Estrogen Receptor Gene Expression Research Articles

Estrogenic activity of Derris scandens stem extracts and its major compounds using MCF-7 cell proliferation assay and estrogen-related gene expression.

Derris scandens, which contains isoflavones and prenylated derivatives, has analgesic and anti-inflammatory properties and is an ingredient in traditional Thai medicine for perimenopause and menopause. However, the estrogenic activity of D. scandens has not yet been explored. Therefore, this study aimed to examine the estrogenic activity of the stem extract of D. scandens and its isoflavone derivatives. In this study, we conducted a proliferation assay in MCF-7 cells and qRT-PCR to assess gene expression. We found that the relative cell proliferation of compounds (1 μM) was ranked in the following order as compared to 0.1 nM 17β-estradiol (100%): genistein (97.84%) > derrisisoflavone A (83.17%) > genistein-7-O-[α-rhamnopyranosyl-(1→6)-glucopyranoside] (GTG) (69.55%) > 6,8-diprenylgenistein (51.91%) > lupalbigenin (18.72%). Furthermore, co-treatment with 1 µM lupalbigenin and 0.1 nM 17β-estradiol was performed, which decreased cell proliferation to 80.38%. In vitro results suggest that lupalbigenin has an estrogen-antagonistic effect. At a dose of 1 µM, genistein had the strongest efficacy in increasing the expression of human estrogen receptor β (ERβ) by 4.0-fold compared to the control. Furthermore, GTG augmented the gene expression of human estrogen receptor α (ERα) and ERβ by 1.5- and 3.4-fold, respectively. Prenylated derivatives of genistein (derrisisoflavone A, 6,8-diprenylgenistein, and lupalbigenin) significantly suppressed the gene expression of human androgen receptor (AR). The administration of crude extract at 10 µg/mL significantly suppressed AR (0.6-fold) and transmembrane protease serine 2 (0.1-fold) expression, but did not significantly affect ERα and ERβ gene expression. This herbal medicine may be safe for estrogen-exposed breast cancer patients.

The Expression of Adipogenic Marker Is Significantly Increased in Estrogen-Treated Lipedema Adipocytes Differentiated from Adipose Stem Cells In Vitro.

Lipedema is a chronic, idiopathic, and painful disease characterized by an excess of adipose tissue in the extremities. The goal of this study is to characterize the gene expression of estrogen receptors (ERα and ERβ), G protein-coupled estrogen receptor (GPER), and ER-metabolizing enzymes: hydroxysteroid 17-beta dehydrogenase (HSD17B1, 7, B12), cytochrome P450 (CYP19A1), hormone-sensitive lipase (LIPE), enzyme steroid sulfatase (STS), and estrogen sulfotransferase (SULT1E1), which are markers in Body Mass Index (BMI) and age-matched non-lipedema (healthy) and lipedema ASCs and spheroids. Flow cytometry and cellular proliferation assays, RT-PCR, and Western Blot techniques were used to determine the expression of ERs and estrogen-metabolizing enzymes. In 2D monolayer culture, estrogen increased the proliferation and the expression of the mesenchymal marker, CD73, in hormone-depleted (HD) healthy ASCs compared to lipedema ASCs. The expression of ERβ was significantly increased in HD lipedema ASCs and spheroids compared to corresponding healthy cells. In contrast, ERα and GPER gene expression was significantly decreased in estrogen-treated lipedema spheroids. CYP19A1 and LIPE gene expressions were significantly increased in estrogen-treated healthy ASCs and spheroids, respectively, while estrogen upregulated the expression of PPAR-ϒ2 and ERα in estrogen-treated lipedema-differentiated adipocytes and spheroids. These results indicate that estrogen may play a role in adipose tissue dysregulation in lipedema.

LSD1 inhibitor hinders the demethylation of FOXA1 to inhibit prostate cancer progression

Abnormal activation of androgen receptor (AR) occurs in prostate cancer (PC) progression and metastasis. Lysine-specific demethylase 1 (LSD1), the first demethylating enzyme, regulates AR-mediated transcriptional activity. Considering the effect of Forkhead box protein A1 (FOXA1) on the expression of AR, estrogen receptor (ER) and tumor suppressor genes, this study investigated the demethylation of FOXA1 upon treatment with LSD1 inhibitors and assessed the biological behaviors of PC cells. PC cells were cultured and infected with viruses. After transient transfection, CWR22-RV1-Cas9 cells were selected by puromycin with expression of LSD1 detected by Western blot. Apart from measurement of formaldehyde production, immunoprecipitation and chromatin immunoprecipitation (ChIP) were performed, followed by ATAC-seq detection, and Western blot. The data indicated the association between LSD1-binding sites and high levels of FOXA1. LSD1 inhibitor treatment resulted in a dramatic decline in overall FOXA1 binding, significantly reducing chromosomal accessibility and also increasing lysine-methylated FOXA1 level, but it failed to affect H3K4me2 levels at LSD1-FOXA1 occupied sites. Overexpression of LSD1-WT obtained reverse outcome. Besides, LSD1 inhibition diminished binding of FOXA1 and restored lysine-methylation of FOXA1 in methylation-deficient cells with mutant K270R. Moreover, silencing of LSD1 suppressed CWR22-RV1 tumor growth, resulting in increased H3K4me2 and decreased AR-FL/V7 gene expression. K270me is demethylated by LSD1. LSD1 inhibitor disrupts FOXA1 chromatin association, blocks FOXA1 K270-demethylation and hinders AR binding, thereby suppressing PC cell growth.

