Hypoxia induces the expression of protease-activated receptor (PAR)-1 through the hypoxic inducible factor (HIF)-1α in MDA-MB-231 breast cancer cells

Introduction: The casual relationship between tumor hypoxia, inflammation and coagulation has been recently reappraised. Recently, there has been considerable interest in understanding the biological significance of PAR-1, the prototypical thrombin receptor, in neoplastic diseases since aberrant expression of PAR-1 is often associated with tumor progression. Aim: The aim of the present study was to study whether hypoxia and HIF-1a could be involved in the regulation of the expression of PAR-1 in breast cancer cells. Materials and Methods: MDA-MB-231 breast cancer cells were exposed to an hypoxic microenvironment (2% O2). Western blot analysis was employed to determine HIF-1a expression as well as the phosphorylation of p42/p44 extracellular-regulated protein kinases. RNA interference was performed to inhibit either HIF-1a or PAR-1 expression and the inhibition was verified by both qRT-PCR and Western blot analysis. Cell death and survival were evaluated by fluorimetric assays based on calcein Am and ethidium homodimer-1 staining. Results: In our model hypoxia enhanced the expression of PAR-1 in MDA-MB-231 breast cancer cells and such expression was associated with hypoxia-inducible factor (HIF)-1a accumulation. Treatment of HIF-1a with short interfering RNA resulted in a dramatic reduction of PAR-1 expression in MDA-MB-231 cells, confirming a significant role for HIF-1 in the hypoxia-dependent induction of PAR-1. We next provide evidence for a major role of PAR-1 activation in breast cancer cell proliferation and survival, since treatment of PAR-1 with short interfering RNA resulted in an increased cell death. Finally, we found that hypoxia-induced PAR-1 overexpression in MDA-MB-231 cells caused an increased phosphorylation of p42/p44 extracellularregulated protein kinases in response to PAR-1-specific ligands, thus confirming the role of PAR-1 in cell survival. Conclusion: Our results suggest that PAR-1 activation plays a unique role in protecting breast cancer cells from cell death in a hypoxic microenvironment.