Estrogen Receptor Transcriptional Activity Research Articles

Abstract PS17-31: Investigating the estrogen receptor Y537S mutation in transgenic models of luminal B breast cancer

Abstract Breast cancer is the most prevalent cancer in women and over two-thirds of cases express the estrogen receptor (ER). Significantly, metastatic ER positive breast cancer is the leading cause of breast cancer mortality. Despite the success of endocrine therapy to treat ER-positive breast cancers, resistance to treatment is a major problem and relapse commonly occurs though patients initially respond well to treatment. The ER Y537S mutant represents one of the most common gain-of-function mutations detected in the metastatic biopsies of patients with ER-positive breast cancer. Previous in-vitro analyses have shown that the ER Y537S mutant exhibits enhanced ER transcriptional activity by adopting a ligand-independent agonist confirmation. In our study, we seek to provide a deeper understanding of the role of this mutant by modeling the human ER Y537S mutant (ER Y541S in mice). Our lab has generated a Cre-inducible knock-in mouse model expressing the activating point mutation driven by the endogenous ER promoter, and it has been shown that when expressed ubiquitously the mutated ER leads to dramatic developmental effects. In order to further our observations in the context of endocrine resistant metastatic breast cancer, we characterize mamary tumour growth of the ER Y541S mutant in combination with luminal B models of mammary tumourigenesis. Consistent with the notion that these ER mutations occur seldom in primary tumours, preliminary data shows that tumour onset and tumour outgrowth is unaffected by the activating point mutation. When analysing tumours from both ER mutant and wildtype ER control animals, it was found that tumours with mutant ER display more basal-like cytokeratins at the RNA level via RNA sequencing and at the protein level via immunohistochemistry. This expression pattern is often associated with poorer prognosis in the clinic. Through ongoing analyses, we also seek to better understand our model through tumour regression studies (doxycycline withdrawal), treatment with tamoxifen and blocking the main source of estrogens via ovariotomies. By studying the ER Y537S mutation in models of luminal B breast cancer, we hope to investigate the factors leading to tumour recurrence and provide a deeper understanding of potential escape pathways to therapy. Citation Format: Gabriella Johnson, Joshua Roccamo, Cynthia Lavoie, Dongmei Zuo, Virginie Sanguin-Gendreau, Adrian Lee, Zheqi Li, Steffi Oesterreich, William Muller. Investigating the estrogen receptor Y537S mutation in transgenic models of luminal B breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-31.

Comparative analysis of the AIB1 interactome in breast cancer reveals MTA2 as a repressive partner which silences E-Cadherin to promote EMT and associates with a pro-metastatic phenotype

Steroid regulated cancer cells use nuclear receptors and associated regulatory proteins to orchestrate transcriptional networks to drive disease progression. In primary breast cancer, the coactivator AIB1 promotes estrogen receptor (ER) transcriptional activity to enhance cell proliferation. The function of the coactivator in ER+ metastasis however is not established. Here we describe AIB1 as a survival factor, regulator of pro-metastatic transcriptional pathways and a promising actionable target. Genomic alterations and functional expression of AIB1 associated with reduced disease-free survival in patients and enhanced metastatic capacity in novel CDX and PDX ex-vivo models of ER+ metastatic disease. Comparative analysis of the AIB1 interactome with complementary RNAseq characterized AIB1 as a transcriptional repressor. Specifically, we report that AIB1 interacts with MTA2 to form a repressive complex, inhibiting CDH1 (encoding E-cadherin) to promote EMT and drive progression. We further report that pharmacological and genetic inhibition of AIB1 demonstrates significant anti-proliferative activity in patient-derived models establishing AIB1 as a viable strategy to target endocrine resistant metastasis. This work defines a novel role for AIB1 in the regulation of EMT through transcriptional repression in advanced cancer cells with a considerable implication for prognosis and therapeutic interventions.

