Oncogene (2014) 33, 5569–5581 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc

REVIEW Mitochondrial ion channels as oncological targets

L Leanza1, M Zoratti2, E Gulbins3 and I Szabo1

Mitochondria, the key bioenergetic intracellular organelles, harbor a number of proteins with proven or hypothetical ion channel functions. Growing evidence points to the important contribution of these channels to the regulation of mitochondrial function, such as ion homeostasis imbalances profoundly affecting energy transducing processes, reactive oxygen species production and mitochondrial integrity. Given the central role of mitochondria in apoptosis, their ion channels with the potential to compromise mitochondrial function have become promising targets for the treatment of malignancies. Importantly, in vivo evidence demonstrates the involvement of the proton-transporting uncoupling protein, a mitochondrial potassium channel, the outer membrane located porin and the permeability transition pore in tumor progression/control. In this review, we focus on mitochondrial channels that have been assigned a definite role in cell death regulation and possess clear oncological relevance. Overall, based on in vivo and in vitro genetic and pharmacological evidence, mitochondrial ion channels are emerging as promising targets for cancer treatment.

Oncogene (2014) 33, 5569–5581; doi:10.1038/onc.2013.578; published online 27 January 2014 Keywords: mitochondria; ion channels; direct targeting; pharmacology

MITOCHONDRIA; ION HOMEOSTASIS AND APOPTOSIS; with the idea that the perturbation of ion homeostasis within IMPACTS ON SIGNALING AND CANCER mitochondria leads to cancer cell death via reactive oxygen The mitochondrial inner membrane (IMM) surrounding the matrix species (ROS) production, mitochondrial calcium overload and permits the formation of an electrochemical proton gradient that metabolism changes, but other mechanisms of action might also drives aerobic adenosine triphosphate (ATP) synthesis, and the exist. Indeed, mitochondria are crucial for maintaining intracellular outer membrane (OMM) delimits an intermembrane space (IMS) Ca2 þ homeostasis and produce ROS, which are two important between the OMM and IMM. Proteins with fundamental roles in factors for cancer cell survival and proliferation. A detailed cell death, such as cytochrome c and SMAC/Diablo, can be overview of the above processes is beyond the scope of this released from the IMS upon receipt of a proapoptotic signal and review; thus, we briefly describe the general mechanisms by can lead to commitment of the cell to apoptosis. The released which ion channels may affect ROS production, mitochondrial serine protease HtrA2/Omi, apoptosis-inducing factor and endo- calcium homeostasis and metabolism. nuclease G also contribute to the executive phase of apoptosis.1 The matrix-negative difference in electrical potential across the Much effort has been devoted to identifying drugs capable of IMM (DCm, ranging between À 150 and À 180 mV) is maintained inducing cancer cell death by stimulating OMM rupture/ by the proton pumps of the respiratory chain (RC). Consequently, permeabilization and release of these proteins.2 Mitochondrial cations (K þ and Ca2 þ ) flow from the IMS (where the concentra- ion channels are therapeutically relevant to cancer because they tion of ions is comparable to cytosolic concentrations because the can influence both OMM and IMM permeability. Members of the OMM is permeable to these ions) to the matrix when a permeation B-cell lymphoma 2 (Bcl-2) family and MAC (mitochondrial pathway opens, as was demonstrated several years ago by apoptosis-induced channel) are directly involved in OMM experiments with ionophores such as valinomycin. The electrical- permeabilization, but they do not function as bona fide ion equivalent anion outflow is considered much less relevant channels. Excellent reviews are available on their role in this because the anion pool in the mitochondrial matrix is limited process;3,4 therefore, the present review focuses on the compared with the cytosolic K þ pool. To compensate for charge mitochondrial voltage-dependent anion channel (VDAC, also movement (unless permeating counterions are also present, known as porin) among the OMM channels. Selective induction which is feasible in experimental settings but unusual under of IMM permeabilization in cancer cells is also a potential physiological conditions), the RC must increase the rate of proton therapeutic strategy because mitochondrial energy production transfer from the matrix to the IMS. To increase RC activity becomes compromised when the IMM becomes freely permeable according to the chemiosmotic model, the transmembrane to solutes, which leads to cell death. electrochemical proton gradient (Dm~H, composed of mainly As a consequence of changes in ion flux across the IMM, DCm) must decrease. Thus, passive charge flow and Dm~H (DCm) alterations of mitochondrial function occur that produce severe are coupled, and the opening of K þ or Ca2 þ channels in the IMM effects on the overall fitness, including ATP production, of these will lead to depolarization. Clearly, this model also functions in organelles. Most available data on ion channels or ion flux reverse; closing cation channels is expected (and observed) to pathways that modulate cancer cells can be ultimately reconciled lead to an increase of a Dm~H (DCm) (that is, hyperpolarization).

