Cancer Cell Metabolism: There Is No ROS for the Weary Chi V

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IN THE SPOTLIGHT Cancer Cell Metabolism: There Is No ROS for the Weary Chi V. Dang Summary: Using a high-throughput short-hairpin RNA library screen targeting 222 metabolic nodes, Ros and colleagues identified 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), a glycolytic enzyme that shunts glucose into the pentose phosphate pathway for NADPH production, as a critical node for the survival of prostate cancer cells. Blocking PFKFB4 induces reactive oxygen species and cancer cell death, suggesting that PFKFB4 could be therapeutically targeted. Cancer Discov; 2(4); 304–7. ©2012 AACR. Commentary on Ros et al., p. 328 (1).

Cancer cells are on the go, adsorbing lipids and picking up Cancer cells with deregulated MYC are addicted to glucose nutrients for deregulated self-replication, which frequently and glutamine, whereas healthy cells diminish the expres- culminates in the death of the host. In this issue of Cancer sion of MYC under nutrient- or oxygen-deprived conditions. Discovery, Ros and colleagues (1) document the dependence Hence, the proto-oncogene responds to nutrient availabil- of prostate cancer cells on 6-phosphofructo-2-kinase/fruc- ity, whereas the deregulated oncogene, through gene ampli- tose-2,6-biphosphatase 4 (PFKFB4), a glycolytic enzyme that fication or chromosomal translocation, for example, severs plays an important role in attenuating reactive oxygen species its ties to the external world, inducing a constitutive growth (ROS) for the weary cancer cell to survive and in lipid synthe- program that is addicted to nutrients. In this regard, it is sis for the cancer cell to replicate. notable that MYC expression is elevated in patients with pros- Normal mammalian cells, such as cells of the gut or bone tate cancer and that MYC plays a central role in murine mod- marrow, can replicate at significant rates for tissue ho- els of prostate cancer and maintenance of human prostate meostasis. Tissue stem cells or immune cells stimulated by cancer, such that knockdown of MYC expression results in growth factors or cytokines undergo a signal transduction diminished prostate cancer cell proliferation (5). and transcriptional program that permits both cell mass The import of glucose and glutamine or other amino acids accumulation through macromolecular synthesis (e.g., is essential for growing cells to generate ATP and provides car- lipid, protein, and nucleic acid synthesis) and acquisition bon skeletons as building blocks for ribosomes, membranes, of a commensurate bioenergetic supply. Nutrient-depleted, various cellular organelles, and replication of the genome lowered energy conditions trigger AMP-activated protein [Fig. 1A (3)]. Glucose is converted to pyruvate and in turn kinase, which diminishes cellular ATP consumption and into acetyl-CoA for mitochondrial oxidation, or into lactate stimulates self-eating or autophagy to recycle cellular com- under hypoxia. Glucose, upon conversion to glucose-6-phos- ponents as energy. phate, is also catabolized by the pentose phosphate pathway In nutrient-replete conditions, glucose modulates tran- (PPP) to produce NADPH, which provides the key reductive scription through its conversion to intracellular hexose- power for biosynthesis and redox balance and ribose for nu- phosphate and hexosamine, whereas glutamine import is cleic acid synthesis (Fig. 1B). Glutamine, however, enters the required for activation of mTORC1 to stimulate cell growth tricarboxylic acid (TCA) cycle via conversion to glutamate (2, 3). Growth factor-receptor signaling further bolsters the and then to α-ketoglutarate, which is further catabolized by cell growth program by activating mTORC2 and AKT, which the TCA cycle [Fig. 1B (6)]. The byproducts of glucose and can directly stimulate glycolysis, and early response genes glutamine catabolism are ROS resulting from electron leak- such as MYC (Fig. 1A). The MYC gene produces a pleiotropic age from the electron transport chain, carbon dioxide, water, transcription factor that stimulates ribosome biogenesis, and lactate that are all disposed by different mechanisms nucleotide synthesis, DNA replication, and the import of (Fig. 1B). ROS, being inherently genotoxic, are titrated by lay- both glucose and glutamine to support the bioenergetic ers of safeguards, including NADPH-glutathione, peroxire- needs of a growing cell (4). doxins, superoxide dismutase, and catalase. As such, rapidly proliferating cancer cells must deal with the ROS load to Author’s Affiliation: Abramson Cancer Center, Abramson Family Cancer survive. Research Institute, Division of Hematology-Oncology, Department of Ros and colleagues (1) undertook a genetic screen with a Medicine, Perelman School of Medicine, University of Pennsylvania, short-hairpin RNA library targeting 222 genes involved in Philadelphia, Pennsylvania metabolism to determine whether prostate cancer cell lines, Corresponding Author: Chi V. Dang, Abramson Cancer Center, Abramson Family Cancer Research Institute, Division of Hematology-Oncology, as compared with a normal prostate cell line, are dependent Department of Medicine, Perelman School of Medicine, University of on critical metabolic nodes. They found that among 3 pros- Pennsylvania, Philadelphia, PA 19072. Phone: 215-662-3929; Fax: 215- tate cancer cell lines (DU145, PC3, and LNCaP) that all dis- 662-4020; E-mail: [email protected] play a dependency on glucose for viability, 2 critical genes doi: 10.1158/2159-8290.CD-12-0069 were required for their survival under lipid-limiting culture ©2012 American Association for Cancer Research. conditions. One gene is PRKAB1, encoding the β1 subunit

