Mitochondrial Dysfunction in Gliomas Christos D. Katsetos, MD, PhD, FRCPath,*,†,‡,§ Helen Anni, PhD,¶ and Pavel Dráber, PhD# Mitochondrial (mt) dysfunction in gliomas has been linked to abnormalities of mt energy metabolism, marked by a metabolic shift from oxidative phosphorylation to glycolysis (“Warburg effect”), disturbances in mt membrane potential regulation and apoptotic signaling, as well as to somatic mutations involving the Krebs cycle enzyme isocitrate dehydrogenase. Evolving biological concepts with potential therapeutic implications include interaction between microtubule proteins and mitochondria (mt) in the control of closure of voltagedependent anion channels and in the regulation of mt dynamics and the mt-endoplasmic reticulum network. The cytoskeletal protein βIII-tubulin, which is overexpressed in malignant gliomas, has emerged as a prosurvival factor associated in part with mt and also as a marker of chemoresistance. Mt-targeted therapeutic strategies that are discussed include the following: (1) metabolic modulation with emphasis on dichloroacetate, a pyruvate dehydrogenase kinase inhibitor; (2) tumor cell death via apoptosis induced by tricyclic antidepressants, microtubulemodulating drugs, and small molecules or compounds capable of inflicting reactive oxygen species–dependent tumor cell death; and (3) pretreatment mt priming and mt-targeted prodrug cancer therapy. Semin Pediatr Neurol 20:216-227 C 2013 Elsevier Inc. All rights reserved.

Introduction

G

liomas account for most central nervous system tumors in children. In pediatric patients, these are broadly classified as low-grade (World Health Organization [WHO] grades I and II) and high-grade gliomas (WHO grades III and IV). Pilocytic astrocytomas (WHO grade I) are focal and relatively circumscribed, and depending on their anatomical

Supported in part by a grant from the Philadelphia Health Education Corporation and St. Christopher’s Hospital for Children Reunified Endowment (CDK), funds from the St. Christopher’s Foundation for Children (CDK), a grant from Sbarro Health Research Organization (HA), Grant LH12050 from the Ministry of Education, Youth and Sports of the Czech Republic (PD), and by Institutional Research Support (RVO 68378050) *Department of Pediatrics, Drexel University College of Medicine, Philadelphia, PA. †Department of Pathology and Laboratory Medicine, Drexel University College of Medicine, Philadelphia, PA. ‡Department of Neurology, Drexel University College of Medicine, Philadelphia, PA. §Section of Neurology, St. Christopher’s Hospital for Children, Philadelphia, PA. ¶Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA. #Laboratory of the Biology of Cytoskeleton, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic. Address reprint requests to Christos D. Katsetos, MD, PhD, FRCPath, Section of Neurology, St. Christopher’s Hospital for Children, 3601 A Street, Philadelphia, PA 19134. E-mail: [email protected]

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1071-9091/13/$-see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.spen.2013.09.003

location, they are curable with gross total resection and no further therapy. Conversely, the diffuse gliomas (WHO grades II-IV) are noncircumscribed tumors exhibiting infiltrative patterns of growth and central nervous system spread. Diffuse pediatric gliomas exhibit distinct molecular characteristics as compared with their adult counterparts.1 High-grade gliomas (WHO grades III and IV) are less common in children compared with adults, but as a group, they are also refractory to currently available treatments and carry a poor prognosis. High-grade thalamic gliomas constitute a clinically distinct subgroup of deep-seated, highly invasive, and largely inoperable tumors.2 Diffuse intrinsic pontine glioma in children is a deadly form of brain cancer, with median life expectancy of less than 12 months, which is managed by focal palliative radiotherapy.3 There is strong consensus that there is a desperate and urgent need for new innovative treatment strategies for diffuse pediatric gliomas in general and thalamic and pontine gliomas in particular. Mitochondria (mt) play key roles in cellular energy metabolism, generation of free radicals, and apoptosis. Mitochondrial (mt) abnormalities have been recognized in cancer.4-7 In previous years, a number of review articles have offered insights into diverse aspects of mt biology and genetics and their significance in tumorigenesis, tumor progression, and therapeutic targeting.4,8,9 Mt dysfunction in gliomas involves abnormalities of energy metabolism, changes in mt membrane potential regulation, and disruption of apoptotic signaling pahways8,10 as well as on mutations in the citric acid cycle

Mitochondrial dysfunction in gliomas enzyme isocitrate dehydrogenase (IDH).11,12 The full extent of mt abnormalities in gliomas is a rapidly expanding area of investigation. This review is an appraisal of our current understanding of mt abnormalities in cancer in general and brain gliomas in particular, with emphasis on the relationship of these abnormalities with cellular systems and molecular pathways involved in tumorigenesis and therapeutic targeting.

