Hypoxic Stress Response Research Articles

Invited review: decoding the microRNA response to hypoxia

microRNAs (miRNAs) were discovered nearly two decades ago by researchers who sought to understand how basic developmental mechanisms work in the nematode Caenorhabditis elegans. Since the identification of conserved miRNA families in higher eukaryotes, there has been an explosion of interest into how these tiny RNA molecules function. miRNAs are 20-24 nucleotide non-coding RNA molecules that predominantly regulate transcripts of target genes through translational inhibition. Much recent interest has focused on the influence of miRNAs on homeostatic regulation, and in particular, hypoxic responses. The ability to sense and respond to hypoxia is of fundamental importance to aerobic organisms and dysregulated oxygen homeostasis is a hallmark in the pathophysiology of cancer, neurological dysfunction, myocardial infarction, and lung disease. miRNAs are ideal mediators of hypoxic stress responses as they are able to modify gene expression both rapidly and reversibly. This enables miRNA-mediated gene regulatory circuits to modify metabolic networks with immaculate precision and control. Therefore, one may consider miRNAs as molecular rheostats which effect tuning and switching of regulatory circuits to facilitate survival and adaptation to hypoxic conditions. Such miRNA-mediated regulatory circuits would provide flexible and conditional alternatives to "conventional" transcriptional regulation. Here, I review recent discoveries that have boosted our understanding of miRNA regulation of hypoxia and discuss where future breakthroughs in this area may be made.

Abstract P2-07-06: Role of CaMKK2-AMPK Signaling in Breast Cancer Metabolic and Hypoxic Stress Responses

Abstract Background: Adenosine monophosphate-activated protein kinase (AMPK) is a major regulator of energy homeostasis at both the cellular and whole-body levels. AMPK is activated by elevated intracellular AMP level and by phosphorylation on threonine 172 (T172) in the activation loop of the α catalytic subunit by upstream kinases, LKB1 and Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2). Once activated, AMPK switches on ATP-producing pathways while switching off ATP-consuming processes. Recent studies suggest that AMPK also controls processes relevant to tumor development, including protein synthesis, cell cycle progression, cell growth, survival and stress-induced autophagy. The purpose of our study is to determine the role of CaMKK2 as an AMPK upstream kinase in breast cancer cells, as well as its function in regulating cell proliferation, migration and survival. We chose the prototypical ERα + MCF-7 breast cancer cell line as our research model since this cell line does not express CaMKK1, which has similar functions to CaMKK2 but cannot activate AMPK. Materials and Methods: Illumina cDNA expression array was preformed on MCF-7, T-47D and MDA-MB-231 breast cancer cell lines, and normalized by rank-invariant method. MCF-7 cells with suppressed CaMKK2 or LKB1 expression were established using lentiviral gene specific shRNA interference. Stable knockdown cells (shNon-Target, shCaMKK2, shLKB1) were subject to acute and chronic stress conditions (i.e. ionomycin, 2-deoxy-D-glucose, hypoxia) and signaling events were evaluated using immunoblot analysis. Proliferation, anchorage-independent growth and motility of the knockdown cells were determined by MTT, soft agar formation and in vitro migration assay. Xenograft tumor studies were performed by inoculating estradiol-supplemented athymic nude mice with shNon-Target, shCaMKK2 and shLKB1 MCF-7 cells into the mammary fat pad. Tumor growth was determined by external caliper measurements twice a week. Forty-five days post-injection, mice were euthanized, tumors removed, weighed and prepared for histological, protein and RNA analyses. Results: CaMKK2 and LKB1 mRNAs are expressed at a similar level in the ERα + and ERα — breast cancer cell lines tested in the Illumina cDNA expression array. CaMKK2 activated AMPK phosphorylation (T172) under increased intracellular Ca2+ level induced by ionomycin. Under acute stress conditions, CaMKK2 functioned as the predominant AMPK upstream kinase. However, when cells are subject to long-term metabolic and hypoxic stress, CaMKK2 and LKB1 were both required for AMPK activation. Loss of CaMKK2 in MCF-7 cells promoted a moderate growth advantage under metabolic stress, increased soft agar colony formation and elevated cell migration capacity. CaMKK2 knockdown xenografts demonstrated less ductal structure formation, decreased E-Cadherin expression and reduced angiogenesis, as well as reduced VEGF and Glut1 mRNA levels in vivo. Conclusions: We have demonstrated that Ca2+-CaMKK2 signaling plays an important role in regulating breast cancer cell proliferation, migration and survival pathways. These findings suggest that Ca2+ activation through CaMKK2 promotes AMPK signaling, survival and angiogenesis in response to stress conditions in breast cancer cells. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-07-06.

