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Oncogene (2008) 27, 6994–7002 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc REVIEW Drosophila asymmetric division, polarity and cancer J Januschke1 and C Gonzalez1,2 1 Cell Division Group, IRB-Barcelona, PCB, c/Baldiri Reixac 10-12, Barcelona, Spain and 2Institucio Catalana de Recerca i Estudis Avanc¸ats, Passeig Lluis Companys 23, Barcelona, Spain A limited number of adult stem cells (SCs) maintain the homoestasis of different tissues through the lifetime of the individual by generating differentiating daughters and renewing themselves. Errors in the SC division rate or in the fine balance between self-renewal and differentiation might result in tissue overgrowth or depletion, two potentially lethal conditions. A few types of SCs have been identified in Drosophila. These include the SCs of the adult intestine and malpighian tubes, (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006; Singh et al., 2007), the prohematocytes that maintain the population of cells involved in the immunoresponse (Lanot et al., 2001; Lemaitre and Hoffmann, 2007), the SC of the follicle epithelia in the ovary (Nystul and Spradling, 2007), germ line SCs (GSCs) of both sexes (Fuller and Spradling, 2007) and neuroblasts (NBs), the fly neural SCs (Yu et al., 2006; Chia et al., 2008; Knoblich, 2008). Drosophila SCs have proved a fruitful model system to unveil some aspects of the molecular logic that sustains SC function. This review focuses on results obtained in the last few years from the study of NBs, particularly from the standpoint of the possible functional connection between asymmetric SC division and cancer. Oncogene (2008) 27, 6994–7002; doi:10.1038/onc.2008.349 Keywords: stem cells; asymmetric division; centrosomes; cell-of-origin; neuroblasts; cancer Tumours in Drosophila A recent and increasing body of literature suggests that adult stem cells (SCs) might be the cells-of-origin for certain human cancers, but conclusive evidence is still lacking (Reya et al., 2001; Beachy et al., 2004; Huntly and Gilliland, 2005; Kelly et al., 2007). Interestingly, such a connection was suggested by results obtained in Drosophila 30 years ago (Gateff, 1978). Naturally occurring tumours have not been reported in Drosophila or other insects (Harshbarger and Taylor, 1968; Mechler et al., 1991), although neither have they been conclusively ruled out. However, experimentally induced loss of function for a number of Drosophila genes results in Correspondence: Professor C Gonzalez, Cell Division Group, IRBBarcelona, PCB, c/Baldiri Reixac 10-12, 08028 Barcelona, Spain. E-mail: [email protected] the growth of tumours that range from benign hyperplasias to malignant neoplasms that are invasive and lethal to the host (Gateff, 1978; Woodhouse et al., 1998), thus showing that tumours can develop from fly cells. Notably, tumours caused by one of such fly tumour suppressors (TSs), lethal giant larvae (lgl), were observed to originate from larval NBs (Gateff and Schneiderman, 1967). This and other types of tumours described in Drosophila over the years strongly suggest that failed key developmental decisions may be the initial cause of tumour formation (Potter et al., 2000; Harris, 2005). Interestingly, lgl mutant alleles are widespread in Drosophila wild-type populations, probably due to increased fitness under environmental stress conditions (Golubovsky et al., 2006). It is, therefore, reasonable to suspect that flies in these populations might be affected by tumours caused by the loss of heterozygosity for mutant alleles of lgl, something that, no doubt, could be extrapolated to other fly populations and TS genes. The first Drosophila TSs, were identified in situ by the massive growth of the brain in mutant third-instar larvae (Gateff, 1978). However, most mutant alleles of TS genes cause early lethality, and homozygous individuals do not reach such a late stage. In these cases, TS activity is detected by the overgrowth of clones of mutant cells induced in heterozygous individuals (Bilder, 2004). Dozens of genes are now known to fulfil this definition, particularly in imaginal discs tissue. However, the tumours caused by the loss of function for most of these TSs are not invasive and cease to proliferate as they undergo terminal differentiation together with the surrounding wild-type tissue. Typically, the final consequence of mutation in such TSs is limited to an ectopic lump of dead cuticle that has no effect on the performance and lifespan of the affected individual (Watson et al., 1994; Bilder, 2004). Therefore, such tissue overgrowths bear little resemblance to