ANTICANCER RESEARCH 27: 3999-4010 (2007)

Overexpression of Wild-type THR‚1 Suppresses the Growth and Invasiveness of Human Papillary Thyroid Cancer Cells

ILYA SEDLIAROU1, MICHIKO MATSUSE1, VLADIMIR SAENKO2, TATIANA ROGOUNOVITCH1, YUKA NAKAZAWA1, NORISATO MITSUTAKE1, HIROYUKI NAMBA1, YUJI NAGAYAMA3 and SHUNICHI YAMASHITA1,2

Departments of 1Molecular Medicine, 2International Health and Radiation Research, and 3Medical Gene Technology, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan

Abstract. Background: Thyroid hormone receptors (THRs) and point mutations of the RAS and BRAF genes which are are transcription factors which regulate cell growth and cumulatively detected in 70% of cases without gross differentiation. The compromised function of THRs has been overlapping in one tumor nodule. RET/PTC rearrangements reported in several human malignancies, suggesting their account for 3-43% of cases in adult series (6), while RAS and implication in carcinogenesis. Materials and Methods: Using BRAF alterations occur with a frequency of 7-26% (7, 8) and an adenoviral delivery system, THR‚1 was expressed in human 24-69% (9, 10), respectively. Although an intracellular papillary thyroid cancer cell lines and corresponding biological signaling differs among oncogenic proteins, all of them and molecular changes were analyzed. Results: THR‚1- converge the signals to the mitogen-activated protein kinases dependent transactivation activity is diminished in thyroid (MAPK) ERK1/2 (p42/p44) contributing to the enhanced cancer cell lines. Its restoration suppresses the proliferation, cell growth (11, 12). causes G1 arrest, depresses DNA synthesis and decreases On the other hand, molecular alterations involving invasiveness of cancer cells paralleled by the attenuation of inactivation of tumor suppressors in PTC are less studied. AKT and MAPK signaling, and reduction of cyclin D1 levels. Several works have demonstrated that p53, p73, p15INK4b, Conclusion: The alteration of THR‚1 functioning is an p16INK4a and maspin may be involved in thyroid important change in human well-differentiated thyroid carcinogenesis due to compromised mRNA or protein carcinomas, affecting tumor cell growth and contributing to the expression, or mutation (13-17). Recent investigations malignant potential of cancer cells. suggest that thyroid hormone receptors (THRs) may function as tumor suppressors in various types of human Thyroid cancer is a common endocrine malignancy with an malignancies, such as hepatocellular, renal and colon incidence of approximately 0.5-10 cases per 100,000 carcinomas (18-21). In papillary thyroid cancer, mutations of population (1). In about 90% of patients, tumors are THRB1 and THRA1 have been reported at high frequencies diagnosed as papillary thyroid carcinomas (PTCs). Although and have often been associated with loss of function (22). long-term prognosis of PTC is generally favorable, recurrence Thyroid hormone receptors are transcription factors rates of up to 40% and cancer-related mortality of up to 12% that belong to the steroid/thyroid nuclear receptor have been reported in high risk patient groups (2-5). superfamily. They act in a ligand-inducible manner by Several major oncogenic events have been implicated in binding to DNA response elements in regulatory regions the pathogenesis of PTC such as RET/PTC rearrangements of target genes. Potentially, THR mutation(s) can recast gene expression profile in a cell resulting in a tendency to tumorigenesis. In an animal model, there is strong evidence of the ability of the functionally impaired THR‚1 Correspondence to: Prof. Shunichi Yamashita, Department of to cause the development of malignant thyroid tumors. Molecular Medicine, Atomic Bomb Disease Institute, Nagasaki The knock-in mouse with a mutant thyroid hormone University Graduate School of Biomedical Sciences, 1-12-4 receptor-beta gene (THRB1PV) has been found to Sakamoto, Nagasaki 852-8523, Japan. Tel: +81 95 849 7116, Fax: spontaneously develop thyroid cancer and distant +81 95 849 7117, e-mail: [email protected] metastases resembling human follicular thyroid carcinoma Key Words: Papillary thyroid carcinoma, thyroid hormone receptor (23). However, the role and effects of an intact THR‚1 in beta, cell growth, cell cycle, invasiveness. thyroid cancer cells remains largely unclear.

