Estrogen Suppression Research Articles

The Relationship Between Estrogen and the Decline in Delta Power During Adolescence.

During adolescence, there is a precipitous decrease in slow-wave sleep (SWS) and its spectral correlate, delta power, which may reflect cortical reorganization. The temporal association between the decrease in delta power and puberty suggests that sex steroids may initiate these changes. This association has not been previously investigated. To determine whether estrogen triggers the adolescent decline in delta power, we compared delta power in 14 girls with central precocious puberty (CPP) and 6 age-matched, prepubertal controls. Five CPP participants were re-studied 7-14 months after pubertal suppression to determine if the changes in delta power are reversible after restoring a prepubertal hormonal milieu. The change in delta power was also compared between CPP participants and five historic controls from a longitudinal polysomnographic study. CPP participants (6.7-10.5 years) spent 30% of the night in SWS. Delta power (3.7 × 106 ± 2.7 × 105 µV2) predominated in the first 2 non-rapid eye movement episodes and decayed exponentially (tau 0.006 minutes). Age-matched controls demonstrated similar sleep staging (24% SWS) and delta dynamics (3.3 × 106 ± 5.1 × 105 µV2, tau 0.004 minutes). Four out of 5 CPP participants had a significant decrease (26%) in delta power after hormone suppression (p < .05), similar to historic controls. Using an innovative model of girls with CPP studied before and after estrogen suppression, the effects of puberty on the decline in delta power were dissociated from those of chronologic age. The current studies suggest that increased estrogen does not cause the adolescent decline in delta power and indicate that neurodevelopmental changes per se or other factors associated with puberty drive these sleep changes.

Abstract P1-08-03: Identification and characterization of a novel endoxifen substrate, PKCβ1, and its interaction with the estrogen receptor

Abstract Background: The primary mechanism by which tamoxifen (Tam) and its metabolites exert their biologic effects is through estrogen receptor (ER) binding and inhibition of ER signaling. We and others demonstrated that endoxifen (Endx) has greater antitumor activity in vitro and in vivo compared to Tam and the first-in-human Endx phase I study demonstrated its antitumor activity in patients with prior progression on Tam (Goetz SABC 2015). PKCs are a family of serine/threonine-specific protein kinases that regulate signaling pathways involved in cell proliferation and tumorigenic transformation. Our prior protein docking studies suggested endoxifen may be a substrate for PKCs. Here we report the effects of Tam and Endx on PKCβ1 binding, kinase activity, as well as interactions between PKCβ1 and ERα. Methods: Surface Plasmon Resonance (SPR, Biacore T200, GE Healthcare) was used to evaluate binding of Tam, N-desmethyl Tam (NDMT), 4-HT, and Endx to PKCβ1 and PKCβ2. The effects of Tam and Endx on PKCβ1 kinase activity were determined. Proliferation and colony formation in MCF7 parental and PKCβ1 overexpressing cells were evaluated. siRNA silencing was used to knockdown PKCβ1 expression in the following cells: MCF7 aromatase expressing cells that were either sensitive (MCF7/AC1) or resistant to letrozole (MCF7/AC1 L-resistant); T47D; and MDA-MB-361. Coimmunoprecipitation assay and DUOlink in situ proximity ligation were used to investigate the interaction between PKCβ1 and ERα. Results: Endx more potently inhibited PKCβ1 kinase activity compared to Tam ( IC50 350 nM vs 47.8 μM ) with KDs for PKCβ1 binding as follows: Endx (100 nM ), Tam ( 2 μM ), 4-HT ( 2 μM ) and NDMT (&amp;gt; 7 μM ). None of the SERMs exhibited PKCβ2 binding. In the MCF7/AC1 and MCF7/AC1 L-resistant cells, PKCβ1 knockdown resulted in ERα degradation and potently inhibited cell proliferation. These results were confirmed in T47D and MDA-MB-361 cells. Notably, PKCβ1 knockdown in MCF7/AC1 cells resulted in significantly greater E2 induced proliferation comparing siRNA knockdown vs. control. To further explore these effects, we evaluated the effects of PKCβ1 overexpression in MCF7 cells and demonstrated that PKCβ1 overexpression reduced cell proliferation and colony formation compared to parental MCF-7 cells without affecting ERα protein stability. Coimmunoprecipitation assays in transient transfected MCF-7 cells with exogenous PKCβ1 as well as PKCβ1 expressing MDA-MB-231 cells transiently or stably transfected with ERα demonstrated PKCβ1 and ERα interaction, with confirmation by Duolink assay that this interaction occurs in the cytoplasm. Conclusions: Our findings demonstrated that endoxifen binds and inhibits PKCβ1 at relevant concentrations achieved in the endoxifen clinical trial studies. PKCβ1 interacts with cytoplasmic ERα and PKCβ1 knockdown inhibits cell proliferation and enhances ERα turnover. However, in PKCβ1 overexpressing cells, PKCβ1 may exhibit tumor suppressive effects. These data suggest a complex interaction between PKCβ1 and ERα and that endoxifen's effects on PKCβ1 may alter drug response of endocrine therapy. Further studies are ongoing to characterize the role of PKCβ1 and its role in ER biology and response to endoxifen. Citation Format: Guo C, Kuffel MJ, Kudgus RA, Huang Z, Bode AM, Cheng J, Suman VJ, Reid JM, Bruinsma ES, Subramaniam M, Ames MM, Hawse JR, Goetz MP. Identification and characterization of a novel endoxifen substrate, PKCβ1, and its interaction with the estrogen receptor [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-08-03.

