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BACKGROUND Activated macrophages require increased oxygen to destroy foreign bodies, leading to an increase in the levels of reactive oxygen species (ROS). Therefore, macrophages would experience hypoxic and oxidative stress conditions at the same time. Thus, this study was aimed to evaluate the mechanism of the activated macrophages to overcoming this dual condition. METHODS The activated macrophages were harvested from the intraperitoneal cavities of 18 BALB/c mice immunized with 2% sheep red blood cells (SRBCs). The macrophage suspension was divided into four groups: control, 24, 48, and 72 hours after-immunization groups. The expressions of hypoxia-inducible factor (HIF)-1α, HIF-2α, and cytoglobin (Cygb), as markers for hypoxic condition, were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and enzymelinked immunosorbent assay (ELISA), whereas peroxisome proliferator-activated receptor gamma coactivator (PGC-1α) protein as a marker for mitochondrial biogenesis and aerobic metabolism was measured with ELISA. The analysis of oxidative stress was conducted with the water-soluble tetrazolium salt test. RESULTS The HIF-1α mRNA expression was the highest at 24 hours, whereas the HIF-2α mRNA showed no increased expression during the observation. The Cygb mRNA decreased after 24 hours. The highest expressions of HIF-1α and HIF-2α proteins were detected at 72 hours, whereas the Cygb protein expression increased since 24 hours. The PGC-1α protein expression increased at 72 hours. The WST test showed the highest ROS level at 24 hours. CONCLUSIONS The macrophages were activated by SRBCs underwent dual hypoxia and oxidative stress condition simultaneously to overcome the foreign bodies. The macrophages overcame these stress conditions by increasing their aerobic metabolism. [ABSTRACT FROM AUTHOR]

Background: FoxM1 plays important regulatory roles in a variety of diseases. However, the functional role of FoxM1 and mechanisms responsible for its expression in gastrointestinal stromal tumor (GIST) is not thoroughly understood.Methods: FoxM1 protein expression and biological function were examined in human GIST tissues and cells using immunohistochemistry, quantitative real-time PCR, western blot, CCK-8, wound-healing- and Matrigel invasion assays, respectively. The role of hypoxia-inducible factor (HIF) signaling in FoxM1 expression was investigated using chromatin immunoprecipitation and luciferase reporter and in vivo tumor growth assays.Results: FoxM1 was highly expressed in highly proliferative and migratory/invasive GIST specimens. Upregulation of FoxM1 was positively correlated with the expression of HIF-1α and HIF-2α in GIST specimens, and hypoxia-induced FoxM1 expression in GIST cells. Functionally, ectopic expression of FoxM1 significantly promoted GIST cell proliferation, cell cycle progression, migration and invasion, whereas the knockdown of endogenous FoxM1 of hypoxic GIST cells had the opposite effects. Molecularly, FoxM1 was transcriptionally regulated by HIF-2α under normoxia, whereas it was upregulated by both HIF-1α and HIF-2α under hypoxia. The xenograft tumor data further confirmed the regulated effect of HIF-1α and HIF-2α on FoxM1, and demonstrated that the simultaneous downregulation of both HIF-1α and HIF-2α inhibited GIST tumor growth.Conclusions: Our data demonstrated the critical role of FoxM1 in promoting GIST progression and uncovered a novel HIF-1α/HIF-2α-FoxM1 axis. These findings identify FoxM1 as a possible new molecular target for designing novel therapeutic treatments to control GIST progression. [ABSTRACT FROM AUTHOR]

Advanced breast cancer may aggressively progress under hypoxic conditions, leading to therapy resistance and metastasis, and the effects of neoadjuvant therapy on tumor hypoxia remain unclear. The current study aimed to evaluate the expressions of hypoxia-inducible factor-1 alpha (HIF-1α) and hypoxia-inducible factor-2 alpha (HIF-2α) in advanced breast cancer cells following neoadjuvant treatment and their correlation with clinical characteristics. In total, 48 breast cancer specimens were collected before and after neoadjuvant therapy (28 treated with chemotherapy, 20 with hormonal therapy). Total RNA was isolated from 50 mg specimens. The mRNA levels of HIF-1α and HIF-2α were measured using qRT-PCR. The 18S rRNA was used as housekeeping gene. This study demonstrates that HIF-2α mRNA expression was significantly lower than those before therapy, whereas that of HIF-1α remained unchanged. We categorized the data based on clinical characteristics, and found that the increased expression of HIF-1α was associated with reduced patient survival. Moreover, high expressions of HIF-1α and HIF-2α were found in patients aged >40 years having breast cancer of low histopathological grades. Thus, we conclude that high expressions of HIF-1α and HIF-2α may be used as biomarkers for the prediction of patient survival following neoadjuvant therapy in advanced breast cancer. [ABSTRACT FROM AUTHOR]

Background: Lymphocytes activated by immunization must increase their metabolism to meet the energy requirements for mitosis, differentiation, and protein synthesis, which may subject the cell to conditions of relative hypoxia and oxidative stress. This study was conducted to investigate the increase in the levels of transcription factors involved in both conditions. Methods: Male Balb/c mice were divided into the following four groups, each consisting of six animals: the control and three experimental groups. The experimental groups were immunized by injection of 0.2 ml of 2% sheep red blood cells (SRBC) suspended in phosphate-buffered saline (PBS). Lymphocytes were harvested from the spleens of each group at time intervals of 24-, 48-, and 72-h post-immunization. The buffy coat from splenocytes was separated using Ficoll Histopaque as the medium. The lymphocytes were separated from adherent cells by incubating the purified splenocytes in microtubes for 2-h. Cells were lysed by three freeze-thaw cycles (-80°C and 37°C) and used to analyze the levels of HIF-1α and HIF-2α (mRNA and protein), Nrf2 (protein), and glutathione peroxidase (GPx) activity. Results: The treatment caused an increase in GPx activity and HIF-1α protein concentration 24-h post-immunization, whereas the HIF-1α mRNA levels remained static. Elevated Nrf2 protein levels were detected within 48-h after treatment. Meanwhile, the HIF-2α mRNA and protein levels increased within72-h after immunization. Conclusion: Immunization with SRBC suspension induced relative hypoxia, elevated reactive oxygen species (ROS), and oxidative stress in the lymphocytes as indicated by the increase in both HIF-1α and HIF-2α protein and mRNA levels, GPx activity, and Nrf2 protein levels. [ABSTRACT FROM AUTHOR]

Abstract Tumor hypoxia is associated with several features of aggressive glioma growth, including migration, invasion, and stemness. Most of the cellular adaptation to hypoxia is mediated by the hypoxia-inducible factors HIF-1α and HIF-2α, but regulation of these factors by both oxygen-dependent and –independent mechanisms in brain tumors is only partially understood. Here, we show that the p75 neurotrophin receptor (p75NTR) is stabilized at hypoxia in murine glioma in vivo , as well as in primary human glioma cultures in vitro. Expression of p75NTR resulted in increased stabilization of HIF-1α and HIF-2α, and RNAi or pharmacologic targeting of p75NTR diminished HIF stabilization and HIF-dependent signaling at hypoxia. Consequentially, p75NTR inhibition resulted in decreased migration, invasion, and stemness in response to hypoxia, suggesting that p75NTR is a central regulator of hypoxia-induced glioma aggressiveness. Together, our findings support the literature that identifies p75NTR as a potential therapeutic target in brain tumors. Highlights • p75NTR levels are increased in hypoxic glioma cells in vivo and in vitro. • p75NTR contributes to HIF-1 and HIF-2 stability at hypoxia. • Hypoxia-induced glioma stemness and migration depends in part on p75NTR. [ABSTRACT FROM AUTHOR]

