The TET2/E-cadherin/?-catenin Regulatory Loop Confers Growth And Invasion In Hepatocellular Carcinoma Cells

Hepatocellular carcinoma(HCC)is a leading cause of cancer-related deaths worldwide.Despite recent progress in the diagnosis and treatment of HCC,the outcome for advanced HCC still remains very poor.Therefore,it is of great urgency and importance to identify valuable biomarkers predicting clinical outcomes and effective targets for HCC treatment.In this study,we found that TET2 is highly expressed in hepatocellular carcinoma cells,and is negatively correlated with e-cadherin expression pattern.Proteins of the TET(Ten-Eleven-Translocation)family have become a focus of substantial interest,since the initial description of their enzymatic activity.The three mammalian TET proteins(TET1,TET2 and TET3)are Fe2+-and 2-oxoglutarate-dependent dioxygenases that successively oxidize 5-methylcytosine(5mC)to 5-hydroxymethylcytosine(5hmC),5-formylcytosine(5fC)and 5-carboxylcytosine(5caC)in DNA.TET proteins have roles in diverse biological processes,including epigenetic regulation of gene transcription,embryonic development,stem cell function and cancer.TET proteins also can play unique roles partly due to their specific interactions with other regulators,allowing them to regulate gene expression independent of DNA hydroxymethylation.As an important link of TET proteins to disease,TET2 frequently acquires function loss in different types of cancers,notably myeloid neoplasms.However,the expression pattern and role of TET2 in HCC has not been assessed.Furthermore,no studies have investigated whether TET2 dysregulation affects clinical outcomes in patients with HCC.Therefore,the objective of this study was to examine the expression pattern,function and clinical significance of TET2 expression in HCC.We found TET2 protein was overexpressed in HCC tissues,compared to paired normal tissues.Experimental data indicated TET2 interacts with HDAC1 to repress E-cadherin expression,then facilitates ?-catenin nuclear translocation and promotes progression of HCC.We investigated the mRNA level of TET2 expression in 97 human HCC tissue samples and 22 normal hepatic tissue samples by qRT-PCR.The results showed that TET2 mRNA was up-regulated in HCC tissues when compared with normal hepatic tissues.Further analysis showed that TET2 mRNA level was associated with worse BCLC stage in HCC.Immunohistochemistry assay was conducted to examine the protein level of TET2 expression in the same tissues.We observed that TET2 protein level was also up-regulated in HCC tissues and correlated with BCLC stage.These results indicated that TET2 expression was up-regulated in HCC tissues and cells,suggesting a potential role of TET2 in HCC progression.Notably,the high TET2 protein level was associated with larger tumor size,multiple tumor nodules,poorer differentiation grade,vascular invasion,BCLC stage and recurrence,and the high TET2 expression group of HCC patients had significantly poorer OS than the low TET2 expression group.To explore the role of TET2 in HCC,we tested TET2 expression in a series of HCC cell lines and found that TET2 mRNA and protein levels in Huh7,HepG2,SMMC7721,MHCC-97L and MHCC-97H were higher than that in normal human hepatic epithelial cell line L-02,Then,we generated stable TET2 knockdown using shRNAs in highly invasive HCC cell lines SMMC7721 and MHCC-97H tested by western blot.We found that TET2 knockdown inhibited the proliferation of SMMC7721 and MHCC-97H cells in vitro,whereas,ectopic TET2 overexpression enhanced the proliferation of HepG2 cells.We further investigated its role in vivo using nude mice with HCC xenografts.We injected subcutaneously into the oxter of athymic mice with TET2 knockdowned SMMC7721 cells and control cells.We found that TET2 knockdown significantly delayed SMMC7721 xenograft tumors growth.These data indicated that TET2 promotes HCC cells growth in vitro and in vivo.Results from transwell assay showed that TET2 knockdown efficiently inhibited the invasion potential of SMMC7721 cells and MHCC-97H,Inversely,the invasion potential of HepG2 cells was dramatically enhanced after TET2 overexpression.These results indicated that TET2 is involved in the invasion of HCC cells.Expectedly,we found that TET2 knockdown resulted into E-cadherin up-regulation in mRNA and protein levels in SMMC7721 and MHCC-97H cell lines.E-cadherin expression is usually epigenetically silenced because of promoter DNA hypermethylation,suggesting TET2 regulated E-cadherin expression beyond its function on decreasing DNA methylation level here.To investigate whether here TET2 regulated E-cadherin expression via modulating histone acetylation status,we applied MS-275,a selective inhibitor of class I HDACs.We found MS-275 treatment also up-regulated E-cadherin mRNA and protein levels in SMMC7721 and MHCC-97H cell lines.Consistently,E-cadherin protein was lowly expressed in HCC cells expressing high TET2.Expectedly,E-cadherin up-regulation in MS-275 treated HCC cells was accompanied with increase of H3K9Ac and H4K16Ac levels at the E-cadherin promoter determined with ChIP-qPCR assay.In agreement,TET2 knockdown also resulted into increase of H3K9Ac and H3K16Ac levels at the E-cadherin promoter.Conversely,TET2 overexpression decreased the E-cadherin mRNA and protein levels in HepG2 cells.TET2 overexpression decreased the H3K9Ac and H4K16Ac levels at the E-cadherin promoter.Expectedly,coimmunoprecipitation(CoIP)result indicated that TET2 could associate with HDAC1 in SMMC7721 and MHCC-97H cell lines.We found HCC cells with high TET2 protein level but with low E-cadherin protein level,have more ?-catenin transactivation.TET2 knockdown inhibited the ?-catenin transactivation in SMMC7721 and MHCC-97H cell lines evaluated by TCL/LEF-1 transcriptional activity measured by the TCF/LEF Reporter Assay(luc)Cignal Lenti Reporter Assays.MS-275 treatment also inhibited the ?-catenin transactivation in SMMC7721 and MHCC-97H cell lines.Inversely,ectopic TET2 expression promoted the P-catenin transactivation in HepG2 cells.Because of its overexpression and roles in HCC cells,we wondered what transcription factor directly regulated TET2 transcription.We screened potential transcription factors located within a two kb region upstream of the transcription start site of TET2 gene using JASPAR database(http://jaspar.binf.ku.dk)and suggested three TCF7L2(TCF4)binding sites at the potential promoter,suggesting ?-catenin signaling maybe feedback regulated TET2 expression in HCC cells.To validate this hypothesis,?-catenin was knockdowned using specific shRNAs in SMMC7721 and MHCC-97H cell lines.?-catenin knockdown reduced TET2 mRNA and protein levels.To investigate whether ?-catenin/TCF4 directly binded to the TET2 promoter,we performed chromatin immunoprecipitation(ChIP)of ?-catenin and TCF4 in HCC cells followed by qPCR of the TET2 promoter.Experimental results indicated that ?-catenin and TCF4 were mainly enriched at site A and site C in SMMC7721 and MHCC-97H cell lines.?-catenin knockdown reduced the enrichment of p-catenin and TCF4 at TET2 promoter in SMMC7721 and MHCC-97H cell lines.To examine whether ?-catenin can activate the TET2 promoter,we transfected wild-type(WT)or mutant-type(deletion of the binding motif)TET2 promoter reporter constructs into control or p-catenin knockdowned SMMC7721 and MHCC-97H cell lines.?-catenin knockdown repressed WT but not mutant TET2 promoter activity.These data suggested that TET2 was transcriptionally regulated by ?-catenin signaling in HCC cells.