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WHY DO HAVE HIGH AEROBIC ?

Robert A. Gatenby* and Robert J. Gillies ‡ Abstract | If occurs by somatic evolution, then common components of the 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 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 in pre-malignant lesions. However, upregulation of glycolysis leads to microenvironmental acidosis requiring evolution to phenotypes resistant to acid-induced 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 reported by Warburg in the 1920s3, leading him to the as occuring by somatic evolution, because it seems for- hypothesis that cancer results from impaired mitochon- mally analogous to Darwinian processes, wherein pheno- drial metabolism. Although the ‘Warburg hypothesis’ typic properties are retained or lost depending on their has proven incorrect, the experimental observations of contribution to individual fitness. According to this increased glycolysis in tumours even in the presence of model, traits that are found in invasive cancers must arise have been repeatedly verified4. as adaptive mechanisms to environmental proliferative Following Warburg’s initial observation, interest in constraints during carcinogenesis1.Conversely, the com- the metabolic property of cancers has varied over time. mon appearance of a phenotypic property in cancer pop- Intense investigation in the 1960s was followed by a ulations is presumptive evidence that it must confer a steep decline concomitant with the widespread applica- selective growth advantage. tion of newer molecular techniques. The atmosphere of A curious, but common, property of invasive cancers the day was summarized by Sidney Weinhouse, who is altered glucose metabolism. Glycolysis — literally lysis said “Since our perspectives have broadened over the of glucose — first requires the conversion of glucose to years, the burning issues of glycolysis and respiration in pyruvate (FIG. 1) and then to the waste product lactic cancer now flicker only dimly”5. acid. In most mammalian cells, glycolysis is inhibited by However, interest in tumour metabolism has been *Departments of Radiology the presence of oxygen, which allows mitochondria to rekindled, mainly because of the widespread clinical and Applied Mathematics, oxidize pyruvate to CO2 and H2O. This inhibition is application of the imaging technique positron- University of Arizona, termed the ‘Pasteur effect’,after Louis Pasteur, who first emission tomography (PET) using the glucose ana- Tucson, Arizona 85721, USA. 18 6–8 ‡ demonstrated that glucose flux was reduced by the logue tracer fluorodeoxyglucose (FdG) . FdG PET Departments of Radiology 2 and Biochemistry and presence of oxygen .This metabolic versatility of mam- imaging of thousands of oncology patients has Molecular Biophysics, malian cells is essential for maintenance of unequivocally shown that most primary and metasta- University of Arizona, production throughout a range of oxygen concentra- tic human cancers show significantly increased glucose Tucson, Arizona 85721, USA. tions. Conversion of glucose to in the pres- uptake (FIG. 2).For many cancers, the specificity and Correspondence to R.A.G. e-mail: rgatenby@ ence of oxygen is known as aerobic glycolysis or the sensitivity of FdG PET to identify primary and 9 radiology.arizona.edu ‘Warburg effect’.Increased aerobic glycolysis is uniquely metastatic lesions is near 90% .Sensitivity is lowered doi:10.1038/nrc1478 observed in cancers. This phenomenon was first because FdG PET has difficulty resolving lesions less

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HEXOKINASES Summary Enzymes that catalyse the transfer of phosphate from ATP •Widespread clinical use of 18fluorodeoxyglucose positron-emission tomography has demonstrated that the glycolytic to glucose to form glucose-6- phenotype is observed in most human cancers. phosphate. This is the first reaction in the metabolism of • The concept of carcinogenesis as a process that occurs by somatic evolution clearly implies that common traits of the glucose and prevents efflux of malignant phenotype, such as upregulation of glycolysis, are the result of active selection processes and must confer a glucose from the cell. significant, identifiable growth advantage. •Constitutive upregulation of glycolysis is likely to be an adaptation to hypoxia that develops as pre-malignant lesions HYPOXIA Refers to a low oxygen level. This grow progressively further from their blood supply. At this stage, the blood supply remains physically separated from means different levels to the growing cells by an intact basement membrane. different investigators, but for •Increased acid production from upregulation of glycolysis results in microenvironmental acidosis and requires further radiation biologists hypoxia adaptation through somatic evolution to phenotypes resistant to acid-induced toxicity. occurs at levels less than 0.1% oxygen in the gas phase. •Cell populations that emerge from this evolutionary sequence have a powerful growth advantage, as they alter their Normoxia refers to normal levels environment through increased glycolysis in a way that is toxic to other phenotypes, but harmless to themselves. The of oxygen (>10%) and anoxia environmental acidosis also facilitates invasion through destruction of adjacent normal populations, degradation of refers to no oxygen. 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 steps14–16. 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 uptake17,18.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 often19–22,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- HEXOKINASES I and II10,11 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 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 Glucose Lactate siveness indicate that the glycolytic phenotype confers 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+ Sodium–hydrogen 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 per glucose, whereas complete oxidation produces 38 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, 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

