REVIEWSCEREBELLARHAEMANGIOGBLASTOMASBox 1 | Genes induced by hypoxia Oxygen transport and iron metabolism•Ceruloplasmin| erythropoietin| ferritin light chain| heme oxygenase-1| transferrin| transferrin receptorAngiogenesis•Adrenomedullin| angiopoietin-2| cyclooxygenase-2| endothelin-1and-2| fibroblast growth factor-3| hepatocytegrowth factor| histone deacetylase | monocyte chemotactic protein-1| nitric oxide synthase| osteopontin| placentalgrowth factor | Tie-2(an angiopoietin receptor) | transforming growth factor (TGF)-α, TGF-β1,TGF-β3| vascularendothelial growth factor (VEGF)-A | VEGF receptor-1Glycolysis and glucose uptake•Aldolase-A | enolase-1|glucose transporter-1,-3 (GLUT1,GLUT3) | glyceraldehyde-3-phosphate dehydrogenase|hexokinase-1;hexokinase-2| lactate dehydrogenase-A| phosfructokinase-C | phosfructokinase-L |phosphoglyceratekinase-1| pyruvate kinase-MTranscription factors•Annexin V| BCL-interacting killer (BIK) | cyclin G2| differentiated embryo-chondrocyte expressed gene 1 (DEC1)|FOS| heat-shock factor| hypoxia-inducible factor (HIF)-1α;HIF-2α| insulin-like growth factor (IGF) binding protein-1,-2,-3| JUN | KIP1 |lipocortin| nuclear factor-κB (NF-κB) | NIP3 | NIX | transgelin| transglutaminase-2| WAF1Metabolism/pH/neurotransmitters•Acetoacetyl CoA thiolase| adenylate kinase-3| aminopeptidase-A | carbonic anhydrase-9,-12 | phosphoribosylpyrophosphate synthetase| spermidine N1-acetyltransferase| tyrosine hydroxylase |α-adrenergic receptorGrowth factors/cytokines•IGF-2| interleukin-6| interleukin-8| intestinal trefoil factor| macrophage inhibitory factor| platelet-derived growthfactor-B| staniocalcinStress-response pathways•150-kDa ORP (oxygen-regulated protein) | glucose-related protein | growth arrest- and DNA damage-induced gene (GADD153) |human apurinic apyrimidinic site endonuclease (HAP-1) | thioredoxinCell adhesion,extracellular matrix,cytoskeleton and proteases/coagulation•CD99| collagen-5α1| Ku70| Ku80| low-density lipoprotein receptor-related protein| metalloproteinases| matrixmetalloproteinase-13| neuronal cell-adhesion molecule L1(L1CAM) | plasminogen activator inhibitor-1| proline-4hydroxylase| tissue factor (TF) | urokinase receptor| vimentin| α-integrinNon-malignant proliferations ofvascular stromal cells in thecentral nervous system.The HIF-1pathwayOne way that cells respond to reduced oxygen levels isthrough hypoxia-inducible transcription factor 1 (HIF-1).HIF-1 is a heterodimer that consists oftheSummary •Hypoxia is a reduction in the normal level oftissue oxygen tension,and occurs duringacute and chronic vascular disease,pulmonary disease and cancer.It induces atranscription programme that promotes an aggressive tumour phenotype.•Hypoxia is associated with resistance to radiation therapy and chemotherapy,but isalso associated with poor outcome regardless oftreatment modality,indicating that itmight be an important therapeutic target.•Hypoxia-inducible factor-1α(HIF-1α) is a key transcription factor that is induced byhypoxia and regulated by a proline hydroxylase.•Pathways that are regulated by hypoxia include angiogenesis,glycolysis,growth-factorsignalling,immortalization,genetic instability,tissue invasion and metastasis,apoptosis and pH regulation.•Most ofthe hypoxia-induced pathways promote tumour growth,but apoptosis is alsoinduced by hypoxia.The balance ofthese pathways might be critical for the effects ofhypoxia on tumour growth.•Drugs that inhibit HIF-1αexpression antagonize HIF-1αinteraction with CBP/p300 orblock downstream function ofgenes such as vascular endothelial growth factor andcyclooxygenase-2 have potentially important roles in tumour therapy.Hypoxia can alsobe used to activate therapeutic gene delivery to specific areas oftissue.hypoxic response factor HIF-1αand the constitu-tively expressed aryl hydrocarbon receptor nucleartranslocator (ARNT) (also known as HIF-1β).In theabsence ofoxygen,HIF-1 binds to hypoxia-responseelements (HREs),thereby activating the expression ofnumerous hypoxia-response genes,such as the pro-angiogenic growth factor vascular endothelial growthfactor (VEGF)8(BOX 1;FIG.1).The redox active apurinic/apyrimidinic endonuclease-1 has been shown to keepHIF-1αin a reduced state9that is necessary for itstranscriptional function10.In the presence ofoxygen,HIF-1αis bound to thetumour suppressor Von Hippel–Lindau (VHL) pro-tein.This interaction causes HIF-1αto become ubiq-uitylated and targeted to the proteasome,where it isdegraded11–14.Mutations in VHL that are associatedwith renal cancerand CEREBELLAR HAEMANGIOGBLASTOMASprevent this ubiquit-ylation,resulting in an accumu-lation ofHIF-1αand continuous activation ofhypoxia-response genes15,16.Recently,two groups have reported that a prolylhydroxylase (a tetramer containing two hydroxylaseunits and two protein disulphide isomerase subunits) ispart ofthe mechanism by which cells sense hypoxia andregulate HIF-1αexpression17,18.The enzyme,whichrequires oxygen,ferrous iron and 2-oxoglutarate forVOLUME 2 | JANUARY 2002 | 39NATURE REVIEWS |CANCER© 2001 Macmillan Magazines Ltd
