NF-kB p53 and warburg effect

Nuclear factor-k B,p53,and mitochondria:regulation of cellular metabolism and the Warburg effect Rene´e F.Johnson and Neil D.PerkinsInstitute for Cell and Molecular Biosciences,Newcastle University Medical School,Catherine Cookson Building,Framlington Place,Newcastle Upon Tyne NE24HH,UKAmong the characteristics acquired by many tumour cells is a shift from using oxidative phosphorylation to using glycolysis for ATP production.Although the nu-clear factor(NF)-k B family of transcriptional regulators have important roles in tumorigenesis,their ability to function as regulators of metabolism has only been recently investigated.This has revealed the importance of crosstalk between NF-k B,the p53tumour suppressor and other crucial cell signalling pathways.This review discusses the mechanisms through which NF-k B regu-lates tumour cell metabolism and the important role of p53in determining the consequences of NF-k B activity.It also proposes a model in which NF-k B contributes to the shift to glycolytic ATP production through regulation of both nuclear and mitochondrial gene expression. Metabolic adaptation in tumoursCancer cells acquire a set of characteristics that facilitate growth and survival.These‘hallmarks of cancer’include evasion of growth suppressors;sustained proliferative sig-nalling;increased resistance to cell death;induction of angiogenesis;limitless replicative potential;and tissue invasion and metastasis[1].Following the renewed inter-est in metabolic changes in cancer cells,in recent years,it has become clear that alterations in metabolism should also be included among the essential characteristics of tumour cells[1–3].Indeed,metabolic reprogramming dur-ing tumorigenesis is intimately integrated with many of these characteristics and is regulated by many of the same effectors[4].Although it wasfirst reported in1927by Otto Warburg that tumours prefer to use glycolysis instead of oxidative phosphorylation[5],there has recently been a revival of interest in tumour cell metabolism.The use of glycolysis even in the presence of oxygen,termed the‘Warburg effect’[6],provides the tumour cell with ATP in a less efficient manner than that produced by oxidative phosphorylation. However,the tumour cell also benefits because using glycol-ysis to break down glucose also provides substrates for anabolic processes including the pentose phosphate path-way,lipid metabolism,and amino acid/nucleotide synthesis through the citric acid cycle[4].It might also confer a growth advantage in hypoxic conditions,allowing tumour cells to survive in the absence of oxygen[7].Warburg hypothesised that this defect in cellular respiration and the subsequent shift to glycolysis is the initiating step in tumorigenesis[6]. However,except for a small subset of cancers in which mutation of one the genes encoding the tricarboxylic acid (TCA)cycle enzymes results in tumourigenesis,it is not yet clear whether altered metabolism results in cancer forma-tion or if these alterations arise after cancer development[8] (Box1).In addition,the use of different model systems and cell lines can make it difficult to determine how generali-sable these effects may be to real tumours of different origins (Box2).Either way,this aspect of tumour cell biology has been diagnostically exploited,in that tumours and metas-tases can be monitored noninvasively using positron emis-sion tomography(PET),which detects the uptake of the radioactive hexokinase substrate2-18F-2-deoxyglucose (FDG)by highly glycolytic tumour cells[9].Furthermore, the increased use of glycolysis for cancer cell cellular energy production may also prove to be a potential therapeutic target,although attempts to inhibit glycolysis in cancer cells have not yet proved to be successful.To a great extent,this metabolic shift results from altered expression of the genes and proteins that regulate glycolysis and oxidative phosphorylation.These gene ex-pression changes are due,at least in part,to the altered activities of key transcription factors,including NF-k B and hypoxia inducible factor(HIF)-1,together with the loss of tumour suppressors such as p53.Although we have chosen to focus on crosstalk between p53,NF-k B,and HIF,it should be noted that these are not the only factors resulting in altered energy metabolism in cancer cells.Other changes,such as in the levels of reactive oxygen species (ROS),cytokines and chemokines,and aberrant AKT ac-tivity,all alter the tumour microenvironment and subse-quently tumour energy metabolism[10,11].Moreover, processes that result in an increase in the rate of glycolysis also result in changes in mitochondrial metabolism through a process known as the Crabtree effect[12].p53p53is a transcription factor that regulates a diverse range of cellular functions including apoptosis,senescence,DNA repair,cell proliferation and autophagy[13].Mutation or suppression of p53,a frequent occurrence in cancer,results in loss of control of these crucial functions,thus promoting tumourigenesis and leading to p53being referred to as aCorresponding author:Perkins,N.D.