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Supplemental Figure 1. Tamoxifen treatment induces expression of NFκBB-related genes. Supplementary Figure 2. Immune cell composition and numbers in the tumor microenvironment do not significantly change upon tamoxifen treatment. Supplementary Figure 3. NFκB pathway and EMT/stemness factors in tamoxifen-tolerant persisters in T47D cells. Supplementary Figure 4. Expression of p65 and p50 proteins in MCF-7 CRISPR/Cas9 knock out clones. Supplementary Figure 5. NFκB pathway is not directly activated by tamoxifen. Supplementary Figure 6. Sorted NFκB+ vs NFκBâ^' or unsorted tamoxifen-tolerant cells based on GFP expression were reseeded for another round of clonogenic assay and the %GFP+ population was quantified in the second round.

Supplemental Table 3. GSEA enrichment plots

Supplemental Table 2. DE Genes

Supplemental Table 1. Patient characteristics.

Supplemental Materials and Methods and Figure Legends

Approximately 70% of breast cancers are estrogen receptor (ER) positive and can be treated with endocrine therapy. Unfortunately, 30-40% of tumors will recur, often as therapy-resistant metastatic disease. One contributor to disease recurrence is a population of cells with stem-like features (i.e. breast cancer stem cells, BCSCs). Previously, we have shown that positive crosstalk between two critical transcription factors, ER and NFκB, can contribute to the expansion of ER+ BCSCs, as indicated by increased mammosphere (MS) forming efficiency and BCSC markers. NFκB has been previously described as a major player in BCSC biology. However, the role of ER, as well as the interaction between ER and NFκB, in driving BCSCs remains unclear. We found in MS that both ER and NFκB are endogenously active and required for MS formation. To investigate whether ER and NFκB work together or independently, we made dual ERE and NFκB-RE reporter cell lines. We confirmed activation of both ER and NFκB in MS, but with a high degree of heterogeneity as indicated by 4 different cell populations (ERE+/NFκB-RE+, ERE+/NFκB-RE-, ERE-/NFκB-RE+ and ERE-/NFκB-RE-). Interestingly, we found >90% of MS arose from ERE+ cells while NFκB-RE+ cells failed to from MS, suggesting that ER but not NFκB activity was sufficient to initiate MS development. Moreover, ERE+ cells, with or without NFκB-RE activity, formed more secondary MS and expressed higher levels of stem cell-associated genes than ERE- cells, indicating ERE+ cells are more stem-like. To study ER and NFκB function in more detail, we utilized single cell RNA sequencing of MS. We identified 4 clusters with clusters 1 and 2 enriched for stem cell gene signatures. Bioinformatic analysis revealed Cluster 2 to be enriched for ER, proliferation, and DREAM complex signatures, and we confirmed these genes were enriched in ERE+ cells. However, we also found that ER activity is associated with activation of the DREAM complex in standard cell culture. This suggests that ER regulation of proliferation via the DREAM complex may not be specific for BCSCs. In contrast to Cluster 2, Cluster 1 was enriched with ER and NFκB signatures, as well as an ER-NFκB crosstalk signature. In addition, Cluster 1 was enriched with pathways related to adaptive stress response such as: oxidative phosphorylation, unfolded protein response, and protein secretion. As some studies have reported activation of stress pathways is associated with maintenance of stemness, we hypothesize that ER and NFκB may work together to promote stem features through activation of multiple stress pathways to increase survival and growth in an anchorage-independent environment. To determine whether either Cluster 1 or Cluster 2 derived signatures are associated with clinical outcome, we first interrogated single cell RNA sequencing data from a metastatic ER+ PDX model. The Cluster 2 signature was enriched in proliferative cell populations found in both primary and metastatic tumors. In contrast, the Cluster 1 signature was enriched only in a metastatic tumor cell population. We also showed that expression of signatures derived from Cluster 1 or 2 in ER+ breast cancer patient tumors predict poor relapse free survival in the METABRIC dataset. However, only co-expression of both signatures predicted poor disease-free survival. Taken together, our findings suggest there are two unique subpopulations of stem-like cells, one of which is dependent on ER driven proliferation, whereas the other population relies on an ER-NFkB mediated stress response for survival and self-renewal. Furthermore, both of these population signatures in patients’ tumors are associated with more aggressive disease and poor outcome. Citation Format: Svetlana E. Semina, Mark Maienschein-Cline, Elaine T. Alarid, Carol A. Sartorius, Jonna Frasor. Identification of novel ER and ER-NFκB driven stem-like cell populations in ER+ breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-11-01.

