RESUMEN

Secretoglobins (SCGBs) are cytokine-like small molecular weight secreted proteins with largely unknown biological functions. Three SCGB proteins, SCGB1A1, SCGB3A1, and SCGB3A2, are predominantly expressed in lung airways. To gain insight into the possible functional relationships among the SCGBs, their protein and mRNA expression patterns were examined in lungs during gestation and in adult mice, using Scgb3a1-null and Scgb3a2-null mice as negative controls, by immunohistochemistry and by qRT-PCR analysis, respectively. The three SCGBs exhibited unique spatiotemporal expression patterns during embryogenesis. The lack of Scgb3a1 or Scgb3a2 did not affect expression of the other Scgb genes as determined by mRNA measurements. Moreover, the lack of Scgb3a1 or Scgb3a2 did not affect development of the pulmonary neuroepithelial bodies during embryogenesis, while the lack of Scgb3a2 may have resulted in slightly fewer ciliated cells than in the wild-type. These results suggest that SCGB1A1, SCGB3A1, and SCGB3A2 each may possess its own unique biological function.

Asunto(s)

Epitelio/metabolismo , Regulación de la Expresión Génica , Pulmón/citología , Proteínas/genética , Secretoglobinas/genética , Uteroglobina/genética , Animales , Diferenciación Celular , Ratones , Análisis Espacio-Temporal

RESUMEN

Achaete-scute homologue-1 or ASCL1 (MASH1, hASH1) plays roles in neural development and pulmonary neuroendocrine (NE) differentiation, and it is expressed in certain lung cancers. This study was aimed to assess whether and/or how ASCL1 plays a role in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced pulmonary NE hyperplasia and carcinogenesis in hamsters. Hamsters were injected 3 times weekly with either NNK or solvent alone (control) for treatment periods of 6 and 24 weeks, both without and with 6-week recovery. Immunohistochemical analysis was carried out to examine the expressions of ASCL1, CGRP (calcitonin gene-related peptide), secretoglobin SCGB1A1 (club [Clara] cell specific 10 kD protein, CC10, CCSP), synaptophysin (SYP), and PCNA (proliferating cell nuclear antigen). The number of ASCL1-expressing NE foci per airway increased from 0.8 in controls to 1.6 and 2.0 during NNK exposure for 6 and 24 weeks, respectively, and the number of cells per foci doubled after NNK exposure. Most ASCL1-expressing cells in NEBs (neuroepithelial bodies) were also CGRP immunoreactive; NNK enhanced this co-expression with CGRP, a NE marker with known proliferation-promoting properties. NNK also increased PCNA expression within NE foci. NNK-induced tumors showed no immunoreactivity for NE markers. This study confirms ASCL1 as an excellent marker for pulmonary NE cells and demonstrates CGRP co-expression in ASCL1-positive NEB cells participating in NNK-induced NE hyperplasia.

RESUMEN

INTRODUCTION: Because small-cell lung carcinomas (SCLC) are seldom resected, human materials for study are limited. Thus, genetically engineered mouse models (GEMMs) for SCLC and other high-grade lung neuroendocrine (NE) carcinomas are crucial for translational research. METHODS: The pathologies of five GEMMs were studied in detail and consensus diagnoses reached by four lung cancer pathology experts. Hematoxylin and Eosin and immunostained slides of over 100 mice were obtained from the originating and other laboratories and digitalized. The GEMMs included the original Rb/p53 double knockout (Berns Laboratory) and triple knockouts from the Sage, MacPherson, and Jacks laboratories (double knockout model plus loss of p130 [Sage laboratory] or loss of Pten [MacPherson and Jacks laboratories]). In addition, a GEMM with constitutive co-expression of SV40 large T antigen and Ascl1 under the Scgb1a1 promoter from the Linnoila laboratory were included. RESULTS: The lung tumors in all of the models had common as well as distinct pathological features. All three conditional knockout models resulted in multiple pulmonary tumors arising mainly from the central compartment (large bronchi) with foci of in situ carcinoma and NE cell hyperplasia. They consisted of inter- and intra-tumor mixtures of SCLC and large-cell NE cell carcinoma in varying proportions. Occasional adeno- or large-cell carcinomas were also seen. Extensive vascular and lymphatic invasion and metastases to the mediastinum and liver were noted, mainly of SCLC histology. In the Rb/p53/Pten triple knockout model from the MacPherson and Jacks laboratories and in the constitutive SV40/T antigen model many peripherally arising non-small-cell lung carcinoma tumors having varying degrees of NE marker expression were present (non-small-cell lung carcinoma-NE tumors). The resultant histological phenotypes were influenced by the introduction of specific genetic alterations, by inactivation of one or both alleles of specific genes, by time from Cre activation and by targeting of lung cells or NE cell subpopulations. CONCLUSION: The five GEMM models studied are representative for the entire spectrum of human high-grade NE carcinomas and are also useful for the study of multistage pathogenesis and the metastatic properties of these tumors. They represent one of the most advanced forms of currently available GEMM models for the study of human cancer.

