Alterations in genomic profiles during tumor progression in a mouse model of follicular thyroid carcinoma
Hao Ying1,
Hideyo Suzuki1,
Hiroko Furumoto1,
Robert Walker2,
Paul Meltzer2,
Mark C. Willingham3 and
Sheue-Yann Cheng1,4
1 Laboratory of Molecular Biology, National Cancer Institute, Winston-Salem, NC 27157-1072, USA
2 Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA
3 Department of Pathology, Wake Forest University School of Medicine, Winston-Salem, NC 27157-1072, USA
4 To whom correspondence should be addressed Email: sycheng{at}helix.nih.gov
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Abstract
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The molecular genetics underlying thyroid carcinogenesis is not well understood. We have recently created a mutant mouse by targeting a mutation (PV) into the thyroid hormone receptor ß gene (TRßPV mouse). TRßPV/PV mice spontaneously develop follicular thyroid carcinoma through pathological progression of hyperplasia, capsular and vascular invasion, anaplasia and eventually metastasis to distant organs. TRßPV/PV mice provide an unusual opportunity to study the alterations in gene regulation that occur during thyroid carcinogenesis. To this end, we profiled the genomic changes in the thyroids of TRßPV/PV mice at 6 months of age, at which time metastasis had begun. From arrays of 20 000 mouse cDNAs, 185 genes were up-regulated (217-fold) and 92 were down-regulated (220-fold). Functional clustering of named genes with reported functions (100 genes) indicated that
39% of these genes were tumor-, metastasis/invasion- and cell-cycle-related. Among the activated tumor-related genes identified, cyclin D1, pituitary tumor transforming gene-1, cathespin D and transforming growth factor
were also found to over-express in human thyroid cancers. Analyses of the gene profiles suggested that the signaling pathways mediated by thyrotropin, peptide growth factors, transforming growth factor-ß, tumor necrosis factor-
and nuclear factor-
B were activated, whereas pathways mediated by peroxisome proliferation activated receptor
were repressed. These results indicate that complex alterations of multiple signaling pathways contribute to thyroid carcinogenesis. The critical genes associated with thyroid follicular carcinogenesis uncovered in the present study could serve as signature genes for diagnostic purposes, as well as for possible therapeutic targets.
Abbreviations: DAD-1, defender against cell death-1; EGF, epidermal growth factor; FTC, follicular thyroid carcinoma; Gs
, guanine nucleotide stimulatory factor a subunit; IGF, insulin-like growth factor; I
B, inhibitor of
B; LpL, lipoprotein lipase; LPS, lipopolysaccharide; MGI IDs, Mouse Genome Informatics Accession Identification Numbers; NF-
B, nuclear factor
B; PAX8, paired box gene 8; PPAR
1, peroxisome proliferator activated receptor
1; PTTG-1, pituitary tumor transforming gene-1; RTH, thyroid hormone resistance syndrome; TGFß, transforming growth factor ß; TNF-
, tumor necrosis factor-
; TR, thyroid hormone receptor; TSH, thyrotropin
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Introduction
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Thyroid cancers in humans consist of an array of several different histologic and biologic types (papillary, follicular, medullary, clear cell, anaplastic, Hurtle cell and others) (1), but the majority of clinically important human thyroid cancers are papillary or follicular. The molecular genetic events underlying these thyroid carcinomas are not clearly understood. Rearrangements of the RET tyrosine kinase receptor gene (RET/PTC) are found in 2.634% of papillary carcinomas in the adult population (2). Over-expression of RET/PTCs in thyroid cells of transgenic mice results in tumors with histologic and cytologic characteristics similar to those of human papillary carcinoma, thus providing evidence for the involvement of RET/PTCs in the initiation of papillary carcinoma (35).
Less is known about the molecular genetic events underlying follicular thyroid carcinoma (FTC). Accumulated evidence indicates that FTC arises through an oncogenic pathway distinct from that of papillary carcinoma, probably from the point of clonal initiation (2). The major differences between the molecular genetics of these two types of carcinomas are a higher prevalence in FTC of activating mutations of all three RAS genes and a greater disposition to develop DNA copy abnormalities (2,6). Still, no evidence supports the possibility that mutations of the RAS genes initiate FTC. Recently, Kroll et al. reported the identification of a chromosomal rearrangement t(2:3)(q13;p25), yielding a PAX8PPAR
1 fusion gene, exclusively in FTC (7,8). When fused to PAX8, PPAR
1 not only loses its ability to stimulate thiazolidinedione-induced transcription, but also acts to inhibit PPAR
1 transcriptional activity (7). However, how the loss of PPAR
1 transcriptional activity affects the normal functions of thyroid follicular cells remains unknown.
Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that regulate cell proliferation, differentiation and development. They are members of the nuclear receptor superfamily. Two genes, TR
and TRß, which are located on human chromosomes 17 and 3, respectively, give rise to four thyroid hormone (T3) binding receptors (TR
1, TRß1, TRß2 and TRß3) and two non-T3 binding receptors (TR
2 and TR
3). Recent studies indicate that mutations of the TR genes are closely associated with several human cancers. Mutated TRs were found in human hepatocellular carcinoma (9), renal clear cell carcinoma (10), and papillary thyroid carcinoma (11). The majority of these TR mutants lose their transcriptional activities and exhibit strong dominant negative activity (11). More recently, bi-allelic inactivation of the TRß gene was identified in 100% of the 11 primary breast tumors examined, thereby raising the possibility that the TRß gene could act as a tumor suppressor gene in breast tumorigenesis (12).
We recently created a mutant mouse by targeting a mutation (PV) to the TRß gene locus (TRßPV mice) (13). TRßPV was derived from a patient (PV) with thyroid hormone resistance syndrome (RTH) (14). RTH patients manifest the symptoms of dysfunction of the pituitarythyroid axis with high circulating levels of thyrotropin (TSH) along with increased circulating levels of thyroid hormones (T3 and T4) (14). The only reported homozygous RTH patient died at a young age from unknown causes (15). Patient PV has one mutant TRß gene allele and manifests severe RTH (16). PV has a unique mutation in exon 10, a C-insertion at codon 448, which produces a frame shift of the C-terminal 14 amino acids of TRß1. PV has lost T3-binding completely and exhibits potent dominant negative activity (17).
Remarkably, as TRßPV/PV mice age, they spontaneously develop follicular thyroid carcinoma (18). Histologic evaluation of thyroids of 514-month-old mice showed capsular invasion (91%), vascular invasion (74%), anaplasia (35%) and metastasis to the lung (30%), but not to the local lymph nodes (18). Thus, TRßPV/PV mice provide an unusual opportunity to dissect the molecular genetic events underlying tumor progression and metastasis of FTC and to identify potential molecular targets for its possible prevention and treatment. As a first step toward this goal, we used the cDNA microarrays to profile genomic alterations during thyroid carcinogenesis. From the arrays of 20 000 mouse cDNAs, 185 genes were up-regulated (217-fold) and 92 were down-regulated (220-fold). Among the named genes undergoing changes,
39% were tumor-, metastasis- and cell-cycle-related, signifying global molecular and genetic changes during carcinogenesis of the thyroid. Alterations of cellular signaling pathways were also identified, an indication that complex changes of cellular processes are associated with carcinogenesis.
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Materials and methods
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RNA isolation
The animal study protocol used in the present study has been approved by the National Cancer Institute Animal Care and Use Committee. The mice harboring the TRßPV gene were created by introducing the PV mutation onto the TRß gene locus via homologous recombination as described (13). Genotyping was carried out using RTPCR as described previously (13). The total RNA of thyroid glands from TRßPV/PV or wild-type siblings was prepared using TRIzol (Invitrogen, Carlsbad, CA) according to manufacturer's instructions.
Microarrays, hybridization and scanning
The mouse arrays contained 20 000 cDNAs. Hybridization, scanning and image analysis were performed as described previously (www.nhgri.nih.gov/DIR/microarray) (1921). Briefly, fluorescent labeled cDNA was synthesized from
20 µg of pooled RNA of five mice by oligo(dT)-primed polymerization in the presence of aminoallyl-dUTP (Amersham Pharmacia Biotech, Piscataway, NJ) and coupled with either Cy-3 or Cy-5. Image analyses were performed using DeArray software (Signal Analytics, Vienna, VA) (20,21). The two fluorescent images (red and green channel) obtained from one array constitute the raw data from which differential gene expression ratio values were calculated. The ratios of the red intensity to the green intensity (R/G) for all targets were determined, and the data were stored in a FileMaker Pro database (FileMaker, Santa Clara, CA).
Northern blot analysis
Total RNA isolated as indicated above (5 µg) was resolved by gel electrophoresis and transferred onto membranes (Hybond-N+, Amersham Pharmacia Biotech), which were hybridized with the cDNA probes for cyclin D1 or decorin after being labeled with [
-32P]dCTP using a random primer hexamer protocol. For normalization, the blots were stripped and rehybridized with a [
-32P]dCTP-labeled GAPDH cDNA. After quantification by NIH image 1.61, the intensities of the mRNA bands were normalized against the intensities of GAPDH mRNA.
