IJC - 國立中興大學web.nchu.edu.tw/pweb/users/hpliu/research/12751.pdf · products were cloned into a TA vector and sequenced. The sequencing results were analyzed with the Vector

Glutamate receptor, ionotropic, kainate 2 silencing by DNAhypermethylation possesses tumor suppressor function ingastric 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, Taiwan2 Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan3Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan4 Department of Pathology, Chang Gung Memorial Hospital, Lin-Kou, Taoyuan, Taiwan5 Department of Life Science, Institute of Molecular Biology, National Chung-Cheng University, Chia-Yi, Taiwan6 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 5 20.44). 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.

Gastric cancer is one of the most common human cancersworldwide and is a major upper gastrointestinal tract malig-nant disease with poor prognosis for patients suffering frommore advanced stages of the disease.1 In the 2009 TaiwanCancer Registry Report, this cancer was ranked as having theseventh highest incidence among cancers. Surgery combined

with chemotherapy is a common treatment for gastric cancer.Factors such as diet, tobacco use and Helicobacter pyloriinfection have been reported as the major risk factors for gas-tric carcinoma.2

Numerous studies have sought to identify biomarkers ca-pable of improving the prognosis for patients with gastriccancer.3 Similar to other cancers, the development of gastriccancer is a multistep process involving a variety of geneticand epigenetic modifications. Some of the cellular moleculesrelated to gastric cancer development and progression seemto be regulated via DNA methylation.4 DNA methylation fre-quently occurs at CpG islands, which are short stretches ofGC-rich sequences frequently located on promoters and inthe first exon of genes.5 Such methylation can have a pro-found effect on the expression of tumor suppressor genes invarious cancers. Therefore, aberrant DNA methylation of tu-mor suppressor genes may be useful as a good biomarker forcancers.6 In gastric cancer, genes such as p14ARF, p16INKaa,7

MGMT, hMLH18 and APC9 reportedly play tumor-suppres-sor roles and are regulated by DNA modification. During thedevelopment of gastric cancer, it is believed that some tumorsuppressor genes undergo DNA hypermethylation, leading totheir decreased expression.10

Glutamate receptors are membrane proteins responsiblefor mediating most excitatory neurotransmissions in themammalian central nervous system (CNS).11 They are

Key words: gastric cancer, GRIK2, tumor suppressor gene,

hypermethylation

Additional Supporting Information may be found in the online

version of this article

The first two authors contributed equally to this work.

Grant sponsor: Ministry of Education, National Science Council;

Grant numbers: NSC 94-2314-B-182A-188, 94-3112-B-182-005, 95-

2320-B-182-001, 97-3112-B-182-008; Grant sponsor: Chang Gung

Memorial Hospital, Taiwan; Grant numbers: CMRPD150961,

CMRPG360221, CMRPG360262

DOI: 10.1002/ijc.24958

History: Received 23 Apr 2009; Accepted 29 Sep 2009; Online 12

Oct 2009

Correspondence to: Yu-Sun Chang, Chang Gung Molecular

Medicine Research Center and Graduate Institute of Biomedical

Sciences, Chang Gung University, 259 Wen-Hwa 1st Road,

Kwei-Shan, Taoyuan, 333 Taiwan, Tel: 886-3-211-8800 x5131;

886-3-211-8683, Fax: 886-3-211-8683, E-mail: [email protected]

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Int. J. Cancer: 126, 2542–2552 (2010) VC 2009 UICC

International Journal of Cancer

IJC

classified into 2 major groups: the metabotropic glutamate re-ceptor (mGlu) group, which is composed of mGlu1-8, andthe ionotropic glutamate receptors, which include the N-methyl-D-aspartate (NMDA), a-amino-3-hydroxy-5-methyl-iso-xazole-4-propionate (AMPA) and kainate glutamatereceptors. Glutamate receptor, ionotropic, kainate 2 (GluR6or GRIK2) is 1 of the 5 members of the kainate glutamate re-ceptor subgroup, which also includes GluR5-7, KA1 andKA2. Although kainate receptors are distributed throughoutthe CNS, their physiological significance is not yet known.GRIK2, located on chromosome 6q16.3-q21, is a 17-exongene12 that may be transcribed into at least 6 different splicevariants.13 Under physiological conditions, GRIK2 forms ahomomeric receptor channel14 or a heteromeric receptorchannel with GluR5.15 Recent studies have indicated that theionotropic glutamate receptors, NMDA2A and NMDA2B,may play tumor-suppressor functions in esophageal cancer,16

gastric cancer17 and colorectal carcinoma.18 The expressionlevel of these NMDA receptors is regulated at least in partthrough changes in DNA methylation. However, no func-tional analysis has suggested that kainate receptors may playa tumor-suppressor role or that their expression might beregulated by DNA methylation in cancer cells.

