International experts present innovative therapeutic strategies to treat cancer patients and prevent disease progression
Extracellular Targeting of Cell Signaling in Cancer highlights innovative therapeutic strategies to treat cancer metastasis and prevent tumor progression. Currently, there are no drugs available to treat or prevent metastatic cancer other than non-selective, toxic chemotherapy. With contributions from an international panel of experts in the field, the book integrates diverse aspects of biochemistry, molecular biology, protein engineering, proteomics, cell biology, pharmacology, biophysics, structural biology, medicinal chemistry and drug development.
A large class of proteins called kinases are enzymes required by cancer cells to grow, proliferate, and survive apoptosis (death) by the immune system. Two important kinases are MET and RON which are receptor tyrosine kinases (RTKs) that initiate cell signaling pathways outside the cell surface in response to extracellular ligands (growth factors.) Both kinases are oncogenes which are required by cancer cells to migrate away from the primary tumor, invade surrounding tissue and metastasize. MET and RON reside on both cancer cells and the support cells surrounding the tumor, called the microenvironment. MET and RON are activated by their particular ligands, the growth factors HGF and MSP, respectively. Blocking MET and RON kinase activation and downstream signaling is a promising therapeutic strategy for preventing tumor progression and metastasis. Written for cancer physicians and biologists as well as drug discovery and development teams in both industry and academia, this is the first book of its kind which explores novel approaches to inhibit MET and RON kinases other than traditional small molecule kinase inhibitors. These new strategies target key tumorigenic processes on the outside of the cell, such as growth factor activation by proteases. These unique strategies have promising potential as an improved alternative to kinase inhibitors, chemotherapy, or radiation treatment.
Table of Contents
List of Contributors xiii
Preface xvii
1 Discovery and Function of the HGF/MET and the MSP/RON Kinase Signaling Pathways in Cancer 1
Silvia Benvenuti, Melissa Milan and Paolo M. Comoglio
1.1 Introduction 1
1.2 MET Tyrosine Kinase Receptor and its Ligand HGF: Structure 1
1.2.1 The Invasive growth Program 2
1.2.2 MET Mediated Signaling 4
1.2.2.1 MET Down-regulation 7
1.2.3 Cross-talk between MET and Other Receptors 7
1.2.4 MET Activation in Human Cancers 9
1.2.4.1 MET, Hypoxia and Ionizing Radiations 10
1.2.4.2 MET Expression in Cancer Stem Cells: a Paradigm of Inherence 11
1.2.4.3 Oncogene Addiction and Oncogene Expedience 11
1.2.5 Targeting HGF/MET as a Therapeutic Approach in Human Cancer 12
1.2.5.1 HGF Antagonists 13
1.2.5.2 Tyrosine Kinase Inhibitors 15
1.2.5.3 Anti-MET Monoclonal Antibodies 17
1.2.5.4 Alternative MET Blocking Strategies 18
1.2.6 Primary and Secondary Resistance 18
1.2.6.1 MET Role in Resistance to Anticancer Agents 19
1.2.6.2 Mechanism of Resistance to MET Inhibitors 19
1.2.6.3 Combinatorial Therapeutic Strategies 20
