Expression Of HIF-1 Target Genes Research Articles

Abstract 1578: Small molecule HIF-2a inhibitors down regulate its activity in vivo and ameliorate the vhl zebrafish phenotype.

Abstract Hypoxia Inducible Factor (HIF) target genes are involved in several cellular processes including angiogenesis, erythropoiesis, metabolism and stem cell proliferation. The von Hippel-Lindau (VHL) tumor suppressor protein targets HIF for degradation in conditions of normal oxygen tension. Loss-of-VHL leads to constitutive expression of HIF. We have discovered small molecules that repress HIF-2α translation by enhancing the binding of Iron-Regulatory Protein 1 (IRP1) to the Iron-Responsive Element (IRE) within the 5’-UTR of HIF-2α mRNA and showed that these small molecules specifically down-regulate HIF-2α expression in vitro. To validate the HIF-2α inhibitors in vivo, first we tested their efficacy to inhibit acute hif induction in wild-type (wt) zebrafish embryos challenged with the hypoxia mimetic DMOG. Treatment of wt embryos with 100μM of DMOG up-regulated the expression of zebrafish 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 HIF-2α inhibitor 76 (10nM) significantly decreased the DMOG-induced up-regulation of HIF target genes and resulted in decrease of DMOG-induced erythrocytosis and blood vessel sprouting. Furthermore, we tested the efficacy of this HIF-2α inhibitor in a vhl zebrafish model (homozygougous mutants harbouring inactivating vhl mutations), which recapitulates aspects of human VHL disease (erythrocytosis, abnormal vascular proliferation in sites such as retina and brain, early lethality). Treatment with inhibitor 76 (10nM, 100nM and 1μM) down regulated the expression of the HIF-target genes phd3, epo and vegf in a dose response manner and rescued the erythrocytosis and sprouting phenotype of vhl embryos. To image and quantify the blood vessel abnormalities of vhl embryos, we generated a Fli-GFP::vhl+/- line and showed that compound 76 improves the abnormal connections between the intersegmental vessels of the Fli-GFP::vhl embryo tail, as well as their human hemangioblastoma-resembling brain vessel proliferation. Finally, we showed that treatment of vhl embryos with compound 76 rescues early lethality. Our data demonstrate that HIF-2α inhibitor 76 is active in vivo at low concentrations and encourage further development and preclinical testing of this HIF-2α inhibitor for treatment of VHL-associated lesions, such as renal cell carcinoma, hemangioblastoma and neuroendocrine tumors. Citation Format: Ana Metelo, Li Xiang, Rania Baker, Lee Kamentsky, Anne Carpenter, Randall Peterson. Small molecule HIF-2a inhibitors down regulate its activity in vivo and ameliorate the vhl zebrafish phenotype. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1578. doi:10.1158/1538-7445.AM2013-1578

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.

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.

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.

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.

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.

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.

Abstract 1839: Phosphodiesterase 4 regulates lung tumor growth through HIF regulation, proliferation, and angiogenesis

Abstract Lung cancer is the leading cause of cancer death worldwide. Recent data suggest that cyclic nucleotide phosphodiesterases (PDEs) are relevant in various cancer pathologies. Here, we elucidate the pathophysiological role of PDE4 and its therapeutic prospects in lung cancer. We exposed 10 different non-small cell lung cancer cell lines (adenocarcinoma, squamous, and large cell carcinoma) to hypoxia and assessed the expression and activity of PDE4 by quantitative 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 determined various potential hypoxia-responsive elements (HRE) in PDE4A and PDE4D. Silencing of hypoxia-inducible factor subunits (HIF1A and HIF2) by siRNA reduced hypoxic induction of PDE4A and PDE4D. Vice versa, using a PDE4 inhibitor (PDE4i), a cAMP-elevating agent, cAMP analogs, PKA activator/inhibitor, and EPAC activator, we demonstrated that PDE4-cAMP-PKA/EPAC axis regulates HIF signaling as measured by HRE reporter gene assay and expression of HIF target genes (LDHA, PDK1, VEGFA). Notably, PDE4i or PDE4A-/PDE4D-selective siRNA reduced human lung tumor cell proliferation in vitro and colony formation and altered the expression of cell-cycle regulators. Moreover, PDE4i treatment reduced hypoxia-induced VEGF secretion in human cells. In vivo, PDE4i inhibited tumor xenograft growth in athymic nude mice by altering proliferation and angiogenesis. Collectively, our findings suggest that PDE4 is overexpressed in lung cancer, exerts crosstalk with HIF pathways, and promotes lung cancer progression; thus, PDE4 may represent a therapeutic target for lung cancer therapy. 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 1839. doi:1538-7445.AM2012-1839