REVIEWSdevelopment25.Hif-2αknockout embryos die because ofadrenal insufficiency,although they can survive withadrenal catecholamine replacement therapy26.Anotherstudy reported that Hif-2α-null embryos show vasculardisorganization throughout the yolk sac and embryoproper27.The differences between these two models isnot yet clearly understood.The HIF-1 complex is also involved in tumorigene-sis.Mouse hepatoma cell lines that express mutatedforms ofARNT form much smaller tumours thatexpress only low levels ofVEGF and do not becomehighly vascularized28.ARNT,however,also interactswith other transcription factors,such as the aryl hydro-carbon receptor,so these results are not definitively dueto loss ofHIF-1 function.Several studies have associated HIF-1αexpressionwith human cancer progression.Histological analyseshave shown that an increased level ofintracellularHIF-1αis associated with poor prognosis and resis-tance to therapy in head and neck cancer,ovarian can-cerand oesophageal cancer29,30.HIF-1αlevelsincreased in the cytoplasm and the nucleus ofcellsstained in various solid tumours29.In a separate study,HIF-1αwas overexpressed in colon,breast,gastric,lung,skin,ovarian,pancreatic,prostateand renal carci-nomas,and associated with cell proliferation30.HIF-1αmRNA is also upregulated early during wound healingand experimental skin carcinogenesis31.HIF-2αexpression is also increased in some cancercell types,such as renal cancer cells and cerebellar hae-mangiogblastomas32.Although there are fewer studieson the role ofHIF-2αin cancer development,someindicate that this protein is mainly expressed in thestromal macrophagesrather than in the epithelial can-cer cells29.Stromal cells might mediate a differentresponse to hypoxia compared with normal epithelialor cancer cells.Hypoxia-regulated pathwaysNormoxiaProlyl hydroxylaseO2Iron chelationUbE1UbCUL2RBX1E2Elongin-BElongin-CVHLOHHIF-1αHIF-αAnti-HIF-1α RNACBP/p300HIF-1αARNTHREPol IIcomplexTarget geneUbDegradationProteasomeinhibitorsHypoxiaNucleusFigure 1 |HIF-1 pathway.In the presence of oxygen (O2), prolyl hydroxylase post-translationallymodifies hypoxia-inducible transcription factor (HIF)-1α, allowing it to interact with the vonHippel–Lindau (VHL) complex. Prolyl hydroxylase contains an iron moiety, so iron chelation inhibitsthis activity. VHL is part of a larger complex that includes elongin-B, elongin-C, CUL2(REF.16),RBX1and a ubiquitin-conjugating enzyme (E2). This complex, together with a ubiquitin-activatingenzyme (E1), mediates the ubiquitylation (Ub) of HIF-1α95,96. The Ub modification targets HIF-1αfordegradation, which can be blocked by proteasome inhibitors. In the absence of oxygen, prolylhydroxylase cannot modify HIF-1α,and the protein remains stable. Stabilized HIF-1αis translocatedto the nucleus, where it interacts with cofactors such as aryl hydrocarbon receptor nucleartranslocator (ARNT), CBP/p300 and the DNA polymerase II (Pol II) complex to bind to hypoxia-responsive element (HREs) and activate transcription of target genes97. ARNT2(REF.98)and MOP3(REF.99)are other proteins that have been shown to heterodimerize with HIF-1α (not shown). Anatural HIF-1αantisense mRNA has been found in renal cancer100.activity,covalently modifies HIF-1α19,converting it to ahydroxylated form.This form ofHIF-1αcan then inter-act with the VHL protein.Under hypoxic conditions,however,this post-translational modification no longeroccurs.HIF-1αremains stable and can upregulateexpression ofits target genes.It is interesting that 2-oxoglutarate is involved inHIF-1αregulation,because there is evidence thatmitochondria,which produce this molecule,mightalso be involved in oxygen sensing,possibly by releas-ing free radicals that modify HIF-1α20.The role ofthemitochondria in the hypoxia response,however,iscontroversial and has not been confirmed21,22.The reg-ulation ofHIF-1αby prolyl hydroxylase also explainsprevious observations that desferrioxamine,an ironchelator,can activate HIF-1α.Desferrioxamineinhibits 2-oxoglutarate’s prolyl hydroxylase activity,leading to stabilization ofHIF-1α.There are now threeknown prolyl hydroxylases that modify HIF,and fur-ther research is required to determine the expressionpattern,mechanisms ofregulation and role ofthisenzyme in cancer pathogenesis23,24.HIF-1 in embryonic and tumour developmentHypoxia-inducible genes regulate several biologicalprocesses,including cell proliferation,angiogenesis,metabolism,apoptosis,immortalization and migra-tion(BOX 1).Cancer cells have a variety ofmecha-nisms to take advantage ofsome ofthese responses(for example,angiogenesis induction),and to evadeothers (for example,apoptosis).Many ofthe knownoncogenic signalling pathways33overlap withhypoxia-induced pathways (TABLE 1;FIG.2).Expressionprofiling studies have highlighted many ofthe genesthat are regulated by hypoxia and by HIF-1α34–37.They include angiogenic factors,proliferation andcell-adhesion