Hypoxia-inducible factors: central regulators of the tumor phenotype
Introduction
Hypoxia occurs when available oxygen falls below 5%, triggering a complex cellular and systemic adaptation mediated primarily through transcription by hypoxia-inducible factors (HIFs). HIF-1α was first identified as a crucial regulator of erythropoietin expression in response to low oxygen [1]. HIF-2α and HIF-3α have also been described, with HIF-3α, also known as IPAS (inhibitory PAS domain protein), functioning as an inhibitor of transcription [2, 3]. More than 100 HIF target genes have been identified in a variety of systems (Figure 1). These include genes that encode angiogenesis-promoting factors such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor, glycolytic enzymes such as aldolase A and phosphoglycerate kinase, and cell cycle regulators such as p21 and p27, in addition to other proteins involved in extracellular matrix remodeling, differentiation and apoptosis [4, 5, 6, 7]. HIF-1α and HIF-2α, complexed with the β-subunits ARNT and (more rarely) ARNT2, bind DNA at hypoxia response elements (HREs) [8, 9]. The biological significance and transcriptional effects of HIF-3α remain somewhat obscure, and only HIF-1α and HIF-2α are discussed further in this review.
HIF-α subunits are continuously transcribed and translated, and their stability is regulated by oxygen availability. Under normoxic conditions, two prolines (at positions 402 and 564 in human HIF-1α, and 405 and 531 in human HIF-2α) in the HIF-α oxygen-dependent degradation domain (ODD) are hydroxylated by a family of oxygen-dependent proline hydroxylases (PHD1, PHD2 and PHD3) [10, 11, 12, 13], enabling binding and ubiquitylation by the von Hippel-Lindau (VHL) tumor suppressor, a component of an E3 ubiquitin ligase complex [14] (Figure 2). Interaction of HIF-α subunits with the transcriptional co-activator p300 is also regulated by oxygen levels, and binding is inhibited by oxygen-dependent asparaginyl hydroxylation (asparagines 803 in human HIF-1α, and 851 in human HIF-2α) of the HIF transactivation domain by factor-inhibiting HIF (FIH) [15, 16].
VHL disease is a hereditary cancer syndrome marked by clear-cell renal carcinoma (RCC), pheochromocytoma and hemangioblastoma. The VHL tumor suppressor protein (pVHL) is required for normoxic degradation of the HIF-α subunits and can also target atypical protein kinase Cλ and some subunits of RNA polymerase for degradation [17]. The pathological stabilization of HIF-2α under normoxia is necessary for the growth of VHL-null RCC and hemangioblastoma. Re-expression of pVHL in an VHL−/− RCC cell line blocks xenograft formation in nude mice. However, xenograft growth is rescued by expression of a normoxically stable mutant of HIF-2α [18, 19], but not stabilized HIF-1α [20]. Liver-specific deletion of Vhl is sufficient to generate hemangiomas in transgenic mice. This effect still occurs in mice with a combined Vhl and Hif-1α deletion, but is abrogated if Vhl is deleted in combination with the common β-subunit Arnt [21]. By contrast, pheochromocytoma results from an HIF-independent effect of pVHL on JunB [22•]. The HIFs also play an important role in non-inherited malignancies. There is substantial clinical data associating HIF-α protein expression with poor outcomes in patients with a broad range of sporadic cancers. These include adenocarcinoma of the breast, lung and gastrointestinal tract, as well as central nervous system (CNS) malignancies and squamous cell tumors of the cervix, head and neck [5]. Data from mouse allograft studies have been less consistent. In some cases, disruption of Hif-1α inhibited allograft growth [23, 24], but in others it promoted it [25, 26]. Consistent inhibition of tumor growth has been observed following the stabilization of HIF-1α in normoxia due to Vhl loss [20, 27, 28, 29]. Similarly, overexpression of either HIF-1α or HIF-2α in glioma models correlates with decreased tumor growth [25, 30].
Section snippets
Regulation of HIF stability and expression
The normoxic degradation of the HIF-α subunits is well characterized, but its inhibition under hypoxia is an area of active investigation, and remains controversial. Given that oxygen is required for hydroxylation, it is a limiting substrate under anoxic (0% O2) conditions. However, HIF-α proteins are stabilized in a reactive oxygen species (ROS)-dependent fashion well above this threshold. Early evidence showed that inhibitors of mitochondrial ROS generation were able to block hypoxic HIF-α
Control of HIF-α translation
The mammalian target of rapamycin (mTOR) kinase responds to nutrient and growth factor availability to regulate translation. Normoxic HIF-α expression is promoted by disruption of mTOR regulation, resulting from increased HIF-α translation rates despite unaltered levels of degradation. This is likely to occur in many tumors that show hyperactivation of receptor tyrosine kinases, and thus translation [42], but is also seen in several inherited tumor syndromes. Loss of the tuberous sclerosis 2
HIF-1α versus HIF-2α
Discovered first and expressed ubiquitously, HIF-1α is by far the best characterized α-subunit. HIF-2α expression is limited to endothelium, kidney, heart, lung and gastrointestinal epithelium, and some cells of the CNS [3, 46, 47]. Differences exist in their targets, with HIF-1α uniquely activating glycolytic enzyme genes and HIF-2α preferentially activating VEGF, transforming growth factor-α (TGFα), lysyl oxidase, Oct4 and Cyclin D1 [7, 48, 49••, 50, 51, 52•]. Similarly, the effects of Hif-1α
HIF transcriptional targets
A series of microarray studies have defined HIF target gene families [6, 7, 50, 60, 61, 62, 63]. Erythropoeisis, angiogenesis, and glycolytic metabolism are controlled through multiple gene targets, with differential activation being based on cell type and which HIF-α subunit is expressed. Continued analysis is expanding our understanding of how some of these responses are mediated. HIF-1α-mediated induction of glycolytic metabolism has been well appreciated, but the inhibition of aerobic
HIF and cancer therapy
Pharmacologic inhibition of the HIF target VEGF has proven efficacy as a cancer therapeutic [73] and has generated interest in targeting global HIF activity. Direct approaches such as inhibition of p300-mediated co-activation [74] and DNA binding [75] are being explored, as is HIF inhibition through repression of its translation. HIF-α subunits appear to be particularly sensitive to translational regulation, because the use of pharmacological mTOR inhibitors can block HIF-α expression even
Conclusion
In addition to important roles in development, hematopoiesis, and ischemic disease, the HIFs also have a broad range of effects on tumor biology. They are directly responsible for tumor angiogenesis and metastasis, and contribute substantially to metabolic changes, the evasion of apoptosis, and genomic instability. Despite the appreciation of their relevance to tumor biology, novel targets and mechanisms are reported frequently. Their pharmacological inhibition represents an opportunity and a
Update
In a recently published report, Holmquist-Mengelbier and colleagues [81•] describe a novel difference between HIF-1α and HIF-2α, with important effects in neuroblastoma. They describe the stabilization of HIF-2α at relatively mild levels of hypoxia (5% oxygen) and over prolonged time courses, compared with HIF-1α. Using a tissue microarray, they show a strong correlation between HIF-2α protein expression, vascularization and poor clinical outcomes. It will be of great interest to assess the
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
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