Abstract 3938: BNip3 limits HIF-1α stabilization and metastasis in a mouse model of breast cancer through effects on mitochondrial integrity and ROS generation

Abstract BNIP3 is a hypoxia-inducible mitochondrial protein that has been implicated in mitochondrial fragmentation and mitophagy. BNIP3 protein is also up-regulated at pre-malignant stages of various different human cancers, including breast cancer and down-regulated later through a variety of mechanisms. To examine the role of BNip3 in tumor progression to metastasis, we crossed mice carrying a targeted deletion in the mouse BNip3 gene to the MMTV-PyMT mouse model of breast cancer. All analyses were performed with mice on a pure FVB/N genetic background. Loss of BNip3 promoted S phase entry that was associated with increased primary tumor growth. There was highly significant increase in the numbers of lung metastases and a reduced latency to metastasis in the MMTV-PyMT;BNip3-/- mice compared to control MMTV-PyMT;BNip3+/+ mice. BNip3 null tumors showed increased invasive properties and nuclear grade at earlier stages, a phenotype that was transplantable to wild-type host mice arguing that the increased invasiveness was intrinsic to BNip3 null tumor cells. BNip3 null tumor cells exhibited abnormal mitochondria, reduced mitochondrial membrane potential, increased ROS levels and increased hypoxia. Their invasive pathology was also associated with increased HIF-1α levels, increased HIF target gene expression and increased CD31-positive blood vessel formation. Quenching ROS limited Hif-1α stabilization, reduced angiogenesis and metastasis in MMTV-PyMT;BNip3-/- mouse tumors to wild-type levels. These results suggest that BNip3 is a metastasis suppressor by virtue of its role in maintaining mitochondrial integrity that mitigates against the metastasis promoting activity of reactive oxygen and hypoxia. Work on this project is supported by NIH/NCI RO1-CA131188 and by Department of Defense BRCP W81XWH-09-1-0587. 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 3938. doi:1538-7445.AM2012-3938

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

Isolation, identification, and biological evaluation of HIF-1-modulating compounds from Brazilian green propolis

The tumor microenvironment is characterized by hypoxia, low-nutrient levels, and acidosis. A natural product chemistry-based approach was used to discover small molecules that modulate adaptive responses to a hypoxic microenvironment through the hypoxia-inducible factor (HIF)-1 signaling pathways. Five compounds, such as baccharin (3), beturetol (4), kaempferide (5), isosakuranetin (6), and drupanin (9), that modulate HIF-1-dependent luciferase activity were identified from Brazilian green propolis using reporter assay. Compounds 3, 9 and 5 reduced HIF-1-dependent luciferase activity. The cinnamic acid derivatives 3 and 9 significantly inhibited expression of the HIF-1α protein and HIF-1 downstream target genes such as glucose transporter 1, hexokinase 2, and vascular endothelial growth factor A. They also exhibited significant anti-angiogenic effects in the chick chorioallantoic membrane (CAM) assay at doses of 300ng/CAM. On the other hand, flavonoids 4 and 6 induced HIF-1-dependent luciferase activity and expression of HIF-1 target genes under hypoxia. The contents (g/100g extract) of the HIF-1-modulating compounds in whole propolis ethanol extracts were also determined based on liquid chromatography–electrospray ionization mass spectrometry as 1.6 (3), 14.2 (4), 4.0 (5), 0.7 (6), and 0.7 (9), respectively. These small molecules screened from Brazilian green propolis may be useful as lead compounds for the development of novel therapies against ischemic cardiovascular disease and cancer based on their ability to induce or inhibit HIF-1 activity, respectively.

