Ras-dependent carbon metabolism and transformation in mouse fibroblasts

Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy.
Oncogene (Impact Factor: 8.46). 09/2006; 25(39):5391-404. DOI: 10.1038/sj.onc.1209528
Source: PubMed


Mutational activation of ras genes is required for the onset and maintenance of different malignancies. Here we show, using a combination of molecular physiology, nutritional perturbations and transcriptional profiling, that full penetrance of phenotypes related to oncogenic Ras activation, including the shift of carbon metabolism towards fermentation and upregulation of key cell cycle regulators, is dependent upon glucose availability. These responses are induced by Ras activation, being specifically reverted by downregulation of the Ras pathway obtained through the expression of a dominant-negative Ras-specific guanine nucleotide exchange protein. Our data allow to link directly to ras activation the alteration in energy metabolism of cancer cells, their fragility towards glucose shortage and ensuing apoptotic death.

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Available from: Ferdinando Chiaradonna, May 15, 2014
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    • "Intracellular ATP levels were measured using CellTiter Glo luciferin-luciferase assay (Promega, Madison, WI, USA) as described in [23]. Mitochondrial potential was analyzed by staining cells with 20 nM JC-1 (5,5 í® í° ,6,6 í® í° -tetrachloro-1,1 í® í° ,3,3 í® í° -tetraethylbenzimidazolylcarbocyanine iodide, Life Technologies) for 10 minutes. "
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    ABSTRACT: Cancer cells generally rely mostly on glycolysis rather than oxidative phosphorylation (OXPHOS) for ATP production. In fact, they are particularly sensitive to glycolysis inhibition and glucose depletion. On the other hand mitochondrial dysfunctions, involved in the onset of the Warburg effect, are sometimes also associated with the resistance to apoptosis that characterizes cancer cells. Therefore, combined treatments targeting both glycolysis and mitochondria function, exploiting peculiar tumor features, might be lethal for cancer cells. In this study, we show that glucose deprivation and mitochondrial Complex I inhibitors synergize in inducing cancer cell death. In particular, our results reveal that low doses of Complex I inhibitors, ineffective on immortalized cells and in high glucose growth, become specifically cytotoxic on cancer cells deprived of glucose. Importantly, the cytotoxic effect of the inhibitors on cancer cells is strongly enhanced by forskolin, a PKA pathway activator, that we have previously shown to stimulate OXPHOS. Taken together, we demonstrate that induction in cancer cells of a switch from a glycolytic to a more respirative metabolism, obtained by glucose depletion or mitochondrial activity stimulation, strongly increases their sensitivity to low doses of mitochondrial Complex I inhibitors. Our findings might be a valuable approach to eradicate cancer cells.
    International Journal of Cell Biology 04/2013; 2013(1):243876. DOI:10.1155/2013/243876
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    • "In order to critically analyze the molecular basis of the change of carbon metabolism in cancer cells, we compared two cell lines that had been previously characterized (Chiaradonna et al. 2006a, b ) , that is NIH3T3 mouse fi broblasts and NIH3T3 cells transformed by an activated form of the K-ras oncogene. Ras proteins are intracellular switches whose activation state (i.e. "
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    ABSTRACT: Oxidative stress is among the major causes of toxicity due to interaction of Reactive Oxygen Species (ROS) with cellular macromolecules and structures and interference with signal transduction pathways. The mitochondrial respiratory chain, specially from Complexes I and III, is considered the main origin of ROS particularly under conditions of high membrane potential, but several other sources may be important for ROS generation, such as mitochondrial p66(Shc), monoamine oxidase, α-ketoglutarate dehydogenase, besides redox cycling of redox-active molecules. ROS are able to oxidatively modify lipids, proteins, carbohydrates and nucleic acids in mitochondria and to activate/inactivate signalling pathways by oxidative modification of redox-active factors. Cells are endowed with several defence mechanisms including repair or removal of damaged molecules, and antioxidant systems, either enzymatic or non-enzymatic. Oxidative stress is at the basis of ageing and many pathological disorders, such as ischemic diseases, neurodegenerative diseases, diabetes, and cancer, although the underlying mechanisms are not always completely understood.
    Advances in Experimental Medicine and Biology 01/2012; 942:93-136. DOI:10.1007/978-94-007-2869-1_5 · 1.96 Impact Factor
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    • "To directly assess the role of oncogenic K-Ras in the previously described metabolic alterations of transformed cells, we performed several metabolic analyses by taking advantage of a cell line, NIH3T3-GEF-DN (reverted cells, R), stably expressing a dominant-negative Guanine Exchange Factor CDC25 (GEF-DN) that specifically attenuates oncogenic K-Ras activation (Vanoni et al, 1999; Bossu et al, 2000). Notably, R cells exhibit changes consistent with a regression of cellular transformation, including Ras-GTP levels, morphology, anchorage-independent growth, reduction of Ras-dependent tumor formation in nude mice, glucose and glutamine dependence and mitochondrial dysfunction to a phenotype similar to that observed in N cells (Chiaradonna et al, 2006b; Gaglio et al, 2009). "
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    ABSTRACT: Oncogenes such as K-ras mediate cellular and metabolic transformation during tumorigenesis. To analyze K-Ras-dependent metabolic alterations, we employed 13 C metabolic flux analysis (MFA), non-targeted tracer fate detection (NTFD) of 15 N-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. [-] [-].
    Molecular Systems Biology 08/2011; 7(1):523. DOI:10.1038/msb.2011.56 · 10.87 Impact Factor
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