Enhanced expression of MycN/CIP2A drives neural crest
toward a neural stem cell-like fate: Implications for
priming of neuroblastoma
Laura Kerosuo
a
, Pushpa Neppala
a,b
, Jenny Hsin
a
, Sofie Mohlin
c
, Felipe Monteleone Vieceli
a
, Zsofia Török
a
, Anni Laine
b
,
Jukka Westermarck
b,d
, and Marianne E. Bronner
a,1
a
Department of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125;
b
Turku Centre for Biotechnology, University of
Turku and Åbo Akademi University, FI-20014 Turku, Finland;
c
Pediatric Oncology, Department of Clinical Sciences Lund, Lund University, SE-22381 Lund,
Sweden; and
d
Institute of Biomedicine, University of Turku, FI-20014 Turku, Finland
Contributed by Marianne E. Bronner, June 13, 2018 (sent for review January 3, 2018; reviewed by Angela Nieto and Carol Thiele-Galetto)
Neuroblastoma is a neural crest-derived childhood tumor of the
peripheral nervous system in which MycN amplification is a hallmark
of poor prognosis. Here we show that
MycN
is expressed together
with phosphorylation-stabilizing factor
CIP2A
in regions of the neu-
ral plate destined to form the CNS, but MycN is excluded from the
neighboring neural crest stem cell domain. Interestingly, ectopic ex-
pression of MycN or CIP2A in the neural crest domain biases cells
toward CNS-like neural stem cells
that express Sox2. Consistent with
this, some forms of neuroblastoma have been shown to share
transcriptional resemblance with CNS neural stem cells. As high
MycN/CIP2A
levels correlate with poor prognosis, we posit that a
MycN/CIP2A-mediated cell-fate bias may reflect a possible mech-
anism underlying early priming of some aggressive forms of neu-
roblastoma. In contrast to
MycN
, its paralogue
cMyc
is normally
expressed in the neural crest stem cell domain and typically is
associated with better overall survival in clinical neuroblastoma,
perhaps reflecting a more
“
normal
”
neural crest-like state. These
data suggest that priming for some forms of aggressive neuro-
blastoma may occur before neural crest emigration from the CNS
and well before sympathoadrenal specification.
neuroblastoma initiation
|
MycN
|
CIP2A
|
neural crest
|
Sox2
N
euroblastoma is the most common extracranial solid tumor
in childhood. Typically occurring before the age of 2 y with a
prevalence of 2.5
–
5 cases per 100,000 people (1), neuroblastoma is
thought to be a neural crest-derived tumor of sympathetic ganglia,
most commonly located in the adrenal glands. Amplification of
the transcription factor
MycN
occurs in
∼
20% of all neuroblas-
toma cases and is associated with aggressive disease with a poor
prognosis (2
–
4). Given the early onset of neuroblastoma, it has
been speculated that tumor initiation may reflect abnormal de-
ployment of events occurring at early stages of nervous system
development. In recent years, research in the field has focused on
tumorigenic changes in sympathoadrenal precursors. In contrast,
little attention has been given to the possible involvement of
earlier events in neural crest development in neuroblastoma onset.
The neural crest is a transient population of multipotent stem
cells that is induced during gastrulation at the neural plate border,
a region between the neural plate (the future CNS) and the
nonneural ectoderm (the future epidermis). After neural tube
closure, premigratory neural crest cells are initially contained with
the dorsal midline of the forming CNS. Subsequently, neural crest
cells undergo an epithelial-to-mesenchymal transition (EMT) to
delaminate from the dorsal neural tube and initiate migration
toward various destinations within the body. Upon localization at
their final sites, they differentiate into a myriad of different cell
types, including the neurons and glia of the peripheral nervous
system (PNS), melanocytes, and endocrine cells, as well as facial
bone and cartilage (5).
The Myc family of transcription factors is involved in many
important normal cellular events such as cell-cycle progression,
self-renewal, and RNA biogenesis, but these proto-oncogenes are
also associated with tumor growt
h and polyploidy in several types
of cancer (6). During early nervous system development,
MycN
is
excluded from the neural crest stem cell region and instead is
expressed in adjacent neural precursors fated to become part of the
CNS, whereas its paralogue
cMyc
is endogenously expressed in the
neural crest (7). Later during neural development, MycN has been
associated with the maintenance of neural fate (8, 9), as it is
expressed by slowly proliferating neural stem cells (radial glial pro-
genitor cells) (10), and is required
for neural progenitor expansion
and differentiation in the CNS (8, 9). In the peripheral nervous
system, MycN also promotes neural fate and differentiation (11, 12).
Following neural crest EMT from the CNS,
MycN
is expressed
only at very low levels in migrating neural crest cells (9, 13) and
appears to be further down-regulated before the cells coalesce to
form ganglia. Later, it has been reported to be reexpressed in dif-
ferentiating sympathetic ganglia
after the onset of the expression of
proneural genes such as
ASCL1 (MASH1/HASH1)
and lineage-
determining factors such as
Phox2B
and
Hand2
(14
–
21). Some
data suggest that the initiation of
MycN
expression in the ganglia is
concomitant with
Phox2a
expression, followed by
Gata2/3
,the
Trk
genes, and the noradrenergic enzymes tyrosine hydroxylase (TH)
and dopamine beta hydroxylase (D
β
H) that are associated with
Significance
Neuroblastoma is a neural crest-derived pediatric cancer that de-
velops in the embryonic peripheral nervous system (PNS). Studies
of PNS progenitors have failed to uncover how tumors initiate or
fully recapitulate the most aggressive forms of the disease. Pre-
vious transcriptome analysis re
veals similarity between some
neuroblastoma samples and neural stem cells. Here, we show that
ectopic expression of MycN in t
he neural crest domain of the
developing neural tube biases neural crest stem cells toward a
more CNS neural stem cell-like fate and thus results in improperly
specified neural crest cells. This may play a role as a priming event
for tumor initiation, thus providing useful insights into under-
standing the mechanism behind neuroblastoma formation.
Author contributions: L.K., P.N., S.M., J.W., and M.E.B. designed research; L.K., P.N., J.H.,
F.M.V., Z.T., and A.L. performed research; L.K. and S.M. contributed new reagents/analytic
tools; L.K., P.N., J.H., S.M., and F.M.V. analyzed data; and L.K., S.M., J.W., and M.E.B. wrote
the paper.
Reviewers: A.N., Instituto de Neurociencias de Alicante, Consejo Superior de Investiga-
ciones Científicas-Universidad Miguel Her
nández; and C.T.-G., National Institutes
of Health.
The authors declare no conflict of interest.
Published under the
PNAS license
.
1
To whom correspondence should be addressed. Email: mbronner@caltech.edu.
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1800039115/-/DCSupplemental
.
Published online July 18, 2018.
www.pnas.org/cgi/doi/10.1073/pnas.1800039115
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terminal differentiation and functionality of sympathetic neurons
(22
–
24), although this remains controversial (9, 25
–
27). Post-
natally, MycN is not expressed in the sympathetic ganglia (16).
Importantly, overexpression of MycN in mouse sympathoadrenal
progenitors in vivo is not sufficient for tumor formation but in-
stead results in increased neural differentiation (28). However,
neuroblastoma-like tumors were reported after enforced MycN
expression in migrating neural crest cells (29), suggesting that
premature exposure of neural crest cells to high MycN levels may
be important for neuroblastoma initiation.
Like MycN, CIP2A (cancerous inhibitor of protein phosphatase
2A) is overexpressed in several cancer types (30
–
34) and has been
shown to play a role during CNS development as well as in the
testis (35, 36). Although CIP2A is known to stabilize cMyc by
shielding it from protein phosphatase 2A (PP2A)-mediated deg-
radation (37), it has not previously been associated with MycN.
Here, we tackle the potential links between MycN/CIP2A func-
tion in early nervous system devel
opment and neuroblastoma. In the
embryo, we find that both
MycN
and
CIP2A
are coexpressed in
the forming CNS. However, upon initiation of
cMyc
expression in
the neural crest stem cell domain of the neural tube (7, 13),
CIP2A
shifts and is coexpressed with
cMyc
instead of
MycN
. As in the early
embryonic CNS,
MycN
and
CIP2A
are coexpressed in high-risk
neuroblastoma. Interestingly,
ectopic expression of MycN in the
neural crest domain biases neura
l crest progenitors toward a more
CNS-like neural stem cell identity,
so that MycN-expressing neural
crest cells may lack a normal neural crest identity. Similarly, some
neuroblastomas have a transcrip
tional resemblance to CNS neural
stem cells (38). This raises the intriguing possibility of a fate bias
from presumptive PNS to more CNS-like cells, leading to improper
differentiation of neural crest ce
lls, perhaps contributing to the
priming of tumor initiation in neuroblastoma.
