Abstract 4985: The essential role of eEF-2 kinase in determining fate of glioma cells under ER stress

Abstract

Abstract Endoplasmic reticulum (ER), an organelle that plays an essential role in protein folding and post-translational modification, can be stressed a variety of stimuli and pathological conditions. To recover normal ER function, an evolutionarily conserved response, termed unfolded protein response, is triggered. When ER homeostasis fails to be re-established, persistent ER stress can induce apoptosis. Recently, it has been demonstrated that ER stress can also induce autophagy, another mechanism for removing unfolded proteins that cannot be eliminated via the ubiquitin/proteasome system, thereby reducing ER stress. However, the precise mechanisms by which the ER stress-induced autophagy is regulated and the functional association between apoptosis and autophagy in tumor cells under ER stress remains unclear. We report here that eukaryotic elongation factor-2 kinase (eEF-2K) is a critical mediator of the ER stress-induced autophagy. We found in the human glioma cells (T98G and LN229) that eEF-2K and autophagy were simultaneously activated following treatment with the ER stress inducers, thapsigargin and tunicamycin; silencing of eEF-2K expression blunted the autophagic response to ER stress. To explore the pathway linking eEF-2K activation and ER stress, we tested the role of REDD1, a stress-induced protein involved in repression of the mTOR signaling. We demonstrated that under ER stress, silencing of REDD1 blocked the phosphorylation of eEF-2, the only known substrate of eEF-2K, indicating that REDD1 is required for transducing the signal for eEF-2K activation. As phosphorylation of eEF-2K at different sites can either activate or inhibit the activity of the enzyme, we next determined the roles of phosphorylation of the kinase in regulating autophagy. We showed that phosphorylation of eEF-2K at Ser398 was essential for the induction of autophagy, while phosphorylation at Ser 366 and Ser78 exerted an inhibitory effect on the kinase activity and autophagy. We further observed that suppression of the ER stress-activated autophagy via silencing of eEF-2K expression aggravated ER stress and promoted apoptotic cell death. Moreover, inhibiting eEF-2K by either RNAi or NH125, a small molecule inhibitor of the enzyme, rendered the glioma cells 2∼3-fold more sensitive to curcumin and velcade, two cytotoxic agents that trigger anti-tumor activities by inducing ER stress. Our study indicates that REDD1-eEF-2K pathway is essential for inducing autophagy and for determining fate of tumor cells under ER stress, and suggests that inhibiting the eEF-2K-mediated autophagy can enhance ER stress and lead to greater apoptotic response, thereby potentiating the efficacy of anticancer agents. These results underscore the potential of eEF-2K as a novel target for cancer therapy. 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 4985. doi:1538-7445.AM2012-4985