[Truncated abstract] Prostate cancer is a significant cause of morbidity and mortality in the male population, and despite its high prevalence, there have been few advances in prostate cancer management during the past 20 years. The primary treatment for prostate cancer centres around inhibition of androgen signalling, halting tumour growth through down-regulation of important proliferative genes under control of the androgen receptor (AR). Frequently, prostate cancer progresses to an androgen-independent state that is resistant to AR-targeting therapies and is characterised by persistent AR expression and re-expression of ARregulated genes. Understanding the regulation of AR in prostate cancer is therefore of paramount importance for the development of new treatments to control advanced disease. The AR is regulated post-transcriptionally at the level of mRNA stability, and a UC-rich cis-element ("ARUC") within the 3' untranslated region (3'UTR) of the AR mRNA has been identified, which is bound by an mRNA-stabilising protein, HuR. HuR is a member of a family of four related mRNA-binding proteins that includes HuD, which is typically expressed in neuronal tissue. In this thesis, HuD expression was examined immunohistochemically in a cohort of 12 prostate specimens and was detected at low levels in benign prostatic hyperplasia (BPH), with up-regulated expression in prostate cancers, in particular in high-grade tumours. HuD mRNA expression was also confirmed in the LNCaP prostate cancer cell line using reverse-transcription (RT)-PCR. Like HuR, HuD binds U-rich mRNA and RNA electrophoretic mobility shift assays (REMSA's) confirmed HuD binding to the UC-rich ARUC motif in the AR mRNA 3'UTR, an association that could be disrupted by introduction of point mutations into the ARUC motif. ... To determine mRNA targets of HuR which may be important in prostate cancer proliferation or progression, an oligonucleotide micro-array analysis was performed on 22Rv1 cells that had been treated with siRNA directed against HuR. This identified a number of growth-associated signalling pathways in which multiple genes were down-regulated, including the mitogen-activated protein kinase (MAPK), focal adhesion, vascular endothelial growth factor (VEGF) and Wingless type/MMTV integration (Wnt) signalling pathways, as well as ubiquitin-mediated proteolysis. Further qPCR and IP-RT-PCR analysis of a subset of the regulated genes that contained 3'UTR HuR consensus binding sites confirmed mRNA down-regulation following HuR siRNA treatment and in vivo interaction with HuR of the CREBBP, PCAF, ELK1 and PAK2 transcripts. In summary, results presented in this thesis have identified aberrant upregulation of expression of the RNA-binding protein HuD in prostate cancer, which may contribute to the maintenance of AR expression via binding and stabilisation of the AR mRNA. HuR has been identified as an important protein for the control of AR gene expression and prostate cancer cell proliferation. Furthermore, HuR regulates expression of a range of key genes involved in transcription and cellular signalling, implicating it as an important potential target for therapeutics.
|Qualification||Doctor of Philosophy|
|Publication status||Unpublished - 2007|