Polymorphisms in the promoter region of estrogen receptor ß gene in endometrial cancer.

INTRODUCTION: The development of endometrial cancer is known to be affected by estrogens. Thus, genetic variations like single nucleotide polymorphisms (SNPs) in genes involved in estrogen biosynthesis, metabolism, and signal transduction might affect risk for endometrial cancer. In this study, we tested the hypothesis that polymorphisms in the promoter of ESR2 gene may be associated with susceptibility to this disease. METHODS: We compared the frequency of three SNPs in the promoter region of ESR2 gene (rs2987983, rs3020450, and rs3020449) in 135 women with endometrial cancer and 135 healthy women serving as controls by means of allele-specific tetra-primer PCR. RESULTS: Regarding allele frequency, allele positivity or genotype frequencies of these SNPs we did not observe any significant difference between healthy women and women with endometrial cancer. CONCLUSION: Our data clearly suggest that the tested SNPs in the promotor region of human ESR2 gene are not associated with the development of endometrial cancer.

Effects of a combined treatment with tamoxifen and estrogen receptor ß agonists on human breast cancer cell lines.

INTRODUCTION: Coexpression of estrogen receptors (ER) α and ß is present in about half of all breast cancer cases. Whereas ERα is a well-established target for endocrine therapy with the selective estrogen receptor modulator tamoxifen, the applicability of ERß as target in breast cancer therapy is unclear. In this study, we examined the effects of two synthetic ERß agonists alone and in combination with tamoxifen on ERα/ß-positive breast cancer cells. METHODS: We treated MCF-7 and T-47D breast cancer cells with the ERß agonists ERB-041 and WAY-200070 and measured the effects on cell growth. In addition, transcriptome analyses were performed by means of Affymetrix GeneChip arrays. RESULTS: When given alone, ERß agonists ERB-041 and WAY-200070 did not affect the growth of MCF-7 or T-47D cells. In contrast, addition of these drugs to tamoxifen increased its growth-inhibitory effect on both cell lines. This effect was more pronounced under serum-free conditions, but was also observed in the presence of serum in T-47D cells. Transcriptome analyses revealed a set of genes regulated after addition of ERß agonists including S100A8 and CD177. CONCLUSION: The observed enhanced growth-inhibitory effects of a combination of tamoxifen and ERß agonists in vitro encourage further studies to test its possible use in the clinical setting.

Expression of SCUBE2 gene declines in high grade endometrial cancer and associates with expression of steroid hormone receptors and tumor suppressor PTEN.

SCUBE2 (Signal peptide-CUB-epidermal growth factor-like domain-containing 2) gene codes for a cell-surface glycoprotein. In breast cancer, SCUBE2 transcript levels are part of important prognostic and predictive gene signatures and are linked to expression of estrogen receptor α (ERα). To elucidate the role of this gene in endometrial cancer, we compared SCUBE2 expression in malignant and normal endometrial tissue specimens. We then examined its correlation with steroid hormone receptors and PTEN and compared it to SCUBE2 expression in breast cancer samples. Expression of SCUBE2 was found to be decreased in G3 endometrial cancer when compared to postmenopausal endometrium or to G1 tumors (p < 0.05). In postmenopausal endometrium, SCUBE2 transcript levels were more than twice as high as in premenopausal women. In breast cancer, SCUBE2 expression was found to be notably reduced particularly in ERα-negative G3 tumors. Both in endometrial and breast cancer we observed a significant positive correlation of SCUBE2 transcript levels with expression of ERα, PR and PTEN. Our data suggest that SCUBE2, like in breast cancer, associates with ERα and might have a potential as prognostic or predictive marker in endometrial cancer.

Network analysis of icb-1 gene function in human breast cancer cells.

