Pure Estrogen Receptor Antagonist Research Articles

Abstract PO4-27-07: A Phase 3, randomized, open-label study of upfront camizestrant vs standard endocrine therapy as adjuvant treatment for ER-positive/HER2-negative early breast cancer with intermediate-high or high risk of recurrence (CAMBRIA-2)

Abstract Background: Camizestrant is a next-generation oral selective estrogen receptor (ER) degrader and pure ER antagonist being investigated in early and advanced breast cancer (BC). In postmenopausal women with advanced ER-positive/human epidermal growth factor receptor 2 (HER2)-negative BC and disease recurrence or progression on or after ≤1 endocrine therapy (ET) in the advanced setting, camizestrant significantly prolonged progression-free survival compared with fulvestrant in the Phase 2 SERENA-2 trial. Camizestrant may also provide benefit in the early BC setting. Following locoregional therapy (surgery ± radiotherapy), standard adjuvant treatment of ET with or without chemotherapy and/or a cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) has shown significant benefit in decreasing the risk of recurrence of stage I–III ER-positive/HER2-negative BC. However, BC recurrence as incurable metastatic disease is still common. Thus, adjuvant therapeutic options with improved clinical outcomes are needed. The CAMBRIA-2 study (NCT05952557) aims to assess whether upfront camizestrant can improve outcomes compared with standard ET as adjuvant treatment in patients with ER-positive/HER2-negative early BC with an intermediate-high or high risk of recurrence after definitive locoregional therapy. Trial design: This ongoing Phase 3, randomized, open-label study is enrolling women (pre-, peri-, or postmenopausal) and men with ER-positive/HER2-negative (HER2-negative status defined as immunohistochemistry 0, or 1+, or in situ hybridization-negative) early BC who are at intermediate-high or high risk of recurrence (as defined in the protocol) after having completed definitive locoregional therapy (surgery ± radiotherapy) ± (neo)adjuvant systemic chemotherapy, and who have no evidence of disease. Patients may have received up to 12 weeks of (neo)adjuvant ET prior to randomization. Patients are randomized (1:1) to receive camizestrant 75 mg ± abemaciclib ± luteinizing hormone-releasing hormone (LHRH) agonist or standard ET of the investigator’s choice (any aromatase inhibitor or tamoxifen) ± abemaciclib ± LHRH agonist for up to 7 years. The primary endpoint is invasive BC-free survival (IBCFS) (Standardized Definitions for Efficacy End Points [STEEP] 2.0 criteria). Secondary endpoints include invasive disease-free survival (IDFS) and distant relapse-free survival (DRFS) (STEEP 2.0 criteria), overall survival, safety, and health-related quality of life. Primary endpoint analysis will use a stratified log-rank test adjusting for stratification factors, assuming a two-sided significance level of 5%. Approximately 5500 patients will be randomized. Clinical trial identification: NCT05952557 Editorial acknowledgment: Writing assistance was provided by Clare Davis of BOLDSCIENCE Inc., funded by AstraZeneca. Legal entity responsible for the study: AstraZeneca Funding: This study was supported by AstraZeneca. Citation Format: Sibylle Loibl, Yeon Park, Sara Tolaney, Ioanna Gioni, Simon Johnston, Teresa Klinowska, Ingrid A. Mayer, Raquel Nunes, Barbara Pistilli, Mary Stuart, Angela Quintana, Andrew Walding, Michael Gnant. A Phase 3, randomized, open-label study of upfront camizestrant vs standard endocrine therapy as adjuvant treatment for ER-positive/HER2-negative early breast cancer with intermediate-high or high risk of recurrence (CAMBRIA-2) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-27-07.

Abstract RF01-01: SERENA-3: A randomized pre-surgical window of opportunity study assessing dose and duration of camizestrant treatment in post-menopausal women with ER-positive, HER2-negative primary breast cancer

