Download WHY DO CANCERS HAVE HIGH AEROBIC GLYCOLYSIS?

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WHY DO CANCERS HAVE HIGH
AEROBIC GLYCOLYSIS?
Robert A. Gatenby* and Robert J. Gillies ‡
Abstract | If carcinogenesis occurs by somatic evolution, then common components of the cancer
phenotype result from active selection and must, therefore, confer a significant growth advantage.
A near-universal property of primary and metastatic cancers is upregulation of glycolysis, resulting
in increased glucose consumption, which can be observed with clinical tumour imaging. We
propose that persistent metabolism of glucose to lactate even in aerobic conditions is an
adaptation to intermittent hypoxia in pre-malignant lesions. However, upregulation of glycolysis
leads to microenvironmental acidosis requiring evolution to phenotypes resistant to acid-induced
cell toxicity. Subsequent cell populations with upregulated glycolysis and acid resistance have a
powerful growth advantage, which promotes unconstrained proliferation and invasion.
*Departments of Radiology
and Applied Mathematics,
University of Arizona,
Tucson, Arizona 85721, USA.
‡
Departments of Radiology
and Biochemistry and
Molecular Biophysics,
University of Arizona,
Tucson, Arizona 85721, USA.
Correspondence to R.A.G.
e-mail: rgatenby@
radiology.arizona.edu
doi:10.1038/nrc1478
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The multistep process of carcinogenesis is often described
as occuring by somatic evolution, because it seems formally analogous to Darwinian processes, wherein phenotypic properties are retained or lost depending on their
contribution to individual fitness. According to this
model, traits that are found in invasive cancers must arise
as adaptive mechanisms to environmental proliferative
constraints during carcinogenesis1. Conversely, the common appearance of a phenotypic property in cancer populations is presumptive evidence that it must confer a
selective growth advantage.
A curious, but common, property of invasive cancers
is altered glucose metabolism. Glycolysis — literally lysis
of glucose — first requires the conversion of glucose to
pyruvate (FIG. 1) and then to the waste product lactic
acid. In most mammalian cells, glycolysis is inhibited by
the presence of oxygen, which allows mitochondria to
oxidize pyruvate to CO2 and H2O. This inhibition is
termed the ‘Pasteur effect’, after Louis Pasteur, who first
demonstrated that glucose flux was reduced by the
presence of oxygen2. This metabolic versatility of mammalian cells is essential for maintenance of energy
production throughout a range of oxygen concentrations. Conversion of glucose to lactic acid in the presence of oxygen is known as aerobic glycolysis or the
‘Warburg effect’. Increased aerobic glycolysis is uniquely
observed in cancers. This phenomenon was first
reported by Warburg in the 1920s3, leading him to the
hypothesis that cancer results from impaired mitochondrial metabolism. Although the ‘Warburg hypothesis’
has proven incorrect, the experimental observations of
increased glycolysis in tumours even in the presence of
oxygen have been repeatedly verified4.
Following Warburg’s initial observation, interest in
the metabolic property of cancers has varied over time.
Intense investigation in the 1960s was followed by a
steep decline concomitant with the widespread application of newer molecular techniques. The atmosphere of
the day was summarized by Sidney Weinhouse, who
said “Since our perspectives have broadened over the
years, the burning issues of glycolysis and respiration in
cancer now flicker only dimly”5.
However, interest in tumour metabolism has been
rekindled, mainly because of the widespread clinical
application of the imaging technique positronemission tomography (PET) using the glucose analogue tracer 18fluorodeoxyglucose (FdG)6–8. FdG PET
imaging of thousands of oncology patients has
unequivocally shown that most primary and metastatic human cancers show significantly increased glucose
uptake (FIG. 2). For many cancers, the specificity and
sensitivity of FdG PET to identify primary and
metastatic lesions is near 90%9. Sensitivity is lowered
because FdG PET has difficulty resolving lesions less
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HEXOKINASES
Summary
Enzymes that catalyse the
transfer of phosphate from ATP
to glucose to form glucose-6phosphate. This is the first
reaction in the metabolism of
glucose and prevents efflux of
glucose from the cell.
HYPOXIA
Refers to a low oxygen level. This
means different levels to
different investigators, but for
radiation biologists hypoxia
occurs at levels less than 0.1%
oxygen in the gas phase.
Normoxia refers to normal levels
of oxygen (>10%) and anoxia
refers to no oxygen.
