HIF-1 Target Genes Research Articles

Cross-talk between constitutive androstane receptor and hypoxia-inducible factor in the regulation of gene expression

Hypoxia inducible factor (HIF) and 5′-AMP-activated protein kinase are often activated under similar physiological conditions. Constitutive androstane receptor (CAR) translocates into the nucleus in accordance with 5′-AMP-activated protein kinase and thus confers transactivation. The aim of the present study was to investigate a possible link between CAR and HIFα. Phenobarbital (PB), a typical CAR activator, increased the gene expression of HIF-target genes in the livers of mice, including erythropoietin, heme oxygenase-1 and vascular endothelial growth factor-a. PB induced an accumulation of nuclear HIF-1α and an increase in the HIF-responsive element-mediated transactivation in HepG2 cells. Cobalt chloride, a typical HIF activator, induced the gene expression of CAR-target genes, including cyp2b9 and cyp2b10, an accumulation of nuclear CAR and an increase in the PB-responsive enhancer module-mediated transactivation in the mouse liver. Immunoprecipitation-immunoblot and chromatin immunoprecipitation analyses suggest that CAR binds to the PB-responsive enhancer module with HIF-1α in the liver of untreated mice and that the complex dissociates upon PB treatment. Taken together these results suggest that CAR and HIF-α interact and reciprocally modulate the functions of each other.

Hypoxia-inducible factor prolyl 4-hydroxylases: common and specific roles

Hypoxia-inducible transcription factor (HIF), an αβ dimer, is the key inducer of hypoxia-responsive genes that operate both during normal development and pathological processes in association with decreased oxygen availability. The products of HIF target genes function in, e.g., hematopoiesis, angiogenesis, iron transport, glucose utilization, resistance to oxidative stress, cell proliferation, survival and apoptosis, extracellular matrix homeostasis, and tumorigenesis and metastasis. HIF is accumulated in hypoxia, whereas it is rapidly degraded in normoxic cells. The oxygen-sensing mechanism behind this phenomenon is provided by HIF prolyl 4-hydroxylases (HIF-P4Hs, commonly known as PHDs and EglNs) that require oxygen in their reaction. In normoxia, two prolines in the oxygen-dependent degradation domain of the HIFα subunit become hydroxylated by the HIF-P4Hs. The 4-hydroxyproline residues formed serve as recognition sites for the von Hippel-Lindau E3 ubiquitin ligase complex and result in subsequent ubiquitination and instant proteasomal degradation of HIFα in normoxia. The HIF-P4H reaction is inhibited in hypoxia. HIFα evades degradation and forms a functional dimer with HIFβ, leading to activation of the HIF target genes. The central role of HIF-P4Hs in the regulation of the hypoxia response pathway has provided an attractive possibility as a drug candidate for treatment of, e.g., severe anemias and ischemic conditions, and several companies are currently carrying out clinical studies on the use of HIF-P4H inhibitors to treat anemia in patients with a kidney disease. Therefore, it is important to understand the effects of individual HIF-P4H isoenzymes on the hypoxia response and potential other pathways in vivo. The common and specific functions of the HIF-P4H isoenzymes are discussed in this review on the basis of available data from cell biological studies and gene-modified animals.

Role of hypoxia inducible factor-1α for interferon synthesis in mouse dendritic cells

Dendritic cells (DCs) are an important link between innate and adaptive immunity. DCs get activated in inflamed tissues where oxygen tension is usually low, which requires the transcription factor hypoxia inducible factor (HIF)-1 for cellular adaptation. To investigate whether the HIF-1 transcriptional complex plays a pivotal role in the function of DCs, we compared the effects of exogenous inflammatory stimuli and hypoxia on HIF-1α in bone marrow-derived DCs from wild type and myeloid-specific HIF-1α knock-out mice. We showed that the Toll-like receptor ligands lipopolysaccharides and cytosine-phosphatidyl-guanines significantly induce HIF-1α mRNA and protein, leading to elevated HIF-1 target gene expression of vascular endothelial growth factor. In contrast, polyinosinic:polycytidylic acid did not show comparable effects. Furthermore the potential to up-regulate inflammatory cytokines critically influences DC function. Our data demonstrate that HIF-1α protein is needed for adequate production of interferon-α and -β. In co-cultures of DCs and cytotoxic T cells, we observed that DCs lacking active HIF-1α protein induce significantly less CD278 and granzyme B mRNA in T cells. We conclude that HIF-1α plays a crucial role in DC interferon production and T cell activation, linking the innate and adaptive immune system.

