RESUMEN
Genome-wide identification of the mechanism of action (MoA) of small-molecule compounds characterizing their targets, effectors, and activity modulators represents a highly relevant yet elusive goal, with critical implications for assessment of compound efficacy and toxicity. Current approaches are labor intensive and mostly limited to elucidating high-affinity binding target proteins. We introduce a regulatory network-based approach that elucidates genome-wide MoA proteins based on the assessment of the global dysregulation of their molecular interactions following compound perturbation. Analysis of cellular perturbation profiles identified established MoA proteins for 70% of the tested compounds and elucidated novel proteins that were experimentally validated. Finally, unknown-MoA compound analysis revealed altretamine, an anticancer drug, as an inhibitor of glutathione peroxidase 4 lipid repair activity, which was experimentally confirmed, thus revealing unexpected similarity to the activity of sulfasalazine. This suggests that regulatory network analysis can provide valuable mechanistic insight into the elucidation of small-molecule MoA and compound similarity.
Asunto(s)
Algoritmos , Antineoplásicos/farmacología , Terapia Molecular Dirigida , Antineoplásicos/química , Epistasis Genética , Estudio de Asociación del Genoma Completo , Neoplasias/tratamiento farmacológico , Bibliotecas de Moléculas PequeñasRESUMEN
By analyzing gene expression data in glioblastoma in combination with matched microRNA profiles, we have uncovered a posttranscriptional regulation layer of surprising magnitude, comprising more than 248,000 microRNA (miR)-mediated interactions. These include â¼7,000 genes whose transcripts act as miR "sponges" and 148 genes that act through alternative, nonsponge interactions. Biochemical analyses in cell lines confirmed that this network regulates established drivers of tumor initiation and subtype implementation, including PTEN, PDGFRA, RB1, VEGFA, STAT3, and RUNX1, suggesting that these interactions mediate crosstalk between canonical oncogenic pathways. siRNA silencing of 13 miR-mediated PTEN regulators, whose locus deletions are predictive of PTEN expression variability, was sufficient to downregulate PTEN in a 3'UTR-dependent manner and to increase tumor cell growth rates. Thus, miR-mediated interactions provide a mechanistic, experimentally validated rationale for the loss of PTEN expression in a large number of glioma samples with an intact PTEN locus.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/metabolismo , MicroARNs/metabolismo , Humanos , Análisis Multivariante , Oncogenes , Fosfohidrolasa PTEN/genética , Interferencia de ARNRESUMEN
MEF2B encodes a transcriptional activator and is mutated in â¼11% of diffuse large B cell lymphomas (DLBCLs) and â¼12% of follicular lymphomas (FLs). Here we found that MEF2B directly activated the transcription of the proto-oncogene BCL6 in normal germinal-center (GC) B cells and was required for DLBCL proliferation. Mutation of MEF2B resulted in enhanced transcriptional activity of MEF2B either through disruption of its interaction with the corepressor CABIN1 or by rendering it insensitive to inhibitory signaling events mediated by phosphorylation and sumoylation. Consequently, the transcriptional activity of Bcl-6 was deregulated in DLBCLs with MEF2B mutations. Thus, somatic mutations of MEF2B may contribute to lymphomagenesis by deregulating BCL6 expression, and MEF2B may represent an alternative target for blocking Bcl-6 activity in DLBCLs.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Linfoma de Células B Grandes Difuso/genética , Proteínas de Dominio MADS/genética , Mutación , Factores Reguladores Miogénicos/genética , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Linfocitos B/metabolismo , Linfocitos B/patología , Ciclo Celular/genética , Proliferación Celular , Análisis por Conglomerados , Biología Computacional , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Perfilación de la Expresión Génica , Centro Germinal/metabolismo , Centro Germinal/patología , Humanos , Linfoma Folicular/genética , Linfoma Folicular/metabolismo , Linfoma de Células B Grandes Difuso/metabolismo , Proteínas de Dominio MADS/química , Proteínas de Dominio MADS/metabolismo , Factores de Transcripción MEF2 , Ratones , Simulación del Acoplamiento Molecular , Factores Reguladores Miogénicos/química , Factores Reguladores Miogénicos/metabolismo , Unión Proteica , Conformación Proteica , Proto-Oncogenes Mas , Sumoilación/genética , Transcripción GenéticaRESUMEN
