Levels Of ERbeta Research Articles

A Role for ER-Beta in the Effects of Low-Density Lipoprotein Cholesterol and 27-Hydroxycholesterol on Breast Cancer Progression: Involvement of the IGF Signalling Pathway?

Cholesterol—in particular, high levels of low-density lipoprotein (LDL) and its metabolite, 27-hydroxycholesterol (27-OHC)—is correlated with increases in the risks of breast cancer and obesity. Although the high expression of LDL/27-OHC has been reported in breast cancer, its effects and mechanism of action remain to be fully elucidated. In this study, we found that the effects of LDL on cell proliferation were mediated by the activation of the cytochrome P450 enzyme, sterol 27 hydroxylase, and cholesterol 27-hydroxylase (CYP27A1) in both ER-α-positive and ER-α-negative breast cancer cells. We found that treatment with 27-OHC only increased cell growth in oestrogen receptor-α (ER-α)-positive breast cancer cells in an ER-α-dependent manner, but, interestingly, the effects of 27-OHC on cell migration and invasion were independent of ER-α. Using ER-α-negative MDA-MB-231 cells, we found that 27-OHC similarly promoted cell invasion and migration, and this was mediated by oestrogen receptor β (ER-β). These results suggest that 27-OHC promotes breast cancer cell proliferation in ER-α-positive breast cancer cells via ER-α, but migration and invasion are mediated via ER-β in ER-α positive and negative cell lines. The addition of LDL/27OHC increased the production of IGF-I and the abundance of IGF-IR in TNBC. We further found that modulating ER-β using an agonist or antagonist increased or decreased, respectively, levels of the IGF-I and EGF receptors in TNBC. The inhibition of the insulin-like growth factor receptor blocked the effects of cholesterol on cell growth and the migration of TNBC. Using TCGA and METABRIC microarray expression data from invasive breast cancer carcinomas, we also observed that higher levels of ER-beta were associated with higher levels of IGF-IR. Thus, this study shows novel evidence that ER-β is central to the effects of LDL/27OHC on invasion, migration, and the IGF and EGF axes. Our data suggest that targeting ER-β in TNBC could be an alternative approach for downregulating IGF/EGF signalling and controlling the impact of LDL in breast cancer patients.

Abstract P139: An estrogen receptor beta agonist liquiritigenin potentiates inhibition of hormone-dependent breast-cancer growth by cholesterol biosynthesis inhibitor RO 48-8071

Abstract Hormone-dependent breast cancer represents approximately 70% of all diagnosed breast cancers. Estrogens and progestins stimulate breast cancer cell proliferation, angiogenesis and metastasis. Estrogen receptor (ER)-alpha positive breast cancers are treated with anti-estrogens and aromatase inhibitors, but resistance to these agents invariably develops during the course of therapy. Our goal was to identify novel treatment strategies to suppress breast cancer growth and avoid drug resistance. Cholesterol biosynthesis plays a vital role in tumor cell growth. Cholesterol is an integral part of membrane structure and is also a precursor of steroid hormones that are synthesized by tumor cells and cause cellular proliferation by interacting with specific receptors. Cancer cells have elevated levels of cholesterol, which could be responsible for anti-hormone resistance. During studies to determine the effects of the cholesterol biosynthesis inhibitor RO 48-8071 ([4’-[6-(Allylmethylamino)hexyloxy]-4-bromo-2’-fluorobenzophenone fumarate] (RO), which inhibits oxidosqualene cyclase (OSC), an enzyme that acts downstream of HMG-CoA reductase to convert 2, 3-monoepoxysqualene to lanosterol (a key step in the biosynthesis of cholesterol), it was serendipitously discovered that RO degraded ER-alpha (ERa), and elevated levels of ER-beta (ERb), a known tumor suppressor. We tested RO alone and in combination with liquiritigenin (LQ), a selective agonist for ERb, to ascertain their inhibitory effects against tumor cell progression. RO or LQ alone reduced significantly the in vitro viability of MCF-7 and BT-474 breast-cancer cells. BT-474 cells were more sensitive than MCF-7 cells to LQ (IC50 0.15 uM vs 0.25 mM, respectively). RO (5-10 uM) + LQ (0.1-0.3 mM) treatment reduced cell viability in an additive manner in MCF-7 cells and in a synergistic manner in BT-474 cells. Combination treatment was significantly more effective than treatment with a single agent. Administration of RO (10 mg/kg/iv) or LQ (20 mg/kg/ip) significantly inhibited growth of tamoxifen-resistant BT-474 tumor xenografts in vivo; combination treatment was even more effective. Both agents appeared to be non-toxic in vivo. Mechanistically, RO, LQ, or RO + LQ reduced ERa but induced ERb expression in tumor xenografts. Both compounds significantly reduced expression of markers of angiogenesis and increased apoptosis in tumor xenografts; use of RO + LQ significantly enhanced the effects observed with a single agent. The anti-tumor properties of RO may in part be due to an off-target effect that reduces ERa and increases ERb, the latter then interacting with LQ to more effectively promote its anti-proliferative effects. The potent anti-tumor properties of RO and LQ, particularly when used together, suggest that this combination has substantial potential as a means by which to clinically manage breast-cancer growth, including in disease types that are tamoxifen resistant. Citation Format: Yayun Liang, Salman Hyder. An estrogen receptor beta agonist liquiritigenin potentiates inhibition of hormone-dependent breast-cancer growth by cholesterol biosynthesis inhibitor RO 48-8071 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P139.