Estrogenic activity of capsule coffee using the VM7Luc4E2 assay

Coffee brewed from capsule machines may contain estrogenic chemicals migrated from plastic, but the estrogenic activity of capsule coffee has not been evaluated. This study evaluated the estrogenic activity of capsule coffee using the VM7Luc4E2 estrogen receptor transcriptional activation assay. Estrogenic potentials of six capsule coffee samples were calculated using relative maximum amplitude response of E2 (>15%RME2 indicative of estrogenic activity) and estradiol equivalent factor (EEF). Estrogenic chemical content was determined using ultra-performance liquid chromatography with tandem mass spectrometry. All capsule coffee samples possessed estrogenic activity (48–56%RME2). EEFs were 6–7 orders of magnitude lower than that of E2, (1.2 × 10−7−1.7 × 10−6), indicating substantially weaker estrogenic potencies. Bisphenol A, bisphenol F, benzophenone, 4-nonylphenol, dibutyl phthalate, and dimethyl terephthalate were detected in capsule coffee. Capsule coffee exhibited estrogenic activity in vitro, and its estrogenic chemical content is likely driving its estrogenicity, warranting further investigations to fully understand the degree to which they are related and to predict the estrogenic potential based on the concentration of estrogenic chemicals.

Cyclic depsipeptide mycotoxin exposure may cause human endocrine disruption: Evidence from OECD in vitro stably transfected transcriptional activation assays

The presence of cyclic depsipeptide mycotoxins in foods and feedstuffs could potentially cause endocrine disrupting effects on humans and wildlife by their inhibition of active steroidogenesis. Therefore, we attempted to assess the human estrogen receptor (ER) and androgen receptor (AR) agonistic/antagonistic effects of representative cyclic depsipeptide mycotoxins, enniatin A1 (ENN A1), and enniatin B1 (ENN B1), by OECD Performand Based Test Guideline (PBTG) No.455, VM7Luc ER transcriptional activation (TA) assay and OECD TG No. 458, 22Rv1/MMTV_GR-KO AR TA assay. No tested cyclic depsipeptide mycotoxins were found to be ER and AR agonists in VM7Luc ER TA and 22Rv1/MMTV_GR-KO AR TA assays. On the other hand, ENN A1, and ENN B1 exhibited the ER and AR antagonistic effects with IC30 and IC50 values in both TA assays. These two cyclic depsipeptide mycotoxins, which were determined as ER and AR antagonists by two in vitro assays, bound to ERα, and AR. Then ENN A1, and ENN B1 inhibited the dimerization of ERα, and AR. These results, for the first time indicated that ENN A1, and ENN B1 could have potential endocrine disrupting effects mediated by interaction of ERα and AR using international standard testing methods to determine the potential endocrine disrupting chemical.

A Novel Dual Antibody Staining Assay to Measure Estrogen Receptor Transcriptional Activity

Activity of the canonical estrogen receptor (ER) pathway is equivalent to functional activity of the nuclear ER transcription factor. Monoclonal antibodies (MoAbs) that identify nuclear ER in cells and tissue samples are frequently used to assess ER transcriptional activity, however, it remains unclear if this approach is sufficiently predictive of ER pathway activity. This study uses ER-positive breast cancer cell lines (MCF7 and T47D) in which ER transcriptional activity was quantified using an mRNA-based ER pathway activity assay. The relationship between ER activity and nuclear ER staining with ER MoAbs was then investigated. Confirming earlier findings, the results show that while the presence of ER in the cell nucleus is a prerequisite for ER activity, it is not predictive of ER transcriptional activity. There were remarkable differences in the behaviours of the antibodies used in the study. EP1 and 1D5 showed reduced nuclear staining when ER was transcriptionally active, while staining with H4624 was independent of ER activity. To improve discrimination between active and inactive nuclear ER based on ER staining, a method was developed which consists of dual ER MoAb immunofluorescent staining, followed by generation of a digital image with a standard digital pathology scanner. Then a cell nucleus detection algorithm and per cell calculation of the nuclear H4624/EP1 fluorescence intensity ratio was applied, where a high H4624/EP1 ratio predicts an active ER pathway. With this method, the EP1 and 1D5 antibodies are interchangeable. We hypothesize that the transcriptional activation of ER hides the epitope recognized by MoAbs EP1 and 1D5, while H4624 binds an ER epitope that remains accessible during ER pathway activation. The method described in this study should add substantial value to the assessment of ER pathway activity for biomedical research and diagnostics.

