Nicotine Induced Epithelial-mesenchymal Transition By O-GlcNAc Modification In Breast Cancer Cells

Breast cancer has occupied the highest incidence of female malignant tumors in the world,seriously affecting women's health.Studies have shown that smoking can increase women's risk of breast cancer by 10%-30%.As an important carcinogenic component in tobacco,nicotine can activate multiple intracellular signaling pathways through nicotine receptors on the cell surface,thereby inducing the invasion and metastasis of tumor cells,including the process of epithelial-mesenchymal transition(EMT)of tumor metastasis.Therefore,nicotine may promote the process of metastasis and malignant transformation in breast cancer.The EMT markers,such as E-cadherin,N-cadherin,and Vinment have been proved to be able to undergo O-linked N-acetylglucosamine(O-GlcNAc)modification,suggesting that OGlcNAcylation may be involved in nicotine Induced breast cancer EMT process.OGlcNAcylation is different from the classical protein glycosylation modification.It is a reversible monosaccharide modification mainly present in soluble cytoplasm and nuclear protein.Although it has been reported in the literature that O-GlcNAcylation is involved in the regulation of numerous physiological and pathological processes,the mechanism by which OGlcNAcylation regulates nicotine-induced breast cancer metastasis has not been elucidated.In this paper,with the help of Western blot,RT-qPCR,protein immunoprecipitation,chromatin immunoprecipitation(ChIP)and other experimental methods,taking breast cancer cells MCF-7,T47 D,MDA-MB-231 as research objects,we found that nicotine induce the upregulation of protein O-GlcNAc modification in breast cancer cells.The enhanced O-GlcNAc modification activates Snail protein and inhibits E-cadherin expression,thereby promoting nicotine-induced EMT process and enhancing cell migration ability.Further,this article demonstrates that nicotine increases the sugar donor uridine diphosphate Nacetylglucosamine(UDP-GlcNAc)by activating glutamine fructose hexaphosphate transaminase(GFAT),a key enzyme in the hexosamine synthesis pathway(HBP).Intracellular accumulation increases the level of O-GlcNAc modification of intracellular proteins.Through experiments such as reporter gene and ChIP,it was confirmed that the transcriptional regulatory complex CCAAT / enhancer binding protein ?(CEBPB)/ C / EBP homologous protein(CHOP)regulates GFAT transcription during this process.In addition,the transcription repressor CHOP can undergo O-GlcNAcylation under the action of nicotine,which dissociates the CEBPB / CHOP heterodimer,which in turn promotes the DNA binding activity of CEBPB,allowing CEBPB to bind to the promoter region of GFAT and Activate its transcription.The up-regulated GFAT can further activate the HBP pathway,forming positive feedback,enhancing nicotineinduced EMT activation process and breast cancer cell invasion ability.In addition,an o-glcnac glucosyltransferase inhibitor Amentoflavone(AF)was obtained through structure-based virtual screening of natural product compound libraries using drug Discovery studio 4.5.By inhibiting the enzyme activity of OGT,AF reduced the modification of protein O-GlcNAc.The results of molecular dynamics simulation show that AF can bind stably in the substrate binding pocket of OGT protein and form hydrogen bonds with several amino acids near the pocket.The results of intracellular activity test showed that AF could effectively inhibit the o-glcnac modification of the protein and had good OGT inhibitory activity.In summary,this article analyzes the changes in O-GlcNAcylation of breast cancer cells before and after nicotine stimulation,explains the relationship between O-GlcNAcylation and breast cancer metastasis,and obtains O-GlcNAcylation to regulate nicotine-induced breast cancer metastasis invasion Mechanism.In this paper,a new natural product O-GlcNAc inhibitor AF was screened by structure-based virtual screening method,and its O-GlcNAc modification inhibitor activity was further verified.The results of this paper provide new ideas for breast cancer research,as well as new small mole cule tools for the biological function of O-GlcNAc modification.