Extracellular Targeting of Cell Signaling in Cancer...Extracellular Targeting of Cell Signaling in Cancer Strategies Directed at MET and RON Receptor Tyrosine Kinase Pathways Edited

Extracellular Targeting of Cell Signaling in Cancer

Extracellular Targeting of Cell Signaling in Cancer

Strategies Directed at MET and RON Receptor Tyrosine Kinase Pathways

Edited by

James W. JanetkaWashington University School of MedicineUSA

Roseann M. BensonConsultantUSA

This edition first published 2018© 2018 John Wiley & Sons Ltd

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Library of Congress Cataloging-in-Publication Data

Names: Janetka, James W., 1968- editor. | Benson, Roseann M., editor.Title: Extracellular targeting of cell signaling in cancer : strategies

directed at MET and RON receptor tyrosine kinase pathways / edited by James W. Janetka, Roseann M. Benson.

Description: First edition. | Hoboken, NJ : John Wiley & Sons, 2018. | Includes bibliographical references and index. |

Identifiers: LCCN 2018000527 (print) | LCCN 2018007450 (ebook) | ISBN 9781119300205 (pdf) | ISBN 9781119300212 (epub) | ISBN 9781119300182 (cloth)

Subjects: LCSH: Metastasis–Treatment–Technological innovations. | Cancer–Treatment–Technological innovations. | Tumor suppressor proteins–Research. | Protein-tyrosine kinase.

Classification: LCC RC269.5 (ebook) | LCC RC269.5 .E97 2018 (print) | DDC 362.19699/40072–dc23

LC record available at https://lccn.loc.gov/2018000527

Cover design by WileyCover image: ©Lightspring/Shutterstock

Set in 10/12pt WarnockPro by SPi Global, Chennai, India

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v

List of Contributors xiiiPreface xvii

1 Discovery and Function of the HGF/MET and the MSP/RON Kinase Signaling Pathways in Cancer 1Silvia Benvenuti, Melissa Milan and Paolo M. Comoglio

1.1 Introduction 11.2 MET Tyrosine Kinase Receptor and its Ligand HGF: Structure 11.2.1 The Invasive growth Program 21.2.2 MET Mediated Signaling 41.2.2.1 MET Down-regulation 71.2.3 Cross-talk between MET and Other Receptors 71.2.4 MET Activation in Human Cancers 91.2.4.1 MET, Hypoxia and Ionizing Radiations 101.2.4.2 MET Expression in Cancer Stem Cells: a Paradigm of Inherence 111.2.4.3 Oncogene Addiction and Oncogene Expedience 111.2.5 Targeting HGF/MET as a Therapeutic Approach in Human Cancer 121.2.5.1 HGF Antagonists 131.2.5.2 Tyrosine Kinase Inhibitors 151.2.5.3 Anti-MET Monoclonal Antibodies 171.2.5.4 Alternative MET Blocking Strategies 181.2.6 Primary and Secondary Resistance 181.2.6.1 MET Role in Resistance to Anticancer Agents 191.2.6.2 Mechanism of Resistance to MET Inhibitors 191.2.6.3 Combinatorial Therapeutic Strategies 201.3 RON Tyrosine Kinase Receptor and its Ligand MSP 211.3.1 Discovery and Structural Biology 211.3.2 RON Mediated Signaling 251.3.3 Cross-talk between RON and other Receptors 261.3.4 RON Activation in Human Cancers 261.4 Targeting MSP/RON as a Therapeutic Approach in Human Cancer 271.5 Concluding Remarks 28 Acknowledgements 29 References 29

Contents

Contentsvi

2 The Role of HGF/MET and MSP/RON Signaling in Tumor Progression and Resistance to Anticancer Therapy 45Lidija Klampfer and Benjamin Yaw Owusu

2.1 Introduction 452.2 HGF/MET Signaling in Cancer 472.3 MSP/RON Signaling in Cancer 522.4 Cross-talk between MET and RON Signaling Pathways 532.5 HGF/MET and MSP/RON Signaling Elicit Resistance to Cancer Therapy 552.6 Conclusions and Perspectives 58 References 58

3 HGF Activator (HGFA) and its Inhibitors HAI-1 and HAI-2: Key Players in Tissue Repair and Cancer 69Hiroaki Kataoka and Takeshi Shimomura

3.1 Introduction 693.2 Discovery of HGFA 703.2.1 Tissue Injury-induced Activation of HGF 703.2.2 Identification of HGFA as a Serum Activator of pro-HGF 713.3 Synthesis of HGFA Zymogen in vivo 713.4 Molecular Structure of HGFA 723.4.1 The Gene Encoding pro-HGFA: HGFAC 723.4.2 ProHGFA Protein and its Activation 723.4.3 Structure Biology of HGFA 743.5 Substrates of HGFA in vivo 753.6 Regulation of HGFA Activity by Endogenous Inhibitors 763.6.1 HGF Activator Inhibitor-1 (HAI-1): a Cell Surface Regulator

of HGFA Activity 763.6.2 HGF Activator Inhibitor-2 (HAI-2) 783.6.3 Protein C Inhibitor (PCI; SERPINA5) 783.7 Proposed Biological Functions of HGFA in vivo 783.8 Roles of HGFA in Cancer 803.8.1 Enhanced Activation of pro-HGF and pro-MSP in Cancer Tissues 803.8.2 Possible Roles of HGFA in Cancer Progression 803.9 Conclusions and Future Perspectives of HGFA Research in Cancer 82 References 83

4 Physiological Functions and Role of Matriptase in Cancer 91Fausto A. Varela, Thomas E. Hyland and Karin List

4.1 Introduction 914.2 Discovery of Matriptase 914.3 Biochemical and Functional Characteristics of Matriptase – Inhibitors,

Substrates and Structure 924.3.1 Endogenous Polypeptide Matriptase Inhibitors 924.3.2 Matriptase Substrates 944.3.3 Matriptase Structure 954.4 Physiological and Pathophysiological Functions of Matriptase 96

Contents vii

4.4.1 Matriptase in Epidermal Development and Homeostasis 964.4.2 Matriptase in the Gastrointestinal Tract 974.4.3 Matriptase in Thymocytes and Salivary Glands 984.4.4 Matriptase in Placental/Embryonic Development 984.4.5 Matriptase in Neural Tube Closure 994.4.6 Pathways requiring Matriptase 994.4.7 Matriptase in Viral Infection 1014.5 Role of Matriptase in Cancer 1014.5.1 Studying Matriptase in Cultured Cancer Cells and Tumor