1.3 RON Tyrosine Kinase Receptor and its Ligand MSP 21
1.3.1 Discovery and Structural Biology 21
1.3.2 RON Mediated Signaling 25
1.3.3 Cross-talk between RON and other Receptors 26
1.3.4 RON Activation in Human Cancers 26
1.4 Targeting MSP/RON as a Therapeutic Approach in Human Cancer 27
1.5 Concluding Remarks 28
2 The Role of HGF/MET and MSP/RON Signaling in Tumor Progression and Resistance to Anticancer Therapy 45
Lidija Klampfer and Benjamin Yaw Owusu
2.1 Introduction 45
2.2 HGF/MET Signaling in Cancer 47
2.3 MSP/RON Signaling in Cancer 52
2.4 Cross-talk between MET and RON Signaling Pathways 53
2.5 HGF/MET and MSP/RON Signaling Elicit Resistance to Cancer Therapy 55
2.6 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 69
Hiroaki Kataoka and Takeshi Shimomura
3.1 Introduction 69
3.2 Discovery of HGFA 70
3.2.1 Tissue Injury-induced Activation of HGF 70
3.2.2 Identification of HGFA as a Serum Activator of pro-HGF 71
3.3 Synthesis of HGFA Zymogen in vivo 71
3.4 Molecular Structure of HGFA 72
3.4.1 The Gene Encoding pro-HGFA: HGFAC 72
3.4.2 ProHGFA Protein and its Activation 72
3.4.3 Structure Biology of HGFA 74
3.5 Substrates of HGFA in vivo 75
3.6 Regulation of HGFA Activity by Endogenous Inhibitors 76
3.6.1 HGF Activator Inhibitor-1 (HAI-1): a Cell Surface Regulator of HGFA Activity 76
3.6.2 HGF Activator Inhibitor-2 (HAI-2) 78
3.6.3 Protein C Inhibitor (PCI; SERPINA5) 78
3.7 Proposed Biological Functions of HGFA in vivo 78
3.8 Roles of HGFA in Cancer 80
3.8.1 Enhanced Activation of pro-HGF and pro-MSP in Cancer Tissues 80
3.8.2 Possible Roles of HGFA in Cancer Progression 80
3.9 Conclusions and Future Perspectives of HGFA Research in Cancer 82
References 83
4 Physiological Functions and Role of Matriptase in Cancer 91
Fausto A. Varela, Thomas E. Hyland and Karin List
4.1 Introduction 91
4.2 Discovery of Matriptase 91
4.3 Biochemical and Functional Characteristics of Matriptase – Inhibitors, Substrates and Structure 92
4.3.1 Endogenous Polypeptide Matriptase Inhibitors 92
4.3.2 Matriptase Substrates 94
4.3.3 Matriptase Structure 95
4.4 Physiological and Pathophysiological Functions of Matriptase 96
4.4.1 Matriptase in Epidermal Development and Homeostasis 96
4.4.2 Matriptase in the Gastrointestinal Tract 97
4.4.3 Matriptase in Thymocytes and Salivary Glands 98
4.4.4 Matriptase in Placental/Embryonic Development 98
4.4.5 Matriptase in Neural Tube Closure 99
4.4.6 Pathways requiring Matriptase 99
4.4.7 Matriptase in Viral Infection 101
4.5 Role of Matriptase in Cancer 101
4.5.1 Studying Matriptase in Cultured Cancer Cells and Tumor Grafting Models 108
4.5.2 In vivo Cancer Studies using Genetic Models 111
4.5.2.1 Squamous Cell Carcinoma 111
4.5.2.2 Colitis-associated Colon Carcinogenesis 112
4.5.2.3 Breast Cancer 112
4.6 Conclusions 114
References 114
5 The Cell-Surface, Transmembrane Serine Protease Hepsin: Discovery, Function and Role in Cancer 125
Denis Belitškin, Shishir Mani Pant, Topi Tervonen and Juha Klefström
5.1 Biology of Hepsin 125
5.1.1 Discovery of Hepsin 125
5.1.1.1 Cloning of Hepsin, HPN Gene 125
5.1.1.2 Assigning Hepsin to Type II Transmembrane Serine Protease Family 126
