RESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Drosophila melanogaster/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Neoplasias/tratamiento farmacológico , Animales , Benzamidas/farmacología , Benzamidas/uso terapéutico , Disponibilidad Biológica , Proliferación Celular/efectos de los fármacos , Citidina Trifosfato/biosíntesis , Difenilamina/análogos & derivados , Difenilamina/farmacología , Difenilamina/uso terapéutico , Drosophila melanogaster/citología , Glutamina/metabolismo , Isoxazoles/farmacología , Isoxazoles/uso terapéutico , Farmacogenética , Proyectos PilotoRESUMEN
We have shown previously that mutations in the apico-basal cell polarity regulators cooperate with oncogenic Ras (Ras(ACT)) to promote tumorigenesis in Drosophila melanogaster and mammalian cells. To identify novel genes that cooperate with Ras(ACT) in tumorigenesis, we carried out a genome-wide screen for genes that when overexpressed throughout the developing Drosophila eye enhance Ras(ACT)-driven hyperplasia. Ras(ACT)-cooperating genes identified were Rac1 Rho1, RhoGEF2, pbl, rib, and east, which encode cell morphology regulators. In a clonal setting, which reveals genes conferring a competitive advantage over wild-type cells, only Rac1, an activated allele of Rho1 (Rho1(ACT)), RhoGEF2, and pbl cooperated with Ras(ACT), resulting in reduced differentiation and large invasive tumors. Expression of RhoGEF2 or Rac1 with Ras(ACT) upregulated Jun kinase (JNK) activity, and JNK upregulation was essential for cooperation. However, in the whole-tissue system, upregulation of JNK alone was not sufficient for cooperation with Ras(ACT), while in the clonal setting, JNK upregulation was sufficient for Ras(ACT)-mediated tumorigenesis. JNK upregulation was also sufficient to confer invasive growth of Ras(V12)-expressing mammalian MCF10A breast epithelial cells. Consistent with this, HER2(+) human breast cancers (where human epidermal growth factor 2 is overexpressed and Ras signaling upregulated) show a significant correlation with a signature representing JNK pathway activation. Moreover, our genetic analysis in Drosophila revealed that Rho1 and Rac are important for the cooperation of RhoGEF2 or Pbl overexpression and of mutants in polarity regulators, Dlg and aPKC, with Ras(ACT) in the whole-tissue context. Collectively our analysis reveals the importance of the RhoGEF/Rho-family/JNK pathway in cooperative tumorigenesis with Ras(ACT).
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Genes ras , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Lesiones Precancerosas/enzimología , Proteínas de Unión al GTP rho/metabolismo , Animales , Diferenciación Celular , Línea Celular , Proliferación Celular , Forma de la Célula , Supervivencia Celular , Células Clonales , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Ojo/citología , Ojo/crecimiento & desarrollo , Ojo/ultraestructura , Genes de Insecto/genética , Humanos , Sistema de Señalización de MAP Quinasas , Lesiones Precancerosas/patología , Proteína Quinasa C/metabolismo , Reproducibilidad de los Resultados , Factores de Intercambio de Guanina Nucleótido Rho , Regulación hacia Arriba/genéticaRESUMEN
Statins are a class of drugs widely used for lowering high cholesterol levels through their action on 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme in the synthesis of cholesterol. We studied the effects of two major statins, simvastatin and atorvastatin, on five Candida species and Aspergillus fumigatus. The statins strongly inhibited the growth of all species, except Candida krusei. Supplementation of Candida albicans and A. fumigatus with ergosterol or cholesterol in aerobic culture led to substantial recovery from the inhibition by statins, suggesting specificity of statins for the mevalonate synthesis pathway. Our findings suggest that the statins could have utility as antifungal agents and that fungal colonization could be affected in those on statin therapy.