Sorafenib potently inhibits papillary thyroid carcinomas harboring RET/PTC1 rearrangement

Abstract

Purpose: Papillary thyroid carcinomas (PTC) are the most common type of thyroid malignancy with one of the two mutations, RET/PTC rearrangement or BRAF mutation. Both mutations are able to activate the MEK/ERK signaling transduction pathway and result in the activation of transcription factors that regulate cellular proliferation, differentiation, and apoptosis. Sorafenib (Nexavar, BAY 43-9006) is a multikinase inhibitor, and in this study, we tested its effects on PTC cells carrying either mutation.

Experimental design: The effects of sorafenib on cell proliferation and signaling were evaluated in vitro on PTC cells using growth curves, cell cycle analysis, and immunoblotting. Using an orthotopic mouse model, we determined the antitumor effects of sorafenib in vivo.

Results: The concentration needed for 50% growth inhibition (GI(50)) by sorafenib was 0.14 mumol/L for the PTC cells with the RET/PTC1 rearrangement, and 2.5 mumol/L for PTC cells with a BRAF mutation, both readily achievable serum concentrations. After 3 weeks of oral administration of sorafenib (80 mg/kg/d) in mice, small (94% reduction compared with controls) or no tumor growth was detected in mice inoculated with PTC cells bearing the RET/PTC1 rearrangement, whereas the tumor volume of the orthotopic tumor implants of PTC cells with a BRAF mutation was reduced 53% to 54% (as compared with controls).

Conclusions: PTC cells carrying the RET/PTC1 rearrangement were more sensitive to sorafenib than PTC cells carrying a BRAF mutation. Because RET/PTC rearrangements are unique to thyroid carcinomas, our findings support the clinical evaluation of sorafenib for patients with PTC and the identification of patients most likely to respond to sorafenib treatment.

Fig. 1

Inhibition of the growth of PTC cells treated with sorafenib. A, cells with the RET/PTC1 rearrangement were treated with sorafenib for up to 4 d. Sorafenib was added to the cells on days 0 and 2. Each day, the cells were counted according to absorption at 570 nm (Abs 570) after staining with MTTusing a microplate reader. Cells treated with DMSO only were used as positive controls (No drug). Sorafenib concentrations were 0.5 (3.6× GI₅₀), 0.05 (0.36 × GI₅₀), or 0.005 μmol/L (0.036× GI₅₀). B, cells with a BRAF mutation were treated with 5 (2× GI₅₀), 1.5 (0.6× GI₅₀), or 0.15 μmol/L (0.06×GI₅₀) of sorafenib for up to 4 days as described for cells with the RET/PTC1r earrangement.

Fig. 2

Expression of p-MEK1/2, p-ERK1/2, and cleaved PARP in PTC cells after treatment with sorafenib.The expression of p-MEK1/2 (A and B), p-ERK1/2 (A and B), and PARP (C and D) was detected usingWestern blot analysis.Total MEK1/2 and ERK1/2 were used as loading controls and cells treated with DMSO only were used as positive controls for normal expression of p-MEK1/2 and p-ERK1/2, respectively. A, cells with the RET/PTC1rearrangement were treated with 0.01, 0.1, 1, or 3 μmol/L of sorafenib for 2 h (top); or treated with 1 μmol/L of sorafenib for 0.5, 2, 6, 8, 15, 24, or 48 h (bottom). B, cells with a BRAF mutation were treated with sorafenib at 0.1, 1, 5, or 10 μmol/L for 2 h (top); or treated with 10 μmol/L of sorafenib for 0.5, 2, 6, 8, 24, or 30 h (bottom). C, PTC cells with a BRAF mutation were treated with 5 μmol/L of sorafenib for 1to 4 d.The expression of cleaved and uncleaved PARP was detected byWestern blot analysis. Cells treated with DMSO only were used as controls, and actin was used as a loading control. D, tumor extracts (mice tumor) were prepared from two independent animal studies with or without sorafenib treatment for each experiment (first and second experiments) and cleaved PARP was detected by Western blot analysis. Protein extracts from PTC cells before inoculation into mice (cell extract) were used as a negative control.Total and cleaved PARP were shown and actin was used as a loading control. Tumor samples from the second animal experiment with mice inoculated with PTC cells carrying the RET/PTC1 rearrangement (left), and no tumor was detected from the first experiment.Tumor samples from both experiments with mice inoculated with PTC cells carrying a BRAF mutation (right).

Fig. 3

Cell cycle analysis of PTC cells treated with sorafenib. PTC cells with the RET/PTC1rearrangement (A) or a BRAF mutation (B) were treated with 0.5 and 1.5 μmol/L or1.5 and 5 μmol/L sorafenib for 24 h, respectively. Cells not treated with sorafenib (0 μmol/L) were used as a control. After flow cytometry, the percentage of cells in the G₁/G₀, G₂-M, and S phase are shown.The percentage of cells in sub-G₁ (apoptotic) phase was too small to show on the graph and was not included.