Human EWS-FLI protein levels and neomorphic functions show a complex, function-specific dose-response relationship in Drosophila.

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.

TrxT and dhd are dispensable for Drosophila brain development but essential for l(3)mbt brain tumour growth.

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.

Oxidative Stress Is Associated with Overgrowth in Drosophila l(3)mbt Mutant Imaginal Discs.

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.

Human EWS-FLI protein recapitulates in Drosophila the neomorphic functions that induce Ewing sarcoma tumorigenesis.

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.

Genome-wide phenotypic RNAi screen in the Drosophila wing: phenotypic description of functional classes.

The Drosophila genome contains approximately 14,000 protein-coding genes encoding all the necessary information to sustain cellular physiology, tissue organization, organism development, and behavior. In this manuscript, we describe in some detail the phenotypes in the adult fly wing generated after knockdown of approximately 80% of Drosophila genes. We combined this phenotypic description with a comprehensive molecular classification of the Drosophila proteins into classes that summarize the main expected or known biochemical/functional aspect of each protein. This information, combined with mRNA expression levels and in situ expression patterns, provides a simplified atlas of the Drosophila genome, from housekeeping proteins to the components of the signaling pathways directing wing development, that might help to further understand the contribution of each gene group to wing formation.

Genome-wide phenotypic RNAi screen in the Drosophila wing: global parameters.

We have screened a collection of UAS-RNAi lines targeting 10,920 Drosophila protein-coding genes for phenotypes in the adult wing. We identified 3653 genes (33%) whose knockdown causes either larval/pupal lethality or a mutant phenotype affecting the formation of a normal wing. The most frequent phenotypes consist of changes in wing size, vein differentiation, and patterning, defects in the wing margin and in the apposition of the dorsal and ventral wing surfaces. We also defined 16 functional categories encompassing the most relevant aspect of each protein function and assigned each Drosophila gene to one of these functional groups. This allowed us to identify which mutant phenotypes are enriched within each functional group. Finally, we used previously published gene expression datasets to determine which genes are or are not expressed in the wing disc. Integrating expression, phenotypic and molecular information offers considerable precision to identify the relevant genes affecting wing formation and the biological processes regulated by them.

EGFRAP encodes a new negative regulator of the EGFR acting in both normal and oncogenic EGFR/Ras-driven tissue morphogenesis.

Activation of Ras signaling occurs in ~30% of human cancers. However, activated Ras alone is insufficient to produce malignancy. Thus, it is imperative to identify those genes cooperating with activated Ras in driving tumoral growth. In this work, we have identified a novel EGFR inhibitor, which we have named EGFRAP, for EGFR adaptor protein. Elimination of EGFRAP potentiates activated Ras-induced overgrowth in the Drosophila wing imaginal disc. We show that EGFRAP interacts physically with the phosphorylated form of EGFR via its SH2 domain. EGFRAP is expressed at high levels in regions of maximal EGFR/Ras pathway activity, such as at the presumptive wing margin. In addition, EGFRAP expression is up-regulated in conditions of oncogenic EGFR/Ras activation. Normal and oncogenic EGFR/Ras-mediated upregulation of EGRAP levels depend on the Notch pathway. We also find that elimination of EGFRAP does not affect overall organogenesis or viability. However, simultaneous downregulation of EGFRAP and its ortholog PVRAP results in defects associated with increased EGFR function. Based on these results, we propose that EGFRAP is a new negative regulator of the EGFR/Ras pathway, which, while being required redundantly for normal morphogenesis, behaves as an important modulator of EGFR/Ras-driven tissue hyperplasia. We suggest that the ability of EGFRAP to functionally inhibit the EGFR pathway in oncogenic cells results from the activation of a feedback loop leading to increase EGFRAP expression. This could act as a surveillance mechanism to prevent excessive EGFR activity and uncontrolled cell growth.

Context-Dependent Tumorigenic Effect of Testis-Specific Mitochondrial Protein Tiny Tim 2 in Drosophila Somatic Epithelia.

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.

Ras2, the TC21/R-Ras2 Drosophila homologue, contributes to insulin signalling but is not required for organism viability.

