Promoter methylation reduces C/EBPdelta (CEBPD) gene expression in the SUM-52PE human breast cancer cell line and in primary breast tumors.
CCAAT/Enhancer Binding Proteins (C/EBPs) are a highly conserved family of leucine zipper proteins that regulate cell growth and differentiation. C/EBPdelta functions in the initiation and maintenance of mammary epithelial cell G(0) growth arrest and 'loss of function' alterations in C/EBPdelta gene expression have been reported in human breast cancer and in rodent carcinogen-induced mammary tumors. The molecular mechanism underlying reduced C/EBPdelta gene expression in mammary tumorigenesis, however, is unknown. In this report we demonstrate that C/EBPdelta gene expression is undetectable in the SUM-52PE human breast cancer cell line and that silencing of SUM-52PE C/EBPdelta gene expression is due to epigenetic promoter hypermethylation (26/27 CpGs methylated). The hypermethylated SUM-52PE C/EBPdelta gene promoter is associated with reduced levels of acetylated Histone H4, consistent with a closed, transcriptionally inactive chromatin conformation. Treatment with 5'-aza-cytidine and trichostatin A (TSA) re-activates cytokine-induced SUM-52PE C/EBPdelta gene expression. C/EBPdelta gene expression is reduced to virtually undetectable levels in 32% (18/57) of primary human breast tumors. Site-specific CpG methylation was observed in 33% (6/18) of the low C/EBPdelta expressing primary breast tumors. CpG methylation adjacent to the C/EBPdelta proximal promoter Sp1 site was associated with reduced C/EBPdelta expression in a primary breast cancer sample. Electromobility shift assays (EMSA) demonstrated a significant reduction in binding to oligos containing the CpG methylation 5' to the Sp1 binding site. These results demonstrate a direct link between C/EBPdelta gene promoter hyper- and site specific-methylation and reduced C/EBPdelta gene expression in breast cancer cell lines and primary breast tumors.
Tang
D
D
Department of Veterinary Biosciences and Ohio State Comprehensive Cancer Center, Ohio State University, Columbus, Ohio 43210, USA.
Sivko
G S
GS
DeWille
J W
JW
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001374:Azacitidine / Q000494:pharmacology; D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D051743:CCAAT-Enhancer-Binding Protein-delta / Q000235:genetics* / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D019175:DNA Methylation; D015536:Down-Regulation; D024202:Electrophoretic Mobility Shift Assay; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016192:Resting Phase, Cell Cycle; D015398:Signal Transduction; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D000964:Antimetabolites, Antineoplastic; C497252:CEBPD protein, human; D012333:RNA, Messenger; D051743:CCAAT-Enhancer-Binding Protein-delta; D001374:Azacitidine