Hypoxia is a fundamental stimulus that impacts cells, tissues, organs, and physiological systems. The discovery of hypoxia-inducible factor-1 (HIF-1) and subsequent identification of other members of the HIF family of transcriptional activators has provided insight into the molecular underpinnings of oxygen homeostasis. This review focuses on the mechanisms of HIF activation and their roles in physiological and pathophysiological responses to hypoxia, with an emphasis on the cardiorespiratory systems. HIFs are heterodimers comprised of an O 2 -regulated HIF-1α or HIF-2α subunit and a constitutively expressed HIF-1β subunit. Induction of HIF activity under conditions of reduced O 2 availability requires stabilization of HIF-1α and HIF-2α due to reduced prolyl hydroxylation, dimerization with HIF-1β, and interaction with coactivators due to decreased asparaginyl hydroxylation. Stimuli other than hypoxia, such as nitric oxide and reactive oxygen species, can also activate HIFs. HIF-1 and HIF-2 are essential for acute O 2 sensing by the carotid body, and their coordinated transcriptional activation is critical for physiological adaptations to chronic hypoxia including erythropoiesis, vascularization, metabolic reprogramming, and ventilatory acclimatization. In contrast, intermittent hypoxia, which occurs in association with sleep-disordered breathing, results in an imbalance between HIF-1α and HIF-2α that causes oxidative stress, leading to cardiorespiratory pathology.
Proc Amer Assoc Cancer Res, Volume 46, 2005 SY06-4 In many human cancers, rapid cell proliferation in associated with the elaboration of tumor vasculature which is structurally and functionally abnormal, resulting in perfusion that is characterized by marked spatial and temporal heterogeneity. Studies of human cancers that are accessible to direct PO2 measurement by Eppendorf microelectrodes, such as cancers of the uterine cervix or head and neck, have led to two important findings. First, intratumoral oxygen concentrations are markedly reduced relative to surrounding normal tissue. Second, the risk that a tumor will invade surrounding tissue, metastasize to distant sites, fail ot respond to radiation and/or chemotherapy, and result in patient mortality is significantly increased in those tumors with the greatest degree of hypoxia (PO2 < 10 mm Hg). Hypoxia-inducible factor 1 (HIF-1) activates the transcription of genes involved in critical aspects of cancer biology, including angiogenesis (vascular endothelial growth factor [VEGF], placental growth factor), cell proliferation and survival (insulin-like growth factor [IGF] 2, transforming growth factor [TGF] α ), glucose metabolism (glucose transporter 1 and 3, hexokinase 1 and 2, lactate dehydrogenase A), and invasion/metastasis (C-MET, CXCR4, matrix metalloproteinase 2, urokinase plasminogen activator receptor). HIF-1 is a heterodimer, consisting of a constitutively-expressed HIF-1β subunit and a HIF-1α subunit, the expression of which is oxygen- and growth factor-regulated. A related protein, designated HIF-2α, is also oxygen-regulated and can dimerize with HIF-1β. Overexpression of HIF-1α in human colon or pancreatic cancer cells or transformed mouse embryo fibroblasts (MEFs) results in increased xenograft growth in nude mice. HIF-1α overexpression leads to increased tumor growth by at least two different mechanisms. HIF-1α overexpression in colon cancer cells induces increased tumor vascularization. In contrast, HIF-1α overexpression in pancreatic cancer cells results in increased glycolytic metabolism and protection against hypoxia-induced apoptosis. In transformed MEFs, loss of HIF-1 activity is associated with decreased resistance to oxygen deprivation, radiation, and chemotherapeutic agents that induce double-strand DNA breaks. Immunohistochemical studies of human cancer biopsies have revealed that HIF-1α is overexpressed in the vast majority of human primary cancers and metastases. In cancers of the brain, breast, colon, endometrial, head and neck, lung, ovary, and pancreas, HIF-1α overexpression is associated with increased microvessel density and/or VEGF expression. In breast and prostate, HIF-1α overexpression occurs at the pre-invasive stage (ductal carcinoma in situ and prostatic intraepithelial neoplasia, respective). Most importantly, HIF-1α overexpression is associated with increased risk of mortality in gastrointestinal stromal cell tumor of the stomach, oligodendroglioma, and cancers of the breast, cervix, endometrium, oropharynx, and ovary. In the case of breast and cervical cancer, HIF-1α overexpression in early stage disease is associated with increased mortality. HIF-2α overexpression is associated with increased risk of mortality in head and neck cancer and malignant melanoma. HIF-1α