Ras1 (Ras85D) and Ras2 (Ras64B) are the Drosophila orthologs of human H-Ras/N-Ras/K-Ras and R-Ras1-3 genes, respectively. The function of Ras1 has been thoroughly characterised during Drosophila embryonic and imaginal development, and it is associated with coupling activated trans-membrane receptors with tyrosine kinase activity to their downstream effectors. In this capacity, Ras1 binds and is required for the activation of Raf. Ras1 can also interact with PI3K, and it is needed to achieve maximal levels of PI3K signalling in specific cellular settings. In contrast, the function of the unique Drosophila R-Ras member (Ras2/Ras64B), which is more closely related to vertebrate R-Ras2/TC21, has been only studied through the use of constitutively activated forms of the protein. This pioneering work identified a variety of phenotypes that were related to those displayed by Ras1, suggesting that Ras1 and Ras2 might have overlapping activities. Here we find that Ras2 can interact with PI3K and Raf and activate their downstream effectors Akt and Erk. However, and in contrast to mutants in Ras1, which are lethal, null alleles of Ras2 are viable in homozygosis and only show a phenotype of reduced wing size and extended life span that might be related to reduced Insulin receptor signalling.

The histone code reader PHD finger protein 7 controls sex-linked disparities in gene expression and malignancy in Drosophila.

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.

The effect of rotation upon dental structure components following orthodontic fix appliance.

BACKGROUND AND AIMS: The purpose of the study is to evaluate through a FEM (Finite Element Method) the effects of the rotation movement upon a complex structure (enamel- pulp -alveolar bone, PDL), for external load. METHOD: The progressive action of a fixed orthodontic device on three teeth: first molar, first and second premolar is modeled and simulated with the components placed on the buccal and palatal surfaces of the tooth. For the reproduction of a situation similar to the real one, the loading of the model was performed through a nodal force applied at a height of the crown, of various amplitudes, F = 1 N; 2, 3 and 4 N. The values of stress are: maximum stress of the whole structure and the shearing effect for the pulp. RESULTS: The characteristics of the material are Young's E modulus and Poisson's ratio of the components of the modeled structure. The most stressed elements of the structure are the pulp and the ligament, revealed by von Mises stress. The elements of the structure are mainly stressed in fiber compression in the direction of the moment's action given by the orthodontic forces and in fiber stretching in the opposite direction. Out of the orthodontic movements: translation, tipping, intrusion-extrusion, rotation is the most dangerous. CONCLUSIONS: The accumulated stress effect in the pulp becomes dangerous. The orthodontic movements given by dental force values higher than 1.5-2N are to be avoided.

An in vivo genetic screen in Drosophila identifies the orthologue of human cancer/testis gene SPO11 among a network of targets to inhibit lethal(3)malignant brain tumour growth.

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.

Tay bridge is a negative regulator of EGFR signalling and interacts with Erk and Mkp3 in the Drosophila melanogaster wing.

The regulation of Extracellular regulated kinase (Erk) activity is a key aspect of signalling by pathways activated by extracellular ligands acting through tyrosine kinase transmembrane receptors. In this process, participate proteins with kinase activity that phosphorylate and activate Erk, as well as different phosphatases that inactivate Erk by de-phosphorylation. The state of Erk phosphorylation affects not only its activity, but also its subcellular localization, defining the repertoire of Erk target proteins, and consequently, the cellular response to Erk. In this work, we characterise Tay bridge as a novel component of the EGFR/Erk signalling pathway. Tay bridge is a large nuclear protein with a domain of homology with human AUTS2, and was previously identified due to the neuronal phenotypes displayed by loss-of-function mutations. We show that Tay bridge antagonizes EGFR signalling in the Drosophila melanogaster wing disc and other tissues, and that the protein interacts with both Erk and Mkp3. We suggest that Tay bridge constitutes a novel element involved in the regulation of Erk activity, acting as a nuclear docking for Erk that retains this protein in an inactive form in the nucleus.

Genetic annotation of gain-of-function screens using RNA interference and in situ hybridization of candidate genes in the Drosophila wing.

