cancer as a “mitochondriopathy”

Review Article

2007 MedUnion Press − http://www.mupnet.com 71

Cancer as a “Mitochondriopathy”Anna M Czarnecka, Antonella Marino Gammazza, Valentina Di Felice,Giovanni Zummo, and Francesco Cappello1

Postgraduate School of Molecular Medicine, Medical University of Warsaw, Warszawa, Poland [A. M. Czarnecka];Human Anatomy Section, Department of Experimental Medicine, University of Palermo, Palermo, Italy [A. M.Gammazza, V. D. Felice, G. Zummo, F. Cappello]

Mitochondria are subcellular organelles, whose well-known function is to produce ATPthrough the oxidative phosphorylation system. Alterations in respiratory activity andmitochondrial DNA (mtDNA) appear to be a general feature of malignant cells. Thepresence of mtDNA mutations has been reported in various cancer cells, and theabundance of mtDNA damage is consistent with the intrinsic susceptibility to constitu-tive oxidative stress. Research about the functional aspects of mtDNA mutations incancer development and therapeutic response is likely to be fruitful and to havesignificant clinical and prognostic impact. Although many studies to date have beenfocused on the identification and characterization of altered mtDNA, it is not clear ifthese accumulated mutations are the cause or the consequence of the carcinogenicprocess. This article provides a brief summary of our current understanding of mito-chondrial pathobiology in cancer development.

Journal of Cancer Molecules 3(3): 71-79, 2007.

Keywords

mtDNA

mitochondria

cancer

prohibitin

homoplasmy

Introduction

Mitochondria are eukaryotic organelles involved in manymetabolic pathways, the most important of which is togenerate most of the cellular energy (ATP) through theoxidative phosphorylation system (OXPHOS2). Mitochondriaare also essential in the processing of important metabolicintermediates in various pathways like carbohydrates, aminoacids, and fatty acids [1-3]. In addition, mitochondria aresemi-autonomous organelles that perform an essentialfunction in the regulation of cell death, signalling and freeradical generation [4-7]. Phylogenetically, mitochondriaarose from an endosymbiotic relationship between a glyco-lytic proto-eukaryotic cell and an oxidative bacterium [8].Recently it has been proposed that mitochondria may havederived from hydrogenosomes, that are organelles synthe-size ATP under anaerobic conditions in amitochondriateprotists which produce H2 and CO2 [9]. As endosymbioticorganelles, mitochondria possess a double-membranestructure and contain their own genome along with their owntranscription, translation, and protein assembly machinery[2]. Nevertheless, the mitochondrial function involves aninterplay between mitochondrial and nuclear genomes [4,10].

The term “mitochondrial medicine” was coined as early as1962 by Rolf Luft in his clinical report on nonthyroid hyper-

Received 4/24/07; Revised 6/5/07; Accepted 6/6/07.1Correspondence: Dr. Francesco Cappello, Human Anatomy Section,Department of Experimental Medicine, University of Palermo, via delVespro 129, 90127 Palermo, Italy. Phone: 39-91-6553518. Fax: 39-91-6553518. E-mail: [email protected]: OXPHOS, oxidative phosphorylation system; mtDNA,mitochondrial DNA; AMPK, AMP-activated kinase; SCO2, Synthesis ofCytochrome c Oxidase 2; COX, cytochrome c oxidase; D-loop,displacement loop; nDNA, nuclear DNA; ROS, reactive oxygen species;HSP, heat-shock protein; PHB, prohibitin; IM, inner mitochondrialmembrane; AAA, ATP-dependent protease; mtTFA, mitochondrialtranscription factor A.

metabolism [11]. It was in 1988 when Holt et al. [12] reportedthe association of human myopathy with a huge deletion ofmitochondrial DNA (mtDNA), while Wallace et al. [13] pub-lished a paper on point mutations in the mtDNA of patientswith Leber’s hereditary optic neuropathy. Disruption ofmitochondrial function in these syndromes suggested anumber of novel mechanisms that can be grouped asmitochondrial pathologies, and mtDNA has been describedas a veritable Pandora’s box of pathogenic mutations. Thenumber of known mutations, deletions, and duplications ofmtDNA exceeded 300 a few years ago and the number ofknown polymorphisms exceeded 1000 [14,15]; new muta-tions are annotated every month. Population studies in UKhave shown that at least one in 8000 adults harbours apathogenic mtDNA mutation [16].

