RESUMO
Precise control of gene expression underpins normal development. This relies on mechanisms that enable communication between gene promoters and other regulatory elements. In embryonic stem cells (ESCs), the cyclin-dependent kinase module Mediator complex (CKM-Mediator) has been reported to physically link gene regulatory elements to enable gene expression and also prime genes for induction during differentiation. Here, we show that CKM-Mediator contributes little to three-dimensional genome organization in ESCs, but it has a specific and essential role in controlling interactions between inactive gene regulatory elements bound by Polycomb repressive complexes (PRCs). These interactions are established by the canonical PRC1 (cPRC1) complex but rely on CKM-Mediator, which facilitates binding of cPRC1 to its target sites. Importantly, through separation-of-function experiments, we reveal that this collaboration between CKM-Mediator and cPRC1 in creating long-range interactions does not function to prime genes for induction during differentiation. Instead, we discover that priming relies on an interaction-independent mechanism whereby the CKM supports core Mediator engagement with gene promoters during differentiation to enable gene activation.
Assuntos
Complexo Mediador , Complexo Repressor Polycomb 1 , Diferenciação Celular/genética , Quinases Ciclina-Dependentes/metabolismo , Complexo Mediador/genética , Complexo Repressor Polycomb 1/genética , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismoRESUMO
The genome consists of regions of transcriptionally active euchromatin and more silent heterochromatin. We reveal that the formation of heterochromatin domains requires cohesin turnover on DNA. Stabilization of cohesin on DNA through depletion of its release factor WAPL leads to a near-complete loss of heterochromatin domains. We observe the opposite phenotype in cells deficient for subunits of the Mediator-CDK module, with an almost binary partition of the genome into dense H3K9me3 domains, and regions devoid of H3K9me3 spanning the rest of the genome. We suggest that the Mediator-CDK module might contribute to gene expression by limiting the formation of dense heterochromatin domains. WAPL deficiency prevents the formation of heterochromatin domains, and allows for gene expression even in the absence of the Mediator-CDK subunit MED12. We propose that cohesin and Mediator affect heterochromatin in different ways to enable the correct distribution of epigenetic marks, and thus to ensure proper gene expression.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Heterocromatina/metabolismo , Complexo Mediador/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Transporte/genética , Linhagem Celular , Sequenciamento de Cromatina por Imunoprecipitação , Epigênese Genética , Técnicas de Inativação de Genes , Humanos , Complexo Mediador/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/genética , RNA-Seq , CoesinasRESUMO
Estrogen receptor α (ERα) is a key transcriptional regulator in the majority of breast cancers. ERα-positive patients are frequently treated with tamoxifen, but resistance is common. In this study, we refined a previously identified 111-gene outcome prediction-classifier, revealing FEN1 as the strongest determining factor in ERα-positive patient prognostication. FEN1 levels were predictive of outcome in tamoxifen-treated patients, and FEN1 played a causal role in ERα-driven cell growth. FEN1 impacted the transcriptional activity of ERα by facilitating coactivator recruitment to the ERα transcriptional complex. FEN1 blockade induced proteasome-mediated degradation of activated ERα, resulting in loss of ERα-driven gene expression and eradicated tumor cell proliferation. Finally, a high-throughput 465,195 compound screen identified a novel FEN1 inhibitor, which effectively blocked ERα function and inhibited proliferation of tamoxifen-resistant cell lines as well as ex vivo-cultured ERα-positive breast tumors. Collectively, these results provide therapeutic proof of principle for FEN1 blockade in tamoxifen-resistant breast cancer. SIGNIFICANCE: These findings show that pharmacologic inhibition of FEN1, which is predictive of outcome in tamoxifen-treated patients, effectively blocks ERα function and inhibits proliferation of tamoxifen-resistant tumor cells.
Assuntos
Neoplasias da Mama/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Receptor alfa de Estrogênio/metabolismo , Endonucleases Flap/metabolismo , Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/genética , Feminino , Endonucleases Flap/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Tamoxifeno/uso terapêuticoRESUMO
PURPOSE: The prognostic and predictive values of the MAPK/AKT/ERα phosphorylation axis (pT202/T204MAPK, pT308AKT, pS473AKT, pS118ERα and pS167ERα) in primary tumours were assessed to determine whether these markers can differentiate between patient responses for switching adjuvant endocrine therapy after 2-3 years from tamoxifen to exemestane and continued tamoxifen monotherapy in the Intergroup Exemestane Study (IES). METHODS: Of the 4724 patients in IES, 1506 were managed in a subset of centres (N = 89) participating in PathIES. These centres recruited 1282 (85%, 1282/1506) women into PathIES of whom 1036 had phospho-marker data. All phospho-markers were analysed by immunohistochemistry staining. Multivariable Cox proportional hazards models of the phospho-markers for disease-free survival (DFS) and overall survival (OS) were adjusted for clinicopathological factors. Treatment effects on the biomarker expression were determined by interaction tests. Benjamini-Hochberg adjustment for multiple testing with a false discovery rate of 10% was applied (pBH). RESULTS: Phospho-T202/T204MAPK, pS118ERα and pS167ERα were all found to be correlated (pBH = 0.0002). These markers were not associated with either DFS or OS when controlling for the established clinicopathological factors. Interaction terms between the phospho-markers and treatment strategies for either DFS or OS were not statistically significant (pBH > 0.05 for all). CONCLUSIONS: This PathIES study confirmed previously described associations between the phosphorylation site markers of AKT, MAPK and ERα activity in postmenopausal breast cancer patients. No prognostic correlations between the phosphorylation markers and clinical outcome were found, nor were they predictive for clinical outcomes among patients who switched therapy over those treated with tamoxifen alone.