Abstract P2-09-06: Addition of ovarian function suppression to endocrine therapy in premenopausal women with early breast cancer: A meta-analysis

Abstract Introduction The Suppression of Ovarian Function Trial (SOFT) and the Tamoxifen and EXemestane Trial (TEXT), together with the Austrian Breast Cancer Study Group (ABCSG-12)and the Eastern Cooperative Oncology Group 3193 (E-3193 trials, have examined adding ovarian function suppression (OFS) using gonadotropin–releasing hormone (GnRH) agonists to endocrine therapy in premenopausal women with early stage breast cancer. However, OFS with GnRH agonist plus aromatase inhibitor has subsequently been found to not consistently reduce estradiol levels to optimal target in the SOFT-EST study (JCO 2016; 34:1584) and implications of these study findings for clinical practice are controversial. Therefore, to further examine this issue, we conducted a systematic literature review and meta-analysis of available evidence. Methods We updated a recently published literature review of randomized clinical trials (JCO 2016; 34:1689) through June 1, 2016 using the keywords “breast cancer,” “ovarian function suppression,” “tamoxifen,” and “aromatase inhibitor”. Of 683 of records identified through database searching, the 4 studies named above met inclusion criteria and were included in the quantitative synthesis. Information on disease-free survival (DFS) and overall survival was examined using hazard ratio (HR) and 95% confidence interval (CI). Because of significant heterogeneity in one performed meta-analyses, the random-effects (DerSimonian and Laird) method was used to estimate the combined RR for studies (Open MetaAnalyst). Results Combining ABCSG-12, SOFT and TEXT trial findings, OFS addition to aromatase inhibitor resulted in 65 fewer DFS events across the 3 trials compared to OFS addition to tamoxifen (350 DFS events vs. 415 DFS events, respectively, HR 0.89, 95% CI 0.57-1.39, P=0.62, Tau2 =0.09, heterogeneity P&amp;lt;0.01). In contrast, 30 more deaths were seen with OFS plus aromatase inhibitor use (155 deaths vs. 125 deaths, respectively, HR 1.31, 95% CI 0.93 -1.84, P=0.12, Tau2 =0.03, heterogeneity P=0.18). Combining SOFT and E-3193 trials to compare OFS addition to tamoxifen to tamoxifen alone use, more concordance between DFS events and overall survival is seen. There were 24 fewer DFS events across the 2 trials with OFS addition to tamoxifen compared to tamoxifen alone (160 DFS events vs. 184 DFS events, respectively, HR 0.83, 95% CI 0.67-1.03, P=0.09, Tau2 =0, heterogeneity P=0.94) and there were 14 fewer deaths in the OFS plus tamoxifen compared to tamoxifen alone group (58 deaths vs. 72 deaths, respectively, HR 0.76 95% CI 0.53 -1.07, P=0.12, Tau2 =0, heterogeneity P=0.78). Conclusion In conclusion, the apparent discordance between DFS and overall survival in the aromatase inhibitor and OFS trials and the results suggesting incomplete and/or intermittent estrogen suppression with GNRH analogs in the SOFT-EST trial suggest that adoption of OFS plus aromatase inhibitor use as adjuvant therapy in premenopausal women with early stage breast cancer may be premature. Longer term survival analyses of the endocrine therapy trials are needed before reliable risks and benefits of aromatase inhibitor plus OFS and tamoxifen plus OFS use as adjuvant therapy can be determined. Citation Format: Chlebowski R, Pan K, Col NF. Addition of ovarian function suppression to endocrine therapy in premenopausal women with early breast cancer: A meta-analysis [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-09-06.

