Tumor cell metabolism

Abstract

Cancer is a genetic disease that is caused by mutations in oncogenes, tumor suppressor genes and stability genes. The fact that the metabolism of tumor cells is altered has been known for many years. However, the mechanisms and consequences of metabolic reprogramming have just begun to be understood. In this review, an integral view of tumor cell metabolism is presented, showing how metabolic pathways are reprogrammed to satisfy tumor cell proliferation and survival requirements. In tumor cells, glycolysis is strongly enhanced to fulfill the high ATP demands of these cells; glucose carbons are the main building blocks in fatty acid and nucleotide biosynthesis. Glutaminolysis is also increased to satisfy NADPH regeneration, whereas glutamine carbons replenish the Krebs cycle, which produces metabolites that are constantly used for macromolecular biosynthesis. A characteristic feature of the tumor microenvironment is acidosis, which results from the local increase in lactic acid production by tumor cells. This phenomenon is attributed to the carbons from glutamine and glucose, which are also used for lactic acid production. Lactic acidosis also directs the metabolic reprogramming of tumor cells and serves as an additional selective pressure. Finally, we also discuss the role of mitochondria in supporting tumor cell metabolism.

Introduction

Cancer is a genetic disease that involves dynamic changes in the genome and is the result of several complex events. Changes in a cell, that direct transformation toward a malignant phenotype, include gain-of-function mutations that activate oncogenes, loss-of-function mutations that inactivate suppressor genes and mutations that inactivate stability genes. Stability genes include those responsible for mitotic recombination and chromosomal segregation. There are six distinctive characteristics that a cell acquires during its progression into malignancy: limitless replicative potential, sustained angiogenesis, avoidance of apoptosis, self-sufficiency in growth signals, insensitivity to anti-growth signals and tissue invasion and metastasis. These hallmarks have been studied extensively.^1,2 Two emerging characteristics have recently been added to the list: evasion of immune destruction² and reprogramming of energy metabolism.^2-5 Although the latter is the most evident change in tumor cell metabolism, it is only part of the large picture of metabolic reprogramming. Besides energy, tumor cells require building block molecules, NADPH and NADH cofactors for housekeeping, growth and proliferation in a changing microenvironment. Tumor cells undergo metabolic reprogramming, which involves changes in the metabolic fluxes, to satisfy large demands for ATP, NADPH, NADH and carbon skeletons.

To carry out replicative division, a cell must duplicate its genome, proteins and lipids and assemble these elements into daughter cells.⁶ The increased rate of cell division in cancer requires metabolic pathways to be redesigned, giving rise to the tumor cell metabolism.

Otto Warburg originally observed increased rates of glycolysis with high lactate production in tumor ascites,⁷ but this was only part of a more complex phenomenon. Tumor cells may reprogram their metabolism based on microenvironmental changes, such as acidosis and substrate and oxygen availability. To this end, tumor cells enhance glycolysis and glutaminolysis to fulfill their ATP and NADPH demands, respectively. Fatty acid and nucleotide biosynthesis mainly use glucose as a carbon source; whereas the Krebs cycle is mostly replenished by glutamine carbons, for which mitochondria are essential (Fig. 1).

Figure 1.

Comparison between the metabolism of tumor and normal cells. The main anaplerotic precursors in normal and tumor cells are shown in ovals. In tumor cells, glucose supports cellular growth through nucleotide and lipid biosynthesis. About 90% of R5P and 60% of fatty acids are glucose derived. Glutamine supports cells via anaplerosis of the Krebs cycle and NADPH regeneration, accompanied by lactate production. About 60% of lactate is glutamine derived.

A better understanding of metabolic reprogramming may lead to the identification of important control points that will help in diagnosis or that will be specific targets for the control of the disease.

Normal and Tumor Cell Proliferation

Mammalian cellular proliferation is not autonomous; in fact, normal cells enter the cell cycle only when given instructions by a finely controlled process that depends on neighbor cells, chemical signals in the microenvironment and the cell itself. In normal cells, an extracellular stimulus, such as growth factors, interacts with the corresponding receptor to promote an intracellular signaling cascade, which can induce cellular duplication. In cancer cells, genetic mutations increase the number of growth factor receptors. Intracellular signaling pathways that promote cellular proliferation may be constantly turned on or amplified, allowing malignant cells to self-stimulate growth and proliferation.⁸ To support this continuous cell proliferation, the biosynthetic capabilities of tumor cells are increased, including fatty acid and nucleotide synthesis. In contrast, β-oxidation of fatty acids is suppressed and futile cycles are minimized. These changes increase the metabolic autonomy of the transformed cells, allowing them to acquire an enhanced anabolic phenotype.⁶

