Mitochondria and Cancer: Is There a Morphological Connection?

E Alirol1 and JC Martinou

Department of Cell Biology, University of Geneva, Gene`ve, Switzerland

Mitochondria are key players in several cellular functions mitochondrial remodelling (Olichon et al., 2003). including growth, division, energy metabolism, and Mitofilin is another protein that has recently been apoptosis.The mitochondrial network undergoes constant involved in cristae morphology (John et al., 2005). remodelling and these morphological changes are of direct Although models for mitochondrial fusion are still relevance for the role of this organelle in cell physiology. incomplete, the mechanism proceeds through three Mitochondrial dysfunction contributes to a number of steps: docking, fusion of the outer mitochondrial human disorders and may aid cancer progression.Here, membrane (OMM) and fusion of the IMM. All three we summarize the recent contributions made in the field of steps involve GTP hydrolysis, and the IMM fusion mitochondrial dynamics and discuss their impact on our requires an intact membrane potential (Mattenberger understanding of cell function and tumorigenesis. et al., 2003). Other players have been identified in yeast Oncogene (2006) 25, 4706–4716. doi:10.1038/sj.onc.1209600 but no mammalian homologs to them have been described so far (Tieu and Nunnari, 2000). Keywords: mitochondrial morphology; apoptosis; The mitochondrial fission pathway involves at least energy metabolism; cancer two proteins: dynamin related protein 1 (Drp1) and Fis1. Drp1 is a cytosolic GTPase, which has been proposed to couple GTP hydrolysis to membrane constriction and fission (Smirnova et al., 2001). Fis1 is an outer membrane protein evenly distributed on the Introduction surface of mitochondria (James et al., 2003). It is thought to recruit Drp1 to punctuate structures on Mitochondria play essential and diverse roles in the mitochondria and is thus considered as the limiting physiology of eukaryotic cells. Not only do they provide factor in the fission process (Stojanovski et al., 2004). energy, but they also participate in numerous metabolic The mechanism of constriction is not understood reactions and play central roles in apoptosis (Desagher but the current model postulates that Drp1 homo- and Martinou, 2000). Impairments of mitochondrial oligomerizes and forms a ring around the mitochon- functions have been implicated in a wide variety of drial tubule (Okamoto and Shaw, 2005). Drp1 complexes human pathologies, among which cancer and age- might generate mechanical force via conformational related diseases (Wallace, 2005). changes, leading to membrane constriction, similar to In the past years, a number of groups showed that dynamin (Danino et al., 2004). Modification of lipid mitochondria are dynamic structures that undergo composition is likely to be involved, and additional fusion and fission events continuously throughout the proteins may participate in mitochondrial fission. For life of a cell (Okamoto and Shaw, 2005). Mounting example, endophilin B1, a putative fatty acyl transfer- evidence indicate that mitochondrial dynamics have ase, has been shown to act downstream of Drp1 in the roles beyond maintenance of morphology, and impact control of mitochondrial morphology (Karbowski et al., on both cell death and cell metabolism (Chen and Chan, 2004) and MTP18 is a novel mitochondrial protein that 2005). In mammals, mitochondrial fusion is driven by is able to trigger mitochondrial fragmentation in a two GTPases of the outer membrane named Mitofusins Drp1-dependent fashion (Tondera et al., 2004). 1 and 2 (Mfn1, Mfn2) (Santel and Fuller, 2001; Chen GDPAP1, which is expressed in Schwann cells and et al., 2003; Eura et al., 2003). The Mitofusins appear to neurons, has also been reported to mediate mitochon- play distinct roles in fusion and the Mfn1 presumably drial fission (Niemann et al., 2005). works together with Opa1, a GTPase of the inner Membrane fusion and fission engage rupture and mitochondrial membrane (IMM) to promote fusion deformation of lipid bilayers, and involve destabilized (Cipolat et al., 2004). While its exact function remains monolayer intermediates (Chernomordik and Kozlov, unclear, Opa1 seems also involved in further intra- 2003). The two membranes are first brought in close contact, then some hydrophobic monolayer discontinu- Correspondence: Dr J-C Martinou, Department of Cell Biology, ity appears that enables the rupture of the bilayer and University of Geneva, Quai Ernest-Ansermet 30, 1211 Geneva 4, the formation of a fusion stalk intermediate. Successive Switzerland. E-mail: [email protected] steps include the opening of a pore and its expansion, 1Current address: Venetian Institute of Molecular Medecine, Via Orus connecting the two aqueous volumes (Kozlovsky et al., 2, 35 129Padova, Italy Mitochondria and cancer E Alirol and JC Martinou 4707 2002). These processes involve specific proteins that and a hallmark of most, if not all, types of cancer establish local intermembrane contacts and generate (Hanahan and Weinberg, 2000; Green and Evan, 2002). local membrane stresses. Absence or excess of one of The inactivation of the p53 protein is the most common these proteins could in theory result in failure of specific strategy developed by tumour cells to evade apoptosis. steps and accumulation of intermediates. p53 is central in the DNA damage response and triggers As summarized in Table 1, disruption of the fission/ cell death by both transcription-dependent and fusion machinery strongly alters organelle function -independent mechanisms (Slee et al., 2004; Yee and affecting both programmed cell death and energy Vousden, 2005). Other examples of apoptosis evasion production. Several lines of evidence suggest that include disruption of Fas death receptor pathway, mitochondria contribute to neoplastic transformation upregulation of the widespread antiapoptotic family of by changing cellular energy capacities, increasing inhibitors of apoptosis proteins (IAPs) or downregula- mitochondrial oxidative stress and modulating apop- tion of caspases (Muschen et al., 2000; Teitz et al., 2000; tosis. A shift in the rate of mitochondrial fission or Yang et al., 2003). The PI3K/Akt/ PTEN pathway was fusion may provide new mechanistic explanation for the also shown to play a role in abrogating apoptosis. Akt/ aetiology of cancer, and may offer additional strategies PKB is activated in a wide variety of cancers and this for therapeutic intervention. activation leads to the phosphorylation of a number of Here, we discuss a possible role played by mitochon- key apoptotic proteins thereby inactivating them (Testa dria in oncogenesis and explore how misregulation of and Bellacosa, 2001). fission and fusion machinery may influence apoptosis, cell metabolism and tumour formation. Mitochondrial membrane permeabilization Many death signals converge to mitochondria which respond by releasing numerous proteins in the cytosol Mitochondrial apoptosis and its subversion in oncogenesis (Patterson et al., 2000), among which several have apoptogenic properties (e.g. cytochrome c, SMAC/ The possibility that apoptosis is implicated in cancer DIABLO, AIF, Endo G and Omi/HtrA2). In many cell formation was first raised in 1972 when Kerr et al. types mitochondrial membrane permeabilization (1972) described massive apoptosis in tumoural cell (MMP) is considered as the point of nonreturn in the populations. In the following years, the antiapoptotic cell suicide (Ferri and Kroemer, 2001). However, in protein Bcl2 was shown to be a tumour-specific marker sympathetic neurons for example, this event appears to which expression was enhanced in many lymphoma be reversible and death can be circumvented down- (Tsujimoto et al., 1984; Bakhshi et al., 1985; Crescenzi stream of cytochromic c release (Martinou et al., 1999; et al., 1988; Vaux et al., 1988; Nunez et al., 1990), Deshmukh et al., 2000; Tolkovsky et al., 2002). Whether opening the investigation of apoptosis in cancer at the it is also the case in cancer cells remains to be molecular level. Suppression of apoptosis is among the determined. Upon release, cytochrome c triggers the minimal requirements for a cell to become cancerous, apoptosome formation and initiates the caspase cascade

