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Activation of tumor cell proliferation by thyroid hormone in a mouse model of follicular thyroid carcinoma

Abstract

Thyroid cancers are the most common malignancy of the endocrine system in humans. To understand the molecular genetic events underlying thyroid carcinogenesis, we have generated a mouse model that spontaneously develops follicular thyroid carcinoma similar to human thyroid cancer (ThrbPV/PV mouse). This mutant mouse harbors a dominant-negative mutated thyroid hormone receptor β (denoted PV). The PV mutation was identified in a patient with resistance to thyroid hormone (TH). ThrbPV/PV mice exhibit highly elevated serum thyroid-stimulating hormone levels and increased TH. We have previously shown that thyroid-stimulating hormone is required, but not sufficient to induce metastatic follicular thyroid cancer in ThrbPV/PV mice. However, whether the elevated TH also contributes to the thyroid carcinogenesis of ThrbPV/PV mice was not elucidated. To understand the role of TH in thyroid carcinogenesis, we blocked the production of TH by treating ThrbPV/PV mice with propylthiouracil (ThrbPV/PV-PTU mice) and compared the development of thyroid cancer in ThrbPV/PV-PTU and untreated ThrbPV/PV mice. We found that thyroid tumor growth was reduced by 42% in ThrbPV/PV-PTU mice as compared with ThrbPV/PV mice. Analysis by bromodeoxyuridine-nuclear labeling showed decreased incorporation of bromodeoxyuridine in thyroid tumor cells of ThrbPV/PV-PTU mice, indicative of decreased tumor cell proliferation. However, cleaved-caspase 3 staining showed no apparent changes in apoptosis of tumor cells in ThrbPV/PV-PTU mice. Molecular studies identified a marked attenuation of the PI3K–AKT–β-catenin signaling pathway that led to decreased protein levels of cyclin D2, thereby decreasing tumor cell proliferation in ThrbPV/PV-PTU mice. Furthermore, matrix metalloproteinase-2, a downstream target of β-catenin and a key regulator during tumor invasion and metastasis, was also decreased. Thus, the present study uncovers a critical role of TH in promoting the thyroid carcinogenesis of ThrbPV/PV mice via membrane signaling events. Importantly, these findings suggest that anti-thyroid drugs could be considered as possible therapeutic agents of thyroid cancer.

Introduction

Thyroid carcinoma, the most common endocrine malignancy, is on the rise worldwide (Davies and Welch, 2006; Rego-Iraeta et al., 2009; Sassolas et al., 2009; Sipos and Mazzaferri, 2010). Thyroid cancers are mainly follicular cell-derived neoplasms, which include papillary thyroid carcinoma (80% of thyroid carcinomas), follicular thyroid carcinoma (FTC, <10%), poorly differentiated thyroid carcinoma (5%) and undifferentiated thyroid carcinoma/anaplastic thyroid carcinoma (<1%) (Nikiforov, 2004; Puxeddu et al., 2011). Several genomic abnormalities, including gene mutations (for example, rat sarcoma oncogene, RAS and v-raf murine sarcoma viral oncogene homolog B1, BRAF) and genomic rearrangements (for example, rearranged during transfection proto-oncogene/papillary thyroid carcinoma (RET-PTC) and paired box-8/peroxisome proliferator-activated receptor gamma (PAX8/PPARG) fusion gene), have been associated with thyroid cancers (Nikiforova and Nikiforov, 2009). However, how these genetic changes lead to thyroid cancers is not fully understood.

To understand the molecular mechanisms underlying thyroid carcinogenesis, we have created a mouse model (ThrbPV/PV mouse) that spontaneously develops FTC (Kaneshige et al., 2000; Suzuki et al., 2002). These mutant mice bear a knock-in mutated Thrb gene (ThrbPV) that encodes TRβPV, which has completely lost thyroid hormone (TH) binding and transcription activity (Kaneshige et al., 2000). The TRβPV mutant was identified in a patient with resistance to thyroid hormone (Parrilla et al., 1991). Extensive characterization of altered signaling pathways during thyroid carcinogenesis of the ThrbPV/PV mouse has identified activation of several tumor promoters such as β-catenin, cyclin D1, pituitary tumor transforming gene (PTTG), steroid receptor coactivator-3 (SRC-3) and phosphatidylinositol 3-kinase (PI3K), and the repression of tumor suppressors such as PPARγ (Ying et al., 2003, 2006, 2008; Furuya et al., 2006; Kim et al., 2007; Guigon et al., 2008). These altered signaling pathways during thyroid carcinogenesis of ThrbPV/PV mice are consistent with the changes reported in human thyroid cancers (Boelaert et al., 2003; Weinberger et al., 2007; Saji and Ringel, 2010). Thus, the ThrbPV/PV mouse is a valid mouse model to elucidate the molecular mechanisms underlying thyroid carcinogenesis.

