Agents For Prevention Of Breast Cancer Research Articles

Virtual Screening and Network Pharmacology-Based Study to Explore the Pharmacological Mechanism of Vitis Venifera (Grapes) for Anti-Breast Cancer Treatment

Background: Breast cancer is a significant global concern, with its burden increasing worldwide. Projections for the coming decades indicate that a substantial proportion of the disease's incidence and mortality will affect underprivileged groups. There is enormous potential in natural products for developing therapeutic agents for breast cancer prevention and treatment. However, research evidence is essential to establish the effectiveness of these organic remedies. Objectives: Using a network ethnopharmacological framework, this study aims to assess the anti-breast cancer effects of Vitis vinifera (grapes), traditionally believed to have anticancer properties. Methods: The study utilized polypharmacological screening to gather information about Vitis vinifera (grapes) from multiple databases. It investigated the relationship between specific bioactives found in Vitis vinifera and targets associated with breast cancer using Binding DB. Results: The network analysis revealed ten potential bioactives interacting with 17 targets related to breast cancer. Among these, quercetin and kaempferol showed a higher number of interactions with the identified targets in the study. Conclusions: The research conducted an in-silico investigation of the cellular mechanisms by which specific bioactive phytoconstituents from Vitis Vinifera influence the inhibition of several targets involved in breast cancer.

A randomized Phase I pre-operative window trial of transdermal endoxifen in women planning mastectomy: Evaluation of dermal safety, intra-mammary drug distribution, and biologic effects

Breast cancer prevention only requires local exposure of the breast to active drug. However, oral preventive agents entail systemic exposure, causing adverse effects that limit acceptance by high-risk women. Drug-delivery through the breast skin is an attractive option, but requires demonstration of dermal safety and drug distribution throughout the breast. We formulated the tamoxifen metabolite (E/Z)-endoxifen for transdermal delivery and tested it in a placebo-controlled, double-blinded Phase I trial with dose escalation from 10 to 20 mg daily. The primary endpoint was dermal toxicity. Thirty-two women planning mastectomy were randomized (2:1) to endoxifen-gel or placebo-gel applied to both breasts for 3–5 weeks. Both doses of endoxifen-gel incurred no dermal or systemic toxicity compared to placebo. All endoxifen-treated breasts contained the drug at each of five sampling locations; the median per-person tissue concentration in the treated participants was 0.6 ng/g (IQR 0.4–1.6), significantly higher (p < 0.001) than the median plasma concentration (0.2 ng/mL, IQR 0.2–0.2). The median ratio of the more potent (Z)-isomer to (E)-isomer at each breast location was 1.50 (IQR 0.96–2.54, p < 0.05). No discernible effects of breast size or adiposity on tissue concentrations were observed. At the endoxifen doses and duration used, and the tissue concentration achieved, we observed a non-significant overall reduction of tumor proliferation (Ki67 LI) and significant downregulation of gene signatures known to promote cancer invasion (FN1, SERPINH1, PLOD2, PDGFA, ITGAV) (p = 0.03). Transdermal endoxifen is an important potential breast cancer prevention agent but formulations with better dermal penetration are needed.

Abstract P2-08-01: Licochalcone A from licorice reprograms metabolic and antioxidant pathways in the breast leading to a tumor preventive environment

Abstract Background: Increased adiposity is a risk factor for postmenopausal breast cancer. It is often accompanied by chronic low-grade inflammation and elevated levels of reactive oxygen species, which drive breast tumorigenesis. Risk reducing drugs such as selective estrogen receptor modulators and aromatase inhibitors, which have demonstrated efficacy, have had a significantly low acceptance among women at high risk for breast cancer. This hesitancy is mainly due to the adverse side effects of these medications such as vasomotor symptoms, osteoporosis, thromboembolism, and uterine cancer. Therefore, alternative strategies with lower toxicity and greater acceptability are needed. We have previously shown that licochalcone A (LicA) from licorice (Glycyrrhiza inflata), which has osteogenic effects, suppresses aromatase expression and activity, enhances the activity of detoxifying enzymes, and reduces estrogen genotoxic metabolism in cell lines and animal models. However, its effects on the breast tissue of high-risk women are understudied. We hypothesize that LicA creates a tumor preventive environment in the breast by locally modulating adipogenesis and antioxidant/anti-inflammatory responses leading to decreased proliferation. Methods: We prepared microstructures from fresh tissue of contralateral unaffected mastectomy specimens of 6 postmenopausal women with incident unilateral breast cancer. We exposed these to DMSO (control) and LicA (5 µM) for 24 h. Employing total RNA sequencing, we examined differential gene expression between treated and control samples. Up-regulated and down-regulated genes were analyzed using Enrichr gene ontology (GO) pathway analysis. Enriched pathways with combined enrichment scores &amp;gt; 4 and FDR &amp;lt; 0.05 were considered statistically significant. Metabolism flux analysis was performed (FDR &amp;lt; 0.05). Live cell imaging to monitor proliferation of pre-malignant DCIS.COM, DCIS.COM/ER+ PR+; and malignant MDA-MB-231 (ER- PR-), MCF-7 (ER+ PR+), MCF-7aro, and BRCA1 defective HCC-1937, and HCC3153 cells was conducted using IncuCyte. Single versus repeated dosing of various concentrations of LicA were also evaluated. Results: We observed significant (P &amp;lt; 0.05) upregulation up to 8-fold of antioxidant genes, consistent with significant upregulation of NRF2, the major regulator of antioxidant pathways. This was accompanied by significant (P &amp;lt; 0.05) downregulation of NF-kB dependent inflammatory pathways. In addition, we observed the significant (P &amp;lt; 0.05) downregulation, ranging from 4 to 32-fold of cholesterol biosynthesis and transport, steroid hormone biosynthesis, as well as lipid metabolism genes, consistent with the profound downregulation of SREBF1 and SREBF2, which encode the master regulator of adipogenesis, SREBP. Metabolic flux results demonstrated a robust increase (FDR &amp;lt; 0.05) in the pentose phosphate shunt and NAD(P)H generation without enhancing ribose 5 phosphate formation, confirming an antioxidant and anti-proliferative environment. Likewise, LicA suppressed proliferation of pre-malignant and malignant cells dose- and time-dependently. Repeated dosing of lower concentrations of LicA (&amp;lt; 10 µM) demonstrated sustained antiproliferative effects even in the aggressive cancer cell lines. Conclusion: Our data suggest that LicA can generate a tumor-preventive breast microenvironment by reprogramming metabolic pathways involved in steroid and lipid homeostasis and antioxidant responses. These observations along with its low toxicity, suggest that LicA is a good candidate for further investigation as a breast cancer prevention agent. Citation Format: Atieh Hajirahimkhan, Elizabeth T. Bartom, Sriram Chandrasekaran, Susan Clare, Seema Khan. Licochalcone A from licorice reprograms metabolic and antioxidant pathways in the breast leading to a tumor preventive environment. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-08-01.

