RESUMEN
The human genome encodes several hundred microRNA (miRNA) genes that produce small (21-23n) single strand regulatory RNA molecules. Although abnormal expression of miRNAs has been linked to cancer progression, the mechanisms of this dysregulation are poorly understood. Malignant mesothelioma (MM) of pleura is an aggressive and highly lethal cancer resistant to conventional therapies. We and others previously linked loss of the 9p21.3 chromosome in MM with short time to tumor recurrence. In this study, we report that MM cell lines derived from patients with more aggressive disease fail to express miR-31, a microRNA recently linked with suppression of breast cancer metastases. We further demonstrate that this loss is due to homozygous deletion of the miR-31-encoding gene that resides in 9p21.3. Functional assessment of miR-31 activity revealed its ability to inhibit proliferation, migration, invasion, and clonogenicity of MM cells. Re-introduction of miR-31 suppressed the cell cycle and inhibited expression of multiple factors involved in cooperative maintenance of DNA replication and cell cycle progression, including pro-survival phosphatase PPP6C, which was previously associated with chemotherapy and radiation therapy resistance, and maintenance of chromosomal stability. PPP6C, whose mRNA is distinguished with three miR-31-binding sites in its 3'-untranslated region, was consistently down-regulated by miR-31 introduction and up-regulated in clinical MM specimens as compared with matched normal tissues. Taken together, our data suggest that tumor-suppressive propensity of miR-31 can be used for development of new therapies against mesothelioma and other cancers that show loss of the 9p21.3 chromosome.
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Eliminación de Gen , Mesotelioma/genética , Mesotelioma/patología , MicroARNs/genética , Secuencia de Bases , Ciclo Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Reparación del ADN/genética , Replicación del ADN/genética , Perfilación de la Expresión Génica , Genómica , Humanos , Mesotelioma/diagnóstico , MicroARNs/metabolismo , Invasividad Neoplásica/genética , Fosfoproteínas Fosfatasas/metabolismo , Reproducibilidad de los Resultados , Telómero/genéticaRESUMEN
The largest gene knock-down experiments performed to date have used multiple short interfering/short hairpin (si/sh)RNAs per gene. To overcome this burden for design of a genome-wide siRNA library, we used the Stuttgart Neural Net Simulator to train algorithms on a data set of 2,182 randomly selected siRNAs targeted to 34 mRNA species, assayed through a high-throughput fluorescent reporter gene system. The algorithm, (BIOPREDsi), reliably predicted activity of 249 siRNAs of an independent test set (Pearson coefficient r = 0.66) and siRNAs targeting endogenous genes at mRNA and protein levels. Neural networks trained on a complementary 21-nucleotide (nt) guide sequence were superior to those trained on a 19-nt sequence. BIOPREDsi was used in the design of a genome-wide siRNA collection with two potent siRNAs per gene. When this collection of 50,000 siRNAs was used to identify genes involved in the cellular response to hypoxia, two of the most potent hits were the key hypoxia transcription factors HIF1A and ARNT.
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Algoritmos , Silenciador del Gen , Modelos Genéticos , Red Nerviosa , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Alineación de Secuencia/métodos , Análisis de Secuencia de ARN/métodos , Secuencia de Bases , Simulación por Computador , Diseño Asistido por Computadora , Biblioteca de Genes , Modelos Estadísticos , Datos de Secuencia MolecularRESUMEN
Considerable progress has been made in exploiting the enormous amount of genomic and genetic information for the identification of potential targets for drug discovery and development. New tools that incorporate pathway information have been developed for gene expression data mining to reflect differences in pathways in normal and disease states. In addition, forward and reverse genetics used in a high-throughput mode with full-length cDNA and RNAi libraries enable the direct identification of components of signaling pathways. The discovery of the regulatory function of microRNAs highlights the importance of continuing the investigation of the genome with sophisticated tools. Furthermore, epigenetic information including DNA methylation and histone modifications that mediate important biological processes add to the possibilities to identify novel drug targets and patient populations that will benefit from new therapies.
