On Getting There from Here

Studies on a variety of interesting biological problems, ranging from circadian rhythm to cancer cell growth to longevity, have begun to give evidence that the physiological state of cells and tissues reflects both the cell’s regulatory systems and its state of intermediary metabolism. It is appreciated that the regulatory state of a cell or tissue, as driven by transcription factors and signaling pathways, can impose itself upon the dynamics of the metabolic state. It follows that the reciprocal must also be the case, that the metabolic state will feed back to impose itself on the regulatory state. An appreciation and understanding of this reciprocity may be required to crack open problems in biological research that have heretofore been insoluble.

For the past 30 years, research in the biological sciences has been dominated by molecular biology. The successes of this approach have shaped our understanding of innumerable domains of biology. But any field that becomes sufficiently muscular can overshadow other credible approaches to scientific inquiry. One field etiolated by the cloud of molecular biology has been metabolism. The vast majority of discoveries made by molecular biologists over the past several decades required no attention to the metabolic state of a cell. Molecular biologists needed no distracting thoughts about the metabolic state of a cell to discover microRNAs, the reprogramming of somatic cells into pluripotent stem cells, or gene rearrangement as the underlying basis for the generation of antibody diversity.

One simple way of looking at things is to consider that 9 questions out of 10 could be solved without thinking about metabolism at all, but the 10th question is simply intractable. As the saying goes, you simply “can’t get there from here” to answer this 10th question if you are ignorant about the dynamics of metabolism. This, I propose, is where we are now finding pregnant opportunities in the field of experimental biology. The low-lying fruit that could be picked by molecular biologists without having to consider the metabolic state of a cell, tissue, or organism is largely gone. The more sticky problems that required attention to the dynamics of metabolism and that were pushed aside for decades now loom as interesting and important challenges.

Consider a prime example of how molecular biologists have begun to embrace the importance of metabolic regulation. Cancer researchers have long known the enigmatic ability of tumor cells to undertake aerobic glycolysis, the so-called Warburg effect (1, 2). It makes sense that cancer cells would be highly glycolytic, yet why would these cells choose to dispose of the terminal product of glycolysis? Instead of allowing pyruvate to be converted into acetyl–coenzyme A (CoA) via the spectacularly beautiful pyruvate dehydrogenase enzyme complex within mitochondria, cancer cells instead convert pyruvate into lactate through the lactate dehydrogenase enzyme, and then simply excrete it—blindly giving away exceptional energetic value stored in the lactate hydrocarbon. Any cell that wants to grow—and there is nothing cancer cells care more about than growth—would be crazy to waste hydrocarbon; this would be akin to a motorist driving down the New Jersey Turnpike throwing away gasoline. The spendthrift waste of lactate likewise deprives the cancer cell of huge amounts of acetyl-CoA to be used for the synthesis of lipids, sterols, and other cellular building blocks. Despite progress, attention, and plenty of hype, it is safe to conclude that the famous Warburg effect remains a mystery.

Cancer researchers now recognize that regulatory proteins, such as the hypoxia-inducible transcription factors, can directly regulate the expression of genes encoding glycolytic enzymes (3). They now pay attention to how their favorite regulatory proteins, including the Myc and p53 transcription factors, help set the metabolic state of cells. The fact that these masterful transcription factors participate in dictating the metabolic state of a cell is now beginning to be accepted. The equally compelling corollary, however, remains largely unappreciated. That is, if the regulatory state (transcription factors, signaling pathways, etc.) is accepted to control the metabolic state, is it not also unconditionally certain that the metabolic state will reciprocally control the regulatory state itself? Understanding this reciprocity, and digging to the bottom of it, is where the future lies. Perhaps fittingly, this research will require the sophistication of scientists having genuine skills in the study of enzymology and intermediary metabolism.

Whole-genome sequencing efforts of individual tumors, now numbering in the thousands— yet soon to be numbered in the hundreds of thousands—are providing unbiased views of the myriad of “oncogenotypes” that underlie human cancer. Instead of ignoring mutations that happen
to fall in the genes encoding metabolic enzymes, scientists seem keener than ever to find and study such mutations. This change may reflect the recognition by cancer researchers that mutations that alter the function of metabolic enzymes might help to resolve the enigmatic, aerobic glycolytic state of certain cancer cells. It is equally likely that an understanding of how cancer cells veer away from normality concerning intermediary metabolism might lead to the conceptualization of new and inventive strategies for therapeutic intervention.

For example, a set of recurrent genetic lesions believed to influence the metabolic state of glioblastoma cancer cells have been identified in the genes encoding either of the two isoforms of isocitrate dehydrogenase (4, 5). Perplexingly, most, if not all, of these lesions mutate a single arginine residue in either of the two isoforms of the enzyme (IDH1 or IDH2). The precise selectivity of the mutational events, coupled with the observation that only one allele of either enzyme appears to be mutated in human cancer, pointed to the possibility that the lesions might be causing the enzymes to adopt a new catalytic function. Indeed, the mutated forms of the IDH1 and IDH2 enzymes exhibit a reduced affinity for isocitrate and are endowed with a new catalytic function wherein a-ketoglutarate is converted in an NADPH (nicotinamide adenine dinucleotide phosphate, reduced)– dependent manner to 2-hydroxyglutarate (6).

