Reviews

Mitochondrial DNA variation

and cancer
Piotr K. Kopinski   1,2,4, Larry N. Singh   2,4, Shiping Zhang2, Marie T. Lott2 and
Douglas C. Wallace   2,3 ✉
Abstract | Variation in the mitochondrial DNA (mtDNA) sequence is common in certain tumours.
Two classes of cancer mtDNA variants can be identified: de novo mutations that act as ‘inducers’
of carcinogenesis and functional variants that act as ‘adaptors’, permitting cancer cells to thrive
in different environments. These mtDNA variants have three origins: inherited variants, which
run in families, somatic mutations arising within each cell or individual, and variants that are also
associated with ancient mtDNA lineages (haplogroups) and are thought to permit adaptation to
changing tissue or geographic environments. In addition to mtDNA sequence variation, mtDNA
copy number and perhaps transfer of mtDNA sequences into the nucleus can contribute to
certain cancers. Strong functional relevance of mtDNA variation has been demonstrated in
oncocytoma and prostate cancer, while mtDNA variation has been reported in multiple other
cancer types. Alterations in nuclear DNA-​encoded mitochondrial genes have confirmed the
importance of mitochondrial metabolism in cancer, affecting mitochondrial reactive oxygen
species production, redox state and mitochondrial intermediates that act as substrates for
chromatin-​modifying enzymes. Hence, subtle changes in the mitochondrial genotype can have
profound effects on the nucleus, as well as carcinogenesis and cancer progression.

Ever since Otto Warburg reported that solid tumour cells By 2006, sufficient cancer mtDNA sequence variants
exhibit “aerobic glycolysis” more than 80 years ago1,2, had been reported for a meta-​analysis17. Surprisingly,
there has been interest in the role of the mitochondrion this meta-​analysis revealed that a significant proportion
in cancer. When the basic biochemistry of mitochon- of mtDNA variants reported in cancer were the same
drial oxidative phosphorylation (OXPHOS) was elu- as those observed as being polymorphisms in various
cidated, efforts were invested in determining whether human populations. This finding led to the hypothesis
cancer cells harbour mitochondrial OXPHOS defects. that there might be two classes of cancer mtDNA
1
Howard Hughes Medical
However, by the mid-1970s it had become clear that mutations: severe mutations that contribute to cancer
Institute, University of
Pennsylvania, Philadelphia,
consistent OXPHOS defects were not a general feature induction and milder mutations important in cancer cell
PA, USA. of all tumours3, and interest in the role of mitochondria adaptation. In this Review we present evidence supporting
2
Center for Mitochondrial in cancer declined. this hypothesis.
and Epigenomic Medicine, Between the 1960s and the 1980s, researchers have Still, the mechanisms by which mtDNA mutations
The Children’s Hospital of described the presence of mitochondrial DNA (mtDNA) contribute to the pathophysiology of tumorigenesis
Philadelphia, Philadelphia,
in cells and elucidated the basic rules of mtDNA genet- remain obscure. Certainly, some mtDNA mutations alter
PA, USA.
ics. It ultimately became clear that the mitochondrial reactive oxygen species (ROS) production and redox
3
Department of Pediatrics,
Division of Human Genetics,
genome encompasses hundreds to thousands of copies status, which contribute to tumour cell growth18–20. In
The Perelman School of of mtDNA per cell, in addition to roughly 1,000–2,000 addition, it was speculated that changes in the mtDNA
Medicine, University of nuclear DNA (nDNA)-​encoded mitochondrial genes. and nDNA mitochondrial genes could change the
Pennsylvania, Philadelphia, Mammalian mtDNA was found to code for 13 essential mitochondrial metabolites, which in turn could alter
PA, USA.
subunits for OXPHOS as well as the tRNAs and ribo­ the epigenome and nDNA gene expression20–22. Proof of
4
These authors contributed somal RNAs required for their translation via mitochon- this conjecture came with the discovery that mutations
equally: Piotr K. Kopinski,
Larry N. Singh.
drial protein synthesis4–6. Extensive analysis of human in certain nDNA-​coded mitochondrial tricarboxylic
✉e-​mail: wallaced1@ mtDNA variation has revealed three classes of pheno­ acid cycle enzyme genes cause cancers by modifying
email.chop.edu typically relevant types of variation7,8: recent germ­ the epigenome21,23,24, and we reported that mutations
https://doi.org/10.1038/ line mutations9–11, somatic mutations12–14 and ancient in the mtDNA can result in epigenome modifications in
s41568-021-00358-​w adaptive polymorphisms15,16. osteosarcoma cells25,26. The objective of this Review is to

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Heteroplasmy discuss the inductive and adaptive functions of mtDNA inherited, 16.5-​kb, circular molecule that encodes seven
The simultaneous presence variation in certain cancers (Fig. 1) and to summarize of the 45 polypeptides of OXPHOS complex I (also
of two or more mitochondrial newly emerging mechanisms for these phenomena. known as NADH dehydrogenase), one of the 11 poly-
DNA genotypes in one cell. peptides of complex III (also known as the bc1 complex),
Myoclonic epilepsy and
mtDNA variation and metabolism three of the 13 polypeptides of complex IV (also known
ragged red fibre (MERRF) Most cellular energy is produced by mitochondria in as cytochrome c oxidase (COX)), two of the 18 poly-
syndrome the form of ATP, whose production requires oxygen peptides of complex V (also known as H+-​translocating
A progressive disease including and an oxidizable carbon source (for example, glucose). ATP synthase), the 22 tRNAs and 12S and 16S ribo-
seizures, muscle spasm and
In the process of metabolizing glucose into water somal RNA for mitochondrial protein synthesis and a
weakness, lack of muscle
control, abnormal sensations in
and carbon dioxide while producing ATP, several 1,122-nucleotide ‘control region’ (Fig. 3). All of the remain­
limbs and fat growth (lipomas). high-​energy intermediates are formed. The generation of ing mitochondrial functions are encoded in the nDNA,
these inter­mediates is directly linked to the status of the including all four subunits of OXPHOS complex II (also
mitochondrial genome, as demonstrated by cancer cell known as succinate dehydrogenase (SDH))7.
mutations in mitochondrial genes affecting tricarboxylic There are three main classes of clinically relevant
acid cycle enzymes that alter mitochondrial metabolite mtDNA variants. The first class involves maternally
levels20,22 (Fig. 2). inherited variants that arise in the female germ line.
The first mtDNA sequence was published in 1981 Initially these variants appear within a cell with pre-
(ref.4) and was subsequently corrected to produce the dominately normal mtDNAs generating a mixed cyto­
revised Cambridge Reference Sequence (NC_012920)5. plasm of variant and reference mtDNAs, a state known
The revised Cambridge Reference Sequence, which as ‘heteroplasmy’. Severely deleterious mtDNA muta-
is European maternal lineage (haplogroup) H2a2a1, is tions can cause significant physiological changes while
used as the reference sequence for most clinical mtDNA in the heteroplasmic state. Examples of such variants
studies27. For certain phylogenetic studies, the Recon­ are the tRNALys (MT-TK) m.8344A>G mutation, which
structed Sapiens Reference Sequence is used as the root causes myoclonic epilepsy and ragged red fibre (MERRF)
sequence for tree construction28. mtDNA is a maternally syndrome10,29, the tRNALeu(UUR) (MT-TL1) m.3243A>G

a Inductive
mtDNA Mutation

Mitochondrion

Healthy cell Tumour cell

Normal tissue Primary tumour
b Adaptive
Primary TME with limited nutrients Seeding event Sites of metastasis

Normal
growth Unsuccessful
seeding

Accelerated
growth

Treatment

Sensitivity Resistance Successful seeding

Fig. 1 | Mitochondrial DNA variants can play inductive and adaptive roles in oncogenesis. a | The appearance of
mitochondrial DNA (mtDNA) variants can contribute to the induction of primary tumours, as exemplified by oncocytic
tumours with complex I mutations194. b | Mutations in mtDNA can permit adaptation to new environments and can
originate from ancient polymorphisms, can pre-​exist at low heteroplasmy in normal tissue or can arise de novo. These may
contribute to accelerated growth (blue), enable distant metastasis (green) and potentially confer treatment resistance
(pink). TME, tumour microenvironment.

