almost a century ago , otto warburg made a very significant observation that would start a long - lasting , heated discussion .
he observed that cancer cells , unlike many other cells in the body , opt for glycolysis rather than mitochondrial respiration , even in the presence of oxygen ( referred to here as the warburg effect ) .
warburg proposed that the aerobic glycolysis phenotype that he observed stemmed from the fact that cancer cell mitochondria are irreversibly dysfunctional .
he believed , in fact , that dysfunctional mitochondria are required and necessary to start all the biochemical events that eventually result in transformation to the cancerous state .
his findings went hand - in - hand with pasteur 's postulations . in 1861 , pasteur reported that yeast cells upregulate glycolysis under hypoxic conditions .
given that the inner regions of solid tumors are hypoxic because of anomalous vascularization , pasteur 's effect seemed to explain warburg 's observation .
however , the biochemist weinhouse was not convinced by warburg 's explanation of cancer initiation by damaged mitochondria . as a pioneer of isotope tracer usage in biochemistry
, he found that cancer cells are able to oxidize glucose and fatty acids to carbon dioxide at levels comparable to those of normal cells .
he argued that the reverse was true : cancer cells have reduced mitochondrial activity as a consequence of heightened glycolytic flux , which is known to inhibit mitochondria  the so - called crabtree effect . to this day
, the field has not been able to reach a conclusive decision on this matter . to explore the relationship between these two views
, we use the chicken - and - egg analogy : it is difficult to determine whether mitochondrial dysfunction emerges first , thereby forcing cells to rely on glycolysis , or whether the reverse occurs , whereby increased glycolytic flux takes place first , which in turn suppresses mitochondrial respiration .
, we will discuss the two different points of view in relation to glycolysis , pyruvate metabolism , and the krebs cycle .
as warburg noticed , cancer cells have elevated levels of glucose uptake compared with non - cancer cells .
these findings have been confirmed by using recent technological developments that allow non - invasive monitoring of glucose uptake in vivo , called 2-fluoro-6-deoxyglucose positron emission tomography ( fdg - pet ) . in this technique ,
a traceable glucose analog is used that is recognized and taken up by glucose transporters ( gluts ) but can not be used for downstream glycolytic reactions .
hence , the glucose analog accumulates in cells and can be visualized and quantified by pet .
, glucose uptake has been shown to increase in certain tumors , and higher fdg uptake has been correlated with poor cancer prognosis [ 911 ] .
multiple observations outlined below , however , suggest that glycolytic flux in cancer cells is upregulated as a result of multiple changes in signaling pathways and not necessarily as a result of impaired mitochondrial function .
glycolysis begins with cellular uptake of glucose via gluts on the cell surface ( figure 1 ) . in 1974
, hatanaka proposed that cells upregulate gluts in order to meet increased glucose demand upon transformation .
later studies showed that , indeed , levels of gluts , especially the high - affinity gluts 1 and 3 , were upregulated in a plethora of tumor types ( nicely reviewed in ) .
moreover , glut1 transcription is upregulated in response to hypoxia   and inhibition of mitochondrial respiration , both conditions in which cells need to divert the metabolic flux from mitochondrial respiration to glycolysis . furthermore , in tumors with high insulin signaling , glut4 is enriched at the cell membrane as a consequence of elevated pi3k / akt signaling ( reviewed in ) and glut1 transcription is upregulated via the serine / threonine kinase akt .
the human genome actually has three families of gluts , namely slc2a , slc5a , and slc50a , with a total of 27 members .
these findings could suggest that upregulation of glucose uptake and hence glycolytic flux is a primary alteration in cancer and not a consequence of impaired mitochondrial function .
that said , activity of gluts is also strongly driven by activation of amp - activated protein kinase ( ampk ) , and ampk can be activated by an adenosine triphosphate ( atp ) decrease caused by mitochondrial dysfunction . therefore , increased glucose uptake could also result from mitochondrial dysfunction .
