The Drosophila tumour suppressor Lgl and Vap33 activate the Hippo pathway through a dual mechanism.

The tumour suppressor, Lethal (2) giant larvae [Lgl; also known as L(2)gl], is an evolutionarily conserved protein that was discovered in the vinegar fly Drosophila, where its depletion results in tissue overgrowth and loss of cell polarity. Lgl links cell polarity and tissue growth through regulation of the Notch and the Hippo signalling pathways. Lgl regulates the Notch pathway by inhibiting V-ATPase activity via Vap33. How Lgl regulates the Hippo pathway was unclear. In this current study, we show that V-ATPase activity inhibits the Hippo pathway, whereas Vap33 acts to activate Hippo signalling. Vap33 physically and genetically interacts with the actin cytoskeletal regulators RtGEF (Pix) and Git, which also bind to the Hippo protein (Hpo) and are involved in the activation of the Hippo pathway. Additionally, we show that the ADP ribosylation factor Arf79F (Arf1), which is a Hpo interactor, is involved in the inhibition of the Hippo pathway. Altogether, our data suggest that Lgl acts via Vap33 to activate the Hippo pathway by a dual mechanism: (1) through interaction with RtGEF, Git and Arf79F, and (2) through interaction and inhibition of the V-ATPase, thereby controlling epithelial tissue growth.

A protocol to study three-dimensional genome structure in individual mutant preimplantation mouse embryos.

Hi-C studies the three-dimensional structure of the genome by detecting genome-wide chromatin regions that are in spatial proximity within the nucleus. We developed single-blastocyst Hi-C in mutant mouse embryos to genotype them on sequence. We describe steps for embryo fixation and nuclei permeabilization, after which chromatin is digested and re-ligated having incorporated a biotin-labeled nucleotide at the ligation junction. After cross-link reversal, we then detail purification of immobilized chimeric DNA ligations, library generation, sequencing, and genome-wide analysis of interactions. For complete details on the use and execution of this protocol, please refer to Andreu et al. (2022).1.

Establishment of 3D chromatin structure after fertilization and the metabolic switch at the morula-to-blastocyst transition require CTCF.

The eukaryotic genome is organized in 3D at different scales. This structure is driven and maintained by different chromatin states and by architectural factors, such as the zinc finger protein CTCF. Zygotic genome structure is established de novo after fertilization, but its impact during the first stages of mammalian development is unclear. We show that deletion of Ctcf in mouse embryos impairs the establishment of chromatin structure, but the first cell fate decision is unperturbed and embryos are viable until the late blastocyst. Furthermore, maternal CTCF is not necessary for development. Gene expression changes in metabolic and protein homeostasis programs that occur during the morula-to-blastocyst transition depend on CTCF. However, these changes do not correlate with disruption of chromatin but with binding of CTCF to the promoter of downregulated genes. Our results show that CTCF regulates both 3D genome organization and transcription during mouse preimplantation development, but as independent processes.

Chromatin dynamics through mouse preimplantation development revealed by single molecule localisation microscopy.

Most studies addressing chromatin behaviour during preimplantation development are based on biochemical assays that lack spatial and cell-specific information, crucial during early development. Here, we describe the changes in chromatin taking place at the transition from totipotency to lineage specification, by using direct stochastical optical reconstruction microscopy (dSTORM) in whole-mount embryos during the first stages of mouse development. Through the study of two post-translational modifications of Histone 3 related to active and repressed chromatin, H3K4me3 and H3K9me3 respectively, we obtained a time-course of chromatin states, showing spatial differences between cell types, related to their differentiation state. This analysis adds a new layer of information to previous biochemical studies and provides novel insight to current models of chromatin organisation during the first stages of development.

PTP61F Mediates Cell Competition and Mitigates Tumorigenesis.