genes.Proliferation.Hypoxia induces expression ofvariousgrowth factors that are known to promote cell prolif-eration.This proliferation is normally involved ininitiating cell migration and regeneration after acuteor chronic hypoxia damage.HIF-1αinduces produc-tion ofgrowth factors such as transforming growthfactor-βand platelet-derived growth factor34–37(BOX 1).The p42/p44mitogen-activated protein kinases,www.nature.com/reviews/cancerHIF-1 is required for normal embryogenesis,becausemice lacking Hif-1αor its homologue Hif-2α(EPAS)both die in utero.Hif-1αknockout embryos die at an early stage and undergo abnormal vascular | JANUARY 2002 | VOLUME 240© 2001 Macmillan Magazines LtdREVIEWSofapoptosis.The PI3K pathway is inhibited by thephosphoinositide phosphatase PTEN,and mutations inPTEN enhance HIF-1-activated responses41.PTEN reg-ulates cell growth and proliferation,and is deleted ormutated in several human cancers,including glioblas-toma,endometrialtumours and prostate cancer.So,PTEN mutations might promote tumour growth bysynergistically promoting HIF-mediated responses.HIF-1 also seems to interact with the oncogenic RASpathway,because loss ofHIF-1αnegatively affectstumour growth in HRAS-transformed cell lines42.However,this negative effect is not due to deficient vas-cularization.Despite differences in VEGF expression,vascular density is similar in wild-type and HIF-1α-nulltumours.This indicates that other pathways down-stream ofHIF-1 are involved in tumorigenesis — possi-bly those relating to the anabolic effects ofglycolysis43.Experiments involving HIF-1α-null embryonic stem-cell-derived tumours have demonstrated oppositeresults,showing reduced vascularization and increasedtumour growth.Another HIF-1-mediated pathway,such as hypoxia-induced apoptosis,might be reduced inthese cells44.Others have observed that HIF-1αdeletioninhibits both cell growth and angiogenesis45.The HIF-1-mediated hypoxia response is therefore complex,anddifferent pathways are likely to be activated in differentcell types.The most well-studied HIF-1α-activated growth fac-tors regulate endothelial-cell proliferation and blood-vessel formation.HIF-1 activates transcription ofVEGFand one ofits receptors,VEGF receptor 1(VEGFR1/FLT-1).VEGF is a key angiogenic factor thatis secreted by cancer cells and normal cells in response tohypoxia.Its receptors — VEGFR1 and VEGFR2— areprimarily expressed on endothelial cells.Hypoxia-induced angiogenesis is blocked by inhibitors ofonco-gene signalling pathways,such as agents that inhibitRAS,epidermal growth factor receptor,and the receptortyrosine kinase ERBB2(HER2/neu).This indicates thatthere is crosstalk between oncogenic and hypoxia-response pathways(FIG.2).HIF-1 activation can also leadto reduced expression ofanti-angiogenic proteins suchas THROMBOSPONDIN-1 and -2(REFS 46,47).Glycolysis.Under hypoxic conditions,cells switchtheir methods ofglucose metabolism from the oxy-gen-dependent tricarboxylic acid (TCA) cycle to gly-colysis,the oxygen-independent metabolic pathway.Hypoxic cancer cells use glycolysis as a primary mech-anism ofATP production,and cellular transformationhas been associated with induction ofglycolysis7,48.Glycolysis provides only two ATP molecules for eachglucose molecule,in contrast to the TCA cycle,whichprovides 38 ATP molecules.HIF-1 has been shown toregulate expression ofall the enzymes in the glycolyticpathway,as well as expression ofthe glucose trans-porters GLUT1and GLUT3(REF.49),which mediatecellular glucose uptake.Recent studies indicate thatincreased glycolysis is a normal response to prolifera-tion,and that migrating cells also use this pathway asan energy source43.The intermediary metabolites ofVOLUME 2 | JANUARY 2002 | 41Table 1 | Factors that regulate HIF-1αexpression or functionPathwayLigands of tyrosine kinase receptors (EGF, IGF1 and IGF2, insulin, PDGF)Ligands of other receptors(thrombin, angiotensin)Amplified receptor ERBB2Kaposi’s sarcoma virus G-protein- coupled receptorp42/p44 MAPKRAS expressionSmall G-protein RAC1PTEN inhibitionv-SRCDiacyl glycerol kinase Hepatitis B virus protein X Action on HIF-1αIncrease expressionIncrease proteinIncreases translationIncreases transcriptional activityPhosphorylates HIF-1αIncreases proteinIncreases proteinIncreases proteinIncreases HIF-1αmRNAIncreases proteinIncreases VEGF transcriptionReferences104105106107384910841109110111EGF, epidermal growth factor; HIF, hypoxia-inducible transcription factor; IGF, insulin-like growthfactor; MAPK, mitogen-activated protein kinase; PDGF, platelet-derived growth factor; VEGF,vascular endothelial growth factor.THROMBOSPONDINSA multigene family ofextracellular proteins that inhibitangiogenesis through severalmechanisms,includingupregulation ofTGF-βanddecreasing the cellular responseto VEGF.which regulate cell proliferation in response to extra-cellular growth factors,have been shown to phospho-rylate HIF-1αand activate transcription ofHIF-1target genes38.This pathway has also been