Mitochondrial Dysfunction in Gliomas Mitochondrial Structural Abnormalities Heterogeneous morphologic abnormalities have been identified in glioma cells and tumor blood vessels at the ultrastructural level, including mt swelling with partial or total cristolysis and mt remodeling by the opposing processes of fusion (mixing) and fission (breaking down).13,14 These changes are attributed directly or indirectly to cellular or biochemical and molecular processes linked to hypoxiaresistant and hypoxia-sensitive tumorigenic phenotypes, effects of the hypoxia-inducible factor-1α (HIF-1α), increased expression of glycolytic protein isoforms, fatty acid synthase, and survivin.14 The ultrastructural changes, particularly the finding of cristolysis, are consistent with alterations of mt bioenergetics pointing to a critical compromise of oxidative phosphorylation (OXPHOS) in glioma cells.13,14 Another potentially important mt-linked mechanism, which may contribute to the invasive properties of glioma cells, is the relationship of activated mt with endoplasmic reticulum (ER) and also the presence of mt in tumor filopodia and invadopodia.15

Abnormal Energy Metabolism Cancer cells are capable of adapting to bioenergetic stress by engaging their mt pathway for their growth and survival requirements.9 Malignant neoplasms are characterized by major changes in energy metabolism, typified by a shift from dual oxidative and glycolytic metabolic pathways to a predominantly—if not solely—glycolytic metabolism referred to as the “Warburg effect”.8,10,11,16,17 Glioma cells use glycolysis, as opposed to OXPHOS, even in the presence of oxygen to generate adenosine triphosphate (ATP) to meet the energy needs for the synthesis of nucleotides, fatty acids, and amino acids.9,18 This decoupling of mt from the OXPHOS pathway (“Warburg effect”) is a core feature shared by many cancers.10,17-20 Unlike normal glial cells, neoplastic glial (glioma) cells cannot metabolize ketones and fatty acids for energy production after glucose deprivation.10 In malignant gliomas, cell populations with differential mt metabolism were found in different parts of the tumor, reflecting a metabolic heterogeneity or functional adaptation.21 Furthermore, attempted fatty acid metabolism by mt in glioma cells triggers the production of abnormally high levels of reactive oxygen species (ROS) leading to apoptotic cell death.10 Accordingly, glucose deprivation and high ketogenic diets may

217 provide the rationale for a therapeutic approach specifically targeting glioma tumor cells while sparing normal glia from bystander cellular damage.10 Citrate is an essential metabolite required in mt energy metabolism and macromolecular synthesis in the cytosol. In hypoxia, a major source of citrate is glutamine. Glutamine-derived α-ketoglutarate (α-KG) undergoes reductive carboxylation by the nicotinamide adenine dinucleotide phosphate (NADPH)-linked mt isoform of IDH, IDH2, to give rise to isocitrate, which is subsequently isomerized to citrate.22 The increased IDH2-dependent carboxylation of glutaminederived α-KG in hypoxia is associated with an increased synthesis of 2-hydroxyglutarate (2-HG) in cells with wildtype IDH1 and IDH2. Reductive glutamine carboxylation is associated with activation of HIF-1 in maintaining synthesis of citric acid and cell growth under hypoxic conditions. Preferential reductive metabolism of glutamine-derived α-KG owing to constitutive activation of HIF-1 is also encountered under normoxic conditions.22 The capacity for reductive carboxylation of α-KG and ability to produce acetyl-CoA are compromised in gliomas and other tumor types, which are characterized by IDH1 and IDH2 mutations, under conditions of hypoxia or impaired mt function or both.23 Mutant IDH tumors are associated with increased production of 2-HG.24,25 In renal cell carcinomas with mutations in the Krebs cycle enzyme fumarate hydratase, the major pathway of citrate formation is a reductive, glutamine-dependent pathway of carboxylation instead of oxidative metabolism.26

Regulatory Protein Adenine Nucleotide Translocase Isoform 2 (ANT2) One of the key regulatory proteins involved in mt bioenergetics of tumors is the ANT2.16 ANT is a mt protein that facilitates the exchange of adenosine diphosphate (ADP) and ATP across the mt inner membrane. The human ANT comprises 4 isoforms (ANT1-4), each with a specific, developmentally defined cell type expression and tissue distribution. ANT1 is specific to skeletal muscle and brain, whereas ANT2 is expressed predominantly in actively proliferating and poorly differentiated cells.16 ANT1 and ANT3 export the ATP produced by OXPHOS from the mt into the cytosol, whereas the ANT2 isoform is linked to glycolytic metabolism and is thus regarded as a cancer biomarker.16

Apoptotic Signaling—Mitochondrially Mediated Apoptosis Mt represent a focal point in cell death. Abnormalities of the intrinsic mt-dependent apoptotic pathway have been described in various cancer types including gliomas.27 In the context of glial tumors, cell death signaling is associated with generation of ROS and mt and other signaling pathways.28 Overexpression of the Bcl-2 protein family has been reported to be linked to radiotherapy and chemotherapy resistance in different cancer types.29,30 These proteins are closely related to mt membranes and their interaction mediates

218 mt release of anti-apoptotic factors, which requires cardiolipin, a phospholipid associated with mt membranes.10 Cardiolipin abnormalities in glioma cells are accompanied by metabolic derangements, with perturbation of the mt respiratory chain or electron transport chain (ETC) and disruption of the apoptotic pathway.10 Oxidative or other cellular stresses are accompanied by mt permeability transition pore (mPTP) opening in the inner membrane of mt. In the context of cancer, mPTP can be used as an executioner to specifically induce cell death.31 Although the molecular identity of the mPTP is unclear, cyclophilin D plays a key regulatory role in the pore opening.31 Aside from their role in DNA damage, toxicity, and cell death, ROS are also important signaling regulators acting in concert with redox-sensitive proteins to regulate cell homeostasis during stress. Beyond mt-mediated apoptosis, mitophagy, the selective autophagic elimination of mt has been shown to be dysregulated in development and cancer.32 Collectively, redox deregulation, autophagic signaling, and mitophagy are factors that can enhance cell survival.32

