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120 | FEBRUARY 2001 | VOLUME 2 www.nature.com/reviews/genetics

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The gliomas are a large collection of primary braintumours that have morphology and gene-expressioncharacteristics similar to GLIA, ASTROCYTES and OLIGODEN-

DROCYTES (and their precursors), which together supportthe functions of neurons in the brain. Because thesetumours arise in the central nervous system (CNS) andaffect surrounding brain structures, patients withgliomas commonly develop symptoms that includeheadaches and seizures, or focal neurological alterationsthat cause weakness of one limb or language distur-bance. Gliomas are usually detected by computedtomography (CT) and magnetic resonance imaging(MRI) scans. Most gliomas arise sporadically and arenot inherited within families; however, patients withgliomas frequently have a family history of diverse can-cer types. About 30,000 patients in the United States arenewly diagnosed with a glioma each year.

Grossly, glioblastoma multiformes (GBMs) are het-erogeneous intraparenchymal masses that show evidenceof necrosis and haemorrhage. Microscopically, they con-sist of several cell types: the glioma cells proper, hyper-proliferative endothelial cells, macrophages and trappedcells of the normal brain structures that are overrun bythe invading glioma1,2. Several histological characteristicsare used to grade and define gliomas (BOX 1). Theseinclude regions of necrosis, which are surrounded bydensely packed tumour cell nuclei and are referred to asbeing ‘pseudopalisading’. In addition, the blood vesselsboth within and adjacent to the tumour are

HYPERTROPHIED. Furthermore, the nuclei of tumour cellsare extremely variable in size and shape, a characteristiccalled ‘nuclear pleomorphism’. Tumour cells characteris-tically invade the adjacent normal brain parenchyma,migrating through the white matter tracts to collectaround blood vessels, neurons and at the edge of theBRAIN PARENCHYMA in the SUBPIAL region. These structures

GLIOMAGENESIS: GENETICALTERATIONS AND MOUSE MODELSEric C. Holland

Glioblastoma multiforme is the most malignant of the primary brain tumours and is almostalways fatal. The treatment strategies for this disease have not changed appreciably for manyyears and most are based on a limited understanding of the biology of the disease. However,in the past decade, characteristic genetic alterations have been identified in gliomas that mightunderlie the initiation or progression of the disease. Recent modelling experiments in mice arehelping to delineate the molecular aetiology of this disease and are providing systems toidentify and test novel and rational therapeutic strategies.

Departments ofNeurosurgery, Neurologyand Cell Biology, MemorialSloan Kettering CancerCenter, 1,275 York Avenue,New York, New York 10021USA. e-mail:[email protected]

Box 1 | Glioma grade and prognosis

Gliomas are the most common primary tumours in the brain and are divided into four clinical grades on the basis of their histology and prognosis. Patients withthe most malignant grade 4 gliomas, or glioblastomamultiforme (GBM), have a mean survival of about 1year, whereas patients with grade 3 or ANAPLASTIC

gliomas survive for 2–3 years, and those with grade 2gliomas can survive for as long as 10–15 years. Grade 1gliomas, also known as pilocytic astrocytomas, arecurable by surgery and might represent a separatedisease from the gliomas of other grades. GBMs eitherarise de novo or progress from lower grade to highergrade over time; the tumours of patients originallydiagnosed with a lower grade lesion will often progressto a GBM before their death. Glioma histologycorrelates with grade and survival, and includesincreased cell density and varied nuclear appearance in the lower grade gliomas, and further vascularproliferation and regional necrosis in the anaplasticgliomas and GBMs.

GLIA

The specialized connective tissueof the central nervous system.It is made up of glial cells such as astrocytes, oligodendrocytesand ependymal cells.

ASTROCYTE

One of the three main cell typesin the brain, the others beingneurons and oligodendrocytes.Astrocytes act as the scaffold thatmaintains the brain structureand that supports the functionsof both neurons andoligodendrocytes.

OLIGODENDROCYTES

One of the three main cell types that make up the brainparenchyma, the other twobeing neurons and astrocytes.Oligodendrocytes producemyelin, which insulates axons to alter the conductionproperties of neurons.

© 2001 Macmillan Magazines Ltd

NATURE REVIEWS | GENETICS VOLUME 2 | FEBRUARY 2001 | 121

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type of tumour that arises might be a result of both theactivity of specific pathways and the cell-of-origin inwhich these alterations occur.

Glioma biology is complex, and a full description ofall the tumorigenic processes that occur are beyond thescope of this article. Excellent reviews on glioma angio-genesis and invasion have been previously published4,5.In this review, I focus on the effects that genetic alter-ations in gliomas have on pathways that are involved insignalling during normal CNS cell development. Thedifferentiation status of the cell-of-origin for thesetumours might affect the outcome of such alterations insignalling. Distinguishing between those alterations thatcause the formation of these tumours and those that areepiphenomena of the tumour progression processrequires the technology to transfer these alterations tothe appropriate cell types in vivo.Various mouse model-ling strategies are being used at present to identify whichof these alterations are capable of inducing the forma-tion of gliomas and the role they have in the process.

Glioma cell-of-originMany cancer types, including gliomas, resemble undif-ferentiated cells in their gene expression and phenotypiccharacteristics. Some of the signalling pathways thataffect the differentiation and proliferation of glial prog-enitors are altered in gliomas. Therefore, a better under-standing of the roles that these pathways have in glial celldevelopment might provide insight into glioma biology,their relationship to glial cell development and potentialtherapeutic strategies for this disease. Glioma cells mostfrequently resemble immature astrocytes, immatureoligodendrocytes, or mixtures of the two cell types, intheir morphology and gene-expression characteristics.The cell type that gives rise to gliomas is not known,although there has been a significant amount of specula-tion on this point. It is a common, although unproven,idea that astrocytomas arise from astrocyte precursors,whereas the oligodendrogliomas arise from oligoden-drocyte precursors, and that mixed gliomas arise fromprogenitors of both astrocytes and oligodendrocytes.

