Thyroid hormone receptors are tumor suppressors in a mouse model of metastatic follicular thyroid carcinoma

Abstract

Aberrant expression and mutations of thyroid hormone receptor genes (TRs) are closely associated with several types of human cancers. To test the hypothesis that TRs could function as tumor suppressors, we took advantage of mice with deletion of all functional TRs (TRalpha1(-/-)TRbeta(-/-) mice). As these mice aged, they spontaneously developed follicular thyroid carcinoma with pathological progression from hyperplasia to capsular invasion, vascular invasion, anaplasia and metastasis to the lung, similar to human thyroid cancer. Detailed molecular analysis revealed that known tumor promoters such as pituitary tumor-transforming gene were activated and tumor suppressors such as peroxisome proliferator-activated receptor gamma and p53 were suppressed during carcinogenesis. In addition, consistent with the human cancer, AKT-mTOR-p70(S6K) signaling and vascular growth factor and its receptor were activated to facilitate tumor progression. This report presents in vivo evidence that functional loss of both TRalpha1 and TRbeta genes promotes tumor development and metastasis. Thus, TRs could function as tumor suppressors in a mouse model of metastatic follicular thyroid cancer.

Figure 1

Kaplan–Meier survival curve for TRα1^−/− TRβ^−/− mice up to 20.9 months of age. The analysis was performed with log-rank (Mantel–Cox) test by using StatView 5.0. The 50% survival age was 9.3 months (n=94).

Figure 2

Thyroid weights of TRα1^−/− TRβ^−/− mice (n=6–25) at the ages of 2–5, 6–9 and 10–13 months. Thyroid glands of TRα1^−/− TRβ^−/− mice were dissected and weighed. The data are presented as the ratios of thyroid weight to body weight (mg/g).

Figure 3

Hematoxylin and eosin (H&E) staining of thyroids and lungs of representative sections from TRα1^−/− TRβ^−/− mice. Histological sections from tissues of mice showed evidence of capsular invasion in thyroid (a) (arrow), vascular invasion in thyroid (b) (arrow), anaplasia in thyroid (c) and metastatic thyroid carcinoma lesions in lung (d) (arrow).

Figure 4

Quantitative analysis of age-dependent occurrence frequency (%) of capsular invasion (a), vascular invasion (b), anaplasia (c) and lung metastasis (d) of TRα1^−/− TRβ^−/− (n=6–14) mice. Sections of thyroids and lungs from TRα1^−/− TRβ^−/− mice were stained with Hematoxylin and eosin (H&E) and analyzed for age-dependent pathological progression. The data are expressed as the percentage of occurrence frequency of the mice examined. The designation (#) indicates 0 occurrence frequency (%).

Figure 5

Activation of TSH–TSHR downstream pathway (A) and cyclin-CDK4–Rb pathway (B) in TRα1^−/− TRβ^−/− mice. For western blot analysis, 30 μg of thyroid extract was used. Two representative results from 4–6 WT (lanes 1, 2, 5 and 6) and TRα1^−/− TRβ^−/− mice (lanes 3, 4, 7 and 8) are shown for p-CREB and total CREB (A), cyclin D1, CDK4 and p-Rb (B). GAPDH was used as loading controls.

Figure 6

Activation of growth signaling pathways in TRα1^−/− TRβ^−/− mice. For western blot analysis, 30 μg of thyroid extract was used. Two representative results from 4 to 6 WT (lanes 1, 2, 5 and 6) and TRα1^−/− TRβ^−/− mice (lanes 3, 4, 7 and 8) are shown for p-AKT and total AKT (A), p-mTOR(S2448), p-mTOR(S2481), total mTOR, p-p70^S6K and total 70^S6K (B), p53 and MDM2 (C) and PPARγ (D). GAPDH was used as loading controls.

Figure 7

Activation of pathways involved in invasion and angiogenesis. Thyroid extract (30 μg) was used in the western blot analysis, as described in Materials and methods section. Two representative results from 5 to 7 WT (lanes 1, 2, 5 and 6), TRα1^−/− TRβ^−/− mice (lanes 3, 4, 7 and 8) are shown for ILK and MMP-2 (A), VEGF, VEGFR2 and p-VEGFR2 (B), and PTTG (C). GAPDH was used as loading controls.

Figure 8

The loss of normal TR functions results in complex alterations of multiple signaling pathways, thereby contributing to thyroid carcinogenesis in TRα1^−/− TRβ^−/− mice. TRs act through genomic and nongenomic actions to regulate key cellular effectors to maintain normal cellular functions in growth, proliferation, cell motility and migration. TRs through T3 repress the expression of TSHα and TSHβ genes in the pituitary. Deficiency of TRs in the pituitary results in the loss of negative regulation and thus increases expression of TSH. Although little is known regarding the precise mechanisms by which TRs regulate other key effectors, such as VEGFR2 and others indicated in this figure (see also Discussion section), the loss of functional TRs results in disarrays of multiple signaling pathways to contribute to thyroid carcinogenesis. The phenotypic manifestation of tumor development and progression because of the loss of normal functions of TR is consistent with the notion that TRs have key tumor suppressor roles.