RESUMEN
Ewing sarcoma (EwS) is a cancer that arises in the bones and soft tissues, typically driven by the Ewing's sarcoma breakpoint region 1-Friend leukemia virus integration 1 (EWS-FLI) oncogene. Implementation of genetically modified animal models of EwS has proved difficult largely owing to EWS-FLI's high toxicity. The EWS-FLI1FS frameshift variant that circumvents toxicity but is still able to perform key oncogenic functions provided the first study model in Drosophila. However, the quest for Drosophila lines expressing full-length, unmodified EWS-FLI remained open. Here, we show that EWS-FLI1FS's lower toxicity is owed to reduced protein levels caused by its frameshifted C-terminal peptide, and report new strategies through which we have generated Drosophila lines that express full-length, unmodified EWS-FLI. Using these lines, we have found that the upregulation of transcription from GGAA-microsatellites (GGAAµSats) presents a positive linear correlation within a wide range of EWS-FLI protein concentrations. In contrast, rather counterintuitively, GGAAµSats-independent transcriptomic dysregulation presents relatively minor differences across the same range, suggesting that GGAAµSat-dependent and -independent transcriptional upregulation present different kinetics of response with regards to changing EWS-FLI protein concentration. Our results underpin the functional relevance of varying EWS-FLI expression levels and provide experimental tools to investigate, in Drosophila, the effect of the EWS-FLI 'high' and 'low' states that have been reported and are suspected to be important for EwS in humans.
Asunto(s)
Proteínas de Fusión Oncogénica , Proteína Proto-Oncogénica c-fli-1 , Proteína EWS de Unión a ARN , Animales , Proteína EWS de Unión a ARN/metabolismo , Proteína EWS de Unión a ARN/genética , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína Proto-Oncogénica c-fli-1/genética , Humanos , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patología , Animales Modificados Genéticamente , Drosophila/genética , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismoRESUMEN
Expression of the Drosophila cancer-germline (CG), X-linked, head-to-head gene pair TrxT and dhd is normally germline-specific but becomes upregulated in brain tumours caused by mutation in l(3)mbt. Here, we show that TrxT and dhd play a major synergistic role in the emergence of l(3)mbt tumour-linked transcriptomic signatures and tumour development, which is remarkable, taking into account that these two genes are never expressed together under normal conditions. We also show that TrxT, but not dhd, is crucial for the growth of l(3)mbt allografts, hence suggesting that the initial stages of tumour development and long-term tumour growth may depend on different molecular pathways. In humans, head-to-head inverted gene pairs are abundant among CG genes that map to the X chromosome. Our results identify a first example of an X-linked, head-to-head CG gene pair in Drosophila, underpinning the potential of such CG genes, dispensable for normal development and homoeostasis of somatic tissue, as targets to curtail malignant growth with minimal impact on overall health.
Asunto(s)
Neoplasias Encefálicas , Encéfalo , Proteínas de Drosophila , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/crecimiento & desarrollo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Humanos , Regulación Neoplásica de la Expresión Génica , TranscriptomaRESUMEN
While testing for genome instability in Drosophila as reported by unscheduled upregulation of UAS-GFP in cells that co-express GAL80 and GAL4, we noticed that, as expected, background levels were low in most developing tissues. However, GFP-positive clones were frequent in the larval brain. Most of these clones originated from central brain neural stem cells. Using imaging-based approaches and genome sequencing, we show that these unscheduled clones do not result from chromosome loss or mutations in GAL80. We have named this phenomenon 'Illuminati'. Illuminati is strongly enhanced in brat tumors and is also sensitive to environmental conditions such as food content and temperature. Illuminati is suppressed by Su(var)2-10, but it is not significantly affected by several modifiers of position effect variegation or Gal4::UAS variegation. We conclude that Illuminati identifies a previously unknown type of functional instability that may have important implications in development and disease.