Asunto(s)

Carcinoma Neuroendocrino/patología , Ingeniería Genética/métodos , Neoplasias Pulmonares/patología , Pulmón/ultraestructura , Neoplasias Experimentales , Animales , Carcinoma Neuroendocrino/genética , Neoplasias Pulmonares/genética , Ratones , Ratones Noqueados , Microscopía Electrónica , Células Tumorales Cultivadas

RESUMEN

Lung cancer remains one of the leading causes of cancer-related deaths worldwide with a 5-year survival rate of less than 20%. One approach to improving survival is the identification of biomarkers to detect early stage disease. In this study, we investigated the potential of the stem cell and progenitor cell marker, Musashi1 (Msi1), as a diagnostic marker and potential therapeutic target for lung cancer. Functional studies in A549 bronchioalveolar carcinoma and NCI-H520 squamous cell carcinoma cells revealed that Msi1 was enriched in spheroid cultures of tumor cells and in the CD133+ cell population. Downregulation of Msi1 by lentivirus-mediated expression of an Msi1 shRNA reduced spheroid colony proliferation. Growth inhibition was associated with reduced nuclear localization of ß-catenin and inhibition of the processing of intracellular Notch. In primary lung cancer, Msi1 protein expression was elevated in 86% of 202 tissue microarray specimens, and Msi1 mRNA was increased in 80% of 118 bronchoscopic biopsies, including metastatic disease, but was rarely detected in adjacent normal lung tissue and in non-malignant diseased tissue. Msi1 was expressed in a diffuse pattern in most tumor subtypes, except in squamous cell carcinomas, where it appeared in a focal pattern in 50% of specimens. Thus, Msi1 is a sensitive and specific diagnostic marker for all lung cancer subtypes.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Neoplasias Pulmonares/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al ARN/metabolismo , Carcinoma Pulmonar de Células Pequeñas/diagnóstico , Esferoides Celulares/metabolismo , Antígeno AC133 , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD/metabolismo , Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Proliferación Celular , Regulación hacia Abajo , Femenino , Glicoproteínas/metabolismo , Humanos , Neoplasias Pulmonares/diagnóstico , Masculino , Persona de Mediana Edad , Neoplasias de Células Escamosas/metabolismo , Células Madre Neoplásicas/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Péptidos/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Proteínas de Unión al ARN/biosíntesis , Proteínas de Unión al ARN/genética , Receptores Notch/metabolismo , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Vía de Señalización Wnt/genética , beta Catenina/metabolismo

RESUMEN

Multiple cells contribute to the function of lungs. Pulmonary neuroendocrine cells (PNECs) are important for the regulation of breathing and carcinogenesis, although they represent only a small population of the airway lining. Achaete-Scute homologue-1 (Ascl1), a proneural basic helix-loop-helix transcription factor, is critical for the development of PNECs. We postulated that Ascl1-defined cells (ASDCs) may be progenitors, and traced their fate during development and injury repair. R26R-stop-lacZ (Rosa) reporter mice were crossed with Ascl1-Cre or Ascl1-CreERTM mice, in which the Ascl1 promoter drives the expression of Cre or inducible Cre recombinase, respectively. ASDCs and their descendants will be permanently labeled. The labeled cells were characterized by immunohistochemistry, using highly specific differentiation markers. Lineage studies revealed a population that proliferates before the pseudoglandular stage, and widely contributes to different compartments. When ASDCs were labeled on Embryonic Day 9.5, they gave rise to both airway and alveolar cells, but when labeled on Embryonic Day 11.5, they only gave rise to airway cells. In postnatal naphthalene injury, ASDCs contributed to regenerating Clara cells. In conclusion, Ascl1-defined cells in the lung represent a novel multipotent lineage, indicating a close relationship of neuroendocrine cells with other cell types.

