Oliver Treeck

Estrogen receptor 1 exerts antitumoral effects on SK-OV-3 ovarian cancer cells

Estrogen receptor (ER) beta1 and its splice variants are expressed both in ovary and ovarian cancer. We studied the role of ERbeta1 and two of its splice variants in regulation of gene expression, cellular proliferation, apoptosis, and migration of an ovarian cancer cell line. In this study, we transfected SK-OV-3 ovarian cancer cells with vectors coding for ERbeta1 or its splice variants ERbeta-delta125 and ERbeta-delta1256, and tested their response to estrogen and tamoxifen in comparison with the untransfected cells. Heterologous expression of ERbeta1, but not of the exon-deleted ERbeta variants resulted in notably slower cell growth of SK-OV-3 ovarian cancer cells, an effect accompanied by more than tenfold increase of cyclin-dependent kinase inhibitor p21(WAF1) transcript levels and a significant reduction of cyclin A2 mRNA levels. SK-OV-3 cells stably overexpressing ERbeta1 ligand independently also exhibited an increased apoptosis rate and a significantly decreased motility, an effect accompanied by upregulation of fibulin 1c. Our data demonstrate that ERbeta1, but not the exon-deleted isoforms tested exerts multiple antitumoral effects on SK-OV-3 ovarian cancer cells even in the absence of estradiol or functional ERalpha.

Estrogen receptor beta exerts growth-inhibitory effects on human mammary epithelial cells

Estrogen receptor β (ERβ) is widely expressed in mammary epithelium. ERβ expression is reported to decline during carcinogenesis of the breast and other tissues. In this study, we examined the consequences of a loss of ERβ expression in mammary epithelial cells. We knocked down ERβ transcript levels in human mammary epithelial MCF-10A cells and in MCF-7 breast cancer cells by means of stable transfection with a specific shRNA plasmid. ERβ knockdown resulted in a significant growth increase of both cell types in a ligand-independent manner. This effect was accompanied by elevated cyclin A2 expression in MCF-10A cells and by decreased expression of growth-inhibitory p21/WAF and epithelial cell marker cytokeratine 8 in both cell lines. Transfection of ERβ shRNA did not alter the absent proliferative estrogen response of MCF-10A cells, but conferred sensitivity to selective estrogen receptor modulator tamoxifen to this cell line. In contrast, ERβ knockdown diminished estrogen responsiveness of MCF-7 breast cancer cells and also weakened the effect of tamoxifen on this cell line. These ligand-dependent effects only observed in MCF-7 cells exhibiting a high ERα/β ratio were accompanied by smaller estrogenic repression of p21/WAF expression, an impaired tamoxifen-triggered induction of this gene and by relative downregulation of ERα and cyclin A2 transcript levels. Our data suggest that ERβ exerts antiproliferative effects both on MCF-10A and MCF-7 cells in a ligand- and ERα-independent manner by regulation of p21/WAF or cyclin A2 gene expression. Knockdown of ERβ in both cell types was sufficient to significantly decrease transcript levels of epithelial cell marker cytokeratin 8. The results of this study support the hypothesis that ERβ acts as a tumor suppressor in mammary epithelium.

Effects of exon-deleted estrogen receptor β transcript variants on growth, apoptosis and gene expression of human breast cancer cell lines

Estrogen receptor β gene codes for a variety of transcript isoforms resulting from alternative splicing, which are expressed both in mammary gland and in breast cancer cells. We studied the function of two exon-deleted ERβ isoforms recently identified by our group in comparison to ERβ1 in regulation of growth, apoptosis and gene expression of two breast cancer cell lines with different ERα status. Overexpression of ERβ1, but not of the exon-deleted variants exerted strong antitumoral effects both on ERα-positive MCF-7 and ERα-negative SK-BR-3 cells. ERβ1 overexpression slowed growth of MCF-7 and SK-BR-3 cells in the absence of E2 and also inhibited E2-triggered growth stimulation of MCF-7 cells, but overexpression of the exon-skipped variants did not affect cell growth. Whereas overexpression of ERβ1 triggered an increased basal and tamoxifen-induced apoptosis of MCF-7 and SK-BR-3 cells, the isoforms ERβδ125 or ERβδ1256 did not affect cellular tamoxifen response. The observed lack of function of the exon-deleted variants in terms of regulation of proliferation was accompanied both by their inability to affect expression of cyclins D1 and A2, p21 (WAF1) and PR and their disability to modulate estrogen response element (ERE) activation. In contrast, our results demonstrating antitumoral effects of ERβ1 on breast cancer cells with different ERα-status support the hypothesis that ERβ is able to exert antitumoral actions both on ERα-positive and -negative breast cancer cells.

