Abstract 4094: Identification of novel breast cancer methylation biomarkers by global demethylation, cDNA microarray, and quantitative methylation analysis

Aberrant DNA methylation is commonly found in breast cancer. Hypermethylation of CpG islands located in promoter regions of genes may result in translational gene silencing and thus play an important role in carcinogenesis. It has been documented that multiple genes involved in cell cycle regulation, apoptosis, DNA repair, hormone regulation, cell adhesion and invasion, angiogenesis, and cellular growth-inhibitory signaling pathways are hypermethylated in breast cancer. To identify additional hypermethylated genes, we conducted a global demethylation experiment using breast cancer cell lines, followed by gene expression microarray and DNA methylation analyses. Breast cancer MCF-7 cells were treated with 5-aza-2′-deoxycytidine (5-aza-dC) and trichostatin A (TSA). Gene expression profiles between 5-aza-dC/TSA treated and untreated cells were compared using the Affymetrix GeneChip Human Genome U133 Plus 2.0 Array. Expression levels of 195 genes were up-regulated at least two-fold by demethylation treatment of the MCF-7 cells. Expression levels of 16 cancer-related genes were selected for confirmation in six additional breast cancer cell lines using quantitative real-time PCR. Expression of 13 of these genes (APLP1, CARHSP1, CDKN2D, CKMT1A, FSCN1, GPX3, HBEGF, HIC2, HSPA2, NOV, S100A6, SERPINI1, and ZNF238) was confirmed to be significantly increased after 5-aza-dC/TSA treatment. Promoter methylation status of these 13 genes was examined in seven breast cancer cell lines using Sequenom MassARRAY quantitative methylation analysis. Promoter CpG island methylation of 6 genes was observed in all 7 breast cancer cell lines. The ZNF238 and HSPA2 genes had the most methylated CpG sites (each has 11 methylated CpG sites) and highest percentage of methylation in the promoter region tested in the study. In summary, we identified promoter hypermethylation in 6 cancer-related genes, including ZNF238 and HSPA2, in breast cancer cell lines. These genes may play a role in breast carcinogenesis. Further studies using human breast cancer samples are warranted to confirm these findings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4094. doi:1538-7445.AM2012-4094