Download A Mutator Phenotype in Cancer

[CANCER RESEARCH 61, 3230 –3239, April 15, 2001]
Perspectives in Cancer Research
A Mutator Phenotype in Cancer1
Lawrence A. Loeb2
Departments of Pathology and Biochemistry, The Joseph Gottstein Memorial Cancer Research Laboratory, University of Washington, Seattle, Washington 98195-7705
Abstract
We have proposed that an early step in tumor progression is the
expression of a mutator phenotype resulting from mutations in genes that
normally function in the maintenance of genetic stability. There is new
and strong experimental evidence that supports the concept of a mutator
phenotype in cancer. As technologies for chromosomal visualization and
DNA advance, there are increasing data that human cancer cells contain
large numbers of mutations. First, I will review the concept of a mutator
phenotype. Second, I will present the recent evidence that individual
cancer cells contain thousands of mutations. Third, I will explore potential
target genes that are required for maintenance of genetic stability in
normal cells and ask if they are mutated in cancer cells. Fourth, I will
address the timing of a mutator phenotype; is it an early event during
tumor progression? Do tumors already contain cells that harbor mutations rendering them resistant to most chemotherapeutic agents? Lastly, I
will speculate on the theoretical and practical implication of a mutator
phenotype in cancer and consider the possibility of cancer prevention by
delay, i.e., a reduction in mutation rates early during carcinogenesis might
slow the progression of tumors.
Introduction
The maintenance of a species, and perhaps of an individual, requires that DNA replication be exceptionally accurate. Information
contained in DNA must be accurately copied and transmitted to each
daughter cell. The fact that the DNA in cancer cells is different from
the DNA in normal cells indicates that carcinogenesis involves substantial errors in DNA replication, deficits in DNA repair, and alterations in chromosomal segregation.
In normal cells, DNA replication and the partitioning of chromosomes are exceptionally accurate processes. During every division
cycle, each daughter cell receives a full and exact complement of
genetic information. Each parental cell duplicates its genome containing approximately 6 ⫻ 109 nucleotides and then partitions the DNA
equally between the two newly generated daughter cells. The fidelity
of this transfer is precise in both germ-line and normal somatic cells.
Errors in these processes result in mutations. In germ-line cells, a few
mutations are necessary to permit a species to adapt to environmental
changes. In principle, somatic cells might be able to tolerate the
production of large numbers of mutations, because these mutations are
not passed on to subsequent generations and thus would not accumulate within the gene pool of a species. However, when these somatic
mutations are expressed, they cause diseases that curtail the life of
individuals. In humans, cancer is the most prominent of these diseases. The concept of a mutator phenotype in cancer was initially
formulated (1) to account for the disparity between the rarity of
mutations in normal cells and the large numbers of mutations present
in a variety of human malignancies. This hypothesis has evolved in
Received 11/6/00; accepted 2/16/01.
1
This work was funded by Grants CA80993 and CA74184 from the National Cancer
Institute.
2
To whom requests for reprints should be addressed, at The Joseph Gottstein Memorial Cancer Research Laboratory, Box 357705, Departments of Pathology and Biochemistry, University of Washington, Seattle, WA 98195-7705. E-mail: laloeb@u.
washington.edu.
accord with our increasing ability to dissect carcinogenic processes at
the molecular level (1– 4). It seems pertinent now to summarize recent
data and to consider the theoretical and practical implications of a
molecular phenotype in cancer.
There are multiple DNA synthetic transactions that could be altered
in normal cells and generate a mutator phenotype; these include both
errors in DNA synthesis and inadequate repair of DNA damage. Here
we define a mutation as any change in the nucleotide sequence of
cellular DNA. Errors in DNA synthesis are generated by misincorporation of nucleotides by DNA polymerases. Mutations are produced
when the number of these misincorporated nucleotides exceeds the
capacity of cells to excise and repair the lesions. DNA damage is
produced by reactive molecules generated in cells by normal metabolic processes, as well as by exogenous environmental agents. Cells
have evolved multiple mechanisms for the excision and repair of
DNA damage. Nevertheless, the amount of DNA damage can exceed
the cell’s capacity for DNA repair, and the residual nonrepaired
lesions can be a dominant source of mutations during DNA replication.
Somatic mutations can also result from errors in the partitioning of
chromosomes during cell division. Inequalities in chromosomal partitioning result in aneuploidy, a change in chromosome number,
which is one of the most common hallmarks in cancer cells. Unfortunately, the genes responsible for chromosomal segregation are only
now beginning to be identified; as a result, partitioning errors will be
considered only briefly in this prospectus.
A Mutator Phenotype in Cancer
Considering the rarity of mutations in normal cells and the large
numbers of mutations observed in human cancers, it has been proposed that the spontaneous mutation rate in normal cells is not
sufficient to account for the number of mutations found in human
cancers (1). Simply stated, cancer cells exhibit a mutator phenotype.
This phenotype is the result of mutations in genes that function in the
maintenance of genomic stability. It is manifested by increases in
mutation rates and in genetic evolution of cancer cells that drives
tumor progression.
