RESUMEN
Alterations in transcriptional regulators can orchestrate oncogenic gene expression programs in cancer. Here, we show that the BRG1/BRM-associated factor (BAF) chromatin remodeling complex, which is mutated in over 20% of human tumors, interacts with EWSR1, a member of a family of proteins with prion-like domains (PrLD) that are frequent partners in oncogenic fusions with transcription factors. In Ewing sarcoma, we find that the BAF complex is recruited by the EWS-FLI1 fusion protein to tumor-specific enhancers and contributes to target gene activation. This process is a neomorphic property of EWS-FLI1 compared to wild-type FLI1 and depends on tyrosine residues that are necessary for phase transitions of the EWSR1 prion-like domain. Furthermore, fusion of short fragments of EWSR1 to FLI1 is sufficient to recapitulate BAF complex retargeting and EWS-FLI1 activities. Our studies thus demonstrate that the physical properties of prion-like domains can retarget critical chromatin regulatory complexes to establish and maintain oncogenic gene expression programs.
Asunto(s)
Proteínas de Unión a Calmodulina/química , Proteínas de Unión a Calmodulina/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína EWS de Unión a ARN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Sarcoma de Ewing/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Células Madre Mesenquimatosas/metabolismo , Repeticiones de Microsatélite , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Priónicas/metabolismo , Dominios Proteicos , Sarcoma de Ewing/patologíaRESUMEN
The interaction of RB with chromatin is key to understanding its molecular functions. Here, for first time, we identify the full spectrum of chromatin-bound RB. Rather than exclusively binding promoters, as is often described, RB targets three fundamentally different types of loci (promoters, enhancers, and insulators), which are largely distinguishable by the mutually exclusive presence of E2F1, c-Jun, and CTCF. While E2F/DP facilitates RB association with promoters, AP-1 recruits RB to enhancers. Although phosphorylation in CDK sites is often portrayed as releasing RB from chromatin, we show that the cell cycle redistributes RB so that it enriches at promoters in G1 and at non-promoter sites in cycling cells. RB-bound promoters include the classic E2F-targets and are similar between lineages, but RB-bound enhancers associate with different categories of genes and vary between cell types. Thus, RB has a well-preserved role controlling E2F in G1, and it targets cell-type-specific enhancers and CTCF sites when cells enter S-phase.
Asunto(s)
Cromatina , Proteína de Retinoblastoma , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Factores de Transcripción E2F/genética , Factores de Transcripción E2F/metabolismo , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F1/metabolismo , Regiones Promotoras Genéticas , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Factor de Transcripción AP-1/genéticaRESUMEN
Chromosomal rearrangements resulting in the fusion of TMPRSS2, an androgen-regulated gene, and the ETS family transcription factor ERG occur in over half of prostate cancers. However, the mechanism by which ERG promotes oncogenic gene expression and proliferation remains incompletely understood. Here, we identify a binding interaction between ERG and the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complex, which is conserved among other oncogenic ETS factors, including ETV1, ETV4, and ETV5. We find that ERG drives genome-wide retargeting of BAF complexes in a manner dependent on binding of ERG to the ETS DNA motif. Moreover, ERG requires intact BAF complexes for chromatin occupancy and BAF complex ATPase activity for target gene regulation. In a prostate organoid model, BAF complexes are required for ERG-mediated basal-to-luminal transition, a hallmark of ERG activity in prostate cancer. These observations suggest a fundamental interdependence between ETS transcription factors and BAF chromatin remodeling complexes in cancer.