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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 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 Figure 2 | Positron-emission tomography imaging with 18 fluorodeoxyglucose of a patient with lymphoma. The sense. That is, although a pre-malignant lesion such as a mediastinal nodes (purple arrow) and supraclavicular nodes polyp or carcinoma in situ might have a vascular stroma, (green arrows) show high uptake of 18fluorodeoxyglucose the hyperplastic epithelia are physically separated from (FdG), showing that tumours in these nodes have high levels their blood supply by a basement membrane. This is of FdG uptake. The bladder (yellow arrow) also has high illustrated in FIG. 5, as the hyperplastic epithelium of a activity, because of excretion of the radionuclide. 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- 60 cinogenesis and development of the malignant pheno- Normoxia type actually occur in an avascular environment. As a result, substrates, such as oxygen and glucose, must dif- 50 Hypoxia fuse from the vessels across the basement membrane and

through layers of tumour cells, where they are metabo- )

–1 40 lized. This process of diffusion and consumption was modelled by Krogh as early as 1919 through reaction–diffusion equations that showed that oxygen 30 mg protein

concentrations decreased with distance from a capillary –1 such that oxygenated cells were limited to a distance of 20 W less than 150 µm from a blood vessel27.In the 1950s, (nmol min empirical studies by Thomlinson and Gray showed that Glucose consumption rate P viable tumour cells were not observed at distances 10 greater than 160 µm from blood vessels, consistent with Krogh’s calculations28.Subsequent experimental studies 0 in WINDOW CHAMBERS in animal models have demonstrated MCF-7 MDA-MB-231 that near-zero partial pressures of oxygen (pO2) are Figure 3 | Pasteur and Warburg effects in non-invasive 29,30 observed at distances of only 100 µm from a vessel . and metastatic breast cancer cell lines. In both cell lines, Therefore, pre-malignant lesions, provided their glucose consumption is reduced in the presence of oxygen basement membranes remain intact, will inevitably — the Pasteur effect (P). However, the more aggressive cell line, MDA-MB-231, has much higher glucose consumption WINDOW CHAMBER develop hypoxic regions near the oxygen diffusion limit, A metal chamber with a glass as persistent proliferation leads to a thickening of the in the presence of oxygen than the MCF-7 cells with a non-invasive phenotype — the Warburg effect (W). This is window that is placed on the epithelial layer, pushing cells ever more distant from dorsal skin of an animal. This consistent with positron-emission tomography scans with their blood supply, which remains on the other side of 18 allows in vivo tumour growth to fluorodeoxyglucose, which show that higher glucose be continuously observed the basement membrane. At this penumbral layer, uptake correlates with more aggressive phenotypes and microscopically. microenvironmental selection forces will favour poorer clinical outcomes.

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33 a 7.4 14 and/or growth factors .For example, in many tissues, pre-malignant lesions are initiated by in 12 pH HRAS or KRAS genes, which alter cellular responses to 7.2 pO 2 10 growth factors34.From this, it follows that mutations 8 affecting substrate use cannot be early events in carcino- 7.0 pH

mm Hg genesis because they would not confer a selective growth 6 2 advantage in an environment in which proliferation is 4 pO 6.8 not limited by substrate availability. 2 The evolutionary models show, however, that clonal expansion of pre-malignant tumour populations is 6.6 0 33 0 100 200 300 400 eventually limited by substrate availability ,as cell prolif- Distance (mm) eration, unconstrained by normal tissue interactions, carries the population increasingly far from its blood 7.0 b supply (see above). In FIG. 5, note the distances between 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 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

