Oncogene (2011) 30, 3985–4003 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc REVIEW Mitochondrial respiratory chain complexes: apoptosis sensors mutated in cancer

A Lemarie1,2 and S Grimm3

1INSERM U1037–Cancer Research Center of Toulouse (CRCT), Department of Experimental Therapeutics, Institut Claudius Regaud, Toulouse, France; 2Faculte´ des Sciences Pharmaceutiques, Universite´ Toulouse III, Toulouse, France and 3Department of Experimental Medicine and Toxicology, Imperial College London, Hammersmith Campus, London, UK

Mutations in cancer cells affecting subunits of the Of note, electron leakage can arise all along the RC and respiratory chain (RC) indicate a central role of oxidative lead to reactive oxygen species (ROS) formation phosphorylation for tumourigenesis. Recent studies have (Figure 1). suggested that such mutations of RC complexes impact Deficiencies in the RC are frequently found in cancer apoptosis induction. We review here the evidence for cells. Among the various causes are numerous mutations this hypothesis, which in particular emerged from work in the mitochondrial genome (mtDNA) affecting sub- on how complex I and II mediate signals for apoptosis. units in RCC I, III and IV (Polyak et al., 1998; Ishikawa Both protein aggregates are specifically inhibited for et al., 2008), and in nuclear-coded components of apoptosis induction through different means by exploiting complex II (Baysal et al., 2000; Bayley et al., 2005; with protease activation and pH change, two widespread Hensen and Bayley, 2011) whose assembly factors are but independent features of dying cells. Nevertheless, likewise mutated (Ghezzi et al., 2009; Hao et al., 2009). both converge on forming reactive oxygen species for These alterations lead to specific defects of the the demise of the cell. Investigations into these mito- respiratory chain, which are described in detail below chondrial processes will remain a rewarding area for and depicted in Figure 2. Equally important for RC unravelling the causes of tumourigenesis and for discover- inhibition is the global metabolic shift from OxPhos to ing interference options. glycolysis (Gatenby and Gillies, 2004; Pelicano et al., Oncogene (2011) 30, 3985–4003; doi:10.1038/onc.2011.167; 2006), notably in hypoxia during the early stages of published online 30 May 2011 tumourigenesis (Papandreou et al., 2006; Gogvadze et al., 2010). Keywords: apoptosis; respiratory chain; reactive oxygen species; mitochondria MtDNA mutations in cancer affecting RCC I, III and IV Most RCCs are multimeric enzyme aggregates com- posed of several mtDNA- and nuclear-encoded proteins Respiratory chain defects in cancer (Figure 1). MtDNA-derived subunits of RCC I (7/45 subunits: NADH dehydrogenase subunits ND1 to ND6 The mitochondrial respiratory chain (RC) consists, as and ND4L), III (1/11 subunits: cytochrome b) and IV detailed in Figure 1, of four membrane-bound, multi- (3/13 subunits, cytochrome c oxidase subunits COXI to meric RC complexes (RCCs) and catalyzes the oxidation III) have been found altered in various types of cancers of reducing equivalents, mainly nicotinamide adenine (Brandon et al., 2006; Lu et al., 2009), although they, dinucleotide (NADH), using the terminal electron like their nuclear-encoded counterparts, can also lead acceptor oxygen (O2) in the inner mitochondrial to neurodevelopmental impairment, neurodegenerative membrane (IMM) (Lenaz and Genova, 2010). The diseases or cardiomyopathies when mutated (Benit electron transfer mediated by the RC is connected to the et al., 2009; Distelmaier et al., 2009; Diaz, 2010; final complex, ATP synthase (complex V), which Schapira, 2010). Consequently, the actual contribution generates ATP. This coupling between RC and ATP of these mutations to tumourigenesis is an important synthesis is called oxidative phosphorylation (OxPhos). question—do these mutations act as a significant cause or do they merely constitute a side effect of tumour Correspondence: Dr A Lemarie, INSERM U1037–CRCT, Department development, caused by, for example, the combined of Experimental Therapeutics, Institut Claudius Regaud, Universite´ effect of the intrinsic oxidative stress in cancer cells Toulouse III, 20-24 rue du Pont Saint Pierre, 31052 Toulouse Cedex, France. (Pelicano et al., 2004; Gogvadze et al., 2010) and the low E-mail: [email protected] or Professor S Grimm DNA repair capacity for mtDNA (Penta et al., 2001)? Department of Experimental Medicine and Toxicology, Imperial A confounding factor is the multiplicity of the mtDNA, College London, Hammersmith Campus, Du Cane Road, London which is present in these organelles in hundreds of W12 0NN, UK. E-mail: [email protected] copies. Sequencing of the mtDNA revealed hetero- Received 4 February 2011; revised and accepted 5 April 2011; published plasmy between normal and cancer cells (He et al., online 30 May 2011 2010). Few papers have so far described the precise Respiratory chain complexes and apoptosis A Lemarie and S Grimm 3986

Figure 1 Representation of the mitochondrial respiratory chain complexes and the oxidative phosphorylation system. The four complexes of the respiratory chain and the ATP synthase are schematized and the electron/proton pathways along these complexes are indicated. RCC I and II are the two main entries of reducing equivalents in the RC. RCC I (NADH:ubiquinone oxidoreductase) receives electrons from NADH and transfers them from a flavin mononucleotide (FMN) cofactor to the eight RCC I-embedded iron– sulphur (Fe-S) clusters to eventually reduce ubiquinone (coenzyme Q (CoQ or Q)) to ubiquinol (QH2) in the IMM. This reaction is coupled to the translocation of protons across the IMM from the matrix into the intermembrane space (IMS). RCC II (succinate:ubiquinone oxidoreductase) also contributes to the QH2 pool via the transfer of electrons from succinate, a tricarboxylic acid (TCA) cycle intermediate, to the complex II-embedded cofactor FAD and then to several Fe-S clusters. QH2 transfers its electrons to RCC III (ubiquinol:cytochrome c oxidoreductase) through two heme centres (cytochromes b and c1) and one Fe-S cluster. The energy generated by the electrons carried by RCC III likewise induces a translocation of protons across the IMM. Finally, RCC III delivers its electrons to cytochrome c (Cyt C), which transfers them to RCC IV (cytochrome c oxidase). This terminal RCC possesses four redox centres (two copper atoms and two cytochromes a and a3) and uses the energy generated by the electron transfer to translocate protons but also to reduce O2 in H2O at the matrix side (Belevich and Verkhovsky, 2008; Lenaz and Genova, 2010). All along the RC, electron leakage can arise and lead to reactive oxygen species (ROS) formation. The main sites for these ROS ‘hot spots’ are RCC I and III, but also RCC II (Lenaz and Genova, 2010), as indicated in the figure. The electron transfer is then coupled to the final complex, ATP synthase (complex V), which uses the protomotive force created by RCC I, III and IV to generate ATP at the matrix side. This coupling between RC and ATP synthesis is called oxidative phosphorylation (OxPhos). The number of mitochondrial DNA (mtDNA)- and nuclear DNA (nDNA)-coded subunits of each complex are also indicated.