Among a wide variety of pathological events, defects inmitochondrial function have long been suspected to con-tribute to the development and progression of cancer. Inearly 1920s, Warburg pioneered the research on alterationsof the mitochondrial respiratory chain in the context ofcancer and proposed a mechanism to explain how theyevolved during the carcinogenic process. In his series oflandmark publications, he hypothesized that a key event incarcinogenesis could be the development of an "injury" tothe respiratory machinery, resulting in compensatoryincreases in glycolytic ATP production. In fact, malignantcells produce their ATP through glycolytic mechanismsrather than through oxidative phosphorylation and in benigntumours the rate of aerobic glycolysis is only 1/3 that inmalignant tumours [17-19]. Other experiments have sug-gested that aerobic glycolysis is highly active in malignantcancers. Since high aerobic glycolysis activity must becorrelated with glucose absorption by the cell, in vivomeasurements have been performed and the data revealedan high glucose intake of cancerous tissues, in gliomas,meningiomas and sarcomas, in comparison with normaltissues [20-22].

Czarnecka et al. J. Cancer Mol. 3(3): 71-79, 2007

72 Print ISSN 1816-0735; Online ISSN 1817-4256

Recently, the Warburg’s theory on glucose metabolism incancer cells has been revised. Many authors supposed thatduring the tumorigenic process the use of the glycolyticpathway is more a need for cancer cells rather than aconsequence of DNA mutations [23-26]. In particular, it hasbeen supposed that cancer cells prefer to use glycolysis asan ATP source to reach a good quantity of energy in order toinhibit energy-sensing enzymes such as AMP-activatedkinase (AMPK). AMPK, in turn, can activate by phosphoryla-tion tumor suppressor genes like p53 and TSC2 (TuberousSclerosis Complex 2 gene)[24,25]. This seems to be amechanism to gain replicative advantage through therepression of replicative senescence [24]. Moreover, Fantinet al. [27] and Bui et al. [23] stated that in cancer cells bothoxidative phosphorilation and glycolysis were active, sincesuppressing glycolysis most tumor cells had a substantialreverse capacity to produce ATP. The coexistence of theglycolytic and oxidative metabolism is also supported byrecent advances on the role of p53 in tumor cells [26]. In fact,they demonstrated that p53 regulated the balance betweenthe utilization of respiratory and glycolytic pathways bytrans-activating Synthesis of Cytochrome c Oxidase 2(SCO2) expression, required for the assembly of mitochon-drial DNA-encoded cytochrome c oxidase (COX) II subunit(MTCO2 gene) into the COX complex [26]. Hence, mutationsof p53 gene, usually found in various cancers, might affectCOX complex assembly and activity [26].

To date the nature of mitochondrial failure in cancer cellsinvolves abnormal ultrastructure and metabolic deregulation[1]. First of all, alternations in aerobic energy metabolismhave been assigned to the disruption of the expression ofnucleus-encoded fumarate hydratase and succinate dehy-drogenase enzymes (complex II of the respiratory chain)[28].Moreover, since several subunits of the respiratory chaincomplex are encoded by mtDNA [29], mutations in themtDNA may result in altered structures of mitochondrialproteins and thus disrupted OXPHOS, which might shiftmetabolism towards anaerobic respiration. The mtDNAalterations are frequently observed in tumour cells, butevidence for direct linkage of respiratory deficiency in aspecific tumour type with a specific mtDNA mutation is stillmissing [28].

However, several authors stated that the majority ofmtDNA mutations observed in tumours might not be in-volved in carcinogenesis. Salas et al. [31] in a recent articlesuggested that many mutations found in mtDNA might beonly the result of sequencing errors due to degraded DNA orextremely low quantities of DNA from old frozen samples orinadequately stored samples used in many chemical studies.On the other hand, some authors reported that mutations inthe mtDNA displacement loop (D-Loop) region were frequentin both benign and malignant thyroid tumours and could notbe considered as a marker of malignancy [32], while othersmentioned that mtDNA mutations might have a role in thedevelopment of cancer but not in the progression [33].

