BACKGROUND: Hypoxic niches in solid tumors harbor therapy-resistant cells. Hypoxia-activated prodrugs (HAPs) have been designed to overcome this resistance and, to date, have begun to show clinical efficacy. However, clinical HAPs activity could be improved. In this study, we sought to identify non-pharmacological methods to acutely exacerbate tumor hypoxia to increase TH-302 activity in pancreatic ductal adenocarcinoma (PDAC) tumor models. RESULTS: Three human PDAC cell lines with varying sensitivity to TH-302 (Hs766t > MiaPaCa-2 > SU.86.86) were used to establish PDAC xenograft models. PDAC cells were metabolically profiled in vitro and in vivo using the Seahorse XF system and hyperpolarized 13C pyruvate MRI, respectively, in addition to quantitative immunohistochemistry. The effect of exogenous pyruvate on tumor oxygenation was determined using electroparamagnetic resonance (EPR) oxygen imaging. Hs766t and MiaPaCa-2 cells exhibited a glycolytic phenotype in comparison to TH-302 resistant line SU.86.86. Supporting this observation is a higher lactate/pyruvate ratio in Hs766t and MiaPaCa xenografts as observed during hyperpolarized pyruvate MRI studies in vivo. Coincidentally, response to exogenous pyruvate both in vitro (Seahorse oxygen consumption) and in vivo (EPR oxygen imaging) was greatest in Hs766t and MiaPaCa models, possibly due to a higher mitochondrial reserve capacity. Changes in oxygen consumption and in vivo hypoxic status to pyruvate were limited in the SU.86.86 model. Combination therapy of pyruvate plus TH-302 in vivo significantly decreased tumor growth and increased survival in the MiaPaCa model and improved survival in Hs766t tumors. CONCLUSIONS: Using metabolic profiling, functional imaging, and computational modeling, we show improved TH-302 activity by transiently increasing tumor hypoxia metabolically with exogenous pyruvate. Additionally, this work identified a set of biomarkers that may be used clinically to predict which tumors will be most responsive to pyruvate + TH-302 combination therapy. The results of this study support the concept that acute increases in tumor hypoxia can be beneficial for improving the clinical efficacy of HAPs and can positively impact the future treatment of PDAC and other cancers.

Hypoxia regulates many hundreds of genes with important roles in ischemic and neoplastic disorders. Central to this response are the hypoxia inducible transcription factors (HIF). This work aimed to better understand the direct transcriptional response to HIF by mapping HIF-binding sites across the genome using chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-seq). ChIP-seq for HIF in MCF-7 breast cancer cells under hypoxic conditions revealed more than 400 high-stringency HIF-binding sites genome-wide. Each member of the HIF heterodimer was present with near complete concordance. Binding of the two principle isoforms revealed a high degree of overlap with no differences in the DNA-binding motif. HIF-binding was associated with upregulation, but not downregulation of genes indicating that it functions as a transcriptional activator but not as a repressor. HIF-binding occurred preferentially at gene promoters, but was also present at promoter-distant sites, which were also associated with gene regulation, implicating long-range interactions in hypoxic gene activation. HIF-binding was associated with markers of open chromatin and active enhancers that were present in normoxia, indicating that HIF-binding sites are already “prepared” to bind HIF before the hypoxic stimulus. Analysis of normoxic and hypoxic RNA pol2 and H3K4me3 signals revealed distinctive hypoxia-inducible changes unique to HIF-binding genes. Comparable numbers of HIF-binding sites were observed in a second cell line (von Hippel-Lindau defective 786-O renal cancer cells) as in MCF-7 breast cancer cells, although approximately 65% were unique to 786-O cells. These unique sites were more frequently promoter-distant. Correlation with expression analyses from renal tumours indicated that many HIF-binding genes were upregulated in renal cancer. One such RCC unique promoter-distant HIF-binding site was identified at an intergenic locus on chromosome 11q13.3 that has been associated with renal cancer in Genome-Wide Association Studies. The HIF-binding site was in high linkage disequilibrium with the disease associated SNP and had the epigenetic hallmarks of an enhancer. Analysis of pan-genomic expression analyses identified the cell-cycle regulator cyclin D1 as highly HIF-regulated, and a physical association between the HIF-binding site and the CCND1 promoter could be determined. Furthermore, in a renal cancer cell line heterozygous at this locus, the RCC-protective allele disrupted HIF-binding leading to an allelic imbalance in cyclin D1 expression.