Gain-of-function screens in Drosophila are an effective method with which to identify genes that affect the development of particular structures or cell types. It has been found that a fraction of 2-10% of the genes tested, depending on the particularities of the screen, results in a discernible phenotype when overexpressed. However, it is not clear to what extent a gain-of-function phenotype generated by overexpression is informative about the normal function of the gene. Thus, very few reports attempt to correlate the loss- and overexpression phenotype for collections of genes identified in gain-of-function screens. In this work we use RNA interference and in situ hybridization to annotate a collection of 123 P-GS insertions that in combination with different Gal4 drivers affect the size and/or patterning of the wing. We identify the gene causing the overexpression phenotype by expressing, in a background of overexpression, RNA interference for the genes affected by each P-GS insertion. Then, we compare the loss and gain-of-function phenotypes obtained for each gene and relate them to its expression pattern in the wing disc. We find that 52% of genes identified by their overexpression phenotype are required during normal development. However, only in 9% of the cases analyzed was there some complementarity between the gain- and loss-of-function phenotype, suggesting that, in general, the overexpression phenotypes would not be indicative of the normal requirements of the gene.

Role of the Drosophila non-visual ß-arrestin kurtz in hedgehog signalling.

The non-visual ß-arrestins are cytosolic proteins highly conserved across species that participate in a variety of signalling events, including plasma membrane receptor degradation, recycling, and signalling, and that can also act as scaffolding for kinases such as MAPK and Akt/PI3K. In Drosophila melanogaster, there is only a single non-visual ß-arrestin, encoded by kurtz, whose function is essential for neuronal activity. We have addressed the participation of Kurtz in signalling during the development of the imaginal discs, epithelial tissues requiring the activity of the Hedgehog, Wingless, EGFR, Notch, Insulin, and TGFß pathways. Surprisingly, we found that the complete elimination of kurtz by genetic techniques has no major consequences in imaginal cells. In contrast, the over-expression of Kurtz in the wing disc causes a phenotype identical to the loss of Hedgehog signalling and prevents the expression of Hedgehog targets in the corresponding wing discs. The mechanism by which Kurtz antagonises Hedgehog signalling is to promote Smoothened internalization and degradation in a clathrin- and proteosomal-dependent manner. Intriguingly, the effects of Kurtz on Smoothened are independent of Gprk2 activity and of the activation state of the receptor. Our results suggest fundamental differences in the molecular mechanisms regulating receptor turnover and signalling in vertebrates and invertebrates, and they could provide important insights into divergent evolution of Hedgehog signalling in these organisms.

A conserved function of the chromatin ATPase Kismet in the regulation of hedgehog expression.

The development of the Drosophila melanogaster wing depends on its subdivision into anterior and posterior compartments, which constitute two independent cell lineages since their origin in the embryonic ectoderm. The anterior-posterior compartment boundary is the place where signaling by the Hedgehog pathway takes place, and this requires pathway activation in anterior cells by ligand expressed exclusively in posterior cells. Several mechanisms ensure the confinement of hedgehog expression to posterior cells, including repression by Cubitus interruptus, the co-repressor Groucho and Master of thick veins. In this work we identified Kismet, a chromodomain-containing protein of the SNF2-like family of ATPases, as a novel component of the hedgehog transcriptional repression mechanism in anterior compartment cells. In kismet mutants, hedgehog is ectopically expressed in a domain of anterior cells close to the anterior-posterior compartment boundary, causing inappropriate activation of the pathway and changes in the development of the central region of the wing. The contribution of Kismet to the silencing of hedgehog expression is limited to anterior cells with low levels of the repressor form of Cubitus interruptus. We also show that knockdown of CHD8, the kismet homolog in Xenopus tropicalis, is also associated with ectopic sonic hedgehog expression and up-regulation of one of its target genes in the eye, Pax2, indicating the evolutionary conservation of Kismet/CHD8 function in negatively controlling hedgehog expression.

The leading edge during dorsal closure as a model for epithelial plasticity: Pak is required for recruitment of the Scribble complex and septate junction formation.

