Metabolic changes in cancer cells upon suppression of MYC
© Anso et al; licensee BioMed Central Ltd. 2013
Received: 1 October 2012
Accepted: 3 January 2013
Published: 4 February 2013
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© Anso et al; licensee BioMed Central Ltd. 2013
Received: 1 October 2012
Accepted: 3 January 2013
Published: 4 February 2013
Cancer cells engage in aerobic glycolysis and glutaminolysis to fulfill their biosynthetic and energetic demands in part by activating MYC. Previous reports have characterized metabolic changes in proliferating cells upon MYC loss or gain of function. However, metabolic differences between MYC-dependent cancer cells and their isogenic differentiated counterparts have not been characterized upon MYC suppression in vitro.
Here we report metabolic changes between MYC-dependent mouse osteogenic sarcomas and differentiated osteoid cells induced upon MYC suppression. While osteogenic sarcoma cells increased oxygen consumption and spare respiratory capacity upon MYC suppression, they displayed minimal changes in glucose and glutamine consumption as well as their respective contribution to the citrate pool. However, glutamine significantly induced oxygen consumption in the presence of MYC which was dependent on aminotransferases. Furthermore, inhibition of aminotransferases selectively diminished cell proliferation and survival of osteogenic sarcoma MYC-expressing cells. There were minimal changes in ROS levels and cell death sensitivity to reactive oxygen species (ROS)-inducing agents between osteoid cells and osteogenic sarcoma cells. Nevertheless, the mitochondrial-targeted antioxidant Mito-Vitamin E still diminished proliferation of MYC-dependent osteogenic sarcoma cells.
These data highlight that aminotransferases and mitochondrial ROS might be attractive targets for cancer therapy in MYC-driven tumors.
Tumor cells display increased glucose metabolism to meet the anabolic demands required for cell proliferation . Glycolytic intermediates fuel anabolic pathways that synthesize NADPH, ribose, phospholipids, triacylglycerols, and serine . However, aerobic glycolysis by itself is not able to supply all the metabolites required for cell proliferation. The mitochondrial tricarboxylic acid cycle (TCA cycle) provides additional metabolites that funnel into lipid, amino acid and nucleotide synthesis. TCA cycle-dependent biosynthesis requires constant replenishment of carbons into the TCA via multiple anaplerotic pathways . Pyruvate derived from glucose can provide acetyl-CoA and oxaloacetate to initiate the TCA while glutamine can also replenish the TCA metabolites through the process of glutaminolysis, where glutamine is converted to glutamate by glutaminases (GLSs), which then enters the TCA cycle by conversion into alpha-ketoglutarate by the aminotransferases or glutamate dehydrogenase . Additionally, glutamine serves as an important nitrogen donor for assembly of amino acids, nucleotides and nicotinamide. Collectively, the metabolism of glucose and glutamine can provide nearly all the necessary carbon and nitrogen required for optimal cell proliferation and growth.
In the past decade, there have been multiple mechanisms uncovered that regulate the metabolism of glucose and glutamine for cell proliferation in normal and cancer cells [5, 6]. One mechanism, by which both normal and cancer cells meet their metabolic demands for cell proliferation, is through activation of c-MYC (herein termed MYC) . Normal cells induce MYC upon cell surface receptor-dependent signaling to stimulate aerobic glycolysis and glutaminolysis to promote cell proliferation, while cancer cells have deregulated MYC allowing proliferation to occur in a cell-autonomous manner [8, 9]. For example, MYC increases glycolysis in part through the regulation of lactate dehydrogenase A (LDHA) and glutaminolysis by upregulating expression of GLS [10–12]. MYC also regulates mitochondrial metabolism through induction of genes such as TFAM, which is required for replication and maintenance of mitochondrial DNA and mitochondrial biogenesis . A consequence of increased mitochondrial metabolism is the generation of reactive oxygen species (ROS) that are required to drive tumorigenesis [14, 15].
