The vascular network delivers oxygen (O2) and nutrients to all cells within the body. It is therefore not surprising that O2 availability serves as a primary regulator of this complex organ. Most transcriptional responses to low O2 are mediated by hypoxia-inducible factors (HIFs), highly conserved transcription factors that control the expression of numerous angiogenic, metabolic, and cell cycle genes. Accordingly, the HIF pathway is currently viewed as a master regulator of angiogenesis. HIF modulation could provide therapeutic benefit for a wide array of pathologies, including cancer, ischemic heart disease, peripheral artery disease, wound healing, and neovascular eye diseases. Hypoxia promotes vessel growth by upregulating multiple pro-angiogenic pathways that mediate key aspects of endothelial, stromal, and vascular support cell biology. Interestingly, recent studies show that hypoxia influences additional aspects of angiogenesis, including vessel patterning, maturation, and function. Through extensive research, the integral role of hypoxia and HIF signaling in human disease is becoming increasingly clear. Consequently, a thorough understanding of how hypoxia regulates angiogenesis through an ever-expanding number of pathways in multiple cell types will be essential for the identification of new therapeutic targets and modalities.
angiogenesis; hypoxia; HIFs; vascular diseases; cancer; anti-angiogenic therapies
O2 deprivation (hypoxia) and cellular proliferation engage opposite cellular pathways, yet often coexist during tumor growth. The ability of cells to grow during hypoxia results in part from crosstalk between hypoxia inducible factors (HIFs) and the proto-oncogene c-Myc. Acting alone, HIF and c-Myc partially regulate complex adaptations undertaken by tumor cells growing in low O2. However, acting in concert, these transcription factors reprogram metabolism, protein synthesis and cell cycle progression, to “fine tune” adaptive responses to hypoxic environments.
Low oxygen levels are a defining characteristic of solid tumors, and responses to hypoxia contribute substantially to the malignant phenotype. Hypoxia-induced gene transcription promotes characteristic tumor behaviors including angiogenesis, invasion, metastasis, de-differentiation and enhanced glycolytic metabolism. These effects are mediated, at least in part, by targets of the Hypoxia Inducible Factors (HIFs). The HIFs function as heterodimers, made up of an oxygen-labile α-subunit and a stable (β-subunit, also referred to as ARNT. HIF-1α and HIF-2α stimulate the expression of overlapping as well as unique transcriptional targets, and their induction can have distinct biological effects. New targets and novel mechanisms of dysregulation place the HIFs in an ever more central role in tumor biology, and have led to development of pharmacological inhibitors of their activity.
Lung cancer is the most common and most deadly cancer worldwide. Because of the aggressive and metastatic nature of many forms of the disease, it is frequently diagnosed late and responds poorly to the therapies currently available. Although our understanding of the molecular origins and evolution of lung cancer is still incomplete, recent research has yielded several developments that may offer opportunities for new, targeted and effective therapy. In this review we first discuss the prevalence and origins of lung cancer, with emphasis on non-small-cell lung cancer and adenocarcinoma, together with current treatments and their efficacy. We then look at a selection of recent papers which between them shed new light on possible therapeutic opportunities, including a novel synthetic interaction with the Kras gene and genomic or proteomic profiling studies that may pave the way for personalized treatment for lung cancer based on specific “signatures” of protein and gene expression.
Lung cancer remains the foremost cause of cancer deaths worldwide. Despite advances in both detection and treatment, diagnosis is often late and the prognosis for patients poor. Our understanding of the molecular basis and progression of lung cancer remains incomplete, hampering the design and development of more effective diagnostic tools and therapies for this devastating disease. However, the last twelve months have witnessed the publication of several studies that represent significant advances in our knowledge of lung cancer, and may represent important steps on the road to effective new therapies. In this review we aim to summarize these recent developments, and give our perspectives on the therapeutic possibilities they may offer in the future.
