Gastric cancer cell line Hs746T harbors a splice site mutation of c-Met causing juxtamembrane domain deletion Yoshinari Asaoka a, * , Motohisa Tada b , Tsuneo Ikenoue a , Motoko Seto a , Mitsuho Imai a , Koji Miyabayashi a , Keisuke Yamamoto a , Shinzo Yamamoto a , Yotaro Kudo a , Dai Mohri a , Yoshihiro Isomura a , Hideaki Ijichi a , Keisuke Tateishi a , Fumihiko Kanai b , Seishi Ogawa c , Masao Omata a , Kazuhiko Koike a a Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan b Departments of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan c Cancer Genomics Project, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan article info Article history: Received 17 March 2010 Available online 21 March 2010 Keywords: Receptor tyrosine kinase c-Met Gastric cancer Gene amplification Splice site mutation abstract Receptor tyrosine kinases (RTKs) are involved in oncogenesis and disease progression for many cancers. Inhibitors targeting them are vigorously developed and some of them are tested in the clinical setting. Amplifications of certain RTKs (c-Met, FGFR2 and ErbB2) have been associated with human gastric cancer progression. According to our genome-wide scans of genetic lesions in 34 gastric cancer cell lines using high-density single-nucleotide polymorphism genotyping microarrays, we confirmed that the c-met locus was amplified in four gastric cancer cell lines (Hs746T, MKN45, NUGC4 and SNU5). It was reported that somatic mutation is occasionally detected in tumor samples of a certain type of cancer with gene amplification. Previous reports showed gastric cancers harbored mutations of FGFR2 and ErbB2, but c-Met oncogenic mutation had not yet been reported. We performed mutational analysis of the cytoplas- mic domains of c-Met using the genome DNA of the gastric cancer cell lines, and found that Hs746T cells had a splice site mutation of exon 14. By cDNA sequencing and Western blotting, we showed that the mutation caused juxtamembrane domain deletion. Previously, this mutation had been detected only in lung cancer specimens and this deletion resulted in the loss of Cbl E3-ligase binding causing decreased ubiquitination and delayed down-regulation. In conclusion, four gastric cancer cell lines harbored ampli- fication of c-met locus, and among them, Hs746T had a putative oncogenic mutation with amplification. This information will be useful for screening of inhibitors targeting gastric cancer with c-Met aberration. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction Receptor tyrosine kinases (RTKs) are key molecules in signal- ing pathways leading to growth and differentiation of normal cells [1]. Aberrant expression of RTKs as reflected by the aber- rant tyrosine phosphorylation in gastric cancer cells was re- ported [2]. Three RTKs, c-Met, FGFR2 and ErbB2, were most extensively studied in gastric cancers, as they were frequently involved in this type of cancer. While c-Met is overexpressed/ amplified in advanced gastric cancers regardless of tumor differ- entiation, FGFR2 activation is more specifically associated with poorly differentiated variants. By contrast, amplification of the ErbB2 gene is often detected in well-differentiated gastric carci- nomas [3]. Thus, subversion of different signal pathways may contribute to the progression of different types of gastric can- cers. Inhibitors targeting RTKs are now coming on the clinical setting against several cancer types. One of the most famous RTK inhibitors is EGFR inhibitor, gefitinib, which was revealed to dramatically effective to lung cancer with EGFR constitutively active mutation [4,5]. On the other hand, monoclonal antibody against EGFR cetuximab is applicable to the treatment of colo- rectal cancers, which do not necessarily harbor mutation of the gene [6]. In the case of gastric cancers, trastuzumab, monoclonal antibody against ErbB2, being used for the treatment of breast cancers with ErbB2 amplification [7,8], is getting around to apply for gastric cancer and has recently been proved to have clinical benefit [9]. Inhibitors of c-Met and FGFR2 also become available for clinical trials [10,11]. For screening of these novel drugs, the information of cell lines about gene alteration, especially ampli- fication and mutation of RTKs, is useful. We have performed genome-wide scans of genetic lesions in 34 gastric cancer cell lines using high-density single-nucleotide polymorphism geno- typing microarrays and reported several novel and minute geno- mic alterations [12]. In this paper, we also used these data to investigate which cell lines had gene amplification of c-met lo- cus. In addition, we performed mutational analysis of c-Met to investigate whether they harbored oncogenic mutation. 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.03.120 * Corresponding author. Fax: +81 3 3814 0021. E-mail address: yasa-tky@umin.ac.jp (Y. Asaoka). Biochemical and Biophysical Research Communications 394 (2010) 1042–1046 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc