RESUMO
BACKGROUND: Tumour dormancy, a resistance mechanism employed by cancer cells, is a significant challenge in cancer treatment, contributing to minimal residual disease (MRD) and potential relapse. Despite its clinical importance, the mechanisms underlying tumour dormancy and MRD remain unclear. In this study, we employed two syngeneic murine models of myeloid leukemia and melanoma to investigate the genetic, epigenetic, transcriptomic and protein signatures associated with tumour dormancy. We used a multiomics approach to elucidate the molecular mechanisms driving MRD and identify potential therapeutic targets. RESULTS: We conducted an in-depth omics analysis encompassing whole-exome sequencing (WES), copy number variation (CNV) analysis, chromatin immunoprecipitation followed by sequencing (ChIP-seq), transcriptome and proteome investigations. WES analysis revealed a modest overlap of gene mutations between melanoma and leukemia dormancy models, with a significant number of mutated genes found exclusively in dormant cells. These exclusive genetic signatures suggest selective pressure during MRD, potentially conferring resistance to the microenvironment or therapies. CNV, histone marks and transcriptomic gene expression signatures combined with Gene Ontology (GO) enrichment analysis highlighted the potential functional roles of the mutated genes, providing insights into the pathways associated with MRD. In addition, we compared "murine MRD genes" profiles to the corresponding human disease through public datasets and highlighted common features according to disease progression. Proteomic analysis combined with multi-omics genetic investigations, revealed a dysregulated proteins signature in dormant cells with minimal genetic mechanism involvement. Pathway enrichment analysis revealed the metabolic, differentiation and cytoskeletal remodeling processes involved in MRD. Finally, we identified 11 common proteins differentially expressed in dormant cells from both pathologies. CONCLUSIONS: Our study underscores the complexity of tumour dormancy, implicating both genetic and nongenetic factors. By comparing genomic, transcriptomic, proteomic, and epigenomic datasets, our study provides a comprehensive understanding of the molecular landscape of minimal residual disease. These results provide a robust foundation for forthcoming investigations and offer potential avenues for the advancement of targeted MRD therapies in leukemia and melanoma patients, emphasizing the importance of considering both genetic and nongenetic factors in treatment strategies.
Assuntos
Modelos Animais de Doenças , Melanoma , Neoplasia Residual , Animais , Melanoma/genética , Melanoma/patologia , Camundongos , Leucemia/genética , Leucemia/patologia , Variações do Número de Cópias de DNA , Sequenciamento do Exoma , Camundongos Endogâmicos C57BL , Proteômica , Transcriptoma , Perfilação da Expressão Gênica , MultiômicaRESUMO
Epithelial ovarian cancers (EOCs) are a heterogeneous collection of malignancies, each with their own developmental origin, clinical behavior and molecular profile. With less than 5% of EOC cases, mucinous ovarian carcinoma is a rare form with a poor prognosis and a 5-year survival of 11% for advanced stages (III/IV). At the early stages, these malignant forms are clinically difficult to distinguish from borderline (15%) and benign (80%) forms with a better prognosis due to the large size and heterogeneity of mucinous tumors. Improving their diagnosis is therefore a challenge with regard to the risk of under-treating a malignant form or of unnecessarily undertaking radical surgical excision. The involvement of microRNAs (miRNAs) in tumor progression and their potential as biomarkers of diagnosis are becoming increasingly recognized. In this study, the comparison of miRNA microarray expression profiles between malignant and borderline tumor FFPE samples identified 10 down-regulated and 5 up-regulated malignant miRNAs, which were validated by individual RT-qPCR. To overcome normalization issues and to improve the accuracy of the results, a ratio analysis combining paired up-regulated and down-regulated miRNAs was performed. Although 21/50 miRNA expression ratios were significantly different between malignant and borderline tumor samples, any ratio could perfectly discriminate the two groups. However, a combination of 14 pairs of miRNA ratios (double ratio) showed high discriminatory potential, with 100% of accuracy in distinguishing malignant and borderline ovarian tumors, which suggests that miRNAs may hold significant clinical potential as a diagnostic tool. In summary, these ratio miRNA-based signatures may help to improve the precision of histological diagnosis, likely to provide a preoperative diagnosis in order to adapt surgical procedures.
