The Mechanism Of Bisecting GlcNAc Modification In Regulating Breast Cancer Progress And Chemoresistance

Breast cancer(BC),the most frequent malignancy among women worldwide,is highly heterogeneous.As the major component of the tumor microenvironment,small extracellular vesicles(sEV)secreted by BC cells can transfer their cargos to recipient cells,and further facilitate distant metastasis,malignancy progression and chemoresistance of BC.Aberrant level of bisecting GlcNAc structures have been found to closely associate with BC progression.This study mainly focuses on the mechanism underlying regulation of migratory and chemoresistance mediated by bisecting GlcNAc,and reveals the essential role of bisecting GlcNAc structure in BC progression and chemoresistance.To explore the biological function of bisecting GlcNAc and its synthase MGAT3,bisecting GlcNAc levels was elevated by transfection of MGAT3 gene or treatment with Forskolin.Elevated bisecting GlcNAc levels suppressed the proliferation,clone formation and migration ability of BC cells,and increased cell apoptosis.Furthermore,metastasis to lung of BC cells in mice was significantly suppressed by introduction of bisecting GlcNAc.Collectively,our study demonstrated that bisecting GlcNAc modification suppressed malignant phenotypes of BC cells,especially migratory ability.In the following study,we found that high-metastatic BC cell derived sEV could enhance migratory ability of low-metastatic recipient cells,and bisecting GlcNAc modification diminished the pro-metastatic function of sEV.Integrin ?1 was identified to be the target glycoprotein bearing bisecting GlcNAc by glycoproteomic analysis and coimmunoprecipitaion.Integrin ?1 was detected to be presented on sEV and transferred to MCF7 cells by sEV interlization.Low levels of bisecting GlcNAc facilitated the binding of vesicular integrin ?1 to galectin 3,activated FAK/AKT signaling and increased in migratory ability of recipient cells.However,high levels of bisecting GlcNAc levels weakened the binding of vesicular ?1 to galectin-3,and suppressed the activation of FAK/AKT signaling and the prometastatic function of sEV.BC patient serum derived sEV were designated as “high-sEV” or“low-sEV” on the basis of high vs.low bisecting GlcNAc levels.Migratory ability was greater in low-sEV-treated treated MCF7 than in high-sEV-treated MCF7.Incidence,numbers and areas of lung metastasis nodules was significantly enhanced by BC cells derived sEV,but not by sEV with higher level of bisecting GlcNAc.In summary,our study illustrated that the prometastatic function of sEV was regulated by the bisecting GlcNAc modification,which was mediated by vesicular-integrin ?1.Meanwhile,bisecting GlcNAc levels of integrin ?1,which was calculated by dividing mRNA expression of MGAT3 by which of integrin ?1,was closely correlated with overall survival of BC patients using TCGA database.Finally,effects of bisecting GlcNAc modification on the chemoresistance of BC cells was investigated.We found that chemotherapeutic treatment resulted in the decreased levels of MGAT3 and bisecting GlcNAc in BC cells,which was resulted from accelerated degradation of ubiquitylated MGAT3 through proteasome pathway.BC cells with high bisecting GlcNAc levels were more sensitive to chemotherapeutics.The efflux of chemotherapy drugs of MGAT3-transfected drug-resistant cells(termed 7/Adr-M3)was slower than which of parental cells.Using glycoproteomic analysis,p-glycoprotein(P-gp)was identified as target glycoprotein modified with bisecting GlcNAc.High bisecting GlcNAc levels on P-gp induced translocation into the cytoplasm and degradation,resulting in the decreased P-gp expression and cell chemoresistance.These findings indicated that the localization and expression levels of P-gp were determined by its bisecting GlcNAc modification,which was closely correlated to the drug resistance.In conclusion,the pro-metastatic function of sEV was diminished by high bisecting GlcNAc modification on vesicular integrin ?1.High levels of bisecting GlcNAc on P-gp suppressed the chemoresistance of BC cells.Our findings may provide an essential basis for diagnosis and treatment of BC.