Medulloblastoma: Molecular Genetics and Animal Models · Medulloblastoma: Molecular Genetics and Animal Models Corey Raffel Department of Neurologic Surgery, Mayo Clinic, 200 First

Medulloblastoma: Molecular Genetics and Animal Models

Corey Raffel

Department of Neurologic Surgery, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA

Abstract

Medulloblastoma is a primary brain tumor found in the

cerebellum of children. The tumor occurs in associa-

tion with two inherited cancer syndromes: Turcot

syndrome and Gorlin syndrome. Insights into the

molecular biology of the tumor have come from

looking at alterations in the genes altered in these

syndromes, PTC and APC, respectively. Murine mod-

els of medulloblastoma have been constructed based

on these alterations. Additional murine models that,

while mimicking the appearance of the human tumor,

seem unrelated to the human tumor’s molecular

alterations have been made. In this review, the clinical

picture, origin, molecular biology, and murine models

of medulloblastoma are discussed. Although a great

deal has been discovered about this tumor, the genetic

alterations responsible for tumor development in a

majority of patients have yet to be described.

Neoplasia (2004) 6, 310–322

Keywords: Medulloblast, cerebellar dysfunction, CNS tumors, primitive neuroectoder-

mal tumors, external granular cell layer.

Introduction

Medulloblastoma is a primary brain tumor that occurs in the

cerebellum of children and young adults. The nomencla-

ture for this tumor is somewhat controversial. The name

‘‘medulloblastoma’’ was given by Bailey and Cushing [1]

in 1925; they suggested that these tumors arise from a

hypothesized central nervous system (CNS) precursor cell

called a medulloblast. Such a cell has never been identi-

fied, leading Rorke [2] to include medulloblastoma in a

group of histologically similar CNS tumors, which she

called primitive neuroectodermal tumors (PNETs). Some

of the controversies regarding nomenclature have been

addressed by recent studies of gene expression using

array analysis (see below). Because these tumors arise

in the cerebellum, patients present with symptoms of

cerebellar dysfunction, including balance problems and

incoordination. In addition, because the tumor frequently

fills the fourth ventricle and blocks the normal circulation of

cerebrospinal fluid (CSF), patients often present with

symptoms and signs of hydrocephalus, including head-

ache, vomiting, diplopia, and papilledema. Medulloblasto-

mas are best demonstrated by magnetic resonance

imaging (MRI), although characteristic findings can also

be seen on computed tomography (Figure 1). Because

these tumors have a propensity to spread through the CSF to

distant CNS sites, patients should have MRI of the entire brain

and spine. Medulloblastoma has an incidence of two to five

cases per 10,000 population per year, resulting in about 240

new cases per year in the United States [3]. Most medulloblas-

tomas arise sporadically, although medulloblastoma may arise

rarely as part of an inherited cancer syndrome (see below).

Grossly, medulloblastomas most often arise in the roof of

the fourth ventricle. They grow to invade the cerebellar vermis

and fill the ventricle, often invading through the ependyma in

the floor of the ventricle to enter the brainstem (Figure 1). Less

commonly, the tumor arises in the cerebellar hemisphere.

Grossly, the tumors appear to be relatively well circumscribed.

The tumor may contain areas of calcification or necrosis.

Microscopically, the tumor cells are small with little cytoplasm

(Figure 2A). They occur in sheets and may show signs of

neuronal or glial differentiation. Histologic subtypes of medul-

loblastoma have been described and include the desmoplastic

variant and the large cell variant. The desmoplastic variant is

composed of islands of larger, pale cells in a sea of smaller,

more typical medulloblastoma cells. In addition, an abundant

collagenous matrix is present. In the large cell variety, the cells

are larger and more pleomorphic. Microscopically, the tumor is

invasive at its edges, although penetration into the surrounding

cerebellum is somewhat limited. Immunhistochemical staining

is frequently positive for synaptophysin and NeuN, indicating

that neuronal differentiation and may be regionally positive for

glial fibrillary acid protein (GFAP), indicating astrocytic differ-

entiation in some parts of the tumor.

Current Therapy of Medulloblastoma

Currently, patients with medulloblastoma are best treated with

surgical removal of the tumor, and adjuvant radiation therapy

and chemotherapy (for review, see Refs. [4–7]). Many pro-

spective, randomized trials have demonstrated that patients do

better if most—if not all—of the tumor is removed. Radiation

therapy, at a dose of at least 55 Gy to the tumor, has also been

shown to prolong survival and result in cures. Because the

tumor has a propensity to spread in the CSF, an additional

35 Gy is given to the entire brain and spinal cord. The

devastating effects on intellect caused by the radiation

Address all correspondence to: Corey Raffel, MD, PhD, Department of Neurologic Surgery,

Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA. E-mail: [email protected]

Received 17 November 2003; Revised 17 November 2003; Accepted 3 December 2003.

Copyright D 2004 Neoplasia Press, Inc. All rights reserved 1522-8002/04/$25.00

DOI 10.1593/neo.03454

Neoplasia . Vol. 6, No. 4, July/August 2004, pp. 310–322 310

www.neoplasia.com

REVIEW ARTICLE

therapy in young children have been well documented. The

younger is the child at the time of irradiation, the worse is

the intellectual outcome. For this reason, children under the

age of 3 years are often treated with chemotherapy alone.

