Abstract 4885: mTOR complex 2 is involved in regulation of c-FLIP stability and sensitization of TRAIL-induced apoptosis

The mammalian target of rapamycin (mTOR), a serine-threonine kinase related tightly to the phosphoinositide 3-kinase family, acts as a central molecule in regulation of cell proliferation and survival through forming two complexes primarily with raptor (mTOR complex 1; mTORC1) or rictor (mTOR complex 2; mTORC2). Compared with the mTORC1, relatively little is known about the biological functions of mTORC2. However, it is clear that both of the mTOR signaling pathways are dysregulated in human cancers, including lung cancer. Accordingly, novel ATP-competitive mTOR kinase inhibitors such as PP242 that inhibit both mTORC1 and mTORC2 have been developed for cancer therapy. In this study, we focus on addressing whether mTORC2 regulates apoptosis, particularly induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a potential cancer therapeutic protein tested in clinical trials. We found that PP242 synergized with TRAIL to augment apoptosis in human lung cancer cells. PP242 reduced the level of the short form of c-FLIP (FLIP S ) and survivin with minimal effects on the expression of DR4 and DR5. Enforced expression of FLIP S , but not survivin, attenuated PP242/TRAIL-induced apoptosis; this suggests that FLIP S downregulation contributes to synergistic induction of apoptosis by the PP242 and TRAIL combination. Mechanistic studies revealed that PP242 decreased FLIP S stability, increased FLIP S ubiqutination and facilitated FLIP S degradation, suggesting that PP242 decreases FLIP S levels through promoting its degradation via an ubiqutination and proteasome-dependent mechanism. Interestingly, knockdown of rictor, but not raptor, mimicked PP242 in decreasing FLIP S levels and sensitizing cells to TRAIL. Moreover, rictor knockdown decreased the stability of FLIP S . These data together suggest that it is mTORC2 inhibition that mediates FLIP S downregulation and subsequent sensitization of TRAIL-induced apoptosis. Our findings hence provide the first evidence showing that mTORC2 positively regulates FLIP S expression, thereby, connecting mTORC2 signaling to regulation of the death receptor-mediated apoptosis pathway. (This study was supported by the Georgia Cancer Coalition Distinguished Cancer Scholar award and NIH/NCI R01 CA118450. FR Khuri and SY Sun are Georgia Cancer Coalition Distinguished Cancer Scholars) 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 4885. doi:1538-7445.AM2012-4885