Tumor-promoting function of apoptotic caspases by an amplification loop involving ROS, macrophages and JNK in Drosophila
Larval imaginal discs in Drosophila are single-cell layered sacs of epithelial cells that develop into the adult appendages such as eyes and wings , and are frequently used as genetic models for growth control and tumor development . Maintenance of apical-basal polarity of epithelial cells is critical for suppression of neoplastic tumor development ( Elsum et al . , 2012; Bergstralh and St Johnston , 2012; Martin-Belmonte and Perez-Moreno , 2011 ) . Mutations in genes encoding components of the Scribble complex including scribble ( scrib ) , lethal giant larvae ( lgl ) and discs large ( dlg ) disrupt apical-basal polarity in epithelial cells which can trigger malignant neoplastic tumor growth ( Bergstralh and St Johnston , 2012; Elsum et al . , 2012; Bilder et al . , 2000; Gateff , 1978; Bilder and Perrimon , 2000 ) . Drosophila larvae entirely mutant for scrib fail to respond to stop signals of growth , fail to pupariate and continue to grow as larvae ( Gateff , 1994; Wodarz , 2000 ) . They die as giant larvae with severely overgrown imaginal discs . However , scrib mutant cells ( clones ) in otherwise wild-type imaginal discs are eliminated by cell competition mediated by neighboring wild-type cells ( Brumby and Richardson , 2003; Menéndez et al . , 2010; Igaki et al . , 2009; Uhlirova et al . , 2005; Ohsawa et al . , 2011; Leong et al . , 2009; Chen et al . , 2012; Vaughen and Igaki , 2016 ) . Mechanistically , in response to cell competition , Eiger , the Tumor Necrosis Factor alpha ( TNFα ) -like ligand in Drosophila , triggers Jun N-terminal kinase ( JNK ) activation and apoptosis in scrib mutant cells ( Igaki et al . , 2009; Brumby and Richardson , 2003; Uhlirova et al . , 2005; Cordero et al . , 2010; Ohsawa et al . , 2011; Leong et al . , 2009; Igaki et al . , 2006; Chen et al . , 2012 ) . This tumor-suppressing function is dependent on Eiger and JNK through induction of apoptosis . Inhibition of Eiger or JNK restores the growth potential of scrib mutant cells which can then form large tumor masses in imaginal discs ( Brumby and Richardson , 2003; Igaki et al . , 2009; Uhlirova et al . , 2005; Chen et al . , 2012 ) . However , if additional oncogenic mutations such as RasV12 are introduced into scrib mutant cells ( referred to a scrib−/− RasV12 ) , they can unleash their full malignant potential ( Brumby and Richardson , 2003; Pagliarini and Xu , 2003 ) . scrib−/− RasV12 mosaic eye/antennal imaginal discs display all neoplastic features observed in human tumors including unrestricted growth , failure to differentiate , tissue invasion and organismal lethality ( Pagliarini and Xu , 2003; Brumby and Richardson , 2003 ) . scrib−/− RasV12 clones occupy a large portion of the mosaic disc and trigger multi-layered overgrowth of the entire disc compared to wild-type controls ( Figure 1H , I ) . scrib−/− RasV12 mutant cells also invade other tissues , most notably the ventral nerve cord ( VNC ) in the brain ( Figure 1H , I ) ( Pagliarini and Xu , 2003 ) . The scrib−/− RasV12 condition in ey-FLP-induced eye imaginal disc mosaics is 100% lethal . 95% of ey-FLP-induced scrib−/− RasV12 mosaic animals die as larvae; the remaining animals die during pupal stages . Interestingly , RasV12 inhibits the apoptotic activity of JNK and converts the tumor-suppressor function of Eiger and JNK in scrib−/− cells into a tumor-promoting one in scrib−/− RasV12 cells ( Enomoto et al . , 2015; Cordero et al . , 2010; Uhlirova et al . , 2005; Igaki et al . , 2006; Uhlirova and Bohmann , 2006 ) . Therefore , the aggressive tumor growth of scrib−/− RasV12 mutant clones becomes dependent on Eiger and JNK ( Igaki et al . , 2006; Uhlirova and Bohmann , 2006; Brumby et al . , 2011 ) . Mechanistically , it is not understood how RasV12 promotes this oncogenic switch of Eiger and JNK . Caspases are Cys-proteases that mediate the mechanistic events of apoptotic cell death ( Shalini et al . , 2015; Xu et al . , 2009; Fuchs and Steller , 2011; Salvesen et al . , 2016 ) . They are synthesized as zymogens and depending on the length of their prodomains can be classified into initiator and effector caspases . Initiator caspases such as mammalian Caspase-9 or its Drosophila ortholog Dronc are controlled by upstream signaling events and when activated initiate apoptosis by activating effector caspases such as mammalian Caspase-3 or its Drosophila ortholog DrICE ( Fuchs and Steller , 2011; Shalini et al . , 2015; Salvesen et al . , 2016 ) . Caspases induce apoptosis of many cells to maintain homeostatic conditions , and are also thought to be critical for tumor suppression by eliminating malignant cells . However , caspases can also have tumor-promoting roles , for example through apoptosis-induced proliferation ( AiP ) , a caspase-driven process by