Mitochondria and Cytoskeleton Coupling Revealed by Proteomic Studies Proteomics is a high-throughput technique that analyzes proteins in different cell populations to identify their functional role and molecular biomarkers of diseases with translational potential in the bedside.33 This approach provides a plethora of data regarding protein identification, quantification, modifications, and subcellular localization and protein-protein interactions and networks. Posttranslational protein modifications (glycosylation, phosphorylation, acetylation, ubiquitination, adduction with metabolic products, etc.) are useful dynamic measures of cellular states.34 Cell lines, patient biopsies, and biological fluids (serum, plasma, cerebrospinal fluid, and interstitial tumor fluid) as well as cells or biofluids from animal disease models have been used as starting material for proteomics. The proteomics approach has provided tools for understanding and treating diseases, such as diagnostic and predictive disease biomarkers and protein signatures, as well as for assessment of therapeutic treatment decision and response, and identification of novel targets for diseases.35 In the past decade, proteomic studies of cell lysates have identified a number of proteins that have provided insights into glioma pathophysiology.36,37 In an early study,38 the proteomic pattern of different gliomas was compared among glioblastomas, anaplastic astrocytomas, astrocytomas, and normal brain tissues. The investigators identified and validated differences in expression of a panel of proteins that distinguished the normal brain from glioma tissue and high- vs lowgrade astrocytomas. Besides the known suspects in cancer biology of cell cycle–regulating proteins (eg, p21) that are increased in diffuse gliomas, a number of other proteins including guanosine-5′-triphosphate (GTP)-binding and signal transduction proteins (eg, in the MAPK/ERK kinase 1 pathway), as well as metabolic enzymes (eg, glutamate dehydrogenase 1, phosphoglycerate mutase 1, and phosphopyruvate hydratase), molecular chaperones (eg, HSP27 and GRP-78),

C.D. Katsetos, H. Anni, and P. Dráber and cytoskeletal components (eg, p81) are involved.38 Recently, IQGAP1, a cell membrane protein associated with tumor progression, was selected from targeted proteomic studies of microdissected tissues and found to be differentially expressed across the grade spectrum of diffuse astrocytic gliomas (WHO grades II-IV).39 In a related study, primary vs secondary glioblastomas were distinguished using a set of proteins identified by proteomics in homogeneous tissuemicrodissected cells.40 Among the differentially regulated proteins, enolase 1 and centrosome-associated protein 350 were found to be involved in primary glioblastomas.40 Interactions between cytosolic glycolytic enzymes enolase and phosphoglycerate mutase with cytoskeletal components have been found in normal tissue,41 and the observed protein increase in the context of glioblastomas suggests a structural and functional cellular dysregulation. In a recent study,42 it was found that the proteome of glioblastomas was enriched not only in cytoskeletal proteins (3- to 6-fold increase of vimentin, α- and β-tubulin, β-actin, and glial fibrillary acidic protein), but also mt (aldehyde dehydrogenase, and manganese superoxide dismutase) and ER chaperone proteins (GRP-75, GRP-57, and HSP27). Because mt control proliferation and apoptosis, the upregulation of key mt and ER proteins and their cross talk43 has significant implications in the pathobiology of glioblastoma.10 Mt dysfunction in cancer cells may be causal to tumor formation or be part of cancer-mediated metabolic reprograming.5 Nevertheless, energy production by mt is severely deregulated in many cancers. Collectively, these findings suggest that there is a relationship between mt dynamics and key cellular processes such as metabolism, apoptosis, and autophagy in cancer cells.6 The technical limitations inherent to the acquisition of data on deregulated mt proteins by proteomics in whole cell lysates can be addressed by studies of subproteomes and in particular of the mt proteome.44 The challenge ahead, with more than 1000 proteins comprising the mammalian mt proteome and dual localization of 15% of them,45 is to develop targeted approaches to explore diseaserelated intracellular compartmentalization, such as in the case of class III β-tubulin (see below).46

Mitochondrial DNA Mutations Oncogene expression and mtDNA mutations have been proposed as mechanisms of mt dysfunction in cancer.19,20,27 However, these mechanisms cannot fully explain the mt dysfunction in apoptosis, and there is absence of mtDNA mutations in chemically induced mouse glioma.47 Somatic mtDNA mutations have been reported in medulloblastomas.48

IDH Mutations and Oncometabolite 2-HG The field of cancer bioenergetics took a new impetus in 2008 after the discovery of mutations in the isocitrate dehydrogenase gene IDH1 in human glioblastomas.11 IDH enzymes catalyze oxidative decarboxylation of isocitrate to α-KG thus reducing NADPþ to NADPH.25,29 IDH1 is functionally compartmentalized in the cytosol and peroxisomes, whereas IDH2 and IDH3 are both localized in the mt.25

