HIF-1 Target Research Articles

Differential Contribution of N- and C-Terminal Regions of HIF1α and HIF2α to Their Target Gene Selectivity.

Cellular response to hypoxia is controlled by the hypoxia-inducible transcription factors HIF1α and HIF2α. Some genes are preferentially induced by HIF1α or HIF2α, as has been explored in some cell models and for particular sets of genes. Here we have extended this analysis to other HIF-dependent genes using in vitro WT8 renal carcinoma cells and in vivo conditional Vhl-deficient mice models. Moreover, we generated chimeric HIF1/2 transcription factors to study the contribution of the HIF1α and HIF2α DNA binding/heterodimerization and transactivation domains to HIF target specificity. We show that the induction of HIF1α-dependent genes in WT8 cells, such as CAIX (CAR9) and BNIP3, requires both halves of HIF, whereas the HIF2α transactivation domain is more relevant for the induction of HIF2 target genes like the amino acid carrier SLC7A5. The HIF selectivity for some genes in WT8 cells is conserved in Vhl-deficient lung and liver tissue, whereas other genes like Glut1 (Slc2a1) behave distinctly in these tissues. Therefore the relative contribution of the DNA binding/heterodimerization and transactivation domains for HIF target selectivity can be different when comparing HIF1α or HIF2α isoforms, and that HIF target gene specificity is conserved in human and mouse cells for some of the genes analyzed.

An allosteric peptide inhibitor of HIF-1α regulates hypoxia-induced retinal neovascularization

Retinal neovascularization (NV), a leading cause of vision loss, results from localized hypoxia that stabilizes the hypoxia-inducible transcription factors HIF-1α and HIF-2α, enabling the expression of angiogenic factors and genes required to maintain homeostasis under conditions of oxygen stress. HIF transcriptional activity depends on the interaction between its intrinsically disordered C-terminal domain and the transcriptional coactivators CBP/p300. Much effort is currently directed at disrupting protein-protein interactions between disease-associated transcription factors like HIF and their cellular partners. The intrinsically disordered protein CITED2, a direct product of HIF-mediated transcription, functions as a hypersensitive negative regulator that attenuates the hypoxic response by competing allosterically with HIF-1α for binding to CBP/p300. Here, we show that a peptide fragment of CITED2 is taken up by retinal cells and efficiently regulates pathological angiogenesis in murine models of ischemic retinopathy. Both vaso-obliteration (VO) and NV were significantly inhibited in an oxygen-induced retinopathy (OIR) model following intravitreal injection of the CITED2 peptide. The CITED2 peptide localized to retinal neurons and glia, resulting in decreased expression of HIF target genes. Aflibercept, a commonly used anti-VEGF therapy for retinal neovascular diseases, rescued NV but not VO in OIR. However, a combination of the CITED2 peptide and a reduced dose of aflibercept significantly decreased both NV and VO. In contrast to anti-VEGF agents, the CITED2 peptide can rescue hypoxia-induced retinal NV by modulating the hypoxic response through direct competition with HIF for CBP/p300, suggesting a dual targeting strategy for treatment of ischemic retinal diseases and other neovascular disorders.

Neuroprotective Effect of HIF Prolyl Hydroxylase Inhibition in an In Vitro Hypoxia Model.

A novel potent analog of the branched tail oxyquinoline group of hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors, neuradapt, has been studied in two treatment regimes in an in vitro hypoxia model on murine primary hippocampal cultures. Neuradapt activates the expression of HIF1 and HIF2 target genes and shows no toxicity up to 20 μM, which is more than an order of magnitude higher than its biologically active concentration. Cell viability, functional activity, and network connectivity between the elements of neuronal networks have been studied using a pairwise correlation analysis of the intracellular calcium fluctuations in the individual cells. An immediate treatment with 1 μM and 15 μM neuradapt right at the onset of hypoxia not only protects from the death, but also maintains the spontaneous calcium activity in nervous cells at the level of the intact cultures. A similar neuroprotective effect in the post-treatment scenario is observed for 15 μM, but not for 1 μM neuradapt. Network connectivity is better preserved with immediate treatment using 1 μM neuradapt than with 15 μM, which is still beneficial. Post-treatment with neuradapt did not restore the network connectivity despite the observation that neuradapt significantly increased cell viability at 1 μM and functional activity at 15 μM. The preservation of cell viability and functional activity makes neuradapt promising for further studies in a post-treatment scenario, since it can be combined with other drugs and treatments restoring the network connectivity of functionally competent cells.

