developed to treat JAK- mutated myeloproliferative disorders [21,22] are currently in clinical trials for a variety of cancers. AZD1480 is a potent small-molecule JAK1/2 inhibitor [23] that is under phase I clinical trials for the treatment of myeloproliferative diseases. We investigated the effects of AZD1480 on IL-6/JAK and RET- dependent signaling (STAT3, ERK/MAPK and PI3K/ AKT) as well as its biological effects in human thyroid cancer models (cell lines and a xenograft model). AZD1480 efficiently inhibited the growth and tumorigenesis of thyroid cancer cell lines harboring oncogenic RET alterations, likely through inhibition of PI3K/AKT signaling, supporting the use of this inhibitor for patients with thyroid cancer, particularly those with advanced MTC, for whom there are no effective therapeutic options. Results AZD1480 blocks the growth of thyroid cancer cell lines harboring RET oncogenic alterations In this study, we determined the sensitivity of thyroid cancer cell lines harboring oncogenic forms of RET to JAK1/2 inhibitor, AZD1480. Specifically, we analyzed PTC-derived TPC-1 (RET/ PTC1 rearrangement), MTC-derived MZ-CRC1 (RET M918T mutation) and TT (RET C634W mutation) cell lines. As comparison, we treated the same cell lines with a MEK1/2 inhibitor, AZD6244, which has been shown to have low efficacy in RET-mutated cells [24], in contrast to BRAF-mutated cells. In accordance, we used two other thyroid cancer cell lines, K1 (PTC- derived) and C643 (anaplastic thyroid carcinoma- derived) that harbor BRAFV600E and HRASG13R mutations, respectively, as controls of AZD6244 efficacy. Cells were treated over 5 consecutive days with AZD6244, AZD1480 or a combination of both drugs, and cell density was determined. AZD1480 inhibited the growth of all RET-mutated/rearranged cell lines after 1 (MZ-CRC1, TT) and 2 days (TPC-1) of treatment (p,0.0001; Fig. 1A) and minimally decreased the growth of C643, while having no effect on K1 (Fig. S1). We observed that AZD6244 minimally decreased the growth of C643 and MZ-CRC1 after 4/ 5 days of treatment (p,0.001; Fig. 1A and S1), had no effect on the growth of TT (Fig. 1A) and decreased TPC-1 growth by 50% after 5 days of treatment. In contrast, AZD6244 efficiently inhibited the growth of BRAF- mutant K1 cell line (Fig. S1). No additive or synergistic effect of combined inhibition of MEKs and JAKs was observed. AZD1480 did not inhibit the growth of a non-malignant rat thyroid cell line, PCCl3 (Fig. 1A). The IC50’s for AZD1480 were determined to be in the high nM range (,200–450 nM) for these cell lines (Fig. 1B and S2), and decreased as a function of time (48 and 72 hours), suggesting a cytostatic effect (Fig. 1B). Given the sensitivity of RET-mutated/rearranged cell lines to AZD1480, we further analyzed the cell cycle profile of TPC-1, MZ-CRC1 and TT treated for 72 hours with AZD1480 (Fig. 2A). The JAK inhibitor led to a G1 arrest in TPC-1 (94% compared with 79% in the control, p = 0.01). In the three cell lines, the percentage of cells in S-phase was decreased after AZD1480 treatment (TPC-1: 2% vs. 8% in the control, p = 0.01; MZ-CRC1: 4% vs. 11%, p = 0.004; TT: 6% vs. 11%, p = 0.09). Similarly, the proportion of cells in the G2/M was also decreased in TPC-1 cells treated with the JAK inhibitor (1% vs. 13% in the control, p = 0.01). In MZ-CRC1 and TT, a significant increase in the subG1 population (indicative of cell death by apoptosis) was detected (18% vs. 2%, in DMSO-treated, p = 0.04 and 11% vs 0.6%, p,0.0001, respectively) after 72 hours of AZD1480 treatment. To confirm the effect of AZD1480 in apoptosis, the cell lines were treated with AZD1480 for 48 hours and stained with TUNEL