AZD1480 Inhibits Thyroid Tumorigenesis Figure 1. AZD1480 inhibits the growth of RET-activated thyroid cancer- derived cell lines. (A) TPC-1, MZ-CRC1 and TT cell lines were treated with AZD6244 (1 mM), AZD1480 (1 mM), and a combination of both drugs (1 mM each) for the indicated time. A rat- derived non malignant thyroid cell line, PCCl3, was treated with AZD1480 (1 mM) or control DMSO. Growth was determined by the sulphorhodamine B assay. (B) Cell lines were treated for 48 or 72 hours with different concentrations of AZD1480 (0.1, 0.5 and 1 mM) and cell density was determined. Data are shown as mean 6 SE of three independent experiments. ***p,0.0001. doi:10.1371/journal.pone.0046869.g001 AZD1480 and/or AZD6244, for 24 hours. The expression and phosphorylation levels of RET as well as of the main effectors of the JAK/STAT3, ERK/MAPK and PI3K/AKT pathway, namely phospho-STAT3 Tyr705, phospho-ERK1/2 Thr202/ Tyr204 and phospho-AKT Ser473/phospho-S6 Ser235/236, respectively, were examined by western-blot analysis. AZD1480 and AZD6244 effectively decreased the levels of their downstream targets phospho-STAT3 and phospho-ERK1/2, respectively, in all of the cell lines (Fig. 5A, B). MZ-CRC1 did not express phospho-ERK1/2 at basal levels (Fig. 5A). Additionally, AZD1480 reduced the levels of phospho-ERK1/2 in PCCl3- RET/PTC3 and TT, as well as of phospho-AKT, phospho-S6 and phospho-RET in all of the cell lines (Fig. 5A, B). In contrast, AZD6244 treatment increased phospho-STAT3 in TPC-1 cells, increased phospho-AKT and phospho-S6 in MZ-CRC1 cells and increased phospho-RET in PCCl3-RET/PTC3 cells (Fig. 5A, B). There is no evidence to date demonstrating a functional association between RET and JAKs. To determine whether the decreased phospho-RET was due to off-target effects of AZD1480, we knockdown JAK1/2 expression in TPC-1, MZ-CRC1 and TT PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e46869