Effects And Underlying Mechanisms Of Extracellular PDI On Vitronectin-mediated Breast Cancer Metastasis

Objective:Vitronectin,an important component in the extracellular matrix,plays an important role in cancer metastasis by binding toα_Ⅴβ₃integrins on breast cancer cells.However,native plasma vitronectin does not bind toα_Ⅴβ₃integrins.The aim of the present study is to identify the roles of extracellular protein disulfide isomerase（PDI）in vitronectin-mediated breast cancer metastasis.Methods:（1）Immunofluorescence staining and flowcytometry were performed to confirm the surface expression of PDI andα_Ⅴβ₃integrins on breast cancer cells.（2）PDI release were examined by western blot in the supernatant of activated cells.（3）Immunofluorescence staining was used to detect the difference of vitronectin binding on breast cancer cells between wt-PDI（active）and inert-PDI（inactive）treated plasma.（4）MTT assay was used to examine the effects of PDI inhibitor CCF642 on vitronectin-induced proliferation of breast cancer cells.（5）Wound healing assay and Transwell assay were used to examine vitronectin-induced tumor cell migration in the presence or absence of CCF642.（6）Western Blot was performed to examine the signaling events involved in vitronectin-induced metastasis.Results:（1）PDI andα_Ⅴβ₃integrins existed on the surface of MDA-MB-468 and 4T1breast cancer cells.（2）When activated with thrombin,the breast cancer cells rapidly released PDI into the supernatant.（3）Compared to inert-PDI,wt-PDI treated plasma had significantly increased vitronectin binding on MDA-MB-468 and 4T1 cells.（4）Under normoxic and hypoxic conditions,vitronectin promoted the proliferation of MDA-MB-468 and 4T1 cells,while such effects was attenuated by PDI inhibitor CCF642.（5）Under normoxic and hypoxic conditions,the effects of vitronectin on tumor cell migration was inhibited by CCF642.（6）Under normoxic and hypoxic conditions,vitronectin activated the AKT/m TOR/4EBP1 signaling pathway,which was also inhibited by CCF642.Conclusion:Extracellular PDI promotes vitronectin-mediated breast cancer metastasis by increasing vitronectin binding to tumor cells and activating the AKT/m TOR/4EBP1 signaling pathway.