Systems biology approaches are extensively used to model and reverse engineer gene regulatory networks from experimental data. Conversely, synthetic biology allows "de novo" construction of a regulatory network to seed new functions in the cell. At present, the usefulness and predictive ability of modeling and reverse engineering cannot be assessed and compared rigorously. We built in the yeast Saccharomyces cerevisiae a synthetic network, IRMA, for in vivo "benchmarking" of reverse-engineering and modeling approaches. The network is composed of five genes regulating each other through a variety of regulatory interactions; it is negligibly affected by endogenous genes, and it is responsive to small molecules. We measured time series and steady-state expression data after multiple perturbations. These data were used to assess state-of-the-art modeling and reverse-engineering techniques. A semiquantitative model was able to capture and predict the behavior of the network. Reverse engineering based on differential equations and Bayesian networks correctly inferred regulatory interactions from the experimental data.
Asunto(s)
Redes Reguladoras de Genes , Técnicas Genéticas , Modelos Genéticos , Saccharomyces cerevisiae/genética , Biología de Sistemas/métodos , Biología Computacional/métodos , Galactosa/metabolismo , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Glucosa/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
Mechanisms that maintain cancer stem cells are crucial to tumour progression. The ID2 protein supports cancer hallmarks including the cancer stem cell state. HIFα transcription factors, most notably HIF2α (also known as EPAS1), are expressed in and required for maintenance of cancer stem cells (CSCs). However, the pathways that are engaged by ID2 or drive HIF2α accumulation in CSCs have remained unclear. Here we report that DYRK1A and DYRK1B kinases phosphorylate ID2 on threonine 27 (Thr27). Hypoxia downregulates this phosphorylation via inactivation of DYRK1A and DYRK1B. The activity of these kinases is stimulated in normoxia by the oxygen-sensing prolyl hydroxylase PHD1 (also known as EGLN2). ID2 binds to the VHL ubiquitin ligase complex, displaces VHL-associated Cullin 2, and impairs HIF2α ubiquitylation and degradation. Phosphorylation of Thr27 of ID2 by DYRK1 blocks ID2-VHL interaction and preserves HIF2α ubiquitylation. In glioblastoma, ID2 positively modulates HIF2α activity. Conversely, elevated expression of DYRK1 phosphorylates Thr27 of ID2, leading to HIF2α destabilization, loss of glioma stemness, inhibition of tumour growth, and a more favourable outcome for patients with glioblastoma.
Asunto(s)
Glioblastoma/metabolismo , Glioblastoma/patología , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Células Madre Neoplásicas/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/antagonistas & inhibidores , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Hipoxia de la Célula , Línea Celular Tumoral , Proteínas Cullin/metabolismo , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Masculino , Ratones , Células Madre Neoplásicas/patología , Oxígeno/metabolismo , Fosforilación , Fosfotreonina/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Ubiquitinación , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasas DyrKRESUMEN
FGF signaling is a potent inducer of lacrimal gland development in the eye, capable of transforming the corneal epithelium into glandular tissues. Here, we show that genetic ablation of the Pea3 family of transcription factors not only disrupted the ductal elongation and branching of the lacrimal gland, but also biased the lacrimal gland epithelium toward an epidermal cell fate. Analysis of high-throughput gene expression and chromatin immunoprecipitation data revealed that the Pea3 genes directly control both the positive and negative feedback loops of FGF signaling. Importantly, Pea3 genes are also required to suppress aberrant Notch signaling which, if gone unchecked, can compromise lacrimal gland development by preventing the expression of both Sox and Six family genes. These results demonstrate that Pea3 genes are key FGF early response transcriptional factors, programing the genetic landscape for cell fate determination.
