Liu Dingxie, Hu Shuiying, Hou Peng, Jiang David, Condouris Stephen, Xing Mingzhao
Division of Endocrinology and Metabolism, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA.
Clin Cancer Res. 2007 Feb 15;13(4):1341-9. doi: 10.1158/1078-0432.CCR-06-1753.
The V600E BRAF mutant plays an important role in the pathogenesis of papillary thyroid cancer (PTC) and is associated with loss of expression of thyroid iodide-metabolizing genes. This study was done to investigate the restorability of expression of these genes by suppressing the BRAF/extracellular signal-regulated kinase kinase (MEK)/mitogen-activated protein (MAP) kinase pathway in V600E BRAF-harboring thyroid cells and to explore the mechanisms involved.
We used inducible expression of V600E BRAF, small interfering RNA transfection, and MEK-specific inhibitor to alter the MAP kinase pathway activities and subsequently examined the changes in expression, promoter activities, and methylation status of thyroid genes.
MEK inhibitor U0126 or cessation of V600E BRAF expression in PCCL3 cells restored expression of thyroid genes silenced by induced expression of V600E BRAF. U0126 also restored the expression of these genes in V600E BRAF-harboring PTC-derived NPA cells. Knockdown of BRAF by specific small interfering RNA restored expression of some of these genes in NPA cells. Luciferase reporter assay using thyroid-stimulating hormone receptor gene as a model showed that the promoter activity was modulated by the MAP kinase pathway. Promoter methylation in association with DNA methyltransferase expression played a role in gene silencing by MAP kinase pathway in NPA cells.
We showed the restorability of expression of thyroid iodide-metabolizing genes silenced by V600E BRAF, and linked this process to gene methylation in PTC cells. The results provide clinical implications that therapeutic targeting at the BRAF/MEK/MAP kinase pathway may be a good approach in restoring thyroid gene expression for effective radioiodine therapy for BRAF mutation-harboring PTC.
V600E BRAF突变在甲状腺乳头状癌（PTC）的发病机制中起重要作用，并与甲状腺碘代谢基因表达缺失有关。本研究旨在通过抑制携带V600E BRAF的甲状腺细胞中的BRAF/细胞外信号调节激酶激酶（MEK）/丝裂原活化蛋白（MAP）激酶途径，研究这些基因表达的恢复能力，并探讨其中涉及的机制。
我们利用V600E BRAF的诱导表达、小干扰RNA转染和MEK特异性抑制剂来改变MAP激酶途径的活性，随后检测甲状腺基因的表达、启动子活性和甲基化状态的变化。
MEK抑制剂U0126或PCCL3细胞中V600E BRAF表达的停止恢复了因V600E BRAF诱导表达而沉默的甲状腺基因的表达。U0126也恢复了携带V600E BRAF的PTC来源的NPA细胞中这些基因的表达。通过特异性小干扰RNA敲低BRAF可恢复NPA细胞中部分这些基因的表达。以促甲状腺激素受体基因为模型的荧光素酶报告基因检测表明，启动子活性受MAP激酶途径调节。与DNA甲基转移酶表达相关的启动子甲基化在NPA细胞中通过MAP激酶途径导致基因沉默中起作用。
我们证明了被V600E BRAF沉默的甲状腺碘代谢基因表达的恢复能力，并将这一过程与PTC细胞中的基因甲基化联系起来。结果提供了临床启示，即靶向BRAF/MEK/MAP激酶途径进行治疗可能是恢复甲状腺基因表达以对携带BRAF突变的PTC进行有效放射性碘治疗的良好方法。