Abstract Background Camizestrant, a next-generation oral selective estrogen receptor (ER) degrader (SERD) and pure ER antagonist, has demonstrated statistically significant and clinically meaningful progression-free survival (PFS) benefit over fulvestrant at both 75 and 150 mg once-daily doses in the Phase 2 SERENA-2 (NCT04214288) study in post-menopausal women with ER+ HER2- advanced breast cancer. SERENA-3 (NCT04588298) was established to explore the biological effects of three camizestrant dose levels in post-menopausal women with ER+ HER2- primary breast cancer. Methods Post-menopausal women with ER+ HER2- primary breast cancer scheduled to undergo curative intent surgery were randomly assigned in Stages 1 and 2 to pre-surgical treatment with camizestrant at 75, 150 or 300 mg once daily for 5 to 7 days or, in a sequential Stage 3, randomly assigned to camizestrant at 75 or 150 mg once daily for 12 to 15 days. Image-guided tumor biopsies were accessed as pre-treatment samples, while on-treatment ultrasound-guided tumor biopsies were obtained on the last day of camizestrant treatment. The primary objective was to explore the effect of camizestrant on ER expression by comparison of pre-treatment and on-treatment biopsies as assessed by immunohistochemistry (IHC) H-score. Secondary objectives included assessment of Ki67 and progesterone receptor (PgR) expression, and also of safety and tolerability, with exploratory pharmacodynamic (PD) assessment using other transcriptomic and proteomic technologies. The study was undertaken in 17 centers across 3 countries (Georgia, Mexico and the United Kingdom). Results 135 patients were randomized and received treatment. 76 patients received camizestrant for 5 to 7 days at doses of 75 mg (n=30), 150 mg (n=33), and 300 mg (n=13); 59 received camizestrant for 12 to 15 days at doses of 75 mg (n=30) and 150 mg (n=29). Baseline disease characteristics, and ER, Ki67 and PgR levels were well balanced across arms and stages. Pharmacokinetic (PK) observations in all stages were commensurate with steady-state predictions derived from previous studies. PD data for ER and Ki67 are shown in Table 1. Camizestrant reduced ER H-score by approximately 65% irrespective of dose or duration. While the 75 mg dose reduced Ki67 score to a lesser degree than 150 mg and 300mg after 5–7 days exposure, after 12–15 days exposure 75 and 150 mg reduced Ki76 score to a similar substantial degree (82% reduction). PD results assessed by additional transcriptomics and proteomics assays were concordant with the IHC analyses. No serious adverse events were reported. All treatment-emergent adverse events were grade 1, apart from one patient with a grade 2 visual impairment, and one patient with grade 3 diarrhea. Conclusions SERENA-3 is the first study investigating the effect of multiple dose levels of camizestrant with different treatment durations on ER, Ki67 and PgR expression in post-menopausal women with primary ER+ HER2˗ breast cancer. SERENA-3 demonstrated that the 75 mg dose of camizestrant achieves maximal levels of ER degradation and Ki67 suppression. Together with SERENA-2, this provides strong evidence supporting 75 mg as the optimal dose of camizestrant, including for the early disease setting, and highlights the additive value of a specifically designed multi-dose pre-surgical PD study for optimal dose selection. Table 1 Citation Format: John Robertson, Teimuraz Gogitidze, Zaza Katashvili, Juan Enrique Bargallo Rocha, Ekaterine Arkania, Iain Moppett, Kwok-Leung Cheung, Gia Nemsadze, Maxine Ajimi, Itziar Irurzun-Arana, Justin Lindemann, Teresa Klinowska, Alastair Mathewson, Christopher Morrow, Myria Nikolaou, Giorgi Dzagnidze. SERENA-3: A randomized pre-surgical window of opportunity study assessing dose and duration of camizestrant treatment in post-menopausal women with ER-positive, HER2-negative primary breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr RF01-01.

Abstract P3-07-13: The next generation oral selective estrogen receptor degrader (SERD) camizestrant (AZD9833) is active against wild type and mutant estrogen receptor α

Abstract Endocrine therapy forms the backbone treatment for patients with estrogen receptor (ER) positive tumors in both the adjuvant and metastatic setting. Aromatase inhibitors (AI) are the most common endocrine treatment option. Mutation of ESR1, the gene encoding ERα, is a common mechanism of resistance to AIs which leads to ligand independent activity of ERα. Camizestrant (AZD9833) is a next generation SERD and pure ER antagonist that is in Phase 3 trials (SERENA-4: NCT04711252; SERENA-6: NCT04964934). Here we report the preclinical and clinical activity of camizestrant in patients with ESR1 wild-type (ESR1wt) and mutant (ESR1m) tumors. The binding affinities of camizestrant, fulvestrant, and estradiol to wt ERα and ERα variants with mutations in the ligand binding domain were assessed. All three compounds exhibited reduced binding to mutant forms of ERα compared with wt ERα; the Y537S mutation had the greatest impact on binding. This was reflected in requirement for greater concentrations of camizestrant and fulvestrant to degrade and antagonize mutated ERα and to impact cellular proliferation in MCF-7 cells that expressed Y537S ESR1m compared to ESR1wt MCF-7 cells. Furthermore, while a 3 mg/kg dose of camizestrant achieved a maximal anti-tumor effect in a ESR1wt patient derived xenograft model, a 10 mg/kg was required for maximal effect in a D538G ESR1m model. Considering this difference between ESR1m and ESR1wt, pharmacokinetic/pharmacodynamic modelling of preclinical data predicted that a camizestrant dose of 75 mg would be maximally efficacious in patients with ESR1m tumors. Indeed, analysis of ESR1m circulating tumor DNA levels in patients from the SERENA-1 (NCT03616587) Phase 1 trial showed a clear effect of 14 days treatment with 75 mg camizestrant resulting in a >2-fold reduction in ESR1m variant allele frequency in 12/13 (92%) cases with complete clearance of ESR1m ctDNA in 7/13 (54%) cases. Interestingly, the clinical activity of camizestrant was higher in heavily pretreated patients with metastatic breast cancer with ESR1m tumors compared to those with no detectable mutation (ESR1m not detected). At a camizestrant dose of 75 mg, median progression-free survival was 8.3 months (maturity 12/15) in patients with ESR1m tumors compared to 5.6 months (8/9) in those with ESR1m not detected (data cut-off 6 October 2021). Camizestrant-induced ERα degradation was seen in both groups (mean reduction in H-score 42% in ESR1m tumors (n= 12 evaluable pairs) and 46% in tumors with ESR1m not detected (n=7)). Whole transcriptome analysis revealed a trend towards higher ERα activity at baseline in ESR1m tumors compared to ESR1m not detected; ERα activity reduced on treatment in both groups. Consistent with the clinical activity data, camizestrant induced more profound reductions in cell proliferation in ESR1m tumors compared to ESR1m not detected tumors (as seen by greater reductions in Ki67-positive tumor cells). These data demonstrate the activity of camizestrant in patients with ESR1m tumors. Clinical activity along with degradation and antagonism of the ERα is also seen in patients with tumors in which ESR1 mutations are not detected. In this heavily pre-treated Phase 1 patient population from SERENA-1, ESR1m may be a predictive biomarker to enrich for patients with maintained endocrine sensitivity. The SERENA-6 trial is investigating the efficacy and safety of camizestrant plus a CDK4/6 inhibitor in patients with metastatic breast cancer and detectable ESR1m. We acknowledge Helen Heffron, PhD, from InterComm International who provided medical writing support funded by AstraZeneca. Citation Format: Christopher Morrow, Larissa Carnevalli, Richard D. Baird, Tim Brier, Carmela Ciardullo, Natalie Cureton, Mandy Lawson, Robert McEwen, Myria Nikolaou, Anne Armstrong, Begoña Bermejo, Emiliano Calvo, Eva Ciruelos, Javier Garcia-Corbacho, Erika Hamilton, Jason Incorvati, Peter Kabos, Mafalda Oliveira, Manish R Patel, Manuel Ruiz-Borregó, Nicholas Turner, Chris Twelves, Christos Vaklavas, Danielle Carroll, Steven Ching, Nevena Cvetesic, Michelle DuPont, Lisa Gibbons, Alastair Mathewson, Rhiannon Maudsley, Pablo Morentin Gutierrez, Avinash Reddy, Jaime Rodriguez-Canales, Susana Ros, Dhivya Sudhan, Andy Sykes, David Whitson, Teresa Klinowska, Justin Lindemann. The next generation oral selective estrogen receptor degrader (SERD) camizestrant (AZD9833) is active against wild type and mutant estrogen receptor α [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 P3-07-13.

