ERα Protein Expression Research Articles

Influence of genistein and diadizine on regularity of estrous cycle in cyclic female Wistar rat: interaction with estradiol receptors and vascular endothelial growth factor.

Isoflavones are estrogenic compounds that exist in soy, clover, and peanuts. They are selective estrogen receptor modulators. The study was planned to explain the interactions of isoflavones with estrogen receptors alpha (ERα), beta (ERβ), and vascular endothelial growth factor (VEGF) expressions in ovarian and uterine tissues during different stages of the estrous cycle of regular cyclic female Wistar rats. Thirty-two regular cyclic females were divided equally into control group: fed casein-based diet and isoflavones group: fed casein-based diet and gavaged 50 mg/kg/day soy isoflavones extract 40%. The regularity of estrus cycles was monitored. Final body weight (FBW), weight gain (BWG), and ovarian and uterine weights were estimated. Histopathology and immunohistochemistry for ERα, Erβ, and VEGF in ovarian and uterine tissues were performed. All females (100%, n = 16) in control group showed regularity in estrous cycle compared to 62.5% (n = 10) in isoflavones group. Estrus and diestrus phases revealed prolongation and shortening in isoflavones rats than control, respectively. Nonsignificant variation was noted in the duration of the whole cycle of both groups. FBW and BWG significantly decreased however, ovarian and uterine weights increased significantly in all estrous phases of isoflavones group than control. Histopathology demonstrated an increase in number of follicles/ovaries besides, hyperplasia and proliferation of luminal epithelium with hydropic degeneration in the isoflavones group. Also, uterine connective tissue stroma showed edema in the isoflavones group during all estrous phases. Immunostaining percentages of ERα, Erβ, and VEGF protein expression were significantly elevated in the isoflavones group during all estrous phases. Isoflavones induced irregularity of the estrous cycle that was encountered by increased and altered ERα, Erβ, and VEGF expressions in ovarian and uterine tissues.

Effect of vitamin D3 on hyperplasia of mammary glands in experimental rats.

1,25(OH)2D3/vitamin D3 receptor (VD3/VDR) signaling pathway can inhibit the occurrence of breast cancer in many ways. We used vitamin D3 to interfere with Hyperplasia of mammary glands (HMG) model rats, and to explore the intervention effect of vitamin D3 on HMG. We divided 42 female rats into six groups randomly: blank control group, hyperplasia model group, negative control group, and vitamin D3 (VD3) groups of low-dose (LVD, 5 µg/kg), medium-dose (MVD, 10 µg/kg), and high-dose (HVD, 20 µg/kg). We established HMG model in all groups except for the blank control group, then different dose of VD3 was intraperitoneal injected for VD3 groups and normal saline (NS) was given to the negative control group. After the experiment, the body weights, heights and diameters of nipples, and the thickness of the mammary gland of rats were measured. The serum content of sex hormone and VD3 were detected by enzyme-linked immunosorbent assay (ELISA). The tissues of mammary glands were analyzed by hematoxylin and eosin (HE) stain, and the expression of estrogen receptor α (ERα), progesterone receptor (PR), and VDR were detected by immunohistochemical (IHC) stain. Similarly, the total protein expression of ERα, PR, and VDR were measured by western blot. Compared with the hyperplasia group, rats in the VD3 groups displayed a marked decrease of the thickness of the mammary glands and the height and diameter of the nipples. The serum estrogen (E2), testosterone (T), luteinizing hormone (LH), and VD3 was markedly decreased in all VD3 groups (P<0.05). The IHC results showed that ERα and PR was decreased in all three VD3 groups; however, VDR was increased. Western blot demonstrated that both ERα and PR were reduced in VD3 groups, while the VDR level was significantly enhanced. Overall, the findings suggested that VD3 could be used in HMG treatment. Supplementation of VD3 could markedly decrease the mammary gland thickness, nipple diameter, and nipple height of rats, accompanied by the decrease of serum E2, T, and LH. Intervention with VD3 can lead to decreased expression of ERa and PR, in conjunction with the increase of VDR.

Effect of 17β-estradiol on the proliferation of condylar chondrocytes.

