RESUMEN
Estrogen Receptor alpha (ERα) plays a major role in most breast cancers, and it is the target of endocrine therapies used in the clinic as standard of care for women with breast cancer expressing this receptor. The two methods ChIP-seq (chromatin immunoprecipitation coupled with deep sequencing) and RIME (Rapid Immunoprecipitation of Endogenous Proteins) have greatly improved our understanding of ERα function during breast cancer progression and in response to anti-estrogens. A critical component of both ChIP-seq and RIME protocols is the antibody that is used against the bait protein. To date, most of the ChIP-seq and RIME experiments for the study of ERα have been performed using the sc-543 antibody from Santa Cruz Biotechnology. However, this antibody has been discontinued, thereby severely impacting the study of ERα in normal physiology as well as diseases such as breast cancer and ovarian cancer. Here, we compare the sc-543 antibody with other commercially available antibodies, and we show that 06-935 (EMD Millipore) and ab3575 (Abcam) antibodies can successfully replace the sc-543 antibody for ChIP-seq and RIME experiments.
Asunto(s)
Anticuerpos , Secuenciación de Inmunoprecipitación de Cromatina/métodos , Receptor alfa de Estrógeno/inmunología , Inmunoprecipitación/métodos , Especificidad de Anticuerpos , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Células MCF-7RESUMEN
Estrogen Receptor-ß (ERß) has been implicated in many cancers. In prostate and breast cancer its function is controversial, but genetic studies implicate a role in cancer progression. Much of the confusion around ERß stems from antibodies that are inadequately validated, yet have become standard tools for deciphering its role. Using an ERß-inducible cell system we assessed commonly utilized ERß antibodies and show that one of the most commonly used antibodies, NCL-ER-BETA, is non-specific for ERß. Other antibodies have limited ERß specificity or are only specific in one experimental modality. ERß is commonly studied in MCF-7 (breast) and LNCaP (prostate) cancer cell lines, but we found no ERß expression in either, using validated antibodies and independent mass spectrometry-based approaches. Our findings question conclusions made about ERß using the NCL-ER-BETA antibody, or LNCaP and MCF-7 cell lines. We describe robust reagents, which detect ERß across multiple experimental approaches and in clinical samples.
Asunto(s)
Anticuerpos Antineoplásicos/farmacología , Receptor beta de Estrógeno/inmunología , Mama/efectos de los fármacos , Mama/metabolismo , Línea Celular Tumoral , Doxiciclina/farmacología , Receptor beta de Estrógeno/genética , Receptor beta de Estrógeno/metabolismo , Femenino , Humanos , Inmunohistoquímica , Indicadores y Reactivos , Masculino , Péptidos , Próstata/efectos de los fármacos , Próstata/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Chromatin immunoprecipitation-sequencing (ChIP-Seq) is a powerful tool which combines the established method of ChIP with next-generation sequencing (NGS) to determine DNA-binding sites of a protein of interest on a genome-wide level, importantly, allowing for de novo discovery of binding events. Here we describe ChIP-seq using the well-established example of estrogen receptor-α mapping in the MCF7 breast cancer cell line.
Asunto(s)
Neoplasias de la Mama/metabolismo , Inmunoprecipitación de Cromatina , Cromatina/metabolismo , Receptor alfa de Estrógeno/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ADN , Sitios de Unión , Neoplasias de la Mama/genética , Cromatina/genética , Biología Computacional , Bases de Datos Genéticas , Receptor alfa de Estrógeno/genética , Femenino , Humanos , Células MCF-7 , Reacción en Cadena de la Polimerasa , Unión Proteica , Flujo de TrabajoRESUMEN
The forkhead protein, FOXA1, is a critical interacting partner of the nuclear hormone receptors, oestrogen receptor-α (ER) and androgen receptor (AR), which are major drivers of the two most common cancers, namely breast and prostate cancer. Over the past few years, progress has been made in our understanding of how FOXA1 influences nuclear receptor function, with both common and distinct roles in the regulation of ER or AR. Recently, another level of regulation has been described, with the discovery that FOXA1 is mutated in 1.8% of breast and 3-5% prostate cancers. In addition, a subset of both cancer types exhibit amplification of the genomic region encompassing the FOXA1 gene. Furthermore, there is evidence of somatic changes that influence the DNA sequence under FOXA1 binding regions, which may indirectly influence FOXA1-mediated regulation of ER and AR activity. These recent observations provide insight into the heterogeneity observed in ER and AR driven cancers.
