GCNT2 Induces Epithelial-mesenchymal Transition And Promotes Migration And Invasion In Esophageal Squamous Cell Carcinoma Cells

Background & Objective:Esophageal cancer(EC)is one of the most common malignant cancers worldwide.Histologically,there are two main forms of EC: esophageal squamous cell carcinoma(ESCC)and adenocarcinoma.Approximately 70% of global ESCC cases occur in China,where it is ranked as the fourth most lethal cancer,and about 95% of the cases are esophageal squamous cell carcinoma(ESCC).Despite continuous efforts in development of chemotherapy and radiotherapy in the last decades,the overall 5-year survival rate of ESCC is approximately 10-20%,which is largely due to diagnosis in later stages accompanied by local invasion and distant metastasis.Tumor metastasis is one of the leading causes of treatment failure and death of patients with ESCC.However,the underlying molecular mechanisms of metastasis in ESCC remain elusive.Therefore,there is an urgent need for in-depth studies and identification of novel effective therapeutic targets in ESCC.Cell surface carbohydrates play a significant role in cell proliferation and invasion.The I-branching ?-1,6-N-acetylglucosaminyltransferase(GCNT2),one of cell surface carbohydrates,can synthesize I-branched polylactosamine chains(I?antigen)by catalyzing the transfer of GlcNAc from uridine diphosphate-GlcNAc with a galactose ?1-6 linkage of linear lactosamine chains.It has been reported that GCNT2 is overexpressed in human cancers,such as breast cancer and prostate cancer.Besides,GCNT2 is implicated in regulation of epithelial-mesenchymal transition(EMT),migration,invasion and metastasis of cancer cells.However,the role of GCNT2 in ESCC remains enigmatic.In this study,we will examine the expression and significance of GCNT2 in ESCC to identify a novel prognostic biomarker for patients with ESCC,and further investigate the effect of GCNT2 on ESCC.Methods:The protein expression of GCNT2 was assessed by Immunohistochemical(IHC)assay in ESCC tissues and matched adjacent non-tumor tissues,and its relationship with clinicopathological parameters and prognosis of ESCC patients was analyzed.qRT-PCR was applied to measure the mRNA level of GCNT2 in ESCC cells.Western blotting analysis was used to detect the expression of relative proteins in ESCC cells.Then,the subcellular localization and expression of EMT-related proteins in ESCC cells were verified by immunofluorescence assay.Trypan blue exclusion assay and plate colony formation assay were performed to investigate the effect of GCNT2 on proliferation.In addition,the role of GCNT2 on the migration and invasion of ESCC cells were evaluated by wound healing assay and transwell assay.Results:IHC analysis of tissue microarrays indicated that the expression of GCNT2 was significantly higher in ESCC tissues than in para-tumor tissues.Moreover,high expression of GCNT2 was associated with poor prognosis in patients with ESCC.We found that enforced expression of GCNT2 increased the expression of N-cadherin and Vimentin,whereas the expression of E-cadherin decreased in KYSE30 and EC9706 cells.Notably,overexpression of GCNT2 promoted the migration and invasion,but had no effect on proliferation of ESCC cells.Conversely,knockdown of GCNT2 decreased the expression of N-cadherin and Vimentin,increased the expression of E-cadherin and inhibited the migration and invasion in KYSE150 and EC109 cells.In addition,overexpression of GCNT2 increased the protein level of MMP-2 in KYSE30 and EC9706 cells,and the expression of MMP-2 was decreased after knockdown of GCNT2 in KYSE150 and EC109 cells.Furthermore,Western blotting analysis revealed that GCNT2 increased the phosphorylation of AKT.Conclusions:In summary,our findings showed that GCNT2 was highly expressed in patients with ESCC and predicted adverse outcome.Overexpression of GCNT2 induces EMT and promotes migration and invasion in ESCC cells.Therefore,GCNT2 may act as a candidate prognostic indicator of outcome and a novel target in ESCC patients.