Polymorphisms in the promoter region of ESR2 gene and breast cancer susceptibility

Genetic variations like single nucleotide polymorphisms (SNPs) in genes involved in estrogen biosynthesis, metabolism and signal transduction have been suggested to affect breast cancer susceptibility. In this study we tested the hypothesis that polymorphisms in the promoter of ESR2 gene may be associated with increased risk for breast cancer. We analyzed three SNPs in the promoter region of human ESR2 gene by means of allele-specific tetra-primer PCR. A total of 318 sporadic breast cancer cases and 318 age-matched controls were included in the study. With regard to homozygous genotypes, women with sporadic breast cancer more frequently carried the CC genotype of ESR2 promoter SNP rs2987983 (OR 1.99, p=0.005). Calculation of allele positivity demonstrated that presence of T allele of this SNP was more frequent in healthy women. Our data suggest that a SNP in the promoter region of ESR2 gene might be able to affect breast cancer risk. These results further support the emerging hypothesis that ERbeta is an important factor in breast cancer development.

Endometrial expression of estrogen receptor β and its splice variants in patients with and without endometriosis.

BackgroundThe role of estrogen receptor beta (ERβ) in pathogenesis of endometriosis remains to be elucidated. In this study, we have examined the expression of the four main ERβ transcript isoforms in human endometrial tissue in women with or without endometriosis.MethodsTotal RNA was isolated from native endometrial tissue and transcript levels of ERα, β1, β2, β4, β5 were analyzed by means of RT-PCR. We compared the results with regard to menstrual cycle phase as well as to presence or absence of endometriosis. We prospectively harvested the endometrium of ten women without endometriosis (five for each cycle phase) and eight patients with endometriosis (five in the proliferative phase, three in the secretory phase).ResultsERα, β1, β2, and β5 transcripts were detected in both cycle phases. During the proliferative phase, healthy women had a significantly higher ERα/ERβ1-ratio than patients with endometriosis. Irrespective of the cycle phase, ERα-mRNA level was significantly higher than transcript levels of ERβ isoforms.ConclusionsERα, β1, β2, and β5 are expressed in human endometrium. The individual receptors differed in terms of expression strength but there was no relevant change during the cycle. The decreased ERα/ERβ1-ratio in proliferative endometrium of endometriosis patients suggest that ERβ1 might be involved in the pathogenesis of endometriosis. Further studies should be undertaken to substantiate the role of ERβ in endometrial pathology.

Influence of insulin resistance on adiponectin receptor expression in breast cancer

OBJECTIVEnAdipositas and insulin resistance are modifiable risk factors for breast cancer. Adiponectin seems to be an important linkage of these associations. In this study, we investigated the relationship between intratumoral adiponectin receptor expression and insulin resistance as well as intratumoral insulin/IGF receptor expression in breast cancer specimen.nnnMETHODSnBreast cancer tissue and fasting serum were collected from 26 female patients. After microdissection of frozen samples, RNA was isolated and expression of insulin receptor, IGFR1, IGFR2, AdipoR1 and AdipoR2 was measured on mRNA level by means of real time RT-PCR. Fasting insulin, glucose and c-peptide serum levels were analysed by ELISA. Insulin resistance was calculated using the HOMA model.nnnRESULTSnWe were able to confirm AdipoR1 and AdipoR2 expression, respectively, in breast cancer specimen. Actually, neither insulin serum level nor whole-body insulin resistance showed any effect on insulin/IGF or adiponectin receptor expression in breast cancer. A strong positive correlation between insulin as well as IGF1 receptor and AdipoR1, but not AdipoR2, expression could be observed. Interestingly, AdipoR2 expression significantly correlated with vascular and lymphovascular invasion of breast cancer.nnnCONCLUSIONnWe propose a close relationship between the intratumoral insulin signalling system and AdipoR1 but not AdipoR2 expression. As AdipoR2 but not AdipoR1 expression seems to correlate with invasiveness, we assume different functions of the two adiponectin receptors in breast cancer.

Estrogen receptor β gene polymorphisms and susceptibility to uterine fibroids

Uterine fibroids are the most common benign tumors of the female genital tract. Steroid hormones, especially estradiol and progesterone, play an important role in the pathobiology of this frequent disease. Recent studies suggested that both expression levels and polymorphisms of estrogen receptor (ER) α and β might affect development of uterine fibroids. In this study, we tested whether single nucleotide polymorphisms (SNPs) in the promoter of estrogen receptor β gene (ESR2) are associated with susceptibility to uterine fibroids. For this purpose, we compared the frequency of three SNPs in the promoter region of ESR2 gene (rs2987983, rs3020450 and rs3020449) in 101 women with uterine fibroids and 102 healthy women serving as controls by means of allele-specific tetra-primer polymerase chain reaction (PCR). Regarding allele frequency, allele positivity, genotype and haplotype frequencies of these SNPs we did not observe any significant difference between healthy women and women with uterine fibroids. In conclusion, our data clearly suggest that the tested SNPs in the promotor region of human ESR2 gene are not associated with the development of uterine fibroids.

