THE ROLE OF FIBROBLAST GROWTH FACTOR-9 IN MALIGNANT MESOTHELIOMA · 1.4 Fibroblast Growth Factors...
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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<0.001.

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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<0.05 MM vs other groups
in both populations). Pleural fluid FGF-9 can differentiate MM from metastatic cancers
(area under curve, AUC 0.8303) and from all non-MM fluids (benign and metastatic
cancers combined, AUC 0.8228). The AUC for combined use of FGF-9 and mesothelin
were 0.8848 (MM vs metastatic cancer) and 0.9002 (MM vs non-MM fluids). It was also
found that in 22 out of 25 samples that had detectable FGF-9 levels in the MM pleural
fluids, the median FGF-9 levels were 6-fold higher in pleural fluids compared to plasma
samples highly suggesting the pleural origin of FGF-9.
FGF-9 induces dose- and time-dependent proliferation of all six human and murine MM
cell lines tested. This proliferation could be inhibited by JNK and p38 inhibitors. ERK
inhibition reduced murine MM cell proliferation only. FGF-9 only induces a dose- and
time-dependent release of IL-8 (or MIP-2), VEGF and MCP-1 from all ten human and
murine MM cell lines tested. Treatment with p38, JNK and ERK inhibitors antagonised
the cytokine release by varying degree. FGF-9 also induces MM cell invasion/migration
using two separate methods (Matrigel assay and scratch assay) in vitro.
FGF-9 shRNA knockdown MM cells were used to assess the necessity of FGF-9 in MM
growth using validated murine models. MM growth was significantly retarded in Balb/c
mice inoculated with FGF-9 shRNA cells compared to the parent cells and control MM
cells transfected with scrambled vectors in both the subcutaneous tumour model
(p<0.001) and if tumours were injected intraperitoneally (p<0.05 in tumour nodules).

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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 using Trypan Blue
4.4 Time-dependent MM cell proliferation
4.5 IL-8 and MIP-2 release from human and murine MM cells
4.6 MCP-1 release from human and murine MM cells
4.7 VEGF release from human and murine MM cells
4.8 Time-dependent cytokine release from human and murine MM cells
4.9 Optimisation of neutralisation antibody concentration
4.10 Inhibition of cytokine release from murine MM cells using antibody neutralisation
4.11 FGF-9 mediated MM cell proliferation is inhibited by ERK, JNK and p38 pathway
inhibitors
4.12 Inhibition of cytokine release from human and murine MM cells

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4.13 Phosphorylation of proteins downstream of FGF-9 signalling pathway
4.14 FGF-9 stimulates MM cell invasion
4.15 FGF-9 stimulates MM cell migration
5.1 FGF-9 shRNA knockdown cells have lower levels of FGF-9 protein
5.2 FGF-9 shRNA knockdown cells have lower levels of FGF-9 mRNA
5.3 Knockdown of FGF-9 in MM cells significantly retards their tumour growth in
heterotopic model
5.4 shRNA knockdown of FGF-9 significantly reduced MM tumour growth in
intraperitoneal model of disease
5.5 shRNA knockdown of FGF-9 had similar MM tumour growth in athymic model of
disease
6.1 Agarose gel showing the band intensities for different conditions for a PCR reaction
6.2 Sequence alignment for FGF-9 exon 1
6.3 Sequence alignment and amino acid translation for FGF-9 exon 3
6.4 Expression and distribution of FGFR2 in the human NO36 and murine AB1 MM cell
lines
6.5 Expression and distribution of FGFR3 in the human NO36 and murine AB2 MM cell
lines
6.6 Amino acid substitution occurring in exon 6 of FGFR2
6.7 Amino acid substitution occurring in exon 11 of FGFR2
6.8 Amino acid substitution occurring in exon 6 of FGFR2 in tumour biopsy

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LIST OF TABLES
1.1 Structure and phylogeny of FGF-9 ligands
2.1 Primary antibodies
2.2 Secondary and tertiary antibodies
2.3 Primers for real-time PCR
2.4 Primers used for PCR for the mutational sequencing analysis in human samples

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ACKNOWLEDGEMENT
Conducting this PhD project and writing up this thesis would be totally
impossible if it is not for my supervisor, Winthrop Professor YC Gary Lee. I would like
to express my heartfelt thanks and appreciation to him for his invaluable guidance,
inspiration, help, support and advice provided throughout the entire years.
I also owe my deepest gratitude to Professor Jeanette Creaney for her technical
advice in the mutational sequencing studies and support in grant writing as well as
supplying pleural fluid samples, the tissue microarray slides and the tumour samples
required for my research work.
I would also like to thank the post-doctoral scientists, Dr Sally Lansley and Dr
Julius Varano for providing me with refreshing insights on this project and Dr Bahareh
Badrian, Dr Scott Fisher and Dr Connie Jackaman for their technical help. I would also
like to thank Prof Nick de Klerk for his help in statistical analysis and Dr Amanda Segal
for her advice in immunohistochemistry.
I am also greatly indebted to my colleagues from the Lung Institute of Western
Australia and Tumor Immunology group, for their encouragement and support. Special
thanks goes out to Miss Hui Min Cheah and Dr Rabab Rashwan for the friendship and
moral support given, Miss Justine Leon and Miss Sarah Wong for assisting with the
pleural fluid samples collection and Miss Samantha Woo for teaching me in vivo
techniques.
Last but not least, my heartfelt thanks to my family and my husband, Chin Fei
Low, for their continuous words of encouragement and unwavering love and for
believing in me that I can achieve a PhD.

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Financial support
I would like to acknowledge the financial support of the following organisations:
• The University of Western Australia for the Scholarship for International
Research Fees, University International Stipend
• The Lung Institute of Western Australia for the LIWA PhD Top-Up
Scholarship
Candidate’s contribution in the research work
I hereby declare that this submission is my own work and that, to the best of my
knowledge, it contains no material that to any substantial extent, has been accepted for
the award of a degree or a diploma from any university. I also declare that the
intellectual content of this thesis was the product of my own work. These studies were
performed at Lung Institute of Western Australia, University of Western Australia.
I would like to acknowledge the collaboration and assistance received during the
course of this project. In Chapter 3, the scoring of the tissue microarray was performed
blindly by Professor Jenette Creaney, National Centre for Asbestos Related Disease,
Perth, Australia. The FGF-9 levels quantitated in the pleural fluids from the Oxford
cohort were performed by our collaborators in the Oxford Pleural Unit, Oxford, UK. The
Matrigel cell invasion assay in Chapter 4 was assessed by our collaborator, Dr
Mulugeta Worku, University College London, UK. Development of FGF-9 transfected
clones in Chapter 5 was performed by our collaborators, Sophia Karabela and
Professor George Stathopoulos, University of Patras, Greece. The inoculation of
transfected cells into animal models was performed blindly by our group colleague, Dr
Sally Lansley and the number of tumour nodules in the intraperitoneal model was
counted blindly by Dr Sally Lansley and Dr Julius Varano, Pleural Disease Unit, Lung
Institute of Western Australia, Perth, Australia.

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PUBLICATIONS ARISING FROM THIS THESIS
Abstracts Published in Scientific Journals
1. A. L. Tan, M. Worku, H. E. Davies, E. Mishra, R. Sadler, J. Creaney, S. M
Lansley, J. Varano, S. Karabela, G. Stathopoulous, R. J. O. Davies, Y. C. G.
Lee. Fibroblast Growth Factor-9 is Up-regulated in Malignant Mesothelioma and
May Be Involved in the Disease Pathobiology. Am J Respir Crit Care Med 185;
2012: A6354
2. A. L. Tan, M. Worku, H. E. Davies, E. Mishra, R. Sadler, J. Creaney, S. M
Lansley, J. Varano, S. Karabela, G. Stathopoulous, R. J. O. Davies, Y. C. G.
Lee. Fibroblast Growth Factor-9 is Upregulated and May Be Involved in
Mesothelioma Disease Pathobiology. Respirology 17 (S1); 2012: TO-097
3. A. L. Tan, M. Worku, H. E. Davies, E. Mishra, R. Sadler, J. Creaney, S. M
Lansley, R. J. O. Davies, Y. C. G. Lee. The Potential Biological Role of
Fibroblast Growth Factor-9 in Malignant Mesothelioma. Journal of Thoracic
Oncology 6; 2011:P2.043
4. A. L. Tan, M. Worku, H. E. Davies, E. Mishra, R. Sadler, J. Creaney, S. M
Lansley, R. J. O. Davies, Y. C. G. Lee. Fibroblast Growth Factor-9 and Its
Potential Biological Role in Malignant Mesothelioma. Am J Respir Crit Care
Med 183; 2011: A2361
5. A. L. Tan, M. Worku, H. E. Davies, E. Mishra, R. Sadler, S. M Lansley, R. J. O.
Davies, Y. C. G. Lee. The Potential Biological Role of Fibroblast Growth Factor-
9 in Malignant Mesothelioma. Respirology 16 (S1); 2011: TO-034
6. A. L. Tan, M. Worku, H. E. Davies, E. Mishra, R. Sadler, J. Creaney, S. M
Lansley, R. J. O. Davies, Y. C. G. Lee. Malignant Pleural Mesothelioma:
Potential Biological Role of Fibroblast Growth Factor (FGF) -9. Am J Respir Crit
Care Med 181; 2010: A4347
7. A. L. Tan, M. Worku, H. E. Davies, E. Mishra, R. Sadler, S. M Lansley, R. J. O.
Davies, Y. C. G. Lee. Fibroblast Growth Factor (FGF) -9 is Upregulated in
Malignant Mesothelioma. Respirology 15 (S1); 2010: TP124

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Publication Peripheral to this Thesis
1. Varano Della Vergiliana JF, Lansley S, Tan AL, Creaney J, Lee YC, Stewart
GA. Mesothelial cells activate the plasma kallikrein-kinin system during pleural
inflammation. Biol Chem 392 (7); 2011:633-42
Publications in Preparation
1. A. L. Tan, M. Worku, H. E. Davies, E. Mishra, R. Sadler, J. Creaney, S. M
Lansley, J. Varano, S. Karabela, G. Stathopoulous, R. J. O. Davies, Y. C. G.
Lee. Fibroblast Growth Factor-9 is Up-regulated in Malignant Mesothelioma and
Is Involved in the Disease Pathobiology.
2. A. L. Tan, J. Creaney, Y. C. G. Lee. The Fibroblast Growth Factor-9 and Its
Receptors: No Significant Mutations Detected in Patients with Malignant
Mesothelioma

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AWARDS AND SCHOLARSHIPS
2012
• UWA Graduate Research School Travel Funding, University of Western Australia
- to support my travel to the American Thoracic Society meeting in the USA 2012
• LIWA Travel Grant, Lung Institute of Western Australia
- to top-up the amount to support my travel to the TSANZ Annual Scientific Meeting
2012 in Canberra
• TSANZ Travel Award, The Thoracic Society of Australia & New Zealand
- to support my travel to the TSANZ Annual Scientific Meeting 2012 in Canberra
2011
• PhD project featured in Australian Society for Medical Research Media Release 2011
• School of Medicine and Pharmacology Research Symposium Oral Presentation Award,
School of Medicine and Pharmacology, University of Western Australia
- for the best oral presentation during the School of Medicine and Pharmacology
Research Symposium
• Telethon Institute for Child Health Research Award, Telethon Institute for Child Health
- for the best oral presentation in Australian Society for Medical Research Symposium
• The Thoracic Society of Australia & New Zealand Ann Woolcock Young Investigator
Award Finalist, The Thoracic Society of Australia & New Zealand
• Slater and Gordon International Travel Grant, Slater and Gordon
- to support my travel to the American Thoracic Society meeting in the USA in 2012
• LIWA Travel Grant, Lung Institute of Western Australia
- to top-up the amount to support my travel to the 14th World Conference on Lung
Cancer 2011 in The Netherlands
• LIWA PhD Top-Up Scholarship (2011-2014), Lung Institute of Western Australia
- to support my postgraduate studies at the University of Western Australia

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2010
• UWA Convocation Postgraduate Research Travel Award, Graduate Research School
of The University of Western Australia
- to support my travel to the 14th World Conference on Lung Cancer 2011 in The
Netherlands
• The Thoracic Society of Australia & New Zealand WA meeting Young Investigator
Award Finalist
• TSANZ Travel Award, The Thoracic Society of Australia & New Zealand
- to support my travel to the TSANZ Annual Scientific Meeting 2010 in Brisbane
• LIWA Travel Grant, Lung Institute of Western Australia
- to top-up the amount to support my travel to the TSANZ Annual Scientific Meeting
2010 in Brisbane
• Scholarship for International Research Fees (SIRF), Safety-Net Top-Up Scholarship
and University International Stipend (UIS) (2010-2013), University of Western Australia
- to support my postgraduate studies at the University of Western Australia

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LIST OF ABBREVIATIONS
Chemicals and reagents
BSA Bovine serum albumin
DAPI 4’,6-Diamidino-2-phenylindole
DMEM Dulbecco’s modified eagle medium
DMSO Dimethyl sulfoxide
DTT Dithiothreitol
ECL Enhanced chemi-luminescenece
EDTA Ethylenediaminetetraacetic acid
ELISA Enzyme linked immunosorbent assay
FBS Fetal bovine serum
FITC Fluorescein isothiocyanate
HRP Horseradish peroxidase
PBS Phosphate buffered saline
PVDF Polyvinylidene difluoride
SDS Sodium dodecylsulphate
SDS-PAGE Sodium dodecylsulphate polyacrylamide gel electrophoresis
TBS Tris buffered saline
Tw Tween
WST-1 Water soluble tetrazolium – 1 salts
Units and measurements
°C Degree celcius
µg Microgram
µL Microlitre
bp Base pairs
Da Daltons
g Gram
kDa Kilodalton

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L Litre
mg Miligram
mins Minutes
mL Mililitre
MW Molecular weight
ng Nanogram
nM Nanomolar
r.p.m. Revolutions per minute
Others
ARE Adenylate/uridylate-rich element
CO2 Carbon dioxide
ddH2O Double distilled water
DNA Deoxyribonucleic acid
ECM Extracellular matrix
EGF Epidermal growth factor
EGFR Epidermal growth factor receptor
EMA Epithelial membrane antigen
EPP Extrapleural pneumonectomy
ERK Extracellular-signal regulated kinase
FGF Fibroblast growth factor
FGF-9 Fibroblast growth factor -9
FGFR Fibroblast growth factor receptor
FGFR-1 Fibroblast growth factor receptor -1
FGFR-2 Fibroblast growth factor receptor -2
FGFR-3 Fibroblast growth factor receptor -3
FGFR-4 Fibroblast growth factor receptor -4
GAPDH Glyceraldehyde 3-phosphate dehydrogenase
H2O Water

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H2O2 Hydrogen peroxide
HER2 Human epithelial growth factor receptor 2
HSPG Heparan sulfate proteoglycan
ICAM-1 Intercellular adhesion molecule
IFN-γ Interferon gamma
Ig Immunoglobulin
IgG Immunoglobulin G
IL Interleukin
JNK c-Jun N-terminal kinase
MAPK Mitogen activated protein kinase
MCP-1 Monocyte chemotactic protein -1
MIP-2 Macrophage inflammatory protein-2
MKP3 Mitogen activated protein kinase phosphatase 3
MM Malignant mesothelioma
MMP Metalloproteinase
MPF Megakaryocyte potentiating factor
mRNA Messenger ribonucleic acid
NO Nitric oxide
PCR Polymerase chain reaction
P/D Pleurectomy/decortication
PDGF Platelet derived growth factor
PI3K Phosphatidyllinositol-3 kinase
ROC Receiver operating curve
RT-PCR Reverse transcriptase polymerase chain reaction
SFM Serum free media
SMRP Soluble mesothelin-related peptide
SNT-1/FRS2 Fibroblast growth factor receptor substrate 2
TGF-β Transforming growth factor beta
TIMP Tyrosine inhibitor of metalloproteinase

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TKI Tyrosine kinase inhibitor
TNF-α Tumour necrosis factor alpha
Tyr Tyrosine
UCC Urothelial cell carcinoma
VCAM-1 Vascular cellular adhesion molecule
VEGF Vascular endothelial growth factor
VEGFR Vascular endothelial growth factor receptor
WT Wild type
WT-1 Wilms’ tumour antigen 1

1
____________________________________________
GENERAL INTRODUCTION
CHAPTER 1

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1.0 GENERAL INTRODUCTION
1.1 The Structure of Mesothelium
The mesothelium is an extensive monolayer of predominantly elongated, flattened,
squamous-like mesothelial cells. The mesothelium lines three serosal cavities; the
pleural, peritoneal and pericardial cavities, as well as the internal organs within
these cavities. Mesothelial cells can also be found in the lining that surrounds the
testes. With a few exceptions, this monolayer of cells is mostly always found at the
same anatomical site and is conserved between different species (Minot, 1890,
Whitaker et al., 1982, Odor, 1954).
Mesothelial cells are supported by connective tissue stroma on a thin basement
membrane. The diameter of a cell is approximately 25 µM and its cytoplasm is
elevated over a central round or oval nucleus (Mutsaers, 2004). Mesothelial cells
contain organelles such as vesicles and vacuoles, microtubules and
microfilaments, a few mitochondria, a poorly developed Golgi apparatus and a few
rough endoplasmic reticulum (Mutsaers, 2004). Microvilli are found on the luminal
surface of the mesothelial cells and they trap glycoproteins rich in hyaluronic acid
to reduce the friction and provide a slippery surface between two adjacent organs
(Andrews, 1973, Wang, 1974). There are also cell-to-cell junctions such as tight
junctions, adherens junctions, gap junctions and desmosomes in between the cells
which are vital for cell surface polarity and semi-permeable diffusion (Mutsaers,
2004).
Mesothelial cells are fragile and shedding of mesothelial cells is common in
humans. As a result, mesothelial cells can be found in pleural fluids (Peng et al.,
1994). These shed-cells are round or oval and are rich in organelles. They are

3
capable of transforming themselves into macrophages-like cells which can
phagocytose (Efrati and Nir, 1976) and so, they may have an immunological role
(Bakalos, 1974).
1.2 The Functions of Mesothelium
In the early years, as mesothelial cells secrete surfactant and lubricating
glycoproteins, it was thought that this monolayer could provide a protective barrier
as well as a slippery surface for frictionless movement of organs and tissues within
the cavity. However, in recent years, the role of the mesothelium has been
extended to the transportation of fluids and particulates, regulation of leukocyte
migration in response to inflammation and tissue repair (Figure 1.1).
1.2.1 Non-adhesive Surface and Protective Barrier
An ultrastructural analysis revealed that mesothelial cells produce and secrete a
lubricant-like substance, similar to the type II pneumocytes, which decreases the
friction and allows free movement of opposing organs (Dobbie J.W., 1988).
In addition to that, mesothelial cells secrete glycosaminoglycans, primarily
hyaluronan, on both the surface and within the pinocytic vesicles (Roth, 1973).
There is an association between the increased concentration of hyaluronan and
the existence of cuboidal mesothelial cells in pleural effusions (Wang and Lai-
Fook, 1998, Satoh K, 1987). In vitro studies have shown that mesothelial cells
synthesize hyaluronan to form “coats” (Castor C.W., 1969, Honda et al., 1986,
Ohashi Y., 1988, Breborowicz A., 1998, Yung et al., 2000, Heldin and Pertoft,
1993). The hyaluronan coats are up-regulated following injury (Yung et al., 2000,

4
Baumann M.H., 1996), possibly to serve as a protective barrier against viral
infections and the cytotoxic effects of lymphocytes (Heldin and Pertoft, 1993).
1.2.2 Transport of Fluids and Particulates
Furthermore, mesothelial cells are involved in the transport and movement of fluids
and particulates across the serosal cavities. The presence of many microvilli on
the luminal surface of the mesothelial cell increases the surface area of the cell to
boost absorption (Odor, 1954). Moreover, the microvilli are covered with glycocalyx
which contains glycosaminoglycans that bind to fluids, further aiding adsorption
(Wang, 1974).
It has been shown that the transport of fluids through the mesothelium occurs via
intracellular junctions and stomata (Leak and Rahil, 1978, Whitaker et al., 1982)
while on the other hand, particulates and solutes are actively transported across
the mesothelium layer by pinocytic vesicles (Fedorko and Hirsch, 1971, Leak and
Rahil, 1978, Fukuta, 1963).
1.2.3 Inflammation
The mesothelium plays an active role in inflammation. In the presence of bacterial
endotoxins and cytokines (Antony et al., 1993), asbestos (Boylan et al., 1992) or
instilled agents (Antony et al., 1995), mesothelial cells secrete various pro- and
anti-inflammatory and immunomodulatory mediators such as prostaglandins,
chemokines, cytokines, nitric oxide, reactive oxygen and nitrogen species and
growth factors (Mutsaers, 2002). The secreted mediators help to restore the
normal serosal architecture and function.

5
For instance, mesothelial cells produce nitric oxide (NO) and reactive nitrogen (NO.
radical) and oxygen (O2-. radical, hydrogen peroxide (H2O2) and OH. radical)
species in vitro in response to asbestos, cytokines and bacterial products (Chen et
al., 2000, Owens and Grisham, 1993, Tracey et al., 1995, Choe et al., 1998).
1.2.4 Tumour Cell Adhesion, Proliferation and Growth
The mesothelium layer can also act as a site for tumour adhesions following injury
or trauma to the mesothelium (Cunliffe and Sugarbaker, 1989). This can be
prevented when the tumour cells adhere to the hyaluronic acid coat secreted by
the mesothelial cells (Jones et al., 1995). During surgical resection, the removal of
hyaluronan and other glycosaminoglycans can cause the tumour cells to attach
and re-seed onto the serosal surface again (Hofer et al., 1998, Van Den Tol et al.,
1998). On the contrary, other studies have shown that adhesion of the tumour cells
to the hyaluronan coat of mesothelial cells can contribute to the metastasis of
ovarian and colorectal cancers. This is because the tumour cells adhering to the
hyaluronic acid on the mesothelium are capable of migrating through the
mesothelium to invade the organs at distal sites (Catterall et al., 1997, Catterall et
al., 1999, Cannistra, 1994).
1.2.5 Leukocyte Migration
Furthermore, mesothelial cells are involved in leukocyte migration. The influx of
leukocytes from the vascular compartment to the serosal cavity contributes to
inflammation (Light, 1990, Brauner A., 1993). Leukocyte migration to the site of
inflammation occurs when (Topley N., 1996):
1. There is a site of activation

6
2. The resident cells are activated to generate signals that initiate a response
3. There is a chemotactic gradient to direct the migration of leukocytes to the site
of inflammation
4. Adhesion molecules such as the intercellular adhesion molecule (ICAM-1),
vascular cellular adhesion molecule (VCAM-1), E-cadherin, N-cadherin are up-
regulated to allow the leukocyte transverse the mesothelial cell monolayer into
the serosal cavity (Zeillemaker et al., 1996, Pelin et al., 1994, Jonjić N., 1992,
Liberek, 1996, Cannistra, 1994).
1.2.6 Tissue Repair
Like epithelial cells, mesothelial cells are capable of secreting a variety of growth
factors and extracellular matrix (ECM) molecules that play a role in inflammation
and tissue repair. The secreted growth factors, such as transforming growth factor
beta (TGF-β), platelet-derived growth factor (PDGF), fibroblast growth factors
(FGFs) and vascular endothelial growth factor (VEGF) stimulate cell migration to
the wound site and promote cell proliferation while the repair cells migrate over
ECM molecules that are already exposed at the wound site to cover the injury and
allow the wound to heal (Mutsaers et al., 1997, Martin et al., 1992).
Mesothelial cells secrete metalloproteinases (MMP) and tissue inhibitors of
metalloproteinases (TIMP) to regulate ECM turnover so that the wounded tissue
can regenerate and re-establish its normal function (Marshall et al., 1993, Rougier
et al., 1997, Ma et al., 1999).

7
Figure 1.1: The functions of mesothelial cells.
The figure above summarises the functions of mesothelial cells. Mesothelial cells can act as a protective barrier, facilitate leukocyte
migration in response to inflammation, regulate particulate and fluid transport, produce surfactant that allows smooth movement
between internal organs, secrete inflammatory mediators, cytokines and growth factors to participate in inflammation and may be
involved in the prevention of tumour adhesion (Mutsaers, 2004).

8

9
1.3 Malignant Mesothelioma
Mesothelial cells can undergo neoplastic transformation and become malignant
mesothelioma (MM). The transformation effects are detrimental as the median
survival for this aggressive tumour is approximately 9 – 12 months (Bograd et al.,
2011). MM is highly resistant to conventional therapies such as chemotherapy,
surgical resection and thoracic radiation alone or in combination.
1.3.1 Causes
The neoplastic transformation of normal, healthy mesothelial cells to MM is
predominantly caused by the exposure to asbestos dust fibres (Yarborough, 2006),
with the duration and quantity of exposure and the type of asbestos fibres inhaled,
as the key determining factors for the development of this malignancy.
Asbestos is widely used as an insulating material due to its fire-resistant
properties. The fibres can be classified into two main subtypes; i) the amphiboles
which contain long and thin fibres and includes crocidolite (blue asbestos) and
amosite (brown asbestos) and ii) serpentine fibres which are curly and includes the
chrysotile or white asbestos (Churg, 1994, Luus, 2007). Approximately 95% of
asbestos globally mined were the latter form (Kamp and Weitzman, 1999).
Epidemiology evidence have suggested that amphiboles are much more potent
than the serpentine fibres in causing MM, with the ratio of quantitative risks being
1:100:500 for chrysotile, amosite and crocidolite (Hodgson and Darnton, 2000).
The reason being is that the half-life of the thin-walled sheets in the chrysotile
fibres is 0.3 – 11 days while the double-chained chemistry of amphibole fibres
have a half-life from 500 days to infinity (Bernstein and Hoskins, 2006). As a result,

10
the chrysotile fibres can be cleared from the lungs much more rapidly than the
amphiboles fibres, hence eliciting a lower carcinogenic potential (Rudd, 2010).
1.3.2 Pathogenesis
As MM has a long latency period of up to several decades between the time of
asbestos exposure and the development of the tumour, it was postulated that there
is a somatic genetic event occurring in the normal, healthy mesothelial cells to
transform them to MM cells (Zervos et al., 2008).
There are many processes by which the asbestos fibres can affect the pleura. The
shape of the fibres, in particular, the ratio of the length to the width of the fibres,
can influence how deep the asbestos fibres penetrate into the lung (Robinson and
Lake, 2005) to irritate and damage the pleura and induce MM (Sebastien P.,
1980). Besides, the asbestos fibres can damage the mitotic spindle in the
mesothelial cells, thereby disrupting the mitotic events, resulting in DNA damage
and hence causing cancer (Ault et al., 1995). DNA damage can also occur due to
the reaction with iron-related reactive oxygen species generated from the asbestos
fibres (Kamp et al., 1995). In addition, the asbestos fibres can activate the
mitogen-activated protein kinase (MAPK) and the extracellular signal-regulated
kinases (ERK) 1 and 2 downstream signalling pathways, leading to the expression
of proto-oncogenes that give rise to Fos-Jun and activator protein 1 families
(Zanella et al., 1996), causing MM.
The metastatic events of MM can be due to the response of MM cells to varying
growth factors such as TGF-β (Fitzpatrick D.R., 1994, Marzo A.L., 1997), PDGF-A
and -B (Versnel M.A., 1991, Garlepp Mj, 1993) and epidermal growth factor (EGF)

11
(Dazzi H., 1990). As mesothelial cells produce these growth factors to stimulate
tissue repair, the over-expression of these growth factors by mesothelial cells can
lead to aberrant autocrine signalling that promotes increased and uncontrolled cell
proliferation. Studies have shown that MM cells produce angiogenic factors such
as VEGF (Masood et al., 2003) and FGF-2 (Kumar-Singh et al., 1999) for the
formation of new blood vessels (Hanahan D., 2000) as a continuous supply of
nutrients is required for tumour growth. There is a correlation between increased
vascularity in MM biopsies with a poor prognosis (Edwards et al., 2003, Kumar-
Singh et al., 1999) and in vivo studies have shown that antagonising VEGF activity
(Merritt et al., 2004) and disrupting the FGF-2 signalling using alpha-Tocopheryl
succinate can impede MM growth (Neuzil et al., 2007).
Besides that, ninety percent of MMs express telomerase. Telomerase is an
enzyme which allows the cells to avoid telomere shortening and hence continue
cell division leading to uncontrolled cell proliferation (Dhaene et al., 2000).
In addition, the uncontrolled cell division in MM could also be a result of the
resistance of the cells to apoptosis due to the absence of tumour suppressor
genes that are involved in the p53 pathway that is implicated in MM. Under normal
circumstances, the presence of ligands activating death receptors or the absence
of growth factors leading to the activation of caspase death signalling pathway
(Riedl and Shi, 2004), causes cells to undergo apoptosis. However, in MM, there is
a possibility that the anti-apoptotic molecule, Bcl-xL, is elevated, causing cells to
evade apoptosis and continue proliferating.

12
1.3.3 Epidemiology
Between the 1940s and 1979, an estimated 27 million individuals were exposed to
asbestos fibres in the United States and Europe where asbestos was used in the
shipbuilding and construction industries. However, the association between MM
and asbestos was not described until 1960 when Wagner et al. first noticed this
disease in the South African miners (Wagner et al., 1960). In the 1970s, workplace
regulations were implemented in the United States to limit the exposure to
asbestos. However, because of the long latency period between asbestos
exposure and the development of MM (Carbone et al., 2002), the incidence rate of
MM in men who were exposed to asbestos dramatically increased only in the last
30 years of the 20th century, with 2000 cases diagnosed in 2000 in the United
States alone (Price and Ware, 2004). The incidence in women remained the same
at approximately 500 cases each year (Price and Ware, 2004). Unfortunately, the
workplace regulations were only implemented in the United Kingdom a decade
later. As a result, the incidence of MM continues to rise and is not expected to
peak until 2015 (Hodgson et al., 2005). As there is no strict regulations to ban the
use of asbestos in most developing countries, the incidence rate of MM and
asbestos-related disease will continue to increase dramatically (Joshi and Gupta,
2004, Le et al., 2011).
1.3.4 Histological Subtypes
There are three main histological subtypes of MM; the epitheloid subtype accounts
for over 60% of MM tumours, the sarcomatoid subtype, about 10% and the rest are
the biphasic (mixed) (Suzuki, 2001). The histology of the epitheloid subtype is very
similar to the other lung carcinomas, making the diagnosis and distinction between
the two challenging (Klebe et al., 2008, Rudd, 2010). This subtype is the least
aggressive of the cell types and hence offers the best prognosis. Under the

13
microscope, the epitheloid cell type displays several patterns, including
tubulopapillary and glandular structure (Rudd, 2010). Each individual cell is usually
cube-shaped and has an easily identifiable nucleus (Inai, 2008).
In contrast, sarcomatoid MM has the worst prognosis. Microscopic analysis
normally shows a spindle cell or storiform structure with elongated nuclei (Inai,
2008). The biphasic subtype has both epitheloid and sarcomatoid components in
the same tumour. The prognosis for the biphasic MM is intermediate between
those of the epitheloid and sarcomatoid MM (Inai, 2008).
1.3.5 Clinical Features
Patients diagnosed with MM usually present with pleural effusions, associated with
breathlessness and persistent, aching, dull and non-pleuritic chest pain (Lee et al.,
2000, Antman, 1981, Elmes and Simpson, 1976, Oels et al., 1971, Antman, 1980).
Other commonly associated symptoms include cough, hemoptysis and weight
loss. By the time these patients present with symptoms, the disease is usually
extensive within the serosal cavity.
1.3.6 Diagnosis
It is often hard to diagnose patients with MM both from a clinical and pathological
perspective because the symptoms they present with are usually non-specific.
Patients typically have to undergo several medical tests including repeated
cytological examinations, serial chest X-rays or computed tomography (CT) scans
and often no definitive diagnosis could be established despite these investigations.
It is not unusual that a definite diagnosis can only be established after a
considerable delay. Nevertheless, findings of a unilateral pleural effusion or

14
thickening together with a history of asbestos exposure should alert the clinician to
the possibility of MM but the definitive diagnosis cannot be made without a
pathological diagnosis.
The International Mesothelioma Interest Group (IMIG) reported that to diagnose
MM, a confirmatory evidence of pleural tissue analysis by immunohistochemistry is
necessary in patients with a cytologic diagnosis suspicious of MM. The evidence
must also be supported by both clinical and radiological data (Husain et al., 2009).
On the contrary, the British Thoracic Society (2007) guidelines accept a cytological
diagnosis if the clinical, radiological and cytological reports support the diagnosis
for MM. A biopsy is needed only if the diagnosis is not clear after tests on pleural
effusions and CT scans are conducted (British Thoracic Society Standards of Care
Committee, 2001).
There is a consensus noting that the observation of neoplastic invasion of
mesothelial cells can be used to differentiate between the benign and malignant
mesothelial proliferation (Kao et al., 2011a). For a pathological diagnosis, fine
needle aspirations have its own limitations and thoracoscopic biopsies including
adjacent and underlying adipose tissue are sometimes needed (Attanoos and
Gibbs, 2008, Kao et al., 2011b). Immunohistochemistry is then conducted on the
sample to test for the expression of cell surface markers to distinguish the
epithelial form of MM from the reactive mesothelial proliferations. It can also be
used to test the cells in pleural effusions (Whitaker, 2000). Markers such as
calretinin and Wilms' tumour 1 antigen (WT1) are used to confirm that the tissue
obtained is of mesothelial origin. A positive expression of epithelial membrane
antigen (EMA; also known as CA15-3 and mucin-1) in a thick peripheral
distribution suggests the malignancy of the tissue (Wolanski et al., 1998, Saad et

15
al., 2005) while a positive staining for cytokeratin can help distinguish MM from
sarcoma and melanoma. Besides that, the tissue should also be stained with two
markers with negative diagnostic value (anti-Ber-EP4, a membrane marker; anti-
thyroid transcription factor-1, a nuclear marker; or monoclonal anti-
carcinoembryonic antigen, anti-B72-3, anti-MOC-31, anti-oestrogen/progesterone,
anti-EMA, cytoplasmic staining) to validate the diagnosis (Scherpereel et al.,
2010). In addition to immunohistochemistry, electron microscopy can differentiate
MM from adenocarcinoma or to distinguish desmoplastic or sarcomatoid MM from
fibrous pleuritis (Segal A, 2002).
There are considerable debates on the role of screening for early diagnosis of MM.
On one hand, the prognosis of MM is very poor and the disease may be treated
more effectively if diagnosed earlier. On the other hand, as there is no cure for
MM, it can be argued that early detection would make no difference to outcome.
Any screening test must prove to have good sensitivity, specificity and positive
predictive values. In addition, the screening test should also be non-invasive or
minimally invasive and the benefits of screening to the patients outweigh the risks
of the screening test (Scherpereel et al., 2010). Biomarkers are useful screening
tools that can screen at-risk populations, establish earlier diagnosis as well as
monitor the treatment outcomes in MM (Ray and Kindler, 2009). Only a small
proportion of those exposed to asbestos will develop MM (Carbone et al., 2012). A
recent study conducted by Testa et al. uncovered mutations occurring in the gene
encoding for BRCA1 associated protein-1 (BAP1) that may predispose asbestos-
exposed individuals to MM (Testa et al., 2011). Germline mutations were found
occurring in the gene encoding for BAP-1 in cases of high incidence of MM. In
addition, somatic mutations were found in familial MM, suggesting that perhaps

16
individuals who are at high risk of developing MM can be screened for mutations
occurring in BAP-1 gene to allow early intervention.
There is currently a challenge to find a biomarker for MM as there is no single
biomarker that can accurately discern MM from the other malignancies with great
sensitivity and specificity. The expression levels of a panel of biomarkers which
include mesothelin, megakaryocyte potentiating factor (MPF) and osteopontin, has
been tested in pleural effusions, serum and urine with variable levels of success.
Mesothelin or soluble mesothelin-related peptide (SMRP) is a glycoprotein that
plays a role in cell adhesion. SMRP is expressed at high concentrations in patients
with MM, lung, ovarian and pancreatic cancers (Ray and Kindler, 2009, Creaney et
al., 2010). At the time of diagnosis, more than 60 percent of MM patients have
elevated levels of SMRP. In a cross-sectional study, the serum SMRP levels were
raised in 84 percent of MM patients but only in less than 2 percent of lung cancer
patients (Robinson et al., 2003). Moreover, the pleural effusions SMRP levels were
increased in MM patients compared to benign and other non-MM groups (Pass et
al., 2008, Creaney et al., 2007). A high SMRP level is correlated with a poor
prognosis (Wheatley-Price et al., 2010) and the levels increase with the
progression of MM and decrease with its regression or following the resection of
the tumour, proving that it can be used as a disease monitoring tool. It may have a
role as a screening tool as some previously healthy persons who had been
exposed to asbestos and have elevated levels of SMRP, were reported to develop
MM within one to six years after their initial blood tests (Robinson et al., 2003,
Creaney et al., 2010, Ray and Kindler, 2009). A high false-positive rate was
reported in a prospective-screening study of over 500 asbestos-exposed subjects
(Park et al., 2008), although the study was short-termed and no subjects actually

17
developed MM. In addition, SMRP levels are sensitive in detecting the epitheloid
but not the sarcomatoid subtypes (Campbell and Kindler, 2011).
MPF is a soluble protein produced by proteolytic cleavage of the mesothelin
precursor protein (Onda et al., 2006). Serum MPF levels were elevated in 51 of 56
patients with MM but the basal levels were maintained in 70 healthy control
subjects. Postsurgical MPF correlated well with the degree of surgical debulking,
suggesting that MPF could be used to monitor treatment effect (Onda et al., 2006).
In addition, a study conducted on 507 patients consisting of healthy control
subjects (n = 101), healthy asbestos-exposed subjects (n = 89), patients with
benign asbestos-related disease (n = 123), benign respiratory disease individuals
(n = 46), lung cancer (n = 63) and MM (n = 85), showed that the receiver operator
curve (ROC) for serum MPF (0.85) was similar to that of serum mesothelin (0.87)
in distinguishing patients with MM from non-MM (Hollevoet et al., 2010).
Osteopontin is also glycoprotein that mediates cell-matrix interactions, that is
expressed by certain cancers, including lung, breast, colorectal, gastric and
ovarian cancers. Unlike mesothelin or SMRP, osteopontin is also increased in both
epitheloid and sarcomatoid subtypes of MM. This glycoprotein is also involved in
the cell signalling cascades that are associated with asbestos-induced
carcinogenesis. Hence, it can distinguish MM patients from patients with asbestos-
related diseases that were not malignant (Pass et al., 2005). Unfortunately,
changes in osteopontin did not appear to parallel disease response to SMRP
(Wheatley-Price et al., 2010).

18
In a direct diagnostic comparison of the above three biomarkers based on a
specificity of 95%, the sensitivity for detecting MM was 73% for SMRP, 45% for
MPF and 47% for osteopontin. Unfortunately, combining the three biomarkers did
not improve the sensitivity (Creaney et al., 2008).
The interpretation of the results using a panel of markers still remains very
subjective (Hammar, 2006, Husain et al., 2009). Mesothelin levels are low in the
non-epitheloid MM and a recent meta-analysis study found that despite having a
good specificity, the poor sensitivity of mesothelin suggests a limited value as a
screening marker for the early diagnosis for MM (Hollevoet et al., 2012). In
addition, there is a high false positive for the use of osteopontin as a screening
marker (Grigoriu et al., 2007). As a result, it is prudent that a reliable assay is
developed or a novel biomarker that can discriminate MM from benign and non-
MM cancers is discovered to achieve an accurate and rapid diagnosis.
At the time of writing, a new study was also conducted to assess the sensitivity
and specificity of fibulin-3 as a biomarker for MM (Pass et al., 2012). It was found
that plasma fibulin-3 was significantly higher in patients with MM compared to
patients without MM but exposed to asbestos. In addition, the fibulin-3 levels in
pleural fluids from patients with MM were also significantly higher than in pleural
fluids from non-MM patients. The study concluded that plasma fibulin-3 can be
used to differentiate asymptomatic asbestos-exposed patients from patients with
MM. Both the pleural fluid and plasma fibulin-3 levels can also distinguish MM from
non-MM and benign fluids (Pass et al., 2012).

19
1.3.7 Prognosis
MM is an aggressive tumour (Scott B., 2000) which often leads to significant
symptoms and has a very poor prognosis of up to 9 – 12 months (Bograd et al.,
2011). The prognosis is worse in those who have a poor performance status, have
a sarcomatoid histology, are in the advance stage of the disease, are males, have
elevated white cell counts, anaemia, thrombocytosis, or high standardised uptake
value ratios on PET (Pass et al., 2005, O’byrne et al., 2004, Herndon et al., 1998).
The expression of cyclooxygenase-2 and VEGF and hence increased vascularity,
as well as hypermethylation of the P16INK4a gene and evidence of SV40 virus, a
cofactor in MM pathology, in the tumour had also been linked to a worse prognosis
(Pass et al., 2005, O’byrne et al., 2004, Herndon et al., 1998).
Grigoriu et al. showed that MM patients who recorded a high mesothelin level
(>3.5 nmol/L) only had a median survival of 7 months while those who had lower
levels of mesothelin had 19 months to live (Grigoriu et al., 2007). Similarly, patients
with high serum osteopontin levels had a significantly shorter prognosis with a
median of 5 months compared to those who had a lower level of osteopontin that
had a median survival of 15 months (Cappia et al., 2008, Grigoriu et al., 2007).
Recently, there have been a growing number of studies looking at the use of
VEGF as a prognostic factor for MM (Hirayama et al., 2011, Demirag et al., 2005,
Strizzi et al., 2001). It has been reported that MM patients had higher levels of
VEGF in pleural effusions when compared to patients diagnosed with non-
malignant pleuritis (Strizzi et al., 2001) or lung cancer (Hirayama et al., 2011).
There was a significant inverse correlation between VEGF and patient survival
(Demirag et al., 2005, Strizzi et al., 2001) and a positive correlation between VEGF
levels and tumour stage (Hirayama et al., 2011, Demirag et al., 2005).

