Distinct Neural Stem Cell Populations Give Rise
to Disparate Brain Tumors in Response to N-MYC
Fredrik J. Swartling,1,2,* Vasil Savov,2,5Anders I. Persson,1,5Justin Chen,1Christopher S. Hackett,1Paul A. Northcott,3
Matthew R. Grimmer,1Jasmine Lau,1Louis Chesler,4Arie Perry,1Joanna J. Phillips,1Michael D. Taylor,3
and William A. Weiss1,*
1University of California, Departments of Neurology, Pathology, Pediatrics, Neurosurgery, Brain Tumor Research Center and Helen Diller
Family Comprehensive Cancer Center, San Francisco, CA 94158, USA
2Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, SE-75185 Uppsala, Sweden
3The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada
4The Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK
5These authors contributed equally to this work
*Correspondence: firstname.lastname@example.org (F.J.S.), email@example.com (W.A.W.)
The proto-oncogene MYCN is mis-expressed in various types of human brain tumors. To clarify how
developmental and regional differences influence transformation, we transduced wild-type or mutationally
stabilized murine N-mycT58Ainto neural stem cells (NSCs) from perinatal murine cerebellum, brain stem,
and forebrain. Transplantation of N-mycWTNSCs was insufficient for tumor formation. N-mycT58Acerebellar
and brain stem NSCs generated medulloblastoma/primitive neuroectodermal tumors, whereas forebrain
NSCs developed diffuse glioma. Expression analyses distinguished tumors generated from these different
regions, with tumors from embryonic versus postnatal cerebellar NSCs demonstrating Sonic Hedgehog
(SHH) dependence and SHH independence, respectively. These differences were regulated in part by the
transcription factor SOX9, activated in the SHH subclass of human medulloblastoma. Our results demon-
strate context-dependent transformation of NSCs in response to a common oncogenic signal.
Medulloblastoma (MB), the most common malignant primary
brain tumor of childhood, arises in the cerebellum. Classic, des-
moplastic (nodular), and large cell/anaplastic (LC/A) pathologies
are described (Eberhart et al., 2004) and transcriptional profiling
has identified distinct subgroups characterized by signaling
through WNT, Sonic Hedgehog (SHH), or other signaling path-
of SHH signaling occurs in ?25% of tumors (Browd et al., 2006;
Kessleretal.,2009;PolkinghornandTarbell, 2007).Patients with
WNT-driven tumors show a favorable outcome when compared
and 4 have the worst outcomes (Eberhart et al., 2004; Ellison
et al., 2011; Pfister et al., 2009; Polkinghorn and Tarbell, 2007).
ciated with aggressive LC/A tumors and poor survival. Expres-
sion of MYCN is high in SHH-driven human tumors, and murine
N-MYC can potentiate preclinical models of MB driven by
activated SHH signaling. In contrast, MYCN-amplified tumors
are predominantly of non-SHH MB subtypes (Korshunov et al.,
2011); and mis-expression of MYCN occurs in the majority
of MB in all histopathologies (Northcott et al., 2009; Pomeroy
et al., 2002; Swartling et al., 2010). Neither gain nor mis-expres-
sion of MYCN isexclusive to MB,asMYCN isalso amplified and/
or mis-expressed in malignant glioma, the most common
Medulloblastoma (MB)/primitive neuroectodermal tumors (PNETs) and glioma represent the most common primary malig-
nant brain tumors in children and adults. These tumors presumably arise from a population of cells that share with normal
NSCs, a requirement for self-renewal and multilineage differentiation. N-myc, a proto-oncogene implicated in both normal
brain development and brain tumors, induced a Sonic Hedgehog (SHH)-dependent program in normal NSCs derived from
prenatal, and a SHH-independent program in NSCs derived from postnatal hindbrain. Orthotopic transplantation of N-myc-
transduced NSCs generated MB/PNETs from hindbrain NSCs, and glioma from forebrain NSCs. Thus, the diversity of brain
tumors in patients may be controlled by a limited set of transcription factors, reflecting both regional and temporal devel-
opmental restrictions among normal NSCs.
Cancer Cell 21, 601–613, May 15, 2012 ª2012 Elsevier Inc. 601
primary brain tumor of adults, and typically localized to forebrain
(cerebrum) rather than hindbrain (cerebellum) (Brennan et al.,
2009; Hui et al., 2001; Perry et al., 2009).
The cell of origin for both MB and glioma is debated. One
candidate is the neural stem cell (NSC) with self-renewal
capacity and potentialto generate progenitors and differentiated
bellum or cerebrum both during development and in adulthood
(Lee et al., 2005; Reynolds and Weiss, 1992). During both normal
cerebellar development and MB tumorigenesis, N-MYC drives
proliferation of granule neuron precursors (GNPs). GNPs are
marked by transcription factors including MATH1 and PAX6,
and are derived from radial glia or NSCs of the developing cere-
bellum(Hatton etal.,2006;Knoepfler etal.,2002;Linetal.,2001;
Schu ¨ller et al., 2008; Yang et al., 2008). Constitutive deletion of
N-myc is embryonic lethal (Charron et al., 1992; Stanton et al.,
ventricular zone (VZ), a markedly reduced cortex, and a diminu-
tive cerebellum (Knoepfler et al., 2002). These results indicate
a role for N-MYC in regulating normal forebrain and hindbrain
To understand whether changes in normal developmental
programs could influence transformation, we transduced
N-myc into forebrain, cerebellar, or brain stem tissues isolated
at distinct developmental time points. We studied mechanisms
for tumor development and compared expression profiles
among the disparate brain tumors generated by this common
oncogene. In the analysis we included our recently described
murine MYCN-driven MB model in which the Glutamate
Transporter 1 (GLT1) promoter drove a bidirectional Tetracy-
cline-Response Element (TRE), directing expression of MYCN
and Luciferase (GTML) (Swartling et al., 2010).
MB Spheres Are MYCN-Dependent and Express
We isolated brain tumors from nine mice from the transgenic
GTML model, and derived tumor spheres in neurobasal (NB)
media supplemented with epidermal growth factor (EGF) and
fibroblast growth factor (FGF). Eight representative sphere lines,
GTML2-9, arose independently of SHH (Swartling et al., 2010),
and showed elevated expression of MYCN protein and Lucif-
erase (LUC) (Figure 1A and Figure S1A available online). SHH-
dependent GTML tumors arise at <5% frequency. A single line
(GTML1) was isolated from a SHH-dependent GTML tumor
and showed low levels of MYCN mRNA (described previously
as GTML-T7 [Swartling et al., 2010]). While MYCN-low GTML1
cells showed no response to doxycycline (dox), proliferation of
MYCN-high GTML spheres was blocked after 3–5 days of dox
treatment, reflecting MYCN dependence (Figures 1B and 1C,
Relative expressionRelative expression
s l l e
c f o . o
o i s
e r p
v i t a l e
No. of cells
No. of cells40k
No. of cells
Day1 Day3 Day5Day1 Day3 Day5
Day1 Day3 Day5Day1 Day3 Day5
Figure 1. GLT1 Drives MYCN-Dependent, Predominantly SHH-
Negative Medulloblastoma and Is a Fate Determinant for NSCs
(A) Immunoblot of MYCN in GTML MB spheres (passages 6–10).
(B and C) Proliferation of MYCN-low (GTML1) and MYCN-high (GTML2)
spheres as indicated.
(D and E) All GTML sphere lines except GTML1 were insensitive to
the SMO inhibitor cyclopamine (5 mM). Tomatidine, 5 mM, was used as
(F–I) Relative expression of neuronal markers Ngn1 and Syp; and Olig2
and Sox9thatare expressedin glial cells inembryonic (E16 WT),and postnatal
(P0 WT) normal neurospheres and tumor spheres GTML1-4.
(J and K) GLT1-positive origin shown by X-gal fate mapping in NSCs isolated
from postnatal (P0) cerebellum and postnatal (P0) forebrain from a Glt1-tTA;
Error bars: ±SD. Scale: 100 mm. See also Figure S1.
N-MYC-Induced Brain Tumors
602 Cancer Cell 21, 601–613, May 15, 2012 ª2012 Elsevier Inc.
Figures S1B–S1D). This proliferation block, associated with loss
of Cyclin D2, did not recover after dox washout, as compared to
controls (Figure S1D and data not shown). These observations
are consistent with our earlier in vivo observations in GTML
mice that withdrawal of MYCN in tumors led to senescence
(Swartling et al., 2010).
To confirm SHH independence of MYCN-positive GTML2-9
tumor spheres, we cultured spheres with the SMO inhibitor
cyclopamine (Taipale et al., 2000), which blocks signaling down-
stream from SHH. GTML1 SHH-dependent spheres responded
to SMO inhibition. However, no significant differences in prolifer-
ation were observed after treating GTML2-9 spheres with
cyclopamine (Figures 1D and 1E and data not shown), or with
SHH-N or SMO agonists that activate SHH signaling (data not
shown). These data are consistent with tumors arising through
a SHH-independent pathway. However, it remains possible
that signaling downstream of SMO could contribute to tumor
Compared to WT cerebellar spheres and SHH-dependent
GTML1 spheres, GTML2-4 tumor spheres with high levels of
MYCN protein showed elevated levels of mRNA for Ngn1 and
Syp (Figures 1F and 1G). NGN1, an inducer of neurogenesis
and a marker of immature GABAergic cells originating from the
roof of the fourth ventricle, is typically expressed in MATH1-
negative, classic MB (Farah et al., 2000; Salsano et al., 2007).