• Widespread clinical use of 18fluorodeoxyglucose positron-emission tomography has demonstrated that the glycolytic
phenotype is observed in most human cancers.
• The concept of carcinogenesis as a process that occurs by somatic evolution clearly implies that common traits of the
malignant phenotype, such as upregulation of glycolysis, are the result of active selection processes and must confer a
significant, identifiable growth advantage.
• Constitutive upregulation of glycolysis is likely to be an adaptation to hypoxia that develops as pre-malignant lesions
grow progressively further from their blood supply. At this stage, the blood supply remains physically separated from
the growing cells by an intact basement membrane.
• Increased acid production from upregulation of glycolysis results in microenvironmental acidosis and requires further
adaptation through somatic evolution to phenotypes resistant to acid-induced toxicity.
• Cell populations that emerge from this evolutionary sequence have a powerful growth advantage, as they alter their
environment through increased glycolysis in a way that is toxic to other phenotypes, but harmless to themselves. The
environmental acidosis also facilitates invasion through destruction of adjacent normal populations, degradation of
the extracellular matrix and promotion of angiogenesis.
• We propose that the glycolytic phenotype, by conferring a powerful growth advantage, is necessary for evolution of
invasive human cancers.
than 0.8 cm3, and specificity is lowered because other
tissues, notably immune cells, also avidly trap FdG.
When these limitations are accounted for, it can be reasonably surmised that virtually all invasive cancers
avidly trap FdG.
The increased glucose uptake imaged with FdG PET
is largely dependent on the rate of glycolysis. FdG
uptake and trapping occurs because of upregulation of
glucose transporters (notably GLUT1 and GLUT3) and
10,11
HEXOKINASES I and II
Although metabolic control over
glycolytic rate can be applied at many steps in the glycolytic pathway12,13, most studies in cancer support the
hypothesis that control over glycolytic flux primarily
Blood vessel
Glucose
HbO2
Glucose
O2
Anion
exchanger
HCO3–
H+
Glucose
transporter
36 ATP
Lactate
Mitochondrion
Lactate
Glucose
Monocarboxylate H+
transporter
2 ATP
Hexokinase
Glucose-6phosphate
Pyruvate
H+
Sodium–hydrogen
exchanger
Figure 1 | Glucose metabolism in mammalian cells. Afferent blood delivers glucose and oxygen
(on haemoglobin) to tissues, where it reaches cells by diffusion. Glucose is taken up by specific
transporters, where it is converted first to glucose-6-phosphate by hexokinase and then to
pyruvate, generating 2 ATP per glucose. In the presence of oxygen, pyruvate is oxidized to HCO3,
generating 36 additional ATP per glucose. In the absence of oxygen, pyruvate is reduced to lactate,
which is exported from the cell. Note that both processes produce hydrogen ions (H+), which
cause acidification of the extracellular space. HbO2, oxygenated haemoglobin.
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resides at the transport and phosphorylation steps14–16.
FdG PET imaging also allows quantitation of glucose uptake. These studies have consistently correlated
poor prognosis and increased tumour aggressiveness
with increased glucose uptake 17,18. In addition,
hypoxic tumours, which require increased glycolysis
to survive, are often19–22, but not always23, more invasive and metastatic than those with normal oxygen
levels. These results demonstrate the clinical importance of glucose metabolism and have moved the glycolytic phenotype from a laboratory oddity to the
mainstream of clinical oncology.
Cells derived from tumours typically maintain their
metabolic phenotypes in culture under normoxic conditions, indicating that aerobic glycolysis is constitutively
upregulated through stable genetic or epigenetic
changes. Consistent with the FdG PET results, the glycolytic rate in cultured cell lines seems to correlate with
tumour aggressiveness. For example, non-invasive
MCF-7 breast cancer cells have much lower aerobic
glucose consumption rates compared with the highly
invasive MDA-mb-231 breast cancer cell line (FIG. 3).
These observations indicate that altered metabolism of glucose by tumours is more than a simple
adaptation to HYPOXIA. We suggest that the nearuniversal observation of aerobic glycolysis in invasive
human cancers, its persistence even under normoxic
conditions and its correlation with tumour aggressiveness indicate that the glycolytic phenotype confers
a significant proliferative advantage during somatic
evolution of cancer and must, therefore, be a crucial
component of the malignant phenotype.
