RESUMO
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.
Assuntos
Proteínas de Drosophila , Neoplasias , Animais , Adenosina Trifosfatases/metabolismo , Polaridade Celular , Drosophila/metabolismo , Drosophila melanogaster , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Via de Sinalização Hippo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismoRESUMO
Elevated Ras signalling is highly prevalent in human cancer; however, targeting Ras-driven cancers with Ras pathway inhibitors often leads to undesirable side effects and to drug resistance. Thus, identifying compounds that synergise with Ras pathway inhibitors would enable lower doses of the Ras pathway inhibitors to be used and also decrease the acquisition of drug resistance. Here, in a specialised chemical screen using a Drosophila model of Ras-driven cancer, we have identified compounds that reduce tumour size by synergising with sub-therapeutic doses of the Ras pathway inhibitor trametinib, which targets MEK, the mitogen-activated protein kinase kinase, in this pathway. Analysis of one of the hits, ritanserin, and related compounds revealed that diacyl glycerol kinase α (DGKα, Dgk in Drosophila) was the critical target required for synergism with trametinib. Human epithelial cells harbouring the H-RAS oncogene and knockdown of the cell polarity gene SCRIB were also sensitive to treatment with trametinib and DGKα inhibitors. Mechanistically, DGKα inhibition synergises with trametinib by increasing the P38 stress-response signalling pathway in H-RASG12V SCRIBRNAi cells, which could lead to cell quiescence. Our results reveal that targeting Ras-driven human cancers with Ras pathway and DGKα inhibitors should be an effective combination drug therapy.
Assuntos
Antineoplásicos , Neoplasias , Animais , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno , Drosophila , Linhagem Celular Tumoral , Transdução de Sinais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Neoplasias/tratamento farmacológicoRESUMO
Precise organ size control is fundamental for all metazoans, but how organ size is controlled in a three-dimensional (3D) way remains largely unexplored at the molecular level. Here, we screen and identify Drosophila Ptp61F as a pivotal regulator of organ size that integrates the Hippo pathway, TOR pathway, and actomyosin machinery. Pathologically, Ptp61F loss synergizes with RasV12 to induce tumorigenesis. Physiologically, Ptp61F depletion increases body size and drives neoplastic intestinal tumor formation and stem cell proliferation. Ptp61F also regulates cell contractility and myosin activation and controls 3D cell shape by reducing cell height and horizontal cell size. Mechanistically, Ptp61F forms a complex with Expanded (Ex) and increases endosomal localization of Ex and Yki. Furthermore, we demonstrate that PTPN2, the conserved human ortholog of Ptp61F, can functionally substitute for Ptp61F in Drosophila. Our work defines Ptp61F as an essential determinant that controls 3D organ size under both physiological and pathological conditions.
Assuntos
Actomiosina , Proteínas de Drosophila , Animais , Humanos , Proteínas de Drosophila/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/genética , Tamanho do Órgão , Transativadores/metabolismo , Proteínas Nucleares/metabolismo , Drosophila/metabolismo , Proteínas Tirosina Fosfatases não ReceptorasRESUMO
Oncogenic mutations in the small GTPase RAS contribute to ~30% of human cancers. In a Drosophila genetic screen, we identified novel and evolutionary conserved cancer genes that affect Ras-driven tumorigenesis and metastasis in Drosophila including confirmation of the tetraspanin Tsp29Fb. However, it was not known whether the mammalian Tsp29Fb orthologue, TSPAN6, has any role in RAS-driven human epithelial tumors. Here we show that TSPAN6 suppressed tumor growth and metastatic dissemination of human RAS activating mutant pancreatic cancer xenografts. Whole-body knockout as well as tumor cell autonomous inactivation using floxed alleles of Tspan6 in mice enhanced KrasG12D-driven lung tumor initiation and malignant progression. Mechanistically, TSPAN6 binds to the EGFR and blocks EGFR-induced RAS activation. Moreover, we show that inactivation of TSPAN6 induces an epithelial-to-mesenchymal transition and inhibits cell migration in vitro and in vivo. Finally, low TSPAN6 expression correlates with poor prognosis of patients with lung and pancreatic cancers with mesenchymal morphology. Our results uncover TSPAN6 as a novel tumor suppressor receptor that controls epithelial cell identify and restrains RAS-driven epithelial cancer.