HEAT SHOCK PROTEIN 90 INHIBITOR 17-AAG SUPPRESSES THE GROWTH OF ENDOMETRIOSIS IN VITRO AND IN VIVO BY INHIBITING ESTROGEN RECEPTOR TRANSCRIPTIONAL ACTIVITY

To investigate the effect of HSP90 inhibitor 17–AAG on the growth of endometriosis in vitro and in vivo. Pharmacologic interventions in human endometriotic stromal cells and in an experimental mouse model of endometriosis. Patient recruitment was carried out at the Sixth affiliated Hospital of Sun Yat-sen University from January 2018 to May 2020. Ectopic endometrium (endometriotic tissue, n=15) was collected from patients with ovarian endometriosis undergoing laparoscopy.Primary cultured human endometriotic stromal cells were prepared. Cells were incubated with 10nM, 100nM, 1μM and 10μM 17-AAG for 24 h. The 3-(4,5-dimethylthiaziazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, colorimetric 5'-bromo-2'-deoxy-uridine (BrdU) incorporation assay and Caspase-Glo luminescent-based assays were used to test the cell viability, proliferation ability and apoptosis. Transcriptional activity of estrogen receptor (ER) was measured by luciferase activity after transient transfection of an ERE-tk-Luc reporter construct that contained the consensus estrogen-responsive elements (EREs) sequences. Furthermore, we modeled endometriosis in 30 female C57BL6 mice by intraperitoneal injection of allogeneic endometrial fragments. We divided the mice to 10ug/g and 30ug/g 17-AAG group, control group and vehicle group. 17-AAG inhibited the viability and proliferation of human primary endometriotic stromal cells in a dose-dependent manner from the concentration of 10nM and 100nM 17-AAG respectively. The caspase-3 activities were enhanced significantly in endometriotic stromal cells from 100nM to 10μM 17-AAG treatments. Transcriptional activity of ER were also inhibited significantly by 17-AAG in a dose-dependent way. In mouse model, no obvious weight loss was observed in all groups, and there was no significantly difference in daily weight between those four groups after 30 days of administration. Compared with the model control and vehicle group, the size of ectopic lesions in the treatment group was significantly reduced. The serum TNF-α level was significantly decreased in the treatment group, but there was no statistical difference in the level of serum AMH/E2/P of mice in each group. HSP90 inhibitor 17-AAG controls the growth of endometriosis by inhibiting transcriptional activity of ER. HSP90 inhibitor could provide a hopeful way for better understanding of estrogen signaling on the pathogenesis of endometriosis, as well as for potential clinical application on endometriosis pharmacotherapy.

Human and Aquatic Toxicity Potential of Petroleum Biodegradation Metabolite Mixtures in Groundwater from Fuel Release Sites.

The potential toxicity to human and aquatic receptors of petroleum fuel biodegradation metabolites (oxygen‐containing organic compounds [OCOCs]) in groundwater has been investigated as part of a multi‐year research program. Whole mixtures collected from locations upgradient and downgradient of multiple fuel release sites were tested using: 1) in vitro screening assays for human genotoxicity (the gamma‐H2AX assay) and estrogenic effects (estrogen receptor transcriptional activation assay), and 2) chronic aquatic toxicity tests in 3 species (Ceriodaphnia dubia, Raphidocelis subcapitata, and Pimephales promelas). In vitro screening assay results demonstrated that the mixtures did not cause genotoxic or estrogenic effects. No OCOC‐related aquatic toxicity was observed and when aquatic toxicity did occur, upgradient samples typically had the same response as samples downgradient of the release, indicating that background water quality was impacting the results. This information provides additional support for previous work that focused on the individual compounds and, taken together, indicates that OCOCs from petroleum degradation at fuel release sites are unlikely to cause toxicity to human or freshwater receptors at the concentrations present. Environ Toxicol Chem 2020;39:1634–1645. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

OR12-05 MDC1 Is a Novel Estrogen Receptor Co-Regulator in Invasive Lobular Carcinoma of the Breast

