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Functional analysis of the p53 pathway in neuroblastoma cells
using the
small-molecule MDM2 antagonist nutlin-3∗
Tom Van Maerken,1,2 Ali Rihani,1 Daniel Dreidax,3 Sarah De
Clercq,4 Nurten Yigit,1 Jean-
Christophe Marine,4 Frank Westermann,3 Anne De Paepe,1 Jo
Vandesompele,1 Frank
Speleman1
1Center for Medical Genetics, Ghent University Hospital, Ghent,
Belgium
2Department of Clinical Chemistry, Microbiology and Immunology,
Ghent University
Hospital, Ghent, Belgium
3Department of Tumor Genetics, German Cancer Research Center,
Heidelberg, Germany
4Laboratory for Molecular Cancer Biology, VIB-UGent, Ghent,
Belgium
Running title: Analysis of the p53 pathway in neuroblastoma
Keywords: neuroblastoma, p53, nutlin-3, p14ARF
Abbreviations: CI, confidence interval; qPCR, quantitative
real-time PCR; qRT-PCR,
quantitative real-time reverse transcription PCR
∗Grants: Research Foundation – Flanders (FWO), Concerted
Research Actions – UGent (GOA), Interuniversity Attraction Poles –
Belgium (IUAP), and Emmanuel van der Schueren Foundation. T. Van
Maerken has conducted the study as Ph.D. fellow of the FWO.
Correspondence: Tom Van Maerken, Center for Medical Genetics, Ghent
University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium.
Phone: 32-9-332-0352; Fax: 32-9-332-6549. E-mail:
[email protected] Conflict-of-interest disclosure: None.
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Abstract
Suppression of p53 activity is essential for proliferation and
survival of tumor cells. A direct
p53-activating compound, nutlin-3, was used in this study,
together with p53 mutation
analysis, to characterize p53 pathway defects in a set of 34
human neuroblastoma cell lines.
We identified 9 cell lines (26%) with a p53 loss-of-function
mutation, including 6 missense
mutations, 1 nonsense mutation, 1 in-frame deletion, and 1
homozygous deletion of the 3′ end
of the p53 gene. Sensitivity to nutlin-3 was highly predictive
of absence of p53 mutation.
Signaling pathways downstream of p53 were functionally intact in
23 out of 25 cell lines with
wild-type p53. Knockdown and overexpression experiments revealed
a potentiating effect of
p14ARF expression on the response of neuroblastoma cells to
nutlin-3. Our findings shed light
on the spectrum of p53 pathway lesions in neuroblastoma cells,
indicate that defects in
effector molecules downstream of p53 are remarkably rare in
neuroblastoma, and identify
p14ARF as a determinant of the outcome of the response to MDM2
inhibition. These insights
may prove useful for the clinical translation of evolving
strategies aimed at p53 reactivation
and for the development of new therapeutic approaches.
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Introduction
The p53 transcription factor plays a critical role in the
cellular defense against malignant
transformation by promoting cell cycle arrest, DNA damage
repair, apoptosis, and senescence
in response to stress signals (1). Tumor cells therefore
invariably acquire aberrations that
permit them to escape from p53-mediated growth control. It is
estimated that approximately
50% of all human cancers harbor inactivating mutations in the
TP53 (p53) gene, whereas
defects in upstream or downstream components of the p53 pathway
are believed to account
for loss of p53 activity in the other half of malignancies.
Dissection of the p53 pathway
defects in individual tumor types is important, since improved
understanding of the
mechanisms behind p53 inactivation may guide the development of
targeted therapeutic
strategies.
Neuroblastoma is an aggressive childhood tumor of neural crest
origin, that has a lethal
outcome in the majority of high-risk patients (2). A remarkable
feature is that p53 is rarely
mutated at diagnosis and only in a minority of neuroblastoma
tumors at relapse, as
demonstrated by a recent study that found mutation rates of 2%
and 15%, respectively (3).
Conflicting data exist regarding p53 pathway signaling in
neuroblastoma cells. The DNA
damage-induced G1 checkpoint function and apoptotic activity of
p53 have been reported to
be impaired by cytoplasmic p53 sequestration (4-6), which may be
caused by p53
hyperubiquitination (7). Furthermore, wild-type p53 in
neuroblastoma cells may be in a
conformation refractory to integration into transcriptional
complexes, resulting in reduced
transcriptional activity (8). In contrast, others have
demonstrated a normal DNA-binding and
transactivation capacity of the p53 protein and an intact p53
signal transduction pathway in
neuroblastoma cells with wild-type p53 (9-11). No study has yet
systematically investigated
the functional integrity of the p53 pathway in neuroblastoma
cells on a larger scale, as the
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reports mentioned above relied on the use of only one to five
neuroblastoma cell lines to
judge on p53 functionality.
