Download Tumorigenesis: the adaptation of mammalian cells to sustained

Carcinogenesis vol.26 no.8 pp.1323--1334, 2005
doi:10.1093/carcin/bgi079
Advance Access publication March 31, 2005
COMMENTARY
Tumorigenesis: the adaptation of mammalian cells to sustained stress environment
by epigenetic alterations and succeeding matched mutations
Tatiana V.Karpinets1, and Brent D.Foy2
Introduction
1
The genome of cancer cells usually displays a large number of
mutations, but causative mechanisms underlying the origin of
these mutations are still debated (1). A recent model of tumorigenesis (2) suggests that cells may generate mutations by
themselves as a result of the mutator response (MR), an
error-prone cell-cycle progression with participation of errorprone polymerases and aberrant mitosis. According to the
model, the MR may be activated in the cellular environment
with persistent production of proliferative and stress-related
survival signals. This environment, referred to as the sustained
stress environment (SSE), gives rise to arrested cells because
of DNA damage or replicative senescence. The induction of
intracellular proliferative signal in the arrested cells in combination with the blockade of apoptosis by stress-related
survival signals can abrogate the cell-cycle arrest and activate
error-prone cell-cycle progression thereby generating mutations in the genome of emerged cells. The objective of this
paper is to show the role of epigenetic alterations (EA) in
inducing the conditions for activation of the MR in SSE and
in providing a tagging system that may direct mutations in the
MR to specific genes in the genome. The combination of these
epigenetic effects ensures the high adaptive potential of mutational modifications in the genome of tumor cells and can
explain many features in cancer origin and progression.
Epigenetic control of gene activity is widespread in the
genome of eukaryotic cells and leads to persistent gene
silencing or gene expression. This control is implemented by
changes in the methylation status of DNA (the methylation of
cytosine residues at 50 carbon) and by chromatin modifications
(e.g. histone acetylation, phosphorylation, methylation and
ubiquitination). The epigenetic modifications efficiently control the metastable state of gene expression in the genome by
inducing stable silencing of some genes and promoting activation of others (3,4). The epigenetic control of gene expression
is heritable through cell division, but reversible, because it
does not involve DNA sequences. DNA methylation is the
best studied EA. It is commonly implicated in the programming of the genome during embryonic development providing
its stability by silencing genes of inactive X chromosome,
ensuring parent-of-origin specific expression of imprinted
genes, regulating tissue specific expression, and silencing of
interspersed repetitive retrotransposons and heterochromatic
satellite repeat sequences (5).
Recent studies reveal the involvement of DNA methylation
and histone modifications in the reprogramming of the genome
of mammalian cells during many age-related human diseases
including cancer. These EA have been mainly elucidated by
comparing cellular genomes in healthy and pathological states
and therefore, terms hypermethylation and hypomethylation
refer to the degree of methylation when compared with the
healthy state. Well known changes in methylation related to
cancer are summarized in several recent reviews (3,4,6--8).
Department of Plant Sciences, University of Tennessee,
2431 Center Drive Knoxville, TN 37996-4500, USA and 2Department of
Physics, Wright State University, 3640 Colonel Glenn Hwy,
Dayton, OH 45435, USA
To whom correspondence should be addressed. Tel: þ1 865 974 0710;
Fax: þ1 865 974 5365;
Email: [email protected]
Recent studies indicate that during tumorigenic transformations, cells may generate mutations by themselves
as a result of error-prone cell division with participation
of error-prone polymerases and aberrant mitosis. These
mechanisms may be activated in cells by continuing
proliferative and survival signaling in a sustained stress
environment (SSE). The paper hypothesizes that longterm exposure to this signaling epigenetically reprograms
the genome of some cells and, in addition, leads to their
senescence. The epigenetic reprogramming results in:
(i) hypermethylation of tumor-suppressor genes involved
in the onset of cell-cycle arrest, apoptosis and DNA
repair; (ii) hypomethylation of proto-oncogenes associated with persistent proliferative activity; and (iii) the
global demethylation of the genome and activation of
DNA repeats. These epigenetic changes in the proliferating cells associate with their replicative senescence and
allow the reprogrammed senescent cells to overcome the
cell-cycle arrest and to activate error-prone replications.
It is hypothesized that the generation of mutations in the
error-prone replications of the epigenetically reprogrammed cells is not random. The mutations match epigenetic alterations in the cellular genome, namely gain of
function mutations in the case of hypomethylation and
loss of functions in the case of hypermethylation. In
addition, continuing proliferation of the cells imposed by
signaling in SSE speeds up the natural selection of the
mutant cells favoring the survival of the cells with mutations that are beneficial in the environment. In this way,
a stress-induced replication of the cells epigenetically
reprograms their genome for quick adaptation to stressful environments providing an increased rate of mutations, epigenetic tags to beneficial mutations and quick
selection process. In combination, these processes drive
the origin of the transformed mammalian cells, cancer
development and progression. Support from genomic,
biochemical and medical studies for the proposed hypothesis, and its implementations are discussed.
Abbreviations: EA, epigenetic alternations; EP, epigenetically
reprogrammed; MR, mutator response; SSE, sustained stress environment.
Carcinogenesis vol.26 no.8 # Oxford University Press 2005; all rights reserved.
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T.V.Karpinets and B.D.Foy
They include (i) tumor or age related hypermethylation of
specific CpG islands and coding regions resulting in silencing
of previously active genes, (ii) changes in methylation of
differentially methylated regions leading to loss of imprinting
and (iii) hypomethylation of previously silent genes and
repetitive sequences resulting in chromosomal instability,
mitotic recombination, chromosomal loss and aneuploidy.
The exact cellular mechanism that provides epigenetic transformations is not established, but many observations indicate a
relationship between the level of gene activity in the genome,
the methylation state of related DNA and chromatin modifications. Several lines of evidence suggest that gene expression is
inversely correlated with DNA methylation (9,10). Mammalian cells tend to methylate promoter sequences of those genes
in the genome that cease their participation in cellular activity.
