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[Cell Cycle 2:3, 202-210, May/June 2003]; © 2003 Landes Bioscience
Review
Multistep Carcinogenesis
A Chain Reaction of Aneuploidizations
Peter Duesberg*
Ruhong Li
Department of Molecular and Cell Biology; Donner Laboratory; UC Berkeley;
Berkeley, California USA
*Correspondence to: Peter Duesberg; Department of Molecular and Cell Biology;
Donner Laboratory; UC Berkeley; Berkeley, California 94720 USA; Tel.:
510.642.6549; Fax: 510.643.6455; Email: [email protected]
Received 04/05/03; Accepted 04/10/03
Previously published online as a Cell Cycle Paper in Press at:
http://www.landesbioscience.com/journals/cc/toc.php?volume=2&issue=3
KEY WORDS
Aneuploidy-dependent malignancy, Cancerspecific aneusomies, Genetic instability, Aneuploidydependent karyotype instability, Aneuploidydependent gene mutation, Mutator genes, Nonmutagenic carcinogens, Drug-resistance, Reversible
phenotypes, Immortality
We are indebted to the Abraham J. and Phyllis Katz Foundation (New York), Robert
Leppo (philanthropist, San Francisco), an American foundation that prefers to be
anonymous, other private sources, the Deutsche Krebshilfe and the Forschungsfonds
der Fakultaet for Klinische Medizin Mannheim (Germany) for support.
202
ABSTRACT
Carcinogenesis is a multistep process in which new, parasitic and polymorphic cancer
cells evolve from a single, normal diploid cell. This normal cell is converted to a prospective
cancer cell, alias "initiated", either by a carcinogen or spontaneously. The initiated cell
typically does not have a new distinctive phenotype yet, but evolves spontaneously—over
months to decades—to a clinical cancer. The cells of a primary cancer also evolve spontaneously towards more and more malignant phenotypes. The outstanding genotype of
initiated and cancer cells is aneuploidy, an abnormal balance of chromosomes, which
increases and varies in proportion with malignancy. The driving force of the spontaneous
evolution of initiated and cancerous cells to ever more abnormal phenotypes is said to be
their "genetic instability". However, since neither the instability of cancer phenotypes nor
the characteristically slow kinetics of carcinogenesis are compatible with gene mutation,
we propose here that the driving force of carcinogenesis is the inherent instability of
aneuploid karyotypes. Aneuploidy renders chromosome structure and segregation errorprone, because it unbalances mitosis proteins and the many teams of enzymes that
synthesize and maintain chromosomes. Thus, carcinogenesis is initiated by a random
aneuploidy, which is induced either by a carcinogen or spontaneously. The resulting
karyotype instability sets off a chain reaction of aneuploidizations, which generate
ever more abnormal and eventually cancer-specific combinations and rearrangements
of chromosomes. According to this hypothesis the many abnormal phenotypes of cancer
are generated by abnormal dosages of thousands of aneuploid, but un-mutated genes.
Carcinogenesis is a very rare process in which a normal cell is converted to a cancer cell via
multiple “steps” or “stages” of cellular evolution, which correspond to various pre-neoplastic
and neoplastic phenotypes and genotypes.1-4 The clinical and biological phenotypes of
the multiple steps of carcinogenesis include hyperplasia, dysplasia, abnormal morphology,
anaplasia or dedifferentiation, drug- and multi-drug resistance, immortality, altered histocompatibility including even transplantability to some heterologous species and susceptibility
to some heterologous viruses, abnormal metabolism, autonomous growth, invasiveness,
and metastasis.1,5-12
Based on various genetic and cytogenetic markers, including structurally altered or
marker chromosomes, most cancers are clonal, i.e., derived from a single cell.3,13-15
However, a minority is polyclonal.1,16,17 Despite their clonal origin, “the structure and
behavior of tumors are determined by numerous, abnormal characters that, within wide
limits, are independently variable, capable of highly varied combinations and assortments
and liable to independent progression.”18,19 As a result of this “genetic instability”3 hardly
any two cells from a given cancer are ever the same.1,20-26,62,137 Indeed, the specific
karyotypes and phenotypes of individual cancer cells can vary at rates of up to a few
percent per cell generation and are thus many orders of magnitude less stable than would
be expected from conventional gene mutations.11,12,24,27-29 But the mechanism of this
genetic instability of cancer cells is still a matter of debate.30-33
In further contrast to gene mutation, the kinetics of carcinogenesis are exceedingly
slow. After a sufficient, alias “initiating,” dose of carcinogen it takes a cell a minimum of
months and many cell generations to develop clinically detectable cancerous phenotypes
in experimental rodents.2,14,34,192 Likewise, experimental transformation of normal,
diploid rodent cells to tumorigenic counterparts in vitro takes a minimum of months.23,35
Even the highest survivable doses of carcinogens, supplemented by treatments with tumor
promoters, can only accelerate experimental carcinogenesis a few-fold.2,34,36,37,177,192
Carcinogenesis in humans takes even decades from an occupational or accidental
exposure to a dose of carcinogen that is sufficient to eventually cause cancer.1,5,14,38-40
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MULTISTEP CARCINOGENESIS
Transformation of diploid human cells with carcinogens in vitro
takes about half a year, and is much less efficient than transformation
of rodent cells.41,42 Spontaneous carcinogenesis in humans from
unknown initiation any time after birth to clinical phenotypes is estimated to take over 50 years, because the risk of spontaneous cancer
rises exponentially, over 1000-fold, from negligible levels under the
age of 50 to over 400 cases per million people per year at the age
of 80.14,43-45 By contrast, classical gene mutation generates a new and
final phenotype within one cell generation, as for example in bacteria.