Much of our understanding of MYC has come from examining metabolic pathways required for cell proliferation upon MYC loss or gain of function [12, 13, 16]. In the current study we took the opposite approach - whereby metabolic changes were examined when MYC was suppressed in osteosarcoma cells highly dependent on MYC for their tumorigenic potential . MYC suppression in these genetically engineered mouse osteosarcoma cells results in differentiation into osteocytes. This allowed us to compare metabolic differences between differentiated and proliferating cells of the same genetic background. Our results indicate the induction of glutaminolysis as the major metabolic difference observed between MYC- dependent osteosarcoma cells and osteocytes. Furthermore, mitochondrial-targeted antioxidants diminished proliferative capacity of osteosarcoma cells without having detrimental effects on osteocytes.
MYC-dependent osteogenic sarcoma cells were isolated from a transgenic mouse as previously described . These cells in the presence of doxycycline undergo differentiation into osteocytes. Osteosarcoma cells were cultured in high glucose Dulbecco’s modification of Eagle’s medium (DMEM) supplemented with 5% penicillin/streptomycin, 10% fetal bovine serum (FBS) and HEPES buffer. For nutrient deprivation experiments, glucose and glutamine-free DMEM was supplemented with 10% dialyzed serum in the presence of 10 mM glucose and/or 4 mM glutamine. Rotenone, antimycin A, oligomycin, FCCP, aminooxyacetic acid (AOA), N-acetylcysteine (NAC), beta-phenylethyl isothiocyanate (PIETC) and buthionine sulfoximine (BSO) were purchased from Sigma (St. Louis, MO, USA). Antibodies against MYC and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against VDAC1, GOT2 and GPT2 were purchased from Abcam (Cambridge, MA, USA). Antibody against GLS1 was prepared in the Matés laboratory.
Oxygen consumption rate (OCR) was measured using the 24 well Extracellular Flux Analyzer XF24 (Seahorse Bioscience, North Billerica, MA, USA) according to the manufacturer’s protocol. Cells were equilibrated with DMEM lacking bicarbonate and HEPES at 37°C for one hour in an incubator lacking CO2. Basal OCR was measured followed by sequential treatments with oligomycin A (5 μM), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, 10 μM) and antimycin A (2 μM) + rotenone (2 μM). Measurements were normalized to cell number in each well. A minimum of four wells were utilized per condition in any given experiment. The spare respiratory capacity was calculated as previously described .
Mitochondrial ROS production was measured using a redox sensitive GFP probe (roGFP2) targeted to the mitochondrial matrix or cytosolic compartments. Cells were infected with adenovirus containing roGFP2 as previously described . As internal controls, samples were fully reduced with 1 mM dithiothreitol (DTT) and fully oxidized with 1 mM H2O2. Upon oxidation the roGFP2 gains excitability at 405 nm while losing excitability at 488 nm. Percent oxidized probe was determined with the equation:
where R is sample without DTT or H2O2 added; RDTT fully reduced sample, and RH2O2 is fully oxidized.
Cells were trypsinized and fixed in ethanol 70% overnight at −20°C. Subsequently, cells were resuspended in a PBS solution containing 50 μg/mL propidium iodide (PI) and 0.1 mg/mL RNase A and incubated 40 minutes at 37°C. Then, the cell pellet was resuspended in PBS and analyzed using a FACS flow-cytometer (Becton Dickinson, Franklin Lakes, NJ USA). Cell death was determined by incubating cells in 0.1 μg/mL PI. Data were analyzed with CellQuest software.
Cells were stained with 100 nM tetramethylrhodamine, ethyl ester (TMRE) for 30 minutes in PBS at 37°C. The cells were trypsinized and washed with PBS. As control, cells were treated with the uncoupling agent FCCP at 50 μM for 10 minutes before staining. Median fluorescence intensity (MFI) values were corrected by FCCP background in each cell type. Data were analyzed in a Beckton Dickinson LSR Fortessa cell analyzer (Franklin Lakes, NJ USA and analyzed with FlowJo (Ashland, OR, USA) software.
Concentrations of glucose, lactate, and glutamine were determined by incubating cells in DMEM with 10% dialyzed FBS and supplemented with 10 mM D-glucose and 2 mM L-glutamine. After six hours, 0.6 mL aliquots of medium were analyzed using an automated electrochemical analyzer (BioProfile Basic-4 analyzer; NOVA Biomedical, Waltham, MA, USA). Metabolic rates were determined by normalizing absolute changes in metabolite abundances to protein content as previously described . Isotopic labeling was performed in DMEM with 10% dialyzed FBS supplemented with either 10 mM D-[U-13C]glucose and 2 mM L-glutamine, or 2 mM L-[U-13C]glutamine and 10 mM D-glucose. After six hours, metabolites were extracted with 50% methanol and analyzed using an Agilent 6970 gas chromatograph and an Agilent 5973 (Santa Clara CA, USA) mass selective detector. Analysis of 13C enrichment and mass isotopomer distribution was performed as previously described .