which apoptotic cells produce mitogenic signals for proliferation of neighboring surviving cells ( Mollereau et al . , 2013 ) ( reviewed in [Fogarty and Bergmann , 2017; Ryoo and Bergmann , 2012] ) . There are two types of AiP . During ‘genuine’ AiP , apoptotic cells release mitogenic factors before completing the apoptotic program . This type of AiP has been described for regeneration and wound healing both in vertebrates and invertebrates ( Tseng et al . , 2007; Fan and Bergmann , 2008; Chera et al . , 2009; Li et al . , 2010 ) . ‘Genuine’ AiP may also be involved in human pathologies such as cancer , and may account for increased cell proliferation and repopulation of tumors following cytotoxic treatments ( chemo- or radiotherapy ) which induces massive apoptosis ( reviewed in [Fogarty and Bergmann , 2017; Ichim and Tait , 2016] ) . Caspases play significant tumor-promoting roles in these settings ( Li et al . , 2010; Huang et al . , 2011; Donato et al . , 2014; Cheng et al . , 2015; Zhang et al . , 2015; Kurtova et al . , 2015 ) . The second type is ‘undead’ AiP . Here , the apoptosis pathway is induced upstream , but the execution of apoptosis is blocked . In Drosophila , apoptosis inhibition is achieved experimentally by expression of the effector caspase inhibitor p35 which very specifically inhibits DrICE ( Hay et al . , 1994; Meier et al . , 2000; Hawkins et al . , 2000 ) . Therefore , because these cells have initiated the apoptotic process and contain active Dronc , but cannot die , they are referred to as ‘undead’ . In ‘undead’ cells , non-apoptotic functions of active Dronc can now be examined , one of which is the release of mitogenic signals for induction of AiP which can lead to hyperplastic overgrowth ( Wells et al . , 2006; Kondo et al . , 2006; Huh et al . , 2004; Ryoo et al . , 2004; Martín et al . , 2009; Pérez-Garijo et al . , 2009; Pérez-Garijo et al . , 2004; Fan et al . , 2014; Rudrapatna et al . , 2013; Pérez-Garijo et al . , 2005 ) . ‘Undead’ states of cells may also be present under pathological conditions . For instance , it has been proposed that RasV12 can maintain apoptotic cells in an ‘undead’-like state promoting tumorigenesis ( Hirabayashi et al . , 2013 ) . Mechanistically , we have shown that AiP-mediated hyperplastic overgrowth of ‘undead’ tissue depends on a Dronc-initiated feedback amplification loop which involves reactive oxygen species ( ROS ) – specifically extracellular ROS produced by the membrane-bound NADPH oxidase Duox – , activation of macrophage-like hemocytes , secretion of Eiger by hemocytes , Eiger-dependent activation of JNK in epithelial disc and further activation of Dronc by JNK ( Fogarty et al . , 2016 ) ( reviewed by [Diwanji and Bergmann , 2017a; Diwanji and Bergmann , 2017b] ) . Therefore , similar to the scrib−/− RasV12 case , Eiger and JNK signaling have proliferation- and growth-promoting functions in this ‘undead’ AiP model . These similarities prompted us to investigate the role of ROS and caspases for tumor growth of scrib−/− RasV12 clones in Drosophila . We report that oncogenic Ras switches the pro-apoptotic activity of caspases into a tumor-promoting one and thereby maintains scrib−/− RasV12 cells in an ‘undead’-like state . Consistently , inhibition of caspases blocks tumor growth and tissue invasion . The tumor-promoting function of apoptotic caspases is dependent on the generation of intra- and extracellular ROS which are required for neoplastic behavior of scrib−/− RasV12 clones . Furthermore , caspase-induced ROS are essential for the recruitment and activation of hemocytes at scrib−/− RasV12 mosaic discs . Hemocytes signal back to tumorous epithelial cells to stimulate JNK signaling which further promotes caspase activity . Thus , these events constitute a feedback amplification loop which is necessary for neoplastic activity of scrib−/− RasV12 cells . This work extends previous models about the conversion of Eiger and JNK signaling from anti-tumor to pro-tumor roles by oncogenic Ras and identifies caspases as essential components of this switch . In conclusion , although apoptotic caspases are usually considered to be tumor suppressors , under certain conditions , for example in the presence of oncogenic RasV12 in scrib mutant cells , they can also adopt a tumor-promoting role .
