Non-muscle Myosin ⅡA(NMⅡA)Heavy Chain Phosphorylation Promotes Breast Cancer Metastasis

Objective:Breast cancer is the second leading cause of cancer-related deaths in women worldwide,and the incidence has continued to rise in recent years.Metastasis and recurrence are the main causes of death of breast cancer patients,which seriously endanger the lives and health of patients.Metastatic breast cancer is easy to recur and has a poor prognosis,with a survival period of only 8 months to 3 years.Therefore,exploring the key molecules related to metastasis is of great significance for improving the survival rate of patients with metastatic breast cancer.Non-muscle myosin ⅡA(Non-muscle myosin ⅡA,NMⅡA)is one of the actin skeletons of cells and is closely related to cell migration,contraction and cytokinesis.Recent studies have found that NMⅡA can promote the migration of cells(including cancer and non-cancer cells)and plays an important role in the metastasis of breast cancer,but its exact molecular mechanism is still unclear.The latest study found that the phosphorylation of the NMⅡA heavy chain S1943 is particularly critical for the invasion of cancer cells,but so far there is no literature report on the role of NMⅡA’s heavy chain phosphorylation in breast cancer migration and its mechanism.Therefore,this topic aims to study whether NMⅡA promotes breast cancer metastasis through heavy chain phosphorylation,and to explore its molecular mechanism.Methods:1.Immunohistochemistry method to determine the expression difference of NMⅡA in primary and metastatic breast cancer and adjacent tissues;Western blot and immunofluorescence methods to determine the expression difference of NMⅡA in breast cancer MCF-7,MDA-MB-231 and MCF-7/MX cells.2.Western blot and immunofluorescence methods were used to determine the differences in the expression of phosphorylated NMⅡA(p-NMⅡA)in MCF-7,MDA-MB-231 and MCF-7/MX cells;overexpression of NMⅡA wild in MCF-7/MX cells Type and heavy chain phosphorylation site mutant type,G418 screening polyclonal cell lines,select blank group(Control),negative control(Vector),NMⅡA wild-type overexpression group(NMⅡA-WT),heavy chain phosphorylation site The mutant NMⅡA overexpression group(NMⅡA-S1943A)was used for follow-up experiments;immunofluorescence and Western blot were used to identify the overexpression effect and efficiency;Transwell migration and invasion experiments were used to determine the migration and invasion capabilities of the cells in the above groups.3.The spheroidization experiment measures the spheroidizing ability of the cells in each group of Control,Vector,NMⅡA-WT and NMⅡA-S1943A;the clonogenic experiment measures the clonogenic ability of the above-mentioned groups of cells;Western blot measures the CSCs(Cancer stem cells)in each group of cells.4.Western blot was used to determine the expression differences of non-phosphorylated(active)β-catenin and β-catenin in the cells of Control,Vector,NMⅡA-WT and NMⅡA-S1943A;immunofluorescence method was used to determine the expression differences of active β-catenin.After treating MCF-7/MX with Wnt pathway inhibitor ICG-001(5,10 μM)for 1d,Western blot was used to determine the expression changes of Wnt pathway related proteins,NMⅡA,p-NMⅡA and CSCs markers.Results:1.NMⅡA is highly expressed in metastatic breast cancer and highly aggressive breast cancer cells.Compared with normal tissues adjacent to cancer,the expression of NMⅡA protein was up-regulated in primary breast cancer(p <0.001),and it was further up-regulated in metastatic breast cancer(p <0.001);compared with breast cancer MCF-7cells,NMⅡA protein was up-regulated in highly aggressive MDA-MB-231 cells(p <0.05),and down-regulated in MCF-7/MX cells(p <0.05).2.NMⅡA heavy chain phosphorylation promotes the migration and invasion of breast cancer cells.Compared with MCF-7 cells,p-NMⅡA protein in highly aggressive MDA-MB-231 cells was up-regulated(p <0.05),and p-NMⅡA protein in MCF-7/MX cells was down-regulated(p <0.05);and Compared with the Control group,the expression level of NMⅡA in the cells of the NMⅡA-WT group(p <0.01)and the NMⅡA-S1943 A group was up-regulated(p <0.001);compared with the Control group,the p-NMⅡA protein in the NMⅡA-WT group was up-regulated(p <0.05),the p-NMⅡA protein in the cells of the NMⅡA-S1943 A group was down-regulated(p <0.05);compared with the Control group,the migration(p <0.01)and invasion ability of the cells in the NMⅡA-WT group were significantly enhanced(p < 0.001),the migration and invasion ability of NMⅡA-S1943 A group cells were significantly weakened(p <0.001).3.NMⅡA heavy chain phosphorylation enhances the stemness of breast cancer cells.Compared with the Control group,the number of spheroidization and colony formation of cells in the NMⅡA-WT group increased(p <0.05),and the number of spheroidization(p<0.01)and colony formation of cells in the NMⅡA-S1943 A group decreased(p <0.05);Compared with the NMⅡA-WT group,the number of spheroiding and cloning formation of the NMⅡA-S1943 A group was significantly reduced(p <0.001);compared with the Control group,the CSCs markers(Sox2,Oct4,Klf4)expression was up-regulated(p<0.01);compared with the NMⅡA-WT group,the expression of CSCs markers in the NMⅡA-S1943 A group was down-regulated(p <0.01,p <0.05,p <0.001).4.NMⅡA heavy chain phosphorylation forms a feedback loop with the Wnt/β-catenin pathway.Compared with the Control group,the expression of active β-catenin and the downstream proliferation protein Survivin of the Wnt pathway in the NMⅡA-WT group was up-regulated(p <0.05);compared with the NMⅡA-WT group,the active β-catenin of the NMⅡA-S1943 A group was(p <0.01),Survivin(p <0.01)and c-Myc protein expression were significantly reduced(p <0.001);Wnt pathway inhibitor ICG-001(5,10 μM)significantly inhibited the expression of active β-catenin,β-catenin,p-NMⅡA,NMⅡA and CSCs markers(p <0.05;p <0.001).Conclusion:1.NMⅡA promotes breast cancer metastasis.2.NMⅡA heavy chain phosphorylation promotes breast cancer metastasis.3.NMⅡA heavy chain phosphorylation and Wnt/β-catenin pathway form a feedback loop to enhance breast cancer cell stemness and promote metastasis.