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Havcr-1 and the Prevention of Metastatic Disease in Human Prostate Cancer

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Havcr-1 and the Prevention of Metastatic Disease in Human Prostate Cancer HAVcR-1 and the Prevention of Metastatic Disease in Human Prostate Cancer by Emily Jacqueline Ann Telford A Dissertation Submitted to Cardiff University in Candidature for the Degree of Doctor of Philosophy Cardiff- China Medical Research Collaborative (CCMRC) Cardiff University Henry Welcome Building Heath Park CF14 4XN United Kingdom March 2019 I Declaration and Statements DECLARATION This work has not been submitted in substance for any other degree or award at this or any other university or place of learning, nor is being submitted concurrently in candidature for any degree or other award. Signed Date: 14/03/2019 STATEMENT 1 This thesis is being submitted in partial fulfilment of the requirements for the degree of PhD Signed Date: 14/03/2019 STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated, and the thesis has not been edited by a third party beyond what is permitted by Cardiff University’s Policy on the Use of Third-Party Editors by Research Degree Students. Other sources are acknowledged by explicit references. The views expressed are my own. Signed Date: 14/03/2019 STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available online in the University’s Open Access repository and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed Date: 14/03/2019 II Acknowledgments I would like to extend my gratitude to my supervisors, Professor Wen Jiang, Dr Stephen Hiscox and Dr Tracey Martin for their support and guidance throughout my PhD. I would also like to thank my funding bodies, Cardiff University and Cancer Research Wales. There are so many people that have helped me throughout my time at the Cardiff- China Medical Research Collaborative. I would like to mention Dr Andrew Sanders, Dr Sioned Owen and Miss Fiona Ruge who have always been available to offer advice and support throughout my PhD. I would also like to thank all the other PhD students I have worked alongside, without the company, conversation and joy that you all provided, my time in Cardiff would not have been the same. I am lucky in that I have also been surrounded by amazing people outside of CCMRC. My family and friends have provided a remarkable amount of love and support throughout my life. I am forever grateful for all the tough times you have helped me through and all the opportunities that I have been able to seize because you have all been there for me. My mother, Pat Telford, deserves a special mention, for I wouldn’t have achieved this without the unwavering belief she has in me as well as the support and guidance she has given me throughout my life. III Publications Abstracts Martin TA, Telford E and Jiang WG. HAVCR1 is a potential prognostic factor in human breast cancer. Anticancer Research. 2015 July; 35 (7) :4327. Reviews Telford EJA, Jiang WG and Martin TA. HAVcR-1 involvement in cancer progression. Histology and Histopathology. 2017 Feb; 32 (2): 121-128 IV Conference Attendance Poster Presentations China UK Cancer (CUKC) Conference in Cardiff in July 2015 Cancer Research Wales 50th Anniversary Symposium in Cardiff in March 2016 Oral Presentations Capital International Cancer Conference 2015 in Beijing in October 2015 CUKC Conference in Beijing in April 2017 V Abbreviations (cc)RCC (clear cell) Renal Cell Carcinoma ACTH Adrenocorticotropic Hormone ADAM A disintegrin and metalloprotease ADC Antibody Drug Conjugate ADT Androgen Deprivation Therapy AF Activation Function AGMK African green monkey kidney cells AJs Adherens Junctions ANOVA Analysis of Variance AP2 Activator Protein-2 AR Androgen Receptor AR-Vs Androgen Receptor Splice Variants ATAD2 ATPase family AAA domain-containing protein 2 ATCC American Tissue Culture Collection ATP Adenosine Triphosphate BAD BCL-2 antagonist of cell death bFGF Basic Fibroblast Growth Factor BM40 Basement Membrane Protein 40 BPE Benign Prostatic Enlargement BPE Bovine Pituitary Extract BPH Benign Prostatic Hyperplasia BRCA1/2 Breast cancer susceptibility gene 1/2 BSA Bovine Serum Albumin VI CAPZB Capping Actin Protein of Muscle Z-Line Beta Subunit CAR Coxsackievirus and Adenovirus Receptor c-Cbl casitas B-lineage lymphoma CCL21 C-C motif ligand 21 CCR7 C-C motif chemokine receptor 7 CD44 Cluster of Differentiation 44 CE Cryptic Exon CHEK2 Checkpoint kinase 2 CPE Clostridium perfringens enterotoxin CPPS Chronic Pelvic Pain Syndrome CRH Corticotropin-releasing hormone CRPC Castrate Resistant Prostate Cancer CT scan Computerised Tomography Scan CT Calcitonin C-terminus Carboxyl-terminus CTL Cytotoxic T-leucocyte CXCL12 C-X-C motif ligand 12 CXCR4 C-X-C chemokine receptor 4 CYP17 Cytochrome p17 DAPI 4’,6-diamidino-2-phenylindole