In recent years there has been resurgence in interest in metabolism as a possible area for development of novel anti-cancer agents. This has been fuelled by advances in technology and understanding of cellular metabolism and how targeting this area could be of therapeutic benefit.

The drivers behind these advances have come from academic-led research pushing our understanding of cancer cell metabolism and the interplay between these pathways and other cancer related signalling events linked to changes in oncogene or tumour suppressor gene status/function. In the past few years this spiralling interest has lead to a rapid increase in the number of publications on metabolism, multiple focused cancer metabolism conferences and the renewed interest from the pharmaceutical industry in this area of anti-cancer research.

This interest has lead to the establishment of several new companies focused on this area and to the generation of partnerships between the Pharma industry and research organisations to understand this complicated field and hunt for new targets. For example, Agios Pharmaceuticals was founded by key academic experts in cancer, oncogenic signalling and metabolism research (Lewis Cantley, Craig Thompson and Tak Mak) to focus on cancer metabolism as a new approach to cancer treatment and has an active pipeline of metabolism targets including isocitrate dehydrogenase (IDH1/2)1 and Pyruvate kinase-M2 (PKM2) 2, targets which will be discussed in more detail later. Agios has also recently entered into a $130 million strategic alliance with Celgene to explore novel drug targets in Cancer metabolism. Cornerstone Pharmaceutical was also founded through academic researchers (Paul Bingham and Zuzana Zachar) with a focus on targeting cancer metabolism and has a candidate drug, CPI-613, which targets pyruvate dehydrogenase3 currently in multiple Phase I/II clinical trials. Another biotech, Advanced Cancer Therapeutics, has invested in research at the James Graham Brown Cancer Centre and the University of Louisville Research Foundation to pursue cancer metabolism targets for example; PFKFB3, a glycolytic regulator4 and choline kinase  component of lipid metabolism pathways5. Likewise AstraZeneca is midway through a multi-project three-year alliance with Cancer Research Technology, Cancer Research UK’s commercialisation and development arm to work on a portfolio of targets selected from Cancer Research UK’s biological research in the emerging field of cancer metabolism.

Many potential drug targets within the metabolic field are currently being evaluated as potential therapeutics in all stages of the drug development progress from early development, through pre-clinical development and into clinical trials although as yet no agent targeting a core metabolic pathway has been approved for cancer6. Figure 1 summaries some of the core metabolic pathways and some of the agents in development looking to target different areas of cancer metabolism. In order to fully understand why there are so many agents and pathways under consideration in cancer metabolism it is important to investigate the main drivers for targeting cancer metabolism. The rest of this review will therefore consider why cancer metabolism could make an attractive area for therapeutic intervention and how our understanding of these pathways and advances in technology/knowledge is driving the hunt for new therapeutics.

Cancer metabolism and the Warburg Effect
Dividing cells require ATP to maintain energy status, increased biosynthetic intermediates and maintenance of cellular redox status. In order to meet these needs carbohydrates, proteins, nucleotide and lipid alterations are required. Both cancer cells and rapidly proliferating normal cells require some of these adaptations for proliferation, however cancer cells must implement these processes in very different and often harsh or stressful environments where nutrients supplies may be low and where redox balance, pH and oxygen levels may not be maintained. Cancer cells have found ways to adapt to these dynamic situations and regulate their metabolic status in order to survive, grow and even prosper.

The links between cancer and altered metabolism is not a new phenomenon. Otto Warburg, a Nobel prize-winning scientist (for the discovery of cytochrome oxidase) noted more than 80 years ago that in tumour tissue slices ATP is generated from glucose via aerobic glycolysis which is an oxygen independent process rather than by oxygen dependent oxidative phosphorylation even when oxygen is present7,8. This switch in ATP generation which has been termed the Warburg Effect is initially paradoxical as while ATP generation is more rapid via glycolytic pathways, far less ATP is generated (2 molecules ATP/molecule glucose) than via oxidative phosphorylation (up to 36 molecules ATP/molecule glucose).

In more recent times this switch has been attributed to the ability of glycolytic pathways to supply essential intermediate components and co-factors via branches off of the core glycolysis pathway including via the pentose phosphate pathway which supplies NADPH for redox balance and lipid metabolism and ribose-5-phosphate for nucleotide synthesis and via the serine biosynthesis pathway (Figure 1)9,10. Alongside this it has been postulated that glycolytic adaption could be the result of adaptations to hypoxic conditions during early tumour development and in order to generate ATP and metabolites at a higher rate when glucose is not limiting11. What is clear is the Warburg shift demands that tumour cells implement an abnormally high rate of glucose uptake to meeting their increased demands for biosynthesis, energy and reducing equivalents.

The advent of Flurodexoyglucose-positron emission topography (18F-FDG-PET) imaging12, in which a radioactive glucose analogue is used to assess glucose uptake, has confirmed that many tumour types have high glucose uptake and is now used as part of clinical diagnostic packages for tumours. 18F-FDG (2-dexoy-2-(18F)fluoro-D-glucose), first synthesised in the 1970s is a glucose analogue which enters the cell in normal ways and is phosphorylated like normal glucose to prevent it being released again but cannot then be further processed by glycolytic pathways before radioactive decay and so is a good reflection of glucose uptake in the body13,14. This ability to monitor glucose within tumours and surrounding tissues and the clear increases in glucose uptake and utilisation in tumours confirms aspects of Warburg’s hypothesis and underlies the important of glucose metabolism in many cancers15,16.