0250-7005/2007 $2.00+.40 3999 ANTICANCER RESEARCH 27: 3999-4010 (2007)

In this work we analyzed the effects of adenovirus-mediated Recombinant adenoviruses. Inserts from the wild-type and mutant THR‚1 expression in papillary thyroid cancer cell lines using THRB1-pHMCMV6 and GFP-pHMCMV6 plasmids were recloned cell growth, cell cycle distribution, intracellular signaling into pAdHM4CMV (28). The plasmids were linearized with PacI and transfected into human embryonal kidney HEK293 cells cascades and cell invasiveness as biological end-points. (American Type Culture Collection, VA, USA). Recombinant adenoviruses (the wtTHRB1-Ad, mutTHRB1-Ad and GFP-Ad) were Materials and Methods propagated and purified by CsCl density-gradient centrifugation (29). The concentration of viral particles was determined by Materials. The following vectors were used: p3FLAG-CMV14 from measuring the absorbance at 260 nm (30). Sigma (MO, USA); pGL2-Control and pRL-CMV from Promega Early cytotoxicity of adenoviruses in TPC1 and NPA cells was (WI, USA), while pHMCMV6 and pAdHM4CMV were prepared estimated using the Cell Counting Kit (Wako, Japan) according to in our University. Antibodies to phospho-p44/42MAPK, the manufacturer’s protocol. Briefly, the cells were plated in T3- 3 p44/42MAPK, phospho-p90RSK, phospho-AKT-1, AKT, phospho- depleted medium in 96-well plates (2x10 cells/well). One day after GSK-3‚, cyclin D1, p21WAF1/CIP1, secondary anti-mouse and plating, the cells were infected with wild-type and mutant THRB1- anti-rabbit HRP-conjugated antibodies were purchased from Cell Ad and GFP-Ad at 50-800 multiplicities of infection (MOI). Signaling (MA, USA), the antibodies to THR‚1 from Santa Cruz Twenty-four hours later, reagent was added to the medium and (CA, USA), anti-FLAG M2 and GFP from Sigma, and anti-MMP- optical density was read at 450 nm in an ImmunoMini NJ-2300 1 and MMP-9 from Daiichi Fine Chemical Co. (Toyama, Japan). (System Instruments, Japan) microplate reader. A similar technique was used to evaluate long-term cell viability. Cell cultures. TPC1 and NPA cells lines initially provided by Dr. J. To evaluate the transduction efficacy, the cells were infected Fagin (Memorial Sloan-Kettering Cancer Center) were with wild-type and mutant THRB1-Ad or GFP-Ad at 50-800 MOI maintained in Dulbecco’s modified Eagle’s medium (DMEM) in the absence or presence of T3. GFP expression in infected cells with 5% fetal bovine serum. Primary human thyrocyte cultures was evaluated using an Eclipse TE2000-U fluorescent microscope were established from surgically excised normal thyroid gland and (Nikon, Japan). THRB1-Ad-infected cells were subjected to maintained in DMEM/F-12 (1:2 v/v) supplemented with 3% fetal immunofluorescent staining using an anti-FLAG M2 antibody bovine serum (Gibco, Invitrogen Corp., NY, USA) (24). For the (Sigma) (31). The viral dose yielding target expression in at least experiments addressing THR‚1 function, serum was treated with 90% of infected cells without evidence of early cytotoxicity was Dowex 1 X 8 resin (Fluka, Germany) to deplete thyroid determined (400 MOI). hormones (25, 26). This procedure reduced T3 concentration to undetectable levels. In the experiments requiring the presence of Reporter plasmids and reporter assay. pGL2-Control plasmid was