Invasive Lobular Carcinoma (ILC) is the 2nd most common histotype of breast cancer, but is critically understudied. ~95% of ILC are estrogen receptor (ER) positive, and previous studies demonstrate the importance of estrogen in ILC etiology. However, retrospective studies show that anti-estrogens are substantially less effective in ILC than in ER+ Invasive Ductal Carcinoma (IDC). This strongly suggests that regulation of ER function is unique in ILC, and we hypothesize that this is due to an ILC-specific cohort of ER co-regulators. We performed Rapid Immunoprecipitation Mass Spectrometry of Endogenous Proteins (RIME) to determine ILC-specific ER-interacting proteins, and identified Mediator of DNA Damage Checkpoint 1 (MDC1) as a novel ER co-regulator in ILC cells. We confirmed ER:MDC1 interaction by co-immunoprecipitation and proximity ligation assays (PLA); interaction was specifically observed in ILC cell lines but not IDC cell lines. Consistent with co-regulator function, we found MDC1 is essential for ER-driven proliferation of ILC cells. MDC1 knockdown dysregulates transcription of ER target genes in ILC cells (e.g. IGFBP4, WNT4). Moreover, RNA-seq analysis showed that in ILC cell line MDA MB 134VI, >50% of ER target genes require MDC1 for their regulation. To understand how MDC1 controls ER transcriptional activity, we performed ChIP-qPCR and found that MDC1 controls ER binding to DNA in ILC cells. Further, MDC1 controls binding of the pioneer factor FOXA1 to DNA, and Dual PLA studies of ER:MDC1 and ER:FOXA1 interaction revealed that MDC1 knockdown decreased ER:FOXA1 interaction. MDC1 canonically functions in DNA damage response, but our preliminary data suggest MDC1 is decoupled from its canonical role in DDR in the context of ER co-regulator activity in ILC cells. Together, these data suggest MDC1, independent of its role in DDR, acts as a novel ER co-regulator in ILC and regulates ER:DNA binding and ER transcriptional function to drive ILC cell proliferation and survival.

Decreased ER dependency after acquired resistance to CDK4/6 inhibitors.

Cyclin-dependent kinase (CDK) 4/6 inhibitors represent a significant advancement in the treatment of estrogen receptor (ER)-positive human epidermal growth factor receptor 2-negative advanced breast cancer. However, mechanisms of alterations after acquired resistance to CDK4/6 inhibitors and the optimal treatment options are still not established. Abemaciclib-resistant cell lines were established from the models of estrogen deprivation-resistant cell lines which retained ER expression and activated ER function derived from MCF-7 breast cancer cell lines. Ribocilib-resistant cell lines were established in the same method as previously reported. Both abemaciclib- and ribociclib-resistant cell lines showed decreased ER expression. ER transcriptional activity was maintained in these cell lines; however, the sensitivity to 4-hydroxytamoxifen and fulvestrant was almost completely lost. These cell lines did not exhibit any ERα gene mutation. Abemaciclib-resistant cell lines demonstrated low sensitivity to other CDK4/6 inhibitors; sensitivities to PI3K inhibitor, mTOR inhibitor, and chemotherapeutic drugs were maintained. Dependence on ER signaling appears to decrease after the development of acquired resistance to CDK4/6 inhibitors. Further, CDK4/6 inhibitor-resistant cells acquired cross-resistance to other CDK4/6 inhibitors, PI3K/Akt/mTOR inhibitor therapy and chemotherapeutic drugs might serve as optimal treatment options for such breast cancers.

Abstract P3-10-13: Rac inhibition as an alternative approach to treating hormone dependent breast cancer

Abstract Hormone positive breast cancer remains one of the leading causes of cancer mortality in women. Despite the advancement in endocrine therapy and the addition of cyclin dependent kinase (CDK) 4/6 inhibitors, patients eventually become refractory to treatment creating a desperate need for the development of alternative therapies. Previously, we discovered HACE1, the E3 ubiquitin ligase that targets the small GTPase Rac1 for proteasomal mediated degradation. HACE1 is commonly lost in estrogen receptor (ER) positive and HER2 positive breast cancers and results in the hyperactivation of the Rac signaling pathway. Here, we show that Rac1 resides in the estrogen receptor transcriptional complex and acts as a critical estrogen receptor transcriptional co-activator. The dependency of Rac on ER transcription is further exemplified by the treating ER+ breast cancer cells with a Rac inhibitor where transcriptional profiling identified ER target genes were preferentially down-regulated. Thus, we confirm the dependency of Rac1 in ER+ breast cancers and show that Rac1 is necessary for estrogen receptor transcriptional activity. Importantly, we show that a novel Rac1 inhibitor is capable of reducing estrogen receptor transcriptional activity as well as impairing cellular proliferation and migration. Moreover, Rac inhibition is shown to enhance the anti-tumor effects of fulvestrant both in vitro and in vivo, including in a patient-derived-xenograft (PDX) model of ER+ disease. Lastly, we demonstrate that the administration of a novel Rac inhibitor is well-tolerated after chronic dosing in both small and large animal species. Together these data suggest a new potential pathway for treating hormone dependent breast cancer. Citation Format: Erik T Goka, Jun Sun, Dayrelis T Mesa Lopez, Marc E Lippman. Rac inhibition as an alternative approach to treating hormone dependent breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-13.

