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Am J Cancer Res. 2011; 1(1): 1–13.
Published online Sep 30, 2010.
PMCID: PMC3090007
NIHMSID: NIHMS244262

Genomic profiling of genes contributing to metastasis in a mouse model of thyroid follicular carcinoma

Abstract

Metastasis is the major cause of thyroid cancer-related death. However, little is known about the genes involved in the metastatic spread of thyroid carcinomas. We have created a mouse that spontaneously develops metastatic follicular thyroid carcinoma (FTC). This mouse harbors a targeted mutation (denoted TRβPV) in the thyroid hormone receptor β gene (ThrbPV/PV mice). Our recent studies show that the highly elevated level of thyroid stimulating hormone (TSH) in ThrbPV/PV mice promotes proliferation of thyroid tumor cells, but requires the collaboration of the oncogenic action of TRβPV to empower the tumor cells to undergo distant metastasis. To uncover genes destined to drive the metastatic process, we used cDNA microarrays to compare the genomic expression profile of laser capture microdissected thyroid tumor lesions of ThrbPV/PV mice with that of hyperplastic thyroid cells of wild-type mice having elevated TSH induced by treatment with the anti-thyroid drug propylthiouracil (WT-PTU mice). Analyses of microarray data indicated that the expressions of 150 genes were significantly altered between ThrbPV/PV and WT-PTU mice (87 genes had higher expression and 63 genes had lower expression in ThrbPV/PV mice than in WT-PTU mice). Thirty-six percent of genes with altered expression function as key regulators in metastasis. The remaining genes were involved in various cellular processes including metabolism, intracellular trafficking, transcriptional regulation, post-transcriptional modification, and cell-cell/extracellular matrix signaling. The present studies have uncovered novel genes responsible for the metastatic spread of FTC and, furthermore, have shown that the metastatic process of thyroid cancer requires effective collaboration among genes with diverse cellular functions. Importantly, the present studies indicate that the tumor cells in the primary lesions are endowed with the genes destined to promote metastasis. Thus, our study has provided new insights into the understanding of the metastatic spread of human thyroid cancer.

Keywords: Metastasis, thyroid cancer, mouse model, microarray, gene expression

Introduction

Thyroid cancers constitute the most frequent endocrine neoplasia, and their incidence has risen over the past several decades. Thyroid cancers in humans consist of an array of different histologic and biological types (papillary, follicular, medullary, clear cell, anaplastic, Hurthle cell, and others) [1], but the majority of clinically important human thyroid cancers are of the papillary and follicular types. Papillary thyroid carcinomas commonly metastasize to lymph nodes and are often multifocal, whereas follicular carcinomas show blood-borne metas-tases. While overall survival of patients with these types of tumor is generally better than for many other cancers, approximately 30% of patients do not survive beyond 20 years, even with successful primary surgical therapy. Recurrence of the tumor with metastasis becomes the major cause for thyroid cancer-related death. The genetic basis for this invasive or metastatic behavior is poorly understood. Animal models exhibiting metastatic spread would be useful in elucidating the molecular basis of metastatic thyroid cancer.

Accordingly, we have generated a mouse model (ThrbPV/PV mice) that spontaneously develops metastatic follicular thyroid cancer (FTC). This mouse harbors a knockin dominant negative mutation, known as PV, in the Thrb gene locus [2]. The PV mutation was identified in a patient suffering from resistance to thyroid hormone (RTH) [3]. PV has a frame-shift mutation in the carboxyl-terminal 14 amino acids, resulting in a complete loss of thyroid hormone (T3) binding activity and transcriptional capacity [3]. Similar to an RTH patient who had two mutated THRB genes [4], ThrbPV/PV mice exhibit highly elevated serum thyroid stimulating hormone (TSH) [2, 5]. Remarkably, as ThrbPV/PV mice age, their thyroids undergo pathological changes from hyperplasia to capsular and vascular invasion, anaplasia, and eventual metastasis to the lung and sometimes to the heart [5]. The pathological progression, route, and frequency of metastasis in ThrbPV/PV mice are similar to that in human FTC.