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A Glucose

R T K Ribose-5- PI3K phosphate

NADP+ NADPH

mTORC2 Lactate Pyruvate AKT Acetyl-CoA Oxaloacetate MYC Citrate Malate

Fumarate Isocitrate GTP Ribosome biogenesis Succinate a-Ketoglutarate mTORC1 Glutamate

Glutamine Leucine

B p53 Glucose

Glucose-6-phosphate Ribose-5- phosphate TIGAR GPI PFKFB4 NADP+ NADPH Fructose-6-phosphate

PFKFB3 PFK1 Fructose-2,6-bisphosphate

Fructose-1,6-bisphosphate

ROS Pyruvate Lactate

Acetyl-CoA

Figure 1. A, diagram depicting receptor signaling and nutrient import. Receptor signaling, through phosphoinositide 3-kinase (PI3K), stimulates mTORC2 and AKT as well as the MYC oncogene, which stimulates ribosome biogenesis and biomass accumulation for cell proliferation. Glucose and glutamine import stimulated by MYC induces mTORC1 and a collateral pathway of activating protein synthesis and ribosome biogenesis. Glutamine is shown converted to glutamate and then to α-ketoglutarate for further mitochondrial oxidation. Glucose is converted to lactate or to acetyl-CoA, which is further oxidized in the mitochondrion. Glucose is also shown shunted to the PPP to produce NADPH and ribose-5-phophate. B, glycolytic conversion of glucose to pyruvate and its conversion to ribose-5-phosphate via the pentose phosphate pathway. Glucose is transported into the cell and phosphorylated to glucose-6-phosphate by hexokinases, and then to fructose-6-phosphate by glucose phosphate isomerase. Fructose-6- phosphate is phosphorylated by PFK1, which is positively allosterically regulated by fructose-2,6-phosphate (F2,6P), to fructose-1,6-phosphate and subsequently metabolized to pyruvate for further oxidation in the mitochondrion, a rich source of ROS. The levels of F2,6P are regulated by the PFK2 family members, PFKFB4 and TIGAR (a target of p53) that dephosphorylates F2,6P and PFKFB3 that generates F2,6P from fructose-2- phosphate. Increased PFKFB4 or TIGAR lowers F2,6P and hence decreases PFK1 activity, shunting glucose-6-phosphate into the pentose phosphate pathway for production of ribose-5-phosphate and NADPH. NADPH reduces glutathione and thereby inhibits ROS. of AMP-activated protein kinase, and the other, PFKFB4, is a ligands, such as the negative regulator citrate produced from member of the phosphofructokinase 2 (PFK2) family. the TCA cycle or the positive regulator 2,6-fructose Phosphofructosekinase 1 (PFK1) is a rate-limiting glyco- bisphosphate (2,6-FBP) produced by the PFK2 family lytic enzyme that converts fructose-1-phosphate to fructose- members (Fig. 1B). The PFK2 enzymes have dual kinase and 1,6-bisphosphate (1,6-FBP). 1,6-FBP is then converted to phosphatase activities, producing or degrading 2,6-FBP. glyceraldehyde-3-phosphate and dihydroxyacetone-3-phos- There are 4 known PFK2 members [PFKFB1-4 (7)]. phate, which are further catabolized to pyruvate for mito- PFKFB3 and PFKFB4 are both activated by hypoxia, but chondrial oxidation. PFK1 is highly regulated by allosteric their biochemical functions are distinctly different, with

