Hyperplastic Alveolar Nodules Research Articles

Responsiveness of Brca1 and Trp53 Deficiency-Induced Mammary Preneoplasia to Selective Estrogen Modulators versus an Aromatase Inhibitor in Mus musculus.

An intervention study initiated at age 4 months compared the impact of tamoxifen (25 mg), raloxifene (22.5 mg), and letrozole (2.5 mg) administered by 60-day release subcutaneous pellet on mammary preneoplasia prevalence at age 6 months in conditional genetically engineered mouse models with different Breast cancer 1 (Brca1) gene dosages targeted to mammary epithelial cells and germline Tumor protein P53 (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 nongenetically engineered Brca1, Trp53, Brca2, or Death-associated protein kinase 3 (Dapk3) alleles. Two-month 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 hyperplastic alveolar nodules (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. Cancer Prev Res; 10(4); 244-54. ©2017 AACR.

Abstract 2286: Spontaneous mammary cancer development in genetically engineered mice with only one copy of Brca1 disrupted in combination with Trp53 haploinsufficiency

Abstract Background: Women carrying one mutated copy of BRCA1 are disposed to developing breast cancer. BRCA1-mutation-related cancers often harbor TRP53 mutations. Previously established genetically engineered mouse (GEM) models with spontaneous mammary cancer development have both copies of Brca1 disrupted in combination with Trp53 haploinsufficiency. Here we characterized cancer development in GEM with only one copy of Brca1 disrupted in mammary epithelial cells using Cre-Lox technology in combination with germ-line Trp53 haploinsufficiency. Methods: From our Brca1f11fT11/MMTV-Cre/Trp53+/− breeding program we noted mammary cancer development in female Brca1f11/WT11/MMTV-Cre/Trp53+/− mice. The time-course of mammary hyperplasia and cancer development was characterized in these mice. Mice were euthanized at age 6 months (n = 9) or when the largest tumor reached 1 cm3 or at 12 months if no tumor developed (n = 12). Cancers were resected, inguinal mammary glands processed for histology and thoracic mammary glands flash frozen or processed for primary cell culture. Hyperplastic alveolar nodules (HANs) and branching patterns were detected using inguinal mammary gland whole mounts. Histology was read on H&E stained slides and immunohistochemistry (IHC) performed for ERα, PGR and HER2 on cancer specimens. Results: Thirty-three percent of the mice developed palpable mammary tumors by age 12 months. Forty-four percent of the mice exhibited HANs at age 6 months and 56% at age 12 months. One mouse developed a female reproductive tract cancer. Three mice developed single palpable mammary adenocarcinomas. One mouse developed two cancers, one palpable spindloid and one non-palpable anaplastic carcinoma. Two adenocarcinomas have completed IHC studies and are ER/PGR/HER2 negative. Multilayered ductal hyperplasia was present only in 12-month-old mice (89%). Two-layered lobular-type hyperplasia was found in 33% of 6-month-old and 100% of 12-month-old mice. Primary cell cultures were established from both cancers attempted. Conclusions: Here we report development of a Brca1 insufficiency mouse model more representative of disease development in women who carry only one allele with a BRCA1-mutation. Similar to models with both Brca1 copies disrupted, Trp53 haploinsufficiency was required for cancer progression. The spectrum of mammary cancer histology was similar to that previously reported when both Brca1 alleles were disrupted. This model represents a tool for investigation of preventive strategies in the face of Brca1 and Trp53 haploinsufficiency and study of possible interactions between this genetic background and genetic and environmental factors that may promote cancer development. Citation Format: Sahar Alothman, Svenja Groeneveld, Ahmad Alamri, Bhaskar Kallakury, Priscilla A. Furth. Spontaneous mammary cancer development in genetically engineered mice with only one copy of Brca1 disrupted in combination with Trp53 haploinsufficiency. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2286. doi:10.1158/1538-7445.AM2015-2286

Abstract 5576: 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 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

Abstract P4-11-01: Effect of letrozole is superior to tamoxifen in limiting preneoplasia in both ERa and aromatase over-expressing mice

