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The purpose of this study was to identify critical pathways promoting survival of tamoxifen-tolerant, estrogen receptor α positive (ER+) breast cancer cells, which contribute to therapy resistance and disease recurrence. Gene expression profiling and pathway analysis were performed in ER+ breast tumors of patients before and after neoadjuvant tamoxifen treatment and demonstrated activation of the NF-κB pathway and an enrichment of epithelial-to mesenchymal transition (EMT)/stemness features. Exposure of ER+ breast cancer cell lines to tamoxifen, in vitro and in vivo, gives rise to a tamoxifen-tolerant population with similar NF-κB activity and EMT/stemness characteristics. Small-molecule inhibitors and CRISPR/Cas9 knockout were used to assess the role of the NF-κB pathway and demonstrated that survival of tamoxifen-tolerant cells requires NF-κB activity. Moreover, this pathway was essential for tumor recurrence following tamoxifen withdrawal. These findings establish that elevated NF-κB activity is observed in breast cancer cell lines under selective pressure with tamoxifen in vitro and in vivo, as well as in patient tumors treated with neoadjuvant tamoxifen therapy. This pathway is essential for survival and regrowth of tamoxifen-tolerant cells, and, as such, NF-κB inhibition offers a promising approach to prevent recurrence of ER+ tumors following tamoxifen exposure.Implications:Understanding initial changes that enable survival of tamoxifen-tolerant cells, as mediated by NF-κB pathway, may translate into therapeutic interventions to prevent resistance and relapse, which remain major causes of breast cancer lethality.

Accumulating evidence suggests that our ability to predict chemical effects on breast cancer is limited by a lack of physiologically relevant in vitro models; the typical in vitro breast cancer model consists of the cancer cell and excludes the mammary microenvironment. As the effects of the microenvironment on cancer cell behavior becomes more understood, researchers have called for the integration of the microenvironment into in vitro chemical testing systems. However, given the complexity of the microenvironment and the variety of platforms to choose from, identifying the essential parameters to include in a chemical testing platform is challenging. This review discusses the need for more complex in vitro breast cancer models and outlines different approaches used to model breast cancer in vitro. We provide examples of the microenvironment modulating breast cancer cell responses to chemicals and discuss strategies to help pinpoint what components should be included in a model.

Aromatase inhibitors are the preferred treatment for certain women with estrogen receptor (ER)-positive breast cancer, but evidence suggests that women with obesity experience aromatase inhibitor resistance at higher rates. To compare how stromal cells derived from women who are lean or obese influence response to the aromatase inhibitor (anastrazole), we incorporated patient-derived stroma in a previously characterized MCF7-derived in vitro duct model. Coculture with adipose stromal cells enabled the metabolism of testosterone (T) to E(2), which induced estrogen response element activity, epithelial proliferation, and hyperplasia in MCF7 cells. The effects of T were inhibited by the ER antagonist tamoxifen and aromatase inhibitor anastrazole and were increased by the aromatase inducer dexamethasone. Primary mammary adipose stromal cells derived from women with obesity displayed increased aromatase mRNA compared with lean controls. MCF7-derived ducts cocultured with obese stromal cells exhibited higher maximal aromatization-induced ER transactivation and reduced anastrazole sensitivity, a difference not seen in 2-dimensional coculture. Finally, tamoxifen was more effective than anastrazole at reducing aromatization-induced ER transactivation and proliferation. These findings suggest that patient-specific responses to hormone therapies can be modeled and studied organotypically in vitro and add to evidence advocating obesity as a parameter to consider when identifying treatments for patients with ER-positive breast cancer.—Morgan, M. M., Arendt, L. M., Alarid, E. T., Beebe, D. J., Johnson, B. P. Mammary adipose stromal cells derived from obese women reduce sensitivity to the aromatase inhibitor anastrazole in an organotypic breast model.

In mammals, the grainyhead-like transcription factor (GRHL) family is composed of three nuclear proteins that are responsible for driving epithelial cell fate: GRHL1, GRHL2, and GRHL3. GRHL2 is important in maintaining proper tubulogenesis during development and in suppressing the epithelial-to-mesenchymal transition. Within the last decade, evidence indicates both tumor-suppressive and oncogenic roles for GRHL2 in various types of cancers. Recent studies suggest that GRHL2 may be especially important in hormone-dependent cancers, as correlative relationships exist between GRHL2 and various steroid receptors, such as the androgen and estrogen receptors. Acting as a pioneer factor and coactivator, GRHL2 may directly affect steroid receptor transcriptional activity. This review will highlight recent discoveries of GRHL2 activity in cancer and in maintaining the epithelial state, while also exploring recent literature on the role of GRHL2 in hormone-dependent cancers and epigenetics.

