Hypoxia-inducible Factor-1 Transcriptional Activity Research Articles

UCHL1-dependent control of hypoxia-inducible factor transcriptional activity during liver fibrosis.

Liver fibrosis is the excessive accumulation of extracellular matrix proteins that occurs in most types of chronic liver disease. At the cellular level, liver fibrosis is associated with the activation of hepatic stellate cells (HSCs) which transdifferentiate into a myofibroblast-like phenotype that is contractile, proliferative and profibrogenic. HSC transdifferentiation induces genome-wide changes in gene expression that enable the cell to adopt its profibrogenic functions. We have previously identified that the deubiquitinase ubiquitin C-terminal hydrolase 1 (UCHL1) is highly induced following HSC activation; however, the cellular targets of its deubiquitinating activity are poorly defined. Here, we describe a role for UCHL1 in regulating the levels and activity of hypoxia-inducible factor 1 (HIF1), an oxygen-sensitive transcription factor, during HSC activation and liver fibrosis. HIF1 is elevated during HSC activation and promotes the expression of profibrotic mediator HIF target genes. Increased HIF1α expression correlated with induction of UCHL1 mRNA and protein with HSC activation. Genetic deletion or chemical inhibition of UCHL1 impaired HIF activity through reduction of HIF1α levels. Furthermore, our mechanistic studies have shown that UCHL1 elevates HIF activity through specific cleavage of degradative ubiquitin chains, elevates levels of pro-fibrotic gene expression and increases proliferation rates. As we also show that UCHL1 inhibition blunts fibrogenesis in a pre-clinical 3D human liver slice model of fibrosis, these results demonstrate how small molecule inhibitors of DUBs can exert therapeutic effects through modulation of HIF transcription factors in liver disease. Furthermore, inhibition of HIF activity using UCHL1 inhibitors may represent a therapeutic opportunity with other HIF-related pathologies.

The factor inhibiting HIF regulates T cell differentiation and anti-tumour efficacy.

T cells must adapt to variations in tissue microenvironments; these adaptations include the degree of oxygen availability. The hypoxia-inducible factor (HIF) transcription factors control much of this adaptation, and thus regulate many aspects of T cell activation and function. The HIFs are in turn regulated by oxygen-dependent hydroxylases: both the prolyl hydroxylases (PHDs) which interact with the VHL tumour suppressor and control HIF turnover, and the asparaginyl hydroxylase known as the Factor inhibiting HIF (FIH), which modulates HIF transcriptional activity. To determine the role of this latter factor in T cell function, we generated T cell-specific FIH knockout mice. We found that FIH regulates T cell fate and function in a HIF-dependent manner and show that the effects of FIH activity occur predominantly at physiological oxygen concentrations. T cell-specific loss of FIH boosts T cell cytotoxicity, augments T cell expansion in vivo, and improves anti-tumour immunotherapy in mice. Specifically inhibiting FIH in T cells may therefore represent a promising strategy for cancer immunotherapy.

Molecular characterization and expression of Megalobrama amblycephala (Wuchang bream) lysine monomethylase set7 and its potential role in hypoxia adaptation

Previously, we reported that the mammalian lysine monomethylase SET7 impairs the transcriptional activity of hypoxia-inducible factor (HIF), and set7-deficient zebrafish exhibit enhanced tolerance to hypoxia stress. Megalobrama amblycephala, an extensively farmed endemic species in China, is highly sensitive to hypoxia stress, which has posed a serious threat to its intensive and large-scale culture. Therefore, it is meaningful to identify key candidate genes related to the hypoxia tolerance trait in Megalobrama amblycephala. In this study, we found that set7 is highly conserved across species. The luciferase reporter assay showed that Megalobrama amblycephala set7 inhibits the transcriptional activity of hif-1αa/b and hif-2αa/b. Furthermore, using the fluorescent glucose analog 2-NBDG, we observed that overexpression of Megalobrama amblycephala set7 inhibited glucose uptake in EPC cells under hypoxic conditions. Our findings may provide clues to understand the function of Megalobrama amblycephala set7 in the hypoxia signaling pathway, and provide a potential target for genetic breeding of hypoxia tolerant species.