Warburg Effect or Aerobic Glycolysis

In mammalian cells, glucose is the preferred substrate for energy-yielding metabolism. Under normal conditions, glycolysis provides two moles of pyruvate, ATP and NADH per mole of consumed glucose. In the mitochondria, pyruvate enters the Krebs cycle to generate NADH, the electron donor of the oxidative phosphorylation (OXPHOS) pathway, which yields 18-fold more ATP than glycolysis. Moreover, glucose provides several metabolites used as building blocks for the biosynthesis of macromolecules that are required for cellular proliferation. In normal cells, some intermediaries of the Krebs cycle are also used for biosynthetic purposes and other molecules such as pyruvate, glutamate and fatty acids guarantee the continued replenishment of carbon skeletons in this cycle⁵ (anaplerotic pathways, Fig. 1).

In the 1920s, Otto Warburg demonstrated that tumor ascites had a high glucose consumption rate and produced lactate, in spite of sufficient oxygen availability to oxidize glucose completely. This phenomenon was termed the Warburg effect or aerobic glycolysis and it is considered a distinctive characteristic of aggressive tumors. The Warburg effect was originally proposed to be a result of a permanent impairment of oxidative metabolism.⁷ However, the role of mitochondria in tumor cells has been controversial because many tumor cell lines with high proliferative rates do not have defects in their oxidative metabolism.⁹ Although lactate dehydrogenase A (LDH-A) is overexpressed in several tumors including gastric cancer, head and neck carcinoma, lung cancer and others,^10-12 it has been shown that LDH-A knocked out tumor cells maintain a substantial capacity to produce ATP by oxidative phosphorylation.¹³

Mitochondria participate in important cellular processes, such as ATP production, reactive oxygen species (ROS) generation, apoptosis and the synthesis of metabolites that serve as building blocks for biomolecules. As such, a mitochondrial dysfunction might favor tumor development if this impairment leads to a decrease in apoptosis, higher production of ROS or the activation of a hypoxia-like pathway under normoxic conditions.¹⁴ Nevertheless, it is not known how tumor growth would be supported in the changing conditions of the tumor microenvironment with a permanent impairment of the mitochondria. Thus, mitochondrial impairment would be a disadvantageous strategy for survival, given the catabolic and anabolic demands of proliferating tumor cells.¹⁵ Throughout this review, studies will be discussed that explain how the mitochondria become a key factor in the development and maintenance of cancer.

The idea that tumors have a particular metabolic phenotype that is associated with increased glycolysis is supported by molecular and functional data.⁵ Microarray data sets collected from several studies have consistently shown most of the genes involved in glucose transport and glycolysis to be upregulated in different types of tumors.^9,16,17 Glucose transporters are overexpressed in hepatocarcinomas, breast cancer, neuroendocrine carcinomas, lymphoblastic leukemia and others.^18-21 It has been proposed that the high glucose uptake in cancer cells favors cell proliferation more than energy production because glucose metabolism mainly supports fatty acid and ribose-5-phosphate synthesis^5,22 (Fig. 1). Glycolytic genes like TPI1, PGK1, ENOα, PK-M2, ALDO-A, GPI and PDHA1 were also found to be significantly upregulated in most of the 20 different types of common cancer examined, regardless of their tissue origin.¹⁷ The substantial increase in the glycolytic flux of cancer cells is caused, in part, by the activation of the regulatory bi-functional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) enzyme.²³ Fructose-2,6-bisphosphate (F2,6BP) is synthesized by the PFKFB enzyme; F2,6BP is an allosteric activator of 6-phosphofructo-1-kinase (PFK-1), which is a rate-limiting enzyme that is an essential control point in the glycolytic pathway. PFK-1 activity is strongly inhibited by ATP; however, F2,6BP can relieve this inhibition and enhance glucose uptake and glycolytic flux.²³ In addition, it has been reported that in vitro inhibition of aconitase, a Krebs cycle enzyme, favors ATP synthesis by phosphorylation at the substrate level or the oxidation of glutamine.²⁴

