REVIEWS

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.

The multistep process of carcinogenesis is often described
as occuring by somatic evolution, because it seems for-
mally analogous to Darwinian processes, wherein pheno-
typic 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 com-
mon appearance of a phenotypic property in cancer pop-
ulations 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
*Departments of Radiology the presence of oxygen, which allows mitochondria to
and Applied Mathematics, oxidize pyruvate to CO2 and H2O. This inhibition is
University of Arizona, termed the Pasteur effect, after Louis Pasteur, who first
Tucson, Arizona 85721, USA.
demonstrated that glucose flux was reduced by the
Departments of Radiology
and Biochemistry and presence of oxygen2. This metabolic versatility of mam-
Molecular Biophysics, malian cells is essential for maintenance of energy
University of Arizona, production throughout a range of oxygen concentra-
Tucson, Arizona 85721, USA. tions. Conversion of glucose to lactic acid in the pres-
Correspondence to R.A.G.
e-mail: rgatenby@ ence of oxygen is known as aerobic glycolysis or the
radiology.arizona.edu Warburg effect. Increased aerobic glycolysis is uniquely
doi:10.1038/nrc1478 observed in cancers. This phenomenon was first
reported by Warburg in the 1920s3, leading him to the
hypothesis that cancer results from impaired mitochon-
drial 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 Warburgs 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 applica-
tion 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 dimly5.
However, interest in tumour metabolism has been
rekindled, mainly because of the widespread clinical
application of the imaging technique positron-
emission tomography (PET) using the glucose ana-
logue tracer 18fluorodeoxyglucose (FdG)68. FdG PET
imaging of thousands of oncology patients has
unequivocally shown that most primary and metasta-
tic 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

NATURE REVIEWS | C ANCER VOLUME 4 | NOVEMBER 2004 | 8 9 1

REVIEWS

HEXOKINASES
Enzymes that catalyse the
transfer of phosphate from ATP
to glucose to form glucose-6-
phosphate. 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.
Summary
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 resides at the transport and phosphorylation steps1416.
tissues, notably immune cells, also avidly trap FdG. FdG PET imaging also allows quantitation of glu-
When these limitations are accounted for, it can be rea- cose uptake. These studies have consistently correlated
sonably surmised that virtually all invasive cancers poor prognosis and increased tumour aggressiveness
avidly trap FdG. with increased glucose uptake 17,18. In addition,
The increased glucose uptake imaged with FdG PET hypoxic tumours, which require increased glycolysis
is largely dependent on the rate of glycolysis. FdG to survive, are often1922, but not always23, more inva-
uptake and trapping occurs because of upregulation of sive and metastatic than those with normal oxygen
glucose transporters (notably GLUT1 and GLUT3) and levels. These results demonstrate the clinical impor-
10,11
HEXOKINASES I and II Although metabolic control over tance of glucose metabolism and have moved the gly-
glycolytic rate can be applied at many steps in the gly- colytic phenotype from a laboratory oddity to the
colytic pathway12,13, most studies in cancer support the mainstream of clinical oncology.
hypothesis that control over glycolytic flux primarily Cells derived from tumours typically maintain their
metabolic phenotypes in culture under normoxic condi-
tions, indicating that aerobic glycolysis is constitutively
upregulated through stable genetic or epigenetic
Blood vessel changes. Consistent with the FdG PET results, the gly-
colytic rate in cultured cell lines seems to correlate with
Glucose HbO2 tumour aggressiveness. For example, non-invasive
MCF-7 breast cancer cells have much lower aerobic
glucose consumption rates compared with the highly
Anion invasive MDA-mb-231 breast cancer cell line (FIG. 3).
Glucose O2 exchanger
HCO3 These observations indicate that altered metabo-
H+ lism of glucose by tumours is more than a simple
Glucose 36 ATP Lactate adaptation to HYPOXIA. We suggest that the near-
transporter Mitochondrion universal observation of aerobic glycolysis in invasive
Mono-
human cancers, its persistence even under normoxic
carboxylate H+ conditions and its correlation with tumour aggres-
transporter siveness indicate that the glycolytic phenotype confers
Glucose Lactate
2 ATP a significant proliferative advantage during somatic
Hexokinase evolution of cancer and must, therefore, be a crucial
Glucose-6- Pyruvate
phosphate component of the malignant phenotype.
H+
Sodiumhydrogen At first glance, this hypothesis seems at odds with
exchanger
an evolutionary model of carcinogenesis, because the
proliferative advantage of the glycolytic phenotype is
Figure 1 | Glucose metabolism in mammalian cells. Afferent blood delivers glucose and oxygen not immediately apparent. First, anaerobic metabo-
(on haemoglobin) to tissues, where it reaches cells by diffusion. Glucose is taken up by specific lism of glucose is inefficient it produces only 2 ATP
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,
per glucose, whereas complete oxidation produces 38
generating 36 additional ATP per glucose. In the absence of oxygen, pyruvate is reduced to lactate, ATP per glucose (FIG. 1). Second, the metabolic prod-
which is exported from the cell. Note that both processes produce hydrogen ions (H+), which ucts of glycolysis, such as hydrogen ions (H+), cause a
cause acidification of the extracellular space. HbO2, oxygenated haemoglobin. spatially heterogeneous but consistent acidification of

