Characterization of Differential Gene Expression in Adrenocortical Tumors Harboring B-Catenin (CTNNB1) Mutations
Julien Durand, Antoine Lampron, Tania L. Mazzuco, Audrey Chapman, and Isabelle Bourdeau
Division of Endocrinology, Department of Medicine, Research Centre, Centre Hospitalier de l’Université de Montréal, Montreal, Quebec, Canada
Background: Mutations of -catenin gene (CTNNB1) are frequent in adrenocortical adenomas (AA) and adrenocortical carcinomas (ACC). However, the target genes of B-catenin have not yet been identified in adrenocortical tumors.
Objective: Our objective was to identify genes deregulated in adrenocortical tumors harboring CTNNB1 genetic alterations and nuclear accumulation of B-catenin.
Methods: Microarray analysis identified a dataset of genes that were differently expressed be- tween AA with CTNNB1 mutations and wild-type (WT) tumors. Within this dataset, the expression profiles of five genes were validated by real time-PCR (RT-PCR) in a cohort of 34 adrenocortical tissues (six AA and one ACC with CTNNB1 mutations, 13 AA and four ACC with WT CTNNB1, and 10 normal adrenal glands) and two human ACC cell lines. We then studied the effects of suppress- ing B-catenin transcriptional activity with the T-cell factor/B-catenin inhibitors PKF115-584 and PNU74654 on gene expression in H295R and SW13 cells.
Results: RT-PCR analysis confirmed the overexpression of ISM1, RALBP1, and PDE2A and the down- regulation of PHYHIP in five of six AA harboring CTNNB1 mutations compared with WT AA (n = 13) and normal adrenal glands (n = 10). RALBP1 and PDE2A overexpression was also confirmed at the protein level by Western blotting analysis in mutated tumors. ENC1 was specifically overex- pressed in three of three AA harboring CTNNB1 point mutations. mRNA expression and protein levels of RALBP1, PDE2A, and ENC1 were decreased in a dose-dependent manner in H295R cells after treatment with PKF115-584 or PNU74654.
Conclusion: This study identified candidate genes deregulated in CTNNB1-mutated adrenocortical tumors that may lead to a better understanding of the role of the Wnt-B-catenin pathway in adrenocortical tumorigenesis. (J Clin Endocrinol Metab 96: E1206-E1211, 2011)
M utations of B-catenin (CTNNB1) are found in 15-26.9% of adrenocortical adenomas (AA) (1-3) and in up to 30.8% of adrenocortical carcinomas (ACC) (1, 3).
Glycogen synthase kinase-3ß (GSK-3ß) forms a com- plex with two ß-catenin-binding proteins, adenoma- tous polyposis coli, and axin. In the absence of WNT signaling, ß-catenin is recruited into a complex that fa-
cilitates its phosphorylation by casein kinase 1 and ac- tivates GSK-3ß, making ß-catenin available for degra- dation (4). In contrast, WNT activation inhibits GSK-3ß activity; consequently, ß-catenin accumulates and forms complexes with T-cell factor (TCF)/lymphoid en- hancer factor proteins in the nucleus. CTNNB1 mutations affect specific serine and threonine residues localized in exon 3 that are essential for the targeted degradation of B-catenin.
Abbreviations: AA, Adrenocortical adenoma; ACC, adrenocortical carcinoma; DMSO, di- methylsulfoxide; GSK-3ß, glycogen synthase kinase-30; NA, normal adrenal gland; RT- PCR, real-time PCR; WT, wild type.
Printed in U.S.A.
Copyright @ 2011 by The Endocrine Society
doi: 10.1210/jc.2010-2143 Received September 22, 2010. Accepted April 19, 2011.
First Published Online May 11, 2011
The TCF/B-catenin antagonist PKF115-584 inhibits the pro- liferation of H295R ACC cells, supporting a role of WNT signaling in adrenocortical tumors (5). Furthermore, consti- tutive activation of ß-catenin in the adrenal cortex of trans- genic mice leads to benign aldosterone-secreting tumor de- velopment and promotes malignancy (6).
