MNK2−/− mouse embryonic fibroblasts more readily undergo apoptosis after removal of serum compared to wild-type cells [29]. Identification of substrates proposed the possible involvement of MNK in transcriptional and translational regulation, inflammatory responses, proliferation and survival [18, 61]. Mitogen- and stress-activated protein kinases 1 and 2 (MSK-1 and MSK-2, also termed RSK-B) play a versatile role in transcription and translational regulation, and therefore they can affect inflammatory responses and neuronal processes [5, 205]. The exact physiological roles of MSKs remain obscure as MSKs seem dispensable for mammalian development, and knockout mice do not exhibit an obvious phenotype under normal conditions (reviewed in Gerits et al. [63]). Perturbed MSK activity was reported in two clinical conditions. Enhanced MSK1 and MSK2 activity was detected in lesional psoriatic epidermis compared to non-lesional skin [58], while a deficit in MSK-1 expression was monitored in the striatum of post-mortem Huntington’s disease patients [181]. The molecular mechanisms for MSKs in these disorders are, however, not understood. Mitogen-activated protein kinase-activated protein kinases 2 (MK2) and MK3 possess 75% sequence identity and are directly activated by p38 MAPK (reviewed in Gaestel [59] and Ronkina et al. [176]). Although both enzymes are ubiquitously expressed, the protein and activity levels of MK2 are more prominent than MK3 [176, 177]. MK2 knockout mice are fertile, viable, of normal size and depict no specific behavioural defects, but these animals display increased stress resistance and survive LPS-induced endotoxic shock [109]. Additional studies revealed that MK2 participates in cytokines production, endocytosis, cytoskeleton architecture, cell migration, cell cycle control, cell survival, and transcriptional regulation (reviewed in Gaestel [59] and Ronkina et al. [177]). Moreover, MK2 may play a

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45

contributing role in infections and in inflammatory diseases such as rheumatoid arthritis, atherosclerosis, and asthma [61]. MK3 deficient mice are viable, fertile and possess an apparent wild-type phenotype under normal condition [176]. However, studies in a MK2-deficient background indicated that MK3 can compensate for the loss of MK2 [176]. This suggests that MK2 and MK3 are functionally closely related enzymes. Unexpectedly, MK2/MK3 double knock-out mice showed no obvious embryonic defects and are viable [176]. Mitogen-activated protein kinase-activated protein kinase 5 (MK5) and its human homologue p38-regulated/activated protein kinase (PRAK) were originally described as in vitro p38 substrates that could be activated by p38 through phosphorylation of Thr-182 [143, 144]. However, MK5 seems to be mainly activated by the atypical ERK3 and ERK4 MAPK [1, 99, 186, 188], while the connection with p38 remains controversial [59]. The biological functions of MK5 are poorly understood, as MK5 knockout mice bred onto different backgrounds display either no obvious phenotype or are embryonic lethal [59]. Recent studies demonstrated that PRAK/MK5 is involved in ras-induced senescence, tumour suppression, inhibition of cell proliferation, rearrangements of the cytoskeleton, and anxiety-related behaviour [64, 65, 108, 116, 198].

4.2 MAPKAPK and Cancer A causative role for several components of the MAPK signalling pathways in cancer is well documented [36, 43, 44, 124, 173, 220]. Far less investigation has been performed on the putative contribution of MAPKAPK in oncogenesis, but these kinases contain properties of either oncogenes or tumour suppressors. In this section we will briefly outline some of these oncogenic properties of the MAPKAPK, while in Section 4.3 we will focus on the implications of MAPKAPK in metastasis.

4.2.1 Mutations in the MAPKAPK-encoding Genes in Cancer Tissue Screening the human kinome, i.e. the complete set of protein kinase genes in the genome, for somatic mutations in different cancers, including breast, lung, colorectal, gastric, testicular, ovarian, renal, melanoma, glioma, and acute lymphoblastic leukaemia revealed single nucleotide substitutions in the genes for the MAPKAPs (see Table 4.1). The impact of these mutations on the functions of the MAPKAPK enzymes and the prevalence of these mutants in cancer tissue have not been investigated in detail, nor has a causative role in tumorigenesis or metastasis been established.

RPS6KA1 RPS6KA3

RPS6KA2

RPS6KA6

RPS6KA5 RPS6KA4

RPS6KA5 MKNK1 MKNK2 MAPKAPK2 MAPKAPK3

MAPKAPK5 12q24.12-q24.13

RSK1 RSK2

RSK3

RSK4

MSK1 MSK2, RSKB

MSK1 MNK1 MNK2 MAPKAPK-2, MK2 MAPKAPK-3, MK3

MAPKAPK-5, MK5, PRAK

None c.1246A>G c.1822C>T (hom) c.1448A>G c.931G>A c.1280C>G c.1581+1G>A (IVS16+1 G>A) c.2195G>A c.772T>A c.419A>G none c.707C>T c.1137_1138GG>TT None None None None c.82C>T c.314A>C None

Mutation

P28S p.E105A

p.S236L p.L379_E380>Lstop

p.R732Q p.S258T p.Y140C

p.I416V p.L608F p.Y483C p.E311K p.S427stop

Amino acid change

Brain Ovary

Breast Lung

Breast Brain Stomach Skin Lung Ovary Large intestine LUNG Lung

Cancer tissue

[70, 86] [70, 86]

[70, 193] [70]

[225] [70, 86] [70] [70] [39, 70] [70] [70] [39, 70] [39, 70]

Reference

Nucleotide changes are represented as “c.” indicating that nucleotide +1 in the coding DNA reference sequence is the A of the ATG translation initiation codon. A “p.” is used to indicate description of amino acid change at the protein level, meaning that the translation initiator Methionine is numbered as +1. Amino acid changes are indicated by the one letter code. “Stop” represents nonsense mutation.

14q31-q32.1 1p33 19p13.3 1q32 3p21.3

14q31-q32.1 11q11-q13

Xq21.1

6q27

1p Xp22.2-p22.1

Gene name

Protein

Chromosome location

Table 4.1 Somatic mutations in genes encoding MAPKAPK

46 A. Shiryaev et al.

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4.2.2 MAPKAPK Expression Levels in Cancers RSK1 and 2 protein levels are often upregulated in prostate and breast cancer compared to normal tissues [32, 192]. On the other hand, RSK3 expression is reduced or absent in ovarian tumours and cell lines, suggesting a role as tumour suppressor [14]. The activities of MNK1 and MNK2 are enhanced in certain breast cancer cell lines that overexpress HER2 [30]. Increased phosphoMK2 and phosphoMK3 levels have also been reported in thyroid carcinomas compared to normal tissue [162], while the expression level of MK5 in ovary, prostate and stomach tumour tissue was elevated compared to levels in normal tissue [66]. The biological implication of anomalous MAPKAPK expression in these cancers remains unclear.

4.2.3 MAPKAPK and Cell Cycle One hallmark of cancer cells is uncontrolled cell cycle regulation [75]. Some MAPKAPK have the potential to interfere with the cell cycle. Inhibition of RSK by the specific inhibitor SL0101 and depletion of RSK1 or RSK2 produced a block in the G1 phase of the cell cycle in mammalian cells [23, 192], while rsk2−/− mouse embryonic fibroblasts accumulate at the G1 phase [28]. RSK3 can reduce cell proliferation and cause G1 arrest in ovarian cancer cells, suggesting a role as tumour suppressor [14]. Depletion of RSK4 strongly reduced the mRNA levels of p21Cip1 and abolished p53-dependent G1 cell arrest induced either by conditional activation by p53 or by DNA damage via ionizing irradiation [11]. On the other hand, Thakur et al. [201] reported that overexpression of RSK4 in the human breast cancer cell line MDA-MB-231 suppressed cell proliferation, triggered accumulation in G0 /G1 phase, and enhanced expression of p21Cip1 . RSK can affect the cell cycle progression through several mechanisms, including controlling the activity of the cell cycle regulator proteins Bub1, Emi2, Cdc25, LKB1, c-FOS, cyclindependent kinase (CDK) inhibitor p27Kip1 and Myt1 through direct phosphorylation, and through stimulation of cyclin B synthesis (reviewed in Kostenko et al. [107]). A role for MSK in cell proliferation derives from studies using the MSK inhibitor Ro31-8220 [185] or knockdown of MSK1 [103], but the molecular mechanisms remain elusive. A few reports suggest a possible involvement of MNKs in cell cycle regulation and cancer. Treatment of the AU565 breast cancer cell lines, which displays enhanced MNK activity, or prostate PC3 cells with the MNK inhibitor CGP57380 (see Table 4.2) prevented colony formation or decreased the rate of proliferation [13, 30], while dominant negative MNK1 restrained the growth of leukaemia cells [30]. Mice transplanted with hematopoietic stem cells expressing activated MNK1 displayed accelerated tumorigenesis compared to mice reconstituted with cells expressing kinase-dead MNK1 [216]. Another group found that overexpression of a dominant negative MNK1 inhibited cell proliferation [216, 226]. All these observations point to a role of MNK in cell cycle regulation, but the

MSK IC50 = 8 nM IC50 = 8.3 μM for MSK1 IC50 = 5 μM for MSK1 60% inhibition with 50 μM 91% inhibition with 0.1 μM IC50 = 1.8 nM

Ro 318220 Y 27632 HA1077 Flavokavain A

A-443654

(1H-Imidazo [4,5-c]pyridin-2-yl)-1,2, 5-oxadiazol-3-ylamine compound 27

IC50 = 2.8 mM for RSK2 IC50 = 3 mM for RSK2 IC50 = 19 μM for RSK2 IC50 = 15 μM for RSK2 60% inhibition at 10 μM

H89 Ro 318220 Y 27632 HA1077 SB415286 IC50 = 120 nM

73% at 10 μM

SU 6656

H89

Aurora B, PIM3 Lck, Src, S6K1 Aurora B, PLK1, MELK, MST2 AMPK, Aurora B, Aurora C, BRSK2, CaMKKα, CaMKKβ, CHK2, DYRK1A, LCK, MST2, PHK, Src, SRPK1 PKA S6K1,MSK1, PKCα MSK MSK1,ROCK-II,PRK2 CaMKKβ, ERK8, GSK3β, DYRK1A, HIPK2, PIM3, SRPK1 PKA, S6K1, ROCK-II, PKBα, RSK2 S6K1, RSK2, PKCα RSK2 RSK2, ROCK-II, PRK2 Aurora B, Dyrk1A, IKKβ, MK3, MK5 DYRK1A, PKA, PKBα, PKBβ, PRK2 Not tested

IC50 = 89 nM IC50 = 15 nM 99% at 0.1 μM

SL0101 Fmk BI-D1870

RSK

Other targets

Inhibitory activity

Compound

MAPKAPK

Table 4.2 MAPKAPK inhibitors, their specificities, and use in clinical trial

No

No

No No No No

No

No No No No No

No

No No No

Clinic trials

[9]

[7]

[38] [38] [38] [57]

[5, 38]

[5, 38] [38] [38] [38] [7]

[7]

[7, 192] [7, 34] [7, 183]

References

48 A. Shiryaev et al.

MK5

MK3

MK2

IC50 = 2.5–10 μM 96% inhibition with 50 μM

Pyrranopyridine derivative Flavokavain A IC50 = 1.9 μM 90% inhibition with 10 μM 91% inhibition with 50 μM

Rottlerin EGCG

Flavokavain A

Aurora B, Dyrk1A, IKKβ, MK3, MSK1

MK3 and MK5 at 10x higher concentrations Not tested Aurora B, Dyrk1A, IKKβ, MK5, MSK1 MK2 DYRK1A

IC50 = 5.4 μM Not tested IC50 < 10 nM

BX 795

Rottlerin MK2i (KKKALNRQLGVAA peptide) Pyrrolopyridine derivatives

65% inhibition with 1 μM (MNK1)

CGP 57380 (4-amino-5-(4-fluoroanilino)pyrazolo[3,4-d]pyrimidine) CK1–7 AMPK,CK1δ, ERK8, PIM1, PIM3, SGK1 Aurora B, Aurora C, ERK8, IKKε, TBK1, MARK3, PDK1 MK5 Not tested

81% inhibition with 1 μM

Compound C

74% inhibition with 25 μM (MNK1) 81% inhibition with 0.1 μM (MNK2)

AMPK, CaMKKα, CaMKKβ, CK2, DYRK2, DRK3, ERK8, HIPK2, PIM2, PIM3, AMPK, DYRK1A, DYRK2, DYRK3, ERK8, HIPK2, MELK, PIM2, PIM3 BRSK2, CK1δ

90% inhibition with 1 μM

ST0609

MNK

Other targets

Inhibitory activity

Compound

MAPKAPK

Table 4.2 (continued)

No Phase Ib, Phase II No

No No

No

No No

No

No

No

No

No

Clinic trials

[57]

[38] [6, 33]

[40] [57]

[3]

[38] [125]

[7]

[7]

[7, 104]

[7]

[7]

References

4 Mitogen-Activated Protein Kinase-Activated Protein Kinases and Metastasis 49

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A. Shiryaev et al.

precise molecular mechanism by which MNK affects the cell cycle is not known. However, phosphorylation of eIF4E by MNK was required for cell cycle progression [13, 216], indicating the involvement of eIF4E. Therefore, overexpression of a dominant negative MNK1 that lacks kinase activity may result in insufficient eIF4E phosphorylation and cell cycle arrest, while cells displaying enhanced MNK activity prevail over cell cycle control. Moreover, inhibition of MNK1 with CPG57380 resulted in selective downregulation of mRNAs encoding proteins involved in cell cycle progression such as CDK2, CDK8 and CDK9 [13], underscoring a role for MNK in cell cycle regulation. Early observations revealed that growth-factor induced proliferation coincided with strong increase in MK2 activity, while MK2 could inhibit Ras-induced proliferation of NIH3T3 cells, suggesting a role for MK2 in cell cycle progression [25, 129, 168]. Cells treated with siRNA targeting MK2 eliminated G1 arrest and G2 /M and S phase checkpoints [130]. MK2 interferes with cell cycle progression through phosphorylation of Cdc25 and as such controlling the activity of check point kinases Chk1 and Chk2 [122, 130, 170, 227] or by modulating the stability of p53 by phosphorylating MDM2 [215]. Overexpression of MK3 resulted in de-repression of the gene encoding p14/p19ARF , which induced both G1 and G2 arrest, and slowed down cell cycling [210]. Wild-type MK5, but not a kinase dead mutant could prevent p21RAS -induced proliferation in NIH3T3 cells [25]. MK5-mediated inhibition of proliferation may depend on MK5’s ability to phosphorylate and thereby stimulate the transcriptional activity of p53, and as a consequence increase the expression of the cyclin-dependent protein kinase inhibitor p21Cip1/Waf1 , a target gene of p53 [116, 198].

4.2.4 MAPKAPK and Cell Survival RSKs positively regulate cell survival in different cell types by phosphorylation/inactivation of pro-apoptotic proteins such as BAD and DAPK (reviewed in Anjum and Blenis [4]). Also MSK and MNK are involved in cell survival, but the mechanisms are incompletely understood (reviewed in Arthur [5] and Buxade et al. [18]). MK2 deficient mouse cell show increased apoptosis after UV irradiation [215], while neurons from MK2−/− mice were more resistant to lipopolysaccharideinduced apoptosis [202]. A plausible role of MK3 and MK5 in apoptosis has not been described so far.

4.3 MAPKAPK and Metastasis Metastasis is a complicated process that allows tumour cells to abandon their place of origin and to colonize at distant organs. MAPKAPK may contribute to metastasis by affecting cell motility through regulating the activity of cytoskeleton modelling proteins in a phosphorylation-dependent manner. Moreover, MAPKAPK have been

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shown to regulate the expression of several proteins implicated in metastasis. These two mechanisms will be discussed in this section.

4.3.1 MAPKAPK and Regulation of the Cytoskeleton The cytoskeleton of human cells is composed of three classes of proteins comprising actin microfilaments, tubulin microtubules, and intermediate filaments [68, 151, 152]. Reorganization of the cytoskeleton is required in many cellular processes including alteration of cell shape, movement of organelles, endo- and exocytosis, cell division, extracellular matrix assembly, and cell migration [115, 118, 160, 208]. The latter process occurs during embryogenesis, immune response, and tissue repair and regeneration. However, abnormal cell migration contributes to disease progression such as cancer invasion and metastasis and requires reorganization of the cytoskeleton, including the formation of protrusive structures such as filopodia, lamellipodia and invadopodia [101, 128, 230]. 4.3.1.1 HSP27 Actin remodelling, including variety of length and spatial conformation is conferred by the action of actin-binding proteins [194]. The MAPKAPK MK2, MK3, and MK5 all seem to be able to affect actin remodelling and cell migration by targeting actin-binding proteins. The mechanisms by which these MAPKAPK affect actin rearrangements will be outlined here. Actin filaments consist of growing ends (so called barbed or “+” ends) and pointed or “−“ ends (Fig. 4.2a). One of the actin-binding proteins is the small heat shock protein 27 (HSP27) [135]. In its unphosphorylated form, HSP27 binds to barbed ends of polar actin microfilaments thereby inhibiting actin polymerization (Fig. 4.2b). Phosphorylation of HSP27 results in release of HSP27 from the barbed ends and concomitantly enhances reorganization of F-actin [117]. MK2, MK3 and MK5 are genuine HSP27 kinases that are implicated in F-actin remodelling and cell motility (reviewed in Kostenko and Moens [106]). Interestingly, HSP27 and phosphoHSP27 levels are enhanced in many tumours compared to healthy tissue [20], suggesting that increased (phospho)HSP27 levels may be implicated in invasiveness. MK5 can also affect HSP27-mediated F-actin polymerization and cell migration through an indirect mechanism requiring 14-3-3 proteins [199]. The group of Gaestel showed that phosphorylated HSP25, the murine homologue of HSP27, can interact with the seven known isoforms of 14-3-3 proteins (β, γ, ε, ν, σ, η, and ζ) in vitro [206]. This may protect phosphoHSP27 against protein phosphatases. Overexpression of MK5 in HeLa cells stimulated motility. This MK5-induced cell migration was inhibited by overexpression of 14-3-3ε protein which was shown to bind MK5 in vivo and to inactivate MK5 [199]. Hence, MK5 can extricate 14-3-3ε from phosphoHSP27 resulting in free phosphoHSP27 which can be dephosphorylated by protein phosphatases. Non-phosphorylated HSP27 will bind the barbed ends of F-actin and prevent polymerization.

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F F-actin polymerization

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Fig. 4.2 Regulation mechanisms of actin polymerization. (a) New G-actin monomers are added at the barbed or plus end of the growing filamentous F-actin chain, while depolymerisation occurs at the pointed or minus ends. (b) Spontaneous assembly of actin in vivo is prevented by monomer sequestering proteins and capping proteins. HSP27 behaves in its unphosphorylated form as an actin cap-binding protein and can inhibit actin polymerization. Phosphorylation of HSP27, e.g. by the MAPKAPK MK2, MK3, or MK5, increases the rate and extent of actin polymerization. Protein phosphatase 2A has been shown to act as an in vivo HSP27 phosphatase (reviewed in Kostenko and Moens [106]). (c) Cofilin can increase the number of barbed ends by severing existing filaments. (d) The ARP2/3 complex allows branching of actin filaments. MK2 can phosphorylate the p16-Arc subunit of the ARF2/3 complex. (e) MK5, HSP27 and cofilin can all bind 14-3-3ε. Phosphorylated cofilin and phosphorylated HSP27 bind 14-3-3ε which protects them from dephosphorylation. MK5 may usurp 14-3-3ε, releasing 14-3-3ε from cofilin and HSP27 and subsequently allowing dephosphorylation of these proteins. Dephosphorylation of cofilin leads to activation and hence severing of F-actin, while dephosphorylation of HSP27 will prevent F-actin polymerization as depicted in (b). (f) The CapZ-CapZIP complex prevents F-actin polymerization by binding the barbed ends. MK2-mediated phosphorylation of CapZIP releases the CapZ–CapZIP complex from F-actin allowing polymerization to occur

4.3.1.2 Cofilin Another actin-binding protein whose activity may be regulated by MAPKAPs is cofilin. Cofilins are small proteins (15–20 kDa) that in their unphosphorylated (i.e. active) state increase actin dynamics by depolymerizating filaments from their pointed ends (Fig. 4.2c). They are essential regulators of actin dynamics during cell migration by severing existing actin filaments. This increases the number of available barbed (+) ends allowing rapid actin polymerization and enhances the rate of F-actin depolymerization, allowing recycling of G-actin [42, 101, 152]. Inhibition of cofilin activity in carcinoma cells either by siRNA-mediated depletion or by constitutive phosphorylation due to overexpression of the cofilin kinase LIM domain kinase (LIMK) prevented cell motility, while increased cofilin levels are detected in invasive subpopulations of tumour cells (reviewed in Wang et al. [212], Yamaguchi and Condeelis [230], and Kelley et al. [101]). These finding underscore that cofilin activity is required for invasion. The activity of cofilin is regulated by

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phosphorylation. Phosphorylation of cofilin leads to inactivation of the protein and decreases its association with actin. In its phosphorylated state, cofilin can bind 14-3-3 proteins, thereby protecting itself from protein phosphatases (Fig. 4.2e). Phosphorylated cofilin 1 and cofilin 2 were found to interact with 14-3-3ε [123], and this prevents dephosphorylation by protein phosphatases [67]. Although cofilin 1 and 2 are not direct substrates for MAPKAPK, they may be indirectly controlled by MK5. MK5 is able to bind 14-3-3ε in vivo [199] and may thus usurp 14-3-3ε, allowing dephosphorylation and activation of cofilin 1 and 2, along with F-actin rearrangements (Fig. 4.2e). In conclusion, abnormal MK5 expression or activity in neoplastic cells may stimulate dynamic F-actin remodelling and promote cell migration through direct phosphorylation of HSP27 or/and through affecting the phosphorylation/activity state of the F-actin remodelling proteins HSP27 and cofilin by sequestering 14-3-3ε. 4.3.1.3 Arp2/3 Several other proteins involved in F-actin architecture are substrates for MAPKAPK. MK2 interacts and phosphorylates the A isoform of the p16 subunit (p16-Arc) of the actin-related protein-2/3 (Arp2/3) complex at Ser-77 [191]. As this complex controls actin nucleation and branching (Fig. 4.2d), and actin-based motility by driving the formation of lamellipodia [161, 127], MK2 may affect metastasis through phosphorylation of p16-Arc A. However, further studies are necessary in order to unequivocally determine if phosphorylation of p16-Arc by MK2 regulates cellular functions dependent on the actin cytoskeleton [191]. 4.3.1.4 CapZIP CapZ, a heterodimer composed of two subunits CapZa and CapZb, is thought to regulate actin filament assembly and organization by binding the barbed ends of the filaments [21]. CapZ-interacting protein (CapZIP) can be phosphorylated at Ser-179 and Ser-244 by MK2 or MK3 in vitro, while phosphospecific antibodies confirmed MK2/MK3-mediated phosphorylation of Ser-179 in cell culture [54]. CapZIP may thus in a phosphorylation dependent manner affect the ability of CapZ to remodel actin filament (Fig. 4.2f). Indeed, phosphorylation of CapZIP terminates actin filament elongation [194]. Hence, perturbed CapZIP phosphorylation by aberrant MK2 or MK3 activity may influence the cytoskeleton and cell migration. Experimental proof comes from studies with chemokines. These are cytokine-like proteins that regulate leukocyte transport by mediating adhesion of leukocytes to endothelial cells, initiation of transendothelial migration, angiogenesis, and tissue invasion [17, 22, 238]. Several results indicate that tumour cells during the process of metastasis might use chemokine-mediated mechanisms that are similar to those for regulating leukocyte transport [136, 137]. In particular, Muller and colleagues have shown that leukocyte chemoattractant receptor CXC chemokine receptor 4 (CXCR4) and its ligand CXC chemokine ligand 12 (CXCL12 or stromalcell-derived factor 1; SDF-1) govern the pattern of breast-cancer metastasis in a

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mouse model. Binding of CXCL12 to CXCR4 receptor activates several signalling cascades, including the p38 MAPK pathway [97, 174]. Rousseau et al. [179] delineated the p38 MAPK-dependent pathway by which chemotactic agents stimulate cell migration. They found that ligand binding to the CXCR4 receptor elicits the sequential activation of PAK1/PAK2, p38α MAPK and MK2, which then may phosphorylate several substrates like CapZIP, HSP27 (3.1.1), and LSP1 (see Section 4.3.1.5). Signalling of two other chemokines, CXCL4 and CXCL10, via the CXCR3-B receptor increased the enzymatic activity of p38 MAPK and coincided with MK2-mediated phosphorylation of HSP27. The CXCR3-B isoform is the chemokine receptor responsible for inhibition of microvascular cell growth [121], and p38 MAPK is a signalling pathway downstream of CXCR3-B, that contributes to mediating the angiostatic effects of CXCL10 and CXCL4 [159]). The biological relevance of CXCL4/10-CXCR3-B-p38MAPK-MK2-HSP27 connection for metastasis remains unexplored. 4.3.1.5 Lymphocyte-Specific Protein 1 The F-actin bundling cytoskeletal protein lymphocyte-specific protein 1 (LSP1) is involved in cell migration and is a genuine MK2 substrate [95]. The gene encoding LSP1 is a breast cancer susceptibility gene [62], but whether anomalous LSP1 phosphorylation by MK2 may be a contributing event in breast cancer metastasis merits investigation. 4.3.1.6 Filamin A The cytoskeletal protein filamin A, a membrane-associated protein that cross-links actin filaments, is essential for cell locomotion [55]. Previous studies demonstrated that filamin A phosphorylation at serine-2152 is required for p21-activated kinase 1 (Pak1)-mediated membrane ruffling as mutation of this residue into alanine prevented cell shape changes. RSK1 and RSK2 can also phosphorylate filamin A at serine-2152 in vivo and elicit migration of human melanoma cells [224]. Strikingly, inhibition of RSK caused a 60% decrease in filamin A-dependent migration of human melanoma cells, while human melanoma cell lines that do not express detectable filamin A levels did not migrate [19, 224]. Hence RSK may play an essential role in cell motility and aberrant RSK expression or activation may be implicated in tumour cell invasiveness. 4.3.1.7 5-Lipoxygenase 5-lipoxygenase (5-LO) is a key enzyme in the biosynthesis of proinflammatory leukotrienes [165]. 5-LO can form complexes with cytoskeletal proteins, including actin [98] and coactosin-like protein, which is a member of the ADF/cofilin group of actin-binding proteins [166]. MK2 and probably MK3 can phosphorylate 5-lipoxygenase in vitro, and this modification potentiated the enzymatic activity of

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5-LO [217]. However, a possible relationship between MK2/3-mediated phosphorylation of 5-LO and its interactions with the cytoskeleton, effect on cell motility, and metastasis in malignant conditions has not been established yet. 4.3.1.8 Vimentin Vimentin, a major intermediate filament protein, acts as an organizer of proteins involved in migration and cell adhesion [91]. Vimentin levels are upregulated in several tumours and RNA interference studies or the use of inhibitors and dominant-negative vimentin mutants that disrupt vimentin filaments support a role for vimentin in metastasis [79, 219]. Phosphorylation induces disassembly of vimentin and structural changes of the intermediate filament network. Although phosphorylation of vimentin may be involved in invasiveness and metastasis, the exact molecular mechanisms remain to be elucidated [90, 92, 190]. MK2 can phosphorylate vimentin at Ser-38, Ser-50, Ser-55, and Ser-82, but while phosphorylation by other kinases correlated with disassembly of vimentin filaments, phosphorylations by MK2 retained vimentin’s ability to form filament, at least in vitro [26]. The authors suggested that MK2 might work synergistically with other kinases to disrupt intermediate filaments. Whether MK2-mediated phosphorylation of vimentin kindles the invasiveness of neoplastic cells remains to be determined. Vimentin has also been shown to interact with 14-3-3ε, but the functional importance is not completely solved [184]. Hypothetically, MK5 may sequester 14-3-3ε, but the consequence for vimentin’s role in metastasis remains elusive. 4.3.1.9 Myosin Myosins are a family of motor proteins that hydrolyse ATP to drive movements along actin filaments. Myosins typically contain light chains (MLC) and heavy chains [111, 145, 187]. The MLC of myosin type II has been shown to be in vitro substrates of MK5 [144], and RSK-2 [197]. Phosphorylation of the MLC is a key mechanism for regulation of actin-myosin contractility and anomalous MLC phosphorylation may contribute to metastasis (reviewed in Olson and Sahai [149]). However, a biological implication of MK5- or RSK-2-mediated phosphorylation of MLC in metastasis remains to be determined In conclusion, aberrant activity of MAPKAPK may interfere with reorganization of the cytoskeleton and cell motility, and hence promote cell migration/metastasis.

4.3.2 MAPKAPK and Gene Regulation Some of the MAPKAPK substrates are transcription factors [5, 18, 23, 59, 205], enabling MAPKAPK to influence the expression of genes, including genes whose products are implicated in metastasis of tumour cells. MAPKAPK not only exert their effect at the transcriptional level by modulating the activity of transcription

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factors, but can also regulate at the post-transcriptional levels by affecting the stability of mRNA. Examples of both mechanisms are discussed below. 4.3.2.1 E-cadherin In many primary tumours with invasive properties, intercellular adhesion is reduced, often because of the loss of E-cadherin, a critical component of cell-cell adhesive interactions [158]. E-cadherin is a calcium-dependent transmembrane protein (for recent review see Baranwal and Alahari [10]). The cytoplasmic tail of E-cadherin is tethered, via α-catenin and β-catenin, to the actin cytoskeleton. Loss of E-cadherin expression is a requisite for cell de-adhesion and migration during the epithelial to mesenchymal transition and is common in carcinomas [200]. One of the regulatory pathways of the E-cadherin expression involves MSK1. Ordóñez-Morán and colleagues have shown by the use of chemical inhibitors, dominant-negative mutants and small interfering RNA, that the activity of RhoA-ROCK, and p38MAPKMSK1 activation is necessary for the induction of CDH1/E-cadherin transcription and acquisition of an adhesive epithelial phenotype, and moreover, for the inhibition of β-catenin-TCF transcriptional activity by 1,25(OH)2 D3 (active vitamin D metabolite). They have confirmed that 1,25(OH)2 D3 increased the level of active, phosphorylated p38MAPK and MSK1 without affecting their total cellular content in colon cancer cells [150]. Furthermore, 1,25(OH)2 D3 increased phosphorylation of the transcription factors cAMP response element-binding protein (CREB) and activating transcription factor 1 (ATF1), that are MSK1 substrates [221]. Finally, the finding that knockdown of MSK1 or MSK2 by siRNA decreased the induction of E-cadherin is further evidence that these kinases mediate 1,25(OH)2 D3 action [150]. Additional studies are necessary to identify the particular role of transcription factors CREB and ATF1 in transcription of E-cadherin. 4.3.2.2 CD44 CD44 is a transmembrane glycoprotein that binds hyaluronic acid and that mediates adhesion of leukocytes and activation of T-cells [76, 141, and references therein]. CD44 is also the receptor for matrix metalloproteinase 9 (see Section 4.3.2.4) and plays an important role among other cell-cell and cell-ECM adhesion molecules employed by cancer (for review see Jothy [96]). Originally described as a protein associated with the activation and homing of lymphocytes, CD44 became relevant to metastasis by the study showing its involvement in the metastatic dissemination of pancreatic cancer cells in a rat model [71] and due to its involvement in both cell-cell and cell-ECM adhesion processes [15, 138]. Many cancer cell types as well as their metastases express high levels of CD44 [213, 232]. Robbins and coworkers proposed that in androgen-independent prostate cancer cells the ERK1/2 and p38 pathways converge to MNK1, which enhances transcription of CD44 [172]. This suggests that MNK1 may play a role in metastasis of certain prostate cancers through stimulating the expression of CD44. Further efforts are needed to describe the full pathway of CD44 transcription regulation.

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4.3.2.3 Urokinase Plasminogen Activator The serine protease urokinase plasminogen activator (uPA), when bound to its specific cell surface receptor uPAR, initiates the activation of metalloproteinases and efficiently converts plasminogen to the active serine protease plasmin. Activated plasmin in turn initiates the destruction of various extracellular matrix proteins [154]. In particular, uPA was shown to be involved in tissue remodelling events in non-pathological conditions. For instance, a coordinated expression of uPA and uPAR is required for trophoblast implantation [102]. Substantial evidence has been obtained that uPA and uPAR are overexpressed in malignancies including breast, ovary, and prostate tumours, and this is associated with disease progression, metastasis and poor prognosis [50, 80]. MK2 can increase the expression of uPA at the post-transcriptional level [74, 83]. In their work, Huang and colleagues showed that p38 MAPK activity had little effect on uPA and uPAR promoter activities, suggesting that p38 MAPK signalling pathway is not required for uPA and uPAR transcription. However, administration of the potent p38 MAPK inhibitor SB203580 reduced the half-life of uPA and uPAR transcripts from over 12 h to 2–3 h in BT549 cells, demonstrating that p38 MAPK activity is required for the stability of uPA and uPAR mRNA [83]. It is well known that adenosine/uridine-rich elements (ARE) in 3 -untranslated region (UTR) of mRNAs can affect the stability of transcripts significantly [49]. The 3 -UTR of uPA mRNA contains a minimum ARE consensus sequence [140] and previous studies have pointed to a role of MK2 in stabilization of cytokine mRNA stability through phosphorylation/inactivation of the ARE-destabilizing protein tristetraprolin [81, 142, 223]. Using β-globin reporter gene constructs containing uPA mRNA 3 -UTR or ARE-deleted 3 -UTR, it was demonstrated that p38 MAPK/MK2 signalling pathway regulates uPa mRNA stability through a mechanism involving the ARE [74]. In conclusion, anomalous MK2 activity in tumour cells may cause enhanced uPA protein levels due to stabilization of uPA transcripts and this may promote metastasis. The observation that inhibition of p38 MAPK concomitantly reduced VEGF-induced migratory ability of human umbilical embryonic cells and uPA expression underscores the MK2-uPA link in metastasis [234]. The expression of uPA seems also to be regulated by MSK1. MSK1-induced expression occurs at the transcriptional level as a result of chromatin remodelling induced by histone H3 phosphorylation by MSK1 [195].

4.3.2.4 Matrix Metalloproteinases 2 and 9 Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that play a major role in remodelling of the extracellular matrix [134]. Aberrant MMP activity is associated with pathogenic processes, including invasion and metastasis [41]. Indeed, overexpression of many MMPs, including MMP-1, -2, -7, -9, -13, and -14 is positively associated with tumour progression and metastasis [41]. Zhang and colleagues reported that VEGF binding to its receptor Flt-1 (VEGFR-1) but not KDR (VEGFR-2) triggered the expression of MMP2 and MMP9 through the p38 MAPK/MK2 signalling pathway [236]. The fine mechanism by which MK2

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influences MMP2 and MMP9 expression was not addressed, but as a consequence tumour cells with perturbed MK2 activity may be more invasive. 4.3.2.5 Transforming Growth Factor β Transforming growth factor β (TGFβ), a 25 kDa homodimeric extracellular cytokine, is an important regulator of cell adhesion and motility in a variety of cell types [189]. It contributes to tumour progression by inducing an epithelial to mesenchymal transition (EMT) and cell migration [8]. Work by Xu and coworkers showed that TGFβ signals through p38 MAPK and MK2. TGFβ-activated MK2 can phosphorylate HSP27 (see Section 4.3.1.1) and subsequently stimulate MMP2 (see Section 4.3.2.4), and promoted cell invasion in human prostate cancer [229]. The precise mechanism of MMP2 activation by HSP27 remains to be solved, but it is tempting to speculate that signal transducer and activator of transcription 3 (STAT3) is involved. STAT3 is known to be implicated in cell invasion and migration and to upregulate MMP2 transcription via direct interaction with the MMP2 promoter [228]. Moreover, HSP27 physically interacts with STAT3 [175]. A plausible scenario could be following: TGFβ activated the p38MAPK/MK2 pathway resulting in HSP27 phosphorylation. Phospho-HSP27 binds to and potentiates the transcriptional activity of STAT3, which increases MMP2 expression. Similarly, the p38 MAPK/MK2/HSP27 pathway was described for growth arrestspecific gene 6 (GAS6)-induced cytoskeletal reshuffling and migration. GAS6, which binds to the Ark (adhesion-related kinase) receptor, has growth-factor like properties [72]. Stimulation of immortalized gonadotropin-releasing hormone neuronal cells with GAS6 resulted in increased MK2 activity, HSP27 phosphorylation, and actin cytoskeletal reorganization and migration, while inhibition of p38MAPK with SB203580 or overexpression of dominant negative p38α blocked GAS6/Ark-mediated migration of cells [2].

4.3.2.6 Chemoattractant Receptors Thakur and colleagues discerned that overexpression of RSK4 in highly invasive and metastatic MDS-MB-231 breast cancer cell line resulted in significantly reduced chemotaxis against CXCL12 as well as a significantly reduced chemoinvasion together with suppression of soft agar colony formation [201]. Even more important, when RSK4-overexpressing cells or vector control cells were injected into the mammary fat pads of female SCID mice, animals injected with RSK4-overexpressing cells showed much smaller tumour size with pseudocapsule, indicative of noninvasive growth, whereas mice with vector control cells had higher volume and weight and showed penetrating growth in stromal tissue. The precise mechanism of RSK4 inhibition of invasion is not clear, but overexpression of RSK4 led to reduced expression of CXCR4 and increased expression of Claudin2 (CLDN2) proteins and vice versa after silencing of RSK4 [201]. CLDN2 is a transmembrane protein essential for tight junctions of epithelial tissue, and loss or reduced expression of it has been

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implicated in EMT and metastasis [146]. RSK4-mediated CLDN2 expression may therefore hamper metastasis. Another chemokine receptor, CCR7, is important for lymphatic invasion of cancer cells and is overexpressed in metastatic breast cancer cells [136]. MSK1 has been shown to regulate the expression of CCR7 and to reverse the inhibitory effect of the plant steroidal lactone tubocapsanolide A on migration of MDA-MB-231 cells [153].

4.3.2.7 Human Epidermal Growth Factor Receptor 2 Human epidermal growth factor receptor 2 (HER2; also known as ErbB-2 or c-neu) is amplified and/or overexpressed in ∼20–30% of breast cancer patients [82]. Patients having elevated HER2 protein levels exhibit poor prognosis as overexpression of HER2 is associated with aggressive tumour growth and metastatic activity [88, 237]. Increased phosphorylation and activity of the MNKs correlate with HER2 overexpression in certain breast cancer cell lines, and inhibition of the MNKs reduces colony formation in soft agar suggesting that MNKs may have a role in proliferation or invasiveness of breast cancer [30]. The exact role of MNK in metastasis of HER2 positive tumours and the mechanism by which MNK regulates HER expression necessitates further investigation.

4.3.2.8 Cyclooxygenese-2 Cyclooxygenase (COX) is the rate-limiting enzyme for the production of prostaglandins and thromboxanes from free arachidonic acid. Two COX isoenzymes have been described: COX-1, which is constitutively and ubiquitously expressed, and the inducible COX-2 [100]. Mounting evidence documents increased levels of COX-2 in carcinomas of the colon, stomach, breast, oesophagus, lung, liver, and pancreas [24, 87, 147, 182]. In contrast, the levels of COX-1 are mostly similar in normal and tumour tissues [182]. Elevated expression of COX-2 and its main product prostaglandin E2 has been shown to increase tumour invasiveness and enhance metastatic potential [47, 48, 222, 233]. COX-2 is regulated by both transcriptional and post-transcriptional mechanisms. ARE within the 3 -UTR of COX2 mRNA can affect both mRNA stability and protein translation [45]. It was originally shown that COX-2 mRNA stability is regulated in part by p38MAPK pathway [78, 93]. Later studies suggest that p38MAPK-induced stabilization of the COX-2 transcripts is mediated by MK2 [196] and probably involves a similar mechanism as for uPA (described in Section 4.3.2.3). The RNA-binding protein heterogeneous nuclear ribonucleoprotein A0 (hnRNP A0) can be phosphorylated by MK2 at Ser84 in vitro and this residue became phosphorylated in LPS-stimulated cells leading to its binding to ARE and stabilization of specific mRNAs, including COX-2 [178]. Alternatively, HSP27 may be involved in COX-2 mRNA stabilization, as β-globinCOX-2 transcripts were partially stabilized by constitutively active MK2 or by a phosphomimicking HSP27 mutant [120].

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Both RSK and MSK can stimulate COX-2 expression at the transcription level [52, 155]. Eliopoulos et al. showed that LPS enhanced COX-2 mRNA levels in an RSK1- and MSK1-dependent manner, while Pathal et al. demonstrated upregulation of COX-2 transcript by activation of the Toll-like receptor 2 (TLR2), and subsequent signalling through ERK and p38 MAPK which both converge to MSK1. MSK phosphorylates CREB [5], which is a key modulator of COX-2 transcription [204]. The TRL-MSK-COX-2 pathway may be relevant for metastasis because TRLs are expressed on a wide variety of tumours and TLRs activation not only promotes tumour cell proliferation and resistance to apoptosis, but also enhances tumour cell invasion and metastasis. The latter processes involves upregulating MMPs and integrins (for recent review see Huang et al. [84]), but might also involve MAPKAPK-regulated transcription of the cox-2 gene [52]. 4.3.2.9 p75NTR The p75NTR neurotrophin receptor is a member of the tumour necrosis factor family and has been identified as a metastasis suppressor [113]. Expression of p75NTR was found to be very low in metastatic human prostate cancer cell lines [113, 139], while p75NTR inhibits expression of uPA, MMP-2 and MMP-9 in the human prostate cancer PC3 cells [139]. This suggests that p75NTR may prevent metastasis by reducing the expression of uPA, MMP-2, and MMP-9. It was recently shown that siRNAmediated knockdown of MK2 or MK3 separately or together in different prostate cancer cell lines resulted in decreased p75NTR levels. The authors suggested that MK2- and MK3- mediated stabilization of p75NTR mRNA probably involves the RNA binding protein HuR [164]. Thus reduced MK2 or/and MK3 levels may promote metastasis of prostate tumour cells due to destabilization of p75NTR transcripts resulting in elevated levels of uPA, MMP-2, and MMP-9. Whether MK2 and MK3 levels are anomalous in metastasising prostate cancer cells remains to be examined. 4.3.2.10 p27KIP1 The cyclin-dependent kinase inhibitor p27KIP1 is a tumour suppressor that acts by regulating G0 to S phase transition [31]. In addition to its effects on the cell cycle, p27KIP1 can also regulate cell migration through the small GTPAse RhoA [12]. p27KIP1 was shown to bind RhoA in vitro and to prevent the interaction of RhoA with its activator guanosine-nucleotide exchange factor (GEF). This abrogated RhoA-dependent ROCK1 activation and increased cell motility [12]. The study by Larrea et al. [119] provide evidence that phosphorylation of p27KIP1 at threonine198 by RSK1 may facilitate the interaction of p27KIP1 with RhoA and reduce actin cytoskeleton stability. It would be relevant to examine the phosphorylation pattern of p27KIP1 in metastasising tumour cells overexpressing RSK. 4.3.2.11 p53 The tumour suppressor p53 is well known to prevent cancer progression by inhibiting proliferation and inducing apoptosis of tumour cells [209, 211]. In

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addition, p53 mutations are associated with decreased E-cadherin expression and increased cancer invasiveness [16]. Several p53-mutant mouse models have been generated and tumours developing in these animals metastasize [148]. Another report showed that wild-type p53 is able to suppress cancer cell invasion by inducing MDM2-mediated Slug degradation. Slug is a member of the Snail family of transcriptional repressors, and is capable of repressing E-cadherin expression [214]. Recently, p53 was shown to be directly phosphorylated by MK5 at Ser37, leading to its activation in Ras-dependent senescence and transcriptional activation of p53 [198]. Thus MK5 may manipulate the function of p53 and e.g. enhance the expression of E-cadherin in a p53-dependent fashion, thereby affecting metastasis. It would be really interesting to clarify the possible involvement of MAPKAPK in the regulation of p53 activity linked to metastasis. In conclusion, a second major mechanism by which MAPKAPK may be implicated in metastasis is by increasing the expression of genes whose products are involved in metastasis. A direct proof for this mechanism was recently provided by a study identifying genes whose expression is controlled by RSK [46]. The authors found by the use of siRNA knockdown of individual RSKs and RSK inhibitors flm, BI-D1870 and SL0101 (see Table 4.2), that RSK1 and RSK2 affected the expression of 228 genes in epithelial cells derived from different tissues, predominantly through the transcription factor FRA-1. About 25% of the genes encoded proteins with established roles in motility and invasion such as uPA and UPAR, VEGF-1 and its receptor Flt-1, MMPs, CD44 which were discussed in this review. They also observed RSK-dependent TGFβ accumulation, a signalling molecule implicated in metastasis (see Section 4.3.2.5). In addition, upregulation of laminin322, integrins, RhoC, tubulin, actinin was monitored (a complete list is found in the supplementary data of the article by Doehn and colleagues). Enhanced expression of these proteins was confirmed in invading carcinoma cells. Moreover, ectopic expression of constitutive active RSK rendered the cells highly motile, while treatment of cells with specific RSK inhibitors or siRNA-mediated depletion of RSK1 and RSK2 greatly suppressed invasive migration [46].

4.4 Role of MAPKAPK in Tumour Angiogenesis Triggered by VEGF The growth and metastasis of solid tumours critically depends on their ability to develop their own blood supply, a process known as tumour angiogenesis [77]. Another role for MAPKAPK in metastasis derives from studies with vascular endothelial growth factor (VEGF). VEGF is a potent stimulator of angiogenesis, proliferation, migration and tubule formation of endothelial cells [56]. VEGF exerts its angiogenic effects through the receptor tyrosine kinases, which are expressed on the surface of endothelial cells, and whose activation leads to stimulation of various intracellular signalling cascades. One of these cascades comprises p38-MK2-LIMK1-cofilin that plays an essential role in VEGF-induced actin

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reorganization, cell migration, and tube formation [105]. In particular, MK2, a major downstream kinase of p38, directly activated LIMK1 by phosphorylating Ser-323 in the consensus sequence motif for MK2 substrates. Moreover, depletion of MK2 by siRNA abolished VEGF induced activation of LIMK1 and inhibited endothelial cell migration. Accordingly, filopodium formation was reduced in MK2−/− macrophages compared to wild-type macrophages, and migration of MK2-deficient embryonic fibroblasts and smooth muscle cells in response to VEGF was diminished [110]. These observations support a role for MK2 in VEGF-triggered actin reorganization.

