Intratumor hypoxia has long been considered a driving force of tumor progression and a negative prognostic factor in human cancers. The discovery of hypoxia inducible factors (HIFs), which mediate transcriptional responses to changes in oxygen levels, has renewed enthusiasm for the discovery and development of targeted therapies exploiting the hypoxic tumor microenvironment. In spite of an ever increasing number of putative small molecule inhibitors of HIF, only few are progressing through preclinical and early clinical development. In this review, we will focus primarily on: 1) HIF inhibitors that have been more recently described and 2) small molecules targeting HIF that are being tested in early clinical trials or that are already approved for use in patients. A rigorous “validation” of HIF targeted therapies in relevant preclinical models and eventually in pharmacodynamic-based early clinical trials is essential for “credentialing” HIF-1 as a legitimate target that can be pharmacologically modulated in cancer patients.
HIF-1; hypoxia; cancer therapy
Hypoxia is a common condition found in a wide range of solid tumors and is often associated with poor prognosis. Hypoxia increases tumor glycolysis, angiogenesis and other survival response as well as invasion and metastasis by activating relevant gene expressions through hypoxia-inducible factors (HIFs). HIF-1α and HIF-2α undergo oxygen-dependent regulation and their overexpression is frequently associated with metastasis and poor clinical outcomes. Recent studies show that each step of the metastasis process, from the initial epithelial-mesenchymal transition to the ultimate organotropic colonization, can potentially be regulated by hypoxia, suggesting a master regulator role of hypoxia and HIFs in metastasis. Furthermore, modulation of cancer stem cell self-renewal by HIFs may also contribute to the hypoxia-regulated metastasis program. Hypoxia-induced metastatic phenotype may be one of the reasons for the modest efficacy of antiangiogenic therapies and may well explain the recent provocative findings that antiangiogenic therapy increased metastasis in preclinical models. Multiple approaches to targeting hypoxia and HIFs, including HIF inhibitors, hypoxia-activated bioreductive prodrugs and gene therapies may become effective treatments to prevent or reduce metastasis.
Most solid tumors and their metastases experience periods of low oxygen or hypoxia, which is of major clinical significance as it promotes both tumor progression and resistance to therapy. Critical mediators of the hypoxic response are the hypoxia-inducible factors HIF-1α and HIF-2α. The HIFs are nonredundant and regulate both overlapping and unique downstream target genes. Here, we describe a novel mechanism for the switch between HIF-1α– and HIF-2α–dependent transcription during tumor hypoxia caused by the hypoxia associated factor (HAF). HAF is overexpressed in a variety of tumors and its levels are decreased during acute hypoxia, but increased following prolonged hypoxia. We have previously identified HAF as an E3 ubiquitin ligase that binds and ubiquitinates HIF-1α by an oxygen and pVHL-independent mechanism, thus targeting HIF-1α for proteasomal degradation. Here, we show that HAF also binds to HIF-2α, but at a different site than HIF-1α, and increases HIF-2α transactivation without causing its degradation. HAF, thus, switches the hypoxic response of the cancer cell from HIF-1α–dependent to HIF-2α–dependent transcription and activates genes involved in invasion such as MMP9, PAI-1, and the stem cell factor OCT-3/4. The switch to HIF-2α–dependent gene expression caused by HAF also promotes an enriched tumor stem cell population, resulting in highly aggressive tumors in vivo. Thus, HAF, by causing a switch from a HIF-1α– to HIF-2α–dependent response to hypoxia, provides a mechanism for more aggressive growth of tumors under prolonged hypoxia.