PO-521 Expression of oestrogen receptor beta as a predictive marker of platinum and taxane-based chemotherapy in ovarian cancer patients

IntroductionPredictive markers of platinum-based chemotherapy efficacy in patients with ovarian cancer have been widely discussed in recent years. Oestrogen receptors (ER) are involved in regulation of proliferative factors responsible for tumour growth, so they could predict the effectiveness of anticancer chemotherapy. The aim of the study was to determine the predictive value of ER-beta expression in a cohort of ovarian cancer patients treated with platinum and taxanes. The level of ER-beta expression was correlated with progression-free survival and also with a number of disease relapses during 40 months of monitoring.Material and methodsThe quantitative immunofluorescence flow cytometry analysis was performed to detect ER-beta in 34 serous ovarian cancer surgical specimens. All the patients were treated with the first line platinum and taxane-based regimen. The primary anti-ER-beta (ab14C8) and the secondary antibodies (DyLight650, ab98729) were used for the analysis. The level of ER-beta expression was calculated by Kolmogorov-Smirnov statistical test as the ratio (%) of specifically fluorescent cells to the number of cells incubated only with secondary antibodies. Association of ER-beta expression levels with progression-free survival was analysed using the Kaplan-Meier method and log-rank tests.Results and discussionsER-beta were revealed in all tumour specimens tested and the median value of the expression level was 41,5%. Patients were dichotomized in groups with low and high level of ER-beta expression, below- and above-median value respectively. Significant differences were shown between these groups. First, median of progression-free survival was larger in the high-level expression group as compared to the low-level expression group – 25,5 vs 8 months. Second, the disease rate during 40 months of monitoring was lover in the high-level expression group as compared the low-level expression group – 6 vs 15 respectively.ConclusionA quantitative index of the ER-beta level expression in tumour tissue predicts the efficacy of the first line platinum and taxane-based chemotherapy in ovarian cancer patients. Progression-free survival was about 3.0 times longer and the number of the disease relapses during 40 months of monitoring was 1.5 times less in patients with high level of ER-beta expression. The study was supported in part by RSF 17-75-10212.

The sharp decline of beta estrogen receptors expression in long-lasting ulcerative-associated carcinoma

Objective. Colorectal carcinoma is an important cause of death in inflammatory bowel diseases, thus requiring surveillance for dysplasia in long-standing ulcerative colitis (UC). Females show a lower incidence probably related to hormonal factors; therefore, a role of estrogen receptors (ERs) has been supposed in carcinoma-associated colitis (CAC) development. Our aim was to identify ER beta/alpha expression in long-lasting pancolitis through each grade of dysplasia to carcinoma and, furthermore, to investigate the simultaneous epithelial apoptosis/proliferation. Materials and methods. Forty-eight patients affected by long-lasting pancolitis were retrospectively investigated. Samples were divided into four groups: UC, low-grade dysplasia/high-grade dysplasia (UC-HGD), and CAC. Normal colon samples were used as controls. ER-beta, ER-alpha, Ki-67, and TUNEL expression (labeling/H index) were evaluated by immunohistochemistry. Results. ER-beta expression revealed an impressive reduction in CAC (10.4 ± 5.1; p < 0.001) compared to controls and UC (34.3 ± 3.1 and 26.8 ± 7.8, respectively), meanwhile ER-beta level in LGD (29.4 ± 3.7) was comparable to UC. As far ER-beta/ER-alpha mean value ratio revealed a progressive reduction. Ki67 demonstrated a progressive significant increase from UC until CAC (37.9 ± 6.4 < 45.7 ± 6.2 < 60.6 ± 5.2 < 71.1 ± 5.1; p < 0.001). Apoptotic index (TUNEL) revealed a strong fall in UC-HGD and CAC. Conclusions. ER-beta fall could be considered as a biomarker of UC-dysplasia progression. It occurs in HGD and overt neoplasia, while in LGD shows a normal expression. At the moment, we are unable to use this tool in the clinical practice to predict tumor progression, but it would be appropriate to encourage ER expression investigations in large samples for the interesting perspectives of application.

