Estrogen Receptor mRNA Research Articles

Microarray-based determination of ER, PR and HER2 receptor status: validation and comparison with IHC assessments.

Abstract Abstract #3007 Background
 In breast cancer patients the level of expression of estrogen receptor (ER), progesterone receptor (PR) and HER2 is predictive for prognosis and/or treatment response. However, differences in assessment methods and interpretation can substantially affect the accuracy and reproducibility of the results. Previously, we have determined the association between immunohistochemistry (IHC) and mRNA levels for ER, PR and HER2, and have confirmed the accuracy of microarray readout on >400 samples. In the current study we describe the use of this microarray based readout on prospectively collected samples. We compared these readouts with multiple IHC and fluorescent in situ hybridization (FISH) assessments generated in various hospitals and a CLIA-certified reference laboratory and developed a microarray based test called TargetPrint™.
 Methods
 Gene expression data for ER, PR and HER2 were obtained by analysis of 100 breast carcinomas that have been collected prospectively within the RASTER study. Samples were stratified as receptor positive or negative using thresholds for ER, PR and HER2 mRNA levels. IHC assessment was performed (1) according to local standards of the hospital from where the sample originated, (2) by the central laboratory of the Netherlands Cancer Institute, and (3) at an independent reference laboratory using FDA-approved procedures and ASCO/CAP guidelines. A tumor was classified positive for ER and PR when ≥10% of tumor cells showed positive staining. HER2 IHC status was scored as 0, 1+, 2+ or 3+; a score of 3+ was considered positive. In case of 2+ samples, a FISH was performed to assess final HER2 amplification status. The cohort used in this study was pre-selected to include about two-third ER and PR positive samples and one-third HER2 positive samples.
 Results
 Multiple microarray readouts were highly reproducible (Pearson correlation 0.991) and resulted in 67, 61 and 39 percent positive samples for ER, PR and HER2, respectively. Comparison of microarray results with IHC (including FISH for HER2) performed at the three centers indicated highly similar results for receptor readout with a concordance of 92, 93 and 92% for ER; 84, 81 and 86% for PR; and 93, 95 and 94% for HER2. Overall misclassification rates between microarray and IHC readout were low for ER (0.08) and HER2 (0.06) and quite low for PR (0.14), and were comparable to the misclassification rates between the three IHC methods.
 Conclusion
 A microarray-based assessment of ER, PR and HER2 in relation to mRNA levels gives results comparable to multiple IHC methods and FISH and provides an objective and more quantitative assessment of tumor receptor status than IHC alone. Using TargetPrint™ for microarray readouts for hormone and HER2 receptor in addition to standard IHC will improve molecular characterization of breast cancer tissue. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3007.

The RNA binding protein HuR/ELAV1 regulates the estrogen receptor and epidermal growth factor receptor beta expression in BT 474 cells.

Abstract Abstract #3076 The RNA binding protein HuR has been shown to increase the mRNA stability of the majority of transcripts to which in binds. Recent data has linked the cytoplasmic distribution of HuR to the tumorigenesis process in many cancer types. HuR levels were found to be higher in more metastatic, higher grade breast tumors, suggesting that it may play a role for stabilization of growth-promoting transcripts in breast cancer cells. Previously, our lab has shown that the DNA methyltransferase inhibitor 5-aza- 2'deoxycytidine (AZA) and histone deacetylase inhibitor Trichostatin A (TSA) decreased the cytoplasmic accumulation of HuR in MDA-MB-231 and MCF-7 cells. Therefore, to examine the role of HuR in the more tumorigenic, estrogen receptor (ER) positive, Tamoxifen resistant BT 474 cells were treated with AZA/TSA or siRNA probes specifically targeting HuR expression. The BT 474 cells had a baseline increase in the cytoplasmic distribution of HuR as compared to MCF-7 and MDA-MB-231 cells. The half-life of ER mRNA was also increased. Following AZA/TSA treatment for 24-96 hours, cytoplasmic HuR was noticeably lower after 48 hours of treatment. Subsequent mRNA levels of both ER and epidermal growth factor beta (erbB2/HER2) were also found to drop with treatment. siRNA inhibition of HuR had a similar effect on the mRNA levels. Biotin pulldown assays using probes specific for the ER 3' UTR showed HuR binding was decreased with AZA/TSA treatment. These were similar findings as previously reported in MCF7 cells. Since ER and erbB2 are central to hormonal therapy, Tamoxifen sensitivity was assessed using BT 474 cells transfected with siRNA probes specifically targeting HuR expression. Cellular viability following Tamoxifen treatment was significantly decreased when HuR expression was inhibited and Tamoxifen sensitivity was restored. Similar studies investigating the role of HuR in herceptin resistance are currently ongoing. Taken together, our data suggests that HuR plays a key role in both de novo and acquired Tamoxifen resistance. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3076.