lung cancer; adenocarcinoma; egfr; kras; chemotherapy; synthetic lethal; genomic profiling; customized therapy; cancer stem cells; hypoxia-inducible factor
Oxygen deprivation, or hypoxia, has profound effects on cell metabolism and growth. Cells can adapt to low O2 in part through activation of hypoxia-inducible factor (HIF). We report here that hypoxia inhibits mRNA translation by suppressing multiple key regulators including eIF2α, eEF2, and the mTOR effectors 4EBP1, p70S6K, and rpS6, independent of HIF. Hypoxia results in energy starvation and activation of the AMPK/TSC2/Rheb/mTOR pathway. Hypoxic AMPK activation also leads to eEF2 inhibition. Moreover, hypoxic effects on cellular bioenergetics and mTOR inhibition increase over time. Mutation of the TSC2 tumor suppressor gene confers a growth advantage to cells by repressing hypoxic mTOR inhibition and hypoxia-induced G1 arrest. Together, eIF2α, eEF2 and mTOR inhibition represent important HIF-independent mechanisms of energy conservation which promote survival under low O2 conditions.
Hypoxia; mTOR; eIF2; eEF2; AMPK; TSC2; Rheb
Intra-tumoral hypoxia (low oxygen [O2] level) is an independent indicator of unfavorable patient diagnosis, and increasing evidence demonstrates that hypoxia contributes to a more aggressive tumor phenotype. Adaptation to hypoxia is predominantly regulated by two structurally related hypoxia inducible factors, HIF-1α and HIF-2α, which activate the expression of genes involved in proliferation, metabolism, angiogenesis, and metastasis. While highly homologous, HIF-1α and HIF-2α have been shown to have different roles in tumorigenesis dependent on specific tumor microenvironments. Here we summarize recent studies on HIF-2α and discuss the potential mechanisms whereby it contributes to tumor aggressiveness.
Hypoxia Inducible Factors (HIFs) regulate adaptive responses to changes in oxygen (O2) tension during embryogenesis, tissue ischemia, and tumorigenesis. Because HIF deficient embryos exhibit a number of developmental defects, the precise role of HIF in early vascular morphogenesis has been uncertain. Using para-aortic splanchnopleural (P-Sp) explant cultures we show that deletion of the HIF-β subunit (ARNT) results in defective hematopoiesis and the inhibition of both vasculogenesis and angiogenesis. These defects are rescued upon the addition of wild type Sca-1+ hematopoietic cells or recombinant VEGF. Arnt−/− embryos exhibit reduced levels of VEGF protein and increased numbers of apoptotic hematopoietic cells. These results suggest that HIF coordinates early endothelial cell emergence and vessel development by promoting hematopoietic cell survival and paracrine growth factor production.
HIF; ARNT; hypoxia; splanchnopleural; endothelial cells; hematopoiesis; vasculogenesis; angiogenesis; VEGF
Myeloid cells provide important functions in low oxygen (O2) environments created by pathophysiological conditions, including sites of infection, inflammation, tissue injury and solid tumors. Hypoxia-inducible factors (HIFs) are principle regulators of hypoxic adaptation, regulating gene expression involved in glycolysis, erythropoiesis, angiogenesis, proliferation and stem cell function under low O2. Interestingly, increasing evidence accumulated over recent years suggests an additional important regulatory role for HIFs in inflammation. In macrophages, HIFs not only regulate glycolytic energy generation, but also optimize innate immunity, control pro-inflammatory gene expression, mediate bacterial killing and influence cell migration. In neutrophils, HIF-1α promotes survival under O2-deprived conditions and mediates blood vessel extravasation by modulating β2 integrin expression. Additionally, HIFs contribute to inflammatory functions in various other components of innate immunity, such as dendritic cells, mast cells and epithelial cells. This review will dissect the role of each HIF isoform in myeloid cell function and discuss their impact on acute and chronic inflammatory disorders. Currently, intensive studies are being conducted to illustrate the connection between inflammation and tumorigenesis. Detailed investigation revealing interaction between microenvironmental factors such as hypoxia and immune cells is needed. We will also discuss how hypoxia and HIFs control properties of tumor-associated macrophages and their relationship to tumor formation and progression.
