Hypoxia-inducible Factor-1α Target Research Articles

Genetic and pharmaceutical targeting of HIF1α allows combo-immunotherapy to boost graft vs. leukemia without exacerbation graft vs. host disease

Despite potential impact on the graft vs. leukemia (GVL) effect, immunotherapy targeting CTLA-4 and/or PD-1 has not been successfully combined with bone marrow transplant (BMT) because it exacerbates graft vs. host disease (GVHD). Here, using models of GVHD and leukemia, we demonstrate that targeting hypoxia-inducible factor 1α (HIF1α) via pharmacological or genetic approaches reduces GVHD by inducing PDL1 expression on host tissue while selectively inhibiting PDL1 in leukemia cells to enhance the GVL effect. More importantly, combination of HIF1α inhibition with anti-CTLA-4 antibodies allows simultaneous inhibition of both PDL1 and CTLA-4 checkpoints to achieve better outcomes in models of mouse and human BMT-leukemia settings. These findings provide an approach to enhance the curative effect of BMT for leukemia and broaden the impact of cancer immunotherapy.

Efficacy, mechanism, and safety of melatonin-loaded on thermosensitive nanogels for rabbit VX2 tumor embolization: A novel design.

Transarterial chemoembolization (TACE) has been widely used for hepatocellular carcinoma.Reducing hypoxia in the tumor microenvironment after TACE remains a challenge as tumor progression is common in post-TACE patients due to the hypoxic tumor microenvironment. In this study, melatonin loaded on p(N-isopropyl-acrylamide-co-butyl methylacrylate) (PIB-M) was used for tumor embolism. Two types of human hepatoma cell lines were used to explore the mechanism by which melatonin prevents the growth and metastasis of cancer cells in vitro. A VX2 rabbit tumor model was used to evaluate the efficacy, mechanism, and safety of PIB-M in vivo. We found that under hypoxic condition, melatonin could inhibit tumor cell proliferation and migration by targeting hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor A (VEGF-A) in vitro. In vivo, PIB-M inhibited tumor growth and metastasis in rabbit VX2 tumors by promoting apoptosis of tumor cells and targeting related angiogenic proteins and vascular permeability proteins. A high concentration of melatonin in the PIB-M group could be maintained in tumor tissue for 72 h after embolization. The liver and kidney functions were most damaged on the first day but recovered to normal on the seventh day after embolization in the PIB-M group. This novel method may open avenues for reduction of tumor growth and metastasis after TACE and is efficacy and safety, which may be used for treatment for other solid tumors and clinical translation.

Targeting Hypoxia-Inducible Factor-1α for the Management of Hepatocellular Carcinoma.

Hypoxia-inducible factor 1 alpha (HIF-1α) is a transcription factor that regulates the cellular response to hypoxia and is upregulated in all types of solid tumor, leading to tumor angiogenesis, growth, and resistance to therapy. Hepatocellular carcinoma (HCC) is a highly vascular tumor, as well as a hypoxic tumor, due to the liver being a relatively hypoxic environment compared to other organs. Trans-arterial chemoembolization (TACE) and trans-arterial embolization (TAE) are locoregional therapies that are part of the treatment guidelines for HCC but can also exacerbate hypoxia in tumors, as seen with HIF-1α upregulation post-hepatic embolization. Hypoxia-activated prodrugs (HAPs) are a novel class of anticancer agent that are selectively activated under hypoxic conditions, potentially allowing for the targeted treatment of hypoxic HCC. Early studies targeting hypoxia show promising results; however, further research is needed to understand the effects of HAPs in combination with embolization in the treatment of HCC. This review aims to summarize current knowledge on the role of hypoxia and HIF-1α in HCC, as well as the potential of HAPs and liver-directed embolization.

BTN3A1 promotes tumor progression and radiation resistance in esophageal squamous cell carcinoma by regulating ULK1-mediated autophagy

