RESUMO
BACKGROUND: High-throughput sequencing (HTS) has become the gold standard approach for variant analysis in cancer research. However, somatic variants may occur at low fractions due to contamination from normal cells or tumor heterogeneity; this poses a significant challenge for standard HTS analysis pipelines. The problem is exacerbated in scenarios with minimal tumor DNA, such as circulating tumor DNA in plasma. Assessing sensitivity and detection of HTS approaches in such cases is paramount, but time-consuming and expensive: specialized experimental protocols and a sufficient quantity of samples are required for processing and analysis. To overcome these limitations, we propose a new computational approach specifically designed for the generation of artificial datasets suitable for this task, simulating ultra-deep targeted sequencing data with low-fraction variants and demonstrating their effectiveness in benchmarking low-fraction variant calling. RESULTS: Our approach enables the generation of artificial raw reads that mimic real data without relying on pre-existing data by using NEAT, a fine-grained read simulator that generates artificial datasets using models learned from multiple different datasets. Then, it incorporates low-fraction variants to simulate somatic mutations in samples with minimal tumor DNA content. To prove the suitability of the created artificial datasets for low-fraction variant calling benchmarking, we used them as ground truth to evaluate the performance of widely-used variant calling algorithms: they allowed us to define tuned parameter values of major variant callers, considerably improving their detection of very low-fraction variants. CONCLUSIONS: Our findings highlight both the pivotal role of our approach in creating adequate artificial datasets with low tumor fraction, facilitating rapid prototyping and benchmarking of algorithms for such dataset type, as well as the important need of advancing low-fraction variant calling techniques.
Assuntos
Benchmarking , Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Neoplasias/genética , Mutação , Algoritmos , DNA de Neoplasias/genética , Análise de Sequência de DNA/métodos , Biologia Computacional/métodosRESUMO
isomiRs, the sequence-variants of microRNA, are known to be tissue and cell type specific but their physiological role is largely unknown. In our study, we explored for the first time the expression of isomiRs across different Stage I epithelial ovarian cancer (EOC) histological subtypes, in order to shed new light on their biological role in tumor growth and progression. In a multicentric retrospective cohort of tumor biopsies (n = 215) we sequenced small RNAs finding 971 expressed miRNAs, 64% of which are isomiRs. Among them, 42 isomiRs showed a clear histotype specific pattern, confirming our previously identified miRNA markers (miR192/194 and miR30a-3p/5p for mucinous and clear cell subtypes, respectively) and uncovering new biomarkers for all the five subtypes. Using integrative models, we found that the 38% of these miRNA expression alterations is the result of copy number variations while the 17% of differential transcriptional activities. Our work represents the first attempt to characterize isomiRs expression in Stage I EOC within and across subtypes and to contextualize their alterations in the framework of the large genomic heterogeneity of this tumor.
Assuntos
MicroRNAs , Neoplasias Ovarianas , Humanos , Feminino , MicroRNAs/genética , MicroRNAs/metabolismo , Carcinoma Epitelial do Ovário/genética , Variações do Número de Cópias de DNA , Estudos Retrospectivos , Perfilação da Expressão Gênica , Neoplasias Ovarianas/patologiaRESUMO
OBJECTIVE: Copy number variations (CNVs) play crucial roles in physiological and pathological processes, including cancer. However, the functional implications of somatic CNVs in tumor progression and evolution remain unclear. This study focuses on identifying CNV alterations with high pathogenic potential that drive and sustain tumorigenesis, distinguishing them from passenger alterations that accumulate during tumor growth. Our goal is to explore the variability of CNVs across different tumor types and infer their impact on tumor cell functions. METHODS: Starting from 7352 copy number profiles across 33 different cancer types, we infer the pathogenicity of each CNV and perform both intra- and inter-tumor analyses to predict the functional impact of different genomic patterns. We evaluate the actionability of genes belonging to altered regions and we correlate the presence of pathogenic regions with genome instability patterns and patients' survival. RESULTS: Our analysis uncovered large heterogeneity among different tumors suggesting in many cases distinct genetic drivers of tumorigenesis. Recurrent genomic alterations frequently coincide with dysfunctional homologous recombination pathways and negative regulation of the immune system. In certain tumors, the number of pathogenic CNVs emerged as a prognostic biomarker, highlighting their significance in cancer progression. CONCLUSION: This study contributes to elucidate the functional impact of pathogenic CNVs in tumor progression and sheds light on their potential as prognostic markers in specific cancer types.