Background/Aims: Liver disease is a leading cause of high mortality and morbidity worldwide. The aim of the present study is to investigate the regulatory role of prolyl hydroxylase-2 (PHD2)-hypoxia-inducible factor-2a (HIF-2α) axis on nonalcoholic fatty liver disease (NAFLD) and to explore the potential mechanisms by which endothelial (EC)-specific PHD2 deficiency regulates hepatic steatosis and fibrosis. Methods: In the endothelial-specific PHD2 knockout (PHD2ECKO) mouse fed with normal diet or high fat diet (HFD), liver lipid accumulation and fibrosis were measured by Oil Red O and Masson trichrome staining. The fat and body weight (FW/BW) ratio and glucose tolerance were measured. The expression of HIF-2α, atrial natriuretic peptide (ANP), angiopoietin-2 (Ang-2), and transforming growth factor-b (TGF-β) were analyzed by western blot analysis. Results: The steatosis and fibrosis were significantly increased in the PHD2ECKO mice. FW/BW ratio was significantly increased in the PHD2ECKO mice. Moreover, knockout of endothelial PHD2 resulted in an impairment of glucose tolerance in mice. Western blot analysis showed that the expression of HIF-2α in liver tissues was not significantly increased. Interestingly, the expression of ANP was decreased, and Ang-2 and TGF-β levels were significantly increased in the liver of PHD2ECKO mice. The FW/BW ratio was also significantly increased in the PHD2ECKO mice fed with HFD for 16 weeks. Feeding HFD resulted in a significant increase in hepatic steatosis in the control PHD2f/f mice, but did not further enhance hepatic steatosis in the PHD2ECKO mice. Conclusions: We concluded that the endothelial PHD2 plays a critical role in hepatic steatosis and fibrosis, which may be involved in the regulation of ANP and Ang-2/TGF-β signaling pathway, but not the HIF-2α expression. [ABSTRACT FROM AUTHOR]

Background/Aims: Although some studies showed that HIF-2α expression was correlated with an unfavorable prognosis in colorectal cancer (CRC), the prognostic results remain conflicting in CRC. The present study was performed to evaluate the association between HIF-2α expression and the clinicopathological features of this disease and to examine the potential prognostic role of HIF-2α expression in CRC. Methods: Pooled odds ratios (ORs) or hazard ratios (HRs) were calculated from available publications, The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) datasets. Trial sequential analysis (TSA) was used to estimate the required sample information. Results: HIF-2α protein expression was more frequent in CRC than in normal colonic tissues (OR = 150.49, P < 0.001), higher in male than female CRC patients (OR = 1.47, P = 0.008), and lower in high-grade than low-grade CRC (OR = 0.49, P = 0.029). TSA verified the reliability of the above results. HIF-2α expression was not linked to the prognosis of CRC in overall survival (OS), disease-specific survival (DSS), metastasis-free survival, and relapse-free survival, and no significant correlation was found between HIF-2α alteration and OS or disease-free survival (DFS) of CRC. Expression of both HIF-2α and vascular endothelial growth factor (VEGFA, VEGFB, or VEGFC) was associated with a poor metastasis-free survival of CRC (HR = 6.95, HR = 113.51, and HR = 8.11, respectively). No association was observed between HIF-2α expression and DFS in other cancers, but HIF-2α expression was correlated with a worse DFS of CRC (HR = 1.23, P = 0.037). Moreover, HIF-2α expression was linked to a good survival benefit in some cancers (B-cell lymphoma and lung adenocarcinoma: OS, multiple myeloma: DSS, breast cancer: distant metastasis-free survival, liposarcoma: distant recurrence-free survival) (all HRs < 1, Ps < 0.05). Conclusions: HIF-2α expression may be associated with the carcinogenesis of CRC, which is higher in males than in females, negatively linked to tumor differentiation, and correlated with a worse DFS of CRC. Additional prospective studies are needed. [ABSTRACT FROM AUTHOR]

Presence of perivascular neuroblastoma cells with high expression of hypoxia inducible factor (HIF)-2α correlates with distant metastasis and aggressive disease. Regulation of HIFs are traditionally considered to occur post-translationally, but we have recently shown that HIF-2α is unconventionally regulated also at the transcriptional level in neuroblastoma cells. Regulatory factors binding directly to EPAS1 (encoding HIF-2α) to promote transcription are yet to be defined. Here, we employ the novel CRISPR/Cas9-based engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) – mass spectrometry (MS) methodology to, in an unbiased fashion, identify proteins that associate with the EPAS1 promoter under normoxic and hypoxic conditions. Our enChIP analysis resulted in 27 proteins binding to the EPAS1 promoter in neuroblastoma cells. In agreement with a general hypoxia-driven downregulation of gene transcription, the majority (24 out of 27) of proteins dissociate from the promoter at hypoxia. Among them were several nucleosome-associated proteins suggesting a general opening of chromatin as one explanation to induced EPAS1 transcription at hypoxia. Of particular interest from the list of released factors at hypoxia was the highly divergent homeobox (HDX) transcription factor, that we show inversely correlates with HIF-2α in neuroblastoma cells. We propose a putative model where HDX negatively regulates EPAS1 expression through a release-of-inhibition mechanism. [ABSTRACT FROM AUTHOR]

Recent studies indicate that erythropoietin (EPO) is present in many areas of the brain and is active in the restoration of impaired neurons. In this study, we examined the presence of EPO and its role in bulbospinal neurons in the rostral ventrolateral medulla (RVLM). Hypoxia is often accompanied by a high blood pressure (BP). We hypothesized that EPO is produced in response to hypoxia in RVLM neurons and then activates them. To investigate whether RVLM neurons are sensitive to EPO, we examined the changes in the membrane potentials (MPs) of bulbospinal RVLM neurons using the whole cell patch-clamp technique during superfusion with EPO. A brainstem-spinal cord preparation was used for the experiments. EPO depolarized the RVLM neurons, and soluble erythropoietin receptor (SEPOR), an antagonist of EPO, hyperpolarized them. Furthermore, hypoxia-depolarized RVLM neurons were significantly hyperpolarized by SEPOR. In histological examinations, the EPO-depolarized RVLM neurons showed the presence of EPO receptor (EPOR). The RVLM neurons that possessed EPORs showed the presence of EPO and hypoxia-inducible factor (HIF)-2α. We also examined the levels of HIF-2α and EPO messenger RNA (mRNA) in the ventral sites of the medullas (containing RVLM areas) in response to hypoxia. The levels of HIF-2α and EPO mRNA in the hypoxia group were significantly greater than those in the control group. These results suggest that EPO is produced in response to hypoxia in RVLM neurons and causes a high BP via the stimulation of those neurons. EPO may be one of the neurotransmitters produced by RVLM neurons during hypoxia. [ABSTRACT FROM AUTHOR]

