HIF-1α Response Research Articles

Proteasomal degradation of O-GlcNAc transferase elevates hypoxia-induced vascular endothelial inflammatory response†.

Hypoxia induces vascular inflammation by a mechanism not fully understood. Emerging evidence implicates O-GlcNAc transferase (OGT) in inflammation. This study explored the role of OGT in hypoxia-induced vascular endothelial inflammatory response. Hypoxia was either induced (1% O2 chamber) or mimicked by exposure to hypoxia-mimetic agents in cultured endothelial cells. Hypoxia increased hypoxia-inducible factor (HIF-1α) and inflammatory response (gene and protein expression of interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1, and E-selectin) but, surprisingly, reduced OGT protein (not mRNA) levels. Hypoxia-mimetic CoCl2 failed to reduce OGT when proteasome inhibitors were present, suggesting proteasome involvement. Indeed, CoCl2 enhanced 26S proteasome functionality evidenced by diminished reporter (Ub(G76V)-GFP) proteins in proteasome reporter cells, likely due to increased chymotrypsin-like activities. Mechanistically, β-TrCP1 mediated OGT degradation, since siRNA ablation of this E3 ubiquitin ligase stabilized OGT. Administration of the oxidative stress inhibitors reversed both proteasome activation and OGT degradation. Furthermore, up-regulation of OGT by stabilization, overexpression, or activation mitigated CoCl2-elicited inflammatory response. These observations were recapitulated in a mouse (C57BL/6J) model mimicking hypoxia, in which lung tissues presented higher levels of HIF-1α, proteasome activity, and inflammatory response, but lower levels of OGT (n = 5/group, hypoxia vs. normoxia, P < 0.05). However, administration of an activator of OGT (glucosamine: 1 mg/g/day, vehicle: saline, ip, 5 days) abolished the up-regulation of proteasome activity and inflammatory response (n = 5/group, the treated vs. untreated hypoxia groups, P < 0.05). 26S proteasome-mediated OGT reduction contributed to hypoxia-induced vascular endothelial inflammatory response. Modulation of OGT may represent a new approach to treat diseases characterized by hypoxic inflammation.

2‐Methoxyestradiol confers neuroprotection and inhibits a maladaptive HIF‐1α response after traumatic brain injury in mice

HIF-1α is pivotal for cellular homeostasis in response to cerebral ischemia. Pharmacological inhibition of HIF-1α may reduce secondary brain damage by targeting post-translational mechanisms associated with its proteasomal degradation and nuclear translocation. This study examined the neuroprotective effects of 2-methoxyestradiol (2ME2), the involved HIF-1α-dependent response, and alternative splicing in exon 14 of HIF-1α (HIF-1α∆Ex14) after traumatic brain injury (TBI) in mice. Intraperitoneal 2ME2 administration 30 min after TBI caused a dose-dependent reduction in secondary brain damage after 24 h. 2ME2 was physiologically tolerated, showed no effects on immune cell brain migration, and mitigated trauma-induced brain expression of neuropathologically relevant HIF-1α target genes encoding for Plasminogen activator inhibitor 1 and tumor necrosis factor alpha. Moreover, TBI-induced expression of pro-apoptotic BNIP3 was attenuated by 2ME2 treatment. Alternatively, spliced HIF-1α∆Ex14 was substantially up-regulated from 6 to 48 h after TBI. In vitro, nuclear location and gene transcription activity of HIF-1α∆Ex14 were impaired compared to full-length HIF-1α, but no effects on nuclear translocation of the transcriptional complex partner HIF-1β were observed. This study demonstrates that 2ME2 confers neuroprotection after TBI. While the role of alternatively spliced HIF-1α∆Ex14 remains elusive, the in vivo data provide evidence that inhibition of a maladaptive HIF-1α-dependent response contributes to the neuroprotective effects of 2ME2. We examined neuroprotective effects of 2-methoxyestradiol (2ME2) and the hypoxia-inducible factor 1-α (HIF-1α) response following traumatic brain injury in mice. Early 2ME2 administration reduced the secondary brain damage and neuronal HIF-1α probably involving ubiquitin proteasome system-mediated degradation. The up-regulation of neuropathological HIF-1α target genes and pro-apoptotic BNIP3 protein was attenuated. We propose that the inhibition of a maladaptive HIF-1α response may contribute to 2ME2-mediated neuroprotection.