It has also been know for more than 50 years that many tumours have increased rates of glutamine uptake and consumption. In fact many cancer cells cannot survive without exogenous glutamine and display a glutamine addiction17. As was seen with glucose, the initial assumption was that the metabolism of glutamine by tumours is inefficient. However, recent studies have shown that glutamine is a key initial substrate in many processes essential for cancer cell maintenance and growth. Products of glutamine metabolism have been found to be essential for the generation of acetyl CoA (the starting block of lipid synthesis), for NADH generation (for lipid synthesis and redox balance), for glutathione synthesis (for redox balance) and for serine synthesis (for nucleotide and protein synthesis)(Figure 1)18,19. A large proportion of glutamine is also converted to lactate in a process which generates NADPH an essential reducing equivalent in lipid and nucleotide synthesis and in redox balance. The multi-step conversion of glutamine to lactate helps to explain high lactate levels in tumours even when glycolytic flux has been slowed to generate key intermediates via branched pathways20,21 (Figure 1).

For both glucose and glutamine metabolism improved imaging techniques coupled with enhanced methods to monitor metabolic flux and identify metabolites (nmr and mass spectroscopy) has really enabled researchers to elucidate what is happening to the key metabolic start points in cancer cells compared with other tissues and help to understand how cancer cells adapt to use these effectively to maintain growth and survival22,23. The advent of technologies such as RNAi, alongside advances in genomic and proteomic profiling, metabolic modelling and improved access to tumour samples, have proved invaluable in allowing researches to really probe metabolic functionality in cancer. Coupled with the enhanced understanding into the fates of glucose and glutamine this has driven advances in understanding the complex nature of metabolic pathways in cells and how these are deregulated in cancers24,25.

Cancer metabolism pathways: drivers and their potential metabolic targets
In recent years what has really catapulted cancer metabolism right back into the spotlight is the understanding of the mechanism by which metabolic adaptations are controlled and regulated in tumours by known oncogenic signalling mechanism25. Alongside this has been the discovery that within several metabolic components there are cancer-related mutations (ie IDH1/2)26,27 or cancer- specific isoforms (ie PK-M2)28 that are critically linked to progression of certain tumour types. The ability to sequence large sample banks of tumours and understand the data has unlocked many of the secrets of different cancers and helped us begin to understand what drives tumour formation and progression with deregulation of cellular energetic now recognised as one of the hallmarks of cancer29.

It is now clear that many oncogenic (Myc18,30,31, PI3k/AKT32-34, Ras10,35) and tumour suppressor proteins (p5336-38, PTEN39,40, LKB141,42) directly affect the expression, regulation and activity of key components of metabolic pathways and it is now believed that these tumourigenic alterations act in part to drive cancer progression via promoting metabolic adaptation towards enhanced glucose and glutamine dependence (see Table 1). For example, Myc has been shown to upregulate glutaminolysis via increasing the expression key components of the glutamine metabolic pathway43 and to enhance oxidative metabolism of glucose via increased pyruvate dehydrogenase44 and lactate dehydrogenase expression/activity45. HIF111,46-48, a transcription factor which can be upregulated in hypoxic conditions or by enhanced oncogenic (ie Myc49,50) or decreased tumour suppressor (ie Von Hippel-Lindau51) function is also known to upregulate the expression of many metabolic enzymes including glucose transporters (GLUT448), metabolic regulators (PFKFB352,53, PFKFB452, pyruvate dehydrogenase kinase (PDK1)44,54) and glycolytic enzymes (hexokinase- 2 (HK2)44, PK-M255, lactate dehydrogenase (LDHa)56, monocarboxylate transporter 4 (MCT4)57). This suggests that metabolic adaptations also play a role in maintaining tumour growth and survival in hypoxic conditions. Akt also increases glycolysis by increasing glucose transporter expression32 and facilitating hexokinase translocation to the mitochondria where it functions to initiate glycolytic flux34,58. The tumour suppressor protein p5359,60 has also been shown to regulate expression of various glycolytic proteins including upregulation of hexokinase and of a protein called TIGAR which acts as a negative regulator of glycolysis61,62. p53 also promotes oxidative phosphorylation via upregulation of SCO263 and suppresses glycolysis via expression of PTEN, a negative regulator of the PI3K pathway64. Therefore, while loss of p53 may drive acquisition of the glycolytic phenotype it will be important to understand metabolic regulation in p53 wild type and mutant tumours.

Understanding the interplay between oncogenes and tumour suppressors and metabolic pathways will be key to deciphering how metabolism integrates into tumour initiation and progression and in looking for potential therapeutic targets with clear clinical stratification. The rest of this review will take a stripped-down look at metabolism and introduce a couple of potential metabolism targets which are currently being extensively investigated both by academia and industry.

The basic stages of glucose and glutamine metabolism in cancer
At its simplest level the complex process of glucose and glutamine metabolism can be split into four key phases (Figure 2). The first phase is the uptake of these essential metabolic substrates into the cell via transporter proteins. The process actively imports high concentrations of glucose or glutamine into the cell ready for utilisation and in cancers predominantly involves the transporters GLUT1 and GLUT4 for glucose65 and ASCT2(SLC1A5) for glutamine66. GLUT1 and GLUT4 have been found to be overexpressed in many cancers and to be upregulated by ras and myc signalling65,67. Currently there are no clear inhibitors of GLUT1/4 published although the natural dihydrochalcone, Phloretin is reported to have GLUT inhibitory activity and be effective in inducing apoptosis in in vivo cancer models68. ASCT2 is also overexpressed in some tumours69 and reported to be directly upregulated by myc18,70 although as yet no clear inhibitors of this transporter have been reported. The oncogenic upregulation of glucose and/or glutamine transporters in cancer helps to explain how tumours adapt to facilitate their increased reliance on glucose and/or glutamine and how tumours can accumulate high levels of these metabolites.

