The oncoprotein H-RasV12 increases mitochondrial metabolism
© Telang et al; licensee BioMed Central Ltd. 2007
Received: 24 August 2007
Accepted: 01 December 2007
Published: 01 December 2007
Neoplastic cells increase glycolysis in order to produce anabolic precursors and energy within the hypoxic environment of a tumor. Ras signaling is activated in several cancers and has been found to regulate metabolism by enhancing glycolytic flux to lactate. We examined the effects of sequential immortalization and H-RasV12-transformation of human bronchial epithelial cells on the anabolic fate of fully-labeled 13C-glucose-derived carbons using two-dimensional total correlated spectroscopic analysis-nuclear magnetic resonance spectroscopy (2D TOCSY-NMR).
We found that the introduction of activated H-RasV12 into immortalized human bronchial epithelial cells unexpectedly increased tricarboxylic acid cycle activity as measured by the direct conversion of 13C-glucose carbons into the anabolic substrates glutamate/glutamine, aspartate and uridine. We then observed that immortalization and H-RasV12-transformation of bronchial epithelial cells caused a stepwise increase in oxygen consumption, a global measure of electron transport chain activity. Importantly, ectopic expression of H-RasV12 sensitized immortalized cells to the ATP-depleting and cytotoxic effects of electron transport perturbation using the complex I inhibitor rotenone.
Taken together, these data indicate that the oncoprotein H-RasV12 increases mitochondrial metabolism and provide new rationale for the targeting of the tricarboxylic acid cycle and electron transport chain as anti-neoplastic strategies.
Biosynthesis of proteins, nucleic acids, lipids and complex carbohydrates requires coupling to the hydrolysis of nucleoside triphosphates (ATP and GTP in protein biosynthesis, CTP and UTP in lipid and carbohydrate biosynthesis), as well as the incorporation of carbon and nitrogen from metabolic precursors. Maintaining Na+/K+ ion gradients and other ion gradients for transport consumes a large fraction of the ATP generated in resting (G0/G1) cells, and activating macromolecule biosynthesis in preparation for cell division requires the production of additional ATP equivalents. This need can be met under aerobic conditions by increasing the flux of acetyl coenzyme A into the tricarboxylic acid cycle (and via anaplerotic reactions to replenish carbon used for biosynthesis) derived from glucose, amino acid and fatty acid oxidation. However, cancer cells have a tendency to increase the glycolytic flux even under aerobic conditions, and secrete a large fraction of the glucose carbons as lactate [1–7]. This implies a bypass of oxidative phosphorylation, such that glycolysis alone accounts for a substantial fraction of the ATP generation. This enhanced aerobic glycolysis is known as the Warburg effect [8, 9]. However, the precise mix of fuels that drive cancer and normal epithelial cells is controversial, and may well be dependent on cell type and the growth conditions [3, 10].
The Ras family of GTPases (H-, K- and N-Ras) function to transduce signals from receptor tyrosine kinases (e.g. epidermal growth factor receptor) that promote cell survival and proliferation . Activating mutations of the Ras GTPases that cause insensitivity to inhibitory GTPase-activating proteins are among the most common genetic alterations detected in human cancers . Given the central signaling role of Ras in cell proliferation and tumorigenesis, it is not surprising that these oncoproteins also mediate neoplastic metabolism. Ectopic expression of oncogenic Ras in immortalized cells has been found to: (i) increase glucose uptake and lactate production; (ii) increase ribose-5-phosphate, a glucose-derived anabolic precursor of DNA and RNA; and (iii) increase sensitivity to the glycolytic inhibitors, 2-deoxyglucose and oxamate [12–14]. However normal, non-immortalized human bronchial epithelial cells supplemented with growth factors were recently observed to consume glucose and secrete lactate at a similar rate as RasV12-transformed human bronchial epithelial cells . Accordingly, the precise downstream metabolic effects of Ras signaling within the setting of immortalized cells and in comparison to matched primary cells have not been well established.
