title: Gene silencing of EREG mediated by DNA methylation and histone modification in human gastric cancers.
abstract: Epiregulin (EREG) induces cell growth by binding to the epidermal growth factor receptor (EGFR). Expression of EREG affects sensitivity to cetuximab a chimeric monoclonal antibody that inhibits the EGFR signaling pathway. The mechanism through which EREG is regulated is largely unknown, but a methyl-array study previously performed by our group revealed that EREG is methylated in gastric cancer cells. In this study, we found that EREG gene expression was low in 7 out of 11 gastric cancer cells and this downregulation was mediated by aberrant CpG methylation of the EREG promoter. Treatment with 5-aza-CdR restored EREG expression and demethylated CpG sites in the EREG promoter. Compared with DNA methyltransferase 1 (DNMT1), knock-down of DNA methyltransferase 3b (DNMT3b) significantly increased the expression of EREG and led to the demethylation of specific CpG sites in the EREG promoter, suggesting that DNMT3b primarily regulates CpG methylation and silencing of the EREG gene. EREG methylation was observed in 30% (4/13) of human primary gastric tumor tissues we evaluated. In addition to DNA methylation, results from a chromatin immunoprecipitation assay demonstrated that transcriptional levels of EREG were associated with the enrichment of active histone marks (H3K4me3 and AcH3) and of a repressive mark (H3K27me2). Treatment with 5-aza-CdR dynamically increased the low occupancy of H3K4me3 and AcH3, while decreasing the high enrichment of H3K27me2, indicating that dynamic histone modifications contribute to EREG regulation in addition to DNA methylation. Finally, the combination of 5-aza-CdR and cetuximab exerted a synergistic anti-proliferative effect on gastric cancer cells. Taken together, the results of our study showed for the first time that EREG is epigenetically silenced in gastric cancer cells by aberrant DNA methylation and histone modification.
authors.affiliation: Cancer Research Institute, Seoul National University College of Medicine, South Korea.
authors.forename: Jiyeon
authors.initials: J
authors.lastname: Yun
authors.forename: Sang-Hyun
authors.initials: SH
authors.lastname: Song
authors.forename: Jinah
authors.initials: J
authors.lastname: Park
authors.forename: Hwang-Phill
authors.initials: HP
authors.lastname: Kim
authors.forename: Young-Kwang
authors.initials: YK
authors.lastname: Yoon
authors.forename: Kyung-Hun
authors.initials: KH
authors.lastname: Lee
authors.forename: Sae-Won
authors.initials: SW
authors.lastname: Han
authors.forename: Do-Youn
authors.initials: DY
authors.lastname: Oh
authors.forename: Seock-Ah
authors.initials: SA
authors.lastname: Im
authors.forename: Yung-Jue
authors.initials: YJ
authors.lastname: Bang
authors.forename: Tae-You
authors.initials: TY
authors.lastname: Kim
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage; D061067:Antibodies, Monoclonal, Humanized; D001374:Azacitidine / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000494:pharmacology; D045744:Cell Line, Tumor; D000068818:Cetuximab; D018899:CpG Islands; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics; D019175:DNA Methylation; D000077209:Decitabine; D004815:Epidermal Growth Factor / Q000037:antagonists & inhibitors* / Q000235:genetics*; D044127:Epigenesis, Genetic / Q000187:drug effects; D066260:Epiregulin; D066246:ErbB Receptors / Q000378:metabolism; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D013274:Stomach Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism*; D023041:Xenograft Model Antitumor Assays; D000097584:DNA Methyltransferase 3B
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D001374:Azacitidine; D000068818:Cetuximab; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D000077209:Decitabine; D004815:Epidermal Growth Factor; D066260:Epiregulin; D066246:ErbB Receptors; D006657:Histones; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D034741:RNA, Small Interfering; D000097584:DNA Methyltransferase 3B; C587880:EREG protein, human; C587881:Ereg protein, mouse; C000619380:DNMT1 protein, human; C000619381:Dnmt1 protein, mouse