Allosteric inhibition of hypoxia inducible factor-2 with small molecules

Hypoxia Inducible Factors (HIFs) are heterodimeric transcription factors induced in many cancers where they frequently promote the expression of many protumorigenic pathways. Though transcription factors are typically considered “undruggable”, the PAS-B domain of the HIF-2α subunit contains a large cavity within its hydrophobic core that offers a unique foothold for small-molecule regulation. Here we identify artificial ligands that bind within this pocket and characterize the resulting structural and functional changes caused by binding. Notably, these ligands antagonize HIF-2 heterodimerization and DNA-binding activity in vitro and in cultured cells, reducing HIF-2 target gene expression. Despite the high identity between HIF-2α and HIF-1α, these ligands are highly selective and do not affect HIF-1 function. These chemical tools establish the molecular basis for selective regulation of HIF-2, providing potential therapeutic opportunities to intervene in HIF-2-driven tumors such as renal cell carcinomas.

Chronic activation of vasopressin V2 receptor signalling lowers renal medullary oxygen levels in rats

In the present study, we aimed to elucidate the effects of chronic vasopressin administration on renal medullary oxygen levels. Adult Sprague Dawley or vasopressin-deficient Brattleboro rats were treated with the vasopressin V2 receptor agonist, desmopressin (5ng/h; 3d), or its vehicle via osmotic minipumps. Immunostaining for pimonidazole and the transcription factor HIF-1α (hypoxia-inducible factor-1α) were used to identify hypoxic areas. Activation of HIF-target gene expression following desmopressin treatment was studied by microarray analysis. Pimonidazole staining was detected in the outer and inner medulla of desmopressin-treated rats, whereas staining in control animals was weak or absent. HIF-1α immunostaining demonstrated nuclear accumulation in the papilla of desmopressin-treated animals, whereas no staining was observed in the controls. Gene expression analysis revealed significant enrichment of HIF-target genes in the group of desmopressin-regulated gene products (P=2.6*10(-21) ). Regulated products included insulin-like growth factor binding proteins 1 and 3, angiopoietin 2, fibronectin, cathepsin D, hexokinase 2 and cyclooxygenase 2. Our results demonstrate that an activation of the renal urine concentrating mechanism by desmopressin causes renal medullary hypoxia and an upregulation of hypoxia-inducible gene expression.

Activation of hypoxia-inducible factor-1 in pulmonary arterial smooth muscle cells by endothelin-1

Numerous cellular responses to hypoxia are mediated by the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 plays a central role in the pathogenesis of hypoxic pulmonary hypertension. Under certain conditions, HIF-1 may utilize feedforward mechanisms to amplify its activity. Since hypoxia increases endothelin-1 (ET-1) levels in the lung, we hypothesized that during moderate, prolonged hypoxia ET-1 might contribute to HIF-1 signaling in pulmonary arterial smooth muscle cells (PASMCs). Primary cultures of rat PASMCs were treated with ET-1 or exposed to moderate, prolonged hypoxia (4% O(2) for 60 h). Levels of the oxygen-sensitive HIF-1α subunit and expression of HIF target genes were increased in both hypoxic cells and cells treated with ET-1. Both hypoxia and ET-1 also increased HIF-1α mRNA expression and decreased mRNA and protein expression of prolyl hydroxylase 2 (PHD2), which is the protein responsible for targeting HIF-1α for O(2)-dependent degradation. The induction of HIF-1α by moderate, prolonged hypoxia was blocked by BQ-123, an antagonist of ET-1 receptor subtype A. The effects of ET-1 were mediated by increased intracellular calcium, generation of reactive oxygen species, and ERK1/2 activation. Neither ET-1 nor moderate hypoxia induced the expression of HIF-1α or HIF target genes in aortic smooth muscle cells. These results suggest that ET-1 induces a PASMC-specific increase in HIF-1α levels by upregulation of HIF-1α synthesis and downregulation of PHD2-mediated degradation, thereby amplifying the induction of HIF-1α in PASMCs during moderate, prolonged hypoxia.