thyroid hormone, medium was supplemented with T3 (Sigma, used as a reporter vector in the transcriptional activation assay. MO, USA) at 10–7 M (22). Thyroid hormone responsive element 5'-CAGGTCATGACCTC-3' was inserted upstream of the SV40 promoter between SacI and Cloning of THRB1 cDNA and construction of the expression vectors. XhoI sites (the pGL2-TRE vector). pRL-CMV plasmid with the Total RNA was extracted from TPC1 and NPA cells and primary Renilla luciferase gene served as an internal control for thyrocytes using Trizol reagent (Invitrogen, CA, USA) according normalization in the transfection experiments. to the manufacturer's instructions. One microgram of RNA was For transcription activation assay, COS7 cells were grown in T3- reverse transcribed with MuLV Reverse Transcriptase (Applied depleted medium for 24 hours. The transfection was carried out in Biosystems, UK) using random hexamers. The coding region of triplicates in 24-well plates with 0.2 Ìg of pGL2-TRE/pRL-CMV THRB1 was amplified with the forward, 5'-AAATATGCGG mixture (weight ratio 100:1) and 0.2 Ìg of wild-type or mutant CCGCATGACTCCCAACAGTATGACAGAA-3', and reverse, 5'- THRB1-pHMCMV6 or GFP-pHMCMV6 using Lipofectamine TCTAGAATCCTCGAACACTTCCAGGAAC-3', primers. PCR (Invitrogen) for 6 hours, then the transfection mixture was replaced products were sequenced and compared with the reference wild- with 0.5 ml of 5% T3-depleted medium and T3 was added to the type THRB1 sequence. appropriate wells. Forty-eight hours after transfection, cells were Wild-type THRB1 amplicon from primary thyrocytes was ligated lysed and a luciferase assay was performed with the Dual into p3FLAG-CMV14 vector. FLAG-tagged THRB1 was amplified Luciferase Reporter Assay System (Promega). from the above vector template using forward, 5'-AAATATGCG To analyze the functional status of endogenous THR‚1 in GCCGCATGACTCCCAACAGTATGACAGAA-3', and reverse, TPC1, NPA cells and also in primary human thyrocytes, the cells 5'-GGTACCCTACTTGTCATCGTCATCCTTGTA-3', primers. were grown in T3-depleted medium as described above and The amplicon containing THRB1-FLAG was recloned into the transfected with 0.4 Ìg of pGL2-TRE/pRL-CMV mixture (weight pHMCMV6 expression vector between NotI and KpnI sites ratio 100:1). T3 was then added to the appropriate experimental (THRB1-pHMCMV6) and sequenced to ensure wild-type structure. wells and the Dual Luciferase Reporter Assay was performed 48 To generate mutant THR‚1, the K411E mutation (an amino hours after T3 treatment. To evaluate the function of exogenous acid substitution in the ligand-binding domain) was introduced into wtTHR‚1, TPC1 and NPA were infected with wtTHRB1-Ad and wild-type THRB1-pHMCMV6 vector using the QuickChange XL then analyzed under the same experimental conditions. site-directed mutagenesis kit (Stratagene, CA, USA). The numbering of amino acid residues of THR‚1 was based on the Western blotting. NPA and TPC1 cells were grown in T3-depleted consensus nomenclature (27). In the control construct, the GFP medium for 24 hours and infected with wtTHRB1-Ad, mutTHRB1- gene was inserted into the pHMCMV6 vector between NotI and Ad and GFP-Ad at 400 MOI for 6 hours. T3 was added to the KpnI sites (GFP-pHMCMV6). medium at the final concentration of 100 nM. Cells were harvested

