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Find video protocols related to scientific articles indexed in Pubmed.
Genetic Disruption of Lactate/H+ Symporters (MCTs) and their Subunit CD147/BASIGIN Sensitizes Glycolytic Tumor Cells to Phenformin.
Cancer Res.
PUBLISHED: 11-19-2014
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Rapidly growing glycolytic tumors require energy and intracellular pH (pHi) homeostasis through the activity of two major MonoCarboxylate Transporters, MCT1 and the hypoxia-inducible MCT4, in intimate association with the glycoprotein CD147/BASIGIN (BSG). To further explore and validate the blockade of lactic acid export as an anticancer strategy, we disrupted, via Zinc Finger Nucleases, MCT4 and BASIGIN genes in colon adenocarcinoma (LS174T) and glioblastoma (U87) human cell lines. First, we showed that homozygous loss of MCT4 dramatically sensitized cells to the MCT1 inhibitor AZD3965. Second, we demonstrated that knockout of BSG lead to a decrease in lactate transport activity of MCT1 and MCT4 by 10- and 6-fold, respectively. Consequently, cells accumulated an intracellular pool of lactic and pyruvic acids, magnified by the MCT1 inhibitor decreasing further pHi and glycolysis. As a result, we found that these glycolytic/MCT-deficient cells resumed growth by redirecting their metabolism towards OXPHOS. Thirdly, we showed that in contrast to parental cells, BSG-null cells became highly sensitive to phenformin, an inhibitor of mitochondrial complex I. Phenformin addition to these MCT-disrupted cells in normoxic and hypoxic conditions induced a rapid drop in cellular ATP inducing cell death by 'metabolic catastrophe'. Finally, xenograft analysis confirmed the deleterious tumor growth effect of MCT1/MCT4 ablation, an action enhanced by phenformin treatment. Collectively, these findings highlight that inhibition of the MCT/BSG complexes alone or in combination with phenformin provides an acute anticancer strategy to target highly glycolytic tumors. This genetic approach validates the anticancer potential of the MCT1 and MCT4 inhibitors in current development.
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Targeting tumour hypoxia to prevent cancer metastasis. From biology, biosensing and technology to drug development: the METOXIA consortium.
J Enzyme Inhib Med Chem
PUBLISHED: 10-28-2014
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Abstract The hypoxic areas of solid cancers represent a negative prognostic factor irrespective of which treatment modality is chosen for the patient. Still, after almost 80 years of focus on the problems created by hypoxia in solid tumours, we still largely lack methods to deal efficiently with these treatment-resistant cells. The consequences of this lack may be serious for many patients: Not only is there a negative correlation between the hypoxic fraction in tumours and the outcome of radiotherapy as well as many types of chemotherapy, a correlation has been shown between the hypoxic fraction in tumours and cancer metastasis. Thus, on a fundamental basis the great variety of problems related to hypoxia in cancer treatment has to do with the broad range of functions oxygen (and lack of oxygen) have in cells and tissues. Therefore, activation-deactivation of oxygen-regulated cascades related to metabolism or external signalling are important areas for the identification of mechanisms as potential targets for hypoxia-specific treatment. Also the chemistry related to reactive oxygen radicals (ROS) and the biological handling of ROS are part of the problem complex. The problem is further complicated by the great variety in oxygen concentrations found in tissues. For tumour hypoxia to be used as a marker for individualisation of treatment there is a need for non-invasive methods to measure oxygen routinely in patient tumours. A large-scale collaborative EU-financed project 2009-2014 denoted METOXIA has studied all the mentioned aspects of hypoxia with the aim of selecting potential targets for new hypoxia-specific therapy and develop the first stage of tests for this therapy. A new non-invasive PET-imaging method based on the 2-nitroimidazole [(18)F]-HX4 was found to be promising in a clinical trial on NSCLC patients. New preclinical models for testing of the metastatic potential of cells were developed, both in vitro (2D as well as 3D models) and in mice (orthotopic grafting). Low density quantitative real-time polymerase chain reaction (qPCR)-based assays were developed measuring multiple hypoxia-responsive markers in parallel to identify tumour hypoxia-related patterns of gene expression. As possible targets for new therapy two main regulatory cascades were prioritised: The hypoxia-inducible-factor (HIF)-regulated cascades operating at moderate to weak hypoxia (<1% O2), and the unfolded protein response (UPR) activated by endoplasmatic reticulum (ER) stress and operating at more severe hypoxia (<0.2%). The prioritised targets were the HIF-regulated proteins carbonic anhydrase IX (CAIX), the lactate transporter MCT4 and the PERK/eIF2?/ATF4-arm of the UPR. The METOXIA project has developed patented compounds targeting CAIX with a preclinical documented effect. Since hypoxia-specific treatments alone are not curative they will have to be combined with traditional anti-cancer therapy to eradicate the aerobic cancer cell population as well.
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Conventional techniques to monitor mitochondrial oxygen consumption.
Meth. Enzymol.
PUBLISHED: 05-28-2014
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Following several key discoveries on hypoxia-inducible factors, we have observed an explosion of studies investigating how the hypoxic microenvironment provokes bioenergetic alterations. This is particularly relevant for cancer cells, as they are often exposed to hypoxic conditions in the course of tumor progression. Thus, interest in the measurement of oxygen consumption at the tissue, cell, or mitochondrion level has been revived. Here, we describe the basic principles of cellular respiration and survey some of the conventional methods for measuring O2 consumption in intact or permeabilized cells.
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Gene disruption using zinc finger nuclease technology.
Methods Mol. Biol.
PUBLISHED: 05-20-2014
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Zinc finger nucleases are reagents that induce DNA double-strand breaks at specific sites that can be repaired by nonhomologous end joining, inducing alterations in the genome. This strategy has enabled highly efficient gene disruption in numerous cell types and model organisms opening a door for new therapeutic applications. Here, we describe the disruption of CD147/basigin by this technique in a human cancer cell line.
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Biochemical Titration of Glycogen In vitro.
J Vis Exp
PUBLISHED: 12-05-2013
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Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive(1).
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Disrupting proton dynamics and energy metabolism for cancer therapy.
Nat. Rev. Cancer
PUBLISHED: 08-24-2013
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Intense interest in the Warburg effect has been revived by the discovery that hypoxia-inducible factor 1 (HIF1) reprogrammes pyruvate oxidation to lactic acid conversion; lactic acid is the end product of fermentative glycolysis. The most aggressive and invasive cancers, which are often hypoxic, rely on exacerbated glycolysis to meet the increased demand for ATP and biosynthetic precursors and also rely on robust pH-regulating systems to combat the excessive generation of lactic and carbonic acids. In this Review, we present the key pH-regulating systems and synthesize recent advances in strategies that combine the disruption of pH control with bioenergetic mechanisms. We discuss the possibility of exploiting, in rapidly growing tumours, acute cell death by metabolic catastrophe.
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Quantitative in vivo characterization of intracellular and extracellular pH profiles in heterogeneous tumors: a novel method enabling multiparametric pH analysis.
Cancer Res.
PUBLISHED: 06-10-2013
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Acid production and transport are currently being studied to identify new targets for efficient cancer treatment, as subpopulations of tumor cells frequently escape conventional therapy owing to their particularly acidic tumor microenvironment. Heterogeneity in intracellular and extracellular tumor pH (pHi, pHe) has been reported, but none of the methods currently available for measuring tissue pH provides quantitative parameters characterizing pH distribution profiles in tissues. To this intent, we present here a multiparametric, noninvasive approach based on in vivo (31)P nuclear magnetic resonance (NMR) spectroscopy and its application to mouse tumor xenografts. First, localized (31)P NMR spectrum signals of pHi and pHe reporter molecules [inorganic phosphate (Pi) and 3-aminopropylphosphonate (3-APP), respectively] were transformed into pH curves using established algorithms. Although Pi is an endogenous compound, 3-APP had to be injected intraperitoneally. Then, we developed algorithms for the calculation of six to eight quantitative pH parameters from the digital points of each pH curve obtained. For this purpose, each pH distribution profile was approximated as a histogram, and intensities were corrected for the nonlinearity between chemical-shift and pH.
