High Expression of ErbB Family Members and Their Ligands in Lung Adenocarcinomas That Are Sensitive to Inhibition of Epidermal Growth Factor Receptor Nobukazu Fujimoto, 1 Marie Wislez, 1,4 Jie Zhang, 1 Kentaro Iwanaga, 1 Jennifer Dackor, 5 Amy E. Hanna, 1 Shailaja Kalyankrishna, 1 Dianna D. Cody, 2 Roger E. Price, 2 Mitsuo Sato, 6 Jerry W. Shay, 6 John D. Minna, 6 Michael Peyton, 6 Ximing Tang, 1 Erminia Massarelli, 1 Roy Herbst, 1 David W. Threadgill, 5 Ignacio I. Wistuba, 1,3 and Jonathan M. Kurie 1 Departments of 1 Thoracic/Head and Neck Medical Oncology, 2 Imaging Physics, and 3 Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas; 4 UPRESEA 3493, CHU Saint-Antoine, Universite Paris VI, Paris, France; 5 Department of Genetics, Curriculum in Genetics and Molecular Biology, Lineberger Cancer Center, University of North Carolina, Chapel Hill, North Carolina; and 6 Harold Simmons Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas Abstract Recent findings in tumor biopsies from lung adenocarcinoma patients suggest that somatic mutations in the genes encoding epidermal growth factor receptor (EGFR ) and Kirsten ras (KRAS ) confer sensitivity and resistance, respectively, to EGFR inhibition. Here, we provide evidence that these genetic mutations are not sufficient to modulate the biological response of lung adenocarcinoma cells to EGFR inhibition. We found high expression of ErbB family members, ErbB ligands, or both in three models that were sensitive to EGFR inhibition, including alveolar epithelial neoplastic lesions in mice that develop lung adenocarcinoma by oncogenic KRAS , human lung adenocarcinoma cell lines, and tumor biopsies from lung adenocarcinoma patients. Thus, lung adenocarci- noma cells that depend on EGFR for survival constitutively activate the receptor through a combination of genetic mutations and overexpression of EGFR dimeric partners and their ligands. (Cancer Res 2005; 65(24): 11478-85) Introduction Somatic mutations in epidermal growth factor receptor (EGFR ), the gene encoding the EGFR, have been found in the tumors of 10% to 40% of patients with non–small cell lung cancer (NSCLC; refs. 1–4). These mutations activate the EGFR tyrosine kinase and are associated with adenocarcinoma histology, female gender, and a nonsmoking history. Lung cancer patients with EGFR mutations frequently experience rapid and sustained shrinkage of primary and metastatic disease after treatment with the EGFR tyrosine kinase inhibitors (TKI) gefitinib or erlotinib (1–4). In a phase III trial, treatment with erlotinib conferred a survival benefit (5), but the proportion of patients who experienced this benefit exceeded the expected frequency of EGFR mutations. In addition, a small proportion of patients whose tumors shrank in response to EGFR TKIs had no evidence of EGFR mutations (1–5). Together, these findings suggest that factors other than EGFR mutations confer sensitivity to EGFR inhibition. EGFR forms homodimers and heterodimers with the other ErbB family members ErbB2, ErbB3, and ErbB4 (6). These dimeric complexes have distinct ligand binding and signaling activities (7). For example, ErbB3 is unique in that it lacks a functional kinase domain. Despite this deficiency, ErbB3 undergoes transphosphor- ylation in complex with other ErbBs and activates downstream kinases, such as phosphatidylinositol 3-kinase (PI3K), in response to ligand binding. Recent studies have implicated ErbB3 in the sensitivity of NSCLC cell lines to EGFR inhibition (8, 9). Together, these findings suggest that aberrant expression of ErbB family members contributes to the responsiveness of NSCLC cells to EGFR inhibition. A recent report (10) linked de novo resistance to treatment with EGFR TKIs in lung adenocarcinoma patients with somatic mutations in Kirsten ras (KRAS ). A growing body of evidence indicates that KRAS mutations are important in the development of lung adenocarcinoma. They occur in 30% to 50% of lung adenocarcinomas (11, 12) and are mutually exclusive from mutations in EGFR (13). Mice that express mutant KRAS develop lung adenocarcinoma rapidly and with high penetrance (14–17). In this study, we investigated the role of KRAS mutations in the resistance of lung adenocarcinoma to EGFR TKIs. We examined Kras LA1 mice, which develop lung adenocarcinoma through somatic activation of a KRAS allele carrying an activating mutation in codon 12 (G12D; ref. 17). Alveolar epithelial cells in this mouse model recapitulate the series of morphologic stages through which human atypical alveolar hyperplasia (AAH) evolves into adenocar- cinoma. Our findings suggest that the presence of KRAS mutations is not sufficient to confer resistance to EGFR inhibition and reveal a novel mechanism of response to EGFR inhibition that is potentially relevant to lung cancer patients. Materials and Methods Animal experiments. Animal experiments were compliant with the guidelines of The University of Texas M.D. Anderson Cancer Center. The Kras LA1 mice were provided by Dr. Tyler Jacks (Massachusetts Institute of Technology, Cambridge, MA), and gefitinib was provided by AstraZeneca (Wilmington, DE). Four-month-old Kras LA1 mice were randomly allocated to treatment with vehicle (PBS with 0.05% Tween 80 in PBS by oral gavage daily), low-dose gefitinib (100 mg/kg/d), or high-dose gefitinib (250 mg/kg/d). Treatment was given for 28 days with a 4-day intermission at 14 days to minimize dermatologic toxicity. Mice treated with high-dose Note: N. Fujimoto and M. Wislez contributed equally to this work. M. Wislez is a postdoctoral fellow of La Fondation de France and La Societe de Pneumonologie de Langue Francaise. Supplementary data for this article are available at Cancer Research Online (http:// cancerres.aacrjournals.org/). Requests for reprints: Jonathan M. Kurie, The University of Texas M.D. Anderson Cancer Center, Box 432, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713- 792-6363; Fax: 713-796-8655; E-mail: jkurie@mdanderson.org. I2005 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-1977 Cancer Res 2005; 65: (24). December 15, 2005 11478 www.aacrjournals.org Research Article