Mitochondrial genomic instability in cancerThe human mitochondrial genome has been completely

sequenced and each gene has been characterized [34,35].Human mtDNA is a double-stranded, closed-circularmolecule of approximately 16.6 Kb, which corresponds to amolecular weight of approximately 10 million Da. In mostcells, it represents only about 0.5-1 % of total DNA content[36,37]. The normal mtDNA state is thought to be a super-coiled structure that is poorly associated with proteins incomparison with nuclear DNA (nDNA). The two strands ofmtDNA can be distinguished due to their different G + Tcontent and can be separated by density in denaturinggradients, giving a heavy or H-strand and a light or L-strand

[2,35]. Each mitochondrion contains 2-10 copies of itsgenome and a mammalian cell typically contains 200–2000mitochondria. As a consequence of mtDNA multiplication inthe cell, mitochondrial genomes can tolerate very high levels(up to 90%) of damaged DNA through complementation withthe remaining wild type [2]. The mitochondrial genomeencodes a small but essential number of polypeptides of theOXPHOS. The coding sequences for two rRNAs, 22 tRNAsand 13 polypeptides, subunits of four OXPHOS complexes,are contiguous and without introns. The tRNAs are regularlyinterspersed between the rRNA and protein-coding genes,playing a crucial role in RNA maturation from the polycis-tronic transcripts. A single major 1-Kb long non-codingregion, called the D-loop region, contains the main regula-tory sequences for transcription and replication initiation [2].The D-loop is a triple-stranded structure in which a nascentH-strand DNA segment of 500-700 bp remains annealed tothe parental L-strand. This is the region most variable insequence. Besides the D-loop, mtDNA is much morevariable in sequence than the nDNA, and it is more vulner-able to mutations. The higher rate of mutation is accompa-nied by a higher ratio of nonsynonymous to synonymoussubstitutions in mtDNA than in nuclear genes [39]. There-fore, there are many characteristics that distinguish mtDNAfrom nDNA, including not only a higher rate of mutation, butalso the use of a divergent genetic code, transmission bymaternal inheritance, the phenomenon of polyploidy andabove all, a specific organization and expression machinery[38].

Clonal expansion of a single somatic mtDNA mutation hassubstantial implications for a cell. Mutations and deletionsin mtDNA, that have been observed in various solid tumoursand haematological malignancies, are associated withabnormal expression of mtDNA-encoded proteins [36](Table1). The hypothesis of an involvement of mitochondrialgenome alterations in the process of cancer developmentcame from the research of D. A. Clayton and J. Vinograd,who had shown that the size of mtDNA was correlated withthe disease progression of leukaemia [40,41]. It has beenproposed that deregulation of mtDNA genetic activity maycontribute to bioenergetic imbalances; in fact, it is knownthat some rapidly growing tumours display alterations ofoxidative metabolism and high rates of aerobic glycolysis.Combinations of different defects in the respiratory chaincomplexes (I, III, IV, and V) might have substantial physi-ological effects on the production of carcinogenic reactiveoxygen species (ROS) for example by deregulation of theOXPHOS [42]. Growing molecular evidence suggests thatcancer cells exhibit increased intrinsic ROS stress (due inpart to oncogenic stimulation), increased metabolic activityand mitochondrial malfunctions, which turn on the viciouscircle of oncogenesis [43]. Given the critical role of mito-chondria in apoptosis, it is conceivable that mutations ofmtDNA in cancer cells could significantly affect the cellularapoptotic response to anticancer agents and promote multi-drug resistance [44]. At the same time, studies have shownthat cell lines derived from human solid tumours lost theircapacity for anchorage-independent growth and tumori-genicity when injected into nude mice, they were recoveredonly if mtDNA from normal human cells was introducedbefore the injection [45]. Taken together, it is evident thatmtDNA mutations are clinically relevant and that mitochon-drial targeting agents may have potential therapeutic impli-cations. This hypothesis has fuelled an explosion in theresearch on mitochondria and mtDNA in recent years, even ifthe role of mitochondria in tumorigenesis remain unclarified[30,46]. To date, a limited number of mtDNA mutations areknown that can be classified as pathological markers. Theseinclude a 4977-bp deletion in Hürtle cell thyroid carcinoma,mutations of complexes I and IV in thyroid carcinoma arising

