Nutlin-3, an Hdm2 antagonist, inhibits tumor adaptation to hypoxia by stimulating the FIH-mediated inactivation of HIF-1alpha.
The interplay among hypoxia-inducible factor 1-alpha (HIF-1alpha), 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-1alpha-p53-Hdm2 interplay. The effect of nutlin-3 on HIF-1alpha function was examined by reporter analyses, immunoprecipitation and immunoblotting. Nutlin-3 downregulated HIF-1alpha, 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-1alpha 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-1alpha, Hdm2, FIH and p300 could be potential targets for treating tumors overexpressing HIF-1alpha.
Lee
Yoon-Mi
YM
Department of Pharmacology, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul, Korea.
Lim
Ji-Hong
JH
Chun
Yang-Sook
YS
Moon
Hyo-Eun
HE
Lee
Myung Kyu
MK
Huang
L Eric
LE
Park
Jong-Wan
JW
D051784:Aryl Hydrocarbon Receptor Nuclear Translocator / Q000276:immunology; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D017930:Genes, Reporter; D045325:HCT116 Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors* / Q000187:drug effects / Q000235:genetics*; D007093:Imidazoles / Q000494:pharmacology*; D007680:Kidney Neoplasms; D008113:Liver Neoplasms; D006899:Mixed Function Oxygenases; D010879:Piperazines / Q000494:pharmacology*; D010957:Plasmids; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000502:physiology; D012097:Repressor Proteins / Q000235:genetics / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007093:Imidazoles; D010879:Piperazines; D012097:Repressor Proteins; D042461:Vascular Endothelial Growth Factor A; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; C482205:nutlin 3; D006899:Mixed Function Oxygenases; C439790:HIF1AN protein, human; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2