4000 Sedliarou et al: Tumor Suppressor Role of THR‚1 in PTC Cells after 2 h to exclude the transient effects of T3 as a 0 h point and also collected 24, 48 and 72 h later. For Western blotting, the cells were lysed, 10-40 Ìg of protein were resolved in a 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Millipore, MA, USA) by semidry blotting. After incubation with an appropriate primary antibody, the antigen-antibody complexes were visualized using HRP- conjugated secondary antibody and enhanced chemiluminescence system (Amersham Biosciences, NJ, USA). For quantitative evaluation of band intensities, image analysis was carried out using Gel-Pro Analyzer ver.3.1. software (Media Cybernetics, MD, USA). At first, the optical density (OD) of each band of interest was normalized against that of corresponding ‚-actin band. Then, the normalized OD of the 0 h time-point band in each experimental group was assigned the value of 1.0 and the relative optical density (ROD) of other bands was recalculated in relation to it.

Bromdeoxyuridine (BrdU) incorporation assay and cell cycle analysis. Figure 1. The transactivation activity of exogenous wild-type and mutant TPC1 and NPA cells were plated, grown and infected with THR‚1 and GFP in COS7 cells. Results are shown as the mean fold adenovirus as described for Western blotting experiments. The BrdU activation in relation to the GFP control in the absence of T3. The assay was performed at 0 and 72 h time-points. Briefly, the cells were experiment was repeated three times using 6 wells for each pHMCMV6 pulsed with bromdeoxyuridine for 30 min, fixed with ethanol, vector-based expression construct. Each bar indicates the mean and denatured with HCl and incubated with FITC-conjugated anti-BrdU standard deviation. antibody using the BD Biosciences Pharmingen FITC BrdU Flow Kit (BD Biosciences, CA, USA) according to manufacturer’s recommendations. Any floating cells were pooled together with the cells detached from plates by trypsin. Flow cytometry was carried out The transcriptional activation of the luciferase reporter using a FACScan machine (Becton-Dickinson, USA). after transfection of any vector was low in the absence of T3. For flow cytometric cell cycle analysis, cells were infected with Addition of T3 to the medium caused a 5.7-fold increase of adenoviral vectors at 400 MOI and grown for 72 h in the presence the reporter signal in wtTHRB1-transfected cells while the or absence of T3. Cells harvested from subconfluent cultures were combined with preliminary collected floating cells, fixed with mutant THR‚1 displayed a compromised transactivation, ethanol, treated with ribonuclease A (0.1 mg/ml, 30 min at room retaining about 35% of the wtTHR‚1 activity (Figure 1), in temperature) and stained with propidium iodide (25 Ìg/ml). line with the previous findings (22). The transcriptional activity of GFP was not significantly affected by the presence Migration assay. To assess the invasiveness, 4x103 cells were seeded or absence of T3. on Matrigel pre-coated filters (12 Ìm pores; Iwaki, Japan) in 24- well plates, as described by Hwang et al. (32), in T3-depleted medium. The cells were infected with wtTHRB1-Ad, mutTHRB1- Status of THR‚1 in PTC cell lines and primary thyrocytes. The Ad, GFP-Ad at 400 MOI and T3 was added to the appropriate direct sequencing of THRB1 cDNA from TPC1, NPA and wells. After 72 hours of incubation, the cells on the interior of the primary thyrocytes revealed the wild-type structure of the inserts were removed by swabbing, the exterior was stained with coding region in all three cell types. However, the functional hematoxylin and clung cells were counted under bright field activity of endogenous THR‚1 differed between the cells microscopy in four random fields. studied. The transfection of cultures with the pGL2- TRE/pRL-CMV plasmids in the absence of T3 revealed the Statistical analysis. Statistical significance was evaluated using one- way analysis of variance (ANOVA) followed by Fisher’s PLSD or steady low basal activity of luciferase in all of them. The unpaired t-test as appropriate. A p-value not exceeding 0.05 was addition of T3 to the medium caused a 8-fold increase of considered significant. activity in primary thyrocytes, while only a 2- and 3-fold increase in TPC1 and NPA cells, respectively. The wild-type Results THRB1 gene transfer evoked marked changes in the receptor’s functional activity 48 hours after infection. In the Transactivation activity of wild-type and mutant THR‚1. To wtTHRB1-transduced cells, the addition of T3 caused 17- analyze the ability of recombinant THR‚1 to transactivate a and 13-fold increases of the reporter signal in TPC1 and responsive gene, experiments were performed on COS7 NPA, respectively, compared with the corresponding control cells which lack endogenous THR‚1. The wild-type and cells which had not received T3 (Figure 2A). mutant THRB1 or GFP in pMCMV6 vector were co- After adenoviral infection, the expression of exogenous transfected with the pGL2-TRE/pRL-CMV plasmid mixture THR‚1 and GFP in transduced cells was confirmed by in the presence or absence of T3. Western blotting. Ectopic accumulation of proteins became

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Figure 2. Expression and functional activity of THR‚1 in the papillary thyroid carcinoma cells and primary thyrocytes. (A) Transactivation activity of THR‚1 in TPC1, NPA and primary thyrocytes before and after adenoviral transduction at 400 MOI. Results are shown as the mean fold activation in relation to basal luciferase activity in the absence of T3 for each type of cells. The bars indicate the mean and standard deviation. (B) Time-course of exogenous THR‚1 and GFP accumulation in TPC1 and NPA cells after infection with adenoviral vectors at 400 MOI. Immunofluorescent staining of NPA cells with an anti-FLAG antibody 72 h after infection with wtTHRB1-Ad at 400 MOI in the absence (C) or presence (D) of T3 (magnification x400). Note that medium supplementation with T3 causes wtTHR‚1 translocation from the cytoplasm to the nucleus. Similar data were obtained in TPC1 cells. noticeable 24 h after infection and reached a maximum 72 h mutant THRB1-Ad effectively delivered genes into TPC1 later. As expected, the molecular weight of FLAG-tagged and NPA cells. The transduction of more than 90% cells THR‚1 was higher than that of endogenous THR‚1 (Figure was observed at 400 MOI without signs of early cytotoxicity. 2B). The efficacy of adenoviral gene transfer to PTC cell In the absence of T3, exogenous THR‚1 was mostly lines was determined by fluorescence for GFP-Ad and by localized in the cytoplasm. Addition of T3 to the medium imunofluorescence for THRB1-Ad. GFP-Ad, wild-type and caused THR‚1 translocation to the nucleus (Figure 2C, D).

4002 Sedliarou et al: Tumor Suppressor Role of THR‚1 in PTC Cells

Figure 3. Wild-type THR‚1 inhibits proliferation of papillary thyroid carcinoma cells in the presence of T3. TPC1 and NPA cells were transduced at 400 MOI and counted 72 h later. The experiment was reproduced three times using 6 wells for each vector. Each bar indicates the mean and standard deviation. *Significant difference, p<0.05.