To study the effects of 17β-estradiol (E2) on the regulation of the proliferation of condylar chondrocytes and provide a preliminary discussion on the role of phosphorylate-mammalian target of rapamycin (p-mTOR) in this regulatory process. Condylar chondrocytes were isolated from 6-week-old female rats for primary culture. Drug treatment with different concentrations of E2 and/or rapamycin (RAPA) was carried out on second-generation cells. Cell Counting Kit 8 was used to measure the cell viability of condylar chondrocytes after culture for 24, 48, or 72 h, and reverse transcription-polymerase chain reaction (RT-PCR) was applied to detect the relative gene expression of estrogen receptor alpha (ERα), estrogen receptor beta (ERβ), collagen type Ⅱ (COLⅡ), autophagy-related gene 6 (Beclin-1), and autophagy-related gene 5 (ATG-5). Western blot was employed to determine the relative protein expression of ERα, ERβ, Beclin-1, lipid-modified light chain 3B (LC3-Ⅱ), and p-mTOR. E2 could significantly promote the proliferation of chondrocytes cultured in vitro, and maximum promotion was achieved at a concentration of 10-8 mol·L-1. RAPA could significantly inhibit cell proliferation. E2 at aconcentration of 10-8 mol·L-1 could greatly improve the gene expression levels of ERα and COLⅡ (P<0.01) with the protein levels of ERα and p-mTOR (P<0.05), and decrease the gene expression levels of Beclin-1 and ATG-5 (P<0.05) with the protein levels of Beclin-1 and LC3-Ⅱ (P<0.05). RAPA could also enhance the relative protein expression of Beclin-1 and LC3-Ⅱ (P<0.01), and reduce the expression of p-mTOR (P<0.01). Treatment with the ERα antagonist significantly reduced the expression of p-mTOR in cells (P<0.01). At a concentration of 10-8 mol·L-1, E2 could effectively activate the phosphorylation of mTOR through the ERα-p-mTOR pathway, inhibit cell autophagy, and promote the proliferation of condylar chondrocytes.

Enhanced IFNα Signaling Promotes Ligand-Independent Activation of ERα to Promote Aromatase Inhibitor Resistance in Breast Cancer.

Simple SummaryInterferon alpha (IFNα) signaling is highly upregulated in ER+ breast cancers that become resistant to estrogen deprivation therapy. This study uncovers how enhanced (IFNα)/JAK-STAT signaling directly influences estrogen receptor (ERα) activation in the absence of estrogen. We found that inhibiting IFNα signaling downregulates expression and activation of ERα. In addition, STAT1 and ERα directly interact and regulate a key interferon-stimulated gene, IFITM1, in AI-resistant breast cancer cells. We demonstrate that crosstalk occurs between IFNα and ERα pathways, which contributes to aggression and survival of AI-resistant breast cancer, thus representing a novel mechanism of acquired AI resistance.Aromatase inhibitors (AIs) reduce estrogen levels up to 98% as the standard practice to treat postmenopausal women with estrogen receptor-positive (ER+) breast cancer. However, approximately 30% of ER+ breast cancers develop resistance to treatment. Enhanced interferon-alpha (IFNα) signaling is upregulated in breast cancers resistant to AIs, which drives expression of a key regulator of survival, interferon-induced transmembrane protein 1 (IFITM1). However, how upregulated IFNα signaling mediates AI resistance is unknown. In this study, we utilized MCF-7:5C cells, a breast cancer cell model of AI resistance, and demonstrate that these cells exhibit enhanced IFNα signaling and ligand-independent activation of the estrogen receptor (ERα). Experiments demonstrated that STAT1, the mediator of intracellular signaling for IFNα, can interact directly with ERα. Notably, inhibition of IFNα signaling significantly reduced ERα protein expression and ER-regulated genes. In addition, loss of ERα suppressed IFITM1 expression, which was associated with cell death. Notably, chromatin immunoprecipitation experiments validated that both ERα and STAT1 associate with ERE sequences in the IFITM1 promoter. Overall, hyperactivation of IFNα signaling enhances ligand-independent activation of ERα, which promotes ER-regulated, and interferon stimulated gene expression to promote survival in AI-resistant breast cancer cells.

Ameliorative Effects of Pueraria lobata Extract on Postmenopausal Symptoms through Promoting Estrogenic Activity and Bone Markers in Ovariectomized Rats.

