Exploring DNA methylation changes in promoter, intragenic, and intergenic regions as early and late events in breast cancer formation

Abstract

Background: Breast cancer formation is associated with frequent changes in DNA methylation but the extent of very early alterations in DNA methylation and the biological significance of cancer-associated epigenetic changes need further elucidation.

Methods: Pyrosequencing was done on bisulfite-treated DNA from formalin-fixed, paraffin-embedded sections containing invasive tumor and paired samples of histologically normal tissue adjacent to the cancers as well as control reduction mammoplasty samples from unaffected women. The DNA regions studied were promoters (BRCA1, CD44, ESR1, GSTM2, GSTP1, MAGEA1, MSI1, NFE2L3, RASSF1A, RUNX3, SIX3 and TFF1), far-upstream regions (EN1, PAX3, PITX2, and SGK1), introns (APC, EGFR, LHX2, RFX1 and SOX9) and the LINE-1 and satellite 2 DNA repeats. These choices were based upon previous literature or publicly available DNA methylome profiles. The percent methylation was averaged across neighboring CpG sites.

Results: Most of the assayed gene regions displayed hypermethylation in cancer vs. adjacent tissue but the TFF1 and MAGEA1 regions were significantly hypomethylated (p ≤0.001). Importantly, six of the 16 regions examined in a large collection of patients (105 - 129) and in 15-18 reduction mammoplasty samples were already aberrantly methylated in adjacent, histologically normal tissue vs. non-cancerous mammoplasty samples (p ≤0.01). In addition, examination of transcriptome and DNA methylation databases indicated that methylation at three non-promoter regions (far-upstream EN1 and PITX2 and intronic LHX2) was associated with higher gene expression, unlike the inverse associations between cancer DNA hypermethylation and cancer-altered gene expression usually reported. These three non-promoter regions also exhibited normal tissue-specific hypermethylation positively associated with differentiation-related gene expression (in muscle progenitor cells vs. many other types of normal cells). The importance of considering the exact DNA region analyzed and the gene structure was further illustrated by bioinformatic analysis of an alternative promoter/intron gene region for APC.

Conclusions: We confirmed the frequent DNA methylation changes in invasive breast cancer at a variety of genome locations and found evidence for an extensive field effect in breast cancer. In addition, we illustrate the power of combining publicly available whole-genome databases with a candidate gene approach to study cancer epigenetics.

Fig. 1

Example of how some gene regions were chosen for examination in this study on the basis of available RRBS DNA methylation profiles for breast cancer cell lines and normal cell cultures and tissues visualized in the UCSC Genome Browser [13]. a The EN1 gene structure with exons as heavy horizontal bars; b, the aligned CpG islands in the illustrated region.; c, DNA methylation (ENCODE/RRBS/HudsonAlpha) profiles for the indicated cell cultures and normal tissues using an 11-color, semi-continuous scale (see color key) to indicate the average DNA methylation levels at each monitored CpG site; d, aligned transcription results indicating that the non-transformed breast cancer cell line is not transcribing this gene irrespective of its lack of DNA methylation. Paradoxically, normal myoblasts are transcribing it despite some upstream DNA methylation. All data are from ENCODE [19]

Fig. 2

Mean percent methylation and 95 % error bars by gene and tissue type for the DNA regions listed in Table 1. a DNA methylation analysis of samples from the Breast Cancer Care in Chicago study (2005-2008) as determined by our bisulfite pyrosequencing. Control samples (reduction mammoplasty) from unaffected women are represented by green bars, cancer-adjacent, histologically normal samples by blue bars and cancer samples by red bars. b Bioinformatic analysis of DNA methylation of breast cancer samples and paired non-cancerous adjacent samples from The Cancer Genome Atlas (TCGA). Paired non-cancerous adjacent samples are represented by blue bars and cancer samples by red bars. In both panels, promoter sequences are displayed first, followed by upstream sequences, then introns and lastly, DNA repeats