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Response of CAIX and CAXII to in vitro re-oxygenation and clinical significance of the combined expression in NSCLC patients.
Lung Cancer
PUBLISHED: 02-28-2013
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The disorganized neo-vasculature in tumours causes fluctuations in the concentration of oxygen, which contributes to tumour development and metastatic potential. Although hypoxic regulation of the expression of the carbonic anhydrases CAIX and CAXII is well established, the effect of re-oxygenation on these proteins remains to be elucidated. A549 and H1975 human lung cancer cell lines were exposed to hypoxia for 24 h and then re-oxygenated. CAIX or CAXII expression and cell cycle progression at different time-points were monitored. A549-shCA9 cells were analyzed for cell cycle progression in the same conditions. We demonstrate for the first time an association between the stability of CAIX and restoration of the S/G2 phase of hypoxia-arrested cells subjected to re-oxygenation. In exchange, we have found that the loss of CA9 did not cause a decreased progression into S/G2 phase during re-oxygenation, but rather affected the hypoxic growth arrest. We previously demonstrated that CAIX expression is a poor prognostic factor and that CAXII expression is a good prognostic factor in non-small cell lung cancer (NSCLC) patients. We further detail the relevance of the combined expression of these proteins for predicting outcome in a large population of NSCLC patients after long-term follow-up. The high CAIX/low CAXII expression sub-group was associated with a high cumulative incidence of relapse and with poor overall survival of NSCLC patients (P < 0.0001). Our results demonstrate a critical role for re-oxygenation on CAIX and CAXII levels that may select for an aggressive lung cancer phenotype. These findings suggest that CAIX and CAXII play dual roles in tumour progression and emphasize their significant prognostic and potential therapeutic value.
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Hypoxia promotes tumor cell survival in acidic conditions by preserving ATP levels.
J. Cell. Physiol.
PUBLISHED: 01-30-2013
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The efficacy of targeting pH disruption to induce cell death in the acidic and hypoxic tumor microenvironment continues to be assessed. Here we analyzed the impact of varying levels of hypoxia in acidic conditions on fibroblast and tumor cell survival. Across all cell lines tested, hypoxia (1% O(2)) provided protection against acidosis induced cell death compared to normoxia. Meanwhile severe hypoxia (0.1% O(2)) removed this protection and in some cases exacerbated acidosis-induced cell death. Differential survival between cell types during external acidosis correlated with their respective intracellular pH regulating capabilities. Cellular ATP measurements were conducted to determine their contribution to cell survival under these combined stresses. In general, hypoxia (1% O(2)) maintained elevated ATP levels in acidic conditions while severe hypoxia did not. To further explore this interaction we combined acidosis with ATP depletion using 2-deoxyglucose and observed an enhanced rate of cell mortality. Striking results were also observed with hypoxia providing protection against cell death in spite of a severe metabolic stress induced by a combination of acidosis and oligomycin. Finally, we demonstrated that both HIF1? and HIF2? expression were drastically reduced in hypoxic and acidic conditions indicating a sensitivity of this protein to cellular pH conditions. This knockdown of HIF expression by acidosis has implications for the development of therapies targeting the disruption of cellular pH regulation. Our results reinforce the proof of concept that acidosis and metabolic disruption affecting ATP levels could be exploited as a tumor cell killing strategy.
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TLR2 and RIP2 pathways mediate autophagy of Listeria monocytogenes via extracellular signal-regulated kinase (ERK) activation.
J. Biol. Chem.
PUBLISHED: 10-27-2011
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Listeria monocytogenes is a facultative intracellular pathogen that invades both phagocytic and non-phagocytic cells. Recent studies have shown that L. monocytogenes infection activates the autophagy pathway. However, the innate immune receptors involved and the downstream signaling pathways remain unknown. Here, we show that macrophages deficient in the TLR2 and NOD/RIP2 pathway display defective autophagy induction in response to L. monocytogenes. Inefficient autophagy in Tlr2(-/-) and Nod2(-/-) macrophages led to a defect in bacteria colocalization with the autophagosomal marker GFP-LC3. Consequently, macrophages lacking TLR2 and NOD2 were found to be more susceptible to L. monocytogenes infection, as were the Rip2(-/-) mice. Tlr2(-/-) and Nod2(-/-) cells showed perturbed NF-?B and ERK signaling. However, autophagy against L. monocytogenes was dependent selectively on the ERK pathway. In agreement, wild-type cells treated with a pharmacological inhibitor of ERK or ERK-deficient cells displayed inefficient autophagy activation in response to L. monocytogenes. Accordingly, fewer bacteria were targeted to the autophagosomes and, consequently, higher bacterial growth was observed in cells deficient in the ERK signaling pathway. These findings thus demonstrate that TLR2 and NOD proteins, acting via the downstream ERK pathway, are crucial to autophagy activation and provide a mechanistic link between innate immune receptors and induction of autophagy against cytoplasm-invading microbes, such as L. monocytogenes.
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Distinct deregulation of the hypoxia inducible factor by PHD2 mutants identified in germline DNA of patients with polycythemia.
Haematologica
PUBLISHED: 09-20-2011
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Congenital secondary erythrocytoses are due to deregulation of hypoxia inducible factor resulting in overproduction of erythropoietin. The most common germline mutation identified in the hypoxia signaling pathway is the Arginine 200-Tryptophan mutant of the von Hippel-Lindau tumor suppressor gene, resulting in Chuvash polycythemia. This mutant displays a weak deficiency in hypoxia inducible factor ? regulation and does not promote tumorigenesis. Other von Hippel-Lindau mutants with more deleterious effects are responsible for von Hippel-Lindau disease, which is characterized by the development of multiple tumors. Recently, a few mutations in gene for the prolyl hydroxylase domain 2 protein (PHD2) have been reported in cases of congenital erythrocytosis not associated with tumor formation with the exception of one patient with a recurrent extra-adrenal paraganglioma.
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CD147 subunit of lactate/H+ symporters MCT1 and hypoxia-inducible MCT4 is critical for energetics and growth of glycolytic tumors.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 09-19-2011
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Malignant tumors exhibit increased dependence on glycolysis, resulting in abundant export of lactic acid, a hypothesized key step in tumorigenesis. Lactic acid is mainly transported by two H(+)/lactate symporters, MCT1/MCT4, that require the ancillary protein CD147/Basigin for their functionality. First, we showed that blocking MCT1/2 in Ras-transformed fibroblasts with AR-C155858 suppressed lactate export, glycolysis, and tumor growth, whereas ectopic expression of MCT4 in these cells conferred resistance to MCT1/2 inhibition and reestablished tumorigenicty. A mutant-derivative, deficient in respiration (res(-)) and exclusively relying on glycolysis for energy, displayed low tumorigenicity. These res(-) cells could develop resistance to MCT1/2 inhibition and became highly tumorigenic by reactivating their endogenous mct4 gene, highlighting that MCT4, the hypoxia-inducible and tumor-associated lactate/H(+) symporter, drives tumorigenicity. Second, in the human colon adenocarcinoma cell line (LS174T), we showed that combined silencing of MCT1/MCT4 via inducible shRNA, or silencing of CD147/Basigin alone, significantly reduced glycolytic flux and tumor growth. However, both silencing approaches, which reduced tumor growth, displayed a low level of CD147/Basigin, a multifunctional protumoral protein. To gain insight into CD147/Basigin function, we designed experiments, via zinc finger nuclease-mediated mct4 and basigin knockouts, to uncouple MCTs from Basigin expression. Inhibition of MCT1 in MCT4-null, Basigin(high) cells suppressed tumor growth. Conversely, in Basigin-null cells, in which MCT activity had been maintained, tumorigenicity was not affected. Collectively, these findings highlight that the major protumoral action of CD147/Basigin is to control the energetics of glycolytic tumors via MCT1/MCT4 activity and that blocking lactic acid export provides an efficient anticancer strategy.