We have previously reported that a small-molecule MDM2
antagonist, nutlin-3, is capable of
inducing potent antitumor effects against neuroblastoma cells
and xenografts with wild-type
p53, which may provide a new opportunity for targeted
therapeutic intervention (12, 13).
Nutlin-3 is designed to compete with p53 for binding into a
hydrophobic pocket on the
surface of MDM2 (14). The resulting disruption of the
interaction between both proteins
releases p53 from negative control by MDM2, which functions as
an E3 ubiquitin ligase to
promote p53 proteasomal degradation and as an inhibitor of p53
transcriptional activity.
Treatment with nutlin-3 thus leads to stabilization and
activation of p53 and, if downstream
effectors are functionally intact, to a robust p53 response.
The availability of a direct and selective p53 activator makes
it possible to systematically
search for defects in p53 and its downstream signaling
components. Here, we set out to
examine the nature of p53 pathway defects in a large panel of
neuroblastoma cell lines using
nutlin-3 as a tool for interrogating the functionality of the
p53 pathway.
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Materials and Methods
Cell culture and nutlin-3 treatment
Human neuroblastoma cell lines were obtained between 1993 and
2010 from Peter Ambros
(STA-NB-1.2, STA-NB-3, STA-NB-8, STA-NB-9, STA-NB-10), Garrett
Brodeur (NGP,
NLF, NMB), Susan Cohn (NBL-S, SHEP), Valérie Combaret (CLB-GA),
Thomas Look
(SJNB-1, SJNB-8, SJNB-10), John Lunec [SK-N-BE(1n),
SK-N-BE(2c)], Sven Påhlman
(SH-SY5Y), Patrick Reynolds (CHP-134, CHP-901, CHP-902R,
SMS-KAN, SMS-KCNR),
and Rogier Versteeg (GICIN-1, IMR-32, LA-N-1, LA-N-2, LA-N-5,
LA-N-6, N-206, SK-N-
AS, SK-N-FI, SK-N-SH, TR-14), or established in our laboratory
(UHG-NP). The
authenticity of the cell lines was verified during this study by
array comparative genomic
hybridization and short tandem repeat genotyping. Cell culturing
and treatment with nutlin-3
(Cayman Chemical, Ann Arbor, MI) were performed as previously
described (12).
p53 mutation analysis
Sequencing of the p53 coding region was performed as previously
described (12).
Cell viability analysis
Cells were seeded in duplicate or triplicate wells of a 96-well
plate (104 cells per well) and
incubated for 6 h before treatment was initiated. Treatment
typically consisted of exposure to
0, 2, 4, 8, 16, and 32 µM nutlin-3 for 24, 48, and 72 h, except
for experiments with inducible
model systems, in which the inducing agent or a negative control
was applied for 16 h prior to
incubation with nutlin-3. Cell viability was measured using a
luminescent ATP-based assay
(CellTiter-Glo, Promega, Madison, WI).
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Analysis of caspase-3 and caspase-7 activity
Cells were plated in duplicate or triplicate wells of a 96-well
plate (104 cells per well) and
incubated for 6 h prior to treatment, which was performed in a
similar way as described for
the cell viability experiments. The combined activity of
caspase-3 and caspase-7 was
determined using the Caspase-Glo 3/7 assay (Promega).
Cell cycle and hypodiploidy analysis
Measurements of cell cycle phase distribution and hypodiploid
(sub-G1) DNA content were
performed as previously described (13).
Quantitative real-time reverse transcription PCR (qRT-PCR)
Cells were treated with 0 or 8 µM nutlin-3 for 24 h (or, in the
case of an inducible model
system, with the inducing agent or a negative control for 16 h
and then with 0 or 8 µM nutlin-
3 for an additional 24 h). Total RNA extraction, DNase
treatment, cDNA synthesis, and
SYBR Green I qRT-PCR were performed as previously described
(13). Primer sequences are
available in the RTPrimerDB database (15): BAX (RTPrimerDB ID
#814), BBC3 (PUMA;
#3500), CDKN1A (p21WAF1/CIP1; #631), GAPDH (#3), SDHA (#7), and
UBC (#8). Expression
levels of the p53 target genes BAX, PUMA, and p21WAF1/CIP1 were
calculated using qbasePLUS
software version 1.5 (Biogazelle, Ghent, Belgium) (16). Levels
of GAPDH, SDHA, and UBC
were used for normalization.
