Hypoxia-inducible Factor-1 Transcriptional Activity Research Articles

Abstract 229: Restoring physiological levels of ascorbate alleviates HIF-1 activation and reduces tumour growth in Gulo-/- mice

Abstract Background: Hypoxia-inducible factor-1 (HIF-1) upregulates the expression of hundreds of genes involved in adaption of tumours to the hypoxic microenvironment. HIF-1 levels and transcriptional activity are regulated by hydroxylase enzymes, which require ascorbate as a cofactor. Adequate supplementation of cells with ascorbate has been shown to reduce HIF-1 activation in vitro, and we hypothesise that a similar activity could affect tumour growth in vivo. In this study we investigated the effect of dietary ascorbate on tumour growth in Gulo-/- mice, a model of the human ascorbate deficiency condition. Methods: C57/Bl6 Gulo-/- mice were supplemented with 3300 mg/L, 330 mg/L or 33mg/L of ascorbate in their drinking water for one month before subcutaneous, syngeneic tumour implantation with either B16-F10 melanoma, or Lewis lung carcinoma (LL/2), and then for duration of the experiment. Levels of HIF-1α, and its targets, carbonic anhydrase-IX (CA-IX), and glucose transporter-1 (GLUT-1) were analysed by western blotting, and vascular endothelial growth factor (VEGF) levels by ELISA. Ascorbate concentrations of tissue, plasma and tumours were measured using high pressure liquid chromatography with electrochemical detection (HPLC-EC). Results: Across all organs and plasma samples, ascorbate content was in the physiological range (up to levels seen in wild-type mice) and was correlated with dietary ascorbate intake. Tumour ascorbate also reflected ascorbate intake and optimal ascorbate levels (3300 mg/L) significantly increased the time for LL/2 tumours to reach 200 mm3 (lag growth) (13.3±2.6 days), compared to these same tumours in mice supplemented with either 330 mg/L (9.1±2.6 days) or 33 mg/L (9.8±1.39 days) of ascorbate (p<0.001). Similarly, tumours took significantly longer to grow from 200-800 mm3 (log phase) when mice were supplemented with 3300 mg/L compared to lower supplementation (p<0.05). B16-F10 tumours showed no difference in the lag growth period between ascorbate treatments (p=0.49). However, supplementing mice with 3300 mg/mL of ascorbate lead to a significant reduction in the tumour growth rate during log phase (3.9±1.1 days), compared to mice supplemented with either 330 mg/L (2.0±0.90 days) or 33mg/L (2.1±0.71 days) (p<0.001). Levels of HIF-1α protein in tumours decreased as dietary ascorbate supplementation increased for both LL/2 (p<0.001) and B16F10 (p<0.001). Tumour ascorbate was significantly correlated with low levels of CA-IX, GLUT-1 and VEGF levels in both LL/2 and B16 tumours (all p<0.05). Conclusions: The data from the current study support the hypothesis that restoration of optimal intracellular ascorbate levels is associated with reduced HIF-1 levels and slower tumour growth. This data may have implications for the management of cancer, although clinical trials are required to assess whether human tumour levels of ascorbate can similarly be manipulated. Citation Format: Elizabeth J. Campbell, Stephanie M. Bozonet, Bridget A. Robinson, Margreet CM Vissers, Gabi U. Dachs. Restoring physiological levels of ascorbate alleviates HIF-1 activation and reduces tumour growth in Gulo-/- mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 229. doi:10.1158/1538-7445.AM2014-229

Abstract 1014: Novel selective HIF1 alpha inhibitor: Well tolerated with excellent efficacy in renal cell cancer xenograft studies