Agents with demonstrated efficacy against medulloblastoma

include DNA alkylating agents such as ethylnitrosoureas

(BCNU and CCNU) and the platinum derivatives (cis-plati-

num and carbo-platinum). Chemotherapy is also used in

Medulloblastoma: Molecular Genetics and Animal Models Raffel 311

Neoplasia . Vol. 6, No. 4, 2004

Figure 1. MRI of medulloblastoma. This T1-weighted, gadolinium diethylenetriamine pentaacetic acid (DTPA)–enhanced MR scan of a patient with a

medulloblastoma demonstrates a mass (large area of white) in the cerebellum, which fills the fourth ventricle and displaces it anteriorly.

Figure 2. (A) Photomicrograph of a human desmoplastic medulloblastoma, demonstrated as small, tightly packed cells with little cytoplasm. A pale island is seen in

the center of the image as an area of less tightly packed cells. Hematoxylin and eosin staining, �400. (B) Photomicrograph of murine tumor of the cerebellum from

a Ptc+/� mouse. Note the similarity to the human tumor. Hematoxylin and eosin staining, �400. (Image courtesy of Dr. Cynthia Wetmore, Rochester, MN.)

conjunction with radiation therapy in older patients, especial-

ly those with CSF metastases at presentation.

Cerebellar Development

The cerebellum develops from neuronal precursors located

in the rhombic lip of the fetal brain (for review, see Ref. [8]).

These precursors migrate tangentially to form the external

granular cell layer (EGL) of the developing cerebellum.

Purkinje neurons and Bergman glia arise from precursors

in the subventricular zone andmigrate toward the EGL. In the

EGL, the granular cell precursors continue to proliferate in

the outer zone. Postmitotic neuronsmove to the inner zone of

the layer and then migrate along Bergmann glial fibers to

finally reside in the internal granular cell layer (IGL). The EGL

eventually disappears as all cell division ceases and all

postmitotic neurons move to the IGL. The number of neurons

that finally reside in the IGL is large. In fact, there are more

neurons in the IGL than in the rest of the brain combined.

The sonic hedgehog (SHH) signaling pathway plays an

essential role in cerebellar development [9,10]. The receptor

for SHH, patched (PTC), is a membrane-associated protein

containing 12 transmembrane domains [11,12]. Associated

with PTC in the membrane is the effector molecule, smooth-

ened (smo) [13]. PTC appears to function to inhibit signaling

by smo. Binding of SHH to PTC releases this inhibition,

resulting in activation of the intracellular components of the

pathway. The main effector of SHH in the cell may be gli1,

a transcriptional activator [14] (Figure 3). In the developing

cerebellum, PTC is expressed by neuronal precursors in the

EGL [9,10]. SHH is produced by Purkinje neurons that lie

beneath the EGL. In vitro, SHH has been shown to be a

potent mitogen for EGL precursors. Blocking of SHH signal-

ing in vivo leads to hypoplastic cerebella in which granule

neurons are greatly reduced or absent. These results strong-

ly suggest that EGL neuronal precursors are stimulated to

divide by SHH signaling. The factors that lead to differenti-

ation andmigration of the postmitotic neurons are not clear at

this time, although b-FGF has been shown to block the SHH-

induced proliferation of EGL cells in vitro. Figure 4 summa-

rizes role of the SHH pathway in the development of the

cerebellum.

Other signaling pathways have been implicated in the

development of the cerebellum. Both neurotrophins and their

receptors have been found to be expressed in the developing

cerebellum. The temporal pattern of expression suggests

that brain-derived neurotrophic factor (BDNF) and its recep-

tor, trkB, may be involved in the division of cells in the EGL,

and that NT3 and its receptor, trkC, may be involved in the

terminal differentiation of these cells into internal granular

312 Medulloblastoma: Molecular Genetics and Animal Models Raffel


Figure 3. The SHH/PTC pathway. By binding to the membrane-bound PTC receptor, SHH removes the inhibition of v-smo mediated by PTC. This ultimately results

in increased gli-mediated transcription. Su-fu and PKA are downstream inhibitors of this process.

layer neurons [15,16]. This model is of interest in light of data

suggesting that the expression of trkC in medulloblastoma is

a marker for better prognosis [17,18].

Medulloblastoma: Cell of Origin

Medulloblastomas are observed to differentiate along glial

and neuronal pathways in situ, suggesting that these tumors

are derived from primitive, pluripotent, neuroepithelial stem

cells. This conclusion is supported by studies of PNET cell

lines that demonstrate expression of specific, developmen-

tally regulated proteins in PNETs [19]. Medulloblastomas

have been demonstrated to express zic, a gene normally

expressed only in the EGL of the developing cerebellum

and its derivatives, suggesting that medulloblastoma arises

from EGL precursor cells [20,21]. Given the rapid prolif-

erative capacity of EGL precursors and the pattern of

gene expression seen, EGL cells and their derivatives seem,

by far, the most likely origin of medulloblastoma. Other

cerebellar cells, such as Purkinje cells, basket neurons, or

glial cells, are unlikely to be cells of origin for medulloblas-

toma. Evidence that other stem cells in the cerebellum may

give rise to medulloblastoma comes from murine models of

human medulloblastoma (see below). For example, a model

of medulloblastoma has been generated using the GFAP

promoter to drive expression of the RecA recombinase,

resulting in tissue-specific inactivation of RB1 [22]. In this

work, cells expressing GFAP were identified in the develop-

ing cerebellum, although the vast majority of EGL precursors

did not express GFAP.