Assuntos
Variações do Número de Cópias de DNA , Neoplasias , Humanos , Neoplasias/genética , Genoma , Carcinogênese/genética , GenômicaRESUMO
Therapies for epilepsy mainly provide symptomatic control of seizures since most of the available drugs do not target disease mechanisms. Moreover, about one-third of patients fail to achieve seizure control. To address the clinical need for disease-modifying therapies, research should focus on targets which permit interventions finely balanced between optimal efficacy and safety. One potential candidate is the brain-specific enzyme cholesterol 24-hydroxylase. This enzyme converts cholesterol to 24S-hydroxycholesterol, a metabolite which among its biological roles modulates neuronal functions relevant for hyperexcitability underlying seizures. To study the role of cholesterol 24-hydroxylase in epileptogenesis, we administered soticlestat (TAK-935/OV935), a potent and selective brain-penetrant inhibitor of the enzyme, during the early disease phase in a mouse model of acquired epilepsy using a clinically relevant dose. During soticlestat treatment, the onset of epilepsy was delayed and the number of ensuing seizures was decreased by about 3-fold compared to vehicle-treated mice, as assessed by EEG monitoring. Notably, the therapeutic effect was maintained 6.5 weeks after drug wash-out when seizure number was reduced by about 4-fold and their duration by 2-fold. Soticlestat-treated mice showed neuroprotection of hippocampal CA1 neurons and hilar mossy cells as assessed by post-mortem brain histology. High throughput RNA-sequencing of hippocampal neurons and glia in mice treated with soticlestat during epileptogenesis showed that inhibition of cholesterol 24-hydroxylase did not directly affect the epileptogenic transcriptional network, but rather modulated a non-overlapping set of genes that might oppose the pathogenic mechanisms of the disease. In human temporal lobe epileptic foci, we determined that cholesterol 24-hydroxylase expression trends higher in neurons, similarly to epileptic mice, while the enzyme is ectopically induced in astrocytes compared to control specimens. Soticlestat reduced significantly the number of spontaneous seizures in chronic epileptic mice when was administered during established epilepsy. Data show that cholesterol 24-hydroxylase contributes to spontaneous seizures and is involved in disease progression, thus it represents a novel target for chronic seizures inhibition and disease-modification therapy in epilepsy.
Assuntos
Epilepsia do Lobo Temporal , Epilepsia , Animais , Colesterol/metabolismo , Colesterol 24-Hidroxilase/metabolismo , Modelos Animais de Doenças , Epilepsia/tratamento farmacológico , Epilepsia/metabolismo , Epilepsia do Lobo Temporal/metabolismo , Hipocampo/metabolismo , Humanos , Camundongos , Piperidinas , Piridinas , RNA/metabolismo , Convulsões/metabolismoRESUMO
The reason why CD4(+) T helper 17 (Th17) cells, despite their well-known pathogenic role in chronic inflammatory disorders, are very rare in the inflammatory sites remains unclear. We demonstrate that human Th17 cells exhibit low ability to proliferate and to produce the T cell growth factor interleukin-2 (IL-2), in response to combined CD3 and CD28 stimulation. This was due to the upregulated expression of IL-4-induced gene 1 (IL4I1) mRNA, a secreted L-phenylalanine oxidase, which associated with a decrease in CD3ζ chain expression and consequent abnormalities in the molecular pathway that allows IL-2 production and cell proliferation. High IL4I1 mRNA expression was detectable in Th17 cell precursors and was strictly dependent on Th17 cell master gene, the retinoid acid related orphan receptor (RORC). Th17 cells also exhibited RORC-dependent CD28 hyperexpression and the ability to produce IL-17A after CD28 stimulation without CD3 triggering. Our findings suggest that the rarity of human Th17 cells in inflamed tissues results from RORC-dependent mechanisms limiting their expansion.