Asunto(s)
Diferenciación Celular/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Aparato Lagrimal/crecimiento & desarrollo , Factores de Transcripción/metabolismo , Animales , Células Epidérmicas/fisiología , Células Epiteliales/fisiología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Aparato Lagrimal/citología , Ratones , Ratones Noqueados , Técnicas de Cultivo de Órganos , Receptores Notch/metabolismo , Factores de Transcripción SOX/genética , Factores de Transcripción SOX/metabolismo , Factores de Transcripción/genéticaRESUMEN
microRNAs (miRNAs) play key roles in cancer, but their propensity to couple their targets as competing endogenous RNAs (ceRNAs) has only recently emerged. Multiple models have studied ceRNA regulation, but these models did not account for the effects of co-regulation by miRNAs with many targets. We modeled ceRNA and simulated its effects using established parameters for miRNA/mRNA interaction kinetics while accounting for co-regulation by multiple miRNAs with many targets. Our simulations suggested that co-regulation by many miRNA species is more likely to produce physiologically relevant context-independent couplings. To test this, we studied the overlap of inferred ceRNA networks from four tumor contexts-our proposed pan-cancer ceRNA interactome (PCI). PCI was composed of interactions between genes that were co-regulated by nearly three-times as many miRNAs as other inferred ceRNA interactions. Evidence from expression-profiling datasets suggested that PCI interactions are predictive of gene expression in 12 independent tumor- and non-tumor contexts. Biochemical assays confirmed ceRNA couplings for two PCI subnetworks, including oncogenes CCND1, HIF1A and HMGA2, and tumor suppressors PTEN, RB1 and TP53. Our results suggest that PCI is enriched for context-independent interactions that are coupled by many miRNA species and are more likely to be context independent.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , MicroARNs/metabolismo , Neoplasias/genética , ARN Neoplásico/metabolismo , Humanos , Neoplasias/metabolismoRESUMEN
The sequential use of signaling pathways is essential for the guidance of pluripotent progenitors into diverse cell fates. Here, we show that Shp2 exclusively mediates FGF but not PDGF signaling in the neural crest to control lacrimal gland development. In addition to preventing p53-independent apoptosis and promoting the migration of Sox10-expressing neural crests, Shp2 is also required for expression of the homeodomain transcription factor Alx4, which directly controls Fgf10 expression in the periocular mesenchyme that is necessary for lacrimal gland induction. We show that Alx4 binds an Fgf10 intronic element conserved in terrestrial but not aquatic animals, underlying the evolutionary emergence of the lacrimal gland system in response to an airy environment. Inactivation of ALX4/Alx4 causes lacrimal gland aplasia in both human and mouse. These results reveal a key role of Alx4 in mediating FGF-Shp2-FGF signaling in the neural crest for lacrimal gland development.