Asunto(s)
Proteínas de Drosophila , Células-Madre Neurales , Animales , Drosophila/genética , Drosophila melanogaster/genética , Mutación/genética , Expresión Génica , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genéticaRESUMEN
Neural stem cells (NSCs) divide asymmetrically to balance their self-renewal and differentiation, an imbalance in which can lead to NSC overgrowth and tumor formation. The functions of Parafibromin, a conserved tumor suppressor, in the nervous system are not established. Here, we demonstrate that Drosophila Parafibromin/Hyrax (Hyx) inhibits ectopic NSC formation by governing cell polarity. Hyx is essential for the asymmetric distribution and/or maintenance of polarity proteins. hyx depletion results in the symmetric division of NSCs, leading to the formation of supernumerary NSCs in the larval brain. Importantly, we show that human Parafibromin rescues the ectopic NSC phenotype in Drosophila hyx mutant brains. We have also discovered that Hyx is required for the proper formation of interphase microtubule-organizing center and mitotic spindles in NSCs. Moreover, Hyx is required for the proper localization of 2 key centrosomal proteins, Polo and AurA, and the microtubule-binding proteins Msps and D-TACC in dividing NSCs. Furthermore, Hyx directly regulates the polo and aurA expression in vitro. Finally, overexpression of polo and aurA could significantly suppress ectopic NSC formation and NSC polarity defects caused by hyx depletion. Our data support a model in which Hyx promotes the expression of polo and aurA in NSCs and, in turn, regulates cell polarity and centrosome/microtubule assembly. This new paradigm may be relevant to future studies on Parafibromin/HRPT2-associated cancers.
Asunto(s)
Proteínas de Drosophila , Células-Madre Neurales , Animales , Polaridad Celular , Centrosoma/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Humanos , Células-Madre Neurales/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The loss-of-function conditions for an l(3)malignant brain tumour (l(3)mbt) in larvae reared at 29 °C results in malignant brain tumours and hyperplastic imaginal discs. Unlike the former that have been extensively characterised, little is known about the latter. Here we report the results of a study of the hyperplastic l(3)mbt mutant wing imaginal discs. We identify the l(3)mbt wing disc tumour transcriptome and find it to include genes involved in reactive oxygen species (ROS) metabolism. Furthermore, we show the presence of oxidative stress in l(3)mbt hyperplastic discs, even in apoptosis-blocked conditions, but not in l(3)mbt brain tumours. We also find that chemically blocking oxidative stress in l(3)mbt wing discs reduces the incidence of wing disc overgrowths. Our results reveal the involvement of oxidative stress in l(3)mbt wing discs hyperplastic growth.
Asunto(s)
Proteínas de Drosophila , Discos Imaginales , Animales , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Discos Imaginales/metabolismo , Estrés Oxidativo , Alas de Animales/metabolismoRESUMEN
Ewing sarcoma (EwS) is a human malignant tumor typically driven by the Ewing sarcoma-Friend leukemia integration (EWS-FLI) fusion protein. A paucity of genetically modified animal models, partially owed to the high toxicity of EWS-FLI, hinders research on EwS. Here, we report a spontaneous mutant variant, EWS-FLI1FS, that circumvents the toxicity issue in Drosophila. Through proteomic and genomic analyses, we show that human EWS-FLI1FS interacts with the Drosophila homologues of EWS-FLI human protein partners, including core subunits of chromatin remodeling complexes, the transcription machinery, and the spliceosome; brings about a massive dysregulation of transcription that affects a significant fraction of known targets of EWS-FLI in human cells; and modulates splicing. We also show that EWS-FLI1FS performs in Drosophila the two major neomorphic activities that it is known to have in human cells: activation of transcription from GGAA microsatellites and out competition of ETS transcription factors. We conclude that EWS-FLI1FS reproduces in Drosophila the known oncogenic activities of EWS-FLI that drive EwS tumorigenesis in humans. These results open up an unprecedented opportunity to investigate EWS-FLI's oncogenic pathways in vivo in a genetically tractable organism.
