Reactivation of the silenced thyroid hormone receptor β gene expression delays thyroid tumor progression

Abstract

That a knock-in mouse harboring a dominant-negative thyroid hormone receptor (TR)-β (Thrb) mutation develops metastatic thyroid cancer strongly suggests the involvement of TRβ in carcinogenesis. Epigenetic silencing of the THRB gene is common in human cancers. The aim of the present study was to determine how DNA methylation affected the expression of the THRB gene in differentiated thyroid cancer (DTC) and how reexpression of the THRB gene attenuated the cancer phenotypes. We used methylation-specific PCR to examine the expression and promoter methylation of the THRB gene in DTC tissues. Thyroid cancer cells with hypermethylated THRB were treated with the demethylating agents 5'-aza-2'-deoxycytidine (5'-aza-CdR) and zebularine to evaluate their impact on the cancer cell phenotypes. THRB mRNA expression in DTC was 90% lower than in normal controls, and this decrease was associated with a higher tumor/lymph node staging. The promoter methylation level of the THRB gene had a significant negative correlation with the expression level of the THRB gene. Treatment of FTC-236 cells with 5'-aza-CdR or zebularine induced reexpression of the THRB gene and inhibited cell proliferation and migration. FTC-236 cells stably expressing TRβ exhibited lower cell proliferation and migration through inhibition of β-catenin signaling pathways compared with FTC-236 without TRβ. 5'-Aza-CdR also led to suppression of tumor growth in an in vivo xenograft model using FTC-236 cells consistent with the cell-based studies. These finding indicate that TRβ is a tumor suppressor and could be tested as a potential therapeutic target.

Fig. 1.

Down-regulation of THRB gene in DTC tissues. A, Relative mRNA expression of THRB and PAX8 in DTC (n = 21), nodular hyperplasia (n = 8), follicular adenoma (n = 8), and normal thyroid tissue (n = 11). The data are presented as mean ± se and were analyzed by Student's t test. B, Relative THRB mRNA expression of individual tissue samples from DTC and normal thyroid determined by real-time RT-PCR assay. C, Relative THRB and PAX8 mRNA expression according to lower (n = 12) and higher (n = 9) tumor (T) staging in DTC tissues. The relative expression of mRNA in different groups was evaluated by examining the P value for trend via linear regression analysis. D, Relative THRB and PAX8 mRNA expression according to the absence (n = 10) or presence (n = 11) of lymph node (N) metastasis in DTC tissues. T staging and N staging were defined according to the sixth edition of the American Joint Committee on Cancer/International Union Against Cancer tumor-node-metastases (TNM) classification.

Fig. 2.

THRB is hypermethylated in DTC. A, Representative results of THRB promoter methylation in cancer and normal thyroid tissue. The amounts of methylated (M) or unmethylated (U) THRB promoter from bisulfite-treated genomic DNA of each tissue were determined by specific PCR analysis. B, The ratio of methylated to unmethylated THRB promoter from normal (n = 7) and cancer tissues (n = 18). The data are presented as medians and analyzed by the Mann-Whitney U test. C, The relative THRB methylation ratios of normal and DTC tissues according to lower (n = 10) and higher (n = 8) tumor (T) staging. The relative THRB methylation ratios of normal and DTC tissues are shown according to lymph node (N) staging [the absence (n = 9) or presence (n = 9) of lymph node (N) metastasis]. The relative ratio of methylated and unmethylated THRB in different groups was evaluated by examining the P value for trend via linear regression analysis. D, Representative results of Western blot analysis of TRβ and GAPDH as the loading control in normal and cancer tissues. E, Quantification of relative protein expression of TRβ after normalization via use of GAPDH as the loading control. The data are presented as medians and analyzed by the Mann-Whitney U test.

Fig. 3.

