Anette Springwald

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.

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.

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.

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.

Expression of Cysteine Protease Cathepsin L is Increased in Endometrial Cancer and Correlates With Expression of Growth Regulatory Genes

Proteases contribute to tumor invasion and metastasis by degrading basement membranes and extracellular matrix (ECM). In this study, we compared gene expression levels of two proteases, cysteine protease Cathepsin L2 (CTSL2) and matrix metalloproteinase MMP11, in human endometrium and endometrial cancer. Our data demonstrate CTSL2 transcript levels to be strongly elevated in endometrial cancer, particularly in G3 tumors. Furthermore, we observed a highly significant positive correlation of CTSL2 with expression of growth regulatory genes Ki-67, cyclin B1, MYBL2, p21/WAF, and HER2 receptor tyrosine kinase. Our data suggest that CTSL2 might be involved in progression of endometrial cancer.

Effects of a combined treatment with GPR30 agonist G-1 and herceptin on growth and gene expression of human breast cancer cell lines.

Expression of G-protein-coupled receptor 30 (GPR30) is present in HER2-overexpressing breast cancer. In this study, we examined to what extent GPR30-agonist G-1 would affect the antitumoral action of trastuzumab (Herceptin). Combined treatment with both drugs exerted an additive growth-inhibitory effect on breast cancer cells which was accompanied by a significant decline of cyclin A2 expression both on the protein and the mRNA level. Combined treatment also resulted in expression changes of c-fos, cyclin D1, or p21/WAF-1. The results of our study encourage further attempts to test the relevance of these in vitro data in the clinical setting.

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 ERα, four single, double or triple exon-skipped mRNA isoforms of ERβ, and four new transcript variations of PR gene. Sequence analysis suggested that at least four of them, ERαΔ5/6, ERαΔ5/6/7, PRΔ7, and PRΔ6/7 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 PRΔ6/7, ERαΔ5/6/7, ERαΔ3/4/5, and ERβΔ1-0N. 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.

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 (ERalpha) expression and a significant decrease of ERbeta expression on the mRNA level. Expression of ERalpha-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 ERalpha transcript levels but sustaining high levels of ERbeta, reducing both activation of ERalpha 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.

Single Nucleotide Polymorphisms in Human Gene icb-1 and 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 women—323 breast cancer cases and just as many controls—were 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.

Silencing of the icb-1 gene inhibits the induction of differentiation-associated genes by vitamin D3 and all-trans retinoic acid in gynecological cancer cells.

Icb-1 (C1orf38) is a human gene initially described by our group to be involved in differentiation processes of cancer cells. To further elucidate the function of the icb-1 gene in differentiation of breast and endometrial cancer cells, we knocked down its expression by means of shRNA transfection. Knockdown of icb-1 inhibited the vitamin D3-induced up-regulation of E-cadherin expression in both MCF-7 and HEC-1B cells. Induction of E-cadherin expression by all-trans retinoic acid (ATRA) was also blocked in both cell lines expressing icb-1 siRNA. Examination of icb-1 and E-cadherin expression in 66 breast cancer tissue samples revealed a significant positive correlation between the two genes. In MCF-7 cells, silencing of the icb-1 gene inhibited the ATRA- and the vitamin D3-induced up-regulation of lactoferrin and estrogen receptor β expression. The data of our knockdown study suggest that icb-1 may act as a mediator of differentiation signals in breast cancer cells induced by ATRA or vitamin D3. These findings together with the observed co-expression of icb-1 with E-cadherin in breast cancer samples support an important role of the icb-1 gene in cancer cell differentiation.