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1.
Clin Cancer Res ; 29(13): 2480-2493, 2023 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-36598859

RESUMO

PURPOSE: Epithelioid hemangioendothelioma (EHE) is a vascular sarcoma caused by the WWTR1(TAZ)-CAMTA1 (TC) gene fusion. This fusion gene has been observed in almost all reported EHE cases and functions as a constitutively activated TAZ. Sequencing of human tumors has, however, identified additional secondary mutations in approximately 50% of EHE, most commonly the loss of tumor suppressor CDKN2A. In this study, the effect of loss of CDKN2A in EHE tumorigenesis was evaluated. EXPERIMENTAL DESIGN: Mice bearing a conditional TC allele were paired with a conditional Cdkn2a knockout allele and an endothelial-specific Cre. Histologic characterization and single-cell RNA-seq of the resultant tumors were performed. EHE cell lines were established through ex vivo culture of tumor cells and evaluated for sensitivity to TEAD inhibition and trametinib. RESULTS: Loss of Cdkn2a within EHE was associated with more aggressive disease, as displayed by earlier tumor-related morbidity/mortality and enhanced tumor cell proliferation. As no previous EHE cell lines exist, we attempted, successfully, to expand EHE tumor cells ex vivo and produced the first EHE cell lines. These cell lines are "addicted" to the TC oncoprotein, replicate the EHE transcriptional profile, and generate EHE tumors when injected into immunodeficient mice. CONCLUSIONS: CDKN2A loss enhances the tumorigenicity of EHE in vivo and enabled the generation of the first cell lines of this disease. These cell lines replicate key facets of the human disease phenotype. Therefore, these cell lines and allograft tumors generated after implantation serve as robust model systems for therapeutic testing of compounds directed at either EHE or other TAZ-driven cancers.


Assuntos
Hemangioendotelioma Epitelioide , Animais , Humanos , Camundongos , Proteínas de Ligação ao Cálcio/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Fusão Gênica , Hemangioendotelioma Epitelioide/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Transativadores/genética , Fatores de Transcrição/genética , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional
2.
Clin Cancer Res ; 28(14): 3116-3126, 2022 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-35443056

RESUMO

PURPOSE: A consistent genetic alteration in vascular cancer epithelioid hemangioendothelioma (EHE) is the t(1;3)(p36;q25) chromosomal translocation, which generates a WWTR1(TAZ)-CAMTA1 (TC) fusion gene. TC is a transcriptional coactivator that drives EHE. Here, we aimed to identify the TC transcriptional targets and signaling mechanisms that underlie EHE tumorigenesis. EXPERIMENTAL DESIGN: We used NIH3T3 cells transformed with TC (NIH3T3/TC) as a model system to uncover TC-dependent oncogenic signaling. These cells proliferated in an anchorage-independent manner in suspension and soft agar. The findings of the cell-based studies were validated in a xenograft model. RESULTS: We identified connective tissue growth factor (CTGF) as a tumorigenic transcriptional target of TC. We show that CTGF binds to integrin αIIbß3, which is essential for sustaining the anchorage-independent proliferation of transformed NIH3T3/TC cells. NIH3T3/TC cells also have enhanced Ras and MAPK signaling, and the activity of these pathways is reduced upon CTGF knockdown, suggesting that CTGF signaling occurs via the Ras-MAPK cascade. Further, pharmacologic inhibition of MAPK signaling through PD 0325901 and trametinib abrogated TC-driven anchorage-independent growth. Likewise, for tumor growth in vivo, NIH3T3/TC cells require CTGF and MAPK signaling. NIH3T3/TC xenograft growth was profoundly reduced upon CTGF knockdown and after trametinib treatment. CONCLUSIONS: Collectively, our results demonstrated that CTGF and the Ras-MAPK signaling cascade are essential for TC-mediated tumorigenesis. These studies provided the preclinical rationale for SARC033 (NCI 10015-NCT03148275), a nonrandomized, open-label, phase II study of trametinib in patients with unresectable or metastatic EHE.


