Oncogene (2011) 30, 883–895 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc REVIEW Adenine translocase family: four isoforms for apoptosis modulation in cancer

C Brenner1,2,3, K Subramaniam4, C Pertuiset1,2,3 and S Pervaiz4,5,6,7

1Univ Paris-Sud, Chaˆtenay-Malabry, France; 2INSERM UMR-S 769, Chaˆtenay-Malabry, France; 3PRES UniverSud Paris, Chaˆtenay-Malabry, France; 4Department of Physiology, Apoptosis, ROS and Cancer Biology Program, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; 5NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore; 6Duke-NUS Graduate Medical School, Singapore and 7Singapore-MIT Alliance, Singapore

Mitochondria have important functions in mammalian release of several pro-apoptotic factors including cyto- cells as the energy powerhouse and integrators of the chrome c, AIF, smac/DIABLO and caspases from the mitochondrial pathway of apoptosis. The adenine nucleo- to the cytosol. Collectively, these tide translocase (ANT) is a family of proteins involved in events contribute to the initiation of the final stages of cell death pathways that perform distinctly opposite cell death. MMP is currently considered as the point-of- functions to regulate cell fate decisions. On the one hand, no-return in the lethal cascade and a promising ANT catalyzes the export from therapeutic target, as its pharmacological or genetic the to the intermembrane space with inhibition prevents cell death under many circumstances the concomitant import of ADP from the intermembrane (Costantini et al., 2000b; Bouchier-Hayes et al., 2005; space to the matrix. On the other hand, during periods of Fulda et al., 2010). stress, ANT could function as a lethal pore and trigger the A number of pathological states, most prominent process of mitochondrial membrane permeabilization, being cancer, has been associated with or results from a which leads irreversibly to cell death. In human, ANT is mitochondrial dysfunction and impairment in MMP encoded by four homologous genes, whose expression induction. Mitochondria can contribute to oncogenesis is not only tissue specific, but also varies according to the through a variety of mechanisms, including mutations pathophysiological state of the cell. Recent evidence of nuclear or mtDNA-encoded mitochondrial tumor revealed a differential role of the ANT isoforms in suppressors, metabolite accumulation (for example apoptosis and a deregulation of their expression in cancer. succinate and fumarate), production of reactive oxygen In this review, we introduce the current knowledge of species (ROS), apoptosis resistance, hypoxia-like path- ANT in apoptosis and cancer cells and propose a novel way activation and bioenergetic dysfunction (Gottlieb classification of ANT isoforms. and Tomlinson, 2005). Thus, in certain tumor cells, the Oncogene (2011) 30, 883–895; doi:10.1038/onc.2010.501; decreased adenosine triphosphate (ATP) production published online 15 November 2010 through mitochondrial oxidative phosphorylation is balanced by an increased ATP synthesis by glycolysis Keywords: cancer; cell death; mitochondria; ADP/ATP to maintain a vital energetic homeostasis, as first carrier; mitochondrial membrane permeabilization described by Warburg et al. (1924) and Warburg (1956). However, non-Warburg tumor cells in which oxydative phosphorylation is used primarily to generate Introduction ATP rather than glycolysis have also been previously described, notably in gliomas cells (Bouzier et al., 1998; Mitochondria have important functions in mammalian Beckner et al., 2005; Griguer et al., 2007; Rodrı´guez- cells, such as serving the energy powerhouse and Enrı´quez et al., 2010). Among the proteins responsible integrators of the intrinsic pathway of apoptosis for mitochondrial dysfunction in cancer, the mito- (Kroemer et al., 1998, 2007; Desagher and Martinou, chondrial adenine nucleotide translocase (ANT) or 2000). Apoptosis implies the translocation or the ADP/ATP carrier has been suggested to have an association of cellular proteins to the membrane of important function (Faure Vigny et al., 1996; Halestrap mitochondria, such as proteins triggering the mito- and Brenner, 2003; Chevrollier et al., 2005a; Brenner chondrial membrane permeabilization (MMP) and the and Grimm, 2006). ANT is an integral protein inserted into the mito- chondrial inner membrane (IM) through six transmem- Correspondence: Dr C Brenner, INSERM UMR-S 769, Faculte´de brane helices as revealed by 3D-structure analysis Pharmacie, Univ Paris-Sud 5, Rue J.-B. Cle´ment, Chaˆtenay-Malabry (Pebay-Peyroula et al., 2003). Its structure is similar to Cedex F-92290, France. E-mail: [email protected] that predicted for other mitochondrial carriers and then Received 30 July 2010; revised 22 September 2010; accepted 25 allows the electrogenic exchange of two substrates, September 2010; published online 15 November 2010 namely ADP and ATP, between the matrix and the ANT isoforms family C Brenner et al 884 intermembrane space (Pfaff and Klingenberg, 1968; proteins that coordinate mitochondrial outer membrane Pfaff et al., 1969). Therefore, this nucleotide-dependent permeabilization via still debated molecular mechanisms function implies a central role in energetic metabolism (for review, see Chipuk and Green, 2008). More and energetic compartmentation via the coupling of recently, a third major breakthrough has been made mitochondrial ATP synthesis to cytosolic delivery. in terms of role of ANT in apoptosis. Thus, ANT is Then, ANT antiport function is critical for many cell expressed as four tissue-specific isoforms, ANT1 to processes, notably in tumor cells, which have increased ANT4, and we and others postulated that each ANT metabolic requirements for DNA and protein synthesis isoform might have a specific role in influencing cell fate, (Gottlieb and Tomlinson, 2005). notably in cancer cells (Bauer et al., 1999; Zamora et al., In 1996, the discovery that ANT also functions as a 2004a; Le Bras et al., 2006; Gallerne et al., 2010). In this non-specific pore upon stimulation by ions and a variety review, we recapitulate our knowledge of the role that of molecules, such as calcium (Ca2 þ ) and ROS, as the various isoforms of ANT play in apoptosis, examine shown by reconstitution of native purified ANT into the expression and the function of the four ANT liposomes, constituted the first important breakthrough isoforms in cancer cells and discuss recent evidence to (Brustovetsky and Klingenberg, 1996; Ru¨ck et al., propose a novel classification of ANT isoforms and 1998). Later, a second breakthrough highlighted the their function in cancer cell death. critical involvement of ANT in the execution of the mitochondrial apoptosis pathway in various pathophy- siological and experimental models, such as ischemia– reperfusion, intoxication and chemotherapy (Marzo ANT: a bi-functional protein et al., 1998a; Cao et al., 2001; Haouzi et al., 2002; Malorni et al., 2009). For instance, ANT could influence ANT, as many proteins involved in cell death signaling, cell death through cooperation with pro-apoptotic harbors two (opposite) functions, both of which are proteins such as Bax, viral proteins (for example viral intricately involved in the control and regulation of protein R (Vpr) from HIV-1, PB1-F2 from Influenza) or cell fate (Halestrap and Brenner, 2003; Kroemer, et al., through participation in a mitochondrial polyprotein 2007). complex, the so-called permeability transition pore complex (PTPC) in diverse model systems such as A vital ADP/ATP translocase function human carcinoma cell lines, cardiomyocytes and neu- ANT constitutes the major protein in the mitochondrial rons (Crompton, 1999; Cao et al., 2001; De Giorgi et al., IM, in which it represents around 10% of the proteins. 2002; Brenner and Grimm, 2006) (Figure 1). However, It catalyzes the energetic process of ATP export from while experimental evidence clearly established a role of the mitochondrial matrix to the intermembrane space, ANT in the so-called MMP in some models of cancer, and conversely, the import of ADP from the intermem- other studies also showed that MMP could be ANT brane space to the matrix (Pfaff and Klingenberg, 1968; independent, but controlled by the Bcl-2 family Pfaff et al., 1969). The transport is electrogenic of proteins (Antonsson et al., 1997; Eskes et al., 1998). and drives the stoichiometric exchange of ATP4À and Bcl-2 family is composed of pro- and anti-apoptotic ADP3À. The enzymatic function of ANT has been

