Abstract
Energy metabolism refers to all the reactions involved in generating adenosine triphosphate (ATP) from nutrients, including both aerobic respiration (oxygen present), anaerobic respiration (fermentation) as well as fatty acid and amino acid metabolism. Under normoxic conditions, normal cells use aerobic respiration to oxidize glucose via mitochondrial oxidative phosphorylation. Cancer cells, on the other hand, prefer to ferment glucose into lactate even with oxygen present (Warburg effect) and show altered lipid and amino acid metabolism. This chapter discusses the metabolic features of neuroblastoma (NB), which enable the tumor to maintain the ATP levels and biomass necessary for supporting high proliferation and highlights new metabolic targets in NB therapy.
Keywords
Cancer metabolism, Glycolysis, Ketogenic diet, Metabolic targeting, Mitochondrion, Neuroblastoma, Oxidative phosphorylation, Warburg effect
Acknowledgments
This work was supported by Austrian Research Promotion Agency (822782/THERAPEP) and the Children’s Cancer Foundation Salzburg.
Introduction
Most cancer cells reprogram their metabolism and pathways of nutrient acquisition to ensure high levels of energy supply and biomass production, while maintaining specific metabolic reduction-oxidation (redox) states, for achieving high proliferation.
Mitochondria are the cell organelles involved in aerobic respiration, many essential metabolic and catabolic pathways, calcium homeostasis, redox balance, and regulation of apoptosis .
Mitochondrial energy metabolism consists of pyruvate oxidation, β-oxidation of fatty acids, tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). Redox equivalents (NADH, FADH 2 ) are oxidized in the respiratory chain, which consists of four mitochondrial OXPHOS complexes. The released energy is used to pump protons across the mitochondrial inner membrane. Complex V of the OXPHOS uses the generated proton gradient to synthesize adenosine triphosphate (ATP) .
In the presence of oxygen, normal cells generally metabolize glucose through the initial steps of glycolysis in the cytoplasm followed by oxidation of pyruvate via the TCA cycle and OXPHOS in mitochondria.
Cancer cells, in contrast, show fundamental metabolic changes, exhibiting higher glucose uptake and a shift from OXPHOS to glycolysis, known as the Warburg effect . This phenomenon has been utilized for diagnostic and even prognostic purposes in positron emission tomography (PET) . The shift from mitochondrial to glycolytic energy metabolism produces about 18 times less ATP per molecule of glucose, which, at first glance, seems counterproductive in terms of energy yield. But, in fact, glycolysis produces energy approximately 100 times faster than OXPHOS . In addition, the glycolytic pathway provides many biosynthetic precursors, which can be routed into other metabolic pathways .
The paradigm that the reduced OXPHOS capacity is only caused by hypoxia changed almost 70 years after Warburg’s discovery. Recent observations indicate that both genetic alterations and tumor microenvironment are also involved, resulting in a wide spectrum of metabolic plasticity . As a result, cancer cells can metabolize a variety of nutrients, such as glucose, amino acids, and fatty acids in different cellular pathways for producing energy and building blocks . Generally, the metabolic features in neuroblastoma (NB) are also characterized by high dependency on glucose metabolism and alterations of the OXPHOS system. Furthermore, common genetic changes in NB also contribute to these metabolic features ( Fig. 7.1 ).
Alterations in the OXPHOS System in NB
Neuroblastic tumors show notable deficiencies in the OXPHOS system. In ganglioneuroma, a severe loss of mitochondria accompanied by a general reduction of all OXPHOS complexes has been noted , whereas NB exhibit an overall decrease of the OXPHOS complexes but normal mitochondrial mass. A loss of mitochondria seems generally associated with stroma-rich tumors as seen in ganglioneuroma and certain types of Wilms’s tumors . Human NB and normal kidney tissue exhibited equal citrate synthase activity and porin levels (also known as voltage dependent anion channel 1, VDAC1), two markers of mitochondrial mass . Additionally, mitochondrial quantification by MitoTracker Green showed similar amounts of mitochondria in NB, cerebral cortex, and retinal ganglion cells .
Succinate dehydrogenase (SDH; or complex II) was the most severely reduced OXPHOS enzyme in human NB . All four subunits of complex II are encoded by the nuclear genome. Inactivating mutations in SDH, fumarate hydratase subunits and assembly factors lead to a large increase in the levels of metabolites, such as succinate and fumarate, that are referred to as “oncometabolites,” a relatively new term . It was demonstrated that the accumulation of succinate and fumarate in NB cells leads to the stabilization and activation of the hypoxia-inducible transcription factors (HIFs) via epigenetic regulation . HIFs are known as master regulators for adapting to low oxygen availability.