Historical Perspective
The multiplicity of chromosomal aberrations found in many human
tumors was noted by pathologists more than 100 years ago and has
served to identify malignant cells and to stratify the aggressiveness of
certain cancers (5). Chromosomal aberrations include translocations,
gene amplifications, deletions, and insertions; these can be classified
as mutations that involve the repositioning, addition, or omission of
millions of nucleotides. The large numbers of these alterations in
certain cancers, coupled with the morphological and physiological
heterogeneity of cancer cells within individual tumors, were important
observations that underpin the concept of a mutator phenotype in
cancer. We initially proposed that the multiple mutations found in
tumor cells would result from mutations in genes that guarantee the
fidelity of DNA synthesis or the adequacy of DNA repair (1). Muta-
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tions in bacterial DNA polymerases were known to produce mutations
throughout the genome (6). Individuals with rare inherited diseases
such as xeroderma pigmentosum (7) or ataxia telangiectasia (8) exhibit a high incidence of certain cancers, and cells from these individuals exhibit decreased survival upon exposure to the same agents
that cause cancers in these individuals. Despite the presence of multiple “hot spots,” DNA damage is primarily a random process; if the
damage is not repaired, it could serve as a potent source of mutations.
By chance these mutations might be in genes required for the maintenance of genetic stability. Mutations in genetic stability genes could
produce additional mutations throughout the genome. Among these
would even be mutations in other genetic stability genes, leading to a
cascade in mutations as cancers evolve.
At about the same time, Nowell (9) proposed that a mutator
phenotype in cancer is the result of repetitive rounds of clonal selection in which mutations in a single cell impart a selective growth
advantage, allowing its progeny to proliferate and populate the tumor.
Tumor progression results from sequential proliferation of more aggressive sublines. Successive rounds of clonal expansion drive tumor
progression and result in multiple mutations. It seems likely that both
mechanisms contribute to the multiple mutations found in cancer; but
surprisingly, the two mechanisms could act in synergy. Mutation
accumulation and clonal selection may be inextricably linked (see
below).
Mutations Are Infrequent in Normal Human Somatic Cells
Normal human cells accurately replicate DNA every time they
divide. The overall mutation rate in somatic human cells has been
estimated at 1.4 ⫻ 10⫺10 nucleotides/cell/division or 2.0 ⫻ 10⫺7
mutations/gene/cell division (10). Considering that each cell contains
⬃70,000 genes, we estimate that each cell accumulates one mutant
gene during the life span of an individual (3). However, we lack
information on mutation rates in somatic cells in different tissues and
in different mammalian species. It is likely that somatic mutation rates
in human cells are lower than in rodent cells, and this may contribute
to the resistance of human cells toward transformation in vitro (11,
12). If this disparity between humans and rodents is manifested in
cancer precursor cells, it suggests that carcinogen testing in rodents is
not an adequate model for human susceptibility, although it is currently the best available.
A large body of evidence indicates that most tumors originate in
one or a few stem cells. These cells have a remarkable plasticity,
allowing them to differentiate into a variety of cell types, including
those that are malignant. On the basis of the assumption that the
accuracy of DNA replication in stem cells is similar to that in human
somatic cells, we estimated that each stem cell amasses one to two
mutant genes (assuming 100 cell divisions during a human life span;
Ref. 13). However, based on a Poisson probability distribution, there
would be a few stem cells that would contain as many as 12 mutations.
The slope of the exponential increase of cancer as a function of age
suggests that there are 6 –12 cancer-causing events (14, 15), each of
which is rate limiting for tumor progression. If one assumes that these
rate-limiting events are mutations, then it is conceivable that normal
mutation rates can account for the age increase in cancer. Each of
these mutations would have to offer a selective growth advantage,
even if it occurred on a single allele. However, if we restrict our
analysis to genes that have been shown to be associated with human
cancers (an estimated 100 known oncogenes and tumor suppressor
genes; Ref. 16), then the normal mutation rates would produce only
two or three mutations in cancer-associated genes. In either case, the
mutation rate in normal cells cannot account for the thousands of
mutations being found in human cancer cells.
Multiple Mutations Are Frequent in Human Tumors
Historically, cytological observations on variations in chromosome
number and integrity in cancers have always implied that cancers
contain multiple mutations. In only rare instances are specific chromosomal aberrations diagnostic of particular malignancies. More informative cytogenetic methods that are being developed may reveal
additional examples of tumor-specific chromosomal abnormalities.
Nevertheless, the majority of chromosomal abnormalities are not
tumor specific but instead may indicate the manifestations of an
underlying instability in cancer cells.
Chromosomal Instability
Chromosomal aberrations in cancer cells have been recognized as a
criterion for cancer and for grading tumors to predict prognosis. With
our increasing ability to characterize chromosomes at the molecular
level, there has been a expanding literature on multiple chromosomal
alterations in different tumors. Two techniques have been particularly
revealing:
(a) Comparative genomic hybridization measures differences in
hybridization between fluorescent labeled fragments of tumor and
normal DNA using metaphase chromosomes as a scaffold. A large
variety of tumors have been shown to exhibit changes in DNA copy
number (17). Some breast cancers exhibit as many as 15 changes in
DNA copy number (18), and an even greater number were exhibited
in high-grade ovarian cancers (19). Even benign tumors such as
uterine leiomyomas (20) exhibit changes in DNA copy number.
Comparative genomic hybridization can detect deletions or amplifications only if they are 1–10 megabases (21); presumably, with the
development of more sensitive techniques, a greater number of
changes may be detected in cancers. These studies are carried out
using bulk DNA, and thus the only changes observed are those that
occur in most of the population. However, a PCR-based strategy for
global amplification of DNA from a single cell has affirmed the
multiplicity of changes in DNA copy number in cancers and can be
used to map clonal evolution (22).