Pueraria lobata (Willd.) Ohwi, known as kudzu, is one of the most popular traditional medicines in Asian countries. It has been widely used as a natural alternative to hormone replacement therapy for treating postmenopausal symptoms. This study aimed to investigate the estrogenic effect of P. lobata extract (PE) against postmenopausal osteoporosis in ovariectomized (OVX) rats. OVX rats were treated with PE (25–1600 mg/kg) for 8 weeks. Biochemical parameters, estradiol, and bone turnover markers (e.g., osteocalcin, C-terminal telopeptide fragment of type I collagen, deoxypyridinoline, and pyridinoline) were measured in plasma samples. In addition, estrogen receptor-alpha (ER-α) protein expression and morphology of uterine were evaluated. Long-term treatment with PE did not cause liver damage in OVX rats. PE supplementation reduced body weight gain in obese rats with high lipid accumulation induced by ovariectomy. Furthermore, PE exhibited a protective effect against insulin resistance, hyperlipidemia, and hepatic lipid peroxidation. PE treatment increased uterine weight and thickness of the uterine layers in cases of uterus atrophy due to removal of ovaries. The levels of bone turnover markers, which were significantly increased in OVX rats, were decreased by PE treatment. Western blotting analysis showed that ER-α protein expression was upregulated in PE-treated rats compared with OVX rats. These results suggest that PE could be a promising alternative functional food for improving menopausal symptoms.

Differential expression of estrogen receptor subtypes in ovarian high-grade serous carcinoma and clear cell carcinoma.

PurposeTo investigate the role of estrogen receptors (ERs) in high‐grade serous carcinoma (HGSC) and clear cell carcinoma (CCC) of the ovary and evaluate ERs as prognostic biomarkers for ovarian cancer.MethodsThis study included 79 patients with HGSC (n = 38) or CCC (n = 41) treated at our institution between 2005 and 2014. Immunohistochemistry examined protein expression of ERα, ERβ, and G protein‐coupled estrogen receptor‐1 (GPER‐1); relationships between ERα, ERβ, and GPER‐1 with patient survival were evaluated. Additionally, cell proliferation assay and phosphokinase proteome profiling were performed.ResultsIn HGSC patients, expression of ERα, cytoplasmic GPER‐1, or nuclear GPER‐1 was associated with poor progression‐free survival (PFS) (P = .041, P = .010, or P = .013, respectively). Cytoplasmic GPER‐1 was an independent prognostic factor for PFS in HGSC patients (HR = 2.83, 95% CI = 1.03‐9.16, P = .007). ER expressions were not associated with prognosis in CCC patients. GPER‐1 knockdown by siRNA reduced the cells number to 60% of siRNA‐control‐treated cells (P < .05), and GPER‐1 antagonist, G‐15 inhibited two HGSC cell lines proliferation (KF and UWB1.289) in a dose‐dependent manner. Phosphoprotein array revealed that GPER‐1 silencing decreased relative phosphorylation of glycogen synthase kinase‐3.ConclusionsHigh GPER‐1 expression is an independent prognostic factor for PFS in HGSC patients, and GPER‐1 may play a role in HGSC cell proliferation.

Abstract 2125: Roles of KAT6A and KAT6B in the biology of estrogen positive breast cancer

Abstract KAT6A and KAT6B are histone acetyl transferase (HAT) that are controlling gene expression by transferring acetyl groups to histones. Yu et al (Oncogene, 2017 36, 2910-2918) have shown that KAT6A has a role in breast cancer development by activating the ERα promoter and was found frequently amplified in 11% and is overexpressed in 15% of breast cancers. Altogether, these data support KAT6A as an attractive target in estrogen receptor driven breast cancer. KAT6B is a close homolog that shares 91% identity in the acetyl coA binding site. While the prevalence of gene amplification for KAT6B in breast cancer is only 1-2%, it is often overexpressed. In this study, we are exploring how KAT6A and KAT6B are contributing to ERα expression and the growth of KAT6A amplified breast cancer cell lines. To decipher each protein's role, we have designed specific siRNA against KAT6A and KAT6B and we tested their action either individually or in combination in the KAT6A gene amplified breast cancer cell lines T-47D, CAM-1 and ZR-75. Following treatment of T-47D, CAMA-1 and ZR75 cell lines with KAT6A or B siRNA we observed a decrease of ERα gene and protein expression for all siRNAs. The effect was more pronounced when using KAT6A over KAT6B and the effect of both siRNA A/B were additive. Phenotypically, the impact on cell line proliferation by applying clonogenic and incucyte assays, mirrors what is observed on ERα gene and protein expression. The effect on cellular proliferation of KAT6A is more prominent than KAT6B silencing, while the combined KAT6A + KAT6B silencing being more impactful than KAT6A silencing alone. In conclusion, our data show that KAT6A and KAT6B share similar function in the control of ER gene and protein expression. This study suggests that KAT6A and KAT6B may be paralogs, one compensating for the loss of the other. This translates into a more pronounced antiproliferative effect when both genes are silenced concomitantly. Citation Format: Isabelle Meaux, Dimitri Gorge-Bernat, Angele Paz, Catherine Geslin, Laurent Debussche, Jürgen Moll, Christophe Henry. Roles of KAT6A and KAT6B in the biology of estrogen positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2125.

