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Metabolism regulates differentiation A reverse genetic engineering approach identifies metabolic enzymes and their cellular pathways as potential regulators of myoblast differentiation. Targeting these metabolic nodes has provocative implications for drug discovery and therapeutic efficacy.
Timothy E McGraw & Vivek Mittal
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myoblasts, progenitor cells that give rise to skeletal muscle. To establish a system for investigating the role played in muscle differentiation by alterations in carbon metabolism, Bracha et al.3 developed an RNAi-based reverse genetic approach to target about 50 enzymes involved in carbon metabolism; they did not, however, include the more ‘conventional’ signal-transduction proteins (such as kinases). As a model system the authors used cultured C2C12 myoblasts, which can be induced to differentiate into multinucleate myotubes under conditions of reduced serum. The screen identified enzymes that when knocked down induced
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C2C12 differentiation in the presence of serum. Knockdown of Pgk1, H6pd and Acl directly altered cellular carbon metabolism and helped to provide unique insights into the influence of the cellular metabolite milieu on differentiation. When individually knocked down, the three enzymes revealed by the screen induced spontaneous differentiation of C2C12 myoblasts (Fig. 1a). These enzymes are involved in diverse metabolic pathways: glycolysis (Pgk1), production of NADPH in the endoplasmic reticulum lumen (H6pd), and lipogenesis and cholesterol biosynthesis (Acl). One interpretation of these data is that changes in metabolites
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utrients and metabolism influence cellular differentiation in unicellular organisms, including species of yeast and Streptomyces. Reduced availability of nitrogen and carbon sources initiates a morphological differentiation process that involves a dimorphic switch characterized in yeast by pseudohyphal differentiation1,2. In metazoans, however, the role of carbon metabolism in cellular differentiation remains elusive. An RNA interference (RNAi)-based screen has now revealed a role for three metabolic enzymes, phosphoglycerate kinase (Pgk1), hexose-6phosphate dehydrogenase (H6pd) and ATP citrate lyase (Acl), in the differentiation of
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Figure 1 | RNAi screens reveal that cell differentiation is regulated by key metabolic enzymes. (a) Schematic representation of key metabolic steps involved during the conversion of glucose to acetyl-CoA. RNAi screens have identified three key metabolic enzymes whose individual suppression promotes differentiation of C2C12 myoblasts. Acl, ATP citrate lyase; G6P, glucose 6-phosphate; HK, hexokinase; PG, phosphoglycerate; Pgk, phosphoglycerate kinase; sh, short hairpin RNA. (b) A hypothetical model illustrating the way that suppression of metabolic enzymes may have a potential in differentiation therapy. For example, anticancer therapies target cancer cells (magenta) but leave unperturbed the cancer stem cells (blue) from which tumors regrow. However, suppression of metabolic enzymes identified by Bracha et al. may promote the differentiation of cancer stem cells, which will then be likely to respond better to conventional anticancer therapeutics. 176
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news & views along these pathways influence differentiation. To investigate changes in metabolites that occur upon ‘standard’ differentiation of C2C12 to myotubes, the authors used a mass spectrometry– based analysis to profile intracellular metabolites of C2C12 cells differentiated by serum withdrawal. They observed changes in a number of metabolites, including some alterations consistent with reduced Pgk1 and H6pd activities. The metabolic profiling correlated with the screening results. This link between metabolism and cell differentiation was not completely unanticipated because during metamorphosis in insects, cellular differentiation is accompanied with pronounced downregulation of metabolic pathways including glycolysis and fatty acid oxidation4. Although determination of the exact mechanistic link between knockdown of the specific enzymes, resultant changes in metabolites and the induction of differentiation awaits future studies, the report of Bracha et al. illustrates the