ERα Protein Research Articles

Estrogen Receptor-Beta2 (ERβ2)-Mutant p53-FOXM1 Axis: A Novel Driver of Proliferation, Chemoresistance, and Disease Progression in High Grade Serous Ovarian Cancer (HGSOC).

Simple SummaryHigh grade serous ovarian cancer (HGSOC) is the most common and lethal subtype of ovarian cancer without effective therapeutic options. The high prevalence of mutations (~96%) in tumor suppressor p53 is a hallmark of HGSOC. Estrogen receptor-beta (ERβ) has been reported to be another important player in HGSOC, although the pro-versus anti-tumorigenic role of its different isoforms remains unclear. The aim of this study was to analyze the crosstalk between ERβ and mutant p53 and its impact on the pro-tumorigenic processes in HGSOC. Using the HGSOC cell line models and patient tumor tissue specimens, we demonstrated functional interaction between the ERβ2 isoform and mutant p53 and their ability to co-dependently increase FOXM1 gene transcription, decrease cell death, increase cell proliferation, and mediate resistance to carboplatin treatment. Furthermore, high levels of ERβ2 as well as FOXM1 correlated with worse patient survival. Collectively, our data suggest that the ERβ2-mutant p53-FOXM1 axis could be a novel therapeutic target for HGSOC.High grade serous ovarian cancer (HGSOC) is the most common and lethal subtype of epithelial ovarian cancer. Prevalence (~96%) of mutant p53 is a hallmark of HGSOC. Estrogen receptor-beta (ERβ) has been reported to be another important player in HGSOC, although the pro-versus anti-tumorigenic role of its different isoforms remains unsettled. However, whether there is functional interaction between ERβ and mutant p53 in HGSOC is unknown. ERβ1 and ERβ2 mRNA and protein analysis in HGSOC cell lines demonstrated that ERβ2 is the predominant isoform in HGSOC. Specificity of ERβ2 antibody was ascertained using cells depleted of ERβ2 and ERβ1 separately with isoform-specific siRNAs. ERβ2-mutant p53 interaction in cell lines was confirmed by co-immunoprecipitation and in situ proximity ligation assay (PLA). Expression levels of ERβ2, ERα, p53, and FOXM1 proteins and ERβ2-mutant p53 interaction in patient tumors were determined by immunohistochemistry (IHC) and PLA, respectively. ERβ2 levels correlate positively with FOXM1 levels and negatively with progression-free survival (PFS) and overall survival (OS). Quantitative chromatin immunoprecipitation (qChIP) and mRNA expression analysis revealed that ERβ2 and mutant p53 co-dependently regulated FOXM1 gene transcription. The combination of ERβ2-specific siRNA and PRIMA-1MET that converts mutant p53 to wild type conformation increased apoptosis. Our work provides the first evidence for a novel ERβ2-mutant p53-FOXM1 axis that can be exploited for new therapeutic strategies against HGSOC.

Abstract P5-05-10: Inhibition of FASN as a potential treatment of advanced therapy-resistant breast cancer

Abstract INTRODUCTION: The majority of breast cancers are estrogen receptor (ERα) positive making endocrine therapy a mainstay for these patients. Unfortunately, resistance to endocrine therapy is a common occurrence. Fatty acid synthase (FASN) is a key enzyme in lipid biosynthesis and is overexpressed in more aggressive and therapy-resistant tumors, including breast cancers. METHODS: The effect of the FASN inhibitor TVB-3166 on ERα expression and cell growth was characterized in tamoxifen-resistant and fulvestrant-resistant cell lines, xenografts, and patient explants. Subcellular localization of ERα was assessed using subcellular fractionations. Palmitoylation and ubiquitination of ERα were assessed by immunoprecipitation. ERα and p-eIF2α protein levels were analyzed by western blotting after treatment with TVB-3166 with or without the addition of palmitate or BAPTA. RESULTS: TVB-3166 treatment leads to a marked inhibition of proliferation in tamoxifen-resistant and fulvestrant-resistant cells compared to the parental cells. Additionally, TVB-3166 significantly inhibited tamoxifen-resistant breast tumor growth in mice and decreased proliferation of primary tumor explants compared to untreated controls. FASN inhibition significantly reduced ERα levels most prominently in endocrine resistant cells and altered its subcellular localization. Furthermore, we showed that the reduction of ERα expression upon TVB-3166 treatment is mediated through the induction of endoplasmic reticulum stress. CONCLUSION: Our preclinical data provide evidence that FASN inhibition by TVB-3166 presents a promising therapeutic strategy for treatment of endocrine-resistant breast cancer. Further clinical development of FASN inhibitors for endocrine resistant breast cancer should be considered. Citation Format: Henriette U Balinda. Inhibition of FASN as a potential treatment of advanced therapy-resistant breast cancer [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 P5-05-10.