By monitoring the flow of 13C from fully labeled glucose into metabolites, it is possible to determine the major pathways that are influenced by immortalization and transformation, including the activity of glycolysis, the oxidative and non-oxidative pentose phosphate pathways, the tricarboxylic acid cycle and pyrimidine biosynthesis. Herein, we report that the introduction of oncogenic H-RasV12 into immortalized bronchial epithelial cells increases the conversion of 13C from labeled glucose into several shunt products of the tricarboxylic acid cycle. Furthermore, we find that H-RasV12 increases oxygen consumption and sensitizes the immortalized cells to electron transport chain disruption. Importantly, the observed increase in mitochondrial metabolism caused by H-RasV12 may prove useful for the development of agents that selectively disrupt the metabolism of neoplastic cells.
Glucose consumption and lactate secretion by normal, hT/LT-immortalized and H-RasV12-transformed human bronchial epithelial cells
Intracellular pyrimidine and purine ribose synthesis from glucose are similar in primary, immortalized and transformed bronchial epithelial cells
Increased conversion of glutamate/glutamine from glucose by Ras-transformed cells
Increased 13C-glucose derived aspartate and uridine in Ras-transformed cells
hT/LT/Ras-transformed cells consume high oxygen and are especially sensitive to anoxia
The high enrichment of 13C-glucose-derived carbons into glutamate/glutamine, aspartate and uridine in the H-RasV12-transformed bronchial epithelial cells provides unambiguous evidence that the tricarboxylic acid cycle is highly active in these cells. That we observed increased pooling of the 13C-glucose-derived products from the tricarboxylic acid cycle in the hT/LT/Ras-transformed bronchial epithelial cells suggests either that H-RasV12 causes increased synthesis or decreased utilization of these anabolic precursors. The NHBE, hT/LT and hT/LT/Ras cells were allowed to double twice prior to extraction and NMR analysis, and we thus anticipate that the relative anabolic utilization of these precursors is not decreased by H-RasV12. Coupled to the observations that oxygen consumption is increased by H-RasV12 and that the H-RasV12-transformed cells are especially sensitive to electron transport perturbation by rotenone, the observed increase in 13C-enrichment of the glutamate/glutamine, aspartate and uridine supports an increased activity of the tricarboxylic acid cycle rather than decreased utilization of the detected anabolic substrates.
The increased 13C-enrichment of glutamate/glutamine and aspartate from glucose indicate increased TCA activity. The high glycolytic flux in these cells requires a means of regenerating cytoplasmic NAD+. The fermentation of pyruvate to lactate, followed by secretion of lactate into the medium, can sustain only that part of glycolytic flux that results in lactate production. Any additional glycolysis that produces pyruvate that enters the TCA (for example) needs a second means of regenerating NAD+. This may stem from the activity of the aspartate/malate NADH shuttle which previously has been found to be active in neoplastic cells . Mitochondrial and cytoplasmic aspartate aminotransferases produce aspartate and glutamate from oxaloacetate and α-ketoglutarate respectively and are activated early in carcinogen-induced transformation in vivo . Furthermore, the concentration of aspartate and glutamate are increased in human colon and gastric adenocarcinomas relative to matched normal tissues . The aspartate/malate shuttle functions to transfer electrons via NADH produced from glycolysis into the mitochondria for electron transport. Given that activated H-RasV12 increases oxygen consumption and confers sensitivity to electron transport perturbation, we speculate that flux through this NADH shuttle may be increased by oncogenic Ras and future studies will address this hypothesis.
