Estrogen Response Research Articles

Identification of Estrus in Sows Based on Salivary Proteomics.

Simple SummaryThe conception rate of sows is an important factor affecting the efficiency and income of pig farms. At present, the traditional estrus identification methods used in production, such as the external observation method and back pressure reaction method, have the disadvantages of being time-consuming and laborious, with low accuracy and high requirements for technicians. In addition, the abnormal estrus status of sows, such as continuous estrus and short estrus, will also lead to misjudgment during estrus inspection, which will affect the conception rate of sows and reduce production efficiency. Therefore, the purpose of this study was to explore a new method for estrus identification, to provide an important reference for accurate estrus identification of sows by screening salivary proteins related to estrus, and to provide a reference for the development of kits/strips for estrus identification of sows.The estrus cycle of multiparous Large White sows was divided into three stages to solve the problems of heavy workload and low accuracy of the traditional estrus identification method in pig production. Saliva protein was extracted from the oral saliva of multiparous sows. Label-free quantitative proteomics was used to detect salivary proteome, and MaxQuant software was used for quality control. Results showed that 246 proteins were identified in the three stages, where 40 proteins were significantly different (p < 0.05). The total proteins identified were enriched by STEM software and the protein function was annotated by using the ClueGO plug-in in the Cytoscape software. The results were enriched to eight different trends. The annotated items were related to protein synthesis and processing and estrogen response. Gene ontology and the Kyoto Encyclopedia of Genes and Genomes enrichment analysis of differential proteins involved in the pathways and entries included oocyte meiosis, response to estradiol, and oogenesis. Further interaction analysis showed that an interaction occurred between P00355, F1SHL9, P28491, F1SDR7, F2Z558, F1RYY6, and F2Z5G3 proteins. The findings served as a basis for revealing the changes in salivary protein content in the sow estrus cycle and provided a reference for the development of an estrus identification kit/test strip in the next step.

Abstract 2206: Pilot phase I clinical trial of RANKL inhibition and breast tissue gene expression in high-risk premenopausal women with dense breasts

Abstract Background: Identifying pathways that can be targeted to reduce breast density and breast cancer incidence is an unmet need, especially in premenopausal women. Receptor activator of nuclear factor-κB ligand (RANKL) signaling mediates the major proliferative response of mammary epithelium to progesterone and is positively associated with breast density but there are no clinical trial data on the impact of RANKL inhibition on breast tissue markers in women with dense breasts. We, therefore, determined the impact of RANKL inhibition on breast tissue gene expression in high-risk premenopausal women with dense breasts. Study design: Pilot phase I clinical trial of 9 healthy high-risk pre-menopausal women (≥35 years of age) with dense breasts performed at Washington University School of Medicine, St. Louis, MO from July 2018-December 2018. Fifty-five percent of participants had a positive family history of breast cancer in a first degree relative. Intervention: Participants were given a single dose of subcutaneous RANKL antibody, denosumab (60mg), at baseline (Day 1). On the same day, prior to the denosumab injection, participants had an ultrasound guided core needle biopsy and a blood draw. All study participants returned for repeat core needle biopsy and blood draw after 60 days (Day 60). Outcome Measures: Changes in breast tissue gene expression between Day 1 and Day 60 with a focus on immune/inflammatory and hormone markers. Gene expression was profiled using NanoString nCounter platform. We performed pathway enrichment analysis and used Cell Maker Enrichment library from EnrichR to identify differentially expressed genes. Genes with a p-value&amp;lt;0.05 on differential expression analysis were considered statistically significant for utilization in pathway enrichment analysis. Libraries utilized for pathway enrichment using the EnrichR platform included the ChEA transcription factor library, GO Biological Process library, and Cellular Components library. Results: Macrophage markers, including CCR5, CD86, CD300A, and CD84 and targets downstream of IRF8: (p-value =4.73e-7), neutrophil degranulation/neutrophil activation in the immune response (p-value=6.0e-21), and the secretory granule membrane pathways (p-value=2.4e-14) were downregulated in breast tissues on Day 60. Pathways involved in progesterone metabolism (p-value=0.0003), estrogen response (p-value=0.0014) and fatty acid metabolism, (p-value=0.0001) were significantly upregulated on Day 60. Specifically, genes involved in steroid hormone metabolism such as DHRS2 (p-value=3.13e-05), and AKR1B15: p-value=4.12e-05) were upregulated on Day 60. Conclusions: A single 60 mg dose of subcutaneous denosumab injection was associated with alterations in pathways involved in hormone, immune and inflammatory signaling in the breast tissues of healthy premenopausal women with dense breasts. Citation Format: Michael Waters, Kay Jayachandran, Debbie Bennet, Jin Zhang, Katherine Weilbaecher, Ian S. Hagemann, Graham A. Colditz, Catherine M. Appleton, Adetunji T. Toriola. Pilot phase I clinical trial of RANKL inhibition and breast tissue gene expression in high-risk premenopausal women with dense breasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2206.