Mimicry of microbially-derived butyrate reveals templates for potent intestinal epithelial HIF stabilizers

ABSTRACT Microbiota-derived short-chain fatty acids, including butyrate (BA), have multiple beneficial health effects. In the colon, BA concentrations range from 10 to 20 mM and up to 95% is utilized as energy by the mucosa. BA plays a key role in epithelial-barrier regulation and anti-inflammation, and regulates cell growth and differentiation, at least in part, due to its direct influence on stabilization of the transcription factor hypoxia-inducible factor (HIF). It remains unclear whether BA is the optimal metabolite for such a response. In this study, we explored metabolite mimicry as an attractive strategy for the biological response to HIF. We discovered that 4-mercapto butyrate (MBA) stabilizes HIF more potently and has a longer biological half-life than BA in intestinal epithelial cells (IECs). We validated the MBA-mediated HIF transcriptional activity through the induction of classic HIF gene targets in IECs and enhanced epithelial barrier formation in vitro. In-vivo studies with MBA revealed systemic HIF stabilization in mice, which was more potent than its parent BA metabolite. Mechanistically, we found that MBA enhances oxygen consumption and that the sulfhydryl group is essential for HIF stabilization, but exclusively as a four-carbon SCFA. These findings reveal a combined biochemical mechanism for HIF stabilization and provide a foundation for the discovery of potent metabolite-like scaffolds.

ABCE1 selectively promotes HIF-1α transactivation of angiogenic gene expression

BackgroundCopper (Cu), by inhibiting the factor inhibiting HIF-1 (FIH-1), promotes the transcriptional activity of hypoxia-inducible factor-1 (HIF-1). ObjectiveThe present study was undertaken to understand the molecular mechanism by which Cu inhibits FIH-1. MethodsHuman umbilical vein endothelial cells (HUVECs) were treated with dimethyloxalylglycine (DMOG) resulting in HIF-1α accumulation and the FIH-1 protein complexes were pulled down for candidate protein analysis. The metal binding sites were predicted by both MetalDetector V2.0 and Metal Ion-Binding Site Prediction Server, and then the actual ability to bind to Cu in vitro was tested by both Copper-Immobilized metal affinity chromatography (Cu-IMAC) and Isothermal Titration Calorimetry (ITC). Subsequently, subcellular localization was monitored by immunocytochemistry, GFP-fusion protein expression plasmid and Western blotting in the nuclear extract. The interaction of candidate protein with HIF-1α and FIH-1 was validated by Co-Immunoprecipitation (Co-IP). Finally, the effect of candidate protein on the FIH-1 structure and HIF-1α transcriptional activity was analyzed by the InterEvDock3 web server and real-time quantitative RT-PCR. ResultsATP-binding cassette E1 (ABCE1) was present in the FIH-1 complexes and identified as a leading Cu-binding protein as indicated by a number of possible Cu binding sites. The ability of ABCE1 to bind Cu was demonstrated in vitro. ABCE1 entered the nucleus along with FIH-1 under hypoxic conditions. Protein interaction analysis revealed that ABCE1 prevented FIH-1 to bind iron ions, inhibiting FIH-1 enzymatic activity. ABCE1 silencing suppressed the expression of Cu-dependent HIF-1 target gene BNIP3, not that of Cu-independent IGF-2. ConclusionThe results demonstrate that ABCE1, as a Cu-binding protein, enters the nucleus under hypoxic conditions and inhibits FIH-1degradation of HIF-1α, thus promoting HIF-1 transactivation of angiogenic gene expression.

Increased transferrin protects from thrombosis in Chuvash erythrocytosis.

