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Am J Physiol Gastrointest Liver Physiol 287: G7–G17, 2004;
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Theme
Inflammation and Cancer
IV. Colorectal cancer in inflammatory bowel disease: the role of inflammation
Steven H. Itzkowitz and Xianyang Yio
Gastrointestinal Division, Mount Sinai School of Medicine, New York, New York 10029
multifocal and, once found, marks the entire colon as being at
heightened risk of neoplasia, thereby warranting surgical removal of the entire colon and rectum. These differences in
morphology and biological behavior not only make clinical
cancer surveillance in IBD patients more challenging than in
the general population, but they raise the important question of
how chronic inflammation contributes to the development of
CRC. The “adenoma-carcinoma” sequence found in the sporadic setting becomes the “inflammation-dysplasia-carcinoma”
sequence in IBD. The object of this theme article is to first
review the clinical and molecular features of CRC in IBD and
then discuss how inflammation may contribute to CRC pathogenesis.
CLINICAL FEATURES OF COLITIS-ASSOCIATED CRC
(IBD), both ulcerative
colitis (UC) and Crohn’s colitis, are at increased risk of developing colorectal cancer (CRC). Indeed, IBD ranks among the top
three high-risk conditions for CRC, together with the hereditary
syndromes of familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC). Unlike the latter
two conditions that have a well-defined genetic etiology, CRC risk
in IBD appears to be related more to chronic inflammation of the
gastrointestinal mucosa than to any clear-cut genetic predisposition.
Regardless of the underlying condition, essentially all CRCs
develop from a dysplastic precursor lesion. In sporadic CRC,
the dysplastic precursor is the adenomatous polyp (adenoma),
a discrete focus of neoplasia that is typically removed by
simple endoscopic polypectomy. In contrast, dysplasia in patients with IBD can be polypoid or flat, localized, diffuse, or
Compared with sporadic colorectal carcinoma (SCC), CRC
arising in patients with IBD has several distinguishing clinical
features. Colitis-associated colorectal cancer (CAC) affects
individuals at a younger age than the general population. They
more often have a mucinous or signet ring cell histology, there
is a higher rate of two or more synchronous primary CRCs, and
in some studies, they demonstrate a more proximal distribution
in the colon. Curiously, these same features are found in CRCs
arising in individuals with HNPCC, although there has been no
clear genetic etiology to CAC. A germline hMSH2 mutation (a
gene responsible for HNPCC) was reported to be more frequent in UC patients who developed high-grade dysplasia
(HGD) and cancer than in those who did not (11), but this has
not been substantiated by other investigators (60). The fact that
a positive family history of CRC confers a twofold greater risk
of developing CRC to the patient with IBD can be viewed as
evidence for a genetic etiology, but this same degree of risk
attributed to a positive family history of CRC also applies to
the general population in whom no clear genetic etiology has
yet been elucidated. There must be other factors besides
genetic predisposition that contribute to cancer development
in IBD.
Several lines of evidence implicate chronic inflammation as
a key predisposing factor to CRC in IBD (46). First, the risk for
developing CRC increases with longer duration of colitis.
Although, for some reason, CRC is rarely encountered before
seven years of colitis; thereafter, the risk increases at a rate of
⬃0.5–1.0% per year. Second, the extent of colitis is another
important risk factor; the more colonic surface that is involved
with colitis, the greater the colon cancer risk. Paradoxically,
however, patients who have inflammation limited only to the
rectum do not have an appreciably increased risk of cancer.
Third, the risk of CRC is much greater in the small subset of
Address for reprint requests and other correspondence: S. H. Itzkowitz, GI
Division, Box 1069, Mount Sinai School of Medicine, One Gustave Levy
Place, New York, NY 10029 (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
inflammatory bowel disease; colorectal cancer; colitis; oxidative
stress; animal models
PATIENTS WITH INFLAMMATORY BOWEL DISEASE
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Itzkowitz, Steven H., and Xianyang Yio. Inflammation and Cancer. IV. Colorectal cancer in inflammatory bowel disease: the role of
inflammation. Am J Physiol Gastrointest Liver Physiol 287: G7–G17,
2004; 10.1152/ajpgi.00079.2004.—Patients with ulcerative colitis and
Crohn’s disease are at increased risk for developing colorectal cancer.
To date, no known genetic basis has been identified to explain
colorectal cancer predisposition in these inflammatory bowel diseases.
Instead, it is assumed that chronic inflammation is what causes cancer.
This is supported by the fact that colon cancer risk increases with
longer duration of colitis, greater anatomic extent of colitis, the
concomitant presence of other inflammatory manifestations such as
primary sclerosing cholangitis, and the fact that certain drugs used to
treat inflammation, such as 5-aminosalicylates and steroids, may
prevent the development of colorectal cancer. The major carcinogenic
pathways that lead to sporadic colorectal cancer, namely chromosomal instability, microsatellite instability, and hypermethylation,
also occur in colitis-associated colorectal cancers. Unlike normal
colonic mucosa, however, inflamed colonic mucosa demonstrates
abnormalities in these molecular pathways even before any histological evidence of dysplasia or cancer. Whereas the reasons for this are
unknown, oxidative stress likely plays a role. Reactive oxygen and
nitrogen species produced by inflammatory cells can interact with key
genes involved in carcinogenic pathways such as p53, DNA mismatch
repair genes, and even DNA base excision-repair genes. Other factors
such as NF-␬B and cyclooxygenases may also contribute. Administering agents that cause colitis in healthy rodents or genetically
engineered cancer-prone mice accelerates the development of colorectal cancer. Mice genetically prone to inflammatory bowel disease
also develop colorectal cancer especially in the presence of bacterial
colonization. These observations offer compelling support for the role
of inflammation in colon carcinogenesis.