Although GRIK2 has been implicated in several neurologi-cal diseases, such as Huntington’s disease,19 autosomal reces-sive mental retardation,20 autism21 and manic-depressive ill-ness,22 little is known about its role in tumor progression. Areport by Sinclair et al. (2004) suggested that GRIK2 may bea candidate tumor-suppressor gene for acute lymphocyticleukemia (ALL), but no subsequent study has examined therelevant biological functions or regulation mechanisms. Weshow for the first time that GRIK2 is regulated by DNAmethylation in gastric cancer and further report that colonyformation and cell migration are suppressed in gastric cancercell lines expressing GRIK2, strongly indicating that GRIK2 isa tumor-suppressor gene.

Material and MethodsCell culture, clinical samples and 50-aza-dC treatment

AGS cells cultured in Ham’s F12 medium containing 10% fe-tal bovine serum (FBS). TMC1 cells were maintained inRPMI medium containing 10% FBS. AZ521 and KATO IIIcells were kindly provided by Dr. Lin KH of Chang GungUniversity (Taiwan) and were cultured in RPMI containing10% FBS. Twenty-seven paired gastric cancer/normal adja-cent tissue samples were collected from patients between

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

P values were calculated using Pearson Chi-square test.1statistically significant when <0.05.

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2000 and 2003 and were obtained from the Chang Gung Me-morial Hospital (CGMH) Tumor Bank (Taoyuan, Taiwan)(Table 1). Two normal adult stomach genomic DNA samples(N24 and N33) were purchased from BioChain Institute(USA). This study was reviewed and approved by the institu-tional review board and ethics committee of CGMH.Informed consent was obtained from all patients and healthycontrols.

One day before experiments, gastric cancer cell lines(AGS, AZ521 and TMC1) were seeded in 10-cm dishes at aconfluence of 20–30% per dish. KATO III cells were seededin 25-T flasks at a density of 20–30% per flask. For 50-aza-dCtreatment, cells were treated with 5 lM 50-aza-dC (Sigma,USA) for 5 days. The culture medium was replaced every24 hr with fresh culture medium containing 50-aza-dC.

Differential methylation hybridization and CpG microarray

Differential methylation hybridization (DMH) was performedas previously described.23 In brief, 2 lg genomic DNA wasdigested with MseI, and the digested DNA fragments wereligated with the synthetic adaptor, H24/H12 (50-AGGCAACTGTGCTATCCGAGGGAT-30, 50-TAATCCCTCGGA-30). The resulting fragments were further digested with theCG methylation-sensitive enzymes, BstUI and HpaII (NewEngland Biolabs). After amplification by PCR using H24 asthe primer, the amplicons were purified using a NucleoSpinExtract II kit (Macherey-Nagel, Germany), labeled with Cy3dye and hybridized with a homemade CpG microchip con-taining 9216 CpG-rich DNA clones (adapted from that ofDr. Tim Hui-Ming Huang, Ohio State University, OH).24,25

Resequencing of these plasmid clones confirmed that thislibrary contains 3,832 individual clones (http://163.25.91.176).The microarray was scanned using a Gene Pix 4200 scanner(Axon, CA, USA), and the results were analyzed using theGenePix 6.0 software.

Duplicate CpG microarray data from the 4 gastric cancercell lines (experimental group) and 2 purchased normalstomach tissues (control) were analyzed using the GenedataExpressionist Pro3.0 software (Genedata AG, Basel, Switzer-land). The signals from these samples were normalized byglobal median normalization, with the median intensity valueset to 250 (cancer cell line >500 and normal <250). Becausecancer cells presumably have a higher methylation level andtherefore a higher CpG microarray signal intensity than nor-mal tissues, we selected genes whose normalized intensity val-ues were 2-fold or more greater in the gastric cancer cell linescompared with the normal controls. In addition, genes whosep value was <0.001 (student t test) were selected. Further-more, redundant CpG clones and clones whose sequencesdid not match those of a known promoter region (2 kbupstream or downstream from the transcription start site)were excluded from this study. In the end, we identified 107genes that were hypermethylated in gastric cancer cell linescompared with normal stomach tissues. Our detailed analysisstrategy is shown in Supporting Information Figure S1.

