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1.
Nucleic Acids Res ; 41(22): 10241-53, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24049075

RESUMO

The ETS domain transcription factor Elk-1 stimulates expression of immediate early genes (IEGs) in response to mitogens. These events require phosphorylation of Elk-1 by extracellular signal-regulated kinase (ERK) and phosphorylation-dependent interaction of Elk-1 with co-activators, including histone acetyltransferases and the Mediator complex. Elk-1 also recruits ERK to the promoters of its target genes, suggesting that ERK phosphorylates additional substrates in transcription complexes at mitogen-responsive promoters. Here we report that MED14, a core subunit of the Mediator, is a bona fide ERK substrate and identify serine 986 (S986) within a serine-proline rich region of MED14 as the major ERK phosphorylation site. Mitogens induced phosphorylation of MED14 on S986 at IEG promoters; RNAi knockdown of MED14 reduced CDK8 and RNA polymerase II (RNAPII) recruitment, RNAPII C-terminal domain phosphorylation and impaired activation of IEG transcription. A single alanine substitution at S986 reduced activation of an E26 (ETS)-responsive reporter by oncogenic Ras and mitogen-induced, Elk-1-dependent transcription, whereas activities of other transcriptional activators were unaffected. We also demonstrate that Elk-1 can associate with MED14 independently of MED23, which may facilitate phosphorylation of MED14 by ERK to impart a positive and selective impact on mitogen-responsive gene expression.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Complexo Mediador/metabolismo , Mitógenos/farmacologia , Regiões Promotoras Genéticas , Ativação Transcricional , Proteínas Elk-1 do Domínio ets/metabolismo , Animais , Genes Precoces , Genes ras , Células HEK293 , Células HeLa , Humanos , Complexo Mediador/genética , Camundongos , Mutação , Células NIH 3T3 , Fosforilação
2.
bioRxiv ; 2024 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-37645893

RESUMO

Tumors may contain billions of cells including distinct malignant clones and nonmalignant cell types. Clarifying the evolutionary histories, prevalence, and defining molecular features of these cells is essential for improving clinical outcomes, since intratumoral heterogeneity provides fuel for acquired resistance to targeted therapies. Here we present a statistically motivated strategy for deconstructing intratumoral heterogeneity through multiomic and multiscale analysis of serial tumor sections (MOMA). By combining deep sampling of IDH-mutant astrocytomas with integrative analysis of single-nucleotide variants, copy-number variants, and gene expression, we reconstruct and validate the phylogenies, spatial distributions, and transcriptional profiles of distinct malignant clones. By genotyping nuclei analyzed by single-nucleus RNA-seq for truncal mutations, we further show that commonly used algorithms for identifying cancer cells from single-cell transcriptomes may be inaccurate. We also demonstrate that correlating gene expression with tumor purity in bulk samples can reveal optimal markers of malignant cells and use this approach to identify a core set of genes that is consistently expressed by astrocytoma truncal clones, including AKR1C3, whose expression is associated with poor outcomes in several types of cancer. In summary, MOMA provides a robust and flexible strategy for precisely deconstructing intratumoral heterogeneity and clarifying the core molecular properties of distinct cellular populations in solid tumors.

3.
Cancers (Basel) ; 16(13)2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-39001492

RESUMO

Tumors may contain billions of cells, including distinct malignant clones and nonmalignant cell types. Clarifying the evolutionary histories, prevalence, and defining molecular features of these cells is essential for improving clinical outcomes, since intratumoral heterogeneity provides fuel for acquired resistance to targeted therapies. Here we present a statistically motivated strategy for deconstructing intratumoral heterogeneity through multiomic and multiscale analysis of serial tumor sections (MOMA). By combining deep sampling of IDH-mutant astrocytomas with integrative analysis of single-nucleotide variants, copy-number variants, and gene expression, we reconstruct and validate the phylogenies, spatial distributions, and transcriptional profiles of distinct malignant clones. By genotyping nuclei analyzed by single-nucleus RNA-seq for truncal mutations, we further show that commonly used algorithms for identifying cancer cells from single-cell transcriptomes may be inaccurate. We also demonstrate that correlating gene expression with tumor purity in bulk samples can reveal optimal markers of malignant cells and use this approach to identify a core set of genes that are consistently expressed by astrocytoma truncal clones, including AKR1C3, whose expression is associated with poor outcomes in several types of cancer. In summary, MOMA provides a robust and flexible strategy for precisely deconstructing intratumoral heterogeneity and clarifying the core molecular properties of distinct cellular populations in solid tumors.