Ischemic preconditioning increased the intestinal stem cell activities in the intestinal crypts in mice

BackgroundIschemic preconditioning (IPC) can protect against ischemia–reperfusion injury in the small intestine. Because intestinal stem cells (ISCs) control the recovery and growth of intestinal villi, this study investigated whether IPC had any effects on the activity of ISCs. Materials and methodsThe small intestines of mice were treated with IPC, laparotomy only (sham), or no surgery. The crypt fractions were isolated and the characteristics of ISCs among various groups were compared. The regenerative ability and the number of organoids grown from various crypt fractions were compared. The expression of hypoxia-inducible factor-1α (HIF-1α) and the related proteins of the Wnt-/β-catenin pathway in the crypt fractions were studied. ResultsThe IPC group had higher messenger RNA levels of various stem cell markers than the sham group at days 1 and 2 after surgery. The IPC group exhibited greater regenerative activity and more crypt organoids than the sham group (P < 0.05). The expression of HIF-1α, β-catenin, and phosphoglycogen synthase kinase 3β was increased in the IPC-treated crypt fractions in vivo and cultured crypt organoid cells with deferoxamine-mimicked hypoxia in vitro. ConclusionsIPC significantly upregulated the activity of ISCs, possibly through the HIF-1α response and Wnt-/β-catenin signaling pathway.

Abstract 2416: Weekly EZN-2208 (pegylated SN-38) in combination with bevacizumab in patients with refractory solid tumors.

Abstract Background: Anti-angiogenic therapies such as bevacizumab (Bev) have been shown to up-regulate hypoxia-inducible factor-1α (HIF-1α), a possible mechanism of resistance. Camptothecin analogues, including SN-38, have been shown to reduce the expression and transcriptional activity of HIF-1α in preclinical models. Thus, co-administration of pegylated SN 38 (EZN 2208) could offset the induction of HIF-1α following administration of Bev, resulting in synergistic anti-tumor effects. We are conducting a trial of the combination of EZN 2208 (E) with Bev in patients with refractory solid tumors to determine the safety and tolerability of the combination, and to evaluate modulation of HIF-1α protein in tumor biopsies following administration of study drugs. Methods: Eligible pts have refractory advanced solid tumors that have progressed following standard therapy; ≥ 18 yrs of age; ECOG PS 0-2; adequate organ function. Bev at 5mg/kg is administered on D-7 and D15 for cycle 1 only and on D1, 15 for each subsequent cycle; q28d cycles. E(9mg/kg) is administered on D1, 8, and 15. Tumor biopsies and dynamic contrast enhanced MRI (DCE-MRI) are obtained on D1 prior to EZN administration (7 days post Bev) and on C2D15 post Bev and E. An additional DCE-MRI is also performed at baseline prior to Bev on D-7. Tumor biopsies are analyzed for HIF-1α protein levels using a validated immunoassay, and HIF-1α response genes, Hexokinase 2(HK), vascular endothelial growth factor A(VEGF), pyruvate dehydrogenase kinase, isozyme 1(PDK1), carbonic anhydrase IX(CA9), solute carrier family 2 (GLUT1), are assessed using RTPCR. Results: Twelve pts have been enrolled to date; median age 50 (range 27-76 yrs); median # prior therapies 3 (1-6); Dx: Soft tissue sarcoma (4), colorectal cancer (2), parotid gland cancer (1), malignant hurthle cell tumor (1), HCC (1), melanoma (1), gastrointestinal stromal tumor (1), Head and neck cancer (1). Grade 3/4 toxicities (#pts): neutropenia (8), leucopenia (3), lymphopenia (2), hypertension (1). Eight pts are evaluable for response; prolonged stable disease was observed in 2 pts: HCC 16 cycles, desmoplastic round cell tumor 7 cycles. HIF-1α protein and mRNA levels of HIF-1α dependent genes were assessed in 5 pts who had paired biopsies. Reduction in HIF-1α protein levels compared to baseline (post Bev alone) was seen in 4 of 5 pts (range 24% - 64%), with evidence of modulation of mRNA in 3 of 5 pt tumors evaluated. Quantitative analysis of DCE MRI from 3 pts revealed changes in Ktrans and kep maps following drug administration. Correlation of observed changes with tumor response is ongoing. Conclusions: This clinical trial provides preliminary proof of mechanism demonstrating modulation of HIF-1α levels in tumors following administration of a camptothecin analog, EZN 2208. Accrual is ongoing to establish safety, efficacy, and further proof of target modulation. Citation Format: Woondong Jeong, Sook Ryun Park, Annamaria Rapisarda, Michelle Eugeni, Robert Kinders, Alice Chen, Giovanni Melillo, Baris Turkbey, James H. Doroshow, Shivaani Kummar. Weekly EZN-2208 (pegylated SN-38) in combination with bevacizumab in patients with refractory solid tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2416. doi:10.1158/1538-7445.AM2013-2416