Acute sex hormone manipulations improve spatial performance and increase hippocampal receptor mRNA expression in the zebra finch (Taeniopygia guttata)

Event Abstract Back to Event Acute sex hormone manipulations improve spatial performance and increase hippocampal receptor mRNA expression in the zebra finch (Taeniopygia guttata) Olivia Haggis1*, Simone Meddle1, Nicola Clayton2 and Sue Healy3 1 University of Edinburgh, United Kingdom 2 Cambridge University, United Kingdom 3 University of St Andrews, United Kingdom In general, testosterone (T) and oestrogen (E) improve spatial memory in mammals both during development and throughout life. Little is known about steroid actions in birds in relation to spatial cognition and the hippocampus. The avian hippocampus is important for spatial memory and contains both androgen (AR) and oestrogen receptors (ER) and the enzyme aromatase that converts T to E. Our aim was to investigate the activational effects of sex steroids in spatial cognition and hippocampal morphology in the adult zebra finch. We manipulated steroid levels by providing food to which steroid had been added. Cognitive abilities were then tested with a spatial one-trial associative memory task. Brains were collected for in situ hybridization for androgen receptor (AR), oestrogen receptor (ER) and aromatase mRNA expression. T administration significantly increased plasma T levels in both males and females, peaking at 30 minutes and returning to baseline levels within 60 minutes. Ingestion of T or E 30 minutes prior to testing significantly improved spatial performance in both males and females after five days. This improvement was blocked when the aromatase inhibitor fadrozole was given in addition to T. Artificially increasing plasma T levels significantly increased hippocampal AR and ER mRNA expression in both sexes after 30 minutes. Males had significantly higher levels baseline levels of aromatase mRNA expression compared to females. T treatment increased expression in both sexes after 30 minutes. Taken together the data suggest that T, likely through conversion to E, is capable of improving spatial cognition in birds. Conference: 41st European Brain and Behaviour Society Meeting, Rhodes Island, Greece, 13 Sep - 18 Sep, 2009. Presentation Type: Poster Presentation Topic: Poster presentations Citation: Haggis O, Meddle S, Clayton N and Healy S (2009). Acute sex hormone manipulations improve spatial performance and increase hippocampal receptor mRNA expression in the zebra finch (Taeniopygia guttata). Conference Abstract: 41st European Brain and Behaviour Society Meeting. doi: 10.3389/conf.neuro.08.2009.09.171 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 09 Jun 2009; Published Online: 09 Jun 2009. * Correspondence: Olivia Haggis, University of Edinburgh, Edinburgh, United Kingdom, olivia.haggis@ed.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Olivia Haggis Simone Meddle Nicola Clayton Sue Healy Google Olivia Haggis Simone Meddle Nicola Clayton Sue Healy Google Scholar Olivia Haggis Simone Meddle Nicola Clayton Sue Healy PubMed Olivia Haggis Simone Meddle Nicola Clayton Sue Healy Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Effect of maternal fatness on fetal steroids and semi-quantitative real-time PCR expression of receptor genes in sheep

Sexual differentiation of the brain occurs between d 30 and 70 in the fetal lamb. The objective of this experiment was to determine if maternal fatness affects fetal steroid production and expression of their receptors which may ultimately alter endocrine systems postnatally. Fetuses were collected from ewes fed at either 100% (Control; n = 5) or 150% (Fat; n = 6) of NRC recommendations from 60 d prior to breeding until collection at 75 d of gestation. Hypothalamic and amygdala neural tissues were collected from twin male/female fetuses. Serum concentrations of testosterone were greater ( P < 0.001) in male fetuses compared to female fetuses. Further, male fetuses from Fat ewes had greater ( P < 0.05) serum concentrations of testosterone than male fetuses from Control ewes, but differences in testicular steroidogenic enzyme mRNA were not detected ( P = 0.18). Quantity of hypothalamic mRNA for estrogen receptor (ER) β tended ( P = 0.1) to be influenced by a sex by treatment interaction. Messenger RNA for ER-β was greater in female fetuses than male fetuses from Control ewes ( P = 0.05). Although amount of ER-β mRNA did not differ among male fetuses ( P = 0.7), amounts tended to be less ( P = 0.07) in female fetuses from Fat ewes compared to those from Control ewes, and did not differ ( P ≥ 0.8) from male fetuses. Hypothalamic ER-α mRNA tended ( P = 0.1) to be less in fetuses from Fat ewes compared to Control fetuses but was not influenced ( P = 0.3) by fetal sex or their interaction. Amount of mRNA for hypothalamic progesterone receptor tended ( P = 0.06) to be greater in male fetuses than female fetuses and tended to be less ( P = 0.06) in fetuses from Fat ewes than in Control fetuses, but did not differ by any sex by treatment interaction ( P = 0.6). Hypothalamic RNA for the androgen receptor did not differ by sex, dam nutritional treatment, or the interaction. Likewise, amygdala RNA for the estrogen or androgen receptor did not differ ( P ≥ 0.3) by sex, treatment, or their interaction. Dam fatness appears to decrease the expression of progesterone receptor, ER-α, and decrease amount of ER-β in the female fetuses while increasing circulating concentrations of testosterone in male fetuses. Altered expression of hypothalamic receptor genes by the uterine environment may affect adult responses to stress, sexual behavior and/or the pattern of gonadotropin release in response to gonadal steroids.

