Differential efatutazone's impact on mammary neoplasia dependent upon Brca1 dose

in Endocrine-Related Cancer
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The impact of oral peroxisome proliferator-activated receptor gamma agonist (PPARG) efatutazone exposure through diet on mammary preneoplasia and cancer development was evaluated in genetically engineered mice carrying one vs two disrupted Brca1 alleles targeted to mammary epithelial cells in the background of germ-line Trp53 (p53) haploinsufficiency. Brca1WT/fl11/Cre/p53+/ and Brca1fl11/fl11/Cre/p53+/ female mice were randomly assigned to receive efatutazone-containing diet (30 mg/kg) or equivalent diet without efatutazone at age 2 months. Efatutazone exposure significantly increased the percentage of mice with tertiary branching (Fig. 1A). Palpable

 

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    Efatutazone exposure starting at age 2 months significantly reduced mammary preneoplasia. (A) Bar graphs comparing percentages of Brca1WT/fl11/Cre/p53+/ and Brca1fl11/fl11/Cre/p53+/mice demonstrating tertiary branching on fourth (inguinal) mammary gland whole mounts (WMs) following exposure to efatutazone or control diet. *P < 0.05, Fisher’s exact test, one-sided. WM number (n) analyzed, age (months (m), mean ± s.e.m.) and weight (grams (g), mean ± s.e.m.) for each cohort: Brca1WT/fl11/Cre/p53+/ Efatutazone (E): n = 12 (9.0 ± 0.7 m, 38.0 ± 1.9 g). Control (C): n = 12 (10.1 ± 0.5 m, 36.9 ± 2.4 g). Brca1fl11/fl11/Cre/p53+/ E: n = 12 (8.4 ± 0.3 m, 40.3 ± 2.2 g). C: n = 11 (8.9 ± 0.3 m, 40.6 ± 2.4 g). WMs prepared at the time of necropsy for experimental endpoints designated in IACUC-approved protocol. All available fourth gland WMs analyzed. Mean ages and weights at necropsy were not statistically significantly different between cohorts. (B) Kaplan–Meier survival plot of all Brca1WT/fl11/Cre/p53+/ mice on efatutazone (green) and control (blue) and Brca1fl11/fl11/Cre/p53+/mice on efatutazone (red) and control (black) diets enrolled in study. *Log-rank test for trend chi-Square P = 0.0005 between both Brca1WT/fl11/Cre/p53+/ and Brca1fl11/fl11/Cre/p53+/ cohorts. Cohort numbers (n), median age (m) from survival plot: Brca1WT/fl11/Cre/p53+/ E: n = 17 (12.1 m). C: n = 15 (12.1 m). Brca1fl11/fl11/Cre/p53+/ E: n = 17 (9.1 m). C: n = 19 (9.2). Censored points for death due to non-palpable-tumor-related health reasons prior to age 12 months indicated by vertical ticks (skin ulcer, rectal prolapse, leg paralysis, shaking, lymphoma, found dead). Median survival was not statistically significant between efatutazone and control cohorts for either genotype. (C) Representative phase-contrast images of mammospheres (indicated by black arrows) that formed from primary mammary epithelial cells isolated from Brca1WT/fl11/Cre/p53+/ and Brca1fl11/fl11/Cre/p53+/ on E and C diets. Images taken at 4×. Scale bar = 0.1 mm. (D) Box and whisker plots (mean and range) illustrating a reduction in mammosphere numbers forming from primary mammary epithelial cells isolated from efatutazone-exposed Brca1fl11/fl11/Cre/p53+/ mice as compared to Brca1fl11/fl11/Cre/p53+/ mice on control diet. bP < 0.05 Mann-Whitney U test, two-tailed. Twelve wells plated and analyzed per mouse. Cohort numbers (n), mean age (m) and weight (g): Brca1WT/fl11/Cre/p53+/ E: n = 7 (4.2 ± 0.0 m, 31.5 ± 1.5 g). C: n = 6 (3.9 ± 0.1 m, 34.6 ± 1.8 g). Brca1fl11/fl11/Cre/p53+/ E: n = 4 (3.9 ± 0.1 m, 32.3 ± 1.7 g). C: n = 5 (4.2 ± 0.0 m, 37.3 ± 2.9 g). Mammosphere numbers forming from Brca1WT/fl11/Cre/p53+/ and Brca1fl11/fl11/Cre/p53+/ mice on control diet were significantly lower than numbers forming from MMTV-Cre (Cre)c and wild-type (WT)a,d mice on standard facility diet. P < 0.05 Kruskal–Wallis Test, Dunn’s multiple comparison. MMTV-Cre: n = 3 (4.2 ± 0.0 m, 31.2 ± 5.1 g). WT: n = 3 (5.2 ± 0.0 m, 25.6 ± 0.6 g). (E) Box and whisker plots (mean and range) illustrating that mammospheres derived from efatutazone-exposed Brca1fl11/fl11/Cre/p53+/ were significantly smaller than those obtained from efatutazone-exposed Brca1WT/fl11/Cre/p53+/ mice. aP < 0.05 Kruskal–-Wallis Test, Dunn’s multiple comparison. Wells and cohorts analyzed were the same as those analyzed for mammosphere numbers (Panel D). (F) Scatter plots (mean ± s.e.m. indicated) comparing numbers of HANs per WM from Brca1WT/fl11/Cre/p53+/ and Brca1fl11/fl11/Cre/p53+/mice following exposure to efatutazone or control diet. *P < 0.05, Mann–Whitney U test, one-tailed. WMs and cohorts analyzed were the same as those analyzed for tertiary branching (Panel A). (G) Bar graphs comparing percentages of Brca1WT/fl11/Cre/p53+/ and Brca1fl11/fl11/Cre/p53+/mice demonstrating HANs on WM following exposure to efatutazone or control diet. *P < 0.05, Fisher’s exact test, one-sided. WMs and cohorts analyzed were the same as those analyzed for tertiary branching (Panel A).

