Genomic screening for genes upregulated by demethylation revealed novel targets of epigenetic silencing in breast cancer.
Breast cancer arises through the accumulation of multiple genetic alterations and epigenetic changes such as methylation, which silences gene expression in a variety of cancers. In the present study, we applied genomic screening to identify genes upregulated by the demethylating agent 5-aza-2'-deoxycytidine (DAC) in a human breast cancer cell line (MCF7). We identified 288 genes upregulated and 29 genes downregulated more than fivefold after treatment with DAC, and gene ontology analyses revealed the genes to be involved in immune responses, apoptosis, and cell differentiation. In addition, real-time PCR analysis of ten genes silenced in MCF7 cells confirmed that they are upregulated by DAC, while bisulfite-pyrosequencing analysis confirmed that nine of those genes were silenced by methylation. We also found that treating MCF7 cells with DAC restored induction of DFNA5 by p53, as well as by two other p53 family genes, p63gamma and p73beta. Introduction of NTN4 into MCF7 cells suppressed cell growth, indicating that NTN4 has tumor suppressive activity. In primary breast cancers, we detected cancer-specific methylation of NTN4, PGP9.5, and DKK3, suggesting that methylation of these genes could be useful markers for diagnosis of breast cancer. Thus, DNA methylation appears to be a common event in breast cancer, and the genes silenced by methylation could be useful targets for both diagnosis and therapy.
Department of Molecular Biology, Cancer Research Institute, Sapporo Medical University, Sapporo 060-8556, Japan.
Tomoko
T
Fujikane
Noriko
N
Nishikawa
Minoru
M
Toyota
Hiromu
H
Suzuki
Masanori
M
Nojima
Reo
R
Maruyama
Masami
M
Ashida
Mutsumi
M
Ohe-Toyota
Masahiro
M
Kai
Toshihiko
T
Nishidate
Yasushi
Y
Sasaki
Tousei
T
Ohmura
Koichi
K
Hirata
Takashi
T
Tokino
D048868:Adaptor Proteins, Signal Transducing; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D018925:Chemokines; D047369:Chromatin Immunoprecipitation; D019175:DNA Methylation / Q000187:drug effects*; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors; D000077209:Decitabine; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D015894:Genome, Human; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D009414:Nerve Growth Factors / Q000235:genetics / Q000378:metabolism; D000075385:Netrins; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D043222:Ubiquitin Thiolesterase / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation
D048868:Adaptor Proteins, Signal Transducing; D014408:Biomarkers, Tumor; D018925:Chemokines; C498624:DKK3 protein, human; D004791:Enzyme Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; C498815:NTN4 protein, human; D009414:Nerve Growth Factors; D000075385:Netrins; D012333:RNA, Messenger; C482860:UCHL1 protein, human; D000077209:Decitabine; D015254:DNA Modification Methylases; D043222:Ubiquitin Thiolesterase; D001374:Azacitidine