Hypoxia regulates gene expression via the transcription factor HIF (Hypoxia-Inducible Factor). Little is known regarding HIF expression and function in primary bone sarcomas. We describe HIF expression and phenotypic effects of hypoxia, hypoglycaemia and HIF in Ewing's sarcoma and osteosarcoma.
HIF-1α and HIF-2α immunohistochemistry was performed on a Ewing's tumour tissue array. Ewing's sarcoma and osteosarcoma cell lines were assessed for HIF pathway induction by Western blot, luciferase assay and ELISA. Effects of hypoxia, hypoglycaemia and isoform-specific HIF siRNA were assessed on proliferation, apoptosis and migration.
17/56 Ewing's tumours were HIF-1α-positive, 15 HIF-2α-positive and 10 positive for HIF-1α and HIF-2α. Expression of HIF-1α and cleaved caspase 3 localised to necrotic areas. Hypoxia induced HIF-1α and HIF-2α in Ewing's and osteosarcoma cell lines while hypoglycaemia specifically induced HIF-2α in Ewing's. Downstream transcription was HIF-1α-dependent in Ewing's sarcoma, but regulated by both isoforms in osteosarcoma. In both cell types hypoglycaemia reduced cellular proliferation by ≥ 45%, hypoxia increased apoptosis and HIF siRNA modulated hypoxic proliferation and migration.
Co-localisation of HIF-1α and necrosis in Ewing's sarcoma suggests a role for hypoxia and/or hypoglycaemia in in vivo induction of HIF. In vitro data implicates hypoxia as the primary HIF stimulus in both Ewing's and osteosarcoma, driving effects on proliferation and apoptosis. These results provide a foundation from which to advance understanding of HIF function in the pathobiology of primary bone sarcomas.
Osteosarcoma has one of the worst prognoses in adolescents; only 20–60% of patients have high rates of histological necrosis with intensive neoadjuvant chemotherapy. In this study, we investigated the prognostic values of hypoxia-inducible factor 1 α (HIF-1α), apurinic endonuclease 1 (APE1), vascular endothelial growth factor (VEGF) and cycloogenase-2 (COX-2) protein expression and their predictive value of tumor necrosis rate and prognosis, as well as their interrelationships. Formalin-fixed paraffin-embedded tissue samples were obtained from 49 patients with osteosarcoma. Immunohistochemistry assays were performed in pre-chemotherapy samples to determine HIF-1α, VEGF, APE1 and COX-2 protein expression levels and hematoxylin and eosin staining was performed in post-operative samples to determine the tumor necrosis rate. Univariate and multivariate analyses were used to assess the impact of protein expression on prognosis. HIF-1α was significantly correlated with every protein we tested: VEGF (P=0.032), APE1 (P<0.001) and COX-2 (P<0.001). HIF-1α protein expression had a significant impact on disease-free survival (P=0.006). Expression of HIF-1α had a sensitivity of 64.7% and a specificity of 71.9% for a poor pathological response (<90% tumor necrosis) versus a good pathological response (≥90% tumor necrosis). In conclusion, expression of HIF-1α is a predictor of tumor response to neoadjuvant chemotherapy and outcome in osteosarcoma, and correlates with VEGF, APE1 and COX-2.
hypoxia-inducible factor 1 α; apurinic endonuclease-1; vascular endothelial growth factor; cycloogenase-2; osteosarcoma
Hypoxia-inducible factor 1 (HIF-1) alpha and its downstream targets carbonic anhydrase IX (CAIX) and vascular endothelial growth factor (VEGF) are key factors in the survival of proliferating tumor cells in a hypoxic microenvironment. We studied the expression and prognostic relevance of HIF-1α and its downstream targets in phyllodes tumors and fibroadenomas of the breast.
The expression of HIF-1α, CAIX, VEGF and p53 was investigated by immunohistochemistry in a group of 37 primary phyllodes tumors and 30 fibroadenomas with known clinical follow-up. The tumor microvasculature was visualized by immunohistochemistry for CD31. Proliferation was assessed by Ki67 immunostaining and mitotic counts. Being biphasic tumors, immunoquantification was performed in the stroma and epithelium.
Only two fibroadenomas displayed low-level stromal HIF-1α reactivity in the absence of CAIX expression. Stromal HIF-1α expression was positively correlated with phyllodes tumor grade (P = 0.001), with proliferation as measured by Ki67 expression (P < 0.001) and number of mitoses (P < 0.001), with p53 accumulation (P = 0.003), and with global (P = 0.015) and hot-spot (P = 0.031) microvessel counts, but not with CAIX expression. Interestingly, concerted CAIX and HIF-1α expression was frequently found in morphologically normal epithelium of phyllodes tumors. The distance from the epithelium to the nearest microvessels was higher in phyllodes tumors as compared with in fibroadenomas. Microvessel counts as such did not differ between fibroadenomas and phyllodes tumors, however. High expression of VEGF was regularly found in both tumors, with only a positive relation between stromal VEGF and grade in phyllodes tumors (P = 0.016). Stromal HIF-1α overexpression in phyllodes tumors was predictive of disease-free survival (P = 0.032).