Assuntos
Adenocarcinoma Mucinoso , MicroRNAs , Neoplasias Císticas, Mucinosas e Serosas , Neoplasias Ovarianas , Lesões Pré-Cancerosas , Feminino , Humanos , MicroRNAs/genética , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Carcinoma Epitelial do Ovário , Adenocarcinoma Mucinoso/diagnóstico , Adenocarcinoma Mucinoso/genética , Adenocarcinoma Mucinoso/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismoRESUMO
Receptor-interacting protein kinase 3 (RIPK3) can induce necroptosis, apoptosis, or cell proliferation and is silenced in several hematological malignancies. We previously reported that RIPK3 activity independent of its kinase domain induces caspase-mediated p65/RelA cleavage, resulting in N-terminal 1-361 and C-terminal 362-549 fragments. We show here that a noncleavable p65/RelA D361E mutant expressed in DA1-3b leukemia cells decreases mouse survival times and that coexpression of p65/RelA fragments increases the tumorigenicity of B16F1 melanoma cells. This aggressiveness in vivo did not correlate with NF-κB activity measured in vitro. The fragments and p65/RelA D361E mutant induced different expression profiles in DA1-3b and B16F1 cells. Stemness markers were affected: p65/RelA D361E increased ALDH activity in DA1-3b cells, and fragment expression increased melanoma sphere formation in B16/F1 cells. p65/RelA fragments and the D361E noncleavable mutant decreased oxidative or glycolytic cell metabolism, with differences observed between models. Thus, p65/RelA cleavage initiated by kinase-independent RIPK3 activity in cancer cells is not neutral and induces pleiotropic effects in vitro and in vivo that may vary across tumor types.
Assuntos
Melanoma , NF-kappa B , Animais , Apoptose , Caspases/metabolismo , Camundongos , NF-kappa B/metabolismo , Fosforilação , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/farmacologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismoRESUMO
GREM1 (gremlin1) is a known inhibitor for BMP15 (bone morphogenetic protein 15) family, but its genetic diversity in sheep is unknown. The present study was conducted to analyze the polymorphism of GREM1 gene using PCR- single-strand conformation polymorphism (SSCP) and DNA sequencing methods and to assess the possible association of GREM1 gene polymorphism with reproductive traits in Awassi ewes. A total of 224 ewes, 124 producing singles and 100 producing twins, were included in the study. Two SSCP patterns were detected in two amplified loci within the exon 2. Two exonic novel single nucleotide polymorphism (SNP)s were identified, c.74 T > G (the silent SNP p.Met123 =) and c.30 T > A with (the missense SNP p.Ile237Phe). Statistical analyses indicated a non-significant (P > 0.05) association of p.Met123 = with the analyzed reproductive traits of fecundity, prolificacy, litter size, and twinning rate. Meanwhile, p.Ile237Phe SNP exhibited a highly significant (P < 0.01) association with the measured reproductive traits, in which ewes with TA genotype (with p.Ile237Phe SNP) exhibited higher litter size, twinning ratio, fecundity, and prolificacy than those with TT genotype (without p.Ile237Phe SNP). The deleterious impact of p.Ile237Phe SNP was observed by the means of ten different state-of-the-art in silico tools that predicted a highly damaging effect of p.Ile237Phe SNP on the structure, function, and stability of gremlin1. In conclusion, the results of our study suggest that p.Ile237Phe SNP has a remarkable negative impact on the gremlin1 structure, function, and stability. Since gremlin1 is a known inhibitor of reproductive performance, a consequent higher reproductive performance was observed in ewes with damaged gremlin1 (with p.Ile237Phe SNP) than those with non-damaged gremlin1 (without p.Ile237Phe SNP). Therefore, it can be stated that the implementation of the novel p.Ile237Phe SNP in the GREM1 gene could be a useful marker in marker-assisted selection. This manuscript is the first one to describe GREM1 gene variations in sheep.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/genética , Tamanho da Ninhada de Vivíparos/genética , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , Reprodução/genética , Ovinos/genética , Animais , FemininoRESUMO
The lipase E hormone-sensitive (LIPE) enzyme is one of the lipolytic enzymes, and it plays a key role in the regulation of adipose tissue deposition. This study was conducted to investigate the possible association between the LIPE gene variations and the main body weight measurements in Awassi sheep. A total of 160 of sexually mature Awassi rams (Ovis aries) that aged between 2 and 3 years were included in the present study. Genomic DNA was extracted and two specific PCR amplicons were designed to amplify two coding regions within the LIPE gene. Genotyping experiments were performed using polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP). Two different SSCP banding patterns were identified, CC and CD in exon 2, and AA and AT in exon 9. Five novel single-nucleotide polymorphisms (SNPs) were detected by sequencing, namely g.151C > A and g.198C > T in exon 2, and g.213G > C, g.226G > T, and g.232A > C in exon 9. Haplotype block analysis showed strong linkage disequilibrium values between the two SNPs in exon 2 and the three SNPs in exon 9. Association analysis of haplotypes with carcass traits demonstrated a significantly higher dressing percentage (P < 0.05) and lower fat tail weight (FTW) in CACT and GCGTAC haplotypes made these haplotypes more favorable for human consumption. The current research is the first one to report a tight association between the LIPE genetic polymorphism and the dressing percentage and FTW traits, suggesting a pivotal role played by these co-inherited SNPs in the metabolism of carcass traits in sheep.