Grafting Models 1084.5.2 In vivo Cancer Studies using Genetic Models 1114.5.2.1 Squamous Cell Carcinoma 1114.5.2.2 Colitis-associated Colon Carcinogenesis 1124.5.2.3 Breast Cancer 1124.6 Conclusions 114 References 114

5 The Cell-Surface, Transmembrane Serine Protease Hepsin: Discovery, Function and Role in Cancer 125Denis Belitškin, Shishir Mani Pant, Topi Tervonen and Juha Klefström

5.1 Biology of Hepsin 1255.1.1 Discovery of Hepsin 1255.1.1.1 Cloning of Hepsin, HPN Gene 1255.1.1.2 Assigning Hepsin to Type II Transmembrane Serine Protease Family 1265.1.2 Hepsin Gene and Protein 1265.1.2.1 Expression, Regulation and Structure 1265.1.2.2 Hepsin Activation and Activity 1305.1.3 Physiological Functions of Hepsin 1315.1.3.1 Growth Factor Activation 1315.1.3.2 Serine Protease Cascades 1325.1.3.3 Cell Proliferation and Motility 1325.1.3.4 Epithelial Integrity 1335.1.3.5 Organ Development 1355.2 Hepsin in Cancer 1375.2.1 Gain of Oncogenic Function 1375.2.1.1 Genetic Alterations 1375.2.1.2 Altered Subcellular Localization 1385.2.1.3 Oncogenic Hepsin Function in vivo 1405.2.1.4 How HPN Promotes Cancer 1415.2.2 Targeting Hepsin in Cancer 1435.3 Future Prospects 1445.3.1 Hepsin’s Role as Guardian of Epithelial Integrity 1445.3.2 Cancer Disease Progression and Metastasis 1455.3.2.1 Uncontrolled Proteolysis 145 Acknowledgements 146 References 146

Contentsviii

6 Targeting HGF with Antibodies as an Anti-Cancer Therapeutic Strategy 155Dinuka M. De Silva, Arpita Roy and Donald P. Bottaro

6.1 Introduction 1556.2 HGF Biology 1566.2.1 HGF Gene Organization and mRNA Transcripts 1566.2.2 HGF Protein Isoforms and Proteolytic Processing 1566.2.2.1 HGF Isoforms 1566.2.2.2 HGF Activation by Proteolytic Processing 1596.2.3 Key HGF Interactions: Heparan Sulfate Proteoglycans and Met 1606.2.3.1 Heparan Sulfate Proteoglycans 1606.2.3.2 Met and Key Intracellular Effectors 1616.2.4 Major Sites of HGF Expression: Tissues and Organs 1626.2.5 HGF Function in Development and Adulthood 1626.2.5.1 hgf or met altered Mice: Embryogenesis 1636.2.5.2 hgf or met altered Mice: Late Development and Adulthood 1636.3 HGF in Cancer 1646.3.1 Lung Cancer 1656.3.2 Hepatocellular Carcinoma 1656.3.3 Genitourinary Malignancies 1666.3.4 Breast Cancer 1676.3.5 Colorectal and Gastric Carcinomas 1676.3.6 Papillary Thyroid Carcinoma 1686.3.7 Brain Tumors 1686.3.8 Melanoma 1696.3.9 Head and Neck Squamous Cell Carcinoma 1696.3.10 Other Malignancies 1696.4 Anti-HGF Monoclonal Antibodies as Anti-Cancer Therapeutic

Candidates 1706.4.1 Rilotumumab 1706.4.2 Ficlatuzumab 1746.4.3 TAK-701 1756.5 Conclusions and Future Directions 176 Acknowledgements 177 References 177

7 MET and RON Receptor Tyrosine Kinases as Therapeutic Antibody Targets for Cancer 199Mark Wortinger, Jonathan Tetreault, Nick Loizos, and Ling Liu

7.1 MET as a Therapeutic Antibody Target for Cancer 1997.2 Challenges in Developing MET Therapeutic Antibodies 2007.3 Anti-MET Antibody Clinical Diagnostics 2037.4 Anti-MET Antibodies in the Clinic 2047.4.1 Onartuzumab – Roche 2047.4.2 Emibetuzumab – Eli Lilly 2067.4.3 ABT-700 – AbbVie 208

Contents ix

7.4.4 SAIT301 – Samsung 2087.4.5 ARGX-111 – Argenx 2097.4.6 Sym-015 – Symphogen 2107.5 Additional anti-MET Antibodies 2107.5.1 DN-30 – University of Turin Medical School 2107.5.2 Other Preclinical Stage anti-MET Antibodies 2107.6 Summary– anti-MET Antibodies 2117.7 RON as a Therapeutic Antibody Target for Cancer 2117.8 Conclusions and Future Outlook 216 References 216

8 Inhibitory Antibodies of the Proteases HGFA, Matriptase and Hepsin 229Daniel Kirchhofer, Charles Eigenbrot, and Robert A. Lazarus

8.1 Anti-Serine Protease Antibodies for Therapeutic Applications 2298.2 Antibodies can Inhibit Trypsin-Fold Serine Proteases in Diverse Ways 2308.2.1 Orthosteric Inhibition (Active Site Binding) 2318.2.2 Allosteric Inhibition 2318.2.3 Exosite Inhibition 2318.2.4 Inhibition of Zymogen Activation 2318.2.5 Cofactor Inhibition 2318.2.6 Inactivation of Oligomeric Serine Proteases 2328.2.7 Comparison of Abs with Natural Occurring Protein Modes

of Inhibition 2328.3 Introduction to Antibodies against HGFA, Matriptase and Hepsin 2338.4 Inhibitory HGFA Antibodies 2348.5 Inhibitory Matriptase Antibodies 2388.6 Inhibitory Hepsin Antibodies 2398.7 Conclusion 240 References 240

9 Inhibitors of the Growth-Factor Activating Proteases Matriptase, Hepsin and HGFA: Strategies for Rational Drug Design and Optimization 247James W. Janetka and Robert A. Galemmo, Jr

9.1 Introduction 2479.1.1 Proteolytic Control of HGF/MET Oncogenic Signaling 2479.1.2 Proteolytic Control of MSP/RON Kinase Signaling 2489.1.3 The Identification of HGF and MSP Converting Enzyme Activity 2499.2 Small Molecular Weight Inhibitors of HGFA, Matriptase and Hepsin 2519.2.1 Mechanism-based Inhibitors derived from Substrate Sequences 2519.2.2 Approved Drugs as Starting Points for Inhibitor Design 2579.2.3 Retro-Engineering Inhibitors of Related Proteases 2589.3 Improving Drug-like Properties of the Current Inhibitors: Lessons

from the Oral Anti-Coagulants 2649.4 Conclusion 269 References 270

Contentsx

10 Cyclic Peptide Serine Protease Inhibitors Based on the Natural Product SFTI-1 277Blake T. Riley, Olga Ilyichova, Jonathan M. Harris, David E. Hoke and Ashley M. Buckle

