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High Basal Levels of Functional Toll-Like Receptor 3 (TLR3) and Noncanonical Wnt5a Are Expressed in Papillary Thyroid Cancer and Are Coordinately Decreased by Phenylmethimazole Together with Cell Proliferation and Migration

Edison Biotechnology Institute and College of Osteopathic Medicine (K.D.M., N.H., C.J.L., R.M., W.B.K., L.D.K.), Ohio University, Athens, Ohio 45701; The Ohio State University, Arthur G. James Cancer Center and Richard J. Solove Research Institute (M.S.), Columbus, Ohio 43210; The Howard Hughes Medical Institute (A.D.K., R.T.M.) and Department of Hematology (A.D.K.), University of Washington, Seattle, Washington 98195; and Department of Internal Medicine (W.B.K.), Asan Medical Center, University of Ulsan College of Medicine, Seoul 138-736, Korea

Address all correspondence and requests for reprints to: Leonard D. Kohn, Konneker Research Building, Room 077, The Ridges, Building 25, Ohio University, Athens, Ohio 45701. E-mail: kohnl{at}ohiou.edu.

	Abstract

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High basal levels of TLR3 and Wnt5a RNA are present in papillary thyroid carcinoma (PTC) cell lines consistent with their overexpression and colocalization in PTC cells in vivo. This is not the case in thyrocytes from normal tissue and in follicular carcinoma (FC) or anaplastic carcinoma (AC) cells or tissues. The basally expressed TLR3 are functional in PTC cells as evidenced by the ability of double-strand RNA (polyinosine-polycytidylic acid) to significantly increase the activity of transfected NF-B and IFN-ß luciferase reporter genes and the levels of two end products of TLR3 signaling, IFN-ß and CXCL10. Phenylmethimazole (C10), a drug that decreases TLR3 expression and signaling in FRTL-5 thyrocytes, decreases TLR3 levels and signaling in PTC cells in a concentration-dependent manner. C10 also decreased Wnt5a RNA levels coordinate with decreases in TLR3. E-cadherin RNA levels, whose suppression may be associated with high Wnt5a, increased with C10 treatment. C10 simultaneously decreased PTC proliferation and cell migration but had no effect on the growth and migration of FC, AC, or FRTL-5 cells. C10 decreases high basal phosphorylation of Tyr705 and Ser727 on Stat3 in PTC cells and inhibits IL-6-induced Stat3 phosphorylation. IL-6-induced Stat3 phosphorylation is important both in up-regulating Wnt5a levels and in cell growth. In sum, high Wnt5a levels in PTC cells may be related to high TLR3 levels and signaling; and the ability of phenylmethimazole (C10) to decrease growth and migration of PTC cells may be related to its suppressive effect on TLR3 and Wnt5a signaling, particularly Stat3 activation.

	Introduction

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THYROID CANCER ACCOUNTS for a large proportion of endocrine-related cancer deaths each year (1). Although the origin of the vast majority of these cancers is the follicular epithelium (1), there are three morphological subtypes: papillary thyroid carcinoma (PTC), follicular carcinoma (FC), and anaplastic carcinoma (AC) (1). PTC is the least aggressive and is usually associated with a more favorable prognosis, the basis for which is unclear (1). PTC, unlike FC or AC, is often associated with immune cell infiltrates that surround the transformed thyrocytes, can resemble immune cell infiltrates in Hashimoto’s thyroiditis, and are associated with the more favorable prognosis (2).

The Wingless (Wnt) family of secreted glycoproteins control early developmental processes including cellular migration, differentiation, and proliferation (reviewed in Ref. 3). The best characterized are canonical Wnts, whose signaling pathway involves glycogen synthase kinase-3-dependent and -independent regulation of cytoplasmic ß-catenin levels. Increases in ß-catenin result in nuclear localization and activation, binding to lymphoid enhancer-binding (LEF)/T-cell factor (TCF) transcription factors, and expression of downstream genes, some of which control growth (4, 5, 6). Noncanonical (ß-catenin-independent) Wnt signaling is less well characterized but is also thought to modulate cell proliferation by inducing the release of intracellular Ca²⁺ and activating both protein kinase C (PKC) (7, 8, 9) and calcium-calmodulin kinase II (CaMKII). Constitutive activation of noncanonical Wnt signaling is associated with tumorigenesis, because noncanonical Wnt5a is up-regulated in many types of human cancers (10, 11, 12, 13). Although a recent study suggests that Wnt5a activation of PKC contributes to enhanced motility and invasiveness of metastatic melanoma (11), Wnt5a is categorized as a nontransforming Wnt based on studies in the C57MG mouse mammary epithelial cell (14, 15, 16) and the observation that loss of function of Wnt5a leads to elevation of ß-catenin, whereas gain of function of Wnt5a suppresses ß-catenin signaling (17, 18). Thus, Wnt5a is a tumor suppressor in some leukemias, and its loss of expression elevates ß-catenin signaling and reduces CaMKII signaling (19). A recent report suggested that there was an elevated level of noncanonical Wnt5a in most or all PTC but in only some FC (20). The significance of this was not clear, nor was the basis for its high basal levels. In this report, we further explore this phenomenon and are able to relate the high basal Wnt5a expression to high basal Toll-like receptor 3 (TLR3) expression and signaling.