ER/AR Multi-Conformational Docking Server: A Tool for Discovering and Studying Estrogen and Androgen Receptor Modulators.

The prediction of the estrogen receptor (ER) and androgen receptor (AR) activity of a compound is quite important to avoid the environmental exposures of endocrine-disrupting chemicals. The Estrogen and Androgen Receptor Database (EARDB, http://eardb.schanglab.org.cn/) provides a unique collection of reported ERα, ERβ, or AR protein structures and known small molecule modulators. With the user-uploaded query molecules, molecular docking based on multi-conformations of a single target will be performed. Moreover, the 2D similarity search against known modulators is also provided. Molecules predicted with a low binding energy or high similarity to known ERα, ERβ, or AR modulators may be potential endocrine-disrupting chemicals or new modulators. The server provides a tool to predict the endocrine activity for compounds of interests, benefiting for the ER and AR drug design and endocrine-disrupting chemical identification.

Influence of genistein and diadizine on regularity of estrous cycle in cyclic female Wistar rat: interaction with estradiol receptors and vascular endothelial growth factor.

Isoflavones are estrogenic compounds that exist in soy, clover, and peanuts. They are selective estrogen receptor modulators. The study was planned to explain the interactions of isoflavones with estrogen receptors alpha (ERα), beta (ERβ), and vascular endothelial growth factor (VEGF) expressions in ovarian and uterine tissues during different stages of the estrous cycle of regular cyclic female Wistar rats. Thirty-two regular cyclic females were divided equally into control group: fed casein-based diet and isoflavones group: fed casein-based diet and gavaged 50 mg/kg/day soy isoflavones extract 40%. The regularity of estrus cycles was monitored. Final body weight (FBW), weight gain (BWG), and ovarian and uterine weights were estimated. Histopathology and immunohistochemistry for ERα, Erβ, and VEGF in ovarian and uterine tissues were performed. All females (100%, n = 16) in control group showed regularity in estrous cycle compared to 62.5% (n = 10) in isoflavones group. Estrus and diestrus phases revealed prolongation and shortening in isoflavones rats than control, respectively. Nonsignificant variation was noted in the duration of the whole cycle of both groups. FBW and BWG significantly decreased however, ovarian and uterine weights increased significantly in all estrous phases of isoflavones group than control. Histopathology demonstrated an increase in number of follicles/ovaries besides, hyperplasia and proliferation of luminal epithelium with hydropic degeneration in the isoflavones group. Also, uterine connective tissue stroma showed edema in the isoflavones group during all estrous phases. Immunostaining percentages of ERα, Erβ, and VEGF protein expression were significantly elevated in the isoflavones group during all estrous phases. Isoflavones induced irregularity of the estrous cycle that was encountered by increased and altered ERα, Erβ, and VEGF expressions in ovarian and uterine tissues.

ERα-Targeting PROTAC as a Chemical Knockdown Tool to Investigate the Estrogen Receptor Function in Rat Menopausal Arthritis.

Proteolytic targeting chimeras (PROTACs) is a rapid and reversible chemical knockout method. Compared with traditional gene-editing tools, it can avoid potential genetic compensation, misunderstandings caused by spontaneous mutations, or gene knockouts that lead to embryonic death. To study the role of estrogen receptor alpha (ERα) in the occurrence and progression of menopausal arthritis, we report a chemical knockout strategy in which stable peptide-based (PROTACs) against ERα to inhibit their function. This chemical knockdown strategy can effectively and quickly inhibit ERα protein in vivo and in vitro. In the rat menopausal arthritis model, this study showed that inhibiting estrogen function by degrading ERα can significantly interfere with cartilage matrix metabolism and cause menopausal arthritis by up-regulating matrix metalloproteinase (MMP-13). The results of this study indicate that ERα is a crucial estrogen receptor for maintaining cartilage metabolism. Inhibition of ERα function by PROTACs can promote the progression of osteoarthritis.

A Genome-Edited ERα-HiBiT Fusion Reporter Cell Line for the Identification of ERα Modulators Via High-Throughput Screening and CETSA.