In Vitro anti-metastasis of Perilla frutescens leaf water extract on aggressive human breast cancer cells

Background: Perilla frutescens is a long-established plant that is often used in foods and traditional medicines in Asian countries. The perilla leaf contains a considerable number of bioactive substances, such as phenolics and flavonoids, which have been demonstrated to possess anticancer activity in vitro and in vivo. Objectives: We aimed to study anti-metastatic activity, anti-invasion activity, and anti-migration activity of perilla leaf water extract (PLW) at 90°C for 1-5 min in MDA-MB-231 aggressive human breast cancer cells. Materials and methods:Dry perilla leaves were extracted using hot water for 1-5 min to obtain crude extract and then lyophilized for PLW powder. PLW was evaluated for total phenolic, total flavonoid, and rosmarinic acid (RA) contents by Folin-Ciocalteau reagent, aluminum chloride colorimetric assay, and ultra-high-pressure liquid chromatography, respectively. Antioxidant activity of PLW was determined by DPPH and ABTS assays. MTT assay was performed to evaluate the cytotoxicity of PLW on MDA-MB-231 cells. Effective PLW was further determined its inhibitory effect on human breast cancer cell metastasis by a Boyden chamber-based transmembrane assay, the MMP-9 activity, and the proteolytic type IV collagenase activity. Results: PLW by 5-min infusion showed the highest amount of total phenolic and flavonoid contents, as well as RA. Moreover, by the 5-min infusion, PLW had the highest antioxidant capacity when compared to PLW by infusions for 1-4 min. Following that, cytotoxicity testing revealed that the PLW is not toxic to MDA-MB-231 cells after a 24-hr exposure. The PLW at non-toxic doses (12.5-100 µg/mL) intensely presents an inhibitory effect on cell invasion and migration. The gelatinolytic activity showed that the PLW at concentrations of 12.5-100 µg/mL decreases MMP-9 activity in a dose-related manner. Furthermore, after treatment with the PLW, the proteolytic type IV collagenase activity was reduced considerably in a dose-related manner. Conclusion: Our findings further showed that the PLW samples inhibit proteolytic enzymes involved in basement membrane breakdown, which might explain the anti-invasion and anti-migration properties of breast cancer cells. From the result, the application of perilla leaf might be developed as an herbal tea and used as an anti-metastatic agent for breast cancer prevention and treatment.

Abstract 717: Title: Modulation of local cholesterol biosynthesis and response to oxidative stress in high-risk postmenopausal breast tissue with licochalcone A from licorice

Abstract Background: The risk of breast cancer increases with age, despite a drastic decline in circulatory estrogen after menopause. Women at increased risk can reduce this by use of anti-endocrine agents, but often decline these drugs to their adverse effects. Therefore, alternative strategies with greater acceptability are needed. We have previously shown that licochalcone A (LicA) from licorice (Glycyrrhiza inflata) suppresses aromatase expression and activity, enhances the activity of detoxifying phase II metabolism enzymes, and reduces estrogen genotoxic metabolism in cell lines and animal models. However, its effects on the breast tissue of high-risk women have not been studied. We hypothesize that LicA creates a tumor preventive environment in the breast by locally modulating cholesterol and steroid hormone biosynthesis, and antioxidant/anti-inflammatory response. Methods: We prepared microstructures from fresh tissue of contralateral unaffected mastectomy specimens of 6 postmenopausal women with incident unilateral breast cancer. We exposed these microstructures to DMSO (control) and LicA (5 µM) for 24 h. After total RNA sequencing, we examined differential gene expression between treated and control samples using the limma R package. Enrichment results were generated by analyzing the up-regulated and down-regulated gene sets using Enrichr. A gene ontology (GO) pathway analysis was performed. The enriched pathways with combined enrichment scores &amp;gt; 4 and FDR &amp;lt; 0.05 were considered statistically significant. We performed confirmatory qPCR experiments in samples obtained from 18 additional women. Results: We observed significant (P &amp;lt; 0.05) upregulation up to 8-fold of antioxidant genes NQO1, HMOX1, G6PD, SQSTM1, PGD, and GPX2. Significant upregulation of Nrf2, the transcription factor involved in the activation of antioxidant enzymes, was evident. In addition, we observed the significant (P &amp;lt; 0.05) downregulation, ranging from 4 to 32-fold of cholesterol biosynthesis and transport genes, MSMO1, FDPS, EBP, SQLE, MVD, MVK, IDI1, DHCR7, NSDHL, FDFT1, LSS, ABCA1, ABCC3, ABCG1, steroid hormone biosynthesis genes HSD17B7, STS, CYP1A1, and CYP1B1, as well as lipid metabolism genes ACAT2, ACYL, FADS1, FADS2, ELOV6, CYP51A1, and LDLR. Some of the genes are shared between these pathways. The significant upregulation of the regulators of these pathways, SREBF1 and SREBF2 provided further confirmation. Conclusion: Our data suggest that LicA can lead to a tumor-preventive breast microenvironment by reducing cholesterol and steroid hormones and lowering oxidative stress mediators. These observations along with the popularity of natural products, suggests that LicA might be a good candidate to be studied further as an alternative breast cancer prevention agent. Citation Format: Atieh Hajirahimkhan, Elizabeth Bartom, Susan Clare, Seema A. Khan. Title: Modulation of local cholesterol biosynthesis and response to oxidative stress in high-risk postmenopausal breast tissue with licochalcone A from licorice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 717.