Chronic hypoxia impairs muscle function in the Drosophila model of Duchenne's muscular dystrophy (DMD).

Duchenne's muscular dystrophy (DMD) is a severe progressive myopathy caused by mutations in the DMD gene leading to a deficiency of the dystrophin protein. Due to ongoing muscle necrosis in respiratory muscles late-stage DMD is associated with respiratory insufficiency and chronic hypoxia (CH). To understand the effects of CH on dystrophin-deficient muscle in vivo, we exposed the Drosophila model for DMD (dmDys) to CH during a 16-day ascent to the summit of Mount Denali/McKinley (6194 meters above sea level). Additionally, dmDys and wild type (WT) flies were also exposed to CH in laboratory simulations of high altitude hypoxia. Expression profiling was performed using Affymetrix GeneChips® and validated using qPCR. Hypoxic dmDys differentially expressed 1281 genes, whereas the hypoxic WT flies differentially expressed 56 genes. Interestingly, a number of genes (e.g. heat shock proteins) were discordantly regulated in response to CH between dmDys and WT. We tested the possibility that the disparate molecular responses of dystrophin-deficient tissues to CH could adversely affect muscle by performing functional assays in vivo. Normoxic and CH WT and dmDys flies were challenged with acute hypoxia and time-to-recover determined as well as subjected to climbing tests. Impaired performance was noted for CH-dmDys compared to normoxic dmDys or WT flies (rank order: Normoxic-WT ≈ CH-WT> Normoxic-dmDys> CH-dmDys). These data suggest that dystrophin-deficiency is associated with a disparate, pathological hypoxic stress response(s) and is more sensitive to hypoxia induced muscle dysfunction in vivo. We hypothesize that targeting/correcting the disparate molecular response(s) to hypoxia may offer a novel therapeutic strategy in DMD.

Targeting mTOR pathway: A new concept in cancer therapy

This article highlights the current knowledge of mTOR biology and provides new insights into the role of mTOR in different cancers. An active mTOR coordinates a response in cell growth directly through its effects on cell cycle regulators and indirectly by sustaining nutrient supply into the cell through the production of nutrient transporters and also through the promotion of angiogenesis. A primary way that mTOR exerts its regulatory effects on cell proliferation is by controlling the production of cyclin D1. mTOR increases the translation of hypoxia-inducible factor 1 (HIF-1)/HIF-2. The HIF transcription factors drive the expression of hypoxic stress response genes, including angiogenic growth factors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor β (PDGF-β), and transforming growth factor a (TGF-α). mTOR also increases the surface expression of nutrient transporters proteins. An increase in these proteins results in greater uptake of amino acids and other nutrients by the cell leading to adequate nutrient support to abnormal cell growth and survival. There is also emerging evidence that mTOR activation may play a role in promoting cell survival through the activation of antiapoptotic proteins that contribute to tumor progression. Given that the mTOR pathway is deregulated in a number of cancers, it is anticipated that mTOR inhibitors will have broad therapeutic application across many tumor types. Until now, no treatment demonstrated Phase III evidence after disease progression on an initial VEGF-targeted therapy in advanced renal cell carcinoma. Everolimus is the first and only therapy with Phase III evidence after failure of VEGF-targeted therapy. Everolimus is a once-daily, oral inhibitor of mTOR (mammalian target of rapamycin) indicated for the treatment of advanced renal cell carcinoma in patients, whose disease has progressed on or after treatment with VEGF-targeted therapy.