TLR are a family of cell surface receptors involved in the recognition of pathogen-associated signature molecules that signal the activation of innate and then adaptive immunity (21, 22, 23, 24, 25). Although the TLR family consists of more than 10 members (25), in humans, TLR3 had been reported to be restricted primarily to dendritic cells of the immune system (26). We, however, have recently shown that TLR3 is not only present and functional in thyrocytes but also that TLR3 were overexpressed in the thyrocytes of all patients examined with Hashimoto’s thyroiditis (17). Pancreatic islets of patients with insulitis and type 1 diabetes are also associated with overexpressed TLR3 (27). Because in both cases, disease expression is associated with immune cell infiltrates (21), because immune cell infiltrates are frequent in PTC but not FC or AC, and because we had shown TLR3 existed in PTC cells (21), we questioned whether TLR3 was overexpressed and functional in PTC but not FC or AC and whether it could increase the activity of genes potentially important in attracting immune cells. We further questioned what relationship it might have to Wnt5a expression, PTC growth, and PTC cell motility. We hypothesized that overexpressed TLR3 might be a phenomenon restricted to PTC, related to noncanonical Wnt5a expression and function, and potentially relevant to the more benign growth and metastatic characteristics of PTC.

TLR3 recognize double-stranded (ds)RNA and activate genes that increase inflammatory cytokines and costimulatory molecules important for immune cell interactions (25). In thyrocytes (21), like immune cells (25, 28, 29, 30, 31), the dsRNA activates two distinct signal pathways (25, 28, 32). One is the nuclear factor-B (NF-B)/MAPK signal path; the other involves IFN-regulatory factor (IRF)-3 and production of IFN-ß. The result is activation of signal transducers and activators of transcription (STATs) and up-regulation of genes important for attracting immune cells, such as the chemokine CXCL10 (25, 28, 32), and for interacting with immune cells, such as major histocompatibility genes (21, 33). We have shown that phenylmethimazole (C10), a more potent derivative of a drug used to treat Graves’ disease, methimazole (21), can inhibit dsRNA signaling in thyrocytes by its ability to inhibit the TLR3-mediated IRF-3/IFN-ß pathway and can inhibit STAT activation in FRTL-5 thyrocytes (21). We took advantage of this C10 activity to explore the relationship between TLR3 and Wnt5a in PTC cells.

In this report we show that basal TLR3 and Wnt5a are coordinately overexpressed in PTC but not FC or AC cells. We show that C10 coordinately decreases TLR3 and Wnt5a expression and signaling in PTC cells, suggesting that they are interrelated signal systems. We link the phenomena to the growth and migration of PTC cells.

	Materials and Methods

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Materials
For the in situ tissue studies, Vectastain Universal Quick Kit (Vector Laboratories, Burlingame, CA) antigen unmasking solution and 3,3'-diaminobenzidine substrate kit were used. The anti-phospho-Stat3 (S⁷²⁷) antibody was from BioSource International (Camarillo, CA), the anti-phospho-Stat3 (Y⁷⁰⁵) was from Cell Signaling Technologies (Beverley, MA), and anti-ß-catenin and anti-vascular endothelial growth factor (anti-VEGF) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Polyinosine-polycytidylic acid (poly-I:C) (a synthetic dsRNA), the endotoxin-free Escherichia coli DNA, and the human TLR3 expression vector were purchased from Invivogen (San Diego, CA). TNF-, IFN-ß, IL-6, and IL-1ß were from Biosource International. C10 was a gift of the Interthyr Corp. (Athens, OH). C-10 was prepared as 200 mM stock solution in dimethylsulfoxide (DMSO). The source of all other materials was the same as previously reported or is noted below (21).

Cells
Papillary carcinoma cell line NPA-87, FC cell line WRO 82-1, and AC cell line ARO 81-1 were kindly provided by Dr. Guy Juillard (University of California, Los Angeles, CA). Papillary carcinoma cell lines BHP 18-21, BHP 10-3, BHP 2-7, and BHP 7-13 were kindly provided by Dr. Jerome Hershman (University of California, Los Angeles, CA). All cell lines were grown in RPMI 1640 medium supplemented with 2 g/liter sodium bicarbonate, 1.4 mM sodium pyruvate, 0.14 mM nonessential amino acids, and 10% fetal bovine serum (pH 7.2).

RNA isolation and Northern analysis
Total RNA was isolated using the RNeasy mini kit (QIAGEN Inc., Valencia, CA) according to the manufacturer’s instructions. For Northern analysis, 20 µg total RNA per sample was run on denaturing agarose gels, blotted onto Nytran membranes (Schleicher & Schuell, Keene, NH) using capillary transfer, UV cross-linked, and hybridized. Probes were labeled with [-³²P]dCTP using the Ladderman labeling kit (Takara, Madison, WI). The probe for TLR3 was the human whole cDNA obtained from the Invivogen human TLR3 expression vector. The probe for Wnt5a was obtained from a human Wnt5a expression vector graciously provided by Drs. Aimee Kohn and Randall Moon (University of Washington School of Medicine, Seattle, WA). The probe for GAPDH has been described (33). The probe for IRF-1 has been previously described (33). Membrane hybridization and washes were performed as previously described (21, 33). Northern blots were developed using either a BAS 1500 bioimaging analyzer (Fuji Photo Film Medical Systems USA, Stamford, CT) or an SRX-101 x-ray film processor (Konica Minolta Photo Imaging USA Inc., Mahwah, NJ).