Steroid profile and ovarian gene expression in Genyatremus luteus (Teleostei, Perciformes: Haemulidae) during sexual maturation

The aim of the present study was to assess histological features, sex steroid profile, and ovarian gene expression throughout the ovarian maturation of Genyatremus luteus in Brazilian Coastal Equatorial Amazon. Specimens were categorized into different gonadal maturation stages–immature (n = 7), maturing (n = 7), mature (n = 7), and spawned (n = 7)–based on histological features of ovarian follicles and on oocyte development. Plasma E2, 11-KT and 17α-OHP levels were measured through enzyme-linked immunosorbent assays. The gene expression of estrogen (er) and LH (lhr) receptors in the ovary was analyzed through real-time polymerase chain reaction. The highest plasma E2 concentration was found in mature specimens (P< 0.05). Plasma 17α-OHP concentrations have significantly increased from the immature to the final maturation stage (P< 0.05). Maturational reproductive stage did not have any effect on plasma 11-KT concentrations. Er expression in ovaries has significantly increased throughout ovarian maturation (P < 0.05), and it remained high at mature and spawned stages. Lhr expression showed similar trend throughout the maturation process, although its peak was observed at spawned stage (P< 0.05). In conclusion, the gene expression profile of er and lhr and plasma E2 and 17α-OHP concentrations are proportional to oocyte development; they play important role in regulating ovarian development and ovulation in G. luteus.

Female sex protects against renal edema, but not lung edema, in mice with partial deletion of the endothelial barrier regulator Tie2 compared to male sex.

The endothelial angiopoietin/Tie2 system is an important regulator of endothelial permeability and targeting Tie2 reduces hemorrhagic shock-induced organ edema in males. However, sexual dimorphism of the endothelium has not been taken into account. This study investigated whether there are sex-related differences in the endothelial angiopoietin/Tie2 system and edema formation. Adult male and female heterozygous Tie2 knockout mice (Tie2+/-) and wild-type controls (Tie2+/+) were included (n = 9 per group). Renal and pulmonary injury were determined by wet/dry weight ratio and H&E staining of tissue sections. Protein levels were studied in plasma by ELISA and pulmonary and renal mRNA expression levels by RT-qPCR. In Tie2+/+ mice, females had higher circulating angiopoietin-2 (138%, p<0.05) compared to males. Gene expression of angiopoietin-1 (204%, p<0.01), angiopoietin-2 (542%, p<0.001) were higher in females compared to males in kidneys, but not in lungs. Gene expression of Tie2, Tie1 and VE-PTP were similar between males and females in both organs. Renal and pulmonary wet/dry weight ratio did not differ between Tie2+/+ females and males. Tie2+/+ females had lower circulating NGAL (41%, p<0.01) compared to males, whereas renal NGAL and KIM1 gene expression was unaffected. Interestingly, male Tie2+/- mice had 28% higher renal wet/dry weight ratio (p<0.05) compared to Tie2+/+ males, which was not observed in females nor in lungs. Partial deletion of Tie2 did not affect circulating angiopoietin-1 or angiopoietin-2, but soluble Tie2 was 44% and 53% lower in males and females, respectively, compared to Tie2+/+ mice of the same sex. Renal and pulmonary gene expression of angiopoietin-1, angiopoietin-2, estrogen receptors and other endothelial barrier regulators was comparable between Tie2+/- and Tie2+/+ mice in both sexes. Female sex seems to protect against renal, but not pulmonary edema in heterozygous Tie2 knock-out mice. This could not be explained by sex dimorphism in the endothelial angiopoietin/Tie2 system.

Cardioprotective effects of calorie restriction against inflammation and apoptosis in ovariectomized obese rats: Role of classical estrogen receptors and SIRT1

