E2-induced Cell Proliferation Research Articles

Combination of Ligustri Lucidi Fructus with Ecliptae Herba and their phytoestrogen or phytoandrogen like active pharmaceutical ingredients alleviate oestrogen/testosterone-induced benign prostatic hyperplasia through regulating steroid 5-α-reductase

BackgroundBenign prostatic hyperplasia (BPH) is a urinary system disease with high prevalence among the middle and elder men. In BPH, proliferation of prostate cells and the imbanlance between androgen and estrogen are both important inducers. Previous studies have demonstrated that compounds from Ligustri Lucidi Fructus (LLF) and Ecliptae Herba (EH) are of phytoestrogenic or phytoandrogenic activities. The combination of LLF with EH at the ratio of 1:1 on crude drugs quantity is called Erzhi formula (EZF), which is used for in vivo research of our study. PurposeThis study aimed to investigate potential mechanisms of EZF and its active pharmaceutical ingredients on BPH in vitro and in vivo. MethodsTherapeutic effects of EZF was evaluated in E2/testosterone (1:100) induced BPH rats model. The pathological changes of prostate, concentrations of testosterone, DHT, E2, PSA in rats’ plasma and prostate were detected. The expressions of PCNA, AR, ERα, ERβ, SRD5A1, SRD5A2 were measured in BPH rat prostates and E2-stimulated human benign prostatic epithelial cells (BPH-1). ResultsEZF treatment significantly attenuated rat prostate enlargement, alleviated BPH pathological features, and decreased the expression of PCNA. The up-regulation of AR, ERα, SRD5A1/2 expressions, and down-regulation of ERβ expression at prostate of rat BPH model were significantly blocked by EZF administration. The expression levels of testosterone, DHT, E2, PSA were strongly inhibited by EZF treatment. At the cellular level, ligustrosidic acid and echinocystic acid inhibited E2-induced BPH-1 cell proliferation and PCNA expressions, which were consistent with the results in vivo. And these two ingredients also down-regulated the expressions of AR, ERα, SRD5A1/2 and up-regulated the expression of ERβ in BPH-1 cells. ConclusionEZF, ligustrosidic acid from LLF and echinocystic acid from EH showed inhibitive effects on BPH via down-regulating prostatic AR, ERα, SRD5A1/2 expressions and up-regulating ERβ expression.

In silico identification of novel inhibitors targeting the DNA-binding domain of the human estrogen receptor alpha

The human estrogen receptor alpha (ERα) is an important regulator in breast cancer development and progression. The frequent ERα mutations in the ligand-binding domain (LBD) can increase the resistance of antiestrogen drugs, highlighting the need to develop new drugs to target ERα-positive breast cancer. In this study, we combined molecular docking, molecular dynamics simulations and binding free energy calculations to develop a structure-based virtual screening workflow to identify hit compounds capable of interfering with the recognition of ERα by the specific response element of DNA. A druggable pocket on the DNA binding domain (DBD) of ERα was identified as the potential binding site. The hits binding modes were further analyzed to reveal the structural characteristics of the DBD–inhibitor complexes. The core structure of the lead molecules was synthesized and was found to inhibit the E2-induced cell proliferation in MCF-7 cell lines. These findings provide an insight into the structural basis of ligand-ERα for alternate sites beyond the LBD-based pocket. The core structure proposed in this study could potentially be used as the lead molecule for further rational optimization of the antiestrogen drug structure with stronger binding of DBD and higher activity.

Overexpression of PELP1 in Lung Adenocarcinoma Promoted E2 Induced Proliferation, Migration and Invasion of the Tumor Cells and Predicted a Worse Outcome of the Patients.

