Preface Hypoxia inducible factors (HIFs) are broadly expressed in human cancers, and HIF1α and HIF2α were previously suspected of promoting tumor progression through largely overlapping functions. However, this relatively simple model has now been challenged in light of recent data from genome-wide analyses of human tumors, genetically engineered mouse models of cancer, and systems biology approaches that reveal unique and sometimes opposing HIFa activities in both normal physiology and disease. These effects are mediated in part through regulation of unique target genes, as well as direct and indirect interactions with important oncoproteins and tumor suppressors, including MYC and p53. As HIF inhibitors are currently under clinical evaluation as cancer therapeutics, a more thorough understanding of unique roles performed by HIF1α and HIF2α in human neoplasia is warranted. This Review summarizes our rapidly changing understanding of shared and independent HIF1α and HIF2α activities in tumor growth and progression, and the implications for using selective HIF inhibitors as cancer therapeutics.

Transcriptional activation of erythropoietin, glycolytic enzymes, and vascular endothelial growth factor occurs during hypoxia or in response to cobalt chloride (CoCl 2 ) in Hep3B cells. However, neither the mechanism of cellular O 2 sensing nor that of cobalt is fully understood. We tested whether mitochondria act as O 2 sensors during hypoxia and whether hypoxia and cobalt activate transcription by increasing generation of reactive oxygen species (ROS). Results show (i) wild-type Hep3B cells increase ROS generation during hypoxia (1.5% O 2 ) or CoCl 2 incubation, (ii) Hep3B cells depleted of mitochondrial DNA ( 0 cells) fail to respire, fail to activate mRNA for erythropoietin, glycolytic enzymes, or vascular endothelial growth factor during hypoxia, and fail to increase ROS generation during hypoxia; (iii) 0 cells increase ROS generation in response to CoCl 2 and retain the ability to induce expression of these genes; and (iv) the antioxidants pyrrolidine dithiocarbamate and ebselen abolish transcriptional activation of these genes during hypoxia or CoCl 2 in wild-type cells, and abolish the response to CoCl 2 in °cells. Thus, hypoxia activates transcription via a mitochondria-dependent signaling process involving increased ROS, whereas CoCl 2 activates transcription by stimulating ROS generation via a mitochondria-independent mechanism.

Multicellular organisms initiate adaptive responses when oxygen (O(2)) availability decreases, but the underlying mechanism of O(2) sensing remains elusive. We find that functionality of complex III of the mitochondrial electron transport chain (ETC) is required for the hypoxic stabilization of HIF-1 alpha and HIF-2 alpha and that an increase in reactive oxygen species (ROS) links this complex to HIF-alpha stabilization. Using RNAi to suppress expression of the Rieske iron-sulfur protein of complex III, hypoxia-induced HIF-1 alpha stabilization is attenuated, and ROS production, measured using a novel ROS-sensitive FRET probe, is decreased. These results demonstrate that mitochondria function as O(2) sensors and signal hypoxic HIF-1 alpha and HIF-2 alpha stabilization by releasing ROS to the cytosol.

During the last century, it has been established that regions within solid tumors experience mild to severe oxygen deprivation, due to aberrant vascular function. These hypoxic regions are associated with altered cellular metabolism, as well as increased resistance to radiation and chemotherapy. As discussed in this Timeline, over the past decade, work from many laboratories has elucidated the mechanisms by which hypoxia-inducible factors (HIFs) modulate tumor cell metabolism, angiogenesis, growth, and metastasis. The central role played by intra-tumoral hypoxia and HTF in these processes has made them attractive therapeutic targets in the treatment of multiple human malignancies.Oxygen (O 2 ) is required for aerobic metabolism to maintain intracellular bioenergetics and serve as an electron acceptor in many organic and inorganic reactions. Hypoxia, defined as reduced O 2 levels, occurs in a variety of pathological conditions, including stroke, tissue ischemia, inflammation, and the growth of solid tumors. The beginnings of hypoxia research in tumor biology can be traced back to observations made in the early 20 th century by Otto Warburg who demonstrated that, unlike normal cells, tumor cells favor glycolysis, independent of cellular oxygenation levels. He postulated that tumor growth is caused by mitochondrial dysfunction in neoplastic cells, forcing them to generate energy through glycolysis (reviewed in 1 ). This hypothesis appears to be incorrect, but a number of other molecular mechanisms promoting "aerobic glycolysis" have been proposed including mutations and epigenetic changes in genes encoding tumor suppressors (e.g. p53), oncogene activation (e.g. c-Myc), and hypoxic adaptations {Denko, 2008 #6606; Gatenby, 2004 #6608; Deberardinis, 2008 #6609.