shown toactivate HIF-2α39.Phosphatidylinositol 3-OH kinase (PI3K) activity isalso increased in some cell types under hypoxic condi-tions40.PI3K is one ofthe key downstream mediators ofmany tyrosine kinase signalling pathways and isinvolved in regulating cell proliferation and suppressionHypoxiaOncogene signallingSRCRASProtein kinase CPI3KHIF-1αPhosphorylated,stabilizedTranscription factorsAP-1ETSCREBRNA stabilization-HuRNucleusHIF-1αARNTHREVEGF(Angiogenesis)GLUT1(Glucosetransport)LDH-A(Glycolytic pathway)NOSEPO(Eythropoiesis)Hypoxia-regulated genesFigure 2 |Other factors involved in HIF-1 activation of hypoxia-response genes. Underhypoxic conditions, hypoxia-inducible transcription factor (HIF)-1αis phosphorylated andstabilized through oncogenic signalling pathways involving SRC, RAS, protein kinase C andphosphatidylinositol 3-OH kinase (PI3K). In the nucleus, HIF-1αcan also interact withtranscription factors such as AP-1, ETSand the cyclic AMP-response-element-binding protein(CREB) to activate transcription. RNA-binding proteins, such as HuR, help to stabilizemRNA101,102. HIF-1α-activated genes include vascular endothelial growth factor (VEGF), whichpromotes angiogenesis; glucose transporter 1 (GLUT1), which activates glucose transport;lactate dehydrogenase (LDH-A), which is involved in the glycolytic pathway; and erythropoietin(EPO), which induces erythropoiesis. HIF-1αalso activates transcription of nitric oxide synthase(NOS)103, which promotes angiogenesis and vasodilation.NATURE REVIEWS |CANCER© 2001 Macmillan Magazines LtdREVIEWS(HDACs) — implicated in alteration ofchromatinassembly and tumorigenesis — are also activated byhypoxia.A specific HDAC inhibitor,trichostatin A(TSA),reduces hypoxia-induced angiogenesis in theLewis lung carcinoma model53.Apoptosis.Hypoxia induces apoptosis by a number ofHIF-1-mediated and -independent pathways (FIG.3).Hypoxic conditions reduce proliferation and increaseapoptosis in wild-type embryonic stem cells,but not inHIF-1α-null cells44.HIF-1αactivates expression oftwopro-apoptotic proteins — NIXand NIP3— in a widerange ofcell lines54–56.The mechanism by which NIP3causes cell death seems to be a combination ofbothnecrosis and apoptosis,termed ‘aponecrosis’.NIP3 is amember ofthe BBL-2 family that localizes to mitochon-dria,although this is not essential for cell death55.NIP3-mediated cell death is independent ofAPAF-1,caspaseactivation,cytochrome crelease,and nuclear transloca-tion ofapoptosis-inducing factor.Cells with active NIP3have phenotypes that are typical ofnecrosis — earlyplasma-membrane permeability,mitochondrial dam-age,extensive cytoplasmic vacuolation and mitochon-drial autophagy.NIP3 has been proposed to mediatenecrosis-like cell death by opening mitochondrial poresand inducing mitochondrial dysfunction.HIF-1αhas also been shown to promote p53-depen-dent apoptosis57(FIG.3).When tumour cells with andwithout Trp53(the gene that encodes p53 in mice)mutations are combined and grown in vivoin mice,Trp53-mutant cells survive better in hypoxic areas thando wild-type cells,indicating that p53 is involved inhypoxia-induced cell death58.Under hypoxic conditions,p53 is stabilized to a form that blocks transcription butthen requires further modification by phosphorylationto cause apoptosis59.HIF-1αhas been reported to pro-mote p53-dependent apoptosis,which is mediated byAPAF-1 and caspase-9(REF.60).Early studies showed thatp53 directly interacts with HIF-1αand blocks HIF-1α’sability to activate transcription.Certain mutations inp53were reported to remove this block61.The ability ofHIF-1αto interact with p53 depends on the phosphory-lation status ofHIF-1α.During hypoxia-induced apop-tosis,only the phosphorylated HIF-1αbinds ARNT57.Bycontrast,the dephosphorylated form ofHIF-1αbindsp53 and induces apoptosis.These results indicate that the functions ofHIF-1αvary with its phosphorylation status and that dephos-phorylated HIF-1αmight mediate apoptosis by bindingand stabilizing p53,or p53 might prevent HIF-1αfromactivating transcription ofanti-apoptotic genes.p53 hasalso been reported to target HIF-1αto a degradationpathway that involves MDM2(REF.62).However,not allthese experiments have been reproduced,and HIF-1αfunctions in both p53-positive and -null cells.Tumour cells have developed many mechanisms toevade HIF-1-mediated cell death under hypoxic con-ditions.For example,induction ofthe anti-apoptoticgene IAP2occurs by a HIF-1-independent hypoxia-driven pathway in cultured cancer cells63.But when docancer cells undergo changes that allow them to evadewww.nature.com/reviews/cancerIGFBP-3IGF-1RIGF-1PI3KAKTBADNIXHIF-1αARNTNucleusp53Cytochrome c-independentcell deathBAXNIP3Cytochrome cAPAF-1Procaspase-9BCL-2ApoptosisCaspase-3Caspase-9Figure 3 |Hypoxia regulation of cell-death pathways.HIF-1 (a complex of HIF-1αand ARNT)activates transcription of many pro-apoptotic genes, some of which are shown here. HIF-1 alsointeracts with the tumour suppressor p53 to promote p53-dependent