Identification of functional hypoxia inducible factor response elements in the human lysyl oxidase gene promoter.

Human lysyl oxidase (LOX) is a hypoxia-responsive gene whose product catalyzes collagen crosslinking and is thought to be important in cancer metastasis and osteoarthritis. We previously demonstrated that LOX was upregulated by hypoxia inducible factor 2 (HIF-2) more strongly than hypoxia inducible 1 (HIF-1). Here, we further investigated the response of the LOX gene and LOX promoter to HIFs. LOX mRNA, measured by real time reverse transcriptase-PCR, was strongly up-regulated (almost 40-fold), by transfection of HEK-293T cells with a plasmid encoding the HIF-2α subunit of HIF-2, but only three-fold by a plasmid encoding HIF-1α. LOX protein was detectable by Western blot of cells transfected with HIF-2α, but not with HIF-1α. Analysis of a 1487 bp promoter sequence upstream of the human LOX gene revealed 9 potential hypoxia response elements (HREs). Promoter truncation allowed the mapping of two previously unidentified functional HREs, called here HRE8 and HRE7; -455 to -451 and -382 to -386 bp, respectively, upstream of the start codon for LOX. Removal or mutation of these HREs led to a substantial reduction in both HIF-1α and HIF-2α responsiveness. Also, expression of LOX was significantly inhibited by a small molecule specific HIF-2 inhibitor. In conclusion, LOX is highly responsive to HIF-2α and this is largely mediated by two previously unidentified HREs. These observations enhance our understanding of the regulation of this important gene involved in cancer and osteoarthritis, and suggest that these conditions may be targeted by HIF-2 inhibitors.

Inactivation of the tumor suppressor gene von Hippel-Lindau (VHL) in granulocytes contributes to development of liver hemangiomas in a mouse model.

BACKGROUND: Mutations in the tumor suppressor gene von Hippel-Lindau (VHL) underlie a hereditary cancer syndrome-VHL disease-and are also frequently observed in sporadic renal cell carcinoma of the clear cell type (ccRCC). VHL disease is characterized by malignant and benign tumors in a few specific tissues, including ccRCC, hemangioblastoma and pheochromocytoma. The etiology of these tumors remains unresolved. METHODS: Conditional inactivation of the VHL gene in mouse (Vhlh) was generated to examine the pathophysiological role of the VHL gene function. Specific cell populations were isolated by fluorescence-activated cell sorting (FACS) and bone marrow transplants were performed to identify the Vhlh-inactivated cells responsible for the phenotype. RESULTS: Previously we showed that inactivation of Vhlh in a subpopulation of kidney distal tubule cells resulted in hyperplastic clear-cell lesions and severe inflammation and fibrosis. Here, we show that this knockout mouse strain also develops Hif-2α-dependent vascular overgrowth (hemangioma) and extramedullary erythropoiesis in the liver. However, Vhlh inactivation was not detected in the liver parenchyma. We instead demonstrate that in these mice, Vhlh is inactivated in liver granulocytes and that hemangiomas are partially rescued in knockout mice reconstituted with wild-type hematopoietic stem cells, indicating the involvement of bone-marrow-derived leukocyte. Interestingly, bone marrow from knockout mice failed to generate the liver phenotype in wild-type recipients, suggesting that an additional cell type that is not derived from the bone marrow is involved in the development of the hemangioma phenotype. CONCLUSION: These results support the idea that the development of a full-blown VHL disease phenotype requires inactivation of the VHL gene not only in the tumor proper, but also in the stromal compartment.

Belzutifan Efficacy and Tolerability in Patients with Sporadic Metastatic Clear Cell Renal Cell Carcinoma.

