Estrogen-regulated Gene Expression Research Articles

Abstract P6-12-02: Connecting Fluctuations in Estrogen Production, Release, and Action with Hormone-Dependent Gene Expression in Breast Cancers

Abstract Estrogens are steroid hormones that play a key role in a wide range of physiological and pathological processes. Estrogen receptor-alpha, ERɑ, functions primarily as a nuclear transcription factor (TF) through ligand-dependent activation by 17β-estradiol (E2), the predominant naturally-occurring estrogen. This results in dimerization, DNA binding to cis-regulatory elements called enhancers, and gene regulatory function of ERɑ. In vivo, E2 production and action fluctuates during reproductive cycles and possibly even in a circadian manner. In tissues and cells, E2-regulated gene expression is dynamic, with rapid induction of enhancer formation and target gene expression (or repression), followed by enhancer “decommissioning” and cessation of the gene regulatory effects. The effects of sustained E2 production and signaling, fluctuations in E2 levels, and removal of E2 on estrogen-regulated gene expression are unknown. Classical experiments exploring the gene regulatory effects of E2 involve stimulation of cultured cells throughout the duration of the experiment with a continuous and typically high dose of hormone, with the cells harvested at the end of the stimulation period. While this has allowed us to understand the molecular underpinnings of signal-dependent transcription, it has not allowed analysis of physiological fluctuations in estrogen signaling involving periods of stimulation (i.e., the presence of hormone) and non-stimulation (i.e., the absence of hormone). In our studies, we are applying a combination of next generation sequencing techniques and fluorescence microscopy to investigate signal-dependent responses to fluctuating and short duration E2 exposures using ERɑ-positive MCF-7 human breast cancer cells after E2 treatment and removal. Our initial results suggest that different E2 target genes respond differently to low dose E2 treatment and E2 withdrawal. Moreover, for some E2 target genes, sustained E2 treatment may not be required for a complete E2 transcriptional response. With these studies, we hope to connect physiological aspects of E2 production, release, and action with the molecular mechanisms of E2-dependent gene expression - an aspect of our understanding of estrogen signaling that has been lacking. Citation Format: Hyung Bum Kim, William L. Kraus. Connecting Fluctuations in Estrogen Production, Release, and Action with Hormone-Dependent Gene Expression in Breast Cancers [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 P6-12-02.

RF03 | PMON297 Connecting Fluctuations in Estrogen Production, Release, and Action with Hormone-Dependent Gene Expression in Breast Cancers

Abstract Estrogens are steroid hormones that play a key role in a wide range of physiological and pathological processes. Estrogen receptor-alpha, ERɑ, functions primarily as a nuclear transcription factor (TF) through ligand-dependent activation by 17β-estradiol (E2), the predominant naturally-occurring estrogen. This results in dimerization, DNA binding to cis-regulatory elements called enhancers, and gene regulatory function of ERɑ. In vivo, E2 production and action fluctuates during reproductive cycles and possibly even in a circadian manner. In tissues and cells, E2-regulated gene expression is dynamic, with rapid induction of enhancer formation and target gene expression (or repression), followed by enhancer "decommissioning" and cessation of the gene regulatory effects. The effects of sustained E2 production and signaling, fluctuations in E2 levels, and removal of E2 on estrogen-regulated gene expression are unknown. Classical experiments exploring the gene regulatory effects of E2 involve stimulation of cultured cells throughout the duration of the experiment with a continuous dose of hormone, with the cells harvested at the end of the stimulation period. While this has allowed us to understand the molecular underpinnings of signal-dependent transcription, it has not allowed analysis of physiological fluctuations in estrogen signaling involving periods of stimulation and non-stimulation. In our studies, we are applying a combination of next generation sequencing techniques and fluorescence microscopy to investigate signal-dependent responses to fluctuating E2 levels using ERɑ-positive MCF-7 human breast cancer cells after E2 treatment and removal. Our initial results suggest that different E2 target genes respond differently to E2 withdrawal. Moreover, for some E2 traget genes, sustained E2 treatment may not be required for a complete E2 transcriptional response. With these studies, we hope to connect physiological aspects of E2 production, release, and action with the molecular mechanisms of E2-dependent gene expression - an aspect of our understanding of estrogen signaling that has been lacking. This work is supported by grants from the NIH/NIDDK (DK058110) and CPRIT (RP160319) to W.L.K. Presentation: Saturday, June 11, 2022 1:12 p.m. - 1:17 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

RF22 | PMON05 Parabens Promote Pro-Tumorigenic Effects in Luminal Breast Cancer Cell Lines With Diverse Genetic Ancestry