RT-PCR
DNA was removed from total RNA using the DNA-free kit (Ambion, Inc., Austin, TX) according to the manufacturer’s instructions. A total of 1 µg RNA was then used to synthesize cDNA using the Advantage RT-for-PCR kit (BD Biosciences, Palo Alto, CA) according to the manufacturer’s protocol. A total of 50 ng cDNA was subsequently used for PCR of TLR3, ß-actin, CXCL10, E-cadherin, and Wnt5a, and 250 ng cDNA was used for PCR of IFN-ß. The primers used for TLR3, ß-actin, Wnt5a, Wnt 1, and Wnt3a have been previously described (34, 35, 36). The 5' and 3' primers for CXCL10 were, respectively, 5'-GCCTACAGCAGAGGAACCTCCAGTCTCAGC-3' and 5'-CTTCACTACCCTCTCCGTCGGAGACACACC-3'. IFN-ß primers were, respectively, 5'-TGGCAATTGAATGGGAGGCTTG-3' and 5'-TCCTTGGCCTTCAGGTAATGCAGA-3'. E-cadherin primers were 5'-TACTGTGTAGAGAAGACGCC-3' and 5'-AGAATGAAGCGGAGGCAACT-3', respectively. PCR conditions for TLR3, ß-actin, CXCL10, E-cadherin, Wnt5a, Wnt1, and Wnt3a are as follows: 94 C for 5 min followed by 35 cycles of 94 C for 30 sec, 55 C for 30 sec, 72 C for 1 min, and a final cycle of 72 C for 7 min IFN-ß PCR conditions were 94 C for 3 min, followed by 35 cycles of 94 C for 10 sec, 58 C for 30 sec, 72 C for 1 min, and a final cycle of 72 C for 10 min.

Western blot analysis
Whole-cell lysates were prepared in lysis buffer containing 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, and a mixture of protease inhibitors (phenylmethylsulfonyl fluoride, leupeptin, and pepstatin A) from Pierce Chemical Co. (Rockford, IL). Nuclear proteins were isolated using the NE-PER nuclear and cytoplasmic extraction reagents (Pierce) according to the manufacturer’s instructions. Thirty micrograms of protein were resolved on denaturing gels using the Nu-PAGE System (Invitrogen, Carlsbad, CA). All proteins were transferred to nitrocellulose membranes, and subsequent antibody binding was revealed using enhanced chemiluminescence (ECL)-plus reagents (Amersham Pharmacia Biotech, Piscataway, NJ).

Primary antibody to total ß-catenin was against theC-terminal portion (amino acids 680–781) of human ß-catenin (Santa Cruz Biotechnology), and antibody to active ß-catenin was against amino acid residues 36–44 of human ß-catenin of which Ser37 and Thr41 were both dephosphorylated (Upstate USA Inc., Charlottesville, VA). Primary antibody against phospho-Stat3 (S⁷²⁷) was from BioSource, and phospho-Stat3 (Y⁷⁰⁵) and ß-actin antibodies were from Cell Signaling Technologies (Beverly, MA). Primary antibody against VEGF was from Santa Cruz Biotechnology. After blocking with 5% nonfat milk/Tris-buffered saline solution with 0.1% Tween 20, nitrocellulose membranes were incubated with 1% nonfat milk/Tris-buffered saline solution with 0.1% Tween 20 solution containing primary antibody (1:1000 dilution) overnight at 4 C. After washing, membranes were incubated with the same solution containing secondary antibodies conjugated with horseradish peroxidase (1:5000) for 2 h at room temperature before membranes were reacted with ECL-plus reagent for 1 min and then exposed to x-ray film.

Luciferase assays and plasmids
These have been previously described in detail (21). In brief, the human IFN-ß promoter sequence was amplified from human genomic DNA (Clontech, Palo Alto, CA) using Ex Taq polymerase (Takara, Madison, WI) and the following primers: hIFN-ß (–125) KpnI (5'-CAGGGTACCGAGTTTTAGAAACTA CTAAAATG-3') and hIFN-ß (+34) XhoI (5'-GTACTCGAGCAAAGGCTTCGAAAG G-3'). The fragment was digested with KpnI and XhoI and then ligated into a similarly digested pGL3 Basic (Promega, Madison, WI) vector. NF-B-luc was purchased from Stratagene. For transfection studies, cells were grown in 10-cm dishes to about 80% confluence and then exposed to a plasmid-Lipofectamine 2000 mixture as described by the manufacturer (Invitrogen). Cells were transfected for 24 h with 100 ng of the indicated constructs or 3 ng pGL3 basic luciferase reporter vector as control. All cells were also transfected with phRL-TK (Int-) vector (Promega), which contains wild-type Renilla luciferase (Rluc) as an internal transfection control. Luciferase assays were conducted with the dual-luciferase reporter assay system (Promega) on a Lumat LB 9507 tube luminometer. In every experiment, each condition was run in at least triplicate wells.

Quantification of cell growth
Cells were evenly seeded and grown on sterile glass slides. Cells were then treated with varying concentrations of C10 for 24 h. In one method, cell growth was quantified by counting the number of cells per unit area. Four different areas, corresponding to the same area on each slide, were sampled. In a second method, cell growth was quantified using the MTT-based in vitro toxicology assay kit from Sigma Aldrich (St. Louis, MO).

