ERα Status Research Articles

Significance of estrogen receptor subtypes in breast tumorigenesis and progression.

The aim of the study was to investigate the effects of estrogen receptor (ER) subtypes (ERα and ERβ) on breast cancer development and progression. The expression level of ERα and ERβ in breast cancer tissues and paired normal breast tissues were detected by Western blot analysis and immunohistochemistry (IHC) staining. The features of ERα and ERβ status in cancer tissues or normal breast tissues and the correlations between clinicopathological characteristics and prognosis were analyzed. The expression levels of ERα and ERβ in breast cancer tissues are significantly lower than those in the paired normal tissues. The expression of ERβ is decreased more than that of ERα. ERα expression levels in cancer tissues are associated with tumor diameter, tumor-node-metastasis (TNM) stage, and progesterone receptor (PR) status. However, ERβ expression levels in cancer tissues are not correlated with clinicopathological factors of patients with breast cancer. In conclusion, ER subtypes might play different roles in the development of breast cancer.

Association of promoter methylation of ERα and ERβ with sporadic breast cancer--a study from North India.

Methylations in estrogen receptor (ER) α and ERβ are known to be involved in the pathogenesis of breast cancer. Here, we explore the role of promoter methylation of estrogen receptors, ERα and ERβ, in sporadic breast cancer cases from a North Indian population. To this end, association between ERα and ERβ methylation status along with different clinicopathological parameters and its correlation with protein expression was examined. Four hundred eighty paired breast cancer tissue samples and adjacent normal controls from 240 sporadic breast cancer patients were included, and their clinical and demographic profiles were recorded. ERα and ERβ methylation was determined by methylation-specific polymerase (MSP) chain reaction. Our findings demonstrate that methylation of ERα and ERβ occurs in high frequency and appears to be a mechanism of gene silencing in our population. Furthermore, on performing stratified analysis, we observed strong associations between ERα/ERβ methylation and ER, PR, and HER2 status, tumor size, clinical stage, and triple negative tumors. Thus, our study not only highlights the role of ERα/ERβ methylation in breast cancer but also suggests the ERα/ERβ methylation pattern as a biomarker for assessing breast cancer risk.

Absence of estrogen receptor alpha (ESR1) gene amplification in a series of breast cancers in Taiwan.

Immunohistochemical expression of ERα, encoded by the ESR1 (estrogen receptor 1) gene located at 6q25.1, is the most important determinant of responsiveness to endocrine therapy in breast cancer. The prevalence and significance of ESR1 amplification in breast cancer remain controversial. We set out to assess ESR1 status and its relevance in breast cancer in Taiwan. We tested tissue samples from 311 invasive carcinomas in a tissue microarray for ESR1 status by fluorescent in situ hybridization (FISH) and chromogenic in situ hybridization (CISH). In order to examine its association with ERα and ESR1 status, HER2 status was determined by FISH. Of the carcinomas, 58.8% (183/311) was ERα positive. None of the carcinomas showed amplification of ESR1 by either method, whereas 24.1% (75/311) of the carcinomas harbored HER2 amplification. Of the carcinomas, 9.6% (26/301) showed ESR1 gain (1.3 ≤ ratio ESR1/chromosome 6 < 2) by FISH and 10% (24/299) by CISH. FISH and CISH results showed a good correlation (κ-coefficient = 0.786). ESR1 gain by FISH and CISH was significantly associated with high-grade (P = 0.0294 and 0.0417, respectively) but not with ERα expression, HER2 status, or overall survival. ERα positivity was significantly associated with better overall survival (P = 0.039). HER2 amplification was significantly related with poor overall survival (P = 0.002). Our data confirm that in breast cancer, HER2 amplification is a frequent genetic aberration and a negative prognostic factor, and show that ESR1 amplification is not a key genetic abnormality in the tumorigenesis of breast cancer in Taiwan.

Significance of ERα, HER2, and CAV1 expression and molecular subtype classification to canine mammary gland tumor.