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PFKFB3 having more kinase activity and PFKFB4 having fraction of lipids through its conversion to α-ketoglutarate more phosphatase activity (Fig. 1B). This duo therefore and subsequent reductive carboxylation to isocitrate, which is provides a yin-yang control over the levels of 2,6-FBP that then converted to citrate for lipid synthesis via ATP citrate ly- potently activates PFK1 to drive glycolysis; in the absence of ase (10, 12). Furthermore, under limiting glucose levels in the 2,6-FBP, glucose is shunted away from PFK1 toward the PPP. hypoxic tumor microenvironment, glutamine fills in the TCA The PFK2 node is also regulated by p53, which transacti- cycle as well as supports the synthesis of glutathione (13). vates TIGAR, producing a PFK2 member that also has greater Notwithstanding these nuances, the article by Ros and col- phosphatase than kinase activity (7). Hence, p53 activation leagues (1) and earlier studies from the laboratories of Cheng shunts glucose toward the PPP (Fig. 1B). and Vousden (7) and Clem and colleagues (14) underscore the The PPP converts glucose to ribulose-5-phosphate through importance of the PFK2 family of enzymes in tumor metabo- glucose-6-phosphate dehydrogenase and 2 other enzymes in lism, which might be exploitable for cancer therapy. the oxidative phase of the PPP, which produces NADPH from Indeed, Clem and colleagues (14) suggested that inhibition NADP+ (Fig. 1). Ribulose-5-phosphate is then converted in of PFKFB3 could be therapeutic via diminishing glycolytic the nonoxidative phase of the PPP to ribose-5-phosphate flux, but Ros and colleagues (1) could not corroborate the im- for nucleic acid synthesis or to other intermediates such as portance of PFKFB3 for prostate cancer cell survival. In this glyceraldehyde-3-phosphate or fructose-6-phosphate, which regard, it will be of significant interest whether highly specific can enter glycolysis. As such, the PFK2 enzymes play an inhibitors of PFKFB4 could be therapeutic in a subset of hu- important role in the switch away from glycolysis toward the man prostate and other cancers. The challenge for targeting pentose phosphate pathway, resulting in the production of cancer cell metabolism is being able to precisely profile the NADPH, which is important both for dampening oxidative cancer cell metabolome according to the type of cancer and stress through increased glutathione levels and for fatty acid to identify the metabolic Achilles’ heels. In fact, the oncogenic synthesis. The unbiased screen reveals that PFKFB4 is critical drive and organ site profoundly influence the cellular usage of for prostate cancer cell line survival, particularly for redox glucose or glutamine, as recently reported by Yuneva and col- homeostasis, because treatment of prostate cancer cell lines leagues (15). The heterogeneous tumor microenvironment and with an antioxidant could partially rescue their demise from the ability of cancer cells to handle ROS through endogenous diminished PFKFB4 expression (1). antioxidant systems are expected to profoundly affect the re- Intriguingly, the necessity of PFKFB4 for cell survival sponse of cancer cells to metabolic inhibitors. Hence, there is extends beyond prostate cancer because other cancer cell no good ROS for the weary cancer cell, which also produces lines are also sensitive to knockdown of PFKFB4 expression. various toxic byproducts such as lactate and carbon dioxide Why PFKFB4 levels are elevated in prostate and other can- that have been exploited for therapy through inhibition of lac- cer cell lines is not known. Diminished PFKFB4 expression, tate transporters, MCT1 and MCT4, or carbonic anhydrase however, does not seem to dramatically affect growth of the CA9 (16). Insights from previous studies and the article by Ros normal human prostate epithelial cell line RWPE1. Until and colleagues (1) suggest that blocking the cellular exhaust there are further studies, it seems premature to suggest that pipes is, therefore, a compelling strategy to interrupt cancer PFKFB4 is not essential for normal cell proliferation in light cell metabolism, in addition to disrupting the fuel lines (3). of a significant overlap in the metabolic profiles of normal T cells and that of lymphoma. MYC induces glucose and Disclosure of Potential Conflicts of Interest glutamine metabolism in normal T-cell mitogenesis and pro- No potential conflicts of interest were disclosed. liferation of neoplastic lymphocytes, suggesting a significant overlap between normal and neoplastic cell metabolism (8). Author Contributions The difference, however, is that deregulated oncogene expres- Writing, review, and/or revision of the manuscript: C. V. Dang sion renders tumor cells addicted to nutrients (4). The article by Ros and colleagues (1) further documents a Acknowledgments difference in the metabolism of prostate cancer cells versus The author thanks Brian Altman for his comments. the normal prostate, which exhibits a truncated TCA cycle that produces vast amounts of citrate. Citrate is secreted into Grant Support the seminal fluid, presumably providing some protective This work is supported by the National Cancer Institute, the measures to the sperm cells. All 3 prostate cancer cell lines Leukemia and Lymphoma Society of America, a Stand Up To Cancer studied by Ros and colleagues (1) are highly glycolytic. It Dream Team Translational Cancer Research Grant, a Program of the is notable, however, that RWPE1 is also glycolytic and does Entertainment Industry Foundation (SU2C-AACR-DT0509), and not display the truncated TCA cycle of normal prostate. Ros the Abramson Family Cancer Research Institute. and colleagues (1) also report the lack of significant dependence of these prostate cancer cell lines on glutamine, which Received February 20, 2012; accepted February 21, 2012; pub- contrasts with some previous published studies; particularly, lished online April 11, 2012. Gao and colleagues (9) documented the dependency of PC3 cells on glutamine and particularly on glutaminase, which References shunts glutamine into the TCA cycle. . 1 Ros S, Santos CR, Moco S, Baenke F, Kelly G, Howell M, et al. It is also notable that glutamine appears to be a signifi- Functional metabolic screen identifies 6-phosphofructo-2-kinase/ cant substrate for the TCA cycle, particularly under hypoxia fructose-2,6-biphosphatase 4 as an important regulator of prostate (10–13). In fact, glutamine can contribute to a significant cancer cell survival. Cancer Discov 2012;2:328–43.

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Chi V. Dang

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