Abstract OT2-09-01: Pilot study of denosumab in BRCA1/2 mutation carriers scheduling for risk-reducing salpingo-oophorectomy

Abstract Background: Denosumab is a monoclonal antibody that inhibits RANKL and is approved for the prevention of fractures in patients with osteoporosis or bone metastases. The RANKL signaling pathway is also involved in BRCA1-associated mammary tumorigenesis via a progesterone-induced paracrine effect of RANKL on luminal progenitor cells. Pre-clinical studies have demonstrated that RANKL inhibition resulted in reduced proliferation of mammary tumors. Early findings from an ongoing pre-surgical study demonstrated that denosumab treatment resulted in decreased Ki67 proliferation index in benign breast tissue. Based on these data, denosumab is being pursued as a potential preventive agent for breast cancer in BRCA1 mutation carriers. While promising, the effect of RANKL inhibition on gynecologic tissues such as the ovaries and fallopian tubes, in which progesterone has a protective effect, is unknown. Trial design: We will conduct a multicenter, open-label randomized pilot study of presurgical administration of denosumab versus no treatment in premenopausal women with BRCA1/2 mutations undergoing risk-reducing salpingo-oophorectomy (RRSO). A total of 60 women will be randomized 1:1 to Arm 1) 3-4 doses of 120 mg denosumab subcutaneously every 4 weeks or Arm 2) No treatment. Participants will be stratified by 1) BRCA1 versus BRCA2 mutation status and 2) Use of hormonal contraceptives within the past 3 months (yes/no). Assuming a 10% unevaluable rate, we expect to have 54 evaluable participants (27 per arm). Eligibility criteria: 1) Premenopausal women (defined as &amp;lt; 3 months since last menstrual period OR serum follicle-stimulating hormone (FSH) &amp;lt; 20 mIU/mL), age &amp;gt; 18 years; 2) Documented germline pathogenic mutation or likely pathogenic variant in the BRCA1 or BRCA2 gene; 3) Plan for RRSO with or without hysterectomy; 4) ECOG performance status ≤ 1 (Karnofsky ≥ 70%); 5) Normal organ and marrow function; 6) Negative pregnancy test and use of adequate contraception; 7) Willingness to take supplemental oral calcium and vitamin D3; 8) Dental examination within 6 months of enrollment and no evidence of active dental issues; 9) Ability to understand and willingness to provide informed consent. Specific aims: Our primary objective is to compare the effect of denosumab to no treatment on Ki67 expression in the fimbrial end of the fallopian tube. Secondary objectives are to assess Ki67 in ovary and endometrium; cleaved caspase-3, RANK/RANKL, ER/PR, CD44, and STAT3/pSTAT3 expression in fallopian tube, ovary, and endometrium; gene expression profiling in the fallopian tube and ovary; serum markers (progesterone, estradiol, C-terminal telopeptide) and denosumab levels; and toxicity. Statistical methods: The primary endpoint is post-treatment Ki67 expression in the fimbrial end of the fallopian tube in the denosumab arm compared to the no treatment arm. Assuming a standard deviation of 5.0%, we will have 82% power to detect a 4.0% absolute difference (or effect size of 0.8) in Ki67 proliferation index between the denosumab and no treatment groups by applying a 2-sample t-test at a 0.05 significance level. Target accrual: 60 participants, to be activated in Summer 2018. Citation Format: Trivedi MS, Samimi G, Wright JD, Holcomb K, Garber JE, Horowitz NS, Arber N, Friedman E, Wenham RM, House M, Parnes H, Lee JJ, Abutaseh S, Vornik LA, Heckman-Stoddard BM, Brown PH, Crew KD. Pilot study of denosumab in BRCA1/2 mutation carriers scheduling for risk-reducing salpingo-oophorectomy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT2-09-01.

Downregulation of HDAC2 and HDAC3 via oleuropein as a potent prevention and therapeutic agent in MCF-7 breast cancer cells.