Nuclear ß-catenin binds to TCF/lymphoid enhancer fac- tor family members and trans-activates its target genes, such as c-Myc and cyclin D1 in colon cancer (7). ß-catenin target genes are unknown in adrenocortical tumors. To identify genes linked to CTNNB1 mutations, we investigated the gene ex- pression profiles of AA harboring CTNNB1 mutations com- pared with wild-type (WT) tumors. Within the dataset, we studied the expression of five genes that were differently ex- pressed between mutated and nonmutated tumors. More- over, we report the expression variation of these genes after treatment with TCF/B-catenin inhibitors in the CTNNB1- mutated ACC H295R cell line.
Materials and Methods
Adrenocortical tissues and human adrenocortical cancer cell lines
We studied 34 adrenocortical tissues, including three AA with CTNNB1 missense mutations [two cortisol-secreting (S45P and S37C) and one aldosterone-secreting (T41A)], three AA with CTNNB1 deletion mutations [two cortisol-secreting (26,943 del 55 bp and 27,127 del 6 bp) and one aldosterone-secreting (26,995 delExon3 (271 bp)], 13 WT AA (five aldosterone-se- creting and eight cortisol-secreting), five ACC (one with CT- NNB1 S45P mutation and four WT), and 10 normal adrenal glands (NA). A pool of commercially available RNA isolated from 62 NA (Clontech Laboratories, Palo Alto, CA) was also investigated. The study was approved by the Institutional Ethics Committee of Centre Hospitalier de l’Université de Montréal, and all patients provided informed, written consent. The human ACC cell lines SW13 and H295R were obtained from the Amer- ican Type Culture Collection (Manassas, VA). H295R cells har- bor an activating missense mutation in exon3 of the CTNNB1 gene (S45P) (1, 2).
Real-time PCR (RT-PCR) validation of five genes differentially expressed in adrenocortical tumors harboring CTNNB1 mutations
Microarray analysis identified a dataset of genes differentially expressed in three AA with CTNNB1 point mutations, showing high nuclear ß-catenin accumulation compared with four AA with WT CTNNB1 and no nuclear accumulation ß-catenin (Supple- mental Methods, published on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org). The dataset was deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE28476 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE28476).
Within this dataset, the expression profiles of five genes were validated in a cohort of 34 adrenocortical tissues and two human ACC cell lines by RT-PCR with Quantitect SYBR
green RT-PCR kit (QIAGEN, Valencia, CA) and Rotor-Gene 6000 cycler (Corbett Research, Sydney, New South Wales, Australia). Primer sequences are described in Supplemental Table 1. An initial denaturation step of 5 min at 95 C was followed by 45 cycles of 95 C for 30 sec, 60 C for 30 sec, and 72 C for 30 sec. Standard curves were charted to confirm 100% efficiency, and melting curve analysis was conducted. mRNA levels were normalized to 18S rRNA. The Pfaffl method (8) normalized expression values with the pool of NA. For each tissue, RT-PCR was repeated at least twice for two independent RNA extractions or three times when only one RNA extraction was available. Averages were calculated, and error was computed from SD. One-way ANOVA and Bonferroni’s multiple- comparison test ascertained statistical significance with a cutoff P value of 0.05.
Protein extraction/immunoblotting
All protein extractions were performed by lysing cells/tissues in RIPA protein lysis buffer on ice for 40 min and then spinning at 15,000 rpm for 15 min before Western blotting was under- taken as described previously (2). Blots were probed with mouse anti-ß-catenin (1:2000) (BD Transduction Laboratories, Frank- lin Lakes, NJ), mouse anti-actin (1:2000) (Sigma-Aldrich, St. Louis, MO), mouse anti-ralbp1 (1:500), mouse anti-pde2a (1: 1000), and mouse anti-enc1 (1:300) (Abnova, Taipei City, Tai- wan) antibodies. Blots were probed with antimouse horseradish peroxidase (Sigma) at 1:5,000-10,000 for 1 h and developed with enhanced chemiluminescence Western blotting detection reagent (GE Healthcare, London, UK).