Mitochondrial dysfunction in gliomas DNA sequence analysis of glioblastomas revealed mutations in the gene encoding the cytosolic form of the enzyme IDH1.50 Somatic mutations in the IDH1 gene were subsequently found in most astrocytomas, oligodendrogliomas, and oligoastrocytomas corresponding to WHO grades II and III.49,51,52 The highest frequencies of heterozygous somatic IDH1 mutations occurring at nucleotides that code for arginine, R132 are present in 470% of diffuse gliomas (WHO grade II and III) and in secondary glioblastomas (88%) (WHO grade IV).49,51,52 IDH1 mutations are infrequent in primary glioblastomas.49,53 The spectrum of WHO grade II gliomas tested includes diffuse astrocytomas (68%), oligodendrogliomas (69%), and oligoastrocytomas (78%).49 A subset of gliomas without IDH1 mutations have mutations in IDH2, the mt homolog of IDH1.24 The IDH2 mutations that have been identified in gliomas occur at the analogous residue to IDH1-R132, that is, IDH2-R172, where R is arginine.24 Mutations in IDH2 are present in grades II and III gliomas at a low frequency.25 Among histologic types within the WHO II and III glioma spectrum, it has been reported that IDH1 mutations are strongly associated with astrocytic gliomas, whereas IDH2 mutations have a proclivity for oligodendrogliomas.52 Overall, IDH1 and IDH2 mutations are more frequent in younger patients and are associated with a more favorable prognosis.51,52 Both IDH1 and IDH2 mutations are accompanied by loss of normal enzyme function and inability to convert isocitrate to α-KG.23,51 In addition to gliomas, somatic IDH1 and IDH2 mutations are also observed in other cancers, such as acute myelogenous leukemia. These mutations are characterized by gain of function reflected in the production and accumulation of the oncometabolite 2-HG, which serves as a biomarker.24,25 Excess accumulation of 2-HG can also result from germline mutations in D- and L-2-HG dehydrogenase genes (D2HGDH and L2HGDH).25 Interestingly, a weak association exists between brain tumors and hereditary 2-hydroxyglutaric aciduria, a rare inherited metabolic disorder, caused by homozygous inactivating germline mutation in D2HGDH gene.25 Screening of 47 glioblastoma samples for changes in IDH1, IDH2, IDH3, D2HGDH, and L2HGDH genes revealed that mutations in IDH3, D2HGDH, and L2HGDH are infrequent in glioblastomas.25 Among recurrent IDH2 mutations at Arg-172 and Arg-140 residues, IDH2-R172 mutations give rise to greater 2-HG accumulation than IDH2-R140 mutations.54 Aside from the better known mutations in the 3 active site arginine residues, notably IDH1-R132, IDH2-R172, and IDH2-R140, 3 additional IDH1 mutations associated with production of 2-HG have been reported. These include IDH1-R100, detected in a glioma from an adult; IDH1-G97, where G is glycine identified in a pediatric glioblastoma; and IDH1-Y139, where Y is tyrosine.55 Even though it has been suggested that a common feature of IDH mutations is the production of 2-HG, non-2-HGproducing IDH mutations can also occur in a variety of human cancers.55 Moreover, additional rare IDH mutations have been

219 reported in lymphomas and thyroid cancer, which are not accompanied by elevation of 2-HG but may still exhibit loss of function owing to reduced wild-type enzymatic activity.55 A recent study has unraveled that in contrast to cytosolic IDH1 mutation in which the resultant cellular 2-HG production is dependent on the activity of a retained wild-type IDH1 allele, 2-HG production owing to mt IDH2 mutations is independent of wild-type mt IDH function.54 IDH is posttranslationally modified by acetylation, and the regulated site of acetylation has been mapped to Lys-413 residue.56 Acetylated IDH2 is accompanied by a profound loss in activity. Sirtuins (SIRTs) are commonly known as NADþ dependent class III histone deacetylase enzymes. Specifically, SIRT3 fully restores maximum IDH2 activity. Given that IDH2 inactivation plays a role in cancer, SIRT3 may be a potential regulator of IDH2-dependent functions in cancer cell metabolism.56 A recent meta-analysis study has shown that IDH mutations are closely linked to the genomic profile of gliomas, including changes in O6-methylguanine-DNA methyltransferase promoter hypermethylation, epidermal growth factor receptor amplification, codeletion of chromosomes 1p/19q, and TP53 gene mutation.57

Mitochondrial p53 Pathway p53 is one of the most well-studied mutated tumor suppressors in human cancers, including gliomas, which has diverse cellular roles, including the regulation of apoptosis, cell cycle arrest, senescence, DNA repair, and genetic stability.58 In addition to its well-known role as a transcription factor, p53 also directly participates in the intrinsic apoptosis pathway by interacting with members of the Bcl-2 family to induce mt outer membrane permeabilization.58 A fraction of p53 translocates to mt, specifically in response to death signals through the induction of mt-mediated apoptosis.59 Mutant p53 is apparently always present at the mt, independent of apoptotic signal, and previous studies in human colorectal cancer and glioblastoma cell lines have shown that mt p53 levels parallel total cellular levels independent of apoptosis.59

Mitochondria and Microtubule Interactions in Cancer Tubulin Regulation of mt Voltage-Dependent Anion Channels (VDAC) Mt metabolism requires inward and outward flux of hydrophilic metabolites, including ATP, ADP, and respiratory substrates, through VDACs located in the mt outer membrane.60 The interaction between cytoskeletal proteins and mt can play a regulatory role in mt function, and to that end, the microtubule protein tubulin may control VDACs.61 One of the first histochemical indications that tubulin is associated with mt wall was provided by staining of the brain sections with the monoclonal antitubulin antibody TU-0162 directed to α-tubulin.63 It was shown by immunohistology and immunoelectron microscopy that the aforementioned antibody stained

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Figure 1 Localization of mitochondria along microtubules in glioblastoma T98G cell and in neurites of neuroblastoma SH-SY5Y cells. To generate long neurites, the SH-SY5Y cells were incubated in the presence of 10 μM all-trans retinoic acid for 6 days. Microtubules were detected in formaldehyde-fixed and Triton X-100–extracted cells either by anti-α-tubulin mouse monoclonal antibody TU-0163 (A, red in merge) or anti-βIII-tubulin mouse monoclonal antibody TU-20144 (D, red in merge). Mitochondria were stained with rabbit monoclonal antibody (Abcam Cat. No. ab137057) to mitofilin, mitochondria inner membrane protein (B and E; green in merge). Panels C and F are double stained (merge) images DNA was stained with DAPI (C and F; blue). Scale bar, 20 μm in (C) and 50 μm in (F). DAPI, 4′,6-diamidino-2-phenylindole. Photography by Dr Eduarda Dráberová (Institute of Molecular Genetics, AS CR, Prague).

