Oncogene (2005) 24, 1277–1283
& 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00
www.nature.com/onc
Low expression of Wnt-5a gene is associated with high-risk neuroblastoma
Etienne Blanc1, Gwenaëlle Le Roux1, Jean Bénard1,2 and Gilda Raguénez*,1
1
Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8126, Université Paris-Sud, Institut Fédératif de Recherche
54, Institut Gustave-Roussy (IGR), 39, rue Camille Desmoulins, 94805 Villejuif Cedex, France; 2Département de Biologie, Service de
Génétique, Institut Gustave-Roussy, Villejuif, France
Disseminated forms of neuroblastoma (NB), a tumor
derived from neuroectodermal tissue, pose a major
therapeutic challenge for pediatric oncology. By performing a comparative cDNA array analysis of metastatic
neuroblasts versus primary xenograft from the human
IGR-N-91 NB model, we were able to identify a set of
downregulated developmental genes in metastatic neuroblasts. One of these genes was Wnt-5a, a member of the
Wnt signaling pathway, known to be involved in the
development of neural crest cells. Since we also found a
significant decrease in Wnt-5a mRNA in unfavorable
versus favorable categories in 37 primary NB tumors
(Po0.007), we wondered whether retinoic acid (RA),
which has a role in neural crest induction and differentiation, might reverse the aberrant negative regulation of
Wnt-5a in metastatic malignant neuroblasts. Following
treatment with 10 lM RA for 6 days, the MYCNamplified IGR-N-91 cell lines underwent neuronal differentiation as assessed by reduced MYCN gene expression
and neuritic extension. In these conditions, data showed
an upregulation of Wnt-5a and PKC-h isoform expressions. Our study highlights, for the first time, the
involvement of Wnt-5a, which has a role in embryonic
and morphogenetic processes, in the response of malignant
neuroblasts to RA. In conclusion, we demonstrated that
RA, which is used in the treatment of high-risk NB
patients with recurrent/residual disease in the bone
marrow, is able to upregulate Wnt-5a gene expression.
Oncogene (2005) 24, 1277–1283. doi:10.1038/sj.onc.1208255
Published online 13 December 2004
Keywords: Wnt-5a; metastatic neuroblastoma; retinoic
acid; differentiation
Neuroblastoma (NB), an embryonal malignancy, originates from sympathetic neurons, which are derived from
the neural crest. NB represents the most common solid
tumor of early childhood. Both metastatic NB in
children aged up to 1 year at diagnosis and MYCNamplified NB are high-risk NB (HR-NB) and account
for more than 50% of all cases. These HR-NB are very
often refractory to conventional dose-intensive therapy.
*Correspondence: G Raguénez; E-mail: [email protected]
Received 26 March 2004; revised 8 October 2004; accepted 8 October
2004; published online 13 December 2004
Indeed, most HR-NB patients present a dramatic
prognosis (about 25% overall survival at 5 years)
despite intensive high-dose and myeloablative chemotherapy, with a rescue therapy involving autologous
hematopoietic stem cell transplantation, followed by a
maintenance regimen of 13-cis-retinoic acid (RA)
(Matthay et al., 1999; Reynolds et al., 2003). We are
still lacking information on the proteins or genes
involved in HR-NB dissemination.