Oxygen (O2) is an essential nutrient that serves as a key substrate in cellular metabolism and bioenergetics. In a variety of physiological and pathological states, organisms encounter insufficient O2 availability, or hypoxia. In order to cope with this stress, evolutionarily conserved responses are engaged. In mammals, the primary transcriptional response to hypoxic stress is mediated by the Hypoxia-inducible factors (HIFs). While canonically regulated by prolyl hydroxylase domain-containing enzymes (PHDs), the HIFα subunits are intricately responsive to numerous other factors including Factor Inhibiting HIF-1α (FIH1), sirtuins, and metabolites. These transcription factors function in normal tissue homeostasis and impinge on critical aspects of disease progression and recovery. Insights from basic HIF biology are being translated into pharmaceuticals targeting the HIF pathway.
MXI1; c-MYC; MAX; HIF-1α; HIF-2α; VHL; renal cell carcinoma
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.
Spermatogenesis, a process involving the differentiation of spermatogonial stem cells into mature spermatozoa, takes place throughout masculine life. A complex system in the testis, including endocrine signaling, physical interactions between germ and somatic cells, spermatocyte meiosis, and timely release of spermatozoa, controls this cycle. We demonstrate herein that decreased O2 levels and Epas1 activation are critical components of spermatogenesis. Postnatal Epas1 ablation leads to male infertility, with reduced testis size and weight. While immature spermatogonia and spermatocytes are present in Epas1Delta/Delta testes, spermatid and spermatozoan numbers are dramatically reduced. This is not due to germ cell-intrinsic defects. Rather, EpasDelta/Delta Sertoli cells exhibit decreased ability to form tight junctions, thereby disrupting the blood-testis barrier necessary for proper spermatogenesis. Reduced numbers of tight junction complexes are due to decreased expression of multiple genes encoding tight junction proteins, including TJP1 (ZO1), TJP2 (ZO2), and occludin. Furthermore, Epas1Delta/Delta testes exhibit disrupted basement membranes surrounding the seminiferous tubules, causing the premature release of incompletely differentiated germ cells. We conclude that low O2 levels in the male gonad regulate germ cell homeostasis in this organ via EPAS1.
Postnatal deletion of Epas1 in the mouse testis impairs the integrity of the blood-testis barrier required for proper spermatogenesis.
developmental biology; hypoxia; Sertoli cells; signal transduction; sperm; spermatogenesis; testis; tight junctions
Circadian oscillations in mammalian physiology and behavior are regulated by an endogenous biological clock. Here we show that loss of the PAS protein MOP3 (also known as BMAL1) in mice results in immediate and complete loss of circadian rhythmicity in constant darkness. Additionally, locomotor activity in light–dark (LD) cycles is impaired and activity levels are reduced in Mop3−/− mice. Analysis of Period gene expression in the suprachiasmatic nucleus (SCN) indicates that these behavioral phenotypes arise from loss of circadian function at the molecular level. These results provide genetic evidence that MOP3 is the bona fide heterodimeric partner of mCLOCK. Furthermore, these data demonstrate that MOP3 is a non-redundant and essential component of the circadian pacemaker in mammals.
The transcriptional response to hypoxia is primarily mediated by two hypoxia-inducible factors – HIF-1α and HIF-2α. While these proteins are highly homologous, increasing evidence suggests they have unique transcriptional targets and differential impact on tumor growth. Furthermore, non-transcriptional effects of the HIF-α subunits, including effects on the Notch and c-Myc pathways, contribute to their distinct functions. HIF-2α transcriptional targets include genes involved in erythropoiesis, angiogenesis, metastasis, and proliferation. Therefore, HIF-2α contributes significantly to both normal physiology as well as tumorigenesis. Here we summarize the function of HIF-2α during development as well as its contribution to pathologic conditions such as tumors and vascular disease.
