Role of ERalpha in the differential response of Stat5a loss in susceptibility to mammary preneoplasia and DMBA-induced carcinogenesis

Abstract

Deregulated estrogen signaling is evidently linked to breast cancer pathophysiology, although the role of signal transducer and activator of transcription (Stat)5a, integral to normal mammary gland development, is less clear. A mouse model of mammary epithelial cell-targeted deregulated estrogen receptor alpha (ERalpha) expression [conditional ERalpha in mammary epithelium (CERM)] was crossed with mice carrying a germ line deletion of Stat5a [Stat5a-/-] to investigate interactions between ERalpha and Stat5a in mammary tissue. CERM, CERM/Stat5a+/-, CERM/Stat5a-/-, Stat5a+/-, Stat5a-/- and wild-type (WT) mice were generated to test the roles of ERalpha and Stat5a on pubertal differentiation and cancer progression with and without exposure to the chemical carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). Only CERM/Stat5a-/- mice demonstrated delayed pubertal terminal end bud differentiation. Without DMBA exposure, Stat5a loss abrogated ERalpha-initiated hyperplastic alveolar nodule (HAN) development and, similarly, Stat5a-/- mice did not develop HANs. However, although Stat5a loss still reduced ERalpha-initiated HAN prevalence following DMBA exposure, Stat5a loss without deregulated ERalpha was associated with an increased HAN prevalence compared with WT. Progression to ERalpha(+) and ERalpha(-) adenocarcinoma was found in all CERM-containing genotypes (CERM, CERM/Stat5a+/-, CERM/Stat5a-/-) and ERalpha(+) adenocarcinoma in the Stat5a-/- genotype. The mammary epithelial cell proliferative index was increased only in CERM mice independent of Stat5a loss. No differences in apoptotic indices were found. In summary, Stat5a cooperated with deregulated ERalpha in retarding pubertal mammary differentiation and contributed to ERalpha-initiated preneoplasia, but its loss did not prevent development of invasive cancer. Moreover, in the absence of deregulated ERalpha, Stat5a loss was associated with development of both HANs and invasive cancer following DMBA exposure.

Fig. 1.

Loss of Stat5a alleles in combination with deregulated ERα led to a delayed terminal end bud differentiation. Graphs representing the number of TEBs in WT, CERM, CERM/Stat5a+/−, CERM/Stat5a−/−, Stat5a+/− and Stat5a−/− mice at 2 (A), 4 (B) and 12 months of age (C). Horizontal lines between genotypes demonstrating statistically significant differences in TEB number (*P < 0.05); mean ± SEM indicated.

Fig. 2.

Deregulated ERα led to an increase in HAN prevalence, which was abrogated with loss of Stat5a alleles. (A) Graph representing the percentage of mice with HANs at 12 months of age in nulliparous WT, CERM, CERM/Stat5a+/−, CERM/Stat5a−/−, Stat5a+/− and Stat5a−/− mice. Horizontal lines between genotypes demonstrating statistically significant differences in TEB number (*P < 0.05); mean ± SEM indicated. (B) Representative mammary gland whole mounts of WT, CERM, CERM/Stat5a+/− and CERM/Stat5a−/− mice at 12 months of age (bars represent 2 mm). Inserts represent areas of mammary gland with HANs in CERM and CERM/Stat5a+/− mice or without HAN in WT and CERM/Stat5a−/− mice. Arrows indicate HANs. (C) Representative hematoxylin and eosin of HAN from CERM mouse (left panel, bar represents 100 μm). HAN is positive for nuclear-localized Stat5a and Ki67 by immunohistochemistry (right panels, bars represent 20 μm).

Fig. 3.

Prevalence of HANs and mammary adenocarcinomas following DMBA exposure at 4 months of age. Graphs representing the percentage of mice with HANs (A) and mammary adenocarcinoma (B) in the mammary gland of DMBA-treated WT, CERM, CERM/Stat5a+/−, CERM/Stat5a+/−, Stat5a+/− and Stat5a−/− mice; mean ± SEM indicated.

Fig. 4.

Characterization of ERα, PR, Stat5a, Stat5b and cyclin D1 expression. (A) Representative histological sections of DMBA-induced mammary carcinomas stained with hematoxylin and eosin and immunohistochemistry for ERα, PR, Stat5a, Stat5b and cyclin D1 of DMBA-induced mammary carcinomas. Adenocarcinomas and adenosquamous carcinomas are indicated. Bars represent 50 μm. Arrows indicate epithelial cells with representative staining. (B) Representative sections of cyclin D1 labeled normal appearing ducts following DMBA exposure. Bars represent 10 μm. Arrows indicate epithelial cells with representative staining. (C) Western blot of Stat5b expression (92 kDa) using protein lysates from the mammary glands of CERM/Stat5a−/−, CERM/Stat5a+/− and CERM mice. Actin was used as loading control. Each lane represents one separate mouse. (D). Representative Stat5b immunohistochemistry in CERM and CERM/Stat5a−/− mammary glands. Arrows indicate epithelial cells with representative staining. (E). Immunoprecipitation of Stat5b (92 kDa) from mammary glands of CERM/Stat5a−/−, CERM/Stat5a+/−, CERM and WT mice was performed with (+) and without (−) Stat5b antibody. Blot was probed with phospho-Stat5, followed by western blot with Stat5b after complete stripping of proteins. Each lane represents one separate mouse.

Fig. 5.

Stage-dependent effects of Stat5a with respect to differentiation, hyperplasia and cancer formation in mouse models with and without ERα deregulation. Deregulated ERα (A) and loss of Stat5a demonstrated persistent TEBs by 4 months of age, abrogation of HAN development at 12 months of age and upon DMBA insult, mammary carcinoma development at 12 months of age. In contrast, loss of Stat5a without deregulated ERα (B) did not demonstrate persistent TEBs at 4 months of age; however, upon DMBA insult, it did lead to mammary carcinoma development. All groups demonstrated normal terminal ductal ends. Groups that demonstrated abnormal findings including persistent TEBs, HANs and cancer are indicated. For TEBs: normal: (≤1%), +: (1.1-2.5%), ++: (2.6-5%), +++: (5.1-7.5%), ++++: (7.6-10%). For HANs: no HAN: (0%), +: (0.1-15%), ++: (15.1-25%), +++: (25.1-35%) and ++++: (≥35.1%).