Estrogen Receptor Gene Research Articles

Molecular expression and identification of caprine estrogen receptor gene 1 for fertility status in bucks

Estrogen and its receptors are essential for sexual development and reproduction. Oestrogen receptor alpha (ERα) is a nuclear receptor activated by the hormone oestrogen. In male, ERα is encoded by the gene ESR1 (oestrogen receptor1) responsible for better fertility. ESR1 is involved in the reabsorption of luminal fluid during the transit of spermatozoa from the testis to the head of the epididymis which is important for their survival and maturation during epididymal storage. The absence of ESR1 leads to reduced epididymal sperm content, reduced sperm motility and fertilizing ability. The present study was undertaken to investigate the expression and presence of ESR1 gene in fertile and low-fertile male goat breeds. We identified ESR1 gene through various molecular tools. Genotyping was carried out by high resonance melting analysis using Roche Light Cycler 480(LC-480) system and found three different genotypes. Genotypic frequency-AA (blue-0.67), BB(Red-0.2), AB(Green-0.08) with allele frequency A(0.71 and B (0.29). The predominance of this gene in head of epididymis in fertile bucks was confirmed by SDS-PAGE, Western blotting and immunohistochemistry. From the results, we corroborated that the present study provides a useful and effective way to predict male fertility in goat breeds, which in turn increases the percentage of fertility in flock leading to more number of offspring in a kidding season.

Sleep and Methylation of Estrogen Receptor Genes, ESR1 and GPER, in Healthy Middle-Aged and Older Women: Findings from the Women 40+ Healthy Aging Study.

PurposeSleep problems in middle-aged and older women are very common and have been associated with menopause-related changes in estrogen levels. However, not all women experience sleep problems as they enter perimenopause, and epigenetic mechanisms might contribute to the differences in sleep quality within this population. In this study, we hypothesized that increased methylation of two estrogen receptor (ER) genes (ESR1 and GPER) would be associated with increased sleep problems in healthy pre-, peri-, and postmenopausal women, either directly or indirectly through the experience of vasomotor symptoms (VMS).Materials and MethodsIn 130 healthy women aged 40–73 years, we assessed DNA methylation from dried blood spots (DBS). Women rated their sleep quality using the Pittsburgh Sleep Quality Index (PSQI), and VMS using the Menopause Rating Scale (MRS).ResultsHigher percentage methylation of ESR1 was associated with increased sleep problems, mediated by VMS, even after controlling for age, menopausal status, body mass index, estradiol levels, depressive symptoms, and caffeine consumption. There was no significant association between GPER methylation and either sleep problems or VMS.ConclusionThe study findings support an association between increased ESR1 methylation and sleep problems through increased VMS among healthy women aged 40–73 years.

Abstract A19: Detection of ESR1 mutations in plasma cell-free DNA from metastatic ER-positive breast cancer patients resistant to hormone therapy

Abstract About 70% of breast cancers (BC) express estrogen receptor α (ER) and most of these are sensitive to endocrine therapies (ET). However, a significant fraction of patients with ER-positive (ER+) BC who benefit from adjuvant ET recur with metastatic disease after prolonged exposure to these therapies. Even patients with ER+ metastatic BC who respond to ET will eventually progress with antiestrogen-resistant hormone-independent disease. Activating mutations in the estrogen receptor gene ESR1 have been described in advanced ER+ BC resistant to ET, particularly in metastatic patients exposed to therapy with aromatase inhibitors (AI). ESR1 mutations frequently occur in a hotspot region within the ligand-binding domain of ER, constitutively activating the receptor in a ligand-independent manner and thus explaining the acquired resistance to hormonal therapy. Recent studies have reported the detection of ESR1 mutations in codons 536, 537, and 538 in circulating tumor DNA (ctDNA) of metastatic BC patients, ensuring the establishment of a causal relationship among ESR1 mutations and acquired resistance to AI therapy. Thereby we evaluated the presence of ESR1 mutations in plasma cell-free DNA (cfDNA) of metastatic ER-positive BC patients who progressed on AI therapy. Among patients with no ESR1 mutations detected, we questioned the presence of any other mutated gene that could be related to acquired resistance to ET. To answer these questions, we first investigated the presence of ESR1 recurrent mutations (L536R, Y537C/N/S, D538G) by Droplet Digital PCR (ddPCR) in ctDNA of 24 ER+ metastatic BC patients after acquired resistance to therapy with AI. ESR1 mutations were detected in 25% (6/24) of the patients, with ESR1 D538G and Y537N being the most frequent, and four patients showed polyclonal disease with more than one ESR1 mutation. The longest interval between the end of aromatase inhibitor treatment and the detection of ESR1 mutations was 2.3 years, corroborating with some studies that showed that these mutations remain readily detectable in ctDNA many months and years after stopping therapy. cfDNA from patients with no ESR1 mutations detected were analyzed by targeted cfDNA sequencing (cfDNA seq) of a 151-cancer gene panel. We were able to detect mutations in genes already known to be associated to ET resistance such as PIK3CA and AKT. We found one patient with AKT E17K mutation and four patients (4/12, 33%) with detectable PIK3CA-mutated ctDNA, with three patients with E545K mutation and one with H1047L mutation. Mutations in PIK3CA are frequently observed in BC and have been suggested as a predictive biomarker for PI3K-selective inhibitor treatment, alpelisib. Based in our results, we can conclude that detection of tumor genetic alterations in plasma cfDNA may have important clinical implications for personalized medicine, including monitoring of recurrence and therapy resistance and therapeutic guidance. Citation Format: Cibele Masotti, Franciele H. Knebel, Rudinei D. M. Linck, Julianne Lima, Andrea K. Shimada, João Victor Alessi, Bruna M. Zucchetti, Dimitrios Kleftogiannis, Louise Barber, Marco Gerlinger, Artur Katz, Max S. Mano, Anamaria A. Camargo, Fabiana Bettoni. Detection of ESR1 mutations in plasma cell-free DNA from metastatic ER-positive breast cancer patients resistant to hormone therapy [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A19.

