Published OnlineFirst October 19, 2010; DOI: 10.1158/0008-5472.CAN-10-2014
Published OnlineFirst on October 19, 2010 as 10.1158/0008-5472.CAN-10-2014
Priority Report
Cancer
Research
Transcriptome Analysis Reveals that p53 and β-Catenin
Alterations Occur in a Group of Aggressive
Adrenocortical Cancers
Bruno Ragazzon1,2, Rossella Libé1,2,3,4, Sébastien Gaujoux1,2,5, Guillaume Assié1,2,3, Amato Fratticci1,2,
Pierre Launay1,2, Eric Clauser1,2, Xavier Bertagna1,2,3,4, Frédérique Tissier1,2,3,6,
Aurélien de Reyniès7, and Jérôme Bertherat1,2,3,4
Abstract
Adrenocortical carcinoma (ACC) is a rare disease with an overall poor but heterogeneous prognosis. This
heterogeneity could reflect different mechanisms of tumor development. Gene expression profiling by transcriptome analysis led to ACC being divided into two groups of tumors with very different outcomes. Somatic
inactivating mutations of the tumor suppressor gene TP53 and activating mutations of the proto-oncogene
β-catenin (CTNNB1) are the most frequent mutations identified in ACC. This study investigates the correlation
between p53 and β-catenin alterations and the molecular classification of ACC by transcriptome analysis of
51 adult sporadic ACCs. All TP53 and CTNNB1 mutations seemed to be mutually exclusive and were observed
only in the poor-outcome ACC group. Most of the abnormal p53 and β-catenin immunostaining was also found
in this group. Fifty-two percent of the poor-outcome ACC group had TP53 or CTNNB1 mutations and 60% had
abnormal p53 or β-catenin immunostaining. Unsupervised clustering transcriptome analysis of this pooroutcome group revealed three different subgroups, two of them being associated with p53 or β-catenin alterations, respectively. Analysis of p53 and β-catenin target gene expressions in each cluster confirmed a profound
and anticipated effect on tumor biology, with distinct profiles logically associated with the respective pathway
alterations. The third group had no p53 or β-catenin alteration, suggesting other unidentified molecular defects.
This study shows the important respective roles of p53 and β-catenin in ACC development, delineating subgroups of ACC with different tumorigenesis and outcomes. Cancer Res; 70(21); 8276–81. ©2010 AACR.
Introduction
Adrenocortical carcinoma (ACC) is a rare and aggressive
tumor with an overall poor prognosis: the 5-year survival rate
is generally below 35% (1, 2). Despite this, overall poor prognosis outcomes and survival vary greatly. This heterogeneity
is partially explained by the tumor stage at diagnosis, but
also probably reflects intrinsic tumor biology and different
mechanisms of tumor development.
Authors' Affiliations: 1Institut Cochin, Université Paris Descartes, CNRS
(UMR 8104); 2Inserm, U1016; 3Assistance Publique Hôpitaux de Paris,
Hôpital Cochin, Department of Endocrinology, Reference Center for
Rare Adrenal Diseases; 4INCa Comete Network; 5Assistance Publique
Hôpitaux de Paris, Hôpital Cochin, Department of Digestive and
Endocrine Surgery; 6Assistance Publique Hôpitaux de Paris, Hôpital
Cochin, Department of Pathology; and 7Programme Cartes d'Identité
des Tumeurs, Ligue Nationale Contre Le Cancer, Paris, France
Note: Supplementary data for this article are available at Cancer
Research Online (http://cancerres.aacrjournals.org/).
B. Ragazzon, R. Libé, and S. Gaujoux contributed equally to this work.
Corresponding Author: Jérôme Bertherat, Service des Maladies
Endocriniennes et Métaboliques, Hôpital Cochin, 27, rue du Faubourg
Saint-Jacques, 75014 Paris, France. Phone: 33-1-58-41-18-95;
Fax: 33-1-46-33-80-60; E-mail: [email protected].
doi: 10.1158/0008-5472.CAN-10-2014
©2010 American Association for Cancer Research.
8276
Most sporadic adrenocortical tumors (ACT) are monoclonal,
suggesting that a somatic genetic defect occurs early in tumorigenesis. Studies of rare genetic syndromes associated with ACC
[i.e., Beckwith-Wiedemann due to insulin-like growth factor II
(IGF2) overexpression, or Li-Fraumeni syndrome due to inactivating mutations of the tumor suppressor gene TP53] have
greatly facilitated progress and increased our understanding
of sporadic ACTs (3). IGF2 overexpression occurs in >85% of
ACC (4, 5). TP53 somatic mutations are present in about a third
of sporadic adult ACCs (6–8). The occurrence of a somatic TP53
mutation is associated with a worse survival (1), and allelic losses
at or around the TP53 locus (17p13) are observed in >80% of ACC
(4). The Wnt/β-catenin signaling pathway plays an important
role in adrenal cortex development (9). Immunohistochemical
studies have shown that the distribution of the β-catenin protein
is abnormal in ACC, suggesting activation of this pathway (10).
