The role of mitochondrial DNA in breast tumors

The

role

of

mitochondrial

DNA

in

breast

tumors

Marjolein

J.A.

Weerts,

Stefan

Sleijfer

and

John

W.M.

Martens

DepartmentofMedicalOncologyandCancerGenomicsNetherlands,ErasmusMCCancerInstitute,ErasmusUniversityMedicalCenter,Rotterdam, TheNetherlands

Somatic

variation

in

mitochondrial

DNA

(mtDNA)

has

been

described

in

primary

breast

tumors,

including

single-nucleotide

variants

and

variation

in

the

number

of

mtDNA

molecules

per

cell

(mtDNA

content).

However,

there

is

currently

a

gap

in

the

knowledge

on

the

link

between

mitochondrial

variation

in

breast

cancer

cells

and

their

phenotypic

behavior

(i.e.,

tumorigenesis)

or

outcome.

This

review

focuses

on

recent

findings

on

mtDNA

content

and

mtDNA

somatic

mutations

in

breast

cancer

and

the

potential

biological

impact

and

clinical

relevance.

Introduction

Ourknowledgeonthegeneticmakeupofbreastcancerhasbeen

rapidlyexpandedbymassiveparallelsequencingofprimarytumor

specimens [1]. With this technique, major somatic alterations

includingsingle-nucleotidevariants,smallinsertionsordeletions,

copy numbervariationsandlargestructuralvariantshave been

characterized in the tumor chromosomes. So far, almost 100

tumor-driving genes have been identified [1]. Also, mutational

signatures of base substitutions and rearrangements have been

identified,pinpointingthe processesshapingbreastcancer

gen-omesandpavingthewaytowardnewdiagnosticsandtreatments

[e.g., poly ADP ribose polymerase (PARP) inhibitors for cases

carryingtumorgenomesignaturesinducedbydefective

homolo-gous-recombination-basedDNAdouble-strandbreakrepair].

Often forgotten or overlooked in these findings isthe

mito-chondrialDNA(mtDNA).Mitochondriaareessentialinmultiple

cellularprocesses,withenergyproductionandinitiationof

apo-ptosis evident in the hallmarks of cancer [2]. mtDNA encodes

proteinsessentialfortheoxidativephosphorylationsystem and

thusmitochondrialfunction.Nearlyacenturyago,themetabolic

switchfromoxidativephosphorylationtofermentationoftumor

cells,eveninthepresenceofoxygen,wasdescribed[3].Despitethe

widelyrecognizedimportanceofmitochondriaincancerandthe

roleofmtDNAinmitochondrialfunction,sofaronlyafewstudies

haveexploredmtDNAinlargecohortsofhumancancers[4–8].

Thisreview focusesonrecentfindings onmtDNA content and

mtDNA somaticmutations inbreast cancer and their potential

biologicalimpactandclinicalrelevance.

mtDNA

HumanmtDNAisgene-dense;itisonly_~17000basepairsinsize

butencodes13proteins,aswellastworibosomalRNAsand22

transferRNAsfunctioninginthemitochondrialprotein

trans-lation apparatus (gene density of 1 per~0.45 kilobases). By

contrast, the 23 chromosomes of the nuclear DNA (nDNA)

comprise~3billionbasepairsand>20000genes(genedensity

of1per~120kilobases).CharacteristicformtDNAisthat

multi-plecopiesofmtDNAcanresideinasinglemitochondrion,and

that multiple mitochondria can reside in a single cell. As a

result,thenumberofmtDNAmoleculespercell(mtDNA

con-tent)ishighlyvariablebetweentissuetypes[9–11].Generally,

themtDNAcontentisdependent ontheenergy demandofa

cell;for example,skeletal muscleandliver cells(high energy

demand) harbor thousands of mtDNA molecules but blood

lymphocytes (lower energy demand) contain only hundreds

ofmtDNA molecules [12].Thiscell-type-specific mtDNA

con-tentis assumed tobefairly stable underphysiological

condi-tionsbutcanbealteredbystresssuchasexogenoustoxins[13]

or viral infection [14]. Also, the mutation rate of mtDNA is

severalordersofmagnitudehigherthanthatofnDNA[15,16],

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SCREEN

Correspondingauthor:Weerts,MarjoleinJ.A. (m.weerts@erasmusmc.nl)

