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1.  Second-generation HIF-activated oncolytic adenoviruses with improved replication, oncolytic, and anti-tumor efficacy 
Gene therapy  2010;17(12):1430-1441.
There is a need to develop more potent oncolytic adenoviruses that exhibit increased anti-tumor activity in patients. The HYPR-Ads are targeted oncolytic adenoviruses that specifically kill tumor cells which express active hypoxia-inducible factor (HIF). While therapeutically efficacious, the HYPR-Ads exhibited attenuated replication and oncolytic activity. To overcome these deficiencies and improve anti-tumor efficacy, we created new HIF-activated oncolytic Ads, HIF-Ad and HIF-Ad-IL4, which have two key changes: (i) a modified HIF-responsive promoter to regulate the E1A replication gene and (ii) insertion of the E3 gene region. The HIF-Ads demonstrated conditional activation of E1A expression under hypoxia. Importantly, the HIF-Ads exhibit hypoxia-dependent replication, oncolytic, and cellular release activities and potent anti-tumor efficacy, all of which are significantly greater than the HYPR-Ads. Notably, HIF-Ad-IL4 treatment led to regressions in tumor size by 70% and extensive tumor infiltration by leukocytes resulting in an anti-tumor efficacy that is up to 6-fold greater than the HYPR-Ads, HIF-Ad, and wild-type adenovirus treatment. These studies demonstrate that treatment with a HIF-activated oncolytic adenovirus leads to a measurable therapeutic response. The novel design of the HIF-Ads represents a significant improvement compared to first-generation oncolytic Ads and has great potential to increase the efficacy of this cancer therapy.
doi:10.1038/gt.2010.100
PMCID: PMC2978277  PMID: 20664541
hypoxia; hypoxia-inducible factor (HIF); adenovirus; tumor; oncolytic; virotherapy; interleukin-4
2.  Hypoxia and hypoxia-inducible factors (HIFs): master regulators of metastasis 
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.
doi:10.1158/1078-0432.CCR-10-1360
PMCID: PMC3005023  PMID: 20962028
3.  Identification of Chemical Compounds that Induce HIF-1α Activity 
Toxicological Sciences  2009;112(1):153-163.
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.
doi:10.1093/toxsci/kfp123
PMCID: PMC2910898  PMID: 19502547
cobalt sulfate heptahydrate; 7-diethylamino-4-methylcoumarin; 7,12-dimethylbenz(a)anthracence; HIF-1α; inducers; iodochlorohydroxyquinoline; NTP 1408 compound library; o-phenanthroline; qHTS
4.  The Critical Impact of HIF-1α on Gastric Cancer Biology 
Cancers  2013;5(1):15-26.
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.
doi:10.3390/cancers5010015
PMCID: PMC3730315  PMID: 24216696
HIF-1α; hypoxia; gastric cancer
5.  Identification of small molecule compounds that inhibit the HIF-1 signaling pathway 
Molecular Cancer  2009;8:117.
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1476-4598-8-117
PMCID: PMC2797767  PMID: 20003191
6.  Microenvironment and Radiation Therapy 
BioMed Research International  2012;2013:685308.
Dependency on tumor oxygenation is one of the major features of radiation therapy and this has led many radiation biologists and oncologists to focus on tumor hypoxia. The first approach to overcome tumor hypoxia was to improve tumor oxygenation by increasing oxygen delivery and a subsequent approach was the use of radiosensitizers in combination with radiation therapy. Clinical use of some of these approaches was promising, but they are not widely used due to several limitations. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that is activated by hypoxia and induces the expression of various genes related to the adaptation of cellular metabolism to hypoxia, invasion and metastasis of cancer cells and angiogenesis, and so forth. HIF-1 is a potent target to enhance the therapeutic effects of radiation therapy. Another approach is antiangiogenic therapy. The combination with radiation therapy is promising, but several factors including surrogate markers, timing and duration, and so forth have to be optimized before introducing it into clinics. In this review, we examined how the tumor microenvironment influences the effects of radiation and how we can enhance the antitumor effects of radiation therapy by modifying the tumor microenvironment.
doi:10.1155/2013/685308
PMCID: PMC3591225  PMID: 23509762
7.  Hypoxia and angiogenesis: regulation of hypoxia-inducible factors via novel binding factors 
Experimental & Molecular Medicine  2009;41(12):849-857.