Hypoxia is one of the fundamental biological phenomena that are intricately associated with the development and aggressiveness of a variety of solid tumors. Hypoxia-inducible factors (HIF) function as a master transcription factor, which regulates hypoxia responsive genes and has been recognized to play critical roles in tumor invasion, metastasis, and chemo-radiation resistance, and contributes to increased cell proliferation, survival, angiogenesis and metastasis. Therefore, tumor hypoxia with deregulated expression of HIF and its biological consequence lead to poor prognosis of patients diagnosed with solid tumors, resulting in higher mortality, suggesting that understanding of the molecular relationship of hypoxia with other cellular features of tumor aggressiveness would be invaluable for developing newer targeted therapy for solid tumors. It has been well recognized that cancer stem cells (CSCs) and epithelial-to-mesenchymal transition (EMT) phenotypic cells are associated with therapeutic resistance and contribute to aggressive tumor growth, invasion, metastasis and believed to be the cause of tumor recurrence. Interestingly, hypoxia and HIF signaling pathway are known to play an important role in the regulation and sustenance of CSCs and EMT phenotype. However, the molecular relationship between HIF signaling pathway with the biology of CSCs and EMT remains unclear although NF-κB, PI3K/Akt/mTOR, Notch, Wnt/β-catenin, and Hedgehog signaling pathways have been recognized as important regulators of CSCs and EMT. In this article, we will discuss the state of our knowledge on the role of HIF-hypoxia signaling pathway and its kinship with CSCs and EMT within the tumor microenvironment. We will also discuss the potential role of hypoxia-induced microRNAs (miRNAs) in tumor development and aggressiveness, and finally discuss the potential effects of nutraceuticals on the biology of CSCs and EMT in the context of tumor hypoxia.
Hypoxia; HIF; CSC; EMT; miRNAs
Hypoxia inducible factor-1 (HIF-1) monitors the cellular response to the oxygen levels in solid tumors. Under hypoxia conditions, HIF-1α protein is stabilized and forms a heterodimer with the HIF-1β subunit. The HIF-1 complex activates the transcription of numerous target genes in order to adapt the hypoxic environment in human cancer cells. In gastric cancer patients, HIF-1α activation following extended hypoxia strongly correlates with an aggressive tumor phenotype and a poor prognosis. HIF-1α activation has been also reported to occur via hypoxia-independent mechanisms such as PI3K/AKT/mTOR signaling and ROS production. This article argues for the critical roles of HIF-1α in glucose metabolism, carcinogenesis, angiogenesis, invasion, metastasis, cell survival and chemoresistance, focusing on gastric cancer.
HIF-1α; hypoxia; gastric cancer
Human solid tumors contain hypoxic regions that have considerably lower oxygen tension than normal tissues. They are refractory to radiotherapy and anticancer chemotherapy. Although more than half a century has passed since it was suggested that tumour hypoxia correlates with poor treatment outcomes and contributes to recurrence of cancer, no fundamental solution to this problem has been found. Hypoxia-inducible factor-1(HIF-1) is the main transcription factor that regulates the cellular response to hypoxia. It induces various genes, whose function is strongly associated with malignant alteration of the entire tumour. The cellular changes induced by HIF-1 are extremely important therapeutic targets of cancer therapy, particularly in therapy against refractory cancers. Therefore, targeting strategies to overcome the HIF-1-active microenvironment are important for cancer therapy. To Target HIF-1-active/ hypoxic tumor cells, we developed a fusion protein drug, PTD-ODD-Procaspase-3 that selectively induces cell death in HIF-1-active/hypoxic cells. The drug consists of the following three functional domains: the protein transduction domain (PTD), which efficiently delivers the fusion protein to hypoxic tumor cells, the ODD domain, which has a VHL-mediated protein destruction motif of human HIF-1α protein and confers hypoxia-dependent stabilization to the fusion proteins, and the human procaspase-3 proenzyme responsible for the cytocidal activity of the protein drug. In vivo imaging systems capable of monitoring HIF-1 activity in transplanted human cancer cells in mice are useful in evaluating the efficiency of these drugs and in study of HIF-1-active tumor cells.
