Expression Of HIF-1α Target Genes Research Articles

Abstract 6959: Acetylcholine potentiates invasion and migration of human pancreatic cancer cells under hypoxia through α7 nAChR-mediated increases in HIF-1α expression

Abstract The most significant nerve-related problem in clinical pancreatic cancer management is perineural invasion (PNI), characterized as the neoplastic invasion of cancer cells into or surrounding the nerves. It is a characteristic feature of pancreatic ductal adenocarcinoma (PDAC) associated with a poor prognosis, tumor recurrence, and emergent pain. However, why PDAC is associated with a higher incidence of PNI remains unclear. HIF-1 (hypoxia-inducible factor-1) regulates the expression of ~1000 genes involved in angiogenesis, metastasis, tumor growth, chemoresistance and radioresistance, underscoring the growing interest in targeting HIF-1 for cancer control. In the present study, we investigated the molecular mechanisms underlying neurotransmitter acetylcholine (Ach)-potentiated HIF-1α expression and subsequent increase in invasion and migration of human pancreatic cancer cells in hypoxia (2% O2). The expression of various acetylcholine receptors in six types of pancreatic cancer cell lines (MIA-PaCa-2, PANC-1, HPAF-II, Capan-1, AsPC-1, and BxPC-3 cells) was observed using qPCR, and α7 nAChRs was observed to be expressed in all pancreatic cancer cells. Therefore, we chose MIA-PaCa-2 cells, expressing high levels of α7 nAChRs, and HPAF-II cells, expressing low levels of α7 nAChRs, for loss- and gain-of-function studies, respectively. In vitro assays showed that, under hypoxic conditions, Ach caused a concentration-dependent increase in α7 nAChRs-mediated HIF-1α expression, enhancing expression of HIF-1α target genes, and promoting invasion and migration of human pancreatic cancer cells. We further found that Ach increased HIF-1α protein stability in pancreatic cancer cells under hypoxic conditions. This effect was mediated by YAP signaling, as evidenced by the fact that Ach-induced increases in HIF-1α protein stability were blocked by siRNA-mediated knockdown of YAP. Consistent with these findings, an investigation of the relationship between α7 nAChRs and clinicopathological features in pancreatic cancer patients using publicly available datasets showed that high levels of α7 nAChRs are correlated with a poorer prognosis in pancreatic cancer patients. Collectively, these findings identify novel mechanisms by which Ach potentiates invasion and migration of pancreatic cancer cells and provide further evidence that HIF-1α is a potential anticancer target in Ach-associated pancreatic cancer. Citation Format: Yunmi Cho, Eun-Taex Oh, Ha Gyeong Kim, Heon Joo Park. Acetylcholine potentiates invasion and migration of human pancreatic cancer cells under hypoxia through α7 nAChR-mediated increases in HIF-1α expression [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 6959.

Metabolic priming by multiple enzyme systems supports glycolysis, HIF1α stabilisation, and human cancer cell survival in early hypoxia

Adaptation to chronic hypoxia occurs through changes in protein expression, which are controlled by hypoxia-inducible factor 1α (HIF1α) and are necessary for cancer cell survival. However, the mechanisms that enable cancer cells to adapt in early hypoxia, before the HIF1α-mediated transcription programme is fully established, remain poorly understood. Here we show in human breast cancer cells, that within 3 h of hypoxia exposure, glycolytic flux increases in a HIF1α-independent manner but is limited by NAD+ availability. Glycolytic ATP maintenance and cell survival in early hypoxia rely on reserve lactate dehydrogenase A capacity as well as the activity of glutamate-oxoglutarate transaminase 1 (GOT1), an enzyme that fuels malate dehydrogenase 1 (MDH1)-derived NAD+. In addition, GOT1 maintains low α-ketoglutarate levels, thereby limiting prolyl hydroxylase activity to promote HIF1α stabilisation in early hypoxia and enable robust HIF1α target gene expression in later hypoxia. Our findings reveal that, in normoxia, multiple enzyme systems maintain cells in a primed state ready to support increased glycolysis and HIF1α stabilisation upon oxygen limitation, until other adaptive processes that require more time are fully established.

LncRNA LUCAT1 Promotes Glioblastoma Progression by Enhancing HIF1α Activity.