The serine/threonine kinase Akt, also known as protein kinase B, is one of the most frequently activated protein kinases in cancer. The activation of Akt can increase the total cellular ATP level through the coordinated regulation of both glycolytic and oxidative metabolism, with a concomitant increase in oxygen consumption.¹⁵ To augment glucose metabolism, Akt induces the translocation of glucose transporters to the cell surface, activates hexokinase (HK) and inhibits glycogen synthase kinase-3.²⁵ Akt activation also favors tumorigenesis by decreasing apoptotic susceptibility due to the increased HK-mitochondria interaction.¹⁵ Activation of phosphatidylinositol 3 kinase (PI3K)/Akt also makes tumor cells glucose-dependent because the energy obtained from fatty acids (β-oxidation) is suppressed.²⁶ Rapidly proliferating tumor cells require high levels of ATP for growth and proliferation, but they also require carbon skeletons for macromolecule biosynthesis. When these cells use and increase aerobic glycolysis to obtain ATP, they also conserve carbon skeletons because there is no CO₂ production in glycolysis. If tumor cells were mainly generating ATP through the Krebs cycle and OXPHOS, they would lose more carbon in the form of CO₂. However, the Krebs cycle must function to some extent to fulfill its anabolic role.

Tumor Cell Metabolism in Hypoxic and Normoxic Conditions

It has been shown that the microenvironment of premalignant epithelial cells inevitably develops hypoxia and acidosis.²⁷ These conditions serve as selective forces that govern cellular adaptation during the delayed stage of tumor evolution, which explains the extreme variability in gene expression patterns.³ In fact, the partial pressure of oxygen (pO₂) inside the tumor is frequently lower than the surrounding normal tissue. Intratumoral hypoxia is associated with a larger risk of local spread, metastasis, failure of the treatment and mortality for the patient^28-30 (Fig. 2). Hypoxia selects cells with a fundamental metabolic adaptation in which glycolysis is uncoupled from the respiratory chain and becomes the main ATP production source.³¹ Hypoxia also increases the resistance of tumor cell mitochondria to apoptosis by acting as a selective pressure on mitochondrial apoptotic pathways.³²

Figure 2.

Schematic representation of the tumor microenvironment in which glucose and oxygen gradients are inevitably formed, with the highest concentrations close to the blood vessels. Left side: metabolic symbiosis is represented, with the lactate transporter MCT1 playing a key role. Under lactic acidosis, aerobic tumor cells express MCT1 and preferentially take up lactate, which is transformed into pyruvate. This metabolite enters the mitochondria to produce ATP, allowing the tumor cells most distant from the blood vessels to acquire glucose and, consequently, produce lactic acid. Right side: metabolic symbiosis is disrupted via the inhibition of MCT1 expression. Aerobic tumor cells take up glucose; the tumor cells most distant from the blood vessels cannot consume glucose and consequently die. HIF-1, hypoxia-inducible factor 1; MCT1, monocarboxylate 1 transporter; GLUT1, glucose transporter 1; LDHA, lactate dehydrogenase A; PDK, pyruvate dehydrogenase kinase; PDH, pyruvate dehydrogenase; MCT4, monocarboxylate 4 transporter.

Metabolic reprogramming of tumor cells changes the metabolic fluxes, enhancing glycolysis and restructuring the Krebs cycle in response to the reduced availability of oxygen. One of the main regulatory mechanisms underlying aerobic glycolysis involves hypoxia-inducible factor (HIF-1). The activation of HIF-1 regulates a pleiotropic response that involves the altered expression of approximately 450 genes involved in glycolysis, lactate production and lactate/proton extrusion, angiogenesis, metastasis and iron metabolism. In tumor cells, HIF-1 induces the upregulation of some isoforms of glycolytic enzymes that are different from those found in non-malignant cells. These include glucose transporters (GLUT1, GLUT3), glycolytic enzymes (HKI, HKII, PFK-1, ALDO-A, ALDO-C, PGK1, ENO-α, PK-M2, PFKFB-3) and enzymes related to lactate production and lactate/proton extrusion (LDH-A, MCT4).^33,34 Hypoxia also induces the expression of the pyruvate dehydrogenase kinases (PDK1 and PDK3 isoforms),^26,35 which have different activities and specificities for the phosphorylation and inactivation of the three isoforms of pyruvate dehydrogenases (PDHA1, PDHA2 and PDHB),^36,37 which transform pyruvate into acetyl-CoA (Fig. 2). Increasing evidence has associated HIF-1 function with metastatic characteristics, such as the epithelial to mesenchymal transition, cell detachment, invasion and tumor cell seeding;^28,30 HIF-1 overexpression and prevalence is also correlated with the severity of the cancer.^3,4,38,39