Regions of severe oxygen deprivation (hypoxia) arise in tumors due to rapid cell division and aberrant blood vessel formation. The hypoxia-inducible factors (HIFs) mediate transcriptional responses to localized hypoxia in normal tissues and in cancers and can promote tumor progression by altering cellular metabolism and stimulating angiogenesis. Recently, HIFs have been shown to activate specific signaling pathways such as Notch and the expression of transcription factors such as Oct4 that control stem cell self renewal and multipotency. As many cancers are thought to develop from a small number of transformed, self-renewing, and multipotent "cancer stem cells," these results suggest new roles for HIFs in tumor progression.

Citrate is a critical metabolite required to support both mitochondrial bioenergetics and cytosolic macromolecular synthesis. When cells proliferate under normoxic conditions, glucose provides the acetyl-CoA that condenses with oxaloacetate to support citrate production. Tricarboxylic acid (TCA) cycle anaplerosis is maintained primarily by glutamine. Here we report that some hypoxic cells are able to maintain cell proliferation despite a profound reduction in glucose-dependent citrate production. In these hypoxic cells, glutamine becomes a major source of citrate. Glutamine-derived α-ketoglutarate is reductively carboxylated by the NADPH-linked mitochondrial isocitrate dehydrogenase (IDH2) to form isocitrate, which can then be isomerized to citrate. The increased IDH2-dependent carboxylation of glutamine-derived α-ketoglutarate in hypoxia is associated with a concomitant increased synthesis of 2-hydroxyglutarate (2HG) in cells with wild-type IDH1 and IDH2. When either starved of glutamine or rendered IDH2-deficient by RNAi, hypoxic cells are unable to proliferate. The reductive carboxylation of glutamine is part of the metabolic reprogramming associated with hypoxia-inducible factor 1 (HIF1), as constitutive activation of HIF1 recapitulates the preferential reductive metabolism of glutaminederived α-ketoglutarate even in normoxic conditions. These data support a role for glutamine carboxylation in maintaining citrate synthesis and cell growth under hypoxic conditions. C itrate plays a critical role at the center of cancer cell metabolism. It provides the cell with a source of carbon for fatty acid and cholesterol synthesis (1). The breakdown of citrate by ATP-citrate lyase is a primary source of acetyl-CoA for protein acetylation (2). Metabolism of cytosolic citrate by aconitase and IDH1 can also provide the cell with a source of NADPH for redox regulation and anabolic synthesis. Mammalian cells depend on the catabolism of glucose and glutamine to fuel proliferation (3). In cancer cells cultured at atmospheric oxygen tension (21% O 2 ), glucose and glutamine have both been shown to contribute to the cellular citrate pool, with glutamine providing the major source of the four-carbon molecule oxaloacetate and glucose providing the major source of the two-carbon molecule acetyl-CoA (4, 5). The condensation of oxaloacetate and acetyl-CoA via citrate synthase generates the 6 carbon citrate molecule. However, both the conversion of glucose-derived pyruvate to acetyl-CoA by pyruvate dehydrogenase (PDH) and the conversion of glutamine to oxaloacetate through the TCA cycle depend on NAD + , which can be compromised under hypoxic conditions. This raises the question of how cells that can proliferate in hypoxia continue to synthesize the citrate required for macromolecular synthesis.This question is particularly important given that many cancers and stem/progenitor cells can continue proliferating in the setting of limited oxygen availability (6, 7). Louis Pasteur first highlighted the impact of hypoxia on nutrient metabol...