Asunto(s)
Carcinogénesis/genética , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Nucleares/genética , Proteínas de Fusión Oncogénica/genética , Neoplasias de la Próstata/genética , Serina Endopeptidasas/genética , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Animales , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular , Cromatina/química , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Células HEK293 , Humanos , Masculino , Ratones Transgénicos , Proteínas Nucleares/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Organoides/metabolismo , Organoides/patología , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Unión Proteica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ets , Serina Endopeptidasas/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulador Transcripcional ERG/genética , Regulador Transcripcional ERG/metabolismoRESUMEN
Various types of repetitive sequences are dysregulated in cancer. In Ewing sarcoma, the oncogenic fusion protein EWS-FLI1 induces chromatin features typical of active enhancers at GGAA microsatellite repeats, but the function of these sites has not been directly demonstrated. Here, by combining nascent transcription profiling with epigenome editing, we found that a subset of GGAA microsatellite repeats is transcriptionally active in Ewing sarcoma and that silencing individual repeats abolishes local nascent transcription and leads to markedly reduced expression of putative target genes. Epigenome silencing of these repeat sites does not affect gene expression in unrelated cells, can prevent the induction of gene expression by EWS-FLI1, and, in the case of a GGAA repeat that controls SOX2 expression from a distance of 470 kb, is sufficient to impair the growth of Ewing sarcoma xenografts. Using an experimental approach that is broadly applicable to testing different types of repetitive genomic elements, our study directly demonstrates that specific repeat microsatellites can have critical gene regulation functions in cancer and thus represent tumor-specific vulnerabilities that may be exploited to develop new therapies.
Asunto(s)
Neoplasias Óseas/genética , Elementos de Facilitación Genéticos , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Repeticiones de Microsatélite , Proteínas de Fusión Oncogénica/metabolismo , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína EWS de Unión a ARN/metabolismo , Sarcoma de Ewing/genética , Animales , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Células Cultivadas , Cromatina/metabolismo , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Humanos , Células Madre Mesenquimatosas/metabolismo , Ratones , ARN no Traducido/biosíntesis , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patología , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Transcripción Genética , Células Tumorales Cultivadas , Proteínas de Pez CebraRESUMEN
Tumor-stromal communication within the microenvironment contributes to initiation of metastasis and may present a therapeutic opportunity. Using serial single-cell RNA sequencing in an orthotopic mouse prostate cancer model, we find up-regulation of prolactin receptor as cancer cells that have disseminated to the lungs expand into micrometastases. Secretion of the ligand prolactin by adjacent lung stromal cells is induced by tumor cell production of the COX-2 synthetic product prostaglandin E2 (PGE2). PGE2 treatment of fibroblasts activates the orphan nuclear receptor NR4A (Nur77), with prolactin as a major transcriptional target for the NR4A-retinoid X receptor (RXR) heterodimer. Ectopic expression of prolactin receptor in mouse cancer cells enhances micrometastasis, while treatment with the COX-2 inhibitor celecoxib abrogates prolactin secretion by fibroblasts and reduces tumor initiation. Across multiple human cancers, COX-2, prolactin, and prolactin receptor show consistent differential expression in tumor and stromal compartments. Such paracrine cross-talk may thus contribute to the documented efficacy of COX-2 inhibitors in cancer suppression.
Asunto(s)
Carcinogénesis/metabolismo , Prolactina/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Transducción de Señal/fisiología , Células del Estroma/metabolismo , Animales , Carcinogénesis/efectos de los fármacos , Celecoxib/farmacología , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa 2/farmacología , Dinoprostona/metabolismo , Modelos Animales de Enfermedad , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Masculino , Ratones , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Receptores X Retinoide/metabolismo , Transducción de Señal/efectos de los fármacos , Células del Estroma/efectos de los fármacos , Células del Estroma/patología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Intrinsically disordered regions (IDRs) in DNA-associated proteins are known to influence gene regulation, but their distribution and cooperative functions in genome-wide regulatory programs remain poorly understood. Here we describe DisP-seq (disordered protein precipitation followed by DNA sequencing), an antibody-independent chemical precipitation assay that can simultaneously map endogenous DNA-associated disordered proteins genome-wide through a combination of biotinylated isoxazole precipitation and next-generation sequencing. DisP-seq profiles are composed of thousands of peaks that are associated with diverse chromatin states, are enriched for disordered transcription factors (TFs) and are often arranged in large lineage-specific clusters with high local concentrations of disordered proteins and different combinations of histone modifications linked to regulatory potential. We use DisP-seq to analyze cancer cells and reveal how disordered protein-associated islands enable IDR-dependent mechanisms that control the binding and function of disordered TFs, including oncogene-dependent sequestration of TFs through long-range interactions and the reactivation of differentiation pathways upon loss of oncogenic stimuli in Ewing sarcoma.