Radiotherapy is one of the most effective treatments for esophageal squamous cell carcinoma (ESCC); however, radioresistance is a clinical problem that must urgently be solved. Here, we found that butyrophilin subfamily 3 member A1 (BTN3A1) is upregulated in ESCC tumor tissues compared with nontumor tissues. We also evaluated BTN3A1 expression in patients with ESCC receiving adjuvant radiotherapy. The results demonstrated that BTN3A1 upregulation predicts a poor prognosis for ESCC patients. BTN3A1 overexpression promotes ESCC cell proliferation in vitro and in vivo. Moreover, BTN3A1 knockdown sensitized ESCC cells to radiation. We further explored the mode of death involved in BTN3A1-mediated radioresistance. Previous studies have shown that apoptosis, autophagy, necrosis, pyroptosis and ferroptosis are important for the survival of ESCC cells. We performed an RT-PCR array and western blotting (WB) to identify the mode of death and revealed for the first time that BTN3A1 promotes cell radioresistance by activating autophagy. In addition, by performing immunoprecipitation and mass spectrometry analyses, we found that BTN3A1 regulated the expression of UNC-51-like autophagy activating kinase 1(ULK1) and promoted its phosphorylation to subsequently initiate autophagy. Chromatin immunoprecipitation (ChIP) and luciferase reporter assay results indicated that BTN3A1 is a novel direct target of hypoxia inducible factor-1α (HIF-1α). HIF-1α, a transcription factor, promotes BTN3A1 transcription upon irradiation. Overall, the present study is the first to show that BTN3A1 plays a key role in radioresistance and that targeting BTN3A1 might be a promising strategy to improve radiotherapy efficacy in patients with ESCC.

Hypoxia-Induced Autophagy Enhances Cisplatin Resistance in Human Bladder Cancer Cells by Targeting Hypoxia-Inducible Factor-1α.

Purpose To investigate the effect of hypoxia on chemoresistance and the underlying mechanism in bladder cancer cells. Methods BIU-87 bladder cancer cell line was treated with cisplatin under hypoxic and normoxic conditions and tested using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, and Western blotting. All the data were expressed as mean ± standard error from three independent experiments and analyzed by multiple t-tests. Results Apoptosis of bladder cancer cells caused by cisplatin was attenuated in hypoxic conditions. Hypoxia enhanced autophagy caused by cisplatin. The autophagy inhibitor and HIF-1α inhibitor can reverse the chemoresistance in hypoxic condition. Apoptosis and autophagy of bladder cancer cells were downregulated by HIF-1α inhibitor YC-1. Hypoxia-induced autophagy enhanced chemoresistance to cisplatin via the HIF-1 signaling pathway. Conclusion Resistance to cisplatin in BIU-87 bladder cancer cells under hypoxic conditions can be explained by activation of autophagy, which is regulated by HIF-1α-associated signaling pathways. The hypoxia–autophagy pathway may be a target for improving the efficacy of cisplatin chemotherapy in bladder cancer.

Long Noncoding RNA Expression Profiling Reveals Upregulation of Uroplakin 1A and Uroplakin 1A Antisense RNA 1 under Hypoxic Conditions in Lung Cancer Cells.

Hypoxia plays important roles in cancer progression by inducing angiogenesis, metastasis, and drug resistance. However, the effects of hypoxia on long noncoding RNA (lncRNA) expression have not been clarified. Herein, we evaluated alterations in lncRNA expression in lung cancer cells under hypoxic conditions using lncRNA microarray analyses. Among 40,173 lncRNAs, 211 and 113 lncRNAs were up- and downregulated, respectively, in both A549 and NCI-H460 cells. Uroplakin 1A (UPK1A) and UPK1A-antisense RNA 1 (AS1), which showed the highest upregulation under hypoxic conditions, were selected to investigate the effects of UPK1A-AS1 on the expression of UPK1A and the mechanisms of hypoxia-inducible expression. Following transfection of cells with small interfering RNA (siRNA) targeting hypoxia-inducible factor 1α (HIF-1α), the hypoxia-induced expression of UPK1A and UPK1A-AS1 was significantly reduced, indicating that HIF-1α played important roles in the hypoxia-induced expression of these targets. After transfection of cells with UPK1A siRNA, UPK1A and UPK1A-AS1 levels were reduced. Moreover, transfection of cells with UPK1A-AS1 siRNA downregulated both UPK1A-AS1 and UPK1A. RNase protection assays demonstrated that UPK1A and UPK1A-AS1 formed a duplex; thus, transfection with UPK1A-AS1 siRNA decreased the RNA stability of UPK1A. Overall, these results indicated that UPK1A and UPK1A-AS1 expression increased under hypoxic conditions in a HIF-1α-dependent manner and that formation of a UPK1A/UPK1A-AS1 duplex affected RNA stability, enabling each molecule to regulate the expression of the other.

Hypoxia regulates the differentiation and anti-tumor effector functions of γδT cells in oral cancer.

