Conserved metabolic energy production pathways govern Eiger/TNF-induced nonapoptotic cell death

Hiroshi Kandaa, Tatsushi Igakib,c, Hideyuki Okanoa, and Masayuki Miurad,e,1

a Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan; bDepartment of Cell Biology, Global Center of Excellence (G-COE), Kobe University Graduate School of Medicine, Hyogo 650-0017, Japan; cPrecursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, Saitama 332-0012, Japan; dDepartment of Genetics, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan; and eCore Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Tokyo 113-0033, Japan

Edited by Kathryn V. Anderson, SloanKettering Institute, New York, NY, and approved October 12, 2011 (received for review February 28, 2011)

Caspase-independent cell death is known to be important in physiological and pathological conditions, but its molecular regulation is not well-understood. Eiger is the sole y ortholog of TNF. The ectopic expression of Eiger in the developing eye primordium caused JNK-dependent but caspase-independent cell death. To understand the molecular basis of this Eiger-induced nonapoptotic cell death, we performed a large-scale genetic screen in Drosophila for suppressors of the Eiger-induced cell death phenotype. We found that molecules that regulate metabolic energy production are central to this form of cell death: it was dramatically suppressed by decreased levels of molecules that regulate cytosolic glycolysis, mitochondrial -oxidation of fatty acids, the tricarboxylic acid cycle, and the electron transport chain. Importantly, reducing the expression of energy production-related genes did not affect the cell death triggered by proapoptotic genes, such as reaper, hid, or debcl, indicating that the energy production-related genes have a specic role in Eiger-induced nonapoptotic cell death. We also found that energy production-related genes regulate the Eiger-induced cell death downstream of JNK. In addition, Eiger induced the production of reactive oxygen species in a manner dependent on energy production-related genes. Furthermore, we showed that this cell death machinery is involved in Eigers physiological function, because decreasing the energy production-related genes suppressed Eiger-dependent tumor suppression, an intrinsic mechanism for removing tumorigenic mutant clones from epithelia by inducing cell death. This result suggests a link between sensitivity to cell death and metabolic activity in cancer.
necroptosis

| chromosomal deciency screen | tumor | scribble

family proteins: Eiger and its receptor, Wengen (912). The ectopic expression of Eiger induces cell death through the activation of c-Jun N-terminal kinase (JNK) signaling both in vivo (9, 10) and in vitro (12). Intriguingly, Eiger-induced cell death seems far less sensitive to the baculovirus-derived pan-caspase inhibitor p35 (9, 10) than other apoptotic cell deaths (13). Thus, the Eigerinduced cell death signaling in Drosophila can provide a powerful genetic model system for studying the conserved mechanism of TNF-induced, caspase-insensitive cell death signaling in vivo. Although several downstream molecules that mediate EigerWengen signaling have been identied (912, 1418), little is known about the mechanism by which these molecules mediate nonapoptotic cell death. In mammals, the signaling cascade of nonapoptotic cell death has been studied since the identication of necroptosis, which is induced by TNF in the presence of caspase inhibitor (1921). However, no comprehensive genetic investigation to elucidate the mechanisms of nonapoptotic cell death, programmed necrosis, or necroptosis in vivo has been reported. Therefore, to examine the genetic control of nonapoptotic cell death, we chose a forward genetic screen for Eiger/ TNF-induced cell death signaling in Drosophila. In this study, we found that Eiger-induced nonapoptotic cell death signaling requires molecules related to the production of metabolic energy downstream of activated JNK. The ectopic activation of Eiger signaling resulted in the overproduction of reactive oxygen species (ROS) downstream of JNK signaling. We also found that this cell death system is, at least in part, used as an intrinsic tumor suppression system in endogenous Eiger signaling to remove tumorigenic scrib mutant cells from epithelia. Results
Eiger-Induced Cell Death Does Not Require the Canonical Caspase Activation Pathway in Drosophila Developing Eyes. We previously

ecent studies have revealed that nonapoptotic cell death is important in both physiological and pathological conditions; however, its molecular mechanism is still largely unknown. Agonistic ligands of death receptors, such as tumor necrosis factor- (TNF), FasL, and TNF-related apoptosis-inducing ligand (TRAIL), generally induce caspase-dependent cell death, the typical form of apoptosis (1). Intriguingly, these ligands can also induce nonapoptotic cell death when caspases are inhibited or cannot be activated efciently (2, 3). A surprising nding that implied the importance of nonapoptotic cell death was that, except in the brain, mice mutant for core regulators of apoptosis do not exhibit severe embryonic tissue-sculpting defects such as interdigital tissue removal, a classic example of physiological cell death (although bax/bak/ double mutant mice have some remaining interdigital tissue) (4). Interestingly, nonapoptotic cell death is observed in the interdigital region of apaf-1 mutant mice (5, 6). These results indicate that nonapoptotic cell death may function physiologically to remove unnecessary tissue or as an alternative mechanism to remove cells when the apoptotic machinery is inhibited. Caspaseindependent or nonapoptotic cell death has also been observed in oncogenic ras-induced cell death (7) and under other pathological conditions (8). We and other groups previously reported that the Drosophila genome encodes one pair of TNF/TNF receptor (TNFR) superwww.pnas.org/cgi/doi/10.1073/pnas.1103242108

showed that the sole Drosophila TNF superfamily ligand, Eiger, induces massive cell death when it is ectopically expressed in the developing eye primordium (eye imaginal disc). This cell death requires the activation of the Drosophila JNK, Basket (Bsk) (9, 10). Intriguingly, although Eiger has been shown to activate caspases (9, 10, 22, 23), the Eiger-induced eye phenotype was only slightly suppressed by coexpression of the pan-caspase inhibitor, p35 (Fig. 1 B and E) (9, 10), whereas p35 almost completely suppressed the cell death phenotype triggered by the ectopic expression of the proapoptotic gene reaper or head involution defective (hid) (Fig. 1 C, D, F, and G). This nding suggested that the Eiger-induced cell death is only partly dependent on caspases. Therefore, we rst sought to clarify the

Author contributions: H.K., T.I., and M.M. designed research; H.K. performed research; H.K., T.I., and M.M. analyzed data; and H.K., T.I., H.O., and M.M. wrote the paper. The authors declare no conict of interest. This article is a PNAS Direct Submission.
1

To whom correspondence should be addressed. E-mail: miura@mol.f.u-tokyo.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1103242108/-/DCSupplemental.