Oncogene Respiratory chain complexes and apoptosis A Lemarie and S Grimm 3987

Figure 2 Mutations of the respiratory chain components in cancer. The four complexes of the respiratory chain are schematized and the various mutations found in cancer cells that impact RC complex subunits and assembly factors are highlighted: ND1-6 and ND4L (nicotinamide adenine dinucleotide (NADH) dehydrogenase subunits 1–6 and 4L); SDHA–D (succinate dehydrogenase subunits A–D), SDHAF2 (SDH assembly factor 2); cytochrome b; COXI–III (cytochrome c oxidase subunits I–III). mechanisms of how mtDNA mutations are linked to further confirmed through the method of transmito- tumourigenesis and what consequence they have on RC chondrial cybrids (Ohta, 2006), in which mitochondria functions. Only recently have different groups supported from tumour cells are transferred into normal cells and the likelihood of a causative effect on tumour progres- allow study the mtDNA mutations independently of sion. In thyroid cancer cell lines (Abu-Amero et al., the tumoral nuclear DNA. Using this technique, it was 2005) and renal oncocytomas (Gasparre et al., 2008), shown that mutations in mtDNA of complex I (ND1) for example, mtDNA mutations in the complex I and III (cytochrome b) led to a considerable decrease of subunits ND2 and ND4–6 were shown to be associated both enzymatic activities and to ROS overproduction in with complex I deficiency and enhanced proliferation thyroid carcinoma (Bonora et al., 2006). In accordance, (Gasparre et al., 2008). By the same token, human cells another study established that the ND6 mutation carrying an overexpressed ND5 mutant were asso- confers a high metastatic potential to its cybrid and ciated with a drop in oxygen consumption, increased was associated with a profound complex I deficiency ROS production, potentiated glucose dependency and and an overproduction of ROS (Ishikawa et al., 2008). enhanced tumour growth (Park et al., 2009). MtDNA Finally, another argument for the relevance of mtDNA mutations of the complex III subunit COXI were found mutations in cancer progression is made by a cybrid in 11 to 12% of all prostate cancer patients screened model with a mutant form of ATP6, the mtDNA-coded in one study and associated with a complete loss of subunit of ATP synthase (2/16 mtDNA-coded subunits, the COXI protein in tumoral tissues and an increased ATPase 6 and 8; Figure 1). This model showed the tumour growth rate (Petros et al., 2005). Recognizing positive contribution of the ATP6 mutant to tumour the inefficient DNA repair and the heteroplasmy of the growth in mice through a decreased respiration rate, mtDNA in the mitochondria and hence the complex a higher proliferation in culture and a significant interrelation of cause and effect, additional techniques resistance to apoptosis (Shidara et al., 2005). In conclu- were employed to study the role of mitochondrial sion, it appears that a major tumourigenic route links changes on carcinogenesis. The involvement of mtDNA mtDNA mutations to (1) the decrease of one or more mutations in RC dysfunction and tumourigenesis was RCC activities, (2) an impaired electron flux along the

Oncogene Respiratory chain complexes and apoptosis A Lemarie and S Grimm 3988 RC associated with a reduced oxygen consumption, (SDH5), a SDH assembly factor involved in the addi- (3) an increase in the electron leakage outside the tion of the flavin-adenine nucleotide (FAD) prosthetic affected RC leading to an increase of the basal ROS group of SDHA (Hao et al., 2009; Bayley et al., 2010). production and to the subsequent alterations of Finally, in 2010, Gimenez-Roqueplo and colleagues mtDNA, (4) a global resistance to apoptotic processes (Burnichon et al., 2010) detected the first mutation in (Kwong et al., 2007) and finally to (5) an enhanced SDHA leading to PGL. Both of these studies high- proliferation and growth instigating the development of light that at least in some PGL cases SDHA or SDHA- tumour formation (Lu et al., 2009). related genes such as SDHAF2 can act as tumour suppressors. Of note, another complex II assembly factor (SDHAF1) was also recently identified but no Nuclear DNA mutations in cancer affecting complex II involvement in PGL/PH was shown. Rather, SDHAF1 Complex II (succinate dehydrogenase (SDH) or succi- mutations were discovered in two families with infantile nate coenzyme Q (CoQ) reductase (SQR)) of the leukoencephalopathy (Ghezzi et al., 2009). mitochondrial respiratory chain is composed of only An important common characteristic of these SDH four subunits (SDHA, SDHB, SDHC and SDHD) and mutations is their effect on the presence of complex II in is the only complex to be fully encoded by nuclear DNA. the IMM of carrier patients. It appears that mutations This heteroligomer, which is also a component of the in SDHA (Burnichon et al., 2010), SDHB (Gimenez- tricarboxylic acid cycle, contains tumour-suppressor Roqueplo et al., 2003; van Nederveen et al., 2009), proteins (Baysal et al., 2000; Niemann and Muller, SDHC (van Nederveen et al., 2009), SDHD (Gimenez- 2000; Astuti et al., 2001). Indeed, germ-line loss- Roqueplo et al., 2001; Douwes Dekker et al., 2003; van of-function mutations of SDHB, SDHD (cytochrome Nederveen et al., 2009) and SDHAF2 (Hao et al., 2009) bS (cybS)) and, more rarely, SDHC (cytochrome bL all lead to the complete absence or at least a dramatic (cybL)) predispose carriers to head and neck paragan- decrease of the intact complex II and its enzymatic gliomas (PGLs), extra-adrenal PGLs and pheochromo- activity, both in PGL/PH tumours (van Nederveen cytomas (PHs). These SDHB/C/D mutations account et al., 2009) and in gastrointestinal stromal tumours for 15% of all hereditary cases (Rutter et al., 2010) and, (Janeway et al., 2011). Interestingly, even in the absence as they implicate loss of heterozygosity by somatic loss of mutations in the coding sequence of complex II of the normal allele, SDHB/C/D are defined as classical subunits, individual SDH genes can also be markedly tumour-suppressor genes (Baysal, 2008). It is note- downregulated in PGL/PH (Blank et al., 2010; worthy that mutations of the same subunits have also Feichtinger et al., 2011) and in other types of cancers been found in 11–16% of patients with apparently (Habano et al., 2003; Putignani et al., 2008), suggesting sporadic PGL (Amar et al., 2005; Burnichon et al., 2009) that downregulation of RCC proteins in tumours can but appear to be absent in sporadic PH (Waldmann have similar effects as mutations in the protein sequence. et al., 2009; Feichtinger et al., 2011). Mutated SDHB in those tumours is regarded as a high-risk factor for malignancy, poor prognosis and metastasis (Amar et al., Metabolic shift and the general RCC inhibition in cancer 2007; Boedeker et al., 2007; Gimenez-Roqueplo et al., Although various RCC subunits are mutated in some 2008). The neuroendocrine tumours PGL and PH are cancers, in most tumours types the RCC genes are intact considered as relatively rare with a yearly incidence of but the whole respiratory chain is nevertheless restrained 1 to 2 per 1 000 000, respectively (Baysal, 2008), but by the metabolic shift of cancer cells from oxidative complex II mutations have also been found in other phosphorylation to glycolysis, an effect that can still be types of cancers. SDHB mutations, for example, have observed at later stages of the tumour, even when the been associated with renal cell carcinomas (Neumann oxygen supply is restored. This so-called Warburg effect et al., 2004; Ricketts et al., 2008) and neuroblastoma may be an adaptation to the growth conditions during (Schimke et al., 2010), and SDHB or SDHD mutations early tumourigenesis (Gatenby and Gillies, 2004) and with papillary thyroid cancer (Neumann et al., 2004). on a molecular level could be caused by the upregulation Mutations in SDHB, SDHC and SDHD have also been of hypoxia-inducible factor-1a (HIF1a) in response detected in hereditary PGL-associated gastrointestinal to ROS and/or succinate accumulation (reviewed in stromal tumours (McWhinney et al., 2007) and sporadic Gottlieb and Tomlinson, 2005; Bayley and Devilee, gastrointestinal stromal tumours (Janeway et al., 2011). 2010). It was hypothesized by Crabtree that glucose Surprisingly, although numerous germ-line mutations accumulation through the glycolytic shift in tumour cells were found in the iron–sulphur (Fe-S) subunit of can further decrease oxidative phosphorylation, thereby complex II (Ip, SDHB) and in its two small mem- setting in motion a self-amplifying process (Wojtczak, brane-anchoring subunits SDHC and SDHD (Bayley 1996; Diaz-Ruiz et al., 2011). Different mechanisms et al., 2005), initially no mutation in its large flavopro- were evoked, as summarized in Table 1, notably ROS tein catalytic subunit (Fp, SDHA) could be detected overproduction during glucose catabolism, altering in patients. Mutated forms of SDHA were only found mitochondrial proteins (Yang et al., 1997). in a subset of individuals with Leigh syndrome (LS), Concerning ROS, it is noteworthy that direct altera- a neurodegenerative mitochondrial encephalopathy tion of the different RCCs by superoxides, hydrogen (Rutter et al., 2010). Only in 2009, Rutter et al. (2010) peroxides or hydroxyl radicals can be observed in identified in PGL a relatively rare mutation of SDHAF2 isolated mitochondria (Zhang et al., 1990) or in intact

