Abrogating K458 Acetylation Enhances HNF4α-induced Differentiation Therapy For Hepatocellular Carcinoma

Background and Objective According to the report of global cancer data,liver cancer,ranking as the sixth most common cancer,is the third leading cause of cancer-related deaths with an estimated number of 905,677 new cases and 830,180 deaths occurring in 2020.Clinically,the pathological type of 90% liver cancer is identified as hepatocellular carcinoma.,while other types of liver cancer,such as intrahepatic cholangiocarcinoma or hepatic hemangioma,are less common.The pathophysiology of HCC is a complex multistep process,in which the interplay of various factors is at the origin of the early steps of hepatocyte malignant transformation and HCC development.Over 90% HCC cases occur in the setting of chronic liver disease.Cirrhosis resulting from any aetiology is the strongest risk factor for HCC.Until 2020,only sorafenib and lenvatinib were approved as the first-line therapies for advanced HCC.However,the efficacy of these drugs is limited resulting in the frequent recurrence and high mortality of HCC.Thus,the development of novel therapeutic strategy for HCC treatment is urgently needed.Hepatocyte nuclear factor 4α(HNF4α),belonging to the nuclear hormone receptor superfamily(NR2A1),is a liver-enriched transcription factor which could bind to the promoter of 12% of genes expressed in the adult liver.HNF4α plays a crucial role in hepatocyte differentiation and the maintenance of hepatic function.The expression of HNF4α was deregulated in HCC samples and the decrease of HNF4α expression correlated with poor prognosis.Our previous study has demonstrated that the enforced expression of HNF4α exerted a striking suppression effect on HCC via inducing the differentiation of hepatoma cells into hepatocytes,and suppressing hepatocyte EMT and cancer stem cell generation.In addition to HCC,HNF4α also exhibits a strong therapeutic effect on chronic liver diseases,including NASH,liver fibrosis and chronic hepatic failure.Upregulation of HNF4α in hepatocytes improves the hepatic function and impedes the progression of adipose deposition in hepatocyte.Moreover,a recent study showed that delivery of HNF4 A m RNA encapsulated-LNP induced a robust inhibition of fibrogenesis in mice.These studies suggest that enforced expression of HNF4α could be a promising strategy for therapy of chronic liver diseases,include HCC.Recent studies report that posttranslational modifications(PTMs)of the HNF4α protein,including phosphorylation,acetylation,methylation,are involved in the regulation of HNF4αtranscriptional activity.An acetylation of HNF4α at lysine 458 has been identified as an important PTM for HNF4α-mediated transcriptional control.Moreover,recent study showed that the deacetylation of HNF4α at lysine 458 by a NAD+ dependent deacetylase SIRT2 activated HNF4α activity and prevents liver steatosis and metabolic disorders in NAFLD mice.The mutation of HNF4α 458 lysine(K)to glutamine(Q)can mimic acetylation,while the mutation of lysine(K)to arginine(R)can inhibit acetylation.Based on the above research background,this study aims to investigated the effect of manipulating the acetylation status of HNF4α at lysine 458 on the differential therapy of HCC.Methods 1.The acylation of HNF4α-K458 affects the biological characteristics of HCC cells(1)Effect of the acylation of HNF4α-K458 on cell proliferation Huh7 and Hep 3B were infected with Ad-GFP,Ad-HNF4α,Ad-K458 Q or Ad-K458 R for 12 hours.Then the cells were plated into a 96-well plate at 3 × 103/well.The number of metabolically active cells was determined using the Cell Counting Kit-8(CCK8)every day for 1 week.(2)Effect of the acylation of HNF4α-K458 on the migration and invasion of HCC cells We utilized transwell chambers(BD Bioscience)without or with Matrigel according to the manufacturer’s instructions to perform In vitro migration and invasion assays.HCC cells infected with adenovirus were plated in the upper chamber without 10% FBS.Then,10% FBS supplemented with medium was administrated in the lower chamber.After 48 h,the cells on the lower membranes were stained with 0.1% crystal violet.The area of positive staining was measured by Image analysis software.2.The acylation of K458 regulates HNF4α induced differentiation in HCC cells(1)Effect of the acylation of HNF4α-K458 on HNF4α transcriptional activity RNA from Huh-7 and Hep 3B cells infected with adenovirus were extracted with TRZOL reagent.Real-Time PCR was used to detect the downstream gene expression of HNF4α.The adenovirus-infected cells were planted into a 96-well plate at 8000/ well,then the cells were co-transfected with the reporter gene plasmid PGL3-promoter-HNF4α and SV40 vector for 24 h.Luciferase reporter gene assays were performed using the DualLuciferase Reporter Assay System according to the manufacturer’s instructions.