hypoxia-inducible factor 1 (HIF-1); tumour hypoxia; hypoxia responsive element (HRE); protein transduction domain (PTD); bioluminescence; in vivo imaging
Hypoxia-inducible factor-1 (HIF-1) is the major hypoxia-regulated transcription factor that regulates cellular responses to low oxygen environments. HIF-1 is composed of two subunits: hypoxia-inducible HIF-1α and constitutively-expressed HIF-1β. During hypoxic conditions, HIF-1α heterodimerizes with HIF-1β and translocates to the nucleus where the HIF-1 complex binds to the hypoxia-response element (HRE) and activates expression of target genes implicated in cell growth and survival. HIF-1α protein expression is elevated in many solid tumors, including those of the cervix and brain, where cells that are the greatest distance from blood vessels, and therefore the most hypoxic, express the highest levels of HIF-1α. Therapeutic blockade of the HIF-1 signaling pathway in cancer cells therefore provides an attractive strategy for development of anticancer drugs. To identify small molecule inhibitors of the HIF-1 pathway, we have developed a cell-based reporter gene assay and screened a large compound library by using a quantitative high-throughput screening (qHTS) approach.
The assay is based upon a β-lactamase reporter under the control of a HRE. We have screened approximate 73,000 compounds by qHTS, with each compound tested over a range of seven to fifteen concentrations. After qHTS we have rapidly identified three novel structural series of HIF-1 pathway Inhibitors. Selected compounds in these series were also confirmed as inhibitors in a HRE β-lactamase reporter gene assay induced by low oxygen and in a VEGF secretion assay. Three of the four selected compounds tested showed significant inhibition of hypoxia-induced HIF-1α accumulation by western blot analysis.
The use of β-lactamase reporter gene assays, in combination with qHTS, enabled the rapid identification and prioritization of inhibitors specific to the hypoxia induced signaling pathway.
In the Western world, endometrial cancer (EC) is the most common malignant tumor of the female genital tract. Solid tumors like EC outgrow their vasculature resulting in hypoxia. Tumor hypoxia is important because it renders an aggressive phenotype and leads to radio- and chemo-therapy resistance. Hypoxia-inducible factor-1α (HIF-1α) plays an essential role in the adaptive cellular response to hypoxia and is associated with poor clinical outcome in EC. Therefore, HIF-1 could be an attractive therapeutic target. Selective HIF-1 inhibitors have not been identified. A number of nonselective inhibitors which target signaling pathways upstream or downstream HIF-1 are known to decrease HIF-1α protein levels. In clinical trials for the treatment of advanced and/or recurrent EC are the topoisomerase I inhibitor Topotecan, mTOR-inhibitor Rapamycin, and angiogenesis inhibitor Bevacizumab. Preliminary data shows encouraging results for these agents. Further work is needed to identify selective HIF-1 inhibitors and to translate these into clinical trials.
Glioblastomas, like other solid tumors, have extensive areas of hypoxia and necrosis. The importance of hypoxia in driving tumor growth is receiving increased attention. Hypoxia-inducible factor 1 (HIF-1) is one of the master regulators that orchestrate the cellular responses to hypoxia. It is a heterodimeric transcription factor composed of α and β subunits. The α subunit is stable in hypoxic conditions but is rapidly degraded in normoxia. The function of HIF-1 is also modulated by several molecular mechanisms that regulate its synthesis, degradation, and transcriptional activity. Upon stabilization or activation, HIF-1 translocates to the nucleus and induces transcription of its downstream target genes. Most important to gliomagenesis, HIF-1 is a potent activator of angiogenesis and invasion through its upregulation of target genes critical for these functions. Activation of the HIF-1 pathway is a common feature of gliomas and may explain the intense vascular hyperplasia often seen in glioblastoma multiforme. Activation of HIF results in the activation of vascular endothelial growth factors, vascular endothelial growth factor receptors, matrix metalloproteinases, plasminogen activator inhibitor, transforming growth factors α and β, angiopoietin and Tie receptors, endothelin-1, inducible nitric oxide synthase, adrenomedullin, and erythropoietin, which all affect glioma angiogenesis. In conclusion, HIF is a critical regulatory factor in the tumor microenvironment because of its central role in promoting proangiogenic and invasive properties. While HIF activation strongly promotes angiogenesis, the emerging vasculature is often abnormal, leading to a vicious cycle that causes further hypoxia and HIF upregulation.
