Estrogen effects on epithelial proliferation and benign proliferative lesions in the postmenopausal primate mammary gland

Abstract

Proliferative lesions of the mammary gland are risk markers and potential precursors for the development of breast cancer in postmenopausal women. In this study we evaluated mammary epithelial proliferation and proliferative lesions in a group of 63 aged postmenopausal macaques randomized by social group to receive one of three experimental diets for 8 months: (1) control; (2) control with 17beta-estradiol (E2) at the human equivalent dose of 1.0 mg per day; and (3) control with the soy phytoestrogen equol (EQ) at the human equivalent dose of 105 mg per day. In normal mammary epithelium, treatment with E2 but not EQ resulted in greater proliferation, epithelial area, and progesterone receptor expression (P<0.05 for all). Mammary lesions included columnar cell change (26/63), columnar cell hyperplasia with and without atypia (13/63), atypical ductal hyperplasia (6/63), and atypical lobular hyperplasia (3/63). Lesions were most common within terminal ductal lobular units. The prevalence of columnar cell hyperplasia (total and atypical cases) was higher in animals treated with E2 compared to control (P<0.05 for both). Compared to normal mammary epithelium, columnar cell lesions (CCLs) showed greater constitutive expression of estrogen receptor-alpha across all groups (P<0.001) and greater expression of progesterone receptor in response to E2 (P<0.01). Independent of treatment, animals with CCLs on histology had greater gene expression of estrogen receptor-alpha and markers of estrogen receptor activity (trefoil factor 1) and proliferation (gene for Ki67 antigen) at a site contralateral to the CCL (P<0.05 for all). These findings demonstrate that the terminal ductal lobular units of the postmenopausal mammary gland contain morphologically distinct cell populations that may hyperrespond to E2 exposure, resulting in specific types of hyperplastic lesions.

Figure 1

Treatment effects on normal mammary gland epithelium. (a), whole mount images showing atrophic postmenopausal mammary gland epithelium with small lobular size in the control (con) and equol (EQ) groups compared to the estradiol (E2) group; arrowhead indicates a focus of cystic change; bars = 1.5 cm. (b), effects of E2 and EQ on epithelial density, measured by histomorphometry. (c), immunolabeling of normal epithelial cells for estrogen receptor alpha (ESR1), progesterone receptor (PGR), and the proliferation marker Ki67. Vertical bars indicate standard errors. * P < 0.05 and *** P < 0.001 vs. respective control group by Wilcoxon Rank Sum test.

Figure 2

Morphologic features of mammary gland epithelial lesions. (a), normal duct with flattened cuboidal epithelium (left). (b) – (c), columnar cell change (CCC) variants showing luminal cells with ovoid to elongate nuclei aligned perpendicular to the basement membrane. (d), terminal ductal lobular unit (TDLU) with dilation of terminal ducts lined by columnar luminal cells. (e) – (f), CCC variants without atypia. (g), columnar cell hyperplasia (CCH) without atypia showing luminal cells in >2 layers with partial luminal obstruction. (h) CCH with tufting and apical secretory blebs (consistent with human “columnar alteration with prominent secretory snouts,” or CAPSS lesion). (i) – (j) CCH with low-grade monomorphic nuclear atypia and a flat apical surface (consistent with human “flat epithelial atypia,” or FEA lesion). (k) – (l), atypical ductal hyperplasia (ADH) lesions with micropapillary-type architecture. (m), atypical lobular hyperplasia (ALH) showing multiple layers of secretory acinar cells, bordered below by small normal lobuloalveolar acini. (n) – (o), ductal carcinoma in situ (DCIS) lesions with cells arranged in sheets, micropapillae, and fenestrated bridges.

Figure 3

Prevalence of mammary epithelial lesions following control (con), estradiol (E2), and equol (EQ) treatment. CCC, columnar cell change; CCH, columnar cell hyperplasia; ADH, atypical ductal hyperplasia; ALH, atypical lobular hyperplasia. Numbers on bars indicate the number of animals identified with each type of lesion. * P < 0.05 vs. control group by Fisher’s exact test.

Figure 4

Immunolabeling of columnar cell lesions for estrogen receptor alpha (ESR1), progesterone receptor (PGR), and the proliferation marker Ki67. (a), labeling indices across treatments, stratified by lesion type. CCLs included columnar cell change (CCC) and columnar cell hyperplasia (CCH) lesions. Vertical bars indicate standard errors. * P < 0.05, ** P < 0.01, and *** P < 0.001 compared to respective normal epithelium values by Wilcoxon Rank Sum test. (b), representative immunostaining of normal duct (top row) and CCH (bottom row) for ESR1, PGR, and Ki67 in an E2-treated animal.

Figure 5

Relative expression of genes related to estrogen receptor activity and proliferation in the mammary gland. (a), effects of estradiol (E2) and equol (EQ) treatment on expression of estrogen receptor alpha (ESR1), estrogen receptor beta (ESR2), progesterone receptor (PGR), trefoil factor 1 (TFF1), and Ki67 antigen (MKI67). (b), gene expression sorted by columnar cell lesion (CCL) status. All gene expression values were corrected for internal control gene expression and then expressed relative to control group values; lesion status effects were adjusted for any treatment group effects. * P < 0.05, ** P < 0.01, and *** P < 0.001 compared to respective control values.