The mammalian target of rapamycin (mTOR) positively regulates cell growth and survival, primarily through forming a complex either with raptor (mTORC1) or with rictor (mTORC2). While mTORC1 is well known to exert its biological functions primarily via inhibition of cap-dependent protein translation, little is known about the mechanisms by which mTORC2 regulates cell survival and growth. Mcl-1 is an anti-apoptotic Bcl-2 family protein and functions as an oncogenic protein. It is a short-lived protein that undergoes GSK3/FBXW7-dependent ubiquitination and proteasomal degradation. mTORC1 positively regulates Mcl-1 translation while mTORC2 regulation of Mcl-1 stability has not been reported and thus is the focus of this study. We found that inhibition of mTORCs with mTOR kinase inhibitors (mTORKinibs) including INK128, AZD8805, Torin1 and PP242 decreased Mcl-1 levels in cancer cells, particularly those sensitive to mTORKinibs. mTORKinibs did not alter Mcl-1 mRNA levels, but decreased its protein stability. Moreover, mTORKinib-induced Mcl-1 reduction could be rescued by proteasome inhibition (e.g.,with MG132). Hence, it is clear that mTORKinibs enhance Mcl-1 degradation, resulting in Mcl-1 reduction. Suppression of GSK3 using GSK3 chemical inhibitors (e.g.,SB216763 and CHIR99021) or gene silencing approaches prevented Mcl-1 reduction or degradation. This indicated that a GSK3-dependent mechanism is involved in mTORKinib-induced Mcl-1 reduction. Although both the F-box E3 ubiquitin ligases, FBXW7 and β-TrCP, have been suggested to mediate GSK3-dependent degradation of Mcl-1, we observed that FBXW7 knockdown but not β-TrCP attenuated or abolished the ability of mTORKinibs to reduce Mcl-1 levels, demonstrating a critical role of FBXW7 in mediating mTORKinib-induced Mcl-1 degradation. These results demonstrate that mTORKinibs reduces Mcl-1 levels by promoting GSK3-dependent and FBXW7-mediated proteasomal degradation of Mcl-1. Through genetic approaches, we further showed that knockdown of rictor, but not raptor expression reduced Mcl-1 levels and promoted Mcl-1 degradation. In agreement, enforced expression of an ectopic rictor, but not raptor, elevated Mcl-1 levels and enhanced Mcl-1 stability. Furthermore, Mcl-1 reduction induced by rictor knockdown could be rescued by inhibition of GSK3 or FBXW7. Thus, it is apparent that genetic inhibition of rictor, but not raptor, can replicate the effects of mTORKinibs on inducing GSK3-dependent and FBXW7-mediated Mcl-1 degradation. Collectively, we conclude that mTORC2 may positively regulate Mcl-1 stability by suppressing GSK3-dependent and FBXW7-mediated proteasomal degradation of Mcl-1. Our current study highlights a novel biological function of mTORC2 in regulating protein degradation. Further investigation of the detailed mechanism by which mTORC2 stabilizes Mcl-1 protein is warranted.

Citation Format: Junghui Koo, Ping Yue, Fadlo R. Khuri, Shi-Yong Sun. mTOR complex 2 (mTORC2) positively regulates Mcl-1 stability by suppressing GSK3-dependent and FBXW7-mediated proteasomal degradation of Mcl-1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 349. doi:10.1158/1538-7445.AM2014-349