Abstract 1025: Mechanism of antitumor effect of Sorafenib in anaplastic thyroid cancer cell lines

Abstract

Introduction: Anaplastic thyroid cancer (ATC) is a rare refractory disease, found in 1-3% of thyroid cancer. Because of its highly malignant potential, patients often become lethal within 6 months, nevertheless for intensive multimodal therapies. Recent observations indicated the strong efficacy of multi-kinase inhibitor to control disease progression of ATC. Frequent BRAF mutation (40-60%) in tumors of ATC was reported in Japan, indicating BRAF gene alteration as possible molecular target. Moreover, the secretions of VEGF were often found in ATC cell lines, suggesting the important role of VEGF-mediated tumor neo-vascularization in the aggressive progression of ATC. We, thus, examined the antitumor effect of Sorafenib, a multi-kinase inhibitor demonstrating its effect by inhibiting both BRAF and VEGF signal. Materials & Methods: Human ATC cell lines having four different gene mutations were used (OCUT-4; BRAF mutation, OCUT-6, ACT-1; NRAS mutation, OCUT-2; BRAS and PI3KCA mutation). The effect of Sorafenib on cellular viability was evaluated by MTT assay. In order to investigate the mechanism of cellular damage by Sorafenib, cell cycle was evaluated by flowcytometry, and the signal transduction of RAF/MEK pathway was examined by Western blotting. The amount of VEGF secretion from cells was measured by ELISA. Expression of VEGF-receptor in cells was evaluated by Western blotting. Furthermore, the effects of Sorafenib on human vascular endothelial cells were investigated by recruiting HUVEC (Kurabo, Japan) stimulated with VEGF and conditioned medium of the cancer cells. Results: The effect of Sorafenib appears most strongly in OCUT-4 cell with BRAF mutation compared with other cell lines. However, the impairment of cellular viability was shown at relatively high concentration (>100 nM). OCUT-4 showed G1 phase arrest by Sorafenib exposure. The specific effect of BRAF inhibition was confirmed by the suppression of MEK and ERK phosphorylation in OCUT-4. The suppression was not observed in OCUT-6, a cell line with NRAS mutation. The paradoxical increase of MEK phosphorylation was found in this cell line, instead. Every cell line secreted different amount of VEGF, and the proliferation of HUVEC was stimulated by their conditioned mediums similarly as by VEGF. The stimulation of proliferation was suppressed both by anti-VEGF antibody and Sorafenib. Discussion: In the present study, we clearly demonstrated that Sorafenib inhibits growth of human vascular endothelium mediated by VEGF secretion from cancer cells, as well as direct cell cycle arrest on ATC cells. A high concentration of Sorafenib may be needed to show inhibition on the RAF/MEK pathway. Furthermore, the inhibition of the pathway was not effective when additional growth stimulatory pathway than RAF was activated as in case of OCUT-2, or RAS gene were mutated. However, in any case, cancer cell growth mediated by autocrine system was inhibited clearly by Sorafenib. Citation Format: SAE ISHIHARA. Mechanism of antitumor effect of Sorafenib in anaplastic thyroid cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1025.