What pathways might be expected to be altered in a cell impeded for the production of aketoglutarate and concomitantly endowed with increased intracellular production of 2-hydroxyglutarate? A logical guess would be the family of dioxygenase enzymes that use a-ketoglutarate as an essential cofactor and, simultaneously, are inhibited in the presence of 2-hydroxyglutarate. Included among this family of dioxygenase enzymes are the prolyl-hydroxylase enzymes that modify and negatively regulate the HIF-1a (hypoxiainducible factor 1a) andHIF-2a transcription factors in oxygenated cells (7, 8). Partial elimination of these dioxygenase enzymes could be interpreted to lead to the activation of the hypoxia response pathway, thereby accounting for the activation of transcription of genes encoding glycolytic enzymes just as happens in the absence of the Von Hippel–Lindau (VHL) tumor suppressor gene (9, 10). This interpretation is very likely oversimplistic. Attenuation of a-ketoglutarate concentrations
and accumulation of increased amounts of 2-hydroxyglutarate almost certainly lead to the inhibition of other dioxygenase enzymes, of which mammalian cells have scores of isoforms. Intriguingly, some of these additional dioxygenase enzymes have been implicated in the control of histone demethylation (11–13), leaving open the possibility that changes in the metabolic state might impose alterations in the epigenetic state of cancer cells.

The drift of this thinking is concordant with the recent discovery of mutations found in renal cell cancers in the mitochondrial enzyme fumarate hydratase that converts fumarate to malate (14). These mutations are rarer and of a recessive nature—wherein both alleles of the gene
encoding fumarate hydratase must be inactivated. Because fumarate is known to be an inhibitor of the aforementioned family of dioxygenase enzymes, it is conceptually logical to hypothesize that the accumulation of excessive amounts of fumarate might inactivate the prolyl-hydroxylase
enzymes that normally keep the HIF transcription factors in an inactive state (and, perhaps, likewise impose alterations in the epigenetic state of cancer cells). Finally, the same reasoning might apply to rare mutations in the human genes encoding mitochondrial succinate dehydrogenase and its assembly factors that are believed to contribute to the formation of paragangliomas and pheochromocytomas (15, 16). The exciting angle on these studies of cancer-causing mutations in the genes encoding isocitrate dehydrogenase, fumarate hydratase, and succinate dehydrogenase is that they formally predict the concept that the metabolic state of a cell can indeed exert control over its regulatory state, thereby confirming the reciprocal relationship between the two.

One enduring complication that shows little sign of resolution concerns how cancer cells are grown and studied in tissue culture plates. We routinely grow cancer cells under conditions of unlimited access to glucose—not to mention oxygen, vitamins, known and unknown growth factors present in serum, and every nutritional fertilizer imaginable. The growth environment of cancer cells within tumors, especially solid tumors, could hardly be more different than that of cells being grown under standard, tissue culture conditions. It is reasonable to suspect that cancer cells weave their way through exceptionally selective oncogenetic gymnastics to achieve a growth-permissive metabolic state. If so, what features of this metabolic state can be anticipated to be preserved and studied when cells are practically grown in Karo syrup or alongside floating logs of Snickers Bar candy? Returning to the “you can’t get there from here” theme, it is predictable that we will have to find more biologically sound ways to grow cancer cells in culture to favorably use them in efforts to discover therapeutics that might exploit their unique metabolic state.

The resurrection of research involving or including metabolism is clearly upon us—that is the good news. The bad news is that the field was sufficiently snuffed over the past several decades that we have precious few scientists who have been trained to genuinely understand intermediary metabolism. Just because we can now pronounce the names of the metabolic enzymes whose encoding genes and mRNAs show up on our ChIP-Seq (chromatin immunoprecipitation–sequencing) and DNA microarray lists does not necessarily mean that we can put two and two
together. Despite the handicap of not being able to field an experienced team at this point, it is encouraging to see favorable trends that may enable negotiation of discovery routes that were, until now, largely obscure.

References and Notes

1. O. Warburg, K. Posener, E. Negelein, Biochem. Z. 152, 319 (1924).
2. O. Warburg, Science 123, 309 (1956).
3. N. V. Iyer et al., Genes Dev. 12, 149 (1998).
4. D. W. Parsons et al., Science 321, 1807 (2008).
5. H. Yan et al., N. Engl. J. Med. 360, 765 (2009).
6. L. Dang et al., Nature 462, 739 (2009).
7. A. C. R. Epstein et al., Cell 107, 43 (2001).
8. R. K. Bruick, S. L. McKnight, Science 294, 1337 (2001).
9. P. H. Maxwell et al., Nature 399, 271 (1999).
10. M. Ohh et al., Nat. Cell Biol. 2, 423 (2000).
11. Y. Tsukada et al., Nature 439, 811 (2006).
12. J. R. Whetstine et al., Cell 125, 467 (2006).
13. P. A. Cloos et al., Nature 442, 307 (2006).
14. J. S. Isaacs et al., Cancer Cell 8, 143 (2005).
15. M. A. Selak et al., Cancer Cell 7, 77 (2005).
16. H. X. Hao et al., Science 325, 1139 (2009).
17. I thank M. Brown, R. Bruick, B. Tu, and J. Rutter for helpful editorial comments.

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