Reactive oxygen species and calcium signalling in the induction of hypoxia-mediated epithelial-mesenchymal transition

Introduction: Hypoxia is a hallmark of the cancer microenvironment and induces epithelial-mesenchymal transition (EMT), a process whereby epithelial cells acquire more migratory and invasive characteristics. Hypoxia can be associated with altered calcium (Ca²⁺) signalling through the production of reactive oxygen species (ROS), however, this association has not been fully characterised in breast cancer cells.

Aims: To assess the remodelling of Ca²⁺ signaling and ROS production in a hypoxia model of EMT in MDA-MB-468 breast cancer cells and to define the role of ROS and specific Ca²⁺ -related proteins in EMT induction in this model.

Methods: To induce EMT, MDA-MB-468 breast cancer cells were incubated in 1% O₂. Levels of ROS were assessed using the cell-permeable fluorogenic probe 2',7'-dichlorodihydrofluorescin diacetate (DCFH-DA). Quantitative RT-PCR or immunoblotting were used to assess mRNA or protein levels of EMT markers and Ca²⁺-related proteins. Dharmacon ON-TARGETplus SMARTpool siRNA was used to silence specific Ca²⁺ channels.

Results: Hypoxia increased the mRNA (24 h) and/or protein levels (48 h) of the EMT markers vimentin, N- cadherin, snail, twist and increased intracellular ROS levels (12 h). Among the fifty Ca²⁺-related proteins assessed, four underwent significant mRNA up-regulation (3 to 10-fold) with hypoxia. Chelation of ROS with 10 mM N-acetylcysteine (NAC) significantly altered the expression pattern of these Ca²⁺-related proteins and some EMT markers (N-cadherin). Silencing of TRPC1 channel, one of the four up-regulated Ca²⁺-related proteins, significantly decreased hypoxia-induced vimentin protein expression.

Discussion: These results implicate an important role for ROS and Ca²⁺ signalling in the induction of hypoxia-mediated EMT in breast cancer cells.