Quantitative real-time RTPCR
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-RTPCR Reaction Mix SYBR, 2 µl resolution solution, 0.4 µl LightCycler-RTPCR 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 s; 95°C for 15 s, 58°C for 30 s and 72°C for 30 s; 6595°C with a heating rate of 0.1°C/s and cooling step to 40°C. The primers used are as follows. Pituitary tumor transforming gene-1: forward primer 5'-CCT GAT GAT GCC TAC CCA G-3'; reverse primer 5'-CCC TTA CCA GAT TCC CAT GA-3'. Thyrotropin receptor: forward primer 5'-ACTGATCGCAAAAGACACCT; reverse primer 5'-TGTAGTCATAGTGGCTCTCG. Lipoprotein lipase: forward primer 5'-TGCCATGACAAGTCTCTGAAG-3'; reverse primer 5'-ATGGGCCATTAGATTCCTCA-3'. GAPDH: forward primer 5'-CCCTTCATTGACCTCAACTACAT-3'; reverse primer 5'-ACAATGCCAAAGTTGTCATGGAT-3'. Cathepsin D: forward primer 5'-ATCTTGGGCATGGGCTACC-3'; reverse primer 5'-GGCTGGACACCTTCTCACAA-3'. Transforming growth factorß (TGFß)-induced 68 kDa protein: forward primer 5'-CCGGGAAGGGGTCTACACTG-3'; reverse primer 5'-CCTCCTCGGTCTTCCTGCTAAT-3'. Defender against cell death 1: forward primer 5'-CATGTCGGCGTCTGTGGTGTC-3'; reverse primer 5'-CGTGCTGGCAAAGAGGAAGTCA-3'. Heme oxygenase 1: forward primer 5'-TTTCCGCATACAACCAGTGAGT-3'; reverse primer 5'-CCAGTGAGGCCCATACCAG-3'. Follistatin-like: forward primer 5'-AACCCATCCTTCAACCCTCCTG-3'; reverse primer 5'-TGGCCACCCTCATTTCCTTTAT-3'. ß-catenin: forward primer 5'-TGAAGGCGTGGCAACATAC-3'; reverse primer 5'-ATCAGGCAGCCCATCAACT-3'.
Statistical analysis
The data from the quantitative real time PCR and northern blot analyses are expressed as mean ± SEM. Differences between groups were examined for statistical significance using Student's t-test. P < 0.05 is considered statistically significant.
Immunohistochemistry of cyclin D1 in tissues
Mouse tissues were fixed by immersion in 3.7% formaldehyde for 2 h at room temperature, followed by serial dehydration into ethanol, xylene and routine processing into paraffin blocks. Five micron sections were prepared and attached to Fisher Plus slides, and then treated with a commercial antigen retrieval reagent (Vector Labs, Burlingame, CA) for 1 h in a 95°C water bath. Following blocking in 1% bovine serum albumin (BSA) with phosphate-buffered saline (PBS), sections were incubated with mouse monoclonal anti-cyclin D1 (NCL-cyclin D1-GM; Novocastra, Newcastle upon Tyne, UK) at a 1:25 dilution in 1% BSAPBS overnight at 4°C. The anti-cyclin D1 antibody was documented to react both with human and mouse proliferating tissues (Figure 2). The second step reagent used (goat anti-mouse IgG) was an affinity-purified horseradish peroxidase (HRP) conjugate (Jackson ImmunoResearch, West Grove, PA) used at 25 µg/ml in BSAPBS for 30 min at room temperature. HRP reactions were developed using a standard diaminobenzidine-peroxide step and the sections were counterstained with 0.03% light green, which stains the cytoplasm of cells but does not interfere with detection of nuclear reactivity.

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Fig. 2. Immunohistochemical detection of cyclin D1 in mouse and human tissues. Paraffin sections of human (A) breast cancer, (B) mouse transplanted S180 tumor, (C) normal mouse thyroid and (D) hyperplastic mouse thyroid from a TRßPV/PV mouse, were processed for peroxidase immunohistochemistry as described in Materials and methods. The brown reaction product of the peroxidase label shows nuclear reactivity in both human and mouse tumor cells (A and B), the absence of reactivity in normal thyroid follicular epithelial cells (C) and the scattered and variable reactivity of potentially neoplastic thyroid epithelial cells from a TRßPV/PV mouse (D). (Magnification bar = 37 µm).
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Results
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cDNA microarray analysis
As a first step to understand the molecular genetics underlying thyroid follicular carcinoma, we compared the genomic profiles of thyroids of TRßPV/PV and wild-type siblings at the age of 6 months, at which time metastasis has begun (18). The cDNA arrays consist of 20 000 mouse genes. RNA was prepared from the thyroids of TRßPV/PV and age-matched wild-type mice. Only genes with ratios >2 were selected for further analysis. We found 185 genes that were up-regulated and 92 genes that were down-regulated. Among the up-regulated genes, 69 (37%) were named and 116 (63%) were unnamed. A slightly higher percentage of the named genes (39 genes; 42%) was detected in the down-regulated genes.
