Succinate Dehydrogenase and Fumarate Hydratase: Linking Mitochondrial Dysfunction and Cancer

Home , Cytochrome C, Mitochondrial matrix, SDHA, Succinate dehydrogenase

Oncogene (2006) 25, 4675–4682 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc REVIEW Succinate dehydrogenase and fumarate hydratase: linking mitochondrial dysfunction and cancer

A King, MA Selak and E Gottlieb

Cancer Research UK, The Beatson Institute for Cancer Research, Glasgow, UK

The phenomenon of enhanced glycolysis in tumours has complexes (I–IV) of the electron transport chain been acknowledged for decades, but biochemical evidence (ETC) and ATP synthase (complex V) (Figure 1). to explain it is only just beginning to emerge. A significant Electron transport from NADH and FADH2 along hint as to the triggers and advantages of enhanced the ETC generates a proton gradient across the glycolysis in tumours was supplied by the recent discovery mitochondrial inner membrane – the mitochondrial that succinate dehydrogenase (SDH) and fumarate membrane potential – that is harnessed by the enzyme hydratase (FH) are tumour suppressors and which ATP synthase to convert ADP þ Pi to ATP, with each associated, for the first time, mitochondrial enzymes and molecule of NADH or FADH2 resulting in three or two their dysfunction with tumorigenesis. Further steps molecules of ATP, respectively. Thus, under normal forward showed that the substrates of SDH and FH, oxygen and glucose conditions and continuous energy succinate and fumarate, respectively, can mediate a requirements, most of the ATP in most cell types is ‘metabolic signalling’ pathway. Succinate or fumarate, produced by oxidative phosphorylation. which accumulate in mitochondria owing to the inactiva- Energy metabolism denotes the reactions from which tion of SDH or FH, leak out to the cytosol, where they the cell obtains and expends energy. Although tightly inhibit a family of prolyl hydroxylase enzymes (PHDs). controlled energy metabolism is fundamental to survi- Depending on the PHD inhibited, two newly recognized val, many types of cancer paradoxically exhibit abnor- pathways that support tumour maintenance may ensue: mal glycolysis or mitochondrial activity in the face of affected cells become resistant to certain apoptotic signals high growth and proliferation rates. In fact, in cancer and/or activate a pseudohypoxic response that enhances cells, ATP can be predominantly derived from glyco- glycolysis and is conveyed by hypoxia-inducible factor. lysis, a significantly less efficient route of ATP produc- Oncogene (2006) 25, 4675–4682. doi:10.1038/sj.onc.1209594 tion, compared to oxidative phosphorylation (Gatenby and Gillies, 2004). When glycolysis prevails, pyruvate Keywords: mitochondria; tumour suppressor genes; is reduced to lactate in order to reoxidize NADH hypoxia inducible factor; apoptosis to NAD þ that is required for sustained glycolysis (Figure 1). Although this apparently anaerobic glycolysis under aerobic conditions is now widely recognized, its biochemical causes and biological consequences in tumours are not (Gatenby and Gillies, 2004; Zu and Background Guppy, 2004). Research to elucidate the mechanisms that lead to enhanced glycolysis in tumours and explain Enhanced glycolysis in cancer cells its advantages to their growth and survival will Glycolysis is the cytosolic pathway that converts glucose undoubtedly help us target energy metabolism pathways to pyruvate. Under aerobic conditions, pyruvate enters to produce cancer therapeutics. the mitochondria to be oxidized by pyruvate dehydro- Increased glycolysis by tumour cells under aerobic genase and enzymes of the tricarboxylic acid (TCA) conditions could be due to one or more of several cycle (Figure 1). Although glycolysis and the TCA cycle mechanisms. Broadly, these can originate from either produce only a small amount of ATP from oxidation of enhancing signals that directly increase glycolysis or each glucose molecule, two major bioenergetic products, from inhibiting energy metabolism by the mitochondria, NADH and FADH2, result from these processes and, in rendering glycolysis the major source of ATP. Glycoly- the presence of oxygen, supply reducing equivalents for tic enzymes are induced by oncogenes (Dang and ATP synthesis by oxidative phosphorylation. The Semenza, 1999; Plas and Thompson, 2005) or by the oxidation of NADH and FADH2 and the subsequent hypoxia-inducible transcription factor (HIF) (Maxwell, phosphorylation of ADP to form ATP take place in 2005a), whereas oxidative phosphorylation can be the inner mitochondrial membrane by the protein inhibited by mutations in mitochondrial DNA (Carew and Huang, 2002; Modica-Napolitano and Singh, 2004) Correspondence: Dr E Gottlieb, Cancer Research UK, the Beatson or a dysfunctional TCA cycle owing to loss of function Institute for Cancer Research, Glasgow, UK. of mitochondrial tumour suppressor genes (Eng et al., E-mail: [email protected] 2003; Gottlieb and Tomlinson, 2005). This review Succinate dehydrogenase and fumarate hydratase A King et al 4676