Abstract Background: Breast cancer develops as genetic changes give rise to preneoplastic lesions, which may or may not progress to invasive breast cancer. Studies suggest that estrogens may promote this process. Targeted over-expression of both estrogen receptor (Esr1) and aromatase (CYP19A1) to mammary epithelial cells (MECs) of genetically engineered mice results in the development of mammary hyperplasia and invasive cancer. Both of these genetic interventions lead to altered estrogen signaling pathways with increased expression levels of progesterone receptor (PGR) and cyclin D1. Tamoxifen is currently the only FDA-approved drug for chemoprevention of invasive breast cancer in premenopausal high-risk women but while effective it does not absolutely prevent cancer development. Purpose: Test the impact of letrozole and tamoxifen in both tet-op-CYP19A1/MMTV-rtTA mice and tet-op-Esr1/MMTV-rtTA. Methods: Female tet-op-CYP19A1/MMTV-rtTA and tet-op-Esr1/MMTV-rtTA mice were implanted subcutaneously with a pellet containing either letrozole 2.5mg (60-day release), tamoxifen 25mg (60-day release), or placebo at age 10m. Mice were necropsied at age 12m and mammary tissue collected for morphological, histological, and protein expression studies. Whole mounts and hematoxylin and eosin stained slides were used to evaluate growth abnormalities, hyperplastic alveolar nodules (HANs) or cancer. Immunohistochemistry and western blot analyses were used to compare and quantitate changes in protein expression. Results: Letrozole treatment resulted in no HAN detection in both Esr1 and CYP19A1 over-expressing mice. Tamoxifen reduced HAN prevalence to 14 (compared to 60%) in CYP19A1 over-expressing mice; HAN prevalence persisted in 56% (as compared to 67%) in Esr1 over-expressing mice. The reduction in preneoplasia prevalence in letrozole treated mice was accompanied by a statistically significant reduction in the proliferative index in both CYP19A1 and Esr1 over-expressing mice. However, reductions found following tamoxifen exposure were not statistically significant. Letrozole significantly reduced the percentage of ERα positive nuclei and the modified Allred score for cyclin D1 in CYP19A1 over-expressing mice, but not in Esr1 over-expressing mice as compared to control mice. In Esr1 over-expressing mice tamoxifen exposure was associated with a significant increase in the percentage of ERα positive nuclei that was not seen in the CYP19A1 over-expressing mice. PGR expression differences were not statistically significant for either treated group. Conclusions: The impact of letrozole was greater than tamoxifen in reducing prevalence of preneoplasia and MEC proliferation rates in both ERα and aromatase over-expressing mice. These results suggest that letrozole should be considered as a chemopreventive agent based on individual estrogen signaling prognostic factors. Support: P30CA051008, RO1CA112176, T32CA009686-15, KG080359. Support: P30CA051008, RO1CA112176, T32CA009686-15, KG080359. Content solely responsibility of authors and does not necessarily represent official NCI/NIH views. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-11-01.

The PPARγ Agonist Efatutazone Increases the Spectrum of Well-Differentiated Mammary Cancer Subtypes Initiated by Loss of Full-Length BRCA1 in Association with TP53 Haploinsufficiency

Peroxisome proliferator-activated receptor gamma (PPARγ) agonists have anticancer activity and influence cell differentiation. We examined the impact of the selective PPARγ agonist efatutazone on mammary cancer pathogenesis in a mouse model of BRCA1 mutation. Mice with conditional loss of full-length BRCA1 targeted to mammary epithelial cells in association with germline TP53 insufficiency were treated with efatutazone through the diet starting at age 4 months and were euthanized at age 12months or when palpable tumor reached 1 cm(3). Although treatment did not reduce percentage of mice developing invasive cancer, it significantly reduced prevalence of noninvasive cancer and total number of cancers per mouse and increased prevalence of well-differentiated cancer subtypes not usually seen in this mouse model. Invasive cancers from controls were uniformly estrogen receptor α negative and undifferentiated, whereas well-differentiated estrogen receptor α-positive papillary invasive cancers appeared in efatutazone-treated mice. Expression levels of phosphorylated AKT and CDK6 were significantly reduced in the cancers developing in efatutazone-treated mice. Efatutazone treatment reduced rates of mammary epithelial cell proliferation and development of hyperplastic alveolar nodules and increased expression levels of the PPARγ target genes Adfp, Fabp4, and Pdhk4 in preneoplastic mammary tissue. Intervention efatutazone treatment in mice with BRCA1 deficiency altered mammary cancer development by promoting development of differentiated invasive cancer and reducing prevalence of noninvasive cancer and preneoplastic disease.

Abstract 197: Reduction in lobular but not ductal hyperplasia by the PGR inhibitor ORG33628 in a CYP19A1 overexpressing mouse model of breast cancer risk.