Dorsal closure (DC) of the Drosophila embryo is a model for the study of wound healing and developmental epithelial fusions, and involves the sealing of a hole in the epidermis through the migration of the epidermal flanks over the tissue occupying the hole, the amnioserosa. During DC, the cells at the edge of the migrating epidermis extend Rac- and Cdc42-dependent actin-based lamellipodia and filopodia from their leading edge (LE), which exhibits a breakdown in apicobasal polarity as adhesions are severed with the neighbouring amnioserosa cells. Studies using mammalian cells have demonstrated that Scribble (Scrib), an important determinant of apicobasal polarity that functions in a protein complex, controls polarized cell migration through recruitment of Rac, Cdc42 and the serine/threonine kinase Pak, an effector for Rac and Cdc42, to the LE. We have used DC and the follicular epithelium to study the relationship between Pak and the Scrib complex at epithelial membranes undergoing changes in apicobasal polarity and adhesion during development. We propose that, during DC, the LE membrane undergoes an epithelial-to-mesenchymal-like transition to initiate epithelial sheet migration, followed by a mesenchymal-to-epithelial-like transition as the epithelial sheets meet up and restore cell-cell adhesion. This latter event requires integrin-localized Pak, which recruits the Scrib complex in septate junction formation. We conclude that there are bidirectional interactions between Pak and the Scrib complex modulating epithelial plasticity. Scrib can recruit Pak to the LE for polarized cell migration but, as migratory cells meet up, Pak can recruit the Scrib complex to restore apicobasal polarity and cell-cell adhesion.

A cautionary tale on genetic screens based on a gain-of-expression approach: The case of LanB1.

Gain of function screens have being frequently used to search for genes affecting a particular adult character or developmental process. These experiments are made possible by the adoption of the Gal4/UAS system to flies, and by the design of P elements bearing UAS sequences. We recently published two screens in which a large number of newly generated P-UAS insertions were crossed with Gal4 drivers expressed in the pupal veins and in the central region of the wing disc. From the data obtained in these and other screens, it seems that a gain-of-function phenotype is a rare occurrence observed only for about 5-8% of insertion sites. Insertions affecting the expression of signaling molecules were particularly enriched in the screens. In contrast, gain-of-function phenotypes due to insertions not belonging to this class appear to be caused by multiple protein-specific mechanisms that could only be unraveled after extensive analysis. We present some data concerning the overexpression of LamB1, a gene encoding the B subunit of Laminin trimers in Drosophila, and show that Notch protein subcellular localization and signaling is compromised in cells overexpressing LanB1.

Drosophila laminins act as key regulators of basement membrane assembly and morphogenesis.

Laminins are heterotrimeric molecules found in all basement membranes. In mammals, they have been involved in diverse developmental processes, from gastrulation to tissue maintenance. The Drosophila genome encodes two laminin alpha chains, one beta and one Gamma, which form two distinct laminin trimers. So far, only mutations affecting one or other trimer have been analysed. In order to study embryonic development in the complete absence of laminins, we mutated the gene encoding the sole laminin beta chain in Drosophila, LanB1, so that no trimers can be made. We show that LanB1 mutant embryos develop until the end of embryogenesis. Electron microscopy analysis of mutant embryos reveals that the basement membranes are absent and the remaining extracellular material appears disorganised and diffuse. Accordingly, abnormal accumulation of major basement membrane components, such as Collagen IV and Perlecan, is observed in mutant tissues. In addition, we show that elimination of LanB1 prevents the normal morphogenesis of most organs and tissues, including the gut, trachea, muscles and nervous system. In spite of the above structural roles for laminins, our results unravel novel functions in cell adhesion, migration and rearrangement. We propose that while an early function of laminins in gastrulation is not conserved in Drosophila and mammals, their function in basement membrane assembly and organogenesis seems to be maintained throughout evolution.

Drosophila Axud1 is involved in the control of proliferation and displays pro-apoptotic activity.

Cell division rates and apoptosis sculpt the growing organs, and its regulation implements the developmental programmes that define organ size and shape. The balance between oncogenes and tumour suppressors modulate the cell cycle and the apoptotic machinery to achieve this goal, promoting and restricting proliferation or, in certain conditions, inducing the apoptotic programme. Analysis of human cancer cells with mutation in AXIN gene has uncovered the potential function of AXUD1 as a tumour suppressor. It has been described that Human AXUD1 is a nuclear protein. We find that a DAxud1-GFP fusion protein is localised to the nucleus during interphase, where it accumulates associated to the nuclear envelope, but becomes distributed in a diffused pattern in the nucleus of mitotic cells. We have analysed the function of the Drosophila AXUD1 homologue, and find that DAxud1 behaves as a tumour suppressor that regulates the proliferation rhythm of imaginal cells. Knocking down the activity of DAxud1 enhances the proliferation of these cells, causing in addition a reduction in cell size. Conversely, the increase in DAxud1 expression impedes cell cycle progression at mitosis through disturbance of Cdk1 activity, and induces the apoptosis of these cells in a JNK-dependent manner.