Study On The Mechanism Of CAV1 Regulating Integrin α6β4 Expression To Promote Lung Metastasis Of Breast Cancer Through Exosomes

Background and objective:Breast cancer(BC)is prone to lung metastasis,and caveolin-1(CAV1)plays an important role in lung metastasis of breast cancer.Breast cancer-derived exosomes assemble signaling molecules to domesticate pre-metastatic sites for breast cancer metastasis.Integrin α6β4,a cell surface receptor of the integrins family,is recognized and bound by surfactant-associated proteins(SFTPC).Meanwhile,integrin α6β4 is widely expressed in exosomes,which not only promotes tumor invasion and migration,but also influences the direction of tumor metastasis.In order to further investigate the regulatory mechanism of CAV1 in lung metastasis from breast cancer,we will proved that CAV1 regulates the expression of integrin α6β4 in cells and exosomes in breast cancer cells.We also attempted to demonstrate that changes in CAV1 affect the expression of SFTPC in lung epithelial cells and block the recognition and binding of integrin α6β4 to the lungs,thereby fully elucidate the mechanism by which CAV1 regulates the expression of integrin α6β4 through exosomes to promote lung metastasis of breast cancer.Our work provides theoretical reference for the research and clinical application of targeted lung metastasis in breast cancer.Methods:1.CAV1 regulates the expression of integrin α6β4/Src/FAK and integrin α6β4recognition protein SFTPC in lung epithelial cells.Immunoprecipitation was used to detect the interaction of CAV1 and integrinα6β4 with SFTPC.After knocking down or overexpressing CAV1 in breast cancer cells,western blot,RT-qPCR and immunofluorescence was performed to evaluate the expression of integrin α6β4 and signal pathway molecule Src/FAK in BC cells and SFTPC in lung epithelial cells.2.In vitro breast cancer exosomes were co-incubated with lung epithelial cells to explore the effect and mechanism of CAV1 on the proliferation,invasion and migration of lung epithelial cells.Stable breast cancer cell lines with CAV1 overexpression and CAV1 knockdown were constructed,and the exosomes of each group were extracted to detect the expression of integrin α6β4 in exosomes.The exosomes of BC cells were co-incubated with lung epithelial cells that expressed SFTPC normally or had SFTPC knocked out.Confocal microscopy was applied to evaluate the uptake of exosomes by lung epithelial cells.Proliferation,invasion and migration of exosome cultured cells were detected by CCK-8,transwell and wound healing tests.Western blot analysis of the activation of Src/PI3K(the downstream signal of integrin α6β4)and EMT index after co-incubation.3.Lung metastasis model of breast cancer was established in vivo.Lung metastasis of breast cancer and activation of Src/PI3 K downstream signal of lung integrin α6β4 in nude mice after exosome acclimation were detected by HE staining and IHC.Result:1.Co-immunoprecipitation results showed that integrin β4 subunits and SFTPC could be precipitated by CAV1.Western blot and RT-qPCR results demonstrated that the expression of integrin α6β4 in breast cancer cells and exosomes was up-regulated(or down-regulated)with CAV1 overexpression(or CAV1 knockdown),and the expression and activation of integrin signaling pathway molecule Src/FAK were positively correlated with CAV1.When CAV1 was knocked down,SFTPC expression in lung epithelial cells was down-regulated.2.The results of co-incubation experiment between breast cancer exosomes and lung epithelial cells indicated that the uptake rate of lung epithelial cells to breast cancer exosomes overexpressing CAV1 was higher.When CAV1 of breast cancer was knocked down or CAV1 of lung epithelium was downregulated(down-regulated expression of SFTPC),the uptake of exosomes by lung epithelial cells of breast cancer decreased.After co-incubation with CAV1-overexpressed breast cancer exosomes,the proliferation,invasion and migration of lung epithelial cells were significantly increased,and the expression of downstream integrin α6β4 signaling molecule p-Src/p-PI3 K and EMT indicators were up-regulated.When CAV1 of breast cancer was knocked down or CAV1 of lung epithelium was down-regulated,cell proliferation,invasion and migration were decreased,and the expression of p-Src/p-PI3 K and EMT indicators of downstream integrin α6β4 signaling molecules were decreased.3.The results of lung metastasis model of breast cancer in vivo showed that the lung metastasis of nude mice acclimated with breast cancer exosomes overexpressing CAV1 was obvious,and the activation degree of Src/PI3 K was higher in the downstream signaling molecule of lung integrin α6β4.Conclusion:1.CAV1 regulate the expression of integrin α6β4 in breast cancer cells,regulating the expression of integrin signaling molecule Src/FAK and integrin α6β4by recognition protein SFTPC in lung epithelial cells.2.Integrin α6β4 recognize and bind to SFTPC through exosomes,promote proliferation,invasion and migration of lung epithelial cells,promote cell EMT,and activate Src/PI3 K signal downstream of integrin α6β4.3.The regulatory effect of CAV1 can promote lung metastasis of breast cancer in vivo and activate Src/PI3 K signal downstream of integrin α6β4.