Spheres from GTML 2-4 showed low levels of glial-lineage
transcription factors Olig2 and Sox9 (Figures 1H and 1I), both
of which are also typically low in human MB, and high in cultured
normal human cerebellar NSCs (Alcock and Sottile, 2009;
de Bont et al., 2008; Ligon et al., 2004). These marker data
collectively verify that GTML2-4 spheres resemble SHH-
negative human MB.
Freshly isolated GTML tumor cells can be transplanted ortho-
topically into the brains of immunocompromised mice to regen-
erate MB (Swartling et al., 2010). While SHH-dependent GTML1
tumor spheres did not grow orthotopically, spheres from SHH-
independent tumors (GTML2 and GTML3) generated lethal
tumors (Figure S1E). Transplanted GTML2 tumor spheres grew
significantly more rapidly than transplanted GTML3 tumor
spheres consistent with higher levels of MYCN protein in
GTML2 cells (Figures S1D and S1E).
To determine whether NSCs isolated from normal cerebellum
could represent cells of origin for GTML MB, we crossed
Glt1-tTA mice to TRE-Cre:Rosa26-lsl-LacZ reporter animals,
enabling us to visualize LacZ as a marker of cell fate. Indeed,
NSCs cultured as spheres from these triply transgenic mice at
postnatal ages were b-galactosidase positive, suggesting that
the GLT1 promoter is active and that GTML tumors could origi-
nate from normal NSCs (Figures 1J and 1K).
Transduction of N-mycT58Ainto the GFAP-Positive NSCs
GFAP is a stem cell and astroglial marker (Doetsch et al., 1999)
and shows tight coexpression with GLT1 both in cerebellar and
cerebral cells in vivo (Schmitt et al., 1996) as well as in our
NSC cultures (Figure S1F). We therefore investigated how
GFAP-positive populations of NSCs would respond to N-myc,
using mice transgenic for the avian tv-a retroviral receptor driven
by the GFAP promoter (Gtv-a) (Holland et al., 2000). We
dissected cells at embryonic day 16 (E16) and postnatal day 0
(P0) from three different brain regions of Gtv-a mice (Figure 2A):
luminal parts of forebrain ventricular zone (<0.1 mm), total
cerebellum, and the top layer (<0.1 mm) isolated from the
ventricular zone region of the dorsal brain stem. This isolated
region encompassed both VZ brain stem cells and the lower
rhombic lip (LRL) structure that contains candidate cells of origin
for MB (Gibson et al., 2010).
Dissected cells were cultured in neurobasal media for 1 week
and subsequently transduced with harvested RCAS viruses
containing either a green fluorescent protein (GFP) reporter,
Flag-tagged wild-type N-mycWT, or mutationally stabilized
Flag-tagged N-mycT58A(Figures S2A–S2E). After differentiation
for 72 hr without growth factors, most GFP-transduced NSCs
from both E16 and P0 cerebellar and forebrain NSCs remained
positive for the NSC marker GFAP, but were negative for the
neuronal marker, TUJ1, confirming selective transduction of
GFAP-positive NSCs (Figure 2B and Figure S2F).
To further characterize the types of cells transduced, we
sorted GFP-positive and -negative NSC fractions from E16
cerebellum, and P0 cerebellum and forebrain, 72 hr after trans-
duction with RCAS-GFP. The percentage of GFP-positive cells
ranged from 2.5% up to 8% of the entire cell population (Fig-
ure 2B and Figure S2F). Interestingly, GFP-sorted single NSCs
from these conditions produced significantly more secondary
spheres than GFP-negative NSCs after 2 weeks in culture (Fig-
ure S2G). These data are consistent with selective transduction
of GFAP-positive cells within these primary cultures, showing
increased self-renewal as compared with more differentiated
populations of cells.
We next analyzed cells transduced with RCAS-N-myc
constructs. N-mycT58A-transduced cerebellar NSCs stained
intensely for Flag on western blot as compared to N-mycWT-
transduced cells (Figure 2C), likely reflecting stabilization of
N-MYC protein due to decreased proteasomal degradation
(Salghetti et al., 1999). As compared to cerebellar NSCs trans-
duced with GFP or N-mycWT, NSCs transduced with N-mycT58A
and GTML2 tumor spheres were both resistant to blockade of
new protein synthesis by cycloheximide (CHX, Figures S2H–
S2K) consistent with stabilization of N-MYCT58Aor overexpres-
sion of MYCN in these cells.
moderate proliferation when cultured on coated plates, trans-
duction of N-mycT58Ainto NSCs from P0 cerebellum promoted
increased proliferation, associated with high levels of the fourth
ventricular zone marker NGN1 (Figures 2C and 2D). Similarly,
transduction of N-mycT58Ainto P0 forebrain NSCs, as well as
E16 NSCs from both cerebellum and forebrain, promoted
proliferation, as compared to both vector GFP control and
N-mycWT-transduced NSCs (Figure 2E and data not shown).
Because NSCs in the brain stem may also generate brain
tumors (Gibson et al., 2010), we next tested VZ-derived NSCs
proliferation in E16 and P0 brain stem cultures in this system
(data not shown), prompting us to test VZ-derived NSCs from
E14 dorsal brain stem/LRL (henceforth called E14 LRL). Trans-
duction of E14 LRL NSCs cells with N-mycT58Aled to increased
proliferation as compared to GFP- and N-mycWT-transduced
NSCs (Figure 2F).
N-MYC-Induced Brain Tumors
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Culturing cerebellar NSC for a week in neurobasal media
typically removes any residual GNPs (Klein et al., 2005; Sutter
et al., 2010). Transduction of N-mycWTor N-mycT58Aretroviruses
into purified P0 GNPs did not drive proliferation, presumably
because very few PAX6-positive GNPs also expressed GFAP
and tv-a, thus evading viral transduction (Figures S2L–S2M).
To clarify whether N-myc could drive self-renewal and substi-
tute for growth factors, we cultured NSCs and tumor spheres in
limiting dilution for 1 week with or without EGF and FGF (ten
cells/well). To distinguish effects on NSCs from effects on neural
progenitors, 100 cells/well were replated in a secondary sphere
formation assay for a second week. N-mycWT-, N-mycT58A-, and
GFP-transduced control secondary spheres retained strict
growth factor dependence and showed no significant differ-
ences in sphere number (Figures 2G–2I, Figures S2N–S2P). By
contrast, GTML tumor spheres generated secondary spheres
even when cultured without growth factors (Figure 2J). MYCN
blocked differentiation in GTML tumor spheres, as spheres
with high MYCN protein levels neither differentiated nor
extended processes upon growth factor starvation or doxycy-
cline treatments (data not shown). In contrast and as expected,
SHH-dependent GTML1 cells and control cerebellar NSCs
transduced with GFP virus showed multipotent differentiation
into both GFAP-positive astrocytes and TUJ1-positive neurons
(Figure 2B, Figure S2F and data not shown).
An N-MYC Neuronal Program Depends on Both
Developmental Age and Regional Origin
To characterize the potential for N-myc to induce distinct
differentiation programs as a function of both NSC age and
Day3 Day5 Day7Day3 Day5 Day7
No. of cells
2º spheres 2º spheres
Day3 Day5 Day7
tv-a receptor on a GFAP+ cell
tv-a receptor on an infected GFAP+ cell
uninfected GFAP- cell lacking tv-a
only GFAP+ cells
isolated NSCs cultured
Brain regions isolated from perinatal mice expressing
tv-a receptor from the GFAP promoter (Gtv-a)
Figure 2. NSC-Selective Infection with N-mycT58AAlters Proliferation and Differentiation
(A) NSCs isolated from E16 and P0 forebrain (ventricular zone), E16 and P0 hindbrain (total cerebellum), and E14, E16, and P0 brain stem (ventricular zone/lower
rhombic lip) from mice transgenic for Gtv-a were cultured for 7 days in NB media on low-affinity plates with one or two passages. RCAS viruses containing GFP,
N-mycWT(N-MYC), or N-mycT58A(T58A) came from supernatants of DF-1 virus-producing cells (see Figure S2). NSCs were transduced with RCAS viruses mixed
1:1 with fresh NB media. Cells were infected (72 hr), dissociated, and cultured in fresh NB media. Cells were orthotopically transplanted in nude mice 7 days after
(B) Selective infection of GFAP-positive P0 cerebellar NSCs, evidenced by coexpression of RCAS-GFP (green) with GFAP (blue) 72 hr after growth factor
depletion. TUJ1 (red). Scale: 25 mm.
(C) Immunoblot of FLAG-tagged N-mycWTor N-mycT58Ain P0 cerebellar NSCs. NGN1 and b-actin control are also shown.
(D–F) Proliferation at3,5, and 7 daysafter transduction with GFP,N-mycWT,or N-mycT58Aof P0 NSCsisolated from cerebellum (D), forebrain (E), and dorsal brain
stem/lower rhombic lip (F), respectively.
(G–J) Growth factor independence assay showing sphere diameter (as indicated) and number of secondary spheres in P0 cerebellar NSCs transduced with GFP,
N-mycWT, or N-mycT58Aand in GTML2 spheres. Although not indicated, there are significantly more spheres irrespective of size in ‘‘GF conditions’’ (FGF, EGF, or
FGF+EGF) as compared to ‘‘no GF conditions’’ when analyzing GFP-, N-MYC-, and T58A-transduced NSCs, respectively, when using Student’s t test (p < 0.05).