Influence of Evolution in Tumor Biobanking on the Interpretation of Translational Research

Translational cancer research increasingly relies on human tissue biospecimens and this has coincided with a shift in tissue biobanking approach. Newer biobanks (post year 2000) deploy standard operating procedures to reduce variability around biospecimen collection. Because current translational research is based on pre-2000 and post-2000 era biospecimens, we consider whether the collection era may influence gene expression data. We compared the range of breast tumor collection times from pre-2000 and post-2000 era biobanks and compared estrogen receptor (ER) protein expression with collection time. We then collected 10 breast tumor biospecimens under a standardized protocol and examined whether the expression of c-myc and ER was influenced by storage on ice < or = 24 hours. The range of collection times achieved at a pre-2000 versus post-2000 era biobank differed. Thirty-two percent of biospecimens were cryopreserved within 30 minutes at the pre-2000 era biobank versus 76% at the post-2000 era biobank. Collection time and ER protein level was inversely correlated (r = -0.19, P = 0.025; n = 137). We observed a wide range in initial c-myc and ER mRNA levels (50- versus 130-fold). Although mRNA levels of both genes declined with increasing collection time, the rate of change differed because c-myc was significantly reduced after 24 hours (mean reduction to 79% of initial) versus ER (94% of initial). The overall shift in biobanking around the year 2000 is reflected in the ranges of collection times associated with pre-2000 and post-2000 era biobanks. Because collection time can differentially alter gene expression, the biospecimen collection era should be considered in gene expression studies.

Timing is everything: Order of administration of 5-aza 2′ deoxycytidine, trichostatin A and tamoxifen changes estrogen receptor mRNA expression and cell sensitivity

Restoration of estrogen receptor (ER) expression using epigenetic inhibitors re-establishes expression of the estrogen receptor (ER) and restores tamoxifen sensitivity in ER negative breast cancer cells. We tested if order of administration of the DNMT (5-aza 2′ deoxycytidine/AZA) or HDAC (trichostatin A/TSA) inhibitors and tamoxifen affected ER re-expression and tamoxifen sensitivity. Treatment with AZA followed by co-administration of TSA plus tamoxifen resulted in the greatest ER re-expression and tamoxifen sensitivity, although sensitivity was not increased as robustly as expected. This could be due to increased cytoplasmic levels of HuR, suggesting that cytoplasmic HuR levels are central to tamoxifen responsiveness.

Evidence for the expression of estrogen receptors in osteogenic cells isolated from hen medullary bone

Medullary bone is a unique tissue in female birds and forms in the cavity of long bones. This bone displays rapid remodeling in response to circulating estrogen levels, suggesting that the osteoblasts in this bone are highly sensitive to estrogen. The present study examined expression of two estrogen receptor (ER) mRNAs in osteogenic cells of medullary bone of white Leghorn hens in vitro. At day 3, isolated cells from the hen medullary bone expressed alkaline phosphatase activity. Using immunocytochemistry, ER protein was demonstrated in the nuclei of these cells. RT-PCR analysis revealed that ER-alpha mRNA was constantly expressed from day 3 to day 15 of culture, while ER-beta mRNA was not detected throughout the culture period. These results indicate that estrogen may act via ER-alpha, but not ER-beta, on osteogenic cells of the avian medullary bone.