this nicely illustrates the fact that glycolytic flux and mitochondrial function are so intertwined that it is difficult to determine what is cause and what is consequence .
dhap , dihydroxyacetone phosphate ; f-1,6-bisp , fructose-1,6-bisphosphate ; fruc-6-p , fructose-6-phosphate ; gluc-6-p , glucose-6-phosphate ; glut , glucose transporter ; hif-1 , hypoxia - induced factor 1 ; hk , hexokinase ; pep , phosphoenolpyruvate ; pfk , phosphofructokinase ; pg , phosphoglycerate ; pgam1 , phosphoglycerate mutase 1 ; pgi , phosphoglucose isomerase ; pk , pyruvate kinase ; pyr , pyruvate ; ppp , pentose phosphate pathway ; tigar , tp53-induced glycolysis and apoptosis regulator ; vdac , voltage - dependent anion channel ; vegf , vascular endothelial growth factor . after uptake into the cell , the next step in glycolysis is phosphorylation of glucose to glucose-6-phosphate by hexokinase ( hk ) ( figure 1 )
. there are four isoforms of hk and upon transformation , isoform ii , the isoform with the highest enzymatic activity , becomes the prevalent isoform in the cell   and this is due in part to hif1 ( hypoxia - induced factor 1 )-dependent transcriptional upregulation .
hki and especially hkii are known to interact with voltage - dependent anion channels ( vdacs ) on the mitochondrial outer membrane of rapidly proliferating cells .
this interaction is important for the inhibition of apoptosis by blocking cytochrome c release into the cytoplasm .
for these reasons , the vdac - hkii interaction is a potential target for cancer therapy .
there are data showing that the vdac - hkii interaction favors glycolysis by inhibiting a negative feedback of hkii by its own product and by stabilizing hkii protein .
thus , hkii offers a mechanistic explanation for the warburg effect independently of mitochondrial dysfunction .
glucose-6-phosphate is next converted to fructose-6-phosphate by phosphoglucose isomerase ( pgi ) ( figure 1 ) .
expression of pgi is induced in response to hif-1 and vascular endothelial growth factor ( vegf ) signaling , both of which are often deregulated in tumors .
secreted pgi is a tumor marker , as it can be detected in serum and urine of patients with cancer .
pgi is also called amf ( autocrine motility factor ) because treatment of fibrosarcoma cells with purified pgi protein induces cell migration , implicating it in metastasis .
furthermore , mere pgi gain of function is sufficient to drive cell proliferation in 3t3 fibroblasts .
secondly , the fact that atp , its own substrate , allosterically inhibits phosphofructokinase 1 and 2 ( pfk1 and 2 ) [ 33 - 35 ] offers an explanation for the pasteur effect : inhibition of glycolysis by mitochondrial respiration .
thirdly , this is a decision point for glucose to enter further into glycolysis or to be diverted into the pentose phosphate pathway ( ppp ) .
the ppp has two branches : an irreversible , oxidative branch that starts with glucose-6-phosphate and generates reduced nicotinamide adenine dinucleotide phosphate ( nadph ) and a reversible , non - oxidative branch that interconverts 5-carbon sugars and produces no nadph ( figure 1 ) .
hence , changes in pfk activity influence how much glucose enters the oxidative branch of ppp , yielding reducing equivalents for fatty acid biosynthesis and reactive oxygen species ( ros ) defense ( figure 1 ) .
one critical factor determining pfk1 activity is the level of fructose-2 - 6-biphosphate , which is the product of the bifunctional enzyme phosphofructokinase 2/fructosebisphosphatase ( pfkb ) .
pfkb has four isozymes that are expressed in a tissue - specific manner ; however , pfkbp3 , which has the highest kinase activity , is upregulated in high - grade astrocytomas and malignant breast and colon tumors , tipping the scale of the bidirectional reaction in favor of fructose-2,6-bisphosphate production .