The division, differentiation, and function of stem cells and multipotent progenitors are influenced by complex signals in the microenvironment, including oxygen availability. Using a genetic "knock-in" strategy, we demonstrate that targeted replacement of the oxygen-regulated transcription factor HIF-1␣ with HIF-2␣ results in expanded expression of HIF-2␣-specific target genes including Oct-4, a transcription factor essential for maintaining stem cell pluripotency. We show that HIF-2␣, but not HIF-1␣, binds to the Oct-4 promoter and induces Oct-4 expression and transcriptional activity, thereby contributing to impaired development in homozygous Hif-2␣ KI/KI embryos, defective hematopoietic stem cell differentiation in embryoid bodies, and large embryonic stem cell (ES)-derived tumors characterized by altered cellular differentiation. Furthermore, loss of HIF-2␣ severely reduces the number of embryonic primordial germ cells, which require Oct-4 expression for survival and/or maintenance. These results identify Oct-4 as a HIF-2␣-specific target gene and indicate that HIF-2␣ can regulate stem cell function and/or differentiation through activation of Oct-4, which in turn contributes to HIF-2␣'s tumor promoting activity.[Keywords: HIF; hypoxia; HIF-2␣; Oct-4; VEGF; TGF-␣; stem cells; cancer] Supplemental material is available at http://www.genesdev.org.

The Ah receptor (AHR) is a ligand-activated transcription factor that mediates a pleiotropic response to environmental contaminants such as benzo [a]pyrene and 2, 3,7,8-tetrachlorodibenzo-p-dioxin. In an effort to gain insight into the physiological role of the AHR and to develop models useful in risk assessment, gene targeting was used to inactivate the murineAhr gene by homologous recombination. Ahr-l mice are viable and fertile but show a spectrum of hepatic defects that indicate a role for the AHR in normal liver growth and development. TheAhr-1-phenotype is most severe between 0-3 weeks ofage and involves slowed early growth and hepatic defects, including reduced liver weight, transient microvesicular fatty metamorphosis, prolonged extramedullary hematopoiesis, and portal hypercellularity with thickening and fibrosis.The Ah receptor (AHR) is a ligand-activated transcription factor that regulates a biphasic pleiotropic response to a variety of structurally related environmental contaminants (1, 2). Upon binding polycyclic aromatic hydrocarbons (PAHs), such as benzo [a]pyrene, the AHR increases the expression of xenobiotic metabolizing enzymes, including the cytochrome P450IA1, P450IA2 and P450IB1-dependent monooxygenases, the glutathione S-transferase Ya subunit, quinone oxidoreductase, and UDP-glucuronosyltransferase (3-8). In response to more potent halogenated aromatic agonists like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the AHR induces xenobiotic metabolism and also mediates a spectrum of toxic responses, including thymic involution, teratogenesis, tumor promotion, wasting, and epithelial hyperplasia and metaplasia (9-11).The AHR is a member of a family of transcription factors containing basic/helix-loop-helix and PAS homology domains (bHLH-PAS) (1). In response to agonist binding within the PAS domain, the cytosolic AHR undergoes a conformational change, translocates to the nucleus, dissociates from the 90-kDa heat shock protein, and dimerizes with a second bHLH-PAS protein known as the Ah receptor nuclear translocator (ARNT) (12-16). This heterodimer interacts with dioxin-responsive enhancer elements upstream of target genes and activates their transcription. Despite our mechanistic understanding of the role of the AHR in regulating xenobiotic metabolism, we still have little understanding of how the AHR mediates the toxic responses of halogenated agonists and why such responses are produced only by high affinity, poorly metabolized ligands such as TCDD.To examine the importance of the AHR in normal vertebrate biology, we have used gene targeting to create mice with a mutation at theAhr locus. We anticipated that such a mutant could provide insights into additional physiological roles of the AHR and would represent a powerful model to understand the toxicology of halogenated aromatic pollutants like TCDD.