Estrogen Breast Cancer Research Articles

NJK14013, a novel synthetic estrogen receptor-α agonist, exhibits estrogen receptor-independent, tumor cell-specific cytotoxicity.

Estrogens act through interactions with estrogen receptors (ERs) to play diverse roles in various pathophysiological conditions. A number of synthetic selective estrogen receptor modulators (SERMs), such as tamoxifen and raloxifene, have been developed and used to treat ER-related diseases, including breast cancer and osteoporosis. Here, we identified a novel compound, bis(4-hydroxyphenyl)methanone-O-isopentyl oxime, designated NJK14013, as an ER agonist. NJK14013 activated ER-dependent transcription in a concentration-dependent manner, while suppressing androgen receptor-dependent transcriptional activity. It induced the activation-related phosphorylation of ER and enhanced the transcription of growth regulation by estrogen in breast cancer 1 (GREB1), further supporting its ER-stimulating activity. NJK14013 exerted anti-proliferative effects on various cancer cell lines, including an ER-negative breast cancer cell line, suggesting that it is capable of suppressing the growth of cancer cells independent of its ER-modulating activity. In addition, NJK14013 treatment resulted in significant apoptotic death of MCF7 and Ishikawa cancer cells, but did not induce apoptosis in non-cancer human umbilical vein endothelial cells. Collectively, our findings demonstrate that NJK14013 is a novel SERM that can activate ER-mediated transcription in MCF7 cells and suppress the proliferation of various cancer cells, including breast cancer cells and endometrial cancer cells. These results suggest that NJK14013 has potential as a novel SERM for anticancer or hormone-replacement therapy with reduced risk of carcinogenesis.

Therapeutic advances in BIG3-PHB2 inhibition targeting the crosstalk between estrogen and growth factors in breast cancer.

Our previous studies demonstrated that specific inhibition of the BIG3-PHB2 complex, which is a critical modulator in estrogen (E2) signaling, using ERAP, a dominant negative peptide inhibitor, leads to suppression of E2-dependent estrogen receptor (ER) alpha activation through the reactivation of the tumor suppressive activity of PHB2. Here, we report that ERAP has significant suppressive effects against synergistic activation caused by the crosstalk between E2 and growth factors associated with intrinsic or acquired resistance to anti-estrogen tamoxifen in breast cancer cells. Intrinsic PHB2 released from BIG3 by ERAP effectively disrupted each interaction of membrane-associated ERα and insulin-like growth factor 1 receptor beta (IGF-1Rβ), EGFR, PI3K or human epidermal growth factor 2 (HER2) in the presence of E2 and the growth factors IGF or EGF, followed by inhibited the activation of IGF-1Rβ, EGFR or HER2, and reduced Akt, MAPK and ERα phosphorylation levels, resulting in significant suppression of proliferation of ERα-positive breast cancer cells in vitro and in vivo. More importantly, combined treatment with ERAP and tamoxifen led to a synergistic suppression of signaling that was activated by crosstalk between E2 and growth factors or HER2 amplification. Taken together, our findings suggest that the specific inhibition of BIG3-PHB2 is a novel potential therapeutic approach for the treatment of tamoxifen-resistant breast cancers activated by the crosstalk between E2 and growth factor signaling, especially in premenopausal women.

Functional evaluation of genetic variants associated with endometriosis near GREB1.