Error bars: ± SD. See also Figure S2.
N-MYC-Induced Brain Tumors
604 Cancer Cell 21, 601–613, May 15, 2012 ª2012 Elsevier Inc.
regional origin, we analyzed the glial markers SOX9 (Stolt et al.,
2003) and GFAP, 72 hr after transduction of N-mycT58Ainto
cerebellar NSCs at E16 and P0 and forebrain NSCs at P0.
N-mycT58A-transduced NSCs all showed increased N-myc
mRNA levels as by Affymetrix exon array analysis of distinct
N-mycT58Ashowed reduced mRNA expression of Gfap and
protein levels of SOX9 as compared to E16 cerebellar NSCs,
whereas P0 forebrain NSCs showed elevated levels of Gfap
and SOX9 (Figure 3A).
As SOX9 has been reported to also act downstream of the
SHH/SMO to induce and maintain NSCs (Scott et al., 2010),
we next asked whether N-mycT58Aaffected SHH signaling.
GFP-transduced control NSCs from E16 and P0 cerebellum
and forebrain all responded to SMO inhibition through cyclop-
amine treatment (Figures 3B and 3C), consistent with reports
that forebrain NSCs, cerebellar radial glia, and cerebellar NSCs
are sensitive to SHH signals in vivo (Ahn and Joyner, 2005;
Huang et al., 2010). While N-mycT58A-transduced E16 cerebellar
cells and N-mycT58A-transduced P0 forebrain cells retained
sensitivity to cyclopamine, N-mycT58A-transduced P0 cerebellar
and E16 forebrain cells, like GTML tumor spheres, were resistant
(Figures 3D and 3E).
mycT58A-transduced cerebellar cultures, we again used Affyme-
trix exon arrays to analyze expression of Smo, a coreceptor
for the SHH signal; Sfrp1, a marker of SHH-driven MB; Gli1,
a marker of SHH activation, and Gli3, a marker of SHH repres-
sion. Expression of Smo was unchanged in comparing P0
and E16 cultures (Figures S3A and S3B). Cyclopamine-resistant
P0 cultures showed repression of SHH signaling. Both Sfrp1
and Gli1 were decreased, while Gli3 was upregulated in P0
N-mycT58Acultures, as compared to E16 cerebellar NSCs.
promotes SHHindependence (downstreamofSMO)inpostnatal
cerebellar cells, in embryonic forebrain cells, and in GTML
N-mycT58AInitiates Brain Tumors from NSCs
and whether the differentiative programs shown in Figure 3
would persist in vivo, we transduced embryonic and postnatal
NSCs with N-mycWTor N-mycT58Aand transplanted cells
orthotopically into the brains of nude mice. In contrast to
N-mycWTNSCs, N-mycT58ANSCs from E16 and P0 cerebellum
or E16 and P0 forebrain all formed brain tumors (Figures 4A
and 4B). N-mycT58Acerebellar NSCs generated tumors at higher
incidence and penetrance than the N-mycT58Aforebrain NSCs
(Figures 4A and 4B). We also analyzed LRL-derived NSC.
Consistent with our in vitro data, N-mycT58AE16 and P0 LRL
NSCs failed to generate brain tumors, while N-mycT58A–
transduced E14 LRL NSCs generated brain tumors in 40% of
mice when transplanted orthotopically (Figure 4C).
Orthotopic transplantation of N-mycT58AP0 cerebellar NSCs
into cerebellum resulted in massive tumors with morphology of
human MB, including Homer Wright (neuroblastic) rosettes,
and prominent cell wrapping characteristic of LC/A tumors
(Figures 4D and 4E and Figure S4A)). The histology of N-mycT58A
P0 cerebellar tumors also resembled GTML tumors (Swartling
et al., 2010), sometimes showing LC/A histopathology (Figures
P0 forebrain NSCs into cerebrum induced aggressive forebrain
tumors (Figure 4F) that were more invasive than the cerebellar
tumors (Figure 4G). The P0 forebrain tumors resembled diffuse
gliomas in terms of distant spread of individual tumor cells along
white matter tracts (Figure 4G and Figure S4D). E14 LRL tumors
were typically characterized by marked cellular and nuclear
pleomorphism (Figures 4H and 4I).
While no differences in proliferation (KI67) and vascularity
(CD34) were found among the P0 forebrain and P0 cerebellar
tumors, P0 cerebellar tumors were significantly more apoptotic
than P0 forebrain tumors (Figures S4E–S4J). P0 cerebellar
tumors were generally negative or showed only scattered cells
positive for the astrocytic markers GFAP, OLIG2, and SOX9
(Figures 4J–4L) and moderate immunoreactivity for the neuronal
marker SYP (Figure 4M). In contrast, P0 forebrain tumor cells
were strongly positive for GFAP, OLIG2, and SOX9 (Figures
4N–4P) and negative for SYP (Figure 4Q). LRL tumors showed
an intermediate phenotype with few scattered cells positive for
the astrocytic markers GFAP, moderate positivity for OLIG2
No. of cells
Day3 Day5 Day77yaD5yaD3yaD1yaDDay1
7.69 8.47 (log2)
GFP T58A GFP T58A
No. of cells
No. of cells80k
Day3 Day5 Day7Day1
No. of cells80k
Day3 Day5 Day7Day1
P0 GFP E16 GFP
5.89 8.04 (log2)
Figure 3. The N-mycT58A-Driven Neuronal Program Depends on
Regional Origin and NSC Age
(A) Levels of N-myc and Gfap from Affymetrix exon arrays (log2values) and
immunoblots of SOX9 and b-actin in E16 and P0 cerebellar and P0 forebrain
NSCs transduced with GFP or N-mycT58A, respectively.
(B–E) Proliferation (7 days) of cerebellar and forebrain NSCs transduced with
GFP (B and C) or N-mycT58A(D and E) and treated with cyclopamine (cyc) or
tomatidine (tom) in 5 mM final concentrations for E16 and P0 ages, respec-
Error bars: ±SD. *p < 0.05 from Student’s t test. See also Figure S3.
N-MYC-Induced Brain Tumors
Cancer Cell 21, 601–613, May 15, 2012 ª2012 Elsevier Inc. 605
and SOX9 (Figures 4R-4T) and scattered islands of SYP-positive
cells (Figure 4U).
The histology of N-mycT58A
cerebellar, E16 forebrain NSCs generally resembled N-mycT58A
tumors induced from P0 cerebellar and P0 forebrain NSCs,
respectively (Figures S4K and S4L). The majority of tumors
derived from E16 cerebellar NSCs were SOX9 positive (Fig-
ure S4M). Most embryonic forebrain tumors showed distinct
areas of glial differentiation (Figure S4N). Lack of nuclear
b-catenin expression in LRL tumors (Figure S4O) suggests
a WNT-independent tumor subtype, as compared to a recently
reported model for MB also derived from LRL cells originating
from brainstem (Gibson et al., 2010). These and tumors from
E16 or P0 cerebellum were similarly insensitive to the GSK3b
inhibitor TWS119 at 1 mM (data not shown) demonstrating WNT
independence, although it remains possible that N-mycT58A
tumors induced from E16
0 100200 300 Days
P0 N-MYC (n=20)
E16 T58A (n=20)
P0 T58A (n=25)
100200 300 Days
E16 N-MYC (n=15)
P0 N-MYC (n=20)
E16 T58A (n=20)
P0 T58A (n=30)
E16 N-MYC (n=15)
0 100 200 300 Days
E14 T58A (n=10)
E16 T58A (n=10)
P0 T58A (n=10)
CerebellumForebrain Brain stem/LRL
Figure 4. N-mycT58AInduces Brain Tumors
from Both Cerebellum and Forebrain
(A–C) Survival of mice orthotopically transplanted
with 100,000 NSCs isolated from forebrain, cere-
bellum, and brain stem/LRL, transduced with
N-mycWTor N-mycT58Aviruses. n = number of
(green colors) or N-mycT58AE16 and P0 brain
stem/LRL (red and green, respectively) generated
(D–I) Histopathology of representative P0 cere-
bellar (D and E), P0 forebrain (F and G), and E14
brain stem/LRL (H–I) N-mycT58Atumors (H&E
features, including cell wrapping in a tumor with
prominent LC/A features (D, arrowhead) and
Homer Wright rosettes (E, arrowhead). Forebrain
tumors displayed diffuse invasion of tumor cells
along white matter tracts (arrowhead) (G). LRL
tumors displayed marked cellular and nuclear
pleomorphism (I, arrowhead). Box in H (2003)
denotes region shown at higher magnification in I
N-mycT58A-induced cerebellar, forebrain, and LRL
tumors demonstrating greater neuronal differenti-
ation in cerebellar tumors, greater glial differenti-
ation in forebraintumors,
differentiation in LRL tumors. *Tumor region in
pictures where an adjacent normal brain is
Scale: 100 mm. See also Figure S4 and Table S1.
late and destabilize b-catenin.
Regional Origin Exerts a Dominant
Effect on Brain Tumor Type
NSCs into forebrain VZ generates mostly
et al., 2005). To determine if the regional
brain niche was responsible for the
generated tumor phenotypes, we in-
jected N-mycT58A-transduced P0 cere-
bellar NSCs into the forebrain and N-mycT58A-transduced P0
forebrain NSCs into the hindbrain. We studied the same markers
as in Figure 4 and quantified positive cells (Table S1). We
included three controls each of site-matched tumors for E16
and P0 cerebellum, tumors from E16 forebrain, P0 forebrain,
and E14 brain stem, and three GTML tumors.