Endocrine biomarkers in low-grade serous ovarian cancers (LGSC) and serous ovarian tumors of low malignant potential (LMP).

e18045 Background: LGSC and serous ovarian LMP tumors are rare entities along a disease continuum. Because they are relatively chemotherapy resistant, there is interest in targeted treatment, including endocrine therapy. In studies of endocrine agents for treatment of recurrent ovarian cancer, tumor estrogen receptor (ER) status alone does not effectively predict outcomes. The objective of this study is to utilize protein and gene expression from ER and downstream targets to identify signaling components that will act as prognostic and predictive biomarkers. Methods: A single institution tumor registry was queried for patients with LGSC or serous LMP tumors between 1993-2016. Formalin-fixed, paraffin embedded (FFPE) tissue was obtained. Clinicopathologic data was collected. Immunohistochemistry (IHC) was performed and scored as dichotomous (+/-) and continuous (H-score). NanoString nCounter platform was used for gene expression of a predesigned endocrine biomarker panel. Wilcoxon Rank Sum was used to compare mRNA and protein expression. Survival analyses were performed using Cox proportional hazard (PH) method with elastic net to fit the prediction model for the gene panel. Results: Tissue was analyzed from 23 LGSC, 71 serous LMP tumors, and 2 recurrences. At diagnosis, 86% of patients with LMP tumors had stage I disease vs 78% of patients with LGSC had stage III/IV disease. There were 7 (10%) LMP and 13 (56.5%) LGSC recurrences. Median initial PFS was 76 and 37 months and median OS was 88 and 82 months for LMP and LGSC, respectively. Fourteen received endocrine therapy; nine for treatment of recurrent LGSC. Median therapy was 21 months, with 3 complete responses, 2 partial responses and 1 stable disease. All tumors were ER+ (median H-score 210) and 89% were PR+ (median H-score 130). ER H-scores and ESR1 expression levels were not significantly different between LGSC and LMP tumors (p = 0.1942, p = 0.0893). PR H-scores were higher in LMP tumors (p < 0.00001) but PGR expression was not significantly different (p = 0.6581). In the Cox PH analysis, ER H-score was a significant predictor of both PFS and OS (p < 0.001, p = 0.004). The Cox model identified independent sets of gene expression associated with PFS and OS in both the LGSC and LMP cohorts (c-indices 0.679, 0.570, 0.626, 0.689). Conclusions: Endocrine therapy is an active area of interest in the treatment of ovarian neoplasms. ER H-score and gene expression of downstream ER targets could improve upon the use of hormone receptors to determine risk of recurrence and guide use of adjuvant endocrine therapy.

X-ray crystal structure analysis of elacestrant (RAD1901), a novel selective estrogen receptor degrader (SERD), bound to estrogen receptor alpha ligand binding domain.

e15647 Background: Antiestrogens are a mainstay of treatment for estrogen receptor positive (ER+) breast cancer in both the adjuvant and the advanced/metastatic settings. Elacestrant is a mixed activity selective estrogen receptor (SER) alpha (ERα) antagonist, acting as a SER modulator (SERM) at low doses and a SER degrader (SERD) at high doses. It has shown activity in hormone sensitive wild type (WT) ERα and insensitive estrogen receptor gene 1 (ESR1) mutation-harboring (Y537S and D538G) ERα breast cancer, both in preclinical models and in clinical studies. It also possesses a unique pharmacology compared to other competitive ER antagonists in its ability to cross the blood brain barrier. Competitive ERα antagonists are typically comprised of a core that sits in the ligand binding pocket and an arm that manipulates the structure to achieve SERM or SERD activities. In these molecules, the arm is attached in the same position as the triphenylethylene core of tamoxifen. However, elacestrant possesses a novel site of attachment. As such, we hypothesized that elacestrant adopts an alternative binding orientation in the ERα ligand binding pocket to achieve its unique pharmaceutical profiles. Methods: X-ray crystallography was used to solve a co-crystal structure of elacestrant in complex with WT ERα ligand binding domain to 2Å. Results: Overall, elacestrant promotes the formation of a canonical ERα ligand binding domain antagonist conformation, whereby helix 12 (H12) is docked into the activating function-2 cleft. However, elacestrant adopts a novel vector in the ERα ligand binding pocket that places it in close proximity to helix 12. As a result, it forms a bifurcated hydrogen bond that is not observed in other competitive antiestrogens and samples a chemical space known to increase H12 mobility and induce SERD activity. This novel vector also places it near positions 537 and 538, the two most common sites of somatic mutation. Conclusions: The high-resolution x-ray crystal structure of elacestrant suggests that the unique binding mode it adopts enables novel pharmacology and positions it to achieve potency in the WT and activating somatic ERα mutated breast cancer setting.