Do DNA variants in the growth regulation by estrogen in breast cancer 1 (GREB1) region regulate endometrial GREB1 expression and increase the risk of developing endometriosis in women? We identified new single nucleotide polymorphisms (SNPs) with strong association with endometriosis at the GREB1 locus although we did not detect altered GREB1 expression in endometriosis patients with defined genotypes. Genome-wide association studies have identified the GREB1 region on chromosome 2p25.1 for increasing endometriosis risk. The differential expression of GREB1 has also been reported by others in association with endometriosis disease phenotype. Fine mapping studies comprehensively evaluated SNPs within the GREB1 region in a large-scale data set (>2500 cases and >4000 controls). Publicly available bioinformatics tools were employed to functionally annotate SNPs showing the strongest association signal with endometriosis risk. Endometrial GREB1 mRNA and protein expression was studied with respect to phases of the menstrual cycle (n = 2-45 per cycle stage) and expression quantitative trait loci (eQTL) analysis for significant SNPs were undertaken for GREB1 [mRNA (n = 94) and protein (n = 44) in endometrium]. Participants in this study are females who provided blood and/or endometrial tissue samples in a hospital setting. The key SNPs were genotyped using Sequenom MassARRAY. The functional roles and regulatory annotations for identified SNPs are predicted by various publicly available bioinformatics tools. Endometrial GREB1 expression work employed qRT-PCR, western blotting and immunohistochemistry studies. Fine mapping results identified a number of SNPs showing stronger association (0.004 < P < 0.032) with endometriosis risk than the original GWAS SNP (rs13394619) (P = 0.034). Some of these SNPs were predicted to have functional roles, for example, interaction with transcription factor motifs. The haplotype (a combination of alleles) formed by the risk alleles from two common SNPs showed significant association (P = 0.026) with endometriosis and epistasis analysis showed no evidence for interaction between the two SNPs, suggesting an additive effect of SNPs on endometriosis risk. In normal human endometrium, GREB1 protein expression was altered depending on the cycle stage (significantly different in late proliferative versus late secretory, P < 0.05) and cell type (glandular epithelium, not stromal cells). However, GREB1 expression in endometriosis cases versus controls and eQTL analyses did not reveal any significant changes. In silico prediction tools are generally based on cell lines different to our tissue and disease of interest. Functional annotations drawn from these analyses should be considered with this limitation in mind. We identified cell-specific and hormone-specific changes in GREB1 protein expression. The lack of a significant difference observed following our GREB1 expression studies may be the result of moderate power on mixed cell populations in the endometrial tissue samples. This study further implicates the GREB1 region on chromosome 2p25.1 and the GREB1 gene with involvement in endometriosis risk. More detailed functional studies are required to determine the role of the novel GREB1 transcripts in endometriosis pathophysiology. Funding for this work was provided by NHMRC Project Grants APP1012245, APP1026033, APP1049472 and APP1046880. There are no competing interests.

Calcium efflux pump, PMCA2, in human breast tissue with lactational change and as a therapeutic target in breast cancer

Calcium pumps and channels modulate cell proliferation and apoptosis by regulating intracellular calcium (Ca2+). The plasma membrane Ca2+ ATPase isoform, PMCA2, is a calcium efflux mechanism that extrudes Ca2+ from the cytosol into the extracellular space. PMCA2 has a restricted expression, including expression in cochlear hair cells and cerebellar Purkinje cells. PMCA2 expression is increased in mouse mammary glands during lactation where it plays a major role in the excretion of Ca2+ into milk; however, PMCA2 expression has not been assessed in human breast tissue exhibiting lactational changes. Our previous studies have shown that PMCA2 mRNA levels are elevated in some breast cancer cell lines and that pan-PMCA antisense attenuates the proliferation of MCF-7 breast cancer cells. However, the consequences of silencing PMCA2 in breast cancer cells are still not well understood. Our study assessed PMCA2 expression in breast tissue exhibiting lactational change and in human malignant breast tissue samples. The role of PMCA2 in the proliferation of breast cancer cells was also evaluated. Immunohistochemistry using a rabbit anti-PMCA2 antibody showed membranous PMCA2 expression in the luminal epithelium of breast tissue exhibiting lactational change. PMCA2 expression was assessed in human breast tumor samples assembled into tissue microarrays. Nine of 96 breast tumours (9.4%) showed membranous PMCA2 staining. PMCA2 expression did not significantly correlate with the breast cancer pathological markers, estrogen, progesterone or HER2 receptor status. High-content imaging demonstrated that PMCA2 silencing in MDA-MB-231 breast cancer cells is associated with a reduction in cell number and an inhibition of the percentage of S-phase positive cells. The effect of PMCA2 silencing combined with various cytotoxics (cisplatin, doxorubicin or mitomycin C) on cell proliferation was assessed in MDA-MB-231 cells using a kinetic imaging system (IncuCyte). The results showed that PMCA2 silencing promotes the effects of some cytotoxics. These findings indicate that PMCA2 protein expression is elevated during human lactation and in some breast cancers. Inhibitors of PMCA2 may represent a novel therapeutic strategy for some breast cancers.