Asunto(s)
Cromatina , ADN , Análisis de Secuencia de ADNRESUMEN
The development of targeted therapies offers new hope for patients affected by incurable cancer. However, multiple challenges persist, notably in controlling tumor cell plasticity in patients with refractory and metastatic illness. Neuroblastoma (NB) is an aggressive pediatric malignancy originating from defective differentiation of neural crest-derived progenitors with oncogenic activity due to genetic and epigenetic alterations and remains a clinical challenge for high-risk patients. To identify critical genes driving NB aggressiveness, we performed combined chromatin and transcriptome analyses on matched patient-derived xenografts (PDXs), spheroids, and differentiated adherent cultures derived from metastatic MYCN nonamplified tumors. Bone marrow kinase on chromosome X (BMX) was identified among the most differentially regulated genes in PDXs and spheroids versus adherent models. BMX expression correlated with high tumor stage and poor patient survival and was crucial to the maintenance of the self-renewal and tumorigenic potential of NB spheroids. Moreover, BMX expression positively correlated with the mesenchymal NB cell phenotype, previously associated with increased chemoresistance. Finally, BMX inhibitors readily reversed this cellular state, increased the sensitivity of NB spheroids toward chemotherapy, and partially reduced tumor growth in a preclinical NB model. Altogether, our study identifies BMX as a promising innovative therapeutic target for patients with high-risk MYCN nonamplified NB.
Asunto(s)
Proteína Proto-Oncogénica N-Myc , Neuroblastoma , Esferoides Celulares , Neuroblastoma/genética , Neuroblastoma/patología , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/metabolismo , Humanos , Proteína Proto-Oncogénica N-Myc/genética , Proteína Proto-Oncogénica N-Myc/metabolismo , Animales , Esferoides Celulares/patología , Esferoides Celulares/metabolismo , Esferoides Celulares/efectos de los fármacos , Ratones , Ensayos Antitumor por Modelo de Xenoinjerto , Línea Celular Tumoral , Regulación Neoplásica de la Expresión GénicaRESUMEN
The tumor suppressor gene hypermethylated in cancer 1 (HIC1), which encodes a transcriptional repressor, is epigenetically silenced in many human tumors. Here, we show that ectopic expression of HIC1 in the highly malignant MDA-MB-231 breast cancer cell line severely impairs cell proliferation, migration, and invasion in vitro. In parallel, infection of breast cancer cell lines with a retrovirus expressing HIC1 also induces decreased mRNA and protein expression of the tyrosine kinase receptor EphA2. Moreover, chromatin immunoprecipitation (ChIP) and sequential ChIP experiments demonstrate that endogenous HIC1 proteins are bound, together with the MTA1 corepressor, to the EphA2 promoter in WI38 cells. Taken together, our results identify EphA2 as a new direct target gene of HIC1. Finally, we observe that inactivation of endogenous HIC1 through RNA interference in normal breast epithelial cells results in the up-regulation of EphA2 and is correlated with increased cellular migration. To conclude, our results involve the tumor suppressor HIC1 in the transcriptional regulation of the tyrosine kinase receptor EphA2, whose ligand ephrin-A1 is also a HIC1 target gene. Thus, loss of the regulation of this Eph pathway through HIC1 epigenetic silencing could be an important mechanism in the pathogenesis of epithelial cancers.
