Department of Hematology and Medical Oncology, Emory University School of Medicine and Winship Cancer Institute, Atlanta, Georgia 30322, USA.
Cancer Res. 2013 Mar 15;73(6):1946-57. doi: 10.1158/0008-5472.CAN-12-3710. Epub 2013 Jan 14.
The mTOR positively regulates cell proliferation and survival through forming 2 complexes with raptor (mTOR complex 1; mTORC1) or rictor (mTOR complex 2; mTORC2). Compared with the mTORC1, relatively little is known about the biologic functions of mTORC2. This study focuses on addressing whether mTORC2 regulates apoptosis, particularly induced by TRAIL (TNFSF10). Using the mTOR kinase inhibitor, PP242, as a research tool, we found that it synergized with TRAIL to augment apoptosis of cancer cells. PP242 reduced the abundance of the short form of c-FLIP (FLIP(S), CFLAR(S)) and survivin (BIRC5). Enforced expression of ectopic FLIP(S), but not survivin, attenuated augmented apoptosis induced by PP242 plus TRAIL. Thus, it is FLIP(S) downregulation that contributes to synergistic induction of apoptosis by PP242 plus TRAIL. PP242 decreased FLIP(S) stability, increased FLIP(S) ubiquitination, and facilitated FLIP(S) degradation. Moreover, knockdown of the E3 ligase Cbl (CBL) abolished PP242-induced FLIP(S) reduction. Thus, PP242 induces Cbl-dependent degradation of FLIP(S), leading to FLIP(S) downregulation. Consistently, knockdown of rictor or mTOR, but not raptor, mimicked PP242 in decreasing FLIP(S) levels and sensitizing cells to TRAIL. Rictor knockdown decreased FLIP(S) stability, whereas enforced expression of rictor stabilized FLIP(S). Moreover, silencing of Cbl abrogated FLIP(S) reduction induced by rictor knockdown. Collectively we conclude that it is mTORC2 inhibition that results in FLIP(S) downregulation and subsequent sensitization of TRAIL-induced apoptosis. Our findings provide the first evidence showing that mTORC2 stabilizes FLIP(S), hence connecting mTORC2 signaling to the regulation of death receptor-mediated apoptosis.
mTOR 通过与 Raptor（mTOR 复合物 1；mTORC1）或 Rictor（mTOR 复合物 2；mTORC2）形成 2 种复合物，正向调节细胞增殖和存活。与 mTORC1 相比，人们对 mTORC2 的生物学功能知之甚少。本研究旨在探讨 mTORC2 是否调节细胞凋亡，特别是 TRAIL（TNFSF10）诱导的细胞凋亡。我们使用 mTOR 激酶抑制剂 PP242 作为研究工具，发现它与 TRAIL 协同作用增强癌细胞的凋亡。PP242 降低了 c-FLIP（FLIP(S)，CFLAR(S)）和 survivin（BIRC5）的短型的丰度。异位表达外源性的 FLIP(S)，但不是 survivin，可减弱由 PP242 和 TRAIL 协同诱导的凋亡。因此，FLIP(S)下调有助于 PP242 和 TRAIL 协同诱导细胞凋亡。PP242 降低了 FLIP(S)的稳定性，增加了 FLIP(S)的泛素化，并促进了 FLIP(S)的降解。此外，Cbl（CBL）E3 连接酶的敲低消除了 PP242 诱导的 FLIP(S)减少。因此，PP242 诱导 Cbl 依赖性的 FLIP(S)降解，导致 FLIP(S)下调。一致地，敲低 Rictor 或 mTOR，但不是 Raptor，可模拟 PP242 降低 FLIP(S)水平并使细胞对 TRAIL 敏感。Rictor 敲低降低了 FLIP(S)的稳定性，而强制表达 Rictor 稳定了 FLIP(S)。此外，沉默 Cbl 消除了 Rictor 敲低诱导的 FLIP(S)减少。综上所述，我们的结论是 mTORC2 抑制导致 FLIP(S)下调，随后 TRAIL 诱导的细胞凋亡增加。我们的研究结果首次证明了 mTORC2 稳定了 FLIP(S)，从而将 mTORC2 信号与死亡受体介导的细胞凋亡的调节联系起来。