5.1.2 Hepsin Gene and Protein 126
5.1.2.1 Expression, Regulation and Structure 126
5.1.2.2 Hepsin Activation and Activity 130
5.1.3 Physiological Functions of Hepsin 131
5.1.3.1 Growth Factor Activation 131
5.1.3.2 Serine Protease Cascades 132
5.1.3.3 Cell Proliferation and Motility 132
5.1.3.4 Epithelial Integrity 133
5.1.3.5 Organ Development 135
5.2 Hepsin in Cancer 137
5.2.1 Gain of Oncogenic Function 137
5.2.1.1 Genetic Alterations 137
5.2.1.2 Altered Subcellular Localization 138
5.2.1.3 Oncogenic Hepsin Function in vivo 140
5.2.1.4 How HPN Promotes Cancer 141
5.2.2 Targeting Hepsin in Cancer 143
5.3 Future Prospects 144
5.3.1 Hepsin’s Role as Guardian of Epithelial Integrity 144
5.3.2 Cancer Disease Progression and Metastasis 145
5.3.2.1 Uncontrolled Proteolysis 145
6 Targeting HGF with Antibodies as an Anti-Cancer Therapeutic Strategy 155
Dinuka M. De Silva, Arpita Roy and Donald P. Bottaro
6.1 Introduction 155
6.2 HGF Biology 156
6.2.1 HGF Gene Organization and mRNA Transcripts 156
6.2.2 HGF Protein Isoforms and Proteolytic Processing 156
6.2.2.1 HGF Isoforms 156
6.2.2.2 HGF Activation by Proteolytic Processing 159
6.2.3 Key HGF Interactions: Heparan Sulfate Proteoglycans and Met 160
6.2.3.1 Heparan Sulfate Proteoglycans 160
6.2.3.2 Met and Key Intracellular Effectors 161
6.2.4 Major Sites of HGF Expression: Tissues and Organs 162
6.2.5 HGF Function in Development and Adulthood 162
6.2.5.1 hgf or met altered Mice: Embryogenesis 163
6.2.5.2 hgf or met altered Mice: Late Development and Adulthood 163
6.3 HGF in Cancer 164
6.3.1 Lung Cancer 165
6.3.2 Hepatocellular Carcinoma 165
6.3.3 Genitourinary Malignancies 166
6.3.4 Breast Cancer 167
6.3.5 Colorectal and Gastric Carcinomas 167
6.3.6 Papillary Thyroid Carcinoma 168
6.3.7 Brain Tumors 168
6.3.8 Melanoma 169
6.3.9 Head and Neck Squamous Cell Carcinoma 169
6.3.10 Other Malignancies 169
6.4 Anti-HGF Monoclonal Antibodies as Anti-Cancer Therapeutic
Candidates 170
6.4.1 Rilotumumab 170
6.4.2 Ficlatuzumab 174
6.4.3 TAK-701 175
6.5 Conclusions and Future Directions 176
Acknowledgements 177
References 177
7 MET and RON Receptor Tyrosine Kinases as Therapeutic Antibody Targets for Cancer 199
Mark Wortinger, Jonathan Tetreault, Nick Loizos, and Ling Liu
7.1 MET as a Therapeutic Antibody Target for Cancer 199
7.2 Challenges in Developing MET Therapeutic Antibodies 200
7.3 Anti-MET Antibody Clinical Diagnostics 203
7.4 Anti-MET Antibodies in the Clinic 204
7.4.1 Onartuzumab – Roche 204
7.4.2 Emibetuzumab – Eli Lilly 206
7.4.3 ABT-700 – AbbVie 208
7.4.4 SAIT301 – Samsung 208
7.4.5 ARGX-111 – Argenx 209
7.4.6 Sym-015 – Symphogen 210
7.5 Additional anti-MET Antibodies 210
7.5.1 DN-30 – University of Turin Medical School 210
7.5.2 Other Preclinical Stage anti-MET Antibodies 210
7.6 Summary– anti-MET Antibodies 211
7.7 RON as a Therapeutic Antibody Target for Cancer 211
7.8 Conclusions and Future Outlook 216
References 216
8 Inhibitory Antibodies of the Proteases HGFA, Matriptase and Hepsin 229
Daniel Kirchhofer, Charles Eigenbrot, and Robert A. Lazarus
8.1 Anti-Serine Protease Antibodies for Therapeutic Applications 229
8.2 Antibodies can Inhibit Trypsin-Fold Serine Proteases in Diverse Ways 230
