P2Y(2)R-Mediated PAK1 Activation Is Involved in ESM-1 Overexpression in RT-R-MDA-MB-231 through FoxO1 Regulation

SIMPLE SUMMARY: Radiotherapy is an important treatment to treat triple-negative breast cancer (TNBC; ER(−), PR(−), HER2(−)) patients. However, the radioresistance of BC cells remains the main obstacle to radiotherapy efficacy, which leads to increased tumor relapse and metastasis. Previously, we discovered that endothelial cell-specific molecule-1 (ESM-1) is the most increased gene in radiotherapy-resistant (RT-R)-TNBC cells compared to their parental cells and determined that ESM-1 plays a critical role in the tumorigenesis of RT-R-TNBC cells through the regulation of several genes related to tumor growth, progression and metastasis. Therefore, in this study, we aim to identify the mechanism by which ESM-1 is overexpressed in RT-R-MDA-MB-231 cells. Our results demonstrate, for the first time, that ESM-1 overexpressed in RT-R-MDA-MB-231 cells is regulated through the P2Y(2)R-PAK1-FoxO1 signaling pathway. Our findings suggest that targeting this pathway may be a new therapeutic target for patients with TNBC who acquire RT-R and may improve their prognosis. ABSTRACT: ESM-1, overexpressed in several cancer types, is a potential cancer diagnostic and prognostic indicator. In our previous study, we determined that RT-R-TNBC cells were more aggressive than TNBC cells, and this difference was associated with ESM-1 overexpression. However, the mechanism explaining upregulated ESM-1 expression in RT-R-TNBC cells compared to TNBC cells was unclear. Therefore, we aimed to identify the mechanism by which ESM-1 is overexpressed in RT-R-MDA-MB-231 cells. RT-R-MDA-MB-231 cells were treated with various ESM-1 transcription factor inhibitors, and only the FoxO1 inhibitor downregulated ESM-1 expression. FoxO1 nuclear localization was modulated by JNK and p38 MAPKs, which were differentially regulated by PKC, PDK1 and PAK1. PAK1 profoundly modulated JNK and p38 MAPKs, whereas PKC and PDK1 affected only p38 MAPK. P2Y(2)R activated by ATP, which is highly released from RT-R-BC cells, was involved in PAK1 activation, subsequent JNK and p38 MAPK activation, FoxO1 induction, and ESM-1 expression in RT-R-MDA-MB-231 cells. These findings suggest for the first time that ESM-1 was overexpressed in RT-R-MDA-MB-231 cells and regulated through the P2Y(2)R-PAK1-FoxO1 signaling pathway.