Scratch assays
Scratch assays were performed as previously described (11). Briefly, cells were grown to confluency, a scratch was made in the confluent layer of cells using a sterile pipette tip, treated (or not), and analyzed for ability to heal the scratch. Scratch assays were documented using digital photography.

Patients and tissue samples
De-identified tissue specimens were obtained from individuals treated at the Ukrainian Center of Endocrine Surgery in Kiev as previously noted (17). The project was approved by the Institutional Research Committee at the Ukrainian Center of Endocrine Surgery in Kiev, and informed consent was obtained. Thyroid lesions were classified as PTC, FC, or AC based on their histology (37). Normal thyroid tissue was largely from the contralateral glands of patients undergoing thyroid surgery for the tumors. After fixation in 10% formalin and embedding in paraffin, 5-µm-thick serial sections were made for each specimen. The 5-µm sections were stained with hematoxylin and eosin.

Immunohistochemical analysis
Tissue sections were dewaxed, soaked in alcohol, and treated in antigen unmasking solution in the microwave for 10 min followed by incubation in 3% hydrogen peroxide for 15 min to inactivate endogenous peroxidase activity. Sections were then incubated at 4 C overnight with anti-TLR3 (1:100 dilution) or anti-Wnt5a (5 µg/ml). Immunostaining was done using the Vectastain Universal Quick Kit according to the manufacturer’s instructions. 3,3'-Diaminobenzidine was used to visualize peroxidase staining. A negative control involved omission of antiserum.

Statistics
All experiments were replicated at least three times on different groups of cells. All data are expressed as mean ± SD. Student’s t tests were used to evaluate statistical significance between poly-I:C-stimulated vs. untreated groups in Fig. 3, A and B. Data in Figs. 4 and 9 were evaluated for statistical significance using one-way ANOVA, and statistical significance for comparison of means of different groups was calculated using Bonferroni post hoc analyses.

View larger version (16K): [in this window] [in a new window]   FIG. 3. TLR-3 is functional in PTC cells. A, Treatment with poly-I:C activates NF-B in NPA thyrocytes. B, Treatment with poly I:C activates IFN-ß in NPA thyrocytes. In A and B, NPA cells were transiently transfected with 100 ng luciferase reporter plasmid pNF-B Luc or pIFN-ß Luc and 2 ng of internal control plasmid phRL-Tk. Twenty-four hours after transfection, cells were incubated with poly-I:C (100 µg/ml), IL-1ß (1 ng/ml), or TNF- (10 ng/ml). Luciferase activity was measured using the dual luciferase assay system (Promega). Error bars represent SD. *, P < 0.0001 between untreated and poly-I:C groups. C, The mRNA levels of IFN-ß increase after dsRNA incubation and transfection. NPA cells were treated or transfected with the indicated amounts of poly-I:C for 24 h, and IFN-ß mRNA levels were assessed by semiquantitative RT-PCR. D, CXCL10 is expressed basally in NPA thyrocytes but not in WRO or ARO thyrocytes. E, CXCL10 is up-regulated by IFN-ß and dsRNA but not by dsDNA. Semiquantitative RT-PCR was used to measure mRNA levels of IFN-ß and CXCL10.

View larger version (19K): [in this window] [in a new window]   FIG. 4. C10 inhibits poly-I:C-induced NF-ß activation in NPA thyrocytes. NPA cells were transiently transfected with 100 ng luciferase reporter plasmid pNF-B Luc and 2 ng internal control plasmid phRL-Tk. Twenty-four hours after transfection, cells were incubated with poly-I:C (100 µg/ml) or poly-I:C in addition to 0.25% DMSO or indicated amounts of C10 for 6 h. Luciferase activity was measured using the dual luciferase assay system (Promega). Error bars represent SD. *, P < 0.01 between groups as indicated.

View larger version (25K): [in this window] [in a new window]   FIG. 9. C10 inhibits cellular proliferation of NPA thyrocytes. Cellular proliferation was measured after a 24-h treatment with the indicated amounts of C10, using either traditional cell-counting methods (A) or the MTT-based in vitro toxicology assay kit (Sigma) (B) according to the manufacturer’s instructions. The relative number of cells per well was quantified by measuring the absorbance at 570 nm of the solution resulting from the solubilization of the formazan crystals formed during the assay. Error bars represent SD. *, P < 0.05 between groups as indicated.

	Results

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View larger version (97K): [in this window] [in a new window]   FIG. 1. TLR3 is overexpressed in human PTC and colocalizes with overexpressed Wnt5a in thyrocytes. A, Immunostaining of 20 separate thyroid tumors, six loci each, revealed that TLR3 was positive in all 12 PTC studied, two representative immunostains being depicted in the right two panels. Immunostaining of normal tissue present in contralateral lobes of patients with PC did not reveal TLR3 as indicated by the representative data in the left-most panel. Similarly, we did not observe TLR3 immunostaining in four different follicular thyroid carcinoma specimens as illustrated with the picture in the second panel from the left. B, TLR3 and Wnt5a immunostaining of the same PTC at the border of the tumor panels a and c and in the center of the tumor in panels b and d. This is representative of all PTC tumors examined. Immunostaining is described in Materials and Methods.