Assuntos
Androstadienos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Estrogênio/metabolismo , Tamoxifeno/uso terapêutico , Adulto , Idoso , Androstadienos/administração & dosagem , Androstadienos/efeitos adversos , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Fosforilação , Prognóstico , Tamoxifeno/administração & dosagem , Tamoxifeno/efeitos adversos , Resultado do TratamentoRESUMO
Tamoxifen, a small-molecule antagonist of the transcription factor estrogen receptor alpha (ERα) used to treat breast cancer, increases risks of endometrial cancer. However, no parallels of ERα transcriptional action in breast and endometrial tumors have been found that might explain this effect. In this study, we addressed this issue with a genome-wide assessment of ERα-chromatin interactions in surgical specimens obtained from patients with tamoxifen-associated endometrial cancer. ERα was found at active enhancers in endometrial cancer cells as marked by the presence of RNA polymerase II and the histone marker H3K27Ac. These ERα binding sites were highly conserved between breast and endometrial cancer and enriched in binding motifs for the transcription factor FOXA1, which displayed substantial overlap with ERα binding sites proximal to genes involved in classical ERα target genes. Multifactorial ChIP-seq data integration from the endometrial cancer cell line Ishikawa illustrated a functional genomic network involving ERα and FOXA1 together with the enhancer-enriched transcriptional regulators p300, FOXM1, TEAD4, FNFIC, CEBP8, and TCF12. Immunohistochemical analysis of 230 primary endometrial tumor specimens showed that lack of FOXA1 and ERα expression was associated with a longer interval between breast cancer and the emergence of endometrial cancer, exclusively in tamoxifen-treated patients. Our results define conserved sites for a genomic interplay between FOXA1 and ERα in breast cancer and tamoxifen-associated endometrial cancer. In addition, FOXA1 and ERα are associated with the interval time between breast cancer and endometrial cancer only in tamoxifen-treated breast cancer patients. Cancer Res; 76(13); 3773-84. ©2016 AACR.
Assuntos
Neoplasias da Mama/genética , Neoplasias do Endométrio/genética , Receptor alfa de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Elementos de Resposta/genética , Tamoxifeno/uso terapêutico , Antineoplásicos Hormonais/uso terapêutico , Biomarcadores Tumorais/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Imunoprecipitação da Cromatina , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Receptor alfa de Estrogênio/genética , Feminino , Fator 3-alfa Nuclear de Hepatócito/genética , Humanos , Técnicas Imunoenzimáticas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
The advent of genome-wide transcription factor profiling has revolutionized the field of breast cancer research. Estrogen receptor α (ERα), the major drug target in hormone receptor-positive breast cancer, has been known as a key transcriptional regulator in tumor progression for over 30 years. Even though this function of ERα is heavily exploited and widely accepted as an Achilles heel for hormonal breast cancer, only since the last decade we have been able to understand how this transcription factor is functioning on a genome-wide scale. Initial ChIP-on-chip (chromatin immunoprecipitation coupled with tiling array) analyses have taught us that ERα is an enhancer-associated factor binding to many thousands of sites throughout the human genome and revealed the identity of a number of directly interacting transcription factors that are essential for ERα action. More recently, with the development of massive parallel sequencing technologies and refinements thereof in sample processing, a genome-wide interrogation of ERα has become feasible and affordable with unprecedented data quality and richness. These studies have revealed numerous additional biological insights into ERα behavior in cell lines and especially in clinical specimens. Therefore, what have we actually learned during this first decade of cistromics in breast cancer and where may future developments in the field take us?