Abstract In the United States, Black women are 39% more likely to die from breast cancer compared to White women (AACR Cancer Disparities Progress Report 2020). Furthermore, Black women are at a higher risk of developing breast cancer under the age of 40 than any other racial or ethnic group. While the underlying cause of these disparities is multifactorial, exposure to endocrine disrupting chemicals (EDCs) in hair and personal care products has been associated with increased risk of breast cancer. Parabens are known EDCs that are commonly used as preservatives in hair and other personal care products. The Environmental Working Group scores methylparaben (MP), propylparaben (PP), and butylparaben (BP) moderate (MP) to high (BP and PP) on a hazardous scale of chemicals found in personal care products. Furthermore, these parabens are restricted in personal care products with set maximum concentration standards in Japan, the European Union, and Southeast Asia. Studies have shown that parabens impact breast cancer cell proliferation, death, migration/invasion, and metabolism, as well as gene expression in vitro. However, these studies were conducted using cell lines of European ancestry; to date, no studies have utilized breast cancer cell lines of West African ancestry to examine effects of parabens. We hypothesize that, like what has been shown for breast cancer cell lines with European ancestry, parabens will promote pro-tumorigenic effects in breast cancer cell lines with West African ancestry. To test this hypothesis, we treated MCF-7 (European ancestry) and HCC1500 (West African ancestry) luminal breast cancer cell lines with biologically relevant doses of either MP, PP, or BP. We also co-treated with the estrogen receptor (ER) antagonist ICI 182,780 to determine whether observed effects are mediated by ER. We found BP increases HCC1500 cell viability, but not MCF-7 cell viability. This effect was not blocked by co-treatment with ICI 182,780. We also observed that BP and PP, but not MP, increased expression of estrogen-regulated genes in both MCF-7 and HCC1500. The increase in gene expression was reduced with co-treatment of ICI 182,780 in both cell lines, suggesting that these effects are ER-mediated. Preliminary studies suggest that parabens promote migration of both cell lines, with a potentially greater effect on HCC1500 cells. Taken together, these data demonstrate that parabens, particularly BP and PP, promote pro-tumorigenic effects in diverse luminal breast cancer cell lines. These findings have future translational relevance as we are part of a community-led initiative called Bench to Community. Bench to Community brings together basic researchers in endocrinology and social-behavioral scientists, community stakeholders including breast cancer survivors to develop interventions to reduce adverse exposures to EDCs in hair and other personal care products in Black women. Presentation: Sunday, June 12, 2022 12:42 p.m. - 12:47 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Abstract PD1-04: Estrogen-mediated mechanisms in estrogen receptor-positive breast cancer at the single cell level

Abstract Background: Estrogen typically promotes the progression of hormone-dependent breast cancer through activation of estrogen receptor (ER)-α encoded by ESR1. While estrogen-induced tumor suppression in ER+ breast cancer has been clinically observed as an unexpected outcome of aromatase inhibitor (AI)-resistance, the molecular mechanisms have not yet been fully defined. Methods: Characterization of estrogen regulation in the two ER+ breast cancer patient-derived xenograft (PDX) models with opposite responses to estrogen offered us an unprecedented opportunity to assess how 17β-estradiol (E2) modulates ER+ cancer. We succeeded in establishing estrogen-stimulating (SC31) and estrogen-suppressing (GS3) PDX models. In vivo tumor promotion or suppression by estrogen was confirmed through experiments by implanting E2 pellets in mice carrying SC31/GS3, and then single cell analysis was performed using SC31/GS3 tumors. To investigate whether GS3 would change its response to E2, we performed intermittent E2 treatment every 28 days (E2 pellet on/off every 28 days) and whole-genome RNA sequencing using GS3 tumors. Results: SC31 and GS3 behaved oppositely regarding estrogen-mediated tumor growth. Immunohistochemistry indicated that the number of ERα+ cells and Ki-67+ cells were increased in SC31 and decreased in GS3 after E2 treatment, but progesterone receptor+ cells appeared in both SC31 and GS3 after E2 treatment at the protein level. The E2-induced suppression of GS3 involves ERα, not ERβ, which was wild-type and not amplified. Single cell RNA sequencing analysis of these PDXs had revealed that E2 upregulated the expression of estrogen-regulated genes (e.g., PGR and AREG) in both SC31 and GS3. However, E2 treatment induced cell cycle promotion in SC31, while E2 induced cell cycle arrest in GS3. These gene-expression changes occurred in both ESR1+ cells and ESR1- cells, demonstrating for the first time the influence of estrogen on ESR1- cells in ER+breast tumors. This result suggests that 100% ER positivity is not essential for endocrine response. E2 also upregulated a tumor suppressor gene, IL24, only in GS3. More IL24+ cells were ESR1+ and in G1 phase than IL24- cells. Hallmark apoptosis gene sets were upregulated and the hallmark G2M checkpoint gene set was downregulated in IL24+ cells after E2 treatment. After three rounds of intermittent E2 treatment on GS3, an E2 independent growth developed. Approximately 60% of genes in the intermittent E2-treated sample had the same trend as E2-treated samples (mainly ER target genes), in which 40% of the genes behaved similarly to the placebo-treated sample (mainly cell cycle progression genes). Furthermore, lower levels of IL24 were linked to estrogen independence. Conclusions: Estrogen/ERα signaling increases the expression of estrogen-regulated genes, but it can modulate HR+ tumor growth in different manners. While E2 should activate ERα and regulate ESR1+ cells, our findings regarding ESR1- cells were new and suggested crosstalk between ESR1+ cells and ESR1- cells in both estrogen-stimulating and -suppressing ER+ tumors. Furthermore, our studies demonstrate the potential roles of tumor suppressor gene, IL24 in HR+ cancer. Our findings point to the need to identify biomarkers for patients with estrogen-suppressing tumors who can benefit from E2 treatment after AI resistance; measurements of ER and PR expression are insufficient. Expression of IL24 in AI-resistant tumors may be one such indicator for favorable response to E2 therapy. Citation Format: Hitomi Mori, Kohei Saeki, Gregory Chang, Jinhui Wang, Xiwei Wu, Pei-Yin Hsu, Noriko Kanaya, George Somlo, Masafumi Nakamura, Andrea Bild, Shiuan Chen. Estrogen-mediated mechanisms in estrogen receptor-positive breast cancer at the single cell level [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 PD1-04.

PARP-1 Regulates Estrogen-Dependent Gene Expression in Estrogen Receptor α-Positive Breast Cancer Cells.

