Metabolomics (2008) 4:1–12 DOI 10.1007/s11306-007-0101-3
ORIGINAL ARTICLE
Robust metabolic adaptation underlying tumor progression Pedro Viza´n Æ Sybille Mazurek Æ Marta Cascante
Received: 8 August 2007 / Accepted: 14 December 2007 / Published online: 9 January 2008 Ó Springer Science+Business Media, LLC 2007
Abstract Tumor metabolism represents the end point of many signal cascades recruited by oncogenic activation. Energy metabolism of cancer cells attracted the attention of biochemists over eight decades ago. For example, high consume of glucose and high lactate production under aerobic conditions make up one of the most fundamental characteristics of cancer cells and has been exploited for diagnosis. At the same time, study of the metabolic status of tumor cells during tumor progression reveals characteristic adaptations during carcinogenesis. Although these metabolic adaptations are not the main defects that cause cancer, they may confer advantages to survive. In this review, we discuss the main metabolic hot spots and their relationship with main tumor progression events. An accurate metabolic map of the many tumor phenotypes could offer new options in the treatment of cancer. Keywords Tumor metabolism Warburg effect Regulation of glucose metabolism in tumors Acidosis and tumor progression Biosynthetic pathways in tumoral metabolism Alternative to glycolytic energy conversion
P. Viza´n M. Cascante (&) Department of Biochemistry and Molecular Biology, Faculty of Biology (edifici nou annexe planta -2), Associated Unit to CSIC, Institute of Biomedicine of University of Barcelona (IBUB), Diagonal 645, 08028 Barcelona, Spain e-mail:
[email protected] S. Mazurek Institute of Biochemistry & Endocrinology, Veterinary Faculty, University of Giessen, 35392 Giessen, Germany S. Mazurek ScheBo Biotech AG, 35394 Giessen, Germany
Isoenzyme expression in tumor cells Tumor metabolism and apoptosis
1 Introduction A unified view of the cellular network is currently emerging. This grasps the complexity, robustness and versatility of cells in adjusting their intracellular machinery in response to changes in their environment, food availability and developmental state behavior. This integrated view involves various levels of cell organization, including genes, proteins, metabolites and their interactions. Intimate and direct links between signal transduction, energetics and metabolism are essential drivers of cell fate (Ockner 2004). The main traditional dogma of molecular biology assigned a gene to a protein, which constituted the functional unit of biological behavior. However, the genome sequence and functional genomics have not yet been able to explain the genotype-to-phenotype gap. Complementary to this reductionist vision, Systems Biology, defined as a comprehensive quantitative analysis of the manner in which all the components of a biological system interact functionally over time (Aderem 2005), has emerged in recent years to explain biological phenomena, not on a gene-by-gene basis, but through the net interactions of all cellular and biochemical components within a cell or organism. This extended view clearly pointed out that the cellular regulatory hierarchy and information flow runs two ways, from genome to metabolome and vice versa (Cho et al. 2006; Hornberg et al. 2006; Kell 2006; Liu 2005; Kitano 2002; Oltvai and Barabasi 2002). For example, genome transcription factor-controlled information retrieval is strongly influenced by the state of the metabolome (Oltvai and Barabasi 2002). Thus, whereas genes and
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proteins set the stage for what happens in the cell, metabolites are responsible for the fine tuning of all cell reactions and can regulate processes as cell signaling, energy transfer and cell-to-cell communication (Schmidt 2004). In fact, metabolomics may amplify changes in the proteome, since a small change in a protein concentration may result in large changes in metabolite concentrations as has been proven by metabolic control analysis (Kell 2006; Urbanczyk-Wochniak et al. 2003; Cascante et al. 2002; Raamsdonk et al. 2001). In this context, metabolic status could be considered as the end point and a main actor in many molecular events. Thus, study of the metabolome has become a powerful tool in the fields of biotechnology (Oksman-Caldentey and Saito 2005), natural compounds (Pierens et al. 2005; Yang et al. 2004; Ott et al. 2003), nutrition (Trujillo et al. 2006), biomedicine (van der Greef et al. 2006; German et al. 2005), and especially in cancer studies (Malyankar 2007; Morvan and Demidem 2007; Jordan and Cheng 2007; Claudino et al. 2007; Denkert et al. 2006; Boros et al. 2002). Altered metabolic profiles of cancer cells attracted the attention of biochemists over eight decades ago. It is known from Warburg’s research 70 years ago (Warburg et al. 1924) that high rates of glucose uptake and increased levels of glycolysis despite reduced levels of oxygen consumption make up one of the most fundamental characteristics of cancer cells. In this review, we discuss the main metabolic hot spots and their relationship with main tumor progression events.