Extensive molecular analyses of altered signaling pathways during thyroid carcinogenesis further validated that the ThrbPV/PV mouse is a pre-clinical mouse model of FTC. As found in human FTC, ThrbPV/PV mice exhibit similar aberrant signaling pathways that include constitutive activation of phosphatidyl inositol 3-kinase (PI3K)/Akt [6, 7], repression of peroxisome proliferator-activated receptor γ signaling [8, 9], and aberrant accumulation of both the pituitary tumor transforming gene (PTTG) [10, 11] and β-catenin [12]. Thus, the ThrbPV/PV mouse model faithfully recapitulates the molecular aberrations found in human thyroid cancer and is suitable for further identification by genomic profiling of genes that contribute to metastasis.

We have recently shown TSH is required for thyrocyte proliferation, but not sufficient for metastatic thyroid carcinogenesis of ThrbPV/PV mice [13]. This finding has facilitated the search for metastatic genes during thyroid carcinogenesis of ThrbPV/PV mice. In the present study, we compared the genomic expression profiles of laser capture microdissected thyroid lesions of ThrbPV/PV mice that have highly elevated TSH levels with that of hyperplastic follicular cells of wild-type mice that also have highly elevated TSH induced by propylthiouracil treatment (WT-PTU). By doing so, the identification of genes responsible for metastatic process was therefore simplified, as the expression of genes induced by TSH would not be considered. Indeed, we found distinctly different genomic expression profiles between thyroid cancer cells of ThrbPV/PV mice and hyperplastic follicular cells of WT-PTU mice. Among the 19 metastatic genes identified, 18 novel metastatic genes were uncovered in thyroid tumor cells. The identification of these novel genes suggests that the tumor cells in the primary lesions are endowed with the genes destined to promote metastasis. Furthermore, in addition to altered expression of genes acting in the metastatic process, we have also found altered expression of genes involved in various cellular processes including metabolism, intracellular trafficking, transcriptional regulation, post-transcriptional modification, and cell-cell/extracellular matrix (ECM) signaling. These results show that the metastatic process requires the collaboration of metastatic genes with diverse cellular signaling pathways for tumor cells to invade and to migrate to distant target sites.

Materials and methods

Animals

The animal protocols used in the study were approved by the NCI Animal Care and Use Committee. Mice harboring the TRβPV gene (ThrbPV/pv mice) were generated as previously described [2]. To generate mice with a high TSH level, wild-type (WT) siblings of ThrbPV/PV mice were fed with an iodine-deficient diet supplemented with 0.15% propylthiouracil (PTU) (Harlan Teklab) ad libitum starting at the age of 2 months. Thyroid tissues were collected from mice when they reached 10 months old and stored at -80°C for further analyses.

Laser capture microdissection, RNA extraction and amplification

Laser capture microdissection (LCM) was performed on the thyroid sections that were verified by histopathological analysis. Briefly, sections (5- to 8-μm) were cut from the OCT (optimal cutting temperature) compound blocks (cat. no. 4583, Tissue Tek, Sakura Finetek USA, Inc.) on PEN (polyethylene naphthalate) membrane slides (cat no. LCM 0522, MDS Analytical Technologies). LCM was then performed using an ArcturusXT (Arcturus Engineering, Inc.) or a Veritas (Arcturus Engineering, Inc) machine. Captured cells attached to the polymer film surface on the CapSure Macro LCM caps (Arcturus Engineering, Inc.) were used for RNA extraction with PicoPure kit (cat. no. KIT#0202, Arcturus Biosciences, Inc.) according to the manufacturer's instructions.

