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1.
Cell Death Dis ; 14(11): 784, 2023 11 30.
Article in English | MEDLINE | ID: mdl-38036520

ABSTRACT

Medulloblastoma is a cancerous brain tumor that affects mostly children. Among the four groups defined by molecular characteristics, Group 3, the least well characterized, is also the least favorable, with a survival rate of 50%. Current treatments, based on surgery, radiotherapy, and chemotherapy, are not adequate and the lack of understanding of the different molecular features of Group 3 tumor cells makes the development of effective therapies challenging. In this study, the problem of medulloblastoma is approached from a metabolic standpoint in a low oxygen microenvironment. We establish that Group 3 cells use both the mitochondrial glycerol-3 phosphate (G3PS) and malate-aspartate shuttles (MAS) to produce NADH. Small molecules that target G3PS and MAS show a greater ability to decrease cell proliferation and induce apoptosis specifically of Group 3 cells. In addition, as Group 3 cells show improved respiration in hypoxia, the use of Phenformin, a mitochondrial complex 1 inhibitor, alone or in combination, induced significant cell death. Furthermore, inhibition of the cytosolic NAD+ recycling enzyme lactate dehydrogenase A (LDHA), enhanced the effects of the NADH shuttle inhibitors. In a 3D model using Group 3 human cerebellar organoids, tumor cells also underwent apoptosis upon treatment with NADH shuttle inhibitors. Our study demonstrates metabolic heterogeneity depending on oxygen concentrations and provides potential therapeutic solutions for patients in Group 3 whose tumors are the most aggressive.


Subject(s)
Cerebellar Neoplasms , Medulloblastoma , Child , Humans , NAD/metabolism , Medulloblastoma/genetics , Cerebellar Neoplasms/genetics , Hypoxia , Oxygen , Malates/metabolism , Aspartic Acid/metabolism , Tumor Microenvironment
2.
bioRxiv ; 2023 Oct 20.
Article in English | MEDLINE | ID: mdl-37905067

ABSTRACT

Medulloblastoma (MB) is the most prevalent brain cancer in children. Four subgroups of MB have been identified; of these, Group 3 is the most metastatic. Its genetics and biology remain less clear than the other groups, and it has a poor prognosis and few effective treatments available. Tumor hypoxia and the resulting metabolism are known to be important in the growth and survival of tumors but, to date, have been only minimally explored in MB. Here we show that Group 3 MB tumors do not depend on the canonical transcription factor hypoxia-inducible factor-1α (HIF-1α) to mount an adaptive response to hypoxia. We discovered that HIF-1α is rendered inactive either through post-translational methylation, preventing its nuclear localization specifically in Group 3 MB, or by a low expression that prevents modulation of HIF-target genes. Strikingly, we found that HIF-2 takes over the role of HIF-1 in the nucleus and promotes the activation of hypoxia-dependent anabolic pathways. The exclusion of HIF-1 from the nucleus in Group 3 MB cells enhances the reliance on HIF-2's transcriptional role, making it a viable target for potential anticancer strategies. By combining pharmacological inhibition of HIF-2α with the use of metformin, a mitochondrial complex I inhibitor to block respiration, we effectively induced Group 3 MB cell death, surpassing the effectiveness observed in Non-Group 3 MB cells. Overall, the unique dependence of MB cells, but not normal cells, on HIF-2-mediated anabolic metabolism presents an appealing therapeutic opportunity for treating Group 3 MB patients with minimal toxicity.

3.
Oncogenesis ; 9(7): 68, 2020 Jul 24.
Article in English | MEDLINE | ID: mdl-32709889

ABSTRACT

Cancer cells are characterized by the Warburg effect, a shift from mitochondrial respiration to oxidative glycolysis. We report here the crucial role of cyclin D1 in promoting this effect in a cyclin-dependent kinase (CDK)4/6-independent manner in multiple myeloma (MM) cells. We show that the cyclin D1 oncoprotein targets hexokinase 2 (HK2), a major glycolysis regulator, through two original molecular mechanisms in the cytoplasmic and nuclear compartments. In the cytoplasm, cyclin D1 binds HK2 at the outer mitochondrial membrane, and in the nucleus, it binds hypoxia-inducible factor-1α (HIF1α), which regulates HK2 gene transcription. We also show that high levels of HK2 expression are correlated with shorter event-free survival (EFS) and overall survival (OS) in MM patients. HK2 may therefore be considered as a possible target for antimyeloma therapy.

