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      Oncogenic K-Ras decouples glucose and glutamine metabolism to support cancer cell growth

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          Abstract

          • A systems approach using 13C metabolic flux analysis (MFA), non-targeted tracer fate detection (NTFD), and transcriptional profiling was applied to investigate the role of oncogenic K-Ras in metabolic transformation.

          • K-Ras transformed cells exhibit an increased glycolytic rate and lower flux through the oxidative tricarboxylic acid (TCA) cycle.

          • K-Ras transformed cells show a relative increase in glutamine anaplerosis and reductive TCA metabolism.

          • Transcriptional changes driven by oncogenic K-Ras suggest control nodes associated with the metabolic reprogramming of cancer cells.

          Abstract

          The ras and myc oncogenes drive pleiotropic changes in cell signaling, nutrient uptake, and intracellular metabolism ( Chiaradonna et al, 2006b; Yuneva et al, 2007; Wise et al, 2008; Vander Heiden et al, 2009). Mutated ras proteins, identified in 25% of human cancers ( Bos, 1989; Downward, 2003), correlate with an increased rate of glucose consumption, lactate accumulation, altered expression of mitochondrial genes, increased ROS production, and reduced mitochondrial activity ( Bos, 1989; Downward, 2003; Vizan et al, 2005; Chiaradonna et al, 2006a; Yun et al, 2009; Baracca et al, 2010; Weinberg et al, 2010). Furthermore, K-Ras transformed cancer cells are dependent upon glucose and glutamine availability, since their withdrawal induces apoptosis and cell-cycle arrest, respectively ( Ramanathan et al, 2005; Telang et al, 2006; Yun et al, 2009). However, the precise metabolic effects downstream of oncogenic Ras signaling as well as the mechanisms by which intracellular glucose and glutamine metabolism change have not been completely elucidated.

          In this report, we have investigated the reprogramming of central carbon metabolism in cancer cells and its regulation by the K- ras oncogene, applying a systems level approach using 13C metabolic flux analysis (MFA), non-targeted tracer fate detection (NTFD), and transcriptional profiling. These data reveal a coordinated decoupling of glycolysis and the tricarboxylic acid (TCA) cycle. K-Ras transformed mouse and human cells exhibited a high glucose to lactate flux and relatively lower oxidative metabolism of pyruvate. Such changes were supported by increased expression of glycolytic genes as well as several pyruvate dehydrogenase kinases. In contrast to glucose, the contribution of glutamine carbon to TCA cycle intermediates through both oxidative and reductive metabolism was significantly increased upon K-Ras transformation. Despite this increase in glutamine anaplerosis, oxidative TCA flux was significantly decreased. Additionally, we observed elevated levels of glutamine-derived nitrogen in various biosynthetic metabolites in transformed cells, including amino acids, 5-oxoproline, and the nucleobase adenine. Consistent with these changes, we detected increased transcription of genes associated with glutamine metabolism and nucleotide biosynthesis in cells expressing oncogenic K-Ras.

          Taken together, these findings indicate an important role of oncogenic K-Ras in cancer cell metabolism. The observed decoupling of glucose and glutamine metabolism enables the efficient utilization of both carbon and nitrogen from glutamine for biosynthetic processes. In accord with these alterations, oncogenic K-Ras induces gene expression changes that may drive this metabolic reprogramming. Finally, these results may enable the identification of metabolic and transcriptional targets throughout the network and allow more effective cancer therapies.

          Abstract

          Oncogenes such as K- ras mediate cellular and metabolic transformation during tumorigenesis. To analyze K-Ras-dependent metabolic alterations, we employed 13C metabolic flux analysis (MFA), non-targeted tracer fate detection (NTFD) of 15N-labeled glutamine, and transcriptomic profiling in mouse fibroblast and human carcinoma cell lines. Stable isotope-labeled glucose and glutamine tracers and computational determination of intracellular fluxes indicated that cells expressing oncogenic K-Ras exhibited enhanced glycolytic activity, decreased oxidative flux through the tricarboxylic acid (TCA) cycle, and increased utilization of glutamine for anabolic synthesis. Surprisingly, a non-canonical labeling of TCA cycle-associated metabolites was detected in both transformed cell lines. Transcriptional profiling detected elevated expression of several genes associated with glycolysis, glutamine metabolism, and nucleotide biosynthesis upon transformation with oncogenic K-Ras. Chemical perturbation of enzymes along these pathways further supports the decoupling of glycolysis and TCA metabolism, with glutamine supplying increased carbon to drive the TCA cycle. These results provide evidence for a role of oncogenic K-Ras in the metabolic reprogramming of cancer cells.

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          Akt stimulates aerobic glycolysis in cancer cells.