The hypoxia-inducible transcription factors (HIF)-1α and -2α play a critical role in cellular response to hypoxia. Elevated HIF-α expression correlates with poor patient survival in a large number of cancers. Recent evidence suggests that HIF-2α appears to be preferentially expressed in neuronal tumor cells that exhibit cancer stem cell characteristics. These observations suggest that expression of HIF-1α and -2α is differentially regulated in the hypoxic tumor microenvironment. However, the underlying mechanisms remain to be fully investigated. In this study, we investigated the transcriptional regulation HIF-1α and -2α under different physiologically relevant hypoxic conditions. We found that transcription of HIF-2α was consistently increased by hypoxia, whereas transcription of HIF-1α showed variable levels of repression. Mechanistically, differential regulation of HIF-α transcription involved hypoxia-induced changes in acetylation of core histones H3 and H4 associated with the proximal promoters of the HIF-1α or HIF-2α gene. We also found that, although highly stable under acute hypoxia, HIF-1α and HIF-2α proteins become destabilized under chronic hypoxia. Our results have thus provided new mechanistic insights into the differential regulation of HIF-1α and -2α by the hypoxic tumor microenvironment. These findings also suggest an important role of HIF-2α in the regulation of tumor progression under chronic hypoxia.
hypoxia; hypoxia-inducible factor; HIF-1α and HIF-2α; transcription; promoter
Hypoxia plays an important role in the tumor microenvironment by allowing the development and maintenance of cancer cells, but the regulatory mechanisms by which tumor cells adapt to hypoxic conditions are not yet well understood. MicroRNAs are recognized as a new class of master regulators that control gene expression and are responsible for many normal and pathological cellular processes. Studies have shown that hypoxia inducible factor 1 (HIF1) regulates a panel of microRNAs, whereas some of microRNAs target HIF1. The interaction between microRNAs and HIF1 can account for many vital events relevant to tumorigenesis, such as angiogenesis, metabolism, apoptosis, cell cycle regulation, proliferation, metastasis, and resistance to anticancer therapy. This review will summarize recent findings on the roles of hypoxia and microRNAs in human cancer and illustrate the machinery by which microRNAs interact with hypoxia in tumor cells. It is expected to update our knowledge about the regulatory roles of microRNAs in regulating tumor microenvironments and thus benefit the development of new anticancer drugs.
microRNA; hypoxia; HIF1; human cancer; angiogenesis; apoptosis; cell cycle; cancer metastasis; chemoresistance; radioresistance
Angiogenesis is essential for promoting growth and metastasis of solid tumors by ensuring blood supply to the tumor mass. Targeting angiogenesis is therefore an attractive approach to therapeutic intervention of cancer. Tumor angiogenesis is a process that is controlled by a complex network of molecular components including sensors, signaling transducers, and effectors, leading to cellular responses under hypoxic conditions. Positioned at the center of this network are the hypoxia-inducible factors (HIFs). HIF-1 is a major transcription factor that consists of two subunits, HIF-1α and HIF-1β. It mediates transcription of a spectrum of gene targets whose products are essential for mounting hypoxic responses. HIF-1α protein level is very low in the normoxic condition but is rapidly elevated under hypoxia. This dramatic change in the cellular HIF-1α level is primarily regulated through the proteosome-mediated degradation process. In the past few years, scientific progress has clearly demonstrated that HIF-1α phosphorylation is mediated by several families of protein kinases including GSK3β and ERKs both of which play crucial roles in the regulation of HIF-1α stability. Recent research progress has identified that Polo-like kinase 3 (Plk3) phosphorylates HIF-1α at two previously unidentified serine residues and that the Plk3-mediated phosphorylation of these residues results in destabilization of HIF-1α. Plk3 has also recently been found to phosphorylate and stabilize PTEN phosphatase, a known regulator of HIF-1α and tumor angiogenesis. Given the success of targeting protein kinases and tumor angiogenesis in anti-cancer therapies, Plk3 could be a potential molecular target for the development of novel and effective therapeutic agents for cancer treatment.
