RESUMO
Melanoma is a tumour of transformed melanocytes, which are originally derived from the embryonic neural crest. It is unknown to what extent the programs that regulate neural crest development interact with mutations in the BRAF oncogene, which is the most commonly mutated gene in human melanoma. We have used zebrafish embryos to identify the initiating transcriptional events that occur on activation of human BRAF(V600E) (which encodes an amino acid substitution mutant of BRAF) in the neural crest lineage. Zebrafish embryos that are transgenic for mitfa:BRAF(V600E) and lack p53 (also known as tp53) have a gene signature that is enriched for markers of multipotent neural crest cells, and neural crest progenitors from these embryos fail to terminally differentiate. To determine whether these early transcriptional events are important for melanoma pathogenesis, we performed a chemical genetic screen to identify small-molecule suppressors of the neural crest lineage, which were then tested for their effects on melanoma. One class of compound, inhibitors of dihydroorotate dehydrogenase (DHODH), for example leflunomide, led to an almost complete abrogation of neural crest development in zebrafish and to a reduction in the self-renewal of mammalian neural crest stem cells. Leflunomide exerts these effects by inhibiting the transcriptional elongation of genes that are required for neural crest development and melanoma growth. When used alone or in combination with a specific inhibitor of the BRAF(V600E) oncogene, DHODH inhibition led to a marked decrease in melanoma growth both in vitro and in mouse xenograft studies. Taken together, these studies highlight developmental pathways in neural crest cells that have a direct bearing on melanoma formation.
Assuntos
Melanoma/genética , Melanoma/patologia , Crista Neural/enzimologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Transcrição Gênica , Substituição de Aminoácidos , Animais , Animais Geneticamente Modificados , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Linhagem da Célula/efeitos dos fármacos , Di-Hidro-Orotato Desidrogenase , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Genes p53/genética , Humanos , Isoxazóis/farmacologia , Isoxazóis/uso terapêutico , Leflunomida , Melanoma/tratamento farmacológico , Melanoma/enzimologia , Camundongos , Crista Neural/efeitos dos fármacos , Crista Neural/metabolismo , Crista Neural/patologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Ratos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/patologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
Melanoma skin cancer is a potentially deadly disease in humans and has remained extremely difficult to treat once it has metastasized. In just the last 10 years, a number of models of melanoma have been developed in the zebrafish that are biologically faithful to the human disease and have already yielded important insights into the fundamental biology of melanoma and offered new potential avenues for treatment. With the diversity and breadth of the molecular genetic tools available in the zebrafish, these melanoma models will continue to be refined and expanded upon to keep pace with the rapidly evolving field of melanoma biology.
Assuntos
Modelos Animais de Doenças , Melanoma/patologia , Animais , Melanoma/genética , Peixe-ZebraRESUMO
Molecular genetics approaches in zebrafish research are hampered by the lack of a ubiquitous transgene driver element that is active at all developmental stages. Here, we report the isolation and characterization of the zebrafish ubiquitin (ubi) promoter, which drives constitutive transgene expression during all developmental stages and analyzed adult organs. Notably, ubi expresses in all blood cell lineages, and we demonstrate the application of ubi-driven fluorophore transgenics in hematopoietic transplantation experiments to assess true multilineage potential of engrafted cells. We further generated transgenic zebrafish that express ubiquitous 4-hydroxytamoxifen-controlled Cre recombinase activity from a ubi:cre(ERt2) transgene, as well as ubi:loxP-EGFP-loxP-mCherry (ubi:Switch) transgenics and show their use as a constitutive fluorescent lineage tracing reagent. The ubi promoter and the transgenic lines presented here thus provide a broad resource and important advancement for transgenic applications in zebrafish.