(b) Measurements of loss of heterozygosity in tumors have been
analyzed using microsatellite markers that scan only a small fraction
of the genome. Nevertheless, some primary breast cancers exhibited
⬎20 regions with loss of heterozygosity (23). Because the probes used
in this study sampled only 0.01% of the genome, we calculate that
some of these tumors might contain as many as 200,000 regions with
loss of heterozygosity (24). This calculation is biased, because the
number of mutations detected in small regions may not be representative of that which occurs throughout the entire genome, and also the
target sequences examined were those demonstrated previously to be
altered frequently in human tumors. Nevertheless, if these changes are
in any way representative of other changes throughout the genome,
one must conclude that in many cancers there are tens of thousands of
mutations.
It has been argued that there are two types of genetic instability,
microsatellite instability and chromosomal instability (25, 26). Cancers that exhibit extensive microsatellite instability are those with
mutations or inactivation of mismatch repair genes; those that exhibit
predominantly chromosomal instability are those with mutations that
affect the partitioning of chromosomes during mitosis. However, both
microsatellite instability and chromosomal instability may be manifestations of a mutator phenotype. Both of these phenomena may be
easy to detect; microsatellite sequences are subjected to slippage
during copying and are present in thousands of copies, and chromosomal alterations can be visualized cytologically, because they can
involve millions of nucleotide units in DNA (27). In contrast, random
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changes in one or a few adjacent nucleotides in DNA could occur
more frequently, yet would be very difficult to detect.
Aneuploidy
Aneuploidy is a change in chromosomal number resulting from
unequal partitioning of chromosomes during cell division. It occurs
frequently in solid tumors, and the generation of aneuploidy has been
proposed as the initiating event in carcinogenesis (28, 29). Aneuploidy provides a one-step process that generates thousands of genetic
alterations that could destabilize the genome. However, diseases such
as Down’s syndrome present a striking enigma; cells contain an extra
copy of chromosome 21 with some 200 genes (30), and yet these cells
differentiate to produce an individual. Although individuals with
Down’s syndrome exhibit an elevated incidence of leukemia, if anything they have a decreased incidence of most solid tumors (31).
Furthermore, one still has to face the proverbial dilemma of whether
aneuploidy generates genomic instability, or whether genomic instability generates aneuploidy, or are both manifestations of an underlying mutator phenotype. In Barrett’s esophagus, alterations in p53,
p16, and increased tetraploidy (4N) precede aneuploidy (32, 33), and
genetic instability in ulcerative colitis precedes tumor detection (34).
Microsatellite Instability
The extensiveness of cancer-associated genetic instability was first
heralded by findings of mutations in repetitive sequences. Peinado et
al. (35) used random oligonucleotides as arbitrary primers in PCR
reactions and observed additional products of different sizes using
DNA obtained from human colon tumors compared with those obtained using DNA from adjacent normal tissues. The tumor-specific
PCR products with altered lengths encompassed segments with repetitive nucleotide sequences. These investigators sampled only a small
fraction of the genome, and yet within their limited vista they observed large numbers of alterations in DNA sequence. Upon extrapolating these results to the whole genome, they concluded that some
tumors contained as many 100,000 mutations. The tumor cells with
the greatest numbers of changes in the lengths of repetitive sequences
were subsequently shown to possess mutations in mismatch repair
genes (36). Altered repetitive sequences were mainly detected in
segments between genes (microsatellites) and occurred predominantly
in HNPCC.3 Mutations in repetitive sequences within exons were
subsequently demonstrated (37, 38). These results were confirmed
recently and extended to sporadic colon cancers using inter (simple
sequence repeat)-PCR (39). These authors estimate that many sporadic colon cancers contain tens of thousands of mutations, and that
many of these mutations are already present in premalignant polyps.
In the case of HNPCC, instability is mediated by mutations in mismatch repair genes that function in correcting replication error. Many
sporadic colon (40) and sporadic gastric (41) cancers that do not
appear to contain mutations in mismatch repair genes exhibit hypermethylation of the promoter region of MLH1, thus silencing mismatch
repair by an epigenetic mechanism. A large number of nonhereditary
cancers have been shown to exhibit alterations in microsatellite repeats to a lesser extent but do not appear to involve mutations in
mismatch repair genes. It remains to be established whether the source
of this microsatellite instability in non-HNPCC tumors is the result of
decreased expression of mismatch repair genes or is attributable to
mutations in other genes involved in maintaining genetic stability.
The cause(s) of microsatellite instability remains to be determined.
In general, mutations are generated when the rate of mutation pro3
The abbreviation used is: HNPCC, hereditary nonpolyposis coli.
duction exceeds the cell’s capacity for correction. In vitro studies
indicate additions and deletions at repetitive sequences are generated
by slippage of DNA polymerases during copying, and that the resultant looped out DNA is excised by the mismatch repair system. Thus,
enhanced slippage or deficits in mismatch repair could alter this
dynamic equilibrium and result in microsatellite instability. Enhanced
slippage by polymerases could be the result of DNA damage within
repetitive sequences. In vitro (42) and in Escherichia coli (43), damage to DNA containing repetitive sequences by oxygen-reactive species results in mutations within repeats. Sequence analysis of the
mutated DNA indicates that mutations are exclusively deletions and
additions within the repeats. Damage to the same plasmids containing
repetitive sequences by agents that do not generate oxygen reactive
species produces few, if any, mutations within the repeats (42). Other
less direct evidence suggests a role for oxygen-reactive species in the
generation of microsatellite instability. In yeast, mutations in the
mismatch repair genes, MSH2 or MSH6, can result in a 60,000-fold
increase in reversion rates of mutant genes, and this increase requires
growth in the presence of oxygen (44). Exposure of human lung cell
lines containing a plasmid with a (CA)n to oxygen free radical
generating systems resulted in a 27-fold increase in frameshift mutations, mostly located within the repetitive sequence (45). If DNA
damage within the repetitive sequence by reactive oxygen species
induces mutations, then a reduction of oxygen-reactive species in
precancer or cancer cells by oxygen radical scavengers might reduce
mutagenesis during tumor progression.