Medulloblastoma: Karyotypic Abnormalities

Only one karyotypic abnormality has been found to be typical

of medulloblastoma—the presence of an isochromosome

17q, present in about 50% of tumors [23]. The breakpoint

has been localized to 17p11.2, but no tumor-specific gene

rearrangement has been identified. Despite multiple studies,

no tumor-suppressor gene that can be implicated in the

development of medulloblastoma has been found on chro-

mosome 17p. Specifically, no alteration in p53 has been

identified with more than 100 tumors investigated to date.

The breakpoint for the rearrangement has been mapped

to 17p11.2 [24]. Other less common karyotypic abnormali-

ties, including loss of heterozygosity (LOH) on chromosome

9q, have been identified in about 20% of medulloblastomas.

Interestingly, the loss of 9q in medulloblastoma has been

correlated with the desmoplastic subtype [25].



Figure 4. The role of SHH in cerebellar development. Granule neuronal precursors (A–D) migrate tangentially from the rhombic lip and may use the SHH pathway

in transient autocrine manner. Purkinje neurons and later-born Bergmann glia (B) derive from the ventricular zone and migrate toward the EGL. SHH from the

Purkinje neurons induces Bergmann glia maturation (C). In the later EGL, granule neuronal precursors proliferate in the outer zone, utilizing SHH secreted from

Purkinje neurons. At the same time, mature glia send their extensions toward the inner EGL (D) and these or other cortical cells may provide factors that promote

the differentiation if granule neurons, antagonizing the effects of SHH. Granule cells then migrate on glial fibers across the molecular and Purkinje layers to form the

IGL. Maintained autocrine SHH signaling in the EGL (E) may result in the development of cerebellar tumors. (Reprinted with permission from Ref. [3].)

Analyses of Gene Expression in Medulloblastoma

Two recent reports of gene expression in medulloblastoma

have added insights to the basic biology of these tumors. In

the first of these, medulloblastoma samples were divided into

two groups, depending upon whether CSF dissemination of

tumor was seen at the time the patient presented with the

tumor [26]. Gene expression was analyzed on Affymetrix

G110 cancer arrays, which are enriched for genes thought to

be important in cancer biology. Fifty-nine genes that had

increased expression in the metastatic tumors compared to

the nonmetastatic tumors were identified. An additional 29

genes were identified, which had decreased expression in

the metastatic tumors. Two of theses genes, PDGFRa and

SPARC, were shown by immuohistochemical staining to be

expressed differentially between metastatic and nonmeta-

static tumors. In addition, antibodies to PDGFRa blocked

tumor cell migration in in vitro assays. These investigators

were able to use the pattern of gene expression to predict

which tumors presented with CSF metastases in a blinded

group of tumors. These results suggest that there may be

genes important in the progression of medulloblastoma, or

that alterations in different pathways may be responsible for

the differences in prognosis seen between tumors present-

ing without and with CSF dissemination.

In the second study, a group of ‘‘embryonal tumors’’ of the

CNS, including medulloblastomas, extracerebellar PNETs,

atypical teratoid/rhabdoid tumors, and malignant gliomas,

was presented [27]. Gene expression was analyzed on

Affymetrix HuGene FL arrays, containing 5920 known genes

and 897 expressed sequence tags. The first finding from this

data set was that each tumor type could be easily distin-

guished from the others based on gene expression pattern.

Especially interesting was the ability to distinguish medullo-

blastomas from other CNS PNETs, suggesting that the

lumping of these tumors together, as proposed by Rorke,

may be inappropriate. The medulloblastomas often

expressed genes normally expressed in the EGL, lending

credence to the hypothesis that EGL precursors represent

the cell of origin of medulloblastoma. Second, these inves-

tigators were able to distinguish ‘‘classic’’ medulloblastomas

from desmoplastic medulloblastomas. The important genes

with increased expression in the desmoplastic tumors were

those involved in the SHH/PTC pathway (see below). Lastly,

using an eight-gene model, these investigators were able to

accurately predict outcome in their patients with medullo-

blastoma. Genes associated with favorable clinical outcome

included TRKC and other genes characteristic of cerebellar

differentiation. In contrast, these genes were underex-

pressed in tumors with poor prognosis and genes involved

in cell proliferation were overexpressed.

