Claus Lattrich

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.

GPR30 gene polymorphisms are associated with progesterone receptor status and histopathological characteristics of breast cancer patients.

G-protein coupled receptor GPR30 has been demonstrated to mediate estrogenic effects on essential features of human breast cancer cells. Polymorphisms in GPR30 gene might therefore affect breast cancer susceptibility or tumor characteristics. This is the first study examining allele and genotype frequencies of GPR30 single nucleotide polymorphisms (SNPs) in breast cancer patients. A total of 257 sporadic breast cancer cases and 247 age-matched controls were genotyped for three GPR30 polymorphisms by means of allele-specific tetra-primer PCR. Comparison of the breast cancer case and the control group with regard to the SNP allele, genotype and haplotype frequencies did not show significant differences. In contrast, the GPR30 SNPs tested were significantly associated with tumor size, histological grading, nodal status and progesterone receptor (PR) status. The A allele of SNP rs3808351 was significantly less frequent in patients with large or G3 tumors, T allele of SNP rs11544331 less frequently occurred in patients with positive nodal status, suggesting that both SNPs might exert protective effects regarding aggressive breast cancer entities. Both homozygous GG genotype of promoter SNP rs3808350 and T allele of missense SNP rs11544331 were inversely associated with PR-negativity, suggesting that they might exert protective effects regarding development of PR-negative cancer. In conclusion, the results of this study support the important role of GPR30 in breast cancer and encourage functional studies on the molecular mechanisms underlying the association of GPR30 polymorphisms with PR status and tumor growth.

Role of estrogen receptor β in gynecological cancer.

Estrogen receptor β is expressed in normal and neoplastic ovarian and endometrial tissues. Recent studies indicate that levels of this receptor decline in ovarian tumorigenesis, like in breast or prostate cancer. Furthermore, ERβ expression has been associated with good prognosis in ovarian cancer. In contrast, previous studies on the role of this receptor in endometrial cancer suggested that ERβ might play different roles in the carcinogenesis of the ovary and endometrium. Besides its possible role as a prognostic factor, ERβ might be a potential target for the treatment of ovarian and endometrial cancer.

Additive effects of trastuzumab and genistein on human breast cancer cells.

Soy isoflavone genistein, a tyrosine kinase inhibitor and agonist of estrogen receptor-&bgr; (ER&bgr;), is known to have antitumoral properties. Given that ER&bgr; often is coexpressed with HER2 in breast cancer, both functions of genistein might be able to enhance the antitumoral action of trastuzumab. In this in-vitro study, we tested whether combined treatment with genistein and trastuzumab exerts additive effects on breast cancer cells. HER2-overexpressing breast cancer cell lines were treated with genistein alone and in combination with trastuzumab. The effects of this treatment on proliferation and gene expression were analyzed. Treatment with high-dose genistein (10 &mgr;mol/l) significantly increased the growth-inhibitory effect of trastuzumab on HER2-overexpressing, ER&agr;/&bgr;-positive BT-474 breast cancer cells. Combinatory treatment using lower doses of trastuzumab exerted similar effects as a single treatment with standard doses of this drug. In contrast, this effect was absent in ER&agr;-negative SK-BR-3 cells. Similar results were obtained after cotreatment with the ER&bgr; agonist, 2,3-bis(4-hydroxyphenyl)propionitrile. The growth-inhibitory effect of both drugs was accompanied by an increased expression of the putative tumor suppressor ER&bgr; variant, cx, and their combination further elevated mRNA levels of this receptor. In conclusion, genistein significantly enhanced the antitumoral effect of trastuzumab on BT-474 breast cancer cells in vitro. The relevance of these data particularly for women with HER2-overexpressing and ER&agr;/&bgr;-positive breast cancer has to be verified in animal or clinical studies.