Asunto(s)
Factores de Transcripción de Tipo Kruppel/metabolismo , Receptor EphA2/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Progresión de la Enfermedad , Regulación hacia Abajo/genética , Fibroblastos/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Células HEK293 , Histona Desacetilasas/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/deficiencia , Factores de Transcripción de Tipo Kruppel/genética , Glándulas Mamarias Humanas/citología , Glándulas Mamarias Humanas/metabolismo , Glándulas Mamarias Humanas/patología , Regiones Promotoras Genéticas/genética , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/metabolismo , TransactivadoresRESUMEN
HIC1 (hypermethylated in cancer 1) is a tumor suppressor gene epigenetically silenced or deleted in many human cancers. HIC1 is involved in regulatory loops modulating p53- and E2F1-dependent cell survival, growth control, and stress responses. HIC1 is also essential for normal development because Hic1-deficient mice die perinatally and exhibit gross developmental defects throughout the second half of development. HIC1 encodes a transcriptional repressor with five C(2)H(2) zinc fingers mediating sequence-specific DNA binding and two repression domains: an N-terminal BTB/POZ domain and a central region recruiting CtBP and NuRD complexes. By yeast two-hybrid screening, we identified the Polycomb-like protein hPCL3 as a novel co-repressor for HIC1. Using multiple biochemical strategies, we demonstrated that HIC1 interacts with hPCL3 and its paralog PHF1 to form a stable complex with the PRC2 members EZH2, EED, and Suz12. Confirming the implication of HIC1 in Polycomb recruitment, we showed that HIC1 shares some of its target genes with PRC2, including ATOH1. Depletion of HIC1 by siRNA interference leads to a partial displacement of EZH2 from the ATOH1 promoter. Furthermore, in vivo, ATOH1 repression by HIC1 is associated with Polycomb activity during mouse cerebellar development. Thus, our results identify HIC1 as the first transcription factor in mammals able to recruit PRC2 to some target promoters through its interaction with Polycomb-like proteins.
Asunto(s)
Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas Represoras/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cerebelo/embriología , Cerebelo/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteína Potenciadora del Homólogo Zeste 2 , Femenino , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Proteínas de Neoplasias , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Proteínas Represoras/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Dedos de ZincRESUMEN
The transcriptional repressor HIC1 (Hypermethylated in Cancer 1) is a tumor suppressor gene inactivated in many human cancers including breast carcinomas. In this study, we show that HIC1 is a direct transcriptional repressor of ß-2 adrenergic receptor (ADRB2). Through promoter luciferase activity, chromatin immunoprecipitation (ChIP) and sequential ChIP experiments, we demonstrate that ADRB2 is a direct target gene of HIC1, endogenously in WI-38 cells and following HIC1 re-expression in breast cancer cells. Agonist-mediated stimulation of ADRB2 increases the migration and invasion of highly malignant MDA-MB-231 breast cancer cells but these effects are abolished following HIC1 re-expression or specific down-regulation of ADRB2 by siRNA treatment. Our results suggest that early inactivation of HIC1 in breast carcinomas could predispose to stress-induced metastasis through up-regulation of the ß-2 adrenergic receptor.
Asunto(s)
Neoplasias de la Mama/patología , Movimiento Celular , Factores de Transcripción de Tipo Kruppel/metabolismo , Receptores Adrenérgicos beta 2/genética , Estrés Fisiológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/fisiopatología , Adhesión Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/deficiencia , Factores de Transcripción de Tipo Kruppel/genética , Invasividad Neoplásica , Metástasis de la Neoplasia , Interferencia de ARN , ARN Interferente Pequeño/genética , Receptores Adrenérgicos beta 2/deficiencia , Estrés Fisiológico/genéticaRESUMEN
The tumor suppressor gene HIC1 (Hypermethylated In Cancer 1) encodes a transcriptional repressor involved in the regulation of growth control and DNA damage response. We previously demonstrated that p57Kip2; a member of the CIP/KIP family of CDK (cyclin dependent kinase) inhibitors (CKI); is a direct target gene of HIC1 in quiescent cells. Here we show that ectopic expression of HIC1 in MDA-MB-231 cells or its overexpression in BJ-Tert fibroblasts induces decreased mRNA and protein expression of p21 (CIP1/WAF1) another member of this CKI family that plays essential roles in the p53-mediated DNA damage response. Conversely, knock-down of endogenous HIC1 in BJ-Tert through RNA interference up-regulates p21 in basal conditions and further potentiates this CKI in response to apoptotic etoposide-induced DNA damage. Through promoter luciferase activity and chromatin immunoprecipitation (ChIP), we demonstrate that HIC1 is a direct transcriptional repressor of p21. Thus, our results further demonstrate that HIC1 is a key player in the regulation of the DNA damage response.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Daño del ADN/genética , Regulación de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Genes Reporteros , Células HEK293 , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Luciferasas/genética , Interferencia de ARN , Proteínas Represoras/genéticaRESUMEN