outer mt membranes of Bergmann glia in rat cerebellum.64 Further studies revealed that tubulin is an inherent component of mt membranes where it is associated with the VDAC, the main component of the mPTP.65 As compared with soluble cytosolic tubulin, mt tubulin is enriched in acetylated and tyrosinated α-tubulin and also in the class III β-tubulin isotype (βIII-tubulin).65,66 Tubulin is present in mt isolated from different human cancer and nonneoplastic cell lines and could play a role in apoptosis by way of interaction with the mPTP65 or other hitherto unknown mechanisms, or both. Microtubules also secure mt positioning and their translocation. Localization of mt along microtubules in glioblastoma T98G cell and in neurites of neuroblastoma SH-SY5Y cells is shown in Figure 1. VDACs are formed by highly conserved proteins and are the most abundant channels of the mt outer membrane accounting for its permeability to hydrophilic metabolites like ATP, ADP, and respiratory substrates.67 VDAC closure is an important mechanism for regulation of mt metabolism both in the context of apoptosis and cell survival.67 In human glioma xenograft cells, changes of mt outer membrane permeabilization are preceded by a metabolic shift from mt OXPHOS to aerobic glycolysis.68 Tubulin has emerged as a potent regulator of VDAC, which constitutes a major pathway for ATP or ADP and other metabolites across the mt outer membrane.69 VDAC permeability for the mt respiratory substrates is regulated by dimeric tubulin and channel phosphorylation.69 The formation of the mt membrane potential (ΔΨ) depends on flux of respiratory substrates, ATP, ADP, and Pi through VDAC.70 Dimeric tubulin induces reversible blockage of VDAC reconstituted into a planar lipid membrane and dramatically reduces

respiration of mt.69 It has been proposed that inhibition of VDAC by free tubulin limits mt metabolism in cancer cells.70 Compounds that increase cellular-free tubulin and decrease mt ΔΨ include microtubule destabilizers, rotenone, colchicine, and nocodazole.69,70 In contrast, the microtubule stabilizer paclitaxel decreases cellular-free tubulin and increases mt ΔΨ.70 Moreover, a decrease in mt ΔΨ and VDAC conductance is produced by protein kinase A.67,69,70 Conversely, an increase in mt ΔΨ and VDAC conductance with resultant hyperpolarization is produced by glycogen synthase kinase 3β.69,70 Tubulin-dependent closure of VDACs represents a new mechanism contributing to the suppression of mt metabolism underpinning the Warburg phenomenon.60 Free tubulin inhibits VDAC1 or 2 and limits mt metabolism in human hepatocellular cancer (HepG2) cells, whereas reversal of tubulin-VDAC interaction by erastin antagonizes the Warburg effect and restores oxidative mt metabolism.71 In summary, a previously unknown mechanism of regulation of mt energetics is that governed by VDAC interaction with tubulin at the mt-cytosol interface.72 Immediate pathophysiological implications from this interaction include, but are not limited to, cellular and molecular aspects relating to serine-threonine kinase signaling pathways, Ca2þ homeostasis, and microtubule dynamics in cancer cells.69,72

Microtubules and Mitochondrial Dynamics Novel biochemical pathways in mt biology include mt fusion, fission, and organellar motility along microtubules and microfilaments (mt dynamics).7,66 An interaction between microtubules and mt may play a role in cell motility, invasion, and

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metastasis. Asymmetric compartmentalization (anterior localization) of mt correlates with faster migration velocities and increased directional persistence in migrating epithelial cancer cells.73 Similarly, functional disruption of mt-microtubule links by the knockdown of the outer mt membrane protein mt Rho GTPase (MIRO) significantly decreases the velocity and directional persistence of cancer cells.73 γ-Tubulin is a minor member of tubulin family.74 It is essential for nucleation of microtubules from microtubule organizing centers, such as the centrosomes or the Golgi complex, and is present in the form of γ-tubulin small or large complexes.75 γ-Tubulin is encoded by 2 genes.76 Although γ-tubulin 1 is ubiquitously expressed, γ-tubulin 2 is concentrated in brain.77 Both γ-tubulin variants are however capable of nucleating microtubules.78 Substantial changes in mt morphology have been reported in primary cultures of striatal spiny neurons isolated from Tubg2knockout mouse and has been suggested that γ-tubulin 2 with DRP1 protein regulates mt morphology.79 γ-Tubulin is associated with cellular membranes in various cell types,80-82 including human glioblastoma cell lines.83,84 Interestingly, γ-tubulin is associated with detergentresistant membranes (lipid rafts) in mouse embryonal carcinoma cells P19 undergoing neuronal differentiation.85 The presence of αβ-tubulin dimers in rafts has been frequently reported, especially for neural tissue and myelin, although estimates of the amounts vary widely.86 Recent data from our laboratory indicate that both γ-tubulin isoforms are associated with mt when SH-SY5Y extracts are fractionated by differential centrifugation (Sulimenko and Dráber, unpublished data). The elucidation of functional relevance of γ-tubulin association with mt thus deserves further investigation.