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Evaluación Preclínica de Medicamentos/métodos , Genómica/métodos , Animales , Biología Computacional , Epigénesis Genética , Expresión Génica , Predisposición Genética a la Enfermedad/genética , Humanos , MicroARNs/análisisRESUMEN
The completion of the sequencing of the human genome, and those of other organisms, is expected to lead to many potential new drug targets in various diseases, and it is predicted that novel therapeutic agents will be developed against such targets. The role of functional genomics in modern drug discovery is to prioritize these targets and to translate that knowledge into rational and reliable drug discovery. Here, we describe the field of functional genomics and review approaches that have been applied to drug discovery, including RNA profiling, proteomics, antisense and RNA interference, model organisms and high-throughput, genome-wide overexpression or knockdowns, and outline the future directions that are likely to yield new drug targets from genomics.
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Diseño de Fármacos , Genómica/métodos , Oligonucleótidos Antisentido/farmacología , Proteoma/genética , Animales , Genómica/tendencias , HumanosRESUMEN
We have synthesized a histone deacetylase inhibitor, NVP-LAQ824, a cinnamic hydroxamic acid, that inhibited in vitro enzymatic activities and transcriptionally activated the p21 promoter in reporter gene assays. NVP-LAQ824 selectively inhibited growth of cancer cell lines at submicromolar levels after 48-72 h of exposure, whereas higher concentrations and longer exposure times were required to retard the growth of normal dermal human fibroblasts. Flow cytometry studies revealed that both tumor and normal cells arrested in the G(2)-M phase of the cell cycle after compound treatment. However, an increased sub-G(1) population at 48 h (reminiscent of apoptotic cells) was observed only in the cancer cell line. Annexin V staining data supported our hypothesis that NVP-LAQ824 induced apoptosis in tumor and transformed cells but not in normal cells. Western blotting experiments showed an increased histone H3 and H4 acetylation level in NVP-LAQ824-treated cancer cells, suggesting that the likely in vivo target of NVP-LAQ824 was histone deacetylase(s). Finally, NVP-LAQ824 exhibited antitumor effects in a xenograft animal model. Together, our data indicated that the activity of NVP-LAQ824 was consistent with its intended mechanism of action. This novel histone deacetylase inhibitor is currently in clinical trials as an anticancer agent.
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Antineoplásicos/toxicidad , Neoplasias del Colon/tratamiento farmacológico , Inhibidores de Histona Desacetilasas , Ácidos Hidroxámicos/toxicidad , Animales , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , División Celular/genética , Línea Celular Tumoral , Neoplasias del Colon/patología , Inhibidores Enzimáticos/toxicidad , Fluorouracilo/uso terapéutico , Histona Desacetilasas/aislamiento & purificación , Humanos , Cinética , Masculino , Ratones , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Trasplante HeterólogoRESUMEN
The dynamic balance between histone acetylation and deacetylation plays a significant role in the regulation of gene transcription. Much of our current understanding of this transcriptional control comes from the use of HDAC inhibitors such as trapoxin A (TPX), which leads to hyperacetylated histone, alters local chromatin architecture and transcription and results in tumor cell death. In this study, we treated tumor cells with TPX and HDAC1 antisense oligonucleotides, and analysed the transcriptional consequences of HDAC inhibition. Among other genes, the small GTPase RhoB was found to be significantly upregulated by TPX and repressed by HDAC1. The induction of RhoB by HDAC inhibition was mediated by an inverted CCAAT box in the RhoB promoter. Interestingly, measurement of RhoB transcription in approximately 130 tumor-derived cell lines revealed low expression in almost all of these samples, in contrast to RhoA and RhoC. Accumulating evidence indicates that the small GTPase Rho proteins are involved in a variety of important processes in cancer, including cell transformation, survival, invasion, metastasis and angiogenesis. This study for the first time demonstrates a link between HDAC inhibition and RhoB expression and provides an important insight into the mechanisms of HDAC-mediated transcriptional control and the potential therapeutic benefit of HDAC inhibition.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Inhibidores de Histona Desacetilasas , Neoplasias Pulmonares/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Péptidos , Proteína de Unión al GTP rhoB/metabolismo , Acetilación/efectos de los fármacos , Antibacterianos/farmacología , Secuencia de Bases , Factor de Unión a CCAAT/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Transformada , Inhibidores Enzimáticos/farmacología , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Datos de Secuencia Molecular , Proteínas de Unión al GTP Monoméricas/genética , Regiones Promotoras Genéticas , Proteínas Represoras/antagonistas & inhibidores , Células Tumorales Cultivadas , Regulación hacia Arriba , Proteína de Unión al GTP rhoB/genéticaRESUMEN
Dynamic changes in the post-translational modification pattern of histories such as acetylation, deacetylation, phosphorylation, methylation and ubiquitination are thought to provide a code for correct regulation of gene expression by affecting chromatin structure and interaction with regulatory factors. Our studies focus on the role of histone deacetylases (HDACs) in transcriptional regulation and addressing functional differences of class I and class II HDACs. To identify genes that were transcriptionally regulated by specific HDACs, genome scale expression profiles were performed in cancer cells following the inhibition of three HDAC family members by specific oligonucleotides. The modulated genes identified in this study represented a wide range of modifications in different cellular pathways. In addition, treatment of cancer cells with a HDAC inhibitor was found to induce the expression of the small GTPase RhoB through an inverted CCAAT box in the RhoB promoter. These studies identified a specific transcription element involved in HDAC-mediated gene transcription and genes that are transcriptionally regulated by specific HDACs, providing important insight into the potential therapeutic benefit of HDAC inhibition.