Mitochondria and Cancer


Table 1: Mutations of mtDNA occurred in solid and haematological malignancies

Malignancies Mutated genes or regionsBreast cancer D-loop [129], 16S rRNA [130], ND1 [131], ND4 [131], ND5 [131], Cyt. b [131]Ovarian cancer D-loop [132], 12S rRNA [133], 16S rRNA [133], Cyt. b [133]

Colorectal cancer 12S rRNA [134], 16S rRNA [134], ND1 [135], ND4 [136], ND5 [135], Cyt. b [137], COX I [138],COX III [137]

Gastric cancer D-loop [139], ND1 [140], ND5 [140]Hepatic cancer D-loop [141], ATPase 6 [141]Oesophageal cancer D-loop [142]

Prostate cancer D-loop [143], RNR1 [143], RNR2 [143], 12S rRNA [144], 16S rRNA [144], ND3 [143], ND4 [143],ND4L [143], Cyt. b [145], COX I [145], ATPase 6 [145]

Thyroid tumour ND1 [146], ND2 [147], ND5 [147], COX I [148], ATPase 6 [149]Bladder, head and neck, and lung cancers D-loop [150], ND2 [150], ATPase 8 [150], COX II [150]Leukaemia D-loop [151]

from C cells, and a 264-bp deletion (nt. 3323-3588) in renalcell carcinoma. Further analysis revealed that accumulationof mtDNA mutations is correlated with tumour aggressive-ness and multi-drug resistance [4,19,36,40,47,48] or withpoor prognosis and acute symptoms and signs [49]. Theincreasing ease with which the mitochondrial genome canbe analysed, and the availability of a consensus humansequence, have both helped to recognize mtDNA mutationsas a frequent event in carcinogenesis. However, it is difficultto estimate the true prevalence of mtDNA related cancerowing to many clinical guises, presentations and the in-volvement of numerous causative mutations.

The mtDNA mutation rate is 1000 times higher than thenuclear genome. The evolution rate of mtDNA is much fasterthan that of the nuclear genome and several reasons areinvoked to explain this fact: mtDNA is less protected byproteins and is physically associated with the mitochondrialinner membrane where damaging ROS are generated andappears to have less-efficient repair mechanisms than thenucleus [19,47,50]. Also, malfunction of mismatch repair(MMR) and slipped strand misspairing (SSM) should beconsidered as factors for mitochondrial genome instability[50,51].

The proposal that mtDNA mutations and respiratory dys-function may be linked directly to carcinogenesis via apop-totic or ROS–mediated pathways is challenging, but urgentlyneeds experimental evidence in cancer cells, preferably inspecimens from cancer patients. Moreover, to date ROSover-production has been associated with mitochondrialdisease such as NARP (neurogenic muscle weakness, ataxiaand retinitis pigmentosa), MELAS (mitochondrial encepha-lomyopathy lactic acidosis, and stroke-like episodes),MERRF (myoclonic epilepsy and ragged-red fibers), LHON(Leber hereditary optic neuropathy), and KSS (Kearns-Sayresyndrome)[52].

Mitochondrial DNA in the nucleus

Along with the abundance of mtDNA mutations in cancercells, pro-oncogenic conditions become worse whenevertranslocation of mtDNA to the nucleus occurs. Non-pathogenic mtDNA translocation has taken place during theprocess of evolution. Endosymbiotic theory states thatmitochondria originated as bacterial intracellular symbionts,the size of the mitochondrial genome was gradually reducingover a long period, owing, among other things, to genetransfer from the mitochondria to the nucleus [53]. Thestructure of the genomic mitochondrial sequence ho-