4.
Biochim Biophys Acta Bioenerg ; 1858(8): 665-673, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28283400

ABSTRACT

The voltage-dependent anion channel (VDAC) is a pore located at the outer membrane of the mitochondrion. It allows the entry and exit of numerous ions and metabolites between the cytosol and the mitochondrion. Flux through the pore occurs in an active way: first, it depends on the open or closed state and second, on the negative or positive charges of the different ion species passing through the pore. The flux of essential metabolites, such as ATP, determines the functioning of the mitochondria to a noxious stimulus. Moreover, VDAC acts as a platform for many proteins and in so doing supports glycolysis and prevents apoptosis by interacting with hexokinase, or members of the Bcl-2 family, respectively. VDAC is thus involved in the choice the cells make to survive or die, which is particularly relevant to cancer cells. For these reasons, VDAC has become a potential therapeutic target to fight cancer but also other diseases in which mitochondrial metabolism is modified. This article is part of a Special Issue entitled Mitochondria in Cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux.


Subject(s)
Mitochondrial Membranes/metabolism , Mitochondrial Proteins/physiology , Neoplasm Proteins/physiology , Neoplasms/metabolism , Voltage-Dependent Anion Channels/physiology , Animals , Apoptosis/physiology , Biological Transport , Calcium Signaling , Energy Metabolism , Hexokinase/metabolism , Humans , Mitochondria/metabolism , Mitochondrial Proteins/antagonists & inhibitors , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/genetics , Mitophagy/physiology , Molecular Targeted Therapy , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/chemistry , Neoplasm Proteins/genetics , Structure-Activity Relationship , Substrate Specificity , Voltage-Dependent Anion Channels/antagonists & inhibitors , Voltage-Dependent Anion Channels/chemistry , Voltage-Dependent Anion Channels/genetics
5.
Front Oncol ; 6: 193, 2016.
Article in English | MEDLINE | ID: mdl-27625993

ABSTRACT

The voltage-dependent anion channel (VDAC) is the main interface between the cytosol and mitochondria of cells. It plays a crucial role in both mitochondrial metabolism and cell death. The main basic function of this channel is to mediate and gate the flux of small ions, metabolites, and adenosine triphosphate. Changes in its structure, and thus conformation, are expected to affect its activity and modulate the ability of cancer cells to expand. In this review, we describe a novel mechanism by which mitochondria of cells in hypoxia, a low level of oxygen, protects from apoptosis. In hypoxia, some mitochondria become enlarged due to hyperfusion. These mitochondria possess a truncated form of VDAC1 (VDAC1-ΔC), which is linked to the higher metabolic capacity and the greater resistance to cell death of hypoxic cells. However, not all of the VDAC1 protein is truncated, but the amount of the full-length form is diminished compared to the amount in normoxic cells. First, we describe how such a decrease effects cell proliferation, respiration, glycolysis, and other processes. Second, we report on a novel mitochondrial-endolysosomal crosstalk that leads to VDAC1 truncation. By pharmacological targeting of VDAC1-ΔC, the production of energy could be turned off and the sensitivity to cell death restored. This could counteract the favorable microenvironment that gives cancer cells a growth advantage and thereby disrupts the balance between life and death, which is controlled by VDAC1.

6.
Adv Exp Med Biol ; 772: 101-10, 2014.
Article in English | MEDLINE | ID: mdl-24272356

ABSTRACT

Finding new therapeutic targets to fight cancer is an ongoing quest. Because of insufficiencies in tumor vasculature, cells often are exposed to a hostile microenvironment that is low in oxygen (hypoxic) and nutrients. Thus, tumor cells face the challenge of finding new sources of energy and defying apoptosis, which allow them to survive, grow, and colonize other tissues. Eradicating specifically these hypoxic cells is one of the many goals of anticancer therapies. The mitochondrial voltage-dependent anion channel (VDAC) is a protein at the crossroads of metabolic and survival pathways. As its name suggests, VDAC is involved in ion transport as well as adenosine triphosphate and NAD(+) transport. We recently reported the presence in tumor cells of a novel hypoxia-induced form of VDAC. This form, a C-terminal truncated protein (VDAC1-ΔC), was associated in some cancer cell lines with a high output of adenosine triphosphate and a strong resistance to chemotherapy-induced apoptosis. Furthermore, VDAC1-ΔC was detected in tissues of 50 % of 46 patients with lung cancer. This review examines the significance of this new form of VDAC1 for anticancer therapy.