Plk3; Tumor angiogenesis; Tumor suppression; HIF-1α; PTEN
Hypoxia, a reduction in partial oxygen pressure, is a salient property of solid tumors. Hypoxia drives malignant progression and metastasis in tumors and participates in tumor resistance to radio- and chemotherapies. Hypoxia activates the hypoxia-inducible factor (HIF) family of transcription factors, which induce target genes that regulate adaptive biological processes such as anaerobic metabolism, cell motility and angiogenesis. Clinical evidence has demonstrated that expression of HIF-1 is strongly associated with poor patient prognosis and activation of HIF-1 contributes to malignant behavior and therapeutic resistance. Consequently, HIF-1 has become an important therapeutic target for inhibition by small molecules. Herein, we describe the design and synthesis of small molecules that inhibit the HIF-1 signaling pathway. Many of these compounds exhibit inhibitory activity in the nanomolar range. Separate mechanistic studies indicate that these inhibitors do not alter HIF-1 levels, but interfere with the HIF-1α/HIF-1β/p300/CBP complex formation by interacting with p300 and CBP.
drug development; cancer; hypoxia; hypoxia-inducible factor; transcription factor
The hypoxia-inducible factors 1α (HIF-1α) and 2α (HIF-2α) have extensive structural homology and have been identified as key transcription factors responsible for gene expression in response to hypoxia. They play critical roles not only in normal development, but also in tumor progression. Here we report on the differential regulation of protein expression and transcriptional activity of HIF-1α and -2α by hypoxia in immortalized mouse embryo fibroblasts (MEFs). We show that oxygen-dependent protein degradation is restricted to HIF-1α, as HIF-2α protein is detected in MEFs regardless of oxygenation and is localized primarily to the cytoplasm. Endogenous HIF-2α remained transcriptionally inactive under hypoxic conditions; however, ectopically overexpressed HIF-2α translocated into the nucleus and could stimulate expression of hypoxia-inducible genes. We show that the factor inhibiting HIF-1 can selectively inhibit the transcriptional activity of HIF-1α but has no effect on HIF-2α-mediated transcription in MEFs. We propose that HIF-2α is not a redundant transcription factor of HIF-1α for hypoxia-induced gene expression and show evidence that there is a cell type-specific modulator(s) that enables selective activation of HIF-1α but not HIF-2α in response to low-oxygen stress.
Hypoxia-inducible factor-1 (HIF-1) is a key mediator of oxygen homeostasis that was first identified as a transcription factor that is induced and activated by decreased oxygen tension. Upon activation, HIF-1 upregulates the transcription of genes that promote adaptation and survival under hypoxic conditions. HIF-1 is a heterodimer composed of an oxygen-regulated subunit known as HIF-1α and a constitutively expressed HIF-1β subunit. In general, the availability and activity of the HIF-1α subunit determines the activity of HIF-1. Subsequent studies have revealed that HIF-1 is also activated by environmental and physiological stimuli that range from iron chelators to hormones. Preclinical studies suggest that HIF-1 activation may be a valuable therapeutic approach to treat tissue ischemia and other ischemia/hypoxia-related disorders.
The focus of this review is natural product-derived small molecule HIF-1 activators. Natural products, relatively low molecular weight organic compounds produced by plants, animals, and microbes, have been and continue to be a major source of new drugs and molecular probes. The majority of known natural product-derived HIF-1 activators were discovered through pharmacological evaluation of specifically selected individual compounds. The combination of natural products chemistry with appropriate high-throughput screening bioassays could provide an alternative approach to discover novel natural product-derived HIF-1 activators. Potent natural product-derived HIF-1 activators that exhibit a low level of toxicity and side effects hold promise as new treatment options for diseases such as myocardial and peripheral ischemia, and as chemopreventative agents that could be used to reduce the level of ischemia/reperfusion injury following heart attack and stroke.
