SNO Is a Probable Target for Gene Amplification at 3q26 in Squamous-Cell Carcinomas of the Esophagus Issei Imoto,* Atiphan Pimkhaokham,* Yoji Fukuda,* Zeng-Quan Yang,* Yutaka Shimada,† Nobuo Nomura,‡ Hisamaru Hirai,§ Masayuki Imamura,† and Johji Inazawa* ,1 *Department of Molecular Cytogenetics, Division of Genetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan; †Department of Surgery, Surgically Basic Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan; ‡Facility for RI Experiments, Kazusa DNA Research Institute, Kisarazu, Japan; and §Department of Hematology and Oncology, Faculty of Medicine, University of Tokyo, Tokyo, Japan Received July 13, 2001 Amplification of the 3q26 region appears to occur frequently among esophageal squamous cell carcino- mas (ESCs). We examined ESC cell lines for amplifica- tion and expression levels of four genes in this region: SNO and EVI1, which encode proteins antagonizing transforming growth factor- signaling, and two other putative target genes, TERC and PIK3CA. Amplifica- tion of SNO was accompanied by significant increases in its expression level, suggesting that this gene is activated in an amplification-dependent manner. SNO was also amplified in 5 of 44 primary ESCs (11.4%). However, expression levels of EVI1, TERC, and PIK3CA did not correlate with their copy-numbers, even though EVI1 and TERC showed the same ampli- fication pattern as SNO. Taken together, the data sug- gest that of the four candidates, SNO is the most prob- able target in the 3q26 amplicon for involvement in the progression of ESC. © 2001 Academic Press Key Words: amplification; 3q26; SNO; EVI1; PIK3CA; TERC; esophageal squamous cell carcinoma. Gene amplification is a frequent event in numerous types of human cancer, and typically results in overex- pression of specific target genes that then confer a selective advantage upon cancer cells (1). In esopha- geal squamous cell carcinomas (ESCs), several onco- genes, e.g. EGFR, MYC, and CCND1, are known to be activated by gene amplification mechanism (2, 3). Re- cently, additional chromosomal amplicons have been identified in ESCs by comparative genomic hybridiza- tion (CGH) analysis (4 – 6). However, the specific gene targets within these amplicons remain largely un- known. In order to provide novel and important in- sights into the pathogenesis of ESC and design the best protocols for its clinical management, it is necessary to explore and characterize candidate genes present in the amplified regions. One region that is often amplified in ESCs lies within 3q. Earlier we identified the minimal overlap- ping region of amplification at 3q26 in primary tumors and cell lines by CGH analysis (4, 6). Amplification around 3q26 also has been found consistently in tu- mors of the cervix (7, 8), ovary (8, 9), lung (8, 10, 11), head and neck (12), and prostate (13). Although CGH analysis cannot always define the target region nar- rowly, 3q26 seems to lie in the center of the amplified regions in most cases, suggesting that this region may harbor gene(s) activated by the amplification mecha- nism and involved in the carcinogenesis of tumors in question. Several groups of investigators focused on this chromosomal region have adopted a positional candidate-gene approach to search for target genes in different types of cancers (10, 11, 13, 14). Although recent studies have provided convincing evidence that two genes located in the 3q26, TERC and PIK3CA, show oncogenic activity in some tumors (14 –16), nei- ther of those genes has ever been proven to be the target in ESCs. EVI1 and SNO, which negatively regulate the TGF- signaling pathway (17, 18), have been mapped at 3q26. TGF- is a potent inhibitor of proliferation and induces apoptosis in epithelial cells; indeed, disruption of TGF- signaling is observed frequently in various types of human cancers, including ESC (19). Although mutations of SMAD4 and TGF- receptor type II (TG- FBR2) are generally the most common alterations found among TGF- signaling components in cancer cells (20, 21), they are rare in ESC (22–24). Therefore, different components may be involved in disruption of the TGF- signaling pathway in esophageal tumors. 1 To whom correspondence should be addressed at Department of Molecular Cytogenetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Ja- pan. Fax: +81-3-5803-0244. E-mail: johinaz.cgen@mri.tmd.ac.jp. Biochemical and Biophysical Research Communications 286, 559 –565 (2001) doi:10.1006/bbrc.2001.5428, available online at http://www.idealibrary.com on 559 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.