Asunto(s)

Lesión Pulmonar Aguda/patología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular , Pulmón/patología , Células Madre/fisiología , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/fisiopatología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Femenino , Expresión Génica , Pulmón/embriología , Masculino , Ratones , Ratones Transgénicos , Naftalenos , Células Neuroendocrinas/metabolismo , Células Neuroendocrinas/fisiología , Regeneración , Estadísticas no Paramétricas , Células Madre/metabolismo

RESUMEN

Clara cells are non-ciliated, secretory bronchiolar epithelial cells that serve to detoxify harmful inhaled substances. Clara cells also function as stem/progenitor cells for repair in the bronchioles. Clara cell secretory protein (CCSP) is specifically expressed in pulmonary Clara cells and is widely used as a Clara cell marker. In addition CCSP promoter is commonly used to direct gene expression into the lung in transgenic models. The discovery of CCSP immunoreactivity in plasma membranes of airway lining cells prompted us to explore the possibility of enriching Clara cells by flow cytometry. We established a novel and simple method for the isolation of CCSP-expressing cell Clara cells using a combination of mechanical and enzymatic dissociation followed by flow cytometry sorting technology. We showed that ∼25% of dissociated cells from whole lung expressed CCSP. In the resulting preparation, up to 98% of cells expressed CCSP. Notably, we found that several common stem cell markers including CD44, CD133, Sca-1 and Sox2 were expressed in CCSP(+) cells. Moreover, CCSP(+) cells were able to form spheroid colonies in vitro with 0.97‰ efficiency. Parallel studies in vivo confirmed that a small population of CCSP(-)expressing cells in mouse airways also demonstrates stem cell-like properties such as label retention and harboring rare bronchioalveolar stem cells (BASCs) in terminal bronchioles (TBs). We conclude that CCSP(+) cells exhibit a number of stem cell-like features including stem cell marker expression, bronchosphere colony formation and self-renewal ability. Clara cell isolation by flow cytometry sorting is a useful method for investigating the function of primary Clara cells in stem cell research and mouse models.

Asunto(s)

Células Madre/citología , Células Madre/metabolismo , Uteroglobina/metabolismo , Animales , Bronquios/citología , Membrana Celular/metabolismo , Células Cultivadas , Citometría de Flujo , Inmunohistoquímica , Pulmón/citología , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

Our previous data suggested that the human basic helix-loop-helix transcription factor achaete-scute homologue-1 (hASH1) may stimulate both proliferation and migration in the lung. In the CNS, cyclin-dependent kinase 5 (Cdk5) and its activator p35 are important for neuronal migration that is regulated by basic helix-loop-helix transcription factors. Cdk5/p35 may also play a role in carcinogenesis. In this study, we found that the neuronal activator p35 was commonly expressed in primary human lung cancers. Cdk5 and p35 were also expressed by several human lung cancer cell lines and coupled with migration and invasion. When the kinase activity was inhibited by the Cdk5 inhibitor roscovitine or dominant-negative (dn) Cdk5, the migration of lung cancer cells was reduced. In neuroendocrine cells expressing hASH1, such as a pulmonary carcinoid cell line, knocking down the gene expression by short hairpin RNA reduced the levels of Cdk5/p35, nuclear p35 protein, and migration. Furthermore, expression of hASH1 in lung adenocarcinoma cells normally lacking hASH1 increased p35/Cdk5 activity and enhanced cellular migration. We were also able to show that p35 was a direct target for hASH1. In conclusion, induction of Cdk5 activity is a novel mechanism through which hASH1 may regulate migration in lung carcinogenesis.

Asunto(s)

Adenocarcinoma , Quinasa 5 Dependiente de la Ciclina , Proteínas de Unión al ADN , Neoplasias Pulmonares , Proteínas del Tejido Nervioso , Factores de Transcripción , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 5 Dependiente de la Ciclina/genética , Quinasa 5 Dependiente de la Ciclina/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Células HEK293 , N-Metiltransferasa de Histona-Lisina , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Invasividad Neoplásica , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Purinas/farmacología , Roscovitina , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Lung cancer is the leading cause of cancer-related deaths in the world. Achaete-scute complex homolog-1 (Ascl1) is a member of the basic helix-loop-helix (bHLH) transcription factor family that has multiple functions in the normal and neoplastic lung such as the regulation of neuroendocrine differentiation, prevention of apoptosis and promotion of tumor-initiating cells. We now show that Ascl1 directly regulates matrix metalloproteinase-7 (MMP-7) and O(6)-methylguanine-DNA methyltransferase (MGMT). Loss- and gain-of-function experiments in human bronchial epithelial and lung carcinoma cell lines revealed that Ascl1, MMP-7 and MGMT are able to protect cells from the tobacco-specific nitrosamine NNK-induced DNA damage and the alkylating agent cisplatin-induced apoptosis. We also examined the role of Ascl1 in NNK-induced lung tumorigenesis in vivo. Using transgenic mice which constitutively expressed human Ascl1 in airway lining cells, we found that there was a delay in lung tumorigenesis. We conclude that Ascl1 potentially enhances DNA repair through activation of MMP-7 and MGMT which may impact lung carcinogenesis and chemoresistance. The study has uncovered a novel and unexpected function of Ascl1 which will contribute to better understanding of lung carcinogenesis and the broad implications of transcription factors in tobacco-related carcinogenesis.