Assuntos
Tecido Adiposo/metabolismo , Hereditariedade , Lipase Lipoproteica/genética , Carne/análise , Polimorfismo de Nucleotídeo Único , Carneiro Doméstico/fisiologia , Animais , Iraque , Desequilíbrio de Ligação , Lipase Lipoproteica/metabolismo , Masculino , Carneiro Doméstico/metabolismoRESUMO
BACKGROUND: Metastatic melanoma is one of the most aggressive forms of cancer in humans. Among its types, mucosal melanomas represent one of the most highly metastatic and aggressive forms, with a very poor prognosis. Because they are rare in Caucasian individuals, unlike cutaneous melanomas, there has been fewer epidemiological, clinical and genetic evaluation of mucosal melanomas. Moreover, the lack of predictive models fully reproducing the pathogenesis and molecular alterations of mucosal melanoma makes its treatment challenging. Interestingly, dogs are frequently affected by melanomas of the oral cavity that are characterized, as their human counterparts, by focal infiltration, recurrence, and metastasis to regional lymph nodes, lungs and other organs. In dogs, some particular breeds are at high risk, suggesting a specific genetic background and strong genetic drivers. Altogether, the striking homologies in clinical presentation, histopathological features, and overall biology between human and canine mucosal melanomas make dogs invaluable natural models with which to investigate tumor development, including tumor ætiology, and develop tailored treatments. METHODS: We developed and characterized two canine oral melanoma cell lines from tumors isolated from dog patients with distinct clinical profiles; with and without lung metastases. The cells were characterized using immunohistochemistry, pharmacology and genetic studies. RESULTS: We have developed and immunohistochemically, genetically, and pharmacologically characterized. Two cell lines (Ocr_OCMM1X & Ocr_OCMM2X) were produced through mouse xenografts originating from two clinically contrasting melanomas of the oral cavity. Their exhaustive characterization showed two distinct biological and genetic profiles that are potentially linked to the stage of malignancy at the time of diagnosis and sample collection of each melanoma case. These cell lines thus constitute relevant tools with which to perform genetic and drug screening analyses for a better understanding of mucosal melanomas in dogs and humans. CONCLUSIONS: The aim of this study was to establish and characterize xenograft-derived canine melanoma cell lines with different morphologies, genetic features and pharmacological sensitivities that constitute good predictive models for comparative oncology. These cell lines are relevant tools to advance the use of canine mucosal melanomas as natural models for the benefit of both veterinary and human medicine.
Assuntos
Melanoma/diagnóstico por imagem , Melanoma/genética , Neoplasias Bucais/diagnóstico por imagem , Neoplasias Bucais/genética , Neoplasias Cutâneas/diagnóstico por imagem , Neoplasias Cutâneas/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Cães , Relação Dose-Resposta a Droga , Feminino , Humanos , Melanoma/tratamento farmacológico , Camundongos , Camundongos Nus , Neoplasias Bucais/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Melanoma Maligno CutâneoRESUMO
CAG triplet expansions in Ataxin-2 gene (ATXN2) cause spinocerebellar ataxia type 2 and have a role that remains to be clarified in Parkinson's disease (PD). To study the molecular events associated with these expansions, we sequenced them and analyzed the transcriptome from blood cells of controls and three patient groups diagnosed with spinocerebellar ataxia type 2 (herein referred to as SCA2c) or PD with or without ATXN2 triplet expansions (named SCA2p). The transcriptome profiles of these 40 patients revealed three main observations: i) a specific pattern of pathways related to cellular contacts, proliferation and differentiation associated with SCA2p group, ii) similarities between the SCA2p and sporadic PD groups in genes and pathways known to be altered in PD such as Wnt, Ephrin and Leukocyte extravasation signaling iii) RNA metabolism disturbances with "RNA-binding" and "poly(A) RNA-binding" as a common feature in all groups. Remarkably, disturbances of ALS signaling were shared between SCA2p and sporadic PD suggesting common molecular dysfunctions in PD and ALS including CACNA1, hnRNP, DDX and PABPC gene family perturbations. Interestingly, the transcriptome profiles of patients with parkinsonian phenotypes were prevalently associated with alterations of translation while SCA2c and PD patients presented perturbations of splicing. While ATXN2 RNA expression was not perturbed, its protein expression in immortalized lymphoblastoid cells was significantly decreased in SCA2c and SCA2p versus control groups assuming post-transcriptional biological perturbations. In conclusion, the transcriptome data do not exclude the role of ATXN2 mutated alleles in PD but its decrease protein expression in both SCA2c and SCA2p patients suggest a potential involvement of this gene in PD. The perturbations of "RNA-binding" and "poly(A) RNA-binding" molecular functions in the three patient groups as well as gene deregulations of factors not yet described in PD but known to be deleterious in other neurological conditions, suggest the existence of RNA-binding disturbances as a continuum between spinocerebellar ataxia type 2 and Parkinson's disease.