Results
Expression Pattern of
CIP2A
Overlaps with
MycN
in the Early Neural
Plate and Closing Neural Tube.
CIP2A is a known stabilizing factor
for cMyc, which is important for neural crest specification (7, 13).
However, we found that
CIP2A
expression starts in the developing
neural plate (Fig. 1
A
) at the gastrula stage [Hamburger Hamilton
(HH) stage 4] and well before the onset of
cMyc
expression, which
initiates only at the time of neural tube closure. This prompted us
to examine other potential binding partners for CIP2A. Although
not previously implicated as a CIP2A-binding partner, in situ hy-
bridization shows that
CIP2A
and
MycN
have very similar ex-
pression patterns during the early neurulation stages (Fig. 1
A
),
whereas
cMyc
is absent from the neural plate (7, 13). At stage
HH7,
CIP2A
and
MycN
are expressed at high levels in the neural
plate but are absent from the neural plate border, which will give
rise to the neural crest as highlighted by Pax7 immunostaining. As
the neural tube closes (starting at stage HH8 in the cranial region),
CIP2A
and
MycN
are coexpressed throughout the neural tube
except in the dorsum, where cMyc expression initiates. Finally, at
stage HH9, as the cranial neural crest initiates migration,
CIP2A
expression shifts from the neural region to neural crest and is
apparent in the premigratory neural crest cells, overlapping with
cMyc
, but is down-regulated in other parts of the neural tube that
remain marked by
MycN
expression (Fig. 1
B
and
C
and
SI Ap-
pendix
,Fig.S1
A
). Along the entire body axis,
MycN
and
CIP2A
are
expressed in the neural plate and neural tube, whereas
cMyc
ex-
pression is absent before the premigratory neural crest stage (
SI
Appendix
,Fig.S1
B
). Migrating neural crest cells (stage HH10)
express high levels of
cMyc
and
CIP2A
but much lower levels of
MycN
mRNA, which remains strongly expressed in the more
ventral parts of the neural tube that will form the CNS.
CIP2A
expression was also detected in the ectoderm at these later stages
(Fig. 1
B
and
C
and
SI Appendix
, Fig. S1
B
). Taken together, the
results show that
CIP2A
expression overlaps with
MycN
during
early nervous system development before
cMyc
expression but
subsequently is coexpressed with
cMyc
in premigratory and mi-
grating neural crest cells.
MycN
Is Not Expressed in the Forming Peripheral Ganglia.
Since neu-
roblastoma tumors are typically found in sympathetic and adrenal
sites, we next examined the expression pattern of
MycN
at the end
of neural crest migration and onset of their condensation into
peripheral ganglia. To this end, we performed in situ hybridization
using a specific probe that gave strong expression in other parts of
the embryo (limb buds, neural plate, neural tube, and others).
Despite long exposure times, we failed to detect
MycN
expression
either in migrating neural crest cells or condensed dorsal root or
sympathetic ganglia (Fig. 2
A
and
B
) from stage HH12 (equivalent
to human
∼
E24/week 3.5) to E4 (equivalent to human E40
–
42/
week 6). Similarly at E7 (equivalent to human E50
–
52/week 7
–
7.5)
the TH
+
sympathetic ganglia did not express
MycN
(Fig. 2
C
and
D
). These results suggest that
MycN
is largely absent from the
peripheral nervous system during the early stages of embryogen-
esis corresponding to the first trimester in human development.
Effects of the Loss of MycN or CIP2A on Early Nervous System Development
and the Neural Crest.
To investigate their developmental role, we next
performed loss-of-function experi
ments using translation-blocking
morpholinos (Mo) against
CIP2A
or
MycN
. These were electro-
porated into the epiblast so that a blocking morpholino was intro-
duced on one side of the embryo, and a control morpholino (CoMo)
was introduced on the contralateral side as an internal control (
SI
Appendix
,Fig.S2
A
). To demonstrate morpholino efficacy, we com-
pared the ability of CIP2AMo or MycNMo versus the control
morpholino to block expression of
a construct containing the trun-
cated 5
′
UTR and ORF, including the morpholino-recognition se-
quence, of their respective genes driving expression of RFP. Both
morpholinos produced efficient and specific loss of the corresponding
construct (
SI Appendix
,Fig.S2
B
–
D
).
The results show that the loss of CIP2A disrupts the induction
and early specification of neural crest cells. Expression of the
neural plate border markers
Msx1/2
and Pax7 at stage HH7 was
reduced in over 80% of the embryos (
n
=
39) (Fig. 3
A
–
C
and
H
).
In a transverse section,
Msx1/2
expression was nearly completely
missing on the CIP2AMo side of the neural plate border at stage
HH7 (Fig. 3
B
and
B
′
). Slightly later in development (stage
HH8), expression of the neural crest-specifier gene
FoxD3
was
severely reduced, with
>
90% of embryos displaying a strong
phenotype (
n
=
12) (Fig. 3
E
,
E
′
, and
H
) as viewed in whole
mounts. In contrast, embryos electroporated with the CoMo
alone showed no phenotype and little loss of neural crest marker
expression (stage HH7,
n
=
19 and stage HH8,
n
=
20), displaying
only normal variation between the two sides (Fig. 3
A
,
D
,and
H
).
To further demonstrate the specificity of CIP2A knockdowns,
we performed rescue experiments. To this end, CIP2A was
expressed together with the CIP2A morpholino. We observed a
dose-dependent effect, with 88% of the embryos rescued to
normal with 1
μ
g/
μ
L of CIP2A (
n
=
8) (Fig. 4
B
and
SI Appendix
,
Fig. S2
E
) and 57% rescued with 0.5
μ
g/
μ
L(
n
=
7).
Next we examined the effects of the loss of MycN, a member
of the Myc family that is expressed in in the neural plate (the
future CNS) in a similar fashion to CIP2A. Similar to the loss of
CIP2A, the loss of MycN also caused a deficit of neural crest
precursors at the neural plate border (stage HH7,
n
=
39) as
shown in a transverse section (Fig. 3
F
and
H
). Slightly later,
when the neural tube is closed (stage HH8,
n
=
37), the expression
of
FoxD3
in premigratory neural crest cells in the dorsal neural tube
was clearly affected also (Fig. 3
G
and
H
;stageHH7:67%strong,
28% mild, and 5% no phenotype; stage HH8: 82% strong, 5% mild,
and 11% no phenotype). The phenotype was penetrant throughout
the embryo from cranial to trunk levels. These results suggest that
knockdown of both MycN and CIP2A impacts neural crest devel-
opment although neither gene is expressed in the presumptive
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Kerosuo et al.
neural crest at this early developmental time point. Thus, we spec-
ulate that both MycN and CIP2A are required for proper develop-
ment of the neural plate and thus the neural stem cells that form the
CNS, and that disruption of this pro
cess secondarily affects subse-
quent neural crest development in the neural plate border (39).
Epistatic Relationship Between MycN and CIP2A.
To examine
whether CIP2A and MycN may be functionally associated, we
asked whether the loss of CIP2A, predicted to lead to faster
degradation of MycN, can be compensated by the overexpression
of MycN. To this end, we knocked down CIP2A using a mor-
pholino and coinjected MycN to test whether this was sufficient
to rescue the phenotype. Indeed, 75% of the embryos (
n
=
12)
were rescued by coinjection of 0.75 or 1
μ
g/
μ
L MycN (Fig. 4
A
,
A
′
,
and
B
)and62%(
n
=
4) were rescued with 0.5
μ
g/
μ
L MycN. In-
terestingly, a small percentage (
<
20%) of the embryos showed a
slight increase in the expression of the neural crest marker
FoxD3
(Fig. 4
B
). These results reveal a dose-dependent ability of MycN
to rescue the effects of loss of CIP2A, consistent with an epistatic
interaction.
Ectopic MycN or CIP2A Causes a Shift in Neural Crest Identity Toward
a More CNS-Like Fate.
Since amplification of MycN is a hallmark
of neuroblastoma with poor prognos
is, we next examined the effects
of elevated MycN or CIP2A in the neural plate border and pre-
sumptive neural crest domain, sites where MycN is not endoge-
nously expressed. The results show that overexpression of either
CIP2A or MycN significantly reduce
d the expression of neural crest
markers
FoxD3
and Pax7 at all axial levels from cranial to anterior
trunk (Fig. 5
A
,
B
,and
D
). Instead, ectopic MycN in the dorsal
neural folds led to enhanced CNS stem cell identity as shown by
increased Sox2 throughout the neural tube (from ventral to dorsal,
Fig. 5
C
and
D
,and
“
Sox2 all
”
in Fig. 5
G
).
