Dysregulation of microRNA expression drives aberrant DNA hypermethylation in basal-like breast cancer

Abstract

Basal-like breast cancers frequently express aberrant DNA hypermethylation associated with concurrent silencing of specific genes secondary to DNMT3b overexpression and DNMT hyperactivity. DNMT3b is known to be post-transcriptionally regulated by microRNAs. The objective of the current study was to determine the role of microRNA dysregulation in the molecular mechanism governing DNMT3b overexpression in primary breast cancers that express aberrant DNA hypermethylation. The expression of microRNAs (miRs) that regulate (miR-29a, miR-29b, miR-29c, miR-148a and miR-148b) or are predicted to regulate DNMT3b (miR‑26a, miR-26b, miR-203 and miR-222) were evaluated among 70 primary breast cancers (36 luminal A-like, 13 luminal B-like, 5 HER2‑enriched, 16 basal-like) and 18 normal mammoplasty tissues. Significantly reduced expression of miR-29c distinguished basal-like breast cancers from other breast cancer molecular subtypes. The expression of aberrant DNA hypermethylation was determined in a subset of 33 breast cancers (6 luminal A-like, 6 luminal B-like, 5 HER2-enriched and 16 basal-like) through examination of methylation‑sensitive biomarker gene expression (CEACAM6, CDH1, CST6, ESR1, GNA11, MUC1, MYB, TFF3 and SCNN1A), 11/33 (33%) cancers exhibited aberrant DNA hypermethylation including 9/16 (56%) basal-like cancers, but only 2/17 (12%) non-basal-like cancers (luminal A-like, n=1; HER2-enriched, n=1). Breast cancers with aberrant DNA hypermethylation express diminished levels of miR-29a, miR-29b, miR-26a, miR-26b, miR-148a and miR-148b compared to cancers lacking aberrant DNA hypermethylation. A total of 7/9 (78%) basal-like breast cancers with aberrant DNA hypermethylation exhibit diminished levels of ≥6 regulatory miRs. The results show that i) reduced expression of miR-29c is characteristic of basal-like breast cancers, ii) miR and methylation-sensitive gene expression patterns identify two subsets of basal-like breast cancers, and iii) the subset of basal-like breast cancers with reduced expression of multiple regulatory miRs express aberrant DNA hypermethylation. Together, these findings strongly suggest that the molecular mechanism governing the DNMT3b-mediated aberrant DNA hypermethylation in primary breast cancer involves the loss of post-transcriptional regulation of DNMT3b by regulatory miRs.

Figure 1.

(A) Gene expression patterns of methylation-sensitive genes for primary breast cancers and (B) miR expression patterns and miR scores for primary breast cancers. (A) Black boxes indicate a measured level of expression for an individual gene that is below the median level of expression for the dataset, and white boxes indicate a measured level of expression for an individual gene that is above the median level of expression for the dataset. The numbers at the bottom of each column indicate the gene expression score which reflects the number of methylation-sensitive genes expressed at diminished levels in an individual cancer. (B) Black boxes indicate a level of expression for an individual miR below the median value for the dataset, and white boxes indicate a level of expression of an individual miR that is above the median value for the dataset. The numbers at the bottom of each column indicate the miR score which represents a measure of the number of miRs expressed at diminished levels in an individual breast cancer. Individual breast cancers are designated with: B, basal-like; H, HER2-enriched; LA, luminal A-like; and LB, luminal B-like.

Figure 2.

Differential miR expression in basal-like breast cancers with and without aberrant DNA hypermethylation. (A) Black bars represent average miR expression among basal-like breast cancers exhibiting aberrant DNA methylation (n=9), and grey bars represent average miR expression among basal-like breast cancers that lack aberrant DNA hypermethylation (n=7). Comparison of the observed expression levels between groups of basal-like breast cancers was accomplished using an unpaired t-test (two-tailed). The levels of expression for miR-29a and miR-26a were significantly different between basal-like breast cancers that exhibit and lack aberrant DNA hypermethylation (p= 0.03). (B–G) Black bars represent miR expression levels for individual basal-like breast cancers that exhibit aberrant DNA hypermethylation, and grey bars represent miR expression levels for individual basal-like breast cancers that lack aberrant DNA methylation. The black dashed line represents the optimal threshold value determined by Baysian analysis for correct assignments related to aberrant DNA hypermethylation status of individual breast cancers. Each real-time assay was performed in triplicate and the error bars represent SEM. (B) miR-29b, (C) miR-26a, (D) miR-26b, (E) miR-29a, (F) miR-148a, and (G) miR-148b.

Figure 3.

miR expression patterns correlate with promoter methylation status among basal-like breast cancers. Correlation of miR expression patterns (miR expression score) with gene expression levels based on qPCR results (gene expression score) for methylation-sensitive genes among basal-like breast cancers that exhibit or lack aberrant DNA hypermethylation. Closed circles indicate individual basal-like breast cancers with aberrant DNA hypermethylation and open circles indicate individual basal-like breast cancers lacking aberrant DNA hypermethylation.

Figure 4.

Co-regulation of miR expression among primary breast cancers. Primary breast cancers demonstrate statistically significant relationships between miR expression levels suggesting co-regulation of miRs that regulate DNMT3b. The black line represents the linear regression trend line (p-values are indicated). Association of expression between (A) miR-26a and miR-26b; (B) miR-29a and miR-26a; (C) miR-29a and miR-29b; (D) miR-29a and miR-26b; (E) miR-29b and miR-26b; and (F) miR-148b and miR-26b.