In order to identify metastatic-related genes in NB,
we performed a cDNA array analysis of malignant
neuroblasts derived from a human NB experimental
model (IGR-N-91 model), which had been previously
characterized in our laboratory and derived from an
HR-NB (Ferrandis et al., 1994). Briefly, an in vitro NB
cell line, IGR-N-91, when injected subcutaneously in
nude mice, yielded a primary tumor xenograft (PTX),
and metastatic neuroblasts in two sites, myocardium
(Myoc) and bone marrow (BM). Then PTX, Myoc, and
BM neuroblasts were subcultured to establish the three
respective cell sublines. We have reported data showing
that this experimental model is appropriate for studying
aggressive disease and chemoresistance mechanism in
HR-NB (Blanc et al., 2003). All IGR-N-91-derived
neuroblasts harbor a consistently high MYCN amplification but, when compared to PTX, BM and Myoc
human neuroblasts elicit a concomitant increase in
MYCN and P-gp/MDR1 expressions (Blanc et al.,
2003). Our data showed that the MycN protein activates
the transcription of the MDR1 gene and contributes to
the drug resistance acquisition, which occurs throughout
the HR-NB’s metastatic process. Using this model, the
analysis of gene expression profiles provided 18 significant differentially expressed genes in metastatic BM
and Myoc compared to PTX neuroblasts. The candidate
genes listed in Figure 1a include development genes:
sonic hedgehog (SHH), disheveled 1 (DVL1), winglesstype 5a (WNT-5A), notch Drosophila homolog of 4
(NOTCH4); signaling genes: interleukin 13 (IL13),
basigin (BSG), linker for activation of T cells (LAT);
cell adhesion genes: caveolin-1 (CAV1), chondroitin
sulfate proteoglycan 1 (CSPG1), cadherin-associated
protein-related (CAPR), integrin alpha-8 (ITGA8); and
detoxication genes: superoxide dismutase 1 (SOD1),
glutathione S-transferase y2 (GST-y2), cell cycle and
protein/RNA turnover genes cyclin A2 (CCNA2), cyclin
B1 (CCNB1), CDC28-asociated protein CKS1 (CSK1),
E2F transcription factor 3 (E2F3), and ribosomal S6
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kinase 1 (RSK1). Interestingly, some developmental
genes are coordinately downregulated in metastatic
neuroblasts (BM and Myoc). These genes belong to
the Hedgehog (Shh), Wnt (DVL1 and Wnt-5a) and
Notch (Notch 4) signaling pathways, well known for
their role during development in orchestrating patterning, organogenesis and cell fate in embryogenic processes, and their recently identified involvement in
human cancer (Taipale and Beachy, 2001). Indeed,
when deregulated, many genes may be associated with
specific human cancers via key regulator gene alterations. For example, oncogenic mutations have been
described for b-catenin and APC in colon carcinoma
(Polakis, 2000), Notch in T-cell leukemia (Ellisen et al.,
1991), and Shh in medulloblastoma (Wechsler-Reya and
Scott, 1999).
The neural crest origin of NB focused our interest on
Wnt-5a, a member of the Wnt family, which was
identified as downregulated in our IGR-N-91 metastatic
model. Quantitative RT–PCR and Western blot analyses confirmed that a significant decrease of Wnt-5a
gene expression occurred in BM and Myoc neuroblasts
when compared to PTX neuroblasts (Figure 1b).
Furthermore, Wnt-5a has been shown to be upregulated
in many human cancers (Iozzo et al., 1995) as well as in
the metastatic dissemination of melanoma (Weeraratna
et al., 2002). In contrast, it has been shown to be
downregulated in invasive ductal breast cancer (Jönsson
et al., 2002). No gene amplification or rearrangement of
the Wnt-5a gene has been reported in these cancers. We
therefore used the results obtained from our IGR-N-91
metastatic model to extend the measurement of Wnt-5a
gene expression by QRT–PCR into a set of 37 primary
NB tumors. On the basis of clinical and biological
criteria, including the MYCN copy number, and the
overall survival at 2 years after diagnosis, two main
groups of NB tumors were defined as having a favorable
(F) or unfavorable (Uf) prognosis (Table 1). The first
group of 25 patients with an F prognosis encompassed
stage 1, 2 and 3 tumors (n ¼ 13) and stage 4o1 year of
age tumors (n ¼ 12). The second group, which included
12 HR-NB patients with an Uf prognosis, encompassed
stage 4>1 year of age tumors, including MYCNnonamplified (n ¼ 8) and MYCN-amplified patients
(n ¼ 4), associated with a dramatic prognosis or a fatal
outcome. A significantly reduced Wnt-5a gene expression appears to be correlated with this highly aggressive
HR-NB tumor class (Po0.007, Wilcoxon’s test)
(Figure 1c). However, Wnt-5a role in NB metastatic
process cannot be now established unless Wnt-5a stable
transfectants are performed in NB cell lines followed by
related functional assays in nude mice.
It is well established that Wnt members are secreted as
glycoproteins, and involved in the proliferation of stem
cells and patterning decisions of the nervous system
throughout development, including the specification of
the neural crest (Ikeya et al., 1997, Garcia-Castro et al.,
2002). Wnt-5a is a potential tumor suppressor gene as it
has been seen in experiments showing that ectopic Wnt5a, when injected into athymic nude mice, prevents and
reverses tumorigenesis (Olson et al., 1997). Wnt-5a is an
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Table 1 Characteristics of NB tumors and clinical outcome of
patients
Neuroblastic tumors Stage MYCN n
copy
Localized
1–2–3
Metastatic o1 year
4
Metastatic >1 year
4
4
1
1
1
6–300
13
12
8
4
Overall
survival
Alive
Alive
Alive
Alive
(12/13)
(11/12)
(5/8)
(2/4)
Clinical
classification
F
F
Uf
Uf
Human neuroblastic tumors were obtained from the ‘Centre de
Ressources Biologiques’ (Institut Gustave Roussy, Villejuif, France).
