) levels are known to vary widely across sub-domains of solid tumors, due to rapid cell division and aberrant tumor angiogenesis and blood flow. Although extended exposure to complete O2
deprivation (anoxia) can result in necrosis, viable hypoxic cancer cells often surround necrotic zones. Tumor hypoxia has long been associated with increased malignancy, poor prognosis and resistance to radiotherapy and chemotherapy (reviewed in1,2
), prompting intensive research into cellular responses to O2
deprivation. Particular interest has been focused on the mechanisms by wh ich hypoxic tumor cells alter their transcriptional profiles to modulate glycolysis, proliferation, survival and invasion to persist under conditions of hypoxic stress3
The Hypoxia Inducible Factor (HIF) transcription factors mediate the primary transcriptional responses to hypoxic stress in normal and transformed cells. HIFs are heterodimeric complexes composed of bHLH-PAS proteins including an O2
-Iabile alpha subunit (HIF1α, HIF2α, or HIF3α) and a stable beta subunit (HIF1β, also known as ARNT), which together bind hypoxia responsive elements (HREs) containing a conserved RCGTG core sequence (see Box 1
). Hypoxic HIF activity is controlled primarily through post-translational modification and stabilization of HIF1α and HIF2α subunits, so that HIFa protein levels and overall HIF transcriptional activity increase as cells become more hypoxic. The central molecular mechanisms underlying the O2
-lability of HIFa subunits were first elaborated in 2001 by multiple groups, and are the subject of several recent reviews4,5
). Briefly, HIFa subunits are modified by HIF-specific prolyl-hydroxylases (PHDs) in the presence of O2
, leading to normoxic proteasomal degradation mediated in part by the Von Hippel Lindau tumor suppressor protein (pVHL) (Box 1
). It is also important to note that elevated oncogenic signaling in cancer cells can induce HIFa expression through O2
-independent mechanisms including increased transcription and/or translation of HIFα mRNAB6
Box 1. O2-dependent HIF regulation
Using molecular O2
and 2-oxoglutarate as substrates, HIF
prolyl hydroxlase (PHD) enzymes4
hydroxylate two specific proline residues that reside in the 02-dependent degradation domain (ODD) of HIF-a proteins. These hydroxylation events occur on P402 and P564 in HIF1α, and P405 and P531 in HIF2α, respectively, and are required for the Von Hippel-Lindau (pVHL) tumor suppressor protein, the recognition component of an E3-ubiquitin ligase, to bind and degrade HIFα subunits under normoxic conditions. Hypoxia inhibits PHD activity through a number of mechanisms, including substrate limitation (reviewed in4
), resulting in HIFα stabilization, heterodimerization with HIF1β/ARNT, and increased HIF
transcriptional activity. Hypoxic conditions also inhibit a second hydroxylation of a conserved HIFα C-terminal asparagine residue by the FIH hydroxylase, an event that blocks the interaction between HIFα and the transcriptional co-activators p300/CBp149-151
. Thus, whereas PHD-mediated hydroxylation destabilizes HIFα subunits, FIH-mediated hydroxylation inhibits their transcriptional activity.
HIF1α was first described by Semenza and colleagues in 1995, and was shown to playa central role in mediating O2
-dependent transcriptional responses7
. The identification of HIF2α by independent groups in 1997 (initially called endothelial PAS protein 1 (EPAS1)8
, HIF-related factor (HRF)9
, HIF1α-like factor (HLF)10
, and member of PAS family 2 (MOP2)11
) indicated that HIF regulation was more complex. Whereas HIF1α appears to be expressed in nearly all cell types, RNA in situ hybridization on mouse embryos revealed that HIF2α expression is more restricted, and particularly abundant in blood vessels. This observation led to the hypothesis that the primary role of HIF2α is to modulate vascular endothelial cell (Ee) function, an idea supported in part by the close correlation of HIF2α and VEGF mRNA expression patterns8
. A more complex view emerged as HIF2α protein expression was identified in multiple cell types in hypoxic rat kidney, lung, and colonic epithelia, as well as hepatocytes, macrophages, muscle cells and astrocytes12
, indicating that both HIF1α and HIF2α are co-expressed in a large number of cell types.
The majority of HIF transcriptional responses have been attributed to HIF1α and HIF2α; however, a third HIFα subunit (HIF3α) has also been described13
. HIF3α mRNA is differentially spliced to produce multiple isoforms that either promote or inhibit the activity of other HIF complexes, although little is yet known about the impact of HIF3α on hypoxic tumor progression14-17
. Similarly, a second ARNT protein (ARNT2) has been identified18
and shown to regulate neuronal development19
and exhibit overlapping activity with ARNT20
; however, its activity in human cancer cells has not been studied in depth21
. Although it will be important to determine whether (and how) HIF3α and ARNT2 affect HIF-mediated responses in cancers, the available evidence suggests that HIF1α and HIF2α account for the vast majority of HIF-dependent effects on tumor growth and progression described to date.
Elevated expression of HIF1α and HIF2α protein has been observed in a broad array of human cancer cell types, and associated with poor prognosis in many cases (). Particular attention has been focused on renal clear cell carcinomas (RCCs), approximately 90% of which lose function of the Von Hippel-Landau tumor suppressor protein (pVHL), which binds prolyl-hydroxylated HIFα subunits and targets them for ubiquitin-mediated proteolysis22
). pVHL-deficient RCC cell lines consequently cannot degrade HIFα subunits in an 02-dependent manner, and have been used extensively to investigate the roles of HIF1α and HIF2α in tumor growth.
Correlation between HIFa protein expression and poor prognosis in human cancers*
The observations summarized in have led to the general view that elevated HIFα protein expression in tumor cells, whether induced by hypoxia or aberrant oncogenic signaling, actively drives tumor growth and progression by regulating the expression of critical target genes. Disparate correlations have been observed in some tumor types; for example, HIF1α expression has been associated with both better and worse prognosis in separate analyses of renal and non-small cell lung cancers (see ). The basis of these apparent discrepancies is not understood, but may reflect the consequences of HIF activity in different cancer subtypes, or at different stages of tumor progression. In some tumors, including gastric cancers and glioma, only one HIFα subunit is correlated with prognosis, suggesting it plays a particularly important role or predominant role in these tumor cell types. Interestingly, multiple recent studies have also revealed unexpected tumor suppressive activities of HIF1α and HIF2α in specific contexts23-26
. Although initially viewed as having largely overlapping functions, there is now mounting evidence that HIF1α and HIF2α can promote highly divergent – even opposing – outcomes when expressed in the same cell type. It appears that HIF1α and HIF2α mediate these disparate responses partly through independent regulation of distinct target genes, but also through direct and indirect interactions with complexes containing important oncoproteins and tumor suppressors.