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Predoctoral Program in Human Genetics [J. B. J.], Departments of Oncology [P. M. H., J. J. S., G. P., R. H. H., S. E. K.], Pathology [R. H. H., G. P., S. E. K.], and Biostatistics [G. P.], Johns Hopkins University, Baltimore, Maryland 21231
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Introduction |
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The biology of the mitochondria suggests that its genome may be an attractive target for mutations that could drive tumorigenesis. Mitochondria generate ROS, which at slightly elevated concentrations are highly mitogenic molecules that are also mutagenic to the nuclear and mitochondrial genomes (3) . Mitochondria are also known to play an important role in regulating or effecting apoptosis (reviewed in Ref. 4 ). Furthermore, it has long been known that mitochondria of rare oncocytic tumors can accumulate to a high density (5) , and more commonly, that tumors tend to up-regulate glycolysis, in turn leading to a lesser dependence on the mitochondria for oxidative phosphorylation (6) .
The "mitochondrial bottleneck" is the presumptive process by which the maternal oocyte contributes a homogeneous population of mtDNA genomes to the zygote, resulting in an organism with only one mitochondrial genotype, i.e., homoplasmy (7) . Recently, however, several groups have identified homoplasmic variants and somatic mutations of mtDNA in various tumor types (8, 9, 10) . It has proven empirically easier to detect known mtDNA mutations in clinical exfoliative samples of cancer patients as compared with detection of mutations of nuclear genes, although the physical basis of this observation remained unexplored. In the current study, we investigate the mtDNA mutations of pancreatic cancer to address the evidence of a role in tumorigenesis and discuss the physical basis, benefits, and limitations of mtDNA analysis as a diagnostic tool.
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Materials and Methods |
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MnSOD Sequencing.
A panel of 100 pancreatic cancer xenografts was analyzed by PCR for the
presence of homozygous deletions of the MnSOD locus using
gene-specific PCR primers. This panel was evaluated for LOH at 6q25.2
using polymorphic dinucleotide repeat markers D6S1579,
D6S1581, D6S305, D6S1599, and D6S1719 (Research
Genetics, Huntsville, AL). In those samples exhibiting LOH, all five
exons were amplified using intronic or 5' untranslated region primers
and were directly sequenced to examine the complete coding regions. PCR
primers and sequencing primers used in this project are available upon
request.
Complete Mitochondrial Genome Sequencing.
The entire 16.5-kb mitochondrial genome was amplified and sequenced in
samples from five xenografted primary pancreatic adenocarcinomas and
the cell lines AsPc1, BxPc3, CAPAN1, CAPAN2, COLO357, Hs766T, Su86.86,
CFPAC1, MiaPaCa 2, and Panc1. Total cellular DNA was prepared for each
sample as described (12)
. To avoid amplification of
nuclear mitochondrial pseudogenes, the mitochondrial genome was
amplified as 10 overlapping 1–3-kb PCR products (9)
.
Direct automated sequencing of amplified templates was performed on
Beckman CEQ2000 and ABI377 machines. Resulting sequence data files were
analyzed and compared with the standard Anderson mtDNA sequence
(13)
using the Sequencher analysis program (Gene Codes,
Ann Arbor, MI). Homoplasmic deviations from the standard sequence were
compared with the on-line Mitomap database of previously published
mtDNA mutations and
polymorphisms.4
Confirmation of those mutations that were not described previously as
polymorphisms was performed by sequencing in the reverse direction on
independent PCR products. To determine which mutations were somatic,
DNA samples from normal nonneoplastic tissues corresponding to each
tumor were sequenced as above. Limiting dilution PCR, followed by
allele-specific oligonucleotide hybridization, was used to detect the
potential presence of low levels of mutant mtDNA in normal tissue. This
(digital PCR) was performed similarly to methods described but used
sequence-specific dot blot hybridization for allele discrimination
(14)
.
mtDNA Mass Comparison.
To determine the relative copy number (mass) of mtDNA upon comparison
of two sets of paired pancreatic cancer xenografts and surrounding
normal tissue, Southern blot hybridizations were performed. To detect
mtDNA, a 1-kb probe from the mitochondrial mtND1 gene was
labeled by random primer method with [32P]dCTP
and applied to blots of 10 µg of EcoRI-digested cellular
DNAs using standard protocols. Total nDNA was relatively quantitated by
reprobing the same blot with a random primer-labeled CotI
DNA probe (Bio-Rad, Hercules, CA). The relative amounts of both mtDNA
and nDNA were estimated by PhosphorImager and image analysis
densitometry using ImageQuant software (Molecular Dynamics, Sunnyvale,
CA).