Von Hippel-Lindau protein (VHL) is essential to hypoxic regulation of cellular processes. VHL promotes proteolytic clearance of hypoxia-inducible transcription factors (HIFs) that have been modified by oxygen-dependent HIF-prolyl hydroxylases. A homozygous loss-of-function VHLR200W mutation causes Chuvash erythrocytosis, a congenital disorder caused by augmented hypoxia-sensing. Homozygous VHLR200W results in accumulation of HIFs that increase transcription of the erythropoietin gene and raise hematocrit. Phlebotomies reduce hematocrit and hyperviscosity symptoms. However, the major cause of morbidity and mortality in Chuvash erythrocytosis is thrombosis. Phlebotomies cause iron deficiency, which may further elevate HIF activity and transferrin, the HIF-regulated plasma iron transporter recently implicated in thrombogenesis. We hypothesized that transferrin is elevated in Chuvash erythrocytosis, and that iron deficiency contributes to its elevation and to thrombosis. We studied 155 patients and 154 matched controls at steady state and followed them for development of thrombosis. Baseline transferrin was elevated, and ferritin reduced in patients. VHLR200W homozygosity and lower ferritin correlated with higher erythropoietin and transferrin. During 11 years of follow-up, risk of thrombosis increased 8.9-fold in patients versus controls. Erythropoietin elevation, but not hematocrit or ferritin, correlated with thrombosis risk. Unexpectedly, transferrin elevation associated with reduced rather than increased thrombosis risk. The A allele of the promoter EPO single nucleotide polymorphisms (SNP), rs1617640, associated with elevated erythropoietin and increased thrombosis risk, whereas the A allele of the intronic TF SNP, rs3811647, associated with higher transferrin and protection from thrombosis in patients. Our findings suggest an unexpected causal relationship between increased transferrin and protection from thrombosis in Chuvash erythrocytosis.

Role of MicroRNAs in Regulation of Cellular Response to Hypoxia.

Hypoxia causes changes in transcription of the genes that contribute to adaptation of the cells to low levels of oxygen. The main mechanism regulating cellular response to hypoxia is activation of hypoxia-inducible transcription factors (HIF), which include several isoforms and control expression of more than a thousand genes. HIF activity is regulated at various levels, including by small non-coding RNA molecules called microRNAs (miRNAs). miRNAs regulate cellular response to hypoxia by influencing activation of HIF, its degradation, and translation of HIF-dependent proteins. At the same time, HIFs also affect miRNAs biogenesis. Data on the relationship of a particular HIF isoform with miRNAs are contradictory, since studies have been performed using different cell lines, various types of experimental animals and clinical material, as well as at different oxygen concentrations and durations of hypoxic exposure. In addition, HIF expression may be affected by the initial resistance of organisms to lack of oxygen, which has not been taken into account in the studies. This review analyzes the data on the effect of hypoxia on biogenesis and functioning of miRNAs, as well as on the effect of miRNAs on mRNAs of the genes involved in adaptation to oxygen deficiency. Understanding the mechanisms of relationship between HIF, hypoxia, and miRNA is necessary to develop new approaches to personalized therapy for diseases accompanied by oxygen deficiency.

Mebendazole Treatment Disrupts the Transcriptional Activity of Hypoxia-Inducible Factors 1 and 2 in Breast Cancer Cells

Breast cancer is the most diagnosed cancer in women in the world. Mebendazole (MBZ) has been demonstrated to have preclinical efficacy across multiple cancers, including glioblastoma multiforme, medulloblastoma, colon, breast, pancreatic, and thyroid cancers. MBZ was also well tolerated in a recent phase I clinical trial of adults diagnosed with glioma. The mechanisms of action reported so far for MBZ include tubulin disruption, inhibiting angiogenesis, promoting apoptosis, and maintaining stemness. To elucidate additional mechanisms of action for mebendazole (MBZ), we performed RNA sequencing of three different breast cancer cell lines treated with either MBZ or vehicle control. We compared the top genes downregulated upon MBZ treatment with expression profiles of cells treated with over 15,000 perturbagens using the clue.io online analysis tool. In addition to tubulin inhibitors, the gene expression profile that correlated most with MBZ treatment matched the profile of cells treated with known hypoxia-inducible factor (HIF-1α and -2α) inhibitors. The HIF pathway is the main driver of the cellular response to hypoxia, which occurs in solid tumors. Preclinical data support using HIF inhibitors in combination with standard of care to treat solid tumors. Therefore, we tested the hypothesis that MBZ could inhibit the hypoxia response. Using RNA sequencing and HIF-reporter assays, we demonstrate that MBZ inhibits the transcriptional activity of HIFs in breast cancer cell lines and in mouse models of breast cancer by preventing the induction of HIF-1α, HIF-2α, and HIF-1β protein under hypoxia. Taken together, our results suggest that MBZ treatment has additional therapeutic efficacy in the setting of hypoxia and warrants further consideration as a cancer therapy.

