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 (Thrb(PV/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). Thrb(PV/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 Thrb(PV/PV) mice. However, whether the elevated TH also contributes to the thyroid carcinogenesis of Thrb(PV/PV) mice was not elucidated. To understand the role of TH in thyroid carcinogenesis, we blocked the production of TH by treating Thrb(PV/PV) mice with propylthiouracil (Thrb(PV/PV)-PTU mice) and compared the development of thyroid cancer in Thrb(PV/PV)-PTU and untreated Thrb(PV/PV) mice. We found that thyroid tumor growth was reduced by ∼42% in Thrb(PV/PV)-PTU mice as compared with Thrb(PV/PV) mice. Analysis by bromodeoxyuridine-nuclear labeling showed decreased incorporation of bromodeoxyuridine in thyroid tumor cells of Thrb(PV/PV)-PTU mice, indicative of decreased tumor cell proliferation. However, cleaved-caspase 3 staining showed no apparent changes in apoptosis of tumor cells in Thrb(PV/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 Thrb(PV/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 Thrb(PV/PV) mice via membrane signaling events. Importantly, these findings suggest that anti-thyroid drugs could be considered as possible therapeutic agents of thyroid cancer.

Figure 1

Comparison of serum levels of TT4 and TSH in Thrb^PV/PV mice with or without PTU treatment. Serum TT4 (a) and TSH (b) of Thrb^PV/PV (n=10) and Thrb^PV/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.

Figure 2

Inhibition of TH production by PTU treatment suppresses the development of FTC in Thrb^PV/PV mice. (a) Thyroid glands from Thrb^PV/PV (n=12) and Thrb^PV/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 Thrb^PV/PV and Thrb^PV/PV-PTU mice was significant (P<0.005) as determined by the unpaired t-test analysis. (b) Pathological progression of FTC in Thrb^PV/PV mice with or without PTU treatment. Sections of thyroids and lungs from Thrb^PV/PV and Thrb^PV/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.

Figure 3

Inhibition of TH production by PTU decreases proliferation of thyroid tumor cells of Thrb^PV/PV mice with no apparent effect on apoptosis. (A) Thyrocyte proliferation in wild type (WT) (a, b), Thrb^PV/PV (c, d) or Thrb^PV/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, Thrb^PV/PV and Thrb^PV/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 Thrb^PV/PV (a) and Thrb^PV/PV-PTU (b) mice. (D) No alteration in the protein level of apoptosis regulator Bcl-2 in thyroids of Thrb^PV/PV (lanes 1–3) and Thrb^PV/PV-PTU (lanes 4 and 5) mice. Total extracts were prepared from thyroids of Thrb^PV/PV and Thrb^PV/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.

Figure 4

Ablation of TH by PTU decreases activation of the integrin αvβ3–AKT signaling pathway in thyroids of Thrb^PV/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 Thrb^PV/PV mice (Aa, lanes 1 and 2), a decrease of integrin αv was observed in Thrb^PV/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 Thrb^PV/PV-PTU mice as compared with Thrb^PV/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.

Figure 5

Increased PTEN expression in thyroid tumors of PTU-treated Thrb^PV/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 Thrb^PV/PV-PTU mice (Aa, lanes 4 and 5) than that in Thrb^PV/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 Thrb^PV/PV-PTU mice than in Thrb^PV/PV mice.

Figure 6

Downregulation of AKT activation suppresses the β-catenin activity in the FTC of Thrb^PV/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 Thrb^PV/PV and Thrb^PV/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.

Figure 7

A proposed model illustrating the effects of reduced TH in thyroid cancer development in Thrb^PV/PV-PTU mice. In Thrb^PV/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.