The extracted total RNA was amplified with the use of a MessageAmpTMII aRNA amplification kit (AM 1751, Ambion) following the kit's protocol. In short, RNA (0.1-1.0 ng) was subjected to two rounds of amplification, and enriched aRNA was labeled with biotin-11-UTP for microarray hybridization. The quantity, integrity, and quality of biotinylated aRNA were assessed by Nanodrop (Thermo Scientific) and 2100 Bioanalyzers (Agilent Technologies). Mouse Pax8 gene was used as a positive control to validate the thyroid tissue specificity by RT-PCR. The primers for murine Pax8 were FP: 5'-CAC CTT CGT ACG GAC ACC TT-3' and RP: 5'-GTT GCG TCC CAG AGG TGT AT-3'.

Microarray analysis

Biotinylated-aRNA samples from three individual mice of each group were used in hybridization of the GeneChip Mouse Genome 430 2.0 array (Affymetrix, Santa Clara, CA) and scanned on an Affymetrix GeneChip scanner 3000. Data were collected using Affymetrix GCOS software. Statistical and clustering analyses were performed with Partek Genomics Suite software using the robust multichip average (RMA) normalization algorithm. Differentially expressed genes were identified with ANOVA analysis. Genes that were up- or down-regulated more than 1.5 fold and with a p <0.05 were considered significant. Significant genes were analyzed for enrichment of pathways and functions using the DAVID bioinformatics database (http://david.abcc.ncifcrf.gov/) [14, 15] and Ingenuity Pathway Analysis (IPA, Ingenuity Systems, Inc., Redwood City, CA).

Real time RT-PCR validation of microarray data

Selected genes from microarray data were chosen for real time RT-PCR validation. A total 50 ng of RNA extracted from thyroids of ThrbPV/PV or WT-PTU mice was used in the real-time RT-PCR. The reactions were performed with the Quan-tiTech SYBR RT-PCR kit (Qiagen, Germantown, MD) on an ABI 7900HT system. In each group, four to six samples with triplicates were tested on the target genes. Data were analyzed using Prism V5 (GraphPad Software, Inc., La Jolla, CA). Primers used in RT-PCR are listed in Supplementary Table 1.

Results

Analysis of gene expression profiles of hyperplastic follicular cells of WT-PTU mice and thyroid tumor cells of ThrbPV/PV mice

Array data were obtained from laser capture microdissected thyroid samples of age-matched male wild-type (WT) mice, WT-PTU mice, and ThrbPV/PV mice (n=3 for each type of mice). Figure 1A shows the results of principal component analysis (PCA) of the gene expression profiles from WT mice with normal TSH levels, WT-PTU mice with highly elevated TSH levels, and ThrbPV/PV mice with highly elevated TSH levels. The three-dimensional projection of the top three principal components of PCA, capturing 66.7% of total variance, shows clear separation of the three groups (Figure 1A). The well-segregated three clusters of data derived from WT, WT-PTU, and ThrbPV/PV mice, respectively, allowed us to compare the changes in gene expression due to TSH (compare WT with WT-PTU mice) or due to combined effects of TSH and TRβPV (compare WT with ThrbPV/PV mice). Subsequent comparison of gene expression profiles between the TSH-mediated effect and the effects due to combined actions of TSH and TRβPV allowed us to sort out the gene expression profiles due to the oncogenic actions of TRβPV critical for metastasis. Comparison of the array data between ThrbPV/PV and WT-PTU mice showed that 150 genes (Supplementary Table 2) exhibited altered expression (>1.5-fold change, p <0.05). In ThrbPV/PV mice, 87 genes were up-regulated and 63 genes were down-regulated. Among the up- and down-regulated genes, 13 (14.9%) and 8 (16%), respectively, were unnamed genes. A heat map displays the top 50 differentially expressed genes in the thyroid tumor cells of ThrbPV/PV mice by hierarchical clustering (Figure 1B). It is clear that the gene expression profiles mediated by TSH alone in WT-PTU were strikingly distinct from those mediated by the combined actions of TSH and TRβPV in ThrbPV/PV mice.