Involvement of hypoxia-inducible factor activity in inevitable air-exposure treatment upon differentiation in a three-dimensional keratinocyte culture.

Formation of the human skin epidermis can be reproduced by a three-dimensional (3D) keratinocyte culture system, in which air-exposure is inevitable upon initiation of differentiation. In the continuous submerged culture without air-exposure, even with a differentiation-compatible medium, several keratinocyte-specific proteins were not induced resulting in the formation of aberrant epidermal layers. To clarify the mechanism by whichair-exposure promotes keratinocyte differentiation, we performed a comparative analysis on biological properties between submerged and air-liquid interphase culture systems. By transcriptomic analysis, hypoxia-inducible factor (HIF)-related genes appeared to significantly change in these cultured cells. In submerged culture, the transcriptional activity of HIF on its canonical response element was enhanced, while air-exposure treatment drastically reduced the transcriptional activity despite the high HIF protein level. Regulating HIF activity through reagents and genetic manipulation revealed that the reduced but retained HIF-transcriptional activity was essentially involved in differentiation. Furthermore, we showed, for the first time, that artificial supplementation of oxygen in the submerged culture system could restore keratinocyte differentiation as observed in the air-exposed culture. Thus, we mechanistically evaluated how HIF regulates the air-exposure-dependent differentiation of keratinocytes in a 3D culture system.

Pyruvate dehydrogenase phosphatase 1 (PDP1) stimulates HIF activity by supporting histone acetylation under hypoxia.

Cancer cells, when exposed to the hypoxic tumour microenvironment, respond by activating hypoxia-inducible factors (HIFs). HIF-1 mediates extensive metabolic re-programming, and expression of HIF-1α, its oxygen-regulated subunit, is associated with poor prognosis in cancer. Here we analyse the role of pyruvate dehydrogenase phosphatase 1 (PDP1) in the regulation of HIF-1 activity. PDP1 is a key hormone-regulated metabolic enzyme that dephosphorylates and activates pyruvate dehydrogenase (PDH), thereby stimulating the conversion of pyruvate into acetyl-CoA. Silencing of PDP1 down-regulated HIF transcriptional activity and the expression of HIF-dependent genes, including that of PDK1, the kinase that phosphorylates and inactivates PDH, opposing the effects of PDP1. Inversely, PDP1 stimulation enhanced HIF activity under hypoxia. Alteration of PDP1 levels or activity did not have an effect on HIF-1α protein levels, nuclear accumulation or interaction with its partners ARNT and NPM1. However, depletion of PDP-1 decreased histone H3 acetylation of HIF-1 target gene promoters and inhibited binding of HIF-1 to the respective hypoxia-response elements (HREs) under hypoxia. Furthermore, the decrease of HIF transcriptional activity upon PDP1 depletion could be reversed by treating the cells with acetate, as an exogenous source of acetyl-CoA, or the histone deacetylase (HDAC) inhibitor trichostatin A. These data suggest that the PDP1/PDH/HIF-1/PDK1 axis is part of a homeostatic loop which, under hypoxia, preserves cellular acetyl-CoA production to a level sufficient to sustain chromatin acetylation and transcription of hypoxia-inducible genes.

NARF is a hypoxia-induced coactivator for OCT4-mediated breast cancer stem cell specification.

Hypoxia is a key characteristic of the breast cancer microenvironment that promotes expression of the transcriptional activator hypoxia-inducible factor 1 (HIF-1) and is associated with poor patient outcome. HIF-1 increases the expression or activity of stem cell pluripotency factors, which control breast cancer stem cell (BCSC) specification and are required for cancer metastasis. Here, we identify nuclear prelamin A recognition factor (NARF) as a hypoxia-inducible, HIF-1 target gene in human breast cancer cells. NARF functions as an essential coactivator by recruiting the histone demethylase KDM6A to OCT4 bound to genes encoding the pluripotency factors NANOG, KLF4, and SOX2, leading to demethylation of histone H3 trimethylated at lysine-27 (H3K27me3), thereby increasing the expression of NANOG, KLF4, and SOX2, which, together with OCT4, mediate BCSC specification. Knockdown of NARF significantly decreased the BCSC population in vitro and markedly impaired tumor initiation capacity and lung metastasis in orthotopic mouse models.