BACKGROUND AND OBJECTIVE: Belzutifan, a hypoxia-inducible factor 2 alpha inhibitor, was approved initially for patients with von Hippel-Lindau disease and more recently for sporadic, metastatic clear cell renal cell carcinoma (ccRCC) based on the results of LITESPARK-005. There is a paucity of data regarding real-world experience with belzutifan in patients with sporadic, metastatic ccRCC. This study aims to describe clinical outcomes with belzutifan in patients with sporadic, metastatic ccRCC. METHODS: A retrospective study of 22 patients who received belzutifan at MD Anderson Cancer Center prior to the Food and Drug Administration approval was conducted. Progression-free survival (PFS) and objective response rate (ORR) were assessed by a blinded radiologist using Response Evaluation Criteria In Solid Tumors (RECIST) version 1.1. PFS and overall survival (OS) were measured from belzutifan initiation. KEY FINDINGS AND LIMITATIONS: The median follow-up time was 14.9 mo. Most patients had International Metastatic RCC Database Consortium intermediate-risk disease, more than three metastatic sites, and a median of five prior lines of treatment at initiation of belzutifan; all patients received prior immune checkpoint therapy (ICT) and vascular endothelial growth factor receptor tyrosine kinase inhibitors (VEGFR-TKIs). The median PFS was 8.51 mo (95% confidence interval [CI] 0-18.4) and ORR was 36.4%. The median OS was 14.72 mo (95% CI 7.34-22.10). Of 22 patients, four (18.2%) patients required dose reductions and three (13.6%) patients discontinued belzutifan because of adverse drug events (ADEs). The most common ADEs were anemia (77.3%; 17/22) and hypoxia (36.4%; 8/22). There were no treatment-related deaths. CONCLUSIONS AND CLINICAL IMPLICATIONS: In a heavily pretreated cohort of patients with sporadic, metastatic ccRCC, belzutifan had meaningful clinical activity and was well tolerated. These real-world results add to the results of LITESPARK-005 and support the use of belzutifan after progression on ICT and VEGFR-TKIs. PATIENT SUMMARY: Belzutifan is a new medicine used to treat a type of clear cell kidney cancer that has spread to other parts of the body (metastasized). A study at MD Anderson Cancer Center followed 22 patients who were treated with belzutifan, and found that it worked to control the cancer for almost 9 mo and caused the cancer to shrink in 36% of patients. This study confirms that belzutifan can be effective and safe, even after other treatments have not worked.

High-Altitude Hypoxia Induces Excessive Erythrocytosis in Mice via Upregulation of the Intestinal HIF2a/Iron-Metabolism Pathway.

Excessive erythrocytosis (EE) is a preclinical form of chronic mountain sickness (CMS). The dysregulation of iron metabolism in high-altitude hypoxia may induce EE. The intestinal hypoxia-inducible factor 2 alpha (HIF2a) regulates the genes involved in iron metabolism. Considering these findings, we aimed to investigate the function and mechanism of intestinal HIF2α and the iron metabolism pathway in high-altitude EE mice. C57BL/6J mice were randomized into four groups: the low-altitude group, the high-altitude group, the high-altitude + HIF2α inhibitor group, and the high-altitude + vehicle group. In-vitro experiments were performed using the human intestinal cell line HCT116 cultured under hypoxic conditions for 24 h. Results showed that high-altitude hypoxia significantly increased the expression of intestinal HIF2α and iron metabolism-related genes, including Dmt1, Dcytb, Fpn, Tfrc, and Fth in EE mice. Genetic blockade of the intestinal HIF2α-iron metabolism pathway decreased iron availability in HCT116 cells during hypoxia. The HIF2α inhibitor PT2385 suppressed intestinal HIF2α expression, decreased iron hypermetabolism, and reduced excessive erythrocytosis in mice. These data support the hypothesis that exposure to high-altitude hypoxia can lead to iron hypermetabolism by activating intestinal HIF2α transcriptional regulation, and reduced iron availability improves EE by inhibiting intestinal HIF2α signaling.

Differential Expression of HIF1A, EPAS1, and VEGF Genes in Benign and Malignant Ovarian Neoplasia.