Thyroid hormones (T3 and thyroxine, T4) have a critical role in development, growth and differentiation, and in maintaining metabolic homeostasis. They bind to thyroid hormone nuclear receptors (TRα and TRβ) to regulate target gene transcription (Cheng et al., 2010). In addition to mediating genomic actions, increasing lines of evidence also suggest that TH can initiate extranuclear functions through cytosolic thyroid hormone receptors (TRs) (Cheng et al., 2010) and/or via plasma membrane receptors (for example, integrin αvβ3) (Bergh et al., 2005; Davis et al., 2009). TH induces proliferation of several cancer cell lines, including breast, brain, pancreas and thyroid (Lin et al., 2007, 2009; Verga Falzacappa et al., 2007; Glinskii et al., 2009; Yalcin et al., 2010). The ThrbPV/PV mouse, similar to a homozygous patient with resistance to thyroid hormone (Ono et al., 1991), exhibits highly elevated thyroid-stimulating hormone (TSH) in the face of elevated TH (Kaneshige et al., 2000). Recently, using a mouse-genetics approach, we have clearly demonstrated that TSH signaling is essential, but not sufficient to induce the development of metastatic FTC (Lu et al., 2010). However, it is unclear whether elevated TH levels also affect the thyroid carcinogenesis of ThrbPV/PV mice. To explore the role of TH in thyroid carcinogenesis, we treated ThrbPV/PV mice with the goitrogen propylthiouracil (PTU) to block TH synthesis. We monitored the spontaneous development of thyroid cancer in these ThrbPV/PV-PTU mice and compared it with that in untreated ThrbPV/PV mice. Here we show that inhibition of TH synthesis by PTU treatment decreased thyroid tumor growth and delayed tumor progression in ThrbPV/PV mice. The decreased tumor cell proliferation was attributed to a marked attenuation of the PTEN-PI3K-AKT signaling pathway, resulting in reduction of cyclin D2 to decrease tumor cell proliferation. Reduction of β-catenin-matrix metalloproteinase-2 (MMP-2) signaling contributed to delayed tumor progression. Thus, the present study shows that TH, via membrane signaling pathways, acts to promote thyroid carcinogenesis in a mouse model of thyroid cancer.

Results

Inhibition of thyroid hormone (TH) production by propylthiouracil (PTU) leads to the inhibition of follicular thyroid carcinoma (FTC) development in ThrbPV/PV mice

Our previous studies have shown that the development of FTC in ThrbPV/PV mice requires the TSH proliferation signal, whereas its progression, specifically metastasis, is attributed to the mutated TRβ (Lu et al., 2010, 2011). However, whether elevated TH levels also contribute to the thyroid carcinogenesis of ThrbPV/PV mice is unclear. To investigate the role of TH in FTC development in ThrbPV/PV mice, we treated ThrbPV/PV mice with PTU to block TH synthesis starting at 2 months and continuing on the PTU diet until the mice were killed for analyses. To confirm the effect of PTU, we measured serum levels of total T4 (TT4) and TSH in ThrbPV/PV and ThrbPV/PV-PTU mice (Figure 1). Consistent with the previous report (Kaneshige et al., 2000), an elevated serum level of TT4 was found in ThrbPV/PV mice (Figure 1a, TT4=19.52±1.44 μg/dl, n=10), and the TH level was greatly suppressed (Figure 1a, ThrbPV/PV-PTU mice, TT4=1.64±0.12 μg/dl, n=10), even lower than that of wild-type mice (WT; TT4=2.51±0.19 μg/dl, n=28, data not shown in the graph). However, PTU treatment did not affect serum TSH levels of ThrbPV/PV mice, as indicated in Figure 1b, in that no statistically significant differences were detected (8002±2263 ng/ml, n=10 and 12 790±3506 ng/ml, n=8 for ThrbPV/PV mice and ThrbPV/PV-PTU, respectively).

Figure 1
figure1

Comparison of serum levels of TT4 and TSH in ThrbPV/PV mice with or without PTU treatment. Serum TT4 (a) and TSH (b) of ThrbPV/PV (n=10) and ThrbPV/PV-PTU mice (n=8–10) at the ages of 8–12 months were determined as described in Materials and methods. P values are indicated. NS, not significant.