Breast cancer is the most common malignancy in the world with the highest rate of morbidity and mortality. Due to the several side effects of chemotherapy and radiotherapy, recent studies have focused on the use of herbal medicines. Epidemiological reports have shown the inverse relationship between breast cancer risk and intake of olive. Oleuropein (OLE) is a polyphenolic compound in virgin olive oil with antineoplastic properties and it is well tolerated by humans. Recent reports have shown that OLE has effects on the control of cancer by modulating epigenetics, such as histone deacetylase (HDAC) inhibition. However, the epigenetic mechanisms of OLE anticancer properties are yet to be properly investigated. Therefore, this study aimed to determine the therapeutic effects of OLE through the modulation of histone deacetylase 2 (HDAC2) and histone deacetylase 3 (HDAC3) expression in breast cancer cell line. MCF-7 cells were tested with and without OLE, and also the cell viability, apoptosis, and migration were examined. HDAC2 and HDAC3 expression genes were assessed by quantitative real-time polymerase chain reaction. It was found that OLE decreased the expression of both HDAC2 and HDAC3 (P < 0.05), induced apoptosis and retarded cell migration and cell invasion in a dose-dependent manner (P < 0.05). These results showed that OLE is a potential therapeutic and preventive agent for breast cancer.

Non-hormonal Chemoprevention

Potential strategies for prevention of breast cancer include lifestyle modification, preventive therapies (i.e., chemoprevention), and surgical prevention. Several therapies (such as selective estrogen response modifiers and aromatase inhibitors) have been shown to prevent breast cancer. However, tolerability and toxicity limit use and these therapies only prevent estrogen receptor–positive (ER) disease. There is emerging data regarding prevention properties of several non-hormonal agents. These agents generally have lower toxicities and seem to be effective for reduction of both ER+ and ER− disease and are often approved for use in other diseases. The purpose of this review is to discuss the data supporting the potential role of these non-hormonal agents for breast cancer prevention. Several studies have demonstrated the potential of aspirin, vitamin D, metformin, and bisphosphonates to prevent breast cancer. The outlined studies support biological mechanisms and show epidemiologic associations but randomized controlled trials are necessary before any of these agents can be recommended for breast cancer prevention. Given the wide spread use of these agents, significant consideration to trial design is needed (i.e., focus on high-risk populations and use of biomarker endpoints). Another important consideration may be the investigation of combinations of agents as suggested by others.

Ethanol Extracts of Coleus tuberosusFlesh and Peel as a Potential Source of Natural Antioxidant and Breast Cancer Prevention Agent

This study examines the effects of ethanol extracts of Coleus tuberosus flesh and peel on T47D cancer cells. Antioxidant potential was evaluated through cellular antioxidant activity experiment, and anti-proliferation activity was evaluated using MTT assay (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide). The effects on cell cycle were evaluated with a flow cytometry, while induction of apoptosis was evaluated based on morphological changes by staining with acridine orange and ethidium bromide. The results indicate that the peel extract had higher cellular antioxidant activity than the flesh extract. The IC50 of celullar antioxidant activity of the flesh and peel extracts were 287.13±10.35 g/ml and 217.86±12.96 g/ml, respectively. The peel extract also had higher anti-proliferative activity. The IC50 of anti-proliferative activity were 887.05±5.03 µg/ml (flesh extract) and 548.18±4.52 µg/ml (peel extract). The peel and flesh extracts can cause cell cycle arrest in the S phase and G2-M phase. The extracts induce apoptosis within T47D cancer cells, showing an orange color. Therefore, these extracts could be used as potential sources of natural antioxidants and breast cancer prevention agents.

Esculentoside A suppresses breast cancer stem cell growth through stemness attenuation and apoptosis induction by blocking IL-6/STAT3 signaling pathway.

Breast cancer stem cells (CSCs) survive in inflammatory microenvironment, their survival are regulated by inflammatory cytokines and signaling pathways. Esculentoside A (EsA), a triterpene saponin derived from the root of Phytolacca esculenta, possesses antiinflammation effects; but whether it has anticancer activity is unknown. The purpose of this study is to test the inhibitory effect of EsA on the growth of breast CSCs and to elucidate its probable mechanisms of action. The proliferation inhibitory effect of EsA on breast CSCs in vitro were determined by cytotoxicity, mammosphere formation inhibition, apoptotic cell detection assays, and in vivo tumor growth inhibition experiment. The possible molecular mechanisms elucidating the inhibitory effect of EsA on breast CSC growth were examined with western blotting. EsA caused proliferation and mammosphere formation inhibition of breast CSCs; induced breast CSCs apoptotic death; suppressed the growth of tumors generated from breast CSCs significantly; the expressions of stemness proteins including ALDH1A1, Sox2, and Oct4 were downregulated; proapoptotic proteins, Bax and cleaved caspase-3 were upregulated, whereas the antiapoptotic protein Bcl-2 was reduced; IL-6/STAT3 pathway proteins including IL-6, phosphorylated STAT3 (Tyr705), and STAT3 (Ser727) were downregulated significantly in EsA-treated breast CSCs and tumor tissues. EsA inhibited breast CSC growth in vitro and in vivo through stemness attenuation and apoptosis induction by blocking IL-6/STAT3 signaling pathway; it might serve as a novel candidate agent for human breast cancer treatment and/or prevention.

Abstract 276: Targeting cancer stem cells with chemopreventive agents for breast cancer prevention