THRB1 inhibits thyroid cancer cell growth in the presence of moderate increase of p21CIP/WAF1 levels in TPC1 and NPA T3. In the presence of T3, cell growth, as determined by the lines, whereas mutTHR‚1 and GFP had no effect (Figure water-soluble tetrazolium-based assay, was suppressed in 4). Thus, inhibition of PTC cell proliferation by wtTHR‚1 TPC1 and NPA cells infected with wtTHRB1-Ad compared correlates with the decline of the level of cyclin D1 and with the control cells infected with either mutTHRB1-Ad or accumulation of p21CIP/WAF1. GFP-Ad. The extent of growth inhibition was 27-35% in The analysis of the cell cycle demonstrated that TPC1 TPC1 and 10-14% in NPA cells (Figure 3). No such and NPA cultures transduced with wtTHR‚1 showed the significant differences were observed in the absence of T3, significant accumulation of cells in the G1-phase in the nor there were differences between mutTHRB1-Ad and presence of T3. This was accompanied by a marked GFP-Ad infected cells under any conditions. reduction in the proportion of cells in the S- and G2/M- To elucidate the mechanisms of growth inhibition, we phases. Such a cell shift was not observed in mutTHRB1- analyzed the level of cyclin D1, a key regulator of the cell Ad or GFP-Ad transduced cells either in the presence or cycle progression from the G1- to the S-phase. Cells absence of T3 hormone (Figure 5A). Note that there were infected with wtTHRB1-Ad, mutTHRB1-Ad and GFP-Ad virtually no changes in the numbers of sub-G1 cells (less grown in T3-depleted medium showed high levels of cyclin than 2%) attesting to the low cytotoxicity of adenoviral D1 in TPC1 and NPA cultures indicative of their high vectors and products of their expression. The BrdU proliferative activity (Figure 4). When T3 was added to the incorporation assay revealed reduced DNA synthesis in the medium, the level of cyclin D1 in cells transduced by cells infected with wtTHRB1-Ad in the presence of T3 72 h mutTHR‚1 and GFP remained unchanged. On the after infection in comparison with mutTHRB1-Ad or GFP- contrary, cells transduced by wtTHR‚1 displayed a Ad (Figure 5 B). Together these findings are consistent with conspicuous reduction of cyclin D1 starting from 48 h after the T3-dependent retardation of PTC cell proliferation infection and reaching a minimum at 72 h. The specifically induced by wtTHR‚1. downregulation of cyclin D1 in the presence of T3 was strong in TPC1 while in NPA cells it was lower, compatible Suppression of AKT and MAP kinase signaling by wild-type with the different extent of cell growth inhibition in the two THR‚1 in the presence of T3. Since AKT and MAP kinase cell lines. In addition, we examined the level of pathways are known to be involved in carcinogenesis of p21CIP/WAF1, which is a cyclin kinase inhibitor involved in thyroid follicular cells, we examined whether THR‚1 can cell cycle regulation. T3-bound wtTHR‚1 caused a interfere with these cascades.

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Figure 4. Expression of wtTHR‚1 in the presence of T3 leads to the down-regulation of cyclin D1 and accumulation of p21 in TPC1 and NPA cells. TPC and NPA cells were infected with adenoviral vectors at 400 MOI in the presence or absence of T3 and lysates were collected at different time-points. The relative optical density is shown after the normalization against ‚-actin and in the relation to the OD of band at 0 h time-point in each experimental group. The experiment was reproduced at least three times; the representative membranes are shown in the figure.

In the presence of T3, overexpression of wtTHR‚1 inhibited ERK1/2 and p90RSK in NPA cells, in which the intrinsic level AKT-1 phosphorylation at Ser473 in TPC1 and NPA cells and of phospho-ERK1/2 is very high compared to TPC1 (data not also reduced the level of the phosphorylated GSK-3‚ (Ser21) shown). In these cells, the effect of wtTHR‚1 on phospho- at 48 h and 72 h after infection. Such effects were observed ERK1/2 and its downstream effectors did not differ from only in the cells infected with wtTHRB1-Ad in the presence of those of mutTHR‚1 or GFP. T3 and they were concordant with the accumulation of THR‚1 protein. The cells infected with mutTHRB1-Ad and GFP-Ad THR‚1 modulates invasiveness of tumor cells and reduces showed no changes in the phosphorylation of AKT-1 or GSK- metalloproteinase expression. Besides the uncontrolled 3‚, regardless of T3 presence (Figure 6). growth, one of the major properties of malignant neoplasms The wtTHR‚1 also inhibited ERK2 (p42) phosphorylation is their ability to metastasize. In this study we evaluated the in TPC1 cells (Figure 6). Subsequently, there was a reduction effects of THR‚1 expression on TPC1 and NPA cell in the phosphorylation of p90RSK, which is a substrate of invasiveness using Matrigel-coated membranes. The results ERK1/2. The changes in the levels of ERK2 and p90RSK demonstrated that 72 h after infection with wtTHRB1-Ad in phosphorylation started 48 h after infection and enhanced up the presence of T3, the number of TPC1 and NPA cells to the 72 h time-point. Mutant THR‚1 and GFP could not passed through the membrane was 38-52% and 18-27% inhibit the phosphorylation of these proteins in TPC1 cells. By lower, respectively, compared with the same cells infected contrast, wtTHR‚1 did not affect the phosphorylation of with GFP-Ad or mutTHRB1-Ad (Figure 7A).