C/EBP Homologous Binding Protein (CHOP) Underlies Neural Injury in Sleep Apnea Model

Obstructive sleep apnea (OSA) is associated with cognitive impairment and neuronal injury. Long-term exposure to intermittent hypoxia (LTIH) in rodents, modeling the oxygenation patterns in sleep apnea, results in NADPH oxidase 2 (Nox2) oxidative injury to many neuronal populations. Brainstem motoneurons susceptible to LTIH injury show uncompensated endoplasmic reticulum stress responses with increased (CCAAT/enhancer binding protein homologous protein (CHOP). We hypothesized that CHOP underlies LTIH oxidative injury. In this series of studies, we first determined whether CHOP is upregulated in other brain regions susceptible to LTIH oxidative Nox2 injury and then determined whether CHOP plays an adaptive or injurious role in the LTIH response. To integrate these findings with previous studies examining LTIH neural injury, we examined the role of CHOP in Nox2, hypoxia-inducible factor-1α (HIF-1α) responses, oxidative injury and apoptosis, and neuron loss. Within/between mice subjects. Laboratory setting. PARTICIPANTSSUBJECTS: CHOP null and wild-type adult male mice. LTIH or sham LTIH. Relative to wild-type mice, CHOP-/- mice conferred resistance to oxidative stress (superoxide production/ carbonyl proteins) in brain regions examined: cortex, hippocampus, and motor nuclei. CHOP deletion prevented LTIH upregulation of Nox2 and HIF-1α in the hippocampus, cortex, and brainstem motoneurons and protected mice from neuronal apoptosis and motoneuron loss. Endogenous CHOP is necessary for LTIH-induced HIF-1α, Nox2 upregulation, and oxidative stress; CHOP influences LTIH-induced apoptosis in neurons and loss of neurons. Findings support the concept that minimizing CHOP may provide neuroprotection in OSA.

Oleuropein, a Secoiridoid Derived from Olive Tree, Inhibits the Proliferation of Human Colorectal Cancer Cell Through Downregulation of HIF-1α

Oleuropein (OL) is the most prominent phenolic compound in the fruit of olive tree. Although OL has shown powerful anticancer activity the underlying action mechanism remains largely unknown. The present study evaluated the effects of OL on hydroxityrosol (HT)-29 human colon adenocarcinoma cells in comparison to hydroxytyrosol, its hydrolysis product, and to elucidate the underlying anticancer molecular mechanisms involved. Cell proliferation was determined using SRB assay. Cell cycle and apoptosis were assessed by flow cytometry and changes in MAPK cascade protein expression, HIF-1α, p53, PPARγ, and NFKβ signaling pathways by Western blot. Although OL showed less potency than HT, in terms of cell growth inhibition, induced significant changes in cell cycle analysis and caused a significant increase in the apoptotic population. Both compounds produced a remarkable decrease in HIF-1α protein and an upregulation of p53 protein expression. However, no significant changes in IkB-α and MAPK cascade protein expressions were observed. HT produced a significant upregulation in peroxisome proliferator-activated receptor gamma (PPARγ) expression whereas OL failed. PPARγ upregulation may be one of the principal mechanisms of the tumor shrinkage by HT. Our novel findings demonstrate that OL limits the growth and induces apoptosis in HT-29 cells via p53 pathway activation adapting the HIF-1α response to hypoxia.

Abstract 19209: Ectonucleoside Triphosphate Diphosphohydrolase-1 (cd39) Attenuates Expression f of P-Selectin Glycoprotein Ligand-1 in Hypoxic Raw Cells via Hif-1α