P-values associated with all pairwise comparisons were based on Student’s t-test for independent groups. Error bars were defined using standard error of the mean (SEM).
MYC’s regulation of cellular metabolism in a cell-autonomous manner has been primarily elucidated by using immortalized proliferating cells with inducible expression of MYC, mitogenic stimulation of quiescent primary fibroblasts or by utilizing naive T cells isolated from wild-type or c-MYC null mice. However, the metabolic differences between isogenic MYC-dependent cancer cells and their differentiated counterparts have not been studied. Furthermore, the metabolic transitions that accompany exit from the cell cycle have only rarely been evaluated. Our present results indicate that MYC-dependent osteosarcoma cells are dependent on glucose metabolism through glycolysis for survival compared to osteocytes, their differentiated counterparts. Glutamine-induced mitochondrial respiration is necessary for cell proliferation and survival of MYC-dependent cancer cells compared to their differentiated counterparts. This is consistent with previous studies where MYC induces glucose and glutamine-dependent metabolism to sustain the anabolic demands due to cell proliferation. Aminotransferases were required for glutamine to sustain mitochondrial metabolism in MYC-dependent cancer cells, consistent with previous findings that these enzymes predominate over glutamate dehydrogenase in glucose-consuming, proliferating cancer cells . Interestingly, the inhibition of these enzymes results in cell death of the MYC-dependent cancer cells but not their differentiated counterparts. We had previously shown that oncogenic Kras-dependent cells require aminotransferases to sustain mitochondrial metabolism and cell proliferation . Furthermore, inhibition of aminotransferases prevents xenograft tumor growth of MDA-MB-231 breast cancer cells and MYC-dependent neuroblastoma cells [33, 34]. This raises the possibility that inhibition of aminotransferases might be an effective strategy to inhibit MYC-dependent tumorigenesis.
Previous studies have suggested that tumor cells have higher levels of ROS compared to normal cells. ROS, in addition to causing genomic instability, can also increase tumorigenesis by activating signaling pathways that regulate cellular proliferation, angiogenesis, and metastasis . The higher levels of ROS in cancers cells can be exploited as an effective selective strategy to kill tumor cells over normal cells. The administration of agents such as PIETC or BSO, which disable antioxidant defense mechanisms in cells, raises ROS to intolerable levels in cancer cells but not to normal cells which exhibit lower basal ROS levels . However, we did not notice substantial differences in ROS levels between MYC- dependent cancer cells and their differentiated counterparts and PIETC or BSO did not have any selective killing of MYC-dependent cancer cells. Nevertheless, MYC-dependent tumor cells were dependent on mitochondrial ROS for cell proliferation as mitochondrial targeted antioxidant MVE drastically reduced cell proliferation. While previous studies have demonstrated that NAC is effective in reducing MYC-dependent tumorigenesis , it remains to be determined whether mitochondrial-targeted antioxidants would also prevent tumorigenesis in vivo.
Dulbecco’s modification of Eagle’s medium
fetal bovine serum
voltage dependent anion channel 1
glutamic oxaloacetic transaminase 2
glutamic pyruvate transaminase 2
reactive oxygen species
redox sensitive green fluorescent protein 2
mitochondrial targeted roGFP2
oxygen consumption rate
green fluorescent protein
tricarboxylic acid cycle
tetramethylrhodamine, ethyl ester
mitochondrial transcription factor A
mitochondrial targeted vitamin E
We thank Drs. Balaraman Kalyanaraman and Joy Joseph for their kind contribution of MVE. This work is supported by NIH (R01CA123067) to NSC and NIH (R01CA157996) and the Robert A Welch Foundation (I1733) to RJD. The work was also supported ARM from NIH Training grant (5T32GM083831). DWF was supported by NIH (R01CA089305) and (R01CA34233). JMM was supported by SAF2010-17573 and CVI-6656.
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