Importantly, the neural crest domain, as indicated by the ex-
pression of Pax7, was significantly reduced in size, suggesting that
ectopic MycN may have neuralized a large proportion of the cells
MycN
CIP2A
MycN
cMyc
CIP2A
transverse sections
whole embryos
Pax7
Pax7
Stage 7
Stage 4
Stage 8
Stage 9
Stage 7
MycN
CIP2A
cMyc
AB
NP
B
B
NPB
NPB
NPB
NC
NC
NT
NT
NC
NC
NT
NT
NT
NC
Stage 4
Stage 7
Stage 8
Stage 9
Stage 10
non-neural ectoderm
neural plate border (St 4-7)
neural plate (St 4-7)
neural crest (St 8-10)
neural tube (St 8-10)
MycN
cMyc
CIP2A
C
N
N
NC
NC
NC
NC
N
time
Expression pattern
E
E
E
cMyc
B
s
s
s
Fig. 1.
Expression patterns of
CIP2A
,
MycN
, and
cMyc
in the developing neural tube and neural crest cells in the chicken embryo. (
A
) Whole-mount in situ
hybridization shows that
CIP2A
and
MycN
, but not
cMyc
, are expressed in the neural plate (NP) during gastrulation throughout the anterior-to-posterior axis.
B, areas shown in panel
B
; s, somites. (
B
) In contrast, the neural plate border (NPB) that later forms the neural crest does not express any of the three genes at
stages HH4
–
7 as also seen in transverse sections. As the neural tube closure begins at stage HH8,
CIP2A
is expressed throughout the entire neural tube (NT),
whereas
MycN
is restricted to the ventral parts that will form the CNS.
cMyc
expression onsets at late stage HH8 in the dorsal neural tube that will become the
neural crest (NC). By stage HH9
CIP2A
and
cMyc
are restricted to the dorsal neural crest area, whereas
MycN
is expressed in the remaining neural tube but not
in the dorsum.
CIP2A
expression is also seen in the nonneural ectoderm (E) after stage HH9. (Scale bars: 20
μ
m.) (
C
) A schematic diagram summarizing the
expression patterns seen in
A
and
B
and
SI Appendix
, Fig. S1
. Red represents the neural crest, blue represents neural stem cells of the future CNS, and gray is
future epidermis. The thin red line for
MycN
represents a much lower expression level of
MycN
in the migrating neural crest compared with the other stages
and genes, respectively.
Kerosuo et al.
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DEVELOPMENTAL
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in the neural crest domain (green arrowheads in Fig. 5
E
), as also
shown by a decrease in overall Pax7 expression on the MycN-
injected side compared with control embryos (Fig. 5
G
). Consis-
tent with this possibility, even the small remaining population of
Pax7
+
neural crest cells had significantly higher Sox2 levels com-
pared with the endogenous Sox2 expression levels on the control
side (Fig. 5
E
and
“
Sox2 dorsal
”
in Fig. 5
G
).
Although very low Sox2 levels are normally present in neural
crest cells (see control side in Fig. 5
E
), quantitative analysis at
single-cell-level resolution revealed a significant increase in the
cells that expressed high or intermediate levels of Sox2 in
the MycN-overexpressing neural crest domain. Conversely, the
number of cells that express no or low levels of Sox2 (normally
75% of the neural crest cells) was reduced to 45% due to en-
hanced MycN expression (see pink arrowheads indicating high
Sox2 levels and purple arrowheads indicating intermediate Sox2
levels in Fig. 5
E
and quantifications in Fig. 5
H
). Ectopic MycN
also caused a significant decrease in the proportion of Sox2
−
cells
[from 42% (SD
=
0.06) on the control side to 20% (SD
=
0.1) on
the MycN-injected side;
P
=
0.003,
n
=
7], consistent with an
overall increase in Sox2 levels on the MycN-injected side (Fig.
5
G
). In contrast, the Pax7 levels in individual cells were decreased
by 25% on average (Fig. 5
I
) but displayed less variance (ranging
from medium to high levels) than Sox2 expression on both the
MycN-overexpressing and the control sides (Fig. 5
E
). Cells
expressing both high Pax7 and high Sox2 levels on the control side
were extremely rare, with only a few found at the border region
between CNS and neural crest domains in seven embryos; this
number was significantly (14-fold) higher following MycN over-
expression (Fig. 5
J
). These results suggest that ectopic MycN shifts
the presumptive neural crest domain toward a more CNS-like
neural stem cell fate. This is consistent with reports showing
that fate changes can occur by tweaking transcription factor levels
in the neural plate border (40). Importantly, these results suggest
that the abnormally high levels of Sox2 do not abolish neural crest
identity but rather result in a mixed identity (high/intermediate
Sox2
+
/Pax7
+
), which we refer to as
“
CNS-like
”
neural crest cells.
CNS-Like Neural Crest Cells Emigrate from the Neural Tube and Are
Migratory.
The results described above raised the intriguing
question of whether these Sox2
+
/Pax7
+
CNS-like neural crest cells
can undergo EMT, migrate, and contribute to the peripheral
nervous system. To address this possibility, we examined embryos
at later times, when neural crest cells were emigrating and mi-
grating through the periphery. The results showed that neural
crest cells with excess MycN were able to undergo EMT to leave
the CNS. Although some migrating MycN-overexpressing cells
lacked Pax7, perhaps reflecting bias toward a neural fate (Fig. 5
F
,
20%
60%
100%
stage 7
CIP2AMo
No phenotype
Mild phenotype
Strong phenotype
Loss of NC
Loss of NC
stage 7
stage 7
stage 8
stage 8
stage 8
MycNMo
ControlMo
CIP2AMo
MycNMo
Pax7
FoxD3
FoxD3
Msx1
FoxD3
Msx1
Msx1
o
M
A
2
P
I
C
o
M
o
C
o
M
o
C
CoMo
o
M
o
C
o
M
A
2
P
I
C
o
M
A
2
P
I
C
o
M
o
C
CoMo
CIP2AMo
CoMo
CIP2AMo
CoMo
MycNMo
MycNMo
CoMo
Msx1
Stage 7
Stage 8 / 8+
n=39
n=28
n=39
n=37 n=19
n=20
FoxD3
o
M
o
C
Stage 7+
Stage 7
Stage 8+
A
B’
C
B’
E’
D
FG
H
BE
o
M
o
C
Fig. 3.
Neural crest is lost upon knockdown of MycN or CIP2A. (
A
–
C
) Whole-
mount images of a chicken embryo electroporated with a control morpho-
lino shows similar expression of the neural plate border marker
Msx1
on
both sides (
A
), while knockdown of CIP2A significantly reduces the expres-
sion of both
Msx1
(
B
and
B
′
) and Pax7 (
C
) along the entire neural axis of the
embryo at stage HH7 (arrowheads). (
D
–
E
′
) The phenotype persists at stage
HH8, as expression of the neural crest specifier gene
FoxD3
is reduced from
anterior to posterior parts of the embryo. (
F
and
G
) Similarly, morpholino-
mediated knockdown of MycN causes a loss of the expression of
Msx-1
(
F
)
and
FoxD3
(
G
). (Scale bars: 20
μ
m.) (
H
) Quantification of the phenotypes.
A
B
C
D
B
D
E4
E7
MycN
HNK-1
TH
DAPI
nt
drg
d
sg
n
da
nt
n
sg
drg
Fig. 2.
MycN
is not expressed in the developing sympathetic ganglia. (
A
)At
day 4 of chicken embryo development,
MycN
is expressed in the dorsal
neural tube and the dermatome (arrowheads), but no
MycN
expression was
detected in the peripheral ganglia as shown by in situ hybridization in the
trunk level. The ganglia are highlighted by HNK immunostaining. (
B
) High-
magnification images of the E4 sympathetic ganglia with no
MycN
expres-
sion. (
C
) At day 7,
MycN
expression is visible in the ventricular apical region
surrounding the neural tube, but no expression is detected in the TH-
immunopositive sympathetic ganglion. (
D
) High magnification of the
E7 sympathetic ganglia with no
MycN
expression (boxed area in panel
C
). d,
dermatome; da, dorsal aorta; drg, dorsal root ganglion; n, notochord; nt,
neural tube; sg, sympathetic ganglion. (Scale bars: 100
μ
m.)
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Kerosuo et al.
blue arrowheads), most remained Pax7
+
(Fig. 5
F
, white arrow-
heads). In addition, there was variability in the MycN-RFP levels,
with some cells expressing high levels of Pax7 having low MycN-
RFP expression (Fig. 5
F
, pink arrowheads). This is expected, since
the expression levels of constructs are variable from cell to cell
after electroporation.