Figure 1
Principal component analysis (PCA) of the gene expression profiles in ThrbPV/PV, WT-PTU, and WT mice (A), and a heat-map of hierarchical clustering in the top 50 genes with altered expression between ThrbPV/PV mice and WT-PTU mice (B). A. Three-dimensional ...

Functional classification of genes with altered expression in thyroid tumor cells of ThrbPV/PV mice

The gene ontology analyses were first performed by using both the Gene Ontology Consortium tool available at http://www.informatics.jax.org and the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 (http://david.abcc.ncifcrf.gov/) [14, 15]. These two approaches provided a comprehensive analysis of molecular functions and classification of each named gene. The functional classification analyzed by DAVID is presented in Supplementary Table 3. This table shows that 24.7% (37/150 total genes) were related to cell development and differentiation during embryogenesis. Twenty-four percent of genes (36/150) were found in the category with functions related to maintaining cell structure, cell movement, or cell-to-cell signaling. About 9% of the identified genes regulated immune response, and approximately 10% regulated metabolism.

In addition, we also searched the PubMed database for each named gene in combination with the following terms: tumor, metastasis, thyroid, thyroid tumor, invasion, mobility, immune response, and stem cells. Combined with results of DAVID and IPA analyses, the altered genes between ThrbPV/PV and WT-PTU were grouped in three major categories (Table 1: Metastasis/ invasion-related genes; Table 2: Tumor-related genes; Table 3: Genes with other cellular functions), signifying molecules with wide diverse functions and complex signaling pathways during metastatic thyroid carcinogenesis of ThrbPV/ PV mice.

Table 1
Metastasis-related genes (n=19) with altered expression in thyroid tumors of ThrbPV/PVmice
Table 2
Tumor-related genes (n=35) with altered expression in thyroid tumors of ThrbPV/PV mice
Table 3
Genes with diverse cellular functions (n=56) that have altered expression in thyroid tumors of ThrbPV/PV mice

Since it is not possible to detail the cellular functions for each of 150 genes that had altered expression within the journal space allowed, only the genes that were well-studied in other cancers are highlighted in each category as shown below. Readers should refer to the Supplemental Tables 2 and and33 for the genes that they are interested in.

Uncovering of novel metastasis-related genes in thyroid tumor cells of ThrbPV/PV mice

Table 1 lists 19 genes that contribute to tumor metastasis in the microdissected thyroid lesions of ThrbPV/PV mice. The gene names and symbols are listed for easy reference. These genes account for 14.4% of the total named genes involved in cell-cell interaction, ECM interaction and signaling, cell migration, and angiogenesis. All these functions are critical for cell migration and invasion. Among these genes, 10 were up-regulated (52.6%), ranging from 1.64- to 5.52-fold; the other 9 genes were down-regulated, ranging from 1.87- to 4.35-fold. These genes are reported to play a role in cell invasion and metastasis in other cancers. However, except for the Nr2f2 gene that was studied as a potential marker for diagnosis of thyroid cancer [16], all other 18 genes listed in Table 1 were uncovered in the present study to play a role in migration and invasion of thyroid tumor cells of mice.

At the top of the list (Table 1) is the extracellular matrix glycoprotein olfactomedin 4 (Olfm4) gene. The Olfm4 mRNA level was 5.52-fold higher in thyroid tumors of ThrbPV/PV mice shown by array analysis, and the increase was further shown by real time RT-PCR analysis (4.14-fold; Figure 2A). Over-expression of Olfm4 has been found in gastric cancer, pancreatic cancer, and colorectal carcinomas [17-19] and serves as an early diagnostic marker for gastric cancer patients [19]. The gene with the second highest activation was the mucin 4 gene (Muc4; 3.85-fold increase by array analysis). Its increased expression was also shown by real time RT-PCR analysis (Figure 2B). Overexpressed Muc4 has been observed in various preneoplastic and neoplastic lesions [20, 21]. In addition to its role in cell adhesion, this membrane protein has also been implicated in the regulation of cellular growth signaling through its interaction with the ErbB family of growth factor receptor tyrosine kinases (RTKs).