cancer (Hanahan and Weinberg, 2000). How can a given type of in situ overgrowth in a fly tissue be characterized as malignant? In Drosophila, as in vertebrate cancer models, the best established assay is to implant the affected tissue into a healthy host (allograft culture, also known as ‘dauer’ culture) (Gateff, 1978; Gonzalez, 2007). Upon implantation, wild-type tissue never overgrows, and benign hyperplasias grow slowly, and do not invade other tissues and retain the capacity for differentiation. Malignant neoplasms, in contrast, Asymmetric division and cancer J Januschke and C Gonzalez 6995 display autonomous growth, the ability to migrate to and colonize distant organs, and result in lethality to the host. Moreover, malignant neoplasms frequently become immortal and can be expanded limitlessly through successive rounds of implantation into healthy hosts. Thus, in addition to its diagnostic potential, allograft culture circumvents the short time window imposed by Drosophila development and can be used to age tumours for months, or years, thus providing a means to test for changes that might occur during longterm tumour progression. Another advantage of allograft culture is that it provides extra time that may be critical for the growth of tumours that might not be able to develop within the time constraints of larval development. Drosophila NBs as SC models Drosophila NBs originate as cells that delaminate from the embryonic neuroepithelium. Acquisition of NB identity imposes a self-renewing asymmetric mitosis mode, whereby each of the two daughter cells acquires one of the two possible developmental fates: NB or ganglion mother cell (GMC; Chia et al., 2008; Doe, 2008; Gonczy, 2008; Knoblich, 2008). GMCs can be considered as intermediate progenitors to use the terminology that is common in vertebrates, that divide, normally just once, to generate cells that eventually differentiate into neurons or glia. Therefore, some of the key processes that characterize SCs occur in Drosophila NBs. Nevertheless, it is worth pointing out that, as in any other model, there are some limitations here. Firstly, NBs are SCs that operate exclusively during development. Unlike adult SCs that ensure homoestasis through the lifetime of the individual, NBs are programmed to disappear once neurogenesis is completed and are not immortal (Truman and Bate, 1988; Ito and Hotta, 1992; Maurange et al., 2008). Secondly, whereas asymmetric NB division is self-renewing, in the sense that one of the daughters retains NB identity and will continue the programme of asymmetric mitoses, self-renewal might not be complete and daughter NBs might be more restricted than their mother NBs in terms of the cell types that they can generate. Such progressive restriction in competence has been demonstrated in embryonic NBs (Isshiki et al., 2001; Pearson and Doe, 2003), and cannot be excluded in larval NBs as well (Doe, 2008). Finally, another potentially significant difference between Drosophila larval NBs and adult SCs is the dependence on an SC niche. The SC niche can be defined as the specific anatomic location that regulates how SCs participate in tissue generation, maintenance and repair (Scadden, 2006). Drosophila GSCs fulfil this definition because they are totally dependent on direct contact with a niche of specialized cells to the extent that GSCs that abandon their niche lose SC identity and differentiating daughter cells that establish ectopic contact with the niche might acquire SC identity (Brawley and Matunis, 2004; Kai and Spradling, 2004). Indeed, NBs occupy defined anatomical positions during development and are in close contact with neighbouring cells: their most recent daughters on the basal side (Truman and Bate, 1988), and cortex glial cells on their apical and lateral sides (Dumstrei et al., 2003). Moreover, NBs are not insensitive to their neighbours and are definitively responsive to signals like Anachronism or Activin, which are secreted by larval glia (Ebens et al., 1993; Zhu et al., 2008) as well as to Notch, and other signalling pathways that control the number of proliferating NBs in the larval brain (Park et al., 2003; Wang et al., 2006; Lee et al., 2006a). However, the presence of a cell niche necessary for maintaining the NB self-renewing asymmetric division programme has not been demonstrated (Doe, 2008). If anything, the ability of isolated embryonic and larval NBs to continue dividing in a self-renewing asymmetric manner, when cultured in vitro strongly argues the contrary (Wu et al., 1983; Broadus and Doe, 1997; Feiguin et al., 1998; Ceron et al., 2006). The asymmetric division machinery of Drosophila