It is proposed that chromatin remodeling as a result of histone
deacetylation and methylation is the primary event in initiating
gene silencing, whereas DNA methylation, a subsequent event,
establishes a permanent state of gene inactivation (11). It was
also postulated that methyl-binding proteins, a component of
the promoter methylation mechanism, have to compete with
transcription factor abundance for the establishment of gene
repression (12). The existence of this competition may explain
why steady cessation of a cellular function and a concomitant
lack of transcription factors steadily decreases gene activity
related to this function. Conversely, a steady presence of the
transcription factors shifts the balance to demethylation of
the promoter. Therefore, while hypermethylation of gene
promoters relates to transcriptional silencing of unwanted
genes, hypomethylation indicates an increase in the level of
gene activity (4).
We present a hypothesis that considers EA in cells imposed
by stress-related proliferative and survival signaling in a
SSE as starting events in cancer development. The hypothesis
describes potential epigenetic mechanisms involved in
priming cells for the generation of beneficial mutations in the
genome and quick adaptation to SSE. We provide support of
the hypothesis from the published literature and discuss its
potential implications.
Hypothesis
Tumorigenesis is considered a process of the adaptation of
mammalian cells to SSE by means of EA of the genome,
subsequent mutations matched to EA and natural selection of
originated mutant cells via apoptosis (Figure 1). The process of
adaptation to SSE involves the emergence of senescent
epigenetically reprogrammed (ER) cells with specific tumorrelated EA in the genome. The EA prime the cells for errorprone replications and provide epigenetic tags in the genome
for beneficial mutations to arise.
Emergence of the ER cells. We hypothesize that tumorrelated EA occur before mutations and reprogram the genome
for their origin. The initiation event for these EA is long-term
exposure of cell tissue to SSE that induces continuing replication of some cells in the tissue. Persistent proliferative and
stress-related survival signals in the environment induce in
these proliferating cells a stable activation of biological processes related to promotion of replication and stress response.
On the other hand, processes related to inhibition of proliferation, activation of cell-cycle arrest and apoptosis, and DNA
repair pathways will be constantly suppressed in these cells.
1324
Fig. 1. Scheme of processes involved in cellular transformation imposed by
SSE.
Most well-known oncogenes are crucial regulators in these
processes. Stable changes in the expression of the genes are
secured in the continuously replicating cells by proper EA in
the genome, hypermethylation in case of upregulation and
hypomethylation in case of downregulation. The EA in the
oncogenes are also accompanied by global demethylation of
the genome and stress-induced activation of DNA repast.
In combination, these EA reprogram the genome for quick
adaptation to stressful environments and give rise to ER
cells bearing tumor-related EA in their genome.
Mutator phenotype of the ER cells. Continuing stressinduced replication of the ER cells leads to their senescence.
In normal cells replicative senescence is accomplished by cellcycle arrest, i.e. cells remain viable, but cannot subsequently
divide. ER cells cannot activate the cell-cycle arrest, apoptosis
or DNA repair, because these processes are silenced epigenetically in the cells. Therefore, they eventually continue the
proliferation in an error-prone mode, regardless of replicative
senescence or DNA damage in the genome.
Tumor-related mutations in the ER cells. We hypothesize
that mutations generated in the error-prone replications are
not random. They match EA in the genome of the ER cells.
Hypermethylation of CpG dinucleotide predisposes the ER
cells to point mutation, frameshift mutation, missense mutation and deletions. These dinucleotides are often dominated in
gene promoters. Therefore, hypermethylation of a promoter
will lead to a loss of gene function. Hypomethylation predisposes cells to chromosomal mutations, rearrangements and
aneuploidy. These mutations usually confer a gain of gene
Tumorigenesis: adaptation to SSE by EA and succeeding mutations
function. In addition, global demethylation imposed by SSE
leads to a variety of mutational effects and to a diversification
of the genome through the activity of DNA repeats. In this
way, EA in the genome of the ER cells (hypermethylation and
hypomethylation imposed by SSE) predispose to generation of
certain stress-associated adaptive mutations in the error-prone
replications. As a result, the ER cells give rise to mutant cells
bearing the tumor-related mutations. The natural selection of
the mutant cells is accelerated by their continuing proliferation
in the stressful environment. It favors the survival of mutant
cells harboring mutations that are most suitable for this environment, i.e. confer immortality and high proliferative activity.
Multiple rounds of error-prone replication fix these mutations
in the genome of progenies and lead to the emergence of
transformed cells. In this way, EA in the genome imposed by
proliferative and survival signaling in SSE drive the tumor
development.
The following experimental findings support the model and
will be discussed in more detail in the following sections.
(i) EA in the genome play a leading role in the origin of
cancer and its progression.
(ii) EA in cells exposed to SSEs are similar to those
indicated at premalignant and early stages of cancer
development.
(iii) Activity of cell-cycle inhibitors is essential to accomplish
stress-induced replicative senescence and their
inactivation in the senescent cells can lead to tumorigenic
transformations.
(iv) Known molecular mechanisms may underlie the causative effects of EA on succeeding mutations in ER cells.
Experimental studies that support mechanisms underlying
the activation of the MR and the natural selection via apoptosis
are presented elsewhere (2).
EA in the genome play a leading role in the origin of cancer
and its progression
According to the proposed hypothesis, the EA induced by
continuing replication in the cellular genome (epigenetic
reprogramming) are indispensable preliminary events for
cancer-related genetic mutation. Two stress-induced epigenetic events are a requisite for cancer development in the
hypothesis. They are (i) epigenetic silencing/activation of
oncogenes involved in proliferation, cell-cycle control and
DNA repair, and (ii) global hypomethylation of the genome.
The epigenetic silencing/activation of oncogenes is a necessary introduction to adaptive genetic mutation in the cell,
because it primes the genome for the error-prone cell division
and provides epigenetic tags to genes that are under selection
pressure in the cellular microenvironment. Global hypomethylation is important for subsequent adaptation of the
cells to a new environment and for a quick acquisition of
new cellular functions, because demethylation of the genome
leads to activation of transposable elements and facilitates
chromosomal mutations in the cells including a chromosome
loss or a gain.