CD39 is a transmembrane enzyme that phosphohydrolyzes ATP and ADP to AMP. CD39 inhibits platelet reactivity and inflammation and thus limits platelet accumulation and leukocyte transmigration to sites of inflammation or injury. P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like glycoprotein on the surface of leukocytes which binds with high affinity to P-selectin, to promote tethering and rolling adhesion of leukocytes to activated endothelial cells and platelets. As this leads to increased leukocyte migration to sites of inflammation or injury, we hypothesized that CD39 may regulate PSGL-1 expression on leukocytes under hypoxic conditions. Murine RAW macrophages transfected with a CD39 overexpression construct (CD39Tx) or a pcDNA3.1 vector (VTx) as control were incubated in hypoxia (0.5% O2) or nomoxia. Subsequent flow cytometry showed 35% more cells expressed PSGL-1 after 6 hours of exposure to hypoxia (H) on VTx cells compared to normoxic (N) VTx control (p &lt; 0.0001). The number of cells expressing PSGL-1 was reduced by 14% in H CD39Tx cells compared with H VTx cells (p = 0.0061). Western blotting showed PSGL-1 antigen expression increased 4.9, 7.0 and 3.9 fold after 2, 6, and 16 hours of hypoxia in VTx cells, respectively, compared to N VTx controls; and PSGL-1 antigen was also attenuated 19%, 30%, and 28% respectively in H CD39Tx cells compared with H VTx cells. Quantitative real-time PCR results showed that PSGL-1 mRNA expression increased by 2.1 fold in 6 hours H VTX cells compared with N VTx cells (p &lt; 0.0001) and decreased 40% in H CD39Tx cells compared with H VTx control (p &lt; 0.0001). The PSGL-1 promoter region contains 3 HIF-1α response elements (HRE). The activity of PSGL-1 promoter increased by 1.9-fold in 6 hours H VTx cells compared with N VTx controls (p &lt; 0.0001) and decreased by 61% in H CD39Tx cells compared with H VTx controls (p &lt; 0.0001). HIF-1α protein expression was also reduced in nuclear protein samples of H CD39Tx cells compared to H VTx cells. These data identify CD39 as a critical regulator of macrophage PSGL-1 expression, acting through the HIF-1alpha pathway to squelch inflammatory cell trafficking under hypoxic/ischemic conditions.

Abstract 267: Hemoglobin, Endothelial Cells, and TLRs. Common Connection?

Introduction: Patients suffering from chronic hereditary hemolytic anemic syndromes, such as sickle cell disease (SCD) and thalassemia, are often at risk for systemic and pulmonary vascular disease. It has been suggested that chronic exposure to cell free hemoglobin (CFH) may contribute to some vascular diseases associated with these syndromes such as pulmonary arterial hypertension. To date, the vasculotoxic effects of CFH have mostly been attributed to its pro-oxidant and nitric oxide scavenging characteristics. However, emerging evidence suggests CFH may contribute to inflammation by directly activating a signaling cascade event by binding to a pattern recognition receptor (PRR) or a toll like receptor (TLR) on vascular endothelial cells. Hypothesis: We hypothesized that CFH would increase the activity of transcription factors, NF-κb and HIF-1α, via a MyD88-dependent pathway. Methods: Human microvascular endothelial cells (HMEC) were transfected with either an NF-κB or HIF-1α luciferase reporter gene and treated with CFH (ferrous, ferric, and ferryl forms) in the presence or absence of SOD, catalase, dexamethasome, MyD88 inhibitor, or, the PHD inhibitor, DMOG. Messenger RNA for HIF-1α and HIF-2 were also measured after treatments. Results: All three states of hemoglobin increased NF-κB and HIF-1α activity in a dose response fashion, with ferryl inducing the greatest activity of both NF-κB and HIF-1α. Time course studies showed that NF-κB and HIF-1α activity tracked together. A unique synergy was noted with co-treatment of ferryl and DMOG. Co-treatment with SOD or catalase did not inhibit the CFH-induced NF-κB or HIF-1α response. Dexamthasome and MyD88 inhibition reduced the CFH-induced NF-κB and HIF-1α activity. Conclusion: Our results support the hypothesis, that CFH may activate a TLR or PRR signaling cascade subsequently activating MyD88-NF-κB and HIF-1α. Our data, that showed SOD and/or catalase did not block CFH effects, suggests that this event is not mediated by CFH pro-oxidant characteristics. CFH-induced HIF-1α was blocked by NF-κB inhibition with either, Dexamethasome or MyD88 inhibition emphasizing the importance of NF-κB in the HIF-1α pathway.

Impaired response of hypoxic sensor protein HIF-1α and its downstream proteins in the spinal motor neurons of ALS model mice

We have recently reported spinal blood flow-metabolism uncoupling in an amyotrophic lateral sclerosis (ALS) animal model using Cu/Zn-superoxide dismutase 1 (SOD1)-transgenic (Tg) mice, suggesting a relative hypoxia in the spinal cord. However, the hypoxic stress sensor pathway has not been well studied in ALS. Here, we examined temporal and spatial changes of the hypoxic stress sensor proteins HIF-1α and its downstream proteins (VEGF, HO-1, and EPO) during the normoxiccourse of motor neuron (MN) degeneration in the spinal cord of these ALS model mice. We found that HIF-1α protein expression progressively increased both in the anterior large MNs and the surrounding glial cells in Tg mice from early symptomatic 14 week (W) and end stage 18W. Double immunofluorescence analysis revealed that HIF-1α, plus GFAP and Iba-1 double-positive surrounding glial cells, progressively increased from 14W to 18W, although the immunohistochemistry in large MNs did not change. Expression levels of VEGF and HO-1 also showed a progressive increase but were significant only in the surrounding glial cells at 18W. In contrast, EPO protein expression was decreased in the surrounding glial cells of Tg mice at 18W. Because HIF1-α serves as an important mediator of the hypoxic response, these findings indicate that MNs lack the neuroprotective response to hypoxic stress through the HIF-1α system, which could be an important mechanism of neurodegeneration in ALS.