At first glance, this hypothesis seems at odds with
an evolutionary model of carcinogenesis, because the
proliferative advantage of the glycolytic phenotype is
not immediately apparent. First, anaerobic metabolism of glucose is inefficient — it produces only 2 ATP
per glucose, whereas complete oxidation produces 38
ATP per glucose (FIG. 1). Second, the metabolic products of glycolysis, such as hydrogen ions (H+), cause a
spatially heterogeneous but consistent acidification of
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the extracellular space, which might result in cellular
toxicity24–26 (FIG. 4a,b). Intuitively, it would seem that the
Darwinian forces prevailing during the somatic evolution of invasive cancers would select against a metabolic
phenotype that is more than an order of magnitude less
efficient than its competitors and that is environmentally poisonous. In other words, the accepted tenet of
‘survival of the fittest’ would seem to generally favour
populations with more efficient and sophisticated substrate metabolism. So, why do tumour populations consistently evolve to the inefficient and potentially toxic
glycolytic phenotype?
We propose that the remarkable prevalence of
upregulated glycolysis in clinical cancers is neither random nor accidental. Rather, it represents an evolved
solution to common environmental growth constraints
during carcinogenesis, and its persistence in primary
and metastatic malignancy indicates that it continues to
confer a proliferative advantage even to fully transformed cells. So, we suggest that increased glycolysis is
an essential component of the malignant phenotype
and, therefore, a hallmark of invasive cancers. Herein we
explore its causes and consequences.
The microenvironment in pre-malignant lesions
WINDOW CHAMBER
A metal chamber with a glass
window that is placed on the
dorsal skin of an animal. This
allows in vivo tumour growth to
be continuously observed
microscopically.
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Figure 2 | Positron-emission tomography imaging with
18
fluorodeoxyglucose of a patient with lymphoma. The
mediastinal nodes (purple arrow) and supraclavicular nodes
(green arrows) show high uptake of 18fluorodeoxyglucose
(FdG), showing that tumours in these nodes have high levels
of FdG uptake. The bladder (yellow arrow) also has high
activity, because of excretion of the radionuclide.
60
Normoxia
Hypoxia
50
Glucose consumption rate
(nmol min–1 mg protein–1)
Although pre-malignant lesions are often characterized
as highly vascularized, this is true only in a macroscopic
sense. That is, although a pre-malignant lesion such as a
polyp or carcinoma in situ might have a vascular stroma,
the hyperplastic epithelia are physically separated from
their blood supply by a basement membrane. This is
illustrated in FIG. 5, as the hyperplastic epithelium of a
carcinoma in situ is clearly delimited from the stroma by
a thin basement membrane. Blood vessels are confined
to the stromal compartment and, therefore, early carcinogenesis and development of the malignant phenotype actually occur in an avascular environment. As a
result, substrates, such as oxygen and glucose, must diffuse from the vessels across the basement membrane and
through layers of tumour cells, where they are metabolized. This process of diffusion and consumption was
modelled by Krogh as early as 1919 through
reaction–diffusion equations that showed that oxygen
concentrations decreased with distance from a capillary
such that oxygenated cells were limited to a distance of
less than 150 µm from a blood vessel27. In the 1950s,
empirical studies by Thomlinson and Gray showed that
viable tumour cells were not observed at distances
greater than 160 µm from blood vessels, consistent with
Krogh’s calculations28. Subsequent experimental studies
in WINDOW CHAMBERS in animal models have demonstrated
that near-zero partial pressures of oxygen (pO2) are
observed at distances of only 100 µm from a vessel29,30.
Therefore, pre-malignant lesions, provided their
basement membranes remain intact, will inevitably
develop hypoxic regions near the oxygen diffusion limit,
as persistent proliferation leads to a thickening of the
epithelial layer, pushing cells ever more distant from
their blood supply, which remains on the other side of
the basement membrane. At this penumbral layer,
microenvironmental selection forces will favour
40
30
20
10
W
P
0
MCF-7
MDA-MB-231
Figure 3 | Pasteur and Warburg effects in non-invasive
and metastatic breast cancer cell lines. In both cell lines,
glucose consumption is reduced in the presence of oxygen
— the Pasteur effect (P). However, the more aggressive cell
line, MDA-MB-231, has much higher glucose consumption
in the presence of oxygen than the MCF-7 cells with a
non-invasive phenotype — the Warburg effect (W). This is
consistent with positron-emission tomography scans with
18
fluorodeoxyglucose, which show that higher glucose
uptake correlates with more aggressive phenotypes and
poorer clinical outcomes.
