Growth Factor-Mediated Induction of HDM2 Positively Regulates Hypoxia-Inducible Factor 1α Expression

Abstract

The hypoxia-inducible factor 1 (HIF-1) transcriptional complex is regulated by cellular oxygen levels and growth factors. The phosphoinosotide 3-kinase (PI-3K)-Akt/protein kinase B (PKB) pathway has been shown to regulate HIF-1 activity in response to oncogenic signals and growth factors. We assessed whether the HDM2 oncoprotein, a direct target of Akt/PKB, could regulate HIF-1α expression and HIF-1 activity under normoxic conditions. We found that growth factor stimulation, overexpression of Akt/PKB, or loss of PTEN resulted in enhanced expression of both HIF-1α and HDM2. Growth factor-mediated induction of HIF-1α was ablated by transient expression of a dominant negative form of Akt/PKB or by treatment with LY294002. Transient expression of HDM2 led to increased expression of HIF-1α. Pulse-chase and cycloheximide experiments revealed that HDM2 did not significantly affect the half-life of HIF-1α. Growth factor-induced HIF-1α and HDM2 proteins were localized to the nucleus, and induction of both proteins was observed in both p53^+/+ and p53^−/− HCT116 cells to comparable levels. Importantly, insulin-like growth factor 1-induced HIF-1α expression was observed in p53-null mouse embryo fibroblasts (MEFs) but was significantly impaired in p53 Mdm2 double-null MEFs, indicating a requirement for Mdm2 in this process. Finally, we showed that phosphorylation at Ser166 in HDM2 contributed in part to growth factor-mediated induction of HIF-1α. Our study has important implications for the role of the PI-3K-Akt/PKB-HDM2 pathway in tumor progression and angiogenesis.

We thank Amato Giaccia (Stanford University, Stanford, Calif.) and Gregg Semenza (Johns Hopkins University Medical School, Baltimore, Md.) for helpful suggestions regarding growth factor induction of HIF-1α. Thanks to Paul Workman (Institute of Cancer Research, Sutton, Surrey, United Kingdom), Christine Blattner (Institute of Genetics and Toxicology, Karlsruhe, Germany), and Neville Ashcroft (DNA Damage and Stability Centre, University of Sussex, Sussex, United Kingdom) for their critical review of the manuscript and helpful discussions. We thank Karen Vousden (Beatson Institute, Glasgow, United Kingdom) for the MEFs. Finally, we thank Alan Lehmann (DNA Damage and Stability Centre, University of Sussex), Nina Perusinghe, and all other members of the Ashcroft laboratory for their input and support.

This work was funded by Cancer Research UK (CRC grant C309/A2984) and the Institute of Cancer Research.