Abstract Traditional anticancer therapies are limited in their efficacy because they often lead to drug-resistant tumors and display toxicity to normal cells. Control of angiogenesis represents one of the key goals in cancer therapy, as exemplified by targeting the VEGF pathway with bevacizumab.Chronic hypoxia, a hallmark of many solid tumors is responsible for the elevated levels of VEGF and other pro-proliferative factors. The cellular response to hypoxia is primarily mediated by a family of transcription factors, among which hypoxia-inducible factor 1 (HIF1) plays a major role. Under normoxia, the oxygen-sensitive α subunit of HIF1 is rapidly and constitutively degraded but is stabilized and accumulates under hypoxia. Upon nuclear translocation, HIF1 controls the expression of over 100 genes involved in angiogenesis, and pro-proliferative mechanisms that promote tumor growth. The specific hypothesis behind the proposed research is that angiogenesis could be effectively controlled by down-regulation of hypoxia-inducible gene expression via a disruption of key transcription factor-coactivator interactions with designed molecules. The team at BioCycive and its collaborators at USC are currently developing such compounds that block hypoxia-inducible transcription of key genes. We have designed and synthesized a novel dimeric epidithiodiketopiperazine, ETP2, which targets pleiotropic coactivator p300 disrupts HIF1α-p300 complex in vitro and effectively down-regulates hypoxia-inducible signaling and gene expression. ETP 2 effectively inhibits HIF1-induced activation of VEGFA, LOX, Glut1, and c-Met genes in a panel of breast and lung cancer cell lines. We have shown excellent antitumor efficacy of both (±)-ETP 2 and meso-ETP 2 in a fully established breast carcinoma model by intravital microscopy. A significant decrease in the vascularization of these tumors upon treatment with ETP 2 was also noticed, which is consistent with the proposed mechanism of inhibition of vascularization via down-regulation of hypoxia-inducible signaling.Our efficacy studies with renal carcinoma cells lines resulted in similar findings, where we successfully reached the primary endpoint of efficacy (reduction in tumor volume of >50%) and secondary endpoint of survival. Nude mice engrafted with 786-O (renal cell carcinoma) were treated with ETPs at 1 mg/kg given IV every three days for 45 days. All tumors were imaged at the end of the study and harvested for histopathology studies. MTD studies clearly indicated that ETP 2 was non-toxic within the range of injected doses, with an average MTD of 21.1 mg/kg. Our preliminary PK studies show that the ETP 2 is a lipophilic molecule, with a plasma half-life of 3.5-5 days. Our results suggest that inhibition of HIF1 transcriptional activity by designed dimeric ETPs could offer an innovative approach to cancer therapy with the potential to overcome hypoxia-induced tumor growth and resistance. Citation Format: Ramin Dubey, Ivan Grishagin, Usha Nagavarapu, Chenera Balan, Shalabh Gupta, Bogdan Z. Olenyuk. Novel selective HIF1 alpha inhibitor: Well tolerated with excellent efficacy in renal cell cancer xenograft studies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1014. doi:10.1158/1538-7445.AM2014-1014

The radiosensitising effect of gemcitabine and its main metabolite dFdU under low oxygen conditions is in vitro not dependent on functional HIF-1 protein

BackgroundRegions within solid tumours often experience oxygen deprivation, which is associated with resistance to chemotherapy and irradiation. The aim of this study was to evaluate the radiosensitising effect of gemcitabine and its main metabolite dFdU under normoxia versus hypoxia and to determine whether hypoxia-inducible factor 1 (HIF-1) is involved in the radiosensitising mechanism.MethodsStable expression of dominant negative HIF-1α (dnHIF) in MDA-MB-231 breast cancer cells, that ablated endogenous HIF-1 transcriptional activity, was validated by western blot and functionality was assessed by HIF-1α activity assay. Cells were exposed to varying oxygen environments and treated with gemcitabine or dFdU for 24 h, followed by irradiation. Clonogenicity was then assessed. Using radiosensitising conditions, cells were collected for cell cycle analysis.ResultsHIF-1 activity was significantly inhibited in cells stably expressing dnHIF. A clear radiosensitising effect under normoxia and hypoxia was observed for both gemcitabine and dFdU. No significant difference in radiobiological parameters between HIF-1 proficient and HIF-1 deficient MDA-MB-231 cells was demonstrated.ConclusionsFor the first time, radiosensitisation by dFdU, the main metabolite of gemcitabine, was demonstrated under low oxygen conditions. No major role for functional HIF-1 protein in radiosensitisation by gemcitabine or dFdU could be shown.