Canine mammary gland tumor (CMT) and human breast cancer (HBC) share many similarities regarding their risk factors, histological features, and behavior. Despite the increasing evidence of molecular marker expression as a prognostic indicator for HBC, few studies have applied this approach to CMT. The aim of the present study is to evaluate the significance of the expression of estrogen receptor-alpha (ERα), human epidermal growth factor receptor 2 (HER2), and caveolin-1 (CAV1) to the behavior and the clinical outcome of CMT. Additionally, the correlation between subtype classification (luminal A, luminal B, HER2-overexpressing, basal-like, and normal-like) and tumor behavior prognosis were assessed. Canine mammary gland tissues were immunohistochemically stained for ERα, HER2, and CAV1 and evaluated and classified into 5 subtypes on the basis of immunoreactivity. Although there were no statistically significant differences in the molecular marker immunoreactivity of different subtypes, the degree of positive staining for ERα, extranuclear ERα, HER2, and CAV1 showed significant correlations (P < 0.05) with the behavior and prognosis of the tumor. The current study indicates the prognostic value of immunohistochemical staining status of ERα, HER2, and CAV1 for CMT. In addition, some trends were seen in subtype classification on the prognosis of the tumor, implying that, although further analysis is needed, there is potential clinical application of 5-subtype classification for CMT.

The atypical ubiquitin ligase RNF31 stabilizes estrogen receptor α and modulates estrogen-stimulated breast cancer cell proliferation

Estrogen receptor α (ERα) is initially expressed in the majority of breast cancers and promotes estrogen-dependent cancer progression by regulating the transcription of genes linked to cell proliferation. ERα status is of clinical importance, as ERα-positive breast cancers can be successfully treated by adjuvant therapy with antiestrogens or aromatase inhibitors. Complications arise from the frequent development of drug resistance that might be caused by multiple alterations, including components of ERα signaling, during tumor progression and metastasis. Therefore, insights into the molecular mechanisms that control ERα expression and stability are of utmost importance to improve breast cancer diagnostics and therapeutics. Here we report that the atypical E3 ubiquitin ligase RNF31 stabilizes ERα and facilitates ERα-stimulated proliferation in breast cancer cell lines. We show that depletion of RNF31 decreases the number of cells in the S phase and reduces the levels of ERα and its downstream target genes, including cyclin D1 and c-myc. Analysis of data from clinical samples confirms correlation between RNF31 expression and the expression of ERα target genes. Immunoprecipitation indicates that RNF31 associates with ERα and increases its stability and mono-ubiquitination, dependent on the ubiquitin ligase activity of RNF31. Our data suggest that association of RNF31 and ERα occurs mainly in the cytosol, consistent with the lack of RNF31 recruitment to ERα-occupied promoters. In conclusion, our study establishes a non-genomic mechanism by which RNF31 via stabilizing ERα levels controls the transcription of estrogen-dependent genes linked to breast cancer cell proliferation.

Abstract P6-04-03: ERb and breast cancer: A potential predictive and prognostic biomarker and novel therapeutic drug target

Abstract Background: Estrogen receptor beta (ERβ), unlike ERα, classically functions as a tumor suppressor in vitro. However, ERβ's biological functions in vivo and predictive/prognostic value in breast cancer are controversial. Methods: Expression of ERβ protein was determined using a well characterized and validated ERβ specific monoclonal antibody that only recognizes the full-length form of this receptor (PPG5/10) in the following 3 cohorts: 1) a cohort with all breast cancer subtypes (n = 182), 2) a prospective NCCTG adjuvant tamoxifen trial for postmenopausal women with ERα positive breast cancer with long-term follow-up (n = 198) and 3) a cohort of 80 triple negative breast cancers (TNBCs). To elucidate the biological functions of ERβ in breast cancer, novel ERβ expressing MCF7 and MDA-MB-231 cell lines were developed and characterized using multiple techniques and were examined for responsiveness to various ERβ targeted therapies. Results: About one-third of all breast tumors, regardless of sub-type, were shown to express nuclear ERβ and this expression was independent of ERα or HER2. In the NCCTG 89-30-52 cohort, breast cancer recurrence rates were significantly associated with ERβ protein expression with 10 year recurrence rates of 25%, 15% and 4% for zero, low or high levels of ERβ expression respectively. Interestingly, in TNBCs, nuclear ERβ was expressed at intermediate or high levels in 24% of tumors. In the triple negative MDA-MB-231 cell line, expression of ERβ led to inhibition of proliferation and induction of apoptosis in response to estrogen and multiple ERβ specific agonists. Conversely, these same treatments induced proliferation of ERβ-expressing MCF7 cells which endogenously express ERα. However, ERβ expression sensitized MCF7 cells to the anti-proliferative effects of anti-estrogens. Microarray analysis and RT-PCR profiling of MDA-MB-231-ERβ cells revealed that estrogen and ERβ agonists highly induced the expression of multiple cystatins, a family of small secreted cysteine protease inhibitors which function as tumor suppressors, and potently inhibited canonical TGFβ signaling. Conditioned media isolated from estrogen or ERβ agonist treated MDA-MB-231-ERβ cells suppressed the proliferation rates and inhibited TGFβ signaling in other TNBC cell lines, effects that were completely reversed following the depletion of cystatins from the conditioned media. Conclusions: These data demonstrate that ERβ is expressed in a substantial proportion of breast cancers and may have value as a predictive and/or prognostic biomarker. Therapeutic targeting of ERβ may have clinical benefit in multiple breast cancer sub-types; however, the specific drug of choice may vary based on ERα status. Specifically, we have demonstrated that ERβ expression in ERα+ MCF7 cells sensitizes them to the effectiveness of anti-estrogens, an observation that was confirmed in women enrolled in a prospective adjuvant tamoxifen trial. In TNBCs, where targeted therapies are lacking, our data suggest that targeting ERβ with either estrogen or ERβ specific agonists will elicit anti-tumor effects through the induction of cystatins and inhibition of TGFβ signaling resulting in tumor regression and improved patient outcomes. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-03.