Tissue homeostasis via the elimination of aberrant cells is fundamental for organism survival. Cell competition is a key homeostatic mechanism, contributing to the recognition and elimination of aberrant cells, preventing their malignant progression and the development of tumors. Here, using Drosophila as a model organism, we have defined a role for protein tyrosine phosphatase 61F (PTP61F) (orthologue of mammalian PTP1B and TCPTP) in the initiation and progression of epithelial cancers. We demonstrate that a Ptp61F null mutation confers cells with a competitive advantage relative to neighbouring wild-type cells, while elevating PTP61F levels has the opposite effect. Furthermore, we show that knockdown of Ptp61F affects the survival of clones with impaired cell polarity, and that this occurs through regulation of the JAK-STAT signalling pathway. Importantly, PTP61F plays a robust non-cell-autonomous role in influencing the elimination of adjacent polarity-impaired mutant cells. Moreover, in a neoplastic RAS-driven polarity-impaired tumor model, we show that PTP61F levels determine the aggressiveness of tumors, with Ptp61F knockdown or overexpression, respectively, increasing or reducing tumor size. These effects correlate with the regulation of the RAS-MAPK and JAK-STAT signalling by PTP61F. Thus, PTP61F acts as a tumor suppressor that can function in an autonomous and non-cell-autonomous manner to ensure cellular fitness and attenuate tumorigenesis.

Cell-to-cell communication mediates glioblastoma progression in Drosophila.

Glioblastoma (GB) is the most aggressive and lethal tumour of the central nervous system (CNS). GB cells grow rapidly and display a network of projections, ultra-long tumour microtubes (TMs), that mediate cell to cell communication. GB-TMs infiltrate throughout the brain, enwrap neurons and facilitate the depletion of the signalling molecule wingless (Wg)/WNT from the neighbouring healthy neurons. GB cells establish a positive feedback loop including Wg signalling upregulation that activates cJun N-terminal kinase (JNK) pathway and matrix metalloproteases (MMPs) production, which in turn promote further TMs infiltration, GB progression and neurodegeneration. Thus, cellular and molecular signals other than primary mutations emerge as central players of GB. Using a Drosophila model of GB, we describe the temporal organisation of the main cellular events that occur in GB, including cell-to-cell interactions, neurodegeneration and TM expansion. We define the progressive activation of JNK pathway signalling in GB mediated by the receptor Grindelwald (Grnd) and activated by the ligand Eiger (Egr)/TNFα produced by surrounding healthy brain tissue. We propose that cellular interactions of GB with the healthy brain tissue precede TM expansion and conclude that non-autonomous signals facilitate GB progression. These results contribute to deciphering the complexity and versatility of these incurable tumours.

New Cellular Dimensions on Glioblastoma Progression.

Gliomas are brain tumors originated from glial cells. The most frequent form of glioma is the glioblastoma (GB). This lethal tumor is frequently originated from genetic alterations in epidermal growth factor receptor (EGFR) and PI3K pathways. Recent results suggest that signaling pathways, other than primary founder mutations, play a central role in GB progression. Some of these signals are depleted by GB cells from healthy neurons via specialized filopodia known as tumor microtubes (TMs). Here, we discuss the contribution of TMs to vampirize wingless/WNT ligand from neurons. In consequence, wingless/WNT pathway is upregulated in GB to promote tumor progression, and the reduction of these signals in neurons causes the reduction of synapse number and neurodegeneration. These processes contribute to neurological defects and premature death.

Tumourigenesis: Using Cytonemes to Engage Mesenchymal Cells in Epithelial Tumours.

A new study in Drosophila shows that inter-tissue communication between epithelial and mesenchymal cells via Notch signalling plays a role in EGFR-driven tumourigenesis of epithelial tissues.

Glioblastoma cells vampirize WNT from neurons and trigger a JNK/MMP signaling loop that enhances glioblastoma progression and neurodegeneration.