10.1 Introduction: Naturally Occurring Polypeptide Serine Protease Inhibitors 277

10.1.1 Serpins 27710.1.2 Standard Mechanism Inhibitors 27810.1.2.1 Kunitz Type 27810.1.2.2 Kazal Type 27810.1.2.3 Bowman–Birk Inhibitor (BBI) Family 27810.2 Selective Inhibitors of Serine Proteases using the Sunflower Trypsin Inhibitor

(SFTI-1) as a Scaffold for Rational Drug Design 27910.2.1 Trypsin 27910.2.2 Chymotrypsin, Neutrophil Elastase and Cathepsin G 28610.2.3 Proteasome 28610.2.4 Matriptase and other Type II Transmembrane Serine Proteases

(TTSPs) 28610.2.5 MASP-1 and MASP-2 28610.2.6 Other KLKs (KLK5, 7, 14) 28710.2.7 KLK4 28710.3 Normal and Pathophysiological Functions of the Human Tissue Kallikrein

(KLK)-related Serine Protease Family 28810.3.1 Physiological Role for KLKs 28810.3.2 KLKs and their Role in Prostate Cancer Pathogenesis 28910.3.3 Kallikrein-related Peptidase 4 as a Point of Therapeutic

Intervention 29010.4 Inhibitors of KLK4 Serine Protease 29110.4.1 Molecular Basis of KLK4 Inhibition by SFTI-1 29110.4.2 Use of SFTI-1 as a Scaffold in Ligand Design and Optimization 29210.4.3 Identification of an Optimal Tetrapeptide Substrate 29210.4.4 SFTI-1FCQR is a Potent Selective Inhibitor of KLK4 29310.4.4.1 Structural Basis for Potency and Selectivity of SFTI-1FCQR Derivative 29310.5 Potential Therapeutic Applications and Challenges 29410.6 Conclusions/Future Directions 297 References 297

11 Screening Combinatorial Peptide Libraries in Protease Inhibitor Drug Discovery 307Marcin Poreba, Paulina Kasperkiewicz, Wioletta Rut and Marcin Drag

11.1 Introduction 30711.2 Proteases Involved in Cancer 30911.2.1 Metalloproteases 30911.2.2 Serine Proteases 31011.2.3 Cysteine Proteases 31111.2.4 Aspartic Proteases 31111.2.5 Threonine Proteases 31211.2.6 Target Protease Substrates and Inhibitors 312

Contents xi

11.3 Identification and Optimization of Preferred Substrates 31311.3.1 Positional Scanning of Substrate Combinatorial Libraries (PS-SCL) 31311.3.2 Peptide Microarrays 31811.3.3 Hybrid Combinatorial Substrate Library (HyCoSuL) 31811.3.4 Counter Selection Substrate Library (CoSeSuL) 32011.3.5 Combinatorial Substrate Synthesis for Aminopeptidase Screening 32011.3.6 Internally Quenched Fluorescent (IQF) Substrates 32111.3.7 Phage Display 32211.3.8 Protease Substrates – Summary 32511.4 Design of Covalent Inhibitors Based on Substrates 32611.4.1 Background and General Characteristics of Inhibitors 32611.4.2 Substrate-based Inhibitor Design and Discovery 32711.4.3 PS-SCL Applied to Inhibitors other than Substrates 32811.4.4 Inhibitors from Phage Display Screening and Directed Evolution

of Proteins 33111.5 Anticancer Drugs – How much Information do We Need? 33411.6 Conclusions 336 Acknowledgements 337 References 337

12 Chemical Probes Targeting Proteases for Imaging and Diagnostics in Cancer 351Pedro Gonçalves and Steven H. L. Verhelst

12.1 Introduction 35112.2 Chemical Probes for Proteases 35212.2.1 Substrate-based Probes 35212.2.2 Activity-based Probes (ABPs) 35612.2.3 Photo-crosslinking probes 35612.2.4 Non-Covalent Probes 35812.3 Molecular Imaging of Cancer 35812.3.1 Imaging Tumors with Substrate-based Probes 35912.3.1.1 Preclinical Model Systems 35912.3.1.2 Clinical Trials 36112.3.2 Imaging Tumors with ABPs 36212.3.2.1 Conventional and multimodal ABPs 36212.3.2.2 Quenched ABPs 36412.3.2.3 Towards Clinical Applications 36512.3.3 Imaging Tumors with Affinity-based Reagents 36612.3.3.1 Preclinical Models 36612.3.3.2 Clinical Trials 36712.4 Conclusions 369 Acknowledgements 370 References 371

13 Cancer Diagnostics of Protease Activity and Metastasis 377Timothy J. O’Brien and John Beard

13.1 Introduction 377

Contentsxii

13.2 The Proteins Identified from Patient Tumor Profiling 38613.2.1 Matriptase 38613.2.2 Hepsin 38713.2.3 KLK7 38713.2.4 KLK6 38813.2.5 KLK8 38813.2.6 TMPRSS3 38813.2.7 MMP-7 38913.3 ELISA Assay Development 38913.4 The Role of Markers for Cancer Surveillance and Tumor Monitoring

(Early Detection) 39013.5 Cell Signaling and the Cancer Cascade 39913.6 Conclusions and Future Prospects 400 References 402

14 Roles of Pericellular Proteases in Tumor Angiogenesis: Therapeutic Implications 411Janice M. Kraniak, Raymond R. Mattingly and Bonnie F. Sloane

14.1 Introduction 41114.2 Initiation of Angiogenesis 41214.3 Mechanisms of New Blood Vessel Formation 41314.3.1 Sprouting Angiogenesis 41414.3.2 Intussesceptive or Non-sprouting Angiogenesis 41514.3.3 Neovasculogenesis 41514.3.4 Vascular Mimicry 41614.4 Pericellular Proteases and Angiogenesis 41714.4.1 Metalloproteinases: MMPs, ADAMs and ADAM-TS 41814.4.1.1 MMPs 41814.4.1.2 ADAMs and ADAM-TS 42214.4.2 Serine Proteases 42414.4.3 Cysteine Cathepsins 42514.4.3.1 Cysteine Cathepsins in Angiogenesis 42614.5 Novel Approaches for Targeting Tumor Angiogenesis 42814.6 Summary 432 Acknowledgements 433 References 433

