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Calcium signalling in epithelial-mesenchymal transition induced by hypoxia in breast cancer cells

I. Azimi,1 F.M. Davis,1 P.A. Kenny,2 E.W. Thompson,3,4,5 S.J. Roberts-Thomson1 and G.R. Monteith,1 1School of Pharmacy, The University of Queensland, St Lucia, QLD 4072, Australia, 2Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, New York, NY, USA, 3St. Vincent’s Institute, Fitzroy, VIC 3065, Australia, 4University of Melbourne Department Surgery, St. Vincent’s Hospital Fitzroy, VIC 3065, Australia and 5Institute of Health and Biomedical Innovation and School of Biomedical Science, Queensland University of Technology, Kelvin Grove, QLD 4059, Australia.

Hypoxia is a hallmark of the cancer microenvironment and induces epithelial-mesenchymal transition (EMT) in breast cancer cells. EMT is a process whereby cancer cells acquire a more invasive phenotype. We have recently characterized the remodelling of calcium (Ca2+) signalling as a consequence of EMT induced by epidermal growth factor (EGF). In this study we aimed to elucidate the remodelling of Ca2+ signalling in a model of EMT induced by hypoxia in MDA-MB-468 breast cancer cells.

The induction of EMT by hypoxia (1% O2) was confirmed via protein level of vimentin (after 48 h hypoxia) and mRNA levels of some of the main EMT markers including vimentin, Snail, Twist, N-cadherin and the ratio of CD44/CD24 (after 24 h hypoxia). The mRNA levels of fifty Ca2+ pumps, channels, sensors and G-coupled receptors in the presence and absence of hypoxia (24 h) were evaluated using real time RT-PCR. This led to identification of four specific targets that were significantly up-regulated in hypoxia compared to normoxia (21% O2). P2Y6 purinergic receptor, as one of these up-regulated targets, showed a three-fold increase in mRNA levels by hypoxia. Selective pharmacological inhibition of P2Y6 significantly reduced cellular migration of the mesenchymal like MDA-MB-231 breast cancer cell line. Gene expression profiling of 458 human breast tumours showed elevated levels of P2Y6 in basal breast cancer subtypes compared to Luminal A and Luminal B subtypes. Furthermore, breast cancer patients with high P2Y6 levels (n = 651) showed reduced overall survival rates compared to patients with low levels of P2Y6 (n = 464) (P = 0.019).

In conclusion this study suggests that hypoxia-induced EMT is associated with alterations in Ca2+ signalling and P2Y6 purinergic receptor expression. Further studies may identify a specific Ca2+ pump, channel or receptor that may represent a target for the mesenchymal phenotype of breast cancer cells and a potential therapeutic strategy for the control of breast cancer metastasis.