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wild-type

Eiger

Reaper

Hid

A
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receptor, Wengen, was identied in the deciency region of SE1, as described previously (11).
Knockdown of Genes Related to Energy Production Suppresses the Eiger-Induced Small Eye Phenotype. We noticed that genes involved

E
p35

K
LacZ Cleaved Caspase 3 Eiger Hid

J
FRT dronc
A8

GMR-GAL4, UAS-eiger

FRT

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Fig. 1. Eiger-induced cell death is caspase-independent in Drosophila eyes. (AG) Genetic interactions between the pan-caspase inhibitor p35 and Eiger, Reaper, or Hid. Note that the coexpression of p35 with Eiger did not signicantly suppress the reduction of the adult eye size. (HJ) Ectopic expression of Eiger did not induce a signicant increase in caspase 3-like activity. (KN) Loss of the Drosophila initiator caspase, dronc, did not suppress the Eiger-induced eye reduction. The dronc-null mutant allele is described in ref. 49. In all images, anterior is to the left.

or predicted to be involved in mitochondrial function were frequently found in the responsible regions of the SE lines (Table S1). For instance, the Eiger-induced small eye phenotype was signicantly suppressed by P-element insertions into or near the loci of cytochrome c-d (cyt.c-d; it encodes one of two isoforms of Drosophila Cyt.c), Indy (a Krebs cycle intermediate transporter at the plasma membrane), Nmd (an ATPase), or CG18317 (a mitochondrial carrier protein) (Table S1). In addition, the locus of Isocitrate dehydrogenase, which encodes a mitochondrial tricarboxylic acid (TCA) cycle enzyme, was involved in a small deletion of Df(3L)66C-I65, which was recovered as the small deletion in SE8 (Table S1). Because the mitochondrion is the major energy-producing organelle, we next evaluated the role of metabolic energy production-related genes in the regulation of Eiger-induced cell death. To examine the contribution of energy homeostasis to Eiger-induced cell death, we knocked down energy production-related genes in Eiger-expressing imaginal discs. Strikingly, the knockdown of genes involved in cytoplasmic glycolysis, the mitochondrial TCA cycle, the -oxidation of fatty acids, or the electron transport chain was effective in suppressing the Eiger-induced small eye phenotype (Fig. 2 A, F, and G). However, the knockdown of these genes had no effect on the caspase-dependent cell death triggered by the overexpression of Reaper, Hid, or Debcl (Fig. 2 BD), indicating that regulation by energy production-related molecules is specic for the Eigerinduced nonapoptotic cell death. These results indicated that the energy production system is a crucial regulator of Eiger signaling.
Energy Production-Related Molecules Act Downstream of JNK Signaling. Because Eiger-induced cell death is mediated by the

requirement for caspase activation in Eiger-induced cell death. Immunostaining for cleaved caspase 3 revealed that the overexpression of Eiger only slightly increased the caspase 3-like protease activity (Fig. 1 HJ). In addition, because p35 blocks the effector caspases drICE and DCP1 but not the initiator caspase Dronc (24, 25), we evaluated Droncs involvement in the Eigerinduced cell death by overexpressing Eiger in a dronc-null mutant background. However, no phenotypic suppression was observed under this condition (Fig. 1 KN), consistent with a previous observation that the overexpression of a dominant negative form of Dronc does not affect the Eiger-induced eye phenotype (9). Thus, Eiger-induced cell death requires little, if any, activation of the canonical caspase pathway in the eye imaginal disc.
Eighteen Deciencies That Dramatically Suppress the Eiger-Induced Eye Phenotype Identied by a Genome-Wide Dominant Modier Screen. To understand the molecular mechanism of the Eiger-