4.5 MAPKAPK Inhibitors The recognition of crucial roles for protein kinases in cancer has led to an exponential increase in the development of small specific inhibitors against protein kinases [69, 132]. The versatile roles of MAPKAPK in metastasis make them attractive therapeutic targets as an alternative or supplement to currently used drugs against cytoskeleton dynamics [133, 171]. Many compounds that have the potential to inhibit the kinase activity of MAPKAPK in cell culture studies have been described, but unfortunately most of them lack specificity and with the exception of one, none have successfully entered clinical trials (see Table 4.1). Several promising MK2 inhibitors have been identified [3,60,125], but none have entered the clinics [61]. The compounds developed by Anderson et al. [3] are highly potent against MK2 (IC50 < 10 nM) and >200-fold higher concentrations were required to inhibit the kinase activity of the MNK1, MNK2, MSK1, and MSK2 by 50%. The IC50 for MK3 and MK5 were 2–300 times higher depending on the compound, making them potential drug candidates for clinical trials. MK5 is the only MAPKAPK that is efficiently inhibited (90% inhibition with 10 μM EGCG) by the green tea compound (–) epigallocatechin gallate [6, 57]. Animal studies and aetiological studies have shown that tea consumption may reduce the risk of cancer [231]. EGCG is being tested by the Louisiana State University (Shreveport, USA) in a Phase II clinical trial to determine its inhibitory effect on c-Met signalling and activation of pathways that contribute to breast cancer progression and in Phase Ib and Phase II clinical trials with prostate cancer and lung cancer patients [33].

4.6 Future Perspectives Invasion and settlement of tumour cells is a complicated process that requires the participation of many proteins (for recent reviews see Chiang and Massagué [27], Kumar and Weaver [114], and Psaila and Lyden [163]). MAPKAPK seem to contribute to metastasis by two major mechanisms. They can stimulate the expression of genes whose products are implicated in the metastasis process, and they can phosphorylate proteins whose activities contribute to cell invasiveness and metastasis. So far, a function of MAPKAPK in the regulation of protein-encoding genes has been

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appreciated as is outlined in this review. However, recent studies have identified microRNAs (miRNAs) with anti- or prometastatic properties (reviewed in Ma and Weinberg [126] and Dumont and Tlsty [51]). As miRNAs are RNA polymerase IIderived transcripts and their promoters share the same general features as those of protein coding genes [35], their expression may be regulated by MAPKAPK that modulate the activity of transcription factors involved in transcription of miRNAs. Consequently, MAPKAPK-mediated expression of miRNAs implicated in metastasis may present an additional putative mechanism of the involvement of MAPKAPK in metastasis. Activation of MAPK is a frequent event in tumour progression and metastasis (see e.g. [73, 85, 124, 169, 207]) but many studies are based on the use of inhibitors for either MAPK kinases (e.g. the MEK inhibitor PD98059) or MAPK (e.g. the p38 MAPK inhibitor SB203580). MAPKAPK act downstream of these kinases and therefore the possible involvement of MAPKAPK in metastasis cannot be excluded by the results obtained with these inhibitors. The use of specific MAPKAPK inhibitors may facilitate studies aimed at elucidating a causative role of MAPKAPK in metastasis. Thus MAPKAPK inhibitors may in the future be used as anti-metastatic drugs or in combination with other protein kinase inhibitors or chemotherapy that are presently being used or have entered clinical trials.

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Chapter 5

Grb2 and Other Adaptor Proteins in Tumor Metastasis Alessio Giubellino and Praveen R. Arany

Abstract Metastatic disease is a major source of cancer morbidity and mortality. Understanding the molecular basis of cancer metastasis will have a considerable impact on cancer therapy. We now understand that uncontrolled cell signaling is of great importance in oncogenesis and tumor progression. A major role in the assembly of signaling proteins into biochemical pathways and networks is played by adaptor proteins. In this chapter we describe specifically the role of these proteins in the process of tumor metastasis and the potential for cancer therapy. Historically considered undruggable targets, protein-protein interactions and adaptor proteins are emerging as rational and viable targets, as exemplified by the development of selective antagonist of the adaptor protein Grb2. Targeting adaptor proteins and cellular miswiring is an emerging and exciting cancer therapeutic opportunity. Keywords Adaptor proteins · Grb2 · Metastasis · Signaling

Contents 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Grb2 as a Paradigm for Adaptor Proteins in Oncogenesis and Tumor Metastasis 5.2.1 The Adaptor Protein Grb2 . . . . . . . . . . . . . . . . . . . . . 5.2.2 Grb2 in Cancer and Tumor Metastasis . . . . . . . . . . . . . . . . 5.2.3 Targeting Grb2 . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Other Adaptor Proteins in Tumor Metastasis . . . . . . . . . . . . . . . . 5.3.1 Shc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.2 IRS Adaptor Proteins in Mammary Tumor Metastasis . . . . . . . . . 5.3.3 Other Grb Proteins in Oncogenesis and in Tumor Metastasis: Grb7, Grb10 and Grb14 . . . . . . . . . . . . . . . . . . . . . . . . .

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A. Giubellino (B) National Cancer Institute, National Institutes of Health, 10 Center Drive, Bldg. 10-CRC, Rm. 1W-5832, Bethesda, MD 20892, USA e-mail: [email protected] W.-S. Wu, C.-T. Hu (eds.), Signal Transduction in Cancer Metastasis, Cancer Metastasis – Biology and Treatment 15, DOI 10.1007/978-90-481-9522-0_5,  C Springer Science+Business Media B.V. 2010

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78 5.3.4 Nck1 and 2 in Cell Motility and Invasion . 5.3.5 CRK and CRKL . . . . . . . . . . . . 5.3.6 NEDD9 in Melanoma and Other Cancers 5.4 Conclusions . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

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5.1 Introduction Metastasis, although the most relevant and important clinical process in oncology, remain the least understood at the molecular level [21]. Despite progressive advancement in our understanding and in the treatment of cancer over the last decade, a total of 1.6 million new cases of invasive cancer are expected in the United States in 2009 and around 600,000 cancer deaths will occur, primarily due to metastatic disease [54]. Patients with metastatic disease have far less favorable survival rates when compared with tumors discovered and treated when are still confined to the primary site of occurrence. Furthermore, metastasis can insidiously appear many years after the primary tumor is removed or treated, due to the presence and growth of undetectable micrometastasis. The reported statistics highlight the substantial need for therapies targeting specifically and preventing this unrelenting disease. Current cancer treatments focus on killing the greatest number of tumor cells using cytostatic agents and targeted therapies, but these regimens offer only sporadic success, with frequent relapse. Thus, to improve survival rates for most cancers, more effective ways of treating micrometastatic disease are required. These improvements will emerge from a better understanding of the molecular basis of metastatic disease. Metastasis is a multistep process [38] in which cells from the primary tumor have to remodel cell-matrix adhesions, detach, became motile and migrate through the extracellular matrix to reach newly formed blood vessels (angiogenesis) and access the blood stream to reach distant sites through extravasation to a new location where proliferation can begin again. A number of signaling pathways are involved in this process. Blocking any stage of this process can potentially be an effective strategy to block the entire chain of metastatic events. Human proteins are typically composed of structural sub-domains that are linked in various combinations into distinct proteins and the thorough understanding of the combination of these domains in multiprotein ensembles is a major challenge to understanding cell signaling and complex biological functions and diseases [100, 101]. The detailed mechanisms that govern multiple protein-protein interactions in the formation of large signaling complexes, and how the pharmacological inhibition of those interactions interfere with complex formation, in a physiological context and in disease, are just beginning to be understood. A major role in the assembly of signaling proteins into biochemical pathways and networks is played by adaptor proteins [98]. Adaptor proteins mostly function as flexible molecular scaffolds that mediate protein-protein interactions. Consequently, they are critical players in highly controlled cellular processes due to their ability

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to simultaneously interact with multiple effectors downstream of activated surface receptors. Although adaptors themselves do not contain any enzymatic or other direct effector function, they are often responsible for bringing effectors into close proximity to their targets. Specificity of such processes is achieved by the particular type of protein binding domains within the adaptor protein, the specific recognition of amino acid sequences in target proteins, the subcellular localization and proximity to the binding partners, and by their post-translational modifications. Moreover, many enzymatically active proteins also contain modular domains that allow them to serve as scaffolding proteins, performing additional functions as adaptor proteins. For example, the proto-oncogene c-Src can affect adhesion [14] and migration [58] by a kinase-independent mechanism; these observations suggest that in tumors where Src expression and activity is often deregulated, strategies to block the adaptor function of Src should be considered alongside inhibition of kinase activity [120]. Adaptor proteins are attractive and tractable molecular targets for therapy in a number of diseases, including cancer. Several reports have pointed out a critical role for adaptor proteins, including Shc [139], Grb2 [42], Nck [134] and Crk [9], in oncogenesis and cancer progression, and some adaptors have been identified among essential genes in cancer [75]. In chronic myelogenous leukemia, for example, Grb2 and CrkL are core components of the interaction network centered on the molecular hallmark Bcr-Abl fusion protein [14] and disruption of Src-homology 2 (SH2) and Src-homology 3 (SH3) domain interactions could have therapeutic potential [56]. SH2 and SH3 domains are common elements in signal transduction proteins, including proteins clearly linked to human diseases, and many of them would be novel targets for cancer therapy. SH2 domains recognize specific phosphotyrosylcontaining sequences on intracellular proteins, with additional flanking amino acid contacts responsible for generating selective binding. SH3 domains binds to prolinecontaining motifs, which often form a poly-proline type II helix (PPII) with a PxxP core consensus motif, but a growing number of binding motifs that do not fit this simple consensus motif has been reported. Other domains play a major role in cell signaling, including PH and PDZ domains. For a thorough list and description of such domains and their role in signal transduction, we refer the reader to more detailed resources [55, 100].

5.2 Grb2 as a Paradigm for Adaptor Proteins in Oncogenesis and Tumor Metastasis 5.2.1 The Adaptor Protein Grb2 The Growth Factor Receptor bound protein 2 (Grb2) is a widely expressed adaptor protein that is essential in several oncogenic signaling pathways. It has a modular structure with one Src homology 2 (SH2) domain and two SH3 domains [74].

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Through its SH2 domain, which is a conserved sequence of 100 amino acids, Grb2 can interact directly with receptor tyrosine kinases, such as the epidermal growth factor receptor (Egfr), the hepatocyte growth factor receptor (Met) and the vascular growth factor receptor (Vegfr), and non-receptor tyrosine kinases, such as focal adhesion kinase (FAK) and Bcr/Abl [32]. The phosphopeptide motif pYXNX (where N is asparagine and X any residue) is the minimal requirement for Grb2 SH2 domain binding. The two carbonyl and amino-terminal Src homology 3 (SH3) domains, which have a conserved sequence of around 50 amino acids, bind proline-rich regions within interacting proteins. The grb2 gene is located on chromosome 17, in a region that is known to be duplicated in leukemias and solid tumors [50]. Grb2 is critical in development [20] and knock-out mice die at a very early embryological stage. To investigate the role in development, Pawson’s group has devised a hypomorphic allele strategy [113] which has shown that Grb2 is critical for epithelial morphogenesis and for processes such as cell motility and vasculogenesis. The canonical model of Grb2 function relies on the widely confirmed observation that Grb2 SH3 domains are constitutively associated with Sos1, a guaninenucleotide exchange factor that promotes GDP–GTP exchange on Ras. Upon growth factor receptor activation and tyrosyl phosphorylation, Grb2 brings Sos1 into close proximity of membrane-bound Ras, thereby activating Ras and the downstream MAPK cascade, a well-know oncogenic signaling convergence point [137]. Interactions with several other proteins connect Grb2 with numerous downstream pathways as discussed below.

5.2.2 Grb2 in Cancer and Tumor Metastasis The role of Grb2 in cancer extends beyond its role as a receptor proximal intermediate in several oncogenic signaling pathways. In chronic myelogenous leukemia (CML), the chimeric Bcr-Abl tyrosine kinase oncoprotein is able to bind the Grb2 SH2 domain through Y177 in the BCR region, linking the fusion protein to the Ras pathway [102]. Inhibition of the ATP binding site of the Abl tyrosine kinase with small molecules, such as Imatinib, is currently the standard of care, but inhibition of Grb2 could become a valid adjuvant therapy or an alternative in patients resistant to other treatments. Several reports have outlined the overexpression of Grb2 in various solid tumors. In breast cancer, in addition to its role as a proximal mediator of ErbB2/Neu signaling, Grb2 itself was found to be overexpressed in several breast cancer cell lines and breast cancer tissue samples [26, 141]. Grb2 is also important in polyomavirus induced mammary carcinoma, and grb2 gene dosage is rate limiting for the onset and development of mammary carcinomas [113], highlighting its critical role in the transformation process. Grb2 is involved in keratinocyte growth factor (KGF) induced motility in MCF-7 breast cancer cells [156] further suggesting that Grb2 can be a valid therapeutic target for pathological processes such as the spread of solid tumors through local invasion and metastasis. In the highly metastatic

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cancer cell line 1-LN, Grb2 was one of the effector proteins significantly induced, together with Sos1, Shc and Raf1, through activation of the α2 -macroglobulin receptor [86]. Another group, using immunohistochemical analysis of tissue microarray from more than one thousand specimens, has reported the overexpression of Grb2, together with overexpression of Her2, in gastric carcinoma, specifically with an increase of Grb2 expression in primary cancer and nodal metastasis when compared with normal gastric mucosa [155]. Moreover, Grb2 overexpression was associated with poor survival rates, supporting a role for Grb2 in tumor cell aggressiveness. The same group has previously investigated Grb2 expression in colorectal cancers and found a significant increase in specimens derived from metastatic lesions [154]. The contribution of Grb2 to tumor cell dissemination is outlined by the several direct and indirect interactions of Grb2 with molecules involved in cytoskeleton remodeling, motility and other cellular processes recapitulated in the multistep cascade of cancer metastasis. In the early phase of the metastatic process a change occurs in the adhesion properties of potentially metastatic cells [18]. Several stimuli, such as those mediated by integrins, can induce FAK autophosphorylation, creating docking sites for proteins containing SH2 domains, including Src. Src can also activate FAK and promote phosphorylation on other tyrosine residues; one of these residues, Y925, occurs within a consensus sequence (pYXNX) for high affinity binding to the SH2 domain of Grb2 [115]. Interestingly, elevated Src activity, as observed during colon cancer progression, specifically promotes phosphorylation on tyrosine Y925, inducing changes in integrin adhesion and deregulation of E-cadherin [5], leading to an E-cadherin/N-cadherin switch [48]. This event is part of an important hallmark of cell transformation and metastasis, namely epithelial-mesenchymal transition (EMT). The interaction of Grb2 with FAK, Shc and other proteins also leads to activation of the Ras and ERK2 pathways; integrin engagement of these pathways induces cell spreading through actin cytoskeleton rearrangement. Indeed, one of the differences between ERK2 pathway activation by growth factor receptors versus via integrin receptor is that the latter requires a functional actin cytoskeleton to signal, while growth factor receptors can signal even when the actin microfilaments are disrupted by cytochalasin D, a potent inhibitor of actin polymerization [88, 116]. Grb2 interacts directly with the actin filament machinery. The interaction with WASp [121], a regulator of actin cytoskeletal rearrangement, is a well documented example. Binding of WASp with Grb2 may result in translocation of WASp from the cytosol to the plasma membrane, where it can interact with membrane bound proteins such as Rac and Cdc42 [2]. Furthermore, Grb2 links the EGF receptor to WASp protein constitutively and this interaction is enhanced upon EGF stimulation. WASp at the membrane also interacts with Nck [114], and together with Grb2, cooperatively stabilizes the actin-nucleating complex. So Grb2 and Nck, both SH2 and SH3 domain containing proteins, can link membrane receptors and membrane-bound proteins to intracellular cytoskeletal regulators, increasing their local concentrations at the membrane and facilitating enzymatic reactions and activation. Several migratory signaling pathways from the cell surface converge on the p21activating kinases (PAKs), which consequently translocate to the leading edge of the

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cell and contributes to motility and invasion. Activation of PAKs and translocation to the plasma membrane are processes that involve the interaction with adaptor proteins such as Nck and Grb2. PAKs are important regulators of actin cytoskeletal dynamics and the role of PAKs in cancer has been widely reported in literature. Interestingly, PAK can directly and specifically interact with Grb2 through a prolinerich motif in the PAK sequence [105]. This interaction is independent of EGF stimulation, but it is increased after stimulation of the EGF receptor and EGFRGrb2-PAK1 interaction is required for EGF induced lamellipodia formation. The complexity of Grb2 involvement in actin-based cell motility is highlighted by the growing list of interacting cytoskeletal proteins, including cortactin [23] promoting the formation of lamellipodia. Cortactin is associated with an invasive phenotype, formation of invadopodia and secretion of MMPs, favoring the spread of cancer cells through tissue [22, 148]. The number of Grb2 interacting proteins involved in the actin and tubulin cytoskeleton is continuously increasing, reinforcing the idea that Grb2 is playing a fundamental role in this context. Many of the pathways in which Grb2 is involved are important in the formation of novel vessel through angiogenesis and lymphangiogenesis. Several growth factors such as VEGF, angiopoietin-1, FGF2 and HGF contribute to the development of new blood vessels in physiologic and in pathological conditions, such as tumor angiogenesis. The vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2/KDR) is phosphorylated in response to its ligand, VEGF-A, which is secreted by many tumor cells, and this activation lead to the direct recruitment of Grb2, Shc and Nck [65]. This occurs also for the VEGF receptor 3 (FLT4L). Besides the direct interaction, Grb2 can also bind indirectly through the intercession of Shc [39]. Other signaling pathways such as those driven by angiopoietin-1 and Fibroblast growth factor-2 (FGF-2) also stimulate angiogenesis through direct and indirect interaction with Grb2, via intermediate proteins such as the FGF receptor substrate 2, Gab1 and Shc [123, 131]. Another mediator of angiogenic signaling is HGF, primarily through direct effect on vascular endothelial cells. Several HGF-driven cellular events in tumor angiogenesis require the activation of multiple signaling for which Grb2 has been demonstrated to be a key intermediate [109, 110]. Besides its role as a critical effector protein in focal adhesion platforms, phosphorylation of FAK through Y925 and Grb2 binding is involved in tumor neo-vascularization. Indeed FAK activation and consequent binding to Grb2 within tumor cells induce the expression of VEGF; elevated VEGF production in tumor cells stimulate cell motility and survival of endothelial cell, without affecting cell proliferation [87]. Moreover, previous studies have demonstrated that hypoxia conditions, like those experienced inside tumors, is able to increase phosphorylation of FAK with consequent Grb2 binding [118].

5.2.3 Targeting Grb2 Several efforts to selectively disrupt the intracellular network of Grb2 confirm the importance of this adaptor protein in several oncogenic signaling pathways. The

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peculiar structure of the SH2 domain of Grb2 has been the focus of intensive studies in an effort to synthesize selective antagonists. Compared with other proteins containing similar domains, the SH2 domain of Grb2 invariably prefers an asparagine at the second position downstream of the phosphotyrosine (pY+2) and requires the ligand peptide to adopt a β-bend configuration [62]. The basis of these requirements became clear when the structure of Src and Grb2 proteins became available for comparison. For the Src kinase, the peptide that binds the SH2 domain has a linear configuration because it allocates in a broad pocket that is open and elongated, while the peptide sequence that binds to the Grb2 SH2 domain is forced into a much deeper pocket and this explain the sequence requirement at pY+2 and the β-bend configuration. Using short phosphotyrosine containing peptides as starting platforms, several potent, cell permeable and phosphatase-resistant compounds have been developed over the last decade. To confer phosphatase resistance the phosphotyrosyl residue has been substituted by phosphonomethyl phenylalanine (Pmp) residue, or similar structures [18]; another approach was to use non-phosphate containing ligands [153]. More recently, Song et al. [126] reported the synthesis of potent inhibitor free of phosphotyrosine or any phosphotyrosyl mimetic, demonstrating that complete replacement of the phosphotyrosyl residue can be accomplished without significant loss of binding affinity. High affinity compounds, able to block EGFR-Grb2 interactions in intact cells [40] were derived through systematic and stepwise substitution of the backbone motif pYxN and mimicking the β-turn conformation. Starting from this platform, potent peptidomimetic inhibitors of SH2 domain interactions have been developed [15, 78]. Macrocyclization to stabilize the β-turn conformation and other modifications have been identified that increase inhibitor affinity and the potential for cell membrane penetration [17]. Using synthetic binding antagonists of the Grb2 SH2 domain, Atabey et al. [3] demonstrated their potent blockade of HGF-stimulated cell motility, matrix invasion, and branching morphogenesis in epithelial and hematopoietic target cell models. The same compounds, without affecting HGF-stimulated mitogenesis, was able to inhibit angiogenesis and vasculogenesis [127]. Testing of a prototypical antagonist in two aggressive tumor models revealed inhibition of tumor metastasis without affecting primary tumor growth in vivo, thus highlighting the critical role of the Grb2 adapter function in motility, invasion and the spreading of solid tumors [41]. The ligand binding surface of the SH3 domains of Grb2 have also been recognized as an important therapeutic target. The ligand of these SH3 domains typically occupies two pockets with two hydrophobic prolines, while a third pocket frequently interacts with basic residues, in a so called polyproline-2 (PPII) conformation [61]. The same conformation is recognized by other proline-recognizing modules such as WW and profilin domains [61], and this similarity has been the subject of several studies to better understand the selectivity of SH3 domains for target proteins. The importance of conformation over sequence for SH3 domain ligand recognition is also gaining acceptance. Although SH3 domains have long been thought to bind

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preferentially to proline rich sequences of the form PXXP in target proteins, several exceptions to this rule have been found [82]. For example, the SH3 domains of Grb2 have also been found to bind a RXXK core consensus motif [8]. Despite doubts regarding low binding affinities reported for SH3/target protein interactions and the basis for SH3 domain selectivity [143], several progress has been made in the development of SH3 domain antagonists [34]. For Grb2 in particular, SH3 domain target selectivity may be increased through two mechanisms, one intrinsic to the SH3 domains themselves and the other through their context in an SH2 domain containing protein. For example, both Grb2 SH3 domains may interact simultaneously with different sites on a single target molecule, e.g. SOS1, thereby increasing both the apparent affinity and selectivity of Grb2-target interaction [82]. Because the Grb2 SH2 domain restricts Grb2 subcellular localization, the pool of potential SH3 domain binding partners is also likely to be limited, further increasing their apparent selectivity. Screening short peptide libraries for peptides binding the SH3 domains of the Caenorhabditis elegans Grb2 homolog Sem-5 yielded a bivalent peptide ligand with nanomolar affinity [36]. Pak1-derived peptides encompassing proline-rich sequences in that protein were found to specifically disrupt Grb2 SH3 domain-Pak1 interactions with relevant impact on growth factor mediated migration and lamellipodia formation [105]. Non-natural amino acids analogs have been substituted at the proline-requiring site of Grb2 SH3 domain ligands [92]. These have provided peptides with nanomolar affinity and reinforced the concept that proline residues may be dispensable in the design of SH3 domain binding antagonists. An adaptation of the target peptide screening approach in developing SH3 domain binding antagonists has been to systematically introduce point mutations in target peptide sequences. High affinity peptides capable of blocking the proliferation of primary blast cell cultures derived from patients with chronic myelogenous leukemia (CML) and Bcr/Abl positive cell lines have been developed using this strategy [59]. Subsequent modifications of these peptides to improve their ability to permeate cells yielded agents that more potently disrupted Grb2 signaling complexes in CML-derived cells [60]. These preclinical studies support the concept that Grb2 SH3 domain binding antagonists could provide a therapeutic alternative for CML patients developing resistance to standard treatments [35]. Enhancing the affinity and selectivity of artificial SH3 domain binding antagonists by exploiting the existence of two SH3 domains in Grb2, dimeric peptides with high affinity binding to both SH3 domains of Grb2 have been designed with the goal of disrupting Grb2-SOS1 interactions [24]. These “peptidimers” inhibited cell growth in vitro and displayed anti-tumor effects in xenograft models, and thus represent the first examples of in vivo activity for this class of compounds [44]. Introducing N-alkylated residues into both monomers of the peptidimer and optimizing the linker improved the affinity for Grb2 to the subnanomolar range [142]. Finally, non-peptidic small molecule inhibitors have also been explored. The first example is the Src signal transduction inhibitor UCS15A that disrupts several SH3 domain mediated interactions, including those of Grb2 [95]. Although target selectivity remains to be improved, this and similar chemical structures may provide

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a platform for the development of small synthetic drugs that potently antagonize specific SH3 domain binding interactions. Further studies are necessary to refine our understanding of the complexity of Grb2 signaling and to better understand how Grb2 is linked to tumor metastasis in different tumor types. A promising and powerful strategy to explore target protein selectivity and mechanism of action of these compounds is the synthesis of tool compounds (e.g. modified with chemical tags, such as biotin) [43]. This and other strategies, such as the use of microarrays for the global analysis of gene expression profiles, will be extensively used to develop pharmacodynamic markers of drug action, a high priority in any modern drug development effort.

5.3 Other Adaptor Proteins in Tumor Metastasis In addition to Grb2, several other adaptor proteins have been reported to be important for triggering tumor formation and metastasis. Some of the most frequently involved in these processes will be summarized as follows.

5.3.1 Shc The role of the adaptor protein Shc (Src homology and collagen homolog) in tyrosine phosphorylation signaling pathways is well recognized. Three shc genes are known in mammals: ShcA (which is ubiquitously expressed), ShcB and ShcC (whose expression is limited to neuronal cells) [76]. The ShcA gene encodes three proteins which are produced by different promoter usage (p66) or by alternative translational initiation (p46 and p52). The modular architecture of the ShcA protein contains two distinct domains that bind phosphotyrosine containing sequences: an amino-terminal PTB domain and a carboxy-terminal SH2 domain. Human ShcA also has a central collagen homology 1 (CH1) domain with three tyrosine phosphorylation sites that transduce both Ras/MAPK-dependent (specifically recruiting the Grb2/Sos1 complex) and independent signals. The 66 kDa isoform of ShcA contains an additional amino-terminal CH-like region (CH2) [107]. Beside activation by several growth factor, cytokine, G-protein coupled and hormone receptors, Shc has been linked in transformation by Polyoma middle T antigen [16] and Bcr/Abl [104], and its hyperphosphorylation has been described in many different type of tumors [10, 130]. The involvement of ShcA in the regulation of cell proliferation, cell survival and angiogenesis clearly imply a role for this adaptor protein in tumorigenesis and cancer progression. In human breast cancer, where around one third of the patients have ErbB2/Neu overexpression or amplification, ShcA binding site on ErbB2 must be retained for transformation [28]. Similarly, several transgenic mouse models have suggested an important role for ShcA during mammary tumorigenesis. For example, mutation of the ShcA binding site within the Polyomavirus middle T (MT) antigen (which

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promotes metastatic mammary tumors in transgenic mouse models) is sufficient to delay mammary tumor onset and progression [149]. In a study of 116 archival primary breast cancer specimens using semiquantitative immunohistochemical analyses, it was found that increased phosphorylation of ShcA at tyrosine 317 and reduced p66ShcA levels strongly correlated with nodal status, disease stage and relapse [29]. Shc proteins have an important role in hormone-regulated cancers as well. Indeed, p66Shc is a promising potential biomarker of cancer prognosis in tumors where steroid hormone activity is important for tumorigenesis and progression, including prostate and breast cancers [1]. In the late stage of breast carcinoma, the tumor recruits novel blood vessels to support its growth (angiogenesis). Also in this stage ShcA covers an important role. As a downstream effector of Met and ErbB2 signaling, ShcA induces VEGF expression [112] and ShcA signaling have proangiogenic effects on MT-induced primary mammary tumors [139]. In addition, angiopoeitin-1 induces recruitment of ShcA to the Tie2 receptor, leading to endothelial cell migration and formation of new blood vessels [4]. The direct interaction of ShcA to the MEMO (Mediator of ErbB2-driven cell motility) protein promotes the formation of lamellipodia, which are associate with an increased motile phenotype [106]. Shc is also involved in tumor cell intravasation and TGF-beta-induced focal adhesion turnover [100]. Several other studies have highlighted the importance of ShcA in the increased migration and invasion observed in breast cancer cells, but its role in several other cancers in vivo need further experimental characterization.

5.3.2 IRS Adaptor Proteins in Mammary Tumor Metastasis Downstream of activated cell surface receptors, the insulin receptor substrate (IRS) proteins function as cytosolic docking proteins. The IRS proteins were originally identified as substrates of the insulin receptor tyrosine kinase, but several other receptors has been linked to them, including vascular endothelial growth factor (VEGF) receptors, a number of integrin receptors, prolactin and growth hormone receptors, and selected cytokine receptors [150, 159]. There are six member of the IRS family. IRS-1 and IRS-2 are largely expressed in several tissues, including muscle, brain, breast and kidney. IRS-4 is more restricted in its expression to brain, thymus and liver. IRS-3 is expressed only in rodens and a paralog has not been identified in humans, while IRS-5 and IRS-6 are only marginally similar to the other member of the family. The structure of IRS-1 and IRS-2 is very similar: both contains a N-terminus that is highly conserved, followed by a plextrin homology (PH) domain and a phosphotyrosine binding (PTB) domain which mediate the binding to insulin receptor and insulin-like growth factor receptor. Numerous tyrosine and serine residues at the C-terminus serve as docking sites for SH2 domain containing proteins [White, 2006], including PI3K, Shp-2 and Grb2.

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Only IRS-1 and IRS-2 are expressed in normal mammary epithelial cells and in breast carcinoma cells. Several evidences support a role of IRS-1 and IRS-2 proteins in breast cancer initiation and progression. The primary involvement of these adaptors in breast cancer is the fact that IRS proteins are major downstream effectors of IGF-IR [45]. Suppression of IRS-1 is able to block IGF-1 stimulated cell growth to a great extent leading to apoptosis in the breast cancer cell line MCF-7 [19] and its overexpression allow growth in condition of serum deprivation. Moreover, IRS-1 increases the aggressiveness of the breast cancer cell line BT-20 [25] and after nuclear translocation it associates with the cell cycle gene promoters c-myc and cyclin D1 [151]. In analogy, more recently IRS-1 has been reported to bind to the androgen receptor, translocate into the nucleus and activate transcriptional activity [71]. Other studies have evaluated the impact of gain or loss of expression of these adaptors in transgenic mouse models. Overexpression of IRS-1 or IRS-2 in a transgenic mouse is sufficient to promote tumorigenesis in the mammary gland [31]. Conversely, knocking-out IRS-1 or IRS-2 in mice do not affect initiation or growth of mammary tumors [77, 90]. These experimental evidences support a redundant role for these proteins in tumor initiation. The roles of IRS-1 and IRS-2 in tumor metastasis seem to be divergent. In the absence of Irs-1 gene expression breast cancer cells have a greater metastatic potential. In support of this evidence, a study reported decreased expression of IRS-1 in grade 3, poorly differentiated breast cancer [117]. However, another study supports a correlation between high IRS-1 expression and poor survival [64]. These discrepancies require follow up studies. A possible explanation of these contrasting results can be that the overall expression of IRS-1 do not reflect its functional status; rather, the cellular localization may be more important in IRS-1 function. Indeed, a recent report identify IRS-1 nuclear expression as a marker of well differentiated, non metastatic breast cancer [132]. Other experimental evidences point out that one mechanism by which IRS-1 can be activated or inactivated is through post-translational modifications, such as serine phosphorylation [77]. In contrast to a negative role of IRS-1, IRS-2 has a clear positive role in breast cancer metastasis, as evidenced by its preferential involvement in cell motility (versus cell proliferation) and tumor cell invasion. Tumors expressing only IRS-2 (and not IRS-1) are highly metastatic and mammary tumor metastasis are significantly reduced in Irs2 -deficient cells [77, 90]. IRS-2 contributes to tumor cell metastasis also through the regulation of cell survival [90]. Another potential mechanism for IRS-2-specific regulation of tumor metastasis is the recruitment of distinct intracellular effectors to a unique IRS-2 binding motifs and the consequent activation of distinct downstream signaling pathways. In conclusion, additional studies are needed to have an exhaustive picture of the role of IRS adaptors in tumor progression. In particular, a promising future direction is the study of IRS-1 and IRS-2 activity, rather than only expression, through the use of phospho-specific antibodies in breast cancer and other malignancies.

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5.3.3 Other Grb Proteins in Oncogenesis and in Tumor Metastasis: Grb7, Grb10 and Grb14 By screening bacterial expression libraries with the phosphorylated C-terminal tail of the EGF receptor, using the CORT (Cloning Of Receptor Targets) method, several SH2 domain containing proteins were characterized [80]. The proteins identified, known as growth factor receptor-bound (Grb) proteins, included PI-3 K (Grb-1) [125], Grb2, Crk (Grb-3) and Nck (Grb-4). Grb7 and Grb10 were discovered as a novel family of SH3 domain proteins with a distinct structure [81]. Indeed the molecular architecture of Grb7 (the prototype of the superfamily of adaptor proteins including Grb10 and Grb14) include an N-terminal proline rich region, a C-terminal SH2 domain and a central segment named GM region (for Grb and Mig) which includes a PH domain with sequence homology to the C. elegans protein Mig-10 [79, 128]. Despite the highly conserved structure homology, the members of the Grb7/10/14 superfamily exhibit a distinctive expression pattern among different tissue types. Interestingly, overexpression of these proteins has been observed in several cancers and correlate with tumor progression [72, 122]. A further level of complexity of this family of proteins (not completely understood and under investigation) is represented by potential different mechanisms of regulation, including phosphorylation and the occurrence of homo and hetero-oligomerization. Grb7 participate in multiple signaling transduction pathways under the control of several tyrosine kinase receptors, including members of the epidermal growth factor receptor family, the insulin receptor, the PDGF receptor, RET and the ephrin receptor EphB1. Other tyrosine phosphorylated non-receptor proteins have the specific binding motifs for Grb7 docking, including FAK, calmodulin and Grb2. All these interactions implicate Grb7 in the metastatic spread of tumor cells, where it is frequently overexpressed. The human Grb7 gene localize on chromosome 17, in proximity of the ERBB2 gene, and several studies show Grb7 involvement in breast cancer in the context of HER-2/neu amplification (co-amplification), both in human cell lines and in small cohorts of primary breast tumors [129, 144]. A subgroup of human breast cancer cell lines was shown to co-express and associate Grb7 with Her3 and Her4 [37]. Grb7 overexpression enhance Her2 and Akt activation and promotes tumor formation in animal models [6]. Messenger RNA (mRNA) levels have been reported to be important in predicting breast cancer prognosis. Indeed, Grb7 was included in a 21-gene set used to predict the prognosis of early-stage breast cancer [97], and in a small set of patients higher expression of Grb7 mRNA correlated with bad prognosis. A larger study which analyzed 319 node-negative and 319 node-positive breast cancer cores using an automated quantitative assay, revealed that high Grb7 expression strongly correlate with decreased survival and it is an independent prognostic marker [89]. The same study also confirmed a strong association between HER2/neu and Grb7 expression. Altogether these data demonstrate that Grb7 define a subgroup of patient with breast cancer with decreased survival, and suggest that Grb7 is a good prognostic biomarker and drug target of tumor progression.

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Grb7 expression was also increased in chronic lymphocytic leukemia (CLL) primary cells, and higher expression correlate with advanced disease stage [47]. The expression of Grb7 in hepatocellular carcinoma (HCC), the most frequent epithelial cancer of the liver, can modulate the invasive phenotype, and high expression of Grb7 protein is associate with clinical progression [51]. The human grb10 gene is located on chromosome 7, in proximity of the Egfr gene and Grb10 protein interacts with several proteins, including the EGF receptor, IGF-I receptor, KIT and VEGF receptor 2. Several connections between Grb10 and cancer have been reported, including increased expression in cervical squamous carcinoma [94] and in human osteosarcoma [71]. In human metastatic malignant melanoma Grb10 have been reported to be dysregulated [85]. In chronic myelogenous leukemia (CML) Grb10 associate with the Bcr/Abl oncogenic tyrosine kinase, in a site different from the Grb2-binding site, and its binding is important for tumorigenesis [7]. Moreover, in a mouse model of mammary carcinoma, using a strategy to identify tumor-associated breast cancer antigens, Grb10 was recognized to be enriched in tumor cells that induced a robust immunity [135]. These finding demonstrate that Grb10 is a valuable therapeutic target in breast cancer. In summary, Grb10 is an adaptor protein that is primarily involved in the growth of several rapidly proliferating tumor cells and further experimental work will better define its role in oncogenesis and tumor progression. Although Grb14 has been more extensively studied in the context of insulin signaling and diabetes, this adaptor protein has an important role in tumorigenesis as well. Besides its role as an intracellular effector of insulin receptor signaling, mediated through the interaction with IRS-1, Grb14 is a receptor proximal effector of oncogenic signaling from other receptor tyrosine kinases, like the EGF, FGF and PDGF receptors. The grb14 gene, which is located close to the ERBB4 gene on chromosome 2, is highly transcribed in human breast and prostate cancer [27]. A high mutation frequency of this gene in human colorectal cancers with microsatellite instability further underscores the potential implication of Grb14 in oncogenesis [33]. Considering the incomplete and fragmented picture we just described, much more experimental work is needed to fully characterize the role of Grb7, Grb10 and Grb14 in tumor formation and in tumor progression. Such studies will clarify the potential use of these adaptors as cancer biomarkers and as therapeutic targets.

5.3.4 Nck1 and 2 in Cell Motility and Invasion The Nck family of SH2/SH3 domain containing adaptor proteins links extracellular signal-induced phospho-tyrosine signaling to downstream regulators of the actin cytoskeleton [16]. The nck gene was isolated from a human melanoma c-DNA library using monoclonal antibodies (MoAb) against the melanoma-associated antigen. In humans the Nck family has two members, Nck-1 and Nck-2, that share 68% amino acid identity. The molecular weigh of both proteins is around 43 KDa and,

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like the adaptors Grb2 and Crk, are both cytosolic molecules exclusively composed of SH2 and SH3 domains. Both these domains share several partners with Grb2 and, to a certain extent, Grb2 and Nck have an interconnecting and complementary role on the actin cytoskeleton machinery. As prominent regulators of cell migration, Nck adaptor proteins are under intense investigation as modulators of motility-related structures, such as podosomes, lamellipodia and invadopodia [49, 96]. These structures are extensively associated with tumor invasion and the metastatic phenotype of tumor cells.

5.3.5 CRK and CRKL The role of Crk in tumors was first elucidated by Claude [1937] [26]. Mayer et al. [83] first characterized Crk as an adaptor protein for its ability to increase tyrosinephosphorylation of other proteins in chicken embryo fibroblasts transformed by chicken tumor virus 10. The increase in phophorylation was noted despite Crk lacking a tyrosine kinase domain and that led to the name viral-‘cellular regulator of kinase’ (v-Crk). The host cell-expressed oncoprotein, essential for malignant transformation, corresponds to the cellular protein c-Crk I and its structure includes one SH2 domain and one SH3 domain. c-Crk II, a second cellular Crk protein, is a splicing isoform of the same gene, and encode a protein with an extra SH3 domain and a linker region containing an important tyrosine residue (Y221) [108]. Binding of the SH2 domain of c-Crk II to the phosphorylated Y221 negatively regulate the binding ability of its N-terminal SH3 domain. The SH2 domain of c-Crk II bind also to several proteins, including paxillin, p130Cas, c-Cbl, EGFR, PDGFR and Gab1. The scaffolding function of Crk allows the binding of various downstream molecules that play key roles in cellular migration. Crk proteins are also important in epithelial-mesenchymal transition and in HGF-induced cell spreading [67]. Several other intracellular proteins interact with the SH3 domains of the Crk proteins. These connections involve Crk in many cellular processes, including cell morphology changes, cell migration, proliferation, differentiation and in immune functional responses. The same interactions, which are involved in numerous oncogenic signaling pathways, are attractive molecular target for several diseases, including cancer [34]. Recent studies suggest that member of a new family of GTPase regulators, the DOCK family, may have a particularly important role in Crk signaling and in the oncogenic role of Crk [34, 53]. Several studies suggest a role for Crk proteins in oncogenesis and tumor progression. Overexpression has been demonstrated in several tumors [73, 84, 93, 136, 147] and correlate frequently with increased cell motility and metastasis, and consequently, with poor prognosis. The role of increased Crk phosphorylation by mutated cRet receptor has also been studied in multiple endocrine neoplasia type 2B (Men 2B) that results in activation of paxillin by tyrosine phosphorylation and fibroblast transformation [11]. A similar mutated RET in the papillary thyroid carcinoma oncoprotein (PTC) has been shown to recruit a protein complex containing

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Gab1, Crk II and C3G resulting in Rap1 mediated stimulation of BRAF kinase and the ERK/MAPKs [30]. Moreover, C3G activation of Rap1 links Crk signaling to modulation of cell adhesion and cell dissemination. A distinct gene, which is located near the CML breakpoint of chromosome 22, encodes for Crk-like (CRKL), a Crk homologous protein. CRKL binds to Bcr-Abl and Tel-Abl (ETV6/Abl) and increases their catalytic activity, resulting in downstream activation of Ras-GTPases and mitogenic ERK MAPK as well as Akt/PKB. These events promote tumor cell survival and growth [145]. More recently, Druker’s group reported the importance of the interactions of CRKL, Grb2 and Cbl with Bcr/Abl in the induction of leukemia in a mouse model [56]. These data imply that targeting the interaction with CRKL could have therapeutic potential in CML. A better understanding of the role of Crk and CRKL proteins is expected in the near future. Understanding the specific cellular localization and the temporal sequence of activation of these adaptor proteins are major challenges which require a rigorous and systematic experimental analysis.

5.3.6 NEDD9 in Melanoma and Other Cancers The scaffold protein NEDD9 (Neural precursor cell expressed, Developmentally Downregulated 9) was originally described in 1992 as predominantly expressed in the early embryonic brain [66]. Independently HEF1 (Human Enhanced of Filamentation 1) [68] and Cas-L (Crk-associated substrate-related protein, Lymphocyte type) [89] proteins were isolated and later recognized to be indistinguishable from the NEDD9 protein. The structure of the NEDD9/HEF1/Cas-L protein (herein called NEDD9) include a N-terminal SH3 domain, a coiled coil domain (which forms a bundle of alpha helices involved in protein interactions) and several SH2 domain binding sites. NEDD9 preferentially localizes at focal adhesion and at the centrosome. As an important downstream effector of FAK and integrin signaling, it is not surprising that NEDD9 is important in multiple aspects of the metastatic program. Indeed, a series of studies have identified NEDD9 as an essential metastatic cellular hub. Using comparative oncogenomics and a series of elegant biochemical and functional assay, Kim et al. [63] were able to identify NEDD9 as a bona fide melanoma metastasis gene. Its role in apoptotic signaling and in cell cycle is well characterized as well [103, 124]. NEDD9 is required also for invasion by glioblastoma cells [91] (345 /id) and breast cancer [52]. More recently, NEDD9 has been linked to resistance to first-line anti-cancer therapy in several tumor types, including imatinibresistant gastrointestinal stromal tumors (GISTs) [69]. The ability of assembling multiple signaling pathways and the discussed role in tumor progression, make NEDD9 an attractive molecular target for cancer metastasis [138]. A better understanding of its role in development and disease, coupled with a better understanding

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of the structural details of its multiple interactions, will be essential to develop potent and selective inhibitors. The identification of NEDD9-interacting peptides able to prevent its degradation imply that targeting NEDD9 is a feasible strategy [119].

5.4 Conclusions At a molecular level, cancer is a disease of aberrant cell signaling. An increasing number of protein-protein interactions involved in cell signaling has been identified as potential targets for the development of anticancer agents. It has become also increasingly important to understand the cell and tissue specific functions of signaling proteins in terms of organization in supramolecular complexes and in terms of the interconnection between different signaling pathways. Clearly there is an urgent need to understand more about defects in signaling pathways in order to design better and effective treatments [99]. The detailed mechanism that govern multiple protein-protein interactions in the formation of large signaling complexes, and how the pharmacological inhibition of those interactions, singularly or in concert, interfere with the formation of these complexes (and consequently cell functions) is just beginning to be understood. One of the key questions in cell signaling is how signaling specificity is generated following stimulation of a common complement of signaling pathways by a given cell surface receptor. Such specificity is believed to be embedded in the presence of non-catalytic modules (such as SH2 and SH3 domain, PH domains, etc.), which direct the protein to the right substrate and help the protein to localize to a particular subcellular compartment. Adaptor proteins, which are mainly composed of these modules, are cellular switches that ultimately specify cellular behaviors and control many aspects of cell function including migration, proliferation, differentiation and death; therefore, adaptor proteins are key proteins to understand why different kind of cells behave differently in response to various external signals. Likewise, in a pathological setting, adaptor proteins are interesting research topics to work out how cell signaling miswiring lead to a disease state. A better understanding of the modular organization of signaling proteins and a detailed structural characterization of the interacting surfaces, will guide us to better understand complex intracellular pathways in health and disease. Recent evidences suggest that posttranslational protein modification of adaptor proteins have an unrecognized role in the modulation of protein-protein interactions mediated by modular domains. For example, Grb2 phosphorylation seems to modulate negatively its signaling [46, 70]. Taken together, these data suggest a far more complex dynamic control of cellular behaviors through adaptor proteins which require more experimental work to be understood. Also, the study of the subcellular localization of adaptor proteins offers the opportunity to better understand their cellular functions and to discover unknown ones. The modular domains, such SH2 and SH3 domains, present in adaptor proteins are characteristic of intracellular signaling protein, but the odd exception exists. For

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instance, Stroll et al. [132, 133] demonstrated that the human melanoma inhibitory activity (MIA) protein acquires a SH3 domain-like configuration in the extracellular space. MIA protein has been associated with progression of melanocytic tumors [12, 140] through inhibition of attachment of melanoma cells to fibronectin [13] and it has been evaluated as a marker in patients with malignant melanoma [12, 57, 146]. Recently the adaptor protein CRKL has been reported to be externalized in several tumor cells and to bind to the PSI domain of β1 integrin in the human prostate adenocarcinoma cell line DU145 [90]; extracellular CRKL appear to have a role in cell growth and migration. It would be interesting to determine if other adaptor proteins are present in the extracellular milieu and to determine if the extracellular localization is relevant to physiological and pathological cell functioning. A major challenge of the post-genomic era is the identification of functions and interaction networks of proteins in health and disease. Recent reports of genomescale protein-protein interaction maps [111, 152] provide an enormous amount of data for biologists and biological chemists. These data will help to prioritize the important interactions in a network, to study the molecular mechanism of several diseases and to prioritize therapeutic targets. We anticipate that the study of adaptor proteins and their role in protein-protein interaction will provide a better understanding of how aberrant network dynamics contribute to disease [99] and reveal novel biomarker and molecular targets. More importantly, these works will generate therapeutic opportunity for several diseases where signaling miswiring occur, including cancer.