Assuntos
Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Células Th17/citologia , Células Th17/metabolismo , Artrite Juvenil/genética , Artrite Juvenil/imunologia , Artrite Juvenil/patologia , Antígenos CD28/metabolismo , Complexo CD3/metabolismo , Proliferação de Células , Criança , Expressão Gênica , Genes fos , Genes jun , Humanos , Inflamação/etiologia , Inflamação/imunologia , Inflamação/patologia , Interleucina-17/biossíntese , Interleucina-2/biossíntese , L-Aminoácido Oxidase/genética , Fatores de Transcrição NFATC/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/imunologia , Células Th1/citologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th17/imunologiaRESUMO
High-grade serous ovarian cancer (HGS-EOCs) is generally sensitive to front-line platinum (Pt)-based chemotherapy although most patients at an advanced stage relapse with progressive resistant disease. Clinical or molecular data to identify primary resistant cases at diagnosis are not yet available. HGS-EOC biopsies from 105 Pt-sensitive (Pt-s) and 89 Pt-resistant (Pt-r) patients were retrospectively selected from two independent tumor tissue collections. Pathway analysis was done integrating miRNA and mRNA expression profiles. Signatures were further validated in silico on a cohort of 838 HGS-EOC cases from a published dataset. In all, 131 mRNAs and 5 miRNAs belonging to different functionally related molecular pathways distinguish Pt-s from Pt-r cases. Then, 17 out of 23 selected elements were validated by orthogonal approaches (SI signature). As resistance to Pt is associated with a short progression-free survival (PFS) and overall survival (OS), the prognostic role of the SI signature was assessed, and 14 genes associated with PFS and OS, in multivariate analyses (SII signature). The prognostic value of the SII signature was validated in a third extensive cohort. The expression profiles of SDF2L1, PPP1R12A and PRKG1 genes (SIII signature) served as independent prognostic biomarkers of Pt-response and survival. The study identified a prognostic molecular signature based on the combined expression profile of three genes which had never been associated with the clinical outcome of HGS-EOC. This may lead to early identification, at the time of diagnosis, of patients who would not greatly benefit from standard chemotherapy and are thus eligible for novel investigational approaches.
Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo I/genética , Cistadenocarcinoma Seroso/tratamento farmacológico , Perfilação da Expressão Gênica/métodos , Proteínas de Membrana/genética , Fosfatase de Miosina-de-Cadeia-Leve/genética , Neoplasias Ovarianas/tratamento farmacológico , Platina/uso terapêutico , Adulto , Idoso , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Gradação de Tumores , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Prognóstico , Estudos Retrospectivos , Análise de Sobrevida , Resultado do TratamentoRESUMO
Aberrant microRNA (miR) expression has an important role in tumour progression, but its involvement in bone marrow fibroblasts of multiple myeloma patients remains undefined. We demonstrate that a specific miR profile in bone marrow fibroblasts parallels the transition from monoclonal gammopathy of undetermined significance (MGUS) to myeloma. Overexpression of miR-27b-3p and miR-214-3p triggers proliferation and apoptosis resistance in myeloma fibroblasts via the FBXW7 and PTEN/AKT/GSK3 pathways, respectively. Transient transfection of miR-27b-3p and miR-214-3p inhibitors demonstrates a cooperation between these two miRNAs in the expression of the anti-apoptotic factor MCL1, suggesting that miR-27b-3p and miR-214-3p negatively regulate myeloma fibroblast apoptosis. Furthermore, myeloma cells modulate miR-27b-3p and miR-214-3p expression in fibroblasts through the release of exosomes. Indeed, tumour cell-derived exosomes induce an overexpression of both miRNAs in MGUS fibroblasts not through a simple transfer mechanism but by de novo synthesis triggered by the transfer of exosomal WWC2 protein that regulates the Hippo pathway. Increased levels of miR-27b-3p and miR-214-3p in MGUS fibroblasts co-cultured with myeloma cell-derived exosomes enhance the expression of fibroblast activation markers αSMA and FAP. These data show that the MGUS-to-myeloma transition entails an aberrant miRNA profile in marrow fibroblasts and highlight a key role of myeloma cells in modifying the bone marrow microenvironment by reprogramming the marrow fibroblasts' behaviour. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Células da Medula Óssea/metabolismo , Exossomos/metabolismo , Fibroblastos/metabolismo , MicroRNAs/metabolismo , Gamopatia Monoclonal de Significância Indeterminada/metabolismo , Mieloma Múltiplo/metabolismo , Actinas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Células da Medula Óssea/patologia , Células Cultivadas , Progressão da Doença , Endopeptidases , Exossomos/genética , Exossomos/patologia , Proteína 7 com Repetições F-Box-WD/genética , Proteína 7 com Repetições F-Box-WD/metabolismo , Feminino , Fibroblastos/patologia , Gelatinases/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Proteínas de Membrana/metabolismo , MicroRNAs/genética , Pessoa de Meia-Idade , Gamopatia Monoclonal de Significância Indeterminada/genética , Gamopatia Monoclonal de Significância Indeterminada/patologia , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Serina Endopeptidases/metabolismo , Transdução de Sinais , Microambiente Tumoral , Regulação para CimaRESUMO
High-grade serous epithelial ovarian cancer (HGS-EOC) is a systemic disease, with marked intra and interpatient tumor heterogeneity. The issue of spatial and temporal heterogeneity has long been overlooked, hampering the possibility to identify those genomic alterations that persist, before and after therapy, in the genome of all tumor cells across the different anatomical districts. This knowledge is the first step to clarify those molecular determinants that characterize the tumor biology of HGS-EOC and their route toward malignancy. In our study, -omics data were generated from 79 snap frozen matched tumor biopsies, withdrawn before and after chemotherapy from 24 HGS-EOC patients, gathered together from independent cohorts. The landscape of somatic copy number alterations depicts a more homogenous and stable genomic portrait than the single nucleotide variant profile. Genomic identification of significant targets in cancer analysis identified two focal and minimal common regions (FMCRs) of amplification in the cytoband 3q26.2 (region α, 193 kb long) and 8q24.3 (region ß, 495 kb long). Analysis in two external databases confirmed regions α and ß are features of HGS-EOC. The MECOM gene is located in region α, and 15 genes are in region ß. No functional data are yet available for the genes in the ß region. In conclusion, we have identified for the first time two FMCRs of amplification in HGS-EOC, opening up a potential biological role in its etiopathogenesis.
Assuntos
Carcinoma Epitelial do Ovário/genética , Cromossomos Humanos Par 3/genética , Cromossomos Humanos Par 8/genética , Variações do Número de Cópias de DNA , Neoplasias Ovarianas/genética , Biópsia , Carcinoma Epitelial do Ovário/patologia , Estudos de Coortes , Biologia Computacional , Bases de Dados Genéticas , Conjuntos de Dados como Assunto , Feminino , Genômica , Humanos , Gradação de Tumores , Neoplasias Ovarianas/patologia , Ovário/patologia , Sequenciamento do ExomaRESUMO
We recently discovered that forebrain activation of the IL-1 receptor/Toll-like receptor (IL-1R1/TLR4) innate immunity signal plays a pivotal role in neuronal hyperexcitability underlying seizures in rodents. Since this pathway is activated in neurons and glia in human epileptogenic foci, it represents a potential target for developing drugs interfering with the mechanisms of epileptogenesis that lead to spontaneous seizures. The lack of such drugs represents a major unmet clinical need. We tested therefore novel therapies inhibiting the IL-1R1/TLR4 signaling in an established murine model of acquired epilepsy. We used an epigenetic approach by injecting a synthetic mimic of micro(mi)RNA-146a that impairs IL1R1/TLR4 signal transduction, or we blocked receptor activation with antiinflammatory drugs. Both interventions when transiently applied to mice after epilepsy onset, prevented disease progression and dramatically reduced chronic seizure recurrence, while the anticonvulsant drug carbamazepine was ineffective. We conclude that IL-1R1/TLR4 is a novel potential therapeutic target for attaining disease-modifications in patients with diagnosed epilepsy.
Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Anticonvulsivantes/administração & dosagem , Epilepsia/terapia , MicroRNAs/administração & dosagem , Receptores Tipo I de Interleucina-1/antagonistas & inibidores , Receptor 4 Toll-Like/antagonistas & inibidores , Animais , Carbamazepina/farmacologia , Cianobactérias , Dipeptídeos/administração & dosagem , Modelos Animais de Doenças , Epilepsia/tratamento farmacológico , Epilepsia/fisiopatologia , Hipocampo/fisiopatologia , Ácido Caínico , Lipopolissacarídeos/administração & dosagem , Masculino , Camundongos Endogâmicos C57BL , Oligonucleotídeos/administração & dosagem , Distribuição Aleatória , Receptores Tipo I de Interleucina-1/metabolismo , Fatores de Tempo , Receptor 4 Toll-Like/metabolismo , para-Aminobenzoatos/administração & dosagemRESUMO
BACKGROUND: Lurbinectedin is a novel anticancer agent currently undergoing late-stage (Phase II /III) clinical evaluation in platinum-resistant ovarian, BRCA1/2-mutated breast and small-cell lung cancer. Lurbinectedin is structurally related to trabectedin and it inhibits active transcription and the DNA repair machinery in tumour cells. METHODS: In this study we investigated whether lurbinectedin has the ability to modulate the inflammatory microenvironment and the viability of myeloid cells in tumour-bearing mice. RESULTS: Administration of lurbinectedin significantly and selectively decreased the number of circulating monocytes and, in tumour tissues, that of macrophages and vessels. Similar findings were observed when a lurbinectedin-resistant tumour variant was used, indicating a direct effect of lurbinectedin on the tumour microenviroment. In vitro, lurbinectedin induced caspase-8-dependent apoptosis of human purified monocytes, whereas at low doses it significantly inhibited the production of inflammatory/growth factors (CCL2, CXCL8 and VEGF) and dramatically impaired monocyte adhesion and migration ability. These findings were supported by the strong inhibition of genes of the Rho-GTPase family in lurbinectedin-treated monocytes. CONCLUSIONS: The results illustrate that lurbinectedin affects at multiple levels the inflammatory microenvironment by acting on the viability and functional activity of mononuclear phagocytes. These peculiar effects, combined with its intrinsic activity against cancer cells, make lurbinectedin a compound of particular interest in oncology.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Carbolinas/farmacologia , Fibrossarcoma/tratamento farmacológico , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Macrófagos , Monócitos/efeitos dos fármacos , Monócitos/fisiologia , Neoplasias Ovarianas/tratamento farmacológico , Microambiente Tumoral/efeitos dos fármacos , Animais , Antineoplásicos Alquilantes/uso terapêutico , Apoptose/efeitos dos fármacos , Carbolinas/uso terapêutico , Caspase 8/metabolismo , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Quimiocina CCL2/biossíntese , Dioxóis/farmacologia , Regulação para Baixo , Feminino , Fibrossarcoma/imunologia , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Células HL-60 , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Humanos , Interleucina-8/biossíntese , Contagem de Leucócitos , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Neovascularização Patológica/prevenção & controle , Tetra-Hidroisoquinolinas/farmacologia , Trabectedina , Microambiente Tumoral/imunologia , Células U937 , Fator A de Crescimento do Endotélio Vascular/biossíntese , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas rho de Ligação ao GTP/genéticaRESUMO
BACKGROUND & AIMS: Hepatic stellate cell (HSC) transdifferentiation into collagen-producing myofibroblasts is a key event in hepatic fibrogenesis, but the transcriptional network that controls the acquisition of the activated phenotype is still poorly understood. In this study, we explored whether the nuclear receptor chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) is involved in HSC activation and in the multifunctional role of these cells during the response to liver injury. METHODS: COUP-TFII expression was evaluated in normal and cirrhotic livers by immunohistochemistry and Western blot. The role of COUP-TFII in HSC was assessed by gain and loss of function transfection experiments and by generation of mice with COUP-TFII deletion in HSC. Molecular changes were determined by gene expression microarray and RT-qPCR. RESULTS: We showed that COUP-TFII is highly expressed in human fibrotic liver and in mouse models of hepatic injury. COUP-TFII expression rapidly increased upon HSC activation and it was associated with the regulation of genes involved in cell motility, proliferation and angiogenesis. Inactivation of COUP-TFII impairs proliferation and invasiveness in activated HSC and COUP-TFII deletion in mice abrogate HSC activation and angiogenesis. Finally, co-culture experiments with HSC and liver sinusoidal endothelial cells (SEC) showed that COUP-TFII expression in HSC influenced SEC migration and tubulogenesis via a hypoxia-independent and nuclear factor kappaB-dependent mechanism. CONCLUSION: This study elucidates a novel transcriptional pathway in HSC that is involved in the acquisition of the proangiogenic phenotype and regulates the paracrine signals between HSC and SEC during hepatic wound healing. LAY SUMMARY: In this study, we identified an important regulator of HSC pathobiology. We showed that the orphan receptor COUP-TFII is an important player in hepatic neoangiogenesis. COUP-TFII expression in HSC controls the crosstalk between HSC and endothelial cells coordinating vascular remodelling during liver injury. TRANSCRIPT PROFILING: ArrayExpress accession E-MTAB-1795.