Abstract OT2-11-05: SERENA-6: A Phase III study to assess the efficacy and safety of AZD9833 (camizestrant) compared with aromatase inhibitors when given in combination with palbociclib or abemaciclib in patients with HR+/HER2- metastatic breast cancer with detectable ESR1m who have not experienced disease progression on first-line therapy

Abstract Background: More than two thirds of patients with metastatic breast cancer (mBC) have hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2−) tumors. Current guidelines recommend combining endocrine therapy (ET), such as an aromatase inhibitor (AI), with an inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6i) as first-line (1L) treatment (Cardoso et al. Ann Oncol 2020). However, drug resistance eventually develops, leading to disease progression. Mutations in the estrogen receptor (ER) alpha gene (ESR1m) result in constitutively active, estrogen-independent ER signaling which can reduce AI efficiency (Reinert et al. Front Oncol 2017). Clinically, ESR1m are associated with acquired resistance to AI as well as more aggressive disease features, including development of visceral metastasis (Gerratana et al. Eur J Cancer 2021). At the initial diagnosis of mBC, the frequency of ESR1m is low (approximately 3%) (Bidard et al. Ann Oncol 2019); however, this increases to 17−35% of patients when disease progresses on an AI + CDK4/6i (Bidard et al. Ann Oncol 2019; Goetz et al. J Clin Oncol 2020). Patients with ESR1m tumors have poor outcomes with subsequent lines of therapy. New approaches are needed to maximize time on 1L treatment with ET + CDK4/6i and prevent further clinical and radiological disease progression. AZD9833 (camizestrant) is a highly potent, next-generation oral selective ER degrader (ngSERD) and pure ER antagonist that has demonstrated antitumor activity in a wide range of ER+ breast cancer cell lines and patient-derived xenograft models, including those with wild type ESR1 (ESR1wt) and the most prevalent ESR1m, D538G and Y537S (Scott et al. AACR 2020; Lawsone et al. AACR 2020). The Phase I SERENA-1 study demonstrated that AZD9833 shows encouraging clinical activity as monotherapy or in combination with a CDK4/6i in heavily pre-treated patients with ER+/HER2− advanced breast cancer whose tumors are ESR1wt or ESR1m (Baird et al. SABCS 2020). SERENA-6 will assess the efficacy of switching patients from AI to AZD9833, while continuing CDK4/6i treatment, once ESR1m are detected but before overt disease progression. Study description: SERENA-6 is an ongoing, randomized, multicenter, double-blind, Phase III trial. Patients with HR+/HER2− mBC who have received at least 6 months of 1L AI (letrozole or anastrozole) + CDK4/6i (palbociclib or abemaciclib) and do not have clinical or radiological disease progression will be enrolled into Step 1, the ESR1m detection phase. During this phase, patients will be monitored regularly for the presence of ESR1m via central circulating tumor DNA analysis. Patients with detectable ESR1m and no overt disease progression (by RECIST v1.1 criteria) will be enrolled into Step 2 double-blind 1:1 randomization to either continue AI plus CDK4/6i, plus a placebo for AZD9833, or switch to AZD9833 (75 mg oral once daily), plus the same CDK4/6i plus a placebo for the AI. The primary endpoint will be investigator-assessed progression-free survival (PFS) per RECIST v1.1 criteria. A key secondary endpoint will be time to second progression or death on a subsequent therapy. Other secondary endpoints will include overall survival, chemotherapy-free survival, objective response rate, clinical benefit rate, patient-reported outcomes, and safety. Enrollment began in June 2021 and is expected at approximately 200 sites across 19 countries. Acknowledgments: We thank Julia Mawer, PhD, of Oxford PharmaGenesis, UK, for medical writing assistance, which was funded by AstraZeneca. Funding: The SERENA-6 trial is funded and overseen by AstraZeneca. Citation Format: François-Clément Bidard, Kevin Kalinsky, Massimo Cristofanilli, Giampaolo Bianchini, Stephen KL Chia, Wolfgang Janni, Cynthia X Ma, Erica L Mayer, Yeon Hee Park, Steven Fox, Xiaochun Liu, Andrew Walding, Cynthia Huang Bartlett, Nick C Turner. SERENA-6: A Phase III study to assess the efficacy and safety of AZD9833 (camizestrant) compared with aromatase inhibitors when given in combination with palbociclib or abemaciclib in patients with HR+/HER2- metastatic breast cancer with detectable ESR1m who have not experienced disease progression on first-line therapy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT2-11-05.