Poly(ADP-ribose) polymerase-1 (PARP-1) has gained considerable attention as a target for therapeutic inhibitors in breast cancers. Previously we showed that PARP-1 localizes to active gene promoters to regulate histone methylation and RNA polymerase II activity (Pol II), altering the expression of various tumor-related genes. Here we report a role for PARP-1 in estrogen-dependent transcription in estrogen receptor alpha (ERα)-positive (ER+) breast cancers. Global nuclear run-on and sequencing analyses functionally linked PARP-1 to the direct control of estrogen-regulated gene expression in ER+ MCF-7 breast cancer cells by promoting transcriptional elongation by Pol II. Furthermore, chromatin immunoprecipitation sequencing analyses revealed that PARP-1 regulates the estrogen-dependent binding of ERα and FoxA1 to a subset of genomic ERα binding sites, promoting active enhancer formation. Moreover, we found that the expression levels of the PARP-1- and estrogen-coregulated gene set are enriched in the luminal subtype of breast cancer, and high PARP-1 expression in ER+ cases correlates with poor survival. Finally, treatment with a PARP inhibitor or a transcriptional elongation inhibitor attenuated estrogen-dependent growth of multiple ER+ breast cancer cell lines. Taken together, our results show that PARP-1 regulates critical molecular pathways that control the estrogen-dependent gene expression program underlying the proliferation of ER+ breast cancer cells. IMPLICATIONS: PARP-1 regulates the estrogen-dependent genomic binding of ERα and FoxA1 to regulate critical gene expression programs by RNA Pol II that underlie the proliferation of ER+ breast cancers, providing a potential therapeutic opportunity for PARP inhibitors in estrogen-responsive breast cancers.

Pharmacological Inhibition of CBP/p300 Blocks Estrogen Receptor Alpha (ERα) Function through Suppressing Enhancer H3K27 Acetylation in Luminal Breast Cancer.

Simple SummaryBreast cancer is the most common cancer in women, affecting 1 in 20 women globally. Breast cancer is divided into four subtypes: luminal A, luminal B, HER2-enriched, and basal-like. Estrogen receptor-positive breast cancer accounts for nearly 80% of all breast cancer cases and most breast cancer deaths occur in patients with breast cancer of luminal subtypes. Estrogen receptor antagonists are the standard-of-care treatment for this type of breast cancer; however, resistance to these treatments is common in the clinic. Therefore, we aimed to explore novel therapeutic strategies for inhibiting estrogen receptor activity in breast cancer. Estrogen receptor relies on two highly related proteins, known as CBP and p300, to function. We report that CBP/p300 inhibitors effectively block estrogen receptor function and inhibit breast cancer cell growth. These findings reveal CBP and p300 as promising new targets for breast cancer treatment.Estrogen receptor alpha (ER) is the oncogenic driver for ER+ breast cancer (BC). ER antagonists are the standard-of-care treatment for ER+ BC; however, primary and acquired resistance to these agents is common. CBP and p300 are critical ER co-activators and their acetyltransferase (KAT) domain and acetyl-lysine binding bromodomain (BD) represent tractable drug targets, but whether CBP/p300 inhibitors can effectively suppress ER signaling remains unclear. We report that the CBP/p300 KAT inhibitor A-485 and the BD inhibitor GNE-049 downregulate ER, attenuate estrogen-induced c-Myc and Cyclin D1 expression, and inhibit growth of ER+ BC cells through inducing senescence. Microarray and RNA-seq analysis demonstrates that A-485 or EP300 (encoding p300) knockdown globally inhibits expression of estrogen-regulated genes, confirming that ER inhibition is an on-target effect of A-485. Using ChIP-seq, we report that A-485 suppresses H3K27 acetylation in the enhancers of ER target genes (including MYC and CCND1) and this correlates with their decreased expression, providing a mechanism underlying how CBP/p300 inhibition downregulates ER gene network. Together, our results provide a preclinical proof-of-concept that CBP/p300 represent promising therapeutic targets in ER+ BC for inhibiting ER signaling.

Estrogen suppresses HOXB2 expression via ERα in breast cancer cells

The expression of HOXB2, a homeobox transcription factor, is altered in a variety of solid tumors. Using an in vivo screen to identify regulators of breast tumor growth in murine mammary fat pads, Boimel and co-workers recently identified HOXB2 as a tumor suppressor. However, the mechanistic underpinnings of its role in breast cancer is not understood. Given the emerging interaction of estrogen-regulated gene expression and altered HOX gene expression network in the pathophysiology of breast cancer, this study addressed the relationship between estrogen signaling and HOXB2 expression. Using a mouse model and human breast cancer cell lines, we show that estrogen suppresses HOXB2 expression. Suppression of HOXB2 by PPT, a known ERα agonist, in MCF-7 and T47D cells indicated the involvement of ERα, which was confirmed by siRNA-mediated ERα knockdown experiments. In-silico analysis of the upstream promoter region revealed the presence of three putative EREs. Chromatin immunoprecipitation experiments showed that upon estrogen binding, ERα engaged with EREs in the 5' upstream region of HOXB2 in MCF-7 and T47D cells. Future investigations should address the implications of estrogen-mediated suppression on the proposed tumor suppressor function of HOXB2.

β-Sitosterol-D-Glucopyranoside Mimics Estrogenic Properties and Stimulates Glucose Utilization in Skeletal Muscle Cells.

