Expression Of HIF-1α Target Genes Research Articles

HEXIM1 down-regulates hypoxia-inducible factor-1α protein stability

We have previously reported on the inhibition of HIF-1α (hypoxia-inducible factor α)-regulated pathways by HEXIM1 [HMBA (hexamethylene-bis-acetamide)-inducible protein 1]. Disruption of HEXIM1 activity in a knock-in mouse model expressing a mutant HEXIM1 protein resulted in increased susceptibility to the development of mammary tumours, partly by up-regulation of VEGF (vascular endothelial growth factor) expression, HIF-1α expression and aberrant vascularization. We now report on the mechanistic basis for HEXIM1 regulation of HIF-1α. We observed direct interaction between HIF-1α and HEXIM1, and HEXIM1 up-regulated hydroxylation of HIF-1α, resulting in the induction of the interaction of HIF-1α with pVHL (von Hippel-Lindau protein) and ubiquitination of HIF-1α. The up-regulation of hydroxylation involves HEXIM1-mediated induction of PHD3 (prolyl hydroxylase 3) expression and interaction of PHD3 with HIF-1α. Acetylation of HIF-1α has been proposed to result in increased interaction of HIF-1α with pVHL and induced pVHL-mediated ubiquitination, which leads to the proteasomal degradation of HIF-1α. HEXIM1 also attenuated the interaction of HIF-1α with HDAC1 (histone deacetylase 1), resulting in acetylation of HIF-1α. The consequence of HEXIM1 down-regulation of HIF-1α protein expression is attenuated expression of HIF-1α target genes in addition to VEGF and inhibition of HIF-1α-regulated cell invasion.

Digoxin as an inhibitor of global hypoxia inducible factor-1α (HIF1α) expression and downstream targets in breast cancer: Dig-HIF1 pharmacodynamic trial.

TPS1144 Background: Unlike normal cells, tumor cells thrive in a hypoxic microenvironment, and intratumoral hypoxia correlates with increased tumor invasiveness, metastasis, and poor prognosis in cancer. The hypoxic microenvironment is primarily mediated through HIF1α, a transcription factor for over 200 genes involved in cellular metabolism, proliferation, and angiogenesis. In pre-clinical models, cardiac glycosides, including digoxin, act as potent inhibitors of HIF1α protein synthesis and expression of HIF1α target genes (Zhang et al, PNAS 09). Methods: Trial design: The proposed study is a randomized, controlled, two arm, pre-surgical study. Eligible patients include women with stage I-III carcinoma of the breast scheduled to undergo definitive surgery, tumor size ≥ 1cm, grade 2/3 or Ki-67 ≥ 10%, normal organ function, and no known cardiac arrhythmias. Participants will receive oral digoxin daily, or no therapy, for 14 days (±4 days) prior to scheduled surgery. Trial Objectives: 1) To evaluate whether daily oral digoxin therapy, as compared to no study drug, reduces HIF1α expression by IHC and mRNA or its target genes (VEGF, CA-9, and GLUT1) in breast cancer tissue. 2) To evaluate whether daily oral digoxin therapy, as compared to no study drug, reduces levels of serum VEGF & PAI-1, reduces tissue Ki-67 expression, and modulates proteomic profiles of breast cancer tissue. 3) To assess safety and tolerability of digoxin therapy in the pre-surgical setting. Statistical methods: The primary hypothesis is that 2 weeks of digoxin therapy will reduce the level of HIF1α expression in breast cancer tissue assessed by pathologists blinded to the treatment assignment. Our preliminary study shows the mean HIF1α expression level is 2.43. A 33% reduction would be considered clinically relevant. Allowing for up to 20% attrition, a total sample size of 64 will provide more than 85% power to detect 33% reduction with a two-sided significance level of 0.05. Target accrual: 64 (32 each arm). Funding and Acknowledgement: Dept. of Defense, Commonwealth Foundation, ASCO YIA. Contact Person: Dr. Vered Stearns, Email: vstearn1@jhmi.edu .ClinicalTrials.gov Identifier: NCT01763931. Clinical trial information: NCT01763931.