No description available.

Os lagostins são crustáceos decápodos límnicos que podem ser encontrados em água corrente, outros preferem água com pouca ou nenhuma corrente, como pequenos riachos, lagos, reservatórios e pântanos. Muitas espécies vivem em galerias subterrâneas com níveis mais baixos de oxigênio; assim, estas espécies podem mostrar adaptações metabólicas às condições hipóxicas. O objetivo desta pesquisa foi comparar o metabolismo de duas espécies de lagostins com diferentes hábitos, Parastacus defossus e Parastacus brasiliensis. P. defossus é uma espécie fossorial, vive em galerias com baixos níveis de oxigênio e P. brasiliensis é encontrado em ambientes lóticos com maiores níveis de oxigênio. Amostragens sazonais foram realizadas da primavera de 2006 ao inverno de 2007 para determinações metabólicas sazonais e posteriormente, amostragens foram realizadas durante o inverno de 2008 para análises metabólicas dos animais submetidos à hipóxia e recuperação pós-hipóxia. P. brasiliensis foi amostrado em Mariana Pimentel, Rio Grande do Sul (Brasil) e P. defossus foi amostrado no Lami, Porto Alegre, Rio Grande do Sul (Brasil). Nos experimentos de hipóxia, grupos de animais foram submetidos à hipóxia por 1, 2, 4 e 8 horas. Períodos de recuperação póshipóxia também foram analisados, após 4 hs de hipóxia, grupos de animais foram colocados em aquários com água aerada e foram removidos em intervalos de 1, 3, 6 e 9 hs. Após esse período foram extraídas amostras de hemolinfa e removidos o hepatopâncreas, o músculo, as brânquias e as gônadas para a determinação de glicose, lactato, glicose livre, glicogênio, proteínas totais, lipídios totais, colesterol total, arginina e arginina fosfato. Os resultados das análises sazonais mostraram diferentes respostas entre as estações do ano e entre as espécies, para todos os parâmetros metabólicos, com exceção das proteínas nas brânquias e do lactato na hemolinfa. As variações metabólicas em P. defossus foram principalmente relacionadas com o período reprodutivo e os períodos de baixa concentração de oxigênio nas galerias, enquanto os resultados em P. brasiliensis sugerem uma alocação significativa dos nutrientes da dieta para o tecido gonadal durante o período reprodutivo, com uma menor transferência das reservas de diferentes tecidos para as gônadas. Em relação ao metabolismo dos animais submetidos à hipóxia foi observado que em ambas as espécies, os níveis de glicose e de lactato aumentaram significativamente em hipóxia. Reduções de glicogênio, lipídios e colesterol foram registradas no hepatopâncreas e no tecido muscular, especialmente de P. defossus. Todos os tecidos de P. defossus e P. brasiliensis mostraram reduções nos níveis de glicose livre, mas essas reduções não foram significativas. Todas as reservas das brânquias anteriores e posteriores, com exceção das reservas de glicogênio, mostraram comportamento semelhante em ambas as espécies. As duas espécies de Parastacus armazenaram e utilizaram arginina fosfato, principalmente P. defossus. Entre os resultados do metabolismo dos animais submetidos à recuperação pós-hipóxia foram observadas que a restauração dos níveis de lactato foi mais rápido em P. defossus quando comparado com P. brasiliensis. Essa espécie restabeleceu suas reservas de glicogênio do hepatopâncreas e do tecido muscular. Já os níveis de glicose livre foram rapidamente restabelecidos em todos os tecidos das duas espécies. Em relação às reservas de arginina fosfato, P. defossus mostrou