892 | NOVEMBER 2004 | VOLUME 4 www.nature.com/reviews/cancer

 REVIEWS

the extracellular space, which might result in cellular

toxicity2426 (FIG. 4a,b). Intuitively, it would seem that the
Darwinian forces prevailing during the somatic evolu-
tion 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 environmen-
tally poisonous. In other words, the accepted tenet of
survival of the fittest would seem to generally favour
populations with more efficient and sophisticated sub-
strate metabolism. So, why do tumour populations con-
sistently evolve to the inefficient and potentially toxic
glycolytic phenotype?
We propose that the remarkable prevalence of
upregulated glycolysis in clinical cancers is neither ran-
dom 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 trans-
formed 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

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 car-
cinogenesis and development of the malignant pheno-
type actually occur in an avascular environment. As a
result, substrates, such as oxygen and glucose, must dif-
fuse from the vessels across the basement membrane and
through layers of tumour cells, where they are metabo-
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
50 Hypoxia
Glucose consumption rate
(nmol min1 mg protein1)

40
lized. This process of diffusion and consumption was
modelled by Krogh as early as 1919 through
reactiondiffusion equations that showed that oxygen 30

concentrations decreased with distance from a capillary

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.
such that oxygenated cells were limited to a distance of
less than 150 m 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 m from blood vessels, consistent with
Kroghs 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 m 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
W
20
P
10
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.

NATURE REVIEWS | C ANCER VOLUME 4 | NOVEMBER 2004 | 8 9 3

REVIEWS
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 | NOVEMBER 2004 | VOLUME 4
a 7.4 14
12
pH
7.2 pO2
10
pO2 mm Hg
8 affecting substrate use cannot be early events in carcino-
pH
7.0
6
4
6.8
2 The evolutionary models show, however, that clonal
6.6 0
0 100 200 300 400
Distance (mm)
7.0
b supply (see above). In FIG. 5, note the distances between
Extracellular pH
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 magnetic-
resonance 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
Evolutionary game theory is a mathematical approach
that analyses strategy dynamics in adaptation to environ-
mental 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 lev-
els 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 sen-
sitivity to these normal tissue constraints. So, prolifera-
tion will follow genetic alterations that reduce sensitivity
to growth constraints generated by other cells, the ECM
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
genesis because they would not confer a selective growth
advantage in an environment in which proliferation is
not limited by substrate availability.
expansion of pre-malignant tumour populations is
eventually limited by substrate availability33, as cell prolif-
eration, unconstrained by normal tissue interactions,
carries the population increasingly far from its blood
blood vessels and the necrotic zone of late-stage carci-
noma in situ. Low oxygen concentrations seem to be the
first substrate limitation confronting neoplastic cell pop-
ulations, as reactiondiffusion 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 late-
stage 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. Oxichypoxic
cycles in tumours have been measured to occur with
periodicities of minutes38, hours39 or days40. For instance,
Gallezs 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 12
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 deliv-
ery to tumours is inconsistent. These temporal cycles are
probably due to a range of physiological mechanisms.
Relatively rapid oxicanoxic 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 regres-
sion due to hypoxia-induced expression of secreted vas-
cular 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
www.nature.com/reviews/cancer

0.16 mm
Figure 5 | Late-stage ductal carcinoma in situ. A 5m-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 m/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
MichaelisMenten 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.
NATURE REVIEWS | C ANCER
N
T
B
S degradation of the ECM and promotion of angiogenesis.
the anoxic episodes. The mechanisms underlying this
upregulation are discussed in the next section.
Although the upregulation of glycolysis is a success-
ful adaptation to hypoxia/anoxia, it also has significant
negative consequences because of increased acid pro-
duction, 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 gly-
colysis 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
MAX M
example, GLUT3) glucose transporters. Such upregu-
lation of glucose transporters has been observed dur-
ing carcinogenesis in oesophageal, gastric, breast and
colon cancers5557.
In summary, we suggest that the glycolytic pheno-
type 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
REVIEWS
oxygen. This constitutive upregulation might occur
through mutations or epigenetic changes such as alter-
ation 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 popula-
tions. Second, acidification of the microenvironment
facilitates tumour invasion both through destruction of
adjacent normal populations and through acid-induced
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 glu-
cose transporters and hexokinases are the key molecules
regulating glycolytic flux. It must be noted that a corol-
lary of the current hypothesis is that the selective advan-
tage conferred by the glycolytic phenotype is insensitive
to the exact mechanism of glycolytic induction.
A key regulator of the glycolytic response is the tran-
scription factor hypoxia-inducible factor-1 (HIF1)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 sys-
tems, constitutively increased HIF1 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 HIF1 because of a muta-
tion in the von HippelLindau (VHL) ubiquitin ligase.
(The wild-type enzyme targets HIF1 for degradation.)
Re-inserting VHL as a transgene in these cells restores
normal HIF1 levels and greatly reduces aerobic glucose
consumption rates61. Although HIF1 strongly links aer-
obic 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,
HIF1 levels can in fact be stabilized by a range of fac-
tors, including cyclooxygenase-2 activity, insulin-like
growth factor 2, ERBB2, epidermal growth factor recep-
tor, phosphatidylinositol 3-kinase, heat-shock protein 90,
microtubule status, thioredoxin and histone deacetylase,
to name a few 6365. Additionally, stabilization of HIF1
in tumours can result from hypoxia-reoxygenation
injury 66, which indicates that its constitutive upregula-
tion might result from the periodic oxichypoxic cycles
that occur in pre-malignant tumours. Consistent with
our somatic-evolution model, lack of HIF1 decreases
survival in response to hypoxia67, leading to selection of
cells with upregulated HIF1.
VOLUME 4 | NOVEMBER 2004 | 8 9 5
REVIEWS