Detection of mtDNA Mutation versus nDNA Mutations.
Two DNA sets, each consisting of a primary pancreatic tumor (C16 and
C19), a xenograft of the tumor (PX16 and PX19), and corresponding
normal duodenum (N16 and N19) from two patients with known mtDNA and
nDNA (K-ras) mutations, were obtained. The neoplastic
cellularity of each sample was estimated by light microscopy. Both
primary cancer samples consisted of 30% or fewer neoplastic cells.
Amplified products containing each of the known mutations were created
using 40 ng of template DNA for each sample. The amplified DNAs were
directly sequenced.
Bioinformatics.
tRNA structure prediction was performed using the tRNAscan-SE program
available on-line (Washington University, St. Louis, MO). Sequence
alignments were performed using public resources (National Center for
Biotechnology Information).
Mathematical Modeling.
To address the possibility that homoplasmic mutations of mtDNA may
arise in the absence of selection, we created a simulation algorithm to
statistically model mtDNA
segregation.5
The algorithm probabilistically generates cell histories under a
variety of conditions. It is written in the publicly available software
package R and is available for
download.6
On the basis of current literature, we started with cells containing an
average of 100 mitochondria, each containing one genome
(15)
. In this model, a single mtDNA genome/cell is mutated
at one position at time zero. The fate of the mutant mtDNA species is
then evaluated after the parent cell completes 2000 cell division
events. At each cell division, a percentage (p) of
mitochondria receive a signal to divide. In our modeling experiment,
p was set to 0.05, 0.5, or 0.95. The parent cell divides
when a sufficient number of new mitochondria (at least twice the
original number) are present. The distribution of mitochondria to the
two daughter cells is random.
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Results |
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. Of these 49 variants, 26 were silent changes within expressed
sequences. The 23 remaining homoplasmic mutations within coding
sequences, or regulatory sequences, exhibit potential functional
significance.
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. Two cell lines (AsPc1 and Su86.86) had no apparent functionally significant homoplasmic mutations, i.e., all variants were previously described polymorphisms, whereas each of the remaining eight cell lines harbored at least one new variant of the coding sequence. Two cell lines (Hs766T and MiaPaCa 2) each contained only one new coding sequence mutation. Three other lines (Panc1, CAPAN2, and BxPc3) each contained two new coding mutations. CFPAC1 contained four new coding mutations, whereas CAPAN1 and COLO357 each, respectively, contained five and six individual homoplasmic potentially functional mutations not reported previously. There were no major deletions or rearrangements of mtDNA in any sample. All new mutations were single bp substitutions; nearly all were transition mutations consistent with damage from ROS (16) . Each of the mutations of a protein coding sequence resulted in a single missense mutation. The functional significance of these mutations was unclear. However, one of the mutations (G9804A) of the mtCO3 gene in cell line BxPc3 is reported as a secondary mutation in Leber hereditary optic neuropathy (17) .
Unfortunately, because normal DNA corresponding to the commercial
cancer cell lines was not available, it was not possible to
differentiate those cell line mutations that arose somatically from
those mutations that were rare constitutional variants. To search for
somatic mutations, the complete mitochondrial genome of five
xenografted pancreatic cancers was sequenced. One hundred twenty-seven
homoplasmic variants were detected, of which 27 were not among the
polymorphisms reported previously. Nearly all of these variants were
also detected in the corresponding normal DNA of the patients. However,
four of the five xenografts were shown to harbor a single somatic
homoplasmic mtDNA mutation (Table 2)
.
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Functional Significance of Somatic Mutations.
Of the four somatic mutations discovered in the xenografted cancers,
one mutation was an A-to-T substitution in the mitochondrial rRNA, of
unknown significance. Two of the somatic mutations occurred in
protein-coding regions of the mitochondrial genome, both coding for
protein subunits of the mitochondrial respiratory chain NADH
dehydrogenase complex, mtND1 and mtND6. The degree of evolutionary
conservation of the affected residues was estimated using a multiple
sequence alignment comprising 105 metazoan mitochondrial ND1
and ND6 sequences (data not shown). The mtND6 serine residue
mutated by the transition at mtDNA position 14603 in tumor sample PX16
was conserved in 71% of metazoan mtDNAs. Mutation of this residue to
phenylalanine may thus significantly affect the activity of this
protein. The second mutation at position 3670 replaces an alanine codon
in the mtND1 gene with threonine. This alanine is conserved
in 74% of metazoans but tolerates a threonine mutation in the Pacific
oyster. The effect of this mutation on complex I activity remains
unclear.