20
1.3.8 Current Therapies
There is currently no cure for MM. Attempts of complete resection of the tumour
through surgery, either extrapleural pneumonectomy (EPP) or
pleurectomy/decortication (P/D) is ineffective as residual microscopic tumours
cannot be completely removed. In addition, surgical resection is often not
advisable for patients in their advanced age, have a medical comorbidity, a
sarcomatoid histology or are in an advanced stage of the disease. In a
Mesothelioma and Radical Surgery (MARS) feasibility study conducted to assess
the clinical outcomes of patients who were randomly assigned to EPP or no EPP, it
was found that the median survival and quality of life scores were considerably
lower in the EPP group compared to the no EPP group (Treasure et al., 2011). Out
of the 112 patients registered, 50 were randomly assigned to both the groups, with
24 to EPP and 26 to no EPP group. After taking into account the factors for not
proceeding to randomisation, it was found that the median survival for the EPP
group (n = 16) was 14.4 months (5.3 – 18.7) and 19.5 months (13.4 to time not yet
reached) for the no EPP group (n = 26). A lower median quality of life scores was
recorded in the EPP group compared to the no EPP group, although no significant
differences between the groups were reported. In fact, there were ten serious
adverse events reported in the EPP group and only two reported in the no EPP
group (Treasure et al., 2011). Despite that, controversies still exist on the road on
EPP (Weder W et al., 2011, Weder W and Opitz I, 2012).
Hence, an approach using chemotherapy drugs remains the mainstay of
treatment. In a phase III trial of pemetrexed in combination with cisplatin (n = 226)
versus cisplatin alone (n = 222) in MM patients, those who received pemetrexed
and cisplatin had a median survival time of 12.1 months compared to 9.3 months
in the cisplatin group (Vogelzang et al., 2003). The response rate in the drug

21
combination group was 41.3% versus 16.7% in the control group (Vogelzang et al.,
2003). Vitamin supplementation in the drug combination group reduced the
toxicities of the drug and did not adversely affect the survival time (Vogelzang et
al., 2003).
However, pemetrexed does not provide a cure and only <50% of patients respond.
Also, not all MM patients are fit to receive chemotherapy drugs. As a result, there
are currently novel molecular targets being studied as a therapy for MM. For
instance, VEGF and its receptors, VEGF receptors 1 – 3, are found to be over-
expressed in MM patients (Demirag et al., 2005). This growth factor promotes MM
cell proliferation in vitro in a dose-dependent manner and the mitogenic effects can
be antagonised by anti-VEGF antibodies (Strizzi et al., 2001). MM patients have
recorded the highest levels of VEGF compared to patients with any other solid
tumours and the levels correlate inversely with their survival (Linder et al., 1998,
Kumar-Singh et al., 1999, Strizzi et al., 2001, Demirag et al., 2005). As a result,
sorafenib, an inhibitor of the tyrosine kinases on VEGFR-2 and PDGFR-b (platelet-
derived growth factor receptor), has been developed. However, the phase II trials
conducted only reported a modest improvement in survival duration of patients
compared to the chemotherapy-naïve patients (Janne et al., 2007, Dubey et al.,
2010).
A double-blinded, placebo-controlled randomised Phase II study reported that the
anti-VEGF monoclonal antibody, bevacizumab, compared to gemcitabine and
cisplastin only slightly improved the progression-free survival to 6.9 months in the
drug group compared to 6.0 months in the placebo group, with the difference not
statistically significant (Ray and Kindler, 2009, Kindler et al., 2012). The median
survival times were 15.6 versus 14.7 months for bevacizumab and placebo

22
respectively but it was not statistically significant either (Ray and Kindler, 2009,
Kindler et al., 2012). An on-going trial by the French Intergroup aims to test the
effects of pemetrexed-cisplatin with bevacizumab in a randomized phase III trial
(Zalcman G. et al., 2012). Other multiple single-arm phase II trials using other
VEGF inhibitors such as SU416, thalidomide, vatalinib (Kindler, 2008), sunitinib
(Nowak et al., 2010) and cediranib (Garland et al., 2009) show modest single-
agent activity.
Although antibody-based therapies targeting molecules found to be over-
expressed in tumours have shown benefits in other types of cancers, no such
benefits of these therapies have been shown in MM in randomised studies to date.
Therefore, there is a prudent need to identify novel therapeutic targets critical for
growth and propagation in MM.
1.3.9 Animal Models
Animal models, in particular rats and mice, have been well established to assess
the carcinogenicity of the various asbestos fibres as well as the pathogenesis of
MM (Davis et al., 1992, Kane, 2006). Genetically engineered mice develop MM
when exposed to asbestos fibres (Kane, 2006). The tumours developed closely
resemble the MM tumours in human in their biological behaviour, histopathology
and molecular alterations, including the latency of the disease, the superficial
growth of tumour on the serosal surface, shedding of tumour cells and growth as
spheroids as well as the accumulation of serosanguinous ascites (Fleury-Feith et
al., 2003, Altomare et al., 2005, Craighead and Kane, 1994). In addition, murine
mesothelial cells respond to asbestos exposure in a very similar way as to the
human mesothelial cells (Davis et al., 1992). As a result, the murine models can be
used to investigate immunotherapy, gene therapy and combined modality

23
treatments. For instance, xenografts of human MM can be used to test
chemotherapeutic agents whereby the experimental data could be translated to the
patients quite easily.
1.4 Fibroblast Growth Factors
Fibroblast growth factors (FGFs) are heparin-binding polypeptides, involved in
various metabolic processes such as cell proliferation, migration, tissue repair, cell
differentiation and morphogenesis (Antoine et al., 2007, Maggie et al., 2009, Ornitz
and Itoh, 2001). There have also been numerous in vivo studies pointing out the
role of FGFs in early embryonic development in mammals (Martin, 1998,
Yamaguchi and Rossant, 1995, Heikinheimo et al., 1994, Lewandoski et al., 2000,
Moon and Capecchi, 2000, Sekine et al., 1999).
1.4.1 Nomenclature of Fibroblast Growth Factors
FGF-1 and FGF-2 that were the first two members identified in the FGF family,
were isolated from bovine pituitary extracts and brain tissue in the 1970s and
found to elicit mitogenic effects (Armelin, 1973, Gospodarowicz, 1974). There are
currently 23 related polypeptides identified in the family, all consisting of similar
structure; having 28 highly conserved and 6 identical amino acid residues (Ornitz,
2000). They also share 13 – 71% of sequence homology. Their molecular weights
range from 17 to 34 kDa (Ornitz et al., 1996). Out of the 23 homologous
polypeptides, FGF-11 to FGF-14 are incapable of activating the FGF signalling
pathway. Instead, they interact with the intracellular domains of voltage-gated
sodium channels (Goldfarb, 2005). Hence, they are called the fibroblast
homologous factors and are classified as the intracellular fibroblast growth factor
subfamily. This leaves 19 members in the FGF family which can be further divided

24
into two subfamilies: the hormone-like and the canonical subfamilies. The
members in the hormone-like subfamily are FGF-15 (or FGF-19 which is the
human orthologue in mouse), FGF-21 and FGF-23. They have a lower affinity to
heparin and act as endocrine factors to regulate metabolism. The members in the
canonical subfamily can be further divided into five groups whose members share
synteny, greater homology and similar binding specificities to receptor. The five
groups are the FGF-1 group (FGF-1 and FGF-2), FGF-4 group (FGF-4 to FGF-6),
FGF-7 group (FGF-3, FGF-7, FGF-10 and FGF-22), FGF-8 group (FGF-8, FGF-17
and FGF-18) and FGF-9 group (FGF-9, FGF-16 and FGF-20) (Itoh and Ornitz,
2008) (Table 1.1).
All of the FGFs are differentially expressed in many, if not all, tissues but the
patterns and timing of expression vary. Each subfamily of FGFs has a similar
pattern of expression and each member of the subfamily has unique sites of
expression. For example, FGF-3, FGF-4, FGF-8, FGF-15, FGF-17 and FGF-19 are
expressed exclusively during embryonic development where as FGF-1, FGF-2,
FGF-5 – 7, FGFs-9 – 14, FGF-16, FGF-18 and FGFs-20 – 23 are found in the
embryonic and adult tissues (Ornitz and Itoh, 2001).

25
Table 1.1: Structure and phylogeny of FGF ligands (Guillemot and Zimmer,
2011).
The 23 human and mouse FGF ligands can be subdivided in canonical,
intracellular and hormone-like FGFs subfamilies (Itoh and Ornitz, 2008). All FGFs
consist of a heparan sulphate proteoglycan binding domain and most have an N-
terminal signal peptide and are secreted via a classical secretory pathway. The
three members of the FGF-9 subfamily (FGF-9, FGF-16 and FGF-20) are
efficiently secreted but have uncleavable signal sequences (Itoh and Ornitz, 2008).
Scheme Family Members
FGF-3 FGF-4
FGF-4
FGF-6 FGF-1 FGF-2
FGF-5
FGF-5 FGF-8
FGF-17
FGF-8
FGF-18 FGF-9 FGF-16
FGF-9
FGF-20 FGF-7 FGF-10
Canonical FGFs
FGF-10
FGF-22 FGF-15/FGF-19
FGF-21 Hormone-like
FGFs FGF-15/FGF-19
FGF-23 FGF-11 FGF-12
FGF-13
Intracellular FGFs
FGF-11
FGF-14
Signal protein
Heparan sulphate proteoglycan binding domain

26
1.5 Fibroblast Growth Factor-9
Our understanding on most aspects of the biology of FGF-9 is limited. FGF-9 was
first identified as a secreted factor in human glioma cells, NMC-G1 (Miyamoto et
al., 1993, Naruo et al., 1993). This growth factor which can be found in abundance
in the rat central nervous system was originally recognised as a mitogen for
primary rat glial cells (Naruo et al., 1993). As a result, it was called the glial
activating factor. Soon after, it was discovered that the glial activating factor share
a sequence homology with the rest of the other FGFs in the family and it was then
termed FGF-9 (Miyamoto et al., 1993).
The earlier studies showed that FGF-9 acts in an autocrine and/or paracrine
fashion to elicit its neuroprotective effect in the central nervous system (Tagashira
et al., 1995). Since then, there is a growing number of studies on FGF-9
expressions in other organs, such as in the normal uterine endometrium (Tsai et
al., 2002) and in the prostate (Giri et al., 1999) where it also acts in the same
manner to elicit its effects as a potent mitogen.
Although FGF-9 has no proliferative effect on the medulloblastoma cell lines
(Duplan et al., 2002) and on human umbilical vein endothelial cells (Naruo et al.,
1993), it is a potent mitogen on many other cells. Hence, abnormal FGF-9
expression and its dysregulated activity is associated with various human
diseases, including brain tumours, prostate cancer, lung adenocarcinomas and
endometriosis (Todo et al., 1998, Wing et al., 2003, Chien-Kai et al., 2009, Giri et
al., 1999). A study conducted by Sumie et al. reported that the over-expression of
this gene in mouse embryonic fibroblast 3T3 cells and in nude mice caused
cellular transformation and tumour formation respectively (Sumie et al., 1997).

27
1.5.1 Fibroblast Growth Factor-9 Gene
The gene encoding human FGF-9 is located on chromosome 11q11-13 (Mattei et
al., 1995) whereas its position on the mouse DNA is on chromosome 14 (Mattei et
al., 1997). The FGF-9 gene has three exons and two large introns (Colvin et al.,
1999). FGF-9 has only a 30% sequence identity with all the other members of the
family (Hecht et al., 1995) (Figure 1.2). There is, however, a greater than 93%
sequence identity between the FGF-9 found in mice, rats and humans, indicating
that this gene is likely to be important for vital biological functions (Miyamoto et al.,
1993).
Figure 1.2: The alignment of amino acid sequence of FGF-9 and other
members of the FGF-9 family (Miyamoto et al., 1993).
The amino acid sequences of the human FGF family, including FGF-9, are aligned
and the well-conserved amino acid regions are shaded in grey. The figure shows
that FGF-9 is very different from the other FGF members in the family.

28
1.5.1.1 Polymorphisms on the Fibroblast Growth Factor-9 Gene
Studies have reported a single nucleotide variant existing on the FGF-9 gene. A
human gonadal dysgenesis study conducted on 21 XY females, 72 XX females
and XY males showed a microsatellite polymorphism event occurring on the 3’
untranslated region of the human FGF-9 gene, which plays a role in regulating
FGF-9 promoter activity and hence modulating the gene expression in human
embryonic kidney and neuroblastoma cells (Chen et al., 2007). The polymorphism
gave rise to four alleles, (TG)13-16, whereby the consequences were cell-type
specific at both the pre- and post-transcriptional levels. One of the alleles was the
(TG)14 allele that was associated with the risk for 46 XY male-to-female sex
reversal, suggesting that any genetic variation in the FGF-9 expression might have
an impact on sex development (Chen et al., 2007).
In addition, a recent report by Harada et al. described a polymorphism event
occurring on the FGF-9 gene in the Eks mutant mice (FGF-9Eks), whereby an
asparagine on position 142 on the chromosome 14 in the mouse DNA, is
substituted with threonine (Harada et al., 2009). Consequently, the FGF-9Eks is
unable to form dimers. Since FGF-9 homodimers have a higher affinity to heparin
as compared to the monomers, FGF-9Eks were unable to bind to heparin, which
has a pivotal role in facilitating the transport of FGF-9 through the extracellular
matrix to the other distant FGFRs (Ornitz and Itoh, 2001, Ornitz, 2000,
Flaumenhaft et al., 1990). Accordingly, the mitogenic effect for FGF-9Eks is
decreased compared to the wild type FGF-9 (Harada et al., 2009). While the wild
type FGF-9 gene activates the ‘b’ and ‘c’ isoforms of FGFR3, the FGF-9Eks protein
activates only the ‘c’ splice variant of FGFR3 which is expressed in the
cartilaginous condensations. As a result of this aberrant signalling, the joint and
suture development is repressed in these mutants and this gave rise to a new

29
gain-of-function phenotype similar to the elbow and knee joint synostosis (Harada
et al., 2009).
1.5.2 Fibroblast Growth Factor-9 mRNA
The stability of FGF-9 mRNA is crucial to understand its implications in diseases. It
was shown that the adenylate/uridylate-rich elements (AREs), located on the FGF-
9 3’-untranslated region, influence the stability of the FGF-9 mRNA. Hence, the
expression level of FGF-9 can be controlled through the rapid degradation of FGF-
9 mRNA (Chen et al., 2010, Xu et al., 1997, Wilson and Treisman, 1988). The
AREs are found in gene transcripts that need to be precisely controlled, such as in
genes that function as growth factors, proto-oncogenes and cytokines where they
play a crucial role in regulating post-transcription of the gene (Chen and Shyu,
1995, Wu et al., 2007, Chen et al., 1995). It was found that the p42 isoform of AU-
rich element binding protein 1 (AUF1) acts as a negative regulator for FGF-9
expression post-transcriptionally by binding to the AREs of FGF-9 and
destabilising the mRNA. Consequently, FGF-9 protein synthesis is impaired.
Subsequently, failure to control this process could result in a prolonged half-life of
FGF-9 mRNA which leads to the sustained expression levels of FGF-9 that can
contribute to disease progression (Chen et al., 2010).
Studies have shown that FGF-9 mRNA can be up-regulated when stimulated by a
variety of factors, such as prostaglandin E2 (Chuang et al., 2006), estrogen (Tsai et
al., 2002) and androgen (Giri et al., 1999), but the mRNA levels will rapidly return
to its basal levels. This implies that the FGF-9 expression is strictly controlled to
avoid excessive production of FGF-9.

30
In a study conducted on the rat retina, it was found that there were high levels of
FGF-9 mRNA at birth but the steady-state mRNA levels decreased thereafter
during periods where terminal differentiation occurs. However, once the terminal
differentiation process was completed, the FGF-9 mRNA levels increased to the
initial levels at birth and remained constant throughout adulthood (Cinaroglu,
2005). It was postulated that low levels of FGF-9 mRNA allowed cells to
differentiate and develop to their final phenotypic status whereas excess cells
underwent apoptosis. Once the differentiation was complete, FGF-9 no longer
functioned as a mitogen but a survival factor to cells (Cinaroglu, 2005).
1.5.3 Fibroblast Growth Factor-9 Protein Structure
The predicted molecular mass of FGF-9 protein, consisting of 208 amino acids, is
23, 532 Da (Santos-Ocampo et al., 1996). FGF-9 can undergo post-translational
modifications whereby the protein is glycosylated prior to secretion (Miyamoto et
al., 1993). Through Western blotting studies, Cinaroglu et al. showed that the
immunoreactive bands of FGF-9 in rat retinal tissue were at 30 and 55 kDa
(Cinaroglu, 2005). Some studies have also found three molecular species of this
protein at 30, 29 and 25 kDa, where it is cleaved at Leu-4, Val-13 and Ser-34
respectively. There are also smaller sized products as a result of proteolytic
digestion (Miyamoto et al., 1993).
The atomic model of FGF-9 consists of one FGF-9 molecule (residues 52-208),
two phosphate ions and 68 water molecules (Plotnikov et al., 2001). Unlike the
other FGF members in the family, FGF-9 forms dimers when in solution, with a Kd
of 680 nm (Plotnikov et al., 2001). The crystal structure of this protein revealed
three copies of four-stranded β-sheet, known as β-trefoil fold, which is similar to
the other FGFs. However, in contrast to FGF-1 and -2, the N- and C- terminals in

31
the FGF-9 regions outside this β-trefoil core are ordered to give a more regular
conformation of the protein (Plotnikov et al., 2001). It was discovered that the
interactions between the N- and C- terminals of each FGF-9 promoter drives the
formation of a dimer complex. There is a significant surface area (>2000A2) that is
buried inside the 2-fold dimer interface that obstructs the receptor binding site of
FGF-9. As a result, it was postulated that the sequences outside of the β-trefoil
core influences the auto-inhibitory mechanism for FGF-9 (Plotnikov et al., 2001)
(Figure 1.3).

32
Figure 1.3: A ribbon diagram of FGF-9 (Plotnikov et al., 2001).
The β strands of FGF9 are labelled according to the conventional strand nomenclature for FGF-1 and FGF-2. CT, C-terminal; NT, N-
terminal.

33
1.5.4 Secretion of Fibroblast Growth Factor-9
In general, most of the FGFs contain a signal sequence located in the amino-
terminal to facilitate its export from the cells. However, FGF-9 is very different from
the other FGF members in the family. Sequencing studies conducted on the FGF-9
protein structure revealed a mildly hydrophobic amino terminal that is atypical
characteristic of a signal peptide (Revest et al., 2000). The non-cleaved signal
sequence is located on the first 33 residues that exports the protein out for
secretion (Revest et al., 2000, Matsumoto-Yoshitomi et al., 1997).
Immunofluorescence studies conducted on FGF-9-transfected COS-1 cells by
Revest et al. illustrated a strong perinuclear staining but a weak reticular staining
over the cytoplasm for mouse FGF-9 (Revest et al., 2000). Further studies
conducted demonstrated that FGF-9 is associated with the Golgi complex and
enters the constitutive secretory pathway (Revest et al., 2000). An
immunohistochemistry study conducted on rat hepatic stellate cells revealed FGF-
9 staining in the intracellular as well as cell surface localisation of FGF-9 (Antoine
et al., 2007). Although FGF-9 is a secreted protein, there have been no studies
that have looked at the half-life of the protein. However, it is believed that the
endogenous heparan sulphate proteoglycan (HSPG) or exogenous heparin can
increase the stability and hence prolong the half-life of the molecule (Santos-
Ocampo et al., 1996).
1.5.5 Physical Interactions between Fibroblast Growth Factor-9 and Heparin
FGF-9 is a classical signalling molecule that is secreted in the extracellular space
where it has a high affinity to HSPGs and binds to it. As a result of this, the
diffusion radius of FGF-9 is limited and this sequesters the FGF-9 in the
extracellular matrix of the connective tissue. It can however be released from such
microenvironmental stores by proteolytic enzymes or by the action of a specific

34
FGF-binding protein (Heinzle et al., 2011). This is evident from a report depicting
that in the absence of exogenous heparin, FGF-9 was found to be present at the
cell surface of rat hepatic stellate cells and detected in the cell lysates but not in
the supernatants, suggesting that FGF-9 was binding to the HSPG located on the
cell surface (Antoine et al., 2007). However, in the presence of heparin, FGF-9 is
efficiently secreted into the supernatants but the immunoreactive bands for FGF-9
were not detected in the matched cell lysates (Antoine et al., 2007).
HSPG plays an important role in the interaction of FGF ligands to their receptors
(Moscatelli, 1987). It not only regulates the distribution of FGFs, but also enhances
the interaction and stability between the ligands and their receptors in the FGF-
FGFR complex, leading to the activation of the FGF receptors (Roghani et al.,
1994, Mansukhani, 1992). This is proven through evidence given by Rapraeger et
al. and Yayon et al. whereby they demonstrated the incapability of FGF-2 to
activate its receptor in the CHO or Swiss 3T3 cells that were genetically,
chemically or enzymatically engineered to lack the endogenous HSPG (Yayon et
al., 1991, Rapraeger et al., 1991).
1.5.6 Biological Function of Fibroblast Growth Factor-9
Although the full functions of FGF-9 have yet to be uncovered, studies to date
have shown that FGF-9 plays a major role in many biological aspects pertaining to
the growth of the cell such as angiogenesis, migration, cell proliferation and
survival.

35
1.5.6.1 Developmental
There are many studies examining the role of FGF-9 during embryonic
development, acting as crucial activating factors that stimulate growth and
migration.
FGF-9 is vital for fetal testicular development as mouse embryos that lack this
gene result in a male-to-female sex reversal (Colvin et al., 2001a). Besides that,
these mice develop a disproportionately shorten small intestine that is associated
with short bowel syndrome (Geske, 2008).
FGF-9 was also found to be involved in epithelial and mesenchymal cell signalling
in the embryonic lung development (Antoine et al., 2007). FGF-9 is expressed in
both the mesothelium and lung epithelium where it acts as a mitogen for sub-
mesothelial mesenchyme (White et al., 2006) and a regulator for FGF-10-mediated
branching morphogenesis during lung development (White et al., 2006). Once the
lung is developed, FGF-9 is no longer expressed in the epithelium but continue to
be expressed in the mesothelium (Colvin et al., 1999). A study on FGF-9 knockout
mice conducted by Colvin et al. described that the lungs was underdeveloped
which led to the death of the mice, further strengthening the role of FGF-9 in
embryonic lung development (Colvin et al., 2001b).
1.5.6.2 Survival
Besides playing a role in somatic cell development in the testis, FGF-9 also
functions as a survival factor for XY germ cell after 11.5 days post coitum in mice
(Dinapoli et al., 2006). FGF-9 also promoted the survival of rat motoneurons that
are located in the embryonic spinal cord (Garcès et al., 2000).

36
Furthermore, Kinkl et al. demonstrated that FGF-9 signalling promoted the survival
of adult pig retinal ganglion cells (Kinkl et al., 2003) and rat retina (Cinaroglu,
2005) which is important to treat retinal degenerative diseases such as glaucoma.
1.5.6.3 Wound Healing and Angiogenesis
FGF-9 also has a role in wound healing. In a study involving young mice, it was
found that FGF-9 mRNA levels were significantly up-regulated at day 2 after
wounding on skin (Komi-Kuramochi et al., 2005). It has not been studied as to how
FGF-9 promotes wound healing. It is believed that like FGFs-1, -2, -7 and -10,
FGF-9 is released from HSPGs in the extracellular matrix through the up-
regulation of FGF-binding protein or when the damaged cell is enzymatically
degraded by proteases (Braun et al., 2004, Podolsky, 1997). This may promote the
proliferation and migration of cells such as keratinocytes and epithelial cells to
close the wound (Abuharbeid et al., 2006). Besides that, FGF-9 is involved in
tissue repair. During acute liver insult, FGF-9 is over-expressed in human hepatic
stellate cells and acts on the primary hepatocytes to repair the injured liver in a
paracrine fashion (Antoine et al., 2007).
The role of FGF-9 in angiogenesis can be demonstrated in a recent study
conducted by Frontini and colleagues where they showed that FGF-9 acts as a
survival factor and mitogen to promote survival and proliferation as well as
migration of smooth muscle cells to nascent microvessels. This allows the new
microvessels to mature, making them long lasting and physiologically active in vivo
(Frontini et al., 2011). In addition, a study conducted by Behr et al. demonstrated
that FGF-9 treatment in FGF-9+/- mice promoted angiogenesis through VEGF-A
and successfully promoted osteogenesis for bone healing (Behr et al., 2010).

37
1.5.6.4 Immune Response
In addition to the above roles, FGF-9 has been shown to play a role in the immune
response in humans (Workalemahu et al., 2004). Workalemahu et al. showed in
their study that γδ T-lymphocytes that play a role in immune response on epithelial
cells to guard against foreign pathogens, had an up-regulated FGF-9 mRNA and
protein levels over their basal levels in response to a mycobacterial antigen
(Workalemahu et al., 2004).
1.5.7 Expression Modulation of Fibroblast Growth Factor-9
There are various in vitro studies conducted to modulate the expression levels of
FGF-9 in the biological system. For instance, it was found that FGF-9 mRNA
expression is up-regulated when endometrial stromal cells were treated with 17β-
estradial (Tsai et al., 2002). The effect was not seen when the cells were treated
with progesterone, giving further evidence of the mitogenic role of FGF-9 in
endometrium during the late proliferative phase (Tsai et al., 2002). FGF-9 mRNA
expression is also up-regulated in lung adenocarcinoma cells following
benzo[a]pyrene treatment, a carcinogen that is an agonist for aryl hydrocarbon
receptor found at high levels in lung adenocarcinoma (Chien-Kai et al., 2009).
On the other hand, it was observed that the FGF-9 mRNA and protein expression
was down-regulated in Parkinson’s disease, where elevated levels of MPP+, a
dopaminergic neurotoxin that can induce oxidative stress, were found. The down-
regulation of FGF-9 expression led to the death of the primary cortical neurons
(Jui-Yen et al., 2009). The effect of H2O2 treatment on the down-regulation of FGF-
9 mRNA expression was antagonised when melatonin, an antioxidant, was co-
administered with FGF-9 (Jui-Yen et al., 2009). It was proposed in that study that

38
oxidative stress could destabilise FGF-9 mRNA or repress the gene promoter
activity by lowering its affinity to transcription factors hence down-regulating its
expression.
The association between oxidative stress and FGF-9 may be organ-specific. What
was observed in the nervous system was not reproduced in the respiratory system.
In human lung adenocarcinoma cells, FGF-9 mRNA levels were increased when
aryl hydrocarbon receptors are activated. There is a positive correlation between
aryl hydrocarbon receptor and intracellular oxidative stress in lung
adenocarcinoma cells (Chien-Kai et al., 2009). However, when transfected BEAS-
2B bronchial epithelial cells over-expressing the receptor were treated with an
antioxidant, FGF-9 mRNA levels remained elevated (Chien-Kai et al., 2009).
Hence, it is proposed that FGF-9 expression in human lung cells is independent of
oxidative stress (Chien-Kai et al., 2009).
1.5.8 Mutations on Fibroblast Growth Factor-9
A recent study revealed six mutations occurring on the FGF-9 gene including one
frameshift, four missense and one nonsense mutation in 10 (six colorectal and four
endometrial) out of 203 tumours and cell lines (Abdel-Rahman et al., 2008). The
mutations were located at the distal part of exon 3, a “hotspot” area, in eight of the
10 samples, but were not detected in the matching normal tissues of the primary
tumours. This suggested that the mutations may be of somatic origins. The first
mutation (c.563delT) was a deletion of one nucleotide from a (T6) repeat tract in
exon 3 that causes a frameshift which creates a premature stop codon that deletes
the last four amino acids (p.L188fxX18 or FGF9∆205-208). The second mutation,
c.424G>T, was a nonsense mutation truncating the distal 67 amino acids (p.E142X
or FGF9∆142-208) whereas the third mutation was a missense mutation (p.R173K)

39
present in the KM12 colon cancer cell line. The last three missense mutations
(p.G84E, p.V192M and p.D203G) were, on the other hand, identified in
endometrial carcinomas. All of these six mutations can cause the following:
1) The FGF9V192M, FGF9D203G and worse, the FGF9∆205-208 mutations negatively
impact ligand-receptor interactions, leading to the decreased activation of the
downstream signalling pathway to phosphorylate MAPKs
2) The FGF9G84E and FGF9∆142-208 mutations interfere with protein folding
3) The FGF9R173K mutation impairs binding of FGF-9 to heparin or HSPG, a
necessary cofactor for FGF signalling
1.5.9 Fibroblast Growth Factor-9 in Cancer
As FGF-9 is a potent mitogen for human glial cells, the uncontrolled activity of
FGF-9 could elicit detrimental effects to the human brain. Using
immunohistochemistry, a moderate to strong immunoreactivity of FGF-9 was
observed in 40 of 49 (82%) cases of human brain tumours examined (Todo et al.,
1998). In vitro studies demonstrated a time-dependent cell proliferation in U87 MG
cells following FGF-9 stimulation after Day 4, with the effects observed in three of
four human meningiomas studies (Todo et al., 1998).
FGF-9 was also found to be up-regulated in prostate cancer (Murphy et al., 2010,
Kwabi-Addo et al., 2004, Li et al., 2008). Positive FGF-9 expression in prostate
cancer cells was observed through immunohistochemistry in 24 of 56 primary
tumours derived from human organ-confined prostate cancer. FGF-9 is also
involved in bone metastasis in prostate cancer in 25 of 25 bone metastasis cases
studied (Li et al., 2008). Furthermore, there was a positive correlation between the

40
presence of FGF-9-positive cancer cells with post-operative recurrence in prostate
cancer. This is backed up by the in vitro evidence that FGF-9 stimulation on
LNCaP cells demonstrated significant cell proliferation as assayed by MTT assay
and cell invasion by Matrigel invasion assay (Teishima et al., 2012).
FGF-9 is also an important mediator of development and progression of ovarian
endometrioid adenocarcinomas (Hendrix et al., 2006). It was found through
microarray and RT-PCR studies that FGF-9 mRNA and protein were up-regulated
in 30 primary ovarian endometrioid adenocarcinomas that had a defected Wnt
pathway, as well as in two ovarian endometrioid adenocarcinomas-derived cell
lines. In vitro studies confirmed that FGF-9 promoted the invasion of epithelial and
endothelial cells as well as the neoplastic transformation of epithelial cells through
a focus formation assay (Hendrix et al., 2006).
In the lung context, it was demonstrated through immunohistochemistry that 86 of
the 146 non-small cell lung cancer samples expressed high levels of FGF-9 and it
was more prevalent in adenocarcinomas compared to squamous cell carcinomas
(odds ratio, 3.81; 95% confidence interval, 1.275-1.509) (Chien-Kai et al., 2009).
Another study reported that FGF-9 mRNA and protein were frequently co-
expressed with FGF-2, FGFR1IIIc and FGFR2IIIc in non-small cell lung cancer cell
lines, in particular, those that have not responded well to gefitinib, an EGFR-
specific tyrosine kinase inhibitor (Marek, 2009). FGF-9 could potently activate adult
lung epithelial cells and drive a progressive oncogenic process. Also, cytological
examination on the ascites fluids collected from mice induced to over-express
FGF-9 for 3 months, gave results that were consistent with metastatic
adenocarcinoma (Betsuyaku et al., 2011).

41
There are no studies investigating the role of FGF-9 in MM. Given the potent
carcinogenic potential exhibited by FGF-9 in other malignancies, it is important that
its role in MM be explored.
1.6 Fibroblast Growth Factor Receptors
The fibroblast growth factor receptors (FGFRs) belong to the receptor tyrosine
kinase subfamily. Alternative splicing of the transcribed receptor genes can occur
and give rise to a variety of receptor isoforms, such as soluble, secreted FGFRs,
FGFRs with truncated COOH-terminal domain and FGFRs with either two or three
Ig-like domains (Haugsten et al., 2010, Rose-John and Heinrich, 1994).
There are four members in the FGFR family, FGFR1 – 4, which share a 64 – 74%
amino acid identity in the extracellular domain and 72 – 85% sequence identity in
the kinase domain region (Jaye et al., 1992, Powers et al., 2000). The genes
encoding these FGFR monomers each contain approximately 20 exons.
RT-PCR experiments on adult rat retinal mRNA demonstrated amplification
products for FGFR1, 2, 3 and 4 at 297-, 247-, 214- and 156- bp respectively
where as western blotting studies revealed the presence of FGFR1 – 4 as multiple
forms between ~80 – 200 kDa (Cinaroglu, 2005, Kirschbaum et al., 2009, Yiangou
et al., 1997).
1.6.1 The Structure of Fibroblast Growth Factor Receptor
A typical FGFR consists of a cleavable signal peptide, an acidic box, intracellular
tyrosine kinase domain, a single transmembrane domain and an extracellular

42
portion containing three immunoglobulin (Ig)-like domains, IgI, IgII and IgIII
(Mckeehan et al., 1998) (Figure 1.4).
The IgI domain is absent in certain isoforms and is thought to be involved in
receptor auto-inhibition while the IgII domain contains a heparin-binding domain
and is hence necessary for ligand binding (Wiesmann et al., 2000).
An alternative splicing event can occur on the second half of the IgIII domain, in
exons 8 and 9 of the receptor, but this event only occurs specifically in FGFR1, 2
and 3, generating either the IIIa, IIIb or the IIIc isoform of the receptor with different
ligand-binding specificities and affinities (Chellaiah et al., 1994, Werner et al.,
1992, Johnson, 1991, Zhang et al., 2006, Ornitz et al., 1996).
The IIIa isoform is a secreted FGF-binding protein whereas the IIIb and IIIc splice
variants are membrane-bound receptors. Studies have suggested that the IIIb
isoform is expressed in cells of epithelial lineages while the IIIc variant is found in
the mesenchyme (Avivi et al., 1993, Yan et al., 1993, Orr-Urtreger et al., 1993). In
contrast, the ligands for the IIIb variant are expressed in the mesenchymal cells
while the IIIc ligands are found in the epithelial cells, generating a paracrine cross-
talk interaction between the mesenchyme and epithelium that is critical for normal
development and homeostasis. It was also reported that activation of the 'c' splice
variant of FGFR2 and FGFR3 elicits a more potent mitogenic effect compared to
the activation of the ‘b’ splice variant (Santos-Ocampo et al., 1996, Ornitz et al.,
1996).

43
On the contrary, the soluble FGFR contains an extracellular ligand-binding domain
of the native FGFR but does not have a tyrosine kinase domain. As a result,
ligands can still bind to the receptor but are unable to activate it, thus 'mopping up'
ligands to prevent them from binding and activating the native FGFRs (Celli et al.,
1998).

44
Figure 1.4: The fibroblast growth factor receptor structure (Mason, 2007).
This figure illustrates the 3- immunoglobulin (Ig) isoforms of the four FGFRs. The extracellular domain contains the three Ig loops (IgI,
IgII and IgIII). The acidic box (AB) is located between IgI and IgII. In some splice variants, the acidic box is absent. The alternatively
splice events occurring in the IgIII domain give rise to the ‘a’ (not shown), ‘b’ and ‘c’ isoforms in FGFRs 1 – 3. There is also a
transmembrane domain (TM) and an intracellular domain consisting of two tyrosine kinase enzyme domain (TKI and TKII). The FGF
ligand-binding site is located at the C-terminal part of IgII and the N-terminal portion of IgIII.

45
1.6.2 The Receptors for Fibroblast Growth Factor-9
An affinity-labelling study conducted by Hetch et al. showed that FGF-9 binds
efficiently and activates the IIIc but not the IIIb isoforms of FGFR2 and FGFR3 and
its binding affinity was largely dependent on the presence of heparin (Hecht et al.,
1995). FGF-9 does not bind to FGFR 1 and FGFR4 (Hecht et al., 1995).
In addition, a radioligand binding assay followed by another binding assay utilising
the extracellular domains of the FGF receptors fused to alkaline phosphatase
conducted by Santos-Ocampo et al. also demonstrated that FGF-9 binds best to
FGFR3IIIc but not FGFR1IIIc. Furthermore, their study also demonstrated that
FGF-9 binds weakly to the IIIb isoforms of both FGFR2 and FGFR3 and was
incapable of activating the FGFR2IIIb isoform (Santos-Ocampo et al., 1996).
Interestingly, they also found that even though FGF-9 could not bind to cell surface
or soluble FGFR1IIIc, the mitogenic assay performed showed that FGF-9 could
partially activate that receptor isoform (Santos-Ocampo et al., 1996).
1.6.3 Fibroblast Growth Factor-9 Signalling Pathway
The FGF-9 molecule forms homodimers and binds to the IgII and IgIII regions of
the receptor. This interaction is stabilised by the HSPG. The binding of the FGF-9
ligand to the receptor, which exists as inactivated monomers, causes the receptor
to undergo homodimerisation (Schlessinger et al., 2000). Activation of the receptor
stimulates the downstream signalling pathway where several signalling proteins
are recruited to the tyrosine auto-phosphorylated sites on the activated FGFR to
transautophosphorylate several other tyrosine residues on the receptors (Klint and
Claesson-Welsh, 1999).

46
There is a critical docking protein called the SNT-1/FRS2 (fibroblast growth factor
receptor substrate 2) which serves as a platform for more tyrosine kinases to bind.
This leads to the phosphorylation of more tyrosine residues on the receptor to
induce the RAS/MAPK and PI3K (phosphatidyllinositol-3 kinase signalling
pathway) (Eswarakumar et al., 2005, Klint and Claesson-Welsh, 1999). As a result,
the phosphorylation state of ERK1/2 in the MAPK pathway can be assessed to
understand the FGF signalling pathway (Thisse and Thisse, 2005).
Activated FGFRs can also phosphorylate and activate PLCγ. The activated PLCγ
produces diacylglycerol and inositol 1,4,5-triphosphate, which in turn, releases the
intracellular calcium storages and activates calcium-dependent members of the
protein kinase C (Klint P. and Claesson-Welsh L., 1999).
The activation of the FGFR pathway causing a chemotactic response,
differentiation, apoptosis, migration and cell proliferation is context-dependent and
tissue-specific as the effects elicited is dependent on the location of the different
isoforms of the receptor in the tissue (Turner and Grose, 2010, Klint and Claesson-
Welsh, 1999).
FGFR signalling is regulated at multiple levels, resulting in a tight control of its
expression levels, its sites of expression and its duration of signalling. FGFR
signalling can be negatively regulated by several proteins such as the MAPK
phosphatase 3 (MKP3), the Sprouty proteins and Sef (similar expression to FGF)
family members (Turner and Grose, 2010). FGFRs are also endocytosed and
degraded in lysosymes to attenuate its signalling (Haugsten et al., 2008) (Figure
1.5).

47
In addition, it was postulated that the FGF-9 homodimer can lead to the obstruction
of critical binding sites on the receptors; a feedback mechanism to modulate the
signal transduction at another level. The signalling specificity of individual FGFR
types is further complicated by the fact that most of the cells express more than
one FGFR variant and FGFRs can also form active heterodimers (Bellot, 1991).

48
Figure 1.5: Signalling through fibroblast growth factor receptors (Mason, 2007).
This figure summarises the FGFR signalling as well as the endogenous agonists and antagonists acting on both the upstream and
downstream of the receptor signalling pathway. The ERK-MAPK pathway is widely studied in the FGFR signalling pathway and is
activated by the Ras downstream of FRS2-SOS-Grb2 complex while the calcium/protein kinase C pathway is activated by activation of
phospholipase C. The potential nuclear targets are those proteins that antagonize or potentiate FGFR signals and those that are
transcription factors. CAM, cell adhesion molecule; CREB, cyclic AMP response element binding protein; FLRT, fibronectin leucine-rich
transmembrane proteins; FRS, FGF receptor substrate; HSPG, heparan sulphate proteoglycan; Ig, immunoglobulin; IP3, inositol tris
phosphate; MAPK, mitogen-activated protein kinase; MKP, MAPK phosphatase; PI3K, phosphatidylinositol-3-kinase; PIP3,
phosphatidylinositol-3-phosphate; PIP4, phosphatidylinositol-4-phosphate; PKB, protein kinase B; PLC , phospholipase C ; SOS, son
of sevenless; TK, tyrosine kinase.

49

50
1.6.4 Mutations of the Receptors for Fibroblast Growth Factor-9
1.6.4.1 Activating Mutations
Mutations of fibroblast growth factor receptor 2 (FGFR2) have been described in
12% of endometrial cancers (Dutt et al., 2008). Mutants such as the N549K and
K659N are highly sensitive to FGFR tyrosine kinase inhibitors, suggesting that
FGFR2 can also be a potential therapeutic target in the treatment of endometrial
cancer (Ornitz and Marie, 2002). Studies have shown that the endometrial cell
lines that harbour these mutations have reduced cell proliferation and decreased
survival following treatment with an FGFR inhibitor, PD173704 or knockdown of
FGFR2 (Dutt et al., 2008, Byron, 2008).
There are many studies associating bladder cancer with FGFR mutations. It was
reported that somatic mutations in the FGFR3 coding sequence occurs in
approximately 50% of bladder cancers (Cappellen et al., 1999). The majority of the
mutations in FGFR3 occur at a single position in the extracellular domain. One of
them is the S249C where an intermolecular cysteine disulphide bridge is formed
causing the aberrant folding of the FGFR3 protein leading to the constitutive
dimerization and hence activation of the receptor (Naski et al., 1996, Di Martino et
al., 2009). Mutations are also commonly found in the transmembrane domain,
such as the Y373C and the G380R (Pandith et al., 2010) and less common in the
kinase domain, such as the K652E (Munro and Knowles, 2003). The effects of the
mutations in the transmembrane and the kinase domains impact less on the
constitutive activation of the receptor (Di Martino et al., 2009, Naski et al., 1996).
FGFR3 can be a useful potential therapeutic target for urothelial cell carcinoma
(UCC), the most common form of bladder cancer, (Pandith et al., 2010, Cappellen

51
et al., 1999, Hernández et al., 2006) as numerous studies have demonstrated the
oncogenic role of the mutationally-activated FGFR3 in UCC. This is evident from
the knockdown studies of mutated FGFR3 or the inhibition of FGFR3 using
monoclonal antibody targeting FGFR3 in mouse xenografts or specific FGFR
inhibitors such as SU5402 and PD173704 in UCC cell lines, which have shown
reduced tumour properties (Bernard-Pierrot et al., 2006, Qing et al., 2009, Miyake
et al., 2009, Tomlinson et al., 2007, Lamont et al., 2011).
Besides bladder cancer, FGFR3 mutations have also been identified in cervical
cancers (Rosty et al., 2005), multiple myeloma and prostate cancers (Hernandez
et al., 2009).
1.6.4.2 Chromosomal Translocations
Besides activating mutations, chromosomal translocations can also occur on the
receptor genes (Figure 1.6). 15% of multiple myelomas harbour a t(4;14)
translocation that links the FGFR3 at 4p16.3 to the immunoglobulin heavy chain of
immunoglobulin H (IgH) locus at 14q32 (Avet-Loiseau et al., 1998, Chesi et al.,
1997). As a result of this, FGFR3 is controlled by the highly active IgH promoter.
This leads to the over-expression of the receptor and hence increased sensitivity
to ligands. As a result, the negative feedback of the signalling and also the
receptor internalisation and hence receptor degradation are decreased leading to
aberrant signalling of FGFR3 (Otsuki T. et al., 1999). FGFR3 translocation in
multiple myeloma is associated with a poor prognosis and it promotes a rapid
conversion from the precursor condition to the full blown multiple myeloma (Avet-
Loiseau et al., 1999).