The CNS stem cell gives rise to neuronal and glialprogenitors, which subsequently give rise to the maturecell types found in the brain, including neurons, oligo-dendrocytes and astrocytes6–9. The stages in these lineageshave been defined by cell morphology and by the expres-sion of specific markers (FIG. 1). Specific signal-transduc-tion pathways have been shown to control the differentia-tion of precursor cells into mature glia. In cell culture,PLATELET-DERIVED GROWTH FACTOR (PDGF) causes the oligo-dendroglial progenitor population to proliferate10, andcooperates with FIBROBLAST GROWTH FACTOR 2 (FGF2) to pre-vent that population’s further differentiation into matureoligodendrocytes11,12. Epidermal growth factor (EGF) andCILLIARY NEUROTROPHIC FACTOR (CNTF) force glial progeni-tors towards astrocytic and oligodendrocytic differentia-tion13,14. Although the effects of these growth factors onglial development are well documented, as are the sig-nalling pathways downstream of their receptors, ourunderstanding of how these pathways control cell fateand proliferation in glial development is still incomplete.

were originally described by Scherer over 60 years agoand are referred to as the secondary structures ofScherer3. The extent to which these tumours invade adja-cent structures is variable; at its extreme, large portions ofthe brain are diffusely infiltrated by individual tumourcells with no clear focus of tumour per se.

Low-grade gliomas are divided into two histologicalvariants: astrocytomas, which consist of cells with largeamounts of cytoplasm and which express the astrocyte-specific marker gene GFAP (which encodes GLIAL FIBRIL-

LARY ACIDIC PROTEIN), and oligodendrogliomas, which havesmall round nuclei, a minimal cytoplasm and which donot express GFAP.Very commonly, these tumours con-tain mixtures of both cell types; a tumour with greaterthan 25–30% of each cell type is described as a ‘mixedglioma’. The frequent occurrence of a mixed gliomamorphology suggests that some similarities existbetween glioma cells and bipotential glial progenitorsthat can differentiate into either oligodendrocytes orastrocytes. Many glioma studies do not distinguish theexact pathological diagnosis of many of these tumoursor take in to account their mixed character. Therefore,correlating specific molecular abnormalities with theregional histological appearance of these tumours is dif-ficult. Low-grade gliomas will often acquire the histo-logical and clinical characteristics of high-grade gliomasover time. This shift in malignancy probably occursthrough the acquisition of further mutations.

One characteristic of gliomas is significant: anincrease in gene and chromosomal deletion and ampli-fication often correlates with increasing clinical grade(BOX 2). Many of the biological pathways involved ingliomagenesis have been recognized by the identifica-tion of genes affected by these mutations. The geneticalterations found in gliomas mostly fall into two func-tional categories. As discussed below, one group of alter-ations activates the signal-transduction pathways down-stream of the tyrosine kinase receptors. The secondgroup of mutations disrupts the cell-cycle arrest path-ways that maintain cells in G1 arrest. Both of thesegroups affect signalling pathways that are altered inmany cancers and are therefore not glioma specific. The

HYPERTROPHY

A pathological increase in thesize of cells or the structure thatthey form.

BRAIN PARENCHYMA

The inner substance of the brainthat is composed primarily ofneurons, oligodendrocytes,astrocytes and blood vessels.

SUBPIA

The region directly below thepia, the membrane that formsthe limiting edge of the brain.Invading glioma cells tend toaccumulate in this region togenerate one of the classicsecondary structures ofhuman gliomas.

ANAPLASTIC

When cells or tissues revert to a more embryonic orundifferentiated form and have an increased capacity to multiply.

GLIAL FIBRILLARY ACIDIC

PROTEIN

(GFAP). An intermediate filamentprotein. The expression of itsgene is limited to astrocytes.

PLATELET-DERIVED GROWTH

FACTOR

(PDGF). A growth factor thatexists as a homodimer orheterodimer of PDGFA andPDGFB chains. This growthfactor binds to and activates the PDGF receptor PDGFRA or PDGFRB.

FIBROBLAST GROWTH FACTOR 2

(FGF2). This ligand binds to the FGF2 receptor, promotes the proliferation ofundifferentiated cells andstimulates angiogenesis.

Box 2 | Genetic alterations in gliomas

Several gene expression alterations and chromosomal abnormalities are commonlyfound in gliomas, and in some cases these mutations correlate with clinical grade. Inthe lower grade gliomas, several growth factors such as platelet-derived growth factor(PDGF), fibroblast growth factor 2 (FGF2) and cilliary neurotrophic growth factor(CNTF), and their receptors, are commonly overexpressed, and p53 is often mutated.The grade 3 (anaplastic) gliomas additionally show an increased occurrence ofdisrupted cell-cycle arrest pathways because of the deletion of INK4A, amplification ofcyclin-dependent kinase 4 (CDK4), or loss of retinoblastoma (RB). In addition to theabove mutations, the grade 4 gliomas, or GBMs, show frequent loss of 10q22–25, achromosomal region that carries several tumour suppressors, notably PTEN (forphosphatase and tensin homologue). Loss of PTEN expression leads to elevated AKTactivity. Amplification and activating mutations in the epidermal growth factorreceptor (EGFR) gene are also seen. Almost all these mutations lead to the disruption ofcell-cycle arrest. None of these specific mutations is found in all gliomas of a givengrade, but their frequent occurrence and correlation with tumour grade highlights theimportance of specific pathways in gliomagenesis. The sum effect of these alterationscontributes to the biology of these tumours.



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abnormally active in glial tumours, have a critical role ingliomagenesis. In the most malignant gliomas — GBMs— activating mutations have been found in the EGFR

gene, which encodes the EGF receptor. Between 30 and50% of GBMs show amplification or activating muta-tion of this gene to produce a constitutively active recep-tor in the absence of EGF ligand20. The pathways thatthe EGFR specifically activates to promote growth ofGBMs are not known at present.