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Regulación de la Expresión Génica/fisiología , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Transcripción Genética/fisiología , Factor de Unión a CCAAT , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/biosíntesis , Ciclinas/genética , Inhibidores de Histona Desacetilasas , Humanos , Péptidos/farmacología , Células Tumorales Cultivadas , Proteína de Unión al GTP rhoB/biosíntesis , Proteína de Unión al GTP rhoB/genéticaRESUMEN
In the current study, we isolated sublines of the human breast adenocarcinoma cell line MDA 435 that exhibited increasing resistance to epothilone A, a microtubule-stabilizing cytotoxic agent. The resistant cells did not express P glycoprotein or multidrug resistance-associated protein (MRP) which are known mediators of multidrug resistance (MDR). Two groups of epothilone A-resistant cells were selected: cells which exhibited low resistance to both epothilone A and Taxol, and cells which exhibit low resistance to Taxol but high resistance to epothilone A. cDNA microarrays of epothilone A-resistant and Taxol-resistant cells were utilized to further characterize epothilone A resistance. Hierarchical clustering of genes according to their levels of expression indicated that the majority of genes which were highly expressed in epothilone A-resistant cells but not in taxol-resistant MDR cells encode known interferon-inducible proteins. Genes whose expression increased with increasing epothilone A resistance include microtubule-associated GTPases, cytoskeletal proteins, cell signalling proteins and a drug metabolising enzyme. The majority of the genes that were repressed in both epothilone A- and Taxol-resistant cells encode proteins regulating cellular growth signalling mechanisms.
Asunto(s)
Antineoplásicos/farmacología , Epotilonas/farmacología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/genética , Adenocarcinoma/genética , Adenocarcinoma/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteínas del Citoesqueleto/biosíntesis , Proteínas del Citoesqueleto/genética , Resistencia a Antineoplásicos/genética , Femenino , GTP Fosfohidrolasas/biosíntesis , GTP Fosfohidrolasas/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Sustancias de Crecimiento/biosíntesis , Sustancias de Crecimiento/genética , Células HL-60/efectos de los fármacos , Humanos , Interferones/farmacología , Masculino , Microtúbulos/efectos de los fármacos , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/fisiología , Paclitaxel/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
We reviewed the phenotypic and molecular characteristics of MCF10DCIS.com and the SUM cell lines based on numerous studies performed over the years. The major signaling pathways that give rise to the phenotype of these cells may serve as a good resource of information when researchers in drug discovery and development use these cells to identify novel targets and biomarkers. Major signaling pathways and mutations affecting the coding sequence are also described providing important information when using these cells as a model in a variety of studies.