1202 1359-6446/ã

mainly attributed to the fidelity of the mitochondrial DNA

polymerase(POLG)[4,17,18].ThepolyploidnatureofmtDNA

combinedwithitsmutationrateinvokestheconceptof

hetero-plasmy–thestatewheregeneticallydifferentmtDNAmolecules

residewithinasinglecellorevenwithinasingle

mitochondri-on.Heteroplasmypatternswithinanindividualcandiffer

be-tweentissues [19–22] (Fig.1), where a heteroplasmicmtDNA

variant can either be present in only one tissue within an

individualorinmultipletissuesbutat variableheteroplasmic

allelefrequencies.

mtDNA

changes

in

cancer

ThemtDNAcontentamonghumancancersappearstobehighly

variable (Fig. 2a). Increases and decreases in mtDNA content

compared with tumor-adjacent tissue have been described for

different tumor types [7]. With respect to mutations, somatic

single-nucleotidevariantsaremorecommonthansomaticsmall

insertionsor deletionsin thetumormtDNA [4–6,8].The major

processshapingthemutationalsignatureofthosesingle

nucleo-tidevariants is replication-coupled[4],owingto the fidelityof

POLG,similartotheprocessshapingthegermlinemtDNA

poly-morphicvariation.Among differenttumortypesthenumberof

somatic mtDNA single-nucleotide variants(mutational burden)

pertumordoesnotvaryextensively(medianbetween0and3per

tumor)(Fig.2b),similartothenumberofheteroplasmicmtDNA

variantsintissueswithinanindividual(medianbetween1and4

pertissue)(Fig.1).This isdifferent comparedwith the nuclear

somaticmutationalburdenpertumor,wherethemedian

muta-tionalburden variesroughly between0.5 and10mutationsper

megabase(dependentontumortype)[23].ThemtDNA

mutation-alburdenisthusintheorderof10-to100-foldgreaterthanthe

nDNA mutational burden in human cancers (not taking into

account the mtDNA content). The somatic mtDNA variants in

primarytumorsappearacross theentiremtDNA.Thereissome

recurrence oncertainpositions,whichcanbeexplainedby the

underlying mutationalprocess (byPOLG); butitcannotbe

ex-cludedthatmutationalselectionisinvolvedgivingselective

ad-vantage to certain positions. Besides the mtDNA content and

somaticmtDNA mutations,themtDNA ‘common deletion’has

alsobeenstudiedincancer.This4977base-pairdeletionoccursat

recurringbreakpointswithinthemtDNAandisobservedinseveral

diseases.Althoughthisdeletionhasbeendetectedintumor

speci-mens,itisalsofrequentlydetectedinnon-neoplasticspecimens

suchastumor-adjacentnormaltissueorblood[24]andits

occur-rencehasbeenrelatedtohumanagingaswell[25,26].

AdefinitelinkbetweenchangesinmtDNAcontentorsomatic

mtDNAsingle-nucleotidevariantsandtumorigenesisor

progres-sionhasneverbeenestablished.Inpreclinicalmodelsdepletionof

mtDNA in tumor cells yields increased and decreased in vitro

tumorigenic phenotypes [27–34], as well as gain and loss of

tumorigenicpotentialininvivomousexenografts[33–38].Only

recentlyhasitbeenshownthatthetumorigenicpotentialofcells

depletedofmtDNAisdependentonrestoringfunctionaloxidative

phosphorylation,viatheacquisitionofwholemitochondriaand

their mtDNA from surrounding cells [37,39,40]. However, the

downregulationofoxidativephosphorylationhasbeenassociated

Skin Myocardial muscle Cerebellum Small intestine Kidney 0 9 8 7 6 5 4 3 2 1 15 14 12 13 11 10 Liver Skele tal muscle Cortex Cerebrum Blood Ovary Large intestine MtDNA v ariants

(Number per tis

sue)

Tissue type

Drug discovery today

FIGURE1

NumberofsomaticheteroplasmicmitochondrialDNA(mtDNA)variantsacross(noncancerous)humantissuetypes.SomaticheteroplasmicmtDNAvariants (numberpertissueperindividual)withindifferenttissuetypesobtainedatautopsy(causesofdeath:32.2%cardiovascular-related,23%traumaticinjuries,21.7% naturalcauses,10.5%intoxicationand12.5%unclearorother)aspublishedbyLietal.[22].Heteroplasmylevelsofvariantsnotshown(between0.5%and94.5% allelefrequency).