During the long lag between initiation and clinical phenotypes,
the prospective cancer cell “progresses” first through various stages of
“characters that have no discernible effect at all on clinical behaviour”,
such as increased susceptibility to transformation by additional
carcinogens, hyperplasia, altered cell morphology and aneuploidy.1
Aneuploidy is defined as an abnormal balance of either intact
chromosomes or segments of chromosomes or both.6,46,84 But once
the first clinical phenotypes appear, e.g., dysplasia and carcinoma in
situ, progression to more malignant phenotypes, such as metastasis,
abnormal morphologies, abnormal metabolism and drug-resistance,
typically occurs at much higher rates.1,13,47 Since the multiplicity of
the malignant phenotypes is not fixed, there is no absolute definition of
a cancer cell.1
Aneuploidy stands out as the most consistent marker of malignancy. A simple aneuploidy is the earliest and most distinctive
pre-neoplastic genotype.3,6,17,26,48-61 By contrast, a complex aneuploidy is the most typical neoplastic genotype.53,57-59,62-64 The more
aneuploid the cell the higher its malignancy.3,13,47,53,62,65-75
According to Foulds, “In general the abnormalities of the karyotypes
were of greater complexity in malignant than in non-malignant
lesions…”.1 Thus, the degree of malignancy is directly proportional
to the degree of aneuploidy.
The instability of the karyotype of cancer cells is also proportional
to the degree of aneuploidy: It increases from undetectable rates of
gains, losses and rearrangements of chromosomes per mitosis in
diploid cells to a few percent in highly aneuploid cells.28,29,50 Even
the low degrees of aneuploidy of pre-neoplastic cells coincide with
karyotype-instability.48,53,60
Thus carcinogenesis is fundamentally different from mutation: It
is very slow, timed from an initiating and sufficient dose of carcinogen.
It is associated with aneuploidy from its initiation. After initiation, it
generates abnormal phenotypes spontaneously, i.e. without further
carcinogens, including eventually cancer-specific phenotypes. These
phenotypes are unstable and thus typically progress from bad to
worse,1 or shift from drug-sensitive to drug-resistant, or from one
morphological grade or metabolic state to another—unlike conventional gene mutations.2,4,11,12
MECHANISMS OF CARCINOGENESIS
Despite over a century of cancer research, the mechanism of
carcinogenesis is still a matter of debate, primarily between two
schools of thought.33,76-80 One school holds that cancer-specific
phenotypes are the result of multiple, specific gene mutations, which
either generate dominant oncogenes, or inactivate recessive tumor
suppressor genes.2,77,81-86 The competing school holds that cancerspecific phenotypes are the results of the thousands of normal genes
whose dosage is altered by reassortments of the corresponding chromosomes or aneuploidy.6,35,76
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ARE THE MULTIPLE STEPS OF CARCINOGENESIS MULTIPLE
MUTATIONS?
Since the risk of mutations increases linearly with age, but the
risk of cancer increases exponentially, over 1000-fold, the proponents
of the mutation hypothesis postulate that between 4 and 7 mutations
are necessary for carcinogenesis.14,40,43-45 Only complete sets of
these genes are thought to cause cancer.14,45,83 However, the problem
of where the huge number of random mutations would come from
that is necessary to mutate 4–7 specific genes has not been solved.