Figure 1 Schematic representation of the metabolic pathways discussed in this review. Pathways are colour-coded: green – glycolysis; red – tricarboxylic acid (TCA) cycle; blue – oxidative phosphorylation; brown – non-essential amino-acid synthesis; pink – pentose phosphate pathway and nucleotide synthesis; purple – fatty acid synthesis. The fumarate hydratase (FH) and succinate dehydrogenase (SDH) tumour suppressors are also depicted. The SDH complex is involved in both the TCA cycle and oxidative phosphorylation and is coloured accordingly. The anaplerotic reaction mediated by pyruvate carboxylase is indicated here owing to its crucial role in restoring depleted TCA cycle intermediates required for amino and fatty acid synthesis. Electron ﬂow within different complexes of the electron transport chain and the mobile electron carriers quinone and cytochrome c are not depicted in this diagram. Solid arrows indicate direct conversion whereas dotted arrows indicate a general pathway.

focuses on a newly discovered biochemical link between different a-subunits of HIFa (HIF1a,2a and 3a) and the loss of mitochondrial tumour suppressors and the two genes encode HIFb subunits (HIF1b/ARNT1 and induction of glycolysis by the HIF pathway. It will ARNT2), enabling combinatorial complexity and the address the contribution of this and alternative path- formation of HIF heterodimers with potentially differ- ways to cancer formation owing to loss of mitochondrial ent activities (Maxwell, 2005b). Under normal oxygen tumour suppressors. levels (normoxia), the protein level of HIFa is very low owing to constant degradation, mediated by a sequence of post-translational modification events. The first is Hypoxia-inducible factor HIFa hydroxylation on two prolyl residues, a reaction In many tumours, oxygen availability becomes limited that is catalysed by HIFa prolyl hydroxylase enzymes (hypoxia) very quickly during cancer development. (PHD1, 2 and 3also known as EglN2, 1 and 3, Intermittent hypoxia may arguably be the only reason respectively) (Dann and Bruick, 2005). Following HIFa for increased tumour glycolysis (Zu and Guppy, 2004). hydroxylation, the von Hippel–Lindau (VHL) gene The major regulator of the response to hypoxia is the product (pVHL) mediates the ubiquitylation of HIFa, HIF transcription factor. Hypoxia-inducible factor is a leading to proteasome-mediated degradation (Schofield heterodimeric complex comprised of HIFa and HIFb and Ratcliffe, 2005). Hypoxia-inducible factora hydro- subunits (Maxwell, 2005b). Three genes encode three xylation by PHD enzymes is oxygen dependent.

Oncogene Succinate dehydrogenase and fumarate hydratase A King et al 4677

Figure 2 Hypoxia-inducible factor (HIF)a homeostasis is controlled by its rate of translation and degradation. Under physiologic conditions, HIFa is constantly degraded in a process dependent on oxygen, prolyl hydroxylation (by PHD) and ubiquitylation (mediated by pVHL). In cancer, elevated HIFa levels can be seen owing to hypoxia (lack in oxygen) or pseudo-hypoxia (PHD inhibition or pVHL mutation) that inhibit HIFa degradation or owing to accelerated translation (potentially mediated by Akt or loss of Tsc complex or LKB1). Proteins coloured in green represent HIF downregulators whereas those in red represent HIF inducers. Interestingly, many oncogenes and tumour suppressor genes are involved in HIF regulation either positively or negatively, respectively, emphasizing the importance of HIF in tumour development and/or sustention. Solid arrows indicate direct interaction whereas dotted arrows indicate an indirect pathway.