Severinia buxifolia-isolated acridones inhibit lung cancer invasion and decrease HIFα protein synthesis involving 5’UTR-mediated translation inhibition

BackgroundLung cancer is one of the most common cancers worldwide and is by far the leading cause of cancer death attributed to its rapid metastasis and poor prognosis. Given that hypoxia-inducible factors (HIFs) are associated with cancer metastasis, discovering agents to inhibit HIF-mediated invasive cancer is highly desired. PurposeThis study aimed to investigate the natural acridone compounds isolated from Severinia buxifolia for the potential to delay hypoxia-induced lung cancer invasiveness by HIF inhibition. MethodsUsing a hypoxia-responsive element (HRE) luciferase reporter, cell migration and invasion assays, real-time PCR, Western blot, and DNA recombinant clones, compound effect on HIF activity, cancer metastasis, HIF-1α mRNA transcription, HIFs protein stability, and HIF-1α translation were observed under hypoxia conditions. ResultsAtalaphyllidine (Sbs-A) and atalaphyllinine (Sbs-B) were found to show the most potent effects on HIF transcriptional activity and HIF-1α protein expression in NSCLC cell line A549, although Sbs-A and Sbs-B might not attribute decreasing HIF-1α mRNA expression to potent inhibition of HIF activity. HIF-1α protein stability was not affected by Sbs-A; also, prolyl hydroxylase and proteasome inhibitors could not reverse the inhibitory effect from compounds. Furthermore, 3 - 10 μM low concentrations of Sbs-A inhibited HIF target gene expression, gelatin zymography activity, and A549 cancer invasion. Ultimately, Sbs-A inhibited HIF-1α 5′UTR-mediated translation independent of oxygen concentration, underlying the mechanism of compounds inhibiting HIF-1α protein expression. ConclusionOur study proposed Severinia buxifolia-isolated acridone compounds inhibited 5’-mRNA HIFA-mediated translation and provided evidence supporting the ability of acridone compounds in targeting HIFα for delayed lung cancer metastasis.

Congenital Erythrocytosis Due to Novel CMTR1K769L Mutation Has Augmented Hypoxia Signaling and Features of Both Primary and Secondary Erythrocytosis

Primary erythrocytoses (PE) include germline gain of function mutations of erythropoietin (EPO) receptor (EPOR) gene and polycythemia vera (PV), a clonal disease due to acquired somatic JAK2 mutations. Both these PEs have intrinsically abnormal early erythroid progenitors (BFU-E) characterized by their in vitro EPO hypersensitive growth (EPORmutations), or even EPO independent growth (the hallmark of PV); both have low plasma EPO (Chapter 58, Williams Hematology, 10e). Secondary erythrocytoses (SE) have intrinsically normal BFU-Es but are caused by increased circulating erythropoiesis stimulators, chiefly EPO. In contrast, congenital Chuvash erythrocytosis is characterized by augmentation of hypoxia inducible factors (HIFs) causing high EPO and hypersensitive BFU-E's EPO responses; thus, it has features of both PE and SE. We report a 7-year-old normoxic girl with history of symptomatic erythrocytosis since birth of unknown etiology, with hemoglobin of 22 g/dL at birth, consistently elevated EPO at 50-75 mU/mL, and normal hemoglobin oxygen affinity. Her BFU-E grew without EPO yet no somatic PV defining mutations were found. A targeted analysis of previously described erythrocytoses genes was negative. Whole genome sequencing, performed under the auspices of Undiagnosed Disease Network, identified a heterozygous missense variant (p. Lys769Leu) of CMTR1(Cap Methyltransferase 1) gene, not listed in the Genome Aggregation Database (gnomAD). Cap methylation on mRNA protects mRNA from being degraded and promotes RNA translation by recruiting translation initiation factors (PMID:30312682). The variant was present in the proband but not in her asymptomatic mother (father was not available for the analysis). This variant was found in leukocytes and in nail DNA, indicating germline origin. Transcriptional X-chromosome inactivation assay (PMID:18641369) revealed polyclonal granulocytes and platelets, confirming that the erythrocytosis is caused by germline mutation, not by somatic mutation-driven clonal hematopoiesis. Because of her high EPO, we hypothesized that CMTR1K769L variant might cause high HIF activity. HIF-mediated gene regulation is tissue- and stage of differentiation-specific. Expression of HIF target genes SLC29A1, VEGFA, EDN1, and LDHA was upregulated in granulocytes while in platelets all target genes except SLC29A1 were upregulated. To test if CMTR1 regulates HIF transcriptional activity, 1) we correlated CMTR1 transcript levels with HIF target gene expression levels in granulocytes and platelets. CMTR1 transcript levels were positively correlated with expression levels of HIF target genes. 2) we tested if CMTR1 overexpression and knockdown alters HIF target gene expression. In erythroid cell lines (K562 and HEL), overexpressed CMTR1 increased SLC2A1 and VEGFA (HIF target genes transcripts) while knockdown of CMTR1 downregulated their expression. This demonstrates that CMTR1 positively regulates HIF transcriptional activity, suggesting that CMTR1K769L accounts for increased plasma EPO and for EPO mediated feature of SE. As erythroid progenitors express endogenous erythropoietin (PMID 9573013) which contributes to their differentiation and proliferation (PMID 10903342), we evaluated if CMTR1K769L also promotes endogenous erythroid EPO accounting for EPO independent growth of CMTR1K769L mutated BFU-Es and thus provides potential mechanism as a feature of PEfor the erythrocytosis. The proband's endogenous EPO transcript levels in in vitro expanded erythroid cells at day 4, when most of cells are erythroid progenitors, were 10 times higher than the mother's see Figure. We present a new cause of erythrocytosis associated with the novel germline mutation of principal epigenetic modifier of RNA, CMTR1 (PMID:33922187), associated with high EPO (SE). Her BFU-E grew without extrinsic EPO (PE) likely due to previously unobserved stimulation of EPO production in erythroid progenitors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mutual Regulation between Redox and Hypoxia-Inducible Factors in Cardiovascular and Renal Complications of Diabetes.

