Role And Relevant Mechanism Of ATP-Citrate Lyase In Regulating The Biological Function Of Hepatocellular Carcinoma

Background&Aims:Liver cancer is the sixth most frequently diagnosed cancer and the fourth leading cause of cancer-related deaths worldwide.Hepatocellular carcinoma(HCC)and intrahepatic cholangiocarcinoma are the most common histological types of liver cancer,accounting for75%-85%and 10%-15%of all cases,respectively.Surgery,liver transplantation or in situ ablation are effective for early-stage HCC patients,but due to the high recurrence rate of liver cancer,the five-year survival rate is very low.For advanced-stage patients,the first-line targeted drugs only yield modest efficacy.This necessitates the search for the intricate molecular mechanisms that drive HCC progression and metastasis,which is critically needed for developing novel therapeutic strategies to improve the clinical outcomes of HCC patients.Unlike most normal cells and tissues,which satisfy their requirement for fatty acids(FA)by importing them from the circulation,tumor cells prefer to undergo high rates of de novo lipogenesis.This type of metabolic reprogramming,has been viewed as a main character of tumors and attracted more and more attention.Generally,the importance of FA synthesis in cancer cell proliferation and survival in vitro has now been described in a number of studies.However,whether autochthonous tumors in vivo possess a similar requirement is less understood.The elevated de novo fatty acid synthesis observed in tumors is reflected by the increased expression of lipogenic enzymes including fatty acid synthase(FASN),acetyl-Co A carboxylase(ACC)and ATP citrate lyase(ACLY)that has been described in a number of cancers.ACLY,which catalyzes citrate to produce acetyl-Co A and oxaloacetate in the cytoplasm,is a key enzyme linking glucose/glutamine metabolism with de novo FA synthesis.A number of studies have shown that ACLY expression is upregulated in a variety of tumors and is associated with tumor growth.Its catalytic product acetyl-Co A is the requisite building block for de novo synthesis of FA and cholesterol.Therefore,expression reduction or enzyme activity inhibition of ACLY can lead to tumor growth repression due to reduced synthesis of fatty acids and cholesterol necessary for cell proliferation.At the same time,Acetyl-Co A is also the donor of acetyl groups for acetylation of histones or non-histone proteins.Acetylation of histones plays an important role in regulating gene expression,DNA replication and DNA damage repair.Interference with ACLY can regulate the acetylation modification of histones by reducing the abundance of acetyl-Co A,and cause site-specific gene expression regulation,so as to regulate cell proliferation,migration and adhesion,etc.Nuclear ACLY-mediated production of acetyl-Co A,promotes histone acetylation,BRCA1 recruitment,and subsequent HR-mediated DNA repair in response to DNA damage.Furthermore,ACLY-mediated production of acetyl-Co A also contributed disturbed lipid metabolism in HBV pre-S2 mutant-induced tumorigenesis.However,so far,the specific effects and related mechanisms of ACLY on the growth and metastasis of HCC have not been reported.Tumor-initiating cells(TICs,or cancer stem cells,CSCs)hypothesis is one of the main theories on tumor development.It proposes that cancer cells differ in their tumorigenic abilities,and only a small subset of stem cell-like cancer cells,known as tumor-initiating cells(TICs,also called CSCs),harbor the characteristics similar to those of normal tissue stem cells,i.e.the potential for self-renewal and polydifferentiation.Since the first identification of CSC by Tsvee Lapidot for AML,more and more researchers have confirmed the validity of the CSC model in various tumor types.Accumulating evidence has revealed that existence of liver tumor-initiating cells(LTICs)in HCC,participating in its tumorigenesis and progression.A number of studies have showed that LTICs are the root of HCC metastasis,recurrence and drug resistance.Targeting LTICs may effectively address metastasis,recurrence,and drug resistance that lead to death in HCC patients.This study aims to explore the role of ACLY in the biological functions of liver cancer and the related mechanisms.On the basis of clarifying the expression of ACLY in human HCC tumor tissues,we analyzed the relationship between the expression level of ACLY and the prognosis and progression of HCC patients;evaluated the regulation of ACLY expression on LTICs,the effect of ACLY expression on the proliferation,migration,invasion and metastasis of HCC tumor cells in vitro and in vivo;and explored the relevant mechanism.These findings will help to further understand the mechanism of the occurrence and progression of HCC,providing new insights and new targets for the diagnosis and treatment of HCC.Methods:1.Expression data of ACLY in HCCDB public database was downloaded and analyzed;m RNA and protein levels of ACLY in HCC tissues were further verified by Quantitative Real-time PCR(q RT-PCR)?Western blot(WB)and immunohistochemistry;2.HCC tissue samples in the HCCDB database were divided into the ACLY high-expression group and the ACLY low-expression group based on the m RNA expression level,and the overall survival period,mortality risk ratio,TNM stage and portal vein invasion ratio of the two groups were compared,respectively;3.The LTICs were enriched by various methods and the expression of ACLY of