(n.d.perkins@).0968-0004/$–see front matterß2012Elsevier Ltd.All rights reserved./10.1016/j.tibs.2012.04.002Trends in Biochemical Sciences,August2012,Vol.37,No.8317tumour suppressor.In undamaged cells,the murine double minute chromosome protein2(MDM2)E3ubiquitin ligase keeps p53in check through rapid ubiquitin-mediated pro-teolysis.In general,induction of p53involves the inactiva-tion of this process through post-translational modification and/or protein binding to MDM2,leading to its dissociation from p53[14].Although induction of p53is most frequently associated with DNA damage and oncogene activation,it is also activated by a wide variety of cellular stresses,includ-ing metabolic changes,limited nutrients,and reduced oxygen tension(hypoxia)[15].For example,p53can be activated by AMP-activated kinase(AMPK)in response to changes in ATP/ADP levels,or by malate dehydrogenase,a TCA cycle enzyme,in response to glucose deprivation[15]. Unmutated p53promotes oxidative phosphorylation and inhibits the Warburg effect,thus contributing to its tumour suppressor functions[15,16].By contrast,the inhibition,loss and/or mutation of p53,which is one of the most common events in tumorigenesis,results in decreased oxygen consumption and increased glycolysis[16].There are a variety of reported mechanisms in different cell types through which p53might achieve these effects [15](Figure1).For instance,in many different cell lines, p53induces expression of TIGAR(TP53-induced glycolysis and apoptosis regulator),a fructose-2,6-bisphosphatase, leading to a drop in fructose-2,6-bisphosphate levels in cells,which in turn leads to inhibition of glycolysis and reduced intracellular ROS levels[15].In mouse models and cell lines,p53has also been shown to increase oxidative phosphorylation through upregulation of the synthesis of cytochrome c oxidase2(SCO2)gene[15].In addition,p53 directly regulates glycolytic genes.For example,p53-re-sponsive elements have been identified in the promoters of the hexokinase(HK)-II and phosphoglycerate mutase (PGM)genes,which both encode enzymes in the glycolytic pathway[15].Moreover,p53has been shown to down-regulate expression of PGM and the glucose transporters (GLUT)1,3,and4[15,17].Interestingly,increased glucose metabolism(through upregulation of HK1or GLUT1)can in turn suppress p53activity,providing a potential mech-anism through which alterations in metabolism can con-tribute to tumorigenesis[18].The metabolic effects of p53 are not restricted to glycolysis and oxidative phosphoryla-tion.Consequences of p53activation also include increased pentose phosphate pathway activity,resulting in an in-crease in the NADPH:NADP ratio and glutathione[15]. In response to glucose starvation,p53can also induce the expression of guanidinoacetate methyltransferase (GAMT),a regulator of creatine synthesis,thus allowing fatty acid oxidation(FAO)to be used as an alternate energy source[19].p53and NF-k B crosstalk regulates tumour cell metabolismNF-k B is a dimeric transcription factor composed offive family members:RelA(p65),p105/p50(NF-k B1),p100/ p52(NF-k B2),c-Rel,and RelB.NF-k B functions as an important regulator of the immune and inflammatory responses[20].NF-k B is induced by a wide variety of inflammatory and immunological stimuli and cell stresses,including cytokines such as tumour necrosis factor(TNF)a,interleukin(IL)-1,bacterial lipopolysac-charide(LPS),low oxygen tension(hypoxia),and DNA damage[20].It plays a crucial role in multiple cellular pathways including cell survival,proliferation,adhesion, and angiogenesis[20].Through these processes,aberrant-ly active NF-k B found can contribute to most,if not all,of the‘hallmarks’of cancer(reviewed in[21,22]).There are two principal pathways leading to NF-k B activation,both of which utilise members of the I k B kinase(IKK)family. Thefirst of these involves IKK b-dependent phosphoryla-tion of the NF-k B inhibitor I k B a,resulting in the ubiqui-tylation and degradation of I k B a.This allows NF-k B complexes(most commonly a RelA/p50heterodimer)to