mologues indicates that during evolution there occurredtransposition of fragments of the mitochondrial genome intothe nucleus, followed by rearrangements and single nucleo-tide substitutions [54] and to date transpositions of mtDNAsequences to the nuclear genome have been documented ina wide variety of individual taxa [55]. If the process isprovoked in normal cells, consequences may be unexpectedand unforeseen. In normal cells, damaged mtDNA is frag-mented and degraded inside mitochondria, but fragmentscan be transported to the cytoplasm. In some cases theymight be translocated to the nucleus and subsequentlyinserted into nDNA. In this process, mtDNA acts as atransposition element to modify the nuclear genome [50,56-59]. The insertion of mtDNA into nDNA might be beneficial,neutral or disrupting for nDNA, compromising cell integrityand/or homeostasis. Kristensen and Prydz in 1986 [60]reported mtDNA insertion into nDNA in HeLa cells, andinitiated research in this field of mitochondrial medicine.Further experiments have revealed chimeric c-Myc/cytochorome oxidase transcripts in HeLa cells [57-59,62]. Since the initial publication, other cancer cell lineshave been screened for nuclear insertions of mtDNA.Cytochrome oxidases I and III, 12S rRNA, and NADH dehy-drogenase pseudo-genes have been discovered in HeLacells, and ND6 gene was detected in hepatoma cells; cyto-chrome oxidase I, II and III genes in high copy number havebeen discovered in meningioma nDNA [50,56,62,63]. Never-theless, further in vivo experiments, with necessary normalcell DNA as controls, are crucial to investigate the role ofmtDNA “transposition” to nucleus.

Homo- and heteroplasmy in cancer cells

Cells are polyploid with respect to mtDNA: most mam-malian cells contain hundreds of mitochondria, each withmultiple copies of mtDNA. If in a given individual with acondition known as homoplasmy (all mtDNA copies areidentical) mutations are introduced and amplified and theycoexist with wild-type mtDNA, heteroplasmy must be defined.At cell division mitochondria and their genomes are ran-domly distributed to daughter cells and hence, starting froman heteroplasmic situation, different levels of heteroplasmymay be found in daughter cell lineages [2,38,40,52,64]. Incancer cells, mutated mtDNA can be homo- or heteroplasmic[19]. Many reports show that some mtDNA mutations incancer cells are homoplasmic, including mutations in thehaplotype polymorphic region. This raises the question ofmechanisms responsible for normal to mutated mtDNA shift



Figure 1: Dual role of PHB in proteindegradation and stabilization insidemitochondria. Functional PHB cooper-ates with mtHSP70 and HSP60/HSP10complexes during mitochondrial foldingand stabilizes polypeptides in their nativeconformational state. PHB may inhibitm-AAA protease, avoiding excessivedegradation of mitochondrial polypep-tides.

in carcinogenesis [50,64]. At least two scenarios areprobable to drive this alteration. First, mutant mitochondriathat have lost certain mtDNA fragments by deletion seem toreplicate more rapidly than normal ones, resulting in anadvantage in intracellular mitochondrial competition (replica-tive advantage). If the competition is intense (high rate ofcell proliferation), heteroplasmic cells possessing both typesof mitochondria give rise to homoplasmic daughter cells,including mutant mitochondria only. According to mathe-matical models, the rate of transfer from wild type to mutatedmitochondria can be affected by factors including theintensity of intracellular competition and the effectivepopulation size [53]. Researchers in several laboratorieshave reported a high frequency of homoplasmic mtDNAmutations in human tumours and proposed that there wasnot only replicative advantage for mutated mtDNA copies[65,66], but also growth and/or tumorigenic advantages for acell containing certain mtDNA mutations [67,68]. On theother hand, it remains possible that homoplasmy arises intumour progenitor cells entirely by chance without anyphysiological advantage or tumorigenic requirement.Through extensive computer modelling, it has been demon-strated that there are sufficient opportunities for a tumourprogenitor cell to achieve homoplasmy through unbiasedmtDNA replication and sorting during cell division [69].Computer models show that random drift can also be asufficient mechanism to explain homoplasmic nature ofcancer cells [19,38]. Relaxed replication phenomenonanalysis in normal cells showed that replicative or metabolicadvantage is not indispensable for homoplasmy to arise[19,64,68]. Although a role for other mechanisms is notexcluded, random processes are sufficient to explain theincidence of homoplasmic mtDNA mutations in humantumours. Depending on how the mutant copies are distrib-uted between stem and transition cells, the mutants aredepleted or enriched and the number of mutants in thelineage fluctuates as a random walk. On average, onlyapproximately 70 generations are required for a mutationthat is destined to become homoplasmic. The number of 70generations is small if compared with the number of celldivision that a tumour progenitor cell is expected to undergo[65,66].