NBs Self-renewing asymmetric division of Drosophila NBs relies on the tight coordination of two processes: (i) the differential sorting of several protein complexes that leads to the generation of cortical asymmetry within these cells (Chia et al., 2008; Gonczy, 2008; Knoblich, 2008) and (ii) the controlled positioning of the plane of cytokinesis that leads to the unequal segregation of those cortical protein complexes between the daughter cells (Gonzalez, 2007). Clearly, both of these processes are necessary, but neither of them is sufficient. Complexes sorted to the apical cortex and inherited by the daughter cell that retains NB identity include the Par and Pins (Partner of inscuteable or Rapsynoid) complexes, which can interact through the bridging protein Insc (Kraut et al., 1996; Schober et al., 1999; Schaefer et al., 2000; Petronczki and Knoblich, 2001). The core components of the Par complex include Bazooka (Baz/Par3), Par6 and Atypical protein kinase C (aPKC; Wodarz et al., 1999; Petronczki and Knoblich, 2001; Rolls et al., 2003). The main partners complexed with Pins are Galphai and Mushroom body defect (Mud; Parmentier et al., 2000; Schaefer et al., 2000; Yu et al., 2003; Izumi et al., 2004, 2006; Bowman et al., 2006; Siller et al., 2006). Both complexes contain known auxiliary modulators (Chia et al., 2008; Knoblich, 2008), the number of which is steadily increasing (Atwood et al., 2007; Chabu and Doe, 2008; Speicher et al., 2008). Proteins sorted to the basal cortex, and hence inherited by the daughter cell that is primed to lose NB identity and to enter the differentiation programme, include Staufen, Prospero and Brain tumour (Brat) that are in a complex with the scaffolding protein Mira, and the Partner of Numb (Pon)-Numb complex (Doe et al., 1991; Ikeshima-Kataoka et al., 1997; Li et al., 1997; Shen et al., 1997; Lu et al., 1998; Betschinger et al., 2006; Lee et al., 2006c; Caussinus and Hirth, 2007). Other elements of this intricate molecular Oncogene Asymmetric division and cancer J Januschke and C Gonzalez 6996 machinery are the protein kinases CDK1 (cdc2), Polo and Aurora A (Tio et al., 2001; Wang et al., 2006, 2007; Lee et al., 2006a), and the cortical proteins Discs large (Dlg), Lgl and Scribbled (Scrib; Ohshiro et al., 2000; Peng et al., 2000; Albertson and Doe, 2003; Humbert et al., 2003). These last three genes, dlg, lgl and scrib were previously known by their TS activity. Sorting the apical and basal protein complexes that control NB asymmetric division would serve no purpose, if cytokinesis did not ensure the unequal distribution of these complexes between the two daughter cells. Controlled orientation of the plane of cleavage, which is governed by the orientation of the mitotic spindle, is therefore an integral part of the asymmetric division machinery (Kraut et al., 1996; Kaltschmidt et al., 2000). Perhaps not surprisingly, some of the cortex-bound polarized cues take part in spindle alignment. In particular, the Pins–Galphai–Mud complex is thought to anchor the aster of one of the spindle poles during mitosis (Bowman et al., 2006; Izumi et al., 2006; Siller et al., 2006). Combined live imaging of spindle assembly and centrosome function has shown that spindle orientation in larval NBs is determined, at least partially, from early interphase, much earlier than any of the known polarized markers can be seen at the apical cortex of the cell (Rebollo et al., 2007; Rusan and Peifer, 2007). Soon after cytokinesis, both centrosomes migrate to the nearest cortex, which roughly coincides with the region where apical markers were last localized. A few minutes later, the two centrosomes start to display a markedly asymmetric behaviour. One of them stays fixed at the apical cortex organizing an aster that will be the main microtubule network during most of the NB interphase. Orientation of the future mitotic spindle can be accurately predicted from the position of this apical aster and can, therefore, be considered predetermined at such an early stage of the cell cycle. The second centrosome, which has little, if any, pericentriolar material (PCM) and does not display any significant microtubule organisation activity, moves extensively throughout the cytoplasm, mainly on the apical side first, more basally later, and until shortly before mitosis, it slows down near the basal cortex, recruits PCM and organizes the second mitotic aster. Thus, the two centrosomes of an NB are very different structurally and functionally. They are also unequal in fate as the apical centrosome remains in the SC, whereas the other centrosome goes into the differentiating daughter. How the