Mammary Cancer Prevention Research Articles

Dietary Supplementation of Inulin Contributes to the Prevention of Estrogen Receptor-Negative Mammary Cancer by Alteration of Gut Microbial Communities and Epigenetic Regulations.

Breast cancer (BC) is among the most frequently diagnosed malignant cancers in women in the United States. Diet and nutrition supplementation are closely related to BC onset and progression, and inulin is commercially available as a health supplement to improve gut health. However, little is known with respect to inulin intake for BC prevention. We investigated the effect of an inulin-supplemented diet on the prevention of estrogen receptor-negative mammary carcinoma in a transgenic mouse model. Plasma short-chain fatty acids were measured, the gut microbial composition was analyzed, and the expression of proteins related to cell cycle and epigenetics-related genes was measured. Inulin supplementation greatly inhibited tumor growth and significantly delayed tumor latency. The mice that consumed inulin had a distinct microbiome and higher diversity of gut microbial composition compared to the control. The concentration of propionic acid in plasma was significantly higher in the inulin-supplemented group. The protein expression of epigenetic-modulating histone deacetylase 2 (Hdac2), Hdac8, and DNA methyltransferase 3b decreased. The protein expression of factors related to tumor cell proliferation and survival, such as Akt, phospho-PI3K, and NF-kB, also decreased with inulin administration. Furthermore, sodium propionate showed BC prevention effect in vivo through epigenetic regulations. These studies suggest that modulating microbial composition through inulin consumption may be a promising strategy for BC prevention.

Gamma-ray Irradiation of Rodent Diets Alters the Urinary Metabolome in Rats with Chemically Induced Mammary Cancer.

In this study, a comparative, untargeted metabolomics approach was applied to compare urinary metabolite profiles of rats fed irradiated and non-irradiated diets. γ-Irradiated and non-irradiated NIH 7001 diet was given orally to animals beginning 5 days after exposure to the carcinogen N-methyl-N-nitrosourea and continued for 120 days. There was a 36% reduction in mammary tumor incidence in rats consuming the γ-irradiated diet, compared to rats receiving the non-irradiated form of the same diet. Urine samples from rats fed with γ-irradiated and non-irradiated diets were analyzed using nanoLC-MS/MS on a Q-TOF mass spectrometer, collecting positive and negative ion data. Data processing involved feature detection and alignment with MS-DIAL, normalization, mean-centering and Pareto scaling, and univariate and multivariate statistical analysis using MetaboAnalyst, and pathway analysis with Mummichog. Unsupervised Principal Component Analysis and supervised Partial Least Squares-Discriminant Analysis of both negative and positive ions revealed separation of the two groups. The top 25 metabolites from variable importance in projection scores >1 showed their contributions in discriminating urines the γ-irradiated diet fed group from non-irradiated control diet group. Consumption of the γ-irradiated diet led to alteration of several gut microbial metabolites such as phenylacetylglycine, indoxyl sulfate, kynurenic acid, hippurate and betaine in the urine. This study provides insights into metabolic changes in rat urine in response to a γ-irradiated diet which may be associated with mammary cancer prevention.

An integrated analysis of the effects of maternal broccoli sprouts exposure on transcriptome and methylome in prevention of offspring mammary cancer.

Broccoli sprouts (BSp), a cruciferous vegetable, has shown promising effects on prevention of many types of cancer including breast cancer (BC). BC has a developmental foundation, and maternal nutrition status may influence an offspring’s risk to BC later in life. What is less understood, however, is the influence of maternal nutrition intervention on reversing epigenomic abnormalities that are essential in BC programming during early development. Our research focused on how maternal exposure to BSp diet prevents offspring BC and investigation of possible epigenetic mechanisms during these processes. Our results showed that maternal feeding of BSp can prevent mammary tumor development in the offspring of a transgenic mouse model. Through comprehensive integrated multi-omics studies on transcriptomic and methylomic analysis, we identified numerous target genes exhibiting significantly differential gene expression and DNA methylation patterns in the offspring mammary tumor. We discovered that maternal exposure to BSp diet can induce both gene and methylation changes in several key genes such as Avpr2, Cyp4a12b, Dpp6, Gria2, Pcdh9 and Tspan11 that are correlated with pivotal biological functions during carcinogenesis. In addition, we found an impact of maternal BSp treatment on DNA methyltransferase and histone deacetylases activity. Our study provides knowledgeable information regarding how maternal BSp diet influences key tumor-related gene expression and the epigenetic changes using a genome-wide perspective. Additionally, these findings provide mechanistic insights into the effectiveness of maternal BSp administration on the prevention of BC in the offspring later in life, which may lead to an early-life BC chemopreventive strategy that benefits the progenies’ long-term health.

Abstract 2571: Paternal epigenetic regulation contributes to the prevention of estrogen receptor-negative mammary cancer with combined broccoli sprouts and green tea polyphenols consumption in transgenic mice

