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.