The next step is the initial processing of these imported metabolises by conversion into the first metabolic product which can act as an entry substrate into the main metabolic processing pathways. This conversion acts to trap the imported metabolite within the cell and to commit it to further processing into downstream intermediates as well as shift equilibrium balances to allow the influx of more glucose or glutamine to meet the cell’s high demand for these metabolites. For glucose, this initial step is conversion to glucose-6- phosphate by the hexokinase’s of which hexokinase 2 appears to predominant in many cancers71 and has shown to be regulated by both AKT58 and mutant p5372. siRNA studies or use of nonhydrolysable glucose mimics which have been shown to inhibit Hexokinase-2 activity, suggest that modulating Hexokinase-2 activity could be of therapeutic benefit in cancer73-75. Two of these potential hexokinase inhibitors; 2-doexyglucose76- 78 and 3-bromopyruvate71,74 have shown promising anti-cancer activity in multiple pre-clinical models but as yet data from clinical trials has not supported their use as clinical anti-cancer agents. For glutamine the first step is conversion of glutamine to glutamate by glutaminase proteins (GLS1/2)79. GLS1 expression has been shown to be increased in several tumour types and to be under indirect control from Myc via myc-dependent regulation of miR23a/b levels31,80. Studies using siRNA technology81 or potential inhibitors of GLS1 (DON82, 96883) have suggested that inhibition of this target could be of benefit in glutamine dependent tumour cells.

The next phase of metabolite processing involves the conversion of these metabolites into a series of intermediates through metabolic cascades. This process is not a liner and intermediates can be further processed through branched pathways (ie tricarboxylic acid cycle, pentose phosphate pathway or serine biosynthesis pathway) to yield additional products (see Figure 1). The net result of all the processing pathways of core metabolism is; the generation of biosynthetic intermediates for the manufacture of lipids, proteins, nucleotides and complexes sugars, the generation of energy in the form of ATP and the generation of key co-factors, ie glutathione, NADH and NADPH, which are essential for the functionality of many protein and have key roles in protecting the cell from oxidative stress. Multiple proteins on these pathways have been shown to be over expressed in cancer, dependent on oncogenic control or in inhibition studies (RNAi or tool compounds) been shown to be involved in cell proliferation and/or survival mechanisms. Proteins which are of potential interest as possible therapeutic targets include the glycolytic enzymes84 (eg Hexokinase-271, Phosphoglycerate kinase-185,86, Phosphoglycerate mutase87,88 and Pyruvate kinasem228,89- 91); the pentose phosphate proteins (eg Glucose-6-phosphate dehydrogenase92-94, transaldolase95 and transketolase96-98) and lipid synthesis/ fatty acid metabolism targets (eg ATP citrate lyase99,100, fatty acid synthase101,102, monoglyceride lipase103,104 and carnitine palmitoyltransferase 1C105). See 6,84,106 for more detailed reviews of some of these.

The final process within centralised metabolism is the export of end-point products/waste from the cell in order to protect the cell from build up of potentially toxic components and also to modify the extracellular vicinity around the cell which may act to assist the cell’s establishment in this environment. In cancer cells the main exported substance from glucose metabolism is lactate which is effluxed via the transporter proteins MCT1 and MCT457,66. Studies have found MCT transporters to be overexpressed in multiple tumour types107- 110 and that chemical or genetic inhibition of MCT function can reduce tumour growth suggesting that molecular targeted therapy against MCT transporters could be a possible mechanism for targeting cancer metabolism57,111. Inhibitors targeting MCT1 have been successfully shown to affect in vitro and in vivo tumour growth and a MCT1 inhibitor designed by AstraZeneca (AZD-3965) is about to enter clinical trials as part of the CR:UK clinical development partnership. For glutamine metabolism it is believed ammonia released during the process of glutaminolysis diffuses into the extracellular environment by as yet undefined mechanisms. Understanding what these mechanisms could be may offer an attractive therapeutic strategy as inhibiting ammonia removal could cause build up of this toxic product and thereby potentially kill the tumour cell.

Therefore even in this stripped-down version of metabolism it is clear that tumours are adapting to maximise the usage of glucose and glutamine to promote survival and even growth in potential hostile environments and targeting these adaptions could be a therapeutic mechanism.

As understanding of cancer metabolism develops, potential therapeutic targets are identified based on their roles in cancer metabolism coupled with cancer specific expression/isoforms, potential mutations and oncogenic control mechanisms. Targets which fit these profiles have been at the forefront of the new push for cancer metabolism drugs and examples include Pyruvate kinase M2 and Isocitrate dehydrogenase.

Pyruvate kinase M2 (PKM2)
Pyruvate kinase (PK) catalyses the conversion of phosphoenolpyruvate (PEP) into pyruvate in a rate limiting and ATP generating step within glycolysis (Figure 3)2. There are multiple isoforms of PK of which the muscle form is of key interest in cancer cells89. PKM1 is found in muscle and brain and is reported to be constitutively active whereas PKM2 is present in embryonic and adult stem cells and controlled by various regulator mechanisms. It has been widely reported that PKM2 is also overexpressed in many tumour cells and a switch from PKM1 to PKM2 expression in cancer has been proposed2,28,89. The PKM2 isoform is generated by alternative splicing of exon 10 on the PK gene, an event shown to be under myc regulation suggesting a potential oncogenic driver for PKM2 expression in cancers30,112. However, the PKM2 expression hypothesis has been recently questioned in a study which used mass-spectroscopy to identify PK isoforms and reported that PKM2 is expressed in normal tissues as well as cancers and that PKM1 had low expression in cancers as well as in normal tissue113. Although the ratio of PKM2 to PKM1 expression was similar between cancers and matched normal tissues, the actual amounts of each protein were much higher in the tumours suggesting that both PKM2 and PKM1 were overexpressed in tumour samples113.

