P2Y2R-mediated transactivation of VEGFR2 through Src phosphorylation is associated with ESM-1 overexpression in radiotherapy-resistant-triple negative breast cancer cells

We previously reported that radiotherapy-resistant (RT-R) triple negative breast cancer (TNBC) cells upregulate the expression of endothelial-specific molecule-1 (ESM-1) compared with TNBC cells. In addition, ESM-1 is involved in an increased proliferation and invasion of RT-R-TNBC cells compared with TNBC cells. It was further identified that, in RT-R-TNBC cells, P2Y(2) purinergic receptor (P2Y(2)R)-mediated activation of p21-activated kinase 1 (PAK1), protein kinase C (PKC), c-Jun N-terminal kinase (JNK) and p38 MAPKs is related to ESM-1 expression via forkhead box O1 (FoxO1) regulation. Notably, it has been reported that P2Y(2)R mediates the transactivation of vascular epithelial growth factor receptor 2 (VEGFR2), and VEGFR2 is known to be involved in ESM-1 expression. Therefore, in the present study, the involvement of VEGFR2 in the P2Y(2)R-mediated ESM-1 upregulation in RT-R-TNBC cells and the relationship between P2Y(2)R and VEGFR2 activation was further examined. Western blotting and reverse transcription-PCR were used to monitor the expression of ESM-1, and the results demonstrated that extracellular ATP treatment regulated the expression of ESM-1 in a P2Y(2)R-dependent manner in RT-R-MDA-MB-231 cells. In addition, extracellular ATP activated Src and VEGFR2 after 5 min of incubation, which was abolished by knockdown of P2Y(2)R expression. VEGFR2 activation in response to ATP was also decreased by inhibiting Src activity, suggesting that ATP-activated P2Y(2)R regulates VEGFR2 phosphorylation via Src activation. Furthermore, ATP-induced ESM-1 expression was decreased by transfection with VEGFR2 small interfering RNA (siRNA). ESM-1-related signaling molecules, PAK1, PKC, JNK and p38 MAPKs, and the transcriptional regulator, FoxO1, which were activated by ATP, were also decreased following transfection with VEGFR2 siRNA. These results suggest that P2Y(2)R-mediated transactivation of VEGFR2 through Src phosphorylation is associated with ESM-1 overexpression in RT-R-TNBC cells.