HIF-1 directly induces TET3 expression to enhance 5-hmC density and induce erythroid gene expression in hypoxia

AbstractIn mammalian cells, cytosines found within cytosine guanine dinucleotides can be methylated to 5-methylcytosine (5-mC) by DNA methyltransferases and further oxidized by the Ten-eleven translocation dioxygenase (TET) enzymes to 5-hydroxymethylcytosine (5-hmC). We have previously shown that hematopoietic stem and progenitor cells (HSPCs) with TET2 mutations have aberrant 5-hmC distribution and less erythroid differentiation potential. However, these experiments were performed under standard tissue culture conditions with 21% oxygen (O2), whereas HSPCs in human bone marrow reside in ∼1% O2. Therefore, to model human erythropoiesis more accurately, we compared 5-hmC distribution and gene expression in hypoxic vs normoxic conditions. Despite TET enzymes having limited O2 as a substrate in hypoxia, 5-hmC peaks were more numerous and pronounced than in normoxia. Among the TET genes, TET3 was upregulated specifically in hypoxia. We identified 2 HIF-1 binding sites in TET3 by chromatin immunoprecipitation of HIF-1α followed by sequencing, and TET3 upregulation was abrogated with deletion of both sites, indicating that TET3 is a direct HIF-1 target. Finally, we showed that loss of one or both of these HIF-1 binding sites in K562 cells disrupted erythroid differentiation in hypoxia and lowered cell viability. This work provides a molecular link between O2 availability, epigenetic modification of chromatin, and erythroid differentiation.

Crosstalk between hydroxytyrosol, a major olive oil phenol, and HIF-1 in MCF-7 breast cancer cells

Olive oil intake has been linked with a lower incidence of breast cancer. Hypoxic microenvironment in solid tumors, such as breast cancer, is known to play a crucial role in cancer progression and in the failure of anticancer treatments. HIF-1 is the foremost effector in hypoxic response, and given that hydroxytyrosol (HT) is one of the main bioactive compounds in olive oil, in this study we deepen into its modulatory role on HIF-1. Our results in MCF-7 breast cancer cells demonstrate that HT decreases HIF-1α protein, probably by downregulating oxidative stress and by inhibiting the PI3K/Akt/mTOR pathway. Strikingly, the expression of HIF-1 target genes does not show a parallel decrease. Particularly, adrenomedullin and vascular endothelial growth factor are up-regulated by high concentrations of HT even in HIF-1α silenced cells, pointing to HIF-1-independent mechanisms of regulation. In fact, we show, by in silico modelling and transcriptional analysis, that high doses of HT may act as an agonist of the aryl hydrocarbon receptor favoring the induction of these angiogenic genes. In conclusion, we suggest that the effect of HT in a hypoxic environment is largely affected by its concentration and involves both HIF-1 dependent and independent mechanisms.

PRDM16 suppresses HIF-targeted gene expression in kidney cancer.

Analysis of transcriptomic data demonstrates extensive epigenetic gene silencing of the transcription factor PRDM16 in renal cancer. We show that restoration of PRDM16 in RCC cells suppresses in vivo tumor growth. RNaseq analysis reveals that PRDM16 imparts a predominantly repressive effect on the RCC transcriptome including suppression of the gene encoding semaphorin 5B (SEMA5B). SEMA5B is a HIF target gene highly expressed in RCC that promotes in vivo tumor growth. Functional studies demonstrate that PRDM16's repressive properties, mediated by physical interaction with the transcriptional corepressors C-terminal binding proteins (CtBP1/2), are required for suppression of both SEMA5B expression and in vivo tumor growth. Finally, we show that reconstitution of RCC cells with a PRDM16 mutant unable to bind CtBPs nullifies PRDM16's effects on both SEMA5B repression and tumor growth suppression. Collectively, our data uncover a novel epigenetic basis by which HIF target gene expression is amplified in kidney cancer and a new mechanism by which PRDM16 exerts its tumor suppressive effects.