HIF-1 is a heterodimeric complex formed by two subunits: HIF-1β, which is constitutively expressed and HIF-1α, which is highly susceptible to oxygen-dependent degradation.³⁸ In the presence of oxygen, HIF-1α is hydroxylated on proline residues by PHD (prolyl hydroxylase domain protein). This oxygen-dependent hydroxylation allows HIF-1α recognition by VHL (von Hippel-Lindau protein), which targets HIF-1α for proteasomal degradation.^40,41 However, tumor cells achieve HIF-1α stabilization under normoxia through genetic alterations to this oxygen-signaling pathway, such as the inactivation of VHL,^39,42 the inhibition of PHD by the accumulation of succinate as a result of mutations in succinate dehydrogenase,^41,43 fumarate hydratase,⁴⁴ or the PHD inactivation by endogenous 2-oxoacid oxaloacetate.⁴⁵ The activation of Akt may also contribute to increased HIF-1α levels under normoxic conditions via an mTORC1-mediated effect on HIF-1α mRNA translation.¹⁵ The HIF-dependent transcriptional mechanism remains incompletely understood. The HIF1α-HIF1β heterodimer, along with the transcriptional coactivator p300/CBP, binds to cognate hypoxia-responsive elements (HREs, consensus 5′-RCGTG-3′), upregulating a plethora of hypoxic genes that carry HREs within their promoters or enhancers.³⁵ However, HIF requires additional coactivators to mediate the induction of hypoxic genes. A molecular mechanism was recently reported by which PK-M2 interacts with HIF-1α in the nucleus and acts as a transcriptional coactivator in HeLa cervical carcinoma and Hep3B hepatoblastoma cells.⁴⁶ Estrogen-related receptors (ERRs) physically interact with HIF and were found to serve as essential cofactors for HIF in mediating the hypoxic response in a breast cancer cell line, although additional elements are necessary in the process.³⁵

Tumor cell populations are heterogeneous in their oxygen concentrations because tumors contain well-oxygenated (aerobic) and poorly oxygenated (hypoxic) regions. Some authors have noted that a tumor cell can experience oxygen oscillations, changing from hypoxic to normoxic conditions and vice versa in short periods of time.^27,29,30 The existence of a “metabolic symbiosis” between aerobic and hypoxic cancer cells has been reported. When tumor cells are cultured under hypoxic conditions, they increase their expression of the glucose transporter GLU-1, which is activated by HIF-1 (Fig. 2). The enhanced glycolytic carbon flux, in turn, increases lactate production, which is secreted to prevent intracellular acidification via the monocarboxylate 4 system (MCT4), which is a lactate transporter coupled to H⁺. Tumor cells, growing under normoxic conditions, take up lactate instead of glucose supporting the concept of a tumor metabolic symbiont. In aerobic cancer cells, the monocarboxylate 1 transporter (MCT1) internalizes lactate, which is converted into pyruvate by the lactate dehydrogenase B enzyme (LDHB). Pyruvate can then enter the Krebs cycle and its products may be used by the OXPHOS pathway for energy production (Fig. 2). When MCT1 is inhibited, aerobic tumor cells consume more glucose than lactate, breaking the metabolic symbiosis, at which point the anaerobic tumor cells die from glucose deprivation³¹ (Fig. 2). This phenomenon was demonstrated in a murine model in which the treatment of lung cancer with an MCT1 inhibitor indirectly induced the death of distant hypoxic tumor cells, causing necrosis in the hypoxic zone.³¹ It has also been shown that MCT1 expression is exclusively found in aerobic regions of human tumor tissue from head, neck, breast and colon cancers. This result is consistent with the overexpression of LDHB for utilizing lactate as an energy substrate.³¹ This phenomenon is of particular clinical importance because the hypoxic zone is known to be especially resistant to chemotherapy and radiation, resulting in treatment failure, disease relapse or finally, the death of the patient⁴⁷ (Fig. 2).

Tumor Cell Metabolism in Acidosis

When tumor cells enhance glycolysis and produce large quantities of lactic acid, they generate an environment that is more toxic to the adjacent normal cells than to the malignant cells because tumor cells have developed survival mechanisms. In addition to lactic acid, CO₂ is a significant source of acidic extracellular pH (pHe) in the tumor microenvironment; in the reaction catalyzed by carbonic anhydrase (CA), CO₂ is hydrated, producing bicarbonate (HCO^-₃) and H⁺. Hypoxia is usually associated with acidosis, but acidosis can also be observed in the tumor microenvironment under normoxic conditions because of the Warburg effect.

