Mitochondrial DNA as a potential tool for early cancer detection

The recent surge in mitochondrial research has been driven by the identification of mitochondria-associated diseases and the role of mitochondria in apoptosis. Both of these aspects have identified mitochondrial analysis as a vital component of medical research. Moreover, mitochondria have been implicated in the process of carcinogenesis because of their vital role in energy production, nuclear-cytoplasmic signal integration and control of metabolic pathways. Interestingly, at some point during neoplastic transformation, there is an increase in reactive oxygen species, which damage the mitochondrial genome. This accelerates the somatic mutation rate of mitochondrial DNA. It has been proposed that these mutations may serve as an early indication of potential cancer development and may represent a means for tracking tumour progression. The purpose of this review is to explore the potential utility that these mutations may afford for the identification and monitoring of neoplasia and malignant transformation where appropriate body fluids or non-invasive tissue access is available for mitochondrial DNA recovery. Specifically, prostate, breast, colorectal, skin and lung cancers are discussed.


Mitochondrial characteristics
There ares everal biological characteristics which cast mitochondria and, in particular,t he mitochondrial genome,a sa biological tool for early detection and monitoring of neoplasia and its potentialp rogression. These vital characteristics are important in cancer research, as not all neoplasias become malignant. 1 Mitochondria are archived in the cytoplasmofthe ovum and as such do not recombine. 2 This genome has an accelerated mutation rate,b yc omparison with the nucleus, 3 and accrues somatic mutations in tumour tissue. 4 Moreover, mitochondrial DNA (mtDNA) has ah igh copyn umberi n comparison withthe nucleararchiveo fD NA. There are potentially thousandsofmitochondrial genomes per cell, which enables detection of important biomarkers, even at lowl evels. In addition, mtDNAc an be heteroplasmic,w hich means that disease-associated mutations occur in asubset of the genomes. The presence of heteroplasmyi sa ni ndication of disease and is found in many human tumours. 5,6 Identification of lowlevels of heteroplasmym ay allowu nprecedented early identification and monitoring of neoplastic progression to malignancy. Mitochondria have beenimplicated in the carcinogenic process because of their role in apoptosis 7 and other aspectsoftumour biology.Deletions in the mitochondrial genome are associated with organelledysfunction and serve as biomarkersa sw ell. 8 Finally,m tDNAi samodest molecule by comparison withits nuclear cellmate.The compact size of the mitochondrial genome allows extensive, economical biological characterisation such as complete genome sequencing. Coding for just 13 enzyme complex subunits, 22 transfer RNAs and two ribosomal RNAs, the mitochondrial genome is packaged in acompact 16,569 base pair (bp) circular molecule. 9 These products participate in the critical electron transportp rocess of ATPproduction. Collectively,mitochondria generate 80 per cent of the chemicalfuel which fires cellular metabolism. As aresult, nuclear investment in the mitochondria is highthat is, several thousand nuclear genes control this organelle in order to accomplish the complex interactions required to maintain anetwork of pathways, which coordinate energy demanda nd supply.

Prostate cancer
Prostate cancer (PCa) is at ransformation of the secretory epithelialc ells lining the ductal network of the prostate gland. In the USA alone,o ver2 30,000 men developed PCa in 2005 ( Facts andF igures,A merican Cancer Society,2 005). The wayi nw hich humanp rostatee pithelialc ells metabolise citrate is strikingly different from other soft tissues. The peripheral zone of the prostate gland, which accounts for the majority of prostate tumours,h as au niquely different metabolism. For this reason, the metabolic characteristics of this tissuea re centralt om tDNA damage and its role in PCa. Unliket he centralz one,t he peripheral zone cells accumulate very high levels of zinc,d ue in partt ot he activity of az inc transporter. 