Role And Mechanism For Ezrin In Breast Cancer Cells Chemotaxis To CCL5

BACKGROUND:Breast cancer is one of the most common female cancers in the world.The poor prognosis of patients with breast cancer is related to tumor metastasis.Metastasis,as a result of several sequential steps,represents a highly organized and organ-selectiveprocess.Tumor cell migration and metastasis share many similarities with the specifichoming of various subsets of hematopoietic cells to specific sites,which is criticallyregulated by chemokines and their receptors.This ligand CCL5 is expressed at high levelsin some tissues (lung,liver,bone marrow,and lymph nodes) that frequently harbor breastcancer metastasis.Expression of the receptors for CCL5 (CCR5) by breast cancer cells isthought to correlate with the progression of the tumor.CCL5 was shown to induce breastcancer cell migration,mediated by the receptors on the breast cancer cells.Ezrin,a memberof the ERM (Ezrin-radixin-moesin) cytoskeleton-associated protein family,is originallythought to be simple cross linkers between membrane proteins and actin filaments.Increasing evidence indicates that Ezrin is regarded as a metastatic determinant and a keycomponent in tumor metastasis.The objective of the present study was to demonstratechemotaxis and invasion of breast cancer cells can be facilitated by CCL5 expression,and apotential role for Ezrin in the process.METHODS:1.Human breast carcinoma cell line MCF-7 was cultured.The invasion assays wereperformed on breast cancer cells in presence of CCL5.Western blot was performed toexamine total and phosphorylation levels of Ezrin expression in the MCF-7 ceils respondedto CCL5 at different times.RT-PCR analysis of CCR5 was performed in the breast cancercell line MCF-7.2.Two pairs of small interfering RNA (siRNA) targeting Ezrin were transfected into thecells.Real-time PCR and Western blotting were used to detect the down-regulation of EzrinmRNA and protein at different time points.Then,the invasion assays were performed on breast cancer cells in presence of CCL5.Western blot was performed to examine total andphosphorylation levels of Ezrin expression in the MCF-7 cells responded to CCL5 atdifferent times.RESULTS:1.The results demonstrate that CCL5 induces MCF-7 migration in a dose andgradient-dependent manner.,with maximal migration observed with a concentration of 20ng/ml.After treatment with CCL5 for a ladder time,the phosphorylated and the totalexpression of Ezrin were markedly increased.Breast cancer cell line MCF-7 expressedCCR5 mRNA.2.Western blot analysis of whole cell lysates revealed a reduction in Ezrin protein andmRNA levels compared with the control cells expressing pSUPER.Stable EzrinsiRNA-expressing MCF-7 clones showed reduced cell invasion significantly comparedwith the cells transfected with pSUPER or the cells with no transfectant,with maximalmigration observed with a concentration of 50ng/ml.Reduced Ezrin expression bysiRNA inhibited the changes in total and phosphorylation levels of Ezrin in MCF-7 cellsresponse to CCL5.RESULTS:Our results indicate that the chemokine CCL5 can induce migration in humanbreast carcinoma cell line MCF-7 in vitro and suggest a potential role for Ezrin in theprocesses of breast cancer cell migration,invasion and possibly metastasis responsed toCCL5.We demonstrate that Ezrin siRNA specifically inhibits Ezrin expression of MCF-7cells and deregulates several functions such as cell motility and cell invasion,indicatingthat Ezrin plays a key role in the development of human breast cancer cells.It's suggestedthat Ezrin may act as a new anti-invasion therapeutic target for human breast cancer.