A Functional Role of the Cyclin-Dependent Kinase Inhibitor 1 (P21WAF1/CIP1) for Neuronal Preconditioning

Hypoxic preconditioning is thought to rely on gene products regulated by hypoxia-inducible factor (HIF)-1. Here, we show that the HIF-1 target gene cyclin-dependent kinase inhibitor 1, p21(WAF1/CIP1), is essential for neuroprotection by hypoxic/aglycemic or erythropoietin preconditioning using wild-type and p21(WAF1/CIP1)-deficient neurons. Furthermore, overexpression of wild-type p21(WAF1/CIP1) or phospho-mutants significantly increased cell death after hypoxia/aglycemia. Moreover, deferoxamine-induced endogenous tolerance did not involve p21(WAF1/CIP1) expression in cortical neurons. Our data suggest that balanced expression and potentially posttranslational regulation of p21(WAF1/CIP1) is required for hypoxic preconditioning.

Cloning and characterization of hypoxia-inducible factor-1 subunits from Ascaris suum — A parasitic nematode highly adapted to changes of oxygen conditions during its life cycle

The parasitic nematode Ascaris suum successfully adapts to a significant decrease in oxygen availability during its life cycle by altering its metabolic system dramatically. However, little is known about the regulatory mechanisms of adaptation to hypoxic environments in A. suum. In multicellular organisms, hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor composed of HIF-1α and HIF-1β subunits, is a master regulator of genes involved in adaptation to hypoxia. In the present study, cDNAs encoding HIF-1α and HIF-1β were cloned from A. suum and characterized. The full-length A. suum hif-1α and hif-1β cDNAs contain open reading frames encoding proteins with 832 and 436 amino acids, respectively. In the deduced amino acid sequences of A. suum HIF-1α and HIF-1β, functional domains essential for DNA-binding, dimerization, and oxygen-dependent prolyl hydroxylation were conserved. The interaction between A. suum HIF-1α and HIF-1β was confirmed by the yeast two-hybrid assay. Both A. suum hif-1α and hif-1β mRNAs were expressed at all stages examined (fertilized eggs, third-stage larvae, lung-stage larvae, young adult worms, and adult muscle tissue), and most abundantly in the aerobic free-living third-stage larvae, followed by a gradual decrease after infection of the host. hif-1 mRNA transcription was not sensitive to the oxygen environment in either third-stage larvae or adult worms (muscle tissue), and was regulated in a stage-specific manner. High expression of hif-1 mRNAs in third-stage larvae suggests its contribution to pre-adaptation to a hypoxic environment after infection of their host. Sequence analysis of 5′-upstream regions of mitochondrial complex II (succinate–ubiquinone reductase/quinol-fumarate reductase) genes, which show stage-specific expression and play an important role in oxygen adaptation during the life cycle, revealed that all subunits except for the adult-type flavoprotein subunit (Fp) possess putative hypoxia-responsive elements (HREs), suggesting that they are hif-1 target genes.

Abstract P1-05-15: Identification of a novel Hypoxia Inducible Factor-1 regulated gene involved in breast cancer growth and metastasis