Ovarian stimulation in patients with breast cancer.

Breast cancer is the most prevalent malignancy among women under 50. Improvements in diagnosis and treatment have yielded an important decrease in mortality in the last 20 years. In many cases, chemotherapy and radiotherapy develop side effects on the reproductive function. Therefore, before the anti-cancer treatment impairs fertility, clinicians should offer some techniques for fertility preservation for women planning motherhood in the future.In order to obtain more available oocytes for IVF, the ovary must be stimulated. New protocols which prevent exposure to increased estrogen during gonadotropin stimulation, measurements to avoid the delay in starting anti-cancer treatment or the outcome of ovarian stimulation have been addressed in this review.There is no evidence of association between ovarian stimulation and breast cancer. It seems that there are more relevant other confluent factors than ovarian stimulation. Factors that can modify the risk of breast cancer include: parity, age at full-term birth, age of menarche, and family history.There is an association between breast cancer and exogenous estrogen. Therefore, specific protocols to stimulate patients with breast cancer include anti-estrogen agents such as letrozole. By using letrozole plus recombinant follicular stimulating hormone, patients develop a multifollicular growth with only a mild increase in estradiol serum levels.Controlled ovarian stimulation (COS) takes around 10 days, and we discuss new strategies to start COS as soon as possible. Protocols starting during the luteal phase or after inducing the menses currently prevent a delay in starting ovarian stimulation.Patients with breast cancer have a poorer response to COS compared with patients without cancer who are stimulated with conventional protocols of gonadotropins.Although many centres offer fertility preservation and many patients undergo ovarian stimulation, there are not enough studies to evaluate the recurrence, breast cancer-free interval or mortality rates in these women.

FOXP1 regulation via the PI3K/Akt/p70S6K signaling pathway in breast cancer cells.

Loss of Forkhead box P1 (FOXP1) protein expression confers a poor prognosis in sporadic and familial breast cancer patients, and the FOXP1 gene maps to a tumor suppressor locus at chromosome 3p14. Although correlation studies have indicated that FOXP1 has a role in tumor suppression, determination of the regulatory mechanism of FOXP1 is required to establish its function in breast cancer. It has previously been identified that FOXP1 is regulated by estrogen in breast cancer and that treatment with bisphenol A is effective for regulating the transformation of the normal human breast epithelial cell line, MCF-10F. In addition, FOXO-regulated activation of FOXP1 inhibits the apoptosis of MCF-10F cells following tamoxifen and Akt inhibitor VIII administration. The present study indicates that FOXP1 regulation occurs via a PI3K/Akt/p70S6 kinase (p70S6K) signaling pathway. Following treatment with wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K)/Akt, MCF7 and MDA-MB-231 breast cancer cells demonstrated decreased FOXP1 protein expression levels; this result was also observed in the small interfering (si)RNA silencing of Akt. By contrast, overexpression of Akt resulted in increased FOXP1 protein expression levels in the MDA-MB-231 cells compared with the control cell lysates. Furthermore, treatment with rapamycin, a specific inhibitor of the mammalian target of rapamycin/p70S6K cascade, resulted in decreased FOXP1 expression in the MCF7 cells, but not in the MDA-MB-231 cells, which were resistant to rapamycin-induced inhibition. In addition, silencing of p70S6K using siRNA produced a marked decrease in FOXP1 expression. These data indicate that FOXP1 protein expression is regulated by a PI3K/Akt/p70S6K signaling cascade in breast cancer.

The role of oestrogen and progesterone receptors in breast cancer - immunohistochemical evaluation of oestrogen and progesterone receptor expression in invasive breast cancer in women.