Assuntos
Hemangioendotelioma Epitelioide , Sarcoma , Adulto , Animais , Proteínas de Ligação ao Cálcio/genética , Carcinogênese/genética , Criança , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Hemangioendotelioma Epitelioide/tratamento farmacológico , Hemangioendotelioma Epitelioide/genética , Humanos , Camundongos , Células NIH 3T3 , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
3.
PLoS One ; 17(4): e0266143, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35417479

RESUMO

Disrupting the formation of the oncogenic YAP/TAZ-TEAD transcriptional complex holds substantial therapeutic potential. However, the three protein interaction interfaces of this complex cannot be easily disrupted using small molecules. Here, we report that the pharmacologically active small molecule aurintricarboxylic acid (ATA) acts as a disruptor of the TAZ-TEAD complex. ATA was identified in a high-throughput screen using a TAZ-TEAD AlphaLISA assay that was tailored to identify disruptors of this transcriptional complex. We further used fluorescence polarization assays both to confirm disruption of the TAZ-TEAD complex and to demonstrate that ATA binds to interface 3. We have previously shown that cell-based models that express the oncogenic TAZ-CAMTA1 (TC) fusion protein display enhanced TEAD transcriptional activity because TC functions as an activated form of TAZ. Utilizing cell-based studies and our TC model system, we performed TC/TEAD reporter, RNA-Seq, and qPCR assays and found that ATA inhibits TC/TEAD transcriptional activity. Further, disruption of TC/TEAD and TAZ/TEAD interaction by ATA abrogated anchorage-independent growth, the phenotype most closely linked to dysregulated TAZ/TEAD activity. Therefore, this study demonstrates that ATA is a novel small molecule that has the ability to disrupt the undruggable TAZ-TEAD interface.


Assuntos
Ácido Aurintricarboxílico , Fatores de Transcrição , Proteínas de Fusão Oncogênica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
ChemMedChem ; 16(18): 2823-2844, 2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34032019

RESUMO

Starting from our previously reported hit, a series of 1,5-diaryl-1,2,3-triazole-4-carbohydrazones were synthesized and evaluated as inhibitors of the YAP/TAZ-TEAD complex. Their binding to hTEAD2 was confirmed by nanodifferential scanning fluorimetry, and some of the compounds were also found to moderately disrupt the YAP-TEAD interaction, as assessed by a fluorescence polarization assay. A TEAD luciferase gene reporter assay performed in HEK293T cells and RTqPCR measurements in MDA-MB231 cells showed that these compounds inhibit YAP/TAZ-TEAD activity to cells in the micromolar range. In spite of the cytotoxic effects displayed by some of the compounds of this series, they are still good starting points and can be suitably modified into an effective and viable YAP-TEAD disruptor in the future.


Assuntos
Antineoplásicos/farmacologia , Hidrazonas/farmacologia , Fatores de Transcrição de Domínio TEA/antagonistas & inibidores , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/antagonistas & inibidores , Triazóis/farmacologia , Proteínas de Sinalização YAP/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Humanos , Hidrazonas/síntese química , Hidrazonas/química , Estrutura Molecular , Relação Estrutura-Atividade , Fatores de Transcrição de Domínio TEA/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/metabolismo , Triazóis/síntese química , Triazóis/química , Proteínas de Sinalização YAP/metabolismo
5.
Elife ; 92020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32369438

RESUMO

Collective cell migration is central to many developmental and pathological processes. However, the mechanisms that keep cell collectives together and coordinate movement of multiple cells are poorly understood. Using the Drosophila border cell migration model, we find that Protein phosphatase 1 (Pp1) activity controls collective cell cohesion and migration. Inhibition of Pp1 causes border cells to round up, dissociate, and move as single cells with altered motility. We present evidence that Pp1 promotes proper levels of cadherin-catenin complex proteins at cell-cell junctions within the cluster to keep border cells together. Pp1 further restricts actomyosin contractility to the cluster periphery rather than at individual internal border cell contacts. We show that the myosin phosphatase Pp1 complex, which inhibits non-muscle myosin-II (Myo-II) activity, coordinates border cell shape and cluster cohesion. Given the high conservation of Pp1 complexes, this study identifies Pp1 as a major regulator of collective versus single cell migration.


Assuntos
Movimento Celular/fisiologia , Proteínas de Drosophila/fisiologia , Proteína Fosfatase 1/fisiologia , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Feminino , Genes/genética , Genes/fisiologia , Masculino , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo
6.
Cell Death Dis ; 11(2): 152, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32102991