In vitro-in silico-based probabilistic risk assessment of combined exposure to bisphenol A and its analogues by integrating ToxCast high-throughput in vitro assays with in vitro to in vivo extrapolation (IVIVE) via physiologically based pharmacokinetic (PBPK) modeling

Combined risk assessment of endocrine effects of bisphenol A (BPA) and its analogues, such as bisphenols S, F, and AF (BPS, BPF, and BPAF), is challenging due to lack of related common toxicity metrics. This study conducted a population-based in vitro-to-in vivo extrapolation using physiologically based pharmacokinetic (PBPK) models coupled with Monte Carlo simulations to convert ToxCast in vitro estrogen receptor (ER) assays to human equivalent doses (HEDs). The ER pathway-based HEDs were compared with HEDs from animal studies and used to assess the combined risks for different populations across different countries/regions in a probabilistic manner. The estimated ER pathway-based HEDs for the four bisphenols (BPs) matched the animal-derived HEDs. The HEDs for the ER gene transcription (the common biological process target among BPs) were 0.40 (2.5th–97.5th percentiles: 0.06–5.42), 4.43 (0.69–53.84), 3.30 (0.51–626.57), and 1.12 (0.16–9.73) mg/kg/day for BPA, BPS, BPF, and BPAF, respectively. Results suggest a potentially moderate concern for combined risks of activating the ER pathway for toddlers and adults with high dietary exposures. This study presents in vitro-based credible HEDs for the four BPs and represents an advancement in the application of in vitro–in silico-based alternative approaches in human health risk assessment.

Coumarin-Fatty Acid Conjugates as Potential ERα/AKT-1 Antagonists for ER Positive Breast Cancer.

Current drugs used for the treatment of hormone-dependent breast cancer function as anti-estrogens in the breast, in addition to Estrogen Receptor (ER) agonists in the uterus, thus elevate a woman's risk of developing uterine cancer. This is due to the lack of selective binding and partial agonistic effect of these drugs towards estrogen receptors. In recent years, therefore, researchers have turned their attention towards antiestrogens devoid of these agonist properties and thus have a mechanism of action different from the existing drugs. In this context, we report here the design, development and in vitro evaluation of some novel pharmacophores containing coumarin and fatty acid scaffolds for their anti-breast cancer activity. A library of coumarin-fatty acid conjugates was designed using structure-based drug design approach. The conjugates which have shown good in silico results were then synthesized, characterized and evaluated for their anti-breast cancer activity by MTT assay, Apoptotic assay, Cell proliferation assay, Estrogen binding assay and Gene expression study. Out of the fifteen compounds screened, two compounds, SAC-2 and LNAC-2, showed good activity with IC50 values 22µg/ml, 25μg/ml, respectively. These compounds suppressed the proliferation of ER overexpressed MCF-7 cells, increased ERα degradation and hence inactivate the ERα pathway. ER binding assay and gene expression RT-PCR study reveal that SAC-2 downregulated the expression of ERα receptor and AKT-1 gene. Compound SAC-2 is a good antagonist to ER and hence has a potential for treating breast cancer and other cancers where AKT plays an important role.

The antioxidant icariin protects porcine oocytes from age-related damage in vitro

ObjectiveIf fertilization does not occur within a specific period, the quality of unfertilized oocytes in the oviduct (in vivo aging) or in culture (in vitro aging) will deteriorate over time. Icariin (ICA), found in all species of Epimedium herbs, has strong antioxidant activity, and is thought to exert anti-aging effects in vitro. We asked whether ICA protects oocytes against age-related changes in vitro.MethodsWe analyzed the reactive oxygen species (ROS) levels and expression of antioxidant, maternal, and estrogen receptor genes, and along with spindle morphology, and the developmental competence and quality of embryos in the presence and absence of ICA.ResultsTreatment with 5 μM ICA (ICA-5) led to a significant reduction in ROS activity, but increased mRNA expression of glutathione and antioxidant genes (superoxide dismutase 1 [SOD1], SOD2, peroxiredoxin 5, and nuclear factor erythroid 2-like 2), during aging in vitro. In addition, ICA-5 prevented defects in spindle formation and chromosomal alignment, and increased mRNA expression of cytoplasmic maturation factor genes (bone morphogenetic protein 15, cyclin B1, MOS proto-oncogene, serine/threonine kinase, and growth differentiation factor-9). It also prevented apoptosis, increased mRNA expression of anti-apoptotic genes (BCL2-like 1 and baculoviral IAP repeat-containing 5), and reduced mRNA expression of pro-apoptotic genes (BCL2 antagonist/killer 1 and activation of caspase-3). Although the maturation and cleavage rates were similar in all groups, the total cell number per blastocyst and the percentage of apoptotic cells at the blastocyst stage were higher and lower, respectively, in the control and ICA-5 groups than in the aging group.ConclusionICA protects oocytes against damage during aging in vitro; therefore, it can be used to improve assisted reproductive technologies.