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I Q III c IV V Histone
Glucose O-Acetyl-
MAS O2 H2O ADP ribose
NAD+ NAD+ ADP ADP HDAC
ANT Sirtuins (I, IIa, IIb, IV)
NADH NADH Pi ATP ATP
NAD+
Oxalo-
Pyruvate Pyruvate mt translation acetate Acetlyhistones
NADH MPC CoA mRNA Acetyl-CoA HATs: GNATs,
NAD+ Citrate
rRNA p300/CBP
tRNA CC ACLY
Lactate Acetyl-CoA mtDNA ATP Histone DNA
Folate SAM
SET DNMT1
Oxalo- Citrate Aconitate
acetate SETD2
CoA DNMT3
Isocitrate DOT1L

NADH NAD+ NADP+ Methyl- Methyl-

IDH3 IDH2 HIF1α histones Hydroxy
methyl-
NAD+ NADH NADPH DNA

Malate α-KG

NAD+ IDH2* NADPH O2 O2 O2

H2O FH CoA IDH1* PHD JmjC TET
NADPH
NADH NADP+
NADP+
Fumarate Succinyl-CoA (R)-2-HG

GDP
GTP
FADH2 FAD
CoA O2 H2O
Mitochondrion Succinate

SDH Q III c IV Proteasomal Nucleus Histone DNA

degradation

Fig. 2 | Mitochondrial biology and metabolism and its relationship to the dehydrogenase is both an enzyme of the TCA cycle and a component of
epigenome. Glycolysis reduces nicotinamide adenine dinucleotide (NAD+) the ETC. In addition to oxidative phosphorylation and the TCA cycle, the
to NADH. The reducing equivalents can be used to reduce pyruvate to mitochondria generate reactive oxygen species and control cellular redox
lactate or shuttled into the mitochondrion, where NADH can be oxidized levels, modulate cytosolic Ca2+, regulate the intrinsic pathway of apoptosis
by mitochondrial NADH dehydrogenase (complex I). Complex I of the and determine the intracellular levels of the intermediates that control
electron transport chain (ETC) transfers the electrons to coenzyme Q (Q), other cellular signal transduction pathways, including the epigenome20,22.
complex III, cytochrome c (c), cytochrome c oxidase (COX; complex IV) and Thus, mitochondrial metabolism impinges upon every aspect of the cell. The
O2 to generate water. The energy released is used to create an inner asterisks indicate mutated proteins. ACLY, ATP citrate lyase; CC, citrate
membrane electrochemical gradient, which is used by the H+-​translocating carrier; CRP, CREB-​binding protein; DNMT, DNA methyltransferase; DOT1L,
ATP synthase (complex V) to condense ADP and inorganic phosphate (Pi) disruptor of telomeric silencing 1-​like protein; FH, fumarate hydratase;
to ATP, which is transported out of the mitochondria via adenine nucleotide GDP, guanosine diphosphate; GNAT, Gcn5-​related N-​acetyltransferase;
translocators (ANTs). Succinate dehydrogenase (SDH; complex II) accepts GTP, guanosine triphosphate; HAT, histone acetyltransferase; HDAC, histone
electrons from succinate to generate fumarate and transfers the electrons deacetylase; (R)-2-​H G, (R)-2-​h ydroxyglytarate; IDH, isocitrate
to coenzyme Q. Pyruvate from glycolysis can enter the mitochondria via dehydrogenase; JmjC, Jumonji C histone demethylase; mtDNA,
mitochondrial pyruvate carrier (MPC). In the mitochondria, pyruvate mitochondrial DNA; NADP, nicotinamide adenine dinucleotide phosphate;
dehydrogenase converts pyruvate to acetyl-​C oA, which feeds the PHD, prolyl hydroxylase; rRNA, ribosomal RNA; SAM, S-​adenosylmethionine;
tricarboxylic acid (TCA) cycle, which in turn generates metabolites such as SET, Su(var)3-9, Enhancer of Zeste and Trithorax protein; SETD2, SET
citrate, α-​ketoglutarate (α-​KG), succinate and fumarate. Thus, succinate domain-​containing protein 2; TET, ten–eleven translocase.

mutation, which causes mito­chondrial encephalomyop­ significantly affecting cellular function. The clas-
athy, lactic acidosis and stroke-like episodes (MELAS) sic example of such a mutation is the mtDNA
syndrome 30, and the MT-​ATP6 m.8993T>G (L156R) MT-​ND4 m.11778G>A (R340H) mutation, which causes
mutation, which causes neurogenic muscle weak- Leber hereditary optic neuropathy9 (Fig. 3).
ness, ataxia and retinitis pigmentosa (NARP) and The second clinically relevant class of mtDNA vari-
Leigh syndrome11. Milder mtDNA mutations must seg- ants occurs in the oocyte, during development, or in the
regate to a nearly pure mutant (homoplasmy) before somatic tissues of the body. These mutations can include

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OC m.15342insT The m.10398A>G variant has been shown to alter the