52
1.6.4.3 Gene Amplification
Another type of mutation is the gene amplification where it is more commonly
found in FGFR2 (Figure 1.6). In gastric cancer, an amplified FGFR2 gene results
in increased sensitivity to FGF-7-dependent stimulation of tumour cell growth
(Nakazawa et al., 2003, Takeda, 2007) and is associated with poor prognosis
(Kunii, 2008).
The amplification of FGFR2 has also been described in triple-negative breast
tumours, which are aggressive breast tumours negative for the oestrogen receptor,
progesterone receptor and HER2 (human EGFR2)/ErbB2 (Turner et al., 2010).
The cell lines derived from these tumours had constitutive activation of FGFR2 and
were sensitive to FGFR2 inhibition using a specific FGFR inhibitor, PD173074, and
to FGFR2 RNAi silencing (Turner et al., 2010). FGFR2 may be used as a novel
therapeutic target for these breast tumours that have FGFR2 gene amplification.
1.6.4.4 Germline Single Nucleotide Polymorphisms
Single nucleotide polymorphism (SNP) can also occur on the genes (Figure 1.6). A
recent genome-wide association study has identified the role of FGFR2 as a breast
cancer susceptibility gene (Easton et al., 2007, Hunter et al., 2007). There was a
correlation between a SNP occurring in the second intron of FGFR2 with an
increased risk of developing ER-positive breast cancer but with little or no effect on
ER-negative breast cancer (Garcia-Closas et al., 2008).

53
Figure 1.6: Mechanisms of pathogenic cancer cell FGF signalling (Turner and Grose, 2010).
FGFs and their receptors can be altered in several ways in malignant diseases. Genomic alteration of FGFR can occur through three
mechanisms, leading to ligand-independent signalling. Firstly, activating mutations can cause ligand-independent dimerization or
constitutive activation of the kinase (shown by the yellow lightning). Secondly, chromosomal translocations generate fusion proteins
leading to dimerization of the fusion protein and constitutive signalling. Thirdly, receptor amplification can contribute to an accumulation
of the receptors (Turner and Grose, 2010).

54

55
1.7 Therapeutic Approaches
Many FGFR tyrosine kinase inhibitors (TKIs) are now in early phases of clinical
trials. As the VEGFR and FGFR kinase domains are structurally similar, the TKIs
developed can dually inhibit both receptors, hence inhibiting angiogenesis and
tumour cell proliferation (Turner and Grose, 2010).
However, the dual inhibition increases the side effects of the TKIs. Consequently,
there are therapeutic antibodies being developed to specifically target individual
FGFRs. This can also reduce the potential toxicity of pan-FGFR inhibition.
Antibodies targeting FGFR3 have been shown to have an anti-proliferative effect
on bladder cancer cells (Martínez-Torrecuadrada et al., 2005, Qing et al., 2009)
and t(4;14) myeloma (Qing et al., 2009).
In addition, there are FGF ligand traps, such as the FP-1039, which are soluble
fusion proteins consisting of an extracellular FGFR1IIIc domain fused to the Fc
domain of IgG1. The ligand trap antagonises the activity of multiple FGF ligands
and receptors, exerting both anti-angiogenic and anti-proliferative effects (Zhang
H. et al., 2007).
1.8 Summary
MM is a universally fatal cancer, predominantly caused by exposure to asbestos.
MM has a long latency period of up to 50 years and many patients diagnosed with
MM have less than a year to survive as there is no effective cure. This fatal
disease is difficult to diagnose and patient prognosis is difficult to predict. There is
thus a desperate need to uncover novel molecules which play key biological roles

56
in MM. Such molecules can potentially be crucial therapeutic targets and their
expressions may also have diagnostic and prognostic values.
FGF-9 is a secreted growth factor, first identified in human glioma cells. FGF-9
activates FGFR2 and FGFR3 and is vital for lung and testicular development. The
over-expression of FGF-9 and its aberrant signalling have been recently described
in prostate, lung and endometrial cancers but its role in MM has not been
investigated.
1.9 Preliminary Results
To identify biologically important molecules that are differentially regulated in MM,
our group conducted global gene profiling experiments using human MM samples.
Uncovering key molecules governing the growth of MM may provide novel
therapeutic targets. These proteins may also serve as biomarkers to aid diagnosis
and/or prognosis prediction in MM, which may predict the treatment response.
Pleural biopsies were collected under direct vision from patients undergoing
thoracoscopy. Only samples that had enough RNA quantity (>5 µg) and RNA
quality as defined by Bioanalyzer, were profiled for the expression of more than
22000 genes using Affymetrix HG-U133A microarray chip. 22 samples (14 MM, 3
pleural metastatic adenocarcinoma and 5 benign pleuritis) were analysed.
Using a stringent cut-off of 4-fold increase over benign controls, 25 genes were
found to be statistically significant in the MM group (Figure 1.7). The global gene
profiling data identified FGF-9 as the leading candidate gene with an mRNA
expression of 17-fold higher in MM compared to benign pleural tissue and pleural

57
adenocarcinoma samples (Figure 1.8). FGF-9 was picked out of the 25 genes as it
was a novel molecule that has not been investigated in MM and its role in other
cancers were just only beginning to be discovered. FGF-9 over-expression was
validated in another second separate cohort of 24 human tissue biopsies using
quantitative real-time PCR. This confirmed once again that FGF-9 was over-
expressed in MM at 35-fold (median) increase over adenocarcinoma or benign
pleuritis samples (Figure 1.9).
The following thesis research project aimed to follow-up on these exciting
preliminary data and to examine the role of FGF-9 in MM using a wide range of
molecular and cellular experiments, in vivo studies, and analyses of several large
cohorts of human pleural fluid samples.
Figure 1.7: Global gene profiling on 22 human pleural tissue samples.
Unsupervised hierarchical clustering separated 13 of 14 MM samples into one
group and the others into adenocarcinoma and benign groups, indicating good
homology of the global gene expression within the samples for each group. Ad =
adenocarcinoma.
Benign Ad Mesothelioma
Expression levels
High Low
Medium Not expressed
Sample size nbenign pleuritis = 5
nmesothelioma = 14 nmetastatic adenocarcinoma = 3

58
Figure 1.8: Quantification of FGF-9 gene expression from global gene
profiling.
Quantification of FGF-9 gene expression determined by Affymetrix microarray data
of individual biopsy samples. Median expression of FGF-9 was 17-fold higher in
the MM group than in controls. *p<0.05 compared to benign and adenocarcinoma
controls using Student’s t-test.
Benign & Ad MM
0.01
0.1
1
10
100
*
FG
F-9
gen
e ex
pre
ssio
n
(Norm
alis
ed i
nte
nsi
ty)
n =8 n=14

59
Figure 1.9: Validation of microarray gene expression results using RT-PCR.
Quantification of candidate gene RNA expression in an independent sample set.
FGF-9 was significantly over-expressed in MM over benign controls by 35-fold,
markedly higher than all the other candidate genes examined.
Ben
ign
RA
LDH
CK
7
CX
AD
RH
GF
SK19
LAP
FGF9
0
10
20
30
40
50
Fo
ld i
ncr
ease
M/B
1.10 Hypothesis and Aims
The up-regulation of FGF-9 mRNA in MM over controls in our pilot data (of my
thesis) leads to the hypothesis that FGF-9 plays a vital role in MM pathobiology
including cell proliferation, invasion and metastasis. Therefore, inhibiting FGF-9
activity will retard MM growth. In addition, FGF-9 can serve as a useful diagnostic
and prognostic biomarker for MM patients. We hypothesised that mutations in the
FGF-9 molecule and/or its receptors may cause aberrant FGF signalling in MM.
The aims are:
1a) To characterise the biological role of FGF-9 in MM cells and tissues
b) To study the biological effects of FGF-9 on MM cells in vitro
c) To examine the effects of silencing FGF-9 in MM cells in vivo

60
2) To determine the diagnostic value of FGF-9 levels in pleural fluids and
plasma of MM patients as a stand-alone biomarker and in conjunction with
existing biomarkers
3) To assess MM tissues for mutations in FGF-9 and its receptors, FGFR2
and FGFR3

61
____________________________________________
MATERIALS AND METHODS
CHAPTER 2

62
2.0 MATERIALS AND METHODS
2.1 Maintenance of Malignant Mesothelioma Cell Lines
2.1.1 Cell Culture
MM cell lines were cultured in cell culture flasks using standard growth medium
containing Dulbecco’s Modified Eagle Medium (DMEM) with high glucose (4500
mg/L) (Invitrogen, Grand Island, NY, USA), penicillin (100 U/mL), streptomycin
(100 µg/mL), L-glutamine (4 mM) (Invitrogen, Waverley, Australia) and fetal bovine
serum (FBS) (10%) (Sigma-Aldrich, St Louis, MO, USA). The cells were incubated
in a 5% CO2-containing humidified atmosphere at 37°C. Once the cells reached
80% confluency, they were trypsinised with 0.25% trypsin-
ethylenediaminetetraacetic acid (EDTA) (2 mL) (Invitrogen, Waverley, Australia)
and passaged at a 1:10 dilution.
Prior to all cell culture experiments, cell number was assessed by Trypan Blue
exclusion method. Cells were seeded in standard growth medium for at least 5
hours for the cells to adhere to the plates before the media was changed to serum-
free media (SFM) the day before experiments were conducted.
2.1.2 Malignant Mesothelioma Cell Lines
Eight human MM cell lines were used in this study. Five of the human MM cell
lines (JU77, LO68, NO36, ONE58 and STY) derived from pleural effusions (Linda
et al., 1991) were obtained from Professor Bruce Robinson (University of Western
Australia). The other three human MM cell lines, CRL-2081, CRL-5820 and CRL-
5915, were obtained from American Type Culture Collection (Rockville, MD, USA).
In addition to the human MM cell lines, six murine MM cell lines; AB1, AB2 and

63
AB22, all derived from mice of Balb/C background, AC29, derived from CBA mice
background and AE5 and AE17, derived from C57/Bl6 mice background were
used. These cell lines were derived from MM developed in mice that were exposed
to asbestos (Davis et al., 1992, Jackaman et al., 2003). All of the above-mentioned
cells were maintained in the standard growth conditions mentioned in Section
2.1.1. The MM cells have been well-characterised by cytological and ultrastructural
(electron microscopy) analyses (Davis et al., 1992, Jackaman et al., 2003, Linda et
al., 1991) and have been used in many published studies (Nasreen et al., 2000,
Galffy et al., 1999b, Linda et al., 1991, Jackaman and Nelson, 2010, Kissick et al.,
2012, Crisanti et al., 2009, Greay et al., 2010, Bielefeldt-Ohmann et al., 1995,
Friedlander et al., 2003).
2.1.3 Cryo-Freezing and Storage of Cells
Cells were trypsinised and centrifuged at 1200 r.p.m. for 10 minutes at room
temperature. The cell pellets were resuspended in pre-chilled serum-free growth
medium containing 10% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St Louis, MO,
USA). 2 x 106 cells/mL was aliquoted into each cryovial and stored at -80°C
overnight before they were transferred to liquid nitrogen for long term storage.
Cells in cryovial were thawed in a 37°C water-bath before they were transferred to
cell culture flasks and maintained in conditions described in Section 2.1.1. The
growth medium was changed the next day to remove non-adherent cells and
passaged as in Section 2.1.1.

64
2.2 Phenotypic Characterisation of Cells and Tissues
2.2.1 Immunofluorescence
2 x 105 human and murine MM cells were seeded in 8-well Lab Tek chamber
slides (In Vitro Technologies). After overnight serum-starvation, the cells were
washed with phosphate buffered saline (PBS) (3x) before fixing and permeabilising
using ice-cold methanol (100%) for 10 seconds. The cells were then rinsed with
PBS and non-specific protein binding was blocked with 10% heat-inactivated FBS
in PBS for 30 minutes at room temperature. Following that, the blocking solution
was aspirated and the cells in the wells were incubated in one of the primary
antibodies (FGF-9, FGFR2 and FGFR3) and their appropriate dilutions (Table 2.1)
overnight at 4°C. Universal Negative Control for N-series Rabbit Primary
Antibodies (Dako Denmark A/S Glostrup, Denmark) (Table 2.1) was used as a
negative control. The following day, the cells were washed in PBS (3x) and then
incubated with a secondary antibody, Alexa Fluor 488 goat-anti rabbit IgG (H+L)
(Invitrogen, Grand Island, NY, USA) (Table 2.2) for 30 minutes in the dark. Then,
the cells were washed with PBS (3x) and incubated with Hoechst 33342 for 15
mins in the dark before they were rinsed with PBS and mounted on slides in
aqueous mount and dried in the dark at 4°C. Images were captured using
AxioSkop 2 Plus fluorescence microscope (Zeiss, Thornwood, NY).
Table 2.1: Primary antibodies
Antibody and catalogue number
Dilution Application Supplier
1:200 Immunofluorescence FGF-9 (sc-7876) 1:250 Immunohistochemistry
Santa Cruz Biotechnology
1:250 Immunofluorescence FGFR-2 (ab10648) 1:1000 Immunohistochemistry
Abcam
1:100 Immunofluorescence FGFR-3 (sc-9007) 1:500 Immunohistochemistry
Santa Cruz Biotechnology
β-actin (ab8226) 1:10 000 Western Blotting Abcam Universal Negative Control for N-series
Rabbit Primary
Depending on dilution of
other
Immunohistochemistry Dako

65
Antibodies (N169987)
primary antibody
used p-ERK (# 4377) 1:1000 Western Blotting Cell Signalling p-JNK (# 9251) 1:1000 Western Blotting Cell Signalling p-p38 (# 9215S) 1:1000 Western Blotting Cell Signalling
Total ERK (sc-154) 1:1000 Western Blotting Cell Signalling Total p38(# 9212) 1:1000 Western Blotting Cell Signalling
Table 2.2: Secondary and tertiary antibodies
Antibody and catalogue number
Dilution Application Supplier
Biotinylated goat anti-rabbit (sc-2040)
1:200 Immunohistochemistry Santa Cruz Biotechnology
Streptavidin-horseradish
peroxidise (554066)
1:200 Immunohistochemistry BD Pharmingen
Alexa Fluor 488 goat-anti rabbit IgG
(H+L) (A11034)
1:400 Immunofluorescence Invitrogen
Hoechst 33342 1:1000 Immunofluorescence Sigma-Aldrich Rabbit anti-mouse-
HRP (ab6728) 1:1000 Western blotting Abcam
Goat anti-rabbit HRP (sc-2004)
1:1000 Western blotting Santa Cruz Biotechnology
2.2.2 Immunohistochemistry
The immunohistochemical staining was performed on formalin-fixed, paraffin-
embedded tissue biopsies of human and mice. After de-paraffinised with xylene
and rehydrated with 100% and then 70% ethanol, the tissue sections were then
subjected to heat-induced epitope retrieval in EDTA/Tris buffer, pH 8.0, (Appendix
I) using a microwave oven at high power for 2 minutes and 30 seconds before the
power was lowered for 15 minutes. Following that, the tissue sections were cooled
to room temperature in the same buffer and then rinsed with distilled water. Excess
peroxidase activity was blocked with the addition of 3% hydrogen peroxide solution
(Dako Denmark A/S Glostrup, Denmark) for 5 minutes at room temperature. The
tissue sections were then washed in distilled water before they were incubated
with 10% FBS/PBS for 30 minutes at room temperature to block non-specific

66
protein binding. Primary antibody incubation was done overnight at 4°C for all the
antibodies (FGF-9, FGFR2 and FGFR3 (Table 2.1)). Immunodetection was
performed on PBS-washed immunoreacted sections using a secondary antibody,
biotinylated goat anti-rabbit (Santa Cruz) (Table 2.2) for one hour followed by
incubation with streptavidin-horseradish peroxidise (BD Pharmingen) (Table 2.2)
for 30 minutes, at room temperature. Sections were then incubated with
SIGMAFAST™ 3,3’-Diaminobenzidine tablets (DAB Peroxidase Substrate Tablet
Set) dissolved in distilled water (Sigma-Aldrich, St Louis, MO, USA) and
counterstained with Mayers Haematoxylin (Sigma-Aldrich, St Louis, MO, USA).
After rinsing with distilled water and then Scott’s tap water substitute (Appendix I),
the sections were dehydrated with increasing concentrations of ethanol (70%, 95%
and 100%) and then xylene. DEPEX was added to each section before mounting.
Images were taken using AxioSkop 2 Plus microscope (Zeiss, Thornwood, NY).
2.2.3 Tissue Microarray Analysis
The tissue microarray staining was performed as stated in Section 2.2.2 with the
exception that the heat-induced epitope retrieval was done in sodium citrate buffer,
pH 6.0 (Appendix I). The slides were scored semi-quantitatively for the intensity of
FGF-9 staining by two independent blinded investigators. The intensity was graded
as not present (0), weak (1) and moderate to strong (2).
2.3 Western Immunoblotting
2.3.1 Protein Extraction
1 x 106 human and mice MM cell lines were plated in 65 x 15 mm dish. Following
the appropriate stimulations, the cells were then scraped on ice using a cell
scraper and lysed with pre-chilled breaking buffer (Appendix I) with the addition of

67
1:10 Protease Inhibitor Cocktail (Sigma-Aldrich, St Louis, MO, USA) and
PhosSTOP Phosphatase Inhibitor Cocktail (Roche, Castle Hill, Australia). The
sample was vortexed, left on ice for 10 minutes and vortexed again before being
centrifuged at 13100 r.p.m. for 10 minutes at 4°C. The supernatants were stored in
-80°C until assayed.
2.3.2 Determination of Protein Concentration
Protein concentrations were determined with the use of BioRad DC Protein Assay
Kit 2 (BioRad Laboratories, Hercules, CA) as per the manufacturer’s protocol with
various concentrations of BSA solutions (2, 0.75, 0.5, 0.25, 0.125, 0.025 mg/mL)
prepared in breaking buffer as standards for this assay.
2.3.3 SDS-PAGE and Immunoblotting
Cell lysates (20 µg) was mixed with NuPage® LDS sample buffer (Invitrogen,
Waverley, Australia) and dithiothreitol (DTT) (Sigma-Aldrich, St Louis, MO, USA).
The samples were then heated at 96°C for 10 minutes to denature the protein. 1X
SDS running buffer (Appendix I) or 1X MOPS buffer (Invitrogen, Waverley,
Australia) was added to the reservoirs and the samples were electrophoresed in
Ready Gel 4 – 20 % Tris-HCl (BioRad Laboratories, Hercules, CA) or NuPAGE®
Novex 4 – 12% Bis-Tris Gel (Invitrogen, Waverley, Australia) at 40 V until the
samples resolved through the stacking gel. Following that, the voltage was
increased to 100 V until the samples reached the bottom of the separating gel.
Transfer buffer (Appendix I) was pre-chilled before use and the polyvinylidene
difluoride (PVDF) membranes (PerkinElmer Life Sciences, Inc, Boston, MA) were
incubated with methanol (100%) for 1 minute, rinsed with distilled water and then
soaked in pre-chilled transfer buffer prior to use. The gels were then electro-

68
transferred onto PVDF membranes at 100V for 1.5 hours. The membranes were
stained with Ponseau S (Sigma-Aldrich, St Louis, MO, USA) to visualise effective
transfer and then blocked in 5% milk in TBS/Tw (Tris-buffered saline/0.1% Tween-
20) (Appendix I) for 1 hour at room temperature. The membranes were then
washed with TBS/Tw and probed with specific primary antibodies directed against
p-ERK, p-JNK, p-p38, total ERK, total p38 (Table 2.1) overnight at 4°C with
continuous rocking. Following washing with TBS/Tw, the blots were incubated in
horseradish peroxidise-conjugated goat anti-mouse or goat anti-rabbit IgG
antibody (Table 2.2) at room temperature for 1 hour. After extensive washing of the
membranes with TBS/Tw, bands corresponding to the BenchMark Prestained
Protein Ladder (Invitrogen) were visualised with an enhanced chemiluminescence
detection kit (Chemiluminescent Peroxidase Substrate, Sigma-Aldrich, St Louis,
MO, USA or Enhanced Chemiluminescence, GE Healthcare) according to the
manufacturer’s instructions. Antibody against β-actin (Table 2.1) was used as a
loading control. Membranes were exposed to Hyperfilm (Amersham Biosciences)
and developed on Agfa 1000 film processor (Raynostix, Louisville, KY).
2.4 Functional Assays
2.4.1 Cell Proliferation WST-1 Assay
Cell proliferation was evaluated by a colorimetric assay, WST-1 (Roche, Castle
Hill, Australia). This assay assesses the cellular viability by measuring the
metabolic conversion of a water-soluble tetrazolium salt, WST-1, into formazan.
Due to the enzymatic activity of mitochondrial dehydrogenases in viable cells, a
soluble dark red product is formed. The amount of formazan formed is directly
proportional to the number of viable cells in the culture and can be quantitated
using a spectrophotometer. This method has been employed in many published
reports (Humar et al., 2002, Nasreen et al., 2006, Khodayari et al., 2011).

69
Cell proliferation assay was conducted using the optimal cell density, 3 x 103
cells/well for both human and murine MM cells. This optimal cell density was
established in our pilot studies. The cells were seeded in a 96-well flat bottom cell
culture plate. After overnight serum-starvation, cells were then stimulated with 10%
FBS media (as a positive control) and with human (R & D Systems, Minneapolis,
MN) or murine (PeproTech Inc., Rocky Hill, NJ) recombinant FGF-9 at 1 – 100
ng/mL in 100 µL with 0% - 0.4% FBS for 48 hours. 0% - 0.4% FBS media was
used as a negative control. Cells were then incubated with WST-1 (1:10) for 0.5 - 1
hour at 37°C as per manufacturer’s instructions before the plate was read by a
scanning multiwell spectrophotometer as above.
2.4.2 Trypan Blue Cell Exclusion Assay
To validate the WST-1 assay above in Section 2.4.1, trypan blue cell exclusion
assay was conducted. Briefly, 1 x 104 human MM cells were seeded into a 12-well
plate. After overnight serum-starvation, cells were then stimulated with 10% FBS
media as a positive control and with human recombinant FGF-9 at 20 ng/mL in a
final volume of 1000 µL in 1% FBS media. 1% FBS media was used as a negative
control. Cells were washed with PBS, trypsinised and counted using the trypan
blue cell exclusion assay method on a hemocytometer at Day 1, 3, 6, 10 and 14.
The media was changed every 3 days.
2.4.3 Matrigel – Invasion Assay
Cell invasion was measured using 24-well Matrigel-invasion chamber plates
(Sigma-Aldrich, St Louis, MO, USA). 1 x 104 human MM cells were seeded in the
upper compartment of the Matrigel-coated inserts in a final volume of 500 µL in
serum-free media. Serum-free media alone (negative control) or 100 ng/mL FGF-9

70
was added to the companion plates. After 16 and 24 hours, non-invasive cells on
the upper surface of the insert were removed with a cotton applicator. The
Matrigel-coated insert membranes were stained with Diff-Quick solution (Andwin
Scientific) and viewed under a microscope. Invading cells were counted under a
10X magnification.
2.4.4 Scratch Assay
1.5 x 105 human and murine MM cells were plated in 6-well plates. Once the cells
are 90% confluent and following overnight serum-starvation, a 1 mL pipette tip was
used to scratch the surface of the well in the middle of the confluent cell
monolayer. Cells were then stimulated with human or murine recombinant FGF-9
at 100 ng/mL in 3 mLs of serum-free media for 0, 6, 8, 24, 48 and 72 hours. Cell
migration was monitored and images were taken using a camera attached to a
microscope. Cell migration was assessed by measuring the width of the gap
between the cells using a ruler and calculated as below:
Percentage increase of cell migration over 0 hour
= Width of gap at n hour – Width of gap at 0 hour
Width of gap at 0 hour
2.4.5 Cytokine and Chemokine Measurements
5 x 104 human and murine MM cell lines were plated in 24-well plates. Following
overnight serum-starvation, cells were then stimulated with human or murine
recombinant FGF-9 at 1 – 1000 ng/mL in a final volume of 500 µL in serum-free
media for 24 hours. In another set of experiments, 2.5 x 104 cells were stimulated
with 100 ng/mL FGF-9, resuspended in serum-free media, for 0, 2, 8, 24, 48 and
72 hours. Supernatants were then harvested and stored in -80°C. The samples
were assayed using commercially available ELISA kits for IL-8 (BenderMed
X 100 %

71
Systems), MIP-2 and VEGF (both from R & D Systems, Minneapolis, MN), IL-10,
MCP-1, TNF-α and IFN-γ (all from eBioscience Inc., San Diego, CA) according to
manufacturer’s instructions.
2.5 Molecular Characterisation of Cells
2.5.1 RNA Isolation
6 x 105 cells per well were plated in 6-well plates. After overnight serum-starvation,
RNA from cells was extracted using RNeasy Mini Kit (Qiagen, Hilden, Germany)
according to the manufacturer’s instructions. RNA was stored at -80°C until
required.
2.5.2 Determination of RNA Concentration
RNA quality was calculated by taking the ratio of the absorbance values at 260 nm
and 280 nm as determined using NanoDrop 2000 (Thermo Scientific). A ratio of
1.8 – 2 is considered satisfactory.
2.5.3 Reverse Transcription and cDNA Synthesis
Up to 5 µg of total RNA was reverse transcribed to complimentary DNA with RT2
First Strand Kit (SABiosciences) according to the manufacturer’s instructions,
except that the final step of adding 91 µL of nuclease-free water was omitted in
order to keep the total volume of cDNA at 20 µL. The sample was kept on ice for
the next step or stored at -20°C until required.

72
2.5.4 Real-time Polymerase Chain Reaction
The primers for FGF-9 and the housekeeping gene, glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) are listed in Table 2.3. FGF-9 and GAPDH mRNA levels
in untreated and treated cells were determined by real-time polymerase chain
reaction using RT2 qPCR Primer Assay protocol from SABiosciences on Applied
Biosystems StepOne Plus™ real-time PCR (Applied Biosystems, Scoresby,
Australia) using the following protocol:
Cycle 1 (Holding stage): 95°C, 10 minutes
Cycle 2 (Cycling stage): Step 1: 95°C, 15 seconds,
Step 2: 60°C, 1 minute
Cycle 3 (Melt curve stage): Step 1: 95°C, 15 seconds
Step 2: 60°C, 1 minute
Step 3: An increment of 0.3°C to 95°C, 15 seconds
The FGF-9 mRNA levels were normalised to GAPDH RNA.
Table 2.3: Primers for real-time PCR
Gene Name
Supplier Catalogue number
Amplicon Size
Reference Position
Mouse FGF-9
SABioscience PPM02979E-200 87 bp 1398
Mouse GAPDH
SABioscience PPM02946E-200 140 bp 309
2.6 Sample Processing and Storage
Pleural fluids or plasma samples collected from patients were spun at 2000 r.p.m.
for 10 minutes and the supernatants were stored in -80°C freezer until assayed.
40 cycles

73
Pleural fluids and plasma samples were collected from tertiary pleural referral
centers: the Oxford Pleural Unit, Oxford, UK, as previously described (Davies et
al., 2009), the Pleural Disease Unit, Sir Charles Gairdner Hospital and from the
National Centre for Asbestos Related Diseases (NCARD) biobank, Perth, Western
Australia.
The ethics committees of Sir Charles Gairdner Hospital and Hollywood Hospital
(Australia) and the Mid- and South-Buckinghamshire and Central Oxford (UK)
Research Ethic Committees approved the collection of the samples and all
participants provided written consent.
2.7 Mutational Sequencing of Cells and Tissues
2.7.1 DNA Isolation
DNA was isolated from up to a maximum of 5 x 106 cultured cell lines using
DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following the
manufacturer’s protocol.
The extraction of DNA from tumour samples was also performed similarly using
the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following the
manufacturer’s protocol with the incubation of the cut pieces of biopsy samples
with Proteinase K done at 56 °C overnight on a shaker platform until the tissue was
completely lysed.

74
2.7.2 Determination of DNA Concentration
DNA concentration and quality was determined using NanoDrop 2000 (Thermo
Scientific) similar to as described in Section 2.5.2. DNA quality was calculated by
taking the ratio of the absorbances at 260 nm and 280 nm. A good quality and
uncontaminated DNA has a ratio of between 1.8 and 2. The concentration of DNA
in ng/µL is obtained by taking the absorbance value at 260 nm multiply by 50 (the
extinction coefficient of DNA).
2.7.3 Polymerase Chain Reaction
The primers for all the exons of FGF-9, FGFR2 and FGFR3 and the optimised
conditions for polymerase chain reaction (PCR) are summarised in Table 2.4. PCR
was performed using HotStar Taq DNA Polymerase (Qiagen, Hilden, Germany) in
a final volume of 25 µL using the following cycle:
Cycle 1: 95°C, 15 minutes
Cycle 2: Step 1: 95°C, 30 seconds
Step 2: T1°C, 30 seconds 35 cycles
Step 3: 72°C, 30 seconds
Cycle 3: 72°C, 5 minutes
Cycle 4: 4 – 10°C, eternity

75
Table 2.4: Primers used for PCR for the mutational sequencing analysis in human samples
Gene Primer name
Sequence 5’ to 3’ [MgCl2] (mM)
Presence of Q solution
T1 °C Size (bp)
Reference
Forward CCTGGGTTGACACCATCATT Exon 1
Reverse tttcaacatgtcatctcatggac
1.5 No 57.9 382
Forward gcttagtgtcctctccaaaacc Exon 2
Reverse acagactcagttgcatttctgg
1.5 Yes 62.3 300
Forward cctatcaatccatcccctaggta
FGF-9
Exon 3
Reverse CCGCGTGAAACCTTTATAGTG
1.5 Yes 62.3 388
(Abdel-Rahman et al., 2008)
Forward TCCCTGACTCGCCAATCTCTTTC Exon 2
Reverse TGCCCCCAGACAAATCCCAAAAC
2.5 No 62.3 341
Forward CACTGACCTTTGTTGGACGTTC Exon 3
Reverse GAGAAGAGAGAGCATAGTGCTGG
2.5 No 62.3 380
Forward TGGAGAAGGTCTCAGTTGTAGAT Exon 4
Reverse AGACAGGTGACAGGCAGAACT
2.5 Yes 62.3 232
Forward CAAAGCGAAATGATCTTACCTG Exon 5
Reverse AGAAATGTGATGTTCTGAAAGC
2.5 No 62.3 291
Forward GCTAGGATTGTTAAATAACCGCC
FGFR2
Exon 6
Reverse AAACGAGTCAAGCAAGAATGGG
1.5 No 57.9 226
(Kan et al., 2002)

76
Forward TGAGTTTGCCTCTCCTCGTGTG Exon 7 (5’)
Reverse CCTTCTACAGTTGCCCTGTTGG
2.5 Yes 62.3 390
Forward GATGTGCTGTAGCAGACCTTTGG Exon 7 (3’)
Reverse ATCATCACAGGCAAAACCTGGG
2.5 Yes 62.3 360
Forward GGTCTCTCATTCTCCCATCCC Exon 8 (IIIa)
Reverse CCAACAGGAAATCAAAGAACC
1.5 No 54.8 325
Forward AATGCTAAGACCTTCCTGGTTGG Exon 9 (IIIb)
Reverse CAGTCTCCCAAAGCACCAAGTC
1.5 No 62.3 284
Forward CCTCCACAATCATTCCTGTGTC Exon 10 (IIIc)
Reverse ATAGCAGTCAACCAAGAAAAGGG
1.5 No 62.3 257
Forward TGCGTCAGTCTGGTGTGCTAAC Exon 11
Reverse AGGACAAGATCCACAAGCTGGC
2.5 Yes 62.3 341
Forward TGACTTCCAGCCTTCTCAGATG Exon 12
Reverse AGTCTCCATCCTGGGACATGG
1.5 No 62.3 252
Forward CCCCATCACCAGATGCTATGTG Exon 13
Reverse TTGATAAGACTCTCCACCCAGCC
1.5 No 60.4 221
Forward TAGCTGCCCATGAGTTAGAGG Exon 14
Reverse ATCTGGAAGCCCAGCCATTTC
1.5 No 62.3 250
Exon 15 Forward TGTTTTGCTGAATTGCCCAAG 1.5 No 54.8 294

77
Reverse TCCACCCAGCCAAGTAGAATG
Forward CTGGCGGTGTTTTGAAATTAG Exon 16
Reverse CCTTTCTTCCTGGAACATTCTG
2.5 No 62.3 242
Forward AGCCCTATTGAGCCTGCTAAG Exon 17
Reverse CCAGGAAAAAGCCAGAGAAAAG
2.5 Yes 62.3 177
Forward GGTTTTGGCAACGTGGATGGG Exon 18
Reverse GGTATTACTGGTGTGGCAAGTCC
1.5 No 60.4 250
Forward ACACCACGTCCCCATATTGCC Exon 19
Reverse CTCACAAGACAACCAAGGACAAG
1.5 No 54.8 243
Forward TCTGCCAAAATTGTTGTTTCTAGT Exon 20
Reverse GGTCTGGAACTCCTGACCTCA
2.5 Yes 54.8 208
Forward TCCCACGTCCAATACCCACATC Exon 21
Reverse TACTGTTCGAGAGGTTGGCTGAG
1.5 No 62.3 196
Forward CGTCCAATACCCACATCTCAAG Exon 22
Reverse TTCCCAGTGCTGTCCTGTTTGG
1.5 No 54.8 363
Forward CGAGGGGGCGTGCCCTGCGCC Exon 1
Reverse AGCACCGTTGGACCCCTCCG
1.5 Yes 62.3 339
Forward AGGGGTCGGGACGCAGGAG
FGFR3
Exon 2
Reverse CCCAACGCCTCTGCCCGCAC
1.5 Yes 62.3 350
(Wüchner et al., 1997)

78
Forward GTCTGTAAACGGTGCCGG Exon 3
Reverse ACCAGAGAGACCCCCAGC
1.5 No 60.4 425
Forward ATCTGGGAGGGGCACCTGGG Exon 4
Reverse GTCCCTCAGCTGCCTGTGAAG
1.5 Yes 62.3 222
Forward GTTCAGAGGGGCCTCTGCTC Exon 5
Reverse AGTGAGCGGAGGCAGCAACC
1.5 Yes 54.8 290
Forward CAGGCGCGGTGGTTGCTGCC Exon 6
Reverse GCACGTCCAGCGTGTACGTCTG
1.5 No 60.4 177
Forward CGGCAGTGGCGGTGGTGGTG Exon 7
Reverse CCAGCCCAGGAGCCCCAGCG
1.5 Yes 54.8 299
Forward TCTCCCACATCCTGCGTC Exon 8
Reverse GGGCCTTGGAGCTGGAGCTC
1.5 Yes 54.8 277
Forward AGGGCGGTGCTGGCGCTCGC Exon 9
Reverse AGACAGTGCGGAGCAGCAGC
1.5 Yes 54.8 228
Forward CAGGCCAGGCCTCAACGCCC Exon 10
Reverse AGGCCTGGCGGGCAGGCAGC
2.5 Yes 60.4 271
Forward CTGTACCTCCACGCCCTGTCGC Exon 11
Reverse CTGTTTCACCCCCACCACC
1.5 Yes 57.9 264
Exon 12 Forward GAGTGGGCGAGTTTGCACATCT 1.5 No 60.4 211

79
Reverse GCCCCCAGCCCTGCTCTGCAC
Forward GTGCAGAGCAGGGCTGGGGGC Exon 13
Reverse GCTCCTCAGACGGGCTGCCAG
1.5 Yes 62.3 240
Forward CTGGCAGCCCGTCTGAGGAGC Exon 14
Reverse CTGCTCCCAGCATCTCAGGGCA
1.5 Yes 62.3 286
Forward GGTGGAGAGGCTTCAGCCCT Exon 15
Reverse GCCAGGCGTCCTACTGGCATGA
1.5 Yes 62.3 217
Forward TCATGCCAGTAGGACGCCTGGC Exon 16
Reverse GGTCCTGGCTCTGCCCAGTTC
1.5 No 54.8 184
Forward CAGCGCAGCCCTGGCCTATTC Exon 17
Reverse CCTGAAGGGCTGCCAGTCCCT
1.5 Yes 62.3 314
Forward GAAGCGGCGGGGCTCACTCCT Exon 18
Reverse ATAGGCGGGTGGCACCAGGC
1.5 Yes 57.9 180
Forward GCGAAGAGGGGCTCGGTGGCAC Exon 19
Reverse CACCAGCAGCAGGGTGGGCTGCTAG
1.5 No 57.9 254

80
2.7.4 Agarose Gel Electrophoresis
2% agarose gels were prepared by heating the agarose solution in a microwave
until the solution turns clear. Ethidium bromide was added to a final concentration
of 0.5 µg/mL. The gel was poured into a casting tray and left to polymerise at room
temperature before being transferred to an electrophoresis tank (BioRad,
Hercules, CA) containing 1X TAE Running Buffer (Appendix I). Samples and a 100
bp DNA ladder (Promega, Madison, WI, USA) were loaded and electrophoresed at
100V until the bands are completely separated. The gel was imaged on a Typhoon
scanner.
2.7.5 Unpurified PD+ Capillary Sequencing
Once the PCR reaction was completed and the samples are electrophoresed in
agarose gel as in Section 2.7.4 to verify the correct products, the DNA samples
were sent to Australian Genome Research Facility (AGRF) for further processing
using the unpurified PD+ capillary sequencing method.
2.7.6 DNA Sequencing Analysis
The results obtained were analysed and compared by alignment using AlignX from
the Vector NTI Advance 11 software package (Invitrogen, Grand Island, NY, USA).
2.8 Molecular Knockdown In Vitro
2.8.1 Short Hairpin RNA Knockdown
AB1 was seeded at a concentration of 1 x 105 cells/well in a 6 well plate for 24
hours and then washed with PBS and incubated with serum-free medium for

81
another 24 hours before transfection. The cells were then left at room temperature
for 20 minutes and the serum-free media was replaced with fresh growth media.
Transfection with the FGF-9 plasmid was carried out using jetPRIMETM (Polyplus-
Transfection) Reagent and jetPRIMETM Buffer with N/P = 8 ratio, followed by the
formula determining the ionic balance within the complexes, where N refers to the
number of nitrogen residues in the jetprime (N/P>3 required) per P (phosphate) of
DNA.
µl of jetPRIME™ to be used
= (µg of oligonucleotide x 3) x N/P ratio c
jetPRIME™ concentration in nitrogen residues x 7.5 mM
Three different shRNAs (sh273, sh496, sh588) and a scrambled vector
(pSUPER.retro.puro) were used to transfect the cells. Each plasmid was added to
a different well. From the formula above, 3.2 µL of jetPRIME was diluted to a final
volume of 100 µL jetPRIMETM Buffer for each shRNA and scrambled vector. 1 µg
of each shRNA and the scrambled vector was added separately into another tube
containing 100 µL of jetPRIMETM Buffer and mixed. Then, the diluted jetPRIMETM
was combined with the diluted plasmid and gently mixed to a final volume of 200
µL before they were added slowly to the cells in a drop-wise fashion. Cells cultured
without any plasmids acted as negative controls. The plates were then gently
agitated in the incubator for another 48 hours. The media was then removed, the
cells were washed with PBS and new medium was added together with 1 µg/mL of
puromycin-dihydrochloride (Applichem, Darmstadt, Germany). This selection
process was repeated every 24 hours. At a time when all of the cells in the

82
negative control wells have died, colonies were picked from the treated wells and
placed in new petri dishes and grown in media with the antibiotic. ELISA and RT-
PCR were performed to select clones with successful knockdown of FGF-9.
.
2.9 In Vivo Inoculation of Short Hairpin Knockdown Cells
2.9.1 Heterotopic Model
FGF-9 shRNA knocked down MM cell clones were tested in vivo. A single injection
of 5x105 cells from selected shRNA targeting FGF-9 (sh588), scrambled vector or
parent controls was administered to the subcutaneous right flank of at least 10
Balb/c mice. Tumour dimensions were measured daily. This was performed by one
blinded investigator. Animals were sacrificed after tumour size reached the
maximum limit of 10 mm x 10 mm. Tumour tissues were harvested for histology.
2.9.2 Orthotopic Model
For the orthotopic experiments, Balb/c mice received a single intraperitoneal
injection of 5x105 cells from selected shRNA targeting FGF-9 (sh588), scrambled
vector or parent controls. The mice were observed daily: their weight loss and
survival recorded. At day 13, the animals were sacrificed, their peritoneal cavities
opened and volume of fluids (if any) measured. Two blinded investigators graded
the tumour load i) by counting the number of tumour nodules; and ii) by removing
and weighing all macroscopic tumours.
FGF-9 expression in tumour tissue was determined by immunohistochemistry. The
histology of the tumour and neighbouring tissues were examined with an
experienced pathologist in MM (Dr Amanda Segal, Perth, WA) who was blinded to
the treatment groups.

83
2.9.3 Athymic Model
For the experiments conducted in athymic mice, a single injection of 5x105 cells
from selected shRNA targeting FGF-9 (sh588), scrambled vector or parent controls
was administered to the subcutaneous right flank of at least 5 nude mice. Tumour
dimensions were measured daily. This was performed by one blinded investigator.
Animals were sacrificed after tumour size reached the maximum limit of 10 mm x
10 mm.
2.10 Statistical analysis
Data are presented as mean ± standard error of the mean if normally distributed
and as median (range) if not. For parametric data, statistical comparisons between
groups were performed using either a Student’s or paired t-test. For multiple group
comparison, one-way analysis of variance (ANOVA) was used followed by a post-
hoc test if appropriate. For non-parametric data, comparison of groups were
performed using Mann-Whitney rank sum test and multiple of group comparisons
were performed using the Kruskal-Wallis ANOVA-on-Ranks. Student-Newman-
Keuls or Dunn’s test was used as the post-hoc test. p<0.05 was considered
significant. Statistics were performed using SigmaPlot version 11.0 (Systat
Software, San Jose, California, USA).