Recently , in a model of ‘undead’ AiP , we showed that Duox-generated ROS are important for activation of hemocytes , JNK signaling and hyperplastic overgrowth ( Fogarty et al . , 2016; Diwanji and Bergmann , 2017a; Diwanji and Bergmann , 2017b ) . Therefore , we examined if ROS have a similar function in the neoplastic scrib−/− RasV12 tumor model in Drosophila . scrib−/− RasV12 clones were induced by MARCM using ey-FLP in eye/antennal imaginal discs , the traditional tissue for this model ( Brumby and Richardson , 2003; Pagliarini and Xu , 2003 ) . The ROS indicator dihydroethidium ( DHE ) strongly labels scrib−/− RasV12 mutant clones in mosaic discs , while wild-type ( wt ) , scrib−/− and RasV12-expressing clones are not labeled by DHE or very little ( Figure 1A–D’; quantified in Figure 1F; see also Figure 2A , A’ ) . Similar results were reported recently ( Katheder et al . , 2017; Manent et al . , 2017 ) . A different ROS indicator , H2DCF-DA , confirms these results ( Figure 1—figure supplement 1A–C , G ) . scrib−/− RasV12 clones display an increased diffuse cytosolic DHE labeling ( Figure 1E , E’ ) . At the boundary of scrib−/− RasV12 clones , several cells stain very intensely for DHE ( Figure 1E , E’; arrow head ) . To examine the function of ROS in this neoplastic tumor model , we reduced their amount either by down-regulating ROS-producing enzymes such as Duox or by overexpressing ROS-removing enzymes such as catalases and superoxide dismutases ( SOD ) . As shown previously , scrib−/− RasV12 occupy a large portion of the eye/antennal imaginal disc and display a strong neoplastic tumor phenotype ( Pagliarini and Xu , 2003 ) ( see also Figure 2F ) . Because expression of the antioxidant enzymes was achieved by the UAS/Gal4 system , we tested first whether increasing the UAS gene dose may modify ( suppress ) the tumor phenotype and expressed an unrelated control gene , UAS-lacZ , in scrib−/− RasV12 clones . However , despite strong expression of β-Gal ( Figure 1—figure supplement 2 ) , the tumor phenotype of scrib−/− RasV12 eye/antennal imaginal discs is not significantly suppressed by addition of an additional UAS-driven transgene ( Figure 1I ) . In contrast , removing extracellular ROS by UAS-Duox RNAi or overexpression of the UAS-hCatS transgene which encodes a secreted human catalase ( Ha et al . , 2005b2005; Ha et al . , 2005a ) , strongly suppressed tumor growth of scrib−/− RasV12 mutant cells ( Figure 1J , K ) suggesting that extracellular ROS are required for tumor growth . Interestingly and in contrast to the ‘undead’ AiP model , removing intracellular ROS by misexpression of intracellular Catalase , SOD1 and SOD2 also strongly suppressed tumor growth ( Figure 1L–N ) . These results suggest that both intra- and extracellular ROS are required for tumor growth of scrib−/− RasV12 clones . Importantly also , reduction of ROS strongly reduces the invasive behavior of scrib−/− RasV12 mutant cells ( Figure 1J–N ) which significantly improves the survival rate of the affected animals . Compared to ey >MARCM scrib−/− RasV12 mutant larvae , of which only 5% reach pupal stages , between 40% and 70% of the ey >MARCM scrib−/− RasV12 larvae expressing antioxidant enzymes develop into pupae ( Figure 1G ) . Expression of the UAS-lacZ control transgene does not improve pupal survival ( Figure 1G ) . Furthermore , we also recovered viable adult ey >MARCM scrib−/− RasV12 mosaic animals expressing antioxidant genes , although at a low rate ( 5–15% of the surviving pupae ) , which was never observed for scrib−/− RasV12 only ( Figure 1P–U ) . Although their eyes and heads are deformed and weakly overgrown often with necrotic patches compared to wt control ( Figure 1O , Q–U ) , these animals live ! Previously , only few examples of surviving scrib−/− RasV12 mosaic animals have been reported . In these examples , viable scrib−/− RasV12 mosaic animals were recovered when essential steps in tumor development such as oncogenic JNK signaling or the cell cycle were inhibited in scrib−/− RasV12 clones ( Brumby and Richardson , 2003; Külshammer et al . , 2015; Külshammer and Uhlirova , 2013 ) . Therefore , the observation that reduction of ROS suppresses tumor growth and enhances organismal survival strongly suggests that ROS play a very significant role for the neoplastic characteristics of scrib−/− RasV12 animals . In ‘undead’ cells , the initiator caspase Dronc ( caspase-9 ortholog ) has been shown to stimulate the production of ROS ( Fogarty et al . , 2016 ) . Therefore , we examined the role of Dronc as well as the effector caspase DrICE ( caspase-3 ortholog ) for generation of ROS and tumorous overgrowth in scrib−/− RasV12 mosaic eye discs . As an additional assay , we expressed the effector caspase inhibitor p35 in scrib−/− RasV12 mutant clones . Removing or inhibiting caspases in scrib−/− RasV12 mutant clones strongly reduced DHE labeling suggesting suppression of ROS generation ( Figure 2A–D’; quantified in Figure 2E ) . Similar results were obtained with a different ROS indicator , H2DCF-DA ( Figure 1—figure supplement 1C–G ) . Consequently , tumor overgrowth and invasion of the VNC is dramatically reduced upon removal or inhibition of caspases in scrib−/− RasV12 mutant cells ( Figure 2F–I ) . Reduction of caspase activity also increases pupal survival ( Figure 2N ) and viable animals with mosaic scrib−/− RasV12 heads and eyes were recovered as adults at a rate of 4–9% of the surviving pupae ( Figure 2J–M ) . The requirement of caspases for generation of ROS and neoplastic behavior suggests that caspases are activated in scrib−/− RasV12 mutant cells . To verify this , we labeled scrib−/− RasV12 mosaic eye discs with cleaved caspase-3 ( CC3 ) antibody which detects activated ( cleaved ) effector caspases and an unknown non-apoptotic substrate of Dronc ( Fan and Bergmann , 2010; Srinivasan et al . , 1998 ) . Indeed , while there is very little CC3 labeling in mosaic control discs ( FRT + ) , scrib−/− RasV12 mutant clones label significantly stronger with CC3 antibody ( Figure 3A–B’ , yellow arrowhead; quantified in Figure 3D ) . In addition to the CC3 labeling in scrib−/− RasV12 clones , there is also staining immediately outside the clones which appears even more intense than the labeling inside the clones ( Figure 3B , B’; white arrows ) . In fact , quantification reveals that this non-autonomous CC3 labeling is 2 to 2 . 5-fold higher than autonomous CC3 labeling in scrib−/− RasV12 clones ( Figure 3D ) . Both , CC3 labeling inside and outside of scrib−/− RasV12 clones are autonomously dependent on the caspases DrICE ( Figure 3C ) and Dronc as well as on ROS ( Figure 3—figure supplement 1 ) . Labeling with the CC3 antibody indicates active Dronc both in apoptotic and non-apoptotic cells ( Fan and Bergmann , 2010; Fan et al . , 2014 ) . Therefore , because of the strong tumor growth of scrib−/− RasV12 mosaics , we wondered if the CC3-positive cells in scrib−/− RasV12 clones are actually apoptotic and labeled scrib−/− RasV12 discs with TUNEL , an apoptotic assay that detects DNA fragmentation , a hallmark of apoptosis downstream of effector caspases ( Gavrieli et al . , 1992 ) . Interestingly , although a few TUNEL-positive cells are detectable within scrib−/− RasV12 clones , the majority of TUNEL-positive cells are located outside the clones ( Figure 3E , E’; arrows ) . Quantification reveals that almost 90% of all apoptotic cells in scrib−/− RasV12 mosaic discs are outside the mutant clones ( Figure 3F ) . These observations allow us to make an important conclusion . Despite detectable caspase ( Dronc ) activity in scrib−/− RasV12 mutant clones by CC3 labeling , this activity does not appear to trigger a significant amount of apoptosis in these clones . In contrast , our genetic analysis suggest that the strong tumor growth phenotype of scrib−/− RasV12 mosaic eye discs is dependent on caspases ( Figure 2 ) suggesting that they have adopted a tumor-promoting function . This is surprising as caspase activity and apoptosis in general are thought to act as tumor suppressors ( Hanahan and Weinberg , 2011; Hanahan and Weinberg , 2000 ) . In fact , caspase activity in scrib−/− single mutant cells does act as a tumor suppressor by killing them ( Brumby and Richardson , 2003; Igaki et al . , 2006; Igaki et al . , 2009; Uhlirova et al . , 2005; Chen et al . , 2012 ) . In contrast , in scrib−/− RasV12 mutant cells , this caspase activity persists , but does not appear to induce a significant amount of apoptosis . Therefore , these data suggest that RasV12 maintains scrib−/− cells in an ‘undead’-like condition , consistent with a previous report ( Hirabayashi et al . , 2013 ) . Furthermore , RasV12 changes the activity of caspases to adopt a tumor-promoting role . Next , we examined the role of ROS for neoplastic growth of scrib−/− RasV12 mosaic discs . One known function of extracellular ROS is the recruitment and activation of Drosophila macrophages ( hemocytes ) in the ‘undead’ AiP model ( Diwanji and Bergmann , 2017a; Fogarty et al . , 2016; Diwanji and Bergmann , 2017b ) . Hemocytes have been shown to be associated with scrib−/− RasV12 mosaic discs ( Cordero et al . , 2010; Pastor-Pareja et al . , 2008; Külshammer and Uhlirova , 2013 ) . Therefore , we tested if ROS contribute to the recruitment and activation of hemocytes to scrib−/− RasV12 mosaic eye imaginal discs . At control discs , hemocytes adhere in small cellular aggregates , mostly at the antennal disc ( Figure 4A , A’ , L ) . In contrast , they are recruited to neoplastic scrib−/− RasV12 tumor sites in large numbers where they cover the eye portion of the disc ( Figure 4B , B’; quantified in Figure 4K ) consistent with previous reports ( Cordero et al . , 2010; Pastor-Pareja et al . , 2008; Külshammer and Uhlirova , 2013 ) . They also change their morphological appearance at neoplastic scrib−/− RasV12 discs . They single out from the cellular clusters and develop cellular protrusions , similar to cytonemes ( Figure 4M , arrows ) . This change in cellular behavior and morphology of hemocytes was also observed in the ‘undead’ AiP model ( Fogarty et al . , 2016 ) . We examined if ROS and caspases are required for hemocyte recruitment to scrib−/− RasV12 mosaic discs . Indeed , reduction of ROS strongly reduces the recruitment of hemocytes to scrib−/− RasV12 mosaic discs ( Figure 