Congenital Cancer Syndromes and Medulloblastoma

Medulloblastomas may occur in association with two differ-

ent inherited cancer syndromes: Gorlin syndrome and Turcot

syndrome. Nevoid basal cell carcinoma syndrome (NBCCS),

also called Gorlin syndrome or basal cell nevus syndrome, is

an autosomal dominant disorder [28]. Affected individuals

develop multiple basal cell carcinomas, multiple odontogenic

keratocysts of the jaws, palmar and plantar dyskeratoses,

and skeletal anomalies, especially rib malformations. In

addition, at least 40 cases of medulloblastoma have been

reported in patients with this syndrome, indicating that about

3% of Gorlin patients develop this tumor [29,30]. The gene

for Gorlin syndrome has been mapped to chromosome

9q22.3 [31,32]. Two studies have reported loss of genetic

markers mapped to 9q in medulloblastoma. In the first study,

16 patients were examined with 12 microsatellite markers

mapping between 9q13 and 9q34 [33]. Two tumors (12.5%)

showed LOH with microsatellite markers in this region. In the

second study, medulloblastomas from 20 patients, 17 with

sporadic tumors and 3 with NBCCS, were investigated with

seven microsatellite markers mapped to 9q22.3 to 9q31 [34].

Both informative tumors from patients with NBCCS showed

LOH for markers in the region; the third patient was not

informative. Three of the 17 sporadic tumors also showed

LOH on 9q. Interestingly, all three of the tumors from patients

with NBCCS in this study were designated desmoplastic me-

dulloblastomas. The other three tumors with LOH on 9q were

among six desmoplastic tumors in the sporadic group. Thus,

all of the tumors with LOH on 9q in this study were desmo-

plastic, raising the possibility that an NBCCS gene mutation

is involved in the development of this subclass of tumor.

The gene at 9q22.3 responsible for NBCCS has been

identified as the PTCH gene, the human homolog of the

Drosophila patched gene [35,36]. The Drosophila gene

encodes a protein with 12 putative transmembrane domains;

it may function as a receptor or transporter [37,38]. The

protein has an essential role in correct patterning of larval

segments and imaginal discs during adult fruit fly develop-

ment; a similar role in humans may explain the congenital

anomalies associated with NBCCS.

PTCH mutations have been described in patients with

NBCCS and in spontaneous basal cell carcinomas. A large

number of such mutations have been reported, including

single base substitutions, insertions ranging from a single

base to 300 bases, and deletions ranging from a single base

to 37 bases [35,36,39–42]. The described mutations are

fairly evenly distributed throughout the PTCH gene; no

‘‘mutational hot spots’’ have been identified.

Turcot syndrome is a hereditary disorder in which affected

individuals have multiple colonic polyps and a brain tumor,

either glioblastoma multiforme or medulloblastoma [43]. In

one study, mutations in the APC gene were identified in

the group of patients with Turcot syndrome who developed

medulloblastoma [44]. The relative risk for developing a

medulloblastoma in patients with Turcot syndrome and

an APC gene mutation is 92 times that in the general

population.

APC functions as a key regulator in a complex develop-

mental pathway (Figure 5). In the cytoplasm, APC associ-

ates with at least seven proteins, including b-catenin,glycogen synthase kinase 3b (GSK-3b), axin1 and axin2,

b-TrCP, the B6 subunit of the PP2A phosphatase, and hDLG

[45–55]. The control of the levels of free b-catenin in the

cytoplasm by APC defines the role of APC as a tumor



suppressor. Normally, free levels of b-catenin are low, as

binding of b-catenin by APC sequesters b-catenin and tar-

gets the protein for degradation. APC only binds b-cateninwhen b-catenin is hyperphosphorylated. b-Catenin is phos-

phorylated by GSK-3b, a serine/threonine kinase. b-Catenincontains four phosphorylation sites for GSK-3b, three serines

and one threonine, all encoded in exon 3 of the b-cateningene. When APC is inactivated by mutation (e.g., in colon

carcinoma), levels of cytoplasmic b-catenin rise. Free

b-catenin associates with members of the Tcf family

[56–58]. Four family members have been described; all

are transcription-regulating proteins with DNA-binding

activity. When b-catenin associates with Tcf, the complex

moves to the nucleus and upregulates the expression of

genes that increase the rate of cell division, either by

stimulating cell proliferation or by inhibiting apoptosis.



Figure 5. The wnt signaling pathway. Binding of wnt to its receptor, frizzled, leads ultimately to translation of �-catenin and Tcf into the nucleus, resulting in

transcription of genes controlled by this transactivator. The large complex involved in regulating free cytoplasmic concentrations of �-catenin contains axin1 and

axin2, APC, and GSK-3�. Phosphorylation of �-catenin by GSK-3� in the complex leads to degradation of �-catenin by the ubiquitin system.

Genetic Alterations in Medulloblastoma

Based on the germline gene alterations found in the inherited

syndromes described above, sporadic medulloblastomas

have been investigated for alterations of the involved genes.

Inactivation of the PTCH locus by deletion and mutation

has been found in about 10% of sporadic medulloblastomas,

suggesting that PTCH functions as a classic tumor suppres-

sor in this subset of tumors [59–64]. An analysis of the

SHH/PTCH pathway reveals that other genes in the path-

way might be altered to give a phenotype similar to that

caused by PTC inactivation. An investigation looking for

these alterations in 15 other pathway genes has revealed

only very rare mutations in v-smo and suppressor of fused

[su(fu)] [60,65,66]. This finding suggests that there is some-

thing unique about PTCH inactivation, or that the PTCH

locus is relatively easy to inactivate through mutation or

deletion.