For a majority of patients with non-small cell lung cancer with EGFR mutations, treatment with EGFR inhibitors (EGFRi) induces a clinical response. Despite this initial reduction in tumor size, residual disease persists that leads to disease relapse. Elucidating the preexisting biological differences between sensitive cells and surviving drug-tolerant persister cells and deciphering how drug-tolerant cells evolve in response to treatment could help identify strategies to improve the efficacy of EGFRi. In this study, we tracked the origins and clonal evolution of drug-tolerant cells at a high resolution by using an expressed barcoding system coupled with single-cell RNA sequencing. This platform enabled longitudinal profiling of gene expression and drug sensitivity in response to EGFRi across a large number of clones. Drug-tolerant cells had higher expression of key survival pathways such as YAP and EMT at baseline and could also differentially adapt their gene expression following EGFRi treatment compared with sensitive cells. In addition, drug combinations targeting common downstream components (MAPK) or orthogonal factors (chemotherapy) showed greater efficacy than EGFRi alone, which is attributable to broader targeting of the heterogeneous EGFRi-tolerance mechanisms present in tumors. Overall, this approach facilitates thorough examination of clonal evolution in response to therapy that could inform the development of improved diagnostic approaches and treatment strategies for targeting drug-tolerant cells. SIGNIFICANCE: The evolution and heterogeneity of EGFR inhibitor tolerance are identified in a large number of clones at enhanced cellular and temporal resolution using an expressed barcode technology coupled with single-cell RNA sequencing.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Receptores ErbB/genética , Receptores ErbB/metabolismo , Recurrencia Local de Neoplasia , Tolerancia a MedicamentosRESUMEN
PcG (Polycomb group) proteins are conserved transcriptional repressors essential to regulate cell fate and to maintain epigenetic cellular memory. They work in concert through two main families of chromatin-modifying complexes, PRC1 (Polycomb repressive complex 1) and PRC2-4. In Drosophila, PRC2 contains the H3K27 histone methyltransferase E(Z) whose trimethylation activity towards PcG target genes is stimulated by PCL (Polycomb-like). In the present study, we have examined hPCL3, one of its three human paralogues. Through alternative splicing, hPCL3 encodes a long isoform, hPCL3L, containing an N-terminal TUDOR domain and two PHDs (plant homeodomains) and a smaller isoform, hPCL3S, lacking the second PHD finger (PHD2). By quantitative reverse transcription-PCR analyses, we showed that both isoforms are widely co-expressed at high levels in medulloblastoma. By co-immunoprecipitation analyses, we demonstrated that both isoforms interact with EZH2 through their common TUDOR domain. However, the hPCL3L-specific PHD2 domain, which is better conserved than PHD1 in the PCL family, is also involved in this interaction and implicated in the self-association of hPCL3L. Finally, we have demonstrated that both hPCL3 isoforms are physically associated with EZH2, but in different complexes. Our results provide the first evidence that the two hPCL3 isoforms belong to different complexes and raise important questions about their relative functions, particularly in tumorigenesis.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Empalme Alternativo , Animales , Proteínas de Unión al ADN/genética , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteína Potenciadora del Homólogo Zeste 2 , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Inmunoprecipitación , Proteínas Nucleares/genética , Complejo Represivo Polycomb 2 , Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Factores de Transcripción/genética , TransfecciónRESUMEN
Repeat elements can be dysregulated at a genome-wide scale in human diseases. For example, in Ewing sarcoma, hundreds of inert GGAA repeats can be converted into active enhancers when bound by EWS-FLI1. Here we show that fusions between EWS and GGAA-repeat-targeted engineered zinc finger arrays (ZFAs) can function at least as efficiently as EWS-FLI1 for converting hundreds of GGAA repeats into active enhancers in a Ewing sarcoma precursor cell model. Furthermore, a fusion of a KRAB domain to a ZFA can silence GGAA microsatellite enhancers genome wide in Ewing sarcoma cells, thereby reducing expression of EWS-FLI1-activated genes. Remarkably, this KRAB-ZFA fusion showed selective toxicity against Ewing sarcoma cells compared with non-Ewing cancer cells, consistent with its Ewing sarcoma-specific impact on the transcriptome. These findings demonstrate the value of ZFAs for functional annotation of repeats and illustrate how aberrant microsatellite activities might be regulated for potential therapeutic applications.