Table 1 Proteins involved in mitochondrial fusion and ﬁssion and their effect on apoptosis and energy metabolism Protein Function Localization Apoptosis Energy metabolism References

DRP-1 Fission cytosol Overexpression sensitizes cells Breckenridge et al. (2003), and OMM to apoptosis; silencing and Frank et al. (2001), dominant negative mutant Lee et al. (2004), reduce MMP induced by several Szabadkai et al. (2004) apoptotic stimuli Fis1 Fission OMM Overexpression triggers MMP Overexpression James et al. (2003), and silencing protects from affects Dcm Lee et al. (2004), several apoptotic stimuli Niemann et al. (2005); Yu et al. (2005) Endophilin B1 Fission Cytosol Silencing and dominant negative mutant Karbowski et al. (2004) and OMM cause separation of OMM and IMM MTP18 Fission Mitochondria Silencing sensitizes cells to UV or Tondera et al. (2004) TNFa treatment. Overexpression triggers MMP GDPAP1 Fission OMM Niemann et al. (2005) Mfns Fusion OMM Overexpression inhibits MMP Silencing affects Dcm and Chen et al. (2005), and Bax/Bak activation respiration. Overexpression Pich et al. (2005), of Mfn2 stimulates OXPHOS Sugioka et al. (2004) Opa1 Fusion IMM Silencing causes MMP and apoptosis Affects Dcm and impairs Chen et al. (2005), and IMS in Fis1 dependent fashion. respiration Griparic et al. (2004), siRNA sensitizes cells to apoptotis Lee et al. (2004), Olichon et al. (2003) Mitoﬁlin Increases ROS production John et al. (2005) and affects Dcm