JNK pathway (9, 10), we next examined the epistasis between the JNK pathway and the energy production-related genes that we identied as suppressors of Eiger. The knockdown of carnitinepalmitoyl transferase-I (CPTI; believed to be the rate-limiting step of fatty acid oxidation), phosphoglycerate kinase (pgk; a transferase in glycolysis), or cyt.c-d (electron transport chain protein) signicantly inhibited cell death (Fig. 3 AE) without affecting JNK activation (Fig. 3 AE), suggesting that these molecules mediate Eiger-induced cell death signaling downstream of JNK.
Eiger-Induced Cell Death Is Accompanied by the Production of ROS. In some cell lines, such as the murine brosarcoma-derived L929, treatment with TNF induces caspase-independent cell death through the production of ROS (26). We therefore examined if Eiger-induced cell death follows oxidative stress using ies that express Glutathione S transferase D1-GFP (gstD-GFP), a ROSinducible gstD promoter-GFP reporter (27, 28). When Eiger was ectopically expressed in the eye imaginal discs of these ies, the cells showed an oxidative stress response, which was revealed by the increase in gstD-GFP expression (Fig. 4 AC). The strength of this response correlated with the strength of the adult eye phenotype (Fig. 4 AC and AC). Furthermore, dihydroethidium staining of these eye discs, in which Eiger was expressed in small patches of the disc, indicated that the cells that ectopically expressed Eiger produced superoxide (O2) (Fig. S2). In addition, the up-regulation of gstD-GFP reporter expression was strongly suppressed by reducing the gene dosage of bsk (Fig. 4 C, C, D, D, and F). Furthermore, the ectopic expression of a JNK kinase kinase, dTAK1, also increased the gstD-GFP signal (Fig. S3). These results suggest that oxidative stress is induced downstream of the activated JNK pathway. We also found that the knockdown of energy production-related genes such as cyt.c-d, GAPDH2, or aconitase signicantly
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induced nonapoptotic cell death in vivo, we conducted a genome-wide screen using a series of chromosomal deciency lines to identify dominant suppressors of Eiger-induced eye reduction (Materials and Methods). We screened more than 80% of the genome and recovered 18 deciency alleles, which we named suppressors of Eiger (SEs 118) (Fig. S1 and Table S1). The responsible regions for the suppression in these deciency alleles were narrowed down by using sets of small, partially overlapping deciencies, and the responsible genes were determined by screening publicly available mutant lines. The Drosophila TNF
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A B C D E F
Indy
dicarboxylates, citrate

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Fig. 2. Energy production-related proteins are specically required to execute Eiger-induced cell death signaling. (AE) Light micrographs of transgenic ies. The genetic background in AE was w; UAS-eigerregg1/+; GMR-GAL4/+ (A), w; GMR-GAL4/+; GMR-rpr/+ (B), w; GMR- hid/+; GMR-GAL4/+ (C), w; GMR-GAL4/+; UAS-debcl/+ (D), and w; GMR-GAL4/+; +/+ (E). Columns an indicate the responsible genes for each UAS-dsRNA line. Anterior is to the left. (F and G) Schematic diagram of glycolysis, -oxidation of fatty acids, and the TCA cycle (F) and diagram of the electron transport chain (G). Responsible genes for RNAi lines that suppressed the Eiger-induced phenotype are shown in red letters. Dashed arrows indicate that the corresponding genes have not been mapped to the chromosome yet or RNAi ies are not publicly available. Complete genotypes and abbreviations are listed in SI Materials and Methods.

attenuated the production of oxidative stress that was induced by the ectopic expression of Eiger to a similar extent as the knockdown of bsk or dTAK1 (Fig. 4G). In contrast, the overexpression of Hid did not increase the gstD-GFP signal (Fig. 4 E, E, and F). These results, together with the ndings shown in Fig. 3, suggest that the energy production-related molecules regulate the production of ROS downstream of JNK in the Eiger-induced cell death pathway.
Energy Production-Related Molecules Are Required for the Physiological Eiger-Induced Death of Tumorigenic Mutant Cells. We next

eliminated from the tissue during the larval and pupal stages (Fig. 5 A, B, E, and F) (31). However, when CPTI or pgk was knocked down, the area of the tumorigenic scrib mutant clones in
GMR-GAL4, UAS-eiger, UAS-dsRNA GMR-GAL4 UAS-lacZ

lacZ

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assessed whether these molecules contribute to the cell death triggered by endogenous Eiger. The imaginal tissue of mutants for evolutionarily conserved tumor suppressors, such as scribble (scrib) or discs large (dlg), overgrow and develop into tumors (29). However, when these tumorigenic mutant cells are surrounded by WT tissue, they do not overgrow but are, instead, eliminated from the tissue through EigerJNK-induced cell death (30, 31). Thus, physiological Eiger signaling functions as an intrinsic tumor suppressor to eliminate tumorigenic mutant cells. We therefore asked if energy production-related molecules regulate the endogenous Eiger signaling during the elimination of tumorigenic scrib mutant clones. To this end, we reduced the expression of CPTI (for fatty acid metabolism) or pgk (for glycolysis) in scrib mutant clones by the Mosaic Analysis with a Repressible Cell Marker (MARCM) method (32). The scrib mutant clones generated in the eye antennal discs were largely
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A
Acridine Orange

Fig. 3. Energy production-related molecules act downstream of the JNK pathway. Eye imaginal discs of wandering third instar larvae stained with an anti-phospho-JNK antibody (AE) and acridine orange (AE). (AE) Eigerinduced activation of JNK was not suppressed by the down-regulation of energy production-related molecules. (AE) The extensive Eiger-induced cell death posterior to the morphogenetic furrow was suppressed by the down-regulation of energy production-related molecules. In all images, anterior is to the left, and arrowheads indicate the morphogenetic furrow.

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gstD-GFP,GMR-GAL4 UAS-lacZ UAS-eigerwk UAS-eigerst bsk1 UAS-eigerst GMR-hid, gstD-GFP

+/+ lacZ

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: p<0.01 : p<0.001

0 gstD-GFP GMR-GAL4 UAS-eiger dsRNA n=

** : p<0.001 + + + + + + + + + + + + + +
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ne CP TI pg k l ac Z CP TI pg k no

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n= 300 245 471 416 297 456

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Fig. 4. Activation of Eiger signaling induces ROS production. (AE) Eye imaginal discs of wandering third instar larvae. Anterior is to the top. Arrowheads indicate the morphogenetic furrow. (A E) Adult eye images of AE. Anterior is to the left. (F) The number of proommatidial clusters that expressed GFP within the single eye discs shown in AE was counted and analyzed statistically. Asterisks indicate statistical signicance, which was determined by Students t test. *P < 0.01; **P < 0.001. wk and st mean weak and strong alleles of UAS-eiger, respectively. (G) RNAi-based knockdown of JNK pathway components or mitochondria-related genes attenuated the production of ROS induced by the overexpression of Eiger. Each bar in the graphs of F and G represents the mean SEM. The number of independent data points in F and G is indicated as n.

