Inhibition of mTOR Kinase by AZD8055 Can Antagonize Chemotherapy-induced Cell Death through Autophagy Induction and Down-regulation of p62/Sequestosome 1
Inhibition of mTOR Kinase by AZD8055 Can Antagonize Chemotherapy-induced Cell Death through Autophagy Induction and Down-regulation of p62/Sequestosome 1
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DOI：
10.1074/jbc.m111.297432
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发表时间：
2011-11-18
影响因子：
4.8
通讯作者：
Sinicrope, Frank A.
中科院分区：
文献类型：
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作者：
Huang, Shengbing;Yang, Zhineng J.;Sinicrope, Frank A.
AZD8055是一种哺乳动物雷帕霉素靶标(MTOR)的ATP竞争性抑制物，可形成两个多蛋白复合体mTORC1和mTORC2，并负向调节自噬。我们证明，AZD8055刺激和增强化疗介导的自噬，表现为LC3I-II转换和泛素结合蛋白p62/Sequestosome 1的下调。AZD8055诱导的自噬是支持生存的，其能力表现为减轻细胞死亡和DNA损伤(p-H2AX)，并通过细胞毒性化疗提高克隆形成存活。用针对Beclin 1或LC3B的siRNA或氯喹可部分逆转AZD8055的细胞保护作用，这种作用不依赖于细胞周期的抑制。通过给予细胞保护的Beclin 1的异位表达证实了自噬的促生存作用。为了确定自噬介导的p62/隔离小体1的下调是否有助于其促生存作用，我们使用shRNA产生了p62基因敲除细胞，该细胞显示出对化疗诱导的细胞死亡和DNA损伤的保护作用。我们还过表达了促进化疗诱导细胞死亡的野生型(Wt)p62，而功能区(PB1，Uba)的突变p62未能做到这一点。异位wt p62促进细胞死亡的能力被AZD8055阻断。AZD8055在Ser(757)处抑制自噬起始蛋白ULK1的磷酸化，并抑制mTORC1(p-mTor Ser(2448)，p70S6K，p-S6，p4EBP1)和mTORC2(p-mTor Ser(2481)，p-AKT Ser(473))的已知靶点。敲除mTOR，但不是Raptor或Rictor，减少了Ser(757)的p-ULK1，并增强了化疗诱导的自噬，导致了类似于AZD8055的细胞保护作用。综上所述，AZD8055抑制mTOR激酶和ULK1的磷酸化以诱导自噬，其促生存作用部分是由于下调p62的表达。
AZD8055 is an ATP-competitive inhibitor of mammalian target of rapamycin (mTOR) that forms two multiprotein complexes, mTORC1 and mTORC2, and negatively regulates autophagy. We demonstrate that AZD8055 stimulates and potentiates chemotherapy-mediated autophagy, as shown by LC3I-II conversion and down-regulation of the ubiquitin-binding protein p62/sequestosome 1. AZD8055-induced autophagy was pro-survival as shown by its ability to attenuate cell death and DNA damage (p-H2AX), and to enhance clonogenic survival by cytotoxic chemotherapy. Autophagy inhibition by siRNA against Beclin 1 or LC3B, or by chloroquine, partially reversed the cytoprotective effect of AZD8055 that was independent of cell cycle inhibition. The pro-survival role of autophagy was confirmed using ectopic expression of Beclin 1 that conferred cytoprotection. To determine whether autophagy-mediated down-regulation of p62/sequestosome 1 contributes to its pro-survival role, we generated p62 knockdown cells using shRNA that showed protection from chemotherapy-induced cell death and DNA damage. We also overexpressed wild-type (wt) p62 that promoted chemotherapy-induced cell death, whereas mutated p62 at functional domains (PB1, UBA) failed to do so. The ability of ectopic wt p62 to promote cell death was blocked by AZD8055. AZD8055 was shown to inhibit phosphorylation of the autophagy-initiating kinase ULK1 at Ser(757) and inhibited known targets of mTORC1 (p-mTOR Ser(2448), p70S6K, p-S6, p4EBP1) and mTORC2 (p-mTOR Ser(2481), p-AKT Ser(473)). Knockdown of mTOR, but not Raptor or Rictor, reduced p-ULK1 at Ser(757) and enhanced chemotherapy-induced autophagy that resulted in a similar cytoprotective effect as shown for AZD8055. In conclusion, AZD8055 inhibits mTOR kinase and ULK1 phosphorylation to induce autophagy whose pro-survival effect is due, in part, to down-regulation of p62.