P2Y(2) receptor activation by nucleotides released from highly metastatic breast cancer cells increases tumor growth and invasion via crosstalk with endothelial cells

Abstract

Introduction: Extracellular nucleotides are released and detectable in a high concentration within the tumor microenvironment. G protein-coupled P2Y(2) nucleotide receptor (P2Y(2)R) is activated equipotently by adenosine triphosphate (ATP) and uridine 5'-triphosphate (UTP), which mediate proinflammatory responses such as cell migration and proliferation. However, the role of P2Y(2)R in the process of cancer metastasis remains unclear. This study aimed to determine the role of P2Y(2)R in the proliferation, migration and invasion of highly metastatic MDA-MB-231 breast cancer cells through crosstalk with endothelial cells (ECs). Methods: ATP release and P2Y(2)R activity between high metastatic breast cancer cell MDA-MB-231 and low metastatic breast cancer cell MCF-7 were compared. Then, the role of P2Y(2)R on tumor growth and invasion via crosstalk with ECs was examined in vitro, using MDA-MB-231 cells and ECs transfected with control-or P2Y(2)R-siRNA, and in vivo, using an animal model injected with control-shRNA- or P2Y(2)R-shRNA-transfected MDA-MB-231 cells. Results: We found that this highly metastatic breast cancer cell line released higher levels of ATP and showed a higher P2Y(2)R activity in comparison to a low metastatic breast cancer cell line, MCF-7. In MDA-MB-231 cells, P2Y(2)R activation by ATP or UTP increased proliferation at 24 or 72 hours, which was abolished by P2Y(2)R knock-down. In addition, the adhesion of MDA-MB-231 cells to ECs and cell migration were both significantly increased by ATP or UTP through the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in MDA-MB-231 or ECs but not in cells where P2Y(2)R was knocked down. Furthermore, ATP- or UTP-mediated activation of P2Y(2)R induced MDA-MB-231 invasion through ECs, increased matrix metalloproteinase-9 (MMP-9) activity and vascular endothelial growth factor (VEGF) production in MDA-MB-231 and induced the phosphorylation of vascular endothelial (VE)-cadherin in ECs. Tumor growth and metastasis to other tissues were dramatically reduced, and body weight was increased in mice injected with P2Y(2)R-shRNA-transfected MDA-MB-231 cells compared to mice injected with control shRNA-transfected MDA-MB-231 cells. Conclusion: This study suggests that P2Y(2)R may play an important role in cancer metastasis via modulation of the crosstalk between cancer cells and ECs.