In line with the premigratory stages (Fig. 5
D
and
E
), shortly
after emigration we noted fewer migrating neural crest cells (
n
=
3) (
SI Appendix
,Fig.S2
F
). With time, however, the numbers of
the neural crest cells observed migrating further laterally increased
(Fig. 5
F
and
K
). Immunostaining showed that migrating MycN-
overexpressing neural crest cells had lost their Sox2 expression (
SI
Appendix
,Fig.S2
F
), in accordance with previous reports (40, 41).
These results suggest that the CNS-like Pax7
+
/MycN
+
neural crest
cells are able to undergo EMT and that migrating MycN-
overexpressing cells matured from Sox2
+
precursors into periph-
eral nervous system progenitors. This is consistent with previous
experiments showing that overexpression of MycN at the migra-
tory stage causes an excess of neural crest cells that predominantly
differentiate into peripheral neurons (11).
Strong Association of High
MycN
and
CIP2A
Levels with Aggressive
Forms of Neuroblastoma.
Next, we asked whether the scenario func-
tioning during early neural plate and neural crest development
might be recapitulated in neuroblastoma. To this end, we examined
potential correlations be
tween the expression of
CIP2A
,
MycN
,and
cMyc
in neuroblastoma samples usin
g three independent datasets:
KOCAK (Fig. 6), SEQC498 (
SI Appendix
,Fig.S3
), and Versteeg88
(
SI Appendix
,Fig.S4
).
All three datasets display substantial mRNA expression levels
of all three transcripts, although the
MycN
levels were in the
highest range (Fig. 6
A
and
SI Appendix
, Fig. S4
A
). The expres-
sion pattern for
MycN
was bimodal, presumably reflecting
MycN
-
amplified versus nonamplified status: In the first group, the
majority of samples had lower expression levels, ranging from
2log
12
to 2log
15
, whereas in the second group the levels ranged
from 2log
15
to 2log
18
. Expression levels of
cMyc
were in the range
between 2log
11
and 2log
15
and were lowest for
CIP2A
, ranging
from 2log
7
to 2log
12
(Fig. 6
A
). When the expression levels of
MycN
were compared in the MycN-amplified versus the nonamplified
group, the results clearly show that the levels in the nonamplified
group are comparable to
cMyc
levels, and thus the levels were
much higher in the MycN-amplified tumors (Fig. 6
B
). As in the
early embryo (Fig. 1), the expressions of the two
Myc
family
members show a strong inverse correlation (Fig. 6
C
and
SI Ap-
pendix
, Fig. S3
A
), suggesting that they are not likely to be
coexpressed in the same neuroblastoma tumors. However,
MycN
and
CIP2A
mRNA expressions show positive correlation in the
complete dataset as well as in stage 4 tumors only, reminiscent of
the situation we observe in the early embryo (Fig. 5) after ectopic
MycN expression in the neural crest domain (Fig. 6
D
and
SI
Appendix
, Figs. S3
B
and S4
B
). However, although this correla-
tion is maintained in both the MycN-amplified and nonamplified
stage 4 tumors in the SEQC498 dataset (
SI Appendix
, Fig. S3
B
),
it is lost in the MycN-amplified cases in the KOCAK dataset
(Fig. 6
E
), suggesting a stronger correlation with the
“
more
physiological
”
MycN expression levels (Fig. 6
B
). Conversely,
expression of
cMyc
and
CIP2A
correlated negatively in the
KOCAK dataset as well as in stage 4 tumors only; however, their
expression was weaker in the MycN-
nonamplified stage 4 tumors
(Fig. 6
F
), whereas their correlation was weaker in the complete
dataset and was lost in stage 4 tumors in the SEQC498 dataset (
SI
Appendix
,Fig.S3
D
). The number of individual tumors in the MycN-
amplified and/or the stage 4 group is low (even in the largest
KOCAK dataset there are 41 and 65 tumors, respectively). Thus,
the sample size may be too low for statistical significance or for
drawing firm conclusions. In line w
ith the other datasets, in the
Versteeg88 dataset
CIP2A
correlates with
MycN
in the MycN-
nonamplified group (
SI Appendix
,Fig.S4
B
) and correlates nega-
tively with cMyc in the MycN-amplified group (
SI Appendix
,Fig.
S4
C
). We also found that
MycN
(as previously known) and
CIP2A
individually correlate significantly with the International
Neuroblastoma Staging System (INSS) stages, i.e., highest ex-
pression is found in the most aggressive stage 4 tumors, whereas
cMyc
shows an opposite declining trend (Fig. 6
G
and
SI Ap-
pendix
, Figs. S3
C
and S4
E
).
Consistent with previously published results, high expression
of both
MycN
(3, 4) and
CIP2A
(42) correlate with poor prog-
nosis in neuroblastoma in all three clinical datasets analyzed here
(Fig. 6
H
and
J
and
SI Appendix
,Figs.S3
E
and
G
and S4
D
).
However, the correlation between CIP2A and outcome is signifi-
cant only in the complete dataset and in the MycN-nonamplified
tumors but is lost in the MycN-amplified tumors and stage 4 tu-
mors (Fig. 6
H
and
SI Appendix
,Fig.S3
E
). In contrast, expression
of
cMyc
was associated with improved patient survival in both the
complete dataset and stage 4 tumors (Fig. 6
I
and
SI Appendix
, Fig.
S3
F
) and thus shows an inverse correlation compared with its
ortholog
MycN
(Fig. 6
J
and
SI Appendix
,Fig.S3
G
). Together,
these results are consistent with functional cooperation between
MycN and CIP2A in the development of clinical neuroblastoma.
CIP2A Promotes SOX2 Expression in MycN-Amplified Neuroblastoma
Cells.
To further explore the functional relationship of MycN and
CIP2A in neuroblastoma, we utilized a panel of neuroblastoma
cell lines with [SK-N-BE(2); NGP cells] or without (SK-N-AS
cells) MycN amplification. Both MycN-amplified neuroblastoma
cell lines were positive for CIP2A and Sox2 but expressed very low
or undetectable levels of cMyc (Fig. 7
A
). Instead, SK-N-AS cells
that harbor normal MycN copy numbers expressed extremely low
protein levels for both MycN and Sox2 but expressed substantial
levels of cMyc (Fig. 7
B
). This reciprocal relationship between
cMyc and MycN is reminiscent of our observations in the dorsal
neural tube of early embryos (Fig. 1) as well as in clinical neuro-
blastoma (Fig. 6).
%
20
CIP2AMo
+ MycN
No rescue
CIP2AMo
+ CIP2A
40
60
80
100
Rescue
more crest
n=12
n=8
CIP2AMo
CoMo
+ MycN
FoxD3
AA’
A’
B
Fig. 4.
CIP2A and MycN cooperate in the developing neural tube. (
A
and
A
′
) Loss of CIP2A is rescued by coexpression of MycN as shown by whole-mount in
situ hybridization for
FoxD3
(
A
) and in a transverse section of the same embryo (
A
′
). (Scale bars: 20
μ
m.) (
B
) Quantification of the rescue experiments.
Kerosuo et al.
PNAS
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DEVELOPMENTAL
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To test for a possible direct interaction between CIP2A and
MycN, we used the proximity ligation assay (PLA) to examine
the physical proximity of these proteins in the SK-N-BE(2) cell
line. The results show that CIP2A and MycN indeed associate
with each other (Fig. 7
C
).
We next addressed whether th
e in vivo cooperation between
CIP2A and MycN in the regulation of Sox2 expression shown in
avian embryos (Fig. 5) was a cell-autonomous process. To this end,
we tested the effects of CIP2A depletion in neuroblastoma cells
using three independent siRNAs. CIP2A depletion caused changes
in cellular responses in a cell-viability assay (Fig. 7
D
), with cells
exhibiting decreased anchorage-
independent growth in a soft agar
assay (Fig. 7
E
). Importantly, CIP2A depletion significantly reduced
Sox2 protein expression in the MycN-
amplified cell lines, consistent
with the in vivo results. Levels of MycN were slightly increased,
possibly due to a compensatory effect caused by a lack of the normal
half-life protected by CIP2A (Fig. 7
A
). Interestingly, in the MycN
wild-type cells that display a more neural crest-like expression
profile (low levels of Sox2/MycN and physiological levels of cMyc),
the loss of CIP2A had no effect on cMyc (Fig. 7
B
), further sug-
gesting that CIP2A expression in
neuroblastoma is connected to
MycN and not to cMyc. These results suggest that the cooperation
of MycN and CIP2A in Sox2 regulation observed both in vivo and in
vitro is cell autonomous. They also confirm that the molecular
features detected in neuroblastom
a cells and in clinical neuroblas-
toma are similar to those observed during early CNS development.