Bisulfite treatment and bisulfite sequencing

Genomic DNA (1 lg) was modified by sodium bisulfite usingthe EZ DNA methylation kit (Zymo Research, USA). TheCpG islands of the GRIK2 promoter region were amplifiedusing primers 50-GTTTGGTAAAATTTTTGTTAGTAAAG-30

and 50-AATTCCTTAAAAATATCCAATCCAC-30. The PCRproducts were cloned into a TA vector and sequenced. Thesequencing results were analyzed with the Vector NTI 9.0software (Invitrogen, USA).

Quantitative methylation-specific PCR

Bisulfite-modified DNA was subjected to real-time quantita-tive methylation-specific PCR (Q-MSP) using a Bio-Rad iCy-cler (Bio-Rad, USA). Each reaction contained 7.5 ll of 2�SYBR Green supermix (Bio-Rad), 0.2 lM of each primer and10 ng of bisulfite-modified DNA in a total volume of 15 ll.The reaction conditions consisted of 95�C for 3 min, fol-lowed by 50 cycles of 95�C for 15 sec, 60�C for 20 sec, 72�Cfor 20 sec and 80�C for 10 sec. A CpG-free region of theACTB gene was used as an internal reference, as previouslydescribed.26 The amount of methylated DNA was determinedfor each sample by plotting the threshold cycle numberagainst a standard curve generated from CpGenomeTM

Universal Methylated DNA (Chemicon, USA). To determinethe relative amount of methylated DNA in each sample, thevalues of the target gene were compared with that of theinternal reference gene to obtain a ratio, which was thenmultiplied by 100 to give a percentage value.

RNA extraction, cDNA synthesis and quantitative

real-time RT-PCR

Cells were cultured in culture medium, and total RNA wasisolated with TRIZOL reagent according to the manufac-turer’s protocol (Invitrogen). Total RNA of human adult nor-mal gastric tissue was purchased from BioChain Institute(USA). cDNA was generated by reverse transcription from1 lg of RNA, using Impron II reverse transcriptase (Prom-ega) and oligo-dT primers. The resulting cDNA was diluted1/20 and subjected to real-time PCR, using a Bio-Rad iCyclerwith 7.5 ll SYBR green super-mix and 30 pmol GRIK2 geneprimers 50-GCTGCTCTAATGTATGATGCTGT G-30 and 50-TGTGATGTTCGCTGGCTTTCC-30. The reaction conditionsconsisted of 95�C for 3 min, followed by 50 cycles of 95�Cfor 15 sec, 60�C for 20 sec, 72�C for 20 sec and 80�C for10 sec. GAPDH was used as an internal control.

Transfection and immunofluorescence

The human GRIK2 (NM_021956.2) expression vectorpCMV6-Entry-GRIK2 (Origene, USA) was transfected intoAGS and AZ521 cells using Lipofectamine 2000 (Invitrogen).The transfected cells were grown on cover slides for 24 hr,fixed with 3.7% formaldehyde for 30 min at room tempera-ture and permeabilized and blocked with 0.1% saponin con-taining 1% bovine serum albumin for 15 min. The cover

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2544 Glutamate receptor, ionotropic, kainate 2 silencing


slides were incubated with an anti-GRIK2 antibody (1:100Chemicon, USA) for 2 hr at room temperature, rinsed twicewith 1� phosphate-buffered saline (PBS) and incubated withan anti-rabbit FITC-conjugated secondary antibody (1:200,Jackson ImmunoResearch Laboratories, USA) for 45 min atroom temperature. For visualization of individual cells, thecover slides were incubated with DAPI solution for 5 minand washed several times with 1� PBS. Finally, the coverslides were mounted with Vectashield reagent (Vector Labo-ratories, CA) and stored at 4�C.

Establishment of stable transfectants

AGS and AZ521 cells were transfected with control vector(pCMV6-Entry) or GRIK2 expression vector (pCMV6-Entry-GRIK2) using Lipofectamine 2000 (Invitrogen). Transfectantswere selected by exposure to G418 (800 lg/ml) for 10 days.Single clones were amplified, and GRIK2 expression wasexamined by Western blotting.

Immunohistochemical staining analysis

Immunohistochemical analysis was performed using an auto-matic immunohistochemistry staining device, according tothe manufacturer’s suggested procedure (Vision BioSystems,VIC, Australia). Tissue sections and normal tissue array(#BN1002, US Biomax) were retrieved using Bond EpitopeRetrieval Solution 1 (pH 6.0) on a Bond-max automatedimmunostainer (Vision BioSystems, VIC, Australia), andstained with anti-GRIK2 antibody (1:100; Chemicon, USA).A polymer detection system (Bond Polymer Refine, VisionBioSystems) was used to reduce nonspecific staining. Tissuesections were treated with liquid 3,30-diaminobenzidine rea-gent, using 30-diaminobenzidine tetrahydrochloride as thechromogen and hematoxylin as the counterstaining reagent.

