Epigenetic modulation of tumor suppressor CCAAT/Enhancer binding protein a activity in lung cancer

Background: Loss of tumor suppressor CCAAT/enhancer-binding protein-alpha (C/EBP alpha) 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/EBP alpha promoter may modulate C/EBP alpha expression in lung cancer. Methods: We analyzed C/EBP alpha expression in 15 human lung cancer cell lines and in 122 human lung primary tumors by northern blotting, immunoblotting, and immunohistochemistry. C/EBP alpha 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/EBP alpha promoter by chromatin immunoprecipitation. Binding of methyl-CpG-binding proteins MeCP2 and MBD2 and upstream stimulatory factor (USF) to the C/EBP alpha 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. Results: DNA methylation and histone acetylation in the upstream region (-1422 to -896) of the C/EBPa promoter were associated with low or absent C/EBPa 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/EBP alpha promoter was detected in C/EBP alpha-nonexpressing cell lines; USF binding was detected in C/EBP alpha-expressing cell lines; however, in C/EBP alpha-nonexpressing cell lines USF binding was detected only after trichostatin A and 5-aza-dC treatment. Conclusions: DNA hypermethylation of the upstream C/EBPa promoter region, not the core promoter region as previously reported, is critical in the regulation of C/EBP alpha expression in human lung cancer.