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Research Article

Discovery of Aberrant Expression of R-RAS by Cancer-Linked DNA Hypomethylation in Gastric Cancer Using Microarrays

Michiko Nishigaki,1 Kazuhiko Aoyagi,1 Inaho Danjoh,2 Masahide Fukaya,1 Kazuyoshi Yanagihara,3 Hiromi Sakamoto,1 Teruhiko Yoshida,1 and Hiroki Sasaki1

1Genetics Division, 2Center of Medical Genomics, and 3Central Animal Laboratory, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo, Japan

Abstract causal role in tumor formation, possibly by promoting chromosomal instability. For gene activation by cancer-linked hypomethylation, in Although hypomethylation was the originally identified epigenetic change in cancer, it was overlooked for many 1983 Feinberg and Vogelstein (8) first reported that human growth a h years in preference to hypermethylation. Recently, gene hormone, -globin,and -globin are methylated in normal tissues activation by cancer-linked hypomethylation has been redis- and become hypomethylated in cancers. It has been rediscovered covered. However, in gastric cancer, genome-wide screening recently that the cancer-linked hypomethylation leads to activation of the activated genes has not been found. By using of genes that are important in cancer (4). A correlation between microarrays, we identified 1,383 gene candidates reactivated hypomethylation and overexpression has been shown for MN/CA9 in at least one cell line of eight gastric cancer cell lines after encoding a tumor antigen in renal cell carcinomas (9), MDR1 in treatment with 5-aza-2Vdeoxycytidine and trichostatin A. Of myeloid leukemias (10), BCL-2 in chronic lymphocytic leukemias the 1,383 genes, 159 genes, including oncogenes ELK1, (11), MAGE-1 in melanomas (12), and SNCG/BCSG1 in breast carcinomas and ovarian carcinomas (13). SNCG, also referred to as FRAT2, R-RAS, RHOB, and RHO6, were further selected as gene candidates that are silenced by DNA methylation in BCSG1, is a member of a neuronal protein family, synuclein, and its normal stomach mucosa but are activated by DNA demethy- expression is highly tissue-specific (14, 15). As described above, it was lation in a subset of gastric cancers. Next, we showed that clearly shown that hypomethylation of the SNCG gene CpG island demethylation of specific CpG sites within the first intron of promotes its aberrant expression in breast and ovarian carcinomas R-RAS causes activation in more than half of gastric cancers. (13). Extensive recent study of pancreatic cancer by microarray Introduction of siRNA into R-RAS–expressing cells resulted in analysis showed that frequent hypomethylation of seven genes r the disappearance of the adhered cells, suggesting that (claudin4, lipocalin2, 14-3-3 , trefoil facter2, S100A4, mesothelin,and functional blocking of the R-RAS–signaling pathway has prostate stem cell antigen) caused overexpression (16). In the early great potential for gastric cancer therapy. Our extensive gene studies, c-MYC, H-RAS, c-FOS,andAFP genes have been reported to list provides other candidates for this class of oncogene. be less hypomethylated in gastric cancer tissues than in noncancer- (Cancer Res 2005; 65(6): 2115-24) ous tissues (17). However, in gastric cancer, genome-wide screening of the activated genes has not been found. In this study, using genome-wide expression analysis, we screened genes activated by Introduction cancer-linked hypomethylation in gastric cancer. DNA hypermethylation in cancer has been observed most often in CpG islands in gene regions and leads to down-regulation of tumor Materials and Methods suppressor genes (1, 2). Recently, histone modifications and silencing Tissue Samples and 5-Aza-2VDeoxycytidine and Trichostatin A before DNA methylation of the p16/INK4A gene has been reported Treatment of Gastric Cancer Cells. Thirty-three gastric cancer tissues (3). Therefore, whether DNA hypermethylation is a cause of and six noncancerous tissues were obtained with informed consent from tumorigenesis remains unestablished. In contrast, very frequent patients who underwent a gastrectomy at the National Cancer Center hypomethylation is seen in both highly and moderately repeated Hospital (Tokyo, Japan). Eight cell lines (HSC39, HSC43, HSC44, HSC58, DNA sequences in many human cancers, including heterochromatic HSC59, HSC60, KATOIII, and OCUM2M) that were derived from gastric DNA repeats, dispersed retrotransposons, and endogenous retroviral cancers were used. The treatment of the eight cell lines consisted of 5- elements (4). Whether genome-wide DNA hypomethylation, aza-2Vdeoxycytidine (5Aza-dC, 1 Amol/L) for 24 hours, followed by addition however, is a cause or consequence of tumorigenesis has been of trichostatin A (TSA), to a final concentration of 1 Amol/L for a further unclear. As possible mechanisms, insertional activation of proto- 24 hours. Treatments with TSA alone and 5Aza-dC alone were also done by using the same amount of the drugs. oncogenes by induction of endogenous retroviral elements (5) and Microarray Analysis. For total RNA isolation, surgical specimens and induction of genomic instability (6) were previously considered. cultured cells were mixed with IsoGen lysis buffer (Nippon Gene Co., Ltd., Recently, mutant mice carrying a hypomorphic DNA methyltrans- Toyama, Japan) at room temperature, extracted with chloroform, and ferase 1 (Dnmt 1) have been reported to develop aggressive T-cell precipitated with a glycogen carrier (20 Ag/AL) in isopropanol. The RNA lymphomas that displayed a high frequency of chromosome 15 pellet was washed with 70% ethanol prepared and dissolved in RNase-free trisomy (7). This article shows that DNA hypomethylation plays a water. We used human U95A version 2 arrays (Affymetrix, Santa Clara, CA) for analysis of mRNA expression levels corresponding to 12,626 transcripts. The procedures were conducted according to the protocols of the supplier. Briefly, 10 Ag of fragmented cRNA were hybridized to the microarrays in 200 ALof Requests for reprints: Genetics Division, National Cancer Center Research a hybridization cocktail at 45jC for 16 hours in a rotisserie oven set at Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan. Phone: 81-3-3542-2511; Fax: 81-3-3541-2685; E-mail: [email protected]. 60 rpm. The arrays were then washed with a nonstringent wash buffer I2005 American Association for Cancer Research. (6Â saline-sodium phosphate-EDTA) at 25jC, followed by a stringent wash www.aacrjournals.org 2115 Cancer Res 2005; 65: (6). March 15, 2005