RESUMEN
Thioredoxins (Trxs) are ubiquitous enzymes that regulate the redox state in cells. In Drosophila, there are two germline-specific Trxs, Deadhead (Dhd) and thioredoxin T (TrxT), that belong to the lethal(3)malignant brain tumor signature genes and to the 'survival network' of genes that mediate the cellular response to DNA damage. Dhd is a maternal protein required for early embryogenesis that promotes protamine-histone exchange in fertilized eggs and midblastula transition. TrxT is testis-specific and associates with the lampbrush loops of the Y chromosome. Here, the first structures of Dhd and TrxT are presented, unveiling new features of these two thioredoxins. Dhd has positively charged patches on its surface, in contrast to the negatively charged surfaces commonly found in most Trxs. This distinctive charge distribution helps to define initial encounter complexes with DNA/RNA that will lead to final specific interactions with cofactors to promote chromatin remodeling. TrxT contains a C-terminal extension, which is mostly unstructured and highly flexible, that wraps the conserved core through a closed conformation. It is believed that these new structures can guide future work aimed at understanding embryo development and redox homeostasis in Drosophila. Moreover, due to their restricted presence in Schizophora (a section of the true flies), these structures can help in the design of small-molecular binders to modulate native redox homeostasis, thereby providing new applications for the control of plagues that cause human diseases and/or bring about economic losses by damaging crop production.
RESUMEN
Asymmetric cell division (ACD) is a strategy for achieving cell diversity. Research carried out over the last two decades has shown that in some cell types that divide asymmetrically, mother and daughter centrosomes are noticeably different from one another in structure, behaviour, and fate, and that robust ACD depends upon centrosome function. Here, I review the latest advances in this field with special emphasis on the complex structure-function relationship of centrosomes with regards to ACD and on mechanistic insight derived from cell types that divide symmetrically but is likely to be relevant in ACD. I also include a comment arguing for the need to investigate the centrosome cycle in other cell types that divide asymmetrically.
Asunto(s)
División Celular Asimétrica , CentrosomaRESUMEN
We have undertaken a study towards understanding the effect of ectopic expression of testis proteins in the soma in Drosophila. Here, we show that in the larval neuroepithelium, ectopic expression of the germline-specific component of the inner mitochondrial translocation complex tiny tim 2 (ttm2) brings about cell autonomous hyperplasia and extension of G2 phase. In the wing discs, cells expressing ectopic ttm2 upregulate Jun N-terminal kinase (JNK) signaling, present extended G2, become invasive, and elicit non-cell autonomous G2 extension and overgrowth of the wild-type neighboring tissue. Ectopic tomboy20, a germline-specific member of the outer mitochondrial translocation complex is also tumorigenic in wing discs. Our results demonstrate the tumorigenic potential of unscheduled expression of these two testis proteins in the soma. They also show that a unique tumorigenic event may trigger different tumor growth pathways depending on the tissular context.
Asunto(s)
Proteínas de Drosophila/metabolismo , Epitelio/patología , Proteínas Mitocondriales/metabolismo , Animales , Apoptosis , Encéfalo/metabolismo , Encéfalo/patología , Carcinogénesis/genética , Carcinogénesis/metabolismo , Proliferación Celular , Drosophila , Proteínas de Drosophila/genética , Expresión Génica Ectópica , Epitelio/metabolismo , Puntos de Control de la Fase G2 del Ciclo Celular , Hiperplasia , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Larva/genética , Larva/metabolismo , Masculino , Proteínas Mitocondriales/genética , Especificidad de Órganos , Testículo/metabolismo , Alas de Animales/metabolismo , Alas de Animales/patologíaRESUMEN
The notable male predominance across many human cancer types remains unexplained. Here, we show that Drosophila l(3)mbt brain tumors are more invasive and develop as malignant neoplasms more often in males than in females. By quantitative proteomics, we have identified a signature of proteins that are differentially expressed between male and female tumor samples. Prominent among them is the conserved chromatin reader PHD finger protein 7 (Phf7). We show that Phf7 depletion reduces sex-dependent differences in gene expression and suppresses the enhanced malignant traits of male tumors. Our results identify potential regulators of sex-linked tumor dimorphism and show that these genes may serve as targets to suppress sex-linked malignant traits.