Icb-1 is a human gene previously described by our group to exert important functions in cancer cells of different origin. We now performed microarray-based gene expression profiling with subsequent network modeling to further elucidate the role of icb-1 in breast cancer cells. Analyzing the effect of icb-1 knockdown on the transcriptome of MCF-7 cells, we found 151 differentially expressed genes exhibiting more than twofold changes, 97 of which were up- and 54 downregulated. Most of the upregulated genes were cancer-related genes associated with poor prognosis, invasion and metastasis, building an oncogenic network of TNF target genes. On the other hand, network analysis identified the downregulated genes to be primarily involved in interferon signaling and cellular apoptosis. Confirming these network data, we observed that cells with reduced levels of icb-1 exhibited an impaired response to the apoptosis inducers tamoxifen, staurosporine, actinomycin, and camptothecin. The data of this study suggest that icb-1 might exert a tumor-suppressor function in breast cancer and that its loss might confer relative resistance of breast cancer cells to apoptotic drugs.

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.

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.

Role of estrogen receptor ß in gynecological cancer.

Estrogen receptor ß 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, ERß 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 ERß might play different roles in the carcinogenesis of the ovary and endometrium. Besides its possible role as a prognostic factor, ERß 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-ß (ERß), is known to have antitumoral properties. Given that ERß 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 µmol/l) significantly increased the growth-inhibitory effect of trastuzumab on HER2-overexpressing, ERα/ß-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 ERα-negative SK-BR-3 cells. Similar results were obtained after cotreatment with the ERß 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 ERß 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 ERα/ß-positive breast cancer has to be verified in animal or clinical studies.

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

BACKGROUND: The role of estrogen receptor beta (ERß) in pathogenesis of endometriosis remains to be elucidated. In this study, we have examined the expression of the four main ERß transcript isoforms in human endometrial tissue in women with or without endometriosis. METHODS: Total RNA was isolated from native endometrial tissue and transcript levels of ERα, ß1, ß2, ß4, ß5 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). RESULTS: ERα, ß1, ß2, and ß5 transcripts were detected in both cycle phases. During the proliferative phase, healthy women had a significantly higher ERα/ERß1-ratio than patients with endometriosis. Irrespective of the cycle phase, ERα-mRNA level was significantly higher than transcript levels of ERß isoforms. CONCLUSIONS: ERα, ß1, ß2, and ß5 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/ERß1-ratio in proliferative endometrium of endometriosis patients suggest that ERß1 might be involved in the pathogenesis of endometriosis. Further studies should be undertaken to substantiate the role of ERß in endometrial pathology.

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

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

HER2 status of circulating tumor cells in patients with metastatic breast cancer: a prospective, multicenter trial.

There is a growing body of evidence that HER2 status can change during disease recurrence or progression in breast cancer patients. In this context, re-evaluation of HER2 status by assessment of HER2 expression on circulating tumor cells (CTCs) is a strategy with potential clinical application. The aim of this trial was to determine the HER2 status of CTCs in metastatic breast cancer patients comparing two CTC assays. A total of 254 patients with metastatic breast cancer from nine German university breast cancer centers were enrolled in this prospective study. HER2 status of CTCs was assessed using both the FDA-approved CellSearch® assay and AdnaTest BreastCancer™. Using the CellSearch assay, 122 of 245 (50%) patients had ≥5 CTCs, and HER2-positive CTCs were observed in 50 (41%) of these patients. Ninety of 229 (39%) patients were CTC positive using AdnaTest BreastCancer, and HER2 positivity rate was 47% (42 of 90). The rate of breast cancer patients with HER2-negative primary tumors but HER2-positive CTCs was 32% (25 of 78) and 49% (28 of 57) using the CellSearch assay and AdnaTest BreastCancer, respectively. Considering only those patients who had CTCs on both tests (n = 62), concordant results regarding HER2 positivity were obtained in 50% of the patients (31/62) (P = 0.96, κ = -0.006). HER2-positive CTCs can be detected in a relevant number of patients with HER2 negative primary tumors. Therefore, it will be mandatory to correlate the assay-dependent HER2 status of CTCs to the clinical response on HER2-targeted therapies.

Icb-1 Gene 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.

Estrogen receptor beta 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) alpha and beta might affect development of uterine fibroids. In this study, we tested whether single nucleotide polymorphisms (SNPs) in the promoter of estrogen receptor beta 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.