Prolactin (PRL) and estrogen cooperate in lobuloalveolar development of the mammary gland and jointly regulate gene expression in breast cancer cells in vitro. Canonical PRL signaling activates STAT5A/B, homologous proteins that have different target genes and functions. Although STAT5A/B are important for physiological mammary function and tumor pathophysiology, little is known about regulation of their expression, particularly of STAT5B, and the consequences for hormone action. In this study, we examined the effect of two estrogenic ligands, 17β-estradiol (E2) and the clinical antiestrogen, ICI182,780 (ICI, fulvestrant) on expression of STAT5 isoforms and resulting crosstalk with PRL in normal and tumor murine mammary epithelial cell lines. In all cell lines, E2 and ICI significantly increased protein and corresponding nascent and mature transcripts for STAT5A and STAT5B, respectively. Transcriptional regulation of STAT5A and STAT5B by E2 and ICI, respectively, is associated with recruitment of estrogen receptor alpha and increased H3K27Ac at a common intronic enhancer 10 kb downstream of the Stat5a transcription start site. Further, E2 and ICI induced different transcripts associated with differentiation and tumor behavior. In tumor cells, E2 also significantly increased proliferation, invasion, and stem cell-like activity, whereas ICI had no effect. To evaluate the role of STAT5B in these responses, we reduced STAT5B expression using short hairpin (sh) RNA. shSTAT5B blocked ICI-induced transcripts associated with metastasis and the epithelial mesenchymal transition in both cell types. shSTAT5B also blocked E2-induced invasion of tumor epithelium without altering E2-induced transcripts. Together, these studies indicate that STAT5B mediates a subset of protumorigenic responses to both E2 and ICI, underscoring the need to understand regulation of its expression and suggesting exploration as a possible therapeutic target in breast cancer., STAT5B expression is regulated by ICI and mediates a subset of protumorigenic responses to ER ligands.

Estrogen receptor α (ER) is the target of endocrine therapies in ER-positive breast cancer (BC), but their therapeutic effectiveness diminishes with disease progression. Most metastatic BCs retain an ER-positive status, but ER expression levels are reduced. We asked how the bone tumor microenvironment (TME) regulates ER expression. We observed ESR1 mRNA and ER protein downregulation in BC cells treated with conditioned media (CM) from patient-derived, cancer-activated bone marrow stromal cells (BMSCs) and the BMSC cell line HS5. Decreases in ESR1 mRNA were attributed to decreases in nascent transcripts as well as decreased RNA polymerase II occupancy and H3K27Ac levels on the ESR1 promoter and/or distal enhancer (ENH1). Repression extended to neighboring genes of ESR1, including ARMT1 and SYNE1. Although ERK/MAPK signaling pathway can repress ER expression by other TME cell types, MAPK inhibition did not reverse decreases in ER expression by BMSC-CM. ESR1 mRNA and ER protein half-lives in MCF7 cells were unchanged by BMSC-CM treatment. Whereas ER phosphorylation was induced, ER activity was repressed by BMSC-CM as neither ER occupancy at known binding sites nor estrogen response element–luciferase activity was detected. BMSC-CM also repressed expression of ER target genes. In cells expressing the Y537S and D538G ESR1 mutations, BMSC-CM reduced ESR1, but expression of target genes PGR and TFF1 remained significantly elevated compared with that of control wild-type cells. These studies demonstrate that BMSCs can transcriptionally corepress ESR1 with neighboring genes and inhibit receptor activity, but the functional consequences of the BMSC TME can be limited by metastasis-associated ESR1 mutations.

In estrogen receptor (ER) positive breast cancers, there is a continual balance between growth and differentiation. Upon activation with estrogen, ER is phosphorylated at serine 118 (pS118-ER). High pS118-ER expression has been observed in low grade and well differentiated tumors. However, this phospho-variant presents a paradox as high expression has also been detected in therapy resistant metastatic tumors. Recently our lab conducted a comprehensive genome wide analysis of ER and pS118-ER through a ChIP-seq study. DNA motif analysis revealed enrichment of the GRHL2 motif near pS118-ER occupancy sites over ER occupancy sites. GRHL2 is a transcription factor that has also been implicated in epithelial differentiation and has been shown to have roles as both a tumor suppressor and oncogene in breast cancer. However, its role in facilitating pS118-ER transcriptional activity is unclear. In these studies, we examine the role of GRHL2 in pS118-ER occupancy through conventional ChIP at GRHL2/pS118-ER co-occupancy sites. GRHL2 occupancy can increase upon estrogen treatment at sites where pS118-ER is also present, but that increase has not been seen at sites lacking pS118-ER occupancy. Using a CRISPR knockout of GRHL2, the role of GRHL2 in pS118-ER DNA binding is being investigated further. In addition, we are using luminal cultures to model breast ducts and investigate the role of GRHL2 in breast cancer epithelial differentiation. These lumens maintain their estrogen responsiveness and GRHL2 expression, which will allow for the study of GRHL2 in a biologically relevant structure. These studies provide the foundation for understanding GRHL2 function in breast cancer.