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Targeting hypoxic tumor cell viability with carbohydrate-based carbonic anhydrase IX and XII inhibitors.
J. Med. Chem.
PUBLISHED: 09-02-2011
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Carbonic anhydrase (CA) enzymes, specifically membrane-bound isozymes CA IX and CA XII, underpin a pH-regulating system that enables hypoxic tumor cell survival and proliferation. CA IX and XII are implicated as potential targets for the development of new hypoxic cancer therapies. To date, only a few small molecules have been characterized in CA-relevant cell and animal model systems. In this paper, we describe the development of a new class of carbohydrate-based small molecule CA inhibitors, many of which inhibit CA IX and XII within a narrow range of low nanomolar K(i) values (5.3-11.2 nM). We evaluate for the first time carbohydrate-based CA inhibitors in cell-based models that emulate the protective role of CA IX in an acidic tumor microenvironment. Our findings identified two inhibitors (compounds 5 and 17) that block CA IX-induced survival and have potential for development as in vivo cancer cell selective inhibitors.
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Deletion of ERK1 and ERK2 in the CNS causes cortical abnormalities and neonatal lethality: Erk1 deficiency enhances the impairment of neurogenesis in Erk2-deficient mice.
J. Neurosci.
PUBLISHED: 01-21-2011
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Intracellular signaling through extracellular signal-regulated kinase (ERK) is important in regulating cellular functions in a variety of tissues including the CNS. Although ERK1 and ERK2 have a very similar substrate profile and amino acid sequences, there are strikingly different phenotypes between Erk1- and Erk2-deficient mice. Thus, the question arose as to whether these two proteins are functional homologs that compensate for each other, or whether they have distinct functions. Here, we generated double knock-out mice deficient for Erk2 in the CNS, with ubiquitous homozygous deletion of Erk1, and compared the phenotypes of these mice with those of monogenic Erk2-deficient mice. Although we did obtain double knock-out newborn pups, they survived for not >1 d. These pups appeared normal just after parturition. However, they had no milk in their stomachs even 6-7 h after birth. Intracerebral hemorrhages with varying location and severity were observed. The ventricular zones and corpus callosum of the double knock-out pups did not develop adequately. Neuronal size and nuclear morphology in some brain regions were markedly aberrant in the double knock-out pups compared with controls, while deficiency in Erk2 only caused a mild phenotype. These results suggest that total ERK1/2 activity governs cellular behaviors to ensure proper brain development.
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The HIF-1-inducible lysyl oxidase activates HIF-1 via the Akt pathway in a positive regulation loop and synergizes with HIF-1 in promoting tumor cell growth.
Cancer Res.
PUBLISHED: 01-14-2011
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Adaptation to hypoxia is a driving force for tumor progression that leads to therapy resistance and poor clinical outcome. Hypoxic responses are mainly mediated by hypoxia-inducible transcription factor-1 (HIF-1). One critical HIF-1 target mediating tumor progression is lysyl oxidase (LOX), which catalyzes cross-linking of collagens and elastin in the extracellular matrix, thereby regulating tissue tensile strength. Paradoxically, LOX has been reported to be both upregulated and downregulated in cancer cells, especially in colorectal cancer. Thus, we hypothesized that LOX might regulate expression of HIF-1 to create a self-timing regulatory circuit. Using human colorectal carcinoma cell lines in which HIF-1 and LOX expression could be modulated, we showed that LOX induction enhanced HIF-1 expression, whereas LOX silencing reduced it. Mechanistic investigations revealed that LOX activated the PI3K (phosphoinositide 3-kinase)-Akt signaling pathway, thereby upregulating HIF-1? protein synthesis in a manner requiring LOX-mediated hydrogen peroxide production. Consistent with these results, cancer cell proliferation was stimulated by secreted and active LOX in an HIF-1?-dependent fashion. Furthermore, nude mice xenograft assays established that HIF-1 potentiated LOX action on tumor growth in vivo. Taken together, these findings provide compelling evidence that LOX and HIF-1 act in synergy to foster tumor formation, and they suggest that HIF-1/LOX mutual regulation is a pivotal mechanism in the adaptation of tumor cells to hypoxia.
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pH control mechanisms of tumor survival and growth.
J. Cell. Physiol.
PUBLISHED: 01-07-2011
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A distinguishing phenotype of solid tumors is the presence of an alkaline cellular feature despite the surrounding acidic microenvironment. This phenotypic characteristic of tumors, originally described by Otto Warburg, arises due to alterations in metabolism of solid tumors. Hypoxic regions of solid tumors develop due to poor vascularization and in turn regulate the expression of numerous genes via the transcription factor HIF-1. Ultimately, the tumor microenvironment directs the development of tumor cells adapted to survive in an acidic surrounding where normal cells perish. The provision of unique pH characteristics in tumor cells provides a defining trait that has led to the pursuit of treatments that target metabolism, hypoxia, and pH-related mechanisms to selectively kill cancer cells. Numerous studies over the past decade involving the cancer-specific carbonic anhydrase IX have re-kindled an interest in pH disruption-based therapies. Although an acidification of the intracellular compartment is established as a means to induce normal cell death, the defining role of acid-base disturbances in tumor physiology and survival remains unclear. The aim of this review is to summarize recent data relating to the specific role of pH regulation in tumor cell survival. We focus on membrane transport and enzyme studies in an attempt to elucidate their respective functions regarding tumor cell pH regulation. These data are discussed in the context of future directions for the field of tumor cell acid-base-related research.
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In vivo pH in metabolic-defective Ras-transformed fibroblast tumors: key role of the monocarboxylate transporter, MCT4, for inducing an alkaline intracellular pH.
Int. J. Cancer
PUBLISHED: 01-06-2011
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We present an investigation of tumor pH regulation, designed to support a new anticancer therapy concept that we had previously proposed. Our study uses a tumor model of ras-transformed hamster fibroblasts, CCL39, xenografted in the thighs of nude mice. We demonstrate, for the first time, that genetic modifications of specific mechanisms of proton production and/or proton transport result in distinct, reproducible changes in intracellular and extracellular tumor pH that can be detected and quantified noninvasively in vivo, simultaneously with determinations of tumor energetic status and necrosis in the same experiment. The CCL39 variants used were deficient in the sodium/proton exchanger, NHE-1, and/or in the monocarboxylate transporter, MCT4; further, variants were deficient in glycolysis or respiration. MCT4 expression markedly increased the gradient between intracellular and extracellular pH from 0.14 to 0.43 when compared to CCL39 wild-type tumors not expressing MCT4. The other genetic modifications studied produced smaller but significant increases in intracellular and decreases in extracellular pH. In general, increased pH gradients were paralleled by increased tumor growth performance and diminished necrotic regions, and 50% of the CCL39 variant expressing neither MCT4 nor NHE-1, but possessing full genetic capacity for glycolysis and oxidative phosphorylation, underwent regression before reaching a 1-cm diameter. Except for CCL39 wild-type tumors, no significant HIF-1? expression was detected. Our in vivo results support a multipronged approach to tumor treatment based on minimizing intracellular pH by targeting several proton production and proton transport processes, among which the very efficient MCT4 proton/lactate co-transport deserves particular attention.
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ERK1 and ERK2 are required for radial glial maintenance and cortical lamination.