Index 447

xiii

John BeardStage I Diagnostics, Inc.USA

Denis BelitškinResearch Programs Unit/Translational Cancer Biology & Institute of Biomedicine, Biomedicum HelsinkiUniversity of HelsinkiFinland

Silvia BenvenutiCandiolo Cancer InstituteItaly

Donald P. BottaroUrologic Oncology BranchCenter for Cancer ResearchNational Cancer InstituteNational Institutes of HealthUSA

Ashley M. BuckleDepartment of Biochemistry and Molecular BiologyBiomedicine Discovery InstituteMonash UniversityAustralia

Paolo M. ComoglioCandiolo Cancer InstituteItaly

Dinuka M. De SilvaUrologic Oncology BranchCenter for Cancer ResearchNational Cancer InstituteNational Institutes of HealthUSA

Marcin DragDepartment of Bioorganic ChemistryFaculty of ChemistryWroclaw University of TechnologyPoland

Charles EigenbrotGenentech, IncDepartment of Structural BiologyUSA

Robert A. GalemmoProteXase Therapeutics, Inc.USA

Pedro GonçalvesKU Leuven – University of LeuvenDepartment of Cellular and Molecular MedicineBelgium

Jonathan M. HarrisInstitute of Health and Biomedical InnovationQueensland University of TechnologyAustralia

List of Contributors

List of Contributorsxiv

David E. HokeDepartment of Biochemistry and Molecular BiologyBiomedicine Discovery InstituteMonash UniversityAustralia

Thomas E. HylandDepartment of PharmacologyBarbara Ann Karmanos Cancer InstituteWayne State University School of MedicineUSA

Olga IlyichovaDepartment of Biochemistry and Molecular BiologyBiomedicine Discovery InstituteMonash UniversityAustralia

James W. JanetkaWashington University School of MedicineDepartments of Biochemistry and Molecular Biophysics and ChemistryUSA

Paulina KasperkiewiczSanford Burnham Prebys Medical Discovery InstituteUSA

and

Department of Bioorganic ChemistryFaculty of ChemistryWroclaw University of TechnologyPoland

Hiroaki KataokaDepartment of PathologyFaculty of MedicineUniversity of MiyazakiJapan

Daniel KirchhoferGenentech, Inc. Department of Early Discovery BiochemistryUSA

Lidija KlampferProteXase Therapeutics, Inc.USA

Juha KlefströmResearch Programs Unit/Translational Cancer Biology & Institute of Biomedicine, Biomedicum HelsinkiUniversity of HelsinkiFinland

Janice M. KraniakDepartment of PharmacologyWayne State University School of MedicineUSA

Robert A. LazarusGenentech, Inc. Department of Early Discovery BiochemistryUSA

Karin ListDepartment of PharmacologyBarbara Ann Karmanos Cancer InstituteWayne State University School of MedicineUSA

Ling LiuLilly Research LaboratoriesEli Lilly and CompanyUSA

Nick LoizosLilly Research LaboratoriesEli Lilly and CompanyUSA

List of Contributors xv

Raymond R. MattinglyDepartment of PharmacologyWayne State University School of MedicineUSA

Melissa MilanCandiolo Cancer InstituteItaly

Timothy J. O’BrienStage I Diagnostics, Inc.USA

Benjamin Yaw OwusuDepartment of PathologyUniversity of Alabama at Birmingham School of MedicineUSA

Shishir Mani PantResearch Programs Unit/Translational Cancer Biology & Institute of Biomedicine, Biomedicum HelsinkiUniversity of HelsinkiFinland

Marcin PorebaSanford Burnham Prebys Medical Discovery InstituteUSA

and

Department of Bioorganic ChemistryFaculty of ChemistryWroclaw University of TechnologyPoland

Blake T. RileyDepartment of Biochemistry and Molecular BiologyBiomedicine Discovery InstituteMonash UniversityAustralia

Arpita RoyUrologic Oncology BranchCenter for Cancer ResearchNational Cancer InstituteNational Institutes of HealthUSA

Wioletta RutDepartment of Bioorganic ChemistryFaculty of ChemistryWroclaw University of TechnologyPoland

Takeshi ShimomuraDepartment of PathologyFaculty of MedicineUniversity of MiyazakiJapan

Bonnie F. SloaneDepartment of PharmacologyWayne State University School of MedicineUSA

Topi TervonenUniversity of HelsinkiFinland

Jonathan TetreaultLilly Research LaboratoriesEli Lilly and CompanyUSA

Fausto A. VarelaDepartment of PharmacologyBarbara Ann Karmanos Cancer InstituteWayne State University School of MedicineUSA

Steven H. VerhelstKU Leuven – University of LeuvenDepartment of Cellular and Molecular MedicineBelgium

List of Contributorsxvi

and

Leibniz Institute for Analytical Sciences ISASAG Chemical ProteomicsGermany

Mark WortingerLilly Research LaboratoriesEli Lilly and CompanyUSA

xvii

Cancer has often been described as “a wound that will not heal.” Interestingly, wound healing (tissue repair) in normal tissues is orchestrated in the extracellular compart-ment by coagulation cascade proteases and cell signaling pathways that are initiated by growth factors or cytokines. Arguably, the most prominent growth factor is hepatocyte growth factor (HGF), the activating ligand for the oncogenic MET receptor tyrosine kinase (RTK). HGF is produced and secreted by hepatocytes and fibroblasts as an inac-tive single‐chain precursor, called proHGF which, in response to tissue injury, is pro-cessed into the two‐chain active form. The activation of proHGF in injured tissue, which is generally limited to the site of injury, is mediated by several pericellular serine proteases, the most efficient being HGF‐Activator (HGFA), matriptase and hespin. This activation of HGF allows for MET‐positive epithelial and endothelial cells to rapidly enter a regenerative phase and escape apoptosis. The tissue injury‐mediated activation of the HGF‐activating proteases also leads to the activation of macrophage stimulating protein (MSP), the ligand for RON kinase that resides on macrophages, and certain endothelial cells. Thus, in addition to tissue repair, these proteases have immunomodu-latory roles through macrophage recruitment and inflammatory processes. In most invasive (advanced stage) cancers, the MET and RON pathways, which are key to wound healing, are dysregulated and aberrantly activated in both tumor cells and the surround-ing stromal tissue in the micro‐environment.

Constitutive activation of the HGF/MET signaling pathway promotes the uncon-trolled growth and survival of cancer cells and stimulates cellular transformations, such as epithelial to mesenchymal transition (EMT), one of the early stages of the spread of cancer. Over 90% of cancer‐related deaths are a result of secondary malignant growths at a distant site from the primary tumor. The invasive spread of cancer is called metas-tasis and, currently, there are no effective therapies for the prevention or treatment of metastatic cancer.