Glioblastoma (GB) is the most lethal brain tumor, and Wingless (Wg)-related integration site (WNT) pathway activation in these tumors is associated with a poor prognosis. Clinically, the disease is characterized by progressive neurological deficits. However, whether these symptoms result from direct or indirect damage to neurons is still unresolved. Using Drosophila and primary xenografts as models of human GB, we describe, here, a mechanism that leads to activation of WNT signaling (Wg in Drosophila) in tumor cells. GB cells display a network of tumor microtubes (TMs) that enwrap neurons, accumulate Wg receptor Frizzled1 (Fz1), and, thereby, deplete Wg from neurons, causing neurodegeneration. We have defined this process as "vampirization." Furthermore, GB cells establish a positive feedback loop to promote their expansion, in which the Wg pathway activates cJun N-terminal kinase (JNK) in GB cells, and, in turn, JNK signaling leads to the post-transcriptional up-regulation and accumulation of matrix metalloproteinases (MMPs), which facilitate TMs' infiltration throughout the brain, TMs' network expansion, and further Wg depletion from neurons. Consequently, GB cells proliferate because of the activation of the Wg signaling target, ß-catenin, and neurons degenerate because of Wg signaling extinction. Our findings reveal a molecular mechanism for TM production, infiltration, and maintenance that can explain both neuron-dependent tumor progression and also the neural decay associated with GB.

Cytonemes, Their Formation, Regulation, and Roles in Signaling and Communication in Tumorigenesis.

Increasing evidence during the past two decades shows that cells interconnect and communicate through cytonemes. These cytoskeleton-driven extensions of specialized membrane territories are involved in cell-cell signaling in development, patterning, and differentiation, but also in the maintenance of adult tissue homeostasis, tissue regeneration, and cancer. Brain tumor cells in glioblastoma extend ultralong membrane protrusions (named tumor microtubes, TMs), which contribute to invasion, proliferation, radioresistance, and tumor progression. Here we review the mechanisms underlying cytoneme formation, regulation, and their roles in cell signaling and communication in epithelial cells and other cell types. Furthermore, we discuss the recent discovery of glial cytonemes in the Drosophila glial cells that alter Wingless (Wg)/Frizzled (Fz) signaling between glia and neurons. Research on cytoneme formation, maintenance, and cell signaling mechanisms will help to better understand not only physiological developmental processes and tissue homeostasis but also cancer progression.

Drosophila Models of Cell Polarity and Cell Competition in Tumourigenesis.

Cell competition is an important surveillance mechanism that measures relative fitness between cells in a tissue during development, homeostasis, and disease. Specifically, cells that are "less fit" (losers) are actively eliminated by relatively "more fit" (winners) neighbours, despite the less fit cells otherwise being able to survive in a genetically uniform tissue. Originally described in the epithelial tissues of Drosophila larval imaginal discs, cell competition has since been shown to occur in other epithelial and non-epithelial Drosophila tissues, as well as in mammalian model systems. Many genes and signalling pathways have been identified as playing conserved roles in the mechanisms of cell competition. Among them are genes required for the establishment and maintenance of apico-basal cell polarity: the Crumbs/Stardust/Patj (Crb/Sdt/Patj), Bazooka/Par-6/atypical Protein Kinase C (Baz/Par-6/aPKC), and Scribbled/Discs large 1/Lethal (2) giant larvae (Scrib/Dlg1/L(2)gl) modules. In this chapter, we describe the concepts and mechanisms of cell competition, with emphasis on the relationship between cell polarity proteins and cell competition, particularly the Scrib/Dlg1/L(2)gl module, since this is the best described module in this emerging field.

Oncogenic dependence of glioma cells on kish/TMEM167A regulation of vesicular trafficking.

