RESUMEN
Triple-negative breast cancer (TNBC) is a heterogeneous and clinically aggressive disease for which there is no targeted therapy. BET bromodomain inhibitors, which have shown efficacy in several models of cancer, have not been evaluated in TNBC. These inhibitors displace BET bromodomain proteins such as BRD4 from chromatin by competing with their acetyl-lysine recognition modules, leading to inhibition of oncogenic transcriptional programs. Here we report the preferential sensitivity of TNBCs to BET bromodomain inhibition in vitro and in vivo, establishing a rationale for clinical investigation and further motivation to understand mechanisms of resistance. In paired cell lines selected for acquired resistance to BET inhibition from previously sensitive TNBCs, we failed to identify gatekeeper mutations, new driver events or drug pump activation. BET-resistant TNBC cells remain dependent on wild-type BRD4, which supports transcription and cell proliferation in a bromodomain-independent manner. Proteomic studies of resistant TNBC identify strong association with MED1 and hyper-phosphorylation of BRD4 attributable to decreased activity of PP2A, identified here as a principal BRD4 serine phosphatase. Together, these studies provide a rationale for BET inhibition in TNBC and present mechanism-based combination strategies to anticipate clinical drug resistance.
Asunto(s)
Azepinas/farmacología , Azepinas/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas Nucleares/antagonistas & inhibidores , Estructura Terciaria de Proteína/efectos de los fármacos , Factores de Transcripción/antagonistas & inhibidores , Triazoles/farmacología , Triazoles/uso terapéutico , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Animales , Unión Competitiva/efectos de los fármacos , Quinasa de la Caseína II/metabolismo , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Cromatina/genética , Cromatina/metabolismo , Resistencia a Antineoplásicos/genética , Epigénesis Genética/efectos de los fármacos , Epigénesis Genética/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genoma Humano/efectos de los fármacos , Genoma Humano/genética , Humanos , Subunidad 1 del Complejo Mediador/metabolismo , Ratones , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Unión Proteica/efectos de los fármacos , Proteína Fosfatasa 2/metabolismo , Proteómica , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Sequencing of DNase I hypersensitive sites (DNase-seq) is a powerful technique for identifying cis-regulatory elements across the genome. We studied the key experimental parameters to optimize performance of DNase-seq. Sequencing short fragments of 50-100 base pairs (bp) that accumulate in long internucleosome linker regions was more efficient for identifying transcription factor binding sites compared to sequencing longer fragments. We also assessed the potential of DNase-seq to predict transcription factor occupancy via generation of nucleotide-resolution transcription factor footprints. In modeling the sequence-specific DNase I cutting bias, we found a strong effect that varied over more than two orders of magnitude. This indicates that the nucleotide-resolution cleavage patterns at many transcription factor binding sites are derived from intrinsic DNase I cleavage bias rather than from specific protein-DNA interactions. In contrast, quantitative comparison of DNase I hypersensitivity between states can predict transcription factor occupancy associated with particular biological perturbations.
Asunto(s)
Desoxirribonucleasa I/química , Redes Reguladoras de Genes , Análisis de Secuencia de ADN/métodos , Factores de Transcripción/química , Secuencias de Aminoácidos , Sitios de Unión , Línea Celular Tumoral , Cromatina/química , Femenino , Regulación de la Expresión Génica , Humanos , Células K562 , Células MCF-7 , Masculino , Nucleosomas/química , Nucleótidos/química , Receptores Androgénicos/química , Proteína p53 Supresora de Tumor/químicaRESUMEN
SUMMARY: Chromatin immunoprecipitation and DNase I hypersensitivity assays with high-throughput sequencing have greatly accelerated the understanding of transcriptional and epigenetic regulation, although data reuse for the community of experimental biologists has been challenging. We created a data portal CistromeFinder that can help query, evaluate and visualize publicly available Chromatin immunoprecipitation and DNase I hypersensitivity assays with high-throughput sequencing data in human and mouse. The database currently contains 6378 samples over 4391 datasets, 313 factors and 102 cell lines or cell populations. Each dataset has gone through a consistent analysis and quality control pipeline; therefore, users could evaluate the overall quality of each dataset before examining binding sites near their genes of interest. CistromeFinder is integrated with UCSC genome browser for visualization, Primer3Plus for ChIP-qPCR primer design and CistromeMap for submitting newly available datasets. It also allows users to leave comments to facilitate data evaluation and update. AVAILABILITY: http://cistrome.org/finder. CONTACT: xsliu@jimmy.harvard.edu or henry_long@dfci.harvard.edu.