Extracellular pH 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 MDA-MB-435 enzymes such as carbonic anhydrases IX and XII in late- 6.4 stage ductal carcinoma in situ, particularly adjacent to areas of necrosis37.We suggest that hypoxia in the Figure 4 | Hyperacidity of tumours. These figures illustrate the micro- and macro-heterogeneity of pH. a | Tumour interstitial pH penumbral region of pre-malignant tumours produces an adaptive landscape that favours a switch to anaerobic and partial pressure of oxygen (pO2) are shown with distance from a vessel wall. These were measured in vivo in MCF-7 breast metabolism, which allows maintenance of metabolic cancer cells using fluorescent ratio imaging. b | The extracellular activities in the absence of oxygen. pH of a MDA-MB-435 breast tumour in mice was imaged with A key factor in this adaptive landscape seems to be 1 the pH indicator IEPA and measured by H magnetic- the exposure of cells near the oxygen diffusion limit to resonance spectroscopy. Part a reproduced with permission from REF.30 © (1997) Nature Publishing Group. Part b an unstable environment due to fluctuations in the reproduced with permission from REF.26 © (2002) Wiley. haemodynamics of distant blood vessels. Oxic–hypoxic cycles in tumours have been measured to occur with periodicities of minutes38, hours39 or days40.For instance, phenotypes that adapt to harsh environments (through Gallez’s group has recently imaged tumour xenografts resistance to hypoxia and acid-induced cell toxicity) using a magnetic-resonance imaging (MRI) technique and successfully compete for scarce resources, such as that is sensitive to oxygenation status41.Analyses showed oxygen and glucose31,32. that fluctuations in signal intensity (oxygenation) occurred with discrete periodicities of 1 and 20 cycles Emergence of the glycolytic phenotype per hour. By contrast, Dewhirst and colleagues used Evolutionary game theory is a mathematical approach microelectrodes to show periodicities of about 1–2 that analyses strategy dynamics in adaptation to environ- cycles per minute42.However, it should be noted that

HAEMATOCRIT mental growth — winners in this game proliferate, MRI, although imaging the whole tumour, is insensitive A measure of the concentration whereas losers become extinct. Recently, this method has to rapid fluctuations, and microelectrode instabilities of red cells in the blood. A been applied to somatic evolution of the malignant render these electrodes insensitive to slower changes. reduced haematocrit decreases phenotype33.This analysis showed that proliferation of Nonetheless, all of these studies show that oxygen deliv- the oxygen-carrying capacity of normal cells is controlled by their interactions with other ery to tumours is inconsistent. These temporal cycles are the blood. cells and the extracellular matrix (ECM), and by the lev- probably due to a range of physiological mechanisms. VASOMOTION els of growth factors. Importantly, cell proliferation and Relatively rapid oxic–anoxic cycles can occur because Rhythmic oscillations in survival in normal tissue is not constrained by substrate of fluctuations in HAEMATOCRIT43 and VASOMOTION44. vascular tone caused by local availability, except under pathological conditions such as Variations occurring over days probably involve VASCULAR changes in smooth muscle. acute vascular occlusion (for example, caused by strokes REMODELLING45,46 or cycles of neoangiogenesis and regres- VASCULAR REMODELLING and myocardial infarcts) or chronic occlusion (as seen in sion due to hypoxia-induced expression of secreted vas- The active process of altering diabetic ulcers). It follows, therefore, that the earliest cular endothelial growth factor (VEGF), which is an structure and arrangement in steps in carcinogenesis require alterations in cellular sen- induction and survival factor for new blood vessels40. blood vessels through cell sitivity to these normal tissue constraints. So, prolifera- From a bioenergetic standpoint, periodic hypoxia will growth, cell death, cell migration and production or degradation tion will follow genetic alterations that reduce sensitivity select for cells in which anaerobic glucose metabolism is of the extracellular matrix. to growth constraints generated by other cells, the ECM constitutively upregulated, as they would better survive

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oxygen. This constitutive upregulation might occur

