Control of Glycolytic Flux by AMP-Activated Protein Kinase in Tumor Cells Adapted to Low pH

Tumor cells grow in nutrient- and oxygen-deprived microenvironments and adapt to the suboptimal growth conditions by altering their metabolic pathways. This adaptation process commonly results in a tumor phenotype that displays a high rate of aerobic glycolysis and aggressive tumor characteristics. The glucose regulatory molecule, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), is a bifunctional enzyme that is central to glycolytic flux and is downstream of the metabolic stress sensor AMP-activated protein kinase (AMPK), which has been suggested to modulate glycolysis and possibly activate isoforms of PFKFB, specifically PFKFB3 expressed in tumor cells. Our results demonstrated that long-term low pH exposure induced AMPK activation, which resulted in the up-regulation of PFKFB3 and an increase in its serine residue phosphorylation. Pharmacologic activation of AMPK resulted in an increase in PFKFB3 as well as an increase in glucose consumption, whereas in contrast, inhibition of AMPK resulted in the down-regulation of PFKFB3 and decreased glycolysis. PFKFB3 overexpression in DB-1 tumor cells induced a high rate of glycolysis and inhibited oxygen consumption, confirming its role in controlling glycolytic flux. These results show that low pH is a physiological stress that can promote a glycolytic phenotype commonly associated with tumorigenesis. The implications are that the tumor microenviroment contributes to tumor growth and treatment resistance.     

Warburg,me and Hexokinase 2: Multiple discoveries of key molecular events underlying one of cancers’ most common phenotypes,the “Warburg Effect”, i.e., elevated glycolysis in the presence of oxygen

As a new faculty member at The Johns Hopkins University, School of Medicine, the author began research on cancer in 1969 because this frequently fatal disease touched many whom he knew. He was intrigued with its viscous nature, the failure of all who studied it to find a cure, and also fascinated by the pioneering work of Otto Warburg, a biochemical legend and Nobel laureate. Warburg who died 1 year later in 1970 had shown in the 1920s that the most striking biochemical phenotype of cancers is their aberrant energy metabolism. Unlike normal tissues that derive most of their energy (ATP) by metabolizing the sugar glucose to carbon dioxide and water, a process that involves oxygen-dependent organelles called “mitochondria”, Warburg showed that cancers frequently rely less on mitochondria and obtain as much as 50% of their ATP by metabolizing glucose directly to lactic acid, even in the presence of oxygen. This frequent phenotype of cancers became known as the “Warburg effect”, and the author of this review strongly believed its understanding would facilitate the discovery of a cure. Following in the final footsteps of Warburg and caught in the midst of an unpleasant anti-Warburg, anti-metabolic era, the author and his students/collaborators began quietly to identify the key molecular events involved in the “Warburg effect”. Here, the author describes via a series of sequential discoveries touching five decades how despite some impairment in the respiratory capacity of malignant tumors, that hexokinase 2 (HK-2), its mitochondrial receptor (VDAC), and the gene that encodes HK-2 (HK-2 gene) play the most pivotal and direct roles in the “Warburg effect”. They discovered also that like a “Trojan horse” the simple lactic acid analog 3-bromopyruvate selectively enters the cells of cancerous animal tumors that exhibit the “Warburg effect” and quickly dissipates their energy (ATP) production factories (i.e., glycolysis and mitochondria) resulting in tumor destruction without harm to the animals.     

Mitochondrial and metabolic alterations in cancer cells

Metabolic alterations have been observed in many cancer types. The deregulated metabolism has thus become an emerging hallmark of the disease, where the metabolism is frequently rewired to aerobic glycolysis. This has led to the concept of “metabolic reprogramming”, which has therefore been extensively studied. Over the years, it has been characterized the enhancement of aerobic glycolysis, where key mutations in some of the enzymes of the TCA cycle, and the increased glucose uptake, are used by cancer cells to achieve a “metabolic phenotype” useful to gain a proliferation advantage. Many studies have highlighted in detail the signaling pathways and the molecular mechanisms responsible for the glycolytic switch. However, glycolysis is not the only metabolic process that cancer cells rely on. Oxidative Phosphorylation (OXPHOS), gluconeogenesis or the beta-oxidation of fatty acids (FAO) may be involved in the development and progression of several tumors. In some cases, these metabolisms are even more crucial than aerobic glycolysis for the tumor survival. This review will focus on the contribution of these alterations of metabolism to the development and survival of cancers. We will also analyze the molecular mechanisms by which the balance between these metabolic processes may be regulated, as well as some of the therapeutical approaches that can derive from their study.     