Subject(s)
Biomarkers, Tumor , Mitochondria/genetics , Neoplasms/therapy , Voltage-Dependent Anion Channel 1/physiology , Animals , Apoptosis/genetics , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Cell Hypoxia/genetics , Humans , Mitochondria/metabolism , Neoplasms/genetics , Neoplasms/metabolism , Protein Isoforms/physiology
7.
Cell Death Dis ; 4: e544, 2013 Mar 14.
Article in English | MEDLINE | ID: mdl-23492775

ABSTRACT

The resistance of hypoxic cells to radiotherapy and chemotherapy is a major problem in the treatment of cancer. Recently, an additional mode of hypoxia-inducible factor (HIF)-dependent transcriptional regulation, involving modulation of a specific set of micro RNAs (miRNAs), including miR-210, has emerged. We have recently shown that HIF-1 induction of miR-210 also stabilizes HIF-1 through a positive regulatory loop. Therefore, we hypothesized that by stabilizing HIF-1 in normoxia, miR-210 may protect cancer cells from radiation. We developed a non-small cell lung carcinoma (NSCLC)-derived cell line (A549) stably expressing miR-210 (pmiR-210) or a control miRNA (pmiR-Ctl). The miR-210-expressing cells showed a significant stabilization of HIF-1 associated with mitochondrial defects and a glycolytic phenotype. Cells were subjected to radiation levels ranging from 0 to 10 Gy in normoxia and hypoxia. Cells expressing miR-210 in normoxia had the same level of radioresistance as control cells in hypoxia. Under hypoxia, pmiR-210 cells showed a low mortality rate owing to a decrease in apoptosis, with an ability to grow even at 10 Gy. This miR-210 phenotype was reproduced in another NSCLC cell line (H1975) and in HeLa cells. We have established that radioresistance was independent of p53 and cell cycle status. In addition, we have shown that genomic double-strand breaks (DSBs) foci disappear faster in pmiR-210 than in pmiR-Ctl cells, suggesting that miR-210 expression promotes a more efficient DSB repair. Finally, HIF-1 invalidation in pmiR-210 cells removed the radioresistant phenotype, showing that this mechanism is dependent on HIF-1. In conclusion, miR-210 appears to be a component of the radioresistance of hypoxic cancer cells. Given the high stability of most miRNAs, this advantage could be used by tumor cells in conditions where reoxygenation has occurred and suggests that strategies targeting miR-210 could enhance tumor radiosensitization.


Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Gene Expression Regulation, Neoplastic/radiation effects , Hypoxia-Inducible Factor 1/genetics , Hypoxia/genetics , Lung Neoplasms/genetics , MicroRNAs/genetics , Apoptosis/radiation effects , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Hypoxia/genetics , Cell Hypoxia/radiation effects , Cell Line, Tumor , DNA Breaks, Double-Stranded , DNA Repair , Gamma Rays , Humans , Hypoxia/metabolism , Hypoxia/pathology , Hypoxia-Inducible Factor 1/metabolism , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , MicroRNAs/metabolism , Mitochondria/genetics , Mitochondria/metabolism , Mitochondria/radiation effects , Radiation Tolerance , Signal Transduction/radiation effects , Transcription, Genetic/radiation effects
8.
Oncogene ; 31(24): 2989-3001, 2012 Jun 14.
Article in English | MEDLINE | ID: mdl-22002313