Abstract Breast cancer accounts for one of the most common causes of cancer-related deaths in women. Despite the available treatment modalities to cure breast cancer, therapeutic resistance and recurrence remain major challenges, owing to the culminating evidence of the presence of cancer stem cell subpopulations in breast tumors. The purpose of the study is to identify chemopreventive and dietary compounds that effectively target cancer stem cells to inhibit breast cancer development and progression. The mammosphere culture system has been proven to enrich cancer stem-like cells, and cells from mammospheres are shown to be more tumorigenic in vivo. By culturing mammospheres from breast cancer cell lines, we have shown that 1,25 vitamin D3, a vitamin D3 analog, BXL0124, pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene), tocopherols and triterpenoids (CDDO-Im) decreased the mammosphere forming efficiency (MFE) in a dose-dependent manner. Vitamin D compounds (1,25D3 and BXL0124) decreased the MFE in SUM159, MCF10DCIS and MCF-7 breast cancer cell lines (dose range 1-100 nM). Pterostilbene, a bioactive component of blueberries, effectively decreased the MFE in the MCF-7 cell line. The size of the mammospheres was found to be smaller with increasing concentrations (10-100 μM). Tocopherols significantly decreased estrogen-stimulated MFE in MCF-7 cells (1-10 μM). CDDO-Im, a synthetic triterpenoid derived from natural oleanolic acid, has also been shown to decrease MFE and sphere size in MCF-7 cells (50-100 nM). The effect on the shape of the spheres was also observed, suggesting the differentiation-inducing properties of these compounds. Taken together, we demonstrate that naturally occurring compounds as well as synthetic analogs derived from natural and dietary compounds significantly inhibit sphere forming efficiency and cancer stemness in breast cancer cells. Our study suggests that chemopreventive compounds, 1,25D3, BXL0124, pterostilbene, tocopherols, and CDDO-Im, are agents with the potential to target cancer stem-like cells to prevent breast cancer. Citation Format: Naing Lin Shan, Jeffrey Yang, Min Ji Bak, Joseph Wahler, Nanjoo Suh. Targeting cancer stem cells with chemopreventive agents for breast cancer prevention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 276.

Abstract 3261: Pilot study of the combination of bazedoxifene and conjugated estrogen to modulate risk biomarkers in women with hot flashes at increased risk for breast cancer

Abstract Uptake of anti-hormonal agents for primary prevention of breast cancer is poor due to concern about side effects, especially induction of menopausal symptoms. A combination of 20 mg bazedoxifene plus 0.045 mg conjugated estrogen is FDA approved (as Duavee®) for treatment of hot flashes and prevention of osteoporosis in postmenopausal women with a uterus. We undertook a pilot study to assess the feasibility of using this formulation as a breast cancer prevention agent in women at increased risk for development of breast cancer. Feasibility was to be assessed by accrual, retention, and documentation of a lack of enhanced proliferation in benign breast tissue acquired by random periareolar fine needle aspiration (RPFNA). Eligibility criteria included risk &amp;gt;2X that of average risk woman for age group, postmenopausal having hot flashes or night sweats and not on systemic hormone replacement, and at least 500 cells on a baseline RPFNA. Women were ineligible if they had LCIS or DCIS, a BRCA1/2 germline mutation, had had a hysterectomy, or had &amp;gt;4% Ki-67 positive cells by immunocytochemistry. Fasting blood draw, digital mammography with Volpara software, and DXA scan for body composition was performed at baseline along with QOL questionnaires. Women then received Duavee® daily for 6 months, followed by repeat of baseline tests. We accrued the first 20 subjects in 14 months. Many of the women followed in our cohort and interested in the trial were not eligible due to prior hysterectomy, prior LCIS or a high penetrance gene mutation. Thus accrual was slower than anticipated. All women have reported improvement in hot flash frequency and intensity. None have discontinued prematurely or had a study related serious adverse event. Fourteen women have completed the 6-month intervention and are evaluable for modulation of biomarkers. There have been no protocol-defined increases in proliferation (to &amp;gt;2% Ki-67 for baseline Ki-67 &amp;lt;1% or doubling if baseline Ki-67 ≥1%), with 10 of 14 paired specimens exhibiting a decrease. Ten women had Volpara fibroglandular assessments pre- and post-study with a median relative decrease of 11% (8 decreased and 2 increased). For the first ten subjects where serum hormones and growth factors were assessed in paired assays, favorable modulation was observed for estradiol, testosterone, SHBG, bioavailable testosterone, IGF-1, and the molar ratio of IGF1:IGFBP3. A primary prevention trial in symptomatic women appears feasible given the favorable initial results. The current pilot will continue to accrue so as to inform the design of a randomized, placebo-controlled Phase II trial of Duavee® in women at risk for breast cancer. Financial support provided by grants from the Breast Cancer Research Foundation (BCRF-16-049, BCRF-17-049). Duavee® provided by Pfizer, Inc. which was not involved in design, conduct, or analysis of the study. Citation Format: Carol J. Fabian, Kandy R. Powers, Jennifer L. Nydegger, Amy L. Kreutzjans, Trina Metheny, Teresa A. Phillips, Lauren Nye, Carola M. Zalles, Bruce F. Kimler. Pilot study of the combination of bazedoxifene and conjugated estrogen to modulate risk biomarkers in women with hot flashes at increased risk for breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3261.

Simultaneous downregulation of miR-21 and miR-155 through oleuropein for breast cancer prevention and therapy.

Breast cancer (BC) is the leading cause of cancer mortality in women worldwide. It recently was proven that miRNAs play a critical role in BC development. The use of natural agents for control of cancer by modulating miRNAs is promising. Oleuropein is a natural polyphenolic agent with anti-neoplastic properties and is well tolerated by humans. This study was undertaken to determine the therapeutic effects of oleuropein through modulation of master oncomiRs (miR-21 and miR-155) in BC cells. The present study provides the first link between miRNA and oleuropein as a mechanism in BC. MCF-7 cells were tested with and without oleuropein and the cell viability, apoptosis, and migration were examined. The effect of oleuropein on miR-21 and miR-155 expression was assessed through qRT-PCR. It was found that oleuropein induced apoptosis and retarded cell migration and invasion in a dose-dependent manner in the human MCF7 BC cell line. It was observed that oleuropein significantly decreased expression of both miR-21 and miR-155 over time in a dose-dependent manner. These results demonstrate that oleuropein is a potential therapeutic and preventive agent for BC. Oleuropein exhibits an anti-cancer effect by modulation of tumor suppressor gene expression, which is targeted by oncomiRs.

