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Gene Expression Profiles and Biomarker Identification for KMT5A Identifies Novel Potential Therapeutic Targets in Prostate Cancer

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Gene Expression Profiles and Biomarker Identification for KMT5A Identifies Novel Potential Therapeutic Targets in Prostate Cancer Gene expression profiles and biomarker identification for KMT5A identifies novel potential therapeutic targets in prostate cancer A Thesis submitted in part requirement for the degree of Doctor of Philosophy Zainab Adnan Hatem Alebady Solid Tumour Target Discovery Group Northern Institute of Cancer Research Newcastle University October 2016 Abstract Prostate cancer (PC), is initially androgen dependent due to the androgenic nature of the organ. Hence, initial therapy comprises androgen depletion via chemical castration in conjunction with an anti-androgen therapeutic. However, patients relapse and the tumours aggressively re-grow in a castrate resistant (CRPC) manner. In CRPC, androgen receptor (AR) signaling remains functional via numerous mechanisms hence the AR remains a viable therapeutic target. However, treatment with current AR targeting therapeutics also results in relapse indicating the potential of targeting AR signaling indirectly by targeting AR co-factors. Recently, KMT5A, a lysine methyltransferase, has been identified as an AR co-activator exclusively in models of CRPC. A number of KMT5A inhibitors have been identified recent years, which would enhance the possibility of targeting KMT5A in PC. This thesis aims to determine the signature of genes that are regulated directly by KMT5A or by combined activities of AR and KMT5A in PC cell lines and to further identify biomarkers for KMT5A activity. These aims were approached using Illumina Human HT-12 arrays to detect KMT5A gene expression profiles in an in vitro cell line model of androgen independent PC (LNCaP-AI cells). Microarray data analysis revealed a number of androgen- regulated genes to be modulated by KMT5A concurrently, and other genes that were found to be regulated by KMT5A activity, and a further cohort of genes that were found to be regulated solely by KMT5A. CDC20 was selected for further study from the identified KMT5A regulated genes as a possible biomarker for KMT5A activity in aggressive PC. KMT5A was found to regulate CDC20 mRNA and protein expression. The enzymatic activity of KMT5A was demonstrated to affect CDC20 expression through the enrichment of the H4K20me1 mark at the CDC20 promoter in androgen-sensitive (LNCaP) and androgen-independent (LNCaP-AI) cells. The regulation of CDC20 by KMT5A expression, therefore identifies CDC20 as putative biomarker for KMT5A activity. KMT5A was also shown to influence CDC20 expression via p53. Knockdown of KMT5A inhibited the mono-methylation of p53 at K382 to enhance p53 activity, demonstrated by increased p21 expression which negatively regulated CDC20 i expression. These findings were confirmed using commercially available KMT5A inhibitors Ryuvidine and UNC0379. In summary, KMT5A inhibition in PC cells using small molecule inhibitors may provide benefit to patients that have relapsed on AR- targeting therapeutics and as such requires further investigation as a potential therapeutic target. CDC20 was identified as a putative biomarker for KMT5A activity which may prove useful to detect effective KMT5A inhibition in these studies. ii Acknowledgements First of all, I would like to thank my supervisors, Prof. Craig N. Robson and Dr. Kelly Coffey, for their continuous help, guidance and support over the last three years. I am very grateful for the opportunity they have given me to become one of their laboratory members. My thanks also extend to all members of the Solid Tumour Discovery Laboratory, both present and past members for their assistance, advice and support, especially Dr. Scott Walker, Dr. Luke Gaughan, Mahsa Azizyan, Claudia –Ryan Mendel, Laura Wilson, Dr. Kasturi Rao, Dr. Stuart Williamson, Dr. Anastasia Hepburn, Dr. Mark Wade and James Grey. I would also like to give special thanks to my country Iraq and my sponsor The Higher Committee for Education Development in Iraq, for awarding me this scholarship to study for my PhD in the UK. My deep gratitude for my parents Mr. Adnan Hatem Alebady and Mrs. Nawal Abdulraheem Fayyad for their unlimited love, help, support and prayers, you have always been the light at the end of the tunnel for me, and your love is my biggest achievement ever. My love and thanks to my sisters and brothers for being in my life and for their unconditional love. At the beginning and the end my greatest appreciations for my lovely husband Mr. Massar Alsamraae and my exquisite son Sohaib for their patience, love and support throughout my PhD journey, without you it would not have been possible. Finally, thanks to all members of staff at the Northern Institute for Cancer Research for creating such an amazing welcoming atmosphere within