For all tumors, we also examined protein expression of
KCNA1, a Kv1.1 voltage-gated potassium channel-encoding
gene associated with Group 4 MB (Taylor et al., 2011) as well
as the LRL and WNT markers OLIG3 and b-catenin, respectively
(Gibson et al., 2010). The profiles of GFAP, Nestin, and SYP in
tumors correlated generally with region from which NSCs were
isolated, rather than region of the brain into which we injected
transduced cells. Like the LRL tumors (Figure S4O), all tumors
examinedwere negativefor nuclearb-catenin (TableS1).Intrigu-
ingly, SOX9 and OLIG2 showed a modest and differential
N-MYC-Induced Brain Tumors
606 Cancer Cell 21, 601–613, May 15, 2012 ª2012 Elsevier Inc.
expression pattern in ‘‘misplaced’’ tumors, with higher levels of
protein expression in misplaced hindbrain tumors and lower
expression levels in misplaced forebrain tumors (Table S1). Our
data still suggest that region of origin exerts a dominant effect
on tumor type.
SOX9 Marks SHH-Dependent MB and Malignant Glioma
Transduction of N-mycT58Ainto murine NSCs led to regional and
age-dependent changesin levels ofSOX9 protein(Figure 3A). To
further clarify the expression of SOX9 in MB, we analyzed array
data from 103 human MBs segregated into WNT, SHH, Group
3, or Group 4 tumors (Northcott et al., 2011; Taylor et al.,
2011). Clustering and expression patterns were consistent with
both SOX9 and MYCN as markers for WNT- and SHH-driven
human tumors (Figure 5A). NGN1 expression was increased in
all subtypes except for the SHH group, marked by the human
MATH1 ortholog, ATOH1, and supported by previous data
discriminating these subclasses (Cho et al., 2011; Kool et al.,
2008; Northcott et al., 2011; Thompson et al., 2006). Consistent
with the findings of SOX9 as a marker of SHH-driven tumors,
(Figure S5), which are typically SHH-dependent (Northcott et al.,
2011). Furthermore, SOX9 was only observed in a few scattered
cells in a representative human classic MB (Perry et al., 2009)
previously reported to lack MYCN amplification (Figure 5B),
while a human MYCN-amplified MB showed high levels of
SOX9 (Figure 5C).
characteristic of aggressive human malignant glioma with
components of primitive neuroectodermal tumors (MG-PNET).
We recently described 53 cases of human MG-PNET, detailing
both poor survival (essentially identical to human high-grade
glioma) and common amplification of MYC and MYCN (43%)
in PNET-like foci (Perry et al., 2009). Since MG-PNET may arise
in the setting of relapsed glioblastoma (GBM), we next analyzed
data from Cancer Genome Atlas Research (TCGA) Network
(2008). While OLIG2 and SOX9 were overexpressed in half
or more than half of the GBM samples, expression of neuronal
markers like NGN1 and SYP were downregulated in most
samples (Table S2). Among 424 human GBMs, 20% overex-
pressed MYCN, consistent with a role for MYCN in the
pathogenesis of some GBMs (Table S2). Irrespective of MYCN
amplification, human MG-PNET samples showed high level
and distinct nuclear expression of SOX9 (Figures 5D and 5E),
consistent with results from glioma cell lines (Swartling et al.,
2009) and TCGA data (Table S2).
Alignment of Murine Tumors and Classification Using
Human MB Subgroup Identifiers
Having demonstrated that SOX9 can mark SHH-driven human
MB, we next applied these observations to murine N-mycT58A
MB and GTML tumors, using Affymetrix exon arrays. Unsuper-
vised clustering revealed that N-mycT58Atumors from E16 cere-
bellum, P0 cerebellum, and P0 forebrain NSCs were closely
aligned to the NSCs from which they were derived (Figure 6).
N-mycT58Atumors and their corresponding NSCs were more
similar to GTML tumors than to normal cerebellum (Figure 6).
Within the N-mycT58Atumor group, the region of origin (cere-
bellum versus forebrain) was a larger separator than the age of
isolation of the initiating cells. Originating NSCs, N-mycT58A
tumors, and GTML tumors were all more similar to each other
than to normal cerebellum. The signature of NSCs versus tumor
was greater than the difference between NSC types, precluding
the use of gene expression signatures to identify the most similar
potential originating cell.
Using identifiers for subtypes of human MB (Northcott et al.,
2011), E16 MB (generated from N-mycT58A-transduced E16
cerebellar NSCs) showed a distinct SHH-pathway profile (Fig-
ure S6A) while both P0 MB (generated from N-mycT58A-
transduced P0 cerebellar NSCs) and GTML tumors presented
significantly higher expression of the Group 4 identifier KCNA1,
as compared to E16 MB. By contrast, the other subgroup iden-
tifiers, DKK1 (WNT), SFRP1 (SHH), and NPR3 (Group 3), did not
significantly delineate these groups (Figure S6B).
E16 N-mycT58ACerebellar Tumors Model
SHH-Dependent MB, while P0 Tumors Model
Expression levels of Sox9, N-myc, and Math1 further distin-
guished the various N-mycT58Atumors using both Affymetrix
arrays as well as quantitative real-time PCR, as compared to
expression levels in adult cerebellum (Figures 7A–7C and
MB - no MYCN
MB - with MYCN
Glioma - no MYCN Glioma - with MYCN
Figure 5. SOX9 Marks a SHH-Dependent
Brain Tumor Profile
(A) Expression analysis of SOX9, MYCN, ATOH1
(human MATH1 ortholog), OLIG2, and NGN1 in
103 primary MB samples clustered in four MB
subgroups, WNT,SHH, Group3, and Group 4with
levels normalized to normal adult cerebella (n = 5)
as described previously (Northcott et al., 2011).
Dark blue represents the lowest and dark red the
(B–E) SOX9 immunostaining in representative
human MB (B and C) and glioma (D and E)
sections, without and with MYCN amplification, as
determined by fluorescent in situ hybridization
(Perry et al., 2009).
Scale: 100 mm. See also Figure S5 and Table S2.
N-MYC-Induced Brain Tumors
Cancer Cell 21, 601–613, May 15, 2012 ª2012 Elsevier Inc. 607
Figures S7A and S7B). E16 MB showed high levels of Sox9
mRNA, N-myc, and of the EGL marker Math1, suggesting
a SHH- rather than a WNT-dependent subtype (Figures 7A–
7C). In contrast, P0 MB displayed lower expression of Sox9,
N-myc and Math1. Similar to P0 MB, E14 LRL tumors (generated
from N-mycT58A-transduced prenatal LRL NSCs) showed low
N-myc and Math1 levels consistent with a SHH-independent
origin (Figures 7A–7C).
Interestingly, forebrain N-mycT58AP0 tumors (P0 glioma)
showed high levels of Sox9 and N-myc and absent Math1
(Figures 7A–7C), consistent with transformation of a forebrain
cell type that shows SHH dependence independently of Math1
(Akazawa et al., 1995). Expression analysis substantiated the
glioma phenotype observed in the forebrain tumors, in which
levels of Gfap were significantly elevated as compared to hind-
brain tumors (Figure S7B). Moreover, Nestin (Nes) was among
the top 30 genes overexpressed in P0 glioma as compared to
P0 MB (Table S3). P0 MB showed a low immunoreactivity of
Nestin, while P0 glioma showed elevated Nestin expression
(Figures S7C and S7D and Table S1). As Nestin is a strong
marker for glioma and upregulated in 99% of human GBM
samples in the TCGA database (Table S2), increased Nestin
expression suggests a glioma-like differentiation pattern in fore-
brain tumors compared to the MB-like pattern seen in cerebellar
In agreement with SOX9 as a marker for SHH dependence,
cultured P0 MB spheres with low levels of SOX9 were resistant
to cyclopamine (Figure 7D). Conversely, both E16 MB and P0
glioma with high SOX9 levels were cyclopamine sensitive (Fig-
ure 7D). Similar dependence on SHH was also observed in
N-mycT58ANSCs prior to orthotopic implantation and tumor
initiates SHH-dependent transformation in both E16 cerebellar
NSCs and P0 forebrain NSCs, while transformation of E16
forebrain NSCs and P0 cerebellar NSCs (and possibly in brain
stem NSCs) occurs through a SHH-independent pathway.
SOX9 Promotes Self-Renewal and Generates
GLI2-Expressing Tumors at Shorter Latency
Our data demonstrate that P0 MBs arise through a SOX9- and
SHH-independent program. To establish whether SOX9 plays
a functional role in transforming P0 cerebellar NSCs, we gener-
ated SOX9 RCAS viruses (Figure S8A) and selectively trans-
duced Gtv-a-positive P0 N-mycT58Acerebellar NSCs or P0 MB
spheres (isolated from individual P0 MBs). Forced expression
of SOX9 suppressed proliferation in P0 N-mycT58Acerebellar
cells and in P0 MB spheres (Figure S8B), which have low levels
of SOX9 (Figures 3A, 4I, and 7A). In contrast, similar forced
expression of SOX9 did not affect E16 MB and P0 glioma, which
have high SOX9 levels at baseline (Figures S8C and S8D).