all pfkb isoforms are upregulated in response to hypoxia in vivo , pfkbp3 being induced to the largest extent .
in addition to pfkb3 , pfkb4 has been also found to be important for cancer cell survival via small interfering rna ( sirna ) screens in two different models : glioma stem - like cells   and prostate cancer cell lines . another critical factor controlling
pfk activity is the p53 tumor suppressor - mediated induction of tigar ( tp53-induced glycolysis and apoptosis regulator ) .
tigar negatively regulates pfk2 activity and lowers fructose-2,6-bisphosphate levels , and thereby reduces pfk1 activity and diverts glucose into the ppp rather than glycolysis . in tumors with p53 loss of function ,
p53 has additional regulatory roles in metabolism , such as downregulating glut1 and glut4 expression at the transcriptional level .
phosphoglycerate mutase 1 ( pgam1 ) is another glycolytic enzyme whose activity is increased in several tumor types , including hepatocellular carcinoma .
its activity is important in metabolic regulation because its substrate , 3-phosphoglycerate , inhibits flux through the oxidative branch of ppp , thereby diverting glucose into glycolysis .
therefore , upregulation of pgam1 is advantageous for cell proliferation , although it renders tumor cells sensitive to oxidative stress due to reduction in nadph production from the oxidative branch of ppp .
so far , we have discussed the steps of glycolysis through which flux is increased in cancer cells . interestingly , cancer cells do not increase flux in all steps of glycolysis .
although flux through the upstream steps is increased , they often exhibit a bottleneck in the following steps of glycolysis leading from phosphoenolpyruvate ( pep ) to the krebs cycle and this is for the reasons discussed below .
pyruvate kinase ( pk ) is an important node of control in cancer cell metabolism .
pk has two isoforms produced by alternative splicing of pkm , namely m1 , and m2 , which are spatially and temporally differentially expressed .
pkm1 is expressed mainly during adulthood , and pkm2 is expressed during embryogenesis . the alternatively spliced transcripts of pklr , the l and r isoforms , on the other hand , are expressed specifically in liver . in 2008
in fact , depletion of pkm2 expression in cancer cell lines and reconstitution with pkm1 led to inhibition of growth and reversal of the warburg phenotype .
this finding was unexpected because at first glance it does not fit into the model that cancer cells have increased glycolytic flux .
pkm2 is the isoform with lower enzymatic activity and therefore it slows down this glycolytic reaction .
however , generating a bottleneck at the end of the glycolytic pathway appears to be necessary for giving intermediates the time to flux through alternative pathways , such as ppp , that are of crucial importance for replenishing building blocks required for growth and proliferation .
ppp flux is especially needed in cancer cells for the production of pentose phosphates , which are used for nucleotide biosynthesis , and for the production of nadph , which is a reducing equivalent needed both for fatty acid and sterol biosynthesis   and for mounting an anti - oxidant response to ros by re - oxidizing glutathione .
since tyrosine kinase signaling is often deregulated in a cancer setting , this regulation could be another way of pushing glycolytic intermediates into anabolic pathways such as the ppp .
another advantage that selective pkm2 expression confers to cancer cells is the accumulation of its substrate , pep .
pep acts as a phosphate donor to phosphorylate pgam1 at the catalytic histidine , thereby increasing its activity .
interestingly , the functional relevance of pkm2 in tumor development was analyzed in a recent study in which mice specifically lacking the pkm2 isoform were found to develop breast tumors and liver metastases induced by brca1 loss of function , indicating that pkm2 per se is not required for tumor development .
tumor cells had compensatory pkm1 expression , although pkm1 levels were heterogenous throughout the tumor . whereas non - proliferating tumor cells exhibited higher pkm1 expression , proliferating cells did not .
the same study also analyzed human tumor samples and reported the presence of tumor samples with no detectable pk expression .
even though further studies are required , these findings hint that proliferating cells can lack pk activity altogether .