Abstract Introduction: Hypoxia is a hallmark of most solid tumors. In response to hypoxic stress tumor cells adapt by regulating survival, metabolism and angiogenesis. The heterodimeric Hypoxia-Inducible Factor (HIF)-1 transcription factor is a master regulator of this response. HIF-1alpha protein is over-expressed in ∼30% of primary breast tumors and ∼70% of metastases, which independently correlates with poor prognosis and decreased survival in patients. Moreover, conditional deletion of Hif1a in the mammary epithelium of the MMTV-polyoma middle T (MMTV-PyMT) transgenic model of breast cancer significantly delays mammary tumor initiation and lung metastasis burden. Methods: Our lab has established primary mammary tumor epithelial cells (MTECs) from late stage carcinomas originating in PyMT+; Hif1α floxed mice (FVB/Nj strain, F11). These MTECs were exposed to either Adenovirus-eta-βgal or -Cre to create wild-type (WT) and knock-out (KO) cells, respectively. PyMT MTECs are transplantable into syngeneic FVB/Nj female recipients. Deletion of HIF-1 activity reduced primary tumor growth by ∼60% and the formation of lung macrometastases originating from mammary fat pad tumors by >90%. Microarray profiling was conducted to identify genes differentially expressed between WT and KO cells cultured at normoxia (21% O2) or hypoxia (0.5% O2) as well as between end-stage WT and KO tumors derived from the parental cell lines. Several genes were down-regulated in both gene sets in response to deletion of Hif1a. One mRNA of particular interest, creatine kinase brain isoform (Ckb) was down-regulated in KO cells >100 fold and >2 fold in end-stage tumors. Pools of stable Ckb knockdown (KD) PyMT cells were generated via lentiviral transduction of two independent shRNA constructs (pLKO.1 shc59 or shc61, each producing >75% knockdown) to determine the effects of Ckb knockdown on cell proliferation, migration and metastasis. Results: No differences in cell proliferation were observed between WT and Ckb KD cells when cultured in standard culture conditions. In contrast, when cells were introduced to the mammary fat pad, initiation of palpable tumors was delayed by >30 days for each shRNA KD line, suggesting that CKB function may be modulated by the tumor microenvironment. A significant decrease in invasion through Matrigel was also observed for each shRNA Ckb cell line. Likewise, when cells were introduced into circulation via tail vein injection, 20% of mice injected with either shc59 or shc61 cells developed lung metastases whereas 100% of mice injected with WT cells developed metastases. Bioinformatic analyses indicate that multiple putative HIF-1 binding sites (HREs) are present in both the mouse and human CKB gene promoters; chromatin immunoprecipiation (ChIP) experiments are in progress to validate that CKB is a direct HIF-1 target gene. Conclusions: HIF-1alpha strongly promotes metastasis, the major cause of mortality in breast cancer patients. Further characterization of genes downstream of HIF-1alpha, such as CKB, that play a key role in driving invasion and tumor initiation may identify new pathways amenable to therapeutic intervention for patients with metastatic breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-15.

Silencing of GLUT‐1 inhibits sensitization of oral cancer cells to cisplatin during hypoxia

During tumor development, cells are exposed to a hypoxic microenvironment. Tumor hypoxia also has a profound influence on the sensitivity of cancer chemotherapy. The objective of this study was to investigate the mechanism of cisplatin (CDDP) resistance of oral squamous cell carcinoma (OSCC) cells under hypoxia by analyzing gene expression profiles to identify key genes and factors involved. Cell viability was measured following culture of the cells in the presence or absence of CDDP, under normoxic or hypoxic conditions, using a CCK-8 assay. Analysis of the expression of HIF target genes in hypoxia-treated cells was performed using an HIF-regulated cDNA plate array. Changes in the mRNA expression of selected HIF target genes were analyzed using RT-PCR, and changes in the protein levels of these genes were analyzed by Western blotting. Tumor cell apoptosis was assessed by flow cytometry. The OSCC cell lines responded differently to CDDP under normoxic and hypoxic conditions. The expression of glucose transporter protein-1 (GLUT-1) was up-regulated in human squamous cell carcinoma of mouth (HSC-2) cells under hypoxia. Furthermore, there was little correlation between the cisplatin sensitivity of human squamous cell carcinoma of tongue (SAS) in normoxia and hypoxia. After GLUT-1 knockdown, CDDP treatment resulted in increased rates of apoptosis under hypoxia as compared with normoxia in cell lines HSC-2, Ca9-22, and SAS (P = 0.025). The results of this study suggest that knockdown of GLUT-1 inhibits sensitization of oral squamous cells to CDDP during hypoxia in HSC-2, Ca9-22, and SAS cells.

Molecular, functional, and gene expression analysis of zebrafish hypoxia-inducible factor-3α

Hypoxia-inducible factors 1-3 (HIF1-3) are transcription factors that regulate gene expression in response to hypoxia. Compared with our extensive understanding of HIF-1 and HIF-2, our knowledge of HIF-3 is limited. In this study, we characterized the zebrafish hif-3α gene and determined its temporal and spatial expression, physiological regulation, and biological activity. We show that the chromosomal location, gene structure, and protein structure of zebrafish hif-3α are similar to its mammalian orthologs. When tagged with enhanced green fluorescent protein and transfected into cultured cells, zebrafish Hif-3α was localized in the nucleus and stimulated reporter gene expression in a hypoxia response element-dependent manner. During early development, hif-3α mRNA was detected in all tissues with higher levels in the head. This expression pattern became more apparent in larvae at the 72, 96, and 120 hours post fertilization stages. In the adult stage, hif-3α mRNA was detected in all examined tissues with the highest levels in the ovary. Hypoxia treatment increased Hif-3α protein levels in both embryos and adults. Hypoxia also increased hif-3α mRNA expression levels, and this regulation was tissue-specific. Expression of a stabilized form of Hif-1α in zebrafish embryos increased the expression of igfbp-1a, a Hif-1 target gene, whereas it did not change hif-3α mRNA levels, suggesting that hif-3α is not a Hif-1α target. These results provide new information about the structural and functional conservation, spatial and temporal expression, and physiological regulation of hif-3α in a teleost model organism.