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Cancer Research buffer [100 mmol/L MES (pH 6.7), 0.1 mol/L NaCl, and 0.01% Tween 20] at RNAi Analysis. All siRNAs used for RNA interference of the R-RAS gene 50jC, stained with streptavidin phycoerythrin (Molecular Probes, Eugene, product were designed with an online siRNA design tool provided by Qiagen OR), washed again with 6Â saline-sodium phosphate-EDTA, stained with (http://www.qiagen.com/siRNA). Among the three siRNAs designed, which biotinylated anti-streptavidin immunoglobulin G, followed by a second had no sequence identities with any other known genes including RAS family staining with streptavidin phycoerythrin and a third wash with 6Â saline- genes, two had a suppressive effect on R-RAS mRNA expression (data not sodium phosphate-EDTA. The arrays were scanned using a GeneArray shown). The target sequence used for subsequent analysis was ACACGAA- scanner (Affymetrix) at 3-Am resolution, and the expression value (average GATCTGCAGTGTGGAT. The negative control siRNA of which sequence has difference) of each gene was calculated using GeneChip Analysis Suite no known homology to mammalian genes was also purchased from Qiagen. version 4.0 software (Affymetrix). The mean of average difference values in HSC58 and HSC59 cells were inoculated in 24-well plates at a density of 3 Â each experiment was 1,000 to reliably compare variable multiple arrays. By 104 for each well. Twenty-four hours after inoculation, 76 pmol of siRNA use of human U95A version 2 arrays, we analyzed the eight treated and were transfected into each well with RNAi Fect Transfection Reagent untreated gastric cancer cell lines twice and the 33 surgical specimens once. according to the instructions of the manufacturer (Qiagen). Fluorescein- To select reactivated genes in at least one cell line of the eight gastric labeled negative control siRNA was used to monitor the efficiency of cancer cell lines after treatment with 5Aza-dC and TSA, we compared the transfection. Transfected cells were trypsinized, stained with trypan blue, and mean of average difference values of duplicated experiments between counted viable cells with a hemocytometer at the indicated time. untreated cells and 24-hour-treated cells. More than 3-fold overexpressed genes were selected by Microsoft Excel. The dendrogram of the clustering analysis was generated using the programs Cluster and Treeview (18). Results Reverse Transcription-PCR and cRNA Slot-Blot Analyses. First, we Genome-wide Screening of Genes Reactivated by Pharma- produced tens of micrograms of cRNA from 1 to 5 AgtotalRNAprepared cologic Unmasking of Methylation and Deacetylation. To from the gastric cancer cell lines and the surgical specimen of gastric cancer identify genes induced by treatment with 5Aza-dC and TSA, we by T7 transcription–mediated RNA amplification. Single stranded cDNAs used eight gastric cancer cell lines, HSC39, HSC43, HSC44, HSC58, were synthesized from 5 Ag cRNA by use of First-strand synthesis kit HSC59, HSC60, KATOIII, and OCUM2M. In previous studies, five (Amersham Biosciences, Piscataway, NJ) with random hexamers. We carried tumor suppressor genes, including CDH1 (19), TIMP2 (20), out PCR in a volume of 25 AL containing 1Â Accuprime PCR buffer I (Invitrogen Corp., Carlsbad, CA), 0.5 Amol/L of each primer, and Accuprime CDKN2D (21), CDKN1C (22), and RUNX3 (23, 24), have been Taq DNA polymerase (Invitrogen, Corpo, Carlsbad, CA). The thermal profile reported to be silenced by DNA methylation in gastric cancer. First, consisted of an initial denaturation at 95jC for 5 minutes followed by as a preliminary test we showed the results of the above four genes, repetitions at 95jC for 1 minute, 56jC for 1 minute, and 72jC for 1 minute, except for RUNX3, of a microarray analysis of less than 1,000 genes with a final extension step at 72jC for 10 minutes. We amplified all of the (Fig. 1). CDH1, TIMP2, CDKN2D, and CDKN1C were found to be genes from 50 ng of cDNA template with multiple cycle numbers (20-30 induced in some of the eight gastric cancer cell lines by treatment cycles) to determine the appropriate conditions for obtaining semiquantita- tive differences in expression levels. Primers for ELK1, R-RAS, RHOB, RHO6, and MSX2 were ELK1,5V-ATGGGGCTTTTCAATAGGGG-3V and 5V-CCAG- GAGTCTTTTGAACCCA-3V; R-RAS,5V-TCTGACTACGACCCCACTATTG-3V and 5V-AAGGGTGGGTATGTGATGTGTC-3V; RHOB,5V-GCCTGCT GATCGTGTTCAGT-3V and 5V-GTCGTAGGCTTGGATGCGCA-3V; RHO6,5V- CGCTCTGAACTCATCTCTTC-3Vand 5V-GGACTTAGGTGAGAAGCATGTG-3V; and MSX2,5V-CCTGTTGAGAGGAATTGATGG-3V and 5V-AAAGGTATACCG- GAGGGAGG-3V. Two micrograms of cRNA for each sample were incubated at 65C for 10 minutes and then blotted onto a nylon membrane, Hybond-N+ (Amersham). Probes were obtained by the above reverse transcription-PCR (RT-PCR) method and then labeled with [32P]dCTP using a DNA labeling kit (Roche Diagnostics, Mannheim, Germany). Methylation Analysis by Genomic Southern Blot. Five micrograms of genomic DNA from gastric cancer cell lines were digested to completion with HpaII or MspI. The restriction products were separated on 2% agarose gels and transferred to the nylon membranes. Two probes were obtained by genomic PCR using two primer sets, 5V-CTGGCTCATGGATTAGGAAT-3V and 5V-CATAATCATGAGCTCTGGCA-3V for probe 1, and 5V-ACCATCCAGTT- CATCCAGGT-3V and 5V-GGTGGAATCTCAAAGGTGCT-3V for probe 2. Bisulfite Sequencing Analysis. A region containing site S1 (Fig. 4) was amplified from bisulfite-treated genomic DNA by PCR using two primer sets, 5V-TTTAAGTAGTTGAGATTATAGG-3V and 5V-CTCTCTTAAAACTA- AATAATCCC-3V for site S1 analysis. The PCR products were applied to the ABI 310 DNA analyzer (Applied Biosystems) using the above primers or nested primers. Methylation-Specific PCR. Bisulfite-treated genomic DNA was amplified with either a methylation-specific or unmethylation-specific primer set at 40 cycles: at 95jC for 5 minutes followed by repetitions at 95jC for 45 seconds, 52jC(methylated)and52jC (unmethylated) for 45 seconds, and 72jCfor 1 minute, with a final extension step at 72jC for 10 minutes. The methylation- specific primers for site S1 were designed using 5V-TTTAAGTAGTTGAGATTA- Figure 1. Microarray results of CDH1, TIMP2, CDKN2D, and CDKN1C genes TAGG-3Vas the forward primer and 5V-AAAAACTAAAAATCAAACGTAAAACCG- in eight gastric cancer cell lines, HSC39, HSC43, HSC44, HSC58, HSC59, 3V asthereverseprimer.Theunmethylation-specificprimersforsiteS1were HSC60, KATOIII, and OCUM2M. Induction by treatment with 5Aza-dC and TSA is found in the four genes in some gastric cancer cell lines. The mean of designed using 5V-TTTAAGTAGTTGAGATTATAGG-3V as the forward primer expression values on duplicated experiments in untreated cells (white) and and 5V-AAAAAAAACTAAAAATCAAACATAAAACCA-3V as the reverse primer. treated cells (black).