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7.4
14
pH
pO2
7.2
12
10
pH
8
7.0
6
4
6.8
pO2 mm Hg
a
2
0
6.6
0
100
200
300
400
Distance (mm)
7.0
Extracellular pH
b
MDA-MB-435
6.4
Figure 4 | Hyperacidity of tumours. These figures illustrate the
micro- and macro-heterogeneity of pH. a | Tumour interstitial pH
and partial pressure of oxygen (pO2) are shown with distance
from a vessel wall. These were measured in vivo in MCF-7 breast
cancer cells using fluorescent ratio imaging. b | The extracellular
pH of a MDA-MB-435 breast tumour in mice was imaged with
the pH indicator IEPA and measured by 1H magneticresonance spectroscopy. Part a reproduced with permission
from REF. 30 © (1997) Nature Publishing Group. Part b
reproduced with permission from REF. 26 © (2002) Wiley.
phenotypes that adapt to harsh environments (through
resistance to hypoxia and acid-induced cell toxicity)
and successfully compete for scarce resources, such as
oxygen and glucose31,32.
Emergence of the glycolytic phenotype
HAEMATOCRIT
A measure of the concentration
of red cells in the blood. A
reduced haematocrit decreases
the oxygen-carrying capacity of
the blood.
VASOMOTION
Rhythmic oscillations in
vascular tone caused by local
changes in smooth muscle.
VASCULAR REMODELLING
The active process of altering
structure and arrangement in
blood vessels through cell
growth, cell death, cell migration
and production or degradation
of the extracellular matrix.
894
Evolutionary game theory is a mathematical approach
that analyses strategy dynamics in adaptation to environmental growth — winners in this game proliferate,
whereas losers become extinct. Recently, this method has
been applied to somatic evolution of the malignant
phenotype33. This analysis showed that proliferation of
normal cells is controlled by their interactions with other
cells and the extracellular matrix (ECM), and by the levels of growth factors. Importantly, cell proliferation and
survival in normal tissue is not constrained by substrate
availability, except under pathological conditions such as
acute vascular occlusion (for example, caused by strokes
and myocardial infarcts) or chronic occlusion (as seen in
diabetic ulcers). It follows, therefore, that the earliest
steps in carcinogenesis require alterations in cellular sensitivity to these normal tissue constraints. So, proliferation will follow genetic alterations that reduce sensitivity
to growth constraints generated by other cells, the ECM
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and/or growth factors33. For example, in many tissues,
pre-malignant lesions are initiated by mutations in
HRAS or KRAS genes, which alter cellular responses to
growth factors34. From this, it follows that mutations
affecting substrate use cannot be early events in carcinogenesis because they would not confer a selective growth
advantage in an environment in which proliferation is
not limited by substrate availability.
The evolutionary models show, however, that clonal
expansion of pre-malignant tumour populations is
eventually limited by substrate availability33, as cell proliferation, unconstrained by normal tissue interactions,
carries the population increasingly far from its blood
supply (see above). In FIG. 5, note the distances between
blood vessels and the necrotic zone of late-stage carcinoma in situ. Low oxygen concentrations seem to be the
first substrate limitation confronting neoplastic cell populations, as reaction–diffusion models and empirical
studies have shown that pO2 decline more rapidly with
distance from blood vessels than do glucose levels25,30,35,36.
Although the presence of hypoxia in pre-malignant
in situ lesions has not been measured directly, it can be
inferred from the frequent observation of necrosis in
these lesions and by demonstration of hypoxia-inducible
enzymes such as carbonic anhydrases IX and XII in latestage ductal carcinoma in situ, particularly adjacent to
areas of necrosis37. We suggest that hypoxia in the
penumbral region of pre-malignant tumours produces
an adaptive landscape that favours a switch to anaerobic
metabolism, which allows maintenance of metabolic
activities in the absence of oxygen.
A key factor in this adaptive landscape seems to be
the exposure of cells near the oxygen diffusion limit to
an unstable environment due to fluctuations in the
haemodynamics of distant blood vessels. Oxic–hypoxic
cycles in tumours have been measured to occur with
periodicities of minutes38, hours39 or days40. For instance,
Gallez’s group has recently imaged tumour xenografts
using a magnetic-resonance imaging (MRI) technique
that is sensitive to oxygenation status41. Analyses showed
that fluctuations in signal intensity (oxygenation)
occurred with discrete periodicities of 1 and 20 cycles
per hour. By contrast, Dewhirst and colleagues used
microelectrodes to show periodicities of about 1–2
cycles per minute42. However, it should be noted that
MRI, although imaging the whole tumour, is insensitive
to rapid fluctuations, and microelectrode instabilities
render these electrodes insensitive to slower changes.