10,11 Zinc levels in these cells are about ten-fold higher than in other cell types. Theh igh zinc levels in the mitochondria inhibit an initialK rebs cycle enzyme,a conitase, therebyp reventingt he isomerisation of citrate to isocitrate, which is required for the complete metabolism of citrate. This precludes the movement of NADH and FADH 2 from the Krebs cyclei nto the electron transportc hain. Interestingly, during the pathway of malignantt ransformation, zincl evels drop and the Krebs cycle becomes active. Peripheral zone prostate cells then become energy efficient, consuming substantial amounts of oxygen, in contrast to many other tumours that aree nergy inefficient. The increased functional capacity of theKrebs cycle in thesetransforming cells might be responsible for the up-regulation of enzymes involved in energy-generatingp athwayss ucha s b -oxidation. 12,13 Hypothetically,t his raises the intriguingp ossibility of high reactive oxygen species (ROS) production, which can cause significant oxidatived amage to the mitochondrial genome. There mayb easudden increase,o rb urst, of ROS, which heavily damagem tDNA, perhaps overwhelming any operational mtDNAr epair mechanisms. Proteomicsa nalysis reveals altered expression of nuclear-encoded complex IV subunits in pre-malignantp rostate epithelial cells. 14 Such protein changes could affect the functionso ft his complex, resulting in defectiveo xidativep hosphorylation. Thus, as tate of reversed electron flowi nt he redoxs eries can arise,l eading to elevated ROSp roduction. Under normal circumstances, elevated ROSi nduces the activity of antioxidant scavengers such as glutathione peroxidase.I ns upport of the pivotal role of ROSinPCa development, however, is the early silencing of the gene encoding glutathione S-transferase-p ( GSTP1)v ia hypermethylationo fi ts promoter in PCa 15 and the presence of high levelso fh ydrogen peroxide in PCac ells compared with normal prostate epithelial cells. 16 An early metabolic switch which mayl ead to increased ROSp roduction in association with al oss of antioxidant mechanisms should culminate in accelerated mtDNA mutations in PCa. Not surprisingly then, the few reports of mtDNAm utations in PCa indicate as tate of hypermutagenesis. 17 -20 The first study of mtDNAmutations in PCa examined largem tDNAd eletions by gel electrophoresis and observedapositivec orrelation between deletion frequency and advancing age. 21 Four other studies involved sequencing regions of the mtDNA genome.T he D-loop and adjacent regions were examined in three studies. 17 -19 In these studies, at otal of 34 substitutions and four small insertions and deletions were detected. In addition, the following mutations were detected in coding genes flankingt he D-loop: one mutation each in the 16S ribosomal RNA gene ( 16SrRNA) and the transfer RNA leucineg ene( tRNA leu ); three in ND4; and four each in ND1, ND5 and CYTB.T he fifth and most recent study of mtDNAa lterations in PCa focused on the COI gene.T his study involved al arge population sample from which at otal of 21 unique nucleotide changes were detected in PCa. Tw om utations, G6261A (six individuals) and A6663G (fivei ndividuals) seem to be mutation hot spots in PCa.
MtDNA alterations in the prostate gland mayh aves ignificant utility for early PCad etection, givent he uniqueness of this molecule and the mode of occurrence of mtDNA mutations in the prostate.T he metabolic and mtDNA alterations in the prostate are likely to be early events in PCa development, since they areo bservedi np re-malignant histological benign-appearing glands. Prostate epithelial cells are present in prostate fluids such as prostate massage fluid, and the high copyn umber of mitochondrial genomes coupled with the accelerated mtDNAm utations in PCa and, in some instances, the homoplasmic nature of the mutations, should offer ad etection advantage over nuclear genome alterations in biofluids. 19,22 Breast cancer Much like prostate cancer,b reast cancer is also at ransformation of the epithelial secretoryc ells lining them ammary ducts. Breast cancer wasd iagnosed in nearly 213,000 women in the USA in 2005 ( Facts andF igures,A merican Cancer Society,2 005). More than ten papersd escribing altered mtDNAinhumanbreast tumourtissue have been published in the twoy earss ince Carewa nd Huang published their comprehensive review of mtDNA defects in cancer. 4 There have been av ariety of mtDNAa lterations found in breast tumour tissue since the first reportpublished by Bianchi et al.about ten yearsa go. 23 While the precise frequency,r egion of the mtDNAg enome characterised and nature of mtDNA mutations associated with breast tumour tissue varies in each report, there is compelling evidence that somatic mutations in mtDNAr epresent informativeb iomarkersf or detecting disease.Anumber of investigatorss uccessfully obtained sufficient quantities of DNA from nipple aspirate fluid (NAF) and from ductal lavage to performm tDNAa nalysis, thus providing the possibility of non-invasivem ethods using mtDNAa sasensitivem eans to detect breast cancer.