Studies in which PKM2 is knockdown or replaced by PKM1 have shown that PKM2 is involved in tumour progression and that PKM2 expression confers a tumourigenic advantage over PKM1 expression89. However, it has also been shown that while PKM1 can efficiently promote glycolysis, PKM2 is characteristically found in an inactive state and is inefficient in promoting glycolysis2,28,89,114. PKM2 exists in two possible conformations, an inactive dimer and more active tetramer (Figure 3). Oncogenic tyrosine kinases (eg fibroblast growth factor receptor kinase) have been found to promote the formation of the inactive dimer via the phosphorylation of tyrosine 705 on PKM290,114. PKM2 activity has also been shown to be negatively regulated by acetylation induced by high levels of glucose115. This data collectively suggests that expression of PKM2 in cancer could actually decrease glycolytic flux. While this was initially thought to be counterintuitive, when considered in terms of the cancer cell’s metabolic needs, a mechanism which slows glycolysis is actually potential advantageous. By slowing glycolytic flux the cancer is able to obtain building block, co-factors and precursors by allowing glucose metabolites to enter subsidiary pathways including pentose phosphate, serine biosynthesis, hexcosamine and glycerol synthesis pathway which support cancer proliferation and survival. Another metabolic role for PKM2 is a proposed direct interaction and stabilisation of Hif1 which in turn acts to promote glycolytic metabolism, angiogenesis and cancer progression55. An alternative glycolytic route which bypasses PK in the conversion of PEP into Pyruvate has recently been identified88,116. This route uncouples ATP and Pyruvate generation and could provide biosynthetic intermediates without potential feedback inhibition of glycolysis from ATP accumulation (Figure 3).

The key question around PKM2 is whether activators or inhibitors or PKM2 kinase activity would be the best strategy and if PK-M2 would really be a cancer specific target. Recent publications have shown that progress is being made in designing tool compounds117,118 to test out the PKM2 hypothesis and it will be interesting to monitor the outcome of studies with these and other PKM2 modulation agents to see if a clear rationale and patient selection strategy can be defined for this complex metabolic target.

Isocitrate dehydrogenase (IDH1/2)
Advances in large scale sequencing technologies has enabled more in-depth profiling of the genetics of multiple metabolism and oncogenic components in far larger sample sets (often more than 200 samples) and in multiple tumour types. Using these techniques it has been found that 60-90% of secondary gliomas (around 5% of primary gliomas)27,119 and 12-18% of acute myeloid leukaemias have mutations in the oxidative phosphorylation/TCA cycle components IDH1 or IDH226,120,121. For gliomas the common mutations are IDH1 Arg132 and IDH2 Arg140 and Arg172 whereas for glioma most mutations are in the IDH2 protein1,27,120. It is also worth noting that the vast majority of these mutations are heterozygous.

Mutations affecting the catalytic sites of IDH1 or 2 are thought to be functionally equivalent and were initially reported to negatively affect IDH catalytic activity by reducing isocitrate binding and the ability to convert isocitrate into alphaketoglutarate (-KG). However, recent mass-spectroscopy data has discovered IDH mutations exhibit an altered catalytic activity and convert - KG (the product of wild type IDH proteins) to 2- hydorxyglutarate (Figure 4)120-122. This altered metabolite is found to be 100-fold increased in glioma or AML patients with IDH mutations suggesting it could act as a clinical biomarker120. Large scale analysis of DNA methylation in human gliomas provided a key insight into the role of this mutation in cancer. This study showed that nearly all IDH1 and IDH2 mutations were associated with a highly specific DNA methylation profile corresponding to the oligodendrocyte subtype of glioma26. Similar changes in DNA methylation profiling were also seen in human AML samples. A decrease in -KG levels (probably due to heterodimerisation between -KG producing WT IDH and -KG metabolising mutant IDH [Figure 3]) coupled with an increase in 2-HG levels is reported to block epigenetic events including histone demethylase and TET1/2 (hydroxylases which usually produce 5-hydorxymethylcytosine) activity and are believed to be the major mechanism by which IDH mutants function in tumours1. 2HG has also been reported to stabilise HIF1 which can regulate many metabolic components and also promote VEGF signalling, a driver of tumour angiogenesis123.

Therefore, IDH mutants represent an attractive target for targeted therapy as they show a unique cancer specific function and generate a potential cancer biomarker in 2HG for disease stratification. However, one intriguing recent piece of evidence is that in IDH mutant appear to have slightly prolonged survival which adds more complexity to this intriguing target124.

Future challenges in targeting cancer metabolism
The challenges around targeting metabolism will involve a clear understanding of how a cancer cell differs in its metabolism to that of a rapidly proliferating normal cell. It has long been known the neurological cells also have high glucose demands and also a reliance on other metabolites linked to some of the canonical metabolism pathways and linked to cancer, for example glutamine and serine. For example, it has been shown in normal Tlymphocytes125 and astrocytes126 that siRNA depletion of metabolic enzymes such as PFKFB3 decreases the proliferation rates of these ‘normal’ cell lines127,128. Therefore it will be important to fully understand the metabolic drivers by which a cancer cells may differ from normal cells and also the potential toxicity risk associated with targeting metabolism.

Another challenge will be around patient stratification/ selection. While many tumours may display high glucose or glutamine uptake/utilisation, this alone is likely to be insufficient as a predictive marker for therapeutic potential of an anti-metabolism agent. It will be important to fully understand how known oncogenic drivers of cancer (ie myc, ras, PI3K) or loss of tumour suppressor genes (ie p53, PTEN, LKB1) impact on metabolic flux and/or regulation of key metabolic points in different tumour types. While a few mutations in metabolism enzymes in cancer have been identified, it is likely that in many tumours the impact of metabolism targets will be a complex interplay between expression, regulation, flux and oncogenic changes. It will also be important to consider that within metabolism pathway there are plenty of opportunities for tumour cells to evade metabolic blocks through bypass pathways and redundancy, so understanding key nodal points and possible combination strategies or synthetic lethality approaches will be essential. Likewise the interplay between metabolism targets and current chemotherapies (many of which create increased demand for biosynthetic intermediates as the tumour attempts to survive these agents) could be an important therapeutic strategy to consider as more metabolism targeting agents reach clinical trials in the future.

Pharma is rapidly adapting to these needs by the way it is approaching this area with ties to leading academic experts in cancer metabolism and utilisation of existing expertise against metabolic disorders for which many companies already have a platform. It is hoped that with this approach and the renewed interest in cancer metabolism the way is paved for a new generation of cancer metabolism therapeutics.