scrib-/** p<0.01 * p<0.05

Tumor Suppression

scrib-/** p<0.01

the eye antennal discs increased signicantly (Fig. 5 BD and I), although strong JNK activation was still observed in these cells (Fig. 5 EH and EH). The knockdown of CPTI or pgk in the WT cells did not increase the clone area or pupal lethality; rather, it decreased the clone area, presumably because of the reduction in energy production (Fig. 5I). Therefore, the increased area of the tumorigenic scrib mutant clone was not an additive effect of the CPTI- or pgk-RNAi. Consistent with this result, a signicantly greater number of animals carrying these cells than control animals died as pupae (Fig. 5J). Together, these results indicated that energy production-related genes are crucial for the endogenous Eiger-JNK-mediated cell death signaling that removes tumorigenic scrib cells from epithelia. Discussion
Ectopic Expression of Eiger Induces Nonapoptotic Cell Death. In this paper, we provide genetic evidence that metabolic energy production-related molecules are central to the Eiger/TNF-induced cell death (Fig. 5K). We also provide evidence that this cell death system is, at least in part, used in endogenous Eiger signaling to remove tumorigenic mutant cells from epithelia as an intrinsic tumor suppression system.
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Fig. 5. Energy production-related molecules are required for the endogenous Eiger-JNKinduced cell death signaling that removes tumorigenic scrib cells. GFP-labeled WT (A, E, and E), scrib mutant clones (B, F, and F), or scrib mutant clones with RNAi constructs against energy production-related genes (C, D, G, G, H, and H) were generated in eye antenna imaginal discs. EH show the anti-phospho-JNK antibody staining (magenta) and MARCM clone (green). EH show the anti-phospho-JNK antibody staining of EH. Arrowheads indicate JNK activation in the clone. AD show eye discs. EH and EH show antenna discs. Anterior is to the left. (I) RNAi of energy production-related genes had a survival effect on scrib cells. (J) Pupal lethality caused by the clones induced in eye antennal discs. Asterisks in I and J indicate statistical signicance, determined by Students t test. *, P < 0.05; **, P < 0.01. Each bar in the graphs of I and J represents the mean SEM. The number of independent data points in I and J is indicated as n. (K) Schematic diagram for the JNK-mediated cell death signaling triggered by Eiger. Eigerstimulated signaling is transduced to the JNK pathway through a Wengen/ dTRAF2/dTAB2/dTAK1-mediated signal (912, 1618). The activated JNK signaling is mediated by the energy metabolism-related pathway as shown in this paper. The deregulation of mitochondrial energy homeostasis, involving ROS production, was induced downstream of the activated JNK and energy production. This molecular mechanism is also used by physiological Eiger signaling to remove tumorigenic scrib cells.

It was recently revealed that at least some nonapoptotic cell deaths can be categorized as necroptosis, in which cells undergo nonapoptotic cell death under apoptosis-decient conditions when treated with agonistic ligands of death receptors, such as TNF, FasL, or TRAIL (33). The Eiger-induced cell death shares features with necroptosis in that it is triggered by TNF family proteins, produces ROS, and is caspase-independent. Furthermore, the Drosophila homolog of a tumor suppressor protein, Cylindromatosis, one of the essential regulators of necroptosis (34, 35), has been shown to regulate JNK activation
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in Eiger-induced cell death signaling (16). However, despite the high conservation of most of the apoptotic machinery, blast search analysis has not identied Drosophila homologs of receptor-interacting protein (RIP) 1 or RIP3, the essential kinases for inducing necroptosis (19, 20, 3639). As we showed in Fig. S3, oxidative stress could be induced downstream of the activated JNK pathway. However, we also found that the knockdown of energy production-related genes such as cyt.c-d or GAPDH2 did not suppress the dTAK1- or HepCA-induced cell death phenotype (Fig. S4). This nding could be caused by the overexpression of dTAK1- or HepCAinduced additional pathways, because we found that dTAK1 or HepCA overexpression induced JNK activation much more strongly than Eiger overexpression (Fig. S5). Because dTAK1 also functions downstream of Imd in the innate immune response, Imd-related signaling was another possible mechanism for mediating Eiger signaling. Therefore, we examined the genetic interaction between Eiger-induced cell death and Imd or Imd-related genes. However, we did not nd any signicant interactions between them (Fig. S6). Therefore, it would be interesting to examine if other proteins can substitute for the functions of RIP1 or RIP3 in Eiger signaling or if y TNF signaling uses other mechanisms to induce nonapoptotic cell death.
TNF-Induced Nonapoptotic Cell Death and Energy Production. It has been reported that TNF-induced nonapoptotic cell death leads to the RIP3-dependent activation of glycogen phosphorylase, which is the rate-limiting enzyme in the degradation of glycogen and therefore, the key molecule for regulating energy production (39). In this context, ROS can be generated by the production of excess energy (39). This nding could explain why the downregulation of energy production-related genes suppressed Eigerinduced cell death. However, we also observed that the amount of ATP in the eye antenna imaginal disc decreased when Eiger was overexpressed, and this decrease in ATP was cancelled by the knockdown of bsk, CPTI, pgk, or cyt.c-d (Fig. S7). This nding suggests that the activation of energy production by Eiger signaling could also trigger another mechanism that decreases the tissue ATP level. It is possible that the tissue loses ATP simply because of the massive cell death caused by Eiger expression. Alternatively, the work by Temkin et al. (40) reported that treatment with TNF and the caspase inhibitor zVAD not only induces nonapoptotic cell death with ROS production but also decreases ATP because of the inhibition of adenine nucleotide translocase (ANT) by RIP1. A similar ANT-dependent inhibition mechanism could be involved in Eiger-induced cell death. Because neither RIP1 nor RIP3 has yet been identied in Drosophila, the mechanism by which the total ATP is regulated in Eiger-induced cell death remains to be elucidated. Drosophila Model of Intrinsic Tumor Suppression. When scrib or dlg mutant cells are induced as clones in otherwise WT eye imaginal discs, most of these mutant cells are eliminated by EigerJNKdependent cell death during development (31, 41). This cell death could involve a caspase-independent mechanism, because the elimination of mutant cells is not fully suppressed by the overexpression of p35 compared with the blockage of JNK signaling (30, 31). Thus, the mode of cell death triggered in scrib mutant clones is analogous to the mode of cell death triggered
1. Guicciardi ME, Gores GJ (2009) Life and death by death receptors. FASEB J 23: 16251637. 2. Laster SM, Wood JG, Gooding LR (1988) Tumor necrosis factor can induce both apoptic and necrotic forms of cell lysis. J Immunol 141:26292634. 3. Vercammen D, et al. (1998) Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor. J Exp Med 187:14771485. 4. Lindsten T, et al. (2000) The combined functions of proapoptotic Bcl-2 family members bak and bax are essential for normal development of multiple tissues. Mol Cell 6: 13891399.

