Anaplastic thyroid cancer (ATC) is a rare orphan disease that accounts for 1–3% of thyroid cancers. ATCs progress rapidly and are extremely aggressive toward both adjacent organs by invasion and distant organs by hematological dissemination. Because of the highly malignant potential, ATC cases often become lethal within 6 months from the initial diagnosis, despite intensive therapeutic efforts (1–3). No standardized therapeutic strategy has been documented to manage ATC, and experimental multimodal therapies with surgery, chemotherapy and/or radiation therapy have been attempted practically. Regrettably, no effective therapeutic method for ATC has been established to date (4,5).

However, several molecular targeted therapies have achieved successful results against ATCs (6–8). Rosove et al (9) reported an impressive case of an ATC patient successfully treated with a selective BRAF^V600E inhibitor, vemurafenib. Possible clinical application of this inhibitor has been demonstrated recently in a phase 2 trial in BRAF^V600E mutation-positive ATC patients, demonstrating an overall response rate of 29% (2/7) (10). In a study by Kim et al (11), BRAF mutation in papillary thyroid cancer (PTC) was found more frequently in East Asian countries compared to the Western countries, and the proportion of PTCs among differentiated thyroid cancers (DTCs) was higher in Japan than Western countries. A considerable proportion of ATCs is thought to be derived from long-lasting DTC, and BRAF mutation was found to be maintained during the phenotypical change from DTC to ATC (12). Although the incidence of BRAF mutation was reported to be relatively less common in ATC (15–24%) than that found in PTC (13–15), a preliminary finding indicated that the rate of BRAF mutation in a population of Japanese ATC patients was high (6 of 14 patients) [Uchino, et al, Proceedings of the 20th Annual Meeting of Japan Association of Endocrine Surgeons, O-11 65, 2008 (In Japanese)]. In addition, six of seven thyroid cancer cell lines in our series have a BRAF mutation (16). These observations suggest that the frequency of BRAF mutation in ATC is much higher in Japan compared to Western countries.

A previous study of our group demonstrated a possible effect of molecular therapies targeting epidermal growth factor receptor (EGFR), although the effect was limited to the cells with a preserved RAS/RAF/MEK pathway (17). Our more recent study demonstrated that part of this EGFR-targeted therapy resistance could be overcome with an mTOR inhibitor, although we again observed that the efficacy was limited to the cells with an altered PI3K/AKT/mTOR pathway (18). These observations indicated the importance of direct targeting to the RAS/RAF/MEK pathway to manage ATC.

Another research group also demonstrated the importance of BRAF mutation in the aggressive characteristics of thyroid cancer and the efficacy of its inhibition on the management of the disease (19). Several studies described important roles of BRAF gene alteration in genome-wide aberrant methylation (20), vascular endothelial growth factor (VEGF) expression (21), and the induction of epithelial-mesenchymal transformation (EMT) (22).

We conducted a preclinical investigation of the efficacy of inhibiting the RAS/RAF/MEK pathway in a series of authentic ATC cell lines harboring a genetic alteration in either BRAF or NRAS (16). Our specific aims were to determine the efficacies of BRAF/MEK inhibitors in ATC cells and to identify possible differences in the mechanism of blockade between the cell lines according to the differences in the genetic alterations of the cell lines, the levels of VEGF secretion, and/or the expression of EMT markers.