Asunto(s)
Inmunoprecipitación de Cromatina , Bases de Datos Genéticas , Secuenciación de Nucleótidos de Alto Rendimiento , Almacenamiento y Recuperación de la Información , Animales , Línea Celular , Desoxirribonucleasa I/metabolismo , Epigénesis Genética , Humanos , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Programas InformáticosRESUMEN
MLL-AF4 acute lymphocytic leukemia (ALL) has a poor prognosis. MicroRNAs (miRNA) are small noncoding RNAs that posttranscriptionally regulate expression of target mRNAs. Our analysis of previously published data showed that expression of miR-128b and miR-221 is down-regulated in MLL-rearranged ALL relative to other types of ALL. Reexpression of these miRNAs cooperatively sensitizes 2 cultured lines of MLL-AF4 ALL cells to glucocorticoids. Target genes down-regulated by miR-128b include MLL, AF4, and both MLL-AF4 and AF4-MLL fusion genes; miR-221 down-regulates CDKN1B. These results demonstrate that down-regulation of miR-128b and miR-221 is implicated in glucocorticoid resistance and that restoration of their levels is a potentially promising therapeutic in MLL-AF4 ALL.
Asunto(s)
MicroARNs/genética , MicroARNs/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Sitios de Unión/genética , Línea Celular Tumoral , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Dexametasona/farmacología , Regulación hacia Abajo , Resistencia a Antineoplásicos/genética , Expresión Génica , Glucocorticoides/farmacología , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , TransfecciónRESUMEN
Rhabdomyosarcoma is the most common soft tissue sarcoma in the pediatric population. As this tumor has an undifferentiated myogenic phenotype, agents that promote differentiation hold particular promise as part of a novel therapeutic approach to combat this type of cancer. In this report, we focus on the contribution of two microRNAs (miRNAs) in rhabdomyosarcomas. Levels of miR-1 and miR-133a are drastically reduced in representative cell lines from each major rhabdomyosarcoma subtype (embryonal and alveolar). Introduction of miR-1 and miR-133a into an embryonal rhabdomyosarcoma-derived cell line is cytostatic, thereby suggesting a tumor suppressor-like role for these myogenic miRNAs. Transcriptional profiling of cells after miR-1 and miR-133a expression reveals that miR-1 (but not miR-133a) exerts a strong promyogenic influence on these poorly differentiated tumor cells. We identify mRNAs that are down-regulated by these miRNAs and propose roles for miR-1 and miR-133a in repressing isoforms of genes that are normally not expressed in muscle. Finally, we show that mRNA targets of miR-1 and miR-133a are up-regulated in rhabdomyosarcomas, suggesting a causative role for these miRNAs in the development of rhabdomyosarcomas. More important, these results point to the promise of enhancing rhabdomyosarcoma therapy using miRNAs as agents that mediate cytostasis and promote muscle differentiation.
Asunto(s)
MicroARNs/metabolismo , Rabdomiosarcoma/genética , Rabdomiosarcoma/patología , Diferenciación Celular/genética , Línea Celular Tumoral , Proliferación Celular , Perfilación de la Expresión Génica , Humanos , MicroARNs/genéticaRESUMEN
RATIONALE: Heart failure is a deadly and devastating disease that places immense costs on an aging society. To develop therapies aimed at rescuing the failing heart, it is important to understand the molecular mechanisms underlying cardiomyocyte structure and function. OBJECTIVE: microRNAs are important regulators of gene expression, and we sought to define the global contributions made by microRNAs toward maintaining cardiomyocyte integrity. METHODS AND RESULTS: First, we performed deep sequencing analysis to catalog the miRNA population in the adult heart. Second, we genetically deleted, in cardiac myocytes, an essential component of the machinery that is required to generate miRNAs. Deep sequencing of miRNAs from the heart revealed the enrichment of a small number of microRNAs with one, miR-1, accounting for 40% of all microRNAs. Cardiomyocyte-specific deletion of dgcr8, a gene required for microRNA biogenesis, revealed a fully penetrant phenotype that begins with left ventricular malfunction progressing to a dilated cardiomyopathy and premature lethality. CONCLUSIONS: These observations reveal a critical role for microRNAs in maintaining cardiac function in mature cardiomyocytes and raise the possibility that only a handful of microRNAs may ultimately be responsible for the dramatic cardiac phenotype seen in the absence of dgcr8.
