Investigating Hypoxic Tumor Physiology Through Gene Expression Patterns
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Oncogene (2003) 22, 5907–5914 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc Investigating hypoxic tumor physiology through gene expression patterns Nicholas C Denko*,1, Lucrezia A Fontana1, Karen M Hudson1, Patrick D Sutphin1, Soumya Raychaudhuri2, Russ Altman2 and Amato J Giaccia1 1Division of Radiation and Cancer Biology, Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA 94305, USA; 2Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA Clinical evidence shows that tumor hypoxia is an relyupon oxygen-dependent energyproduction suffer independent prognostic indicator of poor patient outcome. when oxygen falls below a certain threshold level. Hypoxic tumors have altered physiologic processes, However, too much oxygen can also result in the including increased regions of angiogenesis, increased generation of toxic, damaging radical byproducts. The local invasion, increased distant metastasis and altered net effect of these opposing pressures necessitates apoptotic programs. Since hypoxia is a potent controller the evolution of molecular mechanisms to regulate the of gene expression, identifying hypoxia-regulated genes is cellular availabilityand usage of oxygen. a means to investigate the molecular response to hypoxic The response to low-oxygen conditions has been stress. Traditional experimental approaches have identi- identified in both procaryotes and eucaryotes. Although fied physiologic changes in hypoxic cells. Recent studies the mechanisms for gene regulation in response to low- have identified hypoxia-responsive genes that may define oxygen conditions are diverse, parallel systems have the mechanism(s) underlying these physiologic changes. evolved in different organisms that are transcriptionally For example, the regulation of glycolytic genes by responsive to low-oxygen. This convergent evolution hypoxia can explain some characteristics of the Warburg suggests that the abilityto respond to low-oxygen effect. The converse of this logic is also true. By conditions offers a competitive Darwinian advantage. identifying new classes of hypoxia-regulated gene(s), we Although the magnitude of the ‘hypoxic’ response may can infer the physiologic pressures that require the differ between unicellular and multicellular organisms, induction of these genes and their protein products. theyboth share the principal goal of maintaining the Furthermore, these physiologically driven hypoxic gene critical energylevels required for homeostasis, while expression changes give us insight as to the poor outcome extracellular oxygen concentrations decrease. While this of patients with hypoxic tumors. Approximately 1–1.5% review is focused on the transcriptional changes in the of the genome is transcriptionally responsive to hypoxia. pathophysiologic environment of hypoxic tumor cells, However, there is significant heterogeneity in the tran- the reason whyhypoxicgene regulation evolved is scriptional response to hypoxia between different cell obviouslyin response to a more physiologic condition of types. Moreover, the coordinated change in the expression hypoxia such as occurs during embryonic development of families of genes supports the model of physiologic (Chen et al., 1999a) or during wound healing (Warren pressure leading to expression changes. Understanding the et al., 2001). evolutionary pressure to develop a ‘hypoxic response’ provides a framework to investigate the biology of the hypoxic tumor microenvironment. Mechanisms of gene regulation in response to hypoxia Oncogene (2003) 22, 5907–5914. doi:10.1038/sj.onc.1206703 Keywords: hypoxic gene induction; HIF-1; VHL Hypoxia/anoxia-responsive gene expression has been well characterized in procaryotic organisms such as bacteria as well as in lower and higher eucaryotes such as yeast and mammals, respectively. Unicellular organ- isms have less control over environmental oxygen, so their hypoxia response is designed to utilize efficiently Introduction the decreased levels of oxygen. The facultative anaerobic bacterium Escherichia coli utilizes the fumarate, nitrate Evolution of the transcriptional ‘hypoxic response’ reduction (FNR) protein, which is a bifunctional protein Molecular oxygen is beneficial to many organisms due that acts both as a hypoxic sensor and a hypoxia- to its role in efficient energyproduction. Organisms that responsive transcription factor. Under aerobic condi- tions, FNR exists as an inactive apoprotein, but under hypoxia, it forms an active homodimer that is dependent *Correspondence: NC Denko; E-mail: [email protected] Supplemental data is available at: http://cbrl.stanford.edu/hypoxia/ on a redox-sensitive 4Fe–4S cluster (Green et al., 