[en] Metabolic reprogramming is thought to play a key role in sustaining the survival and proliferation of cancer cells. These changes facilitate for example the uptake and release of nutrients required for nucleotide, protein and lipid synthesis necessary for macromolecule assembly and tumor growth. We applied a 2D-DIGE (Two-Dimensional Differential in-Gel Electrophoresis) quantitative proteomic analysis to characterize the proteomes of mouse astrocytes that underwent in vitro cancerous transformation, and of their normal counterparts. Metabolic reprogramming effects on enzymatic and structural protein expression as well as associated metabolites abundance were quantified. Using enzymatic activity measurements and zymography, we documented and confirmed several changes in abundance and activity of various isoenzymes likely to participate in metabolic reprogramming. We found that after transformation, the cells increase their expression of glycolytic enzymes, thus augmenting their ability to use aerobic glycolysis (Warburg effect). An increased capacity to dispose of reducing equivalents through lactate production was also documented. Major effects on carbohydrates, amino acids and nucleotides metabolic enzymes were also observed. Conversely, the transformed cells reduced their enzymatic capacity for reactions of tricarboxylic acid oxidation, for neurotransmitter (glutamate) metabolism, for oxidative stress defense and their expression of astroglial markers. BIOLOGICAL SIGNIFICANCE: The use of a global approach based on a 2D DIGE analysis allows obtaining a comprehensive view of the metabolic reprogramming undergone by astrocytes upon cancerous transformation. Indeed, except for a few enzymes such as pyruvate carboxylase and glutaminase that were not detected in our initial analysis, pertinent information on the abundance of most enzymes belonging to pathways relevant to metabolic reprogramming was directly obtained. In this in vitro model, transformation causes major losses of astrocyte-specific proteins and functions and the acquisition of metabolic adaptations that favor intermediate metabolites production for increased macromolecule biosynthesis. Thus our approach appears to be readily applicable for the investigation of changes in protein abundance that determine various transformed cell phenotypes. It could similarly be applied to the evaluation of the effects of treatments aimed at correcting the consequences of cell transformation.
Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: themetabolic requirements of cell proliferation. Science 2009;324:1029-33.
Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144:646-74.
Koppenol WH, Bounds PL, Dang CV. Otto Warburg's contributions to current concepts of cancer metabolism. Nat Rev Cancer 2011;11:325-37.
Warburg O. On the origin of cancer cells. Science 1956;123: 309-14.
Warburg O, Wind F, Negelein E. The metabolism of tumors in the body. J Gen Physiol 1927;8:519-30.
DeBerardinis RJ, Mancuso A, Daikhin E, Nissim I, YudkoffM, Wehrli S, et al. Beyond aerobic glycolysis: transformed cells can engage in glutamine metabolism that exceeds the requirement for protein and nucleotide synthesis. Proc Natl Acad Sci U S A 2007;104:19345-50.
Lunt SY, Vander Heiden MG. Aerobic glycolysis: meeting the metabolic requirements of cell proliferation. Annu Rev Cell Dev Biol 2011;27:441-64.
Schulze A, Harris AL. How cancer metabolism is tuned for proliferation and vulnerable to disruption. Nature 2012;491: 364-73.
Soga T. Cancer metabolism: key players in metabolic reprogramming. Cancer Sci 2013;104:275-81.
Bouzier-Sore AK, Pellerin L. Unraveling the complex metabolic nature of astrocytes. Front CellNeurosci 2013;7:179.
Guerrero-Cazares H, Attenello FJ, Noiman L, Quiñones-Hinojosa A. Stem cells in gliomas. Handb Clin Neurol 2012;104:63-73.
Friedmann-Morvinski D, Bushong EA, Ke E, Soda Y, Marumoto T, Singer O, et al. Dedifferentiation of neurons and astrocytes by oncogenes can induce gliomas in mice. Science 2012;338:1080-4.
Pellerin L, Stolz M, Sorg O, Martin JL, Deschepper CF, Magistretti PJ. Regulation of energy metabolism by neurotransmitters in astrocytes in primary culture and in an immortalized cell line. Glia 1997;21:74-83.
