Abstract
Tumor hypoxia often directly correlates with aggressive phenotype, metastasis progression, and resistance to chemotherapy. Two transcription factors [hypoxia-inducible factor-1alpha (HIF-1alpha) and HIF-2alpha] are dramatically induced in hypoxic areas and regulate the expression of genes necessary for tumor adaptation to the conditions of low oxygen; however, the relative contribution of these factors is controversial. We used RNA interference-mediated inactivation of HIF-1alpha or HIF-2alpha followed by microarray analysis to identify genes specifically regulated by either HIF-1 or HIF-2 in hypoxia. We found that, in the MCF7 cell line, the vast majority of hypoxia-responsive genes (>80%) were dependent on the presence of HIF-1alpha. However, a small group of genes were preferentially regulated by HIF-2alpha. Promoter analysis for this group of genes revealed that all of them have putative binding sites for ETS family transcription factors, and 10 of 11 HIF-2alpha-dependent genes had at least one potential hypoxia-responsive element (HRE) in proximity to an ETS transcription factor binding site. Knockdown of ELK-1, the most often represented member of ETS family, significantly reduced hypoxic induction of the HIF-2alpha-dependent genes. Physical and functional interaction between ELK-1 and HIF-2alpha were supported by coimmunoprecipitation of these two proteins, luciferase reporter assay using CITED2 promoter, and binding of ELK-1 protein to the promoters of CITED2 and WISP2 genes in proximity to a HRE. These data suggest that the choice of the target genes by HIF-1 or HIF-2 depends on availability and cooperation of HIFs with other factors recognizing their cognate elements in the promoters.
Highlights
To survive, tumor cells must adapt to the conditions of low oxygen originating from delayed blood vessel development during tumor growth
Cooperation between HIF-2 and ETS Factors in Hypoxia analysis revealed that remaining amounts of HIF-1a or HIF-2a mRNA were close to 10% of their respective levels in control cells transfected with scramble oligonucleotide (Fig. 1A)
To identify genes regulated by HIF-1 or HIF-2, cells were transfected with two different HIF-1a or HIF-2a small interfering RNA (siRNA) oligonucleotides in two independent experiments and compared with hypoxia-treated samples from four independent transfections with scramble
Summary
Tumor cells must adapt to the conditions of low oxygen originating from delayed blood vessel development during tumor growth. The adaptive process involves induction of angiogenesis, increased glucose consumption, and a switch to the glycolytic pathway of energy production. This response is regulated by two transcription factors [hypoxia-inducible factor-1 (HIF-1) and HIF-2; refs. 1, 2], which belong to the basic helix-loop-helix. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). M. Wood is currently at the Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH 03755. J.C. Barrett is currently at the Novartis Institutes of BioMedical Sciences, Inc., Cambridge, MA
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