The expression of Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) has been reported to be dysregulated in non-small cell lung carcinoma, especially in lung adenocarcinoma (LUAD). Therefore, we aimed to investigate the functional and prognostic roles of PELP1 in LUAD in this study. We first immunolocalized PELP1 in 76 cases of LUAD and 17 non-pathological or tumorous lung (NTL) tissue specimens and correlated the findings with the clinicopathological parameters of the patients. We then performed in vitro analysis including MTT, flow cytometry, wound healing, and transwell assays in order to further explore the biological roles of PELP1 in 17-β-estradiol (E2) induced cell proliferation, migration, and invasion of LUAD cells. We subsequently evaluated the prognostic significance of PELP1 in LUAD patients using the online survival analysis tool Kaplan-Meier Plotter. The status of PELP1 immunoreactivity in LUAD was significantly higher than that in the NTL tissues and significantly positively correlated with less differentiated features of carcinoma cells, positive lymph node metastasis, higher clinical stage as well as the status of ERα, ERβ, and PCNA. In vitro study did reveal that E2 promoted cell proliferation and migration and elevated PELP1 protein level in PELP1-high A549 and H1975 cells but not in PELP1-low H-1299 cells. Knock down of PELP1 significantly attenuated E2 induced cell proliferation, colony formation, cell cycle progress as well as migration and invasion of A549 and H1975 cells. Kaplan-Meier Plotter revealed that LUAD cases harboring higher PELP1 expression had significantly shorter overall survival. In summary, PELP1 played a pivotal role in the estrogen-induced aggressive transformation of LUAD and could represent adverse clinical outcome of the LUAD patients.

Metformin regulation of progesterone receptor isoform-B expression in human endometrial cancer cells is glucose-dependent.

Metformin (MET) constitutes the first-line treatment against type 2 diabetes. Growing evidence linking insulin resistance and cancer risk has expanded the therapeutic potential of MET to several cancer types. However, the oncostatic mechanisms of MET are not well understood. MET has been shown to promote the expression of progesterone receptor (PGR) and other antitumor biomarkers in patients with non-diabetic endometrial cancer (EC) and in Ishikawa EC cells cultured in normal glucose (5.5 mM) media. Therefore, the present study aimed to assess the effects of MET on EC cells under conditions simulating diabetes. Ishikawa cells treated with 10 nM 17β-estradiol (E2) and/or 100 µM MET and exposed to normal and high (17.5 mM) concentrations of glucose were evaluated for proliferative and PGR expression status. Under normal glucose conditions, MET attenuated E2-induced cell proliferation and cyclin D1 gene expression, and increased total PGR and PGR-B transcript levels. MET inhibited Ishikawa cell spheroid formation only in the absence of E2 treatment. In E2-treated cells under high glucose conditions, MET showed no effects on cell proliferation and spheroid formation, and increased total PGR but not PGR-B transcript levels. Transfection with Krüppel-like factor 9 small interfering RNA increased PGR-A transcript levels, irrespective of glucose environment. Medroxyprogesterone acetate downregulated PGR-A expression more effectively with metformin under high compared with normal glucose conditions. To evaluate the potential mechanisms underlying the targeting of PGR by MET, E2-treated cells were incubated with MET and the AMPK inhibitor Compound C, or with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), under normal glucose conditions. Compound C abrogated the effects of MET on PGR-B while AICAR increased PGR-B transcript levels, albeit less effectively compared with MET. The present results demonstrate the glucose-dependent effects of MET on PGR-B isoform expression, which may inform the response to progestin therapy in diabetic women with EC.

Interleukin-13 receptor subunit alpha-2 is a target of progesterone receptor and steroid receptor coactivator-1 in the mouse uterus†.

The endometrium, composed of epithelial and stromal cell compartments, is tightly regulated by the ovarian steroid hormones estrogen (E2) and progesterone (P4) during early pregnancy. Through the progesterone receptor (PGR), steroid receptor coactivators, and other transcriptional coregulators, progesterone inhibits E2-induced cell proliferation and induces the differentiation of stromal cells in a process called decidualization to promote endometrial receptivity. Although interleukin-13 receptor subunit alpha-2 (Il13ra2) is expressed in the human and mouse endometrium, its potential role in the steroid hormone regulation of the endometrium has not been thoroughly examined. In this study, we employed PGR knockout mice and steroid receptor coactivator-1 knockout mice (SRC-1-/-) to profile the expression of Il13ra2 in the murine endometrium and determine the role of these transcriptional regulators in the hormone-responsiveness of Il13ra2 expression. Furthermore, we utilized a well-established decidualization-inducing steroidogenic cocktail and a siRNA-based knockdown of IL13RA2 to determine the importance of IL13RA2 in the decidualization of primary human endometrial stromal cells. Our findings demonstrate that Il13ra2 is expressed in the subepithelial stroma of the murine endometrium in response to ovarian steroid hormones and during early pregnancy in a PGR- and SRC-1-dependent manner. Furthermore, we show that knockdown of IL13RA2 before in vitro decidualization of primary human endometrial stromal cells partially compromises the full decidualization response. We conclude that Il13ra2 is a downstream target of progesterone through PGR and SRC-1 and plays a role in mediating the stromal action of ovarian steroid hormones.