Cell-culture experiments using several tumour celltypes, including glioma cell lines, have shown that thesegrowth-factor receptors activate several common sig-nalling pathways, including those that lead to the RASand AKT pathways (see below)21. It follows that at leastsome of the pathways activated by these receptors,whether singly or in combination, might be required forthe induction or progression of these glial tumours.

The RAS pathway is one of the best-studied signal-transduction pathways. RAS is indirectly activated bytyrosine kinase receptors and is active when bound toGTP but inactive when the GTP is hydrolysed to GDP.In its GTP-bound form, RAS activates many path-ways, most notably the RAL, RAC and RAF pathways,which lead to the mitogen-activated protein kinases(MAPKs) ERK, Jun kinase (JNK) and p38 (FIG. 2a)22.The net effect of these pathways is to enhance prolifer-ation, progression through the cell cycle and inhibi-tion of apoptosis. RAS-GTPase activator proteins suchas neurofibromatosis (NF1) inactivate RAS byhydrolysing GTP23. Loss of such proteins leads tochronically elevated RAS activity.

Which, if any, of these effects of RAS are involved inthe formation of gliomas in vivo remains unclear, asthere is conflicting evidence regarding the importanceof its activity in glioma formation. On the one hand,RAS activity was elevated in all 20 GBMs analysed inone study24. On the other hand, unlike other cancertypes, activating mutations in RAS do not occur ingliomas. However, mutations in a gene are not the onlymechanism for pathologically elevated activity of thecorresponding gene product. The level of RAS activity islikely to be controlled by a summation of upstreamstimuli and could be a pivotal criterion for transforma-tion. The experiments that would either prove or dis-prove this possibility have yet to be done.

A second pathway activated by tyrosine kinasereceptors that is known to be involved in glial differen-tiation is the AKT pathway, which is activated throughphosphosphatidylinositol-3-OH kinase (PI(3)K) and ismodulated by the tumour suppressor protein PTEN25

(FIG. 2b). In its active form, AKT (called AKT1 inhumans) has multiple effects, including enhancingmetabolism by activating GLYCOGEN SYNTHASE KINASE 3

(GSK3) and altering translation by activating the kinasecalled MAMMALIAN TARGET OF RAPAMYCIN (mTOR). In addi-tion, AKT inhibits apoptosis by inactivating BAD (aninhibitor of BCL2), causing CASPASE activation to be inhib-ited, and by inactivating the transcription factor FORK-

HEAD (FKHR), which promotes cell death26. Which ofthese multiple activities of AKT are involved in oncoge-nesis remains to be shown. Many GBMs show muta-

It is possible that the cell types that give rise togliomas are progenitors to the mature glia that thetumour cells most closely resemble. Alternatively, theoncogenic alterations that induce glioma formationmight cause mature oligodendrocytes and astrocytes todedifferentiate, thereby allowing these differentiatedcells to serve as the cell-of-origin for gliomas.

Altered growth-factor signallingThe genes that encode growth factors and the receptorsthat control glial cell differentiation frequently have ele-vated expression in gliomas. One common alteration ingliomas of all grades is the overproduction of growthfactors such as FGF2 (REFS 15,16), CNTF17,18 and PDGF19,and their receptors, by mechanisms other than by genet-ic mutation. An excess of growth factor or receptor isproduced, frequently in the same cell, which results inautocrine stimulation and increased activity of path-ways downstream of these receptors. It seems likely thatthese signalling pathways, which control the differentia-tion and proliferation of glial precursors and which are

CILLIARY NEUROTROPHIC

FACTOR

(CNTF). This ligand binds to the CNTF receptor andpromotes oligodendrocyte and astrocyte differentiation.

EPIDERMAL GROWTH FACTOR

RECEPTOR

(EGFR) This receptor is boundby epidermal growth factor and the transforming growthfactor-α.

GLYCOGEN SYNTHASE KINASE 3

(GSK3). A kinase involved inseveral biological processes,including glucose metabolismand signalling through the Wntpathway. GSK3 also functionsdownstream of AKT.

Neuroepithelialstem cells

FGF2 Glial-restrictedprecursors

PDGF, FGF2 O2A progenitors

Oligodendrocytes Type-2 astrocytes

O+, PLP+, GalC+,M+,A–,G–,S–,P–

G+,A+,S+,P– G+,S+,A–,P–

PDGF, FGF2

PDGF CNTF, EGF

N+,F+,E+

Neurons

CNTF PDGFFGF2

CNTF, EGF

Type-1 astrocytes

CNTF, EGF

A+,N+,F+,PLP+,P–

A+,P+,N+,PLP+,F+,V+/–,O–

EGF, FGF2Astrocyteprecursor cells

N+,V+,G–,S–

Figure 1 | Cellular differentiation in the central nervous system. Stages of CNS celldifferentiation that are defined by the expression of specific markers — from stem cell todifferentiated astrocyte, oligodendrocyte and neuron. Growth factors that promote theprogression of one cell type to another are indicated in green, those that inhibit progression of one cell type to another are in red, and those that induce proliferation and maintain cells at a given stage of development are indicated in blue. The glial-restricted precursors give rise to both astrocyte-restricted precursors and O2A progenitors, which develop into botholigodendrocytes and astrocytes. Platelet-derived growth factor (PDGF) signalling drives cellsearly in development towards the O2A progenitor cell type and maintains these cells in aproliferating state. Withdrawal of PDGF and fibroblast growth factor 2 (FGF2), and stimulationby cilliary neurotrophic factor (CNTF) and epidermal growth factor (EGF), drives these cellstowards astrocyte and oligodendrocyte differentiation. Type 1 and type 2 astrocytes differ inmorphology and marker expression. (A, A2B5, a monoclonal antibody that recognizes anepitope on type 2 astrocytes and glial progenitors; E, EGF receptor; F, FGF receptor; G, glialfibrillary acidic protein; GalC, galactocerebroside; M, myelin basic protein, an oligodendrocyte-specific protein component of myelin; N, nestin, an intermediate filament protein expressedpredominantly by CNS progenitors; P, PDGF receptor; PLP, myelin proteolipid protein, acomponent of myelin that is limited to oligodendrocytes and glial progenitors; O, O4, a markerfor oligodendrocyte precursors; S, S100, a marker for astrocytes; V, vimentin.)



