Hypoxia-inducible Transcription Factor Research Articles

The special adaptation to hypoxia facilitated the expansion of the Asian house rat (Rattus tanezumi) into Tibet but not other Rattus species.

Rattus species are thought to live only at altitudes less than 2500 m, but the Asian house rat (R. tanezumi) (RT) has recently expanded to altitudes greater than 3500 m in China. Other Rattus species, especially brown rats (R. norvegicus) (RN), still reach only low altitudes on the Tibetan Plateau. Comparative genomics revealed the positive selection of hypoxia-inducible transcription factors 1 and 2 (HIFs) in RT, with the rapid evolution of HIF pathway genes in RT and Mus musculus (MM) but not RN or R. rattus. Population genomics revealed that genes associated with energy metabolism and oxygen transport were positively selected in RT compared with the other four Rattus species, and two specific substitutions (arginine 31 serine and leucine 33 methionine) were identified in the hemoglobin subunit beta (HBB) in RT. The above results suggested that RT possesses unique genetic adaptations to hypoxia, which was further confirmed by behavioral experiments on RT and RN. Normobaric hypoxia significantly reduced locomotion in RN but not in RT. Moreover, through intraspecific transcriptome analysis, the expression of Hbb and genes related to angiogenesis, oxygen transport, and glycolysis was upregulated, and the expression of genes associated with immunological functions in the liver, lungs, and/or sperm was downregulated in RT compared to those in RN. Interspecific transcriptome analysis further revealed that HIF-1α plays a role in modulating the hypoxic adaptation of RT rather than RN. Our work provides genomic, behavioral, and physiological insights into why RT, but not other Rattus species, could invade the Tibetan Plateau.

HIF3A gene disruption causes abnormal alveoli structure and early neonatal death.

Transcriptional response to changes in oxygen concentration is mainly controlled by hypoxia-inducible transcription factors (HIFs). Besides regulation of hypoxia-responsible gene expression, HIF-3α has recently been shown to be involved in lung development and in the metabolic process of fat tissue. However, the precise mechanism for such properties of HIF-3α is still largely unknown. To this end, we generated HIF3A gene-disrupted mice by means of genome editing technology to explore the pleiotropic role of HIF-3α in development and physiology. We obtained adult mice carrying homozygous HIF3A gene mutations with comparable body weight and height to wild-type mice. However, the number of litters and ratio of homozygous mutation carriers born from the mating between homozygous mutant mice was lower than expected due to sporadic deaths on postnatal day 1. HIF3A gene-disrupted mice exhibited abnormal configuration of the lung such as a reduced number of alveoli and thickened alveolar walls. Transcriptome analysis showed, as well as genes associated with lung development, an upregulation of stearoyl-Coenzyme A desaturase 1, a pivotal enzyme for fatty acid metabolism. Analysis of fatty acid composition in the lung employing gas chromatography indicated an elevation in palmitoleic acid and a reduction in oleic acid, suggesting an imbalance in distribution of fatty acid, a constituent of lung surfactant. Accordingly, administration of glucocorticoid injections during pregnancy resulted in a restoration of normal alveolar counts and a decrease in neonatal mortality. In conclusion, these observations provide novel insights into a pivotal role of HIF-3α in the preservation of critically important structure and function of alveoli beyond the regulation of hypoxia-mediated gene expression.

Hypoxia-Inducible Factor-Dependent and Independent Mechanisms Underlying Chemoresistance of Hypoxic Cancer Cells.

In hypoxic regions of malignant solid tumors, cancer cells acquire resistance to conventional therapies, such as chemotherapy and radiotherapy, causing poor prognosis in patients with cancer. It is widely recognized that some of the key genes behind this are hypoxia-inducible transcription factors, e.g., hypoxia-inducible factor 1 (HIF-1). Since HIF-1 activity is suppressed by two representative 2-oxoglutarate-dependent dioxygenases (2-OGDDs), PHDs (prolyl-4-hydroxylases), and FIH-1 (factor inhibiting hypoxia-inducible factor 1), the inactivation of 2-OGDD has been associated with cancer therapy resistance by the activation of HIF-1. Recent studies have also revealed the importance of hypoxia-responsive mechanisms independent of HIF-1 and its isoforms (collectively, HIFs). In this article, we collate the accumulated knowledge of HIF-1-dependent and independent mechanisms responsible for resistance of hypoxic cancer cells to anticancer drugs and briefly discuss the interplay between hypoxia responses, like EMT and UPR, and chemoresistance. In addition, we introduce a novel HIF-independent mechanism, which is epigenetically mediated by an acetylated histone reader protein, ATAD2, which we recently clarified.