Similarly, the APC gene has been investigated for inac-

tivation in sporadic medulloblastoma. Surprisingly, in light of

the association between germline APC gene mutation and

medulloblastoma seen in Turcot syndrome, APC gene muta-

tions have rarely been identified in spontaneously occurring

medulloblastomas. In one study, 47 sporadic medulloblas-

tomas were examined for mutations in the APC mutation

cluster region, comprising 10% of the gene, where more than

two thirds of the mutations seen in colorectal carcinoma

occur [67]. No tumor had an APC mutation in this region.

However, the entire gene was not investigated in this report.

Interestingly, in the two medulloblastomas in this study that

were removed from patients with Turcot syndrome, the

germline mutation was identified in tumor DNA, but no

mutation was seen in the other APC allele. In a second

study, DNA from 23 medulloblastomas was examined for

deletions in the region of the APC gene [68]. No LOH was

found in this region in any tumor. However, the four micro-

satellite markers used in this study, although the closest to

APC available at the time, were at least 30 to 70 kilobases

from the APC locus. In a third study, two tumors among 46

medulloblastomas were found to have APC mutations [69].

However, the mutations identified were very conservative

(i.e., alanine to valine and valine to isoleucine). The muta-

tions did not occur in defined functional domains of the

protein. The functional consequences of these mutations

are not clear. Taken together, these results indicate that

APC mutations in sporadic medulloblastoma are quite rare

and may have no functional consequence.

Because b-catenin/Tcf complexes are translocated to the

nucleus, nuclear localization of b-catenin, demonstrated

immunohistochemically, has been used as a marker for

tumors with active b-catenin/Tcf transcription [70–73]. Nu-

clear localization of b-catenin occurs in tumors with inacti-

vated APC or with oncogenic b-catenin mutations,

suggesting that this finding identifies tumors with increased

b-catenin/Tcf transcription, regardless of the mechanism

responsible. Immunohistochemical staining for b-cateninhas been performed in medulloblastoma [74]. Nine of 51

sporadic medulloblastomas were found to have nuclear

localization of b-catenin. This result suggests that at least

20% of medulloblastomas have alteration in the control of

b-catenin levels other than APC inactivation.

In colon carcinoma, free b-catenin levels may be in-

creased by two mechanisms. The first, inactivation of APC,

has been described above. The second mechanism involves

mutation of the b-catenin gene (locus CTNNB1) itself [75].

Mutations of b-catenin that alter the GSK-3b phosphorylationsites in exon 3 have been described. These missense

mutations change the serines or threonines that are

GSK-3b phosphorylation sites to cysteine, and prevent

b-catenin from being completely phosphorylated. Cotrans-

fection experiments using a reporter construct and mutated

b-catenin demonstrated that these mutations exerted

a dominant effect, rendering b-catenin/Tcf transcription

resistant to APC-mediated downregulation. Transcriptional

activities of b-catenin/Tcf reporter constructs in cells with

wild-type APC transfected with mutant b-catenin were at

least six times higher than in the same cells transfected with

wild-type b-catenin. Gel shift analysis also demonstrated that

free b-catenin was constitutively bound to Tcf-4 in nuclear

extracts from cells containing mutant b-catenin, even in the

presence of wild-type APC. Functionally similar mutations in

which exon 3 has been deleted from the b-catenin gene have

been described [76]. Taken together, these data indicate that

mutations eliminating a GSK-3b phosphorylation site from

b-catenin result in a protein that is no longer regulated by

APC, but that continues to function as a transactivator when

bound to Tcf. This model predicts that mutated b-cateninfunctions as a dominant oncogene, and this has been shown

to be the case. As predicted by this model, inactivating

mutations of APC and oncogenic mutations in b-cateninoccur in nonoverlapping subsets of colon carcinomas. The

ubiquitin-binding region of b-catenin is also in exon 3. Muta-

tion of this region, leading to lack of b-catenin ubiquitization,

has also been shown to be oncogenic.

Given that germline APC mutations in Turcot syndrome

give rise to an increased incidence of medulloblastoma and

given the rarity of APC mutations in sporadic medulloblas-

toma, sporadic medulloblastomas have been screened for

oncogenic mutations in b-catenin, as these mutations result

in the same phenotype as APC inactivation. Exon 3 of

b-catenin, the location of all GSK-3b phosphorylation sites,

was investigated by direct sequencing in 67 sporadic me-

dulloblastomas [77]. Five tumors were found to have

b-catenin mutations. In three cases, the alteration resulted

in the substitution of a cysteine for a serine at a GSK-3bphosphorylation site. Sequencing of exon 3 in the constitu-

tional DNA from these patients (available for two of the three

patients) failed to show a mutation, indicating that the

mutation identified is tumor-specific. Two additional muta-

tions were found, both of which altered an amino acid in the

ubiquitin-binding region, also in exon 3. Thus, 5 of 67 (7.5%)

tumors had oncogenic mutations in b-catenin. As ex-

pected, those tumors with b-catenin mutations also had

nuclear localization of b-catenin. Similar results have been

reported recently from another group [69], who reported

that of 46 medulloblastomas, four (8.7%) were found to

have oncogenic mutations in b-catenin. The finding that



one quarter of medulloblastomas have nuclear localization

of b-catenin, whereas only 7.5% contain oncogenic

b-catenin mutations, suggests that alterations in this path-

way—other than b-catenin mutation that result in in-

creased b-catenin/Tcf transcription—may be occurring

in a subset of medulloblastoma.