Asunto(s)
Cardiomiopatía Dilatada/genética , Insuficiencia Cardíaca/genética , MicroARNs/genética , Proteínas/genética , Animales , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/patología , Eliminación de Gen , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Ratones , Ratones Noqueados , MicroARNs/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Proteínas/metabolismo , Proteínas de Unión al ARNRESUMEN
Sustained treatment of estrogen receptor (ER)-positive breast cancer with ER-targeting drugs results in ER mutations and refractory unresponsive cancers. Androgen receptor (AR), which is expressed in 80%-95% of ER-positive breast cancers, could serve as an alternate therapeutic target. Although AR agonists were used in the past to treat breast cancer, their use is currently infrequent due to virilizing side effects. Discovery of tissue-selective AR modulators (SARMs) has renewed interest in using AR agonists to treat breast cancer. Using translational models, we show that AR agonist and SARM, but not antagonist, inhibit the proliferation and growth of ER-positive breast cancer cells, patient-derived tissues, and patient-derived xenografts (PDX). Ligand-activated AR inhibits wild-type and mutant ER activity by reprogramming the ER and FOXA1 cistrome and rendering tumor growth inhibition. These findings suggest that ligand-activated AR may function as a non-canonical inhibitor of ER and that AR agonists may offer a safe and effective treatment for ER-positive breast cancer.
RESUMEN
Androgen deprivation therapy for prostate cancer (PCa) benefits patients with early disease, but becomes ineffective as PCa progresses to a castration-resistant state (CRPC). Initially CRPC remains dependent on androgen receptor (AR) signaling, often through increased expression of full-length AR (ARfl) or expression of dominantly active splice variants such as ARv7. We show in ARv7-dependent CRPC models that ARv7 binds together with ARfl to repress transcription of a set of growth-suppressive genes. Expression of the ARv7-repressed targets and ARv7 protein expression are negatively correlated and predicts for outcome in PCa patients. Our results provide insights into the role of ARv7 in CRPC and define a set of potential biomarkers for tumors dependent on ARv7.
Asunto(s)
Empalme Alternativo , Neoplasias de la Próstata Resistentes a la Castración/genética , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Análisis de Matrices Tisulares , Transcripción GenéticaRESUMEN
The process of adipogenesis involves a complex program of gene expression that includes down-regulation of the gene encoding Hes-1, a target of the Notch signaling pathway. To determine if Notch signaling affects adipogenesis, we exposed 3T3-L1 preadipocytes to the Notch ligand Jagged1 and found that differentiation was significantly reduced. This effect could be mimicked by constitutive expression of Hes-1. The block was associated with a complete loss of C/EBPalpha and peroxisome proliferator-activated receptor gamma (PPARgamma) induction and could be overcome by retroviral expression of either C/EBPalpha or PPARgamma2. Surprisingly, small interfering RNA (siRNA)-mediated reduction of Hes-1 mRNA in 3T3-L1 cells also inhibited differentiation, suggesting an additional, obligatory role for Hes-1 in adipogenesis. This role may be related to our observation that both Notch signaling and Hes-1 down-regulate transcription of the gene encoding DLK/Pref-1, a protein known to inhibit differentiation of 3T3-L1 cells. The results presented in this study establish a new target downstream of the Notch-Hes-1 pathway and suggest a dual role for Hes-1 in adipocyte development.
Asunto(s)
Adipocitos/fisiología , Diferenciación Celular/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal/fisiología , Células 3T3 , Adipocitos/citología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Proteínas de Unión al Calcio , Regulación hacia Abajo , Proteínas de Homeodominio/genética , Péptidos y Proteínas de Señalización Intercelular , Proteína Jagged-1 , Proteínas de la Membrana/genética , Ratones , Proteínas/metabolismo , ARN Interferente Pequeño/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores Notch , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteínas Serrate-Jagged , Factor de Transcripción HES-1 , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
While the multiple endocrine neoplasia type 1 (MEN1) gene functions as a tumor suppressor in a variety of cancer types, we explored its oncogenic role in breast tumorigenesis. The MEN1 gene product menin is involved in H3K4 trimethylation and co-activates transcription. We integrated ChIP-seq and RNA-seq data to identify menin target genes. Our analysis revealed that menin-dependent target gene promoters display looping to distal enhancers that are bound by menin, FOXA1 and GATA3. In this fashion, MEN1 co-regulates a proliferative breast cancer-specific gene expression program in ER+ cells. In primary mammary cells, MEN1 exerts an anti-proliferative function by regulating a distinct expression signature. Our findings clarify the cell-type-specific functions of MEN1 and inform the development of menin-directed treatments for breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Elementos de Facilitación Genéticos/genética , Oncogenes , Proteínas Proto-Oncogénicas/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular , Estradiol/farmacología , Receptor alfa de Estrógeno/metabolismo , Femenino , Factor de Transcripción GATA3/metabolismo , Regulación Neoplásica de la Expresión Génica , Genómica , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilación , Regiones Promotoras Genéticas , Unión Proteica/efectos de los fármacos , Transcripción GenéticaRESUMEN