Assuntos
Integrases/metabolismo , Regiões Promotoras Genéticas/genética , Transgenes/genética , Ubiquitina/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Ativação Enzimática/efeitos dos fármacos , Integrases/genética , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologiaRESUMO
Progressive multifocal leukoencephalopathy (PML) induced by JC virus (JCV) is a risk for natalizumab-treated multiple sclerosis (MS) patients. Here we characterize the JCV-specific T cell responses in healthy donors and natalizumab-treated MS patients to reveal functional differences that may account for the development of natalizumab-associated PML. CD4 and CD8 T cell responses specific for all JCV proteins were readily identified in MS patients and healthy volunteers. The magnitude and quality of responses to JCV and cytomegalovirus (CMV) did not change from baseline through several months of natalizumab therapy. However, the frequency of T cells producing IL-10 upon mitogenic stimulation transiently increased after the first dose. In addition, MS patients with natalizumab-associated PML were distinguished from all other subjects in that they either had no detectable JCV-specific T cell response or had JCV-specific CD4 T cell responses uniquely dominated by IL-10 production. Additionally, IL-10 levels were higher in the CSF of individuals with recently diagnosed PML. Thus, natalizumab-treated MS patients with PML have absent or aberrant JCV-specific T cell responses compared with non-PML patients, and changes in T cell-mediated control of JCV replication may be a risk factor for developing PML. Our data suggest further approaches to improved monitoring, treatment and prevention of PML in natalizumab-treated patients.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Imunidade Celular/efeitos dos fármacos , Vírus JC/imunologia , Leucoencefalopatia Multifocal Progressiva/imunologia , Esclerose Múltipla/imunologia , Anticorpos Monoclonais Humanizados/efeitos adversos , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Citomegalovirus/imunologia , Infecções por Citomegalovirus/tratamento farmacológico , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/patologia , Feminino , Humanos , Interleucina-10/imunologia , Leucoencefalopatia Multifocal Progressiva/tratamento farmacológico , Leucoencefalopatia Multifocal Progressiva/patologia , Leucoencefalopatia Multifocal Progressiva/virologia , Masculino , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/patologia , Esclerose Múltipla/virologia , Natalizumab , Fatores de RiscoRESUMO
Intellectual disability is a neurodevelopmental disorder that affects 2-3% of the general population. Syndromic forms of intellectual disability frequently have a genetic basis and are often accompanied by additional developmental anomalies. Pathogenic variants in components of TATA-binding protein associated factors (TAFs) have recently been identified in a subset of patients with intellectual disability, craniofacial hypoplasia, and congenital heart disease. This syndrome has been termed as a TAFopathy and includes mutations in TATA binding protein (TBP), TAF1, TAF2, and TAF6. The underlying mechanism by which TAFopathies give rise to neurodevelopmental, craniofacial, and cardiac abnormalities remains to be defined. Through a forward genetic screen in zebrafish, we have recovered a recessive mutant phenotype characterized by craniofacial hypoplasia, ventricular hypoplasia, heart failure at 96â h post-fertilization and lethality, and show it is caused by a nonsense mutation in taf5. CRISPR/CAS9 mediated gene editing revealed that these defects where phenocopied by mutations in taf1 and taf5. Mechanistically, taf5-/- zebrafish displayed misregulation in metabolic gene expression and metabolism as evidenced by RNA sequencing, respiration assays, and metabolite studies. Collectively, these findings suggest that the TAF complex may contribute to neurologic, craniofacial, and cardiac development through regulation of metabolism.
Assuntos
Anormalidades Craniofaciais , Fatores Associados à Proteína de Ligação a TATA , Proteínas de Peixe-Zebra , Animais , Anormalidades Craniofaciais/genética , Coração , Deficiência Intelectual , Mutação , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Small nucleotide variants in non-coding regions of the genome can alter transcriptional regulation, leading to changes in gene expression which can activate oncogenic gene regulatory networks. Melanoma is heavily burdened by non-coding variants, representing over 99% of total genetic variation, including the well-characterized TERT promoter mutation. However, the compendium of regulatory non-coding variants is likely still functionally under-characterized. We developed a pipeline to identify hotspots, i.e. recurrently mutated regions, in melanoma containing putatively functional non-coding somatic variants that are located within predicted melanoma-specific regulatory regions. We identified hundreds of statistically significant hotspots, including the hotspot containing the TERT promoter variants, and focused on a hotspot in the promoter of CDC20. We found that variants in the promoter of CDC20, which putatively disrupt an ETS motif, lead to lower transcriptional activity in reporter assays. Using CRISPR/Cas9, we generated an indel in the CDC20 promoter in human A375 melanoma cell lines and observed decreased expression of CDC20, changes in migration capabilities, increased growth of xenografts, and an altered transcriptional state previously associated with a more proliferative and less migratory state. Overall, our analysis prioritized several recurrent functional non-coding variants that, through downregulation of CDC20, led to perturbation of key melanoma phenotypes.