Oxidative stress and hypoxia-inducible factors (HIFs) have been implicated in the pathogenesis of diabetic cardiovascular and renal diseases. Reactive oxygen species (ROS) mediate physiological and pathophysiological processes, being involved in the modulation of cell signaling, differentiation, and survival, but also in cyto- and genotoxic damage. As master regulators of glycolytic metabolism and oxygen homeostasis, HIFs have been largely studied for their role in cell survival in hypoxic conditions. However, in addition to hypoxia, other stimuli can regulate HIFs stability and transcriptional activity, even in normoxic conditions. Among these, a regulatory role of ROS and their byproducts on HIFs, particularly the HIF-1α isoform, has received growing attention in recent years. On the other hand, HIF-1α and HIF-2α exert mutually antagonistic effects on oxidative damage. In diabetes, redox-mediated HIF-1α deregulation contributes to the onset and progression of cardiovascular and renal complications, and recent findings suggest that deranged HIF signaling induced by hyperglycemia and other cellular stressors associated with metabolic disorders may cause mitochondrial dysfunction, oxidative stress, and inflammation. Understanding the mechanisms of mutual regulation between HIFs and redox factors and the specific contribution of the two main isoforms of HIF-α is fundamental to identify new therapeutic targets for vascular complications of diabetes.

Essential role for epithelial HIF-mediated xenophagy in control of Salmonella infection and dissemination.

SUMMARYThe intestinal mucosa exists in a state of “physiologic hypoxia,” where oxygen tensions are markedly lower than those in other tissues. Intestinal epithelial cells (IECs) have evolved to maintain homeostasis in this austere environment through oxygen-sensitive transcription factors, including hypoxia-inducible factors (HIFs). Using an unbiased chromatin immunoprecipitation (ChIP) screen for HIF-1 targets, we identify autophagy as a major pathway induced by hypoxia in IECs. One important function of autophagy is to defend against intracellular pathogens, termed “xenophagy.” Analysis reveals that HIF is a central regulator of autophagy and that in vitro infection of IECs with Salmonella Typhimurium results in induction of HIF transcriptional activity that tracks with the clearance of intracellular Salmonella. Work in vivo demonstrates that IEC-specific deletion of HIF compromises xenophagy and exacerbates bacterial dissemination. These results reveal that the interaction between hypoxia, HIF, and xenophagy is an essential innate immune component for the control of intracellular pathogens.