Loss of fibroblast HIF-1α accelerates tumorigenesis.

Solid tumors consist of malignant cells and associated stromal components, including fibroblastic cells that contribute to tumor growth and progression. Although tumor fibrosis and aberrant vascularization contribute to the hypoxia often found in advanced tumors, the contribution of hypoxic signaling within tumor-associated fibroblasts to tumorigenesis remains unknown. In this study, we used a fibroblast-specific promoter to create mice in which key hypoxia regulatory genes, including VHL, HIF-1α, HIF-2α, and VEGF-A, were knocked out specifically in tumor stromal fibroblasts. We found that loss of HIF-1α and its target gene VEGF-A accelerated tumor growth in murine model of mammary cancer. HIF-1α and VEGF-A loss also led to a reduction in vascular density and myeloid cell infiltration, which correlated with improved tumor perfusion. Together, our findings indicate that the fibroblast HIF-1α response is a critical component of tumor vascularization.

Roles for Peroxisome Proliferator-activated Receptor γ (PPARγ) and PPARγ Coactivators 1α and 1β in Regulating Response of White and Brown Adipocytes to Hypoxia

Obese white adipose tissue is hypoxic but is incapable of inducing compensatory angiogenesis. Brown adipose tissue is highly vascularized, facilitating delivery of nutrients to brown adipocytes for heat production. In this study, we investigated the mechanisms by which white and brown adipocytes respond to hypoxia. Brown adipocytes produced lower amounts of hypoxia-inducible factor 1α (HIF-1α) than white adipocytes in response to low O(2) but induced higher levels of hypoxia-associated genes. The response of white adipocytes to hypoxia required HIF-1α, but its presence alone was incapable of inducing target gene expression under normoxic conditions. In addition to the HIF-1α targets, hypoxia also induced many inflammatory genes. Exposure of white adipocytes to a peroxisome proliferator-activated receptor γ (PPARγ) ligand (troglitazone) attenuated induction of these genes but enhanced expression of the HIF-1α targets. Knockdown of PPARγ in mature white adipocytes prevented the usual robust induction of HIF-1α targets in response to hypoxia. Similarly, knockdown of PPARγ coactivator (PGC) 1β in PGC-1α-deficient brown adipocytes eliminated their response to hypoxia. These data demonstrate that the response of white adipocytes requires HIF-1α but also depends on PPARγ in white cells and the PPARγ cofactors PGC-1α and PGC-1β in brown cells.

Abstract 2206: Molecular interaction of HIF-1α and the androgen receptor in prostate cancer

Abstract Background: Prostate cancer is the most common noncutaneous cancer among men in the United States, and its progression is largely controlled by the androgen receptor (AR). Androgen deprivation therapy (ADT) is an initially effective treatment for prostate cancer, but most tumors eventually become castrate resistant. Tumor hypoxia also appears to be associated with a poor prognosis in prostate cancer. HIF-1α regulates the transcription of genes that allow tumor survival and growth in low oxygen conditions. Our laboratory has data showing that in response to castration and anti-androgen therapy in mice, there was a strong transcriptional relationship between HIF-1α and AR, as measured by quantitative RT-PCR, suggesting an interaction between the two proteins. Thus, the purpose of this study was to determine if there is a molecular interaction between HIF-1α and AR in prostate cancer cells. Methods: We used Western blot analysis to examine the expression levels of HIF-1α and AR in LNCaP prostate cancer cells to determine if they are upregulated together at the protein level. Four experimental conditions were tested: control (no treatment), DHT for induction of AR expression, CoCl2 for induction of HIF-1α, and a combination treatment of DHT and CoCl2. Immunoprecipitation experiments were carried out to determine if there is an association between HIF-1α and AR. In addition, cells were fractionated into nuclear and cellular cells extracts, followed by Western blot analysis to determine where in the cells this interaction occurs. Results: Western blot analysis of cell lysates showed synergistic upregulation of both HIF-1α and AR expression only under combined CoCl2 and DHT treatment conditions. In addition, immunoprecipitation experiments showed that HIF-1α and AR exist in a complex with one another, and fractionation experiments indicated this complex occurs in the nucleus. Conclusions: Our results demonstrate that HIF-1α and AR associate with one another in cells. Binding assays are in progress to determine the nature of this interaction. In addition, we are examining the cellular consequences of this protein interaction, as it is possible that upregulation of HIF-1α in response to low androgen contributes to the development of tumor resistance to ADT. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2206. doi:1538-7445.AM2012-2206

