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

Breast Cancer Res Treat. 2006 Jan;95(2):161-70. doi: 10.1007/s10549-005-9061-3. Epub 2005 Dec 2.

Abstract

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.

MeSH terms

  • Antimetabolites, Antineoplastic / pharmacology
  • Azacitidine / pharmacology
  • Base Sequence
  • Breast Neoplasms / genetics*
  • Breast Neoplasms / metabolism
  • Breast Neoplasms / pathology
  • CCAAT-Enhancer-Binding Protein-delta / genetics*
  • CCAAT-Enhancer-Binding Protein-delta / metabolism
  • Chromatin Immunoprecipitation
  • DNA Methylation*
  • Down-Regulation
  • Electrophoretic Mobility Shift Assay
  • Female
  • Gene Expression Regulation, Neoplastic*
  • Humans
  • Molecular Sequence Data
  • Promoter Regions, Genetic / genetics*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Resting Phase, Cell Cycle
  • Signal Transduction
  • Transcription, Genetic
  • Tumor Cells, Cultured

Substances

  • Antimetabolites, Antineoplastic
  • CEBPD protein, human
  • RNA, Messenger
  • CCAAT-Enhancer-Binding Protein-delta
  • Azacitidine