them was analyzed.The protein levels of ACLY and CD133 in tissue microarray was detected and the correlation between them was evaluated.Si RNA interference and ACLY stable interfering cell lines were used to study the effect of ACLY knockdown on the proportion of CD133~+cells and self-renewal ability in common liver cancer cell lines Huh7 and HCC-LM3;4.CCK8 and colony formation were used to detect the effect of ACLY knockdown on short-term proliferation and long-term survival of HCC cells in vitro,respectively.The effect of ACLY silencing on the growth of HCC cells in vivo was studied by subcutaneous tumor transplantation in nude mice;5.Effects of ACLY knockdown on the migration and invasion of HCC cells were studied by transwell migration,wound-healing and transwell invasion assays in vitro.Lung metastasis model of Huh7 was established by tail vein injection in mice,and the effect of ACLY silencing on lung metastasis of HCC cell was analyzed;6.Luciferase reporter assay was performed to detect the effect of ACLY knockdown and overexpression on?-catenin-dependent transcriptional activation,and q RT-PCR and Western blot were used to detect m RNA and protein expression of?-catenin and its target genes,nucleoprotein isolation was performed to detect nuclear?-catenin expression;7.The mechanism of?-catenin protein reduction induced by ACLY knockdown was studied by CHX and proteasome inhibitors.The effect of acetylation on the expression of?-catenin was studied by deacetylase inhibitor,and the expression of acetyl-?-catenin(Lys49)and the interaction between p300 or CBP and acetyl-?-catenin(Lys49)were detected by Co-Immunoprecipitation(Co-IP);8.m RNA expression of common Epithelial-Mesenchymal Transition(EMT)markers was detected after ACLY knockdown and further verified by WB.The effect of overexpression of activated?-catenin on downregulated mesenchymal markers(Vimentin,Fibronectin and N-cadherin)was studied;9.Whether reactivation of Wnt/?-catenin pathway could reverse the down-regulation of self-renewal ability of LTICs and migratory ability of HCC cells was investigated.Results:1.The m RNA and protein expression levels of ACLY in HCC tissues were significantly higher than that in adjacent normal tissues;2.High level of ACLY was significantly associated with poor overall survival,increased mortality risk,increased proportion of late stage(III,IV)patients,and increased rate of portal vein invasion;3.ACLY expression in LTICs subpopulation was significantly higher than that in control cells;In HCC tissues,the high expression ratio of CD133 in the ACLY high expression group was significantly higher than that in the ACLY low expression group;ACLY knockdown remarkably reduced the proportion of CD133~+cells and the ability of self-renewal in Huh7 and HCC-LM3,moreover,expression of LTICs markers were also significantly reduced;4.ACLY knockdown did not significantly affect the short-term proliferation capacity of Huh7 and HCC-LM3 cell lines,but it did affect their long-term survival capacity.In vivo xenograft model assay showed that ACLY knockdown affected the growth of subcutaneous transplanted HCC cells,and interfered with the tumor formation rate,growth speed and final tumor volume and weight;5.Knockdown of ACLY dramatically inhibited the migratory and invasive abilities of HCC cells.In the lung metastasis model,the number of metastasis nodules in lungs was significantly reduced in mice injected with ACLY knockdown cells than in mice injected with control cells;6.ACLY expression influenced activation of Wnt/?-catenin signaling pathway,and expression of target genes of?-catenin.Total protein level of and nuclear?-catenin were reduced by ACLY knockdown;7.ACLY knockdown affected?-catenin stability.Inhibition of proteasome-mediated degradation restored?-catenin expression.Acetylation regulated?-catenin expression,depletion of ACLY down-regulated acetyl-?-catenin(Lys49),and attenuated the interaction between p300 or CBP and?-catenin;8.ACLY knockdown decreased expression of partial EMT markers.Overexpression of activated?-catenin partially reversed downregulated mesenchymal markers;9.Reactivation of?-catenin reversed inhibition of self-renewal ability of LTICs and partially reversed the downregulated migratory ability of hepatocellular carcinoma cells induced by ACLY silencing.Conclusion:ACLY expression was frequently upregulated in primary HCC tissues.High level of ACLY was associated with poor overall survival,increased mortality risk,increased proportion of later-stage patients,and increased rate of portal vein invasion for patients with HCC.ACLY knockdown significantly inhibited the proportion and self-renewal of LTICs,the long-term survival of HCC cells in vitro and the growth of HCC cells in vivo,as well as the migration and invasion of HCC cells in vitro and lung metastasis of HCC cells in vivo.Mechanistically,Wnt/?-catenin pathway,the key signaling in tumor,could be regulated by ACLY expression,and expression of ACLY could affect the stability of?-catenin by affecting its acetylation at lysine 49,overexpression of activated?-catenin could partially reverse the above phenotypic and molecular changes.Our study clarified the role of ACLY in the biological functions of HCC,and innovatively discovered that it regulates the key signaling pathways in tumor by affecting the post-translational modification of specific proteins,which would enrich the association between metabolic molecules and tumors.