Abstract 5375: Precision run-on sequencing (PRO-seq) analysis of a treatment time course in ER+ breast cancer cell lines reveals the transcriptional changes underlying response to complete estrogen receptor antagonist (CERAN) OP-1250

Abstract OP-1250 is an orally-bioavailable complete estrogen receptor antagonist (CERAN) previously shown to shrink wild-type and Y537S mutant estrogen receptor-positive (ER+) tumors in multiple preclinical xenograft models. To better understand the effects of OP-1250 treatment on ER+ breast cancer cells, we used precision run-on sequencing (PRO-seq) to profile the direct transcriptional effects of OP-1250 in two commonly used breast cancer cell lines, MCF7 and CAMA-1. PRO-seq detects nascent RNA, and thus can detect changes at shorter timescales than traditional RNA-seq methods. We conducted PRO-seq 15 minutes, 1 hour, 6 hours and 24 hours after administration of OP-1250, estradiol, fulvestrant and tamoxifen to compare the effects of these compounds across the two cell lines. OP-1250 was administered in the absence of estradiol and after estradiol pretreatment to generate a detailed profile of the time course of its activity. We identified genes that display early transcriptional changes in response to the compounds tested, many of which have been previously identified as estrogen response genes. At later time points we observed transcriptional changes consistent with modulation of pathways involved in cell cycle progression. We show that OP-1250 treatment reverses the estradiol-induced transcriptional changes associated with estrogen receptor activation and chronicle the time course of these changes. Finally, we show greater similarity in the transcriptional changes induced by CERANs OP-1250 and fulvestrant compared to tamoxifen, as well as cell line-specific differences in estrogen response and response to anti-estrogen treatment. These data provide additional insights into the mechanisms of OP-1250 action on ER+ cell lines and patient tumors. OP-1250 is currently in a Phase I/II clinical trial for the treatment of ER+ breast cancer. Citation Format: Alison D. Parisian, Caitlin Miller, Fabian Ortega, Leslie Hodges-Gallagher, Susanna Barratt, Joey Azofeifa, Peter J. Kushner, David Kulp, Cyrus L. Harmon. Precision run-on sequencing (PRO-seq) analysis of a treatment time course in ER+ breast cancer cell lines reveals the transcriptional changes underlying response to complete estrogen receptor antagonist (CERAN) OP-1250 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5375.

Abstract 648: A novel small molecule targeting oncogenic PELP1 demonstrates anti-tumor activity in wild-type and mutant ER-positive breast cancer

Abstract A significant proportion of estrogen receptor positive breast cancers (ER+BC) will initially respond to treatment, but many eventually develop therapy resistance (TR-BC), and progress to incurable metastases. Oncogenic ER coregulators overexpressed in BC can contribute to constitutive, ligand-independent and ligand-dependent signaling which drives growth, resistance to therapy and metastasis. Proline-, glutamic acid, and leucine-rich protein 1 (PELP1), is a known coregulator that plays a critical role in ER oncogenic functions. Its expression is deregulated in BC and is a prognostic indicator of poor BC survival. The lack of a small molecule inhibitor that directly targets PELP1 represents a major knowledge gap. Therefore, we conducted a large scale peptide library screening and identified novel Peptide Inhibitor of PELP1 (PIP1). We demonstrated that PIP1 directly interacts with PELP1, promotes its degradation and has the potential to block PELP1 oncogenic functions in vitro. Using innovative peptidomimetic technology, we modeled PIP1 and synthesized several derivatives as Small Molecule Inhibitors of PELP1 (SMIPs). Using MTT assay and multiple BC cell lines, we identified a lead compound, SMIP34 with an IC50 of 5-10µM and with minimal effect on human mammary epithelial cells. SMIP34 in vitro activity was assessed by colony formation and Matrigel invasion assays. Knockdown of PELP1 using shRNA in BC cells significantly reduced SMIP34 activity, indicating target specificity. Further, MST and biotin-SMIP34 pulldown confirmed direct binding of SMIP34 to PELP1. Using ER+WT, mtER (mutant ER), and TR-BC cell lines we demonstrated that SMIP34 exhibits antiproliferative effects and reduces invasiveness. Mechanistic studies using Western blot analysis confirmed that SMIP34 binding to PELP1 contributes to its degradation via the proteasome pathway. Thus MG132 treatment attenuated SMIP34 mediated degradation. RTqPCR analyses confirmed SMIP34 treatment reduced expression of PELP1 target genes. RNAseq analyses showed SMIP34 treatment altered the expression of genes associated with Estrogen response, Cell cycle and Apoptosis pathways. Cell cycle analyses revealed SMIP34 treatment promoted S phase arrest of BC cell lines. Using ER+WT, mtER, and PDX tumor tissues ex vivo, we demonstrated that SMIP34 significantly decreased tumor proliferation as measured by Ki67 staining. Further, SMIP34 (20mg/kg/IP) treatment in vivo significantly reduced tumor progression in mouse models of ER+WT, mtER, and mtER patient-derived xenograft BC. Our results using in vitro, ex vivo, and in vivo models showcase SMIP34 as a first-in-class inhibitor of oncogenic PELP1 signaling and may serve as a potential therapeutic molecule for treating ER+, mtER, and TR-BC. Supported by NIH 1F31CA257298 (KA) and VA I01BX004545 (RV). Citation Format: Kristin Ann Altwegg, Monica Mann, Dimple Chakravarty, Zexuan Liu, Junhao Liu, Uday P. Pratap, Behnam Ebrahimi, John R. Sanchez, Ben H. Park, Hariprasad Vankayalapati, Gangadhara R. Sareddy, Suryavathi Viswanadhapalli, Ratna K. Vadlamudi. A novel small molecule targeting oncogenic PELP1 demonstrates anti-tumor activity in wild-type and mutant ER-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 648.

