The Role And Molecular Mechanisms Of SIRT1Maintains Self-renewal In Human Liver Cancer Stem Cells

Hepatocellular carcinoma (HCC) is the fifth most prevalent tumor and the thirdlethalcause of cancer-related deaths worldwide. Although surgery and radiofrequency ablation(RFA) are the best option for small tumors at early stage of HCC patients, the tumorrecurrence and resistance to chemotherapy and radiotherapy result in poor outcome of HCC.Studies have demonstrated that a subpopulation of cancer cells, often referred to ascancer stem cells (CSCs) or tumor initiating cells (TICs), are capable of extensiveproliferation, self-renewal, and the increased frequency of tumor formation. The concept ofCSCs has significant clinical implications: CSCs have been shown to be more resistant tochemotherapy and radiotherapy; CSCs have the properties of EMT, metastasis and tumorrelapse after surgery. Therefore, understanding common mechanisms in self-renewal of CSCswill enable further research to focus efforts on therapeutic targets which may be most usefulin developing new approaches of treatment. Our previous study have identified transcriptionfactor Nanog as a novel marker for liver CSCs and demonstrated that Nanog could play acrucial role in regulating self-renewal of liver CSCs via IGF signaling pathway. Nevertheless,the key components and molecular mechanisms contributing to biology of liver CSCs arelargely unknown.In cancer, high expression of SIRT1(Sirtuin1, silent mating type information regulation2homolog-1) has been linked with aggressive progression and poor prognosis of leukemiaand solid tumors. Moreover, SIRT1not only regulates the Histone deacetylase andnon-histone proteins, but promotes chemotherapy resistance by enhancing efflux of drugs, aswell as renders cancer cells resist to the radiation-induced apoptosis. The similarities betweenCSCs and SIRT1enable us to put forward a hypothesis that SIRT1may participate theregulation of liver CSCs. In this study, we will explore the expression and biological functionof SIRT1in liver CSCs.In order to identify the relation between CSCs and SIRT1, we divided this study into three sections. The first section was to examine the SIRT1expression pattern in liver CSCs,then to clarify its functional role for maintenance of self-renewal in vitro and in vivo. Thesecond section was to verify that SIRT1regulated transcription of SOX2in liver CSCs. Thethird one was to discuss the SIRT1expression with drug resistance and survival in liver CSCsand HCC patients.A list of expression date and main conclusions are shown as following1. SIRT1is highly expressed in liver CSCs compared to non-CSCs, suggesting thatthe expression of SIRT1is essential for maintaining growth and progression of liverCSCs.(1) Expression of SIRT1in NanogPoscells of liver CSCs was significantly higher thanthose in NanogNegcells of non-CSCs.(2) Expression of SIRT1in NanogPoscells derived from xenograft tumors was higherthan that in NanogNegcells derived from xenograft tumors in NOD/SCID mice.(3) SIRT1is decreasing during the differentiation of liver CSCs in vitro.2. Knock-down of SIRT1expression in the NanogPoscells inhitiors self-renewal ofliver CSCs and prolong survival times in NOD/SCID mice.(1) Knock-down of SIRT1expression in the NanogPoscells significantly inhibit cellgrowth of liver CSCs.(2) Knock-down of SIRT1expression in the NanogPoscells was significantly inhibitcolony and sphere formation efficiency of liver CSCs, and the difference was statisticallysignificant.(3) Knock-down of SIRT1expression could significantly inhibited tumor’s growth, sizeand weight when subcutaneously implanted in NOD/SCID mice.(4) Kaplan-Merier analysis shown that knock-down SIRT1expression could prolongsurvival times in NOD/SCID mouse compared with control group．3. Overexpression of SIRT1restore self-renewal characteristics in liver non-CSCs.(1) Overexpression of SIRT1in the NanogNegcells increased the efficiencies of colonyand sphere formation significantly.