Abstract Breast cancer is one of the leading causes of cancer-related death among women in the United States. It is widely appreciated that abnormal epigenetic alterations act as one of the primary contributors to breast cancer initiation and progression. Parental, including maternal and paternal, nutritional interventions have shown considerable impact on fetal development leading to different susceptibility of offspring to various chronic diseases. Moreover, combinatorial dietary administration to parents could be more efficacious in ameliorating epigenetic aberrations and changing cancer risk in an individual's later life. The bioactive dietary broccoli sprouts (BSp) and green tea polyphenol (GTP) are important epigenetic modulators that can prevent various cancers. Our study focusses on investigating the effect of paternal dietary BSp and GTP administration on mammary cancer prevention of their offspring. Two spontaneous breast cancer transgenic mouse models, C3(1)/SV40 and Her2/neu, were employed in this study. Male C3(1)/SV40 or Her2/neu transgenic mice were randomly assigned into four groups and treated with: control AIN-93G diet, 26% BSp (w/w) in food pellets, 0.5% GTPs (w/v) in drinking water or combined BSp and GTPs from 3 wks of age until 10 wks of age. Treated male mice of different groups were then mated with non-treated female mice. Female pups were selected after their weaning and tumor growth was monitored weekly until the termination of the experiment. Tumor- and epigenetic-related protein expression and histone modifications (methylation and acetylation) were measured. Our study indicated that paternal BSp or/and GTPs administration suppressed tumor growth, decreased tumor incidence, and delayed tumor latency compared with the control in both mouse models. Overall, paternal dietary intervention displayed more efficacy for mammary tumor prevention in C3(1)/SV40 mice and the combination group in this model also showed synergistically effects. Subsequent analysis with the C3(1)/SV40 mouse tumors demonstrated that paternal BSp or/and GTPs treatments upregulated the expression of tumor suppressor proteins, such as P16 and P53, and down-regulated tumor promoting proteins, such as MYC and BMI1. Moreover, altered DNA methylation, histone methylation and acetylation levels were also observed. These results suggested that paternal exposure to BSp and GTPs may contribute to ER-negative mammary tumor prevention in their offspring through epigenetic regulations and the combined addition of bioactive botanicals could be a more promising approach for coping with breast cancer initiation and progression in humans. Citation Format: Shizhao Li, Huixin Wu, Trygve Tollefsbol. Paternal epigenetic regulation contributes to the prevention of estrogen receptor-negative mammary cancer with combined broccoli sprouts and green tea polyphenols consumption in transgenic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2571.

Abstract PD11-08: Low dose endoxifen represses mouse mammary tumorigenesis: A preclinical study of monotherapy and combination with ulipristal acetate

Abstract Background: Endoxifen (ENX), a major active metabolite of the selective estrogen receptor (ER) modulator tamoxifen (TAM), has been shown to inhibit the growth of ER+ cancer. However, it has not been evaluated for prevention of mammary cancer. We report here the cancer prevention efficacy of ENX as monotherapy and in combination with ulipristal acetate (UPA), a selective progesterone receptor (PR) modulator. We compare these to TAM using the SV40 C3 T-antigen (C3-TAg) mouse, a well-documented mammary cancer prevention model. Methods: C3-TAg female virgin mice aged 7-8 weeks were randomized to no-treatment control or to drug treatment groups (TAM, ENX, UPA and ENX+UPA). Drug pellets were subcutaneously implanted on backside of mice. The doses of drug pellet were 12.5mg/30d for TAM, 9mg/90d for ENX, 10mg/30d for UPA, and combination of ENX (9mg/90d) + UPA (10mg/30d). The dose of ENX was equivalent to the exposure resulting from 20 mg/day, the lowest dose used in a recent Phase I human trial. Pellets were replaced every 30 days or 90 days. Mammary tumor formation was monitored twice weekly by palpation. Tumor latency, multiplicity, and tumor volume were recorded; animals are euthanized at 23 weeks of age, or earlier if tumors reach > 1 cm2. Tumors and mammary glands were formalin fixed and paraffin embedded for histology evaluation by a rodent pathologist. The primary endpoint was a reduction in tumor incidence in drug treated versus control groups. Secondary endpoints included prolongation of latency, reduction in tumor multiplicity, and tumor burden. Statistical significance between groups was calculated with Wilcoxon log-rank test for % tumor-free survival and Mann Whitney test for tumor multiplicity and burden.Results: All mice in the control group developed tumors by 18 weeks of age. Tumor-free survival % of ENX, UPA, and ENX+UPA treated groups were significantly higher than control animals, with the greatest increase in the ENX group (p=0.02). TAM had no effect on tumor-free survival (p=0.32). Median tumor latencies were similar in three treated groups: ENX (112 days), UPA (114 days), and ENX+UPA (111 days) groups, and were significantly delayed compared to the control or TAM group (100 days in both) (p<0.05). Median tumor multiplicity (invasive adenocarcinoma) per animal was significantly lower in ENX group compared to control group (3 vs. 7, p=0.02). UPA and TAM did not significantly reduce tumor multiplicity (4 for TAM and 6 for UPA). We observed non-significant 32% and 23% reduction in median tumor weight of ENX+UPA group and ENX group, respectively compared to control group. Histology evaluation for ENX+UPA group and pathologic size of invasive carcinoma is ongoing, along with the expression of Ki67, ERα and PR of mammary tumors and mammary gland by immunohistochemistry.Conclusions: TAM showed no significant effect on delaying tumorigenesis, consistent with a previous study. However, low dose of ENX (equivalent to 24 mg/day in human) effectively repressed tumor development and growth than TAM or UPA treatment. Our data suggest that ENX is promising for prevention of ER+ mammary tumorigenesis and warrant a dose optimization study for improving efficacy. Citation Format: Oukseub Lee, Minhua Wang, Maarten C. Bosland, Omid Hosseini, Irene Helenowski, Seema A. Khan. Low dose endoxifen represses mouse mammary tumorigenesis: A preclinical study of monotherapy and combination with ulipristal acetate [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD11-08.

Nutritional combinatorial impact on the gut microbiota and plasma short-chain fatty acids levels in the prevention of mammary cancer in Her2/neu estrogen receptor-negative transgenic mice.