Pellerin L, Magistretti PJ. Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization. Proc Natl Acad Sci U S A 1994;91:10625-9.
Martinez-Hernandez A, Bell KP, Norenberg MD. Glutamine synthetase: glial localization in brain. Science 1977;195: 1356-8.
San Martin A, Ceballo S, Ruminot I, Lerchundi R, Frommer WB, Barros LF, et al. A genetically encoded FRET lactate sensor and its use to detect the Warburg effect in single cancer cells. PLoS One 2013;8:e57712.
Unlu M, MorganME, Minden JS. Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 1997;18:2071-7.
Dufour C, Cadusseau J, Varlet P, Surena AL, de Faria GP, Dias-Morais A, et al. Astrocytes reverted to a neural progenitor-like state with transforming growth factor alpha are sensitized to cancerous transformation. Stem Cells 2009;27: 2373-82.
Sharif A, Legendre P, Prevot V, Allet C, Romao L, Studler JM, et al. Transforming growth factor alpha promotes sequential conversion of mature astrocytes into neural progenitors and stem cells. Oncogene 2007;26:2695-706.
Prevot V, Lomniczi A, Corfas G, Ojeda SR. erbB-1 and erbB-4 receptors act in concert to facilitate female sexual development and mature reproductive function. Endocrinology 2005;146:1465-72.
Mathy G, Cardol P, Dinant M, Blomme A, Gerin S, Ghysels B, et al. Proteomic and functional characterization of a Chlamydomonas reinhardtii mutant lacking the mitochondrial alternative oxidase 1. J Proteome Res 2010;9:2825-38.
Shevchenko A, Wilm M, Vorm O, Jensen ON, Podtelejnikov AV, Neubauer G, et al. A strategy for identifying gelseparated proteins in sequence databases by MS alone. Biochem Soc Trans 1996;24:893-6.
Romero-Calvo I, Ocón B, Martínez-Moya P, Suárez MD, Zarzuelo A, Martínez-Augustin O, et al. Reversible Ponceau staining as a loading control alternative to actin in Western blots. Anal Biochem 2010;401:318-20.
Aldridge GM, Podrebarac DM, GreenoughWT, Weiler IJ. The use of total protein stains as loading controls: an alternative to high-abundance single-protein controls in semi-quantitative immunoblotting. J Neurosci Methods 2008;172:250-4.
Board M, Humm S, Newsholme EA. Maximum activities of key enzymes of glycolysis, glutaminolysis, pentose phosphate pathway and tricarboxylic acid cycle in normal, neoplastic and suppressed cells. Biochem J 1990;265:503-9.
van der Helm H. A simplified method of demonstrating lactic dehydrogenase isoenzymes in serum. Clin Chim Acta 1962;7:124-8.
Malmqvist U, Arner A, Uvelius B. Lactate dehydrogenase activity and isoform distribution in normal and hypertrophic smooth muscle tissue from the rat. Pflugers Arch 1991;419:230-4.
Wellner VP, Meister A. Binding of adenosine triphosphate and adenosine diphosphate by glutamine synthetase. Biochemistry 1966;5:872-9.
Bentaib A, de Tullio P, Chneiweiss H, Hermans E, Junier MP, Leprince P. Data in support of metabolic reprogramming in transformed mouse cortical astrocytes: a proteomic study. Data in brief; 2014 1.
Noguchi T, Inoue H, Tanaka T. TheM1-andM2-type isozymes of rat pyruvate kinase are produced from the same gene by alternative RNA splicing. J Biol Chem 1986;261:13807-12.
Luo W, Semenza GL. Emerging roles of PKM2 in cell metabolism and cancer progression. Trends Endocrinol Metab 2012;23:560-6.
Eigenbrodt E, Reinacher M, Scheefers-Borchel U, Scheefers H, Friis R. Double role for pyruvate kinase type M2 in the expansion of phosphometabolite pools found in tumor cells. Crit Rev Oncog 1992;3:91-115.
Eigenbrodt EG H. Glycolysis-one of the keys to cancer? Trends Pharmacol Sci 1980;1:240-5.