F T
efficiency of mitochondrial function42. The other line-
V′ 12S CR OC m.14429delG age migrated east along southern Asian and later moved
rRNA CYTB north to generate macrohaplogroup M. From the two
OC m.3571insC
16S ND6 OC m.13937delAC Eurasian macrohaplogroups, M and N, additional
PC, OC rRNA functional advantageous mutations arose within spe-
m.3664G>A
L PC m.13802C>T cific geographic regions, where they were selected and
OC m.3916G>A MELAS
syndrome ND5 founded distinctive Asian haplogroups8. All three classes
OC m.4063G>A ND1 m.3243A>G
OC m.12425delA of mtDNA variants may contribute to cancer and may
PC m.4142G>A interact with each other to modify cancer risk.
L
M LHON HS
m.11778G>A mtDNA variation in cancer
ND2
ND4 OC m.11872insC
PC m.5910G>A Tumours have altered bioenergetic processes, such
MERRF NARP
syndrome m.8993T>G as increased glucose meta­bolism43,44, altered calcium
PC m.5949G>A W ND4L
m.8344A>G regulation45, altered ROS production18,46, or altered struc-
ND3 R
PC m.6124T>C
COI
ture and interorganelle interaction47. These changes may
COIII G
PC m.6253T>C
PC m.10398A>G result from nDNA or mtDNA pre-​existing or de novo
COII ATP6
mutations, changes in gene copy number or alterations
PC 6340C>T D K in gene expression. Still, all three classes of clinically
OC m.6567C>T
relevant mtDNA variants can contribute to neoplastic
PC m.6663A>G PC m.6924G>T PC m.8932C>T
transformation (Fig. 4).
Fig. 3 | Human mtDNA map showing representative variants. Mitochondrial DNA
(mtDNA) encodes seven (MT-​ND1, MT-​ND2, MT-​ND3, MT-​ND4, MT-​ND4L, MT-​ND5 and Global cancer surveys of mtDNA mutations. The
MT-​ND6) of the 45 subunits of complex I, one (MT-​CYTB) of the 13 subunits of complex III, advent of high-​throughput next-​generation sequencing
three (MT-​COI, MT-​COII and MT-​COIII) of the 13 subunits of complex IV, two (MT-​ATP6 (NGS) greatly increased the amount of cancer mtDNA
and MT-​ATP8) of the 18 subunits of complex V, 22 tRNAs, 12S and 16S ribosomal RNA for sequence data. Both whole-​exome sequence (WES) and
protein translation and a 1,122-​nucleotide ‘control region’ (CR). Likely mtDNA ‘inducer’ whole-​genome sequence (WGS) data have been mined
variants of oncocytoma (OC) and prostate cancer (PC) are displayed around the outside to investigate mtDNA variation in cancer. While NGS
of the ring. These variants fulfil some of the criteria sanctioned by ClinVar as potentially has produced a wealth of mtDNA variant data, care must
‘pathogenic’, including being rarely found in the general population, display of multiple
be taken in interpreting the results from NGS studies.
independent recurrence, display of shifts in heteroplasmy with cancer phenotype and
demonstration of a biochemical defect that can be transferred in cybrids81. Known mtDNA
Important concerns include contamination with nDNA-​
disease mutations inside the ring fulfil all of the ClinVar criteria for pathogenicity. These coded mtDNA pseudogene sequences (NUMTs) and
include Leber hereditary optic neuropathy (LHON), myoclonic epilepsy and ragged red PCR amplification and sequencing errors.
fibre (MERRF) syndrome, mitochondrial encephalomyopathy and stroke-​like episodes Exome sequencing entails capturing exon-​coding
(MELAS) syndrome and neurogenic muscle weakness retinitis pigmentosa (NARP). regions of the nDNA using DNA or RNA oligonucle-
Databases that have collated mtDNA variants include MITOMAP27,195, HmtDB196 and otide baits. The enriched regions are then sequenced
MSeqDR197. Criteria for evaluating mtDNA sequence variants can be found at ClinVar81,198. by NGS. Beyond the targeted exon sequences comple-
mentary to the oligonucleotide baits used in exome
base substitutions and deletions. Such mutations accu- sequencing, additional DNA sequences are also cap-
mulate with age and may contribute to the ageing clock. tured, referred to as ‘off-​target sequences’. Because of the
Deletions31–33 can cause an array of symptoms depend- abundance of mtDNA sequences, considerable numbers
Mitochondrial
encephalomyopathy, lactic
ing on their tissue distribution and heteroplasmy levels, of off-​target mtDNA sequences are generated. However,
acidosis and stroke-​like including Pearson syndrome, Kearns–Sayre syndrome and the mtDNA sequence quality might be variable48, and
episodes (MELAS) chronic progressive external ophthalmoplegia7. legitimate mtDNA sequences can be contaminated by
syndrome The third class of clinically relevant mtDNA vari- mixing with NUMTs. This can be a particularly vexing
A heterogenous phenotype
ants is composed of ancient variants that arose as the concern when one is analysing mtDNA variation from
characterized by epilepsy or
dementia, with characteristic human species migrated throughout Africa and around archival WES and WGS data35.
elevated lactate levels in blood the world. Because these variants are ancient, they All human cells harbour on the order of 1,000
and cerebrospinal fluid, as well have segregated to a homoplasmic state. Those func- NUMTs, encompassing ~400 kb of DNA sequence (17 bp
as episodes of selective loss of tional variants that were beneficial as people moved per 10 kb)49. NUMTs can range from a few nucleotides
vision, sensation and speech.
into new environments were selected and gave rise to up to the size of the entire mtDNA35,50–55. As a result, the
Leigh syndrome regional groups of descendant haplotypes, known as mixing of NUMTs and legitimate mtDNA sequences in
Also known as subacute haplogroups. For example, only two mtDNA haplo- studies can be misinterpreted as heteroplasmy56–59 (Box 1).
necrotizing encephalopathy, types (linked array of mtDNA variants) ‘left’ Africa to Moreover, numtogenesis, the transfer of mtDNA
a disease of typically infantile
‘populate’ Eurasia and the Americas, the descendant sequences to the nDNA, is also activated in tumours49,60.
onset with psychomotor
retardation, floppiness, mtDNAs acquiring additional variants as they moved An analysis of 587 tumour–normal tissue pairs from
brainstem dysfunction and into new environments16,34,35. One of these two mtDNAs patients with various cancers for somatic nDNA–mtDNA
abnormal body movements. was associated with an MT-​ND3 variant, m.10398A>G junction fragments revealed that 12 tumour samples
(A allele, 114Thr), which ‘moved’ north into the tem- (2%) had a total of 25 mtDNA integration events in the
Homoplasmy
The presence of only one
perate zone and thereby gave rise to virtually all of the nDNA that were not present in the corresponding con-
mitochondrial DNA genotype European mtDNAs and half of Asian mtDNAs, generat- trol samples61. In a study of NUMTs in patients with
within one cell. ing the megalineage known as macrohaplogroup N8,36–41. colorectal cancer, sex-​related differences in NUMT

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Leber hereditary optic abundance were detected; 4.5 times more NUMTs were and promote tumorigenesis60. NUMTs may also activate
neuropathy found in tumour DNA compared with healthy normal cancer-related genes such as KCNMA1 (ref.61) or MYC62.
A disease of early adulthood genomes from female patients, and 3.1 times more In addition to potential functional roles, NUMTs
with rapidly progressive central NUMTs were found in tumour DNA compared with in cancer cells can become particularly problematic
loss of vision sequentially in
both eyes.
healthy normal genomes from male patients. These as arbitrary read number cut-​offs and nDNA NUMT
results indicate that the process of numtogenesis, at catalogues may be negated in different tumours. The
Pearson syndrome least in colorectal cancers, is increased in tumorigenesis impact of NUMT contamination can be reduced by
A disease of the newborn and modulated by sex differences. Moreover, increased enriching mtDNA before sequencing by mitochondrial
caused by mitochondrial DNA
numtogenesis was also correlated with increased mor- isolation and exonuclease digestion leaving the cir­
deletion presenting with
anaemia, vomiting and failure tality. These NUMTs tended to integrate into gene-​rich cular mtDNA63,64. For non-​tumour tissues, long-​range
to thrive. regions of the chromosomes and can be associated with PCR amplification of full-​length mtDNA using primer
decreased function in tumours of the YME1L1 gene, pairs that are not encompassed within known NUMTs
which encodes a protein responsible for impeding can be helpful. However, numtogenesis renders choos-
the transfer of mtDNA into the nucleus60. Although the ing primers which avoid NUMTs a challenge. Finally,
role of NUMTs in cellular and human health remains PCR incorporation artefacts can also be mistaken for
a mystery, presumably the insertion of NUMTs into low-​heteroplasmy mutations, although this can be
tumour suppressor genes can disrupt cellular pathways minimized by paired-​end duplex sequencing65.

a Male individuals Female individuals Global genomic analyses of cancer mtDNA mutations.
On the basis of analysis of WES and WGS data gener­
Cancer ated by The Cancer Genome Atlas (TCGA)66 and in
Haplogroups individual tumour-​type studies18,67–77, several groups
• Homoplasmic
• Defined by key have identified mtDNA variants of potential relevance
functional variants in cancer. In general, the criterion used to determine
• Shared by many potential relevance in cancer has been to select mtDNA
people
• Predisposing factor variants found in tumours from patients but not in the
for cancer, e.g. patient’s adjacent normal tissues. This approach has
U: prostate cancer,
D5: breast cancer provided the wealth of potentially cancer-​related vari-
ants discussed below. However, none of the large sur-
vey studies and few of the tumour-​specific studies have
confirmed the pathogenicity of these cancer-​correlated
U D5
mtDNA variants. This lack of confirmation is in part
due to the biological consequences of mtDNA variants
being highly dependent on the context, including tis-
sue type, tumour microenvironment and nDNA geno-
b type. Criteria for assessing the pathogenicity of specific
Familial variants mtDNA variants involve the independently repeated
• Homoplasmic or observation of the mutant in the tumour context,
heteroplasmic
• Common to offspring demonstration of a biochemical defect, transfer of the
of a mother biochemical effects from cell to cell via the mtDNA and
• Associated with
inherited cancer in the absence of nDNA (transmitochondrial cybrid stud-
(m.12425delA: ies)78–80 and disease-​specific associations81. The only can-
nasopharyngeal cer mtDNA mutations that have been subjected to this
cancer)
level of rigorous analysis are those observed in prostate
cancer and oncocytomas18,72–77. The best characterized
of these, along with the best characterized mtDNA dis-
ease mutations for Leber hereditary optic neuropathy,
c
mtDNA MERRF syndrome, MELAS syndrome and NARP, are
Somatic variants presented in Fig. 3. This meagre collection of relatively
• Start as heteroplasmic, may well-​characterized cancer mutations would generally fall
segregate to homoplasmy
• Appear throughout lifetime into our category of ‘inducer’ mtDNA mutations (Fig. 1a).
Mitochondrion of an individual in each cell Beyond these mutations that have been analysed in
• Increase with age depth, hundreds of mtDNA variants reported have been
• Contribute to oncogenesis:
thyroid, kidney observed in tumours but not in normal tissues from the
Healthy cell Tumour cell same individual77,82,66. While the cancer inducer status of
these mutations remains to be established, the clear quan-
titative association between mtDNA mutations and spe-
Fig. 4 | Examples of cancer-relevant classes of mtDNA variants. a | Haplogroup
associations demonstrate increased prevalence of prostate cancers among males of cific classes of tumours renders these surveys relevant for
haplogroup U and breast cancer among females of haplogroup D5. b | Familial inherited assessing the importance of mtDNA mutations in cancer.
variants may cause tumorigenesis72. c | Somatic variants constantly arising in each One survey in which the tumour mtDNA sequences
mitochondrial DNA (mtDNA) molecule of each cell in the body increase in frequency were compared with matched normal samples involved
with age and may contribute to tumorigenesis77. the analysis of the mtDNA sequences from 1,675 cancer