The first evidence suggesting that TH could have a role in FTC development was the decreased weight of thyroid tumors in ThrbPV/PV-PTU mice. As shown in Figure 2a, after PTU treatment for about 6–8 months, the thyroid weight was significantly reduced by 42% in ThrbPV/PV-PTU as compared with ThrbPV/PV mice. Pathohistological analyses of thyroid, heart and lung of moribund mice (age: 8–12 months) revealed a delayed tumor progression in ThrbPV/PV-PTU mice as compared with ThrbPV/PV mice. Figure 2b shows that in ThrbPV/PV mice, the frequencies of occurrence of capsular and vascular invasion were 100% and 83.3%, respectively. In ThrbPV/PV-PTU mice, by contrast, lower frequencies of occurrence of capsular invasion (87.5%) and vascular invasion (62.5%) were detected. Moreover, although 33% of ThrbPV/PV mice developed lung metastasis, only 25% of ThrbPV/PV-PTU mice did so. These data indicate that blocking TH synthesis delayed tumor progression in ThrbPV/PV mice.

Figure 2
figure2

Inhibition of TH production by PTU treatment suppresses the development of FTC in ThrbPV/PV mice. (a) Thyroid glands from ThrbPV/PV (n=12) and ThrbPV/PV-PTU mice (n=11) aged 8–12 months were dissected for comparison. The data are presented as the percentage of thyroid weight versus the body weight. The difference of thyroid weights between ThrbPV/PV and ThrbPV/PV-PTU mice was significant (P<0.005) as determined by the unpaired t-test analysis. (b) Pathological progression of FTC in ThrbPV/PV mice with or without PTU treatment. Sections of thyroids and lungs from ThrbPV/PV and ThrbPV/PV-PTU mice of the same age (8–12 months) were stained with hematoxylin and eosin, and analyzed for pathological progression of hyperplasia, capsular invasion, vascular invasion and lung metastasis. The data are expressed as the percentage of occurrence of total mutant mice examined.

Inhibition of thyroid hormone production by propylthiouracil treatment decreases proliferation of thyroid tumor cells in ThrbPV/PV mice with no apparent effect on apoptosis

Cell proliferation and apoptosis are two major processes that affect tumor growth. To determine which process was responsible for the decreased tumor growth due to the reduction of TH in ThrbPV/PV-PTU mice, we compared the extent of cell proliferation in the thyroids of both groups of mice by bromodeoxyuridine (BrdU) incorporation assay. To perform the assay, mice were intraperitoneally injected with BrdU 2 h before killing. Although no apparent positive signals were detected in the thyroid sections of WT mice (Figure 3A, panels a and b) under the experimental conditions, nuclei with BrdU incorporation were clearly visualized by immunostaining of thyroid sections of ThrbPV/PV mice (panels c and d) and ThrbPV/PV-PTU mice (panels e and f). However, fewer nuclei with incorporation of BrdU were observed in the treated mice. Sections from small intestine and brain tissue of WT mice were used as controls, representing cells with high and low proliferation rates, respectively (Figures 3A, g and h). To quantify the percentage of cells that were undergoing an active cell cycle within a 2-h BrdU-labeling period, we calculated the average ratio of BrdU-positive cells to total cells from 10–12 bright fields at high magnification ( × 400) of each section. The quantitative data are shown in Figure 3B. In WT mice, less than 1% of thyrocytes underwent DNA replication (Figures 3Aa, b and B), but more than 2.3% of cells from ThrbPV/PV mice were involved in the cell cycle (Figures 3Ac, d and B). After PTU treatment, this ratio dropped to 1.2% (52% reduction; Figures 3Ae, f and B), indicating a marked reduction in the proliferation of thyroid tumor cells when TH was decreased in ThrbPV/PV-PTU mice.

Figure 3
figure3

Inhibition of TH production by PTU decreases proliferation of thyroid tumor cells of ThrbPV/PV mice with no apparent effect on apoptosis. (A) Thyrocyte proliferation in wild type (WT) (a, b), ThrbPV/PV (c, d) or ThrbPV/PV-PTU (e, f) mice was revealed by BrdU incorporation assay as described in Materials and methods. Two representative images of BrdU-positive thyrocytes (indicated by arrows) in different groups of mice were shown at × 400 magnification. Small intestine (g) and brain tissues (h) were used as illustrations of highly proliferative and rarely proliferative tissues, respectively. (B) Quantification of BrdU-positive cells in thyroids of WT, ThrbPV/PV and ThrbPV/PV-PTU mice. The percentage of BrdU-positive cells to total cells from 10–12 bright fields under × 400 as shown in (A) of each thyroid slide section was calculated. (C) Immunohistochemical analysis of cleaved caspase-3 in thyroid tumors of ThrbPV/PV (a) and ThrbPV/PV-PTU (b) mice. (D) No alteration in the protein level of apoptosis regulator Bcl-2 in thyroids of ThrbPV/PV (lanes 1–3) and ThrbPV/PV-PTU (lanes 4 and 5) mice. Total extracts were prepared from thyroids of ThrbPV/PV and ThrbPV/PV-PTU mice. Western blot analysis was carried out as described in Materials and methods. Representative results from five to six mice of each group were shown. GAPDH was used as the loading control.