Nonetheless, all of these studies show that oxygen delivery to tumours is inconsistent. These temporal cycles are
probably due to a range of physiological mechanisms.
Relatively rapid oxic–anoxic cycles can occur because
of fluctuations in HAEMATOCRIT43 and VASOMOTION44.
Variations occurring over days probably involve VASCULAR
45,46
REMODELLING
or cycles of neoangiogenesis and regression due to hypoxia-induced expression of secreted vascular endothelial growth factor (VEGF), which is an
induction and survival factor for new blood vessels40.
From a bioenergetic standpoint, periodic hypoxia will
select for cells in which anaerobic glucose metabolism is
constitutively upregulated, as they would better survive
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0.16 mm
N
T
B
S
Figure 5 | Late-stage ductal carcinoma in situ. A 5µm-thick biopsy sample was stained with
haematoxylin and eosin, and digitized with the DMetrix camera system (see online links box) with
a resolution of 0.45 µm/pixel. Blood vessels (blue) are seen in the stroma (S) surrounding the
tumour (T), but the tumour itself — within the ducts and surrounded by the basement membrane
(B) — is avascular. The centre of the tumour is necrotic (N).
VMAX and KM
Terms from the
Michaelis–Menten model.
Applied to transport, Vmax is the
maximum possible rate of
uptake of a specific substrate. Km
is the substrate concentration at
which the substrate uptake is
half of Vmax. Cell populations
with low Km are better adapted
to maintaining substrate uptake
in conditions in which substrate
concentrations are low.
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the anoxic episodes. The mechanisms underlying this
upregulation are discussed in the next section.
Although the upregulation of glycolysis is a successful adaptation to hypoxia/anoxia, it also has significant
negative consequences because of increased acid production, which causes significant decreases in local
extracellular pH. Prolonged exposure of normal cells to
an acidic microenvironment typically results in necrosis
or apoptosis through p53- and caspase-3-dependent
mechanisms47,48. The physiological trigger for apoptosis
might be collapse of the transmembrane H+ gradient
that occurs with intracellular acidosis, but other factors
might have a role49. So, constitutive upregulation of glycolysis requires additional adaptation to the negative
effects of extracellular acidosis through resistance to
apoptosis or upregulation of membrane transporters
to maintain normal intracellular pH. Intracellular pH
is maintained by multiple families of H+ transporters,
which are co-expressed and redundant50,51. Na+–H+
exchange51,52 and vacuolar H+-ATPases53 have both been
observed to be upregulated in cancers, and vacuolar
H+-ATPase might confer resistance to apoptosis54.
Additional adaptations might also be required as
the increased glucose consumption rates further
decrease glucose concentrations. Cellular competition
for this increasingly limited resource will therefore
increase and favour phenotypes with greater numbers
of either high V (for example, GLUT1) or low K (for
example, GLUT3) glucose transporters. Such upregulation of glucose transporters has been observed during carcinogenesis in oesophageal, gastric, breast and
colon cancers55–57.
In summary, we suggest that the glycolytic phenotype initially arises as an adaptation to local hypoxia
(FIG. 6). Persistent or cyclical hypoxia subsequently
exerts selection pressures that lead to constitutive
upregulation of glycolysis, even in the presence of
MAX
M
oxygen. This constitutive upregulation might occur
through mutations or epigenetic changes such as alteration in the methylation patterns of promoters. The
consequences of increased glycolysis require further
adaptation to environments with high acid and low
glucose concentrations.
We propose that this is a crucial evolutionary
sequence in the development of invasive cancer. First, it
results in a phenotype with a powerful proliferative
advantage, in that, through persistent aerobic glycolysis,
it is able to alter the local microenvironment in a way
that is harmless to itself, but fatal to competing populations. Second, acidification of the microenvironment
facilitates tumour invasion both through destruction of
adjacent normal populations and through acid-induced
degradation of the ECM and promotion of angiogenesis.
The underlying molecular, cellular and environmental
dynamics are discussed next.
Molecular mechanisms
The molecular mechanisms leading to constitutive
upregulation of aerobic glycolysis are not well defined.