Assuntos
Melanoma , Humanos , Mutação , Melanoma/genética , Melanoma/metabolismo , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Genoma , Proteínas Cdc20/genética , Proteínas Cdc20/metabolismoRESUMO
Transcriptional and epigenetic characterization of melanocytes and melanoma cells isolated from their in vivo context promises to unveil key differences between these developmentally related normal and cancer cell populations. We therefore engineered an enhanced Danio rerio (zebrafish) melanoma model with fluorescently labeled melanocytes to allow for isolation of normal (wild type) and premalignant (BRAFV600E-mutant) populations for comparison to fully transformed BRAFV600E-mutant, p53 loss-of-function melanoma cells. Using fluorescence-activated cell sorting to isolate these populations, we performed high-quality RNA- and ATAC-seq on sorted zebrafish melanocytes vs. melanoma cells, which we provide as a resource here. Melanocytes had consistent transcriptional and accessibility profiles, as did melanoma cells. Comparing melanocytes and melanoma, we note 4128 differentially expressed genes and 56,936 differentially accessible regions with overall gene expression profiles analogous to human melanocytes and the pigmentation melanoma subtype. Combining the RNA- and ATAC-seq data surprisingly revealed that increased chromatin accessibility did not always correspond with increased gene expression, suggesting that though there is widespread dysregulation in chromatin accessibility in melanoma, there is a potentially more refined gene expression program driving cancerous melanoma. These data serve as a resource to identify candidate regulators of the normal vs. diseased states in a genetically controlled in vivo context.
Assuntos
Melanoma , Peixe-Zebra , Animais , Cromatina/genética , Cromatina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Humanos , Melanócitos/metabolismo , Melanócitos/patologia , Melanoma/genética , Melanoma/patologia , Peixe-Zebra/genéticaRESUMO
The role of a neural crest developmental transcriptional program, which critically involves Sox10 upregulation, is a key conserved aspect of melanoma initiation in both humans and zebrafish, yet transcriptional regulation of sox10 expression is incompletely understood. Here we used ATAC-Seq analysis of multiple zebrafish melanoma tumors to identify recurrently open chromatin domains as putative melanoma-specific sox10 enhancers. Screening in vivo with EGFP reporter constructs revealed 9 of 11 putative sox10 enhancers with embryonic activity in zebrafish. Focusing on the most active enhancer region in melanoma, we identified a region 23 kilobases upstream of sox10, termed peak5, that drives EGFP reporter expression in a subset of neural crest cells, Kolmer-Agduhr neurons, and early melanoma patches and tumors with high specificity. A ~200 base pair region, conserved in Cyprinidae, within peak5 is required for transgenic reporter activity in neural crest and melanoma. This region contains dimeric SoxE/Sox10 dimeric binding sites essential for peak5 neural crest and melanoma activity. We show that deletion of the endogenous peak5 conserved genomic locus decreases embryonic sox10 expression and disrupts adult stripe patterning in our melanoma model background. Our work demonstrates the power of linking developmental and cancer models to better understand neural crest identity in melanoma.
Assuntos
Melanoma/genética , Crista Neural/embriologia , Fatores de Transcrição SOXE/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Modelos Animais de Doenças , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Crista Neural/metabolismoRESUMO
BACKGROUND: Zebrafish pigment cell differentiation provides an attractive model for studying cell fate progression as a neural crest progenitor engenders diverse cell types, including two morphologically distinct pigment cells: black melanophores and reflective iridophores. Nontrivial classical genetic and transcriptomic approaches have revealed essential molecular mechanisms and gene regulatory circuits that drive neural crest-derived cell fate decisions. However, how the epigenetic landscape contributes to pigment cell differentiation, especially in the context of iridophore cell fate, is poorly understood. RESULTS: We chart the global changes in the epigenetic landscape, including DNA methylation and chromatin accessibility, during neural crest differentiation into melanophores and iridophores to identify epigenetic determinants shaping cell type-specific gene expression. Motif enrichment in the epigenetically dynamic regions reveals putative transcription factors that might be responsible for driving pigment cell identity. Through this effort, in the relatively uncharacterized iridophores, we validate alx4a as a necessary and sufficient transcription factor for iridophore differentiation and present evidence on alx4a's potential regulatory role in guanine synthesis pathway. CONCLUSIONS: Pigment cell fate is marked by substantial DNA demethylation events coupled with dynamic chromatin accessibility to potentiate gene regulation through cis-regulatory control. Here, we provide a multi-omic resource for neural crest differentiation into melanophores and iridophores. This work led to the discovery and validation of iridophore-specific alx4a transcription factor.