AimObesity is a metabolic complication linked with bad eating habits and a sedentary lifestyle, and the heart is one of the target organs damaged by it. Estrogen deficiency during menopause worsens the situation. Calorie restriction (CR) can contribute to reducing cardiovascular disease (CVD) in postmenopausal conditions. Thus, the effects of CR on inflammation and apoptosis in ovariectomized rats' hearts with obesity were studied. MethodFemale Wistar rats were categorized into Sham and OVX (ovariectomized) groups and received a standard diet (SD) or high-fat diet (60%HFD) or calorie restriction (30% CR) for 16 weeks. The real-time PCR method was used to evaluate the inflammatory markers and estrogen receptors gene expression. Western-blot and ELISA methods were respectively used for the measurement of apoptosis and SIRT1 protein expression. ResultsHFD led to the elevation of body weight, IL-6 (interleukin-6) and TNF-α (tumor necrosis factor-α) and reduction of IL-10 (interleukin-10) gene expressions, and also an increment in protein levels of cleaved caspase-3, Bax and Bax/Bcl2 ratio and decrement in Bcl-2 in OVX rats (P < 0.001). Additionally, HFD reduced SIRT1 (sirtuin1) protein levels, ERα (estrogen receptor α), and ERβ (estrogen receptor β) gene expressions (P < 0.001). In contrast, CR declined body weight, IL-6 and TNF-α (P < 0.001), increased IL-10 expressions (P < 0.05), decreased cleaved caspase-3 (P < 0.001), Bax (P < 0.01), and Bax/Bcl2 ratio (P < 0.05), enhanced Bcl-2 (P < 0.001), increased SIRT1 (P < 0.05) and ERα (P < 0.001) and ERβ (P < 0.01) expressions. ConclusionCR through the SIRT1 regulation and estrogen receptors attenuate obesity-induced-cardiac inflammation and apoptosis. CR can be a cardioprotective candidate in postmenopausal conditions.

The effect of hesperetin on estrogen receptor gene expression and its relationship with the downstream pathways of estrogen receptor alpha.

Estrogen receptor (ER) is a transcription factor that affects the expression of some genes involved in the progression and development of breast cancer (BC). Hesperetin (Hst) is a flavonoid that inhibits the proliferation of BC cells. In this study, we investigated the effect of Hst on the cell viability of MCF-7 cells and the gene expression of the ERα, ERβ, IL-6, Ps2, and Cyclin D1. In this study, cell viability was determined by MTT assay. The cells were seeded in RPMI-1640 medium and then exposed to different concentrations of Hst (0, 25, 50, 100, 200, and 400 µM) for 24h, and IC50 was calculated. Real-time PCR was used to assess the expression of ERα, ERβ, pS2, Cyclin D1, and IL-6 mRNA. MCF-7 cells were seeded in RPMI-1640 medium and then exposed to different concentrations of Hst (0, 25, 50, 100, and 200 µM) for 24h. Real-time PCR was carried out using a Step One Real-Time PCR System (ABI, USA) and Amplicon SYBR Green reagents. The MTT assay revealed increased cytotoxicity with higher concentrations of Hst, and the IC50 was calculated at 200 µM. Real-time PCR analysis following treatment with Hst showed a significant increase in ERα gene expression at 25 µM of Hst and a decrease in expression at 50, 100, and 200 µM of Hst (p < 0.0001). ERβ gene expression significantly decreased across all concentrations of Hst (p < 0.0001), while IL-6 gene expression decreased significantly in all concentrations (p < 0.0001). pS2 gene expression increased significantly with all concentrations of Hst (p < 0.0001), while Cyclin D1 gene expression did not significantly decrease upon Hst exposure (p > 0.05). The results of our study demonstrate that Hst has the ability to induce cell death in MCF-7 cells. Furthermore, it was observed that Hst reduces the expression of the ER gene and enhances its activity, which can affect the downstream pathways of the ER.

Localizing Hormone Receptor Expression to Cellular Compartments in Idiopathic Subglottic Stenosis.

Idiopathic subglottic stenosis (iSGS) is an unexplained progressive fibrosis of the upper airway. iSGS almost exclusively affects women; as a result, female hormones (estrogen and progesterone) have been proposed to participate in the pathogenesis of iSGS. Our aim was to localize cell-specific gene expression of estrogen receptors (ESR1 and ESR2) and progesterone receptor (PGR) using an established iSGS single-cell RNA sequencing (scRNAseq) cell atlas. Ex vivo molecular study of airway scar and healthy mucosa from iSGS patients. An established scRNAseq atlas consisting of 25,974 individually sequenced cells from subglottic scar (n = 7) or matched unaffected mucosa (n = 3) in iSGS patients was interrogated for RNA expression of ESR1, ESR2, and PGR. Results were quantified and compared across cell subsets, then visualized using Uniform Manifold Approximation and Projection (UMAP). Confirmatory protein assessment of endocrine receptors in fibroblasts from iSGS patients (n = 5) was performed via flow cytometry. The proximal airway mucosa in iSGS patients demonstrates differential expression of endocrine receptors (ESR1, ESR2, PGR). Within airway scar, endocrine receptors are primarily expressed by fibroblasts, immune cells, and endothelial cells. Fibroblasts show strong ESR1 and PGR expression, while immune cells possess RNA for both ESR1 and ESR2. Endothelial cells predominantly express ESR2. Epithelial cells in unaffected mucosa express all three receptors, which are all reduced in airway scar. scRNAseq data localized endocrine receptor expression to specific cell subsets. These results provide the foundation for future work interrogating how hormone-dependent mechanisms promote, sustain, or participate in iSGS disease pathogenesis. NA; Basic science Laryngoscope, 133:3506-3511, 2023.