These results indicate that HIF-1α expression is associated with diminished disease-free survival and may play an important role in stromal progression of breast phyllodes tumors. In view of the absence of stromal CAIX expression in phyllodes tumors, stromal upregulation of HIF-1α most probably arises from hypoxia-independent pathways, with p53 inactivation as one possible cause. In contrast, coexpression of HIF-1α and CAIX in the epithelium in phyllodes tumors points to epithelial hypoxia, most probably caused by relatively distant blood vessels. On the other hand, HIF-1α and CAIX seem to be of minor relevance in breast fibroadenomas.
Expression of vascular endothelial growth factor (VEGF) is induced in cells exposed to hypoxia or ischemia. Neovascularization stimulated by VEGF occurs in several important clinical contexts, including myocardial ischemia, retinal disease, and tumor growth. Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix protein that activates transcription of the human erythropoietin gene in hypoxic cells. Here we demonstrate the involvement of HIF-1 in the activation of VEGF transcription. VEGF 5'-flanking sequences mediated transcriptional activation of reporter gene expression in hypoxic Hep3B cells. A 47-bp sequence located 985 to 939 bp 5' to the VEGF transcription initiation site mediated hypoxia-inducible reporter gene expression directed by a simian virus 40 promoter element that was otherwise minimally responsive to hypoxia. When reporters containing VEGF sequences, in the context of the native VEGF or heterologous simian virus 40 promoter, were cotransfected with expression vectors encoding HIF-1alpha and HIF-1beta (ARNT [aryl hydrocarbon receptor nuclear translocator]), reporter gene transcription was much greater in both hypoxic and nonhypoxic cells than in cells transfected with the reporter alone. A HIF-1 binding site was demonstrated in the 47-bp hypoxia response element, and a 3-bp substitution eliminated the ability of the element to bind HIF-1 and to activate transcription in response to hypoxia and/or recombinant HIF-1. Cotransfection of cells with an expression vector encoding a dominant negative form of HIF-1alpha inhibited the activation of reporter transcription in hypoxic cells in a dose-dependent manner. VEGF mRNA was not induced by hypoxia in mutant cells that do not express the HIF-1beta (ARNT) subunit. These findings implicate HIF-1 in the activation of VEGF transcription in hypoxic cells.
Aims: To measure vascular endothelial growth factor (VEGF-A) mRNA in a large, diverse cohort of tumours and to investigate whether VEGF-A expression is associated with markers of hypoxia, including hypoxia inducible factor 1α (HIF-1α) and carbonic anhydrase IX (CA9).
Methods: The expression of VEGF-A and CA9 was assessed in 5067 fresh frozen human tissue samples and 238 cell lines by DNA microarray analysis. In addition, tissue microarrays were constructed from 388 malignancies to investigate the expression of VEGF-A and HIF-1α by in situ hybridisation and immunohistochemistry, respectively.
Results: VEGF-A was significantly upregulated in primary malignancies of the breast, cervix, colon and rectum, oesophagus, head and neck, kidney, ovary, skin, urinary system, and white blood cells by DNA microarray analysis. However, VEGF-A expression only correlated with CA9 expression in renal tissues. In the tissue microarrays, HIF-1α positive cores showed a significant increase in VEGF-A expression in lung, ovary, soft tissue, and thyroid malignancies.
Conclusions: The expression of VEGF-A is upregulated in a large proportion of human malignancies, and may be associated with markers of hypoxia. VEGF-A expression can be induced in the absence of hypoxia and hypoxia does not always provoke VEGF-A upregulation in tumours.
vascular endothelial growth factor; hypoxia inducible factor one alpha; carbonic anhydrase IX; angiogenesis; neoplasia
Hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) are important angiogenic factors in human cancers. Relative to VEGF-C, prognostic significance of VEGF-D expression and its association with HIF-1α expression remain elusive in esophageal squamous cell cancer (ESCC). We studied expression of HIF-1α and VEGF-D using immunohistochemistry in 85 resected ESCC specimens and correlated results with patients' clinicopathologic parameters and survival. Association between expression of HIF-1α and VEGF-D was investigated using a concordance analysis. High expression of HIF-1α and VEGF-D was observed in 52 (61.2%) and 56 (65.9%) patients, respectively. HIF-1α expression correlated well with tumor stage (P = 0.041), whereas VEGF-D expression correlated with tumor stage (P = 0.027) and N status (P = 0.019). Groups of high HIF-1α and VEGF-D showed worse survivals than those of low expression (P = 0.002 and 0.001, respectively). Multivariate analysis supported expression of HIF-1α and VEGF-D as significant survival predictors (P = 0.044 and 0.035, respectively). A concordance rate of 69.5% was observed between expression of HIF-1α and VEGF-D. In conclusion, protein expression of HIF-1α and VEGF-D are independent prognostic predictors. An association between expression of HIF-1α and VEGF-D suggests that these two angiogenic factors are essential in progression of ESCC.
vascular endothelial growth factor (VEGF)-D; esophageal squamous cell carcinoma; prognosis; survival
Hemangiomas are tumors formed by hyper-proliferation of vascular endothelial cells. This is caused by elevated vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we show that elevated VEGF levels produced by hemangioma endothelial cells are reduced by the mTOR inhibitor rapamycin. mTOR activates p70S6K, which controls translation of mRNA to generate proteins such as hypoxia inducible factor-1 (HIF-1). VEGF is a known HIF-1 target gene, and our data show that VEGF levels in hemangioma endothelial cells are reduced by HIF-1α siRNA. Over-expression of HIF-1α increases VEGF levels and endothelial cell proliferation. Furthermore, both rapamycin and HIF-1α siRNA reduce proliferation of hemangioma endothelial cells. These data suggest that mTOR and HIF-1 contribute to hemangioma endothelial cell proliferation by stimulating an autocrine loop of VEGF signaling. Furthermore, mTOR and HIF-1 may be therapeutic targets for the treatment of hemangiomas.