0.16 mm 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 N 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- T tions. Second, acidification of the microenvironment B facilitates tumour invasion both through destruction of adjacent normal populations and through acid-induced S degradation of the ECM and promotion of angiogenesis. The underlying molecular, cellular and environmental Figure 5 | Late-stage ductal carcinoma in situ. A 5µm-thick biopsy sample was stained with dynamics are discussed next. 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 Molecular mechanisms 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). 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 the anoxic episodes. The mechanisms underlying this regulating glycolytic flux. It must be noted that a corol- upregulation are discussed in the next section. lary of the current hypothesis is that the selective advan- Although the upregulation of glycolysis is a success- tage conferred by the glycolytic phenotype is insensitive ful adaptation to hypoxia/anoxia, it also has significant to the exact mechanism of glycolytic induction. negative consequences because of increased acid pro- A key regulator of the glycolytic response is the tran- duction, which causes significant decreases in local scription factor hypoxia-inducible factor-1α (HIF1α)58. extracellular pH. Prolonged exposure of normal cells to This factor mediates a pleiotropic response to hypoxic an acidic microenvironment typically results in necrosis stress by inducing survival genes, including glucose or through p53- and caspase-3-dependent transporters; angiogenic growth factors (for example, mechanisms47,48.The physiological trigger for apoptosis VEGF); hexokinase II59; and haematopoeitic factors (for might be collapse of the transmembrane H+ gradient example, transferrin and erythropoietin)60.In some sys- that occurs with intracellular acidosis, but other factors tems, constitutively increased HIF1α levels are associated might have a role49.So, constitutive upregulation of gly- with constitutively high glucose consumption rates. This colysis requires additional adaptation to the negative is the case in the renal-cell carcinoma cell line RCC4, effects of extracellular acidosis through resistance to which has constitutively high HIF1α because of a muta- apoptosis or upregulation of membrane transporters tion in the von Hippel–Lindau (VHL) ubiquitin ligase. to maintain normal intracellular pH. Intracellular pH (The wild-type enzyme targets HIF1α for degradation.) is maintained by multiple families of H+ transporters, Re-inserting VHL as a transgene in these cells restores which are co-expressed and redundant50,51.Na+–H+ normal HIF1α levels and greatly reduces aerobic glucose exchange51,52 and vacuolar H+-ATPases53 have both been consumption rates61.Although HIF1α strongly links aer- observed to be upregulated in cancers, and vacuolar obic glycolysis to carcinogenesis62, it would be premature H+-ATPase might confer resistance to apoptosis54. to conclude that the glycolytic phenotype in cancer is Additional adaptations might also be required as invariably due to dysregulation of the HIF system. the increased glucose consumption rates further Although it is termed the hypoxia-inducible factor, decrease glucose concentrations. Cellular competition HIF1α levels can in fact be stabilized by a range of fac- for this increasingly limited resource will therefore tors, including cyclooxygenase-2 activity, insulin-like increase and favour phenotypes with greater numbers growth factor 2, ERBB2, epidermal growth factor recep- VMAX and KM

Terms from the of either high VMAX (for example, GLUT1) or low KM (for tor, phosphatidylinositol 3-kinase, heat-shock protein 90, Michaelis–Menten model. example, GLUT3) glucose transporters. Such upregu- microtubule status, thioredoxin and histone deacetylase, Applied to transport,V is the max lation of glucose transporters has been observed dur- to name a few 63–65.Additionally, stabilization of HIF1α maximum possible rate of uptake of a specific substrate. K ing carcinogenesis in oesophageal, gastric, breast and in tumours can result from hypoxia-reoxygenation m 55–57 66 is the substrate concentration at colon cancers . injury ,which indicates that its constitutive upregula- which the substrate uptake is In summary, we suggest that the glycolytic pheno- tion might result from the periodic oxic–hypoxic cycles half of V .Cell populations max type initially arises as an adaptation to local hypoxia that occur in pre-malignant tumours. Consistent with with low K are better adapted m (FIG. 6).Persistent or cyclical hypoxia subsequently our somatic-evolution model, lack of HIF1 decreases to maintaining substrate uptake α 67 in conditions in which substrate exerts selection pressures that lead to constitutive survival in response to hypoxia , leading to selection of concentrations are low. upregulation of glycolysis, even in the presence of cells with upregulated HIF1α.

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Tumour Normal Interstitial Carcinoma Invasive Metastatic stage epithelium neoplasia in situ carcinoma disease

Physiological Initiation Intermittant HIF1 Glycolytic Acidosis Motility Degradation of state α hypoxia stabilization phenotype 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 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 and darker oranges for more mutations). HIF1α, hypoxia-inducible factor-1α; VEGF, vascular endothelial growth factor.