Figure 2
Validation of microarray results by real time RT-PCR. Total RNA was extracted from thyroids of ThrbPV/PV and WT-PTU mice at the age of 10-12 months, and real time RT-PCR was performed as described in Materials and methods. Fold of changes of the expression ...

The epithelial membrane protein gene 2 (Emp2) also caught our attention (Table 1) (2.4-fold activation by array analysis and 1.38-fold by real time RT-PCR determination; Figure 2C). This gene encodes a tetraspan protein Emp2, a key regulator in cell adhesion and invasion. Emp2 also plays a role in trafficking proteins such as integrin αvβ3 and other key membrane receptors for efficient signaling transduction by membrane receptors [22].

Tumor-related immune response signaling in the microenvironment of thyroid tumor cells could promote metastasis. Mediators of tumor-related immune response may provide the favorable microenvironment for the growth of tumor cells. Table 1 shows the altered expression of interleukin (Il11, also see Figure 2D) and down-regulation of interleukin 13 receptor, α2 (Il13ra2; also see Figure 2E). Interleukin-11 is a pleiotropic cytokine that was reported to be over -expressed in endometrial [23] and colorectal adenocarcinoma [24]. In colorectal adenocarcinoma, IL-11 promotes the tumor cell invasion via PI3K signaling and the p42/p44 MAPK pathway [24]. Consistent with these findings, we have recently reported the increased PI3K-AKT and p38 MAPK signaling during thyroid carcinogenesis of ThrbPV/PV mice [6, 13]. The Il13ra2 gene encodes Il13 receptor α2 (Il13Rα2), a sub-unit of the interleukin 13 receptor complex. In human pancreatic cancer cells and glioblastoma, Il13Rα2 has been considered as a tumor antigen with therapeutic potential [25, 26]. In thyroid tumor cells of ThrbPV/PV mice, the suppression in the expression of Il13ra2 gene could imply weakened immunosuppression that could favor metastatic potential of tumor cells.

The validation of these genes relevant in cell adhesion and invasion revealed that an array of genes shown in Table 1 participated in complex signaling to mediate metastatic thyroid carcinogenesis of ThrbPV/PV mice.

Identification of critical tumor-related genes in thyroid tumor cells of ThrbPV/PV mice

In addition to the metastasis-related genes (Table 1), we identified 35 genes (23.3%; 35/150 total genes) involved in tumor development in the microdissected thyroid lesions of ThrbPV/PV mice (Table 2). About 65.7% of genes in this group were activated, with changes ranging from 1.53- to 10.9-fold; 34.2% were repressed, with changes ranging from 1.49-to 10-fold. These genes are key regulators in embryonic development, differentiation, transcription regulation, and signaling transduction. The altered expression of these genes suggested that they are critical in the oncogenesis of thyroid tumor cells. Of particular interest was the activated expression of the sonic hedgehog homolog (Shh) gene (5.8-fold; Table 2) that was further confirmed by real time RT-PCR analysis (Figure 2F). Shh is a secreted protein that plays a key role in embryogenesis and thyroid morphogenesis [27]. Importantly, it controls cell division of adult stem cells and has been implicated in the development of several cancers by promoting angiogenesis or cancer stem cell renewal [28, 29].

Another gene that plays a pivotal role in tissue regeneration and cancer development is the estrogen-related receptor β (Esrrb) gene that encodes an orphan nuclear receptor, Esrrβ (Nr3b2). Consistent with the array data (4-fold up-regulated; Table 2), real time RT-PCR analysis showed that the mRNA expression of Esrrb was increased nearly 3-fold (Figure 2G). In conjunction with other transcription factors critical for pluripotency of stem cells, namely Oct4 and Sox2, Esrrβ could also act to induce fibroblasts into pluripotent stem cells by targeting genes involved in self-renewal and pluripotency [30]. The findings that Shh and Esrrb were activated in the thyroid lesions of ThrbPV/PV mice raised the possibility that adult cancer stem cells could be involved in the oncogenesis of thyroid cancer.