We also determined the effect of TH on apoptosis of thyroid tumor cells in these mice by assessing the abundance of cleaved caspase 3, a marker of apoptosis, by immunohistochemistry. No apparent changes of cleaved caspase 3 staining were found in ThrbPV/PV-PTU thyroid (Figure 3Cb) as compared with the control group (Figure 3Ca). We also measured the protein levels of B-cell lymphoma 2 (Bcl-2), a key regulator in apoptosis, by western blot analysis (Figure 3D). Similar protein levels of Bcl-2 were found in thyroid extracts of ThrbPV/PV and ThrbPV/PV-PTU mice. In addition, we further determined the protein levels of other apoptotic regulators, including B-cell lymphoma-extra large (Bcl-xL) and Bcl-2-interacting mediator of cell death (Bim). No apparent changes in protein levels of these apoptotic regulators were detected in ThrbPV/PV mice with or without PTU treatment (data not shown). Thus, consistent with cleaved caspase 3 staining, these results showed that a reduction of TH had no effect on apoptosis of thyroid tumor cells in ThrbPV/PV mice. On the basis of these findings, we concluded that reduced proliferation, but not apoptosis, contributed to the reduced thyroid tumor growth of ThrbPV/PV mice when TH was lowered.

Inhibition of thyroid hormone production by propylthiouracil treatment decreases activation of the integrin αvβ3-AKT signaling pathway in thyroids of ThrbPV/PV mice

It has been reported that TH increases proliferation of human thyroid cancer cell lines by activating the integrin αvβ3–MAPK signal transduction pathways (Lin et al., 2007). Similar TH effects via these pathways have also been reported in human glioma cell line (Lin et al., 2009). To dissect the molecular mechanisms of TH that led to the increased cell proliferation in FTC of ThrbPV/PV mice, we first examined the abundance of TH membrane receptor integrin αvβ3 at both transcriptional and translational levels. No differences in the mRNA levels were detected for either the Itgav or the Itgb3 gene between ThrbPV/PV and ThrbPV/PV-PTU mice (Supplementary Figures IA and B). However, western blot analysis showed that the protein level of integrin αv was lower in the thyroid tumor extract of ThrbPV/PV-PTU mice than in that of ThrbPV/PV mice (Figure 4Aa, compare lanes 3 and 4 to lanes 1 and 2). After normalization to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) loading controls (Figure 4Ac), a 60% decrease of integrin αv protein abundance was found in ThrbPV/PV-PTU mice. However, no apparent changes in protein levels of integrin subunit β3 were observed in ThrbPV/PV-PTU mice (Figure 4Ab, compare lanes 3 and 4 with lanes 1 and 2). These results suggest a downregulation of TH membrane receptor in response to decreased TH in thyroid tumor cells of ThrbPV/PV-PTU mice.

Figure 4
figure4

Ablation of TH by PTU decreases activation of the integrin αvβ3–AKT signaling pathway in thyroids of ThrbPV/PV mice. Total thyroid lysates from six to seven mice of each group were analyzed by western blot analysis as described in Materials and methods. The average ratios of band intensity after normalization were shown under the image. The expression levels of integrins were normalized to the protein amount of loading control, GAPDH. Compared with ThrbPV/PV mice (Aa, lanes 1 and 2), a decrease of integrin αv was observed in ThrbPV/PV-PTU mice (lanes 3 and 4), whereas no apparent changes of integrin β3 (Ab) were found. The relative abundance of phosphorylated AKT (p-AKT) or p-GSK3β was normalized to the total protein amount. Reduced p-AKT (Ba, lanes 4 and 5) and p-GSK3β (Bc, lanes 4 and 5) were observed in ThrbPV/PV-PTU mice as compared with ThrbPV/PV mice (lanes 1–3 of Ba and Bc, respectively). No apparent alterations in total protein levels of AKT (Bb) and GSK3β (Bd) were detected.

The PI3K-AKT signaling cascade is a critical pathway reported to be associated with the extranuclear signaling of TH-TRs (Cao et al., 2005; Verga Falzacappa et al., 2007). Direct association of TRβPV with the regulatory subunit p85α of PI3K activates PI3K-AKT signaling in tumor cells of ThrbPV/PV mice (Furuya et al., 2006). Accordingly, we addressed the question of whether TH affected the activated AKT by determining the protein abundance of phosphorylated AKT in thyroid tumors of ThrbPV/PV and ThrbPV/PV-PTU mice. Figure 4B shows that AKT phosphorylation was reduced (33%) in PTU-treated ThrbPV/PV mice (Figure 4Ba, compare lanes 4 and 5 with lanes 1–3). In contrast to the reduced phosphorylated AKT, no changes in the total amount of AKT protein were detected (Figure 4Bb). We also determined the phosphorylation status of glycogen synthase kinase 3β (GSK3β), a direct downstream target of AKT. It is known that GSK3β activity is inhibited by AKT phosphorylation, resulting in increased protein synthesis and proliferation in cells (Liang and Slingerland, 2003). Consistent with decreased activation of AKT (Figure 4Ba), western blot analysis revealed decreased phosphorylated GSK3β (Figure 4Bc, compare lanes 4 and 5 with lanes 1–3) in thyroid tumors of ThrbPV/PV-PTU mice without any change in total GSK3β protein levels (Figure 4Bd). Taken together, these results show that the lowering of TH leads to decreased AKT signaling.

