R. B. Batchu1,2, O. V. Gruzdyn1,2, A. M. Qazi1,2, J. Kaur1,2, P. Konka1,2, D. W. Weaver1, S. A. Gruber1,2 1Wayne State University,Surgery,Detroit, MI, USA 2John D. Dingell VA Medical Center,Detroit, MI, USA
Introduction: Although combination chemotherapy often achieves an initial clinical response, tumor relapse with chemoresistance still remains a major problem in epithelial ovarian cancer (EOC). EZH2, a tumor-promoting methyltransferase, is abnormally elevated in EOC, and mTOR is known to be responsible for chemoresistance. PRAS40 is a regulatory protein which, in its active, un-phosphorylated form, binds to and inhibits the mTOR enzymatic complex (mTORC1). We have previously demonstrated a direct correlation between EZH2 over-expression and chemoresistance in EOC. Here we further elucidate the mechanisms involved in this process by evaluating whether knockdown of EZH2 by RNA interference inhibits mTOR signaling via PRAS40, hypothesizing that this will increase the chemosensitivity of EOC cells.
Methods: Standard protocols were followed for propagation of the SKOV-3 EOC cell line, as well as for Western blot, co-immunoprecipitation, and gene-profile analysis.
Results: Either 1 mm paclitaxel exposure or EZH2-shRNA transfection resulted in a 60% decrease by day 3 (both p<0.02) and 80% decrease by day 5 (both p<0.01) in the number of live EOC cells vs. control, while combined treatment resulted in further significant decreases when compared with either treatment alone on both days (all p<0.02). EZH2 knockdown did not change the levels of mTOR, phosphorylated mTOR (p-mTOR), or PRAS40 by both Western blot and gene- profile analysis (Fig. 1). However, phosphorylation of PRAS40 at its threonine 246 residue was significantly reduced, thereby facilitating interaction with, and inhibition of, mTORC1. This interaction was further confirmed by co-immunoprecipitation experiments demonstrating low levels of unbound, phosphorylated PRAS40.
Conclusion: Our results demonstrate that knockdown of EZH2 enhances the chemosensitivity of paclitaxel, and enhanced interaction of mTOR and PRAS40 results in inhibition of the former, contributing to tumor cell apoptosis. This work provides further insight into the anti-tumor effects of EZH2 knockdown, a potential therapeutic intervention for EOC.