Context Positron emission tomography imaging with 2-deoxy-2-[18F]-fluoro-D-glucose (FDG) is used clinically for initial staging, restaging, and assessing therapy response in breast cancer. Tumor FDG uptake in steroid hormone receptor–positive breast cancer and physiologic FDG uptake in normal breast tissue can be affected by hormonal factors such as menstrual cycle phase, menopausal status, and hormone replacement therapy. Objective The purpose of this study was to determine the role of the progesterone receptor (PR) in regulating glucose and FDG uptake in breast cancer cells. Methods and Results PR-positive T47D breast cancer cells treated with PR agonists had increased FDG uptake compared with ethanol control. There was no significant change in FDG uptake in response to PR agonists in PR-negative MDA-MB-231 cells, MDA-MB-468 cells, or T47D PR knockout cells. Treatment of T47D cells with PR antagonists inhibited the effect of R5020 on FDG uptake. Using T47D cell lines that only express either the PR-A or the PR-B isoform, PR agonists increased FDG uptake in both cell types. Experiments using actinomycin D and cycloheximide demonstrated the requirement for both transcription and translation in PR regulation of FDG uptake. GLUT1 and PFKFB3 mRNA expression and the enzymatic activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were increased after progestin treatment of T47D cells. Conclusion Thus, progesterone and progestins increase FDG uptake in T47D breast cancer cells through the classical action of PR as a ligand-activated transcription factor. Ligand-activated PR ultimately increases expression and activity of proteins involved in glucose uptake, glycolysis, and the pentose phosphate pathway.

Phosphorylation of estrogen receptor α (ER) at serine 118 (pS118-ER) is induced by estrogen and is the most abundant posttranslational mark associated with a transcriptionally active receptor. Cistromic analysis of pS118-ER from our group revealed enrichment of the GRHL2 motif near pS118-ER binding sites. In this study, we used cistromic and transcriptomic analyses to interrogate the relationship between GRHL2 and pS118-ER. We found that GRHL2 is bound to chromatin at pS118-ER/GRHL2 co-occupancy sites prior to ligand treatment, and GRHL2 binding is required for maximal pS118-ER recruitment. pS118-ER/GRHL2 co-occupancy sites were enriched at active enhancers marked by H3K27ac and H3K4me1, along with FOXA1 and p300, compared to sites where each factor binds independently. Transcriptomic analysis yielded four subsets of ER/GRHL2-coregulated genes revealing that GRHL2 can both enhance and antagonize E2-mediated ER transcriptional activity. Gene ontology analysis indicated that coregulated genes are involved in cell migration. Accordingly, knockdown of GRHL2, combined with estrogen treatment, resulted in increased cell migration but no change in proliferation. These results support a model in which GRHL2 binds to selected enhancers and facilitates pS118-ER recruitment to chromatin, which then results in differential activation and repression of genes that control estrogen-regulated ER-positive breast cancer cell migration.

The fibril orientation of type I collagen has been shown to contribute to tumor invasion and metabolic changes. Yet, there is limited information about its impact on tumor cells' behavior in a restrictive growth environment. Restrictive growth environments are generated by the inhibition of a proliferation stimulus during therapy or as an inflammatory response to suppress tumor expansion. In this study, the impact of a type I collagen matrix orientation and fibrous architecture on cell proliferation and response to estrogen receptor (ER) therapy were examined using estrogen-dependent breast tumor cells (MCF-7 and T-47D) cultured in a hormone-restricted environment. The use of hormone-free culture media, as well as pharmacological inhibitors of ER, Tamoxifen, and Fulvestrant, were investigated as hormone restrictive conditions. Examination of cultures at 72 h showed that tumor cell proliferation was significantly stimulated (1.8-fold) in the absence of hormones on collagen fibrous substrates, but not on polycaprolactone fibrous substrates of equivalent orientation. ER inhibitors did not suppress cell proliferation on collagen fibrous substrates. The examination of reporter cells for ER signaling showed a lack of activity, thus confirming a shift toward an ER-independent proliferation mechanism. Examination of two selective inhibitors of α2β1 and α1β1 integrins showed that cell proliferation is suppressed in the presence of the α2β1 integrin inhibitor only, thereby indicating that the observed changes in tumor cell behavior are caused by a combination of integrin signaling and/or an intrinsic structural motif that is uniquely present in the collagen fibrils. Adjacent coculture studies on collagen substrates showed that tumor cells on collagen can stimulate the proliferation of cells on tissue culture plastic through soluble factors. The magnitude of this effect correlated with the increased surface anisotropy of the substrate. This sensing in fibril orientation was further supported by a differential expression pattern of secreted proteins that were identified on random and aligned orientation substrates. Overall, this study shows a new role for electrospun collagen I fibrous substrates by supporting a shift toward an ER-independent tumor cell proliferation mechanism in ER+ breast tumor cells.