The drug-specific properties of hypoxia-inducible factor-prolyl hydroxylase inhibitors in mice reveal a significant contribution of the kidney compared to the liver to erythropoietin induction

AimsKidney disease often leads to anemia due to a defect in the renal production of the erythroid growth factor erythropoietin (EPO), which is produced under the positive regulation of hypoxia-inducible transcription factors (HIFs). Chemical compounds that inhibit HIF-prolyl hydroxylases (HIF-PHs), which suppress HIFs, have been developed to reactivate renal EPO production in renal anemia patients. Currently, multiple HIF-PH inhibitors, in addition to conventional recombinant EPO reagents, are used for renal anemia treatment. This study aimed to elucidate the therapeutic mechanisms and drug-specific properties of HIF-PH inhibitors. Methods and key findingsGene expression analyses and mass spectrometry revealed that HIF-PH inhibitors (daprodustat, enarodustat, molidustat, and vadadustat) alter Epo gene expression levels in the kidney and liver in a drug-specific manner, with different pharmacokinetics in the plasma and urine after oral administration to mice. The drug specificity revealed the dominant contribution of EPO induction in the kidneys rather than in the liver to plasma EPO levels after HIF-PH inhibitor administration. We also found that several HIF-PH inhibitors directly induce duodenal gene expression related to iron intake, while these drugs indirectly suppress hepatic hepcidin expression to mobilize stored iron for hemoglobin synthesis through induction of the EPO-erythroferrone axis. SignificanceRenal EPO induction is the major target of HIF-PH inhibitors for their therapeutic effects on erythropoiesis. Additionally, the drug-specific properties of HIF-PH inhibitors in EPO induction and iron metabolism have been shown in mice, providing useful information for selecting the proper HIF-PH inhibitor for each renal anemia patient.

Isthmin-1 promotes growth and progression of colorectal cancer through the interaction with EGFR and YBX-1

While previous studies have indicated the involvement of Isthmin 1 (ISM1), a secreted protein, in cancer development, the precise mechanisms have remained elusive. In this study, we unveiled that ISM1 is significantly overexpressed in both the blood and tissue samples of colorectal cancer (CRC) patients, correlating with their poor prognosis. Functional experiments demonstrated that enforced ISM1 expression significantly enhances CRC proliferation, migration, invasion and tumor growth. Notably, our investigation reveals an interaction of ISM1 with epidermal growth factor receptor (EGFR), a member of the receptor tyrosine kinase (RTK) family of CRC cells. The binding of ISM1 triggered EGFR activation and initiate downstream signaling pathways. Meanwhile, intracellular ISM1 interacted with Y-box binding protein 1 (YBX1), enhancing its transcriptional regulation on EGFR. Furthermore, our research uncovered the regulation of ISM1 expression by the hypoxia-inducible transcription factor HIF-1α in CRC cells. Mechanistically, we identified HIF-1α as a direct regulator of ISM1, binding to a hypoxia response element on its promoter. This novel mechanism illuminated potential therapeutic targets, offering insights into restraining HIF-1α/ISM1/EGFR-driven CRC progression and metastasis.