Tumour Normal Interstitial Carcinoma Invasive Metastatic

stage epithelium neoplasia in situ carcinoma disease

Physiological Initiation Intermittant Glycolytic

HIF1 Acidosis Motility Degradation of
state hypoxia phenotype
stabilization basement
membrane and
vascularization
Process Proliferation Selection Induction Metabolism Selection

VEGF

Model
Glucose diffusion limit

O2 diffusion limit

Basement
membrane
Blood vessel
Stroma

Figure 6 | Model for cellenvironment 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). HIF1, hypoxia-inducible factor-1; VEGF, vascular endothelial growth factor.

Multiple cellular pathways might lead to the gly-
colytic 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 oxidationreduction
changes by the Sp1 transcription-factor complex68.
GLUT1 can be upregulated directly by MYC13,69 or indi-
rectly 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 sys-
tem. RAS activation of GLUT1 transcription seems to
be mediated through HIF1 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 con-
sistent 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
achieved through multiple mechanisms, including
oncogene activation or stabilization of transcription
factors such as HIF1.
Angiogenesis
We suggest that the glycolytic phenotype evolves in a
microenvironment that is avascular; that is, the evolv-
ing 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 pro-
liferating tumour epithelium. Therefore, even though
late-stage carcinoma in situ can be characterized as
angiogenic, the tumour does not become vascular-
ized 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

896 | NOVEMBER 2004 | VOLUME 4 www.nature.com/reviews/cancer


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.
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
Radioresistance
Drug resistance
Metastasis and invasion
Increased mutation rate
Gene expression induced by hypoxia-
inducible factor
Apoptosis
NATURE REVIEWS | C ANCER
in REF. 79). This will not necessarily result in increased
substrate delivery through diffusion, as substrate con-
centrations in the reactiondiffusion 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 hyperpro-
liferating blood vessels. Once the basement mem-
brane 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-malig-
nant 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 envi-
ronment. Indeed, numerous studies have shown that the
extracellular pH of human and animal tumours is consis-
tently acidic and can reach pH values approaching 6.0
(REFS 80,81) (FIG. 4). We have demonstrated both mathe-
matically and empirically that intratumoral acidosis
results in diffusion of H+ ions along concentration gradi-
ents 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 con-
tinue 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 spon-
taneous transformation of normal diploid fibroblasts83.
The resulting phenotypic diversity enhances the evolu-
tionary potential of the tumour population, which
accelerates malignant progression and adaptation to ther-
apeutic 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
Acidosis
Increased radioresistance
Resistance to anthracyclines
Increased metastases
Increased migration and invasion
Mutagenesis/clastogenesis
Apoptosis
REVIEWS
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 gly-
colysis and resistance to extracellular acidosis in adap-
tation 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 gly-
colysis, 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 expres-
sion 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, lodg-
ing 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 advan-
tage 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 inter-
pretations 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 can-
cers is the result of adaptation to consistent environmen-
tal pressures in pre-malignant lesions, when diffusion
limitations result in gradients of hypoxia and acidosis.
Cellular traits selected by these conditions include con-
stitutive upregulation of glycolysis and resistance to acid-
induced 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 prolif-
eration. 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 glu-
cose uptake is observed to coincide with the transition
from pre-malignant lesions to invasive cancer94,95. These
evolutionary advantages explain the remarkable preva-
lence of the glycolytic phenotype in human cancers and
the otherwise puzzling observation that malignant cells
remain glycolytic even in the presence of normoxia.
VOLUME 4 | NOVEMBER 2004 | 8 9 7
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 con-
trols and adaptation to acidosis in cancer progression.
Despite gaps in our knowledge, the glycolytic pheno-
type 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
relationship and timing between the angiogenic
switch and the glycolytic switch? Can pharmacologi-
cal agents be developed to inhibit emergence of the
glycolytic/acidic phenotype and, therefore, retard the
progression in early lesions? Would alteration in sys-
temic pH perturb tumour growth dynamics and con-
fer relative resistance to tumour development? Finally,
would local or systemic alteration of buffering capac-
ity and balance of extracellular tumour pH reverse the
aggressive tumour phenotype in the absence of any
other change? On this last point, we have tantalizing
evidence that mild renal failure which is typically
accompanied by systemic acidosis is associated
with improved prognosis in patients with metastatic
renal cancer following nephrectomy 96.