Mitochondria-Endoplasmic Reticulum Network Microtubules have a key role in intracellular trafficking and contact between organelles including the ER and mt. Contacts between these 2 organelles are important for regulating the dynamics of mt. The ER-mt junctions are vital in the functional organization of these 2 organelles, including regulation of lipid synthesis, Ca2þ signaling, and control of mt biogenesis and intracellular trafficking.87 Mt and ER track against each other and make site contacts with one another over microtubules that are posttranslationally modified by acetylation.88 ER protein(s) that bind the dynamic ER to microtubule motors remain elusive.87 To date, the most well-characterized complex that regulates mt movement includes the MIRO protein that binds to the mt adaptor protein MILTON, which in turn binds to the microtubule motor protein kinesin 1 heavy chain on microtubules89 (Fig. 2). MIRO is localized in areas of ER-mt contact90 (Fig. 2) and has dual functions acting both as a Raslike GTPase and an EF-hand calcium-binding protein that senses increases in cytosolic Ca2þ. Surges of intracellular Ca2þ precipitate cessation of mt movement on microtubules, and this effect can be abrogated either on MIRO depletion or by expression of a MIRO EF-hand mutant.91 Increased

Figure 2 Endoplasmic reticulum (ER)-mitochondria contact sites and the regulation of mitochondrial dynamics. Microtubule motor protein kinesin 1 interacts with factors that are either associated with mitochondria or the ER. In the case of mitochondria, kinesin 1 binds to the cytoplasmic protein MILTON, which binds to MIRO on the outer mitochondrial membrane. The ER proteins that tether the ER to microtubules are unknown. Mitochondrial constriction occurs at sites of ER-mitochondria contact and is mediated by DRP1. The ER contact promotes constriction of the mitochondria, and mitochondrial fission is promoted by DRP1 and its cofactor MFF. MFF, mitochondrial fission factor. (Adapted with permission from Rowland and Voeltz87). Reprinted by permission from Macmillan Publishers Ltd: (Nature Reviews in Molecular Cell Biology), copyright (2012).

intracellular Ca2þ levels trigger activation of DRP1, which increases mt fission.91 Thus, MIRO is a critical regulator of mt movement.87

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Class III β-Tubulin: A Prosurvival Factor Associated with Mitochondria? The expression of βIII-tubulin in gliomas and its relationship to tumor hypoxia, angiogenesis, and cancer stem cells in glioblastomas has been previously appraised.92-96 Currently, βIIItubulin is considered to be a survival factor, which acts by rescuing tumor cells from cell death signals triggered by diverse classes of DNA-targeting compounds, such as cisplatin, doxorubicin, and etoposide97 and whose expression is increased in oncogenesis and tumor progression, irrespective of the type of chemotherapeutic agents employed.98 As a survival factor, βIII-tubulin plays a role in the cellular adaptation to OS and glucose deprivation, suggesting that the βIII isotype may play an important role in drug resistance.46 Strong cumulative evidence exists indicating that the prosurvival properties of βIII-tubulin are independent of the role of this protein in microtubule dynamics.99,100 It has been suggested that βIII-tubulin acts as a “gateway” for prosurvival signals, which enhance tumor growth and resistance to chemotherapeutic drugs.101 Hence, βIIItubulin does not act alone but in concert with other prosurvival factors through protein-protein interactions in the form of a “prosurvival cascade” that opens possibilities for the development of therapeutic approaches aimed at inhibiting prosurvival signals acting on the microtubule cytoskeleton.101,102 A study using chemoresistant ovarian cancer cells has shown that the function of βIII-tubulin is linked to 2 GTPases exhibiting opposing effects: GBP1 activates the function of βIII-tubulin whereas GNAI1 exerts an inhibitory effect.101 PIM1 is a prosurvival factor and downstream partner of GBP1, which is recruited into the microtubule cytoskeleton under hypoxic conditions. Pathologic specimens from patients with ovarian cancer who lacked immunoreactivity for βIII-tubulin and PIM1 (βIIItubulin- and PIM1-) responded to treatment and were characterized by a long overall survival; in contrast, patients whose specimens displayed a βIII-tubulinþ and PIM1þ profile had unfavorable outcomes.101 A recent study has shown that βIII-tubulin plays a role in drug resistance in glioblastomas.103 Drug-treated clonogenic assays using βIIItubulin knockdown cells resulted in a significant increase in sensitivity to temozolomide and the tubulin-binding agents (epothilone B and paclitaxel) in U87vIII human glioblastoma cells compared with control small interferingRNA transfected cells. Additionally, knockdown of βIIItubulin led to increased apoptosis following temozolomide treatment.103 The cancer environment favors the production of free radicals. The presence of βIII-tubulin in tumor cells may be accounted for by the lack of cys239, which allows the assembly of α/βIII-tubulin dimers in the presence of free radicals.104 It is noteworthy that in ovarian cancer cells in vitro, βIII-tubulin expression is strongly induced by (pseudo)hypoxic signals mediated by HIF-1α through methylation of the 3′ enhancer of the βIII isotype.105 In drug-resistant ovarian cancer cell lines, βIII-tubulin exists in 2 posttranslationally modified forms.

C.D. Katsetos, H. Anni, and P. Dráber Although the lower-molecular-weight form is found in mt, the higher molecular weight (glycosylated and phosphorylated) is recruited into microtubules.46 Both posttranslational modifications are found in drug-resistant tumor cells.46 The role that mt play in the development of chemoresistance in malignant brain tumors has been previously appraised.9 A recent study has shown that 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine induces a time- and dose-dependent alterations in mt and early changes in microtubule proteins, characterized by significant increases in βIII-tubulin and enrichment of detyrosinated (deTyr) tubulin, culminating in microtubule disruption in dopaminergic neurons of C57Bl mice.106 This study has also shown that microtubule stabilizer epothilone D rescued microtubule defects and attenuated nigrostriatal degeneration induced by 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine.106 The relationship and interaction of βIII-tubulin with microtubules associated with ER-mt tracking or mt dynamics and VDAC alterations in glioma cells requires further investigation as it may be exploited for therapeutic targeting.