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Chapter 6

The Role of ROS Signaling in Tumor Progression Wen-Sheng Wu and Jia-Ru Wu

Abstract Reactive oxygen species (ROS) are recently proposed to be involved in tumor metastasis which is a complicated processes including epithelial– mesenchymal transition (EMT), migration, invasion of the tumor cells and angiogenesis around the tumor lesion. ROS generation may be induced intracellularly, in either NADPH oxidase- or mitochondria-dependent manner, by growth factors and cytokines (such as TGFβ and HGF) and tumor promoters (such as TPA) capable of triggering cell adhesion, EMT and migration. As a signaling messenger, ROS are able to oxidize the critical target molecules such as PKC and protein tyrosine phosphates (PTPs), which are relevant to tumor cell invasion. PKC contain multiple cysteine residues that can be oxidized and activated by ROS. Inactivation of multiple PTPs by ROS may relieve the tyrosine phosphorylation-dependent signaling. Two of the down-stream molecules regulated by ROS are MAPK and PAK. MAPKs cascades were established to be a major signal pathway for driving tumor cell metastasis, which are mediated by PKC, TGF-beta/Smad and integrin-mediated signaling. PAK is an effector of Rac-mediated cytoskeletal remodeling that is responsible for cell migration and angiogenesis. There are several transcriptional factors such as AP1, Ets, Smad and Snail regulating a lot of genes relevant to metastasis. AP-1 and Smad can be activated by PKC activator and TGFbeta1, respectively, in a ROS dependent manner. On the other hand, Est-1 can be upregulated by H2O2 via an antioxidant response element in the promoter. The ROS regulated genes relevant to EMT and metastasis include Ecahedrin, integrin and MMP. Comprehensive understanding of the ROS-triggered signaling transduction, transcriptional activation and regulation of gene expressions will help strengthen the critical role of ROS in tumor progression and devising strategy for chemo-therapeutic interventions. Keywords Reactive oxygen species · Tumor progression · TGFβ · HGF · Integrin · PKC · MAPK W.-S. Wu (B) Department of Medical Technology, Tzu Chi-University, No. 701, Chung Yang Rd, Sec. 3, Hualien 970, Taiwan, Republic of China e-mail: [email protected] W.-S. Wu, C.-T. Hu (eds.), Signal Transduction in Cancer Metastasis, Cancer Metastasis – Biology and Treatment 15, DOI 10.1007/978-90-481-9522-0_6,  C Springer Science+Business Media B.V. 2010

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Contents 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Generation of ROS for Triggering Tumor Metastasis . . . . . . . . . . . 6.3 The Intracellular ROS Generation Involved in Tumor Metastasis . . . . . . 6.3.1 The Role of NADPH Oxidase . . . . . . . . . . . . . . . . . . . 6.3.2 The Role of Mitochondria . . . . . . . . . . . . . . . . . . . . 6.4 The Signaling Pathway Triggered by ROS for Tumor Cell Progression . . . 6.4.1 The Direct Signal Target of ROS . . . . . . . . . . . . . . . . . 6.4.2 The Down-Stream Signal Cascades Regulated by ROS . . . . . . . 6.5 The Transcriptional System Regulated by ROS in Tumor Progression . . . . 6.6 Expression of Genes Regulated by ROS for Tumor Progression . . . . . . . 6.7 Involvement of ROS in Cytoskeletal Rearrangement . . . . . . . . . . . . 6.8 Prevention of Tumor Progression by Chemical and Enzymatically Antioxidant 6.9 Summary and Conclusion . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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6.1 Introduction Reactive oxygen species (ROS) including superoxide (O2− ), hydroxyl radical (·OH) and H2O2 are constantly generated in aerobic organisms during intracellular metabolism and in response to environmental stimuli. Classically, ROS were regarded as host defending molecule released by neutrophil for destructing exogenous pathogens such as bacteria. Recently, a lot of evidence indicates that ROS play central role in the key intracellular signal transduction pathway for a variety of cellular process [1–4]. Aberrant ROS signaling may result in physiological and pathological changes, such as cell cycle progression [5] apoptosis, aging [6, 7] ischemia/reperfusion injury [8], and diabetic complication [9, 10]. Previously, elevated oxidative status has been found in many types of cancer cells, which contribute to carcinogenesis [11–14]. Recently, the involvement of ROS signaling in tumor metastasis was highlighted [15, 16]. Metastasis is a complicated pathological processes consisting of several stages. Initially, the primary tumor cell exhibit epithelial–mesenchymal transition (EMT) followed by migration and invasion into the surrounding tissue facilitated by matrix degradation enzyme such as MMP. Some of the tumor cell enter blood circulation (intravasation) and survive in an anchorage independent manner. The circulating tumor cell may then transmigrate through the endothelium (extravasation) at a distant organ loci and finally proliferate into secondary tumor [17, 18]. In addition, angiogenesis may support the growth of the metastatistic tumor cell [19]. In this report, how ROS participate the signal transduction mediating tumor metastasis, especially in the initial stage will be reviewed.

6.2 Generation of ROS for Triggering Tumor Metastasis ROS generation may be induced intracellularly by growth factors and cytokines such as TGFbeta1 and HGF that may be secreted in the tumor microenvironment by

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stromal cells, marcrophage and tumor cells itself [20–27]. TGFbeta1 is a multipotent cytokine capable of triggering tumor progression in addition to it growth regulatory roles [28–30]. ROS generation may be induced by TGFbeta1 in several culture systems not only for cell proliferation and apoptosis but also for EMT and cell migration [25, 31, 32]. HGF is another cytokine known to induce EMT and migration in a variety of cultured systems [33, 34] and has been suggested to be a marker for prognosis in hepatocellular carcinoma [35]. Recently, ROS were demonstrated to mediate signaling of c-met, the HGF receptor, resulting in increased metastatistic capacity [26, 27]. ROS generation may also be induced by the phorbol ester TPA which is a well known tumor promoter and recently found to be a potential inducer of tumor invasion. TPA may trigger migration of several tumor cells such as rectal adenocarcinoma, breast tumor, and prostate cancer cells [36, 37]. The cellular receptor of TPA is PKC which play centrol role in the signal transduction mediate EMT and cell migration [38–40]. Several previous reports demonstrated that ROS can be generated by TPA in a PKC-dependent manner [23]. In our recent study, we further demonstrate that ROS cross react with PKC to induce sustained MAPK signal responsible for cell migration of human hepatoma cell HepG2 induced by TPA [41]. It is well established that integrins is the surface receptor of extracellular matrix (ECM) crucial for cell adhesion and migration. Integrin engagement may trigger outside–in signal for survival and invasion of tumor cells such as malignant melanoma [42, 43]. The integrin signaling may cross talk with PKC or TGFbeta pathway [44, 45]. The small GTPase Rac, well known to be essential for cytoskeletal rearrangement [46], is the most important molecule mediating integrin signaling pathway [47–51]. Mounting evidence demonstrate ROS can be generated by integrin–Rac pathway for migration and invasion [52–54]. Several reports demonstrated that chronic and sustained oxidative stress may trigger EMT and migration in vitro. For example, sustained production of H2O2 by phenazine methosulfate (PMS) activates pro-MMP-2 through NF-kB pathway leading to cell migration and invasion [55]. Also, repeated treatments with low dose of H2O2 resulted in EMT like morphological change of mouse mammary epithelial cell in the matrigel invasion chamber [53]. Collectively, these studies suggested an alternative mechanism in which ROS can be generated extracellularly (such as from chronic inflammation) to trigger tumor progression in vivo.

6.3 The Intracellular ROS Generation Involved in Tumor Metastasis 6.3.1 The Role of NADPH Oxidase Previous studies have demonstrated that membrane-localized NADPH oxidase (Nox) is responsible for generation of the intracellular ROS by receptor binding of numerous peptide growth factors such as PDGF, HGF, insulin, VEGF and TNF alpha triggering cell proliferation, differentiation, and apoptosis [56–58]. Also, the NADPH oxidase-dependent ROS generation was shown to be involved

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in cytoskeletal remodeling, extravasation, angiogenesis and regulation of genes associated with tumor metastasis. For example, activation of NADPH oxidase and generation of ROS were required for induction of the biglycan gene (which is involved in cell adhesion and migration) and for cytoskeleton rearrangement induced by TGF-beta [59, 60]. Recently it is was further established that ROS can be induced in the integrin-mediated pathway in a NADPH oxidase-dependent manner for tumor metastasis [61]. It is worthy of noting that Rac GTPase, which is one of the important mediator of cell migration and invasion [62], is the upstream signal enzyme for NAD(P)H oxidase dependent ROS generation [63]. In addition, two reports demonstrated that ROS derived from Rac1-dependent NAD(P)H oxidase are involved in vascular endothelial growth factor (VEGF)-and angiotensin-1 (Ang-1)-mediated endothelial migration, which is fundamental for angiogenesis [64, 65].

6.3.2 The Role of Mitochondria Before the recent discovery of the noninflammatory NADPH oxidase family members as a cytosolic enzyme for ROS generation [66], the major intracellular ROS were known to be derived from mitochondrial origin. It is worthy of noting that generation of ROS through mitochondria was associated with the integrin-mediated cell shape change [54] and gene expression of MMP (see below) [67]. Also, mitochondria dysfunction was involved in the reduced gene expression of MMP and invasiveness of the oxidative phosphorylation-impaired human osteosarcoma cells [68].

6.4 The Signaling Pathway Triggered by ROS for Tumor Cell Progression 6.4.1 The Direct Signal Target of ROS How ROS exert its cellular effect might better be investigated by identifying its intracellular targets. ROS can act as a second-messenger for regulation of diverse cellular processes by oxidation of cysteine on the critica target molecules including kinases, phosphatases [69], redox sensitive transcription factors [70], cell cycle regulator [71] and cell membrane lipids [1] of which PKC and protein tyrosine phosphotase (PTP) are the most relevant to tumor cell invasion. 6.4.1.1 The Role of PKC Protein kinase C (PKC) is a family of serine/threonine kinases that regulates a variety of cell functions including proliferation, cell cycle, differentiation, cytoskeletal organization, cell migration, and apoptosis [72]. Alteration of the cellular

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environment through disease, injury, or exposure to pro-oxidants, or other insults can induce aberrant PKC activation leading to diseases such as cancer and diabetes [72]. Various PKC isozymes are well known to contribute to the progression of malignant phenotype [73] and serve as a key mediator for cell migration induced by a variety of cytokines [74, 75]. PKC may cross-talk with integrin-mediated signaling for cell spreading [76] and regulation of carcinoma cell chemotaxis [77]. Moreover, PKC has been suggested as a critical target for antimetastatistic therapy [78]. Previous reports demonstrated that ROS were involved in PKC-mediated vascular smooth muscle cells migration induced by bradykinin [79], oleic acid [80, 81] and high glucose [81]. PKC contain multiple cysteine residues that can be oxidized and activated by ROS. On the contrary, PKC activation was required for ROS generation in several systems including diabetic complication [82–85] suggesting that ROS can be either upstream or downstream of PKC. This phenomenon was also demonstrated in our reports implicating the critical role of PKC↔ROS signaling in cell migration of hepatoma cell HepG2 [41]. Recently the essential role of ROS in mediating the cross talk of PKC with integrin signal pathway leading to tumor progression was proposed [86], which is an intriguing issue to be investigated in the future. 6.4.1.2 The Role of PTP The other potential signal targets of ROS implicated in tumor cell progression are protein tyrosine phosphatase (PTPs). PTP is a known negative regulator of receptor tyrosine kinase (RTK) signaling and inactivation of multiple PTPs by ROS may relieve tyrosine phosphorylation-dependent signaling [3, 87]. ROS can rapidly oxidize the catalytic cysteine of target PTPs, effectively blocking their enzyme activity induced by PDGFR [88], insulin [89] and integrin-mediated signaling [52]. Whether inactivation of PTP by ROS is associated with tumor progression was not intensively studied until now. One previous report demonstrated that oxidation/inactivation of PTP by ROS occurred during fibroblast adhesion to matrix [90]. Interestingly, a recent report demonstrate that ROS generated by integrin engagement mediated Rac-dependent down regulation of Rho by inactivating low molecular weight PTP [52]. Also, ROS generated from Rac1-induced NADPH oxidase may modify the PTP–PEST in focal contact resulting in membrane ruffling [91]. Thus the inactivation of PTP by ROS seems to be one of the signal mechanisms for tumor progression.

6.4.2 The Down-Stream Signal Cascades Regulated by ROS 6.4.2.1 The MAPK Cascade One of the important down stream signal cascades involved in tumor invasion is mitogen activated protein kinase (MAPK) including ERK, JNK and p-38 [92–95]. MAPKs were established to be a major signal cascade for EMT and cell migration in

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diverse systems [96] some of which are mediated by PKC, TGF-beta/Smad [97, 98] and integrin-mediated signaling [99]. Previously, it was demonstrated that increased ROS levels enhanced MAP kinase activities for malignant progression of mouse keratinocyte cell lines [100]. Lately, the ROS-activated MAPK signaling was shown to be required for migration of keratinocyte and smooth muscle cell [20, 80, 101]. In addition, ROS was shown to mediate TGF-beta1-induced MAPK activation for EMT in renal tubular epithelial cells [25]. How MAPK was activated by ROS to trigger cell migration is not clear. As described above, PKC may be activated by ROS for a variety of cellular effects. Moreover, PKC is also an upstream kinase of ERK (MAPK) required for cell migration [41, 102, 103]. Thus it is very probable that ROS may activate MAPK via PKC. Alternatively, ROS may activate MAPK by oxidative inactivation of PTP. For example, SHP2, one of the SH2-containing PTP, can be inactivated by ROS in PDGF-treated Rat-1 cell, which is associated with autophosphorylation of PDGFR leading to MAPK activation [88]. Furthermore, inhibition of PTPs by H2 O2 may regulates the activation of distinct MAPK pathways [87]. 6.4.2.2 The p-21 Kinase The p-21 kinase (PAK) is another down stream kinase involved in tumor progression [104] that can be potentially regulated by ROS. PAK is an effector of Rac-GTPase mediating cytoskeletal remodeling [48] and angiogenesis [105]. Previously, ROS was shown to be required for activation of PAK by lysophosphatidic acid (LPA) in vascular smooth muscle cells [106]. Recently, ROS was also shown to be involved in angiopoietin-1 and PDGF-induced PAK activation during endothelial and VSMC cell migration, respectively [65, 107]. Therefore, the possibility that PAK is regulated by ROS for tumor progression cannot be excluded.

6.5 The Transcriptional System Regulated by ROS in Tumor Progression Several transcriptional factors such as AP1, Ets, Smad and Snail are potentially involved in ROS-triggered tumor progression. ROS may activate AP-1 and nuclear factor-kappaB (NF-κB) for maintaining the transformed phenotypes [108]. Furthermore, induction of AP-1 can be triggered by ROS through PKC pathway for expression of urokinase plasminogen activator receptor which is correlated closely with tumor cell invasion and metastasis [109]. The Smad transcriptional factors are key transcriptional factors mediating the effect of TGFβ including EMT and cell migration [110]. Activation of Smad signaling also enhances collagenase-3 (MMP-13) expression and invasion of head and neck squamous carcinoma cells [111]. Recently, ROS was found to be involved in ERK mediated Smad activation during TGF-beta1-induced EMT in proximal tubular epithelial cells [25]. Another metastasis-relevant transcriptional factors potentially regulated by ROS is Snail, a

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well known transcriptional suppressor of the cell–cell adhesion molecule E-cadherin [112, 113]. Interestingly Snail can induce tumor invasion by upregulating MMP [114] and on the contrary, MMP may activate Snail via Rac1-dependent ROS generation to trigger EMT [16].

6.6 Expression of Genes Regulated by ROS for Tumor Progression ROS has been well known as key inducers of MMPs production which are critical for tumor progression due to its extracellular matrix-degrading activity [53, 67]. Moreover, ROS-dependent inhibition of protein phosphatases may be responsible for the upregulation of MMP-1 [115]. In addition, by a microarray analysis, redox regulation of matrix metalloproteinase gene was demonstrated in small cell lung cancer cells [69]. It has been well established that deregulated expression of integrin, the extracellular matrix adhesion receptor, may be associated with tumor cell progression [43, 116–118]. Interestingly, a recent report demonstrated that ROS continually produced outside the cell may up-regulate a set of integrin family members (integrin alpha2, alpha6, and beta3) accompanied with elevated invasive potential of mammary epithelial cells [53]. Expression of genes implicated in EMT and angiogenesis may also be regulated by ROS. For example, E-cadherin, a hallmark cell–cell adhesion molecule known to be down-regulated during EMT [119], may be reduced by TGFβ that trigger ROS-dependent tumor cell progression in renal tubular epithelial cells [25]. Also, ROS may upregulate expression of HIF-1alpha and vascular endothelial growth factor (VEGF), two of the important angiogenesis-related genes, in renal cell carcinoma [120]. In addition, gene expression and secretion of HGF, a well known scatter and motility factor [121, 122], may be induced by exogenous ROS leading to autocrine/paracrine stimulation of invasion of rat ascites hepatoma cell [123]. This suggested a alternative mechanism for ROS to enhance invasive activity via induction of gene expression of growth factors. Given the incomplete profile for ROS-regulated gene expression, microarray or differential/subtractive gene expression system should be employed to identify more of the metastasis-related genes regulated by ROS.

6.7 Involvement of ROS in Cytoskeletal Rearrangement Several recent reports implicated the effects (either direct or indirect) of ROS on cytoskeleton remodeling, an essential step during cell migration. For example, TGF-beta induced cytoskeletal alteration in endothelial cells was mediated by ROS [60]. Also, the pro-oxidant tertbutylhydroperoxide-augmented ROS production may trigger actin–cytoskeleton rearrangement and tight-junctional impairment in a PKC-dependent manner [124]. Interestingly, a recent report demonstrate that ROS generated by integrin engagement may cause stress fiber formation through

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glutationylation of actin [125]. Thus, the role of ROS in cell migration might be associated not only with its critical impact on the signaling pathway but also its oxidative modification of structural protein.

6.8 Prevention of Tumor Progression by Chemical and Enzymatically Antioxidant Because ROS is so implicated in cancer progression, introduction of the chemical or enzymological antioxidants to reduce oxidative stress is a promising antimetastatistic strategy. It has been demonstrated that antioxidant may be effective in the chemoprevention of prostate cancer [126, 127] and targeted or sustained delivery of catalase may inhibit metastatistic tumor growth in vivo [128]. On the other hand, a reduction in several anti-oxidant defense mechanisms, including catalase and glutathione S-transferase mu, correlated with the emergence of the malignant phenotype [129]. Dietary chemoprevention might be a promising cost effective and safe

TGFβ, HGF, TPA, Integrin Engagement

NADPH Oxidase

Mitochondria

ROS

PTP↓

PKC↑ MAPK

SMAD, AP1, Ets, Snail, HIF Adhesion, EMT, Migration, Invasion, Angiogenesis, Cytoskeletal Rearrangement

Tumor Progression

Fig. 6.1 Comprehensive overview of the signaling effect of ROS in tumor progression. Growth factor (HGF), cytokine (TGFβ), the tumor promoter (TPA) and integrin engagement may induce ROS generation via NADPH oxidase or derived from mitochondria. Oxidative activation and/or inactivation of PKC and PTP, respectively, by ROS result in MAPK activation followed by activation of various transcriptional factors including SMAD, AP-1, Ets-1 and Snail. Each transcriptional factor then regulate its target genes leading to EMT, migration, invasion adhesion and angiogenesis

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approach in prevention of ROS-triggered tumor progression. It has been suggested that the antitumor effect of lycopene, which is rich in tomato, involve ROS scavenging [130]. Also, the antioxidant EGCG or other tea polyphenols suppressed tumor promotion through reduction of ROS generation [131]. More experimental models and clinical trials are needed to investigate whether reduction of ROS generation is benefit for anti-metastatistic approach.

6.9 Summary and Conclusion During the past decade the role of ROS in tumor progression are emerging on the molecular level and were implicated as an important messenger for EMT, cell migration and angiogenesis. Until now, most of the studies are fragmental and limited, some are related to the signaling transduction, some focusing on transcriptional level and some in the aspect of gene regulation relevant to metastasis. It is worthy of noting that the molecular events demonstrated separately to be regulated by ROS have close relationship with each other. This strongly suggests that ROS and the aforementioned signaling molecules, transcriptional factors and their target genes are components in the same pathway leading to metastasis (Fig. 6.1). More feasible models, both in vivo and in vitro, might be developed to delineate the whole spectrum that ROS play key role in this process. Importantly, this may be employed as a molecular basis to design a more effective anti-metastatistic strategy. Acknowledgment We thank the National Science Council in Taiwan for financial support of our study [41] cited in this review, and C.-Z. Chung BS for technical assistance.

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Chapter 7

Signal Cross Talks for Sustained MAPK Activation and Cell Migration Mediated by Reactive Oxygen Species: The Involvement in Tumor Progression Chi-Tan Hu, Jia-Ru Wu, and Wen-Sheng Wu

Abstract Signal transduction exerted by the microenvironment around the primary tumor locus may trigger tumor metastasis especially at the migration stage. Sustained mitogen activated protein kinase (MAPK) signaling involved in uncontrolled tumor cell migration rely on the cross talks between integrin, receptor tyrosine kinas (RTK) and protein kinase C (PKC). The molecular mechanisms for cross talking between these migration-related signal cascades leading to sustained cell migration are reviewed, focusing on the focal adhesion scaffold protein paxillin as the platform for signal integration. We proposed reactive oxygen species (ROS) as the critical signal messenger sustaining these signal cascades. For the cross talk of integrin with RTK, ROS may suppress paxillin-associated protein tyrosine phosphatase (PTP-PEST) relieving its negative regulatory effects. For the cross talk of integrin with PKC, PKC itself may phosphorylate integrin or paxillin-associated focal adhesion proteins to induce generation of ROS which may reactivate PKC. In the future, ROS will be validated as the promising therapeutic targets for prevention of tumor metastasis. Keywords Receptor tyrosine kinas (RTK) · Integrin · MAPK · Reactive oxygen species (ROS) · Paxillin · Protein kinase C (PKC)

Contents 7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 The Sustained MAPK Activation for Cell Migration and Invasion . . . . . . . . . 7.3 The Role of Integrin Cascade in Sustained MAPK Signaling During Cell Migration and Invasion . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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W.-S. Wu (B) Department of Medical Technology, Graduate Institute of Medicine, Tzu Chi University, Hualien, Taiwan e-mail: [email protected] W.-S. Wu, C.-T. Hu (eds.), Signal Transduction in Cancer Metastasis, Cancer Metastasis – Biology and Treatment 15, DOI 10.1007/978-90-481-9522-0_7,  C Springer Science+Business Media B.V. 2010

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7.4 Cooperation of Integrin Signaling with RTK and PKC to Enhance ERK Activation and Cell Migration . . . . . . . . . . . . . . . . . . . . . . 7.4.1 The Cross Talk of Integrin with RTK . . . . . . . . . . . . . . . 7.4.2 The Cross Talk of Integrin with PKC for Uncontrolled Cell Migration 7.5 The Role of ROS Signal Mediating Sustained ERK Activation and Cell Migration . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5.1 The ROS Signaling . . . . . . . . . . . . . . . . . . . . . . . 7.5.2 Signal Amplification Triggered by ROS . . . . . . . . . . . . . . 7.5.3 The Proposed Molecular Mechanisms for ROS-Mediated Signal Cross Talk Between Integrin and Other Signal Cascade . . . . . . . 7.6 Conclusion and Perspective . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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7.1 Introduction There are several critical steps in metastasis. Firstly, the primary tumor cells migrate and invade to adjacent tissues followed by penetrating the extracellular matrix and blood vessels to enter circulation. The survived circulating tumor cells then penetrate capillary endothelium and finally proliferate at a distal organ [1]. Recently, a lot of evidence demonstrated the impact of signal transduction exerted by the microenvironment around the primary tumor locus to trigger tumor metastasis especially at the migration stage [2, 3]. The migratory cycle constitute several critical steps including cell polarization, membrane protrusion, adhesion formation and rear retraction that spatially and temporally integrated by a lot of signal molecules and cytoskeletal proteins [4]. A lot of cytokines such as hepatocyte growth factor (HGF) [5], transforming growth factor beta (TGFβ) [6], epidermal growth factor (EGF) [7], and Wnt [8] derived from the mesenchymal, stromal and the tumor cells are implicated in triggering tumor cell migration and invasion. Cell migrations induced by these cytokines and growth factors were mediated by important signal cascades such as mitogen activated protein kinase (MAPK), protein kinase C (PKC), focal adhesion kinase (FAK), Src, and PI3 kinase [4–8]. In this report, the molecular mechanisms for cross talking between major signal cascades leading to sustained MAPK activation and cell migration will be reviewed, especially the role of reactive oxygen species (ROS) are proposed.

7.2 The Sustained MAPK Activation for Cell Migration and Invasion The MAPK family proteins consisting of ERK, JNK and p38 play the most important role in cell migration [9, 10]. ERK(MAPK) [11–19, 20] and p38 (MAPK) [15, 21] may be activated via Ras-Raf-MEK cascade to mediate cell migration triggered by integrin engagement [20] and growth factors or cytokines including hepatocyte growth factor (HGF), epidermis growth factor (EGF), platelet derived

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growth factor (PDGF), and neurotrophic factor. Also, JNK may be activated via CAS/CRK-Rac-PAK cascade [for review 22] to mediate cell migration induced by Wnt5a [23] and integrin engagement [24, 25]. In addition, signal pathways triggered by transforming growth factor (TGF)-β1 and PKC can also activate ERK leading to cell migration [26–28]. One distinct feature of the MAPK-mediated cell migration is the sustained signal activation, in contrast with the transient activation observed in mediating mitogenic response [29, 30]. Previously, McCawley and co-workers suggested that duration of ERK activation is an important determinant for growth factors-mediated responses in keratinocytes [31]. Whereas mitogenic ligands transiently activate ERK for cell proliferation, ligands that stimulate cell migration and invasion triggered sustained activation of these kinases. Also, Krueger and co-workers suggested that cell motility is correlated with the magnitude and the duration of MAPK activation [32]. In fact, sustained ERK activation was often associated with cell migration induced by a lot of growth factors and cytokines [for review 40], such as EGF [28, 33], TGF-β1 [28], FGF [34] and PDGFB [35], and the tumor promoter tetradecanoyl phorbol acetate (TPA) [36] as well as by integrin engagement [37, 38] and oncogenic Ras activation [39]. The requirement of robust and extensive signal activation of ERK(MAPK) for cell migration might reflect its multiple roles in this complicated process. On the one hand, MAPKs may regulate cellular motility by promoting specific gene transcription [for review 47], including early responsive gene AP1 which has been shown to be required for EGF-induced migration of human epidermoid cancer cells. Some late responsive genes including the receptor for urokinase-type plasminogen activator (uPA) and the matrix degradation enzyme matrix metalloproteinases (MMP) which are associated with the formation of lamellipodia extensions and tumor invasion, respectively, were also induced via ERK. In addition, MAPK signaling was required for gene expression of the beta(3)-integrin subunit and the cysteine proteases cathepsin induced by cell adhesion molecule L1 [41]. On the other hand, ERK might also be recruited to cytoskeleton thereby phosphorylating specific cytoskeletal and focal adhesion protein such as paxillin, focal adhesion kinase (FAK), microtubule associated protein (MAP), myosin light chain kinase (MLCK) and calpain to regulate the dynamic of focal adhesion and cytoskeletal reorganization [for review 10]. Whereas ERK may phosphorylate MLCK for contraction of actomyosin fiber it also phosphorylate paxillin for augmented paxillin-FAK association and focal adhesion turn over [42]. Collectively, it appears that sustained ERK(MAPK) activation are required for expression of the migration-related genes coupled with posttranslational modifications of cytoskeletal and focal adhesion proteins, which may keep the migration cycle (4) to continue.

7.3 The Role of Integrin Cascade in Sustained MAPK Signaling During Cell Migration and Invasion One of the most promising mechanism for sustained MAPK signaling is the cross talking between major intracellular signal cascades of which integrin, RTK and PKC mediated-pathway are the most important. Integrins are heterodimeric

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transmembrane adhesion receptor that mediate cell attachment toward extracellular matrix (ECM). After engagement with ECM, integrins cluster at the focal adhesion site which serve as a platform for signal integration [43]. It is well established that integrin-mediated signal cascade can trigger sustained (MAPK) activation for a lot of cellular effects including proliferation, adhesion, migration and metastasis [22, 43–45]. The integrin-mediated signal for cell migration is initiated by interactions of arrays of adaptor and signal proteins in the focal adhesion which are regulated in a spatiotemporal fashion [for review 22]. Firstly, two nonreceptor kinases Src and focal adhesion kinase (FAK) are recruited and activated in the focal adhesion. FAK may autophosphorylate at Y397 for binding to SH2 domain of Src followed by phosphorylation on multiple residues resulting in elevation of its kinase activity. Activated FAK/Src may phosphrylate the adaptor protein P130CAS and scaffold protein paxillin. Paxillin may be phosphorylated at tyrosine residue 31 and 118 between its N-terminal LD1 and LD2 motif generating docking site for P130CAS. This may facilitate the recruitment of another SH2/SH3 adaptor CRK and trigger activation of small GTPases Rac. The MAPK cascades are the most important down stream signaling of the FAK-Src-paxillin-CAS/CRK-Rac axis. The paxillin-associated CRK may trigger the Rac specific guanidine exchange factor (GEF) DOCK180 to activate Rac and its down stream PAK (p21 activated kinase)JNK cascade. CRK may also recruit the RAP1 guanine nucleotide exchange factor C3G to activate RAP1 and the down stream B-Raf-ERK(MAPK) cascade [for review 22]. There are specific features of integrin-ECM engagement that contribute to signal amplification and extension [46]. Firstly, the integrin-matrix complex may not be internalized and their signal effects can be persistent over time. Secondly, integrin often cross talk with other pathway such as receptor tyrosine kinase (RTK) and PKC, which are able to induce cell adhesion, migration and invasion via MAPK.

7.4 Cooperation of Integrin Signaling with RTK and PKC to Enhance ERK Activation and Cell Migration 7.4.1 The Cross Talk of Integrin with RTK Mounting evidence demonstrated that integrins may associate with RTKs to potentiate down stream signaling including MAPK that are necessary for cell migration and metastasis [for review 22, 48–53]. For examples, Beta 4 integrin may associate with ErbB2 and amplify ErbB2 signaling to promote mammary tumorigenesis and metastasis [51]. Also, integrin has been found to cooperate with IGF-I receptor to stimulate sustained MAPK activation for cell migration and division of vascular cells [52]. The detailed mechanisms for cross talking of integrin with RTK have been intensively studied in the past decades. It has been suggested that direct interactions between integrin and RTK occur via some signal kinases and adaptor molecules

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[54–59]. For examples, the integrin linked kinase (ILK) may interacts at focal adhesions with the adaptor called particularly interesting new cys-his (PINCH) [56] which further binds another adaptor protein Nck-2 that can interact with activated EGF and PDGF receptors [57]. Also, Src kinase plays a key role in connecting integrin β4 with ErbB2 signaling that lead to breast carcinoma progression [51]. As described in the previous section, paxillin can be phosphorylated by FAK and Src to serve as the scaffold for signal integration during integrin engagement [54, 60–63]. Interestingly, paxillin-mediated signaling was also involved in RTKtriggered cell migration [64–68]. Tyrosine phosphorylation of paxillin pY31 and pY118 by FAK/Src can also be induced by a lot of growth factors to create docking site for P130CAS/CRK for initiating MAPK cascade leading to cell migration [for review 63]. Consistently, formation of a paxillin/FAK complex was found to be required for ERK activation and migration of bladder cancer cell induced by proepithelin, a PC-derived growth factor [67]. Also, paxillin was associated with and phosphorylated by Pyk2/FAK during vascular endothelial growth factor (VEGF)induced migration of neuroendocrine PC12 cells [68]. Moreover, the scatter factor HGF may induce phosphorylation of paxillin Y118 via Src which serve as a scaffold for organization of Raf-MEK-ERK signal cascade leading to cell migration [69]. The ERK thus activated may in turn phosphorylate paxillin at serine 83 [42], which is required for FAK-paxillin association and focal adhesion turn over, implicating a positive feed back regulation for paxillin/ERK-mediated sustained cell migration. In addition to the conventional P130CAS/CRK focal adhesion proteins, a lot of effectors molecules recruited to paxillin (especially on the LD4 motif in N-terminal domain) for mediating integrin and RTK-triggered signals were identified in recent years. The LD4 motif of paxillin serves as binding site for FAK and G-protein coupled receptor kinase interacting protein 1(GIT-1), the paxillin kinase linker(PKL)/GIT2 and Arf GAP [63]. GIT1 may bind to the Rac exchange factor PAK-interactive exchange factor (PIX) which can induce membrane ruffling via activation of Rac1 [70]. Assembly of a Paxillin-PKL/GIT2-PIX-PAK [71] or Paxillin-GIT1-PIX-PAK [72] complex near the leading edge was not only required for integrin-mediated Rac activation and continuous adhesion turnover in the protrusions of migrating cells but also for lamellipodia formation induced by insulin-like growth factor1 [73] or HGF [74]. On the other hand, it is worthy of noting that the LIM 3–4 motif on C-terminus of paxillin is the binding motif for a tyrosine phosphatase PTP-PEST playing as a negative regulator of paxillin-mediated cell migration [63]. Overexpression of PTP-PEST may inhibit the integrin- and PDGF-stimulated cell migration via desphosphorylating p130 CAS or FAK [75] and suppressing Rac activity [76], respectively. Furthermore, binding of PTP-PEST with paxillin is required for obtaining access to PKL/GIT2, one of the PTP-PEST substrate [77]. Dephosphorylation of PKL by PTP-PEST may disrupt the Paxillin-PKL-PIX-PAK module resulting in attenuation of Rac activity. Taken together, it appears that PTP-PEST may safeguard uncontrolled integrin and RTK signaling and it is tempting to speculate that suppression of the paxillin-associated PTP-PEST is prerequisite for the cross talk of integrin and RTK leading to uncontrolled cell migration.

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7.4.2 The Cross Talk of Integrin with PKC for Uncontrolled Cell Migration Protein kinase C (PKC) family consist of the classical serine/threonine kinases that regulates a variety of cell functions including proliferation, differentiation, cytoskeletal organization, migration, and apoptosis [78]. In the past decades, PKC was well known to play important role in mediating tumor invasion and metastasis [79–85]. Specifically, PKC may activate ERK signaling cascade for migration in of a lot of tumor and nontumor cell. [26, 27, 79, 86–90]. Moreover, PKC may cooperate with integrin-dependent pathway to enhance tumor cell migration, some of which are known to be mediated by sustained ERK activation [80, 90, 91]. Previously, a positive feedback mechanisms has been suggested for cross talk between PKC and integrin for cell spreading and survival [92]. Recent reports also demonstrated that PKC may phosphorylate or associate with integrin to influence localization or cytoskeletal rearrangement resulting in activation of the integrin signal [93, 94]. Furthermore, induction of Rac-GTPase activity [95] and phosphorylation of FAK, paxillin and c-Src [96] were closely associated with PKC-induced cell migration and actin rearrangement. Consistently, integrin αvβ5-dependent serine phosphorylation of paxillin was mediated by PKC [97]. On the other hand, the Src-mediated phosphorylation of tyrosine 31/118 on paxillin was required for cell migration induced by an adaptor protein RACK1 which was known to regulate signal transduction via PKC [98]. One recent study demonstrated that TPA may induce Rac1-dependent translocation of phosphorylated JNK to focal adhesion complex thereby to phosphorylate paxillin at serine 178 (Ser178) which was required for migration of glioblastoma cells [99]. Consistently, we also found TPA can induce phosphorylation of paxillin at serine 178 in a PKC-dependent manner in hepatoma HepG2 cell (unpublished data). Taken together, these observations implicated that the FAK/Src-paxillin-Rac cascade may be involved in cross talk between integrin and PKC for sustained MAPK activation and cell migration. In spite of the aforementioned studies highlighting paxillin as the integration center for mediating the cross talks between integrin and RTK (or PKC), there seems to be some unidentified links for establishing the sustained signal circuits. Recently, the reactive oxygen species (ROS) are emerging as the most promising signal messenger for this event.

7.5 The Role of ROS Signal Mediating Sustained ERK Activation and Cell Migration 7.5.1 The ROS Signaling Reactive oxygen species (ROS) including O–2 , OH and H2 O2 are constantly generated in aerobic organisms. Mounting evidences indicate that ROS play critical roles in signal transduction regulating a lot of pathophyiological processes [100–105].

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Elevated oxidative signaling may be implicated in promotion and progression of a lot of tumors including melanoma [106, 107], colon cancer [108], lung cancer [109], skin cancer [110] and hepatoma [111]. Specifically, growth factors such as HGF [112], the tumor promoter TPA [36, 79], and integrin engagement [112] may induce ROS to trigger cell adhesion, migration and invasion. One of the important upstream signaling components that trigger ROS generation is RacGTPase. Previously, Rac2 was known to be essential component of NADPH oxidase for ROS generation in phagocytic cells [113]. Lately, Rac1 was also found to be associated with NADPH oxidase -dependent ROS production in nonphagocytic cell [114]. It is well established that membrane-localized NADPH oxidase (Nox) is responsible for generation of the intracellular ROS triggered by receptor binding of numerous peptide growth factors such as PDGF, HGF, insulin, VEGF and tumor necrosis factor (TNF) alpha triggering cell proliferation, differentiation, and apoptosis [115–117]. Furthermore, Pro-metastatic signaling induced by c-Met was mediated through Rac-1 activation and ROS generation [107]. In addition, ROS may be generated by integrin-mediated cell adhesion via 5-lipoxgenase or NADPH oxidase [for review 46] and mitochondria [118].

7.5.2 Signal Amplification Triggered by ROS One interesting feature of ROS is its role in signal amplification. ROS may trigger sustained JNK signaling for TNF α-induced cell death [119] and cytotoxicity [120] via the oxidation and inactivation of JNK phosphatase. Also, oxidative inactivation of low molecular weight (LMW)PTP and downregulation of Rho was required for Rac-mediated cell adhesion and migration [112]. Furthermore, ROS was also shown to be involved in cross talking between RTK (such as EGFR and Met) and integrin to potentiate down stream signal component such as FAK, Src and MAPK triggering a variety of cellular effects including cell adhesion and spreading [121, 122]. Our recent work further implicated that ROS may be either upstream or down stream of PKC mediating the cross talk of PKC with integrin leading to sustained ERK activation and cell migration [36 and unpublished results].

7.5.3 The Proposed Molecular Mechanisms for ROS-Mediated Signal Cross Talk Between Integrin and Other Signal Cascade 7.5.3.1 ROS-Mediated Cross Talk of Integrin and RTK To delineate the possible mechanisms for ROS-mediated signal cross talk, the targets of ROS in the signal cascade should be defined firstly. The most important target of ROS involved in the cross talk of integrin and RTK is protein tyrosine phosphatase (PTP), which has previously been known as a negative regulator of both signal cascades. PTP including LMWPTP [112] and SHP2 [123] contain oxidant

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sensitive cystein side. Oxidative inhibition of PTP by ROS was required for RTK [102, 123–125] or integrin-mediated signaling [102, 112, 121, 122] that lead to cell adhesion or survival. As described in the previous section, the paxillin-associated PTP-PEST may negatively regulate integrin signaling via desphosphorylation of focal adhesion proteins [75–76]. One plausible mechanism for sustained ERK (MAPK) signal and cell migration is inactivation of PTP-PEST by ROS at the paxillin-centered focal complex to relieve its negative regulatory effects. Previously, ROS has been found to activate focal adhesion via phosphorylation of paxillin and p130Cas [126]. Moreover, Rac1 was required for activation of NADPH oxidase which may be closely associated with generation of ROS localized to membrane ruffles [127]. These observation strongly suggested that Rac1-NADPH oxidasedependent ROS generation may control lamellipodia dynamic spatially at the leading edge of migrating cells. Interestingly, a recent report demonstrated that PTPPEST can be oxidatively inactivated by expression of the orphan adaptor TRAF4 at focal complex coupled with robust membrane ruffling and migration of endothelial cell [128]. TRAF4 is capable of binding to NADPH oxidase subunit p47phox and controls activation of JNK (MAPK) [129]. An active mutant of TRAF4 may associate with the focal contact scaffold Hic-5 triggering Rac-dependent ROS generation to inactivate PEST-PTP [128]. Hic-5 was previous recognized as a paralog of paxillin sharing the same 11 exons in paxillin gene [130]. The Hic-5 scaffold has also been shown to be associated with PAK/PKL [131], FAK/Pyk2 [132] and PTPPEST [133], which are paxillin-associated focal adhesion proteins as described in the previous section. Taken together, it is tempting to speculate that ROS may be also involved in paxillin-mediated signaling as in Hic5. Under this circumstance, sustained paxillin-mediated signaling resulted from cross talk between integrin and RTK may be achieved by oxidative inactivation of PTP-PEST. In our proposed signal network (Fig. 7.1), either integrin or RTK may induce NADPH oxidasedependent ROS generation via Paxillin-PKL-PIX-PAK-mediated Rac1 activation. The ROS thus generated may be employed for inactivation of PEST-PTP induced by the other signal cascade, and vise versa. Therefore, the negative regulatory effects of PEST-PTP on both signal pathways may be relieved so that a positive feed back signal circuit can be established for sustained MAPK activation. In fact, the ROS mediated positive signal loop has been proposed in the aforementioned report [128] in which NADPH oxidase are both upstream and down stream of paxillin-mediated Rac1 activation. Alternatively, ROS generation induced by RTK and integrin signaling may be combinatory in nature, either additive or synergistic [46], resulting in a critical increase of intracellular ROS up to a threshold level in order that oxidative regulation of PEST-PTP can be continued. 7.5.3.2 ROS-Mediated Cross Talk of Integrin and PKC The role of ROS in mediating cross talk of PKC with integrin may act in an alternative way. Mounting evidence indicated the close association of PKC and ROS in mediating MAPK signal cascade. For example, PKC was known to be involved in H2 O2 -induced suppression of PTP leading to ERK phosphorylation [134]. Also,

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Fig. 7.1 Proposed mechanism for ROS-mediated cross talk of integrin and RTK leading to sustained MAPK activation. On the LD4 domain of paxillin, formation of Paxillin-PKL-PIX-PAKcomplex results in Rac1 activation which may trigger NADPH oxidase-dependent ROS generation. The protein phosphotase PEST-PTP, located on LIM3 and LIM4 domain of paxillin, may dephosphorylate PKL which disrupt binding of PKL with paxillin and suppress Rac activity. PEST-PTP may also dephosphorylate FAK and CAS. For signal cooperation between integrin and RTK, PESTPTP induced by one pathway may be oxidatively inactivated by ROS generated by the other. Thick arrow “ ↑ ” indicate sustained activation of the pathway. “+P ” and “−P” indicate phosphorylation and dephosphorylation, respectively. “----” indicate the blockade of the indicated molecules by PTP-PEST.“ T ” indicate the proposed feed back inhibition of PTP-PEST exerted by ROS

ROS and PKC were both involved in activation of ERK for oleic acid-induced vascular smooth muscle cell migration [135]. Interestingly, PKC contain multiple cystein residues that can be oxidatively activated by ROS [for review 136]. On the contrary, activation of PKC was prerequisite for NADPH oxidase-dependent ROS generation in a lot of biological processes [137–144]. Thus ROS may be either upstream or down stream of PKC. It was well established that PKC and ROS may cooperate to generate amplified signaling responsible for some pathological process such as kidney diseases associated with diabetes mellitus [140, 145]. Interestingly, we found TPA can induce phosphorylation of paxillin at Serine 178/Tyrosine 31, and

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Fig. 7.2 Proposed mechanism for ROS-mediated cross talk of integrin and PKC leading to sustained MAPK activation. The tumor promoter TPA induces membrane translocation and activation of PKC which may associate and phosphorylate integrin, Src or paxillin (at the Ser in LIM3 domain) to trigger Paxillin-PKL-PIX-PAK-mediated Rac1 activation and ROS generation. The ROS thus generated may reactivate PKC and a positive feed back circuit is established for sustained ERK signal. The symbols used were the same as in Fig. 7.1

activation of Rac in both PKC and ROS-dependent manner, which were required for cell migration of hepatoma HepG2 (unpublished data). It is very probable that PKC may cross talk with integrin-paxillin-PKL-PIX-Rac-1 signal axis mediated by ROS. We can speculate that after membrane translocation and activation of PKC (such as induced by the tumor promoter TPA [146]), PKC may phosphorylate integrin or focal adhesion proteins (such as paxillin) to induce Rac1-associated NADPH oxidase-dependent generation of ROS. The ROS thus generated may reactivate PKC and establish a positive feed back loop for sustained activation of the down stream MAPK signal (Fig. 7.2).

7.6 Conclusion and Perspective Cell migration is a complicated dynamic process regulated in a spatiotemporal fashion by a lot of adaptors and signal effectors linked to paxillin at the focal adhesion.

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MAPK is the most important down stream component of paxillin-mediated signal triggered by integrin, RTK and PKC. Cross talk of integrin with PKC or RTK via paxillin-mediated signal might result in sustained MAPK activation and cell migration leading to tumor metastasis. Here we proposed that ROS may be the missing link for connecting integrin with the other two signal cascades. Whereas inactivation of PEST-PTP by ROS is essential for cross talk of integrin with RTK, PKC itself might be activated by ROS for cooperation with integrin. Identification of ROS targets on the focal adhesion may help validating its role in these processes. The most important issue is whether PTP-PEST and PKC may actually be inactivated and activated by ROS, respectively, in a Rac-NADPH oxidase-dependent manner at membrane protrusion for regulation of dynamic focal adhesion turnover. In the future, ROS may be recognized as the most promising therapeutic targets for prevention of tumor metastasis. Acknowledgement We thank the financial support from National Science council in Taiwan for the studies relevant to ROS-mediated signal transduction.