Estrogenic molecules have been reported to regulate glucose homeostasis and may be beneficial for diabetes management. Here, we investigated the estrogenic effect of β-sitosterol-3-O-D-glucopyranoside (BSD), isolated from the fruits of Cupressus sempervirens and monitored its ability to regulate glucose utilization in skeletal muscle cells. BSD stimulated ERE-mediated luciferase activity in both ERα and ERβ-ERE luc expression system with greater response through ERβ in HEK-293T cells, and induced the expression of estrogen-regulated genes in estrogen responsive MCF-7 cells. In silico docking and molecular interaction studies revealed the affinity and interaction of BSD with ERβ through hydrophobic interaction and hydrogen bond pairing. Furthermore, prolonged exposure of L6-GLUT4myc myotubes to BSD raised the glucose uptake under basal conditions without affecting the insulin-stimulated glucose uptake, the effect associated with enhanced translocation of GLUT4 to the cell periphery. The BSD-mediated biological response to increase GLUT4 translocation was obliterated by PI-3-K inhibitor wortmannin, and BSD significantly increased the phosphorylation of AKT (Ser-473). Moreover, BSD-induced GLUT4 translocation was prevented in the presence of fulvestrant. Our findings reveal the estrogenic activity of BSD to stimulate glucose utilization in skeletal muscle cells via PI-3K/AKT-dependent mechanism.

Abstract PD7-02: Estrogen-induced cell cycle arrest as an unexpected outcome of aromatase inhibitor-resistance: Insights from single-cell trajectory analysis of a patient-derived xenograft model

Abstract Background: Estrogen such as estradiol (E2) is known to promote ER+ breast cancer. However, several clinical trials reported the unexpected therapeutic benefit of E2 for aromatase inhibitor (AI)-resistant cases of ER+ postmenopausal breast cancer. The objective of this study is to uncover the mechanisms of E2-induced tumor regression, leading to an unconventional treatment of AI resistance. Methods: An E2-suppressive patient-derived xenograft model (named GS3) was established from an AI resistant ER+/PR-/HER2- brain metastatic breast cancer. Placebo or E2 pellets were implanted in mice carrying GS3 for evaluating the effects of E2. Immunohistochemistry (IHC) and RNA sequencing of GS3 were conducted to decipher molecular changes after E2 treatments. Since the cancer tissue has a heterogeneous structure, the single-cell analysis was further performed to examine gene expression profiles in individual cells. In addition, in vitro cell proliferation analysis was carried out using organoids from GS3. Results: E2 inhibited the growth of GS3 both in vivo and in vitro. ERα and ERβ genes in GS3 are wild-type and not amplified. ERα is involved because E2-mediated inhibition of GS3-organoids can be reversed by the co-treatment of ERα antagonist, not by ERβ antagonist. IHC showed that ER, Ki-67 and CEA expressions decreased and PR expression appeared after E2 treatment. Gene set enrichment analysis (GSEA) using RNAseq results showed that the E2 response gene sets were significantly up-regulated after E2 treatment. However, the cell cycle gene sets and the TNFA/NFKB gene set were down-regulated. GS3 gained an E2 independence after three cycles of intermittent E2 treatment (E2 pellet on/off every 4 weeks; Int-E2). Interestingly, the cell cycle and TNFA/NFKB gene sets were up-regulated after Int-E2 treatment. Single-cell RNAseq analysis revealed that cells from one-week E2-treated and Placebo-treated GS3 were placed in different clusters based on principle component analysis of Highly Variable Genes. Although E2 response genes were up-regulated, the percent of ESR1+ cells decreased after E2 treatment (41.3% vs. 31.5%). The number of cells arrested at the G1 phase increased (+12.5%) after E2 treatment. GSEA using genes expressed in only ESR1+ cells showed that the TNFA/NFKB gene set was significantly down-regulated after E2 treatment. Meanwhile, GSEA using genes expressed in only ESR1– cells showed that cell cycle gene sets were significantly down-regulated. Single-cell trajectory analysis disclosed three major branches; 1) common E2 and placebo, 2) E2, and 3) placebo. In the E2 only branch, the cell cycle arrested at the G1 phase, the E2 response gene sets were up-regulated, and the NFKB gene set was down-regulated in ESR1+ cells. Significantly, E2 response gene sets were also up-regulated and cell cycle genes were down-regulated even in ESR1– cells. In the placebo branch, E2 response gene sets were not up-regulated and cell cycle genes were not down regulated. A group of MKI67+ cells (at G2M phase), including some ESR1+ cells, were present in both E2-treated and placebo-treated tumors. Conclusions: E2-induced suppression is an unexpected outcome of AI resistance. In these cases, elimination of estrogen by AI results in maintaining tumor growth. Analysis of GS3 PDX has revealed that estrogen can induce cell cycle arrest and the expression of estrogen-regulated genes. Our results also suggest the cross-talk between ESR1+ and ESR1- cells as well as potential roles of the TNFA/NFKB pathway. Our findings point out the need of markers for such patients who can benefit from E2 treatment after AI resistance, and measurements of ER and PR expression are not sufficient. An intermittent treatment strategy does not sustain the effect of estrogen-mediated suppression. Citation Format: Hitomi Mori, Kohei Saeki, Gregory Chang, Xiwei Wu, Pei-Yin Hsu, Noriko Kanaya, George Somlo, Shiuan Chen. Estrogen-induced cell cycle arrest as an unexpected outcome of aromatase inhibitor-resistance: Insights from single-cell trajectory analysis of a patient-derived xenograft model [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 PD7-02.