Abstract 82: Regulation Of Frataxin By HIF-1 In The Ischemic Diabetic Heart

Background: Diabetes is at epidemic proportions, with the major form of fatality due to congestive heart failure triggered by myocardial infarction (MI). The impaired insulin signalling in the diabetic heart leads to myocardial energy dysregulation that compromises the cardioprotective mechanism against ischemic injury. Therefore understanding how mitochondrial energetics is altered in the diabetic ischemic heart would greatly advance the knowledge base for improving outcomes from heart failure in diabetic patients. Methods/Findings: We observed that db/db mice (leptin deficient, type 2 diabetic mice) have increased infarction size (>30%) compared to wild type mice after ischemia/reperfusion (IR) injury by TTC stain. We also found that activity of Hypoxia inducible factor-1 (HIF1) is involved in the cardioprotective response to ischemia, is impaired in db/db hearts. HIF1 is known to transcriptionally regulate genes involved in myocardial energetics. We recently found that HIF1 transcriptionally regulates the mitochondrial protein frataxin (Fxn) in cardiomyocytes as determined by luciferase assays (>3 fold). In vitro studies indicate that hypoxic conditions increase Fxn protein expression in cardiomyocytes as determined by western analysis (2 fold). Fxn plays an important role in the Fe-S cluster biogenesis required for aconitase, succinate dehydrogenase and complexes in the mitochondria. Interestingly, we observed decreased expression of Fxn in the ischemic diabetic heart. Conclusion: we postulate that attenuated HIF1-Fxn signalling in ischemic diabetic heart leads to abnormally enlarged infarction size in response to IR. The decline in HIF-1 activity in response to hypoxia was further validated in cardiomyocytes cultured in high glucose media. The significance for Fxn against hypoxic injury was confirmed by utilizing overexpressed Fxn cardiomyocytes via MTT, ATP and aconitase activity assays. Current and future work: currently we are attempting to identify the HIF response element (HRE) in Fxn promoter to further validate the transcriptional activity of HIF1. In addition, we are completing the IR surgeries on HIF1 KO mice to address the cardioprotective nature of HIF1-Fxn signalling against MI.

RLIP76 regulates HIF-1 activity, VEGF expression and secretion in tumor cells, and secretome transactivation of endothelial cells.

This study was undertaken to reveal the mechanisms by which RLIP76 regulates endothelial cell angiogenic responses. RLIP76 is an effector of the angiogenic modulator, R-Ras. RLIP76 is overexpressed in many tumors, required for tumor angiogenesis, and blockade of RLIP76 results in tumor regression in multiple models. We report here that RLIP76 was required for expression and secretion of vascular endothelial growth factor (VEGF) in carcinoma and melanoma cells. Conditioned medium derived from RLIP76-depleted tumor cells, but not control knockdown cells, could not stimulate proliferation, migration, or Matrigel cord formation in endothelial cell cultures, which indicates that RLIP76 regulates angiogenic components of the tumor cell secretome. Recombinant VEGF added to conditioned medium from RLIP76-knockdown tumor cells restored these endothelial cell functions. Transcriptional activity of hypoxia-inducible factor 1 (HIF-1), which drives VEGF expression, was blocked in RLIP76-depleted tumor cells. RLIP76 was required for PI3-kinase activation, known to regulate HIF-1, in these cells. However, HIF-1α expression and nuclear localization were unaffected by RLIP76 knockdown, which suggests that RLIP76 regulates HIF-1 at the functional level. Thus, RLIP76 regulates tumor cell transactivation of endothelial cells via control of VEGF expression and secretion, providing a new important link in the mechanism of tumor cell induction of angiogenesis.

A new twist in neuroendocrine tumor research: Pacak-Zhuang syndrome, HIF-2α as the major player in its pathogenesis and future therapeutic options.