Demethylation agents 5′-aza-CdR and zebularine increase THRB expression through reduced THRB promoter methylation. A, The amounts of methylated (M) or unmethylated (U) THRB promoter determined by methylation-specific PCR analysis in human breast cancer cells (T47D, MCF-7, MDA-468) and thyroid cancer cells (SW1736, FTC-236, K-5, WRO, and FTC-133). Ba, Methylation of THRB promoter was decreased after treatment with the demethylating agents, 5′-aza-CdR (upper panel) and zebularine (lower panel) in the FTC-236 human thyroid cancer cell line; b, relative mRNA expression of THRB was increased by treatment with the demethylating agents in FTC-236 cells (data presented as mean ± sd and compared by Student's t test); c, protein abundance of TRβ was increased by treatment with demethylating agents in FTC-236 cells as shown by Western blot analysis of total cell lysates with or without treatment with demethylating agents as marked (upper panel). GAPDH was used as the loading control (lower panel). C, Relative to vehicle treatment, cell proliferation was inhibited by treatment with the demethylating agents in FTC-236 cells (data presented as mean ± sd). *, P < 0.01. D, Decreased Ki-67-positive cells after treatment with 5′-aza-CdR and zebularine in FTC-236 cells compared with vehicle treatment. Representative microphotographs of Ki-67 immunohistochemistry on paraffin-embedded cells after treatment with vehicle (a), 5′-aza-CdR (b), and zebularine (c). Cell proliferation index was determined by proportion of Ki-67-positive cells in FTC-236 cells (d). E, Relative to vehicle treatment, cell migration of FTC-236 was inhibited by treatment with demethylating agents, as determined by wound-healing assay (data presented as mean ± sd). *, P < 0.01.

Fig. 4.

Demethylating agents 5′-aza-CdR and zebularine decrease cell proliferation by attenuating β-catenin signaling in FTC-236. A, Western blot analysis of p-β-catenin (serine 552), cyclin D1, and GAPDH as the loading control in whole-cell lysates from FTC-236 cells after treatment with demethylating agents. B, Quantification of relative protein abundance of p-β-catenin (serine 552) and cyclin D1 after normalization by using GAPDH as a loading control. C, Western blot analysis of protein abundance of β-catenin in the nuclear and cytoplasm cell fractions. PARP was used as loading control for nuclear fraction and α-tubulin was used as loading control for cytoplasmic fractions of FTC-236 cells. D, Western blot analysis of p-β-catenin (serine 552), total β-catenin, cyclin D1, and GAPDH as the loading control in human tissue lysates of normal thyroid and cancer samples.

Fig. 5.

Stable reexpression of TRβ in the FTC-236 thyroid cancer cells reduces cell proliferation and migration by attenuating β-catenin signaling. A, Western blot analysis of expression of TRβ in stable cells after transfection of control (Neo) or THRB-expressing vector in FTC-236 cells. B, Cell proliferation was lower in TRβ-expressing FTC-236 cells (TRβ9 and TRβ23) than in control FTC-236 cells (Neo1 and Neo4). Data are presented as mean ± se. **, P < 0.05; *, P < 0.01. C, Wound-healing assay showed that reexpressing of the TRβ inhibited cell migration of FTC-236 cells. Data are presented as mean ± se. *, P < 0.01. D, Western blot analysis of p-β-catenin (serine 552), total β-catenin, cyclin D1, and GAPDH in whole-cell lysates from TRβ-expressing FTC-236 cells and control FTC-236 cells. E, Quantification of relative protein abundance of p-β-catenin, total β-catenin, and cyclin D1 after normalization using GAPDH as a loading control. Data presented as mean ± se. #, P < 0.001. F, Western blot analysis of protein abundance of β-catenin and cyclin D1 in the nuclear and cytoplasm cell fractions. PARP was used as loading control for nuclear fraction, and α-tubulin was used as loading controls for cytoplasmic fraction from FTC-236 cells.

Fig. 6.

Demethylating agent 5′-aza-CdR inhibits growth of tumors induced by FTC-236 cells in mouse xenograft models. A, Growth curves of FTC-236-induced tumors. FTC-236 cancer cells were sc inoculated into the right flank of athymic NCr-nu/nu mice. Mice were treated by weekly ip injection of 5′-aza-CdR (5 mg/kg; n = 6) or vehicle control (n = 6) for 30 d. Data are presented as mean ± se. *, P < 0.01; **, P < 0.001. B, Comparison of the weight of FTC-236 cell-induced tumors after treatment of 5′-aza-CdR (n = 6) or vehicle control (n = 6) at study endpoint. The data are represented as mean ± se. The differences are significant (P < 0.001).