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina/tendências , Elementos Facilitadores Genéticos , Feminino , Perfilação da Expressão Gênica/tendências , Estudo de Associação Genômica Ampla/tendências , Genômica/tendências , Humanos , FosforilaçãoRESUMO
PURPOSE: The steroid receptor coactivator SRC3 is essential for the transcriptional activity of estrogen receptor α (ERα). SRC3 is sufficient to cause mammary tumorigenesis, and has also been implicated in endocrine resistance. SRC3 is posttranslationally modified by phosphorylation, but these events have not been investigated with regard to functionality or disease association. Here, we investigate the spatial selectivity of SRC3-pS543/DNA binding over the human genome and its expression in primary human breast cancer in relation with outcome. EXPERIMENTAL DESIGN: Chromatin immunoprecipitation, coupled with sequencing, was used to determine the chromatin binding patterns of SRC3-pS543 in the breast cancer cell line MCF7 and two untreated primary breast cancers. IHC was used to assess the expression of SRC3 and SRC3-pS543 in 1,650 primary breast cancers. The relationship between the expression of SRC3 and SRC3-pS543, disease-free survival (DFS), and breast cancer specific survival (BCSS) was assessed. RESULTS: Although total SRC3 is selectively found at enhancer regions, SRC3-pS543 is recruited to promoters of ERα responsive genes, both in the MCF7 cell line and primary breast tumor specimens. SRC3-pS543 was associated with both improved DFS (P = 0.003) and BCSS (P = 0.001) in tamoxifen untreated high-risk patients, such a correlation was not seen in tamoxifen-treated cases, the interaction was statistically significant (P = 0.001). Multivariate analysis showed SRC3-pS543 to be an independent prognostic factor. CONCLUSIONS: Phosphorylation of SRC3 at S543 affects its genomic interactions on a genome-wide level, where SRC3-pS543 is selectively recruited to promoters of ERα-responsive genes. SRC3-pS543 is a prognostic marker, and a predictive marker of response to endocrine therapy.
Assuntos
Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/metabolismo , Coativador 3 de Receptor Nuclear/metabolismo , Fosforilação/fisiologia , Serina/metabolismo , Animais , Antineoplásicos Hormonais/farmacologia , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Células CHO , Linhagem Celular Tumoral , Cromatina/metabolismo , Cricetulus , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Células MCF-7 , Fosforilação/efeitos dos fármacos , Prognóstico , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Tamoxifeno/farmacologiaRESUMO
Oestrogen receptor α (ERα) is a nuclear receptor that is the driving transcription factor expressed in the majority of breast cancers. Recent studies have demonstrated that the liver receptor homolog-1 (LRH-1), another nuclear receptor, regulates breast cancer cell proliferation and promotes motility and invasion. To determine the mechanisms of LRH-1 action in breast cancer, we performed gene expression microarray analysis following RNA interference for LRH-1. Interestingly, gene ontology (GO) category enrichment analysis of LRH-1-regulated genes identified oestrogen-responsive genes as the most highly enriched GO categories. Remarkably, chromatin immunoprecipitation coupled to massively parallel sequencing (ChIP-seq) to identify genomic targets of LRH-1 showed LRH-1 binding at many ERα binding sites. Analysis of select binding sites confirmed regulation of ERα-regulated genes by LRH-1 through binding to oestrogen response elements, as exemplified by the TFF1/pS2 gene. Finally, LRH-1 overexpression stimulated ERα recruitment, while LRH-1 knockdown reduced ERα recruitment to ERα binding sites. Taken together, our findings establish a key role for LRH-1 in the regulation of ERα target genes in breast cancer cells and identify a mechanism in which co-operative binding of LRH-1 and ERα at oestrogen response elements controls the expression of oestrogen-responsive genes.
Assuntos
Neoplasias da Mama/genética , Receptor alfa de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Neoplasias da Mama/metabolismo , Células COS , Chlorocebus aethiops , Feminino , Células MCF-7 , Elementos de RespostaRESUMO
Estrogen receptor alpha (ERα) is involved in numerous physiological and pathological processes, including breast cancer. Breast cancer therapy is therefore currently directed at inhibiting the transcriptional potency of ERα, either by blocking estrogen production through aromatase inhibitors or antiestrogens that compete for hormone binding. Due to resistance, new treatment modalities are needed and as ERα dimerization is essential for its activity, interference with receptor dimerization offers a new opportunity to exploit in drug design. Here we describe a unique mechanism of how ERα dimerization is negatively controlled by interaction with 14-3-3 proteins at the extreme C terminus of the receptor. Moreover, the small-molecule fusicoccin (FC) stabilizes this ERα/14-3-3 interaction. Cocrystallization of the trimeric ERα/14-3-3/FC complex provides the structural basis for this stabilization and shows the importance of phosphorylation of the penultimate Threonine (ERα-T(594)) for high-affinity interaction. We confirm that T(594) is a distinct ERα phosphorylation site in the breast cancer cell line MCF-7 using a phospho-T(594)-specific antibody and by mass spectrometry. In line with its ERα/14-3-3 interaction stabilizing effect, fusicoccin reduces the estradiol-stimulated ERα dimerization, inhibits ERα/chromatin interactions and downstream gene expression, resulting in decreased cell proliferation. Herewith, a unique functional phosphosite and an alternative regulation mechanism of ERα are provided, together with a small molecule that selectively targets this ERα/14-3-3 interface.