Prescription of Chinese herbal products is associated with a decreased risk of invasive breast cancer.

The finding of a decrease in endometrial cancer incidence among breast cancer survivors following the use of Chinese herbal products (CHPs) has led to speculation that CHPs might play a role in breast cancer prevention.This study provides an overview of breast cancer incidence, comparing CHP users with those who do not use traditional Chinese medicine (TCM), referred to as non-TCM users. The results can provide information to clinicians for counseling women about the preventive use of TCM.A total of 184,386 women (20-79 years of age) were recruited from a nationwide 1-million-person representative sample of those covered by National Health Insurance in Taiwan and were followed from 1999 to 2012. A total of 1853 incidents of invasive breast cancer were diagnosed. The person-year approach with the Poisson assumption was used to estimate the incidence density rate. The age-specific hazard ratios of breast cancer in relation to either CHP or siwutang (SWT) use were calculated with multivariate Cox proportional hazard regression.More than 78% of patients had used a CHP at some point previously. The overall incidence density rate of breast cancer for non-TCM users was estimated at 1.73 per 10,000 patient-years. The corresponding values for CHP and SWT users were lower than those of the non-TCM group (CHP group = 0.85; SWT group = 0.63 per 10,000 patient-years). The covariate adjusted HRs for breast cancer were 0.57 (95% confidence interval [CI] 0.50-0.65) and 0.36 (95% CI 0.28-0.46) in women using CHPs and SWT, respectively. The findings were confirmed using propensity score matching.Consumption of CHPs reduces the incidence of invasive breast cancer. Although the mechanism of action of these products is unclear, their use as a preventive agent for breast cancer is appropriate for many women at an increased risk of breast cancer.

Raloxifene hydrochloride for breast cancer risk reduction in postmenopausal women

ABSTRACTIntroduction: Raloxifene is an estrogen receptor modulator which competes with estrogens for binding to the estrogen receptor. Based on the results of the STAR (Study of Tamoxifen And Raloxifene) trial, raloxifene has been approved by the U.S. Food and Drug Administration for the reduction of breast cancer (BC) risk in postmenopausal women at increased risk.Areas covered: This analysis reviews the activity of raloxifene and the clinical trials for non-BC indications which led to investigate its use as BC preventive agent. We review the trial establishing its efficacy for BC prevention and the meta-analyses including different SERMs for BC prevention.Expert commentary: Compared with tamoxifen, raloxifene has shown a slightly lower efficacy in reducing BC risk and a better safety profile. Raloxifene also offers to postmenopausal women a benefit in terms of osteoporosis. Future research should investigate its use in premenopausal women and in association with other preventive agents.

Abstract 1789: Estrogen receptor alpha and beta gene expression in benign breast tissue of high risk premenopausal women treated with the SERM acolbifene

Abstract Background We conducted a pilot study to assess the feasibility of a 4th generation selective estrogen receptor modulator (SERM) acolbifene as a breast cancer prevention agent in premenopausal women at high risk for development of breast cancer [Cancer Prev Res 8, December 2015]. Favorable modulation of biomarkers was observed in benign breast tissue acquired before and after intervention. This included reduction in proliferation (Ki-67 immunocytochemistry) and decreases in expression of genes that code for ERα, pS2, and PgR. While ERα is over-expressed and stimulatory in breast carcinogenesis, ERβ which would be inhibitory is down-regulated. Thus, a change in the ratio of gene expression might be most informative. We have now analyzed gene expression of ERβ so as to examine whether it, or the ratio of ERα to ERβ, may contribute to assessment of response. Methods Premenopausal women at elevated risk for breast cancer were screened by random periareolar fine needle aspiration (RPFNA) performed during the follicular phase of the menstrual cycle. Eligible women (n = 25) received 6-8 months of open-label acolbifene (20 mg/d), followed by repeat RPFNA. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) was performed on 16 paired specimens for several genes associated with estrogen activity: estrogen receptor 1 (ESR1) for ERα, trefoil factor 1 (TFF1) for pS2, GREB1 for growth regulation by estrogen in breast cancer 1, PGR for progesterone receptor, and estrogen receptor 2 (ESR2) for ERβ. The quantity of each biomarker transcript was expressed relative to the reference transcript HPRT1. Further normalization by epithelial cell markers (KRT19 for cytokeratin 19 or CDH1 for E-Cadherin) did not materially alter the results. Results Significant changes had been previously shown for transcripts that code for pS2 (15 decreases, 1 increases; p = 0.002), ERα (11↓, 5↑; p = 0.013), and PgR (15↓, 1↑; p = 0.001); plus a suggestion of effect of GREB-1α (10↓, 6↑; p = 0.074). ERβ exhibited a general increase, but this was not statistically significant (5↓, 11↑; p = 0.24). However, the reduction of the ratio of ERα to ERβ was statistically significant (15↓, 1↑; p = 0.002). Conclusions Assessment of the ratio of gene expression for ERα to ERβ, and possibly their protein products by immunocytochemistry, may provide a useful surrogate endpoint biomarker by which to monitor response to SERMs used for breast cancer chemoprevention in premenopausal women. Supported by NO1-CN-35135. Citation Format: Bruce F. Kimler, Teresa A. Phillips, Carol J. Fabian. Estrogen receptor alpha and beta gene expression in benign breast tissue of high risk premenopausal women treated with the SERM acolbifene. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1789.