overexpression is also associated with resistance to photodynamic and radiation therapy in early-stage esophageal cancer and oropharyngeal squamous cell carcinoma. Analysis of oropharyngeal squamous cell carcinomas revealed two different patterns of HIF-1α overexpression. In approximately two-thirds of the tumor biopsy sections analyzed, HIF-1α was expressed at high levels in cells surrounding areas of necrosis, representing viable cells that were farthest removed from a blood vessel and subjected to the greatest degree of hypoxia. In the remaining cases, HIF-1α was expressed at high levels throughout the section, including cells immediately adjacent to a patent blood vessel, suggesting that a mechanism other than hypoxia was responsible. In support of this hypothesis, increased HIF-1α expression has been shown to be associated with multiple genetic alterations in human cancer cells. Most dramatic is the effect in clear cell renal carcinoma (RCC) of loss-of-function mutations in the VHL gene, which encodes the von Hippel-Lindau tumor suppressor. VHL is the recognition subunit of an E3 ubiquitin-protein ligase that targets HIF-1α and HIF-2α for proteasomal degradation under non-hypoxic conditions. In the absence of VHL, HIF-1α and HIF-2α are constitutively expressed, resulting in dysregulated expression of HIF-1 target genes such as VEGF. As a result, RCC are among the most highly vascularized human tumors. Dysregulated expression of the HIF-1 target gene TGF-α in RCC cells, which express the epidermal growth factor receptor (EGFR), may establish an autocrine signaling loop. In addition, HIF-1-dependent activation of C-MET and CXCR4 may play critical roles in RCC invasion and metastasis. Thus, in RCC, dysregulated HIF-1 activity provides a molecular basis for at least three of the hallmarks of cancer: sustained angiogenesis, proliferation in the absence of exogenous growth factors, and tissue invasion/metastasis. Loss-of-function for other tumor suppressors, including p53, PTEN, and p14ARF, has been shown result in significant but more modest increases in HIF-1α expression. In addition, increased activity of the EGFR, IGF1R, HER2neu (ERBB2), SRC tyrosine kinases and/or the MAP kinase and phosphatidylinositol 3-kinase signal transduction pathways is associated with increased HIF-1 activity. Remarkably, whereas hypoxia increases HIF-1α stability, MAP kinase and phosphatidylinositol 3-kinase signaling have been shown to increase HIF-1α synthesis. Thus, oncogene gain-of-function may increase HIF-1α expression in a manner that is independent of and additive to that of hypoxia. Taken together, the data presented suggest that increased HIF-1 activity contributes to the increased risks of invasion, metastasis, treatment failure, and mortality that are associated with intratumoral hypoxia. Increased HIF-1 activity may contribute to the process by which oncogenic mutations are selected during the clonal evolution of human cancers. Anti-angiogenic effects of several novel signal transduction inhibitors appear to be due in part to their inhibition of HIF-1 activity. Efforts are currently underway to identify and evaluate selective HIF-1 inhibitors as novel anti-cancer agents.
Erythropoietin (EPO) is the primary humoral regulator of mammalian erythropoiesis. The single-copy EPO gene is normally expressed in liver and kidney, and increased transcription is induced by anemia or cobalt chloride administration. To identify cis-acting DNA sequences responsible for regulated expression, transgenic mice were generated by microinjection of a 4-kilobase-pair (kb) (tgEPO4) or 10-kb (tgEPO10) cloned DNA fragment containing the human EPO gene, 0.7 kb of 3'-flanking sequence, and either 0.4 or 6 kb of 5'-flanking sequence, respectively. tgEPO4 mice expressed the transgene in liver, where expression was inducible by anemia or cobalt chloride, kidney, where expression was not inducible, and other tissues that do not normally express EPO. Human EPO RNA in tgEPO10 mice was detected only in liver of anemic or cobalt-treated mice. Both tgEPO4 and tgEPO10 mice were polycythemic, demonstrating that the human EPO RNA transcribed in liver is functional. These results suggest that (i) a liver inducibility element maps within 4 kb encompassing the gene, 0.4 kb of 5'-flanking sequence, and 0.7 kb of 3'-flanking sequence; (ii) a negative regulatory element is located between 0.4 and 6 kb 5' to the gene; and (iii) sequences required for inducible kidney expression are located greater than 6 kb 5' or 0.7 kb 3' to the gene. RNase protection analysis revealed that human EPO RNA in anemic transgenic mouse liver and hypoxic human hepatoma cells is initiated from several sites, only a subset of which is utilized in nonanemic transgenic liver and human fetal liver.