Presence of estrogen and progesterone receptors in proliferating and involuting infantile hemangiomas

Studies in the literature have demonstrated the presence of sex hormone receptors in infantile hemangiomas (IHs), but further investigation is needed to determine the role of these receptors in their proliferation and involution. To date, there are no studies in the literature that aimed to quantitatively examine the expression of sex hormone receptors throughout the different phases of hemangioma development. The objective of our study was to quantitatively evaluate the expression of estrogen (ER) and progesterone (PR) receptors in the proliferative and involuting phases of IHs through the use of real-time polymerase chain reaction (RT-PCR). Twenty IHs (10 proliferating and 10 involuting) were harvested and prepared for molecular investigation. ER receptor alpha (ERα) and beta (ERβ) and the PR expression were examined by RT-PCR and western blot. RT-PCR analysis demonstrated that mRNA expression of ERα, ERβ, and PR was significantly lower in proliferating versus involuting IH. Western blot analysis revealed increased protein expression of ERα in involuting hemangiomas as compared to proliferating ones. Our study demonstrates the variable expression of ER and PR receptors in proliferating and involuting hemangiomas. Further studies are needed to determine the exact role of these hormone receptors in the growth and involution of IHs.

Tributyltin Exposure Is Associated With Recognition Memory Impairments, Alterations in Estrogen Receptor α Protein Levels, and Oxidative Stress in the Brain of Female Mice.

Tributyltin (TBT) is a persistent organometallic pollutant widely used in several agricultural and industrial processes. TBT exposure is associated with various metabolic, reproductive, immune, and cardiovascular abnormalities. However, few studies have evaluated the effects of TBT on behavior. In the present study, we aimed to investigate whether TBT exposure results in oxidative, neuroendocrine, and behavioral alterations. TBT was administered to adult female mice (250, 500, or 750 ng/kg/day or veh for 14 days), and their recognition memory was assessed. We have also evaluated estrogen receptor (ER)α protein expression and oxidative stress (OS) in brain areas related to memory, as well as the correlation between them. A reduction in short- and long-term recognition memory (STM and LTM) performance, as well as in total exploration time was observed in TBT mice. Reduced ERα protein expression was observed in the prefrontal cortex (PFC) and hippocampus of TBT mice, while an increase in TBARS concentration was observed in the PFC of treated animals. Collectively, these data suggest that TBT exposure impairs recognition memory in female mice as a result of, at least in part, its toxicological effects on ERα expression and OS in specific brain areas related to memory.

Estrogen modulates epithelial progenitor cells in rat vagina.

PurposeThe expression of epithelial progenitor cells (EPCs) in rat vagina was recently reported. The aims were to investigate the effects of estrogen on vaginal EPCs in the oophorectomized female rat model.Materials and MethodsFemale Sprague-Dawley rats (230–240 g, n=30) were divided into 3 groups: control (n=10), bilateral oophorectomy (OVX, n=10), and bilateral OVX followed by subcutaneous injections of 17β-estradiol (50 µg/kg/day, n=10). After 4 weeks, the expression of EPC-specific markers (CD44, estrogen receptor alpha [ERα], and progesterone receptor) were evaluated by immunohistochemistry and Western blot.ResultsThe CD44/ERα double-labeled cells were mainly expressed in basal cell layers and suprabasal layers as shown by confocal immunofluorescence. Confocal microscopy revealed that the number of CD44+/ERα+ cells decreased in the OVX group compared with the controls but was similar to control levels in rats receiving estrogen replacements. The protein expression of CD44 and ERα decreased after OVX and was restored to control levels after estrogen supplementation.ConclusionsMarkers of EPCs were expressed in the vagina, and the expression of resident EPCs was regulated by estrogen. These findings imply that resident EPCs may have an important role in the regeneration of vaginal mucosa by estrogen replacement.