Abstract PD1-05: Aspirin use is associated with lower mammographic density in a large screening cohort

Abstract Background: Current breast cancer prevention agents have substantial side effects and do not prevent estrogen receptor negative (ER-) breast cancer. Aspirin is a promising breast cancer prevention therapy; it is cheap, safe, well tolerated, with strong biologic and epidemiologic evidence for a prevention effect on both ER- and ER+ breast cancers. However, clinical trials to date have failed to corroborate a prevention effect; these results are potentially related to study design (dose, duration of therapy and followup, population treated). We sought to evaluate the effect of aspirin on mammographic density, as breast density is a well-accepted, modifiable risk factor for both estrogen receptor positive (ER+) and ER- breast cancer. Methods: Electronic medical records from the University of Pennsylvania were retrospectively evaluated for women from a core set of 36 primary care/ObGyn practices. Individuals were selected if they had both undergone routine screening mammography during 2012-2013 and had an ambulatory visit within the year prior with a confirmed list of medication use. We selected the medication record closest to the screening exam. Logistic regression was performed to test for associations between clinically-recorded BIRADS breast density and aspirin use, after adjusting for the additional risk factors of age, body mass index (BMI) and ethnicity. Results: We identified 26000 women who fit the above criteria, of whom 19.7% reported current aspirin use and 41% were African American. Mean age was 57.3 (standard deviation [sd], 10.9) and mean BMI was 28.9 (sd, 7.3) kg/m2 for the entire cohort. Aspirin users were significantly older and had higher BMI (see Table). There was an independent, inverse association between aspirin use and mammographic density (Ptrend&amp;lt;0.001). Compared to women with extremely dense breasts, women with fatty (OR=1.73, CI: 1.33-2.25) or scattered fibroglandular (OR=1.50; CI: 1.17-1.92) breasts were more likely to be aspirin users. A dose-response pattern was observed, as there was a lower likelihood of having extremely or heterogeneously dense breasts with increasing aspirin dose (OR=0.62, CI: 0.50-0.76 for &amp;gt;300 mg; OR=0.84, CI=0.77-0.91 for &amp;lt;=300 mg; compared to non-users as reference group). The association between aspirin use and density was more pronounced for women &amp;lt;60 and for African American women (p=0.01). Conclusion: We demonstrate an independent association between aspirin use and lower mammographic density in a large, diverse screening cohort. Our results suggest that this association is stronger for younger and African American women: two groups at greater risk for ER- breast cancer. Future evaluation of this cohort will examine duration of aspirin use, and evaluate an automated measure of breast density. These results and others highlight the potential value and need for a randomized, controlled trial of aspirin as a preventive agent for breast cancer. CharacteristicAspirin Non-UsersAspirin UsersPAge, mean (SD)55.3 (10.2)65.3 (9.8)&amp;lt;0.0001BMI, mean (SD)28.5 (7.2)30.4 (7.6)&amp;lt;0.0001Breast density, no. (%) OR (95% CI)BIRADS 12006 (9.6)861 (16.9)1.73 (1.33 - 2.25)BIRADS 29346 (44.7)2859 (55.9)1.50 (1.17 - 1.92)BIRADS 38480 (40.6)1312 (25.7)1.22 (0.95 - 1.56)BIRADS 41057 (5.1)79 (1.6)1.00 (Reference) Citation Format: Wood ME, Sprague BL, Oustimov A, Synnstvedt MB, Cuke M, Emily CF, Kontos D. Aspirin use is associated with lower mammographic density in a large screening cohort. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD1-05.

Abstract P6-10-10: History of chemoprevention in patients with newly diagnosed breast cancers

Abstract Background: Chemoprevention (including tamoxifen, raloxifene and exemestane) is a strategy to reduce breast cancer incidence in high risk women. Studies have shown at least a 50% decrease in the incidence of breast cancer in users of these drugs. The benefit is limited to a reduction in the incidence of ER/PR positive breast cancer. However, there is a growing population of women who have used these agents for primary breast cancer prevention, and a larger population of breast cancer survivors who have used these drugs as part of their systemic treatment. The purpose of this study was to identify a contemporary cohort of women with newly diagnosed breast cancers who had utilized chemoprevention strategies and describe their patterns of disease. Methods: The Breast Cancer Database at our institution was queried for patients who reported use of chemopreventive drugs and developed breast cancer between 1/2010-4/2014. Patients were divided into primary and secondary chemoprevention groups (no previous history of breast cancer and previous history of breast cancer, respectively). Descriptive statistics were utilized. Results: Out of a total of 1782 patients with newly diagnosed breast cancer, there were 106 (6%) patients who had used a chemopreventive agent. Out of these 106 patients, 91 (86%), had used the drug as part of their systemic therapy for prior breast cancer, with a median of 11 years from the initial diagnosis to the diagnosis of a second breast cancer. The primary chemoprevention group included women with risk based on family history and atypical hyperplasias. The majority of patients (81%) were diagnosed with early stage disease (stage 0, 1). In both groups, the majority of cancers were ductal in origin (84%). Eight of the 15 patients (53%) in the primary chemoprevention group were on treatment at the time of their cancer diagnosis; while 29% of patients in the secondary group were on treatment at the time of diagnosis. In the secondary group, 49% were contralateral second primary breast cancers and 44% were ipsilateral recurrences. Interestingly, the majority of cancers in both groups were ER/PR positive. Conclusions: Our cohort of women who used chemoprevention drugs were overwhelmingly diagnosed with early stage breast cancer, likely reflecting their commitment to screening. The majority of cancers were ER/PR positive. In this group, the choice of cancer treatment will need to be modified in light of prior hormonal treatment. Many of the patients in the secondary group were past users of prevention agents and further work is needed to clarify the duration of benefit of these drugs. In a similar vein, we look forward to research efforts to define the optimal age to initiate primary chemoprevention in high risk women. Citation Format: Jennifer Chun, Freya Schnabel, Shira Schwartz, Karen Hiotis, Amber Guth, Deborah Axelrod. History of chemoprevention in patients with newly diagnosed breast cancers [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-10-10.