Sex-specific multi-level 3D genome dynamics in the mouse brain

The female mammalian brain exhibits sex hormone-driven plasticity during the reproductive period. Recent evidence implicates chromatin dynamics in gene regulation underlying this plasticity. However, whether ovarian hormones impact higher-order chromatin organization in post-mitotic neurons in vivo is unknown. Here, we mapped the 3D genome of ventral hippocampal neurons across the oestrous cycle and by sex in mice. In females, we find cycle-driven dynamism in 3D chromatin organization, including in oestrogen response elements-enriched X chromosome compartments, autosomal CTCF loops, and enhancer-promoter interactions. With rising oestrogen levels, the female 3D genome becomes more similar to the male 3D genome. Cyclical enhancer-promoter interactions are partially associated with gene expression and enriched for brain disorder-relevant genes and pathways. Our study reveals unique 3D genome dynamics in the female brain relevant to female-specific gene regulation, neuroplasticity, and disease risk.

Abstract 766: Genomic and transcriptomic comparison between breast cancers from patients of African and European genetically determined ancestries demonstrates potential for ancestry specific biomarker-informed therapies

Abstract Background: Therapeutic approaches for breast cancer (BC) are informed by tumor subtypes and molecular heterogeneity, both of which may be associated with particular patient ancestries. While patients of African ancestry (AA) exhibit a relatively high mortality rate compared with patients of European ancestry (EA), the biological underpinnings of this disparity are not entirely understood. Here, we present a genomic and transcriptomic comparison between BC tumors from AA and EA patients stratified by clinical features in a real-world cohort. Methods: De-identified records from AA (n=629) and EA (n=2832) patients with BC were selected from the Tempus Oncology Database. All tumors underwent sequencing with the Tempus|xT assay, including targeted-panel DNA and full-transcriptome RNA-seq. Race metadata was obtained from abstracted records. Genetic ancestry was estimated using a set of ancestry-informative markers and a supervised version of the ADMIXTURE algorithm. Mutational prevalence, gene expression, and transcriptional signatures were compared between EA and AA groups, and stratified by stage and subtype. Statistical significance was assessed with Bonferroni correction applied to adjust for multiple testing. Results: Genetic ancestry estimates and race metadata had strong concordance, with 97% and 91% of race data matching genetic ancestry categories in AA and EA, respectively. Compared to EA patients, AA patients were slightly younger (median age 53.8 years vs. 56.1 years) and more likely to have stage IV disease (59.0% vs. 56.5%). The percentage of triple-negative BC (TNBC) patients was much higher among AA (23.5%) compared to EA (14.3%), while the proportion of HR+/HER2− patients was higher in EA (46.4%) compared to AA (36.5%). In HR+/HER2- patients, AA patients had a higher prevalence of KMT2C (24% vs. 14%), GATA3 (21% vs. 14%), and FGFR1 (18% vs. 12%) mutations, and a lower prevalence of PIK3CA (33% vs. 42%) and PTEN (6% vs. 13%) mutations. PRSS23 expression and estrogen response late signaling were significantly downregulated in late-stage HR+/HER2- AA patients, while PTEN signaling was downregulated in stages I-III. Among TNBC, AA patients had a higher prevalence of KMT2C (23% vs. 12%) mutations. LILRA6 and PTEN were significantly downregulated in early-stage TNBC patients of AA. Conclusions: After controlling for stage at diagnosis and subtype, we observed significant differences in BC mutational spectrums, gene expression, and relevant pathway activities between patients with genetically determined AA and EA, particularly within the TNBC and HR+/HER2- subtypes. These findings may guide future development of treatment strategies by providing data for biomarker-informed research and precision cancer care. Citation Format: Minoru Miyashita, Joshua S. Bell, Ezgi Karaesmen, Brooke Rhead, Stephane Wenric, Kristiyana Kaneva, Francisco M. De La Vega, Yonglan Zheng, Toshio Yoshimatsu, Galina Khramtsova, Elisabeth Sveen, Fang Liu, Fangyuan Zhao, Frederick Howard, Nora T. Jaskowiak, Rita Nanda, Dezheng Huo, Olufunmilayo I. Olopade. Genomic and transcriptomic comparison between breast cancers from patients of African and European genetically determined ancestries demonstrates potential for ancestry specific biomarker-informed therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 766.

Abstract 1778: NR2F2 mediates cell growth and endocrine resistance in NF1 loss, ER+ breast cancer

Abstract Genomic loss of in Neurofibromin 1 (NF1) is a recurrent mechanism driving metastatic progression and resistance to endocrine therapy in ER-positive breast cancer, via activation of the MAPK pathway, induction of cyclin D1 expression and modulation of estrogen receptor signaling. To identify specific mediators of tumor growth in NF1 mutant cancers, we performed a genetic knockout (KO) screen in isogenic WT and NF1 KO breast cancer cells. After employing stringent cutoff criteria, we found 28 genes that are specifically essential for NF1 KO cells but not WT cells. Among these genes, NR2F2, encoding an orphan nuclear receptor, was one of the top candidates. We validated that its loss significantly impaired the growth of multiple models of NF1 null, ER+ breast cancer. Loss of NF1 leads to the hyperactivation of Ras which sequentially activates multiple effector pathways, including the MAPK pathway and the PI3K-AKT pathway. We found that MAPK activation upon NF1 loss led to elevated NR2F2 expression levels. A negative correlation between NF1 and NR2F2 expression was further confirmed in public gene expression databases. These data suggested that induction of NR2F2 expression might be one mechanism that mediates endocrine resistance caused by NF1 loss. Consistently, we found that exogenous expression of NR2F2 could confer endocrine resistance while its knockout hypersensitized ER+ breast cancer cells to endocrine therapies and significantly attenuated NF1 loss induced resistance. RNA-seq analysis revealed that estrogen response gene sets were among the most enriched signatures upon NF1 KO, NR2F2 over-expression (OE) or NR2F2 KO. NF1 KO or NR2F2 OE decreased the expression of early estrogen response genes and impaired their E2-induced expression, most of which could be rescued by NR2F2 KO, suggesting a regulatory effect of NR2F2 on ER transcriptional activity. To further define these mechanisms, ongoing integrative analyses of NR2F2 qPlex-RIME, ER and NR2F2 ChIP-Sequencing and ATAC-Sequencing will be presented. Taken together, our studies identify NR2F2 as a MAPK driven transcript whose induction mediates endocrine resistance, and may serve as a novel therapeutic target to overcome anti-estrogen resistant breast cancer growth. Citation Format: Yanyan Cai, Peihua Zhao, Fan Wu, Hong Shao, Pedram Razavi, Guotai Xu, Maurizio Scaltriti, Sarat Chandarlapaty. NR2F2 mediates cell growth and endocrine resistance in NF1 loss, ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1778.