Permeability transition Glycolysis 2+ G6P Ca ROS -induced uncoupling HK Pyruvate VDAC VDAC ROS ROS ANT U ANT UCP CypD OM 2+ OM IM Ca H+ H+ Pyruvate

Fumarate Succinate TCA - + NAD+ H 0 H+ ATP - + m 1/2 O 2 ADP NADH 2 ADP - + II I III IV V ANT Ub e- ROS Cytc H+ H+ H+ ATP ROS

Oxydative phosphorylation Figure 1 Scheme of mitochondrial functions involving the ANT. ANT possess diverse functions including the electrogenic exchange ADP/ATP across the IM, the participation to the PTPC stimulate by Ca2 þ and ROS, a mild uncoupling activity that is stimulated by anion superoxide, fatty acids and peroxydated lipids. All functions are modulated by the transmembrane inner potential, DCm, which is, at least in part, generated by the oxidative phosphorylation and the function of the respiratory complexes I to V. Cytc, cytochrome c; G6P, glucose-6- HK, hexokinase, TCA, tricarboxylic acid cycle; U, calcium uniport; UCP, .

Oncogene ANT isoforms family C Brenner et al 885 extensively characterized in isolated mitochondria from sequence (RRRMMM) facing the mitochondrial matrix various tissues with radiolabeled and in (Pebay-Peyroula et al., 2003) (Figure 2). Interestingly, native ANT-containing proteoliposomes (Vignais et al., the ANT crystal analysis confirmed the existence of a 1973; Lauquin et al., 1979; Brandolin et al., 1980). These threefold repeat of about 100 amino acids and the studies have benefited from the use of various inhibitors binding of the IM-specific lipid cardiolipin, whereas it (for a list of ANT inhibitors, see Table 1), and mainly, is inferred that the monomer is the preferential state to two specific inhibitors, namely carboxyatractyloside transport the nucleotides. CAT binding was observed to (CAT) and bongkrekic acid (BA), which bind different be exclusive to nucleotides, thus suggesting that amino sites within the ANT 3D structure and favors, respec- acids involved in the putative nucleotide-binding site tively, c- and m- conformation of the protein (Hender- are also important for CAT binding. A closed–open son and Lardy, 1970; Vignais et al., 1973; Klingenberg, conformation switch has been proposed to allow for the 2008). The efficiency of the carrier to exchange ADP and transport of nucleotides; however, the exact molecular ATP is moderate and the carrier abundance would enzymatic mechanism of ADP/ATP exchange still reflect an adaptation to the high intracellular demand of remains unclear (Pebay-Peyroula et al., 2003). ATP export (Klingenberg, 2008). It is still a matter As indicated above, adaptation of (most) cancer cells of debate whether ANT can function independently or to intra-tumoral hypoxia is manifested by decreased requires a physical association with a partner such as oxidative phosphorylation and a high glycolytic ATP voltage-dependent anion channel (VDAC) (Beutner production (Warburg, 1956; Gottlieb and Tomlinson, et al., 1998), the phosphate carrier (PiC) (Leung et al., 2005; Bellance et al., 2009). To maintain cellular ATP 2008), the F0F1-ATP synthase (Chen et al., 2004) as well homeostasis, mitochondria regulate the expression of as the intermembrane space in certain several nuclear and mitochondrial genes, including ANT tissues (Beutner et al., 1998) (Figure 1). An association isoforms (Chevrollier et al., 2005a; Bellance et al., 2009). with a mitochondrial partner may improve the channel- Thus, it has been postulated that cancer cells over- ing of ATP to hexokinase and ADP to the matrix, to express ANT2 isoform (Chevrollier et al., 2005a), which answer to the cellular energetic demand, and may be would function in a reverse manner, that is import of particularly relevant in the context of metabolic ATP and export of ADP, to compensate for the low adaptation of mitochondria in cancer cells. efficiency of ATP production via oxidative phosphor- Recently, the crystallization of the bovine heart ANT ylation and to promote cell growth (see below for review with the specific inhibitor, CAT, has confirmed a of the putative promoter regions involved in ANT2 up- particular structural organization based on six trans- regulation). In addition, cytosolic hexokinase II associ- membrane helices with a depression at the intermem- ates with porin or VDAC to transfer cytosolic ATP to brane space surface and a short mitochondrial carrier ANT2. The latter is in close functional interaction with