TLR3 is expressed in cells derived from human papillary but not follicular (WRO) or anaplastic (ARO) thyroid tumors
We next asked whether TLR3 was expressed in cultured NPA cells derived from a PTC but not on WRO thyrocytes derived from a follicular thyroid carcinoma or ARO cells derived from an anaplastic thyroid carcinoma. Of the three human thyroid tumor cell lines, we could demonstrate that only NPA cells contained basal levels of TLR3 mRNA (Fig. 2A). Detectable basal levels of TLR3 mRNA were also measured in four other PTC cell lines: BHP 10-3, BHP 2-7, BHP 7-13, and BHP 18-21 (Fig. 2B). These results, together with the in situ studies of TLR3 protein expression in papillary thyroid cancers (see Fig. 1), suggest that basal TLR3 gene expression is a phenomenon unique to PTC. FRTL-5 cells were used as a positive control for TLR3 expression (data not shown) (21). The coincident expression of Wnt5a RNA was consistent with the immunohistochemistry data and will be further discussed below.

View larger version (41K): [in this window] [in a new window]   FIG. 2. TLR3 and Wnt5a are expressed in cells derived from human papillary but not follicular (WRO) or anaplastic (ARO) thyroid tumors. A and B, The mRNA levels of TLR-3 and Wnt5a in NPA, WRO, and ARO thyrocytes (A) and in BHP 10-3, 18-21, 2-7, and 7-13 thyrocytes (B) as determined by semiquantitative RT-PCR.

To see whether TLR3 was functional, we first incubated NPA cells with poly-I:C and evaluated its ability to increase the NF-B signal path in pNF-B-Luciferase-transfected NPA cells by measuring reporter gene activity (Fig. 3A). Poly-I:C incubation significantly (P < 0.0001) increased NF-B luciferase activity by comparison with untreated cells or cells transfected with control plasmid alone (Fig. 3A). Cells treated with IL-1ß and TNF- served as positive controls for NF-B activation.

We next determined whether the TLR3 signal transduction pathway that is coupled to IRF-3/IFN-ß was functional by using IFN-ß-Luciferase-transfected NPA cells. Poly-I:C incubation significantly (P < 0.0001) increased IFN-ß promoter activity by comparison with untreated cells or cells transfected with control plasmid alone (Fig. 3B). Treatment with IL-1ß served as a positive control for IFN-ß promoter activity.

Consistent with the increase in IFN-ß promoter activity, dsRNA incubation increased IFN-ß mRNA levels as measured by RT-PCR (Fig. 3C). Additionally, consistent with our previous results in FRTL-5 rat thyrocytes (21), we could show that dsRNA transfection was more effective than dsRNA incubation in increasing IFN-ß mRNA levels. The dsRNA transfection effect was not duplicated by dsDNA transfection, which had no apparent ability to increase IFN-ß RNA levels above their basal values in PTC cells (Fig. 3C).

Associated with the basal levels of TLR3 and IFN-ß in PTC, we could detect basal levels of the chemokine CXCL10 in PTC but not follicular (WRO) or anaplastic (ARO) cells (Fig. 3D). As expected, dsRNA but not dsDNA could further increase CXCL10 RNA levels (Fig. 3E). The ability of treatment with IFN-ß to also increase CXCL10 RNA levels is consistent with our previous results (21), which suggested that IFN-ß was a functional intermediate in the dsRNA action and could increase major histocompatibility gene expression as well as chemokines and cytokines needed for attracting immune cells. CXCL10 is largely a product of the IRF-3/IFN-ß/IRF-1 signaling pathway, although its promoter does have NF-B elements (39, 40, 41).

In sum, these data indicate that PTC cells basally express TLR3 RNA and that TLR3 signal systems are functional in these cells. High basal TLR3 levels and TLR3 signals capable of increasing cytokines (IFN-ß; Fig. 3) and chemokines (CXCL10; Fig. 3) in PTC cells in vitro are consistent with the existence of immune cell infiltrates in vivo based on related studies suggesting that elevated TLR3/TLR3 signals in Hashimoto’s and insulitis/diabetes are associated with immune cell infiltrates (21, 42). The absence of basally expressed TLR3 in follicular (WRO) and anaplastic (ARO) carcinoma cells in vitro would be consistent with a paucity of immune cell infiltrates in these tumors in vivo.

TLR3 expression and signaling is inhibited by phenylmethimazole (C10)
We asked whether C10 might decrease the basal levels of TLR3 RNA and TLR3 signaling in PTC cells, because it is effective in this regard in FRTL-5 thyrocytes (21). We could demonstrate concentration-dependent inhibition of poly-I:C-induced NF-ß activation in NPA thyrocytes transfected with the NF-ß-Luciferase construct by comparison with control solvent (DMSO)-treated cells (Fig. 4). Similar results were evident measuring the effect of C10 on poly-I:C-induced promoter activity in IFN-ß-Luciferase-transfected NPA cells (data not shown).

Additionally, Northern analysis revealed that treatment of all PTC cell lines with C10 significantly decreased TLR3 RNA levels. This is illustrated in studies of the NPA and BHP 10-3 cells (Fig. 5A) but was true for all BHP lines (data not shown) and was confirmed by PCR (data not shown). These data, in addition to that in Fig. 1, support the conclusion that the basal levels of TLR3 are abnormally high in the PTC cells, because TLR3 expression levels and TLR3 signaling can be so effectively lowered by C10.

View larger version (16K): [in this window] [in a new window]   FIG. 5. C10 inhibits basal TLR-3 and Wnt5a gene expression in PTC cells. A, Northern analyses of human TLR-3 expression in NPA and BHP 10-3 PTC cell lines. B, Northern analysis of human Wnt5a expression in BHP 10-3 cells. NPA and BHP 10-3 PTC cells lines were treated with indicated amounts of DMSO (solvent) or C10 for 24 h. Cells were then harvested, total RNA isolated, and respective Northern analyses performed.