However, as PHDs possess a high Km for oxygen, HIFa Mitochondrial tumour suppressor genes escapes hydroxylation and degradation under hypoxic conditions (Schofield and Ratcliffe, 2004). Once stabi- Both SDH and FH are TCA cycle enzymes that convert lized, HIFa translocates to the nucleus, where it can succinate to fumarate and fumarate to malate, respec- heterodimerize with HIFb to form an active HIF tively. Succinate dehydrogenase is also a functional transcription factor, leading to the expression of genes member (complex II) of the ETC (Figure 1). Surpris- involved in, among other mechanisms, glucose metabo- ingly, although SDH and FH are ‘housekeeping genes’ lism and angiogenesis – both survival responses to with key bioenergetic roles, mutations in these genes hypoxia in normal and malignant tissues (Figure 2). cause cancer: mutations in subunits B, C or D of SDH Tumour growth and survival depend on the enhance- lead to the development of paraganglioma or phaeo- ment of both glucose metabolism and angiogenesis. chromocytoma and mutations in FH cause leiomyoma, Accordingly, some tumours demonstrate high leiomyosarcoma or renal cell carcinoma (for more HIF activity even under normoxic conditions, indica- details see Gottlieb and Tomlinson, 2005). ting high tumour resilience and poor patient progno- As many of the genetic aspects of mitochondrial sis (Maxwell, 2005a). A pseudohypoxic response in tumour suppressor genes have been discussed in several tumours can be achieved either by accelerated mamma- recent extensive reviews (Baysal, 2003; Eng et al., 2003; lian target of rapamycin (mTOR)-dependent translation Favier et al., 2005; Gottlieb and Tomlinson, 2005), only or by impaired degradation of HIFa (Figure 2). The a brief history of the field will be given and more current mTOR-dependent translation of HIFa is induced by data will be covered in this review. In particular, the loss of the Tsc1, Tsc2 or Lkb1 tumour suppressor attention will be given to recent findings concerning genes and potentially by activation via the Akt pathway the potential mechanisms of tumorigenesis in SDH and (Inoki et al., 2005; Plas and Thompson, 2005). Impaired FH-linked tumours. HIFa degradation is severely manifest in cells deficient This relatively new field of research opened with a for the VHL tumour suppressor gene (Kim and Kaelin, genetic study of the cancer syndrome hereditary para- 2004). More recently, impaired HIFa degradation was ganglioma (HPGL). Paraganglioma are typically benign demonstrated in tumours lacking the tumour suppres- tumours derived from neuronal ectoderm cells along the sors succinate dehydrogenase (SDH) and fumarate sympathetic or parasympathetic nervous systems (Da- hydratase (FH or fumarase), nuclear genes that encode hia, 2006). In 2000, Baysal et al. (2000) discovered that mitochondrial proteins (Isaacs et al., 2005; Selak et al., the gene mutated in a particular HPGL syndrome 2005). associated with the 11q23locus (PGL1) is in fact SDHD

Oncogene Succinate dehydrogenase and fumarate hydratase A King et al 4678 (Baysal et al., 2000). Following this initial discovery, it SDH or FH probably indicate that different mechan- did not take long to identify the genes of two other isms contribute variably to tumour phenotype. Such HPGL-associated loci, PGL4 (1p36) and PGL3 (1q21), mechanisms may also cause the small but significant to be SDHB and SDHC, respectively (Niemann and genotype–phenotype differences observed between the Muller, 2000; Astuti et al., 2001). Germ line hetero- SDH genes. Yet pseudo-hypoxia, the major phenomen- zygous mutations of these genes generally follow the on shown to date to mediate the tumorigenic outcome of classic Knudson’s ‘two hits’ sequence of tumour the loss of mitochondrial tumour suppressors, is a suppressor inactivation and are usually, but not always, common mechanism for both SDH and FH mutations. associated with loss of heterozygosity (LOH) in the Still, the existence of a common mechanism is in keeping tumours (Pawlu et al., 2005). Mutations in the SDH with the fact that SDH and FH catalyse successive tumour suppressor genes are associated not only with reactions of the TCA cycle and thus share a common familial syndromes but are also observed in sporadic pathway (Figure 1). paraganglioma and in phaeochromocytoma, a subtype of paraganglioma derived from catecholamine-secreting Loss of succinate dehydrogenase and fumarate hydratase chromaffin cells, usually