As mentioned above, it is commonly assumed that glucose transporters and hexokinases are the key molecules
regulating glycolytic flux. It must be noted that a corollary of the current hypothesis is that the selective advantage conferred by the glycolytic phenotype is insensitive
to the exact mechanism of glycolytic induction.
A key regulator of the glycolytic response is the transcription factor hypoxia-inducible factor-1α (HIF1α)58.
This factor mediates a pleiotropic response to hypoxic
stress by inducing survival genes, including glucose
transporters; angiogenic growth factors (for example,
VEGF); hexokinase II59; and haematopoeitic factors (for
example, transferrin and erythropoietin)60. In some systems, constitutively increased HIF1α levels are associated
with constitutively high glucose consumption rates. This
is the case in the renal-cell carcinoma cell line RCC4,
which has constitutively high HIF1α because of a mutation in the von Hippel–Lindau (VHL) ubiquitin ligase.
(The wild-type enzyme targets HIF1α for degradation.)
Re-inserting VHL as a transgene in these cells restores
normal HIF1α levels and greatly reduces aerobic glucose
consumption rates61. Although HIF1α strongly links aerobic glycolysis to carcinogenesis62, it would be premature
to conclude that the glycolytic phenotype in cancer is
invariably due to dysregulation of the HIF system.
Although it is termed the hypoxia-inducible factor,
HIF1α levels can in fact be stabilized by a range of factors, including cyclooxygenase-2 activity, insulin-like
growth factor 2, ERBB2, epidermal growth factor receptor, phosphatidylinositol 3-kinase, heat-shock protein 90,
microtubule status, thioredoxin and histone deacetylase,
to name a few 63–65. Additionally, stabilization of HIF1α
in tumours can result from hypoxia-reoxygenation
injury 66, which indicates that its constitutive upregulation might result from the periodic oxic–hypoxic cycles
that occur in pre-malignant tumours. Consistent with
our somatic-evolution model, lack of HIF1α decreases
survival in response to hypoxia67, leading to selection of
cells with upregulated HIF1α.
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Tumour
stage
Normal
epithelium
Physiological
state
Process
Interstitial
neoplasia
Initiation
Intermittant
hypoxia
Proliferation
Carcinoma
in situ
HIF1α
stabilization
Selection
Invasive
carcinoma
Glycolytic
phenotype
Induction
Acidosis
Metabolism
Motility
Metastatic
disease
Degradation of
basement
membrane and
vascularization
Selection
VEGF
Model
Glucose diffusion limit
O2 diffusion limit
Basement
membrane
Blood vessel
Stroma
Figure 6 | Model for cell–environment interactions in carcinogenesis. Early carcinogenesis proceeds from normal tissues
through initiation to a hyperplastic state to interstitial neoplasia, progressing to carcinoma in situ. Until this stage, epithelial cancers are
avascular, as shown by histopathology (FIG. 5). Following breakdown of the basement membrane, cells gain access to existing and
newly formed blood and lymphatic vascular routes for metastasis. The stages of tumour growth and their associated physiological
states are diagrammed, showing that progression from one stage to the next is governed by state processes. Normal epithelial cells
(grey) become hyperproliferative (pink) following induction. As they reach the oxygen diffusion limit, they become hypoxic (blue), which
can either lead to cell death (apoptotic cells shown with blebbing) or adaptation of a glycolytic phenotype (green), which allows cells to
survive. As a consequence of glycolysis, lesions become acidotic, which selects for motile cells (yellow) that eventually breach the
basement membrane. As cancer progression proceeds, the mutations in cells increase (nuclei shown as light orange for one mutation
and darker oranges for more mutations). HIF1α, hypoxia-inducible factor-1α; VEGF, vascular endothelial growth factor.
Multiple cellular pathways might lead to the glycolytic phenotype, so that altered glucose metabolism
might even result in cells with normal HIF levels. For
example, upregulation of glycolytic enzymes can be
coordinated in response to oxidation–reduction
changes by the Sp1 transcription-factor complex68.
GLUT1 can be upregulated directly by MYC13,69 or indirectly by KRAS70. Interestingly, in this latter study,
KRAS activation was only associated with a subset of
GLUT1-positive colon cancers, indicating that it is one
of several mechanisms to activate glycolysis in this system. RAS activation of GLUT1 transcription seems to
be mediated through HIF1α transactivation71.