benefit of this type of screen in identifying potentially new pathways for controlling complex processes such as differentiation. For example, in the profiling studies, no changes in metabolites were identified that supported a direct role for Acl. However, knockdown of Acl will reduce both nuclear and cytoplasmic levels of acetyl-CoA—the former required for chromatin acetylation and the latter for cholesterol and lipid syntheses5—and the authors present data supporting a mechanism linking Acl, chromatin acetylation and cholesterol biosynthesis in myoblast differentiation. Indeed, treatment with statins (inhibitors of cholesterol biosynthesis) or trichostatin (an inhibitor of histone deacetylases) stimulates differentiation of C2C12 myoblasts, supporting the conclusion that
knockdown of Acl promotes differentiation through similar effects. To extend these findings to the differentiation of cells other than C2C12 cells, the authors assessed the effects of Acl inhibition on rhabdomyosarcoma tumor cells. Notably, the statin fluvastatin induced rhabdomyosarcoma cell differentiation, reduced cancer cell proliferation and inhibited anchorageindependent growth. Although the therapeutic potential of this observation can only be realized if statins turn out to alter the growth of rhabdomyosarcoma in vivo, it is noteworthy that Acl inhibition is considered a promising approach to cancer therapy. Indeed, recent studies of Acl suppression in the human lung adenocarcinoma cell line A549 found that this led to reductions in acetyl-CoA levels and glucose-dependent lipid synthesis and limited cell proliferation and survival6. Importantly, Acl knockdown cells formed smaller, more differentiated tumors than the controls. Notably, Acl knockdown caused erythroid differentiation of the human chronic myelogenous leukemia cell line K562. Thus Bracha et al. provide strong evidence in cultured cells that Acl inhibition promotes cellular differentiation. The importance of this finding for normal physiologic adaptations is unknown, however, and comprises an exciting area for further investigations. The intriguing phenomenon of the regulation of cell differentiation by metabolism thus provides new insights with potential implications into the dynamics of growth and development. It also provokes many interesting questions: most importantly, how do cancer cells sense and use nutrients to facilitate tumor development and progression, and how does this phenomenon influence pathologic outcome? The link between metabolism
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and cellular differentiation also has important implications for ‘differentiation therapy’ in cancer patients. In tumors, cancer stem cells are more resistant to conventional chemotherapy than are other, more differentiated cells. Therefore, therapies that can induce differentiation of cancer stem cells so that they can be targeted with more conventional drugs are being heralded as an important anticancer strategy. In this context, it is tantalizing to speculate that perturbation of the metabolic nodes identified by Bracha et al. may force cancer stem cells to differentiate and lose their self-renewal property, thereby rendering them vulnerable to more conventional anticancer therapeutics (Fig. 1b). The proof of principle for differentiation therapy has been the treatment of acute promyelocytic leukemia with all-trans-retinoic acid7. Only limited benefit has been achieved with retinoids in the treatment of solid tumors, however, and this comprises an important area of research. L Timothy E. McGraw is in the Department of Biochemistry and Vivek Mittal is in the Departments of Cardiothoracic Surgery and Cell and Developmental Biology, Weill Medical College of Cornell University, New York, New York, USA. e-mail:
[email protected] or
[email protected] References
1. Madhani, H.D. & Fink, G.R. Trends Cell Biol. 8, 348–353 (1998). 2. Gagiano, M., Bauer, F.F. & Pretorius, I.S. FEMS Yeast Res. 2, 433–470 (2002). 3. Bracha, A.L., Ramanathan, A., Huang, S., Ingber D.E. & Schreiber, S.L. Nat. Chem. Biol. 6, 202–204 (2010). 4. White, K.P. et al. Science 276, 114–117 (1997). 5. Wellen, K.E. et al. Science 324, 1076–1080 (2009). 6. Hatzivassiliou, G. et al. Cancer Cell 8, 311–321 (2005). 7. Petrie, K., Zelent, A. & Waxman, S. Curr. Opin. Hematol. 16, 84–91 (2009).
Competing interests statement
The authors declare no competing financial interests.
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