Mammary gland morphological and gene expression changes underlying pregnancy protection of breast cancer tumorigenesis

Abstract

A full-term pregnancy early in life reduces lifetime risk of developing breast cancer, and the effect can be mimicked in rodents by full-term pregnancy or short-term treatment with exogenous estrogen and progesterone. To gain insight into the protective mechanism, 15 3-mo-old postpubertal virgin Lewis rats were randomly assigned to three groups: control (C), pregnancy (P), or hormone (H). The P group animals underwent a full-term pregnancy, and H group animals were implanted subcutaneously with silastic capsules filled with ethynyl estradiol and megesterol acetate for 21 days. C and P animals were implanted with sham capsules. On day 21 capsules were removed, which was followed by a 49-day involution period, euthanasia, and mammary tissue collection. Global gene expression was measured using Rat Genome 230.2 Arrays. Histological analysis revealed that P and H treatments induced sustained morphological changes in the mammary gland with significantly increased percentages of mammary parenchyma and stromal tissues and higher ratio of stroma to parenchyma. Transcriptome analysis showed that P and H treatments induced sustained global changes in gene expression in the mammary gland. Analysis of commonly up- and downregulated genes in P and H relative to C treatment showed increased expression of three matrix metallopeptidases (Mmp3, 8, and 12), more differentiated mammary phenotype, enhanced innate and adaptive immunity, and reduced cell proliferation and angiogenic signatures. The sustained morphological and global gene expression changes in mammary tissue after pregnancy and hormone treatment may function together to provide the protective effect against breast cancer.

Fig. 1.

Profiles of serum 17-β estradiol (A) and progesterone (B) concentrations in the experimental rats of the control (C), pregnancy (P), and exogenous hormone treatment (H) groups.

Fig. 2.

A: histological changes of the mammary gland of rats at day 49 after a full-term pregnancy (P) or exogenous hormone treatment (H) compared with the control (C) rats (scale bars = 100 μm). Each photomicrograph was randomly selected from an individual animal within the group. B: histological analyses of a mammary gland section using the free-hand area-of-interest (AOI) tool of Image Pro Plus for the areas of parenchyma (ii) or the areas of both parenchymal and stromal tissues (iii). i, The section of ii and iii without using the AOI tool.

Fig. 3.

Three-dimensional principle component analysis plot (PCA) of genome-wide gene expression patterns in the mammary gland of rats at day 49 after a full-term pregnancy (square) or exogenous hormone treatment (diamond) compared with the control (circle) rats.

Fig. 4.

Venn diagram analysis of the differentially up (A)- and down (B)-regulated genes in the mammary gland at day 49 after a full-term pregnancy or 21-day exogenous hormone treatment. The cut-off criteria were set at fold change ≥ 1.4 and P < 0.05.

Fig. 5.

Real-time qRT-PCR analysis of B-cell leukemia/lymphoma 11B (Bcl11b; A) and tenascin-C (Tnc; B) mRNA expression in the mammary gland of rats at day 49 after a full-term pregnancy or exogenous hormone treatment compared with the control rats. Relative expression values (means ± SE) were normalized against housekeeping gene HPRT. Different lowercase letters represent P < 0.05.

Fig. 6.

A: immunohistochemical staining (brown color as indicated by arrows) of the macrophage marker ED1 (left) and the matrix metallopeptidase MMP8 (right) in the mammary gland of rats at day 49 after a full-term pregnancy (P) or exogenous hormone treatment (H) compared with the control (C) rats. One representative staining is shown for each group. Scale bar = 200 μm. B: graphs representing the percentages of ED1-stained areas per unit of parenchyma and stroma. Different lowercase letters represent P < 0.01.

Fig. 7.

Schematic diagram of proposed physiological changes and associated expression changes of key genes in the involuted mammary gland following a full-term pregnancy and exogenous estrogen and progesterone treatment that may contribute to the protection of mammary tumorigenesis.