Hypoxia within the tumor microenvironment (TME) is a key factor contributing to immunosuppression in tumors, co-relating with poor treatment outcome and decreased overall survival in advanced oral cancer (OC) patients. Vδ2 is a dominant subset of gamma delta T cells (γδT cells) present in the peripheral blood which exhibits potent anti-tumor cytotoxicity and is evolving as a key player of anti-cancer cellular therapy. However, the fate of γδT cells in hypoxic oral tumors remains elusive. In the present study, we compared the effect of hypoxia (1% O2 ) and normoxia (21% O2 ) on the expansion, proliferation, activation status, cytokine secretion and cytotoxicity of γδT cells isolated from OC patients and healthy individuals. Hypoxia-exposed γδT cells exhibited reduced cytotoxicity against oral tumor cells. Our data demonstrated that hypoxia reduces the calcium efflux and the expression of degranulation marker CD107a in γδT cells, which explains the decreased anti-tumor cytotoxicity of γδT cells observed under hypoxia. Hypoxia-exposed γδT cells differentiated to γδT17 [γδ T cells that produce interleukin (IL)-17] cells, which corroborated our observations of increased γδT17 cells observed in the oral tumors. Co-culture of γδT cells with CD8 T cells in the presence of hypoxia showed that programmed cell death ligand 1 (PD-L1)high γδT cells brought about apoptosis of programmed cell death 1 (PD-1)high CD8 T cells which could be significantly reversed upon blocking PD-1. Thus, future immunotherapeutic treatment modality for oral cancer may use a combined approach of blocking the PD-1/PD-L1 signaling and targeting hypoxia-inducible factor 1α, which may help in reversing hypoxia-induced immunosuppression.

Role of pyruvate kinase M2 in oxidized LDL-induced macrophage foam cell formation and inflammation

Pyruvate kinase M2 (PKM2) links metabolic and inflammatory dysfunction in atherosclerotic coronary artery disease; however, its role in oxidized LDL (Ox-LDL)-induced macrophage foam cell formation and inflammation is unknown and therefore was studied. In recombinant mouse granulocyte-macrophage colony-stimulating factor-differentiated murine bone marrow-derived macrophages, early (1-6 h) Ox-LDL treatment induced PKM2 tyrosine 105 phosphorylation and promotes its nuclear localization. PKM2 regulates aerobic glycolysis and inflammation because PKM2 shRNA or Shikonin abrogated Ox-LDL-induced hypoxia-inducible factor-1α target genes lactate dehydrogenase, glucose transporter member 1, interleukin 1β (IL-1β) mRNA expression, lactate, and secretory IL-1β production. PKM2 inhibition significantly increased Ox-LDL-induced ABCA1 and ABCG1 protein expression and NBD-cholesterol efflux to apoA1 and HDL. PKM2 shRNA significantly inhibited Ox-LDL-induced CD36, FASN protein expression, DiI-Ox-LDL binding and uptake, and cellular total cholesterol, free cholesterol, and cholesteryl ester content. Therefore, PKM2 regulates lipid uptake and efflux. DASA-58, a PKM2 activator, downregulated LXR-α, ABCA1, and ABCG1, and augmented FASN and CD36 protein expression. Peritoneal macrophages showed similar results. Ox-LDL induced PKM2- SREBP-1 interaction and FASN expression in a PKM2-dependent manner. Therefore, this study suggests a role for PKM2 in Ox-LDL-induced aerobic glycolysis, inflammation, and macrophage foam cell formation.

RAD51 is a potential marker for prognosis and regulates cell proliferation in pancreatic cancer