Although forced expression of SOX9 suppressed proliferation
in N-mycT58Acerebellar cells and in P0 MB spheres, SOX9
actually drove increased self-renewal when culturing these at
a limited dilution. Single cell clones from normal P0 cerebellar
NSCs and P0 cerebellar NSCs transduced with N-mycT58A
were transduced with SOX9, and clones with high levels of
SOX9 were selected (Figure 8A). SOX9 increased self-renewal
in both normal and N-mycT58A-transduced P0 cerebellar NSC
clones, as compared to normal and N-mycT58A–transduced P0
cerebellar NSC clones without forced expression of SOX9 (Fig-
ure 8B). Single cell spheres and spheres generated from an
isolated P0 MB (P0C-T3, also cultured and propagated from
a single cell clone) were dependent on growth factors EGF and
FGF as compared to single cell clones subcloned out from the
GTML2 sphere line (Figure 1A) that generated secondary
spheres essentially independently of growth factors. Forced
Tumors, and Normal Murine Tissues Show
Discrete Expression Signatures
Dendrogram showing unsupervised hierarchical
obtained from: (1) primary and tumor-derived cells
(yellow): four individual GFP-transduced E16
and P0cerebellar (sorted
forebrain NSCs, two N-mycT58A-transduced E16
and P0 cerebellar NSCs (in low passages), and
three cell lines (in low passages) derived from
orthotopic E16 and P0 cerebellar and P0 forebrain
tumors. (2) Orthotopic tumors (red): nine freshly
isolated orthotopic E16 and P0 cerebellar and
P0 forebrain N-mycT58A-tumors. (3) GTML tumors
one cultured GTML tumor cell line (GTML2). (4)
Normal cerebellum (blue): five samples from
P7 GTML double heterozygous total cerebella.
The distance in the clustering determines how well
samples are related to each other. See also
N-MYC-Induced Brain Tumors
608 Cancer Cell 21, 601–613, May 15, 2012 ª2012 Elsevier Inc.
expression of SOX9 was also associated with higher levels of
Gli2 (Figures 8C–8E), suggesting that SOX9 can drive SHH-
signaling in these cells.
Since forced expression of SOX9 drove self-renewal while
blocking proliferation of cells in vitro, we next asked how these
potentially opposing activities would impact tumor formation
in vivo. Forced expression of SOX9 and N-mycT58Ain P0 cere-
bellar NSCs generated orthotopic tumors at shorter latency
and with increased tumor penetrance, as compared with NSCs
expressing N-mycT58Aalone (Figure 8F). The SOX9-expressing
tumor cells demonstrated marked cellular and nuclear pleomor-
phism, numerous mitoses, and numerous apoptotic bodies,
consistent with a primitive neuroectodermal phenotype (Fig-
ure S8E). Focally tumor cells contained abundant cytoplasm
and GFAP immunostaining demonstrated a population of
GFAP-positive tumor cells. (Figures S8E and S8F). These
observations suggest that SOX9 can cooperate with N-MYC to
promote malignant progression acting upstream of GLI2.
Forced expression of N-mycT58Arepressed SOX9 in P0 cere-
bellar NSCs. Is SOX9 similarly repressed in GTML tumors? In
response to dox-mediated withdrawal of MYCN, some cells
within GTML tumors undergo senescence (Swartling et al.,
2010).Consistent withthisresult, doxtreatmentofGTML3tumor
spheres led to death of most cells, while a few cells remained
viable without proliferating (Figures S8G and S8H and data not
shown). In response to withdrawal of MYCN, these viable cells
showed elevated levels of SOX9 at levels similar to SHH-
dependent GTML1 control cells (Figure 8G). This suggests
SOX9 is repressed by forced expression of MYCN also in
No. of cells60k
Day3 Day5 Day7Day1
E16 MB (tom)
E16 MB (cyc)
P0 MB (tom)
P0 MB (cyc)
P0 Glioma (tom)
P0 Glioma (cyc)
Figure 7. N-MYC Drives Both SHH-Dependent and SHH-Indepen-
(A–C) Relative expression of Sox9, N-myc, and Math1 in five disparate
N-MYC-driven murine tumors, E16 MB (n = 4), P0 MB (n = 6), E16 glioma
(n = 4), P0 glioma (n = 6), and E14 LRL tumor (n = 3). n = number of individual
are shown for LRL tumors because of few samples (n = 3). Whiskers go from
minimum to maximum values.
(D) Proliferation of cultures of E16 MB, P0 MB, and P0 glioma isolated from
individual tumors and treated with cyclopamine (cyc) or tomatidine (tom) used
at final concentrations of 5 mM. *Statistical significance (p < 0.05) from
Student’s t test. Error bars: ±SD.
See also Figure S7 and Table S3.
100 200300 Days0
2º spheres (%)
Figure 8. SOX9 Promotes Self-Renewal and N-MYC-Driven Brain
(A) Immunoblot of NSCs generated from single cells of GFP- and SOX9-
transduced P0 cerebellar NSCs from normal (six individual clones) and
N-mycT58Atransduced NSCs (three individual clones).
(B) Limiting dilution assay showing secondary (2?) spheres cultured starting
from one single cell per well (with or without (wo) EGF and FGF). Numbers
indicates mean values (from two experiments) of spheres per well from a total
of 60 wells counted (shown as %). Clone numbers used (from A) are indicated.
Assay results from single clones from a P0 MB tumor (P0C-T3 (Cl.1)) and
from a GTML tumor [GTML2 (Cl.1)] are also included. *Statistical significance
(p < 0.05) from Student’s t test.
(C and D) Relative expression of SOX9 and Gli2 in N-mycT58A-transduced P0
cerebellar NSCs and N-mycT58A-transduced P0 cerebellar NSCs further
transduced with SOX9 (P0C-T58A-SOX9 Cl.3), respectively. For SOX9
expression and clone numbers, see (A). *Statistical significance (p < 0.05) from
Student’s t test.
(E) Immunoblot showing GLI2 protein expression in N-mycT58A-transduced P0
cerebellar normal (P0C-T58A Cl.1) or SOX9-transduced (P0C-T58A-SOX9
(F)Survivalafter orthotopic transplantationof100,000 cellsfromN-mycT58AP0
cerebellar NSCs (P0C-T58A, as used in Figure 4A; n = 30) and N-mycT58AP0
cerebellar NSCs further transduced with SOX9 (P0C-T58A-SOX9, n = 10),
respectively. Same clone (Cl.3) was used as in (C)–(E).
(G) Re-expression of SOX9 in representative GTML spheres (GTML3) treated
with dox (1 mg/ml) after 72 hr as shown in an immunoblot, with GTML1 cells as
controls. *Statistical significance (p < 0.05) from Student’s t test.
Error bars: ±SD.
See also Figure S8.
N-MYC-Induced Brain Tumors
Cancer Cell 21, 601–613, May 15, 2012 ª2012 Elsevier Inc. 609
SHH-independent GTML tumors. Collectively, data in Figure 8
demonstrate that interactions between N-MYC and SOX9
regulate proliferation, differentiation, and self-renewal in P0
N-mycT58AMB, and that similar interactions are relevant to
MYCN-driven GTML MB.
Deletion of N-myc in neural stem and progenitor cells in the
mouse causes reduced cellularity of both forebrain and hind-
brain, suggesting an essential role in the growth and develop-
ment of these structures (Knoepfler et al., 2002). Here, by
orthotopic transplantation of N-mycT58ANSCs, we validate
a reciprocal role for mis-expression of N-myc in the pathogen-
esis of both forebrain and hindbrain tumors. Our data, and the
finding that MYCN is commonly overexpressed in both forebrain
and in hindbrain tumors in humans (Brennan et al., 2009; Eber-
hart et al., 2004; Hui et al., 2001; Perry et al., 2009; Pfister
et al., 2009; Pomeroy et al., 2002), suggest a role for MYCN in
the development of brain tumors.
Our experiments are consistent with recent results suggesting
a relationship between normal NSCs and tumor initiating cells
(Holland et al., 2000; Johnson et al., 2010; Schu ¨ller et al., 2008;
Sutter et al., 2010; Yang et al., 2008). Orthotopic injection of
N-mycT58ADF-1 virus-producing cells failed to generate brain
tumors (Browd et al., 2006 and our unpublished data), perhaps
in part because few, if any, NSCs are successfully transduced
through this approach. We instead generated five different
tumors from distinct NSCs transduced with N-mycT58Aafter
a brief propagation in culture. N-mycT58ANSCs derived from
embryonic cerebellum led to SHH-dependent MB, consistent
with transduction of a GFAP-positive NSC with a propensity to
become a MATH1-positive granule neuron precursor (Marino
et al., 2000; Schu ¨ller et al., 2008; Yang et al., 2008). In contrast,
N-mycT58ANSCs derived from postnatal forebrain led to tumors
with characteristics of malignant glioma, consistent with trans-
duction of a GFAP-positive NSC with a propensity to become
a MATH1-negative, SHH-dependent forebrain cell (Palma
et al., 2005).
N-mycT58ANSCs derived from embryonic forebrain generated
brain tumors at low penetrance. Analysis of a limited number of
these tumors suggests transduction of a GFAP-positive NSC
with a propensity to become a SHH-independent forebrain
glioma. N-mycT58ANSCs derived from postnatal cerebellum led
to SHH-independent MB with low levels of Math1 and Sox9.
Tumors were also generated from the lower rhombic lip or the
dorsal regions of the VZ of the developing brain stem. Interest-
ingly, only E14 LRL NSCs could induce brain tumors, whereas
no tumors were observed from E16 or PO LRL/brain stem NSCs.
