SIRT6 Promotes Autophagy Through Interacting With ULK1 And Competitively Binding To PUMA

Objective: Our previous study identified SIRT6(Silent information regulator 6)was a novel downstream of Akt,and this paper further explored the new function and molecular mechanism of tumor suppressor SIRT6 in colorectal cancer(CRC)cell autophagy and its relationship with apoptosis was studied.Methods:(1)Western blotting detected the expression of p-Akt,SIRT6,PUMA and autophagy-related proteins(p-ULK1,p-Beclin1 and LC3-Ⅱ/Ⅰ)in Midostaurin-treated cells.(2)Immunofluorescence analysis of endogenous/exogenous LC3 aggregation in Midostaurin-treated cells.(3)Co-immunoprecipitation to detect the interaction of endogenous/exogenous SIRT6,PUMA and ULK1 proteins.(4)LC3aggregation and expression of LC3-Ⅱ/Ⅰ was detected by immunofluorescence and western blotting in knockdown/overexpression of SIRT6,or overexpression of PUMA cells;(5)Effects of Midostaurin treatment or SIRT6 knockdown on cell viability and survival,chromatin agglutination,apoptosis was detected by CCK-8,clone formation experiment,Hoechst staining,flow cytometry,western blotting.(6)The effects of the combination treatment of Midostaurin and autophagy inhibitors(CQ and 3-MA)on cell viability and apoptosis were detected by western blotting,EdU staining,CCK-8 and flow cytometry.(7)We constructed a model of xenograft tumor nude mice,and tumor volume and weight were analyzed in mice after the combination of Midostaurin and CQ.(8)The expression of p-Akt,SIRT6,PUMA,ULK1,Beclin1,LC3-Ⅱ/Ⅰ,Ki67,Cleaved-Caspase3 in mice tumors tissue were detected by immunohistochemistry in tumor tissue.(9)The change of interaction between SIRT6,PUMA,and ULK1 was detected by co-immunoprecipitation in tumor tissue before and after treatment.Results:(1)Midostaurin reduced p-Akt expression and upregulated the expression of SIRT6,PUMA and autophagy-related proteins,causing LC3 aggregation.(2)Exogenous GFP-SIRT6 interacted with ULK1.Similarly,exogenous mCherry-ULK1 binds to SIRT6.(3)After Midostaurin treatment,the interaction between SIRT6 with ULK1 increased,while the interaction between PUMA with ULK1 weakened.(3)Intracellular LC3 aggregation increased,and LC3-Ⅱ/Ⅰ expression was upregulated after SIRT6 overexpression.LC3 aggregation decreased and LC3-Ⅱ/Ⅰ expression was downregulated inn SIRT6 knockdown or PUMA overexpression cells.(4)After Midostaurin treatment,cell viability and survival rate were reduced,chromatin agglutination was observed,and apoptosis increased;SIRT6 knockdown inhibited Midostaurin-induced apoptosis.(5)Midostaurin inhibited tumor growth in vivo,the level of p-Akt was downregulated,and the expression of SIRT6,PUMA,ULK1,LC3-Ⅱ/Ⅰ,Cleaved-Caspase3 were upregulated in tumor tissue.(6)The interaction between SIRT6 and ULK1 was enhanced,and the interaction between SIRT6 and PUMA was enhanced,while the interaction between PUMA and ULK1 was weakened in midostaurin-treated mouse tumor tissues.(7)Autophagy inhibitor(3-MA/CQ)and Midostaurin are combined to further reduced cell viability and promote more apoptosis;(8)Midostaurin and CQ synergistically inhibited tumor growth,promoted the activation of caspase3 in tumor tissues,and decreased the expression of Ki67.Conclusions: Midostaurin inhibited the activity of Akt,activated SIRT6-dependent autophagy and apoptosis.SIRT6 promoted autophagy through two pathways:(1)SIRT6 activated autophagy by interacting directly with ULK1,or(2)SIRT6 activated autophagy by releasing PUMA-bound ULK1 via competitively binding to PUMA.Blocking autophagy led to increase in Midostaurin-induced apoptosis.In vivo and in vitro experiments have confirmed that autophagy inhibitor CQ and Midostaurin have a synergistic anti-tumor effect.