Abstract 4503: BAY 87-2243, an inhibitor of HIF-1α activation and stabilization, showed promising anti-tumor efficacy either as monotherapy or in combination with anti-VEGF and chemotherapy agents in preclinical tumor models

Abstract Hypoxia is a hallmark of solid tumors and is associated with local invasion, metastatic spread and resistance to radio- as well as chemotherapy (Vaupel et al., 2004). Furthermore, hypoxia constitutes an independent negative prognostic factor in a diverse range of malignant tumors (Harris, 2002; Vaupel et al., 2004). The cellular adaptation to hypoxia is mediated by a heterodimeric transcription factor hypoxia inducible factor-1 (HIF1). HIF-1α, one subunit of HIF, is constitutively degraded under normoxic conditions, but is stabilized and activated in hypoxic regions of tumors. Nuclear activated HIF-1 controls the expression of >100 genes involved in cellular energy metabolism, neoangiogenesis, anti-apoptotic and pro-proliferative mechanisms promoting invasion and metastasis. It was our hypothesis that small-molecule inhibitor that prevent stabilization and activation of HIF-1 may act as valuable novel cancer therapeutics. Recently, we identified BAY 87-2243 as a small molecule inhibitor of hypoxia-induced HIF-1 activation that specifically suppressed hypoxia-induced HIF-1α stabilization and expression of HIF target genes. Here we present preclinical anti-tumor efficacy of BAY 87-2243 in several xenografts, both as single agent and in combination with standards treatments. BAY 87-2243 dosed orally as single agent demonstrated moderate to high tumor growth inhibition in several subcutaneous tumor models of various histological type such as prostate carcinoma, lung carcinoma, neuroblastoma, colorectal and mammary carcinomas. BAY 87-2243 dosed continuously in combination with the anti-angiogenic agent bevacizumab produced long-term tumor growth control or tumor stasis of either small or large established subcutaneous and orthotopic lung carcinoma xenografts (NSCLC H460 model). In an orthotopic pancreatic carcinoma model (DAN-G) a combination of BAY 87-2243 and gemcitabine resulted in a synergistic improvement of the median survival as compared to either agents alone (p=0.0004). In general, BAY 87-2243 was well tolerated in mice without any significant body weight loss. These promising preclinical results suggest that inhibition of HIF-1 by BAY 87-2243 is an innovative approach to cancer therapy with the potential to overcome hypoxia/HIF-induced tumor resistance mechanisms. Harris AL (2002) Hypoxia-a key regulatory factor in tumor growth. Nat Rev Cancer 2: 38-47 Vaupel P, Mayer A, Hockel M (2004) Tumor hypoxia and malignant progression. Methods Enzymol 381:335-354 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4503. doi:10.1158/1538-7445.AM2011-4503

Abstract 1483: Metastatic characteristics and radiosensitivity of thyroid carcinoma cells depends on HIF-1 and PI3K signaling in vitro and in vivo