Assuntos
Doença de Parkinson/etiologia , Doença de Parkinson/metabolismo , RNA/metabolismo , Ataxias Espinocerebelares/complicações , Ataxias Espinocerebelares/metabolismo , Adulto , Idoso , Ataxina-2/metabolismo , Estudos de Casos e Controles , Progressão da Doença , Feminino , Humanos , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transcriptoma , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
The leucine-rich repeat kinase 2 (LRRK2) G2019S mutation is a common genetic cause of Parkinson's disease (PD). Although patients with sporadic PD and individuals with LRRK2-linked PD display the classical PD phenotype, it is not known whether or not the same biological pathways are deregulated in each context. By using transcriptome profiling, we investigated the deregulation of various biological pathways in a total of 47 peripheral blood mononuclear cell (PBMC) samples from patients with sporadic PD, patients heterozygous for the LRRK2 G2019S mutation compared to healthy controls. We found that the deregulation patterns were indeed similar in PBMCs obtained from patients with sporadic PD and from LRRK2 G2019S carriers, with dysfunctions in mitochondrial pathways, cell survival signaling, cancerization, endocytosis signaling and iron metabolism. Analysis of our PBMC data and other publicly available transcriptome datasets (for whole blood samples) showed that deregulation of the immune system, endocytosis and eukaryotic initiation factor 2 (EIF2) signaling are the main features of transcriptome profiles in PD (since they are also present in the transcriptome of dopaminergic neurons from patients). Transcriptome analysis of PBMCs is thus valuable for (i) characterizing the pathophysiological pathways shared by genetic and sporadic forms of PD and (ii) identifying potential biomarkers and therapeutic targets. This minimally invasive approach opens up tremendous perspectives for better diagnosis and therapy of neurodegenerative diseases because it can be applied from the earliest stages of the disease onwards.
Assuntos
Endocitose/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Sistema Imunitário/fisiopatologia , Doença de Parkinson , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Perfilação da Expressão Gênica , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Masculino , Pessoa de Meia-Idade , Mutação/genética , Doença de Parkinson/genética , Doença de Parkinson/imunologia , Doença de Parkinson/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genéticaRESUMO
Genome-wide analysis of a multi-incident family with autosomal-dominant parkinsonism has implicated a locus on chromosomal region 3q26-q28. Linkage and disease segregation is explained by a missense mutation c.3614G>A (p.Arg1205His) in eukaryotic translation initiation factor 4-gamma (EIF4G1). Subsequent sequence and genotype analysis identified EIF4G1 c.1505C>T (p.Ala502Val), c.2056G>T (p.Gly686Cys), c.3490A>C (p.Ser1164Arg), c.3589C>T (p.Arg1197Trp) and c.3614G>A (p.Arg1205His) substitutions in affected subjects with familial parkinsonism and idiopathic Lewy body disease but not in control subjects. Despite different countries of origin, persons with EIF4G1 c.1505C>T (p.Ala502Val) or c.3614G>A (p.Arg1205His) mutations appear to share haplotypes consistent with ancestral founders. eIF4G1 p.Ala502Val and p.Arg1205His disrupt eIF4E or eIF3e binding, although the wild-type protein does not, and render mutant cells more vulnerable to reactive oxidative species. EIF4G1 mutations implicate mRNA translation initiation in familial parkinsonism and highlight a convergent pathway for monogenic, toxin and perhaps virally-induced Parkinson disease.
