N-linked Glycosylation Modification Of EpCAM Regulates EMT And Affects Cell Adhension In Breast Cancer Cells

Breast cancer is the most common malignant tumor,the domestic incidence morbidity was increasing year by year.Breast cancer account for 7%-8% of all malignancies,the domestic incidence is higher than the average worldwide with 1%-2%.At present,breast cancer surgery,radiotherapy,chemotherapy,etc.It is difficult to satisfy the therapy for their simplification.Therefore,seeking effective treatment of breast cancer has certain clinical significance.EMT(Epithelial-Mesenchymal Transition)refers to the tumor cell metastasis,in cell morphology,accompanied by changes that epithelial cells to mesenchymal cells,invasion into the basement membrane,then into the blood vessels,and spread to the next place.In this process,the tumor cells come off from the original place and then invade into the surrounding tissue,resulting in invasive and migratory ability,then break through the contact between cells,form the cell-matrix adhesion sites,complete the chemotaxis process by proteases in matrix.Some studies have shown that EMT and epithelial cell tumor occurrence and development process is closely related.It has been found that tumor metastasis alter the expression of adhesion molecules(Cell Adhesion Molecules,CAMs).Abnormal expression of CAMs on the surface of tumor cells may weaken the adhesion of tumor cells and fall into extracellular matrix(ECM),which may lead to tumor invasion and metastasis.Epithelial cell Adhesion Molecule(EpCAM)is a transmembrane glycoprotein with a molecular weight of 40 k Da consisting of 314 amino acids,including the N-terminal extracellular domain,transmembrane domain,and C-terminal intracellular domain.EpCAM is different from CAMs classic family members and has potential carcinogenicity.Under normal conditions,in addition to squamous epithelial cells,EpCAM has different degrees of expression in all normal epithelial cells.In Pathological conditions,EpCAM is highly expressed almost in all adenocarcinomas,including breast cancer,lung adenocarcinoma,ovarian cancer,pancreatic cancer and so on.The primary structure of EpCAM shows that EpCAM N-terminal extracellular domain contained three N-linked glycosylation sites,including Asn74、Asn111、Asn198.It has been reported that the removal of three glycosylation sites reduces the half-life of EpCAM from 21 hours to 7 hours,indicating that the N-glycosylation site is essential to maintain EpCAM function.In addition,compared with normal tissues,the difference in glycosylation of EpCAM is an important factor to the variation of tumor function.Therefore,it is speculated that N-glycosylation of EpCAM in breast cancer cells may be the key to activate and regulate EMT signal transduction.The preliminary results of this study are devoted to the regulation of EpCAM on EMT in breast cancer.The experimental results show that transcription factor AP-1regulates the expression of EpCAM through JNK signaling pathway induced by TGF-β1 and affects the occurrence of EMT.In this study,we investigated the mechanism of EpCAM N-glycosylation modification affect EMT in breast cancer cells,and provided a theoretical basis for the treatment of breast cancer.First,EpCAM N-glycosylated mutant plasmids were transiently transfected into two breast cancer cell lines.The results show that EpCAM expressed glycosylated and non-glycosylated proteins in breast cancer cells.The results show that EpCAM N-glycosylated mutant plasmid do not alter the expression and location of EpCAM by immunofluorescence assay.The expression of E-cadherin in EMT is increased and the expression of N-cadherin and Vimention decreased with the expression of EpCAM N-glycosylated mutant plasmid combined with TGF-β1 in breast cancer cells.The results suggest that EpCAM N-glycosylation modulates the regulation of EMT.EpCAM N-glycosylation6mutant plasmids combined with inhibitors of TGF-β1 and ERK1/2 and PI3K/Akt are administered to both breast cancer cell lines.The expression of E-cadherin increased,the expression of N-cadherin and Vimention decreased,p Raf-1,p ERK and p Akt decreased in ERK1/2 and PI3K/Akt signaling pathway.The results show that under the induction of TGF-β1,EpCAM N-glycosylation inhibit the presence of EMT by MAPK/PI3K/Akt signaling pathway.In summary,TGF-β1 induces EpCAM N-glycosylation modification through MAPK and PI3K/Akt signaling pathway to regulate breast cancer cell EMT expression.Integrin,as an important adhesion molecule on the cell surface,participates in the adhesion process between cells and extracellular matrix,involves in transmembrane transduction of extra and intracellular.And then involves in cell biological specific regulation of division,adhesion,apoptosis,etc.Cells can also adjust the affinity of integrin and ligands based on their own status to affect adhesion and migration between the cell and extracellular matrix.The expression of Fibronectin(FN)and integrin β1 protein was down-regulated in the breast cancer cells(MCF-7,MDA-MB-231)for 48 hours.The results show that the protein expression of FN and integrinβ1 are reduced.Further activating the downstream FAK by integrin β1,reduces the protein expression of p FAK and p Scr.It was found that FAK could induce the activation of PI3K/Akt signaling pathway,and FAK specific inhibitor PF-573228 combined with EpCAM N-glycosylation mutation plasmid was used to treat breast cancer cells(MCF-7,MDA-MB-231)for 48 hours,then the results suggest that PI3K/Akt signaling pathway.p Akt,p GSK3β,β-catenin and MMP-2/MMP-9 protein expression decreased.Thus,EpCAM N-glycosylation in breast cancer cells inhibits the binding of integrin β1 with extracellular matrix molecules,adjusts MMP-2/MMP-9 expression by PI3K/Akt signaling pathway and transcription factor β-catenin to regulate expression of MMP-2/MMP-9,which reduces breast cancer cells adhesion of external matrix molecules.In summary,EpCAM N-glycosylation can regulate the occurrence of breast cancer cell EMT and extracellular matrix molecules adhesion.The mechanism of EpCAM N-glycosylation with the regulation of EMT in the development of breast cancer cells iselucidated,and which provides scientific basis for the selection of gene therapy regimen and development of gene therapy medicine,offers a solid theoretical basis for breast cancer.EpCAM as a target for the treatment of breast cancer,given a reliable theoretical basis.