Tissue samples were collected from primary tumors of children as
described previously (Douc-Rasy et al., 2002), and cut into 70-mmthick slices with a cryostat. Hematoxylin–eosin-stained tissue (5 mm)
flanking both slides of the thick tumor slices were controlled
histologically to determine the percentage of tumor cells: only tumor
tissues presenting more than 60% of tumor cells were included in the
study. Staging was performed according to the histological criteria.
PCR amplification of MYCN copy number was performed using a
TaqMan 50 nuclease fluorigenic real-time quantitative PCR assay with
the ABI PRISM 7700 Sequence Detection System (PE Biosystems).
Primers have been described previously (Blanc et al., 2003). Overall 2year survival of patients was about 90% for the favorable (F) group
compared to 58% for the unfavorable (Uf) group
important regulator of morphogenetic movements during embryonal development (Huguet et al., 1995) and,
more specifically, regulates the proliferation of progenitor cells (Yamaguchi et al., 1999). Recently, Wnt-5a
signaling has been implicated in the development of the
pituitary gland and, more specifically, in controlling the
organ’s shape (Cha et al., 2004). Importantly, Wnt-5a
does not appear to signal via the canonical Wnt/bcatenin-dependent signaling pathway but instead stimulates the Wnt/Ca2 þ pathway via intracellular Ca2 þ
release and the activation of two kinases, CamKII
and PKC (Miller et al., 1999; Kühl et al., 2000). The
misregulation of Wnt signaling such as in the Wnt/
b-catenin pathway has been reported in colon carcinomas (Polakis, 2000), and in lung cancer with the loss
of Wnt7a expression (Calvo et al., 2000; Ohira et al.,
2003), while alterations in cell–cell interaction via the
Wnt/Ca2 þ pathway are the main mechanisms involved
in epithelial tumors with metastatic disease (Jönsson and
Andersson, 2001; Jönsson et al., 2002; Weeraratna et al.,
2002).
To determine the respective and potential role of the
Wnt/b-catenin and Wnt/Ca2 þ pathways in the IGR-N91 model, we analysed the b-catenin expression status
and PKC activity. No change in b-catenin expression
was noted between PTX and BM/Myoc metastatic
neuroblasts by Western blot analysis (Figure 2a). Moreover, immunocytochemistry experiments clearly demonstrated that no significant difference was observed in the
expression or nuclear translocation of b-catenin between
PTX and metastatic neuroblasts (Figure 2b). PKC
activity was determined by Western blot analysis using
antibodies probed with phospho-PKC (pan) and a panel
of antiphospho-PKC isoforms. The level of PKC-a/b
isoform activity in these isoforms did not vary, while
the PKC-y isoform level was only decreased in
Myoc neuroblasts when compared to PTX neuroblasts
(Figure 2a). Noteworthy, the PKC-y isoform has been
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E Blanc et al
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shown to be involved in several functions such as cell
cycle progression (Datta et al., 1997), reorganization of
the actin skeleton (Tang et al., 1997) and T-cell
activation (Monks et al., 1997). PKC-y expression has
also been demonstrated in cells of neural origin such as
microglial and astroglial cells (Slepko et al., 1999) and
the LAN-5 NB (Passalacqua et al., 1999).