ABSTRACT

We showed previously that factor-inhibiting hypoxia-inducible factor HIF (FIH) monitors the expression of a spectrum of genes that are dictated by the cell's partial oxygen pressure. This action is mediated by the C-TAD, one of two transactivation domains (TADs) of the hypoxia-inducible factor. Here, we questioned: (1) the function of FIH as a HIF-1 modulator of gene expression in the context of a physiological oxygen gradient occurring in three-dimensional cultures and in tumors and (2) the role of FIH as a modulator of the growth of human tumor cells. We first showed that the expression pattern of HIF target genes that depend on the C-TAD, such as carbonic anhydrase IX, was spacially displaced to more oxygenated areas when FIH was silenced, whereas overexpression of FIH restricted this pattern to more hypoxic areas. Second, we showed that silencing fih severely reduced in vitro cell proliferation and in vivo tumor growth of LS174 colon adenocarcinoma and A375 melanoma cells. Finally, silencing of fih significantly increased both the total and phosphorylated forms of the tumor suppressor p53, leading to an increase in its direct target, the cell cycle inhibitor p21. Moreover, p53-deficient or mutant cells were totally insensitive to FIH expression. Thus, FIH activity is essential for tumor growth through the suppression of the p53-p21 axis, the major barrier that prevents cancer progression.


Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/metabolism , Neoplasms/pathology , Tumor Suppressor Protein p53/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Cell Culture Techniques , Cell Line, Tumor , Cell Proliferation , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Gene Expression Regulation, Neoplastic , Gene Silencing , Humans , Melanoma/genetics , Melanoma/metabolism , Melanoma/pathology , Mixed Function Oxygenases/genetics , Neoplasms/genetics , Neoplasms/metabolism , Oxygen/metabolism , Repressor Proteins/genetics
9.
Bull Cancer ; 98(5): 40-6, 2011 May.
Article in English | MEDLINE | ID: mdl-21609892

ABSTRACT

Mitochondria originated from a distant ancestor: the α-proteobacteria. They evolved over millions of years in a symbiotic relationship in eukaryotic cells by favoring consumption of oxygen by the electron transport chain with production of ATP. Contemporary mitochondria still play a crucial role in providing energy but also in apoptosis. Because of this symbiotic relationship and their pivotal function, mitochondria undoubtedly participate in tumorigenesis. Genetic defects in mitochondrial DNA, blockade of oxidative phosphorylation and mitophagy in tumor cells modify the production of damaging reactive oxygen species and restrain apoptosis. As the environment of tumor cells becomes more and more hypoxic, the hypoxia-inducible factor (HIF) is stabilized and participates in the reprogramming of cell metabolism. Recently, we became interested in asking whether HIF and hypoxia affect mitochondrial function. In this review, we show that hypoxia induces enlargement of mitochondria, due to abnormal fusion, which results in resistance to apoptosis and thus in survival. The role of hypoxia-induced BNIP3 and BNIP3L, proteins recently described as pro-survival proteins, in survival is also discussed.


Subject(s)
Apoptosis/physiology , Cell Hypoxia/physiology , Hypoxia-Inducible Factor 1/physiology , Mitochondria/physiology , Neoplasms/etiology , Adenosine Triphosphate/biosynthesis , Cell Death/physiology , Disease Progression , Humans , Hypoxia-Inducible Factor 1/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Size , Mutation
10.
Cell Death Differ ; 18(3): 465-78, 2011 Mar.
Article in English | MEDLINE | ID: mdl-20885442

ABSTRACT

Following the identification of a set of hypoxia-regulated microRNAs (miRNAs), recent studies have highlighted the importance of miR-210 and of its transcriptional regulation by the transcription factor hypoxia-inducible factor-1 (HIF-1). We report here that miR-210 is overexpressed at late stages of non-small cell lung cancer. Expression of miR-210 in lung adenocarcinoma A549 cells caused an alteration of cell viability associated with induction of caspase-3/7 activity. miR-210 induced a loss of mitochondrial membrane potential and the apparition of an aberrant mitochondrial phenotype. The expression profiling of cells overexpressing miR-210 revealed a specific signature characterized by enrichment for transcripts related to 'cell death' and 'mitochondrial dysfunction', including several subunits of the electron transport chain (ETC) complexes I and II. The transcript coding for one of these ETC components, SDHD, subunit D of succinate dehydrogenase complex (SDH), was validated as a bona fide miR-210 target. Moreover, SDHD knockdown mimicked miR-210-mediated mitochondrial alterations. Finally, miR-210-dependent targeting of SDHD was able to activate HIF-1, in line with previous studies linking loss-of-function SDH mutations to HIF-1 activation. miR-210 can thus regulate mitochondrial function by targeting key ETC component genes with important consequences on cell metabolism, survival and modulation of HIF-1 activity. These observations help explain contradictory data regarding miR-210 expression and its putative function in solid tumors.


Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Lung Neoplasms/genetics , MicroRNAs/metabolism , Mitochondria/pathology , Apoptosis , Carcinoma, Non-Small-Cell Lung/enzymology , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/ultrastructure , Caspase 3/metabolism , Caspase 7/metabolism , Cell Hypoxia/genetics , Cell Line, Tumor , Cell Survival/genetics , Down-Regulation/genetics , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Lung Neoplasms/enzymology , Lung Neoplasms/pathology , Lung Neoplasms/ultrastructure , MicroRNAs/antagonists & inhibitors , MicroRNAs/genetics , Mitochondria/enzymology , Mitochondria/ultrastructure , Mitochondrial Proteins/metabolism , Neoplasm Staging , Phenotype , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Succinate Dehydrogenase/metabolism , Up-Regulation/genetics
11.
Br J Cancer ; 102(11): 1627-35, 2010 May 25.
Article in English | MEDLINE | ID: mdl-20461082

ABSTRACT

BACKGROUND: Carbonic anhydrase IX (CAIX) is an enzyme upregulated by hypoxia during tumour development and progression. This study was conducted to assess if the expression of CAIX in tumour tissue and/or plasma can be a prognostic factor in patients with non-small cell lung cancer (NSCLC). METHODS: Tissue microarrays containing 555 NSCLC tissue samples were generated for quantification of CAIX expression. The plasma level of CAIX was determined by ELISA in 209 of these NSCLC patients and in 58 healthy individuals. The CAIX tissue immunostaining and plasma levels were correlated with clinicopathological factors and patient outcome. RESULTS: CAIX tissue overexpression correlated with shorter overall survival (OS) (P=0.05) and disease-specific survival (DSS) of patients (P=0.002). The CAIX plasma level was significantly higher in patients with NSCLC than in healthy individuals (P<0.001). A high level of CAIX in the plasma of patients was associated with shorter OS (P<0.001) and DSS (P<0.001), mostly in early stage I+II NSCLC. Multivariate Cox analyses revealed that high CAIX tissue expression (P=0.002) was a factor of poor prognosis in patients with resectable NSCLC. In addition, a high CAIX plasma level was an independent variable predicting poor OS (P<0.001) in patients with NSCLC. CONCLUSION: High expression of CAIX in tumour tissue is a predictor of worse survival, and a high CAIX plasma level is an independent prognostic biomarker in patients with NSCLC, in particular in early-stage I+II carcinomas.


Subject(s)
Antigens, Neoplasm/blood , Antigens, Neoplasm/metabolism , Carbonic Anhydrases/blood , Carbonic Anhydrases/metabolism , Carcinoma, Non-Small-Cell Lung/diagnosis , Lung Neoplasms/diagnosis , Adult , Aged , Biomarkers, Tumor/blood , Biomarkers, Tumor/metabolism , Carbonic Anhydrase IX , Carcinoma, Non-Small-Cell Lung/blood , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/mortality , Cell Hypoxia/physiology , Cell Proliferation , Cells, Cultured , Female , Humans , Immunohistochemistry , Lung Neoplasms/blood , Lung Neoplasms/metabolism , Lung Neoplasms/mortality , Male , Middle Aged , Prognosis , Tissue Array Analysis , Up-Regulation
12.
Curr Pharm Des ; 9(7): 531-41, 2003.
Article in English | MEDLINE | ID: mdl-12570801

ABSTRACT

In the last few decades it has become clear that detailed understanding of the mechanisms of angiogenesis, a process leading to growth of new blood vessels, should lead to improved treatment of diseases such as ischemic disorders and cancer where neovascularization is impaired or activated, respectively. In this review, we will outline some of our recent findings concerning the regulation of the vascular endothelial growth factor (VEGF), a key player in angiogenesis and one of its transcription factors, the hypoxia-inducible factor-1 (HIF-1) a master gene product driving adaptation to hypoxia. We will discuss the observation that growth factors and oncogenic transformation via the mitogen-activated protein kinases p42/p44 MAPKs not only activate the VEGF promoter through the Sp1/AP-2 transcriptional factor complex but also phosphorylate HIF-1alpha leading in turn to enhance HIF-1 dependent transcriptional activation of VEGF. The stress-activated protein kinases (SAPK) also contribute to angiogenesis by stabilizing VEGF mRNA. Finally, we will present recent advances into oxygen-sensing, in particular the HIF-hydroxylases that govern HIF-1alpha instability (PHD2) or inactivation (FIH-1). The revelation of these oxygen sensors has provided pharmacologists with new molecular targets for the development of novel therapies to control angiogenesis either positively or negatively.


