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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-1381
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 1 159-168
Copyright © 2006 by The Endocrine Society

Wnt/ß-Catenin Signaling Mediates Antineoplastic Effects of Imatinib Mesylate (Gleevec) in Anaplastic Thyroid Cancer

Annavarapu Srinivas Rao, Natalia Kremenevskaja, Reinhard von Wasielewski, Vladimira Jakubcakova, Shashi Kant, Julia Resch and Georg Brabant

Departments of Gastroenterology, Hepatology, and Endocrinology (A.S.R., N.K., J.R., G.B.) and Pathology (R.v.W.), Institute of Biochemistry (S.K.), Medical School Hannover, and Max-Planck Institute for Experimental Endocrinology (V.J.), D-30625 Hannover, Germany

Address all correspondence and requests for reprints to: Prof. G. Brabant, Department of Gastroenterology, Hepatology, and Endocrinology, Medizinische Hochschule Hannover, Carl-Neuberg Strasse 1, D-30625 Hannover, Germany. E-mail: brabant.georg{at}mh-hannover.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: Dysregulation of Wnt signaling is a key step in neoplastic thyrocyte proliferation. However, it is unclear whether the selective tyrosine kinase (TK) inhibitor, imatinib mesylate, is linked to the Wnt/ß-catenin cascade and is able to modulate the pathway.

Objective: Conflicting data are reported on the therapeutic effects of imatinib in anaplastic thyroid carcinomas (ATCs), but the molecular mechanism of action is unclear. Here, we further delineated the antitumor effects and the potential efficacy of imatinib in dedifferentiated thyroid carcinomas.

Results: Tissue microarray of histologically proven ATCs (n = 12) demonstrated that six of 12 tumors expressed at least one of the imatinib-sensitive TKs. Similarily, imatinib-sensitive TKs were detected in seven of 10 thyroid cancer cell lines derived from metastatic papillary, follicular, and ATCs. Coimmunoprecipitation in ARO cells demonstrated a direct link between c-abl and ß-catenin. Imatinib (10 µM for 48 h) drastically reduced ß-catenin expression and redistributed it from the nucleus to the cell membrane. It stabilized adherens junctions by increasing ß-catenin/E-cadherin binding and reduced the invasive potential of thyroid cancer. Furthermore, imatinib (10 µM for 48 h) attenuated T cell factor/lymphoid enhancer factor activity, reduced cyclin D1 levels and dose-dependently suppressed thyrocyte proliferation by half without affecting apoptosis.

Conclusion: Our data provide a molecular mechanism for the antitumor activity of imatinib that may help to develop it as a therapeutic option in a subset of ATC patients.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANAPLASTIC THYROID CARCINOMAS (ATCs) are very aggressive and usually rapidly fatal tumors with a median survival of only 4–12 months (1, 2, 3). No established, even palliative, treatment is currently available. Local surgical tumor debulking combined with radio-chemotherapy is conventionally used, but the low success rate urges the search for new therapeutic approaches (4, 5, 6).

Tyrosine kinases (TKs) are important for the regulation of growth, differentiation, survival, and motility. Various tumors overexpress TKs or harbor activating TK mutations leading to uncontrolled mitogenic signals to the neoplastic cells (7, 8, 9, 10, 11). Lately, highly potent and selective TK inhibitors have been developed that by blocking TKs serve as an alternative to standard chemotherapy. Imatinib mesylate as an example of this novel class of drugs suppresses the TK activities of c-abl, BCR-ABL, platelet-derived growth factor receptor (PDGFR), and c-kit receptors (12, 13, 14, 15, 16). In patients with chronic-phase chronic myeloid leukemia (CML) as well as with blast crisis, imatinib induces remissions by targeting the ATP-binding site of the kinase domain of c-abl/BCR-ABL (17).

Imatinib is currently undergoing clinical trials for the treatment of patients with c-kit-positive unresectable and/or metastatic malignant gastrointestinal stromal tumors (5–7.6 µM) and Philadelphia chromosome-positive CML (0.5 µM). However, little is known about the effects of imatinib in dedifferentiated epithelial-type tumors, and the mechanism of action in these tumors is unknown (18, 19).