Thatour LRL tumors were only seen atE14 ages correlate with
previous findings of WNT-subtype MB in the murine LRL/dorsal
brainstem generated between E11.5 and E15.5 (Gibson et al.,
2010). Our LRL tumors resembled human anaplastic MB/PNETs
and arose independently of WNT signaling, with negative immu-
noreactivity for nuclear b-catenin and the LRL marker OLIG3.
These tumors might thus arise from a brain stem VZ cell type
distinct from an OLIG3-positive LRL progenitor (Storm et al.,
2009). LRL tumors lacked Math1 expression, and showed
moderate levels of Sox9. The low levels of Math1 are also
characteristic of GTML MB arising in transgenic mice, and are
consistent with transduction of a GFAP/GLT1 double-positive
NSC with potential to become a MATH1-negative, SHH-inde-
Expression analysis of E16 MB revealed elevated levels of
many genes upregulated in the SHH subclass of human MB,
with human Group 4 MB (Taylor et al., 2011). Collectively, our
experiments argue that GFAP-positive NSCs from forebrain
and hindbrain can give rise to both malignant glioma and to at
least two distinct subtypes of MB, a SHH subgroup and
a SHH-independent Group 4 MB subgroup, together represent-
ing 38% and 62% of MYCN-amplified human MB, respectively
of origin for our tumors truly represent self-renewing tripotent
NSCs. Therefore, it remains possible that the cells we refer to
as NSCs, actually represent progenitors with a more limited
potential for differentiation. In either case, our observations raise
questions as to the nature of cells transformed in human tumors.
Perhaps mutations that initiate transformation occur at embry-
onic or postnatal ages, generating partially transformed cells
that persist into childhood or adulthood, later giving rise to
SOX9 acts downstream of SHH signaling, at least in part
because GLI1, a marker of SHH activation, binds directly to
a regulatory element in the SOX9 promoter region, driving
expression (Bagheri-Fam et al., 2006; Vidal et al., 2005). SOX9
is essential for formation and maintenance of multipotent NSCs
and is an effector of SHH signaling (Scott et al., 2010). SOX9
levels were high in SHH-driven human MB, a finding aligned
with our murine data that E16 cerebellar NSCs retain SOX9
expression upon transformation, and respond to SHH inhibition.
Forced expression of SOX9 in P0 N-mycT58Acerebellar NSCs
in P0 tumors. However, SOX9 also promoted self-renewal,
associated with increased expression of Gli2 in P0 N-mycT58A
cerebellar NSCs, leading to increased penetrance of a Gli2-
expressing tumor. These data demonstrate that SOX9 expres-
sion can promote SHH-activation in N-mycT58AMB, and suggest
SOX9 as an important regulator of SHH MB. Further, our obser-
vations are consistent with a model in which N-MYC promotes
in a profile similar to the majority of human MB.
N-mycT58Aforebrain tumors showed significant glial differenti-
ation without microvascular proliferation, pseudopalisading
cells and necrosis. These features are reminiscent of human
MG-PNET, a tumor type that we recently detailed, and which
shows frequent amplification of MYCN (Perry et al., 2009). The
to model these tumors in the mouse collectively support a role
for MYCN in the pathogenesis of malignant glioma. Since MG-
PNET may arise in the setting of pre-existing or recurrent GBM
(Perry et al., 2009), our studies also provide a mouse model
both for the study of tumor biology and for development of
therapies against this highly lethal neoplasm.
Although GBM and MB comprise common malignant brain
tumors, the catalog of human brain tumors is extensive, and
the richness of cell varieties in the CNS is reflected in the many
N-MYC-Induced Brain Tumors
610 Cancer Cell 21, 601–613, May 15, 2012 ª2012 Elsevier Inc.
varieties of brain tumors observed in the human population.
Previous reports demonstrate that NSCs in the forebrain and
hindbrain show developmental restrictions based on the brain
regions from which they were derived (Klein et al., 2005; Merkle
et al., 2007). The murine tumors described here showed striking
similarities with specific subtypes of human brain tumors,
suggesting a pathogenic role for MYCN in a heterogeneous
group of human brain tumors. Together with the recently
published models of MYC-driven MB (Kawauchi et al., 2012;
Pei et al., 2012), we have recapitulated the most aggressive
MYC/MYCN-driven human childhood brain tumors.
Animals and Bioluminescent Imaging
Generation of GTML mice of the FVB/NJ strain has been previously described
(Swartling et al., 2010). Tumor progression of doubly Glt1-tTA/MYCN-TRE
transgenic mice was continuously followed by luciferase signaling using the
IVIS Lumina (Caliper Life Sciences, Mountain View, CA) using Living Image
2.5 software (Caliper Life Sciences, Mountain View, CA). Mice were injected
with 75 mg/kg of sodium luciferin (LUCNA, Gold Biotechnology, St. Louis,
MO) in saline prior to imaging. The mice selected for tumor removal and
neurobasal cell culture all had high scores (>5.0 3 1010photons/cm2sec)
from bioluminescence imaging suggesting a functional bidirectional transgene
expressing luciferase as previously reported (Swartling et al., 2010). For cell
fate experiments, Glt1-tTA mice were crossed with TRE-Cre:R26R-LSL-
LacZ mice obtained from Dr. Robert Blelloch, UCSF. The G-tva mice have
been described before (Holland and Varmus, 1998) and were generously
provided by Dr. Eric Holland and backcrossed at least five generations into
FVB/NJ strain before experimental use. Athymic Nude-Foxn1nu mice were
obtained by Simonsen Laboratories (Gilroy, CA) or Harlan Laboratories
(Venray, The Netherlands) and used in experimental procedures as described
below. Mice were maintained in the Animal Facilities at University of California,
SanFranciscoandatUppsalaUniversity,Uppsala.Allanimal procedures were
performed in accordance with national guidelines and regulations and
approved bytheInstitutional AnimalCare and UseCommitteeof theUniversity
of California in San Francisco and Uppsala Ethical Committee on Animal
Experiments in Uppsala.
Neural Stem/Progenitor Cell and Tumor Cell Culturing
Normal tissue or part of tumor was removed and mechanically dissolved in
cold Hank’s buffer without calcium and magnesium (Cell Culture Facility,
UCSF, Mission Bay, San Francisco). After one more wash in cold Hank’s
buffer, the sample was incubated for 20 min in activated papain (Worthington,
Lakewood, NJ). Tumor cells were gently dissociated and cultured in neuro-
basal media (GIBCO) without vitamin A, supplemented with antibiotics (Cell
Culture Facility, UCSF), B27 without vitamin A (Invitrogen), L-glutamine and
20 ng/ml EGF (Sigma), and 20 ng/ml FGF-2 (Peprotech, Rocky Hill, NJ). Cells
were cultured and propagated on low adhesion plates (Corning) for 1 week,
further passaged, and used in experiments. SVZ neurospheres were estab-
lished from FVB/N WT mice and similarly cultured as described above. Fore-
brains of E16.5 (described as E16) and P0.5 (described as P0) were coronally
sectioned (100 mm thick sections) and sections 2.5–3.5 mm posterior of the
base of olfactory bulb and the dorsal regions closest to the ventricles were
isolated and dissociated as above. Total cerebellum was carefully removed
excluding any parts of meninges, brain stem, or midbrain, dissociated and
similarly cultured as above. Cell layers (?0.05–0.1 mm) of dorsal brain stem
ciated and similarly cultured as above. GNPs were enriched and cultured as
previously described in 10 ng/ml SHH-N (SHH N-terminal peptide, R&D
Systems) containing serum-free media (Swartling et al., 2010).
Expressionanalysis(aspresented inFigure5andFigureS5)wasperformed on
103 primary human medulloblastoma using Affymetrix exon arrays as
previously described (Northcott et al., 2009; Swartling et al., 2010). All tumor
specimens were obtained in accordance with the Research Ethics Board at
the Hospital for Sick Children (Toronto, Canada) and deidentified prior to anal-
MATH1, OLIG2, and NGN1 in four distinct molecular variants described as
WNT, SHH, group C, and group D tumors as previously identified by multiple
unsupervised analyses of these medulloblastoma samples (Northcott et al.,
2010). RNA from tumors was isolated using Trizol (Invitrogen, Carlsbad, CA)
and purified using the RNeasy Mini Kit (QIAGEN, Valencia, CA). For the
GTML tumors, 1 mg of RNA was used as a starting template for the RiboMinus
rRNA subtraction protocol (Invitrogen) followed by the ST labeling protocol
(Affymetrix, Santa Clara, CA). For the transplanted tumors, 100 ng of RNA
was used as a starting template for the Ambion WT Expression protocol
(Applied Biosystems, Carlsbad, CA) followed by the WT Terminal Labeling
protocol (Affymetrix). Labeled samples were hybridized to Affymetrix Mouse
Exon 1.0 arrays. All arrays were normalized together with expression values
calculated using RMA in the XPS package in R. Boxplots were generated in
R using the standard graphics package. Two-way comparisons between
groups were performed using Significance of Microarrays (SAM) (Tusher
et al., 2001).
ncbi.nlm.nih.gov/geo/), with accession number GSE36594.