Treatment of ACC cell lines with antagonists of TCF/B-catenin protein complex
H295R cells were plated at 700,000 cells per well in six-well plates. They were then treated with PKF115-584, generously provided by Novartis (Basel, Switzerland) (1, 2.5, 5, and 10 µM), or PNU74654 (Sigma) (3, 30, and 100 µM) at a final dimethyl- sulfoxide (DMSO) concentration of 0.1% or with DMSO alone (vehicle). After 24 h of treatment, the cells were collected and RNA or protein was extracted.
Results
RT-PCR differential expression of five genes in adrenocortical tumors harboring CTNNB1 mutations
RT-PCR studies confirmed the up-regulation of ISM1 (isthmin1, zebrafish homolog), RALBP1 (RalA- binding protein 1), and PDE2A (phosphodiesterase 2A, cGMP-stimulated) in five of six adrenocortical tumors with CTNNB1 mutations compared with either WT adenomas (P < 0.001) or NA (P < 0.001) (Fig. 1A). It is noteworthy that the sample harboring the 26,943 del 55 bp CTNNB1 deletion presented an opposing trend of ex- pression for the five genes, which is not surprising because, in contrast to the other samples, it showed only very weak cytoplasmic/nuclear accumulation of ß-catenin. Because
A
110
B
Mutant AA
Relative ISMI expression
90
WT AA
DM
PM
NA
70
50
1
2
del6bp
Del
S37C
S45P
Exon3
1
2
20
RALBP1
10
.
0
NA
W TAA
PM
DM
WT
PM
Mutant Adenomas
ACC
ß-actin
100
Relative ENC1 expression
0
10
PDE2A
1
ß-actin
0.1
NA
WTAA
PM
DM
WT
PM
Mutant Adenomas
ACC
Relative RALBP1 expression
6
C
RALBP1
PDE2A
5
6-
4
Relative protein levels
3
5-
2
4-
1
3-
0
NA
WTAA
PM
DM
WT
PM
2-
Mutant Adenomas
ACC
1-
14
Relative PDE2A expression
13
12
0
11
NA
WT AA
Mutant AA
WT ACC
Mutant ACC
10
9
Tissue type
7
6
5
4
0
3
2
L
0
NA
WTAA
PM
DM
WT
PM
Mutant Adenomas
ACC
Relative PHYHIP expression
2
1
0.5
0.4
0.3
0.2
0.1
0.0
NA
W TAA
PM
DM
W T
PM
Mutant Adenomas
ACC
of this different pattern, it was excluded from the follow- ing analysis calculating mean expression.
RALBP1was overexpressed in AA with CTNNB1 mu- tations (n = 5) by a mean factor of 3.6 ± 1.6-fold and 3.7 ± 1.7-fold, compared with NA and WT AA (P < 0.001). PDE2A was overexpressed in comparison with NA (7.6 ± 4) and WT AA (8 ±3.9) (P <0.001) as was ISM1 (43.6 ± 30.8) (P <0.001). ENC1 (ectodermal-neu-
ral cortex 1) was significantly overexpressed only in AA harboring missense mutations, which are mutated sam- ples showing higher nuclear accumulation of ß-catenin (26.12 ± 12.2) (P < 0.001). PHYHIP (phytanoyl-CoA 2-hydroxylase-interacting protein) was underexpressed in tumors with CTNNB1 mutations compared with NA tis- sues (-8.94 ±6.9-fold, P<0.001) and WT AA (-6.46 ± 5.36-fold, P < 0.001).
The ACC sample with CTNNB1 point mutation over- expressed ISM1, PDE2A, RALBP1, and ENC1 compared with WT AA and ACC and NA (63.0, 4.5, 2.2, and 18.0) (Fig. 1). PHYHIP levels were reduced in ACC harboring CTNNB1 point mutation compared with WT AA (86.7- fold) and WT ACC (3.2-fold).