Abstract P6-05-13: Crosstalk of post-translational modifications upon ERalpha activation

Abstract Breast cancer risk correlates highly with sustained estrogen receptor (ER) activities. Estrogen antagonists (e.g. anti-estrogens) and estrogen synthesis blockers (e.g. aromatase inhibitors) are frontline treatment to hinder disease progression in patients with breast cancer because most malignant breast tumors exhibit ERalpha positive expression signature. ERalpha harbors several post-translational modifications such as phosphorylation, acetylation, methylation, ubiquitination, sumoylation, palmitoylation, and others. Dysregulation of such PTMs on ERalpha enables abnormal tumor cell proliferation and promote malignancy. Although individual PTM on ERalpha is extensively deciphered, it remains relatively unclear how PTMs interact with each other to coordinate and execute 1) the recruitment of co-activators and RNA polymerase machinery to chromatin and 2) promoter clearance after transcription activation. Here, we report a novel PTM crosstalk between transcription-coupled phosphorylation and an ubiquitination-like modification on ERalpha. We also reveal its potential roles in regulating ER transcriptional activation and its future application in managing ER positive breast cancer. Citation Format: Kai-Hsiung Chang, Hui-Chin Wen, Sin-Yin Tsai. Crosstalk of post-translational modifications upon ERalpha activation [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-05-13.

Neurotoxic effects of aluminum are associated with its interference with estrogen receptors signaling

Aluminum compounds have been observed in various brain regions, and their accumulation has been associated with many neurodegenerative disorders. Neurotoxic effects of aluminum are attributed to reactive oxygen species generation, induction of apoptosis and inflammatory reactions activation. Metalloestrogen activity of aluminum has also been linked to breast cancer progression and metastasis. In this study, taking into account the anti-apoptotic and anti-oxidant activities of estrogens in neuronal cells, which are mediated by estrogen receptors, the possible estrogenic activity of aluminum in SH-SY5Y neuroblastoma cells was studied. Our results showed that aluminum in the form of aluminum chlorohydrate (ACH) exhibited no effect on estrogen receptors transcriptional activation, and differential effect on estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) protein levels. ACH caused reduction in ERβ protein levels, and increase in its mitochondrial localization. ACH-induced reduction in ERβ protein level may be linked, at least in part, to the ACH-induced increase in ERα protein level. This statement is based on our observations showing aluminum-induced reduction in the E2-induced increase in ERα S118 phosphorylation, in MCF-7 and SH-SH5Y cells. Phosphorylation at S118 residue is known to be associated with inhibition of the ubiquitin-induced proteolytic degradation of ERα, leading to its accumulation. Since it is known that ERα negatively regulate ERβ expression, increase in ERα, may contribute to reduction in ERβ levels and subsequent weakening of its anti-apoptotic and anti-oxidant activity, justified by the observed reduction in procaspase 9, mitochondrial cytochrome c, Bcl-2, Bcl-xL and mitochondrial thioredoxin protein level, as well as by the increase in proapoptotic BAX level, in ACH treated SH-SY5Y cells. In addition, increase in mitochondrial ERβ localization may also trigger mitochondrial metabolism, suppress biosynthetic process of gluconeogenesis, as indicated by the observed reduction in the phosphoenolpyruvate carboxykinase protein level, and eventually lead to increase in reactive oxygen species (ROS) generation, known to be implicated in aluminum induced neurodegeneration. This statement was verified by the observed ACH–induced increase in ERβ mitochondrial localization, induction of the mitochondrial membrane depolarization and increase in ROS production, in neuronal-like differentiated SH-SY5Y cells.