Hypoxia Promotes Osteogenesis but Suppresses Adipogenesis of Human Mesenchymal Stromal Cells in a Hypoxia-Inducible Factor-1 Dependent Manner

BackgroundBone fracture initiates a series of cellular and molecular events including the expression of hypoxia-inducible factor (HIF)-1. HIF-1 is known to facilitate recruitment and differentiation of multipotent human mesenchymal stromal cells (hMSC). Therefore, we analyzed the impact of hypoxia and HIF-1 on the competitive differentiation potential of hMSCs towards adipogenic and osteogenic lineages.Methodology/Principal FindingsBone marrow derived primary hMSCs cultured for 2 weeks either under normoxic (app. 18% O2) or hypoxic (less than 2% O2) conditions were analyzed for the expression of MSC surface markers and for expression of the genes HIF1A, VEGFA, LDHA, PGK1, and GLUT1. Using conditioned medium, adipogenic or osteogenic differentiation as verified by Oil-Red-O or von-Kossa staining was induced in hMSCs under either normoxic or hypoxic conditions. The expression of HIF1A and VEGFA was measured by qPCR. A knockdown of HIF-1α by lentiviral transduction was performed, and the ability of the transduced hMSCs to differentiate into adipogenic and osteogenic lineages was analyzed. Hypoxia induced HIF-1α and HIF-1 target gene expression, but did not alter MSC phenotype or surface marker expression. Hypoxia (i) suppressed adipogenesis and associated HIF1A and PPARG gene expression in hMSCs and (ii) enhanced osteogenesis and associated HIF1A and RUNX2 gene expression. shRNA-mediated knockdown of HIF-1α enhanced adipogenesis under both normoxia and hypoxia, and suppressed hypoxia-induced osteogenesis.Conclusions/SignificanceHypoxia promotes osteogenesis but suppresses adipogenesis of human MSCs in a competitive and HIF-1-dependent manner. We therefore conclude that the effects of hypoxia are crucial for effective bone healing, which may potentially lead to the development of novel therapeutic approaches.

Identification of two small molecule inhibitors of hypoxia-inducible factor 1 with different cell-based screening model

To investigate the inhibitory activity of HIF-1 by triptolide and manasaantin A, two cell-based models with luciferase report gene assay were established. Two cell-based models of HIF-1 were used to evaluate HIF-1 inhibition activity of triptolide and manasaantin A. Secreted VEGF expression induced by hypoxia was detected by ELISA with two compounds. The growth inhibition of different solid tumor cell lines was measured by the MTT assay. The expression of firefly luciferase was induced by hypoxia in U251-HRE and T47D-HRE cells. U251-HRE model was suitable for the detection of HIF-1 inhibition activity of triptolide. The IC50 of triptolide on HIF-1 activity was (3.4 +/- 0.5) x 10(-8) mol x L(-1). The report gene assay using T47D cells co-transfected with pGL2-TK-HRE and pRL-CMV showed more sensitive inhibition activity of HIF-1 on manassantin A than that of detected by U251-HRE model. The IC50 of manassantin A on HIF-1 activity was (2.4 +/- 0.6) x 10(-8) mol x L(-1). HIF-1 target gene VEGF was also inhibited by test compounds on protein level in T47D cells. Manasaantin A showed selective inhibition on the growth of human solid cancer cell lines, especially on breast cancer and pancreatic cancer cells. Meanwhile, triplotide showed strong proliferation inhibition activity on all tested cell lines. It is very important to select a suitable cell-based report gene assay of HIF-1 for screening of different kinds of inhibitor.