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Chapter 8

Insights into the Dynamics of Focal Adhesion Protein Trafficking in Invasive Cancer Cells and Clinical Implications Moulay A. Alaoui-Jamali, Krikor Bijian, and Panagiota Toliopoulos

Abstract The development of cancer metastases is multifactorial and can be affected by a wide range of host, cell, and tissue-microenvironment factors. A crucial cellular event by which primary cancer cells invade neighboring tissue structures and distant organs involves the acquisition of autonomous motile and invasive properties; these can be inherent to a specific cancer cell variant, possibly cancer stem-cell type, or can be acquired during the course of tumor progression. Several transmembrane receptors, focal contact proteins, cell cytoskeleton and motor proteins, and intracellular signaling molecules converge to regulate cell migration and invasion. In particular, great research efforts have pinpointed the central role of focal adhesion dynamics and turnover within distinct subcellular compartments in the regulation of cell traction and retraction forces that drive, essential cell locomotion and invasion of neighboring tissues. Of clinical relevance, several components of the signaling pathways that regulate focal adhesion trafficking and turnover are deregulated in human cancer tissues; overexpression and hyperactivation are more common in invasive cancers compared to benign disease. This chapter provides the reader with an abridged, updated, and clinically relevant overview of signaling pathways that regulate trafficking and turnover of focal adhesion proteins involved in cancer cell migration and invasion. Potential clinical implications of specific proteins that can be exploited as biomarkers and therapeutic targets for the management of metastatic disease are emphasized. For detailed molecular biology of trafficking signaling, we refer the reader to selected seminal reviews in the field. Keywords Integrin focal adhesion · Trafficking · Migration · Invasion

M.A. Alaoui-Jamali (B) Faculty of Medicine, Departments of Medicine and Oncology, Segal Cancer Center of the Jewish General Hospital, McGill University, Montreal, Canada e-mail: [email protected] W.-S. Wu, C.-T. Hu (eds.), Signal Transduction in Cancer Metastasis, Cancer Metastasis – Biology and Treatment 15, DOI 10.1007/978-90-481-9522-0_8,  C Springer Science+Business Media B.V. 2010

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Abbreviations TGN G FA ER N PM ECM LE EE RE Lys

trans-golgi network Golgi focal adhesion endoplasmic reticulum nucleus plasma membrane extracellular matrix late endosome early endosome recycling endosome lysosome

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . Biology of Cancer Cell Migration and Invasion . . . . . . . 8.2.1 Overview . . . . . . . . . . . . . . . . . . . . . 8.2.2 The FA Signaling Network . . . . . . . . . . . . . 8.2.3 Cell Motor Proteins, Proteins Traffics, and Cell Motility 8.3 Trafficking and Turnover of FA Proteins in Cancer Cells . . . 8.4 The Rab-GTPases as Central Regulators of Protein Traffics . . 8.5 Deregulation of Protein Trafficking in Cancer and its Clinical Implications . . . . . . . . . . . . . . . . 8.6 Conclusion and Perspectives . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

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8.1 Introduction Cancer remains a highly prevalent disease in present day society. In some cancers, e.g. testicular, bladder, prostate, and breast cancer, survival rates have improved due to early detection and introduction of novel targeted therapies. Relatively often, however, a significant number of patients present with advanced invasive disease at primary diagnosis. In these patients, the development of metastasis is an ominous event with an unpredictable time-course; metastasis can be observed at primary diagnosis or months, years, or even decades after initial diagnosis. Although metastasis has been recognized as the result of progressive and late genetic events during the multistep theory of carcinogenesis, increasing evidence supports that metastasis formation is not always random; metastatic cells can emerge at an early stage of cancer development due to the presence of genetically programmed cell variants, e.g. cancer stem cell-like, with intrinsic invasive capacity [24, 51, 54, 65, 74, 75, 86, 89, 90, 103, 108, 110].

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The metastatic process can be conceptually compartmentalized into progressive steps, including: (i) local cancer invasion of neighboring tissue structures; (ii) intravasation due to chemotaxis towards capillaries and thin-walled venules, a common route for cancer cell entry into the circulation; (iii) survival and arrest in the capillary beds of distant organs; and (iv) extravasation and growth into the parenchyma of the distant target organ. Therefore, the acquisition of motile properties can be viewed as an early event that initiates cancer cell invasion of neighboring tissue structures. How a primary cancer cell acquires motile and invasive capabilties remains an open question but numerous signal transduction pathways have been implicated as initiators and/or drivers of cancer cell invasion beyond primary sites.

8.2 Biology of Cancer Cell Migration and Invasion 8.2.1 Overview Cell migration is a mechanism by which part of the cell extends to reach a target, or by which the entire cell moves towards a target in an “amoeba type” manner. Movement can occur in at least three forms (i) random (occurs in the absence of environmental stimulus), (ii) kinesis (a random motion that is influenced by chemical stimulus), and (iii) chemotaxis (a motion directed towards a gradient of stimulus). There is increasing evidence that invasive cancer cells are more sensitive to chemotactic signals when compared to non-invasive cells or to adult normal epithelial cells [14, 88]. Chemotaxis-mediated directional cell locomotion requires a defined cell polarity and differential localization of the cell cytoskeleton and adhesion proteins at two poles of a moving cell; this can be an intrinsic property of an invasive cell in response to chemotactic signals. These signals are contributed by the tissue microenvironment, in which cytokines and other soluble chemotactic factors are released. In addition, heterotypic cancer-normal cell interactions can impact several aspects of cancer cell motility and invasion [1, 4, 84, 113, 114]. Cancer cell migration generally begins with integrin activation by extracellular matrix (ECM) ligands; this causes integrin clustering and activation of numerous downstream signaling cascades. During cell migration, motile cells induce formation and stabilization of plasma membrane protrusions, which are mediated by extensive remodeling of the cell cytoskeleton, including actin filaments and microtubules [27, 44, 50, 87, 96, 119]. Once formed, cell protrusions are stabilized by the formation of focal adhesions (FAs); these structures are either small nascent adhesion sites found at membrane protrusions (termed focal complexes) or larger and more stable FA structures that extend underneath the cell body [120, 121]. It has been noted that fibroblastic cells deficient in several FA proteins, including FAK, have been found to have larger and more stable FAs and are generally more adherent and less motile compared to wild-type cells, which generally express smaller and more dynamic FAs [40]. In addition to their heterogeneous size, FA structures can

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be morphologically diverse and can appear as either large lamellipodia (flat lamellar extensions) or spike-like filopodia (pointed protrusions). Once formed, FAs are stabilized by anchoring to the extracellular matrix or by adhering to adjacent cells particularly via focal contacts involving focal adhesion proteins [55]. Indeed, FAs are believed to serve as traction sites at which motor proteins, such as the actomyosin system, can regulate the force transmitted to sites of adhesions via interaction with the actin filaments. This also permits the pull-up of the cell body forward as well as the release and retraction at the trailing edge of the cell rear [87]. Moreover, rapidly migrating cells such as leukocytes have few visible FA clusters, suggesting that small adhesions are probably important for their migration. In other cells, small focal complexes can be observed at the leading edge; these mature into larger focal adhesions that can promote tight cell adherence to ECM, which eventually renders the cells non-migratory or very slowly motile.

8.2.2 The FA Signaling Network The focal adhesion signaling network consists of a vast array of kinases, cytoskeleton, and adaptor proteins, including Src, focal adhesion kinase (FAK), Crk, Neural Wiskott-Aldrich syndrome protein (N-WASP), paxillin, α-actinin, tensin, vinculin, talin, and small Rho-GTPases [96]. Following integrin clustering or activation of growth factor receptors, autophosphorylation of FAK at tyrosine 397 (Y397) is amongst early events of FA activation. Activation at Y397 creates a high affinity site for binding to SH2 domain-containing proteins, such as Src kinase. Src binding to Y397 releases FAK auto-inhibition due to conformational changes [61], which then initiates a cascade of FAK phosphorylations at additional tyrosine residues in the catalytic and C-terminal portion of the protein, rendering the enzyme maximally active. This activation process is critical for FAK interaction with its partners, as well as for the regulation of FA formation and turnover [69, 104]. The mechanisms by which FAK and its partners in FA signaling are assembled in FA sites at cell protrusions are partially established. Mechano-sensors that activate integrins and growth factor receptor-mediated signaling have been implicated in the initiation of FA assembly [5, 25, 76, 96]. In contrast to assembly, FA disassembly is observed both at the leading edge, where it accompanies the formation of new protrusions, and at the cell rear, where it promotes tail retraction. At the front of migrating cells, adhesions at the base of a protrusion disassemble as new adhesions form at the leading edge [56, 109, 117]. Retraction at the rear requires Rho kinase and is a myosin-dependent process. Important regulators for FA assembly and disassembly include Src and FAK [68], proteases such as calpains, small GTPases of the Rho and Rab families [109, 111, 120], dynamin [22], and several cytoskeleton proteins including microtubules [27]. The latter are important regulators of focal tension sensing by adhesions and FA turnover [21], as well as cell polarity [115]. FAK activation is required for microtubule stabilization in part via the Rho-mDia signaling pathway in fibroblasts and stable detyrosinated microtubules are observed

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at the leading edge of migrating cells [28]. In addition to microtubules, FA proteins are closely associated with actin filaments in focal adhesions and the turnover of these structures is an essential part of integrin traffic and cell motility [37, 44].

8.2.3 Cell Motor Proteins, Proteins Traffics, and Cell Motility In addition to FA, cell locomotion involves a large number of motor proteins, which control cell tension and contractility, and cell movement. In particular, the force transmitted to sites of FA derives primarily from the interaction of myosins with actin filaments that attach to these sites. Myosins are implicated in F-actin mediated functions such as cell motility, vesicular trafficking, and intracellular transport of macromolecules [13, 25, 34, 53, 118]. The myosin family is represented by over 18 classes, among which myosin I, II, V and VI, have been implicated in cell motility mediated by F-actin [2, 3, 53, 91, 105, 106]. The general structure of myosins consists of a conserved N-terminal actin binding domain, an ATPase domain (motor or head domain), a neck region containing IQ motifs that bind to myosin light chains, and a C-terminal tail domain that binds to specific cargo. Myosins function as actin-dependent Mg2+ ATPases that utilize the energy liberated by the hydrolysis of ATP to move protein cargos along the cytoskeletal network, actin microfilaments, and microtubules. Among myosin classes, myosin II is of particular importance because of its role in transmitting forces to sites of adhesions. Notable is the interaction of myosin II with actin filaments that attach to FA sites. Myosin II assembly and activity are regulated by myosin light-chain (MLC) phosphorylation, which is regulated by MLC kinase (MLCK), Rho kinase (ROCK), and MLC phosphatase. MLC phosphatase is itself phosphorylated and inhibited by ROCK. MLC phosphorylation activates myosin, resulting in increased contractility and transmission of tension to sites of FA. Of relevance to cancer, myosin IIA assembly is also regulated via the binding of the metastasis factor, mts1 or S100A4; mts1 binds to non-muscle myosin IIA and promotes the monomeric, unassembled state of myosin IIA [58–60]. Phosphorylation on Ser1943 of the NMHC-IIA by CDK2 inhibits S100A4 binding and protects against S100A4-induced inhibition of filament assembly and S100A4-mediated depolymerization of myosin-IIA filaments [16]. These regulatory mechanisms are important for transmitting the strongest force to focal sites at the leading edge and at the retracting regions at the rear of a migratory cell. In these two locations, high tension exerted by myosin II contributes the physical break between the linkage of integrins and the actin cytoskeleton, and contributes to cell detachment [55]. This mechanism seems to play a role both in the mesenchymal and amoeboid types of migration. Interestingly, inhibition of myosin II in cancer cells, or deficiency in activity of myosin II or its regulator PAK impairs cell retraction and migration [106]. In addition to myosins, cancer cells express a large number of motor proteins, including kinesins, and dyneins. Like myosins, these proteins utilize the energy liberated by the hydrolysis of ATP to move their cargo along the cytoskeleton network, actin microfilaments and microtubules [19]; this trafficking process is mediated

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by several classes of adaptor “receptor” molecules, particularly the Rab-GTPases, which interact directly or indirectly with microtubules- or the actin-based motor proteins (addressed below).

8.3 Trafficking and Turnover of FA Proteins in Cancer Cells As noted above, directional cell movement requires: (i) a defined cell polarity and protrusions due to the differential and dynamic localization of cell cytoskeleton and to the focal adhesion proteins at two poles of a moving cell, (ii) a cycle of assembly and disassembly of multiple signaling molecules that occurs in a timeand space (specific cell compartment)-dependent manner. These two phenomena require tightly regulated protein trafficking and recycling that must also occur in a time and compartment space-dependent manner. A great effort has been made to elucidate the trafficking pathways in mammalian cells, and a large repertoire of molecules has been identified. Recycling of cellular proteins has been described in numerous ways; complex mechanisms governing plasma membrane fusion and membrane topology during the secretory and endocytosis process have been elucidated. Conceptually, protein trafficking can be viewed as a continuous flow of new proteins via exocytic pathways and through recycling via endocytic pathways. The former sorts newly synthesized proteins from the ER, through the Golgi apparatus to their final destination either at the plasma membrane or in lysosomes or vacuoles. In contrast, the endocytic pathway, which is essential for nutrient uptake, is involved in receptor internalization. Newly internalized proteins are transported to the early endosome, a tubulo-vesicular network localized to the cell periphery. The proteins destined for recycling are sorted to recycling endosomes and then redirected to the plasma membrane, while those destined for degradation are transported to late endosomes and then to lysosomes (Fig. 8.1). These mechanisms are the driving force for FA formation and turnover in invasive cancer cells. For integrin, for example, it has been demonstrated that exocytosis at the advancing leading edge of a cell assists cell locomotion by providing fresh adhesion receptors. Furthermore, these trafficking receptors are constantly internalized by endocytosis at the retracting end of the cell; the function of this internalization is in part to recycle proteins to the FA sites. In this case, synthesis, assembly, maturation and oligomerization of new proteins begin in the endoplasmic reticulum (ER) through the Golgi complex and post-Golgi carriers. The packaged secretory cargo is then transported to cell compartments via tightly regulated bi-directional trafficking owing mostly to the extensive intracellular fibrillar network that connects the ER-Golgi organelles to the nuclear envelope, cell cytoskeleton, and to plasma cell membrane [43, 62, 63]. In particular, intermediates in the form of vesicles or tubules are generally mobilized to target membranes via molecular motors such as microtubules or actin filaments in order to be transported. Tethering follows; intermediates, e.g. vesicles, are brought in close proximity to the target membrane, and then fusion of the vesicles with the target membrane occurs.

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ECM α β FA sites

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Fig. 8.1 Protein trafficking pathways. A simplified schematic representation of the secretory and endocytic pathways involved in protein traffics. Exocytosis regulates the delivery of newly synthesized proteins, lipids, and carbohydrates to cell surface compartments while the endocytic pathway mediates receptor internalization. The former involves endoplasmic reticulum (ER), Golgi complex (G), and plasma membrane (PM), and tubulovesicular intermediates that mediate intracellular transport between organelles. Secreted cargo molecules are synthesized and assembled in the ER, the largest intracellular compartment, which has intricate tubular connections that extend the nuclear envelope to other cell compartments and cell cytoskeleton. The ER also plays a role in controlling protein quality as incorrectly folded and assembled proteins are retained in or degraded from RE (reviewed in [62]). Cargo molecules destined to be secreted or to arrive at the plasma membrane (PM) leave the ER via distinct routes that bud and translocate as tubular-vesicular structures (pre-Golgi) toward the (-) end of microtubules. Pre-Golgi intermediates also sort and recycle selected components back to the ER (retrograde transport). Once cargo molecules merge with Golgi membranes (Golgi), which are located near microtubule organizing center, they are packaged into post-Golgi transport intermediates (post-Golgi), which deliver them to plasma membrane. Rab-GTPases and their effector proteins are key regulators of these multistep transport systems. In the case of focal adhesion proteins, exocytosis at the advancing edge is believed to assist cell locomotion by providing fresh adhesion receptors and that these receptors are internalized by endocytosis at the retracting end of the cell. For instance, mature FA proteins are assembled at focal adhesion sites of cell protrusion, following activation of integrin and growth factor receptors. Once cell protrusions are stabilized via the formed FA sites, FA are disassembled to allow cell migration. Several mechanisms have been proposed, including receptor internalization and endocytosis, which are generally coupled to FA disassembly, in which the connection to actin is lost and microtubules are targeted to dynamin-dependent internalization sites. Endocytosis occurs via alternative pathways, both clathering-dependent and -independent, and involve several signaling molecules, including dynamins, PKCα, Rab-GTPases, Rho-GTPases, microtubules, Arf6, and others (reviewed in [73]). Internalized receptors are then targeted to caveosomes or endosomes. The proteins destined to recycling are targeted to the perinuclear recycling endosomes (PNRE) and then back to the PM; a process involving Rab-GTPases, PKCa and others. Proteins destined to degradation are targeted to late endosome and lysosomes

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Several proteins that regulate the exocytic pathway have been discussed in an assortment of reviews [9, 62, 122]. In particular, Rab-GTPases and their effectors, including SNARE proteins, rabptins, rabenosyns, HOPS complex, Sec proteins, early endosome antigen 1 (EEA1), TRAPP proteins, golgins, α-actinin, and microtubules have been shown to play a major role in these mechanisms. The transport of intermediates carrying protein cargo occurs from the ER to the cis face of the Golgi complex, formed by an intricate tubular network consisting of microtubules and regulated by proteins such as dynein. Moreover, Golgi membrane tubules are critical for interconnecting adjacent Golgi stacks and for retrograde traffic from Golgi back to the ER. As expected, microtubule disruption prevents peripheral pre-Golgi intermediates from tracking into the Golgi region and can cause Golgi proteins to redistribute to peripheral sites. In the case of the endocytic pathway, several mechanisms have been described, including clathrin-dependent and clathrin-independent endocytosis. The clathrinmediated endocytosis involves receptor clustering in clathrin-coated pits, followed by membrane invagination and vesicle scission, which in turn fuse with tubulovesicular compartments (early endosomes). Subsequently, cargo is sorted in the multi-vesicular body (MVB) to undergo either recycling or degradation in the lysosomes. This route of endocytosis involves proteins such as clathrins, dynamins (mediate constriction and fission of vesicle stalks), Rab-GTPases (mediate vesicle budding, motility, and/or fission), adaptor proteins such as AP2, epsins (EGFR pathway substrate; associate with ubiquitin-conjugated cargos). The second mechanism is the caveolae-mediated endocytosis, which involves caveolins, dynamins, PKC and Src kinases (a form of lipid rafts). Third, clathrin- and caveolin-independent endocytosis involves proteins such as cdc42, PKC, rafts, Rho-GTPases, cortactin, dynamins, and epsins. Other routes of endocytosis include macropinocytosis, which involves Rac, cdc42, ruffles, nexins, Arf6, and Rab-GTPases [73]. Well-investigated endocytic pathways are those of integrins [7], select FA proteins [31], and growth factor receptors particularly those of the EGFR family [73, 97]. In both cases, several studies have provided insights into the mechanisms governing integrin trafficking, where various kinases, modulators of the actin cytoskeleton such as Rho-GTPases, and members of the Rab- and Arf GTPase families have been implicated. Taking integrins as an example, the process of endocytosis is associated with FA disassembly and involves PKCα, which binds to β1 cytoplasmic tail, dynamins, Rab-GTPases, microtubules, and Arf6, which is activated by Arf6 GEF (BRAG2) and regulated by the GTPase-activating protein (GAP) ACAP and the Akt pathway. Noticeably, activated Arf6 has been shown to promote integrin endocytosis [17]. Internalized β1 integrin is then targeted to caveosomes [102] or transferrinpositive endosomes [56] depending on the heterodimer. Recent data has shown that the caveolar and endosomal pathways intersect [78]. Once β1 is in the endosomal compartment, β1 cytoplasmic tail associates with Rab, e.g. Rab21-GTP. β1 recycling from the perinuclear recycling endosomes to the plasma membrane is regulated by other GTPases, including Arf6, Rab11, Rab21, Rab4, PKD1, PKC and microtubules.

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8.4 The Rab-GTPases as Central Regulators of Protein Traffics The Rab-GTPase family contains over 60 members which, like other mammalian GTPases, can switch between active GTP bound conformations to inactive GDP bound conformations via a GTPase activating protein (GAP) [99]. Rab-GTP conformation is necessary for Rab binding to effector proteins, as well as Rabmediated downstream functions [29]. Rab-GTPases exert a broad function in the fusion between transport vesicles and target membranes, nuclear transport, vesicle traffic, mitogenesis, and cytoskeletal organization (reviewed in [122]). Great progress has been made in understanding the structural organization of Rab proteins. In particular, Rab proteins contain hypervariable N and C terminal regions, which participate in protein-protein interactions with Rab effectors or regulatory proteins, as well as in subcellular targeting [82, 83, 99, 124]. Most Rab proteins are geranylgeranylated at or near their C-termini; this regulates their association with membranes [23, 93]. The elements necessary for guanine nucleotide- and Mg2+ -binding as well as GTP hydrolysis are found within the five loops that connect the α helices and the β strands of Rab. [92, 99]. Rab proteins additionally contain two important regions that allow them to adopt two different conformations, denoted switch I and switch II regions. Upon formation of “active Rab” (GTP bound Rab), a broad hydrophobic interface forms between switch regions I and II, resulting in ordered structural features that enable binding and response to effectors and regulators such as GDP/GTP exchange factors and GTPase activating proteins [99, 124]. Recent sequence analysis has also revealed that Rab-GTPases consist of five distinct amino acid stretches termed RabF regions (Rab family regions), which group within and around both switch regions. The RabF1 region is found within loop2/β of switch region I, while RabF2, F3, F4 and F5 dwell within and around switch II region between the β3 and β4 sheet. Likewise, four highly conserved Rab subfamily regions termed RabSF have also been identified [57]. RabSF1, RabSF3, and RabSF4 have all previously been known as the Rab complementary-determining regions I, II, and III and are required for specific binding to effector molecules. Among Rab members, there is distinct as well as overlapping functional specificity owing to Rab protein homologies and structural organization. For instance, the Rab5 family of proteins is responsible for clathrin-coated vesicle formation, vesicular transport to early endosomes, and vesicular homotypic fusion to early endosomes [92, 101]. Rab7, Rab11 and Rab27 play a general role in tethering and fusion of membrane vesicles, as well as in their transport and that of associated effector (cargo) proteins through direct or indirect interactions with microtubules or actinbased motor proteins such as myosins [122]. Rab7 is involved in microtubule-based transport of late endosomes/lysosomes [6]. Moreover, the minus-end microtubulebased motor complex dynein-dynactin is recruited to the Rab7-containing compartments through its effector Rab7-interacting lysosomal protein (RILP) [49], and both Rab7 and Rab27A colocalize in melanosomal membranes. Rab27a, but not Rab7, is excluded from the earliest melanosomes and Rab7, unlike Rab27a, is excluded

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from the mature stage IV melanosomes, supporting a maturation-dependent regulation [49]. Rab11 regulates recycling of endosomes to and from the trans-Golgi network to the plasma membrane, plasma membrane recycling of proteins such as β1 integrins, and transport of other molecules [reviewed in 66]. Rab-mediated vesicle delivery is mediated in part by actin filaments and microtubules, which regulate local and distant vesicle transport respectively [98]. Microtubule-dependent traffic requires plus-end-directed motors of the kinesin superfamily or minus-end-directed motors like cytoplasmic dynein, which are regulated by Rab proteins. Kinesin, for example, acts as a direct effector of Rab6 in vesicle transport, and is also indirectly regulated by Rab proteins [41, 122]. As noted above, integrins are endocytosed from the plasma membrane at the rear of the cell following disassembly of FA proteins and are subsequently recycled within vesicles for re-exocytosis at the cell front [8]. Rab-GTPases have been shown to play a pivotal role in this process by coordinating the recycling and internalization of integrins. For instance, Rab4, Rab-5 and Rab-21 are found to coordinate the recycling and endocytosis of many integrin receptors, including α5β1, αvβ3, α6β1, α6β4, and αLβ2 [8]. Rab11 also promotes the transport and recycling of integrins (i.e. β1 integrin) [45, 85] and Rab25 is found to associate with α5β1 integrin through direct interaction with its β1 integrin cytoplasmic tail [8]. Functional selectivity of Rab proteins depends on their association with Rabassociated proteins (i.e. effectors). Effectors for various Rab proteins have been identified, although their functional characterization remains only partially understood. Rab effectors are a heterogeneous group of proteins consisting of coiled-coil proteins involved in membrane tethering and docking of enzymes or of cytoskeletal associated proteins. These proteins regulate Rab activation, post-translational modification, and intracellular localization [15]. The precise targeting of Rab proteins to distinct compartments is crucial since it determines the localization of downstream effectors. For example, a soluble chaperone-like protein such as REP (Rab escort protein) first recognizes soluble Rabs. REP induces binding of Rab to Rab-GGT (Rab geranylgeranyl transferase) to allow for prenylation of Rab [15]. Following geranylgeranyl modification, REP is responsible for delivering Rab to target membranes [94]. Rab is then recognized by Rab GDI (GTP dissociation inhibitor) which functions in extracting Rab from membranes by binding to several residues within the Rab switch regions I and II. Rab GDI thus has a role in the recycling of Rab proteins [15, 94]. Various Rab effectors have been identified, including Rabaptin-5, Rabaptin-β EEA1, p150, p110β, and rabenosyn-5 [101, 122]. Rabaptin-5 is a Rab5 effector found to play a role in driving early endosome fusion of vesicles. In this case, Rabaptin-5 forms a complex with Rabex-5 (an exchange factor for Rab5), which activates Rab5 and other effectors. The effectors, EEA-1 and rabenosyn-5 cooperate with Rab5 to mediate fusion with endosome membranes. The ability of EEA-1 to bind to Rab5 relies on its N- and C-terminal Rab5 binding sites [98]. Other Rab effectors include Rab11 interacting proteins known as FIPS and identified as class I, II and III FIPs [18]. Rab11 FIPs contain a homologous Rab11 binding domain (RBD) located at or near the C-terminus that binds to Rab GTP with high affinity [18]. Class I FIPs consist of Rip11, Rab-coupling protein and FIP2

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proteins, while class II FIPs consist of FIP3/eferin, and FIP4, and class III of FIPs is composed solely of FIP1. These FIPs generally function with Rab11 subfamilies in the recycling of cargo to the cell surface, delivery of membrane to the cleavage furrow/midbody, and linkage of Rab11 proteins with the molecular motor protein machinery of the cell [39]. More specifically, class I FIPs hold an N-terminal C2 domain, concentrate to recycling endosomes and play a role in controlling plasma membrane recycling [18]. Class II FIPs concentrate to recycling endosomes, the trans-golgi network, and the centrosome and are important in membrane trafficking during cytokinesis.

8.5 Deregulation of Protein Trafficking in Cancer and its Clinical Implications Although aberrant protein trafficking is a common feature exhibited by cancer cells, its implications have only been recently investigated in a clinical setting. In particular, Rab-mediated endocytosis and recycling have been linked to carcinogenesis and cancer progression [11, 12, 100]. In particular, increased levels of many RabGTPases, Rab1, Rab5, Rab25, Rab21 and Rab27, have been observed in several carcinoma tissue types, including tongue, breast, ovarian, and hepatocellular carcinomas in comparison to matched non-tumorous tissues [12, 26, 95, 107]. The expression of Rab5 [58] and Rab25 [11, 45] were also correlated with tumor aggressiveness and metastatic potential of lung and stomach cancer, as well as ovarian and breast cancer, respectively. Moreover, Rab5 mutations that result in loss of function have been associated with carcinogenesis [77]. Rab32 gene silencing through CpG island hypermethylation has also been associated with the progression of frequent microsatellite instability colon cancers [72]. However, molecular mechanisms by which Rab-GTPases are deregulated in cancer and are associated with cancer progression remain to be elucidated. Several mechanisms have been proposed; these include the impact of Rab deregulation on inducing aberrant recycling of focal adhesion proteins, including integrin receptors, which can promote cell migration and invasion [73]. For instance, Rab25 and Rab21 can both promote rapid endocytosis and recycling of integrins to migration fronts in a way that enhances cell chemotaxis and invasion [79–81, 100]. Rab25 also associates with the α5β1 integrin promoting the localization of vesicles that deliver integrin to the plasma membrane at pseudopodia tips as well as the retention of a pool of cycling α5β1 at the cell front [8]. Rab can also mediate deregulated transport and secretion of certain cytokines, which could impact on the expression and activity of angiogenic factors such as VEGF and proteases such as MMP and cathepsins [107], or focal adhesion formation and turnover [42]. Recent studies have also reported that Rab-GTPases can promote genomic amplification associated with tumor aggressiveness, and that the Rab-coupling protein (RCP; also known as RAB11FIP1) can act as a “driver” of the 8p11-12 amplicon associated with mammary carcinogenesis [67, 123]. These connections clearly support that proteins involved in the modulation of protein trafficking can serve as potential tumor biomarkers and/or therapeutic targets.

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Virtually all approaches that interfere with the delivery of proteins from the ER to the Golgi complex, and then to cell compartments can lead to therapeutic implications if proven to prevent cancer progression. Indeed, modulators of protein trafficking pathways are already being used in the laboratory for biochemical purposes. For instance, the fungal metabolite brefeldin A is a small molecule that inhibits the GDP-GTP exchange (activation) of the small GTPase ADP-ribosylation factor (ARF), which is required for coated vesicle formation on Golgi cisternae [10, 30]. Brefeldin A has been reported to trigger apoptosis in multiple myeloma, Jurkat, HeLa, leukemia, colon, prostate and adenoid cystic sarcoma cells [112]. Moreover, Ho et al. have successfully been able to demonstrate the inhibition of MMP-9 secretion, which is required for extracellular matrix degradation and tumor invasion, in brefeldin A-treated human fibrosarcoma HT 1080 cells [33]. Breflate (NSC656202), a soluble prodrug form of brefeldin, is currently being investigated as a potential anti-cancer agent, which has already shown promising pharmacokinetics in mice and dogs [112]. Other new investigational inhibitors have focused on targeting Rab-GTPases [38], dynamin [64], proteasomes [20] or lysosomes [71]. Studies utilizing specific inhibitors of Rab-GTPases, mostly using RNA interference, have shown to inhibit cancer cell progression [12]. However, a recent study by Hooper S et al. identified two HMG-CoA reductase inhibitors, namely lovastatin and simvastatin, as molecules capable of inhibiting carcinoma associated-fibroblastmatrix remodelling required for cancer cell invasion, by inhibiting Rab protein function [38]. Dynasore [64], a non-competitive inhibitor of dynamin 2, which is a TGN-localized GTPase and mechanoenzyme [46] that plays a role in severing postGolgi cargo-containing tubules and participates in severing of the Golgi-derived tubules involved in plasma membrane transport, has been extensively used to study dynamin-dependent endocytic processes that are seen during viral infections [70]. Furthermore, a recent study by Yamada et al. revealed the ability of dynasore to inhibit lung cancer cell invasiveness acting through actin destabilization [116]. Additional molecules capable of interfering with microtubule polymerization and stability, such as the Vinca Alkaloids, cryptophycins, halichondrins, astramustine, colchine, paclitaxel, docetaxel, and the epothilones have all demonstrated the imperative role of protein transport in tumor progression and are currently being studied in clinical trials [reviewed in 48]. Other molecules such as cytochalasins, latrunculins, jasplakinolide and misakinolide (which disrupts the actin cytoskeleton) can also inhibit protein trafficking [32] and have demonstrated to possess anti-cancerous activities [reviewed in 47]. However, most of these pharmacological agents are devoid of relevant pharmacological properties and their toxicity profiles preclude their potential use in the clinic. In addition to these proteins, a common protein modification is ubiquitination, which plays a major role in protein turnover. During this process ubiquitin is attached to a target protein in a multistep process involving specific ubiquitin ligases. The attachment of a polyubiquitin chain to a target protein leads to its eventual destruction by the proteasome. This mechanism is important for the regulation of protein trafficking and protein signaling. The ubiquitin-proteosome complex has recently been the focus for drug development in cancer therapy.

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Bortezomib (Velcade), a clinically approved proteosome inhibitor, is currently in use for the treatment of relapsed multiple myeloma and has shown anti-tumour activity against non-Hodgkin lymphoma, exhibiting its action by downregulating NF-κB signaling. Additional proteosome inhibitors, such as carfilzomib, NPI-0052 and CEP-18770, which target different active sites of the 20S proteosome have already been generated and involved in studies analyzing their anti-cancer abilities [35]. Another key enzyme heavily pursued by investigators in the ubiquitin-proteosome system is the E3 ligase. It has been proposed that E3 ligase inhibition could result in specific target inhibition due to its direct interaction with substrates. Inhibitors such as HL198, targeting the E3 ligase activity of MDM2, thus preventing p53 ubiquitination, have shown great promise and have encouraged the development of additional ligase inhibitors with improved clinical parameters. Other inhibitors such as Nutlins and RITA, prevent the protein-protein interaction between MDM2-p53 and prompt p53 dependent apoptosis in cancer cells [36]. Current efforts in developing molecules capable of modulating the ubiquitin-proteosome system are focusing on inhibiting ubiquitin conjugases and ligases, preventing ubiquitin binding and designing novel proteosome inhibitors.

8.6 Conclusion and Perspectives Our daunting challenge to achieve efficient targeting of protein trafficking signaling in invasive cancer requires a detailed molecular understanding of these pathways. In particular, cell invasion signaling such as FA-mediated cell migration, where a cycle of assembly and disassembly that occurs in a time- and cell compartmentdependent manner, is critical for the initiation and progression of cell migration and must be thoroughly understood. Intracellular trafficking and turnover of focal adhesion complexes at FA sites at plasma membrane protrusions are believed to be central to the transduction of cancer cell invasive signals. This process is controlled by complex protein circuits, which include cytoskeleton proteins, motor proteins, Rab-GTPases, dynamins, and many others. However, this protein network has never been systematically and comprehensively investigated, making drug development especially challenging. With the development of new high-resolution imaging technologies to trace GFPtagged proteins that cycle between the ER, Golgi and plasma membrane and then to endocytic vesicles, it is becoming increasingly possible to address key questions relevant to the biological significance and targeting of the exocytic and endocytic pathways in cancer. There are many questions remaining to be answered. For example, how are FA proteins retained and sorted within different compartments in cancer versus normal cells and how does this differ in non/poorly-invasive versus highly invasive cancer? Is the rate of the secretory pathways distinct between cancer and normal cells or non-invasive and invasive cancer? Are there any selective proteinprotein interactions that can discriminate between cancer versus normal cells? What are the distinct regulatory circuits that must be disrupted in order to achieve a

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meaningful inhibition of protein trafficking and impact on cancer invasion and dissemination? How many of these circuits are there? With the availability of novel highthroughput chemical libraries, it is now becoming possible to better understand the dynamics of protein trafficking modulators and to identify the key molecules that can selectively target the trafficking of those proteins that play a key role during the invasive process.

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Chapter 9

Notch Signaling in Cancer Metastasis Pingyu Zhang and Patrick A. Zweidler-McKay

Abstract The evolutionarily-conserved Notch signaling pathway is not only essential for normal cell fate determination, tissue homeostasis and embryonic development, but also plays critical roles in the pathogenesis of human hematologic and solid malignancies. Notch has been shown to regulate cell proliferation, differentiation and apoptosis in a wide variety of cancer cells and recent studies have implicated Notch signaling in multiple aspects of tumor cell invasion and metastasis. Specifically, Notch signaling appears to contribute to epithelial-mesenchymal transition, tumor cell adhesion and invasion, tumor angiogenesis, and the formation and maintenance of cancer stem cells (or cancer initiating cells). In this chapter, we will review the molecular, cellular and phenotypic effects of Notch signaling on a wide range of cancers and how these contribute to tumor metastasis. Keywords Epithelial-mesenchymal transition · Angiogenesis · Invasion · Cancer stem cell · Cell survival

Contents 9.1 The Notch Signaling Pathway . . . . . . . . . . . . . . . . . . . . 9.2 Notch Signaling Regulates Epithelial-Mesenchymal Transition (EMT) . 9.3 Notch Signaling Contributes to Tumor Cell Invasion and Adhesion . . . 9.4 Notch Plays a Central Role in Tumor Angiogenesis . . . . . . . . . . 9.5 Notch Signaling Is Involved in the Tumor Cell Survival and Proliferation 9.6 The Emerging Role of Notch Signaling in Tumor Stem Cell . . . . . . 9.7 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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9.1 The Notch Signaling Pathway Notch signaling is one of the most evolutionarily-conserved signaling pathways. The original discovery of “notched” wings in Drosophila in 1917 eventually led to the identification of the Notch family of genes conserved across the animal kingdom [113]. Beyond “notched” wings, the Notch pathway has now been implicated in vasculogenesis, neurogenesis, myogenesis, somitogenesis, oogenesis and hematopoeisis in many species including humans [4, 34, 73]. Importantly, Notch signaling regulates numerous cellular processes, including proliferation, differentiation, and self-renewal in a cell-type specific manner. Not surprisingly, dysregulation of Notch signaling are involved in many developmental disorders and diseases [2, 13, 35, 52]. The Notch genes encode single-pass type I transmembrane receptors, which expressed on the cell surface as a heterodimer of a transmembrane/intracellular domain and an extra-cellular ligand-binding domain. In mammals, there are four different but highly homologous Notch receptors (Notch1, Notch2, Notch3 and Notch4) and five Notch ligands, categorized into two families, Delta-like and Jagged (Delta-like1, Delta-like3, Delta-like4, Jagged1, Jagged2)[4]. Ligand binding induces cleavage of the Notch receptors. First, ligand binding induces a conformational change in the Notch extracellular domain, which expose S2 site of Notch receptors to ADAM10/17 metalloproteases and subsequently γ-secretase cleaves the Notch receptor transmembrane domain [27] (Fig. 9.1). These successive proteolytic cleavages release the intracellular domain of Notch (ICN) from the cell membrane, which is transported into nucleus [27, 93]. In canonical Notch signaling, ICN

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Fig. 9.1 Notch signaling. Ligand binding to Notch receptors leads to cleavage by gamma-secretase which allows the intracellular domain of Notch (ICN) to enter the nucleus, recruit co-activators and induce activation of CSL-dependent target genes such as the HES family of transcriptional repressors

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interacts with the ubiquitous transcription factor CSL (CBF/RBP-Jk, Suppressor of Hairless, LAG-1) and upregulates a wide range of Notch/CSL target genes. When Notch is inactive, CSL is bound by histone deacetylase and co-repressors which block gene expression through transcriptional repression [47, 62]. When ICN enters the nucleus, it interacts with CSL and disassociates the co-repressors and recruits co-activators, such as Mastermind-like (MAML) and histone acetyltransferase p300/CBP, to convert CSL to a transcriptional activator, inducing expression of CSL target genes [29, 70]. The best-characterized CSL-dependent Notch target genes include the Hairy/enhancer-of-split (HES) and HES-related repressor (HERP, HRT, HEY) families [41]. HES and HERP family proteins are basic helix-loop-helix (bHLH) containing transcriptional repressors which recruit co-repressors, such as the C-terminal binding proteins (CtBP) and groucho/transducin-like enhancer-ofsplit (TLE) proteins [33]. In addition to the CSL-dependent signaling, other genes have been found to be induced by ICN in a CSL-independent manner. These noncanonical Notch targets include MAPK phosphatase LIP1 in C. elegans [11], pre-T alpha [3, 9], cyclin D1 [83] and p21WAF-CIP1 [66, 78]. Due to the great variety of the Notch target genes, the function of Notch signaling can differ greatly in various cell types and disease states.

9.2 Notch Signaling Regulates Epithelial-Mesenchymal Transition (EMT) Epithelial-mesenchymal transition (EMT) is a series of orchestrated events occurring during normal embryonic development, but it is also involved in primary tumor progression, especially invasion and metastases [40, 101]. In normal metazoan development, EMT is critical for morphogenetic movement and tissues/organ formation, e.g. gastrulation, neural crest and heart development. During the EMT process, cell–cell and cell–extracellular matrix (ECM) interactions, which maintain epithelial structure, are disrupted and the epithelial cell cytoskeleton is reorganized, resulting in enhanced cell mobility. Finally, the loss of epithelial cell polarity and integrity lead to fibroblastoid or mesenchymal phenotype, which displays a loosely organized and weakly adhesive phenotype [46]. Although EMT is essential for normal embryonic development, increasing evidence suggests that dysregulated EMT can be a driving force for carcinoma cells to invade into local and distant tissues [40]. There are several steps for primary tumor cells to achieve distant organ metastasis. First, malignant tumor cells lose the attachment to the original tumor and move into surrounding normal tissue (local invasion). Second, tumor cells migrate into the blood or lymphatic vessels through the basal membrane and disseminate into the system circulation (intravasation). Finally, tumor cells that reach distant organs invade again into surrounding tissue (extravasation) and form metastatic foci [71]. During the pre-invasive process,

160 Fig. 9.2 Notch signaling contributes to EMT in cancer. Notch activates HIF-1α, Snail and Slug to suppress E-cadherin, increases EMT. TGF-β also induces JAG1/HES1 expression and Notch activation through Smad3

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tumor cells may reactivate the EMT program and acquire more mesenchymal traits to facilitate the initial steps of local invasion and ultimately invasion into target organs [72]. Notch signaling has been shown to cooperate with other oncogenic pathways and molecules to induce EMT (Fig. 9.2). Early evidence revealed that Notch activity was required for the transforming growth factor β (TGF-β) induced EMT during cardiac development [103, 115]. It was shown that Notch signaling upregulates the transcriptional repressor Snail, which is a mediator of EMT in various tissues. Induction of Snail represses E- and VE-cadherin through their promoters and induces EMT [103]. Further studies showed that TGF-β activates Notch activity through Smad3 which upregulates both JAG1 (Notch ligand) and HEY1 (Notch effector). Elevated Notch signaling then induces Snail expression which suppresses E-cadherin. HEY1 and JAG1 were necessary for the TGF-β mediated EMT in normal epithelial cells from mammary gland, kidney tubules and epidermis. In addition, Notch also induces EMT in transformed epithelial cells, suggesting a role in EMT of cancer cell [103]. Recent studies by Sahlgren et al. link Notch signaling with hypoxia-induced tumor cell migration and invasion [86, 111]. Their data show that Notch-induced tumor cell EMT under hypoxia through two distinct mechanisms. (1) Notch directly upregulates Snail in a CSL-dependent manner. (2) Notch elevates lysyl oxidase (LOX) expression, through increasing the binding of hypoxia-inducible factor 1α (HIF-1α) to LOX promoter, increasing the stability of Snail, resulting in the upregulation of EMT, migration and invasion of cancer cells. Studies in pancreatic cancer provided further evidence of Notch signaling and EMT associated with a drug-resistant phenotype [111]. The authors found the upregulation of Notch2 and its ligand JAG1 in chemo-resistant pancreatic cancer cells, which have an EMT phenotype. SiRNA silencing of Notch leads to the reversal of EMT and downregulation of EMT markers, such as Vimentin, Slug and Snail, in drug resistant pancreatic cancer cells. In breast cancer, Notch signaling has been shown to promote the EMT through the transcriptional repressor Slug [53]. In this study, Notch inhibition abrogated

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primary breast tumor growth and lung metastasis in a xenograft mouse model. Notch suppressed E-cadherin expression by directly activating Slug expression in breast cancer cells, leading to an EMT phenotype. This Slug-induced EMT was also accompanied by activation of β-catenin and resistance to anoikis, which also contribute to breast cancer metastasis.

9.3 Notch Signaling Contributes to Tumor Cell Invasion and Adhesion In non-epithelial tumors loss of adhesion and invasion to surrounding tissue is also a critical step in their pathogenesis. An increasing number of studies indicate that Notch signaling contributes to tumor cell invasion by decreasing cell adhesion and increasing migration and invasion (Fig. 9.3). In pancreatic cancer, Wang et al. found that Notch1 signaling promotes the pancreatic cancer cell invasiveness through regulation of MMP-9, VEGF, survivin and cyclooxygenase-2 (COX-2) [109]. Overexpression of Notch increased invasiveness in matrigel, and down-regulation of Notch1 by siRNA inhibited cell invasion. Consistent with these data, Notch1 induced expression of MMP9 and VEGF through regulation of DNA-binding activity of NF-κB. Notch1 signaling has been found to strongly induce NF-κB promoter activity and expression of NF-κB subunits. Notch1 can also directly bind to the NF-κB p50 subunit and retain the active NF-κB complex in the nucleus. Through these interactions with NF-κB signaling, Notch1 induced expression of the NF-κB downstream targets MMP9 and VEGF, which are important for cancer cell invasion. Notch signaling has also been studied in osteosarcoma [118]. In a panel of osteosarcoma cell lines, Notch activation and HES1 expression are correlated to the invasive and metastatic potential. Inhibition of Notch signaling by γ-secretase inhibitor (GSI) suppressed cell invasion in Matrigel and overexpression of activated Notch1 or HES1 significantly promoted the invasiveness of osteosarcoma cells.

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Fig. 9.3 Notch regulates tumor cell invasion and metastasis. Notch activation promotes tumor cell invasion and metastasis through N-cadherin, HES1 and NF-κB-induced MMP, VEGF and uPA

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Importantly, blockade of canonical CSL-dependent Notch signaling via dominantnegative Mastermind-like (dnMAM) inhibited in vitro invasion and in vivo pulmonary metastasis of osteosarcoma in an orthotopic mouse model. However, Notch manipulation did not alter the cell proliferation rate in vitro and the primary tumor growth rate of osteosarcoma cells in vivo. This study concluded that the function of Notch signaling in osteosarcoma invasion and metastasis is independent from effects on cell proliferation. Interestingly, recent data showed a feedback mechanism for Notch signaling through reciprocal inhibition of Notch downstream target genes HES1 and DTX1 [120]. The Notch/CSL target gene DTX1, an E3 ubiquitin ligase, binds to and ubiquitinates Notch1, leading to down-regulation of Notch signaling. However, the other Notch/CSL target HES1 represses DTX1 expression via binding to the DTX1 promoter. Through this mechanism, the activity of Notch signaling in osteosarcoma cells is controlled by the balance between HES1 and DTX1. Recent studies suggest that Notch signaling may also contribute to cell proliferation in different osteosarcoma cell lines [23]. In a related tumor, Ewing’s sarcoma, Notch signaling was associated with metastasis in Ewing sarcoma patient samples and inhibition of Notch induced cell differentiation of Ewing sarcoma cells, though its relationship to invasion and metastasis has not yet been explored [5, 91]. Notch signaling has also been identified as a regulator of prostate cancer metastasis. Gene expression analysis between primary and metastatic prostate cancer samples showed that Notch ligand JAG1 is over-expressed in metastatic prostate cancer [51]. Consistent with this, immunohistochemical analysis revealed that JAG1 protein is significantly increased in the metastases of prostate cancer, compared to local tumor and benign prostate tissue [88]. Notch1, but not Notch2/3/4, was also found to be expressed at significantly higher levels in bone metastases-derived prostate cancer cell lines (PCa2B and C4-2B), compared with the non-metastasisderived cell lines (LNCaP and DU145) [116]. Functionally, Notch1 inhibition by siRNA in prostate cancer cell lines significantly decreased cell invasiveness in vitro [12]. Further gene expression analyses demonstrated that expression of urokinase plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP9) was reduced by downregulation of Notch1 [12]. Another study showed that GSI treatment reduced cell mobility of prostate cancer cells [92]. These cells also have the ability to undergo osteoblastic differentiation. Inhibition of Notch/HES1 signaling by GSI blocked the osteoblastic differentiation of prostate cancer cells, and decreased the bone invasion and metastasis [116]. The osteoblastic differentiation process requires cooperation of Notch/HES1 and ERK/Runx2 signaling activation which induces osteocalcin expression [116]. In addition to bone metastasis, high Notch1 expression also was found in prostate cancer cells metastasizing to lymph node in a transgenic mice model for prostate cancer (TRAMP) [96]. Similar to prostate cancer, breast cancer invasion and metastasis are also regulated by Notch signaling. Expression of JAG1 and Notch1 are associated with poor overall survival, reduced disease free survival and lymphatic invasion in large cohorts of breast cancer patients [65, 79]. To elucidate the genes involved in breast cancer brain metastasis, metastatic cells were selected by repeated cell isolation from brain lesions after intravenous injection of primary MDA-MB-435

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cells. Activated Notch signaling was revealed in these highly metastatic cells, and Notch inhibition by GSI treatment or siRNA knock-down of JAG2 and Notch1 significantly reduced cell migration and invasion [65]. Constitutively-active Notch has been shown to transform primary human melanocytes in vitro [77]. Recently it was reported that Notch signaling also promoted primary melanoma lung metastasis in a subcutaneous melanoma mouse model [6]. In melanoma cells, activation of Notch1 signaling upregulates MAPK and AKT pathways and increased cell survival and growth [57, 58]. Interestingly, Notch promoted tumor invasion in melanoma spheroids and cell adhesion through upregulation of N-cadherin [57, 58]. These Notch effects appear to be mediated by β-catenin [6].