Low-magnitude vibration promotes osteogenesis of osteoblasts in ovariectomized osteoporotic rats via the estrogen receptor α

The purpose of this study was to investigate the effect of low-magnitude vibration on osteogenesis of osteoblasts in ovariectomized rats with osteoporosis via estrogen receptor α(ERα). The mRNA expression of osteogenic markers were examined with qRT-PCR, based on which the optimal vibration parameter for promoting osteogenesis was determined (45 Hz × 0.9 g, g = 9.8 m/s2). Then we loaded the optimal vibration parameter on the osteoblasts of ovariectomized rats with osteoporosis. The protein expression of osteogenic markers and ERα were detected with Western blot; the distribution of ERα was examined with immunofluorescence technique. Finally, through inhibiting the expression of ERα with estrogen receptor inhibitor ICI182780, the protein and mRNA expression of osteogenic markers were examined. First, the results showed that low-magnitude vibration could promote the expression of osteogenic markers and ERα in osteoblasts of ovariectomized rats with osteoporosis (P < 0.05), and make ERα transfer to the nucleus. On the other hand, the results also showed that after inhibiting the expression of ERα in osteoblasts of ovariectomized rats with osteoporosis, the protein and mRNA expression of osteogenic marker were decreased (P < 0.05). In our study, low-magnitude vibration played an important role in the osteogenesis of osteoblasts in ovariectomized rats with osteoporosis through increasing the expression and causing translocation of ERα. Furthermore, it provides a theoretical basis for the application of low-magnitude vibration in the prevention and treatment of postmenopausal osteoporosis.

Enhanced in vivo targeting of estrogen receptor alpha signaling in murine mammary adenocarcinoma by nilotinib/rosuvastatin novel combination

The development of resistance to endocrine therapy of estrogen receptor alpha (ERα)-positive breast cancer is inevitable, necessitating the introduction of alternative treatment strategies. Therefore, the current study was carried out to investigate the in vivo efficacy and tolerability of nilotinib/rosuvastatin novel combination against ERα-positive breast carcinoma. Results showed that treatment of tumor-bearing mice with nilotinib/rosuvastatin exerted a significant antitumor activity. Mechanistically, the combination treatment efficiently inhibited the in vivo ERα protein expression, whereas ERα mRNA levels were unaffected suggesting a posttranslational regulation. In addition, the combination treatment markedly downregulated the expression of two ERα downstream target genes: C3 and pS2 confirming the inhibition of ERα signaling in vivo. Further, nilotinib/rosuvastatin combination strongly induced apoptosis evidenced by a marked caspase-3 cleavage and downregulation of tumor nitric oxide levels. Moreover, histopathology showed significant declines in mitotic figures and tumor giant cells implying the in vivo capability of the combination treatment to interfere with cancer cell proliferation and persistence. Of note, the combination treatment abrogated nilotinib-induced hypercholesterolemia and did not adversely affect the liver function or body weight. Overall, the present study provided evidences that warrant further assessment of nilotinib/rosuvastatin combination as an alternative therapeutic modality for ERα-positive breast cancer.

Perinatal exposure to glyphosate or a glyphosate-based formulation disrupts hormonal and uterine milieu during the receptive state in rats

We investigated the effects of perinatal exposure to a glyphosate-based herbicide (GBH) or glyphosate alone (Gly) on female fertility and the hormonal and uterine milieu during the preimplantation period. F0 pregnant rats orally received a GBH or Gly in a dose of 2 mg of glyphosate/kg/day from gestational day (GD) 9 until weaning. F1 females were evaluated to determine the reproductive performance on GD19; and the sex steroid serum levels, the expression of estrogen receptor alpha (ERα), progesterone receptor (PR) and implantation-related genes on GD5 (preimplantation period). GBH and Gly induced preimplantation losses in F1 rats. GBH and Gly groups exhibited higher 17β-estradiol serum levels, without changes in progesterone. Both compounds increased the uterine ERα protein expression, with no differences at transcript level; and only Gly decreased PR mRNA expression. Also, GBH and Gly downregulated Hoxa10 and Lif genes, with no difference in Muc1 and Areg expression. To conclude, perinatal exposure to a GBH or Gly disrupted critical hormonal and uterine molecular targets during the receptive state, possibly associated with the implantation failures. Overall, similar results were found in GBH- and Gly-exposed rats, suggesting that the active principle might be the main responsible for the deleterious effects.

