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1.
PLoS Genet ; 14(10): e1007688, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30325918

RESUMO

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/genética
2.
Int J Mol Sci ; 22(23)2021 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-34884538

RESUMO

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/metabolismo
3.
PLoS Genet ; 9(7): e1003627, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23874226

RESUMO

The capacity of tumour cells to maintain continual overgrowth potential has been linked to the commandeering of normal self-renewal pathways. Using an epithelial cancer model in Drosophila melanogaster, we carried out an overexpression screen for oncogenes capable of cooperating with the loss of the epithelial apico-basal cell polarity regulator, scribbled (scrib), and identified the cell fate regulator, Abrupt, a BTB-zinc finger protein. Abrupt overexpression alone is insufficient to transform cells, but in cooperation with scrib loss of function, Abrupt promotes the formation of massive tumours in the eye/antennal disc. The steroid hormone receptor coactivator, Taiman (a homologue of SRC3/AIB1), is known to associate with Abrupt, and Taiman overexpression also drives tumour formation in cooperation with the loss of Scrib. Expression arrays and ChIP-Seq indicates that Abrupt overexpression represses a large number of genes, including steroid hormone-response genes and multiple cell fate regulators, thereby maintaining cells within an epithelial progenitor-like state. The progenitor-like state is characterised by the failure to express the conserved Eyes absent/Dachshund regulatory complex in the eye disc, and in the antennal disc by the failure to express cell fate regulators that define the temporal elaboration of the appendage along the proximo-distal axis downstream of Distalless. Loss of scrib promotes cooperation with Abrupt through impaired Hippo signalling, which is required and sufficient for cooperative overgrowth with Abrupt, and JNK (Jun kinase) signalling, which is required for tumour cell migration/invasion but not overgrowth. These results thus identify a novel cooperating oncogene, identify mammalian family members of which are also known oncogenes, and demonstrate that epithelial tumours in Drosophila can be characterised by the maintenance of a progenitor-like state.


Assuntos
Carcinogênese , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Sistema de Sinalização das MAP Quinases/genética , Neoplasias Epiteliais e Glandulares/genética , Proteínas Nucleares/genética , Animais , Proliferação de Células , Modelos Animais de Doenças , Proteínas de Drosophila/metabolismo , Neoplasias Oculares/genética , Neoplasias Oculares/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Neoplasias Epiteliais e Glandulares/patologia , Proteínas Nucleares/metabolismo , Proteína Oncogênica p65(gag-jun)/genética , Proteína Oncogênica p65(gag-jun)/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
4.
Pest Manag Sci ; 63(8): 803-8, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17514638

RESUMO

Piperonyl butoxide (PBO) is an insecticide synergist known to inhibit the activity of cytochrome P450 enzymes. PBO is currently used in some insecticide formulations, and has also been suggested as a pretreatment for some pesticide applications. Little is known about how insects respond to PBO exposure at the gene transcription level. The authors have characterised the transcriptional response of the Drosophila melanogaster genome after PBO treatment, using both a custom-designed 'detox' microarray, containing cytochrome P450 (P450), glutathione S-transferase (GST) and esterase genes, and a full genome microarray. A subset of P450 and GST genes is identified, along with additional metabolic genes, that are induced by PBO. The gene set is an extremely similar gene set to that induced by phenobarbital, a compound for which pretreatment is known to confer tolerance to a range of insecticide compounds. The implications of the induction of gene families known to metabolise insecticides and the use of PBO in pest management programs are discussed.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Drosophila melanogaster/efeitos dos fármacos , Glutationa Transferase/metabolismo , Sinergistas de Praguicidas/farmacologia , Butóxido de Piperonila/farmacologia , Animais , Sistema Enzimático do Citocromo P-450/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Genoma de Inseto , Glutationa Transferase/genética , Resistência a Inseticidas/genética , Masculino , Análise de Sequência com Séries de Oligonucleotídeos
5.
FEBS J ; 284(14): 2231-2250, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28544778

RESUMO

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.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulação da Expressão Gênica , Masculino , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas Tirosina Fosfatases não Receptoras/genética , Receptores Proteína Tirosina Quinases/genética , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo
6.
Insect Biochem Mol Biol ; 36(12): 934-42, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17098168