Abstract P2-09-12: A single-blind, randomized, placebo-controlled phase II study to evaluate the impact of oral ibandronate on bone mineral density in osteopenic breast cancer patients receiving adjuvant aromatase inhibitors: Final results of the single-center BONADIUV trial

Abstract Background. Several randomized trials demonstrated aromatase inhibitors (AI) superiority in terms of disease-free survival compared to tamoxifen treatment for postmenopausal hormone receptor-positive breast cancer (BC) patients. Anyway AI toxicity profile due to estrogen suppression is a concern. Pivotal trials demonstrated a significant bone mineral density (BMD) loss due to AI, with a consistent risk of fractures, thus impacting on patients' quality of life. Bisphosphonates represent an effective treatment in postmenopausal osteoporosis fractures prevention. Several studies demonstrated that upfront bisphosphonates therapy prevents bone loss in postmenopausal women receiving adjuvant AI for early-stage BC. However an adequate patients selection for adjuvant bisphosphonates treatment during AI endocrine therapy is still a challenge. We present the final results of the BONADIUV trial, a single-blind, randomized, placebo-controlled phase 2 study designed to evaluate the impact of ibandronate treatment on BMD in osteopenic women taking AI. Methods. Between January 2011 and May 2014, 561 patients underwent a baseline BMD assessment before starting AI as planned adjuvant treatment. Overall 171 osteopenic patients (lumbar spine [LS] and/or trochanter -1&amp;lt; T-score &amp;lt;-2.5), were randomized in a 1:1 ratio to receive either placebo or oral monthly ibandronate (150 mg). All patients receive oral supplementation of calcium and vitamin D3. Study duration was 2 years. Exclusion criteria were: premenopausal status at time of randomization; comorbidities with increased risk of osteoporosis; body mass index &amp;lt;18; chronic use of steroids; previous use of bisphosphonates; psychiatric disorders. Primary endpoint was the mean BMD difference between the two arms at a 2-year follow up. ClinicalTrials.gov identifier: NCT02616744. A total of 72 patients per arm of treatment were needed to obtain an 85% statistical power in order to detect a 2% BMD mean difference between the two arms. Considering a 10% dropout, at least 158 patients were required. Results. A total of 171 patients were randomized in the study. Overall 27 patients (15.8%) withdrew the protocol (17 ibandronate vs 10 placebo arm): the final analysis was performed on 144 patients (72 patients per arm). P-value from Wilcoxon test showed no significant difference between arms at baseline both for LS (p=0.94) and trochanter (p=0.83). At 2-year, osteopenic patients treated with ibandronate gained +18.7% and +15.5% at the LS and trochanter BMD, respectively. Patients treated with placebo lost -13.3% at the LS, and gained +2.9% at the trochanter. Trochanter p-value from covariance analysis showed a mean BMD change significantly in favor of ibandronate arm at 1-year (p=0.012), and borderline at 2-year (p=0.087). Concerning LS, the mean BMD change was significantly in favor of ibandronate arm both at 1-year (p=0.002) and 2-year (p&amp;lt;0.0001). Conclusions. Final results of our study showed that treatment with ibandronate, as compared to placebo, improved BMD change in osteopenic women treated with adjuvant AI, and consistently protected patients' bone loss. Citation Format: Livi L, Saieva C, Desideri I, Scotti V, De Luca Cardillo C, Carta G, Cecchini S, Orzalesi L, Sanchez LJ, Casella D, Bernini M, Nori J, Bianchi S, De Feo ML, Meattini I. A single-blind, randomized, placebo-controlled phase II study to evaluate the impact of oral ibandronate on bone mineral density in osteopenic breast cancer patients receiving adjuvant aromatase inhibitors: Final results of the single-center BONADIUV trial [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-09-12.

Endocrine changes, fetal growth, and uterine artery hemodynamics after chronic estrogen suppression during the last trimester of equine pregnancy.

Equine pregnancy is characterized by very high circulating concentrations of estrogens. The physiological roles of estrogens during equine gestation are largely unknown, although some studies suggest a role in the regulation of uterine artery hemodynamics and a relationship between low circulating estrogen concentrations and late pregnancy loss. The objectives of this experiment were to evaluate the effects of estrogen suppression on uterine artery hemodynamics and on pregnancy outcome. Estrogen synthesis was suppressed using letrozole, a potent aromatase inhibitor. Twelve pregnant mares were randomly assigned to a control (n = 6) or treatment (n = 6; 500 mg letrozole orally every 4 days) group with treatment starting at 240 days of gestation and continuing until parturition. Weekly serum samples were analyzed to determine testosterone, dehydroepiandrosterone sulfate, estradiol, estrone sulfate, progestins, and prostaglandin F2α metabolite concentrations. Ultrasonographic examinations were performed biweekly and measurements included uterine artery hemodynamics (diameter, pulsatility, and resistance indices), fetal growth using the diameter of the fetal eye, and placental evaluation using the combined thickness of the uterus and placenta. At parturition, gestational length, foal weight, and neonatal viability were determined. Letrozole suppressed estrogen synthesis during gestation by approximately 90% compared to control values. This large reduction in circulating estrogens had no effect on uterine artery hemodynamics, normal placental development, maintenance of pregnancy, or neonatal viability. However, neonates from letrozole-treated mares had lower (P < 0.05) birth weights than controls, suggesting that estrogens may play a role in fetal growth that is not mediated through regulation of uterine blood flow.

Estrogen Modulates ubc9 Expression and Synaptic Redistribution in the Brain of APP/PS1 Mice and Cortical Neurons.