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withpoorsurvivalinmultiplecancertypes[41].Regardingsomatic

mutationsinthemtDNAofprimarytumors,onlyaminor

propor-tion(<1%)overlapwithmutationsassociatedwithmitochondrial

diseaseandarethusknowntoaffectmitochondrialfunction[4].

Examples existin the literature where specificmtDNA variants

(mainlyathomoplasmy)orspecifichaplotypeshavebeenshown

to have an effect on tumorigenesis or metastatic potential in

modelsystemsby disruptingoxidativephosphorylation[42–44]

(and reviewedin [45]). The occurrence ofvariants that disrupt

oxidative phosphorylation (present either at hetero- or

homo-plasmy)hastodatenotbeenevaluatedinlargecohortsofhuman

tumorspecimens. It appearsthat mtDNA changescan act asa

modifierintumorcells.Heteroplasmylevelscomplicate

interpre-tationofmtDNAchangesdetectedinhumantumorspecimens.

NegativeselectiononmtDNAmutationsthatpotentiallydamage

mitochondrial activity (e.g., protein truncating) has been

de-scribed– nearly always presentat heteroplasmyand seldomat

homoplasmy–whereasmutationsthatappeartohavenoeffecton

(a) (b) mtDNA content (per tumor , log10 transformed) mtDNA variants

(number per tumor)

-6 -4 -2 0 0 1 2 3 4 6 7 8 9 10 11 12 5

Head & neck

Head & neck

Bladder Bladder Cervical Cervical Prostate Prostate Tumor type Tumor type Lung Lung Breast Breast Uterine Uterine Renal Renal Ovarian Ovarian Colorectal Colorectal Gastric Gastric Adenoid cystic Adenoid cystic Hepatocellular Hepatocellular Melanoma Melanoma

Drug Discovery Today FIGURE2

MitochondrialDNA(mtDNA)contentandnumberofsomaticmtDNAvariantsacrosshumancancertypes.(a)mtDNAcontent(log10transformed)within primarytumorsaspublishedbyRezniketal.[7]and(b)(heteroplasmic)somaticmtDNAvariants(numberpertumorperindividual)withinprimarytumorsas publishedbyJuetal.[4].Heteroplasmylevelsofvariantsnotshown(between3%and100%allelefrequency).

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oxidative phosphorylation can drift toward homoplasmy [4,5].

Nevertheless,positiveselectionhasalsobeendescribedforsomatic

variantsinthetumor[8].Thus,therecurrentlyremainsagapin

knowledgeonsomaticmtDNAvariantsandtheir biological

sig-nificanceintumorformationand/orprogression.

Clinical

relevance

of

mtDNA

content

in

breast

cancer

Inbreastcancer,currentclinicalpracticeappliestraditional

prog-nosticmarkers[46]includingageatdiagnosis,tumorsize,lymph

nodestatusandtumorgradetoclassifyindividualprimarybreast

cancerpatientsfortheirriskofmetastasizingand/ordeath[47–49],

and they are used to determine whether or not an individual

patientis advisedto receiveperioperativetreatment. Moreover,

presenceoftheestrogenreceptor(ER)and/orprogesterone

recep-tor(PR),andamplificationofERBB2(HER2)intheprimarytumor,

classify patients in a certain risk group, but this particularly

provides indication for respectively endocrine and anti-HER2

therapy [50]. Primary breast cancer patients who classify as

high-risk based on the traditional clinicopathological markers

receiveperioperativechemotherapyand/orendocrinetreatment,

intended to eliminate potential micrometastases (curative

set-ting).Recently,for patientswith aninconclusiverisk scorethe

indicationforperioperativesystemictreatmentcanbebasedon

gene-expression profiling [51]. In metastatic breast cancer

patients,chemotherapyisalsoamajorlineofsystemictreatment

intendedtoprolonglife(palliativesetting).Here,

anthracycline-based chemotherapy regimens are frequently given, although

taxane-basedregimensarealsocommonlyused[52].Inaddition

toregimensconsistingoftraditionalcytotoxicagents,alsointhe

metastaticsetting,severallinesofendocrine(combination)and/or

anti-HER2treatmentcanbeappliedbasedonthepresenceofER

and/orPRorERBB2amplificationinthe(metastatic)tumor[53–

55].