Renal hypoxia is one of the currently highlighted pathophysiologic mechanisms of diabetic nephropathy (DN). Both hypoxia-inducible factor-1α (HIF-1α) and HIF-2α are major regulators of renal adaptive responses to hypoxia. This study aims to compare the effects of vildagliptin (a dipeptidyl peptidase-IV inhibitor, DPP-4i) and empagliflozin (a sodium-glucose cotransporter 2 inhibitor, SGLT2i) on the differential expression of renal HIF-1α/2α. Tissue expression of prolylhydroxylase 3 (PHD3), a key regulator of HIF-2α stability, was also highlighted in a diabetic nephropathy rat model. Type 1 diabetes mellitus was induced and diabetic rats were treated with either Vildagliptin or Empagliflozin (10 mg/kg/d each) for 12 weeks. Improvements in the kidney functional and histopathological parameters were addressed and correlated to changes in the renal expression of HIF-1α/2α, and PHD3. Urinary KIM-1 concentration was tested as a correlate to HIF pathway changes. Both vildagliptin- and empagliflozin-treated groups exhibited significant improvement in the functional, pathological, and ultra-structural renal changes induced by chronic diabetes. Compared to the untreated group, renal gene expression of HIF-1α was decreased while that of HIF-2α was increased in both treated groups, with significantly greater effects observed with SGLT2i. Renal PHD3 immune-reactivity was also decreased by both drugs, again with better efficacy for the SGLT2i. Importantly, improvements in the diabetic kidney biochemical and structural biomarkers were significantly correlated to PHD3 reductions and HIF-2α increments. Both DPP-4i and SGLT2i could delay the progression of DN through their differential modulating effects on the PHD3/ HIF-2α pathway with significantly better efficacy for SGLT2i. [Display omitted] • PHD3/HIF2α pathway represents an interesting target for preventing DN progression. • SGLT2i and DPP-4i can halt DN via differential modulation of PHD3/HIF2α pathway. • SGLT2i demonstrated better efficacy, especially on up-regulating HIF2α expression. [ABSTRACT FROM AUTHOR]

Background Immunotherapy is a standard-of-care first-line treatment for advanced clear cell renal cell carcinoma (ccRCC). However, many patients will develop resistance to first-line therapy, and effective second- and later-line treatment options are therefore needed. The von Hippel-Lindau (VHL) gene is inactivated in approximately 90% of patients with RCC and results in the constitutive activation of hypoxia-inducible factor 2α (HIF-2α), a key oncogenic driver in RCC. Belzutifan, a first-in-class HIF-2α inhibitor, has demonstrated antitumor activity with manageable safety in previously treated patients with advanced ccRCC. The cyclin-dependent kinase (CDK) pathway is also associated with poor clinical outcomes in ccRCC. In RCC cell lines, the CDK 4/6 inhibitor palbociclib inhibited cell growth. CDK 4/6 inhibition has shown synergistic antiproliferative effects with HIF-2α inhibition in HIF-2α–dependent VHL -/- RCC cell lines. Palbociclib could therefore potentially enhance the efficacy of belzutifan as combination therapy for previously treated pts with advanced RCC. Methods LITESPARK-024 is a 2-part, open-label, multicenter, phase 1/2 randomized study (NCT05468697). Part 1 is intended to establish the recommended phase 2 dose (RP2D) of belzutifan plus palbociclib using a modified toxicity probability interval design. After the RP2D is established, part 2 will directly compare the safety and efficacy of belzutifan monotherapy with belzutifan + palbociclib in patients with advanced ccRCC. In both parts, patients with measurable disease per RECIST v1.1, a Karnofsky Performance Status score of ≥70%, and histologically confirmed unresectable stage IV RCC with a clear cell component and disease progressing on or after receiving at least 2 systemic treatments (both an anti–PD-1/L1 monoclonal antibody and a VEGF receptor–targeted tyrosine kinase inhibitor, in sequence or in combination) will be enrolled. Up to 30 patients will be enrolled into 3 dose groups in part 1 and will receive belzutifan 120 mg once daily plus palbociclib (75, 100, or 125 mg) daily for 21 consecutive days followed by 7 days off. In part 2, approximately 150 patients will be randomly assigned 2:1 to receive belzutifan 120 mg once daily plus palbociclib RP2D (21 consecutive days/7 days off) or belzutifan 120 mg once daily. Patients will be stratified by International metastatic RCC Database Consortium risk (0 vs 1-2 vs 3-6) and sarcomatoid histology (yes vs no) at randomization in part 2. The primary end point for part 1 is to assess dose-limiting toxicities and adverse events and to determine the RP2D of belzutifan plus palbociclib. The primary end point for part 2 is objective response rate per RECIST v1.1 by investigator assessment. Secondary end points for part 2 are clinical benefit rate, duration of response, and progression-free survival per RECIST v1.1 by investigator assessment; overall survival, and safety and tolerability. Enrollment began in July 2022. ©2023 American Society of Clinical Oncology, Inc. Reused with permission. This abstract was accepted and previously presented at the 2023 ASCO Genitourinary Cancers Symposium. All rights reserved. CDMRP DOD Funding: no [ABSTRACT FROM AUTHOR]

ObjectiveThe development of breast cancer cells is linked to hypoxia. The hypoxia-induced factor HIF-1α influences metastasis through neovascularization. Hypoxia seems to decrease the responsiveness to hormonal treatment due to loss of estrogen receptors (ERs). Obesity is discussed to increase hypoxia in adipocytes, which promotes a favorable environment for tumor cells in mammary fat tissue, whereas, tumor cells profit from good oxygen supply and are influenced by its deprivation as target regions within tumors show. This review gives an overview of the current state on research of hypoxia and breast cancer in human adipose tissue.MethodsA systematic literature search was conducted on PubMed (2000–2016) by applying hypoxia and/or adipocytes and breast cancer as keywords. Review articles were excluded as well as languages other than English or German. There was no restriction regarding the study design or type of breast cancer. A total of 35 papers were found. Eight studies were excluded due to missing at least two of the three keywords. One paper was removed due to Russian language, and one was dismissed due to lack of adherence. Seven papers were identified as reviews. After applying exclusion criteria, 18 articles were eligible for inclusion.ResultsTwo articles describe the impairment of mammary epithelial cell polarization through hypoxic preconditioning. A high amount of adipocytes enhances cancer progression due to the increased expression of HIF-1α which causes the loss of ER α protein as stated in four articles. Four articles analyzed that increased activation of HIF’s induces a series of transcriptions resulting in tumor angiogenesis. HIF inhibition, especially when combined with cytotoxic chemotherapy, holds strong potential for tumor suppression as stated in further four articles. In two articles there is evidence of a strong connection between hypoxia, oxidative stress and a poor prognosis for breast cancer via HIF regulated pathways. Acute hypoxia seems to normalize the microenvironment in breast cancer tissue and has proven to affect tumor growth positively as covered in two articles.ConclusionThis review indicates that the development of breast cancer is influenced by hypoxia. A high amount of adipocytes enhances cancer progression due to the increased expression of HIF-1α.

Background: Hypoxia is a risk factor for non-alcoholic fatty liver diseases, leading to permanent imbalance of liver lipid homeostasis and steatohepatitis. The current study examined the effect of HIF-2α, an oxygen-sensitive heterodimeric transcription factor, on hypoxia-induced dysregulation of lipid metabolism in HepG2 cells. Methods: Studies were conducted in C57BL/6 male mice and human HepG2 cells under hypoxic conditions, transfected with HIF-2α-targeted shRNA. The mRNA and protein expressions of key genes relevant to lipid metabolism were determined via RT-qPCR and western blot, respectively. Intracellular lipid accumulation was determined by Nile red, filipin staining and quantitative assay kits. Results: HIF-2α protein was quantified in both HepG2 cells and C57BL/6 mice under hypoxic conditions. Intracellular lipid accumulation and increased lipid levels induced by hypoxia were significantly reduced by silence of HIF-2α expression, associated with reversed expression of ABCA1 and ADRP, key genes in involved cholesterol excretion and fatty acid uptake respectively. However, HIF-2α had no effect on enzymatic activity and expression of key genes involved in fatty acid β-oxidation or cholesterol metabolism. Conclusion: Inhibition of HIF-2α protein reversed lipid metabolism dysregulation induced by acute hypoxia in HepG2 cells, which suggested that HIF-2α signaling may be relevant to oxygen-dependent lipid homeostasis in the liver.