Estrogen exerts multiple actions in the brain and is an important neuroprotective factor in a number of neuronal disorders. However, the underlying mechanism remains unknown. Studies demonstrate that ubiquitin-conjugating enzyme 9 (ubc9) has an integral role in synaptic plasticity and may contribute to the pathology of neuronal disorders. We aimed to investigate the effects of estrogen on ubc9 and in the Alzheimer's disease brain. Ubc9 protein and mRNA were significantly increased in the cortex and hippocampus of APP/PS1 mice with enhanced SUMOylation. Systemic estrogen administration led to reduced ubc9 expression in ovariectomized APP/PS1 mice and reduced SUMOylation. The inhibition of ubc9 expression by estrogen was found to be dose-dependent in cultured neurons. However, estrogen receptor (ER) antagonist ICI182780 did not block the inhibition of ubc9 expression by estrogen. Furthermore, the reduced expression of ubc9 was not mediated by ERα or ERβ agonists alone or in combination, but by the membrane-impermeable ER agonist E2-bovine serum albumin. The activation of the G protein-coupled ER mediated the inhibition of ubc9 expression of estrogen. A phosphoinositide 3-kinase (PI3K) inhibitor, rather than an extracellular signal-regulated kinase inhibitor, blocked the inhibition of ubc9 by estrogen. Estrogen treatment significantly increased the phosphorylation of PI3K, which suggests that activation of the PI3K pathway by estrogen is required for ubc9 regulation. Further, ubc9 interacted with the synaptic proteins post-synaptic density protein 95 (PSD95) and synaptophysin. Estrogen decreased the interaction of ubc9 with post-synaptic PSD95, but increased the interaction of ubc9 with pre-synaptic synaptophysin. These results suggest that a membrane-bound ER might mediate the estrogen inhibition of ubc9 in cortical neurons, where PI3K plays an important role. We also show that ubc9 can interact with synaptic proteins, which are subject to estrogen regulation.

Aryl hydrocarbon receptor induced intratumoral aromatase in breast cancer.

Aryl hydrocarbon receptor (AhR) inhibits estrogen receptor (ER) pathway, which may suppress estrogen-dependent cell proliferation. However, the correlation between AhR stimulation and intratumoral estrogen synthesis, especially through aromatase, has not been reported to date. In the present study, we examined this correlation in breast cancer cells. We examined AhR and aromatase immunoreactivity in 29 patients with invasive ductal carcinoma. We performed in vitro studies using three breast carcinoma cell lines, MCF-7, T47D, and MDA-MB-231. AhR stimulation induced the mRNA expression of the aromatase gene in vitro in three breast carcinoma cell lines, and increased estrogen synthesis in MCF-7 cell line. Results of microarray analysis showed that AhR-induced aromatase expression was associated with BRCA1 induction. Analysis of patients with breast cancer showed a significant positive correlation between intratumoral AhR and aromatase status. We also compared the effects of AhR stimulation on the induction of intratumoral estrogen synthesis and inhibition of the ER signaling pathway, because AhR exerts contradictory effects on estrogen action in breast carcinoma cells. AhR-induced aromatase expression persisted for a significantly longer duration than AhR-induced ER pathway inhibition. Moreover, breast carcinoma cells treated with an AhR agonist tended to show earlier cell proliferation after removing the agonist than cells not treated with the AhR agonist. The results of the present study suggest that AhR stimulates estrogen-dependent progression of breast carcinoma by inducing aromatase expression under some conditions. These results provide new insights on the possible roles of environmental toxins in breast cancer development.

Ovarian suppression in combination endocrine adjuvant therapy in premenopausal women with early breast cancer.

The benefits of adding ovarian suppression to either tamoxifen or aromatase inhibitors as adjuvant breast cancer therapy in premenopausal women are controversial. Therefore, we performed a systematic literature review and meta-analysis of relevant randomized trials. We identified and combined four qualifying trials reporting disease-free survival (DFS) and overall survival (OS) using meta-analysis. Combining ABCSG-12, SOFT, and TEXT studies, there were 65 fewer DFS events (HR 0.89, 95% CI 0.57-1.39) but 30 more deaths for ovarian suppression plus aromatase inhibitor compared to ovarian suppression plus tamoxifen (HR 1.31, 95% CI 0.93-1.84, P=0.12, τ=0.03, heterogeneity, P=0.18). DFS and OS were more concordant for combined SOFT and E-3193 findings; for ovarian suppression plus tamoxifen compared to tamoxifen alone, there were 24 fewer DFS events (HR 0.83, 95% CI 0.67-1.07, P=0.09, τ 2=0) and 14 fewer deaths (HR 0.76, 95% CI 0.53-1.07). The SOFT Estrogen Substudy demonstrated inconsistent estrogen suppression with combined ovarian suppression and aromatase inhibitor. Given the discordance between DFS and OS and inconsistent estrogen suppression with ovarian suppression plus aromatase inhibitor, adding aromatase inhibitor to ovarian suppression as adjuvant therapy in premenopausal women is premature.