Numerousstudieshave explorednew biomarkersthat might

addtothecurrentlyavailableprognosticandpredictivemodelsin

breastcancer,whereasonlyafewaddressedwhetheralterationsin

mtDNA could be used for this purpose. A decrease in mtDNA

content is frequently observed in primary breast tumors when

compared with tumor-adjacent normal mammary epithelium

(70% of cases) [7,56–60]. No recurrent association has been

described between the mtDNA content in the primary tumor

andanyofthetraditionalprognosticorpredictive

clinicopatho-logicalmarkers[7,56–64].However,we have recentlyevaluated

theassociationbetweentheexpressionofmtDNAintheprimary

tumorandER statusandobserved apositiveassociationintwo

independent(ERBB2balanced)cohorts[65],indicatingthat

met-abolicdifferencesmightbepresentacrossbreastcancersubtypes.

Inlinewith this,downregulationof nuclear-encoded genes

in-volvedinoxidativephosphorylationhasbeendescribedfor

triple-negativebreastcancer[66].NucleareffectsofestrogenthroughER

onmtDNAtranscriptionhavebeendescribed(asreviewedin[67]),

whereERindirectlyupregulatesexpressionofthemitochondrial

transcription factor (TFAM). Our work has also shown that

patients withthe lowestmtDNA contentin theprimary tumor

hadaworseprognosis(10-yeardistantmetastasis-freesurvival)ina

retrospectivecohortofprimarybreastcancerpatientswith

lymph-node-negative disease who did not receive any perioperative

systemic therapy [62]. Additionally, patients with low mtDNA

content showed an improved outcome to adjuvant

anthracy-cline-based chemotherapy in a retrospective cohort of

lymph-node-positivedisease(distantmetastasis-freesurvivalinthe

cura-tivesetting)aswellastofirst-lineanthracycline-based

chemother-apy ina retrospectivecohort ofpatients withadvanced disease

(progression-freesurvivalin thepalliativesetting)[63].Thisdid

notapplytothepatientstreatedwithmethotrexate-based

chemo-therapy in both settings [63].These associationswith outcome

were independent of the above-mentioned clinicopathological

markers[62,63].Themechanisms underlyingtheseassociations

remaintobeestablished.Apossibleexplanationmightbebasedon

thefactthatanthracyclinesinducesevereoxidativestress[68]and

areknowntoaccumulateinmitochondria,wheretheycan

inter-calate and damage mtDNA [69], whereas methotrexate is an

antimetabolite,ultimatelyleadingtoinhibitionofDNAsynthesis,

andinducesonlylowlevelsofoxidativestress[68].Eliminationof

damagedmtDNAhasbeendescribedinresponsetooxidativestress

[70]. Hypothetically tumor cells with low mtDNA content are

moresusceptibletostressinducedtothemitochondriaormtDNA,

such asthose induced by anthracyclines, than cellswith high

mtDNAcontent.Otherstudieshavealsoreportedonbreastcancer

patient disease-free or overall survival in relation to tumorous

mtDNAcontent[57,58,71,72],butthosestudiescontainedrather

heterogeneousgroupswitheitherrelativelysmallsamplesizesor

noinformationaboutsystemictreatmentsadministered,

render-inginterpretationandcomparisonbetweenstudiesdifficult.

Larg-ercohortsofuniformlytreatedpatientsarenecessarytovalidate

these findings and to further unravel the clinical relevance of

mtDNAcontent–orexpression–quantificationinbreastcancer.

Nevertheless, the putative linkbetween lowmtDNA content

andsusceptibilitytoregimensthatinducesevereoxidativestress

inmitochondriaisinteresting.Thisphenomenonprobablydoes

nottypicallyapplyto breastcancerbutcouldalsobeof

signifi-cance to other human cancers treated with similar regimens.

Anthracyclinesarealsofrequentlyusedinthetreatmentof

sarco-maandhematologicalmalignancies,andthesecancer typesare

thereforeofinteresttoevaluatethelinkbetweenmtDNAcontent

in the tumor and outcome after chemotherapy. In addition,

platinum-based chemotherapyalsoinduces oxidativestress and

changestomtDNA[73,74],whereasbleomycinhasbeenshownto

damagemtDNAmoreextensivelythanitdoesnDNA[73,75],and

thus also for these regimens mtDNA content is potentially a

predictive marker. To take this even a little further, we could

speculatethatnotonlythetumorcellsbutalsonontumorcells

withlowmtDNAcontentaremoresusceptibletoanthracyclinesor

otherregimensthatinducesevereoxidativestress.Interestingly,

theendocrineagenttamoxifenhasbeenshowntoinhibitmtDNA

replicationanddecreasethemtDNAcontentintheliverininvivo

model systems [76], similar to antiretroviral therapy which is

known todecrease mtDNA contentin peripheralbloodcellsof

HIV-infectedpatients[77,78].Thiscouldmeanthatheterogeneity

inmtDNAcontentinnontumorcellsmightresultin(i)

intrain-dividual specific side-effects because certain tissues with low

mtDNAcontentareaffectedtoalargerextentand(ii)

interindi-vidualdifferencesintoxicitiesexperiencedbecausesome

individ-ualsmightbemoresusceptibletotreatmentside-effectsbecauseof

endogenouslowermtDNAcontentintheirhealthytissues.