Assuntos
Oncogenes , Neoplasias Pancreáticas , Tetraspaninas , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Genes ras , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Camundongos , Mutação , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Tetraspaninas/genética , Tetraspaninas/metabolismoRESUMO
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.
Assuntos
Carcinogênese/metabolismo , Competição entre as Células , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Neoplasias/prevenção & controle , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/patologia , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Tirosina Fosfatases não Receptoras/genética , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Cachexia, the wasting syndrome commonly observed in advanced cancer patients, accounts for up to one-third of cancer-related mortalities. We have established a Drosophila larval model of organ wasting whereby epithelial overgrowth in eye-antennal discs leads to wasting of the adipose tissue and muscles. The wasting is associated with fat-body remodeling and muscle detachment and is dependent on tumor-secreted matrix metalloproteinase 1 (Mmp1). Mmp1 can both modulate TGFß signaling in the fat body and disrupt basement membrane (BM)/extracellular matrix (ECM) protein localization in both the fat body and the muscle. Inhibition of TGFß signaling or Mmps in the fat body/muscle using a QF2-QUAS binary expression system rescues muscle wasting in the presence of tumor. Altogether, our study proposes that tumor-derived Mmps are central mediators of organ wasting in cancer cachexia.
Assuntos
Tecido Adiposo/metabolismo , Metaloproteinases da Matriz/metabolismo , Músculo Esquelético/metabolismo , Neoplasias/metabolismo , Animais , Membrana Basal/metabolismo , Drosophila/metabolismo , Matriz Extracelular/metabolismo , Atrofia Muscular/metabolismoAssuntos
Carcinogênese/genética , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Drosophila melanogaster , Epigênese Genética/fisiologia , Modelos Biológicos , Animais , Animais Geneticamente Modificados , Pesquisa Biomédica/métodos , Carcinogênese/patologia , Linhagem Celular , Transformação Celular Neoplásica/patologia , Ensaios de Seleção de Medicamentos Antitumorais , Epistasia Genética/fisiologia , Humanos , Pesquisa Translacional Biomédica/métodosRESUMO
The highly conserved c-Jun N-terminal Kinase (JNK) signalling pathway has many functions, regulating a diversity of processes: from cell movement during embryogenesis to the stress response of cells after environmental insults. Studies modelling cancer using the vinegar fly, Drosophila melanogaster, have identified both pro- and anti-tumourigenic roles for JNK signalling, depending on context. As a tumour suppressor, JNK signalling commonly is activated by conserved Tumour Necrosis Factor (TNF) signalling, which promotes the caspase-mediated death of tumourigenic cells. JNK pathway activation can also occur via actin cytoskeleton alterations, and after cellular damage inflicted by reactive oxygen species (ROS). Additionally, JNK signalling frequently acts in concert with Salvador-Warts-Hippo (SWH) signalling - either upstream of or parallel to this potent growth-suppressing pathway. As a tumour promoter, JNK signalling is co-opted by cells expressing activated Ras-MAPK signalling (among other pathways), and used to drive cell morphological changes, induce invasive behaviours, block differentiation, and enable persistent cell proliferation. Furthermore, JNK is capable of non-autonomous influences within tumour microenvironments by effecting the transcription of various cell growth- and proliferation-promoting molecules. In this review, we discuss these aspects of JNK signalling in Drosophila tumourigenesis models, and highlight recent publications that have expanded our knowledge of this important and versatile pathway.
RESUMO
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.
Assuntos
Polaridade Celular , Transformação Celular Neoplásica , Proteínas de Drosophila , Drosophila , Animais , Modelos Animais de DoençasRESUMO
Oncogenic mutations in the small GTPase Ras contribute to ~30% of human cancers. However, Ras mutations alone are insufficient for tumorigenesis, therefore it is paramount to identify cooperating cancer-relevant signaling pathways. We devised an in vivo near genome-wide, functional screen in Drosophila and discovered multiple novel, evolutionarily-conserved pathways controlling Ras-driven epithelial tumorigenesis. Human gene orthologs of the fly hits were significantly downregulated in thousands of primary tumors, revealing novel prognostic markers for human epithelial tumors. Of the top 100 candidate tumor suppressor genes, 80 were validated in secondary Drosophila assays, identifying many known cancer genes and multiple novel candidate genes that cooperate with Ras-driven tumorigenesis. Low expression of the confirmed hits significantly correlated with the KRASG12 mutation status and poor prognosis in pancreatic cancer. Among the novel top 80 candidate cancer genes, we mechanistically characterized the function of the top hit, the Tetraspanin family member Tsp29Fb, revealing that Tsp29Fb regulates EGFR signaling, epithelial architecture and restrains tumor growth and invasion. Our functional Drosophila screen uncovers multiple novel and evolutionarily conserved epithelial cancer genes, and experimentally confirmed Tsp29Fb as a key regulator of EGFR/Ras induced epithelial tumor growth and invasion.
