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Endocrinology Vol. 145, No. 10 4430-4438
Copyright © 2004 by The Endocrine Society

A Tumor Suppressor Role for Thyroid Hormone ß Receptor in a Mouse Model of Thyroid Carcinogenesis

Yasuhito Kato, Hao Ying, Mark C. Willingham and Sheue-Yann Cheng

Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute (Y.K., H.Y., S.-Y.C.), Bethesda, Maryland 20892-4264; and Department of Pathology, Wake Forest University School of Medicine (M.C.W.), Winston-Salem, North Carolina 27157-1072

Address all correspondence and requests for reprints to: Dr. S.-Y. Cheng, Laboratory of Molecular Biology, National Cancer Institute, 37 Convent Drive, Room 5128, Bethesda, Maryland 20892-4264. E-mail: sycheng{at}helix.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have created a knockin mutant mouse by targeting a mutation (PV) into the thyroid hormone receptor ß gene (TRßPV mouse). TRßPV/PV mice, but not TRßPV/+ mice, spontaneously develop follicular thyroid carcinoma. To identify other genetic changes in the TRß gene that could also induce thyroid carcinoma, we crossed TRßPV mice with TRß–/– mice. As TRßPV/– mice (mutation of one TRß allele in the absence of the other wild-type allele) aged, they also spontaneously developed follicular thyroid carcinoma through the pathological progression of hyperplasia, capsular and vascular invasion, anaplasia, and eventually metastasis to the lung, but not to the lymph nodes. The pathological progression of thyroid carcinoma in TRßPV/– mice was indistinguishable from that in TRßPV/PV mice. Analyses of the expression patterns of critical genes indicated activation of the signaling pathways mediated by TSH, peptide growth factors (epidermal growth factor and fibroblast growth factor), TGF-ß, TNF-{alpha}, and nuclear factor-{kappa}B, and also suggested progressive repression of the pathways mediated by the peroxisome proliferator-activated receptor {gamma}. The patterns in the alteration of these signaling pathways are similar to those observed in TRßPV/PV mice during thyroid carcinogenesis. These results indicate that in the absence of a wild-type allele, the mutation of one TRß allele is sufficient for the mutant mice to spontaneously develop follicular thyroid carcinoma. These results provide, for the first time, in vivo evidence to suggest that the TRß gene could function as a tumor suppressor gene. Importantly, these findings present the possibility that TRß could serve as a novel therapeutic target in thyroid cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID HORMONE NUCLEAR receptors (TRs) are ligand-dependent transcription factors critical for growth, development, and differentiation. Alternative splicing of the primary transcripts of the two TR genes, {alpha} and ß, yields four major thyroid hormone (T3) binding isoforms: {alpha}1, ß1, ß2, and ß3. These TR isoforms have an amino-terminal A/B domain, a central DNA-binding domain, and a carboxyl-terminal, ligand-binding domain. They differ in the length and amino acid sequence at the amino-terminal A/B domain, but bind T3 with high affinity to mediate gene regulatory activity. The amino- and carboxyl-terminal regions contain activation functions I and II, respectively, that are important for transcriptional activation (1, 2, 3). The expression of TR isoforms is tissue dependent and developmentally regulated (1, 2).

Early evidence to suggest that mutated TR could be involved in carcinogenesis came from the discovery that TR{alpha}1 is the cellular counterpart of the retroviral v-erbA that is involved in the neoplastic transformation leading to acute erythroleukemia and sarcomas (4, 5). v-erbA is a highly mutated chicken TR{alpha}1 that does not bind T3 and loses the ability to activate gene transcription. It competes with TR for binding to thyroid hormone response elements (TREs) and interferes with the normal transcriptional activity of liganded TR on several promoters (6, 7). Since those early studies, mutated TRs have been reported to associate with several human cancers, including liver (8), kidney (9), pituitary tumors (10, 11, 12), and thyroid (13). Reduced expression of TRß1 mRNA was also implicated in the carcinogenesis of human kidney cancer (14) and papillary thyroid carcinomas (13, 15). Recently, silencing of the TRß gene by hypermethylation and concurrent reduction of TRß1 transcripts were demonstrated in breast cancer (16). These studies raise the possibility that the TRß gene could function as a tumor suppressor.