Abstract 373: Transcriptional and post-transcriptional positive regulatory loops between HIF-1a and the RNA-Binding protein hnRNPA18

Abstract Hypoxia, the scarcity of oxygen, is a hallmark of solid tumors to which they adapt by activating the hypoxia-inducible transcription factor 1 (HIF-1). HIF activates the transcription of genes bearing hypoxia responsive elements (RE) in their promoter regions including genes related to angiogenesis, glucose metabolism, cell proliferation, survival, invasion, and metastasis. The HIF-1 pathway is thus an attractive target to prevent cancer aggressiveness and improve the effectiveness of cancer therapy. HIF-1α activity is known to be mainly regulated through post-translational modification by prolyl hydroxylase domain (PHD) enzymes, but accumulating evidence indicate that it is also regulated by other mechanisms such as transcriptional initiation, translational initiation, protein-protein interaction, post-translational modifications and post-transcriptionally, primarily through the action of trans-acting factors (noncoding RNAs and RNA-binding proteins) that interact with the HIF-1α mRNA to regulate its decay and translational rates. We recently identified the RNA binding protein hnRNP A18 as a regulator of HIF-1α translation under hypoxic conditions. hnRNP A18 is a nuclear stress responsive protein that translocates to the cytosol in response to cellular stress including hypoxia to bind to a recognition motif in the 3’UTR of its targeted transcripts including HIF-1α to stabilize the transcripts and increase their translation. Using a bicistronic reporter plasmid containing a HIF-1α internal ribosome entry site (IRES) we now show that hnRNP A18 can also regulate HIF-1α through its IRES in the 5’UTR. In fact, cells expressing hnRNP A18 significantly increase translation of a reporter CAT protein under the control of HIF-1α IRES in the presence of the hypoxia mimetic agent CoCl2. On the other hand, deletion of hnRNP A18 prevents the translation of the CAT reporter protein even in the presence of CoCl2. Moreover, we also identified several HIF-1α REs in hnRNP A18 promoter. Two of the predicted HIF-1α REs, located at -57 and -226 upstream of the hnRNP A18 start codon were validated as bonafide HIF-1α binding sites by Electromobility Shift Assay with recombinant HIF-1α protein and by Chromatin Immunoprecipitation assay in human pancreatic cancer cells Panc 01. These data thus indicate that a positive regulatory loop between hnRNP A18 and HIF1-1α exist to amplify HIF-1α expression under cellular stress. hnRNP A18 can upregulate HIF1α protein expression by stabilizing its transcript at the 3’UTR and by binding to its IRES. On the other hand, HIF-1α can upregulate hnRNP A18 by binding to its proximal promoter. Targeting hnRNP A18 could thus provide a new mechanism to regulate HIF-1α expression and sensitize cancer cells to therapies. Citation Format: Eduardo Solano-Gonzalez, France Carrier. Transcriptional and post-transcriptional positive regulatory loops between HIF-1a and the RNA-Binding protein hnRNPA18 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 373.

Transcriptome analyses describe the consequences of persistent HIF-1 over-activation in Caenorhabditis elegans.

Metazoan animals rely on oxygen for survival, but during normal development and homeostasis, animals are often challenged by hypoxia (low oxygen). In metazoans, many of the critical hypoxia responses are mediated by the evolutionarily conserved hypoxia-inducible transcription factors (HIFs). The stability and activity of HIF complexes are strictly regulated. In the model organism C. elegans, HIF-1 stability and activity are negatively regulated by VHL-1, EGL-9, RHY-1 and SWAN-1. Importantly, C. elegans mutants carrying strong loss-of-function mutations in these genes are viable, and this provides opportunities to interrogate the molecular consequences of persistent HIF-1 over-activation. We find that the genome-wide gene expression patterns are compellingly similar in these mutants, supporting models in which RHY-1, VHL-1 and EGL-9 function in common pathway(s) to regulate HIF-1 activity. These studies illuminate the diversified biological roles played by HIF-1, including metabolism and stress response. Genes regulated by persistent HIF-1 over-activation overlap with genes responsive to pathogens, and they overlap with genes regulated by DAF-16. As crucial stress regulators, HIF-1 and DAF-16 converge on key stress-responsive genes and function synergistically to enable hypoxia survival.