Western blot analysis

Cells were lysed in RIPA buffer, and 50 lg of total proteinwas resolved on a 10% SDS polyacrylamide gel and electro-transferred to a nitrocellulose membrane. The membrane wasblocked and then incubated with anti-GRIK2 (1:1000; Chemi-con, AB5683) or anti-tubulin (1:10000; MDBio, Taiwan) anti-bodies overnight at 4�C. Horseradish peroxidase-conjugatedimmunoglobulins were used as secondary antibodies, andproteins were detected using an ECL system (Amersham,UK) and X-ray films.

Anchorage-independent colony formation assay

For anchorage-independent assays, 3 � 103 AGS cells and1 � 104 AZ521 cells were stably transfected with GRIK2-expressing or empty vectors were seeded in 6-well plates with0.35% top agar and 0.7% bottom agar. After 4 weeks, fociwere stained with 0.005% crystal violet and counted under adissecting microscope. The experiment was performed at least3 independent times, each time in duplicate.

Cell proliferation assay

AGS (5 � 104) and AZ521 (2.5 � 104) cells were plated on6-well plates in culture medium, and total cell numbers werecounted for 4 days. The experiment was performed at least 3independent times, each time in duplicate.

Wound closure assay

Stably transfected GRIK2-expressing AGS and AZ521 cloneswere grown to confluence in 35-mm dishes containing cul-ture medium. For wounding, the cells were scraped mechani-cally with a sterile 10-ll plastic pipette tip. The wounded sur-face was then rinsed with 1� PBS, the image was captured(AXIOVERT 200 MAT, ZEISS) at time 0 hr, the culture me-dium was refreshed and the cells were cultured for an addi-tional 9, 18 and 24 hr for AGS cells, 48 hr for AZ521 cells.Cells were counted from 10 random fields within the residualwound area under 200� magnification, and averages werecalculated.

Transwell migration assay

Transwell migration assays were performed in a 24-wellTranswell chamber (Corning, USA) fitted with a polycarbon-ate membrane (8-lm pore size). AGS (5 � 104) and AZ521(1 � 105) cells were washed twice with serum-free medium,resuspended in 100 ll of serum-free medium and added tothe upper chamber. The lower chamber contained 10% FBSmedium. After 2.5 (for AGS cells), 7 and 24 hr (for AZ521cells), the migrated cells were fixed and stained for 15 minwith 0.25% crystal violet, 10% formaldehyde and 80% metha-nol, and then washed 5 times with ddH2O for removal ofnonadherent cells. Ten random fields under 100� magnifica-tion were captured for each membrane, and the migratedcells were counted. For spectrophotometric detection ofmigration ability, membranes were stained with crystal violetand soaked in 10% acetic acid, and the OD570 wasmeasured.

Statistical analysis

Comparisons of GRIK2 methylation or gene expression levelsbetween paired tumor and adjacent normal tissue sampleswere performed using the Wilcoxon signed rank test. For theanchorage-independent colony formation, cell proliferationand migration ability assays, statistical significance was exam-ined using t-tests. The cutoff value for Q-MSP was deter-mined by ROC analysis.27 The chi-square test was used tocalculate differences in gender of the patient, tumor stageand methylation status between adjacent normal tissues andtumor tissues. These analyses were performed using SPSS13.0 software (SPSS, Chicago, IL). P values <0.05 were con-sidered statistically significant.

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ResultsHypermethylation of GRIK2 CpG islands in