84
____________________________________________
THE EXPRESSION OF FIBROBLAST GROWTH
FACTOR-9 IN MALIGNANT MESOTHELIOMA
CHAPTER 3

85
3.0 THE EXPRESSION OF FIBROBLAST GROWTH FACTOR-9 IN
MALIGNANT MESOTHELIOMA
3.1 Introduction
MM is a fatal disease that has no effective treatment. As a result, there is an
urgent need to discover new molecules involved in MM disease pathobiology
where a potential to develop new therapeutic targets can be achieved. These
molecules may also have potential diagnostic and prognostic values.
The preliminary pilot experiments as discussed in Section 1.9 uncovered FGF-9
from 22000 genes in the global gene profiling data that was up-regulated in MM
but not in the metastatic adenocarcinoma and benign pleural diseases groups. The
gene over-expression was validated in another second cohort of patients. This
chapter explores the hypothesis that FGF-9 is over-expressed in MM tissues
compared to other non-MM cancers and benign pleural conditions. Human pleural
fluids from a range of diseases as well as human and murine MM tissues and cells
were used to test this hypothesis.
3.2 Results
3.2.1 The Detection of FGF-9 in MM Samples
The preliminary results showed that FGF-9 is over-expressed at the RNA level in
human MM thoracoscopic pleural biopsies using Affymetrix microarray and RT-
PCR. Prior to identifying if FGF-9 is over-expressed in human and murine MM cells
and tissues as well as in human pleural fluids, it is important to examine if FGF-9
can be detectable using other methods. As a result, ELISA to quantitate FGF-9 in

86
the cells and histology examinations using immunofluorescence and
immunohistochemistry were used to detect the presence of FGF-9.
The murine FGF-9 ELISA purchased was separate antibody pairs. Optimisation
was performed for the concentrations of the capture and detection antibodies (from
0.25, 0.5, 1 and 2 µg/mL) and for the best wavelength for reading the plates (at
405 nm and 450 nm). The condition that produces a 4-parameter logistic curve (1
µg/mL for both capture and detection antibodies and a wavelength at 450 nm) was
selected as the optimised condition for murine FGF-9 ELISA (Figures 3.1A and B).

87
Figure 3.1: Optimisation of murine FGF-9 ELISA.
The concentrations of capture and detection antibodies were varied accordingly from 0.25, 0.5, 1 and 2 µg/mL and absorbances were
read on the plate reader at (A) 405 nm and (B) 450 nm. The pair concentrations of capture and detection antibodies at 1 µg/mL read at
450 nm was selected as the optimised conditions for murine FGF-9 ELISA that produces a 4-parameter logistic curve (in red box).

88
3.1A
Capture
Detection
0.5 µg/mL 1 µg/mL 2 µg/mL
0.25 µg/mL
1000 2000 3000 4000 5000
-0.02
-0.01
0.00
0.01
0.02
0.03
[FGF-9 standards] (pg/mL)
Ab
sorb
ance
1000 2000 3000 4000 5000
-0.04
-0.03
-0.02
-0.01
0.00
[FGF-9 standards] (pg/mL)
Ab
sorb
ance
0 1000 2000 3000 4000 5000
0.00
0.01
0.02
0.03
0.04
0.05
[FGF-9 standards] (pg/mL)
Abso
rban
ce
0.5 µg/mL
1000 2000 3000 4000 5000
-0.04
-0.02
0.00
0.02
0.04
0.06
[FGF-9 standards] (pg/mL)
Ab
sorb
ance
1000 2000 3000 4000 5000
-0.07
-0.05
-0.03
-0.01
0.01
[FGF-9 standards] (pg/mL)
Ab
sorb
ance
0 1000 2000 3000 4000 5000
0.0
0.2
0.4
0.6
0.8
[FGF-9 standards] (pg/mL)
Ab
sorb
ance
1 µg/mL
0 1000 2000 3000 4000 5000
0.0
0.1
0.2
0.3
0.4
0.5
[FGF-9 standards] (pg/mL)
Ab
sorb
ance
0 1000 2000 3000 4000 5000
0.0
0.1
0.2
0.3
0.4
[FGF-9 standards] (pg/mL)
Ab
sorb
ance
0 1000 2000 3000 4000 5000
0.0
0.2
0.4
0.6
0.8
[FGF-9 standards] (pg/mL)
Ab
sorb
ance

89
3.1B
Capture
Detection
0.5 µg/mL 1 µg/mL 2 µg/mL
0.25 µg/mL
1000 2000 3000 4000 5000
-0.050
-0.025
0.000
0.025
0.050
0.075
0.100
[FGF-9 standards] (pg/mL)
Ab
sorb
ance
0 1000 2000 3000 4000 5000
-0.13
-0.11
-0.09
-0.07
-0.05
-0.03
-0.01
0.01
[FGF-9 standards] (pg/mL)
Abso
rban
ce
0 1000 2000 3000 4000 5000
0.000
0.025
0.050
0.075
0.100
0.125
0.150
0.175
[FGF-9 standards] (pg/mL)
Ab
sorb
ance
0.5 µg/mL
1000 2000 3000 4000 5000
-0.15
-0.10
-0.05
-0.00
0.05
0.10
0.15
0.20
[FGF-9 standards] (pg/mL)
Abso
rban
ce
1000 2000 3000 4000 5000
-0.25
-0.20
-0.15
-0.10
-0.05
-0.00
0.05
[FGF-9 standards] (pg/mL)
Ab
sorb
ance
0 1000 2000 3000 4000 5000
0.0
0.1
0.2
0.3
0.4
0.5
0.6
[FGF-9 standards] (pg/mL)
Ab
sorb
ance
1 µg/mL
0 1000 2000 3000 4000 5000
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
[FGF-9 standards] (pg/mL)
Abso
rban
ce
0 1000 2000 3000 4000 5000
0.0
0.2
0.4
0.6
0.8
1.0
1.2
[FGF-9 standards] (pg/mL)
Ab
sorb
ance
0 1000 2000 3000 4000 5000
0.0
0.5
1.0
1.5
2.0
2.5
3.0
[FGF-9 standards] (pg/mL)
Ab
sorb
ance

90
Using the optimised FGF-9 ELISA (1 µg/mL for both capture and detection
antibodies), murine cell lysates and culture media supernatants were quantitated
for murine FGF-9. Murine FGF-9 can be detected in both the cell lysates and the
supernatants in all the six murine MM cell lines tested (Figure 3.2). The FGF-9
concentration was significantly higher in cell lysates than in the culture media
supernatants, in keeping with the reported observations in other cell types (see
Section 1.5.5). This confirms the presence of FGF-9 in murine MM cells.
Figure 3.2: The presence of FGF-9 in murine MM cells detected using ELISA.
Cells from different murine MM cell lines were lysed with breaking buffer. Cell
lysates and supernatants were tested and quantitated for murine FGF-9 using
ELISA. Values are expressed as mean ± SEM of five replicates from a
representative experiment conducted twice.
AB1 AB2 AB22 AC29 AE5 AE17
0
10
20
30
40
50
Lysates
Supernatants
Cells
Fold
in
crea
se o
f F
GF
-9 l
evel
s in
cel
ls
* *
*
** **
**
n = 5 per group
*p=0.008, **p<0.001 compared to supernatants of the same cell line, t-test

91
The presence and localisation of FGF-9 in murine and human MM cells was also
confirmed using immunofluorescence using a different antibody from that of the
ELISA (Figure 3.3). In all eight human cell lines (JU77, LO68, NO36, ONE58, STY,
CRL-2081, CRL-5820 and CRL-5915) and three murine cell lines (AB1, AB2 and
AE5) tested, immunofluorescence confirmed the presence of FGF-9 in the nuclei
and cytoplasm of the cells. A stronger fluorescent intensity for FGF-9 in the nuclei
of the cells was detected however the significance of its localisation in the nucleus
is unknown. This is the first evidence of the presence of FGF-9 in human and
murine MM cell lines.

92
Figure 3.3: Expression and distribution of FGF-9 in the human JU77 and murine AB2 MM cell lines.
(A) The expression of FGF-9 in a human MM cell line, JU77, as depicted by the green fluorescence intensity from the FITC-conjugated
antibody. (B) The nuclei of the cells as shown by the blue intensity of DAPI staining. (C) A merge of images A and B illustrates the
distribution of FGF-9 in both the nuclei and cytoplasm of the cells. (D-F) The expression of FGF-9 detected in a murine MM cell line,
AB2. These images are a representative image of all of the eight human and three murine MM cell lines tested. In the negative isotype
control (not shown), the only fluorescence observed is the DAPI staining.
FGF-9-FITC DAPI Merge
JU77
AB2
3.3A
3.3D 3.3F
3.3B 3.3C
3.3E

93
In addition, the presence of FGF-9 was detected by immunohistochemistry on MM
tumour tissues from human pleural biopsies and mice implanted with MM. A
greater brown intensity was present in the tumour tissues but lesser in the stroma,
indicating the abundance of FGF-9 in MM tumour tissues where FGF-9 is the key
protein. The presence of FGF-9 in the surrounding stroma demonstrates that
normal tissues also secrete FGF-9 that may be important in homeostasis of the
tissue.
Figure 3.4: Expression and distribution of FGF-9 in the MM human tissue
biopsy and murine MM tumour.
The expression of FGF-9 in MM tissues was observed by immunohistochemistry
using FGF-9 antibody. The brown staining intensity in (A) human MM tissue biopsy
and (C) murine MM tissue observed under a 20X magnification depicts the
presence of FGF-9 protein in tissues. Images B and D show the negative IgG
isotype control for the FGF-9 staining.
3.4A 3.4B
3.4C 3.4D

94
3.2.2 The Expression of FGF-9 in MM Cells and Tissues
Having established the presence of FGF-9 in MM cells and tissues, FGF-9
expression was further characterised in cells obtained from pleural fluids and
tissues from patients who were diagnosed with metastatic adenocarcinoma (n=29)
and MM (n=121). Samples obtained from patients who had benign diseases
(n=35) served as controls. The specificity of the FGF-9 antibody to FGF-9 is
demonstrated from the negative staining of FGF-9 in the IgG isotype control
(Figures 3.5B and D). Figure 3.5E illustrates that FGF-9 is highly expressed in
metastatic adenocarcinoma and MM, with approximately 76% and 65%
respectively being in the higher intensity (score 2-3) category. This is not the case
for benign pleural diseases whereby only in 37% of patients was FGF-9 highly
expressed. Although the results obtained from this immunohistochemistry study is
only semi-quantitative, the proportions of observations in the different patient
groups and the intensity of FGF-9 staining showed significant differences among
the groups (χ2 = 82.8, df = 2, p<0.001) and confirms the abundance of FGF-9 in
malignant cells and tissues but not in the non-malignant samples.
Figure 3.5: Expression of FGF-9 in cells and tissues on tissue microarray.
Tissue microarray for (A) benign mesothelial cells from pleural fluid given a score
of 1 (low intensity) where as adenocarcinoma tissue (C) were given a score of 3
(high intensity). The scoring of staining intensity was performed by two blinded
investigators. (B & D) The negative isotype controls for the figures A and C
respectively illustrates the specificity of FGF-9 antibody. (E) The bar graph shows
the percentage of scoring intensities for the different diagnoses; benign diseases,
metastatic adenocarcinoma and MM.

95
3.5E
Benign diseases Metastatic adenocarcinoma MM
0
10
20
30
40
50
60
70
80
90
100
2-30-1
Scores
n = 35 n = 29 n = 121
Scores
Per
cen
tage
of
score
s
χ2 = 82.8, df = 2, p<0.001
3.5A
3.5D
3.5B
3.5C

96
3.2.3 The Expression of FGF-9 in Pleural Fluids
To measure and quantitate the levels of FGF-9 in patient fluid samples, it is
prudent to investigate if FGF-9 is stable after repeated freeze-thaw cycles. Figure
3.6 shows that FGF-9 is stable in pleural fluid samples even after seven repeated
freeze-thaw cycles. This indicates that FGF-9 could still be detected and the levels
have not changed from the time of the first pleural fluid collection to the seventh
time that it has been thawed out for experiments. This is the first study that has
demonstrated the stability of FGF-9 after repeated freeze-thaw cycles. However,
as this has only been tested in pleural fluids, it is unknown if FGF-9 is stable in
other bodily fluids.
Figure 3.6: The stability of FGF-9 in pleural fluids following freeze-thaw.
Each of the twelve pleural fluid samples (represented as each line) were aliquoted
into eight different microfuge tubes, labelled A to H (represented as each symbol).
Each following tube is subjected to an additional freeze-thaw cycle, such that tube
A was not thawed, tube B was subjected to one thawing stage, C underwent two
stages of thawing process and tube H had seven repeated freeze-thaw cycles. The
FGF-9 levels were measured using human FGF-9 ELISA. FGF-9 is stable after
seven repeated freeze-thaw cycles in the pleural fluid samples tested.
0 1 2 3 4 5 6 7
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
Freeze-thaw cycles
[FG
F-9
] (p
g/m
L)

97
To compare the FGF-9 levels in pleural fluids, two separate patient cohorts were
tested. 283 prospectively collected, undiagnosed pleural fluids were obtained from
the Oxford Pleural Unit biobank cohort. The median FGF-9 levels in 43 pleural
fluids from MM patients (median 1.66 ng/mL; interquartile range 0.23 - 3.66 ng/mL)
were 7.2-fold higher than 137 patients with metastatic carcinomas (median 0.23
ng/mL, interquartile range 0.08 - 1.78 ng/mL, p<0.05 vs MM) and 4.6-fold higher
than 103 patients with benign pleural diseases (median 0.35 ng/mL, interquartile
range 0.09 - 1.82 ng/mL, p<0.05 vs MM) (Figure 3.7A).
This result was extended to a larger cohort of 639 pleural fluids from Perth,
Western Australia where a strikingly similar pattern was observed (Figure 3.7B). In
this cohort, the median levels of FGF-9 measured blindly in 205 pleural fluids
obtained from MM patients (median 1.58 ng/mL, interquartile range 0.56 – 0.31
ng/mL) were 12.5-fold higher than that of 210 patients with metastatic carcinomas
(median 0.13 ng/mL, interquartile range 0.0 – 0.39 ng/mL, p<0.05 vs MM) and
11.3-fold higher than 224 patients with benign pleural diseases (median 0.14
ng/mL, interquartile range 0.0 – 0.39 ng/mL, p<0.05 vs MM). The data confirms the
over-expression of FGF-9 protein in MM pleural fluid samples compared to
adenocarcinoma and benign pleural diseases and is consistent with the FGF-9
mRNA over-expression reported in the global gene profiling data. The results show
significant promise of FGF-9 as a biomarker that can discern MM from other
cancers and benign pleural diseases effectively.
The patient demographics from the Oxford cohort have been previously published
(Davies et al., 2009, Rahman et al., 2008) while the patient demographics from the
Perth cohort are tabulated in Figure 3.7C. The majority of pleural fluids obtained
from MM patients were of epitheloid subtype (n=80) while there were only 16 and

98
19 patients diagnosed with biphasic and sarcomatoid subtypes respectively. In the
remaining 90 pleural fluids, the diagnosis of MM was made on cytology and not
clarified for its histologies subgroups.
Figure 3.7: Pleural fluids FGF-9 levels in the Oxford and Perth cohorts.
(A) Pleural fluid FGF-9 levels in the Oxford cohort in 43 patients with MM were 7.2-
fold higher than in 137 patients with metastatic carcinomas and 4.6-fold higher
than 103 patients with benign pleural diseases (*p<0.05). (B) Pleural fluid FGF-9 in
the Perth cohort in 205 patients with MM were 12.5-fold higher than 210 patients
with metastatic carcinomas and 11.3-fold higher than 224 patients with benign
pleural diseases (*p<0.05). (C) Pleural fluids obtained from the Perth cohort is
categorised into MM, metastatic carcinomas and benign pleural diseases, which
are further subdivided into smaller groups.

99
3.7A 3.7B
*p<0.05 ANOVA-on-rank, Dunn’s test
Pleural fluid FGF-9 levels in Oxford cohort
[FGF-9] (ng/mL)
0 5 10 15 20
Mesothelioman=43
Carcinomasn=137
Benignn=103
*
*
Pleural fluid FGF-9 levels in Perth cohort
[FGF-9] (ng/mL)
0 2 4 6 8
Mesothelioman=205
Carcinomasn=210
Benignn=224
*
*

100
3.7C
Patient characteristics n Age
(median) % females
Mesothelioma Epitheloid 80 68 18.6 Biphasic 16 67 15.8 Sarcomatoid 19 74.5 9.1 Unknown 90 71 16.2 Total 205
Carcinomas Lung 80 68 45.3 Breast 35 64 100.0 Others 95 71 43.4 Total 210
Benign Asbestos exposure 1 79 0 Cardiac diseases 45 77 44.4 Haemothorax 2 79.5 0 Hepatic causes 13 59 15.4 Idiopathic 58 71 39.7 Idiopathoc pleuropericarditis 4 58 0 Infection 71 65 35.2 Pancreatic diseases 1 60 100 Pneumothorax 3 71 33.3 Post-cardiac surgery 3 73 33.3 Post-operative 3 68 33.3 Renal causes 8 52.5 37.5 Rhematological causes 3 64 33.3 Secondary to abdominal causes 5 39 40 TB pleural effusion 4 67.5 25
Total 224

101
3.2.3.1 The Expression of FGF-9 in the Different MM Histological Subtypes
To elucidate the differential expression levels of FGF-9 in the different histological
subtypes, MM pleural fluids in the Perth cohort were further subdivided into the
three main histological subtypes; the epitheloid, biphasic and sarcomatoid. It was
found that the epitheloid subtype (n=80, median 2130.95 pg/mL, interquartile
range 743.03 - 3804.76 pg/mL) had the highest FGF-9 levels of all three subtypes,
with a median level of 8.6-fold higher than in the sarcomatoid subtype (n=19,
median 254.56 pg/mL, interquartile range 63.94 - 612.31 pg/mL, p<0.05). The
median level of FGF-9 in the biphasic subtype (n=16, median 1711.91 pg/mL,
interquartile range 880.74 - 2648.88 pg/mL) was 7.1-fold higher than in the
sarcomatoid subtype (p<0.05). There were 90 pleural fluid samples whose
subtypes could not be characterised (median 1877.64 pg/mL, interquartile range
714.73 - 3173.90 pg/mL) (Figure 3.8). This data shows that like mesothelin, FGF-9
can only be used to diagnose epitheloid MM but not sarcomatoid MM.

102
Figure 3.8: FGF-9 levels in different MM histology.
FGF-9 levels in the epitheloid subtype was 8.6-fold higher than in the sarcomatoid
subtypes where as FGF-9 levels in the biphasic subtype was 7.1-fold higher than
in the sarcomatoid patients (*p<0.05). FGF-9 expression is significantly lower in
the patients with the sarcomatoid subtype.
Epitheloid Biphasic Sarcomatoid Not specified
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
*
*
n = 80 n = 19n =16 n = 90
MM histology
FG
F-9
lev
els
(pg
/mL
)
*p<0.05 ANOVA-on rank, Dunn’s test

103
3.2.3.2 The Expression of FGF-9 in MM Pleural Fluids and Matched Plasma
The over-expression of FGF-9 protein in MM pleural fluids demonstrates that it
may be used as a diagnostic marker to differentiate MM from other cancers and
benign pleural diseases. However, to serve as an early diagnostic biomarker, it
would have to be detected and also up-regulated in the blood so that a simple
minimally-invasive blood test is suffice to screen and diagnose MM from the early
stage. So, paired pleural fluids and serum samples from 35 patients with MM were
selected randomly from a biobank collected from patients from the Pleural Disease
Unit, Sir Charles Gairdner Hospital to compare the relative abundance of FGF-9 in
the pleural cavity and the systemic circulation. Figure 3.9 shows that in 22 out of
25 samples that had detectable FGF-9 in the pleural fluids, the median FGF-9
levels were 6-fold higher in pleural fluids than in plasma samples. Only 3 MM
patients had higher FGF-9 levels in their plasma samples than in their
corresponding pleural fluids. There were 10 patients who had no detectable levels
of FGF-9 in both their pleural fluids and plasma samples. Only 5 out of 35 plasma
samples tested had a positive FGF-9 level reading implying that FGF-9 is
undetectable in the majority of the plasma samples and hence cannot be used as
an early diagnostic marker nor can it be used as a simple screening test to monitor
the disease as treatment commences. In addition, the higher FGF-9 levels
detected in the pleural fluids compared to the matched plasma samples highly
imply the pleural origin of FGF-9.
It is worth to note that the FGF-9 levels in the pleural fluids from the same patient
measured by ELISA separately for two assays; the freeze-thaw assay (Figure 3.6)
and matched pleural fluids-plasma assay (Figure 3.9); were similar, indicating that
the inter-variable assay is very small.

104
Figure 3.9: FGF-9 levels in matched pleural fluid and plasma of patients
diagnosed with MM.
FGF-9 levels were measured using ELISA in matched pleural fluids and plasma
samples from 35 MM patients. Data was plotted as a log ratio of FGF-9 levels in
pleural fluids to plasma as the y-axis, whereby a log ratio of greater than zero
indicates a higher level of FGF-9 in the pleural fluids than in the corresponding
plasma sample and a log ratio of less than zero indicates otherwise. A log ratio of
zero implies that the FGF-9 levels in the pleural fluids and plasma samples are
equal. FGF-9 is detected in abundance in 22 out of 25 pleural fluids than in plasma
samples. Only three patients had a higher level of FGF-9 in plasma instead of their
pleural fluids. Ten patients had no detectable levels of FGF-9 in both their pleural
fluids and plasma samples.
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
Log r
ati
o o
f F
GF
-9 l
evel
s in
ple
ural
flu
id t
o p
lasm
a
n = 35

105
3.2.3.3 The Correlation between FGF-9 and Mesothelin
To determine if FGF-9 in pleural fluids added diagnostic value to mesothelin,
pleural fluid FGF-9 levels from the Perth cohort were compared with the
mesothelin levels previously measured by Creaney et al. (Creaney et al., 2007).
Figure 3.10 illustrates only a weak correlation between FGF-9 and mesothelin (n =
537, rs = 0.511, 95% CI 0.128 to 0.289, p<0.0001). This implies that FGF-9 and
mesothelin are totally different proteins and that measuring FGF-9 and mesothelin
pleural fluid levels separately will give different magnitudes although they are both
up-regulated in MM.
Figure 3.10: The correlation between FGF-9 and mesothelin.
Each dot represents the FGF-9 levels (pg/mL) in each of the pleural fluid samples
that were correlated with the mesothelin levels (nM), previously measured by
Creaney et al. (Creaney et al., 2007). FGF-9 correlates very weakly with a
Spearman rs value of 0.511 (p<0.0001).
0.1 1 10 100 1000
100
1000
10000
Sample size 537Spearman rs 0.511
95% confidence interval 0.127 to 0.289
p value (two-tailed) p<0.0001
Mesothelin (nM)
FG
F-9
(p
g/m
L)

106
3.2.3.4 The Value of a Diagnostic Marker When Combining FGF-9 and Mesothelin
Since FGF-9 correlates weakly with mesothelin, the area under the receiver
operating characteristic curve (ROC) was measured for FGF-9 and compared with
that for mesothelin. A perfect diagnostic marker will have a ROC value of one.
Figure 3.11A shows that the ROC for FGF-9 to differentiate MM (n=205) from
benign pleural diseases (n=224) is 0.816, while the ROC value for mesothelin is
0.9078. To distinguish MM from other cancers (n=210), FGF-9 was comparable to
mesothelin with a ROC value of 0.8303 and 0.8438 respectively (Figure 3.11B).
Furthermore, FGF-9 was as good as mesothelin in teasing out MM versus a
combined of benign pleural diseases and other cancers with a ROC value of
0.8228 and 0.8775 respectively (Figure 3.11C). Interestingly, combining FGF-9
and mesothelin as a panel of biomarkers increases the diagnostic validity to
differentiate MM versus benign pleural diseases to 0.9175 (Figure 3.11A), MM
versus other cancers to 0.8848 and MM versus non-MM to 0.9002 (Figure 3.11C).
The results are summarised in Figure 3.11D. The data demonstrates the quality of
FGF-9 in combination with mesothelin as a panel of biomarkers to diagnose and
differentiate MM from non-MM.

107
Figure 3.11: The area under the ROC for FGF-9 and mesothelin to differentiate MM from non-MM.
The area under the ROC for FGF-9 and mesothelin to predict MM (n=205) versus (A) benign pleural diseases (n=224) were 0.816 and
0.9078 respectively. Combining the two molecules increases the ROC to 0.9175 (B) other cancers (n=210) were 0.8303 and 0.8438
respectively and combining the two increases the ROC to 0.8848 (C) non-MM were 0.8228 and 0.8775 respectively while combining
the two molecules increases the diagnostic validity to 0.9002 (D) The results are summarised in the table.
3.11A 3.11B
3.11C 3.11D
MM vs Benign Cancer Benign &
Cancer
FGF-9 0.816 0.8303 0.8228
Mesothelin 0.9078 0.8438 0.8775
FGF-9 + Mesothelin 0.9175 0.8848 0.9002
0.0
00.
250.
50
0.7
51.
00S
ens
itivi
ty
0.00 0.25 0.50 0.75 1.001-Specificity
0.0
00.
250.
50
0.7
51.
00S
ens
itivi
ty
0.00 0.25 0.50 0.75 1.001-Specificity
0.0
00.
250.
50
0.7
51.
00S
ens
itivi
ty
0.00 0.25 0.50 0.75 1.001-Specificity
FGF-9
Mesothelin
FGF-9 + Mesothelin
FGF-9
Mesothelin
FGF-9 + Mesothelin
FGF-9
Mesothelin
FGF-9 + Mesothelin

108
3.3 Discussion
This is the first study demonstrating the presence of FGF-9 in MM using three
different methods; ELISA, immunofluorescence and immunohistochemistry. The
detection of FGF-9 was confirmed in two different species, human and murine, and
include all three common histological subtypes of MM: the epitheloid (human CRL-
5820 (Nutt et al., 2010)), sarcomatoid (human JU77 (Delage B. et al., 2011)) and
biphasic subtypes (human CRL-2081 (Delage B. et al., 2011)).
FGF-9 protein is also significantly higher in both cohorts of MM pleural fluids
compared to the other groups totalling to more than 900 samples, further
strengthening our observations from the preliminary pilot experiments using pleural
tissue biopsies.
FGF-9 may aid in differentiating between MM from benign pleural diseases and
metastatic carcinomas when it is measured using ELISA on the pleural fluid
samples, as shown in two different cohorts. FGF-9 was significantly up-regulated
in the MM pleural fluids compared to the metastatic carcinomas and benign
groups. Although the peak concentration of FGF-9 in MM pleural fluids differs
between the cohorts, the overall pattern and fold changes between MM and the
control groups are similar.
Interestingly, FGF-9 correlates very weakly with mesothelin, the current best
biomarker for MM, but its area under the receiver operating curve is still
comparable to that of the mesothelin. FGF-9 may therefore be capturing different
information, separate from mesothelin. Combining both the markers increases the
diagnostic validity to differentiate MM from non-MM. The capability of detecting

109
FGF-9 in pleural fluids presents a minimally invasive method of diagnosing MM
from other groups. Furthermore, FGF-9 is stable in pleural fluids after repeated
freeze-thaw cycles implying that the pleural fluids can be stored for later
assessments or further experiments.
Whether FGF-9 increases with the advancing stages of MM is unknown. There are
some MM patients who have undetectable FGF-9 levels in their pleural fluids and
this may represent early stages of the disease. In future, this may be assessed by
measuring FGF-9 in pleural fluid samples during the disease course longitudinally
in patients with indwelling pleural catheters. The role of FGF-9 as a prognostic
mediator should be assessed as well as its role as a disease monitoring tool for
MM especially in evaluating response to treatment. Future studies should also
explore the correlation between FGF-9 levels and survival.
Like mesothelin (Campbell and Kindler, 2011), FGF-9 is raised predominantly in
epitheloid but not in the sarcomatoid subtypes of MM. This may pose as a
limitation for the diagnostic value for FGF-9 although only 10% of the MM cases
are of sarcomatoid subtypes (Suzuki, 2001). Nonetheless, a positive FGF-9 in
pleural fluid still has a strong positive predictive value for diagnosis of MM. Finding
a separate biomarker sensitive to sarcomatoid MM in the future may allow its
combination with FGF-9 to improve the diagnosis for MM.
The immunohistochemistry findings recorded a high percentage of positively-
stained cells in both MM and other cancers groups. Hence the use of this FGF-9
antibody is sensitive to confirm the presence of FGF-9 but not necessarily specific
enough to discern (by intensity of staining) between MM from other cancers.

110
The source of FGF-9 in pleural fluids has not been defined. The many cell types
present in the pleural cavity can all contribute to the overall FGF-9 accumulation.
However, elevated levels of FGF-9 were only observed in the MM pleural fluids but
not in the metastatic carcinomas and benign pleural fluids highly suggest that MM
cells are the key source of pleural fluid FGF-9. These observations also argue
against other infiltrating cells (for example the inflammatory cells) in the pleural
cavity as the main source of FGF-9 as these cells are present in relative
abundance in metastatic pleural cancers and benign pleuritis. Also, these
inflammatory cells such as macrophages (which were stained positive in
immunohistochemistry) are present in highest quantity in peripheral blood. Yet,
FGF-9 levels in plasma are very low, further arguing against these cells as
principal source of FGF-9. The finding that FGF-9 is significantly higher in pleural
fluid over corresponding plasma is consistent with the hypothesis that FGF-9 was
produced by pleural MM cells and that a fraction of it is absorbed systemically. As
currently FGF-9 cannot be detected in the plasma, it cannot be used as an early
diagnostic test nor can it be used as a minimally invasive method to monitor the
disease. Also, FGF-9 is unable to be used as a screening marker in the 5 – 10% of
MM cases that do not develop pleural fluids.
It is interesting that in MM cell culture, FGF-9 is not secreted into the cell
supernatants but FGF-9 is readily secreted into the pleural fluids in patients. This
implies additional mechanisms present in vivo that causes FGF-9 to be released
out from the cells into the pleural fluids. The exact value of this mechanism is
beyond the scope of this thesis. This result however does present a potential
regulatory step of FGF-9 release which may contribute to its total activity in tissues
or tumours.

111
How confident are we that we are detecting FGF-9 and not other FGFs? Based
from the datasheets provided by the manufacturing companies, the antibody for
FGF-9 used in immunohistochemistry and ELISA is specific to human, rat and
mouse FGF-9. The epitope of the human FGF-9 antibody purchased for
immunohistochemistry corresponds to the amino acids 1 – 208 representing the
full length of human FGF-9. FGF-9 has only a 30% sequence identity with all the
other members of the family (Hecht et al., 1995) and the datasheet provided stated
clearly that the antibody does not cross-react with the other FGFs (FGFs-1, 2, 4 –
7) and even the FGF-16 which is in the same subfamily as FGF-9. Besides, the
primers used in RT-PCR are specific to FGF-9. Information on the Refseq
accession number and reference position provided by the manufacturing company
(Qiagen) was checked using the NCBI database. Products obtained from real-time
experiments were electrophoresed through an agarose gel to confirm that the band
size corresponds to the size information provided in the datasheet. All of these
make us confident in knowing that we are detecting the correct molecule and that
FGF-9 is truly over-expressed in MM.
Colvin et al. have illustrated the presence of FGF-9 in the mesothelium even after
embryonic development (Colvin et al., 1999). Aberrant FGF-9 activity has been
implicated recently in brain tumours, prostate cancers and lung adenocarcinomas
(Todo et al., 1998, Chien-Kai et al., 2009, Giri et al., 1999). Since MM cells in the
pleura are exposed to high concentrations of FGF-9 in the pleural fluids, it is likely
that FGF-9 may play a role in the pathobiology of MM.
In the following chapters, we followed up the above observations to study if FGF-9
contributes to the pathogenesis of MM, using in vitro and in vivo models.

112
____________________________________________
THE BIOLOGICAL ROLE OF FIBROBLAST GROWTH
FACTOR-9 IN MALIGNANT MESOTHELIOMA
CHAPTER 4

113
4.0 THE BIOLOGICAL ROLE OF FIBROBLAST GROWTH FACTOR-9 IN
MALIGNANT MESOTHELIOMA
4.1 Introduction
Since FGF-9 expression is elevated in MM at the RNA and protein levels in five
different cohorts, this molecule may have important biological role in the MM
disease pathobiology. As FGF-9 is secreted by MM cells into the pleural fluids and
since MM cells in the pleura are surrounded with elevated levels of FGF-9 in the
pleural fluids, it is probable that FGF-9 elicits an effect on the MM cells. Hence, the
role of FGF-9 in MM warrants investigation.
This chapter explores the hypothesis that FGF-9 plays a vital role in MM
pathobiology including cell proliferation, invasion and metastasis. Human and
murine MM cells were used to test this hypothesis.
4.2 Results
4.2.1 MM Cells Proliferation In Vitro in Response to FGF-9 Stimulation
FGF-9 is known to promote growth in some cancers such as glioma (Todo et al.,
1998) and lung adenocarcinoma (Chien-Kai et al., 2009). Hence, the mitogenic
role of FGF-9 in MM was examined using a commercially available WST-1 assay.
An optimisation experiment was first performed to select the best cell density for
WST-1 assay. Human NO36 and murine AB1 cell lines were selected for this
optimisation study as they were available at that time and they adhere to plastic
the quickest. Figure 4.1 shows a relatively linear relationship between cell density
and absorbance value for both the human NO36 and murine AB1 cell lines, until
50 x 103 cells/well when the absorbance values increased dramatically and
reached a plateau. 3 x 103 cells/well was then selected as the sub-optimal cell

114
Human NO36
1 3 5 7 9 10 30 50 70 90 100
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
Cell density (x103 cells per well)
Ab
sorb
ance
(A
450nm
- A
650nm
)
Murine AB1
1 3 5 7 9 10 30 50 70 90 100
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
Cell density (x103 cells per well)
Ab
sorb
ance
(A
450nm
- A
650nm
)
density for WST-1 assay to allow for a dramatic increase in cell proliferation that is
still within the detection limit of the plate reader for this assay.
Figure 4.1: Optimisation of cell density for MM cell proliferation.
MM cells (A) human NO36 (B) murine AB1 were plated at a range from 1 to 100 x
103 cells/well in 96-well microtiter plates and incubated for at least 7 hours for cells
to adhere to plastic. Cell proliferation was assessed using WST-1 reagent as
described in the Materials and Methods section. Values are expressed as mean ±
SEM of six replicates from a representative experiment conducted twice.
4.1A 4.1B
n = 6 per group

115
The proliferative effects of FGF-9 on MM cells were then examined on three
human (NO36, ONE58 and STY) and three murine (AB22, AE5 and AE17) MM
cells. Figure 4.2 demonstrates that increasing concentrations of FGF-9 increases
MM cell proliferation in the human and murine MM cell lines, showing that FGF-9 is
an important mitogen factor for MM cells.
Figure 4.2: Dose-dependent MM cell proliferation.
MM cells, three human (A) NO36 (B) ONE58 (C) STY and three murine (D) AB22
(E) AE5 (F) AE17, were stimulated with human or murine recombinant FGF-9 in
increasing log concentrations for 48 hours and cell proliferation was assessed
using WST-1 reagent as described in the Materials and Methods section. Values
are expressed as mean ± SEM of nine replicates from a representative experiment
conducted four times.
4.2A
Human NO36
0 1 10 100 10% FBS
0.0
0.5
1.0
1.5
2.0
2.5
3.0
** ***
***
[FGF-9] (ng/mL)
Fo
ld i
ncr
ease
ov
er s
erum
-fre
e co
ntr
ol
4.2B
Human ONE58
0 1 10 100 10% FBS
0.80
0.85
0.90
0.95
1.00
1.05
1.10
1.15
1.20
*
***
[FGF-9] (ng/mL)
Fo
ld i
ncr
ease
ov
er s
erum
-fre
e co
ntr
ol

116
4.2C
Human STY
0 1 10 100 10% FBS
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
* *
***
[FGF-9] (ng/mL)
Fo
ld i
ncr
ease
over
ser
um
-fre
e co
ntr
ol
4.2D
Murine AB22
0 1 10 100 10% FBS
0.0
0.5
1.0
1.5
2.0
2.5
***
*** ***
[FGF-9] (ng/mL)
Fold
incr
eas
e over
ser
um
-fre
e contr
ol
4.2E
Murine AE5
0 1 10 100 10% FBS
0.0
0.5
1.0
1.5
2.0
2.5
3.0
******
***
[FGF-9] (ng/mL)
Fo
ld i
ncr
ease
ov
er s
eru
m-f
ree
con
tro
l
4.2F
Murine AE17
0 1 10 100 10% FBS
0
1
2
3
4
5
6
***
***
***
[FGF-9] (ng/mL)
Fo
ld i
ncr
ease
ov
er s
eru
m-f
ree
con
tro
l
n = 9 per group
*p<0.05, **p=0.001, ***p<0.001 compared to non-stimulated cells, ANOVA
followed by Student-Newman-Keuls post-hoc test

117
The human and murine MM cell proliferation stimulated by exogenous recombinant
FGF-9 protein was validated using Trypan Blue cell exclusion assay. Figure 4.3
shows the dose-dependent MM cell proliferation of human JU77 in response to
exogenous human recombinant FGF-9 stimulation over two weeks, validating the
mitotic effect of FGF-9 on MM cells as assessed by WST-1.
Figure 4.3: Dose-dependent MM cell proliferation using Trypan Blue.
MM cells, human JU77, were plated at 1 x 104 cells/well in 12-well plates, serum-
starved overnight prior to stimulation with human recombinant FGF-9 resuspended
at 20 ng/mL in a final volume of 1000 µL in 1% FBS media. Cell proliferation was
assessed using trypan blue as described in the Materials and Methods section at
1, 3, 6, 10 and 14 days. Values are expressed as mean ± SEM of six replicates
from a representative experiment conducted twice.
n = 6 per group
*p<0.05, ***p<0.001 compared to non-stimulated cells, ANOVA followed by
Student-Newman-Keuls post-hoc test
0.E+00
2.E+05
4.E+05
6.E+05
8.E+05
1.E+06
1.E+06
1.E+06
1 3 6 10 14
Days
To
tal
nu
mb
er o
f cell
s
Control
20ng/ml FGF9
10% serum
***p = 0.0001
*p = 0.001

118
Following from the dose-dependent cell proliferation, the same cell lines were used
to examine the mitotic effects of exogenous recombinant FGF-9 in a time-course
experiment. Figure 4.4 shows the time-dependent cell proliferation of these cell
lines in response to FGF-9 stimulation.
Figure 4.4: Time-dependent MM cell proliferation.
MM cells, human (A) NO36 (B) ONE58 (C) STY and murine (D) AB22 (E) AE5 (F)
AE17, were stimulated with human or murine recombinant FGF-9 at a
concentration of 100 ng/mL for 0, 24, 48 and 72 hours and cell proliferation was
assessed using WST-1 reagent as described in the Materials and Methods
section. Unstimulated cells served as a negative control. Values are expressed as
mean ± SEM of nine replicates from a representative experiment conducted four
times.
4.4A
Human NO36
0 24 48 72
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
*
******
Duration of stimulation (hours)
Fo
ld i
ncre
ase
ov
er
seru
m-f
ree
co
ntr
ol
4.4B
Human ONE58
0 24 48 72
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
**
******
Duration of stimulation (hours)
Fo
ld i
ncr
ease
ov
er s
eru
m-f
ree
con
tro
l

119
4.4C
Human STY
0 24 48 72
0.8
0.9
1.0
1.1
1.2
1.3
* *
Duration of stimulation (hours)
Fo
ld i
ncre
ase
ov
er
seru
m-f
ree
co
ntr
ol
4.4D
Murine AB22
0 24 48 72
0
1
2
3
4
***
***
Duration of stimulation (hours)
Fo
ld i
ncre
ase
over
seru
m-f
ree
co
ntr
ol
4.4E
Murine AE5
0 24 48 72
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
******
***
Duration of stimulation (hours)
Fo
ld i
ncre
ase
ov
er s
eru
m-f
ree c
on
tro
l
4.4F
Murine AE17
0 24 48 72
0.0
0.5
1.0
1.5
2.0
2.5
∗
∗
******
Duration of stimulation (hours)
Fold
in
cre
ase
over
seru
m-f
ree c
on
tro
l
n = 9 per group
*p<0.05, ***p<0.001 compared to non-stimulated cells, ANOVA followed by
Student-Newman-Keuls post-hoc test

120
4.2.2 Cytokines and Chemokines Release in Response to FGF-9 Stimulation
The release of important cytokines and chemokines known to be playing a role in
the MM pathobiology was also examined using commercially available ELISA kits.
Increasing concentrations of FGF-9 stimulates the release of IL-8 or MIP-2 (murine
homolog of IL-8) (Figure 4.5), MCP-1 (Figure 4.6) and VEGF (Figure 4.7) in MM
cells in both the two species. In addition to that, exogenous FGF-9 stimulated time-
dependent release of the above-mentioned cytokines and chemokines in the same
cell lines tested (Figure 4.8). FGF-9 mediated the release of other cytokines
involved in MM pathobiology represents another means by which FGF-9 regulates
MM proliferation.
Figure 4.5: IL-8 and MIP-2 release from human and murine MM cells.
Five human (A) LO68 (B) NO36 (C) STY (D) CRL-2081 (E) CRL-5820 and three
murine MM cells (F) AB1 (G) AB2 (H) AB22 were stimulated with increasing log
concentrations of human or murine recombinant FGF-9 for 24 hours. IL-8 or MIP-2
present in the supernatants was quantitated using ELISA. Values are expressed
as mean ± SEM of three to eight replicates from a representative experiment
conducted thrice.
4.5A
Human LO68
0 1 10 100 1000
0
20
40
60
80
100
120
140
160
[FGF-9] (ng/mL)
[IL
-8]
(pg/m
L)
***
******
4.5B
Human NO36
0 1 10 100 1000
0
25
50
75
100
125
150
175
∗
∗
∗
[FGF-9] (ng/mL)
[IL
-8]
(pg
/mL
)

121
4.5C
Human STY
0 1 10 100 1000
0
50
100
150
200
250
300
[FGF-9] (ng/mL)
[IL
-8]
(pg/m
L)
***
*** ***
4.5D
Human CRL-2081
0 1 10 100 1000
0
100
200
300
400
500
600
700
800
[FGF-9] (ng/mL)
[IL
-8]
(pg/m
L)
*
*
4.5E
Human CRL-5820
0 1 10 100 1000
0
100
200
300
400
500
600 ∗∗
[FGF-9] (ng/mL)
[IL
-8]
(pg
/mL
)
4.5F
Murine AB1
0 1 10 100 1000
0
20
40
60
80
100
[FGF-9] (ng/mL)
[MIP
-2]
(pg/m
L)*** ***
*
4.5G
Murine AB2
0 1 10 100 1000
0
5
10
15
20
25
30
[FGF-9] (ng/mL)
[MIP
-2]
(pg/m
L)
***
***
***
4.5H
Murine AB22
0 1 10 100 1000
0
20
40
60
80
100
120
140
∗
∗
[FGF-9] (ng/mL)
[MIP
-2]
(pg
/mL
)
n = 3 – 8
*p<0.05, ***p<0.001 compared to non-stimulated cells, ANOVA followed by
Student-Newman-Keuls post-hoc test