There are somatic activating mutations of CTNNB1 (β-catenin)
in several types of benign and malignant ACTs, including ACC
(10–12), and this pathway has recently been shown to play a
role in the development of ACTs in transgenic mice (13).
Studies of the genomics of ACTs have helped improve our
understanding of the pathophysiology and classification of
these tumors (14–16). Transcriptome analysis clearly shows
that the gene profile of benign ACTs differs from that of malignant ACTs (ACCs; reviewed in ref. 17). We and others have
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p53 and β-Catenin in Aggressive Adrenocortical Cancers
Figure 1. Distribution of samples between C1A and C1B and characteristics of the whole cohort. A, DFS (left) and specific OS (right) in the C1A and
C1B clusters. The P value of the log-rank test for differences between survival curves is shown. B, overall molecular characteristics of the whole ACC
cohort: TP53 and CTNNB1 mutation (black rectangles), and abnormal p53 and β-catenin immunohistochemistry (IHC; hatched gray rectangles). Clinical
annotations: specific death and metastasis or relapse (black = yes, white = no). Each molecular and clinical variable is associated with a P value
calculated using Fisher's exact test, measuring its association with the distribution of the sample.
recently reported that transcriptome analysis also identified
two groups of ACCs (14, 16), with contrasted outcome and
survival: one having a poor overall survival (OS) rate (cluster
C1A in ref. 16) and the other with a better survival rate (cluster C1B in ref. 16). The 5-year survival rates were 20% and
91% in the C1A and C1B clusters, respectively (18). This suggests that the molecular alterations occurring in these two
groups of ACC are different and that they modulate the
tumor phenotype.
We have therefore investigated a large cohort of adult
sporadic ACC to determine the relationship between the
molecular classification determined by transcriptome analysis and the two most frequent somatic genetic alterations
described in ACC: inactivating mutations of the tumor suppressor gene TP53 and activating mutations of the protooncogene CTNNB1. As we found a clear correlation between
these genetic alterations and the molecular classification,
we analyzed their effect on gene expression profiles, and
specifically the expression level of the respective target
genes of the two pathways.
Materials and Methods
Tumor samples
Fifty-one tumors (whole cohort) were prospectively
collected, and DNA and RNA were extracted as previously
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described (16) with signed informed consent and approval
by the institutional review board of Cochin Hospital.
DNA preparation, sequencing, and
immunohistochemical studies
The TP53 and CTNNB1 coding sequences of tumor DNA
were sequenced as described previously (10, 19). Immunohistochemical staining for p53 and β-catenin was done using
available paraffin-embedded tissue sections (48 of 51 tumors)
as described previously (10, 19) and considered abnormal
when positive for nuclear staining.
Expression analysis
The expression profiles of 34 ACCs derived from a previous
study (16) were performed with the HG-U133 Plus 2.0
Affymetrix GeneChip arrays. All data are available on
ArrayExpress Web site (http://www.ebi.ac.uk/arrayexpress,
experiment E-TABM-311). Unsupervised clustering analysis
of these 34 microarrays was performed as described (16).
The association between clusters and biological variables
was measured with Fisher's exact test. Prediction analysis
was performed on a series of 51 ACCs analyzed by quantitative reverse transcription-PCR (RT-PCR; ΔCt data) using
the PAM algorithm (pamr R package; ref. 20). The 34 ACCs
(21 C1A and 13 C1B) from the expression profiling series
were used to train several predictors and select a final
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Ragazzon et al.
four-gene predictor (MCM5, VEPH1, PINK1, and SLC2A1); this
was then used to classify the remaining 17 samples. Differential expression was measured with Bayes moderated t test
(limma R package). Survival curves were obtained by the
Kaplan-Meier method. Survival differences were assessed
with the log-rank test.
Results
Tumor characteristics and molecular classification
Among 51 ACCs (whole cohort), 34 were previously used
for microarray analysis (ACC microarray cohort). We could
identify two groups of ACCs with different outcomes: C1A
group had poor outcomes and C1B group had good outcomes (16). We analyzed 41 genes by quantitative RT-PCR
in the whole cohort to identify predictors of malignancy,
disease-free survival (DFS), and OS (16).