Respiratory distress syndrome (RDS) caused by preterm delivery is a major clinical problem with limited mechanistic insight. Late-stage embryonic lung development is driven by hypoxia and the hypoxia-inducible transcription factors Hif-1α and Hif-2α, which act as important regulators for lung development. Expression of the BTB-and kelch-domain-containing (BTB-kelch) protein KLEIP (Kelch-like ECT2-interacting protein; also named Klhl20) is controlled by two hypoxia response elements, and KLEIP regulates stabilization and transcriptional activation of Hif-2α. Based on the available data, we hypothesized an essential role for KLEIP in murine lung development and function. Therefore, we have performed a functional, histological, mechanistic and interventional study in embryonic and neonatal KLEIP−/− mice. Here, we show that about half of the KLEIP−/− neonates die due to respiratory failure that is caused by insufficient aeration, reduced septal thinning, reduced glycogenolysis, type II pneumocyte immaturity and reduced surfactant production. Expression analyses in embryonic day (E) 18.5 lungs identified KLEIP in lung capillaries, and showed strongly reduced mRNA and protein levels for Hif-2α and VEGF; such reduced levels are associated with embryonic endothelial cell apoptosis and lung bleedings. Betamethasone injection in pregnant females prevented respiratory failure in KLEIP−/− neonates, normalized lung maturation, vascularization, aeration and function, and increased neonatal Hif-2α expression. Thus, the experimental study shows that respiratory failure in KLEIP−/− neonates is determined by insufficient angiocrine Hif-2α–VEGF signaling and that betamethasone activates this newly identified signaling cascade in late-stage embryonic lung development.

Oxygen homeostasis in normal and tumor cells is mediated by hypoxia-inducible factors (HIFs), which are active as heterodimer complexes, such as HIF-2α–aryl hydrocarbon receptor nuclear translocator (ARNT) and HIF-1α–ARNT. A series of mutations on the interfaces between HIF-2α and ARNT and on the domain–domain interface within HIF-2α has been reported to exert varying effects on HIF-2α–ARNT dimerization. In the present study, molecular dynamic simulations were conducted to evaluate HIF-2α mutations, namely R171A, V192D, and R171A/V192D, which are not involved in the interaction with ARNT but impede HIF-2α–ARNT dimerization. Our results indicate that these mutations induct local conformation leading to a shortened (by V192D) or widened (by R171A and R171A/V192D) Y91–E346 separation distance, where E346 and Y91 are located on the HIF-2α and interact with ARNT according to electrostatic and geometrical shape complementarity, respectively. [ABSTRACT FROM AUTHOR]

Hypoxia-inducible factor-2α ( HIF-2α) plays an important role in increasing cancer progression and distant metastasis in a variety of tumour types. We aimed to investigate its biological function and clinical significance in human pancreatic ductal adenocarcinoma ( PDAC). A total of 283 paired PDAC tissues and adjacent normal tissues were collected from patients who underwent surgery or biopsy at Sun Yat-sen Memorial Hospital between February 2004 and October 2016. In this study, we noted that HIF-2α expression was significantly up-regulated in PDAC, positively associated with disease stage, lymph-node metastasis and patient survival, and identified as an independent prognostic factor of PDAC patients. We demonstrated that HIF-2α silencing could reduce proliferation, migration and invasion of PDAC cells in vitro. The similar effect on growth was demonstrated in vivo. Furthermore, we noted that knock-down of HIF-2α significantly decreased the expression of glutamate oxaloacetate transaminase 1 ( GOT1). Importantly, we confirmed that the PI3K/ mTORC2 pathway promoted GOT1 expression by targeting HIF-2α. Our study validated HIF-2α was an important factor in PDAC progression and poor prognosis and may promote non-canonical glutamine metabolism via activation of PI3K/ mTORC2 pathway. Targeting HIF-2α represents a novel prognostic biomarker and therapeutic target for patients with PDAC. [ABSTRACT FROM AUTHOR]

Background: Cells adapt to hypoxia by transcriptional induction of genes that participate in regulation of angiogenesis, glucose metabolism and cell proliferation. The primary factors mediating cell response to low oxygen tension are hypoxia inducible factors (HIFs), oxygen-dependent transcription activators. The stability and activity of the α subunits of HIFs are controlled by hydroxylation reactions that require ascorbate as a cofactor. Therefore, deficiency of intracellular vitamin C could contribute to HIFs overactivation. In this study, we investigated whether vitamin C content of human thyroid lesions is associated with HIF-1α and HIF-2α protein levels. Methods: Expression of HIF-1α and HIF-2α as well as vitamin C content was analyzed in thyroid lesions and cultured thyroid carcinoma cell lines (FTC-133 and 8305c) treated with hypoxia-mimetic agent (cobalt chloride) and ascorbic acid. The expression of HIFs and hypoxia–induced glucose transporters were determined by Western blots while quantitative real-time PCR (qRT-PCR) was performed to detect HIFs mRNA levels. Ascorbate and dehydroascorbate levels were measured by HPLC method. Results: We found an inverse correlation between vitamin C level and HIF-1α but not HIF-2α expression in thyroid lesions. These results agree with our in vitro study showing that vitamin C induced a dose - dependent decrease of HIF-1α but not HIF-2α protein level in thyroid cancer cells FTC-133 and 8305C. The decreased HIF-1α expression was correlated with reduced expression of hypoxia-related glucose transporter 1 (GLUT1) in thyroid cancer cells. Conclusion: The results demonstrate that HIF-1α activation is associated with vitamin C content in thyroid lesions. Our study suggests that high tumor tissue ascorbate level could limit the expression of HIF-1α and its targets in thyroid lesions. [ABSTRACT FROM AUTHOR]

Background Hypoxia-induced factors (HIF) has a role in angiogenesis and regulate tumorigenesis of cancer cell. The HIF is the best-identified mechanism that shows imbalance between consumption and oxygen supply in progressing tumor. This study of HIF-2α expression in oral squamous cell carcinoma (OSCC) aimed to investigate the relationship of HIF-2α and pathology characteristics related to its clinical correlation. Methods Fifty-eight samples of OSCC and adjacent tissues were fixed in paraffin for microarray preparation. The tissue array then was stained using primary antibody HIF-2α (NB100-122) and autoprobe II ABC universal staining kit. Each tissue sample was captured using camera microscope, and images were analyzed with Photoshop 6.0 using the CMYK method. A statistical analysis was performed with the two-tailed t -test, Kaplan–Meier and log-rank test using Prism for Windows version 5.0. Results The samples of the non-cancerous matched tissues (NCMTs) paired with their OSCC samples showed HIF-2α overexpression with significance difference p < 0.0001. Although no significant difference was found between HIF-2α expression and overall survival rate, cancer-specific survival rate, and disease-free survival rate, the HIF-2α expression showed statistical significance for overall cancer stages with p = 0.013. In addition, patients with high HIF-2α expression tended to develop recurrence within 2 years compared to the low expression group. Conclusion HIF-2 expression has complicated roles in different cancer types, including OSCC. Our study indicated that HIF-2α overexpression can serve as a good biomarker for cancer status for all tumor stages and may predict an early recurrence within two years. [ABSTRACT FROM AUTHOR]