Hypoxia is associated with poor prognosis in many cancers including glioblastoma (GBM). Glioma stem-like cells (GSCs) often reside in hypoxic regions and serve as reservoirs for disease progression. Long non-coding RNAs (lncRNAs) have been implicated in GBM. However, the lncRNAs that modulate GSC adaptations to hypoxia are poorly understood. Identification of these lncRNAs may provide new therapeutic strategies to target GSCs under hypoxia. lncRNAs induced by hypoxia in GSCs were identified by RNAseq. LUCAT1 expression was assessed by qPCR, RNAseq, Northern blot, single molecule FISH in GSCs, and interrogated in IvyGAP, TCGA, and CGGA databases. LUCAT1 was depleted by shRNA, CRISPR/Cas9, and CRISPR/Cas13d. RNAseq, Western blot, immunohistochemistry, co-IP, ChIP, ChIPseq, RNA immunoprecipitation, and proximity ligation assay were performed to investigate mechanisms of action of LUCAT1. GSC viability, limiting dilution assay, and tumorigenic potential in orthotopic GBM xenograft models were performed to assess the functional consequences of depleting LUCAT1. A new isoform of Lucat1 is induced by HIF1α and NRF2 in GSCs under hypoxia. LUCAT1 is highly expressed in hypoxic regions in GBM. Mechanistically, LUCAT1 formed a complex with HIF1α and its co-activator CBP to regulate HIF1α target gene expression and GSC adaptation to hypoxia. Depletion of LUCAT1 impaired GSC self-renewal. Silencing LUCAT1 decreased tumor growth and prolonged mouse survival in GBM xenograft models. A HIF1α-LUCAT1 axis forms a positive feedback loop to amplify HIF1α signaling in GSCs under hypoxia. LUCAT1 promotes GSC self-renewal and GBM tumor growth. LUCAT1 is a potential therapeutic target in GBM.

SIRT3 inhibition suppresses hypoxia-inducible factor 1α signaling and alleviates hypoxia-induced apoptosis of type B spermatogonia GC-2 cells.

Hypoxia has been reported to be an important factor leading to male infertility, and it has been reported that hypoxia can induce the apoptosis of mouse spermatogenic cells. Sirtuin 3 (SIRT3) has been reported to promote the degradation of hypoxia-inducible factor 1α (HIF-1α), and thus, we hypothesized that SIRT3 may influence hypoxia-induced apoptosis of spermatogonia. In this study, we overexpressed or inhibited SIRT3 in mouse type B spermatogonia GC-2 cells and then subjected the cells to hypoxia or normoxia, before examining hypoxia-responsive gene expression and cell viability. We report that SIRT3 stabilizes hypoxia-inducible factor 1α (HIF-1α) and activates its downstream target gene expression in GC-2 cells. We also show that the SIRT3 inhibitor 3-TYP suppresses HIF-1α target gene expression and alleviates hypoxia-induced apoptosis of GC-2 cells. Our study reveals the critical role and underlying mechanisms of SIRT3 in hypoxia-induced apoptosis of mouse type B spermatogonia GC-2 cells.

Gut fungi enhances immunosuppressive function of myeloid-derived suppressor cells by activating PKM2-dependent glycolysis to promote colorectal tumorigenesis

BackgroundAccumulating evidence implicates that gut fungi are associated with the pathogenesis of colorectal cancer (CRC). Our previous study has revealed that Candida tropicalis (C. tropicalis) promotes colorectal tumorigenesis by enhancing immunosuppressive function of myeloid-derived suppressor cells (MDSCs) and increasing accumulation of MDSCs, but the underlying mechanisms remain unestablished.MethodsBone marrow–derived MDSCs were stimulated with C. tropicalis. RNA-sequencing analysis was performed to screen the differentially expressed genes. Quantitative real-time PCR and western blot were used to measure the expression of related proteins. Co-culture assay of MDSCs and CD8+ T cells was used to determine the immunosuppressive ability of MDSCs. Metabolomic analysis was conducted to detect metabolic reprogramming of MDSCs. Aerobic glycolysis of MDSCs was assessed by extracellular acidification rate (ECAR), glucose consumption and lactate production. A CAC mouse model was induced by AOM and DSS to determine the therapeutic action of TEPP-46. IHC and immunofluorescence were performed to examine the expression of PKM2, PKM2 (p-Y105) and iNOS in human CRC-infiltrated MDSCs.ResultsC. tropicalis facilitates immunosuppressive function of MDSCs by increasing the expression of iNOS, COX2 and NOX2, production of nitric oxide (NO) and reactive oxygen species (ROS). Mechanistically, C. tropicalis facilitates the immunosuppressive function of MDSCs through the C-type lectin receptors Dectin-3 and Syk. C. tropicalis-enhanced immunosuppressive function of MDSCs is further dependent on aerobic glycolysis. On the one hand, NO produced by MDSCs enhanced aerobic glycolysis in a positive feedback manner. On the other hand, C. tropicalis promotes p-Syk binding to PKM2, which results in PKM2 Tyr105 phosphorylation and PKM2 nuclear translocation in MDSCs. Nuclear PKM2 interacts with HIF-1α and subsequently upregulates the expression of HIF-1α target genes encoding glycolytic enzymes, GLUT1, HK2, PKM2, LDHA and PDK1, which are required for the C. tropicalis-induced aerobic glycolysis of MDSCs. Blockade of PKM2 nuclear translocation attenuates C. tropicalis-mediated colorectal tumorigenesis. The high expression of PKM2, PKM2 (p-Y105) and iNOS in CRC-infiltrated MDSCs correlates with the development of human CRC.ConclusionC. tropicalis enhances immunosuppressive function of MDSCs via Syk-PKM2-HIF-1α-glycolysis signaling axis, which drives CRC. Therefore, we identify the Syk-PKM2-HIF-1α-glycolysis signaling axis as a potential therapeutic target for CRC.