Voltage dependent anion channels (VDACs): a brief introduction with a focus on the outer mitochondrial compartment’s roles together with hexokinase-2 in the “Warburg effect” in cancer

In recent years there has been renewed interest and focus on mitochondria of animal and human tissues. This interest commenced in the latter part of the past century and has gained momentum during the first eight years of this new millennium. The well accepted reason is that mitochondria are now recognized to represent not only "power houses", i.e., the ATP production factories of tissues essential for cell life, but in response to a variety of different "cues" may participate significantly also in cell death, both that associated with normal turnover and that associated with disease. Conversely, in cancers (particularly the advanced) their mitochondria interact with hexokinase 2 (HK-2) resulting in suppression of cell death while supporting cell growth via enhanced glycolysis, even in the presence of oxygen (Warburg effect). The identification/elucidation of proteins and mechanisms involved in deciding and/or participating in cell fate (i.e., life, death, or cancer) has focused to a large extent on the mitochondrial outer compartment, which is taken here to collectively include the outer membrane, the space between the inner and outer membranes, and contact regions between these two membranes. Among the established proteins believed to be involved in events related to cell fate are "VDACs" that form the basis of this mini-review series. This brief introductory review focuses mainly on the past discovery by the author and colleagues that VDAC located within the outer mitochondrial compartment and its binding partner HK-2 are pivotal players in the "Warburg effect" in cancer. As one case in point, when glucose is added to liver cytosol (mitochondria-free) the rate of glycolysis is very low. However, upon addition of tumor mitochondria containing VDAC bound HK-2, the low glycolytic rate is increased to a high rate near that catalyzed by the tumor cytoplasm from which the tumor mitochondria were derived.     

Alterations in the glycolytic and glutaminolytic pathways after malignant transformation of rat liver oval cells

Oval cells are liver epithelial cells that proliferate during the early stages of hepatocarcinogenesis induced by a variety of chemicals. The oval cell lines OC/CDE 6 and OC/CDE 22 have been established in our laboratory at two time points (6 and 22 weeks) of the carcinogenic process and have been malignantly transformed by different procedures. During the transformation process, the glycolytic and glutaminolytic flux rates were consistently up-regulated and this process was accompanied by an overproportional increase in the activities of cytosolic hexokinase and 6-phosphogluconate dehydrogenase. In transformed oval cells, a strong correlation between the glycolytic flux rate and glutamine consumption as well as glutamate production was observed. Furthermore, the transport of glycolytic hydrogen, produced by the glyceraldehyde 3-phosphate dehydrogenase-catalyzed reaction, from the cytosol into the mitochondria by means of the malate-aspartate shuttle was enhanced, this being due to alterations in the activities of malate dehydrogenase and glutamate oxaloacetate transaminase. The up-regulation of the glycolytic hydrogen transport and the alterations in the glycolytic enzyme complex led to an enhanced pyruvate production at high glycolytic flux rates. Taken together, our data are further proof that a special metabolic feature (increased glycolysis and glutaminolysis) is characteristic for tumor cells and that the mechanisms by which this metabolic state is induced can be totally different. J. Cell. Physiol. 181:136–146, 1999. © 1999 Wiley-Liss, Inc.     