Genes Cells
PUBLISHED: 09-05-2010
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ERK1/2 is involved in a variety of cellular processes during development, but the functions of these isoforms in brain development remain to be determined. Here, we generated double knockout (DKO) mice to study the individual and combined roles of ERK1 and ERK2 during cortical development. Mice deficient in Erk2, and more dramatically in the DKOs, displayed proliferation defects in late radial glial progenitors within the ventricular zone, and a severe disruption of lamination in the cerebral cortex. Immunohistochemical analyses revealed that late-generated cortical neurons were misplaced and failed to migrate the upper cortical layers in DKO mice. Moreover, these mice displayed fewer radial glial fibers, which provide architectural guides for radially migrating neurons. These results suggest that extracellular signal-regulated kinase signaling is essential for the expansion of the radial glial population and for the maintenance of radial glial scaffolding. Tangential migration of interneurons and oligodendrocytes from the ganglionic eminences (GE) to the dorsal cortex was more severely impaired in DKO mice than in mice deficient for Erk2 alone, because of reduced progenitor proliferation in the GE of the ventral telencephalon. These data demonstrate functional overlaps between ERK1 and ERK2 and indicate that extracellular signal-regulated kinase signaling plays a crucial role in cortical development.
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Implication of the ERK pathway on the post-transcriptional regulation of VEGF mRNA stability.
Methods Mol. Biol.
PUBLISHED: 09-03-2010
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Vascular Endothelial Growth Factor-A (VEGF-A) is one of the most important regulators of physiological and pathological angiogenesis. Constitutive activation of the ERK pathway and over-expression of VEGF-A are common denominators of tumours of different origins. Understanding VEGF-A regulation is of primary importance to better comprehend pathological angiogenesis. VEGF-A expression is regulated at all steps of its synthesis including transcription, mRNA stability, an under estimated way of VEGF regulation and translation. In this chapter, we present the link between VEGF mRNA stability through AU-rich sequences present in its 3-untranslated region (3-UTR) and the ERK pathway. We present several methods that have been used to demonstrate that ERKs increase VEGF mRNA half-life. This mRNA-stabilising effect is partly due to reduction of the mRNA destabilising effects of Tristetraprolin (TTP), an AU-Rich binding protein which binds to VEGF-A mRNA 3-UTR.
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Hypoxia and energetic tumour metabolism.
Curr. Opin. Genet. Dev.
PUBLISHED: 08-17-2010
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The hypoxia-inducible factor (HIF-1), in addition to genetic and epigenetic changes, is largely responsible for alterations in cell metabolism in hypoxic tumour cells. This transcription factor not only favours cell proliferation through the metabolic shift from oxidative phosphorylation to glycolysis and lactic acid production but also stimulates nutrient supply by mediating adaptive survival mechanisms. These include epithelial-mesenchymal transition, angiogenesis, autophagy, and synthesis and storage of lipid and glycogen. HIF-1 also ensures survival by correcting tumour acidosis via increased expression of the carbonic anhydrase CA IX and the lactate/H+ symporter MCT4. The targeting of key HIF-1-mediated steps, responsible for exacerbated glycolysis and pHi-control, and of the guardian of cellular energy AMP-kinase should offer novel therapeutic opportunities to fight cancer.
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Proton dynamics in cancer.
J Transl Med
PUBLISHED: 05-28-2010
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Cancer remains a leading cause of death in the world today. Despite decades of research to identify novel therapeutic approaches, durable regressions of metastatic disease are still scanty and survival benefits often negligible. While the current strategy is mostly converging on target-therapies aimed at selectively affecting altered molecular pathways in tumor cells, evidences are in parallel pointing to cell metabolism as a potential Achilles heel of cancer, to be disrupted for achieving therapeutic benefit. Critical differences in the metabolism of tumor versus normal cells, which include abnormal glycolysis, high lactic acid production, protons accumulation and reversed intra-extracellular pH gradients, make tumor site a hostile microenvironment where only cancer cells can proliferate and survive. Inhibiting these pathways by blocking proton pumps and transporters may deprive cancer cells of a key mechanism of detoxification and thus represent a novel strategy for a pleiotropic and multifaceted suppression of cancer cell growth.Research groups scattered all over the world have recently started to investigate various aspects of proton dynamics in cancer cells with quite encouraging preliminary results. The intent of unifying investigators involved in this research line led to the formation of the "International Society for Proton Dynamics in Cancer" (ISPDC) in January 2010. This is the manifesto of the newly formed society where both basic and clinical investigators are called to foster translational research and stimulate interdisciplinary collaboration for the development of more specific and less toxic therapeutic strategies based on proton dynamics in tumor cell biology.
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The MAPK ERK1 is a negative regulator of the adult steady-state splenic erythropoiesis.
Blood
PUBLISHED: 03-11-2010
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The mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 1 (ERK1) and ERK2 are among the main signal transduction molecules, but little is known about their isoform-specific functions in vivo. We have examined the role of ERK1 in adult hematopoiesis with ERK1(-/-) mice. Loss of ERK1 resulted in an enhanced splenic erythropoiesis, characterized by an accumulation of erythroid progenitors in the spleen, without any effect on the other lineages or on bone marrow erythropoiesis. This result suggests that the ablation of ERK1 induces a splenic stress erythropoiesis phenotype. However, the mice display no anemia. Deletion of ERK1 did not affect erythropoietin (EPO) serum levels or EPO/EPO receptor signaling and was not compensated by ERK2. Splenic stress erythropoiesis response has been shown to require bone morphogenetic protein 4 (BMP4)-dependent signaling in vivo and to rely on the expansion of a resident specialized population of erythroid progenitors, termed stress erythroid burst-forming units (BFU-Es). A great expansion of stress BFU-Es and increased levels of BMP4 mRNA were found in ERK1(-/-) spleens. The ERK1(-/-) phenotype can be transferred by bone marrow cells. These findings show that ERK1 controls a BMP4-dependent step, regulating the steady state of splenic erythropoiesis.
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Overexpression of carbonic anhydrase XII in tissues from resectable non-small cell lung cancers is a biomarker of good prognosis.
Int. J. Cancer
PUBLISHED: 02-19-2010
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The pattern of protein expression in tumors is under the influence of nutrient stress, hypoxia and low pH, which determines the survival of neoplastic cells and the development of tumors. Carbonic anhydrase XII (CAXII) is a transmembrane enzyme that catalyzes the reversible hydration of cell-generated carbon dioxide into protons and bicarbonate. Hypoxic conditions activate its transcription and translation and enhanced expression is often present in several types of tumors. The aim of our study was to assess the prognostic significance of CAXII tumor tissues expression in patients with NSCLC. Five hundred fifty-five tumors were immunostained for CAXII on tissue microarrays (TMA) and the results were correlated with clinicopathological parameters and outcome of patients. CAXII overexpression was present in 105/555 (19%) cases and was associated with tumors of lower grade (p = 0.015) and histological type (p < 0.001), being significantly higher in squamous cell carcinoma. High CAXII expression correlated with better overall and disease-specific survival of patients with resectable NSCLC in univariate (p < 0.001) and multivariate survival analyses (p < 0.001). In conclusion, this is the first study demonstrating that a high CAXII tumor tissue expression evaluated on TMAs is related to a better outcome in a large series of patients with resectable NSCLC.
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A functional role for the p62-ERK1 axis in the control of energy homeostasis and adipogenesis.
EMBO Rep.
PUBLISHED: 01-04-2010
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In vivo genetic inactivation of the signalling adapter p62 leads to mature-onset obesity and insulin resistance, which correlate with reduced energy expenditure (EE) and increased adipogenesis, without alterations in feeding or locomotor functions. Enhanced extracellular signal-regulated kinase (ERK) activity in adipose tissue from p62-knockout (p62(-/-)) mice, and differentiating fibroblasts, suggested an important role for this kinase in the metabolic alterations of p62(-/-) mice. Here, we show that genetic inactivation of ERK1 in p62(-/-) mice reverses their increased adiposity and adipogenesis, lower EE and insulin resistance. These results establish genetically that p62 is a crucial regulator of ERK1 in metabolism.
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HIF-1alpha mediates the induction of IL-8 and VEGF expression on infection with Afa/Dr diffusely adhering E. coli and promotes EMT-like behaviour.
