Induction of thyroid gene expression and radioiodine uptake in thyroid cancer cells by targeting major signaling pathways

Abstract

Context: Radioiodine ablation is commonly used to treat thyroid cancer, but a major challenge is often the loss of radioiodine avidity of the cancer caused by aberrant silencing of iodide-handling genes.

Objectives: This study was conducted to test the therapeutic potential of targeting the aberrantly activated MAPK and PI3K/Akt/mTOR pathways and histone deacetylase to restore radioiodine avidity in thyroid cancer cells.

Experimental design: We tested the effects of specific inhibitors targeting these pathways/molecules that had established clinical applicability, including the MAPK kinase inhibitor RDEA119, mTOR inhibitor temsirolimus, Akt inhibitor perifosine, and histone deacetylase inhibitor SAHA, individually or in combinations, on the expression of iodide-handling genes and radioiodide uptake in a large panel of thyroid cancer cell lines.

Results: The expression of a large number of iodide-handling genes could be restored, particularly the sodium/iodide symporter, TSH receptor, and thyroperoxidase, by treating cells with these inhibitors. The effect was particularly robust and synergistic when combinations of inhibitors containing SAHA were used. Robust expression of sodium/iodide symporter in the cell membrane, which plays the most important role in iodide uptake in thyroid cells, was confirmed by immunofluorescent microscopy. Radioiodide uptake by cells was correspondingly induced under these conditions. Thyroid gene expression and radioiodide uptake could both be further enhanced by TSH.

Conclusions: Targeting major signaling pathways could restore thyroid gene expression and radioiodide uptake in thyroid cancer cells. Further studies are warranted to test this therapeutic potential in restoring radioiodine avidity of thyroid cancer cells for effective ablation treatment.

Figure 1

Effects of various inhibitors on the MAPK pathway, PI3K/Akt/mTOR pathway, and HDAC in thyroid cancer cells. Inhibition of ERK and p70S6K phosphorylation and histone deacetylation was achieved using the MEK-specific inhibitor RDEA119, mTOR-specific inhibitor temsirolimus (CCI779), and HDAC-specific inhibitor SAHA, respectively. Thyroid cancer cells were treated with 0.5 μm RDEA119, 10 nm temsirolimus, or 5 μm SAHA for 30 h. Dimethylsulfoxide or PBS was used as the vehicle control. After treatments, cells were lysed for Western blotting analyses. The effect of inhibition is reflected by the level of phosphorylated ERK and p70S6K and acetylated histone detected with specific anti-phosphorylated ERK (p-ERK), anti-phosphorylated p70S6K (p-p70S6K), and anti-acetylated histone H3 (Ac-histone) antibodies. Immunoblotting with antibodies against t-ERK or β-actin was used for quality control.

Figure 2

Induction of the expression of NIS, TSHR, TPO, and Tg genes in thyroid cancer cells using various inhibitors, individually or in different combinations. A, Eleven authenticated thyroid cancer cells, as indicated, were treated with inhibitors (RDEA119, CCI779, and SAHA) as described in Fig. 1, individually or in different combinations, to induce the expression of NIS, TSHR, TPO, and Tg genes. Data were obtained using real-time RT-PCR and are presented as the mean of values from three assays for the ratios of specific gene expression with natural log conversion. The red color represents the highest expression, green is the lowest expression, and black is the intermediate. B, Effect of TSH stimulation on NIS, TSHR, TPO, and Tg expression induced by specific inhibitors (RDEA119, CCI779, perifosine, and SAHA), individually or in different combinations. Details are as described in Materials and Methods, and data were obtained and are presented as in panel A. Green asterisks show that the basal level of gene expression is low. R, RDEA119; C, CCI779 (temsirolimus); P, perifosine; S, SAHA.

Figure 3

TSH enhancement of expression of NIS, TSHR, and TPO genes induced by various inhibitors, individually or in different combinations, in thyroid cancer cells. Various thyroid cancer cells were treated with specific inhibitors (RDEA119, CCI779, perifosine, and SAHA) as described in Fig. 1 and in Materials and Methods, individually or in different combinations, supplemented with or without 20 mU/ml TSH. Data are presented as the mean ± sd of values from three assays. R, RDEA119; C, CCI779 (temsirolimus); P, perifosine; S, SAHA. *, P < 0.05; **, P < 0.01 for comparison with control.

Figure 4

Immunofluorescent microscopic analysis of NIS expression induced by inhibitors in thyroid cancer cells. After a 30-h combined treatment with RDEA119, perifosine, and SAHA, cells were analyzed by immunofluorescent microscopy using anti-NIS antibody and fluorescein isothiocyanate-coupled secondary antibody. Double immunofluorescence was displayed with the red color representing 7-AAD nuclear staining and the green color representing NIS expression. Cells with the green color alone are intact living cells that do not have 7-AAD nuclear staining. NIS staining in these cells represents NIS protein expression on the cell membrane. Cells with double colors suggest that the cells were not intact and therefore both cell membrane NIS staining and 7-AAD nuclear staining occurred. Control cells did not show significant NIS staining, and the dead cells mostly showed nuclear staining with 7-ADD. R, RDEA119; P, perifosine; S, SAHA.

Figure 5

Radioiodide uptake in thyroid cancer cells induced by inhibitors targeting MEK, Akt, and HDAC in thyroid cancer cells. Thyroid cancer cells C643, K1, and KAT18 were treated with a combination of RDEA119, perifosine, and SAHA as described in Materials and Methods, supplemented with or without 20 mU/ml TSH. Cells were subsequently incubated with 1 μCi ¹²⁵I/1 ml/well on 12-well plates for 1 h. Cells were then washed and harvested for radioactivity measurement using a gamma-counter as described in Materials and Methods. Data are expressed as mean ± sd of values from three assays. R, RDEA119; P, perifosine; S, SAHA; Ctr, control. •••, P < 0.001; ••, P < 0.01; •, P < 0.05, compared with NaClO₄-treated cells. *, P < 0.001, compared with untreated cells.