SHARP1 suppresses breast cancer metastasis by promoting degradation of hypoxia-inducible factors

The molecular determinants of malignant cell behaviours in breast cancer remain only partially understood. Here we show that SHARP1 (also known as BHLHE41 or DEC2) is a crucial regulator of the invasive and metastatic phenotype in triple-negative breast cancer (TNBC), one of the most aggressive types of breast cancer. SHARP1 is regulated by the p63 metastasis suppressor and inhibits TNBC aggressiveness through inhibition of hypoxia-inducible factor 1α (HIF-1α) and HIF-2α (HIFs). SHARP1 opposes HIF-dependent TNBC cell migration in vitro, and invasive or metastatic behaviours in vivo. SHARP1 is required, and sufficient, to limit expression of HIF-target genes. In primary TNBC, endogenous SHARP1 levels are inversely correlated with those of HIF targets. Mechanistically, SHARP1 binds to HIFs and promotes HIF proteasomal degradation by serving as the HIF-presenting factor to the proteasome. This process is independent of pVHL (von Hippel-Lindau tumour suppressor), hypoxia and the ubiquitination machinery. SHARP1 therefore determines the intrinsic instability of HIF proteins to act in parallel to, and cooperate with, oxygen levels. This work sheds light on the mechanisms and pathways by which TNBC acquires invasiveness and metastatic propensity.

Deguelin attenuates reperfusion injury and improves outcome after orthotopic lung transplantation in the rat.

The main goal of adequate organ preservation is to avoid further cellular metabolism during the phase of ischemia. However, modern preservation solutions do rarely achieve this target. In donor organs hypoxia and ischemia induce a broad spectrum of pathologic molecular mechanisms favoring primary graft dysfunction (PGD) after transplantation. Increased hypoxia-induced transcriptional activity leads to increased vascular permeability which in turn is the soil of a reperfusion edema and the enhancement of a pro-inflammatory response in the graft after reperfusion. We hypothesize that inhibition of the respiration chain in mitochondria and thus inhibition of the hypoxia induced mechanisms might reduce reperfusion edema and consecutively improve survival in vivo. In this study we demonstrate that the rotenoid Deguelin reduces the expression of hypoxia induced target genes, and especially VEGF-A, dose-dependently in hypoxic human lung derived cells. Furthermore, Deguelin significantly suppresses the mRNA expression of the HIF target genes VEGF-A, the pro-inflammatory CXCR4 and ICAM-1 in ischemic lungs vs. control lungs. After lung transplantation, the VEGF-A induced reperfusion-edema is significantly lower in Deguelin-treated animals than in controls. Deguelin-treated rats exhibit a significantly increased survival-rate after transplantation. Additionally, a downregulation of the pro-inflammatory molecules ICAM-1 and CXCR4 and an increase in the recruitment of immunomodulatory monocytes (CD163+ and CD68+) to the transplanted organ involving the IL4 pathway was observed. Therefore, we conclude that ischemic periods preceding reperfusion are mainly responsible for the increased vascular permeability via upregulation of VEGF. Together with this, the resulting endothelial dysfunction also enhances inflammation and consequently lung dysfunction. Deguelin significantly decreases a VEGF-A induced reperfusion edema, induces the recruitment of immunomodulatory monocytes and thus improves organ function and survival after lung transplantation by interfering with hypoxia induced signaling.

Roles for Peroxisome Proliferator-activated Receptor γ (PPARγ) and PPARγ Coactivators 1α and 1β in Regulating Response of White and Brown Adipocytes to Hypoxia

Obese white adipose tissue is hypoxic but is incapable of inducing compensatory angiogenesis. Brown adipose tissue is highly vascularized, facilitating delivery of nutrients to brown adipocytes for heat production. In this study, we investigated the mechanisms by which white and brown adipocytes respond to hypoxia. Brown adipocytes produced lower amounts of hypoxia-inducible factor 1α (HIF-1α) than white adipocytes in response to low O(2) but induced higher levels of hypoxia-associated genes. The response of white adipocytes to hypoxia required HIF-1α, but its presence alone was incapable of inducing target gene expression under normoxic conditions. In addition to the HIF-1α targets, hypoxia also induced many inflammatory genes. Exposure of white adipocytes to a peroxisome proliferator-activated receptor γ (PPARγ) ligand (troglitazone) attenuated induction of these genes but enhanced expression of the HIF-1α targets. Knockdown of PPARγ in mature white adipocytes prevented the usual robust induction of HIF-1α targets in response to hypoxia. Similarly, knockdown of PPARγ coactivator (PGC) 1β in PGC-1α-deficient brown adipocytes eliminated their response to hypoxia. These data demonstrate that the response of white adipocytes requires HIF-1α but also depends on PPARγ in white cells and the PPARγ cofactors PGC-1α and PGC-1β in brown cells.