Prevention and therapy of COVID-19 via exogenous estrogen treatment for both male and female patients.

The presented work summarizes the results of studies underlining the crucial role of estrogen receptor (ER) signaling in both innate and adaptive immune responses as well as in tissue repairing processes during respiratory virus infection. Experimental studies justify that among respiratory virus infected mice, a weaker ER signaling leads to increased morbidity and mortality in both males and females. In animal experiments, estrogen treatment silences the inflammatory reactions and decreases virus titers leading to improved survival rate; it seems to be an ideal prevention and therapy against COVID-19. We should overcome the widespread reluctance to estrogen therapy as we have a unique estrogen formula; conjugated estrogens, or conjugated equine estrogens available under the brand name of Premarin deriving from natural sources. Premarin can exert similar ER upregulative and gene repairing power like endogenous estrogen without any risk for adverse reactions. Premarin is capable of stopping the COVID-19 pandemic.

Single-Cell Gene Profiling Reveals Social Status-Dependent Modulation of Nuclear Hormone Receptors in GnRH Neurons in a Male Cichlid Fish.

Gonadotropin-releasing hormone (GnRH) is essential for the initiation and maintenance of reproductive functions in vertebrates. To date, three distinct paralogue lineages, GnRH1, GnRH2, and GnRH3, have been identified with different functions and regulatory mechanisms. Among them, hypothalamic GnRH1 neurons are classically known as the hypophysiotropic form that is regulated by estrogen feedback. However, the mechanism of action underlying the estrogen-dependent regulation of GnRH1 has been debated, mainly due to the coexpression of low levels of estrogen receptor (ER) genes. In addition, the role of sex steroids in the modulation of GnRH2 and GnRH3 neurons has not been fully elucidated. Using single-cell real-time PCR, we revealed the expression of genes for estrogen, androgen, glucocorticoid, thyroid, and xenobiotic receptors in GnRH1, GnRH2, and GnRH3 neurons in the male Nile tilapia Oreochromis niloticus. We further quantified expression levels of estrogen receptor genes (ERα, ERβ, and ERγ) in three GnRH neuron types in male tilapia of two different social statuses (dominant and subordinate) at the single cell level. In dominant males, GnRH1 mRNA levels were positively proportional to ERγ mRNA levels, while in subordinate males, GnRH2 mRNA levels were positively proportional to ERβ mRNA levels. These results indicate that variations in the expression of nuclear receptors (and possibly steroid sensitivities) among individual GnRH cells may facilitate different physiological processes, such as the promotion of reproductive activities through GnRH1 neurons, and the inhibition of feeding and sexual behaviors through GnRH2 neurons.

Glutathione peroxidase‐1 regulates adhesion and metastasis of triple‐negative breast cancer cells via FAK signaling

Triple‐negative breast cancer (TNBC) cells, which do not express genes for estrogen receptor (ER), progesterone receptor (PR), and Her2/neu, develop highly aggressive and metastatic tumors resistant to chemo‐ and hormonal therapies. We found that expression of glutathione peroxidase‐1 (Gpx1) is silenced in the non‐TNBC cells but resumed in the TNBC cell lines. Such restored Gpx1 expression plays a vital role in the metastasis of TNBC cells by regulating cell adhesion. Transcriptomic and signaling pathway analyses demonstrate that depletion of Gpx1 essentially impairs cell adhesion/spreading by down‐regulating FAK/c‐Src activation. Mechanistically, the substrate of Gpx1, H2O2, directly inhibits the FAK kinase activity. As a result, depletion of Gpx1 greatly suppresses lung metastasis of TNBC cells in vivo. Overall, our study identifies that Gpx1 is a redox safeguard of FAK kinase and allows for a therapeutic process to effectively control the metastasis of deadly malignant TNBC.Support or Funding InformationNational Research Foundation of Korea (2018R1A2B3006323 and 2017M3A9B6073098)National R&D Program for Cancer Control (1420280).

Вовлеченность полиморфизма генов рецепторов эстрогенов и прогестерона в развитие миомы матки

Вовлеченность полиморфизма генов рецепторов эстрогенов и прогестерона в развитие миомы матки

Estrogen Receptors and Ubiquitin Proteasome System: Mutual Regulation.