Ovarian cancer (OC) has the highest mortality rate of all gynecological malignancies. Moreover, at the time of the first clinical manifestation, most patients have an advanced stage of the disease. Our study examined differences in mRNA levels of hypoxia-inducible factor 1-alpha (HIF1A); endothelial PAS domain protein 1, also known as hypoxia-inducible factor 2-alpha (HIF2A/EPAS1); and vascular endothelial growth factor A (VEGFA) between cancerous tissue, benign hyperplastic changes in the ovary, and normal tissue. Our cohorts consisted of 52 patients diagnosed with OC (n = 55), benign non-cancerous changes (n = 21), and normal tissue samples (n = 38). The mRNA expression level was evaluated using RT-qPCR. We found that gene expression changes were visible not only in the case-control study, but also along with changes in severity. Additionally, the gene expression was differentiated in age, BMI, menopausal status, and the number of comorbidy-related groups. Furthermore, our findings demonstrate that analyzing the correlation between genes is essential. In a case-to-case and case-to-control study, we observed disturbances in the expression levels of interdependent genes. Our findings suggest that mutual association in the expression of both HIF1A and HIF2A/EPAS1 with VEGFA has prognostic importance for patients with OC. Our observations may help identify patients for clinical trials aimed at inhibiting the hypoxia-induced neovascularization-dependent pathways.

HIF2α promotes tumour growth in clear cell renal cell carcinoma by increasing the expression of NUDT1 to reduce oxidative stress.

BACKGROUND: The key role of hypoxia-inducible factor 2alpha (HIF2α) in the process of renal cancer has been confirmed. In the field of tumour research, oxidative stress is also considered to be an important influencing factor. However, the relationship and biological benefits of oxidative stress and HIF2α in ccRCC remain unclear. This research attempts to explore the effect of oxidative stress on the cancer-promoting effect of HIF2α in ccRCC and reveal its mechanism of action. METHODS: The bioinformatics analysis for ccRCC is based on whole transcriptome sequencing and TCGA database. The detection of the expression level of related molecules is realised by western blot and PCR. The expression of Nucleoside diphosphate-linked moiety X-type motif 1 (NUDT1) was knocked down by lentiviral infection technology. The functional role of NUDT1 were further investigated by CCK8 assays, transwell assays and cell oxidative stress indicator detection. The exploration of related molecular mechanisms is realised by Luciferase assays and Chromatin immunoprecipitation (ChIP) assays. RESULTS: Molecular screening based on knockdown HIF2α sequencing data and oxidative stress related data sets showed that NUDT1 is considered to be an important molecule for the interaction of HIF2α with oxidative stress. Subsequent experimental results showed that NUDT1 can cooperate with HIF2α to promote the progression of ccRCC. And this biological effect was found to be caused by the oxidative stress regulated by NUDT1. Mechanistically, HIF2α transcription activates the expression of NUDT1, thereby inhibiting oxidative stress and promoting the progression of ccRCC. CONCLUSIONS: This research clarified a novel mechanism by which HIF2α stabilises sirtuin 3 (SIRT3) through direct transcriptional activation of NUDT1, thereby inhibiting oxidative stress to promote the development of ccRCC. It provided the possibility for the selection of new therapeutic targets for ccRCC and the study of combination medication regimens.

Aging Reprograms the Hematopoietic-Vascular Niche to Impede Regeneration and Promote Fibrosis.

Regenerative capacity is frequently impaired in aged organs. Stress to aged organs often causes scar formation (fibrosis) at the expense of regeneration. It remains to be defined how hematopoietic and vascular cells contribute to aging-induced regeneration to fibrotic transition. Here, we find that aging aberrantly reprograms the crosstalk between hematopoietic and vascular cells to impede the regenerative capacity and enhance fibrosis. In aged lung, liver, and kidney, induction of Neuropilin-1/hypoxia-inducible-factor 2α (HIF2α) suppresses anti-thrombotic and anti-inflammatory endothelial protein C receptor (EPCR) pathway, leading to formation of pro-fibrotic platelet-macrophage rosette. Activated platelets via supplying interleukin 1α synergize with endothelial-produced angiocrine chemokine to recruit fibrogenic TIMP1high macrophages. In mouse models, genetic targeting of endothelial Neuropilin-1-HIF2α, platelet interleukin 1α, or macrophage TIMP1 normalized the pro-fibrotic hematopoietic-vascular niche and restored the regenerative capacity of old organs. Targeting of aberrant endothelial node molecules might help propel "regeneration without scarring" in the repair of multiple organs.