pyruvate is the critical node where the flux of glucose - derived carbons is determined , either toward lactate which is usually secreted or into mitochondria ( figure 2 ) .
as warburg observed , cancer cells metabolize pyruvate by aerobic glycolysis and produce lactate . here
, we will discuss the mechanisms of aerobic glycolysis induction which do not necessarily stem from dysfunctional mitochondria , which would have pleased weinhouse .
dca , dichloroacetate ; hif1 , hypoxia - induced factor 1 ; ldha , lactate dehydrogenase a ; mct4 , monocarboxylate transporter 4 ; mpc , mitochondrial pyruvate carrier ; pdh , pyruvate dehydrogenase ; pdk , pyruvate dehydrogenase kinase ; tca , tricarboxylic acid .
the first possibility for pyruvate is to be reduced to lactate by lactate dehydrogenase ( ldh ) .
another means of inducing ldha activity in the context of cancer is via phosphorylation at the tyrosine 10 residue . reflecting the importance of the warburg effect for cancer cell survival , loss of function of ldha by means of either sirna depletion or pharmacological inhibition forces mitochondria to respire and slows down proliferation . to maintain intracellular ph homeostasis , cells need to evacuate the resulting lactate to the extracellular space .
indeed , loss of function of the hypoxia - responsive lactate / h symporter mct4 ( monocarboxylate transporter 4 )   impairs tumor growth , thereby rendering mct4 a good therapeutic target .
the second possibility of pyruvate utilization is to send it to mitochondria for further oxidation in the krebs cycle .
the limiting step here is the entry of pyruvate into mitochondria by mitochondrial pyruvate carriers ( mpcs ) ( figure 2 ) .
the activity and chemical inhibition of a mpc were shown in 1974 , but the molecular identities of these carriers remained a secret until 2012 , when they were identified as mpc1 and mpc2 .
the cancer relevance of these carriers was thoroughly investigated very recently [ 6668 ] . in mpc1 loss - of - function tumor models ,
forced expression of the protein leads to activation of mitochondrial pyruvate oxidation , which inhibits anchorage - independent growth of colon cancer cells .
these studies indicate that mpc loss of function could be one means of rewiring cancer metabolism toward aerobic glycolysis , the warburg effect . in tumor types with reduced mpc activity
activation of mpc can activate mitochondrial respiration suggests that mitochondria are not necessarily irreversibly damaged , as warburg claimed . upon entry into mitochondria , pyruvate is converted to acetyl coenzyme a ( coa ) and joins the krebs cycle , during which reduced nicotinamide adenine dinucleotide ( nadh ) and precursors for anabolic pathways are generated ( figure 3a ) .
( b ) a truncated form of the krebs cycle is favored in cancer cells with defective mitochondria or under hypoxic conditions ( or both ) to generate metabolic precursors with reductive carboxylation .
2-hg , 2-hydroxyglutarate ; accoa , acetyl coenzyme a ; acl , atp citrate lyase ; asct2 , sodium - dependent neutral amino acid transporter type 2 ; cs , citrate synthase ; dca , dichloroacetate ; fa , fatty acid ; fad , flavin adenine dinucleotide ; fh , fumarate hydratase ; gls , glutaminase ; hif1 , hypoxia - induced factor 1 ; idh , isocitrate dehydrogenase ; me , malic enzyme ; nad , nicotinamide adenine dinucleotide ; nadh , reduced nicotinamide adenine dinucleotide ; nadp , nicotinamide adenine dinucleotide phosphate ; nadph , reduced nicotinamide adenine dinucleotide phosphate ; oaa , oxaloacetate ; pdh , pyruvate dehydrogenase ; pdk , pyruvate dehydrogenase kinase ; pyr , pyruvate ; sdh , succinate dehydrogenase .
at this stage , there is one more regulation point , pyruvate dehydrogenase ( pdh ) .
pdh is negatively regulated by nadh and acetyl coa and through phosphorylation by pyruvate dehydrogenase kinase ( pdk ) .