2 Warburg effect Tumor cells are characterized by a high glycolytic capacity and a high rate of lactate production even in the presence of oxygen. This metabolic phenomenon is known as the Warburg effect, honoring Otto Warburg, who first described it in the 1920s (Warburg et al. 1924), and clashes with the Pasteur effect, which postulates the inhibition of glycolysis by oxygen, provoking the oxidation of pyruvate in mitochondria (Gatenby and Gillies 2004; Racker 1974). Although some authors have expressed their doubts about the inherent glycolytic phenotype of tumors (Zu and Guppy 2004), tumors’ capacity to take up glucose rapidly is widely used in clinical detection of neoplasias by PET (Czernin and Phelps 2002; Gambhir 2002; Burt et al. 2001; Weber et al. 1999; Hawkins and Phelps 1988). Moreover, correlation between lactate production and the degree of malignancy in solid tumors has led to the suggestion that lactate determination is a new metabolic tool for the improvement of prognosis and therapy in clinical oncology (Walenta et al. 2004).
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Physiologically, however, it is difficult to explain why tumor cells prefer to use a metabolic pathway that produces only 5% of the energy available from glucose. It could be argued that the glycolytic switch represents just a consequence of molecular alterations in tumor cells, since several interactions between oncogenes and metabolic machinery have been described (discussed below). However, at the same time, aerobic glycolysis may confer some important advantages on tumor cells. First of all, it should be noted that tumor cells lose the regulation of external signals to uptake nutrients such as glucose, due to an increased and deregulated expression of glucose transporter proteins (Gu et al. 2006; Macheda et al. 2005). The lack of this limitation has the consequence that the waste of energy caused by fermentation of pyruvate to lactate is not critical. Second, the transfer of the hydrogen on pyruvate catalyzed by lactate dehydrogenase (LDH) is faster than mitochondrial respiration and allows an effective recycling of NAD+, necessary for the glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) reaction (Bui and Thompson 2006; Pfeiffer et al. 2001). Importantly, in contrast to mitochondrial respiration, glycolytic energy regeneration is independent on oxygen supply and allows tumor cells to survive and migrate in hypoxic areas. The observation that cells derived from hypoxic tumors and cultured in normoxic conditions still maintain the glycolytic phenotype, indicates that the driving force of the increased glycolytic rate is not a simple adaptation to hypoxia and that stable genetic mechanisms may cause the glycolytic switch inside tumors (Gatenby and Gillies 2004). In this regard, Fantin et al. (2006) recently reported that attenuation of LDH in mammary tumor cells stimulates mitochondrial respiration and compromises cell tumorigenicity, suggesting that tumor cells still possess the ability for mitochondrial respiration and so defects in mitochondrial respiration are not the cause for the increased aerobic glycolysis. A new aspect was provided by the observation that lactate modulates dendritic cell activation and antigen expression, which suggests an influence of tumor-derived lactate on anti-tumor T-cell response (Gottfried et al. 2006).
3 Regulation of glucose metabolism in tumors Since Otto Warburg described more than 70 years ago the reprogramming of tumoral cells from mitochondrial respiration to aerobic glycolysis, the molecular mechanisms that lead to this phenomenon have been uncovered (Figs. 1 and 2) (Kim and Dang 2006; Kim et al. 2006; Matoba et al. 2006; Plas and Thompson 2005; Kondoh et al. 2005; Ramanathan et al. 2005; Elstrom et al. 2004; Gatenby and Gillies 2004; Lu et al. 2002; Semenza et al. 2001; Osthus et al. 2000; Dang and Semenza 1999).