1Department of Biology, University of Padova, Padova, Italy; 2CNR Institute of Neuroscience and Department of Biomedical Sciences, University of Padova, Padova, Italy and 3Department of Molecular Biology, University of Duisburg-Essen, Essen, Germany. Correspondence: Professor I Szabo, Department of Biology, University of Padova, Viale G Colombo 3, Padova PD, 35138, Italy. E-mail: [email protected] or [email protected] Received 15 August 2013; revised 4 December 2013; accepted 5 December 2013; published online 27 January 2014 Mitochondrial ion channels L Leanza et al 5570 Such alterations may in turn have consequences on the rate of disrupts energy production, releases proapoptotic proteins superoxide formation.5,6 Hyperpolarization lowers the efficacy of into the cytoplasm and enables apoptosis and/or necrosis.31–33 cytochrome c oxidase,7 induces the reduction of RC complexes In addition, mitochondrial Ca2 þ accumulation also leads and intermediates and thereby increases the probability of a one- to enhanced ROS production through multiple potential electron transfer to oxygen at respiratory complex I. Complex-III- mechanisms.34 Although the exact mechanistic role of dependent ROS formation can also be significant at high mitochondrial Ca2 þ homeostasis in tumorigenesis (beyond ROS respiration rates (depolarized conditions) because of a high production and modulation of apoptosis) is not well understood, 2 þ concentration of semiquinone at center ‘O’ (SQo or QP) of the mitochondrial Ca is an important signal transducer that can 8 À bc1 complex. Thus, enhanced superoxide (O2 ) production initiate pathways mediating cell death. Therefore, drugs increasing occurs in both the matrix and IMS.7,9–11 In addition, recent calcium uptake may efficiently kill cancer cells; however, how to data have highlighted that complex II also generates superoxide selectively target cancer cells instead of healthy cells by inducing or hydrogen peroxide molecules at the flavin site IIF when mitochondrial calcium accumulation remains unclear. electrons are supplied by either succinate or the reduced Mitochondrial calcium uptake is important for oxidative ubiquinone pool.12 Irrespective of the site of production, which phosphorylation regulation35 and energy metabolism control might depend on the substrates being oxidized,13 the superoxide because it enhances the rate of NADH production by anion is the precursor of most ROS. ROS can diffuse to different modulating the enzymes of the Szent-Gyo¨rgyi–Krebs cycle and subcellular compartments. Increased ROS production is important fatty acid oxidation pathways.36,37 In general, strategies that for the maintenance and evolution of the cancerous phenotype.14 increase energy and metabolite production in energy-demanding The effects of ROS vary according to the stage of carcinogenesis. cancer cells promote a high glycolytic flux rate and thus In the tumor initiation phase, ROS may cause DNA damage and allow enhanced tumor cell growth.38,39 Some IMM channels, mutagenesis, whereas in established cancer they may act as signal by regulating DC, matrix volume, matrix acidification and mediators through the Akt pathway and stimulate proliferation, ROS production, would be expected to impact the functionality conferring a growth advantage.15 ROS may also inhibit Pyruvate of mitochondrial RC complexes. Indeed, enhanced substrate Kinase M2, the embryonic form produced by cancer cells, thereby oxidation with matrix volume expansion has been diverting reducing equivalents to the pentose phosphate demonstrated,40 and pharmacological activation of calcium- pathway.16 induced potassium channels by NS11021, which affects Conversely, ROS release can induce cell death by damaging mitochondrial matrix volume, leads to enhanced respiratory proteins, lipids and DNA and thus act as anticancer agents.17–19 In control.41 Interestingly, the respiration rate and oxidative addition, the activation of pro-apoptotic kinases, such as apoptosis phosphorylation efficiency were found to be increased in signal-regulating kinase 1 (ASK1) and death-associated protein August rats that are characterized by an increased rate of kinases (DAPK; 5 members),20,21 by ROS is of fundamental ATP-dependent mitochondrial potassium transport as compared importance.15,17–21 Oxidative stress can also activate the pro- with Wistar rats that have lower ATP-dependent potassium apoptotic Bax and permeability transition pore (MPTP) (see channel activity.42 A recent paper reported the physical below),22 whose prolonged opening leads to the loss of the interaction and functional coupling of the mitochondrial large- mitochondrial Dm~H, mitochondrial swelling, cristae remodeling conductance calcium-activated potassium channel (mtBKCa) and and pro-apoptotic factor release.23 Bax and other Bcl-2 family complex IV of the RC.43 Whether this physical interaction also proteins regulate OMM permeabilization through pore formation occurs in other channels and plays a direct role in the regulation and possible regulation of mitochondrial fission and fusion.24 of mitochondrial energy fitness by ion channels is an important In light of this dichotomy, both antioxidant and pro-oxidant and unresolved question. Finally, the changes in matrix approaches have been proposed to antagonize cancer.25–28 volume resulting from channel opening are expected to lead to Antioxidants are expected to be useful for cancer prevention, structural cristae reorganization that has recently been shown whereas pro-oxidants are desirable to potentially and selectively to affect respiratory supercomplex organization and respiration induce cancer cell death. Chronic metabolic oxidative stress, likely efficiency.44 occurring in most cancer cell types, can indeed be exploited Along with IMM channels, a role for the OMM channel VDAC in because these cells exhibit an increased sensitivity to ROS-induced metabolism regulation has become clear; the glycolytic enzyme apoptosis.26 Therefore, therapies that specifically target the RC, hexokinase (HK), when bound to VDAC1, regulates metabolite either directly or indirectly, to further elevate ROS production trafficking (including ATP)45,46 through the OMM channel and should selectively kill cancer cells. Because pharmacological provides cancer cells with metabolic advantages. Furthermore, modulation of mitochondrial ion channel function is expected to VDAC1-bound HK is less sensitive to inhibition by its product, lead to changes in membrane potential, targeting these channels glucose-6-phosphate,47 and avoids product inhibition that further to increase ROS levels above a critical threshold might be one way boosts a high glycolytic rate. Thus, IMM and OMM channels are to induce cancer cell apoptosis, as detailed below. expected to affect mitochondrial metabolism and RC function Furthermore, altered ion fluxes might lead, via hyperpolariza- through multiple mechanisms. Therefore, their modulation may tion, to an increased driving force for Ca2 þ entry into the matrix, lead to cancer cell metabolism alteration, ultimately promoting whereas cation (K þ or Mg2 þ ) channel opening-induced depolar- cell death, as discussed below. In summary, OMM and IMM ization decreases Ca2 þ uptake. Mitochondria have the capacity to channel modulation may affect cancer cell fate through different accumulate Ca2 þ , using the mitochondrial calcium uniporter as a signaling mechanisms. highly selective ion channel,29 to a much higher concentration than found in the cytoplasm. Thus, mitochondria shape cytosolic calcium signals.30 The mitochondrial matrix possesses a Ca2 þ THE IN VITRO AND IN VIVO TARGETING OF MITOCHONDRIAL buffering system and calcium exit pathways (the Na þ /Ca2 þ ION CHANNELS IN CANCER exchanger and transient opening of the permeability transition The mitochondrial channels characterized over the past two pore) that normally prevents excessive [Ca2 þ ] buildup (see, for decades include the outer membrane-localized VDAC and, in the 31 example, Drago et al. ). However, after apoptotic stimuli, IMM, potassium channels such as mtKATP, mtBKCa, mtIKCa, mtKv1.3, sustained Ca2 þ release from the endoplasmic reticulum (‘ER mtTASK-3, the nonselective permeability transition pore MPTP, stress’) may lead to high mitochondrial matrix Ca2 þ levels that chloride channels, the magnesium-permeable Mrs-2, the calcium causes prolonged opening of the permeability transition pore uniporter, uncoupling proteins and proteins that have been 2 þ and subsequent loss of mitochondrial Dm~H. Thus, Ca overload shown to function as ion channels in other membranes but not