Western blot analysis
Western blotting was performed as previously described (12)
using primary antibodies against
p53 (mouse clone DO-1; Calbiochem, San Diego, CA), p21WAF1/CIP1
(mouse clone SX118;
BD Biosciences, San Jose, CA), and BAX (rabbit monoclonal
antibody; Upstate,
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Charlottesville, VA). An anti-β-actin antibody (mouse clone
AC-74; Sigma, St. Louis, MO)
was used to confirm equal loading.
Knockdown and overexpression of CDKN2A (p16INK4a/p14ARF)
See Supplementary Data.
Statistical analysis
See Supplementary Data.
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Results
p53 mutation analysis
The 34 human neuroblastoma cell lines used in this study were
first characterized for
mutations in the p53 gene. Sequencing of the entire coding
region in two overlapping
fragments demonstrated wild-type p53 in 25 cell lines (74%) and
various genetic defects in
the other 9 cell lines (26%) (Table 1). The most frequent
aberrations were missense
mutations, which were located in exon 5 [N-206, SK-N-BE(2c)],
exon 6 (NLF), exon 7
(NMB, SK-N-FI), and exon 10 (LA-N-2) of p53. One cell line,
LA-N-1, was characterized by
a nonsense mutation, resulting in a stop codon at amino acid
residue 182. SJNB-8 cells were
found to possess an in-frame deletion that removes the coding
sequence for amino acids 105-
125. The PCR step prior to the sequencing did not produce an
amplicon for the second part of
the p53 coding region in SK-N-AS cells. It could be shown by
quantitative real-time PCR
(qPCR) that this was due to a homozygous deletion of the 3′ end
of p53 (Supplementary Fig.
S1), in line with previously published findings (17, 18).
Sensitivity to nutlin-3
We next used the selective MDM2 antagonist nutlin-3 to test
whether the p53 pathway was
functional in our series of neuroblastoma cell lines. As
illustrated in Fig. 1A, determination of
the nutlin-3 concentration that causes 50% reduction in cell
viability (IC50 value) provides a
quantitative measure of the functional integrity of the p53
pathway. IC50 values were
established at 24, 48, and 72 h of nutlin-3 treatment and
correlated with the mutation status of
p53 (Fig. 1B). Cell lines with wild-type p53 displayed highly
significantly lower IC50 values
than lines harboring mutant p53 (P=0.004 at 24 h, P
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aberration effectively impaired the function of the p53 protein.
Pronounced reductions in cell
viability after nutlin-3 treatment and corresponding low IC50
values were observed in 23 out
of the 25 cell lines with wild-type p53. This suggests that p53
downstream signaling pathways
are not a major target for p53-inactivating lesions in
neuroblastoma and lends support to the
development of therapeutic strategies aimed at p53
reactivation.
Two cell lines, LA-N-6 and SHEP, were relatively resistant to
nutlin-3 despite the presence of
wild-type p53 (IC50 values comparable to those observed in
neuroblastoma lines with mutant
p53, i.e., IC50 values >32 µM, >30 µM, and >20 µM
nutlin-3 at 24, 48, and 72 h of treatment,
respectively) (Fig. 1B). Of particular interest were SHEP cells,
because their response to
nutlin-3 was strikingly different from that of two closely
related cell lines, SK-N-SH and SH-
SY5Y. Cell line SK-N-SH was originally derived from bone marrow
metastases of a patient
with stage 4 neuroblastoma, and subcloning of these cells has
generated several
morphologically distinct sublines, including SHEP and SH-SY5Y
(19). Fig. 2A demonstrates
that nutlin-3 profoundly suppressed the viability of SK-N-SH and
SH-SY5Y cells, whereas
only mild effects were noted in SHEP cells. Further experiments
were performed to determine
whether the poor nutlin-3 response of SHEP cells was due to
failure to enter apoptosis or to
defective cell cycle arrest. Analysis of caspase-3 and caspase-7
activity indicated that a 24-h
exposure to nutlin-3 induced a dose-dependent apoptotic response
in SK-N-SH and SH-SY5Y
cells (Fig. 2B). In contrast, no increase in caspase-3 and