The Autophagy Gene ATG9A as a HIF Target Essential for Intestinal Epithelial Barrier Function

A pathological hallmark of mucosal diseases such as inflammatory bowel disease (IBD) is disruption of the intestinal epithelium, a selectively permeable barrier that acts to absorb nutrients and water while simultaneously guarding against pathogens and environmental threats. The intestinal epithelium exists in a metabolic state of low oxygen tension termed “physiologic hypoxia” that is essential for its proper function as an absorptive and barrier organ. An important factor in maintaining hypoxic epithelial homeostasis is the transcription factor family HIF, which is stabilized in hypoxic conditions and mediates epithelial homeostatic response to low oxygen tensions. To identify HIF transcriptional targets in epithelia, chromatin immunoprecipitation (ChIP) was performed in Caco‐2 intestinal epithelial cells (IECs) using HIF‐1α and HIF‐2α specific polyclonal antibodies. ChIP‐enriched DNA was hybridized to a custom promoter microarray (termed ChIP‐chip) comprising a genome‐wide set of predicted transcription start site (TSS) flanking sequences at 50‐bp resolution. This unbiased approach identified autophagy as one of the major HIF‐1 targeted pathways. Binding of HIF‐1 to ATG9A, the only transmembrane autophagy protein, was significantly enriched by exposure of IEC to hypoxia. Validation of the ChIP‐chip revealed prominent induction of ATG9A mRNA and protein. Likewise, using luciferase promoter assays, we identified a functional hypoxia response element upstream of the TSS. Hypoxia‐mediated induction of ATG9A was lost in cells lacking HIF‐1. Strikingly, we found that lentiviral‐mediated knockdown of ATG9A in IECs prevents epithelial barrier formation and results in mislocalization of multiple junctional proteins. Extensions of these findings showed that ATG9A knockdown cells have intrinsic differences in junctional protein expression, suggesting that ATG9A is essential for multiple steps of epithelial junction biogenesis. Our findings have novel applicability for inflammatory disorders that involve a compromised epithelial barrier, such as IBD, and suggest that targeting ATG9A may be a rational strategy for future therapeutic intervention.

HIF-2 Complex Dissociation, Target Inhibition, and Acquired Resistance with PT2385, a First-in-Class HIF-2 Inhibitor, in Patients with Clear Cell Renal Cell Carcinoma.

The heterodimeric transcription factor HIF-2 is arguably the most important driver of clear cell renal cell carcinoma (ccRCC). Although considered undruggable, structural analyses at the University of Texas Southwestern Medical Center (UTSW, Dallas, TX) identified a vulnerability in the α subunit, which heterodimerizes with HIF1β, ultimately leading to the development of PT2385, a first-in-class inhibitor. PT2385 was safe and active in a first-in-human phase I clinical trial of patients with extensively pretreated ccRCC at UTSW and elsewhere. There were no dose-limiting toxicities, and disease control ≥4 months was achieved in 42% of patients. We conducted a prospective companion substudy involving a subset of patients enrolled in the phase I clinical trial at UTSW (n = 10), who were treated at the phase II dose or above, involving multiparametric MRI, blood draws, and serial biopsies for biochemical, whole exome, and RNA-sequencing studies. PT2385 inhibited HIF-2 in nontumor tissues, as determined by a reduction in erythropoietin levels (a pharmacodynamic marker), in all but one patient, who had the lowest drug concentrations. PT2385 dissociated HIF-2 complexes in ccRCC metastases, and inhibited HIF-2 target gene expression. In contrast, HIF-1 complexes were unaffected. Prolonged PT2385 treatment resulted in the acquisition of resistance, and we identified a gatekeeper mutation (G323E) in HIF2α, which interferes with drug binding and precluded HIF-2 complex dissociation. In addition, we identified an acquired TP53 mutation elsewhere, suggesting a possible alternate mechanism of resistance. These findings demonstrate a core dependency on HIF-2 in metastatic ccRCC and establish PT2385 as a highly specific HIF-2 inhibitor in humans. New approaches will be required to target mutant HIF-2 beyond PT2385 or the closely related PT2977 (MK-6482).

Bclaf1 is a direct target of HIF-1 and critically regulates the stability of HIF-1α under hypoxia.

Hypoxic stress is intimately connected with tumor progression, with hypoxia-inducible factor-1α (HIF-1α) being a critical regulator in this process. HIF-1α is stabilized in response to hypoxia, which is required for the induction of gene transcriptions important for hypoxic adaptation. Bclaf1 is a multifunctional protein involved in tumorigenesis, however, its role in this process is not well characterized. Here we report Bclaf1 is a direct transcriptional target of HIF-1 and upregulated in multiple cell lines during hypoxia. Importantly, we found Bclaf1 is involved in the stabilization of HIF-1α during long-term hypoxic treatments. Compared with the control cells, the protein level and stability of HIF-1α in Bclaf1 knockdown or knockout cells is greatly compromised after long-term hypoxic treatments, concomitant with the impaired inductions of HIF-1 target gene transcription. Bclaf1 knockout HeLa cells exhibit a reduced tumor growth in mice xenografts, in which the expressions of HIF-1α and its target genes are also decreased. Bclaf1 binds to HIF-1α in the nucleus, and this interaction is required for Bclaf1 to stabilize HIF-1α in hypoxic condition. These results uncover a positive feedback loop, HIF-1-Bclaf1, that sustains HIF-1 activity during long-term hypoxic conditions by binding to and protecting HIF-1α from degradation, and suggest that Bclaf1 may promote tumor progression by enhancing HIF-1α stability.