The mechanisms that regulate angiogenesis in hypoxia or hypoxic microenvironment are modulated by several pro- and antiangiogenic factors. Hypoxia-inducible factors (HIFs) have been established as the basic and major inducers of angiogenesis, but understanding the role of interacting proteins is becoming increasingly important to elucidate the angiogenic processes of a hypoxic response. In particular, with regard to wound healing and the novel therapies for vascular disorders such as ischemic brain and heart attack, it is essential to gain insights in the formation and regulation of HIF transcriptional machineries related to angiogenesis. Further, identification of alternative ways of inhibiting tumor growth by disrupting the growth-triggering mechanisms of increasing vascular supply via angiogenesis depends on the knowledge of how tumor cells develop their own vasculature. Here, we review our findings on the interactions of basic HIFs, HIF-1α and HIF-2α, with their regulatory binding proteins, histone deacetylase 7 (HDAC7) and translation initiation factor 6 (Int6), respectively. The present results and discussion revealed new regulatory interactions of HIF-related mechanisms.
doi:10.3858/emm.2009.41.12.103
PMCID: PMC2802680  PMID: 19942820
angiogenic proteins; anoxia; eukaryotic initiation factors; histone deacetylases; hypoxia-ischemia, brain; neovascularization, pathologic
8.  The Hypoxia-Associated Factor Switches Cells from HIF-1α– to HIF-2α–Dependent Signaling Promoting Stem Cell Characteristics, Aggressive Tumor Growth and Invasion 
Cancer Research  2011;71(11):4015-4027.
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.
doi:10.1158/0008-5472.CAN-10-4142
PMCID: PMC3268651  PMID: 21512133
9.  CaMKII Inhibitor KN-62 Blunts Tumor Response to Hypoxia by Inhibiting HIF-1α in Hepatoma Cells 
In rapidly growing tumors, hypoxia commonly develops due to the imbalance between O2 consumption and supply. Hypoxia Inducible Factor (HIF)-1α is a transcription factor responsible for tumor growth and angiogenesis in the hypoxic microenvironment; thus, its inhibition is regarded as a promising strategy for cancer therapy. Given that CamKII or PARP inhibitors are emerging anticancer agents, we investigated if they have the potential to be developed as new HIF-1α-targeting drugs. When treating various cancer cells with the inhibitors, we found that a CamKII inhibitor, KN-62, effectively suppressed HIF-1α specifically in hepatoma cells. To examine the effect of KN-62 on HIF-1α-driven gene expression, we analyzed the EPO-enhancer reporter activity and mRNA levels of HIF-1α downstream genes, such as EPO, LOX and CA9. Both the reporter activity and the mRNA expression were repressed by KN-62. We also found that KN-62 suppressed HIF-1α by impairing synthesis of HIF-1α protein. Based on these results, we propose that KN-62 is a candidate as a HIF-1α-targeting anticancer agent.
doi:10.4196/kjpp.2010.14.5.331
PMCID: PMC2997420  PMID: 21165333
CaMKII; HIF1-α; Hepatocellular carcinoma; Hypoxia; KN-62
10.  Imaging and Targeting of the Hypoxia-inducible Factor 1-active Microenvironment 
Journal of Toxicologic Pathology  2009;22(2):93-100.
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.
doi:10.1293/tox.22.93
PMCID: PMC3246054  PMID: 22271982
hypoxia-inducible factor 1 (HIF-1); tumour hypoxia; hypoxia responsive element (HRE); protein transduction domain (PTD); bioluminescence; in vivo imaging
11.  STAT3 and HIF1α cooperatively activate HIF1 target genes in MDA-MB-231 and RCC4 cells 
Oncogene  2013;33(13):1670-1679.