Approximately 50% of estrogen receptor (ER) positive breast cancer patients will experience relapse. One explanation for this is the presence of breast cancer stem cells (BCSCs), which have self-renewal capability and can contribute to therapy resistance, metastasis, and relapse. Previously, we have shown that cells with active ER and NFκB gain stem cell properties. They are characterized by higher mammosphere (MS) forming efficiency and increased expression of stem-associated markers. NFκB has been previously described as a player in BCSC biology. However, the role of ER remains unclear since some studies have suggested that BCSCs are ER negative. In order to study the role of ER and NFκB in BCSCs in more detail, we utilized single cell RNA sequencing of MS. Dimensionality reduction of single cells identified 4 clusters with unique transcriptional signatures. In order to determine which cluster of these 4 represents BCSCs, we ran functional enrichment analysis (FEA) using MsigDB as a source of various stem cell signatures. Results of FEA showed 1 of 9 stem cell signatures enriched in Cluster 0. Whereas clusters 1, 2 and 3 demonstrated increasing enrichment of different stem cell signatures, respectively. Further analysis of stem cell signatures and genes revealed that cluster 1 included not only classical stem markers (ALDH1A3, KRT8, CD36), but also was enriched for PI3K and p53 signaling pathways. The cluster 2 stem cell signature was associated with DREAM complex activity and BRCA1 mutation. Cluster 3 was enriched for expression of NANOG and SOX2 targets. We also ran FEA to determine ER and NFκB activity in each population. Cluster 0 showed no activity of either ER or NFκB, whereas Cluster 1 was enriched for both, cluster 2 was enriched for ER only, and cluster 3 was enriched for NFκB only. Taken together this data suggests that ER and NFκB may play different roles in promoting BCSC properties. In order to determine how both pathways effect BCSC features, we made a dual reporter cell line expressing ERE-mCherry and NFκB-RE-GFP. It was found ERE activity was constant throughout MS formation, whereas NFκB-RE activity increased proportionally to MS growth over time, suggesting these pathways have different dynamics and further supporting unique roles for each in MS development. To determine which cell population has the capacity to seed MS, an indication of stemness, we sorted cells by reporter activity and tested MS forming efficiency. We discovered that ERE+ cells, either with or without NFκB activity, are more stem-like, whereas activation of NFκB alone is not sufficient to drive MS development. This finding was verified by stem cell-associated gene expression. These data suggest that ER activity is a key driver for BCSC properties and that NFκB may play a supporting role. Furthermore, a novel mechanism of ER regulating the DREAM complex, which is known to be involved in cancer progression and used to evaluate the risk of recurrence in breast cancer, is suggested as a potential ER-mediated mechanism for driving BCSCs. Taken together, our findings suggest novel roles for ER and NFκB in BCSC biology, which could be exploited to target BCSCs therapeutically in ER+ breast cancer patients in order to improve their outcome. Citation Format: Svetlana E. Semina, Irida Kastrati, Luis Alejo Cruz, Tara McCray, Mark Maienschein-Cline, Elaine T. Alarid, Jonna Frasor. Estrogen receptor (ER) and NFkB activity determines cancer stem cell properties in ER positive breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-04.

Personalized cancer therapy focuses on characterizing the relevant phenotypes of the patient, as well as the patient's tumor, to predict the most effective cancer therapy. Historically, these methods have not proven predictive in regards to predicting therapeutic response. Emerging culture platforms are designed to better recapitulate the in vivo environment, thus, there is renewed interest in integrating patient samples into in vitro cancer models to assess therapeutic response. Successful examples of translating in vitro response to clinical relevance are limited due to issues with patient sample acquisition, variability and culture. We will review traditional and emerging in vitro models for personalized medicine, focusing on the technologies, microenvironmental components, and readouts utilized. We will then offer our perspective on how to apply a framework derived from toxicology and ecology towards designing improved personalized in vitro models of cancer. The framework serves as a tool for identifying optimal readouts and culture conditions, thus maximizing the information gained from each patient sample.

The estrogen receptor (ER) regulates the survival and growth of breast cancer cells, but it is less clear how components of the tissue microenvironment affect ER-mediated responses. We set out to test how human mammary fibroblasts (HMFs) modulate ER signaling and downstream cellular responses. We exposed an organotypic mammary model consisting of a collagen-embedded duct structure lined with MCF7 cells to 17-β estradiol (E2), with and without HMFs in the surrounding matrix. MCF7 cells grown as ductal structures were polarized and proliferated at rates comparable to in vivo breast tissue. In both culture platforms, exposure to E2 increased ER transactivation, increased proliferation, and induced ductal hyperplasia. When the surrounding matrix contained HMFs, the onset and severity of E2-induced ductal hyperplasia was increased due to decreased apoptosis. The reduced apoptosis may be due to fibroblasts modulating ER signaling in MCF7 cells, as suggested by the increased ER transactivation and reduced ER protein in MCF7 cells grown in co-culture. These findings demonstrate the utility of organotypic platforms when studying stromal:epithelial interactions, and add to existing literature that implicate the mammary microenvironment in ER + breast cancer progression.

Receptor levels are a key mechanism by which cells regulate their response to stimuli. The levels of estrogen receptor-α (ERα) impact breast cancer cell proliferation and are used to predict prognosis and sensitivity to endocrine therapy. Despite the clinical application of this information, it remains unclear how different cellular processes interact as a system to control ERα levels. To address this question, experimental results from the ERα-positive human breast cancer cell line (MCF-7) treated with 17-β-estradiol or vehicle control were used to develop a mass-action kinetic model of ERα regulation. Model analysis determined that RNA dynamics could be captured through phosphorylated ERα (pERα)-dependent feedback on transcription. Experimental analysis confirmed that pERα-S118 binds to the estrogen receptor-1 (ESR1) promoter, suggesting that pERα can feedback on ESR1 transcription. Protein dynamics required a separate mechanism in which the degradation rate for pERα was 8.3-fold higher than nonphosphorylated ERα. Using a model with both mechanisms, the root mean square error was 0.078. Sensitivity analysis of this combined model determined that while multiple mechanisms regulate ERα levels, pERα-dependent feedback elicited the strongest effect. Combined, our computational and experimental results identify phosphorylation of ERα as a critical decision point that coordinates the cellular circuitry to regulate ERα levels.—Tian, D., Solodin, N. M., Rajbhandari, P., Bjorklund, K., Alarid, E. T., Kreeger, P. K. A kinetic model identifies phosphorylated estrogen receptor-α (ERα) as a critical regulator of ERα dynamics in breast cancer.

