RESUMO
Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism activated in ~10-15% of cancers, characterized by telomeric damage. Telomeric damage-induced long non-coding RNAs (dilncRNAs) are transcribed at dysfunctional telomeres and contribute to telomeric DNA damage response (DDR) activation and repair. Here we observed that telomeric dilncRNAs are preferentially elevated in ALT cells. Inhibition of C-rich (teloC) dilncRNAs with antisense oligonucleotides leads to DNA replication stress responses, increased genomic instability, and apoptosis induction selectively in ALT cells. Cell death is dependent on DNA replication and is increased by DNA replication stress. Mechanistically, teloC dilncRNA inhibition reduces RAD51 and 53BP1 recruitment to telomeres, boosts the engagement of BIR machinery, and increases C-circles and telomeric sister chromatid exchanges, without increasing telomeric non-S phase synthesis. These results indicate that teloC dilncRNA is necessary for a coordinated recruitment of DDR factors to ALT telomeres and it is essential for ALT cancer cells survival.
Assuntos
Telomerase , Homeostase do Telômero , Homeostase do Telômero/genética , Replicação do DNA , RNA , Sobrevivência Celular/genética , Telômero/genética , Telômero/metabolismo , Telomerase/genética , Telomerase/metabolismoRESUMO
Glioblastoma multiforme (GBM) is the most common and malignant brain tumor in adults. The invasiveness and the rapid progression that characterize GBM negatively impact patients' survival. Temozolomide (TMZ) is currently considered the first-choice chemotherapeutic agent. Unfortunately, over 50% of patients with GBM do not respond to TMZ treatment, and the mutation-prone nature of GBM enables the development of resistance mechanisms. Therefore, efforts have been devoted to the dissection of aberrant pathways involved in GBM insurgence and resistance in order to identify new therapeutic targets. Among them, sphingolipid signaling, Hedgehog (Hh) pathway, and the histone deacetylase 6 (HDAC6) activity are frequently dysregulated and may represent key targets to counteract GBM progression. Given the positive correlation between Hh/HDAC6/sphingolipid metabolism in GBM, we decided to perform a dual pharmacological inhibition of Hh and HDAC6 through cyclopamine and tubastatin A, respectively, in a human GMB cell line and zebrafish embryos. The combined administration of these compounds elicited a more significant reduction of GMB cell viability than did single treatments in vitro and in cells orthotopically transplanted in the zebrafish hindbrain ventricle. We demonstrated, for the first time, that the inhibition of these pathways induces lysosomal stress which results in an impaired fusion of lysosomes with autophagosomes and a block of sphingolipid degradation in GBM cell lines. This condition, which we also recapitulated in zebrafish embryos, suggests an impairment of lysosome-dependent processes involving autophagy and sphingolipid homeostasis and might be instrumental in the reduction of GBM progression.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Adulto , Animais , Humanos , Glioblastoma/metabolismo , Desacetilase 6 de Histona , Peixe-Zebra , Sobrevivência Celular , Proteínas Hedgehog , Temozolomida/farmacologia , Lisossomos/metabolismo , Esfingolipídeos , Linhagem Celular Tumoral , Neoplasias Encefálicas/metabolismo , Resistencia a Medicamentos AntineoplásicosRESUMO
Base Editors are emerging as an innovative technology to introduce point mutations in complex genomes. So far, the requirement of an NGG Protospacer Adjacent Motif (PAM) at a suitable position often limits the base editing possibility to model human pathological mutations in animals. Here we show that, using the CBE4max-SpRY variant recognizing nearly all PAM sequences, we could introduce point mutations for the first time in an animal model with high efficiency, thus drastically increasing the base editing possibilities. With this near PAM-less base editor we could simultaneously mutate several genes and we developed a co-selection method to identify the most edited embryos based on a simple visual screening. Finally, we apply our method to create a zebrafish model for melanoma predisposition based on the simultaneous base editing of multiple genes. Altogether, our results considerably expand the Base Editor application to introduce human disease-causing mutations in zebrafish.