Oncogene Mitochondria and cancer E Alirol and JC Martinou 4708 (Nicholson, 1999). These killer proteases target many vital only BH3. Most of them also display a hydrophobic C proteins, committing the cell to death (Fischer et al., 2003). terminus that allow them to associate with membranes Mitochondrial membrane permeabilization is regu- (Schinzel et al., 2004). Heterodimerization of the Bcl2 lated by members of the Bcl2 family (Lucken-Ardjo- family members seems crucial to the regulation of mande and Martinou, 2005b). These proteins contain at MMP, and structural data has shown that antiapoptotic least one of the four conserved regions called Bcl2- proteins display an hydrophobic groove on their surface, Homology domains (BH1-BH4). Antiapoptotic mem- providing a binding site for the BH3 domain of the bers (e.g. Bcl2, Bcl-xL, Bcl-xW, Mcl-1 and A1/bﬂ-1) proapoptotic members (Petros et al., 2004). display all four BH domains, proapoptotic members The exact mechanism leading to MMP is still unclear, (Bax, Bak and Bok/Mtd) lack BH4, and BH3-only although several models have been proposed for this proapoptotic members (Bim/Bod, Bid, Bad, Bmf, Bik/ process (Figure 1) (Martinou and Green, 2001; Cromp- Nbk, Blk, Noxa, Puma/Bbc3 and Hrk/DP5) harbour ton et al., 2002; Waterhouse et al., 2002). According to

Figure 1 Proposed mechanisms for mitochondrial membrane permeabilization (MMP). (a) Classic models involve either the formation of channels by proapoptotic Bcl2-family members (e.g. Bax or Bak) or opening of the permeabilization transition pore (PTP). Upon apoptosis induction, Bax/Bak undergo conformational changes and oligomerize to form large channels. Apoptotic factors are then released from the mitochondrial inter membrane space (IMS) and cristae compartment into the cytosol where they trigger caspase activation and cell death. Alternatively, death stimuli activate pathway that trigger PTP opening such as Ca2 þ or ceramide, allowing water and solutes to enter the mitochondrial matrix. Subsequent swelling of the organelle provokes the rupture of outer mitochondrial membrane (OMM) and release of apoptogenic factors into the cytosol. (b) An alternative scenario could be that during apoptosis, a membrane-perturbing agent (either tBid, Bax or a protein that controls mitochondria morphology) translocates to the mitochondria and destabilizes the lipid bilayer. The recruitment of enzymes involved in lipids modification, such as lysolipid transferases (e.g. tBid or Endophilin B1) would enable the formation of a lipid pore and further intramitochondrial remodelling by mitochondrial shaping proteins (e.g. Opa1 or Mitofilin), triggering the release of apoptogenic factors. (c) Proposed model for mitochondrial division process and fission induced MMP. Under normal conditions the fission machinery (Fis1, Drp1 and putative proteins implicated in membrane perturbation and lipids modifications) is recruited and assembled on local foci on the OMM. IMM and OMM are brought in close apposition to form a stalk intermediate. Under physiological division, Drp1 assembles into a ring and constricts the mitochondria until it divides. Alternatively failure of mitochondrial fission machinery could result in bilayer destabilization and lipid pore formation as previously shown. Apoptogenic factors would then be released in the cytosol.