In epithelial cells, namely in the thyroid, ß-catenin is a multifunctional protein and has at least two distinct but interdependent functions. It binds as an intracellular stabilizer to cadherins to form the adherens junction (20, 21, 22). A number of studies show that E-cadherin binding may be regulated by various TKs associated with epidermal growth factor receptor, PDGFR, or c-met, which phosphorylate ß-catenin at position Tyr654 (23, 24, 25). This leads to dissociation of ß-catenin from E-cadherin, and by weakening adherens junctions, favors cell migration and the formation of metastases (23, 24, 26). On the other hand, ß-catenin plays a pivotal role in the canonical Wnt signaling pathway. Activation of canonical Wnt signaling stabilizes cytosolic ß-catenin, which translocates to the nucleus, stimulates T cell factor/lymphoid enhancer factor (TCF/LEF) and target genes such as cyclin D1, a mechanism substantiated by the occurrence of frequent ß-catenin mutations in the undifferentiated/ATCs (27, 28, 29, 30, 31, 32, 33, 34).

In the present study, we aimed to characterize the molecular basis of imatinib action. We specifically tried to delineate whether imatinib may modulate the ß-catenin/TCF pathway that is shown in many cell types, including the thyroid, to be of central importance for proliferation control.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Thyroid tissues

Tissues from histologically proven ATCs (n = 12) were obtained from histopathological archives of the Department of Pathology. Normal thyroid tissues (n = 11) were obtained by surgery for benign diffuse goiter or during surgery for a parathyroid adenoma. All patients involved in this study gave informed consent.

Cell culture and reagents

The human papillary (BCPAP, NPA, and TPC-1), follicular (WRO, FTC-133, and FTC-238), and anaplastic (ARO, 8505C, Hth-74, and C-643) thyroid carcinoma cell lines were grown in RPMI 1640 medium (with HEPES and L-glutamine; PAA, Cölbe, Germany) or for C-643, FTC-133, and FTC-238 cell lines in DMEM/HAM’S F-12 medium (with L-glutamine) supplemented with 10% fetal calf serum (FCS), 100 µg/ml streptomycin, and 100 U/ml penicillin. All cells were grown at 37 C in 5% CO2 and 95% air.

The following antibodies were used: anti-c-abl monoclonal antibody (mAb) (Chemicon International, Hofheim, Germany); anti-PDGFR-{alpha} and anti-c-kit mAb (Sigma, Taufkirchen, Germany); anti-ß-catenin, anti-phosphotyrosine, and anti-E-cadherin mAb (BD Transduction Laboratories, Heidelberg, Germany); anti-cyclin D1 polyclonal rabbit antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); goat antirabbit IgG alkaline phosphatase-linked secondary antibody and enhanced chemiluminescence immunoblotting detection reagents (Amersham Biosciences, Freiburg, Germany), {alpha}-tubulin mAb and sheep antimouse IgG alkaline phosphatase-linked secondary antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Imatinib was kindly provided by Novartis Pharma AG (Basel, Switzerland), and stock solutions of imatinib were prepared as 10 mM dimethylsulfoxide and stored at –20 C for future use.

Dimethyl-thiazol-diphenyltetrazolium bromide (MTT) cell proliferation assay

Growth experiments were performed in 24-well plates. Cells at 85–100% confluence were harvested with 1x trypsin/EDTA solution and seeded into a 24-well plate at 1000 cells per well and maintained in 200 µl of 6H medium in a humidified incubator. After 24 h, the cells were incubated with different concentrations of imatinib (0, 1, 2, 4, 8, or 10 µM), and the medium was changed daily. Cell proliferation was determined by MTT (Sigma) colorimetric method after culture for 48 h by the following procedure as described previously (35). Color development was determined using a spectrophotometer (Ultrospec K; Biochrom, Berlin, Germany) at a wavelength of 570 nm.