Asmentioned above, binding of b-catenin by APC leads to

degradation of b-catenin. At least two other proteins are

involved. In humans, these proteins have been called axin1

and axin2 (Figure 5). The protein products of both of these

genes associate with b-catenin, APC, and GSK-3b. Both

proteins appear to facilitate phosphorylation of b-catenin by

GSK-3b. In fact, in the absence of axin, GSK-3b phosphor-

ylates b-catenin poorly. A role for axin1 in bringing GSK-3band b-catenin together by mutual binding has been pro-

posed. Inactivation of axin1 or axin2 would be expected to

increase levels of free b-catenin through decreased degra-

dation and, thus, increase b-catenin/Tcf–mediated tran-

scription. Indeed, axin1 has been shown to be mutated in a

subset of hepatocellular carcinomas [78]. Hepatocellular

carcinoma cell lines with mutated axin1 demonstrated nu-

clear localization of b-catenin. Replacement of axin1 activity

by transfection with wild-type axin1 resulted in apoptosis in

these cell lines.

Medulloblastomas have been examined for axin1 muta-

tions [79,80]. In the first study, of 86 tumors, eight were found

to have mutations in axin1. There were seven deletions that

went from intron to intron and included at least two exons and

one point mutation. In four instances, an LOH analysis

revealed no evidence of loss of the wild-type allele. However,

the second study revealed these large deletions to be a

PCR artifact. The actual frequency of axin1 mutations is

low, with missense mutations found in 2 of 39 tumors

examined. These results suggest that axin1 mutations are

rare in medulloblastoma.

A third genetic alteration found in medulloblastoma is

c-myc or N-myc amplification. Amplification of myc genes

is rare, occurring in only 4% of medulloblastomas. Interest-

ingly, c-myc amplification appears to correlate with the large

cell phenotype, which carries a particularly grim prognosis.

Other Growth Factor Pathways in Medulloblastoma

Gilbertson et al. [81–83] have investigated the expression of

erbB family members in medulloblastoma. They have shown

that erbB2 and erbB4 are frequently expressed together in

medulloblastoma. Interestingly, erbB4, but not erbB2, was

expressed in the developing cerebellum. Expression of

erbB2 and erbB4 was associated with simultaneous expres-

sion of neuregulin1-a, suggesting the possibility of an auto-

crine loop in tumors with expression of all three proteins.

Indeed, erbB2/erbB4 dimerization was identified in tumors.

This group has also shown that novel splice variants of

erbB4 are found frequently in medulloblastoma. Significantly,

coexpression of erbB2 and erbB4 indicated a worse

prognosis and that expression of these receptors with

neuregulin1-a was associated with CSF dissemination at

presentation.

Activation of IGF-1R has also been demonstrated in

medulloblastoma cell lines [84]. Autophosphorylation of this

receptor and induction of c-fos expression in the presence of

exogenous IGF-1 have been shown, indicating a functional

receptor. Tumor growth was inhibited by an anti– IGF-1R

antibody that interferes with ligand binding. Similar results

have been reported by others [85].

Murine Models of Medulloblastoma

In the past, xenografts of established medulloblastoma cell

lines in nude mice have been used as the standard model for

this tumor in translational research. Unfortunately, these

models suffer from a number of problems. First, the two most

commonly used cell lines are not typical of the human tumor.

One of these, TE671 was used for a number of years until

proven to be a sarcoma rather than a medulloblastoma [86].

The second, Daoy, has genetic alterations (such as absence

of wild-type p53 and homozygous deletion of the CDKN2

gene, which encodes the tumor suppressors p16 and p15)

that are more characteristic of other types of brain tumors

than of medulloblastoma [87,88]. This raises the issue of the

use of cell lines to model medulloblastoma. PNETs have

proven difficult to establish in culture. Only a few cell lines

have been well characterized. Thus, it is possible that the

established cell lines are not representative of PNET as a

whole. Established cell lines could represent a subpopulation

of PNET with specific mutations that allow for growth in vitro,

or in vitro passage could select for mutations that occur

during initial passages that allow the tumor to grow in vitro.

Because of the limitations of xenograft models, and in an

attempt to replicate the human tumor, two laboratories have

developed ptch knockout mice [89,90]. Homozygous inacti-

vation of ptch causes embryonic lethality due to CNS system

defects including failure of neural tube closure; overgrowth of

head folds, hindbrain, and spinal cord; and cardiac defects

[89]. Hemizygous ptch mice (ptch +/�) have many of the

features of Gorlin syndrome, including skeletal abnormalities,

neural tube closure defects, a generalized overgrowth, and a

predisposition to tumor development. Indeed, about 30% of

these mice develop tumors in the cerebellum that resemble

human medulloblastoma histologically (Figure 2B). As

expected, expression of gli1 is increased in these tumors,

compared to expression in normal cerebellum, suggesting

activation of the SHH signaling pathway in these tumors.