Transcription Factor YY1 Contributes to Tumor Growth by Stabilizing Hypoxia Factor HIF-1α in a p53-Independent Manner

In response to hypoxic stress, hypoxia-inducible factor (HIF)-1α is a critical transcription factor regulating fundamental cellular processes, and its elevated expression level and activity are associated with poor outcomes in most malignancies. The transcription factor Yin Yang 1 (YY1) is an important negative regulator of the tumor suppressor factor p53. However, the role of YY1 under tumor hypoxic condition is poorly understood. Herein, we show that inhibition of YY1 reduced the accumulation of HIF-1α and its activity under hypoxic condition, and consequently downregulated the expression of HIF-1α target genes. Interestingly, our results revealed that the downregulation of HIF-1α by inhibiting YY1 is p53-independent. Functionally, the in vivo experiments revealed that inhibition of YY1 significantly suppressed growth of metastatic cancer cells and lung colonization and also attenuated angiogenesis in a p53-null tumor. Collectively, our findings unraveled a novel mechanism by which YY1 inhibition disrupts hypoxia-stimulated HIF-1α stabilization in a p53-independent manner. Therefore, YY1 inhibition could be considered as a potential tumor therapeutic strategy to give consistent clinical outcomes independent of p53 status.

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.

Abstract 1661: Knockdown of Nrf2 in cancer cells suppresses tumor growth: implication for the inhibitory role in HIF-1α and ErbB2 signaling

Abstract Nuclear factor-erythroid 2 (Nrf2) is the transcription factor regulating multiple antioxidant proteins and phase 2 detoxifying enzymes, and plays a crucial role in the cytoprotection against oxidative stress-associated damages. However, a potential role of Nrf2 in cancer biology has been emerged recently. In our mouse xenografts study, a stable knockdown of Nrf2 in cancer cells from the colon (HCT116 and HT29) and ovary (SKOV-3) strongly suppressed tumor growth. As an underlying mechanism, it was observed that a vessel formation and vascular endothelial growth factor (VEGF) expression were suppressed in Nrf2-inhibited colon cancer tumors. Suppressive effect of Nrf2 knockdown on angiogenesis was further evidenced by the chicken embryo chorioallantoic membrane assay and endothelial tube formation assay. The subsequent mechanism studies revealed that Nrf2-inhibited HT29 cells failed to accumulate HIF-1α protein under 1% O2 hypoxia, resulting in the limitation of the expression of HIF-1α target genes for angiogenesis. Whereas, a tumor growth suppression shown in SKOV-2 cells was associated with repressed expression of ErbB2 and the consequent increase in p27. Further experimental demonstrations showed that the expression of ErbB2 is inversely related with Nrf2 in other types of cancer cell lines; therefore, the cell growth is attenuated in these Nrf2-inhibited cancer cells. Taken together, our current study suggests that a stable Nrf2 inhibition in cancer cells suppresses tumor growth through the alterations in HIF-1α and ErbB2 signaling. Hence, Nrf2 may be a promising target to control cancer cell growth and chemoresistance. 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 1661. doi:10.1158/1538-7445.AM2011-1661

Hypoxia Inducible Factor–Dependent Regulation of Angiogenesis by Nitro–Fatty Acids

Nitro-fatty acids (NO(2)-FAs) are emerging as a new class of cell signaling mediators. Because NO(2)-FAs are found in the vascular compartment and their impact on vascularization remains unknown, we aimed to investigate the role of NO(2)-FAs in angiogenesis. The effects of nitrolinoleic acid and nitrooleic acid were evaluated on migration of endothelial cell (EC) in vitro, EC sprouting ex vivo, and angiogenesis in the chorioallantoic membrane assay in vivo. At 10 μmol/L, both NO(2)-FAs induced EC migration and the formation of sprouts and promoted angiogenesis in vivo in an NO-dependent manner. In addition, NO(2)-FAs increased intracellular NO concentration, upregulated protein expression of the hypoxia inducible factor-1α (HIF-1α) transcription factor by an NO-mediated mechanism, and induced expression of HIF-1α target genes, such as vascular endothelial growth factor, glucose transporter-1, and adrenomedullin. Compared with typical NO donors such as spermine-NONOate and deta-NONOate, NO(2)-FAs were slightly less potent inducers of EC migration and HIF-1α expression. Short hairpin RNA-mediated knockdown of HIF-1α attenuated the induction of vascular endothelial growth factor mRNA expression and EC migration stimulated by NO(2)-FAs. Our data disclose a novel physiological role for NO(2)-FAs, indicating that these compounds induce angiogenesis in an NO-dependent mechanism via activation of HIF-1α.