Hypoxia-inducible factor-1α (HIF-1α) increases transcription of the vascular endothelial growth factor (VEGF) gene. Inhibition of VEGF abolishes VEGF mediated induction of HIF-1α. Recent reports suggested that HIF-1α also mediated the induction of class III β-tubulin (TUBB3) in hypoxia. TUBB3 confers resistance to taxanes. Inhibition of VEGF may decrease the expression of HIF-1α and TUBB3. This study was undertaken to investigate the roles of vascular endothelial growth factor receptor (VEGFR) in gastric cancer cell behavior and to identify methods to overcome paclitaxel resistance in vitro.
Materials and Methods
The protein expression levels of HIF-1α and TUBB3 were measured in human gastric cancer cell lines (AGS) under normoxic and hypoxic conditions. The relationship between TUBB3 and paclitaxel resistance was assessed with small interfering TUBB3 RNA. AGS cells were treated with anti-VEGFR-1, anti-VEGFR-2, placental growth factor (PlGF), bevacizuamb, and paclitaxel.
Hypoxia induced paclitaxel resistance was decreased by knockdown of TUBB3. Induction of HIF-1α and TUBB3 in AGS is VEGFR-1 mediated and PlGF dependent. Hypoxia-dependent upregulation of HIF-1α and TUBB3 was reduced in response to paclitaxel treatment. Expressions of HIF-1α and TUBB3 were most decreased when AGS cells were treated with a combination of paclitaxel and anti-VEGFR-1. AGS cell cytotoxicity was most increased in response to paclitaxel, anti-VEGFR-1, and anti-VEGFR-2.
We suggest that blockade of VEGFR-1 and VEGFR-2 enhances paclitaxel sensitivity in TUBB3-expressing gastric cancer cells.
Vascular endothelial growth factor; Hypoxia-inducible factor 1 alpha; class III beta-tubulin; paclitaxel
Hypoxia plays a key role in ischaemic and neovascular disorders of the retina. Cellular responses to oxygen are mediated by hypoxia-inducible transcription factors (HIFs) that are stabilised in hypoxia and induce the expression of a diverse range of genes. The purpose of this study was to define the cellular specificities of HIF-1alpha and HIF-2alpha in retinal ischaemia, and to determine their correlation with the pattern of retinal hypoxia and the expression profiles of induced molecular mediators.
We investigated the tissue distribution of retinal hypoxia during oxygen-induced retinopathy (OIR) in mice using the bio-reductive drug pimonidazole. We measured the levels of HIF-1alpha and HIF-2alpha proteins by Western blotting and determined their cellular distribution by immunohistochemistry during the development of OIR. We measured the temporal expression profiles of two downstream mediators, vascular endothelial growth factor (VEGF) and erythropoietin (Epo) by ELISA. Pimonidazole labelling was evident specifically in the inner retina. Labelling peaked at 2 hours after the onset of hypoxia and gradually declined thereafter. Marked binding to Müller glia was evident during the early hypoxic stages of OIR. Both HIF-1alpha and HIF-2alpha protein levels were significantly increased during retinal hypoxia but were evident in distinct cellular distributions; HIF-1alpha stabilisation was evident in neuronal cells throughout the inner retinal layers whereas HIF-2alpha was restricted to Müller glia and astrocytes. Hypoxia and HIF-alpha stabilisation in the retina were closely followed by upregulated expression of the downstream mediators VEGF and EPO.
Both HIF-1alpha and HIF-2alpha are activated in close correlation with retinal hypoxia but have contrasting cell specificities, consistent with differential roles in retinal ischaemia. Our findings suggest that HIF-2alpha activation plays a key role in regulating the response of Müller glia to hypoxia.