Robust metabolic adaptation underlying tumor progression Fig. 1 Effects of oncogenes, transcription factors and tumor suppressors on central metabolism
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lactate Many tumors progress under hypoxic conditions. In these conditions, one of the most important regulators is the hypoxia-inducible transription factor 1 (HIF-1). HIF-1 consists of two subunits, HIF-1a and HIF-1b. HIF-1a subunit is regulated in an oxygen-dependent manner, being stable under hypoxic conditions. HIF-1 is an effector of several oncogenes such as Ras, Src or Her-2 and upregulates survival genes and growth factors like vascular endothelial growth factor (VEGF). At the same time, HIF-1 increases the expression of several glycolytic enzymes (Rimpi and Nilsson 2007; Yasuda et al. 2004; Semenza et al. 2001; Dang and Semenza 1999). Metabolic products of glycolysis such as lactate and pyruvate stimulate the accumulation of HIF-1, regardless of the hypoxic conditions (Lu et al. 2002). Thereby HIF-1 prevents pyruvate conversion to acetyl-CoA due to an induction of pyruvate dehydrogenase kinase 1 (PDHK1)
which phoshorylates and inactivates pyruvate dehydrogenase (PDH) (Papandreou et al. 2006; Kim et al. 2006), the required step for producing acetyl-CoA from pyruvate (Fig. 2). A product of TCA cycle, succinate, has been related to the stabilization of HIF-1 (Fig. 2), which has caused two enzymes in the tricarboxylic acid cycle to be redefined as tumor suppressors. Indeed, succinate dehydrogenase (SDH) and fumarate hydratase (FH) genes, which correspond to nuclear-encoded mitochondrial enzymes in the tricarboxylic acid (TCA) cycle (SDH is also a component of complex II in the mitochondrial respiratory chain), behave as classic tumor suppressors (King et al. 2006). Apart from the generation of reactive oxygen species by the inhibition of SDH, leading to oxidative DNA damage and consequent mutagenesis (Ishii et al. 2007), another biochemical mechanism that explains how mutation in these two
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4 Fig. 2 Regulation of TCA cycle and glutaminolisis network in tumoral cells
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enzymes contributes to tumor progression has been suggested: succinate, the substrate of SDH, might translocate out of the mitochondria and inhibit the activity of prolyl hydroxylases (PHD) in the cytosol (Selak et al., 2005). PHD, which needs a-ketoglutarate as a substrate to produce succinate, hydroxylates the a unit of HIF-1, targeting it for the polyubiquitylation mediated by the von Hippel–Lindau protein and its consequent degradation. Thus, inhibition of PHD by accumulation of succinate in succinate dehydrogenase- or fumarate hydratase-deficient tumors (Koivunen et al. 2007; Hewitson et al. 2007; Isaacs et al. 2005) is a mechanism to stabilize HIF-1 even under normoxic conditions (Gottlieb and Tomlinson 2005). However, the HIF-1 effect on tumor metabolism is still controversial. For example, the activation of HIF after von Hippel–Lindau (VHL) gene inactivation does not promote teratocarcinoma growth (Mack et al. 2003). Moreover, the inhibition of PDH induced by HIF-1 described above may impede lipogenesis (Bui and Thompson 2006). As we remark below, lipogenesis is essential for tumor growth, and the acetyl-CoA required for lipid and cholesterol synthesis is produced in the mitochondria by PDH and then translocated to the cytosol as citrate after its combination with oxaloacetate in the first step of the TCA cycle (Fig. 2). The citrate export from mitochondria is favored due to an inhibition of aconitase, the TCA cycle enzyme which catalyzes the conversion of citrate to isocitrate, by high concentrations of ROS. In normal cells, superoxide radicals are immediately inactivated by high activities of mitochondrial (Mn-dependent) and cytosolic (CuZn-dependent) superoxide dismutase, which are reduced during tumor
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formation. Accordingly, over-expression of manganese superoxide dismutase (MnSOD) enhances the activity of aconitase and inhibits tumor growth (Kim et al. 2001). Furthermore, other molecular mechanisms relating to tumor progression and glycolytic phenotype have been proposed. For example, an oncogene product such as Myc transcription factor has been related to energetic metabolism. Traditionally, numerous studies have focused on Myc capacity to promote cell cycle progression. However, it has been stressed lately that virtually all glycolytic enzymes are induced by Myc (Rimpi and Nilsson 2007; Kim and Dang 2005; Osthus et al. 2000; Dang and Semenza 1999). Among these, regulation by Myc of lactate dehydrogenase A (LDH-A), the last enzyme in anaerobic glycolysis, has received most attention: expression of LDH-A by Myc is crucial for tumoral progression, especially under hypoxic conditions (Shim et al. 1997, 1998). It has also been suggested that common oncogenic activation, such as the PI3K/Akt/mTOR pathway, upregulates glucose metabolism (Plas and Thompson 2005; Elstrom et al. 2004). Interestingly, it has been recently described that increase in NADH levels due to a defect in mitochondrial NADH consumption inactivates the tumor suppressor PTEN (product of phosphatase and tensin homolog gene) through a redox modification mechanism, leading to protein kinase B (Akt) activation (Pelicano et al. 2006). Thus, the glycolytic switch contributes to cell survival and drug resistance in cancer cells through Akt activation, especially in regulation of hexokinases and their relationship in avoiding apoptosis, which will be detailed below (see Sect. 8).