Metabolomic analyses reveal profound differences in glycolytic and tricarboxylic acid cycle metabolism in glucose-responsive and -unresponsive clonal β-cell lines

Insulin secretion from pancreatic β-cells is controlled by complex metabolic and energetic changes provoked by exposure to metabolic fuels. Perturbations in these processes lead to impaired insulin secretion, the ultimate cause of T2D (Type 2 diabetes). To increase our understanding of stimulus-secretion coupling and metabolic processes potentially involved in the pathogenesis of T2D, a comprehensive investigation of the metabolic response in the glucose-responsive INS-1 832/13 and glucose-unresponsive INS-1 832/2 β-cell lines was performed. For this metabolomics analysis, we used GC/MS (gas chromatography/mass spectrometry) combined with multivariate statistics. We found that perturbed secretion in the 832/2 line was characterized by disturbed coupling of glycolytic and TCA (tricarboxylic acid)-cycle metabolism. The importance of this metabolic coupling was reinforced by our observation that insulin secretion partially could be reinstated by stimulation of the cells with mitochondrial fuels which bypass glycolytic metabolism. Furthermore, metabolic and functional profiling of additional β-cell lines (INS-1, INS-1 832/1) confirmed the important role of coupled glycolytic and TCA-cycle metabolism in stimulus-secretion coupling. Dependence of the unresponsive clones on glycolytic metabolism was paralleled by increased stabilization of HIF-1α (hypoxia-inducible factor 1α). The relevance of a similar perturbation for human T2D was suggested by increased expression of HIF-1α target genes in islets from T2D patients.

A pilot trial of oral topotecan (TPT) in patients with refractory advanced solid neoplasms expressing HIF-1α.

e13518 Background: HIF-1α is a transcription factor that mediates tumor angiogenesis and survival. Previous studies have demonstrated that daily administration of topotecan (TPT) - a topoisomerase-1 inhibitor - inhibits HIF-1α expression, angiogenesis, and tumor growth in human xenograft models. The primary aim of this study is to investigate whether TPT inhibits HIF-1α expression in human cancers. Methods: TPT was administered orally at 1.6 mg/m2 daily × 5 for 2 weeks, of a 28-day cycle, to patients with advanced solid tumors expressing HIF-1α in at least 10% of tumor cells. Tumor biopsies were obtained before and after 2 cycles of TPT. The primary endpoint was modulation of HIF-1α expression by IHC. mRNA expression of HIF-1α target genes was assessed by real-time PCR. Radiologic correlative studies (comprising 18FDG-PET, to assess tumor metabolism, and DCE-MRI, to assess blood flow and permeability) were obtained at baseline, after 2 weeks of therapy and post cycle 2. The statistical significance of the difference in HIF-1α values was determined by a Wilcoxon signed rank test. Results: 15 patients, median age 54 (range 25-70), were treated, with n=7 undergoing paired tumor biopsies and being evaluable for the primary endpoint. Disease types: melanoma, colon (n=3), ovarian (n=2), sarcoma (n=2), lung, bladder, adrenocortical (n=2), anal, pancreatic, head and neck. Three patients received TPT 1.6 mg/m2before protocol amended to 1.2 mg/m2because of dose-limiting G3 neutropenia and G4 thrombocytopenia. The median number of cycles received was 2. Only 5 patients proceeded beyond 2 cycles (maximum 6). The median reduction in HIF-1α by IHC was -7.5% (range -50.0 to +5.0%; p=0.16). 6 of 7 patients exhibited declines in K trans on DCE-MRI (p=0.13) and 5 of 7 exhibited Kep changes (p=0.45). One patient obtained a PR and 2 patients had SD. Conclusions: This is the first study evaluating HIF-1α modulation in tumor tissue as a primary clinical trial endpoint in cancer patients. Whilst trends in both HIF-1α modulation and radiologic correlates (kTrans alteration on DC-MRI) were seen, these were not statistically significant due to low patient numbers. No significant financial relationships to disclose.