P066 GB004, A NOVEL GUT-TARGETED PROLYL HYDROXYLASE INHIBITOR FOR INFLAMMATORY BOWEL DISEASE: FIRST-IN-HUMAN, MUTLIPLE-DOSE STUDY IN HEALTHY SUBJECTS WITH GUT BIOPSIES

Abstract Background GB004 is a small molecule prolyl hydroxylase inhibitor (PHDi) that stabilizes hypoxia inducible factors (HIF1-α), key transcription factors involved in the protective cellular responses at the intersection of hypoxia and inflammation. GB004 was selected based on its gut-targeted profile to limit systemic on-target effects associated with HIF1-α stabilization. Consistent with this, orally administered GB004 in a healthy non-human primate model engaged HIF-related genes in the gut, and, in animal models of colitis, demonstrated a significant reduction in disease activity, improvements in histologic measures, and greater exposure in GI tissue relative to plasma. GB004 is in clinical development for treatment of inflammatory bowel disease (IBD) and was shown to be safe in a single ascending dose study. The study described here evaluates the safety, tolerability, and pharmacokinetics (PK) of multiple daily doses of GB004 in plasma and colon biopsies. Methods This was a randomized, double-blind, placebo-controlled, multiple dose, Phase 1a study conducted in healthy subjects at a single site in Canada. Three dose levels of GB004 formulated as a solution or placebo solution were administered orally once a day for 8 days; safety and PK were evaluated. Plasma levels of HIF target genes EPO and VEGF were determined by immunoassays from samples collected at pre-dose, 4, 8, and 12 hours post dose on Day 1 and Day 7. Colon biopsies were obtained one day prior to first dose and at Day 8. Results 42 subjects (20 male and 22 female) were dosed. No serious adverse events or deaths were recorded. The most commonly observed adverse event in GB004-treated subjects was dizziness (31%;10/22); all events were mild and did not result in study drug discontinuation. There were no identified risks of GB004. Following oral dosing, GB004 was rapidly absorbed and eliminated from the systemic circulation, with a median time to maximum concentration of 0.5 hour for all doses. Both Cmax and AUC of GB004 increased in a dose-dependent manner on Day 1 and 7. Concentrations of GB004 measured in colon biopsies were greater than concentrations in the plasma at the time of biopsy. Changes in plasma EPO or VEGF levels were similar for GB004 and placebo with no dose-related effects observed. Conclusions This study demonstrated that multiple daily doses of GB004 solution were safe and tolerable. The PK profile was consistent with its intended preferential exposure in the gut. In support of the gut-targeted exposure, HIF target genes EPO and VEGF were not modulated in plasma. A clinical study of GB004 is ongoing in patients with ulcerative colitis to explore safety, PK, and pharmacodynamics both systemically and within colonic tissue (NCT03860896). A tablet formulation is also being developed.

Suppression of HIF-1α accumulation by betulinic acid through proteasome activation in hypoxic cervical cancer

Betulinic acid (BA) exhibits various biological activities such as anti-bacterial, anti-inflammatory, anti-human papilloma virus (HPV), and anti-cancer activities. HPV infection is associated with a high risk of cervical cancer, which is the leading cause of deaths among women worldwide. Therefore, BA is an attractive therapeutic agent for treating cervical cancer. In this study, we investigated the role of BA in regulating the hypoxia-mediated response in HeLa cells and clarified the underlying mechanism of action. We found that BA inhibited the hypoxia-induced accumulation of HIF-1α without affecting HIF-1α mRNA levels and suppressed the expression of HIF target genes, including VEGF, GLUT1, and PDK1 in HeLa cells. Additionally, BA enhanced the β1, β2, and β5 activities of the proteasome, which resulted in reduced levels of ubiquitinated proteins and HIF-1α protein in HeLa cells. However, BA treatment did not affect the deubiquitinase enzyme activity in HeLa cells. These results indicate that inhibition of HIF-1α accumulation by BA is mediated by activation of the proteasome, and BA is a potential anticancer agent for the regulation of the HIF signaling pathway.