Abstract Background: The Women's Health Initiative demonstrated increased breast cancer risk in women exposed to estrogen with progesterone. Genetically engineered mice with conditional mammary epithelial cell targeted Cytochrome P45019A1 (CYP19A1/aromatase) over-expression (tet-op-CYP19A1/MMTV-rtTA mice) tested the impact of a selective progesterone receptor (PGR) inhibitor, ORG33628, on mammary hyperplasia and cancer development. This model demonstrates increased percentages of PGR+ mammary epithelial cells (MECs) cells at the hyperplastic stage and triple negative (Estrogen Receptor (ER)-, PGR-, Receptor tyrosine-protein kinase erbB-2 (HER2/neu)-) adenocarcinomas. Purpose: Test if ORG33628 reduces mammary hyperplasia and blocks cancer formation in tet-op-CYP19A1/MMTV-rtTA mice. Methods: Female tet-op-CYP19A1/MMTV-rtTA mice were implanted subcutaneously with a pellet containing either ORG33628 7.5mg (60-day release) or placebo at age 10m. A smaller study tested ORG33628 in CYP19A1/MMTV-rtTA/trp53+/- mice. Mice were necropsied at age 12m and mammary tissue examined to determine if ORG33628 altered lobular/ductal hyperplasia prevalence or blocked progression to hyperplastic alveolar nodules (HANs) or cancer. Immunohistochemistry (IHC) was employed to compare MEC proliferative index (PI) (Ki-67) and percentage of MECs expressing nuclear-localized ER/PGR. An uterotropic assay tested if ORG33628 blocked estrogen-induced changes in the uterus/cervix. Ovariectomized 6w old mice were implanted subcutaneously with pellets of 0.72g 17β-estradiol and either ORG33628 or placebo, necropsied 3 days later, and uterine weight, PI and ER/PGR expression compared in uterine and cervical epithelium and stroma. Results: ORG33628 treatment reduced lobular hyperplasia prevalence (Fisher's exact, p<0.05) and percentages of PGR+ MEC (9.3% to 5.2%, t-test, p<0.05), but did not alter ductal hyperplasia or HAN prevalence or block cancer formation. ORG33628 reduced estrogen-induced uterine weight gain (t-test, p<0.05) and PI in cervical epithelium (38.6% to 15%, t-test, p<0.05) in the uterotropic assay. Conclusions: ORG33628 reduced lobular but not ductal hyperplasia and did not prevent progression to HANs or cancer. In normal development, estrogen plays the more important role in ductal development and progesterone in lobular development paralleling the isolated impact of ORG33628 on lobular disease here. These results suggest that reducing ductal hyperplasia may be a prerequisite for cancer prevention or that reducing lobular hyperplasia at 10m in this model is too late due to the presence of triple negative cancer progenitor cells. Support: P30CA051008, RO1CA112176, T32CA009686-15, KG080359. Content solely responsibility of authors and does not necessarily represent official NCI/NIH views. Citation Format: Sarah A. Dabydeen, Weisheng Wang, Edgar S. Diaz-Cruz, Levy Kopelovich, Robert I. Glazer, Priscilla A. Furth. Reduction in lobular but not ductal hyperplasia by the PGR inhibitor ORG33628 in a CYP19A1 overexpressing mouse model of breast cancer risk. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 197. doi:10.1158/1538-7445.AM2013-197

Effects of maternal dietary exposure to cadmium during pregnancy on mammary cancer risk among female offspring

Background:Since heavy metal cadmium is an endocrine disrupting chemical, we investigated whether maternal exposure to cadmium during the pregnancy alters mammary tumorigenesis among female offspring.Methods:From gestation day 10 to day 19, pregnant rat dams were fed modified American Institute of Nutrition (AIN93G) diet containing 39% energy from fat (baseline diet), or the baseline diet containing moderate (75 μg/kg of feed) or high (150 μg/kg) cadmium levels. Some dams were injected with 10 μg 17β-estradiol (E2) daily between gestation days 10 and 19.Results:Rats exposed to a moderate cadmium dose in utero were heavier and exhibited accelerated puberty onset. Both moderate and high cadmium dose led to increased circulating testosterone levels and reduced the expression of androgen receptor in the mammary gland. The moderate cadmium dose mimicked the effects of in utero E2 exposure on mammary gland morphology and increased both the number of terminal end buds and pre-malignant hyperplastic alveolar nodules (HANs), but in contrast to the E2, it did not increase 7, 12-dimethylbenz (a) anthracene-induced mammary tumorigenesis.Conclusions:The effects of in utero cadmium exposure were dependent on the dose given to pregnant dams: Moderate, but not high, cadmium dose mimicked some of the effects seen in the in utero E2 exposed rats, such as increased HANs in the mammary gland.