Hypoxia-inducible factor 1 (HIF-1) is a transcriptional activator composed of HIF-1α and HIF-1β subunits. Several dozen HIF-1 targets are known, including the gene encoding vascular endothelial growth factor (VEGF). Under hypoxic conditions, HIF-1α expression increases as a result of decreased ubiquitination and degradation. The tumor suppressors VHL (von Hippel-Lindau protein) and p53 target HIF-1α for ubiquitination such that their inactivation in tumor cells increases the half-life of HIF-1α. Increased phosphatidylinositol 3-kinase (PI3K) and AKT or decreased PTEN activity in prostate cancer cells also increases HIF-1α expression by an undefined mechanism. In breast cancer, increased activity of the HER2 (also known as neu) receptor tyrosine kinase is associated with increased tumor grade, chemotherapy resistance, and decreased patient survival. HER2 has also been implicated as an inducer of VEGF expression. Here we demonstrate that HER2 signaling induced by overexpression in mouse 3T3 cells or heregulin stimulation of human MCF-7 breast cancer cells results in increased HIF-1α protein and VEGF mRNA expression that is dependent upon activity of PI3K, AKT (also known as protein kinase B), and the downstream kinase FRAP (FKBP-rapamycin-associated protein). In contrast to other inducers of HIF-1 expression, heregulin stimulation does not affect the half-life of HIF-1α but instead stimulates HIF-1α synthesis in a rapamycin-dependent manner. The 5′-untranslated region of HIF-1α mRNA directs heregulin-inducible expression of a heterologous protein. These data provide a molecular basis for VEGF induction and tumor angiogenesis by heregulin-HER2 signaling and establish a novel mechanism for the regulation of HIF-1α expression.
Hypoxia-induced factor-1 alpha (HIF-1α) affects many effector molecules and regulates tumor lymphangiogenesis and angiogenesis during hypoxia. The aim of this study was to investigate the role of HIF-1α in the regulation of vascular endothelial growth factor C (VEGF-C) expression and its effect on lymphangiogenesis and angiogenesis in breast cancer. Lymphatic vessel density (LVD), microvessel density (MVD) and the expressions of HIF-1α and VEGF-C proteins were evaluated by immunohistochemistry in 75 breast cancer samples. There was a significant correlation between HIF-1α and VEGF-C (P = 0.014, r = 0.273, Spearman's coefficient of correlation). HIF-1α and VEGF-C overexpression was significantly correlated with higher LVD (P = 0.003 and P = 0.017, respectively), regional lymph nodal involvement (P = 0.002 and P = 0.004, respectively) and advanced tumor, node, metastasis (TNM) classification (P = 0.001 and P = 0.01, respectively). Higher MVD was observed in the group expressing higher levels of HIF-1α and VEGF-C (P = 0.033 and P = 0.037, respectively). Univariate analysis showed shorter survival time in patients expressing higher levels of HIF-1α and VEGF-C. HIF-1α was also found to be an independent prognostic factor of overall survival in multivariate analysis. The results suggest that HIF-1α may affect VEGF-C expression, thus acting as a crucial regulator of lymphangiogenesis and angiogenesis in breast cancer. This study highlights promising potential of HIF-1α as a therapeutic target against tumor lymph node metastasis.
HIF-1α; VEGF-C; lymphangiogenesis; angiogenesis; breast cancer
Hypoxia inducible factor-1α (HIF-1α) is a proangiogenic transcription factor stabilized and activated under hypoxia. It regulates the expression of numerous target genes, including vascular endothelial growth factor (VEGF) and other cytoprotective proteins. In this study, we hypothesized that bone marrow stem cells (BMSCs) secrete growth factors which protect cardiomyocytes via HIF-1α pathway.
BMSCs were obtained from transgenic mice overexpressing green fluorescent protein (GFP). The study was carried out in vitro using co-culture of BMSCs with cardiomyocytes. LDH release, MTT uptake, DNA fragmentation and annexin-V positive cells were used as cell injury markers. The level of HIF-1α protein as well as its activated form and VEGF were measured by ELISA. The expression of HIF-1α and VEGF in BMSCs were analyzed by quantitative PCR and cellular localization was determined by immunohistochemistry.
LDH release was increased and MTT uptake was decreased after exposure of cardiomyocytes to hypoxia for 24 hours, which were prevented by co-culturing cardiomyocytes with BMSCs. Cardiomyocyte apoptosis induced by hypoxia and H2O2 was also reduced by co-culture with BMSCs. VEGF release from BMSCs was significantly increased in parallel with high level of HIF-1α in BMSCs following anoxia or hypoxia in time-dependent manner. Although no significant up-regulation could be seen in HIF-1α mRNA, HIF-1α protein and its activated form were markedly increased and translocated to the nucleus or peri-nuclear area. The increase and translocation of HIF-1α in BMSCs were completely blocked by 2-methoxyestradiol (2-ME2; 5μmol), a HIF-1α inhibitor. Moreover, the protection of cardiomyocytes by BMSC and VEGF secretion were abolished by neutralizing HIF-1α antibodies in a concentration dependent manner (200~ 3200ng/ml).
