AZD1480 Inhibits Thyroid Tumorigenesis Figure 4. Growth inhibition of TPC-1 cells and tumors by AZD1480 is independent of activated phospho-STAT3 Tyr705. (A) STAT3 was knockdown in TPC-1 cells by a short hairpin. Phospho-STAT3 and STAT3 downregulation were confirmed by western-blotting. (B) TPC-1 shSTAT3 cells were treated with DMSO or AZD1480 (1 mM) and growth inhibition was determined by the SRB assay. Results are mean 6 SE of three 3 independent experiments. (C) TPC-1 shSTAT3 cells were injected subcutaneously in both flanks of nude mice. When tumors reached ,0.5 cm , mice were equally distributed in two groups: one was the control group (vehicle) and the other was treated with AZD1480 at 30 mg/Kg, bi-daily. (C1) Tumor volume was measured at the indicated time points. Results represent mean 6 SE. (C2) Representative H&E and phospho-STAT3 immunostaining of tumors sections from untreated (vehicle) or AZD1480- treated TPC-1 shSTAT3 tumors. Arrows indicate phospho-STAT3- positive murine stromal cells. Magnification: 200x. ***p,0.0001. doi:10.1371/journal.pone.0046869.g004 1, MZ-CRC1 and TT with IC50s ,500 nM, which is 2 to 10 fold below that reported for other cancer cell lines [23,30]. The block in growth was due to a G1 cell cycle arrest in TPC-1 cells, while in MZ- CRC1 and TT, JAK inhibition significantly increased apoptosis. On the other hand, a MEK1/2 inhibitor, AZD6244, failed to alter in vitro growth of MZ-CRC1 and TT. No additive or synergistic effects on in vitro growth were observed by combining both inhibitors. On the contrary, AZD6244 (but not AZD1480) efficiently inhibited the growth of a BRAFV600E- mutant cell line, K1. Both AZD1480 and AZD6244 had a minimal effect on the growth of a RAS- mutant cell line, C643. The insensitivity of RET-activated thyroid cancer cells to MEK inhibition has been previously demonstrated, as opposed to the high sensitivity of thyroid cancer cells expressing BRAFV600E [31]. This resistance might reflect the ability of oncogenic RET to activate alternative signaling pathways, particularly the PI3K/AKT/mTOR pathway [32]. Moreover, AZD6244 caused upregulation of phospho- RET Y1062 in the PCCl3-RET/PTC3 model as well as of mTOR effectors, phospho-S6 and phospho-AKT, in MZ-CRC1. Over- activation of the mTOR pathway in response to MEK inhibition can possibly be explained by relief of feed-back inhibition and has been previously reported in other models [33], where it mediates cell resistance to AZD6244 [34,35]. Furthermore, AZD1480 potently inhibited the in vivo growth of TPC-1 xenografts, resulting in tumor regression, while the PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e46869