The role of the mitochondrial outer membrane in energy metabolism of tumor cells

The outer mitochondrial membrane contains a pore structure which is composed of a 30,000 Da protein, porin. The pore has an internal diameter of 2 nm and exhibits a molecular-sieving exclusion limit between 3000 and 6000 Da. These pores, therefore, provide the exit/entrance port for metabolites moving between mitochondria and the cytosol. Hexokinase binds to porin on the outer surface of mitochondria. The location of hexokinase has evoked a number of theories in which bound hexokinase is given a central role in regulating glycolysis, and, perhaps, the metabolic communication between oxidative and glycolytic metabolism. This is of particular importance in rapidly growing tumor cells in which the aerobic production of lactate and hexokinase activity are highly induced. In the present paper, we summarize the suggested roles of the outer membrane and bound hexokinase in regulation glycolysis of tumor cells. Experiments attempting to elucidate the role of hexokinase binding in the regulation of tumor cell metabolism are presented.     

Mitochondrial metabolism in cancer metastasis

We have recently proposed a new two-compartment model for understanding the Warburg effect in tumor metabolism. In this model, glycolytic stromal cells produce mitochondrial fuels (L-lactate and ketone bodies) that are then transferred to oxidative epithelial cancer cells, driving OXPHOS and mitochondrial metabolism. Thus, stromal catabolism fuels anabolic tumor growth via energy transfer. We have termed this new cancer paradigm the “reverse Warburg effect,” because stromal cells undergo aerobic glycolysis, rather than tumor cells. To assess whether this mechanism also applies during cancer cell metastasis, we analyzed the bioenergetic status of breast cancer lymph node metastases, by employing a series of metabolic protein markers. For this purpose, we used MCT4 to identify glycolytic cells. Similarly, we used TO MM20 and COX staining as markers of mitochondrial mass and OXPHOS activity, respectively. Consistent with the “reverse Warburg effect,” our results indicate that metastatic breast cancer cells amplify oxidative mitochondrial metabolism (OXPHOS) and that adjacent stromal cells are glycolytic and lack detectable mitochondria. Glycolytic stromal cells included cancer-associated fibroblasts, adipocytes and inflammatory cells. Double labeling experiments with glycolytic (MCT4) and oxidative (TO MM20 or COX) markers directly shows that at least two different metabolic compartments co-exist, side-by-side, within primary tumors and their metastases. Since cancer-associated immune cells appeared glycolytic, this observation may also explain how inflammation literally “fuels” tumor progression and metastatic dissemination, by “feeding” mitochondrial metabolism in cancer cells. Finally, MCT4(+) and TO MM20(-) “glycolytic” cancer cells were rarely observed, indicating that the conventional “Warburg effect” does not frequently occur in cancer-positive lymph node metastases.     

Increased glycolysis in skeletal muscle coordinates with adipose tissue in systemic metabolic homeostasis

Insulin-independent glucose metabolism, including anaerobic glycolysis that is promoted in resistance training, plays critical roles in glucose disposal and systemic metabolic regulation. However, the underlying mechanisms are not completely understood. In this study, through genetically manipulating the glycolytic process by overexpressing human glucose transporter 1 (GLUT1), hexokinase 2 (HK2) and 6-phosphofructo-2-kinase-fructose-2,6-biphosphatase 3 (PFKFB3) in mouse skeletal muscle, we examined the impact of enhanced glycolysis in metabolic homeostasis. Enhanced glycolysis in skeletal muscle promoted accelerated glucose disposal, a lean phenotype and a high metabolic rate in mice despite attenuated lipid metabolism in muscle, even under High-Fat diet (HFD). Further study revealed that the glucose metabolite sensor carbohydrate-response element-binding protein (ChREBP) was activated in the highly glycolytic muscle and stimulated the elevation of plasma fibroblast growth factor 21 (FGF21), possibly mediating enhanced lipid oxidation in adipose tissue and contributing to a systemic effect. PFKFB3 was critically involved in promoting the glucose-sensing mechanism in myocytes. Thus, a high level of glycolysis in skeletal muscle may be intrinsically coupled to distal lipid metabolism through intracellular glucose sensing. This study provides novel insights for the benefit of resistance training and for manipulating insulin-independent glucose metabolism.     

