MicroRNA methylomes of normal breast tissue from ER negative and ER positive breast cancer identify progression markers specific for estrogen receptor status.

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Cancer Research


The unique structure and function of normal tissues is known to be regulated by epigenetic mechanisms. Understanding how normal cells in their respective tumor milieus might affect their susceptibility to become not only malignant but acquire breast cancer (BC) subtype-specific phenotypes, may determine tumor clinical behavior outcomes. The goal was to compare genome wide methylation profiles of non-coding miRNAs of breast cancer tissue and normal breast epithelium from estrogen receptor (ER) negative (-) and ER positive (+) tumors, and assess their miRNA methylomes in the context of tumor ER phenotypes as either ER- or ER+. BC tissue from 79 patients (39 ER- and 40 ER+) and normal tissue from 39 of these patients (19 ER- and 20-ER+) were assayed using the Illumina 450K bead array. A sub analysis focused on 2249 miRNA CpGs assigned to 615 unique miRNAs. M-values were computed as a logit function [(log (beta/ (1-beta))] of the methylation beta values. T-tests were used to compare the means of the M-values for the ER+ and ER- groups. The t-test p-values were used to generate adaptive FDR (aFDR) levels and aFDRs of 0.05 or lower were considered to be statistically significant (Tier 1). Tier 1 CpGs were subsequently filtered to select only those with a mean beta ratio between ER+ and ER- of under 0.5 or over 2.0 (Tier 2). The Tier 2 CpGs were further filtered to select only those with a mean beta difference of 0.2 or more (Tier 3). In the tumor cohort, 1224/2249 (54%) CpGs were differentially methylated between ER- and ER+ BC at Tier 1. Of the 1224, 963 (78.7%) were hypermethylated, and 1035 (84.6%) were in promoter regions. The 1224 CpGs at Tier 1, the 24 at Tier 2, and 2 CpGs at Tier 3 were associated with 379, 22 and 2 genes respectively. When the same analysis was performed on normal tissue only (19 ER- and 20-ER+), 76 of the 2249 CpGs had significant aFDR values and none of those met the Tier 2 or Tier 3 criteria. Seventy-one of the 76 (93.4%) were hypermethylated, and 65 (85.5%) were in promoter regions. The 76 significant Tier 1 (aFDR) differentially methylated CpGs were associated with 48 genes of which 43 were common to tumor Tier 1 differentially methylated miRNA genes, 10 were common to tumor Tier 2 genes, and 5 were restricted to normal tissue only. Normal epithelial tissues demonstrated similar differential methylation directionality as their respective tumor counterparts, favoring promoter region localization. Accordingly, the recognition of normal breast tissue-specific epigenetic propensities that align with their tumor phenotypes, suggest the possibility of progression markers specific for ER status as well as markers not associated with progression. This provides insights into our view of possible links between epigenetic programming, progression continuums, and how hormonal receptor subtypes may be determined.





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