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Promoter methylation reduces C/EBPδ (CEBPD) gene expression in the SUM-52PE human breast cancer cell line and in primary breast tumors

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Summary

CCAAT/Enhancer Binding Proteins (C/EBPs) are a highly conserved family of leucine zipper proteins that regulate cell growth and differentiation. C/EBPδ functions in the initiation and maintenance of mammary epithelial cell G0 growth arrest and ‘loss of function’ alterations in C/EBPδ gene expression have been reported in human breast cancer and in rodent carcinogen-induced mammary tumors. The molecular mechanism underlying reduced C/EBPδ gene expression in mammary tumorigenesis, however, is unknown. In this report we demonstrate that C/EBPδ gene expression is undetectable in the SUM-52PE human breast cancer cell line and that silencing of SUM-52PE C/EBPδ gene expression is due to epigenetic promoter hypermethylation (26/27 CpGs methylated). The hypermethylated SUM-52PE C/EBPδ 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/EBPδ gene expression. C/EBPδ 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/EBPδ expressing primary breast tumors. CpG methylation adjacent to the C/EBPδ proximal promoter Sp1 site was associated with reduced C/EBPδ 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/EBPδ gene promoter hyper- and site specific-methylation and reduced C/EBPδ gene expression in breast cancer cell lines and primary breast tumors.

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Abbreviations

C/EBP:

CCAAT/Enhancer Binding Protein

DMBA:

7,12-dimethylbenz[a]anthracene

JAK1, JAK2, TYK2:

Janus Kinases

MSPCR:

Methylation specific PCR

PhIP:

2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine

STAT3:

signal transducer and activator of transcription3

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Authors and Affiliations

  1. Department of Veterinary Biosciences and Ohio State Comprehensive Cancer Center, Ohio State University, Columbus, Ohio, USA

    D. Tang, G.S. Sivko & J.W. DeWille

  2. Department of Veterinary Biosciences, Ohio State University, 1925 Coffey Road, Columbus, Ohio, 43210, USA

    J.W. DeWille

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  1. D. Tang
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  2. G.S. Sivko
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  3. J.W. DeWille
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Correspondence to J.W. DeWille.

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Tang, D., Sivko, G. & DeWille, J. Promoter methylation reduces C/EBPδ (CEBPD) gene expression in the SUM-52PE human breast cancer cell line and in primary breast tumors. Breast Cancer Res Treat 95, 161–170 (2006). https://doi.org/10.1007/s10549-005-9061-3

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