Abstract 3672: Preclinical assessment of MET modulation by a VEGFR inhibitor/MET inhibitor combination that shows additive antitumor efficacy

Abstract Background: Suppression of VEGFR signaling promotes activation of the MET signaling pathway, which may compensate for the VEGFR blockade, potentially leading to cellular invasion and metastasis. These findings have led to the hypothesis that VEGFR inhibitors will be more effective when combined with MET inhibitors in the clinic. This hypothesis is currently being tested in a clinical trial of Pazopanib with Tivantinib (ARQ197) at the NCI. We have replicated this trial in a xenograft model to evaluate the pharmacodynamic (PD) response of MET and HIF-1α using newly developed and validated quantitative assays and relate the biomarker response to anti-tumor efficacy. Methods: Ectopic SNU5 (human gastric cancer cell line) xenografts were staged to 200 mm3 in athymic nude mice and treated for 8 days with vehicle, Pazopanib (100 mg/kg/day, QD), Tivantinib (200 mg/kg/day, QD), and combinations of Pazopanib (QD) with two dose levels of Tivantinib (200 mg/kg/day QD and 400 mg/kg/day BID). Trucut needle biopsies (18Ga) were collected at 4 hours and 24 hours after Tivantinib administration on Day 8 and processed according to procedures developed to minimize analyte degradation. Tumor lysates were evaluated for MET, pY1235MET, pY1356MET, and HIF-1α using ELISA assays. Tumor volumes were measured at baseline and on Day 8. Results: PD-biomarker analysis in biopsy specimens revealed that treatment with Tivantinib alone did not significantly change MET, pY1235MET, and pY1356MET levels. Pazopanib treatment did not significantly increase total MET levels, but significantly increased pY1235MET and pY1356 MET at 4 hours after the Day 8 dose. The Pazopanib-induced activation of pY1235MET and pY1356MET was prevented by Tivantinib (p&amp;lt;0.05) in combination (BID schedule). Analysis of intratumoral levels of HIF-1α is currently underway. Compared to the vehicle treated group, single agent Tivantinib and Pazopanib inhibited tumor growth measured on Day 8 by 19% (p&amp;lt;0.05) and 20% (p&amp;lt;0.05), respectively, whereas the combination of Pazopanib and Tivantinib achieved 38-46% inhibition of tumor growth (p&amp;lt;0.05). The combination regimen was more effective when Tivantinib was administered BID than QD. Conclusions: The combination of Pazopanib with Tivantinib exhibited additive anti-tumor activity that was greater than either agent alone. Enhanced efficacy of combination therapy was associated with Tivantinib block of Pazopanib-induced activation of MET. These results confirm the hypothesis underlying clinical trials of combinations of VEGFR with MET inhibitors. The study is being expanded with additional xenograft models and treatment schedules to confirm and further examine the generality of these findings. Funded by NCI Contract #HHSN261200800001E. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3672. doi:1538-7445.AM2012-3672

Hypoxia-inducible factor-1 up-regulates the expression of Toll-like receptor 4 in pancreatic cancer cells under hypoxic conditions

Background & aimsHypoxia is a common characteristic of solid tumors. Recent studies confirmed that Toll-like receptor 4 (TLR4) plays a significant role in cancer invasion and progression. In this study, the correlation between the expression of TLR4 and the change of the protein level of Hypoxia-inducible factor-1 alpha (HIF-1α) was studied. MethodsWe examined 84 human pancreatic cancer tissues for expression of HIF-1α and TLR4 proteins. Panc-1 cells were exposed to normoxia (20% O2) or hypoxia (<1% O2) or treated with CoCl2. TLR4 protein was analyzed by flow cytometry and immunostaining. Growth studies were conducted on cells with the HIF-1α inhibition isolated from stable transfected cell lines. Finally, TLR4 protein was detected by immunohistochemistry in vivo tumors. ResultsThere was a positive correlation between TLR4 and HIF-1α protein in pancreatic cancer tissues. Hypoxic stress induced TLR4 mRNA and protein expression in Panc-1 cells. Cells transfected with HIF-1α siRNA showed attenuation of hypoxia stress-induced TLR4 expression. In vivo growth decreased in response to TLR4 and HIF-1α inhibiton. Transient HIF-1α siRNA treatment could effectively curb tumor growth in vivo. ConclusionThese results suggest that TLR4 expression in pancreatic cancer cells is up-regulated via HIF-1α in response to hypoxic stress and underscore the crucial role of HIF-1α-induced TLR4 in tumor growth.