Assuntos
Proteínas de Drosophila/genética , IMP Desidrogenase/genética , Neoplasias/genética , Tetraspanina 29/genética , Animais , Animais Geneticamente Modificados , Carcinogênese/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Genes ras , Testes Genéticos/métodos , Humanos , IMP Desidrogenase/metabolismo , Masculino , Camundongos , Neoplasias/metabolismo , Neoplasias/patologia , Oncogenes , Transdução de Sinais , Tetraspanina 29/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Diferenciação Celular , Quinases Ciclina-Dependentes/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Transativadores/metabolismo , Alelos , Animais , Morte Celular , Epistasia Genética , Epitélio/metabolismo , Mutação/genética , Fosforilação , Fase S , Transdução de Sinais , Asas de Animais/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Ácidos/metabolismo , Animais , Proteínas de Transporte/genética , Polaridade Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Epitélio/crescimento & desenvolvimento , Epitélio/metabolismo , Olho/crescimento & desenvolvimento , Olho/metabolismo , Feminino , Proteínas de Membrana/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Proteínas Supressoras de Tumor/genética , ATPases Vacuolares Próton-Translocadoras/genética , Vacúolos/metabolismoRESUMO
The Ras oncogene (Rat Sarcoma oncogene, a small GTPase) is a key driver of human cancer, however alone it is insufficient to produce malignancy, due to the induction of cell cycle arrest or senescence. In a Drosophila melanogaster genetic screen for genes that cooperate with oncogenic Ras (bearing the RasV12 mutation, or RasACT), we identified the Drosophila Src (Sarcoma virus oncogene) family non-receptor tyrosine protein kinase genes, Src42A and Src64B, as promoting increased hyperplasia in a whole epithelial tissue context in the Drosophila eye. Moreover, overexpression of Src cooperated with RasACT in epithelial cell clones to drive neoplastic tumourigenesis. We found that Src overexpression alone activated the Jun N-terminal Kinase (JNK) signalling pathway to promote actin cytoskeletal and cell polarity defects and drive apoptosis, whereas, in cooperation with RasACT, JNK led to a loss of differentiation and an invasive phenotype. Src + RasACT cooperative tumourigenesis was dependent on JNK as well as Phosphoinositide 3-Kinase (PI3K) signalling, suggesting that targeting these pathways might provide novel therapeutic opportunities in cancers dependent on Src and Ras signalling.
Assuntos
Carcinogênese , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteínas ras/metabolismo , Animais , Diferenciação Celular , Polaridade Celular , Olho Composto de Artrópodes/enzimologia , Olho Composto de Artrópodes/metabolismo , Olho Composto de Artrópodes/patologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/metabolismo , Epitélio/enzimologia , Epitélio/metabolismo , Epitélio/fisiopatologia , Feminino , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas pp60(c-src)/fisiologia , Proteínas ras/fisiologiaRESUMO
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.
Assuntos
Carcinogênese/patologia , Drosophila melanogaster/patogenicidade , Neoplasias/patologia , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Genes Supressores de Tumor/fisiologia , Humanos , Neoplasias/genética , Oncogenes/genéticaRESUMO
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.