Assuntos
Proteína 9 Associada à CRISPR , Edição de Genes , Animais , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Genoma/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Extracellular vesicles (EVs) are membranous particles released by all cell types. Their role as functional carrier of bioactive molecules is boosted by cells that actively secrete them in biological fluids or in the intercellular space (interstitial EVs, iEVs). Here we have optimised a method for the isolation and characterization of zebrafish iEVs from whole melanoma tissues. Zebrafish melanoma iEVs are around 140 nm in diameter, as determined by nanoparticle tracking and transmission electron microscopy (TEM) analysis. Western blot analysis shows enrichment for CD63 and Alix in the iEV fraction, but not in melanoma cell lysates. Super resolution and confocal microscopy reveal that purified zebrafish iEVs are green fluorescent protein positive (GFP+), indicating that they integrate the oncogene GFP-HRASV12G used to induce melanoma in this model within their vesicular membrane or luminal content. Analysis of RNA-Seq data found 118 non-coding (nc)RNAs differentially distributed between zebrafish melanoma and their iEVs, with only 17 of them being selectively enriched in iEVs. Among these, the RNA components of RNAses P and MRP, which process ribosomal RNA precursors, mitochondrial RNAs, and some mRNAs, were enriched in zebrafish and human melanoma EVs, but not in iEVs extracted from brain tumours. We found that melanoma iEVs induce an inflammatory response when injected in larvae, with increased expression of interferon responsive genes, and this effect is reproduced by MRP- or P-RNAs injected into circulation. This suggests that zebrafish melanoma iEVs are a source of MRP- and P-RNAs that can trigger inflammation in cells of the innate immune system.
Assuntos
Vesículas Extracelulares , Melanoma , Animais , Vesículas Extracelulares/metabolismo , Inflamação/genética , Inflamação/metabolismo , Melanoma/genética , Melanoma/metabolismo , RNA não Traduzido/metabolismo , Peixe-Zebra/genéticaRESUMO
While zebrafish is emerging as a new model system to study human diseases, an efficient methodology to generate precise point mutations at high efficiency is still lacking. Here we show that base editors can generate C-to-T point mutations with high efficiencies without other unwanted on-target mutations. In addition, we established a new editor variant recognizing an NAA protospacer adjacent motif, expanding the base editing possibilities in zebrafish. Using these approaches, we first generated a base change in the ctnnb1 gene, mimicking oncogenic an mutation of the human gene known to result in constitutive activation of endogenous Wnt signaling. Additionally, we precisely targeted several cancer-associated genes including cbl. With this last target, we created a new zebrafish dwarfism model. Together our findings expand the potential of zebrafish as a model system allowing new approaches for the endogenous modulation of cell signaling pathways and the generation of precise models of human genetic disease-associated mutations.
Assuntos
Oncogenes , Mutação Puntual , Transdução de Sinais , Proteínas de Peixe-Zebra/genética , beta Catenina/genética , Animais , Modelos Animais de Doenças , Edição de Genes , Humanos , Mutação , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo , beta Catenina/metabolismoRESUMO
Zebrafish (Danio rerio) has proven to be a versatile and reliable in vivo experimental model to study human hematopoiesis and hematological malignancies. As vertebrates, zebrafish has significant anatomical and biological similarities to humans, including the hematopoietic system. The powerful genome editing and genome-wide forward genetic screening tools have generated models that recapitulate human malignant hematopoietic pathologies in zebrafish and unravel cellular mechanisms involved in these diseases. Moreover, the use of zebrafish models in large-scale chemical screens has allowed the identification of new molecular targets and the design of alternative therapies. In this review we summarize the recent achievements in hematological research that highlight the power of the zebrafish model for discovery of new therapeutic molecules. We believe that the model is ready to give an immediate translational impact into the clinic.