Surprisingly, two independent studies have failed to demon-

strate any alteration in the wild-type allele in the murine

tumors [91,92]. Given the incidence of the tumors and the

retention of the wild-type allele, the possibility of epigenetic

silencing of the wild-type allele or mutation in other genes

must be considered in this model. Epigenetic silencing is

unlikely, as both studies demonstrate expression of wild-type

mRNA in the tumors. However, a recent study has suggested

that the wild-type ptch allele is silenced through methylation

[93]. Unlike the two studies mentioned above, these inves-

tigatorswere unable to identifywild-typeptchmRNA in tumors

from ptch +/� mice. They show that treatment of tumor cell

lines with an inhibitor of methylation results in downregulation



of the SHH/ptch pathway in tumor cell lines. How to reconcile

this result with the identification of wild-type message in

tumors cells by in situ hybridization is problematic.

The issue of other genes cooperating with hemizygous

inactivation of ptch has been investigated by Wetmore et al.

[94]. When the hemizygous ptch alteration is placed in a

p53-null background, more than 95% of the mice develop

medulloblastoma. In addition, the tumor arises earlier on a

p53-null background when compared to the timing of tumor

development when normal p53 is present. The increase in

tumor development was seen only on a p53-null back-

ground and was not associated with an APC hemizygous

background (Min +/�) or a p19/ARF hemizygous inactiva-

tion background. Given that p53 alterations are rarely seen

in sporadic human medulloblastoma, these results suggest

the possibility that the increased genomic instability of the

p53-null background may lead to mutations in genes that

cooperate with the hemizygous ptch alteration in the forma-

tion of these tumors.

Another murine model of medulloblastoma that depends

upon the overactivity of the SHH pathway has been devel-

oped by Weiner et al. [95]. These investigators have injected

the early cerebellar precursors of E13.5 mice in utero with a

SHH-containing retroviral vector, using a sophisticated ultra-

sound guided technique. When examined at P14 or P21, the

injected mice have retained the EGL, suggesting that con-

tinued expression of SHH in the EGL results in persistent

mitosis or prevents differentiation. In addition, the cerebellum

of injected mice contains nodules of mitotically active tissues

that resemble medulloblastoma. When examined at 13

weeks of age, injected mice have larger tumors that are

exerting mass effect on the cerebellum. Interestingly, given

the role of gli1 in the SHH pathway, identical findings were

seen when fetuses of gli1-null mice were injected with the

retrovirus. This result suggests that gli1 is not required for

tumor development in this model. Perhaps another gli family

member is sufficient.

A similar murine model of medulloblastoma has been

developed using the replication-competent ALV splice

(RCAS)/tv-a system [96]. In this model, a mouse line express-

ing the avian retroviral vector receptor, tv-a, under the control

of the nestin promoter was used. Neonatal mice were injected

with an avian RCAS acceptor vector into the cerebellum.

Three of 32 mice (9%) developed medulloblastoma. An addi-

tional 5 of 32 developed multifocal persistence and hyper-

proliferation of the EGL. When the nestin/tv-a mice were

injected with both a SHH/RCAS vector and a c-myc/RCAS

vector, 9 of 39mice (23%) developedmedulloblastoma. These

results suggest that a nestin-expressing cell in the neonatal

cerebellum is able to serve as a precursor to medullo-

blastoma. Whether the cooperation between SHH and c-myc

relates to the human tumor remains to be investigated.

An interesting model of medulloblastoma has been

reported in lig4-null mice. The lig4 gene encodes a protein

that participates in the repair of DNA double-strand breaks

by the nonhomologous end joining (NHEJ) complex. Mice

null for lig4 die in late embryonic development because of

extensive apoptosis of neurons in the CNS [97]. However,

p53-null, lig4-null mice survive [98]. The double-null mice

develop both lymphomas and medulloblastomas [99]. These

investigators attribute the DNA strand breaks to ‘‘genotoxic

stress’’ and suggest that the development of medullo-

blastoma in the double-null mice is related to mutations in

undefined genes caused by improper repair of DNA damage.

Which genes these may be has not been determined.

A second model of medulloblastoma in mice that com-

bines the p53-null state with defective DNA repair has also

been described [100]. In this model, the DNA repair gene

inactivated is poly(ADP-ribose polymerase) (PARP-1), a

DNA strand break–sensing gene that is activated as an

early response to DNA damage. When p53-null, PARP-1–

null mice were investigated, they were found to have a high

frequency of a cerebellar tumor resembling human medullo-

blastoma. The progression of these tumors was associated

with reactivation of expression of Math-1, a transcription

factor expressed in the EGL. Like the preceding model, this

model suggests that disruption of DNA repair in the EGL can

give rise to medulloblastoma. The genes mutated in this

model have also not been identified.

Two other murine models of medulloblastoma that involve

the inactivation of p53 and pRB1 have been developed. In an

attempt to generate astrocytic tumors, Marino et al. [22]

constructed a mouse with conditional knockout of the RB1

gene using the Cre/LoxP system. The Cre recombinase was

placed under control of the GFAP promoter to establish Cre

expression in astrocytes. When the GFAP-Cre mice were

crossed with RbLoxp/LoxP mice, inactivation of Rb in cells that

express GFAP occurs. When these conditions of RB1

knockout mice were combined with a p53-null background,

the resulting mice developed lymphoma, sarcomas, and

tumors of the cerebellum resembling medulloblastoma.