be retained in the nucleus of the cell[20].This‘classical’pathway is typically induced by TNF,IL-1,LPS and other Toll-like receptor ligands[20].The second pathway is induced by stimuli such as lymphotoxin b,and involvesBox1.Caveats to the Warburg hypothesisAlthough many solid tumours and cancer cell lines display theclassic traits of the Warburg effect,such as enhanced glycolysis andreduced or impaired oxidative phosphorylation,not all cancer cellshave been shown to increase glycolysis(reviewed in[61]).Many,but not all tumours display a PET signal,demonstrating highutilisation of glucose,and the PET signal is often only poorlydiagnostic in early cancer stages[62].Furthermore,although sometumour cells have reduced mitochondrial H+-ATP synthase,sug-gesting decreased oxidative phosphorylation[63],several otherstudies have described tumour cells that display relatively normaloxidative phosphorylation[64].Other studies have suggested that,depending on the tumour microenvironment or pattern of geneexpression,glycolysis and oxidative phosphorylation can be eithersuppressed or restored in the cells,suggesting that tumourmetabolism is not an irreversible characteristic[61,65].Therefore,although the Warburg effect and alterations in cell metabolismundoubtedly play a part in tumour formation and growth,it appearslikely that cancer cells use a wide range of metabolic changes thatare under constant adaptation and alteration,thus satisfying theparticular energy and nutrient requirements that are determined bythe specific conditions encountered by the cell.Box2.Generalisations among different cancer cell lines andacross speciesAs with the majority of scientific and medical research,studies incell metabolism have been conducted using a variety of normal andcancerous cells lines,tissue samples and animal models,and theextent to which the resulting data can be generalised across thesesystems is unknown.Studies using cancer cell lines derived fromprimary tumours that have been grown in the presence of highlevels of oxygen and glucose,often for many years,can incorporatechanges resulting from metabolic adaptation to these cultureconditions,and can display chromosomal aberrations and muta-tions that may not have been present in the original tumour.Thus,care should be taken when interpreting data generated using thesesystems.However,it is often the case that studies have beenconducted and confirmed in vastly different systems or acrossspecies.The studies represented in this review used a wide range ofcell lines,from mouse and human origin,as well as human andmouse tissue samples.It should be noted that it is possible that theresults obtained from these different systems may represent datathat are highly cell and or context specific.However,the use of‘switchable systems’allowing genes to be turned on or off in liveanimals[66],might help to elucidate the common or specific natureof these observations with regard to the Warburg effect andtumorigenesis.Trends in Biochemical Sciences August2012,Vol.37,No.8 318the IKK a -dependent phosphorylation and proteolytic pro-cessing of the p100subunit,generating p52-containing NF-k B complexes [20].Crosstalk between p53and the NF-k B family of tran-scription factors provides a mechanism of integrating two signalling pathways with important roles in tumourigen-esis [20,23,24].Although in many cases this has the ap-pearance of an antagonistic relationship,because the tumour promoting NF-k B inhibits the tumour suppressor p53or vice versa [23,24],these effects are probably better described as being modulatory and context dependent.For example,under some circumstances,p53and NF-k B can cooperatively induce or repress joint target genes and promote apoptosis [22].Furthermore,p53can also be a substrate of IKK,leading to p53degradation through a ubiquitin-dependent but Mdm2-independent pathway [25].Crosstalk between p53and NF-k B also appears to have an important role in the changes associated with tumour cell metabolism.Given the complexity of this cross-talk,it is likely that,dependent on the context,NF-k B and IKK can modulate metabolism through p53in a variety of ways.Here,we discuss some specific examples where p53/NF-k B crosstalk has been shown to affect tumour cell metabolism.Of the NF-k B subunits,only RelA has been clearly dem-onstrated to have an important role in tumour cell metabo-lism.However,effects involving the other subunits cannot be ruled out.Interestingly,depending on the context,RelA can either promote or repress oxidative phosphorylation andthe switch to glycolytic energy