The estrogen receptor α (ERα) is a target of intense pharmacological intervention and toxicological biomonitoring. Current methods to directly quantify cellular levels of ERα involve antibody-based assays, which are labor-intensive and of limited throughput. In this study, we generated a post-translational reporter cell line, referred to as MCF7-ERα-HiBiT, by fusing a small pro-luminescent nanoluciferase (NLuc) tag (HiBiT) to the C-terminus of endogenous ERα in MCF7 cells. The tag allows the luminescent detection and quantification of endogenous ERα protein by addition of the complementary NLuc enzyme fragment. This MCF7-ERα-HiBiT cell line was optimized for quantitative high-throughput screening (qHTS) to identify compounds that reduce ERα levels. In addition, the same cell line was optimized for a qHTS cellular thermal shift assay to identify compounds that bind and thermally stabilize ERα. Here, we interrogated the MCF7-ERα-HiBiT assay against the NCATS Pharmacological Collection (NPC) of 2,678 approved drugs and identified compounds that potently reduce and thermally stabilize ERα. Our novel post-translational reporter cell line provides a unique opportunity for profiling large pharmacological and toxicological compound libraries for their effect on ERα levels as well as for assessing direct compound binding to the receptor, thus facilitating mechanistic studies by which compounds exert their biological effects on ERα.

Enhanced IFNα Signaling Promotes Ligand-Independent Activation of ERα to Promote Aromatase Inhibitor Resistance in Breast Cancer.

Simple SummaryInterferon alpha (IFNα) signaling is highly upregulated in ER+ breast cancers that become resistant to estrogen deprivation therapy. This study uncovers how enhanced (IFNα)/JAK-STAT signaling directly influences estrogen receptor (ERα) activation in the absence of estrogen. We found that inhibiting IFNα signaling downregulates expression and activation of ERα. In addition, STAT1 and ERα directly interact and regulate a key interferon-stimulated gene, IFITM1, in AI-resistant breast cancer cells. We demonstrate that crosstalk occurs between IFNα and ERα pathways, which contributes to aggression and survival of AI-resistant breast cancer, thus representing a novel mechanism of acquired AI resistance.Aromatase inhibitors (AIs) reduce estrogen levels up to 98% as the standard practice to treat postmenopausal women with estrogen receptor-positive (ER+) breast cancer. However, approximately 30% of ER+ breast cancers develop resistance to treatment. Enhanced interferon-alpha (IFNα) signaling is upregulated in breast cancers resistant to AIs, which drives expression of a key regulator of survival, interferon-induced transmembrane protein 1 (IFITM1). However, how upregulated IFNα signaling mediates AI resistance is unknown. In this study, we utilized MCF-7:5C cells, a breast cancer cell model of AI resistance, and demonstrate that these cells exhibit enhanced IFNα signaling and ligand-independent activation of the estrogen receptor (ERα). Experiments demonstrated that STAT1, the mediator of intracellular signaling for IFNα, can interact directly with ERα. Notably, inhibition of IFNα signaling significantly reduced ERα protein expression and ER-regulated genes. In addition, loss of ERα suppressed IFITM1 expression, which was associated with cell death. Notably, chromatin immunoprecipitation experiments validated that both ERα and STAT1 associate with ERE sequences in the IFITM1 promoter. Overall, hyperactivation of IFNα signaling enhances ligand-independent activation of ERα, which promotes ER-regulated, and interferon stimulated gene expression to promote survival in AI-resistant breast cancer cells.

Ameliorative Effects of Pueraria lobata Extract on Postmenopausal Symptoms through Promoting Estrogenic Activity and Bone Markers in Ovariectomized Rats.

Pueraria lobata (Willd.) Ohwi, known as kudzu, is one of the most popular traditional medicines in Asian countries. It has been widely used as a natural alternative to hormone replacement therapy for treating postmenopausal symptoms. This study aimed to investigate the estrogenic effect of P. lobata extract (PE) against postmenopausal osteoporosis in ovariectomized (OVX) rats. OVX rats were treated with PE (25–1600 mg/kg) for 8 weeks. Biochemical parameters, estradiol, and bone turnover markers (e.g., osteocalcin, C-terminal telopeptide fragment of type I collagen, deoxypyridinoline, and pyridinoline) were measured in plasma samples. In addition, estrogen receptor-alpha (ER-α) protein expression and morphology of uterine were evaluated. Long-term treatment with PE did not cause liver damage in OVX rats. PE supplementation reduced body weight gain in obese rats with high lipid accumulation induced by ovariectomy. Furthermore, PE exhibited a protective effect against insulin resistance, hyperlipidemia, and hepatic lipid peroxidation. PE treatment increased uterine weight and thickness of the uterine layers in cases of uterus atrophy due to removal of ovaries. The levels of bone turnover markers, which were significantly increased in OVX rats, were decreased by PE treatment. Western blotting analysis showed that ER-α protein expression was upregulated in PE-treated rats compared with OVX rats. These results suggest that PE could be a promising alternative functional food for improving menopausal symptoms.