This review provides information on the structure of estrogen receptors (ERs), their localization and functions in mammalian cells. Additionally, the structure of proteasomes and mechanisms of protein ubiquitination and cleavage are described. According to the modern concept, the ubiquitin proteasome system (UPS) is involved in the regulation of the activity of ERs in several ways. First, UPS performs the ubiquitination of ERs with a change in their functional activity. Second, UPS degrades ERs and their transcriptional regulators. Third, UPS affects the expression of ER genes. In addition, the opportunity of the regulation of proteasome functioning by ERs—in particular, the expression of immune proteasomes—is discussed. Understanding the complex mechanisms underlying the regulation of ERs and proteasomes has great prospects for the development of new therapeutic agents that can make a significant contribution to the treatment of diseases associated with the impaired function of these biomolecules.

Evaluating the Association Between Dental Fluorosis and Polymorphisms in Bone Development and Mineralization Genes Among Population from a Fluoride Endemic Region of Eastern India.

Close to 12 million people in India are affected by more than the desirable level of fluoride in drinking water that could lead to dental, skeletal, and non-skeletal fluorosis. Dental fluorosis is a developmental defect that results in hypo-mineralization and pronounced porosity of enamel in the affected individuals. As estrogen receptor 1 (ESR1), collagen type 1 alpha 2 (COL1A2), bone γ-carboxyglutamic acid protein (BGLAP), and secreted protein acidic and cysteine rich (SPARC) genes are involved in bone development and mineralization, polymorphisms in these genes could be determining factors in influencing the risk to fluorosis among the exposed individuals in fluoride endemic areas. A case-control study was carried out among a total of 87 individuals (case = 36, control = 51) to examine the association between selected polymorphisms in the ESR1, COL1A2, BGLAP, and SPARC genes and risk of dental fluorosis from a fluoride endemic region of Eastern India. Altogether, 10 single nucleotide polymorphisms (SNPs) in ESR1 (rs2234693, rs2228480, rs3798577, rs2077647, and rs9340799), COL1A2 (rs42524, rs412777), BGLAP (rs1800247), and SPARC (rs6579885, rs4958278) genes were genotyped through PCR-RFLP in these subjects. The association of the SNPs for disease risk estimation was measured by odds ratio with 95% confidence interval. The risk genotypes of none of the 10 SNPs showed statistically significant association with risk of dental fluorosis. Frequencies of the haplotypes in the intragenic SNPs of the ESR1, COL1A2, and SPARC genes did not reveal any statistically significant difference between the case and control groups. The present study is the first of its kind from India that has attempted to investigate possible involvement of genetic factors in influencing the risk to fluorosis among the population from a fluoride endemic region.

Association of estrogen receptor gene variants (ESR1 and ESR2) with polycystic ovary syndrome in Tunisia.

SNV (single nucleotide variation) in estrogen receptor (ESR1 and ESR2) genes are susceptibility markers for complex diseases, such as cancer, metabolic disorders and women infertility. We explored six widely used SNVs in ESR1 (rs2234693, rs9340799, rs3798577, rs3020314) and ESR2 (rs1256049, rs4986938) in polycystic ovary syndrome (PCOS) in women from Tunisia (n=254) compared to controls (n=170). Genotyping was performed by RFLP-PCR or real-time PCR and analyzed in GoldenHelix statistical package. Logistic regression revealed association of rs2234693, rs3798577 and rs3020314 (ESR1) and rs1256049 (ESR2), the association of rs2234693 (C/T) being the strongest with P<4.81×10-6, 2.88×10-5 after Bonferroni correction, OR 0.31, 95%CI (0.18-0.53)). Correlations were found with LH, LH/FSH or hyperandrogenism and even more significant with metabolic syndrome (rs9340799) and hyperglycemia (rs3798577). Among 14 haplotypes reconstructed in ESR1gene, four haplotypes (H1 to H4) were associated with PCOS the strongest being that of H1 (P<0.002) supported by Bonferroni (P<0.033) and permutation tests (P<4 x10-4). In haplotype trend regression, concordant correlations were found with insulin resistance (P<0.033) for H2 and with high blood pressure for H3 (P<0.048). While these data revealed influential role on metabolic rather and hormonal features of PCOS, the association of rs2234693 was the strongest among all ethnic populations studied thus far giving a new insight on estrogen receptor gene variation in distant North African populations and their role in metabolic alteration of PCOS.