Asunto(s)
Neoplasias Encefálicas/patología , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulación de la Expresión Génica/genética , Código de Histonas/genética , Proteínas de Homeodominio/genética , Animales , Femenino , Expresión Génica/genética , Perfilación de la Expresión Génica , Masculino , Proteínas Quinasas/genética , Factores SexualesRESUMEN
Centrobin homologues identified in different species localize on daughter centrioles. In Drosophila melanogaster sensory neurons, Centrobin (referred to as CNB in Drosophila) inhibits basal body function. These data open the question of CNB's role in spermatocytes, where daughter and mother centrioles become basal bodies. In this study, we report that in these cells, CNB localizes equally to mother and daughter centrioles and is essential for C-tubules to attain the right position and remain attached to B-tubules as well as for centrioles to grow in length. CNB appears to be dispensable for meiosis, but flagellum development is severely compromised in Cnb mutant males. Remarkably, three N-terminal POLO phosphorylation sites that are critical for CNB function in neuroblasts are dispensable for spermatogenesis. Our results underpin the multifunctional nature of CNB that plays different roles in different cell types in Drosophila, and they identify CNB as an essential component for C-tubule assembly and flagellum development in Drosophila spermatogenesis.
Asunto(s)
Proteínas de Ciclo Celular/genética , Centriolos/genética , Proteínas de Drosophila/genética , Proteínas Serina-Treonina Quinasas/genética , Espermatogénesis/genética , Animales , Axonema/genética , Cuerpos Basales/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Flagelos/genética , Masculino , Mitosis/genética , Células Receptoras Sensoriales/metabolismo , Espermatocitos/crecimiento & desarrollo , Espermatocitos/metabolismoRESUMEN
Single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) are found at different rates in human cancer. To determine if these genetic lesions appear in Drosophila tumors we have sequenced the genomes of 17 malignant neoplasms caused by mutations in l(3)mbt, brat, aurA, or lgl We have found CNVs and SNPs in all the tumors. Tumor-linked CNVs range between 11 and 80 per sample, affecting between 92 and 1546 coding sequences. CNVs are in average less frequent in l(3)mbt than in brat lines. Nearly half of the CNVs fall within the 10 to 100Kb range, all tumor samples contain CNVs larger that 100 Kb and some have CNVs larger than 1Mb. The rates of tumor-linked SNPs change more than 20-fold depending on the tumor type: at late time points brat, l(3)mbt, and aurA and lgl lines present median values of SNPs/Mb of exome of 0.16, 0.48, and 3.6, respectively. Higher SNP rates are mostly accounted for by C > A transversions, which likely reflect enhanced oxidative stress conditions in the affected tumors. Both CNVs and SNPs turn over rapidly. We found no evidence for selection of a gene signature affected by CNVs or SNPs in the cohort. Altogether, our results show that the rates of CNVs and SNPs, as well as the distribution of CNV sizes in this cohort of Drosophila tumors are well within the range of those reported for human cancer. Genome instability is therefore inherent to Drosophila malignant neoplastic growth at a variable extent that is tumor type dependent.