Dr Neil P Jones is a Principal Target Validation Scientist at Cancer Research Technology and involved in the drug discovery alliance between Cancer Research Technology and AstraZeneca to identify new cancer therapies targeting cancer metabolism. He received his PhD at the University of Southampton in the Cell and Molecular Bioscience Laboratory and has previously worked in lipid signalling pathways and cancer at the Institute of Cancer Research, London.

References
1
Yen, KE et al. Cancer-associated IDH mutations: biomarker and therapeutic opportunities. Oncogene. 29(49): p. 6409-17.

2 Gupta, V and Bamezai, RN. Human pyruvate kinase M2: a multifunctional protein. Protein Sci. 19(11): p. 2031-44.

3 Zachar, Z et al. Non-redox-active lipoate derivates disrupt cancer cell mitochondrial metabolism and are potent anticancer agents in vivo. J Mol Med (Berl).

4 Clem, B et al. Small-molecule inhibition of 6- phosphofructo-2-kinase activity suppresses glycolytic flux and tumor growth. Mol Cancer Ther, 2008. 7(1): p. 110-20.

5 Yalcin, A et al. Selective inhibition of choline kinase simultaneously attenuates MAPK and PI3K/AKT signaling. Oncogene. 29(1): p. 139-49.

6 Vander Heiden, MG. Targeting cancer metabolism: a therapeutic window opens. Nat Rev Drug Discov. 10(9): p. 671-84.

7 Warburg, O, Wind, F and Negelein, E. The Metabolism of Tumors in the Body. J Gen Physiol, 1927. 8(6): p. 519-30.

8 Koppenol, WH, Bounds, PL and Dang, CV. Otto Warburg’s contributions to current concepts of cancer metabolism. Nat Rev Cancer. 11(5): p. 325-37.

9 DeBerardinis, RJ et al. The biology of cancer: metabolic reprogramming fuels cell growth and proliferation. Cell Metab, 2008. 7(1): p. 11-20.

10 Vander Heiden, MG, Cantley, LC and Thompson, CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science, 2009. 324(5930): p. 1029-33.

11 Dang, CV. The interplay between MYC and HIF in the Warburg effect. Ernst Schering Found Symp Proc, 2007(4): p. 35-53.

12 Reivich, M et al. Measurement of local cerebral glucose metabolism in man with 18F-2- fluoro-2-deoxy-d-glucose. Acta Neurol Scand Suppl, 1977. 64: p. 190-1.

13 Busk, M et al. Inhibition of tumor lactate oxidation: consequences for the tumor microenvironment. Radiother Oncol. 99(3): p. 404-11.

14 Fowler, JS and Ido, T. Initial and subsequent approach for the synthesis of 18FDG. Semin Nucl Med, 2002. 32(1): p. 6-12.

15 Shani, J et al. Distribution of 18F-5- fluorouracil in tumor-bearing mice and rats. Int J Nucl Med Biol, 1978. 5(1): p. 19-28.

16 Zhu, A, Lee, D and Shim, H. Metabolic positron emission tomography imaging in cancer detection and therapy response. Semin Oncol. 38(1): p. 55-69.

17 Dang, CV. Glutaminolysis: supplying carbon or nitrogen or both for cancer cells? Cell Cycle. 9(19): p. 3884-6.

18 Dang, CV. Rethinking the Warburg effect with Myc micromanaging glutamine metabolism. Cancer Res. 70(3): p. 859-62.

19 Wise, DR and Thompson, CB. Glutamine addiction: a new therapeutic target in cancer. Trends Biochem Sci. 35(8): p. 427-33.

20 Berardi, MJ and Fantin, VR. Survival of the fittest: metabolic adaptations in cancer. Curr Opin Genet Dev. 21(1): p. 59-66.

21 DeBerardinis, RJ et al. Beyond aerobic glycolysis: transformed cells can engage in glutamine metabolism that exceeds the requirement for protein and nucleotide synthesis. Proc Natl Acad Sci U S A, 2007. 104(49): p. 19345-50.

22 Plathow, C and Weber, WA. Tumor cell metabolism imaging. J Nucl Med, 2008. 49 Suppl 2: p. 43S-63S.

23 Bohndiek, SE and Brindle, KM. Imaging and ‘omic’ methods for the molecular diagnosis of cancer. Expert Rev Mol Diagn. 10(4): p. 417-34.

24 Tennant, DA et al. Metabolic transformation in cancer. Carcinogenesis, 2009. 30(8): p. 1269-80.

25 Cairns, RA, Harris, IS and Mak, TW. Regulation of cancer cell metabolism. Nat Rev Cancer. 11(2): p. 85-95.

26 Figueroa, ME et al. Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation. Cancer Cell. 18(6): p. 553-67.

27 Yan, H et al. IDH1 and IDH2 mutations in gliomas. N Engl J Med, 2009. 360(8): p. 765-73.

28 Mazurek, S. Pyruvate kinase type M2: a key regulator within the tumour metabolome and a tool for metabolic profiling of tumours. Ernst Schering Found Symp Proc, 2007(4): p. 99-124.

29 Hanahan, D and Weinberg, RA. Hallmarks of cancer: the next generation. Cell. 144(5): p. 646-74.

30 Collier, JJ et al. c-Myc is required for the glucose-mediated induction of metabolic enzyme genes. J Biol Chem, 2003. 278(8): p. 6588-95.

31 Dang, CV, Le, A and Gao, P. MYC-induced cancer cell energy metabolism and therapeutic opportunities. Clin Cancer Res, 2009. 15(21): p. 6479-83.

32 Elstrom, RL et al. Akt stimulates aerobic glycolysis in cancer cells. Cancer Res, 2004. 64(11): p. 3892-9.

33 Fan, Y, Dickman, KG and Zong, WX. Akt and c-Myc differentially activate cellular metabolic programs and prime cells to bioenergetic inhibition. J Biol Chem. 285(10): p. 7324-33.