Calycosin induces apoptosis by the regulation of ERβ/miR-17 signaling pathway in human colorectal cancer cells.

Prior studies have suggested that a high intake of isoflavonoids is associated with a protective effect against hormone-related cancers, such as colorectal cancer (CRC). Calycosin, a main component of isoflavones, has been shown to suppress the growth of hormone-dependent tumors through an ERβ-mediated signaling pathway. However, the effects of calycosin on CRC remain unclear. In this study, we aimed to investigate the anti-tumor activities of calycosin on CRC and its potential mechanism. HCT-116 cells were treated with calycosin. Cell proliferation, apoptosis and invasiveness were measured by MTT assay, flow cytometry and transwell invasion assay, respectively. mRNA levels of ER beta (ERβ) and miR-17 were quantified by real-time PCR. Protein expressions of ERβ and phosphatase and tensin homolog deleted on chromosome ten (PTEN) were determined by western blotting. We found that calycosin significantly induced apoptosis, and inhibited proliferation and invasiveness of HCT-116 cells in a dose-dependent manner. In addition, ERβ expression significantly increased in calycosin-treated HCT-116 cells, followed by a decrease of miR-17, and up-regulation of PTEN. Our results indicate that calycosin has an inhibitory effect on CRC, which might be obtained by ERβ-mediated regulation of miR-17 and PTEN expression.

Expression of Ki-67 and estrogen receptor beta in primary malignant melanoma as a potential indicator of regional lymph node positivity

Background Malignant melanoma (MM) is one of the tumors with fastest-growing incidence, also deadliest form of skin cancer. In the early stages of the disease it is extremely difficult to adequately predict risk from hematogenic and lymphatic metastasis. We assumed that the determination of Ki-67 and estrogen receptor beta (ER beta) in cells of MM could predict their biological behavior. Objectives To examine whether the level of expression of Ki-67 and ER beta in cells of MM are in correlation with status of regional lymph nodes (LN), and compare the relationship between melanoma thickness (pT) and status of LN. Methods Expression of Ki-67 and ER beta was examined by immunohistochemistry, and then staining positivity assessed and correlated with relevant clinical and pathological parameters. Results Logistic regression analysis showed that when ER beta levels increase by 1% the risk of positive LN decreases by 7% (OR = 0.930, 95%CI 0.87-0.99, p = 0.036), and when the Ki67 expression increases by 1% that the risk of LN positivity increases by 10% (OR = 1.108, 95%CI 1.02 to 1.19, p = 0.009). Conclusions Correlation between melanoma thickness and the status of LN has less prognostic significance than the relationship between expression of Ki-67 and ER beta and the status of LN. Our study showed a significant prognostic value of Ki67 expression in predicting the behavior of MM, and potential prognostic significance of ER beta.

Homeobox A7 stimulates breast cancer cell proliferation by up-regulating estrogen receptor-alpha

Breast cancer is the most common hormone-dependent malignancy in women. Homeobox (HOX) transcription factors regulate many cellular functions, including cell migration, proliferation and differentiation. The aberrant expression of HOX genes has been reported to be associated with human reproductive cancers. Estradiol (E2) and its nuclear receptors, estrogen receptor (ER)-alpha and ER-beta, are known to play critical roles in the regulation of breast cancer cell growth. However, an understanding of the potential relationship between HOXA7 and ER in breast cancer cells is limited. In this study, our results demonstrate that knockdown of HOXA7 in MCF7 cells significantly decreased cell proliferation and ERα expression. In addition, HOXA7 knockdown attenuated E2-induced cell proliferation as well as progesterone receptor (PR) expression. The stimulatory effects of E2 on cell proliferation and PR expression were abolished by co-treatment with ICI 182780, a selective ERα antagonist. In contrast, overexpression of HOXA7 significantly stimulated cell proliferation and ERα expression. Moreover, E2-induced cell proliferation, as well as PR expression, was enhanced by the overexpression of HOXA7. Neither knockdown nor overexpression of HOXA7 affected the ER-beta levels. Our results demonstrate a novel mechanistic role for HOXA7 in modulating breast cancer cell proliferation via regulation of ERα expression. This finding contributes to our understanding of the role HOXA7 plays in regulating the proliferation of ER-positive cancer cells.

