Epidermal Growth Factor Receptor and Hedgehog Signaling Pathways Are Active in Esophageal Cancer Cells From Rat Reflux Model 1 Guoping Sui, M.D.,† ,2 Pramod Bonde, M.D.,* ,2 Surajit Dhara, Ph.D.,† Apoorv Broor, M.B.B.S.,* Jiaai Wang, B.S.,* Guy Marti, M.D.,* Georg Feldmann, M.D.,† Mark Duncan, M.D.,* Elizabeth Montgomery, M.D.,† Anirban Maitra, M.B.B.S.,† , ‡ and John W. Harmon, M.D.* ,3 *Departments of Surgery, †Pathology, and ‡Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland Submitted for publication November 10, 2004 Background. Advancements in experimental thera- peutics for esophageal cancers have been hampered by the lack of a reliable preclinical model that recapit- ulates the biology of human cancer, including in vivo growth in an animal model. Methods. Bilious reflux was induced by esophago- jejunostomy in Sprague–Dawley rats. Nine of 12 (75%) Sprague–Dawley rats developed squamous or adeno- squamous cancers, and three cell lines were created by in vitro propagation of freshly resected tumors, JA and JB lines from one cancer, and the AMY cell line from another. We subsequently tested the ability of these cell lines to propagate long-term in vitro and form xeno- grafts in vivo, both hallmarks of transformed cells. In addition, we determined the effects of small molecule inhibitors of two important oncogenic pathways—the epidermal growth factor receptor (EGFR) and Hedge- hog (Hh) signaling pathways, in vitro, as a “proof of principle” of using these unique cell lines for develop- ing targeted therapies for esophageal cancer. Mech- anism-based growth inhibition was assessed by down- regulation of activated downstream targets of EGFR in the case of Iressa, and by Hh luciferase reporter activity with cyclopamine. Results. JA, JB, and AMY cell lines were able to grow continuously in vitro and consistently form xenografts in vivo in athymic mice, both subcutaneously, as well as in the “orthotopic” location at the gastroesophageal serosal junction (n  2 mice per line, six of six en- grafted). By histology, the tumors grow in vivo as well- differentiated keratinizing squamous cell carcino- mas. JB cells had the highest expression of EGFR protein and also the most profound response to Iressa (gefitinib), an EGFR inhibitor (IC 50 < 1 M). Growth inhibition by Iressa was mirrored function- ally by down-regulation of activated targets of the EGFR pathway, phospho-ERK1/2 and phospho-MEK levels. AMY cells expressed 900-fold elevation of the Hh ligand, Indian Hh (Ihh), compared with normal esophageal epithelium, whereas expression of another Hh ligand, Sonic Hh (Shh), was not detected. On treat- ment with the specific Hh small molecule inhibitor cyclopamine, AMY cells demonstrated growth inhibi- tion, which was accompanied by significant down- regulation of endogenous Hh luciferase reporter activ- ity at 24 h and increased apoptosis in treated cells. Conclusions. We have established a model of esoph- ageal carcinogenesis, capable of long-term in vitro and in vivo passage, and demonstrated therapeutic poten- tial of targeting the EGFR and Hh pathways in the cell lines created from the rodent cancers. These unique cell lines should provide a platform for rapid preclin- ical validation of novel therapeutics for esophageal cancers. © 2006 Elsevier Inc. All rights reserved. Key Words: esophagus; GERD; EGFR; Hh; cyclopam- ine; Iressa; adenocarcinoma. INTRODUCTION There is a significant increase in the incidence of esophageal adenocarcinoma during the last three de- cades [1]. There is increasing evidence suggesting a link between gastroesophageal reflux disease (GERD) 1 Pramod Bonde is supported by the RVH Research Fellowship, Dowager Countess Eleanor Peel Foundation Fellowship, RRG fund- ing from Queens University and St Jude Scholarship of Society of Cardiothoracic Surgeons of Great Britain and Ireland. Guoping Sui is supported by the Collaborative Genetics Research Training Pro- gram (5D43TW006176-03). Anirban Maitra is supported by NCI R01CA113669. 2 Both authors contributed equally to this work. 3 To whom correspondence and reprint requests should be addressed at Johns Hopkins Bayview Medical Center, Room 5C, Bldg A, 4940 Eastern Avenue, Baltimore, MD. E-mail: jharmon@jhmi.edu. Journal of Surgical Research 134, 1–9 (2006) doi:10.1016/j.jss.2005.12.029 1 0022-4804/06 $32.00 © 2006 Elsevier Inc. All rights reserved.