apoptosis57. HIF-1 activatesthe transcriptional activity of p53, which leads to transcription of many pro-apoptotic proteins, suchas BAX. BAX functions at the mitochondrial membrane to promote release of cytochrome c.Cytosolic cytochrome cinteracts with the apoptotic protease-activating factor-1 (APAF-1),acitvating procaspase-9 conversion to caspase-9. Caspase-9 then activates caspase-3, leadingto apoptosis. The pro-apoptotic protein BADfunctions at the mitochondrial membrane in a similarmanner to BAX, by promoting cytochrome crelease. BAD can be inhibited by the kinase AKT,which is activated by phosphotidylinositol 3-OH kinase (PI3K), which is induced by insulin-likegrowth factor-1 (IGF-1) signalling through the IGF-1 receptor (IGF-1R). IGF-1 signalling is thereforeanti-apoptotic. HIF-1 activates transcription of the pro-apoptotic protein IGF-binding protein-3(IGFBP-3), which blocks IGF-1 signalling. HIF-1 also activates expression of NIP3 and NIX54–56,which induce a mitochondrial-pore permeability transition and cell death through a mechanism thatdoes not involve cytochrome crelease or caspases. Hypoxia has also been reported todownregulate expression of the anti-apoptotic protein BCL-2 in some cell types57.the glycolytic pathway provide the precursors for syn-thesis ofglycine,serine,purines,pyrimidines andphospholipids,all ofwhich are essential for cellgrowth and maintenance ofcells under stress.Tumour cell lines that are deficient in the HIF-1 signalling pathway have lower ATP and glycine concentrations in vivo(A.L.H.,J.Griffiths and M.Stubbs,unpublished observations).TELOMERASEA ribonucleoprotein thatmaintains telomere length.Telomerase activity is repressedin most normal adult humansomatic tissues,limitingreplicative capacity.Reactivationoftelomerase is believed to be anecessary event for the sustainedgrowth ofmost humantumours.FRAGILE SITEA site in a chromosome that issusceptible to chromosomebreakage and fusion with otherchromosomes.Immortalization and genetic instability.Hypoxia alsoaffects cellular DNA and chromosomes in ways thatcould promote transformation.TELOMERASEactivityincreases when cancer and endothelial cells are placedunder hypoxic conditions50,promoting cellularimmortalization.Hypoxia has been shown to inducegene amplification and DNA breaks at FRAGILE SITES51,and to disrupt repair ofDNA damage.Using an assayfor repair that is based on host-cell reactivation ofultraviolet-damaged plasmid DNA,cells exposed tohypoxia and low pH have a diminished capacity forDNA repair compared with control cells grown understandard culture conditions52.Histone deacetylases42| JANUARY 2002 | VOLUME 2© 2001 Macmillan Magazines LtdREVIEWSthe apoptotic programme? Hypoxia occurs in theearly stages oftumour development (before metasta-sis),and is therefore commonly observed in non-inva-sive tumours such as intraductal breast cancer.Theactivation ofpro-apoptotic genes is therefore alsolikely to occur during these early stages oftumourdevelopment.So there might be early selective pressureon cancer cells to escape hypoxia-induced apoptosis.A similar selection process might occur in later stagesoftumour development,promoting the aggressivephenotype that is associated with hypoxia.pH regulation.The metabolic activities ofcancer cellsaffect the overall pH oftumours.Tumours have beenshown to adapt to pH changes and grow at lower pHsthan are found in normal tissues,giving the tumour agrowth advantage.Many proteases are activated underacidic conditions,promoting tumour invasion ofsurrounding tissue.Glycolysis is thought to be the main mechanism bywhich tumours lower their pH,through generation oflactic acid.CARBONIC ANHYDRASES,which reversibly convertcarbon dioxide and water to carbonic acid,might also beinvolved.The activities oftwo isoforms — carbonicanhydrase-9and -12 — were reported to be downregu-lated by VHLand strongly induced by hypoxia in arange oftumor cell lines65,indicating that their transcrip-tion might be regulated by HIF-1.Transcription ofcar-bonic anhydrase-9 is activated by hypoxiaand sup-pressed in normoxia66.This enzyme is expressed inperinecrotic areas in a wide range oftumour types,andhigh expression levels have been associated with poorprognosis67.Although it is clear that lactate produced byglycolysis generates an acidic micro-environment intumours,tumour cells that express defective forms oflactate dehydrogenase still maintain a low extracellularpH68.This indicates that carbonic anhydrase activitymight also contribute to the tumour’s low pH.Growth inhibitory signals.Normal cells undergo cell-cycle arrest under conditions ofsevere hypoxia,but arecapable ofrecovering ifhypoxia is not prolonged.Thecyclin-dependent kinases WAF1(p21) and KIP1(p27)might be involved in mediating hypoxia-related growtharrest.KIP1 was shown to be induced by hypoxia,lead-ing to G1/S arrest69.A similar study reported that WAF1and KIP1 regulate cell-cycle re-entry after hypoxic stress,but are not necessary for hypoxia-induced