Oncogenic MET and RON kinase cell signaling pathways are well‐studied and validate therapeutic targets for metastatic cancer in several tumor types. It has been shown that:

● MET and/or RON signaling are up‐regulated in multiple forms of solid tumors including breast, lung, pancreatic, prostate, colon, bladder, ovarian cancer and glioblastoma;

● MET and/or RON signaling are up‐regulated in hematological malignancies such as multiple myeloma and AML;

Preface

Prefacexviii

● MET and RON are co‐expressed in several tumor types and can form heterodimers (a mechanism to enhance downstream signaling and promote tumor progression); and

● A common resistance mechanism to small molecule or antibody‐based kinase inhibi-tors (e.g. EGFR, HER2, BRAF, MET, PDGFR, VEGFR, IGFR) in cancer patients is up‐regulation of MET and/or RON kinase signaling.

These findings indicate that an enhanced clinical benefit might be possible from tar-geting both MET and RON kinase cell signaling pathways. Unfortunately, to date, most inhibitors of MET and RON kinases have failed to show sufficient efficacy in clinical trials. While the reasons are unclear, it is known that most patients rapidly develop resistance to MET‐targeted inhibitors, in some cases by up‐regulating HGF in the tumor micro‐environment. Subsequently, several other promising therapeutic strate-gies have emerged to inhibit cell‐signaling through MET and RON pathways on the outside of the cell. These alternative approaches to intracellular kinase inhibitors are largely designed to prevent kinase activation and signaling by blocking HGF binding to the receptor. Indeed, inhibitory antibodies to HGF and to extracellular domains of MET (and RON) have been developed by several companies to block the binding of HGF to MET and abrogate cell signaling to the activated receptor. Furthermore, a non‐cleavable form of HGF or ‘HGF decoy’ has been reported in addition to neutralizing antibodies against HGFA, matriptase and hepsin.

HGF is the only known activating ligand for MET, while MSP is the only known acti-vating ligand for RON. The secreted forms of HGF and MSP require post‐translation proteolytic processing to an active form capable of activating MET and RON, respec-tively. Remarkably, both HGF and MSP are activated by these same three serine pro-teases, HGFA, matriptase and hepsin. Increased activity of these proteases (and MET and RON signaling), has been correlated with tumor progression and metastasis in multiple tumor types. In many cases, the increased protease activity is associated with a concurrent down‐regulation of the endogenous serine protease inhibitors, HAI‐1, HAI‐2 and PCI (Protein C Inhibitor), through either decreased expression, silencing or mutation. Accordingly, a ‘triplex’ inhibitor targeting all three HGF/MSP‐activating pro-teases would be capable of blocking both MET and RON cancer cell signaling and pre-venting the proteolytic activation of HGF and MSP. Moreover, matriptase and hepsin have several substrates other than HGF and MSP, such as uPA (urokinase‐type plasmi-nogen activator), and are thus implicated in different proteolytic ‘cancer cascades’, which are important for tumor progression.

The Janetka group at Washington University in St Louis, MO, and the Galemmo group at Southern Research Institute in Birmingham, AL, have most recently reported on the first ‘triplex’ peptide‐based and small molecule protease inhibitors of HGFA, matriptase and hepsin. By preventing the pericellular activation of both the MET and RON ligands and kinase receptor activation, inhibition of protease activity results in decreased cancer cell signaling, survival, migration, EMT and invasion. In addition, these newly developed inhibitors of HGFA, matriptase and hepsin are inventive chemical tools to study cancer cell signaling, tumor progression and metastasis. Excitingly, it has been shown by the Klampfer group at the Southern Research Institute that these inhibitors are, in fact, capable of overcoming and preventing resistance to EGFR and MET targeted kinase inhibitors (both small molecule and antibodies) in colon and lung cancer cells. These inhibitors are potentially pioneer anticancer drugs

Preface xix

for treatment of metastatic cancer, as well as adjuvant therapy for disease progression prevention.

Within the pages of this book, scientists from seven countries working in private industry, the government sector, and academia explore this narrow area of study and illuminate its broad implications within the cancer research field. Specifically, the inves-tigators presented several new promising therapies to address the large unmet medical need of preventing and treating metastatic cancer, with a focus on several therapeutic strategies designed to curtail the activation and binding of HGF and MSP to MET and RON, respectively.

January 2018 James W. Janetka Roseann M. Benson

Extracellular Targeting of Cell Signaling in Cancer: Strategies Directed at MET and RON Receptor Tyrosine Kinase Pathways, First Edition. Edited by James W. Janetka and Roseann M. Benson.© 2018 John Wiley & Sons Ltd. Published 2018 by John Wiley & Sons Ltd.

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1.1 Introduction

MET and RON oncogenes encoding two related tyrosine kinase receptors are among the most important genes involved in the control of the invasive growth genetic pro-gram. Under physiological conditions, such as embryonic development and organ regeneration, the invasive growth program controls the normal tissue development by coordinating, in time and space, several biological events including cellular prolifera-tion, disruption of intercellular junctions, migration through the extracellular matrix (ECM), and protection from programmed cell death (apoptosis). In transformed tissues, MET or RON deregulation results in cancer formation and metastatic dissemination. Upon either ligand stimulation or constitutive receptor activation, cancer cells are induced to leave the primary tumor, degrade the basal membrane, move towards differ-ent organs and generate metastasis (1,2). The two sibling receptors exert a dual role: they are necessary oncogenes for those tumors that rely on MET activity for growth and survival (oncogene addiction) and adjuvant, pro‐metastatic genes for other tumors, where MET activation is a secondary event that exacerbates the malignant properties of already transformed cells (oncogene expedience). In this complex scenario, MET and RON become very attractive candidates for targeted therapeutic intervention.