Phosphorylation of estrogen receptor α (ER) at serine 118 (pS118-ER) is induced by estrogen and is the most abundant post-translational mark associated with a transcriptionally active receptor. Cistromic analysis of pS118-ER from our group found enrichment of the GRHL2 motif near pS118-ER binding sites. In this report we use cistromic and transcriptomic analyses to interrogate the relationship between GRHL2 and pS118-ER. We found that GRHL2 is bound to chromatin at pS118-ER/GRHL2 co-occupancy sites prior to ligand treatment, and GRHL2 binding is required for maximal pS118-ER recruitment. pS118-ER/GRHL2 co-occupancy sites were enriched at active enhancers marked by H3K27ac and H3K4me1, along with FOXA1 and p300. Transcriptomic analysis yielded four subsets of ER/GRHL2 co-regulated genes revealing that GRHL2 can both enhance and antagonize E2-mediated ER transcriptional activity. Gene ontology analysis identified several coregulated genes involved in cell migration. Accordingly, knockdown of GRHL2 combined with estrogen treatment resulted in increased cell migration but no change in proliferation. These results support a model in which GRHL2 binds to select enhancers and facilitates pS118-ER recruitment to chromatin which then results in differential activation and repression of genes that control ER-positive breast cancer cell migration.

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.

Transcriptional regulation of ESR1, the gene that encodes for estrogen receptor α (ER), is critical for regulating the downstream effects of the estrogen signaling pathway in breast cancer such as cell growth. ESR1 is a large and complex gene that is regulated by multiple regulatory elements, which has complicated our understanding of how ESR1 expression is controlled in the context of breast cancer. Early studies characterized the genomic structure of ESR1 with subsequent studies focused on identifying intrinsic (chromatin environment, transcription factors, signaling pathways) and extrinsic (tumor microenvironment, secreted factors) mechanisms that impact ESR1 gene expression. Currently, the introduction of genomic sequencing platforms and additional genome-wide technologies has provided additional insight on how chromatin structures may coordinate with these intrinsic and extrinsic mechanisms to regulate ESR1 expression. Understanding these interactions will allow us to have a clearer understanding of how ESR1 expression is regulated and eventually provide clues on how to influence its regulation with potential treatments. In this review, we highlight key studies concerning the genomic structure of ESR1, mechanisms that affect the dynamics of ESR1 expression, and considerations towards affecting ESR1 expression and hormone responsiveness in breast cancer.

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.

Post-translational modifications are key regulators of protein function, providing signals that alter protein interactions and activity. Phosphorylation of estrogen receptor-α (ER) at serine 118 (pS118-ER) occurs in response to multiple stimuli and is involved in modulating ER-dependent gene transcription. While the effects of pS118 on ER-DNA interactions have been investigated at specific genomic sites, the genome-wide binding profile (cistrome) of pS118-ER remains to be studied. To determine and characterize the pS118-ER cistrome, chromatin immunoprecipitation sequencing (ChIP-seq) was performed on both pS118-ER and ER and their binding profiles were compared. A subset of ER sites was found to be occupied by pS118-ER and although these sites were not found to be enriched in promoter regions relative to all ER sites, the pS118-ER sites were associated with the active enhancer mark H3K27ac as well as genes upregulated, but not downregulated, by estrogen. Additionally, pS118-ER sites were found to be enriched in the DNA binding motif for GRHL2 as well as the estrogen response element (ERE) relative to all ER sites. Utilizing a DNA binding microarray, pS118-ER was found to be more commonly associated with direct DNA binding events compared to indirect binding events. These results suggest a role for pS118-ER at active enhancers and as a regulator of direct ER-DNA interactions. Prevention of this phosphorylation event could serve as a potential treatment for ER-positive breast cancers by reducing the transcriptional activity of ER and decreasing its occupancy on DNA.