gastric cancer cell lines

DMH experiments were performed on DNA from 4 gastriccancer cell lines (AGS, AZ521, KATO III and TMC1) and 2normal adult stomach samples (N24 and N33). The detailedprocedures are described in the ‘‘Material and Methods’’ sec-tion and Supporting Information Figure S1a. In brief, using ahomemade CpG island microarray (comprising �8000 CpGclones corresponding to 3,832 individual genes) adapted fromDr. TH Huang,28 we performed DMH and identified 107genes that were differentially hypermethylated in the 4 gastriccancer cell lines (AGS, AZ521, KATOIII and TMC1) but notin the 2 adult normal stomach tissue samples. Among theseindividual genes, there were 7 probes located within the pro-moter and exon 1 of GRIK2 gene, and all 7 probes wereshown very significant p values. The average GRIK2 probe in-tensity of AGS, AZ521, KATOIII and TMC1 was significantlyhigher than that of N33 and N24 (Supporting Information Fig.S1b). In addition, GRIK2 may be a candidate tumor suppressorgene for ALL.29 Furthermore, several ionotropic glutamatereceptors such as NMDA2A and NMDA2B whose expressionlevel is regulated at least in part through changes in DNAmethylation and may play tumor-suppressor functions in sev-eral solid tumors, including gastric cancer.17 This prompted usto investigate whether this gene could be a tumor-suppressorgene in gastric cancer. The promoter region of GRIK2 (http://www.urogene.org/) contains 2 CpG islands (CpG1, �413 to�212; CpG2, �110 to þ95) comprising a total of 30 CpGdinucleotides (Fig. 1a). Bisulfite sequencing showed that theCpG sites of the GRIK2 gene were densely methylated in AGS(93%), AZ521 (51%) and TMC1 (97%) cells, but only sparselymethylated in KATO III cells (26.6%) and the 2 normal stomachsamples (8.6% in N24 and 20% in N33) (Fig. 1b). These resultswere confirmed by the use of Q-MSP (Fig. 1c). To determinewhether GRIK2 gene expression is regulated by DNA methyla-tion, we used quantitative real-time PCR to compare GRIK2gene expression in gastric cancer cell lines treated with and with-out 50-aza-dC. In AGS, AZ521 and TMC1 cells, but not KATOIII cells, GRIK2 mRNA levels were restored after treatment with50-aza-dC for 5 days (Fig. 1d). These results suggest that GRIK2expression in AGS, AZ521 and TMC1 cells is likely to be regu-lated through DNA methylation of the promoter region.

Hypermethylation of the GRIK2 promoter region

in clinical samples

Because the GRIK2 promoter region was found to be hyper-methylated in 3 of the 4 gastric cancer cell lines, we specu-lated that GRIK2 expression might be decreased in tumor tis-sues. Twenty-seven paired clinical samples were examined forimmunohistochemical staining, Q-MSP and quantitative real-time RT-PCR. As shown in Figure 2a and Supporting Infor-mation Figure S2b, immunohistochemistry showed thatGRIK2 expression was lower in tumor tissues compared with

adjacent normal tissues. Immunohistochemistry analysis ofGRIK2 expression in normal tissue array also supported thatGRIK2 is expressed at higher level in normal stomach samples(Supporting Information Fig. S2a and Supporting InformationTable I). Furthermore, bisulfite sequencing of DNA from thepaired tissue samples showed that the GRIK2 CpG sites weremore heavily methylated in tumor samples vs. adjacent nor-mal tissues (70 vs. 21% in sample #89001, and 78 vs. 20% insample #92024; Fig. 2b). On the basis of these findings, wefurther analyzed 27 pairs of clinical samples, using Q-MSPand quantitative real-time PCR to test whether there was acorrelation between GRIK2 promoter methylation and geneexpression in these samples. As shown in Figure 2c, a statisti-cally significant difference (p < 0.001) was observed betweenthe normal and tumor tissues of the 27 paired samples, indi-cating that GRIK2 gene is differentially methylated in gastriccancer. Across all of the clinical samples, the tumor tissuestended to be hypermethylated, whereas their adjacent normaltissues tended to be hypomethylated (Supporting InformationFig. S3). Using 40.8% methylation (per Q-MSP) as the cutoffvalue, hypermethylation of GRIK2 could be used to reliablydifferentiate tumor samples from adjacent normal samples(Table 1; chi-square analysis, p < 0.001). To test whetherGRIK2 promoter hypermethylation was correlated with lowergene expression in clinical samples, Q-RT PCR was per-formed. As shown in Figure 2d, GRIK2 expression was signifi-cantly lower in tumor tissues compared with adjacent normaltissues across the 27 paired biopsy samples (p ¼ 0.001) andthere was an inverse correlation between GRIK2 methylationand its gene expression level (r ¼ �0.44 and p ¼ 0.022; Fig.2e). Collectively, these data strongly suggest that GRIK2expression is very likely to be regulated through promotermethylation in patients with gastric cancer.