Abstract 3632: Differential DNA methylation in the benign and cancerous prostate tissue of African American and European American men

Abstract In this study, we compare patterns of differential DNA methylation (DNAm) distinguishing tumor and benign prostate tissue in European American (EA) and African American (AA) men to improve our understanding of biological mechanisms that may underlie prostate cancer (PCa) disparities. In the United States, AA men are more likely to be diagnosed with and to die of PCa compared with EA men. Changes in DNAm accompany tumor development and progression, and differences in DNAm across ancestry groups may be an important lens through which to study PCa disparities. We collected paired tumor and histologically benign formalin-fixed paraffin-embedded tissue blocks from 151 (76 AA, 75 EA) PCa patients undergoing prostatectomy at the University of Chicago. DNA was extracted and genome-wide DNAm was measured at ~850,000 CpG sites using the Illumina MethylationEpic Array. After quality control, 682,694 CpGs remained for analysis. We conducted differential methylation analyses, adjusting for batch, age, and individual and identified 29,236 and 38,035 tumor-associated CpGs in AA and EA respectively (p&amp;lt;5x10-8 and Δβ&amp;gt;0.3) with 25,263 CpGs common to both sets. There was a depletion of differentially methylated CpGs in CpG islands and promoters in both groups, but depletion was stronger in AA. Tumor-associated CpGs within islands and in promoters were more likely to be hypermethylated for both groups, though this was stronger in EA. We identified 2,392 genes with differentially methylated promoters common to both ancestry groups; 223 and 1,045 were unique to AA and EA respectively. Shared gene regions included GSTP1, APC, RARB, and GRASP, which have been previously reported. We assigned CpG sites to genes and used Gene Set Enrichment Analysis to identify differentially methylated pathways. The identified pathways include several previously associated with cancer development including the epithelial mesenchymal transition. We also identified ancestry-specific pathways including the early and late estrogen response pathways in AA. Examining the predictive ability of our differentially methylated CpGs, we found that relatively few CpGs (~10) were sufficient to distinguish tumor and benign tissue in AA and EA men (AUC&amp;gt;0.9). Scores constructed from differentially methylated CpGs in EA were able to predict tumor vs benign in AA (and vice versa) with high accuracy. Finally, we examined the association between ancestry and DNAm in both tumor and benign tissue. Here, 89 and 423 ancestry-associated CpGs were identified in tumor and benign respectively. Overall, we find that differential methylation patterns distinguishing tumor and benign tissue are generally similar for EA and AA men. However, differenced in tumor-associated DNAm as well as the presence of ancestry-associated CpGs provide a rich area for future studies exploring the impact of these differences on cancer development in AA and EA men. Citation Format: Meytal B. Chernoff, Marc Gillard, Kathryn Demanelis, Dayana Delgado, Anthony Williams, Donald Vander Griend, Brandon L. Pierce. Differential DNA methylation in the benign and cancerous prostate tissue of African American and European American men [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3632.

Defining transcriptomic profiles of early-stage mucinous breast cancers: A FLEX sub study.

3134 Background: Mucinous breast cancer (MuBC) is a rare subtype of invasive ductal carcinoma (IDC) that accounts for less than 2% of all breast cancers and is associated with a favorable prognosis. Since MuBCs are rare in clinical trials, current treatment guidelines are extrapolated from IDC-no special type (IDC-NST). To provide better understanding of MuBCs and factors contributing to their clinical behavior, we examined the transcriptomic profiles of MuBCs in our FLEX study. Methods: The prospective, observational FLEX Study (NCT03053193) includes stage I-III breast cancer patients who receive MammaPrint (MP)/BluePrint (BP) testing and consent to full transcriptome and clinical data collection. For this study, histologically confirmed MuBCs (n = 102) in the FLEX database were included. All patients examined were ER+/HER2- by immunohistochemistry and Luminal by BP. MuBC was compared with IDC matched for Age, MP, and BP index (n = 97). Differential gene expression analyses (DGEA) were performed with R package ‘limma’ and differentially expressed genes (DEGs) were considered significant if they had an adjusted p &lt; 0.05 and fold change ≥ 2. Results: DGEA comparing MuBC (n = 102) with IDC (n = 97) revealed 60 DEGs, regardless of the genomic risk, of which 42 genes were upregulated and 18 were downregulated in MuBC relative to IDC. Genes associated with MuBC, such as MUC2, TFF1, CARTPT were among the upregulated genes. Of the 102 MuBC patients, 56 were Luminal A (MP Low Risk-LR) and 46 were Luminal B (MP High Risk-HR) by MammaPrint and BluePrint. Comparison of LR MuBC with LR IDC revealed 111 DEGs. Functional enrichment showed upregulation of pathways involved in estrogen response (early &amp; late) and androgen response and a downregulation of the epithelial to mesenchymal transition (EMT) and E2F pathways in LR MuBC compared to LR IDC. DGEA between HR MuBC and HR IDC revealed only 22 DEGs with immune pathways being downregulated in HR MuBC. DGEA comparing LR MuBC with HR MuBC resulted in 63 DEGs, indicating LR and HR MuBC are biologically distinct types. Interestingly in LR MuBC, the tumor suppressor marker SCUBE2 is upregulated. Over expression of SCUBE2 is associated with better prognosis. Conclusions: Although MuBCs are often expected to have low clinical risk, MP revealed that half of the MuBCs examined in this study were MP High Risk (Luminal B). MP low risk MuBC is biologically different from MP low risk IDC, and downregulation of E2F and EMT pathways might lead to favorable prognoses in MP low risk MuBC. MP high risk MuBC showed limited DEGs compared to high-risk IDCs indicating these tumor types are highly genomically similar and likely to benefit from chemotherapy. The downregulation of immune pathways in MP high risk MuBC may lead to immune surveillance escape resulting in metastasis and further investigation is needed. Clinical trial information: NCT03053193.