Abstract 1236: Preclinical characterization of LY3484356, a novel, potent and orally bioavailable selective estrogen receptor degrader (SERD)

Abstract Nearly 70% of newly diagnosed breast cancers are estrogen receptor alpha (ERα) positive, for which endocrine therapy is a primary treatment. However, approximately 40% of those patients on endocrine therapy develop resistance, which includes mutations in ERα (ESR1) that drive constitutive activation of the receptor. One approach to overcome this resistance is to develop a potent degrader and pure antagonist of the estrogen receptor, which can effectively suppress estrogen receptor signaling. Fulvestrant, a pure antagonist and selective estrogen receptor degrader (SERD) is approved for the treatment of HR+/HER2- metastatic breast cancer. However, fulvestrant is limited by poor pharmacokinetics properties, limited exposure and sub-optimal in vivo estrogen receptor degradation. Here we describe the preclinical profile of LY3484356, an oral SERD and pure estrogen receptor antagonist, with potent activity against the wild type and mutant estrogen receptor. LY3484356 has Ki values of 0.64 nM and 2.8 nM against wild type ERα and Y537S mutant ERα proteins, respectively. It is a potent and highly efficient degrader of wild type ERα and Y537N mutant ERα proteins in cells, with IC50 values 3.0 nM and 9.6 nM, respectively. LY3484356 is also a potent inhibitor of ERα-mediated transcription in vitro and in vivo. It inhibits cell proliferation in wild type ERα and ESR1 Y537N mutant breast cancer cell lines, with average IC50 values of 3 nM and 17 nM, respectively. In a panel of breast cancer cell lines, 11 out of 12 ER+ breast cancer cell lines were sensitive to LY3484356 (IC50 less than 100 nM), whereas all ER- cell lines tested were insensitive. LY3484356 has demonstrated sustained and prolonged target inhibition (>75% inhibition of PGR transcription up to 96h after last dose) in ESR1 wild type (MCF7) and ESR1 Y537S mutant (ST941/C) xenograft tumors. Consistent with its profile as a pure antagonist, immature rat studies demonstrated no significant effect on uterine wet weight. LY3484356 demonstrated significant tumor growth inhibition and tumor regressions in wild type ESR1 breast cancer xenograft models such as MCF7, T47D and ZR-75-1, as well as ESR1 mutant breast cancer PDX models. LY3484356 has shown synergy or additivity in combination with CDK4/6 inhibitor abemaciclib, mTOR inhibitor everolimus and PIK3CA inhibitor alpelisib in inhibiting cell proliferation in ER+ breast cancer cell lines in vitro and tumor growth inhibition in relevant xenograft or PDX models in vivo. The first-in-human Phase 1/2 clinical trial of LY3484356 (EMBER, ClinicalTrials.gov NCT04188548) is currently ongoing and a window-of-opportunity study evaluating the pharmacodynamic effects of LY3484356 in early stage breast cancer is expected to begin early 2021. Citation Format: Shripad V. Bhagwat, Baohui Zhao, Weihua Shen, Cecilia Mur, Robert Barr, Lisa J. Kindler, Almudena Rubio, Jolie A. Bastian, Jeffrey D. Cohen, Brian E. Mattioni, Eunice Yuen, Thomas K. Baker, Mark A. Castanares, Dongling Fei, Jason R. Manro, Maria Jose Lallena, Sheng-Bin Peng, Alfonso de Dios. Preclinical characterization of LY3484356, a novel, potent and orally bioavailable selective estrogen receptor degrader (SERD) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1236.

Structure-activity relationship study of estrogen receptor down-regulators with a diphenylmethane skeleton.

Selective estrogen receptor (ER) down-regulators (SERDs) are pure ER antagonists that also induce ER degradation upon binding to the receptor. Although SERDs have been developed for the treatment of ER-positive breast cancers for nearly a decade, their precise mechanism(s) of action and structure-activity relationship are still unclear. Generally, Western blotting is used to examine the effects of SERDs on ER protein levels, but the methodology is low-throughput and not quantitative. Here, we describe a quantitative, high-throughput, luciferase-based assay for the evaluation of SERDs activity. For this purpose, we established stable recombinant HEK-293 cell lines expressing ERα fused with emerald luciferase. We also designed and synthesized new diphenylmethane derivatives as candidate SERDs, and evaluated their SERDs activity using the developed system in order to examine their structure-activity relationship, taking EC50 as a measure of potency, and Emax as a measure of efficacy.