RESUMO

Modifications of metabolic pathways are important in insecticide resistance evolution. Mutations leading to changes in expression levels or substrate specificities of cytochrome P450 (P450), glutathione-S-transferase (GST) and esterase genes have been linked to many cases of resistance with the responsible enzyme shown to utilize the insecticide as a substrate. Many studies show that the substrates of enzymes are capable of inducing the expression of those enzymes. We investigated if this was the case for insecticides and the enzymes responsible for their metabolism. The induction responses for P450s, GSTs and esterases to six different insecticides were investigated using a custom designed microarray in Drosophila melanogaster. Even though these gene families can all contribute to insecticide resistance, their induction responses when exposed to insecticides are minimal. The insecticides spinosad, diazinon, nitenpyram, lufenuron and dicyclanil did not induce any P450, GST or esterase gene expression after a short exposure to high lethal concentrations of insecticide. DDT elicited the low-level induction of one GST and one P450. These results are in contrast to induction responses we observed for the natural plant compound caffeine and the barbituate drug phenobarbital, both of which highly induced a number of P450 and GST genes under the same short exposure regime. Our results indicate that, under the insecticide exposure conditions we used, constitutive over-expression of metabolic genes play more of a role in insect survival than induction of members of these gene families.


Assuntos
Cafeína/farmacologia , Proteínas de Drosophila/genética , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/genética , Indução Enzimática/efeitos dos fármacos , Inativação Metabólica/genética , Inseticidas/farmacologia , Fenobarbital/farmacologia , Animais , Sistema Enzimático do Citocromo P-450/genética , Esterases/genética , Perfilação da Expressão Gênica , Glutationa Transferase/genética
7.
PLoS One ; 10(7): e0132987, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26207831

RESUMO

During tumorigenesis, pathways that promote the epithelial-to-mesenchymal transition (EMT) can both facilitate metastasis and endow tumor cells with cancer stem cell properties. To gain a greater understanding of how these properties are interlinked in cancers we used Drosophila epithelial tumor models, which are driven by orthologues of human oncogenes (activated alleles of Ras and Notch) in cooperation with the loss of the cell polarity regulator, scribbled (scrib). Within these tumors, both invasive, mesenchymal-like cell morphology and continual tumor overgrowth, are dependent upon Jun N-terminal kinase (JNK) activity. To identify JNK-dependent changes within the tumors we used a comparative microarray analysis to define a JNK gene signature common to both Ras and Notch-driven tumors. Amongst the JNK-dependent changes was a significant enrichment for BTB-Zinc Finger (ZF) domain genes, including chronologically inappropriate morphogenesis (chinmo). chinmo was upregulated by JNK within the tumors, and overexpression of chinmo with either RasV12 or Nintra was sufficient to promote JNK-independent epithelial tumor formation in the eye/antennal disc, and, in cooperation with RasV12, promote tumor formation in the adult midgut epithelium. Chinmo primes cells for oncogene-mediated transformation through blocking differentiation in the eye disc, and promoting an escargot-expressing stem or enteroblast cell state in the adult midgut. BTB-ZF genes are also required for Ras and Notch-driven overgrowth of scrib mutant tissue, since, although loss of chinmo alone did not significantly impede tumor development, when loss of chinmo was combined with loss of a functionally related BTB-ZF gene, abrupt, tumor overgrowth was significantly reduced. abrupt is not a JNK-induced gene, however, Abrupt is present in JNK-positive tumor cells, consistent with a JNK-associated oncogenic role. As some mammalian BTB-ZF proteins are also highly oncogenic, our work suggests that EMT-promoting signals in human cancers could similarly utilize networks of these proteins to promote cancer stem cell states.


Assuntos
Carcinogênese/genética , Proteínas de Drosophila/fisiologia , Genes ras/fisiologia , Oncogenes/fisiologia , Receptores Notch/fisiologia , Dedos de Zinco/genética , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Análise em Microsséries , Proteínas Nucleares/química , Proteínas Nucleares/genética , Domínios e Motivos de Interação entre Proteínas/genética
8.
Dis Model Mech ; 6(3): 661-78, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23324326

RESUMO

The Ras oncogene contributes to ≈ 30% of human cancers, but alone is not sufficient for tumorigenesis. In a Drosophila screen for oncogenes that cooperate with an activated allele of Ras (Ras(ACT)) to promote tissue overgrowth and invasion, we identified the GTP exchange factor RhoGEF2, an activator of Rho-family signalling. Here, we show that RhoGEF2 also cooperates with an activated allele of a downstream effector of Ras, Raf (Raf(GOF)). We dissect the downstream pathways through which RhoGEF2 cooperates with Ras(ACT) (and Raf(GOF)), and show that RhoGEF2 requires Rho1, but not Rac, for tumorigenesis. Furthermore, of the Rho1 effectors, we show that RhoGEF2 + Ras (Raf)-mediated tumorigenesis requires the Rho kinase (Rok)-Myosin-II pathway, but not Diaphanous, Lim kinase or protein kinase N. The Rho1-Rok-Myosin-II pathway leads to the activation of Jun kinase (JNK), in cooperation with Ras(ACT). Moreover, we show that activation of Rok or Myosin II, using constitutively active transgenes, is sufficient for cooperative tumorigenesis with Ras(ACT), and together with Ras(ACT) leads to strong activation of JNK. Our results show that Rok-Myosin-II activity is necessary and sufficient for Ras-mediated tumorigenesis. Our observation that activation of Myosin II, which regulates Filamentous actin (F-actin) contractility without affecting F-actin levels, cooperates with Ras(ACT) to promote JNK activation and tumorigenesis, suggests that increased cell contractility is a key factor in tumorigenesis. Furthermore, we show that signalling via the Tumour necrosis factor (TNF; also known as Egr)-ligand-JNK pathway is most likely the predominant pathway that activates JNK upon Rok activation. Overall, our analysis highlights the need for further analysis of the Rok-Myosin-II pathway in cooperation with Ras in human cancers.