Objective: The development of breast cancer cells is linked to hypoxia. The hypoxiainduced factor HIF-1α influences metastasis through neovascularization. Hypoxia seems to decrease the responsiveness to hormonal treatment due to loss of estrogen receptors (ERs). Obesity is discussed to increase hypoxia in adipocytes, which promotes a favorable environment for tumor cells in mammary fat tissue, whereas, tumor cells profit from good oxygen supply and are influenced by its deprivation as target regions within tumors show. This review gives an overview of the current state on research of hypoxia and breast cancer in human adipose tissue. Methods: A systematic literature search was conducted on PubMed (2000–2016) by applying hypoxia and/or adipocytes and breast cancer as keywords. Review articles were excluded as well as languages other than English or German. There was no restriction regarding the study design or type of breast cancer. A total of 35 papers were found. Eight studies were excluded due to missing at least two of the three keywords. One paper was removed due to Russian language, and one was dismissed due to lack of adherence. Seven papers were identified as reviews. After applying exclusion criteria, 18 articles were eligible for inclusion. Results: Two articles describe the impairment of mammary epithelial cell polarization through hypoxic preconditioning. A high amount of adipocytes enhances cancer progression due to the increased expression of HIF-1α which causes the loss of ER α protein as stated in four articles. Four articles analyzed that increased activation of HIF's induces a series of transcriptions resulting in tumor angiogenesis. HIF inhibition, especially when combined with cytotoxic chemotherapy, holds strong potential for tumor suppression as stated in further four articles. In two articles there is evidence of a strong connection between hypoxia, oxidative stress and a poor prognosis for breast cancer via HIF regulated pathways. Acute hypoxia seems to normalize the microenvironment in breast cancer tissue and has proven to affect tumor growth positively as covered in two articles. Conclusion: This review indicates that the development of breast cancer is influenced by hypoxia. A high amount of adipocytes enhances cancer progression due to the increased expression of HIF-1α. [ABSTRACT FROM AUTHOR]

Thyroid hormone plays an important role in brain development and adult brain function, and may influence neuronal recovery after Traumatic Brain Injury (TBI). We utilized both animal and cell culture models to determine the effects of thyroid hormone treatment, post TBI or during hypoxia, on genes important for neuronal survival and neurogenesis. We show that TBI in rats is associated with a reduction in serum thyroxine (T4) and triiodothyronine (T3). A single dose of levothyroxine (T4), one hour after injury, increased serum T4 and normalized serum T3 levels. Expression of genes important for thyroid hormone action in the brain, MCT8 and Type 2 deiodinase ( Dio2 ) mRNA, diminished after injury, but were partially restored with T4 treatment. mRNA from the Type 3 deiodinase ( Dio3 ) gene, which inactivates T4 to reverse T3 (rT3), was induced 2.7 fold by TBI, and further stimulated 6.7-fold by T4 treatment. T4 treatment significantly increased the expression of mRNA from Bcl2, VEGFA, Sox2 and neurotrophin , genes important for neuronal survival and recovery. The cortex, compared to the hippocampus and cerebellum, sustained the greatest injury and had the most significant change in gene expression as a result of injury and the greatest response to T4 treatment. We utilized hypoxia to study the effect of neuronal injury in vitro . Neuroblastoma cells were exposed to reduced oxygen tension, 0.2%, and were compared to cells grown at control oxygen levels of 21%. T3 treatment significantly increased hypoxia inducible factor (HIF)-2α protein, but not HIF-1α. In a hypoxia time course exposure, expression of hypoxia-mediated genes ( VEGF, Enolase, HIF2α, c-Jun ) peaked at least 8 h earlier with T3-treatment, compared to cells grown without T3. The early induction of these genes may promote cellular growth after injury. After hypoxic injury, T3 induced mRNA expression of the genes, KLF9 and hairless , important for T3-mediated brain function. The findings from both in vitro and in vivo studies support a role of thyroid hormone in activating pathways important for neuronal protection and promotion of neuronal recovery after injury. [ABSTRACT FROM AUTHOR]

Background Hypoxia is one of the most pervasive physiological stresses in solid tumors. We have previously demonstrated that tissue factor (TF) pathway inhibitor (TFPI) expression was transcriptionally repressed by the activation of hypoxia inducible factor (HIF)-1α under hypoxic conditions. However, the role of HIF-2α, also known as endothelial PAS domain-containing protein 1 (EPAS1), on TFPI expression remains unclear. Aim To explore the role of HIF-2α/EPAS1 in the regulation of TFPI expression under hypoxia in breast cancer cells. Methods and results Quantitative RT-PCR showed that total TFPI mRNA and protein levels were decreased by the overexpression of HIF-2α/EPAS1 in MCF7 cells. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay indicated a HIF-2α/EPAS1 responsive region located in the TFPI promoter region at − 170 to + 21 relative to the transcriptional start site. Subsequent mutagenesis demonstrated a functional hypoxia response element (HRE) 5′-AAACAGGA-3′ for HIF-2α/EPAS1 within the TFPI promoter located at − 45 to − 38. In breast cancer patients, a positive correlation between HIF-2α/EPAS1 and total TFPI mRNA expression was observed by using gene expression analysis. Conclusions This study provides evidence that HIF-2α/EPAS1 is involved in the regulation of TFPI gene expression in breast cancer cells, suggesting that the activation of coagulation and the increased risk of thrombosis observed in breast cancer patients may correlate with local hypoxic regulation of coagulation factors and their inhibitors. [ABSTRACT FROM AUTHOR]

Summary Hypoxia-inducible factors enhance glioma stemness, and glioma stem cells have an amplified hypoxic response despite residing within a perivascular niche. Still, little is known about differential HIF regulation in stem versus bulk glioma cells. We show that the intracellular domain of stem cell marker CD44 (CD44ICD) is released at hypoxia, binds HIF-2α (but not HIF-1α), enhances HIF target gene activation, and is required for hypoxia-induced stemness in glioma. In a glioma mouse model, CD44 was restricted to hypoxic and perivascular tumor regions, and in human glioma, a hypoxia signature correlated with CD44 . The CD44ICD was sufficient to induce hypoxic signaling at perivascular oxygen tensions, and blocking CD44 cleavage decreased HIF-2α stabilization in CD44-expressing cells. Our data indicate that the stem cell marker CD44 modulates the hypoxic response of glioma cells and that the pseudo-hypoxic phenotype of stem-like glioma cells is achieved by stabilization of HIF-2α through interaction with CD44, independently of oxygen. [ABSTRACT FROM AUTHOR]

Metastasis is a serious risk that may occur during the treatment of hepatocellular carcinoma ( HCC), preventing many patients from being surgical candidates and contributing to poor prognosis. Hypoxia has been proved an important factor of metastasis through the epithelial-mesenchymal transition ( EMT) pathway. Acetyl-CoA synthase 2 ( ACSS2) provides an acetyl group for the acetylation of hypoxia-inducible factor ( HIF)-2α, and this epigenetic modification affects the activity of HIF-2α and the subsequent EMT process. Here, we showed that ACSS2 expression was negatively correlated with HCC malignancy. Knockdown of ACSS2 increased the invasion and migration ability of HCC cells and promoted EMT without increasing the total protein level of HIF-2α, even in hypoxic conditions. The immunoprecipitation assay revealed downregulated acetylation levels of HIF-2α after ACSS2 knockdown in hypoxic conditions, which resulted in enhanced HIF-2α activity. Finally, decreased expression of ACSS2 was found to be related to advanced stage and poor overall survival and disease-free survival rates in a cohort of patients with HCC. In conclusion, ACSS2 plays an important role in the acetylation process of HIF-2α, which effectively modifies the activity of HIF-2α under hypoxic conditions and greatly impacts on the prognosis of patients with HCC. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is the most common joint disease, and in recent years has become a major public health problem. The hall-mark of OA is cartilage destruction with local commitment of subchondral bone and the synovial membrane. Hypoxia-inducible factors (HIFs) are transcriptional factors and key regulators of the cellular response to hypoxia. To date, three members of the human HIF-α protein family have been described: HIF-1α, HIF-2α, and HIF-3α. HIF-1α plays an essential role in the articular cartilage (a hypoxic tissue), as it has a protective effect in the maintenance of the articular cartilage matrix, HIF-2α has a harmful effect on the articular cartilage matrix, and HIF-3α acts as a negative regulator of HIF-1α and HIF-2α. Due to the recent growing interest in the role of HIFs in rheumatic diseases, we focused this review on the potential role of these key regulators in articular cartilage maintenance as the central axis in OA development. [ABSTRACT FROM AUTHOR]

