Drosophila melanogaster

REVIEWS

MODELS OF CANCER

Drosophila melanogaster: a model and a tool to investigate malignancy and identify new therapeutics Cayetano Gonzalez

Abstract | For decades, lower-model organisms such as Drosophila melanogaster have often provided the first glimpse into the mechanism of action of human cancer-related proteins, thus making a substantial contribution to elucidating the molecular basis of the disease. More recently, D. melanogaster strains that are engineered to recapitulate key aspects of specific types of human cancer have been paving the way for the future role of this ‘workhorse’ of biomedical research, helping to further investigate the process of malignancy, and serving as platforms for therapeutic drug discovery.

IRB-Barcelona, c/Baldiri Reixac 10–12, Barcelona, Spain. Institucio Catalana de Recerca i Estudis Avançats (ICREA), Passeig Lluís Companys 23, Barcelona, Spain. e‑mail: gonzalez@ irbbarcelona.org doi:10.1038/nrc3461 Published online 7 February 2013

Despite considerable advances, cancer treatment remains suboptimal, underscoring the need for new models with which to tackle this multifaceted disease. Lower-model organisms such as the vinegar fly Drosophila melanogaster might contribute greatly to this end. All model systems have limitations, and D. melanogaster is no exception. Anatomical and physiological differences between humans and flies are many and major, and might preclude the modelling of key aspects of cancer as it proceeds in vertebrates (BOX 1). However, malignant tumours in mammals and flies are made of cells that derail from their normal course of development, grow out of control, become immortal and invasive, and kill the host. Understanding the molecular bases of these and other malignant traits that are common to both flies and mammals might be facilitated by exploiting a genetically tractable organism such as D. melanogaster that allows for sophisticated functional assays and genome-wide studies at a rate and with a level of detail that are unmatched by current mammalian models (BOX 1). This Review summarizes the contribution of D. mela nogaster to cancer research and highlights likely future developments in this field. The Review mostly covers emerging research and only briefly mentions other aspects of cancer research in flies that have been abundantly reviewed elsewhere. I discuss how data derived from basic research in flies often provide information on proteins that are relevant to human cancer. I also present recent advances in creating fly tumours that recapitulate key aspects of human malignant tumours, and discuss how these are helping us to understand the disease. I discuss new data derived from flies on how asymmetric

division, genome instability, centrosome dysfunction, metabolism and unscheduled gene expression impinge on tumour initiation and cancer progression. I also cover exciting results obtained using D. melanogaster both to optimize the therapeutic effect of known compounds and as a platform to screen for new ones. This new contribution of flies to biomedical research has already delivered drugs that are currently undergoing clinical trials.

The silent contribution of D. melanogaster More than 50% of the proteins that cause human diseases, including cancer, have orthologues in D. melanogaster 1,2. In many cases, conservation is such that the corresponding human genes can rescue the loss of function of their D. melanogaster orthologues. Therefore, it is not surprising that conclusions derived from research in flies are often relevant to biomedicine. This is the basis of what is probably the least appreciated, but most important, contribution to date of D. melanogaster to molecular oncology: a great deal of data generated during decades from basic, seemingly non-applied, research that nonetheless have provided key insights into the singular function of numerous factors and their spatiotemporal coordination in molecular axes that are important for human cancer 3. In many cases, the identification of the protein and its molecular function in D. melanogaster has preceded the discovery of the link to cancer of the corresponding human homologue. For example, Notch was identified in the first half of the twentieth century as a gene, which when missing or mutated, results in a mutant fly with notched wings, and genetic and molecular studies in flies have unveiled a host of Notch modulators and targets,

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REVIEWS At a glance • For a century, seemingly non-applied research carried out in Drosophila melanogaster has provided the first glimpse into the mechanism of action of human cancer-related proteins. • Natural malignant tumours can occur in D. melanogaster. • Tumours can also be experimentally induced in larvae and adult flies either by knocking down fly tumour suppressor genes or by recreating in flies the mutant conditions that are causative of certain human cancer types. Current examples of this ‘a la carte’ design include cancer models of glioblastoma, rhabdomyosarcoma, multiple endocrine neoplasia and leukaemia. • D. melanogaster tumours range from hyperplasias to frankly malignant neoplasias that are invasive and lethal to the host. • Both the presence and the lack of supernumerary centrosomes can cause tumours in the larval brain, but unbalanced karyotypes do not. Thus, in D. melanogaster, Boveri’s hypothesis does not apply, but centrosome dysfunction is linked to cancer. • The origin of the widespread genome instability that is characteristic of cancer cells is likely to be multifactorial. • The loss of cell polarity in cells that divide asymmetrically, as well as in epithelial tissues, is often tumorigenic. • The Aurora and POLO protein kinases are tumour suppressors in the larval brain. • The activation of signalling pathways that sense low calorie intake and inhibit target of rapamycin (TOR) compromises cortical polarity and contributes to tumour growth. • Data derived from D. melanogaster strongly substantiate the view that timely repression of gene expression programmes during development has a pronounced tumour suppression function. • D. melanogaster lethal (3) malignant brain tumour (l(3)mbt) tumours recapitulate the ectopic expression of cancer germline (CG; also known as cancer testis (CT)) genes that are observed in many types of somatic human tumours. In D. melanogaster, inactivation of some CG genes inhibits tumour growth. • D. melanogaster is starting to have an important role in chemical genetics, helping to identify the pathways that are affected by current pharmaceuticals, facilitating the design of more efficient derivatives and serving as a platform for semi-automated screens for new anticancer drugs.