Functional classification of microarray outliers
To gain insights into the cellular pathways associated with tumor progression, we classified the named genes that have reported functions. This classification was accomplished by first using controlled vocabularies provided by the Gene OntologyTM Consortium [Gene Ontology (GO) tool available at http://www.informatics.jax.org]. Most outlier genes have multiple functions as shown in the categories according to Gene OntologyTM Consortium (Tables I
IV). As it is impossible to include all information from the GO search about each outlier gene in Tables I
IV, we have included the Mouse Genome Informatics Accession Identification Numbers (MGI IDs) for each gene assigned by the MGI group, a founding member of the Gene OntologyTM Consortium, for additional information. In addition, we also searched the PubMed database for each of the outlier gene names using the following terms: tumor, thyroid, motility, metastasis, invasion, proliferation, cell cycle and apoptosis. Four broad categories were shown: tumor-related genes (20 genes; Table I), metastasis-associated genes (16 genes; Table II), thyroid-related genes (15 genes; Table III) and cell cycle/apoptosis genes (three genes; Table IV). These 54 genes accounted for 54% of the genes that have reported functions (a total of 100 genes). The remainder of the genes (46 genes) was assigned to a modification of the NCBI Clusters of Orthologous Gene classification by searching the PubMed database by gene name (http://ncbi.nlm.nih.gov/cgi-bin/COG/palog?fun=all). Each gene was categorized with a single and mutually exclusive function (Tables V and VI).
Tumor-related genes
Table I shows the functional classification of the gene outliers based on GO search and Pubmed database search. These genes are involved in important normal biological processes and molecular functions, but were also reported to play critical roles in tumor progression, cell proliferation and oncogenesis. We found 20 genes that accounted for 20% of the named genes with reported functions. About 70% of genes in this group were activated, ranging from 2.1- to 7.9-fold, and 30% were repressed, ranging from 2- to 6-fold. At the top of the list was the cyclin D1 gene, which was activated 7.9-fold.
The cyclin D1 gene is known to over-express in human thyroid carcinomas (22,23). We therefore further determined its expression in the thyroids of TRßPV/PV mice by northern blot analysis (Figure 1). Cyclin D1 was expressed in the wild-type mice, but at a low level (Figure 1A, first six lanes). Its expression was clearly activated in the thyroids of TRßPV/PV mice (Figure 1A, last three lanes). Shown in the lower panel (Figure 1B) are the quantitative data after quantification and normalization with GAPDH. Compared with the expression of cyclin D1 in the thyroids of wild-type mice, a 6-fold activation in the expression of cyclin D1 was detected in the thyroids of TRßPV/PV mice.

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Fig. 1. Increased expression of cyclin D1 mRNA in the thyroids of TRßPV/PV mice determined by northern blotting. Total RNA was prepared from mice at the age of 67 months as described in Materials and methods. (A) Comparison of the mRNA expression of Cyclin D1 (a) and GAPDH (control) (b) in the thyroids of TRßPV/PV mice (n = 3) and wild-type siblings (n = 6). (B) Fold of changes of the expression of Cyclin D1 mRNA (P < 0.001) after normalization of the loading with GAPDH. The P value was determined by the Student's t test.
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The expression of cyclin D1 was also evaluated in normal and hyperplastic thyroid tissues. Figure 2 shows the use of immunohistochemistry to demonstrate cyclin D1 protein levels. The antibody used, originally generated against recombinant human cyclin D1, was initially evaluated using both human and mouse neoplasms to document that this antibody would detect cyclin D1 in tissue from both species. Panel A shows cyclin D1-positive tumor cells in paraffin sections of a comparably processed sample of human breast cancer (brown HRP reaction product with contrasting light green counterstain), and panel B shows tumor cell reactivity in a transplanted mouse sarcoma (S180) (24), documenting that this antibody can detect the antigen in mouse cells. Panel C shows the relative absence of significant cyclin D1 in thyroid follicular cells in a normal mouse thyroid (counterstained with light green), while panel D shows the representative results of variable and scattered positive nuclei in a hyperplastic thyroid from a TRßPV/PV mouse, indicating increased expression of cyclin D1 in the thyroid cells. Taken together, these results not only validate findings from the arrays, but also, importantly, are consistent with the studies from human thyroid cancer (22,23).
Another gene that plays a critical role in tumorigenesis is the pituitary tumor transforming gene-1 (PTTG-1) (Table I). PTTG-1 is originally isolated from GH4 pituitary cells and has been shown to cause in vitro cell transformation and to induce tumor formation in vivo (25). Over-expression of PTTG-1 was detected in human thyroid carcinomas (26), colorectal carcinoma (27), pituitary adenomas (28) and hematopoietic neoplasms (29). We further examined its mRNA expression using quantitative real-time PCR. In concordance with the array results (Table I), PTTG-1 was activated nearly 5-fold (Figure 3A). It is important to point out that the over-expression of PTTG-1 was found in a subset of human follicular thyroid carcinomas (26). Moreover, its over-expression in rat FRTL5 thyroid cells and in primary human thyroid cell cultures causes in vitro transformation and produces a dedifferentiated neoplastic phenotype, thus suggesting the possibility that it may play a role in the early molecular events leading to thyroid carcinoma.