Oncogene (2014) 5569 – 5581 & 2014 Macmillan Publishers Limited Mitochondrial ion channels L Leanza et al 5571 yet directly observed in mitochondrial membranes.48–51 Here, we ATP transport out of mitochondria, leads to a cancer cell metabolic discuss the in vitro and in vivo evidence for ion channels involved advantage (termed the Warburg effect), and it antagonizes cell in cancer cell death obtained through either pharmacological or death through the inhibition of Bax-induced cytochrome c genetic means (Figure 1). Data from animal models clearly release62,63 and/or inhibition of the mitochondrial permeability demonstrate the importance of mitochondrial channels and their transition (MPT).64 HK dissociation from VDAC seems to favor cell potential as therapeutic cancer targets. The possible roles of individual channels in cell death in relation to the processes discussed above are summarized in Figure 2 and are described in greater detail below. Pharmacological targeting of these channels CELL DEATH (summarized in Table 1) and possible pitfalls are also critically discussed.

The outer membrane-localized VDAC The major protein of the OMM, VDAC or porin, is required for Cyt c Pro-death MPTP cancer cell survival and participates in apoptotic signaling. A large detachment/release body of literature examines the role of VDAC in the regulation of Kinases apoptosis.48,52 VDAC is being studied as a cancer-specific target because tumor cells have increased glycolysis and VDAC 53 Glycolytic expression. The role of VDAC1 and the other isoforms, VDAC2 metabolism 54–56 Mito & cytosolic and VDAC3, in cell death is complex, but importantly, in vivo ROS Ca2+ homeostasis evidence shows that in cancer cells, the association of HK with VDAC1 protects against mitochondrial-mediated apoptosis. HK Therefore, disruption of the HK-VDAC1 complex represents an Δψ attractive therapeutic cancer target. Overexpression of m ? hexokinase-1 and 2 (HK2) and their association with VDAC are ? important characteristics of glycolytic cancers.57 The expression of VDACs has also been found to be elevated in cancerous cells compared with normal cells and may be altered with chemotherapy. High VDAC levels are an unfavorable prognostic UCP2 Kv1.3 IKCa MCU VDAC factor;53 moreover, VDAC downregulation by RNA interference reduces cancer growth.58 This evidence may seem at odds with 59–61 Figure 2. Mitochondrial ion channels and their main proposed roles the finding that VDAC overexpression induces apoptosis, but in cell death regulation in cancer cells. Ion channels involved in the it illustrates how the functional meaning of a biological parameter regulation of mitochondrial membrane potential, ROS production, depends on context. VDAC upregulation in cancer goes hand in calcium homeostasis and metabolism are indicated. Mitochondrial hand with HK2 upregulation and can be considered a component channels with well documented role in cell death in cancer cells are of glycolytic upregulation. HK2 binding to VDAC, which allows for shown. See text for details and other possible mechanisms.

+ K Kv1.3 K+

K+ IKCa K+

VDAC c y to + + s K TASK-3 K o l MAC

solutes, solutes, ions MPTP ions IM S

Ca2+ MCU Ca2+

H+? UCPs H+?

matrix IMS

Na+ ASIC1a Na+? matrix IMS

Figure 1. Outer and inner mitochondrial membrane ion channels with documented roles in cell death regulation. For details on MAC, we suggest consulting the recent review by the Kinnally group.4 With regard to other channels, detailed descriptions are provided in the text. For the mitochondrial UCP2, and ASIC1a channels, the nature of the ions transported under physiological conditions remains unknown (question marks). Except for ASIC1a and SK2, the involvement of all other channels in cancer cell death has been documented either by pharmacological or genetic means.

& 2014 Macmillan Publishers Limited Oncogene (2014) 5569 – 5581 Mitochondrial ion channels L Leanza et al 5572 Table 1. Pharmacological modulation of mitochondrial ion channels and use in cancer cells