Clinical benefit of fulvestrant monotherapy in the multimodal treatment of hormone receptor and HER2 positive advanced breast cancer: a case series.

Fulvestrant is a pure estrogen receptor (ER) antagonist approved for the treatment of metastatic ER positive breast cancer in postmenopausal women with disease progression following antiestrogen therapy. The clinical results of fulvestrant demonstrated encouraging activity in tumors in spite of HER2 positivity, but data about its use after progression on anti-HER2 agents are limited. Partial responses and durations of response of 12, 25, and 38 months in three cases with multiple metastases of ER positive and HER2 positive breast cancer were observed; all patients had been treated with 1–4 regimens of an anti-HER2 agent in combination with chemotherapy or an aromatase inhibitor before the initiation of fulvestrant. Fulvestrant is a valuable option with limited toxicity and durable response in metastatic HER2 and ER positive breast cancer after progression on anti-HER2 agents as well. Therapeutic benefit even in extensive skin metastases and (irradiated) brain metastases may be expected. Further investigations are warranted to establish where it fits into the multimodal management of ER and HER positive breast cancer.

Specific stereochemistry of OP-1074 disrupts estrogen receptor alpha helix 12 and confers pure antiestrogenic activity

Complex tissue-specific and cell-specific signaling by the estrogen receptor (ER) frequently leads to the development of resistance to endocrine therapy for breast cancer. Pure ER antagonists, which completely lack tissue-specific agonist activity, hold promise for preventing and treating endocrine resistance, however an absence of structural information hinders the development of novel candidates. Here we synthesize a small panel of benzopyrans with variable side chains to identify pure antiestrogens in a uterotrophic assay. We identify OP-1074 as a pure antiestrogen and a selective ER degrader (PA-SERD) that is efficacious in shrinking tumors in a tamoxifen-resistant xenograft model. Biochemical and crystal structure analyses reveal a structure activity relationship implicating the importance of a stereospecific methyl on the pyrrolidine side chain of OP-1074, particularly on helix 12.

RE: Final Overall Survival: Fulvestrant 500 mg vs 250 mg in the Randomized CONFIRM Trial.

Fulvestrant is a pure estrogen receptor (ER) antagonist devoid of the agonistic properties displayed by tamoxifen in some tissues (1–4). After phase III studies, which demonstrated similar efficacy and an acceptable safety profile for fulvestrant 250mg compared with anastrozole (1,5), fulvestrant 250mg was approved as treatment in postmenopausal women with advanced hormone receptor–positive breast cancer that had progressed or recurred after prior antiestrogen therapy. However, previous preoperative studies showed that short-term exposure to fulvestrant was associated with a dose-dependent reduction in the levels of ER, progesterone receptor, and the cell proliferation–related antigen Ki67 (6,7) for fulvestrant doses up to 250mg. Other phase I and phase III studies also suggested a dose–response effect for fulvestrant (1,5,8). The phase III Comparison of Faslodex in Recurrent or Metastatic Breast Cancer (CONFIRM) trial compared the then-approved dose and dosing schedule of fulvestrant (250mg every 28 days) with a higher-dose regimen (500mg every 28 days plus an additional 500mg on day 14 of the first month only) in postmenopausal women with locally advanced or metastatic ER-positive breast cancer that had recurred or progressed after prior endocrine therapy. The initial results showed that fulvestrant 500mg was associated with a statistically significant increase in progression-free survival (PFS) without increased toxicity, therefore corresponding to a clinically meaningful improvement in benefit vs risk compared with fulvestrant 250mg (9). Based on these data, the 500-mg dose of fulvestrant is now the approved dose in the European Union (approved in March 2010), United States (approved in September 2010), Japan (approved in November 2011), and other countries worldwide. In the CONFIRM study, the assessment of the therapeutic efficacy of both doses of fulvestrant was evaluated by several secondary outcome measures, including overall survival (OS). At the time of the initial analysis, approximately 50% of patients had died. After the reporting of the 50% survival data, which showed a trend in favor of 500mg over 250mg, it was agreed to perform a final survival analysis after 75% of patients had died. Here we report the results of this final OS analysis.

Chemosensitization role of fulvestrant in combination with chemotherapy in postmenopausal hormone receptor positive and human epidermal growth factor negative metastatic breast cancer

In metastatic breast cancer (MBC), hormone receptor positive (HR+), human epidermal growth factor negative (HER2−) subtype accounts for the majority. With various new modalities available to prolong life span in this group of patients, the effect is distant from optimum. Prevalent strategy of treating postmenopausal HR+ HER2− MBC is application of chemotherapy (CT) after progression of disease on endocrine therapy (ET) of several lines. Generally, ET targets HR+ ingredients and CT works better with HR− tumor cells. HR+ MBC, though hormone-sensitive, has HR− portion which reacts poorly to ET. Thus, sequential use of ET and CT neglects its insensitive part and gives rise to drug resistance, while alleviation of tumor burden is the top priority in metastatic setting. Chemohormonal therapy (i.e. concomitant use of ET and chemotherapy) complements for the shortcoming of current therapy strategy targeting both HR+ and HR− ingredients theoretically. Fulvestrant, a pure estrogen receptor antagonist and down-regulator, could be a promising agent using concurrently with CT based on chemosensitizing character shown in preclinical and pilot clinical studies. It is hypothesized in this article that chemohormonal therapy with concurrent fulvestrant and CT would be a promising strategy in postmenopausal HR+ HER2− MBC patients. Proof of this hypothesis would help control evolvement of tumor burden and acquirement of drug resistance over a short period of time.

