The T cell factor/B-Catenin Antagonist PKF115-584 Inhibits Proliferation of Adrenocortical Carcinoma Cells
Mabrouka Doghman, Julie Cazareth, and Enzo Lalli
Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique Unité Mixte de Recherche 6097 and Université de Nice Sophia Antipolis, 06560 Valbonne, France
Context: Mutations of the B-catenin (CTNNB1) gene are frequently found in adrenocortical tumors. This has important consequences to deregulate the expression of transcriptional targets of the Wnt pathway, which may contribute to tumorigenesis.
Objective: The objective of the study was to investigate the effect of the small-molecule inhibitor of the T cell factor (Tcf)/B-catenin complex PKF115-584 on B-catenin-dependent transcription and proliferation of H295R adrenocortical tumor cells, which harbor mutations in CTNNB1 as well as the TP53 tumor suppressor gene.
Main Outcome Measures: Immunofluorescence, transient transfection, proliferation assays, and flow cytometric analyses were used.
Results: Nuclear localization of -catenin and constitutive activation of ß-catenin-dependent tran- scription was observed in H295R cells. PKF115-584 dose-dependently inhibited B-catenin-depen- dent transcription and H295R proliferation, even in the presence of increased steroidogenic fac- tor-1 levels, which augment proliferation in this cell line. The drug had no effect on Hela cells, a cell line in which the B-catenin pathway is not activated. PKF115-584 decreased the percentage of H295R cells in S-phase and increased the percentage of apoptotic cells.
Conclusions: Inhibitors of the Tcf/B-catenin complex may prove useful in the treatment of adre- nocortical tumors in which multiple genetic alterations have accumulated. (J Clin Endocrinol Metab 93: 3222-3225, 2008)
T he prevalence rate of adrenocortical tumors (ACT) may be as high as 1-5%. They can manifest with signs of endo- crine dysfunction, and when malignant, they have a poor prognosis. Genetic alterations present in ACTs include TP53 mutations, loss of heterozygosity at 11p15, inactivating mu- tations of PRKAR1A, and others (1, 2). Recently it has been documented that mutations of CTNNB1 (ß-catenin) are a frequent event in adrenocortical tumors, independently from their benign or malignant phenotype and their hormone se- cretion profile (3, 4). Recent data suggest that the same path- way may also be activated in mouse models of adrenocortical tumor (5, 6).
ß-Catenin is a critical transducer of the canonical Wnt sig-
naling pathway, being stabilized and translocated into the nu- cleus after release from phosphorylation by casein dependent kinase @ 1 and glycogen synthase kinase-3ß triggered by Wnt signaling. Activating mutations of the Wnt signaling pathway are found in many human cancers, in which they have an essential pathogenetic role (7). CTNNB1 mutations in ACT involve sites essential for its targeting to the proteasome and induce its accu- mulation in the nucleus, in which it interacts with Tcf transcrip- tion factors and enhances their transcriptional activity (8). Re- cently a high-throughput screening identified small molecules that antagonize the formation of T cell factor (Tcf)/B-catenin complex and inhibit growth of colon, prostate cancer, and mul- tiple myeloma cell lines (9, 10). These compounds may reveal
Abbreviations: ACT, Adrenocortical tumor; SF-1, steroidogenic factor-1; Tcf, T cell factor.
0021-972X/08/$15.00/0
doi: 10.1210/jc.2008-0247 Received February 4, 2008. Accepted May 29, 2008.
First Published Online June 10, 2008
very useful in the treatment of a variety of cancers. Here we show that one of these Tcf/ß-catenin antagonists, PKF115-584, in- hibits proliferation of the H295R human adrenocarcinoma cell line, which harbors the CTNNB1 S45P mutation along with a homozygous R72P polymorphism and a heterozygous F338L mutation in the TP53 gene (11). Moreover, PKF115-584 can override the effect of increased steroidogenic factor-1 (SF-1) lev- els on H295R proliferation (11).