Abstract PO034: Long-term follow-up for a prospective phase II trial of levonorgestrel intrauterine device as non-surgical treatment for complex atypical hyperplasia and early endometrial cancer

Abstract Background: The incidence of complex atypical hyperplasia (CAH) and early stage endometrioid endometrial cancer (EEC) is increasing. While standard upfront treatment for CAH and EEC is hysterectomy, non-surgical options are needed for the increasing population unable to undergo hysterectomy due to medical comorbidities precluding surgery or desired future fertility. Our recently published phase II trial of levonorgestrel intrauterine device (LIUD) showed substantial response for patients with CAH or grade 1 (g1) EEC, with 83% overall response rate at 12 months of LIUD treatment. Yet, relapse rates have not been well-defined in a large prospective clinical trial population. Further, we propose that estrogen-regulated gene expression biomarkers in the endometrium could reflect differences in estrogen signaling that drive later outcomes. Methods: This study was conducted under an approved IRB protocol at MD Anderson Cancer Center. Follow-up data for complete responders (those without evidence of hyperplasia or cancer following 12 months of LIUD) were abstracted from the medical record to determine long-term outcomes and relapse status (presence of CAH or grade 1 EEC). Estrogen-regulated gene expression biomarkers (PR, EIG121, IGF1, IGF2, RALDH2, survivin) were measured by real-time PCR from baseline endometrial biopsies and compared by relapse status. Results: Follow-up data were available for 30 of 39 participants that experienced complete response with 12 months of treatment with LIUD (median follow-up was 40 months). Eleven of 30 patients (36.7%) with complete response at the end of the 12-month LIUD treatment went on to experience relapse (6/23 with initial diagnosis of CAH and 5/7 g1 EEC). Median time to relapse after complete response was 14.3 months. Relapse occurred in patients with LIUD still in place (n=6) and LIUD removed (n=5). Of 11 cases with relapse, 5 subsequently responded to LIUD (including 3 cases with LIUD previously removed) and 6 went on to have hysterectomy. Two out of 30 (6.7%) complete responders showed progression of their initial g1 EEC lesions following the study: one patient progressed to grade 2 EEC and one patient progressed to grade 2 mixed EEC/clear cell carcinoma. Ten of 30 later had hysterectomy (equally from CAH and g1 EEC cohorts). Finally, although 13/30 patients indicated desired future fertility at baseline, only 3 attempted to conceive, and 1 achieved pregnancy and live birth. When comparing estrogen-regulated gene expression, participants that exhibited relapse had lower pre-treatment IGF1 than those that did not relapse (p=0.028). However, other biomarkers were not significantly different by relapse status. Conclusions: These findings show that although many patients with CAH or grade 1 EEC are initially responsive to LIUD, the response may not be durable. Additional studies are needed to evaluate biomarkers to predict risk of relapse and identify candidate targets for improved therapeutic strategies. Citation Format: Mikayla Waters, Navdeep Pal, Misty Woodall, Jessica Gallegos, Shannon Westin, Melinda Yates. Long-term follow-up for a prospective phase II trial of levonorgestrel intrauterine device as non-surgical treatment for complex atypical hyperplasia and early endometrial cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO034.

Hormonal modulation of ESR1 mutant metastasis.

Estrogen receptor alpha gene (ESR1) mutations occur frequently in ER-positive metastatic breast cancer (MBC), and confer clinical resistance to aromatase inhibitors (AIs). Expression of the ESR1 Y537S mutation induced an epithelial-mesenchymal transition (EMT) with cells exhibiting enhanced migration and invasion potential in vitro. When small subpopulations of Y537S ESR1 mutant cells were injected along with WT parental cells, tumor growth was enhanced with mutant cells becoming the predominant population in distant metastases. Y537S mutant primary xenograft tumors were resistant to the antiestrogen tamoxifen (Tam) as well as to estrogen withdrawal. Y537S ESR1 mutant primary tumors metastasized efficiently in the absence of estrogen; however, Tam treatment significantly inhibited metastasis to distant sites. We identified a 9 gene expression signature which predicted clinical outcomes of ER-positive breast cancer patients, as well as breast cancer metastasis to the lung. Androgen receptor (AR) protein levels were increased in mutant models, and the AR agonist dihydrotestosterone (DHT) significantly inhibited estrogen-regulated gene expression, EMT, and distant metastasis in vivo, suggesting that AR may play a role in distant metastatic progression of ESR1 mutant tumors.

XBP1 increases transactivation of somatic mutants of ESR1 and loss of XBP1 reverses endocrine resistance conferred by gain-of-function Y537S ESR1 mutation

Somatic mutations of the estrogen receptor 1 gene (ESR1) is an emerging mechanism of acquired resistance to endocrine therapy in hormonal breast cancers. Hotspot point mutations in the ligand- binding domain of estrogen receptor α (ER) and genomic rearrangements producing ESR1 fusion genes are the two major types of mutations that are associated with endocrine resistance. The crosstalk between X-box binding protein 1 (XBP1), a key transcription factor of the unfolded protein response (UPR) and ER signalling creates a positive feedback loop that results in increased expression of XBP1 in ER-positive breast cancer. Here we report that XBP1 co-operated with point mutants (Y537S, D538G) and fusion mutants (ESR1-YAP1, ESR1-DAB2) of ESR1 to increase their transcriptional activity. XBP1 was required for optimal expression of estrogen-regulated genes, and up to 40% of XBP1-dependent genes were estrogen-responsive genes. Knockdown of XBP1 in genome-edited MCF7 cells expressing Y537S mutant reduced their growth, re-sensitized them to anti-estrogens and attenuated the constitutive and estrogen-stimulated expression of estrogen-regulated genes. Our study provides a rationale for overcoming endocrine resistance in breast cancers expressing ESR1 mutation by combining the XBP1 targeting agents with anti-estrogen agents.