Western blotting analysis of RALBP1 and PDE2A in adrenocortical tumors
Western blotting analysis confirmed RALBP1 protein overexpression in four of five AA with CTNNB1 muta- tions (3.9 ± 0.7-fold) and in one of one mutated ACC (4.7 ± 0.3-fold). Interestingly, RALBP1 protein levels were elevated in four of four WT ACC (3.1 ± 0.7-fold) and detected at lower levels in WT AA (2 ± 0.6-fold, n = 5) compared with NA (n = 6). PDE2A was up-regulated in mutated AA (five of five) (4.17 ± 0.83-fold) and ACC (one of one) (5.0-fold) but not in WT AA and ACC.
Antagonists of TCF/ß-catenin complex dose- dependently inhibit the expression of selected genes in human adrenocortical cell lines
To support the hypothesis that the five genes studied were functionally related to CTNNB1 mutations, we evaluated the effects of suppressing ß-catenin transcrip- tional activity with antagonists of TCF/B-catenin com- plex, PKF115-58, and PNU74654, in SW13 and H295R (CTNNB1 S45P mutation) cell lines. SW13 cells were considered as controls because they harbor no CTNNB1 mutation (personal data) and express low amounts of B-catenin (Supplemental Fig. 1). ISM1, RALBP1, PDE2A, and ENC1 genes were up-regulated in the H295R ACC cell line (40.6-, 4.8-, 5.3-, and 3.6-fold, respectively) but not in SW13 cells compared with NA (Supplemental Fig. 2). PHY- HIP was very poorly expressed in both cell lines and was not further studied.
In H295R cells, ISM1 showed a dose-dependent de- crease in expression after treatment (PKF115-584, 88 ± 1.1%; PNU74654, 74.1 ± 4.3%) (Fig. 2). Protein in- hibition of RALBP1, PDE2A, and ENC1 varied from 61.8-65% with PKF115-584 and from 19.9-73% with PNU74654 treatment. As controls, we studied AXIN2, a marker of ß-catenin/TCF activity (9), which was re- duced by PKF115-584 (41.3 ± 4.9%) and PNU7654 (47.3 ± 8.2%). In SW13 cells, ISM1, PHYHIP, and PDE2A expression was not determined because of their very low to undetectable levels (Supplemental Fig. 2).
Discussion
Deregulation of ß-catenin results in the constitutive for- mation of TCF/B-catenin complexes and in the altered
expression of TCF-regulated target genes, such as MYC (10) and CCND1 (7), as reported in colon cancer. No studies evaluating presumptive target genes of ß-catenin in human adrenocortical tumors have yet been performed. Using microarray, we initially compared the expression profiles of AA with CTNNB1 mutations with AA with WT CTNNB1. Our approach was previously validated in Wilms’ tumors (11) and endometrioid adenocarcinomas (12) to find new target genes of Wnt/B-catenin. From our preliminary study, we identified five genes as differentially expressed in adrenocortical tumors with CTNNB1 muta- tions. Up-regulation of the ISM1 gene supports ß-catenin involvement in ISM1 transcription, as reported previously in zebrafish (13). Interestingly, RALBP1, which mediates drug resistance in cancers, was overexpressed; RALBP1 down- regulation leads to decreased drug resistance but also to apoptosis and regression of lung and prostate tumors in xenograft models (14). PDE2A, which is a dual-function phosphodiesterase capable of both cGMP and cAMP hy- drolysis (15), was overexpressed in mutated tumors. We confirmed the up-regulation of ENC1 gene in AA and ACC with point mutations, as in four of five WT ACC by RT-PCR. However, tumors with deletion mutations did not show ENC1 overexpression. ENC1 is known to be regulated by TCF/B-catenin in colorectal carcinoma and has recognized ß-catenin/TCF-responsive upstream regu- latory sequences (16). The absence of up-regulation in tissues with deletion mutations may be explained by the fact that various types of mutations do not elicit equivalent stabilization of ß-catenin and transcriptional activation of Wnt target genes (17). PHYHIP was underexpressed in all adrenocortical tumors with CTNNB1 mutations but not in WT AA and in all ACC. This is consistent with a recent transcriptome-profiling study of Giordano et al. (18) who described PHYHIIP down-regulation in ACC. The find- ing of a similar pattern of ENC1 and PHYHIP expression in all ACC may not be surprising because Wnt/ß-catenin signaling is known to be more frequent in ACC than in AA independently of CTNNB1 mutational status (1). Similarly, CTNNB1 mutations have been found to be associated with less differentiated nonsecreting and cortisol-secreting adeno- mas (19). These observations may support common expres- sion characteristics of tumors with CTNNB1 missense mu- tations and ACC.