Subject(s)
DNA-Binding Proteins/physiology , Endothelial Growth Factors/physiology , Intercellular Signaling Peptides and Proteins/physiology , Lymphokines/physiology , Mitogen-Activated Protein Kinases/physiology , Neovascularization, Pathologic/physiopathology , Neovascularization, Physiologic/physiology , Nuclear Proteins/physiology , Cell Hypoxia/physiology , DNA-Binding Proteins/genetics , Endothelial Growth Factors/genetics , Endothelium/enzymology , Gene Expression/physiology , Humans , Hypoxia-Inducible Factor 1 , Hypoxia-Inducible Factor 1, alpha Subunit , Intercellular Signaling Peptides and Proteins/genetics , Lymphokines/genetics , Nuclear Proteins/genetics , Transcription Factors/genetics , Transcription Factors/physiology , Transcriptional Activation , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors
13.
Tumour Biol ; 22(5): 310-7, 2001.
Article in English | MEDLINE | ID: mdl-11553861

ABSTRACT

The liver is one of the organs in which hypoxia helps to regulate gene expression under normal physiological conditions and in diseases such as cirrhosis and cancer. We postulated that the expression/activity of some of the 'liver-enriched' transcription factors, which control liver-specific genes, was sensitive to hypoxia. We tested hepatocyte nuclear factor-1 (HNF-1), HNF-3 and HNF-4, which play key roles in differentiation, development and hepatic gene expression, using HepG2 human hepatoma cells cultured under hypoxic conditions. Severe hypoxia/anoxia downregulated HNF-4 DNA-binding activity while DNA-binding activity of HNF-1 and HNF-3 remained unaffected. These hypoxic conditions also strongly and specifically decreased cell contents of HNF-4 protein, indicating that the decrease in HNF-4 DNA-binding activity was due to the lower amount of protein and not to decreased DNA-binding affinity. Northern analysis indicated that the expression of the hnf-4 gene was also downregulated in HepG2 cells cultured under hypoxic conditions. These results provide evidence that hypoxic stress triggers a cascade of events that inhibits the transactivation potential of HNF-4 in HepG2 cells. This step may be crucial in modulating the expression of a subset of liver genes that are targets for this nuclear receptor. This relationship provides a new route for the investigation of the effects of hypoxia on the liver cell.


Subject(s)
Carcinoma, Hepatocellular/genetics , Cell Hypoxia/physiology , DNA-Binding Proteins , Gene Expression Regulation, Neoplastic , Liver Neoplasms/genetics , Phosphoproteins/genetics , Transcription Factors/genetics , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Biomarkers, Tumor/analysis , Biomarkers, Tumor/genetics , Hepatocyte Nuclear Factor 1 , Hepatocyte Nuclear Factor 1-alpha , Hepatocyte Nuclear Factor 1-beta , Hepatocyte Nuclear Factor 4 , Humans , Nuclear Proteins/metabolism , Phosphoproteins/analysis , Transcription Factors/analysis , Tumor Cells, Cultured
14.
Cancer Res ; 61(6): 2429-33, 2001 Mar 15.
Article in English | MEDLINE | ID: mdl-11289110