Oncogene Mitochondria and cancer E Alirol and JC Martinou 4709 the first model, the opening of a high-conductance studies revealed that apoptosis correlated with a channel, the permeability transtition pore (PTP), would progressive proliferation of intracellular membranes cause loss of mitochondrial membrane potential (Dcm), leading to the formation of apoptotic blebs. This was swelling of the organelle and rupture of the OMM as in contrast to the process of necrosis, as apoptotic cells solute and water enter the mitochondrial matrix (Hale- disintegrate without leaking their intracellular contents strap et al., 2002). It should be noted that Dcm loss is out in the medium, preventing the inflammatory not sufficient to prove PTP involvement, as many other reactions that are induced by necrotic cells (Cristea events can provoke it. Although the composition of the and Degli Esposti, 2004). Modifications of lipid PTP remains to be resolved, the prevalent hypothesis is composition and bilayer curvature of the mitochondrial that it consists of three core proteins, namely the voltage membranes seem to be crucial for permeabilization. dependent anion channel (VDAC), the adenine nucleo- Recent work has highlighted the importance of tBid, tide translocase (ANT) and cyclophilin D (Halestrap which is able to insert into specific lysolipids of the et al., 2002). The PTP is thought to open in response to OMM (Esposti et al., 2001; Goonesinghe et al., 2005) calcium and oxidative stress and Bcl2 family members and to promote a negative membrane curvature of also seem to regulate its activity (Zamzami and synthetic lipidic membranes (Epand et al., 2002a). These Kroemer, 2001). However, recent studies of mitochon- changes presumably prepare the ground for insertion drial permeabilization in ANT and Cyclophilin D and oligomerization of Bax and Bak. tBid is also able to deficient backgrounds tend to show that these proteins modify IMM topology, as incubation of mouse liver are not obligatory components of the channel (Kokosz- mitochondria with tBid induces a profound remodelling ka et al., 2004; Baines et al., 2005; Basso et al., 2005; of the cristae compartment (Scorrano et al., 2002). Nakagawa et al., 2005). Both proteins appear to be Tomographic reconstructions revealed that the cristae regulators of the PTP opening as they influence its become more interconnected and that their junctions sensitivity to Ca2 þ . widen concurring with enhanced mobilization of cyto- An alternative model for MMP proposes that chrome c. The molecular events leading to cristae proapoptotic Bcl2 family members oligomerize and remodelling are not known. tBid could interact with form channels within the OMM (Saito et al., 2000; proteins of the IMM or could directly affect lipids to Epand et al., 2002b). This view is supported by the modulate IMM topology. Indeed, tBid has been shown similarity between these proteins and some pore-form- to bind to cardiolipin, a negatively charged mitochon- ing bacterial toxins. Following a death signal, the drial phospholipid present in the IMM (Lutter et al., proapoptotic proteins Bax and Bak undergo a con- 2000; Liu et al., 2005). Recently, Kim et al. (2004) formational change, and their N terminus becomes reported that binding to cardiolipin is required for tBid exposed. Bax, which is usually in the cytosol or loosely induced cristae remodelling and cytochrome c release. associated with mitochondria, translocates to the Yet these rearrangements might not always be required mitochondria, oligomerizes and inserts in the OMM given the fact that Bid knockout mice do not display causing MMP (Hsu et al., 1997; Desagher et al., 1999; particular deficits in cell death and that Bid is not Antonsson et al., 2000). These events can be promoted expressed in all cells in vivo (Krajewska et al., 2002). by tBid, the activated form of Bid, and inhibited by Bcl2 Other reports show that Bax destabilizes lipid bilayers and Bcl-xL (Desagher et al., 1999; Eskes et al., 2000; (Basanez et al., 1999; Epand et al., 2003) and interacts Cheng et al., 2001). with endophilin B1, a potential lysophosphatic acid Mitochondrial membrane permeabilization generally acyltransferase (Modregger et al., 2003). However, a occurs in a concerted and rapid fashion, affecting most, recent study suggests that the effect of endophilin B1 if not all, mitochondria in the dying cell. The classic effect on membrane curvature could be an experimental models mentioned above are far from being complete, artefact (Gallop et al., 2005). and theories on the molecular mechanism are formu- Lipid bilayer bending and tilt of hydrocarbon chains lated, debated and modified on a regular basis (Lucken- can provide sufficient lateral tension to drive pore Ardjomande and Martinou, 2005a). Recent investiga- opening in a nonbilayer intermediate (Chernomordik tions revealed that during apoptosis, the mitochondrial and Kozlov, 2003). This implies that membrane membrane properties are profoundly modified and the permeabilization is not necessarily mediated by a mitochondrial network undergoes extensive fragmenta- proteinaceous pore, but rather results from the con- tion (Desagher and Martinou, 2000; Cristea and Degli certed actions of proteins and particular lipids. As Esposti, 2004). Mitochondrial membrane permeabiliza- illustrated in Figure 1, during apoptosis the formation tion requires drastic membrane remodelling, and both of discontinuity in the OM can lead to nonspecific lipids and components of the fission/fusion machinery lipid pores. Thus Bax pore forming capacity could may participate in this process. be ascribed to membrane perturbation rather than discrete channel activity (Cristea and Degli Esposti, 2004). Importance of lipids and membrane topology These observations shed a new light on the process of The first insight into the role of membrane lipids in cell mitochondrial membrane permeabilization, suggesting death came from the observation that phosphatidyl- that membrane topology is of direct relevance for serine (PS) became exposed at the cell surface during apoptosis. In addition, several investigations in the field apoptosis (Fadok et al., 1992). Subsequently several of mitochondrial dynamics further strengthened the