1. Bernards, R. & Weinberg, R. A. A progression puzzle.
Nature 418, 823 (2002).
A compelling opinion piece, in which the authors
convincingly argue that the molecular phenotypes of
metastatic cancers arose early during
carcinogenesis. Although somatic evolution is implied
in this work, the environmental nature of the selection
pressures are not discussed.
2. Racker, E. History of the Pasteur effect and its pathobiology.
Mol. Cell. Biochem. 5, 1723 (1974).
3. Warburg, O. Ueber den stoffwechsel der tumoren.
(Constable, London, 1930).
4. Semenza, G. L. et al. The metabolism of tumours: 70 years
later. Novartis Found. Symp. 240, 251260 (2001).
In this timely review, Semenza describes the relation
between HIF1 and the regulation of glycolysis.
5. Weinhouse, S. The Warburg hypothesis fifty years later.
Z. Krebsforsch. Klin. Onkol. Cancer Res. Clin. Oncol. 87,
115126 (1976).
6. Hawkins, R. A. & Phelps, M. E. PET in clinical oncology.
Cancer Metastasis Rev. 7, 119142 (1988).
7. Weber, W. A., Avril, N. & Schwaiger, M. Relevance of
positron emission tomography (PET) in oncology.
Strahlenther. Onkol. 175, 356373 (1999).
8. Gambhir, S. S. Molecular imaging of cancer with positron
emission tomography. Nature Rev. Cancer 2, 683693 (2002).
A well written review on FdG PET imaging.
9. Czernin, J. & Phelps, M. E. Positron emission tomography
scanning: current and future applications. Annu. Rev. Med.
53, 89112 (2002).
A comprehensive review of extant literature. The
authors convincingly document the very high
sensitivity and specificity of FdG PET in diagnosing
and staging diverse types of metastatic cancers.
10. Bos, R. et al. Biologic correlates of 18fluorodeoxyglucose
uptake in human breast cancer measured by positron
emission tomography. J.Clin.Oncol. 20, 379387 (2002).
This well-conducted study quantitatively analysed the
molecular phenotypes of tumours that had either high
or low rates of FdG trapping.
11. Burt, B. M. et al. Using positron emission tomography with
[18F]FDG to predict tumor behavior in experimental
colorectal cancer. Neoplasia (New York) 3, 189195 (2001).
12. Schilling, C. H., Schuster, S., Palsson, B. O. & Heinrich, R.
Metabolic pathway analysis: basic concepts and scientific
applications in the post-genomic era. Biotechnol. Prog. 15,
296303 (1999).
13. Dang, C. V., Lewis, B. C., Dolde, C., Dang, G. & Shim, H.
Oncogenes in tumor metabolism, tumorigenesis, and
apoptosis. J. Bioenerg. Biomembr. 29, 345354 (1997).
One of many papers in this issue of the Journal of
Bioenergetics and Biomembranes that dealt with the
molecular controls of glucose metabolism. In this
review, primary data were presented to support the
importance and molecular controls of the glucose
transporter and its regulation by MYC.
14. Rivenzon-Segal, D., Boldin-Adamsky, S., Seger, D., Seger, R.
& Degani, H. Glycolysis and glucose transporter 1 as
markers of response to hormonal therapy in breast cancer.
Int. J. Cancer 107, 177182 (2003).
One of many papers that demonstrates the important
role of the glucose transporter in regulating glycolytic
flux.
15. Artemov, D., Bhujwalla, Z. M., Pilatus, U. & Glickson, J. D.
Two-compartment model for determination of glycolytic
rates of solid tumors by in vivo 13C NMR spectroscopy. NMR
Biomed. 11, 395404 (1998).
16. Mathupala, S. P., Rempel, A. & Pedersen, P. L. Aberrant
glycolytic metabolism of cancer cells: a remarkable
coordination of genetic, transcriptional, post-translational,
and mutational events that lead to a critical role for type II
hexokinase. J. Bioenerg. Biomembr. 29, 339343 (1997).
Provides a cogent argument for the role of
hexokinase in regulating glycolytic flux and its
regulation by oncogenes and subcellular
localization.
17. Kunkel, M. et al. Overexpression of Glut-1 and increased
glucose metabolism in tumors are associated with a poor
prognosis in patients with oral squamous cell carcinoma.
Cancer 97, 10151024 (2003).
This careful study is one of many that document the
diagnostic importance of GLUT1 and glycolysis in
carcinomas.
18. Mochiki, E. et al. Evaluation of 18F-2-deoxy-2-fluoro-D-
glucose positron emission tomography for gastric cancer.
World J. Surg. 28, 247253 (2004).
19. Postovit, L. M., Adams, M. A., Lash, G. E., Heaton, J. P. &
Graham, C. H. Oxygen-mediated regulation of tumor cell
invasiveness. Involvement of a nitric oxide signaling
pathway. J. Biol. Chem. 277, 3573035737 (2002).
20. He, X. et al. Hypoxia increases heparanase-dependent
tumor cell invasion, which can be inhibited by
antiheparanase antibodies. Cancer Res. 64, 39283933