Effects of chronic exposure to nonylphenol at environmental concentration on thyroid function and thyroid hyperplasia disease in male rats

The aim of this work was to determine whether chronic exposure to nonylphenol (NP), a representative substance of environmental endocrine disruptors (EEDs), at environmental concentration would have toxic effects on thyroid function and thyroid hyperplasia disease. Two hundred SPF Sprague-Dawley rats were divided into five groups (n = 40 per group): blank control group (corn oil), low-dose NP exposure group (0.4 mg/kg/d), medium-dose NP exposure group (4 mg/kg/d), high-dose NP exposure group (40 mg/kg/d), and estradiol control group (E2: 30 μg/kg/d). The rats were treated by gavage for 34 weeks, which were sampled twice (17 weeks and 34 weeks respectively). NP accumulation in the thyroid tissue (F = 52.93, P < 0.001) and serum (F = 5.54, P = 0.00) continuously increased in a significant dose-effect relationship. After NP exposure, the serum FT3 levels exhibited a dose-dependent increasing trend (F = 4.68, P = 0.01), while the serum FT4 level showed an opposite trend (F = 3.93, P= 0.01). Compared with the control group, hyperechoic areas (i.e., calcification points) were observed in the high-dose group. Follicular epithelial stratification was extremely severe, the monolayer cubic epithelial cells became flat, and the area of single follicles was even smaller in the high-dose group. In the high-dose NP group, there were numerous mitochondria that were severely swollen. The rough endoplasmic reticulum was abundant, with obvious expansion and vesiculation. The relative expression of ERα (F = 5.29, P = 0.00), ERβ (F = 10.17, P = 0.00), TRα (F = 7.71, P = 0.00), TRβ (F = 3.52.17, P = 0.02) and HMGB1 (F = 10.16, P = 0.01) proteins in the thyroid tissue in each NP exposure group was increased compared with the control group, and the relative expression of proteins increased if the exposure time was prolonged under the same exposure dose. Chronic exposure to NP at environmental concentration could have toxic effects on thyroid function, and induce thyroid hyperplasia disease in male rats.

Single-cell immunoblotting resolves estrogen receptor-α isoforms in breast cancer.

An array of isoforms of the nuclear estrogen receptor alpha (ER-α) protein contribute to heterogeneous response in breast cancer (BCa); yet, a single-cell analysis tool that distinguishes the full-length ER-α66 protein from the activation function-1 deficient ER-α46 isoform has not been reported. Specific detection of protein isoforms is a gap in single-cell analysis tools, as the de facto standard immunoassay requires isoform-specific antibody probes. Consequently, to scrutinize hormone response heterogeneity among BCa tumor cells, we develop a precision tool to specifically measure ER-α66, ER- α46, and eight ER-signaling proteins with single-cell resolution in the highly hetero-clonal MCF-7 BCa cell line. With a literature-validated pan-ER immunoprobe, we distinguish ER-α66 from ER-α46 in each individual cell. We identify ER-α46 in 5.5% of hormone-sensitive (MCF-7) and 4.2% of hormone-insensitive (MDA-MB-231) BCa cell lines. To examine whether the single-cell immunoblotting can capture cellular responses to hormones, we treat cells with tamoxifen and identify different sub-populations of ER-α46: (i) ER-α46 induces phospho-AKT at Ser473, (ii) S6-ribosomal protein, an upstream ER target, activates both ER-α66 and ER-α46 in MCF-7 cells, and (iii) ER-α46 partitions MDA-MB-231 subpopulations, which are responsive to tamoxifen. Unlike other single-cell immunoassays, multiplexed single-cell immunoblotting reports–in the same cell–tamoxifen effects on ER signaling proteins and on distinct isoforms of the ER-α protein.