Assuntos
Cromossomos Humanos Par 3/genética , Fator de Iniciação Eucariótico 4G/genética , Doença de Parkinson/genética , Biossíntese de Proteínas/genética , Sequência de Bases , Clonagem Molecular , Variações do Número de Cópias de DNA , Análise Mutacional de DNA , Citometria de Fluxo , Ligação Genética , Genótipo , Humanos , Imunoprecipitação , Mitocôndrias/fisiologia , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , LinhagemRESUMO
Germline heterozygous alterations of the tumor-suppressor gene neurofibromatosis-1 (NF1) lead to neurofibromatosis type 1, a genetic disorder characterized by a higher risk to develop juvenile myelomonocytic leukemia and/or acute myeloid leukemia (AML). More recently, somatic 17q11 deletions encompassing NF1 have been described in many adult myeloid malignancies. In this context, we aimed to define NF1 involvement in AML. We screened a total of 488 previously untreated de novo AML patients for the NF1 deletion using either array comparative genomic hybridization (aCGH) or real-time quantitative PCR/fluorescence in situ hybridization approaches. We also applied massively parallel sequencing for in depth mutation analysis of NF1 in 20 patients including five NF1-deleted patients. We defined a small â¼0.3 Mb minimal deleted region involving NF1 by aCGH and an overall frequency of NF1 deletion of 3.5% (17/485). NF1 deletion is significantly associated with unfavorable cytogenetics and with monosomal karyotype notably. We discovered six NF1 variants of unknown significance in 7/20 patients of which only one out of four disappeared in corresponding complete remission sample. In addition, only one out of five NF1-deleted patients has an acquired coding mutation in the remaining allele. In conclusion, direct NF1 inactivation is infrequent in de novo AML and may be a secondary event probably involved in leukemic progression.
Assuntos
Deleção de Genes , Leucemia Mieloide Aguda/genética , Neurofibromatose 1/genética , Neurofibromina 1/genética , Adolescente , Adulto , Idoso , Alelos , Hibridização Genômica Comparativa , Feminino , Frequência do Gene , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Pessoa de Meia-Idade , Taxa de Mutação , Neurofibromina 1/deficiência , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Acute myeloid leukemia (AML) is characterized by blocked differentiation and extensive proliferation of hematopoietic progenitors/precursors. Relapse is often observed after chemotherapy due to the presence of residual leukemic cells, which is also called minimal residual disease (MRD). Subclonal heterogeneity at diagnosis was found to be responsible for MRD after treatment. Patient xenograft mouse models are valuable tools for studying MRD after chemotherapy; however, the contribution of the immune system in these models is usually missing. To evaluate its role in leukemic persistence, we generated an immune-competent AML mouse model of persistence after chemotherapy treatment. We used well-characterized (phenotypically and genetically) subclones of the murine C1498 cell line stably expressing the ZsGreen reporter gene and the WT1 protein, a valuable antigen. Accordingly, these subclones were also selected due to their in vitro aracytidine (Ara-c) sensitivity. A combination of 3 subclones (expressing or not expressing WT1) was found to lead to prolonged mouse survival after Ara-c treatment (as long as 150 days). The presence of residual leukemic cells in the blood and BM of surviving mice indicated their persistence. Thus, a new mouse model that may offer insights into immune contributions to leukemic persistence was developed.
Assuntos
Leucemia Mieloide Aguda , Animais , Citarabina/farmacologia , Citarabina/uso terapêutico , Modelos Animais de Doenças , Progressão da Doença , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Camundongos , Neoplasia Residual/diagnósticoRESUMO
The key role of B cells in the pathophysiology of multiple sclerosis (MS) is supported by the presence of oligoclonal bands in the cerebrospinal fluid, by the association of meningeal ectopic B cell follicles with demyelination, axonal loss and reduction of astrocytes, as well as by the high efficacy of B lymphocyte depletion in controlling inflammatory parameters of MS. Here, we use a spontaneous model of experimental autoimmune encephalomyelitis (EAE) to study the clonality of the B cell response targeting myelin oligodendrocyte glycoprotein (MOG). In particular, 94% of SJL/j mice expressing an I-As: MOG92-106 specific transgenic T cell receptor (TCR1640) spontaneously develop a chronic paralytic EAE between the age of 60-500 days. The immune response is triggered by the microbiota in the gut-associated lymphoid tissue, while there is evidence that the maturation of the autoimmune demyelinating response might occur in the cervical lymph nodes owing to local brain drainage. Using MOG-protein-tetramers we tracked the autoantigen-specific B cells and localized their enrichment to the cervical lymph nodes and among the brain immune infiltrate. MOG-specific IgG1 antibodies were detected in the serum of diseased TCR1640 mice and proved pathogenic upon adoptive transfer into disease-prone recipients. The ontogeny of the MOG-specific humoral response preceded disease onset coherent with their contribution to EAE initiation. This humoral response was, however, not sufficient for disease induction as MOG-antibodies could be detected at the age of 69 days in a model with an average age of onset of 197 days. To assess the MOG-specific B cell repertoire we FACS-sorted MOG-tetramer binding cells and clonally expand them in vitro to sequence the paratopes of the IgG heavy chain and kappa light chains. Despite the fragility of clonally expanding MOG-tetramer binding effector B cells, our results indicate the selection of a common CDR-3 clonotype among the Igk light chains derived from both disease-free and diseased TCR1640 mice. Our study demonstrates the pre-clinical mobilization of the MOG-specific B cell response within the brain-draining cervical lymph nodes, and reiterates that MOG antibodies are a poor biomarker of disease onset and progression.