Gene expression of estrogen receptor, progesterone receptor and microtubule-associated protein Tau in high-risk early breast cancer: a quest for molecular predictors of treatment benefit in the context of a Hellenic Cooperative Oncology Group trial

Estrogen receptor (ER) and progesterone receptor (PgR) protein expression carry weak prognostic and moderate predictive utility for the outcome of early breast cancer patients on adjuvant chemohormonotherapy. We sought to study the predictive significance and correlations of transcriptional profiling of the ER, PgR and microtubule-associated protein Tau (MAP-Tau) genes in early breast cancer. Messenger RNA (mRNA) was extracted from 279 formalin-fixed paraffin-embedded breast carcinomas (T1-3N0-1M0) of patients enrolled in the Hellenic Cooperative Oncology Group (HeCOG) trial HE 10/97, evaluating epirubicin-alkylator based adjuvant chemotherapy with or without paclitaxel (E-T-CMF versus E-CMF). Kinetic reverse transcription polymerase chain reaction (kRT-PCR) was applied for assessment of the expression of estrogen receptor, progesterone receptor and MAP-Tau genes in 274 evaluable patients. Cohort-based cut-offs were defined at the 25th percentile mRNA value for ER and PgR and the median for MAP-Tau. Two hundred and ten patients (77%) were ER and/or PgR-positive by immunohistochemistry (IHC). Positive ER and MAP-Tau mRNA status was significantly associated with administration of hormonal therapy and low grade, while MAP-Tau mRNA status correlated with premenopausal patient status. MAP-Tau strongly correlated with ER and PgR mRNA status (Spearmann r = 0.52 and 0.64, P < 0.001). The observed chance corrected agreement between determination of hormonal receptor status by kRT-PCR and IHC was moderate (Kappa = 0.41) for ER and fair (Kappa = 0.33) for PgR. At a median follow-up of 8 years, univariate analysis adjusted for treatment showed positive ER mRNA status to be of borderline significance for reduced risk of relapse (HR = 0.65, 95% CI 0.41-1.01, P = 0.055) and death (HR = 0.62, 95% CI 0.36-1.05, P = 0.077), while positive MAP-Tau mRNA status was significantly associated with reduced risk of relapse (HR = 0.50, 95% CI 0.32-0.78, P = 0.002) and death (HR = 0.49, 95% CI 0.29-0.83, P = 0.008). In multivariate analysis, only axillary nodal metastases (HR = 2.33, 95% CI 1.05-5.16, P = 0.04) and MAP-Tau mRNA status (HR = 0.46, 95% CI 0.25-0.85, P = 0.01) independently predicted patient outcome. However, MAP-Tau mRNA levels did not predict enhanced benefit from inclusion of paclitaxel in the adjuvant chemotherapy regimen (test for interaction P = 0.99). No correlation was evident between increasing ER and PgR mRNA transcription and increasing benefit from endocrine therapy in 203 ER and/or PgR IHC-positive patients receiving adjuvant hormone therapy (Wald P = 0.54 for ER, 0.51 for PR). ER gene transcription carries weak predictive significance for benefit from endocrine therapy or for outcome, with no apparent dose-response association. The predictive significance is possibly exerted via MAP-Tau gene expression, an ER-inducible tubulin modulator with strong predictive significance for patient outcome. However, MAP-Tau mRNA did not predict benefit from the addition of a taxane to adjuvant chemotherapy. Further study of the biologic function and utility of MAP-Tau for individualising adjuvant therapy is warranted.

Expression of estrogen receptor, progesterone receptor and leukemia inhibitory factor on endometrium during different ovarian stimulation protocols in mice

To evaluate the influence of superovulation by GnRHa protocol and pregnant mare's serum gonadotropin (PMSG) alone on the expression of estrogen receptor (ER), progesterone receptor (PR) and leukemia inhibitory factor (LIF) mRNA on endometrium. Forty-five female ICR mice were randomly allocated into 3 groups:(1) GnRHa+PMSG group: alarelin was give first for desensitizing the pituitary, then superovulation with PMSG; (2) PMSG group: mice were injected with PMSG only; (3) Natural cycle group: mice were given with same volume of saline. Endometrium samples were taken at 48 hours after given hCG or ovulation (control group). ER and PR in glandular cells were detected with SP immunohistochemistry semiquantitatively. Expression of LIF mRNA on endometrium was detected with reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The positive rate(%) and expression intense (AU) of ER and PR on glandular epithelium cells were significantly lower in GnRHa+PMSG group and PMSG group than those in natural cycle group (all P <0.01). The expression of LIF mRNA was significantly lower in GnRHa+PMSG group and PMSG group than that in natural cycle group (all P <0.01); but the expressions of ER, PR and LIF in GnRHa+PMSG group were higher than those in PMSG group. The protocol with GnRHa down regulates the expressions of ER, PR and the LIF mRNA on the mice of secretive phase endometrium, suggesting it may have an adverse effect on the endometrial receptivity in mice, but it may still be better than PMSG alone.