Cancer Res 2005; 65: (6). March 15, 2005 2116 www.aacrjournals.org

Table 1. Gene candidates activated by cancer-associated hypomethylation

c No. Information from the NetAffx* Position on genome b GeneBank Gene symbol Gene region CpG island

1 U78027 GLA chrX:100458942-100469096 chrX:100468973-100469737 2 L12350 THBS2 chr6:169433508-169471769 chr6:169466179-169466679 3 U07736 NQO2 chr6:2945230-2964993 chr6:2945069-2945519 4 AI201243 HKE2 chr6:33365356-33366689 chr6:33364518-33364951 5 AF038844 DUSP14 chr17:32924120-32947707 chr17:32923404-32924596 6 AA675900 FNBP3 chr2:153337351-153346186 — 7 AF003837 JAG1 chr20:10566334-10602590 chr20:10600574-10603611 8 U78190 GCHFR chr15:38843577-38847203 chr15:38842689-38844079 9 AB028944 ATP11A chr13:112,527,882-112,588,428 chr13:112522082-112522353 10 W26480 FADS1 chr11:61323675-61340886 chr11:61339150-61341304 11 X81006 TRIM31 chr6:30178655-30188833 — 12 Z82244 HMOX1 chr22:34101641-34114748 chr22:34101543-34101775 13 AB014537 ZBED4 chr22:48568390-48602951 chr22:48567452-48569004 14 M97935 STAT1 chr2:191659382-191704442 chr2:191704115-191704759 15 U76702 FSTL3 chr19:627417-634389 chr19:626672-626881 16 AB016194 ELK1 chrX:47251174-47266274 chrX:47265847-47266762 17 AA203487 STK17B chr2:196827365-196861781 chr2:196861096-196861889 18 AB002354 KIAA0356 chr17:40869050-40923893 chr17:40923397-40924154 19 AL022318 APOBEC3B chr22:37702905-37713283 — 20 J03824 UROS chr10:127467142-127501757 chr10:127501320-127502187 21 J03060 GBA chr1:152017317-152027561 — 22 AL050374 DKFZP586C1619 chr11:34129111-34335378 chr11:34334806-34337286 23 AC004144 DKFZP434J046 chr19:41237660-41287847 chr19:41236842-41237905 24 X14787 THBS1 chr15:37660572-37676959 chr15:37659820-37660859 25 L11373 PCDHGC3 chr5:140835753-140872730 chr5:140835571-140836804 26 Y17829 HOMER1 chr5:78705543-78845456 chr5:78845104-78846224 27 X52730 PNMT chr17:17349604-17435719 chr17:17435324-17436230 28 D49354 PPFIA1 chr11:69794471-69908148 chr11:69793886-69795145 29 AB018293 MICAL2 chr11:12088714-12241908 chr11:12088390-12089592 30 U91329 KIF1C chr17:4842000-4868720 chr17:4841082-4842522 31 AF064084 ICMT chr1:6215520-6230298 chr1:6229795-6255208 32 L07807 DNM1 chr9:128045217-128097081 chr9:128045019-128046070 33 W28438 C14orf78 chr14:104462269-104470618 — 34 Y10936 20D7-FC4 chr19:50906131-50908104 chr19:50906802-50908020 35 AF084481 WFS1 chr4:6389649-6423064 chr4:6389353-6390254 36 U08998 TARBP2 chr12:52180972-52186480 chr12:52179463-52181580 37 U60205 SC4MOL chr4:166606463-166621472 chr4:166606060-166606668 38 AI553878 SARA1 chr10:71579969-71600272 chr10:71599740-71600593 39 AF091890 RE2 chr1:164785657-164837282 chr1:164836975-164838312 40 AL041124 PP1665 chr11:74823334-74914515 chr11:74913838-74915429 41 D79988 KNTC1 chr12:121536689-121635826 chr12:121536142-121536645 42 X96484 DGCR6 chr22:17268346-17274146 chr22:17267806-17268845 43 D12620 CYP4F3 chr19:15612707-15634634 chr19:15556299-15556535 44 Y08200 RABGGTA chr14:23804586-23810583 chr14:23810405-23810782 45 AB014554 PPFIA3 chr19:54314475-54346090 chr19:54313836-54315447 46 U60644 PLD3 chr19:45546447-45576229 chr19:45546014-45546573 47 L05144 PCK1 chr20:55569543-55574922 — 48 S76638 NFKB2 chr10:104145453-104152257 chr10:104143720-104145547 49 W26631 MAP1A chr15:41597133-41611109 chr15:41590174-41590699 50 AB006628 KIAA0290 chr19:17723336-17760363 chr19:17719372-17738777 51 X12433 ABHD2 chr15:87432628-87541199 chr15:87432551-87433213 52 X56777 ZP3 chr7:75671519-75716038 chr7:75667021-75674533 53 L40388 TRIP15 chr15:47206834-47235073 chr15:47234632-47234868 54 W26324 SLC6A8 chrX:152474239-152482887 chrX:152459482-152460391 55 M14949 RRAS chr19:54830364-54835212 chr19:54835036-54835787 56 M14764 NGFR chr17:44927666-44947360 chr17:44927345-44930315 57 Z19574 KRT17 chr17:37029221-37034335 — 58 AJ012590 H6PD chr1:9229100-9260797 chr1:9228668-9229384