Phosphatase and tensin homolog (PTEN) activity is increased in thyroid tumors of ThrbPV/PV-PTU mice

Phosphatase and tensin homolog (PTEN) has an essential role in opposing PI3K-AKT activation by negative regulation of the intracellular level of phosphatidylinositol-3,4,5-trisphosphate (PIP3). PTEN is also one of the tumor suppressors frequently lost in human cancers (Keniry and Parsons, 2008). We therefore assessed the expression of the Pten gene at both the translational and transcriptional levels. Western blot analysis revealed an elevated PTEN protein level (2.4-fold) in thyroid tumor extracts of ThrbPV/PV-PTU mice (Figure 5Aa, compare lanes 4 and 5 with lanes 1–3). Using real-time RT–PCR, we determined the Pten mRNA expression in tumors of ThrbPV/PV and ThrbPV/PV-PTU mice. Consistent with the increased protein levels of PTEN, a small (20%) but significantly elevated Pten mRNA was observed in ThrbPV/PV-PTU mice as compared with ThrbPV/PV mice (Figure 5B). These results indicate that decreased TH leads to activated expression of the Pten gene at both the mRNA and protein levels.

Figure 5
figure5

Increased PTEN expression in thyroid tumors of PTU-treated ThrbPV/PV mice. (A) Protein level of PTEN was determined by western blot analysis as described in Materials and methods. Representative results from thyroid extracts of six to seven mice are shown for each group. The expression levels of PTEN for each group were normalized to GAPDH loading control and the average ratios were indicated under the images. Results showed that an approximately 2.4-fold increase of PTEN protein level was observed in ThrbPV/PV-PTU mice (Aa, lanes 4 and 5) than that in ThrbPV/PV mice (Aa, lanes 1–3). (B) The mRNA expression level of the Pten gene was measured by real-time RT–PCR. Reactions were performed in 50 ng of total RNA from five mouse thyroid samples from each group. Gapdh gene expression was used as a reference. A higher level of Pten mRNA was detected in ThrbPV/PV-PTU mice than in ThrbPV/PV mice.

Downregulation of AKT activation suppresses the activity of β-catenin to inhibit thyroid growth in ThrbPV/PV mice

Aberrant activation of β-catenin by PI3K/AKT signaling pathways is a frequent event in thyroid cancers (Abbosh and Nephew, 2005; Castellone et al., 2009). Phosphorylation of β-catenin at Ser552 by AKT increases the translocation of β-catenin to nucleus, thereby activating its transcriptional activity to affect β-catenin downstream targets involved in cell proliferation (Fang et al., 2007). To understand whether β-catenin is the effector of activated AKT signaling in thyroid carcinogenesis, we compared the protein abundance of phosphorylated β-catenin at Ser552 in thyroid tumors between ThrbPV/PV and ThrbPV/PV-PTU mice. Figure 6A shows a reduction of phospho-Ser552-β-catenin (Figure 6Aa, compare lanes 4 and 5 with lanes 1–3) in ThrbPV/PV-PTU mice with no changes in total β-catenin (Figure 6Ab). One of the downstream targets of β-catenin critical for cell proliferation is cyclin D2 (Rowlands et al., 2004; Cole et al., 2010). To determine whether the reduced β-catenin affects the cyclin D2 protein level, we evaluated the protein abundance of cyclin D2 in thyroid tumors of ThrbPV/PV and ThrbPV/PV-PTU mice. Indeed, we found that the protein level of cyclin D2 was lower (by 38%) in ThrbPV/PV-PTU mice than in ThrbPV/PV mice (Figure 6Ba, compare lanes 3 and 4 with lanes 1 and 2). We also investigated the alteration of MMP2, a downstream target of β-catenin and a key regulator during tumor invasion and metastasis (Kessenbrock et al., 2010; Sonderegger et al., 2010), by western blot analysis. Consistent with the suppressed activity of β-catenin, a decrease in MMP2 protein level (51%) was also observed in ThrbPV/PV-PTU mice as compared with that in untreated mice (Figure 6Ca, compare lanes 3 and 4 with lanes 1 and 2). These results indicate that reduced β-catenin contributes to decreased tumor cell proliferation, and via MMP-2, delays the progression of invasion and metastasis in ThrbPV/PV-PTU mice.