The Macrophage-Associated LncRNA MALR Facilitates ILF3 Liquid-Liquid Phase Separation to Promote HIF1α Signaling in Esophageal Cancer.

Secretion of TNFα by tumor-associated macrophages stimulates cancer cells to upregulate lncRNA MALR, which induces ILF3 liquid-liquid phase separation and activation of HIF1α signaling to promote cancer progression.

Hypoxia-Inducible Factor-1α in Rods Is Neuroprotective Following Retinal Detachment.

PurposeFollowing retinal detachment (RD) photoreceptors (PRs) sustain hypoxic stress and eventually die. Hypoxia-inducible factor-1α (HIF-1α) plays a central role in cellular adaptation to hypoxia. The purpose of this study is to determine the necessity of HIF-1α on PR cell survival after RD.MethodsExperimental RD was created in mice by injection of hyaluronic acid (1%) into the subretinal space. Mice with conditional HIF-1α knockout in rods (denoted as HIF-1αΔrod) were used. HIF-1α expression in retinas was measured real-time polymerase chain reaction (RT-PCR) and Western blotting. PR cell death after RD was evaluated using TUNEL assay. Optical coherence tomography (OCT) and histology were used to evaluate retinal layer thicknesses and PR cell densities. A hypoxia signaling pathway PCR array was used to examine the expression of HIF-1α target genes after RD.ResultsHIF-1α protein levels were significantly increased after RD, and depletion of HIF-1α in rods blunted this increase. A compensatory increase of HIF-2α protein was observed in HIF-1αΔrod mice. Conditional knockout (cKO) of HIF-1α in rods did not lead to any morphologic change in attached retinas but resulted in significantly increased PR cell loss after RD. HIF-1α cKO in rods altered the responses to retinal detachment for 25 out of 83 HIF-1α target genes that were highly enriched for genes involved in glycolysis.ConclusionsRod-derived HIF-1α plays a key role in the PR response to RD, mediating the transcriptional activity of a battery of genes to promote PR cell survival.

Coxiella burnetii Affects HIF1α Accumulation and HIF1α Target Gene Expression.

HIF1α is an important transcription factor regulating not only cellular responses to hypoxia, but also anti-infective defense responses. We recently showed that HIF1α hampers replication of the obligate intracellular pathogen Coxiella burnetii which causes the zoonotic disease Q fever. Prior to development of chronic Q fever, it is assumed that the bacteria enter a persistent state. As HIF1α and/or hypoxia might be involved in the induction of C. burnetii persistence, we analyzed the role of HIF1α and hypoxia in the interaction of macrophages with C. burnetii to understand how the bacteria manipulate HIF1α stability and activity. We demonstrate that a C. burnetii-infection initially induces HIF1α stabilization, which decreases then over the course of an infection. This reduction depends on bacterial viability and a functional type IV secretion system (T4SS). While neither the responsible T4SS effector protein(s) nor the molecular mechanism leading to this partial HIF1α destabilization have been identified, our results demonstrate that C. burnetii influences the expression of HIF1α target genes in multiple ways. Therefore, a C. burnetii infection promotes HIF1α-mediated upregulation of several metabolic target genes; affects apoptosis-regulators towards a more pro-apoptotic signature; and under hypoxic conditions, shifts the ratio of the inflammatory genes analyzed towards a pro-inflammatory profile. Taken together, C. burnetii modulates HIF1α in a still elusive manner and alters the expression of multiple HIF1α target genes.