The hypothesis that the TRß gene functions as a tumor suppressor was supported by the recent discovery that knockin mutant mice harboring two alleles of mutated TRß genes (TRßPV mouse) spontaneously develop follicular thyroid carcinoma (17). The PV mutation introduced into the TRß gene locus via homologous recombination was identified in a patient with thyroid hormone resistance syndrome (RTH) (18, 19). RTH is a genetic disease caused by mutations of the TRß gene (18). Most RTH patients are heterozygotes with only one mutant TRß allele (18, 20). One reported homozygous RTH patient died at an early age (21). RTH patients manifest the symptoms of dysfunction of the pituitary-thyroid axis with high circulating levels of TSH despite elevated levels of thyroid hormone (18, 20). PV has a unique mutation in exon 10, a C insertion at codon 448, which produces a frameshift of the carboxyl-terminal 14 amino acids of TRß1, resulting in total loss of T3-binding and transcriptional activities (22). TRßPV mice faithfully reproduce human RTH, and PV was shown to interfere with the transcriptional activity of wild-type TRs on T3-regulated genes in several target tissues in vivo (17).

The spontaneous development of follicular thyroid carcinoma in TRßPV/PV mice, but not in TRßPV/+ mice, prompted us to ask whether other genetic alterations in the TRß gene could also lead to the development of thyroid carcinoma. To answer this question, we crossed TRßPV/PV mice with TRß–/– mice and examined the propensity for development of thyroid carcinoma in the offspring. We found that because mice with one mutated TRß allele in the absence of the other wild-type allele (TRßPV/– mouse) aged, they spontaneously developed follicular thyroid carcinoma. The pathological progression of hyperplasia, capsular and vascular invasion, anaplasia, and metastasis was indistinguishable from that of TRßPV/PV mice. Moreover, there is a striking similarity between TRßPV/– and TRßPV/PV mice in the patterns of several altered signaling pathways during carcinogenesis. Thus, the mutation of one TRß allele in the absence of the other wild-type allele is sufficient to induce thyroid carcinoma, further strengthening the hypothesis that the TRß gene could function as a tumor suppressor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mouse strains
The animal protocol used in the present study was approved by the NCI animal care and use committee. Mice harboring the TRßPV gene (TRßPV mice) and mice deficient in TRß (TRß–/– mice) were prepared via homologous recombination, as previously described (17, 23). Genotyping was carried out using PCR as described previously (17, 23). TRßPV mice (on a hybrid background of 129/SvxC57BL/6) and TRß–/– mice (a hybrid background of 129/SvxC57BL/6J strains) (17, 23) were intercrossed to generate wild-type, TRß+/–, TRß–/–, TRßPV/+, TRßPV/–, and TRßPV/PV on the same mixed background for analysis.

Quantitative real-time RT-PCR
The LightCycler RNA Amplification Kit SYBR Green I was used according to the manufacturer’s protocols (Roche, Mannheim, Germany). A typical reaction mixture contained 5.2 µl H2O, 2.4 µl MgCl2 stock solution, 4 µl LightCycler RT-PCR Reaction Mix SYBR, 2 µl resolution solution, 0.4 µl LightCycler RT-PCR Enzyme Mix, 2.5 µl forward primer (2 µM), 2.5 µl reverse primer (2 µM), and 1 µl total RNA (200 ng). The cycles were 55 C for 30 min, 95 C for 30 sec, 95 C for 15 sec, 58 C for 30 sec, 72 C for 30 sec, and 65–95 C with a heating rate of 0.1 C/sec and a cooling step to 40 C. A total of 45 cycles were used in the amplification.

Quantification of the gene expression by real-time PCR was based on the mathematic model developed by Pfaffl (24) and was subsequently adopted by the manufacturer to incorporate into the software in the calculation of the normalized relative values (Roche). The following equation, normalized relative values = ETCpT(C) – CpT(S) x ERCpR(S) – CpR(C), was used in the quantification of the results. The designations are: T, target gene of interest; R, reference; S, samples; C, calibrator; E, amplification efficiency; and Cp (crossing point), cycle number at detection threshold. Real-time PCR efficiencies were calculated from the given slopes from the LightCylcer software provided by the manufacturer. The corresponding real-time PCR efficiencies (E) were calculated according to the equation: E = 10[–1/slope]. Cp is defined as the point at which the fluorescence rises appreciably above the background fluorescence and determined by the second derivative maximum of each curve (see also www.roche-applied-science.com/lighcycler-online/lc_support/lc_tech.htm). The reference used is glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

