Reduced expression of RAS protein activator like-1 in gastric cancer Motoko Seto 1 , Miki Ohta 1 , Tsuneo Ikenoue 1 , Takafumi Sugimoto 1 , Yoshinari Asaoka 1 , Motohisa Tada 2 , Dai Mohri 1 , Yotaro Kudo 1 , Hideaki Ijichi 1 , Keisuke Tateishi 1 , Motoyuki Otsuka 1 , Yoshihiro Hirata 1 , Shin Maeda 1 , Kazuhiko Koike 1 and Masao Omata 1 1 Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan 2 Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan RAS signaling is frequently deregulated in human neoplasms. However, RAS mutations have been found in only a small proportion of human gastric cancers, implicating other mechanisms in the activation of RAS signaling in gastric tumorigenesis. We have previously reported that decreased expression of RAS protein activator like-1 (RASAL1), a member of the RAS-GTPase-activating proteins that switch off RAS activity, contributes to colon tumor progression. In our study, we explored the involvement of decreased RASAL1 expression in gastric tumorigenesis. RASAL1 expression was reduced in 6 of 10 gastric cancer cell lines examined by immunoblotting. Knockdown of RASAL1 increased mitogen-activated protein kinase signaling in response to growth factor stimulation, and the forced expression of RASAL1 reduced proliferation of gastric cancer cells. Immunohistochemical analyses in primary gastric tumors showed that RASAL1 expression was reduced in 23 of 48 (48%) of the gastric cancers but in none of the adenomas (0/10). Methylation of the RASAL1 promoter region and loss of heterozygosity (LOH) at the RASAL1 locus were examined to investigate the causes of RASAL1 silencing. All cell lines with reduced RASAL1 had RASAL1 methylation, and two had LOH. In primary gastric cancers, methylation or LOH was detected in 50% (6/12) of those with reduced RASAL1. Furthermore, RASAL1 expression was restored in some cell lines by histone deacetylase inhibitor treatment. Our findings demonstrate that reduced RASAL1 expression, partly due to genetic and epigenetic changes, contributes to gastric carcinogenesis, and also re-emphasize the importance of RAS signaling in gastric cancer development. It is well known that RAS signaling is deregulated in various human tumors. RAS regulates a signal transduction pathway linking plasma membrane receptors to many essential and rate-limiting signals for growth and differentiation. 1 Like other small GTPases, RAS switches between two conforma- tions, an inactive state (GDP bound) and an active state (GTP bound). The rate of switching is controlled by two classes of proteins. Guanine nucleotide exchange factors cata- lyze the release of GDP, allowing GTP to bind, whereas RAS- GTPase-activating proteins (RASGAPs) enhance the intrinsic RAS-GTPase activity, leading to inactivation through the conversion of GTP into GDP. 2 Several effectors have been implicated in RAS-mediated growth transformation and oncogenesis; these include the RAF/mitogen-activated protein kinase, PI3K/AKT, PLC/PKC and GTPase cascades. 3 RAS has been widely established as an important onco- gene aberrantly activated in many human tumors. 4 Approxi- mately 30% of all human tumors have an activating mutation in a RAS gene. In particular, KRAS mutations are among the most common genetic abnormalities in several types of human neoplasms, including pancreatic cancer, 5,6 colon can- cer 7 and lung cancer, 8 although the wide-ranging frequency of RAS mutations depends on the tumor type. In addition to these oncogenic mutations, signaling through wild-type RAS is also frequently deregulated in tumors, via aberrant cou- pling to activated cell surface receptors. Furthermore, evi- dence is emerging that deregulation of RASGAP may provide an additional mechanism. 9 RAS mutations have been found in only a small proportion of human gastric cancers, 10 Key words: gastric tumor, RAS-GTPase-activating protein, RASAL1 Abbreviations:: 5-aza: 5-aza-2 0 -deoxysytidine; EGF: epidermal growth factor; ERK: extracellular signal-regulated kinase; LOH: loss of heterozygosity; MSP: methylation-specific PCR; NF1: neurofibromin 1; p-ERK1/2: phosphorylated extracellular signal- regulated kinase; PCR: polymerase chain reaction; qRT-PCR: quantitative real-time polymerase chain reaction; RASGAP: RAS- GTPase-activating protein; RASAL1: RAS protein activator like-1; shRNA: short hairpin RNA; TSA: trichostatin A Grant sponsors: Sankyo Foundation of Life Science, Foundation for Advancement of International Science DOI: 10.1002/ijc.25459 History: Received 4 Dec 2009; Accepted 8 Apr 2010; Online 13 May 2010 Correspondence to: Motoko Seto, Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan, Tel: 81-3815-ext. 33070), Fax: 81-3-3814-0021, E-mail: seto@kanto-ctr-hsp.com (or) Tsuneo Ikenoue, Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan, Tel.: 81-3-3815-5411 (ext. 33070), Fax: 81-3-3814-0021, E-mail: ikenoue-2im@h.u-tokyo.ac.jp Cancer Cell Biology Int. J. Cancer: 128, 1293–1302 (2011) V C 2010 UICC International Journal of Cancer IJC