The BRAFT1799A mutation confers sensitivity of thyroid cancer cells to the BRAFV600E inhibitor PLX4032 (RG7204)

Abstract

Aberrant signaling of the Ras-Raf-MEK-ERK (MAP kinase) pathway driven by the mutant kinase BRAF(V600E), as a result of the BRAF(T1799A) mutation, plays a fundamental role in thyroid tumorigenesis. This study investigated the therapeutic potential of a BRAF(V600E)-selective inhibitor, PLX4032 (RG7204), for thyroid cancer by examining its effects on the MAP kinase signaling and proliferation of 10 thyroid cancer cell lines with wild-type BRAF or BRAF(T1799A) mutation. We found that PLX4032 could effectively inhibit the MAP kinase signaling, as reflected by the suppression of ERK phosphorylation, in cells harboring the BRAF(T1799A) mutation. PLX4032 also showed a potent and BRAF mutation-selective inhibition of cell proliferation in a concentration-dependent manner. PLX4032 displayed low IC(50) values (0.115-1.156μM) in BRAF(V600E) mutant cells, in contrast with wild-type BRAF cells that showed resistance to the inhibitor with high IC(50) values (56.674-1349.788μM). Interestingly, cells with Ras mutations were also sensitive to PLX4032, albeit moderately. Thus, this study has confirmed that the BRAF(T1799A) mutation confers cancer cells sensitivity to PLX4032 and demonstrated its specific potential as an effective and BRAF(T1799A) mutation-selective therapeutic agent for thyroid cancer.

Fig. 1

Effects of the BRAF^V600E inhibitor PLX4032 on ERK phosphorylation in thyroid cancer cells. (A) Concentration-dependent responses of PLX4032 in OCUT1 and K1 cells. Cells were treated with PLX4032 at the indicated concentrations for 6 h in culture medium containing 5% FBS. Cell lysates were analyzed by Western blotting with antibodies specific to phospho-ERK (p-ERK), ERK1, and β-actin. ERK1 and β-actin were analyzed simultaneously for quality control of proteins. (B) Time-dependent response of the effects of PLX4032 in OCUT1 and K1 cells. Cells were treated with PLX4032 at 1 μM for the indicated times and Western blotting assay was performed as in Fig 1 A. (C) Effects of PLX4032 on p-ERK in the remaining thyroid cancer cells. Cells were treated with PLX4032 at 1 μM for 24 h. Western blotting assay was performed as in Fig 1A.

Fig. 2

BRAF^T1799A mutation-dependent inhibition of thyroid cancer cells by the BRAF^V600E inhibitor PLX4032. Cells were treated with the indicated concentrations of PLX4032 for 5 days with the drug replenished daily by changing the culture medium containing 5% FBS. MTT assay of cell proliferation was performed at the end of the 5-day treatment Cells in the upper row harbor the BRAF^T1799A mutation, cells in the lower row harbor Ras mutations, and cells in the middle row are wild-type, harboring neither BRAF nor Ras mutations. The values on the y-axis represent the relative cell growth rate, with control growth (in the absence of the drug) being defined as 1.