Elabo-ratingonthis,onecanimaginethatalsonucleareffectsonmtDNA

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contentand/orexpressioncanexertsuchdifferencesonresponse

and/ortoxicity.PerhapssomaticvariantsinESR1thataltertheER

to beconstitutivelyactive alsoinfluence the nucleareffectson

mitochondrialexpression.

Off note, the mtDNA content in peripheral blood of breast

cancer patients is higher compared with controls [79–82] and

decreasedmtDNAcontentinperipheralbloodwasassociatedwith

increased cancer-related fatigue in breast cancer patients [83].

However,becausemtDNAcontentvariesbetweencelltypesand

blood composition was not analyzed, it should be evaluated

whethertheobservedchangesinmtDNAcontentreflectan

alter-ationinbloodcompositioninpatientsversuscontrolsorpatients

suffering fromcancer-related fatigue. Summarizing,we propose

thatthefrequentlyobserveddecreaseinmtDNAcontentinbreast

tumors,potentiallymtDNAexpression,butalsomtDNAcontent

and/orexpressioninnontumortissues,couldbeexploitedtoguide

chemotherapeuticregimendecision-making.

Somatic

mtDNA

variants

in

breast

cancer

Somatic mtDNA variants are frequently observed in primary

breasttumors(70%ofcases)[4,5,61,65].Mostofthesevariants

aresingle-nucleotide variantsandnotsmallinsertions or

dele-tions.Thesevariantsaredistributedalongtheentire

mitochon-drialgenome,showinglargeheterogeneityamongcases,andare

acquiredindependentlyofthethreemajormutationalprocesses

shapingthenDNAwithinbreasttumors[65].Sofar,nosomatic

mtDNAmutationsthatclearlyaffectbreastcancertumorigenesis

or progression have been described; however, some mtDNA

haplogroups have been described as modifiers for metastatic

potential[43,44].

Norecurrentassociationhasbeendescribedbetweenthe

num-ber of somatic mtDNA variants in the primary tumor and the

traditional prognostic or predictive clinicopathologicalmarkers

tumorsize,lymphnodestatus,tumorgrade,ERand/orPRstatus,

orERBB2amplificationstatus.However,anassociationbetween

thenumberofsomaticvariantswithintheprimarybreasttumor

andtheageofdiagnosishasbeendescribed[4,65,84].An

expla-nationforthisassociationisthepresenceofallele-specific

varia-tionamongtissueswithinanindividual[19–22],whichhasalso

beenassociatedwithage[12].Becausethemajorprocess

generat-ingsomaticvariantsasdetectedintumortissuealsoappearstobe

causedbythefidelityofthemitochondrialpolymerase[4,65],the

associationwithanolderageimpliesthatalargeamountofthe

somaticvariantsdetectedinthetumorspecimenareprobablynot

tumor-acquired but already present or acquired in the normal

mammaryepithelialcellfromwhichthetumororiginated.Often

nomatchedmammaryepithelialtissueisavailable,sothe

distinc-tionbetweensomaticvariantsacquiredinthemammaryepithelial

cellsbeforecancerinitiationandthosetrulyacquiredinthetumor

cellscannotbemade.Thisisalimitationfrequentlyoverlooked.

Matchednormalmaterialcommonlyusedinstudiesisblood,but

(somatic)variantsinbloodareunlikelytoadequatelyreflectthe

somatic mtDNA status in the mammary epithelial tissue from

which thecancer arose. Inaddition,the tumor specimen

com-monlycontainsnotonlytumorcellsbutalsoothercellssuchas

surroundingepithelialcellsorimmunecellsinfiltratingthetumor.

Inlinewiththeallele-specificvariationamongtissueswithinan

individual,we haveshown extensivemtDNA heterogeneity

be-tweentumorandtumor-adjacenttissueinasmallpatientcohort

ofbreastandcolorectalcancerpatients[85].