Finally, we tested whether
Sox2
and
MycN
correlate in clinical
neuroblastoma. The expression levels of
Sox2
in the KOCAK and
CIP2A
MycN
Control
Control
p = 0.053
SOX2
contr/
contr side
MycN /
contr side
Pax7
Fluorescence
1
0.6
0
1.4
SOX2
all
dorsal
AA’
A’
BB’
B’
G
**
**
Control
n=9
n=5
y
r
o
t
a
r
g
i
m
e
r
p
y
r
o
t
a
r
g
i
m
Sox2
merge
MycN
e
0.5
1.0
0
1.5
2.0
Pax7
contr/
contr side
MycN /
contr side
n=8
n=3
H
E
Pax7
E
D
Control
merge
Sox2
MycN Control
MycN Control
MycN
Pax7
Dapi
Control
MycN
Control
MycN
F
Pax7
MycN-RFP
merge
NT
J
FoxD3
FoxD3
Sox2
C
MycN-RFP
Cont
Fluorescence
I
0
20
40
60
80
%
**
**
SOX2
contr side
MycN side
n=7
SOX2
SOX2
no/low
medium
high
*
Pax7
1.2
1.6
0.6
0.4
0.2
Fluorescence
raw intensity Pax7/Dapi
contr side
MycN side
n=7
*
K
4
8
12
16
0
Fold increase
contr side
MycN side
n=7
**
*
Pax7
high
/Sox2
high
Sox2 and Pax7 in the neural tube
Expression of Sox2 in Pax7+ cells
Pax7 intensity in neural crest cells
Cells with mixed phenotype
raw intensity Pax7/Dapi
Fig. 5.
Enhanced MycN in the neural crest domain causes a fate bias toward CNS-like neural stem cells. (
A
–
B
′
) Ectopic expression of either CIP2A (
A
and
A
′
)or
MycN (
B
and
B
′
) leads to a significant decrease in the size of the neural crest domain as shown by in situ hybridization for
FoxD3
.(
C
) Electroporation of MycN-
RFP causes an increase in Sox2 protein levels throughout the neural folds. (
D
) This leads to both full and partial neuralization of the Pax7
+
neural crest
domain. (
E
) Enlarged view of the boxed area in
D
highlights the different levels of neuralization due to ectopic MycN: completely neuralized Sox2
+
/Pax7
−
cells
(green arrowheads), partially neuralized Pax7
high
/Sox2
high
cells (pink arrowheads), and Pax7
high
/Sox2
intermediate
cells (purple arrowheads). (
F
) Neural crest cells
are able to emigrate and migrate despite ectopic MycN, and most of the cells also express Pax7 (white arrowheads), while some are MycN
+
/Pax7
−
(blue
arrowheads). Electroporation causes variability as shown by a lower MycN level in some cells (pink arrowheads). e, ectoderm; NT, neural tube. (
G
)
Pax7 expression is significantly decreased on the MycN ectopic side (
P
=
0.0041;
t
test), reflecting the smaller size of the neural crest domain, and Sox2
+
levels
are increased both in the entire neural tube (Sox2 all), and the neural crest domain (Sox2 dorsal) (
P
=
0.0021). (
H
) Ectopic MycN increases Sox2 levels in Pax7
+
neural crest cells (high,
P
=
8.6E-06; medium,
P
=
0.042;
t
test). Conversely, the proportion of normal neural crest cells with no or low Sox2 levels was reduced
from 75 to 45% (
P
=
7.4E-05;
t
test) on the MycN-injected side (
I
), and Pax7 expression decreased by 25% (
P
=
0.0031;
t
test). (
J
) The number of cells with high
levels of both Pax7 and Sox2 is increased by 14-fold (
P
=
0.011;
t
test). (
K
) The MycN-overexpressing side has more Pax7
+
migratory neural crest cells than the
control side. The lines inside the box plots represent the median values. (Scale bars: 50
μ
m.) *
P
<
0.05, **
P
<
0.01.
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Kerosuo et al.
SEQC498 clinical datasets were low, with the majority of the tu-
mors ranging from 2log
5
to 2log
9
, and there is no significant
correlation with
MycN
(
SI Appendix
, Fig. S5
A
and
B
). This is in
line with our finding that Sox2 levels are down-regulated after
emigration from the neural tube during normal development and
in MycN-overexpressing neural crest cells (
SI Appendix
, Fig.
S2
F
). We also tested whether we could detect a correlation be-
tween
MycN
and other commonly known neural stem cells
markers such as
Nestin
or
Mushashi2
, but no significant corre-
lations were noted (
SI Appendix
, Fig. S5
C
–
F
). These results
suggest that the expression of Sox2 in neuroblastoma cell lines
may be a sign of regression into an earlier developmental time
point that is not maintained in tumors in vivo but exists under
certain culture conditions in vitro.
Discussion
Despite extensive efforts and different forms of therapy, treat-
ment of aggressive high-risk neuroblastoma remains a challenge.
A
B
C
FG
HI
J
DE
Fig. 6.
CIP2A and MycN are strongly correlated with a poor prognosis, while cMyc correlates with better survival probability. (
A
) MycN, cMyc, and CIP2A show
substantial expression levels in neuroblastoma with CIP2A displaying the lowest range. The expression of MycN is bimodal, and the expression in the h
ighest
group exceeds the level of
cMyc
and
CIP2A
. Note the different values on the
x
and
y
axes in each image. (
B
) The bimodal expression of
MycN
mRNA in MycN-
amplified (amp) and nonamplified (non amp) tumors shows how the amplified levels are above physiological levels. (
C
–
E
) Expression of
cMyc
and
MycN
shows
negative correlation in clinical neuroblastoma samples (
C
), whereas
MycN
correlates positively with
CIP2A
, as shown both in the complete dataset and in stage
4 tumors only (
D
), but, presumably due to the extremely high expression levels, the correlation is lost in the MycN-amplified stage 4 tumors, whereas strong
correlation is shown in the stage 4 nonamplified group (
E
). (
F
)
CIP2A
and
cMyc
correlate negatively in the complete dataset and stage 4 tumors, but the
correlation is weaker in the MycN-nonamplified stage 4 tumors. (
G
)
CIP2A
expression correlates significantly with the INSS stage, and
cMyc
shows an inverse
correlation. (
H
) Kaplan
–
Meier curves showing a significantly lower survival probability for patients with high
CIP2A
levels in the complete dataset and in
MycN-nonamplified tumors (with more physiological
MycN
expression levels). The correlation is lost in the stage 4 tumors and in the MycN-amplified group. (
I
and
J
) High
cMyc
expression levels (which may represent physiological rather than overexpressed levels) correlate with better survival probability in clinical
neuroblastoma in both the complete and stage 4 datasets (
I
), whereas
MycN
, as previously known, correlates with a poor outcome (
J
). *
P
<
0.05; na, not
available.
Kerosuo et al.
PNAS
|
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|
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|
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DEVELOPMENTAL
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Therefore, understanding the initial steps of tumor formation is
crucial for designing novel approaches for treatment. Neuroblas-
toma originates in neural crest-derived sympathetic nerves and is
associated with reduced neural differentiation capacity and thus
an enhanced stem cell-like profile. The etiology remains unknown,
and it is likely that multiple mechanisms lead to the heterogeneous
group of tumors classified as neuroblastoma. Interestingly, a
transcriptome analysis performed over a decade ago reveals that
some neuroblastomas have twice as many genes in common with
CNS neural stem cells than do normal sympathoadrenal progen-
itors (38). Since cancer cells often express features that mirror
their stem cell origin, this raises the intriguing possibility that
factors that might bias neural crest cells toward a more CNS-like
state may play a role as a priming event in neuroblastoma. During
nervous system development, CNS and PNS precursors arise in
adjacent domains within the forming neural plate, with neural
crest stem cells in the neural plate border and CNS precursors in
the immediately lateral neural plate domain. This led us to in-
vestigate whether misregulation of the events that guide fate de-
termination at the neural plate border could help explain the
initiation mechanism underlying neuroblastoma formation.
The majority of studies on neuroblastoma initiation to date
have focused on finding links to sympathetic ganglia formation. In
contrast, far less attention has been paid to the possibility that
predisposing oncogenic changes in the premigratory and early
migrating neural crest may occur before the cells have migrated to
the site of sympathetic ganglia formation. This prompted us to test
the hypothesis that the initiation of some forms of neuroblastoma
may occur at early time points in nervous system development and
well before specification of the sympathoadrenal lineage. Given
that the great majority of neuroblastoma research is done in
neuroblastoma cell lines that already are malignant, the initial
mechanism(s) that triggered the disease may be masked under a
series of secondary defects. Therefore, we went back to the early
embryo to test the role of both endogenous and ectopic MycN and
CIP2A as possible priming factors.