Objective To study the protein expression of HIF-2α in condylar chondrocytes under the different stress loading, to investigate the possible effects of HIF-2α involved in the mortality of condylar chondrocytes under overloaded- stress. Materials and methods Chondrocytes were isolated from TMJ condylar cartilage and cultured in hypoxia-incubator. Chondrocytes were divided into 4 groups: 0, 1000, 2000, 3000 ustrain group, which was subjected to cyclic tensile strain (CTS) of 0.5 Hz for 2 h. The rate of cell mortality was calculated. Western blot was used to measure the expression of HIF-2α and it’s downstream catabolic factors (MMP3, MMP13, ADAMTS4) in protein levels respectively. Results With the increase of CTS, both of the rate of cell mortality and protein expression of HIF-2α increased significantly (p < 0.05). The same tendency was also found in it’s downstream catabolic factors (MMP3, MMP13, ADAMTS4) in protein levels (p < 0.05). Conclusions The results indicated that elevated expression of HIF-2α may be a possible mechanism related to overloaded- stress induced mortality of condylar chondrocytes. [ABSTRACT FROM AUTHOR]

The present study explored the mechanism of hypoxia-inducible factor (HIF)-2α in proliferation and apoptosis of the osteosarcoma cell line, MG-63. Cells were treated with small interfering RNA (siRNA) against HIF-2α (silenced group) or without siRNA (control group). Cell viability of MG-63 in the silenced and the control groups was determined by MTT assay; cell apoptosis was measured by flow cytometry; the expression of HIF-2α and mitogen-activated protein kinase (MAPK)-p38 were measured by western blotting. According to MTT assay, 48 h after siRNA transfection, compared with the control group, cells in the silenced group significantly declined in quantity and the number of apoptotic cells increased significantly. The expression of HIF-2α and MAPK-p38 were significantly decreased (P<0.05). In conclusion, knockdown of HIF-2α in the osteosarcoma cell line reduced the proliferation of cancer cells and increased apoptosis. These effects likely occurred through the MAPK-p38 signaling pathway. [ABSTRACT FROM AUTHOR]

Inflammation is a significant risk factor for colon cancer. Recent work has demonstrated essential roles for several infiltrating immune populations in the metaplastic progression following inflammation. Hypoxia and stabilization of hypoxiainducible factors (HIFs) are hallmark features of inflammation and solid tumors. Previously, we demonstrated an important role for tumor epithelial HIF-2α in colon tumors; however, the function of epithelial HIF-2α as a critical link in the progression of inflammation to cancer has not been elucidated. In colitis-associated colon cancer models, epithelial HIF-2α was essential in tumor growth. Concurrently, epithelial disruption of HIF-2α significantly decreased neutrophils in the colon tumor microenvironment. Intestinal epithelial HIF-2α-overexpressing mice demonstrated that neutrophil recruitment was a direct response to increased epithelial HIF-2α signaling. High-throughput RNA sequencing (RNA-seq) analysis of HIF-2α-overexpressing mice in conjunction with data mining from the Cancer Genome Atlas showed that the neutrophil chemokine CXCL1 gene was highly upregulated in colon tumor epithelium in a HIF-2α-dependent manner. Using selective peptide inhibitors of the CXCL1-CXCR2 signaling axis identified HIF-2α-dependent neutrophil recruitment as an essential mechanism to increase colon carcinogenesis. These studies demonstrate that HIF-2α is a novel regulator of neutrophil recruitment to colon tumors and that it is essential in shaping the protumorigenic inflammatory microenvironment in colon cancer. [ABSTRACT FROM AUTHOR]

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) isomerizes the nearby proline (Pro) residue when it detects phosphorylated serine (Ser) or threonine (Thr) of target proteins, altering their structure, stability, function, and interaction with other proteins. Hypoxia-inducible factor 2α (HIF-2α), a transcription factor that transactivates many oncogenic genes under hypoxic conditions, harbours the pSer/Thr-Pro motif. We found for the first time that Pin1 binds to HIF-2α physically in normoxic as well as hypoxic conditions in human breast cancer cells. The level of ubiquitinated HIF-2α was significantly raised by Pin1 knockdown, while expression of its mRNA transcript was unaffected. In agreement with this observation, the cycloheximide chase assay demonstrated that Pin1 prolonged the stability of HIF-2α. Serine 672, 696, and 790 of HIF-2α were found to undergo phosphorylation. Of these, the main amino acid involved in the Pin1 binding and HIF-2α stabilization was identified as serine 790, located in the nuclear export signal region of HIF-2α. The tissue array with human breast cancer specimens showed elevated expression of HIF-2α as well as Pin1 compared to adjacent normal tissues. Knockdown of Pin1 or HIF-2α diminished breast cancer cell migration and colony formation. In conclusion, Pin1 stabilizes HIF-2α through direct interaction, which contributes to the growth of breast cancer., Competing Interests: Declaration of competing interest The authors declare that they have no competing interest in relation to the submission and publication of this manuscript., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Glucocorticoids are common anti-inflammatory factors; however, they have been reported to have side effects that delay the wound healing process. In a previous study, we found that mesenchymal stem cells isolated from the adipose tissue of patients with long-term glucocorticoid treatment (sAT-MSC) showed impaired wound healing ability due to the downregulation of SDF-1. In this study, we aimed to clarify the mechanisms by which SDF-1 is regulated in sAT-MSC by focusing on the roles of hypoxia-inducible factors (HIFs). Our data suggested that sAT-MSC showed impairment of HIF-1α and the upregulation of HIF-2α. Notably, HIF-2α impairment resulted in the compensatory overexpression of HIF-1α and its target gene SDF-1, which improved the wound healing ability of sAT-MSC. In addition, using knockdown/knockout heterozygous HIF-2α kd/null mice (kd/null), the functions of HIF-2α in the ischemic wound healing process were clarified. With a 50% reduction in the expression of HIF-2α, kd/null mice showed significantly induced wound healing effects, which are involved in the promotion of the inflammatory phase. Specifically, kd/null mice showed the compensatory overexpression of HIF-1α, which upregulated the expression of SDF-1 and enhanced the recruitment of inflammatory cells, such as neutrophils. Our study highlighted the novel function of HIF-2α in the inflammation phase of the wound healing process through the HIF-1α/SDF-1 axis, suggesting that the physiological state of the impaired expression of HIF-2α is a new concept for wound therapy.

