title: Discovery of epigenetically masked tumor suppressor genes in endometrial cancer.
abstract: Realization that many tumor suppressor genes are silenced by epigenetic mechanisms has stimulated the discovery of novel tumor suppressor genes. We used a variety of research tools to search for genes that are epigenetically silenced in human endometrial cancers. Changes in global gene expression of the endometrial cancer cell line Ishikawa was analyzed after treatment with the demethylating agent 5-aza-2'-deoxycytidine combined with the histone deacetylase inhibitor suberoylanilide bishydroxamide. By screening over 22,000 genes, candidate tumor suppressor genes were identified. Additional microarray analysis and real-time reverse transcription-PCR of normal and cancerous endometrial samples and search for CpG islands further refined the list. Tazarotene-induced gene-1 (Tig1) and CCAAT/enhancer binding protein-alpha (C/ebpalpha) were chosen for further study. Expression of both genes was low in endometrial cancer cell lines and clinical samples but high in normal endometrial tissues. Bisulfite sequencing, restriction analysis, and/or methylation-specific PCR revealed aberrant methylation of the CpG island in the Tig1 gene of all 6 endometrial cancer cell lines examined and 4 of 18 clinical endometrial cancers, whereas the C/ebpalpha promoter remained unmethylated in endometrial cancers. Chromatin immunoprecipitation showed increased acetylated histone H3 bound to both Tig1 and C/ebpalpha genes after treatment with 5-aza-2'-deoxycytidine and/or suberoylanilide bishydroxamide. Forced expression of either TIG1 or C/EBPalpha led to significant growth reduction of Ishikawa cells. Our data suggest that C/ebpalpha and Tig1 function as tumor suppressor proteins in endometrial cancers and that their reexpression may be a therapeutic target.
authors.affiliation: Division of Hematology/Oncology, Cedars-Sinai Medical Center/University of California at Los Angeles School of Medicine, 8700 Beverly Boulevard, Los Angeles, CA 90048, USA.
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Takai
authors.forename: Norihiko
authors.initials: N
authors.lastname: Kawamata
authors.forename: Christine S
authors.initials: CS
authors.lastname: Walsh
authors.forename: Sigal
authors.initials: S
authors.lastname: Gery
authors.forename: Julian C
authors.initials: JC
authors.lastname: Desmond
authors.forename: Sadie
authors.initials: S
authors.lastname: Whittaker
authors.forename: Jonathan W
authors.initials: JW
authors.lastname: Said
authors.forename: Laura M
authors.initials: LM
authors.lastname: Popoviciu
authors.forename: Peter A
authors.initials: PA
authors.lastname: Jones
authors.forename: Isao
authors.initials: I
authors.lastname: Miyakawa
authors.forename: H Phillip
authors.initials: HP
authors.lastname: Koeffler
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000235:genetics; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D018899:CpG Islands / Q000235:genetics; D016889:Endometrial Neoplasms / Q000235:genetics*; D044127:Epigenesis, Genetic / Q000235:genetics*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D016147:Genes, Tumor Suppressor; D006657:Histones / Q000378:metabolism; D006801:Humans; D056892:Mediator Complex; D008745:Methylation; D008875:Middle Aged; D014157:Transcription Factors / Q000235:genetics
chemical_list: D022763:CCAAT-Enhancer-Binding Protein-alpha; D006657:Histones; C416944:MED15 protein, human; D056892:Mediator Complex; D014157:Transcription Factors