EGR1 Inhibits Breast Cancer Metastasis Via Suppressing Epithelial-mesenchymal Transformation

Purpose To explore the effect of EGR1 on epithelial-mesenchymal transformation of breast cancer and the possible mechanism of EGR1 on distant metastasis of breast cancer.MethodsBioinformatics was used to analyze the expression of EGR1 in breast cancer and normal breast tissues,and its correlation with survival.The basic expression of EGR1 was detected by q RT-PCR,and a stable cell line overexpressing EGR1 was constructed.The effects of overexpression of EGR1 on the migration,invasion and EMT phenotype of breast cancer were studied in vitro.The changes of migration and invasion of breast cancer were observed by transwell assay,and the expression of EMT-related molecules was detected by western blot and immunofluorescence experiments.The role of NF κB/p65 signal pathway in the regulation of EMT by EGR1 was further discussed.Through the prediction of Jaspar website,we found that there were binding sites between EGR1 and the promoter region of CD47 molecule.The binding relationship between EGR1 and CD47 promoter region was detected by Ch IP experiment,and the regulation of CD47 molecule by EGR1 and the effect of CD47 molecule on EMT phenotype of breast cancer were detected by western blot experiment.Tumor cells were co-cultured with macrophages differentiated from THP1 and bone marrow-derived macrophages respectively.The effect of EGR1 on phagocytosis of tumor cells by macrophages was detected by flow cytometry.Finally,the regulatory effect of small molecular compound disulfiram on EGR1 in breast cancer was detected by Western blot experiment.In vivo,the negative control cells and cells with stable and high expression of EGR1 were used to construct orthotactic breast cancer graft model and metastasis model to observe the effect of EGR1 on tumor growth and metastasis.For the orthotopic transplantation breast cancer model,we compared the tumor size of the two groups of mice,detected the changes of EGR1,CD47,EMT and NFκB pathway related molecules in the tumor tissue of the two groups by immunohistochemistry.For metastasis model,lung metastasis and liver metastasis in two groups of mice were analyzed.ResultsThrough the analysis of TCGA and GEO database,it was found that the expression of EGR1 in breast cancer tissue was significantly lower than that in normal breast tissue,and the expression of EGR1 was significantly related to the survival of breast cancer patients.The survival of patients with high expression of EGR1 was significantly better than that of patients with low expression of EGR1.The basic expression of EGR1 in normal breast cells MCF10 A and breast cancer cell lines MCF-7,SKBR3,T47 D,MDA-MB-231 and BT549 was detected by q RT-PCR assay.The results showed that EGR1 was highly expressed in MCF10 A,but lower in MCF-7,SKBR3,T47 D,MDAMB-231 and BT549.We selected MDA-MB-231 and BT549 to construct cell lines with stable and high expression of EGR1.Transwell assay showed that EGR1 overexpression weakened the migration and invasion ability of breast cancer cells;Western blot results showed that EGR1 overexpression upregulated epithelial marker E-cadherin,down-regulated mesenchymal markers N-cadherin and Vimentin,and reversed the phenotype of EMT.Further detection of the expression of molecules related to NF κ B/p65 pathway showed that EGR1 could down-regulate NF κ B/p65 signal pathway.TNFα is the activator of NF κ B pathway.EGR1 can inhibit the promoting effect of TNFα on p65 nuclear translocation and further inhibit the EMT phenotype after NF κ B activation.EGR1 can down-regulate the expression of immune checkpoint CD47 and knock down the expression of CD47 can reverse the phenotype of EMT.Bioinformatics analysis showed that EGR1 might bind to the promoter region of CD47 molecule.Ch IP-q PCR assay confirmed their binding.EGR1 regulated the expression of CD47 at the transcriptional initiation level.The co-culture of tumor cells and macrophages confirmed that the overexpression of EGR1 promoted the phagocytosis of macrophages.In addition,Western blot detection showed that the small molecular compound disulfiram could significantly up-regulate the expression of EGR1 in vitro.The in vivo experiment showed that overexpression of EGR1 significantly inhibited the growth of tumor cells.Immunohistochemical results showed that overexpression of EGR1 upregulated epithelial marker E-cadherin,down-regulated mesenchymal marker N-cadherin,and down-regulated p65,CD47 and ki67.The metastasis model showed that compared with NC group,the high expression group of EGR1 significantly reduced the rate of lung and liver metastasis of breast cancer.ConclusionsEGR1 is associated with good prognosis of breast cancer,and EGR1 can inhibit distant metastasis of breast cancer via affecting the progression of EMT.EGR1 can inhibit the progression of EMT in breast cancer by inhibiting the NFκ B/p65 pathway,and directly transcriptionally inhibit CD47 molecules.EGR1 has the great potential to regulate phagocytosis of macrophage in immune microenvironment.The small molecule disulfiram may play a tumor suppressive role by up-regulating the expression of EGR1 in breast cancer cells,which provides a new insight for the treatment of breast cancer.