Abstract 3024: HIF1A and HIF2A differentially contribute to early and adaptive changes following in vivo Vhl inactivation in the kidney

Abstract The von Hippel Lindau (VHL) tumor suppressor gene is a classical tumor suppressor whose biallelic inactivation is a truncal event in clear cell renal cell carcinoma (ccRCC) evolution. VHL is best characterized as the recognition component of an E3 ubiquitin ligase complex that targets hypoxia inducible transcription factors (HIF1A and HIF2A) for conditional oxygen-dependent degradation. HIF activation following VHL inactivation is an early event in ccRCC progression. While the functions of HIF1A and HIF2A in ccRCC biology and progression have been studied, those in VHL-null non-neoplastic cells of the renal tubular epithelium (RTE) and their contribution to tumorigenesis have not been fully explored. Here, using a combination of lineage marking and single-cell RNA sequencing (scRNA-seq), we provide the first description of HIF1A- and/or HIF2A-dependent and -independent consequences of in vivo Vhl inactivation in the RTE with single-cell resolution. We have previously reported on the development of a novel mouse model designed to mark and track cells that have undergone Vhl inactivation in vivo. Using this model, we have shown that Vhl inactivation has extensive, cell-type specific effects on gene expression in cells of the RTE within 3 weeks of recombination. Furthermore, we have shown that over a course of 4-8 months after Vhl inactivation, Vhl-null cells undergo specific adaptive changes in gene expression that encompass genes not regulated early after Vhl inactivation. In the work reported here, we have coupled this mouse model to a tamoxifen-inducible RTE-specific Pax8-CreERT2 and conditional alleles for Hif1a and/or Epas1 (codes for HIF2A). We have analyzed ‘marked’ recombined cells at early (1-3 weeks) or late (4-8 months) timepoints after recombination and compared these cells to time-matched Vhl-null cells using immunohistochemistry, in situ RNA hybridization and scRNA-seq. We found that though both HIF isoforms were present in Vhl-null cells at the early timepoint, expression of genes regulated early after Vhl inactivation was affected more by Hif1a inactivation than Epas1 inactivation. In contrast, expression of genes that are regulated in Vhl-null cells over time was drastically impacted by Epas1 deletion but was unaffected by Hif1a deletion. Interestingly, most of these Epas1-dependent, time-dependent changes in Vhl-null cells involved genes that have not been identified as direct transcriptional targets of HIF. Collectively, our work suggests that HIF1A and HIF2A have temporally distinct contributions to gene expression in Vhl-null cells in the RTE. We are currently analyzing the cell-type specificity of these contributions and ascertaining their relevance to the adoption of ccRCC-associated gene expression profiles. Citation Format: Samvid Kurlekar, Joanna Lima, Ayslan B. Barros, Norma Masson, Sophia Zhai, Christopher W. Pugh, Julie Adam, Peter J. Ratcliffe. HIF1A and HIF2A differentially contribute to early and adaptive changes following in vivo Vhl inactivation in the kidney [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3024.

Abstract 370: BACH1 metastatic activity is regulated by oxygen-dependent proline hydroxylation in triple-negative breast cancer

Abstract Hypoxia, low oxygen availability at the tissue level, is a key driver of metastasis in solid tumors. Oxygen-sensitive activation of hypoxia-induced transcription factors is in part regulated by prolyl hydroxylase domain proteins (PHDs). We and others previously showed that the BTB and CNC homolog 1 (BACH1) transcription factor promotes metastasis in various cancer types. In accompanying work, we show that BACH1 is prolyl-hydroxylated and degraded in an oxygen-dependent manner in triple-negative breast cancer (TNBC). Here we show that BACH1 lacking its hydroxylated proline sites (i.e., mutant BACH1) expressed in TNBC cells exhibits increased DNA binding to a canonical BACH1 binding site as well as greater transcriptional output of metastasis and hypoxia-response-related genes relative to wild-type BACH1. Furthermore, in vivo assays showed that mutant BACH1 promotes greater tumor metastasis of TNBC cells than wild-type BACH1. Our findings suggest that the proline hydroxylation of BACH1 directly regulates BACH1’s transcriptional activity by modulating its DNA-binding activity. Together, these results reveal a mechanism underlying oxygen-sensitive regulation of BACH1 that could potentially contribute to BACH1-mediated pro-metastatic signaling and therapeutic resistance under hypoxia in TNBC. Citation Format: Thomas Li, Long Chi Nguyen, Christopher Dann, Madeline Henn, Dongbo Yang, Emily Shi, Lydia Robinson-Mailman, Kazuhiko Igarashi, Marsha R. Rosner. BACH1 metastatic activity is regulated by oxygen-dependent proline hydroxylation in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 370.