[3H]Thymidine incorporation assays

Imatinib-responsive thyroid carcinoma cells (ARO, FTC-238, and BCPAP) were plated out in flat-bottomed 96-well plates (Corning Costar, Cambridge, MA) at a density of 2 x 102 cells per well for 24 h in standard tissue culture medium. After 48 h incubation at 37 C, the cells in the 96-well plates were subjected to increasing concentrations of imatinib (0, 1, 2, 4, 8, and 10 µM). During the last 18 h, the cells were incubated with [3H]thymidine (0.5 µCi/well), and after harvesting, the incorporated radioactivity (counts per minute) reflecting proliferation of the cells was measured in a ß-counter (LKB Wallac, Turku, Finland).

Caspase-3/7 apoptosis assay

Apoptosis was measured by Apo-ONE homogeneous caspase-3/7 assay according to the manufacturer’s protocol (Promega, Mannheim, Germany). Apo-ONE caspase-3/7 reagent was added to imatinib-treated (10 µM for 48 h) and untreated ARO, Hth-74, and 8505-C cells in 96-well plates in a ratio of 1:1 and incubated for 90 min. Appropriate negative controls and blanks were taken. The fluorescence of each well was measured at an excitation wavelength of 485 ± 20 nm and an emission wavelength of 530 ± 25 nm.

Tissue microarray

Tissue microarray was performed as previously described (35). Multi-tissue blocks were assembled according to tissue array technology developed in the Department of Pathology, Medical School of Hannover. The primary antibodies against c-abl, PDGFR-{alpha}, and c-kit were applied in a 1:200 dilution. Biotinylated secondary antibodies were used for the catalyzed signal amplification technique (Dako, Hamburg, Germany). The final color reaction was carried out using new fuchsin as chromogen and hemalum as light counterstaining. Evaluation of the immunostaining was carried out by two independent observers (R.v.W. and A.S.R.). Positive cases were defined as those with more than 10% of neoplastic cells exhibiting immunoreactivity.

Western blot analysis and immunoprecipitation (IP)

Cells were lysed in 100 µl lysis buffer [50 mM HEPES (pH 7.4), 150 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM sodium orthovandate, 10 mM sodium pyrophosphate, 100 mM NaF, 1% Triton X-100, 10 µg/ml leupeptin, 10 U/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride]. For Western blot analysis, proteins (10 µg/lane) were resolved in 7.5% SDS-PAGE and electrotransferred onto a nitrocellulose membrane (Millipore, Schwalbach, Germany) using standard procedures.

Tyrosine phosphorylation of ß-catenin was detected by performing IP with the antibody to ß-catenin followed by Western blotting with anti-phosphotyrosine antibody. For IP, total cell lysates (800 µg protein) were incubated with 2 µg anti-ß-catenin antibody for 6 h at 4 C. Specific antibody-antigen complexes were collected with 20 µl of protein G-Sepharose beads overnight at 4 C with gentle overhead rotation. The samples were centrifuged, washed, and suspended in SDS-PAGE sample buffer. After SDS-PAGE and transfer of proteins, membranes were incubated with anti-phosphotyrosine antibody. For detection of tyrosine-phosphorylated ß-catenin, the membranes were incubated with alkaline phosphate-conjugated antimouse second antibodies (Dianova, Hamburg, Germany) at 1:5000 for 1 h and washed again. Expressed proteins were visualized with CSPD chemiluminescent reagent (Roche, Mannheim, Germany).

Immunofluorescence and confocal microscopy

ARO cells were seeded on 12-mm diameter glass coverslips and used at a confluence of 70–90%. The control and imatinib-treated ARO cells (10 µM for 48 h) were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 (Fluka Sigma-Aldrich Chemie, Deisenhofen, Germany) in PBS for 5 min at room temperature. Incubations with primary antibodies (mouse monoclonal anti-ß-catenin mAb or mouse monoclonal anti-E-cadherin mAB, were followed by sheep antimouse fluorescein isothiocyanate-conjugated secondary antibodies (Sigma, Muenchen, Germany) for 1 h at room temperature.

