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Mol Biol Rep DOI 10.1007/s11033-015-3857-y


Introduction to the molecular basis of cancer metabolism and the Warburg effect

Darleen C. Ngo Katherine Ververis Stephanie M. Tortorella Tom C. Karagiannis

Springer Science+Business Media Dordrecht 2015

Abstract In differentiated normal cells, the conventional route of glucose metabolism involves glycolysis, followed by the citric acid cycle and electron transport chain to generate usable energy in the form of adenosine triphos- phate (ATP). This occurs in the presence of oxygen. In hypoxic conditions, normal cells undergo anaerobic gly- colysis to yield significantly less energy producing lactate as a product. As first highlighted in the 1920s by Otto Warburg, the metabolism exhibited by tumor cells involves an increased rate of aerobic glycolysis, known as the Warburg effect. In aerobic glycolysis, pyruvate molecules yielded from glycolysis are converted into fewer molecules of ATP even in the presence of oxygen. Evidence indicates that the reasons as to why tumor cells undergo aerobic glycolysis include: (1) the shift in priority to accumulate biomass rather than energy production, (2) the evasion of apoptosis as fewer reactive oxygen species are released by the mitochondria and (3) the production of lactate to further fuel growth of tumors. In this mini-review we discuss

D. C. Ngo K. Ververis T. C. Karagiannis ( &)

Epigenomic Medicine, The Alfred Medical Research and Education Precinct, Baker IDI Heart and Diabetes Institute, 75 Commercial Road, Melbourne, VIC, Australia e-mail:

D. C. Ngo K. Ververis T. C. Karagiannis

Department of Pathology, The University of Melbourne, Parkville, VIC, Australia

S. M. Tortorella

Melbourne School of Land and Environment, Department of Agriculture and Food Systems, The University of Melbourne, Parkville, VIC, Australia

S. M. Tortorella

Department of Chemical and Biomolecular Engineering, The University of Melbourne, Parkville, VIC, Australia

The University of Melbourne, Parkville, VIC, Australia emerging molecular aspects of cancer metabolism and the

emerging molecular aspects of cancer metabolism and the Warburg effect. Aspects of the Warburg effect are analyzed in the context of the established hallmarks of cancer in- cluding the role of oncogenes and tumor suppressor genes.


Glucose metabolism Glycolysis Oncogene

Warburg effect Cancer metabolism

Physiological metabolic pathways

In normal differentiated cells, the metabolism of glucose begins with glycolysis [1] (Fig. 1). After entering a cell via glucose transporters on the plasma membrane, glucose is converted to various intermediates mediated via a multi- enzyme process in the cytosol to ultimately yield two molecules of pyruvate, two molecules of adenosine triphosphate (ATP) and two molecules of reduced nicoti- namide adenine dinucleotide (NADH) [2]. The two mole- cules of pyruvate are subsequently converted to acetyl CoA mediated by pyruvate dehydrogenase [2]. The mitochon- drial phase begins when acetyl CoA combines with ox- aloacetate entering the citric acid cycle, in which three molecules of NADH, one molecule of guanosine triphos- phate (GTP) and one molecule of reduced flavin adenine dinucleotide (FADH 2 ) are created by a series of enzymatic processes in preparation for oxidative phosphorylation [3]. High-energy molecules NADH and FADH 2 are reduced by the inner mitochondrial membrane complexes to create a proton gradient in the intermembrane space of the mito- chondria [4, 5]. As protons are continuously pumped out in the intermembrane space, the last step occurs as oxygen is the final acceptor of electrons driving ATP synthase to be activated. This forces protons back into the mitochondrial matrix against a concentration gradient creating energy in


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Mol Biol Rep Fig. 1 The glycolytic pathway, citric acid cycle, and electron transport chain. Glycolysis

Fig. 1 The glycolytic pathway, citric acid cycle, and electron transport chain. Glycolysis is a ten step multi-enzymatic pathway in which glucose is converted to pyruvate. Enzymes are shown in italics, and boxes indicate Hypoxia Inducible Factor targets. Conversion of pyruvate into acetyl coA allows the entry into the Citric Acid Cycle to generate NADH and FADH2. This process is followed by the Electron Transport Chain in which NADH and FADH2 are oxidised

the form of ATP [4, 5]. Typically, 30–34 molecules of ATP are produced from one molecule of glucose and this pro- cess occurs in the presence of oxygen [2, 6]. When oxygen levels are low or absent, cells utilize anaerobic respiration to metabolize glucose [6, 7]. Following glycolysis, the two molecules of pyruvate are converted to lactate and four molecules of ATP via lactate dehydrogenase [7, 8].