RESUMEN
Estrogen receptor-α (ER) is the driving transcription factor in most breast cancers, and its associated proteins can influence drug response, but direct methods for identifying interacting proteins have been limited. We purified endogenous ER using an approach termed RIME (rapid immunoprecipitation mass spectrometry of endogenous proteins) and discovered the interactome under agonist- and antagonist-liganded conditions in breast cancer cells, revealing transcriptional networks in breast cancer. The most estrogen-enriched ER interactor is GREB1, a potential clinical biomarker with no known function. GREB1 is shown to be a chromatin-bound ER coactivator and is essential for ER-mediated transcription, because it stabilizes interactions between ER and additional cofactors. We show a GREB1-ER interaction in three xenograft tumors, and using a directed protein-protein approach, we find GREB1-ER interactions in half of ER(+) primary breast cancers. This finding is supported by histological expression of GREB1, which shows that GREB1 is expressed in half of ER(+) cancers, and predicts good clinical outcome. These findings reveal an unexpected role for GREB1 as an estrogen-specific ER cofactor that is expressed in drug-sensitive contexts.
Asunto(s)
Receptor alfa de Estrógeno/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Ratones , Ratones SCID , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Mapas de Interacción de Proteínas , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transcripción Genética , Trasplante HeterólogoRESUMEN
Oestrogen receptor-α (ER) is the defining and driving transcription factor in the majority of breast cancers and its target genes dictate cell growth and endocrine response, yet genomic understanding of ER function has been restricted to model systems. Here we map genome-wide ER-binding events, by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), in primary breast cancers from patients with different clinical outcomes and in distant ER-positive metastases. We find that drug-resistant cancers still recruit ER to the chromatin, but that ER binding is a dynamic process, with the acquisition of unique ER-binding regions in tumours from patients that are likely to relapse. The acquired ER regulatory regions associated with poor clinical outcome observed in primary tumours reveal gene signatures that predict clinical outcome in ER-positive disease exclusively. We find that the differential ER-binding programme observed in tumours from patients with poor outcome is not due to the selection of a rare subpopulation of cells, but is due to the FOXA1-mediated reprogramming of ER binding on a rapid timescale. The parallel redistribution of ER and FOXA1 binding events in drug-resistant cellular contexts is supported by histological co-expression of ER and FOXA1 in metastatic samples. By establishing transcription-factor mapping in primary tumour material, we show that there is plasticity in ER-binding capacity, with distinct combinations of cis-regulatory elements linked with the different clinical outcomes.
Asunto(s)
Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica , Receptores de Estrógenos/metabolismo , Secuencia de Bases , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Humanos , Metástasis de la Neoplasia/genética , Pronóstico , Unión Proteica , Secuencias Reguladoras de Ácidos Nucleicos/genética , Análisis de Supervivencia , Tamoxifeno/farmacología , Tamoxifeno/uso terapéutico , Resultado del TratamientoRESUMEN
Estrogen receptor (ER) binds to distal enhancers within the genome and requires additional factors, such as the Forkhead protein FoxA1, for mediating chromatin interactions. We now show that the human Groucho protein, Transducin-like enhancer protein 1 (TLE1), positively assists some ER-chromatin interactions, a role that is distinct from its general role as a transcriptional repressor. We show that specific silencing of TLE1 inhibits the ability of ER to bind to a subset of ER binding sites within the genome, a phenomenon that results in perturbations in phospho-RNA Pol II recruitment. Furthermore, TLE1 is essential for effective ER-mediated cell division. We have discovered a distinct role for TLE1, as a necessary transcriptional component of the ER complex, where it facilitates ER-chromatin interactions.
Asunto(s)
Neoplasias de la Mama/genética , Receptor alfa de Estrógeno/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética , Sitios de Unión , Neoplasias de la Mama/patología , Línea Celular Tumoral , Cromatina/metabolismo , Proteínas Co-Represoras , Estrógenos/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Humanos , Unión Proteica/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
Estrogen receptor-α (ER) is the key feature of most breast cancers and binding of ER to the genome correlates with expression of the Forkhead protein FOXA1 (also called HNF3α). Here we show that FOXA1 is a key determinant that can influence differential interactions between ER and chromatin. Almost all ER-chromatin interactions and gene expression changes depended on the presence of FOXA1 and FOXA1 influenced genome-wide chromatin accessibility. Furthermore, we found that CTCF was an upstream negative regulator of FOXA1-chromatin interactions. In estrogen-responsive breast cancer cells, the dependency on FOXA1 for tamoxifen-ER activity was absolute; in tamoxifen-resistant cells, ER binding was independent of ligand but depended on FOXA1. Expression of FOXA1 in non-breast cancer cells can alter ER binding and function. As such, FOXA1 is a major determinant of estrogen-ER activity and endocrine response in breast cancer cells.