RNA extraction and RT-PCR

Total RNA was prepared from cultured ARO cells by using Tri-Reagent in accordance with the recommendations of the manufacturer (Sigma). mRNA was extracted from total RNA using Oligotex mRNA Mini Kit (QIAGEN, Hilden, Germany). Five micrograms of RNA were oligo-dT reverse transcribed for 35 min at 42 C using Moloney murine leukemia virus reverse transcriptase (GIBCO, Gaithersburg, MD), followed by 5 min of inactivation at 90 C. Real-time PCRs were performed using the Gene Amp 5700 Sequence Detection System (Perkin-Elmer Applied Biosystems, Ueberlingen, Germany) for 2 min at 50 C and 10 min at 95 C, followed by 40 cycles of 15 sec at 95 C and 1 min at 55/58 C (depending on the primer) using SYBR green for the detection of the double-stranded PCR product (Perkin-Elmer Applied Biosystems). Standard curves were generated using serially diluted cDNA probes. The ribosomal protein S9 housekeeping gene was used for normalization.

Transfections

Cells were seeded 1 d before transfection at approximately 50–70% confluency. The cells were cultured overnight in six-well dishes and transfected with dominant-negative TCF-4 (kindly provided by H. Clevers, Utrecht University, Utrecht, The Netherlands) using FuGENE 6 transfection reagent (Roche) following the manufacturer’s instructions. Appropriate empty vector (pCineo construct) was taken in the control well. After 48 h of transfection, the cells were fed with fresh medium in the presence of 1 mg/ml Zeocin (Invitrogen, San Diego, CA). After 2 wk, the cells were picked up and plated in a 60-mm culture dish at a density of one to two cells per dish. The cells were subsequently passaged five times separately in selective medium and dnTCF-4-positive clones were isolated and used for experiments.

Luciferase reporter gene assay

For the detection of Wnt transcriptional activity, luciferase reporter gene analysis was performed in control and imatinib-treated (10 µM for 48 h) ARO cells as described previously (35). Luciferase and ß-galactosidase activities were measured 48 h after transfection according to standard methods and were used to control transfection efficiency (Promega Corp., Madison, WI).

Matrigel invasion assay

The invasive potential of the control and imatinib-treated (10 µM for 48 h) ARO cells was analyzed using modified Boyden Matrigel Invasion Chambers (Costar Corp., Cambridge, MA) as described previously (35). The relative invasive rate was calculated as percent OD of the cells from the top of the membrane to the overall OD from the total cells. Assays were performed in triplicate.

Statistical analyses

Each experiment was performed independently at least thrice, and one representative experiment is presented. The significance of the in vitro data was determined using Student’s t test (two-tailed). P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tissue microarray analysis for the expression of imatinib-sensitive TKs c-abl, PDGFR, and c-kit (CD117)

Imatinib mediates its antiproliferative effects by inhibiting the c-abl, c-kit (CD117), and PDGFR TK activities. To gain more insight into the expression of imatinib-sensitive TKs in thyroid carcinomas, we performed a tissue microarray analysis in a panel of ATCs (n = 12). Our data indicate that six of 12 tumors expressed at least one of the imatinib-sensitive targets (Fig. 1Go, A–C). Nonreceptor TK c-abl was positive in three of 12, PDGFR-{alpha} in two of 12 cases, and c-kit in one of 12 cases, suggesting that imatinib may be effective in a subset of ATCs. We further expanded our analysis into a panel of 10 thyroid cancer cell lines derived from papillary, follicular, and ATC cell lines. Western blot analysis of these thyroid cancer cell lines demonstrated the presence of c-abl in 60% (NPA, TPC-1, FTC-238, WRO, ARO, and BCPAP) (Fig. 1DGo) and PDGFR-{alpha} in 10% (Hth-74) of cases. The remaining 30% (FTC-133, C-643, and 8505-C) of thyroid cell lines were negative for the presence of imatinib-sensitive targets.