most of which are evolutionarily conserved. Decades after its identification in flies, aberrant expression of human NOTCH1 was found to be a causative factor for T cell acute lymphoblastic leukaemia4. Activated Notch signalling has now been implicated in many haematopoietic and solid tumours5–7. Like Notch, the Hedgehog signalling pathway takes its name from the D. melanogaster segment polarity gene hedgehog (hh) and, similar to Notch, its basic circuitry has mostly been worked out in flies. Mutations that deregulate HH signalling are directly implicated in basal cell carcinoma and medulloblastoma8. Indeed, the same applies to the Salvador–Warts–Hippo pathway that has been extensively studied in D. melanogaster and that is also involved in human tumorigenesis. Some of the founding members of this pathway, including Warts (WTS), Expanded (EX) and Fat (FT), were identified through the pioneering work in the discovery of tumour suppressors in D. melanogaster that was carried out by E. Gateff, P. Bryant, A. Shearn, B. Mechler and others9,10. Recently, studies on the control of homeostasis in the adult D. melanogaster midgut have provided new data on the role of this pathway in stem cells11,12, including the identification of the adherens junction tyrosine phosphatase PEZ13 as an evolutionarily conserved binding partner of the upstream Hippo signalling component KIBRA14. A final, interesting note regarding cancer-relevant signalling pathways, is that constitutive activation of Janus

kinase (JAK)–signal transducer and activator of transcription (STAT) was found to be linked to haematopoietic overgrowth in flies earlier than it was found to be involved in human leukaemia15,16. Basic studies carried out in D. melanogaster 40 years ago led to the discovery of the phenomenon known as cell competition by which imaginal disc cells with higher fitness survive and proliferate at the expense of neighbouring cells with lower fitness17. It is now well established that besides normal development, cell competition operates between transformed and non-transformed cells during tumour growth18–21, and research in flies continues to uncover the networks that modulate this process22–28. These studies have now reached a new dimension by the implementation of time-lapse imaging analyses, which afford direct observations of cell elimination as it occurs in the living epithelia in certain mutant backgrounds, and these analyses have revealed an intrinsic tumour-suppression mechanism that eliminates pre-malignant cells from the epithelia29,30. It was also through the seemingly unlikely procedure of screening among a collection of female sterile mutants in flies that some master regulators of the cell cycle such as POLO and Aurora (AUR) were identified31,32. These two serine/threonine kinases are the founding members of the families of AUR and POLOlike kinases (PLKs) that function in many steps of mitosis and that have been linked to a wide range of tumour types in humans33–35 (see the Atlas of Genetics and Cytogenetics in Oncology and Haematology website; see Further information). Their relevance to oncology is underscored by the several inhibitors of these kinases that are currently undergoing clinical trials for cancer therapy 36. There are multiple examples of how studies on D. mel anogaster are unveiling important properties of molecules that are causative of human cancer. For example, research on the fly homologues of the human colon tumour suppressor adenomatous polyposis coli (APC) has shown that in certain cell types this molecule is essential to control spindle orientation37, that APC subcellular localization is not functionally relevant for WNT signalling 38 and that the APC amino terminus can enhance ectopic WNT signalling, which may account for the selective retention of such truncated APC variants in human colorectal carcinomas39. Similarly, studies on D. melanogaster development have discovered a previously unsuspected function of anaplastic lymphoma kinase (ALK) in sparing the central nervous system more than other tissues during nutrient restriction by suppressing amino acid sensing and constitutively activating PI3K signalling 40. Human ALK has been found to be constitutively active in a variety of tumours (see the Atlas of Genetics and Cytogenetics in Oncology and Haematology). A final example of a list that could include hundreds on how basic research in flies sheds light on cancer-relevant proteins is that genetic analysis of D. melanogaster BRCA2 has shown it to be required for homologous recombination and for avoiding the error-prone single-strand annealing pathway, which frequently results in large flanking deletions41. Heterozygous mutations in BRCA2 confer a high risk of breast and other cancers in humans (see the Atlas of Genetics and Cytogenetics in Oncology

NATURE REVIEWS | CANCER

VOLUME 13 | MARCH 2013 | 173 © 2013 Macmillan Publishers Limited. All rights reserved

REVIEWS Genetic screens Experimental technique aimed at identifying mutant genes that cause a particular phenotype.

and Haematology). This is also one of the many examples of how proteins that exert their tumour suppressor activity in human tissues for which there are no analogous tissues in flies (for example, the mammary gland) may also be better understood thanks to research carried out in D. melanogaster. A fly’s eye view of cancer. Special mention must be made of the use of the developing D. melanogaster eye as an in vivo ‘test tube’ to identify genetic interactions of D. melano gaster and human cancer-relevant genes (FIG. 1). This approach, which so convincingly proved its worth in the characterization of the RAS signalling pathway42, has rendered a great deal of data, such as the recent identification of lethal giant larvae (LGL), atypical protein kinase C (aPKC) and Crumbs as upstream regulators of the Hippo pathway43; TAO1 (also known as sterile 20‑like kinase) as a new member of this pathway and potential tumour suppressor 44; the redox regulation of PTEN by Thioredoxin (TRX1; also known as TXN)45; the role that phosphorylation by glycogen synthase kinase 3β (GSK3β) has in the binding of β-catenin by APC46; and the identification of DJ1 (also known as parkinson protein 7), a novel regulator

Box 1 | Drosophila melanogaster positives and negatives Positives • Small size, simple husbandry, short generation time and highly prolific nature. • Complex metazoan that shares many key features with higher organisms, such as a segmented body plan, sensory and motor systems, sexual behaviour, learning and memory ability, innate immunity and systemic responses. • Absence of meiotic recombination in males, simple karyotype, giant polytene chromosomes, synthetic balancer chromosomes, exoskeleton with bristles, wing veins and compound eyes that provide an easy readout for mutant phenotypes. • High extent of homology to the human genome and comparatively low redundancy of the fly genome. • Excellent descriptions of Drosophila melanogaster anatomy, histology and cell types. • Thousands of mutants, enhancer and protein traps, and stock collections of nearly genome-wide double-stranded RNAs for RNA interference (RNAi). • Established D. melanogaster cell lines, some of which are ideally suited for RNAi. • Wealth of techniques that make highly sophisticated experiments possible, including high-resolution microscopy of living cells and organs, and the inactivation or misexpression of almost any gene in a timely and tissue-specific or even cell-specific manner. • Public stock centres and databases. A culture of sharing published strains and reagents that is deeply rooted in the D. melanogaster research community. Negatives • Unlike other model animals, fly strains cannot be kept frozen. • Homologous recombination is cumbersome in flies compared with other model systems. • Absence of tissue types that are present in mammals, such as cartilage, bone and blood. • Absence of mammalian organs. D. melanogaster corpora cardiaca and fat body have some of the functions carried out by the mammalian pancreas, liver and adipose tissue, but are quite different from them. • Lack of an adaptive immune response. • Open circulatory system. The absence of veins and arteries precludes the modelling of some important processes; for example, metastatic intravasation and extravasation. • Only four chromosome pairs. This is a drawback for modelling the effect of aneuploidy because only a few combinations of single chromosome loss or gain are possible, and all are cell lethal.