caspase-7 activity was observed in
nutlin-3-treated SHEP cells. This was confirmed by flow
cytometric analysis of sub-G1 DNA
content after treatment with vehicle control or 8 µM nutlin-3
for 24 h, which showed a nutlin-
3-induced increase in the apoptotic sub-G1 fraction in SK-N-SH
and SH-SY5Y cells, but not
in SHEP cells (Fig. 2C). Flow cytometric cell cycle profiling
further demonstrated a reduction
in the percentage of cells in S phase 24 h after treatment of
SK-N-SH, SH-SY5Y, and SHEP
cells with 8 µM nutlin-3, indicative of cell cycle arrest in all
three cell lines (Fig. 2D). The
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phenotypic effects of nutlin-3 on apoptosis and cell cycle
progression were paralleled by
similar changes in expression levels of p53 target genes. As
shown in Fig. 2E, a 24-h
treatment of SK-N-SH and SH-SY5Y cells with 8 µM nutlin-3
induced an increase in the
mRNA levels of p53 target genes involved in apoptosis (BAX,
PUMA) and cell cycle arrest
(p21WAF1/CIP1). A large increase in p21WAF1/CIP1 expression was
also present in SHEP cells
treated with 8 µM nutlin-3 for 24 h, but expression levels of
the proapoptotic target genes
BAX and PUMA remained considerably lower in nutlin-3-treated
SHEP cells than in nutlin-3-
treated SK-N-SH and SH-SY5Y cells. Similar findings were
observed by Western blot
analysis. Treatment with 8 µM nutlin-3 for 24 h induced p53
accumulation and increased
expression of p21WAF1/CIP1 in all three cell lines, whereas
induction of BAX expression was
observed only in nutlin-3-treated SK-N-SH and SH-SY5Y cells
(Fig. 2F). Taken together,
these data indicate that SHEP cells have an intact cell cycle
checkpoint control mechanism,
but fail to undergo apoptosis in response to treatment with
nutlin-3.
Interestingly, SHEP cells have previously been reported to
contain a homozygous deletion of
the CDKN2A gene on chromosome 9p21, in contrast to SK-N-SH and
SH-SY5Y cells (20).
We confirmed the copy number status of CDKN2A in these three
cell lines by qPCR
(Supplementary Fig. S2). The CDKN2A gene encodes two
structurally distinct growth-
inhibitory proteins, p16INK4a and p14ARF, that are important
regulators of the pRb and p53
tumor suppressor pathways, respectively (21). This raised the
possibility that the homozygous
CDKN2A deletion may underlie the nutlin-3-resistant phenotype of
SHEP cells. Analysis of
the entire panel of 25 neuroblastoma cell lines with wild-type
p53 further revealed that the
presence of homozygous CDKN2A deletion was strongly associated
with a higher IC50 value
at 48 and 72 h of nutlin-3 treatment (P=0.009 and P0.05)
(Supplementary Table S1). MYCN-
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amplified neuroblastoma cell lines with wild-type p53 were
characterized by a lower IC50
value at 72 h of nutlin-3 treatment than wild-type p53
neuroblastoma cell lines without MYCN
amplification (P=0.023), but this difference was not observed
anymore after exclusion of cell
lines with homozygous CDKN2A deletion (P>0.05) (Supplementary
Table S1). No significant
difference in p53 mutation status nor in MDM2 and CDKN2A copy
number status was found
between MYCN-amplified and MYCN-nonamplified neuroblastoma cell
lines (P>0.05)
(Supplementary Table S2).
Effect of CDKN2A knockdown on the response to nutlin-3
A possible involvement of p14ARF and p16INK4a in the nutlin-3
response was first tested by
transient knockdown of the CDKN2A gene in IMR-32 and NGP cells,
two easy-to-transfect
neuroblastoma cell lines that have a good and previously
well-characterized nutlin-3 response
(12), using a pool of siRNAs directed against sequences common
to both p14ARF and p16INK4a
transcripts. The efficiency of CDKN2A knockdown, measured by
qRT-PCR 24 h
posttransfection, is shown in Fig. 3A. Silencing of CDKN2A
resulted in a moderate reduction
in the sensitivity of IMR-32 and NGP cells to nutlin-3, as
demonstrated by cell viability
assays performed after 24, 48, and 72 h of exposure to nutlin-3
(Fig. 3B and C).