ESR1 F404 mutations and acquired resistance to fulvestrant in the plasmaMATCH study.

1009 Background: The selective estrogen receptor modulator (SERD) fulvestrant is commonly used to treat patients with hormone receptor positive advanced breast cancer, although potential mechanisms of acquired resistance are poorly understood. plasmaMATCH cohort A (NCT03182634) investigated the activity of fulvestrant in patients with activating ESR1 mutations in circulating tumor DNA (ctDNA). Here we present analysis of baseline and end-of-treatment (EOT) ctDNA to identify potential resistance mutations to fulvestrant. Methods: Paired baseline and EOT plasma samples from patients enrolled into plasmaMATCH underwent ctDNA sequencing (Guardant360, Guardant Health) to identify acquired mutations. For F404 analysis, MCF-7 cells were transiently transfected with estrogen receptor expression constructs containing either wildtype ESR1 (WT), single ESR1 mutations (D538G, E380Q, F404L), or compound mutations (D538G_F404L, E380Q_F404L), alongside an estrogen response element (ERE)-luciferase reporter construct. Transfected cells were treated with or without fulvestrant and ERE-luciferase activity compared. Results: Of 84 patients enrolled in cohort A, 69 had paired baseline and EOT sequencing. Patients with baseline ESR1 Y537S had shorter progression free survival (PFS), and Y537C longer PFS, than those wild-type for each respective mutation (p = 0.03 and p = 0.04). Patients frequently acquired mutations at EOT (n = 35, 51%), including potentially targetable mutations in 25% (including 3 PTEN, 3 BRCA1/2, 2 PIK3CA, 2 HER2, 1 BRAF). Three (4%) patients acquired ESR1 p.F404 mutations (F404L, F404I, F404V), with 7 mutations in total. Of 26 patients with PFS of ≥16 weeks, 3 patients (12%) acquired ESR1 F404. In 800 patients screened for entry to plasmaMATCH, one harbored a F404 mutation (0.13%), with a prior history of fulvestrant. F404 mutations resided in cis with E380Q in 6/7 assessable mutations. In vitro structural modelling revealed that ESR1 p.F404 resides within the ESR1 ligand binding domain, and contributes to estrogen and fulvestrant binding through a pi-stacking bond between the aromatic ring of phenylalanine and estrogen/fulvestrant, with all F404 mutations disrupting this bond. Transient transfection demonstrated that single mutations D538G, E380Q, F404L and wild-type ESR1 were sensitive to fulvestrant (p &lt; 0.0001, p = 0.0006, p = 0.04 and p = 0.0001), whereas compound mutations D538G_F404L and E380Q_F404L were resistant. Further investigation of relative sensitivity of the F404 mutant ESR1 to other anti-estrogens will be presented. Conclusions: We have identified a novel resistance mechanism to fulvestrant, with F404 mutations acquired in patients with pre-existing activating ESR1 mutations. F404 confers fulvestrant resistance through the loss of a pi-stacking bond and likely reduced fulvestrant binding affinity, identifying a new potential target to overcome endocrine therapy resistance.

Potential estrogenic background in aquatic laboratory cultivations.

Aquatic biotests are important tools targeting various effects in ecotoxicology, including endocrine disruption. Unintentional exposure of bioassay organisms to endocrine disruptors during cultivation or testing may interfere with assessed endpoints. We illustrate this issue on the example of laboratory phytoplankton cultivation, where possible sources of estrogenic compounds have been revealed. Fifty-four blank samples (water and fresh or cultivated growth media) were assessed by in vitro biotests for their estrogenicity, and major known estrogens originating from plastic materials, bisphenol A and alkylphenols, were analyzed in selected samples. The samples of freshly prepared growth medium elicited weak estrogenic response in bioassays and some samples of the aerated media caused responses even above the 50% of maximum of the reference compound (17β-estradiol, E2), while the samples from diverse laboratory water sources did not show significant estrogenic activity. The results identified substances contained in the growth medium as minor but reproducible contributors to estrogenicity in the cultivations. Sporadic but significant effects (up to 4.9 ng E2 equivalent/L) can be ascribed to compounds released from the used plastic materials during aeration of the cultivations. The potential sources of unintentional exposure to estrogenic compounds need to be considered in aquatic cultivations and biotests, since they could impact their outcomes, especially in arrangements assessing reproduction or whole life cycle biotests, or production of bioactive compounds by phytoplankton. The findings emphasize the necessity to assess all relevant blanks, ideally by sensitive high throughput in vitro assays that reflect also unknown pollutants and minimize all potential sources of background contamination. In vitro assays show very good applicability for this purpose since they enable to screen for any background estrogenicity of the used media and materials without the need of analyzing individual compounds, which often might not be known.