EGFR-AS1/HIF2A regulates the expression of FOXP3 to impact the cancer stemness of smoking-related non-small cell lung cancer.

BACKGROUND: Early data showed that FOXP3 could induce epithelial-mesenchymal transition by stimulating the Wnt/ß-catenin signaling pathway in non-small cell lung cancer (NSCLC). However, how the expression of FOXP3 is regulated in NSCLC remains unknown. We thus explored the impacts of the long noncoding RNA EGFR antisense RNA 1 (EGFR-AS1) and hypoxia-inducible factor-2A (HIF2A) on FOXP3 expression and the cancer stemness of NSCLC. METHODS: Lung tissues samples from 87 patients with NSCLC and two NSCLC cell lines were used in this study. The regulation of FOXP3 and lung cancer cell stemness by EGFR-AS1 and HIF2A was determined at molecular levels in NSCLC tissue samples and cultured cells in the presence/absence of the smoking carcinogen, 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (also known as nicotine-derived nitrosamine ketone). The results were confirmed in tumor xenograft models. RESULTS: We found that NNK decreased the expression of EGFR-AS1 in the long term, but increased the expression of HIF2A and FOXP3 to stimulate lung cancer cell stemness. EGFR-AS1 significantly inhibited FOXP3 expression and NSCLC cell stemness, whereas HIF2A obviously promoted both. The enhancement of lung cancer stemness by FOXP3 was, at least partially, via stimulating Notch1, as the inhibition of Notch1 could markedly diminish the effect of FOXP3. CONCLUSIONS: FOXP3, the expression of which is under the fine control of EGFR-AS1, is a critical molecule that promotes NSCLC cancer cell stemness through stimulating the Notch1 pathway.

Abundance of TRAIL attenuated by HIF2α and c-FLIP affects malignancy in renal cell carcinomas.

Dormant cancer cells are starvation-resistant leading to problems in the management of cancer. In renal cell carcinomas (RCCs), starvation-resistant cells are resistant to various currently available therapies. However, targeting hypoxia inducible factor 2-alpha (HIF2-alpha) induces cell death in dormant-like/starvation-resistant RCCs. This study showed that the apoptotic cell death caused by tumor necrosis factor (TNF)-related apoptosis-induced ligand (TNFSF10/TRAIL) was attenuated by CASP8 and FADD-like apoptosis regulator (CFLAR/c-FLIP) following HIF2-alpha activation, despite the high expression of TRAIL in such RCCs. Knockdowns of TRAIL averted apoptotic cell death caused by HIF2-alpha inhibition in starvation-resistant RCCs. Knockdowns of both HIF2-alpha and c-FLIP augmented apoptotic cell death, whereas overexpression of c-FLIP completely averted apoptosis. In addition, high abundance of TRAIL was correlated with poor prognosis in patients with RCC, suggesting that TRAIL, followed by HIF2-alpha and c-FLIP, play a role in the survival and/or progression of malignant RCCs.