(2004).
21. Buchler, P. et al. Hypoxia-inducible factor 1 regulates
vascular endothelial growth factor expression in human
pancreatic cancer. Pancreas 26, 5664 (2003).
22. Postovit, L. M., Adams, M. A., Lash, G. E., Heaton, J. P. &
Graham, C. H. Nitric oxide-mediated regulation of hypoxia-
induced B16F10 melanoma metastasis. Int. J. Cancer 108,
4753 (2004).
23. Krtolica, A. & Ludlow, J. W. Hypoxia arrests ovarian
carcinoma cell cycle progression, but invasion is unaffected.
Cancer Res. 56, 11681173 (1996).
24. Schornack, P. A. & Gillies, R. J. Contributions of cell
metabolism and H+ diffusion to the acidic pH of tumors.
Neoplasia (New York) 5, 135145 (2003).
Determined proton production rates in breast cancer
lines with low and high metastatic capability, and
related these to glycolytic rate. These rates were used
in a reactiondiffusion model to predict steady-state
tumour pH values.
25. Griffiths, J. R., McIntyre, D. J., Howe, F. A. & Stubbs, M.
Why are cancers acidic? A carrier-mediated diffusion model
for H+ transport in the interstitial fluid. Novartis Found. Symp.
240, 4662 (2001).
26. Bhujwalla, Z. M. et al. Combined vascular and extracellular
pH imaging of solid tumors. NMR Biomed. 15, 114119
(2002).
Used spectroscopic imaging to measure the spatial
variations in tumour pH, and these were related to
vascular perfusion measures in the same tumours.
27. Krogh, A. The number and distribution of capillaries in
muscles with calculations of the oxygen pressure head
necessary for supplying the tissue. J. Physiol. 52, 409415
(1919).
Demonstrates the annulus of tissues that can be
oxygenated by a single capillary.
28. Thomlinson, R. H. & Gray, L. H. The histological structure of
some human lung cancers and the possible implications for
radiotherapy. Br. J. Cancer 9, 539549 (1955).
Documents that necrosis in tumours occurs at
distances from blood vessels and that this was
consistent with the oxygen diffusion distances.
29. Dewhirst, M. W., Secomb, T. W., Ong, E. T., Hsu, R. &
Gross, J. F. Determination of local oxygen consumption
rates in tumors. Cancer Res. 54, 33333336 (1994).
30. Helmlinger, G., Yuan, F., Dellian, M. & Jain, R. K. Interstitial
pH and pO2 gradients in solid tumors in vivo: high-resolution
measurements reveal a lack of correlation. Nature Med. 3,
177182 (1997).
Despite its title, this very well conducted study
documents the correlation between pH and
oxygenation as they decrease with distances from
feeding capillaries.
31. Graeber, T. G. et al. Hypoxia-mediated selection of cells with
diminished apoptotic potential in solid tumours. Nature 379,
8891 (1996).
Documents the somatic evolutionary pressure
mediated by hypoxia.
32. Gatenby, R. A. & Gawlinski, E. T. A reaction-diffusion model
of cancer invasion. Cancer Res. 56, 57455753 (1996).
Mathematical methods and empirical evidence were
used to demonstrate the acid-induced tumour-
invasion model for the first time.
33. Gatenby, R. A. & Vincent, T. L. An evolutionary model of
carcinogenesis. Cancer Res. 63, 62126220 (2003).
The formal mathematical development of evolutionary
game theory in carcinogenesis.
34. Fearon, E. R. & Vogelstein, B. A genetic model for colorectal
tumorigenesis. Cell 61, 759767 (1990).
Introduced the molecular genetic changes that occur
during gastrointestinal carcinogenesis and discussed
the concept of clonal outgrowth in this context. There
was no discussion of environmental selection
pressures.
35. Chresand, T. J., Gillies, R. J. & Dale, B. E. Optimum fiber
spacing in a hollow fiber bioreactor. Biotechnol. Bioeng. 32,
983992 (1988).
36. Secomb, T. W. et al. Theoretical simulation of oxygen
transport to tumors by three-dimensional networks of
microvessels. Adv. Exp. Med. Biol. 454, 629634 (1998).
37. Wykoff, C. C. et al. Expression of the hypoxia-inducible
and tumor-associated carbonic anhydrases in ductal
carcinoma in situ of the breast. Am. J. Pathol. 158,
10111019 (2001).
This work shows, with histopathology, the expression
of CA IX and CA XII in carcinoma in situ lesions. These
two carbonic anhydrases are sensitive to hypoxia and
these data are consistent with significant hypoxia in
in situ lesions.
38. Kimura, H. et al. Fluctuations in red cell flux in tumor
microvessels can lead to transient hypoxia and
reoxygenation in tumor parenchyma. Cancer Res. 56,
55225528 (1996).
39. Hill, R. P., De Jaeger, K., Jang, A. & Cairns, R. pH, hypoxia
and metastasis. Novartis Found. Symp. 240, 154165 (2001).
40. Gilead, A. & Neeman, M. Dynamic remodeling of the
vascular bed precedes tumor growth: MLS ovarian