Robust metabolic adaptation underlying tumor progression
Recently, Ashrafian (2006) has suggested an elegant mechanism of glycolysis regulation in tumoral cells related to energetic sensor AMP-activated protein kinase (AMPK). AMPK is sensitive to, and is activated by, increased levels of AMP. Then, AMPK activates the glycolytic pathway, but simultaneously suppresses several anabolic processes that are necessary for tumor growth and negatively regulates proliferation through phosphorylation of tumor suppressor p53. An evolutionary view of tumor progression is needed to understand that adaptations favoring early selection of pre-malignant lesions may not match adaptations required for advanced carcinogenesis. Pre-malignant tumors are still not hypoxic and nutrient supply is adequate. Under such conditions, early tumoral cells are favored by maintenance of AMPK inhibition. However, in late stages of malignancy, tumors often have defective tumor suppressor contingent and apoptotic machinery, then a degree of AMPK activation can be tolerated and AMPKactivated levels reinforce the maintenance of energy demands through glycolysis.
4 Acidosis and tumor progression Tumor cells have to develop invasive and metastatic capacities in order to acquire a full malignant phenotype (Hanahan and Weinberg 2000). Since metastatic process is quite inefficient (Chambers et al. 2002), it has been suggested that the acquisition of the glycolytic phenotype may assist in spreading tumor cells and colonizing new tissues. The acidic environment related to solid tumors and usually associated with an increase in the lactate release could be a key in the advance towards late stages of tumor progression, especially tumor invasion (Gatenby et al. 2006). The high rate of lactate production could be caused by direct influence of HIF-1 on glycolytic enzyme activities. Moreover, HIF-1 does also up-regulate Na+/H+ exchangers and carbonic anhydrases which are another source for extracellular acidifications (Brahimi-Horn et al. 2007). The targeting of these key pH-regulating systems has been consistently proposed as a strategy to force necrotic cell death and tumor regression (Pouyssegur et al. 2006). In general, acidic environment of tumors acts as a micro-evolutionary selective pressure for the accumulation of new tumor mutations. This fact should be observed again under an evolutionary point of view of tumor progression. Normally, acidosis provokes p53-mediated necrosis or apoptosis (Park et al. 2000; Williams et al. 1999), but a high proportion of tumor cells, especially when tumor progression advances, are defective in p53 and apoptotic machinery, thereby becoming ideal candidates to acquire and manifestate new mutations. Moreover, acidosis on its own increases mutation rate (Gatenby and Gillies
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2004; Morita et al. 1992). Regarding invasiveness, extracellular acidosis may also promote angiogenesis through regulation of vascular endothelial growth factor (VEGF) (Shi et al. 2001) and excreted lactate may regulate extracellular matrix compounds (Stern et al. 2002).