Identification and Development of Cyclic Peptide Inhibitors of Hypoxia Inducible Factors 1 and 2 That Disrupt Hypoxia-Response Signaling in Cancer Cells.

Hypoxia inducible factor (HIF) is a heterodimeric transcription factor composed of an oxygen-regulated α subunit and a constitutively expressed β subunit that serves as the master regulator of the cellular response to low oxygen concentrations. The HIF transcription factor senses and responds to hypoxia by significantly altering transcription and reprogramming cells to enable adaptation to a hypoxic microenvironment. Given the central role played by HIF in the survival and growth of tumors in hypoxia, inhibition of this transcription factor serves as a potential therapeutic approach for treating a variety of cancers. Here, we report the identification, optimization, and characterization of a series of cyclic peptides that disrupt the function of HIF-1 and HIF-2 transcription factors by inhibiting the interaction of both HIF-1α and HIF-2α with HIF-1β. These compounds are shown to bind to HIF-α and disrupt the protein-protein interaction between the α and β subunits of the transcription factor, resulting in disruption of hypoxia-response signaling by our lead molecule in several cancer cell lines.

Hypoxia-inducible factor induces cysteine dioxygenase and promotes cysteine homeostasis in Caenorhabditis elegans

Dedicated genetic pathways regulate cysteine homeostasis. For example, high levels of cysteine activate cysteine dioxygenase, a key enzyme in cysteine catabolism in most animal and many fungal species. The mechanism by which cysteine dioxygenase is regulated is largely unknown. In an unbiased genetic screen for mutations that activate cysteine dioxygenase (cdo-1) in the nematode Caenorhabditis elegans, we isolated loss-of-function mutations in rhy-1 and egl-9, which encode proteins that negatively regulate the stability or activity of the oxygen-sensing hypoxia inducible transcription factor (hif-1). EGL-9 and HIF-1 are core members of the conserved eukaryotic hypoxia response. However, we demonstrate that the mechanism of HIF-1-mediated induction of cdo-1 is largely independent of EGL-9 prolyl hydroxylase activity and the von Hippel-Lindau E3 ubiquitin ligase, the classical hypoxia signaling pathway components. We demonstrate that C. elegans cdo-1 is transcriptionally activated by high levels of cysteine and hif-1. hif-1-dependent activation of cdo-1 occurs downstream of an H2S-sensing pathway that includes rhy-1, cysl-1, and egl-9. cdo-1 transcription is primarily activated in the hypodermis where it is also sufficient to drive sulfur amino acid metabolism. Thus, the regulation of cdo-1 by hif-1 reveals a negative feedback loop that maintains cysteine homeostasis. High levels of cysteine stimulate the production of an H2S signal. H2S then acts through the rhy-1/cysl-1/egl-9 signaling pathway to increase HIF-1-mediated transcription of cdo-1, promoting degradation of cysteine via CDO-1.

Hypoxia-inducible factor induces cysteine dioxygenase and promotes cysteine homeostasis in Caenorhabditis elegans.

Dedicated genetic pathways regulate cysteine homeostasis. For example, high levels of cysteine activate cysteine dioxygenase, a key enzyme in cysteine catabolism in most animal and many fungal species. The mechanism by which cysteine dioxygenase is regulated is largely unknown. In an unbiased genetic screen for mutations that activate cysteine dioxygenase (cdo-1) in the nematode Caenorhabditis elegans, we isolated loss-of-function mutations in rhy-1 and egl-9, which encode proteins that negatively regulate the stability or activity of the oxygen-sensing hypoxia inducible transcription factor (hif-1). EGL-9 and HIF-1 are core members of the conserved eukaryotic hypoxia response. However, we demonstrate that the mechanism of HIF-1-mediated induction of cdo-1 is largely independent of EGL-9 prolyl hydroxylase activity and the von Hippel-Lindau E3 ubiquitin ligase, the classical hypoxia signaling pathway components. We demonstrate that C. elegans cdo-1 is transcriptionally activated by high levels of cysteine and hif-1. hif-1-dependent activation of cdo-1 occurs downstream of an H2S-sensing pathway that includes rhy-1, cysl-1, and egl-9. cdo-1 transcription is primarily activated in the hypodermis where it is also sufficient to drive sulfur amino acid metabolism. Thus, the regulation of cdo-1 by hif-1 reveals a negative feedback loop that maintains cysteine homeostasis. High levels of cysteine stimulate the production of an H2S signal. H2S then acts through the rhy-1/cysl-1/egl-9 signaling pathway to increase HIF-1-mediated transcription of cdo-1, promoting degradation of cysteine via CDO-1.

