Normal breast tissue DNA methylation differences at regulatory elements are associated with the cancer risk factor age

Abstract

Background: The underlying biological mechanisms through which epidemiologically defined breast cancer risk factors contribute to disease risk remain poorly understood. Identification of the molecular changes associated with cancer risk factors in normal tissues may aid in determining the earliest events of carcinogenesis and informing cancer prevention strategies.

Methods: Here we investigated the impact cancer risk factors have on the normal breast epigenome by analyzing DNA methylation genome-wide (Infinium 450 K array) in cancer-free women from the Susan G. Komen Tissue Bank (n = 100). We tested the relation of established breast cancer risk factors, age, body mass index, parity, and family history of disease, with DNA methylation adjusting for potential variation in cell-type proportions.

Results: We identified 787 cytosine-guanine dinucleotide (CpG) sites that demonstrated significant associations (Q value <0.01) with subject age. Notably, DNA methylation was not strongly associated with the other evaluated breast cancer risk factors. Age-related DNA methylation changes are primarily increases in methylation enriched at breast epithelial cell enhancer regions (P = 7.1E-20), and binding sites of chromatin remodelers (MYC and CTCF). We validated the age-related associations in two independent populations, using normal breast tissue samples (n = 18) and samples of normal tissue adjacent to tumor tissue (n = 97). The genomic regions classified as age-related were more likely to be regions altered in both pre-invasive (n = 40, P = 3.0E-03) and invasive breast tumors (n = 731, P = 1.1E-13).

Conclusions: DNA methylation changes with age occur at regulatory regions, and are further exacerbated in cancer, suggesting that age influences breast cancer risk in part through its contribution to epigenetic dysregulation in normal breast tissue.

Keywords: 5mC; Aging; Breast cancer; DNA methylation; Epigenetic drift; Epigenetics; Normal breast; Reference-free; Risk factors.

Fig. 1

Subject age is strongly associated with DNA methylation in normal breast tissue independent of cell type. a In the volcano plot, each point represents the associations between DNA methylation and age from cell-type-adjusted multivariable linear models for microarray data (limma) at individual cytosine-guanine dinucleotide (CpG) sites. Increasing -log10 (P value) values on the y-axis show increasing statistical significance and limma effect size on the x-axis positioned away from the zero value reveal the largest DNA methylation changes with age. Significant CpG sites are indicated in red (Q value < 0.01). The gene and gene regions are presented for the five CpG sites with the greatest significance. b Unsupervised clustering of DNA methylation values at age-related CpG sites (Komen, n = 100) visualized alongside CpGs measured in specific cell-types form the Roadmap to Epigenomics data set (n = 691 CpG sites). Each column represents a given tissue sample and each CpG is presented in rows

Fig. 2

Age-related DNA methylation is enriched for regions of chromatin remodeling and transcriptional control. Cytosine-guanine dinucleotide (CpG) sites hypermethylated with age (a) and CpG sites hypomethylated with age (b) are highly enriched at the binding sites of transcription factors

Fig. 3

Relationship between epigenetic clocks and cancer risk factors. a The Horvath epigenetic clock age in normal breast tissue is highly correlated with subject age (P = 2.83E-52). Age acceleration was significantly (P < 0.05) larger in African American women. b DNA methylation age as generated by the epigenetic timer of cancer (epiTOC) tool was not correlated with subject age in normal breast tissue (P > 0.05). Higher DNA methylation age was associated with subject race, as breast tissue from African American and Hispanic women demonstrated increased DNA methylation age (P < 0.05)

Fig. 4

DNA methylation differences between tumor and normal breast tissue at age-related cytosine-guanine dinucleotide (CpG) sites in both ductal carcinoma in situ (DCIS) (a, b) and invasive breast cancer (c, d)