Sirt1 Deacetylates P62 To Prevent Its Ubiquitination-dependent Proteasomal Degradation To Promote Hepatocellular Carcinogenesis

Background:Hepatocellular carcinoma(HCC)is one of the most common malignances.HCC currently ranks sixth in incidence and third in mortality worldwide.As one of the most fatal cancers,HCC has the second highest mortality rate in China with an overall five-year survival rate of less than 20%,which seriously threatens human life and health.Because the molecular mechanism of HCC occurrence and development has not been fully elucidated,the therapeutic methods and efficacy for management HCC are limited.The adaptor protein p62/SQSTM1 is highly expressed in various tomors such as HCC and has a significant positive correlation with poor clinical prognosis.However,the molecular mechanism of abnormally high expression of p62 in HCC has not been fully understand.Post-translational modifications such as acetylation,play crucial roles in protein expression and function.Whether and how p62 is acetylated in tumorigenesis and development remains largely unclear.Objective:In this study,we intend to clarify the molecular mechanism of the acetylation modification of p62 and its role in hepatocellular carcinogenesis.Method:The MTS assay and xenograft mouse model were performed to detect the effect of p62 knocking down in HCC.Liquid Chromatograph Mass Spectrometry(LS-MS)was employed to screen the acetylation sites of p62,and immunoprecipitation(IP)was used to verify the acetylation of p62.Co-immunoprecipitation(Co-IP)and pull down were adopted to clarify the Sirt1 interacts with p62 to deacetylate the acetytransferase GCN5 mediated acetylation of p62.Western Blot(WB)and Quantitative Real-time PCR(q RT-PCR)were used to detect the regulation of Sirt1 on p62 expression in HCC cells.Protein half-life assays and in vivo ubiquitylation assays proved that Sirt1 inhibited Keap1-mediated ubiquitination-dependent degradation of p62.Finally,MTS and diethylnitrosamine(DEN)induced primary liver cancer mice model were used to detect the role of Sirt1 on the regulation of p62 acetylation and protein expression in the hepatocellular carcinogenesis.Results:Herein,we confirmed that p62 was upregulated in hepatocellular carcinoma and its higher expression was associated with shorter overall survival in patients.The knockdown of p62 in hepatocellular carcinoma cells decreased cell growth in vitro and in vivo.Intriguingly,p62 protein stability could be reduced by its acetylation at lysine295,which was regulated by deacetylase Sirt1 and acetyltransferase GCN5.Acetylated p62 increased its association with the E3 ligase Keap1,which facilitated its poly-ubiquitination dependent proteasomal degradation.Moreover,Sirt1 was upregulated to deacetylate and stabilize p62 in hepatocellular carcinoma.Additionally,Hepatocyte Sirt1 conditional knockout mice developed much fewer liver tumors after Diethynitrosamine treatment,which could be reversed by the re-introduction of exogenous-p62.Taken together,Sirt1 deacetylates p62 at lysine 295 to disturb Keap1 mediated p62 poly-ubiquitination,thus upregulating p62 expression to promote hepato-carcinogenesis.Conclusion:Sirt1 interacted with p62 to decrease its acetylation at K295 and disturbed Keap1 mediated p62 poly-ubiquitination,thus up-regulating p62 expression to promote hepato-carcinogenesis.Therefore,targeting Sirt1 or p62 is a reasonable strategy for the treatment of HCC.