The Thirty-Fifth Anniversary of K+ Channels in O2 Sensing: What We Know and What We Don’t Know

On the thirty-fifth anniversary of the first description of O2-sensitive K+ channels in the carotid body chemoreceptors O2 sensing remains a salient issue in the literature. Whereas much has been learned about this subject, important questions such as the identity of the specific K+ channel subtype(s) responsible for O2 sensing by chemoreceptors and the mechanism(s) by which their activities are altered by hypoxia have not yet been definitively answered. O2 sensing is a fundamental biological process necessary for the acute and chronic responses to varying environmental O2 levels which allow organisms to adapt to hypoxia. Whereas chronic responses depend on the modulation of hypoxia-inducible transcription factors which determine the expression of numerous genes encoding enzymes, transporters and growth factors, acute responses rely mainly on the dynamic modulation of ion channels by hypoxia, causing adaptive changes in cell excitability, contractility and secretory activity in specialized tissues. The most widely studied oxygen-sensitive ion channels are potassium channels, but oxygen sensing by members of both the calcium and sodium channel families has also been demonstrated. Given the explosion of information on this topic, in this review, we will focus on the mechanisms of physiological oxygen chemotransduction by PO2-dependent K+ channels, with particular emphasis on their function in carotid body chemoreceptor cells (CBCC) and pulmonary artery smooth muscle cells (PASMC), highlighting areas of consensus and controversy within the field. We will first describe the most well-established concepts, those reproduced in multiple laboratories, and then discuss selected observations or questions that remain unresolved, and that limit our progress in this field.

Dimethyloxalylglycine pretreatment of living donor alleviates both donor and graft liver ischemia-reperfusion injury in rats.

Background: Under the circumstance of the increasing waiting list for liver transplantation, living donor liver transplantation (LDLT) can alleviate the shortage of liver donors to some extent. However, how to reduce both donor and graft ischemia-reperfusion injury (IRI) is still an unsolved problem in LDLT. Hypoxia-induced transcription factor 1 (HIF1) activation is considered an important mechanism of cellular adaptation to hypoxia, and early activation of HIF1 may be a new way to alleviate liver IRI. Therefore, we aimed to investigate the impact of the HIF1 stabilizer dimethyloxalylglycine (DMOG) on IRI and the survival rate of donors and recipients of rat LDLT. Methods: Seventy percent partial liver resection and 30% partial liver transplantation were used to simulate donor and recipient of clinical LDLT. Rats were treated with DMOG (40mg/kg) or with an equivalent amount of saline. The expression of HIF1 and downstream targets was analyzed after 2h of reperfusion. Liver function and histopathology, apoptosis and oxidative stress levels were detected 6h after reperfusion. At the same time, the 7-day survival rate of rats was calculated. Results: DMOG pretreatment significantly reduced IR-induced injury in the donor and recipient, which was manifested by reducing liver function damage and promoting tissue recovery. Meanwhile, compared with the untreated group, the oxidative stress level and the cell apoptosis rate were decreased in the group pretreated with DMOG. In addition, the transcription and expression of HIF1 target genes in the DMOG group were significantly enhanced. Remarkably, DMOG also increased the survival rate of the recipient. Conclusion: This study provides the first evidence that DMOG pretreatment of donors significantly alleviates liver IRI in both donors and recipients and increases the survival rate of recipients in LDLT. Therefore, DMOG may be a promising strategy for improving LDLT in the future.

Whole genome profiling of short-term hypoxia induced genes and identification of HIF-1 binding sites provide insights into HIF-1 function in Caenorhabditis elegans.