Progestin suppressed inflammation and cell viability of tumor necrosis factor-α-stimulated endometriotic stromal cells.

Endometriosis is an estrogen-dependent inflammatory disease. Progestins are a first-line treatment for endometriosis via activation of pituitary progesterone receptors and suppression of systemic estrogen: a less than optimal treatment. Increasing evidence is beginning to show that progestins may also influence local endometriotic cells, which may contribute to their clinical efficacy. Endometrial stromal cells (ESC) isolated from women with endometriosis were cultured with TNF-α to simulate an inflammatory environment. ESC were treated with the progestins, medroxyprogesterone acetate (MPA), norethisterone acetate (NETA), or dienogest (DNG) and cytokine mRNA production, protein secretion, and cell viability measured. DNG, NETA, and MPA suppressed the secretion of interleukin (IL)-6, IL-8, and monocyte chemotactic protein (MCP)-1 from ESC. DNG and NETA only reduced the TNF-α-stimulated mRNA production. All three progestins suppressed TNF-α-stimulated ESC proliferation. Progestins may influence endometriotic stromal cells altering the inflammatory microenvironment and their clinical efficacy.

Abstract 2156: Targeting estrogen receptor mutations for treatment of endocrine therapy resistance in breast cancer

Abstract Approximately 70% of all breast cancers express the Estrogen Receptor (ER) and inhibition of the ER signaling remains the mainstay of systemic therapy in such cancers. The advent of endocrine therapies (ET) have contributed significantly to reduction in the incidence, recurrence and mortality associated with breast cancer. However, approximately one-third of patients with ER-positive breast cancer do not benefit from adjuvant ET, and nearly all patients with ER-positive metastatic breast cancer (MBC) ultimately become refractory to all known ETs. While several mechanisms of resistance to ET have been proposed, there are currently no clinically effective treatments for ET resistance. Thus, ET resistance remains a major obstacle to the effective treatment of a significant subset of ER-positive breast cancers. We have recently reported recurrent somatic mutations in the ligand binding domain (LBD) of the Estrogen Receptor gene (ESR1) in metastatic breast cancer patients with a history of prior treatment with ET. The ESR1 mutations result in constitutive activation of the ER and confer ET resistance when ectopically expressed in ER-positive cell lines. In an attempt to develop novel therapeutic strategies to treat ET-resistant breast cancer, we have evaluated OTX015, a clinical bromodomain and extraterminal (BET) domain inhibitor, in pre-clinical models of ET resistance due to ESR1 mutations. Our studies suggest that BET inhibition may serve as an attractive strategy for treatment of ET resistance due to ESR1 mutations. Citation Format: Prasanna G. Alluri, Jose Larios, Rohit Malik, James Rae, Arul M. Chiinaiyan. Targeting estrogen receptor mutations for treatment of endocrine therapy resistance in breast cancer. [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 2156.

Effects of SULT1A1 Copy Number Variation on Estrogen Concentration and Tamoxifen-Associated Adverse Drug Reactions in Premenopausal Thai Breast Cancer Patients: A Preliminary Study.

Tamoxifen is a pharmacological estrogen inhibitor that binds to the estrogen receptor (ER) in breast cells. However, it shows an estrogenic effect in other organs, which causes adverse drug reactions (ADRs). The sulfotransferase 1A1 (SULT1A1) enzyme encoded by the SULT1A1 gene is involved in estrogen metabolism. Previous research has suggested that the SULT1A1 copy number is linked with the plasma estradiol (E2) concentration. Here, a total of 34 premenopausal breast cancer patients, selected from the Thai Tamoxifen (TTAM) Project, were screened for their SULT1A1 copy number, plasma E2 concentration and ADRs. The mean age was 44.3±11.1 years, and they were subtyped as ER+/ progesterone receptor (PR) + (28 patients), ER+/ PR- (5 patients) and ER-/PR- (1 patient). Three patients reported ADRs, which were irregular menstruation (2 patients) and vaginal discharge (1 patient). Most (33) patients had two SULT1A1 copies, with one patient having three copies. The median plasma E2 concentration was 1,575.6 (IQR 865.4) pg/ml. Patients with ADRs had significantly higher plasma E2 concentrations than those patients without ADRs (p = 0.014). The plasma E2 concentration was numerically higher in the patient with three SULT1A1 copies, but this lacked statistical significance.

Role of dietary bioactive natural products in estrogen receptor-positive breast cancer.