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Table 1. Gene candidates activated by cancer-associated hypomethylation (Cont’d) c No. Information from the NetAffx* Position on genome b GeneBank Gene symbol Gene region CpG island

59 AF062739 FRAT2 chr10:99082244-99084448 chr10:99083397-99084945 60 M31932 FCGR2A chr1:158288275-158302415 — 61 L35240 ENIGMA chr5:176843001-176857208 chr5:176856411-176857945 62 AB001928 CTSL2 chr9:96874492-96881094 chr9:96880412-96881279 63 AB029343 C6orf18 chr6:31218199-31233519 chr6:31233874-31234540 64 U78722 ZNF165 chr6:28154551-28165317 — 65 AL021683 SCO2 chr22:49252143-49254116 chr22:49253753-49260912 66 U93181 SBF1 chr22:49175380-49203598 chr22:49202695-49204001 67 AL049654 PRKCABP chr22:36777903-36796207 chr22:36777688-36778003 68 Z15008 LAMC2 chr1:179887056-179945696 — 69 U79266 HSU79266 chr11:64564981-64568875 chr11:64564907-64565840 70 AJ012737 FLNC chr7:128064594-128093276 chr7:128064395-128065112 71 M97815 CRABP2 chr1:153482483-153488448 chr1:153487932-153489727 72 AL109702 LOC342236 chr15:88174837-88176705 — 73 M15205 TK1 chr17:73681776-73694726 chr17:73694673-73695044 74 U40434 MSLN chr16:750798-758866 — 75 AF070071 MSH5 chr6:31815756-31840601 chr6:31815469-31816315 76 M64925 MPP1 chrX:153570672-153597467 chrX:153597071-153597639 77 AI885381 MGC2650 chr19:50358027-50373325 chr19:50373164-50374996 78 D89078 LTB4R chr14:23852357-23855990 — 79 AF053356 GNB2 chr7:99916026-99921442 chr7:99915527-99918654 80 AF045581 BAP1 chr3:52410067-52419049 chr3:52418718-52420144 81 M12174 ARHB chr2:20568463-20570828 chr2:20568057-20569616 82 AL050196 USP49 chr6:41981070-41996855 chr6:41996847-41997541 83 AF038897 STX16 chr20:56659734-56687986 chr20:56658102-56659728 84 AA418080 LRRC15 chr3:195557281-195571769 — 85 U66684 ABCC10 chr6:43507467-43526141 chr6:43503167-43503872 86 U25988 PSG11 chr19:48203649-48222471 — 87 D89377 MSX2 chr5:174084208-174090507 chr5:174084085-174084970 88 W28504 KIAA0601 chr1:23091328-23155488 chr1:23091121-23091983 89 M91196 ICSBP1 chr16:84490275-84513710 chr16:84489623-84490443 90 Z71929 FGFR2 chr10:123227846-123347907 chr10:123346607-123348275 91 L08599 CDH1 chr16:67328696-67426945 chr16:67328536-67329845 92 X63422 ATP5D chr19:1192749-1195823 chr19:1192620-1193569 93 AF007157 PRNPIP chr1:44,355,836-44,489,973 chr1:44489751-44490206 94 X07820 MMP10 chr11:102146444-102156554 — 95 M77481 MAGEA1 chrX:151965723-151970284 — 96 X66435 HMGCS1 chr5:43325255-43349241 chr5:43348949-43349681 97 D87673 FBXL8 chr16:65751392-65755578 chr16:65751048-65751540 98 U68186 ECM1 chr1:147293656-147299109 — 99 U34584 BIK chr22:41831255-41850215 chr22:41830999-41832108 100 L22475 BAX chr19:54149929-54156866 chr19:54149739-54150305 101 L37792 STX1A chr7:72558191-72578613 chr7:72578040-72578855 102 U69563 RHO6 chr12:47537196-47545920 — 103 Z78388 NP25 chr3:113200276-113215424 — 104 U10694 MAGEA9 chrX:148368961-148374758 — 105 U27193 DUSP8 chr11:1533959-1549726 chr11:1550127-1550954 106 U02388 CYP4F2 chr19:15849834-15869884 — 107 U87459 CTAG1 chrX:153377122-153378779 — 108 AF109401 ARTN chr1:44068085-44071977 chr1:44068162-44068628 109 X02162 APOA1 chr11:116211681-116213334 — 110 J03258 VDR chr12:46521589-46585036 chr12:46584913-46585804 111 U66619 SMARCD3 chr7:150373708-150411468 chr7:150411765-150412183 112 U71364 SERPINB9 chr6:2832505-2848506 chr6:2848175-2848905 113 M34182 PRKACG chr9:68857005-68858562 chr9:68857962-68858461 114 U38980 PMS2L5 chr7:73751552-73766504 chr7:73750917-73751838 115 U10688 MAGEA4 chrX:150755278-150764208 — 116 AF061193 ED1 chrX:68618932-69042340 chrX:68506753-68620084 117 L11329 DUSP2 chr2:96230791-96233053 chr2:96232318-96233328 (Continued on the following page)