Oxygen is essential to all the aerobic organisms. However, during normal development, disease and homeostasis, organisms are often challenged by hypoxia (oxygen deprivation). Hypoxia-inducible transcription factors (HIFs) are master regulators of hypoxia response and are evolutionarily conserved in metazoans. The homolog of HIF in the genetic model organism C. elegans is HIF-1. In this study, we aimed to understand short-term hypoxia response to identify HIF-1 downstream genes and identify HIF-1 direct targets in C. elegans. The central research questions were: (1) which genes are differentially expressed in response to short-term hypoxia? (2) Which of these changes in gene expression are dependent upon HIF-1 function? (3) Are any of these hif-1-dependent genes essential to survival in hypoxia? (4) Which genes are the direct targets of HIF-1? We combine whole genome gene expression analyses and chromatin immunoprecipitation sequencing (ChIP-seq) experiments to address these questions. In agreement with other published studies, we report that HIF-1-dependent hypoxia-responsive genes are involved in metabolism and stress response. Some HIF-1-dependent hypoxia-responsive genes like efk-1 and phy-2 dramatically impact survival in hypoxic conditions. Genes regulated by HIF-1 and hypoxia overlap with genes responsive to hydrogen sulfide, also overlap with genes regulated by DAF-16. The genomic regions that co-immunoprecipitate with HIF-1 are strongly enriched for genes involved in stress response. Further, some of these potential HIF-1 direct targets are differentially expressed under short-term hypoxia or are differentially regulated by mutations that enhance HIF-1 activity.

Detecting Hypoxia-Inducible Factor Levels and Activity.

The hypoxia-inducible transcription factors HIF-1 and HIF-2 regulate the response to hypoxia. Both proteins are dimers of an alpha subunit and a shared beta subunit. Under hypoxic conditions, the alpha subunits are stabilized, and the transactivation ability of the HIF-1 transcription factor is induced. Accordingly, assessment of HIF-1α protein levels and HIF transcriptional activity serve as an indirect indicator of hypoxia. In this series of protocols, I describe three methods to probe the HIF pathway.

STAT3 palmitoylation initiates a positive feedback loop that promotes the malignancy of hepatocellular carcinoma cells in mice.

Constitutive activation of the transcription factor STAT3 (signal transducer and activator of transcription 3) contributes to the malignancy of many cancers such as hepatocellular carcinoma (HCC) and is associated with poor prognosis. STAT3 activity is increased by the reversible palmitoylation of Cys108 by the palmitoyltransferase DHHC7 (encoded by ZDHHC7). Here, we investigated the consequences of S-palmitoylation of STAT3 in HCC. Increased ZDHHC7 abundance in HCC cases was associated with poor prognosis, as revealed by bioinformatics analysis of patient data. In HepG2 cells in vitro, DHHC7-mediated palmitoylation enhanced the expression of STAT3 target genes, including HIF1A, which encodes the hypoxia-inducible transcription factor HIF1α. Inhibiting DHHC7 decreased the S-palmitoylation of STAT3 and decreased HIF1α abundance. Furthermore, stabilization of HIF1α by cyclin-dependent kinase 5 (CDK5) enabled it to promote the expression of ZDHHC7, which generated a positive feedback loop between DHHC7, STAT3, and HIF1α. Perturbing this loop reduced the growth of HCC cells in vivo. Moreover, DHHC7, STAT3, and HIF1α were all abundant in human HCC tissues. Our study identifies a pathway connecting these proteins that is initiated by S-palmitoylation, which may be broadly applicable to understanding the role of this modification in cancer.