The primers for the target genes and the reference gene, GAPDH, are as follows: cyclin D1: forward primer, 5'-TACCGCACAACGCACTTTCT; reverse primer, 5'-TCCACATCTCGCACGTCGGT; TSH receptor: forward primer, 5'-ACTGATCGCAAAAGACACCT; reverse primer, 5'-TGTAGTCATAGTGGCTCTCG; cathepsin D: forward primer, 5'-ATCTTGGGCATGGGCTACC-3'; reverse primer, 5'-GGCTGGACACCTTCTCACAA-3'; TGF-ß-induced 68-kDa protein: forward primer, 5'-CCGGGAAGGGGTCTACACTG-3'; reverse primer, 5'-CCTCCTCGGTCTTCCTGCTAAT-3'; follistatin-like: forward primer, 5'-AACCCATCCTTCAACCCTCCTG-3'; reverse primer, 5'-TGGCCACCCTCATTTCCTTTAT-3'; decorin: forward primer, 5'-CATAACTGCGATCCCTCAAG; reverse primer, 5'-CTTTCGAGAGGGGTGTCCA; defender against cell death 1, forward primer, 5'-CATGTCGGCGTCTGTGGTGTC-3'; reverse primer, 5'-CGTGCTGGCAAAGAGGAAGTCA-3'; peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}): forward primer, 5'-TCTGGCCCACCAACTTCGGA-3'; reverse primer. 5'-CTTCACAAGCATGAACTCCA-3'; lipoprotein lipase: forward primer, 5'-TGCCATGACAAGTCTCTGAAG-3'; reverse primer, 5'-ATGGGCCATTAGATTCCTCA-3'; and GAPDH: forward primer, 5'-CCCTTCATTGACCTCAACTACAT-3'; reverse primer, 5'-ACAATGCCAAAGTTGTCATGGAT-3'.

Histopatholoigcal analysis
Mice were killed at different ages, and the thyroid glands and lungs were dissected, fixed in 10% neutral buffered formalin, and subsequently embedded in paraffin. Five-micrometer-thick sections were prepared and stained with hematoxylin and eosin and analyzed.

Statistical analysis
All data are expressed as the mean ± SEM. Statistical analysis was performed with the use of ANOVA, and P < 0.05 was considered significant. Kaplan-Meier cumulative survival analysis was performed using StatView 5.0 (Abacus Concepts, Inc., San Diego, CA) (25). Log-rank (Mantel-Cox) testing for statistical significance was carried out with the use of PRISM 4.0a (GraphPad, Inc., San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mice with the TRßPV mutation in the absence of wild-type TRß (TRßPV/–) spontaneously develop follicular thyroid carcinoma
Previously, analyses of phenotypes of TRßPV/– mice from the crossing of TRßPV/PV and TRß–/– mice have shown that dysregulation of the pituitary-thyroid axis, impairment in weight gain, and abnormal regulation patterns of several T3 target genes are indistinguishable from those in TRßPV/PV mice (26). Serum TSH levels were found to be similarly elevated 468- to 554-fold in TRßPV/PV and TRßPV/– mice (26), but whether TRßPV/– mice are similar to TRßPV/PV mice in developing thyroid carcinoma was unknown. Examining our Kaplan-Meier cumulative survival plot for TRßPV/– mice (n = 70), we found that as they aged, they began to die at the age of approximately 5 months. Analysis of the data showed that at the age of 11.7 ± 0.3 months, 50% of TRßPV/– mice had died, and none survived beyond the age of 16 months. The survival rate of TRßPV/PV mice (n = 26) was 50% at the age of 11.1 ± 0.7 months (Fig. 1Go). There are no significant differences between the survival rates of these two mutant mice (P = 0.137), although a few TRßPV/– mice began to die at an earlier age than TRßPV/PV mice.



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  		FIG. 1. Kaplan-Meier survival curves for TRßPV/– and TRßPV/PV mice up to 16 months of age. Kaplan-Meier cumulative survival analysis was performed using StatView 5.0 (25 ). Statistical analysis of log-rank (Mantel-Cox) test was carried out using PRISM 4.0a (GraphPad, Inc.). Survival rates of TRßPV/– (n = 70) and TRßPV/PV (n = 26) mice did not differ significantly by log-rank test.

