Abstract 1674: Inhibition of GSK3 reduces p70S6K activity and promotes autophagy independently of the JNK-cJun pathway.

Abstract Considering that a tumor promoting role for GSK3 has been suggested in pancreatic cancer (PC) cells and that GSK3 inhibitors are currently under clinical trials for treatment of different disorders, inhibition of GSK3 activity might represent an attractive therapeutic option for PC patients. However, mechanisms under the control of GSK3 activity in PC cells need to be clarified. We have previously demonstrated that prolonged inhibition of GSK3 activity induces JNK-dependent cell death in human PC cells. Recently, autophagy was proposed to be required for pancreatic tumor growth and inhibition of autophagy was shown to sensitize diverse cancer cells to a wide array of stress conditions. The JNK pathway has now well established roles in both apoptosis and autophagy positioning this pathway at the crossroad of these two processes. Furthermore, inhibition of GSK3 was shown to induce autophagy in prostate cancer and neuroblastoma cells suggesting a link between GSK3 activity and the regulation of autophagy. The AIM of this study was to evaluate whether GSK3 controls autophagy in PC cells and to determine whether inhibition of autophagy sensitizes pancreatic epithelial cells to GSK3 inhibition-induced cell death. METHODS Experiments were performed using the human PC cell lines PANC1 and MIAPaCa2 and the non-tumorigenic HPDE cells. GSK3 activity was inhibited by treatment with GSK3 inhibitors SB216763 and CHIR99021 or by specific shRNA targeting GSK3alpha/beta. Apoptosis was measured by assessment of PARP and caspase-7 cleavages. Autophagy was evaluated by the detection of the membrane-bound LC3B-II isoform and was inhibited by treatment with 3-methyladenine or transfection of a siRNA targeting ATG5. RESULTS 1) Prolonged inhibition of GSK3 (48-72h) induced an apoptotic response in PC cells. 2) Moreover, inhibition of GSK3 in pancreatic epithelial cells triggered an autophagic response and increased the autophagic flux. 3) The inhibition of autophagy sensitized pancreatic epithelial cells to the GSK3 inhibition-induced cell death. 4) The inhibition of GSK3 correlated with activation of the JNK-cJun signaling and reduced levels of phosphorylated p70S6K (Thr389) and S6 (Ser235/236). 5) As opposed to what was observed for the GSK3 inhibition-induced apoptosis, activation of the JNK-cJun pathway was not required for the autophagy response following GSK3 inhibition. Also, downregulation of cJun by shRNA did not prevent the reduced levels of phosphorylated p70S6K following GSK3 inhibition. These results suggest that GSK3 acts on other targets then JNK/cJun to modulate autophagy. CONCLUSION Our results provide evidence that GSK3 inhibition induces autophagy in pancreatic epithelial cells and suggest a protective role of autophagy against GSK3 inhibition-induced cell death. Finally, our observations suggest a potential role for the mTOR/p70S6K signaling in the GSK3-dependent regulation of autophagy. Citation Format: Benoit Marchand, Marie-Josée Boucher. Inhibition of GSK3 reduces p70S6K activity and promotes autophagy independently of the JNK-cJun pathway. [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 1674. doi:10.1158/1538-7445.AM2013-1674