JOURNAL OF CELLULAR PHYSIOLOGY 210:549–559 (2007) Combinatorial Treatment of Non-Small-Cell Lung Cancers With Gefitinib and Ad.mda-7 Enhances Apoptosis-Induction and Reverses Resistance to a Single Therapy LUNI EMDAD, 1,2 IRINA V. LEBEDEVA, 1 ZAO-ZHONG SU, 1 PANKAJ GUPTA, 1 DEVANAND SARKAR, 1 JEFFREY SETTLEMAN, 3 AND PAUL B. FISHER 1,2,4 * 1 Department of Urology, Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, College of Physicians and Surgeons, New York, New York 2 Department of Neurosurgery, Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, College of Physicians and Surgeons, New York, New York 3 Department of Medicine, Harvard Medical School, MGH Cancer Center, Charlestown, Massachusetts 4 Department of Pathology, Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, College of Physicians and Surgeons, New York, New York Activation of the epidermal growth factor receptor (EGFR) contributes to the pathogenesis of non-small-cell lung carcinomas (NSCLC) and gefitinib, a selective reversible EGFR inhibitor, is effective in treating patients with NSCLC. However, clinical resistance to gefitinib is a frequent occurrence highlighting the need for improved therapeutic strategies. Melanoma differentiation associated gene-7 (mda-7)/Interleukin-24 (IL-24) (mda-7/IL-24) displays cancer-selective apoptosis induction when delivered via a replication-incompetent adenovirus (Ad.mda-7). In this study, the effect of Ad.mda-7 infection, either alone or in combination with gefitinib, was analyzed in a panel of NSCLC cell lines carrying wild-type EGFR (H-460 and H-2030) or mutant EGFR (H-1650 and H-1975). While H-2030 and H-1650 cells were sensitive, H-460 and H-1975 cells were resistance to growth inhibition by Ad.mda-7, which was reversed by the combination of Ad.mda-7 and gefitinib. This combination increased MDA-7/IL-24 and downstream effector double-stranded RNA-activated protein kinase (PKR) protein expression, promoting apoptosis induction of NSCLC cells. Inhibition of PKR significantly inhibited apoptosis induction by Ad.mda-7 when administered alone but not when used in combination with gefitinib. The combination treatment also augmented inhibition of EGFR signaling. Our findings indicate that a combinatorial treatment with Ad.mda-7 and gefitinib may provide benefit in the treatment of NSCLC, especially in patients displaying resistance to clinically used EGFR inhibitors. J. Cell. Physiol. 210: 549 – 559, 2007. ß 2006 Wiley-Liss, Inc. Lung cancer is the leading cause of cancer-related mortality in both men and women (Jemal et al., 2003) with 1.2 million new cases diagnosed every year and 1 million deaths recorded worldwide in 2001 (Parkin et al., 2001). In 2002 (the most recent year for which statistics are currently available), lung cancer accounted for more deaths than breast cancer, prostate cancer, and colon cancer combined (Group, USCSW, 2005). Non- small-cell lung carcinomas (NSCLC) account for 80% of all lung cancers. The majority of NSCLC patients present with advanced disease at diagnosis and a large portion of those diagnosed with early stage disease eventually recur with metastasis. Although chemother- apy has recently produced promising results as neoadju- vant and adjuvant strategies for early-stage patients (Depierre et al., 2002), and some progress has been made in the treatment of locally progressive and advanced disease (Hotta et al., 2004), treatment outcomes for NSCLC patients are still considered unsatisfactory. In this context, laboratory and clinical research to define new treatment strategies is warranted. The epidermal growth factor-receptor (EGFR) signal- ing pathway contributes to a number of processes important in cancer development and progression such as proliferation, apoptosis, angiogenesis, and metastatic tumor spread (Aaronson, 1991). Accordingly, targeting the EGFR represents an appealing molecular target- based approach to promote selective cancer therapy. Currently, several strategies are being investigated to target the EGFR, including monoclonal antibodies (MAbs) that prevent ligand binding and small molecules that specifically inhibit EGFR tyrosine kinase activity and subsequently downstream intracellular signaling (Mendelsohn, 1997). Among the most promising series of small molecule selective EGFR tyrosine kinase inhibi- tors are the 4-anilinoquinazolines, 4-[ar(alk)ylamino] pyridopyrimidines and 4-phenylaminopyrrolo-pyrimi- dines (Ciardiello and Tortora, 2001). One of these drugs ß 2006 WILEY-LISS, INC. Contract grant sponsor: NIH; Contract grant numbers: R01 CA097318, R01 CA35675; Contract grant sponsor: Samuel Waxman Cancer Research Foundation; Contract grant sponsor: Chernow Endowment. *Correspondence to: Paul B. Fisher, Departments of Pathology and Urology, Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, College of Physicians and Surgeons, New York, NY 10032. E-mail: pbf1@columbia.edu Received 23 August 2006; Accepted 7 September 2006 DOI: 10.1002/jcp.20906