Oncogene Respiratory chain complexes and apoptosis A Lemarie and S Grimm 3989 Table 1 Association of altered cellular pathways observed in cancer cells and RCC inhibition Pathways deregulated in tumour cells Consequences on oxidative phosphorylation Reference

(1) Glycolysis Mechanisms  Competition for ATP and inorganic phosphate General inhibition (Reviewed in Gogvadze et al., 2010) between oxidative phosphorylation and glycolysis.  Intracellular acidification by lactate General inhibition (Heinz et al., 1981; Rodriguez-Enriquez overproduction. et al., 2001; Akhmedov et al., 2010)  Accumulation of the glycolysis intermediate Inhibition of RCC III and IV (Diaz-Ruiz et al., 2008) fructose 1,6 bisphosphate.  Increase of mitochondrial calcium uptake. Inhibition of ATP synthase (Wojtczak et al., 1999)  ROS overproduction (linked notably to Alteration of RCC by ROS-associated Cf. text glucose catabolism). adducts

(2) NO homeostasis: Deregulated in cancer cells (Wink et al., 2008; Direct inhibition of RCC IV (Mason et al., 2006) Muntane´and la Mata, 2010) (competition with RCC IV substrate O2) Inhibition of SDH and RCC I activities (Clementi et al., 1998; Fiorucci et al.,2004)

(3) Ceramide synthesis: or depending on cancer type Direct RCC III inhibition and subsequent (Gudz et al., 1997; Di Paola et al., 2000) (Ryland et al., 2011) ROS production

Abbreviations: NO, nitric oxide; O2, oxygen; RCC, respiratory chain complex; ROS, reactive oxygen species. cells (Hervouet et al., 2008), leading to post-transla- then amplified, in a feed-back loop, the effect leading to tional modifications of each complex, such as lipid further pseudo-hypoxia and HIF1a stabilization (Dahia peroxidation adducts (4-hydroxynonenal or malondial- et al., 2005; Cervera et al., 2008). Moreover, hypoxia dehyde), carbonylation or nitration of residues and was shown to further inhibit complex II activity and finally to a significant decrease of their respective generate mitochondrial ROS (Paddenberg et al., 2003; activities (Choksi et al., 2007, 2008). In the case of Lluis et al., 2007). Finally, hypoxia and probably HIF1a complex II, for example, it was demonstrated that also inhibit ATP synthase, thereby contributing to during ischaemia/reperfusion, superoxides can induce a OxPhos reduction (Campanella et al., 2009). Of note, loss of SDHA glutathionylation favouring its nitration in addition to HIF1a, p53 is also involved in both by peroxynitrite on tyrosine residues, leading to an glucose metabolism (Yeung et al., 2008) and oxidative impairment of its activity (Chen et al., 2007b, 2008). phosphorylation (Frezza and Gottlieb, 2009), and the Finally, besides ROS, pH, calcium, other second downregulation of p53 activity, as observed in tumours, messengers frequently misregulated in cancer were likely leads to RCC impairment and sustained glycolysis shown to control the RC, notably nitric oxide and (Table 2). ceramide, a sphingolipid involved in mitochondrial apoptotic signalling and in the response to anticancer agents (Siskind, 2005; Ryland et al., 2011) (Table 1). Changes in RCC lead to apoptosis resistance In line with these mechanisms of RC regulation, The various RC alterations and mutations found in RC different cellular signals are able to induce a glycolytic complexes imply that their wild-type forms safeguard response in cancer cells and were also found to inhibit cells against malignant transformation and that the the respiratory chain. Among them, the proglycolytic observed changes abrogate this function. The inhibition role of the already mentioned HIF1a has been of apoptosis is regarded as one of the hallmarks of extensively studied (Denko, 2008), and it is now cancer cells (Hanahan and Weinberg, 2000). It allows established that this transcription factor can modulate the malignant cells to continuously grow despite the oxidative respiration (Solaini et al., 2010), as detailed in various adverse conditions they encounter. Conversely, Table 2. Briefly, HIF1a can unbalance the ratio between apoptosis induction is also the basis for the clinical pyruvate dehydrogenase and lactate dehydrogenase in effects of chemotherapeutic drugs (Wallace and Starkov, favour of the latter (Semenza et al., 1996; Kim et al., 2000). Hence, the balance of cell death inhibition and 2006; Papandreou et al., 2006). Recently, the HIF1a susceptibility within the cancer cell can determine both target BNIP3 (Guo et al., 2001) was shown to mediate a the transformation process and the therapeutic outcome similar global RC inhibition (Rikka et al., 2011). Besides of cancer treatment. Accordingly, many cellular regula- these general RC inhibition mechanisms, it was demon- tors of apoptosis turned out to be mutated in cancers. strated that HIF1a can also reduce RCC IV activity Based on these observations, it was speculated that RCCs through the induction of microRNA-210 (Chen et al., also contribute to apoptosis induction. A good candi- 2010). Moreover, in PGL/PH tumours, which exhibit date to make the link between defective RCC function high levels of HIF1a, this transcription factor was and apoptosis are ROS, as they are often formed shown to exert a negative regulatory loop on complex II when RCCs are partially inhibited. These ROS, which through the downregulation of SDHB expression. This can also be generated in physiological conditions, are

Oncogene Respiratory chain complexes and apoptosis A Lemarie and S Grimm 3990 Table 2 The differential effects of HIF1a and p53 transcription factors towards oxidative phosphorylation and glycolysis in tumour cells Factors altered Effects on glycolysis Effects on OxPhos in cancer cells Mechanisms Reference Targeted Mechanisms of OxPhos inhibition Reference complex

HIF1a Glycolysis (Reviewed in General PDH (feeds TCA cycle with acetyl- (Kim et al., 2006; and hypoxic Denko, 2008) inhibition CoA): HIF1a upregulates PDH kinase 1 Papandreou pathways (PDK1), an inhibitor of PDH activity. et al., 2006) LDH (regenerates NADH for glyco- (Semenza et al., lysis): HIF1a upregulates LDH-A subunit. 1996) - Unbalance between sustained glyco- lysis and inhibited RCC. RCC IV COX activity (Chen et al., HIF1a induces miRNA-210 and the 2010) subsequent downregulation of COX10 (assembly factor for RCC IV). RCC II SDHB expression (Dahia et al., HIF1a downregulates SDHB level. 2005; Cervera et al., 2008) General BNIP3 (Rikka et al., inhibition BNIP3 decreases the level of subunits 2011) of RCC I/II/III/IV via their autophagy- associated proteolytic degradation. ATP IF1 (direct natural ATP-synthase (Campanella synthase inhibitor) et al., 2009) - Inhibition/reversion of ATP-synthase phosphorylation function contributing to OxPhos reduction. p53 Glycolysis via (Bensaad RCC IV SCO2 expression (Matoba et al., TIGAR inhibition: et al., 2006) SCO2 is necessary for the correct 2006) TIGAR inhibits the assembly and activity of complex IV glycolysis activator fruc- tose 2,6 bisphosphate.