Effects of naringin on proliferation, differentiation and maturation of rat calvarial osteoblasts in vitro

To investigate the effects of naringin on the proliferation, differention and maturaion of rat calvarial osteoblasts (ROB). Segregated neonatal SD rat skull, enzyme digestion to obtain ROB. The culture medium was replaced every three days. Serial subcultivation proceeded when cells covered with 80% culture dish. Naringin supplemented into the culture at 1 x 10(-4), 1 x 10(-5), 1 x 10(-6), 1 x 10(-7) mol x L(-1) respectively. MTT method was adopted in proliferation analysis and the activity of ALP was examined after induced 9 days. Search the best concentration and supplemented into the medium, then the osteogenic differentiation markers including the secretion amount of osteocalcin, osteopontin and bone morphogenetic protein-2 were compared between the naringin-supplemented group and the control. Total RNA was isolated and the mRNA level of bFGF, IGF-1, Runx-2, Osterix, ERa and ERbeta was investigated by Real time RT-PCR. Total protein also was isolated and the expression ERa, ERbeta and collagen I was examined by Western blot. After the addition of ICI 182.780, an inhibitor of the estrogen signal pathway, these index also was examined and the changes were compared. The ROB proliferation was motivated by naringin dose-dependently. And it evidently leads to osteogenic process and maturation. 1 x 10(-5) mol x L(-1) is the best concentration. Naringin improved the secretion of osteocalcin, osteopontin, bone morphogenetic protein-2 and collagen I significantly. Besides, it can also enhanced the mRNA level of bFGF, IGF-1, Runx-2, Osterix, ERalpha and ERbeta. While all these effects can be restrained by ICI 182.780. The naringin with final concentration of 1 x 10(-5) mol x L(-1) enhances the osteogenic differentiation and maturation of ROB significantly, while the promoting effects vanished after the addition of ICI 182.780. These results suggesting that naringin is one of the phytoestrogens and have the activity of bone formation may via estrogen signal pathway, it can be developed into a new drug for osteoporosis therapy.

Estrogen Receptor Beta-Mediated Proliferative Inhibition and Apoptosis in Human Breast Cancer by Calycosin and Formononetin

Background: Calycosin and formononetin are two main components of isoflavones. In our previous studies, we have respectively reported their antitumor activities on breast cancer cell MCF-7. To further investigate the feasibility of isoflavones in clinically treating breast carcinoma, here we specifically focused on the comparison between calycosin and formononetin, along with the relevant mechanism. Methods: ER-positive (MCF-7, T-47D) and ER-negative breast cancer cells (MDA-231, MDA-435) were respectively treated with calycosin or formononetin. Cell proliferation and apoptosis were measured by MTT assay and flow cytometry. mRNA levels of ER beta (ERβ) and miR-375 were quantifed by real-time PCR. Expression of ERβ and insulin-like growth factor 1 receptor (IGF-1R), and activation of poly (ADP-ribose) polymerase 1 (PARP-1) were determined by Western blotting. Results: Both calycosin and formononetin impaired proliferation and triggered apoptosis of ER-positive breast cancer cells (MCF-7, T-47D) in a time- and dose-dependent manner, especially in the treatment with calycosin. However, no such effect was observed in ER-negative breast cancer cells, indicating the correlation between isoflavones-induced inhibitory effect and ERs. Thus calycosin and most sensitive MCF-7 cells were used to study the relevant signaling pathway. After the treatment of calycosin, ERβ expression was significantly increased in MCF-7 cells, followed by decrease of IGF-1R, activation of PARP-1 cleavage and downregulation of miR-375. Conclusion: Calycosin has an advantage on inhibiting breast cancer growth in comparison with formononetin, which is obtained by ERβ-mediated regulation of IGF-1R signaling pathways and miR-375 expression.

Changes in Estrogen Receptor ERα and ERβ Expression in Chicken (Gallus domesticus) Adrenal Gland during Short-fasting and Refeeding

Estrogen receptors have been found in the adrenal gland of rodents, monkeys, mares and sheep, indicating a connection between sex steroids and the activity of the adrenal gland. In the present study, the expression of estrogen receptors alpha (ERalpha) and beta (ERbeta) in the chicken adrenal gland during stress induced by 24 h fasting and after refeeding was determined using reverse transcription and the polymerase chain reaction (RT-PCR). The presence of both ER mRNAs in the adrenal gland of all examined groups was found. The relative expression of ERalpha mRNA was higher than ERbeta mRNA. There were no significant differences in ERalpha mRNA expression among the examined groups. On the contrary, we observed changes in ERbeta expression during stress conditions. These findings indicate different pathways of estrogen action in the avian adrenal gland. Furthermore, changes in ERbeta level suggest that this form of estrogen receptor plays a predominant role for estrogen action in the chicken adrenal gland during stress.

