THE ROLE OF FIBROBLAST GROWTH FACTOR-9 IN MALIGNANT MESOTHELIOMA

This thesis is presented for the degree of Doctor of Philosophy

from The University of Western Australia

Ai Ling Tan BSc. (Hons)

The University of Western Australia School of Medicine and Pharmacology

2012

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ABSTRACT

Background: Malignant mesothelioma (MM) is a cancer with no cure and its global

incidence continues to rise. Identifying the key molecules governing the pathogenesis

of MM is urgently needed. Such molecules can potentially provide better understanding

of the disease pathology, unveil therapeutic targets and may have diagnostic or

prognostic relevance.

Fibroblast growth factor-9 (FGF-9) was the leading candidate gene in a pilot global

gene profiling study of our laboratory using prospectively-collected human

thoracoscopic biopsies. The finding was validated in a second cohort. FGF-9

expression was 17- and 35-fold higher in MM over controls (metastatic pleural

carcinomas and benign pleuritis) in those studies. This thesis followed on the data to

explore the biological significance of FGF-9 in MM.

Hypothesis: I hypothesize that

• FGF-9 plays a vital role in MM development including proliferation and invasion;

• Antagonising FGF-9 activity retards MM growth;

• FGF-9 can aid diagnosis of MM;

• Mutations in the FGF-9 and/or its receptors cause aberrant FGF-9 signalling in

MM.

Results: I demonstrated the presence of FGF-9 protein in all 10 human and murine

MM cell lines tested. Using tissue-array of human fluid cells and pleural tissues, FGF-9

immunostaining was significantly stronger in MM (n=121) and metastatic

adenocarcinoma (n=29) over benign controls (n=35), chi-square 82.8, df=2, p

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FGF-9 levels in pleural fluids were measured in two (Oxford and Perth) cohorts of 283

and 639 patients respectively. The median FGF-9 levels in MM were significantly

higher by 7.2- and 12.5-fold over metastatic carcinomas as well as 4.6- and 11.3-fold

over benign pleural fluids in the two cohorts respectively (p

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Gene sequencing was performed on 50 human MM samples to examine for mutations

in the exons of FGF-9 and its two receptors, FGFR2 and FGFR3. No significant and

consistent mutations were found.

Summary: FGF-9 over-expression was confirmed at both RNA and protein levels in

human pleural tissue and fluids in five different cohorts. FGF-9 induces MM cell

proliferation, invasion and release of key cytokines known to promote MM

development. Knockdown of FGF-9 significantly retards MM growth in various animal

models. The role and potential application of FGF-9 as a therapeutic target and clinical

aid for diagnosis/prognosis for MM should be explored.

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CONTENTS

CHAPTER 1 1

1.0 GENERAL INTRODUCTION 2

1.1 The Structure of Mesothelium 2

1.2 The Functions of Mesothelium 3 1.2.1 Non-adhesive Surface and Protective Barrier 3 1.2.2 Transport of Fluids and Particulates 4 1.2.3 Inflammation 4 1.2.4 Tumour Cell Adhesion, Proliferation and Growth 5 1.2.5 Leukocyte Migration 5 1.2.6 Tissue Repair 6

1.3 Malignant Mesothelioma 9 1.3.1 Causes 9 1.3.2 Pathogenesis 10 1.3.3 Epidemiology 12 1.3.4 Histological Subtypes 12 1.3.5 Clinical Features 13 1.3.6 Diagnosis 13 1.3.7 Prognosis 19 1.3.8 Current Therapies 20 1.3.9 Animal Models 22

1.4 Fibroblast Growth Factors 23 1.4.1 Nomenclature of Fibroblast Growth Factors 23

1.5 Fibroblast Growth Factor-9 26 1.5.1 Fibroblast Growth Factor-9 Gene 27 1.5.2 Fibroblast Growth Factor-9 mRNA 29 1.5.3 Fibroblast Growth Factor-9 Protein Structure 30 1.5.4 Secretion of Fibroblast Growth Factor-9 33 1.5.5 Physical Interactions between Fibroblast Growth Factor-9 and Heparin 33 1.5.6 Biological Function of Fibroblast Growth Factor-9 34 1.5.7 Expression Modulation of Fibroblast Growth Factor-9 37 1.5.8 Mutations on Fibroblast Growth Factor-9 38 1.5.9 Fibroblast Growth Factor-9 in Cancer 39

1.6 Fibroblast Growth Factor Receptors 41 1.6.1 The Structure of Fibroblast Growth Factor Receptor 41 1.6.2 The Receptors for Fibroblast Growth Factor-9 45 1.6.3 Fibroblast Growth Factor-9 Signalling Pathway 45 1.6.4 Mutations of the Receptors for Fibroblast Growth Factor-9 50

1.7 Therapeutic Approaches 55

1.8 Summary 55

1.9 Preliminary Results 56

1.10 Hypothesis and Aims 59

CHAPTER 2 61

2.0 MATERIALS AND METHODS 62

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2.1 Maintenance of Malignant Mesothelioma Cell Lines 62 2.1.1 Cell Culture 62 2.1.2 Malignant Mesothelioma Cell Lines 62 2.1.3 Cryo-Freezing and Storage of Cells 63

2.2 Phenotypic Characterisation of Cells and Tissues 64 2.2.1 Immunofluorescence 64 2.2.2 Immunohistochemistry 65 2.2.3 Tissue Microarray Analysis 66