122
Figure 4.6: MCP-1 release from human and murine MM cells.
Human MM cells (A) LO68 (B) NO36 (C) STY (D) CRL-5820 and murine MM cells
(E) AB22 (F) AC29 (G) AE5 (H) AE17 were stimulated with increasing log
concentrations of human or murine recombinant FGF-9 for 24 hours. MCP-1
present in the supernatants was quantitated using ELISA. Values are expressed
as mean ± SEM of three to eight replicates from a representative experiment
conducted thrice.
4.6A
Human LO68
0 1 10 100 1000
0
250
500
750
1000
1250
1500
∗
∗
[FGF-9] (ng/mL)
[MC
P-1
] (p
g/m
L)
4.6B
Human NO36
0 1 10 100 1000
0
2000
4000
6000
8000
∗∗
∗
[FGF-9] (ng/mL)
[MC
P-1
] (p
g/m
L)
4.6C
Human STY
0 1 10 100 1000
0
200
400
600
800
1000
1200
1400
∗
∗
[FGF-9] (ng/mL)
[MC
P-1
] (p
g/m
L)
4.6D
Human CRL-5820
0 1 10 100 1000
0
50
100
150
200
250
300
[FGF-9] (ng/mL)
[MC
P-1
] (p
g/m
L) ***
*

123
4.6E
Murine AB22
0 1 10 100 1000
0
2000
4000
6000
8000
10000
∗
∗
∗
∗
[FGF-9] (ng/mL)
[MC
P-1
] (p
g/m
L)
4.6F
Murine AC29
0 1 10 100 1000
0
500
1000
1500
2000
2500
3000
3500
[FGF-9] (ng/mL)
[MC
P-1
] (p
g/m
L)
*** ***
***
4.6G
Murine AE5
0 1 10 100 1000
0
500
1000
1500
2000
2500
∗
∗
∗
[FGF-9] (ng/mL)
[MC
P-1
] (p
g/m
L)
4.6H
Murine AE17
0 1 10 100 1000
0
500
1000
1500
2000
[FGF-9] (ng/mL)
[MC
P-1
] (p
g/m
L)
***
******
.
n = 3 – 8
*p<0.05, ***p<0.001 compared to non-stimulated cells, ANOVA followed by
Student-Newman-Keuls post-hoc test

124
Figure 4.7: VEGF release from human and murine MM cells.
Human MM cells (A) LO68 (B) NO36 (C) STY (D) CRL-2081 (E) CRL-5820 and
murine MM cells (F) AB1 (G) AB2 (H) AB22 (I) AE5 (J) AE17 were stimulated with
increasing log concentrations of human or murine recombinant FGF-9 for 24
hours. VEGF present in the supernatants was quantitated using ELISA. Values are
expressed as mean ± SEM of three to eight replicates from a representative
experiment conducted thrice.
4.7A
Human LO68
0 1 10 100 1000
0
20
40
60
80
∗ ∗
[FGF-9] (ng/mL)
[VE
GF
] (p
g/m
L)
4.7B
Human NO36
0 1 10 100 1000
0
25
50
75
100
125
150
∗ ∗∗
[FGF-9] (ng/mL)
[VE
GF
] (p
g/m
L)
4.7C
Human STY
0 1 10 100 1000
0
20
40
60
80
∗
∗
[FGF-9] (ng/mL)
[VE
GF
] (p
g/m
L)
4.7D
Human CRL-2081
0 1 10 100 1000
0
200
400
600
800
1000
1200
∗ ∗
[FGF-9] (ng/mL)
[VE
GF
] (p
g/m
L)

125
4.7E
Human CRL-5820
0 1 10 100 1000
0
500
1000
1500
2000
2500
∗
∗
[FGF-9] (ng/mL)
[VE
GF
] (p
g/m
L)
4.7F
Murine AB1
0 1 10 100 1000
0
500
1000
1500
2000
2500
3000
∗
∗
∗ ∗
[FGF-9] (ng/mL)
[VE
GF
] (p
g/m
L)
4.7G
Murine AB2
0 1 10 100 1000
0
400
800
1200
1600
∗
∗
∗ ∗
[FGF-9] (ng/mL)
[VE
GF
] (p
g/m
L)
4.7H
Murine AB22
0 1 10 100 1000
0
400
800
1200
1600
∗
∗
∗ ∗
[FGF-9] (ng/mL)
[VE
GF
] (p
g/m
L)
4.7I
Murine AE5
0 1 10 100 1000
0
200
400
600
800
1000
∗∗
∗
∗
[FGF-9] (ng/mL)
[VE
GF
] (p
g/m
L)
4.7J
Murine AE17
0 1 10 100 1000
0
20
40
60
80
100
120
140
∗
∗
[FGF-9] (ng/mL)
[VE
GF
] (p
g/m
L)
n = 3 – 8
*p<0.05 compared to non-stimulated cells, ANOVA followed by Student-Newman-
Keuls post-hoc test

126
Figure 4.8: Time-dependent cytokine release from human and murine MM
cells.
Human MM cells, CRL-2081, CRL-5820, LO68 and NO36 and murine MM cells,
AB1 and AB22 were stimulated with 100 ng/mL human or murine recombinant
FGF-9 for for 0, 2, 8, 24, 48 and 72 hours. (A, B) IL-8, (C, D, I, J) MCP-1, (E, F, K,
L) VEGF and (G, H) MIP-2 present in the supernatants were quantitated using
ELISA. Values are expressed as mean ± SEM of three to eight replicates from a
representative experiment conducted thrice.
4.8A
Human CRL-2081
0 2 8 24 48 72
0
200
400
600
800
1000
∗ ∗
∗
∗
∗
Duration of stimulation (hours)
[IL
-8]
(pg/m
L)
4.8B
Human CRL-5820
0 2 8 24 48 72
0
200
400
600
800
1000
∗∗
∗
∗
∗
Duration of stimulation (hours)
[IL
-8]
(pg/m
L)
4.8C
Human LO68
0 2 8 24 48 72
0
400
800
1200
1600
∗
∗∗
∗
∗
∗
Duration of stimulation (hours)
[MC
P-1
] (p
g/m
L)
4.8D
Human NO36
0 2 8 24 48 72
0
2000
4000
6000
8000
∗ ∗ ∗∗
∗
Duration of stimulation (hours)
[MC
P-1
] (p
g/m
L)

127
4.8E
Human CRL-2081
0 2 8 24 48 72
0
200
400
600
800
1000
1200
1400
1600
∗∗
∗
∗
Duration of stimulation (hours)
[VE
GF
] (p
g/m
L)
4.8F
Human CRL-5820
0 2 8 24 48 72
0
150
300
450
600
750
900
1050
1200
∗ ∗
∗
∗
∗
Duration of stimulation (hours)
[VE
GF
] (p
g/m
L)
4.8G
Murine AB1
0 2 8 24 48 72
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0 ∗
Duration of stimulation (hours)
[MIP
-2]
(pg/m
L)
4.8H
Murine AB22
0 2 8 24 48 72
0
10
20
30
40
50
60
70
∗
∗
∗
Duration of stimulation (hours)
[MIP
-2]
(pg/m
L)
4.8I
Murine AB1
0 2 8 24 48 72
0
200
400
600
800
1000
∗
∗
∗
Duration of stimulation (hours)
[MC
P-1
] (p
g/m
L)
4.8J
Murine AB22
0 2 8 24 48 72
0
500
1000
1500
2000
2500
3000
∗
∗
∗ ∗
∗
Duration of stimulation (hours)
[MC
P-1
] (p
g/m
L)

128
4.8K
Murine AB1
0 2 8 24 48 72
0
200
400
600
800
∗
∗
∗
∗ ∗
Duration of stimulation (hours)
[VE
GF
] (p
g/m
L)
4.8L
Murine AB22
0 2 8 24 48 72
0
200
400
600
800
1000
1200
1400
∗
∗
∗
∗ ∗
Duration of stimulation (hours)
[VE
GF
] (p
g/m
L)
n = 3 – 8
*p<0.05 compared to non-stimulated cells, ANOVA followed by Student-Newman-
Keuls post-hoc test

129
4.2.3 The Effects of Antibody Neutralisation on Cytokine Release
To clarify the contribution of FGF-9 in cytokine release by MM cells, MM cells were
pre-treated with a monoclonal anti-human FGF-9 antibody prior to FGF-9
stimulation. Prior to that, the IC50 for the FGF-9 antibody to inhibit FGF-9 activity
was determined. The monoclonal anti-human FGF-9 antibody is not only specific
to human, as there have been published studies using this antibody in murine (Li
et al., 2008), rat (Jui-Yen et al., 2009) and pig cells (Kinkl et al., 2003). Murine
AB22 cells were chosen for this optimisation study as this cell line released a high
level of VEGF in response to FGF-9. Figure 4.9 shows that the suboptimal
concentration of FGF-9 antibody against VEGF release was 10 µg/mL.
Figure 4.9: Optimisation of neutralising antibody concentration.
5 x 104 murine AB22 cells were pretreated with 0.1 – 100 µg/mL monoclonal anti-
human FGF-9 antibody or 100 µg/mL mouse IgG2A isotype control resuspended in
serum-free media for 30 minutes before they were stimulated with 100 ng/mL
murine recombinant FGF-9 for 24 hours. Untreated cells served as a negative
control whereas cells treated with 100 ng/mL FGF-9 alone serve as a positive
control. The supernatants were harvested and assayed for VEGF release. Values
are expressed as mean ± SEM of two replicates from an experiment.
Murine AB22
SFM FGF-9 +0.1 +1 +10 +100 +IgG2A
0
200
400
600
800
[FGF-9 Ab] (µg/mL)
Treatment
[VE
GF
] (p
g/m
L)
n = 2 per group

130
Following the pilot optimisation study, murine MM AB22 cells were pretreated with
the suboptimal concentration of FGF-9 antibody or mouse IgG2A isotype control at
10 µg/mL prior to stimulation with 100 ng/mL murine recombinant FGF-9 for 24
hours. Figure 4.10 shows that the FGF-9 antibody significantly reduced the levels
of MIP-2, MCP-1 and VEGF in the AB22 cells. The negative IgG2A isotype control
had only little attenuation effect on the FGF-9 stimulated cytokine release,
demonstrating that the release of cytokine is a result of FGF-9 stimulation. This is
consistent with other reports demonstrating the role of FGF-9 using neutralizing
antibody, in prostate cancer cells (Li et al., 2008, Teishima et al., 2012) and lung
adenocarcinomas (Ueng et al., 2010).

131
Figure 4.10: Inhibition of cytokine release from murine MM cells using
antibody neutralisation.
Murine MM AB22 were pretreated with monoclonal anti-human FGF-9 antibody or
mouse IgG2A isotype control, at a final concentration of 10 µg/mL, for 30 minutes
before stimulated with 100 ng/mL murine recombinant FGF-9 for 24 hours. (A)
MIP-2 (B) MCP-1 and (C) VEGF present in the supernatants were quantitated
using ELISA. Values are expressed as mean ± SEM of six replicates from a
representative experiment conducted twice.
4.10A
MIP-2 (pg/mL)
SFM FGF9 +αFGF9+IgG2A
0
50
100
150
200
250
∗
∗
***
Treatment
4.10B
[MCP-1] (pg/mL)
SFM FGF-9 +αFGF9 +IgG2A
0
4000
8000
12000
16000
***
***
***
Treatment
4.10C
[VEGF] (pg/mL)
SFM FGF9 +αFGF9+IgG2A
0
200
400
600
800
∗
∗
∗
Treatment
n = 6 per group
*p<0.05, ***p<0.001 compared FGF-9-stimulated treatment, ANOVA followed by
Student-Newman-Keuls post-hoc test

132
4.2.4 The Effects of Pathway Inhibitors on FGF-9-Stimulated MM Cells
There have been several studies demonstrating that FGF-9 acts through the ERK
pathway (Antoine et al., 2007, Wing et al., 2005). We were also interested to
examine if FGF-9 could elicit its effects through the JNK and p38 pathways too.
Hence, inhibition studies were conducted in the presence or absence of 3
pharmacological inhibitors of different signalling pathways, PD98059 (ERK
inhibitor), SP600125 (JNK inhibitor II) and SB203580 (p38 inhibitor) (all from Merck
Pty Limited, Kilsyth, Victoria, Australia). FGF-9-stimulated proliferation was
moderately inhibited by the ERK inhibitor in the human cells (Figure 4.11A and B),
but so significantly in the murine cells (Figures 4.11C and D). Both the JNK and
p38 inhibitors significantly inhibited cell proliferation in the two cell lines.
On the other hand, Figures 4.12A and B depicts that the p38 inhibitor was unable
to inhibit IL-8 release in the human MM cell (Figures 4.12A and B), but significantly
reduced MCP-1 and VEGF levels in the human and murine MM cells (Figures
4.12C – F). The ERK and the JNK inhibitors are potent in inhibiting all three
cytokine release caused by FGF-9 stimulation in the human and murine MM cells,
suggesting that FGF-9 could be mediating its effects in cytokine release through
the ERK and JNK pathways.

133
Figure 4.11: FGF-9 mediated MM cell proliferation is inhibited by ERK, JNK
and p38 pathway inhibitors.
MM cells, human (A) NO36 (B) ONE58 and murine (C) AB22 (D) AE17 were
pretreated for 30 minutes with ERK inhibitor, PD98059, JNK inhibitor, SP600125 or
p38 inhibitor, SB203580, at final concentrations of 50 µM, 10 µM and 10 µM
respectively. A vehicle control for the inhibitors, DMSO at the final concentration of
0.1%, was also conducted. The cells were then stimulated with 100 ng/mL human
or murine recombinant FGF-9 for 48 hours and inhibition of cell proliferation was
assessed using WST-1 reagent. Values are expressed as mean ± SEM of nine
replicates from a representative experiment.
4.11A
Human NO36
0
20
40
60
80
100
∗∗
Cel
l pro
life
rati
on
(% o
f F
GF
-9 s
tim
ula
ted r
esp
onse
)
FGF-9 + + + + + DMSO - + - - - PD98059 - - + - - SP600125 - - - + - SB203580 - - - - +
4.11B
Human ONE58
0
20
40
60
80
100
∗
∗
Cel
l pro
life
rati
on
(% o
f F
GF
-9 s
tim
ula
ted r
esponse
)
FGF-9 + + + + + DMSO - + - - - PD98059 - - + - - SP600125 - - - + - SB203580 - - - - +

134
4.11C
Murine AB22
0
20
40
60
80
100
∗
∗
∗
∗
Cell
pro
life
rati
on
(% o
f F
GF
-9 s
tim
ula
ted r
esp
onse
)
FGF-9 + + + + + DMSO - + - - - PD98059 - - + - - SP600125 - - - + - SB203580 - - - - +
4.11D
Murine AE17
0
20
40
60
80
100∗
∗
∗
∗
Cel
l p
roli
fera
tio
n
(% o
f F
GF
-9 s
tim
ula
ted r
esp
on
se)
FGF-9 + + + + + DMSO - + - - - PD98059 - - + - - SP600125 - - - + - SB203580 - - - - +
n = 9 per group
*p<0.05 compared to FGF-9-stimulated cells, ANOVA followed by Student-
Newman-Keuls post-hoc test

135
Figure 4.12: Inhibition of cytokine release from human and murine MM cells.
Human CRL-2081, CRL-5820, LO68, NO36 and murine AE17, AB1, AB22 MM
cells were pretreated with MAPK inhibitors, ERK inhibitor (ERK i), JNK inhibitor
(JNK i) and p38 inhibitor (p38 i), at final concentrations of 50 µM, 10 µM and 10 µM
respectively, for 30 minutes. DMSO was used as a vehicle control. 100 ng/mL
human or murine recombinant FGF-9 was added for 24 hours. (A, B) IL-8, (C, D,
G, H) MCP-1 and (E, F, I, J) VEGF present in the supernatants were quantitated
using ELISA. Values are expressed as mean ± SEM of six replicates from a
representative experiment.
4.12A
Human CRL-2081
FGF-9 +DMSO+ERK i+ JNK i +p38 i
0
20
40
60
80
100 ∗
∗ ∗
Treatment
% I
L-8
rel
eas
e over
FG
F-9
contr
ol
4.12B
Human CRL-5820
FGF-9 +DMSO+ERK i+ JNK i +p38 i
0
20
40
60
80
100
120
∗∗
Treatment
% I
L-8
rele
ase
over
FG
F-9
contr
ol
4.12C
Human LO68
FGF-9 +DMSO+ERK i+ JNK i +p38 i
0
20
40
60
80
100
∗
∗
∗
Treatment
% M
CP
-1 r
ele
ase
over
FG
F-9
contr
ol
4.12D
Human NO36
FGF-9 +DMSO+ERK i+ JNK i +p38 i
0
20
40
60
80
100
∗
∗∗
∗
Treatment
% M
CP
-1 r
eleas
e over
FG
F-9
contr
ol

136
4.12E
Human CRL-2081
FGF-9 +DMSO+ERK i+ JNK i +p38 i
0
20
40
60
80
100
∗
∗
∗
∗
∗
Treatment
% V
EG
F r
ele
ase
over
FG
F-9
contr
ol
4.12F
Human CRL-5820
FGF-9 +DMSO+ERK i+ JNK i +p38 i
0
20
40
60
80
100
∗
∗
∗ ∗
Treatment
% V
EG
F r
eleas
e over
FG
F-9
contr
ol
4.12G
Murine AE17
FGF-9 +DMSO+ERK i + JNK i +p38 i
0
20
40
60
80
100∗
∗
∗
∗
Treatment
%
MC
P-1
rel
ease
over
FG
F-9
con
trol
4.12H
Murine AB22
FGF-9 +DMSO+ERK i + JNK i +p38 i
0
20
40
60
80
100
∗
∗
∗
∗
Treatment
% M
CP
-1 r
elease
ov
er
FG
F-9
co
ntr
ol
4.12I
Murine AB1
FGF-9 +DMSO+ERK i + JNK i +p38 i
0
20
40
60
80
100
∗ ∗
∗
Treatment
% V
EG
F r
ele
ase o
ver
FG
F-9
contr
ol
4.12J
Murine AB22
FGF-9 +DMSO+ERK i + JNK i +p38 i
0
20
40
60
80
100
∗
∗ ∗
∗
Treatment
% V
EG
F r
elease
ov
er
FG
F-9
co
ntr
ol
n = 6 per group
*p<0.05 compared to FGF-9-stimulated cells, ANOVA followed by Student-
Newman-Keuls post-hoc test

137
To validate if the pharmacological inhibitors were specific in inhibiting their target
pathways, Western Blot was conducted to visualise the phosphorylation of the
target proteins, ERK, JNK and p38. Figure 4.13 depicts that in the human CRL-
2081 MM cells and murine AB22, FGF-9 stimulation causes an increase in ERK
phosphorylation and this phosphorylation was reduced by the ERK inhibitor,
demonstrating the specificity of the ERK inhibitor to the target ERK protein. A
similar image was also seen in the JNK phosphorylation in the human MM cells
and in the p38 phosphorylation in the murine MM cells. Surprisingly, JNK protein
phosphorylation was increased in response to the JNK inhibitor in the murine MM
cells. This was also the case for the p38 protein in the human MM cells.
Figure 4.13: Phosphorylation of proteins downstream of FGF-9 signalling
pathway.
Phosphorylation of proteins involved in the ERK, JNK and p38 pathway were
studied using Western blotting assays. The images are a representative image
from experiments conducted thrice. i = inhibitor.
Human CRL-2081 Murine AB22 p-ERK +ERKi +DMSO FGF-9 SFM
+ERKi +DMSO FGF-9 SFM
p-JNK +JNKi +DMSO FGF-9 SFM
+JNKi +DMSO FGF-9 SFM
p-p38 +p38i +DMSO FGF-9 SFM
+p38i +DMSO FGF-9 SFM
Beta actin

138
4.2.5 The Role of FGF-9 in MM Cell Invasion
The role of FGF-9 in MM pathobiology in the context of tumour cell invasion was
also studied using Matrigel transwell assay. It has been shown that FGF-9
promotes invasion in prostate cancer cell (Teishima et al., 2012) as well as
epithelial and endothelial cells (Hendrix et al., 2006). Figure 4.14 shows that FGF-
9 at 100 ng/mL also stimulates MM cell invasion by 24 hours.
Figure 4.14: FGF-9 stimulates MM cell invasion.
Human MM CRL-2081 cells were seeded at a concentration of 1 x 104 cells in the
upper compartment of the Matrigel-coated inserts. 100 ng/mL FGF-9 (or serum-
free media (SFM) as negative control) was added to the companion plates. Cell
invasion was measured 16 and 24 hours post-stimulation. The Matrigel-coated
insert membranes were stained with Diff-Quick solution and invading cells were
counted. Values are expressed as a mean ± SEM of three replicates from a
representative experiment conducted twice.
CRL-2081 invasion in response to
FGF-9 (100 ng/mL) for 16 and 24 hours
0
100
200
300
400
500
600*
Cel
l n
um
ber
(co
un
ted a
cross
the
slid
e
at 1
0X
mag
nif
icati
on
)
n = 3 per group
*p<0.05 compared to non-stimulated cells, ANOVA followed by Student-Newman-
Keuls post-hoc test
SFM FGF-9 FGF-9
24 hours 16 hours 24 hours

139
4.2.6 The Involvement of FGF-9 in MM Cell Migration
MM is also potent factor that stimulates cell migration of endothelial and ovarian
cancer cells (Hendrix and Cho, 2005). Treatment with FGF-9 potently stimulated
cell migration in all human and murine MM cells tested. Representative scratch
assay results are shown in Figure 4.15A. The scratch gap was almost closed by 48
hours in the FGF-9 stimulated cells, which is similar to the positive control cells
that were incubated in 10% FBS media. Figure 4.15B-E shows the percentage
increase of cell migration over 0 hour quantitated on two human and two murine
cell lines, using the formula stated in Section 2.4.4.
Figure 4.15: FGF-9 stimulates MM cell migration.
(A) Representative images of MM cell migration following FGF-9 stimulation over
time. Human (B) ONE58 and (C) STY and (D) murine AB22 and (E) AE17 were
seeded in a tissue culture dish until confluent. A 1000 mL pipette tip was used to
scratch the surface of the culture dish. The cells were stimulated with human or
murine recombinant FGF-9 resuspended at 100 ng/mL and cell migration was
analysed under the microscope. Values are expressed as a mean ± SEM of three
replicates from a representative experiment conducted thrice.

140
0 hr 24 hrs 48 hrs 72 hrs SFM
FGF-9
10% FBS
4.15A

141
4.15B
Human ONE58
24 48 72
0
25
50
75
100
SFMFGF-9FBS***
***
******
***
***
Duration of stimulation (hours)
Per
cen
tag
e in
crea
se o
f
cell
mig
rati
on
ov
er 0
ho
ur
4.15C
Human STY
24 48 72
0
25
50
75
100SFMFGF-9FBS
***
******
******
Duration of stimulation (hours)
Per
cen
tag
e in
crea
se o
f
cell
mig
rati
on
ov
er 0
ho
ur
4.15D
Murine AB22
6 8 24
0
25
50
75
100
FGF-9SFM
FBS
***
***
Duration (hours)
Per
cen
tag
e in
crea
se o
f
cell
mig
rati
on
ov
er 0
ho
ur
4.15E
Murine AE17
24 48 72
0
25
50
75
100FGF-9SFM
FBS***
*** ***
*
Duration (hours)
Per
cen
tag
e in
crea
se o
f
cell
mig
rati
on
ov
er 0
ho
ur
n = 3 per group
*p<0.05, ***p<0.001 compared to non-stimulated cells, ANOVA followed by
Student-Newman-Keuls post-hoc test

142
4.3 Discussion
Our data shows that exogenous FGF-9 not only promotes dose- and time-
dependent cell proliferation but also stimulates migration and invasion in human
and murine MM cell lines tested. Furthermore, increasing concentrations of FGF-9
increase the release of IL-8, MIP-2, VEGF and MCP-1 in these cell lines.
It has been previously reported that FGF-9 does not induce cell proliferation on
endometrial epithelial cells (Tsai et al., 2002) and human umbilical vein endothelial
cells (Hendrix et al., 2006, Miyamoto et al., 1993). Our studies show that FGF-9 is
a potent mitogen factor for MM cells, consistent with other reports on other cell
lines such as in the human oesophageal epithelial cells (Mulder et al., 2009),
human lung fibroblasts (Chien-Kai et al., 2009), human prostate cancer cells
(Teishima et al., 2012) and rat kidney epithelial cells (Hendrix et al., 2006). Our cell
proliferation assay recorded a higher fold change in the murine cell lines compared
to the human cell lines possibly due to the higher growth rate in the murine cell
lines observed during the routine expansion of cell cultures. Like its role in prostate
cancer cells (Teishima et al., 2012), FGF-9 is involved in MM cell invasion
suggesting that this growth factor may be playing a role in tumour spread and
invasion.
MM cells secrete important cytokines known to be playing a role in the MM
pathobiology in response to FGF-9. IL-8 is a known autocrine growth factor for MM
(Galffy et al., 1999a), and would contribute to the potent mitogen effect of FGF-9 in
MM. In addition, FGF-9 may also promote angiogenesis via VEGF stimulation
(Masood et al., 2003). This is consistent with other data illustrating the decreased
VEGF signalling and hence delayed vascularisation in Fgf -/- mice (Hung et al.,
2007). Besides that, FGF-9 may also contribute to the increased vascular

143
permeability and pleural fluid formation by inducing MCP-1 (Stathopoulos et al.,
2008), seen in MM. To our knowledge, there are no studies that have reported the
association between FGF-9 and IL-8, and FGF-9 and MCP-1.
Although MM tumour development is a complex process that is regulated by
signals from numerous growth factors, cytokines and chemokines, which are
influenced by the tumour cell itself and/or the tumour microenvironment, our results
show that FGF-9 may play a part in all of the key processes underpinning MM
tumour growth. The signals generated from the release of IL-8, VEGF and MCP-1
in response to FGF-9 stimulation may be inter-related to each other. IL-8 has been
shown to be involved in angiogenesis and metastasis (Li et al., 2003) while MCP-1
is involved in recruiting immune cells, such as monocytes, to the site of
inflammation, a crucial step in promoting post-ischemic angiogenesis (Li et al.,
1997). Besides, VEGF has been postulated to be an important mediator for pleural
fluid formation and accumulation (Cheng et al., 1999, Kraft et al., 1999, Thickett et
al., 1999). We are not certain if the release of the above-mentioned cytokines were
a direct result of FGF-9 stimulation or if FGF-9 was promoting the release of the
cytokines indirectly via a secondary mediator. This will be a subject of interest in
future studies. Although we cannot detect the release of the other vital mediators in
MM including TNF-α (Stathopoulos et al., 2007), IFN-γ and IL-10 by ELISA in
response to FGF-9 stimulation, we cannot conclude that FGF-9 does not signal to
those cytokines or other mediators that are important in MM such as IL-6 (Monti et
al., 1994), cyclooxygenase-2 (Edwards et al., 2002), transforming growth factor
beta (Fujii et al., 2012) and matrix metalloproteases (Servais et al., 2012).
Our time-dependent cytokine release data demonstrates that FGF-9-induced cells
sustained release of cytokines up to 72 hours indicating an increased production

144
(rather than release of intracellular stores) of the cytokines. Cytokine release was
inhibited by FGF-9 neutralizing antibody demonstrating the specificity of the role of
FGF-9 in the MM pathobiology. This is consistent with the previous studies
showing the potential therapeutic use of FGF-9 neutralizing antibody to abolish
signals generated by FGF-9 (Cinaroglu, 2005, Tsai et al., 2002, Chien-Kai et al.,
2009). Our data further support the need to evaluate the efficacy of FGF-9
neutralizing antibody against MM.
A concentration of 1000 ng/mL of FGF-9 was used in our in vitro studies as the
concentration of FGF-9 at the tumour microenvironment is not known. Despite that,
biological effects were still seen from MM cells at least up to 1000 ng/mL of FGF-9
which shows that the MM cells have a significant ability to respond to FGF-9 at a
concentration 50 times higher than the highest FGF-9 concentration found in our
cohorts of fluids, with no observation signs of necrosis or apoptosis.
The mitogen-activated protein kinase (MAPK) pathways such JNK and ERK
pathways, integrate signals from receptor tyrosine kinases, in this case, FGFRs, to
control cell proliferation and other cellular functions (Mckay and Morrison, 2007).
Hence, we are interested to examine the signal transduction pathways that FGF-9
is involved in, to elicit its effects in MM cells. The present study demonstrates that
whilst FGF-9 could potentially be acting as a mitogen for MM through all three
pathways, the p38 pathway may not be important for cytokine release. This is in
contrast to a previous study reporting that p38 inhibitor does not affect proliferation
in the Muller cell primary cultures (Bugra-Bilge and Cinaroglu, 2005). The disparity
could also be due to the different cell lines used. While inhibiting JNK activity in the
human MM cell line using SP600125 decreased JNK phosphorylation, the same
inhibitor paradoxically increased the phosphorylation of JNK in the murine MM

145
cells. This was also seen when a p38 inhibitor (SB203580) decreased p38
phosphorylation in murine MM cells but not in the human MM cell line tested. This
could be due to the induction of other signalling pathways in response to the
blockade of the JNK and p38 pathways. It was shown that the inhibition of p38 with
SB203580 compound increased the phosphorylation status of p38 in a model of
colitis (Ten Hove et al., 2002). In addition, Kumar et al. reported that the SB203580
compound specifically inhibited the activity of p38 MAPK but not its activation by
upstream map kinase kinase (MAPKK) (Kumar et al., 1999). Further evaluation on
other MM cell lines using different inhibitors that target JNK and p38 proteins and
the other proteins involved in the JNK and p38 signalling cascades is therefore
required.
The FGF family also activates the PI3K as well as the PLCγ signalling cascades
(Eswarakumar et al., 2005, Klint and Claesson-Welsh, 1999). A previous report
has demonstrated that FGF-9 also signals through the PI3K pathway in human
uterine endometrial stromal cells (Wing et al., 2005). To our knowledge, there have
been no studies that have investigated if FGF-9 signals through PLCγ. Evaluation
of these signalling pathways in MM through the examination of downstream
signalling proteins involved and the increase of Ca2+ is warranted in future studies.
There have been studies looking at FGF-9 and sonic hedgehog signalling in lung
development (White et al., 2006, White et al., 2007, Del Moral et al., 2006). As the
role of sonic hedgehog signalling in MM has recently been revealed (Shi et al.,
2012), it will be important to examine if FGF-9 is involved in this signalling pathway
during MM progression. Likewise, interactions between FGF-9 signalling and Wnt
signalling have been shown in lung (Yin et al., 2008) and gonadal development
(Kim et al., 2006) and in the pathology of ovarian endometrioid cancer (Hendrix et
al., 2006). Exploring the interactions between FGF-9 and Wnt signalling in MM is

146
outside the scope of my thesis, but may enhance our understanding of the
complex role of FGF-9 in MM tumour development.
It is unclear how FGF-9 is up-regulated in MM. We speculate that FGF-9 is
produced and secreted in abundance as a result of the malignant transformation of
the benign mesothelial cells to MM cells. Very little is known about the upstream
pathway of FGF-9 in cancers as most studies to date have explored its role during
normal embryogenesis. Scattered studies have emerged recently linking the up-
regulation of FGF-9 mRNA expression in endometrial stromal cells to 17β-estradial
(Tsai et al., 2002) and in lung adenocarcinoma cells to benzo[a]pyrene treatment,
a carcinogen found in cigarette smoke (Chien-Kai et al., 2009). Although asbestos
is the main cause of MM, it is unknown if this mineral can drive up-regulation of
FGF-9 in MM. Searching for stimulation of FGF-9 in the setting of pleural MM
should be explored in future studies.
There are limitations of the experiments presented. The experiments performed
using cell lines bear their intrinsic shortcomings. However, the consistency of
response to FGF-9 among all eight human and six murine cell lines argue strongly
that it is a genuine observation. We have shown for the first time that FGF-9 is
expressed at significant levels in MM tissues and pleural fluids (Chapter 3). In this
study, we mimicked the over-expression of FGF-9 by adding recombinant FGF-9
to MM cells in vitro and have demonstrated that MM cells exhibits a response to
exogenous FGF-9. However, endogenous FGF-9 released by MM cells in vivo
cannot be compared to the exogenous recombinant FGF-9 that we used in our in
vitro studies. Hence, the next chapter explores MM tumour growth in vivo using
MM cells lacking endogenous FGF-9.

147
____________________________________________
THE NECESSITY OF FGF-9 IN MALIGNANT
MESOTHELIOMA TUMOUR GROWTH IN VIVO
CHAPTER 5

148
5.0 THE NECESSITY OF FGF-9 IN MALIGNANT MESOTHELIOMA TUMOUR
GROWTH IN VIVO
5.1 Introduction
In the previous chapter, it was shown that exogenous FGF-9 stimulates MM cell
proliferation, migration, invasion as well as the release of important cytokines that
are crucial for this disease. However, whether FGF-9 has an essential role in MM
development requires exploration. FGF-9 is important during embryonic
development and knockout of FGF-9 is embryonically lethal. As such FGF-9
knockout mice are not available. Hence, a knockdown of FGF-9 in MM cells was
employed to explore the necessity of FGF-9 in MM pathobiology.
5.2 Results
5.2.1 Development of FGF-9 shRNA Knockdown Cells
FGF-9 shRNA knockdown studies were performed on murine AB1 MM cells as
these cells are robust to transfection studies. Four clones were selected from each
shRNA plasmid (sh273, sh496, sh588) and a scrambled vector, making it to a total
of 16 clones that were tested by ELISA for FGF-9 levels. The shRNA clone that
recorded the lowest FGF-9 protein level (sh588) and the scrambled vector that
recorded the highest FGF-9 protein level were selected for further validation by
quantitative real-time PCR. Figure 5.1 shows that FGF-9 protein levels was
successfully reduced by approximately 50% in the FGF-9 shRNA knockdown cells
compared with the scrambled vector controls. Figure 5.2 shows that FGF-9 was
knocked down at the RNA level in the shRNA knockdown cells compared to the
parent and the scrambled vector cell lines control. The data confirms that FGF-9
was successfully knocked-down in the shRNA murine AB1 cell line.

149
Figure 5.1: FGF-9 shRNA knockdown cells have lower levels of FGF-9
protein.
FGF-9 protein levels in cell lysates of FGF-9 shRNA and scrambled control murine
MM cells were determined using ELISA. FGF-9 protein levels in FGF-9 shRNA
knockdown cells were reduced by two fold compared to controls. Values are
expressed as a mean ± SEM of three replicates from a representative experiment
conducted twice.
FGF-9 shRNA Vector
0
1
2
3
4
5
6
7
∗∗∗
Cell lines
FG
F-9
/Tota
l ce
lls
(x10
-5)
(pg/m
L/c
ells
)
n = 3
***p<0.001, t-test

150
Figure 5.2: FGF-9 shRNA knockdown cells have lower levels of FGF-9 mRNA.
FGF-9 mRNA levels in parent, FGF-9 shRNA and scrambled murine MM cell lines
were determined using RT-PCR. FGF-9 mRNA levels in FGF-9 shRNA knockdown
cells were reduced compared to controls. Values are expressed as a mean ± SEM
of six replicates from a representative experiment conducted thrice.
Parent FGF-9 shRNA Vector
0.0
0.5
1.0
1.5
2.0
2.5
*
***
Cell lines
mF
GF
-9 m
RN
A e
xpre
ssio
n
n = 6
*p<0.05, ***p<0.001, ANOVA followed by Student-Newman-Keuls post-hoc test

151
5.2.2 FGF-9 shRNA Knockdown Cells in Heterotopic Model
The successfully transfected cells as well as the parent cell line were then
inoculated into Balb/c mice subcutaneously in an established murine model
(Section 2.9.1). Figure 5.3A shows that there was no adverse effects on the
animals inoculated with the transfected cell lines compared to the parent cell lines.
The animals inoculated with the parent and the vector cell lines developed
significant-sized tumours by Day 17. Mice injected with FGF-9 shRNA knockdown
MM cells had significantly smaller tumours (Figure 5.3B). Figure 5.3C depicts the
representative images of the animals from each group. The parent and vector
groups developed large subcutaneous tumours. On the other hand, there were no
macroscopic tumours seen in the shRNA knockdown group. The data
demonstrates the necessity of FGF-9 in MM tumour development.
Figure 5.3: Knockdown of FGF-9 in MM cells significantly retards their
tumour growth in heterotopic model.
Balb/c mice (n = 10 each group) were injected with 5 x 105 parent MM cell line,
shRNA knockdown MM cells or scrambled vector MM cells. (A) No weight loss
was recorded in mice in all three groups. (B) Tumour dimensions were measured
and animals were sacrificed once tumour size reached the limit of 10 mm x 10 mm
maximum as allowed by the animal ethics committee. Tumour development in
shRNA group was significantly suppressed compared to control groups. (C)
Animals in the parent and vector groups developed large macroscopic tumours
that were not seen in the shRNA knockdown groups.

152
5.3A
0 5 7 10 12 14 17 18
21
22
23
24
25
26
27
28ParentFGF-9 shRNAVector
Days post tumour inoculation
Anim
al W
eight
(g)
n = 10 per group
5.3B
0 5 7 10 12 14 17 18
0102030405060708090
100110
ParentFGF-9 shRNAVector
Days post tumour inoculation
Tum
our
Siz
e (m
m2)
***
***
n = 10 per group
***p<0.001, ANOVA followed by Student-Newman-Keuls post-hoc test

153
5.3C
Parent
FGF-9 shRNA
Vector

154
5.2.3 FGF-9 shRNA Knockdown Cells in Orthotropic Model
To further confirm these findings, an orthotopic model was employed whereby the
Balb/c mice received a single intraperitoneal injection (Section 2.9.2). As
preliminary pilot experiments conducted showed that tumours developed much
quicker in the intraperitoneal model compared to the subcutaneous model (not
shown), the animals were sacrificed at Day 13. Figure 5.4A again shows that the
animal weights were not affected by the intraperitoneal injection of the transfected
cell lines. The number of tumour nodules in the FGF-9 shRNA knockdown group
was significantly lower compared to the parent and the vector groups (Figure
5.4B). Figure 5.4C depicts the representative images of the numerous and larger
tumours in the parent and the vector groups compared to the fewer and smaller
tumour nodules in the shRNA knockdown group. This confirms the finding of the
subcutaneous flank model and strengthens the finding that FGF-9 is a key factor in
the development of MM tumour.

155
Figure 5.4: shRNA knockdown of FGF-9 significantly reduced MM tumour
growth in intraperitoneal model of disease.
Balb/c mice (n = 10 each group) were injected with 5 x 105 parent MM cell line,
shRNA knockdown MM cells or scrambled vector MM cells. (A) No weight loss
was recorded in the animals as a result of inoculation of transfected cell lines. (B)
Mice in the shRNA knockdown group have fewer tumour nodules compared to the
parent and the vector groups. (C) Animals in the parent and vector groups
developed large macroscopic tumours that were not seen in the shRNA
knockdown groups.
5.4A
0 2 6 9 12 13
19
21
23
25
27
29
FGF9 shRNA
Parent
Vector
Days post tumour inoculation
Anim
al
Wei
ght
(g)
n = 10 per group

156
5.4B
Parent FGF9 shRNA Vector
0
10
20
30
40
50
Num
ber
of
Tu
mou
r N
odule
s **
n = 10 per group
*p<0.05, ANOVA followed by Student-Newman-Keuls post-hoc test
5.4C
Parent FGF-9 shRNA Vector

157
5.2.4 FGF-9 shRNA Knockdown Cells in Athymic Model
To examine if the immune response in the host system is involved in tumour
growth suppression in the FGF-9 shRNA knockdown group, we inoculated the
transfected and parent cell lines subcutaneously into nude mice that lack a thymus
and hence an inhibited immune system. Figure 5.5A shows that the animal weights
were not affected by the subcutaneous injection of the transfected cell lines while
Figure 5.5B depicts that the tumours developed in the FGF-9 shRNA knockdown
group were as large as the scrambled vector and the parent groups when
measured at the same time point. The data could possibly answer the role of the
immune system on FGF-9 signalling in MM development.
Figure 5.5: shRNA knockdown of FGF-9 had similar MM tumour growth in
athymic model of disease.
Nude mice (n = 7 each group) were injected with 5 x 105 parent MM cell line,
shRNA knockdown MM cells or scrambled vector MM cells. (A) No weight loss
was recorded in the animals as a result of inoculation of transfected cell lines. (B)
Tumour dimensions were measured and animals were sacrificed once tumour size
reached the limit of 10 mm x 10 mm maximum as allowed by the animal ethics
committee. Tumour development in shRNA group was similar compared to control
groups.