centrosome identifies the presumptive apical cortex is not known (Figure 1). Polarized cortical markers are not detected after mitosis, and the first sign of them getting back to the cortex occurs very late in the cell cycle, when mitosis starts, with the accumulation of Baz in prophase (Siller et al., 2006). It is possible that still unknown polarization markers tag the apical cortex for the entire length of the cell cycle and serve as cues for the migration of the centrosome to the cortex. Alternatively, the positioning of the centrosome at the cell cortex might contribute to triggering the sorting of the apical markers at each cell cycle. Maintenance of the active microtubule organisation centre at its fixed Oncogene Figure 1 The timing of polarity markers during the cell cycle of larval NBs. Markers that are asymmetrically localized at the apical (blue) or basal (red) cortex have been observed during mitosis, from prophase to late telophase. No such markers have been observed during interphase, thus opening the question of how cell polarity is maintained or reestablished from one cell cycle to the next. The microtubule network of these cells (green), that remains highly polarized through most of the interphase with its microtubule organizing centre (orange) tightly bound to the presumptive apical cortex could be part of the answer. NBs, neuroblasts. cortical position requires Pins (Rebollo et al., 2007). However, the mechanism by which Pins might exert this function that is required much earlier than the apical crescent of Pins can be detected remains unclear. A suspected regulator of this process is Polo (Rusan and Peifer, 2007), which also controls the apical sorting of aPKC and Numb (Wang et al., 2007). Interestingly, cultured individual NBs show the same asymmetric centrosome behaviour as NBs observed ‘in toto’, strongly suggesting that the regulation of such stereotyped behaviour does not depend on the cross talk between NBs and their neighbouring cells (Rebollo et al., 2007). Self-renewing asymmetric division of NBs and tumour suppression The possible functional link between failed NB asymmetry and tumour growth was first suggested by the identification of known TS genes, such as lgl or dlg, as key regulators of NB asymmetry (Wodarz, 2005). Direct demonstration of such a link came, when pieces of larval brain tissue mutant for any of several elements that regulate NB asymmetry, including notably all known cell-fate determinants, were shown to develop malignant tumours when transplanted to the abdomen of adult hosts (Caussinus and Gonzalez, 2005). Since then, many more of these proteins have been shown to be TSs, both in allograft culture and in situ in the larval brain (Table 1). Protein complexes sorted to the basal cortex contain cell-fate determinants that negatively regulate NB selfrenewal and promote neuronal differentiation. The transcription factor Prospero is normally segregated to the GMC, where it enters the nucleus and regulates Asymmetric division and cancer J Januschke and C Gonzalez 6997 Table 1 Tumour suppressor activity of asymmetric cell division genes Mutant a Overgrowth In situ Clonal analysis aPKC aPKC GOF pins mira brat References Allograft culture NB duplicationb Immortal, invasive metastatic growth Limited growth potential + + + + + + + pros + + numb lgl dlg mud sas6 sas4 sak sak GOF asl cnn aurA polo + + + + + + + + + + + + + + + + + + + + + + Lee et al., 2006c; Caussinus and Gonzalez, 2005 Lee et al., 2006c Rebollo et al., 2007; Lee et al., 2006c; Caussinus and Gonzalez, 2005 Betschinger et al., 2006; Caussinus and Gonzalez, 2005 Lee et al., 2006b; Betschinger et al., 2006; Bello et al., 2006; Caussinus and Gonzalez, 2005; Gateff, 1978 Lee et al., 2006b; Choksi et al., 2006; Betschinger et al., 2006; Caussinus and Gonzalez, 2005 Lee et al., 2006a; Bowman et al., 2006; Caussinus and Gonzalez, 2005 Lee et al., 2006c; Betschinger et al., 2006; Gateff, 1978 Gateff, 1978 Siller et al., 2006; Bowman et al., 2006; Izumi et al., 2006 Castellanos et al., 2008 Castellanos et al., 2008; Basto et al., 2006 Castellanos et al., 2008 Basto et al., 2008 Castellanos et al., 2008; Rusan and Peifer, 2007 Lucas and Raff, 2007 Castellanos et al., 2008; Lee et al., 2006a; Wang et al., 2006 Castellanos et al., 2008; Wang et al., 2007 a GOF: gain of function, all other situations refer to loss of function. Symmetric NB divisions reported by live imaging. b genes that are crucial for cell-cycle repression or initiation of neuronal differentiation (Knoblich et al., 1995; Broadus et al., 1998; Li and Vaessin, 2000; Choksi et al., 2006). Likewise Brat, which is also sorted into the