Assuntos
Modelos Animais de Doenças , Hematologia/métodos , Hematologia/tendências , Pesquisa Translacional Biomédica , Peixe-Zebra/fisiologia , Animais , Descoberta de Drogas/métodos , Ensaios de Seleção de Medicamentos Antitumorais , Neoplasias Hematológicas/patologia , Hematopoese , Humanos , Pesquisa Translacional Biomédica/métodos , Pesquisa Translacional Biomédica/tendênciasRESUMO
The H2.0-like homeobox transcription factor (HLX) regulates hematopoietic differentiation and is overexpressed in Acute Myeloid Leukemia (AML), but the mechanisms underlying these functions remain unclear. We demonstrate here that HLX overexpression leads to a myeloid differentiation block both in zebrafish and human hematopoietic stem and progenitor cells (HSPCs). We show that HLX overexpression leads to downregulation of genes encoding electron transport chain (ETC) components and upregulation of PPARδ gene expression in zebrafish and human HSPCs. HLX overexpression also results in AMPK activation. Pharmacological modulation of PPARδ signaling relieves the HLX-induced myeloid differentiation block and rescues HSPC loss upon HLX knockdown but it has no effect on AML cell lines. In contrast, AMPK inhibition results in reduced viability of AML cell lines, but minimally affects myeloid progenitors. This newly described role of HLX in regulating the metabolic state of hematopoietic cells may have important therapeutic implications.
Assuntos
Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/fisiologia , Leucemia Mieloide Aguda/metabolismo , Fatores de Transcrição/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Autofagia , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Regulação Leucêmica da Expressão Gênica , Hematopoese , Proteínas de Homeodomínio/genética , Humanos , Células K562 , Leucemia Mieloide Aguda/genética , Potencial da Membrana Mitocondrial , PPAR gama/metabolismo , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Clostridium perfringens toxin TpeL belongs to the family of large clostridial glycosylating toxins. The toxin causes N-acetylglucosaminylation of Ras proteins at threonine35 thereby inactivating the small GTPases. Here, we show that all main types of oncogenic Ras proteins (H-Ras, K-Ras and N-Ras) are modified by the toxin in vitro and in vivo. Toxin-catalyzed modification of Ras was accompanied by inhibition of the MAP kinase pathway. Importantly, TpeL inhibited the paradoxical activation of the MAP kinase pathway induced by the BRAF inhibitor Vemurafenib in the human melanoma cell line SBCL2. The toxin also blocked Ras signaling in a zebrafish embryo model expressing oncogenic H-RasG12V, resulting in a reduction of melanocyte number. By using the binding and translocation component of anthrax toxin (protective antigen), the glucosyltransferase domain of TpeL was effectively introduced into target cells that were not sensitive to native TpeL toxin. To reach a higher specificity towards cancer cells, a chimeric TpeL toxin was engineered that possessed the knob region of adenovirus serotype 35 fiber, which interacts with CD46 of target cells frequently overexpressed in cancer cells. The chimeric TpeL fusion toxin efficiently inhibited Ras and MAP kinases in human pancreatic cancer Capan-2 cells, which were insensitive to the wild-type toxin. The data reveal that TpeL and TpeL-related immunotoxins provide a new toolset as Ras-inactivating agents.
RESUMO
It is now clear that microglia and macrophages are present in brain tumors, but whether or how they affect initiation and development of tumors is not known. Exploiting the advantages of the zebrafish (Danio rerio) model, we showed that macrophages and microglia respond immediately upon oncogene activation in the brain. Overexpression of human AKT1 within neural cells of larval zebrafish led to a significant increase in the macrophage and microglia populations. By using a combination of transgenic and mutant zebrafish lines, we showed that this increase was caused by the infiltration of peripheral macrophages into the brain mediated via Sdf1b-Cxcr4b signaling. Intriguingly, confocal live imaging reveals highly dynamic interactions between macrophages/microglia and pre-neoplastic cells, which do not result in phagocytosis of pre-neoplastic cells. Finally, depletion of macrophages and microglia resulted in a significant reduction of oncogenic cell proliferation. Thus, macrophages and microglia show tumor promoting functions already during the earliest stages of the developing tumor microenvironment.