Cell. Microbiol.
PUBLISHED: 12-21-2009
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Microbes regulate a large panel of intracellular signalling events that can promote inflammation and/or enhance tumour progression. Indeed, it has been shown that infection of human intestinal cells with the Afa/Dr diffusely adhering E. coli C1845 strain induces expression of pro-angiogenic and pro-inflammatory genes. Here, we demonstrate that exposure of cryptic-like intestinal epithelial cells to C1845 bacteria induces HIF-1alpha protein levels. This effect depends on the binding of F1845 adhesin to the membrane-associated DAF receptor that initiates signalling cascades promoting translational mechanisms. Indeed, inhibition of MAPK and PI-3K decreases HIF-1alpha protein levels and blocks C1845-induced phosphorylation of the ribosomal S6 protein. Using RNA interference we show that bacteria-induced HIF-1alpha regulates the expression of IL-8, VEGF and Twist1, thereby pointing to a role for HIF-1 in angiogenesis and inflammation. In addition, infection correlates with a loss of E-cadherin and cytokeratin 18 and a rise in fibronectin, suggesting that bacteria may induce an epithelial to mesenchymal transition-like phenotype. Since HIF-1alpha silencing results in reversion of bacteria-induced EMT markers, we speculate that HIF-1alpha plays a key role linking bacterial infection to angiogenesis, inflammation and some aspects of cancer initiation.
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Oxygen tension regulates pancreatic beta-cell differentiation through hypoxia-inducible factor 1alpha.
Diabetes
PUBLISHED: 12-15-2009
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Recent evidence indicates that low oxygen tension (pO2) or hypoxia controls the differentiation of several cell types during development. Variations of pO2 are mediated through the hypoxia-inducible factor (HIF), a crucial mediator of the adaptative response of cells to hypoxia. The aim of this study was to investigate the role of pO2 in beta-cell differentiation.
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Tumour hypoxia induces a metabolic shift causing acidosis: a common feature in cancer.
J. Cell. Mol. Med.
PUBLISHED: 12-08-2009
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Maintenance of cellular pH homeostasis is fundamental to life. A number of key intracellular pH (pHi) regulating systems including the Na(+)/H(+) exchangers, the proton pump, the monocarboxylate transporters, the HCO(3)(-) transporters and exchangers and the membrane-associated and cytosolic carbonic anhydrases cooperate in maintaining a pHi that is permissive for cell survival. A common feature of tumours is acidosis caused by hypoxia (low oxygen tension). In addition to oncogene activation and transformation, hypoxia is responsible for inducing acidosis through a shift in cellular metabolism that generates a high acid load in the tumour microenvironment. However, hypoxia and oncogene activation also allow cells to adapt to the potentially toxic effects of an excess in acidosis. Hypoxia does so by inducing the activity of a transcription factor the hypoxia-inducible factor (HIF), and particularly HIF-1, that in turn enhances the expression of a number of pHi-regulating systems that cope with acidosis. In this review, we will focus on the characterization and function of some of the hypoxia-inducible pH-regulating systems and their induction by hypoxic stress. It is essential to understand the fundamentals of pH regulation to meet the challenge consisting in targeting tumour metabolism and acidosis as an anti-tumour approach. We will summarize strategies that take advantage of intracellular and extracellular pH regulation to target the primary tumour and metastatic growth, and to turn around resistance to chemotherapy and radiotherapy.
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Hypoxia-induced autophagy: cell death or cell survival?
Curr. Opin. Cell Biol.
PUBLISHED: 11-19-2009
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Hypoxia (approximately 3-0.1% oxygen) is capable of rapidly inducing, via the hypoxia-inducible factor (HIF-1), a cell survival response engaging autophagy. This process is mediated by the atypical BH3-only proteins the Bcl-2/E1B 19kDa-interacting protein 3 (BNIP3/BNIP3L (NIX)) that are induced by HIF-1. These mitochondrial associated BNIP proteins also mediate mitophagy, a metabolic adaptation for survival that is able to control reactive oxygen species (ROS) production and DNA damage. In contrast, severe hypoxic conditions or anoxia (<0.1% oxygen), where the latter is often confused with physiological hypoxia, are capable of inducing a HIF-independent autophagic response, generated via an extreme nutritional stress response implicating the AMPK-mTOR and unfolded protein response (UPR) pathways. The autophagic cell death that is often observed in these extreme stress conditions should be seen as the outcome of failed adaptation.
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ERK nuclear translocation is dimerization-independent but controlled by the rate of phosphorylation.
J. Biol. Chem.
PUBLISHED: 11-17-2009
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Upon activation, ERKs translocate from the cytoplasm to the nucleus. This process is required for the induction of many cellular responses, yet the molecular mechanisms that regulate ERK nuclear translocation are not fully understood. We have used a mouse embryo fibroblast ERK1-knock-out cell line expressing green fluorescent protein (GFP)-tagged ERK1 to probe the spatio-temporal regulation of ERK1. Real time fluorescence microscopy and fluorescence correlation spectroscopy revealed that ERK1 nuclear accumulation increased upon serum stimulation, but the mobility of the protein in the nucleus and cytoplasm remained unchanged. Dimerization of ERK has been proposed as a requirement for nuclear translocation. However, ERK1-Delta4, the mutant shown consistently to be dimerization-deficient in vitro, accumulated in the nucleus to the same level as wild type (WT), indicating that dimerization of ERK1 is not required for nuclear entry and retention. Consistent with this finding, energy migration Förster resonance energy transfer and fluorescence correlation spectroscopy measurements in living cells did not detect dimerization of GFP-ERK1-WT upon activation. In contrast, the kinetics of nuclear accumulation and phosphorylation of GFP-ERK1-Delta4 were slower than that of GFP-ERK1-WT. These results indicate that the differential shuttling behavior of the mutant is a consequence of delayed phosphorylation of ERK by MEK rather than dimerization. Our data demonstrate for the first time that a delay in cytoplasmic activation of ERK is directly translated into a delay in nuclear translocation.
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Activation of HIF-1alpha in exponentially growing cells via hypoxic stimulation is independent of the Akt/mTOR pathway.
J. Cell. Physiol.
PUBLISHED: 10-13-2009
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Accumulation of HIF-1alpha during normoxic conditions at high cell density has previously been shown to occur and can be used to stabilize HIF-1alpha protein in the absence of a specific anaerobic chamber. However, the impact and origin of this pool of HIF-1alpha, obtained under normoxia, has been underestimated. In this study, we have systematically compared the related pools of HIF-1alpha stabilized in normoxia by high cell density to those obtained at low density in hypoxia. At first glance, these two stimuli appear to have similar outcomes: HIF-1alpha stabilization and induction of HIF-1-dependent genes. However, upon careful analysis, we observed that molecular mechanisms involved are different. We clearly demonstrate that density-dependant HIF-1alpha accumulation during normoxia is due to the cells high consumption of oxygen, as demonstrated by using a respiration inhibitor (oligomycin) and respiratory-defective mutant cells (GSK3). Finally and most importantly, our data indicate that a decrease in AKT activity followed by a total decrease in p70(S6K) phosphorylation reflecting a decrease in mTOR activity occurs during high oxygen consumption, resulting from high cell density. In contrast, hypoxia, even at severe low O(2) levels, only slightly impacts upon the mTOR pathway under low cell density conditions. Thus, activation of HIF-1alpha in exponentially growing cells via hypoxic stimulation is independent of the Akt/mTOR pathway whereas HIF-1alpha activation obtained in high confluency is totally dependent on mTOR pathway as rapamycin totally impaired (i) HIF-1alpha stabilization and (ii) mRNA levels of CA9 and BNIP3, two HIF-target genes.
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Cutting edge: Extracellular signal-related kinase is not required for negative selection of developing T cells.
