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1.
Am J Physiol Lung Cell Mol Physiol ; 312(2): L186-L195, 2017 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-27941077

RESUMEN

Bronchopulmonary dysplasia (BPD) is a common complication of premature birth. The histopathology of BPD is characterized by an arrest of alveolarization with fibroblast activation. The Wnt/ß-catenin signaling pathway is important in early lung development. When Wnt signaling is active, phosphorylation of ß-catenin by tyrosine kinases at activating sites, specifically at tyrosine 489 (Y489), correlates with nuclear localization of ß-catenin. We examined fetal lung tissue, lung tissue from term newborns, and lung tissue from infants who died with BPD; we found nuclear ß-catenin phosphorylation at Y489 in epithelial and mesenchymal cells in fetal tissue and BPD tissue, but not in the lungs of term infants. Using a 3D human organoid model, we found increased nuclear localization of ß-catenin phosphorylated at Y489 (p-ß-cateninY489) after exposure to alternating hypoxia and hyperoxia compared with organoids cultured in normoxia. Exogenous stimulation of the canonical Wnt pathway in organoids was sufficient to cause nuclear localization of p-ß-cateninY489 in normoxia and mimicked the pattern of α-smooth muscle actin (α-SMA) expression seen with fibroblastic activation from oxidative stress. Treatment of organoids with a tyrosine kinase inhibitor prior to cyclic hypoxia-hyperoxia inhibited nuclear localization of p-ß-cateninY489 and prevented α-SMA expression by fibroblasts. Posttranslational phosphorylation of ß-catenin is a transient feature of normal lung development. Moreover, the persistence of p-ß-cateninY489 is a durable marker of fibroblast activation in BPD and may play an important role in BPD disease pathobiology.


Asunto(s)
Displasia Broncopulmonar/metabolismo , Displasia Broncopulmonar/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Procesamiento Proteico-Postraduccional , beta Catenina/metabolismo , Actinas/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Dasatinib/farmacología , Fibroblastos/efectos de los fármacos , Humanos , Hiperoxia/complicaciones , Hiperoxia/metabolismo , Hiperoxia/patología , Hipoxia/complicaciones , Hipoxia/metabolismo , Hipoxia/patología , Recién Nacido , Pulmón/efectos de los fármacos , Pulmón/crecimiento & desarrollo , Pulmón/metabolismo , Pulmón/patología , Organoides/efectos de los fármacos , Organoides/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Vía de Señalización Wnt/efectos de los fármacos
2.
Mol Cancer ; 16(1): 126, 2017 07 19.
Artículo en Inglés | MEDLINE | ID: mdl-28724437

RESUMEN

BACKGROUND: Long non-coding RNAs (lncRNAs) play a variety of cellular roles, including regulation of transcription and translation, leading to alterations in gene expression. Some lncRNAs modulate the expression of chromosomally adjacent genes. Here, we assess the roles of the lncRNA CASC15 in regulation of a chromosomally nearby gene, SOX4, and its function in RUNX1/AML translocated leukemia. RESULTS: CASC15 is a conserved lncRNA that was upregulated in pediatric B-acute lymphoblastic leukemia (B-ALL) with t (12; 21) as well as pediatric acute myeloid leukemia (AML) with t (8; 21), both of which are associated with relatively better prognosis. Enforced expression of CASC15 led to a myeloid bias in development, and overall, decreased engraftment and colony formation. At the cellular level, CASC15 regulated cellular survival, proliferation, and the expression of its chromosomally adjacent gene, SOX4. Differentially regulated genes following CASC15 knockdown were enriched for predicted transcriptional targets of the Yin and Yang-1 (YY1) transcription factor. Interestingly, we found that CASC15 enhances YY1-mediated regulation of the SOX4 promoter. CONCLUSIONS: Our findings represent the first characterization of this CASC15 in RUNX1-translocated leukemia, and point towards a mechanistic basis for its action.


Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Leucemia Mieloide Aguda/genética , ARN Largo no Codificante/genética , Factores de Transcripción SOXC/genética , Animales , Línea Celular Tumoral , Proliferación Celular/genética , Niño , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Ratones , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Pronóstico , Regiones Promotoras Genéticas/genética , Translocación Genética/genética , Factor de Transcripción YY1/genética
3.
Blood ; 120(16): 3288-97, 2012 Oct 18.
Artículo en Inglés | MEDLINE | ID: mdl-22936659

RESUMEN

Sézary syndrome (SS) is an aggressive cutaneous T-cell lymphoma (CTCL) of unknown etiology in which malignant cells circulate in the peripheral blood. To identify viral elements, gene fusions, and gene expression patterns associated with this lymphoma, flow cytometry was used to obtain matched pure populations of malignant Sézary cells (SCs) versus nonmalignant CD4(+) T cells from 3 patients for whole transcriptome, paired-end sequencing with an average depth of 112 million reads per sample. Pathway analysis of differentially expressed genes identified mis-regulation of PI3K/Akt, TGFß, and NF-κB pathways as well as T-cell receptor signaling. Bioinformatic analysis did not detect either nonhuman transcripts to support a viral etiology of SS or recurrently expressed gene fusions, but it did identify 21 SC-associated annotated long noncoding RNAs (lncRNAs). Transcriptome assembly by multiple algorithms identified 13 differentially expressed unannotated transcripts termed Sézary cell-associated transcripts (SeCATs) that include 12 predicted lncRNAs and a novel transcript with coding potential. High-throughput sequencing targeting the 3' end of polyadenylated transcripts in archived tumors from 24 additional patients with tumor-stage CTCL confirmed the differential expression of SC-associated lncRNAs and SeCATs in CTCL. Our findings characterize the SS transcriptome and support recent reports that implicate lncRNA dysregulation in human malignancies.


Asunto(s)
Biomarcadores de Tumor/genética , Perfilación de la Expresión Génica , Micosis Fungoide/genética , ARN Largo no Codificante/genética , Síndrome de Sézary/genética , Neoplasias Cutáneas/genética , Citometría de Flujo , Humanos , Micosis Fungoide/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Síndrome de Sézary/patología , Neoplasias Cutáneas/patología , Células Tumorales Cultivadas
4.
Oncogene ; 42(6): 434-448, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36509998

RESUMEN

Small cell lung cancer (SCLC) remains a lethal disease with a dismal overall survival rate of 6% despite promising responses to upfront combination chemotherapy. The key drivers of such rapid mortality include early metastatic dissemination in the natural course of the disease and the near guaranteed emergence of chemoresistant disease. Here, we found that we could model the regression and relapse seen in clinical SCLC in vitro. We utilized time-course resolved RNA-sequencing to globally profile transcriptome changes as SCLC cells responded to a combination of cisplatin and etoposide-the standard-of-care in SCLC. Comparisons across time points demonstrated a distinct transient transcriptional state resembling embryonic diapause. Differential gene expression analysis revealed that expression of the PEA3 transcription factors ETV4 and ETV5 were transiently upregulated in the surviving fraction of cells which we determined to be necessary for efficient clonogenic expansion following chemotherapy. The FGFR-PEA3 signaling axis guided the identification of a pan-FGFR inhibitor demonstrating in vitro and in vivo efficacy in delaying progression following combination chemotherapy, observed inhibition of phosphorylation of the FGFR adaptor FRS2 and corresponding downstream MAPK and PI3K-Akt signaling pathways. Taken together, these data nominate PEA3 transcription factors as key mediators of relapse progression in SCLC and identify a clinically actionable small molecule candidate for delaying relapse of SCLC.


Asunto(s)
Neoplasias Pulmonares , Carcinoma Pulmonar de Células Pequeñas , Humanos , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/patología , Fosfatidilinositol 3-Quinasas/genética , Recurrencia Local de Neoplasia , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Línea Celular Tumoral
5.
Methods Mol Biol ; 2386: 17-26, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-34766262

RESUMEN

Immunofluorescence (IF) on tissue sections allows for the detection of protein species subcellular localization. IF studies further offer the ability to achieve this understanding at the level of single cell granularity. Here, we describe the processes by which tissue is fixed, embedded, sectioned, and subsequently utilized for conducting indirect IF assays. We raise potential opportunities for troubleshooting and optimization at varying stages of the protocol.


