Oxidized ATM-induced Autophagy In Breast CAFs Enriches GPR64 In Exosomes Promoting The Invasion Of Breast Cancer Cells

Object: To understand the role and mechanism of oxidized ATM in breast CAFs on breast cancer cell invasion.Methods: Immunohistochemistry(IHC)assay was employed to measure the levels of p-ATM(s1981)in normal breast tissue,invasive ductal carcinoma tissue without metastasis and with metastasis.Breast CAFs were treated with or without KU60019,a specific inhibitor of ATM,under normoxia or hypoxia for 6h and the conditioned medium(CM)were collected and were then co-cultured with breast cancer cells to evaluate the effects of oxidized ATM in CAFs on the invasion of cancer cells.Phosphoproteomics analysis was performed to identify the phosphorylated proteins in hypoxic CAFs compared with normoxic NFs and bioinformatics strategy was used to screen the target protein of oxidized ATM and the potential biological processes.Western blot,Immunofluorescence assay and Co-immunoprecipitation assay were applied to detect the effect of oxidized ATM in breast CAFs on autophagy and the phosphorylated BNIP3 expression.Ultracentrifugation assay was employed to isolate exosomes from CAFs and NFs.The target proteins in exosomes were measured by Western blot.siRNA interference was used to knock down the target protein expression in breast CAFs to evaluate its effect on the invasion of breast cancer cell.Results: The protein levels of p-ATM(s1981)in invasive ductal carcinoma without metastasis and with metastasis were remarkably higher than that in normal breast tissues.Oxidized ATM induced by hypoxia in breast CAFs enhanced the invasion of breast cancer cells.Oxidized ATM was found to involve in autophagy of hypoxic CAFs.Identified by phosphoproteomics and analyzed using bio-informatics,the target proteins of oxidized ATM may play roles in the CAFs autophagy and regulating exosome secretion of CAF.The BNIP3,a target of oxidized ATM at S135 site,acted a critical role in the autophagy of hypoxic CAFs.Furthermore,we found that the enrichment of GPR64 in exosomes from hypoxic CAFs could trigger the NF-?B signaling pathway in cancer cells thus boosting the cancer cells invasion.Conclusion: Oxidized ATM-induced autophagy in breast CAFs enriches GPR64 in exosomes and plays a pivotal role in promoting the invasion of breast cancer cells.