RESUMEN
PURPOSE: Accurate identification of tissue of origin (ToO) for patients with carcinoma of unknown primary (CUP) may help customize therapy to the putative primary and thereby improve the clinical outcome. We prospectively studied the performance of a microRNA-based assay to identify the ToO in CUP patients. EXPERIMENTAL DESIGN: Formalin-fixed paraffin-embedded (FFPE) metastatic tissue from 104 patients was reviewed and 87 of these contained sufficient tumor for testing. The assay quantitates 48 microRNAs and assigns one of 25 tumor diagnoses by using a biologically motivated binary decision tree and a K-nearest neighbors (KNN). The assay predictions were compared with clinicopathologic features and, where suitable, to therapeutic response. RESULTS: Seventy-four of the 87 cases were processed successfully. The assay result was consistent or compatible with the clinicopathologic features in 84% of cases processed successfully (71% of all samples attempted). In 65 patients, pathology and immunohistochemistry (IHC) suggested a diagnosis or (more often) a differential diagnosis. Out of those, the assay was consistent or compatible with the clinicopathologic presentation in 55 (85%) cases. Of the 9 patients with noncontributory IHC, the assay provided a ToO prediction that was compatible with the clinical presentation in 7 cases. CONCLUSIONS: In this prospective study, the microRNA diagnosis was compatible with the clinicopathologic picture in the majority of cases. Comparative effectiveness research trials evaluating the added benefit of molecular profiling in appropriate CUP subsets are warranted. MicroRNA profiling may be particularly helpful in patients in whom the IHC profile of the metastasis is nondiagnostic or leaves a large differential diagnosis.
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Carcinoma/diagnóstico , Carcinoma/secundario , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Neoplasias Primarias Desconocidas/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/uso terapéutico , Carcinoma/genética , Árboles de Decisión , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Primarias Desconocidas/tratamiento farmacológico , Neoplasias Primarias Desconocidas/genética , Estudios Prospectivos , Resultado del Tratamiento , Adulto JovenRESUMEN
Hepatocellular carcinoma (HCC) is generally a fatal disease due to a paucity of effective treatment options. The identification of oncogenic microRNAs that exert pleiotropic effects in HCC cells may offer new therapeutic targets. In this study, we have identified the human microRNA miR-191 as a potential target for HCC therapy. Inhibition of miR-191 decreased cell proliferation and induced apoptosis in vitro and significantly reduced tumor masses in vivo in an orthotopic xenograft mouse model of HCC. Additionally, miR-191 was found to be upregulated by a dioxin, a known liver carcinogen, and was found to be a regulator of a variety of cancer-related pathways. Our findings offer a preclinical proof of concept for miR-191 targeting as a rational strategy to pursue for improving HCC treatment.
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Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , MicroARNs/genética , Regiones no Traducidas 3'/genética , Animales , Pueblo Asiatico/genética , Carcinógenos/farmacología , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/terapia , División Celular , Dioxinas/farmacología , Regulación Neoplásica de la Expresión Génica , Genes Reporteros , Humanos , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/terapia , Ratones , MicroARNs/efectos de los fármacos , Modelos Animales , Modelos Genéticos , ARN Neoplásico/genética , ARN Neoplásico/aislamiento & purificación , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba , Población Blanca/genéticaRESUMEN
The inability to forecast outcomes for malignant mesothelioma prevents clinicians from providing aggressive multimodality therapy to the most appropriate individuals who may benefit from such an approach. We investigated whether specific microRNAs (miR) could segregate a largely surgically treated group of mesotheliomas into good or bad prognosis categories. A training set of 44 and a test set of 98 mesothelioma tumors were analyzed by a custom miR platform, along with 9 mesothelioma cell lines and 3 normal mesothelial lines. Functional implications as well as downstream targets of potential prognostic miRs were investigated. In both the training and test sets, hsa-miR-29c* was an independent prognostic factor for time to progression as well as survival after surgical cytoreduction. The miR was expressed at higher levels in epithelial mesothelioma, and the level of this miR could segregate patients with this histology into groups with differing prognosis. Increased expression of hsa-miR-29c* predicted a more favorable prognosis, and overexpression of the miR in mesothelioma cell lines resulted in significantly decreased proliferation, migration, invasion, and colony formation. Moreover, major epigenetic regulation of mesothelioma is mediated by hsa-miR-29c* and was shown through downregulation of DNA methyltransferases as well as upregulation of demethylating genes. A single miR has the potential to be a prognostic biomarker in mesothelioma, and validation of these findings as well as investigation of its downstream targets may give insight for potential therapies in the future.