β-estradiol adjusts intestinal function via ERβ and GPR30 mediated PI3K/AKT signaling activation to alleviate postmenopausal dyslipidemia

Decreases in estrogen secretion and estrogen receptor function lead to an increase in the incidence of dyslipidemia and cardiovascular disease (CVD) in postmenopausal women. We previously reported that β-estradiol has a significant regulatory effect on lipids in ApoE−/− mice with bilateral ovariectomy. In the present study, we investigated how β-estradiol regulates intestinal function via estrogen receptors to alleviate postmenopausal dyslipidemia. Ovariectomized ApoE−/− mice were treated with β-estradiol for 90 days, and we found that β-estradiol reduced TC, TG, LDL-c, IL-1β and IL-18 levels in serum and decreased lipid accumulation in the liver. β-estradiol reduced injury and inflammation in the jejunum in ovariectomized mice, and promoted the expression of tight junction-related proteins. Moreover, β-estradiol increased ERα, ERβ, GPR30 and ABCG5 protein expression, and decreased the levels of NPC1L1 and SR-B1 in the jejunum of ovariectomized mice. In Caco-2 cells incubated with cholesterol, β-estradiol up-regulated PI3K/AKT signaling, reduced cholesterol accumulation, suppressed inflammatory signaling, and increased the expression of tight junction-related proteins. ERβ or GPR30 inhibition decreased the protective effect of β-estradiol on cholesterol accumulation, tight junctions, and inflammation in cholesterol incubated Caco-2 cells, while silencing both ERβ and GPR30 completely eliminated the protective effect of β-estradiol. PI3K/AKT inhibition abolished the protective effect of β-estradiol on cholesterol accumulation, tight junction-related protein expression, and inflammation, but had no influence on ERα, ERβ or GPR30 expression in cholesterol incubated Caco-2 cells. Our results provide evidence that β-estradiol regulates intestinal function via ERβ and GPR30 mediated PI3K/AKT signaling activation to alleviate postmenopausal dyslipidemia.

The Interaction Test of Binary Mixtures of Endocrine-Disrupting Chemicals Using In Vitro Bioassays

Typical environmental endocrine-disrupting chemicals (EDCs) such as estradiol valerate (EV), diethylstilbestrol (DES), di-2-ethylhexyl phthalate (DEHP), mono-2-ethylhexyl phthalate (MEHP), and bisphenol A (BPA) have a strong reproductive and developmental toxicity at low concentrations. However, information on their joint toxicity is scarce. In this study, we evaluated the combined effects of EV and other four EDCs (DES, DEHP, MEHP, and BPA) on the human breast MCF-7 cells by detecting the cell proliferation, intracellular reactive oxygen species (ROS) levels, and estrogen receptor alpha (ERα) protein expression using equal concentration ratio method. The results showed that, after exposure for 24, 48, and 72 h, single EV, DES, and BPA can promote the proliferation of MCF-7 human breast cancer cells, and EV has the strongest effect in inducing cell proliferation. DEHP and MEHP cannot induce MCF-7 cell proliferation for all exposure time, while cell proliferation induced by EV was significantly attenuated by DES, BPA, DEHP, and MEHP when they mixed with EV. For intracellular ROS, single EV, BPA, DES, DEHP, and MEHP elevated intracellular ROS levels for different exposure time. Similar to the cell proliferation, DES and BPA decreased intracellular ROS levels induced by EV when they mixed with EV for 24 h. EV, DES, and BPA exposed alone or combined with EV upregulated the ERα protein expression. However, DEHP and MEHP exposed alone or combined with EV had no effect on ERα protein expression, indicating that DEHP or MEHP could attenuate ERα protein expression upregulated by EV. These results showed that the joint toxicity of binary mixtures of EV and other EDCs do not interact in a synergistic fashion in inducing cell proliferation, intracellular ROS levels, and ERα protein expression. These findings have important implications in the human risk assessments of EV mixed with other EDCs in the environment.

Hydroxycitric acid potentiates the cytotoxic effect of tamoxifen in MCF-7 breast cancer cells through inhibition of ATP citrate lyase