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Fig. 3. Comparison of PTTG-1 (A), LpL (B) and TSH receptor (C) mRNA expression in the thyroids of TRßPV/PV mice and wild-type siblings. Total RNAs were prepared from mice at the age of 79 months and quantitative real time RTPCR was performed using 0.2 µg of total RNA as described in Materials and methods. Fold of changes of the expression of mRNA were shown (P < 0.01). The P value was determined by the Student's t test.
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Metastasis-related genes
The products of the genes listed in Table II are integral components of cell cytoskeleton, tight junctions and membrane structures. These 16 genes account for 16% of the named genes with reported functions and are involved in inter-cellular and cell-substrate adhesion that play critical roles in motility, metastasis, and invasion of tumor cells. Most of the genes were activated (75% of the total), ranging from 2- to 14-fold, and the remainder was repressed (25% of the total), ranging from 2- to 6-fold. The identification of an array of genes that involved cell motility, metastasis and invasion is consistent with our morphologic studies in that the tumor cells in the thyroid at this age undergo changes that lead to capsular invasion, vascular invasion and metastasis.
One of the better-characterized down-regulated genes in this group is decorin (Table II). Decorin is a small leucine-rich proteogylcan that may negatively modulate tumor growth by its inhibitory effect on the synthesis and bioactivity of TGFß and by interference with cell-cycle progression via induction of p21WAF1/CIP1 (30). We therefore further determined its expression by northern blot analysis (Figure 4). Consistent with the array results, the expression of decorin was repressed
3-fold. Interestingly, down-regulation of decorin was found to be associated with invasive growth of follicular thyroid carcinoma cells (31,32).

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Fig. 4. Analysis of the expression of decorin mRNA expression by northern blotting. (A) Comparison of the mRNA expression of decorin (a) and GAPDH (control) (b) in the thyroids of wild-type (n = 6) and TRßPV/PV mice (n = 3) at the age of 67 months. (B) Fold of changes of the expression of decorin mRNA (P < 0.001) after normalization of the loading with GAPDH. The P value was determined by the Student's t test.
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Thyroid-related genes
The genes listed in Table III are involved in biological processes and molecular functions that are critical for thyroid hormone synthesis, TSH action, thyroid cancer and abnormal thyroid functions (hyper- or hypothyroidism). Notably, among the up-regulated genes, 6 genes (6/11 up-regulated genes; 55%) play important roles in the synthesis of thyroid hormone. These findings are entirely consistent with the phenotype of TRßPV/PV mice in that these mice have elevated levels of serum concentration of thyroid hormone (13), and an increased expression of these genes is consistent with meeting this functional role. Furthermore, cathepsins (33), glutathione peroxidase (34), caveolin-1 (35) and transforming growth factor
(36) are reported to be associated with thyroid neoplasms. The identification of these thyroid-related genes further supports the array results.
One of the down-regulated genes in the group was lipoprotein lipase (LpL), which was repressed
5-fold (Table III). LpL plays a central role in the overall lipid metabolism and transport and is a direct target gene of the peroxisome proliferator activated receptor (PPAR
1) (37,38). The repression of LpL was further confirmed by real-time PCR (Figure 3B). The decreased expression of LpL predicted that the expression of PPAR
1 mRNA would be repressed in the thyroids of TRßPV/PV mice. Indeed, we recently found that the expression of PPAR
1 mRNA was repressed 2-fold during thyroid carcinogenesis (Ying et al., unpublished data).
Cell-cycle- and apoptosis-related genes
In addition to cyclin D1 shown in Table I, we found the activation of serine/threonine kinase 5 (2.6-fold) that is important for cell-cycle regulation. We also found that a Bcl2-like gene (Bcl21) was repressed (
2-fold) and the defender against cell death 1 was activated (2.6-fold). These two genes are involved in apoptosis (39). The up-regulation of these cell-cycle genes further supports the morphologic observations that the thyroid follicular cells are hyperplastic and that the thyroids were enlarged in TRßPV/PV mice (13,18).
Genes involved in other cellular pathways
Tables V and VI list the remainder of the named genes that have reported functions (46 genes), among which 13% were assigned into the classes of information storage and process (three up-regulated genes, Table V, and three down-regulated genes, Table VI), 50% into cellular processes (17 up-regulated genes, Table V, and six down-regulated genes, Table VI), and 37% into metabolism (seven up-regulated genes, Table V, and 10 down-regulated genes, Table VI). The changes ranged from 13-fold of up-regulation for lipocalin to 13-fold of down-regulation for cytosolic cystein dioxygenase 1. The data shown in Table V indicate that alterations of many cellular processes occurred during thyroid carcinogenesis in TRßPV/PV mice. The 17 poorly characterized named genes are also listed in Table V.