by the overexpression of Eiger in imaginal discs. Our observations suggest that the regulation of energy production could be a crucial determinant of the susceptibility of tumor cells to cytotoxic stimuli. Interestingly, tumor cells frequently produce ATP by glycolysis in the cytosol rather than in the mitochondria, which is known as the Warburg effect (42, 43). Because mitochondrial energy production generates cytotoxic ROS, cancer cells might increase their resistance to cytotoxic stimuli by reducing mitochondrial energy production (43). In this sense, mitochondrial energy production could act as a tumor suppressor. In fact, subunits of a TCA cycle enzyme, Succinate dehydrogenase (Sdh; SdhB, SdhC, and SdhD), are reported to be classical tumor suppressors in pheochromocytoma or paraganglioma (44). Furthermore, a specic isoform of pyruvate kinase, which is involved in glycolysis, is necessary for cellular metabolism to shift to aerobic glycolysis and the promotion of tumorigenesis (45). Similarly, the activity of pyruvate dehydrogenase (PDH), which links the glycolytic pathway to the TCA cycle by transforming pyruvate to acetylCoA, is suppressed in cancer cells, whereas the reactivation of PDH induces cell death in a solid tumor cell line and xenografts (46). Interestingly, we found that the knockdown of Drosophila PDH (which is encoded by CG7010) (47) strongly suppressed Eiger-induced cell death (Fig. 2A, g). Furthermore, the downregulation of genes involved in glycolysis and the -oxidation of fatty acids signicantly suppressed the elimination of scrib cells from imaginal epithelia (Fig. 5). These observations suggest that the regulation of cellular energy production or even the source of energy could be critical for controlling the susceptibility of cancer cells to cytotoxic stimuli such as TNF. Materials and Methods
The deciency kit ies were obtained from Bloomington Stock Center. Transgenic ies for RNAi experiments were obtained from the Vienna Drosophila RNAi Center and National Institute of Genetics. The UAS-eigerregg1 allele was described previously (9, 48). The gstD-GFP reporter y (27, 28) was used to detect the antioxidant response. Experiments were approved by the committee on Living Modied Organisms of Keio University, Kobe University, and The University of Tokyo. Additional details are in SI Materials and Methods. ACKNOWLEDGMENTS. We thank Shu Kondo for sharing unpublished data, Hiroka Aonuma and Takahiro Chihara for helpful support, and Shu Kondo and Hirotaka Kanuka for discussions. We also thank Rieko Shimamura, Toshie Naoi, Naoko Tokushige, and Yuki Yamamoto-Goto for technical support, Shizue Ohsawa for technical advice, Takeshi Yagi for kind support and cooperation, and current and former H.O. and M.M. laboratory members for helpful discussions and comments. We thank Toshiro Aigaki, Sharad Kumar, Helena Richardson, Dirk Bohmann, Darren Williams, Yasushi Hiromi, Hermann Steller, Makoto Nakamura, the Bloomington Stock Center, the Drosophila Genetic Resource Center, the National Institute of Genetics stock center (NIG-FLY), and the Vienna Drosophila RNAi center for y stocks. This work was supported in part by grants from the Japanese Ministry of Education, Science, Sports, Culture, and Technology (MEXT; to H.K., T.I., H.O., and M.M.), the Japan Society for the Promotion of Science (to H.K.), the Keio Gijuku Academic Development Funds (to H.K.), the Keio University Grant-in-Aid for Encouragement of Young Medical Scientists (to H.K.), the Strategic Research Foundation Grant-Aided Project for Private Universities from MEXT (to H.K.), the Global Center of Excellence (G-COE) program for Global Center for Education and Research in Integrative Membrane Biology (to T.I.), the International Human Frontier Science Program (to T.I.), the Japan Science and Technology Agency (to T.I. and M.M.), and grant-in-aid for the G-COE program from MEXT to Keio University (to H.K. and H.O.).