Hexokinase–mitochondrial interactions regulate glucose metabolism differentially in adult and neonatal cardiac myocytes

In mammalian tumor cell lines, localization of hexokinase (HK) isoforms to the cytoplasm or mitochondria has been shown to control their anabolic (glycogen synthesis) and catabolic (glycolysis) activities. In this study, we examined whether HK isoform differences could explain the markedly different metabolic profiles between normal adult and neonatal cardiac tissue. We used a set of novel genetically encoded optical imaging tools to track, in real-time in isolated adult (ARVM) and neonatal (NRVM) rat ventricular myocytes, the subcellular distributions of HKI and HKII, and the functional consequences on glucose utilization. We show that HKII, the predominant isoform in ARVM, dynamically translocates from mitochondria and cytoplasm in response to removal of extracellular glucose or addition of iodoacetate (IAA). In contrast, HKI, the predominant isoform in NRVM, is only bound to mitochondria and is not displaced by the above interventions. In ARVM, overexpression of HKI, but not HKII, increased glycolytic activity. In neonatal rat ventricular myocytes (NVRM), knockdown of HKI, but not HKII, decreased glycolytic activity. In conclusion, differential interactions of HKI and HKII with mitochondria underlie the different metabolic profiles of ARVM and NRVM, accounting for the markedly increased glycolytic activity of NRVM.     

Determining and understanding the control of glycolysis in fast-growth tumor cells. Flux control by an over-expressed but strongly product-inhibited hexokinase

Control analysis of the glycolytic flux was carried out in two fast-growth tumor cell types of human and rodent origin (HeLa and AS-30D, respectively). Determination of the maximal velocity (V(max)) of the 10 glycolytic enzymes from hexokinase to lactate dehydrogenase revealed that hexokinase (153-306 times) and phosphofructokinase-1 (PFK-1) (22-56 times) had higher over-expression in rat AS-30D hepatoma cells than in normal freshly isolated rat hepatocytes. Moreover, the steady-state concentrations of the glycolytic metabolites, particularly those of the products of hexokinase and PFK-1, were increased compared with hepatocytes. In HeLa cells, V(max) values and metabolite concentrations for the 10 glycolytic enzyme were also significantly increased, but to a much lesser extent (6-9 times for both hexokinase and PFK-1). Elasticity-based analysis of the glycolytic flux in AS-30D cells showed that the block of enzymes producing Fru(1,6)P2 (i.e. glucose transporter, hexokinase, hexosephosphate isomerase, PFK-1, and the Glc6P branches) exerted most of the flux control (70-75%), whereas the consuming block (from aldolase to lactate dehydrogenase) exhibited the remaining control. The Glc6P-producing block (glucose transporter and hexokinase) also showed high flux control (70%), which indicated low flux control by PFK-1. Kinetic analysis of PFK-1 showed low sensitivity towards its allosteric inhibitors citrate and ATP, at physiological concentrations of the activator Fru(2,6)P2. On the other hand, hexokinase activity was strongly inhibited by high, but physiological, concentrations of Glc6P. Therefore, the enhanced glycolytic flux in fast-growth tumor cells was still controlled by an over-produced, but Glc6P-inhibited hexokinase.     

BRAIN MITOCHONDRIA : II. The Relationship of Brain Mitochondria to Glycolysis

A mitochondrial fraction prepared from calf brain cortex possessed negligible glycolytic activity in the absence of the enzymes of the high speed supernatant fraction. When mitochondria were added to a supernatant system supplemented with optimal amounts of crystalline hexokinase, a 20 per cent stimulation of glycolysis was observed. The supernatant fraction produced minimal amounts of lactate in the absence of exogenous hexokinase; the addition of mitochondria doubled the lactate production. The substitution of glycolytic intermediates for glucose as substrates as well as the addition of exogenous glycolytic enzymes to the supernatant fraction or supernatant fraction plus mitochondria indicated that the mitochondria contributed mainly hexokinase and phosphofructokinase. By direct assay of all of the enzymes of the glycolytic pathway, only hexokinase and phosphofructokinase were shown to be concentrated in the mitochondrial fraction. All other glycolytic enzymes were found to exhibit higher total and specific activities in the supernatant fraction.     