Genetic lesions in glioblastoma (GB) include constitutive activation of PI3K and EGFR pathways to drive cellular proliferation and tumor malignancy. An RNAi genetic screen, performed in Drosophila melanogaster to discover new modulators of GB development, identified a member of the secretory pathway: kish/TMEM167A. Downregulation of kish/TMEM167A impaired fly and human glioma formation and growth, with no effect on normal glia. Glioma cells increased the number of recycling endosomes, and reduced the number of lysosomes. In addition, EGFR vesicular localization was primed toward recycling in glioma cells. kish/TMEM167A downregulation in gliomas restored endosomal system to a physiological state and altered lysosomal function, fueling EGFR toward degradation by the proteasome. These endosomal effects mirrored the endo/lysosomal response of glioma cells to Brefeldin A (BFA), but not the Golgi disruption and the ER collapse, which are associated with the undesirable toxicity of BFA in other cancers. Our results suggest that glioma growth depends on modifications of the vesicle transport system, reliant on kish/TMEM167A. Noncanonical genes in GB could be a key for future therapeutic strategies targeting EGFR-dependent gliomas.

Phosphorylation of Drosophila Brahma on CDK-phosphorylation sites is important for cell cycle regulation and differentiation.

The SWI/SNF ATP-dependent chromatin-remodeling complex is an important evolutionarily conserved regulator of cell cycle progression. It associates with the Retinoblastoma (pRb)/HDAC/E2F/DP transcription complex to modulate cell cycle-dependent gene expression. The key catalytic component of the SWI/SNF complex in mammals is the ATPase subunit, Brahma (BRM) or BRG1. BRG1 was previously shown to be phosphorylated by the G1-S phase cell cycle regulatory kinase Cyclin E/CDK2 in vitro, which was associated with the bypass of G1 arrest conferred by BRG1 expression. However, it is unknown whether direct Cyclin E/CDK2-mediated phosphorylation of BRM/BRG1 is important for G1-S phase cell cycle progression and proliferation in vivo. Herein, we demonstrate for the first time the importance of CDK-mediated phosphorylation of Brm in cell proliferation and differentiation in vivo using the Drosophila melanogaster model organism. Expression of a CDK-site phospho-mimic mutant of Brm, brm-ASP (all the potential CDK sites are mutated from Ser/Thr to Asp), which acts genetically as a brm loss-of-function allele, dominantly accelerates progression into the S phase, and bypasses a Retinoblastoma-induced developmental G1 phase arrest in the wing epithelium. Conversely, expression of a CDK-site phospho-blocking mutation of Brm, brm-ALA, acts genetically as a brm gain-of-function mutation, and in a Brm complex compromised background reduces S phase cells. Expression of the brm phospho-mutants also affected differentiation and Decapentaplegic (BMP/TGFß) signaling in the wing epithelium. Altogether our results show that CDK-mediated phosphorylation of Brm is important in G1-S phase regulation and differentiation in vivo. ABBREVIATIONS: A-P: Anterior-Posterior; BAF: BRG1-associated factor; BMP: Bone Morphogenetic Protein; Brg1: Brahma-Related Gene 1; Brm: Brahma; BSA: Bovine Serum Albumin; CDK: Cyclin dependent kinase dpp: decapentaplegic; EdU: 5-Ethynyl 2'-DeoxyUridine; EGFR: Epidermal Growth Factor Receptor; en: engrailed; GFP: Green Fluorescent Protein; GST: Glutathione-S-Transferase; HDAC: Histone DeACetylase; JNK: c-Jun N-terminal Kinase; Mad: Mothers Against Dpp; MAPK: Mitogen Activated Protein Kinase; MB:: Myelin Basic Protein; nub: nubbin; pH3: phosphorylated Histone H3; PBS: Phosphate Buffered Saline; PBT: PBS Triton; PFA: ParaFormAldehydep; Rb: Retinoblastoma protein; PCV: Posterior Cross-Vein; Snr1: Snf5-Related 1; SWI/SNF: SWitch/Sucrose Non-Fermentable; TGFß: Transforming Growth Factor ß; TUNEL: TdT-mediated dUTP Nick End Labelling; Wg: Wingless; ZNC: Zone of Non-Proliferating Cells.

