B-RafV600E inhibits sodium iodide symporter expression via regulation of DNA methyltransferase 1

Abstract

B-RafV600E mutant is found in 40-70% of papillary thyroid carcinoma (PTC) and has an important role in the pathogenesis of PTC. The sodium iodide symporter (NIS) is an integral plasma membrane glycoprotein that mediates active iodide transport into the thyroid follicular cells, and B-RafV600E has been known to be associated with the loss of NIS expression. In this study, we found that B-RafV600E inhibited NIS expression by the upregulation of its promoter methylation, and that specific regions of CpG islands of NIS promoter in B-RafV600E harboring PTC were highly methylated compared with surrounding normal tissue. Although DNA methyltransferase 3a and 3b (DNMT3a,3b) were not increased by B-RafV600E, DNMT1 expression was markedly upregulated in PTC and B-RafV600E expressing thyrocytes. Furthermore, DNMT1 expression was upregulated by B-RafV600E induced NF-κB activation. These results led us to conclude that NIS promoter methylation, which was induced by B-RafV600E, is one of the possible mechanisms involved in NIS downregulation in PTC.

Figure 1

Sodium iodide symporter (NIS) expression in papillary thyroid carcinoma (PTC). (a) Immunohistochemical analysis of NIS. B-RafV600E mutant and NIS protein were analyzed by immunohistochemical analysis (left figure), and its staining pattern is summarized in a table (lower table). Twenty-three surrounding normal region and 30 cases of PTC were analyzed NIS and B-RafV600E, respectively. B-Raf was sequenced (right figure). NIS expression was analyzed by real-time PCR (b) and western blotting (c). Twenty-nine cases of surrounding normal region and PTC were analyzed for NIS expression by real-time PCR and 14 cases of fresh tissues were analyzed for NIS protein expression by western blotting.

Figure 2

B-RafV600E inhibited sodium iodide symporter (NIS) expression in primary isolated thyrocytes. (a) B-RafV600E induced Erk1/2 phosphorylation in thyrocytes. (b) B-RafV600E inhibited NIS expression. Thyrocytes were infected with B-RafV600E lentivirus and then selected with 3.5 μg ml⁻¹ puromycin for 1 week. NIS expression was analyzed by real-time PCR (left panel) and western blotting (right panel). (c) B-RafV600E inhibited iodide uptake in thyrocytes. Control and B-RafV600E expressing thyrocytes were analyzed for iodide uptake by using Premo Halide Sensor (Invitrogen) for 1 min, and the results are presented as dot graph (left panel). Three independent experiments are presented as a bar graph (right panel). (d) PLX4032 inhibited B-RafV600E induced NIS downregulation. Schematic drawing of an experiment (upper panel). Thyrocytes were infected with control or B-RafV600E lentivirus for 24 h and selected with 3.5 μg ml⁻¹ puromycin. PLX4032 (20 μM) was applied and the cells were harvested 3 days later.

Figure 3

Sodium iodide symporter (NIS) promoter region was highly methylated in papillary thyroid carcinoma (PTC). (a) Thyroid stimulating hormone (TSH) did not induce NIS expression in B-RafV600E expressing thyrocytes. Thyrocytes were infected with control or B-RafV600E lentivirus for 1 week without TSH in the media. Then, they were treated with TSH (1 mU ml⁻¹) for 24 h and analyzed for NIS expression by real-time PCR. Bar graph indicates five independent experiments. (b) SP1 and PAX8 expressions in surrounding normal region and PTC were analyzed by real-time PCR. (c) Methylation-specific PCR analysis of NIS promoter region. Twenty-four cases of normal and PTC regions were analyzed for CpG islands methylation of NIS promoter. Methylation and unmethylation PCR primer were used for the regions indicated (arrow). Lines indicate CpG islands in NIS promoter. Normal and PTC regions methylation PCR is shown in the right upper panel, and B-RafV600E induced methylation in isolated thyrocytes is shown in the right lower panel, respectively. (d) Bisulfite-treated DNA sequencing analysis of NIS promoter region. Twenty-four cases of surrounding normal and PTC region were analyzed for NIS promoter region CpG islands methylation by bisulfite sequencing. Twenty-two CpG islands in NIS promoter and exon1 region (−191 to +73) were analyzed and methylation status is shown as a percentage. Blue circle indicates unmethylation status and red circle for methylation.

Figure 4

DNA methyltransferase 1 (DNMT1) expression in B-RafV600E harboring papillary thyroid carcinoma (PTC). (a) DNMT1, but not DNMT3a,3b, was highly expressed in B-RafV600E harboring PTC. Twenty-eight cases of normal and PTC were analyzed for DNMTs expression by real-time PCR. (b) Immunohistochemical analysis of DNMT1. Forty-nine cases of normal and PTC were analyzed for DNMT1 expression by immunohistochemistry. Dense nucleal and cytoplasmic staining pattern was observed in B-RafV600E harboring PTC. Comparison of DNMT1 expression between PTCs and normal regions of the same slide is presented as a table. Nor and PTC indicate normal and cancer, respectively. (c) Western blotting of DNMT1. Fourteen cases of B-RafV600E harboring PTC tissue were further analyzed by western blotting. DNMT1 expression is presented as a table. (d) B-RafV600E increased DNMTs activity. DNMTs activity was measured in normal surrounding tissue and B-RafV600E harboring PTC. (e) Comparison between sodium iodide symporter (NIS) and DNMTs expression.

Figure 5

DNA methyltransferase 1 (DNMT1) expression in B-RafV600E expressing thyrocytes. B-RafV600E induced DNMT1 expression, but not DNMT3a,3b. Thyrocytes were infected with control or B-RafV600E lentivirus for 1 week and then analyzed for DNMTs expression by real-time PCR (a), and western blot (b). (c) DNMTs activity in B-RafV600E harboring PTC. DNMTs activity was measured in normal surrounding regions and B-RafV600E harboring PTC, respectively. (d) PLX4032 inhibited B-RafV600E-induced DNMT1 upregulation. Experimental scheme was same as in Figure 2d. Thyrocytes were infected with control or B-RafV600E lentivirus for 24 h and selected with 3.5 μg ml⁻¹ puromycin. PLX4032 (20 μM) was applied and the cells were harvested 3 days later. B-RafV600E induced NF-κB activation. IκBα degradation was accelerated in B-RafV600E expressing thyrocytes (e) and p65 nucleus localization was more frequently observed in B-RafV600E expressing thyrocytes (f).