The functional implications of the build-up of mtDNA mu-tations, both in homoplasmy and in heteroplasmy, have notbeen defined yet, but they may lead to functional alterationsof the mtDNA-encoded proteins. Most of the mtDNA-

encoded proteins are involved in the electron transportchain, and hence these may lead to electron flow disruptionand increased generation of ROS [19].

Mitochondrial protein stabilization in cancercells and apoptosis

In addition to DNA metabolism, protein metabolism incancer cells is also disrupted. As a result of increased ROSgeneration, folding and assembly of OXPHOS proteins isimpaired [17,70,71]. Additionally, this condition is worsenedby unfolded protein stabilization by heat-shock proteins(HSPs)[72]. In mitochondria, interaction with unfoldedpeptides has been reported for heat-shock protein 70(HSP70) and prohibitin (PHB) proteins, for example OXPHOSpeptides not incorporated in the inner mitochondrial mem-brane (IM)(Figure 1). Excess of protein aggregates leads toIM disintegration, increased ROS production, pro-proliferative cell signaling activation, and possibly to celltransformation [73-75]. Oxidative damage in cancer cellsmay be increased by ionizing radiation. In fact, in addition tothe recognized effects of radiation on nDNA, it impairsmitochondrial function through loss of enzymatic activity,oxidative phosphorylation and onset of lipid peroxidation[74]. Since during ROS production misfolded OXPHOSproteins are stabilized in their unfolded state by mitochon-drial HSPs, the use of an HSP-inhibitor [76] or the knockoutof the heat-shock transcription factor 1 (Hsf-1)[77] mayfavour radioresistance. If perturbation of protein metabolismis involved in carcinogenesis, it cannot be excluded thatprotein turnover modifiers may also be involved in thisprocess.

To date many pathways of programmed cell death havebeen described [78]. One of the best known apoptotic celldeath pathways is p53-dependent [79,80]. Induction ofapoptosis is one of the central activities by which p53 exertsits tumour-suppressing function. In transcription-dependentapoptosis, the biphasic kinetics of p53-targeted geneexpression has been described. Puma, Noxa, and Baxexpression appear at 2, 4, and 8 h, respectively, while Bid,Killer/DR5 and p53DinP1 are induced after 20 h [81]. Apop-tosis-related p53 target proteins include pro-caspase-9activator Apaf-1, pro-apoptotic proteins from the Bcl-2 familysuch as Bax and Bid [82,83], Noxa [84], Puma [82], Asc,caspase-6 [85-87], CD95 (Fas/APO-1) and Killer/DR5receptors, as well as the mitochondrial potential uncoupling



Figure 2: Mitochondrial p53 interac-tions. In response to a broad spectrumof apoptotic stimuli, p53 may translocateto mitochondria and promote apoptosisby interacting with HSP60/10, HSP70,polymerase γ, mtTFA, Bcl-2, and Bcl-XL.p53/HSP complexes may interfere withprotein re-folding. The interaction of p53with Bcl-2 family proteins may inducecitochrome c release. The mtTFA/p53complex formation may cause a confor-mational change of mtTFA that isrequired for binding to damaged mtDNAand p53/polymerase γ complex, and mayinhibit mitochondrial protein synthesis.Finally, we can suppose that a p53-PHBinteraction inside mitochondria mayenhance m-AAA activity, leading toprotein degradation.

