Prostate cancer is the second leading cause of death from cancer in Australian men. As prostate cancer cells are reliant on androgens for growth and survival, the standard therapy for metastatic disease is androgen ablation therapy (AAT). AAT inhibits androgen signalling by altering androgen synthesis or prevent binding of androgens to their intracellular mediator, the androgen receptor (AR). Although initially effective, virtually all patients relapse, beyond which there are limited treatment options. The failure of AAT is not necessarily due to a decreased requirement for androgen signalling, but rather the AR is able to maintain signalling and tumour growth in an androgen-depleted environment. Therefore novel strategies that directly target the AR may provide a more effective therapeutic approach. We have endeavoured to suppress AR activity in prostate cancer cells by utilising a dominant negative AR. The most effective dominant negative construct developed, ARi41O, lacks amino acids 39-410 in the AR amino terminal transactivation domain. In studies of transcriptional activity, ARi410 has no intrinsic activity but inhibits the activity of wild type AR (wtAR) and also clinically relevant AR variants, by up to 95%. The objective of this thesis was to characterise the mechanisms of action of ARi410 and assess the functional effects of introducing this dominant negative receptor into prostate cancer cells. To investigate the mechanism by which ARi410 suppresses AR activity, a robust and sensitive AR inhibition assay was developed. This assay revealed that ARi410 is a potent inhibitor of AR activity on three independent AR-regulated promoters, regardless of the level of AR expression. Furthermore, while ARi410 can inhibit AR activity, it does not alter AR protein levels. By using ARi410 variants with mutations and/or deletions in regions of functional importance, the AR inhibition assay was also used to identify the critical regions of ARi410 required for its dominant negative activity. These studies demonstrate that the dominant negative activity of ARi41 0 is ligand-dependent, requires dimerisation through the ligand binding domain (LBD) and an intact DNA-binding domain (DBD). Further investigation into the mechanism of dominant negative activity revealed that ARi410 does not alter the subcellular localisation of AR, as both receptors are predominantly cytoplasmic in the absence of ligand and rapidly co-localise to the nucleus in response to androgens. Furthermore, an interaction between AR and ARi410 was observed in the presence and absence of ligand, and electrophoretic mobility shift assays demonstrated that AR and ARi410 form heterodimers on DNA. These studies led to the conclusion that the mechanism of dominant negative activity by ARi4I0 involves the formation of inactive receptor heterodimers that assemble on DNA and suppress AR activity. To determine the functional consequence of expressing the dominant negative androgen receptor in prostate cancer cells, an adenoviral method of gene delivery was developed. Adenoviral expression of ARi410 in LNCaP prostate cancer cells did not allow assessment of cell viability due to cell-specific toxicity of the viral vectors when expressed long-term. However, short-term expression of ARi410 in LNCaP cells resulted in inhibition of AR signalling, as determined by reduced expression of the androgen regulated genes apolipoprotein D and kallikrein 2. Importantly, this finding is consistent with the inhibitory activity of ARi410 observed using synthetic AR-regulated reporter genes in the AR inhibition assay, and demonstrates that ARi410 can effectively suppress endogenous AR signalling. The results of this thesis indicate that heterodimerisation between AR and ARi410 is the most likely mechanism of dominant negative inhibition of AR function by ARi410, and that the DBD and dimerisation through the LBD are required for optimal dominant negative activity. Furthermore, this thesis has demonstrated that ARi410 is an effective inhibitor of AR signalling and provides a basis for further functional studies and evaluation of the dominant negative androgen receptor in vitro and in vivo. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1338478 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008
| Identifer | oai:union.ndltd.org:ADTP/280786 |
| Date | January 2008 |
| Creators | Centenera, Margaret Mary |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
Page generated in 0.0023 seconds