Channel Cancer cell studies Agents Development stage Observations

VDAC Adenocarcinoma88 Furanonaphthoquinone Preclinical development FNQ acts on VDAC and causes Non-small-cell lung (FNQs)88 alteration of mitochondrial cancer53 membrane potential, NADH- Glioblastoma57 dependent ROS production and HELA cervical cancer cells58 cytochrome c release (in ref. 88) Melanoma65 Prostate cancer cell line65 Breast cancer cell line65 Lymphoblastic leukemia65 Multiple myeloma66 El-4 lymphoma65 Clotrimazole74 Clinical trial for Sickle cell disease Commonly used as fungicide74 (SCD) (see ref. 52 in ref. 74) Inhibits KCa3.1 calcium-dependent potassium channel73 Erastin86 In vitro studies86 RAS/RAF/MEK-dependent action86 PRLX 93936, erastin homolog in Oxidative cell death (not apoptosis) clinical trial for multiple myeloma was induced via VDAC2 or VDAC3 (in (NCT01695590) ref. 88) Methyl jasmonates65 Preclinical development65,66 Plant hormones induce MOMP in Topical application in cancerous isolated mitochondria. skin lesions in humans67 Disruption of the HK–VDAC interaction;189 activation of BAX and Caspase-3 via ROS production66,68 Indirect activation of MPTP 3-Bromopyruvate77 Clinical trial in hepatocellular A pyruvate mimetic carcinoma79 Acts as an inhibitor of glycolysis78 but also as a covalently alkylating agent Pyruvylates certain proteins at cysteine residues79 MPTP Melanoma117 CSA133 Clinical use for different diseases CSA inhibits MPTP98,99 Prostate Breast cancer Lymphoblastic leukemia112 Chronic lymphocytic leukemia113 Acute promyelocytic leukemia105 Multiple myeloma112,113 4-(N-(S- Development for Novel anti-neovascular agent that glutathionylacetyl)amino) phase I clinical trial105 interacts with redox active, phenylarsenoxide mitochondrial protein dithiols in (GSAO)105,106 endothelial cells105 Mastoparan-like Preclinical development107 (in Cell penetrant, mitochondriotoxic sequences107–109 clinical trial but not for cancer) and apoptogenic107 Betulinic acid2,115,190 Phase I/II clinical trials Steroid-like structure. NCT00346502 Induces MOMP in isolated mitochondria2,189,190 Gold complex AUL12111 Preclinical development Inhibits complex I Increases ROS levels leading to activation of GSK3a/b, favoring MPTP opening111 CD437115 Preclinical development Synthetic retinoid (triterpenoid) able Retinoid analog NRX195183 is in to promote MOMP independently of clinical trial (NCT00675870) nuclear receptors189 Berberine117,118 In vitro studies117,118 Induces mitochondrial fragmentation and depolarization, oxidative stress and decreased ATP levels128 Honokiol119,120 In vitro studies on different cancer Induces MPTP-dependent cell cell lines and in clinical trial for death119,120 microbial infection, anxiety, oxidative stress and platelet aggregation119 a-Bisabolol121 In vitro studies on glioma cell Induces intrinsic apoptotic pathway lines;121 through the loss of mitochondrial Preclinical, ex–in vivo studies on inner transmembrane potential and human acute leukemia191 release of cytochrome c121 Shikonin122 In vitro studies122 Induces necroptosis122 Clinical trial on human lung cancer192

Oncogene (2014) 5569 – 5581 & 2014 Macmillan Publishers Limited Mitochondrial ion channels L Leanza et al 5573 Table 1. (Continued )

Channel Cancer cell studies Agents Development stage Observations

Kv1.3 T-cell leukemia cell line133 Psora-4133,134 Preclinical development. Blocks inward K þ flux inducing ROS, Chronic B lymphocytic Methoxypsoralen is in clinical trial mitochondrial depolarization, leukemia (CLL)134 for cutaneous T-cell lymphoma cytochrome c release and cell Osteosarcoma cell line NCT00056056 death133 Melanoma133 Colon rectum cancer193 Breast cancer193 Prostate cancer193 PAP-1133,134 Preclinical development Blocks inward K þ flux inducing ROS, MT depolarization, cyt c release and cell death133 Clofazimine133 Preclinical development, ex–in vivo Induces mtROS production and studies on chronic lymphocytic triggers cell death133 leukemia134 Used clinically to treat leprosy and autoimmune diseases137 73 73 IKCa Colon cancer cells Clotrimazole Clinical trial for Sickle cell disease Systemic application is not possible Melanoma74 (see ref. 52 in ref. 78) because of hepatotoxicity induced by Breast cancer193 nonspecific effects on cytochrome Prostate cancer193 P45074 Pancreas cancer193 TRAM-3473,74,77,78,194 Clotrimazole analog, lacking cytochrome P450 inhibitory effects, able to enhance TRAIL-induced apoptosis via the mitochondrial pathway74 UCP2 Breast cancer168 Genipin168 In vitro and in vivo studies168 Abolishes UCP2-mediated proton leukemia, ovarian, bladder, leak168 esophagus, testicular, colorectal, kidney, pancreatic, lung and prostate tumors162,163 TASK-3 Ovarian cancer158 Zinc and Nonspecific inhibitors of methanandamide plasmamembrane TASK-3 Abbreviations: ATP, adenosine triphosphate; CSA, cyclosporin A; GSK3a/b, glycogen synthase kinase-3a/b; MOMP, mitochondrial outer membrane permeabilization; MPTP, mitochondrial permeability transition pore; ROS, reactive oxygen species; TASK-3, TWIK-related acid-sensitive K þ channel-3; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; UCP2, uncoupling protein-2; VDAC, voltage-dependent anion channel. This table summarizes the pharmacological tools used to elucidate the role of the indicated ion channels in several cancer cell types, with either in vitro or in vivo studies. SK2, MCU and ASIC1a are not mentioned in this table because pharmacological evidence for their involvement in cancer cell death regulation is not available. See text for further details. death through the disruption of aerobic glycolysis and the cell’s chemoresistant cancer cells, remains unknown. Clotrimazole could energy balance by altering the interaction of Bcl-2 family proteins have multiple effects because it also acts as a membrane- with mitochondria and by regulation of ROS production and permeable inhibitor of the KCa3.1 calcium-dependent potassium facilitation of VDAC oligomer formation.48,55 A class of novel channel that has also been localized to the IMM73 and seems to be apoptosis-inducing anticancer drugs, which currently comprises involved in apoptosis regulation.74 In addition, peptides small molecules such as clotrimazole, bifonazole (both fungicides, corresponding to the amino-terminus of both HK-I75 and HK-II62 in the tens-of-micromolar range) and methyl jasmonate (MJ), act and a cell-permeable HK-II-based peptide76 may be potentially by disrupting the HK-VDAC1 complex (Table 1). useful as pharmacological tools. Among these small molecules, MJ has been demonstrated to be The HK-VDAC complex (more specifically, HK) can also be active in preclinical models of melanoma, prostate and breast targeted by the alkylating agent 3-bromopyruvate (3BP) that cancer, lymphoblastic leukemia and multiple myeloma,65,66 also has been reported efficacious in in vitro and in vivo studies (for independently of p53 status and has been tested in at least one example, see reviews in refs 77–80) and possesses strong pilot human study.67 However, the major obstacle for adopting potential as an anticancer agent in humans.79,81 In animal jasmonates as anticancer agents is their relatively high dosage models, 3BP showed high efficacy against advanced-stage needed to exert their action. It should be also noted that different malignant tumors by inhibiting both glycolysis and mechanisms of action from the mechanism described above have mitochondrial energy generation, possibly by interfering with also been envisioned. MJ activates Caspase-3 through ROS the HK-VDAC1 complex.80 Thus, 3BP has been proposed to target production,68 and another study showed that it also metabolism and block energy supplies. In addition, GAPDH,82 downregulates the expression of proliferating cell nuclear components of the RC,83 ER and lysosomes are targets of this antigen.69 Furthermore, MJ can bind to members of the Aldo- compound. Its remarkable specificity for cancer cells may be keto reductase family 1,70 which are potential tumor biomarkers partially attributed to the presence of glycolytic pathway (HK and involved in steroid metabolism.71,72 The fact that MJ does not GAPDH) members among these targets; however, most of 3BP affect nontransformed, intact cells is compatible with most specificity likely stems from facilitated entry into glycolytic cancer suggested mechanisms. Whether MJ can also induce cell death cells via the lactate transporter.84 The transcription factor ATF2, in the absence of the proapoptotic Bax/Bak, which occurs in which elicits oncogenic and tumor-suppressor activities in