Assuntos
Diferenciação Celular/genética , Cromatóforos/metabolismo , Epigênese Genética , Melanóforos/metabolismo , Peixe-Zebra/genética , Animais , Cromatina/metabolismo , Ilhas de CpG , Metilação de DNA , Redes Reguladoras de Genes , Crista Neural/citologia , Crista Neural/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Transcrição Gênica , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/fisiologiaRESUMO
The cell-intrinsic nature of tumor metabolism has become increasingly well characterized. The impact that tumors have on systemic metabolism, however, has received less attention. Here, we used adult zebrafish harboring BRAFV600E-driven melanoma to study the effect of cancer on distant tissues. By applying metabolomics and isotope tracing, we found that melanoma consume ~15 times more glucose than other tissues measured. Despite this burden, circulating glucose levels were maintained in disease animals by a tumor-liver alanine cycle. Excretion of glucose-derived alanine from tumors provided a source of carbon for hepatic gluconeogenesis and allowed tumors to remove excess nitrogen from branched-chain amino acid catabolism, which we found to be activated in zebrafish and human melanoma. Pharmacological inhibition of the tumor-liver alanine cycle in zebrafish reduced tumor burden. Our findings underscore the significance of metabolic crosstalk between tumors and distant tissues and establish the adult zebrafish as an attractive model to study such processes.
Assuntos
Alanina/metabolismo , Fígado/metabolismo , Melanoma/metabolismo , Envelhecimento/patologia , Animais , Animais Geneticamente Modificados , Rastreamento de Células/métodos , Modelos Animais de Doenças , Gluconeogênese/genética , Humanos , Marcação por Isótopo/métodos , Fígado/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Melanoma/genética , Melanoma/patologia , Metabolômica , Peixe-ZebraRESUMO
Melanomas driven by loss of the NF1 tumor suppressor have a high risk of treatment failure and effective therapies have not been developed. Here we show that loss-of-function mutations of nf1 and pten result in aggressive melanomas in zebrafish, representing the first animal model of NF1-mutant melanomas harboring PTEN loss. MEK or PI3K inhibitors show little activity when given alone due to cross-talk between the pathways, and high toxicity when given together. The mTOR inhibitors, sirolimus, everolimus, and temsirolimus, were the most active single agents tested, potently induced tumor-suppressive autophagy, but not apoptosis. Because addition of the BCL2 inhibitor venetoclax resulted in compensatory upregulation of MCL1, we established a three-drug combination composed of sirolimus, venetoclax, and the MCL1 inhibitor S63845. This well-tolerated drug combination potently and synergistically induces apoptosis in both zebrafish and human NF1/PTEN-deficient melanoma cells, providing preclinical evidence justifying an early-stage clinical trial in patients with NF1/PTEN-deficient melanoma.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Inibidores de MTOR/administração & dosagem , Melanoma/tratamento farmacológico , Neurofibromina 1/genética , PTEN Fosfo-Hidrolase/genética , Pirimidinas/administração & dosagem , Sulfonamidas/administração & dosagem , Tiofenos/administração & dosagem , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Everolimo/administração & dosagem , Everolimo/farmacologia , Humanos , Mutação com Perda de Função , Inibidores de MTOR/farmacologia , Melanoma/genética , Melanoma/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Pirimidinas/farmacologia , Sirolimo/administração & dosagem , Sirolimo/análogos & derivados , Sirolimo/farmacologia , Sulfonamidas/farmacologia , Tiofenos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-ZebraRESUMO
Recent genomic and scRNA-seq analyses of melanoma demonstrated a lack of recurrent genetic drivers of metastasis, while identifying common transcriptional states correlating with invasion or drug resistance. To test whether transcriptional adaptation can drive melanoma progression, we made use of a zebrafish mitfa:BRAFV600E;tp53-/- model, in which malignant progression is characterized by minimal genetic evolution. We undertook an overexpression-screen of 80 epigenetic/transcriptional regulators and found neural crest-mesenchyme developmental regulator SATB2 to accelerate aggressive melanoma development. Its overexpression induces invadopodia formation and invasion in zebrafish tumors and human melanoma cell lines. SATB2 binds and activates neural crest-regulators, including pdgfab and snai2. The transcriptional program induced by SATB2 overlaps with known MITFlowAXLhigh and AQP1+NGFR1high drug-resistant states and functionally drives enhanced tumor propagation and resistance to Vemurafenib in vivo. In summary, we show that melanoma transcriptional rewiring by SATB2 to a neural crest mesenchyme-like program can drive invasion and drug resistance in autochthonous tumors.