Role of Persistent Organic Pollutants in Breast Cancer Progression and Identification of Estrogen Receptor Alpha Inhibitors Using In-Silico Mining and Drug-Drug Interaction Network Approaches

Simple SummaryThe role of persistent organic pollutants (POPs) in breast cancer progression and their bioaccumulation in adipose tissue has been reported. We used a computational approach to study molecular interactions of POPs with breast cancer proteins and identified natural and synthetic compounds to inhibit these interactions. Moreover, for comparative analysis, standard drugs and screened compounds were also docked against estrogen receptor alpha (ERα) and identification of the finest inhibitor was performed using in-silico mining and drug-drug interaction (DDI) network approaches. Based on scoring values, short-chained chlorinated paraffins demonstrated strong interactions with ERα compared to organo-chlorines and PCBs. Synthetic and natural compounds demonstrating strong associations with the active site of the ERα protein could be potential candidates to treat breast cancer specifically caused by POPs and other organic toxins and can be used as an alternative to standard drugs.The strong association between POPs and breast cancer in humans has been suggested in various epidemiological studies. However, the interaction of POPs with the ERα protein of breast cancer, and identification of natural and synthetic compounds to inhibit this interaction, is mysterious yet. Consequently, the present study aimed to explore the interaction between POPs and ERα using the molecular operating environment (MOE) tool and to identify natural and synthetic compounds to inhibit this association through a cluster-based approach. To validate whether our approach could distinguish between active and inactive compounds, a virtual screen (VS) was performed using actives (627 compounds) as positive control and decoys (20,818 compounds) as a negative dataset obtained from DUD-E. Comparatively, short-chain chlorinated paraffins (SCCPs), hexabromocyclododecane (HBCD), and perfluorooctanesulfonyl fluoride (PFOSF) depicted strong interactions with the ERα protein based on the lowest-scoring values of −31.946, −18.916, −17.581 kcal/mol, respectively. Out of 7856 retrieved natural and synthetic compounds, sixty were selected on modularity bases and subsequently docked with ERα. Based on the lowest-scoring values, ZINC08441573, ZINC00664754, ZINC00702695, ZINC00627464, and ZINC08440501 (synthetic compounds), and capsaicin, flavopiridol tectorgenin, and ellagic acid (natural compounds) showed incredible interactions with the active sites of ERα, even more convening and resilient than standard breast cancer drugs Tamoxifen, Arimidex and Letrozole. Our findings confirm the role of POPs in breast cancer progression and suggest that natural and synthetic compounds with high binding affinity could be more efficient and appropriate candidates to treat breast cancer after validation through in vitro and in vivo studies.

Molecular Effects of Topical Estrogen on Vaginal Granulation Tissue in Postpartum Women.

The aims of this study were to evaluate the biomolecular properties of vaginal and perineal granulation tissue in postpartum women and assess the potential impact of vaginal estrogen application. We prospectively identified women referred to a subspecialty peripartum clinic between September 2016 and April 2018 who developed symptomatic perineal or vaginal granulation tissue. As part of routine clinical care, granulation tissue was excised from each participant by a urogynecologist and subjected to RNA extraction, real-time quantitative polymerase chain reaction, histologic evaluation, and immunohistochemistry. Serum steroid hormone levels were measured. Comparisons were made between participants who used topical vaginal estradiol (E2) and those who did not (non-E2 controls). Sixteen postpartum women were recruited for this pilot study. More than 30% of patients (n = 5, 31%) had used topical vaginal estradiol (E2) during their postpartum recovery. Histological appearance of granulation tissue evaluated by hematoxylin and eosin staining was similar in women treated with vaginal E2 and non-E2 controls. Both estrogen receptor α (ERα) and ERβ mRNA and ERα protein were readily detectable in the granulation tissue of E2-treated women. Although not statistically significant, participants who used topical E2 developed granulation tissue that exhibited local estrogen-responsive gene upregulation. Serum levels of estrone, E2, dehydroepiandrosterone, progesterone, and testosterone did not differ between vaginal E2-treated patients and controls. Estrogen receptor α seems to be the predominant receptor mediating estrogen action in postpartum perineal and vaginal granulation tissue. Vaginal E2 use does not seem to affect serum levels of estrone, E2, dehydroepiandrosterone, progesterone, and testosterone in postpartum women.