Abstract As in the case of the HOX proteins (described in Chapter 9), the PAX proteins represent a family of transcription factors whose members ap pear to play key roles in the development of both invertebrates and vertebrates (reviewed by Chalepakis et al., 1993; Strachan and Read, 1994; Stuart and Gruss, 1995). Nine PAX genes have been identified to date in mice and humans (Fig. 7.1). Unlike the HOX genes, which are clustered at four loci in the human and mouse genomes, mapping of the PAX genes revealed no clustering even though several of the genes have similar genomic structure and coding sequences. The proteins encoded by these genes have in common a unique 128- amino-acid DNA-binding domain, the paired domain. Although the paired domain contains three regions that are predicted to form a heli ces, there is no sequence similarity to the helix-turn-helix motif present in homeodomains. In addition to the paired domain, which is present in all nine PAX proteins, four of the proteins (PAX3, PAX4, PAX6, and PAX7) also contain a homeodomain that contributes to the affinity and specificity of DNA binding by these proteins. Curiously, three PAX proteins (PAX2, PAXS, and PAX8) contain truncated homeodomains of un known function. PAX 1 and PAX9 lack homeodomain-related sequences altogether and must therefore rely entirely on the paired domain for DNA-binding activity.
Hypoxia is a physiological cue that impacts diverse physiological processes, including energy metabolism, autophagy, cell motility, angiogenesis, and erythropoiesis. One of the key cell-autonomous effects of hypoxia is as a modulator of cell proliferation. For most cell types, hypoxia induces decreased cell proliferation, since an increased number of cells, with a consequent increase in O 2 demand, would only exacerbate hypoxic stress. However, certain cell populations maintain cell proliferation in the face of hypoxia. This is a common pathological hallmark of cancers, but can also serve a physiological function, as in the maintenance of stem cell populations that reside in a hypoxic niche. This review will discuss major molecular mechanisms by which hypoxia regulates cell proliferation in different cell populations, with a particular focus on the role of hypoxia-inducible factors.
Human breast tumors contain regions of hypoxia in which cells that are located far from a functional blood vessel have significantly reduced oxygen concentrations when compared with normal mammary tissue. Breast cancer cells adapt to hypoxic conditions by increasing levels of hypoxia-inducible factors (HIFs), which induce the expression of multiple genes involved in angiogenesis, glucose utilization, resistance to oxidative stress, cell proliferation, resistance to apoptosis, invasion and metastasis. Breast cancer patients with increased HIF expression levels in primary tumor biopsies are at increased risk of metastasis. This is an important finding since 90% of breast cancer deaths are the result of metastasis, primarily to the bone, lungs, liver, brain and regional lymph nodes. Although the prognostic significance of reduced oxygen levels in primary breast tumors of cancer patients is well recognized, the mechanisms underlying hypoxia-induced, HIF-dependent breast cancer metastasis are just beginning to be uncovered. Recent studies have implicated HIF target genes in every step of the metastatic process. Drugs, such as digoxin, show the potential therapeutic effects of blocking HIF activity by decreasing primary tumor growth, vascularization, invasion and metastasis in animal models of breast cancer.
Prostaglandin E(2) (PGE(2)) has been implicated as an inducer of angiogenesis in human colon cancer. Here, we demonstrate that PGE(2) exposure induces the expression of vascular endothelial growth factor (VEGF) mRNA in HCT116 human colon carcinoma cells that is mediated by the transcriptional activator hypoxia-inducible factor 1 (HIF-1). PGE(2) exposure induces the phosphorylation of extracellular signal-regulated kinase (ERK) and AKT. Pharmacologic inhibition of ERK phosphorylation blocks the induction of VEGF mRNA and HIF-1alpha protein expression in response to PGE(2) stimulation. Inhibition of C-SRC tyrosine kinase activity also blocks PGE(2)-induced HIF-1alpha protein and VEGF mRNA expression without blocking ERK phosphorylation. In contrast, phosphorylation of AKT is dependent on ERK and C-SRC activity. Thus, the activity of multiple signal transduction pathways is required for the HIF-1-mediated induction of VEGF expression in colon cancer cells exposed to PGE(2).
