Altered purinergic receptor‐Ca²⁺ signaling associated with hypoxia‐induced epithelial‐mesenchymal transition in breast cancer cells

Abstract

Hypoxia is a feature of the microenvironment of many cancers and can trigger epithelial-mesenchymal transition (EMT), a process by which cells acquire a more invasive phenotype with enriched survival. A remodeling of adenosine 5'-triphosphate (ATP)-induced Ca(2+) signaling via purinergic receptors is associated with epidermal growth factor (EGF)-induced EMT in MDA-MB-468 breast cancer cells. Here, we assessed ATP-mediated Ca(2+) signaling in a model of hypoxia-induced EMT in MDA-MB-468 cells. Like EGF, hypoxia treatment (1% O2) was also associated with a significant reduction in the sensitivity of MDA-MB-468 cells to ATP (EC50 of 0.5 μM for normoxic cells versus EC50 of 5.8 μM for hypoxic cells). Assessment of mRNA levels of a panel of P2X and P2Y purinergic receptors following hypoxia revealed a change in levels of a suite of purinergic receptors. P2X4, P2X5, P2X7, P2Y1 and P2Y11 mRNAs decreased with hypoxia, whereas P2Y6 mRNA increased. Up-regulation of P2Y6 was a common feature of both growth factor- and hypoxia-induced models of EMT. P2Y6 levels were also significantly increased in basal-like breast tumors compared to other subtypes and breast cancer patients with higher P2Y6 levels showed reduced overall survival rates. P2Y6 siRNA-mediated silencing and the P2Y6 pharmacological inhibitor MRS2578 reduced hypoxia-induced vimentin protein expression in MDA-MB-468 cells. P2Y6 inhibition also reduced the migration of mesenchymal-like MDA-MB-231 breast cancer cells. The up-regulation of P2Y6 appears to be a common feature of the mesenchymal phenotype of breast cancer cells and inhibition of this receptor may represent a novel therapeutic target in breast cancer metastasis.

Nucleotides and nucleoside signaling in the regulation of the epithelium to mesenchymal transition (EMT)

“…These observations supported the idea that high expression of the P2Y6 receptor mediated EMT induction and correlated with the high invasivity of breast cancer tumors. In agreement with the in vitro evidence, patients with tumors showing high P2Y6 receptor expression have a poor prognosis [92,101]. Importantly, it was shown that UDP is released in response to chemotherapy, highlighting a putative mechanism for drug resistance [101].…”

“…ATP elicited a reduced Ca 2+ response during EMT in MDA-MB-468 breast carcinoma cells under hypoxic conditions; this response correlated with a lower expression of P2X4, P2X5, P2X7, P2Y1, and P2Y11 purinergic receptors, and an elevated expression of P2Y6 [92]. P2Y6 receptor overexpression has been associated with EMT induced by growth factors and hypoxia; accordingly, the pharmacological inhibition or genetic silencing of this receptor prevented the induction of EMT and the increase in cellular migration [92,101].…”

“…P2Y6 receptor overexpression has been associated with EMT induced by growth factors and hypoxia; accordingly, the pharmacological inhibition or genetic silencing of this receptor prevented the induction of EMT and the increase in cellular migration [92,101]. The role of UDP in the tumor microenvironment activating the P2Y6 receptor and the concomitant metastatic transformation were also shown in breast cancer cell lines MDA-MB-231, Hs578t, BT-549, and MCF-7; these responses were characterized by the expression of MMP-9 and the activation of MAPK and Nf-κB [101].…”

A critical look at the function of the P2Y11 receptor

“…This has been a huge problem in the studies reported so far. To our knowledge, only five articles have been published that investigate P2RY11 mRNA expression without detecting the fusion transcript [17][18][19][20][21]. All other studies have featured primer sets predicted additionally to recognize the fusion transcript (Table S1).…”

Differential effects of two-pore channel protein 1 and 2 silencing in MDA-MB-468 breast cancer cells

“…The MDA-MB-468 breast cancer cell line is a commonly utilized in vitro model for the study of EMT in the context of defining responses to signals from the surrounding tumor microenvironment [11,19,20]. TPC2 but not TPC1 silencing significantly inhibited EGFinduced vimentin expression relative to siNT in MDA-MB-468 cells (Fig.…”