HIF-1; Natural Product; Tissue Ischemia; Therapeutic Angiogenesis; Molecular-Target; Small Molecule Activator; Chemoprevention; Ischemia/Reperfusion Injury
Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor containing an inducibly expressed HIF-1α subunit and a constititutively expressed HIF-1β subunit. Under hypoxic conditions, the HIF-1α subunit accumulates due to a decrease in the rate of proteolytic degradation, and the resulting HIF-1α-HIF-1β heterodimers undergo posttranslational modifications that promote transactivation. Recent studies suggest that amplified signaling through phosphoinositide 3-kinase, and its downstream target, mTOR, enhances HIF-1-dependent gene expression in certain cell types. In the present study, we have explored further the linkage between mTOR and HIF-1 in PC-3 prostate cancer cells treated with hypoxia or the hypoxia mimetic agent, CoCl2. Pretreatment of PC-3 cells with the mTOR inhibitor, rapamycin, inhibited both the accumulation of HIF-1α and HIF-1-dependent transcription induced by hypoxia or CoCl2. Transfection of these cells with wild-type mTOR enhanced HIF-1 activation by hypoxia or CoCl2, while expression of a rapamycin-resistant mTOR mutant rendered both HIF-1α stabilization and HIF-1 transactivating function refractory to inhibition by rapamycin. Studies with GAL4-HIF-1α fusion proteins pinpointed the oxygen-dependent degradation domain as a critical target for the rapamycin-sensitive, mTOR-dependent signaling pathway leading to HIF-1α stabilization by CoCl2. These studies position mTOR as an upstream activator of HIF-1 function in cancer cells and suggest that the antitumor activity of rapamycin is mediated, in part, through the inhibition of cellular responses to hypoxic stress.
c-Myc is frequently overexpressed in tumors and plays an important role in the regulation of cancer metabolism. Hypoxia-inducible factor-1 (HIF1), the master regulator of the hypoxic response, enhances tumorigenesis and influences metabolism via upregulation of the glycolytic pathway and suppression of mitochondrial respiration. Together, deregulated Myc and HIF1 cooperate to lend metabolic advantages to proliferating cancer cells and contribute to the Warburg Effect. Here we show that overexpression of Myc significantly stabilizes the alpha subunit of HIF1 (HIF1alpha) under normoxic conditions and enhances HIF1alpha accumulation under hypoxic conditions in cells. Post-transcriptional regulation of HIF1α by Myc led to the induction of HIF1α gene targets. Normoxic HIF1α protein expression was also dependent on Myc. Functionally; HIF1α expression was required for Myc-induced anchorage-independent growth and cell proliferation. Myc-dependent stabilization of HIF1α involved either disruption of binding to the VHL complex or post-translational protein modifications. Taken together, our findings uncover a previously uncharacterized regulatory relationship between Myc and HIF1 that has important implications for cancer metabolism and development.
Growing tumors are hypoxic and respond to microenvironmental stress through increased expression of the hypoxia inducible factor-1α (HIF-1α) transcription factor, resulting in an adaptive switch to glycolytic metabolism, angiogenic signaling, survival, and metastasis. HIF-1α expression is associated with tumor resistance to cytotoxic therapy and inferior patient outcomes. Pancreatic cancer is the most hypoxic of all solid tumors and remains refractory to current chemoradiotherapy. We have seen nuclear HIF-1α in 88% of human pancreatic ductal carcinoma but in only 16% of normal pancreas. Stroma adjacent to the pancreatic ductal carcinoma also showed HIF-1α in 43% of cases. We investigated the novel selective HIF-1α inhibitor PX-478 on in vitro and in vivo radiation response of human pancreatic cancer models. Inhibition of HIF-1α by PX-478 increased cell killing by radiation. In mice with Panc-1, CF-PAC-1, or SU.86.86 pancreatic xenografts, concurrent administration of PX-478 potentiated the antitumor effects of fractionated radiation, with or without combined treatment with 5-fluorouracil or gemcitabine. Alternative sequencing of PX-478 with fractionated radiotherapy suggests optimal radiosensitization with concurrent or neoadjuvant administration of drug. Early tumor responses to combined PX-478/radiation treatment could be rapidly and repeatedly quantified by vascular imaging biomarkers. Dual-tracer dynamic contrast enhanced-magnetic resonance imaging and ultrasound imaging discriminated response to combined treatment prior to detection of differences in anatomic tumor size at 10 days posttreatment. Therefore, PX-478 is a mechanistically appealing and potentially clinically relevant enhancer of pancreatic cancer radiosensitivity, inhibiting tumor and stromal HIF-1 proangiogenic signaling and reducing the innate radiation resistance of hypoxic tumor cells.