Aim of the studyExpression of oestrogen and progesterone receptors is a very powerful and useful predictor. Because the response rate to hormonal treatment in breast cancer is associated with the presence of oestrogen and progesterone receptors, assessment of the receptor expression profile allows for prediction of breast cancer response to hormonal treatment. The aim of this study was to assess whether the expression of receptors for oestrogen (ER) and progesterone (PR) in the tumour tissue of patients with invasive breast cancer correlated with tumour histological type, histological grade of malignancy, tumour size, and lymph node status.Material and methodsMaterials consisted of histological preparations derived from patients treated for invasive breast cancer. Evaluations were conducted with histopathological and immunohistochemical methods using suitable antibodies.ResultsAmong 231 cases of breast cancer 18 invasive lobular carcinomas (ILC) and 213 invasive ductal carcinomas (IDC) were diagnosed. Taking the histological type of tumour into account, oestrogen receptor-positive reaction was observed in 74.2% of IDC and 77.8% of ILC, and the positive response to PR was observed in 67.1% of IDC and 61.1% of ILC. Considering the histological grade, ER- in the largest percentage (72%) was observed in second-grade (G2) invasive carcinomas. Similarly, PR expression (75%) was found in the largest percentage in second-grade (G2) carcinomas. Based on our own studies and data from literature, it appears that the ER (+) status is an indicator of good prognosis, because it points to a less aggressive cancer, in which overall survival and disease-free time is longer in comparison with ER (–) tumours.ConclusionsDetermination of ER status may, therefore, have significant clinical value and is widely used in routine pathological diagnostics.

Detection of Estrogen Responsive Breast Cancer Circulating Tumor Cells: Assay Development for Anti-Hormone Therapy Resistance

Recent clinical trials with histone deacetylase inhibitors (HDACi) have shown increased progression free survival by re-sensitizing resistant estrogen receptor positive (ER+) breast cancer cells to hormone suppressive therapies (HT). However, these trials lacked a sensitive, specific assay to identify and monitor HDACi/HT sensitive or resistant tumors. We tested detection of ER expression and histone acetylation of chromatin at the growth regulation by estrogen in breast cancer 1 (GREB1) gene, an estrogen-responsive gene involved in ER expression, in circulating tumor cell (CTC) as potential candidate assays for HDACi/HT sensitivity. ER+ and ER- CTC were detected and isolated from breast cancer patient peripheral blood by high speed fluorescence activated cell sorting (FACS) for use in mRNA analysis and anti-acetylated histone-mediated Chromatin Immunoprecipitation (ChIP). cDNA from mRNA and DNA extracted from the ChIP isolates were quantified by real-time PCR for GREB1. CTC isolates from patients who had an ER+ breast cancer primary contained both ER+ and ER- cells. More ER+ than ER- CTC was found in HT sensitive patients compared to HT resistant patients (p = 0.0559). GREB1 was found in acetylated histone chromatin from both ER+ and ER- CTC. The number of ER+ and ER- CTC found in peripheral blood appears to parallel patient outcomes as to their sensitivity to HT. Acetylated histone analysis can detect chromatin containing GREB1 in CTC, suggesting it may be useful as a more specific measure of HDACi effects on breast tumor cells. A larger, longitudinal data set following patients through HT/HDACi trials is needed to confirm these observations and their development for clinical use.

Immunohistochemical determination of estrogen and progesterone receptors in breast cancer: pathological correlation and prognostic indicators

Carcinoma of the breast is the most common malignancy of women globally and the incidence has more risen in recent years. The current study was conducted with the objective of assessing estrogen receptor (ER) and progesterone receptor (PR) reactivity patterns of mammary cancers and to evaluate their association with clinicopathological features. A total of 61 cases of breast carcinoma were examined retrospectively using immunostains for estrogen receptor (ER) and progesterone receptor (PR). Staining pattern and intensity were correlated with histological subtypes and nuclear grades of tumors. The left breast was more commonly involved (57%) and tumor size ranged from 0.5-13.0cm. The predominant morphology was infiltrating ductal carcinoma (85.3%). The majority of the cases presented as grade II (55.3%) lesions with tumor necrosis (70%) and lymph node involvement (71.3%(. Positive nuclear staining for ER and PR was observed in 70.5% and 57.5 % of invasive carcinomas, respectively. In ER+ cases, fifty five cases (90%) gave diffuse immunohistochemical reaction for ER; in the remaining 10%, a focal ER reaction was seen. In PR+ cases, 49 cases (80%) gave diffuse immunohistochemical reaction for PR and In remaining 20% of PR+ tumors, the reaction was heterogeneous. In ductal infiltrative carcinomas the percentage of cases showed ER+ nuclear labeling is higher than those in cases of infiltrative lobular carcinomas. Assessment of ER and PR as prognostic markers for the clinical management of breast cancer patients is strongly advocated to provide best therapeutic options.