& 2014 Macmillan Publishers Limited Oncogene (2014) 5569 – 5581 Mitochondrial ion channels L Leanza et al 5574 melanoma and nonmalignant skin cancer, respectively, has been along with MPT. Some of the MPTP-targeting molecules, such as recently identified as a potent disruptor of the HK1–VDAC1 4-(N-(S-glutathionylacetyl) amino) phenylarsenoxide, are currently complex. When localized to mitochondria in a protein kinase C-E- being evaluated in clinical trials as promising drugs against dependent manner, ATF2 increases mitochondrial permeability refractory tumors.105,106 Mitochondria-penetrating peptides, such and promotes apoptosis.85 as mastoparan-like sequences, peptides of the innate immune The heterocycle erastin has been found to selectively induce system and molecules developed by the Kelley group,107–109 also the death of cells with mutations in the oncogenes HRas, KRas induce MPT and could become candidates for future clinical trials. and BRaf in a VDAC2/3-dependent manner. Apparently, this MPTP can also be indirectly activated (or inhibited) through compound induces oxidative stress involving VDAC through modulatory signaling pathways. For example, in cancer models an unknown mechanism.86 An erastin homolog, PRLX 93936, containing constitutively active extracellular signal-regulated is currently being evaluated in a clinical trial in multiple kinase, this kinase acts through the glycogen synthase kinase- myeloma patients (http://www.cancer.gov/clinicaltrials). Finally, the 3b/Cyclophilin D axis to repress cell death by MPT inducers such anticancer agent furanonaphthoquinone, isolated from Avicennia as arachidonic acid or BH3-mimetic EM20-25.110 Induction of plants, induces caspase-dependent apoptosis via ROS production oxidative stress by the gold complex AUL12 can lead to activation by acting on VDAC. VDAC has also been proposed to mediate exit of glycogen synthase kinase-3a/b that favors MPTP opening.111 A of superoxide anions from the intermembrane space, and its large portion of MPTP-opening inducers (direct or indirect) for closure caused internal oxidative stress and sensitized mito- which in vivo antitumor activities have been reported2 are natural chondria to Ca2 þ /ROS-induced MPT.87 Moreover, the anticancer compounds, including jasmonates,112–114 betulinic acid, the activity of furanonaphthoquinone and ROS production were synthetic retinoid CD437,115,116 berberine,117,118 honokiol,119,120 enhanced by VDAC1 overexpression.88 a-bisabolol121 and shikonin122 (Table 1). Among these com- VDAC has also been proposed to directly participate in OMM pounds, betulinic acid is currently in a phase I /II clinical trial for permeabilization and mediate cytochrome c release, either by dysplastic nevi (http://www.cancer.gov/clinicaltrials). A retinoid oligomer formation48 or by the formation of a large pore analog, NRX 195183, is also in a phase II trial for acute comprising VDAC and Bax/Bak (Tsujimoto et al.89 but see promyelocytic leukemia (http://www.cancer.gov/clinicaltrials). Martinez-Caballero et al.90). Knockdown of VDAC1 prevented A specific, powerful MPTP inhibitor is also available: cyclosporin cisplatin-induced conformational activation of Bax91 or selenite- A, a cyclic endecapeptide.123–125 Cyclosporin A inhibits the MPTP induced cytochrome c release.92 However, Bax-induced through binding to matrix cyclophilin, a peptidyl-prolyl cis–trans cytochrome c release from mitochondria isolated from wild-type isomerase, and acts as an immunosuppressant by inhibiting or VDAC1-, VDAC3- and VDAC1/VDAC3-null cells was reported to calcineurin. Further evidence for the link between MPTP and be identical.93 In any case, the binding of anti-apoptotic Bcl-2 and cancer is illustrated by the observation that patients treated with Bcl-xL to VDAC1 (with resulting porin activity inhibition)94 cyclosporin A to prevent rejection of organ transplants have a produced anti-apoptotic actions.95 A recent study reported that high incidence of cancer.126 constructs consisting of cell-penetrating Antp fused to VDAC1- derived sequences, which prevented the interaction of VDAC1 and HK, Bcl-2 and Bcl-xL, were effective in the selective eradication of B The IMM potassium channel Kv1.3 cells from patients with chronic lymphocytic leukemia.96 As mentioned above, IMM-localized potassium channels are In summary, a systematic search for compounds that act on predicted to participate in the regulation of mitochondrial VDAC to antagonize cancer remains to be performed, but those membrane potential, volume and ROS production. Our research compounds already identified are promising. groups have identified a potassium channel, mtKv1.3, in the IMM of several cell types and have shown with mitochondria from cells expressing or lacking this channel that mtKv1.3 activity indeed has 127,128 Inner membrane-localized permeability transition pore an impact on DCm and on ROS production. Similar to some A number of cytotoxic agents and cellular stressors trigger the other IMM channels,50 mtKv1.3 is the mitochondrial counterpart of loss of IMM permeability. This process is most often caused by the plasma membrane-localized Shaker family potassium channel, MPT that is considered to be a final common pathway of various Kv1.3. Its crucial role in apoptosis became evident because the forms of cell death.97,98 In fact, MPT results in a ‘bioenergetic expression of a mitochondria-targeted Kv1.3 construct was catastrophe’; the transmembrane electrochemical proton gradient sufficient to induce cell death upon apoptotic stimuli in CTLL-2 dissipates, ATP synthesis ceases and respiratory substrates are lost T lymphocytes that lack Kv channels and are otherwise resistant to from the mitochondrial matrix. MPT is caused by the opening of a apoptosis. A physical interaction between Bax and mtKv1.3 has large, Ca2 þ -activated and oxidative stress-sensitive pore (MPTP) been demonstrated in apoptotic cells, and Bax has been shown to 129,130 that creates an IMM permeable to ions and solutes up to 1500 Da inhibit channel activity at nM concentrations. Incubation of molecular weight and leads to matrix swelling. This pore coincides Kv1.3-positive isolated mitochondria with Bax or specific mtKv1.3 with the mitochondrial megachannel that has been studied by inhibitors triggered apoptotic events, including membrane patch clamp and characterized by conductances up to 1.5 nS.99 potential changes (hyperpolarization followed by depolarization MPTP has recently been proposed to be formed by dimers of the because of MPTP opening), ROS production and cytochrome c FoF1 ATP synthase and Cyclophilin D.98,100 MPTP/mitochondrial release; however, Kv1.3-deficient mitochondria were resistant to megachannel opening is considered to account for a substantial these inhibitors. The highly conserved Bax lysine residue 128 was portion of the tissue damage caused by ischemia/reperfusion and shown to protrude into the intermembrane space.131 This residue oxidative stress. is responsible for the inhibitory effect of Bax on Kv1.3 activity In cancer cells, signaling pathways are activated that render because it mimics a crucial lysine residue in Kv1.3-blocking mitochondria more resistant to MPT induction.101–103 Because peptide toxins. Indeed, mutant BaxK128E did not exert its effects chemotherapeutic agents cause oxidative stress, they may on Kv1.3 and mitochondria, and it did not mediate apoptosis in activate signals that induce cell death through MPTP opening.104 Bax/Bak double-knockout mouse embryonic fibroblasts, indicating MPT inducers have potential oncological applications, but that the toxin-like action of Bax on Kv1.3 triggers the above potential side effects involving the nervous system or cardiac described mitochondrial events.130 tissues can be expected. A consistent number of compounds Three membrane-permeable inhibitors of Kv1.3, Psora-4, PAP-1 (often used at relatively high concentrations) have been shown to and clofazimine, are able to induce cell death at mM concentra- induce oxidative stress and/or disruption of Ca2 þ homeostasis tions by directly targeting mtKv1.3 in different cancer cell lines, in