4.
Nucleic Acids Res ; 39(15): 6390-402, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21543455

RESUMO

The ETS (E26) protein Elk-1 serves as a paradigm for mitogen-responsive transcription factors. It is multiply phosphorylated by mitogen-activated protein kinases (MAPKs), which it recruits into pre-initiation complexes on target gene promoters. However, events preparatory to Elk-1 phosphorylation are less well understood. Here, we identify two novel, functional elements in Elk-1 that determine its stability and nuclear accumulation. One element corresponds to a dimerization interface in the ETS domain and the second is a cryptic degron adjacent to the serum response factor (SRF)-interaction domain that marks dimerization-defective Elk-1 for rapid degradation by the ubiquitin-proteasome system. Dimerization appears to be crucial for Elk-1 stability only in the cytoplasm, as latent Elk-1 accumulates in the nucleus and interacts dynamically with DNA as a monomer. These findings define a novel role for the ETS domain of Elk-1 and demonstrate that nuclear accumulation of Elk-1 involves conformational flexibility prior to its phosphorylation by MAPKs.


Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas Elk-1 do Domínio ets/química , Sequência de Aminoácidos , Linhagem Celular , DNA/metabolismo , Dimerização , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Conformação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Deleção de Sequência , Proteínas Elk-1 do Domínio ets/metabolismo
5.
J Invest Dermatol ; 140(1): 164-173.e7, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31580842

RESUMO

The use of microRNAs as biomarkers has been proposed for many diseases, including the diagnosis of melanoma. Although hundreds of microRNAs have been identified as differentially expressed in melanomas as compared to benign melanocytic lesions, a limited consensus has been achieved across studies, constraining the effective use of these potentially useful markers. In this study, we applied a machine learning-based pipeline to a dataset consisting of genetic features, clinical features, and next-generation microRNA sequencing from micro-dissected formalin-fixed paraffin embedded melanomas and their adjacent benign precursor nevi. We identified patient age and tumor cellularity as variables that frequently confound the measured expression of potentially diagnostic microRNAs. By employing the ratios of microRNAs that were either enriched or depleted in melanoma compared to the nevi as a normalization strategy, we developed a model that classified all the available published cohorts with an area under the receiver operating characteristic curve of 0.98. External validation on an independent cohort classified lesions with 81% sensitivity and 88% specificity and was uninfluenced by the tumor content of the sample or patient age.


Assuntos
Biomarcadores Tumorais/genética , Melanócitos/fisiologia , Melanoma/diagnóstico , MicroRNAs/genética , Nevo/diagnóstico , Neoplasias Cutâneas/diagnóstico , Conjuntos de Dados como Assunto , Diagnóstico Diferencial , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Aprendizado de Máquina , Prognóstico , Curva ROC , Sensibilidade e Especificidade , Análise de Sequência de RNA
6.
Cancer Cell ; 33(5): 874-889.e7, 2018 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-29681511

RESUMO

Gliomas comprise heterogeneous malignant glial and stromal cells. While blood vessel co-option is a potential mechanism to escape anti-angiogenic therapy, the relevance of glial phenotype in this process is unclear. We show that Olig2+ oligodendrocyte precursor-like glioma cells invade by single-cell vessel co-option and preserve the blood-brain barrier (BBB). Conversely, Olig2-negative glioma cells form dense perivascular collections and promote angiogenesis and BBB breakdown, leading to innate immune cell activation. Experimentally, Olig2 promotes Wnt7b expression, a finding that correlates in human glioma profiling. Targeted Wnt7a/7b deletion or pharmacologic Wnt inhibition blocks Olig2+ glioma single-cell vessel co-option and enhances responses to temozolomide. Finally, Olig2 and Wnt7 become upregulated after anti-VEGF treatment in preclinical models and patients. Thus, glial-encoded pathways regulate distinct glioma-vascular microenvironmental interactions.


