The Effect And Mechanism Of Atm On The Migration And Invasion Of Breast Cancer Cells

Objective: To investigate the effect and mechanism of ATM on the migration and invasion of breast cancer cells.Methods: The immunohistochemistry was applied on normal breast tissue,invasive ductal carcinoma without or with metastasis tissue.After exposed to 1% oxygen,the levels of p-ATM in BT549 and MDA-MB-231 cells were detected by IF staining and Western Blotting,and the cell migration and invasion potentials of BT549 and MDA-MB-231 were evaluated by transwell assay.Then,the transwell assay was also used to detect the cell migration and invasion abilities of BT549 and MDA-MB-231 cells under treatment with a specific inhibitor of ATM kinase activity,Ku60019,as well as by stably knocked down ATM expression with a specific shATM against ATM.After untreated or treated with Ku60019 under hypoxic condition,quantitative phosphoproteome analysis was performed,then Gene Ontology(GO)and KEGG Pathway analysis were used to screen the target protein related to migration and invasion.Immunoprecipitation experiments were used to test the interaction between ATM and its target proteins and the phosphorylation status of the target proteins.The functions of target protein phosphorylation were tested by specific short hairpin RNA(shRNA),site-directed mutagenesis technique,transwell assay,immunoprecipitation experiment and F-actin stress fiber formation assay.Results: The expression of p-ATM protein is gradually increased in the worse breast tumor tissues.After exposed to 1% oxygen,the levels of p-ATM in BT549 and MDA-MB-231 cells were significantly up-regulated,and the cell migration and invasion abilities were markedly enhanced.In the absence of ATM activity and expression by Ku60019 or shRNA,the cell migration and invasion potentials were significantly decreased.Phosphoproteome data and immunoprecipitation experiment showed that ATM can phosphorylate CTTN on Serine 113.When knockdown CTTN,the cell migration and invasion potentials were significantly suppressed.Site-directed mutagenesis technique and F-actin stress fiber formation assay showed that interaction of Arp2/3 complex and the phosphorylated CTTN S113 can regulate actin polymerization,thus involve in promoting tumor cell migration and invasion.Conclusion: ATM-mediated the phosphorylated CTTN S113 plays a critical role in promoting breast tumor cell mobility and invasion via actin polymerization.