Solid tumors generally grow under hypoxic conditions, a pathophysiological change, which activates the expression of genes responsible for malignant, aggressive, and treatment-refractory properties. Hypoxia inducible factor (HIF) is the chief transcription factor regulating hypoxia-driven gene expression. Therefore, the HIF pathway has become a critical target for cancer therapeutics development. We screened a privileged library of about 10,000 natural-product-like compounds using a cell-based assay for HIF-dependent transcriptional activity and identified several arylsulfonamide HIF pathway inhibitors. Among these compounds, the most potent ones showed an IC50 of ~0.5 μM in the hypoxia-responsive element (HRE)-luciferase reporter system. Further studies are needed to fully elucidate the mechanism of action of this class of compounds and their structure-activity relationship.
drug development; cancer; transcription factor; hypoxia; angiogenesis; glycolysis
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
Drug resistance is a major factor for the limited efficacy of chemotherapy in gastric cancer treatment. Hypoxia-inducible factor-1α (HIF-1α), a central transcriptional factor in hypoxia, is suggested to participate in the resistance. Here, we identified a hypoxia-mimic (cobalt chloride) sensitive gastric cell line BGC-823 to explore whether diosgenin, an aglycone of steroidal saponins, can inhibit cancer cell invasion and survival of solid tumor in a hypoxic mimic microenvironment. We have shown that diosgenin is a potent candidate for decreasing the ability of invasion and survival in cobalt chloride treated BGC-823 cells. In addition, when combined with HIF-1α specific short hairpin RNA (shRNA), diosgenin can inhibit BGC-823 cells more effectively. The anti-invasion role of diosgenin may be related to E-cadherin, integrinα5 and integrin β6. These results suggest that diosgenin may be a useful compound in controlling gastric cancer cells in hypoxia condition, especially when combined with down-regulated HIF-1α.
gastric cancer; HIF-1α; hypoxia; shRNA; diosgenin
Cellular metabolism depends on the availability of oxygen and the major regulator of oxygen homeostasis is hypoxia-inducible factor 1 (HIF-1), a highly conserved transcription factor that plays an essential role in cellular and systemic homeostatic responses to hypoxia. HIF-1 is a heterodimeric transcription factor composed of hypoxia-inducible HIF-1α and constitutively expressed HIF-1β. Under hypoxic conditions, the two subunits dimerize, allowing translocation of the HIF-1 complex to the nucleus where it binds to hypoxia-response elements (HREs) and activates expression of target genes implicated in angiogenesis, cell growth, and survival. The HIF-1 pathway is essential to normal growth and development, and is involved in the pathophysiology of cancer, inflammation, and ischemia. Thus, there is considerable interest in identifying compounds that modulate the HIF-1 signaling pathway. To assess the ability of environmental chemicals to stimulate the HIF-1 signaling pathway, we screened a National Toxicology Program collection of 1408 compounds using a cell-based β-lactamase HRE reporter gene assay in a quantitative high-throughput screening (qHTS) format. Twelve active compounds were identified. These compounds were tested in a confirmatory assay for induction of vascular endothelial growth factor, a known hypoxia target gene, and confirmed compounds were further tested for their ability to mimic the effect of a reduced-oxygen environment on hypoxia-regulated promoter activity. Based on this testing strategy, three compounds (o-phenanthroline, iodochlorohydroxyquinoline, cobalt sulfate heptahydrate) were confirmed as hypoxia mimetics, whereas two compounds (7-diethylamino-4-methylcoumarin and 7,12-dimethylbenz(a)anthracence) were found to interact with HIF-1 in a manner different from hypoxia. These results demonstrate the effectiveness of qHTS in combination with secondary assays for identification of HIF-1α inducers and for distinguishing among inducers based on their pattern of activated hypoxic target genes. Identification of environmental compounds having HIF-1α activation activity in cell-based assays may be useful for prioritizing chemicals for further testing as hypoxia-response inducers in vivo.