Evidences that estrogen receptor α interferes with adiponectin effects on breast cancer cell growth

Adiponectin, the most abundant protein secreted by adipose tissue, exhibits insulin-sensitizing, anti-inflammatory, antiatherogenic, and antiproliferative properties. In addition, it appears to play an important role also in the development and progression of several obesity-related malignancies, including breast cancer. Here, we demonstrated that adiponectin induces a dichotomic effect on breast cancer growth. Indeed, it stimulates growth in ERα+ MCF-7 cells while inhibiting proliferation of ERα− MDA-MB-231 cells. Notably, only in MCF-7 cells adiponectin exposure exerts a rapid activation of MAPK phosphorylation, which is markedly reduced when knockdown of the ERα gene occurred. In addition, adiponectin induces rapid IGF-IR phosphorylation in MCF-7 cells, and the use of ERα siRNA prevents this effect. Moreover, MAPK activation induced by adiponectin was reversed by IGF-IR siRNA. Coimmunoprecipitation studies show the existence of a multiprotein complex involving AdipoR1, APPL1, ERα, IGF-IR, and c-Src that is responsible for MAPK signaling activation in ERα+ positive breast cancer cells. It is well known that in addition to the rapid effects through non-genomic mechanisms, ERα also mediates nuclear genomic actions. In this concern, we demonstrated that adiponectin is able to transactivate ERα in MCF-7 cells. We showed the classical features of ERα transactivation: nuclear localization, downregulation of mRNA and protein levels, and upregulation of estrogen-dependent genes. Thus, our study clarifies the molecular mechanism through which adiponectin modulates breast cancer cell growth, providing evidences on the cell-type dependency of adiponectin action in relationship to ERα status.

Discordance in ERα, PR and HER2 receptor status across different distant breast cancer metastases within the same patient

BackgroundWe studied discordance in estrogen receptor alpha (ERα), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) status between multiple distant metastases from the same breast cancer patient. Material and methodsMultiple distant metastases from 55 female patients were stained for ERα, PR and HER2 by immunohistochemistry and in situ hybridization for confirmation of the HER2 status. ResultsDifferent metastatic sites within the same patient showed discordance in ERα receptor status in 7.3% or 10.9% of patients (using a 10% or 1% threshold for positivity, respectively). For PR, 29.1% or 30.9% of patients showed discordance. Taking ERα and PR together, 36.4% of cases (both thresholds) showed discrepancy between metastases. In 10.9% (10% threshold) or 14.5% of patients (1% threshold), such discordance could have clinical consequences with regard to hormonal treatment. For HER2, there was 3.6% discordance on the immunohistochemical level but 0% on the gene level. ConclusionIn a significant proportion of metastatic breast cancer patients, discordance in ERα and PR receptor status between different metastatic sites was observed. This implies that multiple metastases may need to be biopsied to optimally reassess receptors.