in the adrenal medulla (Got- causes pseudo-hypoxia and hypoxia-inducible factor tlieb and Tomlinson, 2005). The observation that loss of induction function of the SDH complex is mostly associated with paraganglioma, regardless of the SDH subunit involved, The link between the loss of mitochondria tumour suggests that tumorigenesis because of these mutations suppressors and pseudo-hypoxia was indicated by stems from a common biochemical pathway. This is several studies. Initial research by Gimenez-Roqueplo supported by the observation that mutations in either et al. (2001) showed that in some tumours with SDHD SDHB SDHB or SDHD result in the disintegration of the SDH or mutations, the HIF pathway and conse- complex and a complete loss of SDH enzymatic activity quently an angiogenic response are activated. This study (Gimenez-Roqueplo et al., 2001; Gimenez-Roqueplo explained the high vascular density observed in these et al., 2002; Douwes Dekker et al., 2003). However, a paragangliomas. More recently, an extensive gene closer examination of the genotype–phenotype link expression analysis of 76 phaeochromocytomas with revealed several differences between the different SDH different genetic lesions revealed a ‘HIF signature’ genes. SDHC mutations or deletions are rare and have pattern of gene expression in a cluster of phaeochro- SDHB SDHD VHL so far been associated only with familial and sporadic mocytomas with , or mutations et al head and neck paraganglioma (Baysal et al., 2004; (Dahia ., 2005). Interestingly, in the past year, it was Schiavi et al., 2005). In addition, two comprehensive shown that FH-deficient tumours also display high et al comparison studies of tumours with SDHB or SDHD vascularity (Pollard ., 2005a), increased HIFa levels et al mutations have been performed and several obvious and activity (Pollard ., 2005b) and increased differences have been identified (Neumann et al., 2004; expression of glycolytic genes (measured by gene et al Benn, 2006). It is noteworthy that SDHD mutations are profiling) (Vanharanta ., 2006). These independent mostly related to head and neck paraganglioma whereas studies collectively suggested that pseudo-hypoxia, SDHB mutations can be seen at higher frequencies in manifested by high HIF activity under normoxic adrenal and extra-adrenal phaeochromocytoma as well conditions, is an important factor that links mitochon- as in non-paraganglioma tumours. SDHB mutations are drial tumour suppressors and cancer. also associated with higher incidences of malignant and metastatic tumours such as malignant phaeochromocy- Mechanisms of hypoxia-inducible factor induction. The toma and, in some cases, renal cell carcinoma (Vanhar- works described above, while linking the loss of anta et al., 2004). mitochondrial tumour suppressors to HIF activation, In 2002, the FH gene at 1q43was independently do not provide a biochemical rationale for the link. The identified as the fourth mitochondrial tumour suppres- first biochemical explanation was reported in 2005 sor gene in families with the hereditary leiomyomatosis (Selak et al., 2005) and demonstrated that succinate, and renal cell carcinoma (HLRCC) syndrome (Tomlin- the substrate of SDH, can provoke HIF stability. son et al., 2002). Like the SDH genes, FH mutations Succinate is a dicarboxylic acid, capable of crossing lead to loss of function and are associated with loss of the mitochondrial inner membrane (the only real barrier the wild-type allele and FH activity in the tumours to small metabolites in the mitochondria) via the (Tomlinson et al., 2002; Wei et al., 2006). Unlike SDH, dicarboxylate carrier. Selak et al. (2005) showed that somatic FH mutations are rare and are restricted to SDH dysfunction in cells raised the levels of succinate uterus leiomyomas (Lehtonen et al., 2004). and, that succinate serves as an intracellular messenger between the mitochondria and the cytosol. Succinate that accumulates in the mitochondrial matrix owing to SDH dysfunction leaks out into the cytosol where it Mechanisms linking loss of mitochondrial tumour inhibits the activity of HIFa PHDs. As mentioned suppressors to cancer above, HIFa prolyl hydroxylation by PHD is the first step in tagging HIFa for degradation, enabling its It is important to state that the different types of interaction with pVHL. PHDs are a-ketoglutarate- tumours that develop from the dysfunction of either dependent dioxygenases and overall they catalyse the