The WWOX/HIF1A Axis Downregulation Alters Glucose Metabolism and Predispose to Metabolic Disorders.

Recent reports indicate that the hypoxia-induced factor (HIF1α) and the Warburg effect play an initiating role in glucotoxicity, which underlies disorders in metabolic diseases. WWOX has been identified as a HIF1α regulator. WWOX downregulation leads to an increased expression of HIF1α target genes encoding glucose transporters and glycolysis’ enzymes. It has been proven in the normoglycemic mice cells and in gestational diabetes patients. The aim of the study was to determine WWOX’s role in glucose metabolism regulation in hyperglycemia and hypoxia to confirm its importance in the development of metabolic disorders. For this purpose, the WWOX gene was silenced in human normal fibroblasts, and then cells were cultured under different sugar and oxygen levels. Thereafter, it was investigated how WWOX silencing alters the genes and proteins expression profile of glucose transporters and glycolysis pathway enzymes, and their activity. In normoxia normoglycemia, higher glycolysis genes expression, their activity, and the lactate concentration were observed in WWOX KO fibroblasts in comparison to control cells. In normoxia hyperglycemia, it was observed a decrease of insulin-dependent glucose uptake and a further increase of lactate. It likely intensifies hyperglycemia condition, which deepen the glucose toxic effect. Then, in hypoxia hyperglycemia, WWOX KO caused weaker glucose uptake and elevated lactate production. In conclusion, the WWOX/HIF1A axis downregulation alters glucose metabolism and probably predispose to metabolic disorders.

Treatment with the Prolyl Hydroxylase Inhibitor JNJ Promotes Abdominal Aortic Aneurysm Progression in Diabetic Mice

Prolyl hydroxylase domain containing proteins (PHD) rigorously regulate intracellular hypoxia inducible factor-1 (HIF-1) protein expression and activity. Diabetes impairs PHD activity and attenuates abdominal aortic aneurysm (AAA) progression. The extent to which dysregulated PHD activity contributes to diabetes mediated AAA suppression remains undetermined. AAAs were induced in diabetic and non-diabetic male C57BL/6J mice via intra-aortic elastase infusion. A PHD inhibitor (JNJ-42041935, aka "JNJ", 150 mmol/kg) or vehicle alone was administered daily starting one day prior to AAA induction for 14 days. Influences on AAA progression was assessed via ultrasonography and histopathology. Expression of aortic HIF-1α, three of its target genes and macrophage derived mediators were assayed via quantitative reverse transcription polymerase chain reaction. Aneurysmal sections from AAA patients with and without diabetes (two patients in each group) were immunostained for HIF-1α and vascular endothelial growth factor (VEGF)-A. Expression of HIF-1α target genes (erythropoietin, VEGF-A, and glucose transporter-1) was reduced by 45% - 95% in experimental diabetic aortas. Diameter enlargement was similarly limited, as were mural elastin degradation, leukocyte infiltration, and neo-angiogenesis (reduced capillary density and length) on histopathology. Pre-treatment with JNJ prior to AAA initiation augmented aortic HIF-1α target gene expression and aneurysm progression in diabetic mice, along with macrophage VEGF-A and matrix metalloproteinase 2 mRNA expression. No differences were noted in HIF-1α or VEGF-A expression on aortic immunohistochemical staining of human aortic tissue as a function of diabetes status. Small molecule PHD inhibitor treatment reduces or offsets impairment of experimental AAA progression in hyperglycemic mice, highlighting the potential contribution of dysregulated PHD activity to diabetes mediated aneurysm suppression.

LRP5 Regulates HIF-1α Stability via Interaction with PHD2 in Ischemic Myocardium

Low-density lipoprotein receptor-related protein 5 (LRP5) has been studied as a co-receptor for Wnt/β-catenin signaling. However, its role in the ischemic myocardium is largely unknown. Here, we show that LRP5 may act as a negative regulator of ischemic heart injury via its interaction with prolyl hydroxylase 2 (PHD2), resulting in hypoxia-inducible factor-1α (HIF-1α) degradation. Overexpression of LRP5 in cardiomyocytes promoted hypoxia-induced apoptotic cell death, whereas LRP5-silenced cardiomyocytes were protected from hypoxic insult. Gene expression analysis (mRNA-seq) demonstrated that overexpression of LRP5 limited the expression of HIF-1α target genes. LRP5 promoted HIF-1α degradation, as evidenced by the increased hydroxylation and shorter stability of HIF-1α under hypoxic conditions through the interaction between LRP5 and PHD2. Moreover, the specific phosphorylation of LRP5 at T1492 and S1503 is responsible for enhancing the hydroxylation activity of PHD2, resulting in HIF-1α degradation, which is independent of Wnt/β-catenin signaling. Importantly, direct myocardial delivery of adenoviral constructs, silencing LRP5 in vivo, significantly improved cardiac function in infarcted rat hearts, suggesting the potential value of LRP5 as a new target for ischemic injury treatment.