Table 1 Direct regulators of ANT functions Compound Pore ADP/ Cell Reference ATP death exchange

Calcium þþBrustovetsky and Klingenberg (1996); Beutner et al. (1996) ADP, ATP ÀþÀBrustovetsky and Klingenberg (1996) Atractyloside, carboxyatractyloside þþþBeutner et al. (1996) Bongkrekic acid ÀÀÀBrustovetsky and Klingenberg (1996); Beutner et al. (1996) Nitric oxide, peroxynitrite, 4-hydroxynonenal þ ND þ Vieira et al. (2002) Diamide, bis-maleimido-hexane, dithiopyridine þ ND þ Costantini et al. (2000a) Bax þÀþBelzacq et al. (2003) Bcl-2 ÀþÀBelzacq et al. (2003) BaxBH3, Bcl-2BH3 þ ND þ Vieira et al. (2002) Vpr 52-96 þ ND À Jacotot et al. (2001) Lonidamine, verteporfin, arsenic trioxide, CD437 þ ND þ Belzacq et al. (2001a); Belzacq et al. (2001b) Nelfinavir À ND À Weaver et al. (2005) GD3-7-aldehyde þ ND þ Brenner et al. (2010) Fatty acids þ ND þ Jan et al. (2002) Biapigenin, berberine, N-acetyl perfluorooctane þÀþPereira et al. (2008); O’Brien et al. (2008) sulfonamides Quercetin þÀþOrtega and Garcı´a (2009) NH(2)-26-44 tau fragment ND ÀþAtlante et al. (2008) All-trans-retinoic acid þÀþNotario et al. (2003) CO/glutathion ÀþÀQueiroga et al. (2010)

Abbreviations: ANT, adenine nucleotide translocase; ATP, adenosine triphosphate; ND, not determined; ( þ ), activation; (À), inhibition. This table lists small molecules, ions, proteins acting directly on ANT. Effects of ANT activity modulation have been determined into artificial membranes (e.g. liposomes or planar lipid bilayers) in which native ANT has been reconstituted. ANT has been purified from various organs and animals in the presence of Triton X-100 and reconstituted into artificial biomembranes. The pore function has been measured by electrophysiology or by measurement of the release of fluorescent probe or of a metabolite from proteoliposomes. The effect on cell death has been evaluated on cancer cell lines or primary neurons.

Oncogene ANT isoforms family C Brenner et al 886 Ct Nt

CAT Cys 56

Ct Nt

Figure 2 3D-model of bovine ANT1 (P02722) co-crystallized with CAT. (a) Top view of ANT1/CAT complex from the intermembrane space. (b) Lateral view of ANT1/CAT complex. The model has been prepared with the program PyMOL and the published crystal structure of ANT (Pebay-Peyroula et al., 2003). N- and C-terminal extremities (Nt, Ct), which face the intermembrane space are indicated. The ADP/ATP translocase inhibitor CAT is shown in yellow and cardiolipins residues in brown. Two sites proposed to be important for the modulation of the pore function are indicated: the second hydrophilic loop (position 104–116, red), a putative-binding site for Bax, Bcl-2 and Vpr 52–96 and an oxidable cystein (position 56, orange).