In support of a leading role of these mechanisms in tumorigenesis, many studies draw attention to hypermethylation and
hypomethylation taking place at precancerous and early stages
of cancer. Some of these studies are summarized in Tables I
and II. Given a role of EA in the control of a stable state of
gene expression, the early onset of hypermethylation and
hypomethylation of the genes found in the studies indicates
that persistent changes in the related cellular functions occur
before tumorigenic transformations. In concordance with the
hypothesis, studies show early epigenetic silencing of genes
involved in the activation of cell-cycle arrest and apoptosis
including cyclin-dependent kinase inhibitors p16, p14, p15,
p73, APC gene and DAP-kinase (Table I). The other processes
Table I. The role of hypermethylation in the origin of cancer and its progression
Epigenetic alterations
Tissue/organisms under consideration
Ref.
p16
An animal model of lung carcinogenesis and human squamous cell carcinomas
Nonmalignant bronchial epithelial cells from current and former smokers
Early cervical tumor cell populations
Barrett’s epithelia (premalignant state of esophageal adenocarcinoma)
Human primary tumors and normal tissue of breast, colon and bladder
Leukemic cell lines, acute myeloid leukemia and acute lymphoblastic leukemia samples
Multiple myeloma cell lines and patients with malignant plasma cell disorders
Benign and malignant breast samples obtained from breast cancer patients and samples
from unaffected women
Summary of the data on ovarian cancer
Human gastric cancer cell lines and primary tumors
Lymphoid/hematopoietic tumors, potential premalignant specimens, control specimens,
EBV-transformed B lymphoblastoid cultures and lymphoma/leukemia or multiple
myeloma cell lines
Nonneoplastic gastric epithelia
Summary of the data on lung cancer
Breast tumor, paired preoperative serum DNA, normal healthy women and patients
with inflammatory breast disease or nonneoplastic breast tissue specimens
Premalignant lesions in the colon
Normal tissue adjacent to tumors (lung, prostate and pancreatic carcinomas) and
normal tissue from noncancer patients
Summary of the data on the role of BRCA1 hypermethylation in breast and ovarian cancer
Early-onset sporadic colorectal carcinomas
Ovarian primary tumors
A summary of the data on a wide spectrum of human tumors
Comparison of stages of mouse skin tumor progression including premalignant stages
Colorectal cancer progression
13
14
15
16
17
18
19
20
p16 and p14
p15, p16
p16 and p73; SOCS-1; E-cadherin; DAP-kinase
APC gene; RARbeta2 gene; RASSF1A
FNACF
TGF-beta receptor
SHP1; SOCS1; SYK
p16; E-cadherin; hMLH1
MGMT gene; TIMP-3; DAP-kinase; p16 and p15
APC gene; RASSF1A; DAP-kinase
CRBP1, CDH13 (H-cadherin); MINT31
HIN-1
BRCA1 and BRCA2
hMLH1 and hMLH2
hMLH1
MGMT gene promoter
MGMT and MLH1; E-cadherin
SFRP
21
22
23
24
25
26
27, 28
29
30, 31
32
33
34
35, 36
37
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T.V.Karpinets and B.D.Foy
Table II. The role of hypomethylation in the origin of cancer and its progression
Epigenetic alterations
Tissue/organisms under consideration
Ref.
Sat2 (satellite 2), global hypomethylation
Breast adenocarcinomas of known tumor grade and stage, non-neoplastic breast
tissues (mostly fibroadenomas), and a variety of normal somatic tissue controls
The folate/methyl deficient model of multistage hepatocarcinogenesis (rat liver)
Normal gastric mucosa, superficial gastritis and chronic atrophic gastritis specimens
Non-small cell lung cancer samples, primary cancer samples, corresponding normal
lung tissues and bronchial brush specimens from former smokers without lung cancer
Gastric carcinoma specimens, corresponding non-neoplastic mucosae and gastric
carcinoma cell lines
Summary of the experimental data on alteration of DNA methylation in gastrointestinal
carcinogenesis
Human livers of different ages, cirrhosis and hepatocellular carcinoma
Chronic liver disease without hepatocellular carcinoma, non-tumor liver tissues from
patients with carcinoma, carcinoma tissues and control liver tissues
Hepatocellular carcinoma fetal and normal human liver tissues
Comparison of stages of mouse skin tumor progression including premalignant stages
Comparison of colorectal cancer specimens with normal mucosa of non-cancer patients
38
Global hypomethylation
Global hypomethylation
Melanoma antigen-encoding genes
(MAGE-A1, MAGE-A3 and MAGE-B2)
Cyclin D2 promoter
c-myc, c-Ha-ras, c-fos
c-fos
c-myc, c-Ki-ras
c-myc, EGF receptor
Snail
The tumor associated changes in promoter
CpG island methylation of 17 genes
subjected to early hypermethylation in different tissues include
the regulation of cell-to-cell adhesion (E-cadherin and
H-cadherin, MINT31, SYK, TIMP-3, Snail), suppression of
cytokine signaling (SOCS1, TGF-beta, SHP1, HIN-1), DNA
repair (BRCA1, BRCA2, MGMT, hMLH1, hMLH2) and
regulation of cell differentiation (SFRP, APC, RARbeta2,
TGF-beta). Early hypomethylation (Table II) is found for
transcription factors involved in activation of proliferation
(c-myc, c-fos) and transduction of proliferative and survival
signals (EGFR, Ras). Interestingly, when one of the two alleles
is mutated in the germ line of a patient with a familial form of
cancer, hypermethylation is commonly seen as the second
inactivation event (48). Specifically, this epigenetic effect
was reported for p16 gene and E-cadherin (49,50). Additional
evidence on a leading role for DNA methylation at the preneoplastic stages of cancer development was reviewed by Jones
and Laird (51) including the fact that reverse demethylation
of methylated genes suppresses the formation of some tumors.
In summary, although pathways and genes affected by early
EA may be different in different tumor types, in almost all
instances they relate to one of the following processes:
(i) continuing activation of pathways related to replication;
(ii) silencing of pathways and genes with growth-inhibitory
activities including the activation of cell-cycle arrest, apoptosis and cellular differentiation; (iii) silencing of DNA repair
genes. These cellular processes indicate persistent activation
of proliferative and survival signals in the cells even before
premalignant stages of cancer and therefore support the plausibility of epigenetic reprogramming of the cells by these signaling as a preliminary event for tumorigenic transformations.