898 | NOVEMBER 2004 | VOLUME 4 www.nature.com/reviews/cancer


carcinoma spheroids implanted in nude mice. Neoplasia
(New York) 1, 226230 (1999).
41. Baudelet, C. et al. Physiological noise in murine solid tumors
using T2*-weighted gradient echo imaging: a marker for
tumor acute hypoxia? Phys. Med. Biol. 49, 33893411
(2004).
42. Braun, R. D., Lanzen, J. L. & Dewhirst, M. W. Fourier
analysis of fluctuations of oxygen tension and blood flow in
R3230Ac tumors and muscle in rats. Am. J. Physiol. 277,
H551H568 (1999).
43. Dewhirst, M. W. et al. Microvascular studies on the origins of
perfusion-limited hypoxia. Br. J. Cancer Suppl. 27,
S247S251 (1996).
44. Sonveaux, P. et al. Endothelin-1 is a critical mediator of
myogenic tone in tumor arterioles: implications for cancer
treatment. Cancer Res. 64, 32093214 (2004).
45. Patan, S. et al. Vascular morphogenesis and remodeling in a
human tumor xenograft: blood vessel formation and growth
after ovariectomy and tumor implantation. Circ. Res. 89,
732739 (2001).
46. Kiani, M. F., Pries, A. R., Hsu, L. L., Sarelius, I. H. & Cokelet, G. R.
Fluctuations in microvascular blood flow parameters caused
by hemodynamic mechanisms. Am. J. Physiol. 266,
H1822H1828 (1994).
References 3846 document the periodic nature of
tumour oxygenation.
47. Park, H. J., Lyons, J. C., Ohtsubo, T. & Song, C. W. Acidic
environment causes apoptosis by increasing caspase
activity. Br. J. Cancer 80, 18921897 (1999).
48. Williams, A. C., Collard, T. J. & Paraskeva, C. An acidic
environment leads to p53 dependent induction of apoptosis
in human adenoma and carcinoma cell lines: implications for
clonal selection during colorectal carcinogenesis. Oncogene
18, 31993204 (1999).
49. Shrode, L. D., Tapper, H. & Grinstein, S. Role of intracellular
pH in proliferation, transformation, and apoptosis.
J. Bioenerg. Biomembr. 29, 393399 (1997).
References 4749 deal with pH-induced apoptosis.
Grinsteins review concludes that cytoplasmic
acidification is unlikely to be part of the apoptosis
paradigm, but that externally lowered pH might
promote apoptotic cell death.
50. Lee, A. H. & Tannock, I. F. Heterogeneity of intracellular pH
and of mechanisms that regulate intracellular pH in
populations of cultured cells. Cancer Res. 58, 19011908
(1998).
51. Ober, S. S. & Pardee, A. B. Intracellular pH is increased after
transformation of Chinese hamster embryo fibroblasts.
Proc. Natl Acad. Sci. USA 84, 27662770 (1987).
52. McLean, L. A., Roscoe, J., Jorgensen, N. K., Gorin, F. A. &
Cala, P. M. Malignant gliomas display altered pH regulation
by NHE1 compared with nontransformed astrocytes. Am. J.
Physiol. 278, C676C688 (2000).
53. Martinez-Zaguilan, R., Lynch, R. M., Martinez, G. M. &
Gillies, R. J. Vacuolar type proton ATPases are functionally
expressed in the plasma membranes of human tumor cells.
Am. J. Physiol. 265, c1015c1029 (1993).
References 5053 describe mechanisms of pH
regulation that are documented to be upregulated in
cancers.
54. Gottlieb, R. A., Giesing, H. A., Zhu, J. Y., Engler, R. L. &
Babior, B. M. Cell acidification in apoptosis: granulocyte
colony-stimulating factor delays programmed cell death
in neutrophils by up-regulating the vacuolar H+-ATPase.
Proc. Natl Acad. Sci. USA 92, 59655968 (1995).
Demonstrates that vacuolar H+-ATPase activity is
anti-apoptotic.
55. Younes, M., Ertan, A., Lechago, L. V., Somoano, J. &
Lechago, J. Human erythrocyte glucose transporter
(Glut1) is immunohistochemically detected as a late
event during malignant progression in Barretts
metaplasia. Cancer Epidemiol. Biomarkers Prev. 6,
303305 (1997).
56. Sakashita, M. et al. Glut1 expression in T1 and T2 stage
colorectal carcinomas: its relationship to clinicopathological
features. Eur. J. Cancer 37, 204209 (2001).
57. Grover-McKay, M., Walsh, S. A., Seftor, E. A., Thomas, P.
A. & Hendrix, M. J. Role for glucose transporter 1 protein in
human breast cancer. Pathol. Oncol. Res. 4, 115120
(1998).