Solid tumors often exhibit simultaneously inflammatory and hypoxic microenvironments. The ‘signal transducer and activator of transcription-3’ (STAT3)-mediated inflammatory response and the hypoxia-inducible factor (HIF)-mediated hypoxia response have been independently shown to promote tumorigenesis through the activation of HIF or STAT3 target genes and to be indicative of a poor prognosis in a variety of tumors. We report here for the first time that STAT3 is involved in the HIF1, but not HIF2-mediated hypoxic transcriptional response. We show that inhibiting STAT3 activity in MDA-MB-231 and RCC4 cells by a STAT3 inhibitor or STAT3 small interfering RNA significantly reduces the levels of HIF1, but not HIF2 target genes in spite of normal levels of hypoxia-inducible transcription factor 1α (HIF1α) and HIF2α protein. Mechanistically, STAT3 activates HIF1 target genes by binding to HIF1 target gene promoters, interacting with HIF1α protein and recruiting coactivators CREB binding protein (CBP) and p300, and RNA polymerase II (Pol II) to form enhanceosome complexes that contain HIF1α, STAT3, CBP, p300 and RNA Pol II on HIF1 target gene promoters. Functionally, the effect of STAT3 knockdown on proliferation, motility and clonogenic survival of tumor cells in vitro is phenocopied by HIF1α knockdown in hypoxic cells, whereas STAT3 knockdown in normoxic cells also reduces cell proliferation, motility and clonogenic survival. This indicates that STAT3 works with HIF1 to activate HIF1 target genes and to drive HIF1-depedent tumorigenesis under hypoxic conditions, but also has HIF-independent activity in normoxic and hypoxic cells. Identifying the role of STAT3 in the hypoxia response provides further data supporting the effectiveness of STAT3 inhibitors in solid tumor treatment owing to their usefulness in inhibiting both the STAT3 and HIF1 pro-tumorigenic signaling pathways in some cancer types.
doi:10.1038/onc.2013.115
PMCID: PMC3868635  PMID: 23604114
cotranscriptional activation; HIF; hypoxia; STAT3; transcription
12.  Development of an oncolytic Herpes Simplex Virus using a tumor-specific HIF-responsive promoter 
Cancer gene therapy  2010;18(2):123-134.
We exploited the differential activation of hypoxia-inducible factor (HIF)-dependent gene expression in tumors versus normal tissue for the design of a targeted oncolytic Herpes simplex virus type-1 (HSV-1). A gene that is essential for viral replication, ICP4, was placed under the regulation of a HIF-responsive promoter and then introduced into the thymidine kinase locus (UL23) of HSV d120 which contains partial deletions in the two endogenous ICP4 genes. Recombinant HIF-HSV were isolated and their derivation from d120 was verified by expression of a truncated, nonfunctional form of ICP4 protein. Disruption of the UL23 locus was confirmed by loss of thymidine kinase expression and resistance to acyclovir. Unexpectedly, HIF-HSV expressed ICP4 and induced tumor cell lysis at similar levels under normoxia and hypoxia. The lack of HIF-dependent ICP4 transgene expression by HIF-HSV was due to two factors that have not previously been reported- reversion of the ICP4 gene region to its wild-type configuration and increased HIF-transcriptional activity under normoxia when cells were infected with any strain of HSV-1. The findings that an oncolytic HSV-1 is genetically unstable and can activate a tumor-related promoter in a non-specific manner have important implications for any proposed use of this virus in cancer therapy.
doi:10.1038/cgt.2010.62
PMCID: PMC3021095  PMID: 20930860
hypoxia; hypoxia-inducible factor (HIF); herpes simplex virus; HSV; oncolytic; ICP4
13.  Inhibition of SIRT1 Impairs the Accumulation and Transcriptional Activity of HIF-1α Protein under Hypoxic Conditions 
PLoS ONE  2012;7(3):e33433.
Sirtuins and hypoxia-inducible transcription factors (HIF) have well-established roles in regulating cellular responses to metabolic and oxidative stress. Recent reports have linked these two protein families by demonstrating that sirtuins can regulate the activity of HIF-1 and HIF-2. Here we investigated the role of SIRT1, a NAD+-dependent deacetylase, in the regulation of HIF-1 activity in hypoxic conditions. Our results show that in hepatocellular carcinoma (HCC) cell lines, hypoxia did not alter SIRT1 mRNA or protein expression, whereas it predictably led to the accumulation of HIF-1α and the up-regulation of its target genes. In hypoxic models in vitro and in in vivo models of systemic hypoxia and xenograft tumor growth, knockdown of SIRT1 protein with shRNA or inhibition of its activity with small molecule inhibitors impaired the accumulation of HIF-1α protein and the transcriptional increase of its target genes. In addition, endogenous SIRT1 and HIF-1α proteins co-immunoprecipitated and loss of SIRT1 activity led to a hyperacetylation of HIF-1α. Taken together, our data suggest that HIF-1α and SIRT1 proteins interact in HCC cells and that HIF-1α is a target of SIRT1 deacetylase activity. Moreover, SIRT1 is necessary for HIF-1α protein accumulation and activation of HIF-1 target genes under hypoxic conditions.