RESUMEN
Oncogenic fusion proteins generated by chromosomal translocations play major roles in cancer. Among them, fusions between EWSR1 and transcription factors generate oncogenes with powerful chromatin regulatory activities, capable of establishing complex gene expression programs in permissive precursor cells. Here we define the epigenetic and 3D connectivity landscape of Clear Cell Sarcoma, an aggressive cancer driven by the EWSR1-ATF1 fusion gene. We find that EWSR1-ATF1 displays a distinct DNA binding pattern that requires the EWSR1 domain and promotes ATF1 retargeting to new distal sites, leading to chromatin activation and the establishment of a 3D network that controls oncogenic and differentiation signatures observed in primary CCS tumors. Conversely, EWSR1-ATF1 depletion results in a marked reconfiguration of 3D connectivity, including the emergence of regulatory circuits that promote neural crest-related developmental programs. Taken together, our study elucidates the epigenetic mechanisms utilized by EWSR1-ATF1 to establish regulatory networks in CCS, and points to precursor cells in the neural crest lineage as candidate cells of origin for these tumors.
Asunto(s)
Sarcoma de Células Claras , Neoplasias de los Tejidos Blandos , Carcinogénesis/genética , Cromatina/genética , Humanos , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Oncogenes , Proteína EWS de Unión a ARN/genética , Sarcoma de Células Claras/genética , Sarcoma de Células Claras/patología , Neoplasias de los Tejidos Blandos/genéticaRESUMEN
Synovial sarcoma (SyS) is an aggressive mesenchymal malignancy invariably associated with the chromosomal translocation t(X:18; p11:q11), which results in the in-frame fusion of the BAF complex gene SS18 to one of three SSX genes. Fusion of SS18 to SSX generates an aberrant transcriptional regulator, which, in permissive cells, drives tumor development by initiating major chromatin remodeling events that disrupt the balance between BAF-mediated gene activation and polycomb-dependent repression. Here, we developed SyS organoids and performed genome-wide epigenomic profiling of these models and mesenchymal precursors to define SyS-specific chromatin remodeling mechanisms and dependencies. We show that SS18-SSX induces broad BAF domains at its binding sites, which oppose polycomb repressor complex (PRC) 2 activity, while facilitating recruitment of a non-canonical (nc)PRC1 variant. Along with the uncoupling of polycomb complexes, we observed H3K27me3 eviction, H2AK119ub deposition and the establishment of de novo active regulatory elements that drive SyS identity. These alterations are completely reversible upon SS18-SSX depletion and are associated with vulnerability to USP7 loss, a core member of ncPRC1.1. Using the power of primary tumor organoids, our work helps define the mechanisms of epigenetic dysregulation on which SyS cells are dependent.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/genética , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Sarcoma Sinovial/genética , Sitios de Unión , Cromatina/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica , Histonas/metabolismo , Humanos , Complejos Multiproteicos/metabolismo , Organoides , Unión Proteica , Transporte de Proteínas , Sarcoma Sinovial/metabolismo , TranscriptomaRESUMEN
Synovial sarcoma (SyS) is an aggressive neoplasm driven by the SS18-SSX fusion, and is characterized by low T cell infiltration. Here, we studied the cancer-immune interplay in SyS using an integrative approach that combines single-cell RNA sequencing (scRNA-seq), spatial profiling and genetic and pharmacological perturbations. scRNA-seq of 16,872 cells from 12 human SyS tumors uncovered a malignant subpopulation that marks immune-deprived niches in situ and is predictive of poor clinical outcomes in two independent cohorts. Functional analyses revealed that this malignant cell state is controlled by the SS18-SSX fusion, is repressed by cytokines secreted by macrophages and T cells, and can be synergistically targeted with a combination of HDAC and CDK4/CDK6 inhibitors. This drug combination enhanced malignant-cell immunogenicity in SyS models, leading to induced T cell reactivity and T cell-mediated killing. Our study provides a blueprint for investigating heterogeneity in fusion-driven malignancies and demonstrates an interplay between immune evasion and oncogenic processes that can be co-targeted in SyS and potentially in other malignancies.