Mitochondria as Therapeutic Targets in Gliomas Metabolic Modulation-Pyruvate Dehydrogenase Kinase (PDK) Various types of cancer, including the most common and lethal primary brain cancer glioblastoma, develop resistance to cell death, in part because of a switch from mt OXPHOS to cytoplasmic glycolysis.107,108 This metabolic remodeling is accompanied by mt hyperpolarization.108 Previous studies have shown that such mt hyperpolarization, which is a hallmark cancer-specific metabolic and mt remodeling in glioblastoma is rapidly reversed by the small-molecule and orphan drug dichloroacetate (DCA).108 Generic DCA is an orally available drug that inhibits PDK and increases the influx of pyruvate into the mt thus promoting glucose oxidation over glycolysis.109 DCA depolarizes mt, increases mt ROS, and induces apoptosis in glioblastoma cells, as well as in putative cancer stem cells, both in vitro and in vivo.108 A dose-limiting or dose-dependent toxicity is in the form of a reversible peripheral neuropathy; no hematologic, hepatic, renal, or cardiac toxicities are identified. Clinical efficacy has been demonstrated at a dose that does not cause peripheral neuropathy and at serum concentrations of DCA that are sufficient to inhibit PDK II, the target enzyme of DCA, which is highly expressed in glioblastomas.108 DCA also reverses the suppressed mt apoptosis in cancer and results in suppression of tumor growth both in vitro and in vivo.109 PDK is a mt enzyme that is activated in a variety of cancers and results in the selective inhibition of pyruvate dehydrogenase, a complex of enzymes that converts cytosolic pyruvate to mt acetyl-CoA, the substrate for the Krebs cycle.110 Inhibition of PDK with either small interfering RNAs or the orphan drug DCA shifts the metabolism of cancer cells from glycolysis to glucose oxidation and reverses the suppression of mt-dependent

Mitochondrial dysfunction in gliomas apoptosis.110 In addition, PDK inhibition increases the production of diffusible Krebs cycle intermediates and mt-derived ROS, activating p53 or inhibiting proproliferative and proangiogenic transcription factors like nuclear factor of activated T cells and HIF-1α.108,110 Hence, in addition to proapoptotic and antiproliferative effects, mt-targeting metabolic modulators that increase pyruvate dehydrogenase lead to suppression of angiogenesis.111 Moreover, the M2 isoform of pyruvate kinase (PKM2), which is highly expressed in cancer, is associated with suppressed mt function.110 Similar to DCA, activation of PKM2 in many cancers results in increased mt function and decreased tumor growth.110 In summary, the apoptosis resistance linked to the distinctive metabolic profile of cancer (aerobic glycolysis) may be therapeutically targeted by metabolic-modulating drugs, like PDK inhibitors or PKM2 activators, making them promising anticancer agents in the treatment of gliomas.108-112 In a small clinical trial of patients with glioblastoma, DCA was shown to decrease tumor growth and angiogenesis.110

Mitochondrial-Mediated Apoptosis and Tricyclic Antidepressants Various tricyclic antidepressant drugs, such as clomipramine and amitriptyline, can enter the mt of tumor cells causing reduction of oxygen consumption, release of cytochrome c, and activation of the caspase cascade hence exerting their tumoricidal action through apoptosis in vitro; as such, these drugs can be used as potential anticancer agents aimed at targeting mt in gliomas.113,114 Clomipramine and its metabolite norclomipramine have been found to be potent inhibitors of cellular respiration in anaplastic astrocytoma cells in vitro thereby enhancing apoptotic cell death, an effect that is synergistically accentuated by dexamethasone.114 Subsequent studies have shown that imipramine exerts antitumor effects on the U87-MG human glioblastoma cell line by inhibiting PI3K-Akt-mTOR signaling and by inducing autophagic, rather than apoptotic, cell death.115 Desipramine has also been shown to induce autophagy through the PERK-ER stress pathway in C6 rat glioma cells.116 Imipramine blue (IB), an anti-invasive small molecule, inhibits invasion of glioma in vitro by inhibiting NADPH oxidase–mediated ROS generation and altering the expression of cytoskeletal actin regulatory.117 In vivo, liposomal IB (nano-IB) abrogates invasion of glioma, leading to tumor cytoreduction in the RT2 syngeneic astrocytoma rodent model. Combination of nano-IB therapy and nanodoxorubicin chemotherapy prolonged survival compared with nano-IB or nano-doxorubicin alone.117 The clinical use of tricyclic antidepressants in the treatment of glioma has been limited to anecdotal cases.113 Although imipramine holds promise as an adjuvant treatment for diffuse gliomas, a recent epidemiologic study from the University of Nottingham in the United Kingdom using a cohort of 1364 glioma cases showed no significant mortality reduction in patients with glioma treated with tricyclics. However, the authors called for future blinded clinical studies to determine drug efficacy in patients with glioma.118