The fourth mutation is a C-to-T transition at position 15983 in the mitochondrial tRNA for proline. A predicted tRNA structure was created using tRNAscan (data not shown). From this structure, the effect of the mutation would be to change a wild-type U/A stem bp to a U/G bp. Although less stable than U/A pairing, U/G base pairing is often seen in wild-type tRNA structures.
Detection of Mutant mtDNA in Normal Tissue.
The human mitochondrial genome has been sequenced extensively. An
on-line database, Mitomap, lists the compilation of disease-causing
mutations, as well as hundreds of mtDNA polymorphisms that have been
observed4
(24). Most (227 of 353; 64%)
homoplasmic mtDNA variations observed in this study were present in
this database as known mtDNA polymorphisms. Twenty-three (85%) of the
27 of the non-listed homoplasmic variations from the Anderson sequence
in our tumor xenografts were also detected in corresponding normal
tissue. Thus, these variations represent undescribed rare polymorphisms
of mtDNA, i.e., individual constitutional variation. Each
xenograft harbored from 2 to 10 of these new variants in homoplasmy.
In one sample, xenograft PX27, the homoplasmic G-to-A variation from
the Anderson sequence at position 3670 was also detected by direct
sequencing as coexisting with the consensus sequence in the
corresponding normal duodenal tissue (Fig. 1A
and Table 2
). This indicates that during evolution of the
tumor, a preexisting heteroplasmic population of mitotypes had evolved
to apparent homoplasmy. The presence of mutant mtDNAs in corresponding
normal tissues at a level below detection by direct sequencing had not
yet, however, been excluded as a possibility in the other patients.
Therefore, we studied the G-to-A mutation at position 14603 in PX16 by
digital PCR. Of 191 single mtDNA template PCR products from the
corresponding normal DNA, one product contained the mutant allele,
whereas all others contained the wild-type allele (data not shown).
This borderline result was felt to be inconclusive, consistent with a
trivial explanation such as disease metastasis or surgical
contamination during specimen acquisition, as well as with an extremely
low level of constitutional heteroplasmy.
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Detection of mtDNA and nDNA Mutations in Primary Tumors.
A Southern blot was performed to compare the levels of mtDNA in
pancreatic cancer versus normal cells. DNA from two
xenografted pancreatic tumors (PX16 and PX19) were compared with DNA
samples of normal duodenum from the same patients (Fig. 1B)
.
The quantity of mtDNA was 7.7-fold higher than normal in tumor sample
PX19 and 6-fold higher than normal for sample PX16 when
normalized for nDNA levels, as determined by PhosphorImager
densitometry.
Two sets of samples (patients 16 and 19), each composed of a pancreatic
primary cancer sample, a xenograft of this sample, and a corresponding
normal sample from the same patient, were then studied by sequencing.
Both mtDNA and nDNA (K-ras) mutations of these cancers were
known. Xenografts PX16 and PX19 had the homoplasmic mtDNA mutations
described above, while also harboring an nDNA G-to-T mutation in codon
12 of the K-ras gene. The malignant cellularity of both
primary cancers was histologically estimated at 30% or less. PCR
products containing the mutations were amplified from the primary
cancer, xenograft, and normal samples for these two cases. The results
of direct sequencing of these products are shown in Fig. 1C
.
In the case of patient 16, the nuclear K-ras gene mutation
was detectable on one allele in the xenograft sample, absent from the
normal sample, and not visible in the primary cancer because of
contamination by nonneoplastic cells (Fig. 1C)
. In striking
contrast, the mutant A allele (i.e., the mtDNA
mutation) was clearly present in both the xenograft and primary cancer
samples while absent from normal, as expected. Similar results were
seen in patient 16 (data not shown).
MnSOD Sequencing.
To effect a theoretical oncogenic increase in the level of ROS, the
observed mutation of one mtDNA molecule followed by the expansion of
that molecule to become the dominant species within the cell would
appear to be a less efficient means than would mutation of a
nuclear-encoded gene that regulated ROS. For this reason, a sequence
analysis of the MnSOD gene, coding for an enzyme localized
to mitochondria and responsible for neutralizing ROS, was performed. In
a survey of 100 xenografted pancreatic tumors, we found LOH of
chromosome 6q25.2 in 26% of these tumors. Complete sequencing of the
coding sequences of the MnSOD gene was performed on the
tumors having LOH, and no mutations were discovered.