Asunto(s)
Carcinogénesis/genética , Terapia Molecular Dirigida , Proteínas de Fusión Oncogénica/genética , Sarcoma Sinovial/tratamiento farmacológico , Línea Celular Tumoral , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Inhibidores de Histona Desacetilasas/uso terapéutico , Histona Desacetilasas/genética , Histona Desacetilasas/uso terapéutico , Humanos , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Oncogenes/genética , RNA-Seq , Sarcoma Sinovial/genética , Sarcoma Sinovial/patología , Análisis de la Célula IndividualRESUMEN
HIC1 (Hypermethylated In Cancer 1) a tumor suppressor gene located at 17p13.3, is frequently deleted or epigenetically silenced in many human tumors. HIC1 encodes a transcriptional repressor involved in various aspects of the DNA damage response and in complex regulatory loops with P53 and SIRT1. HIC1 expression in normal prostate tissues has not yet been investigated in detail. Here, we demonstrated by immunohistochemistry that detectable HIC1 expression is restricted to the stroma of both normal and tumor prostate tissues. By RT-qPCR, we showed that HIC1 is poorly expressed in all tested prostate epithelial lineage cell types: primary (PrEC), immortalized (RWPE1) or transformed androgen-dependent (LnCAP) or androgen-independent (PC3 and DU145) prostate epithelial cells. By contrast, HIC1 is strongly expressed in primary PrSMC and immortalized (WMPY-1) prostate myofibroblastic cells. HIC1 depletion in WPMY-1 cells induced decreases in α-SMA expression and contractile capability. In addition to SLUG, we identified stromal cell-derived factor 1/C-X-C motif chemokine 12 (SDF1/CXCL12) as a new HIC1 direct target-gene. Thus, our results identify HIC1 as a tumor suppressor gene which is poorly expressed in the epithelial cells targeted by the tumorigenic process. HIC1 is expressed in stromal myofibroblasts and regulates CXCL12/SDF1 expression, thereby highlighting a complex interplay mediating the tumor promoting activity of the tumor microenvironment. Our studies provide new insights into the role of HIC1 in normal prostatic epithelial-stromal interactions through direct repression of CXCL12 and new mechanistic clues on how its loss of function through promoter hypermethylation during aging could contribute to prostatic tumors.
RESUMEN
Polycomb repressive complex 2 (PRC2) allows the deposition of H3K27me3. PRC2 facultative subunits modulate its activity and recruitment such as hPCL3/PHF19, a human ortholog of Drosophila Polycomb-like protein (PCL). These proteins contain a TUDOR domain binding H3K36me3, two PHD domains and a "Winged-helix" domain involved in GC-rich DNA binding. The human PCL3 locus encodes the full-length hPCL3L protein and a shorter isoform, hPCL3S containing the TUDOR and PHD1 domains only. In this study, we demonstrated by RT-qPCR analyses of 25 prostate tumors that hPCL3S is frequently up-regulated. In addition, hPCL3S is overexpressed in the androgen-independent DU145 and PC3 cells, but not in the androgen-dependent LNCaP cells. hPCL3S knockdown decreased the proliferation and migration of DU145 and PC3 whereas its forced expression into LNCaP increased these properties. A mutant hPCL3S unable to bind H3K36me3 (TUDOR-W50A) increased proliferation and migration of LNCaP similarly to wt hPCL3S whereas inactivation of its PHD1 domain decreased proliferation. These effects partially relied on the up-regulation of genes known to be important for the proliferation and/or migration of prostate cancer cells such as S100A16, PlexinA2, and Spondin1. Collectively, our results suggest hPCL3S as a new potential therapeutic target in castration resistant prostate cancers.
RESUMEN
Ewing sarcoma (EwS) is associated with poor prognosis despite current multimodal therapy. Targeting of EWS-FLI1, the fusion protein responsible for its pathogenesis, and its principal downstream targets has not yet produced satisfactory therapeutic options, fueling the search for alternative approaches. Here, we show that the oncofetal RNA-binding protein LIN28B regulates the stability of EWS-FLI1 mRNA in ~10% of EwSs. LIN28B depletion in these tumors leads to a decrease in the expression of EWS-FLI1 and its direct transcriptional network, abrogating EwS cell self-renewal and tumorigenicity. Moreover, pharmacological inhibition of LIN28B mimics the effect of LIN28B depletion, suggesting that LIN28B sustains the emergence of a subset of EwS in which it also serves as an effective therapeutic target.