Mol Biol Rep
DOI 10.1007/s11033-015-3857-y
S.I. : MOLECULAR EVENTS AT THE GENETIC LEVEL IN HEALTH AND DISEASE
Introduction to the molecular basis of cancer metabolism
and the Warburg effect
Darleen C. Ngo • Katherine Ververis •
Stephanie M. Tortorella • Tom C. Karagiannis
Ó 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: tom.karagiannis@bakeridi.edu.au
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
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.
Keywords Warburg effect
Cancer metabolism

Glucose metabolism
Glycolysis
Oncogene
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 (FADH2) are created by a series of enzymatic
processes in preparation for oxidative phosphorylation [3].
High-energy molecules NADH and FADH2 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
123

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
123
Mol Biol Rep
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 FADH2 are
yielded in low concentrations from the citric acid cycle
consequently decreasing the proportion of ATP produced
Mol Biol Rep
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),
123

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].
Conclusion
Understanding the key principles and mechanisms in the
way tumour cells behave in their environment and respond
123
Mol Biol Rep
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.
References
1. Warburg O (1956) On the origin of cancer cells. Science
123(3191):309–314
2. Vander Heiden MG, Cantley LC, Thompson CB (2009) Under-
standing the Warburg effect: the metabolic requirements of cell
proliferation. Science 324(5930):1029–1033
3. Akram M (2014) Citric acid cycle and role of its intermediates in
metabolism. Cell Biochem Biophys 68(3):475–478. doi:10.1007/
s12013-013-9750-1
4. Saraste M (1999) Oxidative phosphorylation at the fin de siecle.
Science 283(5407):1488–1493
5. Fulda S, Galluzzi L, Kroemer G (2010) Targeting mitochondria
for cancer therapy. Nat Rev Drug Discovery 9(6):447–464
6. Lunt SY, Vander Heiden MG (2011) Aerobic glycolysis: meeting
the metabolic requirements of cell proliferation. Annu Rev Cell
Dev Biol 27(1):441–464
7. Kim JW, Dang CV (2006) Cancer’s molecular sweet tooth and
the Warburg effect. Cancer Res 66(18):8927–8930
8. DeBerardinis RJ, Lum JJ, Hatzivassiliou G, Thompson CB
(2008) The Biology of Cancer: metabolic reprogramming fuel
cells growth and proliferation. Cell Metab 7(1):11–20
9. Shaw RJ (2006) Glucose metabolism and cancer. Curr Opin Cell
Biol 18(6):598–608
10. Cairns RA, Harris IS, Mak TW (2011) Regulation of cancer cell
metabolism. Nat Rev Cancer 11(2):85–95
11. Dang CV (2010) Rethinking the Warburg effect with Myc mi-
cromanaging glutamine metabolism. Cancer Res 70(3):859–862
12. Ward PS, Thompson CB (2012) Metabolic reprogramming: a
cancer hallmark even warburg did not anticipate. Cancer Cell
21(3):297–308
13. Levine AJ, Puzio-Kuter AM (2010) The control of the metabolic
switch in cancers by oncogenes and tumor suppressor genes.
Science 330(6009):1340–1344
14. Jones RG, Thompson CB (2009) Tumor suppressors and cell
metabolism: a recipe for cancer growth. Genes Dev
23(5):537–548
15. Gillies RJ, Robey I, Gatenby RA (2008) Causes and conse-
quences of increased glucose metabolism of cancers. J Nucl Med
49(Suppl 2):24S–42S
Mol Biol Rep
16. Elstrom RL, Bauer DE, Buzzai M, Karnauskas R, Harris MH,
Plas DR, Zhuang H, Cinalli RM, Alavi A, Rudin CM, Thompson
CB (2004) Akt stimulates aerobic glycolysis in cancer cells.
Cancer Res 64(11):3892–3899
17. Gatenby RA, Gillies RJ (2004) Why do cancers have high aerobic
glycolysis? Nat Rev Cancer 4(11):891–899
18. Kastan MB, Canman CE, Leonard CJ (1995) P53, cell cycle
control and apoptosis: implications for cancer. Cancer Metastasis
Rev 14(1):3–15
19. Livingstone LR, White A, Sprouse J, Livanos E, Jacks T, Tlsty
TD (1992) Altered cell cycle arrest and gene amplification po-
tential accompany loss of wild-type p53. Cell 70(6):923–935
20. Toshiyuki M, Reed JC (1995) Tumor suppressor p53 is a direct
transcriptional activator of the human bax gene. Cell
80(2):293–299
21. Goh AM, Coffill CR, Lane DP (2011) The role of mutant p53 in