The Warburg Effect and cancer metabolism

As first indicated by Otto Warburg, cancer cells follow a different metabolic path known as aerobic glycolysis


and a high proton concentration accumulates in the inner membrane space of the mitochondria. Direction of electron flow is highlighted through the mitochondrial complexes. Aerobic Glycolysis in Tumour Cells. Aerobic glycolysis in tumour cells occurs in the presence of oxygen. The conversion of pyruvate to ATP and lactate is facilitated by lactate dehydrogenase

(Fig. 1) [1, 7, 9]. Known as the Warburg effect, cancer metabolism by aerobic glycolysis has emerged as a topic of intense research interest [10]. In short, the paradigm sug- gests that even in the presence of oxygen, cancer cells me- tabolize glucose into pyruvate via glycolysis, which is followed by the conversion of pyruvate to lactate and four molecules of ATP [2, 7, 11]. It was noted that the proportion of pyruvate converted into acetyl CoA prior to entering the citric acid cycle, was significantly reduced and therefore entry of acetyl CoA into citric acid cycle was decreased [2]. As a result, high-energy molecules NADH and FADH 2 are yielded in low concentrations from the citric acid cycle consequently decreasing the proportion of ATP produced

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during oxidative phosphorylation [2]. A number of hy- potheses have been suggested to answer the question: Why do cancer cells metabolize glucose via aerobic glycolysis? An important aspect is the generation of biomass to sustain the proliferative capacity of the rapidly dividing cells [12]. There is also evidence suggesting that due to the Warburg effect and the production of lactate cancer cells possess a selective advantage over normal cells in their microenvi- ronment [2]. It has also been shown that lactate is a key driver of angiogenesis and is critical for the development and growth of cancer cells [13]. Studies have shown that oncogenes and tumor suppressor genes play important roles in the switch to aerobic glycolysis in cancer cells and molecular aspects are discussed below [13].

The role of oncogenes and tumor suppressor genes in the Warburg effect

The dysregulation of oncogenes and tumor suppressor genes are key components of carcinogenesis [13]. Activation of oncogenes, typically as a result of mutation can ultimately lead to uncontrolled proliferation of cells leading to carcino- genesis [2, 12]. Similarly, the inactivation of tumor suppressor genes can result in uncontrolled cell growth [12, 14]. Oncogenes include growth factors (for example, insulin- like growth factor, IGF-1; epidermal growth factor, EGF; Her-2), receptor tyrosine kinases such as epidermal growth factor receptor (EGFR), cytoplasmic tyrosine kinases (proto-oncogene c-Src (Src), spleen tyrosine kinase (Syk), Abelson murine leukemia viral oncogene (Abl)), hydrolyse guanosine triphosphate enzymes (GTPases, Ras) and tran- scription factors such as c-Myc (Myc) [13, 15]. As an ex- ample, a well established event in carcinogenesis is aberrant activation of Ras which results in continuous activity of signal transduction pathways that ultimately control the cell cycle, survival and migration in tumor cells [15]. Further, the interaction between the catalytic domain of phos- phatidylinositol-3 kinase (PI3K) and Ras has been shown to activate protein kinase B (Akt), a serine/threonine kinase, to promote cell proliferation, avoid apoptosis as well as to promote aerobic glycolysis and glucose dependence [15, 16]. Myc, also over-expressed in a number of malignancies, combined with hypoxia-inducible factor 1 (HIF-1) has the potential to upregulate glucose transporters, glycolytic en- zymes, lactate dehydrogenase and pyruvate dehydrogenase lipoamide kinase isozyme 1 (PDK1) to enhance aerobic glycolysis in cancer cells [10, 17]. Similarly, the down-regulation or inactivation of tumor suppressor genes has been shown to be important in cancer metabolism and the Warburg effect. Under normal physio- logical conditions, tumor suppressor genes act to regulate metabolic pathways. Classes of tumor suppressor genes