4C–F’ ) . Likewise , the recruitment of hemocytes to scrib−/− RasV12 tumors is impaired by loss of caspase activity ( Figure 4G–I’ ) . Quantification of hemocyte recruitment to ROS-depleted or caspase-inhibited scrib−/− RasV12 discs normalized to GFP ( to account for the reduced size of ROS-depleted or caspase-inhibited scrib−/− RasV12 clones ) , revealed a significant loss of hemocytes compared to scrib−/− RasV12 mosaic discs ( Figure 4K ) . For example , more than 90% of scrib−/− RasV12 discs expressing Duox RNAi are not attached by any hemocyte despite the presence of many small clones ( Figure 4C’ ) . Furthermore , in addition to the significant loss of hemocytes , the few hemocytes that are attached to ROS- and caspase-depleted scrib RasV12 discs ( Figure 4D’–I’ ) , display the naive morphology seen at control discs ( Figure 4N–R ) . These observations provide strong evidence that caspase-dependent generation of ROS is essential for recruitment and activation of hemocytes to scrib−/− RasV12 tumors . As reported previously , JNK activity is strongly induced in scrib−/− RasV12 clones ( Figure 5C ) and is essential for the neoplastic phenotype of scrib−/− RasV12 mosaic animals ( Brumby and Richardson , 2003; Igaki et al . , 2006; Igaki et al . , 2009; Uhlirova et al . , 2005; Leong et al . , 2009; Cordero et al . , 2010 ) . In fact , activation of JNK in oncogenic RasV12 background is sufficient to trigger a neoplastic tumor phenotype similar to the scrib−/− RasV12 condition ( Uhlirova et al . , 2005; Uhlirova and Bohmann , 2006 ) . Therefore , we examined the relation between ROS , caspases and JNK signaling . In a first set of experiments , we blocked JNK signaling by expressing a dominant negative JNK construct ( JNKDN ) in scrib−/− RasV12 mutant clones . In JNKDN-expressing scrib−/− RasV12 mutant clones , caspase activity ( CC3 ) and ROS production are strongly reduced ( Figure 5A–B’ ) . Likewise , the recruitment and activation of hemocytes is strongly impaired at JNKDN; scrib−/− RasV12 discs ( Figure 4J ) consistent with a previous report ( Külshammer and Uhlirova , 2013 ) . These findings suggest that caspase activation , ROS generation and hemocyte activation are dependent on JNK signaling . In the second set of experiments , we examined if there is a dependence of JNK signaling on caspases and ROS using anti-phosphoJNK ( pJNK ) antibody as a JNK activity marker . These labelings revealed a significant loss of JNK activity in caspase-inhibited or ROS-depleted scrib−/− RasV12 clones ( Figure 5D–K’; quantified in Figure 5L ) . Similar results were obtained using MMP1 antibody labeling as an additional JNK marker ( Figure 5—figure supplement 1 ) . These observations suggest that the maintenance of JNK activity requires caspases and ROS . Combined , these results imply that JNK is acting both upstream ( Figure 5A , B ) and downstream ( Figure 5E–L ) of caspases and ROS . The easiest way to explain such a behavior is that caspases , ROS , hemocytes and JNK signaling constitute an amplification loop in scrib−/− RasV12 mutant clones similar to the ‘undead’ AiP model ( Diwanji and Bergmann , 2017a; Fogarty et al . , 2016 ) .
The traditional view of caspases holds that they counter tumorigenesis by eliminating tumor cells and thus mediate a tumor suppressor function ( Hanahan and Weinberg , 2000; Hanahan and Weinberg , 2011 ) . This tumor-suppressing function of caspases has been reported in mammalian systems ( Asselin-Labat et al . , 2011; Ho et al . , 2009 ) and in Drosophila , where , for example , scrib mutant cells undergo caspase-dependent apoptosis in a JNK- and Eiger-dependent manner ( Brumby and Richardson , 2003; Igaki et al . , 2006; Igaki et al . , 2009; Uhlirova et al . , 2005 ) . However , more recent work has suggested that caspases and apoptosis in general can also have the opposite , tumor-promoting function , both in flies and in mammals ( Cheng et al . , 2015; Donato et al . , 2014; Huang et al . , 2011; Kurtova et al . , 2015; Li et al . , 2010; Zhang et al . , 2015 ) ; reviewed by ( Ryoo and Bergmann , 2012; Ichim and Tait , 2016; Fogarty and Bergmann , 2017 ) . Furthermore , in Drosophila it was previously shown that oncogenic Ras switches the tumor-suppressing function of JNK and Eiger in scrib−/− mutant cells to a tumor-promoting one in scrib−/− RasV12 cells ( Igaki et al . , 2006; Cordero et al . , 2010; Uhlirova et al . , 2005; Enomoto et al . , 2015 ) . Mechanistic details about this oncogenic switch have been largely elusive . Our data presented here imply that a critical step for this oncogenic switch is the conversion of caspase activity by oncogenic Ras . Consistent with a previous report in different context ( Hirabayashi et al . , 2013 ) , our results demonstrated that oncogenic Ras can suppress the apoptotic activity of caspases and keeps scrib−/− RasV12 cells in an ‘undead’-like condition . While