Materials and Methods
Cell culture and proliferation assays
H295R cells (American Type Culture Collection, Manassas, VA) were cultured in DMEM/F-12 supplemented with 2% NuSerum (Becton Dick- inson, Oxford, UK), 1% ITS Plus (Becton Dickinson, Franklin Lakes, NJ) and antibiotics. An H295R clone overexpressing SF-1 in a doxycycline- inducible fashion (H295R TR/SF-1) was produced and maintained as de- scribed (11). HeLa cells were cultured in DMEM (4.5 g/liter glucose) sup- plemented with 10% fetal calf serum and antibiotics. To measure proliferation, cells were seeded in duplicate in 24-well plates at the density of 3 × 104 cells/well and cultured in medium without serum in the presence of the indicated concentrations of PKF115-584 (Novartis, Basel Switzer- land) and doxycycline (Sigma-Aldrich, Saint-Quentin Fallavier, France; 1 µg/ml). Cell numbers were counted after 6 d of culture (H295R cells) or 4 d of culture (HeLa). Results are indicated as the average (± SEM) of at least three independent experiments performed in duplicate.
Immunofluorescence
Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, blocked with 2% BSA, and incubated with mouse monoclonal anti-ß-catenin antibody (Transduction Laboratories, Lexing- ton, KY) and rabbit polyclonal anti SF-1 (Millipore, Bisley, UK) overnight at 4 C. Cells were washed with 0.1% Triton X-100 in PBS, incubated with highly cross-absorbed Alexa488-conjugated goat antirabbit and Alexa594- conjugated goat antimouse secondary antibodies (Invitrogen, Cergy-Pon- toise, France), washed again, and mounted in SlowFade Gold antifade (In- vitrogen). Cells were analyzed by confocal microscopy using a DMRBE instrument (Leica, Heidelberg, Germany).
Transient transfection assays
H295R cells were transfected with reporter plasmids TOPflash and its negative control FOPflash (Millipore) to monitor Tcf/B-catenin transcriptional activity, dominant-negative pANTCF4 (a kind gift of H. Clevers; Hubrect Institute, Utrecht, The Netherlands), and pCH110 as a ß-galactosidase expression vector. H295R cells were also transfected with the SF-1-dependent FATE1 promoter luciferase construct (11). Cells were transfected and luciferase assays were per- formed as described (12). Results are indicated as the average (± SEM) of at least three independent experiments performed in duplicate.
Flow cytometry and apoptosis assay
H295R cells were labeled with bromodeoxyuridine using the fluo- rescent in situ cell proliferation kit (Roche, Meylan, France), following the manufacturer’s protocol. DNA was counterstained with propidium iodide (Invitrogen), and cells were analyzed with a FACScan instrument (Becton Dickinson). Apoptosis was assayed using the terminal deoxy- nucleotidyl transferase-based in situ cell death detection kit (Roche).
Pull-down assays and immunoblots
Pull-down assays and immunoblots were performed as described (9). Briefly, bacterially expressed GST-Tcf4 (amino acids 8-54) and GST-
E2F1 (1-171) were incubated overnight at 4 C with H295R extracts in the presence of dimethylsulfoxide, 0.5 or 1 µM PKF115-584, washed three times in a buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 0.5% Nonidet P-40, and 1 mM EDTA. Bound ß-catenin and cyclin A were detected by immunoblot using specific antibodies [Transduction Laboratories and Santa Cruz Biotechnology (Santa Cruz, CA), respec- tively]. CyclinD1, c-Myc, and cyclin E were detected by immunoblot using specific antibodies (Santa Cruz) in H295R cell extracts in basal culture conditions or treated for 24 h with 0.5 or 1 µM PKF115-584.
Results
Previous reports identified the S45P mutation in the ß-catenin gene in H295R cells (3, 4). We confirmed the presence of this mutation in our own stock of cells (data not shown). The S45P mutation is predicted to produce a constitutively active ß-cate- nin. H295R cells display a strong nuclear signal for ß-catenin, which colocalizes with endogenous SF-1 (Fig. 1A). Consistently with this, activity of the ß-catenin-dependent reporter TOPflash is constitutively high in H295R/TR SF-1 cells, which overexpress SF-1 in a doxycycline-inducible fashion (11; Fig. 1B). The Tcf/ ß-catenin antagonist PKF115-584 substantially inhibits TOP- flash activity in H295R/TR SF-1 cells at the concentration of 1 IM, whereas it has only marginal effect at 0.5 MM (Fig. 1B). TOPflash activity is also down-regulated dose-dependently by dominant-negative ANTCF4. Conversely, the drug has no effect on transcription driven by the SF-1-dependent FATE1 promoter (Fig. 1B). Also, PKF115-584 has no effect on nuclear localiza- tion of ß-catenin (supplementary Fig. 1, published as supple- mental data on The Endocrine Society’s Journals Online Web site at http://jcem.endojournals.org). The drug dose-dependently in- hibited Tcf/B-catenin but not E2F/cyclin A interactions (supple- mentary Fig. 2A). Also, PKF115-584 dose-dependently inhib- ited the expression of the ß-catenin target genes cyclin D1 and c-Myc, whereas it had no effect on cyclin E expression (supple- mentary Fig. 2B). These data confirm previous findings con- cerning the mechanism of action of the PKF115-584 inhibitor targeting specifically Tcf/B-catenin interactions and ß-cate- nin-dependent transcription (9, 10).