We tested the suitability of these 41 genes for classifying
the ACC microarray cohort in C1A and C1B groups. A predictor based on the combination of MCM5, VEPH1, PINK1, and
SLC2A1 genes provided the best classification of these tumors; it was therefore used to classify the remaining 17 tumors. Finally, 31 tumors were classified as C1A and 20 were
classified as C1B. As expected, the patients with C1A tumors
had very poor outcomes. DFS and OS, represented by the
Kaplan-Meier curves in Fig. 1A, were lower for the C1A group
(n = 31) than for the C1B group (n = 20; log-rank test: P = 4.91 ×
10−4 for DFS and P = 1.99 × 10−5 for OS), reinforcing our previous results (16).
Somatic TP53 and CTNNB1 mutations and
immunohistochemistry
About the whole cohort, TP53 mutations and nuclear p53
immunostaining were present in 9 (17.6%) and 13 (27%) of
the ACCs, respectively (Figs. 1B and 2A; see Supplementary
Table S1 for details). Seven of the nine ACCs with TP53 mutations had nuclear p53 immunostaining. CTNNB1 activating
mutations and abnormal β-catenin immunostaining were
present in 8 (15.7%) and 12 (25%) of the ACCs, respectively
(Figs. 1B and 2B; see Supplementary Table S1 for details).
Seven of the eight ACCs with CTNNB1 mutations showed
nuclear staining.
Interestingly, TP53 and CTNNB1 mutations, and β-catenin
nuclear staining were exclusively found in the poor-outcome
(C1A) group (29%, 25.8%, and 40% of C1A, respectively) in
comparison with the good-outcome (C1B) group (Fisher's exact test: P < 0.008, P < 0.02, and P < 0.02, respectively). p53
nuclear staining was more frequent in C1A group (36.7%)
than in C1B group (11.1%), but the difference was not statistically significant. The TP53 and CTNNB1 mutations seemed
to be mutually exclusive, only one ACC (no. 9) showed both
TP53 and CTNNB1 mutations.
Figure 2. Abnormal accumulation
of p53 and β-catenin in ACC.
A, p53 immunohistochemistry in a
tumor showing p53 overexpression
(right, dark brown nuclear staining)
and in a tumor without
overexpression (left, blue negative
nuclear staining). B, β-catenin
immunohistochemistry in a tumor
showing an abnormal/strong
cytoplasmic and nuclear
accumulation of β-catenin (right)
and in a tumor with only a
normal membrane location (left).
Magnification, ×200.
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Published OnlineFirst October 19, 2010; DOI: 10.1158/0008-5472.CAN-10-2014
p53 and β-Catenin in Aggressive Adrenocortical Cancers
Figure 3. The three subgroups of the poor-outcome (C1A) group identified by unsupervised analysis. Dendrogram of 34 ACCs based on the top 1%
(n = 547) most varying (robust coefficient of variation) probe sets using Ward linkage, and (1 − Pearson coefficient) as distance. Molecular annotations for
TP53 and CTNNB1 are the same as in Fig. 1 and are associated with a P value calculated using Fisher's exact test, measuring its association with the
assignment of the sample to C1A(p53), C1A(x), or C1A(β-catenin) for the ACC microarray cohort.
There was at least one mutation in the TP53 or CTNNB1
genes in 52% of the poor-outcome (C1A) group, and 60%
of tumors had abnormal p53 and/or β-catenin immunostaining. But 32.2% of these C1A tumors had neither mutations nor abnormal immunohistochemistry.
Subclassification of the poor-outcome ACC group
(C1A cluster) by gene profiling
We carried out a specific unsupervised clustering analysis
of the C1A group of the ACC microarray cohort to understand
more clearly the molecular genetics of the poor-outcome ACC
group. Hierarchical clustering identified three subgroups (Fig. 3).
TP53 mutations and β-catenin nuclear staining were
clearly associated with this molecular subclassification
(Fisher's exact test: P < 0.02 and P < 0.001, respectively):
C1A(p53) group contained all tumors with a TP53 mutation
and all tumors of C1A(β-catenin) group had β-catenin nuclear
staining. We found no association with the third group [C1A(x)].
We investigated the associations of TP53 mutations and
β-catenin nuclear staining with clustering effect target gene
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expression by gene set enrichment analysis for p53 and Wnt/
β-catenin pathways. We compared each subgroup with C1B
tumors. The p53 signaling pathway was enriched in C1A(p53)
(P < 0.006) and the Wnt/β-catenin signaling pathway was
enriched in C1A(β-catenin) (P < 0.005; Supplementary Table S3).
Moreover, global expression of p53-positive target genes was
altered in the C1A(p53) subgroup (Supplementary Table S3;
P < 0.05). Thirty-four percent of p53 target genes were altered;
in particular, RRM2B, TP53INP1, and MDM2 were strongly
diminished (Fig. 4A). Similarly, global expression of β-catenin–
positive target genes was altered in C1A(β-catenin) (Supplementary Table S3; P < 0.005). Fifty-eight percent of β-catenin
target genes were altered; in particular, CLDN1, AXIN2, and
LGR5 were strongly increased (Fig. 4B).