of the tumour suppressor PTEN47, to cite only five from a long list of cancer-relevant discoveries. This assay has also been applied to determine the nature of ALK mutants linked to neuroblastoma. For example, ectopic expression of human ALK(F1174S) in the developing D. melanogaster eye causes a strong rough eye phenotype48, thus revealing the ligand-independent nature of this mutant. By contrast, neither ALK(R1464STOP) nor ALK(F1174L) generates rough eyes in this assay, which suggests that these mutants are ligand-dependent in nature49. These data help to determine whether to treat patients with ALK inhibitors. Another successful application of the rough eye assay in flies revealed that myocyte-specific enhancer factor 2A (MEF2A), MYC-binding protein 2 (MYCBP2), solute carrier family 17 member 5 (SLC17A5), bone morphogenetic protein 2 (BMP2), cytoplasmic polyadenylation elementbinding protein 1 (CPEB1) and cell division cycle 16 (CDC16) genetically interact with E‑cadherin mutants found in patients with diffuse gastric cancer50,51 and might, therefore, be implicated in the progression of the disease. Finally, studies showing that the cytotoxin-associated gene A (CagA) protein of Helicobacter pylori can rescue ommatidia development in the absence of the D. melanogaster GRB2‑associated binding protein (GAB) homologue, Daughter of Sevenless (DOS), provided the first demonstration that CagA functions as a GAB adaptor protein52. CagA is translocated into host cells and is a major risk factor for human gastric adenocarcinoma. These are also examples of how D. melanogaster can be useful for learning about cancer types that do not have a fly equivalent.

Creating cancer in flies Current cancer models in flies, which affect a variety of organs in both adult flies and developing larvae (FIG. 2), fall into one of three classes: natural tumours, tumours caused by mutants obtained by genetic screens and tumours made ‘a la carte’. In many of these models, tumour cells are invasive and can colonize other tissues53. Natural tumours. Because human cancer is a multistage process that often develops over decades it is definitively counterintuitive that anything similar might occur over the very short lifespan of D. melanogaster, which lasts only in the order of 6 to 8 weeks. This seemingly solid argument has led to the generalized assumption that flies do not get cancer. However, recent data have proved this assumption wrong. Tumours of the testis and gut are frequent in wild-type laboratory strains of D. melanogaster 54. Most importantly, the incidence of these tumours increases with age, demonstrating that despite the orders of magnitude difference in lifespan ageing is also a risk factor for tumour development in flies. Larval tumours are also expected to be fairly frequent in wild-type populations55. Understanding the mechanisms of origin and the growth of D. melanogaster natural malignant tumours might unveil principles that apply to human cancer. Genetic screens. D. melanogaster might also help us to understand cancer by the more direct approach of carry ing out genetic screens that interrogate the entire genome for tumour suppressor functions. Pioneering research

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REVIEWS Homologues of these might function as tumour suppressors in mammals by balancing self-renewal and the differentiation of stem cells64.

Eye development

Gain or loss of function of Drosophila melanogaster homologues of human cancer genes Under normal conditions in wild-type flies

Ectopic expression of human cancer genes

Normal eye

Rough eye

Mutations or chemical compounds

Figure 1 | Using eye development for functional assays. The compound Drosophila melanogaster eye consists of a hexagonal array of about 800 ommatidia that are | Cancer Nature Reviews homogeneous in size and regularly spaced. Each ommatidium is composed of eight photoreceptor cells and 12 accessory cells166. Such a reiterative structure makes the adult eye particularly amenable to genetic analysis because even subtle defects in ommatidia development are amplified several hundred-fold in the entire eye, thus leading to the disruption of the normal homogeneous lattice166. Moreover, because the eye is a non-essential organ for organismal survival, gain or loss of function conditions that interfere with cell proliferation, growth, determination or differentiation during eye development can be easily scored in the adult fly as a rough eye phenotype. This assay might be used to study the function of D. melanogaster genes that have homology with human cancer genes. It may also be used to study the effect of expressing human mutant genes that are linked to cancer. Suppression of the rough eye phenotype is a sensitive readout for identifying genes or chemicals that modulate human oncogenes and tumour suppressors. Fly eye images are reproduced, with permission, from REF. 167 © (2008) Macmillan Publishers Ltd. All rights reserved.

D. melanogaster neuroblasts Precursors that generate neural tissue.

Rhabdomyosarcoma Malignancy of muscle myoblasts that fail to exit the cell cycle and do not fuse into syncytial skeletal muscle.

Syncytial skeletal muscle Muscle made of multinucleated cells, also known as fibres, formed by the fusion of thousands of individual myoblast cells.

based on this approach has generated dozens of tumour types of different grades that range from benign hyperplasia to frankly malignant neoplasia56,57. Notably, these studies identified the first tumour suppressor gene, contemporarily with the experiments that gave rise to the term tumour suppressor in human oncology 58–60. An excellent critical review of the contribution of these pioneer studies has been published61. Good, old-fashioned genetic screens are far from exhausted, and new ones are currently being carried out. One of these screens has identified tumour suppressors that affect the recently discovered renal and nephric stem cells (RNSCs). RNSCs are multipotent cells that can generate all cell types of the adult Malpighian tubules, which are the fly equivalent of the mammalian kidney 62. Mutations that compromise scribbled (scrib) or salvador (sav) or that activate RAS in RNSCs cause overgrowth in the lower tubules and ureters62,63. A different approach to the same goal of searching for new functions that are involved in cancer is a genome-wide, RNA interference (RNAi) screen that has identified more than 600 genes that control self-renewal of D. melanogaster neuro blasts, many of which are brain tumour suppressors.