In addition to the alteration of OXPHOS complexes, NB also shows a significant reduction of mitochondrial DNA (mtDNA) copy number compared to normal kidney tissue . In primary NB tumors, several somatic and germline mtDNA mutations with pathogenic potential were reported, although in silico multistep analysis revealed no link between these mutations and OXPHOS reduction or tumorigenesis . Nevertheless, a study on primary and relapsed NB tumors indicates a concordant increase in both nuclear and mtDNA variants during tumor progression . Taken together, the molecular basis of the OXPHOS deficiency in NB remains elusive.
However, a question, which arises, is whether the impairment of OXPHOS in NB is a permanent or a transient phenotype. Based on in vitro and in vivo findings, the answer is controversial. Preventing glycolytic flux in NB cells by replacing glucose with galactose in tissue culture medium, or treatment of NB cells with a glycolysis inhibitor such, as 2-deoxy- d -glucose (2-DG) or iodoacetate, results in increased mitochondrial respiration . However, in vivo experiments did not show an adaptation of OXPHOS enzyme activity in NB tumors to low glucose levels induced by dietary intervention with a high-fat or calorie-restricted diet . The transcriptional coactivator peroxisome proliferator activated receptor γ coactivator 1α (PGC-1α) is a master regulator of nuclear-encoded mitochondrial genes. In NB cells, overexpression of PGC-1α upregulates the expression of genes involved in mitochondrial function, glucose transport and fatty acid metabolism .
Differentiated NB cells show higher OXPHOS activity compared to undifferentiated ones . Moreover, differentiation of NB is associated with the increased cellular content of mitochondria. Inhibition of mitochondrial protein synthesis arrests the neuronal differentiation of NB cell lines, whereas inhibition of mitochondrial ATP synthase does not affect differentiation . Doublecortin-like (DCL) is a microtubule-binding protein crucial for neuroblast proliferation, migration, and differentiation. Knockdown of DCL in NB cells decreases cytochrome c oxidase activity and ATP synthesis and significantly reduces NB growth in vivo .
Glucose Metabolism
Glucose Transport
Glucose is metabolized through several intermediary pathways, which are extensively reprogrammed in cancer cells, including glycolysis, the pentose phosphate pathway, the hexosamine pathway, glycogenesis, and the serine biosynthetic pathway . NB, like most solid cancers, exhibit a similar metabolic switch towards aerobic glycolysis, i.e., a higher amount of glucose utilization and lower level of oxygen consumption . Accordingly, a PET examination with 18 F-fluorodeoxyglucose (FDG) in NBs demonstrates high glucose influx into NB cells . Of clinical interest is the positive correlation between PET-FDG high maximum standardized uptake value (SUVmax) and poor prognosis in NB .
Glucose is taken up by cells through membrane-associated carrier proteins, termed sugar transporters, which are categorized into two families: facilitated glucose transporters (GLUT) and sodium/glucose cotransporters (SGLT). The GLUT family consists of 14 glucose transporter isoforms, with different affinities for glucose and other hexoses. Overexpression of GLUT1 is seen in many human cancers . In NB, a correlation between elevated GLUT1 expression and poor prognosis in patients was shown, suggesting the expression level of GLUT1 is an independent risk factor that can predict overall patient survival . Under low glucose conditions, insulin-like growth factor 1 (IGF-1) and the prosurvival protein Bcl-2 increases translocation of GLUT1 to the NB cell membrane and consequently the survival of the glucose-deprived cells . Similarly, it has been shown that GLUT4 translocation to the plasma membrane of NB cells can be regulated by both insulin- and leptin-signaling pathways .
Hexokinase
High glucose influx into cancer cells is often accompanied by increased glycolytic flux and lactate production. Hexokinase 2 (HK2), the first enzyme of glycolysis, is a key factor supporting the glycolytic phenotype of cancer cells. In NB, HK2 is upregulated in metastatic and stage 4 tumors and is associated with resistance to the chemotherapeutic deferoxamine , whereas differentiated NB cells show lower expression of HK1 and HK2 . Silencing of HK2 in NB cells results in lower engraftment, smaller tumor volumes, and reduced lung metastasis .
Protein Kinase B (AKT) activation has pleiotropic effects on diverse signaling cascades that regulate cell proliferation and survival, angiogenesis, suppression of apoptosis, resistance to standard cancer therapy, cell size, and response to nutrient limitation . It has been demonstrated that activation of AKT signaling stimulates glucose metabolism in NB, and is associated with poor prognosis . Phosphorylation of HK2 by AKT was found to increase the binding of HK2 to the outer mitochondrial membrane and prevent apoptosis in NB cells .
Pyruvate Oxidation
Pyruvate, the product of glycolysis, is converted by pyruvate dehydrogenase (PDH) to acetyl-coenzyme A, which in normal cells under normoxic conditions enters the TCA cycle. In cancer cells, pyruvate is mainly converted to lactate regardless of the presence of oxygen.