Effects of 2,4‐dichlorophenol on the expression of vitellogenin and estrogen receptor genes and physiology impairments in Chinese rare minnow (Gobiocypris rarus)

2,4-Dichlorophenol (2,4-DCP) is known as a toxicant, but unknown as an environmental endocrine disruptor. In the present work, Chinese rare minnow (Gobiocypris rarus) was exposed to 0.03, 0.3, and 3 mg/L 2,4-DCP for 3 d and 0.1, 0.3, and 1 mg/L 2,4-DCP for 21 d, respectively. Endpoints including somatic index, vitellogenin (VTG) mRNA and protein level, estrogen receptor (ER) mRNA and histopathology were measured. In the 3-d exposure experiment, the effect concentrations were 0.3 mg/L and above, and in 21-d exposure, the effect concentrations were 0.1 mg/L and above. When exposed to the effect concentrations, GSI was significantly reduced for both male and female, ER mRNA was upregulated in male and downregulated in female. There were no significant variations of VTG mRNA in both male and female in 3-d exposure and in male in 21-d exposure. However, VTG mRNA in female in 21-d exposure was upregulated, corresponding to an increase of VTG protein in serum. Histopathological observation showed that ovaries were degenerated in the effect concentrations, where follicular atresias were more frequently observed. Because the sex hormones related genes and toxicological endpoints were affected in the dose-dependent manner, the results suggest that 2,4-DCP could be a potential endocrine disruptor and might cause adverse effects in female sex organs through interruption of ER-mediated processes.

Regulation of Estrogen Receptor Alpha Expression in Breast Cancer Cells by Sulforaphane

Sulforaphane (SUL, 1‐isothiocyanato‐4‐(methylsulfinyl) butane) is an isothiocyanate derived from glucoraphanin that is abundant in broccoli and broccoli sprouts and which has a variety of potential chemopreventive actions. SUL is capable of inhibiting cancer at both early (initiation) and late (promotion) stages of experimental tumorigenesis. In this study we analyzed the effects of SUL on proliferation of several human breast cancer cell lines and evaluated the effect of SUL on estrogen receptor alpha (ERα) protein and mRNA expression in MCF‐7 cells. SUL at doses between 10 and 30 μM inhibited proliferation of neoplastic breast cell lines to a greater extent than non‐neoplastic MCF‐10F breast cells. SUL at doses between 2.5 and 30 μM decreased ERα protein expression in MCF‐7 cells. This effect of SUL on ERα expression was apparent under complete and estrogen‐depleted growth conditions and was accompanied by a decrease in progesterone receptor protein expression. At SUL doses of 30μM and greater, SUL treatment was associated with suppression of ERα mRNA transcription. In contrast, at doses of SUL less than 30μM, there was no effect of SUL on ERα mRNA expression. However, the proteasome inhibitor MG132 reversed the suppression of ERα protein levels in MCF‐7 cells treated with SUL at the lower SUL doses. Thus, the downregulation of ERα protein expression appears to be due to multiple dose‐dependent mechanisms involving changes in mRNA expression and proteasome activity. The effect of SUL on hormone‐responsiveness of human breast cancer cells warrants further study.

Study of Endometrial Receptivity during Implantation in Implantation Dysfunction Mouse

Objective To establish the mice model of implantation dysfunction and to study the endometrial receptivity during implantation in implantation dysfunction mouse. Methods Sexually mature female virgin, Kunming mice were randomly assigned to the control group and the model group postcoitally. The model mice at 9 : 00 AM on d 4 of pregnancy(d 4) were injected subcutaneously with mifepristone. All animals were sacrificed at 9 : 00 PM on d 4 and their uterine horns were examined for the presence of implanted embryos. Histopathology of uterine endometrium was observed by light-microscope. The endometrial expressions of estrogen receptor (ER) and progesterone receptor (PR) assessed by immunnohistochemical SP method. The endometrial expressions of ER mRNA and PR mRNA were assessed by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Results Compared with control group, implantation rates and average embryo number significently decreased in model group, the development of endometrium was inhibited. In model group, absorbency and area rate of ER and PR in the gland and stroma were lower than those in control group (P Conclusion The endometrial receptivity and implantation decreased in mifepristone- induced implantation dysfunction mouse.

The contribution of steroidal androgens and estrogens to reproductive maturation of the eastern mud snail Ilyanassa obsoleta