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their products. This will be reviewed below, following a brief
overview of the molecular pathogenesis of CAC.
MOLECULAR PATHOGENESIS OF SPORADIC CRC
To place the molecular pathogenesis of CAC in proper
perspective, it is first helpful to appreciate the molecular events
involved in the development of SCC. SCC arises as a result of
genomic instability. The two main types of genomic instability
that contribute to colon carcinogenesis are chromosomal instability (CIN) and microsatellite instability (MSI), accounting for
85 and 15% of SCC, respectively. Chromosomal instability
results in abnormal segregation of chromosomes and abnormal
DNA content (aneuploidy). As a result, loss of chromosomal
material [loss of heterozygosity (LOH)] often occurs, contributing to the loss of function of key tumor suppressor genes
such as adenomatous polyposis coli (APC) and p53. These
genes can also be rendered nonfunctional by mutation. In either
event, it is the accumulation of molecular disturbances mainly
in tumor suppressor genes that drives the sporadic adenomacarcinoma progression, and therefore this pathway has sometimes been referred to as the “suppressor pathway.” Colon
cancers arising in patients with FAP tend to proceed via this
pathway.
Loss of APC function is typically an early event in SCC
pathogenesis, giving the APC gene the monicker of “gatekeeper” of the colon (Fig. 1). Although some have argued that
APC mutation may not be the universal initiating event but
may instead occur at somewhat later stages of adenoma progression (48), APC still contributes to the process of CIN (32).
During the progression of the adenoma, whereby increases in
adenoma size, degree of dysplasia, and degree of villous
histology take place, other changes in genetic regulation occur,
such as induction of the k-ras oncogene and loss of function of
tumor suppressor genes on chromosome 18q in the region of
the deleted in colon cancer (DCC) and deleted in pancreatic
cancer (DPC4) genes. Loss of p53 gene function occurs late
and is believed to be the defining event that drives the adenoma
to carcinoma.
Fig. 1. Molecular pathogenesis of sporadic colon cancer (top)
and colitis-associated colon cancer (bottom). COX-2, cyclooxygenase-2; CIN, chromasomal instability; MSI, microsatellite
instability; mut., mutation; LOH, loss of heterozygosity; DCC,
deleted in colon cancer; DPC, deleted in pancreatic cancer;
APC, adenomatous polyposis coli.
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IBD patients who also have primary sclerosing cholangitis, an
idiopathic condition characterized by chronic inflammation of
the bile ducts, which predisposes not only to CRC but also to
biliary tract cancer. Fourth, evidence is mounting to suggest
that anti-inflammatory medications, especially 5-aminosalicylates but possibly corticosteroids, can reduce the development
of colorectal dysplasia and cancer in IBD. This situation is
similar to healthy individuals and even those with FAP, in
whom the use of aspirin or other nonsteroidal anti-inflammatory agents has been shown to diminish the growth and subsequent development of colorectal neoplasia.
Despite all of the evidence strongly implicating chronic
inflammation as the culprit, surprisingly little research has
directly addressed the question of whether inflammation per se
correlates with CRC risk in IBD. In fact, historically, the
degree of colitis activity has been considered not to be an
independent risk factor for CRC, probably because of the way
that disease activity was defined. For example, when activity of
disease was measured according to the frequency of clinical
(symptomatic) exacerbations, there did not appear to be a
correlation with CRC risk (25, 64). However, when colitis
activity has been defined histologically, a recent case-control
study found that greater degrees of histologically active inflammation were indeed associated with increased risk of CRC
(74). New evidence also indicates that CRC can arise in areas
of microscopic colitis proximal to areas of gross colitis, suggesting that histological change, even without colonoscopic
alteration, is a better determinant of inflammation for the
purposes of cancer risk (54). It is important to realize though,
that patients with the most severe inflammation often undergo
colectomy early in their disease because they are not responding to medical therapy. As such, they are no longer at risk for
developing CRC. Ironically then, many patients who develop
CRC in IBD have clinically, as well as histologically, quiescent
inflammation. Active inflammation in colorectal mucosa is
characterized by a predominant neutrophilic infiltration with
crypt abscesses and ulceration of the epithelium. Inactive, or
quiescent, inflammation is marked by a predominance of lymphocytes. Thus better insight into the pathogenesis of CAC will
likely come from studying the role of the immune cells and
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MOLECULAR PATHOGENESIS OF COLITIS-ASSOCIATED CRC
Cancers in the setting of IBD are believed to occur by a
progression from no dysplasia to indefinite dysplasia to lowgrade dysplasia (LGD) to HGD to carcinoma (Fig. 1). It is
important to realize that in clinical practice, cancers can arise
without proceeding through each of these steps (47). Similar to
sporadic CRC, colon carcinogenesis in IBD is a consequence
of sequential episodes of somatic genetic mutation and clonal
expansion. However, unlike SCC, where dysplastic lesions
arise in one or two focal areas of the colon, in colitic mucosa,
it is not unusual for dysplasia or cancer to be multifocal,
reflecting a broader “field change.” Careful mapping studies
using DNA aneuploidy as a marker of genomic instability
indicate that individual cell populations are observed in the
same locations of the colon on repeated colonoscopic examinations and became more widely distributed over time, occupying larger areas of the mucosa (72). Moreover, within an
aneuploid area, additional subclones of aneuploidy seem to
emerge from their predecessors. Thus genomic instability of
increasingly dysregulated subclones of cells with clonal expansion of mutant cell populations occurs at the expense of the
normal surrounding epithelium. For the most part, in IBD,
neoplastic lesions arise within areas of the mucosa that have
been involved with colonic inflammation. It is not known
whether the reepithelialization of large patches of colonic
mucosa by abnormal clones is simply a consequence of the
healing response to ulceration caused by chronic inflammation
or whether the epithelial cells of IBD patients have an innate
ability to replace surrounding epithelium. Regardless, aneuploidy is often more widespread than dysplasia, and this
indicates that substantial genomic alterations can occur in
colonic mucosa without disturbing morphology.