Based on the normal, spontaneous gene mutation rate of about
10-6 per mitosis,87 only 1 in 1024–1042 human cells would ever
become cancer cells.6,31,88,89 Since humans consist of 1014 cells,14
only 1 in 1010–1028 humans would ever get cancer. This number
would be even lower, if the mutation rates of the recessive cancer
genes, as for example the hypothetical tumor suppressor genes, are
squared. In other words cancer would hardly exist.
To solve this problem, the proponents of the mutation hypothesis
have postulated that prior to mutation of prospective cancer genes,
another class of cellular genes must be mutated to mutator genes,
which in turn would mutate prospective cancer genes to real cancer
genes.3,32,88,90,91 The presence of such mutator genes, early in
carcinogenesis, could then also explain the spontaneous, or carcinogenindependent, progression of initiated cells to cancer cells. However,
in the words of Breslow and Goldsby, “a mutator gene really begs the
question … one is left with the problem of accounting for such a
high frequency of mutator genes.”92
But, the origin of multiple specific mutations in the same cell is
not the only problem of the mutation hypothesis. According to Cairns’
Cancer, Science and Society, “One of the problems is that mutations
lead to loss of function rather than creation of new function.”14
Creation of cancer-specific phenotypes is particularly hard to reconcile
with mutation because of their multiplicity and extraordinary
complexity. Even ardent proponents of the mutation hypothesis
acknowledge this problem: “our understanding of the biology of these
tumors will become a phenomenology of unlimited complexity.”93
Thus, how could a prospective cancer cell ever survive the “loss
of function” that is associated with the high mutation rates, which
are necessary to generate the 4–7 specific mutations and possibly
further “unlimited complexity” of cancer cells? Since the probability
that any one of the 35,000 human genes191 is mutated is 1:35,000,
but the probability that 4–7 genes are mutated is only 1:(35,000)4-7,
virtually all genes of a prospective cancer cell would have to be
mutated before it could turn into a cancer cell. Suppose then a cell
with a mutator gene has suffered 1000 random mutations, its chance
to become transformed into a cancer cell by 4 specific mutations is
only 1 in 1.5 million, and after it has suffered 5000 random mutations, this chance is still only 1 in 2400. But how could any cell carry
out the many cell cycles, that set apart initiation from completion of carcinogenesis, in the face of so many mutations?
In view of these questions, we have reexamined the merits of the
mutation hypothesis by testing whether it could predict a list of 14
basic facts of carcinogenesis, that are summarized in Table 1 (next
page). Since the hypothesis fails to predict any of these facts, we
conclude that it is not a satisfactory explanation of carcinogenesis.
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MULTISTEP CARCINOGENESIS
Table 1
DISCREPANCIES BETWEEN
THE
PREDICTIONS
OF THE
Predictions of the
mutation hypothesis
GENE MUTATION-CANCER HYPOTHESIS
AND THE
FACTS
Facts
1
Carcinogens are mutagens.
But, half of all known carcinogens, such as polycyclic aromatic hydrocarbons, asbestos,
hormones, mineral oil, hydrazine, solid bodies, nickel and cytoplasmic radiations are not
mutagenic.6,94-96,193
2
Cancer-phenotypes are determined by cancerspecific mutations.
But, about half of all solid cancers of any kind lack hypothetical cancer genes or oncogenes.31,89,97-100,193 In other words oncogenes do “not impart distinct clinical properties
on most tumors.”99 Yet, “phenocopies” of known oncogenes are said to cause those
cancers in which known oncogenes can not be found.97,101
3
Cancer-specific mutations confer selective
advantages for carcinogenesis.
But, cancers contain up to 100 times more selectively neutral or silent mutations than normal
cells, even in tumorsuppressor genes.32
4
Cancer-causing mutations are present in all
transformed cells of a clonal cancer.
But, the mutant oncogene K-ras is non-clonal in human colon cancer,102,103 mutant Kras, H-ras and myc are non-clonal in prostate cancers,104,105 mutant H- and N-ras are
non-clonal in melanoma,106,107 mutant her/EGFR/neu/erbB-2 is non-clonal in esophageal
Barrett's cancers,108 in bladder cancers,109 in malignant gliomas,110,111 and in breast
cancers112 together with other non-clonal, hypothetical oncogenes.113
5
Cancers are maintained by the expression of
cancer genes
But, hypothetical cancer genes are un-expressed in many cancer cells.78,114-116
6
Normal cells are transformed
into cancer cells by oncogenes.