The development of the neural crest and sympathetic
nervous system depends on various factors such as
FGFs and RA signals (Christiansen et al., 2000). During
embryogenesis, RA induces neuroectodermal differen-
Figure 1 (a) Representative differential gene expression in
metastatic NB cell lines, defined by a twofold or greater change
relative to the expression in the PTX cell line. The IGR-N-91 cell
line was established from an involved BM harvested from a highrisk NB (stage 4-NB, 8-year-old boy). Neuroblasts were injected
subcutaneously into nude mice and a PTX was isolated while
metastatic neuroblasts were isolated from the Myoc and BM. These
neuroblasts were further cultured in vitro on bovine corneal
extracellular matrix to establish sublines. These IGR/PTX, IGR/
Myoc and IGR/BM sublines were maintained as reported earlier
(Blanc et al., 2003). The exponentially growing IGR/PTX, IGR/
BM and IGR/Myoc neuroblasts were grown to about 80%
confluency and then harvested for RNA extraction. Gene expression analyses were performed by hybridizing labeled cDNA from
human cell lines onto the Atlast Human Cancer 1.2 arrays
(Clontech Laboratories, Ozyme, France). Total RNA (2 mg) was
reversed transcribed and labeled using [a-32P]dATP. The probe was
then hybridized on cDNA spotted arrays. Hybridization was
performed at 601C with continuous agitation overnight. The
printed surface of membranes was exposed to a PhosphorImager
screen for 24 h, and then scanned at a resolution of 50 mm. Analysis
was performed using the AtlasImage 2.01 software (Clontech).
Global normalization using the sum method was used to compare
the membranes. For positive controls, we used the following
housekeeping genes: b-actin, ubiquitin, and GAPDH (among
others). Experiments were performed twice. PubMed Accession
numbers are: SHH (M81830), DVL1 (NM-00421), WNT-5A (NM003392), NOTCH4 (U95299), IL13 (L06801), BSG (L20471), LAT
(AFO36905), CAV1 (NM-001753), CSPG1 (NM-001135), CAPR
(M94251), ITGA8 (L36531), SOD1 (M13267), GST-y2 (NM00854), CCNA2 (X51688), CCNB1 (M25753), CSK1 (X54941),
E2F3 (Y10479), RSK1 (LO7597). (b) Analysis of Wnt-5a gene
expression in NB cell lines. The RNAs (1 mg) were subjected to realtime RT–PCR analyses using the TaqMan procedure described
previously (Blanc et al., 2003). The primers were determined as
follows: cWnt-5a forward primer, 50 -GCC AAG GGC TCC TAC
GAG AG-30 ; cWnt-5a reverse primer, 50 -AGC CAG GTT GTA
CAC CGT CC-30 ; and the TaqMan probe, FAM50 -AAC CTG
CAC AAC AAC GAG GCC G-30 TAMRA. Ct values (fractional
cycle number at which the fluorescence generated by cleavage of the
probe crosses a fixed threshold) were determined. Calibration
curves plotting Ct against reference cDNA quantity were generated
and cDNA level for the test sample was determined by extrapolation. Control parental IGR-N-91 cells were used. Samples were
normalized using 18S mRNA expression with primers described
previously (Blanc et al., 2003). Duplicates were used in each
individual experiment and results are the mean of three independent experiments7s.d. The difference of Wnt-5a gene expression
level is significant between PTX and BM/Myoc neuroblasts and
noted (**Po0.01) according to an unpaired Student’s t-test.
Controls have been performed without reverse transcriptase and
are negative for every sample (data not shown). (c) Wnt-5a is
decreased in unfavorable neuroblastic tumors. Levels of Wnt-5a
gene expression in favorable (F) versus unfavorable (Uf) neuroblastic tumors were performed by QRT–PCR analysis as described
above. A significantly reduced Wnt-5a level is noted in the Uf
group compared to the F group according to Wilcoxon’s test
(Po0.007)
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Figure 2 Wnt/Ca2 þ signaling is involved in metastatic neuroblasts. (A) Wnt-5a, b-catenin, and phospho-PKC analyses were
performed by Western blotting as described earlier (Blanc et al., 2003). Immunoblots were probed with the following antibodies diluted
at 1/500 for Wnt-5a (R&D Biosystems, France) at 1/5000 for b-catenin (Transduction Laboratories, Lexington, KY, USA), at 1/500
for phospho-PKC (pan), phospho-PKC-a/bII, or phospho-PKC-y antibodies (Cell Signaling Technologies, France). Controls were
performed with b-actin antibody (1/2000, MAB 1501, Chemicon International). Protein concentrations were determined by the BCA
method (Pierce). (B) Immunocytochemistry of b-catenin in PTX (a), BM (b), and Myoc (c) malignant neuroblasts. Cells were fixed for
30 min at room temperature in 1% PAF, rinsed twice in PBS, and blocked for 2 h in PBS containing 0.05% Triton X-100, BSA 2%,
and then incubated for 18 h at 41C with primary antibody using the streptavidin–biotin peroxidase technique. The mouse monoclonal
b-catenin (BD Biosciences, Palo Alto, CA, USA) was diluted at 1/200. A biotinylated anti-mouse IgG was used as secondary antibody
and as a negative control (d)
tiation with the formation of several cell types, including