P2.16 Estrogen Receptors Alpha and Beta as Targets for Non-Small Cell Lung Cancer Hormonal Therapy

ABSTRACT Background Recent investigations demonstrate important role of estrogens in occurrence and progression of non-small cell lung cancer (NSCLC) as well as prognostic value of estrogen receptors (ER) alpha and beta in the tumor tissue. But the data about ER expression in the NSCLC differ significantly because of using semiquantitative methods for estimation only. To answer the question what is the significance of each type of the receptor as potential targets for antiestrogen therapy we have studied ERalpha and ERbeta expression in the NSCLC using the quantitative immunofluorescence methodology developed previously. Materials and methods NSCLC surgical specimens (97) were analyzed by flowcytometry. Primary antibodies were: anti-ERbeta 14C8, mouse IgG2a and IgG1 (Abcam); anti-ERalpha 1D5 (DAKO). Secondary antibody - FIT?-conjugated anti-mouse-IgG (F2772,Sigma). Number of specifically fluorescent cells was analyzed with Kolmogorov-Smirnov statistical approach. Three levels of ERbeta expression were compared: high - more than 50 % of the specifically fluorescent cells; moderate - 30-49 %; low - less than 30 %. Results 1.Expression of ERalpha and beta was reviled in about 90 % of NSCLC specimens. 2.Median of ERalpha level (22 %) was in 1·7-fold less then ERbeta level (37 %, p = 0,000002). 3.In women median of ERbeta level, but not ERalpha, was in 1·6-fold higher than in men (p = 0,008). 4.ERbeta level in men (32 %) and women (50 %) was higher in adenocarcinomas as compared with squamous-cell carcinomas in men (28 %): p = 0,0001 and 0,013 respectively. 5.ERalpha level in men (27 %) and women (22 %) was higher in adenocarcinomas as compared with squamous-cell carcinomas in men (16 %): p = 0,0003 and 0,016 respectively. 6. High and moderate level of ERbeta was revealed in 61 % of cases, while ERalpha - in 28 % only. 7. It was no coexpression of high ERalpha with low ERbeta level or any ERalpha expression with no ERbeta. Conclusion NSCLC is characterized by expression of ERbeta and ERalpha that is higher in adenocarcinomas than in squamous-cell carcinomas. ERbeta have main clinical relevance as potential target for antiestrogen therapy, because of higher level and frequency of expression than ERalpha. Generally, about half of adenocarcinoma and one third of squamous-cell carcinoma patients could benefit from adjuvant antiestrogen therapy. Supported by RFBR (Grants 10-04-00551-?,12-04-00028-?).

Estrogen receptor beta binds Sp1 and recruits a corepressor complex to the estrogen receptor alpha gene promoter

Human estrogen receptors alpha and beta are crucially involved in the regulation of mammary growth and development. Normal breast tissues display a relative higher expression of ER beta than ER alpha, which drastically changes during breast tumorogenesis. Thus, it is reasonable to suggest that a dysregulation of the two estrogen receptor subtypes may induce breast cancer development. However, the molecular mechanisms underlying the potential opposing roles played by the two estrogen receptors on tumor cell growth remain to be elucidated. In the present study, we have demonstrated that ER beta overexpression in breast cancer cells decreases cell proliferation and down-regulates ER alpha mRNA and protein content, along with a concomitant repression of estrogen-regulated genes. Transient transfection experiments, using a vector containing the human ER alpha promoter region, showed that elevated levels of ER beta down-regulated basal ER alpha promoter activity. Furthermore, site-directed mutagenesis and deletion analysis revealed that the proximal GC-rich motifs at -223 and -214 are critical for the ER beta-induced ER alpha down-regulation in breast cancer cells. This occurred through ER beta-Sp1 protein-protein interactions within the ER alpha promoter region and the recruitment of a corepressor complex containing the nuclear receptor corepressor NCoR, accompanied by hypoacetylation of histone H4 and displacement of RNA-polymerase II. Silencing of NCoR gene expression by RNA interference reversed the down-regulatory effects of ER beta on ER alpha gene expression and cell proliferation. Our results provide evidence for a novel mechanism by which overexpression of ER beta through NCoR is able to down regulate ER alpha gene expression, thus blocking ER alpha's driving role on breast cancer cell growth.

Abstract 953: Therapeutic significance of estrogen receptor ≤ agonists in gliomas

Abstract Purpose: Gliomas are the most common type of primary brain tumors with bleak prognosis. Gliomas exhibit gender bias with females at lower risk than males, implicating estrogen mediated protective effects. Estrogen functions are mediated by two ER subtypes: ERbeta, that functions as tumor promoter and ERbeta that function as tumor suppressor. In this study, we examined the status of ERbeta and tested whether natural ERbeta agonists have therapeutic potential on gliomas. Experimental Design: Tumor microarrays (TMAs) were utilized to determine the levels of ERbeta in gliomas. Therapeutic effect of three ERbeta agonists was determined using proliferation, foci formation and cell cycle progression assays. Functionality of ERbeta signaling in gliomas was determined by reporter assays and localization by confocal microscopy. The therapeutic potential of liquiritigenin was determined by systemic administration in a glioma xenograft model. Results: TMA analysis revealed that ERα expression is down regulated in high-grade gliomas. Glioma model cells exhibited detectable ERα with functional ERα signaling. Treatment of glioma cells with ERα agonists resulted in significant decreases in proliferation. Further, ERbeta agonist treatment induced the expression of ERα. Compared to control mice, animals treated with liquiritigenin had 50% reduction in tumor volume and size. IHC analysis of tumors revealed a significant increase in the nuclear ERα expression with a concomitant decrease in cell proliferation in the liquiritigenin-treated group. Conclusion: We found that ERα agonists promote both expression and tumor suppressive functions of ERα. Liquiritigenin, a plant-derived ERα agonist significantly reduced in vivo tumor growth in a xenograft model. Our results suggest that ERα signaling has a tumor suppressive function in gliomas and thus ERα agonists may represent a novel class of drugs for curbing glioma progression. Since ERα agonists are currently in clinical trials and are well tolerated with fewer side effects, identification of an ERα agonist as a therapeutic agent can be readily extended to clinical use with current chemotherapies, providing an additional tool for enhancing survival in glioma patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 953. doi:1538-7445.AM2012-953

