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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.

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