158
5.5A
0 4 6 8 11 13 18
15
16
17
18
19
20
21 ParentFGF-9 shRNAVector
Days post tumour inoculation
Anim
al
Weig
ht
(g)
n = 7 per group
5.5B
0 4 6 8 11 13 18
0
20
40
60
80
100
120
140
ParentFGF-9 shRNAVector
Days post tumour inoculation
Tum
our
Siz
e (m
m2)
n = 7 per group

159
5.3 Discussion
Our results show that the FGF-9 shRNA knockdown cells had significantly retarded
growth compared to the parent and scrambled vector groups in vivo. Tumours
developed in the FGF-9 shRNA knockdown group in both the subcutaneous and
intraperitoneal models were significantly smaller compared to the controls. On the
other hand, in the nude mice model, the tumours developed in the FGF-9 shRNA
knockdown group were as large as the scrambled vectors and the parent groups
when measured at the same time point, thus implying that the retardation of MM
growth seen in FGF-9 knockdown cells is mediated by immune response.
FGF-9 induces significant biological responses in MM cells (Chapter 4). In this
chapter, we tested the necessity of FGF-9 in the pathobiology of MM using shRNA
to inhibit FGF-9 expression in MM cells. The clones were selected through
puromycin selection and the success of the transfection assay was confirmed by
ELISA and quantitative RT-PCR. While there is no obvious phenotypic difference
in the cells in vitro, the results were dramatic when the cells were inoculated into
mice in our in vivo studies.
Knockdown of FGF-9 expression impacts significantly on its growth in vivo but not
in vitro. The fact that FGF-9 shRNA-treated cells grew significantly slower in both
the subcutaneous and the intraperitoneal models suggests that it is unlikely that
the retardation of tumour growth can be explained by local factors related to the
mesothelial environment or blood and nutrient supply in the host system. This
raised the possibility that FGF-9 allows MM cells to escape immune surveillance
and the knockdown of FGF-9 therefore results in immune clearance of the tumour
cells. Indeed the retardation of tumour growth in FGF-9 shRNA cells no longer

160
exist when athymic mice were used. This strongly implies that FGF-9 inhibits the
cell-mediated immunity and its ability to clear MM cells. Further studies are
currently in progress in our lab to tease out the immune mechanisms involved.
The observed effects from FGF-9 shRNA cells should be specific to the down-
regulation of FGF-9. The sequences were confirmed on the NCBI database and
the real-time PCR and ELISA data show a significant reduction in FGF-9 mRNA
and protein in the shRNA knockdown cells. Care was taken to titrate and use the
lowest shRNA concentration that still gave the desirable effects in knocking down
FGF-9 to reduce the potential off-target effects. Our findings that shRNA
knockdown cells caused slower tumour growth in vivo are in keeping with our data
in previous chapters showing that FGF-9 is a key factor for MM tumour
development. We plan to validate our results with other shRNA clones generated
from different shRNA sequences to the non-overlapping regions of FGF-9. In
addition, we are attempting to reconstitute the levels of FGF-9 in the FGF-9 shRNA
knockdown cells by knocking in FGF-9 into the FGF-9 shRNA cells for further
revalidation.
If FGF-9 is essential during MM development, then antagonising FGF-9 should
attenuate MM growth. However, as FGF-9 is crucial to lung development, knockout
of FGF-9 is lethal due to lung hypoplasia and hence intrauterine death of the mice
(Colvin et al., 2001b). In theory, FGF-9 may be knocked out specifically from the
mesothelium in vivo to generate conditional knockout mice which can be subjected
to asbestos inhalation to generate MM (Yin et al., 2011, Lin et al., 2006). Showing
that condition knockout mice had reduced tumour development will help strengthen
our observations.

161
The use of shRNA knockdown of FGF-9 MM cells allows us to provide proof-of-
principle data confirming the essential function of FGF-9 in MM pathogenesis. To
target FGF-9 theoretically, several potential strategies can be attempted. FGF-9
monoclonal antibodies are available although their efficacies in vivo have not been
well established. The pharmacokinetics of these antibodies have not been
thoroughly studied even for common laboratory animals. Future studies will need
to establish the optimal route and dose of delivery, particularly to the pleural
tissues, if these antibodies are tested against MM. Another approach will be to
employ FGF-9 receptor antagonist or small inhibitors to the downstream signalling
of FGF-9. FGF-9 binds to FGFR2 and FGFR3 (Hecht et al., 1995) and activates
the IIIc isoform of the two receptors best (Santos-Ocampo et al., 1996). Hence,
any therapeutic attempts directing at FGFRs must target this isoform. Other FGF
family members can also bind to this isoform (Guillemot and Zimmer, 2011), thus
demonstrating the limitations of using FGFR antagonists to antagonise FGF-9
activity.
It would be interesting in future to examine the effects of FGF-9 over-expression in
normal murine mesothelial cells and assess for malignant transformation via
anchorage-independent growth assay in soft agar (Combes, 1999). Since FGF-9 is
a potent mitogen factor (as shown in Chapter 4), it is hypothesized that over-
expressing the levels of FGF-9 in the normal mesothelial cells would cause the
cells to transform into malignant cells causing them to form colonies in soft agar: a
feature that non-transformed cells are unable to. The malignant potential of the
FGF-9 over-expressed cells can then be tested using murine models.
It is also important to be cautious that our results in knocking down FGF-9,
although impressive in murine studies, still require validation in human MM. Our

162
group will attempt to knock down FGF-9 in human MM cells and test their biology
in vitro and in vivo.
In conclusion, our in vitro findings demonstrating the role of FGF-9 in MM
pathobiology is backed up with this in vivo study, confirming that FGF-9 is a key
factor in MM. Like other cancers caused by genetic mutations, there is a possibility
that there may be mutations occurring in the FGF-9 gene as well as in its receptors
that may lead to MM development. This is explored in the next chapter.

163
____________________________________________
MUTATIONS OF FGF-9 AND ITS RECEPTORS IN
MALIGNANT MESOTHELIOMA PATHOBIOLOGY
CHAPTER 6

164
6.0 MUTATIONS OF FGF-9 AND ITS RECEPTORS IN MALIGNANT
MESOTHELIOMA PATHOBIOLOGY
6.1 Introduction
As FGF-9 plays an important role in MM, it is prudent to investigate if there are any
mutations occurring in FGF-9 and its receptors that may impact on its signalling,
which lead to the development of this disease. One study on 203 tumours and cell
lines have reported six somatic mutations occurring on the FGF-9 gene; one
frameshift, four missense and one nonsense mutation in 10 (six colorectal and four
endometrial) tumour samples (Abdel-Rahman et al., 2008). Mutations of FGF-9 in
MM tumours have not been investigated before.
In addition, mutations in FGFR2 have also been described in 12% of endometrial
cancers (Dutt et al., 2008) while somatic mutations in the FGFR3 coding sequence
have been reported in 50% of bladder cancers (Cappellen et al., 1999), leading to
aberrant protein folding and hence constitutive activation of the receptor (Naski et
al., 1996, Di Martino et al., 2009). All of these lead to the next question whether
there are any mutations occurring in FGF-9 and its receptors, FGFR2 and FGFR3,
which are important in MM and is discussed in this chapter.
6.2 Results
6.2.1 Mutations in FGF-9
To examine if there are any mutations occurring in FGF-9, the conditions for the
PCR reaction using the primers to amplify the three exons in FGF-9 had to be first
optimised for the concentrations of Mg2+, the presence or absence of Q solution
and the melting temperature, T1 °C. Figure 6.1 shows a representative image of a

165
PCR optimisation study conducted by varying the conditions stated above. Each
column represents a different condition. DNA in RNAse-free water was used as a
negative control for the PCR reaction. The condition that gives the best band
intensity, with no smear was selected for later PCR reactions conducted on eight
human MM cell lines (Section 2.7.3). The sequences obtained from each sample
following the unpurified PD+ capillary sequencing method was compared against
each other and the consensus sequence from the NCBI database using Vector
NTI Advance 11 software package. Figure 6.2 shows the representative diagram
from AlignX demonstrating that all the sequences from the 8 MM samples align
with a 100% similarity for FGF-9 exons 1 and 2. Figure 6.3A shows the single point
mutation from A to G in the FGF-9 exon 3 for all the human MM cell lines tested
except for the NO36 cell line. Translation of the DNA sequence gave the same
amino acid sequence demonstrating that the single point mutation was a silent
mutation for FGF-9 (Figure 6.3B). The pilot study did not find any significant FGF-9
mutations in the MM human samples studied.

166
Figure 6.1: Agarose gel showing the band intensities for different conditions for a PCR reaction.
Each column represents a different condition for the PCR reaction. The top half of the gel shows the PCR products for FGF-9 exon 2
while the bottom half is for exon 3. The second column at the conditions of 62°C, 1.5 mM Mg2+ and the presence of Q solution was
selected for the FGF-9 exons 2 and 3 PCR reaction conditions.
Conditions: 62°C 1.5 mM Mg2+ Presence of Q solution
FGF-9 Exon 2
FGF-9 Exon 3

167
Figure 6.2: Sequence alignment for FGF-9 exon 1.
The representative image of a 100% sequence alignment for FGF-9 exons 1 and 2. All eight sequences for the eight human MM cell
lines tested were aligned with the sequence obtained from NCBI database using the AlignX program from the Vector NTI Advance 11
software package. The figure shows a 100% sequence alignment of all the sequences as depicted from the highlighted sequences in
yellow.

168
Figure 6.3: Sequence alignment and amino acid translation for FGF-9 exon 3.
(A) The image of a 98.6% sequence alignment for FGF-9 exon 3. All eight sequences for the eight human MM cell lines tested were
aligned with the sequence obtained from NCBI database using the AlignX program from the Vector NTI Advance 11 software package.
The figure shows one column highlighted in turquoise while the rest of the sequences were highlighted in yellow, demonstrating the
mutation occurring in that point of sequence (in turquoise), hence giving a 98.6% sequence alignment. (B) The amino acid translation
for one of the DNA sequences that were mutated. The amino acid sequence is exactly the same as the one for the wild type,
demonstrating the point mutation is a silent DNA mutation.

169
6.3A

170
6.3B

171
6.2.2 Expression of FGF-9 Receptors in MM Cells
To investigate if there are any mutations occurring in the FGF-9 receptors, it is
important to first determine if the receptors are present in MM cells. The presence
of the FGFR2 and FGFR3, which are the receptors for FGF-9, were identified
using immunofluorescence studies. Figures 6.4A – F demonstrates the strong
fluorescence observed in both the human (NO36) and murine (AB1) MM cell lines
tested for the presence of FGFR2 while Figures 6.5A – F showed the presence of
FGFR3 in both the human (NO36) and murine (AB2) MM cell lines. Although there
is only one paper to date that has reported the basal expression levels of FGFR2
in MM cells using real-time PCR (Stapelberg et al., 2005), the cell lines used were
different from this study. Hence, to our knowledge, this is the first study which
confirms the presence of FGFR2 and FGFR3 in both the human and murine MM
cells using immunofluorescence.

172
Figure 6.4: Expression and distribution of FGFR2 in the human NO36 and murine AB1 MM cell lines.
The expression of FGFR2 in human NO36 and murine AB1 MM cell lines is observed from the green fluorescent intensity in A & D.
The nuclei of the cells are shown by the blue intensity of the DAPI staining in images B & E. Images C & F show the distribution of
FGFR2 in the nuclei and cytoplasm of the cells from the merged images of the FITC and DAPI staining. The images are
representatives from the immunofluorescence studies conducted on eight human and three murine MM cell lines tested.
FGFR2-FITC DAPI Merge
NO36
AB1
6.4D 6.4F 6.4E
6.4A 6.4B 6.4C

173
Figure 6.5: Expression and distribution of FGFR3 in the human NO36 and murine AB2 MM cell lines.
The expression of FGFR3 in human NO36 and murine AB2 MM cell lines is observed from the green fluorescent intensity in A & D.
The nuclei of the cells are shown by the blue intensity of the DAPI staining in images B & E. Images C & F show the distribution of
FGFR2 in the nuclei and cytoplasm of the cells from the merged images of the FITC and DAPI staining. The images are
representatives from the immunofluorescence studies conducted on eight human and three murine MM cell lines tested.
FGFR3-FITC DAPI Merge
NO36
AB2
6.5A
6.5D 6.5F
6.5B 6.5C
6.5E

174
6.2.3 Mutations in FGFR2 and FGFR3 in MM Cell Lines and Tumour Biopsies
After confirming that FGFR2 and FGFR3 are present in MM, mutational
sequencing were carried out to determine if mutations occurred on FGFR2 and
FGFR3 were important in MM disease. An optimisation study was conducted to
decide on the best condition for each primer for the PCR reaction. In the early pilot
experiment conducted on 8 human MM cell lines on all the exons in FGFR2 and
FGFR3, two mutations were found in FGFR2. Figure 6.6A shows an image from
AlignX demonstrating that all but one sequence from the ONE58 human MM cell
line, align with a 100% similarity for FGFR2 exon 6, which codes for the IgII
domain of the receptor. Figure 6.6B shows the DNA mutation identified after
comparing with the consensus sequence obtained from the NCBI database and
Figure 6.6C depicts the amino acid substitution from glycine to serine occurring at
the point of mutation. The second mutation identified, occurred at Exon 11, which
codes for the juxta-membrane region in FGFR2. Figure 6.7A shows the image
obtained from AlignX demonstrating the point mutation from C to T in CRL-2081
and CRL-5820 cell lines. Figure 6.7B depicts the image obtained from ABI
sequence scanner while Figure 6.7C demonstrates the amino acid translation
showing a deletion of arginine amino acid in the two cell lines. There were no
mutations found in the other exons of FGFR2 and FGFR3 (image not shown).
As there were two mutations identified in FGFR2, a further mutational sequencing
analysis was performed on MM tumour biopsies. A total of 50 frozen MM tumour
biopsy samples were obtained from Professor Jenette Creaney, National Centre
for Asbestos Related Diseases, Perth, Western Australia of which 31 of them were
of the epitheloid subtype. There were eight biphasic and four sarcomatoid
subtypes while the histology for the remaining seven was unspecified. Figure 6.8
shows the image from AlignX demonstrating that out of the 50 tumour samples

175
tested, only one tumour (epitheloid subtype) sample showed the same DNA
mutation (G substituted by A) seen in ONE58 MM cell line. No mutations were
found in Exon 11 in the 50 tumour samples tested (image not shown). The data
obtained showed mutations that were not occurring in the hotspots area of the
receptors in other cancers.

176
Figure 6.6 : Amino acid substitution occurring in exon 6 of FGFR2.
(A) A diagram from AlignX software showing the single point mutation occurring in exon 6 of ONE58 MM cell line. All eight sequences
for the eight human MM cell lines tested were aligned with the sequence obtained from NCBI database using the AlignX program from
the Vector NTI Advance 11 software package. The figure shows one column highlighted in turquoise while the rest of the sequences
were highlighted in yellow, demonstrating the mutation occurring in that cell line (ONE58) at that point of sequence (in turquoise). (B)
The DNA mutation and the histogram as shown from ABI sequence scanner software. (C) The amino acid translation and the amino
acid substitution obtained from Vector NTI Advance 11 software package.

177
6.6A

178
6.6B
ONE58 Exon 6 (IgII domain): 72G→A
Mutation: G T G T A G T G G
Wild type: G
6.6C
Type of mutation: amino acid substitution
>rf 1 FGFR2 exon 6
ALEPHYGKCGPI*QGKLYLCGGE*IRVHQSHVPPGCC
>rf 1 ONE58 exon 6
ALEPHYGKCGPI*QGKLYLCSGE*IRVHQSHVPPGCC

179
Figure 6.7 : Amino acid substitution occurring in exon 11 of FGFR2.
(A) A diagram from AlignX software showing the single point mutation occurring in CRL-2081 and CRL-5820 MM cell lines. All eight
sequences for the eight human MM cell lines tested were aligned with the sequence obtained from NCBI database using the AlignX
program from the Vector NTI Advance 11 software package. The figure shows one column highlighted in turquoise while the rest of the
sequences were highlighted in yellow, demonstrating the mutation occurring in those cell lines (CRL-2081 and CRL-5820) at that point of
sequence (in turquoise). (B) The DNA mutation and the histogram as shown from ABI sequence scanner software. (C) The amino acid
translation and the amino acid substitution obtained from Vector NTI software.

180
6.7A

181
6.7B
CRL-2081 and CRL-5820 Exon 11 (Juxta-membrane domain): 124C→T
Mutation: A A C A T G A C C A A
Wild type: C
6.7C
Type of mutation: deletion
>rf 1 FGFR2 exon 11
RLEEKRRLQLPQTTWR*PFTA*GSS*SPVWW*QSSCAE*RTRPRSQTSAASRLCT
S*PNVSPCGD
>rf 1 ONE58 exon 11
RLEEKRRLQLPQTTWR*PFTA*GSS*SPVWW*QSSCAE*RT*PRSQTSAASRLCT
S*PNVSPCGD

182
Figure 6.8: Amino acid substitution occurring in exon 6 of FGFR2 in tumour biopsy.
A diagram from AlignX software showing the single point mutation occurring in one tumour biopsy sample (2005006) that had the
same mutation (G substituted by A) as in ONE58 MM cell line (Figure 6.6). All 50 sequences for the 50 human MM tumour biopsies
tested were aligned with the sequence obtained from NCBI database using the AlignX program from the Vector NTI Advance 11
software package. The figure shows one column highlighted in turquoise while the rest of the sequences were highlighted in yellow,
demonstrating the mutation occurring in the tumour biopsy (2005006) at that point of sequence (in turquoise).

183

184
6.3 Discussion
Based on our mutational analyses data, we report that there are no consistent or
frequent mutations occurring in all the three exons of FGF-9 and in all 19 exons of
FGFR3 in the MM samples that we tested. There are only two distinct mutations
occurring in two separate FGFR2 exon regions in two out of the 58 MM samples
tested. This is the first study carried out to analyse mutations on FGF-9 and both
its receptors in MM. DNA sequencing was carried out in both directions (on both
strands of the DNA) for verification.
Since FGF-9 is important in MM development (Chapters 4 and 5) and because
there have been no studies examining the mutational status of FGF-9 and its
receptors in MM, investigating if there are any mutations occurring in these genes
in MM samples is useful to understand if the mutations are implicated in this
cancer. Had mutations been recognised, it would be interesting to correlate the
mutational status of FGF-9/FGFR with the patient’s prognosis.
In our study, the frequency of the two FGFR2 mutations in the MM samples that
we tested is very low (3.4%) but comparable to the frequencies detected in
endometroid (1 in 46 samples, 2.2%) and serous (1 in 41 samples, 2.4%) ovarian
cancer (Byron et al., 2010) and slightly lower in melanoma (15 of 116 melanoma
cell lines and 8 of 100 uncultured metastases and primary tumours, 10.6%)
(Gartside et al., 2009). While we cannot exclude that a larger cohort may find
mutations, the chances of missing any mutations in our samples are very unlikely.
The low mutational frequency in FGFR2 exons and the lack of mutations in the
FGFR3 exons in our study depicts that the mutations in the exons of FGF-9
receptors are unlikely to be important in MM tumour development. Even the
mutation detected in FGF-9 exon 3 resulted in the same amino acid sequence

185
demonstrating that mutations in FGF-9 exons are very rare. This shows that the
DNA sequence of FGF-9 is conserved, a feature that is very important for a
molecule that has a key role in driving cancer progression. One paper recently
reported six distinct FGF-9 mutations in colorectal and endometrial samples
(Abdel-Rahman et al., 2008). All six mutations were loss-of-function mutations,
demonstrating the tumour-suppressor properties of FGF-9 in these cancers
(Abdel-Rahman et al., 2008). As FGF-9 has a key role in MM tumour development,
it is less likely that any loss-of-function mutations would occur. Mutations however
can result in the gain-of-function, leading to aberrant activity of the growth factor
although no such mutations have been identified in FGF-9 to date. Our data,
together with our results from earlier chapters, further strengthens the importance
of FGF-9 in MM. This allows the possibility of FGF-9 and/or its receptors as
therapeutic targets for MM.
The mutations that we detected are not the common mutations occurring in the
hotspot regions of FGFR2. Previous studies have reported mutations in FGFR2
within the IIIa and IIIc exons hotspots in other diseases (Kan et al., 2002, Kan et
al., 2004, Meyers et al., 1996, Jang et al., 2001). Here we identified only one
mutation (G substituted by A) at exon 6 in FGFR2 in one of eight MM cell lines and
2% of the 50 MM tumour samples tested. Exon 6 codes for IgII domain in FGFR2
which is necessary to induce receptor dimerization following ligand binding
(Wiesmann et al., 2000). The mutation we found causes glycine (wild type) to be
replaced by serine. The second mutation identified occurred at exon 11, which
codes for the juxta-membrane region in FGFR2, resulting in a deletion of arginine
amino acid in two out of eight MM cell lines. However, this mutation was not found
in the 50 MM tumour samples tested. The juxta-membrane region in FGFR is
required and sufficient for the interaction and phosphorylation of FRS2 (Xu et al.,

186
1998, Lin et al., 1998, Ong et al., 2000, Dhalluin et al., 2000). FRS2 is an adapter
protein that links FGFRs to the MAPK signalling pathway (Eswarakumar et al.,
2005, Klint and Claesson-Welsh, 1999). It is not known if the mutations that we
detected in exons 6 and 11 of FGFR2 are germline mutations or a mutation as a
result of MM. This can be verified by examining if the same exons in FGFR2 are
mutated in benign tissues obtained from the same patient. However, this is outside
the scope of my thesis. Nonetheless, the mutations we detected are infrequent in
MM and have not been previously reported in other diseases, suggesting that the
two mutations in the exon regions of the gene may not be important.
This technique proves to be robust. Although the sensitivity of the capillary
electrophoresis method used in this study to sequence DNA has not been
assessed, this method has long been established and used in many previous
studies (Bai et al., 2010, Dragileva et al., 2009, Novak-Weekley et al., 2012).
This mutation study only focused on all the exons of FGF-9 and its receptors.
Future work will need to examine the promoter and the regulatory regions which
may account for increased FGF-9 expression in MM. We will examine for any
methylation status occurring in the promoter region of FGF-9 that could lead to
increase copy numbers and hence the amplification of the molecule. These
experiments to sequence the promoter region of FGF-9 are necessary to unravel
as to why FGF-9 is increased or how the downstream signalling is affected in MM.
Yet, the mutations may not be the only cause for its over-expression in MM. We
cannot rule out the possibility of the up-stream effect of other molecules on FGF-9
signalling that may also implicate on FGF-9 signalling in MM.

187
____________________________________________
SUMMARY
CHAPTER 7

188
7.0 SUMMARY
Given the rising incidence of MM and the lack of effective therapy, there is a
desperate need to identify the key molecules. Such molecules would have a role
as therapeutic, diagnostic and prognostic markers for MM. Understanding the
molecular drivers of MM development may help establish new treatments. Prompt
diagnosis and reliable predictors of survival will significantly benefit patient care.
Our preliminary pilot experiments uncovered that FGF-9 mRNA was significantly
up-regulated in MM compared to metastatic adenocarcinoma and benign pleural
tissues. This thesis tested the general hypothesis that FGF-9 plays a vital role in
MM pathobiology and that its inhibition retards MM growth. FGF-9 is a secreted
growth factor, first identified in human glioma cells (Miyamoto et al., 1993, Naruo et
al., 1993). This growth factor activates FGFR2 and FGFR3 (Hecht et al., 1995) and
is vital for lung development (Colvin et al., 2001b).
Using a wide array of techniques, we have demonstrated, in Chapter 3, the
significant up-regulation of FGF-9 mRNA and protein in MM cells, tissues and
pleural fluids compared to metastatic adenocarcinomas and benign pleural
diseases. Our results are robust and consistent in a large cohort of more than
1000 samples in total. Previous studies have examined the role of FGF-9 in
embryonic development (Colvin et al., 2001a, Geske, 2008, Antoine et al., 2007,
White et al., 2006, Colvin et al., 1999) and in other cancers such as prostate
(Murphy et al., 2010, Kwabi-Addo et al., 2004, Li et al., 2008), lung (Chien-Kai et
al., 2009, Betsuyaku et al., 2011) and endometrial cancers (Hendrix et al., 2006).
We are the first to report its up-regulation in MM.

189
FGF-9 is a member of the FGF family which are heparin-binding polypeptides,
involved in various metabolic processes such as cell proliferation, migration, tissue
repair, cell differentiation and morphogenesis (Antoine et al., 2007, Maggie et al.,
2009, Ornitz and Itoh, 2001). We showed that recombinant FGF-9 stimulated
dose- and time-dependent cell proliferation in the human and murine MM cell lines
tested. In addition, FGF-9 is involved in MM cell migration and invasion.
Stimulation of human and murine MM cells with FGF-9 caused the release of
cytokines important in MM pathobiology (IL-8, MIP-2, VEGF and MCP-1) (Galffy et
al., 1999b, Masood et al., 2003, Hung et al., 2007, Stathopoulos et al., 2008).
FGF-9-induced cell proliferation in MM was mediated via ERK, JNK and p38
pathways.
FGF-9 knockdown MM cells have retarded growth in vivo using a heterotopic
(subcutaneous) model and an orthotopic (intraperitoneal) model. The growth
inhibition was abolished when experiments were conducted in athymic nude mice,
suggesting that FGF-9 induction of MM growth in vivo is partly mediated by
avoidance of immune clearance.
We also examined for mutations in FGF-9 and both its receptors, FGFR2 and
FGFR3 and found no consistent or significant mutations in the exons of these
genes. The highly conserved sequencing results among all samples examined
may indirectly demonstrate the importance of these proteins in MM.
Our results, collectively as a whole, support the idea that FGF-9 plays a vital role in
MM tumour development. Hence, strategies that antagonise FGF-9 activity may be
a valuable approach to treat MM.

190
Our data shows that FGF-9, in pleural fluids, is higher in MM than in non-MM
cases. The clinical utility, if any, of FGF-9 warrants examination. This would
include its diagnostic accuracy and how it can add value to existing diagnostic
tools.
Strategies to target FGF-9 including using an antisense (Morrison, 1991), a soluble
FGFR (Li et al., 1994), neutralising anti-FGF-9 antibody (Hori et al., 1991, Stan et
al., 1995, Murai et al., 1996, Takahashi et al., 1991) and FGF-9 peptides or small
molecule inhibitors should be tested. Their efficacies in vivo, pharmacokinetics in
the pleura, toxicities and many other issues need evaluation.
The possible prognostic role as well as disease monitoring tool of FGF-9 can also
be studied in MM in future. Whether FGF-9 increases with the advancing stages of
MM has not been studied yet. Measurement of FGF-9 in pleural fluids during the
course of the disease is feasible using longitudinal pleural fluid samples collected
through indwelling pleural catheters. We will also examine if FGF-9 can be used to
monitor treatment outcomes. FGF-9 expression in pleural fluids in response to
chemotherapy agents will be investigated.
Although we have demonstrated the role of FGF-9 in MM using exogenous
recombinant FGF-9, we are interested to examine if the FGF-9 present in the
pleural fluids are all active in rendering a response. Hence, we will investigate if
the endogenous FGF-9 present in the pleural fluids is able to elicit similar
responses as the recombinant FGF-9 used in our in vitro assays.

191
It will also be interesting to further define the signalling pathways involved in FGF-
9-FGFR pathway in MM. The cell signalling is a complex network but a further
understanding on the actions of FGF-9 on MM will no doubt provide a greater
insight on the development of MM and may lead to effective interventions for the
treatment of MM. We are interested in examining the interactions of FGF-9
signalling with other signalling pathways involved in MM cancer, such as the Sonic
Hedgehog, Wnt and TGF-β signalling pathways.
In addition, studies are in progress to establish the therapeutic effect of the
different methods in inhibiting FGF-9 in MM in in vivo models. FGF-9 neutralizing
antibody and the receptor antagonists are now commercially available to allow
assessment of the efficacy of the reagents in antagonising FGF-9 activity.
Knocking down FGF-9 in human MM cells and confirming the effects in a xenograft
murine model are warranted.
We will also examine if methylation or genetic defects in the promoter or regulatory
regions of FGF-9 is one of the causes for the abundance of FGF-9 in MM. Studies
will be conducted to investigate if the asbestos mineral can up-regulate FGF-9
mRNA and protein.
While the results on the role of FGF-9 in MM are exciting, the role of FGF-9
receptors needs to be studied. Investigating the expression profile and the
signalling of the different receptor isoforms in MM cells will no doubt provide an
insight on strategies to treat MM.

192
Our findings are new and only represent the tip of the iceberg of a very highly
complex signalling network of FGF-9 in MM involving numerous other signalling
pathways that we have yet to unravel. Despite that, our results are very promising
and may provide the key to understanding the pathogenesis of MM.

i
APPENDIX

ii
GENERAL BUFFERS AND SOLUTIONS
Scott’s tap water substitute
3.5 g sodium bicarbonate
20 g magnesium sulphate
1 L distilled water
Sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0)
2.4 g Tri-sodium citrate (dehydrate)
1 L distilled water
Adjust pH to 6.0 with 1 M HCl and then add 0.5 mL of Tween-20.
10X EDTA/Tris buffer
6.25 g Tris HCl
12.5 g EDTA
8.0 g sodium citrate
1 L H2O
Adjust pH to 8.0 and make it up to a final volume of 2 L
Breaking buffer
50 mM Tris pH 7.5
0.5 mM EGTA
150 mM NaCl
1% v/v Triton X 100
50 mL ddH2O
10% v/v Protease inhibitor cocktail
1 tablet of PhosSTOP phosphatase inhibitor cocktail in 10 mL extraction solution
Protease inhibitor cocktail and PhosSTOP Phosphatase inhibitor cocktail added
immediately prior to use.

iii
Transfer buffer (store at 4°C)
39 mM Glycine
48 mM Tris base
10% Methanol
SDS running buffer
25 mM Tris
250 mM Glycine (electrophoresis grade) (pH 8.3)
0.5% w/v SDS
Tris buffered saline (TBS)
10 mM Tris pH 7.6
500 mM NaCl
1% Tween-20
50X TAE running buffer
242 g Tris base
57.10 mL Glacial acetic acid
5 mM EDTA (pH 8.0)
1 L ddH2O

iv
REFERENCES Abdel-Rahman, W. M., Kalinina, J., Shoman, S., Eissa, S., Ollikainen, M.,
Elomaa, O., Eliseenkova, A. V., Bützow, R., Mohammadi, M. &
Peltomäki, P. 2008. Somatic FGF9 mutations in colorectal and
endometrial carcinomas associated with membranous β-catenin. Human
Mutation, 29, 390-397.
Abuharbeid, S., Czubayko, F. & Aigner, A. 2006. The fibroblast growth factor-
binding protein FGF-BP. The International Journal of Biochemistry & Cell
Biology, 38, 1463-1468.
Altomare, D. A., Vaslet, C. A., Skele, K. L., De Rienzo, A., Devarajan, K.,
Jhanwar, S. C., Mcclatchey, A. I., Kane, A. B. & Testa, J. R. 2005. A
Mouse Model Recapitulating Molecular Features of Human
Mesothelioma. Cancer Research, 65, 8090-8095.
Andrews, P. M., Porter, K.R. 1973. The ultrastructural morphology and possible
functional significance of mesothelial microvilli. . Anat. Rec., 177.
Antman, K. H. 1980. Malignant Mesothelioma. New England Journal of
Medicine, 303, 200-202.
Antman, K. H. 1981. Clinical presentation and natural history of benign and
malignant mesothelioma. Semin Oncol, 8, 313-20.
Antoine, M., Wirz, W., Tag, C. G., Gressner, A. M., Marvituna, M., Wycislo, M.,
Hellerbrand, C. & Kiefer, P. 2007. Expression and function of fibroblast
growth factor (FGF) 9 in hepatic stellate cells and its role in toxic liver
injury. Biochemical and Biophysical Research Communications, 361,
335-341.

v
Antony, V., Godbey, S., Kunkel, S., Hott, J., Hartman, D., Burdick, M. & Strieter,
R. 1993. Recruitment of inflammatory cells to the pleural space.
Chemotactic cytokines, IL-8, and monocyte chemotactic peptide-1 in
human pleural fluids. The Journal of Immunology, 151, 7216-7223.
Antony, V. B., Hott, J. W., Kunkel, S. L., Godbey, S. W., Burdick, M. D. &
Strieter, R. M. 1995. Pleural mesothelial cell expression of C-C
(monocyte chemotactic peptide) and C-X-C (interleukin 8) chemokines.
American Journal of Respiratory Cell and Molecular Biology, 12, 581-8.
Armelin, H. A. 1973. Pituitary Extracts and Steroid Hormones in the Control of
3T3 Cell Growth. Proceedings of the National Academy of Sciences, 70,
2702-2706.
Attanoos, R. L. & Gibbs, A. R. 2008. The comparative accuracy of different
pleural biopsy techniques in the diagnosis of malignant mesothelioma.
Histopathology, 53, 340-344.
Ault, J. G., Cole, R. W., Jensen, C. G., Jensen, L. C. W., Bachert, L. A. &
Rieder, C. L. 1995. Behavior of Crocidolite Asbestos during Mitosis in
Living Vertebrate Lung Epithelial Cells. Cancer Research, 55, 792-798.
Avet-Loiseau, H., Facon, T., Daviet, A., Godon, C., Rapp, M.-J., Harousseau,
J.-L., Grosbois, B., Bataille, R. & Myélome, F. T. I. F. D. 1999. 14q32
Translocations and Monosomy 13 Observed in Monoclonal Gammopathy
of Undetermined Significance Delineate a Multistep Process for the
Oncogenesis of Multiple Myeloma. Cancer Research, 59, 4546-4550.
Avet-Loiseau, H., Li, J.-Y., Facon, T., Brigaudeau, C., Morineau, N., Maloisel,
F., Rapp, M.-J., Talmant, P., Trimoreau, F., Jaccard, A., Harousseau, J.-

vi
L. & Bataille, R. 1998. High Incidence of Translocations
t(11;14)(q13;q32) and t(4;14)(p16;q32) in Patients with Plasma Cell
Malignancies. Cancer Research, 58, 5640-5645.
Avivi, A., Yayon, A. & Givol, D. 1993. A novel form of FGF receptor-3 using an
alternative exon in the immunoglobulin domain III. FEBS Lett, 330, 249 -
252.
Bai, X., Zhang, W., Orantes, L., Jun, T.-H., Mittapalli, O., Mian, M. a. R. &
Michel, A. P. 2010. Combining Next-Generation Sequencing Strategies
for Rapid Molecular Resource Development from an Invasive Aphid
Species, <italic>Aphis glycines</italic>. PLoS ONE, 5, e11370.
Bakalos, D., Constantakis, N., Tsicricas, T. 1974. Distinction of mononuclear
macrophages from mesothelial cells in pleural and peritoneal effusions.
Acta Cytol., 18, 20-2.
Baumann M.H., S. C., Sahn S.A., Kinasewitz G.T. 1996. Pleural macrophages
differentially alter pleural mesothelial cell glycosaminoglycan production.
Exp Lung Res., 22, 101-11.
Behr, B., Leucht, P., Longaker, M. T. & Quarto, N. 2010. Fgf-9 is required for
angiogenesis and osteogenesis in long bone repair. Proceedings of the
National Academy of Sciences, 107, 11853-11858.
Bellot, F., Crumley, G., Kaplow, J. M., Schlessinger, J., Jaye, M. And Dionne, C.
A. 1991. Ligand-induced transphosphorylation between different FGF
receptors. EMBO J, 10, 2849-2854.

vii
Bernard-Pierrot, I., Brams, A., Dunois-Lardé, C., Caillault, A., Diez De Medina,
S. G., Cappellen, D., Graff, G., Thiery, J. P., Chopin, D., Ricol, D. &
Radvanyi, F. 2006. Oncogenic properties of the mutated forms of
fibroblast growth factor receptor 3b. Carcinogenesis, 27, 740-747.
Bernstein, D. M. & Hoskins, J. A. 2006. The health effects of chrysotile: Current
perspective based upon recent data. Regulatory Toxicology and
Pharmacology, 45, 252-264.
Betsuyaku, T., Yin, Y., Miao, J. & M., O. D. 2011. A Mouse Model For Fibroblast
Growth Factor (FGF) 9-Induced Adenocarcinoma Of The Lung. Am. J.
Respir. Crit. Care Med., 183, A5075.
Bielefeldt-Ohmann, H., Marzo, A. L., Himbeck, R. P., Jarnicki, A. G., Robinson,
B. W. S. & Fitzpatrick, D. R. 1995. Interleukin-6 involvement in
mesothelioma pathobiology: inhibition by interferon α immunotherapy.
Cancer Immunology, Immunotherapy, 40, 241-250.
Bograd, A., Suzuki, K., Vertes, E., Colovos, C., Morales, E., Sadelain, M. &
Adusumilli, P. 2011. Immune responses and immunotherapeutic
interventions in malignant pleural mesothelioma. Cancer Immunology,
Immunotherapy, 60, 1509-1527.
Boylan, A. M., Rüegg, C., Kim, K. J., Hébert, C. A., Hoeffel, J. M., Pytela, R.,
Sheppard, D., Goldstein, I. M. & Broaddus, V. C. 1992. Evidence of a
role for mesothelial cell-derived interleukin 8 in the pathogenesis of
asbestos-induced pleurisy in rabbits. The Journal of Clinical
Investigation, 89, 1257-1267.

viii
Braun, S., Auf Dem Keller, U., Steiling H. & Werner, S. 2004. Fibroblast growth
factors in epithelial repair and cytoprotection. Philos Trans R Soc Lond B
Biol Sci. , 359, 753-757.
Brauner A., H. B., Wretlind B. 1993. Interleukin-6 and interleukin-8 in dialysate
and serum from patients on continuous ambulatory peritoneal dialysis.
Am J Kidney Dis., 22, 430-5.
Breborowicz A., W. J., Polubinska A., Wieczorowska-Tobis K., Martis L., and
Oreopoulos D. G. 1998. Role of peritoneal mesothelial cells and
fibroblasts in the synthesis of hyaluronan during peritoneal dialysis Perit
Dial Int. , 18, 382-6.
British Thoracic Society Standards of Care Committee 2001. Statement on
malignant mesothelioma in the United Kingdom. Thorax, 56, 250-265.
Bugra-Bilge, K. & Cinaroglu, A. 2005. Transient Activation of Erk Is Required for
FGF9 Mediated Proliferation of Retinal Muller Cells in vitro. Invest.
Ophthalmol. Vis. Sci., 46, 5143-.
Byron, S. A. 2008. Inhibition of activated fibroblast growth factor receptor 2 in
endometrial cancer cells induces cell death despite PTEN abrogation.
Cancer Res., 68, 6902-6907.
Byron, S. A., Gartside, M. G., Wellens, C. L., Goodfellow, P. J., Birrer, M. J.,
Campbell, I. G. & Pollock, P. M. 2010. FGFR2 mutations are rare across
histologic subtypes of ovarian cancer. Gynecologic Oncology, 117, 125-
129.

ix
Campbell, N. P. & Kindler, H. L. 2011. Update on Malignant Pleural
Mesothelioma. Semin Respir Crit Care Med, 32, 102,110.
Cannistra, S. A., Ottensmeier, C., Tidy, J., Defranzo, B. 1994. Vascular cell
adhesion molecule-1 expressed by peritoneal mesothelium partly
mediates the binding of activated human T lymphocytes. Exp Hematol,
22, 996-1002.
Cappellen, D., De Oliveira, C., Ricol, D., De Medina, S., Bourdin, J., Sastre-
Garau, X., Chopin, D., Thiery, J. P. & Radvanyi, F. 1999. Frequent
activating mutations of FGFR3 in human bladder and cervix carcinomas.
Nat Genet, 23, 18-20.
Cappia, S., Righi, L., Mirabelli, D., Ceppi, P., Bacillo, E., Ardissone, F.,
Molinaro, L., Scagliotti, G. V. & Papotti, M. 2008. Prognostic Role of
Osteopontin Expression in Malignant Pleural Mesothelioma. American
Journal of Clinical Pathology, 130, 58-64.
Carbone, M., Kratzke, R. A. & Testa, J. R. 2002. The pathogenesis of
mesothelioma. Seminars in Oncology, 29, 2-17.
Carbone, M., Ly, B. H., Dodson, R. F., Pagano, I., Morris, P. T., Dogan, U. A.,
Gazdar, A. F., Pass, H. I. & Yang, H. 2012. Malignant mesothelioma:
Facts, Myths, and Hypotheses. Journal of Cellular Physiology, 227, 44-
58.
Castor C.W., N. B. 1969. Characteristics of normal and malignant human
mesothelial cells studied in vitro. Lab Invest, 20, 437-43.

x
Catterall, J., Gardner, M., Jones, L. & Turner, G. 1997. Binding of ovarian
cancer cells to immobilized hyaluronic acid. Glycoconjugate Journal, 14,
647-649.
Catterall, J. B., Jones, L. M. H. & Turner, G. A. 1999. Membrane protein
glycosylation and CD44 content in the adhesion of human ovarian cancer
cells to hyaluronan. Clinical and Experimental Metastasis, 17, 583-591.
Celli, G., Larochelle, W. J., Mackem, S., Sharp, R. & Merlino, G. 1998. Soluble
dominant-negative receptor uncovers essential roles for fibroblast growth
factors in multi-organ induction and patterning. EMBO J, 17, 1642-1655.
Chellaiah, A. T., Mcewen, D. G., Werner, S., Xu, J. & Ornitz, D. M. 1994.
Fibroblast growth factor receptor (FGFR) 3. Alternative splicing in
immunoglobulin-like domain III creates a receptor highly specific for
acidic FGF/FGF-1. Journal of Biological Chemistry, 269, 11620-11627.
Chen, C.-Y. A. & Shyu, A.-B. 1995. AU-rich elements: characterization and
importance in mRNA degradation. Trends in Biochemical Sciences, 20,
465-470.
Chen, C. Y., Xu, N. & Shyu, A. B. 1995. mRNA decay mediated by two distinct
AU-rich elements from c-fos and granulocyte-macrophage colony-
stimulating factor transcripts: different deadenylation kinetics and
uncoupling from translation. Mol. Cell. Biol., 15, 5777-5788.
Chen, J., Chiu, J., Chen, H., Chen, T., Yang, W. & Yang, A. 2000. Human
peritoneal mesothelial cells produce nitric oxide: induction by cytokines.
Peritoneal Dialysis International, 20, 772-777.

xi
Chen, T.-M., Hsu, C.-H., Tsai, S.-J. & Sun, H. S. 2010. AUF1 p42 isoform
selectively controls both steady-state and PGE2-induced FGF9 mRNA
decay. Nucleic Acids Research.
Chen, T.-M., Kuo, P.-L., Hsu, C.-H., Tsai, S.-J., Chen, M.-J., Lin, C.-W. & Sun,
H. S. 2007. Microsatellite in the 3′ untranslated region of human
fibroblast growth factor 9 (FGF9) gene exhibits pleiotropic effect on
modulating FGF9 protein expression. Human Mutation, 28, 98-98.
Cheng, D.-S., Rodriguez, R. M., Perkett, E. A., Rogers, J., Bienvenu, G.,
Lappalainen, U. & Light, R. W. 1999. Vascular Endothelial Growth Factor
in Pleural Fluid*. Chest, 116, 760-765.
Chesi, M., Nardini, E., Brents, L. A., Schrock, E., Ried, T., Kuehl, W. M. &
Bergsagel, P. L. 1997. Frequent translocation t(4;14)(p16.3;q32.3) in
multiple myeloma is associated with increased expression and activating
mutations of fibroblast growth factor receptor 3. Nat Genet, 16, 260-264.
Chien-Kai, W., Han, C., Po-Hung, C., Jinghua Tsai, C., Yu-Chun, K., Jiunn-
Liang, K. & Pinpin, L. 2009. Aryl hydrocarbon receptor activation and
overexpression upregulated fibroblast growth factor-9 in human lung
adenocarcinomas. International Journal of Cancer, 125, 807-815.
Choe, N., Tanaka, S. & Kagan, E. 1998. Asbestos Fibers and Interleukin-1
Upregulate the Formation of Reactive Nitrogen Species in Rat Pleural
Mesothelial Cells. American Journal of Respiratory Cell and Molecular
Biology, 19, 226-236.