Oncogene Mitochondria and cancer E Alirol and JC Martinou 4710 intimate relationship that exists between organelle Indeed, it seems that two pools of cytochrome c exist: a morphology and cell death. minor soluble pool, which is present in the IMS and a major pool confined in the mitochondrial cristae and which binds cardiolipin (Bernardi and Azzone, 1981; Ott Role of fission and fusion in apoptosis et al., 2002; Scorrano et al., 2002). Mitochondrial fission As mentioned earlier, mitochondria undergo frequent is thought to be necessary for the release of the fission and fusion events that regulate their morphology. membrane-attached pool of cytochrome c. During early stages of apoptosis, mitochondria undergo A role for fission and fusion molecules in this process massive fragmentation and it has been proposed that is supported by two observations. Firstly, loss of Opa1 changes in mitochondrial shape contribute to the expression has been reported to trigger apoptosis by mitochondrial membrane permeabilization, perhaps by inducing such intramitochondrial remodelling (Olichon setting free inter membrane space (IMS) proteins for et al., 2003). Secondly, Bik, a BH3-only protein present release (Frank et al., 2001; Karbowski et al., 2002; Reed on the ER, has been shown to induce Drp1-dependent and Green, 2002; Lee et al., 2004). Both Drp1 and Fis1 remodelling of the cristae compartment (Germain et al., have been implicated in this pathway (Perfettini et al., 2005). 2005). Fis1 overexpression not only induces fragmenta- However, there is still some controversy about the tion of the mitochondrial network, but also leads to the role of mitochondrial fission in apoptosis and recent release of cytochrome c from the mitochondria caspase work suggests that depending on the stimuli, fission can activation and cell death (James et al., 2003). Silencing also interfere with MMP (Szabadkai et al., 2004). The of either Drp1 or Fis1 confers resistance to several biochemical and biophysical relationships between Bcl2 apoptotic stimuli and impairs cytochrome c release family members and the fission machinery remain to be suggesting that fission molecules are required for MMP clarified. The observation that these two classes of to occur (Frank et al., 2001; Karbowski et al., 2002; proteins are responsible for membrane perturbation Breckenridge et al., 2003; Lee et al., 2004; Germain and/or the release of proapoptotic factors does not et al., 2005). However, translocation of Bax to the mean that they act in a concerted fashion. Moreover, mitochondria remains unaffected by fission inhibition, colocalization cannot account for functional interaction, suggesting that fission molecules act downstream of Bax as local lipid bilayer properties directly affect the translocation. Mitochondrial fusion proteins also seem recruitment, insertion and activity of proteins. It is, to protect cells from apoptosis, as their overexpression therefore, crucial to determine the contribution of lipids reduces cell death induced by several stimuli (Sugioka rearrangement to mitochondrial fragmentation both in et al., 2004), and their knocking down sensitizes cells to healthy and apoptotic cells. In addition, further apoptosis (Lee et al., 2004). investigations are needed to determine what are the Several models exist to explain how mitochondrial molecular differences between physiological and death- fission could participate in MMP. Karbowski et al. associated fission. Addressing these issues should help us (2004) reported that in addition to its role in apoptosis, understand how mitochondrial fragmentation partici- endophilin B1 regulates mitochondria morphology. pates in programmed cell death and possibly correlates They also showed that upon apoptosis induction, Bax, with tumorigenesis. Drp1 and endophilin B1 translocate to Mfn2-containing foci on the mitochondria (Karbowski et al., 2002, 2004). The model they propose is the following: rather than Therapeutic perspectives forming a ring around mitochondria, Drp1 would As impaired apoptosis plays a central role in oncogen- mediate vesicle scission from focal regions around the esis, the search is accelerating for novel agents that mitochondrial fission sites. This would allow the engage the cell death machinery (Nicholson, removal of membrane by shearing away lipid vesicles 2000; Johnstone et al., 2002). Impaired apoptosis is also from OMM. One possible explanation for cytochrome c a significant impediment to cytotoxic therapy. The release would be that Bax inserts preferentially in these mutations that favour tumour development stifle the narrow curvature vesicles and permeabilize them. response to chemotherapy and radiation, and Endophilin B1 could promote Bax insertion into treatment might select more refractory clones. Never- membranes through its membrane deformation activity theless, most tumour cells still remain sensitive to (Youle and Karbowski, 2005). However, the study from some apoptotic stimuli, and most cytotoxic anticancer Gallop et al. (2005) challenges this model as it shows drugs currently in clinical use induce apoptosis of that endophilins are devoid of lysophosphatidic acid malignant cells (Don and Hogg, 2004). Therapeutic acyl transferase activity. approaches that enhance apoptosis include targeting of Another model for the role of mitochondrial mor- Bcl-2 or the caspase inhibitors IAPs, or engaging the phology in apoptosis postulates that mitochondrial death receptor pathway, by, for example, ligating shaping proteins could regulate cristae remodelling the receptors for TRAIL (Ashkenazi, 2002). Among and favour MMP (Germain et al., 2005; Scorrano, the recent strategies, the ones attempting to target 2005). As mentioned earlier, rearrangements of the mitochondria and trigger MMP are very promising. cristae compartments is thought to increase the suscepti- With much debate surrounding the components of the bility to apoptosis by enhancing the ability of mitochon- PTP and its possible manipulation in cancer treatment, dria to release cytochrome c (Scorrano et al., 2002). many chemotherapeutics efforts have focused on target-