Abstract A68: Involvement of serotonin receptor 4 (5-HTR4) in estrogen receptor beta (ER beta) expression and invasion of peritumoral mast cells in human prostate cancer tissue

Abstract Background: Androgens have been proposed to play pivotal roles in the development of human prostate cancer, and blockades of their actions have been also established as effective treatment of the patients. However, development of hormone-refractory prostate cancer (HRPC) is generally imminent and has emerged as one of the most critical clinical problems in the long-term management of the patients with prostate cancer. We previously postulated the significance of in situ androgen and estrogen production in human prostate cancer tissues in HRPC achievement via androgen receptor (AR) and estrogen receptor beta (ER beta). The role for neuroendocrine differentiation (NED) in prostate cancer tissues has also been considered important in the development of high grade/stage. Carcinoma cells with NED produce and secrete serotonin (5-HT), and increased expression of 5-HT receptor, especially type 4 (5-HTR4), has been proposed to be involved in autocrine/paracrine loops to sustain growth of HRPC. While, mast cell recruitment around the tumor cells has been proposed to be involved during the formation of HRPC, which may also contribute to stimulating effects on angiogenesis in local environment. Mast cells are also known to produce and secret 5-HT. Therefore, our hypothesis is that 5-HT secreted by both carcinoma and mast cells may be involved in autocrine/paracrine environment to develop HRPC via 5-HTR4. In addition, 5-HTR4 expression level is possibly associated with local microenvironment regulated by androgen and estrogen. Experimental design: We evaluated the expression levels of 5-HTR4 as well as the number of peritumoral mast cells and 5-HT positive carcinoma cells in human prostate cancer (n=112) using immunohistochemistry and correlated the findings with clinicopathological features of the patients and the expression levels of AR and ER beta. Results: 5-HTR4 immunoreactivity was detected in 38 cases of prostate cancer (34%). In the cases positive for 5-HTR4, the number of peritumoral mast cells tended to be higher than those negative for the receptor, while 5-HT positive cells were sparsely detectable in all the cases. In addition, 5-HTR4 immunoreactivity was significantly correlated with that of ER beta (P&amp;lt;0.05). The status of 5-HTR4 immunoreactivity was not significantly correlated with clinicopathological parameters of the cases examined including patient age, concentration of serum PSA levels, Gleason score, pT stage, lymph node states, or AR level. Conclusion: These data all suggest that 5-HTR4 plays a functional role in human prostate cancer via 5-HT secreted by both carcimoma and peritumoral mast cells. In addition, estrogens may possibly regulate the roles of 5-HTR4 via ER beta as previously reported in the other tissue, and their association may influence the development of HRPC. Note: This abstract was not presented at the conference because the presenter was unable to attend. Citation Format: Yasuhiro Nakamura, Takashi Maekawa, Kazue Ise, Shigeto Ishidoya, Yoichi Arai, Hironobu Sasano. Involvement of serotonin receptor 4 (5-HTR4) in estrogen receptor beta (ER beta) expression and invasion of peritumoral mast cells in human prostate cancer tissue [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A68.

Differential role of estrogen receptor beta in early versus metastatic non-small cell lung cancer.

Although women have an increased susceptibility to lung cancer, they also have a favorable clinical outcome. This may in part be due to female specific genetic and hormonal factors. In the present study, expression of ER-beta was investigated by immunohistochemistry using tissue samples from two cohorts: non-small cell lung cancer (NSCLC) diagnosed in 1999 in Manitoba and advanced NSCLC patients from the NCIC-CTG BR.18 trial. In the Manitoba cohort assessable tissue samples available in 79 patients (32 females and 47 males) and the majority (75%) had early stage disease. Fifty-one percent of patients expressed high levels of ER-beta (defined by ≥60, the median immunohistochemistry score) and its expression was comparable in males and females. The 3-year overall survival of the group was 53% and males had significantly worse survival compared to females (HR=2.37, 95%CI 1.15–4.91, P=0.02). Higher ER-beta 1 expression was associated with better survival in both univariate (HR=0.41, 95%CI 0.21–0.80, P=0.009) and in multivariate (HR=0.37, 95%CI 0.18–0.77, P=0.008) analysis. In the NCIC-CTG cohort that were more often later stage, assessable tissue samples from 48 cases were available however higher ER beta 1 expression correlated with poorer survival (HR= 1.94, 95%CI 1.01–3.75 P=0.047). These results suggest a differential impact of ER-beta 1 expression on clinical outcome by disease stage, that needs to be explored further and may explain contradictory observations reported in the literature.