Global hypomethylation of the genome was also reported
as an early epigenetic event in several cancers (Table II).
Though, if hypermethylation and hypomethylation of oncogenes are more often considered in the studies as important
events in the origin of tumors, increased demethylation or
hypomethylation strongly relates to progression of cancers
and increased malignancy. A positive correlation between
the degree of global hypomethylation, the grade of malignancy
in tumors and chromosomal alterations are found in many
cancers (52--55). Hypomethylation is very often observed in
DNA repeats (4). The repeats that display tumor-associated
hypomethylation include endogenous retrotransposons,
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39
40
41
42
43
44
45
46
35
47
LINE-1 repeats, Alu repeats, endogeneous retroviruses and
moderately repeated non-viral DNA sequences. It is believed
that hypomethylation is necessary for metastasis and invasion
in cancer development. Several examples of this role of hypomethylation in breast cancer are given in the review of Szyf
et al. (56). These experimental observations indicate a leading
role of EA not only in the origin of cancer, but also in its
progression.
EA in cells exposed to SSEs are similar to those indicated at
premalignant and early stages of cancer development
According to the model the EA indicated in cells at premalignant stages result from their epigenetic reprogramming in SSE.
The reprogramming may be induced by continuing production
of cytokines in this environment because they activate proliferative and stress-related survival signals in the cells. Proliferative signaling activates genes and pathways involved in
cellular replication. Survival signaling suppresses the activation of cell-cycle arrest and apoptosis. Inactivation of DNA
repair pathways may follow persistent suppression of genes
involved in the cell-cycle control (like APC gene and p53) that
mediate DNA repair pathways (57,58). In long-term, these
changes in gene expression lead to tumor-related EA in the
genome.
In support of the role of SSEs in epigenetic reprogramming,
EA in oncogenes (similar to reviewed above), global hypomethylation and activation of DNA repeats are reported in
non-malignant mammalian cells, if they are exposed to wellknown carcinogens (gamma and ultraviolet radiation, tobacco
smoke, toxicants with potent carcinogenic effects, etc.) or to
oncogenic cellular stresses (chronic inflammation, wounding,
oxidative stress, genotoxic stress, etc.) (Table III). These stresses usually impose continuing cellular assault and death and
are therefore accompanied by persistent production of
cytokines. Although studies on EA after exposure to carcinogens are not as extensive as studies in tumor cells, they also
indicate (i) hypermethylation of crucial regulators of cell-cycle
arrest and apoptosis (p16, p53, APC); (ii) hypomethylation of
oncogenes involved in proliferation and stress-related signaling (c-myc and raf ); (iii) global hypomethylation of the genome and activation of DNA repeats. These changes are similar
Tumorigenesis: adaptation to SSE by EA and succeeding mutations
Table III. Epigenetic alterations induced by SSE in mammalian cells
Epigenetic alterations
Nature of stress
Tissue/organisms
Ref.
Hypermethylation of tumor suppressor
genes (p16, APC) and decrease in the
mean methylation index
Hypermethylation of tumor suppressor
gene p16
Hypermethylation and transcriptional
silencing of p16 and p53
Tobacco smoke exposure
Lung tissue of long-time smokers
59
Exposure with cadmium (potent carcinogen)
Cell culture (BALB/c-3T3 cells)
60
Exposure with dioxin (an ubiquitous
environmental contaminant and a
highly potent carcinogen)
Phenobarbital’s exposure
Exposure with carcinogens (arsenite,
dichloroacetic acid and trichloroacetic acid)
Exposure with cadmium
Exposure with nickel
Gamma radiation
Ultraviolet radiation (UVB 280--340 nm)
Genotoxic stress induced by a variety of DNA
damaging agents
Endogenous oxidative stress
Primary human keratinocytes
61
Rodent liver tissue
Variety of tissues, including the liver
62
63, 64
Rat liver cells (TRL1215)
Chinese hamster embryo cells
Four non-malignant cultured cell lines
Rat liver
Mouse cells
65
66, 67
68
69
70
71
Anoxia (may accompany wound healing)
Glucose 6-phosphate dehydrogenase
deficient mice
Rat fibroblasts
72
Chronic inflammation (chronic ulcerative colitis)
Non-neoplastic mucosa
73
Proliferation under stress (cell culturing)
Primary mouse embryonic fibroblasts
74
SV40 virus infection
Human mesothelial cells
75
Hepatitis B virus and hepatitis C virus infection
in liver with chronic inflammation
Liver tissue specimens of human
hepatocellular carcinoma with or
without hepatitis virus infection
Epstein--Barr virus-associated and
negative gastric carcinoma
76
Hypomethylation including raf-gene
Upregulation and hypomethylation of
c-myc and c-jun
Hypomethylation of the genome
Decondensation of the heterochromatin
Dose-dependent hypomethylation
Dose-dependent hypomethylation
Induction of Alu elements (short
interspersed elements)
Induction of long interspersed nuclear
element-1 (LINE-1)
Induction of VL30 elements (a multigene
family within the class of retroviruses
and retrotransposons)
Hypermethylation of mismatch repair
genes and microsatellite instability
Epigenetic silencing of imprinted genes
that are usually induced by tumorigenesis
Progressive aberrant methylation of
protooncogenes (RASSF1A, HPP1, DcR1,
TMS1, CRBP1, HIC-1 and RRAD)
methylated in malignant mesothelioma
Hypermethylation of p16 promoter
Specific high-density methylation of
cyclin-dependent kinase inhibitors
p14 and p16
Epstein--Barr virus-associated gastric carcinoma
to changes in the cellular processes at premalignant and early
stages of cancers indicated in the previous section.