Figure 6
figure6

Downregulation of AKT activation suppresses the β-catenin activity in the FTC of ThrbPV/PV-PTU mice. Protein abundance of phosphorylated-Ser522-β-catenin (Aa), total β-catenin (Ab), cyclin D2 (Ba) and MMP2 (Ca) in thyroid cells of ThrbPV/PV and ThrbPV/PV-PTU mice was determined by immunoblotting. For each sample, 30–40 μg protein of whole thyroid tissue extract was examined. GAPDH was used as the loading controls (Ac, Bb and Cb). Representative results were shown from the analysis of four to six mice in each group. Average ratios of p-Ser522-β-catenin/total proteins or cyclin D2(or MMP2)/GAPDH were indicated for each mouse group.

Discussion

Extensive phenotypic characterization and detailed molecular analyses have shown that thyroid tumor progression and altered signaling pathways in ThrbPV/PV mice mimic those observed in human thyroid cancers (Furuya et al., 2006; Ying et al., 2006, 2008; Guigon et al., 2008; Guigon and Cheng, 2009; Lu et al., 2010). Thus, the ThrbPV/PV mouse provides us with an unusual opportunity to investigate the role of TH in thyroid carcinogenesis. In ThrbPV/PV mice, the expression of a dominant-negative mutated TRβ (that is, PV) results in dysregulation of the pituitary–thyroid axis with elevated TSH in the face of elevated TH (Kaneshige et al., 2000). To understand how TH affects thyroid carcinogenesis, we treated ThrbPV/PV mice with PTU to block the synthesis of TH. As confirmed by hormone assays, ThrbPV/PV-PTU mice exhibited suppressed serum levels of TT4 as compared with the untreated ThrbPV/PV mice (Figure 1). However, PTU had no effect on the synthesis and secretion of TSH given that ThrbPV/PV-PTU mice maintained a TSH level as high as that of ThrbPV/PV mice. The similarly elevated serum levels of TSH would suggest that the thyroids of ThrbPV/PV and ThrbPV/PV-PTU mice received a similar stimulated activity from the TSH–TSH receptor pathway in promoting thyrocyte proliferation. The similarly elevated TSH level led us to conclude that the differences in phenotypes of thyroid tumors found between PTU-treated and untreated ThrbPV/PV mice were mainly conferred by the effects due to changes in TH levels. By decreasing TH levels in ThrbPV/PV mice, we observed reduced growth of thyroid tumors with delayed pathological progression. The reduced tumor size was mainly attributed to the decreased tumor cell proliferation with no changes in apoptotic status (Figures 2 and 3). Thus, TH acts to stimulate thyroid tumor cell proliferation of ThrbPV/PV mice.

To dissect the molecular mechanisms by which TH activated tumor cell proliferation, we focused on the studies of key regulators involved in membrane signaling events. Although TRβ is the major TR isoform in the thyroid, we reasoned that in ThrbPV/PV mice the mutant TRβ, PV, could no longer mediate TH actions. Moreover, we previously discovered that TRβPV acts not only in a dominant-negative fashion by nucleus-initiated events, but also by extranuclear signaling by activating the PI3K-AKT pathway (Furuya et al., 2006, 2007), as well as by increasing the stability of β-catenin (Guigon et al., 2008, 2010), thereby promoting thyroid carcinogenesis in ThrbPV/PV mice (Lu and Cheng, 2011). Accordingly, we ascertained which players in the TRβPV-mediated extranuclear PI3K-AKT pathway were affected by TH. Indeed, we found that the upstream negative regulator of PI3K-AKT signaling, PTEN, was markedly increased when TH was reduced in ThrbPV/PV-PTU mice (Figure 5, also see Figure 7), leading to an attenuation of PI3K-AKT signaling (Figures 4 and 7). Consistently, the protein abundance of phosphorylated β-catenin, a critical downstream key effector of PI3K-AKT signaling, was reduced when TH was lowered, leading to reduced cyclin D2 to decrease cell proliferation. Thus, the present study has uncovered an effect of TH that modulates the activity of the PTEN–PI3K–AKT–β-catenin–cyclin D2 pathway to affect the tumor cell proliferation and carcinogenesis of the thyroid of ThrbPV/PV mice.

Figure 7
figure7

A proposed model illustrating the effects of reduced TH in thyroid cancer development in ThrbPV/PV-PTU mice. In ThrbPV/PV thyroid tumor cells, TRβPV directly interacts with PI3K to activate AKT signaling pathway (Furuya et al., 2006). By suppression of TH in these cells, the membrane receptor integrin αvβ3 was downregulated. In addition, the PTEN activity was activated, which decreased the formation of PIP3. The reduction of PIP3 decreased the p-AKT and its downstream β-catenin and GSK3β signaling cascades to inhibit cell proliferation. The solid arrows indicate the direction of signaling cascade. The broken arrows mark the up and down changes.