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  		FIG. 1. Tissue microarray analysis performed on a panel (n = 12) of ATCs and three ATC cell lines using mAbs against c-abl, c-kit, and PDGFR-{alpha} demonstrated imatinib mesylate-sensitive TKs in six of 12 cases. A–C, Representative photomicrographs of c-abl, PDGFR-{alpha}, and c-kit; D, immunoblot analysis of eight thyroid cancer cell lines to detect imatinib mesylate-sensitive TKs.

 
ß-Catenin and c-abl tyrosine kinase coimmunoprecipitate with each other

Tyrosine phosphorylation of ß-catenin by various TKs prevents its binding to E-cadherin and thus alters its subcellular localization (25, 26, 27, 28). IP and immunoblot analysis in the c-abl-positive cell line, ARO, supported coimmunoprecipitation of ß-catenin and c-abl TK. Moreover, Western blot analysis showed that imatinib (10 µM for 48 h) reduced the coimmunoprecipitated ß-catenin and c-abl levels in ARO cells (Fig. 2AGo).



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  		FIG. 2. A, Detection of the complex of c-abl TK and ß-catenin by immunoprecipitation. Cell lysates of ARO cells treated with or without imatinib mesylate (10 µM for 48 h) were incubated with a c-abl antibody, and immune complexes were precipitated and subjected to Western blot analysis with c-abl and ß-catenin antibodies. B, Immunoblot analysis to show the protein expression of total ß-catenin in control and imatinib mesylate-treated anaplastic and metastatic thyroid cancer cell lines. C, Effect of imatinib mesylate on the proliferation of FTC133 cell line transfected with wild-type and dead kinase c-abl. *, P < 0.05.

 
Imatinib induces dose-dependent loss of total ß-catenin protein in c-abl-expressing thyroid carcinoma cell lines

Imatinib dose-dependently induced a decrease of ß-catenin protein in ARO, BCPAP, and HTh-74 cells as detected by Western blotting of cell lysates using antibody against human ß-catenin. FTC-238 cells expressing c-abl confirmed this effect, whereas in the c-abl-negative FTC133 cells, ß-catenin was unresponsive to imatinib (Fig. 2BGo). To further prove that the reduction in ß-catenin levels is dependent on c-abl, we transfected FTC133 with wild-type and dead kinase c-abl. Transfection of FTC133 with wild-type c-abl renders the cell line sensitive to imatinib, whereas cells transfected with dead kinase c-abl remained insensitive (Fig. 2CGo).

Imatinib stabilizes membranous ß-catenin and E-cadherin binding: a potential mechanism for enhancing adherens junctions

When treating ARO cells with imatinib, tyrosine-phosphorylated ß-catenin dose-dependently decreased (Fig. 3AGo). IP and immunoblotting suggest that in contrast to the tyrosine-phosphorylated forms, the non-tyrosine-phosphorylated ß-catenin binds to E-cadherin (Fig. 3BGo). Confocal analysis and Western blotting support an imatinib-dependent relocalization of ß-catenin (Fig. 3Go, C and D) and E-cadherin (Fig. 3Go, E and F) to the membrane, and this shift was associated with a relative decrease in nuclear ß-catenin staining. These results suggest that imatinib promotes ß-catenin/E-cadherin binding at adherens junctions leading to a reduction in cytosolic/nuclear accumulation of free ß-catenin.



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  		FIG. 3. A, Immunoprecipitation (with anti-ß-catenin mAb) and immunoblot analysis (with phosphotyrosine mAb) reveal a dose-dependent decrease in the tyrosine phosphorylation of ß-catenin in imatinib mesylate-treated (10 µM for 48 h) ARO, Hth-74, and BCPAP cells compared with the control; B, expression of E-cadherin protein in imatinib mesylate-treated (10 µM for 48 h) ARO, Hth-74, and BCPAP cells compared with the control; C and D, effect of imatinib mesylate (10 µM for 48 h) on the localization of ß-catenin in control (C) and imatinib mesylate-treated (10 µM for 48 h) ARO cells (D); E and F, effect of imatinib mesylate on the localization of E-cadherin in control (E) and imatinib mesylate-treated (10 µM for 48 h) ARO cells (F).

