THE ROLE OF GWAS IDENTIFIED 5P15 LOCUS IN PROSTATE CANCER RISK AND PROGRESSION Panchadsaram Janaththani BSc in Molecular Biology and Biotechnology Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Institute of Health and Biomedical Innovation School of Biomedical Sciences Faculty of Health Queensland University of Technology 2019 Keywords 5p15 locus, Iroquois Homeobox 4, IRX4, IRX4lncRNA, Genome-Wide Association Studies, Multiple Nucleotide Length Polymorphism, Single nucleotide polymorphism, Prostate cancer, Long non-coding RNA, Androgens, ERG. The role of gwas identified 5p15 locus in prostate cancer risk and progression i Abstract Prostate cancer is the second most common cause of cancer death in Australian men. Androgens and the androgen receptor (AR) play a critical role in prostate cancer pathogenesis. Genome-wide association studies (GWAS) have led to the identification of 150 loci associated with prostate cancer risk. Through these GWAS, the rs12653946 SNP at the 5p15 locus was found to be significantly associated with prostate cancer risk in multi ethnic populations. The rs12653946 SNP genotype had also been correlated with the expression levels of its downstream gene Iroquois Homeobox 4 (IRX4) and its antisense long non-coding RNA, IRX4lncRNA. Interestingly, expression of IRX4 and IRX4lncRNA was found to be increased following castration in the LNCaP tumour progression mice model. Therefore, we hypothesised that the prostate cancer risk associated 5p15 locus confers its risk via IRX4 and IRX4lncRNA. The aim of this study was to determine the functional role of IRX4 and IRX4lncRNA in prostate cancer progression and to characterise their androgen-mediated expression regulation in prostate cancer cells. The studies in this thesis have shown that IRX4 and IRX4lncRNA were overexpressed in prostate tumour samples compared to adjacent non-malignant tissues. The expression of IRX4 was correlated with the ERG-fusion status, a prostate cancer specific fusion, while IRX4lncRNA expression was correlated with aggressive disease. The transient knockdown of IRX4 in LNCaP prostate cancer cells reduced cell proliferation and migration. Gene microarray analysis of IRX4 knockdown LNCaP samples revealed that the AR pathway was inhibited, suggesting IRX4 may mediate effective androgen signalling. This was further strengthened by an IRX4 immunoprecipitation assay, in which IRX4 was found to be interacting with the AR co-factors including, FOXA1. Similarly, knockdown of IRX4lncRNA reduced proliferation and migration of LNCaP cells, while overexpression of IRX4lncRNA increased PC3 cell proliferation, suggesting a tumour promoting role for this lncRNA. Both IRX4 and IRX4lncRNA expression were up-regulated with androgen (DHT) treatment in VCaP and DuCaP cells, but down-regulated in LNCaP cells. Interestingly, in-silico analysis identified binding of AR and ERG, upstream of IRX4 in VCaP and DuCaP cells, but not in LNCaP cells. Sequencing of this region identified a Multiple- ii The role of gwas identified 5p15 locus in prostate cancer risk and progression Nucleotide Length Polymorphism (MNLP-rs386684493), where a stretch of 47bp sequence was replaced by a novel 21bp sequence. VCaP and DuCaP cells have an intact AR binding site whereas LNCaP cells have a disrupted AR binding site, suggesting this MNLP may be the functional variant guiding AR binding at this locus. The androgen responsiveness of MNLP was further confirmed by a reporter vector assay. Large-scale genetic association studies of this MNLP in ~80,000 men through the PRACTICAL Consortium identified the MNLP as the most significant prostate cancer risk associated variant at this locus. Furthermore, the 21bp/21bp genotype was correlated with lower expression levels of IRX4 and IRX4lncRNA and poor survival outcome in those patients who underwent androgen deprivation therapy. IRX4 expression was also correlated with ERG-fusion status in prostate cancer datasets, which was also confirmed in our prostate cancer tissue samples. ERG knockdown in VCaP and DuCaP cells increased the androgen-mediated up-regulation of IRX4 expression, while ERG overexpression in LNCaP cells reduced IRX4 expression independent of androgen treatment. In summary, our study identified IRX4 as a potential mediator of effective androgen signalling and targeting IRX4 in patients stratified for ERG-fusion status and MNLP genotype may be a novel therapeutic strategy. In addition, this study discovered the functional role of IRX4lncRNA and identifying molecular mechanisms of this lncRNA in prostate cancer pathology may provide insights for the development of biologically meaningful targets for new therapeutics. The role of gwas identified 5p15 locus in prostate cancer risk and progression iii Table of Contents Keywords .................................................................................................................................. i Abstract .................................................................................................................................... ii List of Figures ........................................................................................................................ vii List of Tables ............................................................................................................................ x List of Abbreviations ............................................................................................................... xi Statement of Original Authorship ......................................................................................... xiv Awards and Publications ........................................................................................................ xv Acknowledgements .............................................................................................................. xvii Chapter 1: Literature Review ................................................................................... 1 1.1. Prostate Cancer .................................................................................................................. 1 1.1.1. Prostate cancer staging and grading ..................................................................... 1 1.1.2. Diagnosis ............................................................................................................. 2 1.1.3. Treatment ............................................................................................................. 2 1.2. The role of the androgen receptor in the progression of prostate cancer........................... 3 1.3. The role of TMPRSS2-ERG fusion in prostate cancer ..................................................... 4 1.4. Prostate cancer risk factors ................................................................................................ 6 1.5. Genome-Wide Association Studies (GWAS) ................................................................... 6 1.6. The rs12653946 SNP at the 5p15 locus is associated with prostate cancer risk ............... 7 1.7. Iroquois Homeobox 4 (IRX4) .......................................................................................... 11 1.8. Antisense long non-coding RNA, IRX4lncRNA, at 5p15 locus ....................................... 16 1.9. Long non-coding RNAs (lncRNAs) ................................................................................ 17 1.10. Summary and knowledge gap ....................................................................................... 33 1.11. Hypothesis and Aims..................................................................................................... 34 Chapter 2: Materials and Methods ......................................................................... 35 2.1. Cell culture ...................................................................................................................... 35 2.2. Genomic DNA samples for genetic association studies .................................................. 35 2.3. RNA isolation from FFPE prostate tumour tissues and matched controls ...................... 36 2.4. RNA isolation from cell lines .......................................................................................... 36 2.5. cDNA synthesis ............................................................................................................... 36 2.6. Reverse transcription polymerase chain reaction (RT-PCR) .......................................... 37 2.7. Quantitative RT-PCR (qRT-PCR) ................................................................................... 37 2.8. Western blot .................................................................................................................... 38 2.9. siRNA mediated knockdown ........................................................................................... 38 2.10. Proliferation assay ......................................................................................................... 39 2.11. Migration assay ............................................................................................................. 39 2.12. Statistical analysis ......................................................................................................... 40 iv The role of gwas identified 5p15 locus in prostate cancer risk and progression Chapter 3: The role of Iroquois-Homeobox 4 (IRX4) at the GWAS identified 5p15 locus in prostate cancer .................................................................................