Asunto(s)

Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN , Neoplasias Pulmonares/metabolismo , Metaloproteinasa 7 de la Matriz/metabolismo , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Adenoma/inducido químicamente , Adenoma/metabolismo , Animales , Antineoplásicos Alquilantes/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Transformación Celular Neoplásica/inducido químicamente , Transformación Celular Neoplásica/metabolismo , Cisplatino/farmacología , Daño del ADN , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Inducción Enzimática , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Pulmonares/inducido químicamente , Metaloproteinasa 7 de la Matriz/genética , Ratones , Ratones Transgénicos , Nitrosaminas , Regiones Promotoras Genéticas , Unión Proteica , ARN Interferente Pequeño/genética , Proteínas Supresoras de Tumor/genética , Regulación hacia Arriba

RESUMEN

Achaete-scute homolog-1 (ASH1) is pivotal for the development of pulmonary neuroendocrine (NE) cells. We examined human ASH1 (hASH1) expression across a comprehensive panel of human lung cancer cell lines, primary human lung tumors and normal fetal and post-natal lungs. While hASH1 was a cardinal feature of NE carcinomas, a subgroup of non-NE lung cancers also exhibited expression of this factor. Twenty lung cancer cell lines out of 33 were positive for hASH1 mRNA by reverse transcription PCR, including 6/6 small cell carcinomas (SCLC), 5/5 carcinoids, 6/7 non-SCLC with NE features, and 3/14 other non-SCLC. Among human primary tumors, 2/2 SCLC, 5/5 pulmonary carcinoids, and 10/41 non-SCLC (only 4 of which had NE features) were positive for hASH1 by immunohistochemistry and RNA-RNA in situ hybridization. In normal human fetal lung, the expression of hASH1 and the neural marker synaptophysin was highly concordant in neuroepithelial bodies and solitary NE cells, while the rest of the epithelium was negative. In childhood and adulthood, the markers became progressively discordant, with a majority of hASH1-immunoreactive foci (69%) being negative for synaptophysin in adults, potentially representing dormant NE cell progenitors. We conclude that hASH1 provides an early indication of NE program in human lung.

Asunto(s)

Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Tumor Carcinoide/genética , Carcinoma Neuroendocrino/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Células Pequeñas/genética , Neoplasias Pulmonares/genética , Carcinoma Neuroendocrino/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Células Pequeñas/metabolismo , Línea Celular Tumoral , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Inmunohistoquímica/métodos , Pulmón/metabolismo , Neoplasias Pulmonares/metabolismo , Células Neuroendocrinas/metabolismo , Células Neuroendocrinas/patología , ARN Mensajero/genética , Sinaptofisina , Proteínas de Transporte Vesicular/metabolismo

RESUMEN

Smoking is the leading cause of preventable cancer deaths in the United States. Nicotine replacement therapies (NRT) have been developed to aid in smoking cessation, which decreases lung cancer incidence. However, the safety of NRT is controversial because numerous preclinical studies have shown that nicotine enhances tumor cell growth in vitro and in vivo. We modeled NRT in mice to determine the effects of physiologic levels of nicotine on lung tumor formation, tumor growth, or metastasis. Nicotine administered in drinking water did not enhance lung tumorigenesis after treatment with the tobacco carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Tumors that develop in this model have mutations in K-ras, which is commonly observed in smoking-related, human lung adenocarcinomas. In a transgenic model of mutant K-ras-driven lung cancer, nicotine did not increase tumor number or size and did not affect overall survival. Likewise, in a syngeneic model using lung cancer cell lines derived from NNK-treated mice, oral nicotine did not enhance tumor growth or metastasis. These data show that nicotine does not enhance lung tumorigenesis when given to achieve levels comparable with those of NRT, suggesting that nicotine has a dose threshold, below which it has no appreciable effect. These studies are consistent with epidemiologic data showing that NRT does not enhance lung cancer risk in former smokers.