Assuntos
Linfócitos B/imunologia , Encefalomielite Autoimune Experimental/imunologia , Glicoproteína Mielina-Oligodendrócito/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Autoanticorpos/imunologia , Autoantígenos/imunologia , Linfócitos B/citologia , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/fisiopatologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Glicoproteína Mielina-Oligodendrócito/genéticaRESUMO
We provide an original multi-stage approach identifying a gene signature to assess murine fibroblast polarization. Prototypic polarizations (inflammatory/fibrotic) were induced by seeded mouse embryonic fibroblasts (MEFs) with TNFα or TGFß1, respectively. The transcriptomic and proteomic profiles were obtained by RNA microarray and LC-MS/MS. Gene Ontology and pathways analysis were performed among the differentially expressed genes (DEGs) and proteins (DEPs). Balb/c mice underwent daily intradermal injections of HOCl (or PBS) as an experimental murine model of inflammation-mediated fibrosis in a time-dependent manner. As results, 1456 and 2215 DEGs, and 289 and 233 DEPs were respectively found in MEFs in response to TNFα or TGFß1, respectively. Among the most significant pathways, we combined 26 representative genes to encompass the proinflammatory and profibrotic polarizations of fibroblasts. Based on principal component analysis, this signature deciphered baseline state, proinflammatory polarization, and profibrotic polarization as accurately as RNA microarray and LC-MS/MS did. Then, we assessed the gene signature on dermal fibroblasts isolated from the experimental murine model. We observed a proinflammatory polarization at day 7, and a mixture of a proinflammatory and profibrotic polarizations at day 42 in line with histological findings. Our approach provides a small-size and convenient gene signature to assess murine fibroblast polarization.
Assuntos
Fibroblastos , Fator de Necrose Tumoral alfa , Animais , Cromatografia Líquida , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibrose , Camundongos , Camundongos Endogâmicos BALB C , Proteômica , RNA/metabolismo , Espectrometria de Massas em Tandem , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND & AIM: The key role of environmental factors in the pathogenesis of Inflammatory Bowel Diseases (IBD) is recognized. Aluminum is suspected to be a risk factor for IBD. However, mechanisms linking aluminum exposure to disease development are unknown. We examined the role of aluminum transport and subcellular localisation on human colon susceptibility to aluminum-induced inflammation. METHODS: Human colon biopsies isolated from Crohn's disease (CD) or control patients and Caco-2 cells were incubated with aluminum. The effects of aluminum were evaluated on cytokine secretion and transporter expression. The role of aluminum kinetics parameters was studied in Caco-2 using transport inhibitors and in human colon biopsies by assessing genetic polymorphisms of transporters. RESULTS: Aluminum exposure was shown to induce cytokine secretion in colon of CD but not healthy patients. In Caco-2 cells, aluminum internalisation was correlated with inflammatory status. In human colon, analysis of genetic polymorphisms and expression of ABCB1 and SLC26A3 transporters showed that their decreased activity was involved in aluminum-induced inflammation. CONCLUSIONS: We hypothesize that alteration in detoxifying response would lead to a deregulation of intestinal homeostasis and to the expression of IBD. Our study emphasizes the complexity of gene/environment interaction for aluminum adverse health effect, highlighting at risk populations or subtypes of patients. A better understanding of correlations between gene expression or SNP and xenobiotic kinetics parameters would shift the medical paradigm to more personalized disease management and treatment.