Abbreviations: COX, cytochrome c oxidase; HIF1a, hypoxia-inducible factor-1a; LDH, lactate dehydrogenase; NADH, nicotinamide adenine dinucleotide; OxPhos, oxidative phosphorylation; PDH, pyruvate dehydrogenase; RCC, respiratory chain complex; SCO2, synthesis of cytochrome c oxidase 2; TCA, tricarboxylic acid; TIGAR, TP53-induced glycolysis and apoptosis regulator.

overproduced when the RC is inhibited, as shown for nism of how complex I couples this electron transport to RCC I, II and III (Ott et al., 2007). The precise mole- a vectorial proton translocation remains only incomple- cular processes of how complexes of the RC are inhi- tely understood. Complex I catalyzes the first reaction bited for apoptosis induction are so far only known in the respiratory chain, the oxidation of NADH to for complex I and II (see below). In both cases, free NAD þ , and provides two electrons, which are sequen- electrons are generated from the substrates of the RCC, tially transferred from a flavin mononucleotide cofactor which are then transferred to molecular oxygen to form to the eight Fe-S clusters in complex I to eventually ROS. These molecules then act in a pleiotropic way in reduce ubiquinone to ubiquinol (Figure 1). the cell for apoptosis induction; they crosslink proteins, produce ceramide, oxidize proteins in the mitochondrial permeability transition pore, activate c-Jun N-terminal Role of complex I in apoptosis kinase, oxidize glutathione and damage DNA (Circu Complex I was the first complex of the respiratory chain and Aw, 2010). for which a molecular explanation for its role in apop- tosis induction was found. Green and colleagues (Ricci et al., 2004) used an ingenious method to detect sub- strates of caspase-3. They separated proteins by sodium Complex I in apoptosis dodecyl sulphate–polyacrylamide gel electrophoresis in one dimension, incubated them with caspase-3 and Structure and function of complex I separated them again by sodium dodecyl sulphate– Complex I (NADH dehydrogenase, also called NAD- polyacrylamide gel electrophoresis in the second dimen- H:ubiquinone oxidoreductase) is the largest complex of sion. Those proteins that were not cleaved ended up the respiratory chain with 45 separate polypeptides in on a straight line, whereas those that were cleaved mammalian cells. Two superstructural ‘arms’ have been were detected below it. Using this approach they identified: one that anchors the complex in the IMM identified NADH dehydrogenase (ubiquinone) Fe-S and is responsible for the translocation of protons, and protein 1 (NDUFS1 or p75) of complex I as a substrate the other that protrudes into the matrix and constitutes for caspase-3. The p75 is—as its name implies—a 75 kDa most of the electron transport moiety. The exact mecha- protein and the largest subunit of complex I. It is localized

Oncogene Respiratory chain complexes and apoptosis A Lemarie and S Grimm 3991 in the IMM, but the loop that is cleaved by caspase-3 could also cause a disturbance of complex I assembly seems to be accessible from the intermembrane space of similar to what was observed with complex II (see below). mitochondria. The cleavage of p75 leads to an inhibition An unexpected role of calpain 10 in connection with of complex I, mitochondrial transmembrane potential complex I inhibition was recently uncovered (Arrington (DCM) collapse and ROS production. A dominant- et al., 2006). Calpains are proteolytic enzymes known to negative p75 mutant (p75D255A), which cannot be respond to calcium increase and thereby get activated. cleaved any more, sustained DCM when introduced into Calpain 10 was found in association with mitochondrial cells and led to a delay in the loss of plasma membrane membranes in the intermembrane space and the matrix integrity. Importantly, this mutant also diminished the of this organelle. With NADH dehydrogenase (ubiqui- formation of ROS. Although the most obvious inter- none) flavoprotein 2 (NDUFV2) and ND6, two pretation of these results is that the cleavage of p75 subunits of complex I were identified as substrates for inactivates it and this is underlying the effects on calpain 10. In line with this, calcium increase inhibited apoptosis, it cannot be excluded that the p75 protein complex I, and when a calpain inhibitor was applied, fragments have an additional effect such as acting as a this effect was mitigated. Hence, it is plausible, although dominant-negative variant. In any case, it can be not yet experimentally documented, that upon apoptosis concluded that p75 is a major substrate for caspase-3 induction, the increase in mitochondrial Ca2 þ causes the and crucial for the execution of apoptosis. Interestingly, activation of calpain 10 and the subsequent cleavage this process appears to affect immunological tolerance. of NDUFV2, and ND6 leads to the inactivation Tolerance can be induced by the injection into mice of of complex I similar to the effects seen with caspase-3 dendritic cells that had engulfed apoptotic cells expressing or GzmA. wild-type p75, whereas this was not the case for the Complex I is known to generate ROS in two ways as noncleavable p75D255A mutant (Kazama et al.,2008). observed in isolated mitochondria from various tissues: Hence, the cleavage of p75 by caspases seems to be one is influenced by the NADH/NAD ratio in the cells important for the induction of immune tolerance by that, when increased and the RC is inhibited at the same apoptotic cells, possibly because it leads to inactivation of time, allows the flavin mononucleotide molecule within an immunostimulatory activity in the dying cells and complex I to transfer electrons to molecular oxygen results in tolerance induction. (Kudin et al., 2004; Kussmaul and Hirst, 2006). The Killer lymphocytes release granzymes and serine other scenario is called reverse electron transport when proteases in order to destroy their target cells (Cullen the ubiquinol pool increases under high Dp by, for and Martin, 2008). Cell death by granzyme A (GzmA) is example, respiration on succinate. Electrons are then morphologically apoptotic but caspase inhibition does transferred from QH2 through complex I (Cino and Del not prevent this process, nor does the activation of anti- Maestro, 1989). It is suggestive to assume that the apoptotic Bcl-2 (B-cell lymphoma 2) family members. reduced flavin mononucleotide is then also the site of Interestingly, GzmA fails to disrupt the mitochondrial electron transfer to O2 but this has not yet been outer membrane (MOM), an event that is otherwise unequivocally proven (Murphy, 2009). It is unknown causing apoptosis induction, as apoptogenic factors from whether complex I is using the same processes to the intermembrane space are thought to be required generate ROS during apoptosis when either NDUFS1 for apoptosis induction (Martinvalet et al., 2005). ROS or NDUFS3 is cleaved. have been shown to underlie cell death effect by GzmA An alternative or additional explanation for the as superoxide scavengers block cell death (Martinvalet effects of complex I on cell death could be the reduction et al., 2005). Martinvalet et al. (2008) showed that, of the ATP level, which then can contribute to despite lacking a mitochondrial targeting sequence, apoptosis. In this regard, it is interesting to note that GzmA makes its way into the mitochondrial matrix when the mitochondrial outer membrane is permeabi- through the Tim/Tom twin translocases and cleaves lized under caspase-inhibition conditions, it seems to be NADH dehydrogenase (ubiquinone) Fe-S protein 3 the reduction of the ATP level through the inhibition (NDUFS3), another subunit of RCC I, after lysine 56 mainly of complex I that is then responsible for cell (Martinvalet et al., 2008). NDUFS3 is an Fe-S protein death (Lartigue et al., 2009). but does not contain a transmembrane domain and Hence, complex I is a sensor for caspase-3/GzmA therefore seems to be localized to the matrix part of the activation (Ricci et al., 2004; Martinvalet et al., complex. Although the exact molecular understanding is 2008) and calcium increase via calpain 10 activation still lacking, it has been demonstrated that the cleavage (Arrington et al., 2006) as common features of apop- of NDUFS3 leads to the formation of ROS and to cell totic cells (summarized in Figure 3) and its inactivation death. When the researchers introduced a protease- is necessary for at least some features of apoptosis. resistant mutant of NDUFS3, they observed a reduction of GzmA- and not GzmB-induced cell death. It will be interesting to see if the NDUFS3 fragment generated by GzmA causes ROS formation and apoptosis when Complex II in apoptosis expressed into cells. On the other hand, if it is the inhibition of NDUFS3 that is responsible for the effects, Structure and function of complex II a knockdown of this protein should exert the same effect RCC II is, besides RCC I, the second entry point of on apoptosis. Alternatively, a reduction of the protein reducing equivalents into the RC via FADH2 generated