9.4 Notch Plays a Central Role in Tumor Angiogenesis In response to angiogenic stimulation (such as hypoxia), new blood vessels are generated from pre-existing vessels. This process is called angiogenesis [76]. It has been demonstrated that Notch signaling is critically important for the physiological angiogenesis during embryonic development and pathologic angiogenesis in many diseases [22]. Global and endothelium-specific Notch1 knockout mice display severe vascular remodeling defects, resulting in embryonic lethality [37, 49, 50, 114]. Notch3 is also required for the functional artery formation, differentiation and maturation [19]. However, over-expression of active Notch4 in the mouse vasculature also induced similar angiogenic defects as Notch1 null mice [105], suggesting that Notch signaling should be controlled at a proper dosage for normal angiogenesis. Notch signaling functions at multiple points during angiogenesis, including endothelial cell specification, proliferation, mobility, adhesion, filopodia formation and blood vessel stability (Fig. 9.4) [22]. Recent studies showed that VEGF-induced DLL4/Notch signaling specifies endothelial “tip” versus “stalk” cell differentiation during angiogenesis [36, 59]. With VEGF stimulation, some endothelial cells become tip cells and produce DLL4. Via a process of lateral inhibition, the induced Notch signaling in neighboring cells drives their differentiation to stalk cells [16, 36, 39]. Preliminary evidence suggests that Notch signaling suppresses tip cell differentiation through repressing Flt1, Kdr, Nrp1 and Flt4 gene expression [39, 100]. Notch signaling was also reported to reduce endothelial proliferation in a CSLdependent manner. In the endothelial context, Notch inhibits Kdr-mediated MAPK and PI3K/AKT signaling and leads to enhanced p21WAF1/CIP expression, resulting in reduced cell proliferation and cell cycle arrest [10, 57–59, 100]. Endothelial cell migration is also negatively regulated by Notch signaling, possibly through downregulation of VEGF co-receptor neuropilin-1 (Nrp-1) [54, 90]. Nrp-1 stimulates VEGF-induced endothelial migration, but not proliferation [108]. Notch signaling also regulates endothelial cell adhesion through inducing expression of extracellular matrix molecules such as fibronectin, laminin and collagen [90],

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Fig. 9.4 VEGF-induced Notch signaling is required for endothelial cell specification during angiogenesis. (a) VEGF induces DLL4 expression in endothelial “tip” cells, then DLL4 activates Notch signaling in adjacent cells to prevent them becoming “tip” cells and inducing “stalk” cell differentiation. (b) Tumor cells often over-express JAG1/DLL4 which supports tumor angiogenesis by inducing stalk cell specification of endothelial cells. Inhibition of DLL4 or Notch signaling leads to increased blood vessels, likely through increased tip cells, however these vessels lack stalk cells and are often non-functional and decrease tumor blood flow, thereby leading to inhibition of tumor growth

as well as beta1-integrins enhancing endothelial cell adhesion [38]. To keep an established vessel stable, endothelial cells remain quiescent to prevent abnormal sprouting. Notch downstream target Nrarp reduced endothelial cell proliferation and blood vessel rearrangement in vivo, suggesting a regulatory role of Notch in vascular stability [76]. Evidence has shown that Notch may also cooperate with Wnt signaling to modulate the balance of new vessel formation and vessel stabilization [76]. Angiogenesis is required to support tumor growth with nutrition and oxygen. Evidence reveals that tumor angiogenesis is a dynamic process regulated by the interaction between tumor cells, endothelial and stroma cells [94]. Notch signaling from tumor cells was shown to activate endothelial cell differentiation and trigger tumor angiogenesis in vivo. Zeng et al. reported that Notch ligand JAG1 expressed in head and neck squamous cell carcinoma (HNSCC) promoted tumor angiogenesis by activating Notch signaling in neighboring endothelial cells [117]. Notch inhibition abolished JAG1-induced angiogenesis in these tumors. JAG1 expression was induced via the mitogen-activated protein kinase

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(MAPK) signaling pathway by HGF, EGF, and TGFα in HNSCC cells. JAG1 also affects the microvascular network formation in breast cancer. Estrogen or fibroblast growth factor (FGF) upregulated JAG1 and Notch1 expression in MCF7 breast cancer cells and promoted the vascular formation from endothelial cells [98]. A Notch antagonist (Notch decoy, a soluble form of the Notch receptor) significantly reduced tumor growth, by blocking tumor angiogenesis [30]. JAG1 and Notch1 expression was also correlated with poor survival in breast cancer patients [18, 79, 80]. The Notch ligand DLL4 has also been identified as a critical regulator of tumor angiogenesis. DLL4 is highly expressed in tumor endothelial cells and is induced by VEGF signaling [61, 102]. Inhibition of VEGF in vivo showed significant reduction of tumor growth and DLL4 expression in tumor endothelial cells [61]. This suggested that DLL4 promotes tumor angiogenesis during tumor growth. However, direct experimental manipulation of DLL4 in tumor models reported paradoxical results. Blockade of DLL4 by either an antibody against DLL4 or an antagonistic soluble DLL4 resulted in tumor growth inhibition, but led to increased blood vessel sprouting, branching and blood vessel density [68, 82, 90]. Further examination showed that these overgrown blood vessels were mostly non-functional [90]. This disorganized and non-functional tumor vasculature limits blood delivery and thus suppresses tumor growth. Notch inhibition also produced similar tumor growth inhibition and tumor vessel outgrowth in other tumor models. One explanation for this abnormal blood vessel growth is the dysregulated cell specification between vessel “tip” cells and “stalk” cells during Notch/DLL4 inhibition. Normal VEGF signaling induces DLL4 expression in endothelial “tip” cells, however inhibition of DLL4 or Notch appears to lead to excessive “tip” cell specification with more vessel formation without proper differentiation to vessel “stalk” cells, and thus poor vascular structure and intergity. Conversely, upregulating Notch signaling in tumor endothelial cells by over-expression of JAG1/DLL4 in tumor cells resulted in less tumor vascular formation and density, but stronger functional vessels, which promoted tumor growth in vivo [55]. These studies demonstrate the critical coordinating function of DLL4/Notch signaling in tumor angiogenesis triggered by VEGF signaling. Studies in pancreatic cancer reveal a novel role for Notch signaling in plateletderived growth factor-D (PDGF-D) regulated tumor angiogenesis [110]. Blockade of PDGF-D using siRNA in tumor cells reduced the expression of VEGF via Notch1-induced NF-κB activation, and subsequently inhibited the tube formation of human umbilical vascular endothelial cells in vitro. Although it remains to be verified in vivo, this result suggests that PDGF-induced Notch1 may promote tumor angiogenesis in pancreatic cancer.

9.5 Notch Signaling Is Involved in the Tumor Cell Survival and Proliferation Although not a direct measure of metastasis, tumor cell survival and proliferation provide opportunity for metastasis and are therefore worth discussing in the context

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of Notch signaling. Interestingly, Notch appears to play contrasting roles depending on the specific cellular context. In T cells Notch activation occurs in over half of cases and protects cells against apoptosis through four major signaling pathways (Fig. 9.5). First, Notch activates the expression of pre-Tα, which is critical for the pre-T-cell receptor (pre-TCR)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway [3, 9]. ERK1/2 activation promotes T cell proliferation via inhibition of tumor suppressor E2A [67]. Second, Notch upregulates anti-apoptotic proteins, such as the Inhibitor of Apoptosis (IAP) and Bcl2 family members to enhance cell survival [17, 85]. Third, Notch activates nuclear factor κB (NFκB) by activating IκB kinase via pre-TCR signaling [8, 26, 99, 106]. Fourth, Notch signaling enhances G1/S cell cycle progression in T cells by activation of cyclin D3, CDK4 and CDK6 in a CSL-dependent manner. In other hematologic cancers, such as B-CLL, Notch may promote cell survival through upregulation of CD23a [21, 84]. JAG1 and Notch1 were also reported to activate tumor cell proliferation and survival in Hodgkin lymphoma, anaplastic large cell lymphoma and multiple myeloma cells [44, 45]. However, in a broad range of B-cell malignancies, Notch signaling has been shown to induce cell cycle arrest and apoptosis, suggesting a tumor suppressor function of Notch [122]. Similarly, in AML cells, Notch activation may also inhibit proliferation and induce apoptosis in AML cells [104]. In regard with solid tumors, Notch signaling upregulates cell proliferation in non-small-cell lung cancer (NSCLC), pancreatic cancer, melanoma, glioma and medulloblastoma. In part, Notch utilizes common mechanisms, such as Bcl2 and NFκB, to increase cell survival. However, several additional mechanisms have been identified. It was reported that Notch activated CDK2 and Notch direct target gene cyclin D1 to increase cell cycle entry and proliferation in kidney epithelial cells, leading to oncogenic transformation [83]. Notch promotes proteosome-mediated degradation of the cell cycle inhibitors p21WAF1/CIP1 and p27KIP1 through inducing expression of S phase kinase-associated protein 2 (SKP2) [89]. Also, the Notch target gene HES1 can repress p27KIP1 directly [64], allowing Notch activation to enhance cell growth. Notch signaling has also been linked to Ras-induced

Notch

Fig. 9.5 Notch promotes tumor cell proliferation and protects tumor cells from apoptosis. Notch activates Skip2, HES1, pre-Tα, cyclin D and CDKs to increase cell growth, while activating Bcl2/IAPs and AKT and inhibiting JNK pathways to promote tumor cell survival

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tumorigenesis. Ras activation induced Notch1/4 and DLL1 expression, and blockade of Notch signaling downregulated Ras-induced malignant phenotype in human fibroblast and epithelial cells [112]. Finally, Notch activation blocked apoptosis by regulating c-Jun N-terminal kinase (JNK) [48, 60]. Notch physically interacted with JNK-interacting protein (JIP1) and interfered with JNK and JIP1 interaction, leading to suppression of JNK activation. All of these cell-specific mechanisms of Notch signaling promote or inhibit tumor proliferation and survival, which also contribute to metastasis.

9.6 The Emerging Role of Notch Signaling in Tumor Stem Cell Tumor “stem” cells (also called tumor initiating cells) are a small fraction of cancer cells which are characterized by their “stem cell-like” properties. The “stem celllike” traits include the sustained self-renewal, and ability to generate heterogeneous tumor cells and ability to recapitulate the original tumor from one cell. Tumor stem cells have high tumorigenic ability, therapeutic resistance, tumor angiogenesis and maintenance of tumor growth [15, 81, 107]. Although the concept of tumor stem cells has been proposed for long time, recent advances in the identification of tumor stem cells from several human cancers have provided more convincing evidence for this concept. To date tumor stem cells have been identified from breast, lung, colon, prostate, brain, liver, head and neck, pancreas, skin and hematopoietic malignancies. Based on cancer type-specific markers, the percentage of tumor stem cells in the whole tumor cells is various from 0.1 to 40% [15, 107]. Stem cells in normal tissues often exist in a special microenvironment (called the stem cell niche) where they can maintain their self-renewal capacity and multipotency [69]. Although the origin of tumor stem cells is controversial, many signaling factors that support the normal stem cells are reported to regulate tumor stem cells as well. These signaling factors include many receptor tyrosine kinase (RTKs), bone morphogenetic proteins (BMPs), Hedgehog, Wnt, and Notch signaling [56]. Notch signaling members, including ligands and receptors, are expressed in human embryonic stem (ES) cells and their malignant counterpart embryonic carcinoma (EC) cells [28]. Notch blockade by siRNA of Notch1/2 or GSI reduced the proliferation of human ES and EC cells in vitro, suggesting a critical role of Notch in human ES and EC cell proliferation [28]. The Notch target gene HES1 was reported as a key regulator for the maintenance of the quiescent status of adult stem cell and tumor stem cells [87]. Suppression of HES1 by a dominant negative HES1 (dnHES1) or GSI reduced proliferation and promoted differentiation of rhabdomyosarcoma cells, suggesting that Notch/HES1 may inhibit tumor stem cell differentiation. Notch signaling is also essential for neural stem cell maintenance by promoting self-renewal and inhibiting differentiation of neuronal and glial progenitors [31]. In the brain tumor glioma, tumor stem cells were first isolated using CD133 marker [97]. These CD133+ cells showed similar properties to normal neural stem cells (NSCs) in vitro, including presence of neural stem cell markers and the ability

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to produce differentiated progeny. More importantly, in a NOD-SCID (non-obese diabetic, severe combined immunodeficient) mouse xenograft model, as few as 100 CD133+ cells initiated tumor, whereas 105 CD133- cells did not [7]. In these glioma cells, Notch signaling induced the expression of stem cell marker Nestin and the formation of neurosphere-like colonies in vitro [121]. Functionally, activation of Notch signaling in a K-RAS-induced glioma mouse model promoted proliferation of NSC cells, resulting in glioma formation [95]. Inhibition of Notch signaling by GSI strongly suppressed CD133+ cells proliferation, increased their apoptotic rate, and inhibited their colony-forming ability and xenograft brain tumor initiation [24]. JAG1/Notch activation enhanced the expression of Inhibitor of differentiation 4 (Id4) permitting undifferentiated astrocytes to acquire neural stem cell-like properties, leading to increased malignant transformation of gliomas [42]. Other studies reported that ABCG2, an ATP-binding cassette transporter associated with chemo-resistance and cell proliferation, is highly expressed in tumor stem cells with Notch1 expression [74]. Furthermore, it is reported that microRNA199b-5p targets HES1 to block the medulloblastoma stem cell function and tumor growth [32]. Notch signaling regulates normal human mammary development by affecting the mammary stem and progenitor cells [14] and appears to have an oncogenic role in mammary tumorigenesis. Human breast cancer stem cells have upregulated expression of Notch receptors [1, 25]. Notch activation by DSL peptide increased the secondary mammosphere formation in vitro [20] and JAG induced Notch signaling also mediates proliferation and self-renewal in breast cancer stem cells [75]. Several other cancers reveal a connection between Notch signaling and tumor stem cells. Ovarian cancer-initiating cells demonstrate upregulation of Notch1 and other stem cell markers, such as Nestin, ABCG2, Nanog, CD44 and CD177 [119]. Notch inhibition by GSI significantly reduced colon cancer stem cell population (aldehyde dehydrogenase positive) and blocked colon cancer initiation in vitro and in vivo, suggesting that Notch promotes tumor initiating cell function in colon cancer [14]. Similarly, genetic and pharmaceutical inhibition of Notch signaling in pancreatic cancer blocked the anchorage-independent growth in vitro and xenograft tumor initiation in vivo [63]. Interestingly, a recent study showed that a pancreatic cancer-initiating cell population (CD44+/CD133+) can be depleted by microRNA-34 through targeting Notch1/2, indicating a role for Notch signaling in maintenance of pancreatic cancer stem cells [43].

9.7 Summary Notch signaling regulates a great variety of cellular functions and processes from EMT to stem cell self-renewal in a wide variety of cancers (Fig. 9.6). All of these cellular properties contribute to cancer metastasis and make modulation of Notch signaling a potential therapy to suppress and eliminate cancer metastasis. More work is needed, though the rationale for Notch-directed therapy is sound.

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Notch

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Metastasis Fig. 9.6 Notch signaling contributes to many aspects of cancer metastasis

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90. Scehnet JS, Jiang W, et al. Inhibition of DLL4-mediated signaling induces proliferation of immature vessels and results in poor tissue perfusion. Blood 2007; 109(11): 4753–4760. 91. Schaefer K-L, Eisenacher M, et al. Microarray analysis of Ewing’s sarcoma family of tumours reveals characteristic gene expression signatures associated with metastasis and resistance to chemotherapy. Eur. J. Cancer 2008; 44(5): 699–709. 92. Scorey N, Fraser SP, et al. Notch signalling and voltage-gated Na+ channel activity in human prostate cancer cells: independent modulation of in vitro motility. Prost. Cancer Prost. Dis. 2006; 9(4): 399–406. 93. Selkoe D, Kopan R. Notch and presnilin: regulated intramembrane proteolysis links development and degeneration. Annu. Rev. Neurosci. 2003; 26(1): 565–597. 94. Shi W, Harris A. Notch signaling in breast cancer and tumor angiogenesis: cross-talk and therapeutic potentials. J. Mammary Gland Biol. Neoplasia 2006; 11(1): 41–52. 95. Shih A, Holland E. Notch signaling enhances nestin expression in gliomas. Neoplasia 2006; 8(12): 1072–1082. 96. Shou J, Ross S, et al. Dynamics of Notch expression during murine prostate development and tumorigenesis. Cancer Res. 2001; 61(19): 7291–7297. 97. Singh SK, Hawkins C, et al. Identification of human brain tumour initiating cells. Nature 2004; 432(7015): 396–401. 98. Soares R, Balogh G, et al. Evidence for the Notch signaling pathway on the role of estrogen in angiogenesis. Mol. Endocrinol. 2004; 18(9): 2333–2343. 99. Song LL, Peng Y, et al. Notch-1 associates with IKK[alpha] and regulates IKK activity in cervical cancer cells. Oncogene 2008; 27(44): 5833–5844. 100. Suchting S, Freitas C, et al. The Notch ligand Delta-like 4 negatively regulates endothelial tip cell formation and vessel branching. Proc. Natl. Acad. Sci. 2007; 104(9): 3225–3230. 101. Thiery JP, Sleeman JP. Complex networks orchestrate epithelial-mesenchymal transitions. Nat. Rev. Mol. Cell Biol. 2006; 7(2): 131–142. 102. Thurston G, Kitajewski J. VEGF and Delta-Notch: interacting signalling pathways in tumour angiogenesis. Br. J. Cancer 2008; 99(8): 1204–1209. 103. Timmerman LA, Grego-Bessa JN, et al. Notch promotes epithelial-mesenchymal transition during cardiac development and oncogenic transformation. Gene Dev. 2004; 18(1): 99–115. 104. Tohda S, Murata-Ohsawa M, Sakano S, Nara N. Notch ligands, Delta-1 and Delta-4 suppress the self-renewal capacity and long-term growth of two myeloblastic leukemia cell lines. Int. J. Oncol. 2003; 22(5): 1073–1079. 105. Uyttendaele H, Ho J, et al. Vascular patterning defects associated with expression of activated Notch4 in embryonic endothelium. Proc. Natl. Acad. Sci. USA 2001; 98(10): 5643–5648. 106. Vacca A, Felli MP, et al. Notch3 and pre-TCR interaction unveils distinct NF-κB pathways in T-cell development and leukemia. EMBO J. 2006; 25(5): 1000–1008. 107. Visvader JE, Lindeman GJ. Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat. Rev. Cancer 2008; 8(10): 755–768. 108. Wang L, Zeng H, et al. Neuropilin-1-mediated vascular permeability factor/vascular endothelial growth factor-dependent endothelial cell migration. J. Biol. Chem. 2003; 278(49): 48848–48860. 109. Wang Z, Banerjee S, et al. Down-regulation of Notch-1 inhibits invasion by inactivation of nuclear factor-κB, vascular endothelial growth factor, and matrix metalloproteinase-9 in pancreatic cancer cells. Cancer Res. 2006; 66(5): 2778–2784. 110. Wang Z, Kong D, et al. Down-regulation of platelet-derived growth factor-D inhibits cell growth and angiogenesis through inactivation of Notch-1 and nuclear factor-{kappa}B signaling. Cancer Res. 2007; 67(23): 11377–11385. 111. Wang Z, Li Y, et al. Acquisition of epithelial-mesenchymal transition phenotype of gemcitabine-resistant pancreatic cancer cells is linked with activation of the Notch signaling pathway. Cancer Res. 2009; 69(6): 2400–2407.

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112. Weijzen S, Rizzo P, et al. Activation of Notch-1 signaling maintains the neoplastic phenotype in human Ras-transformed cells. Nat. Med. 2002; 8(9): 979–986. 113. Wharton K, Johansen K, et al. Nucleotide sequence from the neurogenic locus notch implies a gene product that shares homology with proteins containing EGF-like repeats. Cell 1985; 43(3 Pt 2): 567–581. 114. Xue Y, Gao X, et al. Embryonic lethality and vascular defects in mice lacking the Notch ligand Jagged1. Hum. Mol. Genet. 1999; 8(5): 723–730. 115. Zavadil J, Cermak L, et al. Integration of TGF-[beta]/Smad and Jagged1/Notch signalling in epithelial-to-mesenchymal transition. EMBO J. 2004; 23(5): 1155–1165. 116. Zayzafoon M, Abdulkadir SA, et al. Notch signaling and ERK activation are important for the osteomimetic properties of prostate cancer bone metastatic cell lines. J. Biol. Chem. 2004; 279(5): 3662–3670. 117. Zeng Q, Li S, et al. Crosstalk between tumor and endothelial cells promotes tumor angiogenesis by MAPK activation of Notch signaling. Cancer Cell. 2005; 8(1): 13–23. 118. Zhang P, Yang Y, et al. Critical role of Notch signaling in osteosarcoma Clinical Cancer Resarch 2008; 14: 2962–2969. 119. Zhang S, Balch C, et al. Identification and characterization of ovarian cancer-initiating cells from primary human tumors. Cancer Res. 2008; 68(11): 4311–4320. 120. Zhang P, Yang Y, et al. Regulation of NOTCH signaling by reciprocal inhibition of HES1 and Deltex 1 and its role in osteosarcoma invasiveness. Oncogene 2010; 29: 2916–2926 121. Zhang X-P, Zheng G, et al. Notch activation promotes cell proliferation and the formation of neural stem cell-like colonies in human glioma cells. Mol. Cell. Biochem. 2008; 307(1): 101–108. 122. Zweidler-McKay PA, He Y, et al. Notch signaling is a potent inducer of growth arrest and apoptosis in a wide range of B-cell malignancies. Blood 2005; 106(12): 3898–3906.

Chapter 10

New Concepts on the Critical Functions of Cancer- and Metastasis-Initiating Cells in Treatment Resistance and Disease Relapse: Molecular Mechanisms, Signaling Transduction Elements and Novel Targeting Therapies Murielle Mimeault and Surinder K. Batra Abstract New concepts on cancer- and metastasis-initiating cells suggest that these immature cells endowed with a high self-renewal potential and aberrant multilineage differentiation ability can provide critical functions for tumor initiation, metastases at distant tissues and organs, treatment resistance and disease relapse. The malignant transformation of multipotent tissue-resident adult stem/progenitor cells or their early progenies endowed with a self-renewal capacity is generally associated with the accumulation of different genetic and/or epigenetic alterations concomitant with the changes in their local microenvironment, niches. Particularly, the cancer progression to locally invasive and metastatic stages is often accompanied by a down-regulation of tumor suppressor genes combined with a sustained activation of distinct growth factor signaling pathways during the epithelial-mesenchymal (EMT) transition program that provide critical functions in the stringent regulation of self-renewal and/or differentiation of tissue-resident adult stem/progenitor cells. The signaling cascades that are often deregulated in highly tumorigenic cancerand metastasis-initiating cells include hedgehog, epidermal growth factor receptor (EGFR), Wnt/β-catenin, NOTCH, polycomb gene product BMI-1 and/or stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4). The cooperation between these signal transduction elements may play a major role for their sustained proliferation, survival, invasion and metastasis. Importantly, the unique intrinsic properties of cancer stem/progenitor cells, including their high expression levels of DNA repair and detoxifying enzymes, anti-apoptotic factors, and ATPbinding cassette (ABC) multidrug transporters, may also be associated with their resistance to the current clinical cancer therapies and disease recurrence. Therefore, the molecular targeting of distinct deregulated signaling elements in cancer- and metastasis-initiating cells and their progenies is of immense therapeutic interest to overcome the treatment resistance. These novel targeting approaches should

M. Mimeault (B) Department of Biochemistry and Molecular Biology, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198-5870, USA e-mail: [email protected] W.-S. Wu, C.-T. Hu (eds.), Signal Transduction in Cancer Metastasis, Cancer Metastasis – Biology and Treatment 15, DOI 10.1007/978-90-481-9522-0_10,  C Springer Science+Business Media B.V. 2010

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eliminate the total cancer cell mass, and thereby improve the efficacy of the current therapeutic regimens against aggressive, metastatic, recurrent and lethal cancers. Keywords Cancer-initiating cells · Metastasis-initiating cells · Carcinogenesis · Treatment resistance · Target therapies Abbreviations ABC ALDH BTSCs CDK COX-2 CXCR4 ECM EGF EGFR EMT Fzd KIT HA MAPKs MEK MDR MMPs MGMT NF-κB NSCs PI3 K PTCH PTEN Rb RTK SDF-1 SHH SMO TERT TK TR uPA VEGF VEGFR Wnt

ATP-binding cassette aldehyde dehydrogenase brain tumor stem cells cyclin-dependent kinase clyooxygenase 2 CXC chemokine receptor 4 extracellular matrix epidermal growth factor epidermal growth factor receptor epithelial-mesenchymal transition Frizzeled receptor stem cell factor receptor hyaluronan mitogen-activated protein kinase extracellular signal-related kinase kinase multidrug resistance matrix metalloproteinases O6-methylguanine DNA methyltransferase nuclear factor-kappa B neural stem cells phosphoinositide 3 -kinase hedgehog patched receptor phosphatase and tensin homolog deleted on chromosome 10 retinoblastoma receptor tyrosine kinase stromal cell-derived factor-1 sonic hedgehog ligand smoothened co-receptor telomerase reverse transcriptase tyrosine kinase telomere RNA component urokinase-type plasminogen activator vascular endothelial growth factor vascular endothelial growth factor receptor Wingless ligand

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Contents 10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 Molecular Mechanisms Associated With the Malignant Transformation of Tissue-Resident Adult Stem Cells in Cancer- and Metastasis-Initiating Cells During Cancer Initiation and Progression . . . . . . . . . . . . 10.2.1 Molecular Transforming Events in Tissue-Resident Stem/Progenitor Cells and Their Progenies Induced Through the Interplay of Diverse Growth Factors, Cytokines and Chemokines 10.2.2 Molecular Transforming Events in Cancer-Initiating Cells and Their Progenies Induced Through the EMT Process and Tumor-Associated Stromal Remodeling . . . . . . . . . . . 10.3 Intrinsic and Acquired Phenotypes of Cancer- and Metastasis-Initiating Cells Associated With Their Resistance to Current Cancer Treatments . . 10.3.1 Functions of ABC Transporters and Anti-apoptotic Factors in Intrinsic and Acquired Multidrug Resistance Phenotypes of Cancer Stem/Progenitor Cells . . . . . . . . . . . . . . . . 10.4 Novel Targeted Therapies Against Aggressive and Recurrent Cancers . . 10.4.1 Molecular Targeting of Tumor- and Metastasis-Initiating Cells and Their Differentiated Progenies . . . . . . . . . . . . . . 10.4.2 Molecular Targeting of the Local Microenvironment of Tumorand Metastasis-Initiating Cells and Their Differentiated Progenies 10.5 Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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10.1 Introduction Major progress in developing earlier diagnostic tests and the identification of new therapeutic targets in cancer cells in the few last years has led to a significant improvement of the overall survival of cancer patients [1–13]. The therapeutic management for the patients diagnosed with localized cancers by surgical resection, anti-hormonal therapy, radiation and/or chemotherapy generally leads to a complete remission and high curative rate of cancer patients. Unfortunately, the rapid progression of organ-confined cancers to locally invasive or metastatic disease stages may result in resistance to the current conventional treatments, disease relapse and the death of cancer patients in a short-term period [8, 9, 11, 14–19]. This inefficacy of the current treatments against aggressive, locally advanced and metastatic cancers underlines the importance to further establish the oncogenic events and molecular mechanisms associated with the resistance of cancer cells to the current cancer therapies. In this regard, recent lines of experimental evidence revealed that the accumulation of genetic and/or epigenetic alterations in adult stem/progenitor cells may result in their malignant transformation into leukemic or tumorigenic cancer stem/progenitor cells (Fig. 10.1) [15, 20–36]. The highly

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Fig. 10.1 Model of epithelial cancer initiation and progression mediated through tumorigenic and migrating cancer stem/progenitor cells. This scheme shows the molecular events associated with the malignant transformation of tissue-resident adult stem cells into tumorigenic cancer stem cells during cancer initiation through the inactivation of tumor suppressor genes combined with the activation of oncogenic signaling elements. The asymmetric division of cancer stem cells, also termed as tumor-initiating cells, localized in the epithelial compartment into transit-amplifying (TA) cancer progenitor cells which can generate the bulk mass of differentiated cancer cells constituting the solid tumor is indicated. Furthermore, the acquisition of a migratory phenotype by tumorigenic stem/progenitor cells, which may be induced by the sustained activation of distinct growth factor signaling pathways during the epithelial-mesenchymal transition (EMT) program, is also shown. The possible invasion of certain tumorigenic and migrating cancer stem/progenitor cells, also designated as metastasis-initiating cells in the activated stroma, which may lead to their dissemination through the peripheral circulation and metastases at distant tissues is also illustrated

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tumorigenic cancer stem/progenitor cells, also designated as cancer- or tumorinitiating cells, can provide critical roles for primary tumor growth, metastases at distant tissues and organs, resistance to current conventional therapies and disease relapse [10, 15, 16, 24, 25, 28, 29, 37–49]. In support of the major implication of cancer stem/progenitor cells in cancer initiation and progression, the small subpopulations of immature cancer stem/progenitor cells with stem cell-like properties, comprising about 0.1–3% of total tumor cell mass, have been identified in most of the human cancers [15, 21, 27, 28, 37–45, 47, 50–68]. The cancer stem/progenitor cells typically express several specific stem cell-like markers such as telomerase, aldehyde dehydrogenase (ADLH), CD133, CD44, ATP binding-cassette (ABC) multidrug transporters and/or CXC chemokine receptor-4 (CXCR4), but lack differentiation marker expression. The highly tumorigenic cancer stem/progenitor cells were able to give rise in vitro and in vivo to further differentiated tumor cells expressing phenotypes of the original patient’s tumors [21, 27, 28, 38–44, 47, 51–68]. In addition, it has been shown that the cancer progression to locally invasive and metastatic stages is generally associated with the occurrence of distinct molecular events in cancer stem/progenitor cells and their progenies that may contribute to their acquisition of a more malignant behavior (Fig. 10.1) [8, 13, 15–17, 21, 24, 32–34, 40, 42, 45, 46, 69–80]. Particularly, the inactivation of diverse tumor suppressor gene products and stimulation of diverse oncogenic signaling elements in cancer stem/progenitor cells, combined with the changes in their local microenvironment, including the release of soluble factors by host activated stromal cells, may promote cancer development [8, 13, 15, 16, 21, 24, 32–34, 40, 45, 46, 70–83]. Moreover, the acquisition of a migratory phenotype by tumorigenic cancer stem/progenitor cells during the epithelial-mesenchymal transition (EMT) process, may result in their invasion and metastases at distant tissues and organs (Fig. 10.1) [15–17, 21, 32, 33, 40, 72–75, 80, 82–92]. Consistently, the tumorigenic and migrating cancer stem/progenitor cells, also designated as metastasis-initiating cells, have been detected at invasion sites in primary tumors as well as isolated from peripheral blood and secondary tumor samples of cancer patients and metastatic cancer cell lines [40, 42, 44, 47, 51, 54, 58, 62, 66, 72, 82, 93–98]. Importantly, the results from numerous recent studies have also revealed that the tumorigenic cancer stem/progenitor cells may be more resistant than their differentiated progenies to the current clinical anti-cancer therapies [10, 15, 17, 29, 40, 41, 44–48, 61, 63–66, 69, 70, 80, 91, 99–114]. Consequently, the persistence of cancer-initiating cells at the primary and secondary malignant neoplasms after treatment initiation may be responsible for the tumor re-growth and disease relapse. In this matter, we describe here the molecular oncogenic events that frequently occur in cancer stem/progenitor cells versus their differentiated progenies during cancer etiology and progression to locally invasive and metastatic disease stages. Of therapeutic interest, we also discussed the potential therapeutic drug targets in tumor- and metastasis-initiating cells. The information provided should help to design new targeting strategies for eradicating the total cancer cell mass including tumor- and metastasis-initiating cells and improve the current cancer treatments against aggressive, metastatic, recurrent and lethal cancers.

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10.2 Molecular Mechanisms Associated With the Malignant Transformation of Tissue-Resident Adult Stem Cells in Cancer- and Metastasis-Initiating Cells During Cancer Initiation and Progression Numerous recent studies suggest that cancer development may generally derive from the clonal expansion and aberrant differentiation of cancer stem cells (CSCs) and/or their early progenies endowed with a high self-renewal capacity that trigger tumor growth (Fig. 10.1) [16, 17, 33, 74, 75]. Among the cancer types harboring a subpopulation of leukemic or tumorigenic cancer stem/progenitor cells, there are leukemias, lymphomas, sarcomas, melanoma, brain tumors and a variety of epithelial cancers, including skin, head and neck, thyroid, lung, cervical, renal, hepatic, esophageal, gastrointestinal, colon, bladder, pancreatic, prostatic, mammary and ovarian cancers [15, 21, 27, 28, 37–45, 47, 49–64, 66–68, 115]. It has been shown that the small subpopulations of isolated cancer stem/progenitor cells with stem cell-like properties displayed a greater clonogenic potential in vitro and generated leukemias or tumors with a higher incidence as compared to their differentiated progenies in the animal model [21, 27, 28, 38–44, 47, 51–68, 116]. In this regard, it is noteworthy that different cancer subtypes with variable degrees of heterogeneity in functional and/or differentiation phenotypes of cancer cells may harbor distinct subsets and/or a different number of cancer-initiating cells during primary cancer progression and metastasis formation at distant sites as well as before or after therapy initiation and disease recurrence [45, 46, 65, 82, 97, 98, 117–121]. Moreover, the in vivo estimation of the number of cancer-initiating cells that are able to drive cancer formation may be influenced by the experimental cancer cell and animal models used [97, 117, 118]. For instance, a high proportion of human melanoma cells isolated from patients with primary and metastatic melanomas and expressing different phenotypic markers were able to form tumors in highly immunocompromised mouse models in vivo [97]. In addition, the cancer progression is usually associated with the acquisition of a more malignant behavior by leukemic or tumorigenic stem/progenitor cells and their progenies [21, 27, 28, 39, 40, 42–44, 47, 51–61, 63–68, 121].

10.2.1 Molecular Transforming Events in Tissue-Resident Stem/Progenitor Cells and Their Progenies Induced Through the Interplay of Diverse Growth Factors, Cytokines and Chemokines The transition from non-malignant hyperproliferative lesions to well-established cancers has been associated with the occurrence of some oncogenic events in tissueresident adult stem/progenitor cells and their microenvironment resulting in their acquisition of a malignant behavior [16, 17, 33, 49, 74, 75, 78, 82, 121, 122]. The

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transforming events include the stimulation of telomerase and inactivating mutations in numerous tumor suppressor gene products [p16INK4A , pRb, p53 and/or phosphatase and tensin homolog deleted on chromosome 10 (PTEN)]. Moreover, a constitutive activation of diverse growth factors and oncogenic signaling products [Ras, Myc, NF-κB, PI3 K/Akt/mTOR, Bcl-2, survivin and/or fusion proteins resulting from chromosomal rearrangements] frequently occurs during cancer progression (Fig. 10.1) [13, 15, 22, 34, 49, 70, 123–128]. In addition, the up-regulation of distinct oncogenic signaling pathways in tumorigenic cancer stem/progenitor cells during cancer progression may also contribute to their sustained growth, survival and/or invasion [13, 15, 16, 24, 32–34, 45, 46, 49, 73–75, 79–81, 93, 129]. These signaling pathways include hedgehog, epidermal growth factor (EGF)-EGFR system, Wnt/β-catenin, NOTCH, SCF/KIT, hyaluronan (HA)/CD44 receptor, interleukin-4 (IL-4)/IL-4Rα, stromal cell-derived factor-1 (SDF-1)/CXCR4, and/or polycomb group (PcG) proteins (Figs. 10.1 and 10.2). For instance, the stimulation of the HA/CD44 signaling cascade may lead to the activation of multiple growth factor signaling pathways and the up-regulation of distinct anti-apoptotic factors and ABC transporters [130–132]. The growth factor cascades induced through the activation of the HA/CD44 axis includes receptor tyrosine kinases (RTKs) such as EGFR, ErbB2, insulin growth factor 1 receptor-β (IGF1R-β), platelet-derived growth factor-β (PDGFR-β) and/or the c-MET receptor of hepatocyte growth factor (HGF) [130–132]. Hence, these effects mediated through the stimulation of HA-CD44 axis may promote the survival, invasion, multidrug resistance and/or protection against DNA oxidative damages of cancer stem/progenitor cells and their progenies. In general, the cancer development is also accompanied by an enhanced glycolysis in cancer cells including cancer stem/progenitor cells [82, 133–135]. This phenomenon known as the Warburg effect may contribute to the resistance of cancer cells to oxidative stress as well as their survival in intratumoral hypoxic conditions [82, 133–135]. Moreover, the EMT phenomenon, which occurs through the tissue and organ morphogenesis and patterning during embryonic development as well as tissue regeneration and wound healing in adults, is also re-activated during the progression of numerous aggressive cancers. Among them, there are brain, skin, prostate, ovarian, mammary, hepatic, gastrointestinal, pancreatic and colorectal carcinomas [15–17, 21, 32, 33, 40, 72–75, 82–87, 89, 136].

10.2.2 Molecular Transforming Events in Cancer-Initiating Cells and Their Progenies Induced Through the EMT Process and Tumor-Associated Stromal Remodeling The acquisition of a more malignant behavior by tumor-initiating cells and their progenies in primary malignant neoplasms, including a migratory phenotype during the EMT process, represents a determinant factor that may contribute to cancer progression to locally invasive and metastatic cancer subtypes [15–17, 21, 32, 33, 40,

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Fig. 10.2 New cancer therapies by molecular targeting distinct signaling pathways and multidrug transporters involved in aggressive behavior and multidrug resistance phenotype of tumorigenic and migrating cancer stem/progenitor cells. The cytotoxic effects induced by diverse pharmacological agents such as the selective inhibitors of interleukin-4 receptor (IL-4R), receptor tyrosine kinase (RTK) activity (gefitinib or erlotinib), smoothened (SMO) hedgehog signaling element (cyclopamine or IPI-269609), CXC chemokine receptor 4 (CXCR4), telomerase, Akt/mTOR and NF-κB on cancer stem/progenitor cells are indicated. Moreover, the anti-carcinogenic effects induced by a monoclonal antibody (mAb) directed against the IL-4 ligand, CD133 or CD44 stem cell-like marker, sonic hedgehog (SHH) or Wnt ligand and stromal cell-derived factor-1 (SDF-1) are also shown. Particularly, the anti-proliferative, anti-invasive and apoptotic effects induced by these pharmacological agents in cancer stem/progenitor cells through the down-regulation of the expression levels of numerous gene products are indicated. In addition, the potent inhibitory effect induced either by a specific inhibitor of ATP-binding cassette (ABC) multidrug transporters on drug efflux or aldehyde dehydrogenase (ALDH) such as (DEAB) on drug metabolism is also illustrated. These agent types can enhance the intracellular drug accumulation and decreased drug resistance

72–75, 82–91, 136]. The occurrence of the EMT program in cancer stem/progenitor cells and their progenies may lead to changes in their differentiation including a loss of epithelial cell markers concomitant with a gain of mesenchymal phenotypes that promote their migratory ability. This process is generally associated with a disruption of cell-cell junctions, loss of contact inhibition and extensive reorganization of the actin cytoskeleton and remodeling of extracellular matrix (ECM) components that lead to an increase of the motile and invasive abilities of cancer cells (Fig. 10.1) [15–17, 21, 32, 33, 40, 72–75, 82–91, 136]. A complex network of different cascades initiated through different growth factors and their cognate receptors may cooperate to induce a more complete EMT program in cancer cells including cancer stem/progenitor cells (Fig. 10.1) [73, 74, 82, 84–88, 90, 91, 111, 136, 137]. Among them, there are the sonic hedgehog SHH/PTCH/SMO/GLIs,

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EGF/EGFR/Ras/MAPKs, fibroblast growth factor (FGF)/FGFR, Wnt/β-catenin, transforming growth factor-β (TGF-β) superfamily cytokines and PDGF/PDGFR (Fig. 10.2) [73, 74, 82–88, 90, 91, 111, 136–138]. The sustained stimulation of these growth factor pathways may result in an up-regulation of diverse gene products in cancer stem/progenitor cells and their differentiated progenies during the EMT program. The molecules that are frequently altered in cancer cells during the EMT process include a decreased expression of E-cadherin concomitant with an up-regulation of different signaling elements, such as N-cadherin, vimentin, nuclear factor-kappaB (NF-κB), snail, slug, twist, β-catenin, CXCR4 and anti-apoptotic factors [40, 72–74, 82–85, 87, 88, 90–92, 127, 129, 136, 138]. These molecules may contribute to the invasive phenotype of cancer cells and enhanced treatment resistance. In addition, the cancer progression is also accompanied by an extensive tumor stromal remodeling of ECM components and changes in gene expression patterns in activated tumor-associated stromal cells including myofibroblasts and/or stellate cells, as well as infiltrating circulating endothelial progenitor cells (EPCs) and immune cells such as macrophages (Fig. 10.2) [2, 10, 15, 17, 33, 74, 75, 139–141]. The soluble growth factors, cytokines and chemokines released by tumor stromal cells in reactive stroma include EGF, insulin-like growth factor (IGF), hepatocyte growth factor (HGF), TGF-β and SDF-1 as well as matrix metalloproteinases (MMPs) and urokinase plasminogen (uPA) (Fig. 10.1) [10, 15, 17, 33, 72, 74, 75, 88, 136, 139, 140]. Hence, these molecular transforming events may result in the acquisition of an aggressive behavior by cancer stem/progenitor cells, and thereby contribute to their invasion, dissemination through the peripheral circulation, metastasis at distant tissues and organs, resistance to current therapeutic treatments and disease recurrence.

10.3 Intrinsic and Acquired Phenotypes of Cancerand Metastasis-Initiating Cells Associated With Their Resistance to Current Cancer Treatments The resistance of cancer stem/progenitor cells to the current clinical treatments by chemotherapy or radiation has been associated with several intrinsic properties of these immature cancer cells common with their normal counterpart, tissue-resident adult stem/progenitor cells and/or their acquisition of more malignant phenotypes during cancer progression [10, 15, 17, 29, 40, 41, 44–48, 61, 63–66, 69, 70, 80, 91, 99, 100, 102–113]. The molecular mechanisms that may contribute to the resistance of cancer- and metastasis-initiating cells to the current cancer therapies include their slow division and the high expression levels of DNA-repair and detoxifying enzymes [10, 15, 17, 19, 41, 45, 75, 99, 113, 119, 138, 139, 142–157]. More specifically, the elevated expression levels of O6-methylguanine DNA methyltransferase (MGMT) and ALDH may protect these immature cells against the cytotoxic effects induced by different chemotherapeutic drugs (Fig. 10.2) [41, 45, 99, 113,

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119, 138, 153–157]. Moreover, the cancer stem/progenitor cells typically express high expression levels of distinct ABC multidrug transporters and anti-apoptotic factors such as Bcl-2, survivin and NF-kB that may contribute to their multidrug resistance (MDR) phenotype [15–17, 29, 45, 46, 55, 63, 99, 113, 114, 119, 158–165].