Backround. There is increasing evidence of the role of hypoxia or pseudohypoxia in tumorigenesis, including pheochromocytoma (PHEO) and paraganglioma (PGL). (Pseudo)hypoxia leads to activation of hypoxia-inducible transcription factors (HIFs) and thus, promotes the transcription of hypoxia-responsive genes which are involved in tumorigenesis. Recently identified is a new syndrome consisting of multiple and recurrent PGLs or PHEOs, somatostatinoma, and congenital polycythemia, due to somatic hypoxia-inducible factor 2α gene (HIF2A) mutations. PubMed and Web of Science online databases were used to search reviews and original articles on the HIF, PHEO/PGL, and Pacak-Zhuang syndrome. The novel somatic and germline gain-of-function HIF2A mutations described latterly emphasize the role of the HIF-2α in the PHEO/PGL development and these findings designate HIF, especially HIF-2α, as a promising treatment target.

Gramicidin A Blocks Tumor Growth and Angiogenesis through Inhibition of Hypoxia-Inducible Factor in Renal Cell Carcinoma

Ionophores are hydrophobic organic molecules that disrupt cellular transmembrane potential by permeabilizing membranes to specific ions. Gramicidin A is a channel-forming ionophore that forms a hydrophilic membrane pore that permits the rapid passage of monovalent cations. Previously, we found that gramicidin A induces cellular energy stress and cell death in renal cell carcinoma (RCC) cell lines. RCC is a therapy-resistant cancer that is characterized by constitutive activation of the transcription factor hypoxia-inducible factor (HIF). Here, we demonstrate that gramicidin A inhibits HIF in RCC cells. We found that gramicidin A destabilized HIF-1α and HIF-2α proteins in both normoxic and hypoxic conditions, which in turn diminished HIF transcriptional activity and the expression of various hypoxia-response genes. Mechanistic examination revealed that gramicidin A accelerates O(2)-dependent downregulation of HIF by upregulating the expression of the von Hippel-Lindau (VHL) tumor suppressor protein, which targets hydroxylated HIF for proteasomal degradation. Furthermore, gramicidin A reduced the growth of human RCC xenograft tumors without causing significant toxicity in mice. Gramicidin A-treated tumors also displayed physiologic and molecular features consistent with the inhibition of HIF-dependent angiogenesis. Taken together, these results demonstrate a new role for gramicidin A as a potent inhibitor of HIF that reduces tumor growth and angiogenesis in VHL-expressing RCC.

Restriction of Food Intake Prevents Postinfarction Heart Failure by Enhancing Autophagy in the Surviving Cardiomyocytes

We investigated the effect of restriction of food intake, a potent inducer of autophagy, on postinfarction cardiac remodeling and dysfunction. Myocardial infarction was induced in mice by left coronary artery ligation. At 1 week after infarction, mice were randomly divided into four groups: the control group was fed ad libitum (100%); the food restriction (FR) groups were fed 80%, 60%, or 40% of the mean amount of food consumed by the control mice. After 2 weeks on the respective diets, left ventricular dilatation and hypofunction were apparent in the control group, but both parameters were significantly mitigated in the FR groups, with the 60% FR group showing the strongest therapeutic effect. Cardiomyocyte autophagy was strongly activated in the FR groups, as indicated by up-regulation of microtubule-associated protein 1 light chain 3-II, autophagosome formation, and myocardial ATP content. Chloroquine, an autophagy inhibitor, completely canceled the therapeutic effect of FR. This negative effect was associated with reduced activation of AMP-activated protein kinase and of ULK1 (a homolog of yeast Atg1), both of which were enhanced in hearts from the FR group. In vitro, the AMP-activated protein kinase inhibitor compound C suppressed glucose depletion-induced autophagy in cardiomyocytes, but did not influence activity of chloroquine. Our findings imply that a dietary protocol with FR could be a preventive strategy against postinfarction heart failure.

Mechanism for HIF-1 activation by cholesterol under normoxia: A redox signaling pathway for liver damage