Breast cancer is the second leading cause of cancer-related mortality in women. Various nutritional compounds possess anti-carcinogenic properties which may be mediated through their effects on the gut microbiota and its production of short-chain fatty acids (SCFAs) for the prevention of breast cancer. We evaluated the impact of broccoli sprouts (BSp), green tea polyphenols (GTPs) and their combination on the gut microbiota and SCFAs metabolism from the microbiota in Her2/neu transgenic mice that spontaneously develop estrogen receptor-negative [ER(-)] mammary tumors. The mice were grouped based on the dietary treatment: control, BSp, GTPs or their combination from beginning in early life (BE) or life-long from conception (LC). We found that the combination group showed the strongest inhibiting effect on tumor growth volume and a significant increase in tumor latency. BSp treatment was integrally more efficacious than the GTPs group when compared to the control group. There was similar clustering of microbiota of BSp-fed mice with combination-fed mice, and GTPs-fed mice with control-fed mice at pre-tumor in the BE group and at pre-tumor and post-tumor in the LC group. The mice on all dietary treatment groups incurred a significant increase of Adlercreutzia, Lactobacillus genus and Lachnospiraceae, S24-7 family in the both BE and LC groups. We found no change in SCFAs levels in the plasma of BSp-fed, GTPs-fed and combination-fed mice of the BE group. Marked changes were observed in the mice of the LC group consisting of significant increases in propionate and isobutyrate in GTPs-fed and combination-fed mice. These studies indicate that nutrients such as BSp and GTPs differentially affect the gut microbial composition in both the BE and LC groups and the key metabolites (SCFAs) levels in the LC group. The findings also suggest that temporal factors related to different time windows of consumption during the life-span can have a promising influence on the gut microbial composition, SCFAs profiles and ER(-) breast cancer prevention.

Abstract 19: Examination of HJC0152, a putative modulator of glucose and energy metabolism, for mammary cancer prevention

Abstract Breast cancer (BC) is the most common cancer in women in the US. Approximately 30-40% of BC cases are estrogen-receptor (ER)-negative (ENBC); the majority of these are triple-negative (TNBC). No drugs have been shown to effectively prevent ENBC including TNBC. TNBC incidence is higher in young African American and Hispanic/Latina women, thus the need for preventive agents for TNBC is critical. Developing agents targeting non-ER-based oncogenic pathways will therefore offer alternative and potentially more effective approaches for preventing ENBC/TNBC. Glucose metabolism is an essential energy-producing process for normal cells and is aberrant and reprogrammed in cancer cells including BC cells. We recently developed HJC0152, a novel small-molecule glucose metabolism modulator as a potential cancer preventive agent. HJC0152 has been shown to inhibit glycolysis by differentially regulating glycolytic enzyme expression and the function of mitochondrial respiratory complexes in both EPBC and TNBC cells, leading to reduced production of energy molecule adenosine-triphosphate (ATP), induced apoptosis, and decreased proliferation and tumor progression. In this study, we determined the preventive anti-cancer efficacies of long-term and short-term (inhibition of premalignant lesions) HJC0152 administration in mouse models of ENBC and TNBC. HJC0152 was given orally 6 days/wk for 2-10 months in well-established ENBC model, MMTV-erbB2; and TNBC model, C3(1)/TAgSV40. HJC0152 at 50 and 75 mg/Kg doses delayed and partially prevented mammary tumor development in ENBC (MMTV-erbB2) mice (p=0.0034 for all groups). A maximum 6-week delay was achieved for median time to tumor formation (TTF). In the TNBC (C3(1)/TAgSV40) model, HJC0152 significantly delayed mammary tumor development (p=0.0114 for all groups) and achieved 3.3-week delay for median TTF. Up to 21% less mice developed mammary tumors in both models. In addition, TNBC mice developed less number of tumors per mouse than in ENBC mice, and the tumor volume doubling time increased from 6.9 to 9.2 days. In the short-term premalignant lesion study, the number of mammary intraepithelial neoplasia (MIN) lesion (representing human DCIS) was significantly decreased (p=0.034) in the HJC0152-treated MMTV-erbB2 mice. In the HJC0152-treated C3(1)/TAgSV40 mice, the number of hyperplasia and MIN lesions were significantly decreased (p=0.0014 and 0.0203, respectively). These results demonstrate that HJC0152 suppresses mammary tumor development in ENBC and TNBC models via reducing premalignant lesions, even in the most aggressive TNBC model, C3(1)/TAgSV40. There were no significant side effects or toxicity signs observed in both long- and short-term prevention experiments. We conclude that HJC0152 has promising cancer preventive efficacy by targeting non-ER-based signaling pathways such as STAT3 signaling and glucose metabolism. This study was supported by a NIH/NCI PREVENT Program fund. *Current Affiliation: LSUHSC. Citation Format: Hyejin Kim, Xi Liu, Yefei Wen, Jia Zhou, Romaine Fernando, Shizuko Sei, Powel H. Brown, Qiang Shen. Examination of HJC0152, a putative modulator of glucose and energy metabolism, for mammary cancer prevention [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 19.

Abstract P1-12-01: Prevention of BRCA1-associated mammary cancers by selective progesterone receptor modulators (SPRM) in mice