BackgroundThe DNA damage and repair pathway is considered a promising target for developing strategies against cancer. RAD51, also known as RECA, is a recombinase that performs the critical step in homologous recombination. RAD51 has recently received considerable attention due to its function in tumor progression and its decisive role in tumor resistance to chemotherapy. However, its role in pancreatic cancer has seldom been investigated. In this report, we provide evidence that RAD51, regulated by KRAS, promotes pancreatic cancer cell proliferation. Furthermore, RAD51 regulated aerobic glycolysis by targeting hypoxia inducible factor 1α (HIF1α).MethodsTCGA (The Cancer Genome Atlas) dataset analysis was used to examine the impact of RAD51 expression on overall survival of pancreatic cancer patients. Lentivirus-mediated transduction was used to silence RAD51 and KRAS expression. Quantitative real-time PCR and western blot analysis validated the efficacy of the knockdown effect. Analysis of the glycolysis process in pancreatic cancer cells was also performed. Cell proliferation was determined using a CCK-8 (Cell Counting Kit-8) proliferation assay.ResultsPancreatic cancer patients with higher levels of RAD51 exhibited worse survival. In pancreatic cancer cells, RAD51 positively regulated cell proliferation, decreased intracellular reactive oxygen species (ROS) production and increased the HIF1α protein level. KRAS/MEK/ERK activation increased RAD51 expression. In addition, RAD51 was a positive regulator of aerobic glycolysis.ConclusionThe present study reveals novel roles for RAD51 in pancreatic cancer that are associated with overall survival prediction, possibly through a mechanism involving regulation of aerobic glycolysis. These findings may provide new predictive and treatment targets for pancreatic cancer.

Effect of lincRNA-p21 targeting HIF-1α on biological functions of liver cancer cells.

The effects of long intergenic non-coding ribonucleic acid (lincRNA)-p21 targeting hypoxia-inducible factor-1α (HIF-1α) on proliferation, apoptosis and migration of liver cancer cells were investigated. MHCC97H liver cancer cells were infected with control lentivirus (control group) and lincRNA-p21 lentivirus (observation group), and control stable cell lines and lincRNA-p21 stable cell lines were screened and obtained by using puromycin. The expression levels of lincRNA-p21 messenger RNA (mRNA) in the control and observation groups were analyzed via reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Bioinformatics was used to search for the lincRNA-p21 target. The expression of target gene was analyzed via western blotting, and the proliferation, apoptosis, migration and in vivo tumor formation of MHCC97H cells in the control and observation groups were also analyzed. Compared with that in control group, the lincRNA-p21 mRNA level in observation group was increased significantly (P<0.05). It was found via bioinformatic comparison that HIF-1α was one of the targets of lincRNA-p21. Results of Western blotting revealed that the expression level of HIF-1α protein in cells in observation group was significantly downregulated (P<0.05). Besides, the level of vascular endothelial growth factor (VEGF) protein in cells in control group was obviously higher than that in observation group (P<0.05). Compared with those in control group, the cell proliferation and migration capacities in observation group were markedly reduced, but the apoptosis level was significantly increased (P<0.05). According to the in vivo tumor formation assay, the cell proliferation rate in control group was obviously higher than that in observation group (P<0.05). The number of tumor blood vessels in cells in control group was obviously reduced compared with that in observation group (P<0.05). lincRNA-p21 can significantly downregulate the level of HIF-1α, thus downregulating the expression of VEGF and affecting the cell proliferation, apoptosis and migration.

Lipase member H is a downstream molecular target of hypoxia inducible factor-1α and promotes papillary thyroid carcinoma cell migration in BCPAP and KTC-1 cell lines.

BackgroundPapillary thyroid carcinoma (PTC) is the most common type of thyroid carcinoma, which is associated with a high incidence of lymph-node metastasis. Multiple biomarkers have been identified for the precise diagnosis of PTC at an early stage. However, their role in PTC remains poorly elucidated. Previously, we reported that lipase H (LIPH), a membrane-bound protein, was highly expressed in PTC. This study aimed to fully elucidate the causal role of LIPH in the development of PTC and investigated its relationship with lymph-node metastasis in PTC.Materials and methodsQuantitative reverse transcription PCR and immunohistochemistry were used to measure the mRNA and protein expression levels of LIPH in 45 and 6 pairs of PTC tissues and adjacent normal tissues, respectively. Clinical tissue data of 504 PTC tissues and 60 normal thyroid tissues from The Cancer Genome Atlas database were used to analyze the correlation between LIPH expression level and clinical features in PTC. siRNAs were used to knock down genes, while plasmids were used to overexpress genes. Two PTC cell lines (KTC-1 and BCPAP) were used in subsequent cytological function studies. In addition, a hypoxia stress model was constructed using cobaltous chloride hexahydrate reagent, and the protein expression level of the corresponding biomarkers was measured by Western blotting.ResultsThis study revealed that high expression of LIPH in PTC was closely associated with lymph-node metastasis. Our cellular function experiments indicated that LIPH positively correlated with the malignant behavior of PTC cell lines. We further confirmed the role of LIPH in hypoxia and its relationship with the epithelial–mesenchymal transition pathway in PTC.ConclusionLIPH plays an important role in PTC oncogenesis and development, especially in lymph-node metastasis. It can be regarded as a biomarker for the diagnosis and treatment of PTC in the near future.