 
Examination of the killed TRßPV/– mice revealed that a common, obvious abnormality was the marked enlargement of the thyroid glands. The weights of the thyroid glands increased as these mice aged (Fig. 2Go). The average weights were 49.4 ± 7.0 mg (n = 13) and 234 ± 25.1 mg (n = 5) at the ages of 3–6 and 12–15 months, respectively. The average weights of adult thyroid glands of wild-type mice were 5 ± 0.6 mg (n = 11) and 5.7 ± 0.8 mg (n = 6) at the ages of 3–6 and 12–15 months, respectively. These differences represent significant increases of 10- and 41-fold in size of the thyroid glands in TRßPV/– mice at the ages of 3–6 and 12–15 months, respectively (P < 0.001). The marked enlargement of the thyroid glands severely compressed the trachea, thus causing the respiratory distress observed in the moribund TRßPV/– mice. Figure 2Go shows the extent of enlargement of thyroid glands in TRßPV/– and TRßPV/PV mice at the two different ages. There were no significant differences between them at 3–6 months (P = 0.966) or 12–15 months (P = 0.102) of age. Furthermore, no significant correlation between the weights of thyroid glands and serum TSH levels was observed, suggesting that growth factors other than TSH could also contribute to the proliferation of thyrocytes in these mutant mice.



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  		FIG. 2. Age-dependent enlargement of thyroid glands in TRßPV/– and TRßPV/PV mice. Thyroid glands of killed TRßPV/– and TRßPV/PV mice were dissected and compared in the same age groups. The numbers of wild-type, TRßPV/–, and TRßPV/PV mice in the two age groups are 6 and 11, 5 and 13, and 7 and 10, respectively. The P values, determined by ANOVA, are indicated.

 
Histopathological evaluation of the thyroids from the 5- to 16-month-old moribund TRßPV/– mice showed changes consistent with neoplastic progression from papillary hyperplasia to the invasion of the thyroid capsule by follicular elements (Fig. 3AGo, arrow), invasion of vascular spaces within and adjacent to the thyroid (Fig. 3BGo, arrow), focal anaplasia of epithelial cells within the hyperplastic areas (Fig. 3CGo, arrow), and, finally, appearance of distant metastatic lesions in the lung (Fig. 3DGo, arrow). The metastatic lesions in the lungs showed characteristics of follicular morphology (Fig. 3DGo, arrow), including focal accumulations of colloid. No metastases were detected in the lymph nodes.



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  		FIG. 3. Morphological features of thyroid carcinoma in TRßPV/– mice. TRßPV/– mice developed adenomatous hyperplasia of the thyroid, followed by features characteristic of malignant progression of follicular carcinoma: capsular invasion (A, arrow), vascular invasion (B, arrow), foci of anaplasia (C, arrow), and the presence of metastases in the lung (D, arrow). Magnification: A–C, x157; D, x63. Bar: A, 64 µm; D, 160 µm.

 
Hyperplasia was identified by the increased number and cytoplasmic volume of thyroid epithelial cells, often with narrowing of follicular lumens, and the formation of papilla within follicles. Early stages of this process in young mice were mainly manifested by altered epithelial cell cytological features, but at later ages, the degree of papillary hyperplasia and architectural distortion was profound and not difficult to identify. Capsular invasion was identified as the protrusion of collections of follicles and epithelial cells through the identifiable limits of the fibrous capsule (Fig. 3AGo). Although only one such focus was required to classify such glands as showing capsular invasion, it was common to see this event in multiple sites in the same gland. Vascular invasion required the presence of an epithelial follicular structure isolated within the limits of an endothelial-lined space (Fig. 3BGo). These vessels often also showed red blood cells and clear endothelial cell margins. Images in which the endothelium could not be identified or where there was a visible connecting stalk to surrounding stroma were not counted. Only one such image was necessary to classify such glands as showing vascular invasion, but it was common to see more than one such example in thyroids showing this feature. Anaplasia was identified by the detection of single foci of collections of cells with spindle cell morphology within the thyroid (Fig. 3CGo). Such foci were profoundly different from surrounding hyperplastic thyroid structures and were usually present as isolated, single foci. Metastasis was identified as obvious collections of epithelial cells, often with follicular lumens, in the lung (Fig. 3DGo). Previous studies in these mice have shown such metastases to be positive for thyroid differentiation markers using immunohistochemistry (thyroglobulin and the sodium iodide symporter) (27). Although only one such lesion was necessary to classify the mouse as having lung metastases, it was common to find multiple microscopic foci of metastatic cells in the lungs of the same mouse.