Eugenol triggers apoptosis in breast cancer cells through E2F1/survivin down-regulation

BackgroundBreast cancer is a major health problem that threatens the lives of millions of women worldwide each year. Most of the chemotherapeutic agents that are currently used to treat this complex disease are highly toxic with long-term side effects. Therefore, novel generation of anti-cancer drugs with higher efficiency and specificity are urgently needed.MethodsBreast cancer cell lines were treated with eugenol and cytotoxicity was measured using the WST-1 reagent, while propidium iodide/annexinV associated with flow cytometry was utilized in order to determine the induced cell death pathway. The effect of eugenol on apoptotic and pro-carcinogenic proteins, both in vitro and in tumor xenografts was assessed by immunoblotting. While RT-PCR was used to determine eugenol effect on the E2F1 and survivin mRNA levels. In addition, we tested the effect of eugenol on cell proliferation using the real-time cell electronic sensing system.ResultsEugenol at low dose (2 μM) has specific toxicity against different breast cancer cells. This killing effect was mediated mainly through inducing the internal apoptotic pathway and strong down-regulation of E2F1 and its downstream antiapoptosis target survivin, independently of the status of p53 and ERα. Eugenol inhibited also several other breast cancer related oncogenes, such as NF-κB and cyclin D1. Moreover, eugenol up-regulated the versatile cyclin-dependent kinase inhibitor p21WAF1 protein, and inhibited the proliferation of breast cancer cells in a p53-independent manner. Importantly, these anti-proliferative and pro-apoptotic effects were also observed in vivo in xenografted human breast tumors.ConclusionEugenol exhibits anti-breast cancer properties both in vitro and in vivo, indicating that it could be used to consolidate the adjuvant treatment of breast cancer through targeting the E2F1/survivin pathway, especially for the less responsive triple-negative subtype of the disease.

Variation in Use of Estrogen Receptor-α Gene Promoters in Breast Cancer Compared by Quantification of Promoter-Specific Messenger RNA

IntroductionEstrogen receptor (ER)-α expression offers a critical characterization of breast cancer, but risk of recurrence is difficult to predict using only ERα status. The ERα gene has at least 6 transcription start sites, 6 distinct first exons, and probably 6 promoters. To examine whether these promoters have differential effects in breast cancer, we quantified expression of promoter-specific ERα messenger RNA (mRNA), using real-time polymerase chain reaction (PCR) and statistical assessment. Patients and MethodsWe examined variations in the use of breast cancer cell lines and 43 ERα positive (ERα+) breast cancer tissue samples by quantifying promoter-specific mRNA of ERα with real-time PCR analysis using primers and probes specially designed for this study. Moreover, we correlated the results of quantified the promoter-specific mRNA with mRNA of total ERα and related them to clinicopathological factors statistically. We also examined multiregression analyses for promoter-specific mRNAs of ERα. ResultWe found the promoters to be used at almost similar ratios among ERα+ breast cancer cell lines and ERα+ breast cancer tissues. Clinicopathological variations were associated with identical ERα promoter choices. When we examined the contribution of mRNA from 3 promoters in breast cancer tissues to total ERα using multiple regression analysis, we found that only promoter A showed a significant (P < .05) transcript coefficient. ConclusionOur findings imply that the use of ERα promoters as prognostic biomarkers is unfeasible. However, our results suggest that promoter usage of ERα may contribute to its expression in normal development and differentiation of individual or carcinogenesis of breast cancer.

Estrogen Response element-GFP (ERE-GFP) introduced MCF-7 cells demonstrated the coexistence of multiple estrogen-deprivation resistant mechanisms

The acquisition of estrogen-deprivation resistance and estrogen receptor (ER) signal-independence in ER-positive breast cancer is one of the crucial steps in advancing the aggressiveness of breast cancer; however, this has not yet been elucidated in detail. To address this issue, we established several estrogen-deprivation-resistant (EDR) breast cancer cell lines from our unique MCF-7 cells, which had been stably transfected with an ERE-GFP reporter plasmid. Three cell lines with high ER activity and another 3 cell lines with no ER activity were established from cell cloning by monitoring GFP expression in living cells. The former three ERE-GFP-positive EDR cell lines showed the overexpression of ER and high expression of several ER-target genes. Further analysis of intracellular signaling factors revealed a marked change in the phosphorylation status of ERα on Ser167 and Akt on Thr308 by similar mechanisms reported previously; however, we could not find any changes in MAP-kinase factors. Comprehensive phospho-proteomic analysis also indicated the possible contribution of the Akt pathway to the phosphorylation of ERα.On the other hand, constitutive activation of c-Jun N-terminal kinase (JNK) was observed in ERE-GFP-negative EDR cells, and the growth of these cells was inhibited by a JNK inhibitor. An IGF1R-specific inhibitor diminished the phosphorylation of JNK, which suggested that a novel signaling pathway, IGF1R-JNK, may be important for the proliferation of ER-independent MCF-7 cells. These results indicate that ER-positive breast cancer cells can acquire resistance by more than two mechanisms at a time, which suggests that multiple mechanisms may occur simultaneously. This finding also implies that breast cancers with different resistance mechanisms can concomitantly occur and mingle in an individual patient, and may be a cause of the recurrence of cancer.