But, transgenic mice, carrying hypothetical oncogenes in every cell of their body, are
viable and fertile and have either a normal or just a moderately higher than normal risk
of developing a clonal (not polyclonal!) cancer.6,117-122
7
Since only 4–7 gene mutations are thought to be
necessary for carcinogenesis,14,83,84,123 only
small numbers of genes should be abnormally
expressed in cancer cells.
But, thousands of genes are abnormally expressed in cancer cells,114-116 “the sine qua
non of malignant transformation”.4
8
Inheritance of 3–6, of the 4–7 mutations thought to
to be necessary for carcinogenesis, predicts solid
cancer at very young age from a single, somatic
mutation in any of billions of susceptible cells.
But, the incidence of solid cancer at young age is negligible,14,43-45; For key, see Table
2(#6)
9
In view of the low rates of spontaneous gene
mutation, 10-6 per mitosis (see text), mutator genes are
necessary for carcinogenesis—even for the
spontaneous “progression” of “initiated”
cells to cancer cells.
But, “mutator genes” are only detectable in a small minority of cancers.24,29,31,32,124-126
And the gene mutation rates of many cancers are normal.127,128 Moreover, mutator
genes are typically only detectable at late stages of carcinogenesis, suggesting that they
are consequences of neoplastic development.128,129
10
The majority of cancers, ie. those without mutator
genes, are genetically stable, like conventional
mutations.
But, all cancer cells are, more or less, genetically unstable.1,3,11,12,48,60
11
Genetic instability of cancer cells is proportional to
the presence and nature of mutator genes.
Instead, genetic instability of cancers is proportional to the degree of aneuploidy (see
text).28,50,130
12
Congenital mutator genes increase the risk of all cancers.
But, the mutator gene of Xeroderma Pigmentosum, which is postulated to cause skin
cancer, does not increase the risk of non-skin cancers.131; For key, see Table 2(#1)
13
Certain mutant phenotypes of cancer cells, such as
Yet, despite 3 billion years of mutation, none of these desirable phenotypes are ever
immortality, resistance to singular or even multiple
found in normal organisms! For key, see Table 2(#6)
cytotoxic drugs, and resistance to cytotoxic viruses,
should be highly attractive for normal animals and humans.
14
Cancer cells are diploid, differing from normal cells
only in 4–7 specific mutations.
But, unlike all conventional gene mutations, cancers are aneuploid (see below).35,76,77
ARE THE MULTIPLE STEPS OF CARCINOGENESIS MULTIPLE
ANEUPLOIDIZATIONS?
In an effort to find an alternative cancer hypothesis, we decided
to search the literature for facts that are neglected by the mutation
hypothesis. Surprisingly, we did not have to look very long to find
aneuploidy.
But despite a preponderance of evidence in support of the
hypothesis that aneuploidy may cause cancer; there is currently a
strong bias against this hypothesis.132 In the following we describe:
1. the evidence for aneuploidy;
2. the current bias against the aneuploidy hypothesis; and
3. a unifying hypothesis that answers the scientific objections that
have been made against aneuploidy, and that explains all facts of
carcinogenesis.
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1. The Evidence for Aneuploidy as the Cause of Cancer. The
following collection of facts lends correlative, functional and conceptual support to the aneuploidy-cancer hypothesis:
Aneuploidy is ubiquitous in solid cancer.25,62,67,133 This is correlative proof for causation. Aneuploidy is also found in about half of all
leukemias,134,135 the diploid half being hyperplasias.6 In contrast to
the diploid leukemias, the cells of the aneuploid leukemias are
dedifferentiated, immortal, and able to mutate to drug-resistance at
high rates, just like solid cancers.12,136
The degree of malignancy is proportional to the degree of aneuploidy of a cancer: The more aneuploid the karyotype the more
malignant the cancer (see above).3,13,47,59,60,65-75,137
Aneuploidy inevitably causes highly abnormal and complex
phenotypes in lower and higher eukaryotes by altering the dosages
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MULTISTEP CARCINOGENESIS
Figure 1. Multistep carcinogenesis via aneuploidizations. The vertical banded bars are chromosomes, the squares are normal diploid, and the circles are
abnormal, aneuploid cells. Our model proposes that various specific chromosome assortments or aneusomies cause cancer. According to this model, carcinogenesis is initiated by a random aneuploidy, which is induced either by a carcinogen or spontaneously. Since aneuploidy unbalances mitosis proteins
and the many enzymes that synthesize and maintain chromosomes, it renders chromosome segregation error-prone. The resulting karyotype instability sets
off a chain reaction of aneuploidizations that generate ever more abnormal chromosome assortments, including non-viable and eventually cancer-specific
chromosome assortments. The cancer-specific phenotypes—abnormal metabolism and morphology, invasiveness, drug-resistance, immortality and metastasis—are generated by the abnormal dosages of thousands of normal genes. Since aneuploid chromosome assortments are reversible, cancer-specific phenotypes such as drug-resistance can get lost at the same rates at which they are generated; likewise malignancy of non-tumorigenic cancer-normal-cell
hybrids is recovered by loss of chromosomes. The model predicts the heterogeneous phenotypes and karyotypes of cancer cells as mixtures of selectively
advantageous, cancer-specific and selectively neutral, random chromosome assortments. The model further predicts that both malignancy and karyotype
instability are proportional to the degrees of aneuploidy, which is consistent with the literature (see text).