Tumours ‘a la carte’. A third class of cancer models in D. melanogaster includes those designed to recreate in flies the combination of loss and gain of function conditions that are causative of certain human cancer types. The first efforts in this direction yielded the cooperative tumorigenesis models in which the expression of oncogenic mutants, such as RasV12, was complemented with mutants that disrupt cell polarity such as scrib or discs large (dlg), to generate invasive tumours in imaginal discs65–67. SCRIB expression is deregulated in human prostate cancer, and its deficiency in mice promotes prostate neoplasia68,69. Tumours have also been cooperatively produced by combining RAS activation and mitochondrial dysfunction. RAS activation and mitochondrial dysfunction cooperatively stimulate the production of reactive oxygen species (ROS), thus activating JUN N‑terminal kinase (JNK) signalling. The cooperation of oncogenic RAS with JNK inactivates the Salvador– Warts–Hippo pathway, leading to the upregulation of the interleukin‑6 and WNT homologues Unpaired (UPD) and Wingless (WG)70. The combination of RAS activation and mitochondrial dysfunction also exerts an effect in neighbouring cells with activated RAS but normal mitochondrial function, causing them to exhibit metastatic behaviour 29,70. These are important results because mitochondrial function is frequently impaired in human cancer 71. Cooperative hyperplastic growth can also be triggered when cells of the imaginal wing disc that are driven into apoptosis by cellular stress conditions, such as X‑radiation, heat shock or aneuploidy, are kept alive by expressing the caspase inhibitor P35 (REFS 72,73). Other examples of a la carte design include cancer models of glioblastoma, rhabdomyosarcoma, leukaemia and gastric cancer. D. melanogaster models of glioblastoma, the most common and most malignant human brain tumour, have been developed by manipulating pathways that are known to be affected in human glioblastoma, such as constitutive co‑activation of epidermal growth factor receptor (EGFR)–RAS and PI3K signalling pathways in larval glia74,75. Glia thus transformed are highly proliferative and generate multilayered aggregations that develop as malignant immortal neoplasms in allograft assays. Consistent with a high frequency of RB pathway mutations in human glioblastoma, the loss of Rbf1, one of the two RB genes in flies, strongly enhances the development of this type of tumour in D. melanogaster 74. These models hold great potential for further characterizing gliomagenic pathways. A second example is rhabdomyosarcoma, a malignancy of muscle myoblasts, which fail to fuse into syncytial skeletal muscle. Transgenic expression in flies of the human fused transcription factors paired box 3 (PAX3)–forkhead box O1 (FOXO1) or PAX7–FOXO1, which are found in some human rhabdomyosarcomas76, results in cells that detach from myofibres and invade non-muscular tissue compartments77. A screen for suppressors of this phenotype in flies identified rolling pebbles (rols) as a downstream



REVIEWS a

Epithelial tumours following: • Activated RAS and polarity loss65–67 • Activated RAS and mitochondrial dysfunction70 • Cellular stress and caspase inhibition72,73 • Loss of L(2)GL58

Imaginal discs Brain

Glial tumours (model of glioblastoma) following activation of EGFR–RAS and PI3K74,75 Neuroblast tumours following: • Compromised • Loss of AUR or asymmetric division104 POLO109,124–126

Muscle

Neuroepithelial tumours following loss of L(3)MBT145 Myoblast tumours (model of rhabdomyosarcoma) following PAX–FOXO1 expression77

Haemolymph

Tumours from haemotopoietic precursors (model of AML) following AML1–ETO expression82,83

b

Gut Brain

Malpighian tubule Testes or ovaries

Neuroblast tumours following BRAT loss during larval development168

Tumours from ISCs: • Natural tumours54 • EGFR–RAS, UPD–JAK–STAT, SAV–WTS–HPO, WNT–APC89,90,92,93 • Following compromised asymmetric division117 • Following loss of MUS309 (REF. 111)

Natural tumours in testes54 Follicle cell tumours following loss of LKB1 or AMPK in fooddeprived flies134,135

Tumours from RNSCs following RAS activation, loss of SCRIB or loss of SAV63

Figure 2 | Tumours in flies. Currently available tumour types in Drosophila melanogaster Nature Reviews | Cancer affect different organs in larvae (part a) and adult flies (part b). Only some examples are shown. Some D. melanogaster tumours are more or less dysplastic hyperplasias that present during larval development. This type of tumour gives rise to terminally differentiated adult structures and, therefore, bears little resemblance to cancer in mammals. Others, however, display three main characteristic malignant traits: they are neoplastic; refractory to differentiation signals; and, perhaps most importantly, they are immortal, as reflected by their ability to be maintained through successive rounds of biweekly allograft culture in the abdomen of adult host flies. Moreover, allografted tumours of this kind invade the organs of the host, causing the host to die, as do human malignant tumours xenografted into mice. D. melanogaster malignant neoplasms display genomic and centrosomal abnormalities that are also frequent in cancer in mammals. Thus, despite distinctive main differences, malignant neoplasms in D. melanogaster and mammals do share some fundamental traits. AML, acute myeloid leukaemia; AMPK, AMP-activated protein kinase; APC, adenomatous polyposis coli; AUR, Aurora; BRAT, brain tumour; EGFR, epidermal growth factor receptor; FOXO, forkhead box O; HPO, Hippo; ISC, intestinal stem cell; JAK, Janus kinase; L(2)GL, lethal (2) giant larvae; L(3)MBT, lethal (3) malignant brain tumour; MUS309, mutagen sensitive 309; PAX, paired box; RNSC, renal and nephric stem cell; SAV, Salvador; SCRIB, Scribbled; STAT, signal transducer and activator of transcription; UPD, Unpaired; WTS, Warts.