Estrogen and progesterone receptors in breast cancer.

Breast cancer is the most common cancer in women worldwide. The majority of breast cancers show overexpression of estrogen receptors (ERs) and progesterone receptors (PRs). The development of drugs to target these hormone receptors, such as tamoxifen, has brought about significant improvement in survival for women with hormone receptor-positive breast cancers. Since information about ER and PR is vital for patient management, quality assurance is important to ensure accurate testing. In recent guidelines, the recommended definition of ER and PR positivity is 1% or more of cells that stain positive. Semiquantitative assessment of ER and PR is important for prognosis and, hence, management. Even with the development of genomic tests, hormone receptor status remains the most significant predictive and prognostic biomarker.

Cellular stress responses in cancer and cancer therapy.

Cellular stress responses in cancer and cancer therapy.

Yes, Virginia, Chemo Brain is Real

Introduction Twenty years ago, while interviewing a newly assigned patient on our hospital’s oncology unit, she apologized to me for “being stupid.” Startled at this self-deprecating response to a medical history question I had just asked, I put down my pen and met her eyes. “What makes you think you’re stupid?” Embarrassed, she blushed with her answer. “Ever since getting chemo, I feel like I’m forgetting everything. I never used to be like that. Now I can’t remember names of people or things. At work I can’t find the right words or write the drafts I’ve been doing for years . or spell or type. I’m sure I (appear) as though I’m not educated.” This was my first introduction to “chemo brain,” also described as “brain fog.” What we know currently is that this phenomenon is a toxicity of the nervous system causing cognitive impairment in verbal ability, visuospatial ability, problem solving, and affecting concentration and memory. Until around 1995, medical practitioners referred a patients’ concern with these symptoms to the commonly known chemotherapy side effects such as anemia, fatigue, depression, and hormonal changes. Primarily it was breast cancer patients who voiced concerns: studies during early acknowledgment of this phenomenon were noted only as a supplement to pharmaceutical clinical study and not specifically addressed. However, research now indicates that men with prostate cancer and subsequent treatment with hormonal therapy, such as leuprolide and goserelin, can be at risk for cognitive side effects, particularly memory maintenance. Tamoxifen, an oral drug long used to inhibit estrogen in hormone receptorpositive breast cancer, might cause neurological function changes such as concentration and planning ability. Besides chemotherapy and radiation, immunotherapy (interleukin-1 and interleukin-6) can generate increased inflammation and fatigue, both thought to contribute to cognitive dysfunction.

Abstract 32: Heterogeneous expression of Her2, estrogen and progesterone receptors in human breast cancer metastasized to the brain

Abstract Assessment of hormone (estrogen (ER) and progesterone (PR)) and human epidermal growth factor-2 (Her2) receptors expression in breast cancer has been an accepted standard to predict clinical outcome. Importantly, expression of these receptors in primary breast cancer has been an important predictor of visceral organ metastasis. Although many studies of breast cancer have reported risk factors for brain metastasis, including Her2 positivity and ER negativity, currently it is not possible to accurately predict brain metastasis. One possible reason for this impasse could be the expression of the above receptors in different combinations influencing metastasis to the brain (brain mets). To address this possibility, we analyzed six different human brain mets for the expression of Her-2, ER and PR by the fluorescent in situ hybridization (FISH) and immunohistochemistry procedures. To further validate this data, eight different human primary breast tumors were similarly analyzed. The immunohistochemistry analysis showed that brain mets are heterogenous in the expression of these receptors: five were Her2 positive and one negative; four were ER positive and two were negative; five were PR positive and one negative. However, expression of these receptors in their combination is also heterogenous: three brain mets were positive for all of the Her2, ER and PR; one for Her2 positive but negative for ER/PR; one positive for PR but negative for Her2/ER. Similar heterogenous expression of these receptors in primary tumors were also observed using immunohistochemistry procedure. While this data is mostly corroborative to the FISH analysis, the FISH identified receptor positivity could not be established by immunohistochemistry. Additionally, tumors characterized by immunohistochemistry as PR negative were found to express this receptor, albeit in tens of cells among thousands in brain mets. Such differences in results using these two procedures were noted in the Her2 expression in both brain mets and primary tumors. Undoubtedly, FISH is highly sensitive and identifies gene amplification, whereas, immunohistochemistry allows the detection of receptor expression which is heavily dependent on several factors including accessibility of antigen and affinity of the antibody. In conclusion, our analysis suggests that the currently utilized Her2, estrogen and progesterone receptor detection to predict brain mets needs further refinement to allow for better understanding of the mechanisms of metastasis to the central nervous system and treatment of such brain mets. Citation Format: Morgan Labhart, Prema S. Rao, Seshadri Thirumala, Subrahmanyeswara U. Rao. Heterogeneous expression of Her2, estrogen and progesterone receptors in human breast cancer metastasized to the brain. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 32. doi:10.1158/1538-7445.AM2014-32