Nuclear speckles are membrane-less bodies within the cell nucleus enriched in RNA biogenesis, processing, and export factors. In this study we investigated speckle phenotype variation in human cancer, finding a reproducible speckle signature, based on RNA expression of speckle-resident proteins, across >20 cancer types. Of these, clear cell renal cell carcinoma (ccRCC) exhibited a clear correlation between the presence of this speckle expression signature, imaging-based speckle phenotype, and clinical outcomes. ccRCC is typified by hyperactivation of the HIF-2α transcription factor, and we demonstrate here that HIF-2α drives physical association of a select subset of its target genes with nuclear speckles. Disruption of HIF-2α-driven speckle association via deletion of its speckle targeting motifs (STMs)-defined in this study-led to defective induction of speckle-associating HIF-2α target genes without impacting non-speckle-associating HIF-2α target genes. We further identify the RNA export complex, TREX, as being specifically altered in speckle signature, and knockdown of key TREX component, ALYREF, also compromises speckle-associated gene expression. By integrating tissue culture functional studies with tumor genomic and imaging analysis, we show that HIF-2α gene regulatory programs are impacted by specific manipulation of speckle phenotype and by abrogation of speckle targeting abilities of HIF-2α. These findings suggest that, in ccRCC, a key biological function of nuclear speckles is to modulate expression of a specific subset of HIF-2α-regulated target genes that, in turn, influence patient outcomes. We also identify STMs in other transcription factors, suggesting that DNA-speckle targeting may be a general mechanism of gene regulation., Competing Interests: DECLARATION OF INTERESTS A.R. receives royalties from LGC/Biosearch Technologies related to Stellaris RNA-FISH.

Background: Aberrant interplay between epigenetic reprogramming and hypoxia signaling contributes to renal cell carcinoma progression and drug resistance, which is an essential hallmark. How the chromatin remodelers enhance RCC malignancy remains to be poorly understood. We aimed to elucidate the roles of CHD1L in determining hypoxia signaling activation and sunitinib resistance., Methods: The qRT-PCR, western blotting, and immunohistochemistry technologies were used to detect CHD1L expressions. Lentivirus transfection was used to generate stable CHD1L-KD cells. The roles of SIRT7/CHD1L were evaluated by CCK-8, wound healing, transwell assays, xenograft models, and tail-vein metastasis models. Co-immunoprecipitation, Chromatin Immunoprecipitation (ChIP), and luciferase reporter assays were conducted to explore epigenetic regulations., Results: We screened and validated that CHD1L is up-regulated in RCC and correlates with poorer prognosis of patients. CHD1L overexpression notably enhances cell proliferation, migration, and self-renewal capacities in vitro and in vivo. Mechanistically, SIRT7 physically interacts with CHDL1 and mediates the deacetylation of CHD1L. Wild-type SIRT7, but not H187Y dead mutant, stabilizes CHD1L protein levels via attenuating its ubiquitination levels. SIRT7 is increased in RCC and correlates with hazardous RCC clinical characteristics. SIRT7 depends on CHD1L to exert its tumor-promoting functions. Accumulated CHD1L amplifies HIF-2α-driven transcriptional programs via interacting with HIF-2α. CHD1L recruits BRD4 and increases the RNA polymerase II S2P loading. CHD1L ablation notably abolishes HIF-2α binding and subsequent transcriptional activation. CHD1L overexpression mediates the sunitinib resistance via sustaining VEGFA and targeting CHD1L reverses this effect. Specific CHD1L inhibitor (CHD1Li) shows a synergistic effect with sunitinib and strengthens its pharmaceutical effect., Conclusions: These results uncover a CHD1L-mediated epigenetic mechanism of HIF-2α activation and downstream sunitinib resistance. The SIRT7-CHD1L-HIF-2α axis is highlighted to predict RCC prognosis and endows potential targets., (© 2023. Society of Chinese Bioscientists in America (SCBA).)

Opinion Statement: Current treatment options for patients with metastatic renal cell carcinoma (mRCC) are limited to immunotherapy with checkpoint inhibitors and targeted therapies that inhibit the vascular endothelial growth factor receptors (VEFG-R) and the mammalian target of rapamycin (mTOR). Despite significantly improved outcomes over the last few decades, most patients with mRCC will ultimately develop resistance to these therapies, thus highlighting the critical need for novel treatment options. As part of the VHL-HIF-VEGF axis that rests at the foundation of RCC pathogenesis, hypoxia-inducible factor 2α (HIF-2α) has been identified as a rationale target for mRCC treatment. Indeed, one such agent (belzutifan) is already approved for VHL-associated RCC and other VHL-associated neoplasms. Early trials of belzutifan indicate encouraging efficacy and good tolerability in sporadic mRCC as well. The potential inclusion of belzutifan and other HIF-2α inhibitors into the mRCC treatment armamentarium either as a single agent or as combination therapy would be a welcome addition for patients with mRCC., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Summary TLX is known as the orphan nuclear receptor indispensable for maintaining neural stem cells in adult neurogenesis. We report here that neuroblastoma cell lines express high levels of TLX, which further increase in hypoxia to enhance the angiogenic capacity of these cells. The proangiogenetic activity of TLX appears to be induced by its direct binding to the von Hippel-Lindau protein (pVHL), which stabilizes TLX. In turn, TLX competes with hydroxylated hypoxia-inducible factor (HIF-α) for binding to pVHL, which contributes to the stabilization of HIF-2α in neuroblastoma during normoxia. Upon hypoxia, TLX increases in the nucleus where it binds in close proximity of the HIF-response element on the VEGF-promoter chromatin, and, together with HIF-2α, recruits RNA polymerase II to induce VEGF expression. Conversely, depletion of TLX by shRNA decreases the expression of HIF-2α and VEGF as well as the growth-promoting and colony-forming capacity of the neuroblastoma cell lines IMR-32 and SH-SY5Y. On the contrary, silencing HIF-2α will slightly increase TLX, suggesting that TLX acts to maintain a hypoxic environment when HIF-2α is decreasing. Our results demonstrate TLX to play a key role in controlling angiogenesis by regulating HIF-2α. TLX and pVHL might counterbalance each other in important fate decisions such as self-renewal and differentiation, as well as angiogenesis and anti-angiogenesis.

Background and objective CCR7 is closely related with the lymph node metastasis of non-small cell lung cancer. The objective of this work is to investigate the expressions of chemokine receptor CCR7, hypoxiainducible factor 1α (HIF-1α) and hypoxia inducible factor 2α (HIF-2α) protein in non small cell lung cancer and the relationships of their expression, and to study the mechanism of CCR7 upregulation in NSCLC. Methods T he levels of expressions of CCR7, HIF-1α and HIF-2α protein were detected in 94 specimens of human primary non small cell lung cancer by immunohistochemical S-P method. Human lung adenocarcinoma cell line A549 cells were transfected by lipofection with HIF-1α siRNA、HIF-2α siRNA, the change of CCR7 was observed by RT-PCR and immunofluorescence staining. Correlations between the expression of CCR7 and HIF-1α, HIF-2α were respectively analyzed. Results Immunohistochemistry showed that CCR7 was distributed in cytoplasm and/or membrane of tumor cells, HIF-1α, HIF-2α was distributed in nucleus and/or cytoplasm of tumor cells. The levels of expressions of CCR7, HIF-1α and HIF-2α protein were found to be 75.53% (71/94), 54.25% (51/ 94) and 70.21% (66/94) in non small celllung cancer, respectively. the levels of expression of CCR7 protein were closely related to the clinical stages (P 0.05). Furthermore, A significant correlation were found among CCR7, Hif-1α and HIF-2α (r =0.272, P

Cellular adaptation to hypoxia occurs via a complex programme of gene expression mediated by the hypoxia-inducible factor (HIF). The oxygen labile alpha subunits, HIF-1α/-2α, form a heterodimeric transcription factor with HIF-1β and modulate gene expression. HIF-1α and HIF-2α possess similar domain structure and bind to the same consensus sequence. However, they have different oxygen-dependent stability and activate distinct genes. To better understand these differences, we used fluorescent microscopy to determine precise localization and dynamics. We observed a homogeneous distribution of HIF-1α in the nucleus, while HIF-2α localized into speckles. We demonstrated that the number, size and mobility of HIF-2α speckles were independent of cellular oxygenation and that HIF-2α molecules were capable of exchanging between the speckles and nucleoplasm in an oxygen-independent manner. The concentration of HIF-2α into speckles may explain its increased stability compared with HIF-1α and its slower mobility may offer a mechanism for gene specificity.

