Low Shear Stress-induced Breast Cancer Cell Migration Is Mediated By Caveolin-1-dependent Integrin Trafficking

In the process of metastasis,breast cancer cells are constantly subjected to shear stress imposed by blood flow.Studies have shown that low shear stress(LSS)can affect cancer cell adhesion,migration,proliferation and anoikis.Integrin trafficking plays a crucial role in focal adhesions(FAs)turnover and cellular motility.Integrins undergo constant endo/exocytic shuttling to facilitate the dynamic regulation of FAs.However,the effect and the biological consequences of LSS on integrin trafficking remained incompletely understood.Here,we identified caveolin-1-dependentβ1 integrin trafficking as a critical role in low shear stress-induced cell migration.By using immunofluorescence staining,flow cytometry and Western blot,we observed that LSS(1.8 dyn/cm~2)altered the distribution ofβ1 integrin in MDA-MB-231cells but not the expression.Capture-ELISA analysis of biotin-labelled cells revealed that the levels ofβ1 integrin internalization and recycling were improved after LSS exposure.Results from immunofluorescence staining showed that LSS promotedβ1integrin internalization by caveolin-1-dependent route.To further investigate the role of caveolin-1-dependentβ1 integrin internalization in LSS-induced cell migration,time-lapse microscopy was performed examining the FA dynamics of EGFP-vinculin-transfected 231 cells.It was found that LSS could promote FA disassembly and induce caveolin-1(Cav-1)accumulate at FAs with activeβ1 integrin.However,after using Nystatin to block Cav-1-dependent internalization,LSS-induced cell migration and FA disassembly were inhibited.Further studies found that LSS induced cytoskeleton remodeling and down-regulated the acetylation level of microtubule(MT).LSS-induced cytoskeleton remodeling was required for caveolin-1-dependentβ1 integrin internalization.The results of live cell imaging and FRAP experiments showed that LSS-induced tubulin deacetylation improved the MT dynamics and Cav-1 motility.Capture-ELISA analysis showed that LSS-induced tubulin deacetylation promoted caveolin-1-dependentβ1 integrin recycling.By using HDAC6activation and acetyltransferase assays,we also determined that LSS promoted MT deacetylation through activation of HDAC6.Furthermore,ROCK inhibitor Y-27632pretreatment could suppress LSS-induced deacetylation of MT.Using the tumor genomic microarray databases to analyze the survival rate of clinical patients,we found that the high expression of ROCK and HDAC6 contributed to the poor prognosis of breast cancer patients.In summary,these results defined a novel mechanism by which LSS improved Cav-1-dependentβ1 integrin trafficking via cytoskeleton remodeling and ROCK/HDAC6 mediated deacetylation of MT,thereby promoting FAs turnover and cell migration.