Pyruvate dehydrogenase kinase (PDK) is upregulated in a variety of cancers, which, in turn, inactivates PDH and induces a glycolytic phenotype. Hypoxia, via the activation of hypoxia-inducible factor 1α (HIF-1α), induces the expression of the PDK isoform PDK3 . Interestingly, under both normoxic and hypoxic conditions, silencing of PDK4 led to reduced protein expression of HIF-1α . In most tumor cells HIF-1α is stabilized under hypoxic and pseudohypoxic conditions, and is subsequently, responsible for upregulation of glycolysis and inhibition of OXPHOS in cancer cells .
Lactate Production
Lactate production in cancer cells provides carbon equivalents and ATP. The level and activity of lactate dehydrogenase (LDH), the enzyme which converts pyruvate to lactate, has been reported to be significantly higher in the serum of NB patients and is associated with worse prognosis . Moreover, stage-4 NB patients with lung metastasis have elevated levels of LDH . MYCN-amplified NB cells are highly dependent on LDHA activity, as depletion of its expression significantly inhibited their proliferation at normoxia in vitro and suppressed NB growth in xenograft model in vivo .
The lactate produced by cells should be excreted to maintain cellular homeostasis. Lactate efflux is driven by plasma membrane monocarboxylate transporters (MCTs) . The MCT1 expression shows a positive correlation with risk of fatal disease in NB and amplification of the transcription factor MYCN. Inhibition of MCT1 with Lonidamine significantly reduces the viability of NB cells .
Amino Acid Metabolism
Glutamine
In cancer cells, the supply of pyruvate-derived acetyl-CoA to the TCA cycle is diminished; instead, glutamine-derived carbon intermediates replenish the TCA cycle to maintain anaplerotic reactions for amino-acid, nucleotide, and lipid biosynthesis. In addition, glutamine can also be used by cancer cells to generate energy . In this context, MYCN-amplified NB cells are more sensitive to glutamine starvation than are nonamplified cells, and glutamine deprivation in these cells leads to depletion of TCA cycle intermediates and ultimately cell death . Uptake of glutamine in NB cells relies on alanine-serine-cysteine transporter 2 (ASCT2), which doubles as a glutamine transporter . High expression of ASCT2 has been observed in MYCN-amplified NB, which promotes tumor aggressiveness. Indeed, elevated ASTC2 levels correlate with expression of MYCN and ATF4 in high-stage NB . ATF4 is a stress-induced transcription factor. ATF4 is often induced in response to stress signals such as endoplasmic reticulum (ER) stress, nutrient deprivation, oxidative stress, and hypoxia and is frequently upregulated in cancer cells .
Replenishing TCA cycle intermediates from glutamine depends on the activity of mitochondrial glutaminases GLS or GLS2, which convert glutamine to glutamate and an ammonium ion . GLS contributes to NB cells’ migration, invasion, and proliferation . Suppression of GLS2 function in MYCN-amplified NB cells results in a significant reduction of glutamine-dependent anaplerosis and aerobic glycolysis and, subsequently, NB growth inhibition . Furthermore, the expression level of GLS2 correlates with NB cell differentiation and intracellular levels of ATP .
Glutamine is not only involved in amino acid metabolism but is also required for glutathione (GSH) generation, a tripeptide (Glu-Cys-Gly) that serves to neutralize peroxide free radicals. Cancer cell metabolism and mitochondrial dysfunction promote a continuous and elevated production of reactive oxygen species (ROS), which, in turn, promote tumor growth and malignant progression . Depletion of GSH in NB cells with different MYCN status did not induce ROS production or affect cell survival in vitro . However, a study using the TH-MYCN mouse line, a model of high-risk NB with MYCN-amplification, showed that glutathione generation is upregulated at the initiation of tumorigenesis in MYCN-driven NB. Inhibition of GSH biosynthesis in this animal model significantly delayed tumor onset and sensitized the tumor to chemotherapy, suggesting that GSH is needed for tumorigenesis .
Serine
Serine is biosynthesized from intermediates of the glycolytic pathway. The glycolytic intermediate 3-phosphoglycerate is converted to serine in a three-step enzymatic reaction. Serine can provide one-carbon precursors through conversion to glycine, mediated by serine hydroxymethyltransferase (SHMT), which is needed in a variety of biosynthetic pathways . A mitochondrial isoform of this enzyme (SHMT2) is highly expressed in MYCN-amplified and aggressive NBs with unfavorable outcomes for patients. Accordingly, knockdown of SHMT2 significantly reduces the growth of NB xenografts . The TH-MYCN mouse model showed that NB cells with a stem cell-like phenotype acquire reprogrammed metabolism to serine-glycine synthesis, a result of increased ATF4 expression .
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