A modern look at the molecular-biological mechanisms of breast tumours in dogs

High morbidity and increase in the number of registrations of breast tumours in dogs, their wide application as biological models, discussion of numerous questions of oncogenesis, and the lack of a uniform/unified methodological approach to the study of molecular and biological mechanisms of treatment of cancer determine the relevance of the problem of cancer both in humans and in our domestic companions. The analysis of publications allowed us to establish the following patterns of carcinogenesis. The peculiarities of the biological behaviour of breast tumours depend on their pathomorphological structure. Genetic predisposition to breast cancer is characteristic only in the single breed aspect. Environmental factors are of critical relevance to carcinogenesis : chemical pollutants initiate oncogenesis indirectly – by altering the expression of several receptors, impaired endocrine balance and direct mutagenic effects. Reproductive status plays a key role in the initiation and progression of breast tumours by reducing the expression of estrogen, progesterone and prolactin receptor genes. The inflammatory response that accompanies the neoplasia process is characterized by increased production of cytokines, cyclooxygenase-2, interleukins (IL-1, IL-6, IL-8), vascular endothelial growth receptors, and impaired hemostatic status (oxidative stress), which promotes progression of disease. In breast cancer in dogs, genomic instability leads to genomic aberrations, and subsequently, mutations that support the proliferation, survival and dissemination of neoplastic cells. The initiation and progression of mammary gland tumours is provided by cancer stem cells by disrupting the regulation of precursor cell self-renewal, which also predispose to resistance to chemotherapeutic agents, radiation, and hormonal cancer therapy. The analysis of the publications revealed the major markers of carcinogenesis that could potentially be used as biological targets for the design of modern diagnostic strategies and high-performance therapeutic protocols.

Inter-Individual Variation in Response to Estrogen in Human Breast Explants.

Exposure to estrogen is strongly associated with increased breast cancer risk. While all women are exposed to estrogen, only 12% are expected to develop breast cancer during their lifetime. These women may be more sensitive to estrogen, as rodent models have demonstrated variability in estrogen sensitivity. Our objective was to determine individual variation in expression of estrogen receptor (ER) and estrogen-induced responses in the normal human breast. Human breast tissue from female donors undergoing reduction mammoplasty surgery were collected for microarray analysis of ER expression. To examine estrogen-induced responses, breast tissue from 23 female donors were cultured ex- vivo in basal or 10nM 17β-estradiol (E2) media for 4days. Expression of ER genes (ESR1 and ESR2) increased significantly with age. E2 induced consistent increases in global gene transcription, but expression of target genes AREG, PGR, and TGFβ2 increased significantly only in explants from nulliparous women. E2-treatment did not induce consistent changes in proliferation or radiation induced apoptosis. Responses to estrogen are highly variable among women and not associated with levels of ER expression, suggesting differences in intracellular signaling among individuals. The differences in sensitivity to E2-stimulated responses may contribute to variation in risk of breast cancer.

Abstract P6-04-02: Integrative cistromic/transcriptomic profiling identifies a high FOXA1/ER-activated pro-metastatic secretome in endocrine-resistant breast cancer

Abstract Background: The estrogen receptor (ER) plays an evolving role in conferring endocrine resistance (EndoR) via altering chromatin binding and transcriptional reprogramming in ER+ metastatic breast cancer (BC). We have recently reported that high levels of FOXA1 (H-FOXA1), a pioneer factor for ER-chromatin binding and function, promote EndoR and metastatic phenotypes in ER+ BC cells. Here we further investigated the interplay between FOXA1 and ER by cistromic and transcriptomic profiling of ER+ BC cells expressing H-FOXA1 and a consequential pro-metastatic secretome associated with poor outcome of ER+ BC. Methods: FOXA1 and ER cistromes were analyzed by ChIP-seq in MCF7 cells expressing ectopic H-FOXA1 or treated with estrogen or the growth factor EGF. Transcriptomes of ER+ BC cells (MCF7, ZR75-1, and T47D) expressing ectopic H-FOXA1 and our established EndoR cell models (MCF7, ZR75-1, T47D, and 600MPE) were analyzed by RNA-seq. A core gene signature (CGS) and secretome gene sets induced by H-FOXA1 were identified by integrative analyses of cistromic/transcriptomic data and public secretory protein databases. Multivariant proportional hazards modeling and Kaplan-Meier log-rank test were performed to assess prognostic significance of the secretome gene sets using the METABRIC and KMplotter datasets. A transcriptomic dataset of ER+ primary and metastatic BC (The Metastatic Breast Cancer Project) was used to determine secretome expression changes in metastatic vs. primary tumors and the correlation with FOXA1 levels. A tamoxifen-resistant ER+ metastatic mouse model we previously established was used to determine pro-metastatic secretome gene expression. Results: Ectopic H-FOXA1 expanded and reprogramed the FOXA1 cistrome in ER+ BC cells and induced a CGS shared by multiple H-FOXA1-expressing cancer cell models, including prostate and pancreatic cancer cells. H-FOXA1 redirected an estrogen-independent ER cistrome in ER+ BC cells, but had no effect on the FOXA1 cistrome under short-term treatment with estrogen or EGF. Upregulated CGS was enriched for secretome-encoding genes with variance in ER dependency across multiple EndoR cell models. The H-FOXA1/ER-activated (n=10), but not the ER-repressed or -independent secretome, predicted poor disease-free survival in ER+ BC treated with endocrine therapy (METABRIC, n=1,103). Multivariate analyses further identified each of four secretome genes (ST6GALNAC2, SERPINI1, S100P, and CD55) significantly contributing to overall survival of ER+ BC. The prognostic significance of this secretome signature was also observed in relapse-free survival and distant metastasis-free survival of ER+, but not ER- BC, using the KMplotter dataset. The expression levels of the identified secretome were significantly increased in ER+ metastatic (n=147) vs. primary (n=48) tumors (including 31 pairs) and positively correlated with FOXA1 mRNA levels in metastases. Notably, there was no association between the H-FOXA1/ER-regulated pro-metastatic secretome and the ESR1 mutations. Finally, H-FOXA1-induced EndoR metastatic xenograft tumors were associated with elevated secretome gene expression. Conclusions: We identified an H-FOXA1/ER-activated pro-metastatic secretome via integrative analyses of the FOXA1/ER cistromes and transcriptomes of our preclinical models expressing H-FOXA1. This secretome signature predicts poor outcome of ER+ BC treated with endocrine therapy and has no correlation with the ESR1 mutations in ER+ metastatic BC. The increased expression of these secretome genes in ER+ metastatic vs. primary tumors suggests continuing interplay of FOXA1 and ER in promoting pro-metastatic transcriptional programs during ER+ disease progression, which warrants further investigation. Citation Format: Xiaoyong Fu, Resel Pereira, Carmine De Angelis, Sarmistha Nanda, Lanfang Qin, Martin J Shea, Tamika Mitchell, Jamunarani Veeraraghavan, Vidyalakshmi Sethunath, Carolina Gutierrez, Balázs Győrffy, Ofir Cohen, Nikhil Wagle, Agostina Nardone, Rinath Jeselsohn, Myles Brown, Mothaffar F Rimawi, C Kent Osborne, Rachel Schiff. Integrative cistromic/transcriptomic profiling identifies a high FOXA1/ER-activated pro-metastatic secretome in endocrine-resistant 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 P6-04-02.