moreover , oncogenic tyrosine kinases contribute to the reduction of pdh activity in two ways : they phosphorylate pdk to increase its activity   and they phosphorylate and inhibit a phosphatase that acts on pdh . dicholoroacetate ( dca ) is a pharmacological inhibitor of pdk [ 7578 ] , which is used in treatment of lactic acidosis and cancer .
the fact that dca treatment is able to reverse warburg phenotypes   is another indication that mitochondria do not have to be irreversibly damaged .
even though cancer cells tend to use glucose in aerobic glycolysis rather than oxidizing it in the krebs cycle , the krebs cycle is not dispensable altogether .
the krebs cycle constitutes a chain of reactions that are essential for the production of building blocks for growth and proliferation .
as anabolic reactions use substrates from the krebs cycle , thereby depleting the krebs cycle , the cell needs to replenish these intermediates via a set of so - called anaplerotic reactions .
for example , vacanti and colleagues   made the observation that , upon inhibition of pyruvate entry into mitochondria in c2c12 myotubes , cells were able to maintain krebs cycle metabolism without any drastic changes in the levels of intermediates , even though they exhibit a reduction in the mitochondrial oxidation of pyruvate .
cancer cells that divert glucose metabolism away from mitochondria face a situation similar to pyruvate entry inhibition and therefore employ strategies to keep the cycle going , discussed below . by this means , they use mitochondria for biosynthesis rather than degradation
next , glutamate is converted to -ketoglutarate ( -kg ) either by a transamination reaction of the amine group onto another ketoacid or by deamination .
myc , which is activated in most cancer types , increases glutaminolytic activity by increasing expression of gls and the glutamine transporter asct2 ( asc amino acid transporter 2 ) .
if a particular tumor uses this strategy to keep the krebs cycle filled , it renders the cancer cells addicted to glutamine .
the filling of the krebs cycle via glutaminolysis is then used for several cataplerotic reactions important for cancer cell lipid biosynthesis : ( 1 ) malate is converted to pyruvate by malic enzyme ( me ) in the cytosol , thereby yielding nadph ( figure 3a ) , which is a reducing equivalent used for fatty acid biosynthesis ; ( 2 ) oxaloacetate ( oaa ) is converted back to malate and also exported to yield nadph ; and ( 3 ) citrate is used as a source of acetyl coa for fatty acid synthesis   ( figure 3a ) .
the relative contribution of these reactions via me to the nadph pool is quite significant and is roughly equivalent to the nadph production by the ppp .
although glutaminolysis can replenish krebs cycle intermediates , it still requires functional mitochondria and oxygen , which are not always available in the tumor context .
cancer cells use reductive carboxylation in cases of defective mitochondria   and hypoxia   ( figure 3b ) .
this pathway involves nadph - dependent reductive carboxylation of -kg ( that derives from glutaminolysis , see above paragraph ) by isocitrate dehydrogenase ( idh ) into isocitrate , which is the reverse reaction of the conventional krebs cycle .
substrate availability also influences the direction of the reaction : that is , reductive carboxylation or oxidative decarboxylation of -kg . a high nadph / nadp ( nicotinamide adenine dinucleotide phosphate ) ratio , low citrate levels , and
cancer cells need to upregulate their fatty acid biosynthesis upon neoplastic transformation   in order to meet the increased demand of membrane production due to increased proliferation .
citrate production is of importance for fatty acid biosynthesis because the citrate shuttle transports mitochondrial acetyl coa across the membranes to the cytoplasm , where it is converted back to oaa and acetyl coa by atp citrate lyase   ( figure 3a and b ) .
this is one of the reasons why anaplerotic reactions are crucial for supporting growth and proliferation of the tumor cells .
so far , we have discussed mechanisms that promote aerobic glycolysis independently of possible mitochondrial defects . however , there are cases where warburg was right , whereby mutation of genes whose products act in mitochondria are enough to induce transformation . discussed below are examples of such cases .