Lgl reduces endosomal vesicle acidification and Notch signaling by promoting the interaction between Vap33 and the V-ATPase complex.

Epithelial cell polarity is linked to the control of tissue growth and tumorigenesis. The tumor suppressor and cell polarity protein lethal-2-giant larvae (Lgl) promotes Hippo signaling and inhibits Notch signaling to restrict tissue growth in Drosophila melanogaster Notch signaling is greater in lgl mutant tissue than in wild-type tissue because of increased acidification of endosomal vesicles, which promotes the proteolytic processing and activation of Notch by γ-secretase. We showed that the increased Notch signaling and tissue growth defects of lgl mutant tissue depended on endosomal vesicle acidification mediated by the vacuolar adenosine triphosphatase (V-ATPase). Lgl promoted the activity of the V-ATPase by interacting with Vap33 (VAMP-associated protein of 33 kDa). Vap33 physically and genetically interacted with Lgl and V-ATPase subunits and repressed V-ATPase-mediated endosomal vesicle acidification and Notch signaling. Vap33 overexpression reduced the abundance of the V-ATPase component Vha44, whereas Lgl knockdown reduced the binding of Vap33 to the V-ATPase component Vha68-3. Our data indicate that Lgl promotes the binding of Vap33 to the V-ATPase, thus inhibiting V-ATPase-mediated endosomal vesicle acidification and thereby reducing γ-secretase activity, Notch signaling, and tissue growth. Our findings implicate the deregulation of Vap33 and V-ATPase activity in polarity-impaired epithelial cancers.

Modelling Cooperative Tumorigenesis in Drosophila.

The development of human metastatic cancer is a multistep process, involving the acquisition of several genetic mutations, tumour heterogeneity, and interactions with the surrounding microenvironment. Due to the complexity of cancer development in mammals, simpler model organisms, such as the vinegar fly, Drosophila melanogaster, are being utilized to provide novel insights into the molecular mechanisms involved. In this review, we highlight recent advances in modelling tumorigenesis using the Drosophila model, focusing on the cooperation of oncogenes or tumour suppressors, and the interaction of mutant cells with the surrounding tissue in epithelial tumour initiation and progression.

The Scribble Cell Polarity Module in the Regulation of Cell Signaling in Tissue Development and Tumorigenesis.

The Scribble cell polarity module, comprising Scribbled (Scrib), Discs-large (Dlg) and Lethal-2-giant larvae (Lgl), has a tumor suppressive role in mammalian epithelial cancers. The Scribble module proteins play key functions in the establishment and maintenance of different modes of cell polarity, as well as in the control of tissue growth, differentiation and directed cell migration, and therefore are major regulators of tissue development and homeostasis. Whilst molecular details are known regarding the roles of Scribble module proteins in cell polarity regulation, their precise mode of action in the regulation of other key cellular processes remains enigmatic. An accumulating body of evidence indicates that Scribble module proteins play scaffolding roles in the control of various signaling pathways, which are linked to the control of tissue growth, differentiation and cell migration. Multiple Scrib, Dlg and Lgl interacting proteins have been discovered, which are involved in diverse processes, however many function in the regulation of cellular signaling. Herein, we review the components of the Scrib, Dlg and Lgl protein interactomes, and focus on the mechanism by which they regulate cellular signaling pathways in metazoans, and how their disruption leads to cancer.

Drosophila melanogaster Guk-holder interacts with the Scribbled PDZ1 domain and regulates epithelial development with Scribbled and Discs Large.