Posttranslational modifications are key regulators of protein function, providing cues that can alter protein interactions and cellular location. Phosphorylation of estrogen receptor α (ER) at serine 118 (pS118-ER) occurs in response to multiple stimuli and is involved in modulating ER-dependent gene transcription. While the cistrome of ER is well established, surprisingly little is understood about how phosphorylation impacts ER-DNA binding activity. To define the pS118-ER cistrome, chromatin immunoprecipitation sequencing was performed on pS118-ER and ER in MCF-7 cells treated with estrogen. pS118-ER occupied a subset of ER binding sites which were associated with an active enhancer mark, acetylated H3K27. Unlike ER, pS118-ER sites were enriched in GRHL2 DNA binding motifs, and estrogen treatment increased GRHL2 recruitment to sites occupied by pS118-ER. Additionally, pS118-ER occupancy sites showed greater enrichment of full-length estrogen response elements relative to ER sites. In an in vitro DNA binding array of genomic binding sites, pS118-ER was more commonly associated with direct DNA binding events than indirect binding events. These results indicate that phosphorylation of ER at serine 118 promotes direct DNA binding at active enhancers and is a distinguishing mark for associated transcription factor complexes on chromatin.

The Grainyhead-like protein family, composed of GRHL1, GRHL2, and GRHL3, are nuclear transcription factors that regulate epithelial differentiation. GRHL2 has been associated with several nuclear hormone receptors, including progesterone receptor (PR), androgen receptor (AR), and more recently, estrogen receptor (ER). In breast cancer, GRHL2 has been shown to both activate ER-dependent enhancers through FOXA1 and MLL3-mediated deposition of the activating histone mark H3K4me1 and repress enhancers via inhibition of the histone acetyltransferase p300. Cistromic analysis by our group of ER phosphorylated at serine 118 (pS118-ER), a form of transcriptionally active ER, found an enrichment of the GRHL motif near pS118-ER binding sites. Despite these findings, the direct relationship between GRHL2 and ER transcriptional function and how that relationship influences ER-positive breast cancer growth and differentiation is not well-defined. To explore the relationship between GRHL2 and pS118-ER further, we used transcriptomic and cistromic analysis of ER-positive cells lacking GRHL2 to determine the impact of the loss of GRHL2 on cellular and transcriptional responses to estrogen. This analysis identified a subset of genes that are controlled by both GRHL2 and estrogen. In addition, CRISPR engineered T47D cells lacking a portion of the GRHL2 transactivation domain (ΔTAD) demonstrate reduced nuclear ER levels and reduced ER chromatin occupancy. Gene expression analysis of ΔTAD-GRHL2 cells showed increased GRHL3 expression, and ChIP analysis revealed increased Pol II occupancy at the GRHL3 promoter, suggesting that there may be a compensatory mechanism within the GRHL family to regulate the transcriptome. Finally, ΔTAD-GRHL2 mutants reduced growth and colony formation relative to wild-type controls. Together, this work will provide an understanding of how transcriptionally active ER and GRHL2 selectively cooperate to regulate transcription, growth, and differentiation in ER-positive 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.

The nuclear receptor superfamily is a group of transcriptional regulators that control multiple aspects of both physiology and pathology, and are broadly recognized as viable therapeutic targets. While receptor-modulating drugs have been successful in many cases, the discovery of new drug targets is still an active area of research, because resistance to nuclear receptor-targeting therapies remains a significant clinical challenge. Many successful targeted therapies have harnessed the control of receptor activity by targeting events within the nuclear receptor signaling pathway. In this review, we explore the role of nuclear receptor ubiquitylation and discuss how the expanding roles of ubiquitin might be leveraged to identify additional entry points to control receptor function for future therapeutic development.

