Inhibition of Estrogen Signaling Reduces the Incidence of BRCA1-associated Mammary Tumor Formation

Abstract

BRCA1-deficient breast cancer is a very well-known hereditary cancer. However, except for resection of normal mammary glands and ovaries, there is no acceptable measure for proactively preventing tumor development. Importantly, inherited BRCA1 mutations are closely associated with tumors in hormone-responsive tissues. Here, we examined the effects of estrogen on the accumulation of genetic instabilities upon loss of BRCA1, and assessed the contribution of estrogen signaling to the incidence and progression of Brca1-mutated mammary tumors. Our in vitro studies showed that treatment of BRCA1-depleted breast cancer cells with estrogen induced proliferation. Additionally, estrogen reduced the ability of these BRCA1-knockdown cells to sense radiation-induced DNA damage and also facilitated G1/S progression. Moreover, long-term treatment of Brca1-mutant (Brca1^co/coMMTV-Cre) mice with the selective estrogen receptor (ER)-α degrader, fulvestrant, decreased the tumor formation rate from 64% to 36%, and also significantly reduced mammary gland density in non-tumor-bearing mice. However, in vivo experiments showed that fulvestrant treatment did not alter the progression of ER-positive Brca1-mutant tumors, which were frequently identified in the aged population and showed less aggressive tendencies. These findings enhance our understanding of how ER-α signaling contributes to BRCA1-deficient mammary tumors and provide evidence suggesting that targeted inhibition of ER-α signaling may be useful for the prevention of BRCA1-mutated breast cancer.

Keywords: BRCA1; cancer prevention; estrogen; fulvestrant.

Fig 1

Estrogen induces proliferation of BRCA1-deficient mammary gland epithelial cells. (A) Protein expression patterns in MCF7 cells treated with different concentration of E2; β-actin was detected as a loading control. MCF7 cells were transfected with control siRNA or siRNA targeting BRCA1, and treated with the indicated concentrations of E2 (B) for the indicated durations (C), after which survival was estimated using the MTT assay. The numbers represent mean values ± SD. (D) Whole-mount and (E) H&E staining of abdominal mammary glands from 2-month-old Brca1^co/coMMTV-cre and age-matched wild-type (Brca1^co/co) female mice treated with vehicle or E2. The panels at right are magnifications of the boxed areas in the adjacent panels (E). Scale bars: 200 μm.

Fig 2

Estrogen reduces the ability to sense DNA damage and alters G1/S phase transition in BRCA1-knockdown MCF7 cells. (A) MCF7 cells transfected with control siRNA or siRNA targeting BRCA1 were irradiated (0.3 Gy) in the absence or presence of E2 (100 nM), then immunostained for γ-H2AX foci (red) and counterstained with DAPI to detect nuclei (blue). (B). Foci numbers and signal accumulation per cell are shown in the histogram (*P < 0.05, **P < 0.01). (C) MCF7 cells transfected with control siRNA or siRNA targeting BRCA1 were irradiated in the absence or presence of E2 (100 nM), and DNA breakage was measured using Komet software (Andor Technology). (D) Olive tail moments of comet assays were calculated from at least 30 cells for each condition. (E) Representative histograms showing the DNA content of MCF7 cells. MCF7 cells transfected with control siRNA or siRNA targeting BRCA1 were treated with E2 (100 nM) for 1 day and analyzed by flow cytometry with propidium iodide staining. (F) Percentages of cells in each phase of the cell cycle are shown.

Fig 3

Estrogen increases the rigidity of mammary glands in Brca1-mutant mice. (A) Branch software (ver. 1.1) was used to estimate the total length (open) and branch numbers (filled) of ducts between the lymph node and end tip in mammary glands of 12-month-old Brca1^co/coMMTV-Cre mice in the absence or presence of E2 (1.7 mg) for 90 days. (B) The density of the mammary gland was significantly increased in E2-treated mice compared with control mice (**P < 0.01). (C) Representative whole-mount stainings of mammary glands from 12-month-old Brca1^co/coMMTV-Cre mice in the absence or presence of E2 for 90 days. (D) Histological analysis of mammary glands in the absence or presence of E2-beads. (E) Levels of PCNA and cyclin D1 in dense mammary glands were examined in the absence or presence of E2-beads by immunohistochemical analysis. Scale bars: 100 μm.

Fig 4

Inhibition of estrogen signaling by fulvestrant treatment reduces mammary tumor formation in Brca1^co/coMMTV-cre mice. (A) Kaplan-Meier curves showing tumor-free survival of Brca1^co/coMMTV-cre mice. Nine-month-old Brca1^co/coMMTV-cre mice were treated with vehicle (n = 14) or fulvestrant (n = 14) every other week for 5 months. Nine of 14 (64%) vehicle-treated mice and five of 14 (36%) fulvestrant-treated mice spontaneously developed palpable mammary tumors during this period. (B) Protein expression patterns and (C) histological analysis of tumors from vehicle- and fulvestrant-treated mice. Areas highlighted by dotted lines represent necrotic regions. Scale bars: 100 μm.

Fig 5

Fulvestrant treatment decreases the rigidity of mammary glands in Brca1-mutant mice. (A) Branch software (ver. 1.1) was used to estimate the total length (circles) and branch numbers (triangles) of ducts between the lymph node and end tip in mammary glands of 14-month-old non-tumor-bearing mice. (B) The density of the mammary gland was significantly lower in fulvestrant-treated mice than in vehicle-treated mice (*P < 0.05, **P < 0.01). (C) Representative whole-mount stainings of non-tumor-bearing mammary glands from 14-month-old Brca1^co/coMMTV-Cre mice treated with vehicle or fulvestrant for 5 months. (D) Histological analysis of mammary glands from control and fulvestrant-treated mice. Areas highlighted by boxes (left panels) were further analyzed by H&E staining, immunohistochemistry, and trichrome staining.

Fig 6

Inhibition of estrogen signaling fails to suppress the growth of Brca1-mutant breast tumors. (A) Protein expression patterns in BRCA1-deficient HCC1937 cells treated with E2 (100 nM); β-actin was detected as a loading control. (B) HCC1937 cells were treated with the indicated concentrations of FBS, with or without E2 (100 nM), and survival was estimated using the MTT assay. (C) Overview of the allograft model and fulvestrant treatments. Eighteen spontaneously developed mammary tumors were collected from Brca1^co/coMMTV-Cre mice and transplanted into nude mice. Corresponding tumors grown under mock conditions were compared with those treated with fulvestrant. After tumors in any mouse implanted with the same original tumor reached 3 cm³, all mice implanted with that tumor were sacrificed and examined. (D) Graph shows calculated RTVs (RTV of treated tumor/RTV of control tumor X 100) for tumors treated with fulvestrant. (E) Analysis of fulvestrant response-associated biomarkers. Heat map shows selected downregulated and upregulated genes according to the responsiveness to fulvestrant (|R| > 0.6, P < 0.05). Tumor samples are sorted with respect to the fulvestrant response to highlight the correlation between response and gene expression. Highly correlated genes are ranked high in the heat map. Top-ranked genes only were selected for presentation (see Supplementary Tables 1 and 2 for the full gene list). (F and G) Functional GO enrichment analysis of genes that were positively correlated (F) and negatively correlated (G) with fulvestrant responsiveness. A node is an enriched GO term (Bonferroni corrected P < 0.05), and two associated terms connected by a line share many responsive genes (Cohen's kappa score > 0.4). The node label with the highest significance among associated terms is colored.