Abstract Background: It was previously demonstrated that mifepristone (MFP), an SPRM with anti-glucocorticoid receptor (GR) activity, blocks mammary tumor formation in a BRCA1 deficient mouse model (deletion of exon 11). We sought to compare tumor prevention efficacy of MFP with other SPRMs having less GR binding, using a different BRCA1 deficient mouse model (deletion of exon 22-24). We also compared the efficacy of multiple SPRMs on suppression of cell proliferation (Ki67) in benign mammary glands, to evaluate early effects in unaffected glands. Methods: At 4-months-old, the female mice (Blg_Cre Brca1 f22-24/f22-24; p53+/-) were randomized to four treatment groups (n= 14-21 per group): no treatment control, MFP, telapristone acetate (TPA), and ulipristal acetate (UPA) and treated subcutaneously with a pellet containing 30mg of drug. Pellets were replaced every 90 days. The mice were monitored for tumor formation and growth until 15- months of age. When the tumor burden of mice became 1 cm2 or at age 15-months, the mice were euthanized to collect tumors and mammary glands for histopathologic evaluation. Ki67 and PR expression in mammary tissues were measured by immunohistochemistry and images were analyzed by ImageJ/Fiji software. Total RNA extracted from benign and tumor tissue was sequenced to discover SPRM-modulated genes and molecular pathways, in comparison with no-treatment controls. Results: The occurrence of invasive cancers (adenocarcinoma) was similar in MFP-treated and control mice in a time-adjusted analysis (72% of MFP group vs. 74% of controls, p= 0.55). TPA-treated mice displayed a non-significant lower tumor incidence (56 %) compared to control group (p=0.11). However, UPA-treated mice showed significantly less tumor incidence (43%) than controls (p=0.04). These results were supported by suppression of %Ki67 labeling Index (LI) of benign glands. Median %Ki67 LI of benign glands of tumor-affected mice were: controls (18%) = MFP (18%) > TPA (16%) > UPA groups (10 %). Median Ki67% LI of benign glands of tumor-unaffected mice were: MFP (16%) > TPA (13%) > controls (12%) > UPA group (6%). Reduction of cell proliferation by UPA was significant in benign glands of tumor-affected and un-affected mice compared to controls (p=0.007 for both) while reductions in the MFP and TPA groups were not significant. All of the invasive cancers were PR negative; PR is expressed in abnormal and benign lesions adjacent to invasive adenocarcinoma. To determine whether PR expression is related to tumor progression, we are evaluating PR expression in both benign and abnormal glands from all mice. Furthermore, RNA sequencing is underway to determine genes differentially expressed by UPA and MFP treatment compared to no treatment. Conclusions: Our mouse model (BRCA1 Δexon22-24) displayed a spectrum of sensitivity to the three SPRMs we have tested: UPA>TPA>MFP, UPA showing higher potency than TPA. The reason for the lack of efficacy of MFP may relate to its strong affinity to GR, resulting in mixed effects on both PR and GR as a reason for its failure in this model. Understanding mechanisms underlying the different anti-tumor efficacy of UPA and MFP in our model will provide important insights that will aid the clinical development of PR-directed strategies for breast cancer prevention in both BRCA1 mutation carriers. Citation Format: Oukseub Lee, Limin Sun, Maarten C. Bosland, Minhua Wang, Ali Shidfar, Omid Hosseini, Irene Helenowski, Susan E. Clare, Seema A. Khan. Prevention of BRCA1-associated mammary cancers by selective progesterone receptor modulators (SPRM) in mice [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-12-01.

Abstract P5-14-02: Effects of bazedoxifene and/or letrozole in the prevention of chemically-induced mammary cancers

Abstract Bazedoxifene is described as a third-generation selective estrogen receptor modulator (SERM) that has been approved in Europe for the prevention and treatment of postmenopausal osteoporosis. The agent has also been shown to have therapeutic activity against estrogen receptor positive (ER+) breast cancer. Our laboratory has evaluated bazedoxifene for chemopreventive efficacy against mammary cancers when given alone or in combination with the aromatase inhibitor letrozole. Initially, the agents were evaluated alone to determine effective chemopreventive doses in the methylnitrosourea (MNU) ER+ mammary cancer model using female Sprague-Dawley rats. Bazedoxifene was mixed directly into a standard (Teklad, 4% fat) diet, while letrozole was gavaged 7x/week (vehicle was ethanol: PEG 400, 10:90, v/v). When evaluated alone in rats receiving MNU at 50 days of age, bazedoxifene (started one week after MNU) at doses of 100 and 30 mg/kg diet decreased mammary cancer multiplicity by 93 and 88%, respectively. In a similar protocol, letrozole at a dose of 0.1 mg/kg BW/day reduced cancer multiplicity by 89%, while a dose of 0.05 mg/kg BW/day reduced the number by 47%. In a separate study evaluating lower doses of bazedoxifene (5 mg/kg diet) and letrozole (0.04 mg/kg BW/day), mammary cancer multiplicity was decreased by 73% by both agents. When the agents were given in combination at these lower dose levels, cancer number was reduced by 91%. It is obvious that bazedoxifene is as effective as letrozole in the prevention of ER+ mammary cancers. Furthermore, administration of the agents in combination suggests an additive effect. The large increase in body weight gain generally noted in letrozole treated rats was not observed in rats receiving bazedoxifene; of interest, rats receiving the combination also had no increase in body weights. Several published invitro studies suggested that bazedoxifene might also be effective in preventing ER- mammary cancers; e.g., the agent is a novel inhibitor of IL-6/GP130 protein-protein interactions using multiple ligand simultaneous docking and drug repositioning approaches that result in apoptosis. In an initial study using MMTV/Neu mice (N=21/group) that received dimethylbenzanthracene (DMBA), we showed that administering bazedoxifene (15 mg/kg diet) would reduce the mammary tumor multiplicity from 5.4/mouse in controls to 1.1/mouse (an 80% decrease). These studies suggested that bazedozxifene is superior to other similar agents in that both ER+ and ER- mammary cancers are prevented at non-toxic doses. Supported by NCI contract HHSN261201200021I, Task Order HHSN26100007. Citation Format: Dunn BK, Grubbs CJ. Effects of bazedoxifene and/or letrozole in the prevention of chemically-induced mammary cancers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-14-02.