Assuntos
Neoplasias Encefálicas/irrigação sanguínea , Glioma/irrigação sanguínea , Fator de Transcrição 2 de Oligodendrócitos/metabolismo , Oligodendroglia/microbiologia , Proteínas Wnt/metabolismo , Animais , Bevacizumab/farmacologia , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/tratamento farmacológico , Glioma/metabolismo , Humanos , Camundongos , Transplante de Neoplasias , Fator de Transcrição 2 de Oligodendrócitos/genética , Temozolomida/farmacologia , Células Tumorais Cultivadas , Microambiente Tumoral , Proteínas Wnt/genética , Via de Sinalização Wnt/efeitos dos fármacos
7.
Cell Stem Cell ; 21(1): 91-106.e6, 2017 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-28457749

RESUMO

Tissue homeostasis requires the production of newly differentiated cells from resident adult stem cells. Central to this process is the expansion of undifferentiated intermediates known as transit-amplifying (TA) cells, but how stem cells are triggered to enter this proliferative TA state remains an important open question. Using the continuously growing mouse incisor as a model of stem cell-based tissue renewal, we found that the transcriptional cofactors YAP and TAZ are required both to maintain TA cell proliferation and to inhibit differentiation. Specifically, we identified a pathway involving activation of integrin α3 in TA cells that signals through an LATS-independent FAK/CDC42/PP1A cascade to control YAP-S397 phosphorylation and nuclear localization. This leads to Rheb expression and potentiates mTOR signaling to drive the proliferation of TA cells. These findings thus reveal a YAP/TAZ signaling mechanism that coordinates stem cell expansion and differentiation during organ renewal.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Diferenciação Celular , Proliferação de Células , Quinase 1 de Adesão Focal/metabolismo , Incisivo/metabolismo , Fosfoproteínas/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular , Quinase 1 de Adesão Focal/genética , Incisivo/citologia , Camundongos , Camundongos Transgênicos , Fosfoproteínas/genética , Células-Tronco/citologia , Serina-Treonina Quinases TOR/genética , Proteínas de Sinalização YAP
8.
FEBS Lett ; 585(7): 1089-96, 2011 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-21402070

RESUMO

LIMD1 is a tumour suppressor gene (TSG) down regulated in ∼80% of lung cancers with loss also demonstrated in breast and head and neck squamous cell carcinomas. LIMD1 is also a candidate TSG in childhood acute lymphoblastic leukaemia. Mechanistically, LIMD1 interacts with pRB, repressing E2F-driven transcription as well as being a critical component of microRNA-mediated gene silencing. In this study we show a CpG island within the LIMD1 promoter contains a conserved binding motif for the transcription factor PU.1. Mutation of the PU.1 consensus reduced promoter driven transcription by 90%. ChIP and EMSA analysis demonstrated that PU.1 specifically binds to the LIMD1 promoter. siRNA depletion of PU.1 significantly reduced endogenous LIMD1 expression, demonstrating that PU.1 is a major transcriptional activator of LIMD1.


Assuntos
Genes Supressores de Tumor , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Ativação Transcricional , Animais , Linhagem Celular Tumoral , Sequência Consenso/genética , Ilhas de CpG/genética , Técnicas de Silenciamento de Genes , Hematopoese/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM , Dados de Sequência Molecular , Mutagênese , Mutação , Regiões Promotoras Genéticas/genética , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , RNA Interferente Pequeno/genética , Transativadores/química , Transativadores/deficiência , Transativadores/genética
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