Hydroxycitric acid (HCA), a dietary-derived weight loss supplement, competitively inhibits ATP citrate lyase (ACLY). Tamoxifen (TAM) is the most frequently used therapy for estrogen receptor (ER)-positive breast cancer patients, but its application was restricted due to efficacy related issues. Lipid metabolic reprogramming plays a key role in cancer progression and response to treatment. This study will test the hypothesis that targeting lipid metabolic enzymes could enhance TAM effect against breast cancer cells. MCF-7 ER-positive breast cancer cell line was used, and the cytotoxic effect of TAM treatment, alone and in combination with HCA was evaluated. Flowcytometric analysis of apoptosis following TAM and/or HCA treatment was additionally performed. Besides, the effects of TAM and/or HCA on ACLY, acetyl CoA carboxylase alpha (ACC-α) and fatty acid synthase (FAS) expression were investigated. Likewise, expression of ER-α protein through TAM and/or HCA treatment was examined. Cell contents of cholesterol and triglyceride were quantified. Treatment with TAM or HCA significantly reduced cell viability in a concentration-dependent manner whereas co-treatment synergistically reduced cell viability, promoted apoptosis, and decreased the expression of ACLY, ACC-α, and FAS. Intracellular triglyceride and cholesterol were accumulated in response to treatment with TAM and/or HCA. Moreover, either solitary TAM or TAM/ HCA co-treatment increased ER-α protein levels non significantly. Our results revealed that TAM effects on breast cancer are mediated, in part, through the regulation of key genes involved in lipid metabolism. Accordingly, inhibition of ACLY by HCA might be beneficial to enhance the therapeutic index of TAM against breast cancer.

SUN-747 Steroid Hormone Metabolism Mediated Racial Disparity in Men with Benign Prostatic Hyperplasia

Introduction and Objective: Racial disparity in prostate cancer has been well established, with African American (AA) men having higher rates of diagnoses and death from the disease compared to Caucasian American (CA) men. AA men also have a high incidence of benign prostatic hyperplasia (BPH), a disease associated with lower urinary tract symptoms (LUTS) that affect >210 million men worldwide. Furthermore, AA men with BPH have an increased incidence of non-surgical treatment failure, larger prostates at time of surgery, and surgery occurring at a younger age. The use of selective estrogen receptor modulators (SERMs) in the treatment of BPH has been proposed, as an increase in ERα has been associated with disease progression. AA men have higher levels of circulating estrogens as compared to CA leading to an increased prenatal exposure to estrogens. Estrogen exposure has been shown to alter the epigenetic landscape of genes, and this prenatal exposure to estrogens could sensitize the AA men to altered steroid homeostasis leading to an increase susceptibility to BPH and an altered response to treatment. In this study, we examine the prostate expression and localization changes in estrogen receptors (ERα, ERβ) as well as steroid metabolism genes in AA and CA with or without BPH. Methods: To examine the impact of race on BPH, we examined prostate tissue from 66 men. We utilized 21 normal transition zone controls from radical prostatectomies, 8 normal transition zone controls from organ donors, and 37 BPH samples divided between CA and AA men. Using multispectral quantitative multiplex IHC, we examined the steroid hormone related protein expression of ERα, ERβ, CYP7B1, and AKR1C1 on each FFPE tissue section. We quantified the optical density of each protein of interest as well as examined colocalization and coexpression through cell and tissue segmentation. Results: In CA men, there is a dysregulation of ERα:ERβ homeostasis with BPH relative to normal as an increase in ERα and a decrease in ERβ expression was observed. Furthermore, an increase in CYP7B1, an enzyme that degrades ERβ ligands, was also observed. In AA men, we observed no difference between normal and BPH states, however in both normal and BPH prostate tissues, ERα and ERβ were increased relative to CA men. In addition, there is a decrease in AKR1C1, the enzyme that metabolizes DHT to an ERβ ligand. Conclusions: Our study supports the concept that differences in hormone pathways exist between AA and CA men. Understanding how these racial difference in steroid metabolism enzymes as well as ERs between CA and AA men with BPH could enhance treatment strategies for men with BPH.

Exploring the transcriptional impact of ERα in the B cell response of systemic lupus erythematosus

Abstract Systemic lupus erythematosus (SLE) is a heterogeneous, autoimmune disease. Being female is a significant, baseline risk factor for developing lupus. The mechanisms responsible for enhanced risk remain unknown. The nuclear hormone receptor and key transcription factor, estrogen receptor alpha (ERα), has a number of immune gene targets; yet, understanding of its regulation of the immune response is incomplete. Lupus prone mice, expressing a truncated functional ERα knockout, survived longer and had reduced renal disease. However, a complete knockout of ERα was not protective. Our goal is to identify the molecular mechanisms utilized by ERα in regulating the immune response in lupus B cells. We identified ERα associated genes and determined expression differences in B cells isolated from female African American (AA) lupus patients. RNA-seq analysis indicates that 60% of ERα target genes were deferentially expressed and 82% of these genes were upregulated in patients compared to controls. To understand ERα function, full-length ERα and an isoform, ERα46 (lacking the AF-1 activation domain), were transfected into an EBV transformed SLE B cell line and the expression of ERα gene targets, including inflammatory cytokines (IL1β and IL6) examined. Transfection results varied by gene and suggest the interaction between ERα isoforms is complex. The effect of epigenetic modifying agents on ERα protein expression in transfected SLE B cells was also studied. Evidence suggests that an increase in DNA methylation leads to increased protein expression of ERα isoforms. The regulatory role of ERα in lupus B cells is complex, involves epigenetic changes and interplay between isoforms. Results support a role for ERα in the pathogenesis of SLE.