Treating gynecologic malignancies with selective estrogen receptor downregulators (SERDs): promise and challenges

Endometrial and ovarian cancers are estrogen-dependent gynecologic malignancies. Although many are estrogen receptor (ER) positive, treatment with the selective estrogen receptor modulator (SERM) tamoxifen, a tissue selective partial-agonist, has demonstrated only modest clinical benefit. Selective estrogen receptor downregulators (SERDs) are pure ER antagonists showing a benefit for advanced ER positive breast cancer, which has bolstered their potential use for ER positive gynecologic malignancies. We summarize these preclinical and clinical data, suggesting that a subpopulation of patients with endometrial or ovarian cancer exists in which treatment with SERDs results in improved outcome. However, the full potential of SERDs for a gynecologic malignancies will be realized only when the appropriate predictive biomarkers are identified. Additionally, a further understanding ER signaling in the context of ovarian and endometrial tissues that appear to involve c-Src and other kinase pathways is needed to successfully address the emergence of resistance with rationally designed combination therapies.

17β-Estradiol inhibits vascular smooth muscle cell migration via up-regulation of striatin protein

Striatin, an estrogen receptor (ER)-interacting protein, plays an important role in estrogen’s nongenomic actions in vascular endothelial cells. However, the role of striatin in VSMCs is unknown. Here, we investigated the role of striatin in estrogen-regulated VSMCs migration. 17β-Estradiol (E2) at 10 nM largely inhibited VSMCs migration, which was reversed by the silencing of striatin expression. E2 increased striatin protein expression in a dose- and time-dependent manner. ERα agonist PPT, but not ERβ agonist DPN, mimicked the regulatory effect of E2. The regulatory effect of E2 on striatin protein expression was blocked by the pure ER antagonist ICI 182,780 or the mitogen-activated protein kinase inhibitor PD98059, but not by the phosphatidylinositol-3 kinase inhibitor wortmannin or Src inhibitor PP2, suggesting that E2 increased striatin protein expression via extracellular-signal regulated kinase 1/2 (ERK1/2). E2 resulted in phosphorylation of ERK1/2 in a time-dependent manner. The silencing of ERK1/2 largely abolished E2-enhanced striatin expression. Finally, the inhibitory effect of E2 on VSMC migration was reversed by ICI 182,780 or PD98059. Taken together, our results indicate that E2 inhibits VSMC migration by increasing striatin expression via ERα to ERK1/2 pathway, which maybe helpful to understand estrogen’s anti-atherogenic effect in VSMCs.

Wedelolactone induces growth of breast cancer cells by stimulation of estrogen receptor signalling

Wedelolactone, a plant coumestan, was shown to act as anti-cancer agent for breast and prostate carcinomas in vitro and in vivo targeting multiple cellular proteins including androgen receptors, 5-lipoxygenase and topoisomerase IIα. It is cytotoxic to breast, prostate, pituitary and myeloma cancer cell lines in vitro at μM concentrations. In this study, however, a novel biological activity of nM dose of wedelolactone was demonstrated. Wedelolactone acts as agonist of estrogen receptors (ER) α and β as demonstrated by transactivation of estrogen response element (ERE) in cells transiently expressing either ERα or ERβ and by molecular docking of this coumestan into ligand binding pocket of both ERα and ERβ. In breast cancer cells, wedelolactone stimulates growth of estrogen receptor-positive cells, expression of estrogen-responsive genes and activates rapid non-genomic estrogen signalling. All these effects can be inhibited by pretreatment with pure ER antagonist ICI 182,780 and they are not observed in ER-negative breast cancer cells. We conclude that wedelolactone acts as phytoestrogen in breast cancer cells by stimulating ER genomic and non-genomic signalling pathways.

2-methoxyestradiol induces vasodilation by stimulating NO release via PPARγ/PI3K/Akt pathway.

The endogenous estradiol metabolite 2-methoxyestradiol (2-ME) reduces atherosclerotic lesion formation, while the underlying mechanisms remain obscure. In this work, we investigated the vasodilatory effect of 2-ME and the role of nitric oxide (NO) involved. In vivo studies using noninvasive tail-cuff methods showed that 2-ME decreased blood pressure in Sprague Dawley rats. Furthermore, in vitro studies showed that cumulative addition of 2-ME to the aorta caused a dose- and endothelium-dependent vasodilation. This effect was unaffected by the pretreatment with the pure estrogen receptor antagonist ICI 182,780, but was largely impaired by endothelial nitric oxide synthase (eNOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) or by phosphoinositide 3-kinase (PI3K) inhibitor wortmannin (WM). Moreover, 2-ME（10−7 ∼10−5 M）enhanced phosphorylation of Akt and eNOS and promoted NO release from cultured human umbilical endothelial cells (HUVECs). These effects were blocked by PI3K inhibitor WM, or by the transfection with Akt specific siRNA, indicating that endothelial Akt/eNOS/NO cascade plays a crucial role in 2-ME-induced vasodilation. The peroxisome proliferator-activated receptor γ (PPARγ) mRNA and protein expression were detected in HUVECs and the antagonist GW9662 or the transfection with specific PPARγ siRNA inhibited 2-ME-induced eNOS and Akt phosphorylation, leading to the impairment of NO production and vasodilation. In conclusion, 2-ME induces vasodilation by stimulating NO release. These actions may be mediated by PPARγ and the subsequent activation of Akt/eNOS cascade in vascular endothelial cells.

