Endothelin-1 inhibits prolyl hydroxylase domain 2 to activate hypoxia-inducible factor-1alpha in melanoma cells.
BACKGROUND
The endothelin B receptor (ET(B)R) promotes tumorigenesis and melanoma progression through activation by endothelin (ET)-1, thus representing a promising therapeutic target. The stability of hypoxia-inducible factor (HIF)-1alpha 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.


RESULTS
Here we found that in melanoma cells ET-1, ET-2, and ET-3 through ET(B)R, enhance the expression and activity of HIF-1alpha and HIF-2alpha 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-alpha stability by inhibiting its degradation, as determined by impaired degradation of a reporter gene containing the HIF-1alpha oxygen-dependent degradation domain encompassing the PHD-targeted prolines. In particular, ETs through ET(B)R markedly decrease PHD2 mRNA and protein levels and promoter activity. In addition, activation of phosphatidylinositol 3-kinase (PI3K)-dependent integrin linked kinase (ILK)-AKT-mammalian target of rapamycin (mTOR) pathway is required for ET(B)R-mediated PHD2 inhibition, HIF-1alpha, HIF-2alpha, 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-1alpha, ET(B)R expression is associated with low PHD2 levels. In melanoma xenografts, ET(B)R blockade by ET(B)R antagonist results in a concomitant reduction of tumor growth, angiogenesis, HIF-1alpha, and HIF-2alpha 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-1alpha stability and thereby regulates angiogenesis and melanoma cell invasion. These results further indicate that targeting ET(B)R may represent a potential therapeutic treatment of melanoma by impairing HIF-1alpha stability.
Spinella
Francesca
F
Laboratory of Molecular Pathology, Regina Elena National Cancer Institute, Rome, Italy.
Rosanò
Laura
L
Del Duca
Martina
M
Di Castro
Valeriana
V
Nicotra
Maria Rita
MR
Natali
Pier Giorgio
PG
Bagnato
Anna
A
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D065131:Endothelin B Receptor Antagonists; D019332:Endothelin-1 / Q000378:metabolism / Q000494:pharmacology* / Q000627:therapeutic use; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D006900:Hydroxylation / Q000187:drug effects; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000737:chemistry / Q000378:metabolism*; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D008297:Male; D008545:Melanoma / Q000098:blood supply / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D009361:Neoplasm Invasiveness / Q000473:pathology; D009389:Neovascularization, Pathologic / Q000188:drug therapy; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011393:Procollagen-Proline Dioxygenase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D055550:Protein Stability / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D044023:Receptor, Endothelin B / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013379:Substrate Specificity; D000099200:Scaffold Protein ILK; D000099162:Endothelial PAS Domain-Containing Protein 1
D051792:Basic Helix-Loop-Helix Proteins; D065131:Endothelin B Receptor Antagonists; D019332:Endothelin-1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D019869:Phosphatidylinositol 3-Kinases; D011393:Procollagen-Proline Dioxygenase; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D044023:Receptor, Endothelin B; D000099200:Scaffold Protein ILK; D000099162:Endothelial PAS Domain-Containing Protein 1; C497442:HIF1A protein, human; C490190:EGLN1 protein, human