Oncogene Succinate dehydrogenase and fumarate hydratase A King et al 4679 bioenergetics (Rustin and Rotig, 2002). This study also addressed the important question as to why SDHA has never been reported to be a tumour suppressor gene and, in particular, why carriers of an SDHA mutant allele (heterozygous parents or siblings of Leigh syndrome patients) do not develop HPGL. Briere et al. (2005) addressed that puzzle by studying the expression of two different (though highly similar) SDHA type I and II genes and showed that, unlike ﬁbroblasts, the paraganglia system expresses both genes. This indicates that for SDHA mutation carriers, the unlikely situation of inactivating three additional alleles, two of which are the intact wild-type alleles of one gene, needs to occur in order to develop HPGL.

Figure 3 Hypoxia-inducible factor (HIF)a prolyl hydroxylase Advantages of pseudo-hypoxic tumours. Most tumours (PHD) requires oxygen and a-ketoglutarate as cosubstrates and eventually reach a hypoxic state and it is conceivable ferrous iron and ascorbate as cofactors. For that reason, its activity that during rapid proliferation, tumour cells frequently is regulated by oxygen levels and can be inhibited by reactive oxygen species that oxidize its cofactors and by succinate or experience intermittent hypoxic conditions. Therefore, fumarate, which compete with a-ketoglutarate for binding to the tumours that have adapted to an anaerobic state could enzyme. Several studies have linked PHD activity to cancer have a survival advantage. Another aspect of adaptation formation owing to the loss of mitochondrial tumour suppressors. to hypoxia is HIF-mediated neovascularization, seen in Tumour progression owing to PHD inhibition results from high pseudohypoxic tumours such as those with SDH or FH HIF activity or defective apoptosis. deficiency (see above). This may be important for boosting the nutrient supply needed for tumour survival and growth. Lastly, as mentioned above, HIF plays a conversion of a prolyl residue, molecular oxygen and major role in the enhanced glycolysis observed in many a-ketoglutarate to hydroxy-prolyl, carbon dioxide and tumours. In addition to adaptation to anaerobic succinate using ferrous iron and ascorbate as cofactors conditions, enhanced glycolysis is an energetic boost to (Figure 3). Thus, succinate is not only a substrate for the cells, rapidly generating ATP in the cytosol. More- SDH in the mitochondria, but also a product of PHDs over, enhanced glucose breakdown provides building (and, one must add, of other a-ketoglutarate-dependent blocks for the synthesis of nucleotides (via the pentose dioxygenases) in the cytosol. Therefore, the accumulated phosphate pathway), and amino and fatty acids (from succinate, by feedback inhibition of PHDs, leads to glycolytic and TCA cycle intermediates) (Figure 1) HIFa stabilization and activation of the HIF complex. (Owen et al., 2002). Even under conditions in which Importantly, a second work described a mechanistic the TCA cycle is not fully functional, as is the case for link between FH deficiency and HIF activation in SDH- and FH-deficient tumours, the anaplerotic reac- HLRCC tumours (Isaacs et al., 2005). In this work, it tion catalysed by pyruvate carboxylase, which converts was shown that, like succinate (and potentially even pyruvate to oxaloacetate, can operate to replenish TCA better), fumarate, which accumulates in FH-deficient cycle intermediates (Figure 1). Further metabolism of tumours, inhibits PHD activity in the cytosol. Fumarate oxaloacetate by the TCA cycle generates precursors for is not a product of PHD but it is chemically similar to many non-essential amino acids and for fatty acids and succinate. Structurally, owing to a double bond in the may provide reducing equivalents for oxidative phos- centre of the dicarboxylic acid, fumarate is a rigid phorylation. These processes are required for the molecule compared to succinate. It is possible that due anabolic reactions (protein and DNA synthesis) that to its structure, fumarate interacts better with PHDs. lead to growth and proliferation of cancer cells. Subsequent support for the biochemical role of succinate and fumarate in SDH- and FH-deficient tumours came from metabolomic studies of these Resistance to apoptosis tumours (Pollard et al., 2005b). It was clearly demon- Whereas under normal conditions they provide the bulk strated that succinate levels are high in SDH-deficient of the energy needed to sustain cell life, mitochondria HPGL tumours and that both succinate and fumarate may also assume the role of executioner: when levels are high in HLRCC tumours. The increase in the elimination of cancer cells is required, either physio- these metabolites coincided with high levels of HIFa logically or therapeutically, mitochondria can protein (Pollard et al., 2005b). Interestingly, high levels trigger apoptosis, the programmed self-destruction of of succinate and the induction of HIF1a was recently cells (Green and Kroemer, 2005). Many pro- and described in SDHA-deficient fibroblasts (Briere et al., anti-apoptotic proteins have been shown recently to 2005). Although SDHA was not identified as a tumour affect mitochondrial physiology (Hammerman et al., suppressor, bi-allelic mutations of SDHA were described 2004). For example, mitochondria-associated hexoki- in Leigh syndrome, an early-onset, progressive neuro- nase, a key glycolytic enzyme, confers resistance to degenerative disease cased by defective mitochondrial apoptosis (Robey and Hay, 2005). Therefore, defective