The Treatment of Climacteric Symptoms

BACKGROUND Peri- and postmenopausal women commonly suffer from climacteric symptoms. In this article, we provide information to help physicians recognize climacteric symptoms and treat them appropriately. METHODS The information presented here is based on a selective search of the literature for pertinent articles that appeared from 2008 to early 2011, including the German S3 guideline on hormone therapy (HT) during and after menopause, which was published in 2009. RESULTS Perimenopausal women often suffer from climacteric symptoms. Typically, women undergoing menopause complain of heat waves and vaginal dryness. According to randomized controlled trials as well as national and international guidelines, HT is the most effective treatment for vasomotor symptoms and also improves vulvovaginal atrophy; for the latter indication, HT is preferably administered locally. Vaginal estrogen therapy lowers the frequency of recurrent urinary tract infections. However, HT is associated with an increased risk for a number of diseases, including stroke, thromboembolic events, gall-bladder diseases, and breast cancer. Alternative treatments for climacteric symptoms have little or no efficacy. CONCLUSION HT should only be used to treat climacteric symptoms after extensive patient education about its benefits and risks. Participatory decision-making is desirable. The generalized use of HT by all women with climacteric symptoms cannot be recommended.

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.

Effects of estriol on growth, gene expression and estrogen response element activation in human breast cancer cell lines

OBJECTIVE Local application of estradiol (E2) to treat vulvovaginal atrophy in postmenopausal breast cancer patients receiving aromatase inhibitors is known to elevate serum estradiol levels and thereby might counteract breast cancer therapy. Thus, vaginal application of estriol (E3) has been recommended for these patients. However, it is unclear to what extent E3 stimulates breast cancer cell growth. In this study, we examined the effect of E3 on growth and gene expression of two human breast cancer cell lines. METHODS We used an established in vitro cell culture assay and compared the effect of E2 and E3 on growth of the estrogen receptor alpha-positive breast cancer cell lines MCF-7 and T-47D testing a wide range of hormone concentrations of 10(-12)-10(-7)M. E3 effects on gene expression were examined by means of reporter gene assays, RT-qPCR and Western blot analysis. RESULTS E3 acted as a potent estrogen and exerted a mitogenic effect on T-47D and MCF-7 cells at concentrations of 10(-9)M (288 pg/ml) and higher. With regard to activation of an estrogen response element (ERE) in breast cancer cells, effects of E3 were visible at 10(-10)M. The same concentrations of E3 activated expression of the estrogen-responsive gene PR and of the proliferation genes cyclin A2, cyclin B1, Ki-67, c-myc and b-myb, providing molecular mechanisms underlying the observed growth increase. CONCLUSIONS Like E2, low levels of E3 were able to trigger a robust estrogenic response in breast cancer cells. Thus, our data suggest caution regarding use of E3 by breast cancer survivors.

Estrogen receptor β agonists affect growth and gene expression of human breast cancer cell lines.

Expression of estrogen receptor β (ERβ) has been described to reduce growth of cancer cell lines derived from hormone-dependent tumors, like breast cancer. In this study we tested to what extent two ERβ agonists, androgen derivative 3β-Adiol and flavonoid Liquiritigenin, would affect growth and gene expression of different ERβ-positive human breast cancer cell lines. Under standard cell culture conditions, we observed 3β-Adiol to inhibit growth of MCF-7 cells in a dose-dependent manner, whereas growth of BT-474 and MCF-10A cells was suppressed by the maximum concentration (100 nM) only. When treated in serum-free medium, all cell lines except of MDA-MB-231 were responsive to 1 nM 3β-Adiol, and ZR75-1 cells exhibited a dose-dependent antiproliferative response. Providing putative mechanisms underlying the observed growth-inhibitory effect, expression of Ki-67 or cyclins A2 and B1 was downregulated after 3β-Adiol treatment in all responsive lines. In contrast, treatment with lower doses of Liquiritigenin did not affect growth. In MCF-7 cells, the highest dose of this flavonoid exerted proliferative effects accompanied by increased expression of cyclin B1, PR and PS2, indicating unspecific activation of ERα. In conclusion, the ERβ agonists tested exerted distinct concentration-dependent and cell line-specific effects on growth and gene expression. The observed inhibitory effects of 3β-Adiol on breast cancer cell growth encourage further studies on the potential of this and other ERβ agonists as targeted drugs for breast cancer therapy.