Asunto(s)
Factor 10 de Crecimiento de Fibroblastos/genética , Proteínas de Homeodominio/genética , Aparato Lagrimal/crecimiento & desarrollo , Morfogénesis/genética , Cresta Neural/crecimiento & desarrollo , Animales , Apoptosis/genética , Diferenciación Celular/genética , Linaje de la Célula/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Aparato Lagrimal/metabolismo , Mesodermo/crecimiento & desarrollo , Ratones , Células Madre Pluripotentes/metabolismo , Unión Proteica , Factores de Transcripción SOXE/genética , Transducción de SeñalRESUMEN
The Notch1 gene is a major oncogenic driver and therapeutic target in T-cell acute lymphoblastic leukemia (T-ALL). However, inhibition of NOTCH signaling with γ-secretase inhibitors (GSIs) has shown limited antileukemic activity in clinical trials. Here we performed an expression-based virtual screening to identify highly active antileukemic drugs that synergize with NOTCH1 inhibition in T-ALL. Among these, withaferin A demonstrated the strongest cytotoxic and GSI-synergistic antileukemic effects in vitro and in vivo. Mechanistically, network perturbation analyses showed eIF2A-phosphorylation-mediated inhibition of protein translation as a critical mediator of the antileukemic effects of withaferin A and its interaction with NOTCH1 inhibition. Overall, these results support a role for anti-NOTCH1 therapies and protein translation inhibitor combinations in the treatment of T-ALL.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Biosíntesis de Proteínas/efectos de los fármacos , Receptor Notch1/antagonistas & inhibidores , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , Sinergismo Farmacológico , Inhibidores Enzimáticos/uso terapéutico , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Terapia Molecular Dirigida/métodos , Fosforilación/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Receptor Notch1/genética , Receptor Notch1/metabolismo , Transducción de Señal/efectos de los fármacos , Witanólidos/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , eIF-2 Quinasa/metabolismoRESUMEN
OBJECTIVE: Pancreatic ductal adenocarcinoma (PDA) has among the highest stromal fractions of any cancer and this has complicated attempts at expression-based molecular classification. The goal of this work is to profile purified samples of human PDA epithelium and stroma and examine their respective contributions to gene expression in bulk PDA samples. DESIGN: We used laser capture microdissection (LCM) and RNA sequencing to profile the expression of 60 matched pairs of human PDA malignant epithelium and stroma samples. We then used these data to train a computational model that allowed us to infer tissue composition and generate virtual compartment-specific expression profiles from bulk gene expression cohorts. RESULTS: Our analysis found significant variation in the tissue composition of pancreatic tumours from different public cohorts. Computational removal of stromal gene expression resulted in the reclassification of some tumours, reconciling functional differences between different cohorts. Furthermore, we established a novel classification signature from a total of 110 purified human PDA stroma samples, finding two groups that differ in the extracellular matrix-associated and immune-associated processes. Lastly, a systematic evaluation of cross-compartment subtypes spanning four patient cohorts indicated partial dependence between epithelial and stromal molecular subtypes. CONCLUSION: Our findings add clarity to the nature and number of molecular subtypes in PDA, expand our understanding of global transcriptional programmes in the stroma and harmonise the results of molecular subtyping efforts across independent cohorts.
Asunto(s)
Adenocarcinoma/patología , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Regulación Neoplásica de la Expresión Génica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Adenocarcinoma/genética , Adenocarcinoma/cirugía , Carcinoma Ductal Pancreático/cirugía , Estudios de Casos y Controles , Simulación por Computador , Matriz Extracelular/patología , Perfilación de la Expresión Génica , Humanos , Microdisección , Neoplasias Pancreáticas/cirugía , Sensibilidad y EspecificidadRESUMEN
Mutations in the Epidermal growth factor receptor (EGFR) kinase domain, such as the L858R missense mutation and deletions spanning the conserved sequence 747LREA750, are sensitive to tyrosine kinase inhibitors (TKIs). The gatekeeper site residue mutation, T790M accounts for around 60% of acquired resistance to EGFR TKIs. The first generation EGFR TKIs, erlotinib and gefitinib, and the second generation inhibitor, afatinib are FDA approved for initial treatment of EGFR mutated lung adenocarcinoma. The predominant biomarker of EGFR TKI responsiveness is the presence of EGFR TKI-sensitizing mutations. However, 30-40% of patients with EGFR mutations exhibit primary resistance to these TKIs, underscoring the unmet need of identifying additional biomarkers of treatment