UNLABELLED: As a master regulator of chromatin function, the lysine methyltransferase EZH2 orchestrates transcriptional silencing of developmental gene networks. Overexpression of EZH2 is commonly observed in human epithelial cancers, such as non-small cell lung carcinoma (NSCLC), yet definitive demonstration of malignant transformation by deregulated EZH2 remains elusive. Here, we demonstrate the causal role of EZH2 overexpression in NSCLC with new genetically engineered mouse models of lung adenocarcinoma. Deregulated EZH2 silences normal developmental pathways, leading to epigenetic transformation independent of canonical growth factor pathway activation. As such, tumors feature a transcriptional program distinct from KRAS- and EGFR-mutant mouse lung cancers, but shared with human lung adenocarcinomas exhibiting high EZH2 expression. To target EZH2-dependent cancers, we developed a potent open-source EZH2 inhibitor, JQEZ5, that promoted the regression of EZH2-driven tumors in vivo, confirming oncogenic addiction to EZH2 in established tumors and providing the rationale for epigenetic therapy in a subset of lung cancer. SIGNIFICANCE: EZH2 overexpression induces murine lung cancers that are similar to human NSCLC with high EZH2 expression and low levels of phosphorylated AKT and ERK, implicating biomarkers for EZH2 inhibitor sensitivity. Our EZH2 inhibitor, JQEZ5, promotes regression of these tumors, revealing a potential role for anti-EZH2 therapy in lung cancer. Cancer Discov; 6(9); 1006-21. ©2016 AACR.See related commentary by Frankel et al., p. 949This article is highlighted in the In This Issue feature, p. 932.
Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Cromatina/genética , Cromatina/metabolismo , Modelos Animales de Enfermedad , Diseño de Fármacos , Elementos de Facilitación Genéticos , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Expresión Génica , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Imagen por Resonancia Magnética , Ratones , Modelos Moleculares , Conformación Molecular , Terapia Molecular Dirigida , Regiones Promotoras Genéticas , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Benign plexiform neurofibromas in NF1 patients can transform spontaneously into malignant peripheral nerve sheath tumors (MPNSTs). Although mutations in the p53 gene have been found in a subset of MPNSTs and mouse models support a role for p53 mutations in malignant conversion, we found that each of three Schwann cell lines derived from human MPNSTs possessed active p53. One of the lines expressed the Notch intracellular domain (NICD), indicative of ongoing Notch signaling. Consistent with a role in malignancy, NICD was able to transform primary rat Schwann cells. Transformation was robust--NICD-transduced cells generated tumors in nude rats--and was associated with the loss of markers associated with Schwann cell differentiation. These data suggest that aberrant Notch signaling may contribute to the conversion of benign neurofibromas to MPNSTs.
Asunto(s)
Transformación Celular Neoplásica , Proteínas de la Membrana/metabolismo , Células de Schwann/metabolismo , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Neoplasias del Sistema Nervioso Periférico/genética , Neoplasias del Sistema Nervioso Periférico/metabolismo , Estructura Terciaria de Proteína , Ratas , Ratas Desnudas , Receptores Notch , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Since their discovery as key regulators of early animal development, microRNAs now are recognized as widespread regulators of gene expression. Despite their abundance, little is known regarding the regulation of microRNA biogenesis. We show that three highly conserved muscle-specific microRNAs, miR-1, miR-133 and miR-206, are robustly induced during the myoblast-myotube transition, both in primary human myoblasts and in the mouse mesenchymal C2C12 stem cell line. These microRNAs were not induced during osteogenic conversion of C2C12 cells. Moreover, both loci encoding miR-1, miR-1-1, and miR-1-2, and two of the three encoding miR-133, miR-133a-1 and miR-133a-2, are strongly induced during myogenesis. Some of the induced microRNAs are in intergenic regions, whereas two are transcribed in the opposite direction to the nonmuscle-specific gene in which they are embedded. By using CHIP analysis, we demonstrate that the myogenic factors Myogenin and MyoD bind to regions upstream of these microRNAs and, therefore, are likely to regulate their expression. Because miR-1 and miR-206 are predicted to repress similar mRNA targets, our work suggests that induction of these microRNAs is important in regulating the expression of muscle-specific proteins.