Cryptolepine suppresses breast adenocarcinoma via inhibition of HIF-1 mediated glycolysis

As a characteristic transcription factor in solid tumors, hypoxia inducible factor-1 (HIF-1) acts as a master regulator in breast cancer progression. Cryptolepine, as a natural alkaloid, noticeably inhibited HIF-1 transcriptional activity and decreased the protein expression of hypoxia-induced HIF-1α in breast cancer cells. Further study showed that cryptolepine blocked HIF-1-mediated glycolysis and suppressed the expression of multiple glycolysis enzymes, resulting in a decrease in ATP production in hypoxic T47D and 4T1 cells. Meanwhile, cryptolepine displayed potent suppressive effect on tumor growth in a dose-dependent manner. In 4T1 tumor xenografts, cryptolepine reduced HIF-1α protein expression, and thus decreased the levels of both lactate acid and ATP productions. The mechanistic study revealed that cryptolepine could effectively suppress the process of HIF-1α mRNA translation rather than transcription, which was attributed to the inhibition on the phosphorylation of eIF4E regulated by both MAPK and mTOR signaling pathways. Collectively, current findings suggested that cryptolepine possesses the potential to treat breast cancers by modulating HIF-1 both in vitro and in vivo.

Factor inhibiting HIF can catalyze two asparaginyl hydroxylations in VNVN motifs of ankyrin fold proteins

The aspariginyl hydroxylase human factor inhibiting hypoxia-inducible factor (FIH) is an important regulator of the transcriptional activity of hypoxia-inducible factor. FIH also catalyzes the hydroxylation of asparaginyl and other residues in ankyrin repeat domain–containing proteins, including apoptosis stimulating of p53 protein (ASPP) family members. ASPP2 is reported to undergo a single FIH-catalyzed hydroxylation at Asn-986. We report biochemical and crystallographic evidence showing that FIH catalyzes the unprecedented post-translational hydroxylation of both asparaginyl residues in “VNVN” and related motifs of ankyrin repeat domains in ASPPs (i.e., ASPP1, ASPP2, and iASPP) and the related ASB11 and p18-INK4C proteins. Our biochemical results extend the substrate scope of FIH catalysis and may have implications for its biological roles, including in the hypoxic response and ASPP family function.

Essential role for epithelial HIF‐xenophagy axis in control of Salmonella infection and dissemination

The intestinal mucosa exists in a state of “physiologic hypoxia,” in which oxygen tensions of the epithelium and lumen are markedly lower than those encountered in other tissues. Intestinal epithelial cells (IECs) have evolved to maintain mucosal homeostasis in this austere environment in part through the actions of dedicated oxygen‐sensitive transcription factors, including hypoxia‐inducible factors (HIFs). HIFs are composed of a labile α‐subunit that becomes stabilized most notably in hypoxic environments and a constitutive β‐subunit. Using an unbiased chromatin immunoprecipitation (ChIP) promoter array screen for HIF‐1 targets in IEC, we identified autophagy as a major pathway induced by hypoxia and HIF in cultured IECs. Autophagy is a conserved eukaryotic system for degradation of intracellular cargo through the lysosome. Validation of the ChIP screen revealed that HIF promotes expression of multiple autophagy genes in IEC cell lines and further validated in primary murine organoids. One important function of autophagy is to defend against intracellular pathogens, a process termed xenophagy, a crucial innate immune mechanism in the mucosa that serves to control invasion of intracellular bacteria in vivo and to limit extraintestinal dissemination of enteric pathogens. Loss of function analysis revealed that HIF is a central regulator of autophagic flux and that in vitro infection of IECs with SalmonellaTyphimurium, a model intracellular bacterial pathogen, results in stabilization of HIF with the subsequent increase in HIF transcriptional activity that tracks with the clearance of intracellular Salmonella. Studies in vivo revealed that Salmonella­ drives increases in HIF activity using HIF luciferase reporter mice (HIF∆ODD) in combination with streptomycin‐pretreatment model of murine colitis. Further studies in vivo demonstrated that intestinal epithelial‐specific deletion of HIF (Hif1b∆IEC) compromised IEC xenophagy and exacerbated bacterial dissemination to multiple extraintestinal organs, implicating xenophagy‐assisted clearance of Salmonella in mucosal innate immunity.