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    Impact of efatutazone exposure on mammary cancer development in Brca1WT/fl11/Cre/p53+/and Brca1fl11/fl11/Cre/p53+/ mice. (A) Stacked bar graphs comparing numbers of mice euthanized for tumor burden with lipoma alone, lipoma and mammary cancer, or mammary cancer alone. *P < 0.05, 2 × 3 Fisher’s exact. Number (n) of mice and age (m, mean ± s.e.m.) for each cohort: Brca1WT/fl11/Cre/p53+/ E: n = 8 (9.8 ± 0.7 m). C: n = 7 (10.4 ± 0.7 m). Brca1fl11/fl11/Cre/p53+/ E: n = 13 (8.5 ± 0.4 m). C: n = 10. (9.0 ± 0.4). (B) Stacked bar graphs comparing distribution of mammary cancer histology identified in the palpable mammary cancers. Number (n) of mice and age (m, mean ± s.e.m.) for each cohort: Brca1WT/fl11/Cre/p53+/ E: n = 3 (7.7 ± 0.7 m). C: n = 7 (10.4 ± 0.7 m). Brca1fl11/fl11/Cre/p53+/ E: n = 11 (8.6 ± 0.4 m). C: n = 10. (9.0 ± 0.4). Representative hematoxylin and eosin images of triple negative mammary adenocarcinoma (C), anaplastic carcinoma (D and F), sarcomatoid carcinoma (E and G) histology from palpable mammary cancers that developed in Brca1WT/fl11/Cre/p53+/ and Brca1fl11/fl11/Cre/p53+/ mice on efatutazone or control diet. Insets to right show representative images of ER (top), PR (middle) and HER2 (bottom) IHC. (H and I) Representative images of the histology and ER, PR and HER2 IHC of the two palpable ER+/PR+ mammary adenocarcinomas that developed in Brca1fl11/fl11/Cre/p53+/ mice on efatutazone diet. Arrows indicate representative mammary cancer cells demonstrating nuclear-localized ER and PR staining. Inset IHC images taken at 40× (C, D, E, F and G). Larger images taken at 20× (C, D, E, F, G, H and I). Scale bar = 0.1 mm.

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