include: phosphatases, G-protein inhibitors, ubiquitin li- gase, dehydrogenases as well as genes encoding DNA repair and cell cycle checkpoint proteins [15]. A classical example of an effective tumor suppressor gene is p53 [18]. This transcription factor is responsible for arresting the cell cycle to allow for the DNA repair machinery to perform its function to curtail the accumulation of mutations that could potentially lead to tumorigenesis [19]. Failure to repair cellular damage may result in the initiation of apoptosis mediated by p53 [20]. Inactivation or reduced p53 activity is observed in the majority of cancers [21]. In general, p53 inactivation allows cancer cells, even those with accumu- lated damage, to continue to proliferate, increasing the likelihood of carcinogenic accumulation of mutations. Another tumor suppressor, the retinoblastoma protein (RB) has an important role in the cell cycle, regulating prolif- eration [22]. RB controls the entry into the S phase of the cell cycle by binding and interacting with the transcription factor, E2F [23, 24]. In the presence of growth stimuli in the form of a mitogen or growth factor, RB is phosphorylated and therefore inactivated, releasing E2F – triggering the entry into S phase of the cell cycle [25]. Hence, uncontrolled regulation of mitogen release in cancer will ultimately prime cells to constitutively enter S phase causing increased pro- liferation [26]. This defiance of cancer cells in response to growth and anti-growth signals are among the fundamental features that form part of the hallmarks of cancer [27, 28]. In this context, the complex relationship between the Warburg effect and formation of reactive oxygen species (ROS) is interesting. ROS is produced in various sites within a cell, including the mitochondria via the electron transport chain, the endoplasmic reticulum through the Ero1-DPI oxidative folding system and cell membrane bound nicoti- namide adenine dinucleotide phosphate oxidases (NOX) complexes [29]. According to Li et al., ROS are considered to be versatile signaling molecules, which have the ability to regulate cellular activities, ultimately influencing whether a cell survives or undergoes programmed cell death [30]. Numerous studies have shown that the accumulation of ROS has the potential to cause mutations via various cellular pathways including: ATG4–ATG8/LC3, Beclin-1, phos- phatase and tensin homolog (PTEN), p53, phosphotidyl inositol 3 kinase (PI3K–Akt–mTOR) and MAPK signaling [31]. Moreover, emerging research into the role of the tu- mour microenvironment, which involves the release of ROS, appears to also influence the Warburg effect [32].

The tumor microenvironment and its role in cancer metabolism

The tumour microenvironment consists of various com- ponents including: cancer associated fibroblasts (CAFs),


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inflammatory cells, signalling molecules, endothelial cells, blood vessels, stroma and the extracellular (ECM) matrix all of which have been implicated in aberrant cancer metabo- lism [33, 34]. For example, fibroblasts are particularly im- portant, facilitating the structural and functional properties of the extracellular matrix [35]. Activated cytokines and chemokines recruit fibroblasts to the site of injury and fa- cilitate wound healing in particular, tissue remodelling and differentiation into myofibroblasts [35, 36]. Myofibroblasts differ from their quiescent fibroblast form such that myofi- broblasts express a-smooth muscle actin (a-SMA), which is needed to generate force in order to complete ECM and wound contraction [37]. Once the wound healing process has completed, myofibroblasts undergo nemosis, a spe- cialised programmed cell-death followed by degradation by granulation tissue [38]. Fibrosis on the other hand deviates from this response by prolonging fibrous tissue deposition leading to mass accumulation of ECM. These concepts are related to the hypothesis that tumors are essentially wounds that do not heal [39].This leads to the idea that CAFs share similar properties to myofibroblasts, where both express a- SMA in its activated form. However unlike myofibroblasts, CAFs do not undergo nemosis. Instead, the persistent nature of CAFs allows an accumulation of ECM deposition within the stroma of the tumor microenvironment. This phenotype has been observed in solid tumors in the breast, pancreas and prostate [40, 41]. Overall, it is evident that the tumor mi- croenvironment represents an environment conducive to cancer progression. When tumors increase in size, blood vessels supplying nutrients and oxygen relative to the size of the tumor is often not enough to sustain the development of the tumor. As a result of the Warburg effect, reduced oxygen supplied to the tumor acts as a compromise, ultimately leading to hypoxia [42, 43]. When four molecules of ATP lactate are yielded as a result of aerobic glycolysis in cancer cells, lactate is secreted into the tumor microenvironment [44]. The consequence of the release of lactate leads to the de- velopment in growth and proliferation of cancer cells, also increasing the likelihood of metastasis [44, 45]. However, the inhibition of lactate dehydrogenase leads to less con- version of pyruvate to lactate, thus decreased concentra- tions of lactate released into the microenvironment, consequently down-regulated proliferation of cancer cells [45, 46]. Therefore, the development of therapeutic drugs to target lactate dehydrogenase is an area of considerable interest [46, 47].


Understanding the key principles and mechanisms in the way tumour cells behave in their environment and respond


to various signalling molecules under the control oncoge- nes and tumour suppressor genes is important in the de- velopment of potential drug therapies. The observations of Otto Warburg and the renaissance of the field have created new opportunities for drug development; interventions which may modulate the activity of key enzymes in cel- lular metabolism or modify the tumour microenvironment are expected to emerge. To date, therapeutic agents tar- geting metabolism, in particular the Warburg effect in cancer, have been limited, predominantly due to lack of specificity. In looking forward, perhaps combinations of metabolic regulators and conventional chemotherapeutic agents to target different aspects in malignancy may prove to be beneficial.

Acknowledgments TCK is supported by an Australian Research Council Future Fellowship and the Epigenomic Medicine Laboratory is supported by McCord Research. Supported in part by the Victorian Government’s Operational Infrastructure Support Program.



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