Vitreoscilla Hemoglobin (VHb) Overexpression Increases Hypoxia Tolerance in Zebrafish (Danio rerio)

Aquaculture farming may benefit from genetically engineering fish to tolerate environmental stress. Here, we used the vector pCVCG expressing the Vitreoscilla hemoglobin (vhb) gene driven by the common carp β-actin promoter to create stable transgenic zebrafish. The survival rate of the 7-day-old F(2) transgenic fish was significantly greater than that of the sibling controls under 2.5% O(2) (dissolved oxygen (DO), 0.91mg/l). Meanwhile, we investigated the relative expression levels of several marker genes (hypoxia-inducible factor alpha 1, heat shock cognate 70-kDa protein, erythropoietin, beta and alpha globin genes, lactate dehydrogenase, catalase, superoxide dismutase, and glutathione peroxidase) of transgenic fish and siblings after hypoxia exposure for 156h. The expression profiles of the vhb transgenic zebrafish revealed that VHb could partially alleviate the hypoxia stress response to improve the survival rate of the fish. These results suggest that that vhb gene may be an efficient candidate for genetically modifying hypoxia tolerance in fish.

Protection of Cells in Physiological Oxygen Tensions against DNA Damage-induced Apoptosis

Oxygen availability has important effects on cell physiology. Although hyperoxic and hypoxic stresses have been well characterized, little is known about cellular functions in the oxygen levels commonly found in vivo. Here, we show that p53-dependent apoptosis in response to different DNA-damaging agents was reduced when normal and cancer cells were cultured at physiological oxygen tensions instead of the usual atmospheric levels. Different from what has been described in hypoxia, this was neither determined by decreases in p53 induction or its transactivation activity, nor by differences in the intracellular accumulation of reactive oxygen species. At these physiological oxygen levels, we found a constitutive activation of the ERK1/2 MAPK in all the models studied. Inhibition of this signaling pathway reversed the protective effect in some but not all cell lines. We conclude that a stress-independent constitutive activation of prosurvival pathways, including but probably not limited to MAPK, can protect cells in physiological oxygen tensions against genotoxic stress. Our results underscore the need of considering the impact of oxygen levels present in the tissue microenvironment when studying cell sensitivity to treatments such as chemotherapy and radiotherapy.

A database for poplar gene co-expression analysis for systematic understanding of biological processes, including stress responses

Reforestation in the humid tropics and arid zones, where trees are often subject to stresses, is an effective approach for mitigating global warming. Forestation with Populus species that are tolerant to the stresses in such regions has been conducted. The selection of poplar trees with higher stress tolerance leads to more efficient reforestation. The genome-wide bioinformatics approaches of gene function have been used for revealing the mechanisms of biological processes, including such stress tolerance. The decoding of the poplar genome has been followed by the genome-wide identification of genes and then the inference of gene function for systematic understanding of biological processes. To predict gene function in poplar, we analyzed poplar gene expression data using DNA microarray datasets obtained from the Gene Expression Omnibus database and developed a database for poplar gene co-expression analysis. Using the database, we illustrate the steps to retrieve two groups of co-expressed genes that are specifically expressed in experiments of hypoxic stress response in gray poplar, a flooding-tolerant tree species. Our database allows users to extract genes involved in biological processes, such as stress reaction, and then is useful for understanding such mechanisms in tree species.

PERK Regulates the Proliferation and Development of Insulin-Secreting Beta-Cell Tumors in the Endocrine Pancreas of Mice

BackgroundPERK eIF2α kinase is required for the proliferation of the insulin-secreting beta- cells as well as insulin synthesis and secretion. In addition, PERK signaling has been found to be an important factor in determining growth and angiogenesis of specific types of tumors, and was attributed to PERK-dependent regulation of the hypoxic stress response. In this report we examine the role of PERK in regulating proliferation and angiogenesis of transformed beta-cells in the development of insulinomas.MethodologyThe SV40 Large T-antigen (Tag) was genetically introduced into the insulin secreting beta-cells of Perk KO mice under the control of an inducible promoter. Tumor growth and the related parameters of cell proliferation were measured. In late stage insulinomas the degree of vascularity was determined.Principal FindingsThe formation and growth of insulinomas in Perk-deficient mice was dramatically ablated with much fewer tumors, which averaged 38-fold smaller than seen in wild-type control mice. Beta-cell proliferation was ablated in Perk-deficient mice associated with reduced tumor growth. In the small number of large encapsulated insulinomas that developed in Perk-deficient mice, we found a dramatic reduction in tumor vascularity compared to similar sized insulinomas in wild-type mice. Although insulinoma growth in Perk-deficient mice was largely impaired, beta-cell mass was increased sufficiently by T-antigen induction to rescue the hypoinsulinemia and diabetes in these mice.ConclusionsWe conclude that PERK has two roles in the development of beta-cell insulinomas, first to support rapid cell proliferation during the initial transition to islet hyperplasia and later to promote angiogenesis during the progression to late-stage encapsulated tumors.