Oncogene Succinate dehydrogenase and fumarate hydratase A King et al 4680 mitochondrial physiology, compensated for by en- extended to a mammalian system, in which NIH3T3 hanced glycolysis, could possibly render tumour cells mouse fibroblasts were transfected with a murine apoptosis resistant. mutant of SDHC equivalent to the mev-1 mutant, and More direct evidence for the role of mitochondrial high levels of ROS accompanied by increased nuclear tumour suppressors in apoptosis has recently emerged DNA mutagenesis were detected (Ishii et al., 2005). from studies of VHL (Lee et al., 2005). Generally, VHL Interestingly, in addition to mutagenesis, ROS may disease is a cancer predisposition syndrome caused by have another role in the pathology of these tumours. the loss of function of pVHL (Kim and Kaelin, 2004). Reactive oxygen species were reported to inhibit PHD The tumour pattern of VHL patients includes central activity under normoxic conditions by oxidizing the nervous system and retinal hemangioblastomas, clear PHD cofactors ferrous iron and ascorbate (Figure 3) cell renal cell carcinoma and phaeochromocytoma. Lee (Gerald et al., 2004). It is therefore tempting to speculate et al. (2005) focused on specific VHL mutations of type that ROS also play a role in the pseudohypoxic response 2C that can cause only phaeochromocytomas and tried of SDH (and maybe FH)-deficient tumours. to elucidate the cause for their development. Interest- Despite the observations described above, and for the ingly, type 2C VHL mutants can bind and target HIFa reasons listed below, it is likely that the role of ROS in for degradation (Hoffman et al., 2001). This indicates the pathology of FH- and SDH-deficient tumours is that, unlike the case of haemangioblastoma and clear restricted to specific mutations in SDHC. The majority cell renal cell carcinoma, mechanisms other than of tumours derived from SDHB or SDHD mutations pseudo-hypoxia play a role in phaeochromocytoma have a very weak to non-detectable activity of SDH development. Lee et al. (2005) found that apoptosis in (Gimenez-Roqueplo et al., 2001; Gimenez-Roqueplo phaeochromocytoma cells is mediated by c-Jun, which et al., 2002; Douwes Dekker et al., 2003). This indicates activates PHD3(EglN3).To activate c-Jun, pVHL that the SDH complex in these tumours is incapable of eliminates atypical-PKC activity that induces the oxidizing succinate, a required enzymatic reaction for transcription of JunB, the upstream inhibitor of c-Jun ROS generation. Moreover, in two recent studies, (Lee et al., 2005). They also showed that PHD3is an induced HIF1a but no signs of redox stress were important target for succinate-mediated inhibition in identified in tumours with FH mutations or in SDHA- SDH-deficient cells (Lee et al., 2005). They concluded mutated fibroblasts (Briere et al., 2005; Pollard et al., that the block of PHD3-mediated apoptosis is a 2005b). As carriers of SDHC mutations exhibit a common pathway for phaeochromocytoma formation phenotypic profile different to carriers of SDHB or in both SDH and VHL mutants. It remains unclear, SDHD mutations (Schiavi et al., 2005), it is possible that however, which PHD3substrates cause the apoptotic ROS generation has a role in determining phenotypic response. Although this study did not observe HIF outcomes in these tumours. induction in SDH-inhibited cells, it is likely that several targets of PHDs, including HIFa and apoptosis indu- cing factors, contribute to tumour progression owing to the loss of mitochondrial tumour suppressor genes Open questions and future directions (Figure 3). In the relatively short time since the discovery of mitochondrial tumour suppressors, vast genetic and Signalling by reactive oxygen species biochemical knowledge has accumulated to help eluci- Apart from the metabolic signalling, which is mediated date the pathways leading to tumorigenesis. But, as is by succinate and fumarate, other mitochondria-derived usually the case, we still have more questions than molecules have been suggested to trigger the oncogenic answers. The metabolic signalling pathway mediated by signal. These are reactive oxygen species (ROS), which succinate or fumarate points to a common tumorigenic can be generated by the ETC, especially in complex I cause in SDH- or FH-deficient cells. This is in line with and III (Raha and Robinson, 2000). The role of ROS in the physiological role of these enzymes in the TCA cycle. the pathogenesis of HPGL and HLRCC is still being However, this metabolic signalling pathway does not debated. Structural and functional analysis of SDH explain the different tumour types that develop owing to (complex II) from Escherichia coli did indicate the mutations in either protein. Despite their shared potential of ROS production on the SDHA site metabolic pathway, two major biochemical differences (Yankovskaya et al., 2003). This, however, required a can help to tell SDH- and FH-mediated tumorigenesis block in the electron flow from the FAD site on SDHA apart. Succinate is the prime metabolite to inhibit PHD to the iron–sulphur centres on SDHB and further to the in SDH-deficient cells, whereas fumarate seems to be the quinone attached to SDHC and SDHD. It seems likely chief inhibitor in FH-deficient cells. Although both that when tumorigenic mutations in the SDH subunits molecules were shown to mediate HIF induction (Isaacs occur, succinate may still be oxidized to fumarate, but et al., 2005; Selak et al., 2005), it is possible that they electrons accumulate on FAD and are then transferred inhibit different PHDs with different efficiency. In line to molecular oxygen to generate superoxide. This was in with this idea, it was shown that fumarate is a better fact shown to occur in a specific SDHC mutant (mev-1) inhibitor of PHD2 than succinate (Isaacs et al., 2005). of the Caenorhabditis elegans nematode (Ishii et al., As PHD2 is the major HIFa hydroxylase, this 1998; Senoo-Matsuda et al., 2001). This initial work was may suggest that pseudo-hypoxia is more profound in