Effects of UHRF1 on Estrogen Receptor and Proliferation, Invasion and Migration of BCPAP Cells in Thyroid Papillary Carcinoma

To investigate the effects of ubiquitin-like PDH and ring finger domain 1 (UHRF1) on the expression ratio of estrogen receptor (ER) α/ERβ, and to explore the experimental mechanism of UHRF1 affecting the proliferation, invasion and migration of BCPAP cells in papillary thyroid carcinoma. The protein and mRNA expressions of UHRF1, ERα and ERβ in normal thyroid Nthy-ori3-1 cells and thyroid papillary carcinoma BCPAP cells were detected by Western blot and qRT-PCR. BCPAP cells were treated with Scrambled siRNA and UHRF1 siRNA, respectively. The expressions of ER α and ER β mRNAs were detected by qRT-PCR. MTT and Transwell were used to determine the proliferation, invasion and migration in each group of BCPAP cells. Compared with Nthy-ori3-1 cells, the expressions of UHRF1 and ERα proteins and mRNAs in BCPAP cells were significantly up-regulated ( P<0.05), while the expressions of ERβ protein and mRNA were significantly down-regulated ( P<0.05). Compared with the control group and Scrambled siRNA group, the expression of ER α mRNA in BCPAP cells transfected with UHRF1 siRNA was significantly decreased ( P<0.05), while the expression of ER β mRNA was significantly increased ( P<0.05). The proliferation, invasion and migration of BCPAP cells transfected with UHRF1 siRNA were significantly decreased ( P<0.05). UHRF1 upregulates ERα/ERβ expression ratio and promotes proliferation, invasion and migration of BCPAP cells in papillary thyroid carcinoma.

Perfluorooctanoic Acid (in the Presence of Fetal Bovine Serum) Induces Proliferation in ERα Positive and ERα Negative Breast Cancer Cell Lines

Perfluorooctanoic acid (PFOA) is a synthetic chemical belonging to a larger group of fluorotelomers. These compounds have been used in the production of both industrial and consumer products as surfactants and due to their chemical nature are environmentally persistent pollutants. Individuals can be exposed to PFOA through ingesting PFOA‐contaminated water or food. PFOA has been shown to have a half‐life of approximately 4 years in humans. While the long‐term effects of PFOA are largely unknown, there is increasing evidence suggesting it to be an endocrine disruptor. Studies have shown that PFOA binds to and activates peroxisome‐proliferator‐activated receptor α (PPARα), which can regulate the expression of other genes and receptors such as the other PPAR isoforms as well as estrogen receptor α (ERα).Previous experiments in our lab demonstrated that PFOA treatment of MCF‐7 breast cancer cells (an ERα positive cell line) decreased expression of ERα mRNA and protein, and decreased cell viability by ~20% within 48h of treatment compared to DMSO controls. However, these cells were treated in the absence of fetal bovine serum (FBS). When we repeated these experiments without serum withdrawal, we initially noted a tendency towards increased proliferation in MCF‐7 cells treated with 50μM and 100μM PFOA at both 24h and 48h compared to control. To further examine the role of ERα in this PFOA‐induced proliferation, we carried out additional experiments in MCF‐7 cells along with experiments in another ERα‐positive cell line, T47D, as well as an ERα‐negative cell line, MDA‐MB‐231. There was a tendency for increased viability in all three cell lines when treated with PFOA, although only the MDA‐MB‐231 cells and the T47D cells showed a statistically significant (~20%) increase at 48h with 50μM PFOA and 100μM PFOA, respectively. These data suggest that the PFOA‐induced increase in cell viability in these cell lines is not dependent on ERα expression. In addition, the opposing effects of PFOA on proliferation in MCF‐7 cells in the presence and absence of FBS demonstrates the importance of accurately and completely reporting cell culture and treatment conditions.Support or Funding InformationAugusta University Department of Biological Sciences, Augusta University Center for Undergraduate Research and Scholarship