Acidosis promotes the subversion of both cell cycle checkpoints and apoptotic mechanisms.⁴⁸ As a result, after substantial cell mortality, acidosis selects for resistant phenotypes that are able to maintain higher invasion and higher motility for multiple generations.⁴⁹ Acidosis also promotes extracellular matrix degradation, increasing invasiveness.⁴⁹

Acidosis drives a stress response in tumor cells by provoking cell cycle arrest, restricting ribosomal biogenesis (the most energy demanding cellular process) and increasing mitochondrial activity.^50,51 As a result, tumor cells undergo cycles of cellular quiescence and proliferation, depending on nutrient concentrations and pH⁵² (Fig. 2).

Regulators like HIF-1 and Akt may play an important role in acidosis. HIF-1 has been reported to enhance the expression of some intracellular pH (pHi)-regulating systems that help cells cope with acidosis. These systems are vital because the maintenance of intracellular pH homeostasis is fundamental to life.⁵³ There are several key pHi-regulating systems that cooperate in maintaining a pHi permissible for survival. These systems include Na(+)/H(+) exchangers (NHEs), monocarboxylate transporters (MCTs), proton pumps (V-ATPase) and membrane-associated and cytosolic carbonic anhydrases (CAs), among others.⁵³ Some pHi-regulating systems can be overexpressed during acidosis in a hypoxia-independent manner, such as CA9 in glioblastoma cells.⁵⁴ CA9 expression confers a survival advantage on tumor cells exposed to a hypoxic and acidic microenvironment, its expression correlates with poor prognosis in human tumors.⁵⁵

Existing evidence indicates that under normoxic acidosis, tumor cells inhibit proteasomal degradation of HIF-1 via the nucleolar sequestration of VHL until neutral pH conditions are restored.⁵⁶ As a result, HIF-1 enhances glucose transport, glycolysis and the activity of pHi-regulating systems.⁵⁴ HIF activation by an acidosis-dependent mechanism functions in parallel to PHD activity, because pH has been shown not to affect PHD expression.⁵⁷

Other studies have shown that normoxic lactic acidosis in the MCF7 breast cancer cell line promotes a stress response that turns on the global regulator TXNIP. This leads to a significant reduction in glucose transport and glycolysis and OXPHOS returns as the main pathway of ATP production. This phenomenon may be mediated by the suppression of the Akt and HIF-1 pathways.^26,51

Acidosis has also been shown to stimulate inflammatory cells, such as neutrophils and dendritic cells; this inflammatory response is mediated via PI3K/Akt activation.⁵⁸ More studies are needed to elucidate the participation of Akt and HIF-1 in tumor cell survival under acidic conditions, while taking lactate concentration, hypoxia, normoxia and timing into account.

Glutamine Metabolism

Glutamine is the most abundant free amino acid in the body and the main transporter of ammonia in its nontoxic form.⁵⁹ In tumor cells, glutamine metabolism exceeds that of any other nonessential amino acid and plays an important role in the anaplerosis of the Krebs cycle, NADPH regeneration through lactate production and the restoration of the reduced glutathione pool to promote the scavenging of reactive oxygen species (ROS).^60-62 Glutamine can also act as a nitrogen donor for the synthesis of purine and pyrimidine nucleotides, amino acids and other metabolites⁶³ (Fig. 1).

Anaplerosis refers to the replenishment of Krebs cycle metabolites, which are constantly used to build macromolecules during cellular proliferation. A high anaplerotic flux can be an indicator of cellular proliferation and is more specific than a high glycolytic flux because the latter can be induced by either hypoxia or another kind of stress that is independent of macromolecular synthesis.⁶

Glutamine metabolism takes place within the mitochondria. For this reason, it must be transported from the extracellular medium into the mitochondria of tumor cells by specific transporters in the plasma and mitochondrial inner membranes.⁶⁴ The mitochondrial transport of metabolites is usually one or two orders of magnitude faster than plasma-membrane-related transport. Studies using native vesicles isolated from the mitochondrial inner membrane confirmed the existence of a transport system with a high capacity and specificity for L-glutamine, showing cooperation and strong inhibition of some L-glutamine analogs.⁶³ In the mitochondria, the phosphate-activated glutaminase produces glutamate and ammonia by deaminating glutamine. High concentrations of inorganic phosphate inside the mitochondria of tumor cells allow the complete activation of the tumor glutaminase. Experimental evidence supports the direct correlation between glutaminase activity and the proliferative capacity of malignant cells.⁶⁵ Tumor glutaminase reaches its maximum expression and activity just before reaching the maximum proliferation rate.^60,61 Concomitant with higher glutaminase activity, an inhibition of glutamine synthetase has been observed; glutamine synthetase has been considered a “dispensable” enzyme for tumors, except under glutamine depletion conditions.^60,61

