An intervention study initiated at age 4 months compared the impact of tamoxifen (25mg), raloxifene (22.5mg) and letrozole (2.5mg) administered by 60-day-release-subcutaneous-pellet on mammary preneoplasia prevalence at age 6 months in conditional genetically engineered mouse models with different Brca1 gene dosages targeted to mammary epithelial cells and germline Trp53 haploinsufficiency (10-16/cohort). The proportion of unexposed control mice demonstrating mammary preneoplasia at age 6 months was highest in Brca1fl11/fl11/Cre/p53−/+ (54%) mice followed by Brca1WT/fl11/Cre/p53−/+ mice (30%). By age 12 months invasive mammary cancers appeared in 80% of Brca1fl11/fl11/Cre/p53−/+ and 42% of Brca1WT/fl11/Cre/p53−/+ control unexposed mice. The spectrum of cancer histology was similar in both models without somatic mutation of the non-genetically engineered Brca1, Trp53, Brca2 or Dapk3 alleles. Two months exposure to tamoxifen, raloxifene and letrozole significantly reduced estrogen-mediated tertiary branching by 65%, 71% and 78%, respectively, in Brca1fl11/fl11/Cre/p53−/+ mice at age 6 months. However, only letrozole significantly reduced HAN prevalence (by 52%) and number (by 30%) and invasive cancer appeared despite tamoxifen exposure. In contrast, tamoxifen significantly reduced HAN number by 95% in Brca1WT/fl11/Cre/p53−/+ mice. Control mice with varying combinations of the different genetically modified alleles and MMTV-Cre transgene demonstrated that the combination of Brca1 insufficiency and Trp53 haploinsufficiency was required for appearance of preneoplasia and no individual genetic alteration confounded the response to tamoxifen. In summary, although specific antihormonal approaches showed effectiveness, with Brca1 gene dosage implicated as a possible modifying variable, more effective chemopreventive approaches for Brca1-mutation-induced cancer may require alternative and/or additional agents.

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Biology and transcriptomes of non-cancerous human mammary epithelial cells at risk for breast cancer development were explored following primary isolation utilizing conditional reprogramming cell technology from mastectomy tissue ipsilateral to invasive breast cancer. Cultures demonstrated consistent categorizable behaviors. Relative viability and mammosphere formation differed between samples but were stable across three different mammary-specific media. E2F cell cycle target genes expression levels were positively correlated with viability and advancing age was inversely associated. Estrogen growth response was associated with Tissue necrosis factor signaling and Interferon alpha response gene enrichment. Neoadjuvant chemotherapy exposure significantly altered transcriptomes, shifting them towards expression of genes linked to mammary stem cell formation. Breast cancer prognostic signature sets include genes that in normal development are limited to specific stages of pregnancy or the menstrual cycle. Sample transcriptomes were queried for stage specific gene expression patterns. All cancer samples and a portion of high-risk samples showed overlapping stages reflective of abnormal gene expression patterns, while other high-risk samples exhibited more stage specific patterns. In conclusion, at-risk cells preserve behavioral and transcriptome diversity that could reflect different risk profiles. It is possible that prognostic platforms analogous to those used for breast cancer could be developed for high-risk mammary cells.

Background: Reduced BRCA1 and/or p53 function in mammary tissue is associated with tamoxifen resistance in vivo and in vitro. A shift towards tamoxifen agonist activity with reduced BRCA1 function is found in vitro. The goal of this study was to identify which combinations of disrupted Brca1 and p53 gene(s) result in preneoplasia and test for tamoxifen resistance and activity in vivo. Methods: Brca1 homozygous and heterozygous conditional exon 11 knockout and wildtype mice all carrying MMTV-Cre on a p53 wildtype or haploinsufficient background received a 25 mg 60-day constant release tamoxifen pellet (Innovative Research of America, Sarasota, FL) or sham surgery at age 4m, euthanasia/necropsy at age 6m and mammary glands taken for whole mount, histological and western blot (pERK / ERK and pHistone/Histone ratios, cleaved PARP). Cohorts of untreated Brca1f11/WT11/MMTV-Cre/p53+/- and Brca1f11/f11/MMTV-Cre/p53+/- mice were observed to age 12m for mammary cancer. Fisher's exact and Kruskal-Wallis tests were used for statistical analyses. Results: Hyperplastic alveolar nodules at age 6m and cancer development at age 12m were limited to Brca1f11/WT11/MMTV-Cre/p53+/- and Brca1f11/f11/MMTV-Cre/p53+/- mice. Ductal hyperplasia was detected in Brca1f11/WT11/MMTV-Cre/p53+/+, Brca1f11/f11/MMTV-Cre/p53+/+, Brca1f11/WT11/MMTV-Cre/p53+/-, Brca1f11/f11/MMTV-Cre/p53+/- and Brca1WT11/WT11/MMTV-Cre/p53+/- but not wildtype mice. Only Brca1f11/WT11/MMTV-Cre/p53+/+ and Brca1f11/f11/MMTV-Cre/p53+/+ mice showed significant reductions in ductal hyperplasia on tamoxifen. In contrast prevalence of ductal hyperplasia and/or hyperplastic alveolar nodules was not altered by tamoxifen in Brca1WT11/WT11/MMTV-Cre/p53+/-, Brca1f11/WT11/MMTV-Cre/p53+/- or Brca1f11/f11/MMTV-Cre/p53+/- mice. No evidence of tamoxifen activity was found as proliferation measures were either reduced (percentage mammary epithelial cells with nuclear-localized PCNA) or unchanged (pERK / ERK and pHistone/Histone ratios) with tamoxifen treatment in all genotypes. Cleaved PARP was detected in tamoxifen-treated but not control mice. Conclusions: p53 haploinsufficiency in combination with loss of either one or two copies of intact Brca1 was sufficient to induce hyperplastic alveolar nodules and cancer. Tamoxifen resistance was found in all p53 haploinsufficient mice. There was no indication of a shift towards tamoxifen agonist activity, that is, no increase in expression of any proliferative marker with Brca1 deficiency, and apoptosis was activated. Alternative pathways for tamoxifen resistance involving p53 may be responsible for the tamoxifen resistance observed in Brca1 deficient preneoplastic mammary epithelial cells. Citation Format: Sahar J. Alothman, Weisheng Wang, Priscilla A. Furth. Tamoxifen fails to induce regression of mammary preneoplasia in mice lacking either one or two intact BRCA1 genes in combination with p53 haploinsufficiency but without evidence of in vivo agonist activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5576. doi:10.1158/1538-7445.AM2014-5576