Purpose Recent misclassification (false negative) incidents have raised awareness concerning limitations of immunohistochemistry (IHC) in assessment of estrogen receptor (ER) in breast cancer. Here we define a new method for standardization of ER measurement and then examine both change in percentage and threshold of intensity (immunoreactivity) to assess sources for test discordance. Methods An assay was developed to quantify ER by using a control tissue microarray (TMA) and a series of cell lines in which ER immunoreactivity was analyzed by quantitative immunoblotting in parallel with the automated quantitative analysis (AQUA) method of quantitative immunofluorescence (QIF). The assay was used to assess the ER protein expression threshold in two independent retrospective cohorts from Yale and was compared with traditional methods. Results Two methods of analysis showed that change in percentage of positive cells from 10% to 1% did not significantly affect the overall number of ER-positive patients. The standardized assay for ER on two Yale TMA cohorts showed that 67.9% and 82.5% of the patients were above the 2-pg/μg immunoreactivity threshold. We found 9.1% and 19.7% of the patients to be QIF-positive/IHC-negative, and 4.0% and 0.4% to be QIF-negative/IHC-positive for a total of 13.1% and 20.1% discrepant cases when compared with pathologists' judgment of threshold. Assessment of survival for both cohorts showed that patients who were QIF-positive/pathologist-negative had outcomes similar to those of patients who had positive results for both assays. Conclusion Assessment of intensity threshold by using a quantitative, standardized assay on two independent cohorts suggests discordance in the 10% to 20% range with current IHC methods, in which patients with discrepant results have prognostic outcomes similar to ER-positive patients with concordant results.

Nearly 75% of breast tumors express estrogen receptor (ER) and will be treated with endocrine therapies, such as tamoxifen or aromatase inhibitors. Despite their proven success, it is estimated that up to 50% of tumors fail on endocrine therapy and recur as aggressive therapy-resistant tumors. Therefore, preventing recurrence remains a major clinical problem. One hypothesis is that recurrences are driven by cells that survive therapeutic intervention. This poorly understood population is referred to as minimal residual disease that consists of drug-tolerant cells capable of surviving by entering a state of negligible growth. In trying to model tamoxifen tolerance and minimal residual disease in vitro, we have utilized the clonogenic assay where ER+ breast cancer cells' clonal growth and survival in the presence of tamoxifen is examined. Tamoxifen-tolerant cells show activation of the pro-inflammatory NFĸB pathway measured by an NFĸB-reporter, elevated expression of bona-fide target genes, and enhanced nuclear localization of p65 and p50, two major transcription factors of the NFĸB pathway. Tamoxifen does not directly activate the NFĸB pathway, but rather cells with high NFĸB activity proliferate independently of tamoxifen despite retaining ER expression. Mechanistically, NFĸB-dependent ER phosphorylation at S305 residue may contribute to failure to respond to tamoxifen. More importantly blocking NFĸB with multiple NFĸB inhibitors is sufficient to eradicate this population of tamoxifen-tolerant cells, suggesting NFĸB is required and can be exploited for therapy. We find that dimethyl fumarate, an FDA-approved anti-inflammatory and immune-modulatory drug, which we have established as an effective NFĸB inhibitor in breast cancer cells, prevents recurrence modeled both in vitro and in vivo. Altogether this work shows a novel role for the NFĸB pathway in promoting a population of cells that are tamoxifen tolerant and capable of seeding recurrent breast cancer disease. Furthermore, therapeutic targeting by an NFĸB inhibitor such as dimethyl fumarate can be used to eradicate residual disease and prevent breast cancer recurrence. Citation Format: Irida Kastrati, Svitlana D. Brovkovych, Joshua D. Stender, Elaine T. Alarid, Jonna Frasor. NFĸB pathway activation is a key determinant of tamoxifen tolerance and recurrence in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5242.

Estrogens promote cell proliferation and metastases in several human cancers. Here, we describe a different action of estrogens likely to contribute to tumor development-blocking immunosurveillance. In breast cancer cells, increasing concentrations of estrogen induce increasing levels of the granzyme B inhibitor, SerpinB9/proteinase inhibitor 9 (PI-9) and progressively block cell death induced by NK92 natural killer (NK) cells, but do not block killing by a second NK cell line, NKL cells. RNA interference knockdown of PI-9 abolishes estrogen's ability to block NK92 cell-induced cytotoxicity. Expressing elevated levels of estrogen receptor alpha (ERalpha) increases the induced level of PI-9, and makes tamoxifen (TAM), but not raloxifene or ICI 182,780, a potent inducer of PI-9. At elevated levels of ERalpha, induction of PI-9 by estradiol or TAM blocks killing by both NK92 and NKL cells. When the Erk pathway is activated with epidermal growth factor, the concentration of estrogen required to induce a protective level of PI-9 is reduced to 10 pM. Elevated concentrations of estrogen and ER may provide a dual selective advantage to breast cancer cells by controlling PI-9 levels and thereby blocking immunosurveillance. Expressing elevated levels of ERalpha reveals a potentially important difference in the effects of TAM, raloxifene and ICI 182,780 on immunosurveillance in breast cancer.