Assuntos
Neoplasias Encefálicas/patologia , Movimento Celular , Macrófagos/fisiologia , Células-Tronco Neoplásicas/fisiologia , Receptores CXCR4/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Quimiocina CXCL12/metabolismo , Modelos Animais de Doenças , Humanos , Neuroglia/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Peixe-ZebraRESUMO
Ras genes are among the most commonly mutated genes in human cancer; yet our understanding of their oncogenic activity at the molecular mechanistic level is incomplete. To identify downstream events that mediate ras-induced cellular transformation in vivo, we analyzed global microRNA expression in three different models of Ras-induction and tumor formation in zebrafish. Six microRNAs were found increased in Ras-induced melanoma, glioma and in an inducible model of ubiquitous Ras expression. The upregulation of the microRNAs depended on the activation of the ERK and AKT pathways and to a lesser extent, on mTOR signaling. Two Ras-induced microRNAs (miR-146a and 193a) target Jmjd6, inducing downregulation of its mRNA and protein levels at the onset of Ras expression during melanoma development. However, at later stages of melanoma progression, jmjd6 levels were found elevated. The dynamic of Jmjd6 levels during progression of melanoma in the zebrafish model suggests that upregulation of the microRNAs targeting Jmjd6 may be part of an anti-cancer response. Indeed, triple transgenic fish engineered to express a microRNA-resistant Jmjd6 from the onset of melanoma have increased tumor burden, higher infiltration of leukocytes and shorter melanoma-free survival. Increased JMJD6 expression is found in several human cancers, including melanoma, suggesting that the up-regulation of Jmjd6 is a critical event in tumor progression. The following link has been created to allow review of record GSE37015: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=jjcrbiuicyyqgpc&acc=GSE37015.
RESUMO
The prolyl isomerase Pin1 plays a key role in the modulation of proline-directed phosphorylation signaling by inducing local conformational changes in phosphorylated protein substrates. Extensive studies showed different roles for Pin1 in physiological processes and pathological conditions such as cancer and neurodegenerative diseases. However, there are still several unanswered questions regarding its biological role. Notably, despite evidences from cultured cells showing that Pin1 expression and activity may be regulated by different mechanisms, little is known on their relevance in vivo. Using Danio rerio (zebrafish) as a vertebrate model organism we showed that pin1 expression is regulated during embryogenesis to achieve specific mRNA and protein distribution patterns. Moreover, we found different subcellular distribution in particular stages and cell types and we extended the study of Pin1 expression to the adult zebrafish brain. The analysis of Pin1 overexpression showed alterations on zebrafish development and the presence of p53-dependent apoptosis. Collectively, our results suggest that specific mechanisms are operated in different cell types to regulate Pin1 function.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Peixe-Zebra/embriologia , Animais , Peptidilprolil Isomerase de Interação com NIMA/genética , Especificidade por SubstratoRESUMO
Somatic mutations activating MAPK and PI3K signalling play a pivotal role in both tumours and brain developmental disorders. We developed a zebrafish model of brain tumours based on somatic expression of oncogenes that activate MAPK and PI3K signalling in neural progenitor cells and found that HRASV12 was the most effective in inducing both heterotopia and invasive tumours. Tumours, but not heterotopias, require persistent activation of phospho (p)-ERK and express a gene signature similar to the mesenchymal glioblastoma subtype, with a strong YAP component. Application of an eight-gene signature to human brain tumours establishes that YAP activation distinguishes between mesenchymal glioblastoma and low grade glioma in a wide The Cancer Genome Atlas (TCGA) sample set including gliomas and glioblastomas (GBMs). This suggests that the activation of YAP might be an important event in brain tumour development, promoting malignant versus benign brain lesions. Indeed, co-expression of dominant-active YAP (YAPS5A) and HRASV12 abolishes the development of heterotopias and leads to the sole development of aggressive tumours. Thus, we have developed a model proving that neurodevelopmental disorders and brain tumours might originate from the same activation of oncogenes through somatic mutations, and established that YAP activation is a hallmark of malignant brain tumours.