Several genetic alterations of cancer cause induction of cellular proliferation through the epidermal growth factor receptor (EGFR)-Ras-Raf-mitogen activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK; MEK)-ERK-MAPK pathway, including EGFR amplifications (colorectal, pancreatic and lung cancers) and mutations (lung adenocarcinomas and glioblastoma), and activating mutations of Ras (pancreatic, papillary thyroid, colon and lung cancers) and Raf (melanoma, papillary thyroid and colon cancers) . In this study, we have found that activation of this signaling cascade through ectopic expression of H-RasV12 in hT/LT-immortalized bronchial epithelial cells causes an increase in the enrichment of 13C-carbons from glucose into key anabolic precursors produced from tricarboxylic acid cycle intermediates. Based on these observations, we predict that downstream signaling effectors of the EGFR-Ras-Raf-MEK-ERK-MAPK pathway cause activation of key rate-limiting anabolic enzymes required for increased production of these precursors. Furthermore, we postulate that the metabolic rationale for increased pooling of these precursors (i.e. glutamate and aspartate) in H-RasV12- transformed proliferating cells may be related to the need for a ready supply of substrates for the production of amino acids since these precursors are required for the de novo synthesis of several amino acids (i.e. glutamine, proline, arginine, asparagine, methionine, lysine and threonine). In support of this hypothesis, recent studies have demonstrated that Ras/ERK signaling promotes translation initiation by facilitating assembly of the preinitiation complex . Accordingly, increasing the availability of anabolic substrates for the translational machinery may prove to be a key metabolic event activated by oncogenic Ras.
Recent studies by Chen et al. have found that metastatic breast cancer cells utilize aerobic glycolysis, coupled with the tricarboxylic acid cycle and oxidative phosphorylation to generate ATP needed for cellular proliferation . They conducted proteomic analyses of breast cancer cells isolated from a stage IV breast cancer patient before and after metastatic spread to the brain in athymic mice and observed a marked increase in the expression of proteins involved not only in glycolysis but also in the tricarboxylic acid cycle, including aconitate hydratase, isocitrate dehydrogenase and mitochondrial malate dehydrogenase, and in oxidative phosphorylation, including cytochrome c oxidase subunits, NADH-ubiquinone oxidoreductases and ATP synthase chains. Ras is over-expressed in the majority of breast adenocarcinomas examined [24–26] and the observed high protein expression of tricarboxylic acid cycle enzymes provides further support for the conclusion that the activities of the tricarboxylic acid cycle and electron transport chain are increased as a result of H-RasV12-transformation.
Pharmacologic disruption of glycolysis is currently under development as an anti-neoplastic strategy due to the observations that tumor cells metabolize glucose rapidly and are especially sensitive to glucose deprivation . 3-Bromopyruvate and 2-deoxyglucose are two compounds that inhibit the first irreversible enzyme of glycolysis, hexokinase, and suppress tumor growth in vivo [27–29]. In this study, we provide evidence for increased tricarboxylic acid cycle activity, oxygen consumption and energetic reliance on electron transport in H-RasV12-transformed cells relative to matched, normal and immortalized cells. Based on these observations, we conclude that inhibition of mitochondrial metabolism may cause selective anti-neoplastic effects. It is noteworthy that several known cytotoxic agents function by targeting the mitochondria, including arsenite, lonidamine and betulinic acid . In future studies, we will determine the precise downstream Ras effector enzymes that regulate this metabolic shift in order to develop highly targeted anti-metabolites as chemotherapeutic agents.
Cell lines and cell culture
Normal human bronchial epithelial (NHBE) cells were obtained from Cambrex (Walkersville, MD) and NHBE cells expressing telomerase and SV40 large T antigen (hT/LT) and activated Ras (hT/LT/Ras) were gifts from Dr. B. J. Rollins, Dana Farber Cancer Institute. All experiments with NHBE cells were conducted between 4 and 6 passages. NHBE, hT/LT and hT/LT/Ras cells were grown in media containing 1 gm/L (5.5 mM) glucose and formulated with bovine pituitary extract, recombinant human epidermal growth factor, hydrocortisone, insulin, epinephrine, tri-iodothyronine, transferrin, gentamicin, amphotericin B and retinoic acid (BEGM with SingleQuots, Cambrex, Walkersville, MD). All cells were maintained at 37°C in 5% CO2. For some experiments, NHBE, hT/LT and hT/LT/Ras cells were grown in 0% oxygen (and 5% CO2) in a modular incubator chamber at 37°C (Billups-Rothenberg, Del Mar, CA). For certain other experiments, NHBE, hT/LT and hT/LT/Ras cells were treated with 10 μM rotenone (Sigma, St. Louis, MO) for 24 hours. The cells were enumerated by direct visualization using light microscopy. Sizes of NHBE, hT/LT and hT/LT/Ras cells were determined using Imagestream and IDEAS software (both from Amnis, Seattle, WA).
