N-glycosylation Modification Of EpCAM Mediates The Regulation Of Stemness And EMT Of Breast Cancer Cells Under Hypoxic Condition

Objective:Breast cancer is one of the most common cancers in Chinese women,and its incidence is the highest among all female cancers.Global cancer statistics report that the annual incidence of breast cancer in cancer diagnosis accounts for 23%,accounting for14%of global cancer mortality.The main treatment of breast cancer is surgery,radiotherapy and chemotherapy,but these methods have shortcomings and it is difficult to achieve satisfactory results.Therefore,it is significant to find effective methods for clinical treatment of breast cancer.Epithelial Cell Adhesion Molecule（EpCAM）is a transmembrane glycoprotein,which molecular weight is about 40 kd,potentially carcinogenic.In normal conditions,EpCAM is expressed in different degrees in normal epithelial cells except for squamous epithelial cells.Under pathological conditions,EpCAM is highly expressed in many adenocarcinomas,which consist of pancreatic cancer,breast cancer,ovarian cancer,and lung adenocarcinom.EpCAM plays an important role in cell adhesion,stemness,cell signaling,migration,proliferation and differentiation.Studies have shown that EpCAM promotes the growth and metastasis of breast cancer cells.Protein glycosylation is a common and important type of protein posttranslational modification.It refers to the process of forming glycosidic bonds by the protein polypeptide chain and glycan under the action of glycosyltransferase.According to different glycosidic bond binding sites,protein glycosylation is mainly divided into N-glycosylation and O-glycosylation.The primary structure of EpCAM shows that Ep CAM has three N-glycosylation sites（N74,N111,N198）.It is reported that the removal of these three glycosylation sites can reduce the half-life of EpCAM from 21 hours to 7 hours,indicating that the N-glycosylation site is essential for maintaining the function of EpCAM.Previous study found that N-glycosylation mutations of EpCAM can promote breast cancer apoptosis,and enhancing 5-FU drug sensitivity.In addition,the study also found that N-glycosylation of EpCAM mediates EMT of breast cancer.The previous experimental results proved that the N-glycosylation modification of EpCAM play an important role in the development of breast cancer cells,and it also provides a solid theoretical basis for the use of EpCAM as a target for the treatment of breast cancer.Recent studies have found that cancer stem cells is an additional mark of EpCAM,with high tumorigenicity and drug resistance,many malignant tumors contain the cancer stem cell,as a result,this also makes EpCAM tumor targets for the treatment of a more meaningful.Cancer stem cells（CSCs）,a small subpopulation of cancer cells,which possess self-renewal and multilineage differentiation potential.In breast cancer cells,a small subpopulation of cells,which are designated as tumor-initiating cells or breast cancer stem cells（BCSCs）,have the unique property of generating daughter BCSCs,which are capable of infinite proliferation through self-renewal,and transient amplifying cells,which are capable of a limited number of cell divisions and give rise to differentiated breast cancer cells.Only BCSCs are capable of forming a secondary（recurrent or metastatic）tumor.Breast cancer stem cell phenotypes are regulated by the expression of stem pluripotency factors,including OCT4,SOX2 and NANOG.In order to better understand the transfer and design of more effective treatment methods,the molecular mechanisms regulating breast cancer stem cell phenotype need to be described.EpCAM is one of the specific cells of stem cell properties,expression of EpCAM associated with tumor stem cell pluripotency in breast cancer,breast cancer stem cells has the ability of self-renewal and the characteristics of invasive,it is very important to the transfer of early steps.Group pilot study identified N-glycosylation modification of EpCAM in the development of the occurrence of breast cancer cells play an important role,so we speculated that N-glycosylation modification of EpCAM may regulates stemness of breast cancer cells.Recent studies have shown that HIF is required for the specification and/or maintenance of BCSCs under hypoxia conditions.In addition,hypoxic-induced EMT requires HIF-1α,which is also associated with the BCSC phenotype.High level expression of HIF-1 is a