Hormone therapy use and breast tissue DNA methylation: analysis of epigenome wide data from the normal breast study

Abstract

Hormone therapy (HT) is associated with increased risk of breast cancer, strongly dependent on type, duration, and recency of use. HT use could affect cancer risk by changing breast tissue transcriptional programs. We hypothesize that these changes are preceded by changes in DNA methylation. To explore this hypothesis we used histologically normal-appearing breast tissue from the Normal Breast Study (NBS). DNA methylation β-values were obtained using the Illumina HumanMethylation 450 BeadChips for 90 samples including all NBS-participants who used HT within 5 y before surgery. Data were analyzed using the reference-free cell mixture method. Cancer Genome Atlas (TCGA) mRNA-Seq data were used to assess correlation between DNA methylation and gene expression. We identified 527 CpG sites in 403 genes that were associated with ever using HT at genome wide significance (FDR q < 0.05), of these, 68 sites were also significantly associated with duration of use or recency of use. Twelve sites reached significance in all analyses one of which was cg01382688 in ARHGEF4 (p < 1.2x10^-7). Mutations in ARHGEF4 have been reported in breast tumors, but this is the first report of possible breast cancer-related DNA methylation changes. In addition, 22 genes included more than one significant CpG site and a majority of these sites were significantly correlated with gene expression. Although based on small numbers, these findings support the hypothesis that HT is associated with epigenetic alterations in breast tissue, and identifies genes with altered DNA methylation states which could be linked to breast cancer development.

Keywords: DNA methylation; Hormone therapy; epigenetics; epigenome wide association; normal breast tissue.

Figure 1.

Manhattan plot depicting differentially methylated CpG sites (by location and – log₁₀ p-value) from the analysis comparing ever vs. never HT-users (n = 90). Five hundred twenty-seven CpGs passed the FDR cutoff (q < 0.05), represented by the lower black line. CpGs from 3 genes (cg01382688 in ARHGEF4, cg26334888 in FAM3D and cg03472655 in SH2D5), on chromosomes 2, 3, and 1 passed a strict Bonferroni correction of p < 1.2x10⁻⁷ (higher black line).

Figure 2.

Genetic location of significant CpG sites in (a) GRB7 (from left to right: cg14263391, cg08284496, cg11183072 and cg17740645) and (b) CTBP1-AS2 (from left to right: cg20743744, cg23835219 and cg25897951. CpG sites are depicted as ball-and-stick representations. Gene sequence, CpG island location and Chromatin State are adapted from http://genome.ucsc.edu/using the human GRCh73/hg 19 build. Scale is the same in both figures.