OR12-07 Full Antagonism of Breast Cancer Cell Proliferation Can Result from Many Ligand-Induced Conformational Distortions of the Estrogen Receptor Ligand Binding Domain

Although most estrogen receptor alpha (ERα)-positive breast cancers initially respond well to endocrine therapies using aromatase inhibitors (AIs) or antiestrogens, after varying time periods the cancer frequently recurs as metastatic disease. A significant fraction of these recurrences are driven by ERs that have acquired activating mutations in their ligand binding domains (LBDs), giving them constitutive activity and thus resistance to AIs. Because these mutations also reduce the affinity and potency of SERMs and SERDs, expanded efforts have been made to vary the structure of antiestrogens to make them more potent.Typical antiestrogens are comprised of a core element that binds securely in the ligand binding pocket and from which extends a single ring (ring E) having a side chain that sterically interferes with the position of helix-12 by direct antagonism, reorienting it so that it occludes the activation function 2 (AF2) hydrophobic groove for coactivator binding. Through structural studies, we found that bridged oxabicycloheptene-sulfonamide (OBHS-N) core ligands have two rings (E and F) that can be poised to engage in both “direct antagonism” and “indirect antagonism”, the latter of which disrupts the orientation of helix-12 by impinging on helix-11 and the helix-11–12 loop.In this study, we have placed typical antiestrogen side chains on either the E or the F ring of OBHS-N core ligands and characterized their activities in ERα-positive breast cancer cells. All compounds have full antiproliferative activity and reverse estrogen-regulated gene expression, with the antiproliferative potency of each type of side chain having a distinct preference for E- vs F-ring attachment. Conformational analysis using a multiplexed coregulator peptide interaction assay shows that compounds with an E-ring substitution have interaction profiles similar to 4-hydroxytamoxifen and fulvestrant, whereas the F-ring substitution gives a very different pattern, suggesting that the antagonist activity of the two classes rely on different sets of coregulator proteins. A large number of high resolution (better than 2 Å) X-ray crystal structures reveal that this set of novel ER antagonists disrupt the conformation of the ER LBD in a variety of ways, several of which are distinct from those seen with previous antiestrogens such as Tamoxifen and Fulvestrant.Our findings expand design concepts by which ERα ligands can block the activity of this receptor and illustrate how direct and indirect modes of ER antagonism can be combined to facilitate the development of more efficacious antiestrogens for breast cancer treatment and possibly for regulating ER-mediated activities in other estrogen target tissues.

Abstract P6-04-10: Combining direct and indirect modes of antiestrogen ligand antagonism of estrogen receptor activity in breast cancer

Abstract Resistance to endocrine therapies in breast cancer highlights the need to explore new modes for antagonizing the activity of estrogen receptor alpha (ERα) that might lead to clinically more effective antiestrogens. Most clinically approved antiestrogens, however, have conceptually similar overall structures. Typically, this includes a lipophilic ligand core with a single side chain attached to an E-ring that uses direct steric contact to displace helix-12 of the ER ligand binding domain (LBD) from the agonist conformation. Unlike conventional antiestrogens, certain structurally bridged oxabicycloheptene-sulfonamide (OBHS-N) ER ligands have two rings (E and F) onto which side chains can be attached, thereby probing two distinct regions and modes of antagonism within the ER LBD. We attached characteristic ER antagonist side chains onto each of these two rings seeking to displace helix-12 sterically (“direct antagonism”) and/or to distort the ligand binding pocket and thereby alter the conformation of the helix-11/12 loop (a distinct mode, termed “indirect antagonism”). Several of these OBHS-N compounds have full antiproliferative activity on ERα-positive breast cancer cells and reverse estrogen-regulated gene expression, and the inhibitory potency of compounds with each type of side chain shows a distinct preference for E- or F-ring of attachment. Conformational analysis of these ER-ligand complexes using a multiplexed coregulator peptide interaction assay shows that sidechain substitution on the E-ring gives interaction profiles similar to that for hydroxytamoxifen and fulvestrant, whereas F-ring substitution gives a very different pattern. X-ray crystallographic structure analysis confirms that F-ring substituted OBHS-N compounds use a combination of direct and indirect modes of antagonism that both displaces and disorders helix-12 of the ER LBD. Our findings expand design concepts by which ERα ligands can block the activity of this receptor in breast cancers and structurally how direct and indirect modes of inhibition can be combined. This could facilitate the development of more efficacious antiestrogens for breast cancer treatment. Supported by the Breast Cancer Research Foundation (BCRF 18-084 to JAK and BSK) and the National Institutes of Health (R01 CA220284 to JAK, BSK, TI, and KWN; T32 GM070421 Fellowship to VSG). Citation Format: John A Katzenellenbogen, Jian Min, Sathish Srinivasan, Erumbi S. Rangarajan, Jerome C. Nwachukwu, Valeria Sanabria Guillen, Yvonne Ziegler, Shunchao Yan, Kathryn E. Carlson, Yingwei Hou, Sung Hoon Kim, Tina Izard, Rene Houtman, Kendall W. Nettles, Benita S. Katzenellenbogen. Combining direct and indirect modes of antiestrogen ligand antagonism of estrogen receptor activity in 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 P6-04-10.

Red clover and lifestyle changes to contrast menopausal symptoms in premenopausal patients with hormone-sensitive breast cancer receiving tamoxifen.

To determine whether a red clover preparation plus dietary intervention administered to premenopausal women with breast cancer (BC), improves menopausal symptoms due to anti-oestrogen treatment, and hence promotes compliance with tamoxifen, prevents weight gain and is safe. Surgically-treated premenopausal women with oestrogen receptor (ER) positive disease taking tamoxifen were recruited to a prospective double-blind randomized trial (NCT03844685). The red clover group (N = 42) received one oral tablet/day (Promensil® Forte) containing 80mg red clover extract for 24months. The placebo group (N = 39) received one oral tablet/day without active ingredient. All women were encouraged to follow a Mediterranean-type diet and keep active. Outcomes were Menopausal Rating Score (MRS), body mass index (BMI), waist and hip girth, insulin resistance, and levels of cholesterol, triglycerides, and sex hormones. As safety indicators, endometrial thickness, breast density, and effects of patient serum on ER-positive BC cell lines were investigated. MRS reduced significantly (p < 0.0001) with no between-group difference (p = 0.69). The red clover group had significantly greater reductions in BMI and waist circumference (p < 0.0001 both cases). HDL cholesterol increased significantly in both groups (p = 0.01). Hormone levels and insulin resistance changed little. Endometrial thickness remained constant (p = 0.93). Breast density decreased significantly in both groups (p < 0.0001). Proliferation and oestrogen-regulated gene expression didn't differ in cell lines treated with serum from each group. This is the first trial to assess red clover in BC patients on tamoxifen. The preparation proved safe clinically and in vitro, and was associated with reduced BMI and waist circumference, but the diet-lifestyle intervention probably improved the menopausal symptoms.