At present, how TH regulates the expression of the Pten gene is not clear. In addition to an increased protein abundance of PTEN (Figure 5A) when TH was lowered, we found a small but significant increase in the mRNA expression of the Pten gene in ThrbPV/PV-PTU mice (Figure 5B). One possibility that could be considered to account for the increased expression of the Pten gene protein is by the TH membrane receptor, integrin αvβ3, reported by Davis and his group (Bergh et al., 2005; Lin et al., 2009; Cheng et al., 2010). Actions of TH on the integrin αvβ3 receptor have been demonstrated in many cell types with diverse cellular effects, such as T4-simulated angiogenesis on vessel endothelial cells and enhanced proliferation of tumor cells, including breast cancer, glioblastoma and thyroid cancer cell lines (Davis et al., 2004; Mousa et al., 2006; Glinskii et al., 2009; Lin et al., 2009). The present study showed decreased protein abundance of integrin subunit αv, albeit with no changes of β3 protein levels when TH was lowered in ThrbPV/PV-PTU mice (Figure 4A). It is possible that the expression of the Pten gene is affected by dampening integrin αvβ3 signaling, followed by relaying the signals via MAPK phosphorylation cascades (Bergh et al., 2005; Lin et al., 2007) to reach the ultimate downstream nuclear events (yet to be identified). However, this possibility awaits future studies to be tested and validated.

The data from the present molecular study led us to propose a model (see Figure 7) that supports the contention that one mechanism by which TH acted was by integrin-mediated extranuclear signaling. TRβ is the major TR isoform in the thyroid, and is mutated to PV that cannot bind TH in ThrbPV/PV mice. However, at present, we could not exclude the possibility that TH could potentially mediate its proliferative effects by TRα1-initiated nuclear genomic signaling events. However, PV is known to be a potent dominant-negative mutant (Parrilla et al., 1991; Kaneshige et al., 2000). The possibility exists that it could also act to interfere with the transcription activity of TRα1. However, at present, no data are available to clarify the role of TRα1 in mediating TH effects in the thyroid of ThrbPV/PV mice.

Association studies have shown that amplification of the PIK3CA gene and aberrant activation of AKT are frequent in human thyroid cancer (Hou et al., 2007; Saji and Ringel, 2010; Xing, 2010), demonstrating the crucial role of PI3K-AKT signaling in thyroid carcinogenesis. Recently, using a mouse model of FTC in which two alleles of the Pten gene were selectively deleted in the thyroid, Antico-Arciuch et al. (2010) showed a direct role of estrogens in increasing thyrocyte proliferative index by crosstalk with the PI3K pathway. The present study identified TH as a critical hormonal stimulator to promote thyroid carcinogenesis of ThrbPV/PV mice by regulating the PI3K-AKT signaling. The present data have shown that TH acts to promote tumor cell proliferation by PI3K–AKT–β-catenin–cyclin D2 signaling. In view of these findings, anti-thyroid drugs could be considered as potential therapeutic agents for the treatment of thyroid cancer.

Materials and methods

Mouse strain

All aspects of animal care and experimentation were approved by the National Cancer Institute Animal Care and Use Committee. ThrbPV/PV mice used in the present study were the offspring of many generations of intersibling mating over 6 years (more than 30 generations). For the PTU treatment group, ThrbPV/PV mice were fed an iodine-deficient diet supplemented with 0.15% PTU (cat. no.; TD.95125, Harlan Laboratories, Inc., Indianapolis, IN, USA) beginning at the age of 2 months and continuing on the PTU diet until the mice were killed for analyses. Control group mice were fed a normal mouse diet. Moribund mutant mice and PTU-treated littermates were euthanized to harvest tissues for weighing, histological analysis and biochemical studies.

Determination of serum levels of total T4 and thyroid-stimulating hormone by radioimmunoassay

Blood was collected from the mice before killing. The serum level of TT4 was determined by using a Gamma Coat T4 assay radioimmunoassay kit (Dia-Sorin, Stillwater, MN, USA) according to the manufacturer's instructions. TSH levels in serum were measured as previously described (Kaneshige et al., 2000).

Histology and immunohistochemistry

For histological evaluation, thyroid glands were fixed in 10% neutral-buffered formalin and subsequently embedded in paraffin. Five-μm-thick sections were prepared and stained with hematoxylin and eosin for histopathological analysis. Immunohistochemistry was also performed with paraffin sections by standard methods. In brief, de-waxed sections were processed using 0.05% citraconic anhydride buffer (pH 7.4) at 98 °C for 45 min to expose the antigen epitopes. The primary antibody, rabbit anti-cleaved caspase 3 antibody (1:50 dilution, Cell Signaling Technology, Danvers, MA, USA), was incubated with tissues overnight at 4 °C. Peroxidase activity from the secondary antibody was detected by adding substrate 3,3′-diaminobenzidine, and the sections were counterstained with hematoxylin.