Rates of somatic mtDNA mutations in breast cancer range from that reported in a2 002 study by Ta n et al. 24 (74 per cent; 14/19 tested) to the recent results of Ta n et al. 25 looking at am uch larger sample size,w ithar eported rate of 45 per cent heteroplasmy. Zhu et al.f ound mutated mtDNAi n9 3p er cent of breast cancer samples( 14 of 15 tested), withthe frequency of mutations higher in the D-loop than in other loci tested (1.5p er cent versus 0.18p er cent). 26 In addition, Zhu et al.t ested whether NAF provided mtDNA that carried mutations found in the patient'stumour and found that 40 per cent of the NAF samples contained some of the mutations observedi nt he patient'st umour mtDNA. Rosson and Keshgegian found mtDNAm utations in 15 of 15 tumour samplesm icro-dissected by laser capturem icroscopyf rom1 5 different patients. 27 Then umber of mutations found in the D-loop rangedf romo ne to 15 relative to an adjacent normal tissue control mtDNAs equence.A so thersh avef ound that adjacent normal control tissue does not necessarily represent the 'wild-type' and frequently harbours tumour-type mtDNAmutations, the results of Rosson and Keshgegian may under-reportt he true number of mutations in the samples studied. 27 Zhu et al.f ound a4 ,576 bp deletion in mtDNA to be ag ood marker for breast cancer,i nt hat it wasp resent 77 per cent of the time in 39 tumours and 0p er cent in true normals amples. 28 Interestingly,1 3p er cent of the histologically 'normal' adjacent tumour tissue sampless howedt he 4,576 bp deletion, suggestingt hat, while the histology appears normal, the genotype is moving towards the tumour state. Dani et al.found adifferent mtDNAdeletion-the 4,977 base pair deletion-in 24 percent of breast tumours but concluded that this resulted from contaminating adjacent tissue and, in fact, stated that tumours areessentially free of the 4,977base pair deletion. 29 This is in contrast to Zhu et al., who found the 4,977 base pair deletions in breast tumours (18/39,4 6p er cent) to occur at ar ate higher than in adjacent normal tissue (13/39,3 3p er cent) and normal tissue (6/23, 26 per cent). 28 The explanation for this apparent discrepancy remains to be determined. Isaacs et al.i nvestigated the D310 30 mtDNA marker in conjunctionw ithl oss of heterozygosity from 14 women of known BRCA1 status and found three of nine mtDNAm utations in BRCA1 carriers. They further demonstrated that the same DNA typingr esults could be obtained from NAFasw ellasductal lavage samples. 31 D310is of particular note becausei ns omeg roupst his nucleotide is a Taso pposed to aC .I nasimilar study,P arrella et al. looked at the D310 mtDNAm arker and found that three of 20 mtDNAm utations (15 per cent) had mutated in the tumour compared with the normal tissue. 32 They reported that, comparing tumourw ith blood mtDNA,6 1p er cent of the tumours had mtDNAm utations.

Colorectal cancer
Colorectal cancer affected over 145,000 people in theU SA alone in 2005 (Cancer Facts and Figures 2005). More than 95 per cent of colorectal cancersa re adenocarcinomas which develop from adenomatous polyps (adenomas);however,only a small portion of theselesions progress to invasivecancer (1 -10 per cent). Current diagnostic tools include thef aecal occult blood test and the multitarget assayp anel (MTAP), which looks for mutations associated witht he nuclear genome (Jani National Cancer Institute). Deficiencies in DNA repair and ah igher mutation rate in the mitochondria result in the accumulation of mutations that mayp rovide am eans for the early detection of humanc olorectal lesions (polyps or cancer) and mayi ncrease the utility of the MTAP test. For those at risk (familialo ra ge related), ac olonoscopyi st he screening method of choice.
Previous research examining the D-loop or non-coding region (NCR), or performing completes equencing of the mitochondrial genome in colorectal tumours, found that the majority of the somatic mutations were homoplasmic transitionsa nd single bp insertions or deletions, and that there were few large-scale deletions. 33 -36 Habano et al. were among the first to reportm icrosatellite instabilityi n2 0/45 colorectal cancer samples in the NCR of the mitochondrial genome. 37 The nature of thesealterations is consistentwith ROSdamage and lowl evels of DNA repair.F urthermore,s omatic mutations and altered gene expression were found in: 12SrRNA; 16SrRNA;the NADH ubiquinone oxidoreductase subunit genes ND1, ND4, ND5;t he cytochrome oxidase subunit genes COXI, COXII, COXIII;a nd the cytochrome gene CYTB.R ecently,G uleng et al.r eported no mutations in the ND1 and ND5 genes. 38 Aikhionbare et al.investigated the mitochondrial genome with restriction analyses, followedb y the sequencing of length variants for three histological subtypes of colorectal adenomas, in association with tumour and matched surroundingn ormal tissues. 36 Thirty-eight sequence variants for the precursor and cancerous lesions were identified. This method does not detect single nucleotide polymorphisms (SNPs) that do not create ar estriction site, however, meaning that the actual number of alterations could be higher.I nterestingly,n one of their 38 sequencev ariants matched the somatic mutations cited in previous reviews, 4,5 but the regionso fm itochondrial instability were similar. The results of Aikhionbare et al.i ndicate ah igh prevalence of mutations in tumour tissue, withm uch lowern umbersi n pre-cancerous lesions and no mutations in the surrounding normal tissue.Aprogressive patterni si ndicated.