The haplotypes GCA and ACA in ESR1 gene are associated with the susceptibility of recurrent spontaneous abortion (RSA) in Chinese Han: A case-control study and meta-analysis.

The nonstructured abstract were supplied as following: Estrogen receptor is involved in the pathogenesis of recurrent spontaneous abortion (RSA). The ESR1 and ESR2 genes can mediate nongenomic estrogen responses. This study aimed to assess the genetic association between the ESR1 and ESR2 genes polymorphisms and RSA susceptibility in a Chinese Han population. A total of 258 women who had experienced RSA and 264 unrelated healthy women were recruited. Genotypes of the 6 polymorphisms in the ESR1 (rs9340799, rs2234693, and rs3798759) and ESR2 genes (rs207764, rs4986938, and rs1256049) were analyzed using Snapshot technology. No association was detected between the alleles and genotypes of ESR1 rs9340799, rs2234693, and rs3798759 polymorphims and RSA risk (P > .05). Subjects carrying the haplotype of rs9340799A-rs2234693C-rs3798759A had a significantly increased RSA risk in the case group compared with the control group (P = .0005, Padj = .003, odds ratios [95% CI] = 0.35 [0.19–0.65]). However, subjects carrying the haplotype of rs9340799G-rs2234693C-rs3798759A had a significantly decreased RSA risk in the case group compared with the control group (P = .0005, Padj = .003, odds ratios [95% CI] = 2.99 [1.57–5.70]). In addition, no association was found between the alleles, genotypes, and haplotypes of ESR2 rs207764, rs4986938, rs1256049 polymorphisms and RSA risk (P > .05). In conclusion, the haplotype rs9340799A-rs2234693C-rs3798759A of ESR1 might be a risk factor. And the haplotype rs9340799G-rs2234693C-rs3798759A of ESR1 might be a protective factor for RSA in a Chinese Han population.

Characterization of the Estrogen Response Helps to Predict Prognosis and Identify Potential Therapeutic Targets in Cholangiocarcinoma

Cholangiocarcinoma (CCA) is an aggressive malignancy originating from the epithelium of the bile duct. The prognosis of patients is poor regardless of radical resection and chemoradiotherapy. The current classification and prognostic model of CCA are unable to satisfy the requirements for predicting the clinical outcome and exploring therapeutic targets. Estrogen signaling is involved in diverse cancer types, and it has long been established that CCA could be regulated by estrogen. In our study, estrogen response was identified to be significantly and stably correlated with poor prognosis in CCA. Employing several algorithms, CCA was classified into ES cluster A and B. ES cluster B was mainly composed of patients with fluke infection and overlapped with CCA cluster 1/2, and ES cluster A was mainly composed of patients without fluke infection and overlapped with CCA cluster 3/4. COMT and HSD17B1 were identified to be responsible for the differential estrogen response between ES clusters A and B, and the estrogen response may be correlated with the differentiation and cancer stemness of CCA at the single-cell level. Complement activation and the expression of C3 and C5, which are mainly expressed by CCA cells, were significantly downregulated in ES cluster B. An estrogen response risk score (ESRS) model was constructed to predict the prognosis of CCA, followed by a nomogram integrating ESRS and clinical features. Finally, altered pathways, applicable drugs and sensitivity to chemical drugs were analyzed specific to the estrogen response. In summary, our results provide insights into the role of the estrogen response in CCA progression as well as applicable drugs and potential therapeutic targets in estrogen metabolism, the complement system and ESRS-related pathways.

PD16-08 ALTERNATE ANDROGEN-INDEPENDENT ESTROGEN SIGNALING FOR PROSTATIC GROWTH

You have accessJournal of UrologyCME1 May 2022PD16-08 ALTERNATE ANDROGEN-INDEPENDENT ESTROGEN SIGNALING FOR PROSTATIC GROWTH Christina Sharkey, Xingbo Long, Ra'ad Al-Faouri, Zongwei Wang, and Aria F. Olumi Christina SharkeyChristina Sharkey More articles by this author , Xingbo LongXingbo Long More articles by this author , Ra'ad Al-FaouriRa'ad Al-Faouri More articles by this author , Zongwei WangZongwei Wang More articles by this author , and Aria F. OlumiAria F. Olumi More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002548.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Steroid 5α reductase 2 (SRD5A2) is the predominant enzyme responsible for prostatic development and growth. SRD5A2 inhibitors (5ARI) are the only class of benign prostate hyperplasia-related medications that reduce prostate size. However, the progression of lower urinary tract symptoms (LUTS) associated with BPH was only slowed by 34% in those treated with 5ARI. Through epigenetic regulation, we have shown that one-third of adult patients lack expression of prostatic SRD5A2. Here we wished to identify the subpopulation of prostate cells resistant to 5ARI therapy and determine alternative non-androgenic pathways that contribute to prostate growth in the absence of SRD5A2. METHODS: Single prostatic cells of homozygous SRD5A2-/- mice and littermate heterozygous SRD5A2+/- control mice were isolated and processed using the 10x genomics platform for single cell RNA-sequencing (scRNA-seq). A complete transcriptomic profile was obtained to identify cellular subsets and functional differentiation. Specific cell clusters were validated in situ using immunohistochemistry and RNA in situ hybridization. Levels of transcription factors were tested by qPCR. RESULTS: Analysis of transcriptome data for 23,000 single cells revealed 16 subpopulations. Luminal epithelial cells (LE) were further identified as five clusters. In SRD5A2-/- mice, while the total number of LE cells decreased, the percentage of LE2 subcluster increased. LE2, a predominant high expressor of estrogen receptor alpha (ESR1), was predominately located in the anterior prostate with enriched gene ontology (GO) terms of Wnt and MAPK signaling pathways. Zfp143, the transcription factor of ESR1, was dramatically upregulated in the absence of SRD5A2. CONCLUSIONS: Our data demonstrate the heterogeneity of cell populations within the mouse prostate. LE2, with an enhanced estrogen response gene signature, may play an important role in alternative pathways for prostate growth in the absence of SRD5A2. Understanding the underlying androgen-independent pathways contributing to prostate growth can help identify novel therapeutic targets for the management of prostatic diseases. Source of Funding: National Institutes of Health (NIH)/ National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (NIH/R01 DK124502) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e271 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Christina Sharkey More articles by this author Xingbo Long More articles by this author Ra'ad Al-Faouri More articles by this author Zongwei Wang More articles by this author Aria F. Olumi More articles by this author Expand All Advertisement PDF DownloadLoading ...