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tumours had mutations in this pathway, most of whichwere deletions in the INK4A gene (also called INK4AARF

or the cyclin-dependent kinase inhibitor 2A (CDKN2A)gene), which encodes two proteins, p16INK4A and p14ARF

(REFS 30,31). These two proteins control pathways thatlead to the critical proteins: RB, which maintains cells inG1, and p53, which either arrests cells in G1 or inducesapoptosis32–38. In some tumours, homozygous deletionsof INK4A have been found, whereas heterozygous dele-tions have been seen in others. However, even tumourswith heterozygous deletions mostly do not express theremaining wild-type allele, possibly owing to inactiva-tion of the INK4A gene by methylation of its promoteror by other unknown mechanisms30. For these reasons,in ~60% of GBMs, neither p16INK4A nor p14ARF is pro-duced. For the most part, the remaining GBMs (~40%)have loss-of-function mutations in TP53 (referred to asp53 hereafter), the gene that encodes p5339. Thesetumours also either overexpress cyclin-dependentkinase 4 (CDK4), the kinase that phosphorylates andinactivates RB and so promotes cell-cycle progression,or show loss of RB expression. By either mechanism,both the cell-cycle arrest pathways that lead to p53 andRB are disrupted in the majority of these tumours. Thereason for some tumours developing INK4A losswhereas others develop p53 mutations is not clear.However, these data emphasize the importance of thesepathways in the process.

Further emphasis of the importance of cell-cyclearrest disruption in gliomagenesis comes from theoccurrence of mutations in other critical genes in thesepathways. For example, amplification and overexpres-sion of CDK4 is seen in 15% of GBMs. In addition, theamplification of the genes that encode the relatedCDK6, cyclin D1 (the binding partner of CDK4) andMDM2 (which degrades p53) has been reported40. Allof these alterations disrupt normal control mechanismsthat maintain cells in the G1 phase of the cell cycle. It isnoteworthy that CDK4 and MDM2 are located near oneanother on chromosome 12, and co-amplification ofthese genes in glioma cell lines has been reported41.

Some specific mutations are found in both primaryand secondary GBMs, whereas other mutations are pre-dominantly found in one or the other. The reason for thecorrelation between certain mutations and glioma typeand grade is not yet clear but is well described (FIG. 4). Forexample, there is a tendency for primary GBMs that arisein older patients to have deletions in INK4A (on chromo-some 9q26), and to have cells that are diploid or PSEUDO-

DIPLOID42,43. In addition, these primary GBMs frequentlyshow amplification and activating mutations of EGFR(on chromosome 7p12). By contrast, the secondaryGBMs tend to arise initially in younger patients as lowergrade gliomas, to have mutations in p53 (on chromo-some 17p13) and to be aneuploid44. These secondaryGBMs tend to acquire CDK4 amplification (on chro-mosome 12q13) or RB deletions (on chromosome13q13) and generally do not have EGFR alterations45.Both primary and secondary GBMs, but not lowergrade gliomas, frequently show loss of PTEN expression(on chromosome 10q24), either due to mutation or to

tion27 and frequent loss of expression28 of the PTENgene, and the majority of GBMs have elevated AKTactivity29. Although this pathway is activated in mostGBMs, animal modelling in vivo is required to showthat it actually contributes to the induction of the dis-ease (see below). The specific effects of AKT signallingon glial differentiation have not yet been investigated.

Altered cell-cycle arrestThe second group of mutations found in gliomasincludes those that disrupt the pathways that keep thecells in G1 cell-cycle arrest (FIG. 3). Large numbers ofgliomas have been analysed for mutations in genes thatencode proteins involved in cell-cycle arrest pathways.Approximately 25% of low-grade gliomas show muta-tions in genes that encode proteins in this pathway (par-ticularly loss of the retinoblastoma (RB) locus), whereasabout 66% of anaplastic gliomas have such mutations.In the largest group of GBMs analysed, nearly all

INK4A Cell cycleG1 G2

S

M

p16INK4A

p14ARF

Apoptosis

CDK4

MDM2

RB

p53

Figure 3 | INK4A-initiated cell-cycle arrest pathways. p16INK4A and p14ARF control theactivity of retinoblastoma (RB) and p53. RB promotes cell-cycle arrest in G1 and regulatesentry into the S phase of the cell cycle through its effects on E2F. p53 has several effects,including causing G1 and G2 arrest and promoting apoptosis. Loss of p53 function alsopromotes genomic instability. Proteins encoded by genes that are frequently altered in humangliomas are highlighted in red.

Growth factor

SOS GRB2 SHC PIP2 PIP2PIP3

PDKAKTRAS

GSK3FKHR

mTORBADNF-κB

PTENPI(3)K

Cell membraneTyrosine kinasereceptor

RAC

a

b

RALRAF

MEK

ERK

JNKK

JNK

MKK

p38

Figure 2 | Signalling pathways altered by mutations in human gliomas. RAS and AKTsignal-transduction pathways and their tyrosine kinase receptors. a | The binding of RAS toGTP is initiated by activated tyrosine kinase receptors and occurs through the effector andadaptor proteins SHC, GRB2 and SOS. When interacting with the cognate ligand, thesereceptors also activate phosphatidylinositol-3-OH kinase (PI(3)K), which convertsphosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-trisphosphate(PIP3). b | PIP3 localizes AKT to the cell membrane and is required for the activation of AKT by pyruvate dehydrogenase kinase 1 (PDK1) and PDK2. The tumour suppressor PTEN inhibitsthe activation of AKT by converting PIP3 back to PIP2, reducing the availability of this requiredlipid cofactor. Although many of the growth-factor receptors involved in glial cell developmentare reported to activate a similar set of pathways in certain cell types, the relative activation of these pathways in glial cells and gliomas by specific receptors is the subject of intenseinvestigation. (BAD, BCL2-antagonist of cell death; FKHR, forkhead transcription factor; GSK3, glycogen synthase kinase 3; JNK, Jun kinase; mTOR, mammalian target of rapamycin;NF-κB, nuclear factor of kappa light polypeptide gene enhancer in B-cells.)