For NMR experiments, after cells reached 25% confluence, the media was changed to glucose-free Dulbecco's modified Eagle medium (DMEM, Invitrogen, Grand Island, NY) supplemented with SingleQuots (Cambrex) as above and also with 13C labeled glucose (5.5 mM) from a sterile 20% stock solution of [U-13C6]-glucose (98% 13C) (Cambridge Isotopes Laboratories, Andover, MA) in phosphate-buffered saline.
Protein extraction and Western blotting
Cells were treated with 0.25% trypsin-EDTA, washed in PBS, and lysed in 2x RIPA buffer. Protein samples were resolved on a 4–20% gradient SDS-PAGE gel and transferred to a PVDF membrane. Membranes were blocked in TBS-Tween 20 (1%) containing 5% milk. Either rabbit anti-hTERT (1:250, Rockland Immunochemicals, Gilbertsville, PA), mouse anti-β-actin (1:5000, Sigma, St. Louis, MO), rabbit anti-H-Ras (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA) or mouse anti-SV40 large T-antigen antibody (1:1000, Oncogene/Calbiochem, San Diego, CA) were re-suspended in 10 ml of TBS-Tween 20 (5% milk) and incubated with the membrane for 1 hour. Secondary antibodies were goat anti-rabbit or anti-mouse HRP conjugated (1:8000, Pierce Biotechnology, Rockford, IL). All Western blotting experiments were repeated for a total of 4 experiments.
Scanned images were quantified by densitometric analyses using Quantiscan software Version 3.0 (Biosoft, United Kingdom). Values obtained were normalized to α-tubulin) (as a control) and expressed in densitometric units as a percentage of the control. All values represent the mean ± SD of 4 independent experiments.
Lactate and glucose measurements
Lactate concentrations in the media were measured using a lactate oxidase-based assay read at 540 nm (Trinity, Wicklow, Ireland). Glucose concentrations were measured using a hexokinase-glucose-6-phosphate dehydrogenase enzymatic assay read at 340 nm (Sigma, St. Louis, MO). All data are expressed as the mean ± SD of five experiments. Statistical significance was assessed by the unpaired two-tail t- test.
Cells grown in the presence of 13C-labeled glucose were harvested after two population doublings by trypsinization with 0.25% trypsin-EDTA for 60 seconds at 37°C in 5% CO2 and low speed centrifugation. The cells were counted by direct visualization using light microscopy and equal numbers of cells were spun down, washed twice with ice-cold PBS followed by low speed centrifugation (1000 rpm) at 4°C to remove adhering medium, and then flash frozen in liquid N2. The cold pellet was extracted with 10% ice-cold TCA (twice), followed by lyophilization as previously described [17, 18]. Dry cell extract was redissolved in 0.35 ml D2O with 142 μM DSS (2, 2-dimethyl-2-silapentane-5-sulfonate sodium salt) as both a chemical shift reference and as a concentration standard and loaded into a 5 mm Shigemi tube.
Nuclear Magnetic Resonance
All NMR spectra were recorded at 14.1 T on Varian Inova NMR spectrometer at 20°C using a 90° excitation pulse. 1D spectra were recorded using an acquisition time of 2 seconds and a relaxation delay of 3 seconds during with the residual HOD signal was suppressed using a weak transmitter pulse (ca. 20 Hz B1 field). For analyzing the cellular extracts and determining the positional enrichment with 13C we used 2D experiments (TOCSY and HSQC), and analyzing the satellite peaks in the TOCSY as described in detail [31, 32]. TOCSY experiments were recorded with a 6000 Hz spectral width both dimensions, 0.341 s acquisition time in t2 and 0.05 s in t1, a recycle time of 2 s, a 50 ms isotropic mixing time, and a B1 field strength of 8 kHz generated with MLEV-17. The data tables were zero filled to 8192 by 2048 complex points, apodized using an unshifted Gaussian function and 0.5 Hz line broadening exponential in both dimensions prior to double fourier transformation.
Where M(obs), M(true) are the observed and fully relaxed magnetizations, R1 is the spin-lattice relaxation rate constant and D is the recycle time.