Abstract C34: Signaling regulation of epithelial-mesenchymal transition in pancreas cancer cells cultured under hypoxic conditions

Abstract Pancreatic ductal adenocarcinoma (PDAC) tumors exhibit hypoxia due to high oxygen demand and oxygen diffusional limitations (1). Hypoxia has been reported as a driver of epithelial-mesenchymal transition (EMT) (2), which occurs early in pancreas cancer dissemination and promotes resistance to chemotherapy (3,4). Druggable signaling pathways that promote EMT in the hypoxic PDAC microenvironment may therefore represent useful targets for combination therapy. Our lab previously studied the roles of ERK and SHP2 in driving EMT in response to growth factors (5,6). More recently, our lab employed an unbiased data-driven modeling approach and found that the ERK, JNK, and NF-kappaB pathways coordinate to drive robust EMT in PDAC cells treated with combinations of growth factors (unpublished). In the present study, we sought to determine the roles of those pathways in promoting EMT in PDAC cells in response to a shift from normoxic to hypoxic conditions. The immortalized human PDAC cell line HPAF-II, which exhibits baseline epithelial characteristics, was used for these studies. In simple two-dimensional cell culture, a shift from 20% to 1% oxygen for ≤120 hr led to several indications of EMT, including decreased epithelial cell cluster circularity, reduced junctional E-cadherin expression and an increased fraction of vimentin-positive cells (determined by an automated image analysis pipeline), and increased expression of the mesenchymal transcripts VIM, SNAI1, and SNAI2 (determined by RT-qPCR). A hypoxic cellular response was confirmed by immunoblotting, which showed an early (4 hr) peak in HIF-1alpha expression and a relatively late (48 hr) peak in HIF-2alpha expression, as well as increased expression of the HIF target genes PGK1 and SLC2A1. In three-dimensional cultures of HPAF-II cell spheroids in a collagen I matrix, a shift to hypoxia promoted cell invasion into the matrix, a behavior also observed when HPAF-II cells were treated with exogenous growth factors that drive EMT. To probe for the activation of the ERK, JNK, and NF-kappaB pathways, we used a combination of immunoblotting and immunofluorescence microscopy. A shift from 20% to 1% oxygen increased the phosphorylation of ERK and promoted the nuclear localization of c-Jun, a substrate of JNK. Inhibition of MEK or JNK at the same time as the shift to 1% oxygen prevented the loss of junctional E-cadherin that otherwise occurred, with JNK inhibition having the greater effect. JNK inhibition also abrogated loss of CDH1 transcripts and the increase of VIM transcripts otherwise observed in response to hypoxia. Thus, activation of the ERK and JNK pathways in PDAC cells in response to a shift to low oxygen conditions promoted EMT. These pathways may therefore represent useful targets for antagonizing EMT in the hypoxic microenvironment characteristic of PDAC tumors.

Abstract B119: Inhibition of UCHL1 blocks proliferation and metastasis in HIF-1 dependent tumor malignancy

Abstract Hypoxia-inducible factor 1 (HIF-1) is a key transcription factor for tumor malignancy. Recently, ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme, has been reported to stabilize HIF-1α by releasing the pVHL complex conjugated Ub moieties. In this study, we investigated whether inhibition of UCHL1 can be an effective strategy for HIF-1-related tumor chemotherapy. First, we found that UCHL1 inhibition by siRNA or LDN57444, a well-known UCHL1 inhibitor, drastically lowered HIF-1α protein levels in UCHL1-expressing MDA-MB-436 breast cancer cells, by Western blotting analysis and immunostaining experiments. In UCHL1 non-expressing MDA-MB-231 cells, the ectopic expression of UCHL1 significantly increased HIF-1α protein expression levels, which was canceled by the treatment of LDN57444. We further analyzed whether the UCHL1 inhibitor could affect the HIF-dependent transactivation using 5HRE-luciferase reporter assay and RT-qPCR. LDN57444 dose-dependently inhibited the HIF activity and decreased the transcription of HIF downstream genes in MDA-MB-436 cells. Next we tested the effects of the UCHL1 inhibitor on tumor cell migration. LDN57444 significantly blocked cell migration in MDA-MB-436 cells in wound healing and transwell migration assays, suggesting a crucial role of the enzyme in tumor migration. We finally investigated potential roles of UCHL1 in tumor malignancy by using a 3D spheroid culture model, which has been more widely applied in the preclinical studies to provide more physiologically relevant information. Image analysis using open source image processing suites, AnaSP and ReViSP revealed that ectopic expression of UCHL1 in MDA-MB-231 cells significantly upregulated malignancy representing factors such as integrity and volume. Whole cell number of UCHL1 transfected MDA-MB-231 spheroids was also much higher than that of the mock treated spheroids when measured by 3D cell viability assay. Depletion of UCHL1 or inhibition of UCHL1 enzyme activity in MDA-MB-436 cells, on the contrary, downregulated these malignant factors. Besides, in spheroid viability staining and spheroid invasion assay, LDN57444 abolished the UCHL1 mediated cell proliferation and invasiveness as well. Moreover, the ectopic expression of UCHL1 in MDA-MB-231 cells stabilized HIF-1α protein level and promoted the expression of HIF-1 target genes in the 3D model as in the case of 2D monolayer culture. In conclusion, we clearly proved the involvement of UCHL1 in the HIF-1 induced tumor malignancy employing the 3D spheroid culture model. Our research indicates that the UHCL1-HIF-1 pathway is a promising therapeutic target for cancer chemotherapy. Citation Format: Xuebing Li, Akira Hattori, Senye Takahashi, Yoko Goto, Hiroshi Harada, Hideaki Kakeya. Inhibition of UCHL1 blocks proliferation and metastasis in HIF-1 dependent tumor malignancy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B119. doi:10.1158/1535-7163.TARG-19-B119