Using ¹³C NMR spectroscopy, it has been revealed that glutamine is the primary anaplerotic precursor in tumor cells.²² Glutamine replenishes the Krebs cycle at the α-ketoglutarate (α-KG) level and becomes the main source of replenishment for oxaloacetate²² (Fig. 1). Pyruvate carboxylase (PC) can also restore oxaloacetate, using pyruvate as substrate in neuronal and glial tumor cell lines or alanine in hepatic tumors in rats.^66,67 Interestingly, the activity of PC can be suppressed in glioblastoma cells and in C6 glioma cells using glucose as the main substrate.^22,68

A fraction of malate is secreted from the mitochondria to the cytosol, where it is transformed into pyruvate by malic enzymes, simultaneously reducing NADP+ to NADPH (Fig. 1). Pyruvate is mainly transformed into lactate by LDH enzymes, although it can also be used for alanine production. Approximately 60% of glutamine is converted to lactate.²²

In malignant cells, glutaminolytic flux (the transformation of glutamine into lactate with NADPH regeneration) seems to be the main NADPH source. NADPH production is higher than the necessary for fatty acid production, which suggests that the NADPH generated during glutaminolysis may supply other anabolic processes, such as nucleotide biosynthesis or may be used in the cell’s antioxidant defense against a hostile microenvironment (Fig. 1).^4,22,62,69

Glutamine catabolism results in the production of lactate, alanine and ammonia, which maintains nonessential amino acid pools. Some glutamine amino groups are also secreted out of the cell instead of being incorporated into other molecules, a consequence of using glutamine for regenerating NADPH pools.²²

One of the best characterized regulatory mechanisms that induce glutamine catabolism is mediated by c-Myc. The MYC oncogene encodes a master transcription factor, c-Myc, that is frequently deregulated in human cancers.⁷⁰ c-Myc regulates the expression of genes and microRNAs involved in the cell cycle and in glucose and glutamine metabolism.⁵⁹ In addition, it also regulates genes involved in the biogenesis of ribosomes and mitochondria.⁷¹ In MYC-transformed cells, glutamine depletion induces apoptosis as a result of the decrease in Krebs cycle intermediaries.⁷²

c-Myc increases glutamine influx through transcriptional repression by miR-23a/b microRNAs, resulting in an increase in mitochondrial glutaminase expression, glutamine metabolism and the glutamine transporters ASCT2 and SLC7A1.^63,73

c-Myc also induces the expression of lactate dehydrogenase A (LDH-A),⁷¹ possibly to metabolize the accumulated pyruvate generated by the enhanced glycolytic flux in proliferating cells,⁶ as it has been suggested that the tricarboxylic acid cycle is saturable.⁷⁴

Tumor Cells Synthesize Fatty Acids and Nucleotides at High Rates

There are two biosynthetic activities needed for tumor proliferation: (a) fatty acid production for lipid biosynthesis and (b) ribose-5-phosphate (R5P) production for nucleotide biosynthesis.⁷⁵ The biosynthesis of lipids and nucleotides share four important characteristics: (1) utilization of glucose as the main carbon source, (2) consumption of intermediates of the Krebs cycle, imposing the need for mechanisms to replenish the cycle, (3) a need for NAPDH reductive power⁷⁵ and (4) anaplerosis and NADPH regeneration are mainly supported by glutamine metabolism (Fig. 1). Tumor cells require a robust nutrient intake to maintain their anabolic metabolism.²²

Fatty Acid Synthesis

Most normal human tissues use exogenous lipids acquired from the diet for the synthesis of new structural lipids, while de novo fatty acid synthesis is usually suppressed. In contrast, de novo fatty acid synthesis is usually enhanced in tumor cells, leading directly to fatty acid accumulation (Fig. 1). This increase can also affect fundamental cellular processes as well as signal transduction and gene expression because cytosolic proteins, enzymes and membrane-targeted proteins are modified by lipids.^75-77

