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

Estrogen receptor beta (ER beta) is widely expressed in mammary epithelium. ER beta expression is reported to decline during carcinogenesis of the breast and other tissues. In this study, we examined the consequences of a loss of ER beta expression in mammary epithelial cells. We knocked down ER beta 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. ER beta 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 ER beta 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, ER beta 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 ER alpha/beta ratio were accompanied by smaller estrogenic repression of p21/WAF expression, an impaired tamoxifen-triggered induction of this gene and by relative downregulation of ER alpha and cyclin A2 transcript levels. Our data suggest that ER beta exerts antiproliferative effects both on MCF-10A and MCF-7 cells in a ligand- and ER alpha-independent manner by regulation of p21/WAF or cyclin A2 gene expression. Knockdown of ER beta 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 ER beta acts as a tumor suppressor in mammary epithelium

Single Nucleotide Polymorphisms in Human Geneicb-1and Breast Cancer Susceptibility

In this study, we tested the hypothesis that single nucleotide polymorphisms (SNPs) of differentiation-associated human gene icb-1 (C1orf38) may be associated with breast cancer susceptibility. A total of 646 women323 breast cancer cases and just as many controlswere included. Breast cancer patients more frequently carried the homozygous genotype AA of SNP rs1467465 than did healthy women. Analysis of allele positivity revealed that AG or GG genotypes were significantly less frequent in breast cancer patients, suggesting that presence of G allele might have protective effects. Our data suggest that SNP rs1467465 of human gene icb-1 might affect breast cancer susceptibility

Identification of novel transcript variants of estrogen receptor α, β and progesterone receptor gene in human endometrium

The human progesterone receptor (PR) and estrogen receptor genes (ESR1 and ESR2) are known to code for a multitude of transcript variants resulting from alternative splicing. Many of them are translated into nuclear receptor proteins with altered structure and function. Expression of these alternative estrogen and progesterone receptors modulates the cellular response to sexual steroid hormones. Recent studies also suggested their significance in development of hormone-dependent diseases like gynecological cancers. We report identification of 12 new transcript variations of the PR, ESR1, and ESR2 gene in human endometrium which result from differential exon-skipping. We succeeded in cloning of four new double or triple exon-deletion transcript variants of ER alpha, four single, double or triple exon-skipped mRNA isoforms of ER beta, and four new transcript variations of PR gene. Sequence analysis suggested that at least four of them, ER alpha I"5/6, ER alpha I"5/6/7, PR Delta 7, and PR Delta 6/7 are translated into receptor proteins which might exert ligand-independent effects on steroid hormone signalling. Comparison of pre- and post-menopausal endometrium revealed differential expression of PR Delta 6/7, ER alpha I"5/6/7, ER alpha I"3/4/5, and ER beta I"1-0N. We also report differential expression of the exon-skipped isoforms in a panel of human cancer cell lines derived from the breast, ovary, and endometrium. Our identification of additional transcript variations further increases the complexity of steroid hormone receptor gene expression and signalling

Icb-1Gene Expression is Elevated in Human Endometrial Adenocarcinoma and is Closely Associated With HER2 Expression

Human gene icb-1 (C1orf38) has been initially cloned by our group from endometrial adenocarcinoma cells. In this study, we examined icb-1 expression in 90 endometrial cancer and normal endometrial tissue specimens. Expression of icb-1 was significantly (about 3.5-fold) higher in endometrial cancer than in normal endometrium (p <.0001). Determination of various molecular markers revealed that only Ki-67 expression differed between both groups in a similarly significant manner. Furthermore, we observed a highly significant positive correlation of icb-1 transcript levels with c-erbB2 (HER2) expression (p <.0001). Our data strongly encourage further studies on the function of icb-1 in endometrial cancer

Knockdown of ICB-1 gene enhanced estrogen responsiveness of ovarian and breast cancer cells

ICB-1 chromosome 1 open reading frame 38 (C1orf38) is a human gene initially described by our group to be involved in differentiation processes of cancer cells. Recently, we have reported ICB-1 as a novel estrogen target gene and identified an estrogen response element in its promoter. In this study, we examined the role of ICB-1 in regulation of proliferation of breast and ovarian cancer cells. We knocked down its expression in estrogen-dependent MCF-7 breast cancer cells and hormone-unresponsive SK-OV-3 ovarian cancer cells by stable transfection with a specific shRNA plasmid followed by G-418 selection. Knockdown of ICB-1 enabled a considerable estrogen response of SK-OV-3 cells in terms of proliferation. This transformation of SK-OV-3 cells into an estrogen-responsive phenotype was accompanied by upregulation of estrogen receptor α (ERα) expression and a significant decrease of ERβ expression on the mRNA level. Expression of ERα-dependent genes progesterone receptor, pS2, fibulin 1c, and c-fos was elevated in SK-OV-3 cells stably expressing ICB-1 shRNA. In MCF-7 cells, ICB-1 knockdown exerted similar effects on gene expression, supporting a general role of ICB-1 in estrogen responsiveness. Our data suggest that differentiation-associated gene ICB-1 might exert antagonistic actions on cellular estrogen response, which can result in inhibition of estradiol-triggered proliferation. The molecular mechanisms mediating this inhibitory effect of ICB-1 on estrogen signaling are suggested to be limitation of ERα transcript levels but sustaining high levels of ERβ, reducing both activation of ERα target genes and cellular proliferation. The identification of ICB-1 as a new player in endocrine-related cancer encourages further studies on the significance of this gene in cancer development and therapy.</jats:p

Knockdown of ICB-1 gene enhanced estrogen responsiveness of ovarian and breast cancer cells

ICB-1 chromosome 1 open reading frame 38 (C1orf38) is a human gene initially described by our group to be involved in differentiation processes of cancer cells. Recently, we have reported ICB-1 as a novel estrogen target gene and identified an estrogen response element in its promoter. In this study, we examined the role of ICB-1 in regulation of proliferation of breast and ovarian cancer cells. We knocked down its expression in estrogen-dependent MCF-7 breast cancer cells and hormone-unresponsive SK-OV-3 ovarian cancer cells by stable transfection with a specific shRNA plasmid followed by G-418 selection. Knockdown of ICB-1 enabled a considerable estrogen response of SK-OV-3 cells in terms of proliferation. This transformation of SK-OV-3 cells into an estrogen-responsive phenotype was accompanied by upregulation of estrogen receptor alpha (ER alpha) expression and a significant decrease of ER beta expression on the mRNA level. Expression of ER alpha-dependent genes progesterone receptor, pS2, fibulin 1c, and c-fos was elevated in SK-OV-3 cells stably expressing ICB-1 shRNA. In MCF-7 cells, ICB-1 knockdown exerted similar effects on gene expression, supporting a general role of ICB-1 in estrogen responsiveness. Our data suggest that differentiation-associated gene ICB-1 might exert antagonistic actions on cellular estrogen response, which can result in inhibition of estradiol-triggered proliferation. The molecular mechanisms mediating this inhibitory effect of ICB-1 on estrogen signaling are suggested to be limitation of ER alpha transcript levels but sustaining high levels of ER beta, reducing both activation of ER alpha target genes and cellular proliferation. The identification of ICB-1 as a new player in endocrine-related cancer encourages further studies on the significance of this gene in cancer development and therapy. Endocrine-Related Cancer (2010) 17 147-15

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. (C) 2009 Published by Elsevier Ltd