The estrogen receptor α is the key regulator of the bifunctional role of FoxO3a transcription factor in breast cancer motility and invasiveness

The role of the Forkhead box class O (FoxO)3a transcription factor in breast cancer migration and invasion is controversial. Here we show that FoxO3a overexpression decreases motility, invasiveness, and anchorage-independent growth in estrogen receptor α-positive (ERα+) cancer cells while eliciting opposite effects in ERα-silenced cells and in ERα-negative (ERα−) cell lines, demonstrating that the nuclear receptor represents a crucial switch in FoxO3a control of breast cancer cell aggressiveness. In ERα+ cells, FoxO3a-mediated events were paralleled by a significant induction of Caveolin-1 (Cav1), an essential constituent of caveolae negatively associated to tumor invasion and metastasis. Cav1 induction occurs at the transcriptional level through FoxO3a binding to a Forkhead responsive core sequence located at position −305/−299 of the Cav1 promoter. 17β-estradiol (E2) strongly emphasized FoxO3a effects on cell migration and invasion, while ERα and Cav1 silencing were able to reverse them, demonstrating that both proteins are pivotal mediators of these FoxO3a controlled processes. In vivo, an immunohistochemical analysis on tissue sections from patients with ERα+ or ERα− invasive breast cancers or in situ ductal carcinoma showed that nuclear FoxO3a inversely (ERα+) or directly (ERα−) correlated with the invasive phenotype of breast tumors. In conclusion, FoxO3a role in breast cancer motility and invasion depends on ERα status, disclosing a novel aspect of the well-established FoxO3a/ERα interplay. Therefore FoxO3a might become a pursuable target to be suitably exploited in combination therapies either in ERα+ or ERα− breast tumors.

The p53-Estrogen Receptor Loop in Cancer

Tumor suppressor p53 maintains genome stability by regulating diverse cellular functions including cell cycle arrest, apoptosis, senescence and metabolic homeostasis. Mutations in the p53 gene occur in almost all human cancers with a frequency of up to 80%. However, it is only 20% in breast cancers, 18% in endometrial cancers and 1.5% in cervical cancers. Estrogen receptor alpha (ERα) plays a pivotal role in hormone-dependent cancer development and the status of ERα is used for designing treatment strategy and for prognosis. A closer look at the cross-talk between p53 and ERα has revealed that their activities are mutually regulated. This review will summarize the current body of knowledge on p53, ERα and ERβ in cancer. Clinical correlations between estrogen receptors and p53 status have also been reported. Thus, this review will discuss the relationship between p53 and ERs at both the molecular and clinical levels.

Effects of sorafenib on energy metabolism in breast cancer cells: role of AMPK–mTORC1 signaling

In this study, we investigated the effects and the underlying molecular mechanisms of the multi-kinase inhibitor sorafenib in a panel of breast cancer cell lines. Sorafenib inhibited cell proliferation and induced apoptosis through the mitochondrial pathway. These effects were neither correlated with modulation of MAPK and AKT pathways nor dependent on the ERα status. Sorafenib promoted an early perturbation of mitochondrial function, inducing a deep depolarization of mitochondrial membrane, associated with drop of intracellular ATP levels and increase of ROS generation. As a response to this stress condition, the energy sensor AMPK was rapidly activated in all the cell lines analyzed. In MCF-7 and SKBR3 cells, AMPK enhanced glucose uptake by up-regulating the expression of GLUT-1 glucose transporter, as also demonstrated by AMPKα1 RNA interference, and stimulated aerobic glycolysis thus increasing lactate production. Moreover, the GLUT-1 inhibitor fasentin blocked sorafenib-induced glucose uptake and potentiated its cytotoxic activity in SKBR3 cells. Persistent activation of AMPK by sorafenib finally led to the impairment of glucose metabolism both in MCF-7 and SKBR3 cells as well as in the highly glycolytic MDA-MB-231 cells, resulting in cell death. This previously unrecognized long-term effect of sorafenib was mediated by AMPK-dependent inhibition of the mTORC1 pathway. Suppression of mTORC1 activity was sufficient for sorafenib to hinder glucose utilization in breast cancer cells, as demonstrated by the observation that the mTORC1 inhibitor rapamycin induced a comparable down-regulation of GLUT-1 expression and glucose uptake. The key role of AMPK-dependent inhibition of mTORC1 in sorafenib mechanisms of action was confirmed by AMPKα1 silencing, which restored mTORC1 activity conferring a significant protection from cell death. This study provides insights into the molecular mechanisms driving sorafenib anti-tumoral activity in breast cancer, and supports the need for going on with clinical trials aimed at proving the efficacy of sorafenib for breast cancer treatment.