Nitric oxide modulates hypoxia-inducible factor-1 and poly(ADP-ribose) polymerase-1 cross talk in response to hypobaric hypoxia

The physiological response to hypobaric hypoxia represents a complex network of biochemical pathways in which the nitrergic system plays an important role. Previous studies have provided evidence for an interplay between the hypoxia-inducible factor-1 (HIF-1) and poly(ADP-ribose) polymerase-1 (PARP-1) under hypoxia. Here, we evaluate the potential involvement of nitric oxide (NO) in the cross talk between these two proteins. With this aim, we studied comparatively the effect of pharmacological inhibitors of NO production or PARP activity in the response of the mouse cerebral cortex to 4 h of exposure to a simulated altitude of 31,000 ft. Particularly, we analyzed the NO and reactive oxygen species production, the expression of NO synthase (NOS) isoforms, PARP-1 activity, HIF-1α expression and HIF-1 transcriptional activity, the protein level of the factor inhibiting HIF, and, finally, beclin-1 and fractin expression, as markers of cellular damage. Our results demonstrate that the reduction of NO level did not affect reactive oxygen species production but significantly 1) dampened the posthypoxic increase in neuronal NOS and inducible NOS expression without altering endothelial NOS protein level; 2) prevented PARP activation; 3) decreased HIF-1α response to hypoxia; 4) achieved a higher long-term HIF-1 transcriptional activity by reducing factor inhibiting HIF expression; and 5) reduced hypoxic damage. The pharmacological inhibition of PARP reproduced the NOS expression pattern and the HIF-1α response observed in NOS-inhibited mice, supporting its involvement in the NO-dependent regulation of hypoxia. As a whole, these results provide new data about the molecular mechanism underlying the beneficial effects of controlling NO production under hypobaric hypoxic conditions.

Abstract C3: Validation and fitness testing of a quantitative immunoassay for HIF1α in biopsy specimens.

Abstract HIF1α is an important marker of hypoxia in human tumors that is altered in a number of cancers, but a reproducible method to determine changes in HIF1α protein expression in human tumor biopsy specimens has not been available. HIF1α is being used as a pharmacodynamic marker in a clinical trial of metronomic Topotecan (CTEP# 8610, 9534). However, an important limitation for studying the response of HIF1α to cancer therapeutic agents is the lability of the protein, in the presence of oxygen, upon collection of the sample. We have devised a method of specimen collection, handling and extraction that preserves and stabilizes HIF1α levels in tumor biopsies. Employing this specimen handling method allowed validation of a two-site immunoassay for HIF1α quantitation in solid tissue extracts, such as tumor biopsies. Intra-assay variability was less than 10% and inter-assay variability was less than 20%. Accuracy, assessed by spike recovery, was 100 +/− 10%. HIF1α readings declined linearly with decreasing sample load over a range of 1 to 10 μg protein per well. HIF1α is being used as a pharmacodynamic marker in a clinical trial of metronomic Topotecan, so fitness for purpose was demonstrated by quantifying a reduction in HIF1α protein levels following topotecan treatment of a xenograft model. HIF1α was also demonstrated to be upregulated under low oxygen tension culture conditions in DU145 human prostate cancer cells. The HIF1α immunoassay is currently being transferred to the NCI's National Clinical Target Validation Laboratory for use in support of NCI-sponsored early clinical trials. This Research has been funded with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C3.

Physical and functional interactions between Runx2 and HIF-1α induce vascular endothelial growth factor gene expression

