Suppression of BRAF/MEK/MAP Kinase Pathway Restores Expression of Iodide-Metabolizing Genes in Thyroid Cells Expressing the V600E BRAF Mutant

Abstract

Purpose: 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. Experimental Design: 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. Results: MEK inhibitor U0126 or cessation of V600E BRAF expression in PCCL3 cells restored expression of thyroid genes silenced by induced expression ofV600E BRAF. U0126 also restored the expression of these genes inV600E 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. Conclusions: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.Papillary thyroid cancer (PTC) accounts for 80% of all thyroid cancers and is the most common endocrine malignancy, with a currently rapidly increasing incidence (1, 2). Following thyroidectomy, radioiodine ablation is the mainstay of medical treatment for PTC, but patients may fail it when the cancer has lost radioiodine avidity, a primary cause for thyroid cancerrelated morbidity and mortality (3,4). This radioiodine treatment takes advantage of the unique function of thyroid cells to uptake, concentrate, and organify iodide, a substrate normally used for synthesis of thyroid hormones. Several thyroid-specific protein molecules play a key role in this iodide-metabolizing process, including thyroid stimulating hormone receptor (TSHR), sodium iodide symporter (NIS), thyroglobulin (Tg), thyroperoxidase (TPO), and the thyroid gene transcription factors TTF-1 and Pax-8 (5). Loss of expression of the genes for these molecules is common in thyroid cancer and is a sufficient cause for the loss of radioiodine avidity and failure of radioiodine therapy in this cancer (6 -10). Thus, a novel approach leading to restoration of expression of these thyroid iodide-metabolizing genes would provide great hope for those pati...

Potential Utility and Limitations of Thyroid Cancer Cell Lines as Models for Studying Thyroid Cancer

“…Interestingly, several thyroid-specific genes were shown to be silenced on activation of the BRAF=MAPK=MEK pathway, with induced expression of BRAF V600E in rat thyroid cell line PCCL3 (48,49). Suppression of MAPK pathway could restore the expression of these genes.…”

“…Suppression of MAPK pathway could restore the expression of these genes. In case of TSH-R gene, demethylation of the gene associated with its expression occurred with suppression of the BRAF=MAPK=MEK pathway (49). Although the tumorigenic role of these genes is not clear, the silencing of these thyroid-specific genes by methylation can cause failure of clinical radioiodine treatment of thyroid cancer.…”

Epigenetic silencing of TTF-1/NKX2-1 through DNA hypermethylation and histone H3 modulation in thyroid carcinomas

“…Indeed, suppression of the MAPK pathway by knockdown of mutant BRAF or administration of the MEK inhibitor restores expression of iodide-metabolizing genes in human thyroid carcinoma cells expressing the BRAF mutation. 29 It is likely that endogenous epigenetic genes, such as DNA methyl transferases (DNMTs) and HDACs, mediate Epigenetic silencing of TTF-1 in thyroid T Kondo et al epigenetic dysregulation of tumor-suppressor genes in carcinoma cells. 30 Overexpression of DNMT1 and HDACs has been described in several human cancers including colorectal and breast cancers.…”

Molecular characteristics of papillary thyroid carcinomas without BRAF mutation or RET/PTC rearrangement: relationship with clinico-pathological features

“…In vitro studies have shown that expression of RET/PTC (De Vita et al 1998 or BRAF V600E (Mitsutake et al 2005, Liu et al 2007) in PCCl3 rat thyroid cells leads to a down-regulation of expression of thyroid-specific genes. A higher level of expression of matrix-metalloproteases and a higher cell motility have been found in BRAF V600E -expressing cells as compared with RET/PTC3-expressing PCCl3 cells (Mesa et al 2006).…”