The molecular chaperone heat shock protein 90 (Hsp90) is an important target for cancer therapy as it is required for the correct maturation and function of its various client proteins, many of which are known oncogenes. In prostate cancer, targeting Hsp90 is particularly attractive as the androgen receptor (AR), the key mediator of prostate cancer cell growth and survival, depends on Hsp90 for its ligand-binding capacity and stability. Despite promising results in pre-clinical studies, to date Hsp90 inhibitors including 17-allylamino-demethoxygeldanamycin (17-AAG) have demonstrated limited efficacy in clinical trials for advanced prostate cancer. While this is in part due to drug formulation issues, we propose that a treatment strategy that simultaneously targets multiple aspects of AR action will more effectively eliminate AR-dependent prostate cancer cells than single agent treatment strategies. In this study, we markedly enhanced the efficacy of 17-AAG by combining lower doses of this agent with either an antiandrogen (bicalutamide) or a histone deacetylase inhibitor (vorinostat), both of which are inhibitors of AR function and/or expression. Interestingly, comparison of gene expression profiles altered by each combination indicated that these treatments did not markedly enhance abrogation of androgen signalling compared with individual agents. Rather, the combination treatments regulated expression of unique gene sets that were enriched for cell cycle, apoptosis, MAPK and insulin signalling. To further enhance the efficacy of a combinatorial approach involving Hsp90 inhibitors, we investigated two new generation Hsp90 inhibitors, NVP-AUY922 and the orally available NVP-HSP990. We found that both agents were significantly more potent with regards to modulation of Hsp90 client proteins, inhibition of cell proliferation and induction of cell death than 17-AAG in prostate cancer cell lines. To assess the combinatorial approach in human disease, we have developed a unique approach to study human prostate cancer where tumours are cultured as explants, with maintenance of tissue integrity, cell proliferation and androgen signalling. Using this strategy, we observed that human prostate tumour tissue responds to the novel synthetic Hsp90 inhibitors, but not 17-AAG, with a marked reduction in proliferation. Collectively, these findings suggest that these novel Hsp90 inhibitors warrant further clinical investigation in prostate cancer. We therefore propose to conduct a pharmacodynamic study to assess the biological effects of AUY922, when administered to patients with localised prostate cancer prior to surgery.Evidence of molecular efficacy for AUY922 in this study will underpin subsequent clinical trials for this agent in prostate cancer.