[Display omitted] • Chemotherapy promotes the expression of VISTA in tumor cells. • HIF-2α mediates chemotherapy-induced VISTA expression. • VISTA overexpression in melanoma cells promotes immune evasion. • VISTA blockade enhances the therapeutic effect of carboplatin. Tumor cells can evade the innate and adaptive immune systems, which play important roles in tumor recurrence and metastasis. Malignant tumors that recur after chemotherapy are more aggressiveciscis, suggesting an increased ability of the surviving tumor cells to evade innate and adaptive immunity. Therefore, in order to reduce patient mortality, it is important to discover the mechanisms by which tumor cells develop resistance to chemotherapeutics. In the present study we focused on the tumor cells that survived chemotherapy. We found that chemotherapy could promote the expression of VISTA in tumor cells, and that this change was mediated by HIF-2α. In addition, VISTA overexpression on melanoma cells promoted immune evasion, and the application of the VISTA-blocking antibody 13F3 enhanced the therapeutic effect of carboplatin. These results offer an insight into the immune evasion of chemotherapy-resistant tumors, and provide a theoretical basis for the combined application of chemotherapy drugs and VISTA inhibitors to treat tumors. [ABSTRACT FROM AUTHOR]

This article is a summary of the plenary lecture presented by Jared Rosenblum that was awarded the Manger Prize at the Sixth International Symposium on Pheochromocytoma/Paraganglioma held on 19-22 October 2022 in Prague, Czech Republic. Herein, we review our initial identification of a new syndrome of multiple paragangliomas, somatostatinomas, and polycythemia caused by early postzygotic mosaic mutations in EPAS1, encoding hypoxia-inducible factor 2 alpha (HIF-2α), and our continued exploration of new disease phenotypes in this syndrome, including vascular malformations and neural tube defects. Continued recruitment and close monitoring of patients with this syndrome as well as the generation and study of a corresponding disease mouse model as afforded by the pheochromocytoma/paraganglioma translational program at the National Institutes of Health has provided new insights into the natural history of these developmental anomalies and the pathophysiologic role of HIF-2α. Further, these studies have highlighted the importance of the timing of genetic defects in the development of related disease phenotypes. The recent discovery and continued study of this syndrome has not only rapidly evolved our understanding of pheochromocytoma and paraganglioma but also deepened our understanding of other developmental tumor syndromes, heritable syndromes, and sporadic diseases.

Hypoxia-inducible factor-1 (HIF-1) is a critical transcription factor that regulates the expression of genes involved in cellular adaptation to low oxygen levels. Aberrant regulation of the HIF-1 signaling pathway is linked to various human diseases. Previous studies have established that HIF-1α is rapidly degraded in a von Hippel-Lindau protein (pVHL)-dependent manner under normoxic conditions. In this study, we find that pVHL binding protein 1 (VBP1) is a negative regulator of HIF-1α but not HIF-2α using zebrafish as an in vivo model and in vitro cell culture models. Deletion of vbp1 in zebrafish caused Hif-1α accumulation and upregulation of Hif target genes. Moreover, vbp1 was involved in the induction of hematopoietic stem cells (HSCs) under hypoxic conditions. However, VBP1 interacted with and promoted the degradation of HIF-1α in a pVHL-independent manner. Mechanistically, we identify the ubiquitin ligase CHIP and HSP70 as new VBP1 binding partners and demonstrate that VBP1 negatively regulated CHIP and facilitated CHIP-mediated degradation of HIF-1α. In patients with clear cell renal cell carcinoma (ccRCC), lower VBP1 expression was associated with worse survival outcomes. In conclusion, our results link VBP1 with CHIP stability and provide insights into underlying molecular mechanisms of HIF-1α-driven pathological processes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Hypoxia-inducible factors (HIFs) regulate the transcription of genes that mediate the response to hypoxia. HIFs are constantly expressed and degraded under normoxia, but stabilized under hypoxia. HIFs have been widely studied in physiological and pathological conditions and have been shown to contribute to the pathogenesis of various vascular diseases. In clinical settings, the HIF pathway has been studied for its role in inhibiting carcinogenesis. HIFs might also play a protective role in the pathology of ischemic diseases. Clinical trials of therapeutic angiogenesis after the administration of a single growth factor have yielded unsatisfactory or controversial results, possibly because the coordinated activity of different HIF-induced factors is necessary to induce mature vessel formation. Thus, manipulation of HIF activity to simultaneously induce a spectrum of angiogenic factors offers a superior strategy for therapeutic angiogenesis. Because HIF-2α plays an essential role in vascular remodeling, manipulation of HIF-2α is a promising approach to the treatment of ischemic diseases caused by arterial obstruction, where insufficient development of collateral vessels impedes effective therapy. eIF3e/INT6 interacts specifically with HIF-2α and induces the proteasome inhibitor-sensitive degradation of HIF-2α, independent of hypoxia and VHL. Treatment with eIF3e/INT6 siRNA stabilizes HIF-2α activity even under normoxic conditions and induces the expression of several angiogenic factors, at levels sufficient to produce functional arteries and veins in vivo. We have demonstrated that administration of eIF3e/INT6 siRNA to ischemic limbs or cold-injured brains reduces ischemic damage in animal models. This review summarizes the current understanding of the relationship between HIFs and vascular diseases. We also discuss novel oxygen-independent regulatory proteins that bind HIF-α and the implications of a new method for therapeutic angiogenesis using HIF stabilizers.

During prolonged hypoxic conditions, endothelial cells change their gene expression to adjust to the low oxygen environment. This process is mainly regulated by the hypoxia-inducible factors, HIF-1α and HIF-2α. Although endothelial cells do not form sprouts during prolonged hypoxic culturing, silencing of HIF-2α partially restores sprout formation. The present study identifies novel HIF-2α-target genes that may regulate endothelial sprouting during prolonged hypoxia. The gene expression profile of primary human microvascular endothelial cells (hMVECs) that were cultured at 20 % oxygen was compared to hMVECs that were cultured at 1 % oxygen for 14 days by using genome-wide RNA-sequencing. The differentially regulated genes in hypoxia were compared to the genes that were differentially regulated upon silencing of HIF-2α in hypoxia. Surprisingly, KEGG pathway analysis showed that metabolic pathways were enriched within genes upregulated in response to hypoxia and enriched within genes downregulated upon HIF-2α silencing. Moreover, 51 HIF-2α-regulated genes were screened for their role in endothelial sprouting in hypoxia, of which four genes ARRDC3, MME, PPARG and RALGPS2 directly influenced endothelial sprouting during prolonged hypoxic culturing. The manipulation of specific downstream targets of HIF-2α provides a new, but to be further evaluated, perspective for restoring reduced neovascularization in several pathological conditions, such as diabetic ulcers or other chronic wounds, for improvement of vascularization of implanted tissue-engineered scaffolds. [ABSTRACT FROM AUTHOR]