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1.
Nature ; 529(7586): 413-417, 2016 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-26735014

RESUMO

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.


Assuntos
Azepinas/farmacologia , Azepinas/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas Nucleares/antagonistas & inibidores , Estrutura Terciária de Proteína/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Triazóis/farmacologia , Triazóis/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Ligação Competitiva/efeitos dos fármacos , Caseína Quinase II/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Cromatina/genética , Cromatina/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Epigênese Genética/efeitos dos fármacos , Epigênese Genética/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genoma Humano/efeitos dos fármacos , Genoma Humano/genética , Humanos , Subunidade 1 do Complexo Mediador/metabolismo , Camundongos , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteína Fosfatase 2/metabolismo , Proteômica , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Nat Methods ; 11(1): 73-78, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24317252

RESUMO

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.


Assuntos
Desoxirribonuclease I/química , Redes Reguladoras de Genes , Análise de Sequência de DNA/métodos , Fatores de Transcrição/química , Motivos de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Cromatina/química , Feminino , Regulação da Expressão Gênica , Humanos , Células K562 , Células MCF-7 , Masculino , Nucleossomos/química , Nucleotídeos/química , Receptores Androgênicos/química , Proteína Supressora de Tumor p53/química
3.
Bioinformatics ; 29(10): 1352-4, 2013 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-23508969

RESUMO

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.


Assuntos
Imunoprecipitação da Cromatina , Bases de Dados Genéticas , Sequenciamento de Nucleotídeos em Larga Escala , Armazenamento e Recuperação da Informação , Animais , Linhagem Celular , Desoxirribonuclease I/metabolismo , Epigênese Genética , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Software
4.
Blood ; 114(19): 4169-78, 2009 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-19749093

RESUMO

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.


Assuntos
MicroRNAs/genética , MicroRNAs/metabolismo , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Sítios de Ligação/genética , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p27 , Dexametasona/farmacologia , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/genética , Expressão Gênica , Glucocorticoides/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Transfecção
5.
FASEB J ; 24(9): 3427-37, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20466878

RESUMO

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.


Assuntos
MicroRNAs/metabolismo , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Perfilação da Expressão Gênica , Humanos , MicroRNAs/genética
6.
Circ Res ; 105(6): 585-94, 2009 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-19679836

RESUMO

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.


Assuntos
Cardiomiopatia Dilatada/genética , Insuficiência Cardíaca/genética , MicroRNAs/genética , Proteínas/genética , Animais , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/patologia , Deleção de Genes , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Camundongos , Camundongos Knockout , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Proteínas/metabolismo , Proteínas de Ligação a RNA
7.
iScience ; 21: 341-358, 2019 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-31698248

RESUMO

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.

8.
Cancer Cell ; 35(3): 401-413.e6, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30773341

RESUMO

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.


Assuntos
Processamento Alternativo , Neoplasias de Próstata Resistentes à Castração/genética , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Neoplasias de Próstata Resistentes à Castração/metabolismo , Análise Serial de Tecidos , Transcrição Gênica
9.
Mol Cell Biol ; 24(8): 3505-13, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15060169

RESUMO

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.


Assuntos
Adipócitos/fisiologia , Diferenciação Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais/fisiologia , Células 3T3 , Adipócitos/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteínas de Ligação ao Cálcio , Regulação para Baixo , Proteínas de Homeodomínio/genética , Peptídeos e Proteínas de Sinalização Intercelular , Proteína Jagged-1 , Proteínas de Membrana/genética , Camundongos , Proteínas/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Notch , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Serrate-Jagged , Fatores de Transcrição HES-1 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
Cell Rep ; 18(10): 2359-2372, 2017 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-28273452

RESUMO

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.


Assuntos
Neoplasias da Mama/genética , Elementos Facilitadores Genéticos/genética , Oncogenes , Proteínas Proto-Oncogênicas/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Feminino , Fator de Transcrição GATA3/metabolismo , Regulação Neoplásica da Expressão Gênica , Genômica , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Regiões Promotoras Genéticas , Ligação Proteica/efeitos dos fármacos , Transcrição Gênica
11.
Cancer Discov ; 6(9): 1006-21, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27312177

RESUMO

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.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Cromatina/genética , Cromatina/metabolismo , Modelos Animais de Doenças , Desenho de Fármacos , Elementos Facilitadores Genéticos , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Imageamento por Ressonância Magnética , Camundongos , Modelos Moleculares , Conformação Molecular , Terapia de Alvo Molecular , Regiões Promotoras Genéticas , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Oncogene ; 23(5): 1146-52, 2004 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-14762442

RESUMO

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.


Assuntos
Transformação Celular Neoplásica , Proteínas de Membrana/metabolismo , Células de Schwann/metabolismo , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Neoplasias do Sistema Nervoso Periférico/genética , Neoplasias do Sistema Nervoso Periférico/metabolismo , Estrutura Terciária de Proteína , Ratos , Ratos Nus , Receptores Notch , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo
13.
Proc Natl Acad Sci U S A ; 103(23): 8721-6, 2006 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-16731620

RESUMO

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.


Assuntos
Regulação da Expressão Gênica , MicroRNAs/genética , Músculos/metabolismo , Animais , Células Cultivadas , Cromossomos de Mamíferos/genética , Éxons/genética , Genoma/genética , Humanos , Camundongos , MicroRNAs/metabolismo , Proteína MyoD/metabolismo , Mioblastos/metabolismo , Miogenina/metabolismo , Especificidade de Órgãos , Ligação Proteica , Fatores de Tempo
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