223

Microtubule-Modulating Drugs and Mitochondrially Mediated Apoptosis Certain tubulin-binding agents, such as taxanes (paclitaxel), epothilones (patupilone), and vinca alkaloids (vinflunine and vinblastine) cause mt release of cytochrome c leading to initiation and execution of intrinsic apoptotic signaling.119-121 Treatment of human SK-N-SH neuroblastoma cells with vinflunine causes upregulation and translocation of Bax to the mt at different concentrations and Bcl-2 phosphorylation at a high concentration.121 Treatment of human neuroblastoma cells with patupilone, an epothilone B, triggers early increased generation of mt-derived ROS, mt membrane potential collapse, mt morphologic changes, and cytochrome c release from mt culminating in apoptosis.120 Hence, in the context of patupilone treatment, mt assume the dual role of activator and integrator of apoptotic signals.120 The potential involvement of microtubule-sequestered proteins or microtubule-transported proteins or both in apoptotic signaling through the mt intrinsic pathway is an area of major investigative interest.122,123 The apoptosis-inducing factor (AIF), a crucial mediator of cell death, plays a relevant role in noscapine-mediated apoptosis in glioma cells.124 A recent study has shown a distortion in the mt membrane potential after exposure of glioma cell lines to the noscapinoid microtubule-modulating agent EM011 (9-bromonoscapine), and this phenomenon could be associated with the release of the mt resident protein AIF.125 Thus, EM011-mediated apoptosis is associated with transient release of AIF from the mt.125 Importantly, EMO11 inhibits angiogenesis by repressing the HIF-1α axis and cell migration by inhibiting membrane ruffling and impeding the formation of filopodia, lamellipodia, and stress fibers as well as causing centrosomal abnormalities.126 Similarly, apicularen A acetate, a novel antitumor agent, causes loss of mt membrane potential and translocation of AIF from mt, thus inducing cell death in HM7 colon cancer cells.127 In addition, apicularen A acetate significantly decreases tubulin mRNA and protein levels and induces disruption of microtubule networks.127 The cytotoxic or tumoricidal action of certain novel microtubule inhibitors, such as the compound MPT0B214, an aroylquinolone regioisomer, involves apoptotic cell death through the mt-mediated caspase 9–dependent pathway in addition to inhibition of tubulin polymerization through strongly binding to the tubulin’s colchicine-binding site.128

Polyunsaturated Fatty Acids (PUFAs) PUFAs are known to inhibit cell proliferation of tumor cells. Their capacity to interfere with cell proliferation has been linked to ROS production in tumors leading to apoptotic cell death.129 PUFAs can induce loss of bound hexokinase from the mt VDAC in tumor cells, which may result in loss of protection from apoptosis.129 Tumor cells overexpressing Akt activity, including gliomas, are sensitized to ROS damage by the Akt protein and may be good targets for chemotherapeutic agents, which produce ROS, such as PUFAs.129 Cardiolipin peroxidation may be an initial event in the release of

C.D. Katsetos, H. Anni, and P. Dráber

224 cytochrome c from the mt, and enriching cardiolipin with PUFA acyl chains may lead to increased peroxidation and therefore an increase in apoptosis.129

Small-Molecule Mitochondria Targeting of the Stress Response to ROS Oncogene-induced mitogenic signaling and oncogeneinduced (pseudo)hypoxic signaling lead to intracellular accumulation of ROS, which sensitize cancer cells to ROS-induced cell death.130-132 The reprograming of cancer cell metabolism toward aerobic glycolysis compromises ROS production.133 The dependence of cancer cells on glycolysis underlies the concept of nononcogene addiction that is amenable to selective targeting by small molecules.134,135 Targeting of the increased ROS sensitivity of cancer cells has gained acceptance as a concept in cancer therapeutics.136,137 Compounds capable of inflicting ROS-dependent cell death selectively in cancer cells are elesclomol,138 piperlongumine,139 erastin,140 and SMIP004 (N-[4-butyl-2-methyl-phenyl] acetamide).141

Mitochondrial Priming and MitochondriaTargeted Prodrug Cancer Therapy The clinical response to chemotherapeutic agents targeting DNA and microtubules correlates with, and may be partially governed by, the pretreatment proximity of tumor cell mt to the apoptotic threshold, a property called mt priming.142 An assay measuring mt response to peptides derived from proapoptotic BH3 domains of proteins critical for cell death signaling to mt was used to measure priming in tumor cells from patients with hematopoietic and ovarian cancers.142 Patients with highly primed cancers exhibited superior clinical response to chemotherapy compared with poorly primed cancers, which were chemoresistant.142 Mt priming is a promising tool capable of enhancing the efficacy of cytotoxic agents. Another cellular therapeutic approach is prodrug delivery to mt as a means of augmenting the therapeutic efficacy and mitigating the cytotoxicity of the taxanes, paclitaxel, and docetaxel in cancer cells.143

Conclusion and Future Directions Our current understanding of mt dysfunction in gliomas is centered on abnormalities of mt energy metabolism typified by a metabolic shift from OXPHOS to glycolysis (“Warburg effect”), disturbances of mt membrane potential regulation, and apoptotic signaling, as well as somatic IDH mutations. Recent advances in mt biology, with implications in cancer biology and cellular therapeutics, include interaction of microtubule proteins and mt in the control of closure of VDACs in the mt outer membrane as well as in the regulation of mt dynamics and mt-ER network. Mt-targeted therapeutic strategies include metabolic modulation through PDK inhibitors (principally DCA) and mt-mediated apoptosis induced by tricyclic antidepressants, microtubule-modulating drugs

(noscapine and EM011), and small molecules or compounds capable of inflicting ROS-dependent cell death selectively in tumor cells. In addition, pretreatment mt priming and mttargeted prodrug cancer therapy are newly emerging promising approaches, which may be potentially useful in the treatment of gliomas.

Acknowledgments We thank Eduarda Dráberová, PhD, Laboratory of the Biology of Cytoskeleton, Institute of Molecular Genetics, AS CR, Prague, for providing the photomicrographs in Figure 1.

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