Breast cancer hypoxia in relation to prognosis and benefit from radiotherapy after breast-conserving surgery in a large, randomised trial with long-term follow-up

BackgroundBreast-conserving surgery followed by radiotherapy is part of standard treatment for early-stage breast cancer. Hypoxia is common in cancer and may affect the benefit of radiotherapy. Cells adapt to hypoxic stress largely via the transcriptional activity of hypoxia-inducible factor (HIF)-1α. Here, we aim to determine whether tumour HIF-1α-positivity and hypoxic gene-expression signatures associated with the benefit of radiotherapy, and outcome.MethodsTumour HIF-1α-status and expression of hypoxic gene signatures were retrospectively analysed in a clinical trial where 1178 women with primary T1-2N0M0 breast cancer were randomised to receive postoperative radiotherapy or not and followed 15 years for recurrence and 20 years for breast cancer death.ResultsThe benefit from radiotherapy was similar in patients with HIF-1α-positive and -negative primary tumours. Both ipsilateral and any breast cancer recurrence were more frequent in women with HIF-1α-positive primary tumours (hazard ratio, HR0–5 yrs1.9 [1.3–2.9], p = 0.003 and HR0–5 yrs = 2.0 [1.5–2.8], p < 0.0001). Tumour HIF-1α-positivity is also associated with increased breast cancer death (HR0–10 years 1.9 [1.2–2.9], p = 0.004). Ten of the 11 investigated hypoxic gene signatures correlated positively to HIF-1α-positivity, and 5 to increased rate/risk of recurrence.ConclusionsThe benefit of postoperative radiotherapy persisted in patients with hypoxic primary tumours. Patients with hypoxic primary breast tumours had an increased risk of recurrence and breast cancer death.

Understanding the Role of Estrogen Receptor Status in PRODH/POX-Dependent Apoptosis/Survival in Breast Cancer Cells.

Simple SummaryThe estrogen receptor (ER) status and the availability of agonists or antagonists of these receptors determine the processes of growth, differentiation, and proliferation of breast cancer cells. Estrogens and anti-estrogenic compounds have been shown to influence breast cancer cell survival/apoptosis via action through the mitochondrial enzyme proline dehydrogenase/proline oxidase (PRODH/POX). In this review, we highlight the molecular effects of ER stimulation/inhibition in signaling pathways.It has been suggested that activation of estrogen receptor α (ER α) stimulates cell proliferation. In contrast, estrogen receptor β (ER β) has anti-proliferative and pro-apoptotic activity. Although the role of estrogens in estrogen receptor-positive breast cancer progression has been well established, the mechanism of their effect on apoptosis is not fully understood. It has been considered that ER status of breast cancer cells and estrogen availability might determine proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis. PRODH/POX is a mitochondrial enzyme that converts proline into pyrroline-5-carboxylate (P5C). During this process, ATP (adenosine triphosphate) or ROS (reactive oxygen species) are produced, facilitating cell survival or death, respectively. However, the critical factor in driving PRODH/POX-dependent functions is proline availability. The amount of this amino acid is regulated at the level of prolidase (proline releasing enzyme), collagen biosynthesis (proline utilizing process), and glutamine, glutamate, α-ketoglutarate, and ornithine metabolism. Estrogens were found to upregulate prolidase activity and collagen biosynthesis. It seems that in estrogen receptor-positive breast cancer cells, prolidase supports proline for collagen biosynthesis, limiting its availability for PRODH/POX-dependent apoptosis. Moreover, lack of free proline (known to upregulate the transcriptional activity of hypoxia-inducible factor 1, HIF-1) contributes to downregulation of HIF-1-dependent pro-survival activity. The complex regulatory mechanism also involves PRODH/POX expression and activity. It is induced transcriptionally by p53 and post-transcriptionally by AMPK (AMP-activated protein kinase), which is regulated by ERs. The review also discusses the role of interconversion of proline/glutamate/ornithine in supporting proline to PRODH/POX-dependent functions. The data suggest that PRODH/POX-induced apoptosis is dependent on ER status in breast cancer cells.