hypoxia; erythropoiesis; development; renal carcinoma; neuroblastoma
Low levels of oxygen (O2) occur naturally in developing embryos. Cells respond to their hypoxic microenvironment by stimulating several hypoxia-inducible factors (and other molecules that mediate O2 homeostasis), which then coordinate the development of the blood, vasculature, placenta, nervous system, and other organs. Furthermore, embryonic stem and progenitor cells frequently occupy hypoxic ‘niches’ and low O2 regulates their differentiation. Recent work has revealed an important link between factors involved in regulating stem/progenitor cell behaviour and hypoxia-inducible factors, which provides a molecular framework for hypoxic control of differentiation and cell fate. These findings have important implications for the development of therapies for tissue regeneration and disease.
Cellular properties are influenced by complex factors inherent to their microenvironments. While oxygen deprivation (hypoxia) occurs in tumors due to rapid cell proliferation and aberrant blood vessel formation, embryonic cells develop in a naturally occurring hypoxic environment. Cells respond to hypoxia by stabilizing hypoxia-inducible factors (HIFs), which are traditionally viewed to function by altering cellular metabolism and blood vessel architecture. Recently, HIFs have been shown to modulate specific stem cell effectors, such as Notch, Wnt, and Oct4, that control stem cell proliferation, differentiation and pluripotency. Direct molecular links have also been established between HIFs and critical cell signalling pathways such as cMyc and p53. These novel links suggest a new role for HIFs in stem cell and tumor regulation.
Hypoxia-inducible factors (HIF-1, HIF-2); stem cell signalling; cancer pathways
Many organisms activate adaptive transcriptional programs to help them cope with decreased oxygen levels, or hypoxia, in their environment. These responses are triggered by various oxygen sensing systems in bacteria, yeast and metazoans. In metazoans, the hypoxia inducible factors (HIFs) mediate the adaptive transcriptional response to hypoxia by upregulating genes involved in maintaining bioenergetic homeostasis. The HIFs in turn are regulated by HIF-specific prolyl hydroxlase activity, which is sensitive to cellular oxygen levels and other factors such as tricarboxylic acid cycle metabolites and reactive oxygen species (ROS). Establishing a role for ROS in cellular oxygen sensing has been challenging since ROS are intrinsically unstable and difficult to measure. However, recent advances in fluorescence energy transfer resonance (FRET)-based methods for measuring ROS are alleviating some of the previous difficulties associated with dyes and luminescent chemicals. In addition, new genetic models have demonstrated that functional mitochondrial electron transport and associated ROS production during hypoxia are required for HIF stabilization in mammalian cells. Current efforts are directed at how ROS mediate prolyl hydroxylase activity and hypoxic HIF stabilization. Progress in understanding this process has been enhanced by the development of the FRET-based ROS probe, an vivo prolyl hydroxylase reporter and various genetic models harboring mutations in components of the mitochondrial electron transport chain.
reactive oxygen species; prolyl hydroxylase; mitochondria; cellular oxygen sensing; hypoxia; hypoxia inducible factor
The basic helix-loop-helix-Per-ARNT-Sim–proteins hypoxia-inducible factor (HIF)-1α and HIF-2α are the principal regulators of the hypoxic transcriptional response. Although highly related, they can activate distinct target genes. In this study, the protein domain and molecular mechanism important for HIF target gene specificity are determined. We demonstrate that although HIF-2α is unable to activate multiple endogenous HIF-1α–specific target genes (e.g., glycolytic enzymes), HIF-2α still binds to their promoters in vivo and activates reporter genes derived from such targets. In addition, comparative analysis of the N-terminal DNA binding and dimerization domains of HIF-1α and HIF-2α does not reveal any significant differences between the two proteins. Importantly, replacement of the N-terminal transactivation domain (N-TAD) (but not the DNA binding domain, dimerization domain, or C-terminal transactivation domain [C-TAD]) of HIF-2α with the analogous region of HIF-1α is sufficient to convert HIF-2α into a protein with HIF-1α functional specificity. Nevertheless, both the N-TAD and C-TAD are important for optimal HIF transcriptional activity. Additional experiments indicate that the ETS transcription factor ELK is required for HIF-2α to activate specific target genes such as Cited-2, EPO, and PAI-1. These results demonstrate that the HIF-α TADs, particularly the N-TADs, confer HIF target gene specificity, by interacting with additional transcriptional cofactors.