High-Fat Diet Promotes Adipogenesis in Offspring Female Rats Induced by Perinatal Exposure to 4-Nonylphenol.

Both high-fat diet (HFD) and 4-nonylphenol (4-NP) could affect fat formation in adipose tissue individually. We investigated whether HFD promote abnormal adipose tissue formation caused by early exposure to 4-NP in life and preliminarily explore the possible mechanisms involved. The first-generation rats were treated with HFD on postnatal day after pregnant rats exposure to 5 ug/kg/day 4-NP. Then, the second generation rats started to only receive normal diet without 4-NP or HFD. We analyzed organ coefficient and histopathology of fat tissues, biochemical index, and gene level involved in lipid metabolism in female offspring rats. HFD and 4-NP interaction synergistically increased birth weight, body weight, and organ coefficients of adipose tissue in offspring female rats. HFD accelerately aggravated abnormal lipid metabolism and increased the adipocyte mean areas around the uterus of the offspring female rats induced by prenatal exposure to 4-NP. HFD also facilitate the regulation of gene expression involved lipid metabolism in offspring female rats induced by perinatal exposure to 4-NP, even passed on to the second generation of female rats. Moreover, HFD and 4-NP interaction synergistically declined the gene and protein expression of estrogen receptor (ER) in the adipose tissue of second-generation female rats. HFD and 4-NP synergistically regulate the expression of lipid metabolism genes in adipose tissue of F2 female rats and promote adipose tissue generation, leading to obesity in offspring rats, which is closely related to low expression of ER. Therefore, ER genes and proteins may be involved in the synergistic effect of HFD and 4-NP.

Alterations in gonadotropin, prolactin, androgen and estrogen receptor and steroidogenesis-associated gene expression in gander testes in relation to the annual period

Physiological mechanisms of seasonal changes in testicular function in birds are not fully elucidated. The balance between androgens and estrogens and testis sensitivity for gonadotropin and gonadal steroids are still unclear. The aim of the study was to examine: (1) the changes in circulating and intra-testicular steroid hormone levels and their relationship; (2) the mRNA expression of testicular gonadotropin, prolactin (PRL), progesterone (P4), androgen, and estrogen receptors, and (3) key steroidogenesis processes-related genes with immunofluorescent localization of aromatase in gander testes during the annual period. Testes from ganders (n = 25) in the first reproduction season were obtained at five breeding stages, i.e., prebreeding (PrB), peak of reproduction (PR), postbreeding (PoB), nonbreeding (NB), and onset of reproduction (OR). Males were kept under breeding conditions. It was found that plasma P4 levels decreased at the PoB and NB stages, whereas intra-testicular P4 was the highest in the NB stage. Intra-testicular estradiol (E2) levels were higher at the PoB and NB stages than the other stages, whereas testosterone (T) levels showed a nearly opposite pattern. The plasma estradiol–to–testosterone ratios were higher at the PrB, PoB and NB stages compared to other stages. The transcript abundances for luteinizing hormone receptor (LHR), PRL receptor (PRLR), estrogen receptor alpha (ERα), and estrogen receptor beta (ERβ) also change in testicular tissue during the annual period. Moreover, StAR mRNA expression was upregulated at the PoB and NB stages, and CYP11A1 transcript level was the highest at the PoB stage. Stage-dependent changes in the CYP19A1 mRNA and aromatase protein levels with higher abundances of transcript at PoB and NB stages and protein at the NB stage were observed. Localization and immunofluorescent signal intensity for aromatase also differed in relation to the examined stages. It may be suggested that differential E2 levels, as well as aromatase expression and localization across annual stages are responsible for the seasonal activation/inactivation stages of testis spermatogenesis in domestic ganders. These data strongly suggest a role of aromatase in the control of gander steroidogenesis as changes in this enzyme level are associated with alternation in gonadal steroid hormones. In addition, joint action with others hormones, like PRL and LH, seems to be important in the final effect of seasonal reproduction potential.

Abstract 5357: Estrogen receptor gene expression prediction from H&amp;E-stained whole slide images