caspases may still induce apoptosis in a few tumor cells , they now largely promote generation of intra- and extracellular ROS which are required for malignant growth and tissue invasion of surviving neoplastic cells . Evidence that caspases are indeed activated in scrib−/− RasV12 clones is not only provided in this study , but also in a report that showed that small malignant clones ( lgl−/− RasV12 ) undergo caspase-mediated apoptosis and elimination in a similar way as scrib clones ( Menéndez et al . , 2010 ) . Only when mutant clones have reached a certain size , can they develop malignant tumors despite intrinsic caspase activation ( Menéndez et al . , 2010; Ballesteros-Arias et al . , 2014 ) . Our data suggest that JNK activity acts both upstream and downstream of caspases and ROS generation in scrib−/− RasV12 mutant clones ( Figure 5 ) . It is possible that initially , when the scrib−/− RasV12 mutant cells form , a cell competition signal triggers JNK activation in scrib−/− RasV12 cells , similar to the events in scrib−/−-only mutant cells ( Figure 6 ) . In both scrib−/− and scrib−/− RasV12 mutant cells , this JNK activity results in caspase activation ( Figures 3 and 5A ) . However , due to the anti-apoptotic activity of RasV12 , caspases induce only very little apoptosis in scrib RasV12 clones ( Figure 3 ) . Therefore , although caspases are active , most scrib−/− RasV12 cells do not die and thus are in an ‘undead’-like state . Caspases now promote the generation of extracellular ROS through activation of NADPH oxidase Duox . These ROS recruit and activate hemocytes ( Figure 4 ) . It is known that hemocytes can release Eiger ( Cordero et al . , 2010; Parisi et al . , 2014; Fogarty et al . , 2016 ) which signals through its receptor Grindelwald ( Andersen et al . , 2015 ) for further stimulation of JNK activity in scrib−/− RasV12 cells . Thus , we postulate that JNK , caspases , ROS , hemocytes and Eiger constitute an amplification loop ( Figure 6 ) which may be necessary for tumor initiation . Evidence of an amplification loop is provided by the mutual dependence of caspases , ROS and JNK ( Figure 5; Figure 3—figure supplement 1 ) . Similar amplification loops have been described in apoptotic and ‘undead’ cells ( Wells et al . , 2006; Shlevkov and Morata , 2012; Fogarty et al . , 2016 ) . This amplification loop ensures persistent oncogenic signaling in scrib−/− RasV12 cells . This is in striking contrast to scrib−/− mutant cells alone in which JNK signaling triggers linear caspase activation and apoptosis ( Brumby and Richardson , 2003; Igaki et al . , 2006; Igaki et al . , 2009; Uhlirova et al . , 2005 ) . In later stages of tumorigenesis , the amplification loop may have reached full strength and promotes malignant growth and invasion of scrib−/− RasV12 cells ( Figure 6 ) . Amplification loops have also been observed in other neoplastic tumor models in Drosophila . For example , intestinal stem cell tumors form in response to an amplification loop ( Chen et al . , 2016 ) . In a glycolytic tumor model , ROS are also part of a amplification loop that facilitates metabolic reprogramming ( Wang et al . , 2016 ) . Thus , it is possible that tumorigenesis in general depends on amplification loops to sustain oncogenic signaling . There are similarities and differences in the amplification loops of the ‘undead’ hyperplastic AiP model and the neoplastic scrib−/− RasV12 tumor model . In both models , caspases , ROS , hemocytes and JNK are required for growth . However , regarding caspases , the ‘undead’ AiP model only involves the initiator caspase Dronc for growth ( in fact , effector caspases are inhibited by P35 in this model ) ( Fan et al . , 2014; Huh et al . , 2004; Pérez-Garijo et al . , 2004; Pérez-Garijo et al . , 2009; Ryoo et al . , 2004 ) . In contrast , the neoplastic scrib−/− RasV12 model requires both initiator ( Dronc ) and effector ( DrICE ) caspases . Inhibition of either suppresses malignant growth and invasion . Another interesting difference is the differential involvement of ROS . While only extracellular ROS are essential in the ‘undead’ AiP model , neoplastic growth of scrib−/− RasV12 cells requires both intra- and extracellular ROS . Mitochondria are the likely source of intracellular ROS because expression of mitochondrial SOD2 can suppress tumor growth and invasion of scrib−/− RasV12 tumors ( Figure 1N ) . It is unclear if these two populations of ROS are dependent or independent of each other . Recently , it was shown in endothelial cells that Nox-derived ROS can trigger an increase in mitochondrially-derived ROS and loss of mitochondrial membrane potential suggesting a dependence of mitochondrial ROS from Nox-generated ROS ( Shafique et al . , 2017 ) . However , alternatively , it is also possible that intra- and extracellular ROS are produced independently by caspase activity , and both are