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Mesotelioma/genética , MicroARNs/análisis , Neoplasias Pleurales/genética , Adulto , Anciano , Anciano de 80 o más Años , Amianto/envenenamiento , Línea Celular Tumoral , Femenino , Humanos , Masculino , Mesotelioma/etiología , MicroARNs/biosíntesis , MicroARNs/genética , Persona de Mediana Edad , Neoplasias Pleurales/etiología , Pronóstico , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , TransfecciónRESUMEN
Distinguishing hepatocellular carcinoma from metastatic tumors in the liver is of great practical importance, with significant therapeutic and prognostic implications. This differential diagnosis can be difficult because metastatic cancers in the liver, especially adenocarcinomas, may mimic the morphology and immunoexpression of hepatocellular carcinoma. Biomarkers that are specifically expressed in either hepatocellular carcinoma or metastatic adenocarcinoma can therefore be useful diagnostic tools. To find such biomarkers, we studied microRNA expression in 144 tumor samples using custom microarrays. Hsa-miR-141 and hsa-miR-200c, microRNAs that promote epithelial phenotypes, had significantly higher levels in non-hepatic epithelial tumors. In contrast, endothelial-associated hsa-miR-126 showed higher expression levels in hepatocellular carcinomas. Combinations of these microRNAs accurately identified primary hepatocellular carcinoma from metastatic adenocarcinoma in the liver. These findings were validated using quantitative real-time PCR to measure microRNA expression in additional samples. Thus, the tissue-specific expression patterns of microRNAs make them useful biomarkers for the diagnosis of liver malignancies.
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Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Hígado/metabolismo , MicroARNs/genética , Carcinoma Hepatocelular/patología , Diagnóstico Diferencial , Humanos , Hígado/patología , Neoplasias Hepáticas/patología , MicroARNs/metabolismo , Metástasis de la Neoplasia , Curva ROC , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The definitive identification of malignant pleural mesothelioma (MPM) has significant clinical implications, yet other malignancies often involve the lung pleura, confounding the diagnosis of MPM. In the absence of accurate markers, MPM can be difficult to distinguish from peripheral lung adenocarcinoma and metastatic epithelial cancers. MicroRNA expression is tissue-specific and highly informative for identifying tumor origin. We identified microRNA biomarkers for the differential diagnosis of MPM and developed a standardized microRNA-based assay. Formalin-fixed, paraffin-embedded samples of 33 MPM and 210 carcinomas were used for assay development. Using microarrays, we identified microRNAs differentially expressed between MPM and various carcinomas. Hsa-miR-193-3p was overexpressed in MPM, while hsa-miR-200c and hsa-miR-192 were overexpressed in peripheral lung adenocarcinoma and carcinomas that frequently metastasize to lung pleura. We developed a standardized diagnostic assay based on the expression of these microRNAs. The assay reached a sensitivity of 100% and a specificity of 94% in a blinded validation set of 68 samples from the lung and pleura. This diagnostic assay can provide a useful tool in the differential diagnosis of MPM from other malignancies in the pleura.
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Biomarcadores de Tumor/genética , Mesotelioma , MicroARNs/genética , Análisis por Micromatrices/métodos , Neoplasias Pleurales , Regulación Neoplásica de la Expresión Génica , Humanos , Mesotelioma/diagnóstico , Mesotelioma/genética , Mesotelioma/patología , MicroARNs/metabolismo , Análisis por Micromatrices/normas , Neoplasias Pleurales/diagnóstico , Neoplasias Pleurales/genética , Neoplasias Pleurales/patología , Sensibilidad y EspecificidadRESUMEN
PURPOSE: Recent advances in treatment of lung cancer require greater accuracy in the subclassification of non-small-cell lung cancer (NSCLC). Targeted therapies which inhibit tumor angiogenesis pose higher risk for adverse response in cases of squamous cell carcinoma. Interobserver variability and the lack of specific, standardized assays limit the current abilities to adequately stratify patients for such treatments. In this study, we set out to identify specific microRNA biomarkers for the identification of squamous cell carcinoma, and to use such markers for the development of a standardized assay. PATIENTS AND METHODS: High-throughput microarray was used to measure microRNA expression levels in 122 adenocarcinoma and squamous NSCLC samples. A quantitative real-time polymerase chain reaction (qRT-PCR) platform was used to verify findings in an independent set of 20 NSCLC formalin-fixed, paraffin-embedded (FFPE) samples, and to develop a diagnostic assay using an additional set of 27 NSCLC FFPE samples. The assay was validated using an independent blinded cohort consisting of 79 NSCLC FFPE samples. RESULTS: We identified hsa-miR-205 as a highly specific marker for squamous cell lung carcinoma. A microRNA-based qRT-PCR assay that measures expression of hsa-miR-205 reached sensitivity of 96% and specificity of 90% in the identification of squamous cell lung carcinomas in an independent blinded validation set. CONCLUSION: Hsa-miR-205 is a highly accurate marker for lung cancer of squamous histology. The standardized diagnostic assay presented here can provide highly accurate subclassification of NSCLC patients.