17β-Estradiol regulates oxidative stress in prostate cancer cell lines according to ERalpha/ERbeta ratio

Estrogen action is mediated by the two receptor isoforms: estrogen receptor alpha and beta. Both receptors are expressed in human prostate tissue and have different action profiles. ERalpha is positively correlated with the malignancy of prostate cancer, while ERbeta may protect against abnormal prostate cell growth. 17β-Estradiol (E2), at least in part, induces cancerous transformations by causing deleterious mutations through the formation of reactive oxygen species (ROS). The aim was to study the effect of E2 on oxidative stress and the expression of uncoupling proteins (UCPs) and antioxidant enzymes in several prostate cancer cell lines with different ERalpha/ERbeta ratios. The cell prostate lines with a lower ERalpha/ERbeta ratio had lower oxidative stress, which could be partially explained by the increased expression of antioxidant enzymes and UCPs. Moreover, the action of E2 on the expression of antioxidant enzymes and UCPs was dual and dependent on the ERalpha/ERbeta ratio. Treatments with 0.1 nM E2 in cell lines with high ERalpha/ERbeta ratio produced a decrease in antioxidant enzymes and UCPs levels, with an increase in ROS production. These effects disappeared when the treatment was done in the presence of an ERalpha antagonist (MPP). In the cell lines with greatest levels of ERbeta and the lowest ERalpha/ERbeta ratio, E2 treatment caused the up-regulation of antioxidant enzymes and UCPs with a look-up decrease in ROS production. These effects were reversed when the cells were treated with E2 in the presence of an ERbeta antagonist (R,R-THC). On the whole, our results suggest a dual E2 effect; increasing or decreasing oxidative stress in part by modulation of UCPs and antioxidant enzymes according to the abundance ERbeta and ERalpha/ERbeta ratio in prostate cancer cell lines.

Altered Expression and Localization of Estrogen Receptors Alpha and Beta in the Testes of a Cryptorchid Rat Model

To assess the involvement of estrogen in spermatogenesis, we evaluated the expression of estrogen receptors (ERs) alpha (ERalpha) and beta (ERbeta) in the cryptorchid testes in model rats exposed to flutamide during the fetal stage. Cryptorchid model rats were produced by administering flutamide to pregnant Sprague-Dawley rats. To evaluate the sequential change in the expression of ERalpha and ERbeta genes, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using specific primers. Immunohistochemistry with ERalpha and ERbeta antibodies was performed, and the results were evaluated to determine the influences of orchiopexy. Real-time RT-PCR revealed that ERalpha expression in control testes increases with growth and peaks value at 7 weeks and significantly decreases in cryptorchid testes. ERbeta expression was low, and there were no significant differences between both the cryptorchid and control testes groups. Immunohistochemistry revealed that ERalpha protein was present in the spermatids and Sertoli cells of descended testes, and that this protein was strongly expressed in the Leydig cells of cryptorchid testes. ERbeta was detected in multiple cells in both groups. After orchiopexy, ERalpha expression was detected in the spermatids of cryptorchid testes. Spermatogenesis in cryptorchid rats is disrupted. Because the expression of ERbeta was unchanged in both control and cryptorchid testes, we supposed that alteration in ERalpha levels is more closely related to spermatogenic failure than ERbeta levels in the cryptorchid testes. We considered that increased expression of ERalpha in Leydig cells of cryptorchid testes is associated with estradiol level in the testicular tissue, and androgen-estrogen imbalance deteriorates spermatogenesis in cryptorchidism.