Oncogene (2014) 5569 – 5581 & 2014 Macmillan Publishers Limited Mitochondrial ion channels L Leanza et al 5575

ROS

apoptotic Bax cascade

Psora PAP Clofazimine

ax

B

Bax Psora PAP Clofazimine Bax  m OMM MPTP Kv1.3 Cyt c K+ IMM ROS

Figure 3. Proposed mechanism of action of mitochondrial Kv1.3 during apoptosis. Normally mtKv1.3 acts as a conduit for the passive flow of K þ ions across the IMM, that is, as a component of the machinery controlling mitochondrial volume, ion homeostasis and transmembrane potential. In cells challenged with membrane-permeable Kv1.3 inhibitors able to reach the IMM-localized Kv1.3, a transient hyperpolarization and enhanced ROS production occurs. These ROS can stimulate Bax recruitment and/or induce mitochondrial permeability transition as well as promote cytochrome c detachment from the outer IMM surface. Consequently, a loss of cytochrome c occurs (large, red) via Bax oligomers or OMM lacerations that are caused by swelling of the matrix compartment following MPTP induction. The latter event also results in the collapse of the potential and cytoplasmic dispersion of small molecules, such as NADH and Krebs cycle intermediates, hampering respiration. Pharmacological inhibition of mtKv1.3 is sufficient to induce Bax–Bak-independent cell death in cancerous cells that are already subjected to a higher than normal redox stress.