Assuntos
Fator II de Transcrição COUP/metabolismo , Células Estreladas do Fígado/metabolismo , Animais , Fator II de Transcrição COUP/deficiência , Fator II de Transcrição COUP/genética , Comunicação Celular , Movimento Celular , Proliferação de Células , Transdiferenciação Celular , Técnicas de Cocultura , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Estreladas do Fígado/citologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Hipóxia/metabolismo , Hipóxia/patologia , Fígado/lesões , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Fisiológica/genética , Regulação para Cima , Cicatrização/genética , Cicatrização/fisiologiaRESUMO
BACKGROUND: The existence of cancer stem cells (CSCs) within a tumor bulk has been demonstrated for many solid tumors including epithelial ovarian carcinoma (EOC). CSCs have been associated to tumor invasion, metastasis and development of chemoresistant recurrences. In this context, we aim to characterize EOC CSCs from the molecular point of view in order to identify potential biomarkers associated with chemoresistance. METHODS: We isolated a population of cells with stem-like characteristics (OVA-BS4 spheroids) from a primary human EOC cell line under selective conditions. OVA-BS4 spheroids were characterized for drug response by cytotoxicity assays and their molecular profile was investigated by microarray and RT-qPCR. Finally, we performed a gene expression study in a cohort of 74 high-grade serous EOC (HGSOC) patients by RT-qPCR. RESULTS: Spheroids exhibited properties of self-renewal and a pronounced expression of well-known stem cell genes. Moreover, they demonstrated greater resistance towards several anticancer drugs compared to parent cell line, consistent with their higher ABCG2 gene expression. From microarray studies MAL (T-cell differentiation protein) emerged as the most up-regulated gene in spheroids, compared to parent cell line. In HGSOC patients, MAL was significantly overexpressed in platinum-resistant compared to platinum-sensitive patients and resulted as an independent prognostic marker of survival. CONCLUSIONS: This investigation provides an important contribution to the identification of molecular markers of ovarian CSCs and chemoresistance. Successful translation of molecular findings would lead to a better comprehension of the mechanisms triggering chemoresistant recurrences, to the individuation of novel therapeutic targets and to the personalization of treatment regimens.
Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/genética , Neoplasias Císticas, Mucinosas e Serosas/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Progressão da Doença , Feminino , Humanos , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Neoplasias Císticas, Mucinosas e Serosas/metabolismo , Neoplasias Císticas, Mucinosas e Serosas/mortalidade , Neoplasias Císticas, Mucinosas e Serosas/cirurgia , Células-Tronco Neoplásicas , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/cirurgia , Prognóstico , Regulação para CimaRESUMO
The production rate of gene expression data is nothing less than astounding. However, with the benefit of hindsight we can assert that, since we completely ignored the non-coding part of the transcriptome, we spent the last decade to study cell mechanisms having few data in our hands. In this scenario, microRNAs, which are key post-trascriptional regulators, deserve special attention. Given the state of knowledge about their biogenesis, mechanisms of action and the numerous experimentally validated target genes, miRNAs are also gradually appearing in the formal pathway representations such as KEGG and Reactome maps. However, the number of miRNAs annotated in pathway maps are very few and pathway analyses exploiting this new regulatory layer are still lacking. To fill these gaps, we present 'micrographite' a new pipeline to perform topological pathway analysis integrating gene and miRNA expression profiles. Here, micrographite is used to study and dissect the epithelial ovarian cancer gene and miRNA transcriptome defining and validating a new regulatory circuit related to ovarian cancer histotype specificity.
Assuntos
Perfilação da Expressão Gênica , Redes Reguladoras de Genes , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Software , Transcriptoma , Feminino , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismoRESUMO
Human Th17 cells have a limited proliferative capacity compared to other T-cell subsets. We have shown that human Th17 cells display impaired IL-2 production due to IL-4-induced gene 1 (IL4I1) upregulation. Here, we show that in human Th17 cells, IL4I1 also maintains high levels of Tob1, a member of the Tob/BTG (B-cell traslocation gene) antiproliferative protein family, which prevents cell-cycle progression mediated by TCR stimulation. Indeed, Th17 cells exhibited higher levels of Tob1 than Th1 cells in both resting and TCR-activated conditions. Accordingly, the expression of positive regulators of the cell cycle (cyclin A, B, C, and E and Cdk2), as well as of Skp2, which promotes Tob1 degradation, was lower in Th17 cells than in Th1 cells. Tob1 expression in human Th17 cells correlated with both RAR (retinoic acid receptor)-related orphan receptor C (RORC) and IL4I1 levels. However, RORC was not directly involved in the regulation of Tob1 expression, whereas IL4I1 silencing in Th17 cells induced a substantial decrease of Tob1 expression. These data suggest that IL4I1 upregulation in human Th17 cells limits their TCR-mediated expansion not only by blocking the molecular pathway involved in the activation of the IL-2 promoter, but also by maintaining high levels of Tob1, which impairs entry into the cell cycle.