Functional significance of mitochondrial bound hexokinase in tumor cell metabolism. Evidence for preferential phosphorylation of glucose by intramitochondrially generated ATP

Previous studies from this laboratory have shown that mitochondrial bound hexokinase is markedly elevated in highly glycolytic hepatoma cells (Parry, D. M., and Pedersen, P.L. (1983) J. Biol. Chem. 258, 10904-10912). A pore-forming protein, porin, within the outer membrane appears to comprise at least part of the receptor site (Nakashima, R.A., Mangan, P.S., Colombini, M., and Pedersen, P.L. (1986). Biochemistry 25, 1015-1021). In studies reported here experiments were carried out to assess the functional significance of mitochondrial bound tumor hexokinase. Two approaches were used to determine whether the bound enzyme has preferred access to mitochondrially generated ATP relative to cytosolic ATP. The first approach compared the time course of glucose 6-phosphate formation by AS-30D hepatoma mitochondria under conditions where ATP was regenerated endogenously via oxidative phosphorylation or exogenously by added pyruvate kinase and phosphoenolpyruvate. The second approach involved the measurement of the specific radioactivity of glucose 6-phosphate formed following the addition of [gamma-32P]ATP to either phosphorylating or nonphosphorylating AS-30D mitochondria. Both approaches provided results which show that the source of ATP for bound hexokinase is derived preferentially from the ATP synthase residing within the inner mitochondrial membrane compartment rather than from the medium (i.e. from the cytosolic compartment). These results provide the first direct demonstration that the exceptionally high level of hexokinase bound to mitochondria of highly glycolytic tumor cells has preferred access to mitochondrially generated ATP, a finding that may have rather profound metabolic significance for such tumors.     

Energy metabolism transition in multi-cellular human tumor spheroids

It is thought that glycolysis is the predominant energy pathway in cancer, particularly in solid and poorly vascularized tumors where hypoxic regions develop. To evaluate whether glycolysis does effectively predominate for ATP supply and to identify the underlying biochemical mechanisms, the glycolytic and oxidative phosphorylation (OxPhos) fluxes, ATP/ADP ratio, phosphorylation potential, and expression and activity of relevant energy metabolism enzymes were determined in multi-cellular tumor spheroids, as a model of human solid tumors. In HeLa and Hek293 young-spheroids, the OxPhos flux and cytochrome c oxidase protein content and activity were similar to those observed in monolayer cultured cells, whereas the glycolytic flux increased two- to fourfold; the contribution of OxPhos to ATP supply was 60%. In contrast, in old-spheroids, OxPhos, ATP content, ATP/ADP ratio, and phosphorylation potential diminished 50-70%, as well as the activity (88%) and content (3 times) of cytochrome c oxidase. Glycolysis and hexokinase increased significantly (both, 4 times); consequently glycolysis was the predominant pathway for ATP supply (80%). These changes were associated with an increase (3.3 times) in the HIF-1alpha content. After chronic exposure, both oxidative and glycolytic inhibitors blocked spheroid growth, although the glycolytic inhibitors, 2-deoxyglucose and gossypol (IC(50) of 15-17 nM), were more potent than the mitochondrial inhibitors, casiopeina II-gly, laherradurin, and rhodamine 123 (IC(50) > 100 nM). These results suggest that glycolysis and OxPhos might be considered as metabolic targets to diminish cellular proliferation in poorly vascularized, hypoxic solid tumors.     

Modeling cancer glycolysis

Most cancer cells exhibit an accelerated glycolysis rate compared to normal cells. This metabolic change is associated with the over-expression of all the pathway enzymes and transporters (as induced by HIF-1α and other oncogenes), and with the expression of hexokinase (HK) and phosphofructokinase type 1 (PFK-1) isoenzymes with different regulatory properties. Hence, a control distribution of tumor glycolysis, modified from that observed in normal cells, can be expected. To define the control distribution and to understand the underlying control mechanisms, kinetic models of glycolysis of rodent AS-30D hepatoma and human cervix HeLa cells were constructed with experimental data obtained here for each pathway step (enzyme kinetics; steady-state pathway metabolite concentrations and fluxes). The models predicted with high accuracy the fluxes and metabolite concentrations found in living cancer cells under physiological O(2) and glucose concentrations as well as under hypoxic and hypoglycemic conditions prevailing during tumor progression. The results indicated that HK≥HPI>GLUT in AS-30D whereas glycogen degradation≥GLUT>HK in HeLa were the main flux- and ATP concentration-control steps. Modeling also revealed that, in order to diminish the glycolytic flux or the ATP concentration by 50%, it was required to decrease GLUT or HK or HPI by 76% (AS-30D), and GLUT or glycogen degradation by 87-99% (HeLa), or decreasing simultaneously the mentioned steps by 47%. Thus, these proteins are proposed to be the foremost therapeutic targets because their simultaneous inhibition will have greater antagonistic effects on tumor energy metabolism than inhibition of all other glycolytic, non-controlling, enzymes.     

