Endothelin-1 inhibits prolyl hydroxylase domain 2 to activate hypoxia-inducible factor-1α in melanoma cells

Background: The endothelin B receptor (ET_BR) promotes tumorigenesis and melanoma progression through activation by endothelin (ET)-1, thus representing a promising therapeutic target. The stability of hypoxia-inducible factor (HIF)-1a is essential for melanomagenesis and progression, and is controlled by site-specific hydroxylation carried out by HIF-prolyl hydroxylase domain (PHD) and subsequent proteosomal degradation. Principal Findings: Here we found that in melanoma cells ET-1, ET-2, and ET-3 through ET_BR, enhance the expression and activity of HIF-1a and HIF-2a that in turn regulate the expression of vascular endothelial growth factor (VEGF) in response to ETs or hypoxia. Under normoxic conditions, ET-1 controls HIF-a stability by inhibiting its degradation, as determined by impaired degradation of a reporter gene containing the HIF-1a oxygen-dependent degradation domain encompassing the PHD-targeted prolines. In particular, ETs through ET_BR markedly decrease PHD2 mRNA and protein levels and promoter activity. In addition, activation of phosphatidylinositol 3-kinase (PI3K)-dependent integrin linked kinase (ILK)-AKTmammalian target of rapamycin (mTOR) pathway is required for ET_BR-mediated PHD2 inhibition, HIF-1α HIF-2α and VEGF expression. At functional level, PHD2 knockdown does not further increase ETs-induced in vitro tube formation of endothelial cells and melanoma cell invasiveness, demonstrating that these processes are regulated in a PHD2-dependent manner. In human primary and metastatic melanoma tissues as well as in cell lines, that express high levels of HIF-1α ETBR expression is associated with low PHD2 levels. In melanoma xenografts, ET_BR blockade by ETBR antagonist results in a concomitant reduction of tumor growth, angiogenesis, HIF-1α and HIF-2α expression, and an increase in PHD2 levels. Conclusions: In this study we identified the underlying mechanism by which ET-1, through the regulation of PHD2, controls HIF-1α stability and thereby regulates angiogenesis and melanoma cell invasion. These results further indicate that targeting ET_BR may represent a potential therapeutic treatment of melanoma by impairing HIF-1α stability.