Asunto(s)

Adenocarcinoma/etiología , Transformación Celular Neoplásica/patología , Modelos Animales de Enfermedad , Neoplasias Pulmonares/etiología , Mutación/genética , Nicotina/administración & dosificación , Proteínas Proto-Oncogénicas p21(ras)/genética , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Animales , Carcinógenos/toxicidad , Células Cultivadas , Agua Potable , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos A , Ratones Endogámicos C57BL , Nitrosaminas/toxicidad

RESUMEN

Secretoglobin (SCGB) 3A2, also called uteroglobin-related protein (UGRP) 1, is a downstream target for a homeodomain transcription factor NKX2-1, which is critical for the development of lung, thyroid and ventral forebrain. Both SCGB3A2 and NKX2-1 are expressed in airway epithelial cells and the latter also in alveolar Type II cells. NKX2-1 has been used clinically for diagnosis of human pulmonary tumors. Recently, the expression of SCGB3A2 was reported in human carcinomas, suggesting the use of this protein as a tumor marker. In this study, 28 lung tumors from aging B6;129 mice and nine lung adenocarcinomas from CC10TAg transgenic mice that express SV40 large T antigen under the mouse Scgb1a1 (CC10) gene promoter, were subjected to histopathological and immunohistochemical analyses for the expression of NKX2-1 and SCGB3A2. NKX2-1 was expressed in all types of tumors albeit more focally in carcinomas. In contrast, SCGB3A2 normally expressed in Clara cells, was negative in Type II cell hyperplasias and adenomas. However, it was expressed in alveolar Type II cell carcinomas and Clara cell adenocarcinomas. In these carcinomas, SCGB3A2 expression was observed in the portion of the tumor where NKX2-1 expression was reduced or almost abolished. As a comparison, the expression of SCGB3A2 and NKX2-1 from 23 human non-small cell lung carcinoma specimens was also examined. The results demonstrate that SCGB3A2 is a useful marker for diagnosis of pulmonary tumors both in mice and humans.

Asunto(s)

Biomarcadores de Tumor/biosíntesis , Neoplasias Pulmonares/metabolismo , Proteínas/metabolismo , Uteroglobina/biosíntesis , Adulto , Factores de Edad , Animales , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/patología , Ratones , Ratones Transgénicos , Proteínas Nucleares/biosíntesis , Secretoglobinas , Factor Nuclear Tiroideo 1 , Factores de Transcripción/biosíntesis

RESUMEN

Late stage or aggressive cancers exhibit metastatic growth at multiple sites, and the characterization of treatment response in various organs to drugs with potentially wide-ranging efficacy is needed. Tumor cells that induce angiogenesis are a common characteristic of metastatic disease, and clinically, antiangiogenic therapies have shown value in the setting of advanced cancer. However, recent preclinical studies have suggested that exposure to antiangiogenic drugs can increase tumor invasiveness and metastasis, making it important to determine which contexts antiangiogenic therapy is most appropriate. We describe here the effects of cediranib, a receptor tyrosine kinase inhibitor, in a model of advanced prostate cancer metastatic to skeleton and brain. Treatment with cediranib decreased metastatic tumor burden in the brain and bone, decreased cerebral vasogenic edema, and improved survival, despite increasing the invasive histology of brain metastases. Short-duration cediranib treatment given at the time of tumor cell dissemination was sufficient to inhibit the establishment and subsequent growth of bone metastases, although brain metastases were subject to rebound growth after the discontinuation of cediranib. Distinct growth patterns at different organ sites in the same animal showed that certain tumor microenvironments such as bone may be most amenable to interventions by anti-vascular endothelial growth factor (VEGF) therapies. In addition, anti-VEGF treatment may be of utility in decreasing the rapid growth of solid brain metastases and vasogenic edema in patients with advanced cancer, leading to reduced morbidity and associated clinical benefit.

Asunto(s)

Neoplasias Óseas/prevención & control , Neoplasias Encefálicas/prevención & control , Neoplasias de la Próstata/prevención & control , Quinazolinas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Western Blotting , Neoplasias Óseas/irrigación sanguínea , Neoplasias Óseas/secundario , Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/secundario , Humanos , Técnicas para Inmunoenzimas , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Desnudos , Neovascularización Patológica/prevención & control , Neoplasias de la Próstata/irrigación sanguínea , Neoplasias de la Próstata/patología , Tasa de Supervivencia , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