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Box 1 | NUMT and numtogenesis low-​heteroplasmy mutants, which are initially pheno-
typically masked. From these variants, a small subset
Numtogenesis49,60, the transfer of mitochondrial DNA of cellular mtDNAs might drift to higher heteroplasmy
(mtDNA) sequences into the nuclear DNA (nDNA), is an levels, which if they prove advantageous to the cancer
ongoing process55,199, generating mtDNA pseudogenes cell in certain environments become enriched7,8 (Fig. 1b).
(see the figure). nDNA-​coded mtDNA pseudogene
Another large data study analysed mtDNA variation
sequences (NUMTs) become shielded from a higher
mutation rate occurring in the mitochondrial matrix in 1,916 tumours and matched normal tissues across
compared with the nucleus. Without accounting for the 24 cancer types, analysed by WGSs generated by TCGA66.
introduction of the mtDNA sequence into nDNA, NUMT A total of 2,350 tumour-​specific somatic mtDNA muta-
contamination can be misinterpreted as heteroplasmy in tions were found in samples of tumours from 1,231 patients
whole-​exome sequences and whole-​genome sequences. (64% of the patients) as compared with 1,154 normal-
NUMTs can be enriched depending on the source of the cell heteroplasmic variants from 760 patients (40% of
DNA and the way it was isolated58. Disregarding low the patients). While the normal-​cell heteroplasmies clus-
heteroplasmy200 or using computational techniques to tered within the non-​coding D loop, the tumour-​specific
discard sequencing reads that do not uniquely map to the somatic mutations were dispersed across both coding
mtDNA201,202 can be problematic as they also can eliminate
and non-​coding regions of mtDNA. The tumours had
valid low-​heteroplasmy mutations203. One commendable
approach for human genetic diseases is to build NUMT more indels (9%) than normal tissues (3%). Moreover,
libraries and to use these libraries to create computational metastatic and recurrent tumours had more coding
programs to identify and remove the NUMTs50,200,203. region gene variants versus D-​loop variants than pri-
However, if a tumour has undergone numtogenesis, then mary tumours, which is in line with the hypothesis of
the value of NUMT catalogues from a different cell source mtDNA variants providing adaptive features for the
may be limited. disseminating cancer cells (Fig. 1b). The most notable
observation was the differential frequency of functional
mtDNA (high mutation rate)
mtDNA mutations between the different tumour types.
The kidney chromophobe and thyroid tumours had the
highest number of somatic mtDNA mutations, and
nDNA (low mutation rate) their variant heteroplasmic allele frequencies corre-
lated positively with the severity of the mutation class,
indicating positive selection, which was recently vali-
dated by another study77. Also, those non-​synonymous
heteroplasmic alleles that were at low frequency (5%)
Kearns–Sayre syndrome in the normal tissue were found to increase in mtDNA
A disease with typical onset
before the age of 20 years
allele frequency in the tumours, giving a median allele
with abnormal pigmentation frequency in tumours of 59%, as compared with 19%
of the retina and progressive for synonymous alleles. Among the thyroid and kid-
abnormal eye movements, ney tumours, 28 patients were found to have frameshift
as well as cardiac conduction mtDNA sequence integrated into nDNA sequence mutations that were present at level of less than 5% in
abnormalities and cerebellar (low mutation rate)
dysfunction.
the normal tissue but that rose to more than 90% in the
tumour. These tumours were also found to have a higher
Chronic progressive mtDNA copy number than the normal tissues (729 ver-
external ophthalmoplegia sus 165 for patients without these frameshift mutations).
Slowly progressive weakness
of extraocular eye muscles
biopsy samples from 31 tumour types deduced from The study authors concluded, “these results suggest
resulting in abnormal eye 704 WGSs and 971 WESs82. This study identified 1,907 positive selection in tumor cells for disruptive mtDNA
movements and lid lag. somatic substitutions, the vast majority of these base variants present at low levels in normal cells”66.
substitution mutations being transitions. This tran- A third large dataset was used to analyse the mtDNAs
Numtogenesis
sition bias is likely due to replication errors caused by of 38 tumour types in 2,658 cancer and matched control
The transfer of mitochondrial
DNA sequences into the
the preferential oxidative inactivation of the mitochon- pairs from the Pan-​Cancer Analysis of Whole Genomes
nucleus. drial polymerase-​γ (PolgA) proofreading exonuclease consortium77. Transition mutations predominated,
activity83. Indeed, genetic inactivation of the exonucle- more than 85% of the substitutions were hetero­
Transmitochondrial cybrid ase (PolgAD257A/D257A) results in increased mtDNA sub- plasmic, and the D-​loop and MT-ND4 gene were found
A cell line in which the
mitochon­drial and mitochon-
stitutions and deletions84,85. This cancer study reported to be hypermutable. The MT-​ND5 gene was the most
drial DNA encompassed within that of the tumours, 58% harboured at least one somatic mutated across cancers, MT-​ND4 was most mutated in
an enucleated cytoplasmic frag- mtDNA substitution and 31% harboured multiple prostate and lung cancers, and MT-​COI was most mutated
ment is fused to a recipient cell, mutations82. Of the 1,907 substitutions, 60% occurred in breast, cervical and bladder cancers. High-allele-
resulting in a cytoplasmic hybrid
in the 13 protein-​coding mtDNA regions. Of these, 63 frequency truncating mutations were found in kidney
(cybrid) with the nucleus of the
recipient cell but the mitochon-
were nonsense, 4 were loss of stop codon, 878 were mis- chromophobe cancer and kidney papillary, thyroid and
drial and mitochondrial DNA of sense and 110 were insertion–deletion (indel) mutations. colorectal cancers. mtDNA copy number was high-
the donor cell. By contrast, only 208 silent substitutions were observed. est in ovarian cancer and lowest in myeloid cancer.
The ratio of somatic missense mutations to silent sub- Somatic transfer of mtDNAs into the nucleus (that
Oncocytomas
Benign tumours harbouring a
stitutions was 4.2 (878/208), which implies a selective is, somatic numtogenesis) was observed in more than
large numbers of cytoplasmic bias towards acquisition of functional mutations in 5% of lung, skin, breast and uterine tumours, in 16%
mitochondria. tumours82. Presumably cancer cells generate numerous of breast cancers and in 14.6% of squamous cell lung