Abstract Gene expression profiling (GEP) represents an important approach to inform breast cancer treatment. However, access to GEP involves challenges associated with cost, tissue transportation, and turn around time. In this work, we explore the prediction of estrogen receptor gene (ESR1) expression directly from images of hematoxylin and eosin (H&amp;E) stained, formalin-fixed paraffin-embedded (FFPE) breast cancer tissue. Since H&amp;E staining is a fast and inexpensive component of the standard tissue preparation in pathology, this approach is tissue preserving and requires no additional tissue processing. Our method uses a deep multiple instance learning approach to process cropped image patches from whole-slide images (WSI) into a numeric embedding vector summarizing the information in each patch. A gated attention mechanism then aggregates these embeddings into a single prediction for the WSI. We train and tune the model on a site-based split of The Cancer Genome Atlas (TCGA) BRCA dataset, and evaluate it on both a heldout split of TCGA (independent sites) and a separate dataset from a tertiary teaching hospital (TTH). All splits of TCGA have ESR1 value, immunohistochemistry (IHC) estrogen receptor (ER) status, and limited clinical outcome data. The TTH dataset has IHC-based ER status and clinical outcome, but not ESR1 expression. On the TCGA heldout test split, our model’s root mean square error (RMSE) for predicting normalized gene expression counts (TPM) was 2.90 [95% CI: 2.57, 3.23], and the Pearson correlation was 0.57 [95%CI; 0.46, 0.67]. For predicting IHC-based ER status on the same TCGA split, this weakly-supervised ESR1-predicting model had an area under the receiver-operator curve (AUROC) of 0.81 [0.74, 0.87]. This was comparable to a strongly-supervised method directly predicting ER status (AUROC: 0.85 [0.77, 0.92]). Lastly, when evaluated for association with patient outcomes (progression-free interval; PFI) using the independent TTH dataset, this ESR1-predicting model had a concordance index (c-index) of 0.59 [0.52, 0.65]. For comparison, the c-index for PFI using the IHC-based ER status for these cases was 0.61 [0.54, 0.66]. This work further demonstrates the potential to infer gene expression from H&amp;E stained images in a manner that shows meaningful associations with clinical variables. Because obtaining H&amp;E stained images is substantially easier and faster than genetic testing, the capability to derive molecular genetic information from these images may increase access to this type of information for patient risk stratification and provide research insights into molecular-morphological associations. Future work incorporating more comprehensive sets of genes remains a valuable next step. Citation Format: Anvita A. Srinivas, Ronnachai Jaroensri, Ellery Wulczyn, James H. Wren, Elaine E. Thompson, Niels Olson, Fabien Beckers, Melissa Miao, Yun Liu, Cameron Chen, David F. Steiner. Estrogen receptor gene expression prediction from H&amp;E-stained whole slide images. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5357.

Synthesis and evaluation of novel thiazole moiety-containing compounds as antibreast cancer agents.

Progesterone receptor (PR) agonists represent pivotal agents in trapping breast cancer cells through modulating the expression of estrogen receptor (ER). The present investigation aimed to test three novel thiadiazole-containing compounds as antibreast cancer agents. Test compounds were synthesized and abbreviated as 2-{(5-amino-1, 3, 4-thiazole-2-yl) amino}-4-(4-chloro-3-methylphenyl)-4-oxobutanoic acid (TAB), 4-(4-chloro-3-methylphenyl)-4-oxo 2-[(5-sulfanyl-1, 3, 4-thiadiazol-2-yl)] sulfanyl-butanoic acid (TSB) and 4-(4-chloro-3-methylphenyl)-4-oxo 2-[(5-sulfanyl-1, 3, 4-thiadiazol-2-yl)] sulphonyl-botanic acid (TSSB). Molecular docking of the test compounds with PR was simulated. The IC 50 of the test compounds against both Michigan cancer foundation-7 (MCF-7) and HepG2 was determined. Ehrlich solid tumor (EST) was grown in the right thigh of the mouse as a model of breast cancer in vivo . Hepatic and renal functions, besides hematological indicators, were tested. The expression of ER and ER genes in EST was determined using real-time PCR. Immunohistochemistry was carried out for the determination of Ki-67 and cyclin-dependent kinase 1 (CDK-1) in EST. Our results revealed that TAB, TSB and TSSB reduced Ehrlich tumor size by 48, 64 and 52%, respectively, compared to the EST control group. The docking scores achieved by TAB, TSB and TSSB with PR were -9.29, -9.41 and -9.24 kcal/mol, respectively. The most potent compound against MCF-7 was TSB, with an IC 50 of 3.9 g/ml. The administration of test compounds suppressed Ki-67 and CDK1, and the best effect was observed at TSB. Our findings suggest that test compounds are applicants to be antibreast cancer agents.

Expression of estrogen receptors, PELP1, and SRC in human spermatozoa and their associations with semen quality