Cholesterol and chronic activation of hypoxia-inducible factor-1 (HIF-1) have been separately implicated in the pathogenesis and progression of liver diseases. In AML12 hepatocytes increased HIF-1α protein accumulation was evident after 2h of incubation with cholesterol, whereas enhanced HIF-1 transcriptional activity was observed after 6h. Investigations into the molecular mechanism have shown that cholesterol inhibited HIF-1α degradation. Mitochondrial dysfunction and enhanced mitochondrial reactive oxygen species (ROS) generation were observed in 2-h cholesterol-treated cells along with augmented nitric oxide (NO) levels. Further analysis indicated that HIF-1α stabilization at later time (6h), but not after 2h, of incubation with cholesterol was dependent on NO production. To elucidate the role of mitochondrial dysfunction in HIF-1α stabilization, mitochondrial DNA-depleted hepatocytes were prepared. In these cells the ability of cholesterol to activate the HIF-1 pathway was abolished. Similarly, catalase overexpression also attenuated cholesterol-induced HIF-1α accumulation. These results demonstrate that cholesterol promotes HIF-1 activation in a ROS- and NO-dependent manner. Chronic liver activation of HIF-1 by cholesterol may mediate its deleterious effects in the liver.

Transcriptional activation of hypoxia-inducible factor-1 (HIF-1) in myeloid cells promotes angiogenesis through VEGF and S100A8

Emerging evidence indicates that myeloid cells are essential for promoting new blood vessel formation by secreting various angiogenic factors. Given that hypoxia-inducible factor (HIF) is a critical regulator for angiogenesis, we questioned whether HIF in myeloid cells also plays a role in promoting angiogenesis. To address this question, we generated a unique strain of myeloid-specific knockout mice targeting HIF pathways using human S100A8 as a myeloid-specific promoter. We observed that mutant mice where HIF-1 is transcriptionally activated in myeloid cells (by deletion of the von Hippel-Lindau gene) resulted in erythema, enhanced neovascularization in matrigel plugs, and increased production of vascular endothelial growth factor (VEGF) in the bone marrow, all of which were completely abrogated by either genetic or pharmacological inactivation of HIF-1. We further found that monocytes were the major effector producing VEGF and S100A8 proteins driving neovascularization in matrigel. Moreover, by using a mouse model of hindlimb ischemia we observed significantly improved blood flow in mice intramuscularly injected with HIF-1-activated monocytes. This study therefore demonstrates that HIF-1 activation in myeloid cells promotes angiogenesis through VEGF and S100A8 and that this may become an attractive therapeutic strategy to treat diseases with vascular defects.

The Role of Factor Inhibiting HIF (FIH-1) in Inhibiting HIF-1 Transcriptional Activity in Glioblastoma Multiforme

Glioblastoma multiforme (GBM) accounts for about 38% of primary brain tumors in the United States. GBM is characterized by extensive angiogenesis induced by vascular growth factors and cytokines. The transcription of these growth factors and cytokines is regulated by the Hypoxia-Inducible-Factor-1(HIF-1), which is a key regulator mediating the cellular response to hypoxia. It is known that Factor Inhibiting HIF-1, or FIH-1, is also involved in the cellular response to hypoxia and has the capability to physically interact with HIF-1 and block its transcriptional activity under normoxic conditions. Delineation of the regulatory role of FIH-1 will help us to better understand the molecular mechanism responsible for tumor growth and progression and may lead to the design of new therapies targeting cellular pathways in response to hypoxia. Previous studies have shown that the chromosomal region of 10q24 containing the FIH-1 gene is often deleted in GBM, suggesting a role for the FIH-1 in GBM tumorigenesis and progression. In the current study, we found that FIH-1 is able to inhibit HIF-mediated transcription of GLUT1 and VEGF-A, even under hypoxic conditions in human glioblastoma cells. FIH-1 has been found to be more potent in inhibiting HIF function than PTEN. This observation points to the possibility that deletion of 10q23-24 and loss or decreased expression of FIH-1 gene may lead to a constitutive activation of HIF-1 activity, an alteration of HIF-1 targets such as GLUT-1 and VEGF-A, and may contribute to the survival of cancer cells in hypoxia and the development of hypervascularization observed in GBM. Therefore FIH-1 can be potential therapeutic target for the treatment of GBM patients with poor prognosis.