Assuntos
Doença de Crohn , Doenças Inflamatórias Intestinais , Alumínio/toxicidade , Células CACO-2 , Doença de Crohn/genética , Doença de Crohn/metabolismo , Citocinas/genética , Interação Gene-Ambiente , Humanos , Inflamação , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , XenobióticosRESUMO
Genomic multiplication of the alpha-synuclein gene (SNCA) locus is one cause of familial Parkinson disease (PD). We performed detailed genomic, SNCA expression level, clinical, neuropsychological and functional imaging analyses of a parkinsonian kindred with a known duplication of the SNCA locus. We demonstrated that the duplication spanned 4.928 Mb (encompassing 31 known and putative genes) and was the largest to have been described at this locus. The presence of several repetitive long interspersed nuclear elements (LINEs) flanking the potential break area suggested that the duplication resulted from a genomic recombination between LINEs. We sequenced the break junction and confirmed the involvement of L1PA2 and L1PA4 in a non-allelic, homologous recombination. An analysis of mRNA levels in immortalized lymphoblastoid cells and peripheral blood mononuclear cells showed SNCA overexpression in subjects with the duplication, as well as overexpression of 13 other genes highlighting the usefulness of such cell models to study this duplication. Interestingly, abnormal tracer uptake in DaTSCAN(®) imaging correlated with the severity of the clinical symptoms. Our detailed genomic analysis and clinical exploration enabled us to specify the genotype-phenotype relationship, identify a case of presymptomatic PD and gain insight into the role of LINEs in SNCA locus duplication.
Assuntos
Duplicação Gênica , Elementos Nucleotídeos Longos e Dispersos/genética , Doença de Parkinson/genética , Fenótipo , alfa-Sinucleína/genética , Sequência de Bases , Predisposição Genética para Doença , Humanos , Modelos Genéticos , Dados de Sequência Molecular , LinhagemRESUMO
Hematopoietic cancer stem cells preserve cellular hierarchy in a manner similar to normal stem cells, yet the underlying regulatory mechanisms are poorly understood. It is known that both normal and malignant stem/progenitor cells express CD34. Here, we demonstrate that several cell lines (HL-60, U266) derived from hematopoietic malignancies contain not only CD34(-) but also CD34(+) subpopulations. The CD34(+) cells displayed a stem/progenitor-like phenotype since, in contrast to CD34(-) cells, they frequently underwent cellular division and rapidly formed colonies in methylcellulose-based medium. Strikingly, a constant fraction of the CD34(+) and CD34(-) cell subpopulations, when separated, rapidly switched their phenotype. Consequently, both separated fractions could generate tumors in immunocompromised NOD/LtSz-scid/scid mice. Cultures in vitro showed that the proportion of CD34(+) stem/progenitor-like cells in the population was decreased by cell-cell contact and increased by soluble factors secreted by the cells. Using cytokine arrays, we identified some of these factors, notably thymopoietin that was able to increase the proportion of CD34(+) cells and overall colony-forming capacity in tested cell lines. This action of thymopoietin was conserved in mononuclear cells from bone marrow. Therefore, we propose that hematopoietic cancer cell lines containing subpopulations of CD34(+) cells can provide an in vitro model for studies of cancer stem/progenitor cells.
Assuntos
Antígenos CD34/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas Nucleares/metabolismo , Timopoietinas/metabolismo , Animais , Antígenos CD34/genética , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Células Clonais , Células HL-60 , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteínas Nucleares/farmacologia , Timopentina/farmacologia , Timopoietinas/farmacologiaRESUMO
Melanocortin-4 receptor (MC4R) gene plays a key role in the regulation of body weight and energy homeostasis. This study aims to evaluate the association of single nucleotide polymorphisms (SNPs) of the MC4R gene with live body weight and hormonal assays in two breeds of sheep that differ in productive performance, Awassi and Arabi. All known coding sequences of the MC4R gene were covered in this study. DNA samples from 150 animals (Awassi and Arabi breed) were genotyped by PCR-single-strand conformation polymorphism (PCR-SSCP) to assess their pattern of genetic variation. Concerning exon 1, clear heterogeneity was detected with three different SSCP-banding patterns. The sequencing reactions confirmed these variations by detecting the presence of the two novel SNPs, 107G/C and 138A/C, and three genotypes, GC, AC and AA. The 107G/C SNP was detected in GC genotype, while the 138A/C was detected on both GC and AC genotypes. The other SSCP-banding pattern (AA genotype) did not show any detectable unique variation. Both SNPs were closely and strongly linked in both breeds (D' and r2 values were 1.00), which signifies that both loci were co-inherited as one unit. Association analysis indicated that both breeds with GC/AC haplotype showed higher live body weight (37.250 ± 0.790) relative to the GG/AA (30.244 ± 0.968) and CC/CC (47.231 ± 1.230) haplotypes (p < .05). Concerning the genotyping of exon 2, only 362 bp showed heterogeneity with a missense mutation, with no significant association (p > .05) with the measured traits. In conclusion, the two novel SNPs (107G/C and 138 A/C) were highly associated with live body weight in both breeds. Haplotype analysis confirmed that these two novel SNPs were in strong linkage disequilibrium (LD) and could be used as genetic markers for sheep phenotypic trait improvement.