effector of PAX7–FOXO1 (REF. 78). Consistent with the results obtained in D. melanogaster, decreased expression of TANC1, the mammalian orthologue of rols, causes rhabdomyosarcoma cells to fuse, forming differentiated syncytial muscle, and thus losing neoplastic potential. These results point to TANC1 as a target of choice for dif ferentiation therapy. A third example of made‑to‑measure models of human cancer in D. melanogaster recapitulates some aspects of acute myeloid leukaemias (AMLs) that carry a fusion between the AML1 (also known as RUNX1) transcription factor and the transcriptional co-repressor ETO79,80. Remarkably, expression of the human AML1–ETO fusion in D. melanogaster leads to the expansion of the haematopoietic precursors and to the formation of melanotic tumours in the fly. Because the AML1–ETO fusion is not by itself sufficient to cause leukaemia in mice it is suspected that other key factors cooperate with AML1–ETO to cause AML81. Some of these factors might correspond to the modifiers of the AML1–ETO expression phenotype that have been identified in flies, including the protease Calpain B (CALPB) and proteins that control the levels of ROS82,83. Finally, recent studies in flies have shown that expression of the H. pylori protein CagA in a few epithelial cells disrupts tissue integrity and induces apoptosis through the activation of JNK signalling, and that co‑expression of CagA with oncogenic RAS enhances tumour growth and invasion84. These are but a few examples. Other tumour models are available in flies, including germline and intestinal tumours (FIG. 2). Interestingly, work in intestinal tumours has demonstrated a role for Apc and armadillo (the D. melanogaster homologue of CTNNB1 (which encodes β-catenin)) in regulating intestinal stem cell proliferation85–89, and has unveiled a tantalizing synergy between bacterial infection and genetic predisposition in intestinal dysplasia90. Moreover, recent studies have identified different signalling pathways that prevent dysplastic growth by maintaining the balance of self-renewal and differentiation of the intestinal stem cells10–12,91–93.

The unstable genome of cancer cells Dysfunction of the mechanisms that maintain genome integrity are common in cancer cells. Consequently, point mutations, as well as structural and numerical chromosome alterations, are a distinct feature of malignant tumours94. However, it is unclear to what extent such changes might cause, or be a consequence of, cancer 95–97. Investigating the cause. The origin of the widespread genome instability that is characteristic of cancer cells is likely to be multifactorial. D. melanogaster is being used to investigate possible causes (FIG. 3), one of which might be telomere loss. Human telomeres are made of GC‑rich repeats extended by telomerase, which are protected by the shelterin complex; D. melanogaster telomeres are made of retroelements, maintained by transposition, which are capped by the terminin complex 98. Thus, despite notable differences in the molecular components, the same logic to protect the ends of linear chromosomes applies to both

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REVIEWS Mitochondria

2n

DNA replication without mitosis in normal development

Defective export of citrate to the cytoplasm

Single telomere loss Citrate ↓

Acetyl-CoA ↓

Falling Acetyl-CoA levels

8n Ac Ac

Ac

Ac

Defective histone acetylation

Nucleosome Aneuploidy and chromosome breaks

Figure 3 | Different routes to genomic instability. Three routes to genomic instability that have recently been characterized in Drosophila melanogaster are depicted. Genomic Nature Reviews Cancer instability is high in naturally occurring polyploid cells in the adult fly papillae, as |well as in diploid cells following single telomere loss, or following falling citrate levels in the cytoplasm. Ac, acetylation.

Differentiation therapy Reprogramming neoplastic cells to terminally differentiate.

Multipolar spindles Spindles that have more than two poles.

Lagging chromosomes Chromosomes that fail poleward anaphase movement.

revealed that, in D. melanogaster, as in mammals, cells with multiple centrosomes form multipolar spindles but that these are transient and normally become bipolar before mitosis proceeds105. In mammalian cells this often results in lagging chromosomes106,107 that can form micronuclei where asynchronous DNA replication results in DNA damage108. Together, these studies strongly suggest that a variety of events, by no means limited to the ones described here, might contribute to generating the widespread genome instability that characterizes cancer cells.

flies and humans98. Indeed, the term telomere was coined by H. Muller following his discovery of the lethal nature of terminal deletions in D. melanogaster 99. Studies in flies have assayed the effect of single chromosome telomere loss, a condition that is difficult to reproduce in other model systems. Only a few cells affected by telomere loss escape death, but those that do proliferate and display the wide range of chromosomal abnormalities that are common in cancer cells100. A second study has unveiled a link between genome instability and defective intermediary metabolism. Chromosome integrity crucially relies on efficient DNA repair, which is only possible after changes in chromatin structure that are mediated by histone acetylation. Acetyl-CoA, the key metabolite required for acetylation is derived from citrate that is exported from mitochondria. Loss of function of the D. melanogaster mitochondrial citrate transporter Scheggia (SEA) leads to extensive chromosomal breakage101. The same happens in primary fibroblasts on inhibition of the human citrate transporter SLC25A1. A third recent study focused on a type of cells located in the adult rectal papillae of flies. During the course of normal development these cells become polyploid because they undergo rounds of DNA replication without mitosis, but later they enter mitosis. Remarkably, errors in chromosome integrity and segregation are frequent in these wild-type cells. This observation suggests that polyploid mitosis has an intrinsic genome instability activity 102 so that polyploid tumour cells, regardless of their origin, might make an important contribution to genome instability in cancer. Studies in D. melanogaster have also addressed the link between extra centrosomes and aneuploidy. Supernumerary centrosomes are frequent in human and D. melanogaster tumour cells103,104. Live imaging has

Investigating the effect. As in human cancer, aneuploidy is a landmark of malignant growth in D. melanogaster larval brains104. However, a series of mutant conditions that generate cells with a wide range of aneuploid pheno types fail to cause tumorigenesis both in larval brains and in imaginal discs72,109. This result strongly suggests that unbalanced chromosome number is not an efficient tumorigenic event in flies (FIG. 4). It does not discard, however, a possible contribution of aneuploidy to the acquisition of malignant traits, a hypothesis that deserves further testing in D. melanogaster. Conversely, loss of mutagen sensitive 309 (mus309) in adult flies, which causes frequent chromatid exchange events and an increased rate of spontaneous mutagenesis, does significantly increase tumour incidence, hence demonstrating a clear link between genome integrity and tumour suppression110,111. In humans, mutations in BLM, the human homologue of D. melanogaster mus309, cause Bloom syndrome, a rare disorder that is associated with cancer predisposition and premature ageing 112.