We then tested PKF115-584 and PNU74654. Com- pound PKF115-584 alters TCF/B-catenin transcriptional activity in H295R cells (5) and other cancer models (20) by interfering with TCF/B-catenin interaction. We demon- strated that both antagonists down-regulated the overex- pressed ISM1, RALBP1, PDE2A, and ENC1 genes.
This study is a first attempt to detect genes specifically deregulated in CTNNB1-mutated adrenocortical tumors.
| PNU74654 (LM) | PKF115-584 (IM) | |
|---|---|---|
| Vehicle | 30 100 | 1 5 |
A
125-
125
% ISM1 expression
% ISM1 expression
B
100-
100
75-
75-
50
50-
10
25-
25-
0
Vehicle
3.3
30
100
0
Vehicle
1
2.5
5
10
PNU74654 ( AM)
PKF115-584 ( M)
RALBP1
125-
125
% RALBP1 expression
100-
% RALBP1 expression
100-
75-
75-
50-
50-
ß-catenin
25-
25-
0
Vehicle
3.3
30
100
0
PNU74654 ( AM)
Vehicle
1
2.5
5
10
PKF115-584 ( µM)
ENC1
125
125-
% PDE2A expression
100
% PDE2A expression
100-
75-
75-
50
50-
25-
25-
0
0
PDE2A
Vehicle
3.3
30
100
Vehicle
1
2.5
5
10
PNU74654 ( M)
PKF115-584 (AM)
125-
125-
% ENC1 expression
100-
% ENC1 Expression
100-
ß-actin
75-
75
50
50-
25-
25-
0
Vehicle
3.3
30
100
0
Vehicle
1
2.5
5
10
PNU74654 ( AM)
PKF115-584 (AM)
C
| Summary of Results | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Genes | Tissues expression | PKF115-584 inhibitory effect at 10uM after 24 h | PNU 74654 inhibitory effect at 100uM after 24 h | |||||||||
| mRNA | Protein | mRNA | Protein | mRNA | Protein | |||||||
| WT AA | MAA | WT ACC | M ACC | WT AA | MAA | WT ACC | M ACC | |||||
| ISM1 | 12/13 | 1 5/6 | 12/4 | 11/1 | NA | NA | NA | NA | 88.5±0.66% | NA | 74.12 ± 3.31 | NA |
| PDE2A | 1 0/13 | 1 5/6 | 10/4 | 11/1 | 1 0/5 | 1 5/6 | 10/4 | 11/1 | 34 ± 5.02% | 64.1 ± 9.54% | 50 ± 14% | 46.5± 9% |
| ENC1 | 11/13 | 1 3/6 | 13/4 | 11/1 | NA | NA | NA | NA* | 41.8 ± 15% | 61.8 ± 20.8% | 55.15 ±6.77% | 73 ± 14.7% |
| RALBP1 | 11/13 | 1 5/6 | 10/4 | 11/1 | 3/51 | 1 4/6 | 4/4 | 11/1 | 42.1 ±2.5% | 65 ± 9% | 38.1 ± 5% | 19.9 ±0.4% |
| PHYHIP | 41/13 | 15/6 | 14/4 | Į1/1 | NA | NA | NA | NA | No effect | NA | No | NA |
FIG. 2. A, Effects of TCF/B-catenin inhibitors on gene expression of ISM1, RALBP1, PDE2A, and ENC1 in H295R cells studied by RT-PCR. The cells were treated with PKF115-584 (left panels) (1, 2.5, 5 and 10 µM), PNU74654 (right panels) (3.3, 30, and 100 µM), or vehicle (0.1% DMSO) for 24 h. Bars represent the means of two independent experiments, each in triplicate with real-time reactions performed in duplicate. Errors are shown as percent sD. * , P ≤ 0.05; ** , P ≤ 0.01; *** , P ≤ 0.001. B, Western blotting analysis of RALBP1, B-catenin, ENC1, PDE2A, and ß-actin after 24 h treatment with PKF115-584 or PNU74654 in H295R cells. C, Table summarizing all the results at the mRNA and protein levels in various adrenocortical tissues and after treatments of H295R cells with PKF115-584 or PNU74654.