Assuntos
Transformação Celular Neoplásica/patologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Miosina Tipo II/metabolismo , Transdução de Sinais , Proteínas ras/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Apoptose , Antenas de Artrópodes/crescimento & desenvolvimento , Proteínas de Ciclo Celular , Diferenciação Celular , Forma Celular , Transformação Celular Neoplásica/metabolismo , Células Clonais , Drosophila melanogaster/enzimologia , Ativação Enzimática , Técnicas de Silenciamento de Genes , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Larva/metabolismo , Sistema de Sinalização das MAP Quinases , Modelos Biológicos , Proteínas Proto-Oncogênicas c-raf/metabolismo , Pupa/metabolismo , Regulação para Cima , Proteínas rac de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo
9.
Dis Model Mech ; 6(2): 521-9, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22996645

RESUMO

Anti-cancer drug development involves enormous expenditure and risk. For rapid and economical identification of novel, bioavailable anti-tumour chemicals, the use of appropriate in vivo tumour models suitable for large-scale screening is key. Using a Drosophila Ras-driven tumour model, we demonstrate that tumour overgrowth can be curtailed by feeding larvae with chemicals that have the in vivo pharmacokinetics essential for drug development and known efficacy against human tumour cells. We then develop an in vivo 96-well plate chemical screening platform to carry out large-scale chemical screening with the tumour model. In a proof-of-principle pilot screen of 2000 compounds, we identify the glutamine analogue, acivicin, a chemical with known activity against human tumour cells, as a potent and specific inhibitor of Drosophila tumour formation. RNAi-mediated knockdown of candidate acivicin target genes implicates an enzyme involved in pyrimidine biosynthesis, CTP synthase, as a possible crucial target of acivicin-mediated inhibition. Thus, the pilot screen has revealed that Drosophila tumours are glutamine-dependent, which is an emerging feature of many human cancers, and has validated the platform as a powerful and economical tool for in vivo chemical screening. The platform can also be adapted for use with other disease models, thus offering widespread applications in drug development.


Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Drosophila melanogaster/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Neoplasias/tratamento farmacológico , Animais , Benzamidas/farmacologia , Benzamidas/uso terapêutico , Disponibilidade Biológica , Proliferação de Células/efeitos dos fármacos , Citidina Trifosfato/biossíntese , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Difenilamina/uso terapêutico , Drosophila melanogaster/citologia , Glutamina/metabolismo , Isoxazóis/farmacologia , Isoxazóis/uso terapêutico , Farmacogenética , Projetos Piloto
10.
Insect Biochem Mol Biol ; 41(11): 863-71, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21807095

RESUMO

Organisms induce the expression of detoxification enzymes such as cytochrome P450s to deal with xenobiotics encountered in the environment. Research using cell culture systems has identified some of the cis-regulatory elements (CREs) and transcription factors involved in the induction of P450 genes in response to xenobiotic challenges. It was recently found that the CREs required for the basal expression of some P450s are distinct from the CREs involved in their induction. How these CREs mediate induction to xenobiotics in a tissue specific manner is not known. In this paper we show that, in Drosophila melanogaster, the induction response of the P450 gene Cyp6g1 to the xenobiotic Phenobarbital (PB) requires the presence of both tissue specific enhancers and a distinct CRE. The CRE does not drive gene expression but is required for the induction response. Site-directed mutagenesis of sequences within the CRE, sequences similar to mouse PB induction sequences, reduces the level of induction by PB, suggesting some degree of mechanistic conservation between flies and mice. This CRE may represent a unique class of CREs that has no inherent role in the basal transcriptional activity of genes, but is required for induction responses. Variations within this class of CREs may explain the variability of gene induction responses.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Animais , Sistema Enzimático do Citocromo P-450/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Fatores de Transcrição GATA/metabolismo , Trato Gastrointestinal/metabolismo , Larva/metabolismo , Túbulos de Malpighi/metabolismo , Camundongos , Fenobarbital , Elementos Reguladores de Transcrição , Xenobióticos
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