P2Y₂receptor activation by nucleotides released from highly metastatic breast cancer cells increases tumor growth and invasion via crosstalk with endothelial cells

Extracellular nucleotides are released and detectable in a high concentration within the tumor microenvironment. G protein-coupled P2Y₂ nucleotide receptor (P2Y₂R) 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 P2Y₂R in the process of cancer metastasis remains unclear. This study aimed to determine the role of P2Y₂R in the proliferation, migration and invasion of highly metastatic MDA-MB-231 breast cancer cells through crosstalk with endothelial cells (ECs).

ATP release and P2Y₂R activity between high metastatic breast cancer cell MDA-MB-231 and low metastatic breast cancer cell MCF-7 were compared. Then, the role of P2Y₂R on tumor growth and invasion via crosstalk with ECs was examined in vitro, using MDA-MB-231 cells and ECs transfected with control- or P2Y₂R-siRNA, and in vivo, using an animal model injected with control-shRNA- or P2Y₂R-shRNA-transfected MDA-MB-231 cells.

We found that this highly metastatic breast cancer cell line released higher levels of ATP and showed a higher P2Y₂R activity in comparison to a low metastatic breast cancer cell line, MCF-7. In MDA-MB-231 cells, P2Y₂R activation by ATP or UTP increased proliferation at 24 or 72 hours, which was abolished by P2Y₂R 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 P2Y₂R was knocked down. Furthermore, ATP- or UTP-mediated activation of P2Y₂R 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 P2Y₂R-shRNA-transfected MDA-MB-231 cells compared to mice injected with control shRNA-transfected MDA-MB-231 cells.

This study suggests that P2Y₂R may play an important role in cancer metastasis via modulation of the crosstalk between cancer cells and ECs.