Multiple cellular pathways might lead to the gly- achieved through multiple mechanisms, including colytic phenotype, so that altered glucose metabolism oncogene activation or stabilization of transcription might even result in cells with normal HIF levels. For factors such as HIF1α. example, upregulation of glycolytic enzymes can be coordinated in response to oxidation–reduction Angiogenesis changes by the Sp1 transcription-factor complex68. We suggest that the glycolytic phenotype evolves in a GLUT1 can be upregulated directly by MYC13,69 or indi- microenvironment that is avascular; that is, the evolv- rectly by KRAS70.Interestingly, in this latter study, ing tumour cells remain physically separated from KRAS activation was only associated with a subset of their blood supply by a basement membrane, as GLUT1-positive colon cancers, indicating that it is one occurs in in situ tumours. This invokes the diffusion of several mechanisms to activate glycolysis in this sys- of substrates from the vascularized stroma to the pro- tem. RAS activation of GLUT1 transcription seems to liferating tumour epithelium. Therefore, even though be mediated through HIF1α transactivation71. late-stage carcinoma in situ can be characterized as Hexokinase II can be transcriptionally activated by ‘angiogenic’,the tumour does not become vascular- mutant p53 (REF. 72) or through demethylation of its ized until the basement membrane is breached by an promoter73.It is also intriguing to note that transfection invasive cell. In fact, there is emerging evidence that of fibroblasts with H+-ATPase or Na+–H+ exchange the ‘glycolytic switch’ occurs before the ‘angiogenic raises the intracellular pH, makes them tumorigenic switch’; lactic acid has been observed in regions of and leads to marked increases in glycolysis74,75.These invasive gliomas76,77 that lack vessel permeability, as alternative systems for upregulating glycolysis are con- shown by the absence of contrast enhancement with sistent with our basic proposal that the mechanism of MRI78.We do not wish to indicate that angiogenesis induction is not as important as the induction itself. does not have a role in this process. In fact, it is likely That is, the glycolytic phenotype is not a secondary that angiogenic factors, such as VEGF, are produced phenomenon that results from induction of some by the tumour and that this will promote increased other pathway during carcinogenesis. Rather, it is vascularity within the stroma (FIG. 6).However, these directly selected because it provides a growth advantage new vessels remain physically separated from the and acquisition of the glycolytic phenotype might be tumour cells by the basement membrane (see figure 2