Oncogene Mitochondria and cancer E Alirol and JC Martinou 4711 ing the Bcl2 family (Oltersdorf et al., 2005). Other Many scientists in the field of cancer research may cytotoxic drugs acting on mitochondrial lipids have been have considered mitochondria only as a reservoir for shown to induce MMP in vitro and some are being stocking harmful molecules such as ROS and apoptotic tested in preclinical mouse models (Don and Hogg, proteins. However, several recent findings demonstrate 2004). Recent findings on the role of mitochondrial that mitochondrial participation in oncogenesis is far fission machinery in the MMP process may provide from being restricted to apoptosis, and that mitochon- additional possibility for the management of neoplastic drial energy production affects both cell proliferation diseases. and tumour progression.

a Glucose Pyruvate Lactate ATP Complex I Complex III Complex IV synthase H H H H

Complex II cyt c CoQ IMM Matrix

NADH Succinate Fumarate NAD+ 1/2O HO

NADH TCA Pyruvate ADP ATP NAD+

b Respirative phenotype Glucose Pyruvate Lactate

TCA Pyruvate

Glycolytic phenotype

H H H Glucose Pyruvate Lactate H

H H H TCA Pyruvate H

Figure 2 Mitochondrial shape changes according to energy state. (a) Schematic representation of glucose metabolism. In the cytosol, glucose is converted through glycolysis to pyruvate. Pyruvate is then either transformed to lactate via lactic fermentation, or transported into the mitochondrial matrix where it enters the tricarboxylic acid (TCA) cycle. Electrons enter the electron transport chain (ETC) via the NADH produced by the TCA cycle. Complexes I, III and IV couple electron transport to protons pumping. The proton gradient generated is then used by ATP synthase to form ATP. (b) Different metabolic strategies are associated with different mitochondrial morphology. In the case of the respirative phenotype, glucose is converted into pyruvate and further oxidized by mitochondrial respiration (TCA þ ETC). The mitochondrial network appears interconnected and cristae compartment is enlarged. In the case of glycolytic phenotype, the majority of cytosolic glucose is converted into lactate, leading to an acidiﬁcation of the extracellular environment. Mitochondria appear fragmented and undergo matrix expansion.

