Targeting vascular sprouts Lee B. Rivera and Gabriele Bergers Science 344, 1449 (2014); DOI: 10.1126/science.1257071

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in E. coli (9). Similarly, analysis of synthetic lethal screens in yeast led to corrections of the pathways leading to NAD synthesis (7). Also, reconciliation of prediction failure led to the description of gluconate kinase in human cells (10). Many false predictions can be resolved by fixing relatively few components in a model’s reactome. For example, a recent study was able to reconcile 2442 false model predictions from the E. coli GEM by updating the function of just 12 genes (11). A GEM thus provides a platform not only for formalizing and solidifying the understanding of a target organism but also for the systematic discovery of its missing parts and functions. Double-knockout collections being produced for E. coli (where highly quantitative measurements under defined growth conditions can be obtained) should lead to millions of possible growth predictions in just a few years. Such experiments and computational predictions would enable largescale predictions of gene-gene interactions. If such efforts for model organisms are successful and genetic manipulation techniques for less-characterized organisms become readily available, then we can foresee a massive scale-up in phenotypic predictions for a spectrum of organisms. For instance, experimentally constructed transposon libraries with sequencing (TnSeq) can now be used to examine the effect of gene knockouts in different environments for organisms with little to no existing biochemical data (12). A draft GEM for such an organism can thus be improved quickly using the same algorithms that reconcile the differences between predicted and observed growth states mentioned above. Such developments would hopefully make the fundamental underpinnings of microbial physiology as solid as our understanding of microbial genomics has become over the past 15 years. By strategically choosing target organisms from across the phylogenetic tree, this approach would allow the comprehensive discovery of the metabolic processes used by the many diverse organisms on our planet. ■

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REFERENCES

1. A. Bordbar et al., Nat. Rev. Genet. 15, 107 (2014). 2. A. M. Feist, B. O. Palsson, Curr. Opin. Microbiol. 13, 344 (2010). 3. J. Monk et al., Nat. Biotechnol. 32, 447 (2014). 4. T. Baba et al., Mol. Syst. Biol. 2, 2006.0008 (2006). 5. M. Costanzo et al., Science 327, 425 (2010). 6. K. Kobayashi et al., Proc. Natl. Acad. Sci. U.S.A. 100, 4678 (2003). 7. B. Szappanos et al., Nat. Genet. 43, 656 (2011). 8. J. D. Orth, B. Ø. Palsson, Biotechnol. Bioeng. 107, 403 (2010). 9. K. Nakahigashi et al., Mol. Syst. Biol. 5, 306 (2009). 10. Ó. Rolfsson et al., Biochem. J. 449, 427 (2013). 11. D. Barua et al., PLOS Comput. Biol. 6, e1000970 (2010). 12. A. Deutschbauer et al., PLOS Genet. 7, e1002385 (2011). SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/344/6191/1448/suppl/DC1 References for figure 10.1126/science.1253388

ANGIOGENESIS

Targeting vascular sprouts Manipulating metabolism could control angiogenesis By Lee B. Rivera and Gabriele Bergers

A

ngiogenesis, the formation of new blood vessels, has long been recognized as a hallmark of cancer (1). Its functional importance for the manifestation and progression of tumors has been validated by the therapeutic effects of angiogenesis inhibitors. However, the overall effectiveness of several approved therapies that target the proangiogenic pathways controlled by vascular endothelial growth factor (VEGF) have had rather mod-

est and transient effects in the clinic (2). This is because tumors can activate alternative pathways to adapt to vascular growth restrictions. Emerging evidence (3–5) indicates that targeting metabolic pathways in blood vessel endothelial cells may be a new and promising strategy because metabolism in these cells not only fuels vascular expansion, but also regulates the very formation of blood vessels. Endothelial cells that form the inner lining of blood vessels are in general quiescent in the adult but have to quickly become activated to form new vascular branches during normal physiological processes or upon injury. Much effort has been devoted to understanding the molecules that regulate the multiple steps of vessel formation, but how metabolic regulation in endothelial cells affects angiogenesis has remained largely unexplored. Glycolysis apparently provides the necessary energy for endothelial cells to switch from quiescence to angiogenesis, and in addition, directly controls several aspects of vascular sprouting (3). This metabolic pathway is much less efficient than oxidative phosphorylation, yet,