Asymmetric cell division and tumorigenesis Recent, but still limited, evidence suggests a link between failed asymmetric division (BOX 2) and certain human cancers113–116. Such a link was first demonstrated in D. melano gaster larval brain tumours104. New data suggest that perturbing asymmetric cell division or the orientation of division of D. melanogaster intestinal stem cells also results in the formation of tumours in the fly gut 117. Current knowledge of the molecular cell biology of asymmetric cell division has been thoroughly reviewed118–123. Below, I only cover three specific points related to this subject. Boveri revisited. Boveri’s century-old hypothesis (BOX 3) on the tumorigenic potential of cells that have too many centrosomes has remained untested until recently when tumour growth was assayed in flies that were engineered to have cells with abnormal centrosome numbers105,109. The results are intriguing (FIG. 4). Consistent with the hypothesis, tumours often form in larval brains with cells overexpressing Plk4 (also known as Sak), which have too many centrosomes105. However, tumours also form in Sas4‑mutant larval brains that have cells with no centrosomes at all109. Moreover, neither having too many centrosomes, nor having none predisposes cells of the imaginal discs to tumour growth, hence revealing a cell type specificity that is hardly consistent with Boveri’s hypothesis as originally stated. These observations, however, do substantiate a link between centrosome



REVIEWS dysfunction and tumour growth. A revised version of the role of centrosomes in tumorigenesis, which is consistent with the results obtained in flies, proposes that different types of centrosome-related dysfunction can trigger tumorigenesis by compromising the self-renewing asymmetric division of larval neuroblasts56,105,109. AUR and POLO are tumour suppressors in D. melano gaster. Although their corresponding homologues in humans and mice, Aurora kinase A (AURKA) and PLK1, are widely regarded as oncogenes33–35, AUR and POLO behave as tumour suppressors in the D. melanogaster larval brain109,124–126. Such tumour suppressor activity could be linked to the role of AUR and POLO in neuroblast asymmetric division because both kinases are required for proper cortical polarity of aPKC and NUMB124–126. Indeed, AUR and POLO are also key regulators of the centrosome cycle and, as described above, centrosome dysfunction in these cells is potentially tumorigenic. Regardless of the mechanism, the possibility that some of the many functions carried out by these two multitasking kinases might have a tumour suppressor activity in humans, as they do in flies, should not be disregarded. Energy stress and tumour growth. Tumour growth requires energy and, therefore, proteins with key growth-promoting functions, such as AMP-activated protein kinase (AMPK), PI3K and mTOR, are obvious targets to fight cancer 127. This holds true for D. melanogaster where reduction of PI3K activity is also tumour inhibitory 128. However, several studies in flies have unveiled an unexpected facet of the AMPK axis in tumour growth. Larval brains that are mutant for partner of inscuteable (pins; also known as raps), suppressor-of‑G2‑allele-of‑skp1 (Sgt1) or lkb1 have fewer and smaller neuroblasts, as do food-deprived wild-type larval brains. But, surprisingly, in larval brains that are double mutant for pins and Larval brain cell types

Allograft-based tumorigenicity assay

2n (wild type) Non-tumorigenic Aneuploid

Polyploid Two centrosomes (wild type)

Implant into host fly

Tumorigenic

More than two Fluorescent centrosomes larval brain No centrosomes

Figure 4 | Testing Boveri’s hypothesis in flies. Green fluorescent larval brains are Nature Reviews | Cancer allografted in the abdomen of adult flies. For simplicity, only one chromosome pair, of the total four of Drosophila melanogaster, is shown. Wild-type brains, in which mitotically active cells are diploid (2n; shown in blue) and have two centrosomes (red dots), are not tumorigenic. Larval brains carrying different combinations of aneuploid and polyploid cells are not tumorigenic either. However, larval brain tissue carrying cells with either too many or no centrosomes develop tumours.

either Sgt1 or lkb1, neuroblasts overproliferate and cause tumours129–132. In all these cases, neuroblast apical cortical polarity, which is only partially affected in each of these mutants alone, is totally lost 129,131,132. Neuroblasts also overproliferate in pins-mutant brains that are either food deprived or exposed to the mTOR (and the D. melano gaster homologue TOR) inhibitor rapamycin, as well as in pins pi3k double-mutant brains129. These observations demonstrate that pathways that are sensitive to food deprivation and that impinge on TOR through the PI3K and SGT1–HSP90–LKB1–AMPK axes contribute to the generation of normal neuroblast cortical polarity and to the prevention of overgrowth129,131–133. A role for LKB1 signalling through AMPK to coordinate epithelial polarity and proliferation with cellular energy status had been previously documented in D. melanogaster. In well-fed flies, cell polarity is not affected in small clones of ovarian follicle cells that are mutant for either LKB1 or AMPK. However, in food-deprived flies, these clones mislocalize cortical markers and form small tumour-like growths134,135.

