AZD1480 Inhibits Thyroid Tumorigenesis Figure 2. AZD1480 induces G1 blockage and apoptosis of thyroid cancer- derived cell lines. (A) TPC-1, MZ-CRC1 and TT were treated with AZD1480 (1 mM; +) for 72 hours. Cells were subjected to cycle profile analysis by flow cytometry. Results are representative of three independent experiments (mean 6 SE). (B) Cells were treated with AZD1480 (1 mM) for 48 hours and apoptotic cell death was determined by TUNEL. (C) AZD1480 induces p27 upregulation and cyclin D1 and BCL-2 downregulation in thyroid cancer cell lines. TPC-1, MZ-CRC1 and TT cell lines were treated with AZD1480 (1 mM) for 24 hours. Cell lysates were probed with the indicated primary antibodies, by western-blotting. *p,0.05; ***p,0.0001. doi:10.1371/journal.pone.0046869.g002 cells by short interfering (si) RNA. At 48 hours, JAK1, JAK2 and phospho-STAT3 levels were downregulated in all cell lines, with no effects on phospho-RET, phospho-ERK1/2 and phospho-S6 (Fig. 6A). In contrast to AZD1480, JAK1/2 knockdown did not decrease the proliferation of any of the cell lines, as seen by BrdU incorporation (Fig. 6B). We performed an in vitro kinase assay and verified that AZD1480 directly inhibited RET kinase activity: 40% inhibition at 0.001 mM, 90% inhibition at 0.1 mM and 99% at 1 mM (Fig. S3). We examined the phosphorylation levels of STAT3, ERK and S6 in the TPC-1 xenografts treated with vehicle, AZD1480 and AZD6244. Similarly to what observed in TPC-1 and the other RET-activated cell lines in vitro, JAK inhibitor led to a significant decrease of phospho-S6 and phospho-STAT3 levels (p,0.001) in the tumor, while no decrease was observed in vehicle or AZD6244- treated tumors (Fig. 5C). Phospho-ERK levels were unchanged between the control and the AZD1480 groups and were signifi- cantly decreased (p = 0.01) in the AZD6244- treated group (Fig. 5C). Discussion RET gene alterations are oncogenic ‘‘drivers’’ in thyroid cancer pathogenesis, particularly in MTC and PTC. Oncogenic RET is a potent activator of the ERK/MAPK and PI3K pathways and can induce the expression of inflammatory mediators such as CCL2, CXCL-1, GM-CSF, IL-1b and IL-6 [5,25–27]. Moreover, RET/PTC and mutant RET can induce phosphorylation of STAT3 either directly [14,28] or in a JAK-dependent manner [29]. JAKs are tyrosine kinases that mediate IL-6- dependent STAT3 activation, which has been shown to promote cancer progression in numerous examples of solid tumors. Importantly, JAK2 activating mutations are critical in the pathogenesis of myeloproliferative disorders and that has led to the development of JAKs small molecule inhibitors [22,23]. Herein, we investigated the biological effects of a JAK1/2 inhibitor, AZD1480, on the growth of PTC- and MTC- derived thyroid cancer cell lines harboring activating RET/PTC rearrangements and RET mutations, respectively. We observed that AZD1480 inhibited the growth of TPC- PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e46869