Estrogen receptor (ER)-positive breast cancer, including luminal-A and -B, is the most common type of breast cancer. Extended exposure to estrogen is associated with an increased risk of breast cancer. Both ER-dependent and ER-independent mechanisms have been implicated in estrogen-mediated carcinogenesis. The ER-dependent pathway involves cell growth and proliferation triggered by the binding of estrogen to the ER. The ER-independent mechanisms depend on the metabolism of estrogen to generate genotoxic metabolites, free radicals and reactive oxygen species to induce breast cancer. A better understanding of the mechanisms that drive ER-positive breast cancer will help optimize targeted approaches to prevent or treat breast cancer. A growing emphasis is being placed on alternative medicine and dietary approaches toward the prevention and treatment of breast cancer. Many natural products and bioactive compounds found in foods have been shown to inhibit breast carcinogenesis via inhibition of estrogen induced oxidative stress as well as ER signaling. This review summarizes the role of bioactive natural products that are involved in the prevention and treatment of estrogen-related and ER-positive breast cancer.

Abstract P5-12-02: Effect of 5 vs 2.5 mg/day letrozole on residual estrogen levels in post-menopausal women with high BMI - A prospective crossover study

Abstract Background: Some studies have suggested that women with high BMI have less benefit from aromatase inhibitors (AI) vs. tamoxifen as adjuvant treatment for early breast cancer. One possible mechanism for this observation is that complete suppression of estrogen is not achieved in these women with the standard flat dose of AI. We evaluated whether a doubling of letrozole to 5 mg/day for 4 weeks affected residual estrogen levels in this population. Methods: Post-menopausal women with early breast cancer and BMI&amp;gt;25 already taking adjuvant letrozole for at least 3 months were recruited from medical oncology clinics at 4 sites in Toronto, Canada. Fasting blood samples were collected 24 hours following the last dose at baseline (routine use of own letrozole), after 28 days of monitored adherence to a provided supply of letrozole (Femara) 2.5 mg/day (Part A), and after an additional 28 days of letrozole (Femara) 5 mg/day (Part B). Symptom/quality of life questionnaires were completed at the same timepoints. Estradiol and estrone were measured using a high sensitivity liquid chromatography-tandem mass spectrometry assay. One interim analysis for futility and efficacy was planned after 31 eligible patients had completed the study, using estradiol and O'Brien-Fleming boundaries with an inner wedge. Results: 36 patients were enrolled and started on study, and 31 eligible patients completed Parts A and B. The 5 non-completers withdrew because of adverse events (n=4, unlikely related to drug) or withdrawal of consent (n=1). Median age was 62 (range 48 to 77) and BMI 28.3 kg/m2 (Range 25.2 to 42.2 kg/m2). One patient had non-postmenopausal estrogen levels at Day 29 and Day 57 and one patient's blood assay was unsuccessful; both were excluded from further analyses. The predetermined stopping rule for futility was met. Estradiol levels (mean±standard deviation) changed from 2.68±0.40 pg/mL at baseline to 2.67±0.59 pg/mL at Day 29 to 2.70±0.53 pg/mL at Day 57. Mean change from Day 29 to Day 57 was 0.03±0.48 pg/mL (95% confidence interval -0.15 to 0.21 pg/mL). Four patients reported new or increased arthralgias (to NCI CTCAE Grade 2 or 3) while taking letrozole 5 mg/day in Part B. There was no association between changes in estradiol levels and either study non-completion or the development of arthralgias. Estrone results were similar. Conclusion: Increasing letrozole from 2.5 to 5 mg/day did not further suppress estrogen levels in women with BMI&amp;gt;25. It is unlikely that letrozole dosing tailored to body size would improve clinical outcomes. The letrozole 5 mg/day intervention was terminated based on the results of the interim analysis for futility. Citation Format: Cescon DW, Ennis M, Pritchard KI, Townsley C, Warr D, Elser C, Rao L, Stambolic V, Sridhar S, Goodwin PJ. Effect of 5 vs 2.5 mg/day letrozole on residual estrogen levels in post-menopausal women with high BMI - A prospective crossover study. [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 P5-12-02.

The involvement of c-Jun N-terminal kinase signaling pathway in the protective effect of estrogen on attenuating ischemia reperfusion injury in a rat flap model

Objective To observe the protective effect of estrogen against ischemia reperfusion injury in axial flaps, and investigate the role of the c-Jun N-terminal kinase signaling pathway in estrogen′s protective effect. Methods An ischemia reperfusion injury model in the abdominal flap was created in 40 Wistar rats that were randomly divided into 4 groups: control (group A), ischemia reperfusion injury (group B), estrogen (group C) and JUN inhibitor (group D). Seven days postoperatively, gross observation of the flap, measurement of flap survival area and calculation of flap survival rate were carried out. The flap tissues were harvested for hematoxylin-eosin staining to observe histological changes, and for Western blot to quantify JNK, p-JNK and MKP-5 expression. The relationship between flap survival and JNK expression was analyzed. Results Flaps in groups C and D grew well. Flap survival rates in these two groups were significantly higher than that in group B, while pathological changes were milder. Expressions of JNK and p-JNK were significantly lower in flaps of groups C and D than in flaps of group B, while expression of JNK negative regulator MKP-5 was the opposite. Conclusion Estrogen can significantly improve the ischemia reperfusion injury in flaps and increase flap survival rate. The potential mechanism of estrogen′s protective effect can be through regulating Jun N-terminal kinase signaling pathway. Key words: Estrogens; Surgical flaps; Ischemia-reperfusion injury; c-Jun N-terminal kinase pathway

Double-Blind, Randomized Trial of Alternative Letrozole Dosing Regimens in Postmenopausal Women with Increased Breast Cancer Risk.