Assuntos
Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/patologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transativadores/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Aminoacil-tRNA Sintetases/genética , Animais , Neoplasias Encefálicas/genética , Carcinogênese/genética , Carcinogênese/patologia , Proliferação de Células , Sobrevivência Celular , Células Clonais , Modelos Animais de Doenças , Elementos Facilitadores Genéticos/genética , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Genes ras , Glioblastoma/genética , Glioblastoma/patologia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica , Mesoderma/patologia , Células-Tronco Neurais/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Telencéfalo/patologia , Proteínas de Sinalização YAP , Proteínas de Peixe-Zebra/genéticaRESUMO
Here we describe the conditional zebrafish cancer toolbox, which allows for fine control of the expression of oncogenes or downregulation of tumor suppressors at the spatial and temporal level. Methods such as the Gal4/UAS or the Cre/lox systems paved the way to the development of elegant tumor models, which are now being used to study cancer cell biology, clonal evolution, identification of cancer stem cells and anti-cancer drug screening. Combination of these tools, as well as novel developments such as the promising genome editing system through CRISPR/Cas9 and clever application of light reactive proteins will enable the development of even more sophisticated zebrafish cancer models. Here, we introduce this growing toolbox of conditional transgenic approaches, discuss its current application in zebrafish cancer models and provide an outlook on future perspectives.
Assuntos
Modelos Animais de Doenças , Neoplasias/patologia , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Hormônios/fisiologia , Neoplasias/genética , Neoplasias/fisiopatologia , Optogenética , Peixe-ZebraRESUMO
The importance of microRNAs as key molecular components of cellular processes is now being recognized. Recent reports have shown that microRNAs regulate processes as diverse as protein expression and nuclear functions inside cells and are able to signal extracellularly, delivered via exosomes, to influence cell fate at a distance. The versatility of microRNAs as molecular tools inspires the design of novel strategies to control gene expression, protein stability, DNA repair and chromatin accessibility that may prove very useful for therapeutic approaches due to the extensive manageability of these small molecules. However, we still lack a comprehensive understanding of the microRNA network and its interactions with the other layers of regulatory elements in cellular and extracellular functions. This knowledge may be necessary before we exploit microRNA versatility in therapeutic settings. To identify rules of interactions between microRNAs and other regulatory systems, we begin by reviewing microRNA activities in a single cell type: the melanocyte, from development to disease.
Assuntos
Melanócitos/metabolismo , Melanoma/genética , MicroRNAs/metabolismo , Animais , Redes Reguladoras de Genes , Humanos , MicroRNAs/genética , Modelos Biológicos , Processamento Pós-Transcricional do RNA/genéticaRESUMO
Live imaging of the interactions between oncogene-transformed cells and leukocytes in zebrafish reveals that PGE(2) promotes the survival and proliferation of cancer cells. Non-steroid anti-inflammatory drugs, like aspirin, are the effective inhibitors of PGE(2) production and could be used with other anti-tumor agents in the treatment of cancer.
Assuntos
Transformação Celular Neoplásica/metabolismo , Dinoprostona/metabolismo , Leucócitos/metabolismo , Peixe-Zebra/genética , AnimaisRESUMO
Non-coding RNAs (ncRNAs) are involved in an increasingly recognized number of cellular events. Some ncRNAs are processed by DICER and DROSHA RNases to give rise to small double-stranded RNAs involved in RNA interference (RNAi). The DNA-damage response (DDR) is a signalling pathway that originates from a DNA lesion and arrests cell proliferation3. So far, DICER and DROSHA RNA products have not been reported to control DDR activation. Here we show, in human, mouse and zebrafish, that DICER and DROSHA, but not downstream elements of the RNAi pathway, are necessary to activate the DDR upon exogenous DNA damage and oncogene-induced genotoxic stress, as studied by DDR foci formation and by checkpoint assays. DDR foci are sensitive to RNase A treatment, and DICER- and DROSHA-dependent RNA products are required to restore DDR foci in RNase-A-treated cells. Through RNA deep sequencing and the study of DDR activation at a single inducible DNA double-strand break, we demonstrate that DDR foci formation requires site-specific DICER- and DROSHA-dependent small RNAs, named DDRNAs, which act in a MRE11RAD50NBS1-complex-dependent manner (MRE11 also known as MRE11A; NBS1 also known as NBN). DDRNAs, either chemically synthesized or in vitro generated by DICER cleavage, are sufficient to restore the DDR in RNase-A-treated cells, also in the absence of other cellular RNAs. Our results describe an unanticipated direct role of a novel class of ncRNAs in the control of DDR activation at sites of DNA damage.