Comparative proteomic profiles of the hepatopancreas in Fenneropenaeus chinensis response to hypoxic stress

Hypoxia, as one suboptimal environmental condition, can affect the physiological state of shrimp during pond aquaculture. To better understand the mechanism of response to hypoxic stress in Chinese shrimp Fenneropenaeus chinensis, proteome research approach was utilized. Differentially expressed proteins of hepatopancreas in adult Chinese shrimp between the control and hypoxia-stressed groups were screened. By 2-DE analysis, 67 spots showed obvious changes after hypoxia. Using LC-ESI-MS/MS, 51 spots representing 33 proteins were identified including preamylase, arginine kinase, phosphopyruvate hydratase, citrate synthase, ATP synthase alpha subunit, chymotrypsin BI, chitinase, ferritin, C-type lectin receptors, transketolase, formylglutathione hydrolase, formyltetrahydrofolate dehydrogenase, aldehyde dehydrogenase, glutathione peroxidase, cytosolic manganese superoxide dismutase, protein disulfide isomerase, beta-actin, oncoprotein nm23, crustacyanin-C1 and so on. These proteins could be functionally classified into several groups such as proteins related to energy production, metabolism-related proteins, immune-related proteins, antioxidant proteins, chaperones, cytoskeleton proteins and ungrouped proteins. The transcription levels of ten selected genes encode the identified proteins were analyzed by real-time PCR at different sampling times of hypoxia. This study is the first analysis of differentially expressed proteins in the hepatopancreas of shrimp after hypoxia and provides a new insight for further study in hypoxic stress response of shrimp at the protein level.

Breathing and Sleep: Measurement Methods, Genetic Influences, and Developmental Impacts

Sleep-disordered breathing comprises alterations in respiratory rate, rhythm, and depth that present during sleep and may or may not be recognizable in breathing during wakefulness. Primary disorders include repetitive apneas, near apneas (hypopneas), or reductions in overall ventilation during sleep (hypoventilation), all of which lead to reductions in pulmonary gas exchange resulting in arousals, arrhythmia, hypercapnia, acidosis, and/or hypoxic stress responses such as pulmonary hypertension or polycythemia. Because the underlying mechanisms resulting in sleep-disordered breathing and its resulting comorbidities remain unclear, researchers use a variety of animal models to better understand the disorder. These models allow for conditioning paradigms, more detailed measurements of respiratory control, and the use of fewer preparations to provide a detailed picture of the individual components that contribute to breathing patterns. Both noninvasive and reduced methods are applicable with conditioned, inbred, and/or genetically manipulated animals to determine effect size and imply mechanisms. Research in animals has established preclinical models showing that intermediate traits of breathing pattern (e.g., responses to hypoxia, hypercapnia, and reoxygenation) vary according to genetic background and conditioning. Such findings permit new ideas about pathogenesis and prevention and form the rationale for observational and interventional studies in the human population. In this article we focus on methods of investigating respiratory control and applicable rodent models.

Hypoxia-Mediated Induction of the Polyamine System Provides Opportunities for Tumor Growth Inhibition by Combined Targeting of Vascular Endothelial Growth Factor and Ornithine Decarboxylase

Hypoxia is a hallmark of solid tumors, which may offer opportunities for targeted therapies of cancer; however, the mechanisms that link hypoxia to malignant transformation and tumor progression are not fully understood. Here, we show that up-regulation of the polyamine system promotes cancer cell survival during hypoxic stress. Hypoxia was found to induce polyamine transport and the key enzyme of polyamine biosynthesis, ornithine decarboxylase (ODC), in a variety of cancer cell lines. Increased ODC protein expression was shown in hypoxic, GLUT-1-expressing regions of tumor spheroids and experimental tumors, as well as in clinical tumor specimens. Hypoxic induction of the polyamine system was dependent on antizyme inhibitor (i.e., a key positive regulator of ODC and polyamine transport), as shown by RNA interference experiments. Interestingly, depletion of the polyamines during hypoxia resulted in increased apoptosis, which indicates an essential role of the polyamines in cancer cell adaptation to hypoxic stress. These results were supported by experiments in an in vivo glioma tumor model, showing significantly enhanced antitumor effects of the antiangiogenic, humanized anti-vascular endothelial growth factor (VEGF) antibody bevacizumab when used in combination with the well-established, irreversible inhibitor of ODC, alpha-difluoromethylornithine. Our results provide important insights into the hypoxic stress response in malignant cells and implicate combined targeting of VEGF and ODC as an alternative strategy to treat cancer disease.