Table 1Go summarizes the pathological progression of 70 5- to 16-month-old TRßPV/– mice. All mice exhibited hyperplasia, 94.3% had capsular invasion, 72.9% had vascular invasion, 25.7% had anaplasia, and 38.6% had metastasis. Statistical analyses indicated that there were no significant differences between TRßPV/– and TRßPV/PV mice at each stage of the pathological progression, although there was a greater metastasis propensity in TRßPV/– mice (Table 1Go). The pathohistological features and metastasis patterns of TRßPV/– mice were indistinguishable from those observed earlier in the thyroid of TRßPV/PV mice (28) and were consistent with follicular thyroid carcinoma.


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  		TABLE 1. Histopathologic progression of thyroid neoplasia in 5- to 16-month-old mutant mice

 
Alterations of signaling pathways during thyroid carcinogenesis in TRßPV/– mice
Previously, using cDNA microarrays, Ying et al. (29) identified complex alterations of signaling pathways that contributed to the carcinogenesis of 6-month-old TRßPV/PV mice, an age at which metastasis has begun. An up-regulation of the signaling pathways mediated by TSH, the peptide growth factors [IGF, epidermal growth factor (EGF), and fibroblast growth factor], TGF-ß, TNF-{alpha}, and nuclear factor-{kappa}B (NF-{kappa}B) was observed. The pathways mediated by the PPAR{gamma} were repressed (29). The similar progression of thyroid carcinogenesis in TRßPV/– and TRßPV/V mice described above prompted us to ascertain whether these pathways were affected in a similar fashion globally. We therefore compared the mRNA expression of the downstream target genes, ligands, and/or effectors of these signaling pathways in the thyroids of TRßPV/PV and TRßPV/– mice at the early age of 3–4 months and at the older age of 11–12 months.

The TSH signaling pathway was activated in both TRßPV/PV and TRßPV/– mice, as evidenced by activation of cyclin D1, a known TSH downstream target gene (Fig. 4AGo) (30). The expression of cyclin D1 remained activated (~2.5- to- 3-fold compared with that in age-matched, wild-type siblings at the corresponding age) during tumor progression from 3–4 months to 11–12 months of age (Fig. 4AGo and Table 2Go). In older mice, the expression of cyclin D1 in TRßPV/PV mice was 1.5-fold higher than that in TRßPV/– mice (Fig. 4AGo). As another means of examining whether the TSH signaling pathway was activated, we determined the expression of TSH receptor (Fig. 4BGo). Compared with the age-matched wild-type siblings, the expression of TSH receptor was increased 4.11- and 6.39-fold in TRßPV/– and TRßPV/PV mice, respectively, at 3–4 months of age and 6.01- and 11.26-fold, respectively, at 11–12 months of age (Fig. 4BGo and Table 2Go). These data indicate that similar to TRßPV/PV mice, the TSH signaling pathway is activated in TRßPV/– mice. Furthermore, it is important to note that the expression of cyclin D1 and TSH receptor was significantly more pronounced in TRßPV/PV mice than in TRßPV/– mice, especially in older mice (Table 2Go and Fig. 4Go). These findings suggest that TSH signaling pathways in the thyroid are more affected in mice with two mutated alleles than in mice with one mutated allele and deletion of one wild-type TRß allele.



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  		FIG. 4. Comparison of cyclin D1 (A) and TR (B) mRNA expression in the thyroids of TRßPV/PV and TRßPV/– mice and their wild-type siblings. Total RNAs were prepared from mice aged 3–4 (n = 3) and 11–12 months (n = 3), and quantitative real-time RT-PCR was performed with 0.2 µg total RNA, as described in Materials and Methods. The fold change in mRNA expression is shown. Data are expressed as the mean ± SEM. The fold change compared with the wild-type siblings is significant (P < 0.05). Significant differences between the two mutant mice and between the two age groups are shown with P values.

 

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  		TABLE 2. Comparison of mRNA expression of critical genes of several signaling pathways between TRßPV/– and TRßPV/PV mice during thyroid carcinogenesis

 
It has been shown that in addition to TSH, other peptide growth factors, such as IGF-I and EGF, also stimulate the proliferation of thyroid cells (30). Because the expression of the cathepsin D gene is activated by these two growth factors (31), we compared its expression in TRßPV/– and TRßPV/PV mice. The expression of cathepsin D was activated in these two mutant mice (4.0- to 3.5-fold and 1.3- to 2.7-fold activation at 3–4 and 11–12 months of age, respectively; Fig. 5Go and Table 2Go), supporting the idea that the signaling pathways mediated by IGF-I and EGF are activated during thyroid carcinogenesis in these two mutant mice. However, similar to that observed for the expression of cyclin D1 and TSH receptor, the expression of cathepsin D was significantly higher in TRßPV/PV (~2-fold) than in TRßPV/– mice at the age of 11–12 months. Furthermore, its expression in TRßPV/– mice at the age of 11–12 months was 3-fold lower than that at the age of 3–4 months.



