No abstract

SUMMARYAnti-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.

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.

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.

SUMMARYThe 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 (RasACT) 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 (RafGOF). We dissect the downstream pathways through which RhoGEF2 cooperates with RasACT (and RafGOF), 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 RasACT. Moreover, we show that activation of Rok or Myosin II, using constitutively active transgenes, is sufficient for cooperative tumorigenesis with RasACT, and together with RasACT 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 RasACT 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.