Abstract The purpose of this study was to determine the importance of the HIF-1 and PI3K pathways in the development of a) a metastatic phenotype and b) desensitization to radiotherapy, in thyroid carcinomas. In vitro scratch wound migration assays and cell spreading assays were performed under various oxygen tensions in the presence or absence of two PI3K inhibitors PI-103 and GDC-0941 (GDC-0941 is currently in clinical trials). The role of HIF-1α and PI3K on these processes was also assessed via genetic inhibition of HIF-1α and PI3K by use of a dominant negative variant (dnHIF) and by over expressing WT PTEN in PTEN-null FTC133 cells. For in vivo studies, follicular (FTC133) and anaplastic (8505c) thyroid carcinoma cells were manufactured to stably express eGFP and implanted sub-cutaneously. Mice were treated with PI-103 and GDC-0941 by intra-peritoneal injection and oral gavage. Spontaneous metastatic colonization to the lungs was confirmed by viewing eGFP postive colonies cultured from digested lung tissue and further by immuno-blotting for eGFP. For irradiation studies, cells were exposed to 0,2,4,6 GY external beam radiation. Both PI3K and hypoxia increased cell migration, cell adhesion and cell spreading on extra-cellular matrix molecules. These processes are key in the development of a metastatic phenotype. Genetic and pharmacological inhibition of both PI3K and HIF-1α reduced the migratory, adhesive and cell spreading potential of thyroid carcinoma cells under varying oxygen tensions. Additionally, pharmacological and genetic inhibition of PI3K and HIF-1α reduced both PI3K and HIF-1 activity resulting in reduced activation of PI3K target proteins (pAKT, pGSK-3β) and reduced expression of HIF-1 target genes (CA-9, VEGF, LDH-A, GLUT-1). In vivo, PI-103 and GDC-0941 reduced PI3K and HIF-1α activity as well as PI3K and HIF-1 target genes in primary follicular and anaplastic tumors. Importantly, mice bearing dnHIF-FTC133 tumors and those treated with GDC-0941 had reduced number of metastatic colonies in the lungs of follicular thyroid tumor bearing mice. Radiation induced HIF-1α activity and expression. GDC-0941 combined with radiation blocked HIF-1 induction, prolonged DNA double-strand breaks and reduced clonogenic survival suggesting a radio-sensitising effect. These data link PI3K, HIF-1 activation and aggressive disease in thyroid carcinoma and suggest PI3K may be an important therapeutic target. One attractive approach would be combination therapy with external beam radiation. The latter is the first line treatment in patients suffering from anaplastic thyroid carcinoma but is seldom successful. With the known desensitizing effects of HIF-1 and PI3K activity on radiation treatment, a combined approach involving both radiation and a PI3K inhibitor may improve both the therapeutic response within the local tumor and reduce metastatic potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1483. doi:10.1158/1538-7445.AM2011-1483

Abstract 4509: BAY 87-2243 targets hypoxia-induced activation and stabilization of HIF-1α: A novel approach to overcome resistance mechanisms in cancer therapy

Abstract The development of resistance against radio- or chemotherapy is one of the main causes for relapse after treatment and ultimately cancer progression. Local hypoxia within tumors is associated with radio- and chemoresistance as well as aggressive tumor growth and invasion (Vaupel et al., 2004). The transcription factor HIF-1α (hypoxia-inducible factor) is stabilized under low oxygen tension and heterodimerizes with HIF-1ß to regulate the expression of a plethora of genes involved in cellular energy metabolism, neoangiogenesis, anti-apoptotic and pro-proliferative mechanisms promoting tumor progression and metastasis (Calzada et al. 2007). Because hypoxia-induced upregulation of HIF-1α appears to be of pivotal importance in tumor resistance mechanisms during cancer treatment, we screened for inhibitors of hypoxia-induced HIF-1 activation. A HCT116 cell line containing 4X-hypoxia response element-luciferase reporter was used in high-throughput screening of small molecule inhibitors under hypoxic condition (1% O2). Lead optimization resulted in the identification of BAY 87-2243, a highly selective and potent inhibitor of hypoxia-induced HIF-1α stabilization and activation. In vitro characterization showed that BAY 87-2243 specifically suppressed HIF-1 regulated target genes as assayed by qPCR. Analyses addressing the mode of action revealed that BAY 87-2243 acts upstream of VHL and PHD because the compound did not suppress HIF-1α protein stabilization and HIF target gene expression either in the presence of a PHD inhibitor in H460 cells or in VHL-null RCC4 cells. In preclinical animal models, BAY 87-2243 dosed orally was well tolerated at therapeutic doses up to 15 mg/kg and showed moderate to high anti-tumor growth inhibitory activity as monotherapy in various subcutaneous and orthotopic xenograft models. Analysis of tumor samples demonstrated a decrease of nuclear HIF-1α protein level by immunohistochemistry as well as a specific suppression of HIF-1 target genes. These data indicate that specific inhibition of hypoxia-induced HIF-1 activation is achievable with small molecule inhibitors and is a novel approach to cancer therapy. Vaupel P, Mayer A, Hockel M (2004) Tumor hypoxia and malignant progression. Methods Enzymol 381:335-354 M. J. Calzada, L. del Peso, Hypoxia inducible factors and cancer. Clin. Transl. Oncol. 2007, 9(5), 278-289: Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4509. doi:10.1158/1538-7445.AM2011-4509