International audience; The ubiquitin-proteasome pathway has emerged as an important regulatory mechanism governing the activity of several transcription factors. While estrogen receptor alpha (ERalpha) is also subjected to rapid ubiquitin-proteasome degradation, the relationship between proteolysis and transcriptional regulation is incompletely understood. Based on studies primarily focusing on the C-terminal ligand-binding and AF-2 transactivation domains, an assembly of an active transcriptional complex has been proposed to signal ERalpha proteolysis that is in turn necessary for its transcriptional activity. Here, we investigated the role of other regions of ERalpha and identified S118 within the N-terminal AF-1 transactivation domain as an additional element for regulating estrogen-induced ubiquitination and degradation of ERalpha. Significantly, different S118 mutants revealed that degradation and transcriptional activity of ERalpha are mechanistically separable functions of ERalpha. We find that proteolysis of ERalpha correlates with the ability of ERalpha mutants to recruit specific ubiquitin ligases regardless of the recruitment of other transcription-related factors to endogenous model target genes. Thus, our findings indicate that the AF-1 domain performs a previously unrecognized and important role in controlling ligand-induced receptor degradation which permits the uncoupling of estrogen-regulated ERalpha proteolysis and transcription.

A common phenotype in breast cancer is the expansion of the estrogen receptor-alpha (ER+) cell population and an inappropriate elevation of ERalpha protein, the latter predisposing patients for a poorer prognosis than those with lower levels of the receptor. A tetracycline-inducible ERalpha overexpression model was developed in the MCF-7 cell line to assess induction of endogenous gene activation and growth in response to elevations in ERalpha protein. Heightened levels of ERalpha resulted in aberrant promoter occupancy and gene activation in the absence of hormone, which was independent of ligand and AF-2 function. This increased receptor activity required the amino-terminal A/B domain and was not inhibited by tamoxifen, which supports an enhancement of AF-1 function, yet was independent of serine-104, 106, and 118 phosphorylation. Ligand-independent transcription was accompanied by an increase in growth in the absence of hormonal stimulation. The results suggest that elevated levels of ERalpha in breast cancer cells can result in activation of receptor transcriptional function in a manner distinct from classical mechanisms that involve ligand binding or growth factor-induced phosphorylation. Further, they describe a potential mechanism whereby increases in ERalpha concentration may provide a proliferative advantage by augmenting ERalpha function regardless of ligand status.

Proteolysis by the 26S proteasome is an important regulatory mechanism that governs the protein stability of several steroid/nuclear receptors and that has been implicated in the control of receptor transcriptional activation function. Herein, we report that thyroid hormone can prevent estrogen-induced proteolysis of estrogen receptor-alpha (ERalpha) protein in lactotrope cells of the pituitary. The stabilization of ERalpha protein by thyroid hormone represents a selective blockade against estradiol-stimulated degradation, because thyroid hormone (but not glucocorticoid) can protect estrogen-activated ERalpha. Moreover, thyroid hormone treatment does not interfere with signal-induced proteolysis of a separate proteasome target, IkappaBalpha or ERalpha proteolysis induced by ICI182780. Using thyroid hormone as a tool to inhibit ERalpha proteolysis, we examined the effect of loss of this regulatory function on estrogen-induced transcriptional responses. Consistent with earlier reports, estrogen activation of an idealized estrogen response element reporter gene was inhibited. However, thyroid hormone did not prevent induction of prolactin gene expression or the ability of ERalpha to stimulate proliferation. These results demonstrate that estrogen-induced proteolysis of ERalpha is not a general requirement for receptor transcriptional activation function, and they demonstrate that proteolytic regulation is a means by which other endocrine factors can indirectly modulate ERalpha activity.

Estrogen receptor-alpha (ERalpha) is a major therapeutic target of hormonal therapies in breast cancer, and its expression in tumors is predictive of clinical response. Protein levels of ERalpha are tightly controlled by the 26S proteasome; yet, how the clinical proteasome inhibitor, bortezomib, affects ERalpha regulation has not been studied. Bortezomib selectively inhibits the chymotrypsin-like activity of the proteasome. Unlike other laboratory proteasome inhibitors, bortezomib failed to stabilize ERalpha protein at a dose exceeding 90% inhibition of the chymotrypsin-like activity. Unexpectedly, however, chronic bortezomib exposure caused a reduction of ERalpha levels in multiple ER+ breast cancer cell lines. This response can be explained by the fact that bortezomib induced a dramatic decrease in ERalpha mRNA because of direct transcriptional inhibition and loss of RNA polymerase II recruitment on the ERalpha gene promoter. Bortezomib treatment resulted in promoter-specific changes in estrogen-induced gene transcription that related with occupancy of ERalpha and RNA polymerase II (PolII) on endogenous promoters. In addition, bortezomib inhibited estrogen-dependent growth in soft agar. These results reveal a novel link between proteasome activity and expression of ERalpha in breast cancer and uncover distinct roles of the chymotrypsin-like activity of the proteasome in the regulation of the ERalpha pathway.

Basic fibroblast growth factor (bFGF) has been implicated as an embryonic mesoderm-inducing factor. It has also been detected in the mesodermally derived fetal and adult reproductive tracts. To address its possible role in the development of the reproductive tract, male and female fetal genital ridges and urogenital (UG) sinuses were transplanted bilaterally under the kidney capsule of syngeneic host rats; the transplants and the hosts were the same sex. Antiserum to bFGF (anti-bFGF) or normal rabbit serum (NRS) was infused into the right renal artery. The transplants on the left uninfused kidney served as internal controls. Upon vascularization, the transplants grow and differentiate essentially normally, but their overall growth after 14 days was reduced by about 40% compared to their growth in situ. Neutralization of endogenous bFGF with anti-bFGF resulted in approximately 41% inhibition of growth in the male genital ridge, but had no effect on the male UG sinus or any component of the female reproductive tract. The growth suppression of the male genital ridge was accompanied by an inhibition of the differentiation of the epididymis. After 7 days of treatment, development of the epididymis was impaired by 75%, and by 14 days, the organ was absent. Previous studies of fetal reproductive tract development have focused on the role of steroids and Mullerian inhibiting hormone. Our studies suggest that bFGF plays a role in the development of the male reproductive tract, especially the epididymis, and further suggest the possible role of this peptide growth factor in a steroid hormone-dependent process.