Abstract P4-04-15: Hormonal prevention of HER2 positive mammary cancers

Abstract Introduction: Approximately 25-30% of breast cancers overexpress human epidermal growth factor receptor 2 (HER2). HER2-positive breast cancers are more likely to be diagnosed in younger women and are highly aggressive. Currently, there are few treatments options available specifically for HER2-positive breast cancer which includes Herceptin, Kadcyla, Perjeta and Tykerb. There is a higher risk of recurrence of HER2-positive breast cancers compared to HER2-negative breast cancers. Hence it is important to devise effective strategies to prevent/treat HER2-positive breast cancer. We had earlier shown that short-term treatment with pregnancy levels of estrogen plus or minus progesterone was highly effective in inhibiting mammary tumor growth in MMTV. Here we attempt to understand the mechanism behind this protection. Materials and Methods: MMTV-neu transgenic mice were treated for 3 weeks, starting at 7 weeks of age, with 100 μg of estradiol in Silastic capsules. This dose of estradiol used in the Silastic capsules results in pregnancy levels of estradiol in the circulation. The control animals received empty Silastic capsules for the same duration. Mice were palpated weekly for 9 months to monitor for mammary cancer development. A set of mice were sacrificed at 7, 10, 16, 22, 28 and 36 weeks of age. Mammary gland was surgically isolated. Inguinal mammary glands were prepared as wholemounts. A piece of the mammary gland was fixed in formalin for histopathology and the rest was snap frozen in liquid nitrogen and stored at -80oC for further molecular analysis. Pathway focused microarrays (Cancer Pathway Finder, and PI3K-AKT Signaling) were performed and the differential regulated genes were validated using real-time RTPCR and immunoblotting. All data obtained were statistically analyzed using Graphpad Prism version 5.03. The repeated measures analysis of variance was used to evaluate the dose and time response to hormone treatment. Multiple comparisons between groups with significant differences were analyzed using Dunnett post–hoc test. Paired t–test was done to analyze intergroup differences. The values of p <0.05 were considered as statistically significant. Results: Short-term treatment with pregnancy levels of estradiol significantly reduced the mammary cancer incidence and multiplicity, while it significantly increased mammary cancer latency. Pathway focused microarray analysis of mammary glands at different time points demonstrated that mice treated with short-term estradiol has persistent changes in gene expression involved in key signaling pathways involved in cancer. Pro-angiogenic genes (Angpt1, Fgf2, Vegfc), anti-apoptotic genes (Bcl2, Birc3), pro-proliferative/survival genes (Akt, Ccnd1, Igf1r, Mtor, Pi3k) were significantly down-regulated in the mammary gland of short-term estradiol treated mice. On contrary, pro-apoptotic genes (Bad,Casp, Cflar) and anti-proliferative/survival genes (Pten, p53) were up-regulated. Conclusion: Our data demonstrates that short-term treatment with pregnancy levels of estradiol is highly effective in inhibiting mammary carcinogenesis in MMTV-neu mice. This protection against HER2 positive mammary cancers is achieved by altering key signaling pathways involved in cancer growth and progression. Citation Format: Subramani R, Lakshmanaswamy R. Hormonal prevention of HER2 positive mammary cancers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-04-15.

Abstract B74: Prevention of breast cancer by withanolides

Abstract Malignancy of the breast is a leading cause of cancer-related deaths among women globally. Feasibility of breast cancer prevention is illustrated by clinical success with SERMs and aromatase inhibitors for ER+ breast cancers. However, a clinically viable preventive intervention targeting ER-negative breast cancers is still lacking. Constituents of Ayurvedic medicine, which has been practiced in India for thousands of years, continue to draw attention for identification of novel small molecules potentially useful for prevention of chronic diseases including cancer. This presentation summarizes efficacy data, molecular targets, and biomarkers of mammary cancer prevention by a small-molecule steroidal lactone (withaferin A) derived from an Ayurvedic medicinal plant (Withania somnifera). This work was supported by the grant US PHS grant RO1 CA142694-05 from the National Cancer Institute. Citation Format: Shivendra V. Singh. Prevention of breast cancer by withanolides [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B74.

Abstract 5246: Effects of bazedoxifene either alone or in combination with letrozole in the prevention of chemically induced mammary cancers

Abstract Bazedoxifene is a third-generation selective estrogen receptor modulator (SERM) that has been clinically approved in Europe for the prevention and treatment of postmenopausal osteoporosis. The agent has also been shown to have therapeutic activity against estrogen receptor positive (ER+) breast cancer. Our laboratory is currently evaluating bazedoxifene for chemopreventive efficacy when given alone or in combination with the aromatase inhibitor (AI) letrozole. Initially, the agents were evaluated alone to determine effective chemopreventive doses in the methylnitrosourea (MNU) ER+mammary cancer model using female Sprague-Dawley rats. Bazedoxifene was mixed directly into a standard (Teklad, 4% fat) diet, while letrozole was gavaged 7x/week (vehicle was ethanol: PEG 400, 10:90, v/v). When evaluated alone in rats receiving MNU at 50 days of age, bazedoxifene (started one week after MNU) at doses of 100 and 30 mg/kg diet decreased mammary cancer multiplicity by 93 and 88%, respectively. In a similar protocol, letrozole at a dose of 0.1 mg/kg BW/day reduced cancer number (multiplicity) by 89%, while a dose of 0.05 mg/kg BW/day reduced the number by 47%. In a separate study evaluating lower doses (MNU also given to rats at 50 days of age), bazedoxifene at a dose of 5 mg/kg diet and letrozole at a dose of 0.04 mg/kg BW/day both decreased mammary tumor multiplicity by 73%. When the agents were given in combination at these lower dose levels, cancer number was reduced by 91%. An additional study was done in which the agents were given to rats receiving MNU at an older age (100 days). Rats receiving the agents at the original doses beginning one week after MNU (bazedoxifene at 30 mg/kg diet; letrozole at 0.1 mg/kg BW/day) had identical decreases in mammary tumor development (96%). It is obvious that bazedoxifene is as effective as letrozole in the prevention of mammary cancers even when initiated in older rats. Furthermore, administration of the agents in combination suggests an additive effect. The large increase in body weight gain generally noted in letrozole treated rats was not observed in rats receiving bazedoxifene; of interest, rats receiving the combination also had no increase in body weights. These studies indicate that bazedoxifene may be superior to other SERMs in that chemopreventive efficacy is achieved at low doses, which may be associated with less toxicity. Supported by NCI contract HHSN261201200021I, Task Order HHSN26100007. Citation Format: Barbara K. Dunn, Chen Suen, Ronald A. Lubet, Vernon E. Steele, Clinton J. Grubbs. Effects of bazedoxifene either alone or in combination with letrozole in the prevention of chemically induced mammary cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5246. doi:10.1158/1538-7445.AM2017-5246