Abstract 5571: Genome-wide crosstalk of estrogen and progesterone signaling in breast cancer

Abstract Estrogen receptor alpha (ER) and progesterone receptor (PR) are established biomarkers in breast cancer and receptor-targeted therapies are routinely used in breast cancer treatment. Estrogen receptor exists as two subtypes, ERα and ERβ, and PR has two isoforms PRA and PRB. Furthermore, millions of women worldwide use estrogen and progesterone formulations as oral contraceptives and hormone replacement therapies. Although critical crosstalk occurs between estrogen and progesterone signaling, the global nature and mechanistic details of this interaction are not known. Our central hypothesis is that there is genome-wide dominance of progesterone over estrogen signaling in breast cancer. This hypothesis is based on our novel findings that gene expression and proliferation of T47D and ZR75 breast cancer cells upon co-treatment with estrogen and progesterone is similar to progesterone-only treatment and is significantly different from estrogen-only treatment. Furthermore, four derivative T47D sublines that lack either of the PR isoforms, both the isoforms of PR or ER along with PR are being used to study ERα-, PRA- and PRB-specific regulatory networks. ER- and PR-regulated gene networks are defined by correlating ER and PR genome binding data obtained from ChIP-seq with gene expression data of T47D cells acquired using RNA-seq. For instance, genes that are known to be important in breast carcinogenesis, such as GATA3, GREB1, PDZK1, FOXA1 are found to be co-regulated by ER and PR. Subsequently the intersection of identified ER- and PR-regulated gene networks will be studied and their clinical value will be assessed. Transcription cofactors FOXA1 and NF1C are critical for transactivation by ER and PR. Knockdown of these cofactors in T47D cells alters the ER/PR crosstalk in these cells. To validate the clinical value of genes co-regulated by ER and PR in breast cancer, meta-analysis for their enrichment in established breast cancer signatures will be done. Cluster analysis of the expression of these genes in patient tumor samples that differ in clinical outcomes will also be performed. Finally, a Kaplan-Meier survival analysis will be done to study a correlation between expression of these genes and patient survival. These innovative studies will determine the global influence of progesterone over estrogen signaling in breast cancer. The resulting map of ER- and PR-regulated gene networks will lead to the optimization of breast cancer treatments, hormonal contraceptives and hormone replacement therapies. Citation Format: Hari Singhal, Geoffrey Greene. Genome-wide crosstalk of estrogen and progesterone signaling in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5571. doi:10.1158/1538-7445.AM2014-5571

Abstract 4229: The anti-estrogen effect of metformin in ERα+ breast cancer and tamoxifen resistant cell lines