response. Here, we sought to characterize the dynamics of tyrosine phosphorylation upon EGFR TKI treatment of mutant EGFR-driven human lung adenocarcinoma cell lines with varying sensitivity to EGFR TKIs, erlotinib and afatinib. We employed stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative mass spectrometry to identify and quantify tyrosine phosphorylated peptides. The proportion of tyrosine phosphorylated sites that had reduced phosphorylation upon erlotinib or afatinib treatment correlated with the degree of TKI-sensitivity. Afatinib, an irreversible EGFR TKI, more effectively inhibited tyrosine phosphorylation of a majority of the substrates. The phosphosites with phosphorylation SILAC ratios that correlated with the TKI-sensitivity of the cell lines include sites on kinases, such as EGFR-Y1197 and MAPK7-Y221, and adaptor proteins, such as SHC1-Y349/350, ERRFI1-Y394, GAB1-Y689, STAT5A-Y694, DLG3-Y705, and DAPP1-Y139, suggesting these are potential biomarkers of TKI sensitivity. DAPP1, is a novel target of mutant EGFR signaling and Y-139 is the major site of DAPP1 tyrosine phosphorylation. We also uncovered several off-target effects of these TKIs, such as MST1R-Y1238/Y1239 and MET-Y1252/1253. This study provides unique insight into the TKI-mediated modulation of mutant EGFR signaling, which can be applied to the development of biomarkers of EGFR TKI response.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Biomarcadores de Tumor/metabolismo , Receptores ErbB/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Fosfotirosina/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteómica/métodos , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Afatinib , Línea Celular Tumoral , Análisis por Conglomerados , Receptores ErbB/metabolismo , Clorhidrato de Erlotinib/farmacología , Clorhidrato de Erlotinib/uso terapéutico , Humanos , Marcaje Isotópico , Neoplasias Pulmonares/patología , Espectrometría de Masas , Mutación/genética , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Quinazolinas/uso terapéutico , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacos , Tirosina/metabolismoRESUMEN
The functional role of bone morphogenetic protein (BMP) signalling in colorectal cancer (CRC) is poorly defined, with contradictory results in cancer cell line models reflecting the inherent difficulties of assessing a signalling pathway that is context-dependent and subject to genetic constraints. By assessing the transcriptional response of a diploid human colonic epithelial cell line to BMP ligand stimulation, we generated a prognostic BMP signalling signature, which was applied to multiple CRC datasets to investigate BMP heterogeneity across CRC molecular subtypes. We linked BMP and Notch signalling pathway activity and function in human colonic epithelial cells, and normal and neoplastic tissue. BMP induced Notch through a γ-secretase-independent interaction, regulated by the SMAD proteins. In homeostasis, BMP/Notch co-localization was restricted to cells at the top of the intestinal crypt, with more widespread interaction in some human CRC samples. BMP signalling was downregulated in the majority of CRCs, but was conserved specifically in mesenchymal-subtype tumours, where it interacts with Notch to induce an epithelial-mesenchymal transition (EMT) phenotype. In intestinal homeostasis, BMP-Notch pathway crosstalk is restricted to differentiating cells through stringent pathway segregation. Conserved BMP activity and loss of signalling stringency in mesenchymal-subtype tumours promotes a synergistic BMP-Notch interaction, and this correlates with poor patient prognosis. BMP signalling heterogeneity across CRC subtypes and cell lines can account for previous experimental contradictions. Crosstalk between the BMP and Notch pathways will render mesenchymal-subtype CRC insensitive to γ-secretase inhibition unless BMP activation is concomitantly addressed. © 2017 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Asunto(s)
Proteínas Morfogenéticas Óseas/genética , Neoplasias Colorrectales/genética , Transición Epitelial-Mesenquimal , Receptores Notch/genética , Transducción de Señal , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular , Estudios de Cohortes , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/patología , Células Epiteliales/patología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Modelos Biológicos , Fenotipo , Pronóstico , Receptores Notch/metabolismo , Proteínas Smad/genética , Proteínas Smad/metabolismoRESUMEN