Hexokinase II can be transcriptionally activated by
mutant p53 (REF. 72) or through demethylation of its
promoter73. It is also intriguing to note that transfection
of fibroblasts with H+-ATPase or Na+–H+ exchange
raises the intracellular pH, makes them tumorigenic
and leads to marked increases in glycolysis74,75. These
alternative systems for upregulating glycolysis are consistent with our basic proposal that the mechanism of
induction is not as important as the induction itself.
That is, the glycolytic phenotype is not a secondary
phenomenon that results from induction of some
other pathway during carcinogenesis. Rather, it is
directly selected because it provides a growth advantage
and acquisition of the glycolytic phenotype might be
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achieved through multiple mechanisms, including
oncogene activation or stabilization of transcription
factors such as HIF1α.
Angiogenesis
We suggest that the glycolytic phenotype evolves in a
microenvironment that is avascular; that is, the evolving tumour cells remain physically separated from
their blood supply by a basement membrane, as
occurs in in situ tumours. This invokes the diffusion
of substrates from the vascularized stroma to the proliferating tumour epithelium. Therefore, even though
late-stage carcinoma in situ can be characterized as
‘angiogenic’, the tumour does not become vascularized until the basement membrane is breached by an
invasive cell. In fact, there is emerging evidence that
the ‘glycolytic switch’ occurs before the ‘angiogenic
switch’; lactic acid has been observed in regions of
invasive gliomas76,77 that lack vessel permeability, as
shown by the absence of contrast enhancement with
MRI78. We do not wish to indicate that angiogenesis
does not have a role in this process. In fact, it is likely
that angiogenic factors, such as VEGF, are produced
by the tumour and that this will promote increased
vascularity within the stroma (FIG. 6). However, these
new vessels remain physically separated from the
tumour cells by the basement membrane (see figure 2
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CLASTOGENIC
Describing any substance or
processes that increases
alterations in the structure of
chromosomes.
GAP JUNCTIONS
Linked channels through
contiguous cell membranes that
interconnect the cytoplasm of
adjacent cells and allow direct
exchange of ions and small
molecules.
in REF. 79). This will not necessarily result in increased
substrate delivery through diffusion, as substrate concentrations in the reaction–diffusion equation are
unaltered. So, a hallmark of these early cancers is a
failure of angiogenesis to relieve hypoxia because of
the physical separation between vessels and the cells
they feed. This can result in a futile cycle of hyperproliferating blood vessels. Once the basement membrane is breached, the tumour will become vascular
both by co-opting the pre-existing vessels within the
stroma and by promoting new vessel growth directly
into the tumour mass.
Acidosis and invasion
Although the glycolytic phenotype seems to be the result
of adaptation to environmental constraints in pre-malignant lesions, its persistence in primary and metastatic
cancers even in conditions of normoxia indicates that it
continues to provide a strong selective growth advantage
following malignant progression. We suggest, in fact, that
acquisition of the glycolytic phenotype is required for
invasive tumour growth.
A constitutive and persistent increase in glycolysis
results in acute and chronic acidification of the local environment. Indeed, numerous studies have shown that the
extracellular pH of human and animal tumours is consistently acidic and can reach pH values approaching 6.0
(REFS 80,81) (FIG. 4). We have demonstrated both mathematically and empirically that intratumoral acidosis
results in diffusion of H+ ions along concentration gradients into peritumoral normal tissue32. Normal cells,
which lack a mechanism to adapt to extracellular acidosis
(such as a p53 mutation) are unable to survive under
such conditions, whereas the tumour populations continue to proliferate. In addition, acidosis itself can be
mutagenic and CLASTOGENIC82, possibly through inhibition
of DNA repair (for a review, see REF. 80) and can lead to
both inhibition of GAP-JUNCTION conductance and to spontaneous transformation of normal diploid fibroblasts83.
The resulting phenotypic diversity enhances the evolutionary potential of the tumour population, which
accelerates malignant progression and adaptation to therapeutic strategies34 (BOX 1). Finally, under some (but not
all) conditions, low pH stimulates in vitro invasion84 and
in vivo metastasis85. The mechanisms of such induction
Box 1 | Consequences of hypoxia and acidosis
As tumours evolve and become first hypoxic and then acidic, malignant progression is
accelerated and resistance to therapeutic strategies occurs. For further information,
see REF. 36.