Mazurek S, Michel A, Eigenbrodt E. Effect of extracellular AMP on cell proliferation and metabolism of breast cancer cell lines with high and low glycolytic rates. J Biol Chem 1997;272:4941-52.
Mazurek S, Zwerschke W, Jansen-Durr P, Eigenbrodt E. Effects of the human papilloma virus HPV-16 E7 oncoprotein on glycolysis and glutaminolysis: role of pyruvate kinase type M2 and the glycolytic-enzyme complex. Biochem J 2001;356:247-56.
Boros LG, Lee PWN, Brandes JL, Cascante M, Muscarella P, Schirmer WJ, et al. Nonoxidative pentose phosphate pathways and their direct role in ribose synthesis in tumors: is cancer a disease of cellular glucose metabolism? Med Hypotheses 1998;50:55-9.
Mazurek S, Boschek CB, Hugo F, Eigenbrodt E. Pyruvate kinase type M2 and its role in tumor growth and spreading. Semin Cancer Biol 2005;15:300-8.
Miccheli A, Tomassini A, Puccetti C, Valerio M, Peluso G, Tuccillo F, et al. Metabolic profiling by 13C-NMR spectroscopy: [1, 2-13C2]glucose reveals a heterogeneous metabolism in human leukemia T cells. Biochimie 2006;88:437-48.
Christofk HR, Vander Heiden MG, Harris MH, Ramanathan A, Gerszten RE, Wei R, et al. The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature 2008;452:230-3.
Mazurek S. Pyruvate kinase type M2: a key regulator of the metabolic budget system in tumor cells. Int J Biochem Cell Biol 2011;43:969-80.
Markert CL, Shaklee JB, Whitt GS. Evolution of a gene. Multiple genes for LDH isozymes provide a model of the evolution of gene structure, function and regulation. Science 1975;189:102-14.
Lee HK, Xiang C, Cazacu S, Finniss S, Kazimirsky G, Lemke N, et al. GRP78 is overexpressed in glioblastomas and regulates glioma cell growth and apoptosis. Neuro Oncol 2008;10: 236-43.
Fehon RG, McClatchey AI, Bretscher A. Organizing the cell cortex: the role of ERM proteins. Nat Rev Mol Cell Biol 2010; 11:276-87.
Grzendowski M, Wolter M, Riemenschneider MJ, Knobbe CB, Schlegel U, Meyer HE, et al. Differential proteome analysis of human gliomas stratified for loss of heterozygosity on chromosomal arms 1p and 19q. Neuro Oncol 2010;12:243-56.
Zhu X, Morales FC, Agarwal NK, Dogruluk T, Gagea M, Georgescu MM. Moesin is a glioma progression marker that induces proliferation andWnt/beta-catenin pathway activation via interaction with CD44. Cancer Res 2013;73:1142-55.
Eng LF, Ghirnikar RS, Lee YL. Glial fibrillary acidic protein: GFAPthirty-one years (1969-2000). Neurochem Res 2000;25:1439-51.
Tascos NA, Parr J, Gonatas NK. Immunocytochemical study of the glial fibrillary acidic protein in human neoplasms of the central nervous system. Hum Pathol 1982;13:454-8.
Velasco ME, Dahl D, Roessmann U, Gambetti P. Immunohistochemical localization of glial fibrillary acidic protein in human glial neoplasms. Cancer 1980;45:484-94.
Jacque CM, Kujas M, Poreau A, Raoul M, Collier P, Racadot J, et al. GFA and S 100 protein levels as an index for malignancy in human gliomas and neurinomas. J Natl Cancer Inst 1979;62:479-83.
van der Meulen JD, HouthoffHJ, Ebels EJ. Glial fibrillary acidic protein in human gliomas. Neuropathol Appl Neurobiol 1978;4:177-90.
Jacque CM, Vinner C, Kujas M, Raoul M, Racadot J, Baumann NA. Determination of glial fibrillary acidic protein (GFAP) in human brain tumors. J Neurol Sci 1978;35:147-55.