Proteasome-mediated proteolysis modulates the cellular concentration of estrogen receptor-alpha (ERalpha) and is induced by treatment of cells with 17beta-estradiol. Herein, we show that multiple receptor agonists, including 17alpha-estradiol and estriol as well as the antagonist ICI-182780, stimulate proteasome-dependent proteolysis of ERalpha in a process that requires ligand binding to the receptor. Proteolysis of receptor depends on ligand concentration, and there exists a direct correlation between ligand-binding affinity and the half-maximal dose of ligand required to stimulate receptor degradation. Furthermore, introduction of a point mutation into the receptor ligand-binding pocket yields a stable receptor resistant to proteolysis. Interestingly, although all ligands stimulate receptor degradation, the extent to which overall ER levels are affected varies with each ligand and is not related to ligand-binding affinity or activation of transcription. These results demonstrate ligand-specific regulation of ERalpha proteolysis, and they introduce the concept that cellular receptor concentration is governed not only at the level of induction of proteolysis but also by the efficiency with which the receptor is degraded.

Regulation of estrogen receptor (ER) concentration is a key component in limiting estrogen responsiveness in target cells. Yet the mechanisms governing ER concentration in the lactotrope cells of the anterior pituitary, a major site of estrogen action, are undetermined. In this study, we used a lactotrope cell line, PR1, to explore regulation of ER protein by estrogen. Estrogen treatment resulted in an approximate 60% decrease in ER steady state protein levels. Suprisingly, the decline in ER protein was apparent within 1 h of estrogen treatment and occurred in the absence of protein synthesis and transcription. Direct regulation of ER protein was further confirmed by pulse chase analysis, which showed that ER protein half-life was shortened from greater than 3 h to 1 h in the presence of estrogen. The estrogen-induced degradation of ER protein could be prevented by pretreatment with peptide aldehyde inhibitors of proteasome protease whereas inhibitors of calpain and lysosomal proteases were ineffective. Inhibition of proteasome activity maintained ER protein at a level equivalent to control cells not stimulated with estrogen but increased estrogen-binding activity by 1.75-fold. Proteolytic regulation of ER by the proteasome is not limited to pituitary lactotrope cells but is also operational in MCF-7 breast cancer cells, suggesting that this may be a common regulatory pathway used by estrogen. These studies describe a nongenomic action of estrogen that involves nuclear ER: rapid proteolysis of ER protein via a proteasome-mediated pathway.

Targeted expression of oncogenes in transgenic mice can immortalize specific cell types to serve as valuable cultured model systems. Utilizing promoter regions from pituitary genes activated in the gonadotrope/thyrotrope lineage at discrete stages of development, we have demonstrated that targeted oncogene expression in transgenic mice can produce cell lines representing sequential stages of differentiation. Each cell line expresses a specific subset of the genes denoting differentiated function such as the subunits of the glycoprotein hormones, hormone receptors, and transcriptional regulatory proteins. These model systems have allowed detailed investigations into molecular and cellular mechanisms otherwise inaccessible in vivo or in complex primary pituitary cell cultures.

Expression of the estrogen receptor-α (ERα) gene, ESR1, is a clinical biomarker used to predict therapeutic outcome of breast cancer. Hence, there is significant interest in understanding the mechanisms regulating ESR1 gene expression. Proteasome activity is increased in cancer and we previously showed that proteasome inhibition leads to loss of ESR1 gene expression in breast cancer cells. Expression of ESR1 mRNA in breast cancer cells is controlled predominantly through a proximal promoter within ∼400 base pair (bp) of the transcription start site (TSS). Here, we show that loss of ESR1 gene expression induced by the proteasome inhibitor bortezomib is associated with inactivation of a distal enhancer located 150 kilobases (kb) from the TSS. Chromatin immunoprecipitation assays reveal several bortezomib-induced changes at the distal site including decreased occupancy of three critical transcription factors, GATA3, FOXA1, and AP2γ. Bortezomib treatment also resulted in decreased histone H3 and H4 acetylation and decreased occupancy of histone acetyltransferase, p300. These data suggest a mechanism to explain proteasome inhibitor-induced loss of ESR1 mRNA expression that highlights the importance of the chromatin environment at the -150 kb distal enhancer in regulation of basal expression of ESR1 in breast cancer cells.

