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The T cell factor/β-Catenin Antagonist PKF115–584 Inhibits Proliferation of Adrenocortical Carcinoma Cells

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

Address all correspondence and requests for reprints to: Enzo Lalli, M.D., Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique Unité Mixte de Recherche 6097, 660 Route des Lucioles, Sophia Antipolis, 06560 Valbonne, France. E-mail: ninino{at}ipmc.cnrs.fr.

	Abstract

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Context: Mutations of the β-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)/β-catenin complex PKF115–584 on β-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 β-catenin and constitutive activation of β-catenin-dependent transcription was observed in H295R cells. PKF115–584 dose-dependently inhibited β-catenin-dependent transcription and H295R proliferation, even in the presence of increased steroidogenic factor-1 levels, which augment proliferation in this cell line. The drug had no effect on HeLa cells, a cell line in which the β-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/β-catenin complex may prove useful in the treatment of adrenocortical tumors in which multiple genetic alterations have accumulated.

	Introduction

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The prevalence rate of adrenocortical tumors (ACT) may be as high as 1–5%. They can manifest with signs of endocrine dysfunction, and when malignant, they have a poor prognosis. Genetic alterations present in ACTs include TP53 mutations, loss of heterozygosity at 11p15, inactivating mutations of PRKAR1A, and others (1, 2). Recently it has been documented that mutations of CTNNB1 (β-catenin) are a frequent event in adrenocortical tumors, independently from their benign or malignant phenotype and their hormone secretion profile (3, 4). Recent data suggest that the same pathway may also be activated in mouse models of adrenocortical tumor (5, 6).

β-Catenin is a critical transducer of the canonical Wnt signaling pathway, being stabilized and translocated into the nucleus after release from phosphorylation by casein dependent kinase 1 and glycogen synthase kinase-3β 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 accumulation in the nucleus, in which it interacts with Tcf transcription factors and enhances their transcriptional activity (8). Recently a high-throughput screening identified small molecules that antagonize the formation of T cell factor (Tcf)/β-catenin complex and inhibit growth of colon, prostate cancer, and multiple myeloma cell lines (9, 10). These compounds may reveal very useful in the treatment of a variety of cancers. Here we show that one of these Tcf/β-catenin antagonists, PKF115–584, inhibits 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) levels on H295R proliferation (11).

	Materials and Methods

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Cell culture and proliferation assays

H295R cells (American Type Culture Collection, Manassas, VA) were cultured in DMEM/F-12 supplemented with 2% NuSerum (Becton Dickinson, 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 described (11). HeLa cells were cultured in DMEM (4.5 g/liter glucose) supplemented with 10% fetal calf serum and antibiotics. To measure proliferation, cells were seeded in duplicate in 24-well plates at the density of 3 x 10⁴ cells/well and cultured in medium without serum in the presence of the indicated concentrations of PKF115–584 (Novartis, Basel Switzerland) 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-β-catenin antibody (Transduction Laboratories, Lexington, 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-Pontoise, France), washed again, and mounted in SlowFade Gold antifade (Invitrogen). 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/β-catenin transcriptional activity, dominant-negative pNTCF4 (a kind gift of H. Clevers; Hubrect Institute, Utrecht, The Netherlands), and pCH110 as a β-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 performed 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 fluorescent 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 deoxynucleotidyl 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 β-catenin and cyclin A were detected by immunoblot using specific antibodies [Transduction Laboratories and Santa Cruz Biotechnology (Santa Cruz, CA), respectively]. 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

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Previous reports identified the S45P mutation in the β-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 β-catenin. H295R cells display a strong nuclear signal for β-catenin, which colocalizes with endogenous SF-1 (Fig. 1A). Consistently with this, activity of the β-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/β-catenin antagonist PKF115–584 substantially inhibits TOPflash activity in H295R/TR SF-1 cells at the concentration of 1 µM, whereas it has only marginal effect at 0.5 µM (Fig. 1B). TOPflash activity is also down-regulated dose-dependently by dominant-negative NTCF4. 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 localization of β-catenin (supplementary Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://jcem.endojournals.org). The drug dose-dependently inhibited Tcf/β-catenin but not E2F/cyclin A interactions (supplementary Fig. 2A). Also, PKF115–584 dose-dependently inhibited the expression of the β-catenin target genes cyclin D1 and c-Myc, whereas it had no effect on cyclin E expression (supplementary Fig. 2B). These data confirm previous findings concerning the mechanism of action of the PKF115–584 inhibitor targeting specifically Tcf/β-catenin interactions and β-catenin-dependent transcription (9, 10).

View larger version (16K): [in this window] [in a new window]   FIG. 1. PKF115–584 inhibits Tcf/β-catenin-dependent transcription and proliferation of adrenocortical H295R but not of HeLa cells. A, H295R cells were stained with anti β-catenin (red) and anti-SF-1 (green) antibodies and analyzed by confocal microscopy. β-Catenin and SF-1 colocalize in the nuclear compartment (yellow) (magnification, x40). B, H295R/TR SF-1 cells were transiently transfected with TOPflash, its negative control FOPflash (left panel) reporters to monitor Tcf/β-catenin transcriptional activity and with the SF-1-dependent FATE1 promoter luciferase (luc) construct (all at 1 µg). Cells were cotransfected with pCH110 (0.5 µg) as a β-galactosidase expression vector for normalization and, where indicated, with dominant-negative pNTCF4 expression vector (1 µg). The effect of PKF115–584 was measured on TOPflash (0.5 and 1 µM) and FATE1 promoter (1 µM) activities. C, Dose-dependent (0.1, 0.5, and 1 µM) inhibition of H295R/TR SF-1 cell proliferation by PKF115–584. Doxycycline (Dox; 1 µg/ml) was added where indicated (black histograms) to increase SF-1 expression. D, No effect of PKF115–584 (0.1, 0.5, and 1 µM) on proliferation of HeLa cells.

View larger version (13K): [in this window] [in a new window]   FIG. 2. Flow cytometric analysis of cell cycle in H295R cells treated with PKF115–584. The drug was added to cultures for 4 d, and then cells were pulsed with bromodeoxyuridine (BrdU), fixed, counterstained with propidium iodide (PI), and analyzed. The percentage of cells in gate R1 (total), R2 (G1 phase), R3 (S-phase), R4 (G2/M phase), and R5 (sub-G1) is shown for each treatment.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mutations in the CTNNB1 gene are frequently found in adrenocortical tumors (3, 4). For this reason, we investigated the effect of PKF115–584, an inhibitor of the Tcf/β-catenin complex, on adrenocortical cell proliferation using the H295R cell model. These cells are differentiated to produce mineralocorticoid, 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 transcription factor in this cell line increases their proliferation (11). Here we have shown that inhibition of Tcf/β-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 β-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 β-catenin-dependent transcriptional activity may be useful in the treatment of adrenocortical tumors in which multiple genetic alterations have accumulated. However, possible side effects of in vivo use of Wnt pathway inhibitors (10) should be taken into consideration to develop suitable drugs for clinical use.

	Acknowledgments

 
We thank Dr. H. Clevers for the gift of the pNTCF4 expression vector, Novartis Corp. for the gift of the PKF115–584 compound, and Frédéric Brau for help with confocal microscopy.

	Footnotes

 
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.

First Published Online June 10, 2008

Abbreviations: ACT, Adrenocortical tumor; SF-1, steroidogenic factor-1; Tcf, T cell factor.

Received February 4, 2008.

Accepted May 29, 2008.

	References

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