Molluscs exposed to endocrine-disrupting chemicals (EDCs) have exhibited changes in reproductive tract development that are typically associated with androgen or estrogen signaling in vertebrates. However, a role for androgens and estrogens in molluscan reproductive endocrinology has yet to be established. In this study, we investigated putative roles for steroidal androgens and estrogens in recrudescence of the eastern mud snail Ilyanassa obsoleta. Our objectives were to: (1) identify associations among concentrations of testosterone and 17β-estradiol, sex, and reproductive status in mud snails that suggest these hormones are involved in recrudescence; and (2) determine whether mud snails express NR3C4-like (androgen receptor) and NR3A-like (estrogen receptor) mRNAs in a manner indicative of a role in recrudescence. Temporal changes in testosterone levels in males were consistent with a positive role in recrudescence. Such a trend was not evident in females or for 17β-estradiol in either sex. Efforts to identify an androgen receptor from the mud snail using targeted, degenerate RT-PCR were unsuccessful. However, an estrogen receptor (ER) cDNA was identified that is highly similar to known ERs of other molluscs. Studies with the ER of other molluscs have shown that this protein does not actually bind estrogens. We therefore considered the possibility that the mud snail ER may regulate reproductive maturation as a ligand-independent transcription factor based upon its tissue abundance. Males expressed greater levels of ER mRNA than did females over the entire reproductive cycle, and this difference was most evident during recrudescence. ER mRNA levels were significantly elevated during recrudescence in males but not females. In conclusion, testosterone may have a role in male reproductive tract recrudescence; however, this putative activity is independent of a NR3C4-type androgen receptor. The ER also may function in male recrudescence, though apparently independent of 17β-estradiol. The retinoid signaling pathway is discussed as a possible alternative hormone/receptor-mediated signaling pathway that regulates male recrudescence.

Impact of estrogen receptor gene polymorphisms and mRNA levels on obesity and lipolysis – a cohort study

BackgroundThe estrogen receptors α and β (ESR1, ESR2) have been implicated in adiposity, lipid metabolism and feeding behaviour. In this report we analyse ESR1 and ESR2 gene single nucleotide polymorphisms (SNPs) for association with obesity. We also relate adipose tissue ESR1 mRNA levels and ESR1 SNPs to adipocyte lipolysis and lipogenesis phenotypes.Methods23 ESR1 and 11 ESR2 tag-SNPs, covering most of the common haplotype variation in each gene according to HAPMAP data, were analysed by Chi2 for association with obesity in a cohort comprising 705 adults with severe obesity and 402 lean individuals. Results were replicated in a cohort comprising 837 obese and 613 lean subjects. About 80% of both cohorts comprised women and 20% men. Adipose tissue ESR1 mRNA was quantified in 122 women and related to lipolysis and lipogenesis by multiple regression. ESR1 SNPs were analysed for association with adipocyte lipolysis and lipogenesis phenotypes in 204 obese women by simple regression.ResultsNo ESR1 SNP was associated with obesity. Five ESR2 SNPs displayed nominal significant allelic association with obesity in women and one in men. The two ESR2 SNPs associated with obesity with nominal P value < 0.01 were genotyped in a second cohort where no association with obesity was observed. There was an inverse correlation between ESR1 mRNA levels in abdominal subcutaneous (sc) adipose tissue and basal lipolysis, as well as responsiveness to adrenoceptor agonists independent of age and BMI (P value 0.009–0.045). ESR1 rs532010 was associated with lipolytic sensitivity to noradrenaline (nominal P value 0.012), and ESR1 rs1884051 with responsiveness to the non-selective beta-adrenoceptor agonist isoprenaline (nominal P value 0.05). These associations became non-significant after Bonferroni correction.ConclusionESR1 gene alleles are unlikely to be a major cause of obesity in women. A minor importance of ESR2 on severe obesity cannot be excluded. The inverse correlation between ESR1 mRNA levels and lipolytic responsiveness to adrenoceptor agonists implies that low adipose tissue ESR1 levels attenuate catecholamine resistance in sc fat cells of obese women hereby contributing to loss of sc and gain of visceral fat. There is no evidence for a genetic impact of ESR1 on lipolysis or lipogenesis.

CD40 signaling predicts response to preoperative trastuzumab and concomitant paclitaxel followed by 5-fluorouracil, epirubicin, and cyclophosphamide in HER-2-overexpressing breast cancer.

IntroductionWe performed gene expression analysis to identify molecular predictors of resistance to preoperative concomitant trastuzumab and paclitaxel followed by 5-fluorouracil, epirubicin, and cyclophosphamide (T/FEC).MethodsPretreatment fine-needle aspiration specimens from 45 patients with HER-2-overexpressing stage II to IIIA breast cancer were subjected to transcriptional profiling and examined for differential expression of various genes and gene sets. The primary endpoint for tumor response was pathologic complete response (pCR). Correlations between pCR and gene expression were sought.ResultsThe overall pCR rate was 64%. Age, nuclear grade, tumor size, nodal status, quantitative expression of estrogen and HER-2 receptor mRNA, and HER-2 gene copy number showed no correlation with pCR. Results of gene set enrichment analysis suggested that the lower expression of genes involved with CD40 signaling is associated with a greater risk of residual cancer after the preoperative chemotherapy that includes trastuzumab.ConclusionCD40 signaling may play a role in determining response to trastuzumab-plus-T/FEC therapy in patients with HER-2-overexpressing breast cancer.

