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

Abstract

IntroductionExtracellular 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).MethodsATP 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.ResultsWe 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.ConclusionThis study suggests that P2Y2R may play an important role in cancer metastasis via modulation of the crosstalk between cancer cells and ECs.Electronic supplementary materialThe online version of this article (doi:10.1186/bcr3694) contains supplementary material, which is available to authorized users.

Targeting calcium signaling in cancer therapy

“…The P2 receptors are divided into P2X and P2Y groups and each group contains several members with distinct ion selectivity and regulatory properties. Numerous studies have demonstrated that P2 receptors involve in cancer cells and are expressed to a very high level in some cases, such as P2X3, P2X5, P2X7 and P2Y2 and P2Y442, 80, 81, 82, 83 (Table 1). In terms of clinical evaluation, P2X3 receptor overexpression was reported to be associated with poor recurrence-free survival in hepatocellular carcinoma patients 84 .…”

The Dynamics of Apoptotic Cell Clearance

“…P2Y2 is expressed at relatively high levels on numerous myeloid cell populations in mice and humans, including tissue-resident macrophages in the lung and peritoneum of mice (Chen et al, 2006; Gautier et al, 2012; Kaufmann et al, 2005; Kronlage et al, 2010). With high sensitivity to both ATP and UTP (maximal responses in ~0.1μM range) (Junger, 2011), P2Y2 has been shown to promote vascular adhesion, motility, and apoptotic cell clearance in a number of different studies of acute and long term inflammation and adaptive immune responses (Elliott et al, 2009; Idzko et al, 2007; Jin et al, 2014; Klämbt et al, 2015; Ma et al, 2013b; McDonald et al, 2010; Shah et al, 2014; Wang and Kubes, 2016). In the context of cell clearance, depletion of extracellular nucleotides or deletion of P2Y2 in mice was shown to cause a delay in the clearance of apoptotic cells by macrophages in the thymus (Elliott et al, 2009).…”

ATP‐P2Y2‐β‐catenin axis promotes cell invasion in breast cancer cells

“…They also demonstrated that ATP stimulated the adhesion and migration of MDA‐MB‐231 cells to endothelial cells (ECs) through the expression of vascular cell adhesion molecule‐1 and intercellular adhesion molecule‐1 in MDA‐MB‐231 or ECs. Their results suggest that P2Y2 promotes cancer metastasis through the modulation of crosstalk between cancer cells and ECs . However, the pro‐invasion mechanisms of ATP and P2Y2 are still poorly investigated.…”