Carcinogenesis Advance Access originally published online on August 20, 2009
Carcinogenesis 2009 30(10):1768-1775; doi:10.1093/carcin/bgp196
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Nutlin-3, an Hdm2 antagonist, inhibits tumor adaptation to hypoxia by stimulating the FIH-mediated inactivation of HIF-1
Department of Pharmacology
1 Department of Physiology
2 Department of Neurosurgery, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-799, Korea
3 Omics and Integration Research Center, Korea Research Institute of Bioscience and Biotechnology, 52 Eoundong, Yuseong, Daejeon 305-333, Korea
4 Department of Neurosurgery, University of Utah School of Medicine, Salt Lake City, UT 84132, USA
* To whom correspondence should be addressed. Tel: +82 2 740 8289; Fax: +82 2 745 7996; Email: parkjw{at}snu.ac.kr
The interplay among hypoxia-inducible factor 1-alpha (HIF-1
), p53 and human orthologue of murine double minute 2 (Hdm2) has been introduced as a key event in tumor promotion and angiogenesis. Recently, nutlin-3, a small-molecule antagonist of Hdm2, was demonstrated to inhibit the HIF-1-mediated vascular endothelial growth factor production and tumor angiogenesis. Yet, the mechanism by which nutlin-3 inhibits HIF-1 is an open question. We here addressed the mode-of-action of nutlin-3 with respect to the HIF-1–p53–Hdm2 interplay. The effect of nutlin-3 on HIF-1 function was examined by reporter analyses, immunoprecipitation and immunoblotting. Nutlin-3 downregulated HIF-1, which occurred p53-dependently but von Hippel-Lindau-independently. On the contrary, nutlin-3 blunted the hypoxic induction of vascular endothelial growth factor by inactivating HIF-1 even in p53-null cells. The C-terminal transactivation domain (CAD) of HIF-1 was inactivated by nutlin-3, and furthermore, the factor-inhibiting hypoxia-inducible factor (FIH) hydroxylation of Asn803 was required for the nutlin-3 action. In terms of protein interactions, Hdm2 competed with FIH in CAD binding and inhibited the Asn803 hydroxylation both in vivo and in vitro, which facilitated p300 recruitment. Moreover, nutlin-3 reinforced the FIH binding and Ans803 hydroxylation by inhibiting Hdm2. In conclusion, Hdm2 functionally activates HIF-1 by inhibiting the FIH interaction with CAD, and the Hdm2 inhibition by nutlin-3 results in HIF-1 inactivation and vascular endothelial growth factor suppression. The interplays among HIF-1, Hdm2, FIH and p300 could be potential targets for treating tumors overexpressing HIF-1.
Abbreviations: aa, amino acid; CAD, C-terminal transactivation domain; CT, C-terminal; EPO, erythropoietin; FIH, factor-inhibiting hypoxia-inducible factor; HA, hemagglutinin; Hdm2, human orthologue of murine double minute 2; HIF-1, hypoxia-inducible factor 1-alpha; NAD, N-terminal transactivation domain; NES, nuclear export signal; NLS, nuclear localization signal; RT–PCR, reverse transcription–polymerase chain reaction; SDS, sodium dodecyl sulfate; VEGF, vascular endothelial growth factor; VHL, von Hippel-Lindau
Received March 5, 2009; revised July 4, 2009; accepted July 30, 2009.