title: Inhibition of mTOR kinase by AZD8055 can antagonize chemotherapy-induced cell death through autophagy induction and down-regulation of p62/sequestosome 1.
abstract: 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.
authors.affiliation: Mayo Clinic and Mayo Cancer Center, Rochester, Minnesota 55905, USA.
authors.forename: Shengbing
authors.initials: S
authors.lastname: Huang
authors.forename: Zhineng J
authors.initials: ZJ
authors.lastname: Yang
authors.forename: Chunrong
authors.initials: C
authors.lastname: Yu
authors.forename: Frank A
authors.initials: FA
authors.lastname: Sinicrope
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D001343:Autophagy / Q000187:drug effects* / Q000235:genetics; D000071189:Autophagy-Related Protein-1 Homolog; D045744:Cell Line, Tumor; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics; D015536:Down-Regulation / Q000187:drug effects* / Q000235:genetics; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D000076222:Mechanistic Target of Rapamycin Complex 1; D009025:Morpholines / Q000494:pharmacology*; D046912:Multiprotein Complexes; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D000071456:Sequestosome-1 Protein; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C493245:CRTC2 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D009025:Morpholines; D046912:Multiprotein Complexes; D011506:Proteins; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; D014157:Transcription Factors; C546624:(5-(2,4-bis((3S)-3-methylmorpholin-4-yl)pyrido(2,3-d)pyrimidin-7-yl)-2-methoxyphenyl)methanol; C546842:MTOR protein, human; D000071189:Autophagy-Related Protein-1 Homolog; D000076222:Mechanistic Target of Rapamycin Complex 1; D017346:Protein Serine-Threonine Kinases; D058570:TOR Serine-Threonine Kinases; C484754:ULK1 protein, human