The transcriptional activity of estrogen receptor α (ERα), the key driver of breast cancer proliferation, is enhanced by multiple cellular interactions, including phosphorylation-dependent interaction with Pin1, a proline isomerase, which mediates cis-trans isomerization of the N-terminal Ser(P)118-Pro119 in the intrinsically disordered AF1 (activation function 1) domain of ERα. Because both ERα and Pin1 have multiple cellular partners, it is unclear how Pin1 assists in the regulation of ERα transactivation mechanisms and whether the functional effects of Pin1 on ERα signaling are direct or indirect. Here, we tested the specific action of Pin1 on an essential step in ERα transactivation, binding to specific DNA sites. DNA binding analysis demonstrates that stable overexpression of Pin1 increases endogenous ERα DNA binding activity when activated by estrogen but not by tamoxifen or EGF. Increased DNA binding affinity is a direct effect of Pin1 on ERα because it is observed in solution-based assays with purified components. Further, our data indicate that isomerization is required for Pin1-modulation of ERα-DNA interactions. In an unbiased in vitro DNA binding microarray with hundreds of thousands of permutations of ERα-binding elements, Pin1 selectively enhances the binding affinity of ERα to consensus DNA elements. These studies reveal that Pin1 isomerization of phosphorylated ERα can directly regulate the function of the adjacent DNA binding domain, and this interaction is further modulated by ligand binding in the ligand-binding domain, providing evidence for Pin1-dependent allosteric regulation of ERα function. Background: Estrogen receptor α (ERα) activity in breast cancer cells is increased by Pin1, an isomerase that alters protein conformation. Results: Pin1-mediated isomerization increases ERα DNA binding affinity. Conclusion: Pin1 allosterically regulates ERα DNA binding directly via isomerization of phosphorylated receptor. Significance: Pin1 regulation of ERα provides a framework for understanding regulation by intrinsically disordered domains on transcription factor function.

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.

While biochemistry evidently affects the growth rate of cells, many biochemists routinely ignore population variation, just like population geneticists usually ignore causal details of biochemistry that underpin a change in growth rate caused by a mutation. A true EvoSysBio integration requires an explicit mechanism for how molecular reaction rates affect the reproduction rates that determine the fitness of an organism. Here we simulate a very simple and completely explicit Continuous-Time Markov Chain (CTMC) model of cancer cells whose growth rate is affected by the biochemical equilibrium between two molecular complexes. Approximately 70% of breast cancers are of a type that overexpress Estrogen Receptor-alpha (ERα). Cell growth in this type of cancer is inhibited by hormonal therapies that antagonize ERα function as a transcription factor. ERα is encoded by the ESR1 gene, which itself is a target of ERα mediated transcription. When activated by estrogen, ERα binds to the ESR1 promoter, repressing new synthesis of ERα protein. Estrogen binding also induces pathways that lead to degradation of ERα protein. This negative feedback loop is finely tuned to natural levels of estrogen and results in natural levels of growth. In breast cancer, the system is thrown off its natural course such that increased levels of ERα induce levels of cell growth that can lead to cancer. Thus, both genetic changes to the ESR1 promoter, ERα protein degradation, and biochemical changes in estrogen metabolism can effectively cause changes in cell growth rates, which can be seen as the ‘fitness’ of a cancer cell. Predicting cancer cell growth in this system raises a conceptual multi-level simulation problem, because the molecular aspects of this model need to compute the biochemistry in a way that influences growth rates at the cellular level, without resetting growth at each cell division. We present progress towards addressing this simulation challenge in pure mass-action models, which we implemented using the Evolvix model description language. We found that such models can be constructed in more than one way. We explored some candidate model properties that could aid efforts to develop abstractions for more efficiently simulating the common multi-level modeling problems behind many important biological questions. These efforts are ongoing and aim to find efficient ways of encoding and exploring such models in silico. In particular, we are investigating how architecting a new compiler for a general-purpose programming language for biology could improve the efficiency of analyzing the dynamic multi-level simulation scenarios that characterize many questions in EvoSysBio. Progress can be followed at http://evolvix.org.

Heterotypic interactions in cancer microenvironments play important roles in disease initiation, progression, and spread. Co-culture is the predominant approach used in dissecting paracrine interactions between tumor and stromal cells, but functional results from simple co-cultures frequently fail to correlate to in vivo conditions. Though complex heterotypic in vitro models have improved functional relevance, there is little systematic knowledge of how multi-culture parameters influence this recapitulation. We therefore have employed a more iterative approach to investigate the influence of increasing model complexity; increased heterotypic complexity specifically. Here we describe how the compartmentalized and microscale elements of our multi-culture device allowed us to obtain gene expression data from one cell type at a time in a heterotypic culture where cells communicated through paracrine interactions. With our device we generated a large dataset comprised of cell type specific gene-expression patterns for cultures of increasing complexity (three cell types in mono-, co-, or tri-culture) not readily accessible in other systems. Principal component analysis indicated that gene expression was changed in co-culture but was often more strongly altered in tri-culture as compared to mono-culture. Our analysis revealed that cell type identity and the complexity around it (mono-, co-, or tri-culture) influence gene regulation. We also observed evidence of complementary regulation between cell types in the same heterotypic culture. Here we demonstrate the utility of our platform in providing insight into how tumor and stromal cells respond to microenvironments of varying complexities highlighting the expanding importance of heterotypic cultures that go beyond conventional co-culture.