Supplemental Information includes eight figures, three tables, Supplemental
Experimental Procedures and can be found with this article online at
The authors would like to acknowledge Eric Holland, Anna-Marie Kenney, and
Daniel Fults for RCAS constructs, Monica Venere and Robert Blelloch for the
TRE-Cre:R26R-LSL-LacZ transgene, Cynthia Cowdrey and the UCSF Brain
Tumor Research Center Tissue Core for tumor samples, and Kim Nguyen
and Slava Yakovenko for skillful technical assistance. We thank Jan Grawe ´
and BioVis, SciLifeLab at Uppsala University, for help with cell sorting. We
thank David Rowitch and Richard Gilbertson for helpful discussions and Justin
Meyerowitz for critical reading of the manuscript. We acknowledge support
from the Pediatric Brain Tumor Foundation, the Swedish Research Council,
the Swedish Cancer Society, the Swedish Childhood Cancer Foundation,
Ake Wibergs Stiftelse, Lions Cancerforskningsfond, Stiftelsen Lars Hiertas
Minne, Children’s Brain Tumor Foundation, NIHR01 CA133091, CA148699,
and CA159859. This work was also supported by funds from NIH K08
NS063456 to J.J.P. and Hellmann Fellowship Award and the American Brain
Tumor Association Translational Award to A.I.P.
Received: November 10, 2010
Revised: January 26, 2012
Accepted: April 4, 2012
Published: May 14, 2012
Ahn, S., and Joyner, A.L. (2005).In vivo analysisof quiescent adult neural stem
cells responding to Sonic hedgehog. Nature 437, 894–897.
Akazawa, C., Ishibashi, M., Shimizu, C., Nakanishi, S., and Kageyama, R.
(1995). A mammalian helix-loop-helix factor structurally related to the product
of Drosophila proneural gene atonal is a positive transcriptional regulator
expressed in the developing nervous system. J. Biol. Chem. 270, 8730–8738.
Alcock, J., and Sottile,V. (2009).Dynamic distribution and stem cell character-
istics of Sox1-expressing cells in the cerebellar cortex. Cell Res. 19,
Bagheri-Fam, S., Barrionuevo, F., Dohrmann, U., Gu ¨nther, T., Schu ¨le, R.,
Kemler, R., Mallo, M., Kanzler, B., and Scherer, G. (2006). Long-range
N-MYC-Induced Brain Tumors
Cancer Cell 21, 601–613, May 15, 2012 ª2012 Elsevier Inc. 611
upstream and downstream enhancers control distinct subsets of the complex
spatiotemporal Sox9 expression pattern. Dev. Biol. 291, 382–397.
Brennan, C., Momota, H., Hambardzumyan, D., Ozawa, T., Tandon, A.,
Pedraza, A., and Holland, E. (2009). Glioblastoma subclasses can be defined
by activity among signal transduction pathways and associated genomic
alterations. PLoS ONE 4, e7752.
Browd, S.R., Kenney, A.M., Gottfried, O.N., Yoon, J.W., Walterhouse, D.,
Pedone, C.A., and Fults, D.W. (2006). N-myc can substitute for insulin-like
growth factor signaling in a mouse model of sonic hedgehog-induced
medulloblastoma. Cancer Res. 66, 2666–2672.
Charron, J., Malynn, B.A., Fisher, P., Stewart, V., Jeannotte, L., Goff, S.P.,
Robertson, E.J., and Alt, F.W. (1992). Embryonic lethality in mice homozygous
for a targeted disruption of the N-myc gene. Genes Dev. 6 (12A), 2248–2257.
Cancer Genome Atlas Research Network. (2008). Comprehensive genomic
characterization defines human glioblastoma genes and core pathways.
Nature 455, 1061–1068.
Cho, Y.J., Tsherniak, A., Tamayo, P., Santagata, S., Ligon, A., Greulich, H.,
Berhoukim, R., Amani, V., Goumnerova, L., Eberhart, C.G., et al. (2011).
Integrative genomic analysis of medulloblastoma identifies a molecular
subgroup that drives poor clinical outcome. J. Clin. Oncol. 29, 1424–1430.
de Bont, J.M., Kros, J.M., Passier, M.M., Reddingius, R.E., Sillevis Smitt, P.A.,
Luider,T.M.,denBoer, M.L.,and Pieters, R.(2008).Differentialexpression and
prognostic significance of SOX genes in pediatric medulloblastoma and
ependymoma identified by microarray analysis. Neuro-oncol. 10, 648–660.
Doetsch, F., Caille ´, I., Lim, D.A., Garcı ´a-Verdugo, J.M., and Alvarez-Buylla, A.
(1999). Subventricular zone astrocytes are neural stem cells in the adult
mammalian brain. Cell 97, 703–716.
Eberhart, C.G., Kratz, J., Wang, Y., Summers, K., Stearns, D., Cohen, K.,
Dang, C.V., and Burger, P.C. (2004). Histopathological and molecular
prognostic markers in medulloblastoma: c-myc, N-myc, TrkC, and anaplasia.
J. Neuropathol. Exp. Neurol. 63, 441–449.
Ellison, D.W., Dalton, J., Kocak, M., Nicholson, S.L., Fraga, C., Neale, G.,
Kenney,A.M., Brat, D.J.,Perry,
Medulloblastoma: clinicopathological correlates of SHH, WNT, and non-
SHH/WNT molecular subgroups. Acta Neuropathol. 121, 381–396.
A., Yong,W.H.,et al. (2011).
Farah, M.H., Olson, J.M., Sucic, H.B., Hume, R.I., Tapscott, S.J., and Turner,
D.L. (2000). Generation of neurons by transient expression of neural bHLH
proteins in mammalian cells. Development 127, 693–702.
Gibson, P., Tong, Y., Robinson, G., Thompson, M.C., Currle, D.S., Eden, C.,
Kranenburg, T.A., Hogg, T., Poppleton, H., Martin, J., et al. (2010). Subtypes
of medulloblastoma have distinct developmental origins. Nature 468,
Hatton, B.A., Knoepfler, P.S., Kenney, A.M., Rowitch, D.H., de Albora ´n, I.M.,
Olson, J.M., and Eisenman, R.N. (2006). N-myc is an essential downstream
effector of Shh signaling during both normal and neoplastic cerebellar growth.
Cancer Res. 66, 8655–8661.
Holland, E.C., and Varmus, H.E. (1998). Basic fibroblast growth factor induces
cell migration and proliferation after glia-specific gene transfer in mice. Proc.
Natl. Acad. Sci. USA 95, 1218–1223.
Holland, E.C., Celestino, J., Dai, C., Schaefer, L., Sawaya, R.E., and Fuller,
G.N. (2000). Combined activation of Ras and Akt in neural progenitors induces
glioblastoma formation in mice. Nat. Genet. 25, 55–57.
Huang, X., Liu, J., Ketova, T., Fleming, J.T., Grover, V.K., Cooper, M.K.,
Litingtung, Y., and Chiang, C. (2010). Transventricular delivery of Sonic
hedgehog is essential to cerebellar ventricular zone development. Proc.
Natl. Acad. Sci. USA 107, 8422–8427.
Hui, A.B., Lo, K.W., Yin, X.L., Poon, W.S., and Ng, H.K. (2001). Detection of
multiple gene amplifications in glioblastoma multiforme using array-based
comparative genomic hybridization. Lab. Invest. 81, 717–723.
Johnson, R.A., Wright, K.D., Poppleton, H., Mohankumar, K.M., Finkelstein,
D., Pounds, S.B., Rand, V., Leary, S.E., White, E., Eden, C., et al. (2010).
Cross-species genomics matches driver mutations and cell compartments
to model ependymoma. Nature 466, 632–636.
Kawauchi, D., Robinson, G., Uziel, T., Gibson, P., Rehg, J., Gao, C.,
Finkelstein, D., Qu, C., Pounds, S., Ellison, D.W., et al. (2012). A mouse model
of the most aggressive subgroup of human medulloblastoma. Cancer Cell 21,
Kessler, J.D., Hasegawa, H., Brun, S.N., Emmenegger, B.A., Yang, Z.J.,
Dutton, J.W., Wang, F., and Wechsler-Reya, R.J. (2009). N-myc alters the
fate of preneoplastic cells in a mouse model of medulloblastoma. Genes
Dev. 23, 157–170.
Klein, C., Butt, S.J., Machold, R.P., Johnson, J.E., and Fishell, G. (2005).
Cerebellum- and forebrain-derived stem cells possess intrinsic regional
character. Development 132, 4497–4508.
Knoepfler, P.S., Cheng, P.F., and Eisenman, R.N. (2002). N-myc is essential
during neurogenesis for the rapid expansion of progenitor cell populations
and the inhibition of neuronal differentiation. Genes Dev. 16, 2699–2712.
Kool, M., Koster, J., Bunt, J., Hasselt, N.E., Lakeman, A., van Sluis, P., Troost,
D., Meeteren, N.S., Caron, H.N., Cloos, J., et al. (2008). Integrated genomics
identifies five medulloblastoma subtypes with distinct genetic profiles,
pathway signatures and clinicopathological features. PLoS ONE 3, e3088.
Korshunov, A.,Remke,M.,Kool, M., Hielscher, T., Northcott, P.A., Williamson,
ical heterogeneity of MYCN-amplified medulloblastoma. Acta Neuropathol.
Lee, A., Kessler, J.D., Read, T.A., Kaiser, C., Corbeil, D., Huttner, W.B.,
Johnson, J.E., and Wechsler-Reya, R.J. (2005). Isolation of neural stem cells
from the postnatal cerebellum. Nat. Neurosci. 8, 723–729.