Unexpected Impact of a Hepatitis C Virus Inhibitor on 17β-Estradiol Signaling in Breast Cancer.

17β-Estradiol (E2) controls diverse physiological processes, including cell proliferation, through its binding to estrogen receptor α (ERα). E2:ERα signaling depends on both the receptor subcellular localization (e.g., nucleus, plasma membrane) and intracellular ERα abundance. Indeed, the control of ERα levels is necessary for the effects of E2, and E2 itself induces ERα degradation and cell proliferation in parallel. Thus, the modulation of intracellular ERα levels is a critical parameter for E2-induced cell proliferation. Therefore, we used this parameter as a bait to identify compounds that influence ERα levels and E2-dependent proliferation in breast cancer (BC) cells from a library of Food and Drug Administration (FDA)-approved drugs. We found that telaprevir (Tel) reduces ERα levels and inhibits BC cell proliferation. Tel is an inhibitor of the hepatitis C virus (HCV) NS3/4A serine protease, but its effect on E2:ERα signaling has not been investigated. Here, for the first time, we analyzed the effects of Tel on intracellular ERα levels and E2:ERα signaling to cell proliferation in different ERα-expressing BC cell lines. Overall, our findings demonstrate that Tel reduces intracellular ERα levels, deregulates E2:ERα signaling and inhibits E2-induced proliferation in BC cells and suggest the potential drug repurposing of Tel for the treatment of BC.

Stabilization of MORC2 by estrogen and antiestrogens through GPER1- PRKACA-CMA pathway contributes to estrogen-induced proliferation and endocrine resistance of breast cancer cells

ABSTRACT Aberrant activation of estrogen signaling through three ESR (estrogen receptor) subtypes, termed ESR1/ERα, ESR2/ERβ, and GPER1 (G protein-coupled estrogen receptor 1), is implicated in breast cancer pathogenesis and progression. Antiestrogens tamoxifen (TAM) and fulvestrant (FUL) are effective for treatment of ESR1-positive breast tumors, but development of resistance represents a major clinical challenge. However, the molecular mechanisms behind these events remain largely unknown. Here, we report that 17β-estradiol (E2), TAM, and FUL stabilize MORC2 (MORC family CW-type zinc finger 2), an emerging oncoprotein in human cancer, in a GPER1-dependent manner. Mechanistically, GPER1 activates PRKACA (protein kinase cAMP-activated catalytic subunit alpha), which in turn phosphorylates MORC2 at threonine 582 (T582). Phosphorylated MORC2 decreases its interaction with HSPA8 (heat shock protein family A [Hsp70] member 8) and LAMP2A (lysosomal associated membrane protein 2A), two core components of the chaperone-mediated autophagy (CMA) machinery, thus protecting MORC2 from lysosomal degradation by CMA. Functionally, knockdown of MORC2 attenuates E2-induced cell proliferation and enhances cellular sensitivity to TAM and FUL. Moreover, introduction of wild-type MORC2, but not its phosphorylation-lacking mutant (T582A), in MORC2-depleted cells restores resistance to antiestrogens. Clinically, the phosphorylation levels of MORC2 at T582 are elevated in breast tumors from patients undergoing recurrence after TAM treatment. Together, these findings delineate a phosphorylation-dependent mechanism for MORC2 stabilization in response to estrogen and antiestrogens via blocking CMA-mediated lysosomal degradation and uncover a dual role for MORC2 in both estrogen-induced proliferation and resistance to antiestrogen therapies of breast cancer cells. Abbreviations 4-OHT: 4-hydroxytamoxifen; Baf A1: bafilomycin A1; CMA: chaperone-mediated autophagy; E2: 17β-estradiol; ESR: estrogen receptor; FUL: fulvestrant; GPER1: G protein-coupled estrogen receptor 1; HSPA8: heat shock protein family A (Hsp70) member 8; LAMP2A: lysosomal associated membrane protein 2A; MORC2: MORC family CW-type zinc finger 2; PRKACA: protein kinase cAMP-activated catalytic subunit alpha; TAM: tamoxifen; VCL: vinculin

Estrogen induces cell proliferation by promoting ABCG2-mediated efflux in endometrial cancer cells