arrest70.There is evidence that changes in pH are more impor-tant than hypoxia per sein cell death117.Another mecha-nism to prevent cells from proliferating under hypoxicconditions is induction ofdifferentiation.INVOLUCRINis amarker ofsquamous cell differentiation (and thereforeinhibition ofproliferation).Involucrin expression hasbeen shown to co-localize with hypoxic areas ofsquamous-cell cancers71.Other hypoxia-response factorscyclic AMP-response-element-binding protein (CREB)72and nuclear factor-κB (NF-κB)73.These factors seem toact independently ofHIF-1,but further research isrequired to find out how they are regulated.Hypoxia also induces blood-clot formation.Monocytes cultured under hypoxic conditions upreg-ulate expression ofthe transcription factor earlygrowth response-1 (EGR-1),causing expression ofthe cell-surface protein tissue factor (TF),leading tovascular fibrin deposition and blood-clotformation74.Mononuclear phagocytes and vascularsmooth muscle cells also upregulate TF underhypoxic conditions75.Accordingly,increases in coagu-lation,DEEP-VEIN THROMBOSISand PULMONARY EMBOLISMTHROMBOSISare recognized features ofcancer.Bloodclots also induce platelets to release angiogenic factorssuch as VEGF,promoting revascularization oftheclot,but also promoting tumour vascularization.TheEGR-1 pathway is also activated during reoxygena-tion and might therefore be involved in acute intermittent hypoxia.Metal-transcription factor-1is another hypoxia-induced transcription factor.It activates expression ofplacental growth factor(PlGF) — another ligand ofVEGFR1 — and metallothionein76.PlGF has beenshown to synergize with VEGF to promoteangiogenesis77.By upregulating PlGF,endothelial andcancer cells amplify signalling through the VEGFR1.So,to continue growing under hypoxic conditions,tumours take advantage ofa variety ofhypoxia-inducedgrowth-promoting signals,and modify growth-inhibitory signalling events.In any population ofcancercells,different aspects ofthese pathways are affected,andthe subset ofcells that ends up with the optimum profilecan survive and proliferate.Hypoxia-targeted therapiesCARBONIC ANHYDRASESEnzymes that convert carbondioxide to carbonic acid andthen to protons and bicarbonateions.INVOLUCRINA cytoskeletal protein insquamous cells that is involvedin their terminal differentiation.DEEP-VEIN THROMBOSISThe process ofclot formation inthe venous circulation,usually inthe lower limbs or pelvis.PULMONARY EMBOLISMTHROMBOSISThe occlusion ofpulmonaryveins by clots dislodged fromperipheral deep veins,usuallyfrom the lower extremities.ACCELERATED RADIOTHERAPYWITH CARBOGEN ANDNICOTINAMIDEReducing tissue hypoxia.Correction ofhypoxia beforeradiation therapy has been routine for many years,byusing blood transfusion to bring the haemoglobinconcentration in patients above 12 g/l — a concentra-tion associated with better response to therapy78.EPOis given to patients to protect them from chemother-apy-associated anaemia,as well as to patients sufferingfrom anaemia ofchronic disorders.Intriguingly,clinicaltrial results have indicated that EPO might improvethe survival ofcancer patients79.It is possible thatEPO,by restoring tumour oxygenation,allows cancercells to proliferate and therefore become more sensitiveto chemotherapy.EPO might also shut down hypoxia-mediated angiogenesis.Other approaches to improveblood flow and oxygen delivery to tumours includethe use ofACCELERATED RADIOTHERAPY WITH CARBOGEN AND80NICOTINAMIDE.Inhibitors ofRAS also reduce oxygenconsumption by tumours and so increase local oxygenconcentrations81.Hypoxia-activated prodrugs.Another way to exploithypoxia is to induce tumour-specific toxicity using pro-drugs that are only activated under hypoxic conditions82.For example,the drug tirapazamine inhibits DNA repairVOLUME 2 | JANUARY 2002 | 43Experimental technique toimprove blood flow and oxygendelivery to tumours.Although most research on hypoxia has concentrated onthe HIF-1 pathway,there are several other transcriptionfactors that are activated by hypoxia.These include theNATURE REVIEWS |CANCER© 2001 Macmillan Magazines Ltd
REVIEWSMacrophages are also attracted to areas ofhypoxia,and might be developed as a useful mechanism fordelivering therapeutic genes to hypoxic areas.Thesecells can be transfected with adenoviral vectors,andmight be used to deliver therapeutic HRE-containinggenes to hypoxic areas.Genetically engineeredmacrophages have been shown to migrate to hypoxicareas in xenografts and upregulate HRE-driven trans-genes in a hypoxia-dependent manner91.Herpes sim-plex virus thymidine kinase (HSV-TK) gene expressiondriven by the VEGFpromoter was shown to be effec-tive in mediating ganciclovir-induced killing ofhighlymetastatic Lewis lung carcinoma cells under hypoxicconditions in vivo(REF.92).Future directionsTable 2 |Oncogenic signalling inhibitors that also block HIF-1DrugHerceptin, Iressa, herbimycin Calphostin C Wortmannin, LY294002 TargetTyrosine kinasesProtein kinase