Table 2 shows the activation of another oncogene, the hepatocyte nuclear factor 1b gene (Hnf1b) (1.95-fold; Table 2). A 6.0-fold increase in mRNA expression was detected by real time RT-PCR (Figure 2H). The Hnf1b gene encodes a homeobox transcription factor that was recently found aberrantly expressed in several human neoplasms, such as ovarian clear cell carcinoma, and thyroid cancer [31, 32]. In thyroid cancers, HNF1b mRNA and protein were detected in several papillary cancer cell lines containing RET-PTC1 translocation. Its high expression (Figure 2H) suggested its pivotal role in the metastatic thyroid carcinogenesis of ThrbPV/PV mice.

The 10.9-fold activated expression of the fi-brinogen gamma chain (Fgg) gene was highly relevant in the understanding of the metastatic process of thyroid cancer (see also Figure 2I). This gene encodes a subunit (gamma-fibrinogen) of fibrinogen that is associated with human cancers such as hepatocarcinoma [33]. Importantly, fibrinogen is known to function as a ligand to activate integrins α5β1 and αvβ3 signaling to affect downstream signaling to increase cell motility and invasion. Indeed, our recent findings showed that an activated integrin signaling promotes thyroid carcinogenesis of ThrbPV/PV mice [13].

Altered expression of genes with diverse cellular functions in thyroid tumor cells of ThrbPV/PV mice

Analyses of the array data further showed altered expression of 56 genes important in various cellular processes (Table 3). These genes represent 37.3% of total genes identified. Among these genes, 31 were up-regulated and 25 were down-regulated. Twenty-three of them (41%) were regulators with enzymatic activity related to metabolic synthesis, protein/lipid phosphorylation, protein degradation, or DNA modification. Six genes (10.7%) had biological activities related to intracellular trafficking, and there were transporter-related genes. The remaining 24 genes were associated with transcriptional regulation or post-transcriptional modification or served as membrane receptors in mediating cell-cell or cell-extracellular matrix (ECM) interaction. Thus, the metastatic process of thyroid tumor cells requires not only that genes function directly in mediating cell invasion and migration, but also that genes actively participate in a variety of cellular functions.

Indeed, it is known that transformation of cells from a normal to a cancerous state is usually accompanied by reprogramming of metabolic pathways, including those that regulate glycolysis and the production of lipids. The altered gene repertoire of metastasis-related genes in ThrbPV/PV thyroids thus extends to metabolism-related enzymes, such as carbonic anhydrase IV (Ca4) in carbon dioxide hydration, glutamic-oxaloacetic transaminase 1 (Got1) in amino acid metabolism, and pterin-4-a-carbinolamine dehydratase (Pcbd1; Figure 2J) in phenylalanine hydroxylation (Table 3).

In addition to genes related to metabolic enzymes, we also found a set of genes related to intracellular trafficking and transport. Two large families of molecular motors—kinesins and dyneins—drive transport along microtubule filaments. Many molecules are translocated in and out of different cellular compartments via this system, for example, p53, glucocorticoid receptors, and androgen receptors. Two members of molecular motor families, kinesin family member 5C (Kif5c) and dynein cytoplasmic intermediate chain 1 (Dync1i1), were found to be up-regulated in ThrbPV/PV thyroid (Table 3). Several genes involved in vesicle-mediated transport were also preferentially expressed in ThrbPV/PV thyroids, such as golgi-specific brefeldin A resistant guanine nucleotide exchange factor 1 (Gbf1), TBC1 domain family member 20 (Tbc1d20), and Golgi integral membrane protein 4 (Golim4). These changes in the expression may affect re-localization of certain molecules to indirectly regulate their functions.