34 Robey, RB and Hay, N. Is Akt the “Warburg kinase”?-Akt-energy metabolism interactions and oncogenesis. Semin Cancer Biol, 2009. 19(1): p. 25-31.

35 Furuta, E et al. Metabolic genes in cancer: their roles in tumor progression and clinical implications. Biochim Biophys Acta. 1805(2): p. 141-52.

36 Bensaad, K and Vousden, KH. p53: new roles in metabolism. Trends Cell Biol, 2007. 17(6): p. 286-91.

37 Cheung, EC and Vousden, KH. The role of p53 in glucose metabolism. Curr Opin Cell Biol. 22(2): p. 186-91.

38 Gottlieb, E and Vousden, KH. p53 regulation of metabolic pathways. Cold Spring Harb Perspect Biol. 2(4): p. a001040.

39 Yuan, TL and Cantley, LC. PI3K pathway alterations in cancer: variations on a theme. Oncogene, 2008. 27(41): p. 5497-510.

40 Kalaany, NY and Sabatini, DM. Tumours with PI3K activation are resistant to dietary restriction. Nature, 2009. 458(7239): p. 725-31.

41 Shaw, RJ et al. The LKB1 tumor suppressor negatively regulates mTOR signaling. Cancer Cell, 2004. 6(1): p. 91-9.

42 Shaw, RJ et al. The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc Natl Acad Sci U S A, 2004. 101(10): p. 3329-35.

43 Wise, DR et al. Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction. Proc Natl Acad Sci U S A, 2008. 105(48): p. 18782-7.

44 Kim, JW et al. Hypoxia-inducible factor 1 and dysregulated c-Myc cooperatively induce vascular endothelial growth factor and metabolic switches hexokinase 2 and pyruvate dehydrogenase kinase 1. Mol Cell Biol, 2007. 27(21): p. 7381-93.

45 Shim, H et al. c-Myc transactivation of LDHA: implications for tumor metabolism and growth. Proc Natl Acad Sci U S A, 1997. 94(13): p. 6658-63.

46 Semenza, GL. Regulation of Metabolism by Hypoxia-Inducible Factor 1. Cold Spring Harb Symp Quant Biol.

47 Lum, JJ et al. The transcription factor HIF-1 plays a critical role in the growth factordependent regulation of both aerobic and anaerobic glycolysis. Genes Dev, 2007. 21(9): p. 1037-49.

48 Semenza, GL. HIF-1: upstream and downstream of cancer metabolism. Curr Opin Genet Dev. 20(1): p. 51-6.

49 Gordan, JD, Thompson, CB and Simon, MC. HIF and c-Myc: sibling rivals for control of cancer cell metabolism and proliferation. Cancer Cell, 2007. 12(2): p. 108-13.

50 Podar, K and Anderson, KC. A therapeutic role for targeting c-Myc/Hif-1-dependent signaling pathways. Cell Cycle. 9(9): p. 1722-8.

51 Kaelin, WG, Jr. The von Hippel-Lindau tumour suppressor protein: O2 sensing and cancer. Nat Rev Cancer, 2008. 8(11): p. 865-73.

52 Bobarykina, AY et al. Hypoxic regulation of PFKFB-3 and PFKFB-4 gene expression in gastric and pancreatic cancer cell lines and expression of PFKFB genes in gastric cancers. Acta Biochim Pol, 2006. 53(4): p. 789-99.

53 Obach, M et al. 6-Phosphofructo-2-kinase (pfkfb3) gene promoter contains hypoxiainducible factor-1 binding sites necessary for transactivation in response to hypoxia. J Biol Chem, 2004. 279(51): p. 53562-70.

54 Koukourakis, MI et al. Pyruvate dehydrogenase and pyruvate dehydrogenase kinase expression in non small cell lung cancer and tumor-associated stroma. Neoplasia, 2005. 7(1): p. 1-6.

55 Luo, W and Semenza, GL. Pyruvate kinase M2 regulates glucose metabolism by functioning as a coactivator for hypoxia-inducible factor 1 in cancer cells. Oncotarget. 2(7): p. 551-6.

56 Firth, JD, Ebert, BL and Ratcliffe, PJ. Hypoxic regulation of lactate dehydrogenase A. Interaction between hypoxia-inducible factor 1 and cAMP response elements. J Biol Chem, 1995. 270(36): p. 21021-7.

57 Izumi, H et al. Monocarboxylate transporters 1 and 4 are involved in the invasion activity of human lung cancer cells. Cancer Sci. 102(5): p. 1007-13.

58 Majewski, N et al. Hexokinase-mitochondria interaction mediated by Akt is required to inhibit apoptosis in the presence or absence of Bax and Bak. Mol Cell, 2004. 16(5): p. 819-30.

59 Vousden, KH. Functions of p53 in metabolism and invasion. Biochem Soc Trans, 2009. 37(Pt 3): p. 511-7.

60 Vousden, KH and Ryan, KM. p53 and metabolism. Nat Rev Cancer, 2009. 9(10): p. 691-700.

61 Bensaad, K. Cheung, EC and Vousden, KH. Modulation of intracellular ROS levels by TIGAR controls autophagy. EMBO J, 2009. 28(19): p. 3015-26.

62 Bensaad, K et al. TIGAR, a p53-inducible regulator of glycolysis and apoptosis. Cell, 2006. 126(1): p. 107-20.

63 Matoba, S et al. p53 regulates mitochondrial respiration. Science, 2006. 312(5780): p. 1650-3.

64 Stambolic, V et al. Regulation of PTEN transcription by p53. Mol Cell, 2001. 8(2): p. 317-25.

65 Macheda, ML, Rogers, S and Best, JD. Molecular and cellular regulation of glucose transporter (GLUT) proteins in cancer. J Cell Physiol, 2005. 202(3): p. 654-62.