These investigators were able to demonstrate GFAP expres-

sion in the EGL precursors in the cerebellum of normal mice

and inactivation of RB1 in the same cells in the GFAP-Cre,

RbLoxp/LoxP mice. These results suggest that the tumors are

indeed arising from EGL precursors. In addition, alteration of

cell cycle control by RB1 inactivation is implicated in the

development of this tumor in mice.qqq

A different model of medulloblastoma that also involves

inactivation of pRB and p53 has been developed by Krynska

et al. [101]. These investigators constructed a transgenic

mouse line with the early sequences from JC virus that

contain the early promoter and the large T antigen gene.

These mice developed tumors of the cerebellum that resem-

ble medulloblastoma. This model shares functional features

with the model of Marino et al. in that large T antigen binds

and functionally inactivates both p53 and pRb. In addition,

tumors expressing T antigen contained evidence of in-

creased activity of the b-catenin/Tcf transactivator, which

has been implicated in the development of a subset of

human tumors.

Given that human medulloblastomas rarely contain either

p53 or pRB alterations, results from these two models

may not be easily extrapolated to the human condition.

Possibly, these models indicate that EGL precursors are

easily transformed by a number of possible alterations, only



some of which occur in humans. Alternatively, the key event

in the generation of these tumors may not be the alteration in

control of the cell cycle, but may rather relate to the activation

of events such as b-catenin/Tcf–regulated transcription that

have been seen in the human condition.

In summary, a number of murine models of medulloblas-

toma have been described. Some of these are the result of

genetic alterations that are similar to those defined in human

tumors, whereas others seem unrelated to the human

condition. Hopefully, work with these models will lead to

new insights into human medulloblastoma and to better

therapies directed by the molecular alterations. Additional

models with alterations in b-catenin or other wnt pathway

members have yet to be described.

Summary

A great deal of information about the genetic alterations

leading to the pediatric brain tumor medulloblastoma has

been reported. Subsets of tumors with alterations in genes

important in CNS development have been defined and

murine models based on some of these have been gener-

ated. Nonetheless, the percentage of tumors that have

identified gene alterations is less than 50% of the total.

Clearly, more work is needed to define what alterations are

responsible for uncontrolled cell proliferation in a majority

of these tumors.

Glossary

CerebellumA part of the brain involved in control ofcoordination and balance. Made up of twolaterally placed hemispheres connected by acentral portion called the vermis

CSF A clear, colorless fluid made by the brain.Fluid is made in chambers inside the brain(called ventricles), flows through channels inthe brain to get outside the brain, bathes thesurface of the brain and spinal cord, and isabsorbed into the venous bloodstream

HydrocephalusWhen circulation of CSF is blocked, synthesisof the fluid continues, leading to accumulationof the fluid in the chambers in the brain. Thechambers enlarge and exert pressure on thesurrounding brain

PleomorphicHaving a varied appearance related to cellsize, cell shape, and nuclear size

SynaptophysinA protein component of the vesicles thatcontains neurotransmitters in neurons

GFAP An intermediate filament protein whose geneexpression is restricted to astrocytes, one ofthe three main cell types in the brain

SHH An extracellular signaling molecule. Theprotein is cleaved into two parts; the activeamino-terminal portion is also cholestero-lated. Its homolog in Drosophila is hedgehog

PTCH The membrane-bound receptor for SHH. Theprotein contains 12 transmembrane domainsand associates in the membrane with v-smo.The Drosophila homolog is patched

v-smo The effector of SHH signaling. PTCH exertsinhibition of v-smo activity; this inhibition isreleased by SHH binding to PTCH. TheDrosophila homolog is smoothened

gli1 An intracellular effector of SHH signaling.There are three members of the gli family.The Drosophila homolog is cubitus interruptus

NeurotrophinsA family of growth and differentiation factorsrelated to nerve growth factor (NGF) andincluding NGF, BDNF, neurotrophin 3 (NT3),and neurotrophin 4 (NT4)

Neurotrophin receptorsThe trk family of tyrosine kinases. TrkA hasthe highest affinity for NGF, trkB for BDNF,and trkC for NT3

PDGFRAOne of two tyrosine kinase receptors forplatelet-derived growth factor (PDGF)

APC The adenomatous polypi coli gene. Theprotein product of this gene is involved inthe degradation of phosphorylated b-catenin

b-CateninA transcriptional transactivator involved in wntsignaling

Tcf The T-cell factor family contains four mem-bers. They provide the DNA-binding specific-ity for the b-catenin/Tcf transactivator

axin1 A gene whose protein product is part of theAPC complex that degrades phosphorylatedb-catenin

EGFR The membrane-associated tyrosine kinasereceptor for epidermal growth factor andtransforming growth factor-a



IGF-1R The membrane-associated tyrosine kinasereceptor for insulin-like growth factor 1

XenograftGrowth of tissue from one species in adifferent species

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Medulloblastoma: Molecular Genetics and Animal Models · Medulloblastoma: Molecular Genetics and Animal Models Corey Raffel Department of Neurologic Surgery, Mayo Clinic, 200 First