CPI3KReferences11211211311411587116PD98059 MAPKRapamycin FRAP/mTORDiphenylene iodonium MannoheptuloseRedox signallingGlucokinaseFRAP, FKBP/rapamycin-associated protein; MAPK, mitogen-activated protein kinase; PI3K,phosphatidylinositol 3-OH kinase.when activated under hypoxic conditions,and acts syn-ergistically with both radiation therapy and chemother-apy.Tirapazamine,used in combination with platinumtherapy,improves survival ofpatients with non-small-cell lung cancer83.Other non-cytotoxic compounds thatbind to substratesonly under low oxygen conditions canbe radiolabelled and used in combination with positronemission tomography or conventional γ-camera imagingto identify and observe hypoxic areas within tumours84.HIF-1 and HREs.Because activation ofHIF-1 has beenassociated with a variety oftumours and oncogenicpathways,it is a prime target for anticancer therapies.Therapies are under development to block HIF-1αitselfor HIF-1α-interacting proteins.Recent data indi-cate that HIF-1αantisense therapy might act synergis-tically with immunotherapy85.In a mouse model ofthymic T-cell lymphoma,stimulation ofspecific T-cellresponses with a co-activating ligand for T cellsinduced regression oflymphoma but could not curemice.In vivodelivery ofantisense to HIF-1αalone bydirect intratumour injection inhibited tumour growth,but combination ofthe two treatments caused markedtumour regression and a sustained antitumourimmune response85.A gene-therapy strategy to blockthe interaction between HIF-1αand its transcriptionalco-activator CBP/p300led to attenuation ofhypoxia-inducible gene expression and inhibition oftumourgrowth in a mouse xenograft model46.There are also natural antagonists to HIF-1α,such asp34srj (for serine–glycine-rich junction).This protein isinduced by hypoxia and also blocks the interaction ofHIF-1αwith CBP/p300 (REF.86).Small-moleculeinhibitors ofHIF-1αfunction,such as diphenyleneiodonium87 (TABLE 2),have been reported,although itsexact mechanism is not clear.HREs linked to marker genes or prodrug activa-tion systems can be used to selectively activate thera-peutics in hypoxic regions88,.For example,gene-therapy vectors that carry pro-apoptotic oranti-proliferation genes driven by HREs can be selec-tively targeted to cancer cells in hypoxic regions ofthetumour.Anaerobic bacteria provide another methodofdelivering genes specifically to hypoxic cells.Anaerobic bacteria have shown tumour-specific proliferation in animal models and inhibited tumourgrowth90,and clinical trials are underway.| JANUARY 2002 | VOLUME 2The ability to survive under hypoxic conditions is oneofthe fundamental physiological differences betweentumour cells and normal cells,although the qualita-tive and quantitative differences in the hypoxiaresponse by both cell types are not known.In bothtumour and normal cells,hypoxia activates a complextranscriptome that includes pathways downstream ofHIF-1αand other signalling pathways.Many ques-tions need to be answered about how cells sensehypoxia and activate these pathways,and how thepathways are integrated.For example,it is importantto learn more about the function ofoxygen sensorssuch as proline hydroxylase.It might be a target ofoncogenesand also a potential target for anticancerdrugs.There are an increasing number ofproteinsfound to interact with VHL besides HIF-1α,and thesemight also be important in the response to hypoxia.These include fibronectinand heterogeneous nuclearribonucleoprotein (hnRNP)93,94.Also,most pathologyand gene-expression studies are done on primarytumours,and little is known about hypoxia inmetastatic tumours.More research is required to determinethe ratioofthe pro-survival pathways and apoptotic pathwaysthat are activated by cancer cells in response tohypoxia,and how these are regulated.For example,atumour that overexpresses NIP3 instead ofVEGFmight undergo slower growth,because cells wouldundergo high levels ofapoptosis and a small amountofangiogenesis.Therapeutics that disrupt HIF func-tion in these cells might only promote tumourgrowth.It will be important to analyse expressionpatterns ofhypoxia-response genes in human cancercells by microarray analysis,and relate the gene-expression patterns to levels ofapoptosis,angiogenesisand metastasis.An important issue that is difficult to prove in theclinic is whether hypoxia generates an aggressive tumourphenotype or whether an aggressive tumour phenotypegenerates hypoxia.In one case,it is possible that tumoursthat contain activating MYC or RAS mutations have anaggressive phenotype,increasing oxygen consumptionand generating hypoxia — these factors would switch onthe HIF-1 signalling pathway.Alternatively,hypoxictumour conditions might activate expression ofgeneswww.nature.com/reviews/cancer44© 2001 Macmillan Magazines Ltd