1.2 MET Tyrosine Kinase Receptor and its Ligand HGF: Structure

MET oncogene, positioned on chromosome 7q21‐31, is composed of 21 exons encoding a transmembrane tyrosine kinase receptor made of a disulphide‐linked heterodimer (190 kDa), which originates from the proteolytic cleavage, in the post‐Golgi compartment, of a single chain precursor. The heterodimer is formed by a single‐pass transmembrane β chain (145 kDa) and a completely extracellular α chain (45 kDa). The extracellular por-tion contains a SEMA (semaphorin) domain, an atypical motif made by over 500 amino acids, which has a low affinity binding activity for the ligand and is involved in receptor

Discovery and Function of the HGF/MET and the MSP/RON Kinase Signaling Pathways in CancerSilvia Benvenuti, Melissa Milan and Paolo M. Comoglio

Candiolo Cancer Institute, Italy

Extracellular Targeting of Cell Signaling in Cancer2

dimerization; a plexin, SEMA and integrin cysteine‐rich (PSI) domain, which encom-passes about 50 residues and contains 4 disulphide bonds; and 4 immunoglobulin‐plexin‐transcription structures (IPT domain), a characteristic protein‐protein interaction region. A single pass hydrophobic membrane‐spanning domain is followed by the intracellular portion made of a juxtamembrane section followed by a catalytic site and a C‐terminal regulatory tail (Figure 1.1). The juxtamembrane segment is essential for receptor down‐regulation (2). It contains a serine residue (Ser985) that, upon phospho-rylation, is responsible for inhibition of receptor kinase activity, and a tyrosine (Tyr1003) capable of binding the E3‐ubiquiting ligase CBL (cellular homologue of Cas NS‐1 onco-gene), that promotes receptor degradation (3,4). The catalytic site contains two tyrosines (Tyr1234 and Tyr1235) that regulate the enzymatic activity. Finally, the C‐terminal tail encompasses two tyrosines (Tyr1349 and Tyr1356) that, when phosphorylated, generate a docking site able to recruit a vast cohort of intracellular molecules and adaptor proteins responsible for transducing the signaling triggered by the ligand‐receptor interaction (5).The latter two tyrosines have shown to be essential and sufficient to execute MET physi-ological functions (5), and to elicit MET oncogenic potential (6).

MET high affinity ligand is known as the scatter factor (SF) or hepatocyte growth factor (HGF). SF is a factor capable of inducing scatter of epithelial cells, a complex phenomenon that consists of a first step in which cells dissociate one from another and a second phase in which the released cells begin to move (7,8). While HGF is a potent growth stimulator for primary hepatocytes kept in culture (9), the two molecules were later shown to be identical (10). SF/HGF belongs to the plasminogen family of pepti-dases; it contains an amino terminal hairpin loop (HL), followed by four Kringle domains, flanked by an activation portion and a serine‐protease domain (SPH) devoid of proteolytic activity (Figure 1.1). This ligand, synthesized and secreted as a single chain inactive precursor (pro‐HGF) by stromal cells (i.e. fibroblasts), is present in the extracellular environment of almost all tissues. Its activation occurs locally upon pro-teolytic cleavage by proteases that cleave the bond between Arg494 and Val495.

To date, several proteases (present either in the serum or within cells) have been pro-posed as HGF/SF activators, including HGF activator (HGFA) (11), plasma kallikrein and coagulation factors XIIa and XIa (12), matriptase and hepsin (13,14), TMPRSS2 (15), TMPRSS13 (16), urokinase‐type plasminogen activator (uPA), and tissue‐type plasminogen activator (tPA) (17). Among them, HGFA and matriptase, synthesized in turn as inactive precursors, show the most efficient pro‐HGF/SF processing activity (18). Mature HGF is a heterodimer made of a 69 kDa α chain and a 34 kDa β chain linked by a disulfide bond. HGF contains two binding sites with differential affinity for the MET receptor: a high‐affinity site located within the α chain and a low affinity site in the β chain. The low affinity site in the β chain becomes accessible only after pro‐HGF activation, which is essential for receptor dimerization and subsequent activation. Cells of mesenchymal origin are the primary producers and source of HGF in the pericellular environment, which acts on cells expressing the MET receptor (cells of epithelial origin) in a paracrine manner.

1.2.1 The Invasive growth Program

Cancer is a multistep process that results from the accumulation of somatic genetic alterations, which either inactivate tumor suppressor genes (i.e. p53, pRB or APC) or

HGF/MET and MSP/RON Kinase Signaling Pathways in Cancer 3

(A) HGF

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Figure 1.1 MET tyrosine kinase receptor and its ligand HGF: structure.MET is a transmembrane tyrosine kinase receptor made of a disulphide‐linked heterodimer formed by a single‐pass transmembrane β chain and a completely extracellular α chain. The extracellular portion contains a SEMA domain, involved in ligand binding and receptors dimerization; a PSI domain, encompassing four disulphide bonds; and four IPT domains, a protein–protein interaction region. A single pass transmembrane domain is followed by the intracellular portion made of a juxtamembrane section, a catalytic site and a C‐terminal regulatory tail. The juxtamembrane segment contains a serine (serine 985) and a tyrosine (tyrosine 1003) responsible to inhibit receptor kinase activity and promote receptor down‐regulation. The catalytic site contains the ‘catalytic’ tyrosines 1234 and 1235 that regulate the enzymatic activity, while the C‐terminal tail encompasses the ‘docking’ tyrosines 1349 and 1356 that, upon phosphorylation, generate a docking site able to recruit a vast cohort of intracellular adaptors and molecules responsible of triggering the signal transduction cascade.HGF: hepatocyte growth factor; HL: hairpin loop; IPT: immunoglobulin‐plexin transcription domain; K: kringle; PSI: plexin‐semaphorin‐integrin domain; SEMA: semaphorin domain; SPH: serine‐protease domain.

Extracellular Targeting of Cell Signaling in Cancer4

activate dominant proto‐oncogenes (i.e. RAS or PI3K) (19,20). These aberrant events release cells from proliferative control and allow primary tumor formation. The initial tumor growth is followed by invasive dissemination and ultimately metastasis, which is the cause of almost all cancer‐related deaths. The ability of neoplastic cells to invade the surrounding tissues, survive in foreign environments, and settle at distant sites, defines a genetic program known as invasive growth. The invasive growth program also occurs under physiological conditions. Throughout embryogenesis, invasive growth orches-trates complex events such as gastrulation (responsible of originating the mesoderm from the embryonic epithelium), morphogenesis of epithelia, angiogenesis, nervous system formation and myoblasts migration (21). In adult life, invasive growth is neces-sary in normal tissues during acute injury repair (23,24) when cells at the wound edge reprogram themselves and start rapidly dividing prior to migrating towards the cut edge to regenerate the lacking tissue.