Sperm cells are target cells for both estrogens and xenoestrogens. Due to the specific structure of spermatozoa, these hormonal compounds may act on sperm in a non-genomic mechanism only. However, the ESR-mediated signaling pathways are still poorly understood. In this study, we obtained 119 samples from male participants of Caucasian descent who donated semen for standard analysis. We analyzed gene expression of estrogen receptors (ESR1 and ESR2) and their coregulators—proline-, glutamic acid-, and leucine-rich protein 1 (PELP1), and cellular kinase c-Src (SRC). RNA level was established using reverse-transcribed RNA as a template, followed by a polymerase chain reaction. Proteins’ presence was confirmed by western blot and immunocytochemistry techniques. “Normal” values of semen parameters were defined as follows: > 32% sperm with progressive motility, > 4% sperm cells with normal morphology, > 15 × 106 sperm per mL, > 58% live spermatozoa and leukocyte amount < 106 cells per mL, according to WHO 2010 reference. Semen parameters that deviated from these “normal” values were labeled as “abnormal”. Gene expression ratios revealed significant, moderate, and negative correlations for ESR1/ESR2 and weak, negative ESR2/PELP1 correlations in the subgroup of patients with abnormal values of semen parameters. In addition, SRC/PELP1 was moderately and positively correlated in the subgroup with parameters within the reference values established by WHO 2010. Our study showed that both PELP1 scaffolding protein and SRC kinase might influence semen quality via ESRs. It seems that not the expression of a single gene may affect the sperm quality, but more gene-to-gene mutual ratio. Characterization of estrogen-signaling pathway-related genes’ modulated expression in sperm cells could aid in better understanding sperm biology and quality.

Exposure to acetaminophen impairs gametogenesis and fertility in zebrafish (Danio rerio).

Acetaminophen (ACE; paracetamol) is one of the most widely used nonsteroidal anti-inflammatory drugs worldwide and is often found in aquatic systems, where it can act on nontarget species and impair fish reproduction. This study aimed to investigate the effects of chronic exposure to environmentally relevant ACE concentrations (0.5, 5 and 50µg/L) on multiple reproductive parameters in zebrafish (Danio rerio). Gametogenesis was analyzed using histology, morphometry, cell proliferation, and apoptosis. This study also evaluated sex steroids, and prostaglandin E2 (PGE2) levels, gene expression for sex steroids and PGE2 receptors, fertilization rate, and semen quality. In females, exposure to 5 and 50µg/L ACE induced larger and more abundant vitellogenic follicles and increased follicular atresia. In these treatments, males showed a lower proportion and proliferation of undifferentiated spermatogonia and a higher proportion of TUNEL-positive differentiated spermatogonia, spermatids, and spermatozoa, resulting in lower sperm production. ACE increased 17β-estradiol (E2) and reduced 11-ketotestosterone levels in the testis, whereas only E2 increased in the ovaries. In both sexes, gonadal PGE2 levels were reduced. ACE at 50µg/L induced an increase in the gene expression of androgen, estrogen, and PGE2 receptors in the ovaries, and reduced expression in the testes. Results also showed lower egg production and fertilization rate from 28days of exposure with reduced sperm quality. These results demonstrated that ACE impairs the reproductive performance of zebrafish, affecting multiple reproductive parameters, which may be caused by the synergistic action of the imbalance of sex steroids, with a reduction of PGE2 and its receptors.

Identification of estrogen receptor down-regulators for endocrine resistant breast cancer

Resistance to endocrine therapies remains an impediment for the treatment of estrogen receptor (ER) positive breast cancer. ER down regulator Fulvestrant has showed great activity to overcome the endocrine resistance. However, Fulvestrant has poor bioavailability due to the hydrophobicity. Identification of novel ER down regulator is still important. Compounds 172 and 183 are two steroidal compounds with androgen scaffold but significantly down regulated ER in multiple breast cancer cell lines. RT-PCR results indicated that both compounds did not affect ER gene expression. Proteasome inhibitor MG132 could attenuate ER down regulation effect of the compounds, suggesting that the ER down regulation was via ubiquitin-proteasomal pathway. Furthermore, compounds 172 and 183 could downregulate ER in endocrine resistant breast cancer cell model long term estrogen deprivation (LTED) MCF-7 cells. Hydrophobicity of compounds 172 and 183 were determined and showed improved solubility compared to Fulvestrant. All these results suggested that compounds 172 and 183 could be potential lead compounds for drug development for the treatment of endocrine resistance breast cancer.

Gene expression of sex steroid metabolizing enzymes and receptors in the skeletal muscle of migrant and resident subspecies of white-crowned sparrow (Zonotrichia leucophrys).

Circulating sex steroid concentrations vary dramatically across the year in seasonally breeding animals. The ability of circulating sex steroids to effect muscle function can be modulated by changes in intracellular expression of steroid metabolizing enzymes (e.g., 5α-reductase type 2 and aromatase) and receptors. Together, these combined changes in plasma hormones, metabolizing enzymes and receptors allow for seasonally appropriate changes in skeletal muscle function. We tested the hypothesis that gene expression of sex steroid metabolizing enzymes and receptors would vary seasonally in skeletal muscle and these changes would differ between a migrant and resident life history strategy. We quantified annual changes in plasma testosterone and gene expression in pectoralis and gastrocnemius skeletal muscles using quantitative polymerase chain reaction (qPCR) in free-living migrant (Zonotrichia leucophrys gambelii) and resident (Z. l. nuttalli) subspecies of white-crowned sparrow during breeding, pre-basic molt, and wintering life history stages. Pectoralis muscle profile was largest in migrants during breeding, while residents maintained large muscle profiles year-round. Circulating testosterone peaked during breeding in both subspecies. Pectoralis muscle androgen receptor mRNA expression was lower in females of both subspecies during breeding. Estrogen receptor-α expression was higher in the pectoralis muscle, but not gastrocnemius, of residents throughout the annual cycle when compared to migrants. Pectoralis aromatase expression was higher in resident males compared to migrant males. No differences were observed for 5α-reductase 2. Between these two subspecies, patterns of plasma testosterone and androgen receptors appear to be conserved, however estrogen receptor gene expression appears to have diverged.

