IJC International Journal of Cancer Glutamate receptor, ionotropic, kainate 2 silencing by DNA hypermethylation possesses tumor suppressor function in gastric cancer Chi-Sheng Wu1, Yen-Jung Lu2, Hsin-Pai Li1,3, Chuen Hsueh4, Chang-Yi Lu2, Yu-Wei Leu5, Hao-Ping Liu3, Kwang-Huei Lin1,3, Tim Hui-Ming Huang6 and Yu-Sun Chang1,3 1 Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan 2 Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan 3 Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan 4 Department of Pathology, Chang Gung Memorial Hospital, Lin-Kou, Taoyuan, Taiwan 5 Department of Life Science, Institute of Molecular Biology, National Chung-Cheng University, Chia-Yi, Taiwan 6 Department of Molecular Virology, Immunology & Medical Genetics-Human Cancer Genetics, Ohio State University, OH Aberrant DNA methylation is considered a major mechanism for silencing tumor suppressor genes in gastric cancer. We used CpG microarray and differential methylation hybridization strategies to identify potential tumor suppressor genes and recovered glutamate receptor, ionotropic, kainate 2 (GRIK2) as a novel epigenetic target in gastric cancer. Additional experiments showed that the promoter region of GRIK2 was hypermethylated in 3 of the 4 tested gastric cancer cell lines, and its expression was restored by treatment of cells with the DNA methylation inhibitor, 50-aza-dC. In clinical samples, the GRIK2 promoter was differentially hypermethylated in tumor tissues compared with adjacent normal tissues (p < 0.001), and this methylation was inversely correlated with the expression level of GRIK2 mRNA (r 520.44). Functional studies further showed that GRIK2-expressing gastric cancer cell lines showed decreased colony formation and cell migration. Taken together, these results suggest that GRIK2 may play a tumor-suppressor role in gastric cancer. Future studies are warranted to examine whether DNA hypermethylation of the GRIK2 promoter can be used as a potential tumor marker for gastric cancer. Cancer Cell Biology Gastric cancer is one of the most common human cancers with chemotherapy is a common treatment for gastric cancer. worldwide and is a major upper gastrointestinal tract malig- Factors such as diet, tobacco use and Helicobacter pylori nant disease with poor prognosis for patients suffering from infection have been reported as the major risk factors for gas- more advanced stages of the disease.1 In the 2009 Taiwan tric carcinoma.2 Cancer Registry Report, this cancer was ranked as having the Numerous studies have sought to identify biomarkers ca- seventh highest incidence among cancers. Surgery combined pable of improving the prognosis for patients with gastric cancer.3 Similar to other cancers, the development of gastric cancer is a multistep process involving a variety of genetic Key words: gastric cancer, GRIK2, tumor suppressor gene, and epigenetic modifications. Some of the cellular molecules hypermethylation related to gastric cancer development and progression seem Additional Supporting Information may be found in the online to be regulated via DNA methylation.4 DNA methylation fre- version of this article quently occurs at CpG islands, which are short stretches of The first two authors contributed equally to this work. GC-rich sequences frequently located on promoters and in Grant sponsor: Ministry of Education, National Science Council; the first exon of genes.5 Such methylation can have a pro- Grant numbers: NSC 94-2314-B-182A-188, 94-3112-B-182-005, 95- found effect on the expression of tumor suppressor genes in 2320-B-182-001, 97-3112-B-182-008; Grant sponsor: Chang Gung various cancers. Therefore, aberrant DNA methylation of tu- Memorial Hospital, Taiwan; Grant numbers: CMRPD150961, mor suppressor genes may be useful as a good biomarker for CMRPG360221, CMRPG360262 cancers.6 In gastric cancer, genes such as p14ARF, p16INKaa,7 DOI: 10.1002/ijc.24958 MGMT, hMLH18 and APC9 reportedly play tumor-suppres- History: Received 23 Apr 2009; Accepted 29 Sep 2009; Online 12 sor roles and are regulated by DNA modification. During the Oct 2009 development of gastric cancer, it is believed that some tumor Correspondence to: Yu-Sun Chang, Chang Gung Molecular suppressor genes undergo DNA hypermethylation, leading to Medicine Research Center and Graduate Institute of Biomedical their decreased expression.10 Sciences, Chang Gung University, 259 Wen-Hwa 1st Road, Glutamate receptors are membrane proteins responsible Kwei-Shan, Taoyuan, 333 Taiwan, Tel: 886-3-211-8800 x5131; for mediating most excitatory neurotransmissions in the 886-3-211-8683, Fax: 886-3-211-8683, E-mail: [email protected] mammalian central nervous system (CNS).11 They are Int. J. Cancer: 126, 2542–2552 (2010) VC 2009 UICC Wu et al. 2543 Table 1. Clinical characteristics of gastric cancer samples Case no. Hypermethylated Hypomethylated p Gender Female 11 9 2 0.231 Male 16 9 7 Age (yr) <65 13 9 4 1 65 14 9 5 Depth of invasion (pT) T1, T2 8 5 3 1 T3, T4 19 13 6 Lymph node status (pN) N0 6 5 1 0.628 Non N0 21 13 8 Histological type Intestinal 11 6 5 0.393 Diffuse 14 10 4 Mix type 2 2 0 Helicobacter pylori infection Negative 21 15 6 0.367 Positive 6 3 3 Paired samples Adjacent normal 27 2 25 <0.0011 Tumor 27 18 9 Cancer Cell Biology P values were calculated using Pearson Chi-square test. 1statistically significant when <0.05. classified into 2 major groups: the metabotropic glutamate re- Although GRIK2 has been implicated in several neurologi- ceptor (mGlu) group, which is composed of mGlu1-8, and cal diseases, such as Huntington’s disease,19 autosomal reces- the ionotropic glutamate receptors, which include the N- sive mental retardation,20 autism21 and manic-depressive ill- 22 methyl-D-aspartate (NMDA), a-amino-3-hydroxy-5-methyl- ness, little is known about its role in tumor progression. A iso-xazole-4-propionate (AMPA) and kainate glutamate report by Sinclair et al. (2004) suggested that GRIK2 may be receptors. Glutamate receptor, ionotropic, kainate 2 (GluR6 a candidate tumor-suppressor gene for acute lymphocytic or GRIK2) is 1 of the 5 members of the kainate glutamate re- leukemia (ALL), but no subsequent study has examined the ceptor subgroup, which also includes GluR5-7, KA1 and relevant biological functions or regulation mechanisms. We KA2. Although kainate receptors are distributed throughout show for the first time that GRIK2 is regulated by DNA the CNS, their physiological significance is not yet known. methylation in gastric cancer and further report that colony GRIK2, located on chromosome 6q16.3-q21, is a 17-exon formation and cell migration are suppressed in gastric cancer gene12 that may be transcribed into at least 6 different splice cell lines expressing GRIK2, strongly indicating that GRIK2 is variants.13 Under physiological conditions, GRIK2 forms a a tumor-suppressor gene. homomeric receptor channel14 or a heteromeric receptor channel with GluR5.15 Recent studies have indicated that the Material and Methods ionotropic glutamate receptors, NMDA2A and NMDA2B, Cell culture, clinical samples and 50-aza-dC treatment may play tumor-suppressor functions in esophageal cancer,16 AGS cells cultured in Ham’s F12 medium containing 10% fe- gastric cancer17 and colorectal carcinoma.18 The expression tal bovine serum (FBS). TMC1 cells were maintained in level of these NMDA receptors is regulated at least in part RPMI medium containing 10% FBS. AZ521 and KATO III through changes in DNA methylation. However, no func- cells were kindly provided by Dr. Lin KH of Chang Gung tional analysis has suggested that kainate receptors may play University (Taiwan) and were cultured in RPMI containing a tumor-suppressor role or that their expression might be 10% FBS. Twenty-seven paired gastric cancer/normal adja- regulated by DNA methylation in cancer cells. cent tissue samples were collected from patients between Int. J. Cancer: 126, 2542–2552 (2010) VC 2009 UICC 2544 Glutamate receptor, ionotropic, kainate 2 silencing 2000 and 2003 and were obtained from the Chang Gung Me- Bisulfite treatment and bisulfite sequencing morial Hospital (CGMH) Tumor Bank (Taoyuan, Taiwan) Genomic DNA (1 lg) was modified by sodium bisulfite using (Table 1). Two normal adult stomach genomic DNA samples the EZ DNA methylation kit (Zymo Research, USA). The (N24 and N33) were purchased from BioChain Institute CpG islands of the GRIK2 promoter region were amplified (USA). This study was reviewed and approved by the institu- using primers 50-GTTTGGTAAAATTTTTGTTAGTAAAG-30 tional review board and ethics committee of CGMH. and 50-AATTCCTTAAAAATATCCAATCCAC-30. The PCR Informed consent was obtained from all patients and healthy products were cloned into a TA vector and sequenced. The controls. sequencing results were analyzed with the Vector NTI 9.0 One day before experiments, gastric cancer cell lines software (Invitrogen, USA). (AGS, AZ521 and TMC1) were seeded in 10-cm dishes at a confluence of 20–30% per dish. KATO III cells were seeded 0 Quantitative methylation-specific PCR in 25-T flasks at a density of 20–30% per flask. For 5 -aza-dC Bisulfite-modified DNA was subjected to real-time quantita- l 0 treatment, cells were treated with 5 M5-aza-dC (Sigma, tive methylation-specific PCR (Q-MSP) using a Bio-Rad iCy- USA) for 5 days. The culture medium was replaced every l  0 cler (Bio-Rad, USA). Each reaction contained 7.5 lof2 24 hr with fresh culture medium containing 5 -aza-dC. SYBR Green supermix (Bio-Rad), 0.2 lM of each primer and 10 ng of bisulfite-modified DNA in a total volume of 15 ll. Differential methylation hybridization and CpG microarray The reaction conditions consisted of 95C for 3 min, fol- Differential methylation hybridization (DMH) was performed lowed by 50 cycles of 95C for 15 sec, 60C for 20 sec, 72C 23 l as previously described.