Estrogen receptor alpha (ERα), a key driver of growth in the majority of breast cancers, contains an unstructured transactivation domain (AF1) in its N terminus that is a convergence point for growth factor and hormonal activation. This domain is controlled by phosphorylation, but how phosphorylation impacts AF1 structure and function is unclear. We found that serine 118 (S118) phosphorylation of the ERα AF1 region in response to estrogen (agonist), tamoxifen (antagonist), and growth factors results in recruitment of the peptidyl prolyl cis/trans isomerase Pin1. Phosphorylation of S118 is critical for Pin1 binding, and mutation of S118 to alanine prevents this association. Importantly, Pin1 isomerizes the serine118-proline119 bond from a cis to trans isomer, with a concomitant increase in AF1 transcriptional activity. Pin1 overexpression promotes ligand-independent and tamoxifen-inducible activity of ERα and growth of tamoxifen-resistant breast cancer cells. Pin1 expression correlates with proliferation in ERα-positive rat mammary tumors. These results establish phosphorylation-coupled proline isomerization as a mechanism modulating AF1 functional activity and provide insight into the role of a conformational switch in the functional regulation of the intrinsically disordered transactivation domain of ERα.

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.

The selective isolation of a sub-population of cells from a larger, mixed population is a critical preparatory process to many biomedical assays. Here, we present a new cell isolation platform with a unique set of advantages over existing devices. Our technology, termed Immiscible Filtration Assisted by Surface Tension, exploits physical phenomena associated with the microscale to establish fluidic barriers composed of immiscible liquids. By attaching magnetically-responsive particles to a target cell population via immunocapture, we can selectively transport this population across the immiscible barrier and into a separate aqueous solution. The high interfacial energy associated with the immiscible phase / aqueous phase boundaries prevents unwanted cells or other contaminants from inadvertently crossing the immiscible phase. We have demonstrated, using fluorescent particles, stromal cells, and whole blood as “background”, that we can successfully isolate ~70% of a target breast cancer cell population with an average purity of >80%. Increased purity was obtained by coupling two immiscible barriers in series, a modification that only slightly increases operational complexity. Furthermore, several samples can be processed in parallel batches in a near-instantaneous manner without the requirement of any washing, which can cause dilution (negative selection) or significant uncontrolled loss (positive selection) of target cells. Finally, cells were observed to remain viable and proliferative following traverse through the immiscible phase, indicating that this process is suitable for a variety of downstream assays, including those requiring intact living cells.

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.

Estrogen receptor alpha (ER) is a member of the nuclear hormone receptor family, which upon binding estrogen shows increased apparent affinity for nuclear components (tight nuclear binding). The nuclear components that mediate this tight nuclear binding have been proposed to include both ER-DNA interactions and ER-protein interactions. In this paper, we demonstrate that tight nuclear binding of ER upon estrogen occupation requires ER-DNA interactions. Hormone-bound ER can be extracted from the nucleus in low-salt buffer using various polyanions, which mimic the phosphate backbone of DNA. The importance of specific ER-DNA interactions in mediating tight nuclear binding is also supported by the 380-fold lower concentration of the ERE oligonucleotide necessary to extract estrogen-occupied ER from the nucleus compared to the polyanions. We also demonstrate that estrogen-induced tight nuclear binding requires both the nuclear localization domain and the DNA binding domain of ER. Finally, enzymatic degradation of nuclear DNA allows us to recover 45% of tight nuclear-bound ER. We further demonstrate that ER-AIB1 interaction is not required for estrogen-induced tight nuclear binding. Taken together, we propose a model in which tight nuclear binding of the estrogen-occupied ER is predominantly mediated by ER-DNA interactions. The effects of estrogen binding on altering DNA binding in whole cells are proposed to occur through estrogen-induced changes in ER-chaperone protein interactions, which alter the DNA accessibility of ER but do not directly change the affinity of the ER for DNA, which is similar for both unoccupied and occupied ER.

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.

Down-regulation of receptor in response to ligand was one of the earliest functional readouts of steroid hormone action. The loss of total receptor content upon stimulation, referred to initially as receptor "processing," was carefully described with respect to receptor nuclear transformation or tight nuclear binding. It was these early studies that were the first to note a correlation between receptor turnover and induction of gene transcription, leading to the proposal that down-regulation of receptor was involved in mechanisms of transcriptional activation. This idea has now attracted renewed attention with the discovery that ligand-induced "processing" in the form of proteolysis is carried out by the 26S proteasome, a multicatalytic enzyme whose activity is directly coupled to cell-cycle control, signal transduction, and importantly, transcription. Here, we review our current understanding of the mechanism and relevance of proteolysis to receptor function based on general concepts that have emerged from analyses of liganded members of the nuclear receptor family.

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.

Co-culture of multiple cell types within a single device enables the study of paracrine signaling events. However, extracting gene expression endpoints from co-culture experiments is laborious, due in part to pre-PCR processing of the sample (i.e., post-culture cell sorting and nucleic acid purification). Also, a significant loss of nucleic acid may occur during these steps, especially with microfluidic cell culture where lysate volumes are small and difficult to access. Here, we describe an integrated platform for performing microfluidic cell culture and extraction of mRNA for gene expression analysis. This platform was able to recover 30-fold more mRNA than a similar, non-integrated system. Additionally, using a breast cancer/bone marrow stroma co-culture, we recapitulated stromal-dependent, estrogen-independent growth of the breast cancer cells, coincident with transcriptional changes. We anticipate that this platform will be used for streamlined analysis of paracrine signaling events as well as for screening potential drugs and/or patient samples.

Estrogen receptor-alpha (ERα) is an important biomarker used to classify and direct therapy decisions in breast cancer. Both ERα protein and its transcript, ESR1, are used to predict response to tamoxifen therapy, yet certain tumors have discordant levels of ERα protein and ESR1, which is currently unexplained. Cellular ERα protein levels can be controlled post-translationally by the ubiquitin-proteasome pathway (UPP) through a mechanism that depends on phosphorylation at residue S118. Phospho-S118 (pS118-ERα) is a substrate for the peptidyl prolyl isomerase, Pin1, which mediates cis-trans isomerization of the pS118-P119 bond to enhance ERα transcriptional function. Here, we demonstrate that Pin1 can increase ERα protein without affecting ESR1 transcript levels by inhibiting proteasome-dependent receptor degradation. Pin1 disrupts ERα ubiquitination by interfering with receptor interactions with the E3 ligase, E6AP, which also is shown to bind pS118-ERα. Quantitative in situ assessments of ERα protein, ESR1, and Pin1 in human tumors from a retrospective cohort show that Pin1 levels correlate with ERα protein but not to ESR1 levels. These data show that ERα protein is post-translationally regulated by Pin1 in a proportion of breast carcinomas. Since Pin1 impacts both ERα protein levels and transactivation function, these data implicate Pin1 as a potential surrogate marker for predicting outcome of ERα-positive breast cancer.

The extraction and purification of nucleic acids from complex samples (e.g. blood, biopsied tissue, cultured cells, food) is an essential prerequisite for many applications in biology including genotyping, transcriptional analysis, systems biology, epigenetic analysis, and virus/bacterial detection. In this report, we describe a new process of nucleic acid extraction that utilizes “pinned” aqueous/organic liquid interfaces in microchannels to streamline the extraction mechanism, replacing all washing steps with a single traverse of an immiscible fluid barrier, termed Immiscible Filtration Assisted by Surface Tension (IFAST). Nucleic acids in biological samples are bound to paramagnetic particles and then drawn across the IFAST device (or array of IFAST devices) using a magnet. While the strength of the IFAST barrier is suitable for separation of nucleic acids from lysate in its current embodiment, its permeability can be selectively adapted by adjusting the surface tensions/energies associated with the cell lysate, the immiscible phase, and the device surface, enabling future expansion to other non-nucleic acid applications. Importantly, processing time is reduced from 15–45 minutes to less than 5 minutes while maintaining purity, yield, and scalability equal to or better than prevailing methods. Operation is extremely simple and no additional lab infrastructure is required. The IFAST technology thus significantly enhances researchers' abilities to isolate and analyze nucleic acids, a process which is critical and ubiquitous in an extensive array of scientific fields.