Abstract Anti-estrogen therapy is still the most important systemic treatment to manage estrogen responsive breast cancer. Tamoxifen resistance is a major obstacle in treatment of ER-positive breast cancer. Recently, metformin, which is a biguanide derivative which is commonly used in the treatment of type II diabetes, has been emerged as a potential anti-cancer agent. We investigated if metformin had anti-estrogen effect in ER-positive breast cancer cell lines which are MCF-7, MDA-MB-361 as well as tamoxifen resistant cells (TR-MCF-7). Metformin inhibited the cell proliferation and the expression of ERα in dose-dependent manner in MCF-7 and TR-MCF-7 cells. Under stimulation of 17β-estradiol (E2), metformin inhibited the cell proliferation and expression of mRNA and protein of ERα in MCF-7 and TR-MCF-7 cells, while tamoxifen didn't affect E2 induced cell proliferation and ERα expression. This result indicates that metformin, as anti-estrogen agent, is superior to tamoxifen for targeting ER in breast cancer. We also identified that metformin inhibited E2 induced cell proliferation and expression of mRNA and protein of ERα in MDA-MB 361 cell line (ER+/Her-2+). We also analyzed estrogen responsive element (ERE) activity using by luciferase assay and identified metformin suppressed E2-inducible luciferase activity and expression of direct ER-regulated genes, such as c-myc, Cyclin D1 and pS2 and FOXA1, which were not inhibited by tamoxifen. In conclusion, metformin has a direct anti proliferative effect and anti-estrogen effect in MCF-7, TR-MCF-7 as well as MDA-MB-361 cell lines. These results suggest that metformin can be used as anti- estrogen in ER positive breast cancer (luminal A &amp; B) and tamoxifen resistant breast cancer. Citation Format: Jinkyoung Kim, Jiyun Lee, Soonyoung Jang, Chungyeul Kim, Aeree Kim. The anti-estrogen effect of metformin in ERα+ breast cancer and tamoxifen resistant cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4229. doi:10.1158/1538-7445.AM2014-4229

Discordances in ER, PR and HER2 receptors between primary and recurrent/metastatic lesions and their impact on survival in breast cancer patients.

The aim of this study was to evaluate the frequency and prognostic impact of changes in the estrogen (ER), progesterone (PR) and human epidermal growth factor receptor 2 (HER2) status between primary and recurrent/metastatic lesions (RML). We investigated 133 breast cancer patients for ER, PR and HER2 status of primary and RML and their follow-up records. Among 133 patients with RML, discordance rate for ER, PR, and HER2 was 18.8, 33.8, and 6.8%, respectively. ER, PR and HER2 discordance were observed in 20.0, 38.1 and 6.7% of the patients with distant metastasis, and in 14.3, 17.9 and 7.1% of the patients with locoregional recurrence. The mean time between the primary diagnosis and last contact or death was 57 (range 22-78) months and between the recurrence biopsy and last contact or death was 17 (range 1-33) months. Among 133 patients with RML, the ER-discordant cases and ER-loss cases experienced a worse overall survival (OS) (p=0.001 and p=0.016, respectively) and post-recurrence survival (PRS) (p=0.001 and p=0.018, respectively), compared with the respective concordant cases. The HER2-discordant patients and HER2-loss patients had a poorer OS (p=0.008 and p=0.001, respectively) and PRS (p=0.004 and p=0.000, respectively) than the respective concordant cases. Among 105 patients with distant metastasis, ER discordance, ER loss, HER2 discordance and HER2 loss, compared with the respective concordant cases, resulted in a worse OS and PRS (p<0.05 for all). Our findings show an evident change in ER, PR and HER2 between breast primary tumors and relapsing tumors. The unstable status for ER or HER2 in breast cancer seems to be clinically significant and to correlate with a worse prognosis.

Interplay between estrogen and retinoid signaling in breast cancer – Current and future perspectives

All-trans-retinoic acid (RA) is a promising agent for breast cancer treatment, but it induces several adverse effects and the few clinical trials performed up to now in breast cancer patients have provided disappointing results. The combination of RA and antiestrogenic compounds, such as tamoxifen, synergistically decreases the proliferation of breast cancer cells and an interplay between retinoid and estrogen signaling has begun to be unraveled, turning these combinations into an appealing strategy for breast cancer treatment. This review focus on the current knowledge regarding the interplay between retinoid and estrogen signaling in breast cancer and the combinations of RA with antiestrogens, aiming their future utilization in cancer therapy.

Bisphenol AF-induced endogenous transcription is mediated by ERα and ERK1/2 activation in human breast cancer cells.