neurons, glia, and fibroblast-like cells. In undifferentiated human embryonal carcinomas, which do not
express Wnt genes, it has been shown that Wnt
expression occurs upon RA-induced differentiation
(Katoh et al, 1996) and, more specifically, involves a
developmental regulation of the human Wnt-13 (Wakeman et al., 1998). Moreover, the expression of multiple
Wnt signaling molecules changed during the early phase
of neuronal differentiation by RA (Katoh, 2002). Thus,
in order to determine if RA might reverse the aberrant
negative regulation of Wnt-5a in metastatic neuroblasts,
we investigated Wnt-5a gene expression in RA-differentiated neuroblasts of the IGR-N-91 model including
PTX, BM and Myoc cell sublines. Following treatment
with 10 mM RA for 6 days, PTX, BM and Myoc
neuroblasts underwent neuronal differentiation as assessed from neuritic extensions (Figure 3a) and the
increase of GAP43 gene expression in PTX neuroblasts
(Figure 3b). The growth-associated protein 43 (GAP43)
is a neuronal protein with an important role in
cytoskeleton-associated processes involved in the extension and sprouting of nerve growth cones (Aigner et al.,
1995). No variation of GAP43 gene expression was
noted in BM and Myoc metastatic neuroblasts. Indeed,
GAP43 is a recognized marker of terminal neuronal
differentiation and its expression is probably delayed in
metastatic neuroblasts. Furthermore, we observed a
Oncogene
marked reduction in MYCN expression both at the
mRNA and protein levels (Figure 3c and d). Of note, the
MYCN decrease is a specific event, which has been
previously described in RA-differentiated neuroblasts
(Thiele et al., 1985). QRT–PCR and immunocytochemistry showed a significant increase in Wnt-5a gene
expression (Figure 3e and f). This paralleled the increase
in PKC-y levels in RA-differentiated neuroblasts, while
b-catenin levels and localization remained unchanged
(Figure 3g and h). Importantly, it has been evidenced
that PKC-y plays a specific role in the neuronal cells
differentiation program and, for example, is upregulated
in RA-treated PC12 and human NB cells (Sparatore
et al., 2000a, b).
In order to determine whether Wnt-5a alone was able
to change the morphology of neuroblasts, Wnt-5a was
overexpressed in PTX neuroblasts following a transient
transfection with a Wnt-5a plasmid vector for 48 h.
Neither neuritic outgrowth nor reduced MycN levels
were noted in Wnt-5a-transfected PTX (Figure 4a, lane
4), while RA induced a reduced level of MycN and a
significant expression of Wnt-5a (lane 6). To further
investigate whether MycN and Wnt-5a act independently, we studied the effect of 10 mM RA treatment on
the MYCN-nonamplified SH-SY5Y human NB cell line,
a well-established system for studying neuronal differentiation (Körner et al., 1995; Pahlman et al., 1995).
Indeed, it has been shown that RA rapidly drives the
Wnt-5a expression in neuroblastoma
E Blanc et al
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Figure 3 Wnt-5a and PKC-y are upregulated in RA-differentiated neuroblasts. (a) Morphological phenotype of IGR neuroblasts
following RA-induction protocol. Cells (1.5 106) were seeded into 25 cm2 tissue culture flasks in 5 ml of culture medium for 24 h. The
medium was then replaced with a fresh medium containing 10 mM RA or the vehicle DMSO. Cells were harvested 6 days later. The
fresh growth medium and agents were replenished after 2 days. RA (all-trans-RA) was purchased from Sigma (St Louis, MO, USA)
and stored as a 100 mM stock solution dissolved in DMSO. (b) Expression of GAP 43 mRNA in neuroblasts after 10 mM RA induction
for 6 days. RT–PCR was performed using primers as described previously (Raguénez et al., 1999). (c) A significantly reduced MYCN
gene expression is observed by QRT–PCR (***Po0.001) and (d) by Western blot in RA-differentiated neuroblasts. (e) A significant
increase of Wnt-5a gene expression is noted by QRT–PCR in RA-differentiated neuroblasts (***Po0.001). (f) Immunofluorescence
pictures showing a Wnt-5a high expression in RA-differentiated PTX neuroblasts. Cells were fixed for 30 min at 41C in
paraformaldehyde 1%, rinsed three times in PBS, and then incubated for 1 h with the Wnt-5a primary antibody diluted at 1 : 20. The
panels show cells stained with propidium iodide (PI) and Wnt-5a antibody. (g) Western blots of b-catenin and phospho-PKC-y
proteins in RA-differentiated neuroblasts. (h) The analysis of PKC-y mRNA was also performed by RT–PCR using the following
primers: c PKC-y forward primer, 50 -GCA GGA TCC CCA TGT CGC CAT TTC TTC G-30 ; c PKC-y reverse primer, 50 -GCT GTC
GAC CAG GAA ATG CCC TGA GGC TC-30 (Zeidman et al., 1999)
differentiation of SH-SY5Y cells along a sympathetic
chromaffin lineage. Here, the SH-SY5Y cells treated
with 10 mM RA exhibited a morphological change
characterized by an extensive network of neurites
(Figure 4b). As early as 2 days following RA induction,
the Wnt-5a and PKC-y gene expressions were concomitantly increased, while b-catenin levels were found to be
unchanged (Figure 4c and d).