Abstract 2125: Roles of KAT6A and KAT6B in the biology of estrogen positive breast cancer

Abstract KAT6A and KAT6B are histone acetyl transferase (HAT) that are controlling gene expression by transferring acetyl groups to histones. Yu et al (Oncogene, 2017 36, 2910-2918) have shown that KAT6A has a role in breast cancer development by activating the ERα promoter and was found frequently amplified in 11% and is overexpressed in 15% of breast cancers. Altogether, these data support KAT6A as an attractive target in estrogen receptor driven breast cancer. KAT6B is a close homolog that shares 91% identity in the acetyl coA binding site. While the prevalence of gene amplification for KAT6B in breast cancer is only 1-2%, it is often overexpressed. In this study, we are exploring how KAT6A and KAT6B are contributing to ERα expression and the growth of KAT6A amplified breast cancer cell lines. To decipher each protein's role, we have designed specific siRNA against KAT6A and KAT6B and we tested their action either individually or in combination in the KAT6A gene amplified breast cancer cell lines T-47D, CAM-1 and ZR-75. Following treatment of T-47D, CAMA-1 and ZR75 cell lines with KAT6A or B siRNA we observed a decrease of ERα gene and protein expression for all siRNAs. The effect was more pronounced when using KAT6A over KAT6B and the effect of both siRNA A/B were additive. Phenotypically, the impact on cell line proliferation by applying clonogenic and incucyte assays, mirrors what is observed on ERα gene and protein expression. The effect on cellular proliferation of KAT6A is more prominent than KAT6B silencing, while the combined KAT6A + KAT6B silencing being more impactful than KAT6A silencing alone. In conclusion, our data show that KAT6A and KAT6B share similar function in the control of ER gene and protein expression. This study suggests that KAT6A and KAT6B may be paralogs, one compensating for the loss of the other. This translates into a more pronounced antiproliferative effect when both genes are silenced concomitantly. Citation Format: Isabelle Meaux, Dimitri Gorge-Bernat, Angele Paz, Catherine Geslin, Laurent Debussche, Jürgen Moll, Christophe Henry. Roles of KAT6A and KAT6B in the biology of estrogen positive breast cancer [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 2125.

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 (&amp;gt;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.

Abstract 1070: SCR6106, an oral and brain penetrant SERD demonstrated anti-tumor activities in vitro and in vivo

Abstract Benefits of endocrine therapy in treating hormone receptor (HR)-positive (HR+) BC are well recorded in clinic. However, ~40% to 50% of HR+ patients eventually developed to endocrine resistance and disease relapsed. Fulvestrant, a selective estrogen receptor degrader (SERD) is approved to treat HR+ metastatic breast cancer in patients with disease progression after endocrine therapy. However, due to its poor bioavailability, it is given by intramuscular injection, which makes inconvenience in administration. Additionally, approximately 14% of hormone positive patients with advanced breast cancer are diagnosed with brain metastases, while, at present, there are no FDA-approved systemic therapies for the treatment of breast cancer brain metastases. Therefore, to develop a new generated oral and brain-penetrant SERD to overcome these challenges is believed to be needed. SCR6106, discovered and currently being developed by Simcere, is a novel, potent, orally delivered and non-steroidal SERD that both antagonizes and degrades ERα. SCR6106 showed high potency on inducing ERα protein degradation (DC50=0.29 nM) and anti-proliferation (IC50=0.43 nM) in MCF7 cells. In addition, SCR6106, showed high activities on ERα degradation and anti-proliferation in a panel of ER+ breast cancer cell lines. Meanwhile, ER target genes transcription were evaluated and SCR6106 exhibit strong suppressive phenotype in T-47D. SCR6106 demonstrated more potent than fulvestrant on anti-tumor growth in vivo. In MCF7 xenograft model, SCR6106 at 3 mpk QD induced 113% tumor growth inhibition (TGI), compared to fulvestrant 56.8% TGI at 250 mpk. Compared to fulvestrant, SCR6106 demonstrated stronger ERα degradation and inhibition of ER target genes transcription in tumors, which was consistent with anti-tumor activities. SCR6106 displayed good oral bioavailability in multiple pre-clinical species, which supports its oral administration in human. Significantly, SCR6106 possess a high BBB permeability, the B/P ratio is more than 4 in pre-clinical species. The exposure of SCR6106 observed in mouse brain is much higher than the IC90 value of anti-proliferation (MCF7), and SCR6106 demonstrated anti-tumor activities in a MCF-7 intracranial tumor xenograft model. In conclusion, SCR6106 is a novel, potent and oral SERD, which demonstrated anti-tumor activities in vitro and in vivo. It showed high potential ability to cross the blood-brain barrier, and could be used to treat HR+ patients with brain metastasis. Citation Format: Feng Zhou, Lei Liu, Guimei Yang, Peng Gu, Liting Xue, Wenqing Yang, Ping Chen, Renhong Tang. SCR6106, an oral and brain penetrant SERD demonstrated anti-tumor activities in vitro and in vivo [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 1070.