Amplification of MycN remains the strongest single indicator
of poor prognosis in neuroblastoma (43). Previously, the de-
velopmental role of MycN has been examined primarily at later
stages, during neural crest migration and gangliogenesis. In the
spinal ganglia and the developing CNS, MycN promotes neural
fate and differentiation (8, 9). Overexpression of MycN in mi-
grating neural crest cells of chicken embryos increases the pro-
portion of neurons at the expense of other derivatives (11).
Reciprocally, loss of MycN in mouse embryos reduces the size of
the entire nervous system, including peripheral, spinal, and cra-
nial ganglia (12), and decreases the number of mature neurons in
the spinal ganglia (9). Although it has been assumed that
MycN
was expressed in chick and mouse peripheral ganglia during their
formation and maturation (9, 21, 25, 26), we were not able to
detect any
MycN
mRNA in the chick sympathetic ganglia at any
time point from migratory stages to late gangliogenesis (E2
–
E7 corresponding to E50
–
52/week 7
–
7.5 in humans as the latest
time point) (Fig. 2). In contrast to our results, a previous study
proposes that there is weak
MycN
expression in sympathetic
ganglia. However, they also find strong expression of
cMyc
at the
same stage in sympathetic ganglia (25), in line with previous
findings (10) and our results here regarding the reciprocal ex-
pression pattern of these two orthologs in the embryo and in
neuroblastoma. We cannot rule out the possibility that
MycN
is
turned on in the sympathetic ganglia later in gestation and then
is turned off again before birth. Taking the data together, we
speculate that MycN expression in neuroblastoma is ectopic and
may reflect an abnormal priming of the early neural crest toward
a more CNS-like neural stem cell fate.
While MycN overexpression in sympathoadrenal progenitors in
vitro leads to neural lineage commitment and tumor-like charac-
teristics, the onset of this oncogenic program is inhibited in an in
vivo context (28). In contrast, MycN overexpression in migrating
neural crest cells is sufficient for in vivo transformation and for-
mation of tumors with phenotypic resemblance to neuroblastoma
(29), again suggesting that time points before sympathoadrenal
lineage specification may be the root of some forms of neuro-
blastoma. We find that MycN amplification in premigratory neural
crest stem cells within the neural tube biases them toward Sox2
+
neural stem cells (Fig. 5). While some cells are completely ven-
tralized, losing Pax7 expression, others coexpress high Sox2 levels
while retaining Pax7. We hypothesize that these cells may be
“
primed
”
for neuroblastoma formation, as they retain the ability
to undergo EMT and migrate despite their ectopic CNS-like
neural stem cell bias. We speculate that the retention of high
MycN levels in the migrating neural crest cells may abnormally
induce proliferation and the maintenance of neural identity but
that these cells are unable to differentiate normally when they
reach the ganglia due to their neural progenitor state.
We report an interesting reciprocal relationship between
cMyc
in the neural crest domain and
MycN
in the remaining neural tube.
Although we have shown that cMyc expression is critical for the
maintenance of early neural crest identity and self-renewal ca-
pacity (Fig. 1) (7, 44), MycN seems to function primarily in CNS
neural stem cells. Similarly, we show that the expression of
cMyc
and
MycN
in clinical neuroblastoma (Fig. 6) is complementary in a
MycN / CIP2A
Dapi
Control merge
MycN/CIP2A merge
SK-N-BE(2)
1
Viability
Viability
(standardized to scr control)
0.5
0
1.5
scr control
CIP2A siRNA #2
CIP2A siRNA #1
MycN low
MycN Amp
Growth area
(standardized to scr control)
Soft agar colony formation
1
0.8
0.6
0.4
0.2
0
1.2
**
**
scr
#1
#2
SK-N-BE(2)
SK-N-BE(2)
SK-N-AS
*
*
MycN
CIP2A
cMyc
Sox2
GAPDH
SK-N-BE(2)
NGP
MycN Amplified
MycN low
SK-N-AS
scr scr
scr
#2
#2
#3
#2
#3
#1
#1
#1
#3
CIP2A siRNA
CIP2A siRNA
CIP2A siRNA
C
DE
AB
Fig. 7.
Loss of CIP2A impacts proliferation and anchorage-dependent growth. (
A
) Knockdown of CIP2A with three different siRNAs reduces the expression of
Sox2 in MycN-amplified neuroblastoma cell lines. (
B
) SK-N-AS cells with wild-type MycN status express undetectable levels of MycN or Sox2 but substantial
levels of cMyc that are unaffected by the knockdown of CIP2A. (
C
) The PLA shows cobinding of MycN and CIP2A. (Scale bars: 50
μ
m.) (
D
) Knockdown of CIP2A
reduces relative viability in neuroblastoma cell lines [SK-N-BE(2),
P
=
0.00040 and 0.01201; SK-N-AS,
P
=
0.019,
t
test]. (
E
) Loss of CIP2A also decreased
anchorage-dependent growth in SK-N-BE(2) cells (
P
=
0.0049 and 0.0068). *
P
<
0.05, **
P
<
0.01.
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Kerosuo et al.
manner paralleling that in the embryo (Fig. 1). Our results using a
combination of functional studies in the embryo (Fig. 5) together
with neuroblastoma cell lines (Fig. 7) and publicly available tu-
mor datasets (Fig. 6 and
SI Appendix
, Figs. S3 and S4
) suggest
that MycN overexpression (or in many cases amplification)
biases their fate from neural crest toward CNS-like neural stem
cells and that this bias reflects an early developmental event. The
fact that CIP2A partners with MycN only during the early neural
plate stage and not later after neural tube closure or in the mi-
grating neural crest cells further strengthens our hypothesis that
some tumors are primed at an early developmental time point. It
is intriguing to speculate that this early mechanism may reflect
the underlying cause of initiation of the most aggressive forms of
neuroblastoma. Our findings also bring neuroblastoma closer to
other pediatric CNS tumors such as medulloblastoma, raising the
possibility that common mechanisms may underlie the initiation
of these tumors.
Many pediatric malignancies arise from embryonic cell types
that have persisted and give rise to tumors in early childhood
(45). Pediatric tumors are thus unlikely to be driven by the
gradual accumulation of genetic lesions but rather via oncogenic
cells that are predisposed to malignant growth while carrying few
mutations (46). In
Drosophila
, it has been shown that in-
termediate neural progenitors born during a particular time
period are predisposed to malignancy (47). Similarly, we specu-
late here that, rather than immediately initiating rapid tumor
growth, these early events may serve as a priming event for tu-
mor susceptibility so that cells ectopically exposed to high MycN
levels early in development have the potential for metastatic
tumor growth later during ganglia formation.
Neuroblast hyperplasia is detected in normal ganglia before
and around birth. Some of these neuroblasts progress into
neuroblastoma-like tumors upon MycN overexpression under
the TH promoter (45) but do not fully recapitulate the metastatic
disease. It is intriguing to speculate that perhaps, in addition to
the constitutive ectopic MycN expression in the developing
ganglia, the early priming event in the neural plate border we
describe in this study triggers the formation of a full-blown
metastatic neuroblastoma. This also suggests that existing
mouse and zebrafish neuroblastoma models that activate MycN
in the peripheral ganglia may initiate the expression at a time
point that is too late for understanding the initiation of at least
some subgroups of the disease (45, 48
–
52). Finally, neuroblas-
toma occasionally occurs in association with other neural crest-
derived defects (also known as
“
neurocristopathies
”
) such as
Hirschsprung
’
s disease (53
–
56), which results from a failure of
neural crest cells to populate the most distal portions of the in-
testines. This supports the idea that early onset of neuroblastoma
may occur in multipotent neural crest stem cells before their
migration into respective target tissues and perhaps may limit the
pool of migrating cells.
We were intrigued by the fact that CIP2A is known to stabilize
cMyc, but little was known about its interaction with MycN. We
noted that
CIP2A
was expressed throughout the neural plate and
neural tube at a time when
cMyc
is not yet expressed. This
prompted us to study whether CIP2A might initially stabilize
MycN at early times until cMyc is turned on, and indeed, our
study reveals CIP2A as a binding partner of MycN (Figs. 4 and
7). CIP2A has an established role as an oncogene, and its
knockdown leads to down-regulation of several oncogenic driv-
ers (Akt, cMyc, and E2F1) due to PP2A dephosphorylation-
mediated degradation (57). Although the loss of CIP2A does
not compromise mouse viability, it causes defects in neural and
spermatogonial progenitors (35, 36). Here we show that it is, in
collaboration with MycN, also required for the correct formation
of the neural stem cell characteristics of the neuroectoderm at
the neural plate but not later, after neural tube closure (Figs. 2
–
4).