Oncogene Respiratory chain complexes and apoptosis A Lemarie and S Grimm 3992

Figure 3 Complex I in apoptosis induction: schematic diagram of the inhibition of complex I for apoptosis induction. The protein subunits that are cleaved by active caspase-3, granzyme A or putatively by calpain 10 are highlighted along with the disrupted electron flux between the flavin mononucleotide (FMN) group and the different iron–sulphur (Fe-S) clusters (N1a to N6b). As a result, the cellular ATP level drops, the mitochondrial transmembrane potential (DCM) collapses and the electron leakage at complex I is used to generate ROS for apoptosis induction. Each protease pathway is summarized on the diagram and detailed in the text. Tom/Tim: twin translocases present in the mitochondrial outer membrane (MOM)/IMM; MOMP: MOM permeabilization.

by the oxidation of succinate to fumarate in the space (Yankovskaya et al., 2003; Sun et al., 2005). tricarboxylic acid (TCA) cycle. The complex II hetero- Of note, their membrane domains contain as an addi- ligomer provides electrons to reduce CoQ to QH2 tional redox group a heme b bound at the interface (Lenaz and Genova, 2010) (Figure 1). RCC II consists between SDHC and SDHD whose role in complex II of only four subunits, all of which are encoded by function is unknown at the moment in mammals nuclear DNA and as such is the smallest RCC. Another (Lemarie and Grimm, 2009) and not yet fully defined particularity is that this complex is the only one in the in yeast (Oyedotun et al., 2007) and Escherichia coli RC not to pump protons across the IMM (Rutter et al., (Tran et al., 2007). Two CoQ-binding sites appear to 2010). In eukaryotic cells, two components, SDHA and reside at the SDHC/SDHD level: QP (proximal) and QD SDHB, constitute the hydrophobic head that protrudes (distal) (Sun et al., 2005; Rutter et al., 2010). CoQ into the matrix. They form the catalytic core that is reduction at the QP site, which has a high affinity for CoQ able to oxidize succinate (which binds to SDHA) and (Maklashina and Cecchini, 2010), is a two-step process carries electrons from this TCA cycle substrate to the with the formation of a partially reduced semiquinone FAD cofactor in SDHA and finally to the 3 [Fe-S] with the first electron transmitted. Only the second clusters in SDHB (Sun et al., 2005; Rutter et al., 2010). electron then fully reduces semiquinone leading to QH2 At this stage, electrons can be captured in vitro by (Guo and Lemire, 2003; Rutter et al., 2010). The semi- artificial electron acceptors and the corresponding quinone radical, contrary to what happens at RCC I and enzymatic activity is called SDH activity (Lemarie III (Lenaz and Genova, 2010), appears to be stabilized at et al., 2011). This catalytic core is associated with a the QP site for complete reduction, avoiding excessive hydrophobic tail formed by two transmembrane pro- electron leakage under normal physiological conditions teins, SDHC and SDHD, both of which also project a (Guo and Lemire, 2003; Rutter et al., 2010). The entire short segment of amino acids into the intermembrane electron transfer from succinate at the catalytic site of

Oncogene Respiratory chain complexes and apoptosis A Lemarie and S Grimm 3993 SDHA to CoQ at the QP site constitutes the SQR of ROS, which could provide a third explanation as to activity of RCC II, which can, just as the SDH activity, why complex II subunits are disabled in tumourigenesis. be measured in vitro with the appropriate enzymatic The first study suggesting that complex II is a central assay (Lemarie et al., 2011). mediator during the apoptotic process was performed The precise molecular mechanisms of ROS produc- by Green and colleagues (Ricci et al., 2003a). They tion at the complex II level have rarely been explored. observed that during apoptosis, after mitochondrial Indeed, the ability of complex II to generate ROS outer membrane permeabilization, RCCs I and II are was mostly neglected in the 1990s as the common inhibited in a caspase-dependent manner and contribute dogma considered RCCs I and III to be the principal via ROS production and DCM collapse to apoptotic cell sources of mitochondrial ROS (McLennan and Degli death (Ricci et al., 2003a). The direct effect of caspase-3 Esposti, 2000; Ricci et al., 2003b), based on studies of on complex I subunit NDUFS1 was later identified chemical inhibition by rotenone and antimycin A, as mentioned above (Ricci et al., 2004), but the exact, respectively (Lenaz and Genova, 2010). Nevertheless, direct or indirect, mechanism by which succinate- different sites for superoxide production have been dependent respiration was altered remained unknown. evoked, for example, at the SDHB/C/D CoQ-binding Schlisio and colleagues demonstrated that neuronal site (Szeto et al., 2007) possibly through the stabilization apoptosis induced by NGF withdrawal necessitates a of the semiquinone radical (Tran et al., 2006), at the functional complex II, acting as a sensor for apoptosis FAD level in SDHA (Yankovskaya et al., 2003) or induction (Lee et al., 2005). Indeed, nerve growth factor between the SDHA-embedded FAD group and a down- deprivation was shown to activate c-Jun and the stream blockade (Guzy et al., 2008; Lemarie et al., downstream proapoptotic pathway mediated by PHD3 2011). These mechanisms appear to depend on the (EglN3) to induce apoptosis. Although the c-Jun- respective targets of the chemical inhibitors used or on activated PHD3 is a prolyl hydroxylase involved the specific sequence of the mutations analysed. in HIF1a degradation, the c-Jun-mediated apoptosis appeared to be independent of HIF1a and required the kinesin KIF1Bb. However, complex II inhibition (by Role of complex II in apoptosis either SDHD siRNA or chemical inhibitors) led mainly The hypothesis that complex II can contribute to to succinate accumulation and to the succinate-mediated apoptosis developed only relatively recently based on blockade of PHD3 proapoptotic function. This mecha- the role of this tetramer in LS, a neurodegenerative nism was hypothesized to favour tumour progression in disease that is associated with neuronal degeneration surviving neurons (Lee et al., 2005). and necrosis/apoptosis (Finsterer, 2008). This was highlighted in the mid-1990s with the discovery of the first SDHA mutation linked to LS (Bourgeron et al., Complex II as a pH sensor for apoptosis. Through a 1995). This observation was corroborated by various genetic high-throughput screen (Grimm, 2004), our studies showing the proapoptotic effects of specific group isolated various proapoptotic genes, notably complex II inhibitors, notably 3-nitropropionate (irre- SDHC and SDHD (but not SDHA or SDHB). Over- versible inhibitor) and methylmalonate (competitive expression of SDHC appeared to inhibit, in a caspase- inhibitor) in neuronal cells (Pang and Geddes, 1997; independent manner, the SQR activity of complex II McLaughlin et al., 1998). Both reagents target the without impairing its SDH activity. This generated SDHA succinate-binding site and equally inhibit the superoxides at complex II and induced a caspase- SDH and SQR activities (Brusque et al., 2002; Lemarie dependent apoptosis (Albayrak et al., 2003). Moreover, et al., 2011). When the tumour-suppressor gene function complex II-deficient hamster CCL16 B9 cells, which of complex II subunits was discovered for SDHD, appear to not assemble an intact complex II (Lemarie SDHC and SDHB (Baysal et al., 2000; Niemann and and Grimm, 2009) owing to a SDHC mutation Muller, 2000; Astuti et al., 2001), two mechanisms were (Oostveen et al., 1995), were dramatically resistant to evoked to explain the oncogenesis process associated numerous structurally diverse anticancer drugs and with complex II deficiency: (1) the establishment of a cytokines, reagents that were shown to specifically pseudo-hypoxic state leading to HIF1a stabilization in reduce complex II activity before overt apoptotic cell tumours through succinate inhibition of the HIF1a death (Albayrak et al., 2003). Subsequent studies were prolyl hydroxylase (PHD), which favours the glycolytic then dedicated to understand the precise mechanisms by pathway and promotes tumour formation (Gimenez- which the anticancer compounds and cytokines inhibit Roqueplo et al., 2001; Pollard et al., 2005, 2006; Selak complex II during the early stages of apoptosis. We et al., 2005; Lehtonen et al., 2007; Cervera et al., 2008) found that all the reagents reduce complex II specifi- and (2) the long-term generation of sublethal super- cally at the SQR level without impairing the upstream oxides at the complex II level contributing to either SDH activity (Lemarie et al., 2011). Of note, the genomic instability and subsequent tumoral develop- complex II-specific inhibitor thenoyltrifluoroacetate, ment or HIF1a stabilization (Senoo-Matsuda et al., which targets the QP-binding site (Sun et al., 2005), 2001; Ishii et al., 2005; Slane et al., 2006; Guzy et al., recapitulated these effects (Krahenbuhl et al., 1991; 2008). Various key publications subsequently brought to Lemarie et al., 2011), suggesting that this downstream light the possible role of complex II as a proapoptotic block of complex II favours disruption of the electron sensor, through the production of deleterious high levels transfer and leakage to molecular oxygen between the