Abstract P2-04-08: Targeted Expression of the Human Chaperone BAG3 to the Murine Mammary Gland Dysregulates Mammary Gland Development and Differentiation By Unrestricted Expansion of Luminal Cells

Abstract BAG3 is a pro-survival pro-invasion chaperone. We hypothesized that human BAG3 overexpression in murine breast would contribute to mammary tumorigenesis. We generated transgenic mice ectopically expressing hBAG3 in the mammary gland under the control of the Mouse Mammary Tumor Viral promoter (MMTV-hBAG3). hBAG3 was expressed in luminal cells throughout the ductal tree in multiple transgenic lines. Targeted expression of hBAG3 dysregulated mammary gland development by both increasing proliferation and differentiation. hBAG3 mammary glands had increased epithelial elongation at 5 weeks of age, premature glandular differentiation at 10 weeks, and features of ductal hyperplasia at week 13 with increased Ki67 and p-Histone H3 in the luminal compartment. At week 13 hBAG3 induced acinar differentiation. BAG3 pro-survival gain of function was recapitulated post-partum with delayed involution. Mammary glands of MMTV-hBAG3 had alveolar persistence three days after forced involution where non-transgenic controls demonstrated typical involution-driven apoptosis and remodeling. We crossed heterozygous MMTV-hBAG3 to WAP-Int3 mice with absent alveolar development in pregnancy. hBAG3 caused alveolar budding in the WAP-Int3 background. We hypothesized hBAG3 gain of function in the mammary gland would promote tumorigenesis. On aging, mammary glands of heterozygous MMTV-hBAG3 displayed densely crowded acinar structures with atypia, consistent with hyperplastic alveolar nodule (HAN). We subjected MMTV-hBAG3 mice to either multiparity or chemical carcinogenesis. After two pregnancies, most MMTV-hBAG3 animals had hyperplastic mammary ductal lesions (MMTV-hBAG3 6/7, 86%; FVB/N 1/7, 14%). Eighteen weeks post 7,12-dimethylbenz(a)antracene (DMBA) treatment, virgin females from five different transgenic lines had more hyperplastic lesions (MMTV-hBAG3 mean 6.3 lesions/gland, N=13, FVB/N mean 3.6 lesions/gland, N=9; p = 0.04), some with atypical hyperplasia. Transgenic MMTV-hBAG3 females developed ER and PR (+) malignant tumors (5/19), 26% while non-transgenic controls had none (0/12). WAP-Int3 mice developed tumors as early as two pregnancies. Heterozygous WAP-Int3xMMTV-BAG3 had the same tumor frequency but preliminary data suggest increased aggressiveness and metastasis. Collectively, our results indicate that MMTV-hBAG3 promotes proliferation, dysregulates mammary gland development and promotes hyperplasia and tumorigenesis and may provide a novel mouse model to represent the human ER+ luminal breast cancer subtype. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-04-08.

Down-regulation of epithelial cadherin is required to initiate metastatic outgrowth of breast cancer

Reduced epithelial cadherin (E-cad) is a hallmark of invasive carcinomas that have acquired epithelial-mesenchymal transition (EMT) phenotypes. Here we show that down-regulated E-cad expression induced by transforming growth factor-β (TGF-β) and EMT preceded breast cancer outgrowth in three-dimensional (3D) organotypic assays and in the lungs of mice. Pharmacological inhibitors against focal adhesion kinase prevented metastatic outgrowth of newly seeded organoids, but not that of their fully established counterparts. Interrogating the D2-HAN (hyperplastic alveolar nodule) model of breast cancer dormancy and metastasis showed that dormant D2.OR cells produced branched organoid morphologies in 3D-cultures, and expressed robust quantities of E-cad that was uncoupled from regulation by TGF-β. In contrast, metastatic D2.A1 organoids were spherical and wholly lacked E-cad expression. Interestingly, D2.A1 cells engineered to re-express E-cad formed branched organoids, down-regulated β1 integrin expression, and failed to undergo metastatic outgrowth. The tumor-suppressing function of E-cad was inactivated by increased microenvironmental rigidity, and was not recapitulated by expression of an E-cad mutant lacking its extracellular domain. Twist expression, but not that of Snail, reinitiated metastatic outgrowth in dormant D2.OR cells. Our findings show that EMT and its down-regulated expression of E-cad circumvent breast cancer dormancy in part by facilitating β1 integrin expression necessary for metastatic outgrowth.