Asunto(s)
Técnica del Anticuerpo Fluorescente Indirecta , Coloración y Etiquetado , Fijación del Tejido
6.
Commun Biol ; 4(1): 601, 2021 05 20.
Artículo en Inglés | MEDLINE | ID: mdl-34017045

RESUMEN

The respiratory tract is a vital, intricate system for several important biological processes including mucociliary clearance, airway conductance, and gas exchange. The Wnt signaling pathway plays several crucial and indispensable roles across lung biology in multiple contexts. This review highlights the progress made in characterizing the role of Wnt signaling across several disciplines in lung biology, including development, homeostasis, regeneration following injury, in vitro directed differentiation efforts, and disease progression. We further note uncharted directions in the field that may illuminate important biology. The discoveries made collectively advance our understanding of Wnt signaling in lung biology and have the potential to inform therapeutic advancements for lung diseases.


Asunto(s)
Homeostasis , Enfermedades Pulmonares/patología , Enfermedades Pulmonares/terapia , Pulmón/citología , Organogénesis , Regeneración , Vía de Señalización Wnt , Animales , Diferenciación Celular , Progresión de la Enfermedad , Humanos
7.
Nat Med ; 27(5): 806-814, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33958799

RESUMEN

Cystic fibrosis (CF) is a lethal autosomal recessive disorder that afflicts more than 70,000 people. People with CF experience multi-organ dysfunction resulting from aberrant electrolyte transport across polarized epithelia due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CF-related lung disease is by far the most important determinant of morbidity and mortality. Here we report results from a multi-institute consortium in which single-cell transcriptomics were applied to define disease-related changes by comparing the proximal airway of CF donors (n = 19) undergoing transplantation for end-stage lung disease with that of previously healthy lung donors (n = 19). Disease-dependent differences observed include an overabundance of epithelial cells transitioning to specialized ciliated and secretory cell subsets coupled with an unexpected decrease in cycling basal cells. Our study yields a molecular atlas of the proximal airway epithelium that will provide insights for the development of new targeted therapies for CF airway disease.


Asunto(s)
Fibrosis Quística/genética , Fibrosis Quística/patología , Células Epiteliales/citología , Pulmón/patología , Mucosa Respiratoria/patología , Diferenciación Celular/genética , Cilios/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/biosíntesis , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/patología , Perfilación de la Expresión Génica , Humanos , Análisis de la Célula Individual/métodos , Transcriptoma/genética
8.
Cell Stem Cell ; 27(3): 413-429.e4, 2020 09 03.
Artículo en Inglés | MEDLINE | ID: mdl-32721381

RESUMEN

Our understanding of dynamic interactions between airway basal stem cells (ABSCs) and their signaling niches in homeostasis, injury, and aging remains elusive. Using transgenic mice and pharmacologic studies, we found that Wnt/ß-catenin within ABSCs was essential for proliferation post-injury in vivo. ABSC-derived Wnt ligand production was dispensable for epithelial proliferation. Instead, the PDGFRα+ lineage in the intercartilaginous zone (ICZ) niche transiently secreted Wnt ligand necessary for ABSC proliferation. Strikingly, ABSC-derived Wnt ligand later drove early progenitor differentiation to ciliated cells. We discovered additional changes in aging, as glandular-like epithelial invaginations (GLEIs) derived from ABSCs emerged exclusively in the ICZ of aged mice and contributed to airway homeostasis and repair. Further, ABSC Wnt ligand secretion was necessary for GLEI formation, and constitutive activation of ß-catenin in young mice induced their formation in vivo. Collectively, these data underscore multiple spatiotemporally dynamic Wnt-secreting niches that regulate functionally distinct phases of airway regeneration and aging.


