Abstract 3559A: Molecular subtype-specific methylation of the miR-29c promoter in breast cancer correlates with basal-like pattern of gene expression

Abstract

Abstract MicroRNA-29c (miR-29c) has been shown to have altered expression in several different cancer types, including gastric, leukemia, thyroid, and gliomas. Our lab has previously identified miR-29c as a significantly down-regulated miRNA in basal-like breast tumors and demonstrated an association with cell invasion and sensitivity to chemotherapy. However, little is known about the genetic and regulatory factors contributing to the altered expression of miR-29c in breast cancer. Expression of miR-29c was determined using qRT-PCR in breast cancer cell lines of the basal, luminal, and claudin-low subtypes. miR-29c promoter was located using CAGE-tag analysis and ChIP assays to determine transcription start sites and RNA polymerase II binding sites, respectively. CpG islands were predicted using methylation software, and primers were designed to amplify those regions for sequencing after bisulfite treatment. The percent of methylated CpGs was compared to expression levels in breast cancer cell lines and results were validated using primary tumors in the TCGA dataset. miR-29c has lower expression in basal-like cell lines compared to claudin-low and luminal cell lines. To determine if the changes in expression are driven by changes in promoter activity, we first identified the promoter of miR-29c at the region upstream of the gene on Chromosome 1q32. We then identified potential CpG islands in the promoter region. Bisulfite sequencing of DNA from breast cancer cell lines revealed a significant difference in percent of methylation between the different subtypes. The methylation of the promoter in basal, claudin-low, and luminal subtypes was 77.1%, 60.0%, and 16.6%, respectively (p=.002). The percent of methylation was inversely correlated with expression of miR-29c in the luminal and basal subtypes (R2=.68). An analysis of TCGA data for methylation at several CpG islands in the miR-29c promoter region in breast tumors confirmed a statistically significant mean difference of methylation of basal-like tumors versus Her2, luminal A and luminal B tumors. We have identified the location of the miR-29c promoter and determined that miR-29c is expressed lower in basal-like breast cancer cell lines. We have also determined that the miR-29c promoter in basal-like breast cancer cell lines has a higher percentage of methylation at CpG islands compared to luminal, which is correlated with expression differences between these two subtypes. In addition, basal-like and luminal breast tumors from TCGA have patterns of methylation in the promoter region that correlate with our cell line data. More work needs to be done to understand how methylation of mir-29c in basal-like tumors contributes to the aggressiveness of the disease and whether it is a potential predictive biomarker for resistance to chemotherapy. Citation Format: Elizabeth Carla Poli, Rachel Tolbert, Jing Zhang, Yoo-Jeong Han, Olufunmilayo I. Olopade. Molecular subtype-specific methylation of the miR-29c promoter in breast cancer correlates with basal-like pattern of gene expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3559A. doi:10.1158/1538-7445.AM2014-3559A