J. Immunol.
PUBLISHED: 10-06-2009
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Signals initiated through the TCR during development can result in either survival and differentiation or cell death. High affinity signals that induce death elicit a robust yet transient activation of signaling pathways, including Erk, whereas low affinity ligands, which promote survival, generate a gradual and weaker activation of the same pathways. It was recently demonstrated that Erk localizes to distinct cellular locations in response to high and low affinity ligands. Although a requirement for Erk in positive selection is well established, its role in negative selection is controversial and, thus, the importance of Erk relocalization during development is not understood. In this study, we examined the role of Erk in negative selection using mice that are genetically deficient in both Erk1 and Erk2 in T cells. Results from three different models reveal that thymocyte deletion remains intact in the absence of Erk.
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Atypical BH3-domains of BNIP3 and BNIP3L lead to autophagy in hypoxia.
Autophagy
PUBLISHED: 08-18-2009
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Normal and tumor cells subjected to a hypoxic microenvironment show evidence of autophagy. We hypothesize that cells will sense hypoxia as a warning signal to upcoming drastic microenvironmental conditions and that autophagy, acting as a survival mechanism, will provide time for cells to adapt. This work demonstrates for the first time that the atypical BH3-domain of BNIP3 and BNIP3L, two HIF-target genes, can compete with Beclin 1-Bcl-2 and Beclin 1-Bcl-X(L) complexes, releasing Beclin 1 from the complex and then enhancing autophagy. We thus revealed a new role for BH3-only proteins in the cellular response to hypoxia.
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Inhibition of prolyl hydroxylase domain proteins promotes therapeutic revascularization.
Circulation
PUBLISHED: 06-22-2009
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The hypoxia-inducible transcription factor (HIF) subunits are destabilized via the O(2)-dependent prolyl hydroxylase domain proteins (PHD1, PHD2, and PHD3). We investigated whether inhibition of PHDs via upregulating HIF might promote postischemic neovascularization.
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Total ERK1/2 activity regulates cell proliferation.
Cell Cycle
PUBLISHED: 03-29-2009
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Regulating ERK activity is essential for normal cell proliferation to occur. In mammals and most vertebrates ERK activity is provided by ERK1 and ERK2 that are highly similar, ubiquitously expressed and share activators and substrates. By combining single and double silencings of ERK1 and ERK2 we recently demonstrated that the apparent dominant role of ERK2 to regulate cell proliferation was due to its markedly higher expression level than ERK1. The contribution of ERK1 was revealed when ERK2 activation was clamped to avoid compensating over-activation of ERK2. We found no evidences in the literature for insulated isoform-specific modules in the Ras/Raf/MEK signaling cascade that could activate specifically ERK1 or ERK2. Obviously in frogs all signal integration and fine modulation provided by three Ras and three Raf isoforms is conducted by only one MEK and one ERK isoform. In mammals, ERK1 and ERK2 display similar specific activities and are activated respectively to their expression levels. After integrating signals from Ras, Raf and MEK isoforms, ERK1 and ERK2 regulate positively cell proliferation according to their expression levels.
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Hypoxia-induced autophagy is mediated through hypoxia-inducible factor induction of BNIP3 and BNIP3L via their BH3 domains.
Mol. Cell. Biol.
PUBLISHED: 03-09-2009
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While hypoxia-inducible factor (HIF) is a major actor in the cell survival response to hypoxia, HIF also is associated with cell death. Several studies implicate the HIF-induced putative BH3-only proapoptotic genes bnip3 and bnip3l in hypoxia-mediated cell death. We, like others, do not support this assertion. Here, we clearly demonstrate that the hypoxic microenvironment contributes to survival rather than cell death by inducing autophagy. The ablation of Beclin1, a major actor of autophagy, enhances cell death under hypoxic conditions. In addition, the ablation of BNIP3 and/or BNIP3L triggers cell death, and BNIP3 and BNIP3L are crucial for hypoxia-induced autophagy. First, while the small interfering RNA-mediated ablation of either BNIP3 or BNIP3L has little effect on autophagy, the combined silencing of these two HIF targets suppresses hypoxia-mediated autophagy. Second, the ectopic expression of both BNIP3 and BNIP3L in normoxia activates autophagy. Third, 20-mer BH3 peptides of BNIP3 or BNIP3L are sufficient in initiating autophagy in normoxia. Herein, we propose a model in which the atypical BH3 domains of hypoxia-induced BNIP3/BNIP3L have been designed to induce autophagy by disrupting the Bcl-2-Beclin1 complex without inducing cell death. Hypoxia-induced autophagy via BNIP3 and BNIP3L is clearly a survival mechanism that promotes tumor progression.
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Gene regulation in response to graded hypoxia: the non-redundant roles of the oxygen sensors PHD and FIH in the HIF pathway.
J. Theor. Biol.
PUBLISHED: 03-02-2009
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Hypoxia Inducible Factor (HIF), being the master protein involved in adaptation to low pO(2), plays a major role in many physiological and pathological phenomena: development, inflammation, ischemia and cancer. Prolyl hydroxylase (PHD) and factor inhibiting HIF (FIH) are the two oxygen sensors that regulate the HIF pathway. Here we model the regulatory dynamics in an oxygen gradient by a system of differential equations. A part of the work consists in a qualitative analysis, driven independently of the values of the parameters, which explains the non-redundant functional roles of FIH and PHD. In a second part, we use biological experiments to fit the model in a physiologically relevant context and run simulations. Simulation results are confronted with success to independent biological experiments. The combination of biological data and mathematical analysis stresses that FIH is a fine modulator determining whether a given gene should be induced in mildly or in strongly hypoxic areas. Moreover it gives access to other functional predictions that are not directly accessible by pure experiments, for instance the stoichiometry of prolyl-hydroxylation on HIF, and the switch-like properties of the system. Availability: an interactive simulation interface is available at http://sdi.ljad.free.fr/spip.php?article111.
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Multiple division cycles and long-term survival of hepatocytes are distinctly regulated by extracellular signal-regulated kinases ERK1 and ERK2.
Hepatology
PUBLISHED: 01-30-2009
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We investigated the specific role of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 1 (ERK1)/ERK2 pathway in the regulation of multiple cell cycles and long-term survival of normal hepatocytes. An early and sustained epidermal growth factor (EGF)-dependent MAPK activation greatly improved the potential of cell proliferation. In this condition, almost 100% of the hepatocytes proliferated, and targeting ERK1 or ERK2 via RNA interference revealed the specific involvement of ERK2 in this regulation. However, once their first cell cycle was performed, hepatocytes failed to undergo a second round of replication and stayed blocked in G1 phase. We demonstrated that sustained EGF-dependent activation of the MAPK/ERK kinase (MEK)/ERK pathway was involved in this blockage as specific transient inhibition of the cascade repotentiated hepatocytes to perform a new wave of replication and multiple cell cycles. We identified this mechanism by showing that this blockage was in part supported by ERK2-dependent p21 expression. Moreover, continuous MEK inhibition was associated with a lower apoptotic engagement, leading to an improvement of survival up to 3 weeks. Using RNA interference and ERK1 knockout mice, we extended these results by showing that this improved survival was due to the specific inhibition of ERK1 expression/phosphorylation and did not involve ERK2.
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Hypoxia-inducible carbonic anhydrase IX and XII promote tumor cell growth by counteracting acidosis through the regulation of the intracellular pH.
Cancer Res.