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cancers77. As mentioned earlier, the transfer of mtDNA mutant mtDNA72. In another study, WGSs of 12 renal
sequences to the nucleus may have functional relevance, oncocytomas led to the detection of mtDNA muta-
such as the activation of KCNMA1 (ref.61) and MYC62. tions in all 12 samples, including mutations in three or
These studies confirm that mtDNA mutations are more tumours, and with 7 of 11 oncocytomas having
relevant to tumorigenesis, although mtDNA mutations mutant allele frequencies of more than 75%. A third
may have different implications in different tumours. study analysed off-​target mtDNA reads from WESs of
Supportive evidence for a role of mtDNA in tumorigen- 19 renal oncocytomas. Protein-​truncating mutations
esis includes the frequent presence of functional mtDNA were found in 16 tumours, and conserved missense
variants in an individual’s tumour tissue but not stromal mutations were found in the remaining three tumours,
tissue, the observation that heteroplasmic mtDNA muta- all in complex I mtDNA genes. In an independent sam-
tions in cancer cells can become enriched, indicating ple of 16 renal oncocytomas, 69% were found to have
positive selection for mtDNA mutations during tumor- high-​heteroplasmy mutations in the MT-ND1, MT​-ND4
igenesis, and the observation of changes in mtDNA copy and MT-ND5 genes73.
number and numtogenesis in different tumours. Analysis of the mtDNA of the thyroid oncocytic
tumour cell line XTC.UC1 revealed two mtDNA muta-
Individual tumour-​a ssociated mtDNA mutations. tions, a frameshift insertion in MT-ND1 and a missense
Numerous studies have analysed the mtDNA variation substitution in MT-​CYB. These mutations were asso-
of specific tumours17,67–71,86. For certain tumours, the ciated with reduced complex I and complex III activ-
total burden of acquired mtDNA variants may represent ities and ATP synthesis levels101. Oncocytomas have
a biomarker for tumorigenicity. For example, the pre­ high levels of reduced glutathione (GSH) and oxidized
sence of mtDNA mutations in cells associated with pros- glutathione (GSSG), implying compensation for high
tate cancer may be supportive of increased tumorigenic mitochondrial ROS production73,102. As superoxide
potential75. mtDNA mutations associated with pros- anion (O 2·−) and H 2O 2 are important mitogens 103,
tate cancer tend to coexist with nuclear somatic driver complex I gene mutations resulting in elevated ROS
events76, emphasizing the role of mtDNA mutations as production may contribute to oncocytoma neoplastic
co-​initiators of cancer75,76,87. mtDNA mutations serving transformation104.
as tumour initiators are also suggested for thyroid Hüthle A role for mtDNA variation in prostate cancer is
cell carcinoma88, breast cancer89,90, pancreatic cancer91, also well established74. Extensive genetic analysis of
gynaecological malignancies92,93, lung adenocarcinoma prostate cancer has revealed a number of nDNA gene
metastases94,95 and acute myeloid leukaemia96,97. By con- loci whose functions are altered by either mutations or
trast, mtDNA variants may be of limited importance in epigenomic modulation. These include IDH1, PTEN,
glioblastoma98. TP53, MYC, FOXA1 and GSTP1, which could mod-
However, the functional consequences of tumour- ulate mitochondrial functions. Importantly, a sub-
associated mtDNA variants have not been extensively stantial number of somatic mtDNA mutations have
evaluated. The most in-​depth studies on the patho- been identified distributed throughout the mtDNA
physiology of tumour mtDNA mutations have been and affecting both the non-​coding control region and
conducted for oncocytomas and prostate cancer87. gene coding regions18,74–76. Of particular interest is the
Oncocytomas, when stained for COX, show dense accu- repeated appearance of the MT-​ND3 m.10398A>G,
mulations of mitochondria99. Analysis of the mtDNAs G allele, mutation in prostate-​to-​bone metastases105.
of a panel of 45 oncocytomas revealed that 26% had Another mtDNA and nDNA variant interaction is the
high-​heteroplasmy mtDNA mutations in MT-​ND1, co-​occurrence of copy number gains in MYC in associ-
MT-​ND2, MT-​ND4, MT-​ND4L, MT-​ND5, MT-​ND6, ation with control region variants around the mtDNA
MT-​CYB and MT-​ATP6. One tumour harboured both origin of H-​strand replication (m.110–441)76. Several of
an MT-​ATP6 mutation and an MT-​ND5 mutation100. the somatic mutations have been observed in multiple
Analysis of the mtDNA sequence variation of nine tumours in independent work77, and clinical outcomes
patient-​derived oncocytomas revealed multiple mtDNA correlated with the total number of mtDNA variants75,
mutations, including nine oncocytomas harbouring the region of the mtDNA mutated76 and high mtDNA
mtDNA mutations at high heteroplasmy, and seven copy number106. Interestingly, a recent co-​expression
with complex I gene mutations (MT-​ND1, MT-​ND4, analysis of mtDNA and nDNA genes showed OXPHOS
MT-​ND5 and MT-​ND6). In addition to a complex I pathways as the top-​ranked enriched pathway in 8 of
mtDNA mutation, one tumour also harboured a 13 cancer types examined77.
MT-​COI mutation, and all nine tumours had a complex I These studies confirm the functional importance of
defect and inability to grow on obligatory oxidative mtDNA mutations in oncocytomas and prostate cancer.
metabolites69. While most oncocytoma mtDNA muta- They also demonstrate the types of experiments that
tions appear to have arisen within the tumour (Fig. 4c), in will be necessary to validate the functional relevance of
one case an mtDNA complex I mutation was maternally mtDNA variants for other tumours.
transmitted (Fig. 4b). The tumour was homoplasmic for
an MT-​ND5 frameshift mutation, but the same mutation Prostate cancer and mitochondrial ROS. Mitochondrial
was present at low heteroplasmy levels in the patient’s ROS is an important signalling molecule and a potent
normal tissues and the tissues of his two sisters. Thus, mitogen20,103,107,108. The first mitochondrial ROS mol-
a germline heteroplasmic mutation became enriched to ecule is O2·−. Superoxide anion production can occur
homoplasmic in the tumour, indicating selection for the from complex I, ubisemiquinone and/or complex III.