Altered AIB1 or AIB1Δ3 Expression Impacts ERα Effects on Mammary Gland Stromal and Epithelial Content

Amplified in breast cancer 1 (AIB1) (also known as steroid receptor coactivator-3) is a nuclear receptor coactivator enhancing estrogen receptor (ER)α and progesterone receptor (PR)-dependent transcription in breast cancer. The splice variant AIB1Δ3 demonstrates increased ability to promote ERα and PR-dependent transcription. Both are implicated in breast cancer risk and antihormone resistance. Conditional transgenic mice tested the in vivo impact of AIB1Δ3 overexpression compared with AIB1 on histological features of increased breast cancer risk and growth response to estrogen and progesterone in the mammary gland. Combining expression of either AIB1 or AIB1Δ3 with ERα overexpression, we investigated in vivo cooperativity. AIB1 and AIB1Δ3 overexpression equivalently increased the prevalence of hyperplastic alveolar nodules but not ductal hyperplasia or collagen content. When AIB1 or AIB1Δ3 overexpression was combined with ERα, both stromal collagen content and ductal hyperplasia prevalence were significantly increased and adenocarcinomas appeared. Overexpression of AIB1Δ3, especially combined with overexpressed ERα, led to an abnormal response to estrogen and progesterone with significant increases in stromal collagen content and development of a multilayered mammary epithelium. AIB1Δ3 overexpression was associated with a significant increase in PR expression and PR downstream signaling genes. AIB1 overexpression produced less marked growth abnormalities and no significant change in PR expression. In summary, AIB1Δ3 overexpression was more potent than AIB1 overexpression in increasing stromal collagen content, inducing abnormal mammary epithelial growth, altering PR expression levels, and mediating the response to estrogen and progesterone. Combining ERα overexpression with either AIB1 or AIB1Δ3 overexpression augmented abnormal growth responses in both epithelial and stromal compartments.

Abstract B54: Pregnancy and postpartum involution promote tumorigenesis in a preclinical model for triple negative/basal breast cancer

Abstract Young African American women have an increased risk of developing and dying from “triple negative/basal breast cancer” compared to young Caucasian women, but the reason for this is unknown. Insight into this problem may be elucidated by the fact that breast cancers associated with a recent pregnancy are more likely to be metastatic. For all women, having had a recent pregnancy is an independent risk factor for poor prognosis. Epidemiologic data shows increased risk of triple negative/basal breast cancer with increased childbearing in African American women (Millikan et al 2008). We speculate that the hormones of pregnancy combined with the tissue-remodeling of the post-partum breast back to its pre-pregnant state (a normal process called postpartum involution that occurs after childbirth in the absence of breastfeeding, or at weaning in women who breastfeed) provides a two-hit tumor promotional tissue environment that results in increased metastasis of these tumors. Using an innovative, intraductal preclinical model developed in our laboratory, we tested whether a triple negative/basal breast cancer cell line (MCF10ADCIS.com) is promoted by pregnancy or postpartum involution. When these cells are injected into mammary ducts of nulliparous female mice, there is evidence of tumor progression from hyperplastic alveolar nodules to ductal carcinoma in situ to local invasion that is accompanied by progressive loss of the protective myoepithelial cell layer. Further, tumor burden in pregnancy and postpartum involution is much greater than their respective nulliparous controls. Importantly, with pregnancy and postpartum involution the protective myoepithelial cell layer surrounding tumors is compromised at earlier stages than in nulliparous controls. Physiologic changes to adherens junctions and myoepithelial cells occur during normal pregnancy and post-partum involution, respectively. These data suggest that the physiological environments of pregnant and involuting glands promote tumor progression and begin to unfold the biological mechanisms for the effects of parity on aggressive breast cancers. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):B54.

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

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.

Abstract A26: Aromatase over-expression results in the development of ductal growth abnormalities in the mammary gland of a conditional transgenic mouse model

Abstract Estrogens are required for the development of the normal mammary gland; however, studies suggest that estrogen and its metabolites may have mutagenic and carcinogenic potential in the mammary gland. Aromatase is key to estrogen biosynthesis, but its over-expression may play a role in the development of breast cancer by increasing mitotic activity in breast epithelial cells. Therefore, it is highly valuable to have mouse models with increased local estrogen production to use to develop optimal therapeutic and chemopreventive strategies to treat these lesions. The purpose of this study was to evaluate if increased local estrogen production led to the development of mammary gland preneoplasia. To test our hypothesis we developed a conditional transgenic mouse model to control the spatial and temporal aromatase expression in the mammary gland. Mammary glands were collected at 4 and 12 months of age for morphological, histological, gene and protein expression studies in both MMTV-rtTA/tet-op-Aromatase and wild-type mice. RT-PCR analysis of aromatase mRNA confirmed that the gene was conditionally expressed in the mammary gland as compared to control mice. Aromatase activity assays confirmed a significantly higher enzyme activity in the mammary glands of MMTV-rtTA/tet-op-Aromatase compared to wild-type. Mammary glands were evaluated for morphological changes using confocal microscopy, whole-mount and hematoxylin and eosin staining. Aromatase over-expression resulted in a higher incidence of morphological and histological abnormalities such as hyperplastic alveolar nodules, ductal dysplasia, ductal carcinoma in situ, and the unexpected persistence of terminal end buds at 12 months of age. These changes were associated with ERK1/2 and AKT activation. In summary, we generated a novel transgenic mouse system to investigate the role of aromatase over-expression in mammary epithelial cells during mammary gland development. Increased aromatase activity resulted in the development of ductal growth abnormalities and increased ductal density. This model represents a valuable tool to investigate the effect of estrogen over-production in the mammary gland microenvironment at specific times during development. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A26.