F0F1-ATP synthase to form the supercomplex ATP varies depending on the tissue as well as the pathophy- synthasome (Chen et al., 2004). siological state (Halestrap, 2009, Rasola et al., 2010). Irrespective of these premises, Brustovetsky and Klingenberg (1996) used electrophysiological app- A lethal pore function roaches to show that pure native ANT could be The initial description of ANT was exclusively restricted converted into a large channel by Ca2 þ following to its functional involvement in the exchange of reconstitution into liposomes (Brustovetsky and Klin- nucleotides. However, the Ca2 þ -induced opening of a genberg, 1996). The channel activity consisted of multi- large conductance channel through the IM was reported ple substates that are sensitive to ANT inhibitors, ANT in a setup of isolated rat liver mitochondria (Zoratti and ligands and insensitive to cyclosporin A (CsA), a Szabo, 1994, 1995). This channel activity was attributed molecule that maintains PTPC in a closed conformation to PTPC opening and modulated by ANT inhibitors, by inhibition of the interaction of ANT and CypD. CAT and BA, as well as by endogenous ligands, ADP Importantly, this conversion was reversible by Ca2 þ and ATP. These studies constitute the basis for the elimination, indicating that the pore channel conforma- controversial hypothesis that ANT could be the IM tion is not a denatured substate of the protein in which channel of the PTPC in association (or not) with VDAC the global 3D structure has been irreversibly lost and cyclophilin D (CypD) (Zoratti and Szabo, 1994, (Brustovetsky and Klingenberg, 1996). The ADP/ATP 1995). These and other observations provide evidence carrier-pore conformation change may result from the that ANT is located in the contact sites of mitochondrial binding of Ca2 þ to cardiolipin, an IM lipid, which membranes in complex with VDAC and other proteins, disrupts the association of the lipid with ANT (Klingen- such as creatine kinase and CypD (Vyssokikh and berg, 2008). The possibility to induce a conversion into a Brdiczka, 2003). However, despite the use of multiple pore upon ligation has been repeated and extended methodologies, such as co-immunoprecipation, proteo- to several mitochondrial carriers (Rial et al., 2010), mic analysis, co-purification, genetic validation, the revealing a novel inducible function for some members identity and the number of PTPC components are still of this highly conserved class of mitochondrial proteins. elusive (Zoratti et al., 2005; Brenner and Grimm, 2006; However, not all carriers support the induction of Halestrap, 2009; Rasola et al., 2010) and it remains to be mitochondrial permeability transition pore (Crichton seen whether ANT and VDAC are the main compo- et al., 2009). nents of the PTPC or just regulators or accessory PTPC Moreover, despite being less active than the uncou- proteins. Notably, using isolated mitochondria from pling protein 3, a mitochondrial carrier, ANT might mice knock-out for ANT 1/2 or, VDAC1, or VDAC3, import proton across the IM and thus, might have a role or VDAC1/3 and CypD, a permeability transition in mediating mild uncoupling to regulate ROS generated response to the classical inducer Ca2 þ was monitored by the electron transport chain. This supports the and results suggested a limited role for these proteins in hypothesis that ANT participates to a first line of MMP (for discussion, see Rasola et al., 2010). Interest- defense against ROS in response to fatty acids, super- ingly, the PiC has been proposed recently to function as oxide and lipid peroxidation products under conditions the IM channel of PTPC (Leung et al., 2008), but this of high oxidative damage (Figure 1) (Scho¨nfeld et al., awaits genetic demonstration. Finally, an attractive 1996; Khailova et al., 2006; Azzu et al., 2008; Jastroch possibility might be that the composition of this pore et al., 2010; Toime and Brand, 2010). This ANT

Oncogene ANT isoforms family C Brenner et al 887 uncoupling function has undoubtedly important impli- (Bauer et al., 1999) and more precisely, the region 105– cations for pathology and cardioprotection (Nadtochiy 156 of ANT2 has been suggested to be the binding et al., 2006), but remains to be investigated in cancer. domain of ANT with several proteins such as Bax and Interestingly, the functions of ANT could depend on Bcl-2 (reviewed in Jacotot et al., 2006). Therefore, it is the dynamics of the mitochondrial networks, that is the tempting to postulate that direct interaction of ligands balance between the /fission events. with specific amino acids contained within the ANT Thus, it has been shown that the inhibition of sequence provokes a conformational change resulting in mitochondrial fission in the heart by pharmacological a switch from the translocase function to the pore as well as genetic approaches impairs the process of function. The inhibition of the ANT translocase permeability transition and cell death (Gazaryan and function can also induce apoptosis. This has been Brown, 2007; Ong et al., 2010). Thus, this suggests that elegantly shown with the small molecule, MT-21, a inhibition of fission could prevent the lethal ANT pore synthetic epolactaene derivatives, which has a g-lactam opening within the PTPC, but again, this requires ring and an alkyl chain at the N-1 position. MT-21 further investigation in appropriate experimental cancer induces apoptosis in human leukemia HL-60 cells models. (Watabe et al., 2000) and a variety of cell lines Paradoxically, ANT function modulation could in- (C Brenner, unpublished data) through ANT inhibition fluence the bioenergetic metabolism and cell fate and cytochrome c release without any effect on the through pore activation as well as carrier inactivation. permeability of the IM (Watabe et al., 2000; Machida Thus, even if it might be difficult to discriminate which et al., 2002). It was proposed that the mechanisms of function is involved in a particular condition, there is MT-21 apoptosis induction could be useful to design a now a reasonable consensus that ANT can be specifi- unique strategy to fight cancer cells, but unfortunately, cally converted into a pore upon treatment with a large MT-21 cytotoxic effects were abolished by overexpres- panel of proteins and molecules and that, upon a certain sion of Bcl-2 compromising its therapeutic development threshold of stimulus, ANT pore induction can lead to for anticancer chemotherapy. cell death (Table 1). For example, we and others showed Whether the antiport function of ANT is well that ANT cooperates with Bax to modulate the ANT conserved through evolution (Palmieri and Pierri, translocase and pore activity (Belzacq et al., 2003) and 2010), a role for ANT in the control of cell death has to induce pro-apoptotic MMP in various cancer cells also been reported in other eukaryotes, thereby suggest- (Marzo et al., 1998a). Importantly, inhibition of ing an evolutionary conservation of its role in cell this protein–protein interaction by CsA, z-VAD-fmk death execution (Zhivotovsky et al., 2009). Thus, only (a caspase inhibitor) and furosemide (a Bax transloca- ANT, and neither VDAC nor CypD, is necessary tion inhibitor) prevented cell death induction by for mitochondrial outer membrane permeabilization chemotherapy (Verrier et al., 2004). This observation and cytochrome c release in yeast (Pereira et al., 2007) suggests that in colon carcinoma cells treatment by and, in Caenorhabditis elegans, the ANT homolog etoposide, which inhibits the topoisomerase II and, WAN-1 promotes canonical apoptosis by cooperation therefore, generates a genotoxic damage, caspase with the core cell death machinery, namely CED-4/ activation precedes the induction of MMP and cell Apaf-1 and CED-9/Bcl-2 (Shen et al., 2009). death by an unknown mechanism. Nevertheless, this confirmed the observation that caspases can activate permeability transition and the release of cytochrome c Four ANT isoforms for cell fate control and AIF from isolated mitochondria, in a circular self-amplification loop (Marzo et al., 1998b). Further- Until recently, studies based on purified proteins from more, using ANT-containing proteoliposomes and rodent organs and cultured cell lines did not address the simultaneous measurement of both ANT functions, we crucial question of a differential role of ANT isoforms showed that the pore function was favored by Bax and in apoptosis regulation. However, the modification of inhibited by Bcl-2, whereas, in contrast, the carrier function was favored by Bcl-2 and inhibited by Bax the relative expression of ANT isoforms (Halestrap and Brenner, 2003) and the fact that MMP process is (Belzacq et al., 2003). Interestingly, mutants of Bax and inhibited in most cancer cells (Kroemer et al., 2007) Bcl-2 that lack the transmembrane a 5 and 6 helices do not interact with ANT in isolated mitochondria as well prompted us to postulate that each isoform could have a different function in apoptosis, and notably that as in ANT-containing proteoliposomes. In addition, some isoform could be pro-apoptotic and other anti- ANT functions proved to be regulated by oxidative apoptotic. stress (for example thiol oxidation of cystein residue 56) (Costantini et al., 2000a), carbon monoxide (Queiroga et al., 2010) and by interaction with viral proteins such Expression of ANT isoforms in normal and cancer tissues as Vpr and PB1-F2 to modulate cell death (Jacotot The ANT isoforms form a subfamily of mitochondrial et al., 2001; Zamarin et al., 2005) (Figure 2). Pharma- carriers composed of four members to date (Palmieri cological studies with CAT and BA suggest that and Pierri, 2010). Different nuclear genes encode for induction of the pore function might be an irreversible four homologous proteins, ANT1 to ANT4, which are process (C Brenner, unpublished data). Interestingly, all located in the mitochondrial IM. The expression of only the first third of the protein is involved in apoptosis the various isoforms is tissue specific and may depend