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Superoxide production can be stimulated by excessive and others show the importance of ROS production119,120
reduction of the electron transport chain caused by in some cancers.
inhibition of complex IV (ref.19) or complex V (ref.18) or
high levels of succinate in the presence of impaired elec- Tumour-​associated mtDNA copy number variation.
tron transport driving reverse electron transport104,109,110. mtDNA copy number is often regarded as a simple proxy
Superoxide anion produced in the mitochondrial matrix for measuring mitochondrial function in an organism.
is converted to hydrogen peroxide (H2O2) by the mito- For example, cells with decreased mitochondrial func-
chondrial matrix manganese superoxide dismutase. tion might be assumed to have decreased mtDNA copy
Complex III-​generated O2·− in the mitochondrial inter- number, although this assumption would be simplistic.
membrane space is converted to H2O2 by copper–zinc Changes in mtDNA copy number have been reported
superoxide dismutase. Within the mitochondrion, H2O2 to differ between certain tumour types. One survey
is reduced to H2O via glutathione peroxidase using GSH reported the mtDNA copy numbers of 15 tumour types
as the reductant. In turn, GSSG is reduced to GSH by by analysing the number of mtDNA/nDNA NGS reads
glutathione reductase using NADPH as the reductant from WGS and WES studies. This revealed that seven
(NADPH is generated from NADH via nicotinamide of the tumour types had reduced mtDNA copy num-
nucleotide transhydrogenase, using energy derived from ber relative to adjacent normal tissue: bladder, breast,
the mitochondrial inner membrane potential). H2O2 can oesophageal, head and neck squamous cell, clear cell
diffuse from the mitochondria into the cytosol, and is and papillary (though not chromophobe) kidney, and
converted to H2O and O2 by the peroxisomal catalase. liver cancers. By contrast, lung adenocarcinoma had
While typically viewed as harmful molecules that significantly increased mtDNA copy number levels
damage DNA, mitochondrial ROS have also been pro- compared with normal tissue121. Transcriptional profile
posed to modulate HIF1α levels111, which may be one analysis revealed that the transcript levels of enzymes
mechanism by which mitochondrial ROS favour cell of the tricarboxylic acid cycle and electron transport
replication. Proof that mitochondrial ROS production chain (complexes I, II, III and IV), fatty acid β-​oxidation
is important in promoting cancer comes from the sup- and branched-​chain amino acid catabolism pathways
pression of tumorigenesis by introduction of a mito- correlated most closely with the mtDNA copy number.
chondrially targeted catalase112 into mice harbouring By contrast, the expression of mitochondrial genes
the polyoma middle T oncoprotein113 or ApcMin/+ plus was inversely correlated with mtDNA content for pros-
Tfam+/− mutations114. tate cancer. Interestingly, the copy number of genes
The functional relevance of prostate cancer somatic encoding proteins of immune pathways, including those
mtDNA mutations has been established by multiple related to interferon signalling, were negatively corre-
examples of de novo mtDNA mutations being enriched lated with mtDNA copy number122,123 for bladder, breast,
in tumour tissue, and by the transfer of tumour mtDNA oesophageal head and neck squamous cell, and clear
mutations into transmitochondrial cybrids that then cell kidney cancers. The mtDNA content was markedly
displayed increased tumorigenicity. From the mtDNA upregulated in low-​grade gliomas harbouring PTEN or
sequences of prostate tumours from patients under- IDH1 mutations and in endometrial carcinomas with
going radical prostatectomy, a de novo MT-​COI chain TP53 mutations compared with wild-​type samples. For
termination variant, 5949G>A (homoplasmic vari- adrenocortical and kidney chromophobe carcinomas,
ant in tumours, homoplasmic reference in adjacent lower mtDNA copy number is associated with marked
healthy epithelial tissue), was identified. Cancerous reduction in patient survival time121.
cells with this variant lacked COX (complex IV), while Recently, an extensive survey of mtDNA mutation
surrounding healthy cells contained COX, suggesting data suggested that for some cancers, mtDNA variation
positive selection in cancer cells for the mtDNA COX is mutually exclusive with certain nuclear driver muta-
deficiency18. Addition to PC3 prostate cancer cells of the tions. For example, for ovarian adenocarcinoma about
mtDNA MT-​ATP6 variant m.8993T>G had a marked 40% of tumours have only nuclear mutations, and almost
effect. PC3 cybrids harbouring the T allele generated 60% have only mtDNA mutations. Only a small per-
sevenfold larger tumours and increased ROS production centage have both77. Hence, different types of tumours
compared with the cybrids with the reference G allele18. have different bioenergetic requirements, which can be
Variant m.6124T>C (Fig.  3) was also identified and modu­lated by either nDNA or mtDNA genetic variation
transferred to the osteosarcoma 143B mtDNA-​deficient and mtDNA copy number.
(ρ0) cell line, and homoplasmic normal and mutant Why mtDNA copy number changes in tumours
MT-​COI m.6124T>C mtDNA cell lines were generated. is not known, but genetic studies offer evidence that
The m.6124T>C, C allele mutant cybrids had a partial the directionality of the change may be dependent on the
defect in complex IV, increased mitochondrial ROS pro- type of mutations present as well as the tumour type.
duction, increased growth rate and increased tumour For example, thyroid and kidney tumours harbouring
growth in immunodeficient mice19. The mutant and nor- variants that inactivate an mtDNA gene have a mark-
mal mtDNA were also transferred to PC3 cells, and the edly increased mtDNA copy number compared with
m.6214T>C, C allele mutant cybrids again formed larger adjacent normal tissue cells. When a null mtDNA allele
tumours than the 6214T>C, T allele normal cybrids, in became fixed in the tumour relative to the adjacent stro-
association with increased resistance to statin-​induced mal tissue, suggesting an adaptive selection, the median
apoptosis115. Similar exchange experiments have been mtDNA copy number was 729. By contrast, in patients
reported for other cancer cell types116–118. These studies whose tumours did not harbour a fixed loss-​of-​function

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mtDNA allele, the mtDNA copy number was 165. This such associations134. Mitochondrial haplogroup U has
association held even after adjustment for tumour type66. been associated with increased risk of prostate and
Therefore, in some tumours, the change in mtDNA copy renal cancer135, although this association has also been
number may be an adaptive response secondary to the challenged 75,136 . Moreover, haplogroup D4a has
effect of a mutation which confers a growth advantage been associated with increased risk of thyroid cancer132,
for a certain tumour type. haplogroup T has been reported to be associated with
Interestingly, even within a class of tumours, dif- colorectal cancer137 and haplogroup JT has been reported
ferences in mtDNA content can correlate with cancer to be protective of myelodysplastic syndromes138. While
severity. In a TCGA-​based study of tumour samples the apparent lack of reproducibility of haplogroup asso-
from patients with breast cancer at various stages, the ciations might result in discounting these associations,
stage IV cancers that had at least one metastatic site had another possibility is that inconsistent study replication
the lowest mtDNA copy number, even when controlled may reflect the extreme mtDNA haplogroup population
for age of onset. Moreover, mtDNA content of tumour stratification. An association found in one well-​defined
samples from patients with triple-​negative (ER-​negative, regional population may not be transferable to another
PR-​negative and HER2-​negative) breast cancer was sig- population because they harbour different arrays and
nificantly lower than that of non-​tumour tissue. In line frequencies of mtDNA haplogroups.
with this observation, triple-​negative breast cancer cell Common mtDNA variants that may or may not be
lines had markedly reduced mitochondrial respiration associated with haplogroups may also alter cancer risk.
and increased glycolysis124. The control region variants m.16519T>C and m.150T>C
Thus, where mtDNA copy number may be a contrib- have been associated with endometrial cancer139 and
utory factor for the growth of certain tumour types, the human papillomavirus-​p ositive cervical  cancer in
significance of mtDNA copy number changes seems to Chinese women140,141, respectively. This intersection
be context specific. A tumour type may increase mtDNA between functional haplogroup variants and cancer
copy number to function in an aerobic environment. becomes clearer when we examine mtDNA missense
mtDNA copy number may also change in response mutations. Among the repeatedly observed prostate
to mtDNA mutations. It may compensate for reductions cancer somatic mtDNA mutations, several change
in electron transport chain function due to the presence precisely the same mtDNA nucleotide as found in key
of a ‘mild’ mtDNA variant or the mtDNA copy num- nodal variants of the human mtDNA phylogeny, imply-
ber may be modulated in response to increased ROS ing a selective advantage. The most dramatic example is
production125,126. the recurrence of the MT-​ND3 m.10398A>G, G allele
(114Ala) mutation in prostate cancer bone metasta-
Haplogroups, recurrent nodal variants and adap- ses. In a survey of primary prostate cancer, soft tissue
tation. Approximately one quarter of human popu­ metastases, and bone metastases from ten patients,
lation mtDNA haplogroup variants lead to changes seven bone metastases had the m.10398A>G, G allele,
in conserved amino acid or nucleotide functions that 114Ala which was not present in any of the other tissues
have a functional consequence16,127. Moreover, a vari- of the same patient105. This means that the m.10398A>G,
ety of mtDNA haplogroups have been associated with G allele mutation must have arisen de novo and been
cancers86,90 (Table 1). For example, mtDNAs harbouring selected in prostate cancer bone metastases. The same
the MT-​ND3 m.10398A>G, A allele (114Thr) have been MT-​ND3 variant separates the Eurasian macrohaplo­
associated with increased breast cancer risk in African group N (MT-​ND3 m.10398A>G, A allele, 114Thr)
American women54,128–131 and haplogroup D5 for breast from African and Asian macrohaplogroups L and M
cancer in Asian women132. While a subclade of haplo­ (MT-​ND3 m.10389A>G, G allele, 114Ala)8, suggesting
group T, T1a1, has been reported to be protective for that this same variant provided a selective advantage for
BRCA2-​mutation carriers133, other studies of haplogroup a portion of the human population and provides prostate
associations with breast cancer have not confirmed cancer cells with an advantage in the bone environment.
Cells harbouring mtDNA haplogroups containing the
m.10398A>G, A allele differ in their mitochondrial
Table 1 | Haplogroup associations with predisposition to cancer matrix pH from cells with the m.10398A>G, G allele,
and this pH difference correlates with alterations in
Cancer phenotype Haplogroup Effect Refs
mitochondrial calcium levels142. Other population vari-
Breast cancer For the MT-ND3 m.10398A>G ↑ Risk 54,128–131
ants observed in prostate cancer74 involve m.4917A>G,
(Thr114Ala) variant, the A allele, amino
acid 114Thr, is at increased risk, relative which delineates European haplogroup T 16, and
to the G allele, amino acid 114Ala, m.3394T>C, which has been repeatedly selected by
for African American women high-​altitude-​dwelling Tibetans143, likely in response to
Breast cancer M, D5 ↑ Risk 132 decreased oxygen tension, which is a well-​established
feature of prostate cancer144. Furthermore, an MT-​ND5
Breast cancer T1a1 ↓ Risk 133,134
m.13708G>A missense mutation (Ala458Thr) was
Prostate and renal cancer U ↑ Risk 75,135,136
reported in a breast cancer tumour but was absent in the
Thyroid cancer D4a ↑ Risk 132 peripheral blood leukocytes145, yet the 13708 variant is
Colorectal cancer T ↑ Risk 137 the polymorphism at the root of European haplogroup J16.
Hence, the same functional mtDNA mutations
Myelodysplastic syndromes JT ↓ Risk 138
that allowed our human ancestors to adapt to new