Although our data need to be validated in a larger cohort of human adrenocortical samples, they led to the identi- fication of new candidate genes that may contribute to a better understanding of the role of Wnt-ß-catenin in ad- renocortical tumorigenesis. Further investigations are re- quired to determine the functional significance of these genes in the development of AA and ACC.
Acknowledgments
We are grateful to Dr. André Lacroix, Centre Hospitalier de l’Université de Montréal, Montreal, Quebec, Canada, for providing adrenocortical samples. We thank Dr. Anne-Marie Mess-Masson
and members of her laboratory for their assistance in the immuno- histochemical studies as well as the MacDonald Stewart Founda- tion for photographic support. We also thank Mimi Tadjine who performed mutational analysis of adrenocortical tumors at the be- ginning of the project. The editing of our manuscript by Ovid Da Silva and logistical support by the Research Support Office, Centre Hospitalier de l’Université de Montréal, are acknowledged.
Address all correspondence and requests for reprints to: Isa- belle Bourdeau, M.D., Division of Endocrinology, Department of Medicine, Centre Hospitalier de l’Université de Montréal- Hôtel-Dieu, 3850 Saint Urbain Street, Montréal, Québec, Can- ada H2W 1T7. E-mail: isabelle.bourdeau@umontreal.ca.
This study was funded by Grant FRSQ-6519/5360 from Fonds de la Recherche en Santé du Québec (to I.B.) and The Cancer Research Society (to I.B.).
Disclosure Summary: The authors have nothing to disclose.
References
1. Tissier F, Cavard C, Groussin L, Perlemoine K, Fumey G, Hagneré AM, René-Corail F, Jullian E, Gicquel C, Bertagna X, Vacher- Lavenu MC, Perret C, Bertherat J 2005 Mutations of ß-catenin in adrenocortical tumors: activation of the Wnt signaling pathway is a frequent event in both benign and malignant adrenocortical tumors. Cancer Res 65:7622-7627
2. Tadjine M, Lampron A, Ouadi L, Bourdeau I 2008 Frequent mu- tations of ß-catenin gene in sporadic secreting adrenocortical ade- nomas. Clin Endocrinol (Oxf) 68:264-270
3. Masi G, Lavezzo E, Iacobone M, Favia G, Palù G, Barzon L 2009 Investigation of BRAF and CTNNB1 activating mutations in adre- nocortical tumors. J Endocrinol Invest 32:597-600
4. Moon RT, Kohn AD, De Ferrari GV, Kaykas A 2004 WNT and B-catenin signalling: diseases and therapies. Nat Rev Genet 5:691- 701
5. Doghman M, Cazareth J, Lalli E 2008 The T cell factor/ß-catenin antagonist PKF115-584 inhibits proliferation of adrenocortical car- cinoma cells. J Clin Endocrinol Metab 93:3222-3225
6. Berthon A, Sahut-Barnola I, Lambert-Langlais S, de Joussineau C, Damon-Soubeyrand C, Louiset E, Taketo MM, Tissier F, Bertherat J, Lefrançois-Martinez AM, Martinez A, Val P 2010 Constitutive B-catenin activation induces adrenal hyperplasia and promotes ad- renal cancer development. Hum Mol Genet 19:1561-1576
7. Tetsu O, McCormick F 1999 ß-Catenin regulates expression of cy- clin D1 in colon carcinoma cells. Nature 398:422-426