10.3.1 Functions of ABC Transporters and Anti-apoptotic Factors in Intrinsic and Acquired Multidrug Resistance Phenotypes of Cancer Stem/Progenitor Cells The large family of ABC multidrug transporters is comprised of diverse transmembrane proteins constituted of one or two transmembrane spanning domains (TMDs) involved in substrate binding and one or two cytoplasmic nucleotide (ATP)-binding domains (NBDs) [17, 166–171]. The ABC multidrug transporters frequently expressed on primitive cancer stem/progenitor cells include the multidrug resistance 1 (MDR1/ABCB1) encoding P-glycoprotein (P-gp), breast cancer resistant protein (BCRP-1/ABCG2) and multidrug resistance associated proteins (MRPs) (Fig. 10.2) [17, 114, 162, 166–169, 172]. The ABC transporters localized at the plasmic membrane may protect these immature cancer cells from cytotoxic effects induced by diverse structurally and functionally non-related chemotherapeutic drugs and in this manner contribute to their MDR phenotype (Fig. 10.2) [17, 99, 169, 171–173]. In fact, the ABC efflux pumps can actively remove intracellular cytotoxic agents out of cells at the expense of ATP hydrolysis. Certain ABC transporter types, such as ABCA2 and ABCA3, may also be localized in the endolysosomal compartment in cancer cells including cancer stem/progenitor cells [63, 167, 174]. These intracellular ABC transporters found in the lysosomal membrane can mediate a sequestration of intracellular toxic compounds, including chemotherapeutic drugs, and thereby induce a decrease of the intracellular drug concentration that promotes the MDR phenomenon [63, 167, 174]. In regard with this, the high expression of ABC transporters in cancer stem/progenitor cells is notable at the basis of the Hoechst dye exclusion method which is useful to isolate a very small cell fraction designated as the side population “SP”, displaying a high capacity to efflux Hoechst 33342 dye from the bulk cancer cell mass [15, 17, 99, 106, 175–177]. It has been observed that a small subset of SP cells isolated from patient’s tumors or different cancer cell lines, which display the stem cell-like properties and express high levels of distinct ABC drug efflux pumps and anti-apoptotic factors, were more tumorigenic in the animal model in vivo and resistant to chemotherapeutic drugs or irradiation therapy than non-SP cells [15, 45, 63–66, 69, 80, 99, 103–107, 158, 175]. For instance, hepatoma HuH7 SP cells expressing high levels of MDR1/P-gp, BCRP/ABCG2, and CEACAM6 displayed a higher resistance to doxorubicin, 5-fluorouracil and gemcitabine than non-SP cells [80]. A SP cell subpopulation detected in neuroblastoma cells from 15 out of 23 patients (65%), which expressed high levels of BCRP/ABCG2 and ABCA3 transporters also showed a

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greater capacity to efflux cytotoxic drugs, such as mitoxantrone as compared to the non-SP cell fraction [63]. In addition, the acquisition of a more malignant phenotype by cancer stem/progenitor cells during cancer progression or following treatment initiation with chemotherapeutic agents or radiation has also been associated with the occurrence of more aggressive cancers displaying a high rate of growth, metastasis and therapy resistance [91, 99, 110, 111, 166–168, 178]. In this matter, it has been reported that the number of CD44+ /CD24–/low mammosphere cell cultures established from human MCF-7 breast cancer cells was increased after radiotherapy as compared to the adherent breast cancer cell fraction [112]. Moreover, the treatment of lung tumor cells with doxorubicin, cisplatin, or etoposide resulted in the selection of drug surviving cells (DSCs) endowed with a self-renewal capacity and expressing stem cell-like markers such as CD133, CD117, SSEA-3, TRA1–81, Oct-4, and nuclear β-catenin [111]. Lung DSCs also express elevated levels of angiogenic and growth factors such as vascular endothelial growth factor (VEGF), basic FGF (bFGF), IL-6, IL-8, hepatocyte growth factor (HGF), PDGF-BB, granulocyte-colony stimulating factor (G-CSF), and stem cell growth factor-β (SCGF-β) [111]. They also displayed high tumorigenic and metastatic potential in the severe combined immunodeficient (SCID) mice model in vivo [111]. Hence, all these aforementioned intrinsic or acquired properties of cancer- and metastasis-initiating cells may provide them with survival advantages as compared to their differentiated progenies and contribute to their resistance to the current treatments and disease relapse. In respect with this, we are reporting new promising targeting approaches that have been developed for overcoming treatment resistance, eradicating cancer- and metastasis-initiating cells and their differentiated progenies and improving the current cancer therapies.

10.4 Novel Targeted Therapies Against Aggressive and Recurrent Cancers The development of locally advanced, invasive and metastatic cancers that are resistant to the current therapies represents one of the major causes of disease recurrence and cancer-related deaths [8, 9, 11, 14–17]. Therefore, the molecular targeting of distinct oncogenic products in cancer cells involved in tumor progression may constitute more promising therapeutic approaches as compared to monotherapy for aggressive and recurrent cancers [16, 17, 161, 179]. Since recent studies have revealed that a subset of tumorigenic and/or migrating cancer stem/progenitor cells can contribute in a substantial manner to drive tumor growth and metastases at distant tissues and organs, resistance to current conventional therapies and disease relapse, the molecular targeting of these immature cancer cells must be considered (Fig. 10.2) [10, 15–17, 22, 23, 27, 31–33, 161]. Of particular interest, we reviewed recent advances on the development of novel targeting approaches that have been designed to eradicate the total cancer cell mass consisting

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of tumorigenic and migrating cancer stem/progenitor cells and their differentiated progenies. These molecular targeting strategies offer great promise for improving the current treatments against locally invasive, metastatic and recurrent cancers.

10.4.1 Molecular Targeting of Tumor- and Metastasis-Initiating Cells and Their Differentiated Progenies The molecular targeting of deregulated signaling elements that mediate the transforming events occurring in tumorigenic and migrating cancer stem/progenitor cells and their differentiated progenies during cancer progression, and more particularly during the EMT process, represents new promising therapeutic strategies to improve the current cancer therapies [15–17, 32, 33, 74, 75, 161]. Among the potential molecular targets often altered in tumor- and metastasis-initiating cells and their differentiated progenies during cancer progression, there are diverse developmental signaling pathways. These deregulated pathways include hedgehog, EGFR, Wnt/β-catenin, NOTCH, HA/CD44, TGF-β/TGFRβ, IL-6/IL-6R, BMI-1, stem cell factor (SCF)/KIT, ECM/integrin and/or SDF-1/CXCR4 signaling elements (Fig. 10.2 and Table 10.1) [15, 16, 32, 33, 42, 74, 75, 120, 130, 132, 138, 161, 165, 180–189]. It has been reported that the blockade of these tumorigenic pathways by using a specific inhibitor or antagonist, monoclonal antibody (mAb) or antisense oligonucleotides (As) led to a growth inhibition, apoptotic death and/or a reduction of invasiveness or metastatic spread of cancer cells in vitro or in animal models in vivo (Fig. 10.2) [15, 16, 32, 33, 42, 74, 75, 130, 132, 138, 161, 180, 181, 183–187, 190–192]. Importantly, the data from recent studies have indicated that the molecular targeting of these oncogenic elements could be effective to eliminate the total cancer cell mass including tumor-initiating cells in certain cancer types. For instance, it has been observed that the EGFR tyrosine kinase activity inhibitor (TKI), gefitinib or erlotinib induced significant anti-proliferative and cytotoxic effects in vitro on isolated CD133+ /EGFR+ brain tumor-initiating cells from patients with primary gliomablastomas (GBM TICs) (Table 10.1) [121]. Two GBM TICs among seven glioma specimens analyzed, which displayed a high level of Akt activation, were however insensitive to this treatment type [121]. In the same way, the inhibition of smoothened (SMO) hedgehog co-receptor by using cyclopamine or orally bioavailable small-molecule hedgehog inhibitor, IPI-269609 also reduced the number of tumor-initiating cells and their progenies [138, 183, 184, 191, 192]. Moreover, the blockade of the hedgehog cascade by these chemical agents also improved the cytotoxic and anti-metastatic effects induced by the current chemotherapeutic drugs on prostate and pancreatic cancer cell lines as well as gliomasphere cells in vitro and/or in vivo (Table 10.1) [138, 183, 184, 191, 192]. The blockade of the HA-CD44 tumorigenic cascade using a specific anti-CD44 antibody or HA antagonist such as HA oligomers has also been observed to inhibit the survival, growth, mobility, invasion and/or metastases of diverse cancer cells in vitro and/or in vivo [130, 132, 180, 181]. Also, the inhibition of HA-CD44 signaling pathway enhanced the chemosensitivity of the cancer cells. For instance, it has been observed that the inhibition of the CD44 signaling pathway using

Lapitinib plus anti-ErbB2 mAb, trastuzumab

SMO inhibitor (cyclopamine)

EGFR (HER-1)/ErbB2 (HER2)

Hedgehog

Gefitinib or erlotinib

Anti-CD 133 mAbs directed against epitope 1 (AC 133/W6B3C1) or epitope 2 (AC141)

CD133 stem cell-like surface marker

Oncogenic signaling element EGFR

Name of inhibitory agent

Targeted element

Brain cancer

Breast cancer

Brain cancer

Melanoma

Cancer type

Induction of anti-proliferative and cytotoxic effects by gefitinb or erlotinib on EGFR+ /CD133+ tumor-intiating cells from 5 patients with gliomablastomas (GBM TICs). It has been noticed that 2 cases of GBM TICs with high Akt activation were insensitive to both drugs or only sensitive to high concentrations of erlotinib. Treatment of 40 women with erbB-2-positive breast cancer with oral dual TKI of EGFR and erbB-2, lapitinib alone for 6 weeks followed by 6 weeks with standard chemotherapy and anti-erbB2 mAb, trastuzumab caused a complete tumor regression in 63% patients. These date suggest that this treatment type could be effective to reduce the number of CD44+ /CD24low breat cancer-initiating cell-like cells. Long-term cyclopamine treatment eradicated all of the CD133+ gliomasphere cells in GBM tumor samples from patients in culture in vitro and inhibited the formation of gliomasphere cell-derived tumors in vivo. Additive or synergistic proliferative and apoptotic effects induced by low doses of cyclopamine plus temozolomide on human gliomasphere cells in vitro.

Induction of cytotoxic effects in FEMX-1 melanoma cells in vitro and decrease of their metastatic capacity in a mouse model in vivo.

Response of cancer cells to anti-carcinogenic effect induced by inhibitory agent

Table 10.1 Potential therapeutic molecular targets involved in sustained growth and survival of tumor- and metastasis-initiating cells

[191]

[120]

[121]

[193]

References

10 New Concepts on the Critical Functions of Cancer- and Metastasis-Initiating Cells 187

HA/CD44 axis

Targeted element

Prostate cancer

Pancreatic cancer

Pancreatic cancer

Pancreatic cancer

SMO inhibitor (cyclopamine)

SMO inhibitor (cyclopamine)

SMO inhibitor (cyclopamine)

Orally bioavailable small-molecule hedgenog inhibitor, IPI-269609

Brain cancer

Prostate cancer

SMO inhibitor (cyclopamine)

Small HA oligosacccharides

Cancer type

Name of inhibitory agent Complete regression of the in vivo growth of human CWR22RV1 and PC3 prostatic cancer cell line-derived xenografts without sign of tumor recurrence after 58 and 148 days of treatment cessation, respectively. These data suggest that the prostate cancer-initiating cells may be eliminated by cyclopamine treatment. Cytotoxic effects induced in vitro by low concentrations of cyclopamine, and chemotherapeutic drug, mitoxantrone, alone or in combination on CD44+/high and CD44−/low DU145 and PC3 cell fractions. Combined treatment with cyclopamine and gemcitabine of in vivo xenografts established from human Panc 185 pancreatic cells induced a tumor regression and decrease in cancer stem cell-like markers such as CD24 and ALDH. Reduction in the number of putative ALDH-expressing progenitor cells in human E3LZ10.7 pancreatic cell line in vitro and inhibition of metastases of E3LZ10.7 cells in an orthotopic xenograft model in vivo. Reduction of the cell fraction with high ALDH activity detected in primary tumors established from orthotopic implantation of human E3LZ10.7 pancreatic cell line in vivo and inhibition of systemic metastases of E3LZ10.7 cells in an orthotopic xenograft model in vivo. Inhibition of the in vivo growth of spinal cord gliomas formed after engraftment of the SP cell fraction from C6 glioma cell line in rats.

Response of cancer cells to anti-carcinogenic effect induced by inhibitory agent

Table 10.1 (continued)

[132]

[183]

[138]

[192]

[182]

[184]

References

188 M. Mimeault and S.K. Batra

Pancreatic cancer

Liver cancer

Anti-CD44 mAb

Anti-CXCR4 mAb

Cancer type

Name of inhibitory agent

Induction of apoptosis in CD90+ and CD90− cell fractions from MHCC-97L liver cell line but only in CD90+ cells from PLC cell line in vitro. Suppression of local and systemic tumor formation induced by the CD90+ cell fraction from MHCC-97L cell line subcutaneously or orthotopically implanted in liver of nude mice in vivo. Reduction of metastatic capacity of highly metastatic human pancreatic cancer cell line L3.6 pl harborning a CD133+ /CXCR4+ cancer cell subpopulation orthotopically implanted in the pancreas of nude mice in vivo.

Response of cancer cells to anti-carcinogenic effect induced by inhibitory agent

[40]

[42]

References

CXCR4, CXC chemokine receptor 4; EGFR; epidermal growth factor receptor; HA, hyaluronan; mAb, monoclonal antibody; SDF-1, stromal cell-derived factor-1; SMO, smoothened; TKI, tyrosine kinase activity inhibitor.

SDF/CXCR4 axis

Targeted element

Table 10.1 (continued)

10 New Concepts on the Critical Functions of Cancer- and Metastasis-Initiating Cells 189

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anti-human CD44 mAb induced the apoptosis in CD90+ /CD44+ progenitor cells isolated from human hepatocellular carcinoma (HCC) cell lines, MHCC-97L and PLC (Table 10.1) [42]. Furthermore, anti-CD44 mAb treatment also prevented the formation of local and metastatic tumor nodules in the liver and the lungs induced by highly aggressive and tumorigenic CD90+ /CD44+ progenitor cells isolated from the MHCC-97L cell line [42]. Of particular interest, the results from a recent study have also revealed that the use of mAbs directed against two different epitopes of the stem cell-like surface marker, CD133 transmembrane glycoprotein induced the cytotoxic effects in FEMX-I melanoma cells in vitro and markedly decreased their metastatic capacity in the mouse model in vivo (Fig. 10.2 and Table 10.2) [193]. Moreover, it has been observed that the melanoma stem cells express the epigeneticallyregulated cancer testic antigens (CTAs) suggesting that these immature cells could be efficiently targeted by CTA-directed immunotherapeutic strategies [194]. Further investigations to more precisely establish the expression levels and specific functions of the CD133 protein and other stem cell-like markers in normal adult stem/progenitor cells versus cancer stem/progenitor cells is essential to develop more effective molecular targeting-based cancer treatments and immunotherapies without secondary effects on regenerative cells in different tissues. Furthermore, the design of multiple targeted therapies and immunotherapeutic strategies that selectively target distinct oncogenic products and/or cell surface marker(s) expressed on cancer stem/progenitor cells may also represent more potential approaches. These therapeutic strategies could overcome the aggressive behavior acquired by immature tumor-initiating cells during cancer progression as well as the heterogeneity of phenotypic markers expressed on these immature cells in certain cancer subtypes. Other potential molecular therapeutic targets also comprise the gene products that are frequently involved in sustained growth, enhanced survival and invasion during the EMT process and/or drug resistance of cancer stem/progenitor cells and their differentiated progenies [10, 17, 32, 33, 74, 87, 99, 102, 138–140, 165, 195, 196]. These cellular signaling effectors include telomerase, Cripto-1, tenacin C, NF-kB, PI3 K/Akt/mTOR, interleukin-4 (IL-4)/IL-4Rα, Bcl-2, survivin, snail, slug, twist, ABC multidrug efflux pumps and/or ALDH as well as deregulated apoptotic signaling elements such as ceramide and caspases. Importantly, some recent investigations have indicated the potential benefit of targeting these signaling elements to overcome MDR phenotype and radioresistance of cancer cells, including cancer stem/progenitor cells (Fig. 10.2 and Table 10.2) [15, 17, 44, 92, 102, 106, 108, 119, 161, 165, 197–199]. For instance, it has been observed that the inhibition of the hedgehog or EGFR cascade by using cyclopamine or gefitinib may repress the expression levels of ABC transporters in human metastatic prostate PC3 cells and head and neck squamous carcinoma cell lines including the SP cell subpopulation [197, 199]. The treatment of the SP cell fraction from human oral squamous cell carcinoma cell line H357 with a broad spectrum ABC transporter inhibitor, verapamil has also been observed to block the mitoxantrone efflux-mediated via ABCG2 and MPR-1/ABCC1 pumps and restore the growth inhibitory effect induced by mitoxantrone on this cancer stem cell-like population [106]. Importantly, the molecular targeting of putative melanoma-initiating cells by

Colorectal cancer

ALDH1A-targeted shRNA

Melanoma

Anti-ABCB5 mAb

Colorectal cancer

Liver cancer

ABCG2-specific inhibitor, 4-FTC or Pl3 K/Akt/mTOR inhibitor, LY294002 or rapamycin

ALDH1-specific inhibitor, DEAB

Head and neck SCC

Gefitinib

ALDH

Aerodigestive SCC

Verapamil

ABC transporters

Cancer type

Name of inhibitory agent

Targeted element Sensibization of parental human H357 cell line and isolated SP cell fraction expressing MPR-1/ABCC1 and ABCG2 transporters to the growth inhibitory effect induced by mitoxantrone on colonie-forming cell ability in vitro. Activation of EGFR following EGF treatment increased the percentage of ABCG2 expressing SP cells detected by Hoechst dye exclusion technique while gefitinib (lressa) treatment reduced the SP cell number at least in part by decreasing ABCB2 transporter molecules on cell surface. Enhanced sensitivity of the SP cell fraction from MHCC-97L hepato-cellular carcinoma cell line to chemotherapeutic durg, doxorubicin by decreasing ABCG2-mediated drug efflux. Akt signaling inhibition may alter the subcellular localization of ABCB2 transporter. Inhibition of tumor formation and growth of human primary melanoma cell-derived xenografts established in nude mice in vivo associated with a reduction in the number of ABCB5+ melanoma-initiating cells. Enhanced sensitivity of human colorectal tumor lines to cytotoxic effects induced in vitro by bioactive metabolite (4-HC) of chemotherapeutic drug, cyclophosphamide (CPA). Decrease in the number of residual tumorigenic ESA+ /CD44+ /CD166+ CoCSCs detected in human colorectal tumor line-derived xenografts subcutaneously established in mice in vivo after treatment with CPA.

Response of cancer cells to anti-carcinogenic effect induced by inhibitory agent

[119]

[119]

[44]

[198]

[197]

[106]

References

Table 10.2 Potential therapeutic molecular targets involved in survival and drug resistance of tumor- and metastasis-initiating cells and tumor angiogenesis

10 New Concepts on the Critical Functions of Cancer- and Metastasis-Initiating Cells 191

Brain cancer

Reduction of the microvasculature density and tumor growth of vessel-associated CD133+ /nestin+ BTSCs in U87 glioma cell-derived orthotopic xengrafts.

Preferential in vitro inhibition of proliferation and/or colony formation of MCF7 sphere cells or SP cells relative to parental MCF7 cells or the non-SP cell fraction, respectively. Decrease of the viability of CD133+ primary prostatic tumor cells but not CD133+ normal cells from benign prostatic hyperplasia, BPH.

Response of cancer cells to anti-carcinogenic effect induced by inhibitory agent

[207]

[165]

[107]

References

ALDH, aldehyde dehydrogenase; BTSCs, brain tumor stem cells; CPA, cyclophosphamide; DEAB, diethylaminobenzaldehyde; DETC, diethyldithiocarbamate; EGFR; epidermal growth factor receptor; 4-FTC, fumitremorgin C; 4-HC, 4-hydroxycyclophosphamide; MRP-1, multidrug resistance protein-1; PDTC, pyrrolidinethiocarbamate; Pl3 K, phosphatidylinositol 3 -kinase; SCC, squamous cell carcinoma; VEGF, vascular endothelial growth factor.

Anti-VEGF mAb, bevacizumab

Prostate cancer

Parthenolide

Tumor angiogenic signaling VEGF

Breast cancer

Panthenolide PDTC and its analog DETC

NF-kB

Cancer type

Name of inhibitory agent

Targeted element

Table 10.2 (continued)

192 M. Mimeault and S.K. Batra

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using a mAb directed against ABCB5 transporter significantly reversed resistance of G3361 melanoma cells to doxorubicin and induced an inhibitory effect on the growth of melanoma stem cell xenograft-derived tumor in vivo (Table 10.2) [44]. In the same pathway, the inhibition of the Akt signaling element in the SP cell fraction sorted from MHCC-97L hepatocellular carcinoma cells also induced an intracellular translocation of the ABCG2 transporter, attenuated the cellular efflux of doxorubicin and increased doxorubicin-induced cytotoxicity [198]. Additionally, the inhibition of the NF-κB signaling pathway using parthenolide (PTL), pyrrolidinedithiocarbamate (PDTC) and its analog diethyldithiocarbamate (DETC) also preferentially inhibited the proliferation and colony formation of MCF7 mammosphere cell cultures and the verapamil-sensitive SP cell population enriched in breast cancer stem-like cells [108]. Moreover, the in vitro treatment of CD133+ primary prostate cancer cells and their progenitors with PTL significantly reduced their viability, while the normal primary prostate cells from a patient with benign prostatic hyperplasia (BPH) were insensitive to this treatment type [165]. In the same way, the molecular targeting of IL-4, which may protect the tumorigenic CD133+ cancer stem/progenitor cells from human colon carcinoma of apoptotic death, by using anti-IL-4 neutralizing antibody or IL-4Rα antagonist, also sensitized these tumorinitiating cells to the antitumoral effects induced by standard chemotherapeutic drugs in vitro and in vivo (Table 10.2) [102]. Of particular therapeutic interest, the induction of the differentiation of cancer stem/progenitor cells by using agents such as retinoic acid and its synthetic analogues, interferons (IFNs) or histone deacetylase inhibitor, also may represent a promising adjuvant therapeutic strategy [200–203]. For instance, it has been reported that the IFN-α treatment caused a dramatic reduction in the verapamil-sensitive SP cell fraction from diverse ovarian cancer cell lines [203].

10.4.2 Molecular Targeting of the Local Microenvironment of Tumor- and Metastasis-Initiating Cells and Their Differentiated Progenies Since the local microenvironment of cancer stem/progenitor cells and their differentiated progenies also plays an active role in their malignant transformation at the primary neoplasms and metastases at distant sites and angiogenic process, efforts are being made to counteract cancer progression by targeting the host stromal cells. In particular, the targeting of myofibroblasts and immune cells that support the malignant transformation of cancer stem/progenitor cells as well as the use of anti-angiogenic agents targeting endothelial cells involved in tumor neo-angiogenic process may constitute the adjuvant treatments. These therapeutic strategies may be effective in improving the current cancer treatments by counteracting cancer progression to locally invasive and metastatic disease states, and thereby decrease the high fatality rate in cancer patients (Fig. 10.2) [10, 15, 17, 32, 74, 75, 139, 140, 187, 204–212]. More specifically, the combined use of cytotoxic agents targeting cancer stem/progenitor cells plus the selective inhibitors of tumor angiogenesis, such as

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cyclooxygenase-1 or 2 (COX-1 or -2), NF-kB and/or VEGF-VEFGR, may represent more effective strategies to prevent disease relapse [187, 207, 209–215]. As a matter of fact, it has been observed that the treatment of mice-bearing orthotopic U87 glioma cell xenografts with an anti-VEGF mAb called bevacizumab noticeably reduced the microvasculature density and tumor growth [207]. This anti-carcinogenic effect was also accompanied by a decrease in the number of vessel-associated self-renewing CD133+ /nestin+ tumor-initiating cells [207]. In addition, the use of pharmacological agents that are able to interfere with the recruitment, homing and/or adhesion of tumorigenic and migrating cancer stem/progenitor cells on the endothelial surface and stromal components at distant metastatic sites including BM also constitute the potential cancer therapies. These therapeutic strategies could eradicate metastasis-initiating cells and prevent secondary tumor initiation and disease relapse. Particularly, the blockade of chemoattractant gradient systems involved in the migration of cancer cells at distant metastatic sites may constitute the potential therapeutic strategies [10, 15, 33, 74, 75, 129, 139, 140, 161, 216–226]. These chemoattractant systems includes SDF-1/CXCR4, monocyte chemoattractant protein CCL2 and its high affinity receptor CCL2, and endothelial E-selectine and its P-selectine glycoprotein ligand-1 expressed on circulating cancer cells [10, 15, 33, 74, 75, 129, 139, 140, 161, 216–226]. For instance, the use of a mAb directed against chemokine SDF-1 or its cognate receptor CXCR4 expressed on migrating cancer stem/progenitor cells or selective CXCR4 antagonists, such as 14-mer peptide (TN14003) and AMD 3100, has given promising results to counteract the migration of cancer cells at distant sites and metastasis formation (Fig. 10.2) [10, 15, 33, 74, 75, 129, 139, 140, 161, 218–226]. In this matter, in view of the fact that the hypoxia-inducible factor-1α (HIF-1α) and NF-kB may cooperate to induce an up-regulation of CXCR4 transcriptional expression and migration of certain cancer cells, the molecular targeting of these signaling elements could also represent the potential anti-metastatic treatments [227, 228]. Future works on isolated cancer stem/progenitor cells should confirm the potential therapeutic benefit of targeting the SDF-1/CXCR4 axis, HIF-1α and/or NF-kB to prevent the invasion and metastases of tumor-initiating cells. In the same pathway, the targeting of the host microenvironment, including the stromal cells and endothelial cells that are required for the tumor formation by metastasis-initiating cells at distant sites is also of major importance to counteract the metastasis formation and disease relapse [19, 229, 230]. On the other hand, in considering the fact that several epithelial cancers such as lung, breast, prostate, pancreas and kidney cancers typically display a pre-disposition to metastasize at the BM site and induce the formation of osteoblastic and/or osteolytic lesions, the use of inhibitors that are able to interfere with the development of these types of bone damage may also constitute an adjuvant therapy. Among these agent types, there are bisphosphates such as zoledronate that can inhibit osteoclast activity, and thereby reduce the osteolytic bone resorption [19, 230]. These adjuvant treatments could prevent severe bone damage and reduce intense pain resulting from osteoblastic lesions that are associated with bone remodeling mediated by osteoblast-driven new bone overgrowth as well as osteolytic lesions implicating bone lysing.

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10.5 Conclusions and Perspectives Recent advances in basic and clinical oncology have revealed that the tumorigenic and migrating cancer stem/progenitor cells can provide critical functions in tumor formation, metastases at distant sites, treatment resistance and disease relapse. Consequently, the molecular targeting of tumor- and metastasis-initiating cells and their microenvironment may represent a potential strategy for improving the efficacy of the current cancer treatments. Additional investigations to establish new signaling elements deregulated in tumor- and metastasis-initiating cells as well as microenvironmental components regulating their self-renewal, differentiation and/or treatment resistance is important to design novel cancer treatments for reversing the MDR phenotype and preventing disease recurrence. These studies should lead to the identification of molecular therapeutic targets that could be exploited to develop novel combination therapies for treating and even curing the patients diagnosed with locally advanced, metastatic, recurrent and lethal cancers. Acknowledgements The authors on this work are supported by grants from the National Institutes of Health (CA78590, CA111294, CA133774, CA131944 and CA138791). We thank Ms. Kristi L. Berger for editing the manuscript.

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Chapter 11

Involvement of Lipid Rafts in Growth Factor Receptors-Mediated Signaling for Cancer Metastasis Samir Kumar Patra

Abstract Tumour development and metastases are complex multistep processes. Separation from neighbor cells of the host tumor is the first step to metastases. Re-establishment in the host tissue followed by proliferation constitutes the final phase of metastasis. The dynamic nature of membrane lipids and membrane proteins plays some vital roles in cell survival and cell death. Within the membrane there are specialized nano- and micro-domains, known as lipid rafts. It is now proven and widely accepted that rafts are involved in coordinating various signaling pathways, including separation of cells from tissues. There is a stunning list of structural and signaling proteins on lipid rafts proteome. Evaluation of molecular components of rafts lipids along with proteome from various tumors, tissues, cell lines, and model rafts revealed the pleiotropism of lipid rafts. In this chapter I discuss how lipid raft mediate function of growth factors (GFs) and their receptors (GFRs: including EGFR, and TGFβ-R1 and R2) in tumour development and cancer metastasis. Keywords Cancer · Ceramide · Cholesterol · EGFR · Lipid rafts · MAP kinase · Metastasis · Signal transduction · Sphingomyelin · TGFβ Abbreviations ASMase cav-1 ECM EGF EGFR GF GFR HGF

acid sphingomyelinase Caveolin-1 extra-cellular matrix epidermal growth factor EGF receptor growth factor GF receptors hepatocyte growth factor

S.K. Patra (B) Associate Professor, Department of Life Science, National Institute of Technology, Rourkela, Orissa 769008, India e-mail: [email protected]; [email protected]

W.-S. Wu, C.-T. Hu (eds.), Signal Transduction in Cancer Metastasis, Cancer Metastasis – Biology and Treatment 15, DOI 10.1007/978-90-481-9522-0_11,  C Springer Science+Business Media B.V. 2010

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HGF receptor matrix metalloproteinases mitogen activated protein kinase MAP/ERK kinase 1/2 plasma membranes receptor tyrosine kinases sphingomyelin transforming growth factor beta

Contents 11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 The Structure and Function of Lipid/Membrane Rafts . . . . . . . . 11.3 The Role of Lipid Rafts in Re-modeling of the Peri-Cellular Microenvironments for Metastasis . . . . . . . . . . . . . . . . . . 11.4 The Involvement of Lipid Rafts in Cell Migration . . . . . . . . . . 11.5 The Role of Lipid Rafts in Signal Transduction Leading to Metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.5.1 The Role of Membrane Lipid Raft in Epidermal Growth Factor Receptor (EGFR) Mediated Signaling . . . . . . . . . . . 11.5.2 Transforming Growth Factor Beta-(TGFβ) 1 Signaling . . . . 11.6 Conclusion and Perspective . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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11.1 Introduction Metastasis of cancer is one of the most painful phases in the lives of the victims. Following malignant transformation at the genetic and epigenetic levels with concomitant cell cycle dysregulation, a metastatic phenotype is dependent on an altered behaviour at various cellular levels. These may be initiated by interactions between tumour cells and cells of the surrounding connective tissue mediated by cytokines and growth factors. Firstly, reduced expression or loss of functions of adhesion molecules (for example, cadherins), which are responsible for cell-to-cell interactions are observed. Simultaneously, a distinct pattern of integrin-mediated cell membrane-to-matrix interactions are found. These changes result in an increased migratory ability of the cells. Also, the tumor may invade the surrounding tissue by secretion of proteases that degrade extracellular matrix components. Subsequently, the tumor cells may enter circulation (i.e. intravasation) and the alive tumor cells may find a second home in the distant organs followed by restart of proliferation [1–12]. Plasma membrane gives a protection to cytoplasmic ingredients and organelles, perform endocytosis, and increase the mechanical stability of cells during division. Moreover, the flexibility of lipids within the membrane causes decrease of shear

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forces during cell separation, which are required for separation of individual cells from the host tumor during cancer metastasis. The dynamic nature of membrane, alongwith flexibility and uneven distribution of lipids casue formation of specialized plasma membrane nano- and micro-domains. Such domains are predominantly composed of lipids containing cholesterol-sphingolipids, or ceramide-sphingomyelin. Both types of membrane microdomains constitute the platforms for various proteins that can play both structural and signaling role in cell proliferation or induction of cell death [1]. In recent time, lipid rafts has emerged as the specialized domains within the membranes coordinating entity for transmission of various signals (reviewed in [1]). Proteins of the receptor tyrosine kinases (RTK) family members, including EGFR, VEGFR, TGFβR2 and other signaling proteins, including Ras, caveolins and CD44, have been implicated in exhibiting their functions (including mediating cell polarity, motility and migration) through lipid rafts, [1–4, 13–19]. In this contribution, I will discuss about the role of lipid rafts in receptor mediated-cell transformation and metastasis.

11.2 The Structure and Function of Lipid/Membrane Rafts Lipid/membrane rafts are small (10–200 nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes. Caveolae are characterized by flask-like invaginations of the plasma membrane stabilized by the presence of caveolin-1 (cav-1). Conceptually, there are molecular and structural difference between lipid domains/rafts and caveolae. In Caveolae the membrane leaflets are defined, coupled and compact, due to the presence of caveolins; whereas rafts are tiny masses in a single leaflet that can float with massive speed in the phospholipid ocean. Membrane rafts are essential for transcytosis, cholesterol and sphingolipid sorting in the cell, and serves as platforms for sequestration of receptors and assembling the signal transduction machinery. Rafts are also important for organization of the actin cytoskeleton, cell polarity, angiogenesis, and membrane fusion [1–4, 15, 20–30]. Figure 11.1 shows a schematic view of lipid rafts. The rafts composition depends on whether they are derived from the cytoplasmic leaflet, shed membrane fractions, or from organelle membranes of living cells. They are also influenced by the type of signals the cells receive. Proteomic analysis of plasma membrane lipid rafts has produced a stunning list of potential constituents [1, 2, 20, 31–34]. Analyzing the available data on lipid rafts proteome from various cancer cells and of model rafts, it has been proposed earlier that there may be two subsets of raft in membrane: cholesterol-rafts (“chol-rafts”) and ceramide-rafts (“cer-rafts”) [1]. Table 11.1 shows the core-lipid compositions of the two kinds of rafts. “Chol-rafts” are responsible for cellular homeostasis, but when dysregulated “chol-rafts” enhances cell transformation, tumor progression, angiogenesis and metastasis. “Cer-rafts” are implicated for signaling and apoptosis depending on the types of proteins associated with the entities.

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Non-raft

Caveolae

Lipid raft

Non-raft

A

Cytoplasmic leaflet

Cholesterol raft

Outer leaflet

Radiation

Chemical agents; like, EGCG, Edelfosine and Aplidin

Ceramide raft

B Fig. 11.1 (continued)

When tumors are exposed to radiation and drugs for therapeutic intervention, acid sphingomyelinase (aSMase) translocates to membrane surfaces (Fig. 11.1) and hydrolyzes SM, which generates sphingosine and ceramide [1, 35–39]. This elevated ceramide rapidly displaces cholesterol from membrane/lipid-“chol-raft” and forms “cer-raft” (Fig. 11.1). The displaced cholesterol may move to the other parts of the membrane enriched with phospholipid, and be continuously balanced by efflux of cellular cholesterol [1, 39–45]. In addition, cancer cells may shed plasma membrane fractions enriched in sphingomyelin (SM), cholesterol, gangliosides and growth factor receptors to counter hosts immune attack (reviewed in Refs. [1, 2, 20–22; 27–29, 45–50]).

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GM1 ASMase Ceramide Raf1 Sphingosine Ezrin Fas FasL

Cholesterol Phospholipid Sphingolipid A non-raft transmembrane protein EGFR Cav-1

Fig. 11.1 Schematic presentation of lipid rafts [(a) diagrammatic view; and (b) molecular view] and a mechanism for how a proliferative raft (Cholesterol-raft) may be transformed into an apoptotic raft (Ceramide-raft). Lipid/membrane rafts are small heterogeneous, highly dynamic, sterolor ceramide, and sphingolipid-enriched domains that compartmentalize cellular processes [1]. Caveolae are characterized by flask-like invaginations of the plasma membrane stabilized by the presence of caveolin-1 (cav-1) [25, 30]. When a cell receives particular stimuli for a specific function, the scattered rafts converge by lateral diffusion and coalesced to form larger platform. In sketch A of the figure 1 it is distinctly shown that rafts distributed non-homogeneously and remain uncoupled in resting cells. Sphingolipid- and cholesterol enriched membrane microdomains are considered to be floating in an “ocean” of phospholipid, and hence have been termed rafts. It has been proposed that there may be two subsets of raft in membrane: cholesterol-rafts (“cholrafts”) and ceramide-rafts (“cer-rafts”). Table 11.1 shows the core-lipid compositions of the two kinds of rafts. “Chol-rafts” are responsible for cellular homeostasis, but when dysregulated “cholrafts” enhances cell transformation, tumor progression, angiogenesis and metastasis. “Cer-rafts” are implicated for signaling and apoptosis depending on the types of proteins associated with the entities [1]. Lower half of (b) shows a typical composition of cell death associated raft clustering enriched with ceramide. When cells and tumors are exposed to radiation or challenged with therapeutic compounds, like aplidin, green tea catechins (GTCs; for example, EGCG), neutral or acid sphingomyelinase (nSMase or aSMase) translocates to membrane surfaces and hydrolyzes SM, which generates sphingosine and ceramide. This elevated ceramide rapidly displaces cholesterol from membrane/lipid-“chol-raft” and forms “cer-raft”. This newly formed “cer-rafts” serve to sequester proteins of the FAS-DISC (death inducing signaling complex) and related proteins, which immediately triggers “start” signals to death/apoptosis following endocytosis (see, [1] for further explanation)

Cholesterol (Chol) Sphingomyelin (SM) Glycosphingo-lipids (e.g., GM1, GM2) Lysophosphatidic acid (LPA) PIP2

Partners

Cholesterol rafts References

Biosynthesis of individual [1, 2 20–23, 25–28, 46–50] components and sorting, efflux from and influx into cells by transport of lipoproteins cholesterol, endocytic recycling

Mechanism of formation and control Ceramide (Cer) Sphingomyelin (SM) Glycosphingo-lipids (e.g., GM1) Sphingosine

Partners

Ceramide rafts

Table 11.1 Lipid core of two distinct types of rafts

References

Biosynthesis of individual [1, 20, 22, 37, 39–41] components, derived from Chol-rafts by in situ production of ceramide through Sphingomyelin break down by aSMase

Mechanism of formation

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11.3 The Role of Lipid Rafts in Re-modeling of the Peri-Cellular Microenvironments for Metastasis During pre-metastasis stage tumor cells undergo drastic reordering and changes in their membrane lipids and cell adhesion protein components. Gene expression of some of the protein components become reversibly switched off [2, 4], some proteins loss their function. Interestingly, lipid rafts may help to re-model the pericellular microenvironments [1, 2]. Specific glycoproteins of the extracellular matrix such as fibronectin, collagen, laminin, and glycosaminoglycans, coupled with various growth factor (VEGF, EGF, TGF-α, and TGF-β) and GFRs are also involved in this pre-metastatic process. The leading steps of metastasis involve loss of adhesion during which lipid-protein-lipid raft (LPLR) may reorder and interact with extracellular matrix (ECM) to decrease mechanical stability and increase shear forces for separation of individual cells from the tumour [1, 2]. Interestingly, the collagen fibers in tumours are more abundant than that of the normal counterpart. Several collagen and laminin genes (Col3a1, Col5a2, and Col6a3, and Lama5 and Lamc2) involved in ECM assembly are over-expressed in tumours as well as cultured cell lines originating from various adenocarcinomas. The laminins are components of basement membranes that are believed to act as mechanical barrier against carcinoma cell invasion [2, 6, 11–13]. Therefore, the LPLR-dependent remodeling of ECM in the immediate pericellular environment of the cells that are ready to move seems to be a necessary step in local invasion. The other function of lipid raft in tumor invasion involved the action of matrix degradation enzyme. Pre-metastatic tumor cells express high amount of protease to degrade ECM. Proteolysis of ECM proteins modifies integrin-mediated anchorage, focal adhesion and cytoskeletal architecture, triggering signaling molecules such as focal adhesion kinase (FAK) and motility factors, including PKC and AMF/PHI [1, 2, 20, 51–56] leading to tumor invasion. Interestingly, MMPs, one of the most important matrix degradation enzyme is secreted from tumor cell in a membrane rafts-anchored form [57, 58]. The other types of proteinase are urokinase type plasminogen activator (uPA), thrombin and plasmin. In several experimental models uPA has been shown to function through its receptor (uPAR), which is also an adhesion receptor for vitronectin and may interacts with integrin β-chains. It is worth noting that integrins, tetraspanins, uPAR are conjointed within membrane rafts to confine the serine proteinase uPA to the invading pseudopodia [1, 2].

11.4 The Involvement of Lipid Rafts in Cell Migration Many motility factors for cancer cells and non-malignant cells were described first as growth factors (described above). A motility factor converts a cell from static to a motile status, a transition that is characterized by the appearance of membrane ruffling, lamella and pseudopodia. Formation of lamella and pseudopodia depends on functional rafts and the raft associated proteins like cav-1, CD44 and

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EGFR (reviewed in [1], see [43, 44, 59]). Once cells have entered the circulatory compartment, they attain ready access to virtually all organs of the body where blood flow, except the brain. Access to brain depends on the permeability through blood brain barrier. Noticeably, they must be able to survive several stresses, including physical damage from hemodynamic shear forces, and immune-mediated destruction. Activated cells shed fragments of their plasma membrane into the extracellular milieu [1, 2]. Shed membrane vesicle(s) from tumour cells derived from selected areas of plasma membrane appear to be enriched with rafts components: cholesterol, GM1, GM3 and SM, and contain surface antigens and proteases often present in tumour cells. Circulating cancer cells may enhance their survival by coopting/anchoring blood platelets, using them as shields, where lipid rafts may help in the nano-scale fusion of small subdomains of two different types of cells. This assumption needs further experimental evidences.

11.5 The Role of Lipid Rafts in Signal Transduction Leading to Metastasis Lipid components of rafts, including SM, GM1–GM3 and cholesterol individually or in combinations can enhance the metastatic potential of various tumor cells. Hydrolysis of SM within rafts generates ceramide, and ceramide results in dramatic coalescence of sub-domains into large-membrane macrodomain (reviewed in [1]). The macrodomain serves as scaffolding for oligomerization of proteins and transmission of signals across the plasma membrane. Lipid rafts can be endocytosed with growth-factor as well as death-receptor in signaling pathways to enhance their respective activity. Detailed analysis of the MAP kinase and FAS pathways has revealed multiple points of intersections, suggesting a complex network of interactions coordinating cell survival and death [1]. The importance of cytoskeletal organization and mechanical tension for raft mediated signaling thereby provides a mechanism by which these considerations can influence the responses to growth factors as well, providing a convergence point for different classes of extracellular cues. Growth-factor signaling, such as that mediated by EGF, HGF, TGFβ through their cognate receptors using lipid rafts as platforms, can modulate many of these activities either directly or indirectly [1–3, 20, 22, 60–78] resulting in metastatic progression.

11.5.1 The Role of Membrane Lipid Raft in Epidermal Growth Factor Receptor (EGFR) Mediated Signaling The EGF receptor (EGFR) is a 170 kDa transmembrane lipid raft glycoprotein composed of three domains: an extracellular ligand binding domain, a single transmembrane lipophilic region, and an intracellular domain that exhibits intrinsic tyrosine kinase activity [79–84]. Endogenous ligands to EGFR include TGF-α,

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heparin-binding EGF, amphiregulin and betacellulin [85]. It transmits signals upon activation by complex formation with the cognate ligand, EGF, or in some instances activated by crosstalk with the other ligands of receptor tyrosine kinases (RTKs) family, namely TGF-α [86, 87]. On binding of the ligand to EGFR, the ligandreceptor complex undergoes dimerization and internalization [84, 86, 87]. The EGFR family plays an essential role in normal organ development by mediating morphogenesis and differentiation, and plays a crucial role in growth, differentiation, and motility of normal as well as cancer cells. Second cysteine-rich region of EGFR have targeting information for caveolae/rafts [88]. Analyses of detergent-insoluble membrane fractions revealed that the EGF-dependent association of endocytic EGFR proteins with rafts is as efficient as that of signaling effector molecules, such as Grb2 or Shc [84, 87]. Lipid rafts have the ability to assemble both the molecular machineries necessary for intracellular propagation of EGFR effector signals and for receptor internalization. Higher expression of EGFR produces a neoplastic phenotype in tumor cells, which is associated with higher rates of progression from superficial to invasive forms of various cancers [1, 2, 85, 80–92]. Cross-talk between G protein-coupled receptor (GPCR) and EGFR signaling pathways contributes to growth and invasion of head and neck squamous cell carcinoma (HNSCC) [93]. The major pathways that lie down stream of the membrane-associated RTK is activation of Raf-1 by lipid raft associated Ras, which follows phosphorylation mediated activation of MAPK/ERK kinase 1/2 (MEK1/2). Because the deregulated over-activation of MAP kinase pathway is frequently seen in tumor progression of a variety of human cancers, modulation of MAP kinases by disruption of lipid rafts along with the use of some natural compound and other inhibitors may be a therapeutic approach [1, 15, 22, 94–97].

11.5.2 Transforming Growth Factor Beta-(TGFβ) 1 Signaling 11.5.2.1 The Role of TGFβ Signaling in Tumor Suppression Transforming growth factor beta family members (TGFβs) are multifunctional growth factors. TGFβ1 (OMIM, 190180) controls proliferation, differentiation, and other functions in many cell types. It also acts as a negative autocrine growth factor. Dysregulation of TGFβ activation and signaling may result in apoptosis. Many cells synthesize TGFβ and almost all of them have specific receptors for this peptide. TGFβ1, TGFβ2, and TGFβ3 all function through the same receptor signaling systems [98–103]. The role of TGFβ signaling in tumor suppression have been reviewed by Derynck et al. TGFβ1 is most frequently upregulated in tumor cells and is the focus of most studies on the role of TGFβ in tumorigenesis [63]. The autocrine and paracrine effects of TGFβ on tumor cells and the tumor microenvironment exert both positive and negative influences on cancer development. Han et al. also suggested that TGFβ1 induces EMT and invasion via distinct mechanisms: TGFβ1-mediated EMT

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requires functional TGFβR, whereas TGFβ1-mediated tumor invasion cooperates with reduced TGFβR2 signaling in tumor epithelia [104]. 11.5.2.2 Involvement of Lipid Raft in TGFβ Signaling Ma et al. have recently reported the lateral diffusion of TGFβ type I receptor (TGFβR1) in living cells by imaging and tracking individual green fluorescent protein tagged TGFβR-1 on the cell membrane [105]. They found that when coexpressed with TGFβ type 2 receptor (TGFβR2), the mobility of TGFβR1 decreased significantly after stimulation with TGFβ1. However, in the cells that had been depleted of cholesterol with Nystatin or methyl-beta-cyclodextrin, the diffusion rate of TGFβR1 was not changed by TGFβ1 treatment. The observations suggested that lipid rafts in membrane may provide a platform which facilitated the association of both TGFβR1 and TGFβR2 for cell signaling. Chen et al have studied TGFβ responsiveness in cultured cells, which can be modulated by TGFβ partitioning between lipid raft/caveolae- and clathrin-mediated endocytosis pathways [106]. The TGFβR2/TGFβR1 binding ratio of TGFβ on the cell surface has recently been found to be a signal that controls TGFβ partitioning between these pathways. They have studied the effects of cholesterol on TGFβ binding to TGFβ receptors and TGFβ responsiveness in cultured cells and in animals. They demonstrated that treatment with cholesterol, alone or complexed in lipoproteins, decreases the TGFβ R2/TGFβR1 binding ratio of TGFβ while treatment with cholesterol-lowering or cholesterol-depleting agents increases the TGFβR2/TGFβR1 binding ratio of TGFbeta in all cell types studied. Among cholesterol derivatives and analogs examined, cholesterol is the most potent agent for decreasing the TGFβR2/TGFβR1 binding ratio of TGFβ. Cholesterol supplement increases accumulation of the TGFβ receptors in lipid rafts/caveolae as determined by sucrose density gradient ultracentrifugation analysis of cell lysates. Cholesterol/LDL suppresses TGFβ responsiveness and statins/beta-CD enhances it, as measured by the levels of P-Smad2 and PAI-1 expression in cells stimulated with TGFβ. A recent report demonstrated that depletion of cholesterol and thus cholesterol-rich lipid rafts interfered with TGFβinduced EMT and cell migration mediated by MAPK. This establish a novel link between lipid rafts and TGFβ-mediated MAPK activation, an event necessary for TGFβ-directed epithelial plasticity [107].