Oncogene Mitochondria and cancer E Alirol and JC Martinou 4712 Mitochondrial physiology in cell proliferation and tumour fusion machinery has recently been established. When progression the balance is artificially tipped towards fission, an energy defect emerges. Disruption of Mitofusins and/or Mitochondria produce ATP through oxidative Opa1 results in severe cellular defects, including poor phosphorylation cell growth, widespread heterogeneity of mitochondrial In many cell types, cellular ATP is produced primarily membrane potential, and decreased cellular respiration by the oxidation of glucose (Figure 2). This process can (Chen et al., 2005). Importantly, the respiration defect be divided into two major steps. The first, which takes turns out to be reversible upon reintroduction of the place in the cytosol, is glycolysis. In the absence of wild-type fusion proteins. Another recent study reports oxygen, the end product of glycolysis, pyruvate, is that downregulation of Mfn2 in L6E9myotubes reduces converted to lactic acid through cytosolic fermentation. both the activity of the TCA and the ETC, while it In the presence of oxygen, pyruvate is utilized by the enhances glucose transport, glycolysis and lactic fer- second step, which takes place in the mitochondria, and mentation (Pich et al., 2005). Conversely, the over- includes the tricarboxylic acid (TCA) cycle and oxida- expression of Mfn2 stimulates OXPHOS activity, tive phosphorylation (OXPHOS). Further oxidation of suggesting that its role is not limited to the control of pyruvate in the mitochondria leads to the production of organelle shape. Mutations in Mfn2 have been ascribed reducing equivalents (NADH and FADH2). These to Charcot–Marie–Tooth type 2A neuropathy, and compounds fuel the electron transport chain (ETC) altered glucose oxidation could provide a mechanistic located in the IMM. The ETC is composed of four explanation for the aetiology of the disease (Zuchner multimeric complexes and couples the electron transport et al., 2004). Silencing of Mitofilin also affects metabolic to the extrusion of protons across the IMM. This fluxes (John et al., 2005). movement of protons creates an electrochemical gradi- Thus, it appears that mitochondrial morphology is ent (Dcp ¼ Dcm þ DpH) across the IMM. Most of the crucially linked to energy metabolism. Enhanced re- gradient is in the form of an electrical component, Dcm. spiration correlates with an interconnected network and The charge separation across the IMM generates free enlarged cristae compartment, whereas low OXPHOS energy, which is utilized by the ATP synthase to activity and high glycolysis correlates with smaller synthesize ATP. As a toxic by-product, OXPHOS mitochondria displaying reduced intracristae space. In generates reactive oxygen species (ROS). ROS can act an extreme pathological situation, overfragmented both as tumour initiators, by inducing mutations in mitochondrial fail to run the ETC correctly, potentially proto-oncogenes and tumour-supressors, and tumour contributing to oxidative stress. On the contrary, promoters through enhancement of cell proliferation promoting fusion stimulates respiration. It is therefore (reviewed in Behrend et al., 2003; Wallace, 2005). fundamental to maintain balanced mitochondrial fission Impairment of the ETC very often results in excessive and fusion as its disruption could increase the suscept- ROS production and subsequent oxidative stress. It is, ibility to develop metabolic defects and consequent therefore, crucial for the cell to keep watch over diseases. mitochondrial respiration as it can turn into a hazar- dous weapon when it fails. Altered energy metabolism is a hallmark of many types of cancer Mitochondria modulate their shape according to their A curious but common property of invasive cancers is bioenergetic activity altered glucose metabolism. Under aerobic conditions, Substrate availability, and energetic state both modulate glycolysis is inhibited (the so-called Pasteur effect) the mitochondrial network (Figure 2). In the 1960s (Krebs, 1972) and normal mammalian cells rely mainly Hackenbrock (1966, 1968a, b) reported that depending on the mitochondrial OXPHOS for their energy supply. on their respiration activity, the mitochondrial mor- However, cancer cells display a significant increase in phology changed from an orthodox to a condensed glycolysis and lactate production even in the presence of conformation. The condensed conformation, in which oxygen. This phenomenon, termed aerobic glycolysis mitochondria display large intracristae spaces, was was first described in 1956 by the German biochemist associated with state III respiration, when ADP is high and Nobel Laureate Otto Warburg (Warburg, 1956). He and OXPHOS activated. The orthodox conformation, postulated that cancer resulted from impaired mito- corresponding to an expanded matrix and a reduced chondrial metabolism. Although it turned out that intracristae space, was observed under state IV. This mitochondrial dysfunction is not the fundamental cause corresponds to low ADP and minimal O2 consumption. of cancer, aerobic glycolysis certainly confers a growth Further evidence that mitochondria remodel according advantage to tumour cells (Gatenby and Gillies, 2004). to changes in OXPHOS activity came from the work of Firstly, it enables cancer cells to adapt to hypoxic Rossignol et al. (2004) who showed that the mitochon- conditions as the premalignant lesion grows progres- drial network extended within cells and became more sively further from the blood supply. Secondly, the ramified and interconnected in the presence of galactose. glycolytic phenotype contributes to the acidification of As previously mentioned, mitochondria shaping tumour microenvironment, which facilitates tumour proteins seem to affect energy production. A direct invasion (Gatenby and Gawlinski, 1996; Schornack connection between bioenergetics and mitochondrial and Gillies, 2003). This metabolic transition towards