Abstract 3616: Potential therapeutic use of Estrogen Receptor β agonist in the prevention and progression of breast cancer using HER-2/neu mouse model

Abstract Breast cancer is the most common cancer among women worldwide. Hormone-mediated therapy to treat estrogen receptor alpha (ERα) positive breast cancers include the use of ER antagonist, Tamoxifen, aromatase inhibitors and other compounds that degrade ERα. Unlike ERα, ERβ has been shown to have tumor-suppressive function in various cancers, including breast cancer. Recent studies have identified, synthesized, and tested the clinical safety of ERβ-selective agonists. Given the tumor-suppressive properties of ERβ, it may be possible to use these compounds to induce or activate the ERβ and test their role in the chemoprevention and blocking the progression of breast cancer. We have investigated the therapeutic utility of ERβ agonists in the prevention and progression of breast cancer using MMTV-HER2/neu mice transgenic mouse model. MMTV-HER2/neu mice develop premalignant lesions at 4-5 months, and tumors starting at month 7 due to overexpression of the HER2/neu proto-oncogene. MMTV-HER2/neu mice were treated with an ERβ agonist, LY500307 examined the prevention and progression of mammary cancers in these mice. When compared to controls, ERβ agonist-treated mice exhibited a significant decrease in the development of preneoplastic changes. Differential gene expression analysis revealed a significant change in the expression of a number of genes in response to LY500307 treatment. Pathway analysis identified an enrichment for chemokines signaling pathways, particularly TNF, in blocking the development of preneoplastic changes resulting from treatment with ERβ agonists. Our studies also show a decrease in the formation of mammary tumors in HER-2/neu mice with preexisting preneoplastic changes when treated with LY500307. This study suggests that ERβ agonist treatment may be a valuable therapeutic option for the prevention in women at increased risk of breast cancer and in blocking the progression of hormone receptor positive breast tumors. Citation Format: Kumaraguruparan Ramasamy, Cathy Samayoa, Naveen K. Krishnegowda, Ratna K. Vadlamudi, Rajeshwar R. Tekmal. Potential therapeutic use of Estrogen Receptor β agonist in the prevention and progression of breast cancer using HER-2/neu mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3616. doi:10.1158/1538-7445.AM2017-3616

Translation of a Tissue-Selective Rexinoid, UAB30, to the Clinic for Breast Cancer Prevention.

This review focuses on our efforts to translate a low-toxicity retinoid X receptor-selective agonist, UAB30, to the clinic for the prevention of breast cancers. The review is divided into several sections. First, the current status of breast cancer prevention is discussed. Next, preclinical studies are presented that support translation of rexinoids to the clinic for cancer prevention. While current FDAapproved retinoids and rexinoids demonstrate profound effects in treating cancers, they lack sufficient safety for long term use in the high risk population that is otherwise disease free. The review stresses the need to identify cancer preventive drugs that are effective and safe in order to gain wide use in the clinic. Due to the heterogeneity of the disease, UAB30 is evaluated for the prevention of ER-positive and ER-negative mammary cancers. Since selective estrogen receptor modulators and aromatase inhibitors are used clinically to prevent and treat ER-positive breast cancers, preclinical studies also must demonstrate efficacy of UAB30 in combination with existing drugs under use in the clinic. To support an Investigational New Drug Application to the FDA, data on pharmacology and toxicity as well as mutagenicity is gathered prior to human trials. The review concludes with a discussion of the outcomes of human Phase 0/1 clinical trials that determine the safety and pharmacology of UAB30. These studies are essential before this agent is evaluated for efficacy in phase 2 trials. Success in phase 2 evaluation is critical before long-term and costly phase 3 trials are undertaken. The lack of surrogate biomarkers as endpoints for phase 2 evaluation of rexinoid preventive agents is discussed.

Anticancer Potential of Nutraceutical Formulations in MNU-induced Mammary Cancer in Sprague Dawley Rats.