View larger version (25K): [in this window] [in a new window]   FIG. 6. Basal IRF-1 expression is inhibited by C10. NPA thyrocytes were treated as indicated. Total RNA was then isolated, and IRF-1 gene expression was analyzed using Northern blot analysis.

C10 exposed a potential functional relationship between the high basal Wnt5a and TLR3. Thus, C10 decreased expression of Wnt5a RNA levels in all of the PTC cell lines along with TLR3 RNA, as illustrated with the NPA and BHP 10-3 cells (Fig. 5B). The effect of C10 was concentration dependent (Fig. 5B) with 0.5 mM C10 having a maximal effect. The solvent (DMSO) had no effect (data not shown). These results were confirmed by RT-PCR analysis (data not shown).

The possibility that expression of Wnt5a and TLR3 were related was also suggested when NPA cells were transfected with dsRNA. As shown previously in FRTL-5 cells (21) and in PTC cells in Fig. 3, C and E, dsRNA but not dsDNA, could further increase IFN-ß levels and CXCL10 RNA levels, respectively, as well as TLR3 levels. As evidenced in Fig. 7, dsRNA but not dsDNA transfection increased both TLR3 and Wnt5a RNA levels. Thus, there was a coordinate decrease in both TLR3 and Wnt5a RNA with C10 treatment and a coordinate increase with dsRNA transfection, supporting a functional relationship.

View larger version (19K): [in this window] [in a new window]   FIG. 7. TLR3 and Wnt5a are up-regulated by dsRNA transfection in PTC cell lines. dsRNA, but not dsDNA, transfection induces an increase in both TLR3 and Wnt5a mRNA expression as determined by semiquantitative RT-PCR. NPA and BHP 10-3 cell lines were transfected with 10 µg poly-I:C or endotoxin-free E. coli DNA for 24 h.

View larger version (13K): [in this window] [in a new window]   FIG. 8. C10 reverses Wnt5a-associated suppression of E-cadherin expression. E-cadherin is present at high levels basally in WRO thyrocytes but is expressed at very low basal levels in NPA thyrocytes. Treatment of NPA thyrocytes with C10 leads to an increase in E-cadherin expression that coincides with C10 inhibition of Wnt5a expression (Fig. 5B). E-cadherin mRNA was analyzed using semiquantitative RT-PCR.

View larger version (31K): [in this window] [in a new window]   FIG. 10. C10 inhibits migration of NPA cells in culture. NPA cells were grown to confluency, and migration was analyzed using a scratch assay that has been previously described (11 ). After scratching, cells were then treated or not with 0.25% DMSO or 0.5 mM C10 for 24 h.

To test this hypothesis, we analyzed the effects of C10 on Stat3 phosphorylation, which we could detect basally (Fig. 11A). We observed a small decrease in basal phosphorylated Stat3 (Y⁷⁰⁵) and Stat3 (S⁷²⁷) by 2 h of treatment with C10; however, by 4 h, C10 largely decreased phosphorylated Stat3 (Y⁷⁰⁵) and Stat3 (S⁷²⁷) (Fig. 11A). Likewise, we observed a large decrease in IL-6-induced phosphorylated Stat3 (Y⁷⁰⁵) (Fig. 11B) protein levels after C10 treatment. The effect of C10 was not restricted to inhibition of IL-6-induced increases in Stat activation; it inhibited IL-6-induced increases in VEGF as well (Fig. 12). Comparison of PTC, FC, and AC revealed that phospho-Stat3 is present basally only in NPA cells and not in WRO or ARO cells (data not shown). The latter is consistent with our findings that TLR3 and Wnt5a expression and signaling are also unique to PTC.

View larger version (24K): [in this window] [in a new window]   FIG. 11. C10 inhibits basal and IL-6-induced Stat3 phosphorylation. A, NPA, thyrocytes were treated with 0.25% DMSO or 0.5 mM C10 for the indicated, times. Nuclear protein was then extracted, and basal phospho-Stat3 (Y705) and (S727) were analyzed via Western analysis using phospho-Stat3-specific antibodies. B, NPA thyrocytes were pretreated for 1 h with or without 0.25% DMSO or 0.5 mM C10 and then treated or not for 10 min with 100 ng/ml human IL-6 as indicated. Nuclear protein was then extracted, and phospho-Stat3 (Y705) was evaluated by Western analysis.

View larger version (16K): [in this window] [in a new window]   FIG. 12. C10 inhibits IL-6-induced VEGF expression. NPA thyrocytes were pretreated for 1 h with or without 0.25% DMSO or 0.5 mM C10 and then treated or not for 10 min with 100 ng/ml human IL-6 as indicated. Protein was then extracted, and VEGF expression was evaluated by Western analysis.

View larger version (21K): [in this window] [in a new window]   FIG. 13. NPA thyrocytes have similar levels of total ß-catenin but low basal levels of active ß-catenin relative to WRO and ARO cells. Total ß-catenin was measured using an antibody against the C-terminal portion (amino acids 680–781) of human ß-catenin (Santa Cruz Biotechnology). Active ß-catenin was measured using an antibody against amino acid residue 36–44 of human ß-catenin of which Ser37 and Thr41 were both dephosphorylated (Upstate USA). Western analyses were performed as described in Materials and Methods.