PUBLISHED: 01-02-2009
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Acidosis of the tumor microenvironment is typical of a malignant phenotype, particularly in hypoxic tumors. All cells express multiple isoforms of carbonic anhydrase (CA), enzymes catalyzing the reversible hydration of carbon dioxide into bicarbonate and protons. Tumor cells express membrane-bound CAIX and CAXII that are controlled via the hypoxia-inducible factor (HIF). Despite the recognition that tumor expression of HIF-1alpha and CAIX correlates with poor patient survival, the role of CAIX and CAXII in tumor growth is not fully resolved. To understand the advantage that tumor cells derive from expression of both CAIX and CAXII, we set up experiments to either force or invalidate the expression of these enzymes. In hypoxic LS174Tr tumor cells expressing either one or both CA isoforms, we show that (a) in response to a "CO(2) load," both CAs contribute to extracellular acidification and (b) both contribute to maintain a more alkaline resting intracellular pH (pH(i)), an action that preserves ATP levels and cell survival in a range of acidic outside pH (6.0-6.8) and low bicarbonate medium. In vivo experiments show that ca9 silencing alone leads to a 40% reduction in xenograft tumor volume with up-regulation of ca12 mRNA levels, whereas invalidation of both CAIX and CAXII gives an impressive 85% reduction. Thus, hypoxia-induced CAIX and CAXII are major tumor prosurvival pH(i)-regulating enzymes, and their combined targeting shows that they hold potential as anticancer targets.
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Knock-down of hypoxia-induced carbonic anhydrases IX and XII radiosensitizes tumor cells by increasing intracellular acidosis.
Front Oncol
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The relationship between acidosis within the tumor microenvironment and radioresistance of hypoxic tumor cells remains unclear. Previously we reported that hypoxia-induced carbonic anhydrases (CA) IX and CAXII constitute a robust intracellular pH (pH(i))-regulating system that confers a survival advantage on hypoxic human colon carcinoma LS174Tr cells in acidic microenvironments. Here we investigate the role of acidosis, CAIX and CAXII knock-down in combination with ionizing radiation. Fibroblasts cells (-/+ CAIX) and LS174Tr cells (inducible knock-down for ca9/ca12) were analyzed for cell cycle phase distribution and survival after irradiation in extracellular pH(o) manipulations and hypoxia (1% O(2)) exposure. Radiotherapy was used to target ca9/ca12-silenced LS174Tr tumors grown in nude mice. We found that diminishing the pH(i)-regulating capacity of fibroblasts through inhibition of Na(+)/H(+) exchanger 1 sensitize cells to radiation-induced cell death. Secondly, the pH(i)-regulating function of CAIX plays a key protective role in irradiated fibroblasts in an acidic environment as accompanied by a reduced number of cells in the radiosensitive phases of the cell cycle. Thirdly, we demonstrate that irradiation of LS174Tr spheroids, silenced for either ca9 or both ca9/ca12, showed a respective 50 and 75% increase in cell death as a result of a decrease in cell number in the radioresistant S phase and a disruption of CA-mediated pH(i) regulation. Finally, LS174Tr tumor progression was strongly decreased when ca9/ca12 silencing was combined with irradiation in vivo. These findings highlight the combinatory use of radiotherapy with targeting of the pH(i)-regulating CAs as an anti-cancer strategy.
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Mechanical allodynia but not thermal hyperalgesia is impaired in mice deficient for ERK2 in the central nervous system.
Pain
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Extracellular signal-regulated kinase (ERK) plays critical roles in pain plasticity. However, the specific contribution of ERK2 isoforms to pain plasticity is not necessarily elucidated. Here we investigate the function of ERK2 in mouse pain models. We used the Cre-loxP system to cause a conditional, region-specific, genetic deletion of Erk2. To induce recombination in the central nervous system, Erk2-floxed mice were crossed with nestin promoter-driven cre transgenic mice. In the spinal cord of resultant Erk2 conditional knockout (CKO) mice, ERK2 expression was abrogated in neurons and astrocytes, but indistinguishable in microglia compared to controls. Although Erk2 CKO mice showed a normal baseline paw withdrawal threshold to mechanical stimuli, these mice had a reduced nociceptive response following a formalin injection to the hind paw. In a partial sciatic nerve ligation model, Erk2 CKO mice showed partially restored mechanical allodynia compared to control mice. Interestingly, thermal hyperalgesia was indistinguishable between Erk2 CKO and control mice in this model. In contrast to Erk2 CKO mice, mice with a targeted deletion of ERK1 did not exhibit prominent anomalies in these pain models. In Erk2 CKO mice, compensatory hyperphosphorylation of ERK1 was detected in the spinal cord. However, ERK1 did not appear to influence nociceptive processing because the additional inhibition of ERK1 phosphorylation using MEK (MAPK/ERK kinase) inhibitor SL327 did not produce additional changes in formalin-induced spontaneous behaviors in Erk2 CKO mice. Together, these results indicate that ERK2 plays a predominant and/or specific role in pain plasticity, while the contribution of ERK1 is limited.
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Polar opposites: Erk direction of CD4 T cell subsets.
J. Immunol.
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Effective immune responses depend upon appropriate T cell differentiation in accord with the nature of an infectious agent, and the contingency of differentiation depends minimally on TCR, coreceptor, and cytokine signals. In this reverse genetic study, we show that the MAPK Erk2 is not essential for T cell proliferation in the presence of optimum costimulation. Instead, it has opposite effects on T-bet and Gata3 expression and, hence, on Th1 and Th2 differentiation. Alternatively, in the presence of TGF-?, the Erk pathway suppresses a large program of gene expression, effectively limiting the differentiation of Foxp3(+) regulatory T cells. In the latter case, the mechanisms involved include suppression of Gata3 and Foxp3, induction of Tbx21, phosphorylation of Smad2,3, and possibly suppression of Socs2, a positive inducer of Stat5 signaling. Consequently, loss of Erk2 severely impeded Th1 differentiation while enhancing the development of Foxp3(+)-induced T regulatory cells. Selected profiles of gene expression under multiple conditions of T cell activation illustrate the opposing consequences of Erk pathway signaling.
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Glycogen Synthesis is Induced in Hypoxia by the Hypoxia-Inducible Factor and Promotes Cancer Cell Survival.
Front Oncol
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The hypoxia-inducible factor 1 (HIF-1), in addition to genetic and epigenetic changes, is largely responsible for alterations in cell metabolism in hypoxic tumor cells. This transcription factor not only favors cell proliferation through the metabolic shift from oxidative phosphorylation to glycolysis and lactic acid production but also stimulates nutrient supply by mediating adaptive survival mechanisms. In this study we showed that glycogen synthesis is enhanced in non-cancer and cancer cells when exposed to hypoxia, resulting in a large increase in glycogen stores. Furthermore, we demonstrated that the mRNA and protein levels of the first enzyme of glycogenesis, phosphoglucomutase1 (PGM1), were increased in hypoxia. We showed that induction of glycogen storage as well as PGM1 expression were dependent on HIF-1 and HIF-2. We established that hypoxia-induced glycogen stores are rapidly mobilized in cells that are starved of glucose. Glycogenolysis allows these "hypoxia-preconditioned" cells to confront and survive glucose deprivation. In contrast normoxic control cells exhibit a high rate of cell death following glucose removal. These findings point to the important role of hypoxia and HIF in inducing mechanisms of rapid adaptation and survival in response to a decrease in oxygen tension. We propose that a decrease in pO(2) acts as an "alarm" that prepares the cells to face subsequent nutrient depletion and to survive.
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Tie1 deficiency induces endothelial-mesenchymal transition.
EMBO Rep.
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Endothelial-mesenchymal transition (EndMT) has a significant role in embryonic heart formation and in various pathologies. However, the molecular mechanisms that regulate EndMT induction remain to be elucidated. We show that suppression of receptor tyrosine kinase Tie1 but not Tie2 induces human endothelial cells to undergo EndMT and that Slug deficiency reverts this process. We find that Erk1/2, Erk5 and Akt cascades control Slug promoter activity induced by Tie1 deficiency. Interestingly, EndMT is present in human pancreatic tumour. We propose that EndMT associated with Tie1 downregulation participates in the pathological development of stroma observed in tumours.
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Positron emission tomography/computed tomography imaging of residual skull base chordoma before radiotherapy using fluoromisonidazole and fluorodeoxyglucose: potential consequences for dose painting.
Int. J. Radiat. Oncol. Biol. Phys.