protein p53AIP1 [79,88]. At the same time, p53 physicallyinteracts with Bcl-2 family proteins including Bak oligomer,causing permeabilization of the mitochondrial membrane,and rapidly induces cytochrome c release (Figure 2)[89,90].To date p53/Bcl-XL and p53/Bcl-2 complexes have beendescribed [91] and the p53-dependent apoptosis regulatorApaf-1/caspase-9 complex has been recognized [79]. p53promotes apoptosis in response to death stimuli not only bytransactivation of target genes, but also by transcription-independent mechanisms. Recent studies have started todefine the mechanisms of non-transcriptional pro-apoptoticp53 activities, operating within the intrinsic mitochondria-mediated pathway of apoptosis. [79,80]. In response to abroad spectrum of apoptotic stimuli (genotoxic, hypoxic, andoxidative stresses), a fraction of wild-type p53 translocatesto mitochondria (Figure 2)[91,92]. The majority of mitochon-drial p53 localizes to the outer membrane [90], although asubfraction is found in a complex with the major mitochon-drial import proteins such as HSP70 and HSP60 in themitochondrial matrix (Figure 2)[93,94]. The localization ofp53 to mitochondria is rapid (1 h), preceding the release ofcytochrome c and procaspase-3 activation, and is blockedby overexpression of anti-apoptotic Bcl-2 family proteins.Moreover, p53 has been recognized to interact with mito-chondrial transcription factor A (mtTFA)[95] and mitochon-drial polymerase γ (Figure 2)[96]. Human PHB is known toenhance p53 transcription activity inside the nucleus, and itcan be supposed that this interaction may also occur insidemitochondria [97,98]. Human PHB proteins (hPhb1p andhPhb2p) are molecular chaperones localized in the IM and itsname comes from its primary function description (PHB:proteins that hold badly formed subunits). The PHB com-plex has been shown to have a role in the stabilization ofnewly synthesized subunits of mitochondrial respiratoryenzymes [99]. Nevertheless, first reports on PHBs describedthem as negative cell-cycle regulators, and only later ex-pression of hPhb1p and hPhb2p has been correlated withmitochondrial proliferation [73,100] and differentiation,atresia, and luteolysis in the ovary [101]. The palisade modelsuggests that the PHB complex forms a barrel-line structure,similar to HSP60 [102]. hPhb1p and hPhb2p form a 106-Dacomplex, composed of 14 subunits (hPhb1p - 32 kDa,hPhb2p - 34 kDa) in 1:1 ratio [71,73,74,76,77]. PHBs areconstitutively expressed in mammal tissues, overexpressedin regenerating liver, chemically induced in cancer cells,

human endometrial hyperplasia and adenocarcinoma[103,104], hepatocellular carcinoma [105], gastric cancer[106], 13 breast cancer cell lines [103,107], and bladdercancer [103]. In cancer patients, PHBs can be detected inserum and therefore used as a clinical marker for diseaseprogression [108]. The level of PHB expression is down-regulated after cisplatin, doxorubicin or methotrexatetherapy and in the ageing process [77,109].

The etiology of PHB overexpression seems to originatefrom a Myc-binding element found in the promoter region ofPHB, whereas c-myc proto-oncogene is overexpressed inmany cancer cells and may facilitate PHB expression[74,103,110]. Although many cancer cell lines and tumorsamples, including breast, ovarian, hepatic and lung cancer,have been tested for phb gene mutations, only sporadicbreast cancer samples, where loss of heterozygosity (LOH)has been reported [111,112], seem to be positive. It stillneeds to be taken into account that the entire gene sequencehas not been tested [113]. Inside mitochondria, the PHBcomplex seems to modulate the activity of the matrix ATP-dependent protease (m-AAA), belonging to an evolutionaryconserved family of proteases localized both in the matrix,m-AAA, or in the mitochondrial intramembranous space, i-AAA [114-116]. The significantly increased protein degrada-tion in Phb1p (-/-) or Phb2p (-/-) cells may suggest a PHBfunction as a negative regulator of m-AAA proteases. PHBby protein-protein interactions seems to stabilize m-AAAproteases in the low-activity conformation by forming a PHB-AAA supercomplex (Figure 1). PHB seems to modulate theactivity of m-AAA proteases, as degradation of proteins thatare not bound to the IM is increased in mitochondria lackingPhb1p or Phb2p. PHB might also modulate accessibility andconformation of IM proteins − potential substrates of prote-ases. This PHB would prevent premature degradation ofproteins not incorporated into IM and therefore assist inOXPHOS assembly (Figure 1)[103,114-116].