Oncogene ANT isoforms family C Brenner et al 888 on the proliferative capacity of the tissue and its energy ANT Isoforms Predominant features Function requirements in term of glycolysis or oxidative phos- in the 5’ flanking region phorylation (Palmieri and Pierri, 2010). In humans, the four ANT genes have similar structures and encode similar proteins, with among hANT1 OXBOX, REBOX Pro-apoptotic between 80 and 90% between the various isoforms (Deniaud et al., 2005; Le Bras et al., 2005). The initial hANT2 GRBOX, C-BOX Anti-apoptotic classification was based on the measurements of ANT1 to ANT3 mRNA levels in rodents and human organs (Lunardi and Attardi, 1991; Faure Vigny et al., 1996; hANT3 SP1 sites Pro-apoptotic Giraud et al., 1998; Le Bras et al., 2006). ANT1 mRNA level has been found by reverse transcription polymerase chain reaction (RT–PCR) to be the predominant hANT4 COUP-TF binding element Anti-apoptotic isoform in heart, brain and muscle, ANT2 mRNA level Figure 3 Summary of predominant features between promoters being the most abundant isoform in proliferative tissues of the four ANT isoform and the apoptosis-linked function of such as lymphoid cells and the liver, and ANT3 being isoforms. The various regulatory elements linked to the capacity of expressed at low levels independently of the tissue type. the isoform to modulate positively or negatively apoptosis are In breast (MCF7), colon (HT29), cervix (HeLa) and indicated. hepato-carcinoma (HepG2) cells, ANT2 mRNA is more abundant than ANT1 (Lunardi and Attardi, 1991; promoter sequences of the four isoforms of ANT and Faure Vigny et al., 1996; Giraud et al., 1998; Le Bras that can influence functional aspect of the ANT protein et al., 2006). Of note, we recently reported that ANT2 toward the pro- or the anti-apoptotic program are mRNA is significantly overexpressed in primary tissues schematized in Figure 3. derived from patients with cancer of the breast, uterus, As the 50-flanking region of the four ANT genes varies ovary, lung, thyroid gland, bladder and testis as with reference to the binding sites of various transcrip- compared with the respective normal tissue (Le Bras tion factors, activation or repression of the genes could et al., 2006). These studies preceded the identification of be correlated to their methylation status. Thus, each of the fourth isoform, ANT4, by real-time quantitative these human ANT genes contains CpG islands in their PCR; ANT4 mRNA was only found in healthy human proximal promoter regions (Brower et al., 2009b). brain, liver and testis (Dolce et al., 2005). It remains Investigations of the methylation status of the three to be determined how the expression of the four ANT genes, that is ANT1, 2 and 4, in various tissues of isoforms varies relative to each other in primary cancer mice revealed that this modification controls gene tissues. transcription. In comparison with the ANT 4 gene in Recently, the measurement of expression sequence which the CpG island methylation is essential for gene tags (Unigene) established overexpression of ANT2 and repression, the CpG islands of ANT1 and ANT2 are ANT3 mRNA and down-regulation of ANT1 mRNA in hypomethylated, regardless of the gene expression status normal tissues as compared with primary cancer tissues, throughout the tissues of male mice. This study provides suggesting the existence of an ANT isoform signature an insight into the differential regulation of ANT in some cancers. Of note, no reliable information on a isoforms in mammals, whereby both the ANT1 and sufficient number of cancer patients is available for ANT2 genes are capable of active expression, but the association with ANT4 expression in the Unigene ANT4 gene can be repressed throughout somatic tissues. database. Interestingly, this is one of the first reported observations to clearly show a differential usage of CpG island methy- lation within a family of genes (Brower et al.,2009b). Mechanisms of regulation of ANT isoform gene expression and impact on cell death A set of elegant studies unraveled the opposing ANT1 isoform is pro-apoptotic and its expression is association of human ANTl and ANT2 mRNA expres- frequently repressed in cancer sion with cell proliferation. ANT2 mRNA is highly Human ANT1 gene is located on chromosome 4 and is expressed in proliferating cells and absent or low in predominantly expressed in heart and skeletal muscles. non-proliferating cells (either quiescent or terminally It possess OXBOX and REBOX sequence elements in differentiated), whereas ANTl mRNA is highly the promoter region, in addition to the canonical expressed in non-dividing, terminally differentiated transcription start sites, the TATA box and CCAAT skeletal muscle cells, but is absent in proliferating box elements (Figure 4). OXBOX element acts as a myoblasts (Doerner et al., 1997). This suggests that the positive regulatory element exclusively in muscle cells expression of the various ANT genes is different and (Li et al., 1990). Therefore, the OXBOX and its tightly regulated. associated trans-activating factor(s) might account for Accordingly, the analysis of the motifs in the the high ANTl expression in heart and skeletal muscle 0 5 -flanking regions of ANT genes revealed that in association with F0F1-ATP synthase b subunit, a their promoter structure and organization are different. component of the oxidative phosphorylation machinery The important structural features that delineate the (Li et al., 1989; Neckelmann et al., 1989). The relatively