doi:10.1371/journal.pone.0033433
PMCID: PMC3316573  PMID: 22479397
14.  Hypoxia-Inducible Factors Regulate Tumorigenic Capacity of Glioma Stem Cells 
Cancer cell  2009;15(6):501-513.
Summary
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.
Significance
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.
doi:10.1016/j.ccr.2009.03.018
PMCID: PMC2693960  PMID: 19477429
15.  Sphingosine-1-Phosphate as a Regulator of Hypoxia-Induced Factor-1α in Thyroid Follicular Carcinoma Cells 
PLoS ONE  2013;8(6):e66189.
Sphingosine-1-phosphate (S1P) is a bioactive lipid, which regulates several cancer-related processes including migration and angiogenesis. We have previously shown S1P to induce migration of follicular ML-1 thyroid cancer cells. Hypoxia-induced factor-1 (HIF-1) is an oxygen-sensitive transcription factor, which adapts cells to hypoxic conditions through increased survival, motility and angiogenesis. Due to these properties and its increased expression in response to intratumoral hypoxia, HIF-1 is considered a significant regulator of tumor biology. We found S1P to increase expression of the regulatory HIF-1α subunit in normoxic ML-1 cells. S1P also increased HIF-1 activity and expression of HIF-1 target genes. Importantly, inhibition or knockdown of HIF-1α attenuated the S1P-induced migration of ML-1 cells. S1P-induced HIF-1α expression was mediated by S1P receptor 3 (S1P3), Gi proteins and their downstream effectors MEK, PI3K, mTOR and PKCβI. Half-life measurements with cycloheximide indicated that S1P treatment stabilized the HIF-1α protein. On the other hand, S1P activated translational regulators eIF-4E and p70S6K, which are known to control HIF-1α synthesis. In conclusion, we have identified S1P as a non-hypoxic regulator of HIF-1 activity in thyroid cancer cells, studied the signaling involved in S1P-induced HIF-1α expression and shown S1P-induced migration to be mediated by HIF-1.
doi:10.1371/journal.pone.0066189
PMCID: PMC3688870  PMID: 23824493
16.  Hypoxia and the hypoxia-inducible-factor pathway in glioma growth and angiogenesis1 
Neuro-Oncology  2005;7(2):134-153.
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.
doi:10.1215/S1152851704001115
PMCID: PMC1871894  PMID: 15831232
17.  Marine Natural Products as Inhibitors of Hypoxic Signaling in Tumors 
Marine natural products have become a major source of new chemical entities in the discovery of potential anticancer agents that potently suppress various antitumor molecular targets. As a consequence of insufficient vascularization, hypoxic regions form within rapidly growing solid tumor masses. Specific alterations of gene expression in these hypoxic tumor cells help facilitate the survival and metastatic spread of solid tumors. The transcriptional response to cellular hypoxia is primarily mediated by the transcription factor hypoxia-inducible factor-1 (HIF-1) that regulates the expression of more than 100 genes involved in cellular adaptation and survival under hypoxic stress. Clinical studies in cancer patients indicate that HIF-1 activation is directly correlated with advanced disease stages and treatment resistance. HIF-1 has emerged as an important tumor-selective molecular target for anticancer drug discovery. As a result, natural product-based inhibitors of HIF-1 activation have been identified from plants and microorganisms. Recently, structurally unique natural products from marine sponges, crinoids, and algae have been identified as HIF-1 activation inhibitors. The US National Cancer Institute’s Open Repository of marine invertebrate and algae extracts has proven to be a valuable source of natural product HIF-1 inhibitors. Among the active compounds identified, certain marine natural products have also been shown to suppress the hypoxic induction of HIF-1 target genes such as vascular endothelial growth factor (VEGF). Some of these marine HIF-1 inhibitors act by interfering with the generation of mitochondrial signaling molecules in hypoxic cells. However, the precise mechanisms of action for many newly identified marine natural product HIF-1 inhibitors remain unresolved.