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Melanoma/genética , Invasividade Neoplásica/genética , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Ligação à Região de Interação com a Matriz/genética , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Crista Neural/citologia , Fatores de Transcrição/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
There is a lack of appropriate melanoma models that can be used to evaluate the efficacy of novel therapeutic modalities. Here, we discuss the current state of the art of melanoma models including genetically engineered mouse, patient-derived xenograft, zebrafish, and ex vivo and in vitro models. We also identify five major challenges that can be addressed using such models, including metastasis and tumor dormancy, drug resistance, the melanoma immune response, and the impact of aging and environmental exposures on melanoma progression and drug resistance. Additionally, we discuss the opportunity for building models for rare subtypes of melanomas, which represent an unmet critical need. Finally, we identify key recommendations for melanoma models that may improve accuracy of preclinical testing and predict efficacy in clinical trials, to help usher in the next generation of melanoma therapies.
Assuntos
Modelos Animais de Doenças , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Microambiente Tumoral/imunologia , Animais , Humanos , Imunidade/imunologia , Imunoterapia/métodos , Melanoma/patologia , Neoplasias Cutâneas/patologiaRESUMO
Despite sharing oncogenetic mutations, only a small number of cells within a given tissue will undergo malignant transformation. Biochemical and physical factors responsible for this cancer-initiation process are not well understood. Here we study biophysical differences of pre-melanoma and melanoma cells in a BRAFV600E/P53 zebrafish model. The AFM indentation technique was used to study the cancer-initiating cells while the surrounding melanocytes were the control. We observed a statistically significant decrease in the modulus of elasticity (the effective Young's modulus) of cancer-initiating cells compared to the surrounding melanocytes. No significant differences in the pericellular coat surrounding cells were observed. These results contribute to a better understanding of the factors responsible for the initiation of cancer.
Assuntos
Melanoma , Peixe-Zebra , Animais , Transformação Celular Neoplásica , Módulo de Elasticidade , Elasticidade , Melanoma/genética , Microscopia de Força Atômica , Peixe-Zebra/genéticaRESUMO
Importance: Recognizing the presenting and immunopathological features of Kelch-like protein-11 immunoglobulin G seropositive (KLHL11 IgG+) patients may aid in early diagnosis and management. Objective: To describe expanding neurologic phenotype, cancer associations, outcomes, and immunopathologic features of KLHL11 encephalitis. Design, Setting, and Participants: This retrospective tertiary care center study, conducted from October 15, 1998, to November 1, 2019, prospectively identified 31 KLHL11 IgG+ cases in the neuroimmunology laboratory. Eight were identified by retrospective testing of patients with rhomboencephalitis (confirmed by tissue-based-immunofluorescence and transfected-cell-based assays). Main Outcomes and Measures: Outcome variables included modified Rankin score and gait aid use. Results: All 39 KLHL11 IgG+ patients were men (median age, 46 years; range, 28-73 years). Initial clinical presentations were ataxia (n = 32; 82%), diplopia (n = 22; 56%), vertigo (n = 21; 54%), hearing loss (n = 15; 39%), tinnitus (n = 14; 36%), dysarthria (n = 11; 28%), and seizures (n = 9; 23%). Atypical neurologic presentations included neuropsychiatric dysfunction, myeloneuropathy, and cervical amyotrophy. Hearing loss or tinnitus preceded other neurologic deficits by 1 to 8 months in 10 patients (26%). Among patients screened for malignancy (n = 36), testicular germ-cell tumors (n = 23; 64%) or testicular microlithiasis and fibrosis concerning for regressed germ cell tumor (n = 7; 