4004 Sedliarou et al: Tumor Suppressor Role of THR‚1 in PTC Cells

Figure 5. Wild-type THR‚1 causes cell accumulation in the G1-phase of the cell cycle and reduces DNA synthesis in the presence of T3. (A) TPC1 and NPA cells were infected at 400 MOI and the cell cycle was examined after 72 h. (B) DNA synthesis in TPC1 and NPA cells 72 h after infection with adenoviral vectors at 400 MOI. All experiments were reproduced at least three times. *Significant difference, p<0.05.

As to a certain extent the propensity to invade Discussion extracellular matrix depends on the synthesis of matrix metalloproteinases (MMPs) by tumor cells, we examined In our study, we found that the coding region of THRB cDNA the expression levels of MMP-1 and MMP-9. The has a wild-type structure in TPC1 and NPA cells (as well as in transduction of TPC1 and NPA cells with THR‚1 and GFP anaplastic thyroid carcinoma cell lines ARO, FRO, KTC2, and in the presence and absence of T3 had no effect on the in follicular carcinoma cell line, WRO; M. Matsuse, expression of MMP-9 (data not shown), but affected the unpublished data), supporting previous reports by Takano and level of MMP-1. In line with the Matrigel invasion assay, Rocha (33, 34). The transcriptional activation by thyroid wtTHRB1-Ad reduced the MMP-1 level in the presence of hormone receptors involves the concerted action of T3 in both TPC1 and NPA cells whereas mutTHRB1-Ad coactivators, chromatin components, and the basal transcription and GFP-Ad did not (Figure 7 B). machinery. Crucial THR coactivators, which bind the activation

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Figure 6. Effects of wtTHR‚1, mutTHR‚1 and GFP expression on AKT and MAPK/ERK pathways in TPC1 line in the absence and presence of T3. Cells were infected at 400 MOI, T3 was added to the appropriate wells and lysates were collected at different time-points. In the presence of T3, only wtTHR‚1 inhibited the phosphorylation of AKT-1, GSK-3‚, ERK2 and p90RSK in TPC1 (shown in the figure) and NPA (data not shown) cells. In contrast to the suppression of the AKT cascade, no changes were observed in ERK1/2 phosphorylation in NPA cells (data not shown). The relative optical density is shown after the normalization against ‚-actin and in relation to the OD of band at 0 h time-point in each experimental group. Experiments were reproduced three times.

function domain of thyroid hormone receptors, include TIF2, BRAFT1799A point mutation, respectively. In the presence of CBP/p300 and TRAP, and are believed to facilitate the T3, transduction with wtTHR‚1 resulted in the accumulation receptor-chromatin interaction (35, 36). In our experiments, the of cells in the G1-phase, more pronounced in TPC1 than in addition of T3 to the culture of normal thyrocytes resulted in NPA cells, paralleled by the reduced DNA synthesis, the strong activation of luciferase reporter, while T3-induced reduction of cyclin D1 and accumulation of p21CIP/WAF1. Our luciferase signal in TPC1 and NPA cells was much weaker. One results are in line with the findings of another research group possible explanation of attenuated T3 response in cancer cells in a neuroblastoma cell line in which wtTHR‚1 suppressed might be the alterations in the recruitment of coactivators and proliferation, induced morphological differentiation and in the sequential activation of THR‚1-mediated transcription. inhibited tumorigenesis in nude mice (40). The CREBBP (encodes CBP) and EP300 (encodes p300) gene One of the possible mechanisms for the antiproliferative abnormalities have been reported in some tumors (37, 38), as effects of THR‚1 in human papillary thyroid carcinoma cells well as the level of TIF2 has been shown to decrease gradually could likely be attributed to the changes in cell signaling in thyroid carcinomas with progressive dedifferentiation (39). cascades. Both selected cell lines expressed AKT and Therefore, it is possible to speculate that the adenovirus- displayed high levels of phosphorylated AKT-1 when grown mediated overexpession of wtTHR‚1 in cancer cells might either in the absence or presence of T3, indicating a enable to overcome functional block and to restore receptor’s constitutive activation of this pathway. AKT-1 phosphorylates activity via the recruitment of alternative mechanisms. GSK-3‚ and inhibits its activity, thus preventing the We provide novel data demonstrating that the restoration phosphorylation and degradation of ‚-catenin. The latter can of THR‚1 function leads to growth inhibition of TPC1 and translocate to the nucleus and exert its transactivation NPA cells harboring two major genetic alterations involved in function to induce the expression of various genes, including thyroid carcinogenesis, an RET/PTC1 rearrangement and the CCND1, which encodes cyclin D1 (41-43).