Treatment with hypoxia-mimetics protects cultured rat Schwann cells against oxidative stress-induced cell death.

Schwann cell (SC) grafts promote axon regeneration in the injured spinal cord, but transplant efficacy is diminished by a high death rate in the first 2-3 days postimplantation. Both hypoxic preconditioning and pharmacological induction of the cellular hypoxic response can drive cellular adaptations and improve transplant survival in a number of disease/injury models. Hypoxia-inducible factor 1 alpha (HIF-1α), a regulator of the cellular response to hypoxia, is implicated in preconditioning-associated protection. HIF-1α cellular levels are regulated by the HIF-prolyl hydroxylases (HIF-PHDs). Pharmacological inhibition of the HIF-PHDs mimics hypoxic preconditioning and provides a method to induce adaptive hypoxic responses without direct exposure to hypoxia. In this study, we show that hypoxia-mimetics, deferoxamine (DFO) and adaptaquin (AQ), enhance HIF-1α stability and HIF-1α target gene expression. Expression profiling of hypoxia-related genes demonstrates that HIF-dependent and HIF-independent expression changes occur. Analyses of transcription factor binding sites identify several candidate transcriptional co-regulators that vary in SCs along with HIF-1α. Using an in vitro model system, we show that hypoxia-mimetics are potent blockers of oxidative stress-induced death in SCs. In contrast, traditional hypoxic preconditioning was not protective. The robust protection induced by pharmacological preconditioning, particularly with DFO, indicates that pharmacological induction of hypoxic adaptations could be useful for promoting transplanted SC survival. These agents may also be more broadly useful for protecting SCs, as oxidative stress is a major pathway that drives cellular damage in the context of neurological injury and disease, including demyelinating diseases and peripheral neuropathies.

Deoxypodophyllotoxin Inhibits Non-Small Cell Lung Cancer Cell Growth by Reducing HIF-1α-Mediated Glycolysis.

Cancer cell proliferation is a metabolically demanding process that requires high rate of glycolysis to support anabolic growth. Deoxypodophyllotoxin (DPT) is a natural flavonolignan with various pharmacological activities, including antitumor effect. However, whether DPT affects the metabolic reprogramming of cancer cells is unknown. The purpose of this study is to investigate the role of DPT on non-small cell lung cancer (NSCLC) and to explore whether HIF-1α-mediated glycolysis is involved in its mechanism of action.The level of HIF-1α mRNA and protein in NSCLC cells following DPT treatment was detected using qRT-PCR and western blotting, respectively. Cell Counting Kit-8 (CCK-8) and caspase-3 activity assays were performed to analyze cell proliferation and apoptosis. The underlying molecular mechanism was identified by dual luciferase assay, Western blotting, qRT-PCR, glucose consumption, lactate production, and immunoprecipitation. A murine NSCLC model was used to clarify the effect of DPT treatment on tumor cell proliferation. Our findings showed that DPT treatment inhibited NSCLC cell growth in a dose- and time-dependent manner. Further analysis suggested that DPT treatment inhibited HIF-1α signaling pathway by Parkin-mediated protein degradation in NSCLC cells. DPT treatment significantly decreased glucose consumption and lactate production. In addition, DPT treatment reduced the expression of HIF-1α target genes, including GLUT1, HK2 and LDHA, resulting in reduction in glycolysis. We further revealed that DPT-induced cell growth inhibition and increased glucose and lactate levels could be reversed by overexpressing HIF-1α. Additionally, we found that DPT repressed NSCLC growth and GLUT1, HK2 and LDHA expression in vivo. Overall, this study suggested that DPT inhibited NSCLC growth by preventing HIF-1α-mediated glycolysis.

Insulin-Containing Wound Dressing Promotes Diabetic Wound Healing Through Stabilizing HIF-1α.