The MDM2 and CDKN2A Copy-number-variation Influence the TP53-signature-score in Wild-type TP53 Luminal Type Breast Cancer.

The TP53-signature is a multi-gene signature that can predict TP53 structural mutations. It has presented remarkable ability to predict the prognosis of early-stage breast cancer. However, some samples presented discordance with the signature status and structure status. We aimed to investigate whether the mRNA expression levels or copy number variation (CNV) of MDM2 and CDKN2A influence the TP53-signature-score, subtype classification, and prognosis prediction in TP53 wild-type, luminal type early-stage breast cancer samples. We selected TP53 wild-type, luminal type early-stage breast cancer samples from The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohorts. Then, we analyzed the correlation between the TP53-signature-score and mRNA expression levels or CNV of MDM2 and CDKN2A. The samples with MDM2 copy number (CN) amplification or those with CDKN2A CN deep deletion presented higher TP53-signature-score. Moreover, samples with MDM2 CN amplification or those with CDKN2A CN deep deletion had more characteristics of the luminal B type. In addition, they showed lower estrogen response early score, which correlated with response to endocrine therapy in breast cancer. However, MDM2 and CDKN2A mRNA expression did not present the same tendency. Furthermore, samples with MDM2 CN amplification or those with CDKN2A CN deep deletion had a worse prognosis in METABRIC cohort. The MDM2 or CDKN2A CNV may be useful for classifying subtypes and predicting prognosis more accurately in TP53 wild-type, luminal type early-stage breast cancer patients.

Estrogen receptor-related receptors in mandarin fish (Siniperca chuatsi): Molecular cloning, characterization, and estrogen responsiveness

The estrogen-related receptors (ERRs), a group of nuclear receptors, are the first orphan receptor to be identified. In this study, six errs (errα, errβ, errγ, errγA, errγB, errδ) were first cloned from the gonads of mandarin fish (Siniperca chuatsi). Gene cloning results showed the open reading frames (ORF) of both errα, errβ, errγ, errγA, errγB, errδ were 1302 bp, 1434 bp, 1323 bp, 1566 bp and 1308 bp, coding a 432, 476, 439, 520, 434 and 337 amino acid (AA) peptide, respectively. Tissue distribution analysis showed that errα was relatively highly expressed in heart and muscle both in male and female and errγA was highly expressed in brain, stomach and muscle while the errγB was extremely highly expressed in heart and liver both in male and female. The errβ expressed relatively highly in medulla oblongata and gonad of male and female and kidney of male, while errγ expressed relatively highly in stomach of male and female. Gonad expression pattern revealed that errα, errγ, errγA and errγB were all detected from 5 to 180 day after hatching (dah) in both males and females, and errδ began to express after 60 dah. Moreover, errγ, errγA and errγB were highly expressed in 5–10 dah while errα and errδ were mainly expressed in 60–180 dah. Furthermore, luciferase assay showed that errγ could response to estrogen stimulation by modulating estrogen-signaling pathway in mandarin fish.

A 13-Gene Signature Based on Estrogen Response Pathway for Predicting Survival and Immune Responses of Patients With UCEC.

Background: Accumulating evidence suggests that anti-estrogens have been effective against multiple gynecological diseases, especially advanced uterine corpus endometrial carcinoma (UCEC), highlighting the contribution of the estrogen response pathway in UCEC progression. This study aims to identify a reliable prognostic signature for potentially aiding in the comprehensive management of UCEC. Methods: Firstly, univariate Cox and LASSO regression were performed to identify a satisfying UCEC prognostic model quantifying patients’ risk, constructed from estrogen-response-related genes and verified to be effective by Kaplan-Meier curves, ROC curves, univariate and multivariate Cox regression. Additionally, a nomogram was constructed integrating the prognostic model and other clinicopathological parameters. Next, UCEC patients from the TCGA dataset were divided into low- and high-risk groups according to the median risk score. To elucidate differences in biological characteristics between the two risk groups, pathway enrichment, immune landscape, genomic alterations, and therapeutic responses were evaluated to satisfy this objective. As for treatment, effective responses to anti-PD-1 therapy in the low-risk patients and sensitivity to six chemotherapy drugs in the high-risk patients were demonstrated. Results: The low-risk group with a relatively favorable prognosis was marked by increased immune cell infiltration, higher expression levels of HLA members and immune checkpoint biomarkers, higher tumor mutation burden, and lower copy number alterations. This UCEC prognostic signature, composed of 13 estrogen-response-related genes, has been identified and verified as effective. Conclusion: Our study provides molecular signatures for further functional and therapeutic investigations of estrogen-response-related genes in UCEC and represents a potential systemic approach to characterize key factors in UCEC pathogenesis and therapeutic responses.