doi:10.1007/s11101-009-9120-1
PMCID: PMC2901131  PMID: 20622986
cellular signaling; crinoids; gene expression; HIF-1 inhibitors; hypoxia-inducible factor-1; marine natural products; molecular-targeted antitumor agents; sponges; transcription factor; tumor hypoxia; tunicates
18.  The Antioxidant N-Acetylcysteine Prevents HIF-1 Stabilization under Hypoxia In Vitro but Does Not Affect Tumorigenesis in Multiple Breast Cancer Models In Vivo 
PLoS ONE  2013;8(6):e66388.
Intratumoral hypoxia is a poor prognostic factor associated with reduced disease-free survival in many cancer types, including breast cancer. Hypoxia encourages tumor cell proliferation, stimulates angiogenesis and lymphangiogenesis, and promotes epithelial-mesenchymal transition and metastasis. Tumor cells respond to a hypoxic state by stabilizing the Hif-1α subunit of the Hypoxia-Inducible Factor (HIF) transcription factor to promote expression of various tumor- and metastasis-promoting hypoxic response genes. The antioxidant N-acetylcysteine (NAC) was recently shown to prevent Hif-1α stabilization under hypoxia, and has been identified as a potential alternative method to target the hypoxic response in tumors. We utilized three orthotopic syngeneic murine models of breast cancer, the PyMT, EO771 and 4T1.2 models, to investigate the ability of NAC to modulate the hypoxic response in vitro and in vivo. While NAC prevented Hif-1α stabilization under hypoxia in vitro and increased levels of glutathione in the blood of mice in vivo, this did not translate to a difference in tumor growth or the hypoxic state of the tumor compared to untreated control mice. In addition, NAC treatment actually increased metastatic burden in an experimental metastasis model. This work raises questions regarding the validity of NAC as an anti-tumorigenic agent in breast cancer, and highlights the need to further investigate its properties in vivo in different cancer models.
doi:10.1371/journal.pone.0066388
PMCID: PMC3688768  PMID: 23840457
19.  Sulfonamides as a New Scaffold for Hypoxia Inducible Factor Pathway Inhibitors 
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.
doi:10.1016/j.bmcl.2011.06.099
PMCID: PMC3292863  PMID: 21831638
drug development; cancer; transcription factor; hypoxia; angiogenesis; glycolysis
20.  Hypoxia-regulated microRNAs in human cancer 
Acta Pharmacologica Sinica  2013;34(3):336-341.
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.
doi:10.1038/aps.2012.195
PMCID: PMC3587916  PMID: 23377548
microRNA; hypoxia; HIF1; human cancer; angiogenesis; apoptosis; cell cycle; cancer metastasis; chemoresistance; radioresistance
21.  Cobalt stimulates HIF-1-dependent but inhibits HIF-2-dependent gene expression in liver cancer cells 
The international journal of biochemistry & cell biology  2013;45(11):10.1016/j.biocel.2013.07.025.
Hypoxia-inducible factors (HIFs) are transcriptional regulators that mediate the cellular response to low oxygen. Although HIF-1 is usually considered as the principal mediator of hypoxic adaptation, several tissues and different cell types express both HIF-1 and HIF-2 isoforms under hypoxia or when treated with hypoxia mimetic chemicals such as cobalt. However, the similarities or differences between HIF-1 and HIF-2, in terms of their tissue- and inducer-specific activation and function, are not adequately characterized. To address this issue, we investigated the effects of true hypoxia and hypoxia mimetics on HIF-1 and HIF-2 induction and specific gene transcriptional activity in two hepatic cancer cell lines, Huh7 and HepG2. Both hypoxia and cobalt caused rapid induction of both HIF-1α and HIF-2α proteins. Hypoxia induced erythropoietin (EPO) expression and secretion in a HIF-2-dependent way. Surprisingly, however, EPO expression was not induced when cells were treated with cobalt. In agreement, both HIF-1- and HIF-2-dependent promoters (of PGK and SOD2 genes, respectively) were activated by hypoxia while cobalt only activated the HIF-1-dependent PGK promoter. Unlike cobalt, other hypoxia mimetics such as DFO and DMOG activated both types of promoters. Furthermore, cobalt impaired the hypoxic stimulation of HIF-2, but not HIF-1, activity and cobalt-induced HIF-2α interacted poorly with USF-2, a HIF-2-specific co-activator. These data show that, despite similar induction of HIF-1α and HIF-2α protein expression, HIF-1 and HIF-2 specific gene activating functions respond differently to different stimuli and suggest the operation of oxygen-independent and gene- or tissue-specific regulatory mechanisms involving additional transcription factors or co-activators.