Aromatase inhibitors (AI) profoundly suppress estrogen levels in postmenopausal women and are effective in breast cancer prevention among high-risk postmenopausal women. Unfortunately, AI treatment is associated with undesirable side effects that limit patient acceptance for primary prevention of breast cancer. A double-blind, randomized trial was conducted to determine whether low and intermittent doses of letrozole can achieve effective estrogen suppression with a more favorable side-effect profile. Overall, 112 postmenopausal women at increased risk for breast cancer were randomized to receive letrozole at 2.5 mg once daily (QD, standard dose arm), 2.5 mg every Monday, Wednesday, and Friday (Q-MWF), 1.0 mg Q-MWF, or 0.25 mg Q-MWF for 24 weeks. Primary endpoint was suppression in serum estradiol levels at the end of letrozole intervention. Secondary endpoints included changes in serum estrone, testosterone, C-telopeptide (marker of bone resorption), lipid profile, and quality-of-life measures (QoL) following treatment. Significant estrogen suppression was observed in all dose arms with an average of 75% to 78% and 86% to 93% reduction in serum estradiol and estrone levels, respectively. There were no differences among dose arms with respect to changes in C-telopeptide levels, lipid profile, adverse events (AE), or QoL measures. We conclude that low and intermittent doses of letrozole are not inferior to standard dose in estrogen suppression and resulted in a similar side-effect profile compared with standard dose. Further studies are needed to determine the feasibility of selecting an effective AI dosing schedule with better tolerability.

Incomplete Estrogen Suppression With Gonadotropin-Releasing Hormone Agonists May Reduce Clinical Efficacy in Premenopausal Women With Early Breast Cancer.

Background Oophorectomy was the first endocrine therapy for breast cancer but has been largely superseded by gonadotropin-releasing hormone agonists (GnRHas) as a result of their reversible, less invasive suppression of ovarian function. The concept was developed more than two decades ago that GnRHa efficacy in treating estrogen receptor–positive breast cancer in premenopausal women might be improved by combination with an antiestrogen or aromatase inhibitor (AI) to antagonize or inhibit the synthesis of the residual nongonadal estrogen. Now, the results of three phase III studies comparing ovarian function suppression (OFS) with and without tamoxifen or comparing OFS plus tamoxifen with OFS plus AI are available; however, the results are conflicting. Here we consider the endocrine pharmacology of these combination therapies and provide potential explanations for the variable clinical findings as well as a possible means of improving combination therapy involving OFS.

The effect of 7 days of letrozole pretreatment combined with misoprostol on the expression of progesterone receptor and apoptotic factors of placental and decidual tissues from first-trimester abortion: a randomized controlled trial

ObjectiveTo evaluate if letrozole-induced suppression of estradiol reduces progesterone receptor expression and apoptosis in the first-trimester placenta. Study DesignWe performed a double-blinded, randomized, placebo-controlled trial. We randomized 20 women requesting first-trimester abortion with gestation up to 63 days to receive either letrozole 10 mg daily or placebo pretreatment for 7 days before administrating 400 mcg of vaginal misoprostol followed by suction abortion. We collected the placental and decidual tissues on which we performed immunohistochemical staining for progesterone receptor and apoptotic markers (active caspase 3, caspase 3, Bcl2, CD95, fas ligand) and determined H-scores of each based on the intensities of staining. We performed terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay for apoptosis in the samples of four women to confirm the findings from apoptotic markers. ResultsWe excluded one woman in the letrozole group from the analysis because she had passage of abortus after taking letrozole, leaving 19 women (9 in the letrozole group, 10 in the placebo group) for analysis. There was no significant difference in the H-scorings of progesterone receptor and apoptotic markers, as well as proportion of apoptotic cells on TUNEL assay between the two groups. The H-scores for the progesterone receptor were 8.17±2.67 (mean±SD) in the letrozole group and 9.01±2.82 in the placebo group (p=0.36). ConclusionWe did not detect a difference in the expression of progesterone receptor and apoptotic markers in placental and decidual tissues after letrozole pretreatment for 7 days in first-trimester abortion. ImplicationsWe did not confirm the hypothesis that letrozole reduces progesterone receptor expression and induces apoptosis in the first-trimester placenta. Further studies are required to allow better understanding of the mechanism by which estrogen suppression following the use of letrozole can lead to improved abortion rate in the first trimester.