GRIK2 does not alter cell proliferation but does affect

anchorage-independent colony formation

Because GRIK2 expression seems to be regulated by methyla-tion and GRIK2 expression is lower in tumor samples com-pared with adjacent normal tissues, we next sought to deter-mine whether GRIK2 can function as a tumor suppressor. TheGRIK2 expression plasmid, pCMV6-Entry-GRIK2, was trans-fected into AGS cells, and GRIK2 expression was observed byimmunofluorescence and Western blotting (Fig. 3a). GRIK2expression was undetectable in parental AGS cells and cellstransfected with the vector control, pCMV-Entry (Fig. 3a).Similar results were also observed in AZ521 cells (SupportingInformation Fig. S4a). To investigate the biological functionsof GRIK2, we examined the proliferation of an AGS cell linestably expressing GRIK2 (G8), a GRIK2-expressing mixedclone (GM) and 2 AGS cell lines stably transfected with emptyvector (C1 and C2). After 4 days, there was no difference ingrowth rate among these cell lines (Fig. 3b). In contrast, an-chorage-independent colony formation assays performed on avector-transfected mixed clone (CM) and GM showed that thecolony formation ability of CM clones was significantly higher

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Figure 1. Methylation status for the CpG islands of GRIK2 in gastric cancer cell lines. (a) Genomic map of the GRIK2 promoter region. The 2

CpG islands, �413 to �212 and �110 to þ95, contain 30 CpG dinucleotides; þ1 is the GRIK2 transcription start site. Each vertical bar

represents 1 CpG dinucleotide. (b) Bisulfite sequencing analysis of the GRIK2 CpG islands. Bisulfite sequencing was performed with

samples from 4 gastric cancer cell lines (AGS, AZ521, TMC1 and KATO III) and 2 stomach samples from healthy individuals (N24 and N33).

Five individual clones from each sample were analyzed. The open and filled squares represent unmethylated and methylated CpG sites,

respectively. Horizontal bars indicate the positions of the utilized methylation-specific PCR primers. The % methylation of the 30 CpG sites

is shown in parenthesis. (c) Quantitative methylation-specific PCR analysis of GRIK2 methylation in gastric cancer cell lines and stomach

samples from healthy individuals. Results were obtained from 3 individual experiments. (d) Recovery of GRIK2 expression in gastric cancer

cell lines after 50-aza-dC treatment. The relative fold change was calculated by dividing the quantitative RT-PCR data from cells treated with

50-aza-dC by those from untreated cells. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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than that of GM clones after 4 weeks (p ¼ 0.02; Fig. 3c). Simi-lar results were obtained in AZ521 cells (Supporting Informa-tion Fig. S4b). These results suggest that GRIK2 inhibits col-ony formation but does not affect cell proliferation in gastriccancer cell lines.

GRIK2 suppress cell migration

Next, we tested GRIK2 for another tumor-suppressor activity,namely the ability to inhibit cell migration. By using theabove-described GRIK2-expressing AGS cell clones, we per-formed wound healing assays. As shown in Figures 4a and

Figure 2. Analysis of GRIK2 expression and the gene methylation status of GRIK2 CpG islands in clinical samples. (a) Paired sample sets from

patients with gastric cancer were analyzed by IHC. GRIK2 positivity was mainly detected in the adjacent normal tissues (brown). Bar, 100 lm;

400� magnification. (b) Methylation status of GRIK2 CpG islands in paired gastric cancer samples. Hypermethylation of GRIK2 CpG islands was

present in the tumor samples but not the adjacent normal tissues of the samples shown in (a). Bisulfite sequencing analysis of tumor and

adjacent normal tissues from 2 patients with gastric cancer was performed to determine the % methylation of the GRIK2 CpG islands. Open and

filled squares represent unmethylated and methylated CpG sites, respectively. Horizontal bars indicate the positions of the quantitative

methylation-specific PCR primers. (c) GRIK2 gene methylation status, as analyzed by Q-MSP assay of 27 paired clinical samples. Box blots

showed significantly higher % methylation in tumor tissues compared with paired adjacent normal tissues (p < 0.001). (d) Analysis of GRIK2

mRNA expression in 27 paired clinical samples. Box blots showed significantly lower GRIK2 expression in tumor tissues compared with adjacent

normal tissues (p ¼ 0.001). (e) Inverse correlation between GRIK2 methylation and gene expression. The differences in the methylation of GRIK2

CpG islands (Q-MSP of T minus N) and the relative fold change of mRNA expression (T over N) were significantly and inversely correlated (r ¼�0.44, p ¼ 0.022). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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4b, the cell migration ability of CM cells was significantlylower than that of CM or parental AGS cells, as measured af-ter 18 and 24 hr of wound healing. Similarly, the number ofcells migrating to the scraped space was significantly lower inG8 cells vs. C1 cells (p < 0.0001; Fig. 4d). Similar resultswere obtained in AZ521 cells (Supporting Information Figs.S5a and S5b). In a trans-well migration assay measuring thenumber of AGS GRIK2-expressing cells that migratedthrough a polycarbonate membrane over the course of 2.5 hr(Fig. 4e), significantly fewer G8 and GM cells migrated com-pared with the number of migratory C1 and C2 cells (p <

0.001; Fig. 4f). Similar results were obtained in AZ521 cells(Supporting Information Figs. S5c and S5d).