HIF-1α is seen as a major regulator during wound healing and controls many wound healing processes, such as angiogenesis, extracellular deposition, and reepithelialization. A diabetic state plays a vicious effect on wound healing, and the destabilization of HIF-1α is a non-negligible factor. Insulin-loaded silk fibroin microparticles were prepared to release insulin by covering the wounds, and this material was proven to promote wound healing in both in vitro and in vivo studies. In this work, we found that this insulin-containing wound dressing could accelerate diabetic wound healing by promoting reepithelialization, angiogenesis, and extracellular matrix, especially collagen deposition. Meanwhile, HIF-1α was stable and accumulated in insulin-containing dressing to group wound cells, which was significantly unstable in the control group. In further studies, we showed that methylglyoxal (MGO), the main form of advanced glycation end products (AGEs), accumulated significantly and caused the destabilization of HIF-1α in the diabetic state. Insulin could alleviate the MGO-induced HIF-1α unstable state and promote HIF-1α target gene expression and its downstream biological effect such as angiogenesis and wound extracellular matrix deposition.

Licochalcone A inhibits hypoxia-inducible factor-1α accumulation by suppressing mitochondrial respiration in hypoxic cancer cells

Hypoxia-inducible factor (HIF)-1 is an important regulator of the cellular response in the hypoxic tumor environment. While searching for HIF inhibitors derived from natural products that act as anticancer agents, we found that Glycyrrhiza uralensis exerts HIF-1 inhibitory activity in hypoxic cancer cells. Among the five components of G. uralensis, licochalcone A was found to potently suppress hypoxia-induced HIF-1α accumulation and expression of HIF-1α target genes, including GLUT1 and PDK1 in HCT116 cells. Licochalcone A also enhances intracellular oxygen content by directly inhibiting mitochondrial respiration, resulting in oxygen-dependent HIF-1α degradation. Hence, licochalcone A may effectively inhibit ATP production, primarily by reducing the mitochondrial respiration-mediated ATP production rate rather than the glycolysis-mediated ATP production rate. This effect subsequently suppresses cancer cell viability, including that of HCT116, H1299, and H322 cells. Consequently, these results suggest that licochalcone A has therapeutic potential in hypoxic cancer cells.

LncRNA SNHG11 facilitates tumor metastasis by interacting with and stabilizing HIF-1\u03b1

Epigenetic alteration is one of the hallmarks of colorectal cancer (CRC). Many driver genes are regulated by DNA methylation in CRC. However, the role of DNA methylation regulating lncRNAs remain elusive. Here, we identify that SNHG11 (small nucleolar RNA host gene 11) is upregulated by promotor hypomethylation in CRC and is associated with poor prognosis in CRC patients. SNHG11 can promote CRC cell migration and metastasis under hypoxia. Interestingly, the DNA-binding motif of SNHG11 is similar to that of HIF-1α. In addition, SNHG11-associated genes are enriched with members of the HIF-1 signaling pathway in CRC. Mechanistically, SNHG11 binds to the pVHLrecognition sites on HIF-1α, thus blocking the interaction of pVHL with HIF-1α and preventing its ubiquitination and degradation. Moreover, SNHG11 upregulates the expression of HIF-1α target genes, i.e., AK4, ENO1, HK2, and Twist1. Notably, SNHG11 can bind to the HRE sites in the promoter of these genes and increase their transcription. In summary, these results identify a SNHG11/ HIF-1α axis that plays a pivotal role in tumor invasion and metastasis.

Decursin promotes HIF-1α proteasomal degradation and immune responses in hypoxic tumour microenvironment

BackgroundHypoxia and HIF-1α are important regulators of tumour growth and angiogenesis and could be attractive targets for cancer therapeutics. Decursin is an active compound extracted from the roots of Angelica gigas and has been shown to have potent anti-cancer and anti-angiogenic activities. However, whether decursin regulates HIF-1α activity and immune responses under hypoxic conditions is not yet understood. PurposeThe aim of this study was to identify whether decursin exhibits anti-cancer activity by targeting HIF-1α. Study designWe investigated whether decursin regulates HIF-1α protein stability and increases its degradation. In addition, we determined if decursin increases immune responses in tumour microenvironment to identify its hypoxia-associated anti-cancer activities. Materials and methodsWe performed the hypoxia-responsive element promoter–reporter assay, Western blot analysis, immune-fluorescence assay, semi-quantitative RT-PCR and ELISA for VEGF secretion, CCK-8 assay for cell proliferation, TUNEL assay for apoptosis and invasion assay in A549 human lung cancer or HCT116 human colon cancer cells. In vivo Lewis lung carcinoma (LLC) allograft mouse model was used to check tumour growth and immune responses in tumour microenvironment by immunohistochemistry analysis. ResultsWe observed that decursin inhibited HIF-1 activation under hypoxia by down-regulating the protein level of its subunit HIF-1α. It increased oxygen-dependant hydroxylation and ubiquitination of HIF-1α to promote HIF-1α degradation. Decursin also decreased mRNA expression of HIF-1α target genes. Decursin suppressed cancer cell proliferation, induced apoptosis and inhibited cancer cell invasion under hypoxia in cancer cells. In the allograft mouse tumour model, decursin reduced the hypoxic area and HIF-1α and PD-L1 expression. Infiltrating T cells (CD3+), helper T cells (CD4+) and cytotoxic (CD8+) T cells were accumulated, but regulatory T cells (Foxp3) and myeloid-derived suppressor cell-mediated immune suppressors (Arg1) were attenuated by decursin. ConclusionOur results suggest that decursin is a novel HIF-1α inhibitor that functions by promoting its proteasomal degradation and that it also helps improve T cell activation in tumour microenvironment; these findings provide new explanations about its anti-cancer and anti-angiogenic activity mechanisms.