of thousands of normal genes. This was first demonstrated in doublyfertilized sea urchins138 and has since been confirmed in aneuploid
drosophila,139 plants,140 yeast,141,142 mice,143 and humans with
Down’s syndrome and other congenital aneusomies.144-146 The probable basis of the abnormal phenotypes of aneuploidy is the abnormal
dosage and expression of thousands of normal genes.146-148,152
The power of aneuploidy to generate abnormal phenotypes
becomes immediately obvious if one compares the cell to a car factory
and chromosomes to assembly lines. Unbalance these assembly lines
and you get highly abnormal cars from entirely normal parts, e.g.,
cars with five wheels, no brakes, red headlights, etc. Most of these
aneuploid cells would be non-functional, but a few would be able to
out-compete normal cells in the permissive habitat of the body.
The analogy also explains why a gene mutation, an altered assembly
line worker in our analogy, is unlikely to generate the new properties
of cancer cells: Any “activating” mutation82 would be buffered by
the un-mutated genes of the assembly line working at normal
rates.149 Likewise, most inactivating mutations would be buffered,
since nearly all enzymes work in the cell at only a very small fraction
of their capacity.150,151 Only very rare null mutations would bring
the assembly line to a halt, in which case the second allele is usually
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sufficient to maintain normal cell function.152 However, if both
assembly lines cease to work by mutations, the resulting cell is more
likely to die than to develop the highly complex, new phenotypes of
cancer cells (see above). Thus, aneuploidy is the simplest explanation
for the many dominant and abnormal phenotypes of cancer cells.152
Aneuploidy has even been proposed as a cause of cancer over 100
years ago, first by von Hansemann because of suggestive evidence for
its presence in epithelial cancer153 (the exact chromosome number
of humans was only determined in 1956), and then by Boveri based
on the discovery that aneuploidy can cause abnormal phenotypes in
sea urchin embryos.138,154 The first experimental proof was
obtained in 1930 by Winge who showed that the skin cancers
induced in mice treated with tar were indeed aneuploid.137
2. The Current Bias Against Aneuploidy. In view of a series of
new genetic and cytogenetic discoveries (see below), above all the
discovery of heterogeneous and thus apparently unspecific karyotypes
in most cancers, the original aneuploidy hypothesis was abandoned
in the 1960s and 1970s. The resulting bias against the aneuploidycancer hypothesis is now based on the following arguments:
1) Aneuploidy is seen as a consequence or epiphenomenon of
cancer. In the 1960s and 1970s it was discovered (a) that most cancers
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MULTISTEP CARCINOGENESIS
Table 2
PREDICTIONS
OF THE
ANEUPLOIDY-CANCER HYPOTHESIS
AND
EXPERIMENTAL SUPPORT
FROM THE
LITERATURE
Prediction
Proofs of Principle
in Literature
1
Carcinogens function as aneuploidogens.
Refs.49,50,94; Key for Table 1 (#12);
Aneuploidy by UV.181
2
Aneuploidy precedes and coincides with carcinogenesis.
Refs.3,6,17,26,48-56,59,60,182
3
Cancer cells can evolve from aneuploid cells autocatalytically at rates that are dependent on the
degree of aneuploidy (which is enhanced by additional aneuploidogens).
Refs.47,64,152,177
4
Even after a sufficient dose of carcinogen, cancer does not appear for a long time—namely years
to decades—because cancer-specific aneuploidies must evolve from random aneuploidizations.
Refs.5,13,14,25,38-40,64
5
Cancer-specific aneuploidies or aneusomies determine cancer-specific phenotypes.
Refs.13,25,53,64
6
Cancer typically appears only at advanced age (age bias), because aneuploidy is not heritable,183
and because the de novo generation of cancer-specific aneuploidies via random aneuploidization
is very slow.