Molecular studies on oestrogen α and progesterone receptors and histomorphometric analysis of canine uteri following aglepristone treatment.

Aglepristone, a competitive progesterone antagonist, is successfully used in various progesterone-dependent conditions. This study investigated uterine histomorphometric analysis, and expressions of the oestrogen α receptor (ERα) and progesterone receptor (PR) in uteri of bitches following the single dose of aglepristone treatment. Twelve client-owned healthy diestrous bitches were used in the study. The single dose of aglepristone (Alizine® , 10mg/kg) was injected subcutaneously 5days before ovariohysterectomy in the treatment group (n=6); bitches without treatment served as a control group (n=6). Uteri were collected for histomorphometric analysis, ERα and PR gene, and protein expressions studies. The mRNA expressions of ERα and PR were determined by RT-qPCR. Immunohistochemical analysis was used to evaluate the ERα and PR protein expressions using an H-score in five parts of the uterus. The results demonstrated glandular epithelium height significantly decreased (p<.05) and ERα mRNA increased (p<.01) in treated dogs. Of the treated bitches, lower expression levels of ERα were observed in the luminal epithelium, crypt and glandular epithelium, with higher expression in the endometrial stroma and myometrium (p<.05); however, PR expression decreased in the luminal epithelium, crypt and glandular epithelium (p<.01). In conclusion, reduction of the uterine glandular epithelium and ERα mRNA upregulation together with changes in ERα and PR expressions were observed in the treated bitches. However, changes in uterine ERα and PR expressions in the treated bitches depended on tissue layers. The treatment had no effect on serum oestradiol and progesterone levels.

Relative bioavailability of H3B-6545 tablets versus capsules and drug-drug interaction between H3B-6545 and pantoprazole.

e13022 Background: H3B-6545 is a selective, orally available, small molecule antagonist of the estrogen receptor (ER), covalently binding to a cysteine residue at position 530 of both wild-type and the constitutively active mutant ERα proteins. H3B-6545 demonstrated preliminary clinical antitumor activity in breasts cancer patients in phase 1b/2. Methods: This was an open-label phase 1 study to evaluate the relative bioavailability of H3B-6545 from a tablet formulation compared to capsules, and the effect of pantoprazole on the pharmacokinetics of H3B-6545, in healthy post-menopausal women. Subjects were randomized (1:1 ratio) to a combined crossover and fixed sequence 3 periods treatment: A single oral dose (SOD) of 450 mg H3B-6545 fasted on day 1 (capsules or tablets), followed by a 4-day washout; A SOD of 450 mg H3B-6545 fasted on day 5 (crossover formulation from the first period), followed by a 4-day washout; daily oral doses of 40 mg pantoprazole on days 9 to 15 with coadministration of a SOD of 450 mg H3B-6545 (tablets) on day 15. Results: A total of 16 subjects were enrolled and received at least one dose of H3B-6545 and 15 subjects completed all 3 periods. One subject assigned to the tablet/capsule treatment sequence withdrew from the study due to subject decision on day 13 (Period 3), following completion of Period 1 (H3B-6545 tablet) and Period 2 (H3B-6545 capsule). Following a SOD of H3B-6545 capsules alone, tablets alone, or tablets at steady-state QD of pantoprazole, H3B-6545 geometric mean Cmax was about 1070, 1120 and 1330 ng/mL, respectively, AUC was about 16600, 17500 and 18300 ng.h/mL, respectively, half-life was about 11.0, 11.0 and 10.2 hours, respectively, and the median Tmax was about 3.0, 4.0, and 2.0 hours post dose, respectively. The ratio (capsule/tablet) of Cmax and AUC was both about 0.95; steady-state pantoprazole QD increased H3B-6545 Cmax by 20% with no change in AUC. Nine subjects (56.3%) reported at least 1 TEAE during the study with constipation being the most common (43.8%); all TEAEs were mild in severity. There were no SAEs reported. Conclusions: Plasma PK of H3B-6545 is similar between tablets and capsules, and in the absence or presence of pantoprazole. Concurrent use of gastric acid reducers had a minimal effect on H3B-6545 exposure and was not considered clinically meaningful.