Abstract P4-15-01: Acquired ESR1 mutation and persistent expression of estrogen regulated genes in ER+ breast cancers on long-term neoadjuvant treatment with aromatase inhibitors

Abstract Background: Aromatase inhibitors (AI), which block the conversion of androgen to estrogen, are the mainstay therapy for estrogen receptor–positive (ER+) breast cancer (BC). However, many patients relapse. Purpose: To identify molecular alterations associated with long-term neoadjuvant AI therapy of ER+ BC. Methods: We retrospectively identified 137 patients diagnosed with ER+ BC and treated with neoadjuvant AI for at least 1 month at the Royal Marsden Hospital (2003–2016). Paired pre- (diagnosis) and post-AI (surgery) biopsies with &amp;gt;40% invasive cell areas were available from 87 ER+ BC patients. In all samples, we evaluated ER, PR and Ki67 immunostaining, ESR1 hot-spot mutations by droplet digital PCR and expression of 801 genes associated with BC and response to endocrine therapy by NanoString. Results: Mean time on neoadjuvant treatment was 26 weeks (range 5.6–92.3). Cell proliferation remained suppressed in most tumours indicating little evidence for acquired resistance: 56/87 (64%) showed residual Ki67 (Ki67r) &amp;lt;2.7% (complete cell cycle arrest) and 14/87 (11.5%) had Ki67r&amp;gt;10%. This was paralleled by reduced expression of proliferation genes, ER (immunostaining and gene expression) and estrogen-regulated genes (ERG) at surgery compared with the diagnostic samples (all p&amp;lt;0.001). There was a weak positive correlation between AI duration and Ki67r and less reduction in proliferation genes, ESR1 and ERGs expression (p&amp;lt;0.05; r=0.26-0.34). Pathway analysis revealed inhibition of cell cycle (e.g. reduced expression of several cyclins), E2F targets and estrogen response (e.g. reduced expression of ER downstream targets such as FOXM1). CDK genes showed a variable response: CDK1 and CDK2 decreasing, CDK4 increasing and no change in CDK6. Most notably, 6 surgical samples showed ESR1 mutations: one of these cases had the mutation at diagnosis. All 5 acquired mutations were detected in patients treated for &amp;gt;6 months, giving a prevalence in this cohort of 5/34 (15%). Tumours with ESR1 mutations showed less suppression of ERGs (p=0.002) and proliferation (p=0.039) and increased ESR1 (p=0.016) expression at surgery compared with tumours without mutation. Pathway analysis confirmed lack of inhibition of estrogen response (FDR&amp;gt;5%) and less inhibition of cell cycle [enrichment score: -0.49 vs -0.78) and E2F targets (-0.47 vs -0.8) in tumours with mutation. Tumours without ESR1 mutation but with Ki67r&amp;gt;10% also showed reduced ERGs response (p=0.006) compared to tumours with Ki67r&amp;lt;2.7%. Additionally, these tumours showed relative activation of cell cycle, estrogen response, E2F targets pathways and mTORC1 signalling (FDR&amp;lt;1%). Conclusion: Overall most tumours showed no evidence for the emergence of resistant disease after neoadjuvant AI therapy even after many months of treatment. However, we detected an enrichment of ESR1 mutations (15% of cases) after long-term treatment as a putative driver of ERG expression and proliferation and thus reduced AI response. Therefore, mutant ER appears to be associated with ligand-independent ERG activity supporting the clinical validity of dual blockade with a selective ER down-regulator combined with a CDK4/6 inhibitor targeting the RB/E2F axis in this scenario. Citation Format: Leal MF, Haynes BP, Schuster E, Yeo B, Afentakis M, Dodson A, Buus R, Cheang MC, Martin L-A, Dowsett M. Acquired ESR1 mutation and persistent expression of estrogen regulated genes in ER+ breast cancers on long-term neoadjuvant treatment with aromatase inhibitors [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-15-01.

Effects of serum estradiol and progesterone on estrogen-regulated gene expression in breast cancers of premenopausal patients.

Expression of estrogen receptor α in breast cancer is essential for estrogen-dependent growth and partially determines the breast cancer subtype. In premenopausal women, expression of estrogen-regulated genes in estrogen receptor-positive breast cancer tissues are reportedly influenced by the menstrual cycle. We investigated correlations between serum estradiol (E2; tested on the day of surgery) and expression of estrogen-regulated genes and proliferation genes in strongly estrogen receptor α-positive breast cancer tissues from 91 premenopausal women by quantitative reverse transcription-polymerase chain reaction. We also investigated correlations between serum progesterone levels on the day of surgery and mRNA expression of progesterone-regulated genes and proliferation genes. The serum E2 level affected expression of estrogen-regulated genes, including progesterone receptor (P = 0.016, Rs = 0.07) but showed no correlation with expression of genes associated with proliferation. We also observed strong positive correlations between mRNA expression of ESR1 and that of estrogen-regulated genes (P < 0.0001, Rs = 0.329-0.756) and proliferation genes (P < 0.0001, Rs = 0.753-0.843). The serum progesterone level affected expression of RANKL mRNA. However, we observed no correlations between serum progesterone and expression of Wnt-4 or proliferation genes. The serum E2 level on the day of surgery influences estrogen-regulated gene expression moderately in patients found to be strongly positive for estrogen receptor α by immunohistochemistry. Changes in serum E2 levels might influence the results of molecular profiling tests in premenopausal women with breast cancer.