Estrogen receptor splice variants as a potential source of false-positive estrogen receptor status in breast cancer diagnostics

It is well established that only estrogen receptor (ER)-positive tumors benefit from hormonal therapies. We hypothesized that a subgroup of breast cancer patients expresses estrogen receptor α (ERα), but fails to respond to hormonal therapy due to the expression of a non-functional receptor. We analyzed a series of 2,658 ERα-positive HER2-negative breast tumors for ERα and progesterone receptor (PR) status as determined by mRNA expression and for their molecular subtypes (Luminal type vs Basal type, assessed by BluePrint™ molecular subtyping assay). In addition, we assessed the recurrence risk (low vs high) using the 70-gene MammaPrint™ signature. We found that 55 out of 2,658 (2.1 %) tumors that are ERα positive by mRNA analysis also demonstrate a Basal molecular subtype, indicating that they lack expression of estrogen-responsive genes. These ERα-positive Basal-type tumors express significantly lower levels of both ERα and PR mRNA as compared to Luminal-type tumors (P < 0.0001) and almost invariably (94.5 %) have a high-risk MammaPrint™ profile. Twelve of the MammaPrint™ genes are directly ERα responsive, indicating that MammaPrint™ assesses ERα function in breast cancer without considering ERα mRNA levels. We find a relatively high expression of the dominant negative ERα splice variant ERΔ7 in ERα-positive Basal-type tumors as compared to ERα-positive Luminal-type tumors (P < 0.0001). Expression of the dominant negative ERα variant ERΔ7 provides a rationale as to why tumors are of the Basal molecular subtype while staining ERα positive by immunohistochemistry. These tumors may lack a functional response to estrogen and consequently may not respond to hormonal therapy. Our data indicate that such patients are of MammaPrint™ high recurrence risk and might benefit from adjuvant chemotherapy.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-013-2648-1) contains supplementary material, which is available to authorized users.

Abstract 2401: Evaluation of circulating serum Amphiregulin levels in patients with breast cancer.

Abstract Amphiregulin (AREG), a ligand for Epidermal Growth Factor Receptor (EGFR), has been implicated as an effector of ERα signaling in mammary gland development and as a candidate ERα effector in human breast cancers. Analysis of 13 luminal subtype breast cancer cell lines and human breast tumors showed a strong correlation between ERα status and high AREG levels. A pilot study using healthy and breast cancer women's serum showed that circulating AREG levels were higher in the ERα positive patients as compared to healthy individuals. Therefore we hypothesized that Amphiregulin is a potential biomarker and/or therapeutic target for ERα positive breast cancers. To test our hypothesis, we are currently evaluating AREG as a potential serum biomarker of breast cancer progression and as an indicator of response to treatment by analyzing a cohort of breast cancer patients (n=225) stratified by stage of disease and subtypes (hormone receptor status). In this study, we want to determine if tumor-derived AREG is released into the systemic circulation and whether it may be a useful serum biomarker for the presence of breast cancer and for response to therapy. In the first phase, serum Amphiregulin was measured by an ELISA assay to establish the reference range in 125 healthy females. Fifty-five percent of the healthy women had no detectable circulating AREG (n=69), and only ten women had levels exceeding 500 pg/mL. Serum AREG levels did not vary significantly during the menstrual cycle. We determined that the 95th and 90th percentile was &amp;gt;1575 pg/ml and &amp;gt;471 pg/ml, respectively, to use as a comparison against the AREG levels of the breast cancer patients. We are analyzing serum AREG levels in breast cancer patient serum samples of three different cohorts: women with no evidence of disease after more than 1 year post-surgery (Cohort 1), women with locally advanced or stage IV with disease progression (Cohort 2) and women with operable disease that will have surgery and subsequent chemotherapy and/or endocrine therapy (Cohort 3). Each cohort was further stratified by hormone receptor status. In addition to determining the baseline AREG levels, for Cohort 3, AREG levels will be measured post-surgery and at different timepoints during treatment. Interim analysis of the different cohorts showed a significance difference (p&amp;lt;0.05) between healthy individuals and patients with ERα+ breast tumors when comparing detectable versus undetectable AREG levels (threshold of detection: 20 pg/ml). Interestingly, longitudinal sampling of one patient with very high levels (18,300 pg/ml at diagnosis) showed a 37% decline following tumor resection and a 60% decline after 9 months of endocrine therapy. In summary, determining the potential role of AREG as predictive biomarker for therapy and/or as therapeutic target by the completion of this clinical study, will contribute to the quest for better predictive biomarkers in breast cancer and better management of the disease. Citation Format: Esther A. Peterson, Paraic A. Kenny. Evaluation of circulating serum Amphiregulin levels in patients with breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2401. doi:10.1158/1538-7445.AM2013-2401