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  		FIG. 5. Comparison of cathepsin D mRNA expression in the thyroids of TRßPV/PV and TRßPV/– mice and their wild-type siblings. Total RNAs were prepared from mice aged 3–4 months (n = 3) and 11–12 months (n = 3), and quantitative real-time RT-PCR was performed with 0.2 µg total RNA, as described in Materials and Methods. Data are expressed as the mean ± SEM. The fold change compared with the wild-type siblings is significant (P < 0.05). Significant differences between the two mutant mice and between the two age groups are shown with P values.

 
In the thyroids of TRßPV/PV mice at the age of 6 months, the expression of two direct downstream target genes of TGF-ß, TGF-ß-induced 68-kDa protein (p68 ßig-h3) and follistatin-like genes, is activated, indicating that the TGF-ß signaling pathway is activated. The secretory protein p68 ßig-h3 is induced in several types of human cells, including melanoma cells, mammary epithelial cells, keratinocytes, and fibroblasts, upon treatment with TGF-ß (32, 33). The follistatin-like gene encodes a novel protein with unknown function that is induced upon treating mouse osteoblastic MC3T3-E1 cells with TGF-ß (34). We compared the expression of p68 ßig-h3 and the follistatin-like gene in TRßPV/– and TRßPV/PV mice at two different ages (Fig. 6Go and Table 2Go). In mice aged 3–4 months, the expression of p68 ßig-h3 was activated 14.22- and 18.99-fold in the thyroids of TRßPV/– and TRßPV/PV mice, respectively; in mice aged 11–12 months, the extent of the activation was only 25.6- and 26.8-fold. The expression of the follistatin-like gene was also activated (1.5- to 4.17-fold) in these two mutant mice. In TRßPV/PV mice, the expression of the follistatin-like gene was significantly higher than that in TRßPV/– mice (Fig. 6BGo). As TRßPV/PV mice aged, the expression of the follistatin-like gene was reduced ~2-fold (Fig. 6BGo). The activation of this TGF-ß downstream target gene indicates the activation of the TGF-ß-mediated pathway during thyroid carcinogenesis in these two mutant mice. The reduced expression of decorin, a negative regulator of TGF-ß activity (Fig. 7AGo and Table 2Go), also supports this idea.



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  		FIG. 6. Comparison of TGF-ß-induced 68KDa protein (A) and follistatin-like mRNA expression in the thyroids of TRßPV/PV and TRßPV/– mice and their wild-type siblings. Total RNAs were prepared from mice aged 3–4 months (n = 3) and 11–12 months (n = 3), and quantitative real-time RT-PCR was performed with 0.2 µg total RNA, as described in Materials and Methods. Data are expressed as the mean ± SEM. The fold change compared with the wild-type siblings is significant (P < 0.05). Significant differences between the two mutant mice and between the two age groups are shown with P values.

 


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  		FIG. 7. Comparison of decorin (A) and DAD-1 (B) mRNA expression in the thyroids of TRßPV/PV and TRßPV/– mice and their wild-type siblings. Total RNAs were prepared from mice aged 3–4 months (n = 3) and 11–12 months (n = 3), and quantitative real-time RT-PCR was performed with 0.2 µg total RNA, as described in Materials and Methods. Data are expressed as the mean ± SEM. The fold change in mRNA expression is shown. Significant differences between the two mutant mice and between the two age groups are shown with P values.

 
The proposal that the signaling pathway of TNF-{alpha} was activated in TRßPV/PV mice at the age of 6 months was based on the decreased expression and reduced activity of its direct downstream target gene, decorin (29). The promoter of the decorin gene contains a TNF-{alpha} response element, located between nucleotides –188 and –140, and the expression of the decorin gene is negatively regulated by TNF-{alpha} (35, 36). We, therefore, examined the expression of decorin in TRßPV/– mice and compared these expression profiles with those in TRßPV/PV mice at the ages of 3–4 and 11–12 months. The extent and patterns of age-dependent changes were similar in these two mutant mice (Fig. 7AGo and Table 2Go), which supports the idea that the TNF-{alpha}-mediated pathway was activated in both during thyroid carcinogenesis.