reagent, revealing an increase in the number of AZD1480 Inhibits Thyroid Tumorigenesis apoptotic MZ-CRC1 (p = 0.02) and TT (p = 0.1) cells compared to DMSO- treated cells (Fig. 2B). As expected, AZD1480 did not induce any apoptosis in TPC-1 (Fig. 2B). In parallel, we observed increased levels of the cyclin-dependent kinase inhibitor, p27, and decreased levels of cyclin D1 and of the anti-apoptotic protein, BCL-2, in AZD1480- treated cells (Fig. 2C). AZD1480 induces regression of TPC-1 xenografts The effects of the JAK inhibitor on the in vivo growth of TPC-1 cells were evaluated by subcutaneous injection in the flanks of 3 nude mice. When tumors reached ,0.5 cm , the mice were treated with vehicle, AZD1480 or AZD6244 for 16 consecutive days (Fig. 3A). The tumors from control mice and AZD6244- treated mice continued to grow until day 9 and due to their large size, the mice were sacrificed. In contrast, AZD1480- treated mice showed evidence of tumor regression after 4 days and, after 16 days, they measured ,23% of their initial size (Fig. 3A). Immunohistochemical staining of representative tumor sections showed significant phospho-STAT3 downregulation by AZD1480 in tumor cells and stromal cells (endothelial cells). The MEK inhibitor, AZD6244 reduced phospho-ERK1/2 levels in tumors (Fig. 3B). Histologically, most of the tumor mass (90%) from AZD1480- treated tumors was composed of necrotic tissue, while the majority of tumors cells of the control and AZD6244 groups were viable and actively proliferating, as seen by Ki67 staining (Fig. 3C). Further characterization of these tumors revealed a reduction in endothelial cells (Meca-32) following AZD1480 treatment, compared to control and AZD6244 groups (p = 0.06 and p,0.0001, respectively; Fig. 3C). No significant differences were detected in the number of apoptotic cells (TUNEL), whose percentage was low throughout the tumors. AZD1480- mediated growth inhibition is independent of STAT3 JAKs are the principal mediators of IL-6/gp130/STAT3 signaling and, in several cancer models, JAK inhibitors’ anti- tumorigenic effects are mediated by STAT3. In order to determine whether STAT3 was required for JAK inhibitor-mediated growth arrest, we stably reduced STAT3 in TPC-1 cells using a short hairpin, as determined by western-blot and immunohistochemistry (Fig. 4A, C2). Cells were treated with AZD1480 for four consecutive days and in vitro cell growth was monitored, revealing significant growth inhibition of the TPC-1 shSTAT3 cells (p,0.0001; Fig. 4B). In vivo growth was assessed by injecting the shSTAT3 cells subcutaneously and, upon reaching ,0.5 cm3, tumor-bearing mice were treated with vehicle or AZD1480, for 21 days. The control group was sacrificed after 8 days due to the large size of the tumors. AZD1480 treatment induced regression (tumor volume was ,24% of its initial size; p,0.0001) of TPC-1 shSTAT3 tumors (Fig. 4C1). Phospho-STAT3 was confirmed to be reduced in tumor cells of the vehicle- treated mice, but not in stromal cells, while tumor- and stromal- phospho-STAT3 were significantly reduced in AZD1480- treated mice (Fig. 4C2). AZD1480 inhibits RET Y1062 phosphorylation and downstream PI3K/AKT/mTOR signaling Oncogenic RET effector pathways include ERK/MEK, PI3K/ AKT and STAT3 [3]. Given the significant growth suppressive actions of the JAK inhibitor on the oncogenic RET- transformed TPC-1 xenograft independently of STAT3, we hypothesized that AZD1480 may have a direct effect on RET-mediated signaling. We treated TPC-1, MZ-CRC1, TT (Fig. 5A) as well as a model of inducible RET/PTC3 expression in PCCL3 (Fig. 5B), with PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e46869