Metformin Inhibits The Metastasis Of Breast Cancer By Regulating MRTF-A Expression

Breast cancer is a common female malignancy.Metformin is a first-line treatment for type 2 diabetes.In recent years,the anti-tumor effect of metformin has attracted wide attention.However,the molecular mechanism of metformin inhibiting breast cancer metastasis is still unclear.Metformin activates AMPK,a protein kinase involved in cellular energy metabolism.MRTF-A is a transcription cofactor that binds to the CArG site of the target gene promoter to promote the expression of the target gene,which in turn is regulated by β-catenin.In cancer cells,the β-catenin/MRTF-A signaling pathway can enhance the expression of genes related to tumor metastasis and promote the migration of breast cancer cells.This project explored the molecular mechanism by which metformin inhibiting breast cancer metastasis,and clarified the role of β-catenin/MRTF-A in it.First,metformin was used to treat MCF-7 and MDA-MB-231 in breast cancer cells to detect the effect of metformin on breast cancer cell migration.The results of cell scratch test and Transwell test showed that 2.5 mM metformin significantly inhibited the migration of breast cancer cells.Then,the effect of metformin on the expression of migration-related genes was detected by Western blot and RT-Real-time PCR.The results showed that the expression of multiple migration genes such as MYL9 and COLAlwas inhibited,and the expression level of MRTF-A,which regulated the above genes,also decreased.Luciferase reporting experiments were carried out using wild-type and CArG site mutant promoter activity reporter plasmids of the above transportation-related genes to detect the effect of metformin on transportation-related gene promoter activity.The results showed that metformin inhibited the activity of wild-type promoters,while there was no significant inhibitory effect on the corresponding CArG mutant promoters,suggesting that the inhibition of metformin on transport-related gene expression was related to MRTF-A.To verify this hypothesis,breast cancer cells were transfected with the MRTF-A expression plasmids,the results showed that metformin no longer inhibited the expression of migration-related genes,which further proved that metformin inhibited the expression of migration genes in breast cancer cells by inhibiting MRTF-A.In order to explore the molecular mechanism of metformin regulating MRTF-A,this study examined the effect of metformin on AMPK activity and the expression of β-catenin.Results showed that metformin activated AMPK and inhibited the expression of β-catenin.On the contrary,when cells were treated with AMPK inhibitor compound C or siAMPK was used to treat the cells,and the results showed that both the expression of β-catenin and MRTF-A were up-regulated.Knockdown AMPK and catenin genes with specific siRNAs resulted in the decrease of MRTF-A expression.Overexpression of β-catenin in metformin treated cells resulted in no inhibition of MRTF-A expression.Taken together,the above results demonstrated that metformin indirectly inhibited the expression of MRTF-A by activating AMPK which in turn inhibits β-catenin.Transforming growth factor TGF-β is highly expressed in breast cancer,activating the β-catenin/MRTF-A signaling pathway and promoting breast cancer metastasis.The results of this study showed that metformin can weaken the effect of TGF-β on the activation of the β-catenin/MRTF-A pathway.At the cellular level,metformin inhibited TGF-β induced migration of breast cancer cells.At the animal level,the models of xenotransplant breast cancer and lung metastasis were established with nude mice.The body weight and blood glucose levels of nude mice in the metformin group and the control group did not change significantly after continuous administration of metformin for 38 days.Western blot results showed that in the xenotransplant carcinoma tissues,the expression of metastasis-related genes decreased significantly after metformin treatment.Pathological examination showed that in the model of lung metastatic carcinoma,there were significantly fewer metastatic nodules in the lung tissue of nude mice treated with metformin.Metformin was herein demonstrated to inhibit the β-catenin/MRTF-A signaling pathway to reduce lung metastasis of breast cancer cells.In summary,this study for the first time reveals that metformin inhibits breast cancer metastasis by activating AMPK and consequently inhibiting the β-catenin/MRTF-A signaling pathway.This mechanism was verified at molecular,cellular and animal levels.