Kajiwara K, Orita T, Nishizaki T, Kamiryo T, Nakayama H, Ito H. Glial fibrillary acidic protein (GFAP) expression and nucleolar organizer regions (NORs) in human gliomas. Brain Res 1992;572:314-8.
Hara A, Sakai N, Yamada H, Niikawa S, Ohno T, Tanaka T, et al. Proliferative assessment of GFAP-positive and GFAPnegative glioma cells by nucleolar organizer region staining. Surg Neurol 1991;36:190-4.
Zheng L, Roeder RG, Luo Y. S phase activation of the histone H2B promoter by OCA-S, a coactivator complex that contains GAPDH as a key component. Cell 2003;114: 255-66.
Hsiao KC, Shih NY, Fang HL, Huang TS, Kuo CC, Chu PY. Surface alpha-enolase promotes extracellular matrix degradation and tumor metastasis and represents a new therapeutic target. PLoS One 2013;8:e69354.
Kim JW, Dang CV. Multifaceted roles of glycolytic enzymes. Trends Biochem Sci 2005;30:142-50.
Stetak A, Veress R, Ovadi J, Csermely P, Keri G, Ullrich A. Nuclear translocationof the tumormarkerpyruvate kinaseM2 induces programmed cell death. Cancer Res 2007;67:1602-8.
Yang W, Xia Y, Ji H, Zheng Y, Liang J, Huang W, et al. Nuclear PKM2 regulates beta-catenin transactivation upon EGFR activation. Nature 2011;480:118-22.
Bluemlein K, Grüning NM, Feichtinger RG, Lehrach H, Kofler B, Ralser M. No evidence for a shift in pyruvate kinase PKM1 to PKM2 expression during tumorigenesis. Oncotarget 2011; 2:393-400.
Vander Heiden MG, Locasale JW, Swanson KD, SharfiH, Heffron GJ, Amador-Noguez D, et al. Evidence for an alternative glycolytic pathway in rapidly proliferating cells. Science 2010;329:1492-9.
McKenna MC, Waagepetersen HS, Schousboe A, Sonnewald U. Neuronal and astrocytic shuttle mechanisms for cytosolic-mitochondrial transfer of reducing equivalents: current evidence and pharmacological tools. Biochem Pharmacol 2006;71:399-407.
Ross JM, Öberg J, Brené S, Coppotelli G, Terzioglu M, Pernold K, et al. High brain lactate is a hallmark of aging and caused by a shift in the lactate dehydrogenase A/B ratio. Proc Natl Acad Sci U S A 2010;107:20087-92.
Rabow L, Kristensson K. Changes in lactate dehydrogenase isoenzyme patterns in patients with tumours of the central nervous system.? Acta Neurochir (Wien) 1977;36:71-81.
Weinberg F, Chandel NS. Mitochondrial metabolism and cancer. Ann N Y Acad Sci 2009;1177:66-73.
Kondoh H, Lleonart ME, Bernard D, Gil J. Protection from oxidative stress by enhanced glycolysis; a possible mechanism of cellular immortalization. Histol Histopathol 2007;22: 85-90.
Sosa V, Moliné T, Somoza R, Paciucci R, Kondoh H, Lleonart ME. Oxidative stress and cancer: An overview. Ageing Res Rev 2013;12:376-90.
Cosentino C, Grieco D, Costanzo V. ATM activates the pentose phosphate pathway promoting anti-oxidant defence and DNA repair. EMBO J 2011;30:546-55.
Vaughn AE, Deshmukh M. Glucose metabolism inhibits apoptosis in neurons and cancer cells by redox inactivation of cytochrome c. Nat Cell Biol 2008;10:1477-83.
Kondoh H, Lleonart ME, Gil J, Wang J, Degan P, Peters G, et al. Glycolytic enzymes can modulate cellular life span. Cancer Res 2005;65:177-85.
Anastasiou D, Poulogiannis G, Asara JM, Boxer MB, Jiang JK, Shen M, et al. Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular antioxidant responses. Science 2011;334:1278-83.
Pelicano H, Xu RH, Du M, Feng L, Sasaki R, Carew JS, et al. Mitochondrial respiration defects in cancer cells cause activation of Akt survival pathway through a redoxmediated mechanism. J Cell Biol 2006;175:913-23.