Refs.5,14,43-45; Key for Table 1(#8
and #13); Non-heritability of
aneuploidy
7
Congenital chromosomal instability, such as Bloom’s syndrome, Fanconi anemia, or Premature
centromere division, induces cancer at young age, because it accelerates spontaneous aneuploidization.
Refs.184,185
8
Simultaneous aneuploidization of many cells by carcinogens, eg. by tobacco, may cause “field
cancerization”.
Refs.16,17,56,186
9
Tumorigenicity of aneuploid cells in culture is proportional to their degrees of aneuploidy.
Refs.163,187
10
Transplantability of tumor cells is proportional to the degree of aneuploidy, because more
aneuploidy generates more antigenic variation.
Ref.7
11
Genetic instability coincides with aneuploidy.
Refs.29,48,60
12
Genetic instability is proportional to the degree of aneuploidy.
Refs.28,50,152
13
Phenotypes of cancer cells, such as drug-resistance and cell morphology, vary with karyotype variations.
Refs.11-13,62,188
14
Human cancer cells may become resistant to human viruses and susceptible to non-human viruses
via karyotype variations.
Refs.9,10
15
Clonal mutations in cancers derive from pre-neoplastic aneuploidy and non-clonal mutations
from neoplastic aneuploidy.
Ref.50; See Table 1
16
“Immortality” of aneuploid cells derives from variants that evade lethal mutations or toxins by
reassorting chromosomes.
Refs.6,11,12,189
17
Transient suppression of tumorigenicity via fusion of cancer cells with normal cells by unbalancing
cancer-specific chromosome assortments. Tumorigenicity re-emerges via loss of chromosomes (Fig. 1).
Refs.6,119,190; See text
18
Abnormal expression of hundreds or thousands of normal but aneuploid genes – the “sine qua non
of malignant transformation”.4
Refs.114-116
are clonal,1,3,4,14,15 and (b) that their karyotypes are heterogeneous
—a “confusing plethora”155—and thus non-clonal.1,13,24,62,63,119,156
Since these facts were incompatible with the expectation, that a
specific aneuploidy would cause a specific cancer, it was concluded
that aneuploidy must be a consequence, rather than a cause of
cancer.13,25,62,79,119,131,157,158
2) Aneuploidy unknown as source of neoplastic phenotypes.
Aneuploidy is also disregarded as a cause of cancer, because phenotype
alteration by aneuploidy is simply unknown in cancer research. By
contrast, phenotype alteration by gene mutation is canonical
knowledge. For example, Foulds writes in Neoplastic Development in
1975, “The interpretations of the observations [of aneuploidy] on
dysplasia and carcinoma in situ and on progression in these lesions
…are still controversial.”1 Cairns writes in 1981 in Nature, “the
observed changes in karyotype could … be trivial secondary events
that occur after the rate limiting steps of carcinogenesis have been
completed.”131 Cancer Biology, by Ruddon, says about “aneuploidy” in
1987, “Although the more subtle changes in the genome—namely
point mutations, gene deletions, and gene rearrangements—may be
associated with initiation of the malignant transformation process,
gross changes in the number of chromosomes usually occur as tumors
206
progress to malignancy.”13 Mitelman et al. state in 1997, “the pathogenic significance of such abnormalities [aneuploidy] is totally
unknown.”158 Orr-Weaver and Weinberg point out in 1998 “Aneuploidy
has long been speculated to be causally involved in tumorigenesis,
but its importance has not been demonstrated.”159 An editorial in
Science comments in 1999 that it is “still unresolved…whether an
increase in ploidy contributes to, or is a consequence of, tumor
development.”148 Zimonjic et al. deplore in 2001 that aneuploidy,
“confounds attempts to determine the precise cohort of genetic
changes that are required for the transformation of normal human cells
to a tumorigenic state...”.160
In line with this disregard for aneuploidy, the identification of
suspected cancer genes and the analysis of the multiple steps of
carcinogenesis are now routinely conducted in highly aneuploid cell
lines, above all the mouse 3T3 and C3H 10T1/2 lines, which are
assumed to be not tumorigenic because they resemble normal
diploid fibroblasts morphologically.2,82,84,161 However, the relevance of results obtained with aneuploid cell lines to carcinogenesis
must be called into question, because most, if not all of them are
tumorigenic irrespective of their morphology.162,163 Indeed, the
3T3 and C3H cells are already sarcomagenic prior to morphological
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MULTISTEP CARCINOGENESIS
transformation with hypothetical cancer genes or with carcinogens.164 Moreover, complete chromosome balances of multiple cells
of human cancers, which are necessary to distinguish specific from
unspecific aneuploidy, are not even reported in the recent literature,
based on our experience. As a result of this bias, modern textbooks