predictor of early diagnosis,recurrence and metastasis of breast cancer,and can lead to adverse clinical outcomes in human breast cancer.Breast cancer cells under hypoxic condition,HIF activates the transcription of target genes,which play an important role in regulating metabolism,tumor growth,angiogenesis,restructuring,motility,invasion,metastasis and chemotherapy drug resistance.Epithelial mesenchymal transformation（EMT）is a process associated with cancer cells with stem cell characteristics.In cancer,EMT is related to the progress of the tumor to the metastatic phenotype,the key characterized of this process is lossing of epithelial cell polarity,lossing contact cells,obtaining the mesenchymal mark and increasing the cell vitality and access.EMT promotes the movement and invasion of tumor cells and may lead to therapeutic resistance.In malignant tumors,EMT is associated with tumor progression,resulting in more movement and invasive cell subgroups,resulting in more metastatic phenotypes.Studies have shown that N-glycosylation of EpCAM plays an important role in the development of breast cancer.Expression of EpCAM related to cancer stem cell pluripotency,and hypoxia is of great importance for breast cancer stem cell phenotype.Therefore,we explored the regulation role of N-glycosylation of EpCAM on the stemness and EMT of breast cancer cells under hypoxic microenvironment,and we try to find a more effective method for the treatment of breast cancer.Although advances has been made in the treatment of early breast cancer,there is still no effective strategy to prevent or treat metastasis,and metastasis is the major cause of death in breast cancer.Therefore,finding an effective treatment for breast cancer has certain clinical significance.Hypoxia is an important feature of tumor microenvironment.Under hypoxic conditions,tumors have the characteristics of stem cells and stronger invasiveness,increasing the risk of metastasis,and enhancing the resistance to radiotherapy and chemotherapy.Therefore,we studied the characteristics of breast cancer stem cells and the occurrence of EMT under hypoxic microenvironment.First of all,we constructed the hypoxic model of breast cancer to analyze the regulation mechanism of hypoxia on breast cancer stem cells and EMT.At the same time,we also discussed the effect of N-glycosylation of EpCAM on the stemness and EMT of breast cancer cells under hypoxic conditions.Methods:1.Apply CoCl₂ as a chemical hypoxia inducer to construct a hypoxic model of breast cancer cells in vitro;2.The expression of OCT4,SOX2 and NANOG were detected by western blot and immunofluorescence assays;3.By western blot and immunofluorescence experiments,the expression of OCT4,SOX2 and NANOG was detected under hypoxia;4.The effect of hypoxia on the stem cell characteristics of breast cancer was detected by the spheroid formation assay;5.The regulation role of N-glycosylation of EpCAM on the stemness and EMT of breast cancer cells under hypoxic was detected by western blot and immunofluorescence assay;6.The effect of N-glycosylation modification of EpCAM on the regulation of EMT and stemness of the NF-κB signaling pathway was detected by western blot method.Results:1.CoCl₂ induce hypoxic model of breast cancer in vitro;2.EpCAM promotes breast cancer stem cell characteristics;EpCAM increases the expression of HIF-1αand stem cell marker molecules in breast cancer cells;EpCAM enhances the spheroid formation ability of breast cancer cells MCF-7 and MDA-MB-231;3.Hypoxia promotes breast cancer stem cell characteristics;Hypoxia enhances the spheroid formation ability of breast cancer cells MCF-7 and MDA-MB-231;N-glycosylation modification of EpCAM mediates the stemness of breast cancer cells;4.N-glycosylation modification of EpCAM regulates EMT in breast cancer cells under hypoxic condition;5.Hypoxia and N-glycosylation modification of EpCAM mediate nuclear translocation of NF-κB;6.N-glycosylation modification of EpCAM mediates the stemness and EMT through the NF-κB signaling pathway induced by hypoxia.Conclusion:1.Hypoxia induces breast cancer cells MCF-7 and MDA-MB-231 stemness enhancement,and promotes the occurrence of EMT by regulating the expression of E-cadherin,N-cadherin,Vimentin,MMP-2 and MMP-9;2.N-glycosylation modification of EpCAM interfere with the expression of the stem cell marker molecules and prevents the occurrence of EMT through hypoxia-induced NF-κB signaling pathway.