IRGD-guided tamoxifen polymersomes inhibit estrogen receptor transcriptional activity and decrease the number of breast cancer cells with self-renewing capacity

BackgroundTamoxifen (Tam) is the most frequent treatment for estrogen receptor (ER) positive breast cancer. We recently showed that fibronectin (FN) leads to Tam resistance and selection of breast cancer stem cells. With the aim of developing a nanoformulation that would simultaneously tackle ER and FN/β1 integrin interactions, we designed polyethylene glycol-polycaprolactone polymersomes polymersomes (PS) that carry Tam and are functionalized with the tumor-penetrating iRGD peptide (iRGD-PS-Tam).ResultsPolyethylene glycol-polycaprolactone PS were assembled and loaded with Tam using the hydration film method. The loading of encapsulated Tam, measured by UPLC, was 2.4 ± 0.5 mol Tam/mol polymer. Physicochemical characterization of the PS demonstrated that iRGD functionalization had no effect on morphology, and a minimal effect on the PS size and polydispersity (176 nm and Pdi 0.37 for iRGD-TAM-PS and 171 nm and Pdi 0.36 for TAM-PS). iRGD-PS-Tam were taken up by ER+ breast carcinoma cells in 2D-culture and exhibited increased penetration of 3D-spheroids. Treatment with iRGD-PS-Tam inhibited proliferation and sensitized cells cultured on FN to Tam. Mechanistically, treatment with iRGD-PS-Tam resulted in inhibition ER transcriptional activity as evaluated by a luciferase reporter assay. iRGD-PS-Tam reduced the number of cells with self-renewing capacity, a characteristic of breast cancer stem cells. In vivo, systemic iRGD-PS-Tam showed selective accumulation at the tumor site.ConclusionsOur study suggests iRGD-guided delivery of PS-Tam as a potential novel therapeutic strategy for the management of breast tumors that express high levels of FN. Future studies in pre-clinical in vivo models are warranted.

Epigenetic Activation of BRCA1 by Genistein In Vivo and Triple Negative Breast Cancer Cells Linked to Antagonism toward Aryl Hydrocarbon Receptor.

Triple negative breast cancers (TNBC) are the most aggressive and lethal breast cancers (BC). The aryl hydrocarbon receptor (AHR) is often overexpressed in TNBC, and its activation results in the epigenetic silencing of BRCA1, which is a necessary factor for the transcriptional activation of estrogen receptor (ER)α. The dietary isoflavone genistein (GEN) modulates BRCA1 CpG methylation in BC cells. The purpose of this study was to investigate the effect of GEN on BRCA1 epigenetic regulation and AHR activity in vivo and TNBC cells. Mice were administered a control or GEN-enriched (4 and 10 ppm) diet from gestation through post-natal day 50. Mammary tissue was analyzed for changes in BRCA1 regulation and AhR activity. TNBC cells with constitutively hypermethylated BRCA1 (HCC38) and MCF7 cells were used. Protein levels and mRNA expression were measured by Western blot and real-time PCR, respectively. BRCA1 promoter occupancy and CpG methylation were analyzed by chromatin immunoprecipitation and methylation-specific PCR, respectively. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. GEN administered in the diet dose-dependently decreased basal Brca1 methylation and AHR activity in the mammary gland of adult mice. HCC38 cells were found to overexpress constitutively active AHR in parallel with BRCA1 hypermethylation. The treatment of HCC38 cells with GEN upregulated BRCA1 protein levels, which was attributable to decreased CpG methylation and AHR binding at BRCA1 exon 1a. In MCF7 cells, GEN prevented the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-dependent localization of AHR at the BRCA1 gene. These effects were consistent with those elicited by control AHR antagonists galangin (GAL), CH-223191, and α-naphthoflavone. The pre-treatment with GEN sensitized HCC38 cells to the antiproliferative effects of 4-hydroxytamoxifen. We conclude that the dietary compound GEN may be effective for the prevention and reversal of AHR-dependent BRCA1 hypermethylation, and the restoration of ERα-mediated response, thus imparting the sensitivity of TNBC to antiestrogen therapy.

Characterization of Estrogenic and Androgenic Activities for Bisphenol A-like Chemicals (BPs): In Vitro Estrogen and Androgen Receptors Transcriptional Activation, Gene Regulation, and Binding Profiles.