73,147 contrast to the membrane-impermeable high-affinity Kv1.3 membranes of human cancer cells. .IKCa can be selectively inhibitors ShK and margatoxin.132,133 Genetic deficiency or small inhibited by low concentrations of clotrimazole and TRAM-34. This interfering RNA-mediated downregulation of Kv1.3 abrogated the latter drug has been reported to synergistically increase the effects of the membrane-permeable drugs that also killed cells in sensitivity of melanoma cells to the death receptor ligand TRAIL the absence of Bax and Bak, in agreement with the above (tumor necrosis factor-related apoptosis-inducing ligand) via its 74 described model (Figure 3). In vivo, intraperitoneal injection of action on mtIKCa. However, the effects of TRAM-34 on clofazimine in an orthotopic melanoma B16F10 mouse model mitochondrial bioenergetics have not yet been investigated in reduced tumor size by 90%, without causing obvious side detail, but TRAM-34 has been reported to induce hyper- 133 effects. Recently, promising results were obtained with polarization of the mitochondrial membrane (which is expected primary human cancer cells from patients with chronic if an influx of positively charged ions/molecules is inhibited), lymphocytic leukemia, where drugs significantly and exclusively confirming that functional IKCa is expressed in the IMM. decreased the survival of pathologic B cells (which express higher Interestingly, TRAM-34 application induced Bax translocation to levels of Kv1.3 compared with B cells from normal subjects) by 134 the mitochondria, representing an early step of apoptosis. Both inducing the crossing of a critical ROS threshold. Thus, the TRAIL and TRAM-34 are characterized by a relatively good safety selective apoptosis-inducing action of these drugs on tumor cells profile, suggesting that co-administration of these two drugs seems to be related to a synergistic effect of an altered cancer cell 134,135 might be exploited to treat melanoma. redox state and higher Kv1.3 expression. Interestingly, In addition to the intermediate conductance channels, the As2O3, a clinically active antileukemic agent, has been reported presence of small-conductance calcium-activated potassium (SK2/ to inhibit mitochondrial respiratory function, increase ROS 148,149 . À KCa2.2) channels in the IMM has been recently reported. Its generation and enhance the activity of other O2 -producing 136 pharmacological activation in a neuronal cell line exposed to toxic agents against cultured and primary patient leukemia cells. glutamate levels attenuated the loss of the mitochondrial Because clofazimine is already used clinically to treat some 137 transmembrane potential, blocked mitochondrial fission, autoimmune diseases and leprosy and shows an excellent prevented the release of proapoptotic mitochondrial proteins safety profile, targeting mtKv1.3 is a feasible cancer therapy for at and reduced cell death.149 SK2 channel opening prevented least some cancer types. Interestingly, altered Kv1.3 expression is 138 mitochondrial calcium overload and mitochondrial superoxide observed in several different cancer cell lines and tumors, and a formation. It will be interesting to investigate whether the correlation between Kv1.3 expression and cell sensitivity to 139 application of mtSK2 inhibitors alone or in combination with clofazimine has been reported. In addition, the ability of other chemotherapeutics could promote cell death in cancer cells these drugs to induce cell death in the absence of Bax and Bak, 140–142 expressing SK2. similar to other agents, could be useful because To our knowledge, whether the mitochondrial large-conduc- downregulation of these pro-apoptotic proteins represents a 2 þ þ 150 143–146 tance Ca -activated K channel (BKCa/KCa1.1) has a role in common tumor cell resistance mechanism. cancer has not been investigated. However, the channel opener CGS7184 has been reported to induce the glioma cell death that Calcium-dependent potassium channels was accompanied by increased respiration and mitochondrial The activity of an intermediate conductance potassium channel depolarization,151 perhaps downstream of an increased Ca2 þ 152 (IKCa, KCa3.1) has been recorded from the inner mitochondrial release from the ER.