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surprisingly, endothelial cells rarely use the latter process (6, 7), even when they proliferate and migrate to form new sprouts (3). Why would these cells choose an inefficient process to produce sufficient energy? During glycolysis, glucose is metabolized to pyruvate, which is then either converted to lactate, yielding two molecules of adenosine 5ⴕ-triphosphate (ATP), or, in the presence of oxygen, fully oxidized in mitochondria to produce 36 ATP molecules. Therefore, it might be expected that endothelial cells would favor pyruvate oxidation

as they have immediate access to oxygen in blood. But consider that the main purpose of neovascularization is to oxygenate areas that suffer from oxygen deprivation. If endothelial cells relied on pyruvate oxidation, they would deplete oxygen from the blood and thereby reduce the amount of oxygen available to diffuse into the surrounding hypoxic tissue. De Bock et al. (3) investigated whether glycolysis not only fuels vessel growth but somehow regulates it. The study identified 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3; also called PFK2) as the most abundantly expressed glycolytic enzyme in endothelial cells. PFKFB3 converts fructose-6-phosphate to fructose-2,6 bisphosphate, an allosteric activator of phosphofructokinase-1 (PFK-1). Therefore, inhibition of PFKFB3 led to only a partial reduction in glycolytic flux and resulted in cell quiescence. This is in contrast to other approaches for targeting glycolysis that Department of Neurological Surgery, Brain Tumor Research Center, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA. E-mail: [email protected] 27 JUNE 2014 • VOL 344 ISSUE 6191

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1449

INSIGHTS | P E R S P E C T I V E S

A

Oxidative phosphorylation PGC-1α

B Glycolysis

Notch Tip cell (angiogenesis)

Angiogenic cues PFKFB3

3PO

Glucose

Glycolysis

PFKFB3 Fructose-6P

Tip cell Notch PFKFB3

PFK-1

Fructose-2,6BP

Fructose-1,6BP

Pyruvate Stalk cell Notch

Oxidative phosphorylation

Lactate

PFKFB3

Metabolism regulates neovascularization. (A) The glycolysis enzyme PFKFB3, transcriptional coregulator PGC-1α, and the signaling protein Notch control metabolism and the formation of tip cells or stalk cells. Small molecules that induce PGC-1α expression have been identified but not tested for their capacity to inhibit angiogenesis. (B) The small molecule 3PO inhibits formation of angiogenic sprouts by blocking PFKFB3 activity. P (phosphate), BP (bisphosphate).

1450

Notch-inhibitory effect exerted by PFKFB3, PGC-1α induced Notch activation, thereby presumably blocking sprout formation and, hence, promoting the stalk phenotype. Furthermore, PGC-1α blunted angiogenic signaling. Thus, PGC-1α activation may represent an additional metabolic approach to block pathologically angiogenic diseases. Molecules that have the capacity to induce PGC-1α expression have already been identified in genetic screens and include microtubule inhibitors and agents that block protein synthesis (11). Importantly, these molecules can activate PGC-1α–mediated oxidative phosphorylation; however, whether or not pharmacological induction of PGC-1α expression can suppress angiogenesis has not been tested. Nevertheless, recent proof-of-principle experiments support the idea of targeting endothelial cell glycolysis to treat disease-associated angiogenesis. Schoors et al. (4) used the small molecule 3-(3-pyridinyl)-1-(4-pyridinyl)-2propen-1-one (3PO) to block PFKFB3 and test this notion in preclinical models of psoriasis and colitis, where it successfully blocked angiogenesis. These inflammatory disorders are exacerbated by angiogenesis. Interestingly, targeting PFKFB3 also enhanced the efficacy of VEGF-pathway inhibitors in animal models of macular degeneration and retinopathy, two conditions in which angiogenesis causes the disease. Inadequate blockade of angiogenesis by VEGF-pathway inhibitors may be due to glycolysis that parallels VEGF signaling to drive neovascularization. It will be very interesting to see whether blocking PFKFB3-driven glycolysis or ac-