microRNA-558 facilitates the expression of hypoxia-inducible factor 2 alpha through binding to 5'-untranslated region in neuroblastoma.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. Our previous studies have shown that hypoxia-inducible factor 2 alpha (HIF-2α), one member of the bHLH-PAS transcription factor family, facilitates the progression of NB under non-hypoxic conditions. However, the mechanisms underlying HIF-2α expression in NB still remain largely unknown. Herein, through analyzing the computational algorithm programs, we identified microRNA-558 (miR-558) as a crucial regulator of HIF-2α expression in NB. We demonstrated that miR-558 promoted the expression of HIF-2α at translational levels in NB cells through recruiting Argonaute 2 (AGO2). Mechanistically, miR-558 directly bound with its complementary site within 5'-untranslated region (5'-UTR) to facilitate the binding of AGO2 to eukaryotic translation initiation factor 4E (eIF4E) binding protein 1, resulting in increased eIF4E enrichment and HIF-2α translation. In addition, miR-558 promoted the growth, invasion, metastasis, and angiogenesis of NB cells in vitro and in vivo, and these biological features were rescued by knockdown of AGO2, eIF4E, or HIF-2α. In clinical NB specimens, miR-558, AGO2, and eIF4E were highly expressed and positively correlated with HIF-2α expression. Patients with high miR-558, HIF-2α, AGO2, or eIF4E levels had lower survival probability. Taken together, these results demonstrate that miR-558 facilitates the expression of HIF-2α through bindingto its 5'-UTR, thus promoting the tumorigenesis and aggressiveness of NB.

Effect of silencing of HIF-2α gene on chemosensitivity of human breast carcinoma cells / 临床与实验病理学杂志

Purpose To investigate the effects of hypoxia inducible factor-2α (HIF-2ot) siRNA on proliferation and chemotherapy sensitivity of breast carcinoma MCF-7.Methods RNA interference was used to silence the expression of HIF-2α in MCF-7 cells.The changes of HIF-2α gene expression were detected by immunocytochemistry and RT-PCR.Under hypoxia environment simulated by CoCl2,MTT assay and flow cytometry (FCM) were used to measure cell growth inhibition rate and cell apoptosis of MCF-7 cells under different dosages of chemotherapeutic agents (5-fluorouracil,adriamycin,and paclitaxel).Results Expression of HIF-2α in MCF-7 were down-regulated by HIF-2α siRNA (P ＜ 0.05).The proliferation inhibition and apoptosis rates were evidently increased after transfection with HIF-2α siRNA (P ＜ 0.05),chemotherapy drug sensitivity was enhanced.Conclusion HIF-2α siRNA can induce the apoptosis and inhibit the proliferation and enhance the sensitivity of breast carcinoma MCF-7 cell line to chemotherapeutic agents.Blocking HIF-2α maybe a very promising strategy for breast carcinoma gene therapy in combination with chemotherapy.

Effects of hypoxia-inducible factor -2α on the expression of tight junction proteins and permeability in rat glomerular endothelial cells under hypoxia condition / 中华肾脏病杂志

Objective To investigate the role of hypoxia?inducible factor?2α(HIF?2α) in the expression of tight junction proteins and permeability alterations in rat glomerular endothelial cells (rGENCs) under hypoxia condition. Methods The expressions of the HIF?2α and tight junction proteins such as occludin and ZO?1 of rGENCs were examined after exposed to 5%oxygen at different treatment time periods (0 h, 12 h, 24 h and 48 h). Then lentiviral transfection was used to knock down HIF?2α expression in rGENCs. The cells were split into four groups, including i) control group where rGENCs were cultured under normal oxygen conditions, ii) hypoxia group, iii) negative control group where rGENCs were infected with a negative vector, iv) HIF?2α lentivirus transfection group. Group ii, iii and iv were kept in hypoxic chamber (5% O2, 5% CO2 and 90% N2) for 24 h. The expressions of occludin, ZO?1 and HIF?2α were assessed by Western blotting. The permeability of rGENCs was measured using trans?epithelium electrical resistant (TEER) by Millicell? ERS voltohmmeter. Results With the elongation of hypoxia time, the expression of HIF?2α was increased gradually, while the occludin expression was decreased, there was statistically significance difference in each group (all P0.05). And a dramatic decrease in TEER of hypoxia cells was detected as compare with control cells (P0.05). Conclusion Hypoxia may promote HIF?2α expression, which could increase the permeability of rGENCs by reducing the expression of occludin and ZO?1.