Background Sin3A is an evolutionarily conserved transcriptional repressor which regulates gene expression as part of the multi-protein Sin3 repressive complex. It functions as a scaffold upon which proteins with enzymatic activity dock, including chromatin modifying histone deacetylases. Although regulation of transcription by Sin3A has been studied in detail, little is understood about the function of Sin3A in cancer cells. We previously showed that Sin3A is expressed in breast cancer cells and is a repressor of estrogen receptor-alpha (ERα, ESR1) gene expression. Here, we expand our previous studies to elucidate the function of Sin3A in the control of gene expression and growth of breast cancer cells. Results Analysis of gene expression following knockdown of Sin3A revealed changes in both basal and regulated gene transcription. Genes of known importance in breast cancer and estrogen signaling, including ERBB2, PGR, MYC, CLU, and NCOA2, were among those identified as Sin3A-responsive. The mechanism of Sin3A action varied among genes and was found to be mediated through both HDAC1/2 -dependent and -independent activities. Loss of Sin3A inhibited breast cancer cell growth by increasing apoptosis without affecting cell cycle progression. Analysis of both ERα-positive and ERα-negative cell lines revealed that the effects of Sin3A on growth were cell-type specific, as Sin3A expression promoted maximum growth of only the ERα-positive cells, and, notably, Sin3A protein itself was increased by estrogen. Further gene expression experiments revealed that Sin3A repressed expression of key apoptotic genes, including TRAIL, TRAILR1, CASP10, and APAF1, in ERα-positive, but not ERα-negative, cell lines, which could provide a mechanistic explanation for cell-type differences in growth. Conclusions This study identifies Sin3A as a regulator of gene expression, survival, and growth in ERα-positive breast cancer cells. Sin3A regulates the transcription of genes involved in breast cancer and apoptosis and acts through multiple mechanisms not limited to histone deacetylase function. These findings reveal previously undescribed functions of Sin3A in breast cancer and provide evidence for an important role of this transcriptional repressor in ERα-positive tumor cell growth.

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.

Gene expression results from the coordinated actions of transcription factor proteins and coregulators. Estrogen receptor alpha (ERalpha) is a ligand-activated transcription factor that can both activate and repress the expression of genes. Activation of transcription by estrogen-bound ERalpha has been studied in detail, as has antagonist-induced repression, such as that which occurs by tamoxifen. How estrogen-bound ERalpha represses gene transcription remains unclear. In this report, we identify a new mechanism of estrogen-induced transcriptional repression by using the ERalpha gene, ESR1. Upon estrogen treatment, ERalpha is recruited to two sites on ESR1, one distal (ENH1) and the other at the proximal (A) promoter. Coactivator proteins, namely, p300 and AIB1, are found at both ERalpha-binding sites. However, recruitment of the Sin3A repressor, loss of RNA polymerase II, and changes in histone modifications occur only at the A promoter. Reduction of Sin3A expression by RNA interference specifically inhibits estrogen-induced repression of ESR1. Furthermore, an estrogen-responsive interaction between Sin3A and ERalpha is identified. These data support a model of repression wherein actions of ERalpha and Sin3A at the proximal promoter can overcome activating signals at distal or proximal sites and ultimately decrease gene expression.

Polydimethylsiloxane (PDMS) has become a staple of the microfluidics community by virtue of its simple fabrication process and material attributes, such as gas permeability, optical transparency, and flexibility. As microfluidic systems are put toward biological problems and increasingly utilized as cell culture platforms, the material properties of PDMS must be considered in a biological context. Two properties of PDMS were addressed in this study: the leaching of uncured oligomers from the polymer network into microchannel media, and the absorption of small, hydrophobic molecules (i.e. estrogen) from serum-containing media into the polymer bulk. Uncured PDMS oligomers were detectable via MALDI-MS in microchannel media both before and after Soxhlet extraction of PDMS devices in ethanol. Additionally, PDMS oligomers were identified in the plasma membranes of NMuMG cells cultured in PDMS microchannels for 24 hours. Cells cultured in extracted microchannels also contained a detectable amount of uncured PDMS. It was shown that MCF-7 cells seeded directly on PDMS inserts were responsive to hydrophilic prolactin but not hydrophobic estrogen, reflecting its specificity for absorbing small, hydrophobic molecules; and the presence of PDMS floating in wells significantly reduced cellular response to estrogen in a serum-dependent manner. Quantification of estrogen via ELISA revealed that microchannel estrogen partitioned rapidly into the surrounding PDMS to a ratio of approximately 9:1. Pretreatments such as blocking with serum or pre-absorbing estrogen for 24 hours did not affect estrogen loss from PDMS-based microchannels. These findings highlight the importance of careful consideration of culture system properties when determining an appropriate environment for biological experiments.