5 Biosynthetic pathways in tumor metabolism Proliferating cells and especially tumor cells strongly depend on the availability of metabolic precursors for the synthesis of cell building blocks—a regulation mechanism termed the metabolic budget system (Eigenbrodt et al. 1985). In fact, besides energy regeneration glycolytic intermediates serve as precursors for the synthesis of nucleic acids, amino acids and phospholipids. A paradigmatic example is the synthesis of ribose-5-phosphate, the sugar component of nucleic acids, by the pentose phosphate pathway (PPP), a metabolic pathway described as a cycle in 1955 (Horecker 2002; Gunsalus et al. 1955). Ribose-5-phosphate can be synthesized from the glycolytic intermediate glucose-6-phosphate via the oxidative branch of pentose phosphate pathway (PPP) as well as from fructose-6-phosphate and glyceraldehyde-3phosphate via the non-oxidative branch of PPP. The oxidative PPP also regenerates NADPH which is the hydrogen donor in fatty acid synthesis as well as an important regenerator of glutathione (Fig. 1). In several tumor-derived cell lines, the non-oxidative branch of PPP was found to be the main source for ribose-5-phosphate synthesis (Cascante et al. 2000; Boros et al. 1998). Moreover, the formation of NADPH via the oxidative branch of PPP may induce tumor growth by the influence of the redox state of transcription factors such as NF-kB, AP-1, and c-Myb, (Kuo et al. 2000). However, induction of cell proliferation is only successful when both synthetic processes and energy regeneration are in optimal balance. Coordination between nucleic acid synthesis and the energy-regenerating metabolism is mediated by dihydroorotate dehydrogenase (DHODH) within pyrimidine de novo synthesis as well as by glycolytic phosphoglycerate mutase (PGM). DHODH is located in the inner mitochondrial membrane and uses prosthetic flavin and ubichinone as proximal and cytochrome c and molecular oxygen as final electron acceptors instead of NAD and NADP (Hansen et al. 2004). Thus, any dysfunction or inhibition of the mitochondrial electron transport chain may also impair pyrimidine, as well as DNA and RNA synthesis. Moreover, UTP, a product of DHODH, is necessary for the synthesis of complex carbohydrates, such as UDP glucose or UDP-N-acetylglucosamine. CTP is involved in phospholipid synthesis (Loffler et al. 2005). Glycolytic PGM is activated by histidine phosphorylation. In differentiated tissues, phosphate derives from the cofactor glycerate-2,3-bisphosphate. However, in tumor
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cells, expression of glycerate-2,3-bisphosphate mutase, the enzyme which catalyzes the synthesis of the cofactor glycerate-2,3-bisphosphate, is low (Hegde et al. 2001). Therefore, tumor cells need an alternative mechanism for PGM phosphorylation and activation in order to keep glycolysis running. A potential candidate is the nucleoside diphosphate kinase (NDPK) isoenzyme type A also known as Nm23-H1 (Fig. 1). In MCF-7 cells it was shown that NDPK type A phosphorylates and activates PGM using nucleotides, i.e. dCTP as phosphate donor. A cell-penetrating peptide encompassing the phosphorylated histidine residue from PGM promoted the growth arrest of several tumor cell lines (Engel et al. 2004). Strategies to inhibit nucleic acid synthesis have been proposed and inhibitors of purine and pyrimidine synthesis are currently used in chemotherapy (Purcell and Ettinger 2003). Following the same reasoning, the inhibition of PPP has been proposed as a target for cancer therapies, and several studies have already demonstrated in vitro and in vivo that cell proliferation is reduced when key enzymes of PPP are inhibited. (Ramos-Montoya et al. 2006; Rais et al. 1999; Boros et al. 1997). Tumor cells also depend on phospholipid synthesis. An increase in lipogenic enzyme expression has been described for a wide variety of different tumor cells (Swinnen et al. 2006). The stimulation of lipogenic gene transcription through activation of the lipogenic transcription factor sterol regulatory element-binding protein-1 is under the control of the oncogenic PI3K/Akt pathway (Porstmann et al. 2005). This stimulation provokes the activation of key enzymes in the fatty acid synthetic pathways such as fatty acid synthase (FAS), which has already been postulated as a cancer target (Kuhajda 2006; Menendez et al. 2005; De Schrijver et al. 2003). Moreover, also acetyl-CoA carboxylase (ACC) the key enzyme within fatty acid synthesis and ATP citrate lyase (ACL) the enzyme involved in citrate export from mitochondria, have been identified as potential targets against tumor progression (Hatzivassiliou et al. 2005; Brusselmans et al. 2005). Fatty acids can be used for phospholipid synthesis or can be released from tumor cells (Mazurek et al. 2005). The release of fatty acids has certain advantages for tumor cells. Firstly, fatty acids provide an effective hydrogen storage and are an effective way to eliminate surplus hydrogen besides the release of hydrogen as lactate. Secondly, fatty acids are immunosuppressive and may protect tumor cells from immune attacks (Grimm et al. 1994).