5. Chautan M, Chazal G, Cecconi F, Gruss P, Golstein P (1999) Interdigital cell death can occur through a necrotic and caspase-independent pathway. Curr Biol 9:967970. 6. Cand C, Cecconi F, Dessen P, Kroemer G (2002) Apoptosis-inducing factor (AIF): Key to the conserved caspase-independent pathways of cell death? J Cell Sci 115: 47274734. 7. Kitanaka C, Kuchino Y (1999) Caspase-independent programmed cell death with necrotic morphology. Cell Death Differ 6:508515. 8. Yuan J, Kroemer G (2010) Alternative cell death mechanisms in development and beyond. Genes Dev 24:25922602.

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Supporting Information
Kanda et al. 10.1073/pnas.1103242108
SI Materials and Methods Genetic Screen. Fly culture and crosses were carried out at 25 C unless otherwise specied. Canton-S was used as the WT strain. A series of Bloomington deciency kit lines were crossed with UAS-eigerregg1; GMR-GAL4/TM3 transgenic ies, and the F1 progenies were examined for suppression of the small eye phenotype. If a chromosomal deciency region contained a gene that functions downstream of Eiger, the Eiger-induced small eye phenotype should be suppressed. More than 80% of the Drosophila genome was analyzed in the primary screen. The responsible regions for phenotype suppression were narrowed down by using small, overlapping deciencies. The candidates for responsible genes were then screened by examining publicly available mutants. To obtain images of the adult eye, ies were frozen at 80 C for 5 min, and photographs were taken and processed with an Olympus SZX16 camera with Dynamic Eye REAL software (Mitani).
Fly Food. To avoid variations in energy supply from the y food, we

activate ATPase and then quickly frozen. The tissue lysates were centrifuged at 15,300 g for 5 min, and the supernatant was subjected to the ATP assay. The luminouoresence was measured by a Lumat LB9507 (Berthold).
Detailed Genotypes of the Animals Used in Figures.

used freshly cooked y food according to the following recipe in all of the experiments: 400 g dry yeast (Oriental Dry Yeast), 400 g corn our (Nippn), and 80 g agar (Kishida Chemical) were mixed and boiled well in 9 L water. Subsequently, 1,000 g glucose (Nakalai Tesque) were dissolved in 1 L hot water, and added to the mixture; 30 mL propionic acid (Nakalai Tesque) and 50 mL 10% butyl phydroxybenzoic acid in 70% ethanol were added before aliquotting.

Fig. 1A: WT (Canton-S) Fig. 1B: w; UAS-eigerregg1/UAS-lacZ; GMR-GAL4/+ Fig. 1C: w; GMR-GAL4/UAS-lacZ; GMR-rpr/+ Fig. 1D: w; GMR-hid/UAS-lacZ; GMR-GAL4/+ Fig. 1E: w; UAS-eigerregg1/+; GMR-GAL4/UAS-p35 Fig. 1F: w; GMR-GAL4/+; GMR-rpr/UAS-p35 Fig. 1G: w; GMR-hid/+; GMR-GAL4/UAS-p35 Fig. 1H: w; UAS-lacZ/GMR-GAL4 Fig. 1I: w; UAS-eigerregg1/GMR-GAL4 Fig. 1J: w; GMR-hid/+ Fig. 1K: yw eyFLP/+; GMR-hid l(3)* FRT2A/FRT2A Fig. 1L: yw eyFLP/+; GMR-hid l(3)* FRT2A/droncA8 FRT2A Fig. 1M: yw eyFLP/+; GMR-GAL4, UAS-eiger8/+; GMR-hid, l(3)* FRT2A/FRT2A Fig. 1N: yw eyFLP/+; GMR-GAL4, UAS-eiger8/+; GMR-hid, l(3)* FRT2A/ droncA8 FRT2A The transgenic RNAi ies used in the experiments in columns an of Fig. 2 were UAS-lacZ-inverted repeat (IR; a), UASCPTI-IRVDRC4046 (b), UAS- cyt.c-d-IRVDRC17129 (c), UAS-walrus-IRVDRC44378 (d), UAS- pgk-IRVDRC33798 (e), UAS-SdhAIRVDRC110440 (f), UAS- PDH-IRVDRC40410 (g), UAS-GAPDH1IRVDRC31631 (h), UAS-GAPDH2-IRVDRC23645 (i), UAS-aconIRVDRC11767 (j), UAS-Idh-IRVDRC42915 (k), UAS-Indy-IRVDRC9981 (l), UAS-mtacp1-IRVDRC43503 (m), and UAS-Mdh-IRVDRC27535 (n). Fig. 3 A and A : w; UAS-lacZ/+; GMR-GAL4/+ Fig. 3 B and B : w; UAS-eigerregg1/UAS-lacZ-IR; GMR-GAL4/+ Fig. 3 C and C : w; UAS-eigerregg1/+; GMR-GAL4/UAS-CPTIIRVDRC4046 Fig. 3 D and D : w; UAS-eigerregg1/+; GMR-GAL4/UAS-pgkIRVDRC33798 Fig. 3 E and E : w; UAS-eigerregg1/UAS-cyt.c-d-IRVDRC17129; GMR-GAL4/+ Fig. 4 A and A : w; gstD-GFP, GMR-GAL4/UAS-lacZ Fig. 4 B and B : w; gstD-GFP, GMR-GAL4/UAS-eigerwk Fig. 4 C and C : w; gstD-GFP, GMR-GAL4/UAS-eigerst Fig. 4 D and D : w; gstD-GFP, GMR-GAL4/UAS-eigerst, bsk1 Fig. 4 E and E : w; GMR-hid/gstD-GFP Fig. 5 A, E, and E : yw, eyFLP1/+; act > y+ > Gal4, UAS-GFP/+; FRT82B, Tub-Gal80/FRT82B Fig. 5 B, F, and F : yw, eyFLP1/+; act > y+ > Gal4, UAS-GFP/ UAS-lacZ-IR; FRT82B, Tub-Gal80/FRT82B, scrib1 Fig. 5 C, G, and G : yw, eyFLP1/+; act > y+ > Gal4, UASGFP/+; FRT82B, Tub-Gal80/UAS-CPTI-IRVDRC4046 FRT82B, scrib1 Fig. 5 D, H, and H : yw, eyFLP1/+; act > y+ > Gal4, UASGFP/+; FRT82B, Tub-Gal80/UAS-pgk-IRVDRC33798 FRT82B, scrib1 The genotypes of the control animals in which CPTI- or pgkRNAi was induced in the WT clone in Fig. 5 I and J were yw, eyFLP1/+; act > y+ > Gal4, UAS-GFP/+; FRT82B, Tub-Gal80/ UAS-CPTI-IRVDRC4046 FRT82B (CPTI), and yw, eyFLP1/+; act >