Abstract 2851: Metronomic doxorubicin blocks hypoxia inducible factor-mediated responses to hypoxia in sarcomas and augments the effects of anti-VEGF therapy

Abstract Introduction: Levels of hypoxia and hypoxia inducible factors (HIFs) in human sarcomas correlate with tumor progression and metastasis. Prolonged anti-angiogenic therapy of sarcomas can inhibit tumor growth but may also increase hypoxia and HIF activation. The most commonly used chemotherapeutic drug for sarcomas, doxorubicin (dox), was recently found to block HIF binding to DNA at low metronomic doses. Methods: 20 patients with soft tissue sarcomas were treated with anti-VEGF antibody (bevacizumab). The tumors, before and after treatment, were examined for changes in microvessel density using CD31 immunohistochemical staining and global gene expression using microarrays. Patients’ tumors were then treated with a combination of bevacizumab and radiation followed by surgical resection. Expression of specific HIF-target genes in the response to hypoxia and/or dox was examined in vitro in 4 sarcoma cell lines by q-RT-PCR. HT1080 human sarcoma xenografts and sarcomas in genetically engineered LSL-KrasG12D/+Trp53fl/fl mice were treated with anti-VEGF receptor antibody (DC101) and/or metronomic dox and analyzed for levels of hypoxia, HIFs, and HIF-target genes. Results: Bevacizumab alone reduced microvessel density in sarcomas by a median of 53% (p<0.05). Gene set enrichment analysis of microarrays found the Gene Ontology category “Response to hypoxia” (which includes HIF-1a, HIF-2a, and ARNT) was upregulated in sarcomas with a poor response to combination therapy (bevacizumab plus radiation). Dox at 1-10 uM blocked HIF induction of VEGF and CA-9 in all 4 sarcoma cell lines by 83.7-97.0% and 88.6-97.6%, respectively, while inhibition of other HIF-target genes including c-MET and FOXM1 was variable. HT1080 sarcoma xenografts and LSL-KrasG12D/+Trp53fl/fl sarcomas demonstrated increasing hypoxia with larger tumor size and with treatment with an anti-VEGFR-2 antibody (DC101). Increasing hypoxia corresponded to greater nuclear expression of HIF-1α and HIF-2α, and metronomic Dox lowered expression of HIF-depended genes by up to 92.3%. Combining VEGFR-2 antibody and metronomic Dox therapies had a synergistic effect in suppressing tumor growth. Conclusion: Human sarcomas respond to increased hypoxia by expressing HIF-target genes which promote compensatory responses. This hypoxic response may be exacerbated by anti-VEGF therapies and promote resistance to such therapies. Low metronomic doses of dox can block activation of HIF-target genes and works synergistically with anti-VEGF therapies to inhibit tumor growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2851. doi:10.1158/1538-7445.AM2011-2851

Abstract LB-147: 18F-FAZA-PET as a pharmacodynamic biomarker of anti-tumor activity for the novel HIF-1α pathway inhibitor BAY 87–2243 in preclinical tumor models