To unravel whether this potentiating effect of CDKN2A expression
on the response to nutlin-3
was mediated by p14ARF or p16INK4a, NGP cells were infected with
lentiviruses encoding a
p14ARF-specific shRNA, a p16INK4a-specific shRNA, an shRNA
directed simultaneously
against both transcripts, or a negative control shRNA targeting
firefly luciferase, and
subsequently selected with puromycin to eliminate uninfected
cells. qRT-PCR analysis of
p14ARF and p16INK4a expression in the established sublines,
termed NGP-LV-p14, NGP-LV-
p16, NGP-LV-p14/p16, and NGP-LV-luc, respectively, demonstrated
successful transcript-
specific knockdown (Fig. 4A). Treatment of these stable
knockdown cell lines with nutlin-3
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followed by cell viability assays indicated that the influence
of CDKN2A expression on the
nutlin-3 response was primarily attributable to p14ARF (Fig.
4B). These findings were
confirmed by analysis of caspase-3 and caspase-7 activity, which
showed that silencing of
p14ARF decreased the susceptibility of NGP cells to undergo
apoptosis upon nutlin-3 treatment
(Fig. 4C). Quantification of p53 target gene expression
demonstrated that knockdown of
p14ARF, but not p16INK4a, attenuated the p53 transcriptional
response induced by a 24-h
exposure to 8 µM nutlin-3 (Fig. 4D). This was accompanied by a
marked p14ARF shRNA-
induced decrease in the basal mRNA levels of PUMA and
p21WAF1/CIP1 in vehicle-treated cells,
whereas BAX expression was upregulated to a lesser extent by
nutlin-3, rather than basically
suppressed, when p14ARF was silenced.
Effect of CDKN2A overexpression on the response to nutlin-3
We next examined whether overexpression of CDKN2A could enhance
the response of
neuroblastoma cells to nutlin-3. We therefore generated stable
transfectants of an IMR-32
subclone, IMR-5/75, in which transgenic expression of either
p14ARF or p16INK4a or, as a
negative control, lacZ was inducible by addition of
tetracycline. Fig. 5A shows the relative
mRNA expression levels of p14ARF and p16INK4a in these sublines,
designated as IMR-Tet-
p14, IMR-Tet-p16, and IMR-Tet-lacZ, respectively, 24 h after
treatment with tetracycline or
vehicle control. Overexpression of p14ARF resulted in a more
pronounced reduction in cell
viability and stronger caspase-3 and caspase-7 activation
following nutlin-3 treatment,
whereas overexpression of p16INK4a or lacZ had no appreciable
effect on the nutlin-3 response
(Fig. 5B and C). In line with these observations, incubation of
IMR-Tet-p14 cells with 8 µM
nutlin-3 for 24 h induced a more potent p53 transcriptional
response when the cells had been
exposed to tetracycline compared to vehicle control (Fig. 5D).
The expression of PUMA and
p21WAF1/CIP1 in these cells in the absence of nutlin-3 was also
considerably increased by the
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addition of tetracycline. In contrast, switching on transgene
expression in IMR-Tet-p16 and
IMR-Tet-lacZ cells did not affect basal nor nutlin-3-induced
expression levels of p53-
responsive genes.
Finally, similar CDKN2A overexpression experiments were
undertaken in SHEP cells to
investigate whether this manipulation could restore the
sensitivity to nutlin-3. Despite
successful generation of several sublines with
tetracycline-inducible expression of p14ARF and
p16INK4a, we did not observe a reversal or improvement of the
nutlin-3-resistant phenotype of
SHEP cells (Supplementary Fig. S3).
Taken together, our data provide evidence for a dosage effect of
p14ARF expression on the
response of neuroblastoma cells to nutlin-3, but they also
indicate that the homozygous
CDKN2A deletion in SHEP cells is not responsible for the poor
response of these cells to
nutlin-3.
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Discussion
The p53 tumor suppressor protein is at the crossroads of
cellular stress response pathways that
control decisions between life and death. We used here the
selective MDM2 antagonist nutlin-
3 as a tool to gain insight into the mechanisms by which
neuroblastoma cells escape from
p53-mediated growth control. Mutation analysis demonstrated a
p53 gene alteration in 9 out
of 34 neuroblastoma cell lines, which rendered the p53 pathway
nonfunctional in all cases.
Three mutations were located outside the classic hot-spot region
(exons 5-9), indicating that
p53 mutations are best identified by sequencing the entire
coding region. The observed
mutation frequency in our cell line panel (26%) is considerably
higher than the p53 mutation
rate of approximately 1% that was found in early studies of
neuroblastoma tumors (22-27).