6 Isoenzyme expression in tumor cells An interesting aspect of tumor metabolism is the specific expression of isoenzymes, which have different amino acid sequences, but catalyze the same reaction with different
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kinetic and regulatory properties. The expression of these isoenzymes explains certain metabolic characteristics of tumors and, due to their specificity, offers interesting targets for cancer treatment. A paradigmatic example of differential isoenzyme expression in tumor cells is the glycolytic enzyme pyruvate kinase (PK). There are four isoenzymes of PK (type L, R, M1 and M2), but one of them, M2-PK, is predominant in all tissues with high nucleogenesis, such as embryonic cells, stem cells and especially tumor cells (Fig. 1). The upregulation of M2-PK is under the control of Ras and the transcription factors SP1 and SP3 (Mazurek et al. 2005; Schafer et al. 1997). The M-gene, which codes for both the M1 as well as the M2 pyruvate kinase isoenzyme, has two HIF-binding sites, which points to a direct regulation by HIF-1 (Kress et al. 1998). Unlike other PK isoenzymes, M2-PK may shift between a dimeric form, which is characterized by a low affinity to phosphoenolpyruvate (PEP), and a tetrameric form with high PEP affinity. The nearly inactive dimeric form causes an accumulation of all glycolytic intermediates above the pyruvate kinase reaction, which are then available as precursors for synthetic processes such as nucleic acid, phospholipids and amino-acid synthesis. When M2-PK is mainly in the nearly inactive dimeric form energy requirements are supplied by oxygendependent glutaminolysis. The highly active tetrameric form favors the conversion of glucose to lactate with the production of energy. It has been reported that M2-PK is a target of various oncoproteins which induce a dimerization of the enzyme. The amount of the dimeric form of M2-PK in plasma and stool samples has been found to correlate with tumor stages and can be used for early detection of tumors as well as follow up-studies during therapy. Interestingly, the balance between the dimeric and tetrameric form of M2-PK can act as a cycle, depending on the concentrations of various regulating metabolites. For example, high concentrations of glycolytic intermediate fructose-1,6bisphosphate lead to re-association of the nearly inactive dimeric to the highly active tetrameric form M2-PK and a subsequent increase in the flux rate from glucose to lactate. When fructose-1,6-bisphosphate levels drop below a certain value, the tetrameric form dissociates to the dimeric form again. Thus, M2-PK expression offers an integrated explanation for both a high glycolytic flux rate as well as an enhancement of biosynthetic pathways observed in tumor cells [(Mazurek et al. 2005; Mazurek and Eigenbrodt 2003) or visit http://www.metabolic-database.com]. Another relevant glycolytic isoenzyme which is characterized by a tumor specific isoenzyme shift is hexokinase (HK). Four different hexokinase isoenzymes are known. HK I and HK II could be associated with the outer mitochondrial membrane (OMM), where they directly benefit from mitochondrial ATP production and stabilize the OMM voltage-
Robust metabolic adaptation underlying tumor progression
dependent anion channel (VDAC) against apoptotic stimuli (Robey and Hay 2006). In tumor cells the HK isoenzyme type II is predominant and under the control of HIF-1 (Yasuda et al. 2004). The HK isoenzyme type II is less sensitive to inhibition by glucose-6-phosphate, thereby allowing the accumulation of glycolytic phosphometabolites (Fig. 1). Recently, a new isoenzyme of transketolase (TKT), the key enzyme of the non-oxidative branch of the pentose phosphate pathway (PPP), called TKTL1 (tranketolase-like 1), has been described (Coy et al. 2005) (Fig. 1). mRNA levels of this isoenzyme are over-expressed in urothelial, ovarian, colon and gastric cancers (Krockenberger et al. 2007; Langbein et al. 2006; Staiger et al. 2006). Inhibition of TKTL1 expression by RNAi in hepatoma cell line HepG2 has demonstrated that TKTL1 gene influences total transketolase activity and cell proliferation in human hepatoma cells (Zhang et al. 2007). TKTL1 has distinct kinetic properties that permit this isoenzyme to catabolize the monosubstrate reaction from xylulose-5-phosphate, releasing glyceraldehydes-3-phosphate available for glycolysis and acetate, which could be used for fatty acid synthesis. It has been hypothesized that by this mechanism, TKTL1 contributes to the Warburg effect as well as to the enhancement of biosynthetic pathways (Langbein et al. 2006; Coy et al. 2005).
7 Alternative to glycolytic energy conversion In tumor cells the citric acid cycle is truncated due to an inhibition of aconitase by high levels of ROS (Kim et al. 2001). However, tumor cells overexpress phosphate-dependent glutaminase and NAD(P)-dependent malate decarboxylase (malic enzyme), which in combination with the remaining steps of the citric acid cycle from a-ketoglutarate to glutamate, aspartate, CO2, pyruvate, lactate and citrate impart the possibility of a new energy-producing pathway termed glutaminolysis (Perez-Gomez et al. 2005; Rossignol et al. 2004) (Fig. 2). A high capacity of glutaminolysis is linked to neoplastic transformation. An inhibition of glutaminolysis decreases tumor cell proliferation and correlates with phenotypical and functional differentiation of the cells (Lobo et al. 2000; Spittler et al. 1997). Besides the regeneration of energy, glutamine catabolism provides glutamate and aspartate, which, together with glutamine itself, are precursors of nucleic acid and serine synthesis. The glutaminolytic malic enzyme reaction is another source for NADPH production, besides the oxidative PPP.
