AZD1480 Inhibits Thyroid Tumorigenesis Figure 5. AZD1480 induces sustained downregulation of RET tyrosine phosphorylation and PI3K/AKT pathway activation in vitro and in vivo. (A) TPC-1, MZ-CRC1, TT and a (B) PCCl3-RET/PTC3 doxycycline-inducible cell line were treated for 24 hours with AZD1480 (1 mM), AZD6244 (1 mM), or a combination of both drugs. Total cell protein extracts were probed with antibodies for the indicated effectors of the JAK/STAT3, ERK/MAPK and PI3K/AKT signaling pathways as well as for phospho-RET Y1062. In the lower panel of TPC-1, the DMSO (D) lane is non-contiguous to the AZD6444 (6244) lane, in the same gel. (C) TPC-1 xenografts’ representative sections were probed for the activation of STAT3 (phospho-STAT3), ERK/MAPK (phospho-ERK1/2) and PI3K/AKT (phospho-S6) signaling pathways. Quantification is represented graphically (mean 6 SE). *p,0.05; **p,0.001. doi:10.1371/journal.pone.0046869.g005 tumors from AZD6244- treated mice grew slightly more than the control tumors, suggesting that treating RET-mutated thyroid cancers with this inhibitor may promote tumor growth. In TPC-1 tumors, and similarly to the effects in vitro, AZD1480 blocked the proliferation while not significantly affecting apoptosis. However, in vivo, we observed marked PLOS ONE | www.plosone.org 7 October 2012 | Volume 7 | Issue 10 | e46869