Effects of Lactational Exposure to Soy Isoflavones on Reproductive System in Neonatal Female Rats*

To examine effects of lactational exposure to soy isoflavones on female reproductive system, soy isoflavones, 0 (control), 10, 50, 100, 150 or 200 mg/kg body weight, were administered to maternal rats by gavage daily during postnatal days 5-10. Ten female litters were killed on day 11. Uterine and ovary weights, serum oestradiol and progesterone concentrations, proliferating cell nuclear antigen, mRNA expressions of oestrogen receptor, androgen receptor and progesterone receptor in uterus or ovary were measured. When compared to the control group, litters in the 150 and 200 mg/kg body weight groups had significantly higher ovary and uterine weights (P < 0.05). Endometrial thickness was also significantly increased. Isoflavone-treated groups had increased proliferating cell nuclear antigen staining in comparison to the control group. Litters in the 50, 150 and 200 mg/kg body weight groups had significantly higher oestradiol concentration while lower progesterone concentrations than those in the control group (P < 0.05 or P < 0.01). All soy isoflavone-treated groups had androgen receptor mRNA expressions in ovary and uterus comparable to the control group. Progesterone receptor mRNA expression in the 150 and 200 mg/kg body weight groups was significantly lower than that in control group (P < 0.01). However, ovaries in the 150 and 200 mg/kg body weight groups had significantly higher oestrogen receptor mRNA expression while the uterus in these two treatment groups had significantly lower expressions, when compared to the control group (P < 0.05 or P < 0.01). In conclusion, lactational exposure to isoflavones could result in oestrogen-like actions on the reproductive system of neonate female rats, which mechanisms may be, at least, involved with modifications of hormone production and steroid receptor transcription in the reproductive system.

Molecular Basis for Estrogen Receptor Deficiency in BRCA1-Linked Breast Cancer

BRCA1-mutant breast tumors are typically estrogen receptor alpha (ER alpha) negative, whereas most sporadic tumors express wild-type BRCA1 and are ER alpha positive. We examined a possible mechanism for the observed ER alpha-negative phenotype of BRCA1-mutant tumors. We used a breast cancer disease-specific microarray to identify transcripts that were differentially expressed between paraffin-embedded samples of 17 BRCA1-mutant and 14 sporadic breast tumors. We measured the mRNA levels of estrogen receptor 1 (ESR1) (the gene encoding ER alpha), which was differentially expressed in the tumor samples, by quantitative polymerase chain reaction. Regulation of ESR1 mRNA and ER alpha protein expression was assessed in human breast cancer HCC1937 cells that were stably reconstituted with wild-type BRCA1 expression construct and in human breast cancer T47D and MCF-7 cells transiently transfected with BRCA1-specific short-interfering RNA (siRNA). Chromatin immunoprecipitation assays were performed to determine if BRCA1 binds the ESR1 promoter and to identify other interacting proteins. Sensitivity to the antiestrogen drug fulvestrant was examined in T47D and MCF-7 cells transfected with BRCA1-specific siRNA. All statistical tests were two-sided. Mean ESR1 gene expression was 5.4-fold lower in BRCA1-mutant tumors than in sporadic tumors (95% confidence interval [CI] = 2.6-fold to 40.1-fold, P = .0019). The transcription factor Oct-1 recruited BRCA1 to the ESR1 promoter, and both BRCA1 and Oct-1 were required for ER alpha expression. BRCA1-depleted breast cancer cells expressing exogenous ER alpha were more sensitive to fulvestrant than BRCA1-depleted cells transfected with empty vector (T47D cells, the mean concentration of fulvestrant that inhibited the growth of 40% of the cells [IC40] for empty vector versus ER alpha: >10(-5) versus 8.0 x 10(-9) M [95% CI = 3.1 x 10(-10) to 3.2 x 10(-6) M]; MCF-7 cells, mean IC40 for empty vector versus ER alpha: >10(-5) versus 4.9 x 10(-8) M [95% CI = 2.0 x 10(-9) to 3.9 x 10(-6) M]). BRCA1 alters the response of breast cancer cells to antiestrogen therapy by directly modulating ER alpha expression.