Fat atypical cadherin 1 regulating invasion by targeting hypoxia inducible factor-1α in high grade glioma

Objective To investigate the action mechanism of fat atypical cadherin 1 (FAT1) regulating invasion by targeting hypoxia inducible factor-1α (HIF-1α) in high grade glioma under hypoxia. Methods The expression levels of FAT1 and HIF-1α in 48 cases of grade Ⅲ-Ⅳ glioma athological tissue samples in our hospital were detected by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR). The relationship between the expression of FAT1 and HIF-1α was analyzed. The expression of FAT1 and HIF-1α in human glioma cells U87MG and GOS3 under the condition of normal oxygen (20% O2) and hypoxia (1% O2) was detected by FQ-PCR. FAT1 small interfering RNA (siRNA), HIF-1α siRNA and control siRNA were transfected into U87MG and GOS3 cell lines respectively by Lipofectamine 2000. The expression of FAT1 and HIF-1 under the normal oxygen and hypoxia conditions was detected. The invasive abilities of U87MG and GOS3 glioma cells were examined by cell chamber technique. Results The relative expression levels of FAT1 and HIF-1α in 48 cases of pathological tissues of patients with grade Ⅲ-Ⅳ glioma were 4.07 (0.72, 13.42) and 0.94 (0.29, 3.49) respectively. Spearman analysis showed that there was significant correlation between the relative expression levels of FAT1 and HIF-1α (r=0.835, P=0.000). The normal oxygen concentration in GOS3 cell lines FAT1 (0.29±0.03) and HIF-1α (0.34±0.09) relative expression levels were significantly lower than that of U87MG cells (1.08±0.15, 1.00±0.12; t=8.945, 7.621; P=0.010, 0.002); hypoxia condition, U87MG and GOS3 cell lines FAT1 and HIF-1α relative expression levels were significantly higher than the normal oxygen concentration (t=17.994, 8.920, 14.047, 4.037; P=0.000, 0.001, 0.004, 0.016); U87MG cells under hypoxic conditions FAT1 (4.38±0.28) and HIF-1α (2.76±0.32) relative expression levels were significantly higher than that of GOS3 cell line (1.69±0.17, 0.83±0.19; t=14.224, 8.982; P=0.000, 0.001). After transfection of FAT1 siRNA, FAT1 (0.17±0.06, 0.16±0.03, 0.16±0.03, 0.18±0.03) and HIF-1α (0.18±0.03, 0.17±0.04, 0.15±0.04, 0.21±0.02) relative expression levels of U87MG and GOS3 cells in the normal oxygen and hypoxia were significantly decreased (t=7.577, 7.793, 7.793, 7.783, 9.285, 9.260, 9.484, 9.042; P=0.002, 0.013, 0.013, 0.014, 0.009, 0.007, 0.007, 0.011). After transfection of HIF-1α siRNA, FAT1 (0.16±0.04, 0.28±0.03) relative expression levels in the normal oxygen and HIF-1α (0.15±0.03, 0.16±0.02, 0.15±0.03, 0.17±0.02) relative expression levels of U87MG and GOS3 cells in normal oxygen and hypoxia conditions were significantly decreased (t=7.890, 6.834, 9.624, 9.614, 9.624, 9.500; P=0.012, 0.018, 0.008, 0.009, 0.008, 0.010); but the level under hypoxic conditions relative expression of FAT1 (4.27±0.41, 2.72±0.25) were significantly higher than that in normal oxygen conditions (t=12.649, 9.671; P=0.000, 0.001). Under hypoxic conditions, the invasion cell quantity of U87MG cells transfected with FAT1 siRNA and HIF-1α siRNA [(23.2±4.2) and (21.5±3.1) cells] were lower than those of normal oxygen conditions [(57.6±8.1) and (58.7±7.6) cells; t=6.530, 7.850; P=0.003, 0.001]. Conclusion The expression of FAT1 and HIF-1 in high grade gliomas is strongly correlated, and the expression is high under the hypoxia condition. FAT1 can promote the invasion of glioma cells by targeting HIF-1α. Key words: Glioma; Fat atypical cadherin; Hypoxia inducible factor-1α; Invasion

Plasma miR-18 Screens Acute Myocardial Infarction from Healthy Controls by Targeting Hypoxia Inducible Factor 1α