human cancer. J Pathol 223(2):116–126
22. Schaal C, Pillai S, Chellappan SP (2014) The Rb-E2F tran-
scriptional regulatory pathway in tumor angiogenesis and
metastasis. Adv Cancer Res 121:147–182. doi:10.1016/b978-0-
12-800249-0.00004-4
23. Nevins JR, Chellappan SP, Mudryj M, Hiebert S, Devoto S,
Horowitz J, Hunter T, Pines J (1991) E2F transcription factor is a
target for the RB protein and the cyclin A protein. Cold Spring
Harb Symp Quant Biol 56:157–162
24. Chellappan SP, Hiebert S, Mudryj M, Horowitz JM, Nevins JR
(1991) The E2F transcription factor is a cellular target for the RB
protein. Cell 65(6):1053–1061
25. Sherr CJ, McCormick F (2002) The RB and p53 pathways in
cancer. Cancer Cell 2(2):103–112
26. Rigoulet M, Yoboue ED, Devin A (2011) Mitochondrial ROS
generation and its regulation: mechanisms involved in H(2)O(2)
signaling. Antioxid Redox Signal 14(3):459–468. doi:10.1089/
ars.2010.3363
27. Hanahan D, Weinberg RA (2000) The Hallmarks of cancer. Cell
100(1):57–70
28. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next
generation. Cell 144(5):646–674
29. Rigoulet M, Yoboue ED, Anne D (2011) Mitochondrial ROS
generation and its regulation: mechanisms involved in H2O2
signaling. Antioxidants & Redox Signalling 14(3):459–468
30. Li ZY, Yang Y, Ming M, Liu B (2011) Mitochondrial ROS
generation for regulation of autophagic pathways in cancer.
Biochem Biophys Res Commun 414(1):5–8. doi:10.1016/j.bbrc.
2011.09.046
31. Li ZY, Yang Y, Ming M, Bo L (2011) Mitochondrial ROS
generation for regulation of autophagic pathways in cancer.
Biochem Biophys Res Commun 414(1):5–8
32. Subarsky P, Hill RP (2003) The hypoxic tumour microenvironment
and metastatic progression. Clin Exp Metastasis 20(3):237–250
33. Sotgia F, Martinez-Outschoorn UE, Pavlides S, Howell A, Pestell
RG, Lisanti MP (2011) Understanding the Warburg effect and the
prognostic value of stromal caveolin-1 as a marker of a lethal
tumor. Breast Cancer Res 13(4):213
34. Trédan O, Galmarini CM, Patel K, Tannock IF (2007) Drug
Resistance and the Solid Tumor Microenvironment. J Natl Can-
cer Inst 99(19):1441–1454
35. Gabbiani G, Ryan GB, Majne G (1971) Presence of modified
fibroblasts in granulation tissue and their possible role in wound
contraction. Experientia 27(5):549–550
36. Rasanen K, Vaheri A (2010) Activation of fibroblasts in cancer
stroma. Exp Cell Res 316(17):2713–2722. doi:10.1016/j.yexcr.
2010.04.032
37. Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, Brown RA
(2002) Myofibroblasts and mechano-regulation of connective
tissue remodelling. Nat Rev Mol Cell Biol 3(5):349–363. doi:10.
1038/nrm809
38. Cirri P, Chiarugi P (2012) Cancer-associated-fibroblasts and tu-
mour cells: a diabolic liaison driving cancer progression. Cancer
Metastasis Rev 31(1–2):195–208
39. Dvorak HF (1986) Tumors: wounds that do not heal. similarities
between tumor stroma generation and wound healing. N Engl J
Med 315(26):1650–1659
40. Kalluri R, Zeisberg M (2006) Fibroblasts in cancer. Nat Rev
Cancer 6(5):392–401
41. Pietras K, Ostman A (2010) Hallmarks of cancer: interactions
with the tumor stroma. Exp Cell Res 316(8):1324–1331
42. Hsu PP, Sabatini DM (2008) Cancer cell metabolism: Warburg
and Beyond. Cell 134(5):703–707
43. Zhivotovsky B, Orrenius S (2009) The Warburg Effect returns to
the cancer stage. Semin Cancer Biol 19(1):1–3
44. Rattigan Y, Patel B, Ackerstaff E, Sukenick G, Koutcher J, Glod
J, Banerjee D (2012) Lactate is a mediator of metabolic coop-
eration between stromal carcinoma associated fibroblasts and
glycolytic tumor cells in the tumor microenvironment. Exp Cell
Res 318(4):326–335
45. Ristow M (2006) Oxidative metabolism in cancer growth. Curr
Opin in Nutr Metabol Care 9(4):339–345
46. Fantin VR, St-Pierre J, Philip L (2006) Attenuation of LDH-A
expression uncovers a link between glycolysis, mitochondrial
physiology, and tumor maintenance. Cancer Cell 9(6):425–434
47. Brauer H, Makowski L, Hoadley K, Casbas-Hernandez P, Lang
L, Romàn-Pèrez E, D’Arcy M, Freemerman A, Perou C, Troester
M (2013) Impact of tumor microenvironment and epithelial
phenotypes on metabolism in breast cancer. Clin Cancer Res
19(3):571–585
123