Assuntos
Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , L-Aminoácido Oxidase/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Humanos , Modelos Biológicos , Proteínas Quinases Associadas a Fase S/metabolismo , Células Th1/imunologia , Células Th1/metabolismoRESUMO
Denileukin diftitox (DD), a diphtheria toxin fragment IL-2 fusion protein, is thought to target and kill CD25(+) cells. It is approved for the treatment of cutaneous T-cell lymphoma and is used experimentally for the depletion of regulatory T cells (Treg) in cancer trials. Curiously enough, clinical effects of DD did not strictly correlate with CD25 expression, and Treg depletion was not confirmed unambiguously. Here, we report that patients with melanoma receiving DD immediately before a dendritic cell (DC) vaccine failed to develop a tumor-antigen-specific CD4 and CD8 T-cell immune response even after repeated vaccinations. Analyzing the underlying mechanism, so far we found unknown effects of DD. First, DD modulated DCs toward tolerance by downregulating costimulatory receptors such as CD83 and CD25 while upregulating tolerance-associated proteins/pathways including Stat-3, ß-catenin, and class II transactivator-dependent antigen presentation. Second, DD blocked Stat3 phosphorylation in maturing DCs. Third, only activated, but not resting, Treg internalized DD and were killed. Conversely, resting Treg showed increased survival because of DD-mediated antiapoptotic IL-2 signaling. We conclude that DD exerts functions beyond CD25(+) cell killing that may affect their clinical use and could be tested for novel indications.
Assuntos
Antineoplásicos/uso terapêutico , Células Dendríticas/efeitos dos fármacos , Toxina Diftérica/uso terapêutico , Interleucina-2/uso terapêutico , Melanoma/terapia , Neoplasias Cutâneas/terapia , Linfócitos T Reguladores/efeitos dos fármacos , Vacinas Anticâncer , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Terapia Combinada , Células Dendríticas/imunologia , Células Dendríticas/transplante , Citometria de Fluxo , Humanos , Tolerância Imunológica , Teste de Cultura Mista de Linfócitos , Melanoma/imunologia , Microscopia Confocal , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Proteínas Recombinantes de Fusão/uso terapêutico , Neoplasias Cutâneas/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
SUMMARY: Pathway Processor 2.0 is a web application designed to analyze high-throughput datasets, including but not limited to microarray and next-generation sequencing, using a pathway centric logic. In addition to well-established methods such as the Fisher's test and impact analysis, Pathway Processor 2.0 offers innovative methods that convert gene expression into pathway expression, leading to the identification of differentially regulated pathways in a dataset of choice. AVAILABILITY AND IMPLEMENTATION: Pathway Processor 2.0 is available as a web service at http://compbiotoolbox.fmach.it/pathwayProcessor/. Sample datasets to test the functionality can be used directly from the application. CONTACT: duccio.cavalieri@fmach.it SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência com Séries de Oligonucleotídeos , Software , Animais , Humanos , Internet , Camundongos , Ratos , TranscriptomaRESUMO
The activation of TLRs expressed by macrophages or DCs, in the long run, leads to persistently impaired functionality. TLR signals activate a wide range of negative feedback mechanisms; it is not known, however, which of these can lead to long-lasting tolerance for further stimulatory signals. In addition, it is not yet understood how the functionality of monocyte-derived DCs (MoDCs) is influenced in inflamed tissues by the continuous presence of stimulatory signals during their differentiation. Here we studied the role of a wide range of DC-inhibitory mechanisms in a simple and robust model of MoDC inactivation induced by early TLR signals during differentiation. We show that the activation-induced suppressor of cytokine signaling 1 (SOCS1), IL-10, STAT3, miR146a and CD150 (SLAM) molecules possessed short-term inhibitory effects on cytokine production but did not induce persistent DC inactivation. On the contrary, the LPS-induced IRAK-1 downregulation could alone lead to persistent MoDC inactivation. Studying cellular functions in line with the activation-induced negative feedback mechanisms, we show that early activation of developing MoDCs allowed only a transient cytokine production that was followed by the downregulation of effector functions and the preservation of a tissue-resident non-migratory phenotype.
