The Mechanobiological Mechanisms Of Caveolin-1 In Low Shear Stress-induced Breast Carcinoma Cell Adhesion And Migration

Low shear stress(LSS)plays a critical role in the regulation of various aspects of tumor cells functions.Cav-1 represents a modulator of several cancer-associated functions as tumor progression and metastasis.However,the role of Cav-1 in regulating tumor cells response to shear stress remains poorly explored.To investigated the functional role of Cav-1 in promoting cell migration and adhesion following LSS.We used the parallel-plate flow chamber to mimic the LSS that affects tumor cells.Following the exposure to LSS,cells showed an elongated shape and the spread area was increased comparing with the control.Besides,LSS induced cell polarity establishment and more focal adhesion formation to promote cell migration.By using the tumor genomic microarray database R2 platform,we observed that higher levels of Cav-1 mRNA were strongly correlated with poor survival probability in patients and western blot analysis performed further revealed a higher motility of cells corresponding to higher expression of Cav-1 in a certain extent.Fluorescence analysis of TRITC-phalloidin-labeled cells revealed that shCtrl-transfected MDA-MB-231 cells showed considerably more stress fibers traversing the cytoplasm than shCav-1 transfected cells.To further investigate the role of Cav-1 and LSS in cell motility,time-lapse microscopy was performed examining the migration of both shCtrl-and shCav-1-transfected 231 cells.Our results indicated that both two cell types were able to form protrusion and shCtrl-transfected cells showed a markedly increased motility as the morphology quickly changes forming in the front edge of migrating cells the typical fan-shaped lamellipodium.The results of WB indicated that LSS exposure caused a decrease in cofilin and p-MLC levels,while increasing filaminA expression in Cav-1-depleted cells.To investigate the role of LSS in cell adhesion,we analyzed the cell attachment to the extracellular matrix(ECM)of both Cav-1-depleted and control cells after LSS treatment.Binding of shCav-1-transfected cells to collagen I and Matrigel was decreased by 40% and 38%,respectively,in comparison with shCtrl-transfected cells.Also under shear stress exposure,silencing Cav-1 leaded to a decrease in the expression of p-Src(Tyr416),along with Src,FAK,and p-FAK(Tyr397).The results of FRAP showed that FAs were more stable in the shCav-1-transfected cells,suggesting that Cav-1 can regulate cell migration at least in part by promoting FAs dynamics.Finally,the analysis of the distribution of cell cycle showed that shCtrl-and shCav-1-transfected MDA-MB-231 cells did not show significant differences.Our findings provide insights into mechanisms underlying LSS triggered events mediated by downstream Cav-1,including FAK/Src and ROCK/p-MLC pathways,involved in the reorganization of the cytoskeleton,cell motility,FA dynamics and breast cancer cell adhesion.