BACKGROUND: Musashi1 (Msi1) is a conserved RNA-binding protein that regulates the Notch and Wnt pathways, and serves as a stem cell marker in the breast and other tissues. It is unknown how Msi1 relates to other breast cancer markers, whether it denotes tumor initiating cells (TICs), and how it affects gene expression and tumor cell survival in breast cancer cells. RESULTS: Msi1 expression was analyzed in 20 breast cancer cell lines and in 140 primary breast tumors by western blotting and immunohistochemistry, respectively. Lentivirus RNA interference was used to reduce Msi1 expression in breast cancer cell lines MCF-7 and T47D grown as spheroid cultures and to assess stem cell gene expression and the growth of these cell lines as xenografts. In normal human breast tissue, Msi1 was expressed in 10.6% of myoepithelum and 1.2% of ductal epithelium in the terminal ductal lobular unit (TDLU), whereas, less than 0.05% of ductal epithelium and myoepithelium in large ducts outside the TDLU expressed Msi1. Msi1 was expressed in 55% of the breast cancer cell lines and correlated with ErbB2 expression in 50% of the cell lines. Msi1 was expressed in 68% of primary tumors and in 100% of lymph node metastases, and correlated with 5 year survival. Msi1 was enriched in CD133+ MCF-7 and T47D cells and in spheroid cultures of these cells, and Msi1 'knockdown' (KD) with a lentivirus-expressed shRNA decreased the number and size of spheroid colonies. Msi1 KD reduced Notch1, c-Myc, ErbB2 and pERK1/2 expression, and increased p21CIP1 expression, which is consistent with known Msi1 target mRNAs. Msi1 KD also reduced the expression of the somatic and embryonic stem cell markers, CD133, Bmi1, Sox2, Nanog and Oct4. Xenografts of MCF-7 and T47D Msi1 KD cells resulted in a marked reduction of tumor growth, reduced Msi1 and Notch1 expression and increased p21CIP1 expression. CONCLUSION: Msi1 is a negative prognostic indicator of breast cancer patient survival, and is indicative of tumor cells with stem cell-like characteristics. Msi1 KD reduces tumor cell survival and tumor xenograft growth, suggesting that it may represent a novel target for drug discovery.

Asunto(s)

Neoplasias de la Mama/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al ARN/metabolismo , Antígeno AC133 , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Western Blotting , Neoplasias de la Mama/genética , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Inmunohistoquímica , Ratones , Ratones Desnudos , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Proteínas del Tejido Nervioso/genética , Péptidos/genética , Péptidos/metabolismo , Proteínas de Unión al ARN/genética , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esferoides Celulares/citología

Asunto(s)

Neoplasias de la Mama/metabolismo , Células Epiteliales/metabolismo , Receptores Nicotínicos/metabolismo , Fumar/efectos adversos , Fumar/metabolismo , Mama/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/etiología , Neoplasias Pulmonares/metabolismo , Microscopía Confocal , Proteínas del Tejido Nervioso/metabolismo , Contaminación por Humo de Tabaco/efectos adversos , Regulación hacia Arriba

RESUMEN

The basic helix-loop-helix transcription factor achaete-scute homologue-1 (ASH1) plays a critical role in regulating the neuroendocrine (NE) phenotype in normal and neoplastic lung. Transgenic (TG) mice that constitutively express human ASH1 (hASH1) under control of the Clara cell 10-kDa protein (CC10) promoter in non-NE airway lining cells display progressive epithelial hyperplasia and bronchiolar metaplasia or bronchiolization of the alveoli (BOA). However, little is known about the involvement of hASH1 in regeneration of the conducting airway. In this study, we investigated the impact of hASH1 on airway cell injury and repair in the TG mice following an intraperitoneal injection of naphthalene, which specifically ablates bronchiolar Clara cells and induces pulmonary NE cell hyperplasia. We discovered an overall attenuation of NE maturation coupled with increased proliferation in TG mice during post-naphthalene repair. In addition, BOA lesions revealed enhanced epithelial cell proliferation while preserving Clara cell markers CC10 and the principal naphthalene-metabolizing enzyme cytochrome P4502F2. These data suggest that ASH1 may play an important role in maintaining a progenitor phenotype that promotes renewal of both NE and epithelial cells. Moreover, ASH1 may propagate a stem cell microenvironment in BOA where epithelium becomes resistant to naphthalene toxicity.

Asunto(s)

Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Diferenciación Celular/fisiología , Pulmón/efectos de los fármacos , Naftalenos/toxicidad , Células Neuroendocrinas/efectos de los fármacos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Línea Celular Tumoral , Humanos , Inmunohistoquímica , Hibridación in Situ , Pulmón/citología , Pulmón/metabolismo , Ratones , Ratones Transgénicos , Células Neuroendocrinas/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Matrilysin-1 (also called matrix metalloproteinase-7) is expressed in injured lung and in cancer but not in normal epithelia. Bronchiolization of the alveoli (BOA), a potential precursor of lung cancer, is a histologically distinct type of metaplasia that is composed of cells resembling airway epithelium in the alveolar compartment. We demonstrate that there is increased expression of matrilysin-1 in human lesions and BOA in the CC10-human achaete-scute homolog-1 transgenic mouse model. Forced expression of the matrilysin-1 gene in immortalized human normal airway epithelial BEAS-2B and HPLD1 cells, which do not normally express matrilysin-1, promoted cellular migration, suggesting a functional link for BOA formation via bronchiolar cell migration. In addition, matrilysin-1 stimulated proliferation and inhibited Fas-induced apoptosis, while a knockdown by RNA interference decreased cell growth, migration, and increased sensitivity to apoptosis. Western blotting demonstrated increased levels of phospho-p38 and phospho-Erk1/2 kinases after matrilysin-1 expression. Gene expression analysis uncovered several genes that were related to cell growth, migration/movement, and death, which could potentially facilitate bronchiolization. In vivo, the formation of BOA lesions was reduced when CC10-human achaete-scute homolog-1 mice were crossed with matrilysin-1 null mice and was correlated with reduced matrilysin-1 expression in BOA. We conclude that matrilysin-1 may play an important role in the bronchiolization of alveoli by promoting proliferation, migration, and attenuation of apoptosis involving multiple genes in the MAP kinase pathway.

Asunto(s)

Bronquiolos/patología , Neoplasias Pulmonares/patología , Metaloproteinasa 7 de la Matriz/fisiología , Lesiones Precancerosas/patología , Alveolos Pulmonares/patología , Animales , Bronquiolos/enzimología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Humanos , Neoplasias Pulmonares/enzimología , Metaloproteinasa 7 de la Matriz/genética , Ratones , Ratones Transgénicos , Lesiones Precancerosas/enzimología , Alveolos Pulmonares/enzimología

RESUMEN

OBJECTIVE: The Gynecologic Oncology Group (GOG) performed a detailed analysis of p53 overexpression in previously-untreated women with invasive early or advanced stage epithelial ovarian cancer (EOC). METHODS: Women were eligible for the study if they provided a tumor block for translational research and participated in either GOG-157, a randomized phase III trial of three versus (vs.) six cycles of paclitaxel+carboplatin in high-risk, early stage EOC, or GOG-111, a randomized phase III trial of cyclophosphamide+cisplatin vs. paclitaxel+cisplatin in suboptimally-resected, advanced stage EOC. The N-terminal DO-7 p53 antibody was used to examine the expression of the major normal and mutant p53-isoforms. p53 overexpression was defined as >or=10% tumor cells exhibiting nuclear staining. RESULTS: p53 was overexpressed in 51% (73/143) and 66% (90/136) of cases in the GOG-157 and GOG-111 cohorts, respectively. In the GOG-157 cohort, p53 overexpression was not associated with any clinical characteristics or overall survival (OS) but was associated with worse progression-free survival (PFS) (logrank test: p=0.013; unadjusted Cox modeling: p=0.015). In the GOG-111 cohort, p53 overexpression was associated with GOG performance status (p=0.018) and grade (p=0.003), but not with age, stage, cell type or with tumor response and disease status after primary chemotherapy, PFS or OS. Adjusted Cox regression modeling demonstrated that p53 overexpression was not an independent prognostic factor for PFS or OS in either cohort. CONCLUSIONS: p53 overexpression assessed by DO-7 immunostaining is common in early and advanced stage EOC, but has limited prognostic value in women treated with surgical staging and platinum-based combination chemotherapy.

Asunto(s)

Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/terapia , Proteína p53 Supresora de Tumor/biosíntesis , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carboplatino/administración & dosificación , Cisplatino/administración & dosificación , Ciclofosfamida/administración & dosificación , Supervivencia sin Enfermedad , Determinación de Punto Final , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Paclitaxel/administración & dosificación , Factores de Riesgo , Resultado del Tratamiento

RESUMEN

Squamous cell carcinoma accounts for 20% of all human lung cancers and is strongly linked to cigarette smoking. It develops through premalignant changes that are characterized by high levels of keratin 14 (K14) expression in the airway epithelium and evolve through basal cell hyperplasia, squamous metaplasia and dysplasia to carcinoma in situ and invasive carcinoma. In order to explore the impact of K14 in the pulmonary epithelium that normally lacks both squamous differentiation and K14 expression, human keratin 14 gene hK14 was constitutively expressed in mouse airway progenitor cells using a mouse Clara cell specific 10 kDa protein (CC10) promoter. While the lungs of CC10-hK14 transgenic mice developed normally, we detected increased expression of K14 and the molecular markers of squamous differentiation program such as involucrin, loricrin, small proline-rich protein 1A, transglutaminase 1 and cholesterol sulfotransferase 2B1. In contrast, wild-type lungs were negative. Aging CC10-hK14 mice revealed multifocal airway cell hyperplasia, occasional squamous metaplasia and their lung tumors displayed evidence for multidirectional differentiation. We conclude that constitutive expression of hK14 initiates squamous differentiation program in the mouse lung, but fails to promote squamous maturation. Our study provides a novel model for assessing the mechanisms of premalignant lesions in vivo by modifying differentiation and proliferation of airway progenitor cells.