the institute and for their continuous help. ii iii iv List of abbreviations AdoMet S-adenosylmethionine ADT Androgen deprivation therapy AF Activation function APC Anaphase promoting complex APS Ammonium persulphate AR Androgen receptor ARE Androgen response element ATP Adenosine-5'-triphosphate Aurora A Aurora Kinase A BM Basal medium BPH Benign prostate hyperplasia BSA Bovine serum albumin PC Prostate cancer CAFs Cancer-associated fibroblast CBP CREB binding protein Cdk Cyclin dependent kinase cDNA Complementary DNA ChIP Chromatin immunoprecipitation CRPC Castration resistant prostate cancer v CTD C terminal domain DAB 3,3'-Diaminobenzidine DBD DNA binding domain DCC Dextran coated charcoal DDR DNA damage repair DEPC Diethylpyrocarbonate DHT 5 α-dihydrotestosterone DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid dNTP Deoxyribonucleoside triphosphate DSBs Double strand breaks EDTA Ethylenediaminetetraacetic acid EGF Epidermal growth factor EMT Epithelial-mesenchymal transition FCS Foetal calf serum FM Full medium FOXM1 Forkhead box protein M1 GADD Growth arrest and DNA-damage-inducible G-phase Gap phase GR Glucocorticoid receptor H Histone HAT Histone acetyltransferase vi HBD Histone binding domain HDAC Histone deacetylase HGPIN High grade prostatic intraepithelial neoplasia HMT Histone methyl transferase HPRT-1 Hypoxanthine phosphoribosyl transferase 1 HSPs Heat shock proteins HTS High throughput screening IGF Insulin-like growth factor IHC Immunohistochemistry IP Immunoprecipitation ITC Isothermal titration calorimetry JMJD Jumonji domain containing K Lysine kDa Kilo Dalton KGF Keratinocyte growth factor KLK2 Kallikrein-related peptidase 2 KMT Lysine methyltransferase L3MBTL1 Lethal 3 malignant brain tumour 1 LBD Ligand binding domain LEF-1 Lymphoid enhancer-binding factor 1 LH-RH Luteinizing hormone –releasing hormone MAPK Mitogen activated protein kinase vii Me Methyl group miRNA microRNA MMLV Murine moloney leukaemia virus M-phase Mitosis phase mRNA Messenger RNA N NH2 NHEJ Non- homologues end joining NR3C4 Nuclear receptor subfamily 3, group C, member 4 N/S Non silencing NTD N terminal domain Oligo Oligonucleotide p53 Protein 53 PBS Phosphate buffered saline PCNA Proliferating cell nuclear antigen PGS Protein G sepharose PHD Plant homeo domain PI Propidium iodide PIN Prostatic intraepithelial neoplasia PIP PCNA interacting peptide PLK1 Polo like kinase-1 PMSF Phenyl methane sulfonyl fluoride PSA Prostate specific antigen viii QRT-PCR Quantitative real time polymerase chain reaction RNA Ribonucleic acid RTK Receptor tyrosine kinase S Serine SDM Steroid depleted medium SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis SETD8 SET domain containing (lysine methyltransferase) 8 shRNA Short hairpin RNA siRNA Small interfering RNA Skp-2 S phase associated kinase protein 2 SPA Scintillation proximity imaging assay S-phase Synthesis phase SPR Surface plasmon resonance SRC Steroid receptor coactivator TBS Tris buffered saline TCF-4 Transcription factor-4 TEMED Tetramethyl ethylene diamine TMPRSS2 Transmembrane protease serine 2 TNM Tumour, Node and Metastasis TTBS TBS Tween20 Ub Ubiquitination VEGF Vascular endothelial growth factor ix Wnt Wingless WT Wild type x Table of contents Abstract .................................................................................................................................................. i Acknowledgements ............................................................................................................................... ii Chapter 1 . Introduction ...................................................................................................................... 1 1.1. The prostate ..................................................................................................................... 2 1.1.1. Function of the prostate ............................................................................................ 2 1.1.2. Zonal anatomy of the prostate .................................................................................. 2 1.1.3. Histology of the prostate .......................................................................................... 4 1.2. Androgen receptor ........................................................................................................... 6 1.2.1. Structure of androgen receptor ................................................................................. 6 1.2.2. Mechanism of action ................................................................................................ 8 ............................................................................................................................................ 9 1.3. Prostate Diseases ........................................................................................................... 10 1.3.1. Benign prostatic hyperplasia (BPH) ......................................................................
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