Asunto(s)
Neoplasias Encefálicas/genética , Drosophila melanogaster/genética , Inestabilidad Genómica , Envejecimiento/patología , Animales , Línea Celular , Variaciones en el Número de Copia de ADN/genética , Humanos , Larva/genética , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Using transgenic RNAi technology, we have screened over 4000 genes to identify targets to inhibit malignant growth caused by the loss of function of lethal(3)malignant brain tumour in Drosophila in vivo We have identified 131 targets, which belong to a wide range of gene ontologies. Most of these target genes are not significantly overexpressed in mbt tumours hence showing that, rather counterintuitively, tumour-linked overexpression is not a good predictor of functional requirement. Moreover, we have found that most of the genes upregulated in mbt tumours remain overexpressed in tumour-suppressed double-mutant conditions, hence revealing that most of the tumour transcriptome signature is not necessarily correlated with malignant growth. One of the identified target genes is meiotic W68 (mei-W68), the Drosophila orthologue of the human cancer/testis gene Sporulation-specific protein 11 (SPO11), the enzyme that catalyses the formation of meiotic double-strand breaks. We show that Drosophila mei-W68/SPO11 drives oncogenesis by causing DNA damage in a somatic tissue, hence providing the first instance in which a SPO11 orthologue is unequivocally shown to have a pro-tumoural role. Altogether, the results from this screen point to the possibility of investigating the function of human cancer relevant genes in a tractable experimental model organism like Drosophila.
Asunto(s)
Neoplasias Encefálicas/genética , Proteínas de Drosophila/genética , Endodesoxirribonucleasas/genética , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Animales , Daño del ADN , Modelos Animales de Enfermedad , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Endodesoxirribonucleasas/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , MasculinoRESUMEN
Prefoldin is a molecular chaperone complex that regulates tubulin function in mitosis. Here, we show that Prefoldin depletion results in disruption of neuroblast polarity, leading to neuroblast overgrowth in Drosophila larval brains. Interestingly, co-depletion of Prefoldin and Partner of Inscuteable (Pins) leads to the formation of gigantic brains with severe neuroblast overgrowth, despite that Pins depletion alone results in smaller brains with partially disrupted neuroblast polarity. We show that Prefoldin acts synergistically with Pins to regulate asymmetric division of both neuroblasts and Intermediate Neural Progenitors (INPs). Surprisingly, co-depletion of Prefoldin and Pins also induces dedifferentiation of INPs back into neuroblasts, while depletion either Prefoldin or Pins alone is insufficient to do so. Furthermore, knocking down either α-tubulin or ß-tubulin in pins(-) mutant background results in INP dedifferentiation back into neuroblasts, leading to the formation of ectopic neuroblasts. Overexpression of α-tubulin suppresses neuroblast overgrowth observed in prefoldin pins double mutant brains. Our data elucidate an unexpected function of Prefoldin and Pins in synergistically suppressing dedifferentiation of INPs back into neural stem cells.
Asunto(s)
División Celular Asimétrica , Desdiferenciación Celular , Proteínas de Drosophila/fisiología , Inhibidores de Disociación de Guanina Nucleótido/fisiología , Chaperonas Moleculares/fisiología , Animales , Encéfalo/citología , Proteínas de Ciclo Celular , Línea Celular , Polaridad Celular , Proliferación Celular , Drosophila melanogaster/citología , Homeostasis , Larva/citología , Células-Madre NeuralesRESUMEN
Asymmetric division of neural stem cells is a fundamental strategy to balance their self-renewal and differentiation. It is long thought that microtubules are not essential for cell polarity in asymmetrically dividing Drosophila melanogaster neuroblasts (NBs; neural stem cells). Here, we show that Drosophila ADP ribosylation factor like-2 (Arl2) and Msps, a known microtubule-binding protein, control cell polarity and spindle orientation of NBs. Upon arl2 RNA intereference, Arl2-GDP expression, or arl2 deletions, microtubule abnormalities and asymmetric division defects were observed. Conversely, overactivation of Arl2 leads to microtubule overgrowth and depletion of NBs. Arl2 regulates microtubule growth and asymmetric division through localizing Msps to the centrosomes in NBs. Moreover, Arl2 regulates dynein function and in turn centrosomal localization of D-TACC and Msps. Arl2 physically associates with tubulin cofactors C, D, and E. Arl2 functions together with tubulin-binding cofactor D to control microtubule growth, Msps localization, and NB self-renewal. Therefore, Arl2- and Msps-dependent microtubule growth is a new paradigm regulating asymmetric division of neural stem cells.