Assuntos
Peso Corporal/genética , Hormônios Esteroides Gonadais/genética , Polimorfismo de Nucleotídeo Único , Polimorfismo Conformacional de Fita Simples , Receptor Tipo 4 de Melanocortina/genética , Carneiro Doméstico/fisiologia , Animais , Haplótipos , Iraque , Carneiro Doméstico/sangue , Carneiro Doméstico/genéticaRESUMO
RhoH is an unusual member of the Rho family of small GTP-binding proteins in that it lacks GTPase activity. Since the RhoH protein is constantly bound by GTP, it is constitutively active and controlled predominantly by changes in quantitative expression. Abnormal levels of RHOH gene transcripts have been linked to a range of malignancies including acute myeloid leukemia (AML). One of the hallmarks of AML is a block in the normal program of myeloid differentiation. Here we investigate how myeloid differentiation is controlled by the quantitative expression of RHOH. Our analysis demonstrates that increasingly mature myeloid cells express progressively lower levels of RHOH. However, as monocytic myeloid cells terminally differentiate into macrophages, RHOH expression is up-regulated. This up-regulation is not apparent in AML where myeloid differentiation is blocked at stages of low RHOH expression. Nevertheless, when the up-regulation of RHOH is forced, then terminal macrophage differentiation is induced and the Cdc42 and Wnt intracellular signalling pathways are repressed. These results indicate that RHOH induction is a driver of terminal differentiation and might represent a means of effecting AML differentiation therapy. The potential of this therapeutic strategy is supported by forced up-regulation of RHOH reducing the ability of AML cells to produce tumours in vivo.
RESUMO
Despite constant progress in prognostic risk stratification, children with acute myeloid leukemia (AML) still relapse. Treatment failure and subsequent relapse have been attributed to acute myeloid leukemia-initiating cells (LSC), which harbor stem cell properties and are inherently chemoresistant. Although pediatric and adult AML represent two genetically very distinct diseases, we reasoned that common LSC gene expression programs are shared and consequently, the highly prognostic LSC17 signature score recently developed in adults may also be of clinical interest in childhood AML. Here, we demonstrated prognostic relevance of the LSC17 score in pediatric non-core-binding factor AML using Nanostring technology (ELAM02) and RNA-seq data from the NCI (TARGET-AML). AML were stratified by LSC17 quartile groups (lowest 25%, intermediate 50% and highest 25%) and children with low LSC17 score had significantly better event-free survival (EFS: HR = 3.35 (95%CI = 1.64-6.82), P < 0.001) and overall survival (OS: HR = 3.51 (95%CI = 1.38-8.92), P = 0.008) compared with patients with high LSC17 scores. More importantly, the high LSC17 score was an independent negative EFS and OS prognosticator determined by multivariate Cox model analysis (EFS: HR = 3.42 (95% CI = 1.63-7.16), P = 0.001; OS HR = 3.02 (95%CI = 1.16-7.85), P = 0.026). In conclusion, we have demonstrated the broad applicability of the LSC17 score in the clinical management of AML by extending its prognostic relevance to pediatric AML.
Assuntos
Biomarcadores Tumorais/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/patologia , Transcriptoma , Adolescente , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Seguimentos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Lactente , Recém-Nascido , Leucemia Mieloide Aguda/classificação , Masculino , Células-Tronco Neoplásicas/metabolismo , Prognóstico , Taxa de SobrevidaRESUMO
Besides the detection of somatic receptor tyrosine kinases (RTK) mutations in tumor samples, the current challenge is to interpret their biological relevance to give patients effective targeted treatment. By high-throughput sequencing of the 58 RTK exons of healthy tissues, colorectal tumors, and hepatic metastases from 30 patients, 38 different somatic mutations in RTKs were identified. The mutations in the kinase domains and present in both tumors and metastases were reconstituted to perform an unbiased functional study. Among eight variants found in seven RTKs (EPHA4-Met726Ile, EPHB2-Val621Ile, ERBB4-Thr731Met, FGFR4-Ala585Thr, VEGFR3-Leu1014Phe, KIT-Pro875Leu, TRKB-Leu584Val, and NTRK2-Lys618Thr), none displayed significantly increased tyrosine kinase activity. Consistently, none of them induced transformation of NIH3T3 fibroblasts. On the contrary, two RTK variants (FGFR4-Ala585Thr and FLT4-Leu1014Phe) caused drastic inhibition of their kinase activity. These findings indicate that these RTK variants are not suitable targets and highlight the importance of functional studies to validate RTK mutations as potential therapeutic targets.