Effect of Hypoxia-Inducible Factor(HIF)-2 Alpha Silencing on Osteosarcoma MG-63 Cells / 天津医药

Objective To investigate the effect of HIF-2a silencing by transfection of siRNA into MG-63 cells un-der hypoxia. Methods HIF-2αexpression level in MG-63 cells under hypoxia was determined by Western Blot. Small in-terfering RNA (siRNA) was used to construct MG-63/siHIF-2α(siHIF-2α)cells and control MG-63/scramble (NC) cells. The expression levels of HIF-2α, Vascular endothelial growth factor (VEGF), p-Erk/ErK and Mcl-1 in MG-63, NC and si-HIF-2αcells was determined by Western Blot. NC and siHIF-2αcells were cultured under hypoxia. Cell viability was as-sessed by MTT assay. Migration was identified by scratch migration assay. Tumor formation was identified by clone formation assay. Nude mouse subcutaneous xenograft model was used to investigate tumor development in vivo. Results Hypoxia im-proved HIF-2αexpression in MG-63 cells in a time-dependent manner (F=2 037.412,P<0.001). HIF-2αexpression un-der hypoxia in siHIF-2αcells was lower than that in NC cells (P<0.01). Cell viability of siHIF-2αcells under hypoxia for 12 h and 24 h were lower than that in NC cells (P<0.05 or P<0.01). The relative width of scratch in siHIF-2αgroup under hypoxia for 12 h and 24 h were larger than that in NC group (P<0.01 or P<0.01). When cell counts reach 1 000-5 000, the clone formation rates of siHIF-2αcells were lower than that in NC cells (P<0.05 or P<0.01). The expression of VEGF, p-Erk/Erk and Mcl-1 protein under hypoxia in siHIF-2αcells was lower than that in NC cells(P<0.01). Tumor sizes, weights and density of siHIF-2α group in nude mice were suppressed compared with those in NC group (P<0.01). Conclusion Blocking HIF-2αsignal pathway warrants its investigation as a potential strategy in osteosarcoma treatment.

Expression and location of hypoxia inducible factor-1α and -2α in the remnant kidney of 5/6 nephrectomy rats / 中华肾脏病杂志

Objective To investigate the location and expression of hypoxia inducible factor (HIF) subunits in the remnant kidney of 5/6 nephrectomy rats. Methods Remnant kidneys were produced in adult male SD rats by 5/6 nephrectomy. The renal function and histopathological changes were evaluated at week 1, 2, 4, 6, 8 and 12 after operation. Tissues of remnant kidneys were collected to detect the location and expression of HIF-1α and HIF-2α by immunohistochemistry staining and Western blotting. The mRNA levels of HIF targeted genes vascular endothelial growth factor (VEGF) and heme oxygenase-1 (HO-1) were determined by RTPCR. Results (1) 5/6 nephrectomy rats underwent one week of acute renal failure at first[Scr (122.8±22.1) μmol/L] and then developed compensative chronic renal failure [(66.0±3.7)-(66.4±8.4) μmol/L], but the level of Scr increased quickly after week 6 [(66.4±8.4)-(127.8±22.7) μmol/L],concomitantly with progressive tubulointerstitial fibrosis in remnant kidney cortex. (2) In cortex, HIF-1α was expressed only in the atrophic and dilated tubular cells while HIF-2α was located in endothelial, interstitial fibroblasts, and vascular smooth muscle cells. The semiquantitative results of imunohistochemistry and Western blotting revealed that HIF-1α and HIF-2α were both gradually up-regulated during the early stage of remnant kidney, peaked at week 4 and 6, and then gradually down-regulated. (3) The mRNA levels of HIF targeted genes VEGF and HO-1 transiently peeked at week 4 and 6, and then decreased gradually. Conclusions The increased stabilization of HIF-αprotein and transcription of HIF targeted genes at the early stage of this model is a compensation reaction towards hypoxia. The mechanism of decreased expression of HIF-α at the end stage of chronic kidney disease deserves further investigation.