Oncogene ANT isoforms family C Brenner et al 889

OXBOX REBOX TATA CCAAT ANT1 ANT1 -1400 +1

AB C GRBOX TATA ANT2 ANT2 -1196 -1184 -250 -30 +1

TATA CCAAT ANT3 ANT3 -1250 +1

ANT4 ANT4 -586 -212 +1

COUP-TF Binding element SP1 Binding element CpG island AP2 Binding element E2F6- Binding element Oct1 Binding element AP1 binding element SV40 Enhancer element NF1 site

Figure 4 Schematic structural organization of ANT gene isoforms. Positions of various regulatory elements and transcription factor- binding sites have been indicated for the various promoter regions of the four ANT isoforms, ANT1–ANT4. The start site of transcription is indicated þ 1. low level of mitochondrial transcripts in skeletal muscle The ANT2 isoform is an MMP inhibitor and can be is inconsistent with the high level of ANTl and F0F1- up-regulated in hormone-dependent cancer ATP synthase b subunit transcripts. These observations Located on the X chromosome, ANT2 is differentially suggest that transcriptional control of nuclear-encoded expressed in human tumor cells under hypoxia (Chev- OXPHOS genes may be achieved by both tissue-specific rollier et al., 2005b). The 50 region upstream of the start and cell-cycle-specific trans-acting factors (Stepien et al., codon contains the TATA box and three putative AP1 1992). In 1999, a systematic screen for dominant pro- control elements near the transcription start site apoptotic genes led to the identification of ANT1 as a (Figure 4). An AP-2-binding site and five SP1-binding pro-apoptotic factor (Bauer et al., 1999). Apoptosis sites have also been reported. Two of the SP1 elements induced by ANT1 overexpression was accompanied by (conserved AB box) have been shown to activate ANT2 enhanced cytochrome c release, and caspases-9 and -3 transcription, while the third element (C box organiza- activation, indicating that its expression facilitates tion) has been shown to inhibit gene transcription. mitochondria-dependent apoptosis (Bauer et al., 1999, In addition, Luciakova et al. (2008) provided evidence Zamora et al., 2004b). Heterologous overexpression of for an upstream-binding site for NF1, an active ANT1 in cardiomyocytes also significantly induces repressor of the ANT2 gene. ANT2 transcription is apoptosis with multiple consequences, such as a up-regulated in growth-activated cells and down-regu- decrease in NF-kB activity, a down-regulation of Akt lated in cells exiting the cell cycle upon serum restriction activation and Bcl-xL protein levels, a depolarization of or density-dependent growth inhibition. The expression the mitochondrial membrane potential and the up- of human ANT2 gene in highly proliferative cells is regulation of Bax expression (Baines and Molkentin, governed by glycolysis-regulated box, a transcriptional 2009). Supporting a pro-apoptotic role of ANT1 in vivo, element involved in the regulation of glycolytic ATP ANT1 deficiency increases the resistance of mouse brain import into mitochondria (Giraud et al., 1998). Under and neurons to excitotoxic stress (Lee et al., 2009). In aerobic conditions, the specific ROX1 factor binds to addition, ANT1 transfection in breast adenocarcinoma an ROX1-binding motif in the ANT2 promoter region cells (MDA-MB-231 cells) in a nude mice model proved and the ANT2 gene is regulated through heme that to induce apoptosis, to inactivate NF-kB, to increase blocks its expression (Sabova´et al.,1993).Sequence Bax expression and finally to stimulate tumor regres- comparison of the human ANT and the three yeast sion, validating the hypothesis that ANT1 could be a AAC promoters led to the identification of a 13 bp potent therapeutic target for the treatment of cancer motif in the ANT2 promoter: CATTGTTATGATT (Jang et al., 2008a). The pro-apoptotic potential (nt À1196 to À1184 relative to the transcription of ANT1 correlates well with its down-regulation in start) homologous to an AAC3 promoter sequence. human cancers (see above). This precise sequence regulatory element glycolysis-