The invasive growth program consists of several stages, each of them occurring in a specific time and place, all harmoniously orchestrated to allow germ layers in the embryo, and tissues in the adult, to re‐organize. All these events require cells to prolif-erate, migrate, overcome apoptosis, invade the surrounding tissues and re‐organize themselves into new three‐dimensional structures. Epithelial‐mesenchymal transition (EMT) is the mechanism behind the earlier phases of the invasive growth program. During EMT, cells release junctions that maintain the epithelial monolayer structure, change their polarity by means of cytoskeleton rearrangements and attain the ability to move within the extracellular environment. Ultimately, the cells lose their epithelial phenotype to acquire a mesenchymal one. All these events, necessary during embryo-genesis for correct embryo development and in adult tissues to overcome injuries, con-tribute to tumor formation and metastatic spread when aberrantly regulated. MET oncogene in conjunction with its ligand HGF, is one of the key players in the control of the invasive growth program.

1.2.2 MET Mediated Signaling

Under normal circumstances, MET kinase activation and its signaling cascade occurs upon ligand binding. The HGF/MET protein–protein interaction results in:

1) receptor dimerization;2) auto‐phosphorylation of the ‘catalytic’ residues, Tyr1234 and Tyr1235, located

within the kinase activation loop and necessary to switch on receptor activity; and3) trans‐phosphorylation of the ‘docking’ residues, Tyr1349 and Tyr1356, located

within the docking site (Figure 1.1).

Upon phosphorylation, the latter tyrosines recruit several intracellular signaling pro-teins and adaptors by means of their SRC homology 2 (SH2) domains (22) and trigger the broad spectrum of MET‐mediated biological responses. Downstream signaling proteins include the p85 regulatory subunit of phosphatidyl inositol 3‐kinase (PI3K), phospholipase Cγ (PLCγ) (22), SRC homology 2 domain containing transforming protein (SHC) (23), the adaptor growth factor receptor‐bound protein 2 (GRB2) (24), the transcription factor signal transducer and activator of transcription 3 (STAT3) (25),

HGF/MET and MSP/RON Kinase Signaling Pathways in Cancer 5

the v‐crk sarcoma virus CT10 oncogene homolog (CRK) (26), and SRC homology domain‐containing 5’ inositol phosphatase (SHP‐2) (27). In addition, MET associates with the scaffolding protein GRB2‐associated binding protein 1 (GAB1) (28), either directly or indirectly through GRB2. GAB1 lacks intrinsic enzymatic activity. However, with the receptor interaction, GAB1 becomes phosphorylated and provides binding sites for several proteins involved in the MET signaling cascade (2). The different sign-aling proteins and adaptors are responsible for generating MET‐specific biological activities and their harmonic coordination in time and space results in unique biological responses.

Activated MET recruits and activates RAS (rat sarcoma small GTPase) through the specific guanine nucleotide exchange factor SOS (son of sevenless) (31) which, in turn, is engaged by GRB2 and SHC. RAS, in turn, recruits and activates v‐raf murine sarcoma viral oncogene homolog B1 (BRAF). BRAF sequentially activates mitogen‐activated protein kinase effector kinase (MEK) then extracellular signal‐regulated kinase (ERK), Jun N‐terminal protein kinase (Janus kinase 1 JNK) and p38 MAPK, which translocate into the nucleus. Next, p38 modulates the activity of a number of transcription factors to promote cellular proliferation, transformation and differentiation (32). The RAS signaling is also positively reinforced by SHP2, recruited through GAB1, and is respon-sible for prolonging MAPK phosphorylation (29) (Figure 1.2).

GAB1 is used as a scaffolding protein to recruit, among others, the adaptor CRK. MET‐GAB1‐CRK complex results in JNK activation as demonstrated by a loss‐of‐function mutant of CRK where the activation of the JNK pathway by MET is severely impaired. In addition, JNK, through an AP‐1 element in the promoter region, controls the tran-scription of matrix metalloproteinase‐1 (MMP‐1) gene (26). Indeed, the MET‐GAB1‐CRK signaling complex (via JNK) is a crucial event in regulating the tumorigenic phenotype of MET‐transformed cells (Figure 1.2).

In a parallel signaling pathway, MET recruits p85 regulatory subunit of PI3K, directly or indirectly through GAB1, and catalyses the formation of phosphatidylino-sitol (3–5)‐triphosphate (PtdIns(3–5)P3). PtdIns(3–5)P3 constitutes a docking site for AKT (AKT8 virus oncogene cellular homolog). Upon recruitment to the inner side of the plasma membrane, AKT inactivates (by phosphorylation) glycogen syn-thase kinase 3β (GSK3β), which antagonizes the expression of positive cell cycle regulators. AKT activation also results in protection from apoptosis through either inactivation of pro‐apoptotic protein BCL‐2 antagonist of cell death (BAD) or acti-vation of E3 ubiquitin‐protein ligase MDM2 (murine double minute 2) that induces degradation of the pro‐apoptotic protein p53. Finally, AKT activates mammalian tar-get of rapamycin (mTOR), which stimulates protein synthesis and physical cell enlargement (30).

Activated MET receptors also recruit and phosphorylate STAT3 monomers which, upon phosphorylation, homodimerize and translocate into the nucleus and act as tran-scription factors to regulate cellular proliferation, (25) transformation and tubulogene-sis. Tubulogenesis is the formation of branched tubular structures in epithelial cells (25) (Figure 1.2).

Some of the biological processes regulated by HGF/MET, including cellular adhesion and migration, require regulation of cell‐matrix interactions. The effect of HGF on the two major focal adhesion proteins, focal adhesion kinase (FAK) and paxillin, has been

Extracellular Targeting of Cell Signaling in Cancer6

Figure 1.2 MET‐driven signaling and biological activities.HGF/MET interaction results in receptors dimerization, activation and phosphorylation of the ‘docking’ tyrosines. Once phosphorylated, the latter tyrosines recruit several intracellular signaling proteins or adaptors responsible for generating MET‐specific biological activities and their harmonic coordination in time and space results in unique biological responses including: cell growth, differentiation, motility, proliferation, survival, transformation and tubulogenesis.AKT: AKT8 virus oncogene cellular homolog; BAD: BCL‐2 antagonist of cell death; CRK: v‐crk sarcoma virus CT10 oncogene homolog; ERK: extracellular signal‐regulated kinase; FAK: focal adhesion kinase; GAB1: GRB2‐associated binding protein 1; GRB2: growth factor receptor‐bound protein 2; GSK3β: glycogen synthase kinase 3β; HGF: hepatocyte growth factor; JNK: Jun N‐terminal protein kinase; MAPK: mitogen‐activated protein kinase; MDM2: murine double minute 2; mTOR: mammalian target of rapamycin; PI3K: phosphatidyl inositol 3‐kinase; RAS: rat sarcoma small GTPase; SHC: SRC homology 2 domain containing transforming protein; SHP‐2: SRC homology domain‐containing 5’ inositol phosphatase; SOS: son of sevenless; STAT3: signal transducer and activator of transcription 3.