GMC, is a translational repressor with dozens of possible targets, which is required to restrain cell growth (Sonoda and Wharton, 2001; Frank et al., 2002; Loop et al., 2004). Larval brain tissue mutant for prospero or brat develop massive malignant tumours in allograft culture (Caussinus and Gonzalez, 2005) and displays significant overgrowth in situ (Bello et al., 2006; Betschinger et al., 2006; Choksi et al., 2006; Lee et al., 2006c). Sorting of Brat and Pros to the GMC is mediated by the adaptor protein, Mira. Consistently, Mira loss of function also leads to overgrowth in situ (Betschinger et al., 2006) and malignant tumour growth in allograft cultures (Caussinus and Gonzalez, 2005). Another key cell-fate determinant is Numb, best known by its activity as a regulator of Notch signalling (Schweisguth, 2004; Le Borgne et al., 2005). Following its segregation into GMCs, Numb promotes differentiation and inhibits NB self-renewal (Wang et al., 2006; Lee et al., 2006a). Like the loss of function for other cell-fate determinants, allograft culture of Numb mutant clones causes massive malignant tumours (Caussinus and Gonzalez, 2005). Consistently, expression of an activated form of Notch yields increased NB number (Wang et al., 2006), although Notch regulatory signalling seems to be restricted to NBs in the optic lobes and to be inactive in the ventral ganglion (Almeida and Bray, 2005). Some elements of the apical cortex complexes can also, if disrupted, cause overgrowth. Ectopic cortical localization of aPKC inhibits cortical localization of basal protein complexes (Betschinger et al., 2003; Rolls et al., 2003; Smith et al., 2007), and leads to a dramatic increase in NB numbers in situ (Lee et al., 2006b). Consistently, loss of aPKC function leads to NB cellcycle arrest (Rolls and Doe, 2004), and does not result in tumour growth in allograft cultures (Caussinus and Gonzalez, 2005). Spindle misorientation in NBs with otherwise normal cortical polarity can also cause overgrowth, as it has been proposed to occur in mud mutant NBs (Bowman et al., 2006; Izumi et al., 2006; Siller et al., 2006). The behaviour of pins mutant larval brain tissue is intriguing. In situ, loss of Pins function causes a reduction in the number of NBs in late larval stages (Lee et al., 2006b). However, pins mutant NBs have been observed to divide symmetrically by live imaging (Rebollo et al., 2007), and malignant tumours develop from pins mutant tissue in allograft culture (Caussinus and Gonzalez, 2005). The reason for this discrepancy is unknown. Interestingly, all cases of mutants in which symmetric NB division has been observed cause tumour growth, benign or malignant, in allograft culture (Basto et al., 2006, 2008; Siller et al., 2006; Lee et al., 2006a; Lucas and Raff, 2007; Rusan and Peifer, 2007; Castellanos et al., 2008) (Table 1). Finally, some of the main kinases that regulate the process are also TSs. Both AurA and Polo are necessary to constrain aPKC to the apical cortex and regulate the Oncogene Asymmetric division and cancer J Januschke and C Gonzalez 6998 basal localization of Numb, and are required for spindle alignment (Wang et al., 2006, 2007; Lee et al., 2006a). Larval brain mutants for polo or aurA have supernumerary NBs (Wang et al., 2006; Lee et al., 2006a) and develop as malignant tumours when implanted in adult hosts (Castellanos et al., 2008). Cell-of-origin of larval brain tumours Several working models have been put forward as to what are the cells-of-origin of the larval brain tumours and to the mechanistic interpretation of how such a cell gets transformed (Figure 2). The simplest working model suggests direct expansion of NBs (Siller et al., 2006; Lee et al., 2006b). A second model calls on reversion from GMC to NB fate (Betschinger et al., 2006; Lee et al., 2006c). Finally, a third model proposes that overgrowth results from the expansion of transient amplifying cell (Bello et al., 2008; Bowman et al., 2008; Boone and Doe, 2008). Direct NB expansion might apply to cases in which uncoupling of spindle orientation from the apico-basal axis of the cell results in cleavage planes that bisect the NB in two halves that are symmetric, both in terms of the cell-fate determinants that they contain and in size. This has been proposed to occur in mud-mutant NBs (Bowman et al., 2006; Izumi et al., 2006; Siller et al., 2006). This model implies that the presence of apical cortex components like aPKC is read by the system as dominant over the presence of basally localized differentiation promoting determinants, as cells that inherit both retain SC identity. This opens the question of how intermediate