Abstract 4053: The role of ubiquitination in androgen receptor function in prostate cancer
Authors: Darby Steven , Ramsey Hollie , Gaughan Luke , Robson Craig 
Published: August 2014

Introduction: Prostate cancer (PC) is the most common male cancer in the western world. The androgen receptor (AR) plays a central role in PC development and remains the primary target for therapy. Androgen deprivation therapy is currently the standard treatment for PC. However, following a 2-3 year period, a more aggressive cancer usually recurs that is unresponsive to further androgen deprivation and is termed castrate resistant prostate cancer (CRPC). Several AR interacting proteins are components of the ubiquitin-proteasome degradation system, including Mdm2, CHIP and E6AP E3 ligases implicating proteasomal degradation/activation as a key event in AR function. In this study we have investigated the involvement of ubiquitin ligases and deubiquitinase (DUB) enzymes in AR ubiquitination and examined whether altered ubiquitination levels play a critical role in AR function and the development of CRPC.

Methods: In vitro ubiquitination assays were performed with AR as the substrate with multiple purified E3 ligase enzymes in conjunction with wild type or mutant ubiquitin (K7R, K48 and K63). Ubiquitinated AR fragments were excised and subjected to proteomic analysis to determine novel sites of ubiquitination within AR. Mutant recombinant AR proteins were subsequently generated that replaced the putative ubiquitination sites and the effect of residue substitution on AR ubiquitination studied. siRNA library screening, Western blotting and immunoprecipitation studies identified DUBs implicated in AR ubiquitination and downstream events.

Results: In vitro ubiquitination assays utilising lysine null ubiquitin demonstrated monoubiquitination of AR in multiple domains of AR in response to four E3 ligases. K48 ubiquitin (implicated in proteasomal degradation) and K63 ubiquitin (implicated in substrate activation) incorporated into in vitro assays identified differential poly-ubiquitination chain patterns in AR, dependent on which E3 ligase was used in the assay. Proteomic analysis of ubiquitinated AR fragments revealed a novel site of ubiquitination within the N-terminal region AR. AR protein stability was greatly enhanced following mutation of the novel ubiquitination site within the AR. To examine deubiquitination of AR, a siRNA screen was performed and target DUBs identified. Over-expression or knockdown of the target DUBs revealed dramatic effects not only on AR ubiquitination levels but also on protein stability and modulation of downstream AR target genes PSA, NKX3.1, KLK2 and TMPRSS2.

Conclusions: In this study mass spectroscopy revealed a novel site of ubiquitination within the AR and a siRNA screen identified DUB enzymes involved in AR deubiquitination. Modulation of ubiquitination levels of AR was observed following substitution of the novel lysine ubiquitination site or through altering DUB enzyme protein expression. Both events led to stabilisation and accumulation of AR protein levels with subsequent altered downstream gene transcription. Additional experiments are underway to examine the interplay for E3 ligases and DUBs that regulate AR ubiquitination to further elucidate the mechanisms controlling AR signalling in prostate cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4053. doi:10.1158/1538-7445.AM2011-4053