Regulation of GnRH I Receptor Gene Expression by the GnRH Agonist Triptorelin, Estradiol, and Progesterone in the Gonadotroph-Derived Cell Line αT3-1

The secretion of luteinizing hormone (LH) and the GnRH receptor (GnRH-R) concentration are modulated by ovarian steroids and GnRH. To elucidate whether this regulation is due to alterations at the transcriptional level, we examined the GnRH I-R mRNA expression in the gonadotroph-derived cell line α T3-1 treated with different estradiol and progester-one paradigms and the GnRH I agonist triptorelin. αT3-1 cells were treated with different steroid paradigms: 1 nM estradiol or 100 nM progesterone for 48 h alone or in combination. Cells were exposed to 10 nM or 100 pM triptorelin for 30 min, 3 h, 9 h, or, in pulsatile way, with a 5-min pulse per hour. The GnRH I-R mRNA was determined by Northern blot analysis. GnRH I-R mRNA from cells treated with continuous triptorelin decreased in a time- and concentration-dependent manner. Pulsatile triptorelin increased GnRH I-R gene expression. Progesterone alone further enhanced this effect, whereas estradiol and its combination with progesterone diminished it. Continuous combined treatment with estradiol and progesterone lead to a significant decrease of GnRH I-R mRNA by 30% and by 35% for estradiol alone. The addition of 10 nM triptorelin for 30 min or 3 h could not influence that steroideffect. In conclusion, estradiol and progesterone exclusively decreased GnRH I-R mRNA in αT3-1 cells no matter whether they are treated additionally with the GnRH I agonist triptorelin. The enhanced sensitivity of gonadotrophs and GnRH I-R upregulation by estradiel is not due to increased GnRH I gene expression because GnRH I-R mRNA is downregulated by estradiol and progesterone. Other pathways of the GnRH I-R signal transduction might be involved.

Expression of differentiation-associated gene icb-1 is estrogen-responsive in ovarian and breast cancer cell lines.

icb-1 (C1orf38) is a human gene initially described by our group to be upregulated during in vitro differentiation processes of endometrial adenocarcinoma and leukemia cells triggered by different stimuli. We now report presence of a putative imperfect estrogen response element (ERE) in the promoter of icb-1 gene. Given that estrogens are known to regulate cellular differentiation processes of hormone-dependent tissues, we studied whether expression of icb-1 would be regulated by 17-beta (beta) estradiol in breast and ovarian cancer cells. As examined by means of real time PCR, treatment with 17-beta estradiol for at least 24h resulted in a significant increase of icb-1 transcript levels in ERalpha-positive MCF-7 breast cancer and OVCAR-3 ovarian cancer cells, but not in ERalpha-negative SK-BR-3 and SK-OV-3 cells. Upregulation of icb-1 transcript levels was also observed after treatment with specific ERalpha-agonist PPT and was inhibited by co-treatment with pure antiestrogen ICI 182,780 in MCF-7 and OVCAR-3 ovarian cancer cells. Treatment with cycloheximide totally inhibited estrogen effects suggesting that activation of icb-1 gene expression is no ERE-dependent early response but a secondary event requiring protein synthesis. The results of this study demonstrate that transcript levels of differentiation-associated gene icb-1 are estrogen-responsive in breast and ovarian cancer cells in an ERalpha-dependent manner. Whether icb-1 is a mediator of estrogen-triggered cellular differentiation processes has to be determined in further studies.

H-ras Dependent Estrogenic Effects of Epidermal Growth Factor in the Estrogen-Independent Breast Cancer Cell Line MDA-MB-231

A crosstalk between cellular estrogen response and receptor tyrosine kinase signaling has been shown in a variety of estrogen receptor (ER)-positive cell lines. We intended to examine the presence of estrogenic growth factor effects in an ER α-negative breast cancer cell line. By means of reporter gene assays, we investigated the activation of estrogen response elements (EREs) by epidermal growth factor (EGF) in the estrogen-unresponsive cell line MDA-MB-231. Our results demonstrate the H-ras-dependent activation of EREs after EGF treatment in this estrogen-unresponsive cell line, an effect which was not observed in the ERα/β-positive breast cancer cell line MCF-7. In MDA-MB-231 cells, the transcriptional activity of an ERE-containing promotor was enhanced dose dependently by all tested EGF concentrations. This effect could be blocked by co-treatment with the epidermal growth factor receptor (EGFR) inhibitors AG1478 and ZD1839, as well as by co-transfection with a vector coding for a dominant negative H-ras mutant, but not by co-treatment with the pure antiestrogen ICI182,780. Furthermore, expression of constitutively active H-ras was shown to be sufficient to activate EREs in MDA-MB-231 cells. Our results suggest alternative utilization of ERE-mediated gene regulation in an estradiol-unresponsive breast cancer cell line in response to an EGF stimulus. This mechanism was shown to be dependent on EGFR and H-ras activity, but independent of the presence of functional ERα.