Abstract P5-11-02: Dose-escalation study of SAR439859, an oral selective estrogen receptor degrader, in postmenopausal women with estrogen receptor-positive and human epidermal growth factor receptor 2-negative metastatic breast cancer

Abstract Background: Selective estrogen receptor (ER) degraders (SERDs) result in ER competitive antagonism and degradation and can block signaling in ER-dependent tumors resistant to other endocrine therapies. This study investigates SAR439859, a potent oral SERD, as monotherapy and in combination with the cyclin-dependent kinase 4/6 inhibitor, palbociclib, in ER-positive (+) and human epidermal growth factor receptor 2-negative (HER2-) metastatic breast cancer (mBC). We previously presented preliminary results (ASCO 2019). Here we describe safety, pharmacokinetics (PK), ER gene (ESR1)profiling and updated antitumor activity for SAR439859 monotherapy dose escalation. Methods: Part A of this open-label, Phase 1/2 first-in-human study (NCT03284957; TED14856) assessed SAR439859 dose escalation (3 + 3 design) in postmenopausal women with ER+/HER2- mBC treated for ≥ 6 months with prior endocrine therapy and ≤ 3 chemotherapies in the advanced setting. Dose levels ranged from 20-600 mg once daily (QD). Primary objectives: dose-limiting toxicities (DLTs), maximum tolerated dose (MTD), recommended dose. Secondary objectives: safety, PK, tumor response (RECIST v1.1), pharmacodynamic (PD) inhibition of ER occupancy (18F-fluoroestradiol positron emission tomography[18F-FES PET] scan). Exploratory objective: mutation profiling of ESR1 (circulating tumor DNA was assessed for 12 ESR1 mutations at baseline and end of Cycle 2 using multiplex droplet digital polymerase chain reaction). Results: As of May 29, 2019, 16 patients were treated. Median age: 59.5 years (range 40-79); ECOG performance status: 0 (62.5%) or 1 (37.5%); median prior anticancer therapies in the advanced setting: 3 (range 1-8). All patients had ≥ 1 treatment-emergent adverse event (TEAE), mostly grade 1-2; most frequent were hot flushes, diarrhea, constipation, nausea (all 37.5%) and decreased appetite (31.3%). Most frequent TEAEs related to treatment were hot flushes (31.3%), diarrhea, nausea (both 25%), decreased appetite, constipation, night sweats, asthenia (all 18.8%), arthralgia and fatigue (both 12.5%); no event was grade ≥ 3. There were no DLTs at any of the five dose levels (maximum administered dose 600 mg QD); MTD was not reached. In 18F-FES PET scans, signal inhibition &amp;gt; 87% occurred with plasma concentrations &amp;gt; 100 ng/mL. PK profile showed rapid SAR439859 absorption (median tmax ~ 3 hours) and an apparent terminal half-life of ~ 8 hours after first administration. After repeated QD administration, there was no or low accumulation and exposure (Cmax/AUC0-24h) increased with doses up to 600 mg. Food intake did not have a major effect on exposure. Best overall response (BOR) was confirmed partial response (PR) in one patient at 150 mg QD (6.3%) and stable disease (SD) in eight patients (50.0%) including seven (43.8%) with long-term SD (≥ 24 weeks). Seven had progressive disease (43.8%). Tumor shrinkage &amp;gt; 10% was seen in eight patients (50.0%), without any dose dependency. ESR1 mutations were detected at baseline in 11 patients (68.8%), with five patients harboring &amp;gt; 1 mutation. Similar BOR was seen regardless of ESR1 mutation status with SD seen in 54.5% (6/11) of patients with ESR1 mutations and SD/PR seen in 60.0% (SD: 2/5; PR: 1/5) of patients with wild-type ESR1. Conclusions: SAR439859 demonstrated a favorable safety profile and high ER occupancy in pretreated patients with ER+/HER2- mBC. Most patients had ESR1 mutations. Preliminary antitumor activity was encouraging in both ESR1 mutated and wild-type patients. With no DLTs and no MTD, 400 mg QD was selected for expansion cohorts based on safety, PD and PK. Additional data on PK, PD, antitumor activity and the ESR1 mutational profile, and any correlations, will be presented. Funding: Sanofi. Citation Format: Mario Campone, Aditya Bardia, Gary A Ulaner, Sarat Chandarlapaty, Alice Gosselin, Séverine Doroumian, Jeffrey Ming, Marina Celanovic, Hannah M Linden. Dose-escalation study of SAR439859, an oral selective estrogen receptor degrader, in postmenopausal women with estrogen receptor-positive and human epidermal growth factor receptor 2-negative metastatic 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 P5-11-02.