Effects of puerarin on estrogen-regulated gene expression in gonadotropin-releasing hormone pulse generator of ovariectomized rats

Effects of puerarin on the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator function is investigated, for the first time, in ovariectomized rats at the level of mRNA expression of estrogen-responsive genes, e.g., estrogen receptor (ER), GnRH and its receptor (GnRHR). Rats were treated orally for 90 days either with a soy-free diet containing two different doses of puerarin (low dose of 600 mg/kg and high dose of 3000 mg/kg) or estradiol benzoate (E2B) at either low dose (4.3 mg/kg) or high dose (17.3 mg/kg). Levels of mRNA expression in the medial preoptic area/anterior hypothalamus (MPOA/AH), mediobasal hypothalamus/median eminence (MBH/ME) and adenohypophysis were measured by quantitative TaqMan® real-time RT-PCR. Plasma levels of luteinizing hormone (LH) and prolactin (PRL) were measured by radioimmunoassay. In the MPOA/AH, both puerarin and E2B decreased ERα mRNA levels without any significant changes in ERβ and GnRH mRNA levels. Both puerarin and E2B did not significantly alter the expression levels of ERα, ERβ and GnRHR in the MBH/ME. E2B exerted significant effects on the down-regulation of adenohypophyseal GnRHR mRNA transcripts and serum LH levels. Puerarin did not cause significant changes in pituitary GnRHR mRNA transcripts and serum LH and PRL levels. This is the first study to demonstrate that in ovariectomized rat models of ovarian hormone deprivation, puerarin acted as a weak estrogen-active compound in the hypothalamic GnRH pulse generator through the downregulation of MPOA/AH ERα mRNA expression.

Abstract P1-02-03: The Y537S ESR1 mutation carries unique metabolomics profiling in breast cancer

Abstract Background: Estrogen receptor (ESR1) mutations occur at a high frequency in metastatic breast tumors in patients treated with hormonal therapy in the metastatic setting. We do not know if these mutations changed metabolomics and whether these metabolomics change could affect metastasis. Experimental design and methods: We generated ESR1 Y537S homozygous mutations using CRISPR Casp-9 technology. Globe metabolites screening was performed using 6550 Agilent QTOF instrument. Athymic mice were used in tumor xenograft studies. Affymetrix microarrays were performed to compare gene expression changes in Y537S mutant compared with parental cells. Enriched metabolite pathways and gene expression integrated analysis was analyzed by using online analysis tool http://www.metaboanalyst.ca. Results: We generated CRISPR ESR1 Y537S mutation homozygous knock-in clones in MCF-7 cells. In vivo experiments revealed that mutant cells are dominant drivers of metastasis. Transcriptome profiling revealed elevated expression of Hallmark pathways, including EMT and estrogen-regulated gene expression. We performed globe metabolites screening using MCF-7 Y537S and MCF-7 parental and identified 134 metabolites. Serum starvation media was used and estrogen was used as control for both cell lines. As we expected estrogen treatment induced metabolites changes in parental cells. However, metabolites in mutant cells were not changed significantly under estrogen treatment. Interestingly, metabolites in the mutant cells at baseline were remarkably upregulated (78 out of 134 identified total metabolites) indicating mutant cells in serum starvation condition had significantly different metabolomics compared with parental cells. Top upregulated pathways include protein biosynthesis, betaine metabolism and ammonia recycling. Integration of microarray gene expression and metabolites reviewed several metabolomics pathways significantly changed in mutant compared with parental cells including for example aminoacyl-tRNA biosynthesis, arginine and proline metabolism and alanine, aspartate and glutamate metabolism. Conclusion: The Y537S ER mutation is a driver of distant metastasis in ER-positive breast cancer cells. Y537S ER mutant had globe changes of metabolites expression which was confirmed by integrated analysis combining microarray gene expression. The roles of these metabolites need to be studied to correlate with metastasis. Enzymes responsible for converting these metabolites changes could be served as potential therapeutic targets. Citation Format: Gu G, Piyarathna B, Coarfa C, Ellis L, Ando' S, Fuqua S. The Y537S ESR1 mutation carries unique metabolomics profiling in breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-02-03.

Methylmercury promotes breast cancer cell proliferation.

Metalloestrogens are small ionic metals that activate the estrogen receptor (ER). Studies have shown that when metalloestrogens bind to the ER, there is an increase in transcription and expression of estrogen-regulated genes, which induces proliferation of estrogen-dependent breast cancer. Methylmercury (MeHg), a metalloestrogen, is present in the environment and is toxic at moderate to high concentrations. However, at lower concentrations MeHg may promote the proliferation of ER-positive breast cancers and protect cells against pro-apoptotic signals. To investigate the effects of MeHg treatment on breast cancer cells in vitro. MCF7 breast cancer cells were treated with concentrations of MeHg ranging from 1 nM to 100 mM. Hg analysis was used to quantify intracellular mercury concentrations. Cell proliferation and apoptosis were determined by cell counting and Annexin-V staining, respectively. We defined a protocol that maximizes cellular exposure to mercury. Treatment of human ER-positive breast cancer cells with 1 nM MeHg promoted proliferation, while treatment with a concentration of 100 nM induced apoptosis. Clarifying the effects of MeHg on breast cancer will improve our understanding of how environmental toxins affect tumor progression and may lead to the development of future therapeutic strategies.