Abstract 3079: Estrogen signaling promotes cancer stem cell expansion via microRNA-140 targeting of pluripotency-associated factor SOX2 in ERα positive breast cancer cells.

Abstract Multiple reports indicate that the prospective tumor suppressor miR-140 is down regulated in breast tumors compared to normal breast tissues; however, the role of miR-140 in breast tumorigenesis remains unclear. We have identified a novel target for miR-140 in mammary epithelium, the core pluripotency transcription factor SOX2. It is well known that SOX2 regulates the self-renewal of embryonic stem cells, however, less is known concerning possible oncogenic roles of SOX2 in breast cancer. We found that miR-140 and SOX2 target mRNA exhibited inverse expression in breast tumors and normal breast tissues, which was also correlated with estrogen receptor alpha (ERα) status. We investigated the role of ERα signaling in the regulation of miR-140 and SOX2 in breast tumors and found that estrogen stimulation resulted in decreased miR-140 and increased SOX2 expression in mammary epithelium and breast cancer cells. We performed bioinformatics promoter analyses of the miR-140 promoter revealing several predicted estrogen response elements (ERE) in the miR-140 promoter. We examined estrogen-induced activation of these prospective EREs using luciferase reporters for a miR-140 promoter deletion series. Furthermore, we used chromatin-immunoprecipitation to reveal that ERα binds to an ERE flanking the miR-140 promoter, consequently suppressing miR-140 transcription. As estrogen signaling was previously implicated in the regulation of breast cancer stem cells (CSCs), we investigated whether the miR-140/SOX2 pathway might provide a mechanistic link between ERα signaling and CSC maintenance. We found that forced activation of the miR-140/SOX2 pathway decreased mammosphere formation (a surrogate measure of CSC self-renewal) and opposed estrogen-induced expansion of the well-characterized CD44(high)/CD24(low) CSC subpopulation in ERα+ breast cancer cells. These results indicate that an ERα/miR-140/SOX2 pathway critically regulates breast CSC maintenance in ERα+ breast tumors. Citation Format: Yongshu Zhang, Gabriel L. Eades, Yuan Yao, Qinglin Li, Qun Zhou. Estrogen signaling promotes cancer stem cell expansion via microRNA-140 targeting of pluripotency-associated factor SOX2 in ERα positive breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3079. doi:10.1158/1538-7445.AM2013-3079

Abstract 518: Kisspeptin stimulates invasiveness of ERα -negative human mammary epithelial and breast cancer cells.

Abstract Recently, we have shown that KP-10 stimulates invasion of estrogen receptor (ERα)-negative breast cancer cells via transactivation of the epidermal growth factor receptor (EGFR) [Zajac et al. (2011) PLoS ONE 6(6): e21599]. Here we report that KP-10 stimulated cell motility and invasiveness of ERα-negative non-malignant mammary epithelial MCF10A cells that endogenously expressing KISS1R, and MCF10A cells stably expressing KISS1R using three-dimensional (3D) Matrigel cultures. Additionally, exogenous expression of KISS1R in ERα-negative SKBR3 breast cancer cells stimulated invasion in 3D cultures and induced extravasation in vivo using chorioallantoic membrane (CAM) assays. Exogenous KISS1R expression in MCF10A and SKBR3 cells also induced a partial epithelial-to-mesenchymal transition like phenotype as evidenced by the acquisition of a spindle-shaped morphology, intracellular localization of the epithelial marker E-cadherin, increased stress fibre formation and elevated levels of the mesenchymal markers, Snail/Slug, vimentin and N-cadherin. In contrast, KP-10 had no effect on migration and invasion of the ERα-positive T47D and MCF7 breast cancer cells and failed to transactivate EGFR in the ERα-positive cells. This suggested that ERα negatively regulates KISS1R-dependent breast cancer cell migration, invasion and EGFR transactivation. In support of this, we found KP-10-stimulated cell migration, invasion and EGFR transactivation were ablated upon stably expressing ERα in the ERα-negative MDA-MB-231 cells. Lastly, we found that KISS1R was localized at the leading edge of motile cells, where it co-localized with the actin scaffolding protein, IQGAP1. We have identified IQGAP1 as a novel binding partner of KISS1R and have demonstrated that KISS1R regulates EGFR transactivation in breast cancer cells in an IQGAP1-dependent manner. Overall, our data strongly suggest that the ERα status of mammary cells will dictate whether the KISS1R signaling pathway may be a novel clinical target for the treatment of breast cancer metastasis. Citation Format: Moshmi M. Bhattacharya, Dragana Cvetkovic, Magdalena Dragan, Hon Sing Leong, Andy Babwah. Kisspeptin stimulates invasiveness of ERα -negative human mammary epithelial and breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 518. doi:10.1158/1538-7445.AM2013-518