0035 Synchrotron x-ray diffraction of human hair for the early detection of breast pathologies - preliminary results

Goals: Our goal is to investigate the chemopreventive effect of green tea extract (GTE) and tamoxifen (TAM) alone or in combination on mammary carcinogenesis and preneoplastic lesions of C3H/OuJ mouse. Methods: Mammary tumors of C3H/OuJ mouse induced by exogenous MMTV frequently arise from preneoplastic lesions termed hyperplastic alveolar nodules (HANs). Mice were divided into five groups; (1) no treatment as control, (2) 1%GTE orally administered with drinking water, (3) 0.5%GTE orally, (4) TAM pellet subcutaneously administered (10mg /body/3months), (5) 1%GTE orally and TAM subcutaneously in combination. The treatment was started at 10 week-old and observed the tumor formation. At 48 week-old, mice were sacrificed and mammary glands were removed, stained with methylene blue for HAN count, and with silver nitrate for counting Nucleolar Organizer Regions (AgNORs). Results: The tumor incidence of control group, 1%GTE group, 0.5%GTE group, TAM group and combination group was 30%, 25%, 25%, 14% and 0%, respectively, but not significantly different. Average HAN count of control group, 1%GTE group, 0.5%GTE group, TAM group and combination group was 9.2, 6.1, 7.5, 6.0 and 5.8, respectively. HAN formation was significantly reduced in 1%GTE group, TAM group and combination group compared with control group. Average AgNOR count of control group, 1%GTE group, 0.5%GTE group, TAM group and combination group was 2.862/cell, 1.886, 1.993, 1.697 and 1.590, respectively. AgNOR formation was significantly reduced in 1%GTE group, TAM group and combination group compared with control group. Conclusion: These data suggest that GTE and TAM have some potential in chemoprevention of breast cancer, individually and in combination.

0034 Chemopreventive effect of green tea extract and tamoxifen on mouse mammary preneoplastic lesions

Goals: Our goal is to investigate the chemopreventive effect of green tea extract (GTE) and tamoxifen (TAM) alone or in combination on mammary carcinogenesis and preneoplastic lesions of C3H/OuJ mouse. Methods: Mammary tumors of C3H/OuJ mouse induced by exogenous MMTV frequently arise from preneoplastic lesions termed hyperplastic alveolar nodules (HANs). Mice were divided into five groups; (1) no treatment as control, (2) 1%GTE orally administered with drinking water, (3) 0.5%GTE orally, (4) TAM pellet subcutaneously administered (10mg /body/3months), (5) 1%GTE orally and TAM subcutaneously in combination. The treatment was started at 10 week-old and observed the tumor formation. At 48 week-old, mice were sacrificed and mammary glands were removed, stained with methylene blue for HAN count, and with silver nitrate for counting Nucleolar Organizer Regions (AgNORs). Results: The tumor incidence of control group, 1%GTE group, 0.5%GTE group, TAM group and combination group was 30%, 25%, 25%, 14% and 0%, respectively, but not significantly different. Average HAN count of control group, 1%GTE group, 0.5%GTE group, TAM group and combination group was 9.2, 6.1, 7.5, 6.0 and 5.8, respectively. HAN formation was significantly reduced in 1%GTE group, TAM group and combination group compared with control group. Average AgNOR count of control group, 1%GTE group, 0.5%GTE group, TAM group and combination group was 2.862/cell, 1.886, 1.993, 1.697 and 1.590, respectively. AgNOR formation was significantly reduced in 1%GTE group, TAM group and combination group compared with control group. Conclusion: These data suggest that GTE and TAM have some potential in chemoprevention of breast cancer, individually and in combination.

P53 heterozygosity leads to deregulated apoptosis and cell proliferation in the mammary gland of ERα over-expressing mice.