Abstract BAY 87–2243 is a novel small molecule inhibitor of hypoxia inducible factor-1α (HIF-1α) activity. Preclinical studies with BAY 87–2243 in a number of murine tumor models showed moderate to high tumor growth inhibition. The present study describes a non-invasive positron emission tomographic (PET) imaging method that monitors early therapeutic efficacy of this drug. To identify optimal tracer(s) for monitoring BAY 87–2243 anti-tumor activity, female nude mice were implanted on both scapulae with either 3.5×106 human non-small cell lung cancer (H460) or 2×106 human prostate cancer cells (PC3). Both are growth-inhibited by BAY 87–2243. Xenografts were grown to 200 mm3 over 10 days. Four PET tracers were assessed for uptake into tumor xenografts: 18F-FDG, 18F-FPP(RGD)2, 18F-FLT and 18F-FAZA. Tracers were injected via the tail vein. After pre-scan, BAY 87–2243 was orally administered, daily at 9mg/kg to 7 days. Tracer uptake was examined 1 and 3 day(s) after initial administration. Each treatment group for each PET tracer tested consisted of 6 tumors in 3 animals. At each imaging time point, RNA from BAY 87–2243-treated and vehicle-treated H460 tumor xenografts (3 additional animals each) was isolated. HIF target genes were quantified by Real-Time polymerase chain (RT-PCR) reaction. For both 18F-FDG and 18F-FPP(RGD)2, uptake was not significantly altered. However, 18F-FAZA tumor uptake (%ID mean/g) declined by 55–70% (1.21 ± 0.10 %ID/g to 0.35 ± 0.1 %ID/g, n=6, vehicle vs. treatment) in both H460 (p<0.001) and PC3 (p<0.05) models at both 1 and 3 days after drug administration. The decline occurred before any significant difference in tumor volume between drug- and vehicle-treated animals was observed, thus suggesting that decline in 18F-FAZA uptake reflected early changes in tumor biochemistry. A similar observation was noted for 18F-FLT in the H460 model, although the decline was less pronounced (30%; p<0.05 for all time-points). Analysis of HIF target gene expression by RT-PCR revealed that BAY 87–2243 reduced expression of the hypoxia regulated genes CA IX and ANGPTL4 by 99 % and 93 % respectively (p<0.001 for both) which corresponds with reduced 18F-FAZA uptake upon drug treatment. Reduced 18F-FLT uptake was not accompanied by reduced expression of TK1. Expression level of genes associated with glucose metabolism was heterogeneous and did not fully match with unchanged 18F-FDG uptake: GLUT-1 expression was unchanged while HK-2 decreased by 75 % (p<0.01). We detected low ITGB3 expression in untreated xenografts but were unable to detect transcripts after BAY 87–2243 treatment. This findings may explain the lack of change in 18F-FPP(RGD)2 uptake. In conclusion, our pilot studies suggest the suitability of 18F-FAZA-PET to monitor the efficacy of anti-cancer agents targeting the HIF pathway (e.g. BAY 87–2243) as an early PD marker prior to changes in tumor volume Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-147. doi:10.1158/1538-7445.AM2011-LB-147

Induction of hypoxia inducible factor (HIF-1α) in rat kidneys by iron chelation with the hydroxypyridinone, CP94

Hypoxia inducible factor (HIF-1α) is a master regulator of tissue adaptive responses to hypoxia whose stability is controlled by an iron containing prolyl hydroxylase domain (PHD) protein. A catalytic redox cycle in the PHD's iron center that results in the formation of a ferryl (Fe + 4 ) intermediate has been reported to be responsible for the hydroxylation and subsequent degradation of HIF-1α under normoxia. We show that induction of HIF-1α in rat kidneys can be achieved by iron reduction by the hydroxypyridin-4 one (CP94), an iron chelator administered intraperitoneally in rats. The extent of HIF protein stabilization as well as the expression of HIF target genes, including erythropoietin (EPO), in kidney tissues was comparable to those induced by known inhibitors of the PHD enzyme, such as desferrioxamine (DFO) and cobalt chloride (CoCl 2). In human kidney cells and in vitro PHD activity assay, we were able to show that the HIF-1α protein can be stabilized by addition of CP94. This appears to inactivate PHD; and thus prevents the hydroxylation of HIF-1α. In conclusion, we have identified the inhibition of iron-binding pocket of PHD as an underlying mechanism of HIF induction in vivo and in vitro by a bidentate hydroxypyridinone.