The final group of genes that encode enzymatic proteins revealed in the present study is ubiquitination system-related genes. The ubiquitin-proteasome pathway is critical in the degradation of a majority of cellular proteins. Increased deubiquitinating (DUB) enzymes may not only enhance the protein stability, but also affect several biochemical pathways relevant to cancers, such as internalization and degradation of receptor tyrosine kinases, transcription regulation, activation or localization of signaling intermediates, and cell cycle progression [34]. The present array analysis showed one up-regulated DUB enzyme gene (ubiquitin specific peptidase 3, Usp3) and three down-regulated DUBs (OUT domain containing 7A, Otud7a; ubiquitin specific peptidase 37, Usp37; zinc finger, RNA-binding domain containing1, Zranb1) in thyroid tumors of ThrbPV/PV mice (Table 3). Among these DUBs, USP3 has been shown to efficiently de-ubiquitinate histone H2A and H2B. Its depletion can lead to the arrest of the cell cycle in the S-phase [35]. On the basis of these important cellular functions, it is reasonable to propose that they function to facilitate the metastatic process of thyroid tumor cells of ThrbPV/PV mice.

Discussion

Extensive molecular analyses have clearly shown that the ThrbPV/PV mouse is a valid mouse model for dissecting changes in molecular genetics underlying follicular thyroid carcinogenesis. Our recent studies further demonstrated that TSH is necessary for the proliferation of thyrocytes, but not sufficient to drive the metastasis of hyperplastic thyrocytes. The mutant TRβPV is required to empower the hyperplastic thyrocytes to invade and to metastasize to distant sites [13]. This observation has facilitated our genomic profiling of genes contributing to metastatic carcinogenesis of ThrbPV/PV mice. We first compared the gene expression patterns between WT and WT-PTU to identify altered gene expression due to the growth stimulatory effect of TSH. As ThrbPV/PV mice exhibit highly elevated TSH, the comparison of gene expression patterns between WT and ThrbPV/PV mice yielded altered expression of genes due to the combined effects of TSH and TRβPV. By further comparison between TSH-mediated effects (WT-PTU mice) and combined TSH- and TRβPV-mediated effects (ThrbPV/PV mice), we identified the changes in gene expression that mainly reflected the oncogenic actions of TRβPV in promoting the metastatic process. It is clear from the heat map shown in Figure 1B that the gene expression profiles mediated by TSH actions are clearly distinct from those mediated by TRβPV. Therefore, the genes affected by TSH differ from those affected by TRβPV, leading to different pathological consequences. The distinct global changes in gene expression demonstrated in the present study further support our previous conclusions in that without the collaboration of the oncogenic actions of TRβPV, TSH alone is not sufficient to induce metastatic thyroid cancer [13].

By comparing the gene expression profiles of microdissected thyroid cells between WT-PTU and ThrbPV/PV mice, we identified genes that contribute to metastatic thyroid carcinogenesis. A total of 150 genes with distinct expression in the tumor cells of ThrbPV/PV mice were identified. Clustering of the 87 up-regulated and 63 down-regulated genes with known functions showed that 36% of the genes undergoing changes in expression were related to metastasis and carcinogenesis (see Tables 1 & 2). The remaining genes with known functions (about 37.3% of the total) were involved in various cellular processes including metabolism, intracellular trafficking, transcriptional regulation, post-transcriptional modification, and cell-cell/ECM signaling (Table 3). These results clearly indicate that an alteration in global genomic expression is associated with metastatic thyroid cancer. These data further suggest that genes destined to drive the eventual metastasis are present in the primary thyroid lesions.

