Role Of Calpain-YAP Signaling Pathway In The Epithelial To Mesenchymal Transition Of Triple Negative Breast Cancer Cells

Background:Triple negative breast cancer(TNBC)is characterized by a lack of alpha-estrogen receptor,progesterone receptor and HER2 receptor expression,high malignancy,easy metastasis and poor clinical prognosis,and there is no effective targeted therapy.Objective:Triple negative breast cancer(TNBC)is characterized by a lack of expression of alpha-estrogen,progesterone and HER2 receptors.Estrogen(E2)and epithelial growth factor(EGF)are involved in promoting the malignant progression of triple negative breast cancer,but the signal mechanism is not fully clear.The aim of this study was to investigate the effects of E2 and EGF on epithelial-mesenchymal transition(EMT)and the mediation of calmodulin-YAP signaling pathway in triple-negative breast cancer cells MDA-MB-231(231)and MDA-MB-468(468),so as to provide experimental basis for exploring therapeutic targets of breast cancer.Methods:Human triple negative breast cancer(TNBC)cells 231 and 468 were used as cell and molecular experimental model cells and mouse triple negative breast cancer cells 4T1 were used as animal experimental model cells.Cells were cultured routinely and treated with E2/EGF(solvent as control)stimulation.Cells were pretreated with YAP specific inhibitors XMU-MP-1(XMU)and Radicical(RAD)when needed,interfering with intracellular YAP activity or calcium.Protease inhibitors Calpeptin(Cal)and Calpain Inhibitor Ⅲ(CⅠ Ⅲ)pretreated cells;MTT test and plate cloning test were used to investigate cell viability/proliferation;V-FITC/PI fluorescent apoptosis kit was used to detect cell apoptosis;Western blotting test was used to detect the target protein table.The tumorigenicity of the cells in nude mice was detected by subcutaneous tumor-bearing experiment,and the histopathological changes of the tumors were observed by HE staining of pathological sections.Results:(1)E2(10 μM)and EGF(10 μg/L)stimulated the model cells for 24 hours,significantly increased the proliferation of 231 cells by 56.95%,36.47%(*P<0.05),468 cells by 33.86%,31.57%(*P<0.05),231 cells by 106%,328.0%(*P<0.05),468 cells by 234.4%,139.8%(*P<0.05),231 and 468 cells by 139.8%(*P<0.05),and significantly reduced apoptotic cells in the middle and late stages.EMT phenotype of breast cancer 231 cells was changed.The expression of fibronectin(FN)and vimentin(Vim)was up-regulated by 67.65%,44.85%(*P<0.05)and 63.92%,81.27%(*P<0.05),while the expression of E-cadherin(E-Cad)was down-regulated by 68.63%,53.74%(**P<0.01).(2)Cal(10 μM)pretreatment for 2 hours significantly inhibited E2 or EGF-induced cell proliferation.Cal(10 μM)pretreatment decreased the proliferation rate of 231 cells by 30.38%,27.83%(*P<0.05),and C Ⅲ(10 μM)pretreatment decreased the proliferation rate of 231 cells by 40.88%,41.62%(*P<0.05);Cal pretreatment decreased the proliferation rate of 468 cells by 33.22%,30.74%(*P<0.05),and CⅠ Ⅲ pretreatment 468 cells by 33.22%,30.74%(*P<0.05).The rate of colony formation decreased by 38.16%and 34.93%(*P<0.05);the rate of cell colony formation decreased significantly.The rate of colony formation of Cal pretreatment 231 cells decreased by 43.75%,29.31%(*P<0.05),CⅠⅢ pretreatment 231 cells decreased by 68.97%(**P<0.01),42.62%(*P<0.05);the rate of colony formation of Cal pretreatment 468 cells decreased by 32.29%,32.74%(*P<0.05),and Ⅲ pretreatment 468 cells decreased by 68.97%(*P<0.05).The rate decreased by 32.88%and 45.93%(*P<0.05);the expression of FN was significantly down-regulated in EMT,42.36%(*P<0.05),69.42%(**P<0.01),Vim was down-regulated by 65.43%(**P<0.01),55.39%(*P<0.05),and E-Cad was up-regulated by 100.1%(**P<0.01),69.24%(*P<0.05).(3)Cal pretreated model cells significantly decreased the expression of phosphorylated LATS1(p-LATS1)by 51.24%,85.79%(*P<0.05)and phosphorylated YAP(p-YAP)by 94.36%,87.58%(*<0.05)in E2 or EGF-induced 231 cells,and decreased the expression of p-LATS1 by 61.34%,83.49%(*P<0.05)and p-YAP by 66.47%(**P<0.01),59.28%(*P<0.05)in CⅠ Ⅲ pretreated model cells.Promote intranuclear metastasis of YAP.(4)XMU(10 μM)pretreated model cells for 3 hours,significantly inhibited the proliferation of E2 or EGF-induced 231 cells by 40.62%(*P<0.05),41.59%(**P<0.01)and 468 cells by 63.84%(**P<0.01),41.70%(*P<0.05),RAD(10μM)pretreated model cells by 2 hours,and 231 cells by 62.63%(**P<0.01),37.69%(*P<0.05)and 468 cells by 4.The rate of cell colony formation decreased by 61.01%and 36.03%(**P<0.01);the rate of cell colony formation decreased significantly,XMU-induced cell colony formation decreased by 26.45%(*P<0.05),24.63%(**P<0.01)and 468 cell colony formation decreased by 59.83%(*P<0.05),20.42%(**P<0.01);XMU and RAD pretreated cells significantly increased apoptotic cells in the middle and late stages;inhibited cell invasion and EMT phenotype changes,and FN surface.The expressions of Vim and E-Cad were down-regulated by 77.47%(*P<0.05),73.16%(**P<0.01)and 89.36%(**P<0.01),81.48%(**P<0.01),while the expressions of E-Cad were up-regulated by 26.72%and 68.63%(*P<0.05).(5)Intraperitoneal injection of CⅠ Ⅲ(1 mg/kg)significantly inhibited the growth of transplanted 4T1 cells in nude mice.The weight of nude mice decreased by 1.75 times(*P<0.05),the weight of tumors decreased by 3.42 times(*P<0.05),and the volume decreased by 3.48 times(*P<0.05).Conclusion:E2 and EGF promote the proliferation of TNBC cells,enhance their anti-apoptotic ability and induce EMT phenotype.CANP-YAP signaling pathway may be involved in mediating the changes of EMT phenotype and malignant biological behavior induced by E2 and EGF.