This may reflect the fact that cell lines are frequently derived
from progressive or relapsed
tumors, as p53 mutations can develop during chemotherapy and
malignant progression of
neuroblastoma (28). Additionally, older studies may have
underestimated to some extent the
p53 mutation frequency in neuroblastoma tumors, since analysis
was often confined to the
mutational hot-spot region.
Treatment with nutlin-3 was capable of inducing potent
antiproliferative and cytotoxic effects
in 23 out of 25 neuroblastoma cell lines with wild-type p53.
These findings are particularly
relevant in the light of an ongoing debate whether p53 is
functional in neuroblastoma or not
(4-11). Discrepancies between previous studies may be in part
attributed to different treatment
regimens (11) and to whether the p53-inducing stimulus directly
interferes with potential
restraints on p53 activity, such as p53 hyperubiquitination (7).
Our data provide good
evidence of almost uniform functionality of the p53 protein and
its downstream effectors in
neuroblastoma cells with wild-type p53 when the interaction
between p53 and MDM2 is
disrupted by nutlin-3. As a consequence, selective MDM2
inhibitors may prove beneficial for
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treating neuroblastoma patients, provided that wild-type p53 is
present. Our findings of
functional p53 effector pathways also suggest that circumvention
of the p53-driven antitumor
barrier in neuroblastoma cells relies primarily on defects
upstream of p53. Cumulating
evidence indicates that it is precisely an increased activity of
MDM2 which serves as the
predominant mode of p53 inactivation in neuroblastoma (28), but
further study is warranted to
identify the full spectrum of aberrations in regulators of p53
activity.
The presence of a homozygous CDKN2A deletion in the
nutlin-3-refractory SHEP cells but
not in the nutlin-3-sensitive SK-N-SH and SH-SY5Y cells prompted
us to investigate the role
of p14ARF and p16INK4a in the response to nutlin-3. The
nutlin-3-resistant phenotype of SHEP
cells could not be reversed by reintroduction of p14ARF or
p16INK4a, but knockdown and
overexpression experiments in other neuroblastoma cell lines
pointed to a stimulatory effect
of p14ARF expression on the nutlin-3 response. Our data suggest
that a p14ARF-driven increase
in basal expression levels of p53-responsive genes, such as PUMA
and p21WAF1/CIP1,
contributes to this potentiating effect of p14ARF, although
other mechanisms cannot be
excluded. High levels of the MDM2-inhibitory protein p14ARF may
result in a larger fraction
of the nuclear pool of MDM2 molecules being inhibited after
nutlin-3 treatment and thus in
stronger activation of the p53 pathway. Alternatively, p14ARF
may provide a costimulatory
signal for the p53 response independently of MDM2. For instance,
p14ARF may increase p53
protein synthesis (29), inhibit p53 turnover by repressing other
components of the p53
degradation pathway than MDM2 (30), enhance p53 transcriptional
activity (31), or regulate
pathways that crosstalk with p53 signaling (32). We did not aim
to identify the molecular
basis of the cooperation between p14ARF and nutlin-3 in this
study, but rather wish to
comment on the potential clinical implications. Previous studies
using mouse models have
demonstrated that Cdkn2a mutations induce chemoresistance by
disabling p53 (33) and that
loss of p19ARF, the murine homolog of p14ARF, limits the
therapeutic response to the tyrosine
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kinase inhibitor imatinib (34). Based on our findings, it can be
expected that tumor cells may
also gain resistance to nutlin-3 treatment by suppressing
p14ARF. The likelihood of this
scenario is corroborated by data from a switchable p53 knock-in
mouse model of lymphoma
showing that p53 reactivation strongly selects for the emergence
of p53-resistant tumors
through inactivation of either p53 or p19ARF (35). Several
early-phase clinical studies with
selective MDM2 inhibitors and other p53-reactivating compounds
have recently been initiated
(36). Our data indicate that the occurrence of aberrations in
p14ARF should be monitored in
these studies and provide an incentive for the development of
strategies to counter p14ARF
lesions.
The lack of improvement in nutlin-3 sensitivity after
reintroduction of p14ARF into SHEP cells
leaves us with the question of how to explain the resistant
phenotype of these cells. We
provided evidence of intact cell cycle arrest but defective
apoptosis following nutlin-3
treatment of SHEP cells. This cell line is also resistant to
other apoptosis-inducing stimuli,
including irradiation (37, 38) and adenoviral gene therapy (39).