	Discussion

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Fujio et al. (54) have demonstrated that signals modulated by gp130, a common receptor of the IL-6 family of cytokines, up-regulates Wnt5a through Stat3, thereby promoting N-cadherin-mediated cell adhesion in cardiac myocytes. We have shown that IL-6 can activate Stat3 in PTC cells and have observed overexpression of Wnt5a in PTC cells. The hypothesis that emerges is that increased TLR3 signaling, resulting from its high basal expression, may contribute to high levels of activated Stat3 and consequent high levels of Wnt5a levels in PTC. Support for this hypothesis is found in a very recent report by Blumenthal et al. (64) in which they show that bacteria and bacterial lipopolysaccharide, which activates TLR4 and whose signal system is similar to TLR3 (24), can induce the up-regulation of Wnt5a gene expression in human monocytes. The ability of C10 to inhibit TLR3 signaling (21), and concordantly decrease activated Stat3 and Wnt5a RNA levels, supports the conclusion that their expression is similarly regulated. Experiments are in progress with TLR3 siRNA to support this hypothesis and will be presented separately (McCall, K. D., A. Schwartz, A. Weeraratna, J. Wortsman, A. Slomirski, R. Moon, A. D. Kohn, F. Schwartz, D. Goetz, and L. D. Kohn, manuscript in preparation).

Wnt5a-dependent growth and motility have been associated with PKC signaling (7, 8, 9). Of interest in this respect, active ß-catenin levels in PTC cells are low by comparison with follicular (WRO) or anaplastic (ARO) thyroid carcinoma cells, where increased ß-catenin is linked to growth in association with PTEN mutations and mutations preventing ß-catenin degradation (65, 66). In a separate report (67), we have shown that TSH decreases the active form of ß-catenin while increasing the growth of FRTL-5 thyroid cells. Additionally, using FRTL-5 cells, we showed that overexpression of canonical Wnt-1 significantly increased the growth rate without increasing ß-catenin levels. The increased growth was blunted by a PKC inhibitor, staurosporine, in association with inhibition of Stat3 phosphorylation. Combined, these results suggest that 1) canonical and noncanonical Wnt signaling can contribute to enhanced cellular growth via a Ca²⁺/PKC pathway and increases in Stat3 phosphorylation/activation, 2) TLR3-signaled increases in Stat3 might be a major contributor not only to increases in Wnt5a but also to the growth of PTC tumors, and 3) expression of Wnt5a and relatively low levels of active ß-catenin protein in PTC, compared with WRO or ARO cells, suggest that the mechanism of enhanced cellular growth in NPA cells is different from that of WRO or ARO cells, i.e. involvement of a noncanonical rather than a canonical Wnt pathway.

In our separate report (67), we also showed that increased active ß-catenin decreased TPO mRNA and suppressed TPO promoter activity. The target of active ß-catenin suppressive action was a consensus TCF/LEF-binding site 5'-A/T A/T CAAAG-3', –137 to –129 bp on the rat TPO promoter. ß-Catenin overexpression significantly increased complex formation between ß-catenin/TCF-1 and an oligonucleotide containing the TCF/LEF sequence, suggesting that the ß-catenin/TCF-1 complex acts as a transcriptional repressor of the TPO gene. The low levels of active ß-catenin in NPA cells would likely relieve thyroid-specific functional gene suppression, whereas the higher active ß-catenin levels in follicular and anaplastic thyroid cancers might promote growth but would also suppress function. These data may provide a potential explanation for the relative preservation of function in PTC relative to anaplastic or follicular thyroid carcinoma, as well as their very different growth and migration properties.

Both here and in our previous report using FRTL-5 thyrocytes, we show that C10 decreases TLR3 signaling through the IRF-3/IFN-ß/IRF-1 signal path (21). Of interest, IRF-3 activation is related to Akt signaling (68), and Akt signaling is clearly related to Wnt and growth in cells, including thyrocytes (69, 70, 71). We have, however, no present knowledge at this point whether or how C10 plays a significant role in Akt signaling. An important point of action in the IRF-3/IFN-ß signal path is increased IRF-1 gene expression. C10 blocks this increase in thyrocytes and blocks IRF-1 increases induced by the cytokine TNF- in human aortic endothelial cells (44). IRF-1 is clearly related to cancer growth (43, 46, 47, 48), but a role for IRF-1 in Wnt5a signaling is not, to our knowledge, currently known.

The sum of data suggests the model depicted in Fig. 14. An inciting event unique to at least some PTC cells can increase TLR3 and Wnt5a. TLR3 signal generation via TIR domain-containing molecule adapter inducing IFN-ß (TRIF)/Toll/IL-1 receptor-containing TLR adapter (TICAM)-1/IRF-3 increases IFN-ß, which can act as an autocrine/paracrine factor to further increase TLR3 (21). TLR3 induction of both the NF-B and IFN-ß signal pathways contribute to increases in IL-6 (24, 32) and the subsequent activation of Stat3, which can increase Wnt5a. C10 inhibits the TLR3-activated NF-B and IRF-3 signaling, IRF-3-dependent increase in IFN-ß, and the TLR3-induced increase in Stat3 phosphorylation. Phosphorylated Stat3 appears critical to both the increase in Wnt5a as well as the growth and motility of the PTC cells; cytokines and chemokines resulting from overexpressed TLR3 signaling are important in attracting immune cell infiltrates. Although at this point the model in Fig. 14 is speculative, it not only can account for the results presented in this report but also raises several points to consider.