Inhibition of hypoxia-inducible factors limits tumor progression in a mouse model of colorectal cancer

Hypoxia-inducible factors (HIFs) accumulate in both neoplastic and inflammatory cells within the tumor microenvironment and impact the progression of a variety of diseases, including colorectal cancer. Pharmacological HIF inhibition represents a novel therapeutic strategy for cancer treatment. We show here that acriflavine (ACF), a naturally occurring compound known to repress HIF transcriptional activity, halts the progression of an autochthonous model of established colitis-associated colon cancer (CAC) in immunocompetent mice. ACF treatment resulted in decreased tumor number, size and advancement (based on histopathological scoring) of CAC. Moreover, ACF treatment corresponded with decreased macrophage infiltration and vascularity in colorectal tumors. Importantly, ACF treatment inhibited the hypoxic induction of M-CSFR, as well as the expression of the angiogenic factor (vascular endothelial growth factor), a canonical HIF target, with little to no impact on the Nuclear factor-kappa B pathway in bone marrow-derived macrophages. These effects probably explain the observed in vivo phenotypes. Finally, an allograft tumor model further confirmed that ACF treatment inhibits tumor growth through HIF-dependent mechanisms. These results suggest pharmacological HIF inhibition in multiple cell types, including epithelial and innate immune cells, significantly limits tumor growth and progression.

Hypoxia-inducible factor 1 is required for remote ischemic preconditioning of the heart

Both preclinical and clinical studies suggest that brief cycles of ischemia and reperfusion in the arm or leg may protect the heart against injury following prolonged coronary artery occlusion and reperfusion, a phenomenon known as remote ischemic preconditioning. Recent studies in mice indicate that increased plasma interleukin-10 (IL-10) levels play an important role in remote ischemic preconditioning induced by clamping the femoral artery for 5 min followed by 5 min of reperfusion for a total of three cycles. In this study, we demonstrate that remote ischemic preconditioning increases plasma IL-10 levels and decreases myocardial infarct size in wild-type mice but not in littermates that are heterozygous for a knockout allele at the locus encoding hypoxia-inducible factor (HIF) 1α. Injection of a recombinant adenovirus encoding a constitutively active form of HIF-1α into mouse hind limb muscle was sufficient to increase plasma IL-10 levels and decrease myocardial infarct size. Exposure of C2C12 mouse myocytes to cyclic hypoxia and reoxygenation rapidly increased levels of IL-10 mRNA, which was blocked by administration of the HIF-1 inhibitor acriflavine or by expression of short hairpin RNA targeting HIF-1α or HIF-1β. Chromatin immunoprecipitation assays demonstrated that binding of HIF-1 to the Il10 gene was induced when myocytes were subjected to cyclic hypoxia and reoxygenation. Taken together, these data indicate that HIF-1 activates Il10 gene transcription and is required for remote ischemic preconditioning.

Inhibition of the hypoxia-inducible factor pathway by a G-quadruplex binding small molecule

The hypoxia-inducible transcription factor (HIF) co-ordinates the response of tumours to low oxygen by stimulating genes involved in metabolism and angiogenesis. HIF pathway activation is associated with decreased progression-free survival and increased mortality; compounds that target this pathway are potential agents for the treatment of a range of solid tumour malignancies. Renal cancers are likely to be particularly sensitive to inhibition of the HIF pathway since ~80% show constitutive activation of HIF. We have previously described the di-substituted naphthalene derivative, CL67, which binds to a G-quadruplex higher-order structure in the HIF promoter sequence in vitro. We show here that CL67 blocks HIF expression leading to inhibition of HIF-transactivation and down-regulation of downstream target genes and proteins in renal carcinoma cell lines and in a mouse xenograft model of renal cancer. This inhibition is independent of pathways that control HIF abundance through oxygen-dependant degradation and oxygen dependant HIF sub-unit expression.

The circadian clock and the hypoxic response pathway in kidney cancer

The most frequent malignant tumor of the kidney in adults is represented by renal cell carcinoma characterized by high lethality related to presence of metastatic disease at the time of diagnosis. The main characteristic molecular feature of most sporadic renal cell carcinomas is the mutation of the tumor suppressor gene encoding the von Hippel-Lindau protein, with alteration of regulated pathways and activation of hypoxia-inducible transcription factors. Hypoxia-inducible transcription factors are transcriptional regulators of genes controlling mammalian oxygen homeostasis, energy metabolism, neovascularisation, internal pH, cell survival, and migration and are considered powerful promoters of tumor growth. Tight interrelationships have been evidenced between hypoxic response pathway and circadian pathway. Severe deregulation of genes involved in the circadian clock circuitry and response to hypoxia has been found in patients affected by kidney cancer, influencing the process of carcinogenesis, as well as disease progression and outcome. The study of alterations of clock gene expression and hypoxia correlated pathway in kidney cancer may promote the comprehension of pathophysiological mechanisms involved in renal cell carcinoma onset and evolution and may help to exploit more effective therapeutic approaches.

