RANKL/RANK control Brca1 mutation-

Abstract

Breast cancer is the most common female cancer, affecting approximately one in eight women during their life-time. Besides environmental triggers and hormones, inherited mutations in the breast cancer 1 (BRCA1) or BRCA2 genes markedly increase the risk for the development of breast cancer. Here, using two different mouse models, we show that genetic inactivation of the key osteoclast differentiation factor RANK in the mammary epithelium markedly delayed onset, reduced incidence, and attenuated progression of Brca1;p53 mutation-driven mammary cancer. Long-term pharmacological inhibition of the RANK ligand RANKL in mice abolished the occurrence of Brca1 mutation-driven pre-neoplastic lesions. Mechanistically, genetic inactivation of Rank or RANKL/RANK blockade impaired proliferation and expansion of both murine Brca1;p53 mutant mammary stem cells and mammary progenitors from human BRCA1 mutation carriers. In addition, genome variations within the RANK locus were significantly associated with risk of developing breast cancer in women with BRCA1 mutations. Thus, RANKL/RANK control progenitor cell expansion and tumorigenesis in inherited breast cancer. These results present a viable strategy for the possible prevention of breast cancer in BRCA1 mutant patients.

Figure 1

Ablation of Rank in mammary epithelial cells markedly decreases tumor formation in Brca1/p53 mutant female mice. (A) Representative whole mount images (haematoxylin staining, magnification 52×) and paraffin sections (H&E staining, scale bar, 200 μm) of mammary glands from 4-month-old K5Cre;Brca1;p53 double- and K5Cre;Rank;Brca1;p53 triple-knockout littermate mice. (B) Representative images (H&E staining, scale bar, 100 μm) and (C) quantification of low-grade MINs, high-grade MINs and adenocarcinomas in mammary glands from 4-month-old K5Cre;Brca1;p53 and K5Cre;Rank;Brca1;p53 mutant littermates. Data are shown as average number of foci/section of 1 inguinal and 2 thoracic mammary glands per mouse +/− SEM. n ≥ 4 mice/group. ^*P < 0.05, ^***P < 0.001 (2-way ANOVA). (D) Representative images of Ki67 and γH2AX immunostaining of mammary glands from 4-month-old K5Cre;Brca1;p53 double- and K5Cre;Rank;Brca1;p53 triple-knockout littermates. Scale bar, 100 μm.

Figure 2

Genetic deletion or pharmacological inhibition of RANK reduces the incidence of Brca1 deletion-driven mammary tumorigenesis. (A) Onset of palpable mammary tumors in WapCre^C;Brca1;p53 and WapCre^C;Rank;Brca1;p53 mice. Data are shown as percentage of tumor-free mice. (B) Representative histological sections of mammary tumors isolated from WapCre^C;Brca1;p53 and WapCre^C;Rank;Brca1;p53 mice showing different histological grades (H&E) and altered, grade-dependent KRT5/CK5 immunostainings. Scale bar, 100 μm. (C) Schematic of the regimen used to treat MMTV-CreBrca1^{flox11/flox11} mice with control Mu-Fc or RANK-Fc to block RANKL/RANK in vivo. (D) Numbers and percentages of MMTV-Cre Brca1^{flox11/flox11} mice with pre-neoplastic lesions that received subcutaneous (sc) injection of Mu-Fc or RANK-Fc for 6 or 12 months (3 times/week) and were sacrificed for analysis at 9 and 15 month of age. (E) Representative H&E-stained sections of mammary tissue of MMTV-CreBrca1^flox11 mice that received Mu-Fc or RANK-Fc and were sacrificed at 15 month of age. Magnification, 400×.

Figure 3

RANK mediates growth and expansion of Brca1 mutant murine and BRCA1 mutant human mammary progenitor cells. (A) Quantification of Lin⁻CD24⁺CD49f^hi basal mammary epithelial cells in ovariectomized WapCre-negative control, WapCre^C;Brca1;p53 double- and WapCre^C;Rank;Brca1;p53 triple-knockout mice treated with E+P or without hormone treatment (sham). Data are from 10-12-week-old mice, at which age we never observed any evident tumors, and represent mean +/− SEM (n ≥ 3 mice/group). ^*P < 0.05, ^**P < 0.005 (Student's t-test). (B) Representative histologic images and quantification of BrdU immunostaining of mammary glands of ovariectomized WapCre^C;Brca1;p53 double-knockout and WapCre^C;Rank;Brca1;p53 triple-knockout female mice treated with E+P showing reduced mammary epithelial proliferation (reduced BrdU positivity). Quantification data are shown as average numbers of BrdU⁺ cells per duct +/− SEM (n = 4 mice/cohort). ^***P < 0.0001 (Student's t-test). Scale bar, 100 μm. (C) Colony forming capacity of basal progenitors and ER-negative alveolar mammary epithelial progenitor cells from WapCre^C;Rank;Brca1;p53 and WapCre^C;Brca1;p53 knockout mice. Data are shown as number of colonies per 400 sorted cells +/− SEM (n = 3 mice/group). ^*P < 0.05, ns, not significant (Student's t-test). (D, E) Colony forming capacity (CFC) of human mammary progenitor cells. Human mammary progenitor epithelial cells were isolated from three women carrying heterozygous BRCA1 mutations. Single mammary cell preparations were generated from organoids for CFC assays, plated, and were either untreated or treated with the anti-RANKL blocking Ab Denosumab (1 μg/ml). The quantification is shown in D. Data are shown as number of colonies per 5 000 plated cells +/− SEM (n = 3 different BRCA1 carriers per group). ^*P < 0.01 (Paired Student's t-test). Representative images of paired untreated control and anti-RANKL (Denosumab)-treated (1 μg/ml) mammary progenitor colonies are shown in E. Scale bars: upper panels, 5 mm; lower panels, 1mm.

Figure 4

RANK expression in tumor tissues and TNFRSF11A variations linked to BRCA1-mutated status, risk of breast cancer, and cancer progression. (A) Representative images showing high and low RANK expression in invasive breast cancer tissues from BRCA1 mutation carriers. (B) Representative examples of RANK expression in early epithelial pre-neoplastic lesions in women with BRCA1 mutations. Representative images of a flat epithelial atypia and a typical intraductal papilloma are shown. Scale bars are shown. (C) Graph depicting the TNFRSF11A locus including the variants associated with breast cancer risk in BRCA1 mutation carriers, and regulatory evidence from the ENCODE project and human mammary epithelial cells (HMECs). The rs884205 variant and the two in linkage disequilibrium and analyzed in TCGA dataset are marked in red. The exons are marked by blue-filled rectangles and the direction of transcription is marked by arrows in the gene structure. The chromosome 18 positions (base pairs (bp)) and linkage disequilibrium (r²) from a HapMap CEU panel are also shown. (D) Kaplan-Meier survival plots for the indicated TNFRSF11A genotypes (A and B represent the major and minor allele, respectively) in ER-negative and ER-positive breast tumors using TCGA data. (E) Gene Set Expression Analysis (GSEA) graphical output showing the positive correlation between the gene expression in tumors with the minor genotype (associated with poorer prognosis in ER-negative human breast cancer) and gene sets that characterize RANK overexpression in mammary epithelial cells of mice at 8, 24 or 72 h (GSE66174). All three sets were found to be significantly associated (P < 0.01); the score distribution curves overlap. The gene rank corresponds to the 8 h time point and the GSEA enrichment scores are shown.