Comparison Of Exercise-induced Regulation Of Skeletal Muscle Hif-1α Between Endurance Trained And Untrained Individuals

The regulation of gene transcription is essential for muscle adaptations resulting from endurance exercise training. Recent findings implicate hypoxia inducible factor 1α (HIF-1α) in this adaptive process as it regulates genes involved in O2 homeostasis and substrate utilization. Athletes have greater resting levels of muscle HIF-1α inhibitors than untrained individuals, suggesting that suppression of HIF-1α underlies skeletal muscle adaptations to endurance training. However, it is unknown if the exercise-induced expression of HIF-1α and its inhibitors differs between trained and untrained individuals. Further, differences in expression of HIF-1α target genes following acute exercise between trained (ET) and untrained (UT) individuals have yet to be examined. PURPOSE: To compare regulation of HIF-1α and HIF-1α-target genes between ET and UT individuals following acute exercise. METHODS: Five ET and five UT subjects performed an acute bout of cycling consisting of twenty, 10s sprints. Muscle samples were collected pre, post and 3 hours (3H) after exercise and analyzed for HIF-1α, the HIF-1α regulators: PHD2, FIH, VHL and SIRT6, and HIF-1α target genes (BNIP3, PINK1, VEGF, PDK-M, GLUT4, GAPDH). RESULTS: 2x3 repeated measures ANOVA revealed that post-exercise HIF-1α protein was greater in UT compared to ET individuals (.310 ± .020 vs .244 ± .001 AU, p<.05). PHD2 (.056 ± .012 vs .032 ± .023 AU), FIH (.008 ± .001 vs .004 ± .002 AU) and SIRT6 (.04 ± .001 vs .002 ± .001 AU) levels were higher in ET compared to the UT individuals at 3H post exercise (p< .05). Post-exercise fold change values for PDK (2.2 ± .1.2 vs .90 ± .21) and BNIP3 (2.2 ± 1 vs .94 ± .16) were greater in UT compared to ET (p< .05). CONCLUSION: Exercise-induced expression of HIF-1α is blunted in ET individuals compared to UT individuals. This is due to the greater post-exercise expression of HIF-1α regulating proteins. The suppression of HIF-1α in response to exercise in ET individuals is evident at the transcriptional level, as expression of PDK and BNIP3 are lower in ET individuals compared to UT. This suggests that endurance training suppresses transcription of PDK, facilitating ATP-resynthesis via oxidative phosphorylation. The suppression of BNIP3 may reflect a reduction in mitophagy, further supporting mitochondrial function in ET individuals.