De novo fatty-acid synthesis involves three key enzymes: ATP-dependent citrate lyase (ACLY), acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN). ACLY transforms citrate to acetyl-CoA and oxaloacetate, ACC carboxylates acetyl-CoA to form malonyl-CoA, FASN catalyzes the NADPH-dependent condensation of acetyl-CoA and malonyl-CoA to produce long-chain fatty acids.⁷⁶ Normal cells usually maintain FASN expression at low levels. In contrast, several human cancers, including lung, prostate, ovary, breast, endometrium and colon have often shown FASN overexpression.^78-87 This overexpression has been shown to participate in very early tumorigenesis, tumor growth and survival.^76,86 FASN upregulation also protects cells from death by inhibiting the intrinsic pathway of apoptosis.⁸⁰

The siRNA-mediated silencing of FASN or the use of FASN inhibitors, such as cerulenin, C75 and orlistat, has been shown to induce apoptosis in several cancer lines.^80,87,88 Unlike previously studied drugs, FASN inhibition with C93 significantly inhibited growth without causing anorexia or weight loss in the treated animals; hence, it has promising therapeutic potential.⁸⁹

Tumor cells also express high levels of the lipogenic enzymes ACLY and ACC, consistent with the necessity of robust lipid synthesis. The in vivo and in vitro inhibition of the ACLY and ACC enzymes causes diminished cellular proliferation, decreased tumor size and a loss of cellular viability. In addition to its role in fatty acid synthesis, ACLY reinforces the Warburg effect by preventing the accumulation of cytosolic citrate, which is a glycolysis inhibitor.^75,90,91

Glucose provides 60% of the carbon used for fatty acid synthesis.²² The continued export of citrate from the Krebs cycle may cause a carbon deficit, which can be replenished by the anaplerotic flux derived from the metabolism of glutamine to continue fatty acid synthesis and cellular proliferation (Fig. 1). Fatty-acid synthesis requires an NADPH source; this is mainly supplied by the cytosolic malic enzyme (ME1) because the oxidative branch of the pentose phosphate pathway (PPP) has a reduced participation in ribose 5-phosphate synthesis. Under these conditions, glucose 6-phosphate dehydrogenase (G6PDH) cannot support NADPH regeneration.⁶⁹ Previous studies have reported that TGF-β promotes the incorporation of glucose carbon through the nonoxidative branch of the PPP for ribose synthesis in lung tumor cells, causing a decrease in CO₂ production.⁹²

Tumors achieve a high fatty acid biosynthesis rate through multiple effects of oncogenic mutations, especially those involving the PI3K/Akt/mTOR system. Following Akt activation by PI3K, PDK1 or mTORC2, this system stimulates lipogenic gene expression, increasing the nuclear localization of SREBP-1 (sterol response element binding protein-1), which is a transcription factor whose targets include ACLY, ACC, FASN and ME1.^75,76

mTOR (mammalian target of rapamycin) is found in two different complexes within the cell, mTORC1 and mTORC2. mTORC1 is inhibited by rapamycin and its analogs. The disruption of the mTORC2 assembly is observed only in certain cell types after prolonged exposure to these compounds.⁹³ mTORC1 is activated by growth factor signaling through PI3K activation of AKT⁹⁴ and regulates cell mass by increasing protein synthesis.¹⁵ mTORC1 increases the glucose transporter expression in the plasma membrane, allowing the robust transport of the major lipogenic precursor.^75,76 mTOR negatively regulates catabolic processes like autophagy, which can also be a survival mechanism under conditions of metabolic stress.⁹⁴

Nucleotide Synthesis

The abundance of precursors, cofactors and enzymes involved in the nucleotide biosynthesis pathway, determines nucleotide pool levels and the maximal proliferative capacity of a cell. Non-transformed cells use both the oxidative and non-oxidative branches of the PPP to generate ribose 5-phosphate (R5P) and NADPH (Fig. 1). To enhance nucleotide synthesis and DNA repair, non-transformed cells need to increase the availability of fructose 6-phosphate or glyceraldehyde 3-phosphate to convert them in R5P synthesis using the non-oxidative branch of the PPP, which is reversible (Fig. 1). One proposed regulatory mechanism is p53 activation as a consequence of DNA damage, which activates TIGAR (TP53-induced glycolysis and apoptosis regulator) and PGM (phosphoglucomutase). These molecules diminish fructose 2,6 bisphosphate (F2,6BP) levels and phosphofructokinase-1 (PFK-1) activity, leading to an increase in F6P levels that can be channeled into the non-oxidative branch of the PPP.⁷⁵ In contrast, PFK-1 activity is significantly increased in cancer cell lines and primary tumor tissues.^34,62 The p53-inducible TIGAR protein, aside from promoting PPP, helps to lower intracellular ROS levels.⁹⁵