Asunto(s)

Carcinoma de Células Escamosas/genética , Transformación Celular Neoplásica/genética , Queratina-14/genética , Neoplasias Pulmonares/genética , Lesiones Precancerosas/genética , Animales , Western Blotting , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Transformación Celular Neoplásica/metabolismo , Humanos , Inmunohistoquímica , Inmunoprecipitación , Queratina-14/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Transgénicos , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección

RESUMEN

Protein Gene Product 9.5 (PGP9.5) is highly expressed in nervous tissue. Recently PGP9.5 expression has been found to be upregulated in the pulmonary epithelium of smokers and in non-small cell lung cancer, suggesting that it also plays a role in carcinogen-inflicted lung epithelial injury and carcinogenesis. We investigated the expression of PGP9.5 in mice in response to two prominent carcinogens found in tobacco smoke: Naphthalene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). By immunostaining, we found that PGP9.5 protein was highly expressed throughout the airway epithelium in the days immediately following a single injection of naphthalene. In contrast, PGP9.5 was exclusively confined to neurons and neuroendocrine cells in the control and NNK-exposed lungs. Furthermore, we investigated the expression of PGP9.5 mRNA in the lungs by quantitative RT-PCR (qPCR). PGP9.5 mRNA expression was highly upregulated in the days immediately following naphthalene injection and gradually returning to that of control mice 5 days after naphthalene injection. In contrast, exposure to NNK did not result in a significant increase in PGP9.5 mRNA 10 weeks after exposure. No increased expression of two other neuroendocrine markers was found in the non-neuroendocrine epithelial cells after naphthalene exposure. In contrast, immunostaining for the cell cycle regulator p27(Kip1), which has previously been associated with PGP9.5 in lung cancer cells, revealed transient downregulation of p27(Kip1) in naphthalene exposed airways compared to controls, indicating that the rise in PGP9.5 in the airway epithelium is related to downregulation of p27(Kip1). This study is the first to specifically identify the carcinogen naphthalene as an inducer of PGP9.5 expression in non-neuroendocrine epithelium after acute lung injury and further strengthens the accumulating evidence of PGP9.5 as a central player in lung epithelial damage and early carcinogenesis.

Asunto(s)

Pulmón/efectos de los fármacos , Naftalenos/toxicidad , Ubiquitina Tiolesterasa/genética , Animales , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/análisis , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Hiperplasia , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/metabolismo , Masculino , Ratones , Sistemas Neurosecretores/patología , Nitrosaminas/toxicidad , ARN Mensajero/análisis , Ubiquitina Tiolesterasa/análisis

RESUMEN

Despite the importance of airspace integrity in vertebrate gas exchange, the molecular pathways that instruct distal lung formation are poorly understood. Recently, we found that fibrillin-1 deficiency in mice impairs alveolar formation and recapitulates the pulmonary features of human Marfan syndrome. To further elucidate effectors involved in distal lung formation, we performed expression profiling analysis comparing the fibrillin-1-deficient and wild-type developing lung. NeuroD, a basic helix-loop-helix transcription factor, fulfilled the expression criteria for a candidate mediator of distal lung development. We investigated its role in murine lung development using genetically targeted NeuroD-deficient mice. We found that NeuroD deficiency results in both impaired alveolar septation and altered morphology of the pulmonary neuroendocrine cells. NeuroD-deficient mice had enlarged alveoli associated with reduced epithelial proliferation in the airway and airspace compartments during development. Additionally, the neuroendocrine compartment in these mice manifested an increased number of neuroepithelial bodies but a reduced number of solitary pulmonary neuroendocrine cells in the neonatal lung. Overexpression of NeuroD in a murine lung epithelial cell line conferred a neuroendocrine phenotype characterized by the induction of neuroendocrine markers as well as increased proliferation. These results support an unanticipated role for NeuroD in the regulation of pulmonary neuroendocrine and alveolar morphogenesis and suggest an intimate connection between the neuroendocrine compartment and distal lung development.

Asunto(s)

Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Regulación de la Expresión Génica , Pulmón/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Línea Celular , Proliferación Celular , Células Epiteliales/citología , Fibrilina-1 , Fibrilinas , Perfilación de la Expresión Génica , Heterocigoto , Humanos , Ratones , Proteínas de Microfilamentos/metabolismo , Modelos Biológicos , Fenotipo , Transfección