separately required for neoplastic transformation . Furthermore , while we mostly focused from the point-of-view of ‘undead’ AiP , it is also possible that ‘genuine’ AiP contributes to the tumor growth in scrib−/− RasV12 . Consistent with this notion is the observation that there are a few apoptotic cells in scrib−/− RasV12 mutant clones ( Figure 3E’ , F ) . Genuinely apoptotic cells can also produce ROS in Drosophila imaginal discs ( Santabárbara-Ruiz et al . , 2015 ) . Therefore , it is tempting to speculate that some of the differences between the ‘undead’ AiP model and the scrib−/− RasV12 model are due to a combination of ‘undead’ and ‘genuine’ AiP in scrib−/− RasV12 . This may explain the differences in caspase requirement , the differences in ROS production and the different outcomes of growth – hyperplastic vs . neoplastic – between the ‘undead’ AiP and the scrib−/− RasV12 tumor models . Future work will address this important question . Another important question for future studies will be to address how RasV12 switches the activity of caspases from tumor-suppressors to tumor-promoters . A known target of survival signaling by RasV12 is the pro-apoptotic gene Hid which acts upstream of caspase activation ( Bergmann et al . , 1998; Kurada and White , 1998 ) . Because Eiger and JNK can induce expression of hid ( Moreno et al . , 2002 ) , it is possible that Hid activity is inhibited by oncogenic Ras . However , while we do not exclude this possibility , it alone may not be sufficient to explain the altered caspase activity because inhibition of hid would result in loss of caspase activity . However , caspases are still active in scrib−/− RasV12 mutant cells ( Figure 3B , D ) and they are also able to induce apoptosis at least in a small amount of mutant cells ( Figure 3E , F ) . Therefore , it is possible that RasV12 modifies caspase activity in a different manner – directly or indirectly – for non-apoptotic ROS generation . Oncogenic Ras is mediating many steps in the tumorigenic process of scrib−/− RasV12 tissue . It changes the transcriptome of these cells and modifies the downstream activities of JNK ( Atkins et al . , 2016; Külshammer et al . , 2015 ) . However , as shown in this work , a critical step mediated by RasV12 is the modification of caspase activity – directly or indirectly – in the early stage of tumorigenesis in a way that the cells survive . At present it is unclear if the caspase-modulating activity of RasV12 is dependent on transcription . There may not be enough time for a transcriptional response by the cell to escape the apoptotic activity of caspases . Consistently , work by others has suggested that changes in the transcriptome alone does not fully explain the neoplastic phenotype of scrib−/− RasV12 cells and that other potentially non-transcriptional processes are involved ( Atkins et al . , 2016 ) . Modification of caspase activity may be one of these non-transcriptional processes . Non-apoptotic functions of caspases have been reported ( Shalini et al . , 2015; Fogarty and Bergmann , 2017; Mukherjee and Williams , 2017; Nakajima and Kuranaga , 2017 ) . However , it is largely unknown how cells survive in the presence of activated caspases during non-apoptotic processes . It is possible that a reduction of caspase activity below a certain apoptotic threshold is sufficient for survival . Other models include changes in the subcellular localization of caspases or interaction with modifying factors such as Tango7 ( D'Brot et al . , 2013 ) . Interesting in this respect is a recent study which showed that mitochondrially-derived SCSβ restricts caspase activity for spermatid maturation in the Drosophila testis ( Aram et al . , 2016 ) . Since mitochondria release apoptotic signaling molecules such as cytochrome c and Smac during apoptosis ( Fuchs and Steller , 2011 ) , it may also be possible that they release signals such as SCSβ or related factors which modulate the activity of caspases for non-apoptotic functions . More work is necessary to address these essential questions for understanding of tumor initiation and progression .
The scrib allele used is scrib2 ( also known as scrib673 ) ( Bilder and Perrimon , 2000 ) . The recombinant UAS-RasV12 FRT82B scrib2 ( Chen et al . , 2012; Pérez et al . , 2015 ) line was a kind gift of Madhuri Kango-Singh ( U Dayton , OH , USA ) . The MARCM system ( Lee and Luo , 1999 ) with ey-FLP ( Newsome et al . , 2000 ) was used to generate mosaics of eye/antennal imaginal discs and experimental clones were marked by GFP . In ( Figure 1—figure supplement 1 ) , we used a modified MARCM system that marks clones with myrRFP ( Chabu and Xu , 2014 ) . The wt control line ( FRT + ) is FRT82B ( Xu and Rubin , 1993 ) . The following transgenes are all inserted on chromosome 2 and were crossed into the scrib−/− RasV12 background for analysis: UAS-lacZ ( Bloomington , BL3955 ) , UAS-Duox