production.Strikingly,p53can determine which of these effects occurs (Figure 2).For example,Mauro et al.have demonstrated that,upon glucose starvation of mouse embryo fibroblasts (MEFs),RelA acti-vates the p53gene promoter and consequently induces p53RNA and protein levels [26].Consequently,this RelA-de-pendent increase in p53levels leads to an increase in oxidative phosphorylation and a decrease in glycolysis [26].These metabolic effects are due,at least in part,to upregulation of the p53target gene SCO2,a subunit of the mitochondrial cytochrome C oxidase (COX)complex,which is the terminal and crucial component of the electron trans-port chain that catalyses the transfer of electrons from cytochrome c to molecular oxygen (Figure 1).This complex is required for aerobic ATP production through mainte-nance of the proton gradient across the inner mitochondrial membrane [27].In this system,loss of RelA reduced oxidative phos-phorylation and also resulted in the upregulation of genes normally repressed by p53,including the glycolytic en-zyme PGM2and glucose transporters GLUT1,GLUT3,and GLUT4[26](Figure 1).Through these p53-dependent effects,RelA is able to suppress H-Ras-induced transfor-mation in vitro [26].Although the study of Mauro et al.mostly used MEFs,it also demonstrated that RelA can promote metabolic adaptation of CT-26human colorectal cancer cells in a mouse xenograft model.Moreover,loss of RelA further suppressed the growth of tumours chal-lenged with the diabetes drug metformin,an inhibitormtDNAmtDNAmtDNAGlut 1, 3 and 4GlucoseRRM2Bp53I κB αRelARelAp53GlucoseGlucose 6 phosphate Fructose 6-phosphatep53SCO2p53RelAFructose 1,6-bisphosphateGlyceraldehyde 3-phosphate Acetyl CoATCA cycleISCO1/2miR210HIFPDK1HIF NADH FADH1,3-Diphosphoglycerate3-Phosphoglycerate2-PhosphoglyceratePhosphoenolpyruvatePyruvateLactateHIFRelAp53HIFp53TIGARp53HIFHIFHIFT i BSFigure 1.Regulation of metabolic pathways by p53,NF-k B,and HIF.Summary of how the p53,NF-k B,and HIF signalling pathways can regulate the expression and/or activity of key components of the glycolytic and TCA pathways,together with oxidative phosphorylation.Trends in Biochemical Sciences August 2012,Vol.37,No.8319of mitochondrial complex I,beyond treatment with met-formin alone.Therefore,the ability of RelA to induce p53 and SCO2upon glucose starvation adds to a growing list of pathways through which NF-k B can behave more akin to a tumour suppressor rather than its more commonly de-scribed function as a tumour promoter[22].In contrast to these results,RelA can also promote glycolysis.Kawauchi et al.have reported that,upon loss of p53,RelA induces expression of the glucose transporter GLUT3in tumour cells,resulting in increased glucose consumption and lactate production[17].Moreover,in p53-deficient mousefibroblast cells,this process is re-quired for oncogenic Ras-induced cell transformation and acceleration of aerobic glycolysis.Such effects are consistent with reports suggesting that at least in some Ras-dependent tumour models,such as an H-Ras-driven lung adenocarcinoma model,the ability of NF-k B to func-tion as a tumour promoter requires loss of p53[28],al-though another study has suggested that this is not the case[29].These studies provide good examples of the context-dependent duality of function of NF-k B function in cancer,where depending on the context,it can exhibit both tumour-promoting and tumour-suppressing charac-teristics[22].A common theme appears to be that loss of tumour suppressors leads to loss of control of NF-k B, leaving its tumour-promoting functions to dominate. Regulation of mitochondrial function by NF-k B and p53 As described above,a major mechanism through which NF-k B and p53can exert effects on cellular metabolism is through their well-established roles as regulators of nucle-ar gene expression(Figure3).However,there is also evidence that NF-k B and p53have direct effects within mitochondria,the site of oxidative-phosphorylation-depen-dent ATP generation.The vast majority of the>1500 mitochondrial proteins are encoded by nuclear genes and imported from the cytosol[30]but there are some crucial exceptions that are expressed from the mitochon-drial genome(Figure3).This exists as an approximately 16.5-kb closed,covalent,circular DNA structure,encoding 13proteins,22tRNAs,and two ribosomal RNAs[31] (Figure3).All mitochondrially encoded proteins are in-volved in oxidative phosphorylation[31].Mitochondrial gene transcription occurs bidirectionally from three pro-moters located in the D-loop region of the genome.Tran-scription of the light