Oncogene ANT isoforms family C Brenner et al 890 regulated box confers the binding with specific ROX1- The ANT3 isoform is pro-apoptotic in cellula, binding factors that critically regulate the expression but can be up-regulated in cancer of the gene in highly proliferating cells (Giraud Non-rodent mammals including human, cow and dog et al., 1998). have an additional ANT3 gene, which is encoded at Increased levels of ANT2 transcripts that occur in the pseudoautosomal region of X chromosome and is response to serum induction are regulated at the level of ubiquitously expressed. The promoter region of the transcription and transcript stability is unaltered. To ANT3 isoform contains the classical TATA and date, three distinct suppressor regions in the ANT2 CCAAT elements, in addition to as many as 16 SP1- promoter could contribute to regulation under physio- binding sites (Figure 3). There are also 13 potential SPl- logical conditions like response to growth activation in a binding sites within approximately 1250 bp of the tissue-specific manner (Stepien et al., 1992; Doerner transcription start site. The presence of numerous SP1- et al., 1997) and in response to differentiation signals in binding sites could favor apoptosis in a positive manner various cell types, including muscles (Lunardi et al., (Deniaud et al., 2006), because SP1 induces inhibition of 1992). The three suppressor regions in the human ANT2 cell cycle progression that precedes apoptosis and a promoter region might have different physiological transcriptional response targeting genes containing roles. The proximal Sp1 suppressor (Li et al., 1996) Sp1-binding sites in its promoter region, suggesting and one upstream suppressor region appear to influence Sp1-driven gene transcription (Deniaud et al., 2006). promoter activity in exponentially growing cells, Regarding the role of ANT3 in apoptosis, Zamora et al. whereas a distal region of the ANT2 promoter (nucleo- (2004a, b) showed that ANT3 exerts a similar effects to tides –1237 to À546) is required for the down-regulation ANT1 upon overexpression in HeLa cells, in contrast of the gene (Barath et al., 2004). Of note, as almost all with the lack of apoptotic activity of ANT2 (Bauer the SP1-binding sites in the ANT2 promoter are et al., 1999; Zamora et al., 2004a). Both studies claimed epigenetically masked by methylated CpG islands, it is that the effects of ANT1 and ANT3 result from protein– plausible that the transcriptional activation of the gene protein interaction within the PTPC complex because is deregulated. Intrigued by the enhanced ANT2 content the cytotoxic effects of ANT3 can be prevented by the in some cancer cells (Faure Vigny et al., 1996; Le Bras inhibitors of PTPC (CsA) and ANT (BA), respectively. et al., 2006), we and others examined the possibility It was suggested that ANT3 exerts its lethal effects that ANT2 could have cytoprotective effects, that is through an interaction with the matrix protein CypD. proliferative and/or anti-apoptotic effects. Thus, it has Both studies reported a lack of cytotoxicity of the ANT2 been speculated that (1) ANT2 could function in a isoform. Paradoxically, Unigene database reports an up- reverse manner to import ATP into the mitochondrial regulation of ANT3 mRNA in numerous human matrix to compensate for the reduced mitochondrial tumors. These findings are counter-intuitive and, there- ATP production in cancer cells (Chevrollier et al., fore, require further investigations to unravel the specific 2005a) and (2) could interact preferentially with Bcl-2 to factors associated with ANT3 and its pro-apoptotic favor a stabilization of mitochondrial membranes activity. through the inhibition of MMP (Belzacq et al., 2003). Thus, we observed that on the one hand the decrease of ANT2 levels by small-interfering RNA knock-down The ANT4 isoform is anti-apoptotic in human cancer sensitized cells to apoptosis induction (Le Bras et al., cell lines 2006), but on the other hand increased ANT2 levels by The ANT4 isoform expression is tightly controlled stable overexpression in various cell lines (for example depending on organism and tissue (Dolce et al., 2005). HT29 and HeLa cells) promoted chemoresistance Interestingly, ANT4 gene is methylated at a long stretch (Gallerne et al., 2010). For instance, ANT2 down- of nucleotides of almost 300 bases just upstream of the regulation renders HeLa cells resistant to Lonidamine, predicted transcription initiation site in the promoter an anti-cancer agent whose effect is based on the region (À212) (Figure 4). It has been shown earlier that induction of the mitochondrial apoptosis pathway in mouse embryonic stem cells, ANT4 is selectively through the ANT targeting (Ravagnan et al., 1999; expressed and uniquely restricted to developing gametes Belzacq et al., 2001a; Gallerne et al., 2010). Accordingly, in adult mice, and its promoter hypomethylation suppression of ANT2 by small-interfering RNA in was observed only in testis, whereas hypermethylated human breast cancer cells induces apoptosis and was detected in somatic cells (Rodic et al., 2005); ANT4 prevents tumor growth in vitro and in vivo (Jang et al., gene promoter has a conserved E2F6-binding element, 2008b). Thus, the ANT2-mediated cytoprotective effects TCCCGC, essential for repression of the ANT4 in are strikingly opposite to ANT1 lethal effects. In a somatic tissue (Kehoe et al., 2008). Consistent with these limited study on human patients mRNA, we found that findings, addition of a CpG-demethylating agent, 5-aza- ANT2 is frequently overexpressed in hormone-depen- deoxycitidine, to fibroblasts increased gene expression of dent cancers, including ovary, thyroid, testis, breast and ANT4, but not the other isoforms (ANT1 and ANT2), cervix correlating with the presumed regulation of an thus indicating that unlike ANT4, CpG island DNA anti-apoptotic protein in cancer. However, a more methylation perhaps is not a critical factor in the tissue- important study with a larger number of patients specific expression of ANT1 and ANT2 (Rodic et al., is required to validate the expression level of ANT2 as 2005; Brower et al., 2009a). Thus, the ANT4 is a a potential biomarker. pluripotent stem cell- and germ cell-specific isoform of