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Box 2 | Epigenomic regulation of cancer via mitochondrial metabolites In triple-​negative breast cancer cell lines, reduced
mtDNA content was associated with reduced expression
Mutations in nuclear DNA tricarboxylic acid cycle enzymes have been shown to alter of epithelial splicing regulatory protein compared with
mitochondrial metabolites and change nuclear gene expression. non-​triple-​negative breast cancer cell lines (ESRP1, a
Isocitrate dehydrogenase (IDH) mutations, oncometabolites and chromatin gene highly expressed in epithelial cells but expressed at
IDHs catalyse oxidative decarboxylation of isocitrate to α-​ketoglutarate (α-​KG) in low levels in mesenchymal cells)124. Depletion of mtDNA
the tricarboxylic acid cycle (IDH2 and IDH3) or cytosol (IDH1). IDH1 and IDH2 are content caused decreased expression of ESRP1, favour-
NADPH-​linked enzymes that can catalyse the reverse reaction, reductive carboxylation ing epithelial–mesenchymal transition148. The question
of α-​KG back to isocitrate86. then becomes how do mtDNA variants regulate nuclear
IDH1 or IDH2 mutations can result in a gain of function21,163,164 resulting in production
gene expression and whether this is relevant to cancer.
of the oncometabolite (R)-2-​hydroxyglutarate ((R)-2-​HG)). (R)-2-​HG promotes neoplastic
transformation24,162,170–173 by acting on three classes of α-​KG-​dependent dioxygenases
While ROS, redox86,103,114 and calcium149,150 regulation
(α-​KGDDs): (1) the Egl nine homologue (EGLN) family regulators of hypoxia-​inducible have all been discussed in relation to the pathophysio­
factor (HIF), (2) the Jumonji C (JmjC) family of histone-​demethylating enzymes and (3) logy of cancer, the changes observed in the macrohaplo­
the ten–eleven translocation (TET) methylcytosine family of DNA-​demethylating group L versus macrohaplogroup N (haplogroup H)
deoxygenases (Fig. 2). cybrids imply a much more fundamental interaction
(R)-2-​HG inhibition of JmjC histone demethylases alters nuclear gene expression165,166. between these two genomic compartments. Insight into
(R)-2-​HG also affects the TET proteins, which hydroxylate 5-​methylcytosine (5mC). the basis of this interaction comes from the discovery
(R)-2-​HG inhibits the catalytic activity of TET2 (refs164,170), resulting in hypermethylation that the age-​related accumulation of somatic mtDNA
in gliomas and leukaemias167,168, and in acute myeloid leukaemia IDH1 or IDH2 mutations (PolgAD257A/D257A) in the intestinal crypt cells
mutations and TET2 mutations are mutually exclusive168,204.
impairs OXPHOS and results in induction of the serine
Succinate and fumarate and HIF1α stabilization synthesis pathway and enhanced tumorigenesis151. In
Succinate dehydrogenase (SDH) inactivation causes paragangliomas205, pheochromo- addition, mutations in nDNA-​coded mitochondrial
cytomas206,207, gastrointestinal stromal tumours208,209, renal cell carcinomas210, neuro­ genes of the tricarboxylic acid cycle enzymes isocitrate
blastoma development211,212 and sporadic breast cancer213, and results in the accumulation
dehydrogenase (IDH), SDH and fumarate hydratase
of succinate180,214. Fumarate hydratase (FH) loss-​of-​function mutations cause hereditary
leiomyomatosis and renal cell carcinoma syndrome215, and result in the accumulation of
(FH) can cause cancer (Box 2). This results from an
both fumarate and succinate214,216,217. alteration of the level and nature of mitochondrial
SDH loss-​of-​function mutations cause abnormal mitochondrial morphology218, meta­bolites, which in turn modulate the epigenome to
increased reactive oxygen species production, oxidative DNA damage and genomic modify gene expression86,152,153 (Fig. 2).
instability111. Mutations in the mitochondrial chaperone TRAP1 inhibit SDH and are
associated with cancer, leading to HIF activation219. Succinate also inhibits the EGLN Mutations in nDNA mitochondrial genes modulate the
family HIF1α prolyl hydroxylases, which leads to stabilization and activation of HIF1α180 cancer epigenome. Regulation of nuclear gene expression
(Fig. 2). Succinate inhibits all three classes of α-​KGDDs177,178,180,220. Elevated succinate has been shown to involve the epigenetic modification
level inhibits both the JmjC and the TET family α-​KGDDs, resulting in a hypermethylator of the DNA and histone proteins154. All major histone-
chromatin status175,176,181,185. Fumarate is also capable of inhibiting the three families of
modifying and DNA-​modifying mechanisms use metab­
α-​KGDDs and contributing to oncogenesis23,188.
olites that arise from mitochondria (Fig.  2). Histone
acetylation requires citrate-​derived acetyl-​CoA and can
environments may be permitting cancer cells to adapt be removed by NAD-​dependent sirtuins, while histone
to new environments within the body17. and DNA methylation require S-​adenosylmethionine and
can be reversed by enzymes using α-ketoglutarate
Mitonuclear signalling (α-KG) as a substrate, which in turn are inhibited
Evidence of mtDNA variation occurring in cancers by succinate and fumarate 155,156. A whole class of
prompted investigations of the potential mechanisms chromatin-remodelling proteins is ATP dependent157.
by which mtDNA variation might influence tumour Mutations in the genes encoding IDH1 and IDH2,
development. For example, the significance of the recur- which catalyse conversion of isocitrate to α-​KG, have
rence of the MT-​ND3 m.10398A>G (A allele, 114T, been associated with gliomas 158–160, acute myeloid
macrohaplogroup N) variant versus the m.10398A>G leukaemias161 and other solid tumours21,162–164. α-​KG
(G allele, 114Ala, macrohaplogroups L and M) variant serves as a cofactor of α-​KG-​dependent dioxygenases,
was explored by comparing transmitochondrial cybrids including Jumonji C (JmjC) histone demethylases165,166
with the same nucleus but harbouring African macro­ and the ten–eleven translocation (TET) family of dioxy­
haplogroup L mtDNAs (L cybrids) or European genases as a substrate for DNA demethylation167,168
macrohaplogroup N mtDNAs (H cybrids). Interestingly, (Fig. 2). The IDH gene mutations localized to the gene
the L cybrids had lower mtDNA copy numbers, region encoding the active site can have a gain-of-
decreased ATP turnover rates and lower ROS production function effect, causing the mutant enzyme to produce
than the H cybrids. In gene expression array studies, the (R)-2-​hydroxyglytarate (2-​HG). 2-​HG competitively
L cybrids were found to have reduced expression of inhibits TET and JmjC class enzymes, resulting in altered
the complement pathway and innate immunity genes, gene expression, inhibiting differentiation and promoting
but higher expression of inflammation-​related genes146. neoplastic transformation24,162,169–173 (Box 2; Fig. 2).
This may reflect the recent observation that oxidation of Mutations in the gene encoding SDH cause pheo-
mtDNA can modulate the innate immune system122,147. chromocytomas and paragangliomas174–176 (Box 2). SDH
Alteration in the immune function associated with the mutations cause the accumulation of succinate, which
m.10398A>G, G allele (114Ala) could then be beneficial reversibly175,176 inhibits TET and JmjC enzymes177–181.
to prostate cancer cells within bone metastases. Similar results were obtained in gastrointestinal stromal