Discussion
Gene profiling allowed new classifications of cancers, unrevealing new genetic alterations. We and others have used
transcriptome analysis to develop a new classification of
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Ragazzon et al.
Figure 4. Expression of p53- and
β-catenin–positive target genes
in each C1A subgroup. A and
B, three examples of p53 targets
(RRM2B, TP53INP1, and MDM2)
and three examples of β-catenin
targets (CLDN1, AXIN2, and LGR5).
Each panel contains three box plots
representing the distributions of
the log intensity values (microarray
data) for the following groups
of ACC: C1A(p53) (red box),
C1A(β-catenin) (green box), and
C1B (gray box). *, P < 0.05;
**, P < 0.01; ***, P < 0.001
(limma test).
these rare and clinically heterogeneous ACC tumors, with
clinical relevance, for diagnosis and prognosis (14, 16).
We now identify a subgroup of poor-outcome ACCs (C1A)
that are associated with frequent somatic genetic alterations
observed in these tumors: TP53 inactivating mutations and
CTNNB1 activating mutations, which would be late event
promoting the development of aggressive tumors. Moreover,
these mutated ACCs represent the majority of the C1A group.
p53 and β-catenin immunohistochemistry suggest that other
genetic alterations influence the protein profile, as previously
suggested (10, 19). More importantly, unsupervised cluster
analysis showed that the gene profiles of the tumors with
p53 and β-catenin alterations were different, which supports
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Cancer Res; 70(21) November 1, 2010
this molecular classification and its effect on tumorigenesis.
The C1A(p53) cluster contained all the tumors with a TP53
mutations, and all the tumors in the C1A(β-catenin) cluster
had altered β-catenin pathway. The remaining tumors of this
aggressive C1A cluster were all assigned to a specific cluster
of ACC, in which there were neither p53 nor β-catenin alteration associations. This group was enriched in cell cycle and
metabolism genes (data not shown). This suggests that there
are other molecular defects that remain to be identified. By
the same token, the tumors in the C1A(p53) and C1A(β-catenin)
clusters in which no genetic alterations were identified might
harbor genetic or epigenetic alterations of the genes controlling the same pathways.
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p53 and β-Catenin in Aggressive Adrenocortical Cancers
Analysis of the expression of the p53 and β-catenin target
genes in their respective clusters clearly showed that these
alterations have a major influence on tumor biology. Some
of these target genes (Supplementary Table S2) have been implicated in oncogenesis. The fact that the changes in the expressions of these genes are specific to each C1A subcluster
indicates that the tumor biology and cancer development mechanisms of these subclusters are different. Clinically, all these
tumors are associated with a poor outcome. This agrees well
with previous observations about TP53 mutations in ACC (1).
It also suggests that alterations to the Wnt/β-catenin pathway
are a poor prognostic factor in ACC. The IGF2 overexpression
that occurs in >85% of ACC (see review in ref. 3) is widespread
among both the C1A and C1B ACCs (data not shown). Therefore, although IGF2 is important in adrenal cortex oncogenesis,
additional molecular defects are needed for the development
of the most aggressive tumors.
This study provides new insights into the molecular classification of ACC. It points to the major effect of TP53 and
CNNTB1 mutations on tumor biology. This approach should
help identify new genetic alterations that occur in ACC without
mutations of these two genes. This new vision of the molecular classification of ACC should also guide the development of
new therapeutic strategies by identifying those tumors most
likely to respond to drugs targeting specific signaling pathways.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
We thank Dr. Owen Parkes for editing the English text.
Grant Support
Programme Hospitalier de Recherche Clinique grant PHRC060251, Recherche
Translationnelle DHOS/INCA 2009 grant RTD09024, and Contrat d'Initiation à la
Recherche Clinique grant CIRC05045. B. Ragazzon was the recipient of fellowships from the Conny-Maeva foundation and S. Gaujoux was the recipient of a
research fellowship from the Fond d'Etude et de Recherche du Corps Medical,
AP-HP, Paris, France.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 06/04/2010; revised 08/09/2010; accepted 08/31/2010; published
OnlineFirst 10/19/2010.
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Published OnlineFirst October 19, 2010; DOI: 10.1158/0008-5472.CAN-10-2014
Transcriptome Analysis Reveals that p53 and β-Catenin
Alterations Occur in a Group of Aggressive Adrenocortical
Cancers
Bruno Ragazzon, Rossella Libé, Sébastien Gaujoux, et al.
Cancer Res Published OnlineFirst October 19, 2010.
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