Bone marrow stem cells protect cardiomyocytes by up-regulation of VEGF via activating HIF-1α.
bone marrow stem cells; HIF-1α; VEGF; cardiomyocytes; hypoxia; HIF-1α neutralizing antibody
Hypoxia-inducible factor 1-alpha (HIF-1α) is a subunit of HIF-l and thought to be able to protect hypoxic cells from apoptosis or necrosis under ischemic and anoxic conditions. This study aimed to investigated whether recombinant adenovirus vector over-expressing HIF-lα could affect apoptosis-related proteins (Bcl-2 and Bax) and vascular endothelial growth factor (VEGF) in a rat spinal cord injury (SCI) model. A total of 60 male SD rats were divided into 4 groups: Sham, Control, Ad-Blank and Ad-HIF-1α groups. 1, 3, 7, 14, 28 days after surgery, the behavioral recovery was evaluated with BBB scales. Then, rats were sacrificed and the spinal cord was collected for detection of Bcl-2, Bax and VEGF expressions by immunohistochemistry. Results showed the Bcl-2, Bax, VEGF and HIF-lα expressions increased in animals with SCI, but the increase in Bcl-2, VEGF and HIF-lα expressions were higher in Ad-HIF-1α group when compared with other groups, but Bax expression decreased significantly. In addition, administration of Ad-HIF-1α significantly reduced apoptotic cells and promoted the recovery of neurological function. In conclusion, administration of Ad-HIF-1α after SCI could ameliorate neuronal apoptosis and promote angiogenesis in rats. Our study provides a basis for further exploration of the relationship between HIF1α and SCI.
HIF-1α; spinal cord injury; apoptosis; vascular endothelial growth factor
Hypoxia-inducible factor-1α (HIF-1α) has been reported to transactivate the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2), which are frequently overexpressed in numerous types of cancer and are known to be significant regulators of angiogenesis. Few studies have investigated the role of these factors in solid tumors, particularly chordomas, which are rare tumors that are thought to originate from notochordal remnants. To clarify whether HIF-1α is involved in angiogenesis in chordoma tissues, we examined the expression of HIF-1α, VEGF and MMP-2 with immunohistochemistry using a tissue microarray containing 35 chordoma samples. The results indicated that HIF-1α, VEGF and MMP-2 are expressed in the majority of chordoma samples. VEGF expression was significantly correlated with HIF-1α and MMP-2 expression, as well as with microvessel density (MVD). However, the prognosis of the chordoma patients was not significantly associated with the expression of these factors, but was associated with MVD. The results therefore showed that there is a correlation between the expression of HIF-1α, VEGF and MMP-2 in chordomas and that the angiogenic process is a potential therapeutic target for chordomas.
chordoma; hypoxia-inducible factor-1α; vascular endothelial growth factor; matrix metalloproteinase-2
Tumour-associated angiogenesis is partly regulated by the hypoxia-inducible factor (HIF) pathway. Endocrine tumours are highly vascularised and the molecular mechanisms of their angiogenesis are not fully delineated. The aim of this study is to evaluate angiogenesis and expression of HIF-related molecules in a series of patients with pancreatic endocrine tumours (PETs). The expression of vascular endothelial growth factor (VEGF), HIF-1α, HIF-2α and carbonic anhydrase 9 (CA9) was examined by immunohistochemistry in 45 patients with PETs and compared to microvascular density (MVD), endothelial proliferation, tumour stage and survival. Microvascular density was very high in PETs and associated with a low endothelial index of proliferation. Microvascular density was significantly higher in benign PETs than in PETs of uncertain prognosis, well-differentiated and poorly differentiated carcinomas (mean values: 535, 436, 252 and 45 vessels mm−2, respectively, P<0.0001). Well-differentiated tumours had high cytoplasmic VEGF and HIF-1α expression. Poorly differentiated carcinomas were associated with nuclear HIF-1α and membranous CA9 expression. Low MVD (P=0.0001) and membranous CA9 expression (P=0.0004) were associated with a poorer survival. Contrary to other types of cancer, PETs are highly vascularised, but poorly angiogenic tumours. As they progress, VEGF expression is lost and MVD significantly decreases. The regulation of HIF signalling appears to be specific in pancreatic endocrine tumours.
pancreas; endocrine tumours; angiogenesis; Hypoxia; HIF
Angiogenesis is a critical step required for sustained tumor growth and tumor progression. The stimulation of endothelial cells by cytokines secreted by tumor cells such as vascular endothelial growth factor (VEGF) induces their proliferation and migration. This is a prominent feature of high-grade gliomas. The secretion of VEGF is greatly upregulated under conditions of hypoxia because of the transcription factor hypoxia-inducible factor (HIF)-1α, which controls the expression of many genes, allowing rapid adaptation of cells to their hypoxic microenvironment. Flavopiridol, a novel cyclin-dependent kinase inhibitor, has been attributed with antiangiogenic properties in some cancer cell lines by its ability to inhibit VEGF production. Here, we show that flavopiridol treatment of human U87MG and T98G glioma cell lines decreases hypoxia-mediated HIF-1α expression, VEGF secretion, and tumor cell migration. These in vitro results correlate with reduced vascularity of intracranial syngeneic GL261 gliomas from animals treated with flavopiridol. In addition, we show that flavopiridol downregulates HIF-1α expression in the presence of a proteasome inhibitor, an agent that normally results in the accumulation and overexpression of HIF-1α. The potential to downregulate HIF-1α expression with flavopiridol treatment in combination with a proteasome inhibitor makes this an extremely attractive anticancer treatment strategy for tumors with high angiogenic activity, such as gliomas.