Background: Previously established genetically engineered mouse (GEM) models with spontaneous mammary cancer development have both Brca1 alleles disrupted withTrp53 haploinsufficiency (Brca1f11/f11/MMTV-Cre/Trp53+/-). Cell culture plates with nanoimprinted scaffolds (SCIVAX Life Sciences, Inc) purportedly increase concordance between in vitro/in vivo results but reports are limited to human cancer cell lines. Here we characterized hyperplasia and cancer development in Brca1f11/WT11/MMTV-Cre/Trp53+/- mice and established conditions for 3D culture of primary mammary epithelial cells (MEC) using the imprinted plates. Methods: Time-course of mammary hyperplasia and cancer development was defined in female Brca1f11/WT11/MMTV-Cre/Trp53+/- (n = 23) and Brca1f11/f11/MMTV-Cre/Trp53+/- (n = 13) mice euthanized at age 6 and 12 months (m) or when largest tumor reached 1 cm3. Mammary tissue was taken at necropsy for histology (age 6/12m) and primary MEC culture (age 6m) using EpiCult-B (StemCell Technologies) on nanoimprinted scaffold plates. Primary normal (wild-type, n = 2) and precancerous MEC (Brca1f11/f11/MMTV-Cre/Trp53+/-, n = 2; Brca1f11/WT11/MMTV-Cre/Trp53+/-; n = 2) were cultured. Optimal conditions for spheroid growth were established by comparison of spheroid formation from thoracic vs. inguinal mammary glands with different numbers of plated cells and percentage FBS after 7 days culture. Endpoints included sphere number, size and presence/absence of concurrent monolayer growth. Spheres were harvested for histological examination. Hematoxylin and eosin (H&E) stained sections were read for histology and immunohistochemistry (IHC) for Pan-Cytokeratin (Pan-CK) and Ki67 performed for in vivo/in vitro comparisons. Results: Forty-two percent of Brca1f11/WT11/MMTV-Cre/Trp53+/- mice developed mammary cancer by age 12m compared to 100% of Brca1f11/f11/MMTV-Cre/Trp53+/- mice. Cancers appeared by age 6m only in Brca1f11/f11/MMTV-Cre/Trp53+/- mice. Mammary cancers in both models were carcinomas of either adeno, anaplastic or sarcomatoid types. All were Pan-CK positive and proliferation rates were similar between the two genetic models. Spheres developed from all primary MEC attempted. Optimal conditions for sphere formation were plating of 10,000 cells obtained from inguinal mammary gland per well in 5% FBS EpiCult-B. Conclusions: Here we report development of spontaneous mammary cancers in Brca1 insufficiency mice that are more representative of disease development in women who carry only one allele with a BRCA1-mutation and established conditions for use of cell culture plates with nanoimprinted scaffolds for 3D culture of normal and preneoplastic primary murine mammary epithelial cells. Supported by NCI, NIH 1RO1CA112176 (PAF). Citation Format: Sahar J. Alothman, Weisheng Wang, Bhaskar V. Kallakury, Priscilla Furth. Histological and 3D morphological evaluation of mammary cancers and primary cells from genetically engineered mice with only one copy of Brca1 disrupted in combination with Trp53 haploinsufficiency. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 656.