Role of hypoxia-inducible factor (HIF)-1 alpha-versus HIF-2 alpha in the regulation of HIF target genes in response to hypoxia, insulin-like growth factor-1, or loss of von Hippel-Lindau function: Implications for targeting the HIF pathway

Abstract

Overexpression of hypoxia-inducible factors (HIF), HIF-1 alpha and HIF-2 alpha, leads to the up-regulation of genes involved in proliferation, angiogenesis, and glucose metabolism and is associated with tumor progression in several cancers. However, the contribution of HIF-1 alpha versus HIF-2 alpha to vascular endothelial growth factor (VEGF) expression and other HIF-regulated target genes under different conditions is unclear. To address this, we used small interfering RNA (siRNA) techniques to knockdown HIF-1 alpha and/or HIF-2 alpha expression in response to hypoxia, insulin-like growth factor (IGF)-I, or renal carcinoma cells expressing constitutively high basal levels of HIF-1 alpha and/or HIF-2 alpha due to loss of von Hippel-Lindau (VHL) function. We found that HIF-1 alpha primarily regulates transcriptional activation of VEGF in response to hypoxia and IGF-I compared with HIF-2 alpha in MCF-7 cells. We also observed a reciprocal relationship between HIF-1 alpha and HIF-2a expression in hypoxia in these cells: HIF-2 alpha siRNA enhanced HIF-1 alpha-mediated VEGF expression in MCF-7 cells in response to hypoxia, which could be completely blocked by cotransfection with HIF-1 alpha siRNA. In contrast, in renal carcinoma cells that constitutively express HIF-1 alpha and HIF-2 alpha due to loss of VHL function, we found that high basal VEGF, glucose transporter-1, urokinase-type plasminogen activator receptor, and plasminogen activator inhibitor-1 expression was predominantly dependent on HIF-2 alpha. Finally, we showed that a newly identified small-molecule inhibitor of HIF-1, NSC-134754, is also able to significantly decrease HIF-2 alpha protein expression and HIF-2 alpha-regulated VEGF levels in renal carcinoma cells. Our data have important implications for how we target the HIF pathway therapeutically.