RESUMO

Glioblastoma (GBM) is the most prevalent primary malignant brain tumor and is associated with extensive tumor cell infiltration into the adjacent brain parenchyma. However, there are limited targeted therapies that address this disease hallmark. While the invasive capacity of self-renewing cancer stem cells (CSCs) and their non-CSC progeny has been investigated, the mode(s) of migration used by CSCs during invasion is currently unknown. Here we used time-lapse microscopy to evaluate the migratory behavior of CSCs, with a focus on identifying key regulators of migration. A head-to-head migration assay demonstrated that CSCs are more invasive than non-CSCs. Time-lapse live cell imaging further revealed that GBM patient-derived CSC models either migrate in a collective manner or in a single cell fashion. To uncover conserved molecular regulators responsible for collective cell invasion, we utilized the genetically tractable Drosophila border cell collective migration model. Candidates for functional studies were generated using results from a targeted Drosophila genetic screen followed by gene expression analysis of the human homologs in GBM tumors and associated GBM patient prognosis. This strategy identified the highly conserved small GTPase, Rap1a, as a potential regulator of cell invasion. Alteration of Rap1a activity impaired the forward progress of Drosophila border cells during development. Rap1a expression was elevated in GBM and associated with higher tumor grade. Functionally, the levels of activated Rap1a impacted CSC migration speed out of spheres onto extracellular matrix. The data presented here demonstrate that CSCs are more invasive than non-CSCs, are capable of both collective and single cell migration, and express conserved genes that are required for migration and invasion. Using this integrated approach, we identified a new role for Rap1a in the migration of GBM CSCs.


Assuntos
Neoplasias Encefálicas/metabolismo , Movimento Celular/fisiologia , Glioblastoma/patologia , Células-Tronco Neoplásicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/fisiologia , Regulação Neoplásica da Expressão Gênica/genética , Glioblastoma/diagnóstico , Glioblastoma/metabolismo , Humanos , Células-Tronco Neoplásicas/patologia , Prognóstico
7.
Mol Biol Cell ; 27(12): 1898-910, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27122602

RESUMO

Migrating cells need to overcome physical constraints from the local microenvironment to navigate their way through tissues. Cells that move collectively have the additional challenge of negotiating complex environments in vivo while maintaining cohesion of the group as a whole. The mechanisms by which collectives maintain a migratory morphology while resisting physical constraints from the surrounding tissue are poorly understood. Drosophila border cells represent a genetic model of collective migration within a cell-dense tissue. Border cells move as a cohesive group of 6-10 cells, traversing a network of large germ line-derived nurse cells within the ovary. Here we show that the border cell cluster is compact and round throughout their entire migration, a shape that is maintained despite the mechanical pressure imposed by the surrounding nurse cells. Nonmuscle myosin II (Myo-II) activity at the cluster periphery becomes elevated in response to increased constriction by nurse cells. Furthermore, the distinctive border cell collective morphology requires highly dynamic and localized enrichment of Myo-II. Thus, activated Myo-II promotes cortical tension at the outer edge of the migrating border cell cluster to resist compressive forces from nurse cells. We propose that dynamic actomyosin tension at the periphery of collectives facilitates their movement through restrictive tissues.


Assuntos
Miosina Tipo II/metabolismo , Miosinas/fisiologia , Citoesqueleto de Actina/metabolismo , Actomiosina/metabolismo , Animais , Movimento Celular/fisiologia , Forma Celular/fisiologia , Microambiente Celular , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Oogênese , Transdução de Sinais/genética
8.
Dev Dyn ; 242(5): 414-31, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23335293

RESUMO

BACKGROUND: Cell motility is essential for embryonic development and physiological processes such as the immune response, but also contributes to pathological conditions such as tumor progression and inflammation. However, our understanding of the mechanisms underlying migratory processes is incomplete. Drosophila border cells provide a powerful genetic model to identify the roles of genes that contribute to cell migration. RESULTS: Members of the Hedgehog signaling pathway were uncovered in two independent screens for interactions with the small GTPase Rac and the polarity protein Par-1 in border cell migration. Consistent with a role in migration, multiple Hh signaling components were enriched in the migratory border cells. Interference with Hh signaling by several different methods resulted in incomplete cell migration. Moreover, the polarized distribution of E-Cadherin and a marker of tyrosine kinase activity were altered when Hh signaling was disrupted. Conservation of Hh-Rac and Hh-Par-1 signaling was illustrated in the wing, in which Hh-dependent phenotypes were enhanced by loss of Rac or par-1. CONCLUSIONS: We identified a pathway by which Hh signaling connects to Rac and Par-1 in cell migration. These results further highlight the importance of modifier screens in the identification of new genes that function in developmental pathways.


Assuntos
Movimento Celular/genética , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/embriologia , Perfilação da Expressão Gênica , Proteínas Hedgehog/fisiologia , Ovário/citologia , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/fisiologia , Epistasia Genética/fisiologia , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Masculino , Morfogênese/genética , Morfogênese/fisiologia , Oogênese/genética , Oogênese/fisiologia , Ovário/embriologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
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