BACKGROUND: MicroRNAs (miRNAs) play multiple roles in tumor biology. Interestingly, reports from multiple groups suggest that miRNA targets may be coupled through competitive stoichiometric sequestration. Specifically, computational models predicted and experimental assays confirmed that miRNA activity is dependent on miRNA target abundance, and consequently, changes in the abundance of some miRNA targets lead to changes to the regulation and abundance of their other targets. The resulting indirect regulatory influence between miRNA targets resembles competition and has been dubbed competitive endogenous RNA (ceRNA). Recent studies have questioned the physiological relevance of ceRNA interactions, our ability to accurately predict these interactions, and the number of genes that are impacted by ceRNA interactions in specific cellular contexts. RESULTS: To address these concerns, we reverse engineered ceRNA networks (ceRNETs) in breast and prostate adenocarcinomas using context-specific TCGA profiles, and tested whether ceRNA interactions can predict the effects of RNAi-mediated gene silencing perturbations in PC3 and MCF7 cells._ENREF_22 Our results, based on tests of thousands of inferred ceRNA interactions that are predicted to alter hundreds of cancer genes in each of the two tumor contexts, confirmed statistically significant effects for half of the predicted targets. CONCLUSIONS: Our results suggest that the expression of a significant fraction of cancer genes may be regulated by ceRNA interactions in each of the two tumor contexts.
Asunto(s)
Redes Reguladoras de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ARN , Bases de Datos Genéticas , Humanos , Células MCF-7 , MicroARNs/genéticaRESUMEN
The predominant view of pluripotency regulation proposes a stable ground state with coordinated expression of key transcription factors (TFs) that prohibit differentiation. Another perspective suggests a more complexly regulated state involving competition between multiple lineage-specifying TFs that define pluripotency. These contrasting views were developed from extensive analyses of TFs in pluripotent cells in vitro. An experimentally validated, genome-wide repertoire of the regulatory interactions that control pluripotency within the in vivo cellular contexts is yet to be developed. To address this limitation, we assembled a TF interactome of adult human male germ cell tumors (GCTs) using the Algorithm for the Accurate Reconstruction of Cellular Pathways (ARACNe) to analyze gene expression profiles of 141 tumors comprising pluripotent and differentiated subsets. The network (GCT(Net)) comprised 1,305 TFs, and its ingenuity pathway analysis identified pluripotency and embryonal development as the top functional pathways. We experimentally validated GCT(Net) by functional (silencing) and biochemical (ChIP-seq) analysis of the core pluripotency regulatory TFs POU5F1, NANOG, and SOX2 in relation to their targets predicted by ARACNe. To define the extent of the in vivo pluripotency network in this system, we ranked all TFs in the GCT(Net) according to sharing of ARACNe-predicted targets with those of POU5F1 and NANOG using an odds-ratio analysis method. To validate this network, we silenced the top 10 TFs in the network in H9 embryonic stem cells. Silencing of each led to downregulation of pluripotency and induction of lineage; 7 of the 10 TFs were identified as pluripotency regulators for the first time.
Asunto(s)
Algoritmos , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Neoplasias de Células Germinales y Embrionarias/metabolismo , Células Madre Pluripotentes/metabolismo , Factores de Transcripción/metabolismo , Adulto , Línea Celular Tumoral , Humanos , Masculino , Proteínas de Neoplasias/genética , Neoplasias de Células Germinales y Embrionarias/genética , Neoplasias de Células Germinales y Embrionarias/patología , Células Madre Pluripotentes/patología , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Transcription factors (TFs) act downstream of the major signalling pathways functioning as master regulators of cell fate. Their activity is tightly regulated at the transcriptional, post-transcriptional and post-translational level. Proteins modifying TF activity are not easily identified by experimental high-throughput methods. RESULTS: We developed a computational strategy, called Differential Multi-Information (DMI), to infer post-translational modulators of a transcription factor from a compendium of gene expression profiles (GEPs). DMI is built on the hypothesis that the modulator of a TF (i.e. kinase/phosphatases), when expressed in the cell, will cause the TF target genes to be co-expressed. On the contrary, when the modulator is not expressed, the TF will be inactive resulting in a loss of co-regulation across its target genes. DMI detects the occurrence of changes in target gene co-regulation for each candidate modulator, using a measure called Multi-Information. We validated the DMI approach on a compendium of 5,372 GEPs showing its predictive ability in correctly identifying kinases regulating the activity of 14 different transcription factors. CONCLUSIONS: DMI can be used in combination with experimental approaches as high-throughput screening to efficiently improve both pathway and target discovery. An on-line web-tool enabling the user to use DMI to identify post-transcriptional modulators of a transcription factor of interest che be found at http://dmi.tigem.it.