Effect of Swimming on the Induction of Vitellogenin in Conger Eel (Conger myriaster)

Vitellogenin (VTG) plays a very important role in the development of oocytes. This study aims to explore the effect of swimming on the VTG synthesis of conger eel (Conger myriaster). Circulating water flow and hormone injection regimen were two factors set in the following trials: A1 (no injection, no water flow), A2 (no injection, water flow), B1 (injection, no water flow), and B2 (injection, water flow). The flow velocity of the flowing water was 0.4 m/s (12 h, 60 days). We examined VTG, estrogen receptor (Er) gene expression, and VTG content in the liver and serum on the 30th and 60th days. VTG gene expression in A1 and A2 gradually decreased and was not significantly different between the two groups. The expression of Er gene in A1 was significantly higher than that in A2. The expression of VTG gene in group B1 was significantly higher than that in group B2, whereas the expression level of Er gene in group B1 was slightly higher than that in group B1. The VTG content in the liver in group B1 reached 1,396.93 μg/L, which was significantly higher than that in group B2 (1,302.06 μg/L). Results showed that the flowing water factor can inhibit the expression of VTG and Er genes in the liver and reduce the synthesis of VTG in the liver. Stimulation of flowing water can inhibit the yolk accumulation during the ovarian development of conger eel.

In vivo arterial stiffness, but not isolated artery endothelial function, varies with the mouse estrus cycle

Due to the increasing inclusion of female mice in research, it is important to understand the influence of the estrus cycle on vascular function. In humans, endothelial function and arterial stiffness vary throughout the menstrual cycle; however, these functional changes have yet to be investigated for the mouse estrus cycle. During the estrus cycle, circulating estrogen is highest during the proestrus and estrus phases and lowest during diestrus, while progesterone peaks during estrus. Estrogens bind to estrogen receptor α (ERα), estrogen receptor β (ERβ), and estrogen G‐protein coupled receptor (GPR30) in vascular cells. Activation of ERα and GPR30 increases nitric oxide (NO) bioavailability. The purpose of this study was to determine the effect of the estrus cycle phase on arterial stiffness, vascular endothelial function, and estrogen receptor expression.In young female C57Bl/6J mice (n=23; 5.96 ± 0.04 months), we determined the estrus cycle via vaginal cytology and plasma hormone concentrations. We assessed large artery stiffness via aortic pulse wave velocity (PWV). Endothelial function was assessed by dose responses to acetylcholine (ACh) and insulin in pressurized mesenteric arteries (MAs) and posterior cerebral arteries (PCAs). NO‐mediated dilation was determined by the difference in vasodilation to ACh in the presence vs. absence of the NO synthase inhibitor L‐NAME. Endothelium independent dilation was assessed by the dose responses to sodium nitroprusside. We quantified gene expression of ERα (Esr1), ERβ (Esr2), and GPR30 (Gper1) in MAs and PCAs.Plasma progesterone concentrations were higher for mice in estrus (5.7 ± 1.1 ng/mL) compared to proestrus (2.5 ± 0.6 ng/mL; p=0.04) and tended to be higher in diestrus (3.9 ± 1.1 ng/mL, p=0.14), verifying the cytology classifications. Plasma 17β‐estradiol and estrone were below detection in most mice. There was a higher plasma testosterone concentration during diestrus (0.08 ± 0.02 ng/mL) compared to other stages (estrus 0.02 ± 0.00 ng/mL, p=0.006; proestrus 0.01 ± 0.00 ng/mL, p=0.001).Aortic PWV was lower for mice in estrus compared with mice in diestrus (3.0 ± 0.1 vs. 3.4 ± 0.1 m/s, p=0.005). Aortic PWV for mice in proestrus did not differ from the other stages (3.2 ± 0.1 m/s, p&gt;0.05). The MA and PCA responses to ACh, insulin and sodium nitroprusside, as well as NO‐mediated dilation, were not different between estrus cycle phases (p&gt;0.05). PCA Esr2 gene expression for mice in estrus was 57% lower than diestrus (p=0.008) and 54% lower than proestrus (p=0.04). Gene expression did not differ between estrus cycle phases for Esr1 and Gper1 in either the MA or PCA (p&gt;0.05), and MA Esr2 was not detectable.In summary, we found that the estrus phase in mice is associated with lower in vivo large artery stiffness. In contrast, there were no differences between estrus cycle phase in ex vivo resistance artery endothelial function. Changes to estrogen receptor gene expression during the estrus cycle are limited to ERβ in the cerebral arteries. These results suggest that estrus phase should be considered when measuring in vivo arterial stiffness in young female mice.