The induction of DNA repeats in SSE, in particular, has
strong support in the various studies. In 1984, McClintock
(78) first proposed the hypothesis that retrotransposons may
be considered as stress-induced generators of genomic diversity. By now, there is substantial evidence of activation of DNA
repeats by different kinds of stresses in various organisms
including plants, microbes and humans. It is believed that
DNA methylation and chromatin structure globally repress
the transcription of the repeats, but sustained stress activates
them inducing an opening of the chromatin structure and
demethylation of the repeats (79). Recent genomic studies
have revealed an important role of DNA repeats in the revival
of cryptic genes, in diversification of the genome and in
adaptation to stressful environments (80). Considering the
fact that DNA transposons in human genome hold DNA
records from the genomes of evolutionarily preceding species
(81), we may speculate that demethylation of DNA repeats in
SSE prepare tumor cells for diversification of the genome and
for reactivation of cryptic genes involved in functions required
for cellular survival in this environment.
Thus, we conclude that EA induced by exposure to carcinogens and by oncogenic cellular stresses are similar to those
observed at premalignant and early stages of cancer. Similar to
cancer cells, these EA indicate continuing proliferation of
the cells accompanied by silencing of cell cycle arrest and
apoptosis, and activation of DNA repeats. These observations
77
are consistent with a central feature of the hypothesis that
SSEs have a leading promotional role in the epigenetic reprogramming of the cells and their subsequent tumorigenic
transformations.
Activity of cell-cycle inhibitors is essential to accomplish
stress-induced replicative senescence and their inactivation in
the senescent cells can lead to tumorigenic transformations
Further support for the hypothesis is provided by studies
of replicative senescence. This process depends on the number
of cell divisions and is triggered by short telomeres (the ends of
linear chromosomes) that lose ~50--200 bp per round of replication. SSE is accompanied by cell death and renewal, and
therefore, inevitably speeds up the cellular senescence. Many
studies support the idea in the hypothesis that a stressful
environment favors replicative senescence (82). In the recent
review of Ben-Porath and Weinberg (83), cellular senescence
is considered as a general stress response program of the cell
with important biological functions including the onset of
arrest on proliferation. Stress-related survival signal induced
by Ras/Raf/MEK/ERK pathway caused cellular senescence in
several cell types, and the activity of cell-cycle inhibitors p53
and p16 was indispensable to accomplish senescence by cellcycle arrest. Inactivation of these genes prevents cell-cycle
arrest and results in tumorigenic transformation (7,84--86).
According to the studies in human fibroblasts and mammary
epithelial cells, cell-cycle arrest response in senescent cells is
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T.V.Karpinets and B.D.Foy
maintained primarily by p53. P16 provides a dominant second
barrier to the unlimited growth of human cells (86,87). Analysis of the methylation status of cancer-related genes in nonmalignant senescent human cells from different tissues was
reported by Yuasa (88). This study demonstrates that though
the methylation of the promoters of cancer-related genes may
also take place in normal senescence, age-related methylation
never includes hypermethylation of p53 and p16 genes, which
are crucial for the establishment of cell-cycle arrest in senescent cells. A different output may be observed in senescent
mammalian cells subjected to continuing proliferative and
survival signaling, because this environment leads to epigenetic silencing of genes involved in the onset of cell-cycle arrest
and apoptosis (Tables I--III). This silencing disables the onset
of cell-cycle arrest in the senescent cells and leads to malignancy. Given the experimental observations, we can hypothesize that continuing replication of some cells (ER cells in the
model) in SSE leads to their senescence in a way that cannot be
accomplished by cell-cycle arrest, because, in addition to the
replicative senescence, these cells have epigenetically silenced
genes involved in cell-cycle control. This silencing in combination with persistent proliferative signals predisposes the
ER cells to accomplish their divisions in an error-prone
manner, resulting in the generation of mutations and subsequent malignant transformations of the cells.
Known molecular mechanisms may underlie the causative
effects of EA on succeeding mutations in ER cells
Although mechanisms underlying the causative effects of EA
on the succeeding mutations are poorly understood, some of
them have been demonstrated in both hypermethylation and
hypomethylation. A correlation between the methylation status
of oncogenes and their mutations in tumor cells is also well
documented (53,89,90).
The most prominent evidence of the link between EA and
specific mutations is provided by studies of CpG dinucleotides. Mutagenic mechanisms involving 5-methylcytosine
in CpG dinucleotides are particularly common. Methylated
CpG dinucleotides are mutational hot spots in human genes
including well-known cancer-relevant genes, such as p53 and
retinoblastoma gene (90,91). The best characterized mechanism of G:C!A:T transitions at CpG sites is deamination
of 5-methylcytosine at CpG sites, resulting in substitution of
5-methylcytosine by thymine. This occurs spontaneously or is
factor-mediated, e.g. by cyclobutane pyrimidine dimers after
exposure to UVB and sunlight (92), by some environmental
pollutants (93) or through the action of nitric oxide produced
under conditions of chronic inflammation (94). The factormediated C to T transition mutations are particularly frequent
in some codons of p53 gene at sequences containing
5-methylcytosines. It was also suggested that error-prone
DNA polymerases play an important role in the mutagenic
bypass of the lesions and generation of the mutations (92).
Methylated CpG dinucleotides are mutation hot spots in the
retinoblastoma gene (91). Mutational abnormalities indicated
in Rb gene include G!A substitution in the promoter region
(95). Germline mutations in this gene include frameshift mutations, missense mutation and in-frame deletions (96). Deletions (either homozygous or heterozygous) are a major
mechanism of p16 and p15 genes inactivation (97). Loss of
function mutations in these genes are well documented in
tumors. Frequency of mutations in p16 is second only to
mutation of p53. As it was reviewed above (Table I),
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hypermethylation of cell-cycle inhibitor p16 takes place at
premalignant and early stages of many cancers and also may
be imposed by exposure with carcinogens (Table III).