This study aims to evaluate the expression of plasma miR-18, thereby evaluating its potential use as a biomarker to screen acute myocardial infarction (AMI) patients from healthy controls. Real time PCR was carried out to evaluate the level of miR-18 in AMI patients and healthy controls. ROC analysis was performed to evaluate whether miR-18 could be used as a potential biomarker. Dual luciferase assay was used to identify the potential target of miR-18. We showed novel data that plasma miR-18 was significantly greater in AMI patients than in healthy controls. ROC analysis indicated that plasma miR-18 could screen AMI patients from healthy controls with high sensitivity and specificity. Further study demonstrated that plasma miR-18 was positively correlated with serum cardiac troponin I (cTnI) and creatine phosphokinase-MB (CK-MB) concentrations. Additionally, plasma miR-18 was increased in AMI patients with more stenosed coronary vessels. More importantly, plasma miR-18 was decreased in AMI patients after receiving percutaneous coronary intervention (PCI). Dual luciferase reporter assay indicated that overexpression of miR-18 significantly suppressed the relative luciferase activity of pmirGLO- HIF1α-3´UTR. In summary, enhanced plasma miR-18 could be used as a potential biomarker to screen AMI patients from healthy controls via targeting HIF1α.

Targeting Hypoxia-Inducible Factor-1α/Pyruvate Dehydrogenase Kinase 1 Axis by Dichloroacetate Suppresses Bleomycin-induced Pulmonary Fibrosis.

Hypoxia has long been implicated in the pathogenesis of fibrotic diseases. Aberrantly activated myofibroblasts are the primary pathological driver of fibrotic progression, yet how various microenvironmental influences, such as hypoxia, contribute to their sustained activation and differentiation is poorly understood. As a defining feature of hypoxia is its impact on cellular metabolism, we sought to investigate how hypoxia-induced metabolic reprogramming affects myofibroblast differentiation and fibrotic progression, and to test the preclinical efficacy of targeting glycolytic metabolism for the treatment of pulmonary fibrosis. Bleomycin-induced pulmonary fibrotic progression was evaluated in two independent, fibroblast-specific, promoter-driven, hypoxia-inducible factor (Hif) 1A knockout mouse models and in glycolytic inhibitor, dichloroacetate-treated mice. Genetic and pharmacological approaches were used to explicate the role of metabolic reprogramming in myofibroblast differentiation. Hypoxia significantly enhanced transforming growth factor-β-induced myofibroblast differentiation through HIF-1α, whereas overexpression of the critical HIF-1α-mediated glycolytic switch, pyruvate dehydrogenase kinase 1 (PDK1) was sufficient to activate glycolysis and potentiate myofibroblast differentiation, even in the absence of HIF-1α. Inhibition of the HIF-1α/PDK1 axis by genomic deletion of Hif1A or pharmacological inhibition of PDK1 significantly attenuated bleomycin-induced pulmonary fibrosis. Our findings suggest that HIF-1α/PDK1-mediated glycolytic reprogramming is a critical metabolic alteration that acts to promote myofibroblast differentiation and fibrotic progression, and demonstrate that targeting glycolytic metabolism may prove to be a potential therapeutic strategy for the treatment of pulmonary fibrosis.

RAS protein activator-like 1 is functionally involved in hypoxia resistance in breast cancer cells by targeting hypoxia inducible factor-1α.

RAS protein activator-like 1 (RASAL1) is a member of the RAS GTPase-activating protein family, and previous studies indicate that RASAL1 is involved in the progression of hypoxia resistance in breast cancer cells. In the present study, increased levels of hypoxia inducible factor-1α (HIF-1α) were observed to be accompanied with increased expression of RASAL1 in the breast cancer cell lines MCF-7 and MDA-MB-231 cells under hypoxia. Based on this, it was postulated that RASAL1 may serve a functional role in the development of hypoxia resistant in breast cancer cells. In the present study it was demonstrated that: i) Exogenous expression of RASAL1 in MCF-7 and MDA-MB-231 sensitized its reaction to the treatment of hypoxia, which is associated with its ability to directly reduce HIF-1α expression, inhibit migration activity and decrease the accumulation of reactive oxygen species (ROS); ii) knockdown of RASAL1 reversed its reaction to treatment with hypoxia; iii) RASAL1 directly regulated the expression of HIF-1α through the ROS-mediated, extracellular signal-regulated kinase and Akt pathway. These findings provide direct evidence that the RASAL1/HIF-1α axis may serve an essential role in the hypoxia resistance of breast cancer cells, suggesting that this signaling cohort may serve as a novel therapeutic target for the treatment of breast cancer.