(2) Survival rate of tumors was significantly increased in NOD/SCID mice implantedwith NanogNegcells infected with adenoviral vector expressing SIRT1.4. SIRT1control SOX2expression to derive self-renewal characteristics in liver CSCs.(1) Knock-down of SIRT1in the NanogPoscells was decreased SOX2expression in liverCSCs.1) Knock-down of SIRT1in the NanogPoscells was significantly inhibit SOX2expression levels of liver CSCs; Overexpression of SIRT1in the NanogNegwas significantlyincreased SOX2expression.2) Knock-down SIRT1in the NanogPoscells was decreased SOX2expression and itoccurred in the earlier time than Nanog of liver CSCs.3) Western blot results showed that SIRT1and SOX2were positive correlation in HCCcell lines and patients tumor tissues..(2) Overexpression SOX2in knock-down SIRT1cells could be restore theself-renewal characteristics in liver CSCs.1) Immunofluorescence and western blot datas showed that overexpression SOX2in theNanogPoscells with silencing SIRT1was increased SOX2expression level.2) Overexpression SOX2in NanogPoswith silencing of SIRT1that colony and sphereefficiency were significantly increased compared to silencing SIRT1in liver CSCs.3) Overexpression of SOX2in the NanogPoscells with silencing of SIRT1could generatetumor more efficiently as compared to silencing SIRT1in liver CSCs.(3) Co-knockdown SIRT1and SOX2expression did not significantly decreased SOX2expression level, and colony and sphere formation efficiency in liver CSCs.5. SIRT1regulates SOX2expression via DNMT3A（1）SOX2changes by knockdown SIRT1contributes to the transcription regulation inliver CSCs.1）PSOX2-luc luciferase system assay shown that silencing of SIRT1in NanogPoscellsexpressed decreased levels of SOX2.2) ChIP analysis shown that that NangPoscells displayed a relative enrichment oftrimethylated H3K4(H3K4Me3) compared to the levels with silencing of SIRT1. Meanwhile,the levels of trimethylation of H3K27(H3K27Me3) was an increased in silencing of SIRT1compared to CSCs.3) MeDIP assay shown that the increased level of5mC and decreased of5hmC in liverCSCs with silencing of SIRT1. 4) BSP-PCR assay that the CpGs was a significantly increased in non-CSC or withsilencing of SIRT1relative in liver CSCs.5) Western-blot assay showen that SOX2expression was restored in response to5-azacytidine compared with silencing of SIRT1in liver CSCs.(2) DNA methylation is one of the important epigenetic modifications, knockdownSIRT1was increased SOX2DNA methylation in liver CSCs.1) qRT-PCR and western blot assay showed that with silencing of SIRT1expression inliver CSC resulted in significant increased DNMT3A expression.2) Western blot analysis demonstrated that increase expression of DNMT3A in atime-dependent manner with silencing of SIRT1.3) ChIP analysis showed that DNMT3A and DNMT1bound to SOX2promoter moreefficiently in liver CSC with silencing of SIRT1.(3) SIRT1effectively inhibits DNMT3A acetylation and expression in liver CSCs.1) Co-IP analysis shown that SIRT1interacts with DNMT3A in liver CSCs.2) DNMT3A acetylation relative expression was increased with silencing of SIRT1compared with liver CSCs.6. Modulation of Nanog expression by SIRT1is mediated by SOX2(1) PNanog-luc luciferase system assay shown that silencing of SIRT1in NanogPoscellsresulted in the reduced expression of GFP in NanogPoscells with silencing of SIRT1in liverCSCs.(2) Western blot assay shown that there was decreased expression of GFP in NanogPoscells with silencing of SIRT1in liver CSCs.(3) Western blot assay shown that there was decreased expression of GFP in xenografttumors derived from NanogPoscells with silencing of SIRT1.(4) ChIP analysis shown that SOX2can bind to Nanog promoter, with silencing ofSIRT1reduced SOX2binding to Nanog promoter capacity and overexpression of SOX2increased this ability.7. High SIRT1and SOX2expression correlates with poor prognosis in human HCCpatients.