Recently, it has reported that overeating of lipid-food has led to increase the amount of estrogen in vivo and the incidence of endometrial carcinomas. It is well-known that ATP-binding cassette transporter sub-family G2 (ABCG2) is highly expressed in cancer stem cells (CSCs). CSCs possess the ability for differentiation, tumorigenesis, stem cell self-renewal, and the efflux of anti-cancer drug and these abilities affect malignancy of cancer cells. However, little is known about the relationship between the expression of ABCG2 and malignancy of cancer cells. The present study aimed at understanding the regulatory mechanism underlying 17-β-estradiol (E2)-induced cell proliferation under the control of ABCG2. E2 increased cell viability with a peak at 1 μM and facilitated ABCG2 mRNA expression followed by the increase of ABCG2 expression level at plasma membrane. E2-induced cell proliferation was inhibited by reserpine, an inhibitor of ABCG2, and the ABCG2 siRNA treatment. Thus, these results imply that ABCG2 plays an important role in the promotion of E2-induced cell proliferation in Ishikawa cells.

Glyphosate-based herbicide enhances the uterine sensitivity to estradiol in rats.

In a previous work, we detected that postnatal exposure to a glyphosate-based herbicide (GBH) alters uterine development in prepubertal rats causing endometrial hyperplasia and increasing cell proliferation. Our goal was to determine whether exposure to low-dose of a GBH during postnatal development might enhance the sensitivity of the uterus to an estrogenic treatment. Female Wistar pups were subcutaneously injected with saline solution (control) or GBH using the reference dose (2 mg/kg/day, EPA) on postnatal days (PND) 1, 3, 5, and 7. At weaning (PND21), female rats were bilaterally ovariectomized and treated with silastic capsules containing 17β-estradiol (E2, 1mg/ml) until they were two months of age. On PND60, uterine samples were removed and processed for histology, immunohistochemistry and mRNA extraction to evaluate: i) uterine morphology, ii) uterine cell proliferation by the detection of Ki67, iii) the expression of the estrogen receptors alpha (ESR1) and beta (ESR2), and iv) the expression of WNT7A and β-catenin. GBH-exposed animals showed increased luminal epithelial height and stromal nuclei density. The luminal and glandular epithelium were markedly hyperplastic in 43% of GBH-exposed animals. GBH exposure caused an increase in E2-induced cell proliferation in association with an induction of both ESR1 and ESR2. GBH treatment decreased membranous and cytoplasmic expression of β-catenin in luminal and glandular epithelial cells and increased WNT7A expression in the luminal epithelium. These results suggest that early postnatal exposure to a GBH enhances the sensitivity of the rat uterus to estradiol, and induces histomorphological and molecular changes associated with uterine hyperplasia.

17β‐Estradiol inhibits testosterone‐induced cell proliferation in HepG2 by modulating the relative ratios of 3 estrogen receptor isoforms to the androgen receptor

Sex hormones, especially 17β-estradiol (E2) and testosterone (TEST), play crucial roles in the oncogenesis and progression of liver cancer via hormone-related receptors. As women have a lower rate of hepatocellular carcinoma (HCC) than men, estrogens might attenuate the occurrence and development of HCC. This study aimed to investigate the inhibitory effects and mechanisms of E2 on TEST-induced HCC development; the HepG2 cell line was used as an in vitro model. Five endpoints, including cell viability, cell apoptosis, cell cycle, receptor protein expression, and messenger RNA transcription, were investigated. Different roles and the ratios of androgen receptor (AR) and 3 estrogen receptor (ER) subtypes were also estimated. Cell viability assay showed that co-treatment of E2 and TEST resulted in a significant inhibition of E2-induced or TEST-induced cell proliferation. Flow cytometry analysis revealed that combined treatment of E2 and TEST blocked the cell cycle in the G0/G1 phase as well as induced cell early apoptosis, characterized by decreased cyclin-dependent kinase transcription and the ratio of Bcl-2/Bax. Real-time quantitative polymerase chain reaction and Western blot analysis results further demonstrated that estrogen receptor estrogen receptor α66 (ERα66) and estrogen receptor β (ERβ) were upregulated, whereas AR and estrogen receptorα36 (ERα36) were downregulated, irrespective of whether E2 and TEST were considered separately or together, whereas the combined treatment of E2 and TEST resulted in a decrease in the ERα66/ERβ ratio, the ERα66/ERα36 ratio, and the ERβ/ERα36 ratio, but with an increase in the ERα66/AR ratio, the ERα36/AR ratio, and the ERβ/AR ratio. To sum up, E2 could inhibit TEST-induced cell proliferation by modulating the ratio of different hormone-related receptors.