Integrated estrogenic effects and semi-volatile organic pollutants profile in secondary and tertiary wastewater treatment effluents in North China

Endocrine-disrupting effects on aquatic organisms caused by wastewater discharging have raised extensive concerns. However, the efficiency of various wastewater treatment processes to remove estrogenic activity in effluents and the association with organic micropollutants was not well known. We evaluated the estrogenic activity using a well-characterized in vivo bioassay featuring the Chinese rare minnows (Gobiocypris rarus) and analyzed 886 semi-volatile organic compounds (SVOCs) in effluents from four secondary wastewater treatment plants (SWTP A-D) and a tertiary wastewater treatment plant (TTP E) that utilized various common treatment processes in northern China. The final effluents from SWTPs and TTP E all exhibited estrogenic effects, increasing male fish plasma vitellogenin (VTG) contents and estradiol/testosterone (E2/T) ratios. Key regulating genes in the male fish liver including vtg1, vtg3, era, erβ, and cyp19a were upregulated. TTP E demonstrated high performance in reducing estrogenic activity in the effluents, with a reduction of 64% in integrative biomarkers of estrogenic response (IBR). UV disinfection at SWTPs removed IBR by 14%− 33%, while ozone disinfection at TTP E did not reduce IBR. Several SVOCs including alkanes, chlorobenzenes, and phthalates, detected at ng/L to µg/L level, significantly correlated with effluent estrogenic activity. Our findings suggest the necessity and the potential means to improve the efficiency of current wastewater treatment approaches to achieve better protection for aquatic organisms against the joint effects of mixtures of various categories of micropollutants in effluents.

Network Pharmacology with Experimental Investigation of the Mechanisms of Rhizoma Polygonati against Prostate Cancer with Additional Herbzymatic Activity.

A combination therapy of RhizomaPolygonati (RP) with goji (Lycium chinense) has earned a long history in the prescriptions to promote male health. However, the mechanisms at both molecular and nanoscale quantum levels are unclear. Here, we found that processed RP extract induces apoptosis and cell cycle arrest in cancer cells, thereby inhibiting prostate cancer cell proliferation enhanced by processed goji extract associated with an augment of the nanoscale herbzyme of phosphatase. For network pharmacology analysis, RP-induced PI3K-AKT pathways are essential for both benign prostatic hyperplasia and prostate cancer, and the RP/goji combination induces potent pathways which include androgen and estrogen response, kinase regulation, apoptosis, and prostate cancer singling. In addition, the experimental investigation showed that the prostate cancer cells are sensitive to RP extract for inhibiting colony formation. Finally, the natural compound baicalein found in RP ingredients showed a linked activity of top-ranked signaling targets of kinases including MAPK, AKT, and EGFR by the database of cMAP and HERB. Thus, both the nanozyme and ingredients might contribute to the RP in anti-prostate cancer which can be enhanced by goji extract. The proposed nanoscale RP extract might be of significance in developing novel anti-prostate cancer agents by combining goji compositions and targeted therapy compounds.

Increased prognostic value of clinical-reproductive model in Chinese female patients with esophageal squamous cell carcinoma.

BACKGROUNDIn China, it has been well recognized that some female patients with esophageal squamous cell carcinoma (ESCC) have different overall survival (OS) time, even with the same tumor-node-metastasis (TNM) stage, challenging the prognostic value of the TNM system alone. An effective predictive model is needed to accurately evaluate the prognosis of female ESCC patients.AIMTo construct a novel prognostic model with clinical and reproductive data for Chinese female patients with ESCC, and to assess the incremental prognostic value of the full model compared with the clinical model and TNM stage.METHODSA new prognostic nomogram incorporating clinical and reproductive features was constructed based on univariatie and Cox proportional hazards survival analysis from a training cohort (n = 175). The results were recognized using the internal (n = 111) and independent external (n = 85) validation cohorts. The capability of the clinical–reproductive model was evaluated by Harrell’s concordance index (C-index), Kaplan–Meier curve, time-dependent receiver operating characteristic (ROC), calibration curve and decision curve analysis. The correlations between estrogen response and immune-related pathways and some gene markers of immune cells were analyzed using the TIMER 2.0 database. RESULTSA clinical–reproductive model including incidence area, age, tumor differentiation, lymph node metastasis (N) stage, estrogen receptor alpha (ESR1) and beta (ESR2) expression, menopausal age, and pregnancy number was constructed to predict OS in female ESCC patients. Compared to the clinical model and TNM stage, the time-dependent ROC and C-index of the clinical–reproductive model showed a good discriminative ability for predicting 1-, 3-, and 5-years OS in the primary training, internal and external validation sets. Based on the optimal cut-off value of total prognostic scores, patients were classified into high- and low-risk groups with significantly different OS. The estrogen response was significantly associated with p53 and apoptosis pathways in esophageal cancer.CONCLUSIONThe clinical–reproductive prognostic nomogram has an incremental prognostic value compared with the clinical model and TNM stage in predicting OS in Chinese female ESCC patients.