Selective estrogen-receptor modulators suppress microglial activation and neuronal cell death via an estrogen receptor-dependent pathway

Growing evidence shows that steroid hormones, especially 17β-estradiol (E2), protect neuronal cells by attenuating excess activation of microglia. However, the use of E2 in the clinic is controversial because of its peripheral actions in reproductive organs and its potential to increase risk for endometrial cancer and breast cancer. Selective estrogen-receptor modulators (SERMs) bind to estrogen receptors (ERs), but their effects as ER agonists or antagonists are dependent on the target tissue. SERMs pose very little cancer risk as a result of their anti-estrogen action in reproductive organs, but their action in the brain is not well understood. In this study, we investigated the effects of SERMs tamoxifen (Tam) and raloxifene (Rlx) on microglial activation and subsequent neuronal injury. Tam and Rlx suppressed the increases in proinflammatory cytokines and chemokine expression that were induced by lipopolysaccharide (LPS) in rat primary microglia cultures. The microglial-conditioned media pretreated with Tam or Rlx significantly attenuated cellular injury in SH-SY5Y cells elicited by microglial-conditioned media treated with LPS alone. Rat primary microglia expressed ERα and ERβ primarily in the nucleus, and thus we examined the involvement of ERs in the suppressive action of Tam and Rlx on microglial activation using a pure ER antagonist, ICI182,780. Pretreatment with ICI182,780 abolished the suppressive effects of SERMs on microglial activation, as well as their protective action on SH-SY5Y cells. A luciferase assay using a vector with three estrogen response elements (EREs) revealed that Tam and Rlx activated ERE-mediated transcription in rat primary microglia. Taken together, these results suggest that Tam and Rlx suppress microglial activation and subsequent neuronal cell death via an ER-mediated transcription pathway. SERMs could represent a novel therapeutic strategy for disorders of the central nervous system based on their ability to suppress neuroinflammation.

The Effects of p-nonylphenol on the Myometrial Contractile Activity.

We aimed to investigate the effects and mechanisms of action of p-nonylphenol(p-NP) on uterine contractility in rats. The uterine tissues of female Sprague Dawley rats in diestrus were bathed in isolated organ bath. The effects of vehicle alone (0.1% ethanol), the positive control 17-β-E2 (10(-5) M) and p-NP (10(-9) M, 10(-8) M, 10(-7) M, 10(-6) M) on spontaneous and KCl-induced uterine contractility of rats were studied. Also, the effects of p-NP in combination with actinomycin D (10(-5) M) (gene transcription inhibitor), cycloheximide (10(-4) M) (protein synthesis inhibitor), fulvestrant (10(-6) M) (pure estrogen receptor antagonist), 2-hydroxy-5-nonanoylbenzamide (10(-3) M) (compound 1b, anti-uterotrophic compound) on spontaneous uterine contractions, and with propranolol (20 µM) (β-adrenoceptor antagonist) and noradrenaline (5 µM) on KCl (40 mM) induced contractions were investigated. p-NP exhibited a concentration-dependent inhibition on spontaneous uterine contractions. There was no significant difference between the highest p-NP concentration (10(-6) M) and the positive control 17-β-E2 in terms of % inhibition (p>0.05). The inhibitory effect of p-NP (10(-6) M) on spontaneous contractions was blocked by actinomycin D (p<0.001), cycloheximide (p<0.001), fulvestrant (p<0.001) and compound 1b (p<0.001). 17-β-E2 (10(-5) M) exerted a higher inhibition % on KCl induced contractions than p-NP (10(-6) M). The relaxant effect of p-NP on KCl-induced uterine contractions was inhibited by noradrenaline (p<0.05) but not by propranolol (p>0.05). We suggest that p-NP inhibited uterine contractions similar as 17-β-E2 and genomic pathways are involved and β-adrenoceptors might modulate the activity of p-NP. In addition, compound 1b showed an uterotonic activity and reversed the effect of p-NP.