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Table 1. Gene candidates activated by cancer-associated hypomethylation (Cont’d) c No. Information from the NetAffx* Position on genome b GeneBank Gene symbol Gene region CpG island

118 AF039397 CRYM chr16:21177340-21197103 chr16:21196921-21197320 119 L22005 CDC34 chr19:482733-493086 chr19:482115-483896 120 AL096740 UBE3B chr12:108378159-108437226 chr12:108377852-108378358 121 M11507 TFRC chr3:197264660-197297251 chr3:197296586-197297526 122 X75315 RNPC1 chr20:55399870-55417790 chr20:55397680-55400370 123 AB028974 PEG10 chr7:93937468-93943655 — 124 L02867 HUMPPA chr17:70495322-70513479 chr17:70495217-70496186 125 U34038 F2RL1 chr5:76150610-76166895 chr5:76150319-76151845 126 U82987 BBC3 chr19:52415924-52426291 chr19:52421261-52426646 127 Z71460 ATP6V0A1 chr17:37864388-37928122 chr17:37864273-37864558 128 Z29331 UBE2H chr7:129066946-129186740 chr7:129185984-129186842 129 M19309 TNNT1 chr19:60336009-60352386 — 130 D31764 SNX17 chr2:27505040-27511645 chr2:27504497-27505750 131 X59618 RRM2 chr2:10213333-10221220 chr2:10210878-10214079 132 AB007918 KIAA0449 chr1:197670177-197691744 — 133 AF052432 KATNB1 chr16:56328618-56348657 chr16:56326319-56327474 134 X90908 FABP6 chr5:159589015-159598300 — 135 AB020315 DKK1 chr10:53744084-53747149 chr10:53743996-53744833 136 U48213 DBP chr19:53825759-53832400 chr19:53831969-53832943 137 J04948 ALPPL2 chr2:233097058-233100922 chr2:233097910-233100030 138 M21302 SPRR2B chr1:149878684-149880074 — 139 AF010310 PRODH chr22:17274849-17298620 chr22:17298025-17298394 140 Z54367 PLEC1 chr8:145061309-145121531 chr8:145119282-145124293 141 AB023211 PADI2 chr1:17140559-17191236 chr1:17190958-17191394 142 X70991 NAB2 chr12:55769157-55775525 chr12:55767065-55769383 143 AB014545 KIAA0645 chr22:30474623-30627554 chr22:30470190-30474618 144 U06088 GALNS chr16:87407644-87450841 chr16:87397282-87464888 145 AC005787 FZR1 chr19:3473954-3489325 — 146 U60062 FEZ1 chr11:124820860-124871333 chr11:124870348-124871529 147 U31875 DHRS2 chr14:23175422-23184686 — 148 Y00630 SERPINB2 chr18:59705922-59722100 — 149 AF071476 RGS9 chr17:60564054-60654270 chr17:60563793-60564054 150 AF007134 MAPK8IP1 chr11:45863778-45884591 chr11:45863583-45864385 151 AF090102 KIAA1155 chr2:71321524-71365868 chr2:71365244-71365973 152 AL009179 HIST1H3H chr6:27947602-27948078 chr6:27947678-27948048 153 AL022314 FLJ12242 chr22:35772116-35783930 chr22:35772190-35772738 154 U43916 EMP1 chr12:13240924-13260974 — 155 D87468 ARC chr8:143689412-143692835 chr8:143691433-143693168 156 M37984 TNNC1 chr3:52460158-52463098 — 157 S72043 MT3 chr16:55180768-55182500 chr16:55180570-55181415 158 X03656 CSF3 chr17:35425214-35427591 — 159 X83703 CARP chr10:92661837-92671012 —

*The NetAffx enables researchers to correlate their GeneChip array results with array design and annotation information (http://www.affymetrix.com/ analysis/index.affx). cThe positions were indicated according to the description of UCSC Genome Browser on Human May 2004 Assembly (http://genome.ucsc.edu/ index.html?org=Human). bThe position was indicated when a CpG island was present within 1 kb from the first exon.