MicroRNA-320 regulates autophagy in retinoblastoma by targeting hypoxia inducible factor-1α.

Retinoblastoma (RB) is the most common malignancy in children. Due to refractory mechanisms of chemoresistance and the toxicity of chemotherapies, novel therapies for RB treatment are urgently required. MicroRNA-320 (miR-320) is believed to be associated with the tumorigenesis of RB, although the mechanism remains unclear. Considering the hypoxic intratumoral region, the roles of miR-320 and hypoxia inducible factor-1α (HIF-1α) in the regulation of autophagy were investigated in 30 human RB samples and WERI-RB1 cells. The results demonstrated that HIF-1α was the downstream target of miR-320, and decreased miRNA-320 or HIF-1α lead to the inhibition of autophagy in WERI-RB1 cells. Compared with WERI-RB1 cells that were not transfected, silenced HIF-1α caused a 1.41-fold increase (P<0.01) in p62, a 2.71-fold decrease of Beclin1, and inhibited miRNA-320. Silenced HIF-1α also resulted in 7.29- and 7.43-fold increases in phosphorylated-mechanistic target of rapamycin (mTOR) and mTOR, respectively. In conclusion, the present results suggest that miRNA-320 may regulate the development of autophagy by targeting HIF-1α and autophagy-related proteins in RB under hypoxic conditions.

Transarterial embolization combined with RNA interference targeting hypoxia-inducible factor-1α for hepatocellular carcinoma: a preliminary study of rat model.

To study whether transarterial embolization (TAE) with RNA interference (RNAi) targeting hypoxia-inducible factor-1α (HIF-1α) can improve efficacy of TAE in treating hepatocellular carcinoma (HCC). CBRH-7919 rat hepatoma cell line was used and HCC models of rats were constructed. The siRNA transfection compound was made by mixing specific siRNA and Lipofectamine 2000™. Delivery and transfection of siRNA were administered by injecting iodized oil emulsion (diluted lipiodol and siRNA) via hepatic artery. The expression levels of mRNA and protein were detected using the real-time reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry and western blotting assays, respectively. In vitro experiment, the specific HIF-1α-siRNA was proved to inhibit expression levels of HIF-1α and vascular endothelial growth factor (VEGF) effectively. In animal study, real-time RT-PCR assay showed the average relative mRNA expressions of HIF-1α were 0.31±0.01, 0.65±0.03, 0.46±0.005, and 1.00±0.00 in TAE+siRNA, siRNA, TAE, and control groups, respectively. Western blotting assay showed the average relative protein expressions of HIF-1α were 0.13±0.02, 0.87±0.02, 0.39±0.02, and 1.02±0.01 in TAE+siRNA, siRNA, TAE, and control groups, respectively. Compared with control, TAE, and siRNA groups, TAE+siRNA can significantly inhibit protein expressions of HIF-1α and VEGF (P HIF-1α<0.001; P VEGF<0.001). Overall survival of rats underwent TAE+siRNA was significantly longer than that of rats treated with TAE monotherapy (P=0.001). This animal study showed TAE combined with HIF-1α-RNAi could significantly improve efficacy of TAE in treating HCC by inhibiting expressions of HIF-1α and VEGF after TAE treatment.

Snail Is a Direct Target of Hypoxia-inducible Factor 1α (HIF1α) in Hypoxia-induced Endothelial to Mesenchymal Transition of Human Coronary Endothelial Cells

Endothelial to mesenchymal transition (EndMT) was originally described in heart development where the endocardial endothelial cells that line the atrioventricular canal undergo an EndMT to form the endocardial mesenchymal cushion that later gives rise to the septum and mitral and tricuspid valves. In the postnatal heart specifically, endothelial cells that originate from the endocardium maintain increased susceptibility to undergo EndMT as remnants from their embryonic origin. Such EndMT involving adult coronary endothelial cells contributes to microvascular rarefaction and subsequent chronification of hypoxia in the injured heart, ultimately leading to cardiac fibrosis. Although in most endothelial beds hypoxia induces tip cell formation and sprouting angiogenesis, here we demonstrate that hypoxia is a stimulus for human coronary endothelial cells to undergo phenotypic changes reminiscent of EndMT via a mechanism involving hypoxia-inducible factor 1α-induced activation of the EndMT master regulatory transcription factor SNAIL. Our study adds further evidence for the unique susceptibility of endocardium-derived endothelial cells to undergo EndMT and provides novel insights into how hypoxia contributes to progression of cardiac fibrosis. Additional studies may be required to discriminate between distinct sprouting angiogenesis and EndMT responses of different endothelial cells populations.