The poor sensitivity to death-
inducing triggers might be related to the S-type (substrate-
adherent/Schwannian/melanoblastic) morphology of SHEP cells, as
S-type neuroblastoma
cells seem to be more resistant to apoptosis than N-type
(neuroblastic/neuroendocrine)
neuroblastoma cells (40). Another notable feature is that SHEP
cells have lost the capacity to
form colonies in soft agar and tumors in nude mice (41). One
could therefore wonder whether
the loss of oncogenic signals – which often have a collateral
proapoptotic effect – may result
in desensitization to apoptosis. For instance, SHEP cells lack
expression of the MYCN
oncoprotein, and artificial induction of MYCN expression in
these cells has been shown to
slightly increase the sensitivity to nutlin-3 (42).
Alternatively, SHEP cells may contain high
levels of antiapoptotic proteins, as has been previously
proposed (37). Further study is needed
to pinpoint the exact mechanism underlying the
nutlin-3-resistant phenotype of SHEP cells.
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In conclusion, this study provides several insights into the
spectrum of p53 pathway defects in
neuroblastoma cells that may prove useful for designing new
therapeutic approaches. The
rarity of signaling defects downstream of p53 indicates that
p53-reactivating strategies may
represent an excellent therapeutic tool for treating
neuroblastoma tumors with wild-type p53.
Resistance to nutlin-3 is mostly attributable to the presence of
p53 mutation, which is not
uncommon in neuroblastoma cell lines. This highlights the need
to search for effective p53-
independent anticancer agents or mutant p53-targeting compounds
as a complementary
therapeutic modality. Finally, the finding that p14ARF
expression levels modulate the
sensitivity of neuroblastoma cells to nutlin-3 raises the
possibility that p14ARF may contribute
to the outcome of p53 activation in patients treated with
selective MDM2 inhibitors. It
remains to be determined whether clinical treatment failure with
this new class of anticancer
drugs may result from loss or suppression of p14ARF.
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Acknowledgments
We thank Griet Van Lancker and Xiaoyang Zhang for technical
assistance.
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Table 1. Neuroblastoma cell lines with p53 mutation
Cell line p53 mutation* Previous report
LA-N-1 546C>A (C182X)† Yes (43) LA-N-2 1009C>T (R337C) No
N-206 529C>A (P177T) No NLF 607G>A (V203M) No NMB 733G>A
(G245S) Yes (9) SJNB-8 313-375delGGCAGCTACGGTTTCC
GTCTGGGCTTCTTGCATTCTGGGA CAGCCAAGTCTGTGACTTGCACG
(GSYGFRLGFLHSGTAKSVTCT105-125del)
Yes (44)
SK-N-AS Homozygous deletion of exons 10-11‡ Yes (17, 18)
SK-N-BE(2c) 404G>T (C135F) Yes (45) SK-N-FI 737T>G (M246R)
Yes (12)
*The other neuroblastoma cell lines in this study were wild-type
for p53. †X, termination codon. ‡See Supplementary Fig. S1.
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Figure legends
Figure 1. Sensitivity of neuroblastoma cells to nutlin-3. A,
principle of p53 pathway probing
by determination of the IC50 value of nutlin-3. B, distribution
of IC50 values of nutlin-3 in 34
neuroblastoma cell lines according to p53 mutation status.
Calculated IC50 values above 32
µM fall outside the range of tested nutlin-3 concentrations and
are denoted by dots at 32 µM.
Bars, median IC50 value; solid arrow, SHEP cells; dashed arrow,
LA-N-6 cells.
Figure 2. Impairment of the apoptotic response to nutlin-3 in
SHEP cells, but not in SK-N-
SH and SH-SY5Y cells. A, effect of nutlin-3 treatment for 24,
48, and 72 h on cell viability.
Bars, SD (n=3). B, caspase-3 and caspase-7 activity after a 24-h
exposure to nutlin-3, relative
to a similar amount of viable vehicle-treated cells. Bars, SD
(n=3). C, flow cytometric
analysis of the apoptotic sub-G1 fraction after 0 or 8 µM
nutlin-3 for 24 h. Bars, SD (n=3). D,
flow cytometric analysis of cell cycle phase distribution after
0 or 8 µM nutlin-3 for 24 h.
Results are derived from the same three experiments as those
used for sub-G1 quantification.
E, qRT-PCR analysis of p53 target gene expression after 0 or 8
µM nutlin-3 for 24 h. Bars,
SEM of duplicate wells. F, Western blot analysis of p53,
p21WAF1/CIP1, and BAX expression
after 0 or 8 µM nutlin-3 for 24 h. β-actin is shown as loading
control.