Early and late effects of the immunosuppressants rapamycin and mycophenolate mofetil on UV carcinogenesis

Increased skin cancer risk in organ transplant recipients has been experimentally emulated with enhanced UV carcinogenesis from administering conventional immunosuppressants. However, newer generation immunosuppressive drugs, rapamycin (Rapa) and mycophenolate mofetil (MMF), have been shown to impair angiogenesis and outgrowth of tumor implants. To ascertain the overall effect on UV carcinogenesis, Rapa and MMF were admixed into the food pellets of hairless SKH1 mice receiving daily sub-sunburn UV dosages. With immunosuppressive blood levels neither of the drugs affected onset of tumors (<2 mm), but in contrast to MMF, Rapa significantly increased latency of large tumors (>or=4 mm, medians of 190 vs 125 days) and reduced their multiplicity (1.6 vs 4.5 tumors per mouse at 200 days). Interestingly, tumors (>2 mm) from the Rapa-fed group showed a reduction in UV-signature p53 mutations (39% vs 90%) in favor of mutations from putative base oxidation. This shift in mutation spectrum was not essentially linked to the reduction in large tumors because it was absent in large tumors similarly reduced in number when feeding Rapa in combination with MMF, possibly owing to an antioxidant effect of MMF. Significantly fewer tumor cells were Vegf-positive in the Rapa-fed groups, but a correspondingly reduced expression of Hif1alpha target genes (Vegf, Ldha, Glut1, Pdk1) that would indicate altered glucose metabolism with increased oxidative stress was not found. Remarkably, we observed no effect of the immunosuppressants on UV-induced tumor onset, and with impaired tumor outgrowth Rapa could therefore strongly reduce skin carcinoma morbidity and mortality rates in organ transplant recipients.

P38α blockade inhibits colorectal cancer growth in vivo by inducing a switch from HIF1α- to FoxO-dependent transcription

Colorectal cancer cell (CRC) fate is governed by an intricate network of signaling pathways, some of which are the direct target of DNA mutations, whereas others are functionally deregulated. As a consequence, cells acquire the ability to grow under nutrients and oxygen shortage conditions. We earlier reported that p38alpha activity is necessary for proliferation and survival of CRCs in a cell type-specific manner and regardless of their phenotype and genotype. Here, we show that p38alpha sustains the expression of HIF1alpha target genes encoding for glycolytic rate-limiting enzymes, and that its inhibition causes a drastic decrease in ATP intracellular levels in CRCs. Prolonged inactivation of p38alpha triggers AMPK-dependent nuclear localization of FoxO3A and subsequent activation of its target genes, leading to autophagy, cell cycle arrest and cell death. In vivo, pharmacological blockade of p38alpha inhibits CRC growth in xenografted nude mice and azoxymethane-treated Apc(Min) mice, achieving both a cytostatic and cytotoxic effect, associated with high nuclear expression of FoxO3A and increased expression of its target genes p21 and PTEN. Hence, inhibition of p38alpha affects the aerobic glycolytic metabolism specific of cancer cells and might be taken advantage of as a therapeutic strategy targeted against CRCs.

The zinc chelator, N, N, N′, N′-tetrakis (2-pyridylmethyl) ethylenediamine, increases the level of nonfunctional HIF-1α protein in normoxic cells

The hypoxia-inducible factor-1α (HIF-1α) subunit is activated in response to lack of oxygen. HIF-1α-specific prolyl hydroxylase and factor inhibiting HIF-1α (FIH-1) catalyze hydroxylation of the proline and asparagine residues of HIF-1α, respectively. The hydroxyproline then interacts with ubiquitin E3 ligase, the von Hippel–Lindau protein, leading to degradation of HIF-1α by ubiquitin-dependent proteasomes, while the hydroxylation of the asparagine residue prevents recruitment of the coactivator, cAMP-response element-binding protein (CBP), thereby decreasing the transactivation ability of HIF-1α. We found that the Zn-specific chelator, N, N, N′, N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), enhances the activity of HIF-1α-proline hydroxylase 2 but the level of HIF-1α protein does not fall because TPEN also inhibits ubiquitination. Since the Zn chelator does not prevent FIH-1 from hydroxylating the asparagine residue of HIF-1α, its presence leads to the accumulation of HIF-1α that is both prolyl and asparaginyl hydroxylated and is therefore nonfunctional. In hypoxic cells, TPEN also prevents HIF-1α from interacting with CBP, so reducing expression of HIF-1α target genes. As a result, Zn chelation causes the accumulation of nonfunctional HIF-1α protein in both normoxia and hypoxia.