Bromodeoxyuridine incorporation assay

To measure the thyrocyte proliferation rate, the BrdU incorporation assay was performed. In brief, mice were injected intraperitoneally with BrdU at 50 μg/g of body weight and then killed 2 h after the injection. Tissues were processed and immunohistochemistry was performed on paraffin sections as described above with some modifications. Briefly, de-waxed slides were treated with Antigen Unmasking solution (Vector Laboratories, Burlingame, CA, USA) in the microwave for 20 min after boiling, and stained with mouse anti-BrdU antibody (1:10 dilution, Cat. no.; 347589, Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) overnight at 4 °C. The quantification of BrdU-positive cells was performed on digital images captured under × 400 magnification of each thyroid section. Two randomly selected slides from three thyroids of each group were analyzed. For each slide, ratios of BrdU-positive cells/total cells from 10–12 bright fields under × 400 amplification were calculated.

Western blot analysis

Protein extracts from thyroids dissected from ThrbPV/PV and ThrbPV/PV-PTU mice were prepared as described previously (Lu et al., 2010). About 30–40 μg of protein extract was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the western blot analysis was carried out (Furumoto et al., 2005). Primary antibodies for phosphorylated-Ser473-AKT, total AKT, p-Ser21/9-GSK-3α/β, total GSK-3β, p-Ser552-β-catenin, p-Ser33/37/Thr41-β-catenin, total β-catenin, cleaved caspase 3, cyclin D2 and GAPDH were purchased from Cell Signaling Technology. Antibodies for integrin αv, integrin β3 and MMP-2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Quantitative real-time RT–PCR

Total RNA from five to seven mouse thyroids in each experimental group was extracted by TRIzol (Invitrogen, Carlsbad, CA, USA), followed by RNase-free DNase treatment (Qiagen, Valencia, CA, USA) and column purification (RNeasy Mini Kit, Qiagen). For real-time reverse transcription–PCR (RT–PCR), one-step RT–PCR reactions were performed with 50 ng of total RNA using a QuantiTect SYBR Green RT–PCR kit (Qiagen) in a Roche LightCycler PCR instrument (Roche, Indianapolis, IN, USA) with specific primers as follows: mItgav-F2773: 5′-IndexTermCTGCACGGCAGATACAGAGATC-3′; mItgav-R2935: 5′-IndexTermGCACTTGGCGATTCCACAGC-3′; mItgb3-F582: 5′-IndexTermCAAGCCTGTATCGCCGTACATG-3′; mItgb3-R760: 5′-IndexTermCATCTCGATTACGGGACACGCTC-3′; mPten-F1-1427: 5′-IndexTermGATTACAGACCCGTGGCACT-3′; mPten-R1-1606: 5′-IndexTermTGGCTGAGGGAACTCAAAGT-3′; mGapdh-F2-436: 5′-IndexTermTTGTGATGGGTGTGAACCAC-3′; m-Gapdh-R2-674: 5′-IndexTermGGATGCAGGGATGATGTTCT-3′. The reaction conditions were 50 °C for 20 min; 95 °C for 15 min; 40–45 cycles of 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s; and 65–95 °C with a heating rate of 0.1 °C/s and a cooling step to 40 °C.

Statistical analysis

All data of radioimmunoassays were expressed as mean±s.e.m. The statistical analyses on these data were performed using a one-way analysis of variance followed by Bonferroni's post hoc test for multiple comparisons (Prism, GraphPad, San Diego, CA, USA). P<0.05 was considered as statistically significant. For western blot results, films were scanned and the intensity of each band was quantified by ImageJ Analysis Software (NIH, Bethesda, MD, USA). Data were normalized against GAPDH or calculated as a ratio between phosphorylated and total protein levels. For real-time RT–PCR, the Gapdh gene expression was used as an internal control. The statistical analysis was performed using an unpaired t-test by Prism software (GraphPad). P<0.05 was considered as statistically significant.

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Acknowledgements

This research was supported by the Intramural Research Program of National Institutes of Health, National Cancer Institute, Center for Cancer Research.

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Correspondence to S-Y Cheng.

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Lu, C., Zhu, X., Willingham, M. et al. Activation of tumor cell proliferation by thyroid hormone in a mouse model of follicular thyroid carcinoma. Oncogene 31, 2007–2016 (2012). https://doi.org/10.1038/onc.2011.390

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Keywords

  • thyroid hormone
  • follicular thyroid carcinoma
  • animal model
  • protein kinase B/AKT
  • PTEN

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