Phosphodiesterase-4 promotes proliferation and angiogenesis of lung cancer by crosstalk with HIF

Lung cancer is the leading cause of cancer death worldwide. Recent data suggest that cyclic nucleotide phosphodiesterases (PDEs) are relevant in various cancer pathologies. Pathophysiological role of phosphodiesterase 4 (PDE4) with possible therapeutic prospects in lung cancer was investigated. We exposed 10 different lung cancer cell lines (adenocarcinoma, squamous and large cell carcinoma) to hypoxia and assessed expression and activity of PDE4 by real-time PCR, immunocytochemistry, western blotting and PDE activity assays. Expression and activity of distinct PDE4 isoforms (PDE4A and PDE4D) increased in response to hypoxia in eight of the studied cell lines. Furthermore, we analyzed various in silico predicted hypoxia-responsive elements (p-HREs) found in in PDE4A and PDE4D genes. Performing mutation analysis of the p-HRE in luciferase reporter constructs, we identified four functional HRE sites in the PDE4A gene and two functional HRE sites in the PDE4D gene that mediated hypoxic induction of the reporter. Silencing of hypoxia-inducible factor subunits (HIF1α and HIF2α) by small interfering RNA reduced hypoxic induction of PDE4A and PDE4D. Vice versa, using a PDE4 inhibitor (PDE4i) as a cyclic adenosine monophosphate (cAMP) -elevating agent, cAMP analogs or protein kinase A (PKA)-modulating drugs and an exchange protein directly activated by cAMP (EPAC) activator, we demonstrated that PDE4-cAMP-PKA/EPAC axis enhanced HIF signaling as measured by HRE reporter gene assay, HIF and HIF target genes expression ((lactate dehydrogenase A), LDHA, (pyruvate dehydrogenase kinase 1) PDK1 and (vascular endothelial growth factor A) VEGFA). Notably, inhibition of PDE4 by PDE4i or silencing of PDE4A and PDE4D reduced human lung tumor cell proliferation and colony formation. On the other hand, overexpression of PDE4A or PDE4D increased human lung cancer proliferation. Moreover, PDE4i treatment reduced hypoxia-induced VEGF secretion in human cells. In vivo, PDE4i inhibited tumor xenograft growth in nude mice by attenuating proliferation and angiogenesis. Our findings suggest that PDE4 is expressed in lung cancer, crosstalks with HIF signaling and promotes lung cancer progression. Thus, PDE4 may represent a therapeutic target for lung cancer therapy.

Abstract 4253: In vivo activity of small molecule HIF2α inhibitors

Abstract Hypoxia-inducible factor (HIF) is a central regulator of the cellular response to hypoxia. Hypoxia promotes HIF activity by increasing its stability, transcriptional activity and translation. HIF target genes involved in several cellular processes including proliferation, angiogenesis, metabolism and promotion of the “stem-cell” phenotype. In normoxic conditions, HIF-α is recognized for degradation by the von Hippel-Lindau tumor suppressor protein (VHL). Loss-of-VHL function leads to constitutive expression of HIF regulatory subunits and consequent over-expression of HIF-target genes. We and others have shown that HIF2a is necessary and sufficient for the development of VHL-related renal cell carcinoma. We have developed small molecules that repress HIF-2α translation, in vitro, by enhancing the binding of Iron-Regulatory Protein 1 (IRP1) to the Iron-Responsive Element (IRE) within the 5′-UTR of HIF-2α mRNA (Zimmer M. et al. Molecular Cell 2008;32: 828-848). We showed that these small molecules down-regulate HIF2a expression in vitro, in all renal cell carcinoma cell lines. To validate these small molecules in vivo, we tested their efficacy on wild-type zebrafish embryos. Treatment of 3 days post fertilization zebrafish embryos with 100microM of the hypoxic mimetic DMOG or hypoxia dramatically upregulated the expression of HIF-target genes, such as Vascular Endothelial Growth Factor (Vegf), erythropoietin (Epo) and prolyl-hydroxylase 3 (Phd3). Pre-treatment of embryos with the small molecule HIF2a inhibitor 76 potently decreased the DMOG-induced upregulation of HIF target genes. The IC50 values of these small molecules were significantly less in vivo than in vitro. We are currently testing the efficacy of HIF2a inhibitors in rescuing the VHL zebrafish phenotype and prolonging survival of VHL mutant zebrafish embryos. In addition, data on the efficacy of these inhibitors on the growth of human renal cell carcinoma cells as tumors in the nude mouse xenograft assay will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4253. doi:1538-7445.AM2012-4253