We also show that CIP2A couples with MycN in neuroblastoma
(Figs. 6 and 7), which suggests that this early developmental role is
maintained in neuroblastoma. CIP2A is overexpressed in a large
fraction of all major human cancer types and, in line with previous
findings (37, 57), its inhibition leads to decreased tumor cell via-
bility in neuroblastoma cell lines (Fig. 7), suggesting that CIP2A
may be a potential target for neuroblastoma therapy.
cMyc is famous for its oncogenic properties and is overex-
pressed in multiple cancer types (58). Thus, it is counterintuitive
that its expression is associated with higher survival rates and
good prognosis in neuroblastoma (Fig. 6). There are several
possible explanations for this observation. First, cMyc in neuro-
blastoma may reflect a more normal multipotent neural crest
stem cell state that is capable of responding to cues from the
environment to promote differentiation (7). Second, the overall
expression levels of
cMyc
in neuroblastoma samples are signifi-
cantly lower than the expression levels of
MycN
in the MycN-
amplified tumors (Fig. 6) and thus are likely to be similar to the
endogenous physiological levels during embryonic development.
This is in line with the reports on high
cMyc
levels as a prognostic
marker for the poor outcome in a small percentage of the un-
differentiated subtype of neuroblastoma, NBUD (59, 60), in which
the overexpression of cMyc may have triggered the highly pro-
liferative oncogenic machinery that is not turned on in neural crest
cells during normal development (7). In line with this, a recent
study shows that a subset of high-risk neuroblastomas display up-
regulated
cMyc
due to enhancer hijacking, and overexpression of
cMyc under the D
β
H promoter induced tumor mass growth in
vivo in zebrafish (61). cMyc amplification is extremely rare in
neuroblastoma, but a case study reports undifferentiated mor-
phology, poor survival, and low levels of
MycN
expression in these
tumors (62). These studies further support our hypothesis that
physiological rather than overexpressed cMyc levels are associated
with the better outcome of the disease.
Despite their very different endogenous roles during the de-
velopment of the nervous system, it is important to keep in mind
that all Myc family members have oncogenic properties and,
upon misregulation, can trigger the onset of malignant trans-
formation. In fact, in line with the reports on poor prognosis with
very high cMyc levels, transcriptional profiles of downstream tar-
gets due to increased expression of any Myc member (MycN/
cMyc/lMyc) are very similar in neuroblastoma and other cancer
types, and all correlate with poor capacity to differentiate (63, 64).
It is thus possible that some of the reports on forced MycN/cMyc
overexpression in the sympathetic ganglia reflect this general on-
cogenic capacity instead of resembling the actual initiation process
of neuroblastoma. Our results highlight the normal role of MycN
in early neural development and raise the intriguing possibility
that the balance of CIP2A/MycN binding at the neural plate
border can influence cell-fate decisions in early embryos in a
manner that triggers priming of neuroblastoma cells.
Materials and Methods
Detailed information regarding materials and methods can be found in
SI
Appendix, SI Materials and Methods
. Briefly, whole-mount in situ hybrid-
ization and gain and loss of function experiments were performed on
chicken embryos as previously described (65
–
67). Morpholinos were pur-
chased from Gene Tools LLC (
www.gene-tools.com/
), immunostaining was
performed as described (7), and Western blot lysates from the neuroblas-
toma cell lines SK-N-AS and SK-N-BE(2) were made 2 d after RNAi infection;
the Western blot protocol was carried out as previously described (37, 68).
Fluorescence on the images was quantified by using ImageJ (NIH). The PLA
was performed according to the manufacturer
’
s instructions for the Duolink
kit (DUO92102; Sigma-Aldrich), and cell viability and proliferation was
measured using the WST-1 kit (5015944001; Roche). The statistical analyses
were performed on publicly available clinical neuroblastoma datasets
(KOCAK, SEQC498, and Versteeg88) acquired from the R2 microarray anal-
ysis and visualization platform (
https://hgserver1.amc.nl/cgi-bin/r2/main.cgi
).
Kerosuo et al.
PNAS
|
vol. 115
|
no. 31
|
E7359
DEVELOPMENTAL
BIOLOGY
ACKNOWLEDGMENTS.
We thank Dr. Marie Arsenian-Henriksson for pro-
viding SK-N-BE(2) cells, Dr. Kristina Cole for providing NGP cells, Dr. Ruth
Palmer for providing SK-N-AS cells, and Dr. Edward K. Chan for the mouse
monoclonal CIP2A antibody. This work was funded by NIH Grants HD037105
and DE024157 (to M.E.B.) and by grants from the Jane and Aatos Erkko
Foundation, the Ella and Georg Ehrnrooth Foundation, and the Väre Foun-
dation (to L.K.), the American-Scandinavian Foundation (to P.N.), and the
Sigrid Juselius Foundation (to J.W.).
1. Matthay KK, et al. (2016) Neuroblastoma.
Nat Rev Dis Primers
2:16078.
2. Maris JM (2010) Recent advances in neuroblastoma.
N Engl J Med
362:2202
–
2211.
3. Brodeur GM, Seeger RC, Schwab M, Varmus HE, Bishop JM (1984) Amplification of N-
myc in untreated human neuroblastomas correlates with advanced disease stage.
Science
224:1121
–
1124.
4. Schwab M, et al. (1984) Chromosome localization in normal human cells and neuro-
blastomas of a gene related to c-myc.
Nature
308:288
–
291.
5. Kerosuo L, Bronner-Fraser M (2012) What is bad in cancer is good in the embryo:
Importance of EMT in neural crest development.
Semin Cell Dev Biol
23:320
–
332.
6. Eilers M, Eisenman RN (2008) Myc
’
s broad reach.
Genes Dev
22:2755
–
2766.
7. Kerosuo L, Bronner ME (2016) cMyc regulates the size of the premigratory neural
crest stem cell pool.
Cell Rep
17:2648
–
2659.
8. Knoepfler PS, Cheng PF, Eisenman RN (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.
9. Sawai S, et al. (1993) Defects of embryonic organogenesis resulting from targeted
disruption of the N-myc gene in the mouse.
Development
117:1445
–
1455.
10. Zinin N, et al. (2014) MYC proteins promote neuronal differentiation by controlling
the mode of progenitor cell division.
EMBO Rep
15:383
–
391.
11. Wakamatsu Y, Watanabe Y, Nakamura H, Kondoh H (1997) Regulation of the neural
crest cell fate by N-myc: Promotion of ventral migration and neuronal differentiation.
Development
124:1953
–
1962.
12. Charron J, et al. (1992) Embryonic lethality in mice homozygous for a targeted dis-
ruption of the N-myc gene.
Genes Dev
6:2248
–
2257.
13. Khudyakov J, Bronner-Fraser M (2009) Comprehensive spatiotemporal analysis of
early chick neural crest network genes.
Dev Dyn
238:716
–
723.
14. Alam G, et al. (2009) MYCN promotes the expansion of Phox2B-positive neuronal
progenitors to drive neuroblastoma development.
Am J Pathol
175:856
–
866.
15. Pei D, et al. (2013) Distinct neuroblastoma-associated alterations of PHOX2B impair
sympathetic neuronal differentiation in zebrafish models.
PLoS Genet
9:e1003533.
16. Ke XX, et al. (2015) Phox2B correlates with MYCN and is a prognostic marker for
neuroblastoma development.
Oncol Lett
9:2507
–
2514.
17. Hirsch MR, Tiveron MC, Guillemot F, Brunet JF, Goridis C (1998) Control of norad-
renergic differentiation and Phox2a expression by MASH1 in the central and pe-
ripheral nervous system.
Development
125:599
–
608.
18. Vincentz JW, et al. (2012) A Phox2- and Hand2-dependent Hand1 cis-regulatory el-
ement reveals a unique gene dosage requirement for Hand2 during sympathetic
neurogenesis.
J Neurosci
32:2110
–
2120.
19. Pattyn A, Morin X, Cremer H, Goridis C, Brunet JF (1999) The homeobox gene Phox2b
is essential for the development of autonomic neural crest derivatives.
Nature
399:
366
–
370.
20. Sommer L, Shah N, Rao M, Anderson DJ (1995) The cellular function of MASH1 in
autonomic neurogenesis.
Neuron
15:1245
–
1258.
21. Wakamatsu Y, Watanabe Y, Shimono A, Kondoh H (1993) Transition of localization of
the N-Myc protein from nucleus to cytoplasm in differentiating neurons.
Neuron
10:
1
–
9.
22. Tsarovina K, et al. (2004) Essential role of Gata transcription factors in sympathetic
neuron development.
Development
131:4775
–
4786.
23. Nakagawara A, et al. (1993) Association between high levels of expression of the TRK
gene and favorable outcome in human neuroblastoma.
N Engl J Med
328:847
–
854.
24. Ernsberger U, Reissmann E, Mason I, Rohrer H (2000) The expression of dopamine
beta-hydroxylase, tyrosine hydroxylase, and Phox2 transcription factors in sympa-
thetic neurons: Evidence for common regulation during noradrenergic induction and
diverging regulation later in development.