11.6 Conclusion and Perspective Here, I have discussed the role of lipid raft in growth factors-mediated signaling for cell transformation, cancer progression and metastasis. Realizing that growth factor signaling emanates from membrane, structure and composition of membrane are crucial determinants of tissue development, cell differentiation and homeostasis. It suggests that the loss of the power to sense, respond and adapt appropriately to signal contributes to disease, including cancer. The question is how the membrane adjusts the relocations of proteins, withdrawal of proteins function, and how the

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transbilayer signals are coordinated? The lipid rafts, performing as scaffoldings for cell adhesion and signaling proteins, is the critical determinant. Many, if not all, of the growth factor receptors and their signaling elements (such as Ras-MAPK signaling) are localized in caveolae and lipid rafts. Since lipid rafts are tiny domains in a single leaflet, which can float with massive speed in the phospholipids ocean, rafts are thought to sequester and couple with their cognate leaflet as per signals and type of downstream transducer elements. Defining mechanisms for lipid raft regulated GF signaling is essential for understanding tumor metastasis Acknowledgements Thanks are due to Professor Sunil Kumar Sarangi, Director, National Institute of Technology, Rourkela, India for his generous support with special facilities and funding for cancer epigenetic research at the Department of Life Science. I apologise for many other important contributions that I have not been able to include and discuss in this contribution. This work is dedicated to late Professor M. Judah Folkman for his outstanding contributions to cancer research and for creation of the field of angiogenesis research [see, 108, 109]. I am grateful to Wen-Sheng Wu, Editor of this volume for his generous critique on the manuscript.

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97. Schley PD, Brindley DN, Field CJ. (n-3) PUFA alter raft lipid composition and decrease epidermal growth factor receptor levels in lipid rafts of human breast cancer cells. J. Nutr. 2007; 137, 548–553. 98. Roberts AB, Sporn MB, Assoian RK, et al. Transforming growth factor type beta: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc. Natl. Acad. Sci. USA 1986; 83: 4167–4171. 99. Sporn MB, Roberts AB, Wakefield LM, et al. Transforming growth factor-beta: biological function and chemical structure. Science 1986; 233: 532–534. 100. Blobe GC, Schiemann WP, Lodish HF. Role of transforming growth factor beta in human disease. N. Eng. J. Med. 2000; 342: 1350–1358. 101. Gold LI. Role of transforming growth factor-β (TGF-β) in human cancer. Crit. Rev. Oncog. 1999; 10: 303–360. 102. Muroaka RS, Dumont N, Ritter C, et al. Blockade of TGF-β inhibits mammary tumor cell viability, migration and metastases. J. Clin. Invest. 2002; 109: 1551–1559. 103. Akhurst RJ. TGF-β antagonist: Why suppress a tumor suppressor? J. Clin. Invest. 2002; 109: 1533–1536. 104. Han G, Lu S-L, Li AG, et al. Distinct mechanisms of TGF-beta-1-mediated epithelial-tomesenchymal transition and metastasis during skin carcinogenesis. J. Clin. Invest. 2005; 115: 1714–1723. 105. Ma X, Wang Q, Jiang Y, et al. Lateral diffusion of TGF-beta type I receptor studied by single-molecule imaging. Biochem. Biophys. Res. Commun. 2007; 356: 67–71. 106. Chen CL, Huang SS, Huang JS. Cholesterol modulates cellular TGF-beta responsiveness by altering TGF-beta binding to TGF-beta receptors. J. Cell. Physiol. 2007; 215(1): 223–233. 107. Zuo W, Chen YG. Specific activation of mitogen-activated protein kinase by transforming growth factor-beta receptors in lipid rafts is required for epithelial cell plasticity. Mol. Biol. Cell. 2009; 20(3): 1020–1029. 108. Zetter BR. Timeline: The scientific contributions of M. Judah Folkman to cancer research. Nature Rev. Cancer 2008; 8: 647–654. 109. Klagsbrun M, Moses MA. OBITUARY: M. Judah Folkman (1933–2008) scientist, surgeon and creator of the field of angiogenesis research. Nature 2008; 451: 781.

Chapter 12

Cadherin-Catenin Signaling in Ovarian Cancer Progression Lydia W.T. Cheung, Carman K.M. Ip, and Alice S.T. Wong

Abstract Ovarian cancer is a highly metastatic disease and has the highest mortality rate of all gynecological tumors. In contrast to many other types of cancer that metastasize through lymphatics and/or hematogenous routes, ovarian cancer metastasizes by peritoneal dissemination, which relies on the ability of cancer cells to detach from the primary tumor, adhere to, and eventually invade through the peritoneum. This involves dynamic changes in cell-cell adhesion, which is primarily mediated by cell surface receptors known as cadherins. In this review, we will describe the unique profiles of cadherins with their associated signal molecules, catenins, in ovarian cancer and the roles of these adhesion molecules in disease development, tumor cell progression, and the formation of ascites. We will discuss how cadherins perform these functions and their link to a variety of signaling pathways. Finally, we will review the recent findings regarding the potential of cadherins as new therapeutic targets in the treatment of ovarian cancer. Keywords Adhesion · Angiogenesis · Cadherin · Catenin · Differentiation · Invasion · Motility · Ovarian cancer · Regulation · Signaling

Contents 12.1 Introduction . . . . . . . . . . . . . . . . . . . . 12.1.1 Ovarian Cancer . . . . . . . . . . . . . . . 12.1.2 Cadherins . . . . . . . . . . . . . . . . . 12.2 Expression of Cadherins and Catenins in Ovarian Cancer 12.2.1 Classical Cadherins . . . . . . . . . . . . . 12.2.2 Other Cadherins . . . . . . . . . . . . . . 12.2.3 Catenins . . . . . . . . . . . . . . . . . .

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W.-S. Wu, C.-T. Hu (eds.), Signal Transduction in Cancer Metastasis, Cancer Metastasis – Biology and Treatment 15, DOI 10.1007/978-90-481-9522-0_12,  C Springer Science+Business Media B.V. 2010

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12.3 Role of Cadherins and Catenins in Ovarian Cancer . . . . . . . . . . 12.3.1 Cell Survival . . . . . . . . . . . . . . . . . . . . . . . 12.3.2 Differentiation . . . . . . . . . . . . . . . . . . . . . . 12.3.3 Motility and Invasion . . . . . . . . . . . . . . . . . . . 12.3.4 Adhesion . . . . . . . . . . . . . . . . . . . . . . . . 12.3.5 Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . 12.4 Signaling by Cadherin-Catenin in Ovarian Cancer . . . . . . . . . . 12.4.1 Signaling Through β-catenin . . . . . . . . . . . . . . . . 12.4.2 Signaling Through p120ctn . . . . . . . . . . . . . . . . . 12.4.3 Cross-talk with Receptor Tyrosine Kinases . . . . . . . . . 12.5 Regulation of Cadherin by Hormones, Growth Factors, and CytokinesInvolvement in Ovarian Tumor Progression . . . . . . . . . . . . . 12.5.1 E- and N-cadherin . . . . . . . . . . . . . . . . . . . . 12.5.2 P-cadherin . . . . . . . . . . . . . . . . . . . . . . . . 12.6 Cadherins as Drug Targets . . . . . . . . . . . . . . . . . . . . . 12.7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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12.1 Introduction 12.1.1 Ovarian Cancer Ovarian cancer is the sixth most common cancer and the fifth leading cause of cancer deaths in North American women. The lifetime probability of developing ovarian cancer is approximately 1.7% and the disease accounted for 3% of all new cancer cases and 6% of all cancer deaths in 2008 [1]. Of note, it carries a higher mortality rate than any other gynecological cancers combined. However, unlike many other forms of cancer, the age-adjusted incidence and death rates from ovarian cancer have not changed significantly over the past 25 years [2]. Because of the lack of early symptoms, it is difficult to detect this cancer early when it is still curable. More than three fourths of patients are at advanced stages (FIGO stages III-IV) when the tumor has already metastasized to the pelvis or abdomen at the time of diagnosis. Current therapies have very limited success in treatment of metastatic ovarian carcinomas. The 5-year survival rate of this group of patients is very low. Less than 12% of women with stage IV disease survive 5 years after diagnosis. Ovarian carcinomas are thought to arise from the ovarian surface epithelium (OSE) which is a simple mesothelium that overlies the surface of the ovary. The characteristics of ovarian tumors often resemble the specialized, more complex epithelia of the Mullerian duct derivatives that are embryologically related to the OSE but evolve by distinct pathways. Histologically, ovarian carcinomas are classified as serous (40%) (oviduct-like), endometrioid (20%) (uterine-like), mucinous (10%) (endocervix-like) adenocarcinomas, and several less common types. Ovarian cancer also exhibits a distinctive pattern of metastasis. Unlike most cancers (e.g. breast, colon, and lung cancers) which metastasize via the

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bloodstream, ovarian cancer spreading is usually confined within the peritoneal cavity. Intraperitoneal dissemination is found in more than 60% of the patients and these patients usually have no clinically apparent lymphatic or hematogenous metastases [3]. Intraperitoneal dissemination is frequently associated with ascites formation, which is a voluminous exudative fluid consisting of ovarian cancer cells, lymphocytes, and mesothelial cells. The neoplastic cells in the ascites are thought to be a potential source of secondary tumor growth in ovarian cancer patients. Distant metastasis may occasionally occur at late stage of the disease (stage IV) and are usually associated with very poor survival. These secondary sites include liver, lung, spleen, brain, bone, breast, and skin [4]. Metastasis is a complex, multi-step biological process as characterized by a series of distinct yet interrelated steps [5]. The metastatic dissemination of ovarian tumors starts by (a) direct shedding of the cancer cells into the peritoneal cavity. (b) The exfoliated tumor cells are transported throughout the peritoneal cavity along the peritoneal fluid circulation, which facilitates attachment of the tumor cells onto the peritoneum. (c) Subsequently, invasion, proliferation, and colonization of the tumor cells might occur in the peritoneal cavity (Fig. 12.1). The initial driving force for ovarian tumor progression involves dynamic changes in cell-cell adhesion, which is primarily mediated by cell surface receptors known as cadherins. Primary ovarian tumor Ovary (c) Peritoneal implantation

(a) Detachment

(b) Circulation in peritoneal fluid

Peritoneal metastases Pelvic peritoneum

(d) Migration, invasion, proliferation

Fig. 12.1 Schematic representation of ovarian cancer metastatic process. During metastasis, (a) ovarian tumor cells detach from the primary lesion. (b) These shed cells are passively transported by peritoneal fluid, and subsequent seeding of tumor cells characterized by the (c) adhesion and (d) invasion of tumor cells into the peritoneum, leading to miliary dissemination. These steps likely involve dynamic regulation of both cell-cell and cell-matrix adhesions

12.1.2 Cadherins Cadherins are a family of Ca2+ -dependent transmembrane glycoproteins. The classical cadherin is the best characterized subgroup in the family, including E(epithelial), N- (neural), and P- (placental) cadherin. These cadherins contain five highly homologous extracellular cadherin repeats and a conserved intracellular domain, where it interacts with the actin cytoskeleton. A conserved site at the

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carboxyl termini of cadherins bind to β-catenin or γ-catenin (plakoglobin) in a mutually exclusive fashion which associate with α-catenin [6]. Another armadillo catenin, p120 catenin (p120ctn ) interacts with a more membrane proximal cytoplasmic domain of cadherins (the juxtamembrane domain) and has no interaction with either α-catenin or the actin cytoskeleton [7]. In addition to their adhesive functions, cadherins can modulate signal transduction by interacting with the catenins. For example, β-catenin is an essential intracellular mediator for Wnt signaling pathway through its interaction with the T-cell factor (TCF)/lymphoid enhancer factor family of transcription factors to regulate expression of target genes [8, 9]. p120ctn , originally identified as a Src substrate, was subsequently shown to signal via its activity on the Rho family GTPases (e.g. Rac1, Cdc42, RhoA), which mediate cytoskeletal dynamics in cell migration [10, 11]. Moreover, there is also evidence that cadherins interact with growth factor receptors at the cell surface and modulate growth factor signaling activities [12–14]. These signaling pathways will be discussed later in this chapter.

12.2 Expression of Cadherins and Catenins in Ovarian Cancer Cadherins are expressed in a cell-type specific manner within specific compartments, and their expressions change during differentiation or different stages of neoplastic progression related to the functions of OSE and ovarian cancer [15, 16] (Fig. 12.2; Tables 12.1 and 12.2).

Normal ovary

Benign tumor

Malignant tumor

N-cad E-cad

Cad-11 P-cad, VE-cad, Cad-4, Cad-6

Fig. 12.2 Expression of cadherins during ovarian carcinogenesis. The ovary is surrounded by a single cell layer of mesothelium, which has the potential to undergo metaplastic transformation to a more differentiated state. The expression of cadherins (cad) changes during differentiation and at different stages of neoplastic progression. Light shading indicates weak/no expression, medium shading indicates modest expression, and dark shading indicates strong expression

Present

Absent, but may be present in OSE with family history or metaplastic epithelium

Absent

N.D.

N-cadherin

E-cadherin

P-cadherin

H-cadherin

Expression in normal OSE

Present in malignant tumors Absent/low expression in malignant tumors N.D.

↑ with stage N.D.

N.D.

Epigenetic

N.D.

[46]

[17, 19, (i) Induces 24–33, 55, differentiation in 73–75, 80, early stage 86, 87, 91, (ii) Enhances cell 114, survival 135–137] (iii) Functional loss in late-stage promotes metastasis and peritoneal adhesion Transcriptional control Promotes migration [17, 20, 33] and invasion (i) Epigenetic (ii) Transcriptional control (iii) Post-translational modification

[17–20, 71]

References

Lost of expression predicts poor survival

Functions

Transcriptional control Regulates survival of OSE cells

Regulation

N.D.

Association with Prognostic tumor stage significance

Present in benign, No significant borderline and association malignant throughout tumors stage I to IV Present in benign ↓ with stage tumors and welldifferentiated primary tumors; Absent/reduced expression in advanced stage tumors

Expression in ovarian tumors

Table 12.1 Cadherins in normal ovarian surface epithelium and ovarian tumor cells

12 Cadherin-Catenin Signaling in Ovarian Cancer Progression 229

Present

Present

Cadherin-6

Cadherin-11

N.D.

N.D.

N.D.

N.D.

Association with Prognostic tumor stage significance

Present and higher N.D. expression than OSE Present in No significant malignant association tumors between stage I and II Present and higher ↑ with stage expression than OSE Absent or low Already lost in expression stage I

Expression in ovarian tumors

N.D., not determined; OSE, ovarian surface epithelium

Absent

Cadherin-4

VE-cadherin Present

Expression in normal OSE

Table 12.1 (continued)

N.D.

Increases vascular permeability

Functions

N.D.

N.D.

Transcriptional control N.D.

N.D.

N.D.

Regulation

[20, 33, 54]

[33, 51–53]

[33]

[49, 92]

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Detected in malignant tumors; cytoplasmic localization may occur

p120 catenin

(i) Reduced membranous expression predicts poor survival (ii) Nuclear positivity predicts better survival in EC subtype (i) Membranous expression predicts better survival (ii) Nuclear positivity predicts poorer survival (iii) Nuclear positivity predicts better survival in EC subtype N.D.

↓ with stage

N.D.

↓ with stage

Loss of expression predicts poor survival

Prognostic significance

↓ with stage

Association with tumor stage

(i) Interacts with Src family kinase to control cell dispersal (ii) Regulates Rho family GTPases signaling

May mediate Wnt signaling

Mediates Wnt signaling

(i) Suppresses growth and colony formation capability (ii) Inhibits Wnt signaling

Functions

[20, 70, 90, 102]

[55, 60]

[26, 55, 58–61, 65, 93–95, 97]

[26, 55–56, 76–77]

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N.D., not determined; EC, endometrioid ovarian carcinoma

Reduced in malignant tumors; nuclear localization may occur

Present in benign tumors, reduced/lost in malignant tumors; cytoplasmic localization may occur Reduced/lost in malignant tumors; nucleocytoplasmic localization may occur in EC subtype

γ -catenin

β-catenin

α-catenin

Expression in ovarian tumors

Table 12.2 Catenins in ovarian tumor cells

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12.2.1 Classical Cadherins 12.2.1.1 N-cadherin Like other cells of mesodermal origin, the coelomic epithelium-derived OSE is primarily connected by N-cadherin [17]. During neoplastic progression, the expression of N-cadherin persists in most benign and borderline ovarian tumors, and is heterogenously expressed in ovarian carcinomas [18, 19]. Mucinous and clear cell ovarian tumors express little or no N-cadherin, whereas serous and endometrioid ovarian carcinomas express both N- and E-cadherin [18, 20]. The role of N-cadherin in these histologic subtypes is unclear. However, it has been proposed that the coexpression of E- and N-cadherin characterize epithelial differentiation of cells of mesodermal origin, such as in renal cell carcinomas [21]. Unlike ovarian carcinomas, mesotheliomas express only N-cadherin [22]. This difference illustrates that the propensity to Mullerian epithelial differentiation is a unique feature characteristic of tumors that arise from OSE cells, and is not seen in tumors that arise from extraovarian mesothelium. 12.2.1.2 E-cadherin E-cadherin is expressed by almost all epithelia and plays crucial roles in maintaining the morphological and functional integrity of these tissues. In addition, E-cadherin has the capacity to induce epithelial differentiation in embryonic and adult mesenchymal cells [23]. In relation to what we know about the histological appearance and embryological origin of OSE, it is not surprising that E-cadherin is scanty or absent in the OSE. In fact, the only areas where E-cadherin is expressed in OSE are in the invaginations and inclusion cysts of the ovaries with metaplastic epithelium. In keeping with the acquisition of Mullerian duce-derived epithelial characteristics in the process of malignant transformation, E-cadherin is up-regulated and present in dysplastic lesions, benign tumors, and well-differentiated primary ovarian carcinomas [24–27]. Our finding that E-cadherin is already enhanced in OSE from women with hereditary breast/ovarian cancer syndromes suggests that E-cadherin may have a tumor promoting nature in ovarian cancer and changes in E-cadherin expression might indeed coincide with, or even preceed, the stage of neoplastic progression [17]. The expression of E-cadherin is heterogeneous or undetectable in most borderline tumors [19, 24]. During tumor progression, a decrease of E-cadherin has been found in advanced stage ovarian carcinomas and ascites cells, suggesting a dynamic process of E-cadherin regulation [26, 28, 29]. Reduced E-cadherin level was seen in 64% of tumor samples (n = 104) [30]. In fact, negative E-cadherin expression has been correlated with poorer patient prognosis [19, 30], though there is also report suggesting that E-cadherin level is not predictive of outcomes [31]. Moreover, it has been reported that about 60% peritoneal metastases (n = 10) had lower expression of E-cadherin when compared with their primary lesions (n = 39) [28]. Similarly, there was significantly higher E-cadherin expression in the stage I ovarian tumors than in the peritoneal effusions. In addition, stage I ovarian tumors expressed elevated levels of E-cadherin when compared with metastatic lesions [32, 33].

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In addition to these quantitative changes, there is a progression of changes in the polarity of E-cadherin distribution. In normal, flat-to-cuboidal OSE, E-cadherin is not fully polarized. E-cadherin staining is on both apical and lateral surfaces [27]. In metaplastic, columnar OSE, E-cadherin is polarized and is limited to lateral intercellular spaces. Stratification of OSE is one of the earliest histologic indicators of premalignant changes [34]. In such lesions, as in neoplastic OSE, polarity tends to be lost again and E-cadherin is distributed over all cellular surfaces. 12.2.1.3 P-cadherin P-cadherin was originally identified in mouse placental tissue as a molecule that appeared to act as a connector between the embryo and uterus [35]. In human, P-cadherin is expressed in stratified squamous, pseudo-stratified and transitional epithelia but not in simple epithelia [36]. It is also present in the epithelia of Mullerian duct derivatives [37]. In some carcinomas, for example breast, colon, endometrial and pancreatic cancers, P-cadherin expression is frequently elevated and correlates with high tumor grade, advanced stage, and poor prognosis [38–41]. However, in carcinomas of others, such as gastric cancer and melanoma, P-cadherin diminishes with neoplastic progression [36, 42]. Like E-cadherin, P-cadherin protein is not detected in normal OSE, while high levels of P-cadherin expression is found in ovarian tumors and cancer lines and increased with tumor progression [17, 20, 33]. However, this increase in P-cadherin appears to be associated with concomitant decrease in E-cadherin, suggesting that cadherin switching may play an important role in ovarian cancer metastasis [33].

12.2.2 Other Cadherins 12.2.2.1 H-cadherin H-cadherin (also known as cadherin-13 or T-cadherin) differs from known cadherins in that it is truncated and lacks both transmembrane and cytoplasmic domains [43]. Abnormalities in the H-cadherin gene have been described in several human cancers, including ovarian cancer [44–46]. Kawakami et al. [46] reported that 5 out of 12 high grade invasive ovarian carcinomas showed lost of marker D16S422 in the H-cadherin gene. Three out of 5 these tumors showed very low levels of H-cadherin. Methylation-specific PCR revealed aberrant methylation in the H-cadherin alleles of tumors with low H-cadherin expression [46], suggesting that a combination of hypermethylation and loss of heterozygozity may cause the inactivation of H-cadherin in ovarian tumors. H-cadherin has been shown as a tumor suppressor and inhibits the invasive potential and growth rate of H-cadherin transfected breast carcinoma cells both in vivo and in vitro [43, 47]. However, the biological significance of the alterations of the H-cadherin gene in human ovarian cancer is still an unknown area and remains to be explored.

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12.2.2.2 VE-cadherin Expression of VE-cadherin (cadherin-5) in the mouse ovary was verified by immunohistochemistry [48]. In man, its mRNA level in blood from ovarian cancer patients is found to be significantly higher than healthy women [49]. 12.2.2.3 Cadherin-4 Although cadherin-4 (R-cadherin) cannot be detected in human OSE, it is present in human ovarian tumors [33]. However, its involvement in the development or progression of ovarian cancer has not been documented. 12.2.2.4 Cadherin-6 Cadherin-6 (K-cadherin) was first isolated in the kidney and is thought to be involved in the development and progression of renal cell carcinoma [50]. Expression of cadherin-6 has been demonstrated in the normal human OSE cells [33, 51]. Gene microarray analysis revealed significant higher expression of cadherin-6 in ovarian tumors compared with normal ovary [52]. Moreover, it was preferentially up-regulated in ovarian carcinomas compared to 321 tumor samples of 24 other tissue types, which included carcinomas derived from the endometrium, lung, kidney, cervix and colon, suggesting that this cadherin may play a specific role in ovarian cancer. In support, a cohort study showed that protein expression of cadherin-6 increases with tumor stages, with 33.3% positive staining in stage I (n = 205) and 67.9 % positive staining in stage III (n = 84) [53]. Interestingly, cadherin-6 expression was significantly lower in endometrioid and clear cell ovarian carcinomas than the serous subtype, and it could be positively regulated by a tumor suppressor protein BARX2, homeodomain transcription factor [51]. However, whether cadherin-6 has a direct function in ovarian cancer cells has not been investigated. 12.2.2.5 Cadherin-11 Cadherin-11, initially identified in osteoblasts (OB-cadherin) and often associates with mesenchymal phenotype, is expressed in the normal human OSE [33, 54]. Two independent studies consistently showed rare or no expression of cadherin-11 in ovarian tumor samples [20, 33]. Of note, its expression was already lost in stage I or stage II ovarian tumors [33], suggesting that this cadherin is down-regulated during neoplastic transformation of the OSE.

12.2.3 Catenins 12.2.3.1 α-catenin α-catenin is detected around the lateral membranes of normal OSE and epithelial inclusion cysts [26]. Whilst expression of α-catenin is consistently retained in

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benign ovarian tumors, expression level in malignant tumors is frequently lower than that of the normal OSE and benign lesions. The number of cases demonstrating strong immunostaining for α-catenin was significantly larger in stage I and II tumors (71%) than in stage III and IV tumors (41%) [55]. Reduced, lost or inappropriate expression of α-catenin was observed in about 50% of ovarian tumors and predicts poor prognosis in stage I ovarian epithelial cancer [26, 56]. α-catenin protein is mostly commonly located in the plasma membrane, but cytoplasmic staining is also seen in some tumors [26]. Allelic imbalance is not a mechanism behind this reduced expression [57]. It is therefore likely that the α-catenin gene is inactivated by other mechanisms, such as methylation, mutations or through posttranscriptional regulation. 12.2.3.2 β-catenin β-catenin is expressed in the surface epithelium and inclusion cysts of normal ovaries and this membranous expression is retained in benign tumors [26]. Although increase of β-catenin in the malignant tumors as compared to normal ovarian tissue and benign tumors has been reported [58], most other studies have demonstrated reduced or lost of immunohistochemical β-catenin expression in ovarian carcinomas [26, 59, 60]. For example, Gamallo et al. reported reduced membranous β-catenin staining in 78% of stage I and II ovarian carcinomas (n = 69) [59], while another study by Davies et al. found that 33% (n = 34) of tumors samples had reduced/no expression or cytoplasmic localization of the protein [26]. Moreover, β-catenin level was significantly higher in stage I and II tumors (79%) than in stage III and IV tumors (52%) [55]. Reduced cell surface expression of β-catenin was associated with poor tumor differentiation and poor survival in ovarian cancer patients [60, 61]. Mutations in the β-catenin gene are infrequent in ovarian carcinoma and have so far only been found in the endometrioid subtype of ovarian tumors [59, 62–64]. Previous studies suggested that 16–54% of human ovarian tumor samples in this subtype harbors mutations leading to β-catenin deregulation and its nucleocytoplasmic expression [59, 62, 64]. Nuclear β-catenin positivity has been implicated in a better prognosis, whereas exclusively membranous expression has a worse prognosis [65]. Therefore, the subcellular distribution of β-catenin could be a useful prognostic marker for survival and tumor progression of endometrioid ovarian cancer patients. Deregulation of β-catenin signaling may also occur through altered expression of Wnt components. For instance, there is a significant increase of glycogen synthase kinase 3β in adenocarcinomas as compared to normal OSE and benign adenomas. Adenomatous polyposis coli expression was decreased or absent in ovarian adenocarcinomas [58, 66, 67]. Inactivating mutation of the AXIN1 and AXIN2 genes were detected in primary ovarian endometrioid adenocarcinomas that showed wild-type β-catenin alleles [66].

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12.2.3.3 γ-catenin γ-catenin (also known as plakoglobin) is present in immortalized human OSE [25]. In malignant ovarian tumors, expression of cell surface γ-catenin is reduced and the expression level appears to decrease with increasing tumor stage [55, 60]. Cell surface expression of γ-catenin predicts better prognosis, whereas nuclear γ-catenin expression is associated with a number of indicators of poor prognosis including serous histology and poor tumor differentiation [60]. In contrast, in the subgroup of endometrioid ovarian cancers, nuclear γ-catenin predicts better survival [60]. Interestingly, γ-catenin is overexpressed in chemosensitive ovarian tumors when compared with the chemoresistant counterpart as shown by quantitative proteomics analysis [68]. The regulatory mechanism of γ-catenin in ovarian cancer is unknown. However, it has been shown that mutation of the γ-catenin gene is infrequent in ovarian cancer [69]. 12.2.3.4 p120ctn p120ctn is shown to be present in ovarian cancer samples [20, 70]. Sarrio et al. showed that 83.3% of ovarian tumor samples (n = 84) possessed detectable p120ctn , while the remaining 16.7% showed absent expression of p120ctn [20]. Aberrant localization of p120ctn in the cytosol has been observed in ovarian tumors, and a correlation has been noted between cytoplasmic p120ctn and distant metastases [20, 70].

12.3 Role of Cadherins and Catenins in Ovarian Cancer 12.3.1 Cell Survival In human OSE, we have shown that N-cadherin is an important mediator of cell survival [71]. Disruption of N-cadherin-mediated cell-cell adhesion by neutralizing antibody or N-cadherin-specific siRNA increased the rate of apoptosis, suggesting that the anti-apoptotic effect depends on homophilic adhesive activity of N-cadherin. This is of particular importance since the cyclical ovulatory ruptures and repair during each reproductive cycle is thought to be a factor leading to ovarian carcinogenesis [72]. The identification of N-cadherin in controlling the survival capability of human OSE may be helpful in understanding the development and progression of ovarian cancer. E-cadherin is present in more than 60% of ovarian cancers. E-cadherin-mediated cell-cell adhesion is capable of inducing carcinoma cells to proliferate and survive, which are attenuated in the presence of an E-cadherin-neutralizing antibody SHE 78–7 [73]. Ectopic expression of E-cadherin in SV40 large T antigenimmortalized, E-cadherin negative cells derived from normal OSE conferred anchorage-independence [74]. Expression of a dominant-negative E-cadherin in ovarian carcinoma cells suggests that E-cadherin may function as a survival factor of metastatic ovarian tumor cells during early tumor progression [75].

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Ectopic expression of α-catenin has been shown to suppress the growth and colony formation capability of ovarian cancer cells. It also reduced the ability of these cells to grow as tumors in nude mice. These data imply that α-catenin may act as a growth-regulatory or tumor suppressor gene in ovarian cancer [76]. Notably, nuclear α-catenin was detected, suggesting that α-catenin may exert its effect by modulating transcription, a role in processes other than cell adhesion. In support of this possibility, expression of α-catenin in the nucleus has been shown to inhibit β-catenin/TCF-dependent transcription and disrupt the interaction between the β-catenin/TCF complex and DNA [77]. Chemoresistance is a major obstacle to successful chemotherapy of ovarian cancer. Although many patients have a good response to platinum and taxane combination regimens administered after surgery, about half of these patients tend to acquire resistance to cytotoxic chemotherapeutic agents, and in most the disease remains incurable. Gain of E-cadherin is proposed to contribute to chemoresistance, since ovarian tumor spheroids are more resistant to drug-induced apoptosis and disruption of E-cadherin-mediated cell adhesion in spheroids sensitizes tumor cells to chemotherapeutic agents [78]. γ-catenin has also been suggested to play a role in modulating the chemosensitivity of ovarian cancer cells [68]. The mechanism by which E-cadherin/γ-catenin increases chemoresistance in ovarian cancer remains to be elucidated.

12.3.2 Differentiation In contrast to many other types of cancer, ovarian carcinomas are unique in that an increase in differentiation accompanies neoplastic progression. E-cadherin may contribute to the initiation of this aberrant epithelial differentiation in the early stages of ovarian carcinogenesis. For example, E-cadherin expression is increased in metaplastic OSE and is correlated with an epithelialized phenotype [17, 27]. Expression of E-cadherin in well-differentiated adenocarcinomas was higher than that in poorly/moderately-differentiated adenocarcinomas [28]. Moreover, by ectopically expressing the E-cadherin gene in OSE cells, it initiated early preneoplastic changes by committing the cells to a more complex epithelial phenotype that resembles an aberrant Mullerian-like differentiation along with a range of epithelial differentiation markers including keratin, and CA125 tumor antigen [25, 75]. Expression of E-cadherin is generally reduced in more advanced carcinomas, which correlated to dedifferentiation and invasive capacity of the cancer.

12.3.3 Motility and Invasion To metastasize, cancer cells must detach from each other. They also need to acquire the ability to move and invade the matrix. Although E-cadherin is present in primary ovarian carcinomas, its expression is often reduced or lost in ovarian carcinoma

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metastases. Epithelial to mesenchymal transition (EMT) is an essential morphologic conversion during embryonic development. Increasing evidence also suggests that a similar process occurs during cancer progression by which tumor cells acquire the capacity to migrate, invade, and metastasize [79]. In ovarian carcinomas, tumors with little or no E-cadherin have a more invasive behavior than those expressing it [80, 81]. E-cadherin mutation in ovarian carcinomas is very rare [82]. DNA methylation of the E-cadherin gene was observed in ovarian cancer and correlated with a reduced expression of the protein [83]. Loss of E-cadherin expression is often accompanied by a gain in the expression of mesenchymal cadherins, such as N-cadherin, in a process that is known as cadherin switching [84]. This property is also observed in ovarian tumor cells expressing epidermal growth factor receptor (EGFR), endothelin A receptor, and Met [85–88]. A direct role for N-cadherin during ovarian tumor progression and metastasis development has not been demonstrated. However, the increase in N-cadherin, which has been observed in some cases of ovarian cancer [89], has been suggested to also facilitate in vivo metastatic spread by promoting mesenchymal signaling through its interaction with the surrounding stroma. Cadherin switching also includes other cadherins which replace or are coexpressed with E-cadherin, such as P-cadherin. We have recently shown that the inappropriate expression of this cadherin has a dominant role over E-cadherin and may directly contribute to the invasive phenotype of ovarian cancer cells. Moreover, we have defined a metastatic signaling cascade activated by P-cadherin, which alters trafficking of p120ctn to the cytoplasm, and thereby enhancing activities of Rho GTPases (our unpublished observations). p120ctn has also been shown to interact with Src family kinase Fyn at the cell junction under the stimulation of lysophosphatidic acid (LPA). This association is important for tyrosine phosphorylation of β-catenin with a resultant induction of cell dispersal [90].

12.3.4 Adhesion Adhesion of ovarian cancer cells to the mesothelial cells that line the abdominal cavity is an important step for metastasis to the peritoneal surface. Suppression of E-cadherin has been shown to increase the ability of ovarian cancer cells to adhere to the mesothelial cells lining the cavity [91]. The α5-integrin, an adhesion receptor that mediates cell-extracellular matrix interaction, is up-regulated upon E-cadherin down-regulation, by which may support attachment of ovarian cancer cells to the mesothelium [91].

12.3.5 Angiogenesis Like other cancers, ovarian tumor cells must recruit a new blood supply to progress and continue to grow. In ovarian cancer, increased vascular permeability leads to

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the formation of ascites. Vascular endothelial growth factor (VEGF) derived from ovarian cancer cells has been shown to increase vascular permeability by decreasing VE-cadherin expression [92]. VE-cadherin protein correlates with the vascular permeability associated with ovarian neoplasms.

12.4 Signaling by Cadherin-Catenin in Ovarian Cancer In addition to their adhesive functions, it is now known that cadherins can modulate signal transduction.

12.4.1 Signaling Through β-catenin Because cadherins bind β-catenin tightly and stabilize it at the membrane, changes in cell-cell adhesion may alter β-catenin signaling, suggesting that cadherins may be a negative regulator of β-catenin signaling. Recently, we showed that when activity of the N-cadherin/β-catenin complex is inhibited in OSE, β-catenin is displaced from the adherens junctions at the membrane, which in turn accumulates in the cytoplasm and nucleus. Notably, not only β-catenin is stabilized, but this is sufficient to activate a TCF reporter in the cells, demonstrating activation of β-catenin-dependent signaling [93]. β-catenin phosphorylation, its stabilization and subsequent nuclear activation may also play a role in ovarian cancer progression [94, 95]. Many of the Wnt/β-catenin targets thus far identified are cell cycle regulators (e.g. c-Myc, cyclin D1) and transcription factors (e.g. Twist), and function in a cell autonomous manner, providing insight into the mechanism by which tumor cells deregulate proliferation and inhibit apoptosis [8, 9, 96, 97]. In addition to the known Wnt/β-catenin targets, a recent study by Schwartz et al. [97] has identified several novel Wnt-induced genes in ovarian endometrioid adenocarcinomas by oligonucleotide microarrays. These include CST1, EDN3 and FGF9. CST1 and EDN3 have been associated with metastasis in other studies [98, 99]. FGF9 has been confirmed in a later study by Hendrix et al. [100] to be up-regulated by the Wnt/β-catenin pathway and promotes the development and progression of ovarian endometrioid adenocarcinomas.

12.4.2 Signaling Through p120ctn Similar to β-catenin, cadherins are both necessary and sufficient for localization of p120ctn at the cell membrane [101]. In the cytoplasm, p120ctn interacts with Rho GTPases, which promote cell migration and consequently, invasion and metastasis [10, 11, 102]. In addition to the ability to regulate actin dynamics, these small GTPases may also affect the expression of MMPs and tissue inhibitors of metalloproteinases [103, 104]. Therefore, the Rho GTPases are common mediators of growth factor-induced cancer cell progression.

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We recently found that, in ovarian cancer cells, cytosolic p120ctn plays a crucial role in the regulation of ovarian cancer cell motility and invasiveness. These effects are associated with Rac1 and Cdc42 activation (our unpublished observations). These data support the possibility that cytoplasmic p120ctn has an important function in the migratory effects and propensity for metastasis in ovarian cancer. The nuclear functions of p120ctn and Kaiso are also of interest [105]. Several metastasis-related genes, such as MMP7 and MTA2 (metastasis-associated gene 2), are identified as Kaiso targets. Interestingly, Kaiso can block β-catenin/TCFmediated activation of target genes, suggesting that p120ctn and β-catenin may act in a synergistic manner to activate Wnt targets [106, 107]. Given that p120ctn is involved in the tumor progression of ovarian carcinoma and that it could be a molecular target in cancer therapy, understanding the mechanism of action of Kaiso and its role in signal transduction in ovarian cancer is important.

12.4.3 Cross-talk with Receptor Tyrosine Kinases Since cadherins do not possess intrinsic enzymatic activities, they require recruitment of other signaling molecules to transduce outside-in signaling. In this regard, there is evidence that cadherins could interact with receptor tyrosine kinases (RTKs) at the cell surface and modulate signaling pathways downstream of adherens junctions [12, 13, 108]. For example, E-cadherin is associated with EGFR, thus activating the mitogen-activated protein kinase (MAPK) pathway, and VE-cadherin has been shown to bind to VEGF receptor to regulate endothelial cell survival [12, 13, 109, 110]. N-cadherin has also been found to interact with fibroblast growth factor receptor-1 [14]. E-cadherin-mediated cell-cell adhesion has been shown to enhance the proliferation and survival of ovarian cancer cells by triggering a ligand-independent activation of EGFR, which subsequently induces the phosphorylation of PI3K/Akt and MAPK via Gab1 (Fig. 12.3) [73]. However, direct association between Ecadherin and EGFR cannot be detected. More recently, it has been shown that E-cadherin-mediated adhesion may contribute to PI3K/Akt activation in ovarian carcinoma cells by recruiting the PI3K p85 subunit to the site of cell-cell contacts. This interaction appears to be specific for ovarian cancer; it was not observed in either colon or breast tumor cells [111]. Since ovarian carcinomas maintain E-cadherin during tumor progression, these findings suggest PI3K should be explored further as a therapeutic target in ovarian cancer. We have recently reported activation of insulin-like growth factor-1 receptor (IGF-1R) by P-cadherin, leading to p120ctn activation, Rho GTPase signaling, and tumor cell migration/invasion (Fig. 12.4; our unpublished observations). Possible involvement of IGF-1R is also especially interesting in view of the fact that IGF-1R is frequently overexpressed in ovarian cancer and confers adverse prognosis [112, 113]. Unlike E- and N-cadherin in most cases, IGF-1R regulation by P-cadherin is ligand-independent. How P-cadherin transactivates IGF-1R is not known, but this appears to involve complex formation between P-cadherin and IGF-1R.

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E-cadherin

EGFR Cell Membrane

Cytoplasm

P

Gab 1

MEK

PI3K

MAPK

Akt

Cell proliferation

Fig. 12.3 Cross-talk between E-cadherin and EGFR. E-cadherin-mediated cell-cell adhesion enhances the proliferation and survival of ovarian cancer cells by triggering a ligand-independent activation of EGFR, resulting in activation of the PI3K/Akt and MEK/MAPK signaling pathways via Gab1

12.5 Regulation of Cadherin by Hormones, Growth Factors, and Cytokines- Involvement in Ovarian Tumor Progression There is evidence that implicates hormones, growth factors, and cytokines in the modulation of cadherins to promote ovarian tumor invasion/metastasis.

12.5.1 E- and N-cadherin A range of ligands regulate E-cadherin, including estrogen, which has recently been shown to decrease E-cadherin to promote migration of ovarian cancer cells [114]. Growth factors including EGF , hepatocyte growth factor (HGF) and endothelin-1 peptide are shown to promote ovarian cancer cell metastasis through downregulation of E-cadherin [86, 87]. Increased expression of EGFR and its ligands has been detected in tumors, ascitic fluid, and urine of ovarian cancer patients [115–117]. Moreover, overexpression of EGFR in ovarian tumors is associated with

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IGF1R Cell Membrane

Cytoplasm

P p120ctn

Rac1 p120ctn

Rac1 p120ctn

Cell migration and invasion

p120ctn

p120ctn

Cdc42

Cdc42

Fig. 12.4 P-cadherin signaling to p120ctn through IGF-1R. P-cadherin, which cooperates with IGF-1R in a ligand-independent manner, leads to cytoplasmic translocation of p120ctn , which in turn induces activation of Rac1 and Cdc42 GTPases and the subsequent increase in cell migration and invasion

more aggressive clinical behavior and correlates with a poor prognosis [118, 119]. The effects of EGFR may be due to direct activation or indirect upregulation of alternative cytokines or growth factor receptor pathways, such as interleukin-6 signaling [88]. HGF and endothelin-1 are present in high abundance in the ascites and serum of ovarian cancer patients [120–122] and their receptors are frequently overexpressed in ovarian carcinomas [86, 122–125]. This decreased expression of E-cadherin is often accompanied by an increased expression of N-cadherin which promotes inappropriate survival signals. Repression of E-cadherin by these factors occurs through up-regulation of the transcriptional factors Snail and Slug, which may suppress expression of E-cadherin via binding to E-boxes in the E-cadherin promoter [87, 114, 126, 127]. High expression of Snail, Slug, SIP and Twist have

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been identified in ovarian carcinoma and associated with advanced-stage disease and poor outcomes [128–133]. Post-translational modification of E-cadherin has also been detected in ovarian cancer cells. This may be triggered by shedding of the E-cadherin ectodomain. Soluble E-cadherin was found to be expressed at significantly higher concentration in cystic fluids from cystadenocarcinomas and borderline tumors as compared to that from benign cysts [134]. Engagement of integrins with collagen promotes matrix metalloproteinase-9 (MMP-9)- and Src kinase-dependent E-cadherin ectodomain shedding in ovarian carcinoma cells [135]. Regulation of MMP-9 by EGFR may represent another mechanism for proteolysis of E-cadherin in ovarian cancer [136]. LPA, a known ovarian cancer activating factor, has been shown to induce E-cadherin shedding in an uPA-dependent manner and promotes invasion [137]. Treatment of the cells with recombinant E-cadherin ectodomain (hEcad-Fc) at concentrations found in human ovarian cancer ascites rapidly disrupted the adherens junctions and increased cell dispersion [135, 137], suggesting that soluble E-cadherin may promote ovarian cancer progression and dissemination.

12.5.2 P-cadherin Expression of gonadotropin-releasing hormone (GnRH) receptors and specific GnRH-binding sites has been detected in ovarian carcinoma biopsies, including mucinous and serous subtypes, primary cultures of ovarian carcinomas, and various established ovarian cancer cell lines [138, 139]. In addition to its well-established role in regulating proliferation, our recent findings show that GnRH may be involved in other aspects of ovarian tumor progression through upregulation of P-cadherin expression (our unpublished observations), and a strong correlation between GnRH receptor expression and late-stage tumor progression has been described [140].

12.6 Cadherins as Drug Targets As reviewed above, cadherins could be a promising target for the treatment of ovarian cancer. Unlike other cancers, since E-cadherin is usually up-regulated or increased in ovarian cancers, surface E-cadherin could be a target for antibody therapies. Clinical attempts to inhibit N- and P-cadherin have also been made. ADH-1 (ExherinTM ) is a cyclic pentapeptide that specifically inhibits N-cadherin function. ADH-1 damages tumor blood vessels, but not normal, mature blood vessels, even at very high doses and has been shown to inhibit growth of breast, ovarian, colon, and lung carcinomas [141, 142]. Pre-clinical studies showed a synergistic anti-tumor effect of ADH-1 plus paclitaxel in a xenograft model of ovarian cancer, suggesting that anti-adhesion may improve the effectiveness of chemotherapy [143]. Currently, ADH-1 is being studied in phase II trials in a variety of cancers [142]. PF-03732010 (Pfizer) is a new drug that inhibits P-cadherin and needs to be explored in cancer therapy.

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siRNA is another promising novel approach for gene targeting, leading to the specific silencing of the targeted RNA. Targeted therapy by the delivery of siRNAs has been shown to be effective in xenograft models of ovarian cancer and therapeutic efficacy has been shown in preclinical models [144, 145]. There is emerging evidence suggests that microRNAs (miRNAs) that suppress translation may act as regulators of various aspects of tumor biology and their expressions are often deregulated in human cancers, including ovarian cancer [146, 147]. A recent study by Park et al. [148] has identified the miR-200 family as critical regulators of E-cadherin expression in ovarian cancer patients by targeting the Ecadherin repressors ZEB1 and ZEB2. miR-200c was reported to be overexpressed in ovarian cancer cells and mitigate invasiveness and restore sensitivity to chemotherapeutic agents [149, 150]. Whether targeting these miRNAs will sufficiently reduce tumor metastasis and enhance response to chemotherapy in clinical settings remains to be determined.

12.7 Conclusion Given the unique characteristics of changes in cell adhesion in ovarian cancer, the increasing knowledge about cadherins in ovarian carcinomas has provided a better understanding of factors that may contribute to disease development and metastasis. These studies also make cadherin an attractive target for cancer therapeutics. As new agents become available, further studies are warranted to evaluate antiadhesion as a new biologic approach to treat ovarian cancer and to improve response to conventional chemotherapy. Another particular problem associated with ovarian cancer is the heterogeneity of the disease. The four major histological subtypes (mucinous, serous, endometrioid, and clear cell carcinomas) and low- and high-grade malignancies all have variable clinical manifestations and underlying molecular signatures. The distinct expression patterns of cadherins/catenins in different histological subtypes (Table 12.3) may help to understand the heterogeneity of ovarian cancer at the molecular level and implicate the potential of specific adhesion molecules as candidate markers in early diagnosis and prognosis. Knowledge of the different molecular pathways could lead to more targeted therapeutic interventions.

Table 12.3 Distinct expression profiles of cadherins/catenins in ovarian cancer subtypes

E-cadherin N-cadherin P-cadherin Cadherin-6 β-catenin γ-catenin

Clear cell

Endometrioid

Mucinous

Serous

References

+ − − +

+ + − + Nuclear expression

+ − − −

+ + + +

[20] [18, 20] [20] [53] [59, 62, 64] [60]

Nuclear expression

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Acknowledgments This work was supported by Hong Kong Research Grant Council grants 7599/05 M and 778108 and by HKU Outstanding Young Researcher Award (to A.S.T Wong).

