Studying the roles of androgen receptor (AR) coactivators in prostate cancer progression using dominant-negative mutants and RNA interference

Although the progression of prostate cancer initially is dependent on androgens, tumor progression to an androgen-independent growth eventually occurs in most of patients treated with androgen ablation and/or antiandrogen therapy. Antiandrogens given to antagonize androgen receptor (AR) activity gradually lose their efficacy as antagonists and eventually function as agonists to promote AR-mediated growth of prostate cancer cells. The mechanisms of how antiandrogens acquire this agonist activity during hormonal therapy are largely unknown. In this study, we investigated the roles of AR coregulators, ARA70 and ARA55, in this acquired agonist activity of antiandrogens in prostate cancer cells.; Using an in vitro mutagenesis and a yeast two-hybrid screening we identified dominant negative mutants for ARA70 and ARA55, which can inhibit AR transactivation by inactivating the normal function of wild-type coregulators in cancer cells. By chemical mutagenesis we engineered random point mutations in wild-type proteins and generated libraries of ARA70 and ARA55 mutants. We then screened these libraries to isolate a pool of mutants that did not interact with AR. Those noninteracting mutants were further screened to identify any mutant(s), which could also interrupt AR-coregultor interaction. By this double-negative selection, we have identified a pool of potential dominant-negative mutants in yeast and subsequently screened out the two best dominant-negative mutants dARA70 and dARA55, based on their activity in mammalian cells. Expression of dARA55 or dARA70 inhibits AR transcriptional activity followed by the inhibition of prostate-specific antigen (PSA) and cell growth in prostate cancer cells. Most importantly, expression of these mutants effectively reduces the agonist activity of antiandrogens. RNAi-mediated disruption of ARA70 and ARA55 genes resulted in similar results, further strengthening our results. Our results demonstrate that AR-mediated growth of prostate cancer cells can be regulated by modulating the function of AR coregulators, without changing the AR protein itself. These findings not only demonstrate the important roles of the ARA55 and ARA70 coregulators in the agonist activity of antiandrogens, in promoting AR-mediated growth of prostate cancer cells, it may also provide critical targets for developing therapeutic agents for the antiandrogen therapy that almost always fails in the treatment of the hormone-refractory prostate cancer.