Utility of a PI3K/mTOR Inhibitor (NVP-BEZ235) for Thyroid Cancer Therapy Shu-Fu Lin1*, Yu-Yao Huang1, Jen-Der Lin1, Ting-Chao Chou 3 , Chuen Hsueh2, Richard J. Wong 4 * 1 Department of Internal Medicine, Chang Gung Memorial Hospital, Chang Gung University, Taoyuan, Taiwan, Republic of China, 2 Department of Pathology, Chang Gung Memorial Hospital, Chang Gung University, Taoyuan, Taiwan, Republic of China, 3 Laboratory of Preclinical Pharmacology Core, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America, 4 Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America Abstract Background: We assessed the utility of the dual PI3K/mTOR inhibitor NVP-BEZ235 (BEZ235) as single agent therapy and in combination with conventional chemotherapy for thyroid cancer. Methodology/Principal Findings: Eight cell lines from four types of thyroid cancer (papillary, follicular, anaplastic, medullary) were studied. The cytotoxicity of BEZ235 and five conventional chemotherapeutic agents alone and in combination was measured using LDH assay. Quantitative western blot assessed expression of proteins associated with cell cycle, apoptosis and signaling pathways. Cell cycle distribution and apoptosis were measured by flow cytometry. Murine flank anaplastic thyroid cancers (ATC) were treated with oral BEZ235 daily. We found that BEZ235 effectively inhibited cell proliferation of all cancer lines, with ATC exhibiting the greatest sensitivity. BEZ235 consistently inactivated signaling downstream of mTORC1. BEZ235 generally induced cell cycle arrest at G0/G1 phase, and also caused apoptosis in the most sensitive cell lines. Baseline levels of p-S6 ribosomal protein (Ser235/236) and p27 correlated with BEZ235 sensitivity. Growth of 8505C ATC xenograft tumors was inhibited with BEZ235, without any observed toxicity. Combination therapy of BEZ235 and paclitaxel consistently demonstrated synergistic effects against ATC in vitro. Conclusions: BEZ235 as a single therapeutic agent inhibits thyroid cancer proliferation and has synergistic effects in combination with paclitaxel in treating ATC. These findings encourage future clinical trials using BEZ235 for patients with this fatal disease. Citation: Lin S-F, Huang Y-Y, Lin J-D, Chou T-C, Hsueh C, et al. (2012) Utility of a PI3K/mTOR Inhibitor (NVP-BEZ235) for Thyroid Cancer Therapy. PLoS ONE 7(10): e46726. doi:10.1371/journal.pone.0046726 Editor: Guillermo Velasco, Complutense University, Spain Received April 21, 2012; Accepted September 6, 2012; Published October 15, 2012 Copyright: 2012 Lin 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 work was supported by National Science Council of Taiwan, Republic of China (NSC 98-2314-B-182A-012 -MY3) (http://web1.nsc.gov.tw/) and partly by grants from Chang Gung Memorial Hospital and Chang Gung University (CMRPG370581, CMRPG370582, CMRPG370583, CMRPG3A0161) (http://www. cgmh.org.tw/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: wongr@mskcc.org (RJW); mmg@cgmh.org.tw (SL) Introduction Thyroid cancer is the most common endocrine malignancy, originating from thyroid follicular cells (papillary, follicular, poorly differentiated and anaplastic cancer) or parafollicular C cells (medullary cancer). The incidence of thyroid cancer has increased over the past 3 decades, primarily from an increase in the detection of papillary cancer. In contrast, the incidence of follicular and poorly differentiated thyroid cancer remains unchanged [1,2]. Most patients with well-differentiated cancer, including papillary (PTC) and follicular (FTC) thyroid cancer have a favorable prognosis. Nevertheless, about 5% patients develop radioactive iodine refractory tumors and usually cause death within 5 years [3]. Anaplastic thyroid cancer (ATC) is a rare, highly aggressive, and often fatal disease, with a median survival of just 6 months. Medullary thyroid cancer (MTC) accounts for 3– 5% of thyroid malignancy. The frequency of regional and distant metastatic disease in MTC diminishes survival rates [4,5]. Novel therapies for refractory and aggressive thyroid cancer are needed to improve currently poor outcomes for these patients. The PI3K/mTOR pathway is important for cell metabolism, survival and proliferation. Class IA PI3Ks are heterodimers containing a p85 regulatory and a p110 catalytic subunits which phosphorylate phosphatidylinositol-4,5-biphosphate (PIP2), yield- ing phosphatidylinositol-3,4,5-triphosphate (PIP3). PIP3 combines with phosphoinositide-dependent protein kinase 1 (PDK1) to phosphorylate AKT at Thr308. In addition, the phosphorylation of AKT at Ser 473 by mTORC2 is required for full activity of AKT. Activation of AKT phosphorylates mTORC1, which subsequently phosphorylates S6 kinase1 and 4E-BP1, leading to G1/S cell cycle progression and inhibition of apoptosis. PTEN is a tumor suppressor that dephosphorylates PIP3 and inactivates this pathway [6,7]. Alterations of this signaling pathway frequently occur in malignancies and are potential targets for cancer therapy [6–8]. In thyroid cancer, genetic alterations affecting the PI3K/ mTOR pathway have been identified. PIK3CA (encoding p110a of class IA PI3K) copy number gain correlates with increased PIK3CA protein expression. PIK3CA copy number gain occurs more frequently than genetic mutations of PIK3CA or PTEN in thyroid cancer [9–14]. More PIK3CA/AKT1 mutations and PIK3CA copy PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e46726