Channel catfish (Ictalurus punctatus) leukocytes express estrogen receptor isoforms ERα and ERβ2 and are functionally modulated by estrogens

Estrogens are recognized as modulators of immune responses in mammals and teleosts. While it is known that the effects of estrogens are mediated via leukocyte-specific estrogen receptors (ERs) in humans and mice, leucocyte-specific estrogen receptor expression and the effects of estrogens on this cell population is less explored and poorly understood in teleosts. Here in, we verify that channel catfish (Ictalurus punctaus) leukocytes express ERα and ERβ2. Transcripts of these isoforms were detected in tissue-associated leukocyte populations by PCR, but ERβ2 was rarely detected in PBLs. Expression of these receptors was temporally regulated in PBLs following polyclonal activation by concanavalin A, lipopolysaccharide or alloantigen based on evaluation by quantitative and end-point PCR. Examination of long-term leukocyte cell lines demonstrated that these receptors are differentially expressed depending on leukocyte lineage and phenotype. Expression of ERs was also temporally dynamic in some leukocyte lineages and may reflect stage of cell maturity. Estrogens affect the responsiveness of channel catfish peripheral blood leukocytes (PBLs) to mitogens in vitro. Similarly, bactericidal activity and phorbol 12-myristate 13-acetate induced respiratory burst was modulated by 17β-estradiol. These actions were blocked by the pure ER antagonist ICI 182780 indicating that response is, in part, mediated via ERα. In summary, estrogen receptors are expressed in channel catfish leukocytes and participate in the regulation of the immune response. This is the first time leukocyte lineage expression has been reported in teleost cell lines.

Estrogen receptors regulate the estrous behavior induced by progestins, peptides, and prostaglandin E2

The role of classical estrogen receptors (ERs) in priming female reproductive behavior has been studied previously; however, the participation of this receptor during activation of estrous behavior has not been extensively studied. The purpose of this work was to test the possibility that the facilitation of lordosis behavior in estrogen-primed rats by progesterone (P) and its 5α- and 5β-reduced metabolites, gonadotropin-releasing hormone (GnRH), leptin, prostaglandin E2 (PGE2) and vagino-cervical stimulation (VCS) involves interactions with classical ERs by using the selective ER modulator, tamoxifen. To further assess the role of ERs, we also explored the effects of the pure ER antagonist, ICI182780 (ICI), on estrous behavior induced by P and GnRH. Ovariectomized, estrogen-primed rats (5μg estradiol benzoate 40h earlier) were injected intraventricularly with the above-mentioned compounds, or they received VCS. All compounds and VCS effectively facilitated estrous behavior when tested at 60, 120 or 240min after infusion or application of VCS. Intraventricular infusion of tamoxifen (5μg), 30min before, significantly attenuated estrous behaviors induced in estradiol-primed rats by P, most of its 5α- and 5β-reduced metabolites, GnRH, and PGE2, but not by VCS. Although there was a trend for reduction, tamoxifen did not significantly decrease lordosis in females treated with 5β-pregnan-3,20-dione. ICI also inhibited lordosis behavior induced by P and GnRH at some testing intervals. These results suggest that activation of classical ERs participates in the triggering effects on estrous behavior induced by agents with different chemical structures that do not bind directly to ERs.

Influence of beta‐sitosterol on ER and gene PS2 expression in breast cancer cell line T47D cells (1148.7)

Objective: To observe the effect of beta‐sitosterol on ER and downstream gene PS2 expression in breast cancer cell line T47D cells, and explore its phytoestrogenic mechanism. Methods: mRNA expressions of ER‐alpha,ER‐beta and its downstream gene PS2 in T47D were assessed by RT ‐PCR, protein expressions of ER‐alpha,ER‐beta in T47D were assessed by western‐blot. ICI182 780 was employed to observe the influence on the gene expressions of PS2 and protein expressions of ER‐alpha,ER‐beta. Results: beta‐sitosterol up‐regulated ER‐alpha,ER‐beta mRNA expression.PS2 gene are induced in response to beta‐sitosterol treatment. The effect on PS2 gene expression was inhibited by pure estrogen receptor antagonist ICI182780. Beta‐sitosterol increased the level of T47D cells ER‐alpha, ER‐beta proteins expression, respectively. The effect was completely antagonized by ICI182 780. Conclusion: beta‐sitosterol up‐regulated gene PS2 expression through ER. And beta‐sitosterol possessed phytoestrogenic effect by activating ER. Supporttrd by 111 project (B07007)and NSFC(30772849,81273887).Grant Funding Source: Suooorted by 111 projict(b07007) and NSFC（30772849,81273887）

Psoralidin, a coumestan analogue, as a novel potent estrogen receptor signaling molecule isolated from Psoralea corylifolia

A novel biological activity of psoralidin as an agonist for both estrogen receptor (ER)α and ERβ agonist has been demonstrated in our study. Psoralidin has been characterized as a full ER agonist, which activates the classical ER-signaling pathway in both ER-positive human breast and endometrial cell lines as well as non-human cultured cells transiently expressing either ERα or ERβ. The estrogenic activity was determined using the relative expression levels of either reporter or the endogenous genes dependent on the agonist-bound ER to the estrogen response element (ERE). Psoralidin at 10μM was able to induce the maximum reporter gene expression corresponding to that of E2-treated cells and such activation of the ERE-reporter gene by psoralidin was completely abolished by the cotreatment of a pure ER antagonist, implying that the biological activities of psoralidin are mediated by ER. Psoralidin was also able to induce the endogenous estrogen-responsive gene, pS2, in human breast cancer cells MCF-7. It was observed that activation of the classical ER-signaling pathway by psoralidin is mediated via induction of ER conformation by psoralidin and direct binding of the psoralidin–ER complex to the EREs present in the promoter region of estrogen-responsive genes, as shown by chromatin immunoprecipitation assay results. Finally, molecular docking of psoralidin to the ligand binding pocket of the ERα showed that psoralidin is able to mimic the binding interactions of E2, and thus, it could act as an ER agonist in the cellular environment.