strand produces a single mRNA encoding the NADH dehydrogenase6(MTND6)gene and eight tRNA sequences.The heavy strand has two promoters that either produce an RNA encoding the two mitochondrial rRNA sequences12S and16S rRNA(H1),or a polycistronic transcript encoding the remaining12open reading frames(ORFs),14tRNAs,and2rRNAs(H2).The polycistronic transcript then undergoes subsequent RNA processing to generate separate RNA species[31].Mito-chondrial transcription is carried out by a specialised set of mitochondrial transcription factors,including a specific mitochondrial RNA polymerase,POLRMT[polymerase (RNA)mitochondrial(DNA directed)][31].Mitochondrially encoded genes all have the potential to modulate oxidative phosphorylation,albeit at differing levels,thus,it is crucial that mitochondrial transcription be coordinated with the other cellular signalling pathways that impose their own energy demands upon the cell[32] (Figure3).Signalling pathways can indirectly control mi-tochondrial gene expression through regulation of proteins such as peroxisome proliferator-activated receptor g coac-tivator1(PGC-1)a and nuclear respiratory factor(NRF)-1 and2[31],which in turn regulate the expression of the nuclear-encoded mitochondrial transcription factors.In-deed,following LPS stimulation of hepatocytes the NF-k B subunit RelA,in conjunction with cAMP responsive element binding protein(CREB),can activate the NRF-1 promoter and induce its expression,leading to induced expression of nuclear encoded mitochondrial transcription factors and expanded mitochondrial DNA(mtDNA)copy number[33].A role for NF-kB in mitochondria?Such mechanisms inevitably impose a delay on how quick-ly mitochondrial gene expression responds to changes in cellular signalling.The cell can regulate the effect of mitochondrially encoded proteins by modulating their translation efficiency or activity,which is the case for the mitochondrial H+-ATP synthase and the mRNA of one of its constituent proteins,b-F1-ATPase[34].Howev-er,direct regulation of mitochondrial transcription pro-vides another route through which regulation of oxidative phosphorylation can be integrated with cellular signal-ling.For instance,control can be achieved through the targeted mitochondrial localisation of nuclear transcrip-tion factors such as NF-k B,which then act as the effectorsNF-κB NF-κBInduces p53Induces SCO2(other geneexpression effects)Induces oxidativephosphorylation/represses glycolysisOncogene induced transformationInduces Glut3 (and other genes?) Induces glycolysisNF-κB repressesmitochondrial geneexpressionMitochondriaInhibition of oxidativephosphorylationFigure2.Model of how the p53-dependent regulation of ATP production by NF-k Bmight influence oncogene induced transformation.Nutrient deprivation,in at leastin some cell types,causes NF-k B to induce p53protein expression,which in turnresults in induction of synthesis of SCO2.SCO2then acts to promote oxidativephosphorylation.p53can also prevent RelA localisation to mitochondria.However,upon loss of p53expression or activity in a tumour cell,NF-k B can exhibit differentbehaviour,promoting glycolysis through induction of genes such as Glut3,whilethen being able to localise to mitochondria where it can repress mitochondrialgene expression.The context dependency of these results,and whether they arerestricted to certain cell or tumour types,has yet to be established.Blue arrowssymbolise potentially tumour suppressing pathways,and red arrows indicatepotentially tumour-promoting pathways.Trends in Biochemical Sciences August2012,Vol.37,No.8 320of the major cell signalling pathways (Figure 3).Regula-tion of the mitochondrial genome by nuclear transcription factors might be another mechanism by which these cells can adapt metabolic processes to respond to changes in the microenvironment.Indeed,many reports indicate that a wide variety of such factors can be found in mito-chondria,including signal transducer and activator of transcription (STAT)3,CREB,nuclear hormone receptors [32],NF-k B subunits,and p53[32,35–38].However,this is a controversial area because these factors lack defined mitochondrial targeting sequences (MTSs)and their mechanism of mitochondrial import has not yet been explained (Box 3).Several components of the NF-k B/IKK signalling pathway have been putatively identified in mitochon-dria.These include the NF-k B subunits RelA and p50,the I k B a inhibitor as well as the upstream kinases IKK a ,IKK b ,and IKK g [35–38].Although the exact location of these proteins within the mitochondrion is still contentious,they