The following mechanisms may underlie the induction of
matched mutations in genes with methylated CpG dinucleotides. It is generally agreed that C ! T mutations in CpG
dinucleotides may be caused by spontaneous hydrolytic deamination. Cytosine deamination produces uracil, which can be
removed by glycosylase, whereas deamination of 5-methylcytosine generates a GT mispair that cannot be processed
by glycosylase and escapes proofreading, because it is not
obvious which strand carries the correct genetic information
(98). This is a common phenomenon, and in healthy cells, this
type of replication error is repaired by the DNA mismatch
repair system that is directed specifically to the newly synthesized strand and in this way increases the fidelity of DNA
replication by up to three orders of magnitude (99). According
to the model and experimental evidence considered above
(Tables I--III), epigenetic silencing of cell-cycle inhibitors
imposed by SSE activates an error-prone cellular division
without participation of the repair machinery. As a result, mispairs at methylated CpG dinucleotides cannot be repaired and
therefore produce point mutations or deletions and frameshift
mutations in the case of replication slippage of error-prone
DNA polymerase (100). In this way, hypermethylation status
of CpG dinucleotides, which often characterizes TS genes in
the earliest stage of cancer development, may predispose them
to certain mutations, namely point mutation, frameshift mutations, missense mutation and deletions.
In addition to increased endogenous mutability of 5-methylcytosine, its interaction with carcinogens may also contribute
to certain types of mutations (101). For example, many chemical carcinogens including aromatic hydrocarbons in tobacco
smoke preferentially react with guanines flanked by 5-methylcytosine inducing the G ! T transversion mutations. The
majority of mutations observed in skin cancer are C ! T
transitions or CC ! TT mutations at dipyrimidine sequences
resulting from UV-light-induced cyclobutane pyrimidine
dimer formation. Methylation of cytosine increases this formation by up to 15-fold owing to higher energy absorption of
5-methylcytosine (102). These results suggest that hypermethylation of tumor suppressor genes, besides contributing
to endogenous mutability, can also make these genes vulnerable to environmental mutagens and carcinogens.
The causative effect of hypomethylation on succeeding
mutations is also well documented in tumors. If hypermethylation of CpG islands predisposes affected genes to point
mutations, deletions and frameshift mutations, leading to loss
of gene function, hypomethylation predisposes to chromosomal instability and to reshuffling of the mammalian genome
by the activation of DNA repeats. Hypomethylated repetitive
sequences are mainly located in the vicinity of the centromeres
of chromosomes, comprised of heterochromatin (4). It predisposes cells to increased chromosomal rearrangements in this
region, as takes place in many types of cancer. This type of
mutation in tumors often provides a gain of gene function.
Chromosomal instability has been linked to DNA hypomethylation in the heterochromatin regions of chromosomes
in several papers (52,53,103). For example, correlation of
DNA methylation status in heterochromatin regions with
tumor histology and with chromosome alterations was found
in a study of human breast carcinomas. Hypomethylation of
these regions was associated with the accumulation of a large
Tumorigenesis: adaptation to SSE by EA and succeeding mutations
number of chromosome alterations and promoted the development of breast carcinomas with aggressive histological features (53). Likewise, genomewide DNA hypomethylation is
associated with alterations on chromosome 8 in prostate
carcinoma (52). A high percentage of Wilms tumors, breast
adenocarcinomas and ovarian epithelial carcinomas have
cancer-associated hypomethylation of the main satellite DNAs
in the centromere-adjacent heterochromatin of chromosomes 1
and 16 (satellites Chr1 and Chr16), and it is associated with the
recurrent rearrangements of the satellites in the vicinity of the
centromere. These rearrangements usually lead to 1q gains or
16q losses (104).
The following mechanisms may underlie the effect of hypomethylation on chromosome instability. It was shown that in
lymphoblastoid cell lines hypomethylation in the heterochromatin regions of chromosomes induced DNA uncoiling and
rearrangements at these regions (103). In a study of human
hepatocellular carcinoma samples, hypomethylation altered
the interaction between the CpG-rich satellite DNA and chromatin proteins, resulting in heterochromatin decondensation,
breakage and aberrant 1q formation (55). We may hypothesize
that heterochromatin decondensation and DNA uncoiling in
the centromeric regions of chromosomes lead to abnormalities
in normal chromosome segregation during error-prone
cell-cycle progression in the MR, resulting in karyotypic
abnormalities during mitosis. In this way, the alteration in
chromatin induced by hypomethylation may underlie chromosomal mutations in the ER cells.
Mobility of DNA repeats including retrotransposons and
endogenous retroviruses imposed by their hypomethylation
may also induce a variety of mutational effects. All retroelements are able to form an RNA transcript that may be reversetranscribed and inserted into a new location in the genome.
Different classes of retroelements may have their mechanisms
for RNA formation, reverse transcription and integration
(105), but in all instances they contribute to insertional mutagenesis that may modify promoter or coding gene sequences.
Retrotransposition of long terminal repeats, like LINE1, may
remodel the genome through the transduction of 30 flanking
sequences. Such events have the potential to shuffle, e.g. exons
and promoters, creating new genes or altering the function of
old genes (106). LINE1 is also able to generate processed
pseudogenes and chimeric retrotranscripts (107). Alu elements
generate mutations by the insertion into mature mRNAs via a
splicing-mediated process termed exonization (108). Repetitive human Alu elements may cause deleterious mutations both
by retrotranspositional insertion and by unequal, homologous
recombination (109). The variety of ways in which activated
DNA repeats appear to affect genomic diversity in tumor cells
lends additional support to the hypothesis, i.e. hypomethylation of DNA repeats in SSE prepare the genome for mutational
modifications that are important for subsequent adaptation of
the disseminated tumor cells to new environments.
Changes in methylation status of genes occur throughout the
genome and involve many different genes and different codons
within the gene so that the epigenetic tagging system cannot
guarantee the generation of only beneficial mutations in the
error-prone replication. Rather, this system facilitates the
emergence of beneficial mutations in accord with the EA in
the genome. Subsequent natural selection of the mutants by
apoptosis adjusts the mutational spectra to the environment.
Therefore, a continuing proliferation of the cells, a requisite
of SSE, is very important not only for the epigenetic
reprogramming of the cells but also for a quick shaping of
the mutational spectra in the genome to the needs of the
cellular environment. In support of this mechanism, several
studies present evidence that the cytotoxicity of some carcinogens (like UVB light or tobacco smoke), rather than their
mutagenicity, may drive tumorigenesis (110,111).