A substitution mutation in a conserved domain of mammalian acetate-dependent acetyl CoA synthetase 2 results in destabilized protein and impaired HIF-2 signaling.

The response to environmental stresses by eukaryotic organisms includes activation of protective biological mechanisms, orchestrated in part by transcriptional regulators. The tri-member Hypoxia Inducible Factor (HIF) family of DNA-binding transcription factors include HIF-2, which is activated under conditions of oxygen or glucose deprivation. Although oxygen-dependent protein degradation is a key mechanism by which HIF-1 and HIF-2 activity is regulated, HIF-2 is also influenced substantially by the coupled action of acetylation and deacetylation. The acetylation/deacetylation process that HIF-2 undergoes employs a specific acetyltransferase and deacetylase. Likewise, the supply of the acetyl donor, acetyl CoA, used for HIF-2 acetylation originates from a specific acetyl CoA generator, acetate-dependent acetyl CoA synthetase 2 (Acss2). Although Acss2 is predominantly cytosolic, a subset of the Acss2 cellular pool is enriched in the nucleus following oxygen or glucose deprivation. Prevention of nuclear localization by a directed mutation in a putative nuclear localization signal in Acss2 abrogates HIF-2 acetylation and blunts HIF-2 dependent signaling as well as flank tumor growth for knockdown/rescue cancer cells expressing ectopic Acss2. In this study, we report generation of a novel mouse strain using CRISPR/Cas9 mutagenesis that express this mutant Acss2 allele in the mouse germline. The homozygous mutant mice have impaired induction of the canonical HIF-2 target gene erythropoietin and blunted recovery from acute anemia. Surprisingly, Acss2 protein levels are dramatically reduced in these mutant mice. Functional studies investigating the basis for this phenotype reveal multiple protein instability domains in the Acss2 carboxy terminus. The findings described herein may be of relevance in the regulation of native Acss2 protein as well as for humans carrying missense mutations in these domains.

Rb1/Rbl1/Vhl loss induces mouse subretinal angiomatous proliferation and hemangioblastoma.

Von Hippel-Lindau (Vhl) protein inhibits hypoxia-inducible factor (Hif), yet its deletion in murine retina does not cause the extensive angiogenesis expected with Hif induction. The mechanism is unclear. Here we show that retinoblastoma tumor suppressor (Rb1) constrains expression of Hif target genes in the Vhl-/- retina. Deleting Rb1 induced extensive retinal neovascularization and autophagic ablation of photoreceptors in the Vhl-/- retina. RNA-sequencing, ChIP, and reporter assays showed Rb1 recruitment to and repression of certain Hif target genes. Activating Rb1 by deleting cyclin D1 induced a partial defect in the retinal superficial vascular plexus. Unexpectedly, removing Vhl suppressed retinoblastoma formation in murine Rb1/Rbl1-deficient retina but generated subretinal vascular growths resembling retinal angiomatous proliferation (RAP) and retinal capillary hemangioblastoma (RCH). Most stromal cells in the RAP/RCH-like lesions were Sox9+, suggesting a Müller glia origin, and expressed Lgals3, a marker of human brain hemangioblastoma. Thus, the Rb family limit Hif target gene expression in the Vhl-/- retina, and removing this inhibitory signal generates new models for RAP and RCH.

Physiological and genomic evidence that selection on the transcription factor Epas1 has altered cardiovascular function in high-altitude deer mice.