are thought to be located in the intermembrane space [35]as well as in the mitochondri-al matrix [36,37]where the mtDNA is found.There are,however,limited data on the function of these signallingmolecules in this organelle and most attention has fo-cussed on RelA.NF-k B from mitochondrial extracts from prostate cancer cells and U937monocytic leukaemia cells can bind NF-k B consensus sequences in vitro [36,37];an effect enhanced by treating cells with TNF-related apoptosis-inducing ligand (TRAIL)or TNF a [36,37].Treatment with TRAIL or TNF a is associated with a decrease in the expression of mitochon-drial genes encoding cytochrome c oxidase III and II and cytochrome b ,which is inhibited by expression of a mutant or super-repressor form of the NF-k B inhibitor I k B a [36,37].RelA-dependent repression of mitochondrial gene expres-sion has also been observed in a variety of cancer cell lines,including U-2OS osteosarcoma cells and H1299non small cell lung cancer cells.This observation is true for both endogenous and overexpressed,artificially mitochondrially targeted RelA [38].Using a chromatin immunoprecipitation (ChIP)assay,recruitment of RelA to the mitochondrial genome has been shown;RelA repression of transcription is dependent upon the C-terminal RelA transactivation domain,and is associated with reduced POLRMT binding to mtDNA [38].These results are consistent with RelA exerting a direct effect on mitochondrialtranscription,NucleusCytoplasmMitochondrionNuclear encoded mitochondrial genes(>1000)Nuclear TFsMetabolic regulators glycolytic genesNuclear TFsNF-κB NF-κBDirect targets and crosstalkInflammationstress oncogenesOther nuclear transcription factorsRibosomes translation(i)(iii)(ii)(iv)(v)(vi)ATPMatrix2H+4H+ 2H+Inner mitochondrial membraneInter-membrane space3H+Electron transport chain (ETC)Transcription Complex I Complex III Complex IV ATP synthase Ribosomal RNAKey:D Loop regulatory region C y t bN D 6ND5N D4N D4LN D3C O I I IC O IIC OI N D 1N D 212S 16SMitochondrialDNA 16569 bpA T P8A T P 6L S PH S P 1HSP2(vii)& translationMatrix(I)(II) (III)(IV) (V)3H+T i BSFigure 3.NF-k B can regulate ATP production through effects on both nuclear and mitochondrial gene expression.Multiple stimuli can induce NF-k B nuclear translocation (i).In the nucleus,NF-k B can regulate metabolism either by direct effects on target genes or by inducing or modulating the expression and activity of other transcription factors (TFs)such as HIF-1,p53,and STAT3(ii).NF-k B can potentially influence mitochondrial function through crosstalk with transcription factors such as NRF 1/2and PGC 1a ,which regulate the expression of nuclear-encoded mitochondrial proteins,including mitochondrial transcription factors such as transcription factor A,mitochondrial (TFAM),and polymerase (RNA)mitochondrial (DNA directed)(POLRMT)(iii).mRNAs for the nuclear-encoded mitochondrial factors are then translated in the cytosol (iv).These proteins possess mitochondrial targeting sequences,therefore,they are then translocated into mitochondria through the TOM and TIM complexes (v).This inevitably leads to a delayed response between an initial NF-k B activating signal and any effect on mitochondrial function.However,there potentially exists a parallel pathway in which the RelA NF-k B subunit,as well as other normally nuclear transcription factors,can be translocated directly to mitochondria (vi).This would allow a more direct response to inducing stimuli while also providing a mechanism to integrate mitochondrial and nuclear gene expression.The pathways through which these nuclear factors enter mitochondria are poorly defined (Box 3).Mitochondrial function of nuclear transcription factors could occur either in the matrix or intermembrane space.In the matrix of the mitochondria,transcription from multiple copies of the circular mitochondrial genome is regulated by nuclear-encoded transcription factors,some of which transactivate exclusively mitochondrial genes,and others that can transactivate both nuclear and mitochondrial genes (vii).The RelA NF-k B subunit can repress mitochondrial gene expression,thus contributing to the downregulation of oxidative phosphorylation and switch to glycolysis that is seen in cancer cells.The positions of the three mitochondrial transcriptional promoters (HSP1,2,and LSP)in the D loop region in the mitochondrial genome are shown.The coding regions of individual mitochondrial genes in this figure are not to scale,nor are the positions of mitochondrial tRNAs depicted.Trends in Biochemical Sciences August 2012,Vol.37,No.8321。