View larger version (26K): [in this window] [in a new window]   FIG. 14. Speculative model of C10 action on TLR3 and Wnt5a signaling in PTC. C10’s ability to inhibit TLR3 expression and signaling at multiple loci, i.e. by preventing NF-B, IRF3/IFN-ß, and subsequent IL-6, CXCL10, and other cytokine/chemokine production as well as Stat-3 activation and Wnt5a expression may contribute to the inhibition of PTC cell growth and motility. Double slash marks denote inhibition of the respective pathways shown in this manuscript to be inhibited by C10. Based on already published literature, it is hypothesized that Stat3 is a key mediator of cancer cell growth and Wnt5a migration (11 54 55 56 57 ). Likewise, it is suggested in the literature that chemokines and cytokines that are products of the TLR3 signaling pathway are important for immune cell infiltration of tissues (21 25 28 32 33 ). Thus, the circles with the lines through them denote inhibition of growth, motility, and immune cell infiltration as a downstream effect of the inhibition of TLR3 signaling by C10 of these pathways that are hypothesized, based on the literature, to control cell growth and migration as well as immune cell infiltration.

Third, in FRTL-5 cells, we linked the dsRNA increase in TLR3 to virus infection (21), and viruses have been implicated in Wnt overexpression (72, 73). Two IB kinase-related kinases, Iß kinase (IKK) and TANK binding kinase 1 (TBK1), are implicated in the TLR increase associated with lipopolysaccharide signaling, dsRNA signaling, and viral infection (74, 75). It is unclear whether the same kinases are implicated in the Wnt5a activation pathway.

Last, the PTC cells in this report are associated with either BRAF or Ret/PTC mutations (76, 77). As pointed out in the introductory section, PTC are one of the most frequent of endocrine neoplasms, yet account for few cancer-related deaths. Hashimoto’s thyroiditis is one of the most frequent autoimmune diseases involving the thyroid. Although the mechanism linking malignancy and autoimmunity is not clear, expression of the oncogenic fusion protein RET/PTC3 (RP3) in 95% of patients with Hashimoto’s thyroiditis and in PTC is of interest and provides a potential link between TLR3, Ret/PTC protooncogenes, the immune cell infiltrates, and the improved prognosis (1, 78). Interestingly, the signaling caused by activated RET kinase involves overlapping pathways, some common to the inflammatory response. RP3 alone causes increases in nuclear NF-B activity and secretion of monocyte chemoattractant protein-1 and granulocyte-macrophage colony-stimulating factor (79). Transfer of RP3-expressing thyrocytes into mice in vivo attracted dense macrophage infiltrates and rapid thyroid cell death (79). Furthermore, cytokine synthesis and inflammation was largely abrogated by mutation of RP3 Tyr588; an important protein-binding site for downstream signaling (79). Together, these studies implicate oncogene-induced cytokine-signaling pathways in a new mechanism linking inflammation with cancer and are in accord with the observations herein concerning overexpressed TLR3 in PTC and Hashimoto’s.

It has long been recognized that Wnt5a is overexpressed in many types of human cancers including, but not limited to, malignant melanoma, breast, and prostate cancer (10, 11, 12); however, its role in these cancers is not yet known. Interestingly, the growth of these cancers is thought to be controlled by the activation of Stat3 (57). We have observed that TLR3 is also overexpressed in these cancers (McCall, K., A. Schwartz, A. D. Kohn, F. Schwartz, D. Goetz, and L. D. Kohn, manuscripts in preparation) and based on the hypotheses developed in these studies of PTC, we are currently evaluating the efficacy of C10 as an anticancer agent in vivo in mouse models of human malignant melanoma, breast, and pancreatic cancers where TLR3 and Wnt5a are overexpressed basally (manuscripts in preparation). The model and data have thus opened new doorways for studies of Hashimoto’s, PTC, and other cancers, which appear to involve TLR3/Wnt5a signaling. They raise the possibility that C10 may be an effective therapeutic agent in cancer as well as autoimmune-inflammatory diseases.

	Footnotes

 
Disclosure Statement: K.D.M, N.H., C.J.L, R.M., W.B.K, and A.D.K. have nothing to declare. M.S. has equity interests in Interthyr Corp. R.T.M. is a consultant for Cgen Discovery and receives lecture fees from Merck and Pfizer. L.D.K. has equity interests in Interthyr Corp. and Diagnostic Hybrids, Inc., consults for both, and is an inventor on U.S. Patents 4,609,622, 5,556.754, 5,871,950, and 6,365,616.

First Published Online May 24, 2007

Abbreviations: AC, Anaplastic carcinoma; CaMKII, calcium-calmodulin kinase II; DMSO, dimethylsulfoxide; ds, double-stranded; FC, follicular carcinoma; IRF, IFN-regulatory factor; LEF, lymphoid enhancer-binding; NF-B, nuclear factor-B; PKC, protein kinase C; poly-I:C, polyinosine-polycytidylic acid; PTC, papillary thyroid carcinoma; STAT, signal transducers and activators of transcription; TLR3, Toll-like receptor 3; TCF, T-cell factor; VEGF, vascular endothelial growth factor.

Received April 9, 2007.

Accepted for publication May 16, 2007.

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