Identification of signaling pathways associated with thyroid carcinogenesis
On the basis of the functional clustering of the genes shown in Tables I
VI, we identified several major signaling pathways that were altered during carcinogenesis of the thyroids in TRßPV/PV mice (Table VII). The TSH signaling pathway was activated as evidenced by activation of cyclin D1, a known TSH downstream target gene (40). To further support this conclusion, we have also determined the expression of TSH receptor by quantitative real time PCR because TSH receptor was not present in our mouse arrays. Figure 3C shows that, indeed, compared with the wild-type siblings, the expression of TSH receptor was increased 11-fold in the thyroids of TRßPV/PV mice. The up-regulation of the TSH signaling pathway is consistent with the increased proliferation of thyroid cells mediated by the highly elevated TSH in TRßPV/PV mice (13). At the age of 67 months, the weight of the thyroid in TRßPV/PV mice was increased 18-fold compared with that of wild-type siblings (18).
However, the increased expression of cyclin D1 could also be due to an activation of the Wnt/ß-catenin signaling pathway because cyclin D1 is one of its known downstream target genes (41). Moreover, Ishigaki et al. reported that a cytoplasmic accumulation of ß-catenin protein correlates with over-expression of cyclin D1 in several lines of cultured human thyroid cancer cells and human papillary and follicular cancers (42). We, therefore, determined the expression of ß-catenin in the thyroids of TRßPV/PV mice by quantitative real time PCR. Figure 5A shows that the expression of ß-catenin was increased 3.6-fold in the thyroids of TRßPV/PV mice. The concurrent up-regulation of cyclin D1 and ß-catenin suggests that the Wnt/ß-catenin signaling pathway could also be activated in the thyroids of TRßPV/PV mice.

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Fig. 5. Comparison of ß-catenin (A) and cathepsin D (B) mRNA expression in the thyroids of TRßPV/PV mice and wild-type siblings. Total RNAs were prepared from mice at the age of 79 months and quantitative real time RTPCR was performed using 0.1 µg of total RNA as described in Materials and methods. Fold of changes of the expression of mRNA were shown. The differences are significant (P < 0.001 and 0.01 for ß-catenin and cathepsin D, respectively). The P value was determined by the Student's t test.
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In addition to TSH, other peptide growth factors such as insulin-like growth factors (IGF-1) and epidermal growth factors (EGF) have been shown to stimulate the proliferation of the thyroid cells (40). Our array data indicate the up-regulation of the EGF and IGF-1 signaling pathways given that cathepsin D was activated nearly 3-fold (Table I). It is known that these two growth factors induce the expression of the cathepsin D gene (43). We further analyzed the expression of the cathepsin D gene by quantitative real time PCR. As shown in Figure 5B, consistent with that detected in the array study, the expression of the cathepsin D gene was activated 3-fold as compared with that of the wild-type siblings. In addition, we found that the expression of IGF-1 was also increased 1.6-fold. We did not list it in the tables because the extent of its up-regulation had not met the selection criteria (<2.0). These results further support the conclusion that the IGF-1 signaling pathway was activated.
We propose that the TGFß signaling pathway was also activated; this proposal is based on the findings that two of its downstream target genes were activated. One was the TGFß-induced 68 Da protein (p68 big-h3) that was activated 7.3-fold and the other was the follistatin-like gene that was activated 1.9-fold as shown by the array analyses (Table VII). Furthermore, the expression of decorin (a negative regulator of TGFß activity) was repressed (Figure 4 and Tables II and VII). p68 big-h3 is a secretory protein that is induced in several types of human cells, including melanoma cells, mammary epithelial cells, keratinocytes and fibroblasts, upon treatment with TGFß (44,45). The follistatin-like gene encodes a novel protein with unknown function that is induced upon treating mouse osteoblastic MC3T3-E1 cells with TGFß (46). We further confirmed the activated expression of p68 big-h3 and follistatin-like genes by quantitative real time PCR shown in Figure 6A and B, respectively.

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Fig. 6. Comparison of TGFß-induced 68 kDa protein (A) and fallistatin- like (B) mRNA expression in the thyroids of TRßPV/PV mice and wild-type siblings. Total RNAs were prepared from mice at the age of 79 months and quantitative real time RTPCR was performed using 0.1 µg of total RNA as described in Materials and methods. Fold of changes of the expression of mRNA were shown. The differences are significant (P < 0.001). The P value was determined by the Student's t test.