DBD DNA Binding Domain dEP1 density enhance phosphatase 1 DEPC Diethylpyrocarbonate DHT Dihydrotestosterone VII DMEM Dulbecco’s modified Eagle’s medium DMSO Dimethylsuphoxide DNA Deoxyribonucleic acid DRE Digital Rectal Examination DS Desmosomes EC Extracellular E-Cadherin Epithelial Cadherin ECIS Electric Cell-Substrate Impedance Sensing ECM Extracellular Matrix EDTA Ethylenediaminetetracetic acid EGF Epidermal Growth Factor EGFR Epidermal Growth Factor Receptor ELISA Enzyme-Linked Immunosorbent Assay EMT Epithelial to Mesenchymal Transition EPAC2 Exchange protein directly activated by cAMP2 EPLIN Epithelial Protein Lost In Neoplasm ERK Extracellular Regulated Kinases ERSPC The European Randomised Study for Prostate Cancer E-selectin endothelial selectin ETs E-twenty-six F-actin Filamentous Actin FCS Foetal Bovine Serum FITC Fluorescein isothiocyanate FlnA Filamin-A VIII GAP GTPase activation Proteins GEF Guanine Nucleotide Exchange Factor GEO Gene Expression Omnibus GJs Gap Junctions GK Guanylate kinase GnRH Gonadotropin Releasing Hormone GSK-3β Glycogen Synthase Kinase 3 beta GTP Guanosine Triphosphate HAV Hepatitis A virus HAVcR-1 Hepatitis A virus cellular receptor (HAVcR-1) HGF Hepatocyte Growth Factor HIF-1 Hypoxia Inducible Factor 1 HIFU High Intensity Focused Ultrasound HIV Human immunodeficiency virus HLA Human Leucocyte Antigen HPC1 Hereditary Prostate Cancer 1 HPV Human Papilloma Virus HRE Hormone Response Elements HSP Heat Shock Protein IF Immunofluorescent Ig Immunoglobulin IGF Insulin-like growth factor IHC Immunohistochemical IL Interleukin IX JAMs Junctional Adhesion Molecules JNK c-Jun N-terminal Kinase KIM-1 Kidney injury molecule 1 KLK2 Kallikrein Related Peptidase 2 LB Liquid Broth LBD Ligand Binding Domain LH Luteinizing Hormone LLG1 Lethal Giant Larvae 1 LRP5/6 Low-density Lipoprotein receptor-related protein 5 MAGUK Membrane-Associated Guanylate Kinase MAPK Mitogen-activated Protein Kinase MET Mesenchymal to Epithelial Transition MBD Met Binding Domain MEK1 MAPK/ERK kinase 1 MMP Matrix Metalloproteases MRI Magnetic Resonance Imaging MSR1 Macrophage Scavenger Receptor 1 MT1 Membrane-type 1 MUC1 Mucin 1 N-Cadherin Neural Cadherin NES Nuclear Export Signal NLS Nuclear Localisation Signal N-selectin neuronal selection NTD N-Terminal Transactivation Domain X N-terminus Amino-terminus Pax3 Paired Box 3 P-box Permutation box PBS Phosphate Buffer Saline P-Cadherin Placental Cadherin PCP Paracellular Permeability PCR Polymerase Chain Reaction PEDGF Platelet Derived Growth Factor PKA/C/G Protein Kinase A/C/G PMA Propidium Monozide PMH Phenyl-Methylene Hydantoin PON1 Paraoxonase 1 PP1/2A/2B Protein Phosphatase 1/2A/2B ProtecT Prostate Testing for Cancer and Treatment PSA Prostate Specific Antigen PTB Phosphotyrosine Binding PTP Protein tyrosine phosphatases PVDF Polyvinylidene Fluoride qPCR Quantitative Polymerase Chain Reaction Rac Ras-related C3 botulinum toxin substrate Ras Rat Sarcoma Rb Barrier Resistance R-Cadherin Retinal Cadherin Rho Ras homology XI RNA Ribonucleic Acid RNASEL Ribonuclease L ROCK Rho-associated protein Kinase RPMI Roswell Park Memorial Institute RT Reverse Transcriptase SDS Sodium Dodecyl Sulphate SEM Standard Error of the Mean SF Scatter Factor SFM Serum Free Medium SH3 Src Homology 3 SHBG sex hormone binding globulin sLe sialyl Lewis SPARC Secreted Protein Acidic and Cysteine Rich SPH Serine Proteinase Homology TAU Transcriptional Activation Units TBE Tris-Borate-EDTA Tcf-4 Transcription Factor 4 TER Transepithelial/Transendothelial Resistance TF Transcription Factor TGF-β Transforming Growth Factor β TIM-1 T-cell immunoglobulin and mucin domain containing molecule 1 TJs Tight Junctions TM Transmembrane XII TMPRSS2 Transmembrane protease serine 2 TNM staging Tumour, Lymph Node and Metastasis staging TPBS Tween PBS TPR Translocating promoter region TRITC Tetramethylrhodamine isothiocyante TRUS Trans-rectal Ultrasound TURP Transurethral Resection of the Prostate VEGF Vascular Endothelial Growth Factor WB Western Blotting Analysis WCB Wales Cancer Bank ZO Zonula Occludin ZONAB ZO-1 Associated Nucleic Acid-Binding Protein XIII Summary Introduction: Prostate cancer is a significant burden in the UK, despite continuing research our understanding of disease progression and at present treatment options are still limited. In small studies, Hepatitis A virus cellular receptor (HAVcR-1) has been linked to cancer aetiology and may regulate junctional complexes. Its role in prostate cancer remains unexplored. This study aimed to investigate the expression of HAVcR-1 in prostate cancer samples and explore the cellular and molecular impact of HAVcR-1, with particular focus on junctional complexes, using in vitro models. Methods: Clinical serum samples from prostate cancer patients were tested for HAVcR-1 ectodomain levels through
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