Asunto(s)
Regulación de la Expresión Génica/genética , Procesamiento Proteico-Postraduccional/genética , Factores de Transcripción/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Chromatin immunoprecipitation followed by sequencing of protein-bound DNA fragments (ChIP-Seq) is an effective high-throughput methodology for the identification of context specific DNA fragments that are bound by specific proteins in vivo. Despite significant progress in the bioinformatics analysis of this genome-scale data, a number of challenges remain as technology-dependent biases, including variable target accessibility and mappability, sequence-dependent variability, and non-specific binding affinity must be accounted for. RESULTS AND DISCUSSION: We introduce a nonparametric method for scoring consensus regions of aligned immunoprecipitated DNA fragments when appropriate control experiments are available. Our method uses local models for null binding; these are necessary because binding prediction scores based on global models alone fail to properly account for specialized features of genomic regions and chance pull downs of specific DNA fragments, thus disproportionally rewarding some genomic regions and decreasing prediction accuracy. We make no assumptions about the structure or amplitude of bound peaks, yet we show that our method outperforms leading methods developed using either global or local null hypothesis models for random binding. We test prediction performance by comparing analyses of ChIP-seq, ChIP-chip, motif-based binding-site prediction, and shRNA assays, showing high reproducibility, binding-site enrichment in predicted target regions, and functional regulation of predicted targets. CONCLUSIONS: Given appropriate controls, a direct nonparametric method for identifying transcription-factor targets from ChIP-Seq assays may lead to both higher sensitivity and higher specificity, and should be preferred or used in conjunction with methods that use parametric models for null binding.
Asunto(s)
Biología Computacional/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Proteínas de Homeodominio/genética , Receptor Notch1/genética , Factores de Transcripción SOXB1/genética , Algoritmos , Secuencia de Bases , Línea Celular , Inmunoprecipitación de Cromatina/métodos , Secuencia de Consenso/genética , ADN/genética , Humanos , Proteína Homeótica Nanog , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Alineación de Secuencia , Análisis de Secuencia de ADN/métodosRESUMEN
Administering therapeutics through the oral route is a pervasive and widely approved medication administration approach. However, it has been found that many drugs show low systemic absorption when delivered through this route. Such limitations of oral drug delivery can be overcome by polymeric micelles acting as vehicles. As a result, they improve drug absorption by protecting loaded drug substances from the gastrointestinal system's hostile conditions, allowing controlled drug release at a specific site, extending the time spent in the gut through mucoadhesion, and inhibiting the efflux pump from reducing therapeutic agent accumulation. To promote good oral absorption of a weakly water-soluble medicinal drug, the loaded medicine should be protected from the hostile atmosphere of the GI tract. Polymeric micelles can be stacked with a broad assortment of ineffectively dissolvable medications, improving bioavailability. This review discusses the major mechanism, various types, advantages, and limitations for developing the polymeric micelle system and certain micellar drug delivery system applications. The primary goal of this review is to illustrate how polymeric micelles can be used to deliver poorly water-soluble medications.