Datta K, Babbar P, Srivastava T, Sinha S, Chattopadhyay P. p53 dependent apoptosis in glioma cell lines in response to hydrogen peroxide induced oxidative stress. Int J Biochem Cell Biol 2002;34:148-57.
Sies H. Strategies of antioxidant defense. Eur J Biochem 1993;215:213-9.
Linden A, Gülden M, Martin HJ, Maser E, Seibert H. Peroxideinduced cell death and lipid peroxidation in C6 glioma cells. Toxicol In Vitro 2008;22:1371-6.
Wood ZA, Poole LB, Karplus PA. Peroxiredoxin evolution and the regulation of hydrogen peroxide signaling. Science 2003; 300:650-3.
Wood ZA, Schröder E, Robin HJ, Poole LB. Structure, mechanism and regulation of peroxiredoxins. Trends Biochem Sci 2003;28:32-40.
Noh DY, Ahn SJ, Lee RA, Kim SW, Park IA, Chae HZ. Overexpression of peroxiredoxin in human breast cancer. Anticancer Res 2001;21:2085-90.
Yanagawa T, Ishikawa T, Ishii T, Tabuchi K, Iwasa S, Bannai S, et al. Peroxiredoxin I expression in human thyroid tumors. Cancer Lett 1999;145:127-32.
Kinnula VL, Lehtonen S, Sormunen R, Kaarteenaho-Wiik R, Kang SW, Rhee SG, et al. Overexpression of peroxiredoxins I, II, III, V, and VI in malignant mesothelioma. J Pathol 2002; 196:316-23.
Chang JW, Jeon HB, Lee JH, Yoo JS, Chun JS, Kim JH, et al. Augmented expression of peroxiredoxin I in lung cancer. Biochem Biophys Res Commun 2001;289:507-12.
Yanagawa T, Iwasa S, Ishii T, Tabuchi K, Yusa H, Onizawa K, et al. Peroxiredoxin I expression in oral cancer: a potential new tumor marker. Cancer Lett 2000;156:27-35.
Kim HJ, Chae HZ, Kim YJ, Kim YH, Hwangs TS, Park EM, et al. Preferential elevation of Prx I and Trx expression in lung cancer cells following hypoxia and in human lung cancer tissues. Cell Biol Toxicol 2003;19:285-98.
Kinnula VL, Paakko P, Soini Y. Antioxidant enzymes and redox regulating thiol proteins in malignancies of human lung. FEBS Lett 2004;569:1-6.
Odreman F, Vindigni M, Gonzales ML, Niccolini B, Candiano G, Zanotti B, et al. Proteomic studies on low-and high-grade human brain astrocytomas. J Proteome Res 2005;4:698-708.
Jarvela S, Rantala I, Rodriguez A, Kallio H, Parkkila S, Kinnula VL, et al. Specific expression profile and prognostic significance of peroxiredoxins in grade II-IV astrocytic brain tumors. BMC Cancer 2010;10:104.
Ali-Osman F, Stein DE, Renwick A. Glutathione content and glutathione-S-transferase expression in 1, 3-bis(2-chloroethyl)-1-nitrosourea-resistant human malignant astrocytoma cell lines. Cancer Res 1990;50: 6976-80.
Hara A, Yamada H, Sakai N, Hirayama H, Tanaka T, Mori H. Immunohistochemical demonstration of the placental form of glutathione S-transferase, a detoxifying enzyme in human gliomas. Cancer 1990;66:2563-8.
Evans CG, Bodell WJ, Tokuda K, Doane-Setzer P, Smith MT. Glutathione and related enzymes in rat brain tumor cell resistance to 1, 3-bis(2-chloroethyl)-1-nitrosourea and nitrogen mustard. Cancer Res 1987;47:2525-30.
Britten RA, Green JA, Warenius HM. Cellular glutathione (GSH) and glutathione S-transferase (GST) activity in human ovarian tumor biopsies following exposure to alkylating agents. Int J Radiat Oncol Biol Phys 1992;24:527-31.