Bisphenol AF (BPAF)-induced transcriptional activity has been evaluated by luciferase reporter assay. However, the molecular mechanism of BPAF-induced endogenous transcription in human breast cancer cells has not been fully elucidated. In the present study, we investigated the effect and mechanism of BPAF-induced endogenous transcription detected by real-time PCR in human breast cancer cells. We found that BPAF stimulated transcription of estrogen responsive genes, such as trefoil factor 1 (TFF1), growth regulation by estrogen in breast cancer 1 (GREB1) and cathepsin D (CTSD), through dose-dependent and time-dependent manners in T47D and MCF7 cells. Gene-silencing of ERα, ERβ and G protein-coupled estrogen receptor 1 (GPER) by small interfering RNA revealed that BPAF-induced endogenous transcription was dependent on ERα and GPER, implying both genomic and nongenomic pathways might be involved in the endogenous transcription induced by BPAF. ERα-mediated gene transcription was further confirmed by inhibition of ER activity using ICI 182780 in ERα-positive T47D and MCF7 cells as well as overexpression of ERα in ERα-negative MDA-MB-231 breast cancer cells. Moreover, we utilized Src tyrosine kinase inhibitor PP2 and two MEK inhibitors PD98059 and U0126 to elucidate the rapid nongenomic activation of Src/MEK/ERK1/2 cascade on endogenous transcription. Our data showed that BPAF-induced transcription could be significantly blocked by PP2, PD98059 and U0126, suggesting activation of ERK1/2 was also required to regulate endogenous transcription. Taken together, these results indicate that BPAF-induced endogenous transcription of estrogen responsive genes is mediated through both genomic and nongenomic pathways involving the ERα and ERK1/2 activation in human breast cancer cells.

Ultra-low-dose estriol and Lactobacillus acidophilus vaginal tablets (Gynoflor®) for vaginal atrophy in postmenopausal breast cancer patients on aromatase inhibitors: pharmacokinetic, safety, and efficacy phase I clinical study

Phase I pharmacokinetic (PK) study assessed circulating estrogens in breast cancer (BC) patients on a non-steroidal aromatase inhibitor (NSAI) with vaginal atrophy using vaginal ultra-low-dose 0.03 mg estriol (E3) and Lactobacillus combination vaginal tablets (Gynoflor®). 16 women on NSAI with severe vaginal atrophy applied a daily vaginal tablet of Gynoflor® for 28 days followed by a maintenance therapy of 3 tablets weekly for 8 weeks. Primary outcomes were serum concentrations and PK of E3, estradiol (E2), and estrone (E1) using highly sensitive gas chromatography–mass spectrometry. Secondary outcomes were clinical measures for efficacy and side effects; microscopic changes in vaginal epithelium and microflora; and changes in serum FSH, LH, and sex hormone-binding globulin. Compared with baseline, serum E1 and E2 did not increase in any of the women at any time following vaginal application. Serum E3 transiently increased after the first application in 15 of 16 women, with a maximum of 168 pg/ml 2–3 h post-insertion. After 4 weeks, serum E3 was slightly increased in 8 women with a maximum of 44 pg/ml. The vaginal atrophy resolved or improved in all women. The product was well tolerated, and discontinuation of therapy was not observed. The low-dose 0.03 mg E3 and Lactobacillus acidophilus vaginal tablets application in postmenopausal BC patients during AI treatment suffering from vaginal atrophy lead to small and transient increases in serum E3, but not E1 or E2, and therefore can be considered as safe and efficacious for treatment of atrophic vaginitis in BC patients taking NSAIs.

ESR1 and PGR gene promoter methylation and correlations with estrogen and progesterone receptors in ductal and lobular breast cancer.

The aim of this work was to analyze methylation of the promoter sites of the ESR1 and PGR genes and to determine correlations with immunohistochemical expression of estrogen and progesterone receptors in ductal and lobular breast cancers. An observational, descriptive, molecular study was conducted on 20 ductal and 20 lobular breast cancer samples with immunohistochemical determination of estrogen and progesterone receptor expression. The methylation analysis of ESR1 and PGR promoter sites was carried-out by methylation-specific PCR. For correlation analysis, Kendall's tau coefficient was determined. Positive correlations were found between estrogen and progesterone receptors, estrogen receptor and unmethylated progesterone receptor, progesterone receptor, and unmethylated progesterone receptor. Negative correlations were found between estrogen receptor and methylated progesterone receptor, progesterone receptor and methylated progesterone receptor, methylated and unmethylated estrogen receptor, and methylated and unmethylated progesterone receptor. The results suggest that methylation of promoter sites of ESR1 and PGR is a relatively uncommon event in ductal and lobular breast cancer, and also suggest that the determination of epigenetic states of ESR1 and PGR could represent an alternative or complement to the histopathological expression analysis.