of biology do not even mention aneuploidy in the context of carcinogenesis.84,85,170
3) Aneuploidy said to be not necessary for cancer. The existence
of presumably diploid cancers is cited as an argument that aneuploidy is not necessary for cancer. However, each of these cases fails to
provide adequate documentation either for diploidy or for malignancy
of the respective tumors.8,33,76,79,80,165,166 For example, Zimonjic et
al. state in 2001 that, “a limited set of genes suffices to induce human
cell transformation…[and] that widespread genomic changes involving
large arrays of cellular genes [alias aneuploidy] are not required
…”.160 But not even one in over 46 of their reportedly diploid,
human tumor cells proved to be diploid in a subsequent cytogenetic
analysis conducted by us.77 Further, Loeb et al. exclude aneuploidy as
a cause of cancer—“it is neither a clonal marker nor an initial event”—
based on the normal karyotype of “a premalignant condition”.91
4) Aneuploidy said to be not sufficient for cancer. Since some
hybrids of normal and cancer cells are not tumorigenic, it has been
argued that, unless certain normal genes are mutated, aneuploidy is not
sufficient for carcinogenesis.2,4,119 However, all non-tumorigenic
hybrids readily revert to tumorigenicity by spontaneous loss of chromosomes (see below and Fig. 1).2,4,119 This argument also ignores
that the vast majority of such hybrids, including all the mouse lymphocyte- myeloma hybrids or hybridomas, are immortal and tumorigenic a priori.2,6,84,167
5) Aneuploidy interpreted as support for the gene mutation
hypothesis. Recently aneuploidy has been reconsidered as a cause of
cancer, if only as a mechanism to alter the dosage of hypothetical
cancer genes or to generate new ones by mutation. For example,
Lengauer et al. proposed in 1997 that aneuploidy may “select for
gains of chromosomes containing activated oncogenes and for losses
of chromosomes containing tumour-suppressor genes”,29 and, in a
subsequent study, that it can “accelerate the acquisition of growth
promoting mutations”,48 possibly by rearranging genes at breakpoints
of rearranged chromosomes.30,158 In 2002, Weinberg et al. also
proposed “that aneuploidy facilitates the acquisition of the genetic
alterations that lead to cancer.”168 A recent review in Nature simply
reduces Boveri’s chromosomal theory to the current oncogene theory of
cancer, “if we substitute the word ‘gene’ for ‘chromosome’, this vision
clearly predicted the Nobel Prize-winning discovery of cellular protooncogenes by Harold Varmus and Mike Bishop in the 1970s…”.169
Thus, for all those who have recently been looking for alternative
causes of cancer, aneuploidy proved to be, at best, a diamond in the
rough.33,79,80
3. A Unifying Hypothesis—Multistep Carcinogenesis via a
Chain Reaction of Aneuploidizations. Intrigued by the enormous
mutagenic potential of aneuploidy, its ubiquity in cancer and its
proportionality with the degree of malignancy, we took up the challenge to develop an aneuploidy hypothesis, that:
1. would explain the origin of aneuploidy in cancer;
2. the spontaneous but characteristically slow evolution of an initiated
cell to a cancer cell; and
3. how non-clonal and unstable karyotypes cause cancer.
We think the model, that carcinogenesis results from a chain reaction
of aneuploidizations, meets this challenge (see Fig. 1).
According to our model the various phenotypes of cancer are
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consequences of cancer-specific aneuploidies, which alter the dosages
of thousands of normal genes.64 These cancer-specific aneuploidies
are products of a stepwise karyotype evolution that is initiated by
a random aneuploidy. The initiating aneuploidy is caused either by
a physical or chemical carcinogen, or is spontaneous.6 Since aneuploidy
unbalances, via the corresponding genes, numerous, long-established
teams of synergistic mitosis-proteins, and even the numbers of centrosomes,171-174 it renders chromosome segregation error-prone.6,152,175
Thus aneuploidy generates ever-new karyotypes autocatalytically—
like a chemical chain-reaction. Additional carcinogens and tumor
promoters2 can accelerate this evolution hetero-catalytically.176,177,192
Most of the randomly generated chromosome assortments would be
lethal or less viable than normal ones, but some would be able to
out-compete normal cells in the permissive habitat of the body and
would thus be able to cause cancer. Since most random changes of
chromosomes are unlikely to generate chromosome assortments that
out-perform normal ones, the appearance of a cancer-specific aneuploidy is a rare and most probably a multistep event—analogous to the
evolution of a new species. Indeed, carcinogenesis and phylogenesis
both depend on alterations of chromosome numbers and structures.
