Abstract 1665: Autophagy is a survival mechanism in mediating resistance to androgen receptor signaling inhibitors in castrate resistant prostate cancer cells.
Authors: Nguyen Hao G. , Yang Joy C. , Kung Hsing-Jien , Gao Allen C. , Evans Christopher P. 
Published: August 2014

Introduction and Objective: Macro-autophagy is associated with drug resistance in various cancers and can function as an adaptive response to maintain cell survival under metabolic stresses, including androgen deprivation. Our hypothesis was: 1) autophagy is a cellular mechanism that confers resistance to the androgen receptor signaling inhibitor (ARSI), enzalutamide (MDV-3100) therapy and 2) blocking autophagy circumvents this survival mechanism to improve therapeutic response.

Materials and methods: To determine if autophagy was activated, we examined expression levels of LC3-I/II using Western Blotting in prostate cancer (CaP) cell lines, LNCaP, C4-2B, and CWR22 stably over-expressing LC3-GFP. Flow cytometry and fluorescence microscopy were used to quantify and visualize autophagy and to analyze cell cycle progression and apoptosis. Clonogenic assays were employed to evaluate cell survival. Enzalutamide (ENZA) and biclutamide were used as androgen receptor inhibitors. SiRNA to AMPK was transfected with Lipofectamine 2000.

Results: Androgen deprivation or treatment with androgen receptor signaling inhibitors, ENZA or biclutamide induced autophagy in androgen-dependent and in castration resistant CaP (CRPC) cell lines. The autophagic cascade triggered by AR blockage, correlated with the increased LC3-II/I ratio and ATG-5 expression. Autophagy was observed in a subpopulation of C4-2B cells that developed insensitivity to ENZA after sustained exposure in culture. Using flow cytometry and clonogenic assays we showed that inhibiting autophagy with clomipramine or hydroxychloroquine increased apoptosis and significantly impaired cell viability. This autophagic process was mediated by AMPK activation and the suppression of mTOR through Raptor phosphorylation (Serine 792). Finally, si-RNA targeting AMPK significantly inhibited autophagy and promoted cell-death in CaP cells acutely or chronically exposed to ENZA or androgen deprived culturing condition, suggesting that autophagy is an important survival mechanism in CRPC cells.

Conclusion: These novel data support autophagy as an important mechanism of resistance to the androgen receptor signaling inhibitor in CRPC. Antiandrogen mediated autophagy is dependent on the activation of AMPK pathway and the suppression of mTOR pathway. Blocking autophagy pharmacologically or genetically significantly impairs prostate cancer cell survival, implying the therapeutics potential of autophagy inhibitors in the antiandrogen resistance setting.

Citation Format: Hao G. Nguyen, Joy C. Yang, Hsing-Jien Kung, Allen C. Gao, Christopher P. Evans. Autophagy is a survival mechanism in mediating resistance to androgen receptor signaling inhibitors in castrate resistant prostate cancer cells. [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 1665. doi:10.1158/1538-7445.AM2013-1665