maiores concentrações que P. brasiliensis. As duas espécies mostraram capacidade de restaurar os níveis de arginina fosfato, mas também utilizaram essas reservas durante períodos de recuperação. Nas espécies, as reservas de lipídios totais e colesterol parecem ser uma importante fonte de energia durante a recuperação. / Some species of crayfish live in flowing water, and others prefer water with little or no current such as small streams, lakes, reservoirs, and swamps. Many species live in subterranean burrows with lower oxygen levels, and can show metabolic adaptations to hypoxic conditions. The aim of this study was to compare the metabolism of two crayfish species with different habitats, Parastacus defossus and Parastacus brasiliensis. P. defossus is fossorial, living in burrows with low oxygen levels, and P. brasiliensis lives in lotic environments with higher oxygen levels. Seasonal sampling was conducted from spring 2006 to winter 2007 for seasonal metabolic determinations, and samples were taken during the winter of 2008 for metabolic analyses of the animals subjected to hypoxia and during the post-hypoxia recovery. P. brasiliensis was collected in Mariana Pimentel, Rio Grande do Sul (Brazil) and P. defossus at Lami, Porto Alegre, Rio Grande do Sul. In the hypoxia experiments, groups of animals were subjected to hypoxia for 1, 2, 4, and 8 h. Periods of post-hypoxia recovery were also analyzed; after 4 h of hypoxia, groups of animals were placed in tanks with oxygenated water and were then removed at intervals of 1, 3, 6, and 9 h. The hemolymph was extracted, and the hepatopancreas, muscle, gills, and gonads were removed for determination of glucose, lactate, free glucose, glycogen, total proteins, total lipids, total cholesterol, arginine, and arginine phosphate. The results of the seasonal analysis showed, for all metabolic parameters, different seasonal responses between the species, with the exception of proteins in gills, and of lactate in hemolymph. The metabolic variations in P. defossus were mainly related to reproductive period and periods of low oxygen concentration in the burrows. The results for P. brasiliensis suggested a significant allocation of dietary nutrients to gonadal tissue during the reproductive period, with a smaller transfer of reserves from different tissues to gonads. In both species, glucose and lactate levels increased significantly in hypoxia. Reductions of glycogen, lipids, and cholesterol were recorded in hepatopancreas and muscle tissue, especially of P. defossus. In all tissues of P. defossus and P. brasiliensis were observed reductions in the free glucose levels, but these reductions weren’t significant. All reserves in the anterior and posterior gills, except glycogen, behaved similarly in both species. Both Parastacus species, mainly P. defossus, stored and used arginine phosphate. During post-hypoxia recovery, lactate was restored more rapidly in P. defossus than in P. brasiliensis. P. defossus restored its glycogen reserves in the hepatopancreas and muscle tissue. Free glucose was quickly restored in all tissues of both species. In relation to the reserves of arginine phosphate, P. defossus showed higher concentrations than P. brasiliensis. The two species showed ability to restore this metabolite, but they also used this metabolite during longer periods of recovery. In both species, the reserves of total lipids and cholesterol seemed be an important source of energy during the recovery period.