cobalt sulfate heptahydrate; 7-diethylamino-4-methylcoumarin; 7,12-dimethylbenz(a)anthracence; HIF-1α; inducers; iodochlorohydroxyquinoline; NTP 1408 compound library; o-phenanthroline; qHTS
One of the molecules regulated by the transcription factor, hypoxia inducible factor (HIF), is the hypoxia-responsive hematopoietic factor, erythropoietin (EPO). This may have relevance to the development of renal cell carcinoma (RCC), where mutations of the von Hippel-Lindau (VHL) gene are major risk factors for the development of familial and sporadic RCC. VHL mutations up-regulate and stabilize HIF, which in turn activates many downstream molecules, including EPO, that are known to promote angiogenesis, drug resistance, proliferation and progression of solid tumours. HIFs typically respond to hypoxic cellular environment. While the hypoxic microenvironment plays a critical role in the development and progression of tumours in general, it is of special significance in the case of RCC because of the link between VHL, HIF and EPO. EPO and its receptor, EPOR, are expressed in many cancers, including RCC. This limits the use of recombinant human EPO (rhEPO) to treat anaemia in cancer patients, because the rhEPO may be stimulatory to the cancer. EPO may also stimulate epithelial-mesenchymal transition (EMT) in RCC, and pathological EMT has a key role in cancer progression. In this mini review, we summarize the current knowledge of the role of EPO in RCC. The available data, either for or against the use of EPO in RCC patients, are equivocal and insufficient to draw a definitive conclusion.
Hypoxia, which is a loss of oxygen in tissues, is a common condition in solid tumors due to the tumor outgrowing existing vasculature. Under hypoxic conditions, hypoxia-inducible factor (HIF)-1α rapidly accumulates and transactivates hundreds of genes, such as matrix metalloproteinases (MMPs). MMPs contribute to invasion and metastasis of tumor cells by degrading the surrounding basement membrane and extracellular matrix barriers, which enables the easy migration and spread of cancer cells. We examined whether hypoxia increases tumor cell invasion, and whether increased invasiveness was due to HIF-1α and MMP-9 expression.
Transwell invasion assays were performed to demonstrate whether hypoxia enhance tumor invasion by use of MDA-MB-231 breast cancer cells. An immunofluorescence assay was used to demonstrate expression of HIF-1α and MMP-9 under hypoxic conditions. Luciferase and ChiP assays were performed to demonstrate that MMP-9 promoter activity was regulated by HIF-1α.
HIF-1α was stabilized under hypoxic conditions and stimulated MMP-9 expression, which affected the tumor invasiveness of breast cancer cells. HIF-1α transactivated the MMP-9 promoter by forming a transcriptional unit with p300, thus increasing expression of MMP-9 transcripts. Zymography indicated that MMP-9 had more gelatinase activity under hypoxic conditions than normoxic conditions. Furthermore, the small GTPase Ras was also activated in response to hypoxia, which then aids stabilization of HIF-1α, and in turn upregulates MMP-9 expression. We also demonstrate that MMP-9 is upregulated concurrently with HIF-1α in tumor tissues from patients with breast cancer.
These results suggest that HIF-1α promotes cell invasion through a MMP-9-dependent mechanism and that future antitumor agents could be used to target HIF-1α and MMP-9.
Angiogenesis; Breast neoplasms; Hypoxia-inducible factor 1 alpha subunit; Matrix metalloproteinases
Hypoxia is an inadequate oxygen supply to tissues and cells, which can restrict their function. The hypoxic response is primarily mediated by the hypoxia-inducible transcription factors, HIF-1 and HIF-2, which have both overlapping and unique target genes. HIF target gene activation is highly context specific and is not a reliable indicator of which HIF-α isoform is active. For example in some cell lines, the individual HIFs have specific temporal and functional roles: HIF-1 drives the initial response to hypoxia (<24 hours) and HIF-2 drives the chronic response (>24 hours). Here, we review the significance of the HIF switch and the relationship between HIF-1 and HIF-2 under both physiological and pathophysiological conditions.
Hypoxia; HIF; development; angiogenesis; cancer; stem cells