 
Effect of imatinib on the cell proliferation of thyroid carcinoma cell lines in vitro

All cells (ARO, Hth-74, BCPAP, C-653, and 8505-C) were serum starved overnight and incubated in medium containing 1% FCS and increasing concentrations of imatinib (1, 2, 4, 8, and 10 µM). Cell proliferation was measured over 48 h by MTT assay. Concentrations of imatinib higher than 8 µM elicited a dose-dependent decline in growth of ARO and BCPAP cell lines as illustrated in Fig. 4AGo, whereas none of the cell lines responded to concentrations of imatinib below 4 µM, consistent with previous findings (36). The IC50 of ARO and BCPAP averaged 7.2 and 8 µM, respectively. Hth-74 showed an intermediate sensitivity (IC50, ~12 µM) to imatinib, although it expresses PDGFR (both {alpha} and ß). Of note, however, was the finding that the two cell lines not expressing c-abl (C-653 and 8505-C) had a low sensitivity (IC50 more than 20 µM) to the drug, suggesting that only this subset of ATC cell lines expressing classical imatinib targets express sufficient sensitivity. This is further illustrated in the FTC-133 and FTC-238 cell lines. Whereas FTC-133 derived from a lymph node metastasis lacks c-abl expression and is insensitive to imatinib, FTC-238 derived from a lung metastasis of the same patient expresses c-abl and shows moderate sensitivity to imatinib (Fig. 4AGo). To determine whether imatinib could inhibit growth in clinically relevant conditions, three c-abl-positive ATC cell lines (ARO, BCPAP, and FTC-238) were incubated in medium containing 10% FCS and increasing concentrations of imatinib. Growth was measured over 48 h by [3H]thymidine incorporation assay. After 48 h imatinib treatment (10 µM), proliferation decreased in all three cell lines by approximately 40%, which corroborates closely the results obtained in the MTT assay (Fig. 4BGo).



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  		FIG. 4. A, Antiproliferative effect of imatinib mesylate (10 µM for 48 h) in thyroid cancer cell lines. An MTT proliferation assay was performed 48 h after treatment with imatinib mesylate in different concentrations in seven thyroid cancer cell lines. ARO, BCPAP, and FTC-238 cells were highly sensitive; the Hth-74 cell line was moderately sensitive; and FTC-133, C-643, and 8505-C cells were relatively insensitive to imatinib mesylate treatment. Data points show the mean ± SD (n = 3) in each condition; P < 0.05. B, [3H]Thymidine incorporation assay. Imatinib mesylate-treated (10 µM for 48 h) ARO, BCPAP, and FTC-238 cells in 10% FCS demonstrated about 40% reduction in the tritiated thymidine incorporation as compared with the controls (n = 3). C, Apoptosis measured by Apo-ONE homogeneous caspase-3/7 assay did not show any statistical differences between control and imatinib mesylate-treated (10 µM for 48 h) ARO cells. D, ARO and FTC-133 cells were stably transfected with dnTCF-4 to block the Wnt-dependent pathways. The proliferation of both cell lines (measured by [3H]thymidine incorporation assay) was reduced by about 50% in dnTCF-4-transfected cells compared with the controls. E, To assess the effect of imatinib mesylate (10 µM for 48 h) on ß-catenin-mediated transcription in ARO cells, a ß-catenin-driven TOF-FOP Flash TCF/LEF reporter gene assay was performed. ***, P < 0.001.

 
To further delineate whether imatinib mediates its effect through cytotoxic or cytostatic mechanisms, we directly assayed apoptosis induction by treatment. ATC cells were exposed to imatinib (10 µM) or dimethylsulfoxide vehicle for 48 h in serum-containing medium, and apoptosis was assayed by measuring the activation of caspase-3/7. As illustrated in Fig. 4CGo, imatinib did not produce a statistically significant increase in caspase-3/7 activity in ARO, Hth-74, or 8505-C cell lines, indicating that imatinib acts predominantly by cytostatic rather than cytotoxic mechanisms.