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tumours, where SDH-​mutated tumour samples182–185 association with a decline in histone 4 lysine 16 acetyl-
showed markedly different methylomes compared ation (H4K16ac). The mitochondrial α-​KG level was
with KIT-​mutated ones186, and mutation status corre- low in 20–30% mutant 3243A>G mtDNAs, increased
lated with clinical outcome23. The mechanistic aspect in the middle percentages of 3243G mtDNAs, but then
of accumulation of succinate or fumarate inhibiting his- declined again at 90–100% 3424A>G heteroplasmy lev-
tone and DNA demethylation has been confirmed187,188. els. This was inversely proportional to the levels of his-
Given the relationships between mitochondrial metab- tone 3 lysine 9 trimethylation (H3K9me3), consistent
olites and the epigenome189, as well as between nuclear with α-​KG levels driving JmjC histone demethylases.
genes affecting mitochondrial metabolism and cancer190, [13C]glucose and [13C]glutamine tracing through meta­
one must ask whether mtDNA variants could simi- bolic pathways and onto the histones showed many of the
larly contribute to carcinogenesis by modifying the histone acetylation changes were due to the flux of
epigenome. acetyl-​CoA from the mitochondria and were dependent
on the mitochondrial but not the nuclear NAD+:NADH
Changes in mtDNA heteroplasmy modulate the cancer ratio26. Further studies are required to better characterize
epigenome. The idea that mtDNA variation modulates and validate these phenomena.
the cancer epigenome is supported by comparison of the As the nucleus appears to have a limited repertoire
nDNA methylation patterns of osteosarcoma 143B of transcriptional states, very subtle changes in the
cells, breast cancer MDA-​MB-435 cells and immortal- mtDNA genotype can have profound effects on nDNA
ized breast epithelial MCA 12A cells with and without gene expression via modulation of the epigenome. Shifts
mtDNA. The loss of mtDNA resulted in changes to CpG in nDNA gene expression and metabolism can thus be
island methylation, some of which could be restored produced by de novo acquisition of new mtDNA muta-
when mtDNAs were reintroduced into the cells191. tions, alterations in the mtDNA copy number or a small
In one tumour sample from a patient with renal percentage change of mutant mtDNA heteroplasmy level.
cell carcinoma, the mtDNA tRNALeu(UUR) (MT-TL1)
m.3243A>G, G allele MELAS syndrome mutation was Conclusion
discovered in the tumour tissue at an 89% mutational As is the case for different tissues, different tumour types
load, while the patient’s kidney harboured about 50% have different metabolisms, although all tumours require
m.3243A>G, G allele mutant mtDNAs. The tumour both glycolysis and mitochondrial OXPHOS to meet the
tissue showed a sixfold downregulation of complex I energy demands of the cells193. The developmental ori-
and a twofold downregulation of complex IV compared gin of the tumour and the surrounding stromal tissue
with normal renal cortex, as expected for a mitochon- environment are important in determining the available
drial protein synthesis defect. The mtDNA tRNALeu(UUR) energetic substrates and demands on the tumour cells
(MT-​TL1) m.3243A>G mutant tumour showed down- and thus the energetic system required.
regulation of von Hipple-​Lindau protein (VHL) consis­ For tumours in which mtDNA mutations are prev-
tent with the other renal cell carcinomas (Box 2). Hence, alent, these mutations are likely to contribute to tum-
the mtDNA 3243A>G mutation generated physiologi- origenesis. However, unlike nDNA genetic alterations,
cal alterations comparable to those in other renal cell which are dichotomous, mitochondrial metabolic
carcinomas192. function is modulated by both quantized and quantita-
Epigenomic consequences of mtDNA mutations in tive factors. Mutations in nDNA-​coded mitochondrial
tumours have been demonstrated by introducing the genes such as SDH, IDH1 and IDH2 have two copies
mtDNA tRNALeu(UUR) (MT-​TL1) m.3243A>G muta- and act dichotomously. By contrast, mtDNA is repre-
tion into the ρo osteosarcoma cell line 143B (thymidine sented by hundreds of copies per cell, and a heteroplas-
kinase negative). Clones with mtDNA 3243A>G hetero- mic mtDNA mutation can shift to a higher or lower
plasmy levels of approximately 0%, 20%, 30%, 60%, 70%, percentage mutant, permitting the cells to readily vary
90% and 100% G allele showed phasic transcriptional between oxidative and glycolytic metabolism. Similarly,
changes with increasing heteroplasmy. Compared with the mtDNA copy number can be modulated, providing
0% cells, the 20% and 30% mutant cells had a reduced another adaptive strategy for cancer cell survival and
cell volume, increased mtDNA/nDNA ratio and a proliferation. Finally, variants in both the nDNA and the
sharp drop in mtDNA transcript levels. The 60% and mtDNA mitochondrial genes change mitochondrial
70% mutant cells normalized their cellular volume metabolism, and this in turn modulates the epigenome,
and mtDNA copy number, but showed induction of also permitting energetic adaptation.
genes needed for glycolysis and lactate production, had For these reasons, mtDNA genetic variants cannot be
increased mtDNA transcript levels, and transcriptionally readily classified as being or not being cancer mutations.
upregulated KRAS, MYC, TGFα and ER compared Rather, we propose that cancer-​associated mtDNA muta-
with 0% cells. Finally, the 90% and 100% 3243A>G, tions be divided into ‘inducer’ mutations, which contrib-
G allele cells had high lactate production in association ute to various degrees to neoplastic transformation, and
with a marked decline in mtDNA transcript levels and ‘adaptor’ variants, which permit tumour cells to survive
a sharp drop in mitochondrial respiration, as well as and proliferate in alternative tissue environments.
decreased transcription of VHL25. Metabolites and more mtDNA inducer mutations are envisioned as con-
than 150 histone modifications were also affected. For tributing to the initiation of the transformation genetic
example, the mitochondrial acetyl-​CoA level remained event. Likely examples are those found in oncocytomas
stably high to the 90% mutant, when it declined in and prostate cancer (Fig. 3). Potential inducer mtDNA

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mutations are commonly identified in different tumour tension, or can be increased to compensate for partial
types by their tumour-​specific presence. There are many mitochondrial dysfunction to sustain OXPHOS.
hundreds of candidate inducer mtDNA mutations. Mutations in the nDNA-​c oded mitochondrial
However, to date very few cancer mtDNA mutations have genes IDH1, IDH2, FH and SDH can alter the levels of
been subjected to the rigorous genetic and biochemi- mitochondrial intermediates (acetyl-​CoA, succinate,
cal analysis that is required to conclusively determine fumarate and α-​KG) and lead to the generation of onco-
the pathogenicity of a mitochondrial disease-​causing metabolites that modulate the activities of enzymes
mtDNA mutation81. modifying the epigenome. These metabolites and onco-
The mtDNA adaptor variants are even more dif- metabolites can change nuclear gene expression and
ficult to confidently assign a tumorigenesis or cancer contribute to neoplastic transformation. Although lim-
progression function. This is because some of the same ited in number, studies indicating epigenomic changes
mtDNA mutations that permitted our ancestors to adapt in response to mtDNA are emerging. However, because
to different environments also arise de novo in cancers of the high ploidy of mtDNA, heteroplasmic mtDNA
and may function to permit the cancer cells to adapt to genotypes can change rapidly. Multiple mitochondri-
changing tissue environments. Some adaptive variants ally regulated variables, including ATP for phospho-
can be associated with specific cancers when inherited rylation, NAD+:NADH ratio, ROS, Ca2+ and pH, can all
as mtDNA haplogroups, as seen for the m.10398A>G change with mtDNA heteroplasmy, resulting in dynamic
(A allele, 114Thr) variant carried by macrohaplogroup N changes in cellular signal transduction systems and the
and associated with increased African American epigenome26.
breast cancer risk (Table 1). However, the m.10398A>G Hence, mitochondrial physiology and mtDNA vari-
(G allele, 114Ala) variant can also arise de novo from the ation can impart enormous plasticity to the tumour cell.
patient’s 10398A>G, A allele mtDNA in bone metastases This variation can potentiate the induction of a tumour
of prostate cancer patients105. and assist in its adaption to a changing environment
The mtDNA copy number can also vary. For exam- during metastasis and in response to therapy.
ple, mtDNA copy number can be reduced to modu-
late tumour mitochondrial metabolism at low oxygen Published online 27 May 2021

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