4006 Sedliarou et al: Tumor Suppressor Role of THR‚1 in PTC Cells

Figure 7. Wild-type THR‚1 decreases invasiveness of and MMP-1 expression in thyroid cancer cells. (A) TPC1 and NPA cells were seeded onto the Matrigel- coated filters, infected with adenoviral vectors at 400 MOI and incubated for 72 h in the presence or absence of T3. The results are expressed as the mean and standard deviation of the number of cells per observation field. The assay was reproduced three times in duplicates for each experimental point. * Significant difference, p<0.05. (B) TPC1 and NPA cells were infected with adenoviral vectors at 400 MOI in the presence or absence of T3 and harvested at 72 h. The relative optical density is shown after the normalization against ‚-actin and in relation to the OD of band at 0 h time-point in each experimental group.

In agreement with previous work which showed a In parallel with the inhibition of AKT, we observed an correlation between mutant THR‚1 and enhanced influence of THR‚1on the MAPK/ERK pathway as well. PI3K/AKT signaling (23), here we reciprocally found that Unexpectedly, the effect of wtTHR‚1 on phospho-ERK2 expression of the fully functional THR‚1 produced an levels differed markedly between the cell lines. Both TPC1 inhibitory effect on the AKT cascade manifesting itself in the and NPA cultures showed a high level of ERK1/2 reduction of AKT-1 phosphorylation with subsequent de- phosphorylation, with ERKs much more strongly expressed phosphorylation (i.e. activation) of GSK-3‚. Consequently, and activated in NPA cells. Wild-type THR‚1 was able to depression of the AKT pathway resulted in a reduction of reduce the phosphorylation of ERK2 in TPC1 cells, but no cyclin D1 levels eventually contributing to cell growth such similar result was obtained in NPA cells, indicating that retardation. The down-regulation of cyclin D1 appears to be the suppressive role of THR‚1 may depend on the extent of an important component of the machinery by which THRs pathway activation which in turn is probably associated with control cell proliferation. Given that T3-bound THR‚1 can an underlying oncogenic event. The phosphorylation level of inhibit the transcription of the CCND1 gene through the p90RSK, which is downstream of ERK1/2, was lower in TPC1 Tcf/Lef-1 site (43) recruiting ‚-catenin, THR‚1-dependent cells transduced with wtTHR‚1 in the presence of T3, in repression of AKT-1 pathway may be a key biochemical concert with phospho-ERK2 declination. By contrast, NPA change stipulating this effect. The synchronous declination of cells displayed a high level of phospho-p90RSK over the phospho-AKT-1 and cyclin D1 levels in THR‚1-transduced time-course without any tendency for reduction. TPC1 and NPA cells suggest the involvement of a common In addition to its impact on cell proliferation, THR‚1 mechanism of cell growth diminution in these cell lines. also affected invasiveness of PTC cells. TPC1 and NPA