Bisphenol A (BPA) is a high production volume chemical widely used in plastics, food packaging, and many other products. It is well known that endocrine-disrupting chemicals might be harmful to human health due to interference with normal hormone actions. Recent studies report widespread usage and exposure to many BPA-like chemicals (BPs) that are structurally or functionally similar to BPA. However, the biological actions and toxicity of those BPs are still relatively unknown. To address this data gap, we used in vitro cell models to evaluate the ability of 22 BPs to induce or inhibit estrogenic and androgenic activity. BPA, Bisphenol AF (BPAF), bisphenol Z (BPZ), bisphenol C (BPC), tetramethyl bisphenol A (TMBPA), bisphenol S (BPS), bisphenol E (BPE), 4,4-bisphenol F (4,4-BPF), bisphenol AP (BPAP), bisphenol B (BPB), tetrachlorobisphenol A (TCBPA), and benzylparaben (PHBB) induced estrogen receptor (ER)α and/or ERβ-mediated activity. With the exception of BPS, TCBPA, and PHBB, these same BPs were also androgen receptor (AR) antagonists. Only 3 BPs were found to be ER antagonists. Bisphenol P (BPP) selectively inhibited ERβ-mediated activity and 4-(4-phenylmethoxyphenyl)sulfonylphenol (BPS-MPE) and 2,4-bisphenol S (2,4-BPS) selectively inhibited ERα-mediated activity. None of the BPs induced AR-mediated activity. In addition, we identify that the BPs can bind to ER or AR with varying degrees by a molecular modeling analysis. Taken together, these findings help us to understand the molecular mechanism of BPs and further consideration of their usage in consumer products.

Abstract 4769: Gene expression profiling of response to short- and long-term treatment of LSZ102 in MCF7 cells

Abstract Breast cancer is the most common cancer in women worldwide, and the second most common cause of cancer death in woman in the US. Close to 80% of breast cancer are estrogen receptor (ER) positive and can benefit from antiestrogen treatment. LSZ102 is a novel oral selective estrogen receptor degrader (SERD) in clinical development, which exhibits potent activity in inducing ER degradation, modulating ER-target genes transcription and inhibits the proliferation of breast cancer cells both in-vitro and in-vivo. In this study, we have investigated the effects of short term and prolonged LSZ102 treatment on global gene expression in ER positive MCF-7 breast cancer cells. MCF-7 cells were treated with LSZ102 for 24 hours, 8 weeks and 14 weeks, followed by transcriptome profiling by RNA sequencing. Our findings show LSZ102 effectively inhibited ER transcriptional activity and lead to a sustained suppression of estrogen responsive genes expression in LSZ102-treated cells of all time points. We also observed a cumulative up-regulation of EPAS1, a hypoxia-inducible transcription factor. In addition, we found up-regulation of EGFR and ERBB2 genes and regulated genes in cells with prolonged treatment of LSZ102. In summary, our results suggest a possible role of EPAS1 in eliciting a compensatory growth promoting signals in breast cancer cells adapted to ER inhibition. Citation Format: Choi Lai Tiong Yip, Joshua Korn, David Ruddy, Daniel Rakiec, Darrin Stuart, Alex Gaither. Gene expression profiling of response to short- and long-term treatment of LSZ102 in MCF7 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4769.

Abstract 796: Progestins target mitochondrial respiration to shift metabolic phenotype in luminal breast cancer

Abstract The dichotomous nature of progesterone receptor (PR) in estrogen receptor (ER)-positive breast cancers has confounded its use as an endocrine target. Historically, progestins were used to treat some advanced breast cancers and ongoing clinical trials are testing several PR ligands. PR generally opposes ER activity through multiple mechanisms, most notably by altering global ER transcriptional activity to suppress breast tumor growth. However, progestins can also stimulate expansion of endocrine-resistant cancer stem cells (CSCs). Thus, better understanding of potential consequences of progestins in breast cancer is necessary to gauge therapeutic efficacy. Given that ER-driven tumor growth relies on mitochondrial respiration, we evaluated whether PR modulates cell metabolism machinery as a mechanism of ER antagonism. We used functional and morphometric assays in an ER+ breast cancer cell line and patient-derived tumor models to show that progestin treatment dramatically alters mitochondrial function. Multivariate metabolomics analysis highlights a progestin-associated metabolic signature involving accumulation of multiple TCA cycle intermediates, including fumarate, malate, and succinate. Functional assays indicate that progestins impair mitochondrial oxidative reserve capacity and oxygen consumption while ablating estradiol-induced accumulation of ATP in PR+ but not PR− cells. Morphometric assessment of mitochondria via laser-scanning confocal and transmission electron microscopy indicate a shift away from reticular filamentous mitochondria towards smaller, rounder, more perinuclear mitochondria with altered membrane potential. These results suggest that PR targets mitochondrial respiration as part of a broader strategic shift from ER-driven tumor growth to PR-driven thrifty metabolic dormancy. Whether the latter is indicative of an acquired propensity for glycolytic metabolism in indolent tumor cells, CSCs, or perhaps both, is addressed in ongoing studies. Citation Format: Shawna B. Matthews, Lynsey M. Fettig, Ashley V. Ward, Matthew R. Jackman, Paul S. MacLean, Carol A. Sartorius. Progestins target mitochondrial respiration to shift metabolic phenotype in luminal breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 796.