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Chapter 13

PTP4A3, a Signal Molecule Deregulated in Uveal Melanoma Metastasis Cécile Laurent, Jérôme Couturier, Xavier Sastre-Garau, Laurence Desjardins, Emmanuel Barillot, Sophie Piperno-Neumann, and Simon Saule

Abstract Despite improvements in primary treatment protocols, more than 50% of the patients with uveal melanoma die of late-occurring metastases located in the liver. After diagnosis of metastases the average life expectancy is 6 months and no effective treatment is available at this stage of the disease. Transcriptome analysis linked to comparative genomic hybridization have been used for this particular melanoma to identify a set of genes linked to metastasis that may represent valuable future targets for specific treatments. Keywords Uveal melanoma · Comparative genomic hybridization · PTP4A3 · Metastasis

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . How to Identify Metastasis Inducing Genes? . . . . . . . Overexpression of PTP4A3 in Class 2 Uveal Melanoma Cells The Role of PTP4A3 in Signal Transduction Mediating Tumor Metastasis . . . . . . . . . . . . . . . . . . . . 13.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . .

13.1 13.2 13.3 13.4

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13.1 Introduction The choroid is a layer of highly vascularised tissue surrounding the inner side of the ocular globe. Choroidal blood nourishes the retinal pigment epithelium and the photoreceptors on the outer layer of the retina. The choroid develops from two S. Saule (B) Université Paris-Sud 11, Orsay F-91405, France e-mail: [email protected] W.-S. Wu, C.-T. Hu (eds.), Signal Transduction in Cancer Metastasis, Cancer Metastasis – Biology and Treatment 15, DOI 10.1007/978-90-481-9522-0_13,  C Springer Science+Business Media B.V. 2010

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embryonic tissues, the mesoderm and cranial neural crest cells. The endothelial cells of choroidal blood vessels are derived from the mesoderm, whereas cells including stromal cells, melanocytes and pericytes are derived from neural crest [1]. Uveal melanoma is supposed to occur from uveal melanocytes (located in the iris, ciliary body and choroid) and is the most common intraocular malignancy in adults. Uveal melanoma represent about 4–5% of all melanomas. The incidence of the uveal melanoma increases with age reaching a maximum between the 6th and 7th decade of life. Despite the common embryonic origin of their precursor neural-crest cells, clear differences were found between the clinical outcome and molecular biology of uveal and cutaneous melanomas. Neural crest originating from the trunk neural tube providing melanocytes of the skin behave differently from neural crest providing choroidal melanocytes and originating from the most anterior part of the neural tube [2]. Delamination, transcription factor repertoire and cellular environment are different between anterior and trunk melanocytes. For example, choroidal melanocytes are not expected to transfer their melanosomes to surrounding keratinocytes, a well described function for skin melanocytes [3]. The etiological factors involved in the process of malignant transformation of uveal melanocytes are poorly understood. This is partly because of a lack of significant association with systemic syndromes, the absence of familial involvement in most cases, and the doubtful role of environmental factors such as exposure to sunlight in the emergence of uveal melanoma [4]. A number of clinical and histopathological features have been correlated with survival. These include patient age (>60), tumour anterior margin location, tumour cell type, tumour greatest diameter, mitotic activity, vascular loops or extraocular extension, and monosomy of chromosome 3. To date, comparative global gene analysis of melanoma have revealed that aggressive tumor cells express genes associated with multiple cellular phenotypes [5, 6]. These findings support the hypothesis that aggressive melanoma cells adopt a multipotent plastic phenotype. One example of melanoma cell plasticity is vasculogenic mimicry, with aggressive melanoma cells expressing endothelial associated genes and forming extracellular matrix (ECM) rich vasculogenic-like network [7, 8]. The management of uveal melanomas (Fig. 13.1) has greatly evolved, moving towards more focused and conservative treatments (such as observation, photocoagulation, thermotherapy, radiotherapy). However, unlike the improved survival rates for the numerous cancers in which early detection and management have made advances, the survival rate for uveal melanoma has not increased significantly in the last 20 years [9]. According to recent literature, there is no significant difference in survival between patients treated with enucleation and those treated with conservative methods [10]. To date, no adjuvant therapy has proven efficacy in terms of disease-free and overall survival, following the local treatment of ocular melanoma. A randomized study of adjuvant chemotherapy versus simple follow-up showed no difference in terms of 5 year overall survival rate in 348 patients [11]. The metastatic pattern for uveal melanoma differs from that of skin melanoma. Although at diagnosis over 95% of patients have disease limited to the eye, about

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Fig. 13.1 Representation of the eye. (a) Anatomy of the eye, with tissues containing melanocytes underlined. (b) Uveal melanoma resultings on retinal detachment

50% will develop metastases after a median time of 3 years, and will ultimately die of their disease [11]. Once the disease becomes metastatic, median survival ranges from 2 to 6 months [12], and only 15% of the patients survive more than 1 year. Surgical resection of metastases is feasible only if occurring in limited areas. In retrospective studies, conventional chemotherapy, including dacarbazine R ), cisplatinum or nitroso-urea derivatives, gave response rates of less than (Deticen 10% [12]. Genetic differences may explain the various types of melanomas and their different features. Multivariate analyses of genomic imbalances, showed that cutaneous and uveal melanomas harboured different copy number changes [13]. The most frequently detected imbalances in cutaneous melanomas are loss of 9p (63%) and loss of 10 (41%) whereas in uveal melanoma they are loss of 3 (50%) and gain of 8q (50%). With the advent of high resolution genome-wide techniques of genomic and expression profiling, it is now possible to study tumors on a systematic basis, that may improve the characterization of high risk tumors. Recently, using gene expression profiling, two distinct molecular classes strongly associated with metastatic risk could be identified [14, 15]. We compared the frequencies of imbalances between 38 metastatic monosomic 3 primary tumors and 17 non-metastatic ones. Metastastic tumors predominantly show a gain of the entire 8q, most of them having proximal 8q breakpoints (84%) and a loss of 8p (47%). In non-metastatic tumors, the frequency of 8q gain decreases from 80% at the telomere region to 50% near the centromere, and loss of 8p is rare (12%). A second change concerns chromosome 16, metastatic tumors showing frequent losses of 16q (58%), which are not significantly observed in the non-metastatic ones (18%). Loss of 1p, frequent in the tumours (50%) was not retained in the classifier. Finally, gain of 6p is more frequently noticed in nonmetastatic tumours (41%) than in metastastatic ones (21%). Except gain of 6p, all others chromosomal alterations were significantly accumulated by metastatic tumors over non metastatic ones [16].

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13.2 How to Identify Metastasis Inducing Genes? Identifying a metastasis inducing gene requires an integrative approach to obtain a global analysis of genomic, transcriptomic, miRNA alterations and of proteins phosphorylations, and to find convergent differences between tumors from patients who did not metastasize (called meta0 in our analysis) and those having lead to mestastases (called meta1 in our analysis). One of the advantages of uveal melanoma is the great homogeneity of the tumors, in which only two classes have yet been recognized [14, 15, 17], and therefore we may expect clear-cut differences between samples. Genes that discriminate class 1 (low-risk) from class 2 (high-risk) include highly significant clusters of down-regulated genes on chromosome 3 and upregulated genes on chromosome 8q, which is consistent with previous cytogenetic studies [14, 16]. A study of Chang et al. [6] reported that Class 2 gene expression signature was the most accurate predictor of metastasis and the biomarkers most strongly associated with the class 2 signature included epithelioid cell type, beta-catenin, E-cadherin, and hypoxia-inducible factor 1a. Gene Set Enrichment Analysis showed a significant association between genes expressed in class 2 tumors and those expressed in primitive ectodermal and neural stem cells. Class 2 tumors exhibited epithelial features, such as polygonal cell morphology, up-regulation of the epithelial adhesion molecule E-cadherin, colocalization of E-cadherin and beta-catenin to the plasma membrane, and formation of cell-cell adhesions and acinar structures. One of the top class-discriminating genes was the helix-loop-helix inhibitor Id2, which was strongly down-regulated in class 2 tumors. The class 2 phenotype could be recapitulated by eliminating Id2 in cultured class 1 human uveal melanoma cells and in a mouse ocular melanoma model. Id2 seemed to suppress the epithelial-like class 2 phenotype by inhibiting an activator of the E-cadherin promoter. Consequently, Id2 loss triggered up-regulation of E-cadherin, which in turn promoted anchorage-independent cell growth [8]. This suggests the presence of cancer cells with a primitive neural/ectodermal stem cell-like phenotype that may be responsible for metastasis in these highly aggressive tumors. Mutations in the MAPK pathway (Ras, Raf) have been described as an early event occurring in skin melanomas [18]. Mutations in these genes have not been reported in uveal melanomas, excepted for few cell lines grown in vitro [19]. However, mutations in GNAQ (Guanine nucleotide-binding protein alpha subunit, Q polypeptide) have recently been reported in 49% of uveal melanomas [20], but without link with a metastatic behaviour of the primary tumours, and was the only coding sequence analysed found mutated. This suggested that mutations in the coding sequence are not the preferred way to contribute to uveal melanoma metastasis.

13.3 Overexpression of PTP4A3 in Class 2 Uveal Melanoma Cells Another approach to identify genes involved in the metastatic process is to use a biostatistical approach (using a multivariate Cox model) to define the genetic prognosis parameters. The result is compared to the clinical patient data and the transcriptome

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and CGH data, in order to estimate the adding value of genetic prognosis factors to clinical and genomic variables. From preliminary results we have already obtained a set of genes involved in cell migration differentially expressed between primary uveal tumors that have, or not, metastasized. These genes included the PTP4A3 phosphatase (Fig. 13.2a). PTP4A3 is located on 8q24.3, but over-expression of this gene is not merely the consequence of chromosome amplification (Fig. 13.2b). If we compare the expression level of PTP4A3 in tumors also bearing chromosome 6p in addition to the 8q24 amplification, PTP4A3 is no longer differentially expressed between meta0 and meta1 and its level of expression is rather low (Fig. 13.2b) suggesting that amplification of 6p act negatively on PTP4A3 expression. Clinical correlation, ectopic expression and transient suppression studies in general, all support a functional role for PTP4A3 in cancer cell invasion and metastasis. Gene expression profiles compared in colon cancers that had metastasized to the liver with those in primary tumors and normal colon cells [21] showed that PTP4A3 was over expressed and this over-expression is also observed within other types of cancer [22]. Furthermore, down modulation of PTP4A3 in an in vivo mouse cutaneous model of melanoma was able to reduce metastases development [23]. Therefore, this gene seems to be a good new target in human solid tumors to prevent metastatic development [24].

Fig. 13.2 Intensity mRNA expression level (log2 values) of PTP4A3 gene (Affymetrix U133plus2 arrays). (a) PTP4A3 ordered mRNA expression intensity of 55 samples. PTP4A3 is over-expressed on metastasizing tumors (black points). These tumors are principally altered on chromosome 3 (loss) and chromosome 8q (gain) (red alterations). (b) PTP4A3 ordered mRNA expression intensity of 30 samples. On the subgroup of uveal melanomas tumors altered on chromosome 8q, PTP4A3 is still over-expressed on metastasizing tumors. Thus, this over-expressed is not only a consequence of an amplification of 8q region. Less aggressive tumors seems to be protected by 6p region amplification

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13.4 The Role of PTP4A3 in Signal Transduction Mediating Tumor Metastasis Previously, PTP4A3 have been reported to promote motility and invasion in colon cancer cells by stimulating Rho signaling pathways, using transfection approaches [25]. PTP4A3 promote the activation of the Rho GTPases RhoA and RhoC, and signaling through the Rho effector Rho kinase (ROCK) is necessary for PTP4A3induced invasion and motility. In contrast, Rac activity is reduced but no effect on Cdc42 have been observed [25]. Members of the Rho family of small GTPases are key regulators of cell movement through their actions on actin assembly, actomyosin contractility, and microtubules [26]. Expression of Rho family proteins is deregulated in some tumors and correlates with progression of the disease [27]. The three prototypical members of the family, Rho, Rac and CDC42, have all been linked to cell movement. Rac1 drives motility by promoting lamellipodia formation, whereas RhoA signals to the kinases ROCK1 and 2, promoting the formation of actin stress fibers and generation of the actomyosin contractile force required for cell movement. The highly homologous GTPase RhoC has distinct as well as overlapping functions with RhoA, and its overexpression has recently been closely linked with highly invasive and metastatic forms of melanoma [28]. Moreover, cells derived from RhoC-deficient mice are less motile and invasive than their wild-type counterparts containing RhoC [29], further supporting an important role for RhoC in tumor invasion and metastasis. Amoeboid and mesenchymal modes of movement are distinguished by their different usage of signaling pathways. The amoeboid mode involves signaling through ROCK whereas the mesenchymal mode requires extracellular proteolysis for Rac-dependant actin protrusion to be pushed through channels in the extracellular matrix [30]. Rac signals through WAVE2 to direct mesenchymal movement and suppress amoeboid movement through decreasing actomyosin contractility. Conversely, in amoeboid movement, Rho-kinase signaling activates a Rac GAP (ARH-GAP22), that suppresses mesenchymal movement by inactivating Rac. Rho and Rac interplay determinate different modes of tumor movement [31]. Up-regulation of PTP4A3 also activate the Src kinase, which initiates a number of signal pathways resulting in the phosphorylation of ERK1/2, STAT3, and p130 Cas through negative translational control of Csk, a negative regulator of Src [32]. Interestingly, inhibition of Src family kinase with dasatinib blocks migration and invasion of human melanoma cells [33] further suggesting that tyrosine kinase activity is involved in metastasis development and that the control of this activity is involved in the biological effect of PTP4A3. The MAPK cascade is an important signal transduction pathway triggered by integrin beta1 and PTP4A3 increased the phosphorylation level of Erk1/2. In addition, it was shown that PTP4A3 interacted with integrin beta1 in a yeast two-hybrid system [34]. It has also recently been reported that PTP4A3 is a direct target of p53 [35] upregulated in primary cells subjected to DNA damage. PTP4A3 over expression

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mediate a growth-arrest signal in response to p53 activation. PTP4A3 can act as a stimulator of PI3K-AKT signaling [35, 36], but this action appears to require basal levels of PTP4A3, as increased levels of PTP4A3 led to inhibition of Akt activation and concomitant accumulation of the Cdk2 inhibitors p21 and p27. Thus, as has been suggested with oncogene-induced senescence [37], too much stimulation can lead to shutdown of the pathway stimulated by the offending component. Surprisingly, reduction of PTP4A3 level was also found to be cytostatic, via a p38-dependent stimulation of p19ARF, a well known p53 inducer. Therefore, the transition into S- phase may be limited by either induction or loss of a single stimulator of the PI3K-Akt pathway. Through the use of mouse embryos fibroblasts (MEFs) variously lacking Akt1 and Akt2, Cdk2, Cdk4 or all three retinoblastoma proteins family members, Basak et al. [35] demonstrated that PTP4A3 could arrest cells without any pRb activity, but could not arrest in the absence of Akt or Cdk2. Thus, post-restriction point inhibition of cell cycle with PTP4A3 (as evidenced by insensitivity to pRb loss) depends on the target kinases shut down by highly induced PTP4A3. In the absence of these pro-proliferative kinases, cells may have adapted by expressing/activating PTP4A3 insensitive targets that drive proliferation in a less regulatable manner. Indeed, such a result is consistent with Basak’s observation that several tumor cell lines are insensitive to PTP4A3 over-expression, and this may explain the favorite retention of PTP4A3 expression in metastatic cells. Little is known about PTP4A3 inhibitors, but curcumin, the component of the spice turmeric, shows antitumor effect by selectively down-regulating the expression of PTP4A3/PRL-3 but not its family member (PRL1 and 2), in a p53-independent way. Curcumin inhibited the phosphorylation of Src and stat3 partly through PTP4A3 down-regulation [38]. In regard with tumor metastasis, loss of p53 have been reported to activate cell migration through actin remodeling and p53 has been shown to suppress src-induced podosome and cellular invasiveness [39, 40].

13.5 Conclusion When compared with other tumors, uveal melanomas appear rather homogeneous and only two classes could be evidenced in the primary tumors that exhibited distinct metastatic behavior. Among the genes differentially expressed PTP4A3 which encodes a tyrosine phosphatase, may coordinately function with protein tyrosine kinases in signaling pathways involved in a broad spectrum of fundamental physiological processes. Importantly deregulation of this gene may lead to metastasis in uveal melanomas. Pharmaceutical targeting of PTP4A3 or targets of this protein (although not yet clearly defined) may have critical effects on uveal melanoma metastasis development. Acknowledgments This work was supported by a grant from the Département de Transfert, Institut Curie, from the CNRS, the Association pour la Recherche Contre le Cancer and the INCa, Cancéropole Ile-de-France.

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Chapter 14

Signal Transduction Pathways Involved in Hepatocarcinogenesis and Metastasis of Hepatoma Rajagopal N. Aravalli

Abstract Liver cancer is a complex disease that has poor prognosis as it is often diagnosed at an advanced stage. Due to the differences in the prevalence and a wide range of risk factors as well as heterogeneous phenotypic and genetic traits of affected individuals its incidence varies according to the geographical region around the world. It is not amenable to standard chemotherapy and is resistant to radiotherapy. In most cases, surgical resection and liver transplantation remain the only curative treatment options. Extensive research over the past decade has identified a number of molecular biomarkers as well as cellular networks and signaling pathways affected in liver cancer. In this review, the contribution of various cellular transduction events to cancer formation and progression were discussed. Keywords Liver cancer · Hepatocellular carcinoma · Molecular mechanisms · Signal transduction · Hepatoma

Contents 14.1 Introduction . . . . . . . . . . . . . . . 14.1.1 Risk Factors . . . . . . . . . . . 14.1.2 Stem Cells and Liver Function . . . 14.1.3 Role of Stem Cells in Liver Cancer . 14.2 Cellular Signaling Pathways in Liver Cancer 14.2.1 p53 Pathway . . . . . . . . . . . 14.2.2 Retinoblastoma Pathway . . . . . . 14.2.3 Wnt Signaling . . . . . . . . . . 14.2.4 Ras Pathway . . . . . . . . . . . 14.2.5 JAK/STAT Pathway . . . . . . . .

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14.1 Introduction Primary liver cancer is the fifth most common malignancy in men and eighth in women worldwide. It can be classified into one of several histologically different primary hepatic malignancies, such as cholangiocarcinoma (CCA), hepatoblastoma, haemangiosarcoma, and hepatocellular carcinoma (HCC) [72]. Among these, HCC is the most common liver cancer accounting for 70–85% of cases, with nearly 700,000 deaths occurring worldwide each year [33, 34]. In terms of mortality, it is the third most frequent cause of cancer death in men and sixth in women [72]. HCC normally develops as a consequence of underlying liver disease and is most often associated with cirrhosis. Surgical resection and liver transplantation are current best options to treat liver cancer. However, recurrence or metastasis is quite common in patients who have had a resection and survival rate is 30–40% at 5 years postsurgery [12]. The growing incidence of liver cancer has generated intense research effort to understand the physiological, cellular, and molecular mechanisms of the disease with the hope of developing new treatment strategies.

14.1.1 Risk Factors A variety of risk factors cause liver cancer. Such factors include infection with hepatitis viruses [12, 26, 33], exposure to foodstuffs contaminated with aflatoxin B1 (AFB1 ) [117], and vinyl chloride [13], tobacco [128], heavy alcohol intake [29], non-alcoholic fatty liver disease [44], diabetes [103, 132], obesity [103], diet [141], coffee [66], oral contraceptives [81], and hemochromatosis [45]. In general, these factors vary according to the geographical region. For instance, chronic hepatitis B virus (HBV) infection is common in many Asian countries and Africa, whereas HCV is prevalent in Japan and United States [33]. Such differences in infectivity add complexity to the extrapolation of data obtained from one geographical region and applying it to others.

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14.1.2 Stem Cells and Liver Function The liver is the only organ in the human body that is capable of renewing itself following the loss of the natural tissue. Even after 70% hepatectomy, this remarkable regenerative capacity is achieved due to the proliferation of hepatocytes, and under special circumstances, stem/progenitor cells and bone marrow cells that repopulate the liver [35]. Stem/progenitor cells are critical for the tissue restoration process as they are bipotent and can differentiate into the primary cell types of the liver: hepatocytes and biliary ductal cells (cholangiocytes). To date, a number of stem/progenitor cells have been isolated from human, rodent, hamster, and simian livers [2, 3, 8, 31, 36, 37, 47, 49, 60, 67, 69, 70, 80, 83, 92, 101, 107, 118, 119, 144]. Among these, the human hepatic progenitor cells (HPCs) have been observed in liver specimens with severe hepatocellular necrosis, chronic viral hepatitis, and chronic alcoholic liver disease [106]. Activation of the HPCs occurs in the form of reactive ductules and radiating cords of immature cells with an oval nucleus and a small cytoplasm. One of the most common liver stem cells are “oval cells” [84, 100, 115, 129]. They express markers common to both hepatocytes and cholangiocytes, and, they were shown to differentiate into these cell types in vitro [69]. Oval cells are normally isolated from the liver following treatment with agents such as 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), or from animals fed on cholinedeficient diets and treated with 2-acetylaminofluorene (2-AAF) [110, 112]. They are small cells with a characteristic ovoid nucleus that proliferate in vivo following liver damage when the hepatocytes can no longer divide [111]. They are thought to be localized in biliary ductules (canal of Hering) in normal adult liver, and are bipotential. In diseases such as alcoholic liver disease and HCV infection, their numbers increase and correlate with the severity of the disease [76].

14.1.3 Role of Stem Cells in Liver Cancer Over the years it has been well established that both hepatocytes and cholangiocytes are capable of repopulating the liver tissue following injury [86]. Therefore, the concept of stem/progenitor cell existence in the liver did not gain much recognition until the past decade. Furthermore, growing evidence has demonstrated that the capacity to sustain tumor formation and growth resides in a small proportion of “cancer stem cells” (CSCs) [98, 104]. Subsequent identification of CSCs in a number of tissues including brain [46, 113, 114], prostate [24], breast [1], myeloid [68], gastric [50], colon [93, 105] and lung [65] tissues has reinforced the notion that stem cells might also exist in the liver. In the early studies, embryonic stem cells from murine embryos were shown to differentiate into functional hepatocytes in vitro [41, 22]. Later it was shown that murine as well as human bone marrow-derived mesenchymal stem cells could

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differentiate into hepatocytes both in vitro and in vivo [4, 71]. Studies with bone marrow transplant recipients have shown that these cells could home to liver and differentiate into normal hepatocytes [2, 122]. Several groups have isolated oval cells and established liver progenitor cell lines (LPCs) from choline-deficient diet fed rats [69], c-Met transgenic mice [118], p53 null mice [30], and human liver tumors [99]. CSCs from human cell lines were also obtained [80]. The presence of CSCs and successful isolation of oval cells from cancerous tissue suggests that stem/progenitor cells play key role in tumor formation. Further studies with progenitor cells isolated form uninjured liver tissues may provide insight to understand the molecular events that regulate cellular differentiation of the liver and those that lead to tumor progression.

14.2 Cellular Signaling Pathways in Liver Cancer Extensive epidemiological, biochemical, histological and molecular studies over the years have advanced our understanding on the pathogenesis of HCC, but little is known about molecular mechanisms that lead to carcinogenesis. A number of signaling events occur in tumor cells during hepatocarcinogenesis [6]. Abrupt changes in liver tissues that occur due either to viral infection or exposure to hepatotoxic agents cause significant changes in key cellular signaling pathways and alter gene expression resulting in tumor formation (Fig. 14.1). These pathways, discussed below, are being studied extensively to identify potential biomarkers and molecular targets.

14.2.1 p53 Pathway In most human cancers, the tumor suppressor TP53 gene is either inactivated by a single point mutation or p53 signaling is defective causing cell cycle arrest and

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Liver Cancer Fig. 14.1 Cellular signaling pathways implicated in liver cancer. A variety of risk factors govern the activation of a signal transduction pathways, and some of them activate multiple pathways causing tumor formation and progression

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apoptosis [38, 133]. A number of studies have reported that p53 mutations and inactivation may play a critical role in HCC. For instance, in a clinical study of 16 Chinese patients with HCC it was shown that eight of them had a point mutation at the third base position of codon 249 in TP53 gene. Moreover, the G to T transversion in seven samples and the G to C transversion in others were same as the mutations caused by AFB1 [53]. A mutational hotspot in codon 259 was also found in several human cancer cell lines [52]. In a case-control study, serum hepatitis B surface antigen (HBsAg) and liver AFB1 -DNA adducts were found to be significantly elevated in HCC samples compared to controls [79]. Furthermore, all mutations in the codon 249 occurred in HBsAg-seropositive carriers, and mutations in p53 DNA and protein correlated with the tumor stage, suggesting that they are late events. Thus, detection of mutant p53 in plasma serves as a potential biomarker for AFB1 exposure and presence of HCC. Various molecular epidemiological and immunohistochemical investigations have shown that the inactivation of p53 also occurs in cholangiocarcinoma. Mutations in TP53 gene and increased expression of Mdm2, a protein that binds to p53 and prevents transactivation of p53 responsive genes, in 9 out of 13 CCA suggests that impairment of p53 pathway is crucial for CCA [27]. Furthermore, ligands of peroxisome proliferator-activated receptor-gamma (PPARγ) inhibit growth of human CCA cells via p53-dependent GADD45 pathway [42]. Mutations in p53 were also observed in intrahepatic cholangiocarcinoma of mass-forming type in a study with 40 surgically resected ICC cases [63]. Collectively, these data demonstrate that mutations in p53 and impairment of p53 signaling contribute to the development of liver cancer.

14.2.2 Retinoblastoma Pathway The retinoblastoma protein pRb1 is a tumor suppressor that controls cell cycle progression by repressing the function of E2F transcription factor family of proteins. Intracellular cyclin-dependent kinases (CDKs) phosphorylate pRb and lead to G1/S cell cycle transition [32]. Multiple CDKs can phosphorylate at any of the possible 16 phosphorylation sites on pRb with varying degrees of specificity [39]. It has been demonstrated that the expression of one of the CDK inhibitors p16INK4A , p21(WAF1/CIP1) and p27Kip1 could contribute to carcinogenesis in nearly 90% of HCC cases [9, 43]. p16INK4A is inactivated during the early stages of hepatocarcinogenesis as well as in disease progression. Reduced p21(WAF1/CIP1) expression, which is associated mainly with p53 gene mutation in HCC, also contributes to tumor formation [39, 82]. Furthermore, a strong correlation between loss of pRb and lack of functional p53 was observed in early studies on human tumors [131]. Based upon these findings, and the fact that several DNA tumour viruses encode proteins that inactivate both pRB and p53, it has been suggested that the loss of pRb function often results in p53-dependent apoptosis. A number of studies have demonstrated that the pRb pathway is severely disrupted in HCC patients. For example, in a study of 25 patients with HBV-induced

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HCC it was found that pRb expression was severely altered in eight patients [51]. Another study examined the expression of pRb, cyclin D1 and p16 in 47 HCC specimens and found that 38 of them had either an inactive pRb or p16 expression [9]. This disruption in the pRb pathway in HCC was similar to that observed in other cancers and demonstrates that pRb is a critical player in carcinogenesis.

14.2.3 Wnt Signaling The Wnt signaling pathway, originally identified in Drosphila melanogaster, is deregulated in a number of cancers, including HCC [48]. In most cases, either the inactivation of the tumor-suppressor gene adenomatous polyposis coli (APC) or mutation of the proto-oncogene β-catenin, coupled with the activation of Wnt signaling was observed. This pathway is involved in HCC caused by HBV/HCV infections and alcoholic liver cirrhosis. Upregulation of proteins of this pathway, such as frizzled-7, and dephosphorylation of β-catenin is frequently observed in HCC [85]. Therefore, targeted inactivation of Wnt pathway is a potential therapeutic target for liver cancer. Furthermore, mutations in β-catenin were identified in HCC patients with increased exposure to HCV [54] and AFB1 [28], and mutations in two negative regulators of Wnt pathway, Axin 1 and Axin 2, were also observed in HCC samples [94, 120]. A liver specific disruption of APC gene in mice was shown to result in the activation of Wnt pathway [25]. Thus, Wnt/β-catenin pathway is an important signaling pathway in HCC.

14.2.4 Ras Pathway Human ras proteins H-Ras, N-Ras, K-ras4A and K-Ras4B are small GTP-binding proteins that function as molecular switches to influence cell growth, differentiation and apoptosis [108]. Ras interacts with a downstream serine/threonine kinase Raf-1 leading to its activation and downstream signaling, that includes activation of MAPK kinases (MKKs) MEK1 and MEK2, to regulate proliferation and apoptosis [88]. Activation of Ras and expression of Ras pathway proteins such as p21 were also reported in solid tumors [58, 91] as well as in cell lines [18]. Single point mutations in codon 13 of H-ras, codon 12 of N-ras and codon 61 of K-ras was originally observed in HCC that was caused by exposure to various chemicals such as N-nitrosomorpholine, bleomycin, 1-nitropyrene, and methyl (acetoxymethyl) nitrosamine [10, 11, 20, 21, 23, 130]. It has been suggested that the Ras pathway is important in HCC of rodents but not for human HCC based upon the low mutation rate of Ras in humans [40]. However, in a recent study, it was reported that RASSF1A and NORE1A, members of the RASSF family of Ras inhibitors, are inactivated in human HCC [18]. ERK pathway was also implicated in CCA. It has been shown in seven CCA cell lines recently that cells containing mutation in K-ras proliferate faster and that growth inhibition in these cells can be achieved by using

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MEK and Hedgehog blockers [62]. These newer findings underline the importance of the Ras signaling pathway in liver cancer.

14.2.5 JAK/STAT Pathway Signal transducers and activators of transcription (STATs) comprise a family of transcription factors that are activated by a variety of cytokines, hormones and growth factors [102]. Their activation occurs through tyrosine phosphorylation by Janus kinases (JAK). Activated STATs stimulate the transcription of suppressors of cytokine signaling (SOCS) genes. SOCS proteins, in turn, bind phosphorylated JAKs and their receptors to inhibit this pathway, thereby preventing overactivation of cytokine-stimulated cells [140]. JAK stimulation of STATs activates cell proliferation, migration, differentiation and apoptosis, and deregulation of inhibitors leads to human diseases, including cancer [102]. Inactivation of SOCS-1 and SSI-1, a JAK binding protein, have been reported to occur in HCC [89, 140], as also the ubiquitous activation of JAK/STAT pathway [19] in liver cancer.

14.2.6 MAP Kinase Pathway The intracellular mitogen-activated protein (MAP) kinase family were implicated in diverse cellular processes such as cell survival, differentiation, adhesion, and proliferation [134, 137]. Proteins of HBV, HCV and HEV modulate MAPK signaling by targeting multiple steps along the signaling pathway [97]. For instance, HCV E2 protein activates MAPK pathway in human hepatoma Huh-7 cells and promotes cell proliferation [142]. In human HCC, the expression levels of Spred protein (Sprouty-related protein with Ena/vasodilator-stimulated phosphoprotein homology-1 domain), an inhibitor of the ERK pathway, are deregulated and induced expression of Spred caused inhibited ERK activation, both in vivo and in vitro, resulting in reduced proliferation of cancer cells and low secretion of matrix metalloproteinases (MMP) 2 and 9 [138]. This finding shows a direct correlation of MAPK-ERK pathway activation in HCC, suggesting that Spred could serve a therapeutic target for human HCC.

14.2.7 Stress Response Pathways Heat shock proteins (HSPs) are critical players in cellular stress response. In a recent study, HCC progression was found to be associated with a decrease in serine phosphorylation of HSP27 [136]. In another study, several members of the HSP family were found to be associated with the occurrence of HCC [78], suggesting that HSPs are key players in HCC progression.

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The effect of oxidative stress in liver carcinogenesis, most notably in hemochromatosis and Wilson disease, was shown to be associated with p53 mutations [56]. Under such conditions oxidative stress results in the development of cirrhosis with a 200-fold risk for HCC. Mutations that commonly alter p53 function include G:C to T:A transversions at codon 249 as well as to C:T to A:T and C:G to T:A transversions at codon 250. Furthermore, an elevated level of inducible nitric oxide synthase (iNOS) expression was also reported in this study.

14.2.8 Growth Factors and Their Receptors The molecular dynamics of HCC can also be influenced by proteins and cellular factors of other signaling pathways. For example, vascular endothelial growth factor and fibroblast growth factor play important roles in HCC development [55, 139]. It was reported recently that inflammation is inherently associated with cancer and that a number of cytokines are involved in promoting HCC development and progression, especially during infection with hepatitis viruses [17]. In particular, Th2 cytokines are induced and the levels of Th1 cytokines decrease in metastases. Therefore, modulating the expression of cytokines and the use of inhibitors of inflammatory cytokines might be critical in alleviating HCC progression. In a recent study, it was shown that the use of inhibitors of epidermal growth factor receptor (EGFR) and transforming growth factor-ß (TGF-β) prevented the development of HCC in the rat liver demonstrating the harmful nature of these growth factors if they exist in excessive amounts [16, 109].

14.3 Mechanisms Involved in the Metastasis of Hepatoma Primary tumor cells acquire distinct characteristics as they generate metastatic tumors. This process consists of four distinct steps: invasion, intravasation, extravasation, and metastatic growth [64]. The sequence of events in which tumor cells acquire these characteristics appear to be random but tissue invasion and metastasis are always final steps in malignant tumor formation. During the initial stage of invasion, tumor cells lose their cell-cell adhesion properties and gain motility. They then move around and invade adjacent tissues, following which they penetrate through barriers, such as the endothelial barrier, and enter the systemic circulation stream (intravasation). Subsequently, anchorage-independent cells leave the blood stream by attaching to vessels (extravasation), and form micrometastases in the new tumor environment leading to the metastatic growth [64]. A number of studies have reported on the genes and pathways involved during each of these processes. Migratory and invasive events are regulated by various proteolytic enzymes such as cysteine protease, serine protease, and metalloproteases [121]. These enzymes degrade the extracellular matrix and membranes that surround blood vessels. Matrix metalloproteases (MMP), in turn, are regulated by

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the tumor-stromal interactions through CD147, a member of the immunoglobulin superfamily [127]. In HCC, CD147 promotes invasion and metastasis by inducing the tumor cells to produce MMP by regulating calcium inflow [61]. Recently, it was shown that CD147 interacts with annexin II, a calcium- and phospholipid-binding protein to promote invasion and metastasis of human HCC [143]. It has been well established that the proteins involved in embryonic development are aberrantly expressed in a number of cancers [87]. Among these, the suppression of the transcription factor Twist was demonstrated to inhibit the metastatic potential in epithelial cells, whereas its overexpression induces the epithelial to mesenchymal transition (EMT). EMT, a critical process in embryonic development, was recognized as a hallmark of tumor progression and metastasis [123]. It has been described that the accumulation of β-catenin in the nucleus and disruption of E-cadherin from the cell membrane occur during the EMT [15]. Ras signaling through the activation of PI3K/Akt and MAPK pathway is critical for EMT and TGF-β cooperates with Ras in this process [59]. Furthermore, Snail, a target of TGF-β signaling and Snail family member Slug repress E-cadherin during EMT [14, 123]. Snail induction by Notch and EGFR signaling were also reported [77, 126]. A comprehensive study on independent effects of Snail and Twist proteins in promoting metastatic HCC has showed that although Snail was potent in activating MMPs, HCC cells overexpressing Twist were more invasive, suggested that Twist may be acting independently of MMP [135]. Upregulation of Twist in human HCC was also shown to be associated with elevated levels of vascularization and metastasis [90]. Collectively, these data demonstrate that multiple signaling and transcriptional mechanisms are responsible for tumor progression of HCC.

14.4 Strategies for Early Detection and Treatment of Liver Cancer 14.4.1 Gene and Protein Profiling Molecular profiling of genes, proteins and other molecules will provide us with powerful tools needed to gain insights into the molecular mechanisms underlying carcinogenesis [125]. Knowledge obtained from such studies could be translated to develop new diagnostic, prognostic and therapeutic targets for clinical intervention. As discussed above, the expression of a large number of genes, proteins, and other molecules belonging to diverse cellular processes and pathways are altered in liver cancer. Therefore, no single test or a set of tests is sufficient to provide an accurate assessment on hepatic tumor burden for every clinical situation. Many studies have focused on the changes in global expression profiles for a large number of cellular genes and proteins in hepatocarcinogenesis. Specific gene-expression “signatures” obtained from these studies may help to accurately predict the risk for developing HCC. In addition, they may also assist in identifying potential biomarkers and therapeutic targets for elucidating the molecular mechanisms associated with HCC.

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14.4.2 Development of Experimental Tumor Models In order to understand molecular mechanisms in vivo and to test the efficacy of therapeutic compounds, such as inhibitors of various signaling pathways and other small molecules, there is a need for the development of good animal models that mimic the human disease very closely [5]. Based on the foregoing discussion it is apparent that there is opportunity to improve treatment of HCC. As HCC is genetically heterogeneous, and while this is a difficult task, a number of animal models have been developed over the years for studying HCC [7]. Studies conducted with these models, although helpful, did not result in the development of treatments for liver cancer. Therefore, new experimental models that closely mimic the human condition in terms of physiology, etiology, and clinical setting are needed.

14.4.3 Identification of Therapeutic Targets and Novel Biomarkers In recent years, a number of studies have employed various drug treatments such as small-molecule protein kinase inhibitors, monoclonal antibodies, and antibiotics either alone or in combinations to target cellular signaling pathways [116]. One such study examined whether interferon therapy prevents the development of HCC in patients with chronic HCV infection [95]. Following therapy, these patients were studied between 1 and 7 years and the cumulative incidence of HCC was found to be significantly lower in sensitive cases, but not in patients in advanced stages [95]. Similar results were reported in chronic HCV patients undergoing IFN therapy [57]. These advancements have resulted in the identification of novel molecular biomarkers and the design of therapeutic drugs. While most of these approaches have relied on interfering various signaling pathways, some others have focused on blocking the signals of neoangiogenesis [75, 96]. More recently, sorafenib showed a modest, 3-month survival benefit in selected patients [116]. Prior to this, however, over 100 clinical trials of IV chemotherapy using many drugs or combinations for HCC failed to show any statistically significant survival benefit [124]. Sorafenib is a multikinase inhibitor that was shown to inhibit tumor angiogenesis by targeting the Raf/MERK/ERK pathway and induce apoptosis in the PLC/PRF/5 xenograft model of HCC [74]. The success of sorafenib has resulted in the development of a number of therapeutic compounds, some of which are currently undergoing clinical trials [75]. Several of these drugs have shown to inhibit their target pathway in experimental models, cell lines, and in preclinical models. Integration of molecular biomarkers identified from various different studies with these drug targets would allow us to identify potential candidates for drug response.

14.5 Conclusions Liver cancer is a complex disease with multiple underlying pathogenic mechanisms caused by a variety of risk factors. Despite enormous progress achieved during the

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past several decades, patient survival remains very low. The lack of good molecular markers for tumor diagnosis and treatment assessment has posed a major challenge in health care. One of the objectives of global-scale studies for liver cancer is to determine the factors that contribute to the progression of cancer from normal tissue to metastasis. For this, a thorough understanding of genotype-phenotype relationship based upon the environmental risk factors and host’s genetic and hereditory traits is essential. These phenotypes may result from activation of different oncogenic pathways during tumorigenesis and/or from a different cell of origin. Molecular profiling of genes, proteins and other molecules is aimed at deciphering the genotype-phenotype relationship with the goal of developing new therapies for human disease. Furthermore, stem cells and other progenitor cells isolated from healthy livers might prove critical for creating novel cancer therapeutics.

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Index

A Adaptor proteins, 14–15, 77–93, 140, 144 Angiogenesis, 2, 12–16, 29, 33–34, 54, 62–63, 78, 82–83, 85–86, 104, 106, 108–111, 163–165, 167, 191–193, 211, 213, 226, 238–239, 274

Ceramide, 190, 211–214, 216 Cholesterol, 211–214, 216, 218 Cytoskeleton, 11, 17–18, 42, 44–45, 50–51, 54, 56–57, 61, 63, 81–82, 89–90, 106, 109, 121, 139–144, 148–149, 159, 182, 211, 227–228

C Cadherin, 227–245 E-, 10–11, 25, 28, 57, 62, 81, 109, 160–161, 183, 229–230, 232–234, 236–244, 258, 273 H-, 229, 233 N-, 81, 161, 163, 183, 229, 232, 236, 238–244 P-, 229, 233, 238, 240, 242–244 Cancer -initiating cells, 181–183 stem cell, 137–138, 168, 178, 180, 188, 190, 267 Carcinogenesis, 104, 138, 147, 228, 236–237, 265–275 Catenin α-, 57, 228, 231, 234–235, 237 β-, 57, 161, 163, 175, 181, 183, 185–186, 226, 228, 235, 237, 239–240, 244, 258, 268, 270, 273 γ-, 228, 236–237, 244 Cadherin-, 225–244 p120 (p120ctn ), 228, 231, 236, 238–240, 242 Cell migration, 11, 18–19, 30, 44, 51, 53–56, 59, 61, 63, 86, 90, 105–111, 119–129, 139–143, 147, 149, 160, 163, 215–216, 218, 228, 239–240, 242, 259, 261 Cell survival, 3–4, 18, 25, 34, 43–44, 50, 85, 87, 91, 157, 163, 165–166, 209, 216, 226, 229, 236, 240, 271

E EGFR (epidermal growth factor receptor), 3, 5, 19, 80, 83, 89, 90, 125, 144, 175, 181, 183, 186–187, 190–192, 211, 213, 216–217, 238, 240–243, 272–273 EMT (Epithelial-mesenchymal transition), 1–2, 7, 9–20, 27–29, 59–60, 81, 103–105, 107–111, 159–161, 178–179, 181–183, 190, 217–218, 238, 273 ERK1/2 MAP Kinase, 25–34 Extravasation, 5, 7, 10, 27, 32, 78, 104, 106, 139, 159, 272 F Focal adhesion protein, 121, 137–150 G Gene expression, 18, 28–30, 42, 85, 87, 106, 109, 121, 159, 162–163, 183, 215, 257–259, 268 Grb2, 14–15, 77–93, 217 H Hepatocellular carcinoma (HCC), 15, 89, 105, 190, 193, 266, 268–274 Hepatoma, 105, 107, 109, 124–125, 128, 184, 265–275 HGF (hepatocyte growth factor), 2, 7, 13–15, 19, 28, 80, 82–83, 90, 104–105, 109–110, 120, 123, 125, 165, 181, 183, 185, 216, 241–242

W.-S. Wu, C.-T. Hu (eds.), Signal Transduction in Cancer Metastasis, Cancer Metastasis – Biology and Treatment 15, DOI 10.1007/978-90-481-9522-0,  C Springer Science+Business Media B.V. 2010

283

284 I Inhibitor(s), 11, 17, 19, 28, 31–32, 34, 42, 47–48, 50, 56–58, 61–64, 81, 83–84, 92, 146, 148–149, 161, 164, 166, 168, 182, 186–191, 193–194, 217, 239, 258, 261, 269–272, 274 Integrin, 3–5, 7, 10, 15, 17–19, 29–30, 33, 81, 86, 91, 93, 105–110, 119–129, 139–144, 146–147, 186, 210, 215, 238, 260 Intravasation, 3, 7, 27, 31–32, 86, 104, 139, 159, 210, 272–273 Invasion cancer, 51, 139, 150, 162 local, 27–31, 80–81, 159–160, 215 tumor, 11–12, 90, 105, 107–109, 121, 124, 148, 163, 215, 218, 241, 260 tumor cell, 11, 29, 87, 103, 106, 108, 161–163 L Lipid rafts, 144, 209–219 Liver cancer, 189, 191, 265–275 M MAPK-activating protein kinases, 41–64 cytoskeleton, 51–56 inhibitors, 63 metastasis, 50–62 MAPK (mitogen activated protein kinase), 3, 5, 7, 9, 15–18, 25, 43–45, 55, 58–59, 61, 80, 85, 91, 105, 107–108, 110, 119–129, 159, 163, 165, 217–219, 240–241, 260, 270–271, 273 ERK–, 5, 18, 120–122 MAP kinase, 25–34, 108, 216–217, 258, 271 See also MAPK (mitogen activated protein kinase) Metastasis bone, 6, 8, 17, 162 cancer, 6, 10, 27–34, 54–55, 81, 87, 91, 157–169, 209–219, 233 -initiating cells, 175–195 tumor, 1–7 uveal melanoma, 255–261 Migration cell, 11, 18–19, 30, 44, 51, 53–56, 59, 61, 63, 86, 90, 105–111, 119–129, 139–143, 147, 149, 160, 163, 215–216, 218, 228, 239–240, 242, 259, 261

Index Molecular mechanisms, 3, 30, 33, 44, 47, 56, 125–128, 147, 175–195, 266, 268, 273–274 Motility, 5, 13–14, 16, 26–28, 54, 59, 62, 80–82, 109, 211, 215, 237–238, 260 tumor cell, 3, 18, 30–31, 42–44, 50, 51–53, 55–56, 61, 80, 82–83, 86–87, 89–90, 121, 141–142, 240 N Notch, 28, 157–169, 175, 181, 186, 273 O Ovarian cancer, 15, 47, 168, 193, 225–244 P PKC, 2, 15, 19, 105–110, 120–129, 144, 215 PTP4A3, 255–261 R Reactive oxygen species (ROS), 4, 19, 103–110, 119–129 S Signaling cadherin-catenin, 225–244 ERK1/2 MAP Kinase, 25–35 FA network, 140–141 growth factor receptors-mediated, 209–219 integrin-mediated, 17–19 intraversion, 3 liver cancer, cellularpathways, 268–273 MAPK, 121–122 notch, 157–168 PKC Integrin Signaling, 122–124 reactive oxygen species (ROS), 103–111, 124–128 RTK Integrin Signaling, 122–124 TGF-β/Smad, 16–17 transduction elements, 175–195 tumor Metastasis initiation, 2 tumor progression in circulation, 3–6 Signal transduction(s), 1–8, 11, 14, 17–19, 41–42, 79, 84, 104–105, 120, 124, 157, 216–218, 239–240, 255, 260, 265–275 Sphingomyelin, 209–214 Src, 4–7, 15, 18, 48, 79–81, 83–85, 120, 122–125, 127–128, 140, 143–144, 228, 231, 238, 243, 260–261

Index T Target therapies, 185–194 TGF-, 1, 3, 7, 14–19, 24, 28–29, 121, 160, 183, 186, 215, 272–273 Treatment resistance, 175, 183, 185, 195 Tumor metastasis, 1–7

285 progression, 3–5, 11–12, 14–19, 25–34, 87–91, 103–111, 119–129, 148, 159, 185, 211, 213, 217, 226–227, 232–233, 235, 238, 240–243, 268, 273 U Uveal melanoma, 255–261