Zinc chelators increase the activity of Hypoxia‐inducible factor‐1α in normoxic neuronal cells

The hypoxia-inducible factor-1α (HIF-1α) subunit is activated in response to lack of oxygen. We found that the Zn-specific chelator, N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) enhances the activity of HIF-1α-proline hydroxylase 2 but the level of HIF-1α protein does not fall because TPEN also inhibits ubiquitination. Since the Zn chelator does not prevent FIH-1 from hydroxylating the asparagine residue of HIF-1α its presence leads to the accumulation of HIF-1α that is both prolyl and asparaginyl hydroxylated and is therefore nonfunctional. In hypoxic cells, TPEN also prevents HIF-1α from interacting with CBP, so reducing expression of HIF-1α target genes. As a result, Zn chelation causes the accumulation of nonfunctional HIF-1α protein in both normoxia and hypoxia. [This study was supported by the Neurobiology Research program grant (2004-01969) from the Ministry of Science and Technology.]

Is the hypoxia-inducible factor pathway important in gastric cancer?

Tumour hypoxia is well recognised in oncology to be a key factor resulting in treatment resistance and poor prognosis. Hypoxia leads to the expression of a number of gene products that are involved in tumour progression, invasion and metastasis formation. The most important of these proteins is thought to be hypoxia-inducible factor-1α (HIF-1α), which appears to be a master regulator of the cellular response to hypoxia. HIF-1α expression is associated with a poor prognosis and treatment response in a number of tumour sites. There is some evidence that the HIF-1α pathway might be involved in gastric carcinogenesis. Studies have shown reactive oxygen species from Helicobacter pylori, associated with the development of gastric cancer, stabilise HIF-1α. Non-steroidal anti-inflammatory drugs, shown to reduce the risk of gastric cancer, can decrease HIF-1α expression. Although a large study correlating HIF-1α expression with prognosis is lacking in gastric cancer, the immunohistochemical expression of HIF-1α target genes ( Glut-1, VEGF, CA9, iNOS) is associated with a poor prognosis. In addition, the targeted inhibition of HIF-1α has been shown to inhibit the growth of gastric tumours in animals. Increased understanding of the importance of hypoxia and the HIF-1α pathways may therefore hold the key to prevention strategies, improved selection of patients for adjuvant therapy and new treatments for the disease.

Suppression of Hypoxia-inducible Factor 1α (HIF-1α) Transcriptional Activity by the HIF Prolyl Hydroxylase EGLN1

The cellular response to hypoxia is, at least in part, mediated by the transcriptional regulation of hypoxia-responsive genes involved in balancing the intracellular ATP production and consumption. Recent evidence suggests that the transcription factor, HIF-1alpha, functions as a master regulator of oxygen homeostasis by controlling a broad range of cellular events in hypoxia. In normoxia, HIF-1alpha is targeted for destruction via prolyl hydroxylation, an oxygen-dependent modification that signals for recognition by the ubiquitin ligase complex containing the von Hippel-Lindau tumor suppressor. Three HIF prolyl hydroxylases (EGLN1, EGLN2, and EGLN3) have been identified in mammals, among which EGLN1 and EGLN3 are hypoxia-inducible at their mRNA levels in an HIF-1alpha-dependent manner. In this study, we demonstrated that apart from promoting HIF-1alpha proteolysis in normoxia, EGLN1 specifically represses HIF-1alpha transcriptional activity in hypoxia. Ectopic expression of EGLN1 inhibited HIF-1alpha transcriptional activity without altering its protein levels in a von Hippel-Lindau-deficient cell line, indicating a discrete activity of EGLN1 in transcriptional repression. Conversely, silencing of EGLN1 expression augmented HIF-1alpha transcriptional activity and its target gene expression in hypoxia. Thus, we proposed that the accumulated EGLN1 in hypoxia acts as a negative-feedback mechanism to modulate HIF-1alpha target gene expression. Our finding also provided new insight into the pharmacological manipulation of the HIF prolyl hydroxylase for ischemic diseases.