Roles And Mechanisms Of Extracellular Matrix Protein 1 In Facilitating Invadopodia Formation Of Breast Cancer Cells

Tumor metastasis threatens health and life quality of breast cancer patients greatly.So far,the exact mechanism of breast cancer metastasis has not yet been known thoroughly,and cannot be predicted and intervened in clinic effectively.Therefore,it is of great significance to further study the mechanism of breast cancer invasion and metastasis for effectively curing diseases and prolonging the disease-free survival of patients.Invadopodia are pseudopod-like antennae formed from the cytomembrane of tumor cells,which help tumor cells to penetrate the cross-linked network of extracellular matiix,so that tumor cells can pass through the epithelial and endothelial cell layers and then form metastasis.Extracellular matrix protein 1(ECM1)is an oncogene,which is closely related to tumor metastasis.Recent study found that ECM1 could regulate cytoskeletal remodeling,but the exact mechanism has been still unclear.In the present study,the roles of ECM1 in invadopodia formation and in the invasion and metastasis of breast cancer cells were firstly determined by cellular experiments and in-vivo experimental systems,and then the specific roles and mechanisms of which were explored by molecular experiments.Finally,the roles and mechanism of ECM1 were validated in clinical samples,which provided new clues for prevention and treatment of invasion and metastasis of breast cancer.Cellular experiments.Results from western blot analysis showed that more aggressive cell lines,such as Hs 578T and MDA-MB-231,exhibited high levels of ECM1 expression,whereas less aggressive cell lines,such as MCF-7,T-47D and MDA-MB-453,exhibited almost no ECM1 expression.Cell-matrix adhesion experiments showed that ECM1 promoted breast cancer cells to adhere to extracellular matrix.Chamber-invasive experiments showed that ECM1 facilitated invasive ability of breast cancer cells.Results from three-dimensional matrigel culture showed that ECM1 facilitated invadopodia formation of breast cancer cells.In-vivo experiments.We established a nude mouse model by orthotopic transplantation of breast cancer cell line 4T1.Results showed that Ecml gene knockout could inhibit tumors growth and the formation of spontaneous lung metastasis in orthotopic transplantation of breast cancer in nude mice.Molecular mechanism exploration.Results from western blot assay and immunofluorescence showed that ECMI protein expression was correlated to phosphorylated level of moesin(MSN)protein and membranal aggregation.Co-immunoprecipitation and immunofluorescence assays showed that ECM1 could bind to MSN.By stably overexpressing human wild-type MSN and phospho-rnimetic MSN in breast cancer cells,western blot and three-dimensional matrigel culture confirmed that interaction between ECM1 and MSN as well as MSN phosphorylation were both essential for invadopodia formation of breast cancer cells.Clinical specimen level.Results from immunohistochemistry showed that co-expression of ECM1 and MSN protein in human breast cancer specimens was associated to the aggressive phenotype.Collectively,ECMI acts as a membrane protein,which facilitated invadopodia formation.It recruits MSN to promote its membrane translocation and activation,thus facilitates invadopodia formation and metastasis of breast cancer.Co-expression of ECM1 and MSN correlates with aggressive breast cancer phenotypes.Thus,ECM1 may be used as a potential therapeutic target to overcome breast cancer dissemination.