Abstract 2116: Tumor-specific effects of estrogen drive a murine model of human estrogen receptor-positive breast cancer bone metastasis independent of bone microenvironment changes

Abstract Estrogen receptor α-positive (ER+) breast cancers (BrCAs) have the greatest predilection for forming bone metastases (BMETs). To begin to query ERα's role in osteolytic BrCA BMET progression, tumoral vs bone microenvironment effects of 17β-estradiol (E2) were determined in a human xenograft murine model of ER+ BrCA BMET. For these studies, naïve or 17β-estradiol (E2)-treated female athymic mice aged 4 weeks (young) or 15 weeks (mature) were inoculated with 1x105 human ER+ MCF-7 BrCA cells via the left cardiac ventricle 2 days post-placement of 60-day release E2 pellets (0.05, 0.10, 0.18, 0.36, & 0.72 mg E2). For studies examining E2 effects on bone, MCF-7 cells were not inoculated. Osteolytic lesion formation was assessed by hind limb radiographs, and E2 effects on bone were assessed by DXA (Faxitron), μCT (Scanco), and biochemical markers of bone formation or resorption (P1NP and TRAcP, respectively; Immunodiagnostic Systems). BMET incidence and lesion size in young mice were E2 dose-dependent, achieving 100% incidence at the highest dose tested, with smaller lesions and lower incidence occurring in response to lower doses, and no lesions forming in the absence of E2 even after 8 months. In contrast, E2 effects on the bone microenvironment were not dose dependent, and resulted in identical increases in bone mineral density (BMD) and bone volume (BV/TV), primarily via an increase in bone formation. To further query the importance of tumoral vs bone effects of E2 in driving ER+ BrCA progression, the tumoral effects of an identical E2 dose (0.72 mg) were compared in the skeletally immature young mice, which accrued increased bone (BV/TV) due to increases in bone formation in response to E2, vs skeletally mature mice, where E2-induced increases in bone were less marked and primarily driven by decreases in bone resorption. In these two very different bone milieus, progression of E2-dependent BMET was identical. While the relative roles of E2-induced tumor cell proliferation vs osteolytic factor secretion cannot be distinguished here, the development of osteolytic lesions similar in size to those occurring in ER- models driven by tumoral secretion of osteolytic factors, despite a marked increase in bone volume, suggests that tumoral effects of E2 may drive osteolytic ER+ BrCA BMET progression in a bone-specific fashion. Citation Format: Julia N. Cheng, Jennifer B. Frye, Susan A. Whitman, Andrew G. Kunihiro, Madison M. Egan, Julia A. Brickey, Janet L. Funk. Tumor-specific effects of estrogen drive a murine model of human estrogen receptor-positive breast cancer bone metastasis independent of bone microenvironment changes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2116.

Black cohosh inhibits 17β-estradiol-induced cell proliferation of endometrial adenocarcinoma cells

This study was conducted to investigate the effect of black cohosh (BC) extract on the proliferation and apoptosis of Ishikawa cells. Ishikawa human endometrial adenocarcinoma cells were treated with or without BC (1, 5, 10 and 25 μM) and cell proliferation and cytotoxicity were measured by CCK-8 assays and flow cytometry analysis. Additionally, Ishikawa cells were treated with 17β-estradiol (E2), E2 + progesterone and E2 + BC (5 and 10 μM) and the effect of BC and progesterone on E2-induced cell proliferation was analyzed. BC decreased the proliferation of Ishikawa cells at a dose-dependent rate compared with the control group (p < 0.05). The proliferation of Ishikawa cells increased in the presence of E2, whereas the subsequent addition of progesterone or BC decreased proliferation to the level of the control group (p < 0.05). The inhibitory effect of BC on E2-induced cell proliferation was greater than the inhibitory effect of progesterone. In conclusion, BC induces apoptosis in Ishikawa cells and suppresses E2-induced cell proliferation in Ishikawa cells. BC could be considered a candidate co-treatment agent of estrogen-dependent tumors, especially those involving endometrial cells.