situations are read by the system, for example, how much aPKC a GMC can tolerate. This model might also apply to situations in which aPKC is ectopically localized over most of the NB cortex, as reported in cases like expression of aPKC-CAAX, or the combined loss of lgl and pins function (Lee et al., 2006b). In these cases, the two daughter cells receive aPKC, but no cortically localized cell-fate determinants. Reversion of GMCs to an NB fate has been proposed to explain tumour growth in pros and brat mutants as, in their absence, GMCs cannot start their programme of repression of self-renewal and activation of differentiation pathways (Betschinger et al., 2006; Choksi et al., 2006; Lee et al., 2006c). This model implies that the system interprets the loss of pros or brat as a dominant situation to revert back to SC fate, and ignores other determinants of differentiation like Numb. Finally, some cases of overgrowth have been proposed to result from alterations in the recently identified NB lineage that produces transient amplifying cells (Bello et al., 2008; Bowman et al., 2008; Boone and Doe, 2008). Loss of Brat or Numb function in this lineage is thought to inhibit the maturation of Asense-negative transient amplifying cells into Asense-positive cells causing them to overproliferate uncontrollably (Bowman et al., 2008). Unequivocal identification of the cell-of-origin of these tumours will require data obtained by direct imaging of mutant cells expressing a battery of markers Oncogene Figure 2 Models of the origin of Drosophila larval brain tumours. (a) In most larval NBs (wt) asymmetric division self-renews the NB, and creates a GMC (red) that divides into two daughters that differentiate into neurons (black). Situations like uncoupling of spindle alignment and polarity cues (1) or ectopic widespread cortical localization of aPKC (2) are thought to give rise to equal daughters that retain NB identity. In the absence of pros or brat, the newborn GMC has been proposed to revert back to NB identity (3). In all three cases, the result would be a net increase in the number of NBs. (b) Asymmetric division of certain larval NBs self-renews the NBs and creates transient amplifying cells (white). After maturation (grey), these cells can enter mitosis generating more of their kind and GMCs (red) that divide into two daughters that differentiate into neurons. Loss of Brat or Numb function in this lineage is thought to inhibit the maturation process and to result in the uncontrolled growth of the immature cells (Bowman et al., 2008). aPKC, atypical protein kinase C; GMC, ganglion mother cell; NBs, neuroblasts. for asymmetrically sorted proteins, spindle orientation and cell type identity (Doe, 2008). Whichever the model of overgrowth is, it will be essential to define the nature of the overgrowing cells and what makes them different from normal NBs or GMCs. This might require a comprehensive quantitated understanding of the regulatory circuitry of the system with all its integrating elements, which we do not have at the moment. Genome instability and centrosome dysfunction in larval brain tumours The possibility of creating experimentally induced tumours in Drosophila is being exploited to tackle some fundamental questions pertaining to the origin of tumours. One example is Boveri’s old proposal to explain the origin of human tumours–supernumerary Asymmetric division and cancer J Januschke and C Gonzalez 6999 centrosomes cause chromosome instability that triggers tumourigenesis (Boveri, 2008). Indeed, different types of genome instability (GI), including chromosome instability and centrosomal alterations are common traits in human cancer (Lengauer et al., 1998; Duensing, 2005). However, Boveri’s proposal, as originally stated or in its more modern versions that accommodate for a wider range of centrosome abnormalities, remains hypothetical, and direct experimental proof is still lacking (Nigg, 2006). This hypothesis has recently been tested in Drosophila by assaying the tumourigenic potential of cells that have been genetically manipulated to contain centrosome abnormalities that phenocopy those proposed as potentially tumourigenic (Basto et al., 2008; Castellanos et al., 2008). Several types of centrosome dysfunction, including supernumerary centrosomes, were found to be potent tumourigenic conditions in larval brains kept in allograft culture. Interestingly, both benign tumours that had a limited growth potential, and malignant neoplasms that grew unrestrainedly, spreading over different parts of the host’s anatomy and killing the host were observed (Basto et al., 2008; Castellanos et al., 2008). These