Evolutionary adaptation to extreme environments often requires coordinated changes in multiple intersecting physiological pathways, but how such multi-trait adaptation occurs remains unresolved. Transcription factors, which regulate the expression of many genes and can simultaneously alter multiple phenotypes, may be common targets of selection if the benefits of induced changes outweigh the costs of negative pleiotropic effects. We combined complimentary population genetic analyses and physiological experiments in North American deer mice (Peromyscus maniculatus) to examine links between genetic variation in transcription factors that coordinate physiological responses to hypoxia (hypoxia-inducible factors, HIFs) and multiple physiological traits that potentially contribute to high-altitude adaptation. First, we sequenced the exomes of 100 mice sampled from different elevations and discovered that several SNPs in the gene Epas1, which encodes the oxygen sensitive subunit of HIF-2α, exhibited extreme allele frequency differences between highland and lowland populations. Broader geographic sampling confirmed that Epas1 genotype varied predictably with altitude throughout the western US. We then discovered that Epas1 genotype influences heart rate in hypoxia, and the transcriptomic responses to hypoxia (including HIF targets and genes involved in catecholamine signaling) in the heart and adrenal gland. Finally, we used a demographically-informed selection scan to show that Epas1 variants have experienced a history of spatially varying selection, suggesting that differences in cardiovascular function and gene regulation contribute to high-altitude adaptation. Our results suggest a mechanism by which Epas1 may aid long-term survival of high-altitude deer mice and provide general insights into the role that highly pleiotropic transcription factors may play in the process of environmental adaptation.

The Nuclear Export Inhibitor Selinexor Inhibits Hypoxia Signaling Pathways And 3D Spheroid Growth Of Cancer Cells.

PurposeThe nucleocytoplasmic transport of macromolecules is critical for both cell physiology and pathophysiology. Exportin 1 (XPO1), the major nuclear export receptor, is involved in the cellular adaptation to reduced oxygen availability by controlling the nuclear activity of the hypoxia-inducible factors (HIFs). Recently, a specific inhibitor of XPO1, selinexor (KPT-330), has been identified that inhibits nuclear export of cargo proteins by binding to the XPO1 cargo-binding pocket.Patients and methodsWe used different cancer cell lines from human tissues and evaluated the physiological activity of selinexor on the hypoxia response pathway in two-dimensional (2D) monolayer cell cultures in quantitative real-time (qRT)-PCR experiments and luciferase reporter gene assays. A three-dimensional (3D) tumor spheroid culture model of MCF-7 breast cancer cells was established to analyze the effect of selinexor on 3D tumor spheroid structure, formation and viability.ResultsSelinexor treatment reduces HIF-transcriptional activity and expression of the HIF-1 target gene solute carrier family 2 member 1 (SLC2A1). Moreover, 3D tumor spheroid structure, formation and viability are inhibited in response to selinexor-induced nuclear export inhibition.ConclusionHere, we demonstrate the effect of specific XPO1-inhibition on the hypoxic response on the molecular level in 2D and 3D culture models of MCF-7 cells.

In chronic hypoxia, glucose availability and hypoxic severity dictate the balance between HIF-1 and HIF-2 in astrocytes.

Astrocyte function is an important contributor to cellular viability during brain hypoxia and ischemia. Levels of the hypoxia-inducible transcription factors (HIFs) HIF-1 and HIF-2 are increased in hypoxic conditions and impact the neuroprotective properties of astrocytes. For example, HIF-2 induces levels of erythropoietin (EPO), a neuroprotectant, by astrocytes. In contrast, HIF-1 activity in astrocytes diminishes the viability of neurons in cocultures during hypoxia. Thus, HIF-1 and HIF-2 may have opposing effects on astrocytes. In this study, we explore the balance of HIF-1 and HIF-2 signaling in astrocytes during chronic (1-7 d) hypoxia while altering the degree of hypoxia and glucose availability. In addition, we investigate the effects of these conditions on neuron apoptosis. During exposure to chronic moderate hypoxia (2% O2) and plentiful glucose (10 mM), HIF-2 and EPO abundance increases from d 1 to 7. Similarly, pretreatment with moderate hypoxia markedly increases the abundance of HIF-2 and EPO when astrocytes are subsequently exposed to severe hypoxia (0.5% O2; 24 h) in 10 mM glucose, which inhibits neuron apoptosis in coculture. Although HIF-1 targets the expression increase during the 7 d in chronic moderate hypoxia (2% O2) and limited glucose (2 mM), further exposure to severe hypoxia (0.5% O2; 24 h) induces a decrease of most HIF-1 targets in astrocytes. Notably, in astrocyte exposure to 2% O2 prior to 0.5% O2, the expression of iNOS, an HIF-1-regulated protein, keeps increasing when glucose is limited, whereas EPO and VEGF abundance is suppressed, inducing increased apoptosis of neurons in coculture under limited glucose (2 mM). Thus, both hypoxic severity and glucose abundance regulate the balance of HIF-1 and HIF-2 activity in astrocytes, leading to diverse effects on neurons. These results could have important implications on the adaptive or pathologic role of astrocytes during chronic hypoxia and ischemia.-Guo, M., Ma, X., Feng, Y., Han, S., Dong, Q., Cui, M., Zhao, Y. In chronic hypoxia, glucose availability and hypoxic severity dictate the balance between HIF-1 and HIF-2 in astrocytes.

