Epigenetic Modulation of Tumor Suppressor CCAAT/Enhancer Binding Protein α Activity in Lung Cancer

Abstract

Loss of tumor suppressor CCAAT/enhancer-binding protein-alpha (C/EBPalpha) expression is seen in several human malignancies, including acute myelogenous leukemia and lung cancer. We hypothesized that DNA methylation and histone acetylation of the C/EBPalpha promoter may modulate C/EBPalpha expression in lung cancer. We analyzed C/EBPalpha expression in 15 human lung cancer cell lines and in 122 human lung primary tumors by northern blotting, immunoblotting, and immunohistochemistry. C/EBPalpha promoter methylation was assessed using bisulfite sequencing, combined bisulfite restriction analysis, methylation-specific polymerase chain reaction, and Southern blotting. We examined the acetylation status of histones H3 and H4 at the C/EBPalpha promoter by chromatin immunoprecipitation. Binding of methyl-CpG-binding proteins MeCP2 and MBD2 and upstream stimulatory factor (USF) to the C/EBPalpha promoter was assayed in cell lines that were untreated or treated with histone deacetylase inhibitor trichostatin A and demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) by chromatin immunoprecipitation and by electrophoretic mobility shift assays. DNA methylation and histone acetylation in the upstream region (-1422 to -896) of the C/EBPalpha promoter were associated with low or absent C/EBPalpha expression in 12 of 15 lung cancer cell lines and in 81 of 120 primary lung tumors. MeCP2 and MBD binding to the upstream C/EBPalpha promoter was detected in C/EBPalpha-nonexpressing cell lines; USF binding was detected in C/EBPalpha-expressing cell lines; however, in C/EBPalpha-nonexpressing cell lines USF binding was detected only after trichostatin A and 5-aza-dC treatment. DNA hypermethylation of the upstream C/EBPalpha promoter region, not the core promoter region as previously reported, is critical in the regulation of C/EBPalpha expression in human lung cancer.