RNAi and UAS-hCatS ( a kind gift of Won-Jae Lee ) ( Ha et al . , 2005a; Ha et al . , 2005b2005 ) , UAS-Catalase ( BL24621 ) , UAS-SOD1 ( BL24754 ) , UAS-SOD2 ( BL24494 ) , UAS-p35 ( BL5072 ) , UAS-dronc RNAi and UAS-drICE RNAi ( a kind gift of Pascal Meier ) ( Leulier et al . , 2006 ) . UAS-JNKDN ( aka UAS-bskDN ) ( BL6409 ) is an insertion on X chromosome . Crosses were incubated at either 22° or 25°C . DHE and H2DCF-DA ( both from Invitrogen/Molecular Probes ) labeling of unfixed tissue was performed as described ( Owusu-Ansah et al . , 2008 ) . TUNEL labeling ( Roche ) was done according to the manufacturer’s instructions . Antibody labelings were done on fixed tissue following standard procedures ( Fan and Bergmann , 2014; Fogarty and Bergmann , 2014 ) . The following antibodies were used: cleaved caspase-3 ( CC3; Cell Signaling Technology ) ; NimC ( kind gift of I . Andó ) ( Kurucz et al . , 2007 ) ; MMP1 ( Developmental Studies Hybridoma Bank ( DSHB ) ) and pJNK ( Promega ) . Secondary antibodies were donkey Fab fragments from Jackson Immunoresearch . Eye/antennal cephalic complexes were counterlabeled with the nuclear dye DAPI to visualize tissue outline . Images were taken with a Zeiss LSM700 confocal microscope . For quantification of confocal images , the ‘Record Measurement’ function of Photoshop was used . Clones were outlined and signal intensity determined . Multiple clones of five to ten imaginal discs per genotype obtained in three independent experiments were measured . Analysis and graph generation was done using GraphPad Prism 7 . 03 . The statistical method and the P values are indicated in the figure legends .
Apoptosis and its molecular mediators , the caspases , have long been regarded as tumor suppressors and one hallmark of cancer is ‘Evading Apoptosis’ . However , recent work has suggested that apoptotic caspases can also promote proliferation and tumor growth under certain conditions . How caspases promote proliferation and how cells are protected from the potentially harmful action of apoptotic caspases is largely unknown . Here , we show that although caspases are activated in a well-studied neoplastic tumor model in Drosophila , oncogenic mutations of the proto-oncogene Ras ( RasV12 ) maintain tumorous cells in an ‘undead’-like condition and transform caspases from tumor suppressors into tumor promotors . Instead of killing cells , caspases now promote the generation of intra- and extracellular reactive oxygen species ( ROS ) . One function of the ROS is the recruitment and activation of macrophage-like immune cells which in turn signal back to tumorous epithelial cells to activate oncogenic JNK signaling . JNK further promotes and amplifies caspase activity , thereby constituting a feedback amplification loop . Interfering with the amplification loop strongly reduces the neoplastic behavior of these cells and significantly improves organismal survival . In conclusion , RasV12-modified caspases initiate a feedback amplification loop involving tumorous epithelial cells and macrophage-like immune cells that is necessary for uncontrolled tumor growth and invasive behavior .
Throughout the development and life of an animal , many of its cells die and are removed to make space for new tissues . A group of proteins called caspases play a key role in this cell death process . Chemotherapy and radiotherapy work as cancer treatments because they damage dividing cancer cells to such an extent that caspases are turned on and kill those cells . Unfortunately the tumor occasionally grows back after the treatment , partly because caspases also help to produce signals that boost the growth of any surviving cancer cells . Pérez , Lindblad and Bergmann have now used the fruit fly Drosophila to investigate how caspases encourage tumor cells to grow and spread . The fruit flies all carried a mutation in a gene called Ras , which is often mutated in human cancers . Using a combination of genetic and biochemical experiments Pérez , Lindblad and Bergmann found that mutant Ras prevents cells with active caspases from dying . Instead , the caspases redirect their activity and help cancer cells to produce small chemicals called reactive oxygen species . These chemicals can play many different roles in cancers , but in this setting they attract immune cells to the site of the tumor . The immune cells in turn send other signals back to the cancer cells , which further activate the caspases . Overall , this self-perpetuating signaling loop between the cancer cells and the surrounding immune cells helps the tumors to grow . Future work toward developing new cancer treatments will need to work on ways of enhancing the cell-killing properties of caspases while inhibiting their ability to help tumors to grow . Further experiments will also be needed to find out exactly how the mutant Ras gene protects tumor cells from death .