Synthesis and Evaluation of the Antiproliferative Activities of Derivatives of Carboxyalkyl Isoflavones linked to N-t-Boc-hexylenediamine

The isoflavones biochanin A ( 1a), genistein ( 1b), and daidzein ( 4) at concentrations >20 microM inhibit cell growth of various cancer cell lines. To enhance the antiproliferative activities of these compounds, we synthesized three analogs, 2-[3-carboxy-(6-tert-butoxycarbonylamino)-hexylamino-propyl]-7,5-dihydroxy-4'-methoxyisoflavone ( 3a), 2-[3-[N-[6-(tert-butoxycarbonyl)-aminohexyl]]-caboxamidopropyl]-5,7,4'-trihydroxyisoflavone ( 3b), and 5-{2-[3-(4-hydroxy-phenyl)-4-oxo-4 H-chromen-7-yloxy]-acetylamino}-pentyl)-carbamic acid tert-butyl ester ( 6). When cancer cells expressing predominantly estrogen receptor mRNA of the beta- relative to alpha-subtype were treated with 3a, 3b, or 6, DNA synthesis was inhibited in a dose-dependent manner, ranging from 15 to 3000 nmol/L, with little inhibitory effect in normal vascular smooth muscle cells. Compound 6 was the most potent one, and its antiproliferative effect in cancer cells was modulated by estrogen and by the apoptosis inhibitor Z-VADFK. When tested in vivo, compound 6 decreased tumor volume of ovarian xenografts by 50%, with no apparent toxicity. Compound 6 may be a promising agent for therapy of cancer either alone or in combination with chemotherapeutic agents.

Novel bromomelatonin derivatives suppress osteoclastic activity and increase osteoblastic activity: implications for the treatment of bone diseases

The teleost scale is a calcified tissue that contains osteoclasts, osteoblasts, and bone matrix, all of which are similar to those found in mammalian membrane bone. Using the goldfish scale, we recently developed a new in vitro assay system and previously demonstrated that melatonin suppressed both osteoclastic and osteoblastic activities in this assay system. In mammals, 2-bromomelatonin possesses a higher affinity for the melatonin receptor than does melatonin. Using a newly developed synthetic method, we synthesized 2-bromomelatonin, 2,4,6-tribromomelatonin and novel bromomelatonin derivatives (1-allyl-2,4,6-tribromomelatonin, 1-propargyl-2,4,6-tribromomelatonin, 1-benzyl-2,4,6-tribromomelatonin, and 2,4,6,7-tetrabromomelatonin) and then examined the effects of these chemicals on osteoclasts and osteoblasts. All bromomelatonin derivatives, as well as melatonin, had an inhibitory action on osteoclasts. In particular, 1-benzyl-2,4,6-tribromomelatonin (benzyl-tribromomelatonin) possessed a stronger activity than melatonin. At an in vitro concentration of 10(-10) m, benzyl-tribromomelatonin still suppressed osteoclastic activity after 6 hr of incubation. In reference to osteoblasts, all bromomelatonin derivatives had a stimulatory action, although melatonin inhibited osteoblastic activity. In addition, estrogen receptor mRNA expression (an osteoblastic marker) was increased in benzyl-tribromomelatonin (10(-7) m)-treated scales. Taken together, the present results strongly suggest that these novel melatonin derivatives have significant potential for use as beneficial drug for bone diseases such as osteoporosis.

Human prostate cancer: a direct role for oestrogens.

We have studied the response to oestrogen and expression of oestrogen receptors in responsive LNCaP and androgen non-responsive PC3 human prostate cancer cell lines. Growth of LNCaP cells is significantly stimulated by physiological concentrations of oestradiol; this growth increase appears to be comparable to that induced by either testosterone or dihydrotestosterone. In contrast, oestradiol significantly inhibits the proliferation of PC3 cells. We also present novel evidence for functional oestrogen binding in LNCaP cells. This evidence was first obtained by means of radioligand binding assays and was further corroborated using: (a) immunocytochemical analysis of oestrogen and progesterone receptors; (b) reverse transcriptase polymerase chain reaction of oestrogen receptor mRNAs; and (c) immunofluorescence of the 27 kDa heat shock protein (Hsp27), which has been reported to be a marker of functional oestrogen receptors. There appeared to be significantly and consistently lower levels of oestrogen receptor expressed in PC3 cells than in LNCaP cells. The observation that oestradiol-induced growth of LNCaP cells is completely reversed by the pure anti-oestrogen ICI 182,780 clearly implies that the biological response of these cells to oestradiol is mediated mainly via its own receptor. On the other hand, use of a neutralizing antibody against transforming growth factor (TGF)-beta 1 results in a remarkable increase in the growth of PC3 cells; this effect is almost completely abolished after the addition of oestradiol. This suggests that the oestradiol-induced growth inhibition may be mediated by TGF-beta 1. These results suggest that the current model for hormone-dependence of human prostatic carcinoma should be revised. This is of special concern, because recent data indicate that prostate cancer has become the most prevalent cancer and the second principal cause of cancer death in western countries.