Dynamin II is required for 17β-estradiol signaling and autophagy-based ERα degradation.

17β-estradiol (E2) regulates diverse physiological effects, including cell proliferation, by binding to estrogen receptor α (ERα). ERα is both a transcription factor that drives E2-sensitive gene expression and an extra-nuclear localized receptor that triggers the activation of diverse kinase cascades. While E2 triggers cell proliferation, it also induces ERα degradation in a typical hormone-dependent feedback loop. Although ERα breakdown proceeds through the 26S proteasome, a role for lysosomes and for some endocytic proteins in controlling ERα degradation has been reported. Here, we studied the role of the endocytic protein dynamin II in E2-dependent ERα signaling and degradation. The results indicate that dynamin II siRNA-mediated knock-down partially prevents E2-induced ERα degradation through the inhibition of an autophagy-based pathway and impairs E2-induced cell proliferation signaling. Altogether, these data demonstrate that dynamin II is required for the E2:ERα signaling of physiological functions and uncovers a role for autophagy in the control of ERα turnover.

Expression and functional roles of estrogen receptor GPR30 in human intervertebral disc

Estrogen withdrawal, a characteristic of female aging, is associated with age-related intervertebral disc (IVD) degeneration. The function of estrogen is mediated by two classic nuclear receptors, estrogen receptor (ER)-α and -β, and a membrane bound G-protein-coupled receptor 30 (GPR30). To date, the expression and function of GPR30 in human spine is poorly understood. This study aimed to evaluate GPR30 expression in IVD, and its role in estrogen-related regulation of proliferation and apoptosis of disc nucleus pulposus (NP) cells. GPR30 expression was examined in 30 human adult NP and 9 fetal IVD. Results showed that GPR30 was expressed in NP cells at both mRNA and protein levels. In human fetal IVD, GPR30 protein was expressed in the NP at 12–14 weeks gestation, but was undetectable at 8–11 weeks. The effect of 17β-estradiol (E2) on GPR30-mediated proliferation and interleukin-1β (IL-1β)-induced apoptosis of NP cells was investigated. Cultured NP cells were treated with or without E2, GPR30 antagonist G36, and ER antagonist ICI 182,780. NP cell viability was tested by MTS assay. Apoptosis was determined by flow cytometry using fluorescence labeled annexin-V, TUNEL assay and immumnocytochemical staining of activated caspase-3. E2 enhanced cell proliferation and prevented IL-1β-induced cell death, but the effect was partially blocked by G36 and completely abrogated by a combination of ICI 182,780 and G36. This study demonstrates that GPR30 is expressed in human IVD to transmit signals triggering E2-induced NP cell proliferation and protecting against IL-1β-induced apoptosis. The effects of E2 on NP cells require both GPR30 and classic estrogen receptors.

G protein-coupled estrogen receptor 1 (GPER 1) mediates estrogen-induced, proliferation of leiomyoma cells

G protein-coupled estrogen receptor 1 (GPER-1, formerly known as GPR30) has been proposed as the receptor for estrogen-induced, growth of leiomyomas though its precise mechanisms of action are not clear. We obtained leiomyoma cells (LC) and normal smooth muscle cells from 28 women (n = 28, median age 38 years, median parity 1.0). We incubated them with 17-β estradiol (E2), after blocking, or upregulating, expression of GPER-1 with ICI182,780 (a GPER-1 agonist) and siGPR30, respectively. We evaluated the role of GPER-1 in the mitogen-activated protein kinase (MAPK) signaling pathway using Western blot analysis. We studied cell proliferation with 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide, and, mitotic activity with phosphohistone H3 (PPH3) expression in leiomyoma, and, matched, normal, smooth muscle tissues using standard immunohistochemistry. Downregulation of GPER-1 expression with siGPR30 partially attenuated the E2-activated MAPK signaling pathway (p < 0.01). Upregulation of GPER-1 with ICI182,780 enhanced the E2-activated MAPK signaling pathway (p < 0.01). ICI182,780 enhanced E2-induced proliferation of LC (p < 0.01), while knock down of the GPER-1 gene with GPER-1 small interfering RNA partially inhibited E2-induced cell proliferation (p < 0.01). There were no significant differences in PPH3 expression between LCs and normal smooth muscle tissues (p > 0.05). Neither ICI182,780 nor siGPR30 increased mitosis in LCs (p > 0.05). Our results indicate that GPER-1 mediates proliferation of estrogen-induced, LC by activating the MAPK pathway, and, not by promoting mitosis.