Asunto(s)
Células Madre , beta Catenina , Envejecimiento , Animales , Diferenciación Celular , Proliferación Celular , Ratones , Ratones Transgénicos , Células Madre/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismo
9.
Cell Rep ; 30(7): 2055-2064.e5, 2020 02 18.
Artículo en Inglés | MEDLINE | ID: mdl-32075752

RESUMEN

Mechanisms underpinning airway epithelial homeostatic maintenance and ways to prevent its dysregulation remain elusive. Herein, we identify that ß-catenin phosphorylated at Y489 (p-ß-cateninY489) emerges during human squamous lung cancer progression. This led us to develop a model of airway basal stem cell (ABSC) hyperproliferation by driving Wnt/ß-catenin signaling, resulting in a morphology that resembles premalignant lesions and loss of ciliated cell differentiation. To identify small molecules that could reverse this process, we performed a high-throughput drug screen for inhibitors of Wnt/ß-catenin signaling. Our studies unveil Wnt inhibitor compound 1 (WIC1), which suppresses T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) activity, reduces ABSC proliferation, induces ciliated cell differentiation, and decreases nuclear p-ß-cateninY489. Collectively, our work elucidates a dysregulated Wnt/p-ß-cateninY489 axis in lung premalignancy that can be modeled in vitro and identifies a Wnt/ß-catenin inhibitor that promotes airway homeostasis. WIC1 may therefore serve as a tool compound in regenerative medicine studies with implications for restoring normal airway homeostasis after injury.


Asunto(s)
Pulmón/efectos de los fármacos , Pulmón/metabolismo , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Proteínas Wnt/antagonistas & inhibidores , Vía de Señalización Wnt/efectos de los fármacos , Animales , Bronquios/citología , Bronquios/efectos de los fármacos , Bronquios/metabolismo , Bronquios/patología , Diferenciación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Femenino , Ensayos Analíticos de Alto Rendimiento/métodos , Homeostasis/efectos de los fármacos , Humanos , Pulmón/citología , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología , Bibliotecas de Moléculas Pequeñas/farmacología , Células Madre/citología , Células Madre/patología , Transfección , Proteínas Wnt/metabolismo , beta Catenina/antagonistas & inhibidores , beta Catenina/metabolismo
10.
Cell Rep ; 29(11): 3488-3505.e9, 2019 12 10.
Artículo en Inglés | MEDLINE | ID: mdl-31825831

RESUMEN

Progressive organ fibrosis accounts for one-third of all deaths worldwide, yet preclinical models that mimic the complex, progressive nature of the disease are lacking, and hence, there are no curative therapies. Progressive fibrosis across organs shares common cellular and molecular pathways involving chronic injury, inflammation, and aberrant repair resulting in deposition of extracellular matrix, organ remodeling, and ultimately organ failure. We describe the generation and characterization of an in vitro progressive fibrosis model that uses cell types derived from induced pluripotent stem cells. Our model produces endogenous activated transforming growth factor ß (TGF-ß) and contains activated fibroblastic aggregates that progressively increase in size and stiffness with activation of known fibrotic molecular and cellular changes. We used this model as a phenotypic drug discovery platform for modulators of fibrosis. We validated this platform by identifying a compound that promotes resolution of fibrosis in in vivo and ex vivo models of ocular and lung fibrosis.


Asunto(s)
Células Madre Pluripotentes Inducidas/patología , Fibrosis Pulmonar/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Línea Celular , Células Cultivadas , Descubrimiento de Drogas/métodos , Femenino , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Factor de Crecimiento Transformador beta/metabolismo
11.
Nat Genet ; 46(10): 1060-2, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25194279

RESUMEN

Here we report the discovery of recurrent mutations concentrated at an ultraviolet signature hotspot in KNSTRN, which encodes a kinetochore protein, in 19% of cutaneous squamous cell carcinomas (SCCs). Cancer-associated KNSTRN mutations, most notably those encoding p.Ser24Phe, disrupt chromatid cohesion in normal cells, occur in SCC precursors, correlate with increased aneuploidy in primary tumors and enhance tumorigenesis in vivo. These findings suggest a role for KNSTRN mutagenesis in SCC development.


Asunto(s)
Carcinoma de Células Escamosas/genética , Proteínas de Ciclo Celular/genética , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Mutación Puntual , Neoplasias Cutáneas/genética , Aneuploidia , Animales , Carcinogénesis/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Células Cultivadas , Femenino , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Queratinocitos/trasplante , Ratones Endogámicos NOD , Ratones SCID , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Transfección , Trasplante Heterólogo
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