Abstract P4-11-02: Mammary tumor formation induced by N-methyl-N-nitrosourea (MNU) is accelerated by natural and synthetic progesterone but suppressed by an anti-progesterone CDB4124

Abstract Background: CDB4124, anti-progesterone suppresses the development of carcinogen-induced ER+/PR+ mammary tumors in rats, and may have implications for prevention and treatment of human breast cancer. We hypothesize that progesterone (P4) and medroxyprogesterone acetate (MPA) will accelerate mammary carcinogenesis induced by MNU, however CDB4124 will efficiently suppress tumor formation stimulated by progesterone. Methods: ovary intact female Sprague Dawley rats received a single intraperitoneal injection of 50mg/kg MNU at 4-5 weeks of age. 30mg of CDB4124 and 25mg of P4 or MPA (90 release pellets, Innovative research of America, Inc) were implanted in dorsal area at 3 weeks and 4 weeks after MNU injection, respectively. 10-11 rats were used for each treatment group. Tumor incidence, latency, multiplicity, and burden were recorded weekly. 9 weeks after MNU injection all the mice were euthanized and mammary tumors and glands were fixed in 10% (v/v) neutral buffered formalin. Plasma concentrations of CDB4124 and its metabolite CDB4453 were determined by LC-MS/MS. Results: The first tumor appeared in the control group at 5 weeks, and in the P4 and MPA treated groups at 6 weeks after MNU injection. 7 weeks after MNU injection, mammary tumor incidence of MPA and P4 treated groups were 80% and 50%, respectively compared to 30% in the control group. 9 weeks after MNU injection all MPA treated mice, 80% of P4 treated mice, and 60% of control mice developed tumors. Tumor incidence, latency, multiplicity, and tumor weight were summarized as mean ± SD in Table 1. [Table 1] Tumor latency, incidence, multiplicity, and burden in mammary tumorsTreatmentsLatency (days)Incidence(%)MultiplicityBurden (g)Control53.7 ± 12.9602.34.25 ± 7.02P453.6 ± 9.6802.64.11 ± 5.32MPA50.8 ± 7.71002.56.02 ± 4.85P4 + CDB412459.3 ± 11.53621.38 ± 0.61MPA + CDB412454.3 ± 10.7732.84.19 ± 4.39 Tumor latency of CDB4124 treated groups was increased; tumor incidence and burden (g) of CDB4124 treated groups were decreased compared to P4 and MPA treated groups. In particular, tumor incidence and burden of CDB4124 + P4 treated group were significantly lower than those of the control group. Plasma CDB4124 and CDB4453 were 11.6 ±5.88 ng/mL and 3.4±1.68 ng/mL, respectively. Histopathology of tumors and mammary glands and immuno-histochemical evaluations of Ki67, activated caspase-3, CD34, ER, and PR are currently underway. Conclusions: Our results indicated that natural progesterone promotes MNU- induced mammary tumor formation similar to synthetic progesterone, MPA in rats. Under this tumor permissive environment, CDB4124 provided excellent prevention efficacy, suggesting good potential as breast cancer prevention agent. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-11-02.

A pilot chemoprevention study of isopropanolic black cohosh extract in women with ductal carcinoma in situ.

TPS1609 Background: Recent evidence suggests that black cohosh (Cimicifuga racemosa) may be a potential agent for breast cancer prevention. The active ingredients in black cohosh preparations appear to be triterpene glycosides. Recent preclinical data suggest several mechanisms by which triterpenes may prevent and treat breast cancer, including anti-proliferative, pro-apoptotic, anti-estrogenic, and anti-inflammatory effects. Epidemiologic data demonstrate a significant protective effect of black cohosh against the development of breast cancer in healthy women, and prolonged disease-free survival in breast cancer patients. There is also abundant evidence demonstrating the safety and tolerability of black cohosh in several clinical trials studying its use for treatment of hot flashes. We hypothesize that efficacy of black cohosh can be demonstrated in a pilot pre-operative window trial in a cohort of women with ductal carcinoma in situ (DCIS). Methods: In this trial, we treat women with a 2-5 week pre-operative course of commercial standardized isopropanolic black cohosh extract (20 mg orally twice per day). We aim to demonstrate a reduction in breast epithelial cell proliferation as measured by Ki-67 staining in regions of DCIS using traditional IHC staining and AQUA analysis. We also assess safety and tolerability of black cohosh through monitoring of patient adherence, liver function tests and serum hormone levels. 22 patients will be enrolled onto the trial. Sample size is based on power calculations for the specific study aim of determining the mean change in the levels of Ki67, using a targeted effect size. Assuming a 10% drop-out rate, a sample size of 20 patients will achieve 91% power to detect a 0.8 standard deviation of difference with a two-sided significance level at 0.05 using Wilcoxon signed-rank test. Eligible subjects are pre- and post-menopausal women ≥ 18 years of age newly diagnosed with DCIS histologically confirmed on breast core biopsy, prior to definitive excision. Women who have recently taken any agent known to affect Ki67 levels in the breast (e.g. hormone therapy) are excluded. Enrollment is currently ongoing with 10 of 22 patients accrued. Clinical trial registry number NCT01628536. Clinical trial information: NCT01628536.