The Overexpression and Clinical Significance of AP1S1 in Breast Cancer.

PurposeTo explore the mechanism of AP1S1 in breast cancer.Methods and ResultsIn different datasets, we found that AP1S1 is more highly expressed in breast tumors, which was furthermore verified in our local cohort.Immune infiltration analysis showed that AP1S1 was related to a variety of immune cells. The higher AP1S1 expression was negatively correlated with a variety of immune infiltrating cells, suggesting that AP1S1 may affect cellular immunity.Clinical analysis showed that patients with higher AP1S1 expression had higher estrogen receptor gene expression and were more prone to distant metastasis and lymph node metastasis.The overall survival rate, disease-specific survival rate, and progression-free interval time were worse in the group with higher AP1S1 expression. AP1S1 may be a potential oncogene of breast cancer, and overexpression is related to the poor prognosis of breast cancer.Therefore, a nomogram was constructed, along with correlated gene analysis and functional analysis to further explore the carcinogenic mechanism, practical clinical issues, and value of AP1S1.ConclusionAP1S1 is a potential oncogene of breast cancer.

Abstract P1-06-07: Enhanced differentiation of clinical behavior by breast carcinomas utilizing quantification of estrogen and progestin receptor proteins and gene expression subsets

Abstract Background: IHC analyses of clinical biomarkers for breast cancer management yield semi-quantitative results potentially complicated by variation in methods and interpretation. We are mining a comprehensive database established over four decades that contains deidentified results of quantified protein and RNA biomarkers with clinical outcomes to detect candidates for companion diagnostics and molecular targets. Assays were performed in a Reference Laboratory holding both CLIA and Commonwealth of Kentucky licenses. Methods: This retrospective investigation utilized ER/PR protein levels, expressed as fmol/mg cytosol protein, that were quantified for each of 2339 carcinoma biopsies with FDA-approved kits and cut-off values by radio-ligand binding assay (LBA, NEN/DuPont) and/or enzyme immunoassay (EIA, Abbott Labs). Of these, 454 specimens had both LBA and EIA results recorded. Biomarker results and de-identified clinical outcomes were examined using REMARK criteria. Gene expression, quantified biomarkers, features of primary breast cancers and clinical patient outcomes were analyzed by univariable and multivariable Cox regressions, Fisher’s Exact Test, Kaplan Meier plots and with R software v4.0.0. Microarray results of ~22,000 genes were obtained from RNA of LCM-procured breast carcinoma cells of 247 primary breast biopsies and expression levels of 85 cancer-associated genes were determined by RTqPCR of 278 specimens after RNA extraction and purification with established procedures. Results: Quantified levels of both ER and PR protein were expressed to a greater extent in primary carcinomas from post-menopausal patients compared to cancers from premenopausal women regardless of assay type (p = 0.005). ESR1 expression by microarray was only greater in LCM-procured carcinoma cells of post-menopausal patients while both ESR1 and PGR relative expression levels measured by qPCR were greater in tissue biopsies of post-menopausal women (p = 0.05). Box plots of untransformed EIA/LBA results of either ER (n=454) or PR (n=452) using specimens on which both quantifying assays were performed revealed a significant positive ER bias by EIA (median ratio=1.54) compared to that of PR (median ration=1.13). Transformed data of ratios confirmed this observation. Kaplan Meier plots of patient outcomes with ER or PR or both assessed by each assay type suggested LBA was a slightly better predictor. Microarray analyses of LCM-procured cells revealed 2994 genes distinguishing ER+ from ER- breast cancers of which ~200 genes were identified that differentiated 4 distinct molecular subtypes (2 ER+ and 2 ER-) exhibiting different outcomes upon Kaplan-Meier analyses. Expression levels of the 85 genes (including ERBB2), validated by qPCR with RNA from biopsy tissue sections, were evaluated relative to quantified levels of either ER or PR protein or both in primary breast cancer biopsies. Collectively, use of quantified ER/PR protein or ESR1/PGR expression with clinical outcomes enhanced identification of small molecular signatures and drug development candidates. Conclusions: Measurements of ER, PR and HER2 proteins with tests that quantify their levels as well as by microarray and qPCR assessment of ESR1, PGR and ERBB2 relative gene expression were correlated with prediction of clinical outcomes of breast carcinomas exhibiting various features (e.g., pathology classification, TNBC). Results reinforced importance of assessing levels of the clinically relevant biomarkers in a quantified fashion to enhance their use in breast cancer management and prediction of risk of recurrence. These investigations also revealed numerous over-expressed genes in carcinomas with poor clinical outcomes suggesting candidates for development of novel therapeutics. Citation Format: James L Wittliff, Michael W. Daniels. Enhanced differentiation of clinical behavior by breast carcinomas utilizing quantification of estrogen and progestin receptor proteins and gene expression subsets [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-06-07.