Imatinib inhibits the ß-catenin/TCF-LEF-dependent activity

To establish the importance of a functional activation of Wnt/ß-catenin in the proliferation of thyroid cancer cell lines we selected ARO (c-abl positive) and FTC133 (negative for all imatinib targets). We permanently transfected ARO and FTC-133 cells with dnTCF-4 to block canonical Wnt signaling. The proliferation of both cell lines measured by the [3H]thymidine incorporation activity was reduced by about 50% in dnTCF-4-transfected cells compared with the untransfected controls (P < 0.05) (Fig. 4DGo). These data indicate that irrespective of the c-abl status of the thyroid cancer cells, an active Wnt/ß-catenin signaling is required for their basal proliferative activity in neoplastic thyroid cancer cell lines.

Furthermore, compared with controls, imatinib-treated ARO cells (c-abl positive) showed a 40% decrease in the ß-catenin-dependent TCF/LEF transcription levels, indicating that imatinib inhibits the Wnt/ß-catenin signaling by specifically inhibiting TCF/LEF-dependent transcriptional activity (Fig. 4EGo). Testing a known ß-catenin target gene, cyclin D1, in ARO cells, we found that imatinib (10 µM for 48 h) significantly reduced cyclin D1 mRNA levels compared with the control (n = 3) (Fig. 5AGo). Furthermore, Western blot analysis after treatment with or without imatinib (10 µM for 48 h) in ARO (c-abl-positive) and FTC133 (c-abl-negative) cells confirmed these results and points toward an imatinib-mediated down-regulation of Wnt/ß-catenin signaling (Fig. 5BGo).



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  		FIG. 5. A, RT-PCR analysis was performed on RNA from control and imatinib mesylate-treated (10 µM) ARO cells for the detection of cyclin D1 (a known downstream target gene of the Wnt pathway). Primers were chosen to detect cyclin D1 mRNA transcripts, and the mean of two independent experiments (n = 3) is presented. B, Immunoblot analyses of cyclin D1 in control and imatinib mesylate-treated (10 µM for 48 h) ARO (c-abl-positive) and FTC133 (c-abl-negative) cells. *, P < 0.05.

 
Effects of imatinib on invasiveness of thyroid carcinoma cells

Finally, we tested the invasiveness of the cells by using a Matrigel invasiveness assay in ARO cells treated with imatinib (10 µM for 48 h) or controls. The percent invasion significantly dropped after 48 h of incubation with imatinib (10 µM) (Fig. 6Go).



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  		FIG. 6. Matrigel invasion assay in ARO cells. Imatinib mesylate (10 µM for 48 h) decreases invasion in ARO cells compared with untreated control cells. Data points show the mean ± SE (n = 3) in each condition. *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our data confirm the previous reports that the imatinib-sensitive TKs c-abl, c-kit, and PDGFR are overexpressed in dedifferentiated thyroid carcinomas (36). Because 50% of the primary ATCs and approximately 70% of the cell lines expressed at least one of the imatinib-sensitive TKs, it may be effective at least in a subset of these highly aggressive tumors. To dynamically characterize antiproliferative effects of imatinib, we selected three thyroid carcinoma cell lines positive for c-abl expression and three negative for any imatinib target. In c-abl-expressing ARO cells, a direct link between c-abl and ß-catenin was shown by coimmunoprecipitation. Imatinib reduced the total and tyrosine-phosphorylated ß-catenin in c-abl-positive cells and reduced ß-catenin nuclear translocation and transcriptional activity. This was not detectable in c-abl-negative controls. ß-Catenin is able to induce thyroid growth via activation of TCF-4/LEF-1 and induction of target genes such as c-myc or cyclin D1 (35). In addition, ß-catenin induces antiapoptotic processes and increases the invasive potential of the cells. Recent transfection studies of our group with a dominant negative TCF support the specificity and importance of this mechanism that explains approximately 40–50% of the growth potential of a thyrocyte (35). Furthermore, reinforcement of adherens junctions, deduced by increased coimmunoprecipitation of ß-catenin and E-cadherin, may add to the invasiveness of the tumor cells. Our experiments support this dual mechanism because imatinib clearly reduced the invasive potential in vitro.