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CLASTOGENIC in REF. 79). This will not necessarily result in increased are not known, but might involve the metalloproteinases Describing any substance or substrate delivery through diffusion, as substrate con- and/or cathepsins, which promote the degradation of the processes that increases centrations in the reaction–diffusion equation are ECM and basement membranes86,87. alterations in the structure of unaltered. So, a hallmark of these early cancers is a chromosomes. failure of angiogenesis to relieve hypoxia because of Metastasis GAP JUNCTIONS the physical separation between vessels and the cells So far, we have focused on the role of upregulated gly- Linked channels through they feed. This can result in a futile cycle of hyperpro- colysis and resistance to extracellular acidosis in adap- contiguous cell membranes that liferating blood vessels. Once the basement mem- tation to conditions in early pre-malignant lesions and interconnect the cytoplasm of adjacent cells and allow direct brane is breached, the tumour will become vascular in the evolution of invasive primary cancers. However, exchange of ions and small both by co-opting the pre-existing vessels within the we note that this phenotype might also be crucial in molecules. stroma and by promoting new vessel growth directly the maturation of metastases as well. Upregulated gly- into the tumour mass. colysis, evidenced by increased intratumoral lactate concentrations, is associated with increased incidence Acidosis and invasion of metastasis in cervical and head and neck cancers88,89. Although the glycolytic phenotype seems to be the result Furthermore, a correlation between GLUT1 expres- of adaptation to environmental constraints in pre-malig- sion levels and metalloproteinase expression has also nant lesions, its persistence in primary and metastatic been reported in metastatic cancers90. cancers even in conditions of normoxia indicates that it During the process of metastasis, migratory cells continues to provide a strong selective growth advantage invade the stromal tissue and move to distant sites, lodg- following malignant progression. We suggest, in fact, that ing in pre-capillary arterioles and capillaries91,92.These acquisition of the glycolytic phenotype is required for cells probably also experience periodic hypoxic or anoxic invasive tumour growth. episodes as they proliferate and occlude the intravascular A constitutive and persistent increase in glycolysis space. Therefore, the end stage of the metastasis sequence results in acute and chronic acidification of the local envi- will also favour cells that are glycolytic and resistant to ronment. Indeed, numerous studies have shown that the hypoxia- or acid-induced apoptosis. Any selective advan- extracellular pH of human and animal tumours is consis- tage is important, as the success rate of metastasis is low. tently acidic and can reach pH values approaching 6.0 For example, in a typical lung-colonization assay, as many (REFS 80,81) (FIG. 4).We have demonstrated both mathe- as 105 lung cancer cells are injected into mouse tail veins, matically and empirically that intratumoral acidosis but fewer than 100 cells generally survive to form results in diffusion of H+ ions along concentration gradi- colonies. Cells pre-treated with hypoxia for 24 hours are ents into peritumoral normal tissue32.Normal cells, four times more likely to survive than their normoxic which lack a mechanism to adapt to extracellular acidosis counterparts93.Although there are other possible inter- (such as a p53 mutation) are unable to survive under pretations of these data, we suggest that they support the such conditions, whereas the tumour populations con- hypothesis that the glycolytic phenotype contributes to tinue to proliferate. In addition, acidosis itself can be the efficiency of metastasis by allowing cells to survive mutagenic and CLASTOGENIC82,possibly through inhibition transient hypoxia. of DNA repair (for a review, see REF.80) and can lead to both inhibition of GAP-JUNCTION conductance and to spon- Summary and future directions taneous transformation of normal diploid fibroblasts83. In summary, we suggest that upregulation of glycolytic The resulting phenotypic diversity enhances the evolu- metabolic pathways in the vast majority of invasive can- tionary potential of the tumour population, which cers is the result of adaptation to consistent environmen- accelerates malignant progression and adaptation to ther- tal pressures in pre-malignant lesions, when diffusion apeutic strategies34 (BOX 1). Finally, under some (but not limitations result in gradients of hypoxia and acidosis. all) conditions, low pH stimulates in vitro invasion84 and Cellular traits selected by these conditions include con- in vivo metastasis85.The mechanisms of such induction stitutive upregulation of glycolysis and resistance to acid- induced apoptosis. Mathematical models and empirical observation indicate that the advantages conferred by Box 1 | Consequences of hypoxia and acidosis this combination of phenotypic traits are both sufficient As tumours evolve and become first hypoxic and then acidic, malignant progression is and necessary to promote unconstrained tumour prolif- accelerated and resistance to therapeutic strategies occurs. For further information, eration. Furthermore, both mathematical models and see REF. 36. empirical evidence indicate that diffusion of acid from the tumour into peritumoral normal tissue provides a Hypoxia Acidosis specific mechanism promoting tumour invasion32. Radioresistance Increased radioresistance The crucial importance of the glycolytic phenotype is Drug resistance Resistance to anthracyclines emphasized by studies demonstrating that increased glu- Metastasis and invasion Increased metastases cose uptake is observed to coincide with the transition from pre-malignant lesions to invasive cancer94,95.These Increased mutation rate Increased migration and invasion evolutionary advantages explain the remarkable preva- Gene expression induced by hypoxia- Mutagenesis/clastogenesis lence of the glycolytic phenotype in human cancers and inducible factor the otherwise puzzling observation that malignant cells Apoptosis Apoptosis remain glycolytic even in the presence of normoxia.

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The molecular basis for evolution of the glycolytic relationship and timing between the ‘angiogenic phenotype has been clarified by recent advances in switch’ and the ‘glycolytic switch’? Can pharmacologi- understanding the HIF system, but much additional cal agents be developed to inhibit emergence of the work will be required to fully understand the complex glycolytic/acidic phenotype and, therefore, retard the pathways involved in hypoxic response, metabolic con- progression in early lesions? Would alteration in sys- trols and adaptation to acidosis in cancer progression. temic pH perturb tumour growth dynamics and con- Despite gaps in our knowledge, the glycolytic pheno- fer relative resistance to tumour development? Finally, type could be exploited for treatment at several levels. would local or systemic alteration of buffering capac- As this phenotype emerges early in carcinogenesis, it ity and balance of extracellular tumour pH reverse the might represent a possible target in cancer prevention. aggressive tumour phenotype in the absence of any At later stages, a more complete understanding of the other change? On this last point, we have tantalizing molecular and physiological consequences might lead evidence that mild renal failure — which is typically to targeted therapies. accompanied by systemic acidosis — is associated Finally, this model of carcinogenesis indicates new with improved prognosis in patients with metastatic avenues of investigation. For example, what is the renal cancer following nephrectomy 96.

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