Estrogen receptor α L429 and A430 regulate 17β-estradiol-induced cell proliferation via CREB1

17β-Estradiol (E2)-dependent cell proliferation requires both estrogen receptor α (ERα)-based integrated control of gene transcription and kinase pathways activation. Such coordination of intracellular E2:ERα-dependent signaling mechanisms is finely tuned by receptor association with specific partner proteins. Recently, we identified the leucine (L) 429 and alanine (A) 430 within the ERα ligand binding domain as important residues for receptor non-covalent interaction to ubiquitinated species [i.e., ERα ubiquitin-binding surface (ERα UBS)] and for E2-induced ERα activation. To date, if these two ERα amino acids are involved in the control of E2-dependent pathways required for cell proliferation is unknown. Here, by using stably expressing ERα mutated in L429 and A430 (i.e., L429A,A430G — LAAG) cell lines, we show that L429 and A430 are critical for E2-induced cell proliferation, PI3K/AKT pathway activation, and ERα-mediated transcriptional changes. Moreover, we demonstrate that these two receptor structural determinants direct the E2-induced PI3K/AKT/CREB1 pathway activation and CREB1-mediated transcriptional activity that in turn control the hormone-induced cell proliferation. As a whole, our data demonstrate for the first time that the ERα UBS contributes to the modulation of E2-induced ERα-mediated cell proliferation and provide a novel connection between the receptor structure and the functional molecular mechanisms by which E2:ERα complex can regulate cell processes.

Divide and Conquer May Not Be the Optimal Approach to Retain the Desirable Estrogenic Attributes of the Cyclopia Nutraceutical Extract, SM6Met.

The genus Cyclopia, an indigenous South African fynbos plant used to prepare honeybush tea, contains phytoestrogenic compounds. An extract from C. subternata, SM6Met, displays three desirable estrogenic attributes for future development of a phytoestrogenic nutraceutical, namely, ERα antagonism, ERβ agonism, and also antagonism of E2-induced breast cancer cell proliferation. Activity-guided fractionation of SM6Met was used in an attempt to isolate and identify compounds conferring the desirable estrogenic profile to SM6Met. Initial liquid-liquid fractionation of SM6Met yielded a polar fraction (PF) and a non-polar fraction (NPF), with the desirable estrogenic attributes retained in the NPF. Subsequent high performance counter-current chromatography (HPCCC) fractionation of the NPF yielded three fractions (F1-F3). Interestingly, the fractions revealed separation of the previously demonstrated positive estrogenic attributes of the NPF into separate fractions, with F1 and F2 acting as ERα antagonists, only F2 inducing antagonism of E2-induced breast cancer cell proliferation and only F3 retaining robust ERβ agonist activity. In terms of major polyphenols, quantitative HPLC and liquid chromatography tandem mass spectrometry (LC-MS/MS) indicated that HPCCC fractionation resulted in a divergence of polyphenolic classes, with F1 emerging as the dihydrochalcone-rich fraction and F2 as the flavanone- and benzophenone-rich fraction, while the xanthones, flavones and phenolic acids were retained in F3. F3 was re-engineered into F3R by reassembling the major polyphenols identified in the fraction. F3R could, however, not replicate the effect of F3. In conclusion, although activity-guided fractionation results suggest that retention of all the desirable estrogenic attributes of the original SM6Met in one fraction is not an attainable goal, fractionation is a useful tool to enhance specific desirable estrogenic attributes.

Clathrin Heavy Chain Interacts With Estrogen Receptor α and Modulates 17β-Estradiol Signaling.

17β-estradiol (E2)-induced signaling and control of estrogen receptor (ER)α degradation both play a major role in breast cancer cell proliferation. We recently reported the involvement of lysosomal function in both E2-dependent ERα breakdown and E2-induced cell proliferation and thus hypothesized a role for endocytic proteins in ERα signaling. An small interfering RNA screen identified proteins that regulate intracellular endocytic traffic and whose silencing alters E2-induced ERα degradation. One such protein was the clathrin heavy chain (CHC), whose role in E2:ERα signaling to cell proliferation is unknown. Here, we show that CHC physically interacts with ERα in the cytoplasm of breast cancer cells and regulates E2-induced cell proliferation. Surprisingly, the CHC:ERα interaction is required to sustain E2 signaling but is dispensable for ERα degradation. Our data also demonstrate that many membrane trafficking proteins contribute to the regulation of ERα degradation, thus unraveling the contribution of endocytic proteins in E2:ERα signaling.