doi:10.1016/j.biocel.2013.07.025
PMCID: PMC3855297  PMID: 23958427
HIF-2α; EPO; SOD2; Hypoxia; Cobalt; USF2
22.  A yeast three-hybrid system that reconstitutes mammalian hypoxia inducible factor regulatory machinery 
BMC Cell Biology  2008;9:18.
Background
Several human pathologies, including neoplasia and ischemic cardiovascular diseases, course with an unbalance between oxygen supply and demand (hypoxia). Cells within hypoxic regions respond with the induction of a specific genetic program, under the control of the Hypoxia Inducible Factor (HIF), that mediates their adaptation to the lack of oxygen. The activity of HIF is mainly regulated by the EGL-nine homolog (EGLN) enzymes that hydroxylate the alpha subunit of this transcription factor in an oxygen-dependent reaction. Hydroxylated HIF is then recognized and ubiquitinilated by the product of the tumor suppressor gene, pVHL, leading to its proteosomal degradation. Under hypoxia, the hydroxylation of HIF by the EGLNs is compromised due to the lack of oxygen, which is a reaction cosubstrate. Thus, HIF escapes degradation and drives the transcription of its target genes. Since the progression of the aforementioned pathologies might be influenced by activation of HIF-target genes, development of small molecules with the ability to interfere with the HIF-regulatory machinery is of great interest.
Results
Herein we describe a yeast three-hybrid system that reconstitutes mammalian HIF regulation by the EGLNs and VHL. In this system, yeast growth, under specific nutrient restrictions, is driven by the interaction between the β domain of VHL and a hydroxyproline-containing HIFα peptide. In turn, this interaction is strictly dependent on EGLN activity that hydroxylates the HIFα peptide. Importantly, this system accurately preserves the specificity of the hydroxylation reaction toward specific substrates. We propose that this system, in combination with a matched control, can be used as a simple and inexpensive assay to identify molecules that specifically modulate EGLN activity. As a proof of principle we show that two known EGLN inhibitors, dimethyloxaloylglycine (DMOG) and 6-chlor-3-hydroxychinolin-2-carbonic acid-N-carboxymethylamide (S956711), have a profound and specific effect on the yeast HIF/EGLN/VHL system.
Conclusion
The system described in this work accurately reconstitutes HIF regulation while preserving EGLN substrate specificity. Thus, it is a valuable tool to study HIF regulation, and particularly EGLN biochemistry, in a cellular context. In addition, we demonstrate that this system can be used to identify specific inhibitors of the EGLN enzymes.
doi:10.1186/1471-2121-9-18
PMCID: PMC2346465  PMID: 18402654
23.  Increased anti-tumor activity of bevacizumab in combination with HIF-1 inhibition 
Molecular cancer therapeutics  2009;8(7):1867-1877.