Oncogene Respiratory chain complexes and apoptosis A Lemarie and S Grimm 3994

Figure 4 Complex II in apoptosis induction: schematic presentation of the transition from a functional complex II (a) to the specific release of the SDHA/SDHB subcomplex into the matrix in response to the intracellular and mitochondrial pH (pHi,pHM) acidification .À (b). This complex II dissociation uncouples its two enzymatic activities (SDH and SQR activities) for superoxide (O2 ) production and apoptosis (c). For more detailed information, see text.

still active succinate-binding site of SDHA and the and the SDHC/SDHD anchor (Lemarie et al., 2011). inaccessible QP site to generate superoxides (Lemarie Interestingly, two other cases of complex II dissociation et al., 2011). Furthermore, we provided evidence that into subcomplexes were reported: the first in a patient this specific uncoupling was accomplished through presenting a SDHA mutation and suffering from LS intracellular acidification, which includes a mitochon- (Pagnamenta et al., 2006), and the second in cells drial pH decrease. Early during the apoptotic process responding to ionizing irradiation, which induces ROS the cellular pH drops (Lagadic-Gossmann et al., 2004). in a complex II-dependent manner (Dayal et al., 2009). This seems to impact the interactions within complex II In summary, our results led us to propose the model and leads to the release of the catalytic SDHA/SDHB depicted in Figure 4 in which apoptosis is initiated by subunits from the membrane-anchoring subunits of an early cellular pH change that causes the specific complex II into the matrix, creating an uncoupling disintegration of complex II by the release of the sub- between the still functioning SDHA/SDHB subcomplex complex SDHA/SDHB from the membrane-anchoring

Oncogene Respiratory chain complexes and apoptosis A Lemarie and S Grimm 3995 subunits. As this subcomplex is still enzymatically ingly, both Tcm62 and PHB were shown to act as active (Nakamura et al., 1996; Cecchini, 2003), it can mitochondrial chaperones and to be required for remove electrons from its substrate succinate and mitochondrial respiratory function (Dibrov et al.,1998; transfer them to molecular oxygen. This uncontrolled Klanner et al., 2000; Nijtmans et al., 2000, 2002). This enzymatic activity thereby generates excessive ROS for could indicate that members of this chaperone family can apoptosis induction (Albayrak et al., 2003; Lemarie re-assemble complex II during apoptosis and thereby et al., 2011). Hence, we concluded that complex II is a decrease ROS formation and cell death, making this mitochondrial sensor for pH change as a widespread process subject to additional levels of regulations. initiating event in apoptotic cells (Lagadic-Gossmann et al., 2004) and that its specific inhibition can be suffi- cient for the induction of apoptosis through mitochon- Consequences of complex II-generated ROS for apoptosis drial ROS production. The precise executioners of the and tumourigenesis. Our model depicts complex II as apoptotic cascade mediated by RCC II-induced ROS in being directly targeted by pH acidification. However, it response to anticancer drugs still have to be determined, is also possible that other kinds of signals can produce but caspases seemed to form the apex of this apoptotic the same inhibitory effect, that is, a specific inhibition of cascade (Lemarie et al., 2011). As this process is the SQR activity with minimal impairment of the SDH expected to be curtailed in tumours with metabolically activity leading to ROS generation and to apoptosis. downregulated respiration and in cancer cells with Various studies demonstrated that particular complex II mutations in SDH subunits, the disruption of this subunit mutations or their downregulation also induce essential pH sensor for apoptosis induction can explain apoptosis through ROS production. Pioneer work why these cells can survive adverse growth conditions conducted by Ishii et al. (1998) has shown that a SDHC and form tumours. Concerning apoptotic pathways mutant at the CoQ-binding site (mev-1 mutant) induced mediated by early acidification, it is fair to state that the mitochondrial superoxide production and apoptosis in RCC II sensor function is certainly not the only various models (detailed in Table 3) through the specific mediator of apoptosis induction, as caspases, endonu- inhibition of the SQR activity without SDH function cleases and Bcl-2 family members have also been shown impairment. Other SDHB/C/D mutations at the CoQ- to be directly activated by pH acidification (Lagadic- binding site have been shown to reproduce the effects Gossmann et al., 2004). However, the complex II pH and are described in Table 3. Similar observations were sensor can be sufficient for apoptosis induction and the made with different complex II inhibitors targeting deletion of such a proapoptotic sensor leads to succinate or CoQ-binding sites of SDHA or SDHB/ apoptosis resistance. Of note, it would be interesting C/D, respectively (Table 3). Indeed, the same concen- to extend our observations to scenarios of physio- tration of thenoyltrifluoroacetate, a-tocopheryl succi- pathological acidosis, for example, metabolic acidosis nate and aptenins, which all target the CoQ-binding site, or lactic acidosis associated with hypoxic conditions, can induce a potent inhibition of the SQR activity with and determine whether this likewise induces a complex no or weak SDH alteration and lead to significant ROS II-dependent apoptotic cell death. generation and apoptotic or autophagic cell death Although a direct comparison of complex I- and (Table 3). On the other hand, succinate competitors complex II-dependent mechanisms for apoptosis induc- (malonate derivatives) or a chemical inhibitor (3-nitro- tion has not yet been performed, the effects on complex propionate) curtailed both SQR and SDH activities II could take place earlier during apoptosis as pH and failed to induce similar ROS production and/or change occurs before caspase activation in most apoptosis in the same cell lines (Table 3). Altogether, apoptosis models. Moreover, the subcomplex SDHA/ these results illustrate that specific inhibitors and SDHB remains functional for ROS generation in the mutations of complex II subunits can reproduce the matrix after the dissociation, in contrast to the fast and uncoupling effect between SQR and SDH activities seen almost complete disruption of complex I activity by in the pH-sensor model and generate ROS for apoptosis caspases (Ricci et al., 2004). Therefore, the production induction. of superoxides by complex II could exceed other cellular Other mutations of complex II subunits induce a changes during cell death and produce a particular decrease of the expression of their mRNA or their strong apoptotic signal. Two assembly factors for protein and lead to the depletion of the assembled complex II were discovered lately, one acting on SDHA complex II as revealed for a SDHB mutant in flavination (SDHAF2; Hao et al., 2009) and the other Drosophila (Walker et al., 2006) and for a SDHC putatively on SDHB (SDHAF1; Ghezzi et al., 2009). mutant in hamster CCL16 B9 cells (Lemarie and It can be hypothesized that these proteins render complex Grimm, 2009). Both of these SDHB and SDHC mutants II dissociation reversible and reassemble the subcom- were associated with mild ROS production and with plexes, in contrast to the irreversible NDUFS1 cleavage death or tumourigenesis, respectively (Slane et al., 2006; observed in complex I inhibition (Ricci et al.,2004).In Walker et al., 2006). This downregulation appears to be line with this, Tcm62, another complex II assembly factor very similar to complex II mutations in PGL/PH as discovered in yeast (Dibrov et al., 1998), and prohibitin a large majority of these tumours do not express (PHB), its putative mammalian homologue, have been a correctly assembled complex II (van Nederveen implicated in cell death inhibition following growth et al., 2009). Whether or not these particular mutations factor withdrawal (Vander Heiden et al.,2002).Interest- leading to impairment of complex II assembly, like the