MAMMALIAN TARGET OF

RAPAMYCIN

(mTOR). A protein that isactivated by AKT and whichactivates ribosomal protein S6kinase. S6 kinase alters the abilityof the ribosome to translatespecific mRNAs.

BAD

BAD promotes apoptosis by dimerizing with andinhibiting BCL-2.



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effects have been studied in experimental systems, andfrom such data their potential role in gliomagenesis hasbeen inferred. However, the technologies that allow spe-cific genetic alterations to be generated in vivo have onlyrecently been developed, and experiments to showwhich, if any, of these alterations are actually involved inthe aetiology of gliomas have only just begun.

Standard therapy and animal modelsCurrent standard therapy for gliomas primarily usesDNA-damaging strategies such as radiation and alkylat-ing agents (BOX 3). These are effective in a subset ofglioma patients; however, their tumours eventuallyrecur51. Despite their initial promise in animal studies,novel strategies such as immunotherapy or genetherapy52,53 have not yet resulted in significant improve-ments in patient survival and therefore have notbecome the standard of care. The failure of these treat-ment strategies, which were optimized in animal mod-els, to have a therapeutic benefit in humans is disap-pointing, and to some degree might be related to theanimal models that have been used for this purpose.

The standard rodent models for gliomas used overthe past 25 years to optimize therapy in humans consistof either XENOGRAFTS of human glioma cell lines implant-ed into athymic mice or ALLOGRAFTS of rodent glioma celllines. These models have several characteristics thatmake them good test systems, including defined andreproducible location of tumour formation, rate oftumour growth and time to death. Unfortunately, thehistology of these tumours does not replicate that foundin the human disease, especially as far as the invasivecharacteristics of gliomas are concerned54. Furthermore,clonal cell lines maintained in culture have differentselective pressures on them than do tumour cells that areproliferating in the brain, and therefore one would notexpect genetic and gene-expression alterations to be sim-ilar between them. The use of implantation models isespecially problematic for optimizing immunologicallybased therapies. Perhaps even more important is the factthat these models provide little insight into the aetiologyand underlying biology of gliomas that could be used todesign more rational therapies for this disease.

Genetic-modification strategies. Because of the limita-tions of xenograft models for gliomas, several labora-tories have developed genetically and histologicallyaccurate models of glioma formation by gain-of-func-tion transgenic approaches and targeted-deletionstrategies. Transgenic mice express the gene of interestin all cells that use the promoter that drives a con-struct’s expression55, whereas knockout mice loseexpression of the targeted gene in all cells that wouldnormally express it56. In both cases, large numbers ofcells of a specific cell type are modified by these strate-gies. In germline-modification models, the tissue withthe gene-expression alteration is frequently develop-mentally normal. Tumours are often initiated anddevelop in this cell population from secondary, usuallyunknown, genetic events. These strategies are ideal fordemonstrating components that contribute to the

other mechanisms46,47. The correlation between p53mutation and progression of low-grade gliomas tohigher grade tumours might be partly due to genomicinstability and to the acquisition of additional muta-tions associated with loss of p53.

It is noteworthy that the disruption of cell-cyclearrest pathways, ultimately by elevating the activity ofE2F1 transcription factor and by losing p53 function,has been linked to the dedifferentiation of many celltypes48,49. Additionally, elevated MYC activity, whichoccurs in human gliomas, is also related to loss of dif-ferentiation in cultured cells50. It is possible that, inaddition to enhancing cell proliferation, these alter-ations might promote an undifferentiated phenotypethat is more sensitive to the oncogenic effects of cer-tain abnormally and chronically activated signal-transduction pathways.

These alterations in gliomas have been well docu-mented over the past few years. Furthermore, their

Box 3 | Glioma standard of care

The standard treatment for patients with gliomas has remained unchanged for manyyears. Once a patient is identified as having a lesion that resembles a glioma on amagnetic resonance imaging (MRI) scan, the tumour is either biopsied or surgicallyremoved for diagnostic and histological examination. Not all patients with lower gradegliomas receive radiation therapy. However, all patients with malignant gliomas receiveradiation therapy regardless of the extent to which the visible tumour has beenremoved. Following radiation, the patients receive chemotherapy, usually with drugsthat cause DNA alkylation such as carmustine (BCNU), the triple combination ofprocarbazine, cisplatin and vincristine (PCV), or the newly available temozolomide.When a glioma recurs, surgery might be carried out if the risk and benefits of such anoperation are favourable based on the location and character of the recurrent glioma.At this point, further radiation is not usually an option, and additional chemotherapycan be given with other drugs such as cis-retinoic acid (CRA), which promotes tumourcell differentiation, or with either interleukin-2 (Il-2) or interferon, which enhance theimmune response against the tumour. Despite these efforts, 50% of GBM patients diewithin the first year of diagnosis, and only 20% survive for more than 2 years.

Activates signal-transductionpathways

Disrupts p14ARF

and p16INK4A

pathways

Activates signal-transductionpathways

EGFRamplification(7p12)

Cells of origin

INK4A loss(9q26)

Low-gradeglioma

Anaplasticglioma

GBM(secondary)

GBM(primary)

PTEN loss(10q24)

PDGF, FGF2overexpression

p53 mutation(17p13)

CDK4amplification(12q13)RB loss(13q13)

PTEN loss(10q24)

Activate signal-transductionpathways

Disrupts p14ARF pathway,inhibits apoptosis andpromotes genomic instability

Disrupt p16INK4A pathway

Enhances AKT activity

Figure 4 | Pathways to gliomagenesis. Glioblastoma multiforme (GBM) formation is either denovo (primary GBMs) or due to the progression of a lower grade glioma to a higher grade onethrough the acquisition of additional mutations (secondary GBMs). The mutations listed are asubset of those found in these tumours that have some correlation with glioma grade andGBM type. Also listed are some biological effects of these mutations and changes in geneexpression that might contribute to their roles in gliomagenesis1.