In vitro evaluation of a tetrahydroisoquinoline derivative as a steroid sulfatase inhibitor and a selective estrogen receptor modulator.

Selective estrogen receptor modulators (SERMs) are currently in use in the hormonal therapy of breast cancer. In that respect, a new hormone-related approach is the therapeutical inhibition of steroid sulfatase (STS), which converts inactive, sulfated steroids into active hormones. We investigated the potential of 6-EO-14, a non-steroidal STS inhibitor with SERM potential. The latter compound, which exhibits a sulfamate moiety, releases the phenol derivative 8-EO-14 after the irreversible inhibition of STS. STS was inhibited by 6-EO-14 (IC50 = 0.3 μM), but not 8-EO-14, in HEK-293 cells transfected with an STS expression vector. The SERM potential of 8-EO-14 was assessed in osteoblast-like Saos-2 cells by investigating its effect on cell proliferation and on the activity of alkaline phosphatase (ALP), a specific differentiation marker. Saos-2 cell proliferation was increased by 21 % following 8-EO-14 addition (1 μM), and 8-EO-14 induced ALP activity (31 % increase at 0.1 nM) via estrogen receptor alpha (ERα) similarly to the SERM raloxifene. As compared to estradiol (E2) (100 %), the relative binding affinity of 6-EO-14 and 8-EO-14) for ERα was found to be weak (0.09 and 0.01 %, respectively). When assessed in two estrogen-dependent human breast cancer cell lines (MCF-7 and T-47D), 8-EO-14 did not support MCF-7 cell proliferation, whereas both 8-EO-14 and 6-EO-14 exhibited estrogen-like growth stimulation in T-47D cells. These two compounds were also unable to block E2-induced cell proliferation, suggesting their lack of antiestrogenic activity. Despite the known potency of 6-EO-14 as an STS inhibitor, the observed trophic activity of this new scaffold towards ERα-positive cells needs to be carefully considered prior to its potential utilization as a therapeutic agent.

Abstract 4229: The anti-estrogen effect of metformin in ERα+ breast cancer and tamoxifen resistant cell lines

Abstract Anti-estrogen therapy is still the most important systemic treatment to manage estrogen responsive breast cancer. Tamoxifen resistance is a major obstacle in treatment of ER-positive breast cancer. Recently, metformin, which is a biguanide derivative which is commonly used in the treatment of type II diabetes, has been emerged as a potential anti-cancer agent. We investigated if metformin had anti-estrogen effect in ER-positive breast cancer cell lines which are MCF-7, MDA-MB-361 as well as tamoxifen resistant cells (TR-MCF-7). Metformin inhibited the cell proliferation and the expression of ERα in dose-dependent manner in MCF-7 and TR-MCF-7 cells. Under stimulation of 17β-estradiol (E2), metformin inhibited the cell proliferation and expression of mRNA and protein of ERα in MCF-7 and TR-MCF-7 cells, while tamoxifen didn't affect E2 induced cell proliferation and ERα expression. This result indicates that metformin, as anti-estrogen agent, is superior to tamoxifen for targeting ER in breast cancer. We also identified that metformin inhibited E2 induced cell proliferation and expression of mRNA and protein of ERα in MDA-MB 361 cell line (ER+/Her-2+). We also analyzed estrogen responsive element (ERE) activity using by luciferase assay and identified metformin suppressed E2-inducible luciferase activity and expression of direct ER-regulated genes, such as c-myc, Cyclin D1 and pS2 and FOXA1, which were not inhibited by tamoxifen. In conclusion, metformin has a direct anti proliferative effect and anti-estrogen effect in MCF-7, TR-MCF-7 as well as MDA-MB-361 cell lines. These results suggest that metformin can be used as anti- estrogen in ER positive breast cancer (luminal A &amp; B) and tamoxifen resistant breast cancer. Citation Format: Jinkyoung Kim, Jiyun Lee, Soonyoung Jang, Chungyeul Kim, Aeree Kim. The anti-estrogen effect of metformin in ERα+ breast cancer and tamoxifen resistant cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4229. doi:10.1158/1538-7445.AM2014-4229