Importantly,theeffectofsomaticmtDNAvariantsinthetumor

isbasedontheactualpositionandconsequenceofthevariant(s)

combinedwiththeirheteroplasmylevelwithinthetumorcells,

ratherthanthenumberofvariantspresent.Onlyifvariantshave

aneffectonthemitochondrialoxidativephosphorylationsystem

or anotherphysiologicalprocess do they have the potentialto

influenceapatient’scancer.Somaticvariantsthathavenoeffect

onaphysiologicalprocessaremerelybystanders.Here,somatic

variantsinthetumormightexerteffectsontumorformationand/

orprogression butmightalsoinducethe above-mentioned

sus-ceptibility to a regimen that induces severe oxidative stress in

mitochondria. Elaborating on this, not only somatic but also

germlinemtDNAvariantscanexerteffectsontheoxidative

phos-phorylation system [86–88]. An illustrative example is that of

mtDNA haplogroups(containing certainmtDNA variants) with

decreased mitochondrial respiration system coupling efficiency

selectedforinpopulationsinhabitingcolderclimates,leadingto

decreasedATPgenerationinfavorofincreasedheatproduction.

Perhapsalsocertain mtDNAhaplogroupsexertincreased or

de-creasedresponse and/or riskof toxicity aftercertain anticancer

treatments.

Although it could be attractive at first sight because of the

multiplemtDNAcopiesandhighmutationrate,theuseofsomatic

mtDNAvariantsincancerlineagetracingorasablood-circulating

biomarkeriscomplicatedgiventhelargeheterogeneityofmtDNA

variantswithinanindividual.First,itisdifficult(ifnotimpossible)

topinpointthetrulytumor-specificmtDNAvariants,becauseof

the potential‘foundereffect’.Second, thedynamics ofmtDNA

contentcanexertbottleneckeffectsonsomaticmtDNAvariants

whenthereisa(large)reductioninmtDNAcontent.Obviously,

suchfounder-orbottleneck-effectsnotonlyapplytodifferences

betweenthenormalmammaryepitheliumbutalsotodifferences

betweenprimarytumorandmetastasesoramongmetastases.For

now,itremainstobeelucidatedwhethersomaticmtDNAvariants

havebiologicalsignificanceintumorformationandprogression,

or perhaps also in susceptibility to certain systemic treatment

regimensandcanbeofaddedvalueintheclinic.

Concluding

remarks

PatientsdiagnosedatahigherageharbormoresomaticmtDNA

variantsintheirprimarytumor.Also,thereislargeheterogeneity

in somatic mtDNA variants within primary breast tumors.

Al-thoughmtDNAcontentintheprimarybreasttumorisnot

associ-atedwithanyofthetraditionalclinicopathologicalmarkers,low

mtDNAlevelsintheprimarybreasttumorindicateamore

aggres-sive cancer, which appears more susceptible to

anthracycline-based regimens. The question remains how mtDNA genotype

(somatic and germline haplotype), heteroplasmy, expression

and content areaffectedin metastatic disease.Also, itremains

tobeevaluatedwhethercertaintumor-specificmtDNAvariants,or

mtRNA expression associated with the subtype, are connected

withpatientoutcomeandhaveanyclinicalimportance.

TheroleofmtDNAvariationin(breast)tumorshasbeenlargely

neglected.A complete picture should be obtained by studying

primary tumor and metastatic sites for their (somatic) mtDNA

variation. The effects these variations have on mitochondrial

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activityshouldbeassessedbyfunctionalassays,suchas

measure-mentoftheactivityofthecomplexesoftheoxidative

phosphor-ylation system, generation of ATP or ability to induce the

apoptoticpathway.Whenassessingsucheffects,oneshouldalso

takeintoaccountgermlineandcell-lineage-specificsomatic

vari-ation, tissue distribution, the interactions betweenthe nuclear

genomeand the mitochondrialgenome, andalso tissue

depen-denceonoxidativephosphorylation.Also,moreinsightisneeded

intotheroleofmtDNAvariationsinhealthytissueontoxicities

andtowhatextenttheymightbeassociatedwithspecifictoxicities

andinterindividualdifferences.Althoughthepivotalworkfrom

Warburg describing the crucial role of mitochondriain cancer

alreadydatesfromacenturyago,mitochondriaandtheirmtDNA

deservemoreattentionnow.

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