Histology. Eye discs from third instar larvae were stained by standard immunohistochemical procedures using a rabbit antiphospho-JNK monoclonal antibody (1:100; Cell Signaling) or rabbit anti-cleaved caspase 3 antibody (1:100; Cell Signaling). The secondary antibodies were 555 Alexa anti-mouse IgG (1:1,000; Molecular Probes) and 555 Alexa anti-rabbit IgG (1:1,000; Molecular Probes). The stained samples were mounted with antifade reagent (Slow Fade Gold; Invitrogen). For acridine orange staining, dissected eye discs were incubated with 1.6 M acridine orange (Sigma) solution for 2 min (1). After a brief rinse with PBS, the samples were analyzed by uorescence microscopy. Dihydroethidium Staining. Eye discs of third instar larvae were dissected in PBS and then stained in 5 M dihydroethidium (Invitrogen)/PBS solution for 5 min at room temperature. The discs were then briey rinsed with PBS two times and washed with PBS for 5 min. The uorescence was analyzed under a uorescence microscope. Monitoring of Cellular Reactive Oxygen Species Production by a gstDGFP Reporter. The eye discs of transgenic ies containing a gstD-GFP

reporter construct were dissected and stained using an anti-GFP antibody (1:100; MBL). The secondary antibody was 488 Alexa antirabbit IgG (1:1,000; Molecular Probes). The samples were mounted with VECTASHIELD mounting medium (Vector Laboratories). The number of GFP-positive clusters was counted under a uorescence microscope.
ATP Assay. The amount of ATP in the eye antenna imaginal discs

was analyzed using an ATP determination kit (A22066; Molecular Probes). In brief, the discs were dissected in PBS, and each disc was separately placed into 1 reporter lysis buffer (Promega). The samples were immediately heat-shocked at 95 C to in-

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y+ > Gal4, UAS-GFP/+; FRT82B, Tub-Gal80/UAS-pgkIRVDRC33798 FRT82B (pgk), respectively. Abbreviations in Fig. 2 are as follows: acon, aconitase; CPTI, carnitine palmitoyltransferase I; cyt.c-d, cytochrome c-distal; Idh,
1. Miron M, et al. (2001) The translational inhibitor 4E-BP is an effector of PI(3)K/Akt signalling and cell growth in Drosophila. Nat Cell Biol 3:596e601.

Isocitrate dehydrogenase; Indy, Im not dead yet; IR, inverted repeat; Mdh, malate dehydrogenase; mtacp1, mitochondrial acyl carrier protein 1; Pgk, phosphoglycerate kinase; Sdh, succinate dehydrogenase.

Fig. S1. Suppression of the Eiger-induced small eye phenotype by suppressors of Eiger (SE) alleles identied in the screen. Light micrographs of adult y eyes. For all ies except WT (Canton-S) and GMR-GAL4, UAS-eiger (UAS-eigerregg1/+; GMR-GAL4/ +), the genotype was UAS-eigerregg1/+; GMR-GAL4/+ on the heterozygous background for the deciencies mentioned in the panels. The name, cytology of the deciency region, and Bloomington stock number for each line are summarized in Table S1.

Fig. S2. Activation of Eiger signaling induces the production of superoxide. Eye discs were stained with dihydroethidium (DHE), which is an indicator of superoxide (O2). DHE-positive cells were detected in the clones in which Eiger was overexpressed. Genotypes were yw, eyFLP1/+; act > y+ > Gal4, UAS-GFP/ UAS-lacZ; FRT82B, Tub-Gal80/FRT82B (Upper) and yw, eyFLP1/+; act > y+ > Gal4, UAS-GFP/UAS-eiger12; FRT82B, Tub-Gal80/FRT82B (Lower), respectively. Arrowheads indicate the morphogenetic furrow. Anterior is to the top.

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Fig. S3. Ectopic expression of dTAK1 increases the gstD-GFP signal. Eye discs were dissected, and the gstD-GFP signal was examined. The activation of JNK signaling was detected by the immunostaining of anti-phospho-JNK. Genotypes were as follows: w; GMR-GAL4, gstD-GFP/UAS-lacZ (AC) and w; GMR-GAL4, gstD-GFP/UAS-dTAK1 (DF). Anterior is to the left. Arrowheads indicate the morphogenetic furrow. Yellow arrow indicates the gstD-GFP signal, which was induced by the overexpression of dTAK1.