Abstract P4-04-08: Histological and epigenetic analyses of placenta tissue from breast cancer patients and healthy participants

Abstract Background: Breast cancer is one of the most common malignancies during pregnancy. Guidelines for breast cancer treatment during pregnancy demonstrating that breast cancer during pregnancy can be treated similarly to non-pregnancy-associated breast cancer, except for hormonal and anti-HER2 therapies. Nonetheless, a decreased birth weight is often observed in newborns. Therefore, this project aims to analyze the effects of chemotherapy and the impact of cancer progression on the placenta. Methods: Placentas from breast cancer patients (n=63) and non-cancer participants (n=20) enrolled in Breast Cancer in Pregnancy (BCP) study were collected after delivery and embedded in paraffin. Sections were stained with Hematoxylin-Eosin (HE) and IHC (Ki-67, cPARP, p27Kip1); trophoblast morphology was evaluated by an established scoring system and distribution of immunohistochemical markers was assessed. Additionally, epigenetic analyses (cytosine methylation) for the following genes were performed: LINE-1 (general methylation), IGF2-H19 (birth weight), EPO (hypoxia), BDNF (neurobiological development), glucocorticoid receptor, HSD11B2 (inactivation of glucocorticoids), estrogen receptor, P-glycoprotein, CYP-3A4 (drug metabolism). Results: HE staining revealed significant damage of trophoblast nuclei and membranes in placentas from breast cancer patients compared to controls (mean score of damage 1.9/1.8 vs 0.8/0.7, p&amp;lt;0.001). Simultaneously, mean proliferation index (Ki-67 positive cells/ 1000 trophoblasts) was reduced (36.3 vs 58.0, p&amp;lt;0.001). In contrast, nuclear and cytoplasmic expression of the negative cell cycle regulator p27Kip1 was reduced (mean IRS score 1.0/0.9 vs 4.3/4.8, p&amp;lt;0.001). No evidence of enhanced apoptosis was found. Epigenetic analyses showed significant differences in mean percentage of cytosine methylation of EPO (68.4% vs 71.1%, p&amp;lt;0.05) and hCyp-3A4 (87.8% vs 90.0%, p&amp;lt;0.01) gene. Altered methylation of CpG positions of LINE-1, IGF2-H19, HSD11B2, estrogen receptor and P-glycoprotein gene were found. Conclusions: Placentas from breast cancer patients seem to be harmed in contrast to placentas from normal pregnancies, shown by morphologic abnormalities and a decreased proliferation index. Nevertheless, no increase of apoptotic cells could be demonstrated. Altered expression of efflux pumps or drug-metabolizing enzymes might be a reason for good fetal tolerability of chemotherapy during pregnancy as methylation patterns were changed in P-glycoprotein and CYP-3A4 gene. Citation Format: Karolin Fröhlich, Torsten Plösch, Fenja Seither, Volkmar Müller, Thomas Karn, Elmar Stickeler, Christian Schem, Christine Solbach, Amelie Lupp, Rikst N Verkaik-Schakel, Gitta Turowski, Peter Mallmann, Marion van Mackelenbergh, Bruno Sinn, Valentina Nekljudova, Carsten Denkert, Pamela Schittler, Udo Markert, Sibylle Loibl. Histological and epigenetic analyses of placenta tissue from breast cancer patients and healthy participants [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 P4-04-08.