flavopiridol; proteasome inhibitor; hypoxia; HIF-1α; VEGF; glioma
Hypoxia-inducible factor-1α (HIF-1α) is a highly oxygen sensitive bHLH protein that is part of the heterodimeric HIF-1 transcription factor. Under hypoxic stress, HIF-1 activity is induced to control expression of multiple downstream target genes, including vascular endothelial growth factor (VEGF). The normal epidermis exists in a constant mild hypoxic microenvironment and constitutively expresses HIF-1α and HIF-2α. Expression of HIF-1α and/or HIF-2α has been suggested to correlate with the increased malignant potential of melanocytes, therefore, failures of melanoma therapies may be partially linked to high HIF activity. Notably, melanomas that have the V600E BRAF mutation exhibit increased HIF-1α expression. We have utilized a bioinformatics approach to identify putative hypoxia response elements (HREs) in a set of genes known to participate in the process of melanogenesis (includingTRPM1, SLC45A2, HRAS, C-KIT, PMEL and CRH). While some of the mechanistic links between these genes and the HIF pathway have been previously explored, others await further investigation. Although agents targeting HIF activity have been proposed as novel treatment modalities for melanoma, there are currently no clinical trials in progress to test their efficacy in melanoma.
HIF-1; keratinocytes; melanocytes; melanoma; oncogenesis; skin
Hypoxia inducible factor (HIF)-1 plays an important role in cellular adaptation to hypoxia by activating oxygen-regulated genes such as vascular endothelial growth factor (VEGF) and erythropoietin. Sputum VEGF levels are reported to be decreased in COPD, despite hypoxia. Here we show that patients with COPD fail to induce HIF-1α and VEGF under hypoxic condition because of a reduction in histone deacetylase (HDAC) 7.
Peripheral blood mononuclear cells (PBMCs) were obtained from patients with moderate to severe COPD (n = 21), smokers without COPD (n = 12), and nonsmokers (n = 15). PBMCs were exposed to hypoxia (1% oxygen, 5% CO2, and 94% N2) for 24 h, and HIF-1α and HDAC7 protein expression in nuclear extracts were determined by sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE)/Western blotting.
HIF-1α was significantly induced by hypoxia in each group when compared with the normoxic condition (12-fold induction in nonsmokers, 24-fold induction in smokers without COPD, fourfold induction in COPD), but induction of HIF-1α under hypoxia was significantly lower in patients with COPD than in nonsmokers and smokers without COPD (P < .05 and P < .01, respectively). VEGF messenger RNA detected by quantitative real-time polymerase chain reaction was correlated with HIF-1α protein in nuclei (r = 0.79, P < .05), and HDAC7 protein expression was correlated with HIF-1α protein in nuclei (r = 0.46, P < .05). HDAC7 knockdown inhibited hypoxia-induced HIF-1α activity in U937 cells, and HIF-1α nuclear translocation and HIF-1α binding to the VEGF promoter in A549 cells.
HDAC7 reduction in COPD causes a defect of HIF-1α induction response to hypoxia with impaired VEGF gene expression. This poor cellular adaptation might play a role in the pathogenesis of COPD.
Glioblastomas are lethal cancers characterized by florid angiogenesis promoted in part by glioma stem cells (GSCs). As hypoxia regulates angiogenesis, we examined hypoxic responses in GSCs. We now demonstrate that hypoxia-inducible factor HIF2α and multiple HIF-regulated genes are preferentially expressed in GSCs in comparison to nonstem tumor cells and normal neural progenitors. In tumor specimens, HIF2α co-localizes with cancer stem cell markers. Targeting HIFs in GSCs inhibits self-renewal, proliferation and survival in vitro, and attenuates tumor initiation potential of GSCs in vivo. Analysis of a molecular database reveals that HIF2A expression correlates with poor glioma patient survival. Our results demonstrate that GSCs differentially respond to hypoxia with distinct HIF induction patterns and HIF2α may represent a promising target for anti-glioblastoma therapies.
Recent evidence supports the presence of cancer stem cell populations that contribute to tumor progression through preferential resistance to radiation and chemotherapy, and promotion of tumor angiogenesis, invasion, and metastasis. Therefore, the elucidation of molecular regulators of cancer stem cells may translate into improved anti-neoplastic therapies. Our work demonstrates that cancer stem cells derived from glioblastomas differentially respond to hypoxia with a distinct induction of HIF2α. We find that HIFs are critical to cancer stem cell maintenance and angiogenic drive, and that expression of HIF2α is significantly associated with poor glioma patient survival. These data further suggest that anti-angiogenic therapies can be designed to target cancer stem cell specific molecules involved in neoangiogenesis, including HIF2α and its regulated factors.