Unscheduled gene expression The Polycomb group. The tumour suppressor relevance of timely repression of gene expression programmes during development has long been substantiated by the role of Polycomb group (PcG) proteins in human cancer 136 (see the Atlas of Genetics and Cytogenetics in Oncology and Haematology). Polycomb (PC) itself and many PcG proteins were discovered in D. melanogaster, and research in this organism continues to unveil the role of PcG proteins in cancer. An example is the discovery that the regulation of the PcG epigenetic silencers Pipsqueak (PSQ) and Longitudinals lacking (LOLA) is required for tumour growth in eye imaginal discs in which Notch signalling is constitutively activated by ectopic expression of its ligand Delta137. Besides PC itself, growth of these tumours requires the histone-modifying enzymes RPD3, Suppressor of variegation 3–9 (SU(VAR)3‑9) and Enhancer of zeste (E(Z)). Moreover, although there is little DNA methylation in wild-type flies, downregulation of RBF expression in these tumours correlates with hypermethylation of the corresponding genomic sequence137. These results link the Notch–Delta pathway to epigenetic silencing and to RB function in tumorigenesis. Another PcG protein that has been linked to tumori genesis in humans and that has been studied in detail in D. melanogaster is Polyhomeotic (PH). In the developing D. melanogaster eye, both the loss and the gain of PH function inhibit differentiation and cause overproliferation, by silencing Notch signalling components and causing ectopic activation of the JAK–STAT pathway 138–141. Cancer testis and cancer germline genes. Unlike the soma, which is fated to die, the germline can be passed from one generation to the next, endlessly, and is in this regard an essentially immortal lineage that outlives the individual. This is made possible by mechanisms that protect germline cells against the deleterious effects of transposable elements, free radicals and other insults. Indeed, cancer cells are also immortal. Germline cells

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REVIEWS Box 2 | Asymmetric cell division Mitoses that result in sister cells with different developmental capabilities are referred to as asymmetric. In a particular type of asymmetric mitosis, sometimes referred to as self-renewing, one of the daughter cells retains the identity of the mother cell and carries on dividing. Multiple daughter cells can stem out from a single cell that renews itself in this manner, a strategy that is at the core of tissue growth and homeostasis, and that is suspected to be involved in tumour growth. Drosophila melanogaster neuroblasts are undoubtedly the best-characterized model for this type of division in animal cells. Self-renewing asymmetric division in D. melanogaster neuroblasts relies on the coordination of two processes: the generation of apico-basal cortical polarity, and the orientation of cytokinesis such that the furrow cleaves apart the apical and basal sides of the cortex to each daughter cell. Cortical polarity involves the localization on the apical cortex of several proteins, including the Partitioning defective (PAR) and Partner of Inscuteable (PINS; also known as RAPS) complexes, and the Miranda (MIRA) and Partner of Numb (PON) complexes to the basal cortex. Following cytokinesis, the apical cortex and basal cortex are retained by the neuroblast, and inherited by the differentiating daughter, respectively (reviewed in REFS 118–120).

are also endowed with other conspicuous traits that are found in cancer cells such as the ability to migrate and implant themselves in another tissue, demethylation, angiogenesis induction and immune evasion142. It has long been suspected that malignant transformation might encompass the activation of some of these germline activities that are normally silent in the soma142,143. This view is consistent with the aberrant expression in some human somatic cancers of so‑called cancer germline (CG; also known as cancer testis (CT)) genes the expression of which in healthy individuals is generally restricted to the germ line (see the CTDatabase website; see Further information). CG proteins are being exploited as tags for anticancer vaccines 144. CG proteins could also be exploited as targets for conventional pharmacological inhibition if they actually did have a function in tumour growth, but direct proof of this was lacking until recently. Research in this field might benefit from studies carried out on the D. melanogaster tumour-suppressor gene lethal (3) malignant brain tumour (l(3)mbt)145. D. melanogaster larvae that are mutant for l(3)mbt develop massive malignant brain tumours. Gene expression profiling has revealed that the growth of these tumours is concomitant with the upregulation of more than 100 genes that are known as the MBT signature (MBTS)146. About one-third of the MBTS genes are germline genes, some of which are orthologues of human CG genes146. Functional tests show that at least four of these D. melanogaster CG genes are required for tumour growth in l(3)mbt-mutant flies146. These observations have provided the first experimental

evidence to substantiate a function for CG genes in malignant growth, thus suggesting that the inactivation of CG proteins might also have tumour-suppressing effects in other species146. As a new example of the crucial role of controlled repression of gene expression programmes to prevent cancer, the D. melanogaster l(3)mbt tumour model may help to elucidate how such programmes operate. L(3)MBT forms the LINT complex, together with the novel protein L(3)MBT interacting protein 1 (LINT1) and the transcriptional cofactor Co-repressor for element-1‑silencing transcription factor (CoREST)147. Available data strongly suggest that downregulation of the germline gene programme in the soma requires the binding of the LINT complex to the corresponding promoters147,148.

Using flies to find new drugs and drug targets Despite massive investment by the pharmaceutical industry, the rate of discovery of new therapeutic compounds remains disappointingly slow 149. Because of the advanced understanding of D. melanogaster genetics, cell biology and development, fly-based models, which range from cultured cells or organs to the entire animal, are starting to be added to the panoply of platforms available to identify new compounds. The many features that make D. melanogaster an excellent model for genetic ana lysis are also an advantage for chemical genetics studies. Thorough reviews on the opportunities for therapeutic discovery that exploit fly models of different human diseases have recently been published150,151. Screening in cultured cell lines. The low genome redundancy of flies compared with mammals has facilitated screens on D. melanogaster cell lines that stably express STAT92E, a reporter of JAK–STAT signalling activity, to identify compounds that inhibit UPD-induced transcription. Three such compounds are AUH‑6‑96, MS‑1020 and BOT‑4‑one, which, remarkably, also inhibit the expression of persistently activated JAK3 in Hodgkin lymphoma L540 cells152–154. Another D. melanogaster cell-based screen identified compounds that inhibit the transcriptional effect of misregulated β‑catenin without affecting β‑catenin function in adherens junctions. This was possible by using a D. melanogaster cell line in which WNT–β-catenin signalling was activated by doublestranded RNA-mediated depletion of axin155. Some of the identified inhibitors are specifically cytotoxic to primary cultures of cells derived from human colon tumour biopsy samples155.

Box 3 | Boveri’s hypothesis

Apico-basal cortical polarity An unequal build-up of certain molecules on either the apical or the basal side of the cell.

