AZD1480 Blocks Growth and Tumorigenesis of RET- Activated Thyroid Cancer Cell Lines Joana P. Couto1,2, Ana Almeida1,2,3, Laura Daly4, Manuel Sobrinho-Simo˜ es1,2,5, Jacqueline F. Bromberg4,6*, Paula Soares1,2* 1 Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Cancer Biology, Porto, Portugal, 2 Faculty of Medicine of the University of Porto, Porto, Portugal, 3 Institute of Biomedical Sciences Abel Salazar of the University of Porto, Porto, Portugal, 4 Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America, 5 Department of Pathology, Hospital Sa˜o Joa˜o, Porto, Portugal, 6 Department of Medicine, Weill Cornell Medical College, New York, New York, United States of America Abstract Persistent RET activation is a frequent event in papillary thyroid carcinoma (PTC) and medullary thyroid carcinoma (MTC). In these cancers, RET activates the ERK/MAPK, the PI3K/AKT/mTOR and the JAK/STAT3 pathways. Here, we tested the efficacy of a JAK1/2- inhibitor, AZD1480, in the in vitro and in vivo growth of thyroid cancer cell lines expressing oncogenic RET. Thyroid cancer cell lines harboring RET/PTC1 (TPC-1), RET M918T (MZ-CRC1) and RET C634W (TT) alterations, as well as TPC-1 xenografts, were treated with JAK inhibitor, AZD1480. This inhibitor led to growth inhibition and/or apoptosis of the thyroid cancer cell lines in vitro, as well as to tumor regression of TPC-1 xenografts, where it efficiently blocked STAT3 activation in tumor and stromal cells. This inhibition was associated with decreased proliferation, decreased blood vessel density, coupled with increased necrosis. However, AZD1480 repressed the growth of STAT3- deficient TPC-1 cells in vitro and in vivo, demonstrating that its effects in this cell line were independent of STAT3 in the tumor cells. In all cell lines, the JAK inhibitor reduced phospho-Y1062 RET levels, and mTOR effector phospho-S6, while JAK1/2 downregulation by siRNA did not affect cell growth nor RET and S6 activation. In conclusion, AZD1480 effectively blocks proliferation and tumor growth of activated RET- thyroid cancer cell lines, likely through direct RET inhibition in cancer cells as well as by modulation of the microenvironment (e.g. via JAK/phospho-STAT3 inhibition in endothelial cells). Thus, AZD1480 should be considered as a therapeutic agent for the treatment of RET- activated thyroid cancers. Citation: Couto JP, Almeida A, Daly L, Sobrinho-Simo˜ es M, Bromberg JF, et al. (2012) AZD1480 Blocks Growth and Tumorigenesis of RET- Activated Thyroid Cancer Cell Lines. PLOS ONE 7(10): e46869. doi:10.1371/journal.pone.0046869 Editor: Alfredo Fusco, Consiglio Nazionale delle Ricerche (CNR), Italy Received June 18, 2012; Accepted September 6, 2012; Published October 2, 2012 Copyright: 2012 Couto et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by the Portuguese Foundation for Science and Technology (FCT). Joana P. Couto is supported by an FCT grant SFRH/BD/ 40260/2007. Ana Almeida is supported by an FCT grant SFRH/BD/79135/2011. J. Bromberg is supported by R01-CA87637-06, U54:CA148967, Marjorie and Charles Holloway Foundation, Lerner Foundation, Sussman Family Fund and Astra Zeneca. IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education that is partially supported by the FCT. The funders had no role in study design, data collection and analysis, decision to publish, or manuscript preparation. Competing Interests: Jacqueline Bromberg has received research funding from AstraZeneca. She has had an advisory role at AstraZeneca and Mediummune and received honoria for lectures. There are no patents or products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: psoares@ipatimup.pt (PS); bromberj@mskcc.org (JFB) Introduction The REarranged during Transfection (RET) proto-oncogene encodes for one of the first receptor tyrosine kinases (RTKs) that were found to be involved in cancer [1]. RET ligands belong to the glial cell- derived neurotrophic (GDNF) family and, upon engagement with RET, induce autophosphorylation of intracellu- lar tyrosine residues, to which several adaptors bind [2]. These adaptors mediate the activation of multiple pathways, including the mitogen activated protein kinase (MAPK) signaling pathway, the phosphatidylinositol 3-kinase (PI3K) pathway, the c-jun N- terminal Kinase (JNK) pathway, the p38 pathway, SRC, ERK5, PLC-c and Signal Transducer and Activator of Transcription (STAT)3 [3]. The first oncogenic role of RET was described in the most common endocrine cancer, papillary thyroid carcinoma (PTC) [4], as the result of genomic rearrangements leading to its constitutive activation and to increased cell survival, proliferation and motility [5]. RET/PTC rearrangements are the second most common genetic alteration in PTC, found in 30% of the cases [6]. RET point mutations were also found in medullary thyroid carcinoma (MTC) [7,8], accounting for nearly all hereditary cases and about 50% of sporadic MTC [9]. Oncogenic RET has been implicated in mediating tumor- associated inflammation, as mutant forms of RET induced the expression of IL-8 [10] and other inflammatory molecules [11]. Furthermore, RET/PTC upregulated a set of inflammation- related genes in thyrocytes [12,13] many of which belong to the IL-6/JAK/ STAT3 pathway [14]. IL-6/JAK/STAT3 signaling is triggered by IL-6 coupling to its receptor-complex, comprising a receptor for IL- 6 (IL-6R) and the signal transducing component, gp130 [15]. Subsequent phosphorylation of receptor-associated JAKs mediates tyrosine phosphorylation of STATs, particularly STAT3. Addition- ally, IL-6 activates the ERK/MAPK and PI3K/AKT pathways [16]. Deregulated JAK/STAT signaling (hyperactivation) has been described in a variety of diseases, including cancer [17–20]. Selective JAK1/2 small-molecule inhibitors that have been PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e46869