58. Semenza, G. L. Hypoxia-inducible factor 1: master regulator
of O2 homeostasis. Curr. Opin. Genet. Dev. 8, 588594
(1998).
59. Yasuda, S. et al. Hexokinase II and VEGF expression in liver
tumors: correlation with hypoxia-inducible factor 1 and its
significance. J. Hepatol. 40, 117123 (2004).
NATURE REVIEWS | C ANCER
60. Carmeliet, P. et al. Role of HIF-1 in hypoxia-mediated
apoptosis, cell proliferation and tumour angiogenesis.
Nature 394, 485490 (1998).
61. Robey, I., Lien, A., Welsh, S., Baggett, B. & Gillies, R. J.
HIF-1 and the glycolytic phenotype in tumors. Neoplasia (in
the press).
62. Lu, H., Forbes, R. A. & Verma, A. Hypoxia-inducible factor 1
activation by aerobic glycolysis implicates the Warburg
effect in carcinogenesis. J. Biol. Chem. 277, 2311123115
(2002).
63. Semenza, G. Targeting HIF-1 for cancer therapy. Nature
Rev. Cancer 3, 113 (2003).
References 5863 describe the role of HIF1 in
regulating aerobic and anaerobic glycolysis.
64. Semenza, G. Signal transduction to hypoxia-inducible factor 1.
Biochem. Pharmacol. 64, 993998 (2002).
65. Welsh, S. J., Bellamy, W. T., Briehl, M. M. & Powis, G.
The redox protein thioredoxin-1 (Trx-1) increases hypoxia-
inducible factor 1 protein expression: Trx-1 overexpression
results in increased vascular endothelial growth factor
production and enhanced tumor angiogenesis. Cancer Res.
62, 50895095 (2002).
66. Moeller, B. J., Cao, Y., Li, C. Y. & Dewhirst, M. W. Radiation
activates HIF-1 to regulate vascular radiosensitivity in
tumors: role of reoxygenation, free radicals, and stress
granules. Cancer Cell 5, 429441 (2004).
67. Seagroves, T. et al. Transcription Factor HIF-1 is a necessary
mediator of the Pasteur effect in mammalian cells. Mol. Cell.
Biol. 21, 34363444 (2001).
68. Brand, K. Aerobic glycolysis by proliferating cells: protection
against oxidative stress at the expense of energy yield.
J. Bioenerg. Biomembr. 29, 355364 (1997).
69. Osthus, R. C. et al. Deregulation of glucose transporter 1
and glycolytic gene expression by c-Myc. J. Biol. Chem.
275, 2179721800 (2000).
70. Noguchi, Y. et al. Expression of facilitative glucose transporter 1
mRNA in colon cancer was not regulated by k-ras. Cancer
Letters 154, 137142 (2000).
71. Chen, C., Pore, N., Behrooz, A., Ismail-Beigi, F. & Maity, A.
Regulation of glut1 mRNA by hypoxia-inducible factor-1.
Interaction between H-ras and hypoxia. J. Biol. Chem. 276,
95199525 (2001).
72. Mathupala, S. P., Heese, C. & Pedersen, P. L. Glucose
catabolism in cancer cells. The type II hexokinase promoter
contains functionally active response elements for the
tumor suppressor p53. J. Biol. Chem. 272, 2277622780
(1997).
73. Goel, A., Mathupala, S. P. & Pedersen, P. L. Glucose
metabolism in cancer. Evidence that demethylation events
play a role in activating type II hexokinase gene expression.
J. Biol. Chem. 278, 1533315340 (2003).
74. Gillies, R. J., Martinez-Zaguilan, R., Martinez, G. M.,
Serrano, R. & Perona, R. Tumorigenic 3T3 cells maintain
an alkaline intracellular pH under physiological conditions.
Proc. Natl Acad. Sci. USA 87, 74147418 (1990).
75. Reshkin, S. J. et al. Na/H exchanger-dependent intracellular
alkalinization is an early event in malignant transformation
and play an essential role in the development of subsequent
transformation-associated phenotypes. FASEB J. 14,
21852197 (2000).
76. Li, X. et al. Relationship of MR-derived lactate, mobile lipids
and relative blood volume for in vivo gliomas. Am. J.
Neuroradiol. (in the press).
Describes the observation of increased lactate in non-
enhancing grade III gliomas, indicating that metabolic
upregulation might precede angiogenesis.
77. Nelson, S. J. Multivoxel magnetic resonance
spectroscopy of brain tumors. Mol. Cancer Ther. 2,
497507 (2003).
78. Dafni, H., Landstrom, L., Schechter, B., Kohen, F. &
Neeman, M. MRI and fluorescence microscopy of the acute
vascular response to VEGF165: vasodilation, hyper-
permeability and lymphatic uptake, followed by rapid
