Pregnancy-induced chromatin remodeling in the breast of postmenopausal women

Abstract

Early pregnancy and multiparity are known to reduce the risk of women to develop breast cancer at menopause. Based on the knowledge that the differentiation of the breast induced by the hormones of pregnancy plays a major role in this protection, this work was performed with the purpose of identifying what differentiation-associated molecular changes persist in the breast until menopause. Core needle biopsies (CNB) obtained from the breast of 42 nulliparous (NP) and 71 parous (P) postmenopausal women were analyzed in morphology, immunocytochemistry and gene expression. Whereas in the NP breast, nuclei of epithelial cells were large and euchromatic, in the P breast they were small and hyperchromatic, showing strong methylation of histone 3 at lysine 9 and 27. Transcriptomic analysis performed using Affymetrix HG_U133 oligonucleotide arrays revealed that in CNB of the P breast, there were 267 upregulated probesets that comprised genes controlling chromatin organization, transcription regulation, splicing machinery, mRNA processing and noncoding elements including XIST. We concluded that the differentiation process induced by pregnancy is centered in chromatin remodeling and in the mRNA processing reactome, both of which emerge as important regulatory pathways. These are indicative of a safeguard step that maintains the fidelity of the transcription process, becoming the ultimate mechanism mediating the protection of the breast conferred by full-term pregnancy.

Figure 1

Histological sections of HE-stained ductules in lobules type 1 (Lob 1): a and b, from nulliparous (NP), and c and d from parous (P) women’s breast tissues; e, positive IHC stain for keratin 5/6 in basal cells; f, IHC stain for SMA in myoepithelial cells. DAB with hematoxylin (H) counterstain. Magnification bar: 100 μm.

Figure 2

A. Ki 67 immunoreactivity in the nucleus of epithelial cells in ducts (a) and Lob 1 (b) in the parous breast and in ducts (c) and Lob 1 (d) of the nulliparous breast. DAB-H counterstain; magnification bar: 100 μm. B. Box plot of the proliferative activity (Ki67 index) of ductal and Lob 1 epithelial cells. The Ki67 index in ductal epithelial cells of the nulliparous breast (NP-ducts) was significatively higher than in the Lob 1 (NP-Lob) of the same tissues (Paired t-test) (T=2.22; p<0.03), but it did not differ significatively between ducts (P-ducts) and Lob 1 (P-Lob) in the parous breast. The Ki 67 index in NP-ducts was also significative higher than in ducts and Lob 1 of the parous breast.

Figure 3

A. The H3K9(me2) IHC staining is of higher (+) intensity in the nuclei of the P (b) breast than in NP breast (a); a moderately diffuse (±) stain predominates in most of the NP breast. DAB-H counterstain. Magnification bar: 100 μm. B. Box plot shows a significantly higher number of strongly positive cells (+) in P than in NP breasts (p<0.001); moderately positive (±) cells predominate in the NP tissues (p<0.00001), and negative cells are more numerous in the P cells (p<0.05). C. IHC reaction of H3K27(me3) is of higher (+) intensity in the nuclei of the P (b) breast than in NP breast (a), in which the nuclear stain is faint and finely granular, being mostly circumscribed to the nucleoli. DAB-H counterstain. Magnification bar: 100 μm. D. Box plot shows a significantly higher number of strongly positive cells (+) in P than in NP breasts (p<0.0005); weakly to moderately positive (±) cells predominate in the NP tissues (p<0.00005); negative cells are more numerous in the P than in the NP breast cells (p<0.06).

Figure 4

A. Transcriptionally active chromatin is predominantly expressed in the euchromatin-rich nuclei (EUN) of the nulliparous women’s breast. B. Transcriptionally inactive chromatin is more frequently found in the heterochromatin-rich nuclei (HTN) of the parous breast; its presence is associated with histone 3 methylation at lysines 9 and 27, and transcriptional silencing in heterochromatin complexes.