Asunto(s)
Sistemas de Liberación de Medicamentos , Micelas , Humanos , Polímeros , Disponibilidad Biológica , AguaRESUMEN
Drug-induced Behavioral Signature Analysis (DBSA), is a machine learning (ML) method for in silico screening of compounds, inspired by analytical methods quantifying gene enrichment in genomic analyses. When applied to behavioral data it can identify drugs that can potentially reverse in vivo behavioral symptoms in animal models of human disease and suggest new hypotheses for drug discovery and repurposing. We present a proof-of-concept study aiming to assess Drug-induced Behavioral Signature Analysis (DBSA) as a systematic approach for drug discovery for rare disorders. We applied Drug-induced Behavioral Signature Analysis to high-content behavioral data obtained with SmartCube®, an automated in vivo phenotyping platform. The therapeutic potential of several dozen approved drugs was assessed for phenotypic reversal of the behavioral profile of a Huntington's Disease (HD) murine model, the Q175 heterozygous knock-in mice. The in silico Drug-induced Behavioral Signature Analysis predictions were enriched for drugs known to be effective in the symptomatic treatment of Huntington's Disease, including bupropion, modafinil, methylphenidate, and several SSRIs, as well as the atypical antidepressant tianeptine. To validate the method, we tested acute and chronic effects of tianeptine (20 mg/kg, i. p.) in vivo, using Q175 mice and wild type controls. In both experiments, tianeptine significantly rescued the behavioral phenotype assessed with the SmartCube® platform. Our target-agnostic method thus showed promise for identification of symptomatic relief treatments for rare disorders, providing an alternative method for hypothesis generation and drug discovery for disorders with huge disease burden and unmet medical needs.
RESUMEN
The naked mole rat (NMR), Heterocephalus glaber, the longest-living rodent, provides a unique opportunity to explore how evolution has shaped adult stem cell (ASC) activity and tissue function with increasing lifespan. Using cumulative BrdU labelling and a quantitative imaging approach to track intestinal ASCs (Lgr5+) in their native in vivo state, we find an expanded pool of Lgr5+ cells in NMRs, and these cells specifically at the crypt base (Lgr5+CBC) exhibit slower division rates compared to those in short-lived mice but have a similar turnover as human LGR5+CBC cells. Instead of entering quiescence (G0), NMR Lgr5+CBC cells reduce their division rates by prolonging arrest in the G1 and/or G2 phases of the cell cycle. Moreover, we also observe a higher proportion of differentiated cells in NMRs that confer enhanced protection and function to the intestinal mucosa which is able to detect any chemical imbalance in the luminal environment efficiently, triggering a robust pro-apoptotic, anti-proliferative response within the stem/progenitor cell zone.
Asunto(s)
Células Madre Adultas , Longevidad , Ratones , Humanos , Animales , Mucosa Intestinal/metabolismo , Intestinos , Células Madre Adultas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Ratas TopoRESUMEN
We have developed an inducible Huntington's disease (HD) mouse model that allows temporal control of whole-body allele-specific mutant huntingtin (mHtt) expression. We asked whether moderate global lowering of mHtt (~50%) was sufficient for long-term amelioration of HD-related deficits and, if so, whether early mHtt lowering (before measurable deficits) was required. Both early and late mHtt lowering delayed behavioral dysfunction and mHTT protein aggregation, as measured biochemically. However, long-term follow-up revealed that the benefits, in all mHtt-lowering groups, attenuated by 12 months of age. While early mHtt lowering attenuated cortical and striatal transcriptional dysregulation evaluated at 6 months of age, the benefits diminished by 12 months of age, and late mHtt lowering did not ameliorate striatal transcriptional dysregulation at 12 months of age. Only early mHtt lowering delayed the elevation in cerebrospinal fluid neurofilament light chain that we observed in our model starting at 9 months of age. As small-molecule HTT-lowering therapeutics progress to the clinic, our findings suggest that moderate mHtt lowering allows disease progression to continue, albeit at a slower rate, and could be relevant to the degree of mHTT lowering required to sustain long-term benefits in humans.