8. Pfaffl MW 2001 A new mathematical model for relative quantifi- cation in real-time RT-PCR. Nucleic Acids Res 29:e45
9. Jho EH, Zhang T, Domon C, Joo CK, Freund JN, Costantini F 2002 Wnt/ß-catenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway. Mol Cell Biol 22:1172- 1183
10. He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da Costa LT, Morin PJ, Vogelstein B, Kinzler KW 1998 Identification of c-MYC as a target of the APC pathway. Science 281:1509-1512
11. Zirn B, Samans B, Wittmann S, Pietsch T, Leuschner I, Graf N,
Gessler M 2006 Target genes of the WNT/B-catenin pathway in Wilms tumors. Genes Chromosomes Cancer 45:565-574
12. Schwartz DR, Wu R, Kardia SL, Levin AM, Huang CC, Shedden KA, Kuick R, Misek DE, Hanash SM, Taylor JM, Reed H, Hendrix N, Zhai Y, Fearon ER, Cho KR 2003 Novel candidate targets of ß-catenin/T-cell factor signaling identified by gene expression pro- filing of ovarian endometrioid adenocarcinomas. Cancer Res 63: 2913-2922
13. Weidinger G, Thorpe CJ, Wuennenberg-Stapleton K, Ngai J, Moon RT 2005 The Sp1-related transcription factors sp5 and sp5-like act downstream of Wnt/ß-catenin signaling in mesoderm and neuroec- toderm patterning. Curr Biol 15:489-500
14. Vatsyayan R, Lelsani PC, Awasthi S, Singhal SS 2010 RLIP76: a versatile transporter and an emerging target for cancer therapy. Biochem Pharmacol 79:1699-1705
15. Rosman GJ, Martins TJ, Sonnenburg WK, Beavo JA, Ferguson K, Loughney K 1997 Isolation and characterization of human cDNAs encoding a cGMP-stimulated 3’,5’-cyclic nucleotide phosphodies- terase. Gene 191:89-95
16. Fujita M, Furukawa Y, Tsunoda T, Tanaka T, Ogawa M, Naka- mura Y 2001 Up-regulation of the ectodermal-neural cortex 1 (ENC1) gene, a downstream target of the ß-catenin/T-cell factor complex, in colorectal carcinomas. Cancer Res 61:7722-7726
17. Provost E, Yamamoto Y, Lizardi I, Stern J, D’Aquila TG, Gaynor RB, Rimm DL 2003 Functional correlates of mutations in ß-catenin exon 3 phosphorylation sites. J Biol Chem 278:31781-31789
18. Giordano TJ, Kuick R, Else T, Gauger PG, Vinco M, Bauersfeld J, Sanders D, Thomas DG, Doherty G, Hammer G 2009 Molecular classification and prognostication of adrenocortical tumors by tran- scriptome profiling. Clin Cancer Res 15:668-676
19. Bonnet S, Gaujoux S, Launay P, Baudry C, Chokri I, Ragazzon B, Libé R, René-Corail F, Audebourg A, Vacher-Lavenu MC, Groussin L, Bertagna X, Dousset B, Bertherat J, Tissier F 2011 Wnt/ß-catenin pathway activation in adrenocortical adenomas is frequently due to somatic CTNNB1-activating mutations, which are associated with larger and nonsecreting tumors: a study in cortisol-secreting and -nonsecreting tumors. J Clin Endocrinol Metab 96:E419-E426
20. Gandhirajan RK, Staib PA, Minke K, Gehrke I, Plickert G, Schlösser A, Schmitt EK, Hallek M, Kreuzer KA 2010 Small molecule inhib- itors of Wnt/B-catenin/lef-1 signaling induces apoptosis in chronic lymphocytic leukemia cells in vitro and in vivo. Neoplasia 12:326- 335