P0515BRD4 PROTEIN REGULATES THE RESPONSES TO HYPOXIA TRIGGERED IN THE EXPERIMENAL RENAL DAMAGE

Abstract Background and Aims Renal hypoxia is not only one of the most common causes of acute kidney injury, but also a critical mediator in the transition to chronic kidney disease. When the kidney is exposed to an insufficient supply of oxygen to meet demand, some adaptive mechanisms are triggered by the cells to maintain homeostasis. Induction of HIF-1α transcription factor and activation of unfolded protein response (UPR) pathway, as consequence of ER dysfunction, are both essential to mediate the cell survival. The UPR pathway is regulated by three major protein sensors (IRE1α, PERK and ATF6) which under ER stress initiate the activation of the XBP1, ATF4 and ATF6 transcription factors, respectively. However, inappropriate activation of these mechanisms could lead to the enhanced transcription of genes involved in key processes in renal damage (inflammation, cell death or autophagy). On the other hand, BRD4 is an epigenetic reader that recognizes acetylated lysine residues on histone and other proteins, and mediates the binding of transcription factors to the transcriptional machinery. Our aim was to investigate whether BRD4-mediated epigenetic mechanisms could modulate the response to hypoxia triggered in acute renal damage. Method Tubular epithelial cell line, HK2, was cultured with thapsigargin (Tg) or in hypoxia chamber (1% O2, 5% CO2). In addition, these cells were treated with specific BET proteins inhibitors (JQ1, I-BET762) and with small interfering RNAs (siRNA BRD4, p300), or were subjected to knockdown of ATF4 and XBP1 by CRISPR/cas9 technology. Transcriptional changes were analyzed in each condition by RNA-sequencing. The binding of BRD4 to target genes and recruitment of the transcriptional machinery was analyzed by chromatin immunoprecipitation (ChIP) with specific antibodies against BRD4, RNA PolII, AcH3 and AcH4. Effect of JQ1 inhibitor was assayed in an ischemia/reperfusion injury (IRI) model, and analysis of gene expression, inflammatory cell infiltration, and epigenetic remodeling was carried out by quantitative PCR, IHQ and ChIP assay, respectively. Results Treatment of HK2 cells with BETs inhibitors, previously cultured with Tg or under hypoxia conditions, inhibits the gene expression of the GPR78 ER chaperon, and the XBP1 and ATF4 transcription factors modulating the downstream signaling pathways. Meanwhile, ATF6 expression remains unchanged. Gene silencing with siRNA and ChIP assays reveal that under activation of the UPR pathway or hypoxia, BRD4 recognizes acetylated histones in the GPR78, ATF4 and XBP1 promoters, recruits the pTEF-b complex and activates RNA-pol II allowing the gene transcription. Additionally, inhibition of BRD4 impairs the HIF-1α stabilization, downregulating the expression of hypoxia-induced genes. Results from whole-genome gene expression assays after stable knockdown of XBP1 and ATF4 reveal that most (86%) of the UPR genes regulated by BET proteins are dependent of XBP1 and only 32% by ATF4. Moreover, almost all genes regulated by ATF4 are also XBP1-dependent. This result may be due to the fact that ATF4 regulates IRE1α expression and thus modulates the XBP1 mRNA splicing. Administration of JQ1 in an IRI model supports that blockage of BRD4 ameliorates the renal damage (reducing BUN and creatinine levels) due to a decreased UPR activation and expression of HIF-1α target genes. As consequence, the expression of inflammatory genes and the inflammatory cell infiltration is diminished. Conclusion Our results show that BRD4 protein regulates two key processes, induction of HIF-1α transcription factor and UPR pathway activation triggered by renal hypoxia. Pharmacological inhibition of BET proteins reduces the activation these pathways, ameliorating renal damage and avoiding its progression.

A combination of pyridine-2, 4-dicarboxylic acid diethyl ester and resveratrol stabilizes hypoxia-inducible factor 1-alpha and improves hair density in female volunteers.

The aim of this study was first to demonstrate that a combination of pyridine-2, 4-dicarboxylic acid diethyl ester and resveratrol could synergize in vitro on biological pathways associated with hair growth and then to demonstrate the benefit on hair density in a clinical study. The effects of pyridine-2, 4-dicarboxylic acid diethyl ester and resveratrol directly on the hypoxic inducible factor-1α protein (HIF-1α) and related genes expression were demonstrated on keratinocytes in culture in vitro using western-blot analysis and real time quantitative polymerase chain reaction analysis. The effect of resveratrol against oxidative stress induced by hydrogen peroxide treatment was studied in hair follicle and hair matrix cells in vitro using the sensitive probe Dichloro-dihydro-fluorescein diacetate (DCFH-DA). Finally, a randomized clinical study on hair density was conducted on 79 Caucasian female subjects to assess the effect of this combination of actives. Pyridine-2, 4-dicarboxylic acid diethyl ester and resveratrol stabilized HIF-1a protein and increased the expression of HIF-1α target genes. Resveratrol significantly reduced the oxygen peroxide-induced oxidative stress generated in hair follicle and hair matrix cells. The clinical study showed that a topical treatment with the combination significantly increased the hair density on women from 1.5 months. In addition to the antioxidant properties of resveratrol, the association of pyridine-2, 4-dicarboxylic acid diethyl ester and resveratrol revealed a synergistic effect on the HIF-1α pathway. The results of the clinical study confirmed the importance of such a combination to increase the hair density.