Tubulogenesis

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HGF/MET and MSP/RON Kinase Signaling Pathways in Cancer 7

investigated in epithelial cells. Liu et al. found that HGF increased serine/threonine phosphorylation of paxillin, resulting in the recruitment and activation of FAK and sub-sequent enhancement of cell spreading and adhesion (31).

Finally, HGF/MET pairing stimulates NF‐κB DNA binding and transcriptional activa-tion through phosphorylation of nuclear factor‐κB inhibitor‐a‐kinase (IKK), which in turn phosphorylates nuclear factor‐κB inhibitor‐a (IKB). Upon IKB’s phosphorylation, the nuclear factor‐κB (NF‐κB) released is free to translocate into the nucleus and stimu-late the transcription of various genes, including mitogenic (32) and pro‐survival regu-lators (33).

1.2.2.1 MET Down‐regulationIn non‐transformed cells, MET activation is tightly regulated and receptors are switched off through diverse mechanisms. In one instance, CBL, an E3‐ubiquitin ligase, is recruited to Tyr1003 within the juxtamembrane domain, and mediates ubiquitin trans-fer to MET, which is subsequently internalized by endocytosis and degraded (4). In another instance, tyrosine specific phosphatases, including the non‐receptor protein‐tyrosine phosphatase 1B (PTP1B), T‐cell protein tyrosine phosphatase (TCPT/PTPN2) (34), leukocyte common antigen‐related molecule (LAR/PTPrF) (35), and density enhanced protein tyrosine phosphatase‐1 (DEP‐1/PTPRJ) (36), are involved in MET shutdown, consequently triggering de‐phosphorylation of either the ‘catalytic’ (in the case of PTP1B and TCPT) or the ‘docking’ tyrosines (DEP‐1). Furthermore, recruit-ment of PLCγ results in activation of protein kinase C (PKC) that negatively regulates MET phosphorylation and activity (37,38).

Receptor activation is also controlled upstream through regulation of pro‐HGF pro-teolytic processing into mature HGF in the extracellular environment by proteases, as previously discussed (18,39).

1.2.3 Cross‐talk between MET and Other Receptors

Since MET is a transmembrane receptor exposed on the phospholipidic cellular mem-brane, MET interacts in a dynamic way with other cellular surface receptors, and the output signal originates from the combination and integration of this complex network. Ultimately, the cross‐talk with other receptors generates signals that differ in length and magnitude and produce diverse biological outputs. Many different molecules have been demonstrated to be MET partners, among them integrin α6β4, the adhesive molecules CD44, the plexins B family, FAS and, lastly, several other tyrosine kinase receptors such as RON, EGFR and HER2.

MET is constitutively associated with integrin α6β4in a HGF‐dependent manner: upon ligand binding and receptor activation, the integrin becomes phosphorylated, recruits intracellular signal transducers (i.e. SHC, SHP2 and PI3K) and generates a plat-form necessary to promote the receptor invasive growth program (40). In addition, MET and integrin interact through FAK upon MET induced phosphorylation (41).

MET is also associated with CD44, the transmembrane receptor for hyaluronic acid, responsible for connecting ECM components to the cytoskeleton. It has been described that some CD44 isoforms, generated by alternative splicing, can trigger or enhance MET activation. CD44v3, which contains the alternatively spliced exon 3, binds HGF

Extracellular Targeting of Cell Signaling in Cancer8

with high affinity and is responsible for: (i) concentrating the ligand at the cellular surface; and (ii) presenting it in multimerized complexes that result in receptor over‐activation. In addition, a CD44 isoform containing the exon 6 sequence (CD44v6) is strictly required for ligand dependent MET activation, as it promotes HGF‐MET inter-action through its extracellular domain. It certainly has been demonstrated that CD44v6‐deficient tumor cells were unable to activate MET unless they were transfected with a CD44v6 isoform. Moreover, signal transduction from activated MET to MEK and ERK required the presence of CD44v6 portion, including a binding motif for ERM proteins (45). ERM is a protein family that consists of three closely related members, ezrin, radixin and moesin, which are responsible for cross‐linking actin filaments with plasma membranes and involved in signal transfer. In summary, the interaction between MET and CD44 results in an efficient functional cooperation, which generates tumor growth and metastatic spread.

MET also interacts with Plexins B. Plexins are transmembrane receptors for sema-phorins, a large family of both soluble and membrane‐bound ligands, which were origi-nally identified as axon guidance cues in the nervous system (42). It has been shown that stimulation of Plexin B1 with its natural ligand SEMA4D induces plexin clustering as well as HGF‐independent MET activation, resulting in an enhanced invasive growth response (43).

MET can also associate with death receptor FAS. This interaction with MET prevents FAS homo‐oligomerization and clustering and ultimately results in protection for apop-tosis (44).

Finally, other tyrosine kinase receptors can be MET partners. It was initially shown that MET interacts with RON, a member of the same family of tyrosine kinase receptors (discussed extensively below). It was confirmed that ligand‐induced MET activation results in RON trans‐phosphorylation and vice versa. The trans‐phosphorylation occurs in a direct way, as it does not need the C‐terminal docking site of either receptor and a kinase‐dead RON is sufficient to block MET transforming activity (45). More recently, it was shown that in cancer cell lines displaying MET amplification, RON is specifically trans‐phosphorylated by the sibling receptor and sustains MET‐driven proliferation and clonogenic activity in vitro and tumorigenicity in vivo (46). These data show that, while specific for their ligands, scatter factor receptors cross‐talk and combine forces to trigger specific intracellular signaling cascade (47). Similarly, it was shown that MET interacts with the orphan receptor ROR1 and is responsible for its trans‐phosphoryla-tion (48), highlighting the complexity of these signaling networks regulated by onco-gene receptors. This result suggests that multiple targets are likely targeted during combinatorial therapies.

Similarly, although a direct interaction between MET and HER2 has not been described, it has been shown that the two receptors co‐operate to enhance the malignant phenotype, promoting cell–cell junction breakdown and boosting invasion. This is particularly sig-nificant in cancers where HER2 is over‐expressed and HGF is a physiological growth fac-tor found in the stroma (49), such as breast cancer.

Finally, a functional link between MET and EGFR (frequently co‐expressed in human cancers) has been shown: MET can be trans‐activated following EGFR activation in the absence of its ligand and when concomitantly expressed the two receptors exert a syn-ergistic effect on the activation of the downstream signaling cascade enhancing prolif-eration and motility (50). Moreover, it has been shown that over‐expression of HGF is a