& 2014 Macmillan Publishers Limited Oncogene (2014) 5569 – 5581 Mitochondrial ion channels L Leanza et al 5576 TWIK-related acid-sensitive K þ channel-3 (TASK-3) empty-vector control cells.169 The authors interpreted their results A protein recognized by an antibody against TASK-3 (TWIK-related as a consequence of UCP-induced increased autophagy and acid-sensitive K þ channel-3; KCNK9), a two-pore potassium decreased mitochondrial function. This unexpected observation channel, was identified in the mitochondria of melanoma, warrants further investigation. keratinocytes153 and healthy intestinal epithelial cells.154 The reduction of TASK-3 expression in WM35 melanoma cells Mitochondrial calcium uniporter MCU 155 compromised mitochondrial function and cell survival. TASK-3 The mitochondrial Ca2 þ ‘uniporter’ (MCU), a calcium-selective ion knockdown also decreased human keratinocyte HaCaT cell 29 156 channel, is responsible for low-affinity calcium uptake into the viability following ultraviolet B irradiation. In mitochondria mitochondrial matrix. The recent identification of the protein isolated from HaCaT cells, TASK-3-compatible activity has been 170,171 156 responsible for this activity has produced new perspectives recorded for the first time by electrophysiology (patch clamp). pertaining to the control of Ca2 þ signaling in the cell. In fact, MCU Two aspects of TASK-3 still require investigation: first, whether the may be a very useful tool to influence a number of cellular observed migration and invasion-reducing effects of TASK-3 51 157 calcium-dependent processes, including cell death. For example, overexpression in breast cancer cells and increased apoptosis subthreshold apoptotic stimulation in synergy with cytosolic Ca2 þ induced by TASK-3 blockers (zinc and methanandamide) in waves induced MPTP opening172 and, moreover, MCU ovarian carcinoma158 are correlated with the mitochondrial overexpression resulted in increased apoptosis upon with H2O2 channel expression and, second, whether pharmacological and C2-ceramide challenges.170 In addition, recent data suggest action on mtTASK-3 can affect cancer cell death. However, that in the absence of MICU1, a proposed negative regulator of because no highly specific modulators are available, this task MCU, mitochondria become constitutively loaded with Ca2 þ , remains difficult. resulting in excessive ROS generation and sensitivity to apoptotic stress.173 To avoid calcium overload, MCU activity is also regulated 174 Uncoupling protein (UCP) by a dominant-negative subunit, MCUb. As for a possible relationship between MCU and cancer, the MCU-targeting Uncoupling proteins (UCP1–5) belong to the superfamily of microRNA miR-25 has been shown to affect Ca2 þ homeostasis mitochondrial anion-carrier proteins, and UCP1 has recently been in colon cancer cells through the specific downregulation of MCU. shown to mediate fatty acid-regulated proton transport.159 The The miR-25 caused a strong decrease in mitochondrial Ca2 þ widely expressed UCP-2 has also been proposed to transport uptake and, importantly, conferred resistance to Ca2 þ -dependent protons,160 (for review, see Cannon and Nedergaard161), although apoptotic challenges.175 Thus, MCU could be an important protein its physiological importance and the occurrence of UCP2- for tumorigenesis, and its specific pharmacological activators, if mediated proton leak are not entirely clear and remain identified, might become useful tools. However, the situation is debatable. UCP2 has been suggested to regulate cell survival by likely more complex. MCU overexpression has been observed in decreasing mitochondrial ROS because it might limit the maximal breast cancer samples and has been suggested to provide a value of the proton gradient across the IMM, and a depolarizing survival advantage against some cell death pathways.176 proton leak would be expected to diminish superoxide Pharmacological or small interfering RNA-mediated inhibition of production162,163 (see, for example, Cannon et al.164 for a mitochondrial Ca2 þ entry sensitized cancer cells to positively discussion on this topic). Increased ROS production was charged gold nanoparticles through the induction of endoplasmic observed in UCP2 knockout mice, whereas UCP2 overexpression reticulum stress.177 Further complications arose from recent may contribute to a higher apoptotic threshold promoting cancer work reporting that epidermal growth-induced epithelial– cell survival because it prevents oxidative injury. Indeed, UCP2 mesenchymal transition in breast cancer cells was not associated overexpression, documented in numerous tumor types, including with changes in MCU expression, as assessed by quantitative leukemia, ovarian, bladder, esophageal, testicular, colorectal, reverse transcriptase–PCR.178 Finally, in sharp contrast with the kidney, pancreatic, lung and prostate tumors, has been shown extensive literature suggesting that alterations in mitochondrial to protect cells from oxidative stress165 and even to abolish the calcium play a central role in acute metabolic regulation and apoptosis-inducing effects of chemotherapeutic drugs.166 determination of a threshold for cell death, the basal metabolism Interestingly, UCP2 overexpression has also been proposed to of knockout animals was not markedly altered in the absence of directly contribute to the Warburg phenotype167 and to the MCU.179 Furthermore, although experiments with MCU À / À development of tumors in an orthotopic in vivo model of breast mitochondria confirmed that MCU is required for calcium- cancer.168 In support of this hypothesis, UCP2 expression in MCF7 induced MPTP opening, MCU À / À mice exhibited no evidence of breast cancer cells was shown to lead to a decreased protection from ischemia–reperfusion-mediated injury. In this mitochondrial membrane potential and increased tumorigenic study, tumor incidence in knockout mice was not assessed. The properties. These studies suggest that UCP2 overexpression is forthcoming elucidation of MCU regulation,180–182 as well as the involved in the development of a variety of cancers and that UCP2 creation of a conditional knockout mouse, might provide can be considered as a promising oncological target, although the explanation for these apparently contrasting findings. actual ions transported by UCP2 under physiological conditions are still uncertain. As recently suggested,162 UCP2 may act as an uniporter for pyruvate, presumably promoting pyruvate efflux Acid-sensing ion channel 1a from the matrix and thus restricting glucose availability for In a recent study, the sodium-permeable acid-sensing ion channel mitochondrial respiration162 and promoting mitochondrial fatty 1a (ASIC1a) was documented in mitochondria, although the acid oxidation. Efflux of pyruvate would aid highly glycolytic cells, transport properties of this mitochondrial protein were not in which large amounts of pyruvate would otherwise put defined.183 Interestingly, neurons from ASIC1a À / À mice were enormous redox pressure on the mitochondria. Nevertheless, a resistant to cytochrome c release and inner mitochondrial plant-derived small molecule, genipin, has been identified as an membrane depolarization, and mitochondria from these cells agent capable of suppressing the tumor-promoting functions of displayed an enhanced Ca2 þ retention capacity and oxidative UCP2 presumably by abolishing UCP2-mediated proton leak.168 In stress response. The authors proposed that mitochondrial ASIC1a sharp contrast with the above studies, when highly metastatic may serve as a regulator of MPTP through a still ill-defined MDA-MB-231 breast cancer cells recombinantly overexpressing mechanism, thus contributing to oxidative neuronal cell death. UCP1, UCP2 or UCP3 were injected into the flanks of athymic nude Interestingly, lack of ASIC1 prevented axonal degeneration in mice, in vivo tumor growth was reduced 2.2-fold compared with experimental autoimmune encephalomyelitis; however, MPTP

Oncogene (2014) 5569 – 5581 & 2014 Macmillan Publishers Limited Mitochondrial ion channels L Leanza et al 5577 function was not assessed.184 Unfortunately, no information is publications that have been published on this topic. The work carried out in the currently available on mtASIC1a expression in cancer tissues. authors’ laboratories was supported in part by grants from the Italian Association for Cancer Research (AIRC; Grant 11814 to IS), the EMBO Young Investigator Program grant (to IS), the Progetti di Rilevante Interesse Nazionale (PRIN) program THERAPEUTIC PERSPECTIVES (2010CSJX4F to IS and 20107Z8XBW_004 to MZ), the Fondazione Cassa di Risparmio di Padova e Rovigo (to MZ), the CNR Project of Special Interest on Aging (to MZ), the The concept that mitochondria can serve as promising pharma- DFG Grant Gu 335/13–3 (to EG), the International Association for Cancer Research (to cological targets in oncology is gaining increasing support. One EG) and the Progetto Giovani Studiosi 2012 (to LL). intervention strategy envisions direct targeting of mitochondrial channels to induce the death of unwanted, cancerous cells. Although much work remains in this relatively new field, studies REFERENCES involving VDAC, inner membrane K þ and Ca2 þ channels and the permeability transition pore justify high expectations. To produce 1 Galluzzi L, Kepp O, Kroemer G. Mitochondria: master regulators of danger advances in this field, further progress should focus on under- signalling. Nat Rev Mol Cell Biol 2012; 13: 780–788. 2 Fulda S, Galluzzi L, Kroemer G. Targeting mitochondria for cancer therapy. standing the functions of these and other channels in normal cells, Nat Rev Drug Discov 2010; 9: 447–464. mechanisms involved in cell death induction upon their manip- 3 Bender T, Martinou JC. 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