Mech Dev
92:169
–
177.
25. Kramer M, Ribeiro D, Arsenian-Henriksson M, Deller T, Rohrer H (2016) Proliferation
and survival of embryonic sympathetic neuroblasts by MYCN and activated ALK sig-
naling.
J Neurosci
36:10425
–
10439.
26. Sawai S, Kato K, Wakamatsu Y, Kondoh H (1990) Organization and expression of the
chicken N-myc gene.
Mol Cell Biol
10:2017
–
2026.
27. Edsjö A, et al. (2004) Neuroblastoma cells with overexpressed MYCN retain their ca-
pacity to undergo neuronal differentiation.
Lab Invest
84:406
–
417.
28. Mobley BC, et al. (2015) Expression of MYCN in multipotent sympathoadrenal pro-
genitors induces proliferation and neural differentiation, but is not sufficient for
tumorigenesis.
PLoS One
10:e0133897.
29. Olsen RR, et al. (2017) MYCN induces neuroblastoma in primary neural crest cells.
Oncogene
36:5075
–
5082.
30. Li W, et al. (2008) CIP2A is overexpressed in gastric cancer and its depletion leads to
impaired clonogenicity, senescence, or differentiation of tumor cells.
Clin Cancer Res
14:3722
–
3728.
31. Chen KF, et al. (2010) CIP2A mediates effects of bortezomib on phospho-Akt and
apoptosis in hepatocellular carcinoma cells.
Oncogene
29:6257
–
6266.
32. Côme C, et al. (2009) CIP2A is associated with human breast cancer aggressivity.
Clin
Cancer Res
15:5092
–
5100.
33. Dong QZ, et al. (2011) CIP2A is overexpressed in non-small cell lung cancer and cor-
relates with poor prognosis.
Ann Surg Oncol
18:857
–
865.
34. Khanna A, et al. (2009) MYC-dependent regulation and prognostic role of CIP2A in
gastric cancer.
J Natl Cancer Inst
101:793
–
805.
35. Kerosuo L, et al. (2010) CIP2A increases self-renewal and is linked to Myc in neural
progenitor cells.
Differentiation
80:68
–
77.
36. Ventelä S, et al. (2012) CIP2A promotes proliferation of spermatogonial progenitor
cells and spermatogenesis in mice.
PLoS One
7:e33209.
37. Junttila MR, et al. (2007) CIP2A inhibits PP2A in human malignancies.
Cell
130:51
–
62.
38. De Preter K, et al. (2006) Human fetal neuroblast and neuroblastoma transcriptome
analysis confirms neuroblast origin and highlights neuroblastoma candidate genes.
Genome Biol
7:R84, and erratum (2007) 8:401.
39. Rogers CD, Jayasena CS, Nie S, Bronner ME (2012) Neural crest specification: Tissues,
signals, and transcription factors.
Wiley Interdiscip Rev Dev Biol
1:52
–
68.
40. Roellig D, Tan-Cabugao J, Esaian S, Bronner ME (2017) Dynamic transcriptional sig-
nature and cell fate analysis reveals plasticity of individual neural plate border cells.
eLife
6:e21620.
41. Cimadamore F, et al. (2011) Human ESC-derived neural crest model reveals a key role
for SOX2 in sensory neurogenesis.
Cell Stem Cell
8:538
–
551.
42. Khanna A, et al. (2013) Chk1 targeting reactivates PP2A tumor suppressor activity in
cancer cells.
Cancer Res
73:6757
–
6769.
43. Cohn SL, et al.; INRG Task Force (2009) The International Neuroblastoma Risk Group
(INRG) classification system: An INRG Task Force report.
J Clin Oncol
27:289
–
297.
44. Bellmeyer A, Krase J, Lindgren J, LaBonne C (2003) The protooncogene c-myc is an
essential regulator of neural crest formation in xenopus.
Dev Cell
4:827
–
839.
45. Hansford LM, et al. (2004) Mechanisms of embryonal tumor initiation: Distinct roles
for MycN expression and MYCN amplification.
Proc Natl Acad Sci USA
101:
12664
–
12669.
46. Chen X, Pappo A, Dyer MA (2015) Pediatric solid tumor genomics and developmental
pliancy.
Oncogene
34:5207
–
5215.
47. Narbonne-Reveau K, et al. (2016) Neural stem cell-encoded temporal patterning
delineates an early window of malignant susceptibility in Drosophila.
eLife
5:e13463.
48. Zhu S, Thomas Look A (2016) Neuroblastoma and its zebrafish model.
Adv Exp Med
Biol
916:451
–
478.
49. Corallo D, Candiani S, Ori M, Aveic S, Tonini GP (2016) The zebrafish as a model for
studying neuroblastoma.
Cancer Cell Int
16:82.
50. Weiss WA, Godfrey T, Francisco C, Bishop JM (2000) Genome-wide screen for allelic
imbalance in a mouse model for neuroblastoma.
Cancer Res
60:2483
–
2487.
51. Terrile M, et al. (2011) miRNA expression profiling of the murine TH-MYCN neuro-
blastoma model reveals similarities with human tumors and identifies novel candi-
date miRNAs.
PLoS One
6:e28356.
52. Weiss WA, Aldape K, Mohapatra G, Feuerstein BG, Bishop JM (1997) Targeted ex-
pression of MYCN causes neuroblastoma in transgenic mice.
EMBO J
16:2985
–
2995.
53. Williams P, Wegner E, Ziegler DS (2014) Outcomes in multifocal neuroblastoma as
part of the neurocristopathy syndrome.
Pediatrics
134:e611
–
e616.
54. Roshkow JE, Haller JO, Berdon WE, Sane SM (1988) Hirschsprung
’
s disease, Ondine
’
s
curse, and neuroblastoma
–
Manifestations of neurocristopathy.
Pediatr Radiol
19:
45
–
49.
55. Nemecek ER, Sawin RW, Park J (2003) Treatment of neuroblastoma in patients with
neurocristopathy syndromes.
J Pediatr Hematol Oncol
25:159
–
162.
56. Garavelli L, et al. (2015) Noonan syndrome-like disorder with loose anagen hair: A
second case with neuroblastoma.
Am J Med Genet A
167A:1902
–
1907.
57. Khanna A, Pimanda JE, Westermarck J (2013) Cancerous inhibitor of protein phos-
phatase 2A, an emerging human oncoprotein and a potential cancer therapy target.
Cancer Res
73:6548
–
6553.
58. Dang CV (2012) MYC on the path to cancer.
Cell
149:22
–
35.
59. Wang LL, et al. (2015) Augmented expression of MYC and/or MYCN protein defines
highly aggressive MYC-driven neuroblastoma: A Children
’
s Oncology Group study.
Br
J Cancer
113:57
–
63.
60. Wang LL, et al. (2013) Neuroblastoma of undifferentiated subtype, prognostic sig-
nificance of prominent nucleolar formation, and MYC/MYCN protein expression: A
report from the Children
’
s Oncology Group.
Cancer
119:3718
–
3726.
61. Zimmerman MW, et al. (2018)
MYC
drives a subset of high-risk pediatric neuroblas-
tomas and is activated through mechanisms including enhancer hijacking and focal
enhancer amplification.
Cancer Discov
8:320
–
335.
62. Matsuno R, et al. (January 1, 2018) Rare MYC-amplified neuroblastoma with large cell
histology.
Pediatr Dev Pathol
, 10.1177/1093526617749670.
63. Raetz EA, et al. (2003) Identification of genes that are regulated transcriptionally by
Myc in childhood tumors.
Cancer
98:841
–
853.
64. Fredlund E, Ringnér M, Maris JM, Påhlman S (2008) High Myc pathway activity and
low stage of neuronal differentiation associate with poor outcome in neuroblastoma.
Proc Natl Acad Sci USA
105:14094
–
14099.
65. Acloque H, Wilkinson DG, Nieto MA (2008) In situ hybridization analysis of chick
embryos in whole-mount and tissue sections.
Methods Cell Biology
87:169
–
185.
66. Kerosuo L, Bronner ME (2014) Biphasic influence of Miz1 on neural crest development
by regulating cell survival and apical adhesion complex formation in the developing
neural tube.
Mol Biol Cell
25:347
–
355.
67. Sauka-Spengler T, Barembaum M (2008) Gain- and loss-of-function approaches in the
chick embryo.
Methods Cell Biol
87:237
–
256.
68. Kauko O, et al. (2015) Label-free quantitative phosphoproteomics with novel pairwise
abundance normalization reveals synergistic RAS and CIP2A signaling.
Sci Rep
5:
13099.
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|
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Kerosuo et al.