Besides its chaperone activity, PHB is also classified asanti-apoptotic protein [69]. Its interaction with pRb proteinhas been widely explored [117]. Transcription factor E2F(regulated by pRb) interacts with PHB, which leads to cellcycle arrest [118], as PHB favours E2F inactivation mediatedby pRb, p107 and p130 [119]. PHB inhibits transcriptioninduced by E2F and is considered as an active player of cellsignaling cascades [117,120]. Moreover, the interaction ofPHB with SV40 T-antigen has been reported [119], and PHB



localized in the nucleus was reported to mediate histonedeacetylation [117] and enhance p53 transcription activity[121]. PHB also inhibits DNA synthesis in fibroblasts andHeLa cells. Overexpression of PHB protects B lymphocytesagainst apoptosis induced by topoisomerase inhibitors[107,113]. In cytoplasm, PHB is involved in cellular Ras-p38αMAPK signaling [122]. PHB is overexpressed in responseto vitamin D. The screen for putative transcription factorbinding sites identified two vitamin D receptor (VDR)/retinoidX receptor binding sites in the 1-Kb promoter region of PHB[123]. Moreover, PHB is known to interact with an ATP-dependent protease degrading misfolded proteins and thismay lead to complex formation among p53, m-AAA and PHBinside mitochondria (Figure 2). The reported interactionsseem to be indispensable for cell homeostasis and apoptoticpathway activation. Association of p53 with mtTFA possiblyinduces conformational change of mtTFA that is required forbinding to damaged mtDNA. This interaction is possibleonly if conformational alterations of the mtDNA double-helixstructure occur, such as mismatches due to cisplatintreatment and ROS [118,119]. Because mtTFA may recruitrepair complexes to damaged mtDNA regions, the change inits damage recognition activity may contribute to the avoid-ance of cisplatin-induced apoptosis [117].

All presented data favour a multi-functional mode of PHBin mitochondrial protein metabolism and in nucleo-cytoplasmic signaling pathways. The diverse array offunctions, together with emerging evidence that its functioncan be modulated specifically in certain tissues and in avariety of disease states, including cancer, suggest that thisprotein can be taken as a potential target in cancer therapy[124].

Although many papers focus on the p53-mediated sig-nalling cascades that control cell growth arrest and/orapoptosis, recent studies demonstrated the role of p53 inregulating the expression of several novel genes linked tothe process of glycolysis and oxdidative phosphorylationsuch us phosphoglycerate mutase (PGM), TP53-inducedglycolysis and apoptosis regulator (TIGAR) and SCO2 [125].

Perspectives

Since the initial publications by Warburg over half a cen-tury ago [18], a number of cancer-related mitochondrialdefects have been identified and described in the literature.Growing evidence suggests that cancer cells exhibit in-creased intrinsic mitochondrial stress, due, in part, tooncogenic stimulation, increased metabolic activity andmitochondria-nucleus signalling malfunction [67]. Despitethe increased identification of signatures of mtDNA damagein transformed cells, the phenotypic effects of these geneticchanges remain to be established [126]. While there aremany reports of these phenomena, the mechanisms respon-sible for the initiation and evolution of mtDNA mutations,and their roles in the development of cancer, drug resistance,and disease progression, still remain to be elucidated.Although it is not clear if mtDNA mutations are a conse-quence of carcinogenesis or the cause of it, research intothe identification of altered expression patterns of mito-chondrial proteins in cancer cells has been made possibleby the relatively recent development of mitochondrialfunctional proteomics. The potential of this field may berealized in the identification of new markers and risk as-sessment as well as therapeutic targets [41,67]. Despite thedifficulties with mitochondrial proteomics, it is likely that thecombination of mitochondrial genetic and proteomic ap-proaches will provide an effective double-edged sword in thefight against cancer. A few methods have revealed thatmitochondrial chaperones and p53 are potential targets ofanti-cancer therapies [6,124,127]. It is hoped that this

strategy will provide specific genetic markers and proteinprofiles which will provide early detection, risk assessmentand new targets for treatment [62]. Finally, the recentlyinitiated generation of mouse models for mtDNA-linkeddiseases will help to solve some of those open questions[128].

Acknowledgments

The authors thank Prof. Andrzej Bienkiewicz (Departmentof Oncology, Medical Universitry of Lodz, Poland), TomaszKrawczyk, (Institute Centre of Polish Mother Health, Depart-ment of Clinical Patomorphology, Poland), Jerzy S.Czarnecki (University of Lodz, Poland) and PrzemyslawTomalski (Centre for Brain and Cognitive Development,School of Psychology, Birkbeck College, UK) for criticalreading of the manuscript and fruitful discussions.

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