Metabolic profiling of ‘Conference’ pears under low oxygen stress

Pears ( Pyrus communis L. cv. Conference) may develop core breakdown when stored under low oxygen or elevated carbon dioxide conditions. This physiological disorder is characterized by the development of brown spots due to oxidation of phenolic compounds, and eventually, cavities in the center of the fruit. Based on metabolic profiling of brown and sound tissue using GC-EI-TOF-MS, the hypothesis that this disorder is due to an imbalance between oxidative and reductive processes at the cellular level was investigated. Brown tissue was clearly characterized by a distinctive pattern in changes which included a decrease of malic acid and an increase in fumaric acid and gamma aminobutyric acid (GABA), which indicated a reduced metabolic activity at the level of the Krebs cycle and a putative block of the GABA shunt pathway. Increased gluconic acid concentration might be related to ascorbic acid degradation due to insufficient reducing equivalents or to an impaired pentose phosphate pathway. For the first time, GABA and gluconic acid have been shown to be metabolic markers for core breakdown. The concentrations of other compounds which are believed to be related to hypoxic stress response such as trehalose and putrescine were also considerably higher in brown tissue than in sound tissue. The concentration of some sugars which are typically found in xyloglucans also increased during brown development, possibly indicating cell wall breakdown due to enzymatic processes or chemical reactions of hydroxyl radicals.

HIF-1α subunit and vasoactive HIF-1-dependent genes are involved in carbon monoxide-induced cerebral hypoxic stress response

Hypoxia-inducible transcription factor-1 (HIF-1) is the most important component of cellular and molecular adaptive responses to hypoxia. We aimed to analyze effects of systemic hypoxia and CO exposure on the oxygen-regulated alpha-subunit of HIF-1 and HIF-1-dependent vasoactive target genes in rat brain. Brains of adult Sprague-Dawley rats were investigated after incubation for 3 and 12 h under normoxia, hypoxia (8% O(2)) and CO 0.1% (n = 10 per group). Upon 3 h of exposure, hypoxia and CO-induced accumulation of HIF-1alpha protein in brain homogenates assessed by Western blot analysis. In contrast to hypoxia HIF-1alpha signals decreased markedly during 12 h-exposure to CO. By immunohistochemistry, intensive HIF-1alpha-positive staining was found in neurons of the cortex and hippocampus. Cerebral expression of vasoactive target genes adrenomedullin (ADM) and vascular endothelial growth factor (VEGF) showed up-regulation during both hypoxia and CO exposure indicating functional activation of HIF-1. Hypoxia increased ADM (P < 0.05) and VEGF mRNA levels within 3 h (P < 0.01) which persisted up to 12 h of exposure (ADM, P < 0.05; VEGF, P < 0.001). Similarly, CO inhalation led to early up-regulation of VEGF (3 h: P < 0.05; 12 h: P < 0.01), but a more delayed increase of ADM mRNA levels (3 h: n.s., 12 h: P < 0.01). We suggest that CO-induced oxygen deprivation is a potent stimulus to cerebral HIF-1-regulated hypoxic stress responses even though its effects are more transient than exposure to hypoxia.

Hypoxic stress responses and levetiracetam: Differential regulation of hypoxia-inducible neuroprotective factors in fetal mouse brain

Background: Hypoxia-inducible transcription factors (HIFs) have been characterized as the most important mediators of the molecular responses to hypoxia. Experimental stabilization of HIFs and specific target genes, critically involved in vasoactive, metabolic and cytoprotective adaptive mechanisms to hypoxic-ischemic brain lesions, showed neuroprotective effects in adult as well as developing rodent brain. As the antiepileptic drug levetiracetam (LEV) reduces cellular degeneration in focal cerebral ischemia, we aimed to examine its effects on endogenous adaptive factors in developing hypoxic brain. In vivo data on neuroprotective effects of LEV in developing hypoxic CNS are not available in literature.