BCL-2

BCL-2 inhibits apoptosis byinhibiting caspase activation.

CASPASE

One of a family of proteasesthat are activated specifically inapoptotic cells.

FORKHEAD TRANSCRIPTION

FACTOR

(FKHR). This protein activatescell death. It is inactivated byAKT-dependentphosphorylation, whichrelocalizes it from the nucleusto the cytoplasm.



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gies do not produce local injury.Additional mutations and alterations in gene expres-

sion are likely to occur during tumour progressionregardless of the strategy for tumour induction.However, in some cases the majority of tumour cellsmight evolve so as to continue to depend on the initiat-ing mutations. There are reports of reversible oncogene-sis in mice using tetracycline-induced H-RAS expressionto produce melanomas59 and that of Myc to generatelymphomas60 and skin tumours61. Despite genomicinstability in these tumours, they completely regresswith the removal of the initiating genetic alteration.Whether any of the gliomas are initiated by a singleevent or a by summation of many contributing factorsis unknown. However, identifying the pathways thathave the potential to initiate glioma formation is animportant step towards rational therapeutic approachesfor this disease.

Induction and activated signal transductionMouse models have been used to answer several spe-cific questions regarding the role that elevated signaltransduction has in the formation of gliomas. Forexample, can the chronic activation of the signal-transduction pathways that function during normalglial cell development induce the formation ofgliomas in mice? There are many such examples.These include viral oncogenes that activate signal-transduction pathways to cause glioma formation,such as V-SRC62, V-ERBB (W. Weiss, unpublished observa-tions), V-SIS63, and the polyoma virus middle T anti-gen58 gene. Also, activated forms of downstream com-ponents, such as Ras29 (A. Guha, unpublishedobservations) and Akt29, have been used. In addition,targeted deletions of genes that encode tumour sup-pressors, such as Nf1, that normally inhibit specificsignal-transduction pathways have also been shown tocontribute to gliomagenesis64. Current mouse modelsof glioma formation and how they were generated aresummarized in TABLE 1.

One specific question addressed by these models iswhether autocrine stimulation by one of the growth

induction and progression of tumorigenesis.Furthermore, breeding can be used to combine bothtransgenes and targeted deletions. Most studies tomodel cancer in mice use these germline-modificationstrategies at present. However, because extensivebreeding is required to combine multiple mutations,the analysis of large numbers of permutations ofgenetic alterations is expensive and time consuming.

A second and complementary method for geneticalteration is the somatic-cell gene-transfer strategy57, inwhich combinations of mutations are transferred byretroviral infection to specific cell types postnatally. Theanalysis of permutations of multiple mutations withsuch systems is faster and less labour intensive than withgermline strategies. Because the number of cells that areinfected initially is small, secondary events required fortumour initiation are unlikely to occur. However, if atumour forms, the introduced alterations were probablysufficient to induce tumour formation. This point isstriking in cases where abnormally elevated expressionof a single gene, such as that encoding the POLYOMA VIRUS

MIDDLE T ANTIGEN, in a few hundred astrocytes induces theformation of gliomas at high frequency58.

Both germline and somatic-cell gene-transfer strate-gies have advantages and disadvantages, and compar-ing results from each can be informative. Somatic-cellgene-transfer strategies have the disadvantage thatbecause secondary events are less likely to occur, essen-tial mutations must be experimentally provided toinduce tumour formation. In germline strategies,mutations that contribute to, but are insufficient for,oncogenesis will give rise to tumours by the acquisitionof other mutations, which can then be identified. Insomatic-cell gene-transfer strategies, insufficient com-binations are unlikely to score positively for tumourformation, and therefore might be missed.Furthermore, causing retroviral infection by directinjection into the brain generates injury and inflamma-tion that disrupts the local environment and alters geneexpression in potential target cells. The contributionthat these local effects have on tumour formation insuch models is not clear. By contrast, germline strate-

Table 1 | Mouse models of glioma and their construction

Glioma type Signal-transduction Cell-cycle arrest Somatic or Cell of origin/ Referenceabnormality disruption germline affected cells

Oligo p19ARF deletion g All cells 67

Oligo-astro MTA s Gfap-expressing 58astrocyte

Astro v-Src g Gfap-expressing 62astrocyte

GBM K-ras and Akt s Nestin-expressing 29progenitor

GBM Nf1 (Ras activity) p53 deletion g All cells 64

Mixed Pdgf s Mixed population 63

Glioma models that have histological characteristics similar to: oligodendroglioma (oligo), astrocytoma (astro), mixedoligodendroglioma and astrocytoma (oligo-astro), glioblastoma multiforme (GBM) and a mixture of multiple brain tumour types,including GBM (mixed). Signal-transduction pathways are experimentally activated by expression of the indicated genes. Cell-cyclearrest pathways are experimentally disrupted by the indicated genetic alterations. The strategies used to modify gene expression aregermline integration of a transgene or targeted deletion (g), and somatic-cell gene-transfer with retroviral vectors (s). The cell of originindicates the cell type that is genetically altered by the strategy used in the model. (GFAP, glial fibrillary acidic protein; MTA, polyomavirus middle T antigen; PDGF, platelet-derived growth factor.)

PSEUDODIPLOID

DNA content similar to that ofa diploid cell.

E2F1

A transcription factor that isbound to RB during G1 cell-cycle arrest and is released afterphosphorylation of RB byCDK2 or CDK4. Free E2F1 thenalters gene expression to lead tocell-cycle progression.

XENOGRAFT

Cells derived from one speciesthat are implanted into a host ofanother species; for example,human tumour cells implantedinto a mouse.

ALLOGRAFT

Cells implanted into a host thatare derived from anotherindividual of the same species.