2.3 Western Immunoblotting 66 2.3.1 Protein Extraction 66 2.3.2 Determination of Protein Concentration 67 2.3.3 SDS-PAGE and Immunoblotting 67

2.4 Functional Assays 68 2.4.1 Cell Proliferation WST-1 Assay 68 2.4.2 Trypan Blue Cell Exclusion Assay 69 2.4.3 Matrigel – Invasion Assay 69 2.4.4 Scratch Assay 70 2.4.5 Cytokine and Chemokine Measurements 70

2.5 Molecular Characterisation of Cells 71 2.5.1 RNA Isolation 71 2.5.2 Determination of RNA Concentration 71 2.5.3 Reverse Transcription and cDNA Synthesis 71 2.5.4 Real-time Polymerase Chain Reaction 72

2.6 Sample Processing and Storage 72

2.7 Mutational Sequencing of Cells and Tissues 73 2.7.1 DNA Isolation 73 2.7.2 Determination of DNA Concentration 74 2.7.3 Polymerase Chain Reaction 74 2.7.4 Agarose Gel Electrophoresis 80 2.7.5 Unpurified PD+ Capillary Sequencing 80 2.7.6 DNA Sequencing Analysis 80

2.8 Molecular Knockdown In Vitro 80 2.8.1 Short Hairpin RNA Knockdown 80

2.9 In Vivo Inoculation of Short Hairpin Knockdown Cells 82 2.9.1 Heterotopic Model 82 2.9.2 Orthotopic Model 82 2.9.3 Athymic Model 83

2.10 Statistical analysis 83

CHAPTER 3 84

3.0 THE EXPRESSION OF FIBROBLAST GROWTH FACTOR-9 IN MALIGNANT MESOTHELIOMA 85

3.1 Introduction 85

3.2 Results 85 3.2.1 The Detection of FGF-9 in MM Samples 85 3.2.2 The Expression of FGF-9 in MM Cells and Tissues 94 3.2.3 The Expression of FGF-9 in Pleural Fluids 96

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3.3 Discussion 108

CHAPTER 4 112

4.0 THE BIOLOGICAL ROLE OF FIBROBLAST GROWTH FACTOR-9 IN MALIGNANT MESOTHELIOMA 113

4.1 Introduction 113

4.2 Results 113 4.2.1 MM Cells Proliferation In Vitro in Response to FGF-9 Stimulation 113 4.2.2 Cytokines and Chemokines Release in Response to FGF-9 Stimulation 120 4.2.3 The Effects of Antibody Neutralisation on Cytokine Release 129 4.2.4 The Effects of Pathway Inhibitors on FGF-9-Stimulated MM Cells 132 4.2.5 The Role of FGF-9 in MM Cell Invasion 138 4.2.6 The Involvement of FGF-9 in MM Cell Migration 139

4.3 Discussion 142

CHAPTER 5 147

5.0 THE NECESSITY OF FGF-9 IN MALIGNANT MESOTHELIOMA TUMOUR GROWTH IN VIVO 148

5.1 Introduction 148

5.2 Results 148 5.2.1 Development of FGF-9 shRNA Knockdown Cells 148 5.2.2 FGF-9 shRNA Knockdown Cells in Heterotopic Model 151 5.2.3 FGF-9 shRNA Knockdown Cells in Orthotropic Model 154 5.2.4 FGF-9 shRNA Knockdown Cells in Athymic Model 157

5.3 Discussion 159

CHAPTER 6 163

6.0 MUTATIONS OF FGF-9 AND ITS RECEPTORS IN MALIGNANT MESOTHELIOMA PATHOBIOLOGY 164

6.1 Introduction 164

6.2 Results 164 6.2.1 Mutations in FGF-9 164 6.2.2 Expression of FGF-9 Receptors in MM Cells 171 6.2.3 Mutations in FGFR2 and FGFR3 in MM Cell Lines and Tumour Biopsies 174

6.3 Discussion 184

CHAPTER 7 187

7.0 SUMMARY 188

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LIST OF FIGURES

1.1 The functions of mesothelial cells

1.2 The alignment of amino acid sequence of FGF-9 and other members of the FGF-9

family

1.3 A ribbon diagram of FGF-9

1.4 The fibroblast growth factor receptor structure

1.5 Signalling through fibroblast growth factor receptors

1.6 Mechanisms of pathogenic cancer cell FGF signalling

1.7 Global gene profiling on 22 human pleural tissue samples

1.8 Quantification of FGF-9 gene expression from global gene profiling

1.9 Validation of microarray gene expression results using RT-PCR

3.1 Optimisation of murine FGF-9 ELISA

3.2 The presence of FGF-9 in murine MM cells detected using ELISA

3.3 Expression and distribution of FGF-9 in the human JU77 and murine AB2 MM cell

lines

3.4 Expression and distribution of FGF-9 in the MM human tissue biopsy and murine

MM tumour

3.5 Expression of FGF-9 in cells and tissues on tissue microarray

3.6 The stability of FGF-9 in pleural fluids following freeze-thaw

3.7 Pleural fluids FGF-9 levels in the Oxford and Perth cohorts

3.8 FGF-9 levels in different MM histology

3.9 FGF-9 levels in matched pleural fluid and plasma of patients diagnosed with MM

3.10 The correlation between FGF-9 and mesothelin

3.11 The area under the ROC for FGF-9 and mesothelin to differentiate MM from non-

MM

4.1 Optimisation of cell density for MM cell proliferation

4.2 Dose-dependent MM cell proliferation

4.3 Dose-dependent MM cell proliferation