Severe endometriosis: laparoscopic rectum resection.

AIM: Endometriosis is a frequent benign disease of women in reproductive age. An infiltration of the spatium rectovaginal is rare, but if it occurs, in up to 73% the rectum is involved. If there is the indication for surgery, a partial resection of the rectum might be necessary. This can be performed by a laparoscopic approach. It is the aim of this work to describe a patient population treated for endometriosis in the spatium rectovaginal by laparoscopic surgery. PATIENT POPULATION AND METHODS: A retrospective analysis of data from patients with endometriosis in rectum or sigma, which underwent a laparoscopic partial bowel resection in the years 2005-2006 at the Department of Obstetrics and Gynecology, University of Regensburg, was carried out. RESULTS: Between 2005 and 2006, we performed a laparoscopic partial bowel resection in six patients with endometriosis. The mean age at diagnosis was 36.1 years (range 28-50 years) and 36.5 years (range 30-50 years) at surgery. All patients were nulligravida and 50% of the patients were infertile (since 1-6 years). The interval between the onset of symptoms and surgery ranged from a few weeks up to 2.5 years. Two-thirds of the patients had endocrine treatment before surgery. Three patients had a rectum resection, one a sigma resection and two had a combined rectum- and sigma resection. The mean duration of surgery was 201 min and mean hospital stay was 8 days. We saw one post-surgery bleeding at the enteroanastomosis. In that case two erythrocyte concentrates were necessary and the bleeding was stopped by rectoscopic intervention. All follow-up coloscopies were without pathological findings. One patient had a normal delivery after IVF/ICSI treatment. CONCLUSIONS: If severe endometriosis needs a rectum resection then it can be done laparoscopically. This surgery should be performed in a specialized center. The duration of surgery, hospital stay and time of convalescence are short.

Resumption of Trastuzumab in Patients With Disease Recurrence After (Neo-) Adjuvant Anti-HER2-therapy in Patients With HER2-positive Breast Cancer.

BACKGROUND/AIM: HER2-positive breast cancers eventually relapse in about one third of patients. Is anti-HER2-directed therapy with Herceptin® (trastuzumab) effective in re-treatment? Between 2008 and 2018, 216 patients with recurrent HER2-positive breast cancer (BC) were re-treated with Herceptin (HER) during first-line therapy. This study assessed the effectiveness and tolerability of re-treatment with HER. PATIENTS AND METHODS: After approval from Ethical committee, the NIS was conducted according to German Drug Act. Re-treatment with HER was documented at routine visits starting with a basic observational period of maximum 12 months and a follow-up period of maximum additional four years. RESULTS: HER2-positive BC relapsed after a median of 36.5 months (mos). Patients were re-treated with HER +/- chemotherapy +/- endocrine therapy. HER-containing regimens resulted in median progression-free survival (mPFS) of 12.7 (95%CI=10.5-14.8) mos and overall survival (OS-2) of 31.6 mos (95%CI=28.8-38.4) since recurrence diagnosis. Differentiation of recurrence types (local, visceral, non-visceral) unfolded worst prognosis for patients with visceral metastases. Cardiac monitoring within this non-interventional study (NIS) did not result in new safety concerns. CONCLUSION: Re-therapy with HER in the first-line setting of advanced HER2-positive breast cancer is effective and without unexpected or intensified adverse events.

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.

Detection of an elevated HER2 expression in MCF-7 breast cancer cells overexpressing estrogen receptor beta1.