Obesity, insulin resistance and breast cancer outcomes

There is growing evidence that obesity is associated with poor outcomes in early stage breast cancer. This paper addresses four current areas of focus: 1. Is obesity associated with poor outcomes in all biologic subtypes of breast cancer? 2. Does obesity effect AI efficacy or estrogen suppression in the adjuvant setting? 3. What are the potential biologic underpinnings of the obesity-breast cancer association? 4. Are intervention studies warranted? If so, which interventions in which populations? Research is needed to resolve these questions; intervention trials involving lifestyle interventions or targeting the biology postulated to link obesity and cancer are recommended.

Abstract 4017: Matrix manipulation affects attachment and growth of breast cancer cells in a bone-like microenvironment in vitro

Abstract Breast cancer notoriously metastasizes to the bone, but it is unknown why breast cancer cells colonize there. Breast cancer cells may become dormant in the skeleton until a stimulated to grow. The aim of this project is to better understand the role of the bone matrix in determining dormancy or growth. Breast cancer cells were co-cultured with MC3T3-E1 mouse osteoblasts that formed a collagenous matrix. The matrix was manipulated by fixation, decellularization, and by varying the medium estrogen levels as the osteoblasts grew. MDA-MB-231-BRMS1GFP (BRMS), a metastatic suppressed variant of the human MDA-MB-231 breast cancer cell line, was used because these cells do not grow in the bone, allowing for observation of growth or dormancy. BRMS were added to a matrix produced by differentiated MC3T3-E1 cells. BRMS proliferated more on a culture of osteoblasts and their matrix when fixed with 1% paraformaldehyde than on a live osteoblasts matrix. Why do the cells proliferate more on the fixed matrix? We ruled out the possibility that live osteoblasts and cancer cells competed for growth medium. Another possibility was that BRMS attach differently to the matrices with live or fixed osteoblasts. We compared attachment of BRMS to live, fixed, and decellularized matrices and to tissue culture plastic, and after 60 minutes it was seen that more BRMS cells attached to a fixed matrix compared to the other conditions. Fluorescent microscopy revealed that BRMS had many attachment points on a fixed matrix. On the live osteoblasts matrix, the cancer cells remained attached to one another instead of attaching to the matrix. Experiments will determine if the attachment explains the effect on proliferation by testing for attachment factors such as β-actin and focal adhesion kinase. Breast cancer is more prevalent in post-menopausal women. Estrogen production decreases with menopause, leading to the hypothesis that the effects of estrogen deprivation on bone cause metastasis. Osteoblasts were grown in the presence of ICI, an estrogen inhibitor preventing estrogen from binding. Osteoblasts grown with and without ICI were assayed for alkaline phosphatase (AP), stained with von Kossa (mineralization), and with ChondrexTM (collagenous and noncollagenous proteins). While AP and mineralization levels were unaffected by the estrogen inhibition, collagenous proteins production decreased. We will study breast cancer cell attachment to matrices grown without estrogen. It is hypothesized that proliferation will increase on a matrix with less collagen, similar to the increased proliferation seen on a fixed matrix. Work was supported by the U.S. Army Medical Research &amp; Materiel Command under W81XWH-12-1-0127, the Metavivor Research Foundation, Undergraduate Research Funds from the Eberly College of Science &amp; the Biochemistry &amp; Molecular Biology Summer Undergraduate Research Program. Citation Format: Shelby J. Foster, Donna M. Sosnoski, Andrea M. Mastro. Matrix manipulation affects attachment and growth of breast cancer cells in a bone-like microenvironment in vitro. [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 4017. doi:10.1158/1538-7445.AM2015-4017

Association between self-report adherence measures and oestrogen suppression among breast cancer survivors on aromatase inhibitors

PurposePoor adherence to oral adjuvant hormonal therapy for breast cancer is a common problem, but little is known about the relationship between self-report adherence measures and hormonal suppression. We evaluated the relationship of three self-report measures of medication adherence and oestrogen among patients on aromatase inhibitors (AIs). Materials and methodsWe recruited 235 women with breast cancer who were prescribed AI therapy. Participants self-reported AI adherence by completing the following: (1) a single item asking whether they took an AI in the last month, (2) a modified Morisky Medication Adherence Scale-8 (MMAS-8) and (3) the Visual Analog Scale (VAS). Serum estrone and estradiol were analysed using organic solvent extraction and Celite column partition chromatography, followed by radioimmunoassay. ResultsTen percent of participants reported they had not taken an AI in the last month and among this group, median estrone (33.2pg/ml [interquartile range (IQR)=22.3]) and estradiol levels (7.2pg/mL [IQR=3.3]) were significantly higher than those in participants who reported AI use (median estrone=11.5pg/mL [IQR=4.9]; median estradiol=3.4pg/mL [IQR=2.1]; p<0.001). This relationship held when controlling for race and AI drug type. ConclusionsA single-item monthly-recall adherence measure for AIs was associated with oestrogen serum levels. This suggests that patient-reported monthly adherence may be a useful measure to identify early non-adherence behaviour and guide interventions to improve patient adherence to hormonal treatment.