and in vivo, independently of JAK/STAT3 signaling in cancer cells. We sought to identify the mechanisms explaining the growth inhibitory effects of AZD1480 in vitro and in vivo. In all cell lines, AZD1480 efficiently reduced phospho-STAT3 levels, including the C634W mutant TT cell line, although this oncogenic form of RET was described as activating STAT3 independently of JAKs, through two docking sites on RET (Tyr752 and Tyr928) [28]. We suggest that our results differ due to the use of a different JAK inhibitor, with different potencies, than that used by Schuringa et al. So far, no data have demonstrated a role for JAKs in RET activation (Y1062 phosphorylation) nor on activation of its downstream MAPK and PI3K pathways. We determined that AZD1480 blocked RET Y1062 phosphorylation in TPC-1, MZ- CRC1, TT, as well as in a conditional model of RET/PTC3 expression. Moreover, although AZD1480 did not inhibit the ERK/MAPK pathway in most of our cell lines, it blocked the activation of the PI3K effectors AKT and S6. Similar results were obtained in the AZD1480- treated TPC-1 xenografts, where no differences in ERK/MAPK levels were detected, and phospho-S6 was significantly downregulated. We demonstrated that these effects were independent of JAKs, as phospho-RET, phospho- ERK and phospho-S6 levels did not change upon JAK1/2 knockdown by siRNA. We demonstrated that AZD1480 directly inhibits the kinase activity of recombinant RET in a dose- dependent manner, which likely underlines the inhibitory and mutant-RET specific effects of AZD1480 on the growth and survival of thyroid cancer cells. Indeed, in vitro kinase assays from a previous report have demonstrated that AZD1480 can inhibit ,50% and 90% of RET activity at 0.1 and 1 mM concentrations, respectively [23]. In conclusion, we showed that the JAK1/2 inhibitor, AZD1480, can block the growth and induce cell death of thyroid cancer cell lines harboring distinct forms of oncogenic RET in vitro and in vivo. In these cells, AZD1480 likely inhibits RET directly, leading to the consequent blockade of the PI3K/AKT/mTOR pathway, which seems to be the preferential oncogenic force driving RET- activated cells. Although these effects were independent of STAT3 in thyroid cancer cells, AZD1480 effectively inhibited phospho- STAT3 in the stroma, particularly in endothelial cells. In fact, JAK inhibitors are known modulators of the microenvironment through inhibition of angiogenesis and myeloid cell mobilization in a STAT3- dependent manner [36,38]. Given the significant decrease in the vascularity of AZD1480- treated tumors and consequent tumor necrosis, we suggest that phospho-STAT3 inhibition in the microenvironment (endothelial cells) cooperates with RET inhibition in cancer cells to induce tumor regression. Additionally, we cannot discard that other RET-independent tyrosine kinases (such as FLT1, FGFR [23]) may be affected by AZD1480, contributing to the growth arrest of RET- activated cells and tumors. Importantly, MZ-CRC1, which harbors the M918 RET mutation associated with the MEN2B syndrome, was highly sensitive to the growth inhibitory effects of AZD1480. Patients diagnosed with MEN2B develop rapidly progressive, multifocal MTC with lymph node metastases, usually requiring total thyroidectomy before 1 year of age [39]. The greater sensitivity of MZ-CRC1 to AZD1480 compared with TT (harboring the less aggressive C634W mutation), might be explained by different capacities of MEN2A- and MEN2B- RET mutants to activate downstream pathways, namely the PI3K/AKT pathway [40]. Altogether, these results support the use of AZD1480 to treat aggressive forms of thyroid cancer, particularly MEN2B- MTC. AZD1480 Inhibits Thyroid Tumorigenesis Materials and Methods Ethics statement All the procedures of animal research were included in a protocol (number 0011091) approved by the MSKCC Institution- al Animal Care and Use Committee (IUAC), following the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals and the Guidelines and Policies for Rodent experiment. Cell culture and drugs TPC-1, K1, C643 (provided by Prof. Marc Mareel, Belgium) and TT (purchased from American Type Culture Collection – ATCC) [29,41] cell lines were maintained in RPMI, 10% FBS, 1% PenStrep. PCCl3-RET/PTC3 was provided by Dr James Fagin and was cultured as previously described [13]. MZ- CRC1 (provided by Dr Robert Hofstra, Netherlands) [29] and 293T (ATCC) were maintained in DMEM, 10% FBS, 1% PenStrep. All cell culture reagents were from GIBCO, Invitrogen, Carlsbad, USA. AZD1480 [23] and AZD6244 [24] were gifts from Dennis Huszar and Michael Zinda (AstraZeneca, London, UK). Lentiviral infections and generation of stable cell lines The STAT3 shRNA lentiviral construct was previously described [42]. Viral particles carrying the constructs were generated in 293T-cells. The viral particles in the supernatant were precipitated using a polyethylene glycol virus precipitation solution (System Biosciences LLC, Mountain View, CA, USA). Lentiviral constructs included a transcription cassette encoding enhanced green fluorescent protein [42], that allowed selection of cells by sorting. Whole cell protein extracts and western blotting Cells were lysed in radio-immunoprecipitation assay buffer, supplemented with protease and phosphatase inhibitors (Sigma Aldrich, St. Louis, MO, USA). Proteins were quantified using a modified Bradford assay (Biorad Laboratories, Inc., Hercules, CA, USA), resolved by SDS-PAGE and transferred to Hybond ECL membranes (GE Healthcare, Little Chalfont, England). The primary antibodies for phospho-STAT3 (Tyr705), STAT3, phospho-ERK1/2 (Thr202/Tyr204), ERK1/2, phospho-AKT (Ser473), AKT, phospho-S6 (Ser235/236), and S6 were from Cell Signaling Technologies, Inc, Danvers, MA, USA. Cyclin D1 antibody was from Neomarkers, Fremont, CA, USA. Actin antibody, phospho-RET (Y1062), total RET (C-19) and p27 were from Santa Cruz BT, Santa Cruz, USA, BCL-2 antibody was from DAKO, Glostrup, Denmark, and a-tubulin was from Sigma Aldrich, St. Louis, MO, USA. Peroxidase- conjugated secondary antibodies were from Santa Cruz BT. Bands were visualized by chemiluminescence using ECL detection system (Perkin Elmer, Foster City, USA). In vitro cell growth The cell density was assessed by sulforhodamine (SRB) staining, as previously described [43]. Cells were fixed with 10% trichloroacetic acid and stained with a 0.4% sulphorhoda- mine B (Sigma Aldrich, St. Louis, MO, USA) solution. Absorbances were read in a microplate reader, at 565 nm. The IC50s were determined by nonlinear regression using GraphPad Prism v5 (GraphPad Software Inc., San Diego, CA). Each experimental condition was performed 3 times, each including 6 replicates. PLOS ONE | www.plosone.org 9 October 2012 | Volume 7 | Issue 10 | e46869