The importance of hypoxia-inducible factor (HIF) in promoting angiogenesis and vasculogenesis during wound healing has been demonstrated. It is widely accepted that HIF activity can be promoted by many factors, including hypoxia in the wound or cytokines from inflammatory cells infiltrating the wound. However, there has not been a systematic exploration of the relationship between HIF activity and hypoxia in the burn wound. The location of the hypoxic tissue has not been clearly delineated. The time course of the appearance of hypoxia and the increased activity of HIF and appearance of HIF’s downstream transcription products has not been described. The aim of this study was to utilize pimonidazole, a specific tissue hypoxia marker, to characterize the spatial and temporal course of hypoxia in a murine burn model and correlate this with the appearance of HIF-1α and its important angiogenic and vasculogenic transcription products VEGF and SDF-1. Hypoxia was found in the healing margin of burn wounds beginning at 48 hours after burn and peaking at day 3 after burn. On sequential sections of the same tissue block, positive staining of HIF-1α, SDF-1, and VEGF all occurred at the leading margin of the healing area and peaked at day 3, as did hypoxia. Immunohistochemical analysis was used to explore the characteristics of the hypoxic region of the wound. The localization of hypoxia was found to be related to cell growth and migration, but not to proliferation or inflammatory infiltration.
ypoxia; Hypoxia-inducible factor -10α; Burn; Wound Healing; Ki67 Cell Proliferation; Keratin17
► AAI increases whereas OTA decreases production of proangiogenic VEGF. ► The upregulation of VEGF expression by AAI is caused by induction of SP-1 and HIFs. ► Hypoxia prevents OTA-diminished VEGF production ► The effect of hypoxia on OTA-reduced VEGF is mediated by HIF-2α but not HIF-1α.
Aristolochic acid I (AAI) and ochratoxin A (OTA) cause chronic kidney diseases. Recently, the contribution of hypoxic injuries and angiogenic disturbances to nephropathies has been suggested, but underlying mechanisms have not been fully clarified yet.
In porcine kidney epithelial cell line, LLC-PK1 cells, treatment with non-toxic doses of AAI increased whereas with OTA decreased production of vascular endothelial growth factor (VEGF), the angiogenic factor with well-defined functions in kidney. Moreover, the activity of transcription factors regulating VEGF expression was differentially affected by examined compounds. Activity of hypoxia inducible factors (HIFs) and SP-1 was increased by AAI but diminished by OTA. Interestingly, AP-1 activity was inhibited while NFκB was not influenced by both toxins. Mithramycin A, a SP-1 inhibitor, as well as chetomin, an inhibitor of HIFs, reversed AAI-induced up-regulation of VEGF synthesis, indicating the importance of SP-1 and HIFs in this effect. Additionally, adenoviral overexpression of HIF-2α but not HIF-1α prevented OTA-diminished VEGF production suggesting the protective effect of this isoform towards the consequences exerted by OTA.
These observations provide new insight into complex impact of AAI and OTA on angiogenic gene regulation. Additionally, it adds to our understanding of hypoxia influence on nephropathies pathology.
AA, aristolochic acid; AAI, aristolochic acid I; AAII, aristolochic acid II; AA-ATN, aristolochic acid-induced acute tubular necrosis; AAN, aristolochic acid-induced nephropathy; AdGFP, adenoviral vectors containing GFP cDNA; AdHIF-1,-2α, adenoviral vectors containing HIF-1,-2α cDNA; β-gal, β-galactosidase; BEN, Balkan endemic nephropathy; CKDs, chronic kidney diseases; EMT, epithelial to mesenchymal cell transformation; GFP, green fluorescent protein; HIF, hypoxia inducible factor; HRE, hypoxia responsive element; HRP, horseradish peroxidase; LDH, lactate dehydrogenase; LLC-PK1, porcine kidney epithelial cell line; IARC, The International Agency for Research on Cancer; OTA, ochratoxin A; ROS, reactive oxygen species; RT, room temperature; TGFβ, transforming growth factor β; VEGF, vascular endothelial growth factor; Nephropathy; Kidney diseases; Vascular endothelial growth factor; Angiogenesis; Hypoxia; LLC-PK1
Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor composed of HIF-1α and HIF-1β that is the central regulator of responses to hypoxia. The specific binding of HIF-1 to the hypoxia-responsive element (HRE) induces the transcription of genes that respond to hypoxic conditions, including vascular endothelial growth factor (VEGF). Here we report that expression of HIF-1α is increased in diverse Epstein-Barr virus (EBV)-infected type II and III cell lines, which express EBV latent membrane protein 1 (LMP1), the principal EBV oncoprotein, as well as other latency proteins, but not in the parental EBV-negative cell lines. We show first that transfection of an LMP1 expression plasmid into Ad-AH cells, an EBV-negative nasopharyngeal epithelial cell line, induces synthesis of HIF-1α protein without increasing its stability or mRNA level. The mitogen-activated protein kinase (MAPK) kinase inhibitor PD98059 markedly reduces induction of HIF-1α by LMP1. Catalase, an H2O2 scavenger, strongly suppresses LMP1-induced production of H2O2, which results in a decrease in the expression of HIF-1α induced by LMP1. Inhibition of the NF-κB, c-jun N-terminal kinase, p38 MAPK, and phosphatidylinositol 3-kinase pathways did not affect HIF-1α expression. Moreover, LMP1 induces HIF-1 DNA binding activity and upregulates HRE and VEGF promoter transcriptional activity. Finally, LMP1 increases the appearance of VEGF protein in extracellular fluids; induction of VEGF is suppressed by PD98059 or catalase. These results suggest that LMP1 increases HIF-1 activity through induction of HIF-1α protein expression, which is controlled by p42/p44 MAPK activity and H2O2. The ability of EBV, and specifically its major oncoprotein, LMP1, to induce HIF-1α along with other invasiveness and angiogenic factors reported previously discloses additional oncogenic properties of this tumor virus.