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Discussion |
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But can preexisting mtDNA heteroplasmy also contribute to cancer? It
has been observed that in some heteroplasmic cells, the proportion of
mtDNA molecules represented by each segregating mitotype can undergo
rapid shifts during cell culture (18)
. In fact, certain
mitochondrial genotypes can quickly and reliably become the dominant
mtDNA in a mixed population (9)
. The mechanism underlying
this phenomenon is unknown. In our survey of mutations of the
mitochondrial genome in pancreatic cancer, we identified four
homoplasmic somatic mutations. In one case, the homoplasmic mutation in
the cancer apparently descended from a heteroplasmic population of
normal and mutant mtDNAs in the surrounding normal nonneoplastic
tissue. According to the classic selection model, heteroplasmic mtDNA
mutations may create a subtle functional defect in oxidative
phosphorylation that results in slightly increased ROS levels (Ref.
9
; Fig. 2A
). Low levels of ROS are mitogenic to the cell and possibly
mutagenic to the nDNA (3)
. The mutant mtDNA may contribute
to a selective advantage during tumorigenesis in cells that have a high
copy number of the mutant sequence. It would follow that inherited
mutant mtDNA heteroplasmy could be a new basis for genetic cancer
susceptibility. The appropriate biological and epidemiological studies
would be required to address this possibility. However, there is an
alternative hypothesis to explain homoplasmic mtDNA mutations.
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, it is very unlikely that this mutation
would have any functional alteration. Other studies have even
identified somatic mtDNA mutations that are silent, i.e.,
resulting in usage of an equivalent codon (8, 9, 10)
. It is
difficult to imagine that these mutations can confer enough selective
advantage to drive a process of intramitochondrial selection followed
by intracellular selection, as required to become homoplasmic in the
overall cellular mtDNA population (Fig. 2A)
.
A model of random drift to homoplasmy might instead explain the
presence of mtDNA mutations seen by our group and others (Fig. 2B)
. During development, mutations to the mitochondrial
genome would create intracellular heteroplasmy, a population of many
unique mtDNAs, each present at a low frequency. Because of mtDNA
"population genetics" alone, at some rate, a number of these
mutations will become homoplasmic. Other factors could influence the
frequency of this event. For example, the proposed "nucleoid model"
of mtDNA replication may afford a type of somatic bottleneck, whereby
the number of replicating units in each cellular generation is reduced
to only the few mtDNA molecules that are part of the replicating
nucleoid (19)
. Additionally, unequal partitioning of
daughter mitochondria during cell division could increase the rate at
which mutant alleles evolve to homoplasmy. Tumorigenesis, driven in
large part through genetic and epigenetic changes affecting the nDNA,
involves the clonal expansion of subclones of cells, and with them,
their drifted mitotype.
Our statistical modeling of mtDNA segregation in the absence of selection shows that under biologically reasonable conditions, a single mutant mtDNA has the capability to undergo "genetic drift" to become the homoplasmic mtDNA species. This rate can be increased by secondary events, such as a reduction of total replicating units (as in nucleoid model), or reduced number of total mitochondria (effectively the same as an unequal partitioning of daughter mitochondria), as described above. In our simulation, we studied the segregation of a mutation at only one position in the mitochondrial genome. However, given the mutation rate of mitochondrial DNA and the large size of the genome (16.5 kb), the initial somatic cell of the tumorigenic process is likely to contain a considerable number of mutant mtDNA genomes. This suggests that the most likely outcome of random mtDNA evolution in adult-onset tumors might be homoplasmy for one mtDNA mutation.
The likelihood of this premise can be calculated directly from our mathematical modeling. When we began with one mtDNA mutation, we observed that after 2000 generations, 1% of daughter cells contained the mutant mtDNA in homoplasmy. Therefore, inversely, the probability of not generating a homoplasmic mutant cell is 0.99. If the parental cell contained not just 1 but perhaps >50 different mutant mtDNAs, then the probability of not generating a homoplasmic mutant daughter cell would be at most 0.99 raised to the 50th power, i.e., a <60% chance to avoid homoplasmy for any of the mtDNA mutations. When each cell has such a large likelihood of randomly generating a homoplasmic mutant daughter cell, mathematical modeling hereby supports the hypothesis that most, if not all, of these unique homoplasmic mtDNA mutations seen in our study and others may be explained without invoking selection for mtDNA-based replicative advantage.
More convincing evidence for random drift derives from the observation
that normal heteroplasmic cell populations do evolve to homoplasmy
in vivo. Individuals have been described with constitutional
heteroplasmy (Refs. 20
and 21
and Table 2
).