Targeting Hypoxia-Inducible Factor 1α in a New Orthotopic Model of Glioblastoma Recapitulating the Hypoxic Tumor Microenvironment.

Tissue hypoxia and necrosis represent pathophysiologic and histologic hallmarks of glioblastoma (GBM). Although hypoxia inducible factor 1α (HIF-1α) plays crucial roles in the malignant phenotypes of GBM, developing HIF-1α-targeted agents has been hampered by the lack of a suitable preclinical model that recapitulates the complex biology of clinical GBM. We present a new GBM model, MGG123, which was established from a recurrent human GBM. Orthotopic xenografting of stem-like MGG123 cells reproducibly generated lethal tumors that were characterized by foci of palisading necrosis, hypervascularity, and robust stem cell marker expression. Perinecrotic neoplastic cells distinctively express HIF-1α and are proliferative in both xenografts and the patient tissue. The xenografts contain scattered hypoxic foci that were consistently greater than 50 μm distant from blood vessels, indicating intratumoral heterogeneity of oxygenation. Hypoxia enhanced HIF-1α expression in cultured MGG123 cells, which was abrogated by the HIF-1α inhibitors digoxin or ouabain. In vivo, treatment of orthotopic MGG123 xenografts with digoxin decreased HIF-1α expression, vascular endothelial growth factor mRNA levels, and CD34-positive vasculature within the tumors, and extended survival of mice bearing the aggressive MGG123 GBM. This preclinical tumor model faithfully recapitulates the GBM-relevant hypoxic microenvironment and stemness and is a suitable platform for studying disease biology and developing hypoxia-targeted agents.

The effect of lentiviral vector-mediated RNA interference targeting hypoxia-inducible factor 1α on the uptake of fluorodeoxyglucose ((18)f) in the human pancreatic cancer cell line, patu8988.

Hypoxia can stimulate (18)F-fluorodeoxyglucose ((18)F-FDG) uptake in cultured tumor cells. This study has investigated the effect of lentiviral vector-mediated RNA interference (RNAi) targeting hypoxia-inducible factor 1α (HIF-1α) on the changes in HIF-1 and glucose transporter 1 (Glut-1) expression, the cell growth, and the uptake of (18)F-FDG in the human pancreatic cancer cell line, Patu8988. Lentiviral RNAi vector targeting the HIF-1α gene (LV-HIF-1αRNAi) was constructed and used to treat cells at various concentrations (25-200 nM). The expression changes of HIF-1α and Glut-1 in hypoxic Patu8988 cells after RNAi treatment were determined using real time reverse transcription-polymerase chain reaction (real-time PCR). The inhibition rate of cell proliferation 48 hours after the addition of 10 μL of different concentrations of LV-HIF-1αRNAi (25-200 nM) was assayed using the MTT method. Meanwhile, the cell uptake of (18)F-FDG was also assessed. After RNAi transfection, the relative expression levels of HIF-1α mRNA and Glut-1 under hypoxia were reduced and the relative expression levels of HIF-1α protein also decreased. Compared with the control group, the inhibition rates of cell proliferation under different viral dosages were 5.98%, 15.65%, 26.42%, and 40.81%, respectively, positively correlated with the viral doses (r=0.558, p<0.05). Under hypoxia, Glut-1 mRNA expression in Patu8988 cells treated with 200 nM of LV-HIF-1αRNAi for 24, 48, and 72 hours, respectively, was positively correlated with the inhibition rate of cell proliferation (r=0.618, p<0.05) as well as the inhibition rate of (18)F-FDG uptake (r=0.664, p<0.05), while the latter two displayed a positive correlation with each other too (r=0.582, p<0.05). Under hypoxia, RNAi targeting HIF-1α significantly inhibited the expression of Glut-1 mRNA in Patu8988 pancreatic cancer cells and their uptake of (18)F-FDG. These results suggest that LV-HIF-1αRNAi may form a new treatment for pancreatic cancer, and the effectiveness of the treatment can be readily assessed with (18)F-FDG imaging.