The estrogen receptor (ER) expression and HER2 amplification are important factors in determining the prognosis and therapy of breast cancer. Interactions between the two signaling pathways for example resulted in ERalpha-dependent regulation of HER2 expression in breast cancer cells. In this study, we investigated to what extent ERbeta is able to affect the HER2 expression. For this purpose, we analyzed HER2 levels in ERbeta1-overexpressing clones of the breast cancer cell lines MCF-7 and SK-BR-3 and of the ovarian cancer cell lines SK-OV-3 and OVCAR-3 by both RT-PCR and Western blot analysis. Treatment with ligand 17-beta estradiol diminished the HER2 expression in MCF-7 wild-type cells, an effect partially inhibited by treatment with 4-OH tamoxifen. MCF-7 breast cancer cells stably overexpressing ERbeta1 exhibited elevated >5-fold HER2 mRNA levels and elevated >3-fold HER2 protein levels even in the absence of estradiol. In contrast, ERbeta1 overexpression did not affect HER2 protein levels in the ERalpha-positive OVCAR-3 ovarian cancer cells and in the HER2 overexpressing, hormone-independent SK-BR-3 and SK-OV-3 cells. By demonstrating the elevated HER2 expression in a hormone-dependent breast cancer cell line overexpressing ERbeta1, our data suggest the presence of cross-talk between the two receptors. This is one of the molecular mechanisms underlying the significant ERbeta/HER2 co-expression observed in recent clinical studies.

Estrogen receptor {beta}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.

Apoptotic effects of signal transduction inhibitors on human tumor cells with different PTEN expression.

An important mechanism of antitumoral targeted therapies is the induction of apoptosis in tumor cells. Tamoxifen and trastuzumab (Herceptin), respectively, are able to trigger apoptosis in human breast cancer cells. But, frequently altered apoptotic signal cascades, for instance through PTEN mutations, help tumor cells to escape antitumoral therapy. We studied to what extent the apoptotic effect of signal-transduction inhibitors is dependent on PTEN expression. PTEN expression was analysed by Western blot analysis in tumor cell lines of the breast (BT-474, MCF-7, MDA-MB-231), ovary (BG-1, SK-OV-3) and endometrium (Ishikawa, HEC-1A). Apoptotic effects of tamoxifen, trastuzumab, ZD1839 (Iressa) and different mitogen-activated protein kinase (MAP) inhibitors were measured after 24 h of treatment. Cellular apoptosis was determined by the detection of cytoplasmic histone-DNA complexes. The tested tumor cell lines exhibited a different PTEN expression, ranging from a high expression (ovarian cancer cell line BG-1 and BT-474 breast cancer cells) to a total absence of PTEN expression (endometrial Ishikawa cells). The apoptotic effect of receptor-targeting drugs (tamoxifen, trastuzumab, ZD1839) was dependent both on receptor expression and PTEN expression. When cells were treated with MAPK inhibitors, no correlation between PTEN expression and the apoptosis rate was observed. Our data underline the importance of PTEN expression regarding the induction of apoptosis through various targeted therapies.

Primary metastatic breast cancer in the era of targeted therapy - Prognostic impact and the role of breast tumour surgery.

BACKGROUND: Except for meeting the individual palliative need, the benefit of breast surgery in primary metastatic breast cancer (PMBC), also known as de novo metastatic breast cancer, on long-term outcomes remains controversial. Twenty-four hundred and one patients with metastatic breast cancer, enrolled between 2000 and 2011 in two prospective non-interventional studies on targeted therapy, were screened with respect to this question. METHODS: One study investigated trastuzumab therapy for human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer in addition to mainly first-line chemotherapy. The other observed bevacizumab added to chemotherapy as first-line treatment for mostly HER2-negative disease. RESULTS: Five-hundred and seventy (24%) patients presented with PMBC, and valid information on resection of the primary tumour was available for 568 women. Out of these, 426 (75%) underwent local resection. The latter group was characterised by less overall metastatic burden and a lower proportion of T4 tumours. No major differences were observed with respect to age, hormone receptor and HER2 status, visceral disease and performance status. Numerically, the surgery group showed a slightly favourable progression-free survival (PFS, medians: 13.6 versus 11.8 months; P = 0.18) and overall survival (OS, 34.1 versus 31.7; P = 0.23). However, in multivariable analysis, including all other univariably significant parameters, no trend for better outcome after surgery remained detectable, neither for PFS (hazard ratio 0.99; P = 0.92) nor for OS (0.95; P = 0.71). CONCLUSIONS: Our findings suggest no major survival benefit for local resection in the overall PMBC population treated with modern targeted therapies. However, further analyses are warranted to define specific risk groups, which may benefit from surgical removal of the primary.