2001). welcome.htm In contrast, one mechanism that yeast Saccharomyces cerevisiae uses for hypoxic gene expression is a heme- Functions of hypoxia responsive genes NC Denko et al 5908 regulated repressive system based on the ROX1 blood cells, especially in times of systemic hypoxia, such (repressed in oxygen) molecule. Under aerobic condi- as during travel to high altitudes (Caro, 2001). The tions, abundant heme activates the HAP1 complex that enhancer element responsible for EPO induction under in turn transactivates the ROX1 gene. ROX1 protein hypoxia was identified (Imagawa et al., 1991), and the blocks expression of a large set of hypoxia-responsive protein that bound to this hypoxia-responsive element genes (Becerra et al., 2002). Under hypoxic conditions, was purified, cloned, and found to be a heterodimer heme-deficient HAP1 represses ROX1 transcription, (Wang and Semenza, 1993). One subunit of the dimer and the hypoxia-responsive genes are derepressed was constitutivelyexpressed in cells (HIF-1 b or ARNT), (Zitomer et al., 1997). These are two examples of the and the other component was oxygen labile and diverse mechanisms that have evolved in single-cell stabilized in cells exposed to low oxygen (HIF-1a) procaryotes and lower eucaryotes to respond to low- (Jiang et al., 1996). The molecular mechanism of HIF oxygen concentrations. activation has been recentlyelucidated, largelybecause The mammalian transcriptional response to hypoxia of the functional interaction between HIF-1a and the is considerablymore complicated, relying on multi- tumor suppressor protein von Hippel–Lindau (VHL). protein complexes to regulate several transcription Under normoxic conditions, HIF-1a is rapidlyde- factors, the most well studied being (hypoxia-inducible graded, with a half-life of a few minutes. In contrast, factor 1 (HIF-1)). General stress-responsive transcrip- under hypoxic conditions, HIF-1a becomes stabilized tion factors such as AP-1, NF-kB and Egr1 have also (Maxwell et al., 1999), associates with the HIF-1b been reported to be regulated byhypoxiaand/or subunit, translocates to the nucleus, and binds in a reoxygenation (Faller, 1999; Ten and Pinsky, 2002). sequence-specific manner to a hypoxia-responsive ele- However, the sensitivityof these factors to mild ment in target genes to activate transcription. The hypoxia, and the duration of their transcriptional regulation of HIF-1a stabilityhas been shown to rely response is much less than that of HIF-1. The ‘general heavilyupon the VHL tumor suppressor protein stress responsive’ transcription factors are therefore not (Maxwell et al., 1999). Under normoxia, HIF-1a is well suited to regulating gene expression in the rapidly hydroxylated at proline 564 (Ivan et al., 2001; chronicallyhypoxic tumor microenvironment. Further- Jaakkola et al., 2001) bya novel familyof proline more, the number of genes that are upregulated bynon- hydroxylases (Bruick and McKnight, 2001; Epstein HIF mechanisms in response to chronic hypoxia seems et al., 2001). The hydroxylated form of HIF-1a binds to be small compared to the HIF-responsive genes. to the VHL protein that is part of the multiprotein In contrast to gene induction byhypoxia, several complex containing elongins B and C and CUL1 (Hon mechanisms of gene repression byhypoxiahave also et al., 2002; Min et al., 2002). This complex acts to been reported. Repression can contribute to the hypoxic ubiquitinate HIF-1a and target it to the proteasome for response bydownregulating such genes as the anti- degradation (Maxwell et al., 1999). Under hypoxia, angiogenic thrombospondin (TSP) genes (Tenan et al., HIF-1a is not hydroxylated (Chan et al., 2002), does not 2000). The TSP genes function to block new blood vessel bind to VHL, and becomes stabilized. formation, so theyare presumablydownregulated by Studies of the VHL tumor suppressor have identified hypoxia in order to stimulate angiogenesis (Laderoute a role for hypoxia-regulated genes in modifying et al., 2000). Recent in vitro data have suggested that malignant transformation in human tumors (Kondo hypoxic downregulation of gene expression can be et al., 2002). VHL has been identified as a classic tumor through the induction of the (negative cofactor 2 suppressor gene that requires the loss of both alleles to (NC2)) transcriptional repressor (Denko et al., 2003). generate disease (Friedrich, 1999). The von Hippel– NC2 has been shown to block gene expression by Lindau syndrome is a result of the germline loss of one regulating core promoter action through binding to the VHL allele, with the loss of the second allele resulting in TATA-associated factor TBP (Kamada et al., 2001). In high-frequencyrenal cell carcinoma, pheochromocyto- addition, the P53 tumor suppressor gene has been found mas and hemangioblastomas