Abstract Abstract #2008 Background: Transgenic mouse models are a common experimental tool for unraveling gene function. Breast cancer develops as genetic changes accumulate in the ductal epithelium giving rise to precursor lesions such as atypical ductal hyperplasias, which may progress to ductal carcinoma in situ (DCIS) and eventually invasive breast cancer. This process is associated with mutations in tumor suppressor genes such as p53 resulting in the deregulation of essential genes. p53 plays a role in mediating cell response to various stresses by inducing or repressing genes involved in cell cycle arrest, senescence, apoptosis and DNA repair. Studies have shown that p53 is a predictor for local recurrence of DCIS. About 65% of breast cancers are estrogen receptor alpha (ERα) positive and estrogen dependent. Deregulated expression of ERα in normal breast epithelium has been found in conjunction with breast cancer, leading to the concept that loss of the normal regulatory mechanisms that control expression levels of ERα in normal breast epithelium may increase the risk for the development of breast cancer. This study tests the hypothesis that p53 heterozygosity may lead to an aberrant growth response to hormonal stimuli and development of mammary gland preneoplasia.
 Methodology: To examine if p53 heterozygosity promotes disease in mammary glands with deregulated expression of ERα, a conditional ERα in mammary tissue mouse model (MMTV-rtTA/tet-op-ERα or CERM) was bred to a p53 homozygous mutant (p53-/-) mouse model. For progression studies, mammary glands were collected at 12 months of age for morphological and histological studies both in intact nulliparous mice. A group of mice was ovariectomized at 14 weeks of age and 2 weeks later treated with either placebo or 17β-estradiol for 48 hours to dissect the molecular events attributable to specific hormonal stimulation as compared to normal ovarian function.
 Results: Loss of p53 resulted in a statistically significant higher percentage of hyperplastic alveolar nodules, both by itself and in mice with ERα deregulation in the mammary gland. p53 heterozygosity resulted in a higher incidence of morphological and histological abnormalities such as dilated ducts, ductal dysplasia, ductal carcinoma in situ, and abnormal stroma. These lesions showed higher rates of cell proliferation and lower rates of apoptosis. Phospho-p53 immunohistochemistry showed evidence of DNA damage in the p53 heterozygous mice. There was no significant change in the terminal ductal structures in mammary glands of ovariectomized mice when compared to mice treated with exogenous 17β-estradiol after 48 hrs.
 Conclusions: p53 heterozygosity predisposed mammary tissue to preneoplastic changes both by itself and in collaboration with deregulated ERα expression. The combination of deregulated ERα expression and p53 heterozygosity was associated with more disease than either genetic change by itself. Loss of p53 leads to a deregulation in apoptosis and cell proliferation in the mammary gland of ERα over-expressing mice suggesting that both processes are involved in the development of preneoplasia. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2008.

Abstract A6: Stat5a and pregnancy protection in the CERM mouse model

Abstract A6 INTRODUCTION Estrogen Receptor α (ERα) is a key regulator in normal mammary gland development and acts as a growth factor in breast cancer development. We have developed the CERM mouse model with targeted ERα overexpression and deregulation specifically to the mammary gland. By 4 months of age, the CERM mice develop ERα-initiated mammary ductal hyperplasia and ductal carcinoma in-situ which mimic human disease (Frech et al., 2005). In women, there is evidence that early full-term pregnancy decreases lifetime ERα-positive breast cancer risk. Paradoxically, there is also evidence that breast cancer risk is transiently increased within ten years of early full-term pregnancy (Schedin, 2006). One potential mechanism for the lifetime protective effect is believed to occur via differentiation (Russo et al., 2006). In addition, it is well established that STAT5a, a latent cytoplasmic transcription factor, is highly activated during pregnancy and lactation, and is involved in alveolar proliferation and terminal differentiation (Liu et al, 1996 & 1997). Therefore, we hypothesized that Stat5a contributes to the protective effects of pregnancy. The CERM mice develop hyperplastic alveolar nodules (HANs) by 12 months of age, therefore we postulated that pregnancy would protect against HANs in the CERM mice, which have Stat5a, and that loss of Stat5a alleles would reduce the protective effects of pregnancy. METHODS The CERM mouse model, which has targeted ERα transgene expression to mammary epithelial cells, was bred with the Stat5a knockout mouse model to generate CERM, CERM/Stat5a+/- and CERM/Stat5a-/- mice. The mice went through several pregnancies and lactation cycles before being necropsied at 12-13 months of age. RESULTS At 12 months of age, CERM mice showed an increased incidence of mammary hyperplastic alveolar nodules (HANs) (p= 0.0254) over wild type (WT) mice. That increase is reduced with loss of one Stat5a alleles (CERM/Stat5a+/-) and further reduced to zero (p=0.0022) with loss of 2 Stat5a alleles (CERM/Stat5a-/-). We observed an unexpected finding with parity in the CERM and CERM/Stat5a+/- mice. At 12 months of age, the incidence of HANs was not reduced in multiparous CERM versus nulliparous CERM mice as well as in multiparous CERM/Stat5a+/- versus nulliparous CERM/Stat5a+/- mice. Since the incidence of HANs was zero in the nulliparous CERM/Stat5a-/- mice, the protective effects of pregnancy were not studied with loss of two Stat5a alleles. However, we had generated one 12 month old parous CERM/Stat5a-/- mice, which interestingly developed HANs as opposed to nulliparous CERM/Stat5a-/- mice, which do not develop HANs. Interestingly, additional preliminary data demonstrated that parity in the CERM and CERM/Stat5a+/- mice can lead to the development of different neoplastic lesions that were variably ERα positive or negative. CONCLUSION Pregnancy in our CERM model did not confer protection against HANs development at 12 months of age. This lack of pregnancy protection seemed to occur via a Stat5a-independent ERα-dependent growth pathway. Since Stat5a loss leads to lack of HAN development in nulliparous mice, Stat5a loss seemed to have a different impact in multiparous mice which showed development of HANs. Further studies will determine which signaling pathways are involved in lack of pregnancy protection in our models. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A6.