Asunto(s)
Receptor alfa de Estrógeno/metabolismo , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Cromatina/metabolismo , Femenino , Factor Nuclear 3-alfa del Hepatocito/genética , Humanos , TransfecciónRESUMEN
Retinoic acid receptor-alpha (RAR alpha) is a known estrogen target gene in breast cancer cells. The consequence of RAR alpha induction by estrogen was previously unknown. We now show that RAR alpha is required for efficient estrogen receptor-alpha (ER)-mediated transcription and cell proliferation. RAR alpha can interact with ER-binding sites, but this occurs in an ER-dependent manner, providing a novel role for RAR alpha that is independent of its classic role. We show, on a genome-wide scale, that RAR alpha and ER can co-occupy regulatory regions together within the chromatin. This transcriptionally active co-occupancy and dependency occurs when exposed to the predominant breast cancer hormone, estrogen--an interaction that is promoted by the estrogen-ER induction of RAR alpha. These findings implicate RAR alpha as an essential component of the ER complex, potentially by maintaining ER-cofactor interactions, and suggest that different nuclear receptors can cooperate for effective transcriptional activity in breast cancer cells.
Asunto(s)
Neoplasias de la Mama/metabolismo , Receptores de Estrógenos/metabolismo , Receptores de Ácido Retinoico/metabolismo , ADN/metabolismo , Estrógenos/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ligandos , Unión ProteicaRESUMEN
Crosstalk between the oestrogen receptor (ER) and ERBB2/HER-2 pathways has long been implicated in breast cancer aetiology and drug response, yet no direct connection at a transcriptional level has been shown. Here we show that oestrogen-ER and tamoxifen-ER complexes directly repress ERBB2 transcription by means of a cis-regulatory element within the ERBB2 gene in human cell lines. We implicate the paired box 2 gene product (PAX2), in a previously unrecognized role, as a crucial mediator of ER repression of ERBB2 by the anti-cancer drug tamoxifen. We show that PAX2 and the ER co-activator AIB-1/SRC-3 compete for binding and regulation of ERBB2 transcription, the outcome of which determines tamoxifen response in breast cancer cells. The repression of ERBB2 by ER-PAX2 links these two breast cancer subtypes and suggests that aggressive ERBB2-positive tumours can originate from ER-positive luminal tumours by circumventing this repressive mechanism. These data provide mechanistic insight into the molecular basis of endocrine resistance in breast cancer.
Asunto(s)
Genes erbB-2/genética , Factor de Transcripción PAX2/metabolismo , Receptor ErbB-2/genética , Receptores de Estrógenos/metabolismo , Tamoxifeno/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Resistencia a Antineoplásicos/genética , Estrógenos/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Histona Acetiltransferasas , Humanos , Coactivador 3 de Receptor Nuclear , Factor de Transcripción PAX2/deficiencia , Factor de Transcripción PAX2/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Proteínas Represoras/metabolismo , Tamoxifeno/metabolismo , TransactivadoresRESUMEN
Estrogen receptor (ER)-associated cofactors and cooperating transcription factors are one of the primary components determining transcriptional activity of estrogen target genes and may constitute potential therapeutic targets. Recent mapping of ER-binding sites on a genome-wide scale has provided insight into novel cooperating factors based on the enrichment of transcription factor motifs within the ER-binding sites. We have used the ER-binding sites in combination with sequence conservation to identify the statistical enrichment of Nkx and LEF motifs. We find that Nkx3-1 and LEF-1 bind to several ER cis-regulatory elements in vivo, but they both function as transcriptional repressors of estrogen signaling. We show that Nkx3-1 and LEF-1 can inhibit ER binding to chromatin, suggesting competition for common chromatin-binding regions. These data provide insight into the role of Nkx3-1 and LEF-1 as potential regulators of the hormone response in breast cancer.