ABSTRACT

Hypoxia initiates numerous intracellular signaling pathways important in regulating cell proliferation, differentiation, and death. In this study, we investigated the pathway that hypoxia uses to activate Akt and inactivate glycogen synthase kinase-3 (GSK-3), two proteins the functions of which are important in cell survival and energy metabolism. Severe hypoxia (0.01% oxygen) initiated a signaling cascade by inducing the tyrosine phosphorylation of the platelet-derived growth factor (PDGF) receptor within 1 h of treatment and increasing receptor association with the p85 subunit of phosphatidylinositol 3-kinase (PI 3-K). Hypoxia-induced signaling also resulted in PI 3-K-dependent phosphorylation of Akt on Ser-473, a modification of Akt that is important for its activation. This activation of Akt by hypoxia was substantially diminished in cells that possessed mutations in their PDGF receptor-PI 3-K interaction domain. In addition, Akt activation by hypoxia was resistant to treatment with the growth factor receptor poison suramin but was sensitive to treatment with the PI 3-K inhibitor wortmannin. Activation of Akt by hypoxia resulted in the phosphorylation of GSK-3alpha and GSK-3beta at Ser-9 and Ser-21, two well-documented Akt phosphorylation sites, respectively, that are inactivating modifications of each GSK-3 isoform. In support of the phosphorylation data, GSK-3 activity was significantly reduced under hypoxia. In conclusion, we propose that hypoxia activates a growth factor receptor/PI 3-K/Akt cascade that leads to GSK-3 inactivation, a pathway that can impact cell survival, proliferation, and metabolism.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Oxygen/physiology , Phosphatidylinositol 3-Kinases/metabolism , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/metabolism , Receptor, Platelet-Derived Growth Factor beta/metabolism , Signal Transduction/physiology , Androstadienes/pharmacology , Animals , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Cell Hypoxia/physiology , Cell Line , Dogs , Enzyme Activation , Enzyme Inhibitors/pharmacology , Epithelial Cells/enzymology , Fibrosarcoma/enzymology , Glycogen Synthase Kinase 3 , Glycogen Synthase Kinases , Humans , Kidney/cytology , Kidney/enzymology , Phosphorylation , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins c-akt , Signal Transduction/drug effects , Tumor Cells, Cultured , Tyrosine/metabolism , Wortmannin
15.
Proc Natl Acad Sci U S A ; 95(17): 10188-93, 1998 Aug 18.
Article in English | MEDLINE | ID: mdl-9707622

ABSTRACT

IGFBP-1 is elevated in fetuses with long-term, chronic hypoxia and intrauterine growth restriction. We investigated the hypothesis that hypoxia regulates IGFBP-1 in the human fetus in vivo and IGFBP-1 gene expression and protein in vitro. Umbilical artery IGFBP-1 levels (mean +/- SEM) from term babies with respiratory acidosis (acute hypoxia), normal babies, and those with mixed respiratory/metabolic acidosis (more profound and prolonged hypoxia) were measured using an immunoradiometric assay. IGFBP-1 levels were similar in normal (n = 12) and acutely hypoxic (n = 6) babies (189.1 +/- 71.8 vs. 175.8 +/- 45.9 ng /ml, respectively, P = 0.789). However, with more profound and prolonged hypoxia (n = 19), IGFBP-1 levels were markedly elevated (470.6 +/- 80.0 ng /ml, P = 0.044). To investigate IGFBP-1 regulation by hypoxia in vitro, HepG2 cells were incubated under hypoxia (pO2 = 2%) and normoxia (pO2 = 20%). IGFBP-1 protein and mRNA increased 8- and 12-fold, respectively, under hypoxic conditions. Hypoxia did not affect protein or mRNA levels of IGFBP-2 or -4. IGFBP-5 and -6 mRNAs, undetectable in control cells, were not induced by hypoxia, whereas minimally expressed IGFBP-3 mRNA increased twofold. Investigation into IGFBP-1 gene structure revealed three potential consensus sequences for the hypoxia response element (HRE) in the first intron. To investigate functionality, a 372-bp fragment of IGFBP-1 intron 1, containing putative HREs, was placed 5' to a heterologous hsp70 promoter in a plasmid using luciferase as a reporter gene. Under hypoxia, reporter gene activity increased up to 30-fold. Mutations in the middle HRE abolished reporter activity in response to hypoxia, suggesting that this HRE is functional in the IGFBP-1 hypoxia response. Cotransfection of HRE reporter genes with a constitutively expressing hypoxia-inducible factor 1 plasmid in HepG2 cells resulted in a fourfold induction of reporter activity, suggesting a role for hypoxia-inducible factor 1 in hypoxia induction of IGFBP-1 gene expression. These data support the hypothesis that hypoxia regulation of IGFBP-1 may be a mechanism operating in the human fetus to restrict insulin-like growth factor-mediated growth in utero under conditions of chronic hypoxia and limited substrate availability.