8.2.1 Orthosteric Inhibition (Active Site Binding) 231
8.2.2 Allosteric Inhibition 231
8.2.3 Exosite Inhibition 231
8.2.4 Inhibition of Zymogen Activation 231
8.2.5 Cofactor Inhibition 231
8.2.6 Inactivation of Oligomeric Serine Proteases 232
8.2.7 Comparison of Abs with Natural Occurring Protein Modes of Inhibition 232
8.3 Introduction to Antibodies against HGFA, Matriptase and Hepsin 233
8.4 Inhibitory HGFA Antibodies 234
8.5 Inhibitory Matriptase Antibodies 238
8.6 Inhibitory Hepsin Antibodies 239
8.7 Conclusion 240
References 240
9 Inhibitors of the Growth-Factor Activating Proteases Matriptase, Hepsin and HGFA: Strategies for Rational Drug Design and Optimization 247
James W. Janetka and Robert A. Galemmo, Jr
9.1 Introduction 247
9.1.1 Proteolytic Control of HGF/MET Oncogenic Signaling 247
9.1.2 Proteolytic Control of MSP/RON Kinase Signaling 248
9.1.3 The Identification of HGF and MSP Converting Enzyme Activity 249
9.2 Small Molecular Weight Inhibitors of HGFA, Matriptase and Hepsin 251
9.2.1 Mechanism-based Inhibitors derived from Substrate Sequences 251
9.2.2 Approved Drugs as Starting Points for Inhibitor Design 257
9.2.3 Retro-Engineering Inhibitors of Related Proteases 258
9.3 Improving Drug-like Properties of the Current Inhibitors: Lessons from the Oral Anti-Coagulants 264
9.4 Conclusion 269
References 270
10 Cyclic Peptide Serine Protease Inhibitors Based on the Natural Product SFTI-1 277
Blake 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 277
10.1.2 Standard Mechanism Inhibitors 278
10.1.2.1 Kunitz Type 278
10.1.2.2 Kazal Type 278
10.1.2.3 Bowman–Birk Inhibitor (BBI) Family 278
10.2 Selective Inhibitors of Serine Proteases using the Sunflower Trypsin Inhibitor (SFTI-1) as a Scaffold for Rational Drug Design 279
10.2.1 Trypsin 279
10.2.2 Chymotrypsin, Neutrophil Elastase and Cathepsin G 286
10.2.3 Proteasome 286
10.2.4 Matriptase and other Type II Transmembrane Serine Proteases (TTSPs) 286
10.2.5 MASP-1 and MASP-2 286
10.2.6 Other KLKs (KLK5, 7, 14) 287
10.2.7 KLK4 287
10.3 Normal and Pathophysiological Functions of the Human Tissue Kallikrein (KLK)-related Serine Protease Family 288
10.3.1 Physiological Role for KLKs 288
10.3.2 KLKs and their Role in Prostate Cancer Pathogenesis 289
10.3.3 Kallikrein-related Peptidase 4 as a Point of Therapeutic Intervention 290
10.4 Inhibitors of KLK4 Serine Protease 291
10.4.1 Molecular Basis of KLK4 Inhibition by SFTI-1 291
10.4.2 Use of SFTI-1 as a Scaffold in Ligand Design and Optimization 292
10.4.3 Identification of an Optimal Tetrapeptide Substrate 292
10.4.4 SFTI-1FCQR is a Potent Selective Inhibitor of KLK4 293
10.4.4.1 Structural Basis for Potency and Selectivity of SFTI-1FCQR Derivative 293
10.5 Potential Therapeutic Applications and Challenges 294
10.6 Conclusions/Future Directions 297
References 297
11 Screening Combinatorial Peptide Libraries in Protease Inhibitor Drug Discovery 307
Marcin Poreba, Paulina Kasperkiewicz, Wioletta Rut and Marcin Drag
11.1 Introduction 307
11.2 Proteases Involved in Cancer 309
11.2.1 Metalloproteases 309
11.2.2 Serine Proteases 310
11.2.3 Cysteine Proteases 311
11.2.4 Aspartic Proteases 311
