Correlation Between SMYD3 And AR In Breast Cancer And Mechanism Of SMYD3 On ER-negative Breast Cancer

ObjectiveThe aim of this study was to evaluate expression of SET and MYND domain-containing protein 3(SMYD3)and androgen receptor(AR)as well as their possible prognostic value in breast carcinoma patients and correlation of their expression with other known prognostic factors.And we tested the hypothesis that SMYD3 can activate AR and promote growth of ER-negative breast cancer.Method1.The expression of AR and SMYD3 was immunohistochemicaly analysed in 297primary breast cancer patients who underwent surgery at the Tianjin Medical University Cancer Hospital.SMYD3 and AR expression were correlated with other clinico-pathological parameters and based on the available clinical follow up data survival analysis were performed.2.The protein and mRNA expression of the SMYD3 and AR were analyzed using western blot and qRT-PCR,The cell localization of SMYD3 and AR were analyzed using immunofluorescence assays.3.The SMYD3 plasmid and SMYD3 siRNA plasmid was transfected into SK-BR-3cells and MDA-MB-231 cells,AR protein and mRNA levels were analyzed by Western blot and qRT-PCR.4.The SMYD3 plasmid and SMYD3 siRNA plasmid was transfected into SK-BR-3cells and MDA-MB-231 cells.The MTT were performed to evaluate the effect of SMYD3 on the proliferation of ER-negative breast cancer cells;Wound-healing assays and Transwell assays were performed to evaluate the effect of SMYD3 on the invasion and migration of ER-negative breast cancer cells;Clonogenic assays were conducted to evaluate the effect of SMYD3 on the Clonogenic of ER-negative breast cancer cells.5.Twenty nude mice were randomly divided into four groups.We injected2×10~6/100ul SMYD3 MDA-MB-231 cells,SMYD3 siRNA MDA-MB-231 cells and control MDA-MB-231 cells into subcutaneous of female nude mice,respectively.Tumors were measured every 4 days.After 32 days,the mice were sacrificed and the mouse tumors were removed for immunohistochemical and HE analysis.6.In order to study whether SMYD3 promotes ER-negative breast cancer progression by regulating AR,we analysed SMYD3 siRNA,simultaneous SMYD3 siRNA and AR-upregulated and control MDA-MB-231 cells.The MTT were performed to evaluate the proliferation of the above three groups of cells.The Wound-healing assays and Transwell assays were performed to evaluate the invasion and migration of the above three groups of cells.The Clonogenic assays were conducted to evaluate the Clonogenic of the above three groups of cells.Results1.66.7%(198/297)patients exhibited high expression of SMYD3,AR was positive in69.0%(205/297)of patients and 52.9%(157/297)of the patients show SMYD3 and AR co-expression.SMYD3 high expression was significantly associated with histologic grade(P=0.001),HER2 expression(P=0.021)and ki67 index(P<0.001).AR positive expression was show significantly associated with histologic grade(P=0.023),ER and PR expression(P<0.001)and HER2(P=0.038)expression.Spearman correlation showed that SMYD3 and AR were positively related(r=0.314,P<0.001).SMYD3 expression,SMYD3 and AR co-expression had a worse clinical prognosis,especially in the ER-negative patients.Multivariate cox model showed that SMYD3 and AR co-expression was independent predictors for Disease free survival(DFS)(P=0.001)and overall survival(OS)(P=0.005).2.We observed an enhanced SMYD3 protein and mRNA expression in ER-negative BC tissues,in comparison with their matched noncancerous tissue.The mRNA and protein expression level of SMYD3 or AR were higher in both MDA-MB-231 and SK-BR-3 cells.The results of immunofluorescence shown that both AR and SMYD3can be localized in cytoplasm and nucleus.3.SK-BR-3 cells and MDA-MB-231 cells were transfected with SMYD3 plasmid and SMYD3 siRNA plasmid,AR protein and mRNA levels were analyzed by Western blot and qRT-PCR,respectively.AR protein and mRNA levels increased with SMYD3 up-regulation in both SK-BR-3 cells and MDA-MB-231 cells,in contrast,AR protein and mRNA levels decrease with SMYD3 down-regulation in both SK-BR-3 cells and MDA-MB-231 cells.4.SMYD3 plasmid and SMYD3 siRNA plasmid were transfected into SK-BR-3 cells and MDA-MB-231 cells,Cells that overexpressing SMYD3 exhibited a higher rate of growth compared with control group,on the contrary,decreased expression ofSMYD3 reduced growth of SK-BR-3 cells and MDA-MB-231 cells;The results of wound-healing assays showed that,the distance of cell movement of SMYD3up-regulated group was far from that of control group,while SMYD3 down-regulated group was close to that of control group;The results of Transwell assays showed that,the number of cells in the SMYD3 up-regulation group invading through Matrigel in FBS containing medium was more than that in the control group,while decreased expression of SMYD3 reduced the cells invading through Matrigel in FBS containing medium;Clonogenic assays showed that the number of colonies formed by SMYD3up-regulation cells was more than that control cells,whereas the number of colonies formed by SMYD3 siRNA cells was less than that control cells.5.Mices injected with SMYD3 MDA-MB-231 cells exhibited signifcantly higher tumors growth compared to control cells,Mices injected with SMYD3 siRNA MDA-MB-231 cells exhibited signifcantly lower rates tumor growth compared to control cells.Mouse tumor immunohistochemistry results shown that SMYD3 siRNA group showed decreased expression of SMYD3,AR and Ki67 compared with the control group.SMYD3 up-regulation group showed increased expression of SMYD3,AR and Ki67 compared to the control group.Tumor liver metastases can be seen in the SMYD3 up-regulation group,but there were no metastasis in any group of lungs.6.SMYD3 siRNA decrease AR expression,however,simultaneous SMYD3 siRNA and AR-upregulated increase AR expression compared to SMYD3 siRNA alone.Results of the MTT assay showed that AR expression suppressed the decrease in proliferation caused by down-regulation of SMYD3.Wound-healing assays and Transwell assays showed AR expression suppressed the decrease in invasiveness caused by down-regulation of SMYD3.Clonogenic assays showed that AR overexpression suppressed SMYD3 downregulation-induced decrease in clonogenic ability of MDA-MB-231 cells.ConclusionSMYD3 and AR were expressed at a high frequency in breast cancer,expression of SMYD3 and co-expression of SMYD3 and AR might be used as poor prognostic factors in breast cancer patients,especially in ER-negative subgroup.By demonstrating the SMYD3-mediated oncogenic activity and AR expression in ER-negative breast cancer,we provide a potential avenue for controlling AR-mediated ER-negative breast cancer progression by inhibiting SMYD3expression or activity.