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Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , MicroARNs/genética , Adenocarcinoma/diagnóstico , Adenocarcinoma/genética , Adenocarcinoma/secundario , Adulto , Anciano , Anciano de 80 o más Años , Bioensayo , Carcinoma de Células Grandes/diagnóstico , Carcinoma de Células Grandes/genética , Carcinoma de Células Grandes/secundario , Carcinoma de Pulmón de Células no Pequeñas/secundario , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/secundario , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
MicroRNAs (miRNAs) belong to a class of noncoding, regulatory RNAs that is involved in oncogenesis and shows remarkable tissue specificity. Their potential for tumor classification suggests they may be used in identifying the tissue in which cancers of unknown primary origin arose, a major clinical problem. We measured miRNA expression levels in 400 paraffin-embedded and fresh-frozen samples from 22 different tumor tissues and metastases. We used miRNA microarray data of 253 samples to construct a transparent classifier based on 48 miRNAs. Two-thirds of samples were classified with high confidence, with accuracy >90%. In an independent blinded test-set of 83 samples, overall high-confidence accuracy reached 89%. Classification accuracy reached 100% for most tissue classes, including 131 metastatic samples. We further validated the utility of the miRNA biomarkers by quantitative RT-PCR using 65 additional blinded test samples. Our findings demonstrate the effectiveness of miRNAs as biomarkers for tracing the tissue of origin of cancers of unknown primary origin.
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Biomarcadores de Tumor/genética , Perfilación de la Expresión Génica/métodos , MicroARNs/genética , Neoplasias/diagnóstico , Neoplasias/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Secuencia de Bases , Biomarcadores de Tumor/análisis , Humanos , Datos de Secuencia Molecular , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Células Tumorales CultivadasRESUMEN
Histone deacetylases (HDACs) play a critical role in regulating gene expression and key biological processes. However, how HDACs are involved in innate immunity is little understood. Here, in this first systematic investigation of the role of HDACs in immunity, we show that HDAC inhibition by a small-molecule HDAC inhibitor (HDACi), LAQ824, alters Toll-like receptor 4 (TLR4)-dependent activation and function of macrophages and dendritic cells (DCs). Surprisingly, pan-HDAC inhibition modulates only a limited set of genes involved in distinct arms of immune responses. Specifically, it inhibited DC-controlled T helper 1 (Th1) effector but not Th2 effector cell activation and migration. It also inhibited macrophage- and DC-mediated monocyte but not neutrophil chemotaxis. These unexpected findings demonstrate the high specificity of HDAC inhibition in modulating innate and adaptive immune responses, and highlight the potential for HDACi to alter the Th1 and Th2 balance in therapeutic settings.
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Histona Desacetilasas/fisiología , Inmunidad Innata , Células TH1/inmunología , Células Th2/inmunología , Animales , Quimiotaxis de Leucocito/inmunología , Células Dendríticas/inmunología , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/inmunología , Humanos , Ácidos Hidroxámicos/farmacología , Activación de Linfocitos/inmunología , Macrófagos/inmunología , Ratones , Células TH1/citología , Células Th2/citología , Receptor Toll-Like 4/metabolismoRESUMEN
HDAC inhibitors are promising antitumor drugs with several HDAC inhibitors already in clinical trials. LAQ824, a potent pan-HDAC inhibitor, has been shown to induce cell cycle arrest and cell death. However, the mechanism of its antitumor effects and specially its tumor selectivity are still poorly understood. The focus of this study is to elucidate LAQ824 mediated anti-proliferative effects in lung carcinoma cells and the mechanism underlying the different sensitivity of LAQ824 to cancer and normal cells. In this study, LAQ824 mediated apoptosis was found to occur mainly via activation of the mitochondrial death pathway by inducing Apaf1 and caspase 9 and promoting mitochondrial release of key proapoptotic factors in lung cancer cells, but not in normal fibroblast cells. Using chromatin immunoprecipitation assay, we found that RNA Pol II binding and histone H3 acetylation levels at Apaf1 promoter were increased following LAQ824 treatment, explaining LAQ824 induced expression of Apaf1 in lung cancer cells. Furthermore, we showed that LAQ824 only triggered the release of mitochondrial proapoptotic factors such as cytochrome C (Cyto C) and apoptosis inducing factor (AIF) in lung cancer cells but not in normal blast cells. In addition, LAQ824 was found to induce Bax translocation in lung cancer cell, which may play important role in the induction of the release of mitochondrial proapoptotic factors. These data provide insight into the mechanism underlying the selective induction of apoptosis by LAQ824 in cancer cells.