On the basis of the gene expression profiles and functional clustering, we discerned changes in several signaling networks during metastasis of thyroid tumor cells of ThrbPV/PV mice. The enhancement and activation of integrin-ECM signaling was evidenced by increased expression of the Fgg and Emp2 genes, which is consistent with the elevated integrin-c-Src-Fak activity reported previously in ThrbPV/PV mice [13]. In addition, Mia1, a negative regulator of integrin-MAPK signaling, was found down-regulated concurrently with an increased expression of its negative regulator, Hmga1. Adhesion of tumor cells to ECM is a crucial step in the development of cancerous cell metastasis. In human differentiated or anaplastic thyroid carcinoma cells, different patterns of integrin receptors were identified. In follicular thyroid cancer cell lines, high levels of integrins α2, α3, α5, β1, and β3 and low levels of α1 were found, whereas in papillary thyroid cancer cell lines, a dominant expression of integrins a5 and β1 was identified. In undifferentiated anaplastic thyroid cancer cells, integrins α2, α3, α5, α6, and β1 and low levels of α1, α4, and αV were mainly displayed [36]. Taken together, these findings make it clear that the alteration in integrin-ECM signaling plays a crucial role in increasing thyroid tumor cell invasiveness and in promoting distant metastasis in ThrbPV/PV mice.

It is important to point out that several key molecules, such as Shh, Esrrb, Nr2f2, and Hnf1b, critical in embryonic development were aberrantly activated in thyroid tumor cells of ThrbPV/PV mice. These genes encode transcription factors (Esrrb, Nr2f2, and Hnf1b) or signaling regulator (Shh) whose alteration may influence a set of genes related to cell renewal, differentiation, or proliferation. For example, Esrrb can directly activate Oct4 gene expression to maintain cell pluripotency and self-renewal capability [37]. The discovery that these genes are activated in thyroid cancer cells of ThrbPV/PV mice raises the possibility that cancer-initiating stem cells could play a role in metastatic thyroid carcinogenesis and that their precise functions warrant additional studies.

These different signaling pathways identified in the present study may coordinate with each other to converge into a larger signaling network to affect the metastatic progression of thyroid cancer cells in ThrbPV/PV mice. Three top networks with the highest scores and the greatest number of involved molecules were suggested by analyses of the complete list of genes with altered expression (see Supplemental Table 2). Twenty-five key regulators identified in our array analysis were merged into a network to function mainly in cell death, gene regulation, and cell-to-cell signaling and interaction. In this network, genes related to carcinogenesis, immune responses, and other functions are linked via several important pathways including AKT, NFkB, TGF-β, VEGF, and IFNα signaling. In other two networks, amino acid metabolism can be related to post-translational modification while the post-translational modification is affected by the DNA repair process. These networks could each function individually and/or in a coordinated fashion to bring out the full spectrum of metastatic progression in ThrbPV/PVmice.

The present study clearly shows that many aspects of key cellular functions of thyroid tumor cells underwent changes during metastatic processes. That complex alterations of multiple pathways mediate the metastatic process would suggest therapeutic treatments via targeting one single gene or one isolated pathway may not be adequate to ensure total efficacy. However, the key regulators and signaling pathways uncovered in the present study could be studied further to understand molecular mechanisms in the metastatic progression of thyroid cancer. By doing so, it would be possible to understand how best to prevent and/or treat metastasis by using treatment strategies that affect multi-signaling pathways with coordinated molecular targets.

Acknowledgments

This research was supported by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, NIH. We thank Drs. Xiaolin Wu for assistance in the hybridization of the arrays and preliminary analysis of the data, Jaime Rodriguez-Canales and Jeffrey Hanson, for assistance with the laser capture microdissection experiments.

Supplementay data

Supplementary Table 1
Primer sequences used for determination of gene expression by real time RT-PCR
Supplementary Table 2
Complete list of genes with altered expression (n=150) in thyroid tumors of ThrbPV/PV
Supplementary Table 3
Functional analysis of genes (n=150) with aberrant expression in thyroid tumors of ThrbPV/PV mice

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