19%) were found in 83% of the patients (n = 30). In 2 patients, lymph node biopsy diagnosed metastatic lung adenocarcinoma in one and chronic lymphocytic leukemia in the other. Initial brain magnetic resonance imaging revealed T2 hyperintensities in the temporal lobe (n = 12), cerebellum (n = 9), brainstem (n = 3), or diencephalon (n = 3). Among KLHL11 IgG+ patients who underwent HLA class I and class II genotyping (n = 10), most were found to have HLA-DQB1*02:01 (n = 7; 70%) and HLA-DRB1*03:01 (n = 6; 60%) associations. A biopsied gadolinium-enhancing temporal lobe lesion demonstrated T cell-predominant inflammation and nonnecrotizing granulomas. Cerebellar biopsy (patient with chronic ataxia) and 2 autopsied brains demonstrated Purkinje neuronal loss and Bergmann gliosis, supporting early active inflammation and later extensive neuronal loss. Compared with nonautoimmune control peripheral blood mononuclear cells, cluster of differentiation (CD) 8+ and CD4+ T cells were significantly activated when patient peripheral blood mononuclear cells were cultured with KLHL11 protein. Most patients (58%) benefitted from immunotherapy and/or cancer treatment (neurological disability stabilized [n = 10] or improved [n = 9]). Kaplan-Meier curve demonstrated significantly higher probability of wheelchair dependence among patients without detectable testicular cancer. Long-term outcomes in KLHL11-IgG+ patients were similar to Ma2 encephalitis. Conclusions and Relevance: Kelch-like protein-11 IgG is a biomarker of testicular germ-cell tumor and paraneoplastic neurologic syndrome, often refractory to treatment. Described expanded neurologic phenotype and paraclinical findings may aid in its early diagnosis and treatment.
Assuntos
Autoanticorpos/sangue , Proteínas de Transporte/sangue , Encefalite/sangue , Síndromes Paraneoplásicas do Sistema Nervoso/sangue , Fenótipo , Adulto , Idoso , Autoanticorpos/imunologia , Biomarcadores/sangue , Proteínas de Transporte/imunologia , Encefalite/diagnóstico , Encefalite/imunologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes Paraneoplásicas do Sistema Nervoso/diagnóstico , Síndromes Paraneoplásicas do Sistema Nervoso/imunologia , Estudos Retrospectivos , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
In jawed vertebrates (gnathostomes), Hox genes play an important role in patterning head and jaw formation, but mechanisms coupling Hox genes to neural crest (NC) are unknown. Here we use cross-species regulatory comparisons between gnathostomes and lamprey, a jawless extant vertebrate, to investigate conserved ancestral mechanisms regulating Hox2 genes in NC. Gnathostome Hoxa2 and Hoxb2 NC enhancers mediate equivalent NC expression in lamprey and gnathostomes, revealing ancient conservation of Hox upstream regulatory components in NC. In characterizing a lamprey hoxα2 NC/hindbrain enhancer, we identify essential Meis, Pbx, and Hox binding sites that are functionally conserved within Hoxa2/Hoxb2 NC enhancers. This suggests that the lamprey hoxα2 enhancer retains ancestral activity and that Hoxa2/Hoxb2 NC enhancers are ancient paralogues, which diverged in hindbrain and NC activities. This identifies an ancestral mechanism for Hox2 NC regulation involving a Hox-TALE regulatory circuit, potentiated by inputs from Meis and Pbx proteins and Hox auto-/cross-regulatory interactions.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genes Homeobox/fisiologia , Proteínas de Homeodomínio/metabolismo , Crista Neural/embriologia , Vertebrados/genética , Animais , Animais Geneticamente Modificados , Sítios de Ligação/genética , Linhagem Celular , Sequência Conservada/fisiologia , Elementos Facilitadores Genéticos/genética , Proteínas de Homeodomínio/genética , Lampreias , Camundongos , Células-Tronco Embrionárias Murinas , Crista Neural/metabolismo , Alinhamento de Sequência , Vertebrados/embriologia , Peixe-ZebraRESUMO
The manner in which zebrafish are fed may have important impacts on the behavior of disease models. We examined the effect of different feeding regimens on the rate of overt melanoma tumor onset in a p53/BRAF-dependent model, a commonly used read-out in this and many other cancer models. We demonstrate that increased feeding leads to more rapid melanoma onset. The ability to modulate overt tumor onset rates with this regimen indicates additional flexibility to 'tune' the system to more quickly generate tumors for study and to begin to address questions related to cancer metabolism using the zebrafish model.