tivation of PGC-1α expression could represent a new opportunity to improve anti-angiogenic therapy in cancer (2). Importantly, inhibition of PFKFB3 or induction of PGC-1α expression skews the vasculature toward a homeostatic state; therefore, the manipulation of either factor affects endothelial cells only in the context of angiogenesis, leaving the quiescent vasculature unperturbed. Furthermore, tumor cells, like endothelial cells, favor glycolysis in the so-called Warburg effect, for a number of proposed reasons, from adapting to low-oxygen environments within tumors to shutting down mitochondria and its associated programmed cell death mechanism that would kill cancerous cells. Recent studies have identified targets to block tumor glycolysis with very promising results, but it remains to be determined whether such results are due to effects solely in tumor cells or if effects in the tumor vasculature are actually contributing factors (12). ■ REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

D. Hanahan, R. A. Weinberg, Cell 100, 57 (2000). G. Bergers, D. Hanahan, Nat. Rev. Cancer 8, 592 (2008). K. De Bock et al., Cell 154, 651 (2013). S. Schoors et al., Cell Metab. 19, 37 (2014). N. Sawada et al., Cell Metab. 19, 246 (2014). A. Dobrina, F. Rossi, Biochim. Biophys. Acta 762, 295 (1983). A. Krützfeldt, R. Spahr, S. Mertens, B. Siegmund, H. M. Piper, J. Mol. Cell. Cardiol. 22, 1393 (1990). C. Granchi, F. Minutolo, ChemMedChem 7, 1318 (2012). P. Carmeliet, R. K. Jain, Nature 473, 298 (2011). R. Blanco, H. Gerhardt, Cold Spring Harbor Perspectives in Medicine 3, a006569 (2013). Z. Arany et al., Proc. Natl. Acad. Sci. U.S.A. 105, 4721 (2008). F. L. Muller et al., Nature 488, 337 (2012). 10.1126/science.1257071

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completely shut down the process and result in cell death (8). Vascular sprouting is a dynamic process involving distinct types of endothelial cells. A tip cell that bears extending filipodia moves at the front of a sprouting vessel and is followed by proliferating stalk cells that elongate the sprout (9, 10). VEGF activates tip cells, whereas the signaling protein Notch suppresses them and stimulates stalk cells (see the figure). Not only is PFKFB3 required for tip cell formation, it can override the ability of Notch to promote stalk cell behavior (3). Interestingly, although sproutinducing signals like VEGF and fibroblast growth factor can induce PFKFB3 expression, glycolysis has no effect on signaling by VEGF receptor 2, suggesting growth factor signaling and glycolysis promote tip cell function independently. Yet another reason why endothelial cells favor glycolysis is that glycolytic enzymes are selectively compartmentalized within tip cell filipodia (3). Thus, tip cell protrusions extending into areas of low oxygen tension can rapidly generate local ATP for use in processes with high energy demand such as the assembly of cytoskeletal actin filaments. In line with these findings, Sawada et al. (5) recently discovered a metabolic regulator in endothelial cells that blocks angiogenesis by inducing Notch activity while impeding angiogenic signaling. Peroxisome proliferator-activated receptor gamma coactivator 1–α (PGC-1α) is a transcriptional coactivator involved in mitochondrial biogenesis and activity, and it is highly expressed in endothelial cells of diabetic patients and mice. In contrast to the