HIF-1-regulated vasoactive systems are differentially involved in acute hypoxic stress responses of the developing brain of newborn mice and are not affected by levetiracetam

Hypoxia-inducible transcription factor-1 (HIF-1) is critically involved in adaptive endogenous mechanisms to hypoxic brain injury by transcriptional activation of specific target genes that restore oxygen supply. Exogenously, neuroprotective properties of levetiracetam (LEV) have been suggested in experimental cerebral ischemia and epilepsy. We aimed to elucidate 1) effects of acute hypoxic distress on HIF-1 and vasoactive target genes, and 2) effects of LEV on HIF-1-regulated mechanisms in the brain at early developmental stages. To this end, we studied the impact of hypoxia in the presence or absence of LEV on the O 2-dependent HIF-1α subunit as well as on VEGF and iNOS in the developing brain of normoxic and hypoxic mice. C57BL/6 mice (P0, P7) were treated with saline or LEV (i.p.; 50 mg/kg) 1 h before exposure to either normoxia (21% O 2; N) or hypoxia (8% O 2 of 6 h; H) without reoxygenation. HIF-1α was analyzed by Western blot and immunohistochemistry and mRNA levels were quantified by TaqMan RT-PCR. Hypoxia led to prominent accumulation of cerebral HIF-1α protein in cortical neurons and glial cells and significant up-regulation of VEGF mRNA at P0 (N, 0.018 ± 0.002, vs. H, 0.031 ± 0.003, n = 6; p < 0.05) and P7 (N, 0.096 ± 0.032, vs. H, 0.873 ± 0.069, n = 7; p < 0.001). Interestingly, we detected a significant decrease of iNOS mRNA levels in hypoxic brains. LEV treatment did not alter HIF-1α accumulation either in normoxic or hypoxic brains (P0, P7). Moreover, significant changes of VEGF and NOS mRNA levels did not occur with the exception that hypoxia-induced decreased iNOS levels were not observed in P0 brains. We conclude that acute systemic hypoxia differentially affects expression of HIF-1-regulated vasoactive factors in the newborn mouse brain. Of clinical importance, LEV treatment did not alter crucial HIF-1-regulated neuroprotective mechanisms.

An approach to the hypoxic and oxidative stress responses inKluyveromyces lactisby analysis of mRNA levels

Genome duplication, after the divergence of Saccharomyces cerevisiae from Kluyveromyces lactis along evolution, has been proposed as a mechanism of yeast evolution from strict aerobics, such as Candida albicans, to facultatives/fermentatives, such as S. cerevisiae. This feature, together with the preponderance of respiration and the use of the pentose phosphate pathway in glucose utilization, makes K. lactis a model yeast for studies related to carbon and oxygen metabolism. In this work, and based on the knowledge of the sequence of the genome of K. lactis, obtained by the Génolevures project, we have constructed DNA arrays from K. lactis including a limited amount of selected probes. They are related to the aerobiosis-hypoxia adaptation and to the oxidative stress response, and have been used to test changes in mRNA levels in response to hypoxia and oxidative stress generated by H(2)O(2). The study was carried out in both wild-type and rag2 mutant K. lactis strains in which glycolysis is blocked at the phosphoglucose isomerase step. This approach is the first analysis carried out in K. lactis for the majority of the genes selected.

Gene Expression Patterns in the Hypoxic Murine Placenta: A Role in Epigenesis?

Hypoxia has been identified as a major stress or in placental and fetal development. To test the hypothesis that hypoxic stress responses are associated with gene expression changes, the authors measured gene expression in the mouse placenta in response to 48 hours of hypoxia. Embryonic day 15.5 pregnant mice were exposed to 48 hours of hypoxia (10.5% O(2)), after which the Affymetrix Mouse 430A_2.0 array was used to measure gene expression changes in the placenta. The authors observed 171 probe sets, corresponding to 163 genes, that were regulated by hypoxia (P < .01). Ninety genes were upregulated, and 73 were downregulated. The authors functionally annotated the regulated genes and examined overrepresented functional categories. Among the upregulated and downregulated genes, several overrepresented functional categories were observed. Upregulated genes included those involved in metabolism, oxygen transport, proteolysis, cell death, metabolism of reactive oxygen species, and DNA methylation. Genes involved in transcription, cell cycle regulation, and cell structure were downregulated. Microarray analysis has allowed the description of the genetic responses to hypoxia in the mouse placenta. The observation that hypoxia upregulates reactive oxygen species metabolism, in conjunction with DNA methylation enzymes, suggests that hypoxia may contribute to long-term epigenetic changes in stressed fetal tissues and organs.