Oncogene Succinate dehydrogenase and fumarate hydratase A King et al 4681 FH-deficient tumours. It is possible however, that pseudo-hypoxia alone is insufficient to induce tumor- fumarate cannot match succinate in inhibiting PHD3- igenesis. The latter hypothesis is supported by the dependent apoptosis, as is the case in phaeochromocy- observation that mice transgenic for a non-degradable toma (Lee et al., 2005). Moreover, whereas both cutaneous HIF1a mutant were not reported to develop enzymes catalyse sequential reactions in the TCA cycle, tumours (Elson et al., 2001). SDH is also part of the ETC. As discussed above, It is apparent that the tumorigenic effect of SDH or transporting electrons is a risky business and therefore, FH mutations involves more than one mechanism. Most despite the lack of a clear redox stress signature in of the data available today indicate that succinate- or SDHB- and SDHD-deficient tumours, it is still im- fumarate-mediated inhibition of PHDs is fundamental portant to thoroughly explore the role of ROS in these to this process, but PHD inhibition engages more than tumours. one downstream pathway. This might complicate The importance of PHD in tumours derived from translating our knowledge into an effective treatment. SDH or FH mutations is obvious. Moreover, by Although HIF, a downstream target of some PHDs, is a examining the different pathways described in Figure 2, very attractive target for treating cancer in general it appears that some tumour suppressor genes have a (Hewitson and Schofield, 2004) and several inhibitors curtailing effect on HIF activity. This strongly empha- have already been tested in preclinical trials (Kung et al., sizes the role that HIF plays in oncogenesis. Moreover, 2004; Welsh et al., 2004; Chau et al., 2005), HIF may it may suggest that PHDs themselves are tumour only play a supportive rather than a definitive role in the suppressors though to date, there is no evidence of that. formation or maintenance of these tumours. The role of This may be due to a degree of redundancy in the PHD3in apoptosis has not been fully elucidated and so activity of different PHDs. Interestingly, recent studies downstream candidates for therapeutic targeting have have identified specific familial mutations in PHD2 and not been identified. However, as PHD inhibition by pVHL, each of which leads to erythrocytosis associated succinate and fumarate is accomplished by competing with HIF induction, but none of which is associated with a-ketoglutarate (Figure 3), it could be desirable to with cancer (Ang et al., 2002; Percy, 2006). Perhaps, therapeutically deliver enough a-ketoglutarate into cells cancer in these cases is avoided because HIF induction is to alleviate PHD inhibition and potentially induce relatively low (this was not yet evaluated) or because tumour cell death.

References

Ang SO, Chen H, Hirota K, Gordeuk VR, Jelinek J, Guan Y Gimenez-Roqueplo AP, Favier J, Rustin P, Mourad JJ, Plouin et al. (2002). Nat Genet 32: 614–621. PF, Corvol P et al. (2001). Am J Hum Genet 69: 1186–1197. Astuti D, Latif F, Dallol A, Dahia PL, Douglas F, George E Gimenez-Roqueplo AP, Favier J, Rustin P, Rieubland C, et al. (2001). Am J Hum Genet 69: 49–54. Kerlan V, Plouin PF et al. (2002). J Clin Endocrinol Metab Baysal BE. (2003). Trends Endocrinol Metab 14: 453–459. 87: 4771–4774. Baysal BE, Ferrell RE, Willett-Brozick JE, Lawrence EC, Gottlieb E, Tomlinson IP. (2005). Nat Rev Cancer 5: 857–866. Myssiorek D, Bosch A et al. (2000). Science 287: 848–851. Green DR, Kroemer G. (2005). J Clin Invest 115: 2610–2617. Baysal BE, Willett-Brozick JE, Filho PA, Lawrence EC, Myers Hammerman PS, Fox CJ, Thompson CB. (2004). Trends EN, Ferrell RE. (2004). J Med Genet 41: 703–709. Biochem Sci 29: 586–592. Benn DE. (2006). J Clin Endorcrinol Metab 91: 827–836. Hewitson KS, Schoﬁeld CJ. (2004). Drug Discov Today 9: Briere JJ, Favier J, Benit P, El Ghouzzi V, Lorenzato A, 704–711. Rabier D et al. (2005). Hum Mol Genet 14: 3263–3269. Hoffman MA, Ohh M, Yang H, Klco JM, Ivan M, Kaelin Jr Carew JS, Huang P. (2002). Mol Cancer 1:9. WG. (2001). Hum Mol Genet 10: 1019–1027. Chau NM, Rogers P, Aherne W, Carroll V, Collins I, Inoki K, Corradetti MN, Guan KL. (2005). Nat Genet 37: McDonald E et al. (2005). Cancer Res 65: 4918–4928. 19–24. Dahia PL. (2006). Curr Opin Oncol 18: 1–8. Isaacs JS, Jung YJ, Mole DR, Lee S, Torres-Cabala C, Chung Dahia PLM, Ross KN, Wright ME, Hayashida CY, Santagata YL et al. (2005). Cancer Cell 8: 143–153. S, Barontini M et al. (2005). PLoS Geneti 1: e8. Ishii N, Fujii M, Hartman PS, Tsuda M, Yasuda K, Senoo- Dang CV, Semenza GL. (1999). Trends Biochem Sci 24: Matsuda N et al. (1998). Nature 394: 694–697. 68–72. Ishii T, Yasuda K, Akatsuka A, Hino O, Hartman PS, Ishii N. Dann III CE, Bruick RK. (2005). Biochem Biophys Res (2005). Cancer Res 65: 203–209. Commun 338: 639–647. Kim WY, Kaelin WG. (2004). J Clin Oncol 22: 4991–5004. Douwes Dekker PB, Hogendoorn PC, Kuipers-Dijkshoorn N, Kung AL, Zabludoff SD, France DS, Freedman SJ, Tanner Prins FA, van Duinen SG, Taschner PE et al. (2003). J EA, Vieira A et al. (2004). Cancer Cell 6: 33–43. Pathol 201: 480–486. Lee S, Nakamura E, Yang H, Wei W, Linggi MS, Sajan MP Elson DA, Thurston G, Huang LE, Ginzinger DG, McDonald et al. (2005). Cancer Cell 8: 155–167. DM, Johnson RS et al. (2001). Genes Dev 15: 2520–2532. Lehtonen R, Kiuru M, Vanharanta S, Sjoberg J, Aaltonen Eng C, Kiuru M, Fernandez MJ, Aaltonen LA. (2003). Nat LM, Aittomaki K et al. (2004). Am J Pathol 164: 17–22. Rev Cancer 3: 193–202. Maxwell PH. (2005a). Semin Cell Dev Biol 16: 523–530. Favier J, Briere JJ, Strompf L, Amar L, Filali M, Jeunemaitre Maxwell PH. (2005b). Exp Physiol 90: 791–797. X et al. (2005). Horm Res 63: 171–179. Modica-Napolitano JS, Singh KK. (2004). Mitochondrion 4: Gatenby RA, Gillies RJ. (2004). Nat Rev Cancer 4: 891–899. 755–762. Gerald D, Berra E, Frapart YM, Chan DA, Giaccia AJ, Neumann HP, Pawlu C, Peczkowska M, Bausch B, McWhin- Mansuy D et al. (2004). Cell 118: 781–794. ney SR, Muresan M et al. (2004). JAMA 292: 943–951.