Increased cap-dependent mRNA translation rates are frequently observed in human cancers. Mechanistically, many human tumors often overexpress the cap binding protein eukaryotic translation initiation factor 4E (eIF4E), leading to enhanced translation of numerous tumor-promoting genes. In this issue of the JCI, Graff and colleagues describe potent antitumor effects using second-generation antisense oligonucleotides for eIF4E (see the related article beginning on page 2638). If their results are recapitulated in a clinical setting, this strategy will provide a promising antitumor therapy with broad-reaching applications.
Germ line PERK mutations are associated with diabetes mellitus and growth retardation in both rodents and humans. In contrast, late embryonic excision of PERK permits islet development and was found to prevent onset of diabetes, suggesting that PERK may be dispensable in the adult pancreas. To definitively establish the functional role of PERK in adult pancreata, we generated mice harboring a conditional PERK allele in which excision is regulated by tamoxifen administration. Deletion of PERK in either young adult or mature adult mice resulted in hyperglycemia associated with loss of islet and β cell architecture. PERK excision triggered intracellular accumulation of proinsulin and Glut2, massive endoplasmic reticulum (ER) expansion, and compensatory activation of the remaining unfolded-protein response (UPR) signaling pathways specifically in pancreatic tissue. Although PERK excision increased β cell death, this was not a result of decreased proliferation as previously reported. In contrast, a significant and specific increase in β cell proliferation was observed, a result reflecting increased cyclin D1 accumulation. This work demonstrates that contrary to expectations, PERK is required for secretory homeostasis and β cell survival in adult mice.
Oncogenic Myc alters mitochondrial metabolism, making it dependent on exogenous glutamine (Gln) for cell survival. Accordingly, Gln deprivation selectively induces apoptosis in MYC-overexpressing cells via unknown mechanisms. Using MYCN-amplified neuroblastoma as a model, we identify PUMA, NOXA and TRB3 as executors of Gln-starved cells. Gln depletion in MYC-transformed cells induces apoptosis through ATF4-dependent, but p53-independent, PUMA and NOXA induction. MYC-transformed cells depend on both glutamate-oxaloacetate transaminase and glutamate dehydrogenase to maintain Gln homeostasis and suppress apoptosis. Consequently, either ATF4 agonists or glutaminolysis inhibitors potently induce apoptosis in vitro and inhibit tumor growth in vivo. These results reveal mechanisms whereby Myc sensitizes cells to apoptosis and validate ATF4 agonists and inhibitors of Gln metabolism as potential Myc-selective cancer therapeutics.
Malignant melanoma is characterized by a propensity for early lymphatic and hematogenous spread. The hypoxia-inducible factor (HIF) family of transcription factors is upregulated in melanoma by key oncogenic drivers. HIFs promote the activation of genes involved in cancer initiation, progression, and metastases. Hypoxia has been shown to enhance the invasiveness and metastatic potential of tumor cells by regulating the genes involved in the breakdown of the ECM as well as genes that control motility and adhesion of tumor cells. Using a Pten-deficient, Braf-mutant genetically engineered mouse model of melanoma, we demonstrated that inactivation of HIF1α or HIF2α abrogates metastasis without affecting primary tumor formation. HIF1α and HIF2α drive melanoma invasion and invadopodia formation through PDGFRα and focal adhesion kinase–mediated (FAK-mediated) activation of SRC and by coordinating ECM degradation via MT1-MMP and MMP2 expression. These results establish the importance of HIFs in melanoma progression and demonstrate that HIF1α and HIF2α activate independent transcriptional programs that promote metastasis by coordinately regulating cell invasion and ECM remodeling.