Protein profiles in human ovarian cancer cell lines correspond to their metabolic activity and to metabolic profiles of respective tumor xenografts

Many solid tumors show a large variability in glycolytic activity and lactate accumulation, which has been correlated with different metastatic spread, radioresistance and patient survival. To investigate potential differences in protein profiles underlying these metabolic variances, the highly glycolytic human ovarian cancer cell line OC316 was investigated and compared with the less glycolytic line IGROV-1. Extracellular acidification and oxygen consumption were analyzed with an extracellular flux analyzer. Glycolysis-associated proteins, including specific membrane transporters, were quantified through in-cell western analyses. Metabolic properties of corresponding tumor xenografts were assessed via induced metabolic bioluminescence imaging. Extracellular flux analyses revealed elevated bioenergetics of OC316 cells. Hexokinase II, pyruvate kinase, pyruvate dehydrogenase E1 beta subunit and pyruvate dehydrogenase kinase 1, as well as the glucose transporter 1 and the monocarboxylate transporter 4, were overexpressed in these cells compared with IGROV-1. When generating tumor xenografts in SCID mice, cells maintained their glycolytic behavior, i.e. OC316 showed higher lactate concentrations than IGROV-1 tumors. In summary, a congruency between protein profiles and metabolic properties has been demonstrated in the human ovarian cancer lines investigated. Also, a perpetuation of glycolytic characteristics during the transition from in vitro to the in vivo situation has been documented. This model system could be useful for systematic studies on therapeutic intervention by manipulation of tumor glycolysis and associated pathways.     

Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells

Increased conversion of glucose to lactic acid associated with decreased mitochondrial respiration is a unique feature of tumors first described by Otto Warburg in the 1920s. Recent evidence suggests that the Warburg effect is caused by oncogenes and is an underlying mechanism of malignant transformation. Using a novel approach to measure cellular metabolic rates in vitro, the bioenergetic basis of this increased glycolysis and reduced mitochondrial respiration was investigated in two human cancer cell lines, H460 and A549. The bioenergetic phenotype was analyzed by measuring cellular respiration, glycolysis rate, and ATP turnover of the cells in response to various pharmacological modulators. H460 and A549 cells displayed a dependency on glycolysis and an ability to significantly upregulate this pathway when their respiration was inhibited. The converse, however, was not true. The cell lines were attenuated in oxidative phosphorylation (OXPHOS) capacity and were unable to sufficiently upregulate mitochondrial OXPHOS when glycolysis was disabled. This observed mitochondrial impairment was intimately linked to the increased dependency on glycolysis. Furthermore, it was demonstrated that H460 cells were more glycolytic, having a greater impairment of mitochondrial respiration, compared with A549 cells. Finally, the upregulation of glycolysis in response to mitochondrial ATP synthesis inhibition was dependent on AMP-activated protein kinase activity. In summary, our results demonstrate a bioenergetic phenotype of these two cancer cell lines characterized by increased rate of glycolysis and a linked attenuation in their OXPHOS capacity. These metabolic alterations provide a mechanistic explanation for the growth advantage and apoptotic resistance of tumor cells. oxygen consumption; oxidative phosphorylation; Warburg effect; real time     

Regulation of glycolytic metabolism by autophagy in liver cancer involves selective autophagic degradation of HK2 (hexokinase 2)