inactivation of the growth factor. NMR Biomed. 15, 120131
(2002).
79. Hanahan, D. & Folkman, J. Patterns and emerging
mechanisms of the angiogenic switch during tumorigenesis.
Cell 86, 353364 (1996).
80. Raghunand, N., Gatenby, R. A. & Gillies, R. J.
Microenvironmental and cellular consequences of altered
blood flow in tumors. Br. J. Radiol. 77, S11S22 (2004).
81. Gillies, R. J., Raghunand, N., Karczmar, G. & Bhujwalla, Z.
MR Imaging of the tumor microenvironment. J. Magn.
Reson. Imaging 16, 430450 (2002).
A comprehensive review describing MRI of clinical
and experimental tumours.
REVIEWS
82. Morita, T., Nagaki, T., Fukuda, I. & Okumura, K.
Clastogenicity of low pH to various cultured mammalian
cells. Mutat. Res. 268, 297305 (1992).
83. Ruch, R. J., Klaunig, J. E., Kerckaert, G. A. & LeBoeuf, R.
A. Modification of gap junctional intercellular communication
by changes in extracellular pH in syrian hamster embryo
cells. Carcinogenesis 11, 909913 (1990).
84. Martinez-Zaguilan, R. et al. Acidic pH enhances the invasive
behavior of human melanoma cells. Clin. Exp. Metastasis
14, 176186 (1996).
85. Schlappack, O. K., Zimmermann, A. & Hill, R. P. Glucose
starvation and acidosis: effect on experimental metastasic
potential, DNA content and MTX resistance of murine
tumour cells. Br. J. Cancer 64, 663670 (1991).
86. Rozhin, J., Sameni, M., Ziegler, G. & Sloane, B. F. Pericellular
pH affects distribution and secretion of cathepsin B in
malignant cells. Cancer Res. 54, 65176525 (1994).
87. Montcourrier, P., Silver, I., Farnoud, R., Bird, I. & Rochefort, H.
Breast cancer cells have a high capacity to acidify
extracellular milieu by a dual mechanism. Clin. Exp.
Metastasis 15, 382392 (1997).
88. Brizel, D. M. et al. Elevated tumor lactate concentrations
predict for an increased risk of metastases in head-and-neck
cancer. Int. J. Radiat. Oncol. Biol. Phys. 51, 349353 (2001).
89. Walenta, S. et al. High lactate levels predict likelihood of
metastases, tumor recurrence, and restricted patient survival
in human cervical cancers. Cancer Res. 60, 916921 (2000).
90. Ito, S. et al. Coexpression of glucose transporter 1 and
matrix metalloproteinase-2 in human cancers. J. Natl
Cancer Instit. 94, 10801091 (2002).
91. Al Mehdi, A. B. et al. Intravascular origin of metastasis from
the proliferation of endothelium-attached tumor cells: a new
model for metastasis. Nature Med. 6, 100102 (2000).
A watershed paper describing the
microenvironmental behaviour of lung metastases
using a novel microscopy method. This paper
challenges the paradigm that extravasation is a
necessary component of the metastasis programme.
92. Wong, C. W. et al. Intravascular location of breast cancer
cells after spontaneous metastasis to the lung. Am. J.
Pathol. 161, 749753 (2002).
93. Rofstad, E. K. & Danielsen, T. Hypoxia-induced metastasis
of human melanoma cells: involvement of vascular
endothelial growth factor-mediated angiogenesis. Br. J.
Cancer 80, 16971707 (1999).
Provides clear evidence that pretreatment with acute
hypoxia can increase the efficiency of metastasis.
94. Younes, M., Lechago, L. V. & Lechago, J. Overexpression of
the human erythrocyte glucose transporter occurs as a late
event in human colorectal carcinogenesis and is associated
with an increased incidence of lymph node metastases.
Clin. Cancer Res. 2, 11511154 (1996).
95. Yasuda, S. et al. 18F-FDG PET detection of colonic
adenomas. J. Nucl. Med. 42, 989992 (2001).
96. Gatenby, R. A., Gawlinski, E. T., Tangen, C. M., Flanigan, R. C.
& Crawford, E. D. The possible role of postoperative
azotemia in enhanced survival of patients with metastatic
renal cancer after cytoreductive nephrectomy. Cancer Res.
62, 52185222 (2002).
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 | HIF1 | KRAS | MYC | p53 |
VEGF | VHL
FURTHER INFORMATION
DMetrix digital imaging program: www.dmetrix.com
Access to this interactive links box is free online.
VOLUME 4 | NOVEMBER 2004 | 8 9 9