Abstract 4591: Estrogen receptor beta controls a wide variety of functions in breast cancer cells

Abstract The role of ER alpha as the mediator of estrogen action in breast cancer (BC) cells is well known, however, the role of ER beta is controversial. The majority of BC expressing ER alpha also express ER beta. Some clinical studies suggest that ER beta expression is a good prognosticator; however, in others, especially where expression levels of ER beta &amp;gt; ER alpha, ER beta may be associated with more advanced BC. Previously, we showed that ER beta may regulate a number of signaling pathways (AACR, 2008). Our recent work demonstrates that ER beta has even wider actions in MCF7 and T47D cells stably overexpressing ER beta. Thus, ER beta decreased phospho (p)/ total Stat1, p Stat3, p Stat5/total Stat5. Expression of the oncogenic miR cluster 17-92 was highly reduced (p&amp;lt;0.01) when ER beta overexpressing vs. control cells were compared. ER beta also decreased PPAR gamma, superoxide dismutase, as well as the concentration of the mRNA processing factor, RBM5, and the activated form of AMPK. All these changes were significant (p&amp;lt;0.05) in triplicate measurements. While ER beta overexpression increased migration through Matrigel, it significantly decreased CXCR4 expression (p&amp;lt;0.05, n=4). All of these effects of ER beta were estrogen-independent. On the other hand, estrogen increased CXCR4 expression in MCF7 cells significantly. In our study, we analyzed the expression of ER beta and BAD using tissue microarrays with normal and neoplastic breast tissue. Our immunohistochemical (IHC) data revealed a significant increase of ER beta cytoplasmic staining in neoplastic cells compared to normal cells (p&amp;lt;0.001). In addition, the nuclear expression of ER beta was significantly decreased in neoplastic cells compared to normal cells (p&amp;lt;0.01). From our correlation analysis of IHC data obtained from tissue microarray, we identified that cytoplasmic expression of BAD was negatively correlated with cytoplasmic expression of ER beta. In addition, the nuclear expression of BAD was significantly positively correlated with nuclear ER beta. These results suggest that ER beta has a wide variety of actions in BC cells, as predicted by the regulation of 62 miRs (p&amp;lt;0.01) as determined by Exiqon Inc. Taken together our results suggest that ER beta may have a multitude of actions in breast cancer and that the ratio of ER alpha/beta may determine the prognostic outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4591.

Prognostic Role of Estrogen Receptor α and Estrogen Receptor β in Gastric Cancer

The presence of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) have been reported in cell and tissue level in gastric cancer, but its impact on patients' survival remains unclear. This study was designed to investigate the expression level of ERalpha and ERbeta and to assess clinical significance of ERalpha and ERbeta expression in gastric cancer. The expression level of ERalpha and ERbeta were assessed by reverse-transcriptase polymerase chain reaction (RT-PCR) in 35 surgically resected gastric cancer and corresponding normal tissues and by immunohistochemical staining in 211 surgically resected gastric cancer and match normal tissues. The expression level between ERalpha mRNA expression in gastric cancer tissues and match normal tissues had no statistically significant difference. The ERbeta mRNA level in normal tissues was significantly higher than that observed in gastric cancer tissues (P = 0.001). Neither ERalpha nor ERbeta mRNA expression levels had significant correlation with clinicopathologic parameters. Forty-eight of 211 (22.7%) gastric cancer tissues showed positive expression of ERalpha and ERbeta detected in gastric cancer. ERalpha-positive expression correlated with poorer overall survival (P = 0.014), as did the absence of ERbeta expression in patients with gastric cancer (P = 0.001). In multivariate analysis, the positive expression of ERalpha and the absence of ERbeta were significant independent prognostic factors for overall survival (hazard ratio 2.159, P = 0.013, and hazard ratio 2.016, P = 0.025 respectively). Our results indicated that ERalpha and ERbeta were expressed in both gastric cancer and corresponding normal tissues. ERalpha expression and the absence of ERbeta expression are associated with poor survival.

Estrogen Receptor β Is a Novel Therapeutic Target for Photoaging

One of the many harmful factors faced by the skin is solar UV radiation, which damages skin by inducing chronic low-grade inflammation through increased expression of proinflammatory cytokines, metalloproteinases (MMPs) and cyclooxygenase-2 (COX-2). Estrogen receptors (ERs) alpha and beta are ligand-dependent transcription factors that are expressed in skin, and an ERbeta agonist has previously shown efficacy in vivo in models of pain and inflammation. Because ERbeta does not carry the breast and uterine proliferation liabilities of ERalpha, we decided to explore the possibility of using ERbeta as a target for photoaging. We show that ERbeta-selective compounds suppressed the expression of cytokines and MMPs in activated keratinocytes and fibroblast-based in vitro models of photoaging. Furthermore, in activated dermal fibroblasts, ERbeta-selective compounds also inhibited COX-2. These activities of ERbeta ligands in skin cells correlated with the expression levels of ERbeta and showed reversal by treatment with a potent synthetic ER antagonist. Furthermore, the pharmacology of ERbeta-selective compound was observed in wild-type but not in skin cells obtained from ERbeta knockout mice. Finally, we demonstrate that a synthetic ERbeta agonist inhibited UV-induced photodamage and skin wrinkle formation in a murine model of photoaging. Therefore, the potential of an ERbeta ligand to regulate multiple pathways underlying the cause of photoaging suggests ERbeta to be a novel therapeutic target for the prevention and treatment of photoaging.