Discussion
Support of the proposed hypothesis from stationary state
mutagenesis in bacteria and cell culture models of cancer
The mechanism of cellular adaptation proposed in the hypothesis challenges the foundations of neo-Darwinism, namely
that the environment cannot affect the rate of mutations and
cannot direct mutations to certain genes. By now, a long line
of evidence indicates that in unicellular organisms, such as
microbes, the stressful environment can affect the rate of
mutations and direct mutations to beneficial genes in this
environment. Studies of stationary state mutagenesis in bacterial cells provide the most prominent and well-documented
examples of the effect (112--117). Microorganisms increase
the rate of mutations during the periods of prolonged stress.
Stress limits the growth of microorganisms and induces them
to enter a stationary state and produce adaptive mutations that
confer a growth advantage in the growth-limiting environment. This strategy hastens the evolution of microbial pathogens including antigenic variation (changes in surface antigens
to avoid detection by the host immune system) and antibiotic
resistance (118).
The mechanisms that allow cells to increase their mutation
rates under conditions of sustained stress and produce the
mutations in beneficial genes are especially important in
evolutionary terms. Therefore, it is plausible that eukaryotic
organisms retained these mechanisms in their cells and made
them even more sophisticated. According to our hypothesis
such an adaptive strategy is employed in the somatic cells of
mammals in response to sustained stress and plays an important role in the development of malignances. In this case, a
continuing proliferative and survival signaling in the tissue
microenvironment indicates a poor adjustment of the cellular
genome for the environment and hastens cellular adaptation.
These signals are responsible for the epigenetic reprogramming of some cells leading to an increased rate of tumorrelated mutations in the genome. Numerous studies support
the fundamental role of the cellular environment in the transformations. This role is especially evident when we consider
in vitro culture of multicellular organisms. Rodent cells can
easily undergo ‘spontaneous’ neoplastic transformation in culture and are widely used as in vitro models of cancer
(119,120). A well-known example is the rodent fibroblast. In
a primary culture of mouse embryo cells, a small number of
cells, survived after apoptotic crises by forming immortalized
cell lines, can overgrow the culture. These cells invariably
display abnormal karyotypes and may undergo ‘spontaneous’
transformations (120). This output is consistent with the proposed hypothesis. Culturing of the cells mimics SSE in a
tissue, because of cellular assault and continuing proliferation
of some cells. This signaling epigenetically reprograms
the cells and primes them for malignant modifications in
the genome. This transformation represents an adaptive
response, which is similar to a bacterial one, to constraints on
growth. Although human cells rarely undergo spontaneous
1329
T.V.Karpinets and B.D.Foy
transformation in culture, they also may be transformed by a
stepwise process. For example, the transformation may be
accomplished by immortalization of the cells by DNA viruses,
followed by conversion of the immortalized cells to tumorigenic ones by Ras oncogenes or by using chemicals or chemical agents (121). DNA virus in this transformation provides
sustained activation of c-myc that reproduces a persistent
proliferative signal in the SSE. Ras-activation reproduces a
stress-related survival signal that is necessary to silence genes
involved in the cell-cycle arrest and to activate a global
demethylation of the genome.
Delayed effects of environmental factors on cancer development may be explained in terms of the proposed hypothesis
An important inference of the proposed mechanism is that the
nature of the mutations in cancer cells is not quite stochastic.
The mutations are not just caused by DNA damaging agents
in the cellular genome; the cell itself creates conditions to the
generation of proper mutations by epigenetic modification of
the genome in SSE. From this standpoint, mutations observed
in tumors are not the reason for the disease, but a far-reaching
consequence of cellular exposure to SSE. The mutations
accomplish epigenetic changes in genes imposed by this environment. This mechanism may explain the delayed effects of
toxicant exposure and the bystander effect of radiation on tumor
developments, which are inconsistent with the accepted mechanism of direct DNA damage. It was shown, for example, that
perinatal exposure of diethylstilbesterol (a drug with estrogenic
activity) changed the methylation pattern of the promoters of
several estrogen-responsive genes associated with the development of reproductive organs and resulted in a high incidence of
epithelial cancers of the uterus in mice at 18--24 months of age
(122). Long-term effects were also revealed after the short-term
exposure to arsenic. In this study, progeny of the exposed
Chinese hamster cells showed an increased level of chromosomal rearrangements that were not seen in the parents after the
acute exposure. Moreover, the mutations were related to genome DNA hypomethylation induced by arsenic after treatment
(123). In a study of ionizing radiation, non-irradiated cells
acquired mutagenesis through direct contact with cells whose
nuclei had previously been irradiated with alpha-particles
(124). It was also shown that the delayed effects of the ionizing
radiation (delayed reproductive death or lethal mutation, chromosomal instability and mutagenesis) may be transmitted over
many generations after irradiation through the progeny of
surviving cells (125). Molecular mechanisms underlying these
experimental findings are not known, but it is believed that it
may be a consequence of bystander interactions involving
intercellular signaling and production of cytokines (126), i.e.
factors in the hypothesis that induce sustained proliferative and
stress-related survival signals and epigenetically reprogram
cells. In terms of the presented hypothesis, the exposure with
carcinogens lead to accumulation of the reprogrammed cells
with tumor-related EA. But their transformation may be triggered later by cellular senescence or another SSE. The same
epigenetic mechanism may underlie the causative effect of
parental exposure with carcinogens (pesticides, smoking and
so on) on the development of childhood cancers (127).
Etiology of human cancers in terms of the proposed
hypothesis
The proposed model of tumorigenesis is based on the analysis
of principal common characteristics of different cancers.
1330
According to the model, these common characteristics arise
as a result of sustained proliferative and survival signaling in a
SSE. This signaling may be imposed by continuing production
of cytokines in a tissue subjected to continuing cellular assault,
for example as a result of the exposure with carcinogens.
A similar stress-associated signaling may be induced by
some other environmental and physiological factors including
such well known inducers of cancer as inflammation, hormones and viral infection. The effect of these factors on cancer
development also may be considered in terms of the proposed
model.