POLYOMA VIRUS MIDDLE T

ANTIGEN

Viral gene product thatactivates many signallingpathways that are activated bythe PDGF receptors.

H-RAS

Harvey Ras. Activated Ras alleleinitially isolated from Moloneymouse leukaemia virus.

V-SRC

A virally encoded oncogeneoriginally isolated from theRous sarcoma virus. This geneencodes a deleted and activatedversion of the cellular Src gene.Expression of this gene activatesmultiple signalling pathways.

V-ERBB

A virally encoded oncogeneoriginally isolated from thechicken erythroblastosis virus.This gene encodes an activatedvariant of the EGFR.

V-SIS

A virally encoded oncogeneoriginally isolated from thesimian sarcoma virus. This geneencodes the complete sequenceof the PDGFB chain.Overexpression results inautocrine stimulation of thePDGF receptor.



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human gliomas, even in all cells in the mouse, alonedoes not induce gliomas at high frequency. Althoughmice with targeted disruption of genes that encodecomponents of the cell-cycle arrest pathways do notdevelop gliomas, they often develop other tumourtypes, implying that glia might be generally less sensi-tive to such alterations than other cell types.

For example, the Ink4a locus has been knocked outin mice by targeted disruption65. Ink4a−/− astrocytes inculture are immortal, grow rapidly, and maintain apseudodiploid chromosomal status66. Mice withhomozygous deletion of Ink4a do not produce eitherp16Ink4a or p19Arf (the equivalent of the human p14ARF)in any cell. Nonetheless, these mice develop normallyand breed effectively. Furthermore, despite the deletionof Ink4a in every cell, gliomas have never been observedin these mice. These mice develop lymphomas and sar-comas between 4 and 6 months of life65. Interestingly,mice with specific targeted deletion of the Ink4a exon1B, which results in the specific loss of p19Arf expression,develop gliomas at low frequency. In one series of 39such mice, 3 developed tumours that have the histologi-cal characteristics of oligodendrogliomas by 18–20weeks of life67. The reason for this discrepancy isunclear, but might be due to strain differences betweenthe two knockout lines.

In addition, mice with targeted deletions of p53have also been generated68. Cultured astrocytesderived from these p53−/− mice also grow rapidly asimmortalized populations and show significant chro-mosomal instability with rapid development of aneu-ploidy and chromosomal rearrangements69. Despitethese dramatic effects in vitro, mice with homozygoustargeted deletions of p53 develop and breed normally.They develop lymphomas and sarcomas in the first3–6 months of life, but they only rarely developgliomas, even though no cells in the CNS producep53 (REF. 68). If these p53−/− mice were to survivelonger without the formation of early cancers, theymight develop gliomas in later life. Testing such ahypothesis requires the tissue-specific deletion of p53by using the cre/lox system, with cre driven frombrain-specific promoters. Such experiments are inprogress in several laboratories.

Because loss of Rb causes embryonic lethality70,chimeric mice that contain a mosaic of Rb−/− and Rb+/+

cells have been constructed that do survive past birth.The analysis of these mice indicates that their Rb−/−cellsdo not give rise to tumours. In fact, the Rb−/− cells in thebrain seem to enter the cell cycle abnormally and toapoptose71. Given these findings with targeted deletionsof Rb, p53 and Ink4a, it is not surprising that forcedoverexpression of Cdk4 in astrocytes causes neithertumours nor detectable proliferation of these cells rela-tive to the surrounding normal brain tissue66.

Combined pathways to gliomagenesisAlthough disrupting the cell-cycle arrest pathways is initself insufficient to induce gliomas, such mutations canenhance the oncogenic capacity of certain signallingpathways. One example that demonstrates cooperation

factors involved in glial development can contribute togliomagenesis. This question has been answered bysomatic-cell gene-transfer of Pdgfb in vivo using amouse retroviral vector. Mice infected with vectors thatencode Pdgfb frequently develop highly invasivegliomas with a varied histology63. The mouse retroviralvectors used in these experiments can infect many celltypes, which might contribute to the variable histologyof the resultant gliomas. Cell-type-specific transfer ofPdgfb to astrocytes or glial progenitors might allow thecell-of-origin to be correlated with a specific gliomahistology. These data further emphasize the connectionbetween glial differentiation and gliomagenesis.

A second specific question that can be addressed bythese models regards identifying cooperation betweenthe pathways that lie downstream of tyrosine kinasereceptors in gliomagenesis. Cooperative effects betweenAkt and Ras signalling in the formation of malignantgliomas have been shown with somatic-cell gene trans-fer. In this system, transfer of either an activated form ofK-RAS or Akt alone to neural progenitors is insufficient toform gliomas in vivo. However, the combination of acti-vated Akt and Ras in these cells generates malignantgliomas in mice (FIG. 5) with microvascular proliferationand psuedopalisading necrosis similar to that found inhuman GBMs29. Further dissection of these signal-transduction pathways with glioma formation as a read-out is possible with mouse modelling systems.

Cell-cycle, genomic instability andgliomagenesisMouse modelling also allows us to determine the rela-tive efficiency of mutations that disrupt the cell-cyclearrest pathways at causing oncogenesis. In contrast tothe efficient glioma formation induced by activation ofcertain signal-transduction pathways, alterations incell-cycle arrest pathways are much less effective atinducing gliomas in mice. It seems that disruption ofcell-cycle arrest pathways by mutations found in

a

b c

Figure 5 | Mouse glioma histology. a | Gross appearanceof bilateral mouse glioblastoma multiforme (GBM) induced by combined Ras and Akt signalling (left) compared withnormal mouse brain29. b,c | Histological characteristics that define GBM in humans, as seen in this mouse model. b | Pseudopalisading necrosis (arrow) and c | microvascularproliferation (arrow).

K-RAS

Kirsten Ras. Activated Ras alleleinitially isolated from Kirstenmouse leukaemia virus.