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The array data suggest that the tumor necrosis factor-
(TNF-
) signaling pathway was activated (Table VII) as shown by the 1.5-fold activation in its expression [because this increase in activation did not meet the selection criteria (i.e. it is <2-fold), we did not list it in the tables] and by its activity in the down-regulation of its direct downstream target genes, decorin (Figure 4 and Tables II and VII). The promoter of the decorin gene contains a TNF-
response element, located between nucleotides -188 and -140, and the expression of the decorin gene is negatively regulated by TNF-
(47,48). Additional support for this notion came from the findings that the expression of its activator, the lipopolysaccharide (LPS)-induced tumor necrosis factor-
factor (LPS-induced TNF-
factor) was also activated (Tables V and VII). LPS-induced TNF-
factor binds to the TNF-
gene promoter and activates it transcription (49). Furthermore, we found that the expression of the LPS binding protein was also activated (Tables V and VII). LPS binding protein acts to potentiate cell sensitivity to LPS, leading to enhanced expression of TNF-
(50). Furthermore, analysis of array data uncovered another TNF-
-induced gene, the heme oxygenase 1 gene that was activated 1.8-fold (Table VII) (51). Its activation was further confirmed by quantitative real time PCR (Figure 7A). Taken together, we propose that the TNF-
signaling pathway is activated during carcinogenesis of the thyroids in TRßPV/PV mice.

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Fig. 7. Comparison of heme oxygenase 1 (A) and defender against cell death 1 (B) mRNA expression in the thyroids of TRßPV/PV mice and wild-type siblings. Total RNAs were prepared from mice at the age of 79 months and quantitative real time RTPCR was performed using 0.1 µg of total RNA as described in Materials and methods. Fold changes of the expression of mRNA were shown. The differences are significant (P < 0.05 and 0.01 for heme oxygenase 1 and defender against cell death 1, respectively). The P value was determined by the Student's t test.
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Nuclear factor-kappa B (NF-
B) is a transcription factor that regulates genes important for tumor invasion, metastasis and chemoresistance. When bound to cytoplasmic inhibitor of
B (I
B) proteins,
B remains sequestered in an inactive state. Upon release from the inhibitor, NF-
B translocates to the nucleus and activates gene expression upon exposure of cells to growth factors and cytokines. Analysis of the array data indicates that one of its regulated genes, defender against cell death-1 (DAD-1), is activated (Tables IV and VII). The activation of this gene was further confirmed by quantitative real time PCR (Figure 7B). Homozygous mice deficient in DAD-1 died shortly after they were implanted with the characteristic features of apoptosis, an indication of the critical role of DAD-1 in preventing apoptotic cell death (39). The finding that the expression of DAD-1 was up-regulated suggested the activation of the NF-
B signaling pathway. Moreover, it is important to point out that two NF-
B binding sites are present in the promoter of the cyclin D1 gene (52). Thus, cyclin D1 is a direct target gene of NF-
B transcription factor (52). The activated expression of the cyclin D1 gene found by array (Tables I and VII) and northern blot analyses (Figure 1) supports the notion that the NF-
B signaling pathway is activated in the thyroids of TRßPV/PV mice.
PPAR
is a ligand-dependent nuclear transcription factor involved in many cellular processes such as adipogenesis, inflammation, atherosclerosis, cell-cycle control, apoptosis and carcinogenesis (53,54). Recently, Kroll et al. reported the identification of a chromosomal rearrangement t(2:3)(q13;p25), yielding a PAX8PPAR
1 fusion gene exclusively in follicular carcinomas (7). When fused to PAX8, PPAR
1 not only loses its capability to stimulate thiazolidinedione-induced transcription, but also acts to inhibit PPAR
1 transcriptional activity (7). However, how the loss of PPAR
1 transcriptional activity affects the normal functions of thyroid follicular cells is unclear. We therefore ascertained whether the signaling pathway of PPAR
1 was affected by searching for the downstream target genes of PPAR
because the PPAR
1 cDNA was not represented in our mouse arrays. Indeed, we found that LpL was repressed (Tables III and VII and Figure 3B), indicating that the PPAR
signaling pathway was represssed.
 |
Discussion
|
---|
The availability of the TRßPV/PV mice as a model for FTC (18) provides an unusual opportunity to profile genomic changes during carcinogenesis. From the arrays of 20 000 genes, the expression of 185 genes was activated and 92 genes were repressed. Clustering of the named genes with reported functions show that nearly 39% of genes undergoing changes are related to carcinogenesis (20% tumor-related, 16% metastasis/invasion-related and 3% cell-cycle- and apoptosis-related). Fourteen percent of the genes were related to the thyroid functions. The remainder of the genes were involved in the alterations of various cellular processes and metabolism (Tables V and VI). These results clearly show that global genomic changes are associated with thyroid carcinogenesis.