Oncogene Mitochondria and cancer E Alirol and JC Martinou 4713 glycolysis is also seen in human cancer cell lines such as that inhibiting mitochondrial function results in G1 Hela cells, in which minor amounts of glucose are prone arrest (van den Bogert et al., 1986, 1992; Heerdt et al., to the TCA cycle, the majority being converted to 1997; King and Radicchi-Mastroianni, 2002; Gemin lactate (Reitzer et al., 1979). et al., 2005). A recent study in Drosophila links a defect Mitochondria certainly have a role to play in this in mitochondrial energy production to the activation of metabolic shift, as their physiology is inextricably linked AMPK (Mandal et al., 2005). Taken together this data to energy metabolism. Several groups reported that suggests the existence of an energy checkpoint at G1–S tumour aggressiveness correlates with a low-mitochon- transition controlled by the mitochondrial energy drial respiratory chain activity, and that enhancement of production. OXPHOS can reduce tumour growth (Hervouet et al., Interestingly the G1–S transition also correlates with 2005; Schulz et al., 2006). Furthermore, a number of rearrangements of the mitochondrial network. Studies mitochondrial enzyme deficiencies are linked to inher- in both fibroblasts and osteosarcoma cells show that the ited neoplasia (Gottlieb and Tomlinson, 2005). These mitochondria become filamentous and highly intercon- include succinate deshdrogenase (SDH) genes SDHB, nected during the G1-phase while they appear fragmen- SDHC, SDHD and fumarate hydratase (FH), which all ted in the S-phase (Barni et al., 1996; Margineantu et al., participate in the TCA cycle. In addition SDH is a 2002). This is consistent with the observation that functional member (complex II) of the ETC. The mitochondrial shape change according to energy state. mechanism linking failure of the TCA cycle to tumour During the G1-phase the cell displays elongated development is not clear but two explanations have been mitochondria correlating with increased OXPHOS suggested so far. The first hypothesis postulates that activity. This presumably enables efficient energy SDH and FH deficiencies increase the production of production in the expectation of S-phase, which requires ROS (Messner and Imlay, 2002; Yankovskaya et al., substantial amounts of ATP. At the end of the G1-phase 2003). An alternative model proposes that succinate, the cell evaluates its energy stocks, and takes the which accumulates due to TCA cycle impairment, acts decision either to pause and exit the cell cycle, or to as a signalling molecule and triggers the activation of proceed toward the S-phase. However, maintaining a hypoxia inducible factor 1a (HIF1a) (Selak et al., 2005). high OXPHOS activity during DNA replication might Hypoxia inducible factor 1a promotes adaptation of be dangerous, because of the deleterious effects of ROS cells to low-oxygen consumption and activates the production. This could explain the decreased respiration transcription of glycolytic genes (Firth et al., 1995; activity and the fragmented mitochondrial morphology Semenza et al., 1996), stimulating aerobic glycolysis. observed during the S-phase. It seems thus that mitochondrial dysfunction favours The proposal that according to energy requirements the emergence of the glycolytic phenotype, and that cells modify their metabolism by remodelling the after dwindling in importance for decades, Warburg’s mitochondrial network is challenging but still needs hypothesis is enjoying a resurrection. Although further experimental proof. Preliminary results show that studies are required and warranted, induction of knockingdown components of the fission and fusion mitochondrial respiration might be worth considering machinery not only affects ATP production but also as a mechanism to restrict tumour growth. The impedes cell cycle progression ((Chen et al., 2005) and energetic conversion towards aerobic glycolysis most Parone P and Martinou JC unpublished data). Along probably requires remodelling of mitochondria. As with apoptosis resistance, hyper proliferation is a OXPHOS activation seems to perturb malignant common property shared by many cancers, and cell transformation, it would be interesting to see whether cycle checkpoints are privileged targets of chemother- tumour metabolism is affected by the promotion of apy. Future work will certainly address the importance mitochondrial fusion. of mitochondrial network remodelling in energy production, and establish whether these morphological changes play a role in cell proliferation. Mitochondrial ATP production and cell cycle progression Another common property of cancers is a high proliferation rate, which results both from cellular growth and cell division. Although the energetic Conclusion and future perspectives requirements for cell cycle progression have not been investigated thoroughly, it is highly probable that a Over the past decades cancer research has generated a metabolic checkpoint exists. The status of cellular rich and complex body of knowledge, revealing a energy stores is sensed by the AMP-activated protein multitude of parameters that either cause or favour kinase (AMPK), which is activated upon ATP depletion malignant transformation. Several features, however, (Hardie, 2005). AMP-activated protein kinase has been emerge as essential requisites of tumourigenesis: apop- shown to inhibit mTOR and to coordinate G1-S tosis evasion, limitless cell proliferation, sustained transition with carbon source availability (Kimura angiogenesis and tissue invasion are crucial properties et al., 2003; Jones et al., 2005). In mammals numerous of cancers. Growing evidence show that mitochondria studies demonstrated that the G1-phase is associated are profoundly altered in transformed cells and partici- with an overall enhancement of mitochondrial function pate in these processes. The study of cancer-associated (Van den Bogert et al., 1988; Leprat et al., 1990) and mitochondrial dysfunctions such as OXHOS impair-

Oncogene Mitochondria and cancer E Alirol and JC Martinou 4714 ment and apoptosis evasion has to take into account cells and will help to determine whether the changes in their structural context, including the influence of organelle shape are meaningful for mitochondrial membrane topology on internal diffusion and compar- contribution to oncogenesis. timentation. Mitochondrial dynamics are gaining increasing interest as it becomes indubitable that organelle fission and fusion play a role in diverse biological Acknowledgements processes. Here, we have discussed the importance of We thank D James for careful reading of the manuscript and mitochondrial membrane remodelling in cell death and helpful comments and C Frezza for helping with figures. We energy metabolism, and its potential effect on tumor- thank the Swiss National Foundation (Grant 3100A0-109419/ igenesis. Elucidating the molecular events that control 1) for support. EA is currently supported by a fellowship from mitochondria dynamics will certainly give new insight MFSP CDA 0025/04 awarded to Luca Scorrano, VIMM, into organelle function in both normal and malignant Italy.

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