Clear cell renal cell carcinoma (ccRCC) is an aggressive malignancy characterized by inactivation of the von Hippel-Lindau tumour suppressor gene, the protein product of which mediates degradation of the transcription factor hypoxia-inducible factor (HIF). GAL3ST1 is a sulfotransferase which catalyzes the production of sulfatide, a plasma membrane sulfolipid previously implicated in metastasis. We observed GAL3ST1 overexpression in primary ccRCC tumours relative to matched-normal tissue and subsequently asked if GAL3ST1 was a HIF-responsive gene that facilitates ccRCC metastasis. GAL3ST1 expression was suppressed in ccRCC cells by stable reconstitution of wild-type VHL and also siRNA-mediated knockdown of HIF1alpha and HIF2alpha. Dual luciferase assays and chromatin immunoprecipitation revealed a hypoxia-response element in the GAL3ST1 5’-UTR that appeared to be crucial for HIF-mediated upregulation. Finally, stable knockdown of GAL3ST1 significantly impeded ccRCC cell invasion through an in vitro basement membrane mimic. These results suggest GAL3ST1 is a HIF-responsive gene that promotes tumour cell invasion.

Clear cell renal cell carcinoma (ccRCC) is an aggressive malignancy characterized by inactivation of the von Hippel-Lindau tumour suppressor gene, the protein product of which mediates degradation of the transcription factor hypoxia-inducible factor (HIF). GAL3ST1 is a sulfotransferase which catalyzes the production of sulfatide, a plasma membrane sulfolipid previously implicated in metastasis. We observed GAL3ST1 overexpression in primary ccRCC tumours relative to matched-normal tissue and subsequently asked if GAL3ST1 was a HIF-responsive gene that facilitates ccRCC metastasis. GAL3ST1 expression was suppressed in ccRCC cells by stable reconstitution of wild-type VHL and also siRNA-mediated knockdown of HIF1alpha and HIF2alpha. Dual luciferase assays and chromatin immunoprecipitation revealed a hypoxia-response element in the GAL3ST1 5’-UTR that appeared to be crucial for HIF-mediated upregulation. Finally, stable knockdown of GAL3ST1 significantly impeded ccRCC cell invasion through an in vitro basement membrane mimic. These results suggest GAL3ST1 is a HIF-responsive gene that promotes tumour cell invasion.

<p>Malignant gliomas are highly lethal cancers characterized by florid angiogenesis. Glioma stem cells (GSCs), enriched through CD133 (Prominin1) selection, are highly tumorigenic and therapy resistance. However, the mechanism through which GSCs promote tumor growth was largely unknown. As we noticed that tumors derived from GSCs contain widespread tumor angiogenesis, necrosis, and hemorrhage, we examined thepotential of GSCs to support tumor angiogenesis. We measured the expression of a panel of angiogenic factors secreted by GSCs. In comparison with matched non-GSC populations, GSCs consistently secreted markedly elevated levels of vascular endothelial growth factor (VEGF), which were further induced by hypoxia. In an in vitro model of angiogenesis, GSC-conditioned medium significantly increased endothelial cell migration and tube formation compared with non-GSC glioma cell-conditioned medium. The proangiogenic effects of GSCs on endothelial cells were specifically abolished by the anti-VEGF neutralizing antibody bevacizumab, which is in clinical use for cancer therapy. Furthermore, bevacizumab displayed potent antiangiogenic efficacy in vivo and suppressed growth of xenografts derived from GSCs but limited efficacy against xenografts derived from a matched non-GSC population. As hypoxia is a key regulator of angiogenesis, I further examined hypoxic responses in GSCs to determine the molecular mechanisms underlying their angiogenic drive. I demonstrated that multiple hypoxia response genes, including the hypoxia-inducible factors (HIFs)-1a and -2a(EPAS-1) were differentially expressed in GSCs in comparison to non-stem glioma cells and normal neural progenitors. GSCs preferentially induced HIF2a; and HIF2a-regulated genes under hypoxia in comparison to non-stem glioma cells. In contrast, neural progenitor/stem cells did not induce HIF2a in response to hypoxia suggesting that the HIF2a hypoxic response is not a general stem cell response. Targeting HIF1a or HIF2a in GSCs using short hairpin RNA (shRNA) inhibited neurosphere formation efficiency, indicating a requirement for HIFs in cancer stem cell self-renewal. HIF1a and HIF2a were also necessary for VEGF expression in GSCs, but HIF2a was not required in matched non-stem glioma cells. In vivo experiments determined that knockdown of HIFs significantly attenuated the tumorigenic capacity of GSCs and increased survival of immunocompromised mice. Together, our work provides the first evidence that that GSCs can be a crucial source of key angiogenic factors in cancers due to their differential hypoxia responses. It also suggests that anti-angiogenic therapies can be designed to target GSC-specific molecular mechanisms of neoangiogenesis, including the expression and/or activity of HIF2a.</p> / Dissertation