Silent and Multiple Mutations in Individual Genes
The presence of silent mutations and/or multiple mutations in the
same gene may allow one to assess the results of a mutator phenotype
in the absence of clonal selection. Silent mutations result from the
redundancy of the genetic code and are presumably nonselective
unless they alter splice sites or interact with specific tRNAs that are
rate limiting for protein synthesis. If tumors accumulate thousands of
mutations, then they should also accumulate silent or multiple mutations. Indeed, Strauss (46) made use of the large data set on p53
mutations in human tumors (47) and ascertained that additional silent
mutations were present in mutated p53 genes in human tumors at a
frequency that is at least 20 times greater than would be expected. In
addition, some tumors contain as many as four separate substitutions
within the p53 gene, perhaps the result of misincorporations by an
error-prone DNA polymerase expressed in cancer cells. However, as
pointed out by Strauss (46), there are other interesting interpretations
of this data. The p53 gene may be inherently hypermutable, either
because of the DNA sequence or its position in the chromosome.
However, if other genes are found to be equally mutable, the presence
of silent mutations in tumors provides evidence for the expression of
a mutator phenotype during tumor progression.
Timing of a Mutator Phenotype during Tumor Progression
Experimental evidence on the timing of a mutator phenotype in
cancers has been focused on HNPCC and colon cancers. Most, but not
all, studies in colon cancer suggest that mutations in mismatch repair
genes and the resulting microsatellite instability are early events in the
pathogenesis of these tumors. Huang et al. (48) reported that in
sporadic colon cancers, mutations in mismatch repair genes occur
prior to mutations in the APC gene, a marker for colon cancer. In
addition, microsatellite instability was found at high frequency in
adenomas that presumably predate adenocarcinomas. Shibata et al.
(49), who used laser microdissection to trace tumor evolution from
adenoma to adenocarcinoma, also observed microsatellite sequence
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length alterations in early adenomas, and additional microsatellite
alterations appeared as the tumors progressed to adenocarcinomas.
The differences in the spectrum of mutations in the APC gene in
tumors that exhibit microsatellite instability and in tumors that do not
argue strongly that mismatch repair alterations occur prior to APC
mutations (48). These combined studies support the logical inference
that a mutator phenotype is an early event in the generation of colon
tumors in HNPCC, at least with respect to mutations in mismatch
repair genes. However, Homfray et al. (50) reported no differences in
the frequency or types of mutations in the APC gene between sporadic
colon tumors and colon tumors that contained mutations in mismatch
repair genes (HNPCC). These authors concluded the opposite, that
APC mutations initiate carcinogenesis, and mutations in mismatch
repair genes occur at a later stage (51). Despite the later findings, the
weight of evidence would indicate that mutations in mismatch repair
genes occur early during the course of colon carcinogenesis in tumors
that exhibit microsatellite instability.
Is There a Sequential Order of Mutations That Produce
a Cancer?
Models for the progression of colon cancer have been diagrammed
as an ordered sequence of mutations in different oncogenes and tumor
suppressor genes, each associated with a defined step in tumorigenesis
(52). In other human cancers, data have been presented for an ordered
sequence of changes in regions that exhibit loss of heterozygosity.
Using a laser capture dissection of regions of breast carcinomas that
enriches for tumor cells, Shen et al. (53) presents evidence for an
order in regions of loss of heterozygosity that encompass genes
involved in double-stranded break repair, loss of p53 followed by loss
of BRCA1. The finding that the sequential transfection of four cancerassociated genes into human cells has been shown to result in tumors
has also been considered as an argument in support of an ordered
model for mutagenesis (54). Despite these studies, a sequential order
for mutation has not been established, even in colon cancers in which
delineated morphological and histological steps are clearly delineated.
Moreover, other studies indicate that every different breast cancer
possesses different deletions (23). If cancers are produced as a result
of accumulating large numbers of random mutations, then there are
likely to be many different mutations that can offer a selective
advantage during each step of tumorigenesis. Thus, a mutator phenotype is not in accord with a set order of mutation during tumor
evolution.
Eventual Selection against a Mutator Phenotype
It seems probable that the expression of a mutator phenotype
could be lost in the last stages of tumor progression. Theoretical
arguments indicate that the accumulation of large numbers of
mutants can exceed the error threshold for cell replication and
viability (55). Our recent studies on HIV replication have highlighted this; culturing HIV infected cells in the presence of a
mutagenic nucleoside analogue results in increasing viral mutagenesis and eventually in the ablation of viral infectivity (56). By
analogy, the accumulation of large numbers of mutations in tumor
cells might eventually dampen their replicative potential. As a
result, one might predict that tumor cells would lose their flexibility for adaptation to a changing environment. At this stage,
resistance to chemotherapeutic agents would be based on the large
numbers of mutations already present in the tumor cell population.