Fig. S4. Down-regulation of cyt.c-d or GAPDH2 does not suppress the dTAK1- or HepCA-induced eye phenotype. Light micrographs of transgenic ies. Genotypes were as follows: w; GMR-GAL4, UAS-lacZ-IR/+; UAS-dTAK1/+ (A), w; GMR-GAL4, UAS-cyt.c-d-IRVDRC17129/+; UAS-dTAK1/+ (B), w; GMR-GAL4, UASGAPDH2-IR VDRC23645/+; UAS-dTAK1/+ (C), w; GMR-GAL4, UAS-lacZ-IR/+; UAS-hepCA/+ (D), w; GMR-GAL4, UAS-cyt.c-d-IRVDRC17129/+; UAS-hepCA /+ (E), and w; GMR-GAL4, UAS-GAPDH2-IR VDRC23645/+; UAS-hepCA/+ (F). Anterior is to the left. Animals were raised at 18 C to avoid lethality.

Fig. S5. Overexpression of dTAK1 or HepCA strongly activates JNK. Eye discs were dissected, and the activation of JNK signaling was detected by the immunostaining of anti-phospho-JNK. Genotypes were as follows: w; GMR-GAL4/UAS-lacZ (A), w; GMR-GAL4/ UAS-eigerregg1 (B), w; GMR-GAL4/ UAS-dTAK1 (C), and w; GMR-GAL4/ UAS-hepCA (D). Anterior is to the left. Arrowheads indicate the morphogenetic furrow.

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Fig. S6. Lack of a genetic interaction between Eiger-induced cell death and Imd-related genes. Genotypes were as follows: w; UAS-eigerregg1/UAS-lacZ-IR; GMR-GAL4/+ (A), w; UAS-eigerregg1/+; GMR-GAL4/relE20 (B), w; UAS-eigerregg1/UAS-Imd-IR5516R-1; GMR-GAL4/+ (C), and w; UAS-eigerregg1/UAS-Dredd-IR7486R-2; GMR-GAL4/+ (D).

Fig. S7. Activation of Eiger signaling leads to a reduction in ATP. The eye antenna imaginal discs of third instar larvae were dissected and subjected to an ATP assay. The amount of ATP in individual eye antenna imaginal discs was analyzed separately. Genotypes: UAS-lacZ/+; GMR-GAL4/+ (GMR > lacZ), UAS-eigerregg1/+; GMR-GAL4/+ (), UAS-eigerregg1/UAS-lacZ-IR; GMR-GAL4/+ (lacZ), UAS-eigerregg1/+; GMR-GAL4/UAS-bsk-IRVDRC104569 (bsk), UAS-eigerregg1/+; GMR-GAL4/UAS-pgk-IRVDRC33798 (pgk), UASeigerregg1/+; GMR-GAL4/UAS-CPTI-IRVDRC4046 (CPTI), and UAS-eigerregg1/UAS-cyt.c-d-IRVDRC17129; GMR-GAL4/+ (cyt.c-d). All data are presented as the mean SEM (black bar) of the number of eye discs indicated by n. Asterisks indicate statistical signicance determined by Students t test. **P < 0.005; **P < 0.001.

Table S1.
SE 1 2 3 4 5 6 7 8 BL 3,070 967 1,682 2,471 2,583 3,133 6,299 1,541

Eighteen deciency lines that suppressed the Eiger-induced eye reduction

Allele Df(1)E128 Df(1)C246 Df(2R)or-BR6 Df(2R)M60E Df(2L)cact-255rv64 Df(2L)dp-79b Df(2L)BSC5 Df(3L)66C-G28 Small df None None 2 None 1 1 None 1 Responsible region 17C; 18A 11D-E; 12A01-02 59F3; 60A8-16 60E02-03; 60E11-12 36A8-9; 36D 22A6-22B9 26B1-2; 26D1-2 66C07-10; 66C7-10 Allele see ref. 1 hep1 CG11299BG01215 zip1 bln1 CG18317BG02028 Candidate gene wgn hep CG11299 zip cyt-c-d CG18317 Idh Nmt (Predicted) molecular function TNF receptor JNKK Cytoskeletal protein binding Electron transport Mitochondrial carrier Isocitrate dehydrogenase Glycylpeptide N-tetradecanoyltransferase Transcription factor Transcription factor Transcription factor Cytochrome-b5 reductase Cell death induction Transporter Phosphatase

Nmtj1C7 9 10 1,962 1,990 Df(3R)p-XT103 Df(3R)Tpl10 4 None 85A04-05; 85A06-11 83C01-02; 84B02

11 12

2,611 2,990

Df(3L)vin5 Df(3L)Cat

4 1

68C08-11; 68D6 075B08; 075F01

Scr17 ftzUal2rv3 zen2 CG5946BG01087 W05014 I(3)j14E7j14E7 CG6896BG01140

Scr ftz zen CG5946 hid Indy CG6896

13 14 15 16 17

3,011 3,547 5,411 4,431 1,045

Df(3R)Cha7 Df(3R)L127 Df(3L)Aprt-32 Df(3R)DG2 Df(2L)Mdh

4 None 1 4 None

90F01-04; 91A1-2 99B05-06; 99E04-F01 62B07; 62B12 90F01-04; 91A01-02 30D-F; 31F

bsk1, bsk2 nmdk10909

bsk Nmd (no mitochondrial derivative)

JNK ATPase, carrier

18

950

Df(1)RA2

None

7D18; 8A4

The suppressor of Eiger (SE) number, Bloomington stock number (BL), deciency name, number of overlapping small deciencies, cytology of the determined responsible region, allele name that dominantly suppressed the Eiger-induced eye phenotype, predicted responsible gene of the allele, and molecular function of the putative responsible gene product are listed. Energy metabolism-related molecules are highlighted in red letters.

1. Kanda H, Igaki T, Kanuka H, Yagi T, Miura M (2002) Wengen, a member of the Drosophila tumor necrosis factor receptor superfamily, is required for Eiger signaling. J Biol Chem 277: 28372e28375.

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