Background:Nutraceuticals help in combating some of the major health problems of the century including cancer, and ‘nutraceutical formulations’ have led to the new era of medicine and health.Objective:To develop different nutraceutical formulations and to assess the anticancer potential of nutraceutical formulations in N-methyl-N-nitrosourea (MNU)-induced mammary cancer in Sprague Dawley rats.Materials and Methods:Different nutraceutical formulations were prepared using fine powders of amla, apple, garlic, onion, papaya, turmeric, and wheat grass with and without cow urine distillate. Total phenolic content, acute oral toxicity, and microbial load of nutraceutical formulations were assessed. The anticancer potential of nutraceutical formulations was evaluated against MNU-induced mammary cancer in female Sprague Dawley rats.Results:Improvement in total phenolic content was significant (P < 0.001) after self-fortification process. Toxicity studies showed that the nutraceutical formulations were safe to use in animals. Microbial load was within the limits. Significant longer tumor-free days (P < 0.01), lower tumor incidence (P < 0.01), lower tumor multiplicity (P < 0.05) and tumor burden (P < 0.01) were observed for nutraceutical formulation-treated groups.Conclusion:Combination of whole food-based nutraceuticals acted synergistically in the prevention of mammary cancer. Further, the process of fortification is novel and enhanced the anticancer potential of nutraceutical formulations.SUMMARYNutraceuticals help in combating some of the major health problems of the century including cancer, and ‘nutraceutical formulations’ have led to the new era of medicine and health. In this study, different nutraceutical formulations using fine powders of amla, apple, garlic, onion, papaya, turmeric, and wheat grass with and without cow urine distillate. Total phenolic content, acute oral toxicity, and microbial load of nutraceutical formulations were assessed. The anticancer potential of nutraceutical formulations was evaluated against MNU-induced mammary cancer in female Sprague Dawley rats. Improvement in total phenolic content was observed after self-fortification process. Toxicity studies showed that the nutraceutical formulations were safe to use in animals. Microbial load was within the limits. Longer tumor-free days, lower tumor incidence, lower tumor multiplicity and tumor burden were observed for nutraceutical formulation-treated groups. This suggests that combination of whole food-based nutraceuticals acted synergistically in the prevention of mammary cancer. Further, the process of fortification enhanced the anticancer potential of nutraceutical formulations.Abbreviations used: HMNU: N-methyl-N-nitrosourea, CAM: Complementary and Alternative Medicine, NF: Nutraceutical Formulation, SFNF: Self-Fortitfied Nutraceutical Formulation, NFCUD: Nutraceutical Formulation fortified with Cow Urine Disstillate, SFNFCUD: Self-Fortified Nutraceutical Formulation fortified with Cow Urine Disstillate, CPCSEA: Committee for the Purpose of Control and Supervision of Experiments on Animals, OECD: Organisation for Economic Co-operation and Development, TPC: Total Phenolic Content, ANOVA: Analysis of Variance, GAE: Gallic Acid Equivalent, cfu/g: Colony forming unit per g

Studies on the biochemical and molecular effects of some natural herbs on experimental-induced breast cancer in Wistar rats

Background: Breast cancer is the most frequent malignant tumor in women, and its metastatic state represents the second cause of mortality. To treat cancer, we must prevent tumor cell proliferation and angiogenesis. The usage of herbal medication for treatment of some diseases as diabetes is increased nowadays for their wide safety margin. Aims and Objectives: Therefore, the current study was designed to examine the protective effect of ginger extract (GE) and cinnamon extract (CE) in controlling the mammary gland incidence in female Wistar rats. Materials and Methods: A total of 75 female virgin Wistar rats were allocated into 5 groups. Control group without any treatment; mammary gland group administered 7, 12-dimethyl-benz[a]anthracene at a dose of 20 mg/kg orally in corn oil. Rats in 3-5 served as tumor groups and received GE for group 3 (0.125% in water), CE for group 4 (100 mg/kg), and a mixture of GE plus CE for group 5. GE and CE were administered 2 weeks before cancer induction and continued for 4 months. Serum levels of breast cancer biomarker (CA125) and oxidative stress biomarkers were measured. Tissues of tumors were examined at molecular and histopathological levels. Results: There was a significant increase in the serum levels of CA125 in untreated mammary gland tumor group when compared with untreated healthy rats. There was a significant decrease in the serum levels of glutathione (GSH) peroxidase, catalase, superoxide dismutase, and GSH reductase in the untreated mammary gland tumor groups compared to control healthy rats. Administration of ginger and CE normalized the decrease in antioxidants levels. Induction of mammary gland tumor upregulated the genes associated with tumor incidences such as GST-P, CYP1A1, CYP1B1, and vascular endothelial growth factor-receptor 1 and administration of both herbal plants normalized these changes. Coadministration of GE and CE-induced additive inhibitory effect on genes upregulated due to tumor incidence. Bax expression was downregulated in tumor group and increased after GE and CE expression either alone or in combination. At histopathological levels, breast cancer group showed adenocarcinoma that is decreased with plants extracts administration when compared with breast cancer and control group. Conclusion: Results suggest that extracts of ginger and cinnamon have anticarcinogenic protective activities and support the hypothesis that these plants help in the prevention of mammary cancer by controlling the expression of carcinogenesis-associated genes.

Conformationally Defined Rexinoids and Their Efficacy in the Prevention of Mammary Cancers.

(2E,4E,6Z,8Z)-8-(3',4'-Dihydro-1'(2H)-naphthalen-1'-ylidene)-3,7-dimethyl-2,3,6-octatrienoinic acid (UAB30) is currently undergoing clinical evaluation as a novel cancer prevention agent. In efforts to develop even more highly potent rexinoids that prevent breast cancer without toxicity, we further explore here the structure-activity relationship of two separate classes of rexinoids. UAB30 belongs to the class II rexinoids and possesses a 9Z-tetraenoic acid chain bonded to a tetralone ring, whereas the class I rexinoids contain the same 9Z-tetraenoic acid chain bonded to a disubstituted cyclohexenyl ring. Among the 12 class I and class II rexinoids evaluated, the class I rexinoid 11 is most effective in preventing breast cancers in an in vivo rat model alone or in combination with tamoxifen. Rexinoid 11 also reduces the size of established tumors and exhibits a therapeutic effect. However, 11 induces hypertriglyceridemia at its effective dose. On the other hand rexinoid 10 does not increase triglyceride levels while being effective in the in vivo chemoprevention assay. X-ray studies of four rexinoids bound to the ligand binding domain of the retinoid X receptor reveal key structural aspects that enhance potency as well as those that enhance the synthesis of lipids.