Mucin 1 Oncoprotein Blocks Hypoxia-inducible Factor 1α Activation in a Survival Response to Hypoxia

Resistance of carcinoma cells to hypoxic stress is of importance to the growth of solid tumors. The mucin 1 (MUC1) oncoprotein is aberrantly overexpressed by most human carcinomas; however, there is no known relationship between MUC1 and the hypoxic stress response. The present work has demonstrated that MUC1 attenuates activation of hypoxia-inducible factor-1alpha (HIF-1alpha), a regulator of gene transcription in the response of cells to hypoxic stress. In cells with stable gain and loss of MUC1 function, we have shown that MUC1 up-regulates prolyl hydroxylase 3 (PHD3) expression and promotes HIF-1alpha degradation. PHD activity is attenuated by increases in reactive oxygen species (ROS) generated in the hypoxic stress response. Our results further demonstrate that MUC1 blocks hypoxia-induced increases in ROS and thereby potentiates PHD-mediated HIF-1alpha suppression. Importantly, MUC1 also blocks hypoxia-induced apoptosis and necrosis by suppressing accumulation of ROS. These findings indicate that MUC1 attenuates HIF-1alpha activation in a survival response to hypoxic stress.

The Hypoxic Inducible Stress Response as a Target for Cancer Drug Discovery

All solid tumors experience some degree of hypoxia. The response to the stress of hypoxia is mediated in large part by the hypoxia inducible factor-1 (HIF-1) transcription factor that increases the expression of a variety of genes that allow the tumor to survive and grow in the hostile hypoxic environment. Increased tumor HIF-1 has been correlated with increased angiogenesis, aggressive tumor growth, and poor patient prognosis. This has lead to the current interest in HIF-1 as a cancer drug target. HIF-1 activity in tumors depends on the availability of the HIF-1alpha subunit, the levels of which increase under hypoxic conditions or through the activation of oncogenes and/or inactivation of tumor-suppressor genes. HIF-1alpha level and HIF-1 activity are regulated by multiple pathways involving transcription, translation, post-translational modification, and the interaction with other transcription factors. A number of agents have been reported to block HIF-1 activity, acting on all of these mechanisms. Not all of the agents have been shown to block tumor HIF-1 activity in vivo. Some have shown marked HIF-1 inhibition and anti-tumor activity. There are agents already, or soon to be, tested in the clinic as anti-tumor inhibitors of HIF-1. The challenges will be to determine whether the effects of these agents that are seen is due to HIF-1 inhibition and to identify which patients are most likely to benefit from treatment with HIF-1 inhibitors.

The Unfolded Protein Response: A Novel Component of the Hypoxic Stress Response in Tumors

Hypoxia is a physiologically important endoplasmic reticulum (ER) stress that is present in all solid tumors. Numerous clinical studies have shown that tumor hypoxia predicts for decreased local control, increased distant metastases, and decreased overall survival in a variety of human tumors. Hypoxia selects for tumors with an increased malignant phenotype and increases the metastatic potential of tumor cells. Tumor cells respond to hypoxia and ER stress through the activation of the unfolded protein response (UPR). The UPR is an adaptive response to increase cell survival during ER stress. XBP-1 is a critical transcriptional regulator of this process and is required for tumor growth. Pancreatic ER kinase (PKR-like ER kinase) regulates the translational branch of the UPR and is also important in the growth of tumors. Although the exact mechanism has yet to be elucidated, recent data suggest that the UPR affects tumor growth through protection from apoptosis and may influence angiogenic signaling pathways. Targeting various components of the UPR is a promising therapeutic strategy. Understanding the relationship between hypoxia, the UPR, and tumor growth is crucial to improving current cancer therapies.