Breast cancer cell growth and therapeutic response are manipulated extrinsically by microenvironment signals. Despite recognition of the importance of the microenvironment in a variety of tumor processes, predictive measures that incorporate the activity of the surrounding cellular environment are lacking. In contrast, tumor cell biomarkers are well established in the clinic. Expression of Estrogen Receptor-alpha (ERα) is the primary defining feature of hormonally responsive tumors and is the molecular target of therapy in the most commonly diagnosed molecular subtype of breast cancer. While a number of soluble factors have been implicated in ERα activation, the complexity of signaling between the cellular microenvironment and the cancer cell implies multivariate control. The cumulative impact of the microenvironment signaling, which we define as microenvironmental activity, is more difficult to predict based on the sum of its parts. Here we tested the impact of an array of microenvironments on ERα signaling utilizing a microfluidic co-culture model. Quantitative immunofluorescence was employed to assess changes in ERα protein levels, combined with gene expression and phosphorylation status, as measures of activation. Analysis of microenvironment-induced growth under the same conditions revealed a previously undescribed correlation between growth and ERα protein down-regulation. These data suggest an expanded utility for the tumor biomarker ERα, in which the combination of dynamic regulation of ERα protein and growth in a breast cancer biosensor cell become a read-out of the microenvironmental activity.

Targeted expression of oncogenes in transgenic mice can immortalize specific cell types to serve as valuable cultured model systems. Utilizing promoter regions from a set of genes expressed at specific stages of differentiation in a given cell lineage, we demonstrate that targeted oncogenesis can produce cell lines representing sequential stages of development, in essence allowing both spatial and temporal immortalization. Our strategy was based on our production of a committed but immature pituitary gonadotrope cell line by directing expression of the oncogene SV40 T antigen using a gonadotrope-specific region of the human glycoprotein hormone α-subunit gene in transgenic mice. These cells synthesize α-subunit and gonadotropinreleasing hormone (GnRH) receptor, yet are not fully differentiated in that they do not synthesize the β-subunits of luteinizing hormone (LH) or follicle-stimulating hormone (FSH). This observation lead to the hypothesis that targeting oncogenesis with promoters that are activated earlier or later in development might immortalize cells that were more primitive or more differentiated, respectively. To test this hypothesis, we used an LHβ promoter to immortalize a cell that represents a subsequent stage of gonadotrope differentiation (expression of α-subunit, GnRH receptor, and LH β-subunit but not FSH β-subunit). Conversely, targeting oncogenesis with a longer fragment of the human α-subunit gene (which is activated earlier in development) resulted in the immortalization of a progenitor cell that is more primitive, expressing only the α-subunit gene. Interestingly, this transgene also immortalized cells of the thyrotrope lineage that express both α- and β-subunits of thyroid-stimulating hormone and the transcription factor GHF-1 (Pit-1). Thus, targeted tumorigenesis immortalizes mammalian cells at specific stages of differentiation and allows the production of a series of cultured cell lines representing sequential stages of differentiation in a given cell lineage.

Pituitary regulation of reproductive processes depends on the sensitivity of gonadotrope cells to both positive and negative regulators. Hypothalamic GnRH is the primary stimulus for gonadotropin synthesis and secretion. Therefore, the ability of the gonadotrope to respond to GnRH and the status of GnRH receptors (GnRH-R) are critical in the control of reproduction. In the present study, we address the role of GnRH and two second messenger activators, a phorbol ester (12-O-tetradecanoylphorbol-13-acetate) and forskolin, in the regulation of GnRH-R gene expression in the alpha T3-1 gonadotrope cell line. Using Northern blot analysis to monitor endogenous GnRH-R steady state messenger RNA (mRNA) levels, we found that although GnRH and 12-O-tetradecanoylphorbol-13-acetate do not change GnRH-R mRNA levels, forskolin causes a time-dependent decline. All three treatments stimulate glycoprotein alpha-subunit gene expression. To dissect the molecular mechanism of forskolin action on GnRH-R gene expression, de novo RNA synthesis was inhibited with the transcription inhibitor, actinomycin-D (act-D). Act-D alone does not change GnRH-R message levels. However, in the presence of both act-D and forskolin, GnRH-R mRNA levels decline dramatically. These data demonstrate that forskolin alters GnRH-R posttranscriptionally by destabilizing its mRNA. Our data do not, however, exclude possible direct transcriptional effects. This study suggests that activators of the protein kinase-A pathway may alter gonadotrope sensitivity to GnRH by decreasing GnRH-R gene expression and, therefore, indirectly affect reproductive status.

The quantification of mRNA is a ubiquitous and critical tool for understanding cellular mechanisms in cancer. While RT-PCR is often the endpoint, the success of the analysis depends not only on the PCR reaction, but also on an entire process flow linking living cells to the PCR endpoint. For cultured cells, this process flow includes the culture itself, cell lysis, mRNA extraction and purification, and RT-PCR. While much research has been targeted to streamlining and increasing throughput of the PCR process, the remainder of the process flow has largely been neglected. In this study, we link a new mechanism for purifying mRNA, Immiscible Filtration Assisted by Surface Tension (IFAST), with cell culture on a single platform. IFAST uses an immiscible liquid barrier (e.g., oil) to separate the cell culture / lysis region from an elution buffer. Using paramagnetic particles (PMPs) that selectively bind mRNA, we extracted mRNA from the lysate by using a magnet to draw the PMP-captured mRNA through the immiscible phase. This process, which takes only seconds, replaces multiple washing steps required by current mRNA isolation protocols, which typically take 15-60 minutes to complete. The simplicity of IFAST enables facile integration of culture and mRNA extraction on an easy-to-use chip that requires only a micropipette and magnet to operate. Once proof-of-concept was demonstrated with a single cell type, the platform was expanded to incorporate co-culture of two cell types, with breast cancer cells and stromal cells in separate compartments connected via diffusion ports to allow cytokine exchange. Each compartment has an IFAST device, such that mRNA can be collected independently from each side. RT-PCR expression levels from the integrated culture / IFAST platform had less variance than similar experiments with independent culture and mRNA purification components, possibly due to the elimination of error during transfer. mRNA purified with the integrated device had a yield and purity similar to “gold standard” kits. Breast cancer cells co-cultured with bone marrow stromal cells showed increased proliferation and morphological changes relative to breast cancer cells cultured alone. Additionally, mRNA extracted from these cells using the integrated device showed transcriptional changes consistent with estrogen response, even in hormone-independent conditions. We have developed a new platform linking compartmentalized cell co-culture with NA isolation. This technology simplifies and accelerates two processes ubiquitous in cancer biology, enabling the collection of additional endpoints with finite resources while reducing error associated with manipulation. Furthermore, co-cultures of breast cancer with stroma from a metastatic site induced proliferation and transcriptional signaling associated with pro-growth conditions, illustrating the utility of this platform for studying the metastatic microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4272. doi:1538-7445.AM2012-4272