66 Ganapathy, V, Thangaraju, M and Prasad, PD. Nutrient transporters in cancer: relevance to Warburg hypothesis and beyond. Pharmacol Ther, 2009. 121(1): p. 29-40.

67 Maher, JC et al. Differential sensitivity to 2- deoxy-D-glucose between two pancreatic cell lines correlates with GLUT-1 expression. Pancreas, 2005. 30(2): p. e34-9.

68 Wu, CH et al. In vitro and in vivo study of phloretin-induced apoptosis in human liver cancer cells involving inhibition of type II glucose transporter. Int J Cancer, 2009. 124(9): p. 2210-9.

69 Witte, D et al. Overexpression of the neutral amino acid transporter ASCT2 in human colorectal adenocarcinoma. Anticancer Res, 2002. 22(5): p. 2555-7.

70 Kaadige, MR, Elgort, MG and Ayer, DE. Coordination of glucose and glutamine utilization by an expanded Myc network. Transcr. 1(1): p. 36-40.

71 Pedersen, PL. Warburg, me and Hexokinase 2: Multiple discoveries of key molecular events underlying one of cancer’s most common phenotypes, the “Warburg Effect”, i.e., elevated glycolysis in the presence of oxygen. J Bioenerg Biomembr, 2007. 39(3): p. 211-22.

72 Mathupala, SP, Heese, C and Pedersen, PL. Glucose catabolism in cancer cells. The type II hexokinase promoter contains functionally active response elements for the tumor suppressor p53. J Biol Chem, 1997. 272(36): p. 22776-80.

73 Kurtoglu, M, Maher, JC and Lampidis, TJ. Differential toxic mechanisms of 2-deoxy-Dglucose versus 2-fluorodeoxy-D-glucose in hypoxic and normoxic tumor cells. Antioxid Redox Signal, 2007. 9(9): p. 1383-90.

74 Lampidis, TJ et al. Efficacy of 2-halogen substituted D-glucose analogs in blocking glycolysis and killing “hypoxic tumor cells”. Cancer Chemother Pharmacol, 2006. 58(6): p. 725-34.

75 Maher, JC et al. Hypoxia-inducible factor-1 confers resistance to the glycolytic inhibitor 2- deoxy-D-glucose. Mol Cancer Ther, 2007. 6(2): p. 732-41.

76 Singh, D et al. Optimizing cancer radiotherapy with 2-deoxy-d-glucose dose escalation studies in patients with glioblastoma multiforme. Strahlenther Onkol, 2005. 181(8): p. 507-14.

77 Dwarakanath, BS et al. Clinical studies for improving radiotherapy with 2-deoxy-D-glucose: present status and future prospects. J Cancer Res Ther, 2009. 5 Suppl 1: p. S21-6.

78 Prasanna, VK et al. Differential responses of tumors and normal brain to the combined treatment of 2-DG and radiation in glioablastoma. J Cancer Res Ther, 2009. 5 Suppl 1: p. S44-7.

79 Dang, CV. MYC, microRNAs and glutamine addiction in cancers. Cell Cycle, 2009. 8(20): p. 3243-5.

80 Gao, P et al. c-Myc suppression of miR-23a/b enhances mitochondrial glutaminase expression and glutamine metabolism. Nature, 2009. 458(7239): p. 762-5.

81 Seltzer, MJ et al. Inhibition of glutaminase preferentially slows growth of glioma cells with mutant IDH1. Cancer Res. 70(22): p. 8981-7.

82 Catane, R et al. Azaserine, DON, and azotomycin: three diazo analogs of L-glutamine with clinical antitumor activity. Cancer Treat Rep, 1979. 63(6): p. 1033-8. 83Wang, JB et al. Targeting mitochondrial glutaminase activity inhibits oncogenic transformation. Cancer Cell. 18(3): p. 207-19.

84 Pelicano, H et al. Glycolysis inhibition for anticancer treatment. Oncogene, 2006. 25(34): p. 4633-46.

85 Wang, J et al. A glycolytic mechanism regulating an angiogenic switch in prostate cancer. Cancer Res, 2007. 67(1): p. 149-59.

86 Zieker, D et al. Phosphoglycerate kinase 1 a promoting enzyme for peritoneal dissemination in gastric cancer. Int J Cancer. 126(6): p. 1513-20.

87 Ren, F et al. Quantitative proteomics identification of phosphoglycerate mutase 1 as a novel therapeutic target in hepatocellular carcinoma. Mol Cancer. 9: p. 81.

88 Vander Heiden, MG et al. Evidence for an alternative glycolytic pathway in rapidly proliferating cells. Science. 329(5998): p. 1492-9.

89 Christofk, HR et al. The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature, 2008. 452(7184): p. 230-3.

90 Christofk, HR et al. Pyruvate kinase M2 is a phosphotyrosine-binding protein. Nature, 2008. 452(7184): p. 181-6.

91 Hitosugi, T et al. Tyrosine phosphorylation inhibits PKM2 to promote the Warburg effect and tumor growth. Sci Signal, 2009. 2(97): p. ra73.

92 Cosentino, C, Grieco, D and Costanzo, V. ATM activates the pentose phosphate pathway promoting anti-oxidant defence and DNA repair. EMBO J. 30(3): p. 546-55.

93 Jiang, P et al. p53 regulates biosynthesis through direct inactivation of glucose-6- phosphate dehydrogenase. Nat Cell Biol. 13(3): p. 310-6.

94 Kruger, A and Ralser, M. ATM is a redox sensor linking genome stability and carbon metabolism. Sci Signal. 4(167): p. pe17.

95 Perl, A et al. Oxidative stress, inflammation and carcinogenesis are controlled through the pentose phosphate pathway by transaldolase. Trends Mol Med. 17(7): p. 395-403.

96 Coy, JF et al. Mutations in the transketolaselike gene TKTL1: clinical implications for neurodegenerative diseases, diabetes and cancer. Clin Lab, 2005. 51(5-6): p. 257-73.