Glucose is also the main precursor for R5P synthesis in tumor cells; more than 90% of ribose molecules have been shown to be glucose-derived in the H441 lung tumor cell line.⁹² Some studies have reported that the non-oxidative arm of PPP is the principal pathway for R5P synthesis in tumor cells^25,62,92. after finding a significant overexpression of the transketolase-like 1 (TKTL1) enzyme in progressing tumors.⁹⁶ However, other reports have indicated that both G6PDH and TKTL1 are overexpressed during the cell cycle progression of a human colon cancer cell line⁹⁷ and in (pre)neoplastic lesions in rat liver.⁹⁸ It is likely that cancer cells acquire diverse energy supplies that are differentially regulated during the development of the disease. At the beginning of the (pre)neoplastic lesion, both oxidative and non-oxidative PPP pathways are functional, but when the tumor is well established, it displays a metabolic reprogramming toward the non-oxidative PPP pathway (Fig. 1). More studies, however, are needed to confirm this hypothesis.

Tumors and tumor cell lines lacking p53 generally express PK-M2 (one of the four pyruvate kinase isoenzymes: L, R, M1 and M2)^62,75 and TKTL1 (transketolase-like 1).⁷⁵ The enzyme PK-M2 has submaximal activity that allows the accumulation of glycolytic intermediaries, leading to the redirection of carbon skeletons to the non-oxidative branch of PPP by TKTL1. Fructose 1,6-bisphosphate accumulation suppresses G6PDH, limiting the participation of the oxidative branch in R5P synthesis.⁷⁵ It has been reported that the inhibition of G6PDH is not lethal in normal cells, although it can increase oxidative stress susceptibility. In addition, the overexpression of G6PDH in normal cells stimulates their proliferation.⁹⁹

TKTL1, one of the TKT family members (TKT, TKTL1 and TKTL2), is overexpressed in several tumors, including colon and urothelial cancer, head and neck carcinoma, lung cancer and metastatic tumors.^96,100-102 TKTL1 suppression significantly reduces cell growth and glucose consumption and lactate production in colon carcinoma cells. TKTL1 inhibition also diminishes cell proliferation in several types of cancer cells,¹⁰³ indicating its importance in tumor cell proliferation.

In addition, purine biosynthesis requires other important precursors, such as glycine. Glycine synthesis from serine is favored by 3-phosphoglycerate (3-PG) accumulation, via PK-M2 upregulation.⁶² It has been reported that the concentration of PK-M2 correlates with the clinical stage and severity of the disease in cervical cancer patients.¹⁰⁴ In contrast, PK-M2 expression was significantly higher in patients surviving breast cancer for more than 13 y.¹⁰⁵

Conclusions

Throughout this review, we have described how tumor cells change their metabolism in comparison to non-transformed cells. Tumor cells undergo metabolic reprogramming to modify their metabolic fluxes in response to large demands for, not only ATP, but also NADPH, NADH and carbon skeletons for housekeeping, growth and proliferation, depending on the changes in their microenvironment. However, not all tumor cells pass through the same metabolic states at the same time, and they do not stay in that state indefinitely. The changing tumor microenvironment (e.g., nutrient and oxygen availability and acidosis) appears to exert selective pressures on tumor cells, leading to modifications in their metabolic pathways to survive. Hypoxia has been the best documented selective force that directs metabolic adaptation. Recently, acidosis has also been shown to direct metabolic reprogramming in tumor cells, leading to the inhibition of glycolysis and the use of OXPHOS as the main pathway of ATP production. Although the Warburg effect was originally associated with mitochondrial dysfunction, tumor cells need to maintain functional mitochondria to reprogram their metabolism in the ever-changing microenvironment.

Several regulatory pathways participate in the metabolic reprogramming of tumor cells; HIF-1α and PI3K/Akt/mTOR are the best characterized. Through these regulatory mechanisms, tumor cells increase the levels of several glycolytic enzymes, as well as lipogenic enzymes. Classic oncogenes such as c-Myc have been shown to play a role in metabolic reprogramming.

Cancer development depends on many factors, and for this reason, it is difficult to establish a generalized cancer treatment. The study of the metabolic reprogramming processes that give rise to and support tumor development will allow us to find specific markers to establish an early diagnosis, to support prognostic tools and to find selective treatments for limiting or preventing tumor growth. Thus, survival and quality of life will be improved for cancer patients.