11.2.5 Threonine Proteases 312
11.2.6 Target Protease Substrates and Inhibitors 312
11.3 Identification and Optimization of Preferred Substrates 313
11.3.1 Positional Scanning of Substrate Combinatorial Libraries (PS-SCL) 313
11.3.2 Peptide Microarrays 318
11.3.3 Hybrid Combinatorial Substrate Library (HyCoSuL) 318
11.3.4 Counter Selection Substrate Library (CoSeSuL) 320
11.3.5 Combinatorial Substrate Synthesis for Aminopeptidase Screening 320
11.3.6 Internally Quenched Fluorescent (IQF) Substrates 321
11.3.7 Phage Display 322
11.3.8 Protease Substrates – Summary 325
11.4 Design of Covalent Inhibitors Based on Substrates 326
11.4.1 Background and General Characteristics of Inhibitors 326
11.4.2 Substrate-based Inhibitor Design and Discovery 327
11.4.3 PS-SCL Applied to Inhibitors other than Substrates 328
11.4.4 Inhibitors from Phage Display Screening and Directed Evolution of Proteins 331
11.5 Anticancer Drugs – How much Information do We Need? 334
11.6 Conclusions 336
Acknowledgements 337
References 337
12 Chemical Probes Targeting Proteases for Imaging and Diagnostics in Cancer 351
Pedro Gonçalves and Steven H. L. Verhelst
12.1 Introduction 351
12.2 Chemical Probes for Proteases 352
12.2.1 Substrate-based Probes 352
12.2.2 Activity-based Probes (ABPs) 356
12.2.3 Photo-crosslinking probes 356
12.2.4 Non-Covalent Probes 358
12.3 Molecular Imaging of Cancer 358
12.3.1 Imaging Tumors with Substrate-based Probes 359
12.3.1.1 Preclinical Model Systems 359
12.3.1.2 Clinical Trials 361
12.3.2 Imaging Tumors with ABPs 362
12.3.2.1 Conventional and multimodal ABPs 362
12.3.2.2 Quenched ABPs 364
12.3.2.3 Towards Clinical Applications 365
12.3.3 Imaging Tumors with Affinity-based Reagents 366
12.3.3.1 Preclinical Models 366
12.3.3.2 Clinical Trials 367
12.4 Conclusions 369
Acknowledgements 370
References 371
13 Cancer Diagnostics of Protease Activity and Metastasis 377
Timothy J. O’Brien and John Beard
13.1 Introduction 377
13.2 The Proteins Identified from Patient Tumor Profiling 386
13.2.1 Matriptase 386
13.2.2 Hepsin 387
13.2.3 KLK7 387
13.2.4 KLK6 388
13.2.5 KLK8 388
13.2.6 TMPRSS3 388
13.2.7 MMP-7 389
13.3 ELISA Assay Development 389
13.4 The Role of Markers for Cancer Surveillance and Tumor Monitoring (Early Detection) 390
13.5 Cell Signaling and the Cancer Cascade 399
13.6 Conclusions and Future Prospects 400
References 402
14 Roles of Pericellular Proteases in Tumor Angiogenesis: Therapeutic Implications 411
Janice M. Kraniak, Raymond R. Mattingly and Bonnie F. Sloane
14.1 Introduction 411
14.2 Initiation of Angiogenesis 412
14.3 Mechanisms of New Blood Vessel Formation 413
14.3.1 Sprouting Angiogenesis 414
14.3.2 Intussesceptive or Non-sprouting Angiogenesis 415
14.3.3 Neovasculogenesis 415
14.3.4 Vascular Mimicry 416
14.4 Pericellular Proteases and Angiogenesis 417
14.4.1 Metalloproteinases: MMPs, ADAMs and ADAM-TS 418
14.4.1.1 MMPs 418
14.4.1.2 ADAMs and ADAM-TS 422
14.4.2 Serine Proteases 424
14.4.3 Cysteine Cathepsins 425
14.4.3.1 Cysteine Cathepsins in Angiogenesis 426
14.5 Novel Approaches for Targeting Tumor Angiogenesis 428
14.6 Summary 432
Acknowledgements 433
References 433
Index 447