Dabrafenib is a reversible and potent ATP-competitive inhibitor that selectively inhibits the BRAF^V600E kinase (24). In the OCUT-2 and -4 cell lines, which harbor a BRAF^V600E mutation, dabrafenib clearly inhibited cellular growth by demonstrating G0/G1 arrest. The strongest inhibitory effect was in the OCUT-4 cells, in which the marked activation of a downstream pathway from BRAF gene was observed in the stable culturing condition. The phosphorylations of MEK and ERK were strongly downregulated by exposure to dabrafenib in OCUT-4 cells, causing a significant G0/G1 arrest. In the OCUT-2 line, the mutation in PI3KCA gene in addition to BRAF^V600E mutation (16) and signaling through the PI3K/AKT/mTOR pathway can also be expected to contribute to aberrant cell proliferation to some extent. Nevertheless, the dabrafenib treatment resulted in a degree of growth inhibition by G0/G1 arrest in the OCUT-2 cells that was similar to that observed in the OCUT-4 cells. This observation suggested that the activated MAPK/ERK pathway, and not the PI3K/AKT/mTOR pathway, was the main driver for aggressive cell proliferation in OCUT-2 cell line. The results indicate that the inhibition of BRAF^V600E by dabrafenib might be effective against cancer cells harboring active alterations in both the MAPK/ERK and PI3K/AKT/mTOR pathways.

We observed an upregulation of phosphorylated ERK after dabrafenib exposure in ACT-1 and OCUT-6 cells, which have an NRAS mutation. The mechanism of the upregulation of p-ERK in RAS mutant cells after treatment with a selective BRAF^V600E kinase inhibitor has been investigated. Dimeric complexes with wild-type BRAF, CRAF or kinase-dead BRAF is able to generate excessive downstream signaling under stimulation by mutant RAS enzyme (25). This mechanism resulted in paradoxical phosphorylation in ERK after BRAF inhibition, but did not contribute to the cell cycle progression or cell growth in the present study. However, the VEGF secretion was clearly stimulated in the NRAS mutant cells after dabrafenib treatment in our study. VEGF is well known as a strong inducer of cancer neo-vasculature that contributes to the arrangement of the cancer microenvironment for aggressive growth. Our present findings indicated one of the potential mechanisms of tumor growth in dabrafenib-resistant NRAS mutant cancer cells.

The treatment with trametinib, a reversible allosteric inhibitor of MEK1 and MEK2 activation and kinase activity, resulted in universal growth suppression in all four cell lines independent of the mutational status of BRAF or NRAS. A weak growth-inhibitory effect was observed in the BRAF mutant OCUT-4 cells. After trametinib exposure, inhibition of the phosphorylation of ERK was clear, and cell cycle arrest was obviously identified. Nevertheless, the phosphorylation of MEK was strongly induced in OCUT-4 cells by trametinib treatment, suggesting that resistance to trametinib could be caused by a mechanism other than one downstream of the MAPK/ERK pathway. This hypothesis was also suggested by the result of our dual blockade by dabrafenib and trametinib. There was no additional effect of either inhibitor in combination with the other in the OCUT-4 cell line, suggesting a limited effect of inhibiting the MAPK/MEK pathway. In addition, the OCUT-4 cells showed a different EMT marker expression profile after exposure to the inhibitors. Only this cell line showed an upregulation in the expression of the mRNA of SNAI1.

The expression of EMT markers is thought to have a role in the acquisition of resistance to a cytotoxic drug (26). Our present findings indicated that the phenotypical change through the EMT also contributed to the mechanism of resistance to these inhibitors. Several mechanisms have been confirmed to trigger resistance to BRAF inhibition (27–30). Additional investigations are needed to clarify the involvement of the EMT in the effect of BRAF inhibition.

Dabrafenib and trametinib, as monotherapy or in combination, were approved for the treatment of melanomas by the US Food and Drug Administration. Dabrafenib as a treatment for advanced thyroid cancer resulted in durable responses in BRAF-mutant DTC patients (31). A recent report suggested the re-differentiation of iodine-refractory thyroid cancer after dabrafenib treatment (32). These observations clearly indicate the usefulness of BRAF/MEK inhibitors for the management of advanced and inoperable thyroid cancer. The results of the present study suggest the importance of selecting ATC patients in accord with the mutation status of BRAF and RAS when applying inhibitors (33).

Our present findings demonstrated the efficacy of a mutation-selective BRAF inhibitor and a MEK inhibitor in human ATC cell lines. Our observations indicated the existence of a unique driver gene for the aggressive proliferation of ATC cancer cells, and we observed that a cellular growth inhibitory effect can be expected when appropriate inhibitor(s) are selected.