xii
Chuang, P.-C., Sun, H. S., Chen, T.-M. & Tsai, S.-J. 2006. Prostaglandin E2
Induces Fibroblast Growth Factor 9 via EP3-Dependent Protein Kinase
C{delta} and Elk-1 Signaling. Mol. Cell. Biol., 26, 8281-8292.
Churg, A. 1994. Deposition and Clearance of Chrysotile Asbestos. Annals of
Occupational Hygiene, 38, 625-633.
Cinaroglu, A., Ozmen, Y., Ozdemir, A., Ozcan, F., Ergorul, C., Cayirlioglu, P.,
Hicks, D. & Bugra, K. 2005. Expression and possible function of
fibroblast growth factor 9 (FGF9) and its cognate receptors FGFR2 and
FGFR3 in postnatal and adult retina. Journal of Neuroscience Research,
79, 329-339.
Colvin, J. S., Feldman, B., Nadeau, J. H., Goldfarb, M. & Ornitz, D. M. 1999.
Genomic organization and embryonic expression of the mouse fibroblast
growth factor 9 gene. Dev Dyn, 216, 72 - 88.
Colvin, J. S., Green, R. P., Schmahl, J., Capel, B. & Ornitz, D. M. 2001a. Male-
to-Female Sex Reversal in Mice Lacking Fibroblast Growth Factor 9.
Cell, 104, 875-889.
Colvin, J. S., White, A. C., Pratt, S. J. & Ornitz, D. M. 2001b. Lung hypoplasia
and neonatal death in Fgf9-null mice identify this gene as an essential
regulator of lung mesenchyme. Development, 128, 2095-2106.
Combes, R. 1999. Cell transformation assays as predictors of human
carcinogenicity. ATLA, 27, 745-767.
Craighead, J. E. & Kane, A. B. 1994. The Pathogenesis of Malignant and
Nonmalignant Serosal Lesions in Body Cavities Consequent to Asbestos

xiii
Exposure. The Mesothelial Cell and Mesothelioma, eds. M.-C. Jaurand
and J. Bignon. New York: Marcel Dekker, 79-102.
Creaney, J., Olsen, N. J., Brims, F., Dick, I. M., Musk, A. W., De Klerk, N. H.,
Skates, S. J. & Robinson, B. W. S. 2010. Serum Mesothelin for Early
Detection of Asbestos-Induced Cancer Malignant Mesothelioma. Cancer
Epidemiology Biomarkers & Prevention, 19, 2238-2246.
Creaney, J., Yeoman, D., Demelker, Y., Segal, A., Musk, A. W., Skates, S. J. &
Robinson, B. W. S. 2008. Comparison of Osteopontin, Megakaryocyte
Potentiating Factor, and Mesothelin Proteins as Markers in the Serum of
Patients with Malignant Mesothelioma. Journal of Thoracic Oncology, 3,
851-857 10.1097/JTO.0b013e318180477b.
Creaney, J., Yeoman, D., Naumoff, L. K., Hof, M., Segal, A., Musk, A. W., De
Klerk, N., Horick, N., Skates, S. J. & Robinson, B. W. S. 2007. Soluble
mesothelin in effusions: a useful tool for the diagnosis of malignant
mesothelioma. Thorax, 62, 569-576.
Crisanti, M. C., Wallace, A. F., Kapoor, V., Vandermeers, F., Dowling, M. L.,
Pereira, L. P., Coleman, K., Campling, B. G., Fridlender, Z. G., Kao, G.
D. & Albelda, S. M. 2009. The HDAC inhibitor panobinostat (LBH589)
inhibits mesothelioma and lung cancer cells in vitro and in vivo with
particular efficacy for small cell lung cancer. Molecular Cancer
Therapeutics, 8, 2221-2231.
Cunliffe, W. J. & Sugarbaker, P. H. 1989. Gastrointestinal malignancy:
Rationale for adjuvant therapy using early postoperative intraperitoneal
chemotherapy. British Journal of Surgery, 76, 1082-1090.

xiv
Davies, H. E., Sadler, R. S., Bielsa, S., Maskell, N. A., Rahman, N. M., Davies,
R. J. O., Ferry, B. L. & Lee, Y. C. G. 2009. Clinical Impact and Reliability
of Pleural Fluid Mesothelin in Undiagnosed Pleural Effusions. Am. J.
Respir. Crit. Care Med., 180, 437-444.
Davis, M. R., Manning, L. S., Whitaker, D., Garlepp, M. J. & Robinson, B. W. S.
1992. Establishment of a murine model of malignant mesothelioma.
International Journal of Cancer, 52, 881-886.
Dazzi H., H. P. S., Thatcher N., Wilkes S., Swindell R., Chatterjee A.K.. 1990.
Malignant pleural mesothelioma and epidermal growth factor receptor
(EGF-R): relationship of EGF-R with histology and survival using fixed
paraffin embedded tissue and the F4, monoclonal antibody. Br J Cancer,
61, 924-6.
Del Moral, P.-M., De Langhe, S. P., Sala, F. G., Veltmaat, J. M., Tefft, D.,
Wang, K., Warburton, D. & Bellusci, S. 2006. Differential role of FGF9 on
epithelium and mesenchyme in mouse embryonic lung. Developmental
Biology, 293, 77-89.
Delage B., Luong P., Phillips M., Chmielewska-Kassassir M., Cutts R., Chelala
C., Ghazaly E., Jithesh P. V., Fennell D., Lemoine N. R., Hawkes G.,
Wozniak L., Joel S., Jackson, R. C. & P.W., S. 2011. Arginine deprivation
with pegylated arginine deiminase regulates key metabolic genes in RNA
and DNA synthesis in ASS1-deficient malignant mesothelioma cells:
Clinical implications. . Molecular Cancer Therapeutics, 10, B139.

xv
Demirag, F., Ünsal, E., Yilmaz, A. & Çağlar, A. 2005. Prognostic Significance of
Vascular Endothelial Growth Factor, Tumor Necrosis, and Mitotic Activity
Index in Malignant Pleural Mesothelioma*. Chest, 128, 3382-3387.
Dhaene, K., Wauters, J., Weyn, B., Timmermans, J.-P. & Van Marck, E. 2000.
Expression profile of telomerase subunits in human pleural
mesothelioma. The Journal of Pathology, 190, 80-85.
Dhalluin, C., Yan, K. S., Plotnikova, O., Lee, K. W., Zeng, L., Kuti, M., Mujtaba,
S., Goldfarb, M. P. & Zhou, M.-M. 2000. Structural Basis of SNT PTB
Domain Interactions with Distinct Neurotrophic Receptors. Molecular cell,
6, 921-929.
Di Martino, E., L'hote, C. G., Kennedy, W., Tomlinson, D. C. & Knowles, M. A.
2009. Mutant fibroblast growth factor receptor 3 induces intracellular
signaling and cellular transformation in a cell type- and mutation-specific
manner. Oncogene, 28, 4306-4316.
Dinapoli, L., Batchvarov, J. & Capel, B. 2006. FGF9 promotes survival of germ
cells in the fetal testis. Development, 133, 1519-1527.
Dobbie J.W., P. T., Lloyd J., Johnson R.C. 1988. Phosphatidylcholine synthesis
by peritoneal mesothelium: its implications for peritoneal dialysis. Am J
Kidney Dis., 12, 31-6.
Dragileva, E., Hendricks, A., Teed, A., Gillis, T., Lopez, E. T., Friedberg, E. C.,
Kucherlapati, R., Edelmann, W., Lunetta, K. L., Macdonald, M. E. &
Wheeler, V. C. 2009. Intergenerational and striatal CAG repeat instability
in Huntington's disease knock-in mice involve different DNA repair
genes. Neurobiology of Disease, 33, 37-47.

xvi
Dubey, S., Jänne, P. A., Krug, L., Pang, H., Wang, X., Heinze, R., Watt, C.,
Crawford, J., Kratzke, R., Vokes, E. & Kindler, H. L. 2010. A Phase II
Study of Sorafenib in Malignant Mesothelioma: Results of Cancer and
Leukemia Group B 30307. Journal of Thoracic Oncology, 5, 1655-1661
10.1097/JTO.0b013e3181ec18db.
Duplan, S. M., Théorêt, Y. & Kenigsberg, R. L. 2002. Antitumor Activity of
Fibroblast Growth Factors (FGFs) for Medulloblastoma May Correlate
with FGF Receptor Expression and Tumor Variant. Clinical Cancer
Research, 8, 246-257.
Dutt, A., Salvesen, H. B., Chen, T.-H., Ramos, A. H., Onofrio, R. C., Hatton, C.,
Nicoletti, R., Winckler, W., Grewal, R., Hanna, M., Wyhs, N., Ziaugra, L.,
Richter, D. J., Trovik, J., Engelsen, I. B., Stefansson, I. M., Fennell, T.,
Cibulskis, K., Zody, M. C., Akslen, L. A., Gabriel, S., Wong, K.-K.,
Sellers, W. R., Meyerson, M. & Greulich, H. 2008. Drug-sensitive FGFR2
mutations in endometrial carcinoma. Proceedings of the National
Academy of Sciences, 105, 8713-8717.
Easton, D. F., Pooley, K. A., Dunning, A. M., Pharoah, P. D. P., Thompson, D.,
Ballinger, D. G., Struewing, J. P., Morrison, J., Field, H., Luben, R.,
Wareham, N., Ahmed, S., Healey, C. S., Bowman, R., Meyer, K. B.,
Haiman, C. A., Kolonel, L. K., Henderson, B. E., Le Marchand, L.,
Brennan, P., Sangrajrang, S., Gaborieau, V., Odefrey, F., Shen, C.-Y.,
Wu, P.-E., Wang, H.-C., Eccles, D., Evans, D. G., Peto, J., Fletcher, O.,
Johnson, N., Seal, S., Stratton, M. R., Rahman, N., Chenevix-Trench, G.,
Bojesen, S. E., Nordestgaard, B. G., Axelsson, C. K., Garcia-Closas, M.,
Brinton, L., Chanock, S., Lissowska, J., Peplonska, B., Nevanlinna, H.,

xvii
Fagerholm, R., Eerola, H., Kang, D., Yoo, K.-Y., Noh, D.-Y., Ahn, S.-H.,
Hunter, D. J., Hankinson, S. E., Cox, D. G., Hall, P., Wedren, S., Liu, J.,
Low, Y.-L., Bogdanova, N., Schurmann, P., Dork, T., Tollenaar, R. a. E.
M., Jacobi, C. E., Devilee, P., Klijn, J. G. M., Sigurdson, A. J., Doody, M.
M., Alexander, B. H., Zhang, J., Cox, A., Brock, I. W., Macpherson, G.,
Reed, M. W. R., Couch, F. J., Goode, E. L., Olson, J. E., Meijers-
Heijboer, H., Van Den Ouweland, A., Uitterlinden, A., Rivadeneira, F.,
Milne, R. L., Ribas, G., Gonzalez-Neira, A., Benitez, J., Hopper, J. L.,
Mccredie, M., Southey, M., Giles, G. G., Schroen, C., Justenhoven, C.,
Brauch, H., Hamann, U., Ko, Y.-D., Spurdle, A. B., Beesley, J., Chen, X.,
Mannermaa, A., Kosma, V.-M., Kataja, V., Hartikainen, J., Day, N. E.,
Cox, D. R. & Ponder, B. a. J. 2007. Genome-wide association study
identifies novel breast cancer susceptibility loci. Nature, 447, 1087-1093.
Edwards, J. G., Faux, S. P., Plummer, S. M., Abrams, K. R., Walker, R. A.,
Waller, D. A. & O’byrne, K. J. 2002. Cyclooxygenase-2 Expression Is a
Novel Prognostic Factor in Malignant Mesothelioma. Clinical Cancer
Research, 8, 1857-1862.
Edwards, J. G., Swinson, D. E. B., Jones, J. L., Muller, S., Waller, D. A. &
O’byrne, K. J. 2003. Tumor Necrosis Correlates With Angiogenesis and
Is a Predictor of Poor Prognosis in Malignant Mesothelioma*. Chest, 124,
1916-1923.
Efrati, P. & Nir, E. 1976. Morphological and cytochemical investigation of human
mesothelial cells from pleural and peritoneal effusions: a light and
electron microscopy study. Israel journal of medical sciences, 12, 662-
73.

xviii
Elmes, P. C. & Simpson, M. J. C. 1976. The Clinical Aspects of Mesothelioma.
QJM, 45, 427-449.
Eswarakumar, V. P., Lax, I. & Schlessinger, J. 2005. Cellular signaling by
fibroblast growth factor receptors. Cytokine & Growth Factor Reviews,
16, 139-149.
Fedorko, M. E. & Hirsch, J. G. 1971. Studies on transport of macromolecules
and small particles across mesothelial cells of the mouse omentum: I.
Morphologic aspects. Experimental Cell Research, 69, 113-127.
Fitzpatrick D.R., B.-O. H., Himbeck R.P., Jarnicki A.G., Marzo A.L., Robinson
B.W. 1994. Transforming growth factor-beta: antisense RNA-mediated
inhibition affects anchorage-independent growth, tumorigenicity and
tumor-infiltrating T-cells in malignant mesothelioma. Growth factors, 11,
29-44.
Flaumenhaft, R., Moscatelli, D. & Rifkin, D. B. 1990. Heparin and heparan
sulfate increase the radius of diffusion and action of basic fibroblast
growth factor. J Cell Biol, 111, 1651 - 1659.
Fleury-Feith, J., Lecomte, C., Renier, A., Matrat, M., Kheuang, L., Abramowski,
V., Levy, F., Janin, A., Giovannini, M. & Jaurand, M.-C. 2003.
Hemizygosity of Nf2 is associated with increased susceptibility to
asbestos-induced peritoneal tumours. Oncogene, 22, 3799-3805.
Friedlander, P. L., Delaune, C. L., Abadie, J. M., Toups, M., Lacour, J., Marrero,
L., Zhong, Q. & Kolls, J. K. 2003. Efficacy of CD40 Ligand Gene Therapy
in Malignant Mesothelioma. American Journal of Respiratory Cell and
Molecular Biology, 29, 321-330.

xix
Frontini, M. J., Nong, Z., Gros, R., Drangova, M., O'neil, C., Rahman, M. N.,
Akawi, O., Yin, H., Ellis, C. G. & Pickering, J. G. 2011. Fibroblast growth
factor 9 delivery during angiogenesis produces durable, vasoresponsive
microvessels wrapped by smooth muscle cells. Nat Biotech, 29, 421-427.
Fujii, M., Toyoda, T., Nakanishi, H., Yatabe, Y., Sato, A., Matsudaira, Y., Ito, H.,
Murakami, H., Kondo, Y., Kondo, E., Hida, T., Tsujimura, T., Osada, H. &
Sekido, Y. 2012. TGF-β synergizes with defects in the Hippo pathway to
stimulate human malignant mesothelioma growth. The Journal of
Experimental Medicine.
Fukuta, H. 1963. Electron microscopic study on normal rat peritoneal
mesothelium and its changes in absorption of particulate iron dextran
complex. Acta Pathol Jpn, 13, 309-25.
Galffy, G., Mohammed, K. A., Dowling, P. A., Nasreen, N., Ward, M. J. &
Antony, V. B. 1999a. Interleukin 8. Cancer Research, 59, 367-371.
Galffy, G., Mohammed, K. A., Dowling, P. A., Nasreen, N., Ward, M. J. &
Antony, V. B. 1999b. Interleukin 8: An Autocrine Growth Factor for
Malignant Mesothelioma. Cancer Res, 59, 367-371.
Garcès, A., Nishimune, H., Philippe, J.-M., Pettmann, B. & Delapeyrière, O.
2000. FGF9: A motoneuron survival factor expressed by medial thoracic
and sacral motoneurons. Journal of Neuroscience Research, 60, 1-9.
Garcia-Closas, M., Hall, P., Nevanlinna, H., Pooley, K., Morrison, J., Richesson,
D. A., Bojesen, S. E., Nordestgaard, B. G., Axelsson, C. K., Arias, J. I.,
Milne, R. L., Ribas, G., González-Neira, A., Benítez, J., Zamora, P.,
Brauch, H., Justenhoven, C., Hamann, U., Ko, Y.-D., Bruening, T., Haas,

xx
S., Dörk, T., Schürmann, P., Hillemanns, P., Bogdanova, N., Bremer, M.,
Karstens, J. H., Fagerholm, R., Aaltonen, K., Aittomäki, K., Von Smitten,
K., Blomqvist, C., Mannermaa, A., Uusitupa, M., Eskelinen, M.,
Tengström, M., Kosma, V.-M., Kataja, V., Chenevix-Trench, G., Spurdle,
A. B., Beesley, J., Chen, X., Australian Ovarian Cancer Management, G.,
The Kathleen Cuningham Foundation Consortium for Research into
Familial Breast, C., Devilee, P., Van Asperen, C. J., Jacobi, C. E.,
Tollenaar, R. a. E. M., Huijts, P. E. A., Klijn, J. G. M., Chang-Claude, J.,
Kropp, S., Slanger, T., Flesch-Janys, D., Mutschelknauss, E., Salazar,
R., Wang-Gohrke, S., Couch, F., Goode, E. L., Olson, J. E., Vachon, C.,
Fredericksen, Z. S., Giles, G. G., Baglietto, L., Severi, G., Hopper, J. L.,
English, D. R., Southey, M. C., Haiman, C. A., Henderson, B. E., Kolonel,
L. N., Le Marchand, L., Stram, D. O., Hunter, D. J., Hankinson, S. E.,
Cox, D. G., Tamimi, R., Kraft, P., Sherman, M. E., Chanock, S. J.,
Lissowska, J., Brinton, L. A., Peplonska, B., Hooning, M. J., Meijers-
Heijboer, H., Collee, J. M., Van Den Ouweland, A., Uitterlinden, A. G.,
Liu, J., Lin, L. Y., Yuqing, L., Humphreys, K., Czene, K., Cox, A.,
Balasubramanian, S. P., Cross, S. S., Reed, M. W. R., Blows, F., Driver,
K., Dunning, A., Tyrer, J., Ponder, B. a. J., Sangrajrang, S., Brennan, P.,
Mckay, J., Odefrey, F., Gabrieau, V., Sigurdson, A., Doody, M.,
Struewing, J. P., Alexander, B., Easton, D. F. & Pharoah, P. D. 2008.
Heterogeneity of Breast Cancer Associations with Five Susceptibility Loci
by Clinical and Pathological Characteristics. PLoS Genet, 4, e1000054.
Garland, L. L., Chansky, K., Wozniak, A., Tsao, A., Gadgeel, S., Vershraegen,
C., Da Silva, M., Redman, M. & D., G. 2009. SWOG S0509: A phase II

xxi
study of novel oral antiangiogenic agent AZD2171 (NSC-732208) in
malignant pleural J Clin Oncol 27.
Garlepp Mj, C. T., Mutsaers Se, Manning Ls, Davis M, Robinson Bws 1993.
Platelet-derived growth factor as an autocrine factor in murine malignant
mesothelioma. Eur Respir Rev, 3, 192-4.
Gartside, M. G., Chen, H., Ibrahimi, O. A., Byron, S. A., Curtis, A. V., Wellens,
C. L., Bengston, A., Yudt, L. M., Eliseenkova, A. V., Ma, J., Curtin, J. A.,
Hyder, P., Harper, U. L., Riedesel, E., Mann, G. J., Trent, J. M., Bastian,
B. C., Meltzer, P. S., Mohammadi, M. & Pollock, P. M. 2009. Loss-of-
Function Fibroblast Growth Factor Receptor-2 Mutations in Melanoma.
Molecular Cancer Research, 7, 41-54.
Geske, M. J., Zhang X., Patel, K. K., Ornitz, D. M., & Stappenbeck, T. S. 2008.
Fgf9 signaling regulates small intestinal elongation and mesenchymal
development. Development, 135, 2959-2968.
Giri, D., Ropiquet, F. & Ittmann, M. 1999. FGF9 is an autocrine and paracrine
prostatic growth factor expressed by prostatic stromal cells. Journal of
Cellular Physiology, 180, 53-60.
Goldfarb, M. 2005. Fibroblast growth factor homologous factors: Evolution,
structure, and function. Cytokine & Growth Factor Reviews, 16, 215-220.
Gospodarowicz, D. 1974. Localisation of a fibroblast growth factor and its effect
alone and with hydrocortisone on 3T3 cell growth. Nature, 249, 123-127.
Greay, S., Ireland, D., Kissick, H., Levy, A., Beilharz, M., Riley, T. & Carson, C.
2010. Induction of necrosis and cell cycle arrest in murine cancer cell

xxii
lines by <i>Melaleuca alternifolia (tea tree) oil and terpinen-4-ol.
Cancer Chemotherapy and Pharmacology, 65, 877-888.
Grigoriu, B., Scherpereel, A., Devos, P., Chahine, B., Letourneux, M., Lebailly,
P., Grégoire, M., Porte, H., Copin, M. & Lassalle, P. 2007. Utility of
Osteopontin and Serum Mesothelin in Malignant Pleural Mesothelioma
Diagnosis and Prognosis Assessment. Clinical Cancer Research, 13,
2928.
Guillemot, F. & Zimmer, C. 2011. From Cradle to Grave: The Multiple Roles of
Fibroblast Growth Factors in Neural Development. Neuron, 71, 574-588.
Hammar, S. P. 2006. Macroscopic, Histologic, Histochemical,
Immunohistochemical, and Ultrastructural Features of Mesothelioma.
Ultrastructural Pathology, 30, 3-17.
Hanahan D., W. R. A. 2000. The hallmarks of cancer. Cell, 100, 57-70.
Harada, M., Murakami, H., Okawa, A., Okimoto, N., Hiraoka, S., Nakahara, T.,
Akasaka, R., Shiraishi, Y.-I., Futatsugi, N., Mizutani-Koseki, Y., Kuroiwa,
A., Shirouzu, M., Yokoyama, S., Taiji, M., Iseki, S., Ornitz, D. M. &
Koseki, H. 2009. FGF9 monomer-dimer equilibrium regulates
extracellular matrix affinity and tissue diffusion. Nat Genet, 41, 289-298.
Haugsten, E. M., Małecki, J., Bjørklund, S. M. S., Olsnes, S. & Wesche, J.
2008. Ubiquitination of Fibroblast Growth Factor Receptor 1 Is Required
for Its Intracellular Sorting but Not for Its Endocytosis. Molecular Biology
of the Cell, 19, 3390-3403.

xxiii
Haugsten, E. M., Wiedlocha, A., Olsnes, S. & Wesche, J. 2010. Roles of
Fibroblast Growth Factor Receptors in Carcinogenesis. Molecular
Cancer Research, 8, 1439-1452.
Hecht, D., Zimmerman, N., Bedford, M., Avivi, A. & Yayon, A. 1995.
Identification of fibroblast growth factor 9 (FGF9) as a high affinity,
heparin dependent ligand for FGF receptors 3 and 2 but not for FGF
receptors 1 and 4. Growth factors, 12, 223-33.
Heikinheimo, M., Lawshe, A., Shackleford, G. M., Wilson, D. B. & Macarthur, C.
A. 1994. Fgf-8 expression in the post-gastrulation mouse suggests roles
in the development of the face, limbs and central nervous system. Mech
Dev, 48, 129 - 138.
Heinzle, C., Sutterlüty, H., Grusch, M., Grasl-Kraupp, B., Berger, W. & Marian,
B. 2011. Targeting fibroblast-growth-factor-receptor-dependent signaling
for cancer therapy. Expert Opinion on Therapeutic Targets, 15, 829-846.
Heldin, P. & Pertoft, H. 1993. Synthesis and Assembly of the Hyaluronan-
Containing Coats around Normal Human Mesothelial Cells. Experimental
Cell Research, 208, 422-429.
Hendrix, N. D. & Cho, K. R. 2005. Fibroblast growth factor 9 (FGF9) stimulates
migration of endothelial and ovarian cancer cells. AACR Meeting
Abstracts, 2005, 1331-b-.
Hendrix, N. D., Wu, R., Kuick, R., Schwartz, D. R., Fearon, E. R. & Cho, K. R.
2006. Fibroblast Growth Factor 9 Has Oncogenic Activity and Is a
Downstream Target of Wnt Signaling in Ovarian Endometrioid
Adenocarcinomas. Cancer Res, 66, 1354-1362.

xxiv
Hernandez, S., De Muga, S., Agell, L., Juanpere, N., Esgueva, R., Lorente, J.
A., Mojal, S., Serrano, S. & Lloreta, J. 2009. FGFR3 mutations in
prostate cancer: association with low-grade tumors. Mod Pathol, 22, 848-
856.
Hernández, S., López-Knowles, E., Lloreta, J., Kogevinas, M., Amorós, A.,
Tardón, A., Carrato, A., Serra, C., Malats, N. & Real, F. X. 2006.
Prospective Study of FGFR3 Mutations As a Prognostic Factor in
Nonmuscle Invasive Urothelial Bladder Carcinomas. Journal of Clinical
Oncology, 24, 3664-3671.
Herndon, J. E., Green, M. R., Chahinian, A. P., Corson, J. M., Suzuki, Y. &
Vogelzang, N. J. 1998. Factors Predictive of Survival Among 337
Patients With Mesothelioma Treated Between 1984 and 1994 by the
Cancer and Leukemia Group B. Chest, 113, 723-731.
Hirayama, N., Tabata, C., Tabata, R., Maeda, R., Yasumitsu, A., Yamada, S.,
Kuribayashi, K., Fukuoka, K. & Nakano, T. 2011. Pleural effusion VEGF
levels as a prognostic factor of malignant pleural mesothelioma.
Respiratory Medicine, 105, 137-142.
Hodgson, J. T. & Darnton, A. 2000. The quantitative risks of mesothelioma and
lung cancer in relation to asbestos exposure. Annals of Occupational
Hygiene, 44, 565-601.
Hodgson, J. T., Mcelvenny, D. M., Darnton, A. J., Price, M. J. & Peto, J. 2005.
The expected burden of mesothelioma mortality in Great Britain from
2002 to 2050. Br J Cancer, 92, 587-593.

xxv
Hofer, S. O. P., Shrayer, D., Reichner, J. S., Hoekstra, H. J. & Wanebo, H. J.
1998. Wound-Induced Tumor Progression: A Probable Role in
Recurrence After Tumor Resection. Arch Surg, 133, 383-389.
Hollevoet, K., Nackaerts, K., Thimpont, J., Germonpré, P., Bosquée, L., De
Vuyst, P., Legrand, C., Kellen, E., Kishi, Y., Delanghe, J. R. & Van
Meerbeeck, J. P. 2010. Diagnostic Performance of Soluble Mesothelin
and Megakaryocyte Potentiating Factor in Mesothelioma. American
Journal of Respiratory and Critical Care Medicine, 181, 620-625.
Hollevoet, K., Reitsma, J. B., Creaney, J., Grigoriu, B. D., Robinson, B. W.,
Scherpereel, A., Cristaudo, A., Pass, H. I., Nackaerts, K., Rodríguez
Portal, J. A., Schneider, J., Muley, T., Di Serio, F., Baas, P., Tomasetti,
M., Rai, A. J. & Van Meerbeeck, J. P. 2012. Serum Mesothelin for
Diagnosing Malignant Pleural Mesothelioma: An Individual Patient Data
Meta-Analysis. Journal of Clinical Oncology, 30, 1541-1549.
Honda, A., Ohashi, Y. & Mori, Y. 1986. Hyaluronic acid in rabbit pericardial fluid
and its production by pericardium. FEBS Letters, 203, 273-278.
Hori, A., Sasada, R., Matsutani, E., Naito, K., Sakura, Y., Fujita, T. & Kozai, Y.
1991. Suppression of Solid Tumor Growth by Immunoneutralizing
Monoclonal Antibody against Human Basic Fibroblast Growth Factor.
Cancer Research, 51, 6180-6184.
Humar, R., Kiefer, F. N., Berns, H., Resink, T. J. & Battegay, E. J. 2002.
Hypoxia enhances vascular cell proliferation and angiogenesis in vitro via
rapamycin (mTOR) -dependent signaling. The FASEB Journal, 16, 771-
780.

xxvi
Hung, I. H., Yu, K., Lavine, K. J. & Ornitz, D. M. 2007. FGF9 regulates early
hypertrophic chondrocyte differentiation and skeletal vascularization in
the developing stylopod. Developmental Biology, 307, 300-313.
Hunter, D. J., Kraft, P., Jacobs, K. B., Cox, D. G., Yeager, M., Hankinson, S. E.,
Wacholder, S., Wang, Z., Welch, R., Hutchinson, A., Wang, J., Yu, K.,
Chatterjee, N., Orr, N., Willett, W. C., Colditz, G. A., Ziegler, R. G., Berg,
C. D., Buys, S. S., Mccarty, C. A., Feigelson, H. S., Calle, E. E., Thun, M.
J., Hayes, R. B., Tucker, M., Gerhard, D. S., Fraumeni, J. F., Hoover, R.
N., Thomas, G. & Chanock, S. J. 2007. A genome-wide association
study identifies alleles in FGFR2 associated with risk of sporadic
postmenopausal breast cancer. Nat Genet, 39, 870-874.
Husain, A. N., Colby, T. V., Ordóñez, N. G., Krausz, T., Borczuk, A., Cagle, P.
T., Chirieac, L. R., Churg, A., Galateau-Salle, F., Gibbs, A. R., Gown, A.
M., Hammar, S. P., Litzky, L. A., Roggli, V. L., Travis, W. D. & Wick, M.
R. 2009. Guidelines for Pathologic Diagnosis of Malignant Mesothelioma:
A Consensus Statement from the International Mesothelioma Interest
Group. Archives of Pathology & Laboratory Medicine, 133, 1317-1331.
Inai, K. 2008. Pathology of mesothelioma. Environmental Health and Preventive
Medicine, 13, 60-64.
Itoh, N. & Ornitz, D. M. 2008. Functional evolutionary history of the mouse Fgf
gene family. Developmental Dynamics, 237, 18-27.
Jackaman, C., Bundell, C. S., Kinnear, B. F., Smith, A. M., Filion, P., Van
Hagen, D., Robinson, B. W. S. & Nelson, D. J. 2003. IL-2 Intratumoral
Immunotherapy Enhances CD8+ T Cells That Mediate Destruction of

xxvii
Tumor Cells and Tumor-Associated Vasculature: A Novel Mechanism for
IL-2. J Immunol, 171, 5051-5063.
Jackaman, C. & Nelson, D. J. 2010. Cytokine-armed vaccinia virus infects the
mesothelioma tumor microenvironment to overcome immune tolerance
and mediate tumor resolution. Cancer Gene Ther, 17, 429-440.
Jang, J. H., Shin, K. H. & Park, J. G. 2001. Mutations in fibroblast growth factor
receptor 2 and fibroblast growth factor receptor 3 genes associated with
human gastric and colorectal cancers. Cancer Res., 61, 3541-3543.
Janne, P. A., Wang, X. F. & Krug, L. M. 2007. Sorafenib in malignant
mesothelioma (MM): a phase II trial of the Cancer and Leukemia Group
B (CALGB 30307). J Clin Oncol, 25, 7707.
Jaye, M., Schlessinger, J. & Dionne, C. A. 1992. Fibroblast growth factor
receptor tyrosine kinases: molecular analysis and signal transduction.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1135,
185-199.
Johnson, D. E., Lu, J., Chen, H., Werner, S. & Williams L. T. 1991. The Human
Fibroblast Growth Factor Receptor Genes: A Common Structural
Arrangement Underlies The Mechanisms For Generating Receptor
Forms That Differ In Their Their Immunoglubulin Domains. Mol Cell Biol,
11, 4627-4634.
Jones, L. M. H., Gardner, M. J., Catterall, J. B. & Turner, G. A. 1995. Hyaluronic
acid secreted by mesothelial cells: a natural barrier to ovarian cancer cell
adhesion. Clinical and Experimental Metastasis, 13, 373-380.

xxviii
Jonjić N., P. G., Bernasconi S., Sciacca F.L., Colotta F., Pelicci P., Lanfrancone
L., Mantovani A. 1992. Expression of adhesion molecules and
chemotactic cytokines in cultured human mesothelial cells. J Exp Med,
176, 1165-74.
Joshi, T. K. & Gupta, R. K. 2004. Asbestos in developing countries: magnitude
of risk and its practical implications. Int J Occup Med Environ Health, 17,
179-85.
Jui-Yen, H., Yu-Ting, H. & Jih-Ing, C. 2009. Fibroblast growth factor 9 prevents
MPP<sup>+</sup>-induced death of dopaminergic neurons and is
involved in melatonin neuroprotection <i>in vivo</i> and <i>in vitro</i>.
Journal of Neurochemistry, 109, 1400-1412.
Kamp, D. W., Israbian, V. A., Preusen, S. E., Zhang, C. X. & Weitzman, S. A.
1995. Asbestos causes DNA strand breaks in cultured pulmonary
epithelial cells: role of iron-catalyzed free radicals. American Journal of
Physiology - Lung Cellular and Molecular Physiology, 268, L471-L480.
Kamp, D. W. & Weitzman, S. A. 1999. The molecular basis of asbestos induced
lung injury. Thorax, 54, 638-652.
Kan, R., Twigg, S. R. F., Berg, J., Wang, L., Jin, F. & Wilkie, A. O. M. 2004.
Expression analysis of an FGFR2 IIIc 5′ splice site mutation
(1084+3A→G). Journal of Medical Genetics, 41, e108.
Kan, S.-H., Elanko, N., Johnson, D., Cornejo-Roldan, L., Cook, J., Reich, E. W.,
Tomkins, S., Verloes, A., Twigg, S. R. F., Rannan-Eliya, S., Mcdonald-
Mcginn, D. M., Zackai, E. H., Wall, S. A., Muenke, M. & Wilkie, A. O. M.
2002. Genomic Screening of Fibroblast Growth-Factor Receptor 2

xxix
Reveals a Wide Spectrum of Mutations in Patients with Syndromic
Craniosynostosis. American journal of human genetics, 70, 472-486.
Kane, A. B. 2006. Animal Models of Malignant Mesothelioma. Inhalation
Toxicology, 18, 1001-1004.
Kao, S. C.-H., Reid, G., Van Zandwijk, N., Henderson, D. W. & Klebe, S. 2011a.
Molecular biomarkers in malignant mesothelioma: state of the art.
Pathology - Journal of the RCPA, 43, 201-212
10.1097/PAT.0b013e3283445e67.
Kao, S. C.-H., Yan, T. D., Lee, K., Burn, J., Henderson, D. W., Klebe, S.,
Kennedy, C., Vardy, J., Clarke, S., Van Zandwijk, N. & Mccaughan, B. C.
2011b. Accuracy of Diagnostic Biopsy for the Histological Subtype of
Malignant Pleural Mesothelioma. Journal of Thoracic Oncology, 6, 602-
605 10.1097/JTO.0b013e31820ce2c7.
Khodayari, N., Mohammed, K. A., Goldberg, E. P. & Nasreen, N. 2011.
EphrinA1 inhibits malignant mesothelioma tumor growth via let-7
microRNA-mediated repression of the RAS oncogene. Cancer Gene
Ther, 18, 806-816.
Kim, Y., Kobayashi, A., Sekido, R., Dinapoli, L., Brennan, J., Chaboissier, M.-
C., Poulat, F., Behringer, R. R., Lovell-Badge, R. & Capel, B. 2006.
<italic>Fgf9</italic> and
<italic>Wnt4</italic> Act as Antagonistic Signals to Regulate
Mammalian Sex Determination. PLoS Biol, 4, e187.
Kindler, H. 2008. Systemic Treatments for Mesothelioma: Standard and Novel.
Current Treatment Options in Oncology, 9, 171-179.

xxx
Kindler, H. L., Karrison, T. G., Gandara, D. R., Lu, C., Krug, L. M., Stevenson, J.
P., Jänne, P. A., Quinn, D. I., Koczywas, M. N., Brahmer, J. R., Albain, K.
S., Taber, D. A., Armato, S. G., Vogelzang, N. J., Chen, H. X., Stadler,
W. M. & Vokes, E. E. 2012. Multicenter, Double-Blind, Placebo-
Controlled, Randomized Phase II Trial of Gemcitabine/Cisplatin Plus
Bevacizumab or Placebo in Patients With Malignant Mesothelioma.
Journal of Clinical Oncology, 30, 2509-2515.
Kinkl, N., Ruiz, J., Vecino, E., Frasson, M., Sahel, J. & Hicks, D. 2003. Possible
involvement of a fibroblast growth factor 9 (FGF9)–FGF receptor-3-
mediated pathway in adult pig retinal ganglion cell survival in vitro.
Molecular and Cellular Neuroscience, 23, 39-53.
Kirschbaum, K., Kriebel, M., Kranz, E. U., Pötz, O. & Volkmer, H. 2009.
Analysis of Non-canonical Fibroblast Growth Factor Receptor 1 (FGFR1)
Interaction Reveals Regulatory and Activating Domains of Neurofascin.
Journal of Biological Chemistry, 284, 28533-28542.
Kissick, H. T., Ireland, D. J., Krishnan, S., Madondo, M. & Beilharz, M. W. 2012.
Tumour eradication and induction of memory against murine
mesothelioma by combined immunotherapy. Immunol Cell Biol.
Klebe, S., Mahar, A., Henderson, D. W. & Roggli, V. L. 2008. Malignant
mesothelioma with heterologous elements: clinicopathological correlation
of 27 cases and literature review. Mod Pathol, 21, 1084-1094.
Klint, P. & Claesson-Welsh, L. 1999. Signal transduction by fibroblast growth
factor receptors. Frontiers in Bioscience, 4, 165-177.

xxxi
Klint P. & Claesson-Welsh L. 1999. Signal transduction by fibroblast growth
factor receptors. Front Biosci, 4, D165-77.
Komi-Kuramochi, A., Kawano, M., Oda, Y., Asada, M., Suzuki, M., Oki, J. &
Imamura, T. 2005. Expression of fibroblast growth factors and their
receptors during full-thickness skin wound healing in young and aged
mice. Journal of Endocrinology, 186, 273-289.
Kraft, A., Weindel, K., Ochs, A., Marth, C., Zmija, J., Schumacher, P., Unger,
C., Marmé, D. & Gastl, G. 1999. Vascular endothelial growth factor in the
sera and effusions of patients with malignant and nonmalignant disease.
Cancer, 85, 178-187.
Kumar-Singh, S., Weyler, J., Martin, M. J. H., Vermeulen, P. B. & Van Marck, E.
1999. Angiogenic cytokines in mesothelioma: a study of VEGF, FGF-1
and -2, and TGF β expression. The Journal of Pathology, 189, 72-78.
Kumar, S., Jiang, M. S., Adams, J. L. & Lee, J. C. 1999. Pyridinylimidazole
Compound SB 203580 Inhibits the Activity but Not the Activation of p38
Mitogen-Activated Protein Kinase. Biochemical and Biophysical
Research Communications, 263, 825-831.
Kunii, K. 2008. FGFR2-amplified gastric cancer cell lines require FGFR2 and
Erbb3 signaling for growth and survival. Cancer Res., 68, 2340-2348.
Kwabi-Addo, B., Ozen, M. & Ittmann, M. 2004. The role of fibroblast growth
factors and their receptors in prostate cancer. Endocr. Relat. Cancer, 11,
709-724.

xxxii
Lamont, F. R., Tomlinson, D. C., Cooper, P. A., Shnyder, S. D., Chester, J. D. &
Knowles, M. A. 2011. Small molecule FGF receptor inhibitors block
FGFR-dependent urothelial carcinoma growth in vitro and in vivo. Br J
Cancer, 104, 75-82.
Le, G. V., Takahashi, K. E. N., Park, E.-K., Delgermaa, V., Oak, C., Qureshi, A.
M. & Aljunid, S. M. 2011. Asbestos use and asbestos-related diseases in
Asia: Past, present and future. Respirology, 16, 767-775.
Leak, L. V. & Rahil, K. 1978. Permeability of the diaphragmatic mesothelium:
The ultrastructural basis for “stomata”. American Journal of Anatomy,
151, 557-593.
Lee, Y. C. G., Light, R. W. & Musk, A. W. 2000. Management of malignant
pleural mesothelioma: a critical review. Current Opinion in Pulmonary
Medicine, 6, 267-274.
Lewandoski, M., Sun, X. & Martin, G. R. 2000. Fgf8 signalling from the AER is
essential for normal limb development. Nat Genet, 26, 460 - 463.
Li, A., Dubey, S., Varney, M. L., Dave, B. J. & Singh, R. K. 2003. IL-8 Directly
Enhanced Endothelial Cell Survival, Proliferation, and Matrix
Metalloproteinases Production and Regulated Angiogenesis. The Journal
of Immunology, 170, 3369-3376.
Li, J., Brown, L. F., Laham, R. J., Volk, R. & Simons, M. 1997. Macrophage-
Dependent Regulation of Syndecan Gene Expression. Circulation
Research, 81, 785-796.

xxxiii
Li, Y., Basilico, C. & Mansukhani, A. 1994. Cell transformation by fibroblast
growth factors can be suppressed by truncated fibroblast growth factor
receptors. Molecular and Cellular Biology, 14, 7660-7669.
Li, Z. G., Mathew, P., Yang, J., Starbuck, M. W., Zurita, A. J., Liu, J., Sikes, C.,
Multani, A. S., Efstathiou, E., Lopez, A., Wang, J., Fanning, T. V., Prieto,
V. G., Kundra, V., Vazquez, E. S., Troncoso, P., Raymond, A. K.,
Logothetis, C. J., Lin, S.-H., Maity, S. & Navone, N. M. 2008. Androgen
receptor - negative human prostate cancer cells induce osteogenesis in
mice through FGF9-mediated mechanisms. The Journal of Clinical
Investigation, 118, 2697-2710.
Liberek, T., Topley, N., Luffmann, W., and Williams, J. D. 1996. Adherence of
Neutrophils to Human Peritoneal Mesothelial Cells: Pole of Intercellular
Adhesion. J. Am. Soc. Nephrol, 7, 208-217.
Light, R. W. 1990. Parapneumonic effusions and infections of the pleural space.
In: Light RW (ed.). Pleural Diseases, 2nd edn, 129-49.
Lin, H.-Y., Xu, J., Ischenko, I., Ornitz, D. M., Halegoua, S. & Hayman, M. J.
1998. Identification of the Cytoplasmic Regions of Fibroblast Growth
Factor (FGF) Receptor 1 Which Play Important Roles in Induction of
Neurite Outgrowth in PC12 Cells by FGF-1. Molecular and Cellular
Biology, 18, 3762-3770.
Lin, Y., Liu, G. & Wang, F. 2006. Generation of an Fgf9 conditional null allele.
genesis, 44, 150-154.
Linda, S. M., Darrel, W., Ashleigh, R. M., Michael, J. G., Mark, R. D., Arthur, W.
M. & Bruce, W. S. R. 1991. Establishment and characterization of five

xxxiv
human malignant mesothelioma cell lines derived from pleural effusions.
International Journal of Cancer, 47, 285-290.
Linder, C., Linder, S., Munck-Wikland, E. & Strander, H. 1998. Independent
expression of serum vascular endothelial growth factor (VEGF) and basic
fibroblast growth factor (bFGF) in patients with carcinoma and sarcoma.
Anticancer research, 18, 2063-8.
Luus, K. 2007. Asbestos: mining exposure, health effects and policy
implications. McGill Journal of Medicine, 10, 121-126.
Ma, C., Tarnuzzer, R. W. & Chegini, N. 1999. Expression of matrix
metalloproteinases and tissue inhibitor of matrix metalloproteinases in
mesothelial cells and their regulation by transforming growth factor-β1.
Wound Repair and Regeneration, 7, 477-485.
Maggie, L., Alisa, T., Branka, M. & Ann, M. T. 2009. Fibroblast growth factor-9
inhibits astrocyte differentiation of adult mouse neural progenitor cells.
Journal of Neuroscience Research, 87, 2201-2210.
Mansukhani, A., Dell'era, P., Moscatelli, D., Kornbluth, S.,Hanafusa, H. And
Basilico, C. 1992. Characterization of the murine BEK fibroblast growth
factor (FGF) receptor: activation by three members of the FGF family and
requirement for heparin. Proc Natl Acad Sci USA, 89, 3305-3309.
Marek, L., Ware, K. E., Fritzsche, A., Hercule, P., Helton, W. R., Smith, J. E.,
Mcdermott, L. A., Coldren, C. D., Nemenoff, R. A., Merrick, D. T.,
Helfrich, B. A., Bunn Jr. P. A. & Heasley, L. E. 2009. Fibroblast Growth
Factor (FGF) and FGF Receptor-Mediated Autocrine Signaling in Non-
Small Cell Lung Cancer Cells. Mol Pharmacol, 75, 196-207.