Oncogene Respiratory chain complexes and apoptosis A Lemarie and S Grimm 3996 Table 3 Effects of RCC II mutations and inhibitors on the specific inhibition of complex II activities and on the subsequent cellular signalling Mutation or inhibitor Model Consequences Reference

On RCC II activities On cell signalling

Mutations 8 SDHC (mev-1) Caenorhabditis > Mitochondrial superoxides (Senoo-Matsuda et al., 2001) > (CoQ-binding site) elegans > Apoptosis (Senoo-Matsuda et al., 2003) > Absence of ATP/succinate (Ishii et al., 1998) > <> alterations Ageing SDHC (mev-1) Mouse fibro- SQR inhibition Superoxides and apoptosis (Ishii et al., 2005) > (CoQ-binding site) blasts NIH3T3 > No SDH inhibition In surviving cells: DNA damages > and tumourigenesis in vivo > SDHC (mev-1) Mus musculus > ROS (Ishii et al., 2011) (CoQ-binding site) (in vivo) :> apoptosis SDHC (mev-1) Escherichia coli Superoxides (Zhao et al., 2006) (CoQ-binding site) SDHC/D CoQ-binding Saccharomyces SQR inhibition Superoxides (Szeto et al., 2007) site mutations cerevisiae 8 No SDH inhibition > SQR inhibition Strong superoxide SDHB (pro-211) <> Weak SDH inhibition production and ageing CoQ-binding site C. elegans (Huang and Lemire, 2009) mutations > SQR inhibition Low superoxide :> SDH inhibition production and ageing (same extent)

CoQ-binding site inhibitors TTFA Human cell lines SQR inhibition Mitochondrial superoxides (Lemarie et al., 2011) No SDH inhibition Apoptosis (and/or (Guzy et al., 2008) autophagy) (Chen et al., 2007a) a-TOS Human/rodent SQR inhibition Superoxides (Dong et al., 2008) cell lines Weak SDH inhibition Apoptosis (Dong et al., 2011a) (Dong et al., 2011b) Aptenins Human/bovine SQR inhibition Superoxides (Miyadera et al., 2003) cell lines Weak SDH inhibition (Mbaya et al., 2010)

Succinate-binding site inhibitors 3-NP irreversible Human cell SQR inhibition No or weak ROS production (Guzy et al., 2008) inhibitor lines SDH inhibition Absence of apoptosis (Lemarie et al., 2011) (same extent) (in the same cell lines) Malonate derivatives Human/rat SQR inhibition No or weak ROS production (Guzy et al., 2008) competitors cell lines SDH inhibition Absence of apoptosis (Brusque et al., 2002) (same extent) (in the same cell lines) (Lemarie et al., 2011)

Abbreviations: CoQ, coenzyme Q; 3-NP, 3-nitropropionate; RCC, respiratory chain complex; ROS, reactive oxygen species; SDH, succinate dehydrogenase; SQR, succinate CoQ reductase; a-TOS, a-tocopheryl succinate; TTFA, thenoyltrifluoroacetate.

mutations in the coding sequence for its subunits, are production, except one study showing a moderate mito- associated with ROS production is a critical question, chondrial superoxide production with SDHB shRNA but the detection of ROS and notably of superoxides (Guzy et al., 2008) (Table 4). It is conceivable that in depends on the sensitivity of the method used, the cell those experiments, which did not show oxidative stress, specifics, the culture conditions, the different kinetics the techniques used for ROS detection were not sensitive and the level of ROS production, as well as the mecha- enough or that the kinetics of ROS production did not nisms of their generation. In order to assess whether the correspond to the time points of measurement, notably reduction of complex II assembly through subunit for transient RNAi experiments. However, it is also downregulation can lead to ROS formation and finally possible that there is intervariability between the cellular to apoptosis or tumourigenesis, the data obtained by systems used or in the cellular equipment of antioxidants RNA interference (RNAi) could be instructive (sum- (such as superoxide dismutase, catalase or glutathione) marized in Table 4). We and others have shown that leading to the suppression of ROS. Concerning those depletion of SDHA, SDHB, SDHC or SDHD by SDHA RNAi experiments that did not detect ROS RNAi failed to induce significant apoptosis. This is in overproduction even with the sensitive mitochondria- accordance with our model showing that deletion of targeted probe mitoSOX (Table 4), based on our this apoptosis sensor renders cells resistant to cell observation that an equal inhibition of both SQR and death. In addition, a majority of studies using RNAi SDH activities did not induce superoxide production, it against SDHA/B/C/D failed to detect significant ROS was expected that the suppression of SDHA by RNAi,

Oncogene Respiratory chain complexes and apoptosis A Lemarie and S Grimm 3997 Table 4 Effects of RCC II-targeted RNAi strategies on ROS production and apoptosis RNAi-targeted Type of RNAi ROS detection method Consequences Reference genes

Consequences9 on apoptosis SDHA = SDHB et al. SDHC ; shRNA (transient 72 h) — Absence of apoptosis (Lemarie , 2011) SDHD SDHB shRNA (stable) — Absence of apoptosis (Guzy et al., 2008) SDHC siRNA — Absence of apoptosis (Dong et al., 2008) SDHD siRNA (4–5 days) — Weak caspase 3/7 activity (Puissegur et al., 2011)

Consequences on oxidative stress SDHD siRNA/shRNA DHE (superoxides) (microscopy) Absence of ROS (Selak et al., 2005) (transient 30–48 h) detection (Selak et al., 2006) SDHC siRNA DHE (superoxides) (cytometry) Absence of ROS (Dong et al., 2008) detection SDHB siRNA and shRNA Amplex Red kit (H2O2) Absence of ROS (Cervera et al., 2008) (stable) detection SDHB shRNA (stable) MitoSOX (mitochondria-targeted DHE) Mitochondrial ROS (Guzy et al., 2008) (cytometry) and roGFP (redox-sensitive detection (moderate) GFP probe) (microscopy) SDHA shRNA (stable) MitoSOX (cytometry) and roGFP Absence of mitochondrial (Guzy et al., 2008) (microscopy) ROS detection SDHA siRNA (24 h) MitoSOX (microscopy) Absence of mitochondrial (Piantadosi and Suliman, ROS detection 2008)

Abbreviations: DHE, dihydroethidium; ROS, reactive oxygen species; RNAi, RNA interference; shRNA, small hairpin RNA; siRNA, small interfering RNA.

Table 5 Mechanisms associated with tumourigenesis in complex II-mutated cells RCC II mutation Mechanisms Effects Reference

SDHB (1) Disruption of complex II function as a sensor for apoptosis. (Dong et al., 2008) SDHC Consequence: cell death resistance. (Lemarie et al., 2011) SDHD (2) Succinate accumulation (in vitro and in tumours). (Pollard et al., 2005) Consequence: activation of the HIF1a survival pathway. Tumourigenesis (Selak et al., 2005) et al. (3) Subtle and chronic ROS production (optional). (Smith , 2007) Hypothesis: generation through the remaining matrix SDHA. (Slane et al., 2006) Consequences: cellular damages and/or HIF1a stabilization. (Guzy et al., 2008)

SDHA (rare) (1) Disruption of complex II function as a sensor for apoptosis. SDHAF2 (rare) Consequence: cell death resistance. (2) Succinate accumulation. (Hao et al., 2009) Consequence: activation of the HIF1a survival pathway. Tumourigenesis (Burnichon et al., 2010) et al. (3) Probably no ROS production. (Lemarie , 2011) Hypothesis: the complete disruption of a functional SDHA and of the associated SDH activity does not allow any ROS generation.