Identification of White Adipocyte Progenitor Cells In Vivo

The increased white adipose tissue (WAT) mass associated with obesity is the result of both hyperplasia and hypertrophy of adipocytes. However, the mechanisms controlling adipocyte number are unknown in part because the identity of the physiological adipocyte progenitor cells has not been defined in vivo. In this report, we employ a variety of approaches, including a noninvasive assay for following fat mass reconstitution in vivo, to identify a subpopulation of early adipocyte progenitor cells (Lin(-):CD29(+):CD34(+):Sca-1(+):CD24(+)) resident in adult WAT. When injected into the residual fat pads of A-Zip lipodystrophic mice, these cells reconstitute a normal WAT depot and rescue the diabetic phenotype that develops in these animals. This report provides the identification of an undifferentiated adipocyte precursor subpopulation resident within the adipose tissue stroma that is capable of proliferating and differentiating into an adipose depot in vivo.

Activation of estrogen signaling pathways collaborates with loss of Brca1 to promote development of ERα-negative and ERα-positive mammary preneoplasia and cancer

BRCA1 can regulate estrogen receptor-alpha (ERalpha) activity. This study tested the hypotheses that Brca1 loss in mammary epithelium alters the estrogenic growth response and that exposure to increased estrogen or ERalpha collaborates with Brca1 deficiency to accelerate preneoplasia and cancer development. Longer ductal extension was found in mammary glands of Brca1(f/f;MMTV-Cre) mice during puberty as compared to wild-type mice. Terminal end bud differentiation was impaired in Brca1 mutant mice with preservation of prolactin-induced alveolar differentiation. Exogenous estrogen stimulated an abnormal sustained increase in mammary epithelial cell proliferation and the appearance of ERalpha-negative preneoplasia in postpubertal Brca1 mutant mice. Carcinogenesis was investigated using Brca1(f/f;MMTV-Cre) mice hemizygous for p53. Exogenous estrogen increased the percentage of mice with multiple hyperplastic alveolar nodules. Targeted conditional ERalpha overexpression in mammary epithelial cells of mice that were Brca1 mutant and hemizygous for p53 increased the percentage of mice exhibiting multiple hyperplastic nodules, invasive mammary cancers and cancer multiplicity. Significantly more than half of the preneoplasia and cancers were ERalpha negative even as their initiation was promoted by ERalpha overexpression.

Multistep mouse mammary tumorigenesis through preneoplasia to neoplasia and acquisition of metastatic potential

Human breast tissue can give rise to hyperplasias, atypical hyperplasias, and in situ carcinomas originating in a terminal duct-lobular unit (TDLU). These entities are associated with increased risk of subsequent development of invasive carcinoma. Human breast carcinomas arise via intermediate steps known as precursor or premalignant lesions. However, it is difficult to perform stepwise observation of the progression of human breast cancer. Mouse mammary tissue can give rise to several characteristic types of premalignant hyperplasia and tumor, originating in a duct or acinus, that progress to carcinoma. Three specific types of mouse mammary lesion with premalignant potential have been identified: hyperplastic alveolar nodule (HAN), plaque (PLQ), and ductal hyperplasia (DH). These lesions partially resemble human precursor lesions, and clarification of similarities and differences between the human and mouse lesions could improve our understanding of human breast carcinogenesis. Some invasive breast carcinomas acquire metastatic potential and may cause the death of the patient. Because the most malignant aspect of neoplasia is metastasis, recognition of metastatic phenotypes is particularly important. Mouse mammary carcinomas rarely metastasize, but there exist mouse models of metastasis of mammary carcinoma. In the present review article, we describe morphological, biological, and molecular aspects of premalignant lesions of mice, and we discuss their metastatic phenotypes.