NQO1 Overexpression Potentiates Death Evasion In Hepatocellular Carcinoma Via Sirtuin 6/x-linked Inhibitor Of Apoptosis Protein-dependent Apoptotic Pathway

Background and PurposeHepatocellular carcinoma(HCC)is a highly malignant tumor with a poor prognosis.NQO1 is a cytosolic dehydrogenase that has been implicated in a variety of cancers.The role of NQO1 in hepatocellular carcinoma(HCC)remains elusive.In this study,NQO1 was characterized for its role in HCC development and underlying molecular mechanisms.Methods1.The leading genes that was significantly upregulated in HCC tissues and adjacent nontumoral liver tissues were screened by a genome-wide cDNA array and publicly available gene expression data sets.The correlation between NQO1 expression and clinicopathological features in HCC patients was analyzed by utilizing publicly available data sets.2.The expression level of NQO1 in 60 pairs of hepatoma carcinoma(HCC)and adjacent nontumoral liver and HCC cell lines were analyzed by Western blot,qRT-PCR and immunohistochemistry(IHC).The activity of NQO1 in HCC tissues and adjacent nontumoral liver tissues and HCC cell lines was analyzed by NQO1 activity assay.3.HCC cell lines were transfected with lentivirus expression shNQO1-1,shNQO1-2 and shCont.NQO1 was also knockout using CRISPR/Cas-9 in SMMC-7721 cells.The effect of NQO1 knockdown or knockout on cell proliferation was detected by trypan blue exclusion assay,EdU incorporation assay,colony formation assay and soft agar assay.The effect of NQOl knockdown on apoptosis of HCC cells was tested by flow cytometry and Western blot testing for cleaved caspases and cleaved PARP.4.Immortalized human liver cell line MIHA was transfected with lentivirus expressing NQOl.The effects of NQOl over expression on cell proliferation and apoptosis were analyzed using the same experiments as above.5.Potential apoptotic target genes were selected by using tandem mass tags(TMTs)quantitative mass spectrometry between NQOl knockout cell and control cells.The target gene was furthered tested by Western blot and qRT-PCR.The effects of NQOl knockdown on protein stability of target genes was analyzed by Western blot and CO-IP.The effects of target genes on NQOl-depletion mediated cell proliferation inhibition and apoptosis induction were tested by trypan blue exclusion assay,flow cytometry and Western blot.6.The cell mode results were tested by xenograft tumor model in nude mice.Results1.NQO1 is frequently upregulated in HCC tissues and high NQO1 levels significantly correlated with vascular invasion,tumor grade,and a shorter survival.2.NQO1 knockdown or knockout in HCC cell lines inhibited cell proliferation,colony formation and promoted cell apoptosis,whereas overexpression of NQO1 in immortalized hepatocyte promoted cell growth and decreased doxorubicin-induced apoptosis.3.Mechanism study revealed NQO1 depletion promoted XIAP degradation.The specific melocular mechanism is as fellows:NQO1 directly binds to SIRT6 and NQO1 knockdown or knockout promoted SIRT6 proteasome-mediated degradation,decreased SIRT6 protein levels inhibited AKT deacetylation and its phosphorylation thus leading to the inactivation of AKT.Inactivation of AKT suppressed XIAP phosphorylation and downregulated XIAP stability.4.SIRT6 or XIAP overexpression in NQO1-depleted cell rescued cell proliferation inhibition and apoptosis induction.5.NQO1 depletion also suppressed tumor growth in vivo.ConclusionsWe identified NQO1 as an important pro-tumorigenic factor in the development of HCC.These finds suggest that NQO1 could be a promising therapeutic target for HCC.