with 5Aza-dC and TSA. We also confirmed the microarray data by profiles of 33 primary gastric cancers and 6 normal gastric RNA slot-blot analysis (data not shown). Next, all the RNA samples mucosae. Of the 1,383 genes, 159 genes, which are suppressed in 6 of untreated and treated cells were analyzed using microarrays normal tissues but expressed in at least one gastric cancer tissue, containing 12,625 transcripts. In each cell line, f300 to 500 genes were selected (Table 1). We showed the location of CpG islands were found to be transcriptionally activated by the pharmacologic near the first exon of the 159 genes in Table 1. In concordance with unmasking of methylation and deacetylation. a previous report (25), four tumor antigen genes, MAGEA1, Selection of Transcriptionally Activated Genes in Gastric MAGEA4, MAGEA9, and CTAG1, were found. Gastric cancer mainly Cancer by Cancer-Linked DNA Hypomethylation. To select divides into two types, diffuse and intestinal. In a manner similar to genes showing abnormal expression in gastric cancer due to a recent report for disruption of cell-type-specific methylation at cancer-linked DNA hypomethylation, we compared expression the Maspin gene promoter in undifferentiated thyroid cancers (26), www.aacrjournals.org 2119 Cancer Res 2005; 65: (6). March 15, 2005

Figure 2. Hierarchical clustering analysis with expression data of the 159 demethylated gene candidates in primary gastric cancers. Expression data of the 159 genes in 33 primary gastric cancers including the 16 diffuse type and 17 intestinal type were analyzed by the programs Cluster and Treeview (16). The two clusters consist of 7 of 17 intestinal type cancers (horizontal green bar) and 8 of 16 diffuse type cancers (horizontal red bar). Vertical red bar, gene cluster of the diffuse type; vertical green bar, intestinal type. D-P, diffuse type (red); I, intestinal type (green).

Cancer Res 2005; 65: (6). March 15, 2005 2120 www.aacrjournals.org

Figure 3. A, RT-PCR analysis of four oncogenes, ELK1, R-RAS, RHOB, and RHO6, and one potential oncogene, MSX2, in untreated and treated cells with 5Aza-dC and/or TSA. B, RT-PCR of R-RAS in 6 noncancerous tissues and 33 primary gastric cancer tissues. R-RAS is suppressed in the 6 normal tissues but expressed in 18 of 33 (55%) primary gastric cancers. C, RT-PCR of R-RAS in three laser-captured regions, the pit, isthmus/ neck, and gland, in the gastric mucosa. No R-RAS mRNA is detected in the three regions, whereas H-RAS mRNA is expressed in all of the three regions.

tumor suppressor genes (MSH5 and CDH1 encoding E-cadherin) or FGFR2, together with three oncogenes in the RAS/RHO family, R- tumor suppressor gene-associated genes (BAP1 encoding BRCA1- RAS, RHOB,andRHO6.ANotchligand,JAG1, and a target for the associated protein-1, and three BCL2-associated protein genes, RAS signaling, MSX2, are also potential oncogene candidates. By RT- BIK, BBC3, and BAX) were also found to be activated by DNA PCR analysis, we next investigated reactivation of the six oncogenes hypermethylation. To investigate whether type-specific or subtype- and the two oncogene candidates in eight gastric cancer cell lines by specific DNA hypomethylation occurs, we did a hierarchical treatment with 5Aza-dC and/or TSA. As shown in Fig. 3A, five genes clustering analysis of the 159 genes in the 33 gastric cancer tissues (ELK1, R-RAS, RHOB, RHO6,andMSX2) of the eight genes were including 16 diffuse type and 17 intestinal type. As shown in Fig. 2, reactivated by treatment with 5Aza-dC alone, TSA alone, and both 7 of 17 intestinal type cancers (horizontal green bar) and 8 of 16 5Aza-dC and TSA. Although we have not done analyses to confirm diffuse type cancers (horizontal red bar) were clustered. We also reactivation, at least TK1, STAT1, RABGGTA, ABCC10, MMP10, provided three representative gene clusters in an inset in Fig. 2. The CDC34, FZR1,andMAPK8IP1 are considered to be cancer-related gene cluster of the diffuse type (vertical red bar) contains two genes. Among the five genes, we further analyzed the R-RAS gene known oncogenes, R-RAS and ARHB/RHOB, whereas that of the because a critical promoter region has been previously reported (27). intestinal type (vertical green bar) contains an oncogene, ELK1. First, we did RT-PCR analysis of R-RAS in 6 noncancerous tissues Inthegenelist(Table1),wefoundsixknownoncogenes and 33 primary gastric cancers. R-RAS was suppressed in the 6 including a member of the ETS oncogene family, ELK1,anoncogene normal tissues but expressed in 18 (55%) of 33 gastric cancers (9 in in WNT signaling, FRAT2, an oncogene amplified in gastric cancers, 16 diffuse type cancers, 9 in 17 intestinal type cancers; Fig. 3B). www.aacrjournals.org 2121 Cancer Res 2005; 65: (6). March 15, 2005