Epithelial cell polarity is controlled by components of the Scribble polarity module, and its regulation is critical for tissue architecture and cell proliferation and migration. In Drosophila melanogaster, the adaptor protein Guk-holder (Gukh) binds to the Scribbled (Scrib) and Discs Large (Dlg) components of the Scribble polarity module and plays an important role in the formation of neuromuscular junctions. However, Gukh's role in epithelial tissue formation and the molecular basis for the Scrib-Gukh interaction remain to be defined. We now show using isothermal titration calorimetry that the Scrib PDZ1 domain is the major site for an interaction with Gukh. Furthermore, we defined the structural basis of this interaction by determining the crystal structure of the Scrib PDZ1-Gukh complex. The C-terminal PDZ-binding motif of Gukh is located in the canonical ligand-binding groove of Scrib PDZ1 and utilizes an unusually extensive network of hydrogen bonds and ionic interactions to enable binding to PDZ1 with high affinity. We next examined the role of Gukh along with those of Scrib and Dlg in Drosophila epithelial tissues and found that Gukh is expressed in larval-wing and eye-epithelial tissues and co-localizes with Scrib and Dlg at the apical cell cortex. Importantly, we show that Gukh functions with Scrib and Dlg in the development of Drosophila epithelial tissues, with depletion of Gukh enhancing the eye- and wing-tissue defects caused by Scrib or Dlg depletion. Overall, our findings reveal that Scrib's PDZ1 domain functions in the interaction with Gukh and that the Scrib-Gukh interaction has a key role in epithelial tissue development in Drosophila.

Differential regulation of protein tyrosine kinase signalling by Dock and the PTP61F variants.

Tyrosine phosphorylation-dependent signalling is coordinated by the opposing actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). There is a growing list of adaptor proteins that interact with PTPs and facilitate the dephosphorylation of substrates. The extent to which any given adaptor confers selectivity for any given substrate in vivo remains unclear. Here we have taken advantage of Drosophila melanogaster as a model organism to explore the influence of the SH3/SH2 adaptor protein Dock on the abilities of the membrane (PTP61Fm)- and nuclear (PTP61Fn)-targeted variants of PTP61F (the Drosophila othologue of the mammalian enzymes PTP1B and TCPTP respectively) to repress PTK signalling pathways in vivo. PTP61Fn effectively repressed the eye overgrowth associated with activation of the epidermal growth factor receptor (EGFR), PTK, or the expression of the platelet-derived growth factor/vascular endothelial growth factor receptor (PVR) or insulin receptor (InR) PTKs. PTP61Fn repressed EGFR and PVR-induced mitogen-activated protein kinase signalling and attenuated PVR-induced STAT92E signalling. By contrast, PTP61Fm effectively repressed EGFR- and PVR-, but not InR-induced tissue overgrowth. Importantly, coexpression of Dock with PTP61F allowed for the efficient repression of the InR-induced eye overgrowth, but did not enhance the PTP61Fm-mediated inhibition of EGFR and PVR-induced signalling. Instead, Dock expression increased, and PTP61Fm coexpression further exacerbated the PVR-induced eye overgrowth. These results demonstrate that Dock selectively enhances the PTP61Fm-mediated attenuation of InR signalling and underscores the specificity of PTPs and the importance of adaptor proteins in regulating PTP function in vivo.

Tissue growth and tumorigenesis in Drosophila: cell polarity and the Hippo pathway.

Cell polarity regulation is critical for defining membrane domains required for the establishment and maintenance of the apical-basal axis in epithelial cells (apico-basal polarity), asymmetric cell divisions, planar organization of tissues (planar cell polarity), and the formation of the front-rear axis in cell migration (front-rear polarity). In the vinegar fly, Drosophila melanogaster, cell polarity regulators also interact with the Hippo tissue growth control signaling pathway. In this review we survey the recent Drosophila literature linking cell polarity regulators with the Hippo pathway in epithelial tissue growth, neural stem cell asymmetric divisions and in cell migration in physiological and tumorigenic settings.

Robo-Enabled Tumor Cell Extrusion.

How aberrant cells are removed from a tissue to prevent tumor formation is a key question in cancer biology. Reporting in this issue of Developmental Cell, Vaughen and Igaki (2016) show that a pathway with an important role in neural guidance also directs extrusion of tumor cells from epithelial tissues.