Skin cancer
The incidence of skin cancer has steadily increased over the past decade,w ith more than 1m illion new cases currently being diagnosed each year in the USA and 65,000 in the UK (figure providedb yC ancer Research UK).A partf rom malignant melanoma, non-melanoma skin cancer (NMSC) accountsfor around90per cent of skin cancersand consists of basal cell and squamous cell carcinomas (BCC and SCC, respectively). BCCs are the most common formo fN MSC and arise predominantly from the basal keratinocytes of the epidermis but also from cells in hair follicles and sebaceous Parr et al.

Review REVIEW
glands. They are locally invasiveb ut rarely metastasise.S CCs are also derived from basal keratinocytes; however, in contrast to BCCs, SCCs maym etastasise.C utaneousm alignant melanoma is amalignant tumour of the melanocytes, usually arising in the epidermis, and is the most lethal of the skin cancers.
The cause of the great majority of skin cancers, including malignant melanoma, in pale-skinned individuals is ac ombination of the DNA-damaging effects of ultraviolet radiation (UVR)i ns unlight and the characteristics that determine the response of the body to that radiation. In human skin, mtDNA, rather than nuclear DNA,i sauseful biomarker of UV-induced DNA damage. 39 -41 Moreover, within the field of skin cancer research, there is an additional advantage of using mtDNA, namely that there is no evidence of nuclear excision for ther epair of DNA photoproducts (e.g. cyclobutane pyrimidined imers) in mtDNA. 42 -45 To gether,t hese factorsl ead to accumulation of photodamage in skin mtDNA withoutc ompromising cell function.
These aspects, togetherwiththe fact that UVR is important in the developmento fs kin cancer and has been shown to induce mtDNAd amage in humans kin, 40,46 led to the first detailed study of mtDNAd amage in human NMSC. 47 This work showedc lear differences in the distribution of deletions between the tumour and the histologically normalp erilesional skin.
In the literature,t here are threer ecent studies that have investigated the relationship between mtDNAa nd cutaneous malignant melanoma. 48 -50 Tw oo ft he three studies 48,49 have screened for mtDNAm utations (but not deletionso rd uplications) in theN CR alone, which constitutes less than 7p er cent of the mitochondrial genome. The study by Ta keuchi et al. 48 found that the somatic mutations in the D-loop region of tumoura nd paired plasma did not correlate witht he clinicopathological characteristics. The study by Deichmann et al. 49 found microsatellitei nstability (mtMSI) of mtDNAC n tracts in the primaryt umours.T he larger patients tudy by Poetsch et al. 50 investigated the D-loop,a sw ell as looking for the 4,977 bp common deletion in 61 primarym alignant melanomas and in neighbouring normal skin tissue.P oint mutations were ar are feature,w hereas mtDNAi nstabilityi n the D-loop (mtMSI) wasf ound in 13 perc ent of primary nodular tumours and in 20 per cent of metastases. The 4,977 bp common deletion wasdemonstrated in 10 per cent of melanomas.
The increased frequency of the mtDNAdeletions and point mutations in human skin with increasing UVRe xposure provides am easureo fc umulative UV exposure in human skin. To gether with the mtDNA damage identifiedinthe skin cancer studies, this mayi nt urnp rovide an early detection tool for skin cancer development, as well as providing a method for monitoring the long-terms afety of clinicalU V phototherapyr egimens, since ther isk of skin cancer must be minimised.I nterestingly,m tDNAD -loop alterations can be detected in blood and mayt hereforep otentially act as a circulating DNA melanoma marker. 48 In addition, C n tract alterations occurred in twoo ut of nine primarym elanomas with subsequent metastasis, whereas none of the 20 nonmetastasising cutaneous melanomas were affected. 49 These somatic changesm ay reflect genomic imbalance during tumourp rogression and mayt hereforeb eu sefulf or the assessment of patients' prognosis with as imple,s mall punch biopsy.E arly diagnosis of skin cancer is particularly desirable because of the successful methods of treatment which are currently available to the dermatologist.