The gastric epithelium consists of tubular units of epithelial cells Aberrant Expression of R-RAS in Gastric Cancers by (28). Each unit is divided into three successive regions from surface Demethylation of Specific CpG Sites within the First Intron. to base: pit, isthmus/neck, and gland. The isthmus or neck contains We found two CpG islands (GC content >50%, length >200 bp, stem cells from which mature epithelial cells are derived and and ratio of CpG to GpC >0.6) surrounding the first exon by moved to the other two regions. Mucus-secreting pit cells migrate searching with a UCSC Genome Browser (32, 33). A schematic up from progenitor cells toward the gastric lumen. The neck cells diagram of the structure of the 5V region of the R-RAS gene is migrate down toward the base of the gland where they give rise to shown in Fig. 4A. We first investigated the methylation status in pepsinogen-producing chief cells. To conclude that there is no R- the 5V region of R-RAS in eight gastric cancer cell lines by RAS expression in all of the three regions in the gastric mucosa, we genomic Southern blot analysis with a methylation-sensitive carried out RT-PCR analysis combined with a laser-captured enzyme, HpaII, and a resistant enzyme, MspI. Two probes for the microdissection procedure on the human gastric mucosa as Southern blots are shown in Fig. 4A, and results of Southern blot described in our previous report (29). With this procedure, no R- analysis with probe 2 and RNA slot-blot analysis for quantifying RAS mRNA was detected in the three regions whereas H-RAS R-RAS mRNA levels in each cell line are shown in Fig. 4B. mRNA was expressed in all of the three regions (Fig. 3C). The Southern blot analysis with probe 1 showed no correlation expression of a pit cell marker, MUC5AC (30, 31), was significantly between the methylation status of the MspI/HapII sites and R- higher in the pit cell-enriched sample whereas a proliferating cell RAS expression levels (data not shown), whereas Southern blot marker, PCNA, was detected preferentially in the neck cell-enriched analysis with probe 2 clearly showed two types of correlation sample. This indicates the procedure was appropriately done for (Fig. 4A and B). Four bands, termed B1-4, were detected in our purpose. These results show that R-RAS is silenced in the gastric Southern blot analysis with probe 2. Increased R-RAS mRNA epithelium but activated in more than half of gastric cancers. levels correlated with a shift from B3 to B1 in HSC39, KATOIII,

Figure 4. A, schematic diagram of the structure of the 5V region of the R-RAS gene. Genomic locus of the first exon was identified by UCSC Genome Browser and the 5V CpG island of R-RAS was detected by a CpG Island Searcher (42). Two probes for genomic Southern blot analysis with HpaII and MspI are indicated (probe 1 and probe 2). MspI/HapII sites including sites S1-4 and four bands, termed B1-4, which were detected in Southern blot analysis with probe 2 (B and C), are also indicated. B, genomic Southern blot analysis with HpaII and MspI in eight gastric cancer cell lines using probe 2. H and M, blots with HpaII and MspI, respectively. Each result of RNA slot-blot analysis in eight gastric cancer cell lines is shown (bottom). C, genomic Southern blot analysis with HpaII and MspI in two normal tissues and two primary gastric cancer tissues with R-RAS expression using probe 2. H and M, blots with HpaII and MspI, respectively. Each result of RT-PCR in the above four samples is shown (bottom). Two normal tissues (N1 and N2); two primary gastric cancer tissues [T1 (I-W5) and T2 (D-P(2)2s)]. D, methylation status of the MspI/HapII site S1 and its surrounding CpG sites in gastric cancer cell lines. R-RAS mRNA levels increased depending on the levels of demethylation on site S1 and its surrounding CpG sites in HSC39, KATOIII, HSC43 or OCUM2M, HSC60, and HSC58. E, methylation-specific PCR analysis on site S1 in normal tissues, gastric cancer cell lines, and primary gastric cancers. Two noncancerous tissues (N1 and N2); two primary gastric cancer tissues without R-RAS expression [T4 (I-P(1)11) and T5 (D-PS59)]; and three R-RAS–expressing cancer tissues [T1 (I-W5), T2 (D-P(2)2S), and T3 (I-W80)]. Demethylation on site S1 is found only in cancer cell lines and primary gastric cancers that express R-RAS mRNA. The PCR products in lanes U and M indicate the presence of unmethylated and methylated templates, respectively.

