Abstract Inhibition of the ubiquitin proteasome (UPS) pathway is a valid anti-cancer target but the alternative strategy to increase activity in order to degrade pro-survival proteins is much less developed. Betulinic acid (BA) is a naturally occurring small molecule with multiple mechanisms for increasing apoptosis in cancer cells, making it an attractive anti-prostate cancer agent. There is evidence that BA can activate the UPS pathway to degrade multiple pro-survival proteins such as androgen receptor (AR), but the mechanism is not clear. Our western blot data in prostate cancer (PC) cells indicates that 10 μM BA decreases the levels of multiple pro-survival proteins including AR, cyclins, and cyclin-dependent kinases. Inhibition of the UPS pathway with 1 μM MG132 blocks the BA-mediated decrease of AR and AKT proteins and lowers cell death, suggesting a dependence on the UPS pathway. In TRAMP transgenic mice, BA treatment (10 mg/kg) inhibits tumor growth, increases apoptosis, decreases angiogenesis and proliferation, and lowers AR and cyclin D1 protein levels (immunohistochemistry) without toxic side effects to normal tissues. However, BA does not increase UPS activity (Proteasome-Glo Chymotrypsin-like Cell-Based Assay; Promega) in LNCaP PC cells suggesting an alternative mechanism for increasing protein degradation. BA-mediated inhibition of deubiquitinase (DUB) activity, as determined by DUB-Glo Protease Assay (Promega) and DUB labeling with HA-ubiquitin vinyl sulfone, results in the accumulation of poly-ubiquitinated (Ub) proteins that are recognized by the UPS pathway and degraded. The cyclin D1 T286A mutant that cannot be degraded by the UPS pathway is resistant to BA-mediated degradation. In non-cancer BJ fibroblast, however, BA does not inhibit DUB activity nor increases total poly-Ub proteins and this is associated with no effect on cell death. Our data suggests that BA-mediated inhibition of DUBs and induction of apoptotic cell death specifically in PC but not in non-cancer cells will provide an effective non-toxic and clinically selective agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4679. doi:1538-7445.AM2012-4679