DiscussionPrevious studies have shown that many tumor suppressorgenes are inactivated by epigenetic modification, specificallyby DNA methylation.30 In this study, we used DMH com-bined with CpG microarray analysis28 to identify genes thatwere differentially methylated in gastric cancer, in the hopethat 1 or more of them would prove to be novel candidatetumor suppressors. We demonstrated that GRIK2 promoter

Figure 3. Effects of GRIK2 expression in AGS cells. (a) Image analysis of GRIK2 in transiently expressing cells. One lg of pCMV6-Entry-GRIK2

expression vector or empty vector was transiently transfected into AGS cells. GRIK2-positive cells appeared as green fluorescent cells;

protein expression was confirmed by Western blotting. (b) Cell proliferation assay. AGS clones stably expressing GRIK2 G8, and GRIK2-

expressing mixed clone (GM), and 2 clones stably transfected with control vector alone (C1 and C2) were checked by Western blotting for

GRIK2 expression (G8 and GM cells showed various levels of GRIK2 expression), and then subjected to cell proliferation assays. (c)

Anchorage-independent colony formation by AGS cells. Vector-transfected mixed cells (CM) and GRIK2-expressing mixed cells were grown in

0.35% top agar and maintained in 400 ng/ml G418. Colonies were counted after 4 weeks, and the results from 3 independent experiments

were averaged. GRIK2-expressing cells generated significantly fewer colonies than cells transfected with the control vector (p < 0.05).

[Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Figure 4. Wound closure and migration by AGS cells stably expressing GRIK2. (a) Wound closure assays were performed using AGS cells, AGS

cells expressing mixed clones of GRIK2 (GM) and a mixed vector control (CM). Images were obtained at 0, 18 and 24 hr. Dashed lines indicate

the scraped edges at 0 hr. Cells that had migrated into the area between 2 dashed lines were counted. The relative fold differences in the

numbers of migrated cells are shown in (b). AGS and CM cells showed better migration ability than GM at 18 and 24 hr. (c) Wound closure by

AGS cells stably expressing GRIK2. Images were obtained at 0, 9 and 18 hr. Dashed lines indicate the scraped edges at 0 hr. Cells that had

migrated into the area between the 2 dashed lines were counted. The relative fold difference in the number of migrated cells is given in (d).

C1 cells showed significantly better migration ability than GRIK2-expressing G8 cells at 18 hr (p < 0.001). (e) Trans-well migration assay. AGS

clones stably expressing GRIK2 or vector control were subjected to Trans-well migration assays as described in the ‘‘Material and Methods’’

section. Images were captured at 2.5 hr under 200� magnification. Cells were counted from 10 randomly picked fields and averages were

calculated; results were obtained from 3 independent experiments. The relative fold change in the number of migrated cells is shown, with

the results from C1 cells given as 1. The migration abilities of GRIK2-expressing G8 and GM cells were significantly lower than that of C1 cells

(p < 0.05) (f). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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methylation was inversely correlated with its gene expressionin clinical samples and that ectopic expression of GRIK2inhibited the colony formation and cell migration abilities ofgastric cancer cells. To our knowledge, this is the first reportto demonstrate that GRIK2, a gene belonging to the kainatereceptor subgroup of the ionotropic glutamate receptor fam-ily, is a novel tumor-suppressor gene in gastric cancer.

Studies regarding the association of GRIK2 with cancer arelimited.29 We found that the GRIK2 gene promoter was highlymethylated in the majority (�85%) of gastric cancer tumor tis-sues compared with normal adjacent tissues (as assessed by Q-MSP using 40.8% methylation as a cutoff). For comparison, wealso analyzed the methylation status of dlc1, a previouslyknown tumor suppressor gene31 that was among the genesidentified in our DMH analysis. Among our clinical samples,33% showed hypermethylation of the dlc1 promoter region(data not shown); this was consistent with the results from anearlier study.31 The GRIK2 gene is located on chromosome6q16.3-q21, which is frequently deleted in patients with ALL.29

However, we were able to amplify the genomic sequence ofthis gene from all clinical samples (Fig. 2c), suggesting that thisgene was not commonly deleted in gastric cancer tissues.