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , División Celular/fisiología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/fisiología , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animales , Polaridad Celular/fisiología , Células Cultivadas , Centrosoma/metabolismo , Microtúbulos/fisiología , Células-Madre Neurales/metabolismo , Células-Madre Neurales/fisiología , Unión Proteica/fisiología , Tubulina (Proteína)/metabolismoRESUMEN
This protocol describes a method to allograft Drosophila larval tissue into adult fly hosts that can be used to assay the tumorigenic potential of mutant tissues. The tissue of interest is dissected, loaded into a fine glass needle and implanted into a host. Upon implantation, nontransformed tissues do not overgrow beyond their normal size, but malignant tumors grow without limit, are invasive and kill the host. By using this method, Drosophila malignant tumors can be transplanted repeatedly, for years, and therefore they can be aged beyond the short life span of flies. Because several hosts can be implanted using different pieces from a single tumor, the method also allows the tumor mass to be increased to facilitate further studies that may require large amounts of tissue (i.e., genomics, proteomics and so on). This method also provides an operational definition of hyperplastic, benign and malignant growth. The injection procedure itself requires only â¼1 d. Tumor development can then be monitored until the death of the implanted hosts.
Asunto(s)
Aloinjertos , Técnicas de Cultivo de Célula/métodos , Drosophila/fisiología , Animales , Modelos Animales de Enfermedad , Drosophila/crecimiento & desarrollo , Femenino , Larva , MutaciónRESUMEN
Sensory cilia are organelles that convey information to the cell from the extracellular environment. In vertebrates, ciliary dysfunction results in ciliopathies that in humans comprise a wide spectrum of developmental disorders. In Drosophila, sensory cilia are found only in the neurons of type I sensory organs, but ciliary dysfunction also has dramatic consequences in this organism because it impairs the mechanosensory properties of bristles and chaetae and leads to uncoordination, a crippling condition that causes lethality shortly after eclosion. The cilium is defined by the ciliary membrane, a protrusion of the cell membrane that envelops the core structure known as the axoneme, a microtubule array that extends along the cilium from the basal body. In vertebrates, basal body function requires centriolar distal and subdistal appendages and satellites. Because these structures are acquired through centriole maturation, only mother centrioles can serve as basal bodies. Here, we show that although centriole maturity traits are lacking in Drosophila, basal body fate is reserved to mother centrioles in Drosophila type I neurons. Moreover, we show that depletion of the daughter-centriole-specific protein Centrobin (CNB) enables daughter centrioles to dock on the cell membrane and to template an ectopic axoneme that, although structurally defective, protrudes out of the cell and is enveloped by a ciliary membrane. Conversely, basal body capability is inhibited in mother centrioles modified to carry CNB. These results reveal the crucial role of CNB in regulating basal body function in Drosophila ciliated sensory organs.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Cilios/metabolismo , Drosophila/metabolismo , Animales , Axonema/metabolismo , Cuerpos Basales/metabolismo , Drosophila/citología , Neuronas/citología , Orgánulos/metabolismoRESUMEN
Drosophila larval neuroblasts (NBs) are an excellent model for asymmetric division and cell cycle studies in general. For decades, visualizing relevant structures like centrosomes, chromosomes, or the mitotic spindle relied exclusively on immunofluorescence on fix samples. More recently, improvements on sensitivity and acquisition speed of different confocal systems have made it possible to acquire time-resolved images of combined fluorescent reporters from single larval NBs. Here, we provide protocols to visualize centrosomes and other organelles from both primary cultures of isolated single NBs and ex vivo, whole-mounted larval brains.