Figure 3. Transient silencing of CDKN2A decreases the
sensitivity of IMR-32 and NGP cells
to nutlin-3. A, qRT-PCR assessment of siRNA-mediated CDKN2A
knockdown 24 h
posttransfection, using a primer pair that measures both p14ARF
and p16INK4a. Bars, SEM of
duplicate wells. B and C, effect of CDKN2A knockdown on the
nutlin-3 response. Cells were
transfected with negative control siRNA or CDKN2A siRNA and
subsequently treated with
nutlin-3 for 24, 48, and 72 h, followed by cell viability
analysis. Three independent
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experiments were performed. Dose-response curves at 24 h,
derived from a representative
experiment, are shown as an example in B. Bars, SD of duplicate
wells. RLU, relative
luminescence units. IC50 ratios at 24, 48, and 72 h, defined as
the fold change in the IC50 value
of nutlin-3 after CDKN2A knockdown relative to control
transfection and derived from the
three experiments, are shown in C. All IC50 ratios were >1,
indicating that CDKN2A silencing
suppresses the response to nutlin-3. Bars, 95% confidence
interval (CI).
Figure 4. Stable knockdown of p14ARF attenuates the response of
NGP cells to nutlin-3. A,
qRT-PCR analysis of p14ARF and p16INK4a expression in NGP cells
transduced with
lentiviruses carrying a negative control shRNA (NGP-LV-luc), an
shRNA targeting
simultaneously p14ARF and p16INK4a (NGP-LV-p14/p16), a
p14ARF-specific shRNA (NGP-LV-
p14), or a p16INK4a-specific shRNA (NGP-LV-p16). Bars, SEM of
duplicate wells. B, IC50
values as determined by cell viability assays at 24, 48, and 72
h of nutlin-3 treatment. Three
independent experiments were performed. Bars, 95% CI. C, EC50
values as determined by
caspase-3 and caspase-7 assays at 24, 48, and 72 h of nutlin-3
treatment. The EC50 value is the
half-maximal effective concentration of nutlin-3 for caspase
activation, as defined in the
“Statistical analysis” section. Two independent experiments were
performed. Bars, 95% CI.
D, qRT-PCR analysis of p53 target gene expression after 0 or 8
µM nutlin-3 for 24 h. Bars,
SEM of duplicate wells.
Figure 5. Overexpression of p14ARF increases the sensitivity of
IMR-5/75 cells to nutlin-3. A,
qRT-PCR measurement of p14ARF and p16INK4a expression in
IMR-5/75 cells stably
transfected with a tetracycline-inducible expression vector for
a negative control construct
(IMR-Tet-lacZ), p14ARF (IMR-Tet-p14), or p16INK4a (IMR-Tet-p16).
Cells were treated with 1
µg/mL tetracycline or vehicle control for 24 h. Bars, SEM of
duplicate wells. B, effect of
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p14ARF and p16INK4a overexpression on the cell viability
response to nutlin-3. Cells were
treated with 1 µg/mL tetracycline or vehicle control and
subsequently exposed to nutlin-3 for
24, 48, and 72 h, followed by cell viability analysis. IC50
ratios were determined as the fold
change in the IC50 value of nutlin-3 after tetracycline
pretreatment compared to vehicle
control. IC50 ratios in IMR-Tet-p14 cells were 1 at all nutlin-3
concentrations, indicating that
p14ARF overexpression enhances the apoptotic response to
nutlin-3. Three independent
experiments were performed. Bars, 95% CI. D, qRT-PCR analysis of
p53 target gene
expression after treatment with 1 µg/mL tetracycline or vehicle
control and subsequent
exposure to 0 or 8 µM nutlin-3 for 24 h. Bars, SEM of duplicate
wells.
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Published OnlineFirst April 1, 2011.Mol Cancer Ther Tom Van
Maerken, Ali Rihani, Daniel Dreidax, et al. using the
small-molecule MDM2 antagonist nutlin-3Functional analysis of the
p53 pathway in neuroblastoma cells
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Published OnlineFirst on April 1, 2011; DOI:
10.1158/1535-7163.MCT-10-1090
http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-10-1090http://mct.aacrjournals.org/content/suppl/2011/04/07/1535-7163.MCT-10-1090.DC1http://mct.aacrjournals.org/cgi/alertsmailto:[email protected]://mct.aacrjournals.org/content/early/2011/04/01/1535-7163.MCT-10-1090http://mct.aacrjournals.org/
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