Stoehlmacher J, Park DJ, Zhang W, Groshen S, Tsao-Wei DD, Yu MC, et al. Association between glutathione S-transferase P1, T1, and M1 genetic polymorphism and survival of patients with metastatic colorectal cancer. J Natl Cancer Inst 2002;94:936-42.
Bacolod MD, Johnson SP, Ali-Osman F, Modrich P, Bullock NS, Colvin OM, et al. Mechanisms of resistance to 1, 3-bis(2-chloroethyl)-1-nitrosourea in human medulloblastoma and rhabdomyosarcoma. Mol Cancer Ther 2002;1:727-36.
Usarek E, Kazmierczak B, Bojanowski K, Baranczyk-Kuzma A. Expression of glutathione S-transferase isoenzymes in human gliomas. Pol Merkur Lekarski 2005;18:676-9.
Traverso N, Ricciarelli R, Nitti M, Marengo B, Furfaro AL, Pronzato MA, et al. Role of Glutathione in Cancer Progression and Chemoresistance. Oxid Med Cell Longev 2013;2013: 10.
Shank RP, Bennett GS, Freytag SO, Campbell GL. Pyruvate carboxylase: an astrocyte-specific enzyme implicated in the replenishment of amino acid neurotransmitter pools. Brain Res 1985;329:364-7.
Gamberino WC, Berkich DA, Lynch CJ, Xu B, LaNoue KF. Role of pyruvate carboxylase in facilitation of synthesis of glutamate and glutamine in cultured astrocytes. J Neurochem 1997;69:2312-25.
Cheng T, Sudderth J, Yang C, Mullen AR, Jin ES, Mates JM, et al. Pyruvate carboxylase is required for glutamineindependent growth of tumor cells. Proc Natl Acad Sci U S A 2011;108:8674-9.
Cataldo AM, Broadwell RD. Cytochemical identification of cerebral glycogen and glucose-6-phosphatase activity under normal and experimental conditions. II. Choroid plexus and ependymal epithelia, endothelia and pericytes. J Neurocytol 1986;15:511-24.
Brahimi-Horn MC, Bellot G, Pouyssegur J. Hypoxia and energetic tumour metabolism. Curr Opin Genet Dev 2011;21: 67-72.
Pelletier J, Bellot G, Gounon P, Lacas-Gervais S, Pouyssegur J, Mazure NM. Glycogen synthesis is induced in hypoxia by the hypoxia-inducible factor and promotes cancer cell survival. Front Oncol 2012;2:18.
Atkins RJ, Dimou J, Paradiso L, MorokoffAP, Kaye AH, Drummond KJ, et al. Regulation of glycogen synthase kinase-3 beta (GSK-3beta) by the Akt pathway in gliomas. J Clin Neurosci 2012;19:1558-63.
Favaro E, Bensaad K, ChongMG, Tennant DA, Ferguson DJ, Snell C, et al. Glucose utilization via glycogen phosphorylase sustains proliferation and prevents premature senescence in cancer cells. Cell Metab 2012;16:751-64.
Rosati A, Marconi S, Pollo B, Tomassini A, Lovato L, Maderna E, et al. Epilepsy in glioblastomamultiforme: correlation with glutamine synthetase levels. J Neurooncol 2009;93:319-24.
Akimoto J. Immunohistochemical study of glutamine synthetase expression in normal human brain and intracranial tumors. No To Shinkei 1993;45:362-8.
Boza JJ, Moennoz D, Bournot CE, Blum S, Zbinden I, Finot PA, et al. Role of glutamine on the de novo purine nucleotide synthesis in Caco-2 cells. Eur J Nutr 2000;39:38-46.
Lieth E, LaNoue KF, Berkich DA, Xu B, Ratz M, Taylor C, et al. Nitrogen shuttling between neurons and glial cells during glutamate synthesis. J Neurochem 2001;76:1712-23.
Babu R, Eaton S, Drake DP, Spitz L, Pierro A. Glutamine and glutathione counteract the inhibitory effects of mediators of sepsis in neonatal hepatocytes. J Pediatr Surg 2001;36: 282-6.