The implication of hormones in the origin and development
of many cancers is well documented. It is also known that
many hormones activate both proliferative and survival signaling in cells inducing their proliferation on the one hand and
inhibiting their apoptosis on the other (128). This signaling
recapitulates the SSE characterized by continuing cellular
assault. Although different pathways and genes may be
involved in the signal transduction, both environments will
impose similar cellular responses that include continuing proliferation of the exposed cells and silencing of cell-cycle arrest
and apoptosis. In the long term, it will be accomplished by
epigenetic reprogramming of some cells and their subsequent
malignant transformation. Recent studies support the plausibility of this mechanism in the origin of breast and prostate
cancers. The implication of estrogens in the development of
breast cancer is well established. It is also known that this
hormone activates the estrogen receptor and stimulates cellular
proliferation by activation of AP-1 transcription factors (129)
and cellular survival by activation of MAP kinase signaling
(130). The other example is the development of prostate
cancer. This cancer initially requires androgen for the growth,
and responds to the hormone deprivation therapy. This
responsiveness indicates that cellular apoptotic machinery is
functional at this stage of cancer progression. It was found that
activation of Ras-mediated stress-related survival signaling is
sufficient, and may be necessary for progression of the disease;
it may also induce the development of its androgen independent form (131). These examples indicate the involvement of
proliferative and survival signals, which are crucial components of SSE, in breast and prostate cancers development.
The effect of transforming viruses on cellular processes also
mimics many aspects of SSE. The expression of oncoproteins
encoded by some viruses changes cellular processes in the host
in the same way as continuing proliferative and survival signaling. Pathways involved in activation proliferation, cellcycle arrest and DNA repair are common targets of the viral
infection. A detailed summary of these effects for three wellstudied oncogenic viruses (human T-cell leukemia virus type 1,
hepatitis B virus and human papilloma virus) are given in the
review of Gatza et al. (132). Although viral oncoproteins do
not necessarily affect the same pathways, the overall mechanisms are highly analogous among the viruses. They activate/
inactivate critical cellular processes to promote proliferation,
suppress cell-cycle control and DNA repair. Moreover, they
directly facilitate the activation of error-prone replication
stimulating bypass of G1/S and G2/M checkpoints in the cellcycle progression.
Approximately one-quarter of all malignancies are thought
to arise in part to chronic inflammation (133). Similar to
SSE imposed by carcinogens, this cellular environment leads
to production of reactive oxygen species as an important element of cellular defense system. Therefore, inflammation,
Tumorigenesis: adaptation to SSE by EA and succeeding mutations
regardless of etiology, always causes cellular assault, DNA
damage, cell death and renewal. It creates a tissue microenvironment rich in cytokines and growth factors that induce proliferative and stress-related survival signaling. Recent studies
on the role of inflammation in cancer development are considered by Brower (134). It is believed that nuclear factor—
kappa B (a key transcription factor in infection and inflammation) can provide a link between chronic inflammation and
cancer. It was shown that in the premalignant inflammatory
environment, this transcription factor induces epithelial cell
proliferation by upregulating macrophage-produced cytokines
and also provides a survival signal suppressing cellular apoptosis. In summary, the effect of well-known inducers of
tumorigenesis associated with the etiology of many different
cancers may be described in terms of the proposed hypothesis.
Implications of the hypothesis
The proposed hypothesis may be helpful in the explanation of
some common characteristics of tumor cells associated with
their high adaptive potential. According to the hypothesis, the
increased rate of mutations and their beneficial nature are
crucial components of this potential acquired after epigenetic
reprogramming of the cells in SSE. They allow the cells to
easily overcome subsequent stresses imposed by tumor progression or medication. Metastasis and drug resistance are
examples of implementations of this adaptive potential. Epigenetically reprogrammed and then transformed cells faced
with a shortage of oxygen or nutrients, induced by overcrowding, can easily express those genes in the genome
that increase their mobility and allow them to migrate and
proliferate in new environments. In this way, the tumor cells
acquire the sequential specialized adaptations that confer
metastatic capabilities including the expression of metalloproteinases, angiogenesis, lymphangiogenesis and adhesion
molecules (135). Ability to metastasize overcomes the environmental stress imposed by overcrowding. Similar survival
strategies, which are based on increased mobility, are implemented by some unicellular organisms, like yeasts and slime
mould colonies deprived of nutrients (136). A quick development of drug resistance, which is typical for cancer cells, is
the other illustration of their high adaptability. A wellknown example is a variety of mutational effects employed
in the advanced prostate cancer in response to androgen
ablation therapy (137).
The proposed insight into tumorigenesis may improve and
extend our approaches to diagnostics, prevention and
treatment of cancer. Screening of EA in genes involved in
cell-cycle control, DNA repair and repetitive sequences in
heterochromatin may provide a generalized picture of the
exposure of cells to sustained stress and predisposition to
cancer development. It opens the prospect of early diagnosis
of cancer and its prevention, because EA are reversible and
correction of cellular environment at this stage by proper
treatment or by changing a lifestyle may be the most efficient
way to prevent cancer development. Studies of the effect of
environmental factors on EA and cancer development give
indirect medical support for this conclusion (138). It was
shown that dietary deficiencies, chemicals and genetics may
alter methylation processes and cause tumor formation. Even
environmental exposures in early life (e.g. estrogenic, xenobiotic, nutritional factors, stress) may alter DNA methylation
and in this way modulate the risk of cancer and other chronic
diseases in adulthood (139).
Although many experimental observations reviewed in the
paper support the proposed model of tumorigenesis, direct
evidence of the adaptation of mammalian cells to SSE by EA
and succeeding matched mutations postulated in the model as
the main driving force of tumorigenesis is actually absent.
Additional studies are necessary to validate and refine
the model.
Acknowledgements
We thank Dr Greenwood (Warwick University, UK), Drs Riggs, Roberts,
Rodin, Chen and other colleagues in the City of Hope for the comments on
the paper. This study was supported by a grant from the Air Force Office of
Scientific Research.
Conflict of Interest Statement: None declared.
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Received January 3, 2005; revised March 21, 2005;
accepted March 22, 2005