Oncogene ANT isoforms family C Brenner et al 891 ANT in mouse, and DNA methylation has a primary Open questions and perspectives function in its transcriptional silencing in somatic cells. Moreover, the presence of a COUP-TF-binding se- In conclusion, whether all ANT isoforms behave as quence element confers transcriptional repression, there- ADP/ATP carrier across the IM, a central function for by negatively regulating hormonal induction of target the cell metabolism, recent reports support a novel genes (Cooney et al., 1992). In human cancer cell lines classification based on the differential effect of the (for example HeLa and HT29), ANT4 overexpression various ANT isoforms in cell fate decisions in cancer inhibits apoptotic signaling, especially mitochondria- cells: ANT1 and ANT3 being pro-apoptoptic and mediated cell death (Gallerne et al., 2010). Thus, ANT4 ANT2 and ANT4 being anti-apoptotic. This notion of prevents the loss of mitochondrial transmembrane an individual function of each isoform is crucial for the potential (DCm), the opening of PTPC, the activation understanding of how ANT isoforms and other of effector caspase-9 and nuclear alterations. The anti- mitochondrial proteins (for example Bcl-2, VDAC, apoptotic activity of ANT4 isoform could be mediated CypD, PiC and so on) interact for the regulation of by modulation of the ROS steady-state levels. As in this MMP (Kroemer et al., 2007). It is tempting to postulate experimental cellular model (Gallerne et al., 2010), the that sensitivity of cancer cells to apoptosis could be a various ANT isoforms are present, this suggests a function of the ratio of various isoforms, ANT1 and possible complex regulatory balance between ANT ANT3 being pro-apoptotic and ANT2 and ANT4 being pro- and anti-apoptotic isoforms and other apoptosis anti-apoptotic, but this requires further validation in regulatory proteins (that is Bcl-2, GST, catalase) large cohorts of clinical samples. The recent advances in (Gallerne et al., 2010). Owing to its recent discovery, our understanding of the differential effects of the the specific role of ANT4 in cancer has not yet been various isoforms in cell fate signaling has raised novel investigated. Taken together, promoter analysis questions regarding the molecular determinants of this of the four ANT isoforms provide insights into the functional diversity given the strong sequence homo- differential regulation of ANTs in mammals and logies of the four isoforms. One possibility is that increase our understanding of how these genes devel- functional differences between the proteins could be a oped and their expression was conserved throughout function of an association with different partners and/or evolution, retaining distinct functions in specific different sub-compartmentalization within the mito- organs and tissues. chondrial IM (Figure 5). In this regard, ANT1 and

Death Life Cancer

Bax Bcl-2 Bax Bcl-2 Bcl-2

ROS, Ca2+

ADP ATP

HK VDAC 1 VDAC OM VDAC OM PTPC ANT1 ANT2 ANT ANT3 IM ANT4 IM ADP Pi + H+ CypD ATP PiC ATP ADP 1.5 kDa ANT2 PiC

F0

F1

ATP ADP + Pi

H+

ATP Synthasome Figure 5 Hypothetical scheme of topological distribution of ANT isoforms in subcompartments. ANT isoforms fulfill their ADP/ATP exchange function and pore function depending on their association in polyprotein complexes and their submitochondrial localization. The pro-apoptotic isoform, ANT1, has been found in association with VDAC1 and CypP within the PTPC in IM domains in close contact with the OM (Vyssokikh et al., 2001). Upon death induction, Bax interacts with ANT to favor its lethal pore function, whereas Bcl-2 favors the translocase function and inhibits the pore opening (Marzo et al., 1998a; Belzacq et al., 2003). In contrast, ANT2, an anti-apoptotic isoform, participate to the ATP synthasome in association with the ATP synthase and the PiC in the cristae (Chen et al., 2004). In cancer cells, an overexpression of HKII and its association with VDAC has been observed at the surface of mitochondria contributing to the channeling of nucleotides, favoring the metabolic adaptation and inhibiting cell death (Pastorino et al., 2002).

Oncogene ANT isoforms family C Brenner et al 892 ANT2 do not reside in the same area of IM (Vyssokikh potential; ER, endoplasmic reticulum; MMP, mitochondrial et al., 2001) and are not associated with the same membrane permeabilization; IM, inner membrane; OM, outer polyprotein complexes, that is the PTPC, the ATP membrane; PiC, phosphate carrier; PTPC, permeability synthasome (Beutner et al., 1996; Chen et al., 2004) transition pore complex; ROS, reactive oxygen species; (Figure 5). No information regarding the submitochon- UCP1, uncoupling protein 1; VDAC, voltage-dependent anion channel; Vpr, viral protein R. drial localization of ANT3 and ANT4 are available to date. In addition, differences in the epigenetic modification of ANT isoformes in cancer could help to better understand the differential roles of the Conflict of interest isoforms as discussed for other mitochondrial proteins (Smiraglia et al., 2008). Hence, a complete elucidation of The authors declare no conflict of interest. the complex interplay of the various ANT isoforms in cancer cells might have crucial implications for a better understanding of the bioenergetic metabolism of cancer Acknowledgements cells and the design of novel mitochondrial-targeted anticancer therapies (Fulda et al., 2010). The work of CB and CP are supported by le Centre National de la Recherche Scientifique (CNRS) and l’Agence Nationale de la Recherche (ANR, ANR-08PCVI-0008-01). CB is also Abbreviations supported by l’Institut National pour le Cancer (INCa, 2008- 1-PL BIO-04-CNS ON1) and l’Universite´ Paris Sud ANT, adenine nucleotide translocase; BA, bongkrekic acid; (Programme Attractivite´). CB thanks M Nunez for the help Ca2 þ , calcium; CAT, carboxyatractyloside; CsA, cyclosporin in the preparation of Figure 2 and current and past laboratory A; CypD, cyclophilin D; DCm, mitochondrial transmembrane members for their invaluable contribution.

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