REVIEWSthat promote tumour growth,leading to a more aggres-sive phenotype.These different possibilities could lead todifferent therapeutic results.Ifthe HIF pathway is modi-fied,for example,in those tumour types in which thehypoxia was generating the aggressive phenotype,cor-rection ofanaemia (reoxygenation) and a blockade ofHIF function would reduce tumour growth.Conversely,ifan aggressive phenotype is the cause ofhypoxia,cor-recting anaemia might actually enhance tumour growthby providing more oxygen to the tumour cells,andblockade ofHIF would have little effect.The full profile ofhypoxia-responsive genes islikely to be known soon,as microarray analysis is underway.It will take much longer,however,to fig-ure out the mechanisms by which hypoxia modulatestumour growth and differentiates the tissue- and cell-specific patterns ofresponse,to understand fully howcancer cells manipulate these pathways to promote theirown survival.It is becoming clear that cancer involvesthe dysregulation ofmany signalling pathways,and thatthe greatest therapeutic effects are likely to be achievedby treating patients with several different types ofanti-cancer drugs,just as we use combination therapies forinfections and hypertension.Those inhibiting the effectsofhypoxia are likely to be one component.However,themain hypoxia-response pathways must still be defined.Although — clearly — VEGF is one ofthem,there aremany others to discover.Thomlinson, R. & Gray, L. 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REVIEWSof hypoxia-inducible factor-1α. Mol. Pharmacol.59,1216–1224 (2001).115. Zhong, H. et al. Modulation of hypoxia-inducible factor-1αexpression by the epidermal growth factor/phosphatidylinositol3-kinase/PTEN/AKT/FRAP pathway in human prostatecancer cells: implications for tumor angiogenesis andtherapeutics. Cancer Res. 60, 1541–1545 (2000).Reports that oncogenic signalling pathways regulateHIF-1α, independently of hypoxia. Inhibitors of thesepathways also block hypoxia-induced HIF-1 signalling.116. Board, M., Colquhoun, A. & Newsholme, E. A. High Kmglucose-phosphorylating (glucokinase) activities in a rangeof tumour cell lines and inhibition of rates of tumour growthby the specific enzyme inhibitor mannoheptulose. CancerRes. 55, 3278–3285 (1995).117.Schmaltz, C., Hardenbergh, P. H., Wells, A. & Fisher, D. E.Regulation of proliferation-survival decisions during tumorcell hypoxia. Mol. Cell. Biol. 18, 2845–2854 (1998).Shows that cell death in hypoxia might be mediatedby low pH rather than low oxygen.Online linksDATABASESThe following terms in this article are linked online to:CancerNet:http://cancernet.nci.nih.gov/breast cancer | cervical cancer | colon carcinoma | endometrialtumours | gastric carcinoma | glioblastoma | head and necktumours | oesophageal cancer | ovarian cancer | pancreaticcarcinoma | prostate carcinoma | renal cancer | skin carcinomaGenBank:http://www.ncbi.nlm.nih.gov/HSV-TKLocusLink:http://www.ncbi.nlm.nih.gov/LocusLink/α-integrin | α-adrenergic receptor | acetoacetyl CoA thiolase |adenylate kinase-3 | adrenomedullin | AKT | angiopoietin-2 |annexin V | APAF-1 | ARNT | ARNT2 | BAD | BAX | carbonicanhydrase-9 | caspase-3 | caspase-9 | CBP | CD99 |ceruloplasmin | collagen-5α1 | CREB | CUL2 | cyclin G2 |cyclooxygenase-2 | cytochrome c| DEC1 | EGR-1 | elongin-B |elongin-C | endothelin-1 | endothelin-2 | enolase-1 | epidermalgrowth factor receptor | ERBB2 | erythtopoietin | ETS | ferritinlight chain | fibroblast growth factor-3 | fibronectin | FOS |GADD153 | GLUT1 | GLUT3 | glyceraldehyde-3-phosphatedehydrogenase | HAP-1 | heat-shock factor | heme oxygenase-1 | hepatocyte growth factor | hexokinase-1 | hexokinase-2 | Hif-1α| HIF-1α| Hif-2α| HIF-2α| hnRNP |IAP2| IGF-1 | IGF-2 | IGFbinding protein-1 | IGF binding protein-2 | IGF binding protein-3 |IGF-1R | interleukin-6 | interleukin-8 | intestinal trefoil factor | JUN| Ku70 | Ku80 | KIP1 | lactate dehydrogenase-A | L1CAM |lipocortin | low-density lipoprotein receptor-related protein |macrophage inhibitory factor | matrix metalloproteinase-13 |MDM2 | metal-regulatory transcription factor-1 |metalloproteinases | metallothionein | monocyte chemotacticprotein-1 | MOP3 | NF-κB | NIP3 | nitric oxide synthase | NIX |osteopontin | p300 | p44 mitogen-activated kinase | p53 |phosphoglycerate kinase-1 | phosphoribosyl pyrophosphatesynthetase | PI3K | placental growth factor | plasminogenactivator inhibitor-1 | platelet-derived growth factor | platelet-derived growth factor-B | proline-4 hydroxylase | PTEN |pyruvate kinase-M | RBX1 | spermidine N1-acetyl transferase |SRC | TGF-α| TGF-β1 | TGF-β3 | thioredoxin | Tie-2 | transferrin| transferrin receptor | transgelin | transglutaminse-2 | tyrosinehydroxylase | urokinase receptor | VEGF | VEGFR1 | VEGFR2 |VHL | vimentin | WAF1FURTHER INFORMATIONSRI web site on hypoxia in cancer:http://www.sri.com/pharmdisc/cancer_biology/laderoute.htmlAccess to this interactive links box is free online.AcknowledgementsI thank I. Stratford and C. West for their helpful comments, and L. Richards for administrative assistance.NATURE REVIEWS |CANCERVOLUME 2 | JANUARY 2002 | 47© 2001 Macmillan Magazines Ltd
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