Factor inhibiting HIF (FIH-1) promotes renal cancer cell survival by protecting cells from HIF-1α-mediated apoptosis

Background:Clear cell renal cell carcinoma (CCRCC) is the commonest form of kidney cancer. Up to 91% have biallelic inactivation of VHL, resulting in stabilisation of HIF-α subunits. Factor inhibiting HIF-1 is an enzyme that hydroxylates HIF-α subunits and prevents recruitment of the co-activator CBP/P300. An important question is whether FIH-1 controls HIF activity in CCRCC.Methods:Human VHL defective CCRCC lines RCC10, RCC4 and 786–O were used to determine the role of FIH-1 in modulating HIF activity, using small interfering RNA knockdown, retroviral gene expression, quantitative RT–PCR, western blot analysis, Annexin V and propidium iodide labelling.Results:Although it was previously suggested that FIH-1 is suppressed in CCRCC, we found that FIH-1 mRNA and protein are actually present at similar levels in CCRCC and normal kidney. The FIH-1 inhibition or knockdown in the VHL defective CCRCC lines RCC10 and RCC4 (which express both HIF-1α and HIF-2α) resulted in increased expression of HIF target genes. In the 786-O CCRCC cell line, which expresses only HIF-2α, FIH-1 attenuation showed no significant effect on expression of these genes; introduction of HIF-1α resulted in sensitivity of HIF targets to FIH-1 knockdown. In RCC4 and RCC10, knockdown of FIH-1 increased apoptosis. Suppressing HIF-1α expression in RCC10 prevented FIH-1 knockdown from increasing apoptosis.Conclusion:Our results support a unifying model in which HIF-1α has a tumour suppressor action in CCRCC, held in check by FIH-1. Inhibiting FIH-1 in CCRCC could be used to bias the HIF response towards HIF-1α and decrease tumour cell viability.

Aminoflavone, a Ligand of the Aryl Hydrocarbon Receptor, Inhibits HIF-1α Expression in an AhR-Independent Fashion

Aminoflavone (AF), the active component of a novel anticancer agent (AFP464) in phase I clinical trials, is a ligand of the aryl hydrocarbon receptor (AhR). AhR dimerizes with HIF-1beta/AhR, which is shared with HIF-1alpha, a transcription factor critical for the response of cells to oxygen deprivation. To address whether pharmacologic activation of the AhR pathway might be a potential mechanism for inhibition of HIF-1, we tested the effects of AF on HIF-1 expression. AF inhibited HIF-1alpha transcriptional activity and protein accumulation in MCF-7 cells. However, inhibition of HIF-1alpha by AF was independent from a functional AhR pathway. Indeed, AF inhibited HIF-1alpha expression in Ah(R100) cells, in which the AhR pathway is functionally impaired, yet did not induce cytotoxicity, providing evidence that these effects are mediated by distinct signaling pathways. Moreover, AF was inactive in MDA-MB-231 cells, yet inhibited HIF-1alpha in MDA-MB-231 cells transfected with the SULT1A1 gene. AF inhibited HIF-1alpha mRNA expression by approximately 50%. Notably, actinomycin-D completely abrogated the ability of AF to downregulate HIF-1alpha mRNA, indicating that active transcription was required for the inhibition of HIF-1alpha expression. Finally, AF inhibited HIF-1alpha protein accumulation and the expression of HIF-1 target genes in MCF-7 xenografts. These results show that AF inhibits HIF-1alpha in an AhR-independent fashion, and they unveil additional activities of AF that may be relevant for its further clinical development.