In human neuroblastoma, amplification of the MYCN gene predicts poor prognosis and resistance to therapy. Because hypoxia contributes to aggressive tumor phenotypes, predominantly via two structurally related hypoxia inducible factors, HIF-1α and HIF-2α, we examined hypoxia responses in MYCN amplified neuroblastoma cells. We demonstrate here that HIF-1α, but not HIF-2α, is preferentially expressed in both MYCN amplified neuroblastoma cells and primary tumors in comparison to samples without MYCN amplification. Our results showed that interplay between N-Myc and HIF-1α plays critical roles in neuroblastoma. For example, high levels of N-Myc override HIF-1α inhibition of cell cycle progression, enabling continued proliferation under hypoxia. Furthermore, both HIF-1α and N-Myc are essential for the Warburg effect (aerobic glycolysis) in neuroblastomas by activating the transcription of multiple glycolytic genes. Of note, expression of Phosphoglycerate Kinase 1 (PGK1), Hexokinase 2 (HK2) and Lactate Dehydrogenase A (LDHA), were each significantly higher in MYCN amplified neuroblastomas compared to tumors without MYCN amplification. Interestingly, MYCN amplified neuroblastoma cells are “addicted” to LDHA enzymatic activity, as its depletion completely inhibits tumorigenesis in vivo. Thus, our results provide mechanistic insights explaining how MYCN amplified neuroblastoma cells contend with hypoxic stress and paradoxically how hypoxia contributes to neuroblastoma aggressiveness through combinatorial effects of N-Myc and HIF-1α. These results also suggest LDHA represents a novel, pharmacologically tractable target for neuroblastoma therapeutics.
N-Myc; hypoxia inducible factor; neuroblastoma; tumorigenesis; Warburg effect
The ubiquitin-proteasome pathway (UPP) is involved in regulation of multiple cellular processes. Hypoxia-inducible factor 1α (HIF-1α) is a prototypic target of the UPP and, as such, is stabilized under conditions of proteasomal inhibition. Using carbonic anhydrase IX (CAIX) and vascular endothelial growth factor (VEGF) expression as paradigmatic markers of HIF-1 activity, we found that proteasomal inhibitors (PI) abrogated hypoxia-induced CAIX expression in all cell lines tested and VEGF expression in two out of three. Mapping of the inhibitory effect identified the C-terminal activation domain (CAD) of HIF-1α as the primary target of PI. PI specifically inhibited the HIF-1α CAD despite activating the HIF-1α coactivator p300 and another p300 cysteine/histidine-rich domain 1-dependent transcription factor, STAT-2. Coimmunoprecipitation and glutathione S-transferase pull downs indicated that PI does not disrupt interactions between HIF-1α and p300. Mutational analysis failed to confirm involvement of sites of known or putative posttranslational modifications in regulation of HIF-1α CAD function by PI. Our data provide evidence for the counterintuitive hypothesis that inhibition of HIF-1 function could be responsible for at least some of the antitumor effects of proteasomal inhibition. Further studies of the mechanism of the PI-induced attenuation of HIF-1α will provide important, potentially novel insight into regulation of HIF-1 activity and possibly identify new targets for HIF-directed therapy.
Hypoxia‐inducible factor (HIF) is a common transcription factor for many angiogenic proteins. Retinal pigment epithelial (RPE) cells are an important source of angiogenic factors in the retina. The expression of HIF, its regulation by proline hydroxylase (PHD) enzymes, and its downstream regulation of angiogenic factors like vascular endothelial growth factor (VEGF) and erythropoietin (EPO) was studied in RPE cells in order to determine some of the molecular mechanisms underlying ischaemic retinal disease.
ARPE‐19 cells were cultured for various times under hypoxic conditions. Cellular HIF and PHD isoforms were analysed and quantified using western blot and densitometry. VEGF and EPO secreted into the media were assayed using enzyme‐linked immunosorbent assay (ELISA). Messenger RNA (mRNA) was quantified using real‐time quantitative reverse transcriptase polymerase chain reaction (qPCR). RNA interference was achieved using siRNA techniques.
HIF‐1α was readily produced by ARPE‐19 cells under hypoxia, but HIF‐2α and HIF‐3α could not be detected even after HIF‐1α silencing. HIF‐1α protein levels showed an increasing trend for the first 24 h while HIF‐1α mRNA levels fluctuated during this time. After 36 h HIF‐1α protein levels declined to baseline levels, a change that was coincident with a rise in both PHD2 and PHD3. Silencing HIF‐1α significantly decreased VEGF secretion. Significant production of EPO could not be detected at the protein or mRNA level.
HIF‐1α appears to be the main isoform of HIF functioning in ARPE‐19 cells. Under hypoxia, HIF‐1α levels are likely self‐regulated by a feedback loop that involves both transcriptional and post‐translational mechanisms. VEGF production by human RPE cells is regulated by HIF‐1α. EPO was not produced in significant amounts by RPE cells under hypoxic conditions, suggesting that other cells and/or transcription factors in the retina are responsible for its production.
diabetic retinopathy; VEGF; erythropoietin; hypoxia‐inducible factor; proline hydroxylase