In these individuals, the proportion of each mitotype exhibits extreme
tissue variability (20
, 22)
. For example, Wilson et
al. (22)
examined the relative proportions of two
mitotypes in individual hairs, which are derived from very few stem
cells, from a proband with inherited heteroplasmy. They found that some
hairs had a 1:1 ratio of each mitotype, some hairs had complete
homoplasmy of one mitotype, and some hairs had homoplasmy of the other
mitotype. Thus, adjacent clones of cells exhibited random drift during
development from an initial heteroplasmic state to homoplasmy for
either mtDNA allele. Furthermore, in this study we identified at least
one unique mtDNA variant present in homoplasmy in nearly all samples
sequenced. In conclusion, although selection of subtle mutant mtDNAs
may play a role in tumorigenesis, it is important to consider the
alternative model: that tumor mtDNAs may exhibit a random drift to
homoplasmy (Fig. 2)
.
mtDNA as Marker of Clonality.
Clinical tumor detection methods, designed to identify markers of
nuclear genetic lesions, have often failed because of the difficulty of
detecting mutant DNA amid the contaminating wild-type DNA present in
diagnostic samples. Presumably, the detection of mtDNA lesions would
suffer the same consequence because of the same ratio of contaminating
normal mtDNA. Fliss et al. (8)
reported
recently the facile detection of mtDNA mutations in diagnostic samples,
reliably detecting known mutations of mtDNA against the background of
normal mtDNAs present in bodily fluids. There are two potential
mechanisms by which this could have occurred. One is that mtDNA in
tumor cells might be resistant to degradation, perhaps because of
cellular apoptotic defects or other neoplasm-related differences. The
alternative would be the possibility that the ratio of mutant:normal
mtDNA was altered within the tumors. Cancers, perhaps being composed of
relatively larger cells or because of other physical properties, may
simply have more mtDNA than do normal cells. Our Southern blot data
support the latter hypothesis by demonstrating the level of mtDNA in
two pancreatic cancer samples to be 6–8-fold higher than in normal
duodenal cells, when controlled for the level of nDNA. This supports a
recent study by Lee et al. (23)
demonstrating
an increase in mtDNA copy number in lung fibroblasts as an early event
in response to oxidative stress. Additionally, we confirmed the facile
detection of two known mitochondrial mutations by direct sequencing in
primary tumors. The malignant cellularity of the two primary carcinomas
was low, such that the existence of the known K-ras
mutations could not be established by direct sequencing (Fig. 1C)
. Strikingly, the known mtDNA mutations were readily
detected in the same samples (Fig. 1C)
. In the case of
clinical specimens, where the mtDNA mutations are yet unknown,
unfortunately, mtDNA is so highly polymorphic that is will be difficult
to distinguish the true somatic mutations that designate a tumor from
the potential somatic mutations that might occasionally arise during
development of a specific tissue. To use mtDNA mutations as a means of
detecting clonal populations of cells, it may be necessary to perform
sequencing of the entire mitochondrial genome of both a sample
containing tumor cells as well as a sample of a tissue-specific normal
mtDNA genome of the same patient. This must be done to define the
individual average constitutional mitotype amid the wide variability of
"normal" mitochondrial genomes seen among the human population.
Deviation from the mitotype present in the test sample would indicate
the expansion of an alternate mitotype attributable to clonal expansion
of a cell containing a high proportion of mutant mtDNA. Thus, despite
some caveats, the evolution of homoplasmic mutations in tumors,
combined with the relative increase in mtDNA mass quantity, does
provide an attractive potential diagnostic marker for further
exploration.
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FOOTNOTES |
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1 Supported by the NIH Specialized Program of
Research Excellence in Gastrointestinal Cancer Grant CA 62924. 
2 To whom requests for reprints should be
addressed, at The Johns Hopkins University, 451 CRB, Baltimore, MD
21231. Phone: (410) 614-3314; E-mail: sk{at}jhmi.edu
3 The abbreviations used are: rRNA, ribosomal RNA;
mtDNA, mitochondrial DNA; nDNA, nuclear DNA; ROS, reactive oxygen
species; LOH, loss of heterozygosity; MnSOD, manganese superoxide
dismutase.
4 MITOMAP: A Human Mitochondrial Genome Database;
see Internet address http://www.gen.emory.edu/mitomap.html.
5 G. Parmigiani, J. Jones, and S. E. Kern,
manuscript in preparation.
6 Internet address:
http://biosun01.biostat.jhsph.edu/
gparmigi/mito.html.
Received 10/30/00. Accepted 12/29/00.
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