Isotope enrichment from NMR
For metabolites poorly resolved in 1D spectra, we used the 2D TOCSY method as previously described [31, 32]. The base-planes were carefully corrected, and cross peak volumes were determined by volume integrated using VNMR. This approach has been shown to provide very accurate, and unbiased estimates of the 13C content. The various isotopomer enrichments were calculated by replacing the peak area with peak volumes in equation 3. In these TOCSY experiments, the protons were partially saturated owing to the shorter recycle time (2 s). Thus, the actual peak volumes were corrected according to the differential T1 values of protons attached to 13C or 12C according to Eq. (2). Effective T1 values were determined on standards recorded under the same solvent conditions using the inversion recovery sequence.
Analytical precision was determined by replicate analysis of symmetry related cross peaks, and on spectra transformed using slightly different window functions. The errors were then estimated with respect to the signal to noise ratios of the peaks. The number of biological replicates was 3.
NHBE, hTLT and hT/LT/Ras cells in culture were detached, washed × 1 with PBS and resuspended in complete BEGM medium at 107cells/ml. Oxygen consumption was measured using 5 × 106 cells in 500 μL medium at 37°C using a Clark-type polarographic electrode (Strathkelvin Mitocell MT 200, Motherwell, United Kingdom). A starting O2 concentration of 215 μM was assumed based on O2 solubility at sea level at 37°C [35–37]. Experiments were repeated for a total of five times. The data shown are the results of a single representative experiment.
5 × 105 NHBE, hTLT and hT/LT/Ras cells in culture for 24 hrs in normoxia (21% oxygen) or anoxia (0% oxygen) +/- 10 μM rotenone were washed (while still adherent) with cold PBS ×1. Passive lysis buffer (1X, Molecular Probes, Invitrogen, Carlsbad, CA) was then added directly to the plates and the cells immediately harvested by scraping. The lysates were flash frozen (to -80°C) and thawed (to 37°C) once to accomplish complete lysis and then centrifuged (at 4°C) for 30 seconds to clear the lysates. Intracellular ATP levels were determined using a bioluminescence assay (Molecular Probes) utilizing recombinant firefly luciferase and its substrate, D-luciferin and following manufacturer's instructions. The luminescence was read in a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA) at 560 nm. The ATP values were calculated using an ATP standard curve. The protein concentrations of the lysates were estimated using the bicinchoninic acid (BCA) assay (Pierce Biotechnology, Rockford, IL) and ATP was expressed as pmol per μg protein. All data are expressed as the mean ± s.d. of five experiments. Statistical significance was assessed by the unpaired two-tail t- test.
5 × 105 NHBE, hTLT and hT/LT/Ras cells were plated in normoxia or anoxia with or without 10 μM rotenone for 24 hrs, detached (with 0.25% Trypsin/EDTA) and counted using a hemacytometer. Cell viability was assessed by Trypan blue exclusion. Trypan blue solution was prepared by combining 0.5 ml of Trypan blue (Sigma, St Louis, MO) with 0.3 ml of PBS. Cells in suspension were mixed 1:1 with Trypan blue solution and incubated at room temperature for 5 min. The numbers of unstained (viable), stained (dead) and total cells were counted in a hemacytometer by two independent observers and the percentage of viable cells calculated. The numbers of cells counted in the hemacytometer were between 100–500 (1–5 × 106 cells/microliter). All data are expressed as the mean ± s.d. of five experiments. Statistical significance was assessed by the unpaired two-tail t- test.
tricarboxylic acid cycle
heteronuclear single quantum coherence
- TAg SV40 large:
Total Correlation Spectroscopy.
This work was supported by the following grants: NIH 1P20 RR18733 (JC), Leukemia and Lymphoma Society Translational Research Grant (JC), NIH 1 R01 CA11642801 (JC), P. Morris External Research Program (JC), the KY Challenge of Excellence (ANL) the Brown Foundation (ANL) and a NSF EPSCoR for NMR instrumentation (ANL). We gratefully acknowledge helpful discussions with Otto Grubraw and John W. Eaton.
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