Abstract 4657: Targeting STAT3 for mammary cancer prevention in MMTV/Neu mice employing the antagonist GLG-302

Abstract GLG-302 (S3I-201, NSC 74859) from the National Cancer nstitute chemical libraries was identified as a STAT3 antagonist using structure-based virtual screening with a computer model of the Stat3 SH2 domain bound to its Stat3 phosphotyrosine peptide derived from the x-ray crystal structure of the Stat3 homodimer. In addition to preclinical therapeutic efficacy in breast, glioma, and pancreatic cancer, it ameliorated the resistance to cetuximab and doxorubicin in models of liver cancer. It was well tolerated in mice, rats and dogs in pilot safety studies. Since our laboratories have shown that activated STAT3 is present in normal mammary tissue of young mice and mammary cancers, we evaluated the efficacy of GLG-302, given orally, for its preventive activity in female MMTV-Neu mice that develop spontaneous estrogen receptor negative (ER−) mammary cancers. Previous studies had shown that acute dosing with GLG-302 would suppress the growth of established mammary cancers in mouse models at tolerable doses. We conducted a dose selection study lasting 3 months indicating that doses of GLG-302 as high as 500 mg/kg BW/day (administered by gavage 5X/week) would not alter body weights or induce other signs of toxicity. Proliferation rates (Ki67) in the mammary glands of the mice, determined after 2 weeks and after 3 months of GLG-302 administration, revealed that this dose of the agent caused reductions of 66% and 55%, respectively. A cancer prevention efficacy study was then initiated that included the following groups (15 mice/group); Group 1, GLG-302 (500 mg/kg BW/day); Group 2, GLG-302 (250 mg/kg BW/day); Group 3, GLG-302 (125 mg/kg BW/day); Group 4, Lapatinib (100 mg/kg BW/day) as a positive control; and Group 5, no treatment. Treatment (5X/week) was initiated when the mice were 65 days of age, and will continue for 10 months. Mammary tumors are detected by palpation of the mice 2X/week. The study is currently 5 months after the initial GLG-302 treatment. Average palpable mammary tumor number in each group is: Group 1, 0.07; Group 2, 0.33, Group 3, 0.50, Group 4, 0.08, and Group 5, 1.20. The 94% reduction in the average number of mammary tumors observed in mice receiving the high dose of GLG-302 compared to that in the no treatment group; and is comparable to the reduction observed following treatment with lapatinib. No body weight loss or other toxicity has been observed. In conclusion, IHC data demonstrated that GLG-302 can target the normal mammary epithelial cells of the mouse mammary gland during well-tolerated chronic dosing of the agent. Furthermore, current data in the prevention study has demonstrated a dose-dependent decrease in spontaneous mammary tumors in female MMTV/Neu mice. Additional studies using GLG-302 are ongoing in the dimethylbenzanthracene-induced mammary cancer (ER+) rat model, and will be presented. Studies supported by NCI contract HHSN261201200021I. Citation Format: Robert H. Shoemaker, Michael W. Lovell, John J. Whalen, Fariba Moeinpour, Clinton J. Grubbs. Targeting STAT3 for mammary cancer prevention in MMTV/Neu mice employing the antagonist GLG-302. [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 4657. doi:10.1158/1538-7445.AM2015-4657

Development and characterization of a novel rat model of estrogen-induced mammary cancer.

The ACI rat model of 17β-estradiol (E2)-induced mammary cancer is highly relevant for use in establishing the endocrine, genetic, and environmental bases of breast cancer etiology and identifying novel agents and strategies for preventing breast cancer. E2 treatment rapidly induces mammary cancer in female ACI rats and simultaneously induces pituitary lactotroph hyperplasia and adenoma. The pituitary tumors can result in undesired morbidity, which compromises long-term studies focused on mammary cancer etiology and prevention. We have defined the genetic bases of susceptibility to E2-induced mammary cancers and pituitary tumors and have utilized the knowledge gained in these studies to develop a novel inbred rat strain, designated ACWi, that retains the high degree of susceptibility to E2-induced mammary cancer exhibited by ACI rats, but lacks the treatment-related morbidity associated with pituitary lactotroph hyperplasia/adenoma. When treated with E2, female ACWi rats developed palpable mammary cancer at a median latency of 116 days, an incidence of 100% by 161 days and exhibited an average of 15.6 mammary tumors per rat following 196 days of treatment. These parameters did not differ from those observed for contemporaneously treated ACI rats. None of the E2-treated ACWi rats were killed before the intended experimental end point due to any treatment-related morbidity other than mammary cancer burden, whereas 20% of contemporaneously treated ACI rats exhibited treatment-related morbidity that necessitated premature killing. The ACWi rat strain is well suited for use by those in the research community, focusing on breast cancer etiology and prevention.

Altered Blood Proteome in Girls with High Urine Concentrations of Bisphenol A, Genistein, Mono-Ethyl Hexylphthalate and Mono-Benzyl Phthalate

Children exposed to endocrine disruptors are hypothesized to be susceptible for cancer development later in life. Identifying functional biomarkers of specific exposures may indicate predisposition for this disease. The objectives of this study were to identify protein biomarkers of 1) effect and 2) susceptibility for cancer from the blood of girls exposed to select environmental chemicals. In prepubertal girls, urine concentrations of bisphenol A (BPA), genistein, mono-ethyl hexylphthalate (MEHP) and mono-benzyl phthalate (MBzP) were used to identify girls in the top quintile of exposure for each of these environmental chemicals, and age-matched prepubertal girls with urine analyte concentrations below the median. Blood samples of these girls were depleted of the seven most abundant proteins using human-specific affinity spin columns. Using isobaric Tandem Mass Tags and quantitative mass spectrometry (TMT-MS), 51, 34, 57 and 47 differentially expressed proteins were identified from the blood of prepubertal girls with high urine concentrations of BPA, genistein, MEHP and MBzP, respectively, compared to controls. The data demonstrates the potential of proteomic technology to not only provide biomarkers of effect from aminimally invasive source of biological material, blood, but to identify protein molecules that are intimately involved in the pathobiology of cancer. The differentially regulated cancer associated proteins in girls with high concentrations of BPA and genistein are consistent with reported roles of BPA in carcinogenesis and of genistein in mammary cancer prevention, respectively.