Thus, simultaneous variations of Wnt-5a and PKC-y
may occur in response to RA in malignant neuroblasts
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Figure 4 Wnt-5a and MycN act independently in response to RA. (a) Western blots of MycN and Wnt-5a proteins in Wnt-5a-transfected PTX
neuroblasts. Cells were seeded into six-well plates at a density of 80 000 cells/cm2 for 24 h to reach 80% confluency. The IGR/PTX cells were
transfected for 48 h with 4 mg PCDNA3-Wnt-5a plasmid or 4 mg mock vector using 4 mg/ml Lipofectaminet 2000 (Gibco-BRL). After 8 h, the
medium was replaced with a fresh serum-containing medium. Data obtained from a representative of three independent experiments are shown.
Lane 1: parental PTX; lane 2: PTX þ lipofectamine; lane 3: PTX þ mock vector; lane 4: Wnt-5a-PTX; lane 5: mock vectorPTX þ 10 mM RA;
and lane 6: Wnt-5aPTX þ 10 mM RA. The transfection efficiency (80%) was measured by flow cytometry, which counted the number of cells
expressing a PCDNA3-GFP vector. (b) Morphological phenotype of SH-SY5Y neuroblasts following 10 mM RA for 6 days. The SH-SY5Y cells
were maintained as described previously (Pahlman et al., 1995). (c) RT–PCR analysis of Wnt-5a gene expression in RA-differentiated SH-SY5Y
after RA induction for 2, 4, and 6 days. (d) Western blot analyses of b-catenin and phospho-PKC-y proteins in RA-differentiated SH-SY5Y
neuroblasts
regardless of whether they express MYCN or not. To
sum up, this is the first time that the involvement
of Wnt-5a, an actor in embryonic and morphogenetic processes, has been described in the pathogenesis
of a pediatric embryonal malignancy. Moreover,
RA-induced neural differentiation is independent of
the level of MYCN amplification and is concomitantly associated with increased Wnt-5a and PKC-y
gene expressions. Whether Wnt-5a promoter harbors
RA-responsive elements still remains to be elucidated.
The malignant neuroblast RA response is consistent
with clinical studies showing that retinoid therapy is
effective against residual metastatic NB disease and
improves the event-free survival in high-risk NB
(Reynolds et al., 2003). Further investigations are
required to determine whether Wnt-5a gene upregula-
tion might reflect the ability of malignant neuroblasts to
respond to RA.
Acknowledgements
We thank SFCE, Dr O Hartmann, Dr Dominique ValteauCouanet (Service de Pédiatrie), Dr Serge Koscielny (Département de Santé Publique), and Dr Marie-José Terrier-Lacombe
(Service d’anatomo-pathologie). We are also grateful to
Michel Barrois for his invaluable assistance in designing the
gene probes, Sabrina Cantais and Nadine Béron-Gaillard for
their technical assistance (all from Institut Gustave Roussy).
We also thank Dr Ashani T Weeraratna and Dr Yuan Jiang
from the National Human Genome Research Institute/USA
for donating the PcDNA3-Wnt-5a. Edited by Englishbooster
S.A. This work was supported by the Ligue contre le Cancer,
Comité de Montbéliard.
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