Abbreviations: HIF1a, hypoxia-inducible factor-1a; ROS, reactive oxygen species; SDH, succinate dehydrogenase.

which inhibits both activities, failed to induce ROS residual SDH activity through succinate oxidation overproduction. For the downregulation of SDHB, and then generate an electron leakage to molecular SDHC or SDHD, it was shown by our group and oxygen because of the absence of downstream electron others that the resulting disassembly of complex II did acceptors. This phenomenon could explain the ROS not impair the mitochondrial localization of SDHA production observed in two studies during SDHB (Smith et al., 2007; Favier et al., 2009; Lemarie and downregulation (Smith et al., 2007; Guzy et al., 2008). Grimm, 2009), whereas all the other subunits were To conclude, mutations in complex II subunits found absent (Lemarie and Grimm, 2009; van Nederveen et al., in cancer and associated with complex disassembly 2009). This soluble matrix SDHA, even at low quan- could drive the cell fate towards tumourigenesis by the tities (Favier et al., 2009), could suffice to catalyse a confluence of several effects as detailed in Table 5.

Oncogene Respiratory chain complexes and apoptosis A Lemarie and S Grimm 3998 Interestingly, it appears that, contrary to the SDHA/ RC-based strategies in cancer therapy? SDHB/SDHC/SDHD and SDHAF2 mutants found in tumours, the SDHA and SDHAF1 mutants identified in To conclude, the inhibition of the RC is widespread in LS and leukoencephalopathy, respectively, retain a sig- cancer and intimately connected to apoptosis resistance. nificant complex II activity (20–60% according to the Seemingly contradictory, the inhibition of RCC also studies; Bourgeron et al., 1995; Parfait et al., 2000; Horvath leads to ROS formation and apoptosis induction. This et al., 2006; Levitas et al., 2010) and also show a decreased is achieved by the specific way of how the RCCs are but still productively assembled complex II in mitochondria inhibited for cell death. A case in point is that the (Van Coster et al., 2003; Pagnamenta et al., 2006; Ghezzi downregulation of complex II subunits does not lead to et al., 2009). A SDHA mutant identified in a LS patient is apoptosis (Lemarie and Grimm, 2009), whereas its associated with succinate accumulation, HIF1a stabiliza- specific inhibition by dissociation does (Lemarie et al., tion and superoxide production (Briere et al., 2005), as 2011). How then can cancer researchers exploit the described for the SDHB/C/D mutations in PGL/PH. defects of the respiratory chain for therapeutic gain? Succinate accumulation was alsoobservedinacarrierof Can we reactivate the RC in cells affected by the aSDHAF1mutation(Brockmannet al., 2002; Ghezzi Warburg effect and thereby reactivate the apoptosis et al., 2009). As necrosis or/and apoptosis are character- sensors of the RCC? Dichloroacetic acid is a compound istics of LS (Finsterer, 2008) and as ROS-mediated cell that inhibits the enzyme pyruvate dehydrogenase kinase, death was reported to occur in LS models linked to SDHA thus shifting the metabolism of cancer cells from mutations (Mast et al., 2008; Mbaya et al., 2010), it could glycolysis back to OxPhos. This has been shown to be hypothesized that such SDHA/SDHAF1 mutants lead sensitize malignant cells to apoptosis and consequently to neurodegeneration and not to tumourigenesis owing to dichloroacetic acid has been proposed as a therapeutic the maintenance of the apoptosis sensor function of compound (Bonnet et al., 2007; Michelakis et al., 2008, complex II via the remaining assembled complex II and 2010). Other reagents with similar effects have also been not owing to the HIF1a signalling pathway. Of note, used, for example, the glycolysis inhibitors 3-bromopyr- SDHAF1 mutations, thought to impair the assembly of the uvate and 2-deoxyglucose (Halicka et al., 1995; Xu Fe-S cluster in SDHB, lead to a stronger decrease of SQR et al., 2005), but they all must be carefully assessed for than SDH activity (Ghezzi et al., 2009). It is thus conceiv- toxic side effects as they target a quantitative rather than able that the uncoupling generated at the SDHB level may a qualitative difference in the glycolysis rate between be the origin of an electron leakage associated with ROS cancer and normal cells. generation and neuronal cell death. SDHA mutants appear Another class of mitochondrially targeted anticancer to induce an equal SDH and SQR activity inhibition agents, the so-called mitocans (reviewed in Gogvadze (Bourgeron et al., 1995; Parfait et al., 2000; Levitas et al., et al., 2009; Ralph and Neuzil, 2009; Ralph et al., 2010), 2010) and it could be hypothesized that such SDHA muta- have been designed to specifically use the apoptosis tions generate the observed superoxide production (Briere sensor function of the RCCs to induce cell death in et al., 2005) directly at the SDHA level and not between the cancer cells. Different inhibitors of complex I (for succinate catalytic site and the CoQ-binding site. example, tamoxifen; Moreira et al., 2006), complex II (for example, fenretinide; Cuperus et al., 2010) or complex III (for example, adaphostin; Le et al., 2007) Complex II and cell death in neurons: cell-specific apop- markedly induce ROS and apoptosis in tumour cells. totic mechanisms. In relation to the complex II muta- One of the most promising drugs in this field is the tions found in neurodegenerative diseases like LS, it vitamin E derivative a-tocopheryl succinate (Dong et al., appears that neurons are very sensitive to complex 2007, 2008, 2009) and its mitochondrial targeted II-mediated apoptosis. Different studies demonstrated analogue mitoVES (Dong et al., 2011a, b) developed that inhibition of complex II governs a direct apoptotic by the Neuzil group. These compounds were shown to process in neurons through cell-specific pathways. It was specifically target the QP site of complex II for ROS recently shown that neurons, which require high levels of generation by enzymatic uncoupling (see above) and energy, are sensitive to complex II-induced OxPhos elicit apoptosis induction in tumours in vitro and in vivo. impairment and this results in a rapid ATP decrease The apoptotic cascade initiated by these mitochondrial (Mandavilli et al.,2005;Liotet al., 2009; Mbaya et al., ROS was demonstrated to involve specific Bcl-2 family 2010), contrary to other cell types, which did not show members identified as the Noxa–Bak axis and the sucharapidATPdropinresponsetocomplexIIinhib- subsequent mitochondrial outer membrane permeabili- ition by a SDHC mutant or by the inhibitor thenoyltri- zation and effector caspase activation (Prochazka fluoroacetate (Senoo-Matsuda et al., 2001; Ishii et al., et al., 2010; Dong et al., 2011b). Of note, as for the 2007; Soller et al., 2007; Byun et al., 2008). In neurons, reagents that target glycolysis, a crucial aspect is the this ATP decrease induced by either 3-nitropropionate, specificity of these drugs, and further studies are aimed aptenin or a SDHA mutant causes a profound alteration at increasing their efficiency and decreasing potential of the intracellular and mitochondrial calcium homeo- side effects. stasis, which then leads to an amplification of mitochon- Consequently, based on these exciting new findings drial ROS formation by complex II and probably by and the recent discoveries in this field that we have other sources, DCM loss and finally neuronal apoptotic covered here, it emerges that the precise understanding cell death (Liot et al., 2009; Mbaya et al., 2010). of how the RC contributes to apoptosis induction could

Oncogene Respiratory chain complexes and apoptosis A Lemarie and S Grimm 3999 in the future lead to a more informed and targeted Acknowledgements anticancer treatment. We acknowledge the financial support from Cancer Research UK (to AL and SG) as well as from la Ligue Nationale Contre Conflict of interest le Cancer (Hautes-Pyre´ne´es and Haute-Garonne committees; to AL) and support by the Wellcome Trust, CRUK, MRC, The authors declare no conflict of interest. Breast Cancer Campaign (to SG).

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