Assuntos
Biomarcadores , Polaridade Celular/fisiologia , Transformação Celular Neoplásica , Organogênese , Transdução de Sinais , Animais , Regulação da Expressão Gênica , Humanos , Especificidade de Órgãos , Relação Estrutura-AtividadeRESUMO
The Hippo signaling pathway is a master regulator of organ growth, tissue homeostasis, and tumorigenesis. The activity of the Hippo pathway is controlled by various upstream components, including Expanded (Ex), but the precise molecular mechanism of how Ex is regulated remains poorly understood. Here we identify Plenty of SH3s (POSH), an E3 ubiquitin ligase, as a key component of Hippo signaling in DrosophilaPOSH overexpression synergizes with loss of Kibra to induce overgrowth and up-regulation of Hippo pathway target genes. Furthermore, knockdown of POSH impedes dextran sulfate sodium-induced Yorkie-dependent intestinal stem cell renewal, suggesting a physiological role of POSH in modulating Hippo signaling. Mechanistically, POSH binds to the C-terminal of Ex and is essential for the Crumbs-induced ubiquitination and degradation of Ex. Our findings establish POSH as a crucial regulator that integrates the signal from the cell surface to negatively regulate Ex-mediated Hippo activation in Drosophila.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitina/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas do Citoesqueleto/genética , Sulfato de Dextrana , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Deleção de Genes , Genoma , Intestinos/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteólise , Transdução de Sinais , Células-Tronco/efeitos dos fármacos , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
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.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células Epiteliais/citologia , Olho/citologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Asas de Animais/citologia , Animais , Polaridade Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Células Epiteliais/metabolismo , Olho/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Proteínas de Membrana , Proteínas do Tecido Nervoso/genética , Domínios PDZ , Ligação Proteica , Proteínas Supressoras de Tumor/genética , Asas de Animais/metabolismoRESUMO
Drosophila lethal giant larvae (lgl) encodes a conserved tumor suppressor with established roles in cell polarity, asymmetric division, and proliferation control. Lgl's human orthologs, HUGL1 and HUGL2, are altered in human cancers, however, its mechanistic role as a tumor suppressor remains poorly understood. Based on a previously established connection between Lgl and Fragile X protein (FMRP), a miRNA-associated translational regulator, we hypothesized that Lgl may exert its role as a tumor suppressor by interacting with the miRNA pathway. Consistent with this model, we found that lgl is a dominant modifier of Argonaute1 overexpression in the eye neuroepithelium. Using microarray profiling we identified a core set of ten miRNAs that are altered throughout tumorigenesis in Drosophila lgl mutants. Among these are several miRNAs previously linked to human cancers including miR-9a, which we found to be downregulated in lgl neuroepithelial tissues. To determine whether miR-9a can act as an effector of Lgl in vivo, we overexpressed it in the context of lgl knock-down by RNAi and found it able to reduce the overgrowth phenotype caused by Lgl loss in epithelia. Furthermore, cross-comparisons between miRNA and mRNA profiling in lgl mutant tissues and human breast cancer cells identified thrombospondin (tsp) as a common factor altered in both fly and human breast cancer tumorigenesis models. Our work provides the first evidence of a functional connection between Lgl and the miRNA pathway, demonstrates that miR-9a mediates Lgl's role in restricting epithelial proliferation, and provides novel insights into pathways controlled by Lgl during tumor progression.
RESUMO
In both Drosophila melanogaster and mammalian systems, epithelial structure and underlying cell polarity are essential for proper tissue morphogenesis and organ growth. Cell polarity interfaces with multiple cellular processes that are regulated by the phosphorylation status of large protein networks. To gain insight into the molecular mechanisms that coordinate cell polarity with tissue growth, we screened a boutique collection of RNAi stocks targeting the kinome for their capacity to modify Drosophila "cell polarity" eye and wing phenotypes. Initially, we identified kinase or phosphatase genes whose depletion modified adult eye phenotypes associated with the manipulation of cell polarity complexes (via overexpression of Crb or aPKC). We next conducted a secondary screen to test whether these cell polarity modifiers altered tissue overgrowth associated with depletion of Lgl in the wing. These screens identified Hippo, Jun kinase (JNK), and Notch signaling pathways, previously linked to cell polarity regulation of tissue growth. Furthermore, novel pathways not previously connected to cell polarity regulation of tissue growth were identified, including Wingless (Wg/Wnt), Ras, and lipid/Phospho-inositol-3-kinase (PI3K) signaling pathways. Additionally, we demonstrated that the "nutrient sensing" kinases Salt Inducible Kinase 2 and 3 (SIK2 and 3) are potent modifiers of cell polarity phenotypes and regulators of tissue growth. Overall, our screen has revealed novel cell polarity-interacting kinases and phosphatases that affect tissue growth, providing a platform for investigating molecular mechanisms coordinating cell polarity and tissue growth during development.