GRIK2 is the second ionotropic glutamate receptor familymember to reportedly demonstrate a tumor-suppressor functionin gastric cancer; the first was the NMDA receptor subgroup.16,17

However, the tumor suppression mechanisms of GRIK2 andNMDA receptors seem to differ. NMDAR2A induces apoptosisand abolishes the colony formation ability of colorectal carci-noma cell lines.18 However, NMDAR2A promoted proliferationof MKN45 gastric cancer cells by accelerating cell cycle.32 Incontrast, we found similar cell proliferation levels amongGRIK2-expressing AGS cells and vector controls (Fig. 3b). Inaddition, flow cytometry showed that cell cycle progression wassimilar among the stably transfected clones (data not shown).Thus, GRIK2-mediated tumor suppression seems to be mediatedvia mechanisms other than suppression of cell proliferation.

Notably, reintroduction of GRIK2 expression in gastriccancer cells decreased tumor cell migration. Previous studieshave shown that GRIK2 can be recruited by the cadherin/cat-enin complex through an interaction with b-catenin at Cos-7cell–cell junctions.33 b-Catenin signaling plays a dual role inthe process of cell migration: it links cadherins to the cytoskele-ton, thereby allowing tight intercellular adhesion; and, uponstimulation, it translocates into the nucleus to serve as a tran-scription cofactor for target gene regulation.34 In gastric cancercells, GRIK2 was found to colocalize with b-catenin in GRIK2-expressing AGS cells, but its presence or absence did not seemto affect the amount of b-catenin at AGS cell–cell junctions

(data not shown). Furthermore, the expression level of CyclinD1, a well-defined target gene of the b-catenin signaling path-way, remained unchanged in GRIK2-expressing AGS cells com-pared with parental AGS cells (data not shown). In contrast, aprevious report noted that the interaction of GRIK2 and b-cat-enin in Cos-7 cells was partially blocked by overexpression ofthe binding site-competing b-catenin-associated protein, PSD-95.33 Notably, we cannot rule out the possibility that unidenti-fied nutrients or growth factors in the culture medium mayhave acted as agonists of GRIK2, thereby affecting cell migra-tion activity in our assays. Previously, glutamate insert in cul-ture medium was shown to activate GluR3 (an AMPA receptorsubtype) expressed on T cells, thereby triggering CXCR4-medi-ated T-cell chemotactic migration.35 Future studies will be war-ranted to assess the detailed mechanisms involved in GRIK2-mediated inhibition of cell migration.

Recently, increasing numbers of reports have been dedicatedto the emerging role of neuronal receptors in cancer. Studieshave shown that neuronal receptors, such as metabotropic gluta-mate receptors, not only mediate neurotransmission but alsoparticipate in cellular transformation as oncogenes.36 In thisstudy, we found that the ionotropic GRIK2 may be a tumorsuppressor. The average mRNA expression level of GRIK2 wasabout 90% lower in gastric cancer tissues compared with adja-cent normal tissues (Fig. 2d), whereas the latter level was lessthan one sixth of that found in brain (data not shown). In non-neoplastic gastric mucosa, the expression of GRIK2 was strongin parietal cells and chief cells (Fig. 2a, left upper), whereas itwas weak in mucous epithelium, with slightly increased expres-sion in intestinal metaplasia (Fig. 2a, left lower). GRIK2 proteinsare expressed in multiple organs; a higher level expression isdetected in the normal stomach than other organs (SupportingInformation Fig. 2a and Supporting Information Table I). Inthis study, we found that GRIK2 promoter was hypermethylatedin gastric cancer cell lines and tumor region of clinical samples,but not in 2 healthy stomach samples or adjacent normal ofclinical samples, suggesting GRIK2 promoter methylation maybe an early tumorigenic event. Thus, it is possible that, in thetumor microenvironment, GRIK2-mediated signaling is pre-vented because of no or low expression of the receptor, even ifthe GRIK2 ligands may exist. Because the microenvironment ofa tumor may affect disease development, it would be interestingto investigate the function of GRIK2 in the gastrointestinal tract.This may provide an opportunity to study an unexpected mech-anism for gastric cancer development.

AcknowledgementsWe thank the Bioinformatics Core and Pathology Core of Chang Gung Mo-lecular Medicine Research Center for technical support.

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IJC - 國立中興大學web.nchu.edu.tw/pweb/users/hpliu/research/12751.pdf · products were cloned into a TA vector and sequenced. The sequencing results were analyzed with the Vector