Origins of a Mutator Phenotype
Initial Events
The mutator phenotype hypothesis does not address the sources of
mutations that initiate carcinogenesis. Epidemiological studies have
identified mutagenic environmental agents that are causally associated
with specific human tumors. Except for tobacco, UV irradiation, and
aflatoxin B1, only a few environmental agents have been demonstrated to be causally associated with major human cancers. Most
known carcinogens damage DNA, and if this damage is not repaired,
it can cause mutations throughout the genome including genes that are
required to maintain genetic stability. I should stress that the cause of
nearly half of all human cancers has not been documented; these
cancers are not geographically clustered nor associated with exposure
to specific environmental agents. These cancers could arise from
exposure to multiple environmental agents, each causing a small
increment in mutations. Alternatively, these cancers could be “spontaneous” and emanate from DNA by endogenous reactants (57) including alkyl groups, activated lipids, metal cations, and oxygenreactive species (58). DNA damage by endogenous reactions is
similar to that caused by environmental chemicals and can also
produce random mutations throughout the genome. The extent of
DNA damage by normal reactive metabolites appears to be exceptionally high. It has been estimated that oxygen-reactive species
generate as many as 10,000 DNA damaging events/cell/day (59). A
similar number of lesions is generated by spontaneous depurination,
resulting in the loss of purine bases in DNA and miscoding by the
residual apurinic site (60). In total, it has been estimated that each cell
in our body generates and removes hundreds of thousands of altered
bases from DNA/cell/day (61). These background or “spontaneous”
DNA-damaging processes might not only generate the initial lesions
that confer a mutator phenotype but might also contribute to the
accumulation of mutations during tumor progression, specifically in
cells harboring mutations in DNA repair genes. A number of reports
suggest that tumor DNA contains altered bases in DNA and that these
alterations result from carcinogen exposure. The presence of chemical
alterations in DNA must reflect a steady-state equilibrium in which
the rate of generation is equal to the rate of repair. Thus, the presence
of DNA adducts in cells indicates the incompleteness of DNA repair,
and these residual lesions have the potential to cause somatic mutations.
Target Genes
The documentation that inherited mutations in specific genes are
associated with malignancy suggests that these same genes may be
mutated in somatic cells and cause malignancies. Furthermore, specific polymorphisms in these genes could be associated with increased
incidence of tumors. The fact that individuals with certain polymorphisms may exhibit increased incidence of certain cancers does not
argue against random mutations. The technology for detection of
mutations at specific sites in genes in tumors, and for the detection of
polymorphisms in human populations, is rapidly becoming available.
We should soon have information on the presence of mutations in
many genes in cancer cells, at least when they occur at a specific
location in the majority of cells within a tumor. However, methods for
DNA sequencing are still not yet sufficiently robust to detect random
mutations.
DNA Repair Genes. There are four major generic pathways for
the repair of DNA lesions in human cells: nucleotide excision repair,
base excision repair, mismatch repair, and the direct reversal of
lesions (62). Recent studies have increasingly stressed the overlap and
redundancy of these pathways (63); specific chemical alterations in
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DNA can be repaired by more than one mechanism. Historically, a
rare human inherited disease, xeroderma pigmentosa, provided strong
evidence linking human mutation in DNA repair genes with cancer
(7). Patients with xeroderma pigmentosa are exceptionally prone to
skin cancer after exposure to sunlight, and cultured cells from these
patients are defective in repair of UV-induced DNA damage. These
patients inherit mutations in one of the genes involved in nucleotide
excision repair. Mutations in a least one of four mismatch repair genes
(hMsh2, hMSH6, hMLH1, and hPMS2) are associated with increased
incidence of HNPCC and associated tumors. This association between
mutations in mismatch repair genes and malignancies has also been
demonstrated in mice (reviewed in Ref. 64). We lack evidence that
mutations in base excision repair genes are associated with any
inherited predisposition to cancer.
DNA Polymerases. Until recently, our repertoire of eukaryotic
DNA polymerases was limited to five well-characterized enzymes:
Pol-␣, Pol-␦, and Pol-⑀, each involved in DNA replication; Pol-␤,
associated with base excision repair synthesis; and Pol-␥, responsible
for mitochondrial DNA synthesis. Pol-␤ activity is elevated in many
cancers, and there are scattered reports on mutations in human colon
(65), prostate (66), and bladder (67) cancers. Pol-␦ is probably the
main replicative DNA polymerase in eukaryotic cells; it has an
integral 3⬘35⬘ exonuclease activity that corrects the misincorporation
of noncomplementary nucleotides during DNA synthesis. Goldsby
and Preston4 created mice harboring a point mutation in the exonucleolytic domain of Pol-␦ that in yeast results in a mutator phenotype.
Twenty-three of 48 homozygous mutant mice developed tumors (14
lymphomas, 6 squamous cell carcinomas, and 3 other tumors); no
tumors were detected in control or heterozygous animals. These
results demonstrate that the production of a mutator phenotype by
mutations in DNA polymerases increases the incidence of a variety of
malignancies in mice. They provide strong evidence that a mutator
phenotype can result in cancer but do not necessarily indicate that
most cancers result from a mutator phenotype.
In bacteria, 90% of mutations are dependent on the SOS response
that mediates the induction of multiple genes and is associated with
mutagenic bypass of DNA lesions. Recently, one of these genes,
UmuD⬘2 C (E. coli Pol V), has been shown to encode an error-prone
DNA polymerase (68). Simultaneously, a variant of xeroderma pigmentosum (XP-V), which exhibits an elevated mutation rate in cell
culture, was demonstrated to harbor a mutation in related DNA
polymerases, Pol-␩ 10, allowing yet another related polymerase,
probably Pol-␰ 10, to bypass UV dimers by incorporating incorrect
nucleotides (69, 70). Thus far, some six to eight newly discovered
DNA polymerases have been established that could be involved in
error-prone bypass of lesions in DNA (71). Studies are under way to
determine the expression of these potentially mutagenic enzymes in
human tumors.