We also determined whether the signaling pathway mediated by NF-{kappa}B was similarly activated in TRßPV/– mice as in TRßPV/PV mice (29). NF-{kappa}B is a transcription factor that regulates genes important for tumor invasion, metastasis, and chemoresistance. When bound to cytoplasmic inhibitor of {kappa}B proteins, NF-{kappa}B remains sequestered in an inactive state. Upon release from the inhibitor, NF-{kappa}B translocates to the nucleus and activates gene expression upon exposure of cells to growth factors and cytokines. That the NF-{kappa}B-mediated pathway is activated in TRßPV/PV mice at the age of 6 months was supported by the finding of the activation of not only one of its regulated genes, defender against cell death-1 (DAD-1), but also one of its direct downstream target genes, cyclin D1 (29). Two NF-{kappa}B-binding sites are present in the promoter of the cyclin D1 gene (37). To evaluate whether the NF-{kappa}B-mediated pathway is activated in TRßPV/– mice, the expression of DAD-1 was determined. Similar age-dependent alteration patterns in the expression of DAD-1 were found in TRßPV/– and TRßPV/PV mice (Fig. 7BGo and Table 2Go). However, the expression of DAD-1 was more affected in TRßPV/PV mice than in TRßPV/– mice at the age of 11–12 months, with a 2-fold increase.

PPAR{gamma} is a ligand-dependent nuclear transcription factor involved in such cellular processes as adipogenesis, inflammation, atherosclerosis, cell cycle control, apoptosis, and carcinogenesis (38, 39). Ying et al. (29) found that the expression of both PPAR{gamma} and the lipoprotein lipase was repressed in TRßPV/PV mice at the age of 6 months. Furthermore, the transcriptional activity of PPAR{gamma} was repressed by mutant PV (29, 27). The observation that the expression of one of its direct downstream target genes, lipoprotein lipase, was concurrently repressed further confirmed the repression of the PPAR{gamma} signaling pathway (27, 29). Figure 8Go compares the expressions of PPAR{gamma} and lipoprotein mRNA in TRßPV/– and TRßPV/PV mice at the ages of 3–4 and 11–12 months. We found similar concurrent repression of these two genes in these two mutant mice during thyroid carcinogenesis. As mice aged, the repression of the lipoprotein gene became significantly more pronounced (Fig. 8BGo). These results support the idea that the PPAR{gamma} pathway was repressed in both TRßPV/– and TRßPV/PV mice during thyroid carcinogenesis.



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  		FIG. 8. Comparison of PPAR{gamma} (A) and lipoprotein lipase (B) mRNA expression in the thyroids of TRßPV/PV and TRßPV/– mice and their wild-type siblings. Total RNAs were prepared from mice aged 3–4 months (n = 3) and 11–12 months (n = 3), and quantitative real-time RT-PCR was performed with 0.2 µg total RNA, as described in Materials and Methods. Data are expressed as the mean ± SEM. The fold change compared with the wild-type siblings is significant (P < 0.05). Significant differences between the two mutant mice and between the two age groups are shown with P values.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study found that TRßPV/– mice, in contrast to TRßPV/+ mice, spontaneously develop follicular thyroid carcinoma. The pathological progression in the thyroids of TRßPV/– mice was indistinguishable from that in TRßPV/PV mice. This indicates that one mutated TRß allele in the absence of the other wild-type allele is sufficient to induce thyroid carcinoma. Thus, thyroid carcinoma occurs either when both alleles are mutated or when one allele is mutated and there is ablation of the other wild-type allele. These data suggest that the TRß gene could function as a tumor suppressor. This idea is consistent with recent studies, which indicated that not only mutations of the TRß gene, but also reduced expression of the TRß gene, are associated with several human cancers (9, 13, 14). That the TRß gene could function as a tumor suppressor gene gains additional support from findings that in primary breast cancer and cell lines, the expression of the TRß gene was silenced by hypermethylation with concurrent reduction of TRß transcripts (16).