Impaired macroautophagy/autophagy and high levels of glycolysis are prevalent in liver cancer. However, it remains unknown whether there is a regulatory relationship between autophagy and glycolytic metabolism. In this study, by utilizing cancer cells with basal or impaired autophagic flux, we demonstrated that glycolytic activity is negatively correlated with autophagy level. The autophagic degradation of HK2 (hexokinase 2), a crucial glycolytic enzyme catalyzing the conversion of glucose to glucose-6-phosphate, was found to be involved in the regulation of glycolysis by autophagy. The Lys63-linked ubiquitination of HK2 catalyzed by the E3 ligase TRAF6 was critical for the subsequent recognition of HK2 by the autophagy receptor protein SQSTM1/p62 for the process of selective autophagic degradation. In a tissue microarray of human liver cancer, the combination of high HK2 expression and high SQSTM1 expression was shown to have biological and prognostic significance. Furthermore, 3-BrPA, a pyruvate analog targeting HK2, significantly decreased the growth of autophagy-impaired tumors in vitro and in vivo (p < 0.05). By demonstrating the regulation of glycolysis by autophagy through the TRAF6- and SQSTM1-mediated ubiquitination system, our study may open an avenue for developing a glycolysis-targeting therapeutic intervention for treatment of autophagy-impaired liver cancer.     

Recurrent patterns of DNA copy number alterations in tumors reflect metabolic selection pressures

Copy number alteration (CNA) profiling of human tumors has revealed recurrent patterns of DNA amplifications and deletions across diverse cancer types. These patterns are suggestive of conserved selection pressures during tumor evolution but cannot be fully explained by known oncogenes and tumor suppressor genes. Using a pan‐cancer analysis of CNA data from patient tumors and experimental systems, here we show that principal component analysis‐defined CNA signatures are predictive of glycolytic phenotypes, including ¹⁸F‐fluorodeoxy‐glucose (FDG) avidity of patient tumors, and increased proliferation. The primary CNA signature is enriched for p53 mutations and is associated with glycolysis through coordinate amplification of glycolytic genes and other cancer‐linked metabolic enzymes. A pan‐cancer and cross‐species comparison of CNAs highlighted 26 consistently altered DNA regions, containing 11 enzymes in the glycolysis pathway in addition to known cancer‐driving genes. Furthermore, exogenous expression of hexokinase and enolase enzymes in an experimental immortalization system altered the subsequent copy number status of the corresponding endogenous loci, supporting the hypothesis that these metabolic genes act as drivers within the conserved CNA amplification regions. Taken together, these results demonstrate that metabolic stress acts as a selective pressure underlying the recurrent CNAs observed in human tumors, and further cast genomic instability as an enabling event in tumorigenesis and metabolic evolution.     

Glucose conversion by multiple pathways in brain extract: theoretical and experimental analysis

Experimental and model studies were performed to characterize the flux of glucose metabolism and the sharing of glucose-6-phosphate (Glu6P) by the upper parts of glycolytic and pentosephosphate pathways in the brain extract. A mathematical model based upon the kinetic equations of the individual enzymes was evaluated to fit the experimental data. Glucose is converted to glucose-6-phosphate by hexokinase that controls almost exclusively the glucose metabolism. Experiments showed that this crossroad-metabolite was shared between glycolysis and pentosephosphate pathway in the brain extract in a ratio of 1.5:1. This ratio was favorable to the pentosephosphate pathway by the addition of high excess of exogenous glucose-6-phosphate dehydrogenase, standardly used for the activity assay of hexokinase, but still a significant part (17+/-3%) of the common intermediate was converted into the direction of glycolysis. Stimulation of glucose-6-phosphate formation via moderate (30-50%) increase of hexokinase activity by adding exogenous hexokinase or tubulin resulted in the slight increase of the relative flux into direction of glycolysis. The model correctly described all of these observations. However, when the activity of hexokinase was doubled with exogenous enzyme, significantly less glucose-6-phosphate was converted into direction of glycolysis than predicted. This discrepancy shows that the system did not behave in this case as an ideal one, which could be due to the formation of distinct pools for the intermediate.