Abstract 1322: Clinical candidate galeterone (VN/124-1 or TOK-001) induces the degradation of full-length and splice variant androgen receptors in human prostate cancer cell lines via PI3K-Akt-Mdm2 pathway: implications for prostate cancer therapy.
Authors: kwegyir-Afful Andrew K. , Ramalingam Senthilmurugan , Purushottamachar Puranik , Njar Vincent C. O. 
Published: August 2014

Galeterone (VN/124-1; TOK-001; 3β-hydroxy-17-(1H-benzimidazol-1-yl)androsta-5,16-diene) is a proprietary oral small molecule under clinical development for the treatment of castration resistant prostate cancer (CRPC). Galeterone (hereafter referred to as gal) disrupts androgen receptor (AR) signaling through a unique triple mechanism of action, including inhibition of CYP17 (androgen synthesis inhibition), AR antagonism and degradation of AR. Although we have previously unraveled the mechanisms of CYP17 inhibition and AR antagonism, the mechanism of AR degradation is still being elucidated. Here, we report for the first time the mechanism of gal -induced AR degradation, interestingly, gal also depletes splice variant AR in human prostate cancer cells. We evaluated the effects of gal on full-length (wild-type and mutant) and truncated (splice variant) AR in LNCaP, LAPC4 and CWR22Rv1 cells. Real-time PCR analysis of AR mRNA levels led to the hypothesis that gal's effect on AR occurs through a post-translational mechanism(s). The 26S proteasome inhibitor (MG132) blocked the effect of gal on AR depletion, thus suggesting that gal induces AR degradation via the ubiquitin-proteasome pathway. In addition, gal caused increased phosphorylation of Akt and Mdm2, and pretreatment with the PI3K inhibitor, wortmannin, robustly inhibited gal-dependent AR depletion. Targeted knockdown of Mdm2 with siRNA also inhibited the depletion of AR. Antibody pull-down data also indicated that gal treatment enhanced the formation of complex between Akt, Mdm2 and AR which promote phosphorylation-dependent AR ubiquitination and its degradation by proteasome. The possible involvement of the proteasome-independent pathway (caspase-dependent) was eliminated because AR protein was reduced by gal to similar extents in the presence or absence of the pan caspase inhibitor, z-vad-fmk. Thus, in prostate cancer cells, our data support a model where gal induces phosphorylation of AR and Mdm2 through the PI3K/Akt pathway. AR then undergoes ubiquitination by Mdm2 E3 ligase followed by AR degradation by proteasome. Using Western blot analyses and MTT cell viability assays, gal exhibited strong correlation between its ability to deplete AR and reduce cell viability. We also show for the first time that gal and some new analogs can degrade both full-length and truncated AR3 in CWR22Rv1. In conclusion, because gal is currently being evaluated for clinical use against CRPC, its mechanism of AR degradation is of considerable clinical relevance. These discoveries of gal should generate excitement because it appears to be the only agent currently in clinical trial targeting CRPC progression driven by both full-length and splice variants AR.

Citation Format: Andrew K. kwegyir-Afful, Senthilmurugan Ramalingam, Puranik Purushottamachar, Vincent C. O. Njar. Clinical candidate galeterone (VN/124-1 or TOK-001) induces the degradation of full-length and splice variant androgen receptors in human prostate cancer cell lines via PI3K-Akt-Mdm2 pathway: implications for prostate cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1322. doi:10.1158/1538-7445.AM2013-1322