Reduced Androgen Receptor Expression Accelerates the Onset of ERBB2 Induced Breast Tumors in Female Mice

Androgen receptor (AR) is commonly expressed in both the epithelium of normal mammary glands and in breast cancers. AR expression in breast cancers is independent of estrogen receptor alpha (ERα) status and is frequently associated with overexpression of the ERBB2 oncogene. AR signaling effects on breast cancer progression may depend on ERα and ERBB2 status. Up to 30% of human breast cancers are driven by overactive ERBB2 signaling and it is not clear whether AR expression affects any steps of tumor progression in this cohort of patients. To test this, we generated mammary specific Ar depleted mice (MARKO) by combining the floxed allele of Ar with the MMTV-cre transgene on an MMTV-NeuNT background and compared them to littermate MMTV-NeuNT, Arfl/+ control females. Heterozygous MARKO females displayed reduced levels of AR in mammary glands with mosaic AR expression in ductal epithelium. The loss of AR dramatically accelerated the onset of MMTV-NeuNT tumors in female MARKO mice. In this report we show that accelerated MMTV-NeuNT-dependent tumorigenesis is due specifically to the loss of AR, as hormonal levels, estrogen and progesterone receptors expression, and MMTV-NeuNT expression were similar between MARKO and control groups. MMTV-NeuNT induced tumors in both cohorts displayed distinct loss of AR in addition to ERα, PR, and the pioneer factor FOXA1. Erbb3 mRNA levels were significantly elevated in tumors in comparison to normal mammary glands. Thus the loss of AR in mouse mammary epithelium accelerates malignant transformation rather than the rate of tumorigenesis.

G Protein–Coupled Estrogen Receptor Is Apoptotic and Correlates with Increased Distant Disease-Free Survival of Estrogen Receptor–Positive Breast Cancer Patients

G protein-coupled estrogen receptor 1 (GPER1), previously named GPR30, is a membrane receptor reported to mediate nongenomic estrogen responses. We investigated if GPER1 expression correlates with any clinicopathologic variables and distant disease-free survival (DDFS) in patients with breast cancer, if any prognostic impact of the receptor is dependent on estrogen receptor-α (ER-α) status, and if the receptor impacts apoptotic signaling in ER-positive breast cancer cells. GPER1 expression was analyzed by immunohistochemistry in breast tumors from 273 pre- and postmenopausal stage II patients, all treated with adjuvant tamoxifen for 2 years (cohort I) and from 208 premenopausal lymph node-negative patients, of which 87% were not subjected to any adjuvant systemic treatment (cohort II). GPER1-dependent proapoptotic signaling was analyzed in MCF7 cells with and without GPER1 knockdown, T47D cells, HEK293 cells (HEK), and HEK stably expressing GPER1 (HEK-R). GPER1 positively correlates with ER and progesterone receptor expression. Multivariate analysis showed that GPER1 is an independent prognostic marker of increased 10-year DDFS in the ER-positive subgroup. HEK-R has higher basal proapoptotic signaling compared with HEK including increased cytochrome C release, caspase-3 cleavage, PARP cleavage, and decreased cell viability. Treating HEK-R with the proteasome inhibitor epoxomicin, to decrease GPER1 degradation, further increases receptor-dependent proapoptotic signaling. Also, GPER1 knockdown decreases basal and agonist-stimulated proapoptotic receptor signaling in MCF7 cells. GPER1 is a prognostic indicator for increased DDFS in ER-positive breast cancer, which may be associated with constitutive GPER1-dependent proapoptotic signaling in ER-positive breast cancer cells.