DNA Helicases. Eukaryotic cells contain a surprisingly large number of enzymes that unwind double-stranded DNA prior to being
copied by DNA polymerases. These helicases are potential targets for
a mutator phenotype. Inherited mutations in several of these DNA
helicases are associated with diseases that exhibit a high incidence of
cancer. Recently, the genes mutated in Bloom’s syndrome (72) and
Werner’s syndrome (73) have been established as encoding DNA
helicases belonging to the E. coli RecQ family. Cells with mutations
in these genes exhibit characteristic genetic instabilities (74), sister
chromosome exchanges in Bloom’s syndrome (75), and large deletions in Werner’s syndrome (76) and thus provide further evidence for
the association of genetic instability with cancer. These are uncom4
Personal communication.
mon autosomal recessive disorders, and it is important to determine
whether the more frequent heterozygotes in the population exhibit an
increased incidence of the tumors and if sporadic tumors contain
somatic mutations in these helicases.
Other Target Genes. There are many genes involved in insuring
the stability of the genome. Considering the multiple steps involved in
DNA replication, deoxynucleotide metabolism, checkpoints in the
mitotic cycle, chromosomal segregation, and mitosis, it is apparent
that there are many target genes that, if mutated, could induce a
mutator phenotype. DNA synthesizing complexes have been isolated
from human breast cancer cells that misincorporate nucleotides more
frequently than similar complexes isolated in parallel from normal
cells (77). The error-prone proteins in these complexes need to be
established. Mutations in a number of genes are strongly associated
with human cancers, and many of these genes are involved directly or
indirectly in DNA transactions. For example, half of all cancers
contain mutations in p53 (78). Although there is no strong evidence
that mutations in p53 increase point mutations throughout the genome,
there is considerable evidence that p53 is involved in DNA repair,
apoptosis, and recombination (79, 80), and thus p53 could be a target
gene for a mutator phenotype.
Coupling of Mutation Induction and Clonal Selection
Although it was generally recognized that both enhanced mutagenesis and clonal selection contribute to a mutator phenotype, it is
generally not appreciated that these mechanisms act in synergy. Mao
et al. (81) demonstrated that sequential rounds of selection for the
growth of E. coli in different restricted conditions resulted in a
5000-fold increase in the rate of spontaneous mutagenesis. If the
bacteria were first exposed to a mutagen, the same protocol yielded a
population that was 100% mutators. These findings may be particularly relevant to tumor progression (82). As cancers expand, there is
a series of host-mediated restrictions that must be overcome; these
include reduced oxygen, the requirement for the production of growth
and angiogenic factors, and confinement by adjacent tissues (24, 83).
Each of these restrictions can be overcome by the selection of mutations in specific genes. With each round of selection there is a
“piggy-backing” of mutations in genetic stability genes, and as a result
there is an increase in mutation frequencies throughout the genome.
Thus, as one selects for mutants, one simultaneously selects for
mutators that cause these mutants. With multiple rounds of selection,
there is a progressive enrichment of mutations in genetic stability
genes.
Consequences of a Mutator Phenotype
The hypothesis that cancers express a mutator phenotype and that
this phenotype drives the proliferation of cancers and generates variants for clonal selection (Fig. 1) have both theoretical and practical
consequences. Among the theoretical implications are:
Tumor Progression Is Genetically Irreversible
The concept of a mutator phenotype implies that a large number of
mutations are produced randomly throughout the genome of cancer
cells; only a few of which result in clonal proliferation. This heterogeneity of mutated genes militates against any possibility of reverting
cancer cells to wild-type normal cells. Suppression of these mutations
can occur by other mutations or epigenetic silencing and therefore
reduce the expression of the cancer phenotype, but this would not
re-establish the normal genome. Thus, the classical experiments in
3234
A MUTATOR PHENOTYPE IN CANCER
which tumor cells reverted to normal cells (84) may be at the level of
their phenotype but not their genotype.
One cannot eliminate the possibility that epigenetic mechanisms
drive tumor progression. In particular, the silencing of DNA repair
may facilitate the production of the multiple mutations found in
tumors. Silencing of gene expression may be a requirement for cancer
cells to tolerate the introduction of aneuploidy. Although strong
arguments can be invoked for the role of epigenetic mechanisms in
carcinogenesis, we still lack sufficient knowledge of these mechanisms to design critical experiments to validate these concepts.
The Types of DNA Damage and Mutations Found in a
Tumor Are Unlikely to Yield Clues about Initiating Events
in Carcinogenesis
For solid tumors, it takes 20 years or more from the time of
exposure to a chemical carcinogen until the clinical detection of a
tumor. Thus, it seems improbable that DNA lesions initiating the
carcinogenic process would still be present in tumor cells many
generations later, unless ongoing DNA damage by the same agent is
required for tumor formation. Moreover, the mutations found in a
tumor may not result from the most frequent chemical lesions in
DNA. Most DNA-damaging agents cause a variety of chemical alterations in DNA; the most frequent lesions may not be the most
mutagenic.