A combined FSTRA-shotgun proteomics approach to identify molecular changes in zebrafish upon chemical exposure

The fish short-term reproduction assay (FSTRA) is a common in vivo screening assay for assessing endocrine effects of chemicals on reproduction in fish. However, the current reliance on measures such as egg number, plasma vitellogenin concentration and morphological changes to determine endocrine effects can lead to false labelling of chemicals with non-endocrine modes- of-action. Here, we integrated quantitative liver and gonad shotgun proteomics into the FSTRA in order to investigate the causal link between an endocrine mode-of-action and adverse effects assigned to the endocrine axis. Therefore, we analyzed the molecular effects of fadrozole-induced aromatase inhibition in zebrafish (Danio rerio). We observed a concentration-dependent decrease in fecundity, a reduction in plasma vitellogenin concentrations and a mild oocyte atresia with oocyte membrane folding in females. Consistent with these apical measures, proteomics revealed a significant dysregulation of proteins involved in steroid hormone secretion and estrogen stimulus in the female liver. In the ovary, the deregulation of estrogen synthesis and binding of sperm to zona pellucida were among the most significantly perturbed pathways. A significant deregulation of proteins targeting the transcriptional activity of estrogen receptor (esr1) was observed in male liver and testis. Our results support that organ- and sex-specific quantitative proteomics represent a promising tool for identifying early gene expression changes preceding chemical-induced adverse outcomes. These data can help to establish consistency in chemical classification and labelling.

Assessment of human estrogen receptor agonistic/antagonistic effects of veterinary drugs used for livestock and farmed fish by OECD in vitro stably transfected transcriptional activation assays

The presence of veterinary drug residues in foods and the environment could potentially cause adverse effects on humans and wildlife. Several veterinary drugs were reported to exhibit endocrine disrupting effects via binding affinities to sexual hormone receptors such as estrogen and androgen receptors. Therefore, we confirmed the human estrogen receptor (ER) agonistic/antagonistic effects of 135 chemicals that were used as veterinary drugs in Korea by the official Organization for Economic Cooperation and Development (OECD) in vitro ER transcriptional activation (TA) assay using the VM7Luc4E2 cell line. In the case of ER agonist screening, 7 veterinary drugs (cefuroxime, cymiazole, trenbolone, zeranol, phoxim, altrenogest and nandrolone) were determined to be ER agonists. In addition, only zeranol was found to exhibit weak ER antagonistic activity. These 7 veterinary drugs, which were determined as ER agonists and/or antagonists by an OECD in vitro assay, were also found to have binding affinity to ERs. These results indicate that various veterinary drugs possess potential (anti-)estrogenic effects. However, further study is needed to determine the precise endocrine-disrupting effects of these compounds.

Protonation state effects of estrogen receptor α on the recognition mechanisms by perfluorooctanoic acid and perfluorooctane sulfonate: A computational study

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) have been reported to cause adverse health effects on wildlife as well as humans. Numerous studies demonstrated that PFOA and PFOS could interfere with the transcriptional activation of estrogen receptor α (ERα) by mimicking the function of endogenous ligand, whereas some reports suggested that the two compounds present non-estrogenic activities. These conflicting results bring a confusion to understand their molecular mechanism on the ERα-mediated signaling pathway. To address this issue, we performed the molecular docking and molecular dynamics simulations to elaborate the structural characteristics for the binding of PFOA and PFOS to ERα. Our results indicated that the two opposite binding orientations were modulated by the protonation states of key residue His524. In sub-acidic condition, PFOA and PFOS prefer to form the H-bonding interactions with the protonated His524, whereas Arg394 provided the H-bonding interactions for stable binding in sub-alkaline condition. Conformational analyses implied that the diverse binding modes were closely related to the conformational propensity of ERα for subsequent coactivator recruitment and transcription activation. Generally, our findings provide a flexible strategy to assess the pH impacts of microenvironment on the toxicities of perfluoroalkyl acids by their interactions with proteins.