(1) Immunohistostaining analysis shown that SIRT1protein was mainly localized in thenucleus of cancer cells, positive rates of SIRT1expression in HCC cancerous tissue was62% (92/148). In contrast, SIRT1expression in adjacent noncancerous tissue was31%(48/148).Clinical and pathological analysis of HCC samples, it is showed that expression of SIRT1inHCC was correlated with tumor stage, capsular invasion and vascular thrombus.Kaplan-Merier analysis demonstrated that SIRT1expression in HCC was significantlycorrelated with overall and disease-free survival.(2) Immunohistostaining analysis shown that SOX2protein was mainly localized in thenucleus of cancer cell, and positive of SOX2expression in HCC was33.1%(49/148). Incontrast, SOX2expression in adjacent noncancerous tissue was6.76%(10/148). Clinical andpathological analysis of HCC samples, and results shown that SOX2expression in HCC wascorrelated with tumor interstitial hyperplasia of tumor、intrahepatic metastasis and vascularthrombus. Kaplan-Merier analysis demonstrated that SOX2expression in HCC wassignificantly correlated with overall and disease-free survival.(3) Kaplan-Merier analysis shown that SIRT1and SOX2expression in HCC wassignificantly correlated with overall and disease-free survival. Moreover, when patients werecombination of high SIRT1and SOX2expression has the poorest outcome compared with theother four possible combinations.8. Fundamental function of SIRT1on drug resistance of liver CSCs(1) The survival of liver CSCs was significantly reduced drug resistant clones when TV6or silencing of SIRT1expression was combined with sorafenib, as compared with that inNanogPoscells. Immunofluorescence analysis shown that treatment with sorafenib couldinduce higher level of DNA damage marker γ-H2AX focis with siliencing ofSIRT1expression compare with NanogPoscell.(2) Liver CSCs were treated with TV6or sorafenib could induce drug resistant clones. Incontrast, treatment of liver CSCs by combination of either of TV-6with sorafenib couldcompletely abolish drug resistant clones.(3) The treatment with sorafenib resulted in inhibition of tumor growth in tumor-bearinganimals with NanogPoscells. In contrast, treatment with sorafenib could eradicate tumors intumor-bearing animals with NanogPoscells with silencing of SIRT1expression. Pathologicaland immunohistochemistical analysis shown that dramatic necrosis and high levels ofγ-H2AX were observed in tumors derived from NanogPoscells with silencing of SIRT1expression. 9. Association SIRT1expression with sorafenib treatment with poor prognosis ofHCC patients.Among31HCC cases, Kaplan-Merier analysis shown that positive SIRT1expressionwith sorafenib resulted was significantly correlated with overall and disease-free survival，patients had poor survival time. In contrast, negative SIRT1expression with sorafenibresulted was significantly correlated with overall and disease-free survival, patients hadlonger survival time.In conclusion, the above data suggest that1. SIRT1as a key regulator in the maintenance of self-renewal and tumorigenic potentialin liver CSCs.2. SIRT1maintains self-renewal and tumorigenic potential of liver CSCs through directregulation of SOX2expression.3. The up-regulation of SOX2by SIRT1and subsequently activate the network of Nanogmay regulate self-renewal and tumorigenic potential of liver CSCs.4. SIRT1serves as a potent molecular target for therapy in liver cancer.In summary, our study reported for the first time that the role and the maintenance ofself-renewal mechanisms of SIRT1in liver CSCs, moreover to discuss the function of SIRT1on drug resistance of liver CSCs. We suggested that high levels of SIRT1are needed tomaintain the undifferentiated state of human liver CSCs, which is achieved through tightcontrol of the levels and binding ability of DNM3A and histone modifiers at the regulatoryregions of pluripotency transcription factor SOX2gene. SIRT1may serve as attractivetherapeutic target for treatment of HCC by targeting liver CSCs.