(2)Effect of the acylation of HNF4α-K458 on HNF4α induced hepatic function After Huh-7 and Hep 3B cells infected with adenovirus,the senescence of the HCC cells was examined with a Senescence β-Galactosidase Staining Kit.To assess stored glycogen,the cells were stained with a PAS Reaction Kit according to the manufacturer’s instructions.Fluorescent labeled ac-LDL was added to medium of the cells to detect the uptake of low-density lipoprotein by HCC cells.3.The effect of abrogating K458 acetylation of HNF4α on HCC treatment(1)Abrogating K458 acetylation enhances the inhibitory effect of HNF4α on Huh-7 xenografts Huh-7 cells were subcutaneously injected into the flanks of BALB/c nude mice.Adenovirus treatment was initiated when the tumors were established around 100 mm3.Tumor volume was serially estimated according to the following formula:(Length × Width2)/2.After 8 days,mice were sacrificed and the tumor tissues were fixed with formaldehyde solution and embedded with paraffin.(2)Abrogating K458 acetylation enhances the inhibitory effect of HNF4α on an orthotopic tumor model Huh-7 cells stably expressing luciferase were injected subcutaneously into the flanks of NOD/SCID mice to generate tumor xenografts.The tumor nodules from the subcutaneous xenograft model were cut into 1 mm3 pieces and implanted into the left lobe of the livers of NOD/SCID mice(male,5-week-old)to mimic primary HCC.Ad-HNF4α,Ad-K458 R or Ad-GFP was then injected via the tail vein once each week for 3 weeks.The mice were monitored using an IVIS 200 imaging system.The mice were killed 3 weeks after the transplantation of tumor fragment.The weight of tumors was measured and the tumor tissues were fixed with formaldehyde solution and embedded with paraffin.4.The effect of abrogating K458 on the stability of HNF4α By treating Huh-7 cells with AGK2,an inhibitor of SIRT2,to promote the acetylation of HNF4α in Huh-7 cells,the level of HNF4α protein in HCC cells were detected by Western Blot.Huh7 cells transfected with wild type HNF4α,K458 Q mutant or K458 R mutant were treated with CHX.The level of HNF4α protein in HCC cells were detected by Western Blot.HNF4α of Huh-7 xenografts or an orthotopic tumor model was detected by IHC.5.The effect of abrogating K458 on the transcriptome induced by HNF4α in HCC RNA was purified from tissues of Huh-7 xenograft tumors in nude mice,using TRIZOL reagent(Takara).Samples were sequenced using Illumina Novaseq6000.RNA libraries were prepared for sequencing using standard Illumina protocols.HTSeq v0.6.0 was used to count the reads numbers mapped to each gene.We performed significant analysis and FDR analysis under the following criteria:(i)|log2FC| > 1 and(ii)p value <.05,and then we used DESeq2 algorithm to filter the differentially genes.The biological implications of unique genes in the representative profiles of the differentially gene in the experiment was performed by Gene ontology(GO)analysis.The significant GO categories were identified by Fisher’s exact test,and the p values were corrected by FDR.We utilized Pathway analysis to identify the significant pathways of the differentially genes according to KEGG database.The threshold of significance was determined by the p value and FDR.Fisher’s exact test was used to select the significant pathways.GSEA was applied for cellular pathway analysis.The entire data are accessible at NCBI Gene Expression Omnibus under the accession number GSE225329.6.Statistical analyses SPSS software(26.0 version)was used for statistical analyses.The data of experiments involving only two groups was analyzed by Student’s t test.The unequal variances pairing using the Wilcoxon signed rank test or Mann-Whitney U test.P value <0.05 considered significant.Statistical significance was set at *P<0.05,**P<.01,***P<.001,ns: no statistical significance.Results 1.The acylation of HNF4α-K458 affects the malignancy of HCC cells(1)The acylation of HNF4α-K458 affects the proliferation of HCC cells Enforced wild type HNF4α notably suppressed the proliferation of HCC cells.K458 Q mutant partially abolished while K458 R mutant enhanced the suppressive effect of HNF4α on the proliferation of HCC cells.(2)The acylation of HNF4α-K458 affects the migration and invasion ability of HCC cells.Overexpression of wild type HNF4α suppressed cell migration and invasion.K458 Q mutant partially abolished while K458 R mutant enhanced the suppressive effect of HNF4α on the migration and invasion ability of HCC cells.2.The acylation of K458 regulates HNF4α induced differentiation of HCC cells(1)The acylation of K458 regulates HNF4α-mediated transcriptional activity Luciferase reporter assay revealed that K458 R mutant enhanced the transcriptional activity of HNF4α,while the K458 Q mutant reduced the transcriptional activity of HNF4α.Real-time PCR revealed that the HNF4α-induced downregulation of stemness genes in HCC cells was enhanced by K458 R mutant and partially abolished by K458 Q mutant.K458 R enhanced,whereas K458 Q reduced the transcriptional activity of HNF4α.