Subject(s)
Cell Hypoxia/genetics , Fetal Hypoxia/genetics , Insulin-Like Growth Factor Binding Protein 1/genetics , Models, Biological , Animals , Base Sequence , Cell Line , Consensus Sequence , DNA Primers/genetics , Embryonic and Fetal Development/genetics , Embryonic and Fetal Development/physiology , Female , Fetal Blood/metabolism , Fetal Growth Retardation/etiology , Fetal Growth Retardation/genetics , Fetal Growth Retardation/physiopathology , Fetal Hypoxia/complications , Fetal Hypoxia/physiopathology , Gene Expression , Genes, Reporter , Humans , Infant, Newborn , Insulin-Like Growth Factor Binding Protein 1/blood , Insulin-Like Growth Factor Binding Protein 1/physiology , Mice , Pregnancy , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Transfection
16.
Blood ; 90(9): 3322-31, 1997 Nov 01.
Article in English | MEDLINE | ID: mdl-9345014

ABSTRACT

Tumor angiogenesis, the development of new blood vessels, is a highly regulated process that is controlled genetically by alterations in oncogene and tumor suppressor gene expression and physiologically by the tumor microenvironment. Previous studies indicate that the angiogenic switch in Ras-transformed cells may be physiologically promoted by the tumor microenvironment through the induction of the angiogenic mitogen, vascular endothelial growth factor (VEGF). In this report, we show Ras-transformed cells do not use the downstream effectors c-Raf-1 or mitogen activated protein kinases (MAPK) in signaling VEGF induction by hypoxia as overexpression of kinase-defective alleles of these genes does not inhibit VEGF induction under low oxygen conditions. In contrast to the c-Raf-1/MAP kinase pathway, hypoxia increases phosphatidylinositol 3-kinase (PI 3-kinase) activity in a Ras-dependent manner, and inhibition of PI 3-kinase activity genetically and pharmacologically results in inhibition of VEGF induction. We propose that hypoxia modulates VEGF induction in Ras-transformed cells through the activation of a stress inducible PI 3-kinase/Akt pathway and the hypoxia inducible factor-1 (HIF-1) transcriptional response element.


Subject(s)
Cell Transformation, Neoplastic , DNA-Binding Proteins/physiology , Endothelial Growth Factors/physiology , Genes, ras , Lymphokines/physiology , Neovascularization, Pathologic , Nuclear Proteins/physiology , Phosphatidylinositol 3-Kinases/physiology , Signal Transduction , 3T3 Cells , Animals , Cell Hypoxia , Gene Expression Regulation , Hypoxia-Inducible Factor 1 , Hypoxia-Inducible Factor 1, alpha Subunit , Mice , Transcription Factors/physiology , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors
17.
Cancer Res ; 56(15): 3436-40, 1996 Aug 01.
Article in English | MEDLINE | ID: mdl-8758908

ABSTRACT

Hypoxia can select for cells that have lost their apoptotic potential, thereby making them resistant to adverse conditions. However, long-term survival of transformed cells which have diminished apoptotic sensitivity when exposed to low oxygen conditions would require the activation of their angiogenic program to compensate for an insufficient oxygen supply. In this report, we show that the activity (of oncogenic Ha-ras, either constitutively or transiently, enhances the induction of the angiogenic mitogen, vascular endothelial growth factor (VEGF), by hypoxia. Analysis of the 5' flanking region of the VEGF promoter indicates that a HIF-1-like sequence is to promote a 15-fold increase in reporter gene activity in Ha-ras-transformed cells when exposed to hypoxia, whereas mutations in the same site totally inhibited VEGF induction. Under low oxygen conditions, VEGF induction is inhibited in cells expressing a mutant inhibitory allele of Ha-ras (RasN17), indicating a direct role for Ras in modulating VEGF activity. We propose that the angiogenic switch in Ras-transformed cells may be physiologically promoted by the tumor microenvironment through VEGF induction.


Subject(s)
Cell Transformation, Neoplastic/genetics , Endothelial Growth Factors/biosynthesis , Genes, ras , Lymphokines/biosynthesis , 3T3 Cells/metabolism , 3T3 Cells/physiology , Animals , Cell Hypoxia/physiology , Endothelial Growth Factors/genetics , Gene Expression , Lymphokines/genetics , Mice , Promoter Regions, Genetic , RNA, Messenger/biosynthesis , Rats , Stress, Physiological/metabolism , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , ras Proteins/biosynthesis , ras Proteins/physiology
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