Lung cancer
Lung cancer comprises as pectrum of diseases and is the leading cause of cancer-related deaths among men and women in developed countries. There aret wo major classifications of lung cancer,s mall cell (SCLC)a nd non-small cell (NSCLC) carcinoma. SCLC is divided into three subtypes: small cell, mixed small cell/large cell and combined small cell carcinoma (smallc elll ung cancer combined with neoplastic squamous and/or glandular components). Although SCLC is more responsive to chemotherapya nd radiation, it is usuallyw ell spread at primaryd iagnosis, makingi td ifficult to treat successfully.E venw ith treatment, the overall survival rate at fivey earsi s5-10p er cent. NSCLC is ac onglomeration of at least three distinct pathohistologies: squamous cell carcinoma, adenocarcinoma and large cell carcinoma. Localiseds urgical resection is the standard treatment; however, all newly diagnosed NSCLC patients arep ossible candidates for treatment studies.
Currently,there aremore than 170,000 single lung nodules 'discovered' in patients each year in the USA alone.( Facts and Figures,A merican Cancer Society, 2005) Most of these are found during routinel ung X-rays, meaning that detection techniques are really based on chance. Confirmation of diagnosis includes afollow-up biopsy,computed tomography(CT) scan or 'watchful waiting', where X-rayo rC Ts cans are repeated at intervals to assessc hangesi nt he lesion. The' gold standard'i ss urgical resection; however, 60 per cent of lung nodules are benign,h ence,o verh alf of the patients will undergou nnecessarys urgery. From ad iagnostic perspective, there arep romisingm oleculart ools, such as those detecting telomerase activity in tumour cells. Improvement in the diagnostic strategy for lung cancer is ac ritical need in oncology. Indeed,t he highm ortalitya nd morbidity is due to diagnosis of thed isease well after it has spread. MtDNA mayo ffer an advantage over current techniques. Suzuki et al.scored D-loop mutations in 12 SCLC and 16 NSCLC matched B-lymphoblastoid cells lines. 51 In addition, as ubset of the C n wass equenced in 55 resected NSCLC and corresponding non-malignant lungs.M utations were observedi n1 7o ft he 28 cell lines (61 per cent; eight of 12 SCLC and nineo f1 6N SCLC). Moreover, 95 per cent of the SNPs occurred in the hypervariable regions. In addition, single base substitutions were more frequenti nt he D-loop if the C n wasaffected by an insertion or adeletion. For this reason,o nly the C n wass equenced in the 55 resected samples. Within this group,1 1( 20 per cent) had changes. Likewise,i na ne arlier study,F liss et al.f ound a4 3p er cent mutation rate in six of 14 lung cancer samplesw hen sequencing the entire genome and D-loop only from selected samples. 22 Further work mayi dentifys putum as ar eliable source of mtDNAf or monitoring lung neoplasia.

Conclusions
There is ap ressing need to focus on the treatment, and thereforet he early identification and subsequent monitoring, of pre-cancerous lesions, 1 and the analyses of changes in the mitochondrial genome,a ssociated with neoplasia, holds promise in fulfilling this need. Here we have reviewed five specifict ypes of cancer which have tissue,o rb iofluids, from which mtDNAc an be extracted and analysed from an early detection perspective. Although promising, the early results described here await further work and validation. There are many important questions yett ob ea ddressed: such as the relationship between mtDNAa nd the actuald isease; are mutations causativeo rm erely ar eflection of nuclear instability? And, are these processes independent events? Alterations in the non-codingD -loop suggest genome instability; however, as studies focus moreo nt he codingr egions of the mitochondrial genome,p articularlyi nt he case of nonsynonymous mutations in the genes contributing products to the electron transportp rocess, metabolic implications are evident. Moreover, mutations in mitochondrial transfer RNAs indicate the possibility of ag lobal mitochondrial translational shut down. Importantly,t he wide range in the number and locationo fm itochondrial alterations in patients, within and between studies addressing the samem alignancies, indicates the need for ac oordinated approach among researchers. Finally,the interplaybetween nuclear and mitochondrial genes requires careful investigation and mayh old the final understandingo ft he mitochondrial role in tumourigenesis. 52