Irreversible EGFR Inhibitor EKB-569 Targets Low-LET c- Radiation-Triggered Rel Orchestration and Potentiates Cell Death in Squamous Cell Carcinoma 4 1 Natarajan Aravindan2,4,5, Charles R. Thomas Jr3. , Sheeja Aravindan , Aswathi S. Mohan , Jamunarani Veeraraghavan2,5, Mohan Natarajan 1 * 1 Department of Otolaryngology, Head and Neck Surgery, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America, 2 Department of Radiation Oncology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America, 3 Department of Radiation Medicine, Oregon Health and Science University Knight Cancer Institute, Portland, Oregon, United States of America, 4 Department of Pathology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America, 5 Department of Pediatrics, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America Abstract EKB-569 (Pelitinib), an irreversible EGFR tyrosine kinase inhibitor has shown potential therapeutic efficiency in solid tumors. However, cell-killing potential in combination with radiotherapy and its underlying molecular orchestration remain to be explored. The objective of this study was to determine the effect of EKB-569 on ionizing radiation (IR)- associated NFkB-dependent cell death. SCC-4 and SCC-9 cells exposed to IR (2Gy) with and without EKB-569 treatment were analyzed for transactivation of 88 NFkB pathway molecules, NFkB DNA-binding activity, translation of the NFkB downstream mediators, Birc1, 2 and 5, cell viability, metabolic activity and apoptosis. Selective targeting of IR-induced NFkB by EKB-569 and its influence on cell-fate were assessed by overexpressing (p50/p65) and silencing (DIkBa) NFkB. QPCR profiling after IR exposure revealed a significant induction of 74 NFkB signal transduction molecules. Of those, 72 were suppressed with EKB-569. EMSA revealed a dose dependent inhibition of NFkB by EKB-569. More importantly, EKB- 569 inhibited IR-induced NFkB in a dose-dependent manner, and this inhibition was sustained up to at least 72 h. Immunoblotting revealed a significant suppression of IR-induced Birc1, 2 and 5 by EKB-569. We observed a dose- dependent inhibition of cell viability, metabolic activity and apoptosis with EKB-569. EKB-569 significantly enhanced IR- induced cell death and apoptosis. Blocking NFkB improved IR-induced cell death. Conversely, NFkB overexpression negates EKB-569 -induced cell-killing. Together, these pre-clinical data suggest that EKB-569 is a radiosensitizer of squamous cell carcinoma and may mechanistically involve selective targeting of IR-induced NFkB-dependent survival signaling. Further pre-clinical in-vivo studies are warranted. Citation: Aravindan N, Thomas CR Jr, Aravindan S, Mohan AS, Veeraraghavan J, et al. (2011) Irreversible EGFR Inhibitor EKB-569 Targets Low-LET c-Radiation- Triggered Rel Orchestration and Potentiates Cell Death in Squamous Cell Carcinoma. PLoS ONE 6(12): e29705. doi:10.1371/journal.pone.0029705 Editor: Christina Lynn Addison, Ottawa Hospital Research Institute, Canada Received August 8, 2011; Accepted December 1, 2011; Published December 29, 2011 Copyright: 2011 Aravindan 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, in whole or in part, by National Institutes of Health Grant R01 CA112175 (to M.N.) and funds from the Office of Science (Biological and Environmental Research), United States Department of Energy Grant No. DE-FG03-02ER63449 (to M.N.). 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: natarajan@uthscsa.edu Introduction Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world and accounts for 90% of malignant neoplasias of the upper respiratory system [1]. Despite recent advances in the management of locally advanced HNSCC, the overall survival of patients has improved only marginally over the past three decades [2] mainly due to development of therapy- induced chemo and radioresistance. To that note, in recent years there has been substantial interest in developing novel therapeutic agents that specifically target growth factor pathways that, are dysregulated in tumor cells. Such targeted ‘‘biological’’ agents might offer alternative treatment options for patients refractive to chemoradiotherapy. Also, with unique mechanisms of action and toxic profiles that generally do not overlap, targeted agents and standard therapies can be used in combinations to enhance overall treatment efficacies and prevent dose reduction. Because many solid tumors, including HNSCC have hyper activated epidermal growth factor receptor (EGFR) [3,4], there has been great interest in the use of EGFR inhibitors to control cancer growth. EGFR is a 170 kDa glycoprotein containing an extracellular ligand binding domain, and an intracellular tyrosine kinase (TK) domain [5]. Upon binding to ligands such as EGF or TGFa, EGFR dimerizes with itself (homodimers) or other members of the family such as c-ErbB-2 (heterodimers). Upon dimerization, TK activation increases and receptor gets autopho- sphorylated at tyrosine residues. Phosphorylated EGFR (p-EGFR), like other activated receptor TKs, involved in phosphorylation and activation of several signal transduction pathways including phosphoinositide 3-kinase-AKT, extra cellular signal-regulated kinase 1and 2 (ERK1/2), and the signal transducer and activator of transcription 3 (STAT3). Activation of these signal transduction pathways subsequently activate key transcriptional machineries such as NFkB that promote tumor growth and progression by PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e29705