The significant suppression of tumor proliferation and invasion of c-abl-positive cells fit at least in part with previous studies of Podtcheko et al. (36) postulating an imatinib-dependent antiproliferative activity in p53 mutant thyroid carcinoma cell lines and suggested an important therapeutic potential of the drug. Mitsiades et al. (37) and Dziba and Ain (38), however, questioned whether the high doses of imatinib necessary may not be reached in vivo and queried a specific response comparable to treatment in CML patients (37, 38, 39).

To better characterize the specificity of imatinib action, we employed FTC cells, which are particularly interesting because FTC-133 and FTC-238 cells derived from the same primary tumor differ in their expression of c-abl. The c-abl-positive FTC-238 cells in contrast to the c-abl-negative FTC-133 cells were responsive to imatinib treatment. This response is c-abl specific because transfection of FTC-133 cells with active c-abl but not of the dead c-abl kinase form increased proliferation of FTC-133 cells and rendered them responsive to imatinib therapy. Thus, imatinib activity appears to be critically dependent on the expression of its targets such as c-abl, which fits with recent evidence in CML patients of a potential link between c-abl activation and Wnt/ß-catenin signaling in granulocyte-macrophage progenitor cells (40, 41). Furthermore, we show that imatinib has only limited side effects or toxicity even in high doses. Clinically, Mauro et al. (42) observed no dose-limiting toxicity in patients with chronic myelogenous leukemia after a once-daily dose of 600 mg imatinib [mean plasma concentration, 3.9 µg/ml (7.8 µM)]. Moreover, the Food and Drug Administration has recently approved imatinib for the treatment of malignant gastrointestinal stromal tumors, with recommended doses of 400 or 600 mg daily (39). Although we agree with Mitsiades et al. (37) and Dziba and Ain (38) that the concentrations of imatinib required to display its antiproliferative effects are in the upper range of the current clinically relevant doses, we could not demonstrate any proapoptotic action of imatinib at 10 µM concentration. Thus, imatinib may be effective at least in a subset of ATC patients expressing imatinib-sensitive targets. Even though clinical dose recommendations cannot be deduced from in vitro data, our data outline that the in vitro effects are within the concentration range to be reached in vivo. With the development of better therapeutic options for the inhibition of c-abl-dependant pathways by the recent advent of second-generation TK-targeted therapies such as BMS-354825 and PD173955 with two-log-increased potency relative to imatinib, inhibition of c-abl in dedifferentiated thyroid carcinomas may be used in the near future in this group of tumors where other options are not available (43, 44, 45, 46, 47, 48, 49, 50).

In summary, our data provide a novel molecular mechanism for the antitumor activity of imatinib acting through the ß-catenin signaling pathway. The selective action of imatinib in a subset of thyroid carcinoma cells expressing imatinib-sensitive target genes such as c-abl reflects the need for an immunohistochemical profiling to determine therapeutic options in dedifferentiated thyroid carcinomas. Because in our subset of ATCs and cell lines more than 40% of the tumors are potentially responsive to imatinib, our findings provide a basis for a new therapeutic option in these currently untreatable tumors.


   Footnotes
 
This work was supported by Deutsche Krebshilfe.

First Published Online November 1, 2005

Abbreviations: ATC, Anaplastic thyroid carcinoma; CML, chronic myeloid leukemia; FCS, fetal calf serum; IP, immunoprecipitation; LEF, lymphoid enhancer factor; mAb, monoclonal antibody; MTT, dimethyl-thiazol-diphenyltetrazolium bromide; PDGFR, platelet-derived growth factor receptor; TCF, T cell factor; TK, tyrosine kinase.

Received June 22, 2005.

Accepted October 25, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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