97 Langbein, S et al. Expression of transketolase TKTL1 predicts colon and urothelial cancer patient survival: Warburg effect reinterpreted. Br J Cancer, 2006. 94(4): p. 578-85.

98 Liu, H et al. Fructose induces transketolase flux to promote pancreatic cancer growth. Cancer Res. 70(15): p. 6368-76.

99 Bauer, DE et al. ATP citrate lyase is an important component of cell growth and transformation. Oncogene, 2005. 24(41): p. 6314-22.

100 Hanai, JI et al. Inhibition of lung cancer growth: ATP citrate lyase knockdown and statin treatment leads to dual blockade of mitogenactivated protein kinase (MAPK) and phosphatidylinositol-3- kinase (PI3K)/AKT pathways. J Cell Physiol.

101 Kuhajda, FP. Fatty acid synthase and cancer: new application of an old pathway. Cancer Res, 2006. 66(12): p. 5977-80.

102 Zhan, Y et al. Control of cell growth and survival by enzymes of the fatty acid synthesis pathway in HCT-116 colon cancer cells. Clin Cancer Res, 2008. 14(18): p. 5735-42.

103 Nomura, DK et al. Monoacylglycerol lipase regulates a fatty acid network that promotes cancer pathogenesis. Cell. 140(1): p. 49-61.

104 Ye, L et al. Monoacylglycerol lipase (MAGL) knockdown inhibits tumor cells growth in colorectal cancer. Cancer Lett. 307(1): p. 6-17.

105 Zaugg, K et al. Carnitine palmitoyltransferase 1C promotes cell survival and tumor growth under conditions of metabolic stress. Genes Dev. 25(10): p. 1041-51.

106 Tennant, DA, Duran, RV and Gottlieb, E. Targeting metabolic transformation for cancer therapy. Nat Rev Cancer. 10(4): p. 267-77.

 

107 Pertega-Gomes, N et al. Monocarboxylate transporter 4 (MCT4) and CD147 overexpression is associated with poor prognosis in prostate cancer. BMC Cancer. 11: p. 312.

108 Pinheiro, C et al. Monocarboxylate transporter 1 is up-regulated in basal-like breast carcinoma. Histopathology. 56(7): p. 860-7.

109 Pinheiro, C et al. Increasing expression of monocarboxylate transporters 1 and 4 along progression to invasive cervical carcinoma. Int J Gynecol Pathol, 2008. 27(4): p. 568-74.

110 Pinheiro, C et al. Increased expression of monocarboxylate transporters 1, 2, and 4 in colorectal carcinomas. Virchows Arch, 2008. 452(2): p. 139-46.

111 Kennedy, KM and Dewhirst, MW. Tumor metabolism of lactate: the influence and therapeutic potential for MCT and CD147 regulation. Future Oncol. 6(1): p. 127-48.

112 David, CJ et al. HnRNP proteins controlled by c-Myc deregulate pyruvate kinase mRNA splicing in cancer. Nature. 463(7279): p. 364-8.

113 Bluemlein, K et al. No evidence for a shift in pyruvate kinase PKM1 to PKM2 expression during tumorigenesis. Oncotarget. 2(5): p. 393-400.

114 Dang, CV. PKM2 tyrosine phosphorylation and glutamine metabolism signal a different view of the Warburg effect. Sci Signal, 2009. 2(97): p. pe75.

115 Lv, L et al. Acetylation targets the M2 isoform of pyruvate kinase for degradation through chaperone-mediated autophagy and promotes tumor growth. Mol Cell. 42(6): p. 719-30.

116 Locasale, JW. Vander Heiden, MG and Cantley, LC. Rewiring of glycolysis in cancer cell metabolism. Cell Cycle. 9(21): p. 4253.

117 Vander Heiden, MG et al. Identification of small molecule inhibitors of pyruvate kinase M2. Biochem Pharmacol. 79(8): p. 1118-24.

118 Boxer, MB et al. Identification of activators for the M2 isoform of human pyruvate kinase Version 3.

119 Frezza, C, Tennant, DA and Gottlieb, E. IDH1 mutations in gliomas: when an enzyme loses its grip. Cancer Cell. 17(1): p. 7-9.

120 Gross, S et al. Cancer-associated metabolite 2-hydroxyglutarate accumulates in acute myelogenous leukemia with isocitrate dehydrogenase 1 and 2 mutations. J Exp Med. 207(2): p. 339-44.

121 Ward, PS et al. The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate. Cancer Cell. 17(3): p. 225-34.

122 Dang, L et al. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature, 2009. 462(7274): p. 739-44.

123 Zhao, S et al. Glioma-derived mutations in IDH1 dominantly inhibit IDH1 catalytic activity and induce HIF-1. Science, 2009. 324(5924): p. 261-5.

124 Christensen, BC et al. DNA methylation, isocitrate dehydrogenase mutation, and survival in glioma. J Natl Cancer Inst. 103(2): p. 143-53.

125 Colombo, SL et al. Anaphase-promoting complex/cyclosome-Cdh1 coordinates glycolysis and glutaminolysis with transition to S phase in human T lymphocytes. Proc Natl Acad Sci U S A. 107(44): p. 18868-73.

126 Herrero-Mendez, A et al. The bioenergetic and antioxidant status of neurons is controlled by continuous degradation of a key glycolytic enzyme by APC/C-Cdh1. Nat Cell Biol, 2009. 11(6): p. 747-52.

127 Almeida, A, Bolanos, JP and Moncada, S. E3 ubiquitin ligase APC/C-Cdh1 accounts for the Warburg effect by linking glycolysis to cell proliferation. Proc Natl Acad Sci U S A. 107(2): p. 738-41.

128 Bolanos, JP, Almeida, A and Moncada, S. Glycolysis: a bioenergetic or a survival pathway? Trends Biochem Sci. 35(3): p. 145-9.