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To detect the presence of hypoxic tissue, which is known to increase the radioresistant phenotype, by its uptake of fluoromisonidazole (18F) (FMISO) using hybrid positron emission tomography/computed tomography (PET/CT) imaging, and to compare it with the glucose-avid tumor tissue imaged with fluorodeoxyglucose (18F) (FDG), in residual postsurgical skull base chordoma scheduled for radiotherapy.
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Expression of a truncated active form of VDAC1 in lung cancer associates with hypoxic cell survival and correlates with progression to chemotherapy resistance.
Cancer Res.
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Resistance to chemotherapy-induced apoptosis of tumor cells represents a major hurdle to efficient cancer therapy. Although resistance is a characteristic of tumor cells that evolve in a low oxygen environment (hypoxia), the mechanisms involved remain elusive. We observed that mitochondria of certain hypoxic cells take on an enlarged appearance with reorganized cristae. In these cells, we found that a major mitochondrial protein regulating metabolism and apoptosis, the voltage-dependent anion channel 1 (VDAC1), was linked to chemoresistance when in a truncated (VDAC1-?C) but active form. The formation of truncated VDAC1, which had a similar channel activity and voltage dependency as full-length, was hypoxia-inducible factor-1 (HIF-1)-dependent and could be inhibited in the presence of the tetracycline antibiotics doxycycline and minocycline, known inhibitors of metalloproteases. Its formation was also reversible upon cell reoxygenation and associated with cell survival through binding to the antiapoptotic protein hexokinase. Hypoxic cells containing VDAC1-?C were less sensitive to staurosporine- and etoposide-induced cell death, and silencing of VDAC1-?C or treatment with the tetracycline antibiotics restored sensitivity. Clinically, VDAC1-?C was detected in tumor tissues of patients with lung adenocarcinomas and was found more frequently in large and late-stage tumors. Together, our findings show that via induction of VDAC1-?C, HIF-1 confers selective protection from apoptosis that allows maintenance of ATP and cell survival in hypoxia. VDAC1-?C may also hold promise as a biomarker for tumor progression in chemotherapy-resistant patients.
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ERK1 regulates the hematopoietic stem cell niches.
PLoS ONE
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The mitogen-activated protein kinases (MAPK) ERK1 and ERK2 are among the major signal transduction molecules but little is known about their specific functions in vivo. ERK activity is provided by two isoforms, ERK1 and ERK2, which are ubiquitously expressed and share activators and substrates. However, there are not in vivo studies which have reported a role for ERK1 or ERK2 in HSCs and the bone marrow microenvironment. The present study shows that the ERK1-deficient mice present a mild osteopetrosis phenotype. The lodging and the homing abilities of the ERK1(-/-) HSC are impaired, suggesting that the ERK1(-/-)-defective environment may affect the engrafment of HSCs. Serial transplantations demonstrate that ERK1 is involved in the maintenance of an appropriate medullar microenvironment, but that the intrinsic properties of HSCs are not altered by the ERK1(-/-) defective microenvironment. Deletion of ERK1 impaired in vitro and in vivo osteoclastogenesis while osteoblasts were unaffected. As osteoclasts derive from precursors of the monocyte/macrophage lineage, investigation of the monocytic compartment was performed. In vivo analysis of the myeloid lineage progenitors revealed that the frequency of CMPs increased by approximately 1.3-fold, while the frequency of GMPs significantly decreased by almost 2-fold, compared with the respective WT compartments. The overall mononuclear-phagocyte lineage development was compromised in these mice due to a reduced expression of the M-CSF receptor on myeloid progenitors. These results show that the cellular targets of ERK1 are M-CSFR-responsive cells, upstream to osteoclasts. While ERK1 is well known to be activated by M-CSF, the present results are the first to point out an ERK1-dependent M-CSFR regulation on hematopoietic progenitors. This study reinforces the hypothesis of an active cross-talk between HSCs, their progeny and bone cells in the maintenance of the homeostasis of these compartments.
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ERK1 is important for Th2 differentiation and development of experimental asthma.
FASEB J.
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The ERK1/2 signaling pathway regulates a variety of T-cell functions. We observed dynamic changes in the expression of ERK1/2 during T-helper cell differentiation. Specifically, the expression of ERK1/2 was decreased and increased by IL-12 and IL-4, respectively. To address this subject further, we examined the specific role of ERK1 in Th2 differentiation and development of experimental asthma using ERK1(-/-) mice. ERK1(-/-) mice were unable to mount airway inflammation and hyperreactivity in two different models of asthma, acute and chronic. ERK1(-/-) mice had reduced expression of Th2 cytokines IL-4 and IL-5 but not IL-17A or IFN-?. They had reduced levels of allergen-specific IgE and blood eosinophils. T cells from immunized ERK1(-/-) mice manifested reduced proliferation in response to the sensitizing allergen. ERK1(-/-) T cells had reduced and short-lived expression of JunB following TCR stimulation, which likely contributed to their impaired Th2 differentiation. Immunized ERK1(-/-) mice showed reduced numbers of CD44(high) CD4 T cells in the spleen. In vitro studies demonstrated that Th2 but not Th1 cells from ERK1(-/-) mice had reduced numbers of CD44(high) cells. Finally, CD4 T cells form ERK1(-/-) mice expressed higher levels of BIM under growth factor-deprived conditions and reduced Mcl-1 on stimulation. As a result, the survival of CD4 T cells, especially CD44(high) Th2 cells, was much reduced in ERK1(-/-) mice. We conclude that ERK1 plays a nonredundant role in Th2 differentiation and development of experimental asthma. ERK1 controls Th2 differentiation and survival through its effect on JunB and BIM, respectively.
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Glucose-induced O? consumption activates hypoxia inducible factors 1 and 2 in rat insulin-secreting pancreatic beta-cells.
PLoS ONE
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Glucose increases the expression of glycolytic enzymes and other hypoxia-response genes in pancreatic beta-cells. Here, we tested whether this effect results from the activation of Hypoxia-Inducible-factors (HIF) 1 and 2 in a hypoxia-dependent manner.
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HIF-prolyl hydroxylase 2 inhibition enhances the efficiency of mesenchymal stem cells-based therapies for the treatment of critical limb ischemia.
Stem Cells
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Upregulation of HIF-1?, through prolyl-hydroxylase domain protein (PHDs) inhibition, can be thought of as a master switch that coordinates the expression of a wide repertoire of genes involved in regulating vascular growth and remodelling. We aimed to unravel the effect of specific PHD2 isoform silencing in cell-based strategies designed to promote therapeutic revascularization in patients with critical limb ischemia (CLI). PHD2 mRNA levels were up-regulated whereas that of HIF-1? were down-regulated in blood cells from patients with CLI. We therefore assessed the putative beneficial effects of PHD2 silencing on human bone-marrow derived mesenchymal stem cells (hBM-MSC)-based therapy. PHD2 silencing enhanced hBM-MSC therapeutic effect in an experimental model of CLI in Nude mice, through an up-regulation of HIF-1? and its target gene, VEGF-A. In addition, PHD2-transfected hBM-MSC displayed higher protection against apoptosis in vitro and increased rate of survival in the ischemic tissue, as assessed by Fluorescence Molecular Tomography. Co-transfection with HIF1? or VEGF-A short interfering RNAs fully abrogated the beneficial effect of PHD2 silencing on the pro-angiogenic capacity of hBM-MSC. We finally investigated the effect of PHD2 inhibition on the revascularization potential of ischemic targeted tissues in the diabetic pathological context. Inhibition of PHD-2 with shRNAs increased post-ischemic neovascularisation in diabetic mice with CLI. This increase was associated with an up-regulation of pro-angiogenic and pro-arteriogenic factors and was blunted by concomitant silencing of HIF-1?. In conclusion, silencing of PHD2, by the transient upregulation of HIF-1? and its target gene VEGF-A, might improve the efficiency of hBM-MSC-based therapies. Stem Cells 2013.
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