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effectively activates these signalling pathways in astro-cytes. Tumours are generated in these mice that have thehistology of mixed oligodendrogliomas and astrocy-tomas58. These data imply that astrocytes, if appropri-ately stimulated, can be the cell-of-origin for eithertumour type in vivo, and the pathways activated by mid-dle T antigen are sufficient to achieve that effect.

Applications of glioma mouse modelsOver the past 15 years, experiments to identify the dif-ferences in gene expression between glioma cells andnormal brain tissue have provided essential insightinto the biology of this disease. New techniques, suchas serial analysis of gene expression (SAGE) andmicroarray analysis, are further identifying thousandsof genes that are differentially expressed in thesetumours, which might contribute to the neoplasticphenotype73. It is not possible to test all genes identi-fied in such screens in animal models. However, distin-guishing those alterations that are the epiphenomenaof tumour progression from those that act as the aeti-ology of the disease requires these alterations to bemodelled in vivo. Without animal modelling, it wouldnot be possible to determine the relative importance ofcausative pathways to glioma formation or to identifythe essential targets for therapy.

In addition to unravelling the biology of thesegliomas, these mouse models are likely to providereagents for translational and applied investigations.Once the molecular aetiology for initiation and main-tenance of gliomas has been clarified (SEE BOX 4),important targets for glioma therapy in humans canbe developed. Furthermore, such animal models pro-vide excellent opportunities to test drug combinationsaimed at specific targets in vivo. Given the informationwe now have, rational therapies might include combi-nations of RAS and AKT signalling-pathwayinhibitors, along with CDK inhibitors and agents thatforce a differentiated phenotype on glioma cells.Farnysyltransferase inhibitors that block RAS activity,and inhibitors of mTOR (which is downstream ofAKT), are already in clinical trials; CDK inhibitors arebeing developed by several companies. Retinoic-acidderivatives and histone-deacetylase inhibitors promote

between signal-transduction and cell-cycle arrest path-ways in gliomagenesis is mice in which the loss of Nf1and p53 has been combined. Nf1 loss alone does not ele-vate Ras activity sufficiently to induce gliomas.However, in a p53−/− background, Nf1−/− mice frequentlydevelop high-grade gliomas64. Whether the oncogeniceffect of p53 loss is due to effects on G1 arrest, apoptosisor to genomic instability that leads to other cooperatingmutations is not yet known.

Ink4a loss alone does not result in the formation ofgliomas as discussed above; however, several experi-ments using both transgenic and somatic-cell gene-transfer strategies have demonstrated a contributoryrole for the loss of this tumour suppressor in the genera-tion of gliomas. Gene transfer of a constitutively activeform of Egfr alone does not induce the formation ofgliomas in mice. However, in an Ink4a-deficient back-ground, or combined with p53 loss and Cdk4 overex-pression, the Egfr constitutively active mutation causesglioma-like lesions to form72. These data, as well asunpublished work from several laboratories, indicatethat, in some cases, cell-cycle arrest alterations promotea more malignant phenotype and might give a selectiveadvantage in vivo to cells in which they occur. Thesefindings might partly explain the prevalence of thesemutations in human gliomas.

By using somatic-cell gene-transfer systems, theinteraction between cell-of-origin and signal-transduc-tion pathways can be addressed. Undifferentiated glialprogenitor cells seem more sensitive than differentiatedastrocytes to the oncogenic effects of combined Ras andAkt signalling and give rise to GBM formation29. It ispossible that mutations found in gliomas that promotean undifferentiated glial cell phenotype might haveoncogenic effects partly for that reason.

In addition to addressing relationships between cell-of-origin and the oncogenic potential of specific signal-transduction pathways, mouse modelling can also cor-relate the cell-of-origin with the tumour type thatforms. One example is illustrated by gene transfer of thepolyoma virus middle T antigen to mouse Gfap-expressing astrocytes in vivo. Middle T antigen activatesmany of the same signalling pathways as the PDGFreceptor, and gene transfer to Gfap-expressing cells

Box 4 | Outstanding questions

Several questions regarding basic glioma biology and therapeutic strategies for the disease remain outstanding.In some cases, animal modelling has raised these questions and, in others cases, only animal modelling can answer them.• Is the chromosomal instability seen in gliomas the cause or effect of tumour initiation or progression?

• Are any of the gliomas initiated by a single genetic event or does this occur through a summation of multipleabnormalities?

• For the gliomas that might arise from a single event, do they evolve during tumour progression so as to continue to require the initiating event to maintain a neoplastic phenotype?

• How many parallel pathways exist for each of the required elements of transformation, and would it be possible to block a multi-parallel network if it existed?

• Does the differentiation status of a cell affect the ability of oncogenic signals to initiate glioma formation orprogression? Could therapeutic intervention to force a differentiated status on glioma cells make them lesssensitive to known oncogenic pathways?



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The history of glioma therapy has been stagnant andbleak, with little improvement in treatment strategies orpatient outcomes for many decades. However, with theadvances of modern molecular genetics, there has beena shift in our thinking about what this disease is and

Links

DATABASE LINKS GFAP | PDGF | FGF2 | EGF | CNTF |EGFR | ERK | JNK | p38 | NF1 | PI(3)K | PTEN | AKT1 |GSK3 | mTOR | FHKR |RB | INK4A | p53 | CDK4 | CDK6| cyclin D1 | MDM2 | MYC | Myc | Akt | Nf1 | Pdgfb |Ink4a | p53 | Rb | Egfr | Cdk4 | Gfap | PDGFR | VEGFRFURTHER INFORMATION Massachusetts General HospitalBrain Tumour Center | Clinical trials information forbrain tumoursENCYCLOPEDIA OF LIFE SCIENCES Brain cancers | Brain imaging

how it works. This shift has illuminated rational treat-ment options that, although not yet realized, might bethe light at the end of a long and dark tunnel.



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AcknowledgementsI would like to thank Greg Fuller and Joseph Celestino for help withthe pathology, Chengkai Dai for help with the cell-lineage discussion,and V. K. Rajasekhar for help with the signal-transduction discussion.


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