Proliferation of H295R/TR SF-1 cells is dependent on SF-1 dos- age (11). PKF115-584 dose-dependently inhibits proliferation of H295R/TR SF-1 cells in both basal conditions and conditions of SF-1 overexpression (Fig. 1C). Conversely, PKF115-584 has no effect on proliferation of Hela cells, a cell line that lacks ß-catenin mutation and in which the ß-catenin-dependent transcription is not activated (Refs. 13, 14 and data not shown). Flow cytometric anal- ysis shows that PKF115-584 dose-dependently inhibits entry of H295R cells into S phase and increases the percentage of sub-G1 (apoptotic) cells (Fig. 2). Terminal deoxynucleotidyl transferase- mediated deoxyuridine triphosphate nick end labeling analysis con- firmed the dose-dependent activation of apoptosis by the PKF115- 584 compound (supplementary Fig. 3).
Discussion
Mutations in the CTNNB1 gene are frequently found in ad- renocortical tumors (3, 4). For this reason, we investigated the
A
B
20
1
ß-catenin
SF-1
merge
luc activity
15
0.75
10
0.50
5
0.25
0
0
FOPflash
+
FATE1 prom
TOPflash
+
+
+
+
PKF115-584 (u.M)
0
0.5
1
0
1
AN-TCF4
+
C
D
cell number (% of control)
150
cell number (% of control)
150
T
100
100
50
50
T T
0
0
DOX
+
+
+
+
PKF115-584 (u.M)
0
0.1
0.5
1
PKF115-584 (p.M)
0
0.1
0.5
1
effect of PKF115-584, an inhibitor of the Tcf/B-catenin com- plex, on adrenocortical cell proliferation using the H295R cell model. These cells are differentiated to produce mineralocor- ticoid, glucocorticoid, and androgenic steroids and harbor the S45P CTNNB1 mutation along with TP53 gene mutations (3, 4, 11). In addition, inducible overexpression of the SF-1 tran- scription factor in this cell line increases their proliferation (11). Here we have shown that inhibition of Tcf/B-catenin complex by PKF115-584 reduces H295R proliferation both in basal conditions and when SF-1 is overexpressed. No effect
was detected on proliferation of the HeLa cell line, in which the B-catenin pathway is not activated (Refs. 13, 14 and data not shown). The drug inhibited entry of H295R cells into S phase and induced their apoptosis. Our findings suggest that drugs targeting ß-catenin-dependent transcriptional activity may be useful in the treatment of adrenocortical tumors in which multiple genetic alterations have accumulated. How- ever, possible side effects of in vivo use of Wnt pathway in- hibitors (10) should be taken into consideration to develop suitable drugs for clinical use.
Region % Gated
Region % Gated
Region % Gated
R1
100
R1
100
R1
100
R2
64.13
R2
71.68
R2
70.11
BrdU
10 1
R3
21.60
BrdU
101
R3
9.72
BrdU
101
R3
7.82
R4
13.28
R4
13.99
R4
13.75
R
R5
0.50
:20
R5
3.40
R5
6.87
100
100
100
0
200
400
0
200
400
0
200
400
PI
PI
PI
0
0.5
1
PKF115-584 (u.M)
Acknowledgments
We thank Dr. H. Clevers for the gift of the pANTCF4 expression vector, Novartis Corp. for the gift of the PKF115-584 compound, and Frédéric Brau for help with confocal microscopy.