Assuntos
Citocinas/metabolismo , Células Dendríticas/metabolismo , Regulação da Expressão Gênica , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Diferenciação Celular , Células Cultivadas , Citocinas/genética , Células Dendríticas/imunologia , Células Dendríticas/patologia , Retroalimentação Fisiológica , Regulação da Expressão Gênica/imunologia , Humanos , Tolerância Imunológica , Inflamação , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/imunologia , Interleucina-10/genética , Interleucina-10/metabolismo , Lipopolissacarídeos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Monócitos/patologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Receptores Toll-Like/metabolismoRESUMO
Late diagnosis and the lack of screening methods for early detection define high-grade serous ovarian cancer (HGSOC) as the gynecological malignancy with the highest mortality rate. In the work presented here, we investigated a retrospective and multicentric cohort of 250 archival Papanicolaou (Pap) test smears collected during routine gynecological screening. Samples were taken at different time points (from 1 month to 13.5 years before diagnosis) from 113 presymptomatic women who were subsequently diagnosed with HGSOC (pre-HGSOC) and from 77 healthy women. Genome instability was detected through low-pass whole-genome sequencing of DNA derived from Pap test samples in terms of copy number profile abnormality (CPA). CPA values of DNA extracted from Pap test samples from pre-HGSOC women were substantially higher than those in samples from healthy women. Consistently with the longitudinal analysis of clonal pathogenic TP53 mutations, this assay could detect HGSOC presence up to 9 years before diagnosis. This finding confirms the continual shedding of tumor cells from fimbriae toward the endocervical canal, suggesting a new path for the early diagnosis of HGSOC. We integrated the CPA score into the EVA (early ovarian cancer) test, the sensitivity of which was 75% (95% CI, 64.97 to 85.79), the specificity 96% (95% CI, 88.35 to 100.00), and the accuracy 81%. This proof-of-principle study indicates that the early diagnosis of HGSOC is feasible through the analysis of genomic alterations in DNA from endocervical smears.
Assuntos
Neoplasias Ovarianas , Teste de Papanicolaou , Feminino , Humanos , Teste de Papanicolaou/métodos , Estudos Retrospectivos , Detecção Precoce de Câncer/métodos , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/genética , DNA , Instabilidade GenômicaRESUMO
MOTIVATION: Many models and analysis of signaling pathways have been proposed. However, neither of them takes into account that a biological pathway is not a fixed system, but instead it depends on the organism, tissue and cell type as well as on physiological, pathological and experimental conditions. RESULTS: The Biological Connection Markup Language (BCML) is a format to describe, annotate and visualize pathways. BCML is able to store multiple information, permitting a selective view of the pathway as it exists and/or behave in specific organisms, tissues and cells. Furthermore, BCML can be automatically converted into data formats suitable for analysis and into a fully SBGN-compliant graphical representation, making it an important tool that can be used by both computational biologists and 'wet lab' scientists. AVAILABILITY AND IMPLEMENTATION: The XML schema and the BCML software suite are freely available under the LGPL for download at http://bcml.dc-atlas.net. They are implemented in Java and supported on MS Windows, Linux and OS X.
Assuntos
Biologia Computacional/métodos , Linguagens de Programação , Transdução de Sinais , Software , Gráficos por ComputadorRESUMO
In this study, we present evidence of differential Th17 responses in human monocyte-derived dendritic cells exposed to the pathogenic Candida albicans or the nonpathogenic Saccharomyces cerevisiae. We use different forms of the microorganisms, cells, hyphae, and spores, as a toolbox to dissect the role of surface mannan in the fungal immune response. In contrast to the S. cerevisiae yeast cell-induced Th1 response, dendritic cells stimulated with spores or C. albicans hyphae induce cellular responses shifted toward Th17 differentiation. The differential recognition of specific mannan structures is the master regulator of the discrimination between harmful and harmless fungi. The switch between spores and yeast is crucial for the commensalism of S. cerevisiae and depends on the use of a different receptor repertoire. Understanding the role of cell wall recognition during infection might lead to understanding the boundaries between safety and pathogenicity.