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HSC43 or OCUM2M, HSC60, and HSC58 (type I correlation in Discussion Fig. 4B). Another correlation (type II) is that R-RAS mRNA levels In humans, the Ras family of small GTPases consists of nine increased depending on the shift from B2 to B4 in OCUM2M or closely related proteins and can be further divided into two HSC43, HSC59, and HSC44 (Fig. 4B). subgroups in terms of structural similarity. The first of these Thus, in gastric cancer cell lines, DNA demethylation for R-RAS contains the four classic p21 Ras proteins, H-RAS, N-RAS, K-RAS, activation occurred in a bidirectional manner or in two types; and E-RAS that was previously recognized as a pseudogene of H- however, in primary gastric cancers, one of the two directions or the RAS but has now become a new member (35). The other five two types was only found by Southern blot analysis (data not shown). subgroups consist of RAP, RAL, R-RAS, R-RAS2, and M-RAS. Each Representative data are shown in Fig. 4C.Intwonormaltissues(N1 member has different effects on biological phenotypes. and N2) and two primary gastric cancers with R-RAS mRNA The R-RAS gene was initially cloned as a ras-related gene (27) expression (T1 and T2), R-RAS mRNA levels also increased depending and showed interaction with the Bcl-2 gene product (36). Owing to mainly on the shift from B3 to B1 (Fig. 4C). These data indicated that an actively mutated R-RAS inhibiting Bcl-2-mediated rescue of cancer-linked demethylation on the MspI/HapII site S1 mainly leads apoptosis, it is thought that R-RAS promotes apoptosis signaling to abnormal expression of the R-RAS gene in gastric carcinogenesis. pathway(s) (37). On the other hand, as McCormick (38) pointed By genomic bisulfite sequencing in eight gastric cancer cell out, interaction between R-RAS and Bcl-2 might have a biological lines, we confirmed the methylation status of the MspI/HapII site S1 and its surrounding CpG sites in gastric cancer. As shown in Fig. 4D, R-RAS mRNA levels increased depending on the levels of demethylation on site S1 and its surrounding CpG sites in HSC39, KATOIII, HSC43, HSC60, and HSC58. We further did methylation-specific PCR using bisulfite-modified genomic DNAs in gastric cancer cell lines and primary gastric cancers. In the above five gastric cancer cell lines (HSC39, KATOIII, HSC43, HSC60, and HSC58), demethylation of site S1 was only found in HSC60 and HSC58 depending on the levels of R-RAS mRNA (Fig. 4E). We also found a correlation between the demethylation status of site S1 and R-RAS expression was observed in primary gastric cancers. Representative data were shown in Fig. 4E. Two noncancerous tissues (N1 and N2) and two primary gastric cancer tissues [T4 (I-P(1)11) and T5 (D-PS59)] without R-RAS expression showed no demethylation, whereas three R-RAS– expressing cancer tissues [T1 (I-W5), T2 (D-P(2)2S), and T3 (I-W80)] showed partial demethylation. Suppression of R-RAS by RNAi Inhibited Cell Survival in R-RAS–Expressing Gastric Cancer Cells. By mutations at codons 38 or 87 analogous to positions 12 and 61 of H-RAS for its oncogenic activation, R-RAS has been reported to have oncogenic activity, although its activity is weaker than that of H-RAS or K-RAS (34). Therefore, we investigated whether oncogenic mutation of R-RAS occurs in gastric carcinogenesis. Direct sequencing analysis of genomic and cDNA fragments containing the above two sites showed no mutation in 22 gastric cancers with R-RAS mRNA expression (data not shown). To clarify the biological meanings of aberrant expression of R-RAS mRNA in gastric cancer, we tried suppressing R-RAS in HSC58 cells with the highest level of R-RAS mRNA and HSC59 with no R-RAS mRNA by RNAi. The efficiencies of the siRNA introduction that were determined by use of a fluorescein-labeled negative control siRNA were 80% to 90% in HSC58 cells and 60% to 70% in HSC59 cells (data not shown). In R-RAS-expressing HSC58 cells, the introduction of R-RAS- targeted siRNA significantly decreased the cell number that adhered to a plate at 48 hours after the introduction, but the control siRNA did not (Fig. 5B), whereas no siRNA effect was observed in HSC59 cells (data not shown). Along with this effect in HSC58 cells, aggregated cells disappeared, and shrunken cells were frequently observed (Fig. 5C). Because a decreased level of R-RAS mRNA was Figure 5. A, real-time PCR analysis of R-RAS mRNA after siRNA transfection. detected at 7.5 hours after siRNA transfection (Fig. 5A), phenotypic Decreased R-RAS mRNA was detected at 7.5 hours after siRNA transfection, whereas no change of GAPDH mRNA level was observed even at 24 hours after alterations might be induced by some downstream events rather the transfection. B, effect on cell growth and survival after siRNA transfection. than a direct effect of decreased expression of R-RAS mRNA. In any The survived cell number clearly decreased at 48 hours after R-RAS siRNA transfection. C, morphologic changes of cells by siRNA transfection. case, these results suggest that R-RAS is essential for the survival of Accompanied by decreasing cell number, aggregated cells disappeared and the gastric cancer cells that express R-RAS by DNA demethylation. shrunken cells were observed at 48 hours after transfection of R-RAS siRNA. www.aacrjournals.org 2123 Cancer Res 2005; 65: (6). March 15, 2005

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Cancer Research function other than apoptosis because the Bcl-2 protein gastric cancer therapy. Moreover, methylation-specific PCR preferentially binds to the nucleotide-depleted form of R-RAS analysis of the R-RAS gene could become a conventional tool in vitro. Cell transformed by activated H-RAS can survive in the for identifying target patients for any new drug that may be absence of serum whereas R-RAS-transformed cells seem to die developed in the near future. In the same manner, the gene list containing 159 genes also by an apoptotic-like mechanism in response to the removal of provides many candidates for cancer-related genes that are the serum (39). It has also been shown that R-RAS activates transcriptionally activated by DNA hypomethylation in human integrin-mediated and integrin-independent cell adhesion (40, 41). Suppression of R-RAS expression by RNA interference induced cancers. Further identification of such genes may provide insight into morphologic alteration and cell death (Fig. 5). Because of the results tumor biology and provide novel diagnostic and therapeutic targets. of terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling assay showing that the proportion of apoptotic cells Acknowledgments within the adherent cell population did not alter even after siRNA Received 9/15/2004; revised 12/22/2004; accepted 1/11/2005. transfection (data not shown), cell death might arise immediately Grant support: Program for the Promotion of Fundamental Studies in Health after cell detachment from the substrate by means of R-RAS Sciences of the Pharmaceuticals and Medical Devices Agency; Grant-in-Aid for the 3rd Comprehensive 10-Year Strategy for Cancer Control and for Cancer Research (15-5 and interference by siRNA. Our results are consistent with the model for 16-16) from the Ministry of Health, Labour and Welfare of Japan; Research Grant of the regulation of cell adhesion by R-RAS. The biological significance of Princess Takamatsu Cancer Research Fund; and Research Resident Fellowship award the R-RAS function in tumor tissues should be analyzed in detail. from the Foundation for Promotion of Cancer Research (M. Fukaya). The costs of publication of this article were defrayed in part by the payment of page This study suggests that functional blocking of R-RAS itself charges. This article must therefore be hereby marked advertisement in accordance and/or the R-RAS–signaling pathway has great potential for with 18 U.S.C. Section 1734 solely to indicate this fact.

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