The similarity of the altered signaling pathways in the thyroids of TRßPV/– and TRßPV/PV mice calls attention to the critical contributions of such alterations during carcinogenesis. The identification of the common, altered critical genes in the signaling pathways provides new mechanistic insights into how mutated TRß mutants are involved in thyroid carcinogenesis. The PV mutant has completely lost T3 binding and transcriptional activities (22); thus, the normal functions of TRß are lost in TRßPV/– and TRßPV/PV mice. In addition, PV acts to interfere with the transcriptional activity of wild-type TRs (17, 26, 40). In TRßPV/PV mice, both copies of the TRß genes are mutated; in TRßPV/– mice, one wild-type TRß gene is absent, and there is mutation of the other allele. In the absence of wild-type TRß (w-TRß) in these two mutant mice, PV forms inactive dimers with w-TR{alpha}1, and these dimers compete with w-TR{alpha}1 for binding to TREs and interfere with the normal transcriptional activity of w-TR{alpha}1 (26). By this mechanism, PV could function to repress constitutively and directly or indirectly a certain set of genes that prevent cellular transformation, and/or it could function to activate some tumor-promoting genes to facilitate carcinogenesis in the thyroid. This proposed mechanism is strengthened by the observations that two PV alleles in TRßPV/PV mice exerted significantly more pronounced effects on the expression of the critical genes in the affected signaling pathways (cyclin D1, TSH receptor, cathepsin D, follistatin-like gene, DAD-1, PPAR{gamma}, and lipoprotein lipase) than one PV allele in TRßPV/– mice, suggesting a mutant gene dose effect. However, PV could also act via gain of function to promote carcinogenesis. Detailed analysis of these possibilities await future studies.

The serum TSH levels are similarly high in TRßPV/– and TRßPV/PV mice (26), suggesting that TSH-mediated hyperplasia could be an early step in carcinogenesis. Recent evidence, however, does not support the role of TSH as an initiator of follicular thyroid carcinoma. Somatic activating mutations of the TSH receptor or of the guanine nucleotide stimulatory factor {alpha}-subunit (Gs{alpha}) are known to inappropriately activate adenylyl cyclase activity and stimulate relatively unrestrained growth of thyroid cells. Furthermore, transgenic mice with thyroid-specific expression of the A2 adenosine receptor (41), a mutated Gs{alpha} (42), or cholera toxin A1 (43) develop thyroid hyperplasia and hyperthyroidism, but not carcinomas. In addition, patients with Graves’ disease or congenital hyperthyroidism due to germline mutations of the TSH receptor do not appear to have a higher rate of thyroid malignancy (44). Thus, additional genetic changes would need to occur for transformation of hyperproliferative thyroid cells to cancer cells. The present study suggests that in addition to the mutations of both TRß alleles as in TRßPV/PV mice, the mutation of one TRß allele together with the absence of the other wild-type allele could provide those additional genetic changes that lead to thyroid carcinoma.

Carcinoma of the thyroid is a common clinical problem. The underlying genetic alterations that determine the behavior of advanced stages of this disease and the occurrence of anaplastic foci are largely unknown. In addition to TRßPV/PV mice, TRßPV/– mice, newly identified as a model of thyroid carcinogenesis, should provide an additional tool to uncover potential novel gene expression changes in thyrocytes during carcinogenesis. The usefulness of these mouse models in identifying possible signature genes during thyroid carcinogenesis was revealed by the age-dependent alterations in the extent of expression of the critical genes in the affected signaling pathways. For example, the expression of cathespsin D and lipoprotein lipase in TRßPV/– mice was significantly lower at the age of 11–12 months than at 3–4 months, suggesting other additional genetic changes affecting their expression as tumorigenesis progresses to metastatic spread. Similar age-dependent changes were also observed for the follostatin-like and lipoprotein lipase genes in TRßPV/PV mice. Thus, it is possible to use these mouse models to define signature genes that are associated with metastasis and that affect the long-term survival of patients.


   Footnotes
 
Abbreviations: DAD-1, Defender against cell death-1; EGF, epidermal growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Gs{alpha}, guanine nucleotide stimulatory factor {alpha} subunit; NF-{kappa}B, nuclear factor-{kappa}B; PPAR{gamma}, peroxisome proliferator-activated receptor {gamma}; RTH, thyroid hormone resistance syndrome; TR, thyroid hormone nuclear receptor; TRß, thyroid hormone ß receptor; TRE, thyroid hormone response element; w-TRß, wild-type thyroid hormone ß receptor.

Received May 13, 2004.

Accepted for publication June 24, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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