8 Tumor metabolism and apoptosis One of the main characteristics of malignant tumoral progression is the capacity of tumoral cells to avoid apoptosis
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(Hanahan and Weinberg 2000). Alterations in the apoptotic machinery are a common characteristic in cancer pathology. Since mitochondria, the factory of cell energy, plays an important role in the triggering of apoptosis, the relation between metabolism and apoptosis is a relevant issue. An important implication of the Warburg effect is the concomitant decrease of pyruvate available for mitochondrial oxidative phosphorylation (OXPHOS). The respiratory chain coupled to OXPHOS is a source of high levels of ROS, especially under hypoxic conditions when respiration functions abnormally (Garber 2006). Therefore, it could be argued that the under-utilization of mitochondria may protect tumor cells against oxidative damage. However, ROS generation could be understood as a double-edged sword: it has been reported intrinsic ROS stress in cancer cells (Pelicano et al. 2004), which could be explained by mitochondrial malfunction and may provoke mutagenesis through mitochondrial and nuclear DNA (Wallace 2005; Pelicano et al. 2004), enhancing carcinogenesis. But at the same time, since generation of oxidative stress in response to several stimuli is involved in the triggering of apoptosis (Vrablic et al. 2001), it has been proposed for treatment of cancer (Engel and Evens 2006; Mates and Sanchez-Jimenez 2000). The relationship between glycolysis and apoptosis is not only related to OXPHOS. Several studies have demonstrated that inhibition of glycolysis directly causes more sensitivity to induced apoptosis (Xu et al. 2005; Jeong et al. 2004). Thereby different glycolytic enzymes seem to be directly involved in the apoptotic process. One of the these enzymes is hexokinase (HK), the enzyme that performs the first step of glycolysis, phosphorylating glucose to glucose6-phosphate (G6P). The intracellular location of the hexokinase isoforms type I and II may vary between cytosol or attachment to the outer mitochondrial membrane (OMM). Binding of HK to the outer mitochondrial membrane promotes an open configuration of the voltagedependent anion channel (VDAC), confering hexokinase direct access to mitochondrially generated ATP (Arora and Pedersen 1988). Furthermore, HK may compete with proapoptotic proteins Bak and Bax, thus avoiding Bax/Bakmediated cytochrome c release and apoptosis (Robey and Hay 2006). Additionally, the survival factor Akt, commonly related to oncogenic signaling, provokes its antiapoptotic effects in a glucose-dependent manner that involves hexokinases (Fig. 1) (Majewski et al. 2004b; Rathmell et al. 2003), since disruption of the binding of hexokinase to the mitochondrial membrane impairs the anti-apoptotic effects of growth factors and Akt (Majewski et al. 2004a). Moreover, activity of the major downstream effector of Akt, mTOR (mammalian target of rapamycin), is also dependent on glucose and ATP availability (Dennis et al. 2001) and its inhibition by RAD001 in Akt activated
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mouse model decreases the expression of several glycolytic enzymes (Majumder et al. 2004). Liver cells and pancreatic beta cells express a certain hexokinase isoenzyme called glucokinase (GK). Although GK seems to be incapable of binding mitochondria in the liver (Bustamante et al. 2005; Malaisse-Lagae and Malaisse 1988), it has been located in a protein complex together with the pro-apoptotic factor Bad. Glucose deprivation results in dephosphorylation of Bad and Baddependent cell death. On the contrary, glucose promotes phosphorylation of Bad and, interestingly, in this phosphorylated state, GK has greater activity than with unphosphorylated Bad. Thus, glucose-mediated phosphorylation of BAD might enhance glycolysis, as well as preventing cell death (Danial et al. 2003; Downward 2003). Two other glycolytic enzymes which play a role in apoptosis are glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Dastoor and Dreyer 2001) and the M2-type pyruvate kinase isoenzyme (M2-PK) (Stetak et al. 2007), which are both generally over-expressed in tumor cells. Induction of apoptosis by H2O2, UV light or agents such as staurosporine and MG132 (GAPDH) or somatostatin analogues (M2-PK) is accompanied by nuclear translocation of these enzymes. The anti-apoptotic protein Bcl-2 inhibits GAPDH translocation to the nucleus and is discussed as part of a protective mechanism against apoptosis. However, the presence of GAPDH and