Despite the above argument, there are reports that exposure to a
specific agent produces a specific mutation. This could indicate that
repetitive exposure to a specific agent could be the driving force in
clonal selection as might occur in UV-induced skin cancers (85),
aflatoxin-induced liver cancers (86), and perhaps smoking-associated
lung cancers (87). Exposure to UV induces skin cancers, and the
mutations detected in the p53 gene are found opposite pyrimidine
dimers and involved T3 C or CC3 TT tandem substitutions that are
characteristic of UV damage (88). Areas with aflatoxin contamination
have increased incidence of primary hepatomas, and p53 mutations
occur predominately at codon 249 and involve G3 T substitutions. A
less compelling case is provided by the G3 A transversions found in
the p53 gene in lung cancers (87). Although a similar substitution is
found upon exposure of human epithelial cells to benzo[a]pyrene in
tissue culture, G3 A transitions are also the result of errors by DNA
polymerases. The major question is whether these mutations are
diagnostic of specific adducts produced by the carcinogens, or do they
reflect selection of mutations in a random population that promote
clonal proliferation?
Benign Cancers Contain Multiple Mutations
The arguments suggesting that the acquisition of a mutator phenotype is an early event in carcinogenesis imply that early tumors such
as colon adenomas and uterine hyperplasia and leiomyomas should
contain microsatellite and chromosomal aberrations. Evidence supports this assertion (20, 89). With new techniques it should soon be
possible to recreate the archeology of mutated genes that promote
clonal expansions as tumors progress.
Late in Tumorigenesis, Selection May Be against a
Mutator Phenotype
A mutator phenotype may be an early event that drives tumor
progression, yet it may no longer be present by the time a tumor is
clinically apparent. Although many mutations in cancer cells are
neutral or advantageous, some will be deleterious. Thus, there is likely
to be a critical threshold, after which selection will be against a
mutator phenotype. As a result, tumors may no longer exhibit a
mutator phenotype but will never the less reveal its history, random
mutations throughout their genomes.
Tumors Contain Cells with Preexisting Drug-resistant Mutations
If one considers the likelihood that a mutator phenotype is an early
event in carcinogenesis and the 20 years it takes for a cancer to
become clinically apparent, it seem probable that each tumor, consisting of millions of cells, contains one or more cells that have
already accumulated mutations rendering them drug or antibody resistant (Fig. 1). The preexistence of these mutations was first demonstrated by Tlsty et al. (90); tumor cell lines, but not normal cell lines,
rapidly amplified genes, rendering them resistant to chemotherapeutic
agents. Similarly, it is unlikely that there will be antigens that occur in
all tumors of a given type and render these tumors susceptible to
immunotherapy. The current success of immunotherapy is primarily
based on overexpression of normal antigens by specific tumors. Instead, the concept of a mutator phenotype suggests that immunity may
have to be tailor made to individual tumors.
Practical Implications of a Mutator Phenotype in Cancer
Among the practical implications of a mutator phenotype in cancer
are:
Mutations Are a Marker for Cancer
Although no specific mutation is absolutely diagnostic of a specific
malignancy, the presence of an increased number of mutations may
indicate that cells are already on the path to produce a cancer. Both
microsatellite instability and chromosomal alterations are being investigated as tumor markers in cells in blood, gastric, and bronchial
lavages and pancreatic brushings. In addition, naked DNA is found in
serum, and mutations in this DNA can serve as a marker for tumors
at distant sites (91, 92). With the development of more sensitive and
quantitative techniques, the detection of genetic alterations in cells or
DNA, it may be feasible to identify individuals likely to develop
specific cancers.
Emerging Technologies Should Allow One to Quantitate
Random Mutations
Considering that even if the mutation rate of cancer cells are
elevated 100-fold early in carcinogenesis, one would still only anticipate one nucleotide alteration per million nucleotides sequenced. This
stretches the current level of technology; at best, one may be able to
establish nucleotide sequence changes in a very limited number of
tumors. Improvements in methodologies by 10 –100-fold, coupled
with cloning single copies of a gene, should make it possible to
quantitate random mutations in multiple tumors and to assess whether
mutation accumulation is prognostic for susceptibility to cancer.
Cancers Arise in a Field of Normal Cells Harboring
Multiple Mutations
Recent studies suggest that colon cancers arises amid a field of
premalignant cells that express a mutator phenotype. The studies of
Rabinovitch et al. (34) on chromosomal changes in chronic ulcerative
colitis provide a persuasive demonstration of this concept. They
detected an increased loss of chromosomal arms in cells in rectal
biopsies from patients with ulcerative colitis and with associated
cancers elsewhere in the colon but not in cells from patients with
3235
A MUTATOR PHENOTYPE IN CANCER
Fig. 1. Mutation accumulation during tumor progression. 1, random mutations result when DNA damage exceeds the cell’s capacity for error-free DNA repair. 2, these random
mutations can result in clonal expansion and mutations in mutator genes (M). 3, repetitive rounds of selection for mutants yield additional coselection mutants in mutator genes. 4, from
this population of mutant cancer cells, there is selection for cells that escape the host’s regulatory mechanisms for the control of cell replication, invasion, and metastasis.
ulcerative colitis lacking dysplasia or cancer (34).5 The mutated
mucosal cells are at sites distant from the cancer. The implication is
that the cancers arose within an extensive field of cells expressing a
mutator phenotype. This study is of practical importance because it
may provide a simple procedure for the screening of patients with
ulcerative colitis who are likely to develop colon cancer.
The presence of random point mutations in precancerous tissue has
been reported in both hemochromatosis and Wilson’s disease (93).