Angiogenesis and bone formation are intimately related processes. Hypoxia during early bone development stabilizes hypoxia-inducible factor-1α (HIF-1α) and increases angiogenic signals including vascular endothelial growth factor (VEGF). Furthermore, stabilization of HIF-1α by genetic or chemical means stimulates bone formation. On the other hand, deficiency of Runx2, a key osteogenic transcription factor, prevents vascular invasion of bone and VEGF expression. This study explores the possibility that HIF-1α and Runx2 interact to activate angiogenic signals. Runx2 over-expression in mesenchymal cells increased VEGF mRNA and protein under both normoxic and hypoxic conditions. In normoxia, Runx2 also dramatically increased HIF-1α protein. In all cases, the Runx2 response was inhibited by siRNA-mediated suppression of HIF-1α and completely blocked by the HIF-1α inhibitor, echinomycin. Similarly, treatment of preosteoblast cells with Runx2 siRNA reduced VEGF mRNA in normoxia or hypoxia. However, Runx2 is not essential for the HIF-1α response since VEGF is induced by hypoxia even in Runx2-null cells. Endogenous Runx2 and HIF-1α were colocalized to the nuclei of MC3T3-E1 preosteoblast cells. Moreover, HIF-1α and Runx2 physically interact using sites within the Runx2 RUNT domain. Chromatin immunoprecipitation also provided evidence for colocalization of Runx2 and HIF-1α on the VEGF promoter. In addition, Runx2 stimulated HIF-1α-dependent activation of an HRE-luciferase reporter gene without requiring a separate Runx2-binding enhancer. These studies indicate that Runx2 functions together with HIF-1α to stimulate angiogenic gene expression in bone cells and may in part explain the known requirement for Runx2 in bone vascularization.

Dihydrotestosterone attenuates hypoxia inducible factor-1α and cyclooxygenase-2 in cerebral arteries during hypoxia or hypoxia with glucose deprivation

Dihydrotestosterone (DHT) attenuates cytokine-induced cyclooxygenase-2 (COX-2) in coronary vascular smooth muscle. Since hypoxia inducible factor-1α (HIF-1α) activation can lead to COX-2 production, this study determined the influence of DHT on HIF-1α and COX-2 following hypoxia or hypoxia with glucose deprivation (HGD) in the cerebral vasculature. COX-2 and HIF-1α levels were assessed via Western blot, and HIF-1α activation was indirectly measured via a DNA binding assay. Experiments were performed using cerebral arteries isolated from castrated male rats treated in vivo with placebo or DHT (18 days) followed by hypoxic exposure ex vivo (1% O(2)), cerebral arteries isolated from castrated male rats treated ex vivo with vehicle or DHT (10 or 100 nM; 18 h) and then exposed to hypoxia ex vivo (1% O(2)), or primary human brain vascular smooth muscle cells treated with DHT (10 nM; 6 h) or vehicle then exposed to hypoxia or HGD. Under normoxic conditions, DHT increased COX-2 (cells 51%; arteries ex vivo 31%; arteries in vivo 161%) but had no effect on HIF-1α. Following hypoxia or HGD, HIF-1α and COX-2 levels were increased; this response was blunted by DHT (cells HGD: -47% COX-2, -34% HIF-1α; cells hypoxia: -29% COX-2, -54% HIF-1α; arteries ex vivo: -37% COX-2; arteries in vivo: -35% COX-2) and not reversed by androgen receptor blockade. Hypoxia-induced HIF-1α DNA-binding was also attenuated by DHT (arteries ex vivo and in vivo: -55%). These results demonstrate that upregulation of COX-2 and HIF-1α in response to hypoxia is suppressed by DHT via an androgen receptor-independent mechanism.

Effects of ischemic pre- and postconditioning on HIF-1α, VEGF and TGF-β expression after warm ischemia and reperfusion in the rat liver

BackgroundIschemic pre- and postconditioning protects the liver against ischemia/reperfusion injuries. The aim of the present study was to examine how ischemic pre- and postconditioning affects gene expression of hypoxia inducible factor 1α (HIF-1α), vascular endothelial growth factor A (VEGF-A) and transforming growth factor β (TGF-β) in liver tissue.Methods28 rats were randomized into five groups: control; ischemia/reperfusion; ischemic preconditioning (IPC); ischemic postconditioning (IPO); combined IPC and IPO. IPC consisted of 10 min of ischemia and 10 min of reperfusion. IPO consisted of three cycles of 30 sec. reperfusion and 30 sec. of ischemia.ResultsHIF-1α mRNA expression was significantly increased after liver ischemia compared to controls (p = 0.010). HIF-1α mRNA expression was significantly lower in groups subjected to IPC or combined IPC and IPO when compared to the ischemia/reperfusion group (p = 0.002). VEGF-A mRNA expression increased in the ischemia/reperfusion or combined IPC and IPO groups when compared to the control group (p < 0.05).ConclusionIschemic conditioning seems to prevent HIF-1α mRNA induction in the rat liver after ischemia and reperfusion. This suggests that the protective effects of ischemic conditioning do not involve the HIF-1 system. On the other hand, the magnitude of the HIF-1α response might be a marker for the degree of I/R injuries after liver ischemia. Further studies are needed to clarify this issue.

NF-Y joins E2Fs, p53 and other stress transcription factors at the apoptosis table

NF-Y joins E2Fs, p53 and other stress transcription factors at the apoptosis table