<p>Imbalance between oxygen consumption and supply often makes tumors hypoxic (Bristow and Hill 2008). Tumor hypoxia is significantly correlated with aggressive tumor growth, ineffective response to radiation and chemotherapy, and as a result, poor patient prognosis. Hyperthermia (HT) is a strong adjuvant treatment to overcome these challenges of tumor hypoxia because it causes tumor reoxygenation at temperatures lower than 43ºC (Song, Park, and Griffin 2001). However, the detailed molecular mechanisms of how HT enhances tumor oxygenation have not been elucidated. Here we determine that 1 hour HT activates hypoxia-inducible factor-1 (HIF-1) and its downstream targets, vascular endothelial growth factor (VEGF), lactate dehydrogenase A (LDHA), and pyruvate dehydrogenase kinase 1 (PDK1) in tumors. Consistent with HIF-1 activation and upregulation of its downstream genes, HT also enhances tumor perfusion/vascularization and decreases oxygen consumption rates. As a result, tumor hypoxia is reduced after HT suggesting that these physiological changes contribute to HT-induced tumor reoxygenation. Since HIF-1 is a potent regulator of tumor vascularization and metabolism, our findings suggest that HIF-1 plays a role in HT-induced tumor reoxygenation by transactivating its downstream targets. Mechanistically, we demonstrate that NADPH oxidase-mediated reactive oxygen species (ROS) production upregulates HIF-1 after HT. Further, we determine that this pathway is initiated by increased transcription of NADPH oxidase-1 (NOX1) through the ERK pathway.</p><p>A major research effort at Duke focuses on combinations of HT and doxorubicin in the treatment of locally advanced breast and other cancers. Thus, we investigated whether there are HIF-1 responses to doxorubicin treatment. We reveal that doxorubicin also activates HIF-1. Unlike HT, doxorubicin-induced HIF-1 promotes persistent tumor angiogenesis. We also reveal that the signal transducer and activator of transcription 1 (STAT1)/inducible nitric oxide synthase (iNOS) pathway causes HIF-1&#945; accumulation in an oxygen-independent manner. We show that activated STAT1 upregulates iNOS expression and promotes nitric oxide (NO) production in tumor cells resulting in HIF-1&#945; stabilization. We further determine that both iNOS inhibitor, 1400W and STAT1 inhibitor, epigallocatechin-3-gallate (EGCG) significantly decrease intracellular NO production and suppress doxorubicin-induced normoxic HIF-1&#945; accumulation.</p><p>HIF-1 is often considered a promising therapeutic target because of its role in tumor progression (Semenza 2003) and therapy resistance (Moeller et al. 2004). However, our findings suggest that HIF-1 plays a pleiotropic role in response to HT and chemotherapy. Therefore, to preferentially take advantage of HT-induced HIF-1 activation and also to suppress its deleterious effects induced by chemotherapy or as we have previously reported, by radiation (Moeller et al. 2004), HIF-1 inhibition needs to be carefully regulated in a time-sensitive manner to achieve optimal therapeutic effects.</p> / Dissertation

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references. Also available online.

This study investigated two hypotheses. First, that chronic cocaine treatment would mimic the changes in breathing that are associated with ventilatory acclimatisation to chronic hypoxia (VAH). Second, that pre-treatment with a hypoxic stressor would bring about cross-sensitisation to cocaine. To address the first hypothesis, rats were exposed to either chronically hypoxic or chronically normoxic conditions and treated with either cocaine or saline for a 14 day period. Following this period, acute breathing trials were performed to measure resting ventilation and ventilatory chemoreflexes. The results demonstrated that chronic cocaine treatment did not induce the changes in breathing associated with VAH. To address the second hypothesis rats were exposed to a hypoxic stressor for 10 days (either intermittent hypoxia or chronic hypoxia) after which cocaine sensitisation was measured via locomotor sensitisation trials. The results demonstrated that cross-sensitisation between a hypoxic stress and cocaine was observed for intermittent but not chronic hypoxia.