Address all correspondence and requests for reprints to: Enzo Lalli, M.D., Institut de Pharmacologie Moléculaire et Cellulaire, Centre Na- tional de la Recherche Scientifique Unité Mixte de Recherche 6097, 660 Route des Lucioles, Sophia Antipolis, 06560 Valbonne, France. E-mail: ninino@ipmc.cnrs.fr.
This work was supported by Contrat d’Interface Institut National de la Santé et de la Recherche Médicale-Centre Hospitalier Universitaire de Nice and Association Recherche sur le Cancer.
Disclosure Information: All authors have nothing to declare.
References
1. Koch CA, Pacak K, Chrousos GP 2004 The molecular pathogenesis of hered- itary and sporadic adrenocortical and adrenomedullary tumors. J Clin Endo- crinol Metab 87:5367-5384
2. Stratakis CA 2003 Genetics of adrenocortical tumors: gatekeepers, landscap- ers and conductors in symphony. Trends Endocrinol Metab 14:404-410
3. 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
4. Tadjine M, Lampron A, Ouadi L, Bourdeau I 2008 Frequent mutations of ß-catenin gene in sporadic secreting adrenocortical adenomas. Clin Endocrinol (Oxf) 8:264-270
5. Bielinska M, Genova E, Boime I, Parviainen H, Kiiveri S, Leppäluoto J, Rahman N, Heikinheimo M, Wilson DB 2005 Gonadotropin-induced ad- renocortical neoplasia in NU/J nude mice. Endocrinology 146:3975-3984
6. Bernichtein S, Petretto E, Jamieson S, Goel A, Aitman TJ, Mangion JM, Huhtaniemi IT 2008 Adrenal gland tumorigenesis after gonadectomy in mice is a complex genetic trait driven by epistatic loci. Endocrinology 149:651-661
7. Giles RH, van Es JH, Clevers H 2003 Caught up in a Wnt storm: Wnt signaling in cancer. Biochim Biophys Acta 1653:1-24
8. Katoh M, Katoh M 2007 WNT signaling pathway and stem cell signaling network. Clin Cancer Res 13:4042-4045
9. Lepourcelet M, Chen YN, France DS, Wang H, Crews P, Petersen F, Bruseo C, Wood AW, Shivdasani RA 2004 Small-molecule antagonists of the oncogenic Tcf/B-catenin protein complex. Cancer Cell 5:91-102
10. Sukhdeo K, Mani M, Zhang Y, Dutta J, Yasui H, Rooney MD, Carrasco DE, Zheng M, He H, Tai YT, Mitsiades C, Anderson KC, Carrasco DR 2007 Targeting the ß-catenin/TCF transcriptional complex in the treatment of mul- tiple myeloma. Proc Natl Acad Sci USA 104:7516-7521
11. Doghman M, Karpova T, Rodrigues GA, Arhatte M, De Moura J, Cavalli LR, Virolle V, Barbry P, Zambetti GP, Figueiredo BC, Heckert LL, Lalli E 2007 Increased steroidogenic factor-1 dosage triggers adrenocortical cell prolifera- tion and cancer. Mol Endocrinol 21:2968-2987
12. Doghman M, Arhatte M, Thibout H, Rodrigues G, De Moura J, Grosso S, West AN, Laurent M, Mas JC, Bongain A, Zambetti GP, Figueiredo BC, Auberger P, Martinerie C, Lalli E 2007 Nephroblastoma overexpressed/cys- teine-rich protein 61/connective tissue growth factor/nephroblastoma over- expressed gene-3 (NOV/CCN3), a selective adrenocortical cell proapoptotic factor, is down-regulated in childhood adrenocortical tumors. J Clin Endo- crinol Metab 92:3253-3260
13. Mikheev AM, Mikheeva SA, Liu B, Cohen P, Zarbl H 2004 A functional genomics approach for the identification of putative tumor suppressor genes: Dickkopf-1 as suppressor of HeLa cell transformation. Carcinogen- esis 25:47-59
14. Ueda M, Gemmill RM, West J, Winn R, Sugita M, Tanaka N, Ueki M, Drabkin HA 2001 Mutations of the ß- and y-catenin genes are uncommon in human lung, breast, kidney, cervical and ovarian carcinomas. Br J Cancer 85:64-68