Development of an Oxygen-Sensitive Degradable Peptide Probe for the Imaging of Hypoxia-Inducible Factor-1-Active Regions in Tumors

We aimed to develop a radiolabeled peptide probe for the imaging of hypoxia-inducible factor-1 (HIF-1)-active tumors. We synthesized the peptide probes that contain or lack an essential sequence of the oxygen-dependent degradation of HIF-1α in proteasomes ((123/125)I-DKOP30 or (125)I-mDKOP, respectively). The degradation of probes was evaluated in vitro using cell lysates containing proteasomes. In vivo biodistribution study, planar imaging, autoradiography, and comparison between probe accumulation and HIF-1 transcriptional activity were also performed. The (125)I-DKOP30 underwent degradation in a proteasome-dependent manner, while (125)I-mDKOP was not degraded. Biodistribution analysis showed (125)I-DKOP30 accumulation in tumors. The tumors were clearly visualized by in vivo imaging, and intratumoral distribution of (125)I-DKOP30 coincided with the HIF-1α-positive hypoxic regions. Tumoral accumulation of (125)I-DKOP30 was significantly correlated with HIF-1-dependent luciferase bioluminescence, while that of (125)I-mDKOP was not. (123)I-DKOP30 is a useful peptide probe for the imaging of HIF-1-active tumors.

Regulation of hypoxic responses by Sprouty2 in hepatocellular carcinoma

Sprouty2 (Spry2) is an inhibitor of receptor tyrosine kinases (RTKs) and decreases in its levels have been documented in breast, lung, liver, and prostate cancer. Decreases in Spry2 levels have been correlated with poor patient prognosis in hepatocellular carcinoma (HCC). During tumor growth, cells experience hypoxic periods and adapt to these by altering a variety of cellular processes mainly through the Hypoxia Inducible transcription Factors (HIFs). HIFs are composed of an α subunit (HIF1α or HIF2α) and a β subunit (HIF1β a.k.a. aryl hydrocarbon receptor nuclear translocator (ARNT)). In hypoxia, the HIFα subunits are stabilized, translocate to the nucleus, and dimerize with ARNT. The HIFs have been shown to be upregulated in cancers correlating to poor patient prognosis. We have shown that Spry2 protein levels are elevated in hypoxia. The objective of our study was to determine the role of Spry2 in regulating hypoxic responses in HCC cells as it could have an important impact in tumor growth and progression. We found that endogenous Spry2 by modulating ARNT protein levels alters the transcriptional activity of HIF. Thus, our data suggest that Spry2 regulates HIF activity in HCC cells.Supported by NIH grant GM 073181

Reduction of Obliterative Bronchiolitis (OB) by Prolyl-Hydroxylase-Inhibitors Activating Hypoxia-Inducible Transcription Factors in an Experimental Mouse Model

Purpose Obliterative Bronchitis (OB) is the major limiting factor for long-term survival after lung transplantation. As previously shown, donor treatment with a PHD-inhibitor activating Hypoxia-inducible transcription factors (HIFs) prevents graft injury in an allogenic kidney and aortic allograft transplant model. The aim of this study was to investigate the effect of HIF activation with a PHD-inhibitor on the development of OB. Methods and Materials Fully MHC-mismatched C57BL/6 (H2(b)) donor tracheas were orthotopically transplanted into CBA (H2(k)) recipients. Donor animals received a single dose of PHD-inhibitor 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate (ICA) (40mg/kg i.p.) or vehicle 4h before transplantation (n=5). Animals were harvested 30 days after transplantation and grafts were analyzed by histology, mRNA expression and immunofluorescence. Results Donor preconditioning with ICA resulted in HIF accumulation and induction of HIF target genes: HOI, VEGF, TGFβ, and MIFα, which persisted during different times of ischemia. Vehicle treated controls showed substantial luminal obliteration on postoperative day 30 in contrast to groups pre-treated with ICA [luminal obliteration 29.2 +5% (ICA) vs. 36.7+8% (control), p Conclusions Pre-treatment with ICA substantially reduces Obliterative Bronchiolitis. These data suggest that activation of Hypoxia-inducible transcription factors (HIFs) and hereby adaptation to low oxygen prevents the development of OB and therefore allograft injury. Pharmaceutical inhibition of PHDs appears to be a very attractive strategy for organ preservation that deserves further clinical evaluation.