(2)The acylation of K458 affects HNF4α induced cellular senescence of HCC cells Senescence associated β-galactosidase(SA-β-gal)staining indicated more significant cellular senescence in HCC cells with K458 R mutant overexpression than that of HCC cells with wild type HNF4α overexpression.Consistently,the cellular senescence was decreased in HCC cells with K458 Q mutant overexpression.(3)The acylation of K458 affects HNF4α induced glycogen storage of HCC cells Periodic acid-Schiff(PAS)reactions further revealed that K458 R increased repressed while K458 Q mutant HNF4α-induced glycogen storage of Huh-7 cells.(4)The acylation of K458 affects HNF4α induced ac-LDL uptake of HCC cells K458 R mutant also enhanced HNF4α-induced hepatic function of acetylated lowdensity lipoprotein(ac-LDL)intake,while K458 Q mutant significantly decreased this function.3.The effect of abrogating K458 acetylation of HNF4α on HCC treatment(1)Abrogating K458 acetylation enhance S the inhibitory effect of HNF4α on tumourigenicity The sizes and weight of Huh-7 xenografts treated with Ad-HNF4α were significantly less than those the control xenografts.As expected,the inhibitory effect was more pronounced in xenografts treated with Ad-K458 R.The ki67 staining was remarkably decreased in the xenografts treated with Ad-HNF4α than the xenografts treated with AdGFP and almost diminished in xenografts treated with Ad-K458 R.real-time PCR illustrated that the comparable RNA levels of HNF4α in xenografts treated with Ad-HNFα and Ad-K458 R.(2)Abrogating K458 acetylation enhances the inhibitory effect of HNF4α on orthotopic HCC model Then,we utilized the orthotopic HCC model described before to investigate the antitumor effect of HNF4α or HNF4α-K458 R in vivo.the bioluminescence was reduced in the mice treated with Ad-HNF4α compared with the mice Ad-GFP,while the mice treated with Ad-K458 R showed the weakest luciferase activity.The tumors from the mice were significantly smaller of the mice treated with Ad-HNF4α than that of control mice,and AdK458 R showed more obvious inhibitory effect on the tumors.The ki67 staining also showed the more significant inhibition of Ad-K458 R on proliferation of HCC than that of Ad-HNF4α.Real-time PCR revealed the equal overexpression of HNF4α in tumors treated with Ad-HNF4α and Ad-K458 R.4.The effect of abrogating of K458 acetylation on the stability of HNF4α Compared with DMSO,AGK2 obviously accelerated the degradation of HNF4α in Huh-7 cells.In addition,AGK2 displayed a shorter half-life than DMSO in Huh-7 cells treated with CHX.K458 Q mutant displayed a shorter half-life compared with that of wild type HNF4α,while the degradation of HNF4α protein was dramatically inhibited by K458 R mutation.IHC showed elevated protein levels of HNF4α in xenografts or an orthotopic HCC model treated with Ad-K458 R than treated with Ad-HNF4α.5.The effect of abrogating K458 acetylation on the transcriptome induced by HNF4α in HCC Although we had proved that K458 R mutant enhanced the transcriptional activity of HNF4α,it remained unknown whether K458 R mutation alters the transcriptome induced by HNF4α in HCC.Therefore,we performed an RNA-sequencing with Huh-7 xenografts treated with adenovirus.Heatmap of the whole differential genes showed that K458 R mutant had a similar transcriptome to wild type HNF4α.Pathway analysis illustrated that the pathway regulated by K458 R mutant was almost identical with that of wild type HNF4α,such as Primary bile acid biosynthesis,Glycolysis/Gluconeogenesis and Biosynthesis of unsaturated fatty acid.In addition,gene ontology(GO)analysis revealed that enforced expression of K458 R mutant in HCC did not significantly alter biological process,cellular component and molecular function of HCC cells compared with that of overexpression of wild type HNF4α.However,GSEA indicated that both of wild type HNF4α and K458 R mutant achieved a high enrichment score of HNF4α target genes.Furthermore,a heatmap based on GSEA indicated that K458 R mutant enhanced transcriptional activity in HNF4α target genes compared with wild type HNF4α.Therefore,these dates demonstrated that K458 R mutant enhanced HNF4α transcriptional activity without altering the transcriptome induced by HNF4α in HCC.Conclusions 1)K458R mutant enhanced the suppressive effect of HNF4α on the proliferation,migration and invasion.2)K458R mutant promoted HNF4α induced differentiation of HCC cells.3)K458R mutant elevated the inhibitory effect of HNF4α on HCC in vivo.4)K458R mutant inhibited the degradation of HNF4α in HCC cells.5)K458R mutant did not alter the transcriptome induced by HNF4α in HCC.