Summary
Inhibition of HIF-1 is an attractive therapeutic strategy to target the tumor microenvironment. However, HIF-1 inhibitors may have limited activity as single agents and combination therapies may be required. We tested the hypothesis that HIF-1 inhibition in a hypoxic stressed tumor microenvironment, which could be generated by administration of antiangiogenic agents, may result in a more pronounced therapeutic effect. The activity of bevacizumab, either alone or in combination with the HIF-1α inhibitor topotecan, was evaluated in U251-HRE xenografts. Tumor tissue was collected at the end of treatment and changes in tumor oxygenation, angiogenesis, proliferation, apoptosis, HIF-1α levels, HIF-1 target genes and DNA damage were evaluated. Bevacizumab decreased microvessel-density and increased intratumor-hypoxia, but did not induce apoptosis. Moreover, bevacizumab alone caused a significant increase of HIF-1-dependent gene expression in tumor tissue. Addition of a low dose of daily topotecan to bevacizumab significantly inhibited tumor growth, relative to mice treated with topotecan or bevacizumab alone (p<0.01). The addition of topotecan to bevacizumab was also associated with profound inhibition of HIF-1 transcriptional activity, significant inhibition of proliferation and induction of apoptosis. Importantly, DNA damage induced by topotecan alone was not augmented by addition of bevacizumab, suggesting that increased cytotoxic activity did not account for the increased anti-tumor effects observed.
These results strongly suggest that combination of anti-VEGF antibodies with HIF-1 inhibitors is an attractive therapeutic strategy targeting in the hypoxic tumor microenvironment.
doi:10.1158/1535-7163.MCT-09-0274
PMCID: PMC2745101  PMID: 19584228
HIF-1; topotecan; bevacizumab; hypoxia; angiogenesis; cancer therapeutics
24.  Hypoxia Inducible Factor-1 (HIF-1) Independent Microvascular Angiogenesis in the Aged Rat Brain 
Brain Research  2010;1366:101-109.
Angiogenesis is a critical component of mammalian brain adaptation to prolonged hypoxia. Hypoxia-induced angiogenesis is mediated by hypoxia inducible factor-1 (HIF-1) dependent transcriptional activation of growth factors, such as vascular endothelial growth factor (VEGF). Microvascular angiogenesis occurs over a three week period in the rodent brain. We have recently reported that HIF-1α accumulation and transcriptional activation of HIF target genes in the aged cortex of 24 month F344 rats is significantly attenuated during acute hypoxic exposure. In the present study, we show that cortical HIF-1α accumulation and HIF-1 activation remains absent during chronic hypoxic exposure in the aged rat brain (24 month F344). Despite this lack of HIF-1 activation, there is no significant difference in baseline or post-hypoxic brain capillary density counts between the young (3 month F344) and old age groups. VEGF mRNA and protein levels are significantly elevated in the aged cortex despite the lack of HIF-1 activation. Other HIF-independent mediators of hypoxia inducible genes could be involved during chronic hypoxia in the aged brain. PPAR-γ coactivator (PGC)-1α, a known regulator of VEGF gene transcription, is elevated in the young and aged cortex during the chronic hypoxic exposure. Overall, our results suggest a compensatory HIF-1 independent preservation of hypoxic-induced microvascular angiogenesis in the aged rat brain.
doi:10.1016/j.brainres.2010.09.064
PMCID: PMC3378376  PMID: 20875806
Aging; Angiogenesis; Brain; Hypoxia; HIF-1; Microvessels; PGC-1α; VEGF
25.  Modulating Hypoxia-Inducible Transcription by Disrupting the HIF-1–DNA Interface 
ACS chemical biology  2007;2(8):561-571.
Transcription mediated by hypoxia-inducible factor (HIF-1) contributes to tumor angiogenesis and metastasis but is also involved in activation of cell-death pathways and normal physiological processes. Given the complexity of HIF-1 signaling, it could be advantageous to target a subset of HIF-1 effectors rather than the entire pathway. We compare the genome-wide effects of three molecules that each interfere with the HIF-1–DNA interaction: a polyamide targeted to the hypoxia response element, small interfering RNA targeted to HIF-1α, and echinomycin, a DNA-binding natural product with a similar but less specific sequence preference than the polyamide. The polyamide affects a subset of hypoxia-induced genes consistent with its binding site preferences. For comparison, HIF-1α siRNA and echinomycin each affect the expression of nearly every gene induced by hypoxia. Remarkably, the total number of genes affected by either polyamide or HIF-1α siRNA over a range of thresholds is comparable. The data show that polyamides can be used to affect a subset of a pathway regulated by a transcription factor. In addition, this study offers a unique comparison of three complementary approaches towards exogenous control of endogenous gene expression.
doi:10.1021/cb700110z
PMCID: PMC3060759  PMID: 17708671

Results 1-25 (1001866)