results provide the first direct demonstration of a causative relation between abnormal centrosomes and cancer. However, GI is unlikely to be the key event that initiates these tumours because genome alterations that phenocopy those found in vertebrate tumours are poorly tumourigenic, if at all, in the absence of abnormal centrosomes in Drosophila (Castellanos et al., 2008). Consistently, tumourigenic activity and GI do not seem to correlate. Sak overexpression results in supernumerary centrosomes, but the initial multiple spindle poles cluster in two and eventually bipolar spindles are assembled (Basto et al., 2008), and chromosome segregation occurs faithfully in most cells. Likewise acentriolar dsas4 mutant cells often manage to assemble acentrosomal spindles and equal chromosome segregation occurs in most cells (Basto et al., 2006). Yet, despite the relatively low levels of chromosome instability caused by Sak overexpression or loss of dsas-4, these conditions often cause malignant neoplasms (Basto et al., 2006; Castellanos et al., 2008). It is important to stress that whereas these results strongly suggest that the tumours caused by centrosome dysfunction in Drosophila cannot be accounted for solely by GI, they cannot be taken as evidence to discard a possible role of GI in tumourigenesis. Moreover, GI is a common trait in Drosophila tumours that develop in allograft cuture (Caussinus and Gonzalez, 2005; Castellanos et al., 2008). Regardless of whether the mutation that originated the tumour causes a certain level of GI in situ, like aurA or dsas-4 mutant alleles do, or none at all, like pros, mira and others, tumours that grow in allograft culture eventually display significant levels of chromosomal alterations that affect both chromosome integrity and number (Caussinus and Gonzalez, 2005; Castellanos et al., 2008). As allograft culture per se does not cause GI (Remensberger, 1968), such tumour-associated GI might reflect a downstream consequence of tumourigenesis. It will be interesting to determine whether such tumour-associated GI contributes to the worsening of malignant traits displayed by some of these tumours. It is still uncertain how centrosome dysfunction generates tumours in larval brains, but the simplest hypothesis is that it does so by perturbing the segregation of cell-fate determinants, a condition known to cause tumour growth in these cells (Gonzalez, 2007). Consistent with this interpretation, different degrees of perturbed NB self-renewing asymmetric division have been reported in the centrosome dysfunction conditions that cause tumours in larval NBs, and the extent of such perturbations correlates well with the ability to generate tumours (Basto et al., 2006; Castellanos et al., 2008). Moreover, the tumourigenic activity of centrosome dysfunction is negligible in imaginal discs (Castellanos et al., 2008), which do not grow by the self-renewing asymmetric division of SCs. There are different mechanisms by which centrosome dysfunction can be envisaged to perturb self-renewing NB asymmetric division. An obvious one is by compromising the alignment of the spindle along the apico-basal axis of the NB (Basto et al., 2006; Bowman et al., 2006; Izumi et al., 2006; Siller et al., 2006). Loss of function of some centrosomal proteins that directly control the sorting of some of the elements of the asymmetric self-renewing machinery might also impair this process (Wang et al., 2006, 2007; Lee et al., 2006a). Finally, centrosome dysfunction could affect the mechanisms that establish robust NB polarity after each round of mitosis (Siegrist and Doe, 2005; Rebollo et al., 2007; Rusan and Peifer, 2007). Therefore, rather than being linked to chromosome segregation, the tumourigenic activity of centrosome function in Drosophila seems to be directly related to self-renewing asymmetric SC division, thus adding further support to the view that the initial event that triggers tumourigenesis in this model system might be failed developmental decisions. The possibility that centrosome dysfunction might cause tumours when affecting SC is tantalizing given the increasing evidence suggesting that adult SCs may be the cell-of-origin of certain tumour types (Beachy et al., 2004). Acknowledgements This study was supported by EU and Spanish Grants: ONCASYM-037398 FP6, BFU2006-05813, SGR2005 Generalitat de Catalunya and Consolider-Ingenio2010 CENTROSOME_3D CSD2006-23. References Albertson R, Doe CQ. (2003). Dlg, Scrib and Lgl regulate neuroblast cell size and mitotic spindle asymmetry. Nat Cell Biol 5: 166–170. Almeida MS, Bray SJ. (2005). 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