Oncogene Succinate dehydrogenase and fumarate hydratase A King et al 4682 Niemann S, Muller U. (2000). Nat Genet 26: 268–270. Selak MA, Armour SM, MacKenzie ED, Boulahbel H, Owen OE, Kalhan SC, Hanson RW. (2002). J Biol Chem 277: Watson DG, Mansfield KD et al. (2005). Cancer Cell 7: 30409–30412. 77–85. Pawlu C, Bausch B, Neumann HP. (2005). Fam Cancer 4: Senoo-Matsuda N, Yasuda K, Tsuda M, Ohkubo T, 49–54. Yoshimura S, Nakazawa H et al. (2001). J Biol Chem 276: Percy MJ. (2006). Proc Natl Acad Sci USA 103: 654–659. 41553–41558. Plas DR, Thompson CB. (2005). Oncogene 24: 7435–7442. Tomlinson IP, Alam NA, Rowan AJ, Barclay E, Jaeger EE, Pollard P, Wortham N, Barclay E, Alam A, Elia G, Manek S Kelsell D et al. (2002). Nat Genet 30: 406–410. et al. (2005a). J Pathol 205: 41–49. Vanharanta S, Buchta M, McWhinney SR, Virta SK, Pollard PJ, Briere JJ, Alam NA, Barwell J, Barclay E, Peczkowska M, Morrison CD et al. (2004). Am J Hum Wortham NC et al. (2005b). Hum Mol Genet 14: 2231–2239. Genet 74: 153–159. Raha S, Robinson BH. (2000). Trends Biochem Sci 25: 502–508. Vanharanta S, Pollard PJ, Lehtonen HJ, Laiho P, Sjoberg J, Robey RB, Hay N. (2005). Cell Cycle 4: 654–658. Leminen A et al. (2006). Hum Mol Genet 15: 97–103. Rustin P, Rotig A. (2002). Biochim Biophys Acta 1553: Wei MH, Toure O, Glenn GM, Pithukpakorn M, Neckers L, 117–122. Stolle C et al. (2006). J Med Genet 43: 18–27. Schiavi F, Boedeker CC, Bausch B, Peczkowska M, Gomez Welsh S, Williams R, Kirkpatrick L, Paine-Murrieta G, Powis CF, Strassburg T et al. (2005). JAMA 294: 2057–2063. G. (2004). Mol Cancer Ther 3: 233–244. Schofield CJ, Ratcliffe PJ. (2004). Nat Rev Mol Cell Biol 5: Yankovskaya V, Horsefield R, Tornroth S, Luna-Chavez C, 343–354. Miyoshi H, Leger C et al. (2003). Science 299: 700–704. Schofield CJ, Ratcliffe PJ. (2005). Biochem Biophys Res Zu XL, Guppy M. (2004). Biochem Biophys Res Commun 313: Commun 338: 617–626. 459–465.

Oncogene