Development of the seminal vesicle (SV) is elicited by androgens and is dependent on epithelial-mesenchymal interactions. Androgenic signal transmission from the androgen-receptor-positive mesenchyme to the epithelium has been postulated to involve paracrine factors. Keratinocyte growth factor (KGF), a member of the fibroblast growth factor family, is produced by stromal/mesenchymal cells and acts specifically on epithelial cells. The KGF transcript was detected by reverse transcription-polymerase chain reaction in newborn mouse SVs and by Northern blot analysis of RNA from cultured neonatal SV mesenchymal cells. Newborn SVs placed in organ culture undergo androgen-dependent growth and differentiation. Addition of a KGF-neutralizing monoclonal antibody to this system caused striking inhibition of both SV growth and branching morphogenesis. This inhibition was due to a decline in epithelial proliferation and differentiation, as the mesenchymal layer was not affected by anti-KGF treatment. When KGF (100 ng/ml) was substituted for testosterone in the culture medium, SV growth was approximately 50% that observed with an optimal dose of testosterone (10(-7) M). All of these findings suggest that KGF is present during a time of active SV morphogenesis and functions as an important mediator of androgen-dependent development.

The possibility that insulin-stimulated cartilage growth in hypophysectomized rats involves local production of insulin-like growth factor I (IGF-I) was investigated. Osmotic minipumps with attached catheters were used to infuse insulin into the right hindlimb of rats via the common iliac artery for 7 days starting 14 days after pituitary removal. The left, noninfused limb served as an internal control. Doses of insulin ranging from 0.25 to 50 mU/day caused significant increases in the tibial epiphyseal plate width (TEPW) of the infused limbs. The optimal dose of 1.25 mU/day increased the TEPW by 51 microns. Immunohistochemical analysis with an antiserum to human IGF-I showed that the growth response to the optimal dose was accompanied by the accumulation of IGF-I in the chondrocytes in the tibial plate. Infusion of insulin at a dose of 10 mU/day increased the TEPW by 18.6 +/- 3.0 microns. When the same dose of insulin was infused with the antiserum to human IGF-I, the growth response was completely nullified. These results indicate that cartilage cells in hypophysectomized rats are highly sensitive to the direct growth-promoting action of insulin. Furthermore, the growth effect appears to be mediated by or dependent on local production of IGF-I.

Introduction: Estrogen Receptor (ER) is arguably the most powerful predictive marker in breast cancer. However, a recent incident in Canada revealed a strikingly high false-negative rate and raised awareness of the current limitations in our measurement of ER. The current clinical standard is both subjective and qualitative. Even though guidelines are about to be issued to standardize this assay, there is very little data on the misclassification rate in current practice in the US.Hypothesis: Our hypothesis is that the use of a quantitative assay for ER on a series of retrospective collections may reveal both the level and significance of the misclassification rate.Method: Cell lines with a range of ER expression levels were analyzed by quantitative western blotting in parallel with IF/AQUA analysis (r2 = 0.865), in order to create standard curves for assessment of absolute ER protein concentration in tissue. The optimized assay was then used to quantify ER protein expression in a large cohort of archival breast cancer samples from Yale (1962-1982, n =617).Results: Using a set of standard curves with recombinant ER and cell line controls, we developed a standardized method for quantifying ER as an absolute concentration (pg ER per μg total protein) in formalin-fixed tissue on TMAs. This ER AQUA assay has a range in sensitivity from 50 pg/μg to 1500pg/μg total protein. Quantification of ER protein expression on the Yale archival cohort revealed a unimodal distribution with 49.8% of cases above the 50pg/ug threshold and thus defined as positive. When compared to pathologist performed conventional ER classification, we found a false negative rate of 6.65% and a false positive rate of 10.2%, for a total misclassification rate of 16.6%. Although no response data is available on that cohort, data is under analysis on 4 other cohorts with endocrine therapy treatment information, including an independent Yale cohort, SWOG 9313, NSABP B14, and the TEAM trial.Conclusion: We have developed a quantitative method to measure absolute levels of ER in breast tissue. Use of this assay on a series of cohorts suggests a misclassification rate in the 15% range. The significance of this level of misclassification with respect to response to endocrine therapy is currently under study. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4068.

This article highlights a recent study by Holst et al. in Nature Genetics that finds estrogen receptor-alpha (ER-alpha) amplification in early benign lesions and more advanced invasive carcinomas of the breast, and discusses the potential implications to our present understanding of the role of ER-alpha in breast tumorigenesis.