Hypoxia-Inducible Transcription Factor Target Genes VEGF and TGF-β Modulate Transplant Arteriosclerosis in a Murine Aortic Allograft Model

Purpose Transplant arteriosclerosis is associated with a chronic immune response and influenced by the initial injury caused by ischemia and reperfusion. As previously shown, donor treatment with a PHD-inhibitor activating Hypoxia-inducible transcription factors (HIFs) prevents graft injury in an allogenic aortic transplant model. Therefore, the aim of this study was to investigate the underlying mechanisms leading to decreased levels of transplant arteriosclerosis. Methods and Materials MHC-class-I mismatched C.B10-H2(b)/LilMcdJ donor aortas were transplanted into BALB/c mice. Donor animals received a single dose of PHD-inhibitor 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate (ICA) (40mg/kg i.p.) or vehicle 4h before transplantation. Grafts were kept at different times of cold ischemia from 30 min up to 6h (n=5). Upregulation of HIF target genes was analysed at time of transplantation by mRNA expression analysis. Transplanted grafts were analyzed by histology, mRNA expression and immunofluorescence 30 days after transplantation. Results Vascular lesions were present in both experimental groups, however there was again significantly less intimal proliferation in preconditioned grafts as compared to vehicle treated controls [intimal proliferation 31,3+8% (ICA) vs. 55,3+20% (control), p Conclusions These data confirm activation of Hypoxia-inducible transcription factors and their target genes during ischemia. Adaptation to low oxygen protects from ischemia-reperfusion injury. Pharmaceutical inhibition of PHDs appears to be a very attractive strategy for organ preservation that deserves further clinical evaluation.

Suppression of Tumor Growth by Designed Dimeric Epidithiodiketopiperazine Targeting Hypoxia-Inducible Transcription Factor Complex

Hypoxia is a hallmark of solid tumors, is associated with local invasion, metastatic spread, resistance to chemo- and radiotherapy, and is an independent, negative prognostic factor for a diverse range of malignant neoplasms. The cellular response to hypoxia is primarily mediated by a family of transcription factors, among which hypoxia-inducible factor 1 (HIF1) plays a major role. Under normoxia, the oxygen-sensitive α subunit of HIF1 is rapidly and constitutively degraded but is stabilized and accumulates under hypoxia. Upon nuclear translocation, HIF1 controls the expression of over 100 genes involved in angiogenesis, altered energy metabolism, antiapoptotic, and pro-proliferative mechanisms that promote tumor growth. A designed transcriptional antagonist, dimeric epidithiodiketopiperazine (ETP 2), selectively disrupts the interaction of HIF1α with p300/CBP coactivators and downregulates the expression of hypoxia-inducible genes. ETP 2 was synthesized via a novel homo-oxidative coupling of the aliphatic primary carbons of the dithioacetal precursor. It effectively inhibits HIF1-induced activation of VEGFA, LOX, Glut1, and c-Met genes in a panel of cell lines representing breast and lung cancers. We observed an outstanding antitumor efficacy of both (±)-ETP 2 and meso-ETP 2 in a fully established breast carcinoma model by intravital microscopy. Treatment with either form of ETP 2 (1 mg/kg) resulted in a rapid regression of tumor growth that lasted for up to 14 days. These results suggest that inhibition of HIF1 transcriptional activity by designed dimeric ETPs could offer an innovative approach to cancer therapy with the potential to overcome hypoxia-induced tumor growth and resistance.