This is the reason, why it typically takes a long time to develop cancer.
The end-product of this multistep evolution would be a maximally
aneuploid or near-triploid, and thus also a maximally unstable and
hence adaptable karyotype,152 which is the karyotype of the most
malignant cancer cells.6,62,67,178
The inherent instability of highly aneuploid karyotypes explains
the notorious heterogeneity or non-clonality of the karyotypes of
clonal cancers as superpositions of selectively advantageous, cancerspecific and selectively neutral, random chromosome assortments.
Thus cancer is clonal for aneuploidy and for various specific aneusomies, but not for a unique karyotype.64
Since aneuploidy also unbalances the many teams of DNA synthesis- and DNA maintenance-genes,84,179 and the genes that maintain
nucleotide pools,180 aneuploidy also destabilizes the structure of the
chromosomes and genes of cancer cells50—a state that is collectively called “genetic instability”.3,33 According to our model many gene
mutations of cancer cells would then be consequences of aneuploidy.
Thus, our model predicts that malignancy, karyotypic and genetic
instability are all proportional to the degrees of aneuploidy, which is
consistent with the literature50 (see Table 1, Table 2 and Fig. 1).
Our model also predicts that aneuploid chromosome assortments—but not rearrangments— are reversible. In support of this,
some cancer-specific phenotypes such as drug-resistance can get lost
at the same rates at which they are generated,11,12 and malignancy
of non-tumorigenic cancer-normal-cell hybrids is recovered by loss of
chromosomes (Fig. 1).62,119
Our model is not only relevant to the origin of cancer from
normal cells but also from hyperplasias with balanced karyotypes
that carry a higher than normal risk of aneuploidization, as for
example Barrett’s esophagus61 or the chronic phase of myeloid
leukemia with the Philadelphia chromosome.4,6 However, our model
is not relevant to the origin of diploid hyperplasia. For example, UVlight causes benign,hyperplastic papillomas in a small fraction of the
mice in which it causes carcinomas—compatible with an aneuploidyindepdent mechanism.192
Our model of carcinogenesis confirms and extends the one
proposed by Nowell in 1976.3 Like Nowell, we propose that the
steps of progression of an initiated cell are aneuploidizations, although
Nowell left open whether aneuploidy or gene mutation were causing
cancer, “the genetic-versus-epigenetic debate”. But in contrast to
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Nowell’s model, which holds that “It is certainly clear that visible
alterations in chromosome structure are not essential to the initial
change”, our model proposes that initiation is a random aneuploidy
induced by a carcinogen, along the lines of Boveri’s hypothesis. Most
importantly our hypothesis explains the origin of “genetic instability”
by autocatalytic karyotype variation of aneuploid cells. As observed
by Nowell, “this important characteristic has not been satisfactorily
explained.”3
To prove our model, we have tested 18 of its predictions either by
conducting new experiments or by selecting predicted experimental
results from the large literature on cancer. It is shown in Table 2, that
the literature, including our own work, does indeed support all of
these 18 predictions made by the aneuploidy-cancer hypothesis.
Thus the hypothesis, that multistep carcinogenesis is a chain
reaction of aneuploidizations, can explain and predict all facts of
carcinogenesis, particularly those that are paradoxical in view of the
gene mutation hypothesis (Table 1).
RELEVANCE OF THE ANEUPLOIDY HYPOTHESIS FOR THE
CONTROL OF CANCER
The chromosome hypothesis promises several benefits for the
control of cancer. For example, eliminating from food and drugs
substances that cause aneuploidy could prevent cancer. Identifying
pre-cancerous cells in biopsies based on aneuploidy could revolutionize cancer therapy by eliminating affected tissues before they
turn malignant. Even the treatment of clinical cancer could possibly
be improved based on the risks that particular chromosome combinations may present. In principle this is already being proposed now
based on genome-wide transcription profiles derived from micro-arrays
of thousands of cellular genes.114-116
Acknowledgments
We thank Peter Nowell (University of Pennsylvania, Philadelphia) for a
critical review of the manuscript, David Rasnick, Rainer Sachs, Hung-Hsi
Wu (U.C. Berkeley), Alice Fabarius and Ruediger Hehlmann (Mannheim,
Germany) for information and discussions, and Mikhail Blagosklonny for
inviting this review.
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