CSIG-26. TARGETING HYPOXIC CANCER CELLS IN MEDULLOBLASTOMA

Abstract The goal of this study is to thoroughly assess the role of hypoxia-induced transcription factors (HIFs) in MB growth and examine HIF as a potential therapeutic target. We first examined patient MB tumor by IHC and identified scattered areas with expression of HIF1a and HIF2a. We then established orthotopic human MB xenografts in mice and found hypoxic areas throughout the tumors using pimonidazole detection and they expressed both HIF1a, and HIF2a. There are only few observations of hypoxic areas within experimental MB. To explore the impact of HIFs on MB growth, we exposed MB cell lines (ONS-76; Shh and D556; Group 3) to 1% hypoxia in cell culture. We observed a decrease in cell growth, an increase in HIF1a and HIF2a protein expression, activation of HIF targets (Glut-1, VEGF), and a hypoxia-response element-driven luciferase reporter assay. To further examine the role of individual HIF isoforms, we employed CRISPR/Cas9 to knockout (KO) the HIF1a and/or HIF2a genes in ONS76 MB cells. The HIF reporter activity was partially canceled with single HIF-KO and completely abrogated with double KO and double KO reduced cell growth, indicating that both HIF-1 and HIF-2 are having roles for activating transcription and cell growth. Treatment of MB cells with 64B, an arylsulfonamide HIF/p300 inhibitor abrogated reporter activity by both HIF-1 and HIF-2 and reduced expression of hypoxia-induced genes. To investigate the potential of targeting HIF in vivo, we injected mice brains with wild-type and HIF1a/2a-KO ONS-76 cells and monitored their growth and survival. Mice injected with double KO HIF cells exhibited prolonged survival compared to those with parental cells (n = 5, p< 0.01). Further experiments to expand this approach with other MB cell line and test the anti-tumor effects of 64B in mice are ongoing. These findings support targeting HIFs as a novel therapeutic strategy in MB treatment.

Controlling Trophoblast Cell Fusion in the Human Placenta-Transcriptional Regulation of Suppressyn, an Endogenous Inhibitor of Syncytin-1.

Cell fusion in the placenta is tightly regulated. Suppressyn is a human placental endogenous retroviral protein that inhibits the profusogenic activities of another well-described endogenous retroviral protein, syncytin-1. In this study, we aimed to elucidate the mechanisms underlying suppressyn's placenta-specific expression. We identified the promoter region and a novel enhancer region for the gene encoding suppressyn, ERVH48-1, and examined their regulation via DNA methylation and their responses to changes in the oxygen concentration. Like other endogenous retroviral genes, the ERVH48-1 promoter sequence is found within a characteristic retroviral 5' LTR sequence. The novel enhancer sequence we describe here is downstream of this LTR sequence (designated EIEs: ERV internal enhancer sequence) and governs placental expression. The placenta-specific expression of ERVH48-1 is tightly controlled by DNA methylation and further regulated by oxygen concentration-dependent, hypoxia-induced transcription factors (HIF1α and HIF2α). Our findings highlight the involvement of (1) tissue specificity through DNA methylation, (2) expression specificity through placenta-specific enhancer regions, and (3) the regulation of suppressyn expression in differing oxygen conditions by HIF1α and HIF2α. We suggest that these regulatory mechanisms are central to normal and abnormal placental development, including the development of disorders of pregnancy involving altered oxygenation, such as preeclampsia, pregnancy-induced hypertension, and fetal growth restriction.

HIF-2α Controls Expression and Intracellular Trafficking of the α2-Subunit of Na,K-ATPase in Hypoxic H9c2 Cardiomyocytes.

The Na,K-ATPase (NKA) pump plays essential roles for optimal function of the heart. NKA activity decreases in necropsy materials from ischemic heart disease, heart failure and in experimental models. Cellular adaptation to hypoxia is regulated by hypoxia-induced transcription factors (HIF); we tested whether HIFs are involved in regulating the expression and intracellular dynamics of the α2-isoform of NKA (α2-NKA). HIF-1α and HIF-2α expression was suppressed in H9c2 cardiomyocytes by adenoviral infection, where cells were kept in 1% O2 for 24 h. The silencing efficiency of HIFs was tested on the mRNA and protein expression. We measured the mRNA expression of α2-NKA in HIF-silenced and hypoxia-exposed cells. The membrane and intracellular expression of α2-NKA was measured after labelling the cell surface with NHS-SS-biotin, immunoprecipitation and Western blotting. Hypoxia increased the mRNA expression of α2-NKA 5-fold compared to normoxic cells in an HIF-2α-sensitive manner. The plasma membrane expression of α2-NKA increased in hypoxia by 2-fold and was fully prevented by HIF-2α silencing. Intracellular expression of α2-NKA was not affected. These results showed for the first time that in hypoxic cardiomyocytes α2-NKA is transcriptionally and translationally regulated by HIF-2α. The molecular mechanism behind this regulation needs further investigation.