Differential Roles For STIM1 And STIM2 In TGF-?-induced EMT In Breast Cancer Cells

BackgroundBreast cancer is a major global health threat to women,and metastasis is the critical predictor of cancer-related death.Tumor metastasis follows migration and invasion of tumor cells into non-tumor tissues,and is dependent on epithelial-mesenchymal transitions(EMT),which contribute to phenotypic transformations of epithelial carcinoma cells into mesenchymal-like cells.EMT require complex changes in cell activity through a series of molecular events.Accordingly,numerous biomarkers have been associated with stages of EMT,and these include downregulation of the epithelial markers E-cadherin and Cytokeratin and upregulation of the mesenchymal markers N-cadherin and Vimentin,and the EMT-inducing transcription factors Snail1,Slug,and Twist.Transforming growth factor-?(TGF-p)is a master regulator of EMT,and mediates activation of type ? and type ? receptor complexes,leading to the phosphorylation of Smad2 and Smad3 and EMT are activated.Downregulation of E-cadherin expression in epithelial tumor cells is associated with TGF-?-induced EMT,whereas overexpression of Vimentin alters epithelial cell shapes and increases breast epithelial cell motility.Several studies show that EMT are influenced by Ca2+ influx,which generally follows store-operated calcium entry(SOCE)in breast cancer cells.Stromal interaction molecules(STIM)are important components of SOCE,and both STIM1 and STIM2 are integral type ? membrane proteins of the endoplasmic reticulum(ER).However,STIM1 has also been detected on plasma membranes.STIM2 and STIM1 have differing C-terminal fragments and STIM1 has been characterized as a Ca2+ sensor protein that responds to changes in ER Ca2+ concentrations,aggregates into puncta,and then redistributes to proximal ER-plasma membrane regions,leading to contact activation of Orail channels and SOCE.In addition to STIM1 and Orail,STIM2,Orai2,and Orai3 are expressed ubiquitously and may be regulated by luminal and cytoplasmic Ca2+ levels.Hence,STIM2 likely promotes SOCE following minor changes in ER,although the precise roles of STIM2 remain controversial.ObjectiveWe hypothesized the different roles of STIM1 and STIM2 in SOCE during TGF-?-induced EMT.We discovered that whereas STIM2 is involved in SOCE and ROCE,STIM1 participates only in SOCE during EMT.Taken together,the present data suggest that STIM1 and STIM2 proteins play important roles in TGF-?-induced EMT.Methods1.Kmplot tumor prognosis database was used to detect the survival curve of STIM1 and STIM2 in breast cancer patients.2.Using lentivirus to interference and overexpress STIM1 and STIM2 and then screening with puromycin.3.Western Blot was used to detect the protein content of interfering and overexpressing STIM1 and STIM2 group and observe the effect of interference and overexpression.4.MDA-MB-231 and MCF-7 breast cancer cells in different treatment groups after 24 hours of TGF-? stimulation was observed by Transwell methods.The changes of invasiveness and EMT were observed.5.The EMT markers E-cadherin,Vimentin and Snail1 were detected by RT-qPCR and immunoblotting.The EMT of STIM1 and STIM2 were observed in different treatment groups.6.The level of SOCE in different treatment groups was observed by patch clamp technique.7.Using Confocal to detect the level of SOCE and non-SOCE in different treatment groups.Results1.We found poor prognosis in breast cancer patients with high expression of STIM1 and STIM2.2.Immunoblotting showed the significant results of lentivirus interference and overexpression of STIM1 and STIM2.3.Transwell results showed that the invasion capability of interference with STIM1 and STIM2 was attenuated,and overexpression of STIM1 and STIM2 increased.4.Morphology,RT-qPCR method and immunoblotting results showed interfering with STIM1 and interfering with STIM2 could attenuate EMT,overexpressing STIM1 and STIM2 could enhance EMT.5.Patch clamps and Confocal results showed that interfere with STIM1 and STIM2 could decrease SOCE,overexpressing STIM1 and STIM2 could enhance SOCE.While the non-SOCE level of the interfering and overexpressed STIM1 group were not significantly changed,interfering of STIM2 could decrease non-SOCE,whereas the overexpression of STIM2 could enhanced non-SOCE.ConclusionIn the present study,we demonstrated roles of STIM proteins in TGF-?-induced EMT of breast cells.Our data distinguish between the roles of STIM1 and STIM2 in SOCE during TGF-?-induced EMT.