Both diseases are characterized by an increased incidence of primary
hepatoma. Random (nonclonal) mutations in p53 similar to those
produced by oxygen-reactive species were detected in nontumorous
liver in patients with Wilson’s disease. In the latter study, the assay
used provided a uniquely high sensitivity in detecting one mutation in
107 nucleotides and thus may offer an advantage in examining other
genes for random events. Using the same approach, Hussain et al. (94)
detected p53 mutated alleles in nontumorous colon tissue from patients with ulcerative colitis, further supporting that cancers may arise
in a field of mutated cells. Although these studies are limited to
precancerous diseases associated with inflammation, they provide
additional evidence that mutations in normal tissues precede the onset
of detectable malignancies.
Other evidence supports the concept that regional clonal expansion
of phenotypically normal but genetically altered cells may precede the
development of cancers. The inactivation of an insulin-like growth
factor by deletion of a polydeoxyguanosine tract occurs in liver tissue
5
R. E. Golds and B. Preston, personal communication.
adjacent to hepatocellular carcinomas (95). Regions of loss of heterozygosity have been reported in morphological normal lobes adjacent to breast carcinomas, and clonal areas harboring p53 mutations
are found in normal skin. It may be possible that tumors arise in fields
of cells that exhibit a higher mutation rate attributable to epigenetic
modification. Silber et al. (96) reported that gliomas are surrounded
by normal cells that are defective in the repair of DNA damage by
alkylating agents (mer⫺), implying lack of O6-alkylguanine-DNA
transferase production is a predisposing factor for the development of
brain tumors.
Chemotherapies Directed against Specific Targets or Oncogenes
Are Unlikely to Kill All Cancer Cells
The fact that no single oncogene or suppressor gene is universally
mutated or down-regulated in all cases of solid human cancer reinforces the concept that tumors are heterogeneous. The marked heterogeneity of cell populations within a tumor presents a plethora of
randomly mutated cells, some of which are likely to contain resistant
mutations to chemotherapeutic agents. In the presence of chemotherapy, the mutant tumor cells will have a selective growth advantage
and repopulate the tumor. It seems likely that tumors would also
contain cells that are resistant to antibodies directed against specific
oncogenes. Thus, specific chemotherapies are unlikely to eradicate
100% of cancer cells within a tumor, and one has to rely on host
immunological mechanisms, which in many cases are diminished by
cancer chemotherapy.
3236
A MUTATOR PHENOTYPE IN CANCER
Effects of Increased Mutagenesis
Acknowledgments
The large number of mutations in a tumor suggests the possibility
that the fidelity of DNA replication operates near the error threshold
for cell viability. Further errors in DNA replication may be lethal. In
support of this concept is the high level of apoptosis in tumors.
Furthermore, many drugs used in the treatment of cancer are powerful
mutagens, and enhanced mutagenesis may be a significant factor in
their therapeutic efficacy. Thus, one should consider the testing of
mutagenic nucleoside analogues in the treatment of human malignancies, weighing heavily the problem of inducing second tumors. Of
particular utility would be analogues that are not subject to excision
by human repair enzymes.
I thank Richmond Prehn, Ray Monnat, Ann Blank, Aimee Jackson, and
Peter Rabinovitch for free discussion and critical comments.
References
Prevention of Cancer by Delay
If a mutator phenotype drives the carcinogenic process, it might be
possible to prevent cancer by delay. A 2-fold reduction in mutation
rates could prolong the time between initiation and clinical manifestations of cancer from 20 or more years to 40 or more years. Prevention by delay may be particularly applicable to cancers associated with
prolonged chronic inflammation by either bacteria (gastric cancer;
Ref. 97), viral (primary hepatitis; Ref. 98, 99), immunological insufficiencies (ulcerative colitis; Ref. 100), or unknown etiology (chronic
pancreatitis; Ref. 101). In each disease, the chronic inflammatory
response is associated with increased generation of oxygen-reactive
species by inflammatory cells. These reactive oxidants are generated
by phagocytes to kill bacteria but may also damage host cell DNA.
The elimination of the causative agent or the reduction of the inflammatory response would be predicted to delay the clinical appearance
of the associated tumors.
Summary
This prospectus on a mutator phenotype suggests that cancer in the
adult results from the accumulation of large numbers of somatic
mutations. Mutations occur randomly throughout the genome; among
these are mutations in genes that function in normal cells to maintain
the stability of the genome and to guarantee that it is faithfully copied
and transmitted to progeny during each cell division. Mutations in
genetic stability genes lead to further mutation throughout the genome, including other genes involved in maintaining genetic stability.
Our emphasis has been on DNA polymerases and enzymes involved
in DNA repair, but there are many other potential targets for the
generation of a mutator phenotype. Processes that lead to the further
accumulation of mutations include selection for cells harboring mutator mutants and the constant bombardment of DNA by reactive
chemicals produced by normal metabolic processes.
Current advances in DNA sequencing and related technologies
should now make it feasible to measure the accumulation of random
mutations in human cancer and thus to substantiate the hypothesis of
a mutator phenotype in human cancer. It will now be important to
determine the distribution of mutant genes in individual cells within a
tumor both to trace the evolution of clonal lineages and to identify
genes that are mutated in most cells. Among these deep mutations
should be those that confer a mutator phenotype. The ensemble of
mutated genes within a tumor may define the probability of developing drug resistance or guide drug therapy. Sensitive methods for the
detection of random mutations should allow the monitoring of blood
and human materials for precancerous cells, which could enable early
therapeutic intervention. The most important verification of the mutator phenotype hypothesis may be the demonstration that a reduction
in the multiplicity of random mutations in precancerous cells prevents
the development of malignancies.
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