various cancer types have been found to harbor neomorphic mutations in idh1 and idh2 enzymes , causing them to lose their activity of decarboxylation of isocitrate to -kg , and to gain the activity of converting -kg to the oncometabolite 2-hydroxyglutarate ( 2-hg )   ( figure 3a ) .
the prevalent view on the mechanism of 2-hg - induced tumor formation is that it alters cellular methylation of both dna and proteins .
there is evidence that 2-hg competitively inhibits -kg - dependent dioxygenases , such as methylcysteine hydroxylases and the jumonji c ( jmjc ) family of histone demethylases [ 9799 ] . data also show that 2-hg stabilizes hif-1 by inhibiting prolyl hydroxylases ( phds ) ( also -kg - dependent dioxygenases ) . some enzymes in the krebs cycle function as tumor suppressors .
for example , mutations in succinate dehydrogenase ( sdh ) subunits have been associated with hereditary paraganglioma , and fumarate hydratase ( fh ) mutations were implicated in renal cell cancers and smooth muscle tumors . in both cases , substrates of the enzymes , succinate and fumarate , respectively , have been shown to accumulate .
hypoxia - responsive genes are regulated by the transcription factor hif1. under normoxic conditions , hif1 is constantly hydroxylated at proline residues by phds . upon hydroxylation
, hif1 is readily recognized and ubiquitinated by von hippel - lindau complex that has e3 ubiquitin ligase activity .
phds use -kg as cofactors and generate succinate at the end of the reaction . in the context of shd and fh mutations , cytosolic succinate   and fumarate   concentrations increase and they inhibit the activity of phds , leading to stabilization of hif1 ( figure 3a ) even in the presence of oxygen and induction of an aberrant hypoxic response .
there has been discussion for almost a century about the reasons for the metabolic phenotypes that warburg initially observed , i.e. that cells use glucose via glycolytic reactions instead of respiration , even in the presence of oxygen .
it is a phenomenon that appears counterintuitive and wasteful , given how little atp glycolysis yields compared with oxidative phosphorylation .
the main disagreement revolves around warburg 's proposal that irreversible damage of mitochondria is the main reason for carcinogenesis . in some cases
, the fact that mutations in some krebs cycle enzymes , such as sdh and fh , are able to induce renal tumors indicates that mitochondrial dysfunction can be sufficient for tumor formation . however , there are reports that in some cases cancer cell mitochondrial function is intact . when they are forced to respire
, they are able to . in sum , it seems that mitochondrial dysfunction is usually the consequence of tumor formation , yet there are cases in which mitochondrial dysfunction is the cause of the tumor formation .
indeed , one recent study suggests that glycolysis is needed for sustaining stemness and proliferation , whereas forced activation of mitochondrial respiration induces differentiation and reduces proliferation .
there seems to be a very strong , yet not very well - understood , crosstalk between the regulation of proliferation and a preference for glycolysis .
 compared with normal cells , cancer cells show alterations in many cellular processes , including energy metabolism . 
 studies on cancer metabolism started with otto warburg 's observation at the beginning of the last century . according to warburg , 
 cancer cells rely on glycolysis more than mitochondrial respiration for energy production . considering that glycolysis yields much less energy compared with mitochondrial respiration , warburg hypothesized that mitochondria must be dysfunctional and this is the initiating factor for cancer formation . 
 however , this hypothesis did not convince every scientist in the field . 
 some believed the opposite : the reduction in mitochondrial activity is a result of increased glycolysis . 
 this discrepancy of opinions is ongoing . in this review 
 , we will discuss the alterations in glycolysis , pyruvate metabolism , and the krebs cycle in cancer cells and focus on cause and consequence . 
Introduction
Changes in the glycolytic pathway during tumorigenesis
Pyruvate: the intersection
Hacking the Krebs cycle
Warburg was not totally wrong
Conclusions
Disclosures