M2-PK in the nucleus does not inevitably induce apoptosis. Thus, when GAPDH locates in nucleus, it is also related to gene expression, cell cycle regulation and DNA repair (Carujo et al. 2006; Dastoor and Dreyer 2001; Sirover 1999). Nuclear M2-PK has been found to participate in the phosphorylation of histone 1 (Ignacak and Stachurska 2003). The relationship between p53 and metabolism should be noted. p53 is one of the main apoptotic sensors and one of the most commonly studied tumor suppressor genes. Several studies have recently described its promotion to tumorigenesis not involving cell-cycle regulation and apoptosis, but through energetic interventions. For example, glucose deprivation induces p53 activation through energetic sensor AMPK (Jones et al. 2005). Interestingly, phosphoglucomutase (PGM), a glycolytic enzyme that is not controlled by HIF-1 (Yasuda et al. 2004; Semenza et al. 2001; Dang and Semenza 1999), is a direct transcriptional target of p53 in myocytes (Ruiz-Lozano et al. 1999). It has recently been reported that enhancement of PGM activity immortalizes mouse embryonic fibroblast, with the suggestion that p53 mutation facilitates this process (Kondoh et al. 2005) (Fig. 1). Two other recent studies have stressed the importance of the p53 controlling metabolism. Bensaad et al. (2006) identified TIGAR (TP53-induced glycolysis and apoptosis
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regulator), which is expressed before moderate apoptotic stimuli as a new target of p53. TIGAR shares similarities with the bisphosphatase domain of the bifunctional enzyme, 6-phosphofructo-2-kinase/kinase-2,6-bisphosphatase, which causes a decrease in fructose-2,6-bisphosphate. Fructose-2,6-bisphosphate is a potent allosteric activator of glycolysis through the activation of 6-phosphofructo-1kinase. Thus, TIGAR expression blocks glycolysis, channeling the glycolytic intermediates into the oxidative pentose phosphate pathway (PPP). The enhancement of the PPP increases NADPH generation, which is used to transform glutathione to its reduced form, promoting the scavenging of ROS. Moreover, NADPH blocks activation of caspase-2 (Nutt et al. 2005), thereby inhibiting temporary p53-induced apoptosis. Furthermore, the PPP generation of pentose phosphate intermediates through the PPP could play an important role in DNA repair as nucleotide precursors. A link between p53 and mitochondrial metabolism is described by Matoba et al. (2006), who found that SCO2, a protein required for the assembly of the mitochondrial cytochrome c oxidase (COX II) subunit, is induced in a p53-dependent manner. In eukaryotic cells, mitochondrial COX complex is where most of the molecular oxygen is consumed and its disruption causes a metabolic switch towards glycolysis, as observed in p53-deficient cells.
9 Conclusions and perspectives Tumor metabolism represents the end point of many signal cascades recruited by oncogenic activation. At the same time, study of the metabolic status of tumor cells during tumor progression reveals characteristic adaptations during carcinogenesis. Although it is arguable whether metabolic alterations are simply a consequence of other cellular events such as changes in signaling pathways or protein expression, these alterations are not only a mirror of the physiological changes within tumor cells, but also actively enhance the degree of tumor malignancy. The maintenance of the high glycolytic phenotype gives tumor cells the advantages of surviving and invading in areas with low oxygen supply. Determined support for further investigation is needed to put cancer metabolomics on a level with genomics and proteomics. Vizan et al. (2005) showed that different codon-specific mutations in K-ras cause distinct metabolic phenotypes in NIH3T3 mice fibroblast, which gives us an idea of the sensitivity of central carbon metabolism in response to oncogenic transformation. Thus, differential metabolic adaptations of cancer cells can be new complementary targets in the design of rational combinational treatments in chemotherapy.
Robust metabolic adaptation underlying tumor progression Acknowledgments This work was supported by Grants SAF200501627 from the Spanish Ministery of Education and Science and ISCIII-RTICC (RD06/0020/0046), from the Spanish Ministry of Health and Consumption as well as from the Deutsche Forschungsgemeinschaft (Ma 1760 1-2 and 2-1). We are also grateful to Michael Eaude of the University of Barcelona Language Service for valuable assistance in the preparation of the manuscript.
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