4007 ANTICANCER RESEARCH 27: 3999-4010 (2007) cells transduced with wild-type THR‚1 migrated References significantly slower through the Matrigel coat compared with mutTHR‚1 or GFP-transduced cells. One of the 1 DeLellis RA, Lloyd RV, Heitz PU and Eng C: Pathology and circumstances likely to mediate this effect is the down- Genetics of Tumors of Endocrine Organs. Lyon, IARC Press, p. 320, 2004. regulation of an interstitial collagenase, MMP-1, which 2 Mazzaferri EL and Jhiang SM: Differentiated thyroid cancer breaks down the most abundant extracellular matrix long-term impact of initial therapy. Trans Am Clin Climatol Assoc components, collagens type I and III, and facilitates the 106: 151-168, 1994. spread of cancer cells. Of note, MMP-1 expression is 3 Mazzaferri EL and Jhiang SM: Long-term impact of initial regulated by PI3K/AKT and MAPK/ERK signaling so that surgical and medical therapy on papillary and follicular thyroid the inhibition of one or both of these pathways may have cancer. Am J Med 97: 418-428, 1994. an effect on MMP-1 levels. Forough et al. have shown a 4 DeGroot LJ, Mangklabruks A and McCormick M: Comparison of RA 131I treatment protocols for Graves' disease. J Endocrinol direct link between AKT suppression and reduced MMP-1 Invest 13: 111-118, 1990. activity (44). The inhibition of ERK1/2 phosphorylation has 5 Hay ID: Papillary thyroid carcinoma. Endocrinol Metab Clin been also associated with the reduction of MMP-1 in a North Am 19: 545-576, 1990. human glioblastoma cell line (45). From our experiments, 6 Fagin JA: Perspective: lessons learned from molecular genetic however, it was evident that MMP-1 is not the only factor studies of thyroid cancer-insights into pathogenesis and tumor- affecting thyroid cancer cell migration. The down- specific therapeutic targets. Endocrinology 143: 2025-2028, 2002. regulation of MMP-1 was more pronounced in the 7 Soares P, Trovisco V, Rocha AS, Lima J, Castro P, Preto A, wtTHR‚1-transduced NPA cells than in TPC1 cultures, Maximo V, Botelho T, Seruca R and Sobrinho-Simoes M: BRAF mutations and RET/PTC rearrangements are alternative events in whereas the net reduction in the passage through the the etiopathogenesis of PTC. Oncogene 22: 4578-4580, 2003. membrane was stronger in TPC1 cells, similar to the 8 Namba H, Rubin SA and Fagin JA: Point mutations of ras depression of cell growth and signal transduction. oncogenes are an early event in thyroid tumorigenesis. Mol Results of examining THR‚1 effects in two papillary Endocrinol 4: 1474-1479, 1990. thyroid cancer cell lines with different mutational backgrounds 9 Namba H, Nakashima M, Hayashi T, Hayashida N, Maeda S, demonstrate that wild-type THR‚1 suppresses proliferation Rogounovitch TI, Ohtsuru A, Saenko VA, Kanematsu T and and invasiveness more effectively in the cells harboring Yamashita S: Clinical implication of hot spot BRAF mutation, V599E, in papillary thyroid cancers. J Clin Endocrinol Metab 88: RET/PTC rearrangement than in those with mutant BRAF. It 4393-4397, 2003. is well-known that gene rearrangements are more 10 Cohen Y, Xing M, Mambo E, Guo Z, Wu G, Trink B, Beller U, characteristic for childhood patients with PTC, while BRAF Westra WH, Ladenson PW and Sidransky D: BRAF mutation in point mutations are found with increasing frequencies in adult papillary thyroid carcinoma. J Natl Cancer Inst 95: 625-627, 2003. patients (46). At the same time, older age is an unfavorable 11 Marshall CJ: MAP kinase kinase kinase, MAP kinase kinase and prognostic index of PTC according to various scoring systems, MAP kinase. Curr Opin Genet Dev 4: 82-89, 1994. while BRAF mutation has also been correlated with an 12 Marais R and Marshall CJ: Control of the ERK MAP kinase advanced stage of disease and poorer prognosis (47). cascade by Ras and Raf. Cancer Surv 27: 101-125, 1996. 13 Ferru A, Denis S, Guilhot J, Gibelin H, Tourani JM, Kraimps JL, Although the comparative status of THR‚1 in childhood and Larsen CJ and Karayan-Tapon L: Expression of TAp73 and adult malignancies or in the PTCs harboring RET/PTC and DeltaNp73 isoform transcripts in thyroid tumours. Eur J Surg BRAF mutation is unknown and requires further Oncol 32: 228-230, 2006. investigations, the results of our work imply a possible 14 Boltze C, Hoang-Vu C, Schneider-Stock R, Lehnert H and involvement of THR‚1 to the more effective moderation of Roessner A: Role of the class II tumor suppressor gene maspin in tumors driven by RET/PTC oncogenes and a contribution of thyroid carcinogenesis. Verh Dtsch Ges Pathol 88: 237-245, 2004. receptor to the better prognosis. 15 Boltze C, Zack S, Quednow C, Bettge S, Roessner A and Schneider-Stock R: Hypermethylation of the CDKN2/p16INK4A In conclusion, our results demonstrate the role of THR‚1 promotor in thyroid carcinogenesis. Pathol Res Pract 199: 399- as a tumor suppressor in human PTC and its functional 404, 2003. significance for tumor progression. In this study, we showed 16 Jones CJ, Shaw JJ, Wyllie FS, Gaillard N, Schlumberger M and previously unrecognized effects of wild-type THR‚1 on the Wynford-Thomas D: High frequency deletion of the tumor major signaling pathways in thyroid carcinogenesis and suppressor gene P16INK4a (MTS1) in human thyroid cancer cell biological properties of tumor cells. lines. Mol Cell Endocrinol 116: 115-119, 1996. 17 Chen Y, Zhang P, Li Y, Zhuang J and Gao L: Clinicopathologic Acknowledgements significance of structural alterations of p53 protein in papillary thyroid carcinoma. Zhonghua Bing Li Xue Za Zhi (in Chinese) This study was supported in part through Nagasaki University 21st 31: 217-221, 2002. Century COE Program and by research grants 19390253, 19790651 18 Lin KH, Shieh HY, Chen SL and Hsu HC: Expression of mutant and 18790637 from Ministry of Education, Culture, Sports, Science thyroid hormone nuclear receptors in human hepatocellular and Technology of Japan. carcinoma cells. Mol Carcinog 26: 53-61, 1999.

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Endocr Relat Cancer 13: 455-464, 2006. 33 Takano T, Miyauchi A, Yoshida H, Nakata Y, Kuma K and Amino N: Expression of TRbeta1 mRNAs with functionally Received May 14, 2007 impaired mutations is rare in thyroid papillary carcinoma. J Clin Revised August 1, 2007 Endocrinol Metab 88: 3447-3449, 2003. Accepted September 28, 2007

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