Based on the gene profiles, the changes in several signaling pathways during thyroid carcinogenesis were identified (Table VII). The activation of the TSH signaling pathway is consistent with the increased serum TSH in TRßPV/PV mice and the enlargement of the thyroid glands (13,18), indicative of the stimulation of thyroid growth by TSH. Consistent with that reported in human thyroid cancer (42), our data suggest that the Wnt/ß-catenin signaling pathway is activated. Other peptide growth factors, such as IGF-1, EGF, fibroblast growth factor (FGF) and TGF-
, have been reported to promote thyroid growth in vitro and in vivo (55). Indeed, Table VII shows that the signaling pathways mediated by growth factors, including IGF-1, EGF, FGF and TGF-
, were also activated, thus indicating the contribution of these pathways in promoting thyroid growth in TRßPV/PV mice.
It is of interest to find that the TGF-ß signaling pathway was activated (Table VII). In vitro studies suggest that TGF-ß may play a role as a negative modulator of thyroid growth (5658). It is possible that the activation of the TGF-ß signaling pathway is one of the mechanisms utilized by the thyroid to restrain cells from uncontrolled growth. The up-regulation of inhibitory pathways as a regulator to counteract over-stimulation was also supported by the activation of the TNF-
signaling pathway (Table VII). Administration of TNF-
in rats reduces the TSH mRNA expression, inhibits the uptake of iodide, and reduces the release of T3/T4 by the thyroid. It also blocks the stimulation of incorporation of [3H]thymidine uptake by FRTL-5 and thyroid cells (59,60), thus indicating a negative regulatory role of TNF-
in thyroid functions. Hence, the activation of the TNF-
signaling pathway could be the cellular means to counteract the hyper-functioning thyroids in TRßPV/PV mice.
Very little is known about the role of NF-
B in thyroid carcinogenesis. The array data indicate the up-regulation of DAD-1 mRNA in the thyroids of TRßPV/PV mice (Tables IV and VII). Since DAD-1 is a downstream target of NF-
B, the present study highlights the contribution of the pathway in thyroid carcinogenesis. Mice deficient in DAD-1 undergo increased embryonic apoptosis that leads to lethality by embryonic day 10.5 (61), an indication of the anti-apoptotic role of this gene. The up-regulation of this gene could contribute to the tumorigenesis by prevention of apoptosis of tumor cells.
Among the activated pathways, long-term stimulation of the thyroid by TSH has been implicated in a more frequent incidence of thyroid cancer (62). 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 a subunit (Gs
) are known to inappropriately activate adenyl cyclase activity and to stimulate a relatively unrestrained growth of thyroid cells. However, consensus from clinical studies indicates that autonomously functioning thyroid nodules have a low probability of malignant transformation (63). Furthermore, transgenic mice with thyroid-specific expression of the A2 adenosine receptor (64), a mutated Gs
(65), or cholera toxin A1 (66) develop thyroid hyperplasia and hyperthyroidism, but not carcinomas. In addition, patients with Graves' disease or with congenital hyperthyroidism due to germ-line mutations of the TSH receptor do not appear to have a higher rate of thyroid malignancy. Thus, additional genetic changes would need to occur for the transformation of the hyper-proliferative thyroid cells to cancer cells.
The mutations of the two TRß alleles in the thyroids of TRßPV/PV mice led to an array of genomic changes that resulted in the ultimate development of FTC with distant organ metastasis (18). Genes that were found to be over-expressed in human FTC such as cyclin D1 (22), PTTG-1 (26), cathepsin D (33), glutathione peroxidase 2 (67) and TGF
(68,69) were also uncovered to be activated in the thyroid of TRßPV/PV mice (Tables I and II). Repression of the kit gene was found in the present study (Table I) and also in human FTC (70,71). The identification of the same genes with similar altered expression patterns in human FTC and TRßPV/PV mice further supports the validity of the array approach. At present, it is unclear how the mutant TRß acts in vivo to bring about the genomic changes uncovered in the present studies; it is also unknown how the mutant TRß cooperates with other signaling pathways to result in the transformation of the hyperplastic thyroid cells to tumor cells. It is, however, possible to conclude that in this mouse model, mutations of the two TRß alleles are obligatory for the carcinogenesis to occur. The present study also shows that several activated pathways converge, leading to the up-regulation of the important oncogene, cyclin D1 (Table VII). Thus, it is clear that thyroid carcinogenesis requires the complex coordinated alterations of the cellular pathways. The identification of the tumor- and metastasis/invasion-related genes and the altered signaling pathways associated with carcinogenesis should clear the way for subsequent dissection of the molecular genetic events essential to understanding the molecular basis of follicular thyroid carcinoma.
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Received April 1, 2003;
revised June 9, 2003;
accepted June 21, 2003.