Following David von Hansemann’s description of cancer cells segregating unequal numbers of chromatids to each pole during anaphase, and building on his own seminal studies on the effect of supernumerary centrosomes, Theodor Boveri hypothesized in 1914 that cancer could be caused by cells that accumulate too many centrosomes, thus organizing multipolar spindles which he expected would give rise to aneuploid daughter cells (reviewed in REF. 165). For more than 100 years, Boveri’s hypothesis has inspired a wealth of research into the possible role of centrosomes and centrosomal proteins in cancer. Recent results in different organisms, particularly in flies, suggest that the main assumptions on which this hypothesis was based may not be correct. However, compelling evidence also demonstrates a clear correlation between centrosome dysfunction and cancer.



REVIEWS Screening in whole flies. Perhaps the most appealing and promising types of drug screens that are being implemented with flies are those that are carried out on the living animal. The long and fruitful history of D. melanogaster as a model for genetics makes it a promising model for whole-organism screening. Indeed, screening for compounds that target proteins is not conceptually different from disrupting genes with mutagenic agents, which is the basis of genetic analysis. Both are ‘black-box’ approaches that interrogate the entire proteome, or genome, to identify proteins or genes that are required for a certain process. Screening in whole flies carries inherent advantages that are not matched by in vitro or cell-based screens. An important advantage is that each compound is assayed for its activity against not one, but the entire pool of structural and regulatory elements, including systemic elements, that are required for the process of interest. Moreover, screening in live flies filters out drugs that poison the animal, as well as drugs that a

Growth in drug-containing food

Flotation to bring larvae to the surface Automatic recording of tumour (fluorescent) growth

b Tumour growth (fluorescent; green)

No tumour

Figure 5 | Whole-organism screening in Drosophila Nature | Cancer melanogaster larvae. The method usedReviews in the screen reported in reference REF. 164 is shown (part a). In this screening method, larvae genetically engineered to develop a green fluorescent tumour (part b) are grown in multi-well plates in which each well contains a different small compound. After a few days, high-density medium is added to each well, thus floating the larvae in a single focal plane, away from the food. Fluorescent microscopy is then used to identify the wells in which larvae do not develop tumours (tumours are shown by green dots).

cannot reach their targets owing to lack of stability or because they cannot penetrate anatomical barriers. These are particularly relevant features because drug candidates often fail at preclinical or clinical stages owing to a lack of bioavailability or to toxicity. The use of D. melanogaster to identify new therapeutic compounds has taken off only fairly recently. A good example is the case of the inhibitors of RET oncogenic activity to treat multiple endocrine neo plasia156,157 (reviewed in REF. 158). The same D. melano gaster model of multiple endocrine neoplasia has been taken one step further to identify the pathways that account for the efficacy and dose-limiting toxicity of known active compounds 159 and to design new chemicals optimized for high efficacy and low toxicity. Similar approaches combining chemistry and D. melanogaster genetics hold great potential for optimizing known therapeutics. Screens in live flies are also yielding new drugs for combinatorial therapy. Combining two or more therapies that act synergistically might allow for lower effective dosages and reduced side effects. One such approach currently explored in clinical trials combines radiation with chemotherapy. Until now, the compounds used for this purpose have been chosen on the basis of educated guesses, underscoring the urgent need for large-scale, unbiased screens that might render entirely new and more efficient molecular entities. A pioneer screen to this end has identified known radiosensitizers such as the microtubule-depolymerizing agent maytansinol isobutyrate (also known as NSC 292222)160. Further studies showed that the effect of maytansinol is p53 dependent in D. melanogaster cells and in human cancer cells, and that the combinatorial effect of drug and radiation is additive. Interestingly, whole larvae can tolerate higher doses of drugs such as maytansinol than cultured cells, thus emphasizing the importance of screening in live flies161. The same screen also identified new enhancers of ionizing radiation, such as Bouvardin (also known as NSC 259968)162,163, previously known for its inhibitory activity on protein translation. Bouvardin also enhances the killing effect of X‑rays in human cancer xenografts in mice. These results substantiate the importance of regulation of protein translation in cancer therapy that is still underappreciated, as shown by the fact that only a few translation inhibitors, such as Aplidin (plitidepsin; PharmaMar) and Homoharringtonine (omacetaxine mepesuccinate; ChemGenex), have so far been tested in clinical trials. Efforts are also being made to implement semiautomated, multi-well-plate-based screens of larvae feed with libraries of chemical compounds (FIG. 5). A recent pilot screen to this end has identified Acivicin, a glutamine analogue with known activity against human tumour cells, as a potent inhibitor of RAS-driven D. melanogaster tumour formation164. Genetic analyses in flies suggest that CTP synthase might be a crucial target of Acivicin-mediated inhibition. These results further substantiate the great potential of whole-organism screening in D. melanogaster.

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REVIEWS Perspectives The limitations of D. melanogaster as a model system to study cancer are many and substantial, but it also has important advantages. By being conscious of the limitations but exploiting the advantages, studies in D. melanogaster will contribute to understanding cancer across different fronts. First, as it has done for more than a century, D. melanogaster basic research will carry on its silent contribution: relentlessly unveiling principles of genome organization, gene regulation, cell cycle control, signalling and development that are often relevant to human cancer. In addition, I expect the short-term future to bring a notable increase in the sophistication of D. melanogaster models of cancer that will expand to include tumours derived from other tissues, as well as new specific tumour subtypes. Considerable advances are also expected in elucidating

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Acknowledgements

The author is very grateful to T. T. Su, Y. Zheng, S. Llamazares, E. Scheenaard, J. Januschke, J. Reina, F. Rossi, J. Petrovic, J. Pampalona and G. Pollarollo for critical reading of the manuscript. Research in the author’s laboratory is funded by grants BFU2009‑07975/BMC, BFU2012-32522, CENIT ONCOLOGICA‑20091016, SGR Agaur 2009 CG041413 and ERC‑2011‑AdG 294603.

Competing interests statement

The author declares no competing financial interests.

FURTHER INFORMATION Cayetano Gonzalez’s homepage: http://www.pcb.ub.edu/divisioncelular/ Atlas of Genetics and Cytogenetics in Oncology and Haematology: http://atlasgeneticsoncology.org/ CTDatabase: http://www.cta.lncc.br/ ALL LINKS ARE ACTIVE IN THE ONLINE PDF