Design and synthesis of novel benzothiophene analogs as selective estrogen receptor covalent antagonists against breast cancer.

Endocrine therapy (ET) has benefited patients with estrogen receptor alpha (ERα) positive breast cancer for decades. Selective estrogen receptor modulator (SERM) such as Tamoxifen represents the clinical standard of care (SoC). Despite the therapeutic importance of current SoC agents, 30-50% of prolonged treatment patients inevitably generated resistant tumor cells, usually eventually suffered tumor relapse and developed into metastatic breast cancer (MBC), which was the leading cause of female cancer-related mortality. Among these, most resistant tumors remained dependent on ERα signaling, which reignited the need for the next generation of ERα related agents. We hypothesized that selective estrogen receptor covalent antagonists targeting ERα would provide a therapeutic alternative. In the current work, series of novel benzothiophene hybrids bearing electrophile moieties were synthesized and biologically evaluated. The representative analogue 15c exhibited potent anti-proliferative effect in MCF-7cell lines invitro, and further mechanism studies confirmed the necessity of covalent bonding. More importantly, 15c could attenuate the expression of TFF-1, GREB-1 and downregulate the levels of cellular ERα protein.

The Effects of Tert-butyl Hydroquinone (TBHQ) on Estrogen Receptor Alpha (ERα) and Tumor Suppressor Gene p53 in Breast Cancer Cells

Tert-butyl hydroquinone (TBHQ) is an aromatic compound that is commonly used as a preservative in processed food to prevent rancidity and lengthen shelf life. TBHQ is known to act as an antioxidant by protecting cells from radical oxygen species and thus preventing DNA damage. Although previous studies have found TBHQ to cause cancer cell death at high concentrations, they have also contrastingly found TBHQ, when studied in animal models, to enhance carcinogenic effects. However, the effect of TBHQ on breast cancer has not been thoroughly explored. With the prevalence of breast cancer and the wide use of TBHQ in processed food items, it is imperative that we explore their possible relationship. This study examined the effects of TBHQ, alone and in combination with hormones and anti-hormones, on ERα and p53 expression in both MCF-7 and T-47D breast cancer cell lines. To ensure treatment conditions without the presence of endogenous steroids or growth factors, the cells were cultured with a 5% charcoal-stripped fetal bovine serum (FBS) for six days. Western blot analysis revealed alterations in the expression of ERα and p53 protein levels after 24 hours of treatment with varying concentrations of TBHQ (0.005 to 1 mM). A concentration-dependent decrease of ERα protein levels was observed in both cell lines, with a 49% reduction occurring with 100 µM TBHQ as compared to the control. P53 levels portray a continued increase of expression through concentrations of TBHQ (0.005 to 1 mM), found similarly in both cell lines. To gain further insight into possible similarities between BPS and other known effectors of ERα, the optimal concentration of TBHQ (100 μM) was used in combination with hormones and anti-hormones. Down-regulation of ERα protein levels was observed after 24-hour co-treatment of T-47D & MCF-7 cells with a combination of TBHQ and E2. Antiestrogen ICI with TBHQ showed a significant down-regulation as compared to TBHQ alone, and TBHQ with TAM portrayed no significant differences. A similar trend in the effects on p53 expression was depicted in T-47D and MCF-7 cells. Image cytometric analysis with propidium iodide staining was utilized to quantify cell values and viability changes to further portray the effects of TBHQ on T-47D and MCF-7 cellular growth. The viability assay shows a biphasic effect with increasing concentrations of TBHQ, with a maximum decrease in proliferation seen at a concentration of 100 uM TBHQ. TBHQ alone and in combination with E2 and antiestrogens showed a decreased proliferation compared to the control in T-47D cells. However, cytolocalization of ERα upon treatment with estradiol and TBHQ remained unaltered. Our studies offer a unique perspective on the effects of TBHQ on two different breast cancer cell lines, and provide valuable insight for further exploration of the mechanism of action of TBHQ on tumor suppressor gene and steroid receptors.

Mineralocorticoid and Estrogen Receptors in Endothelial Cells Coordinately Regulate Microvascular Function in Obese Female Mice.

[Figure: see text].