Ligon, K.L., Alberta, J.A., Kho, A.T., Weiss, J., Kwaan, M.R., Nutt, C.L., Louis,
D.N., Stiles, C.D., and Rowitch, D.H. (2004). The oligodendroglial lineage
marker OLIG2 is universally expressed in diffuse gliomas. J. Neuropathol.
Exp. Neurol. 63, 499–509.
Lin, J.C., Cai, L., and Cepko, C.L. (2001). The external granule layer of the
developing chick cerebellum generates granule cells and cells of the isthmus
and rostral hindbrain. J. Neurosci. 21, 159–168.
Marino, S., Vooijs, M., van Der Gulden, H., Jonkers, J., and Berns, A. (2000).
of Rb in the external granular layer cells of the cerebellum. Genes Dev. 14,
Merkle, F.T., Mirzadeh, Z., and Alvarez-Buylla, A. (2007). Mosaic organization
of neural stem cells in the adult brain. Science 317, 381–384.
Northcott, P.A., Fernandez-L, A., Hagan, J.P., Ellison, D.W., Grajkowska, W.,
Gillespie, Y., Grundy, R., Van Meter, T., Rutka, J.T., Croce, C.M., et al.
(2009). The miR-17/92 polycistron is up-regulated in sonic hedgehog-driven
medulloblastomas and induced by N-myc in sonic hedgehog-treated cere-
bellar neural precursors. Cancer Res. 69, 3249–3255.
Bouffet, E., Clifford, S.C., Hawkins, C.E., French, P., et al. (2011).
Medulloblastoma comprises four distinct molecular variants. J. Clin. Oncol.
Palma,V.,Lim,D.A., Dahmane, N., Sa ´nchez, P.,Brionne,T.C., Herzberg, C.D.,
Gitton, Y., Carleton, A., Alvarez-Buylla, A., and Ruiz i Altaba, A. (2005). Sonic
hedgehog controls stem cell behavior in the postnatal and adult brain.
Development 132, 335–344.
Pei, Y., Moore, C.E., Wang, J., Tewari, A.K., Eroshkin, A., Cho, Y.J., Witt, H.,
Korshunov, A., Read, T.A., Sun, J.L., et al. (2012). An animal model of
MYC-driven medulloblastoma. Cancer Cell 21, 155–167.
Perry, A., Miller, C.R., Gujrati, M., Scheithauer, B.W., Zambrano, S.C., Jost,
S.C., Raghavan, R., Qian, J., Cochran, E.J., Huse, J.T., et al. (2009).
Malignant gliomas with primitive neuroectodermal tumor-like components:
a clinicopathologic and genetic study of 53 cases. Brain Pathol. 19, 81–90.
Pfister, S., Remke, M., Benner, A., Mendrzyk, F., Toedt, G., Felsberg, J.,
Wittmann, A., Devens, F., Gerber, N.U., Joos, S., et al. (2009). Outcome
prediction in pediatric medulloblastoma based on DNA copy-number aberra-
tions ofchromosomes 6qand 17q and theMYC and MYCN loci. J.Clin. Oncol.
N-MYC-Induced Brain Tumors
612 Cancer Cell 21, 601–613, May 15, 2012 ª2012 Elsevier Inc.
Polkinghorn, W.R., and Tarbell, N.J. (2007). Medulloblastoma: tumorigenesis, Download full-text
current clinical paradigm, and efforts to improve risk stratification. Nat. Clin.
Pract. Oncol. 4, 295–304.
Pomeroy, S.L., Tamayo, P., Gaasenbeek, M., Sturla, L.M., Angelo, M.,
McLaughlin, M.E., Kim, J.Y., Goumnerova, L.C., Black, P.M., Lau, C., et al.
(2002). Prediction of central nervous system embryonal tumour outcome
based on gene expression. Nature 415, 436–442.
Reynolds, B.A., and Weiss, S. (1992). Generation of neurons and astrocytes
from isolated cells of the adult mammalian central nervous system. Science
Salghetti, S.E., Kim, S.Y., and Tansey, W.P. (1999). Destruction of Myc by
ubiquitin-mediated proteolysis: cancer-associated and transforming muta-
tions stabilize Myc. EMBO J. 18, 717–726.
Salsano, E., Croci, L., Maderna, E., Lupo, L., Pollo, B., Giordana, M.T.,
Consalez, G.G., and Finocchiaro, G. (2007). Expression of the neurogenic
basic helix-loop-helix transcription factor NEUROG1 identifies a subgroup of
medulloblastomas not expressing ATOH1. Neuro-oncol. 9, 298–307.
Schmitt,A.,Asan,E.,Pu ¨schel,B.,Jo ¨ns,T.,andKugler,P.(1996).Expressionof
the glutamate transporter GLT1 in neural cells of the rat central nervous
system: non-radioactive in situ hybridization and comparative immunocyto-
chemistry. Neuroscience 71, 989–1004.
Schu ¨ller, U., Heine, V.M., Mao, J., Kho, A.T., Dillon, A.K., Han, Y.G., Huillard,
E., Sun, T., Ligon, A.H., Qian, Y., et al. (2008). Acquisition of granule neuron
precursor identity is a critical determinant of progenitor cell competence to
form Shh-induced medulloblastoma. Cancer Cell 14, 123–134.
Scott, C.E., Wynn, S.L., Sesay, A., Cruz, C., Cheung, M., Gomez Gaviro, M.V.,
Booth, S., Gao, B., Cheah, K.S., Lovell-Badge, R., and Briscoe, J. (2010).
SOX9 induces and maintains neural stem cells. Nat. Neurosci. 13, 1181–1189.
Stanton, B.R., Perkins, A.S., Tessarollo, L., Sassoon, D.A., and Parada, L.F.
(1992). Loss of N-myc function results in embryonic lethality and failure of
the epithelial component of the embryo to develop. Genes Dev. 6 (12A),
Stolt, C.C., Lommes, P., Sock, E., Chaboissier, M.C., Schedl, A., and Wegner,
M. (2003). The Sox9 transcription factor determines glial fate choice in the
developing spinal cord. Genes Dev. 17, 1677–1689.
Storm, R., Cholewa-Waclaw, J., Reuter, K., Bro ¨hl, D., Sieber, M., Treier, M.,
Mu ¨ller, T., and Birchmeier, C. (2009). The bHLH transcription factor Olig3
marks the dorsal neuroepithelium of the hindbrain and is essential for the
development of brainstem nuclei. Development 136, 295–305.
Sutter, R., Shakhova, O., Bhagat, H., Behesti, H., Sutter, C., Penkar, S.,
Santuccione, A., Bernays, R., Heppner, F.L., Schu ¨ller, U., et al. (2010).
Cerebellar stem cells act as medulloblastoma-initiating cells in a mouse model
and a neuralstemcell signature characterizes a subset of human medulloblas-
tomas. Oncogene 29, 1845–1856.
Swartling, F.J., Ferletta, M., Kastemar, M., Weiss, W.A., and Westermark, B.
(2009). Cyclic GMP-dependent protein kinase II inhibits cell proliferation,
Sox9 expression and Akt phosphorylation in human glioma cell lines.
Oncogene 28, 3121–3131.
Swartling, F.J., Grimmer, M.R., Hackett, C.S., Northcott, P.A., Fan, Q.W.,
Goldenberg, D.D., Lau, J., Masic, S., Nguyen, K., Yakovenko, S., et al.
(2010). Pleiotropic role for MYCN in medulloblastoma. Genes Dev. 24,
Taipale, J., Chen, J.K., Cooper, M.K., Wang, B., Mann, R.K., Milenkovic, L.,
Scott, M.P., and Beachy, P.A. (2000). Effects of oncogenic mutations in
Smoothened and Patched can be reversed by cyclopamine. Nature 406,
Taylor, M.D., Northcott, P.A., Korshunov, A., Remke, M., Cho, Y.J., Clifford,
S.C., Eberhart, C.G., Parsons, D.W., Rutkowski, S., Gajjar, A., et al. (2011).
Molecular subgroups of medulloblastoma: the current consensus. Acta
Neuropathol. 123, 465–472.
Thompson, M.C., Fuller, C., Hogg, T.L., Dalton, J., Finkelstein, D., Lau, C.C.,
Chintagumpala, M., Adesina, A., Ashley, D.M., Kellie, S.J., et al. (2006).
Genomics identifies medulloblastomasubgroups that areenrichedforspecific
genetic alterations. J. Clin. Oncol. 24, 1924–1931.
Tusher, V.G., Tibshirani, R., and Chu, G. (2001). Significance analysisof micro-
arrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci. USA
Vidal, V.P., Chaboissier, M.C., Lu ¨tzkendorf, S., Cotsarelis, G., Mill, P., Hui,
C.C., Ortonne, N., Ortonne, J.P., and Schedl, A. (2005). Sox9 is essential for
outer root sheath differentiation and the formation of the hair stem cell
compartment. Curr. Biol. 15, 1340–1351.
Yang, Z.J., Ellis, T., Markant, S.L., Read, T.A., Kessler, J.D., Bourboulas, M.,
Schu ¨ller, U., Machold, R., Fishell, G., Rowitch, D.H., et al. (2008).
Medulloblastoma can be initiated by deletion of Patched in lineage-restricted
progenitors or stem cells. Cancer Cell 14, 135–145.
N-MYC-Induced Brain Tumors
Cancer Cell 21, 601–613, May 15, 2012 ª2012 Elsevier Inc. 613