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Apoptosis/efectos de los fármacos , Ácidos Hidroxámicos/farmacología , Neoplasias Pulmonares/patología , Mitocondrias/efectos de los fármacos , Acetilación/efectos de los fármacos , Caspasas/metabolismo , Relación Dosis-Respuesta a Droga , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Histonas/metabolismo , Humanos , Neoplasias Pulmonares/genética , Mitocondrias/metabolismo , Transporte de Proteínas/efectos de los fármacos , Factores de Tiempo , Células Tumorales Cultivadas , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Histone deacetylases (HDACs) are catalytic subunits of multiprotein complexes that are targeted to specific promoters through their interaction with different transcriptional repressors causing silencing of the corresponding genes. This study describes the isolation of dHDAC4, a novel, catalytically active class II Drosophila histone deacetylase, and the analysis of its role in embryonic development. In early embryos, dHDAC4 is expressed in several phases. Initial ubiquitous expression becomes localized to an anterior domain, then evolves into a pair-rule-like and finally into a segment-polarity-like pattern. Suppression of dHDAC4 during early embryogenesis by double-stranded RNA interference led to segmentation defects. Analysis of dHDAC4 expression in gap and pair-rule gene mutants demonstrated that hunchback, knirps, and giant activate, while even-skipped suppresses dHDAC4 expression. These data revealed dHDAC4 involvement in the segmentation regulatory pathway and suggested complex transcriptional regulation as a potential mechanism that controls its expression.
Asunto(s)
Drosophila/embriología , Expresión Génica/fisiología , Genes Reguladores/fisiología , Histona Desacetilasas/fisiología , Interferencia de ARN/fisiología , Secuencia de Aminoácidos , Animales , Células Cultivadas , Drosophila/genética , Drosophila/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
BACKGROUND: In Schizosaccharomyces pombe, Hus1 is a component of the radiation sensitive (Rad) machinery that has been identified as playing a role in DNA repair and cell cycle G2/M checkpoint control pathways. Hus1 has been shown to exist in a discrete complex with at least two Rad family members, Rad1 and Rad9. Furthermore, Hus1 is essential for checkpoint activation, since Hus1 mutants fail to arrest the cell cycle in response to DNA damage or unreplicated DNA. To establish the role and relevance of human Hus1 in cell cycle regulation, the authors applied antisense technology to selectively downregulate the expression of Hus1 mRNA. METHODS: Transfection of 2'-O-methoxyethyl-modified Hus1 antisense oligoribonucleotides into human H1299 nonsmall lung carcinoma cells was performed using Lipofectin as the carrier. The authors prepared RNA from transfected cells, and levels of Hus1 expression were analyzed by real time polymerase chain reaction. The growth and viability of cells treated with Hus1 antisense oligonucleotides in the presence or absence of cisplatin were analyzed and compared to controls. RESULTS: Transfection of selected Hus1 antisense oligonucleotides into p53 deficient H1299 cells resulted in significant downregulation of Hus1 mRNA, up to 80%; RNA analyses reveal a maximal Hus1 antisense activity at a concentration of 200 nM with an IC50 determined to be 90 nM. The design and transfection of oligonucleotides containing three mismatches to their corresponding antisense counterparts had no or only minor effects on Hus1 mRNA levels, showing the specificity of Hus1 mRNA downregulation. The cisplatin IC50 in untransfected H1299 cells was found to be 20 microM and could be reduced significantly to only 7 microM after transfection of a Hus1 antisense oligonucleotide. CONCLUSIONS: Experiments addressing the proliferation and viability of transfected H1299 cells suggest that downregulation of Hus1 by specific antisense oligonucleotides sensitizes human cells to treatment with the DNA damaging agent cisplatin.