Abstract 3942: The role of vHL and REDD1 in predicting sensitivity to targeted therapies in renal cell carcinoma

Abstract There are currently 6 FDA approved therapies in metastatic renal cell carcinoma and their introduction has improved both time to progression and survival in these patients. Despite treatment with targeted therapies approximately 20% of patients do not respond and the remaining patients will most likely develop resistance during the first 18 months of treatment. The aim of this study was to identify a link between differences in sensitivity to targeted therapies, the vHL status and the expression of genes involved in the mTOR pathway in both historic and primary renal cancer cell lines. Drug sensitivity and gene expression data was analysed in 6 historic and 10 primary renal cancer cell lines obtained from nephrectomy specimens. Gene expression data was also available from an additional 68 renal tumours. We found that cell lines with mutated or absent vHL showed increased sensitivity to rapamycin (p<0.01) but not to other therapies. Gene expression data showed that the expression of REDD1 (regulated in development and DNA damage responses 1), a known HIF1 target gene which inhibits the mTORC1 pathway in response to hypoxic stress correlates with rapamycin sensitivity (R-squared = 0.64, p=0.05). REDD1 expression was also higher in renal tumours when compared with matched normal tissue (p<0.01) and was found to correlate with CAIX expression (R-squared = 0.47, p<0.01). CAIX expression is associated with reduced survival in renal cell carcinoma and so further analysis is being performed to establish a potential role of REDD1 as a prognostic marker. Our findings suggest that vHL status and the levels of REDD1 expression in renal cancer cell lines can predict sensitivity to rapamycin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3942. doi:1538-7445.AM2012-3942

Abstract 3945: HIFs regulate alternative splicing of HIF target genes

Abstract Alternative splicing is the process in which identical pre-mRNA molecules are spliced in different ways, allowing individual gene to express multiple mRNAs or isoforms that encode proteins with diverse and even antagonistic functions. Alternative splicing occurs in over 70% of human genes, is important in normal development and many diseases including cancer. It is well known that most solid-tumors are hypoxic and the hypoxic microenvironment is a driving force for tumor progression and metastasis by hypoxia-inducible-factor (HIF)-mediated transcriptional programs that increase genes involved in tumor growth, angiogenesis, and metastasis. Here, we report a novel finding that transcriptional factor HIF not only activates gene expression, also regulates RNA splicing of its target genes, generating isoforms that favor tumor growth. Using an Exon Microarray we found that approximately 1000 genes are predicted to undergo hypoxia-mediated AS in Hep3B cells, 100 of those genes are hypoxia inducible genes. Using RT-PCR, RT-qPCR and WB we validated that the first 10 HIF target genes, CA9, ANGPTL4, WNK1, PLOD2, ADM, DUOX2, ENO2, P4HA2, EGLN3, and PDK1 undergo hypoxia-mediate AS, as predicted by exon array. Interestingly, AS of these genes is regulated by HIFs, not by hypoxia since: 1) siRNA knockdown of ARNT, HIF1, or HIF2 inhibits hypoxic induction of HIF target genes as well as inhibits hypoxia-mediated splicing changes; and 2) Normoxic HIF activity by deleting VHL, inhibiting PHD, or expressing normoxic-stable HIF protein led to hypoxic splicing patterns of HIF target genes, demonstrating HIF activity is sufficient to induce alternative splicing of HIF target genes. However, AS of HIF target genes cannot be explained by increased transcription of these genes since no splicing changes were observed when these genes were activated by hybrid protein with HIF transactivation domains replaced by VP16, E2F1, or SP1 transactivation domain. Further experiments indicated that the N-terminal transactivation domains (N-TAD) of HIF1α or HIF2α are responsible for splicing changes of these HIF-target genes. In summary, we have uncovered a novel function of HIFs in the regulation of target gene splicing. The discovery of HIF's role in mRNA splicing of its target genes will change the dogma concerning the role of HIF in cancer progression. We propose that HIFs promote cancer progression not only through transcription activation but also through alteration of mRNA splicing of HIF targets. RO1 CA134687 NIH/NCI (Hu) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3945. doi:1538-7445.AM2012-3945