          Cancer cells frequently display high rates of aerobic glycolysis in comparison to their nontransformed counterparts, although the molecular basis of this phenomenon remains poorly understood. Constitutive activity of the serine/threonine kinase Akt is a common perturbation observed in malignant cells. Surprisingly, although Akt activity is sufficient to promote leukemogenesis in nontransformed hematopoietic precursors and maintenance of Akt activity was required for rapid disease progression, the expression of activated Akt did not increase the proliferation of the premalignant or malignant cells in culture. However, Akt stimulated glucose consumption in transformed cells without affecting the rate of oxidative phosphorylation. High rates of aerobic glycolysis were also identified in human glioblastoma cells possessing but not those lacking constitutive Akt activity. Akt-expressing cells were more susceptible than control cells to death after glucose withdrawal. These data suggest that activation of the Akt oncogene is sufficient to stimulate the switch to aerobic glycolysis characteristic of cancer cells and that Akt activity renders cancer cells dependent on aerobic glycolysis for continued growth and survival.
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            Overexpression of hypoxia-inducible factor 1alpha in common human cancers and their metastases.

            Neovascularization and increased glycolysis, two universal characteristics of solid tumors, represent adaptations to a hypoxic microenvironment that are correlated with tumor invasion, metastasis, and lethality. Hypoxia-inducible factor 1 (HIF-1) activates transcription of genes encoding glucose transporters, glycolytic enzymes, and vascular endothelial growth factor. HIF-1 transcriptional activity is determined by regulated expression of the HIF-1alpha subunit. In this study, HIF-1alpha expression was analyzed by immunohistochemistry in 179 tumor specimens. HIF-1alpha was overexpressed in 13 of 19 tumor types compared with the respective normal tissues, including colon, breast, gastric, lung, skin, ovarian, pancreatic, prostate, and renal carcinomas. HIF-1alpha expression was correlated with aberrant p53 accumulation and cell proliferation. Preneoplastic lesions in breast, colon, and prostate overexpressed HIF-1alpha, whereas benign tumors in breast and uterus did not. HIF-1alpha overexpression was detected in only 29% of primary breast cancers but in 69% of breast cancer metastases. In brain tumors, HIF-1alpha immunohistochemistry demarcated areas of angiogenesis. These results provide the first clinical data indicating that HIF-1alpha may play an important role in human cancer progression.
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              Systematic variation in gene expression patterns in human cancer cell lines.

              We used cDNA microarrays to explore the variation in expression of approximately 8,000 unique genes among the 60 cell lines used in the National Cancer Institute's screen for anti-cancer drugs. Classification of the cell lines based solely on the observed patterns of gene expression revealed a correspondence to the ostensible origins of the tumours from which the cell lines were derived. The consistent relationship between the gene expression patterns and the tissue of origin allowed us to recognize outliers whose previous classification appeared incorrect. Specific features of the gene expression patterns appeared to be related to physiological properties of the cell lines, such as their doubling time in culture, drug metabolism or the interferon response. Comparison of gene expression patterns in the cell lines to those observed in normal breast tissue or in breast tumour specimens revealed features of the expression patterns in the tumours that had recognizable counterparts in specific cell lines, reflecting the tumour, stromal and inflammatory components of the tumour tissue. These results provided a novel molecular characterization of this important group of human cell lines and their relationships to tumours in vivo.
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                Author and article information

                Journal
                Mol Syst Biol
                Molecular Systems Biology
                Nature Publishing Group
                1744-4292
                2011
                16 August 2011
                16 August 2011
                : 7
                : 523
                Affiliations
                [1 ]simpleDepartment of Biotechnology and Biosciences, University of Milano-Bicocca , Milan, Italy
                [2 ]simpleDepartment of Chemical Engineering, Massachusetts Institute of Technology , Cambridge, MA, USA
                Author notes
                [a ]Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, Milan 20126, Italy. Tel.: +39 02 64483515; Fax: +39 02 64483519; lilia.alberghina@ 123456unimib.it
                [b ]Department of Chemical Engineering, Massachusetts Institute of Technology, Room 56-469C, 77 Massachusetts Ave, Cambridge, MA 02139, USA. Tel.: +1 617 258 0398; Fax: +1 617 253 3122; gregstep@ 123456mit.edu
                [*]

                These authors contributed equally to this work

                [†]

                Present address: Department of Bioengineering, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093, USA

                Article
                msb201156
                10.1038/msb.2011.56
                3202795
                21847114
                0c921c9a-c051-46c4-91db-0023ad6bcd85
                Copyright © 2011, EMBO and Macmillan Publishers Limited

                This is an open-access article distributed under the terms of the Creative Commons Attribution Noncommercial Share Alike 3.0 Unported License, which allows readers to alter, transform, or build upon the article and then distribute the resulting work under the same or similar license to this one. The work must be attributed back to the original author and commercial use is not permitted without specific permission.

                History
                : 29 October 2010
                : 08 July 2011
                Categories
                Article

                Quantitative & Systems biology
                ras,metabolic flux analysis,cancer,transcriptional analysis,metabolism

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