Abstract 373: Regulation of Hypoxia by Chromatin Reader Protein Kinase C Binding Protein 1 (PRKCBP1)

PRKCBP1 (also known as RACK7 and Zmynd8) is a polyvalent chromatin reader known to cooperatively bind acetylated and methylated nucleosomes. Recently it has been shown to regulate transcription and cancer progression by coordinating histone methylation modifications affecting enhancer and promoter regions of genes. The role of PRKCBP1 in the cardiac myocardium is unexplored. Hypoxia-inducible factor 1α (HIF-1α) upregulation and stabilization is a common feature of both cancer and myocardial ischemia, promoting cellular functions such as proliferation, glucose metabolism and angiogenesis. Here we investigated the mechanism by which PRKCBP1 modulates the cardiac hypoxic response. We hypothesize that PRKCBP1 inhibits the HIF-1 response in the hypoxic heart by reducing enhancer activity of HIF-1 target genes and altering availability of HIF-1 binding sites. We have found that in transgenic mice with a mutation that increases HIF-induced expression of PRKCBP, the effect of induced oxygen-stable HIF is markedly diminished. These mice did not exhibit the typical HIF-1 over-expression phenotype of dilated vessels, increased heart size and reduced ventricular function. Semi-quantitative rtPCR analysis of mouse neonatal cardiomyocytes transfected with CMV-driven expression plasmids for PRKCBP1 and oxygen-stable HIF-1α showed striking reduction of multiple HIF-1 target genes such as PDK1 (45% reduction relative to Ehbp1) compared to the HIF-1α plasmid alone. RNAi mediated knockdown of PRKCBP1 removed this negative regulation (65% increase). Analysis of human PRKCBP1 and HIF-1α ChIP-seq data indicate that PRKCBP1 binds to the enhancer of 78% of HIF-1 regulated genes. ATAC-seq data suggest that PRKCBP1 affects genome-wide chromatin accessibility, with loci-specific modifications at numerous HIF-1 target genes, such as EGLN3. These data suggest that PRKCBP1 may be acting both by modulating enhancer activity in cis - to HIF-1 target genes and by preventing HIF-1 binding to hypoxia response elements of target genes. We have discovered a new regulator of HIF-1 action that modifies the hypoxic response, likely through chromatin remodeling. This new form of regulation may modify the pathophysiology of ischemia and provide new targets for therapy.

PIN1 transcript variant 2 acts as a long non-coding RNA that controls the HIF-1-driven hypoxic response

The transcription factor HIF-1 induces the expression of genes that are essential for cell survival and oxygen homeostasis in hypoxic conditions. The prolyl isomerase Pin1 plays a role in the regulation of HIF-1α. However, the mechanism by which Pin1 controls HIF-1α remains controversial. Surprisingly, we here show that a PIN1 transcript downregulates HIF-1α as a long non-coding RNA. Pin1-silencing siRNAs augmented the hypoxia-induced expression of HIF-1α, thereby upregulating the expression of HIF-1 target genes. However, the overexpression of Pin1 protein did not inhibit the hypoxic expression of HIF-1α. Pin1 restoration in Pin1-depleted cells also failed to reverse the induction of HIF-1α by Pin1 knockdown. Unexpectedly, HIF-1α was found to be induced by both siRNAs for PIN1 transcript variants 1/2 and that for PIN1 transcript variants 2/3, indicating that the PIN1 transcript variant 2 (PIN1-v2) is responsible for HIF-1α induction. Mechanistically, PIN1-v2, which is classified as a long non-coding RNA due to early termination of translation, was evaluated to inhibit the transcription of HIF1A gene. In conclusion, PIN1-v2 may function in balancing the HIF-1-driven gene expression under hypoxia.