xxxv
Marshall, B. C., Santana, A., Xu, Q. P., Petersen, M. J., Campbell, E. J., Hoidal,
J. R. & Welgus, H. G. 1993. Metalloproteinases and tissue inhibitor of
metalloproteinases in mesothelial cells. Cellular differentiation influences
expression. The Journal of Clinical Investigation, 91, 1792-1799.
Martin, G. R. 1998. The roles of FGFs in the early development of vertebrate
limbs. Genes Dev, 12, 1571 - 1586.
Martin, P., Hopkinson-Woolley, J. & Mccluskey, J. 1992. Growth factors and
cutaneous wound repair. Progress in Growth Factor Research, 4, 25-44.
Martínez-Torrecuadrada, J., Cifuentes, G., López-Serra, P., Saenz, P.,
Martínez, A. & Casal, J. I. 2005. Targeting the Extracellular Domain of
Fibroblast Growth Factor Receptor 3 with Human Single-Chain Fv
Antibodies Inhibits Bladder Carcinoma Cell Line Proliferation. Clinical
Cancer Research, 11, 6280-6290.
Marzo A.L., F. D. R., Robinson B.W., Scott B.. 1997. Antisense oligonucleotides
specific for transforming growth factor beta2 inhibit the growth of
malignant mesothelioma both in vitro and in vivo. Cancer Res, 57, 3200-
7.
Mason, I. 2007. Initiation to end point: the multiple roles of fibroblast growth
factors in neural development. Nat Rev Neurosci, 8, 583-596.
Masood, R., Kundra, A., Zhu, S., Xia, G., Scalia, P., Smith, D. L. & Gill, P. S.
2003. Malignant mesothelioma growth inhibition by agents that target the
VEGF and VEGF-C autocrine loops. International Journal of Cancer,
104, 603-610.

xxxvi
Matsumoto-Yoshitomi, S., Habashita, J., Nomura, C., Kuroshima, K.-I. &
Kurokawa, T. 1997. Autocrine transformation by fibroblast growth factor 9
(FGF-9) and its possible participation in human oncogenesis.
International Journal of Cancer, 71, 442-450.
Mattei, M. G., De Moerlooze, L., Lovec, H., Coulier, F., Birnbaum, D. & Dickson,
C. 1997. Mouse fgf9 (fibroblast growth factor 9) is localized on
chromosome 14. Mamm Genome, 8, 617 - 618.
Mattei, M. G., Penault-Llorca, F., Coulier, F. & Birnbaum, D. 1995. The human
FGF9 gene maps to chromosomal region 13q11-q12. Genomics, 29, 811
- 812.
Mckay, M. M. & Morrison, D. K. 2007. Integrating signals from RTKs to
ERK//MAPK. Oncogene, 26, 3113-3121.
Mckeehan, W. L., Wang, F. & Kan, M. 1998. The heparan sulfate-fibroblast
growth factor family: diversity of structure and function. Prog Nucleic Acid
Res Mol Biol, 59, 135 - 176.
Merritt, R. E., Yamada, R. E., Wasif, N., Crystal, R. G. & Korst, R. J. 2004.
Effect of inhibition of multiple steps of angiogenesis in syngeneic murine
pleural mesothelioma. Ann Thorac Surg, 78, 1042-1051.
Meyers, G. A., Day, D., Goldberg, R., Daentl, D. L., Przylepa, K. A., Abrams, L.
J., Graham, J. M., Jr, M. F., Moeschler, J. B., Rawnsley, E., Scott, A. F.
& Jabs, E. W. 1996. FGFR2 exon IIIa and IIIc mutations in Crouzon,
Jackson-Weiss, and Pfeiffer syndromes: evidence for missense changes,
insertions, and a deletion due to alternative RNA splicing. Am J Hum
Genet, 58, 491-498.

xxxvii
Minot, C.-S. 1890. The Mesoderm and the Coelom of Vertebrates. The
American Naturalist, 24, 877-898.
Miyake, M., Ishii, M., Koyama, N., Kawashima, K., Kodama, T., Anai, S.,
Fujimoto, K., Hirao, Y. & Sugano, K. 2009. PD173074, a selective
tyrosine kinase inhibitor of FGFR3, inhibits cell proliferation of bladder
cancer carrying the FGFR3 gene mutation along with up-regulation of
p27/Kip1 and G1/G0 arrest. Journal of Pharmacology and Experimental
Therapeutics.
Miyamoto, M., Naruo, K., Seko, C., Matsumoto, S., Kondo, T. & Kurokawa, T.
1993. Molecular cloning of a novel cytokine cDNA encoding the ninth
member of the fibroblast growth factor family, which has a unique
secretion property. Mol. Cell. Biol., 13, 4251-4259.
Monti, G., Jaurand, M.-C., Monnet, I., Chretien, P., Saint-Etienne, L., Zeng, L.,
Portier, A., Devillier, P., Galanaud, P., Bignon, J. & Emilie, D. 1994.
Intrapleural Production of Interleukin 6 during Mesothelioma and Its
Modulation by γ-Interferon Treatment. Cancer Research, 54, 4419-4423.
Moon, A. M. & Capecchi, M. R. 2000. Fgf8 is required for outgrowth and
patterning of the limbs. Nat Genet, 26, 455 - 459.
Morrison, R. S. 1991. Suppression of basic fibroblast growth factor expression
by antisense oligodeoxynucleotides inhibits the growth of transformed
human astrocytes. Journal of Biological Chemistry, 266, 728-734.
Moscatelli, D. 1987. High and low affinity binding sites for basic fibroblast
growth factor on cultured cells: absence of a role for low affinity binding

xxxviii
in the stimulation of plasminogen activator production by bovine capillary
endothelial cells. J Cell Physiol, 131, 123 - 130.
Mulder, D. J., Pacheco, I., Hurlbut, D. J., Mak, N., Furuta, G. T., Macleod, R. J.
& Justinich, C. J. 2009. FGF9-induced proliferative response to
eosinophilic inflammation in oesophagitis. Gut, 58, 166-173.
Munro, N. P. & Knowles, M. A. 2003. Fibroblast Growth Factors and Their
Receptors in Transitional Cell Carcinoma. The Journal of urology, 169,
675-682.
Murai, N., Ueba, T., Takahashi, J. A., Yang, H.-Q., Kikuchi, H., Hiai, H.,
Hatanaka, M. & Fukumoto, M. 1996. Apoptosis of human glioma cells in
vitro and in vivo induced by a neutralizing antibody against human basic
fibroblast growth factor. Journal of Neurosurgery, 85, 1072-1077.
Murphy, T., Darby, S., Mathers, M. E. & Gnanapragasam, V. J. 2010. Evidence
for distinct alterations in the FGF axis in prostate cancer progression to
an aggressive clinical phenotype. The Journal of Pathology, 220, 452-
460.
Mutsaers, S. E. 2002. Mesothelial cells: Their structure, function and role in
serosal repair. Respirology, 7, 171-191.
Mutsaers, S. E. 2004. The mesothelial cell. The International Journal of
Biochemistry & Cell Biology, 36, 9-16.
Mutsaers, S. E., Bishop, J. E., Mcgrouther, G. & Laurent, G. J. 1997.
Mechanisms of tissue repair: from wound healing to fibrosis. The
International Journal of Biochemistry & Cell Biology, 29, 5-17.

xxxix
Nakazawa, K., Yashiro, M. & Hirakawa, K. 2003. Keratinocyte growth factor
produced by gastric fibroblasts specifically stimulates proliferation of
cancer cells from scirrhous gastric carcinoma. Cancer Res., 63, 8848-
8852.
Naruo, K., Seko, C., Kuroshima, K., Matsutani, E., Sasada, R., Kondo, T. &
Kurokawa, T. 1993. Novel secretory heparin-binding factors from human
glioma cells (glia-activating factors) involved in glial cell growth.
Purification and biological properties. Journal of Biological Chemistry,
268, 2857-2864.
Naski, M. C., Wang, Q., Xu, J. & Ornitz, D. M. 1996. Graded activation of
fibroblast growth factor receptor 3 by mutations causing achondroplasia
and thanatophoric dysplasia. Nature Genet., 13, 233-237.
Nasreen, N., Mohammed, K. A. & Antony, V. B. 2006. Silencing the receptor
EphA2 suppresses the growth and haptotaxis of malignant mesothelioma
cells. Cancer, 107, 2425-2435.
Nasreen, N., Mohammed, K. A., Dowling, P. A., Ward, M. J., Galffy, G. &
Antony, V. B. 2000. Talc Induces Apoptosis in Human Malignant
Mesothelioma Cells In Vitro. American Journal of Respiratory and Critical
Care Medicine, 161, 595-600.
Neuzil, J., Swettenham, E., Wang, X.-F., Dong, L.-F. & Stapelberg, M. 2007. α-
Tocopheryl succinate inhibits angiogenesis by disrupting paracrine FGF2
signalling. FEBS Letters, 581, 4611-4615.
Novak-Weekley, S. M., Marlowe, E. M., Poulter, M., Dwyer, D., Speers, D.,
Rawlinson, W., Baleriola, C. & Robinson, C. C. 2012. Evaluation of the

xl
Cepheid® Xpert® Flu Assay for Rapid Identification and Differentiation of
Influenza A, Influenza A 2009 H1N1, and Influenza B. Journal of Clinical
Microbiology.
Nowak, A. K., Millward, M., Francis, R. J., Hasani, A., Van Der Schaaf, A. A.,
Seguard, T., Musk, A. W. & J., B. M. 2010. Final results of a phase II
study of sunitinib as second-line therapy in malignant pleural
mesothelioma (MPM). [abstract 7036] J Clin Oncol, 28.
Nutt, J. E., Razak, A. R. A., O'toole, K., Black, F., Quinn, A. E., Calvert, A. H.,
Plummer, E. R. & Lunec, J. 2010. The role of folate receptor alpha
(FR[alpha]) in the response of malignant pleural mesothelioma to
pemetrexed-containing chemotherapy. Br J Cancer, 102, 553-560.
O’byrne, K. J., Edwards, J. G. & Waller, D. A. 2004. Clinico-pathological and
biological prognostic factors in pleural malignant mesothelioma. Lung
Cancer, 45, Supplement, S45-S48.
Odor, D. L. 1954. Observations of the rat mesothelium with electron and phase
microscopes. Am. J. Anat., 95, 433.
Oels, H. C., Harrison, E. G., Carr, D. T. & Bernatz, P. E. 1971. Diffuse
Malignant Mesothelioma of the Pleura: A Review of 37 Cases. Chest, 60,
564-570.
Ohashi Y., H. A., Iwai T., Mori Y. 1988. Stimulatory effect of vanadate on
hyaluronic acid synthesis in mesothelial cells from rabbit pericardium.
Biochem Int., 16, 293-302.

xli
Onda, M., Nagata, S., Ho, M., Bera, T. K., Hassan, R., Alexander, R. H. &
Pastan, I. 2006. Megakaryocyte Potentiation Factor Cleaved from
Mesothelin Precursor Is a Useful Tumor Marker in the Serum of Patients
with Mesothelioma. Clinical Cancer Research, 12, 4225-4231.
Ong, S. H., Guy, G. R., Hadari, Y. R., Laks, S., Gotoh, N., Schlessinger, J. &
Lax, I. 2000. FRS2 Proteins Recruit Intracellular Signaling Pathways by
Binding to Diverse Targets on Fibroblast Growth Factor and Nerve
Growth Factor Receptors. Molecular and Cellular Biology, 20, 979-989.
Ornitz, D. & Itoh, N. 2001. Fibroblast growth factors. Genome Biology, 2,
reviews3005.1 - reviews3005.12.
Ornitz, D. M. 2000. FGFs, heparan sulfate and FGFRs: complex interactions
essential for development. BioEssays, 22, 108 - 112.
Ornitz, D. M. & Marie, P. J. 2002. FGF signaling pathways in endochondral and
intramembranous bone development and human genetic disease. Genes
& Development, 16, 1446-1465.
Ornitz, D. M., Xu, J., Colvin, J. S., Mcewen, D. G., Macarthur, C. A., Coulier, F.,
Gao, G. & Goldfarb, M. 1996. Receptor specificity of the fibroblast growth
factor family. J Biol Chem, 271, 15292 - 15297.
Orr-Urtreger, A., Bedford, M. T., Burakova, T., Arman, E., Zimmer, Y., Yayon,
A., Givol, D. & Lonai, P. 1993. Developmental localization of the splicing
alternatives of fibroblast growth factor receptor-2 (FGFR2). Dev Biol,
158, 475 - 486.

xlii
Otsuki T., Yamada O., Yata K., Sakaguchi H., Kurebayashi J., Nakazawa N.,
Taniwaki M., Yawata Y. & Ueki A. 1999. Expression of fibroblast growth
factor and FGF-receptor family genes in human myeloma cells, including
lines possessing t(4;14)(q16.3;q32. 3) and FGFR3 translocation. Int J
Oncol, 15, 1205-12.
Owens, M. W. & Grisham, M. B. 1993. Nitric oxide synthesis by rat pleural
mesothelial cells: induction by cytokines and lipopolysaccharide.
American Journal of Physiology - Lung Cellular and Molecular
Physiology, 265, L110-L116.
Pandith, A. A., Shah, Z. A. & Siddiqi, M. A. 2010. Oncogenic role of fibroblast
growth factor receptor 3 in tumorigenesis of urinary bladder cancer. Urol.
Oncol.
Park, E.-K., Sandrini, A., Yates, D. H., Creaney, J., Robinson, B. W., Thomas,
P. S. & Johnson, A. R. 2008. Soluble Mesothelin-related Protein in an
Asbestos-exposed Population. American Journal of Respiratory and
Critical Care Medicine, 178, 832-837.
Pass, H. I., Levin, S. M., Harbut, M. R., Melamed, J., Chiriboga, L., Donington,
J., Huflejt, M., Carbone, M., Chia, D., Goodglick, L., Goodman, G. E.,
Thornquist, M. D., Liu, G., De Perrot, M., Tsao, M.-S. & Goparaju, C.
2012. Fibulin-3 as a Blood and Effusion Biomarker for Pleural
Mesothelioma. New England Journal of Medicine, 367, 1417-1427.
Pass, H. I., Lott, D., Lonardo, F., Harbut, M., Liu, Z., Tang, N., Carbone, M.,
Webb, C. & Wali, A. 2005. Asbestos Exposure, Pleural Mesothelioma,

xliii
and Serum Osteopontin Levels. New England Journal of Medicine, 353,
1564-1573.
Pass, H. I., Wali, A., Tang, N., Ivanova, A., Ivanov, S., Harbut, M., Carbone, M.
& Allard, J. 2008. Soluble Mesothelin-Related Peptide Level Elevation in
Mesothelioma Serum and Pleural Effusions. Ann Thorac Surg, 85, 265-
272.
Pelin, K., Hirvonen, A. & Linnainmaa, K. 1994. Expression of cell adhesion
molecules and connexins in gap junctional intercellular communication
deficient human mesothelioma tumour cell lines and communication
competent primary mesothelial cells. Carcinogenesis, 15, 2673-2675.
Peng, M.-J., Wang, N.-S., Vargas, F. S. & Light, R. W. 1994. Subclinical
Surface Alterations of Human Pleura. Chest, 106, 351-353.
Plotnikov, A. N., Eliseenkova, A. V., Ibrahimi, O. A., Shriver, Z., Sasisekharan,
R., Lemmon, M. A. & Mohammadi, M. 2001. Crystal Structure of
Fibroblast Growth Factor 9 Reveals Regions Implicated in Dimerization
and Autoinhibition. Journal of Biological Chemistry, 276, 4322-4329.
Podolsky, D. K. 1997. Healing the epithelium: Solving the problem from two
sides. Journal of Gastroenterology, 32, 122-126.
Powers, C. J., Mcleskey, S. W. & Wellstein, A. 2000. Fibroblast growth factors,
their receptors and signaling. Endocr Relat Cancer, 7, 165-197.
Price, B. & Ware, A. 2004. Mesothelioma Trends in the United States: An
Update Based on Surveillance, Epidemiology, and End Results Program

xliv
Data for 1973 through 2003. American Journal of Epidemiology, 159,
107-112.
Qing, J., Du, X., Chen, Y., Chan, P., Li, H., Wu, P., Marsters, S., Stawicki, S.,
Tien, J., Totpal, K., Ross, S., Stinson, S., Dornan, D., French, D., Wang,
Q.-R., Stephan, J.-P., Wu, Y., Wiesmann, C. & Ashkenazi, A. 2009.
Antibody-based targeting of FGFR3 in bladder carcinoma and t(4;14)-
positive multiple myeloma in mice. The Journal of Clinical Investigation,
119, 1216-1229.
Rahman, N. M., Mishra, E. K., Davies, H. E., Davies, R. J. O. & Lee, Y. C. G.
2008. Clinically Important Factors Influencing the Diagnostic
Measurement of Pleural Fluid pH and Glucose. American Journal of
Respiratory and Critical Care Medicine, 178, 483-490.
Rapraeger, A. C., Krufka, A. & Olwin, B. B. 1991. Requirement of heparan
sulfate for bFGF-mediated fibroblast growth and myoblast differentiation.
Science, 252, 1705 - 1708.
Ray, M. & Kindler, H. L. 2009. Malignant Pleural Mesothelioma. Chest, 136,
888-896.
Revest, J.-M., Demoerlooze, L. & Dickson, C. 2000. Fibroblast Growth Factor 9
Secretion Is Mediated by a Non-cleaved Amino-terminal Signal
Sequence. J. Biol. Chem., 275, 8083-8090.
Riedl, S. J. & Shi, Y. 2004. Molecular mechanisms of caspase regulation during
apoptosis. Nat Rev Mol Cell Biol, 5, 897-907.

xlv
Robinson, B. W. S., Creaney, J., Lake, R., Nowak, A., Musk, A. W., De Klerk,
N., Winzell, P., Hellstrom, K. E. & Hellstrom, I. 2003. Mesothelin-family
proteins and diagnosis of mesothelioma. The Lancet, 362, 1612-1616.
Robinson, B. W. S. & Lake, R. A. 2005. Advances in Malignant Mesothelioma.
New England Journal of Medicine, 353, 1591-1603.
Roghani, M., Mansukhani, A., Dell'era, P., Bellosta, P., Basilico, C., Rifkin, D. B.
& Moscatelli, D. 1994. Heparin increases the affinity of basic fibroblast
growth factor for its receptor but is not required for binding. Journal of
Biological Chemistry, 269, 3976-3984.
Rose-John, S. & Heinrich, P. C. 1994. Soluble receptors for cytokines and
growth factors: generation and biological function. The Biochemical
journal, 300 ( Pt 2), 281-90.
Rosty, C., Aubriot, M., Cappellen, D., Bourdin, J., Cartier, I., Thiery, J. P.,
Sastre-Garau, X. & Radvanyi, F. 2005. Clinical and biological
characteristics of cervical neoplasias with FGFR3 mutation. Molecular
Cancer 4.
Roth, J. 1973. Ultrahistochemical demonstration of saccharide components of
complex carbohydrates at the alveolar cell surface and at the mesothelial
cell surface of the pleura visceralis of mice by means of concanavalin A.
Exp Pathol (Jena). , 8, 157-67.
Rougier, J.-P., Moullier, P., Piedagnel, R., Ronco, P. M. & Guia, S. 1997.
Hyperosmolality suppresses but TGF[beta]1 increases MMP9 in human
peritoneal mesothelial cells. Kidney Int, 51, 337-347.

xlvi
Rudd, R. M. 2010. Malignant mesothelioma. British Medical Bulletin, 93, 105-
123.
Saad, R. S., Cho, P., Liu, Y. L. & Silverman, J. F. 2005. The value of epithelial
membrane antigen expression in separating benign mesothelial
proliferation from malignant mesothelioma: A comparative study.
Diagnostic Cytopathology, 32, 156-159.
Santos-Ocampo, S., Colvin, J. S., Chellaiah, A. & Ornitz, D. M. 1996.
Expression and Biological Activity of Mouse Fibroblast Growth Factor-9.
J. Biol. Chem., 271, 1726-1731.
Satoh K, A. H., Nagai H, Ito M, Sato H, Motomiya M, Konno K. 1987. Acid
glycosaminoglycans in experimental pleural effusions. Lung, 165, 191-9.
Scherpereel, A., Astoul, P., Baas, P., Berghmans, T., Clayson, H., De Vuyst, P.,
Dienemann, H., Galateau-Salle, F., Hennequin, C., Hillerdal, G., Le
Péchoux, C., Mutti, L., Pairon, J.-C., Stahel, R., Van Houtte, P., Van
Meerbeeck, J., Waller, D. & Weder, W. 2010. Guidelines of the European
Respiratory Society and the European Society of Thoracic Surgeons for
the management of malignant pleural mesothelioma. European
Respiratory Journal, 35, 479-495.
Schlessinger, J., Plotnikov, A. N., Ibrahimi, O. A., Eliseenkova, A. V., Yeh, B. K.,
Yayon, A., Linhardt, R. J. & Mohammadi, M. 2000. Crystal structure of a
ternary FGF-FGFR-heparin complex reveals a dual role for heparin in
FGFR binding and dimerization. Mol Cell, 6, 743 - 750.
Scott B., M. S., Lake R., Robinson B.W.S. 2000. Malignant Mesothelioma. In:
Hanson H, ed. Textbook of lung cancer, London: Martin Dunitz, 273-93.

xlvii
Sebastien P., J. X., Gaudichet A., Hirsch A., Bignon J. 1980. Asbestos retention
in human respiratory tissues: comparative measurements in lung
parenchyma and in parietal pleura. clinicopathological, 30, 237-46.
Segal A, W. D., Henderson D, Shilkin K 2002. Pathology of mesothelioma. In:
Robinson BWS, Chahinian AP, eds. Mesothelioma, London: Martin
Dunitz, 143-84.
Sekine, K., Ohuchi, H., Fujiwara, M., Yamasaki, M., Yoshizawa, T., Sato, T.,
Yagishita, N., Matsui, D., Koga, Y., Itoh, N. & Kato, S. 1999. Fgf10 is
essential for limb and lung formation. Nat Genet, 21, 138 - 141.
Servais, E. L., Colovos, C., Rodriguez, L., Bograd, A. J., Nitadori, J.-I., Sima, C.,
Rusch, V. W., Sadelain, M. & Adusumilli, P. S. 2012. Mesothelin
Overexpression Promotes Mesothelioma Cell Invasion and MMP-9
Secretion in an Orthotopic Mouse Model and in Epithelioid Pleural
Mesothelioma Patients. Clinical Cancer Research.
Shi, Y., Moura, U., Opitz, I., Soltzermann, A., Rehrauer, H., Thies, S., Weder,
W., Stahel, R. A. & Felley-Bosco, E. 2012. ROLE OF HEDGEHOG
SIGNALING IN MALIGNANT PLEURAL MESOTHELIOMA. Clinical
Cancer Research.
Stan, A. C., Nemati, M. N., Pietsch, T., Walter, G. F. & Dietz, H. 1995. In vivo
inhibition of angiogenesis and growth of the human U-87 malignant glial
tumor by treatment with an antibody against basic fibroblast growth
factor. Journal of Neurosurgery, 82, 1044-1052.
Stapelberg, M., Gellert, N., Swettenham, E., Tomasetti, M., Witting, P. K.,
Procopio, A. & Neuzil, J. 2005. α-Tocopheryl Succinate Inhibits Malignant

xlviii
Mesothelioma by Disrupting the Fibroblast Growth Factor Autocrine
Loop. Journal of Biological Chemistry, 280, 25369-25376.
Stathopoulos, G. T., Kollintza, A., Moschos, C., Psallidas, I., Sherrill, T. P.,
Pitsinos, E. N., Vassiliou, S., Karatza, M., Papiris, S. A., Graf, D.,
Orphanidou, D., Light, R. W., Roussos, C., Blackwell, T. S. &
Kalomenidis, I. 2007. Tumor Necrosis Factor-α Promotes Malignant
Pleural Effusion. Cancer Research, 67, 9825-9834.
Stathopoulos, G. T., Psallidas, I., Moustaki, A., Moschos, C., Kollintza, A.,
Karabela, S., Porfyridis, I., Vassiliou, S., Karatza, M., Zhou, Z., Joo, M.,
Blackwell, T. S., Roussos, C., Graf, D. & Kalomenidis, I. 2008. A Central
Role for Tumor-derived Monocyte Chemoattractant Protein-1 in
Malignant Pleural Effusion. Journal of the National Cancer Institute, 100,
1464-1476.
Strizzi, L., Catalano, A., Vianale, G., Orecchia, S., Casalini, A., Tassi, G.,
Puntoni, R., Mutti, L. & Procopio, A. 2001. Vascular endothelial growth
factor is an autocrine growth factor in human malignant mesothelioma.
The Journal of Pathology, 193, 468-475.
Sumie, M.-Y., Junko, H., Chisako, N., Ken-Ichi, K. & Tsutomu, K. 1997.
Autocrine transformation by fibroblast growth factor 9 (FGF-9) and its
possible participation in human oncogenesis. International Journal of
Cancer, 71, 442-450.
Suzuki, Y. 2001. Pathology of Human Malignant Mesothelioma. Preliminary
Analysis of 1,517 Mesothelioma Cases. Industrial Health, 39, 183-185.

xlix
Tagashira, S., Ozaki, K., Ohta, M. & Itoh, N. 1995. Localization of fibroblast
growth factor-9 mRNA in the rat brain. Molecular Brain Research, 30,
233-241.
Takahashi, J. A., Fukumoto, M., Kozai, Y., Ito, N., Oda, Y., Kikuchi, H. &
Hatanaka, M. 1991. Inhibition of cell growth and tumorigenesis of human
glioblastoma cells by a neutralizing antibody against human basic
fibroblast growth factor. FEBS Letters, 288, 65-71.
Takeda, M. 2007. AZD2171 shows potent antitumor activity against gastric
cancer over-expressing fibroblast growth factor receptor 2/keratinocyte
growth factor receptor. Clin. Cancer Res., 13, 3051-3057.
Teishima, J., Shoji, K., Hayashi, T., Miyamoto, K., Ohara, S. & Matsubara, A.
2012. Relationship between the localization of fibroblast growth factor 9
in prostate cancer cells and postoperative recurrence. Prostate Cancer
Prostatic Dis, 15, 8-14.
Ten Hove, T., Van Den Blink, B., Pronk, I., Drillenburg, P., Peppelenbosch, M.
P. & Van Deventer, S. J. H. 2002. Dichotomal role of inhibition of p38
MAPK with SB 203580 in experimental colitis. Gut, 50, 507-512.
Testa, J. R., Cheung, M., Pei, J., Below, J. E., Tan, Y., Sementino, E., Cox, N.
J., Dogan, A. U., Pass, H. I., Trusa, S., Hesdorffer, M., Nasu, M.,
Powers, A., Rivera, Z., Comertpay, S., Tanji, M., Gaudino, G., Yang, H. &
Carbone, M. 2011. Germline BAP1 mutations predispose to malignant
mesothelioma. Nat Genet, 43, 1022-1025.

l
Thickett, D. R., Armstrong, L. & Millar, A. B. 1999. Vascular endothelial growth
factor (VEGF) in inflammatory and malignant pleural effusions. Thorax,
54, 707-710.
Thisse, B. & Thisse, C. 2005. Functions and regulations of fibroblast growth
factor signaling during embryonic development. Developmental Biology,
287, 390-402.
Todo, T., Kondo, T., Kirino, T., Asai, A., Adams, E. F., Nakamura, S., Ikeda, K.
& Kurokawa, T. 1998. Expression and Growth Stimulatory Effect of
Fibroblast Growth Factor 9 in Human Brain Tumors. Neurosurgery, 43,
337-346.
Tomlinson, D. C., Hurst, C. D. & Knowles, M. A. 2007. Knockdown by shRNA
identifies S249C mutant FGFR3 as a potential therapeutic target in
bladder cancer. Oncogene, 26, 5889-5899.
Topley N., L. T., Davenport A., Li F.K., Fear H., Williams J.D. 1996. Activation of
inflammation and leukocyte recruitment into the peritoneal cavity. Kidney
Int Suppl, 56, S17-21.
Tracey, W. R., Nakane, M., Kuk, J., Budzik, G., Klinghofer, V., Harris, R. &
Carter, G. 1995. The nitric oxide synthase inhibitor, L-NG-
monomethylarginine, reduces carrageenan-induced pleurisy in the rat.
Journal of Pharmacology and Experimental Therapeutics, 273, 1295-
1299.
Treasure, T., Lang-Lazdunski, L., Waller, D., Bliss, J. M., Tan, C., Entwisle, J.,
Snee, M., O'brien, M., Thomas, G., Senan, S., O'byrne, K., Kilburn, L. S.,
Spicer, J., Landau, D., Edwards, J., Coombes, G., Darlison, L. & Peto, J.

li
2011. Extra-pleural pneumonectomy versus no extra-pleural
pneumonectomy for patients with malignant pleural mesothelioma:
clinical outcomes of the Mesothelioma and Radical Surgery (MARS)
randomised feasibility study. The Lancet Oncology, 12, 763-772.
Tsai, S.-J., Wu, M.-H., Chen, H.-M., Chuang, P.-C. & Wing, L.-Y. C. 2002.
Fibroblast Growth Factor-9 Is an Endometrial Stromal Growth Factor.
Endocrinology, 143, 2715-2721.
Turner, N. & Grose, R. 2010. Fibroblast growth factor signalling: from
development to cancer. Nat Rev Cancer, 10, 116-129.
Turner, N., Lambros, M. B., Horlings, H. M., Pearson, A., Sharpe, R., Natrajan,
R., Geyer, F. C., Van Kouwenhove, M., Kreike, B., Mackay, A.,
Ashworth, A., Van De Vijver, M. J. & Reis-Filho, J. S. 2010. Integrative
molecular profiling of triple negative breast cancers identifies amplicon
drivers and potential therapeutic targets. Oncogene, 29, 2013-2023.
Ueng, T.-H., Chang, Y.-L., Tsai, Y.-Y., Su, J.-L., Chan, P.-K., Shih, J.-Y., Lee,
Y.-C., Ma, Y.-C. & Kuo, M.-L. 2010. Potential roles of fibroblast growth
factor-9 in the benzo(a)pyrene-induced invasion in vitro and the
metastasis of human lung adenocarcinoma. Genotoxicity and
carcinogenicity.
Van Den Tol, P. M., Van Rossen, E. E. M., Van Eijck, C. H. J., Bonthuis, F.,
Marquet, R. L. & Jeekel, H. 1998. Reduction of Peritoneal Trauma By
Using Nonsurgical Gauze Leads to Less Implantation Metastasis of
Spilled Tumor Cells. Annals of Surgery, 227, 242-248.

lii
Versnel M.A., C.-W. L., Hammacher A., Bouts M.J., Van Der Kwast T.H.,
Eriksson A., Willemsen R., Weima S.M., Hoogsteden H.C., Hagemeijer
A., Et Al. 1991. Human malignant mesothelioma cell lines express PDGF
beta-receptors whereas cultured normal mesothelial cells express
predominantly PDGF alpha-receptors. Oncogene, 6, 2005-11.
Vogelzang, N. J., Rusthoven, J. J., Symanowski, J., Denham, C., Kaukel, E.,
Ruffie, P., Gatzemeier, U., Boyer, M., Emri, S., Manegold, C., Niyikiza, C.
& Paoletti, P. 2003. Phase III Study of Pemetrexed in Combination With
Cisplatin Versus Cisplatin Alone in Patients With Malignant Pleural
Mesothelioma. Journal of Clinical Oncology, 21, 2636-2644.
Wagner, J. C., Sleggs, C. A. & Marchand, P. 1960. Diffuse Pleural
Mesothelioma and Asbestos Exposure in the North Western Cape
Province. Br J Ind Med, 17, 260-271.
Wang, N. S. 1974. The difference of pleural mesothelial cells in rabbits. Am.
Rev. Respir. Dis., 110, 623–33.
Wang, P. M. & Lai-Fook, S. J. 1998. Effects of Ventilation on Hyaluronan and
Protein Concentration in Pleural Liquid of Anesthetized and Conscious
Rabbits. Lung, 176, 309-324.
Weder W & Opitz I 2012. Multimodality therapy for malignant pleural
mesothelioma. Ann Cardiothorac Surg, 1, 502-507.
Weder W, Stahel Ra, Baas P, Dafni U, De Perrot M, Mccaughan Bc, Nakano T,
Pass Hi, Robinson Bw, Rusch Vw, Sugarbaker Dj & N., V. Z. 2011. The
MARS feasibility trial: conclusions not supported by data. Lancet Oncol.,
12, 1093-4.

liii
Werner, S., Duan, D. S., De Vries, C., Peters, K. G., Johnson, D. E. & Williams,
L. T. 1992. Differential splicing in the extracellular region of fibroblast
growth factor receptor 1 generates receptor variants with different ligand-
binding specificities. Mol. Cell. Biol., 12, 82-88.
Wheatley-Price, P., Yang, B., Patsios, D., Patel, D., Ma, C., Xu, W., Leighl, N.,
Feld, R., Cho, B. C. J., O'sullivan, B., Roberts, H., Tsao, M. S.,
Tammemagi, M., Anraku, M., Chen, Z., De Perrot, M. & Liu, G. 2010.
Soluble Mesothelin-Related Peptide and Osteopontin As Markers of
Response in Malignant Mesothelioma. Journal of Clinical Oncology, 28,
3316-3322.
Whitaker, D. 2000. Invited review The cytology of malignant mesothelioma.
Cytopathology, 11, 139-151.
Whitaker, D., Papadimitriou, J. M. & Walters, M. N.-I. 1982. The Mesothelium
and Its Reactions: A Review. Critical Reviews in Toxicology, 10, 81-144.
White, A. C., Lavine, K. J. & Ornitz, D. M. 2007. FGF9 and SHH regulate
mesenchymal Vegfa expression and development of the pulmonary
capillary network. Development, 134, 3743-3752.
White, A. C., Xu, J., Yin, Y., Smith, C., Schmid, G. & Ornitz, D. M. 2006. FGF9
and SHH signaling coordinate lung growth and development through
regulation of distinct mesenchymal domains. Development, 133, 1507-
1517.
Wiesmann, C., Muller, Y. A. & De Vos, A. M. 2000. Ligand-binding sites in Ig-
like domains of receptor tyrosine kinases. Journal of Molecular Medicine,
78, 247-260.

liv
Wilson, T. & Treisman, R. 1988. Removal of poly(A) and consequent
degradation of c-fos mRNA facilitated by 3[prime] AU-rich sequences.
Nature, 336, 396-399.
Wing, L.-Y. C., Chen, H.-M., Chuang, P.-C., Wu, M.-H. & Tsai, S.-J. 2005. The
Mammalian Target of Rapamycin-p70 Ribosomal S6 Kinase but Not
Phosphatidylinositol 3-Kinase-Akt Signaling Is Responsible for Fibroblast
Growth Factor-9-induced Cell Proliferation. Journal of Biological
Chemistry, 280, 19937-19947.
Wing, L.-Y. C., Chuang, P.-C., Wu, M.-H., Chen, H.-M. & Tsai, S.-J. 2003.
Expression and Mitogenic Effect of Fibroblast Growth Factor-9 in Human
Endometriotic Implant Is Regulated by Aberrant Production of Estrogen.
J Clin Endocrinol Metab, 88, 5547-5554.
Wolanski, K. D., Whitaker, D., Shilkin, K. B. & Henderson, D. W. 1998. The use
of epithelial membrane antigen and silver-stained nucleolar organizer
regions testing in the differential diagnosis of mesothelioma from benign
reactive mesothelioses. Cancer, 82, 583-590.
Workalemahu, G., Foerster, M. & Kroegel, C. 2004. Expression and synthesis
of fibroblast growth factor-9 in human {gamma}{delta} T-lymphocytes.
Response to isopentenyl pyrophosphate and TGF-{beta}1/IL-15. J
Leukoc Biol, 75, 657-663.
Wu, M.-H., Shoji, Y., Chuang, P.-C. & Tsai, S.-J. 2007. Endometriosis: disease
pathophysiology and the role of prostaglandins. Expert Reviews in
Molecular Medicine, 9, 1-20.

lv
Wüchner, C., Hilbert, K., Zabel, B. & Winterpacht, A. 1997. Human fibroblast
growth factor receptor 3 gene (FGFR3): genomic sequence and primer
set information for gene analysis. Human Genetics, 100, 215-219.
Xu, H., Lee, K. W. & Goldfarb, M. 1998. Novel Recognition Motif on Fibroblast
Growth Factor Receptor Mediates Direct Association and Activation of
SNT Adapter Proteins. Journal of Biological Chemistry, 273, 17987-
17990.
Xu, N., Chen, C. Y. & Shyu, A. B. 1997. Modulation of the fate of cytoplasmic
mRNA by AU-rich elements: key sequence features controlling mRNA
deadenylation and decay. Mol. Cell. Biol., 17, 4611-4621.
Yamaguchi, T. P. & Rossant, J. 1995. Fibroblast growth factors in mammalian
development. Current Opinion in Genetics & Development, 5, 485-491.
Yan, G., Fukabori, Y., Mcbride, G., Nikolaropolous, S. & Mckeehan, W. L. 1993.
Exon switching and activation of stromal and embryonic fibroblast growth
factor (FGF)-FGF receptor genes in prostate epithelial cells accompany
stromal independence and malignancy. Mol Cell Biol, 13, 4513 - 4522.
Yarborough, C. M. 2006. Chrysotile as a cause of mesothelioma: an
assessment based on epidemiology. Crit Rev Toxicol, 36, 165-87.
Yayon, A., Klagsbrun, M., Esko, J. D., Leder, P. & Ornitz, D. M. 1991. Cell
surface, heparin-like molecules are required for binding of basic
fibroblast growth factor to its high affinity receptor. Cell, 64, 841 - 848.
Yiangou, C., Cox, H., Bansal, G. S., Coope, R., Gomm, J. J., Barnard, R.,
Walters, J., Groome, N., Shousha, S., Coombes, R. C. & Johnston, C. L.

lvi
1997. Down-regulation of a novel form of fibroblast growth factor receptor
1 in human breast cancer. Br J Cancer, 76, 1419-1427.
Yin, Y., Wang, F. & Ornitz, D. M. 2011. Mesothelial- and epithelial-derived
FGF9 have distinct functions in the regulation of lung development.
Development, 138, 3169-3177.
Yin, Y., White, A. C., Huh, S.-H., Hilton, M. J., Kanazawa, H., Long, F. & Ornitz,
D. M. 2008. An FGF–WNT gene regulatory network controls lung
mesenchyme development. Developmental Biology, 319, 426-436.
Yung, S., Thomas, G. J. & Davies, M. 2000. Induction of hyaluronan
metabolism after mechanical injury of human peritoneal mesothelial cells
in vitro. Kidney Int, 58, 1953-1962.
Zalcman G., Mazieres J., Scherpereel A., Margery J., Moro-Sibilot D., Parienti
J., Gounant V., Riviere A., Monnet I., Molinier O., Lena H., Friard S.,
Duhamel J., Audigier-Valette G., Robinet C., Creveuil C., Ligeza-Poisson
C. & F., M. 2012. IFCT-GFPC-0701 MAPS trial, a multicenter
randomized phase III trial of pemetrexed-cisplatin with or without
bevacizumab in patients with malignant pleural mesothelioma (MPM). J
Clin Oncol 30.
Zanella, C. L., Posada, J., Tritton, T. R. & Mossman, B. T. 1996. Asbestos
Causes Stimulation of the Extracellular Signal-regulated Kinase 1
Mitogen-activated Protein Kinase Cascade after Phosphorylation of the
Epidermal Growth Factor Receptor. Cancer Research, 56, 5334-5338.
Zeillemaker, A. M., Mul, F. P. J., Hoynck Van Papendrecht, A. a. G. M., Leguit,
P., Verbrugh, H. A. & Roos, D. 1996. Neutrophil adherence to and

lvii
migration across monolayers of human peritoneal mesothelial cells: The
role of mesothelium in the influx of neutrophils during peritonitis. The
Journal of laboratory and clinical medicine, 127, 279-286.
Zervos, M., Bizekis, C. & Pass, H. 2008. Malignant mesothelioma 2008. Curr
Opin Pulm Med, 14, 303-309.
Zhang H., Masuoka L., Baker K., Sadra A., Bosch E., Brennan T., Doberstein
S., Goodworth G., Hestir K., Hollenbaugh D., Long L., Qin M. & Williams
L. T. 2007. FP-1039 (FGFR1:Fc), a soluble FGFR1 receptor antagonist,
inhibits tumor growth and angiogenesis AACR–NCI–EORTC
International Conference, San Francisco.
Zhang, X., Ibrahimi, O. A., Olsen, S. K., Umemori, H., Mohammadi, M. & Ornitz,
D. M. 2006. Receptor Specificity of the Fibroblast Growth Factor Family.
Journal of Biological Chemistry, 281, 15694-15700.