Hypoxia
Acidosis
Radioresistance
Increased radioresistance
Drug resistance
Resistance to anthracyclines
Metastasis and invasion
Increased metastases
Increased mutation rate
Increased migration and invasion
Gene expression induced by hypoxiainducible factor
Mutagenesis/clastogenesis
Apoptosis
Apoptosis
NATURE REVIEWS | C ANCER
are not known, but might involve the metalloproteinases
and/or cathepsins, which promote the degradation of the
ECM and basement membranes86,87.
Metastasis
So far, we have focused on the role of upregulated glycolysis and resistance to extracellular acidosis in adaptation to conditions in early pre-malignant lesions and
in the evolution of invasive primary cancers. However,
we note that this phenotype might also be crucial in
the maturation of metastases as well. Upregulated glycolysis, evidenced by increased intratumoral lactate
concentrations, is associated with increased incidence
of metastasis in cervical and head and neck cancers88,89.
Furthermore, a correlation between GLUT1 expression levels and metalloproteinase expression has also
been reported in metastatic cancers90.
During the process of metastasis, migratory cells
invade the stromal tissue and move to distant sites, lodging in pre-capillary arterioles and capillaries91,92. These
cells probably also experience periodic hypoxic or anoxic
episodes as they proliferate and occlude the intravascular
space. Therefore, the end stage of the metastasis sequence
will also favour cells that are glycolytic and resistant to
hypoxia- or acid-induced apoptosis. Any selective advantage is important, as the success rate of metastasis is low.
For example, in a typical lung-colonization assay, as many
as 105 lung cancer cells are injected into mouse tail veins,
but fewer than 100 cells generally survive to form
colonies. Cells pre-treated with hypoxia for 24 hours are
four times more likely to survive than their normoxic
counterparts93. Although there are other possible interpretations of these data, we suggest that they support the
hypothesis that the glycolytic phenotype contributes to
the efficiency of metastasis by allowing cells to survive
transient hypoxia.
Summary and future directions
In summary, we suggest that upregulation of glycolytic
metabolic pathways in the vast majority of invasive cancers is the result of adaptation to consistent environmental pressures in pre-malignant lesions, when diffusion
limitations result in gradients of hypoxia and acidosis.
Cellular traits selected by these conditions include constitutive upregulation of glycolysis and resistance to acidinduced apoptosis. Mathematical models and empirical
observation indicate that the advantages conferred by
this combination of phenotypic traits are both sufficient
and necessary to promote unconstrained tumour proliferation. Furthermore, both mathematical models and
empirical evidence indicate that diffusion of acid from
the tumour into peritumoral normal tissue provides a
specific mechanism promoting tumour invasion32.
The crucial importance of the glycolytic phenotype is
emphasized by studies demonstrating that increased glucose uptake is observed to coincide with the transition
from pre-malignant lesions to invasive cancer94,95. These
evolutionary advantages explain the remarkable prevalence of the glycolytic phenotype in human cancers and
the otherwise puzzling observation that malignant cells
remain glycolytic even in the presence of normoxia.
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REVIEWS
The molecular basis for evolution of the glycolytic
phenotype has been clarified by recent advances in
understanding the HIF system, but much additional
work will be required to fully understand the complex
pathways involved in hypoxic response, metabolic controls and adaptation to acidosis in cancer progression.
Despite gaps in our knowledge, the glycolytic phenotype could be exploited for treatment at several levels.
As this phenotype emerges early in carcinogenesis, it
might represent a possible target in cancer prevention.
At later stages, a more complete understanding of the
molecular and physiological consequences might lead
to targeted therapies.
Finally, this model of carcinogenesis indicates new
avenues of investigation. For example, what is the
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Acknowledgements
We wish to acknowledge the invaluable contributions of
E. Gawlinski and T. Vincent for their efforts in the mathematical
modelling that led to the insights presented here. We also thank
E. Racker for stimulating this research by posing to us the question
in the title.
Competing interests statement
The authors declare no competing financial interests.
Online links
DATABASES
The following terms in this article are linked online to:
National Cancer Institute: http://cancer.gov/
breast cancer | cervical cancer | colon cancer | gastric cancer |
head and neck cancer | oesophageal cancer
Entrez Gene:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene
caspase-3 | GLUT1 | GLUT3 | HRAS | HIF1α | KRAS | MYC | p53 |
VEGF | VHL
FURTHER INFORMATION
DMetrix digital imaging program: www.dmetrix.com
Access to this interactive links box is free online.
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