RESUMO
In this report, we explored the mechanisms underlying keratinocyte-specific and differentiation-specific gene expression in the skin. We have identified five keratinocyte-specific, open chromatin regions that exist within the 6 kb of 5' upstream regulatory sequence known to faithfully recapitulate the strong endogenous keratin 5 (K5) promoter and/or enhancer activity. One of these, DNase I-hypersensitive site (HSs) 4, was unique in that it acted independently to drive abundant and keratinocyte-specific reporter gene activity in culture and in transgenic mice, despite the fact that it was not essential for K5 enhancer activity. We have identified evolutionarily conserved regulatory elements and a number of their associated proteins that bind to this compact and complex enhancer element. The 125-bp 3' half of this element (referred to as 4.2) is by far the smallest known strong enhancer element possessing keratinocyte-specific activity in vivo. Interestingly, its activity is restricted to a subset of progeny of K5-expressing cells located within the sebaceous gland. The other half of HSs 4 (termed 4.1) possesses activity to suppress sebocyte-specific expression and induce expression in the channel (inner root sheath) cells surrounding the hair shaft. Our findings lead us to a view of keratinocyte gene expression which is determined by multiple regulatory modules, many of which contain AP-2 and/or Sp1/Sp3 binding sites for enhancing expression in skin epithelium, but which also harbor one or more unique sites for the binding of factors which determine specificity. Through mixing and matching of these modules, additional levels of specificity are obtained, indicating that both transcriptional repressors and activators govern the specificity.
Assuntos
Elementos Facilitadores Genéticos/genética , Queratinócitos/fisiologia , Animais , Sequência de Bases , Sítios de Ligação , Diferenciação Celular/genética , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Regulação da Expressão Gênica , Cabelo/fisiologia , Humanos , Queratina-15 , Queratina-5 , Queratinócitos/citologia , Queratinas/genética , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Glândulas Sebáceas/metabolismo , Homologia de Sequência do Ácido Nucleico , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp2 , Fator de Transcrição AP-2 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The establishment of in vitro cultures of zebrafish cancer cells has expanded the potential of zebrafish as a disease model. However, the lack of effective methods for gene delivery and genetic manipulation has limited the experimental applications of these cultures. To overcome this barrier, we tested and optimized vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral and retroviral vector transduction protocols. We show that lentivirus successfully and efficiently transduced zebrafish melanoma cell lines in vitro, allowing antibiotic selection, fluorescence-based sorting, and in vivo allotransplantation. In addition, injection of concentrated lentiviral particles into embryos and tumors established the feasibility of in vivo gene delivery.
Assuntos
Vetores Genéticos/administração & dosagem , Lentivirus/genética , Melanoma/genética , Retroviridae/genética , Transdução Genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Melanoma/patologia , Glicoproteínas de Membrana/genética , Células Tumorais Cultivadas , Proteínas do Envelope Viral/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Cancer is an evolutionary disease, and there is increasing interest in applying tools from evolutionary biology to understand cancer progression. Restriction-site associated DNA sequencing (RADseq) was developed for the field of evolutionary genetics to study adaptation and identify evolutionary relationships among populations. Here we apply RADseq to study tumor evolution, which allows for unbiased sampling of any desired frequency of the genome, overcoming the selection bias and cost limitations inherent to exome or whole-genome sequencing. We apply RADseq to both human pancreatic cancer and zebrafish melanoma samples. Using either a low-frequency (SbfI, 0.4% of the genome) or high-frequency (NsiI, 6-9% of the genome) cutter, we successfully identify single nucleotide substitutions and copy number alterations in tumors, which can be augmented by performing RADseq on sublineages within the tumor. We are able to infer phylogenetic relationships between primary tumors and metastases. These same methods can be used to identify somatic mosaicism in seemingly normal, non-cancerous tissues. Evolutionary studies of cancer that focus on rates of tumor evolution and evolutionary relationships among tumor lineages will benefit from the flexibility and efficiency of restriction-site associated DNA sequencing.