Perhaps not surprisingly, many of the molecular alterations
responsible for SCC development also play a role in colitisassociated colon carcinogenesis. In fact, emerging evidence
suggests that in CAC, the frequency of CIN (85%) and MSI
(15%) is roughly the same as in SCC (87). Distinguishing
features of CAC, however, are differences in the timing and
frequency of these alterations (Fig. 1). For example, APC loss
of function, considered to be a very common early event in
SCC, is much less frequent and usually occurs late in the
colitis-associated dysplasia-carcinoma sequence (4, 68, 84).
Mutations in APC are rarely, if ever, encountered in colitic
mucosa that is negative or indefinite for dysplasia, and fewer
than 14% of tissues manifesting LGD or cancer harbor APC
mutations (4, 68, 86). Likewise, allelic deletion of APC occurs
in fewer than 33% of colitis-associated neoplasms (86).
Greater evidence implicates p53 as playing an instrumental
role in CAC. Allelic deletion of p53 occurs in ⬃50 – 85% of
cancers (13, 89). Indeed, p53 LOH correlates with malignant
progression, occurring in 6% of biopsies without dysplasia, 9%
with indefinite dysplasia, 33% with LGD, 63% with HGD, and
85% with cancer (13). In the setting of colitis, p53 mutations
occur early and are often detected in mucosa that is nondysplastic or only indefinite for dysplasia (10, 13). In carefully
mapped colectomy specimens, p53 mutation was an early
molecular change that occurred before aneuploidy, which, in
turn, preceded p53 LOH (Fig. 1) (10). In fact, a high
frequency of p53 mutations was found in inflamed mucosa
from UC patients who did not have cancer, indicating that
chronic inflammation predisposes to these early mutations
(see below) (43).
Methylation is assuming increasing importance as a mechanism contributing to the genetic alterations in CAC (Fig. 1).
Indeed, methylation of CpG islands in several genes seems to
precede dysplasia and is more widespread throughout the
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Tumors that arise via the CIN/tumor suppressor gene pathway are typically microsatellite stable (MSS). The remaining
15% of sporadic CRCs arise through the MSI pathway. This
pathway is preferred by the vast majority of CRCs that occur
in patients with HNPCC. The MSI pathway involves the
primary loss of function of genes that usuallly repair DNA
base-pair mismatches that occur during the normal process of
DNA replication in dividing cells. Although two of these
genes, human MutL homolog-1 (hMLH1) and human MutS
homolog-2 (hMSH2), are most commonly affected by loss of
function in CRC, several other DNA mismatch repair (MMR)
genes cooperate to repair DNA base mismatches, and the
mutation or loss of any one of them gives rise to DNA
replication errors throughout the genome. These errors preferentially affect target genes such as transforming growth factor
(TGF)␤RII, IGF2R, and BAX, which contain in their coding
regions short nucleotide repeats that are intrinsically unstable
and therefore prone to be copied incorrectly during DNA
replication. The resulting microsatellite instability renders
these genes incapable of normal colonocyte homeostasis resulting in malignant growth. This pathway of colon carcinogenesis has been referred to as the “mutator” pathway because
of the many mutations in key genes involved. Compared with
MSS sporadic colon cancers, MSI sporadic colon cancers are
more likely to be diploid (normal DNA content), located in the
proximal colon, mucinous, poorly differentiated, show lymphocytic infiltration, and associated with a more favorable
prognosis (48). MSI-positive CRCs can be further classified
into those with high (MSI-H) or low (MSI-L) degrees of MSI
depending on how many markers of a consensus panel are
found to be unstable (8).
Epigenetic alterations also contribute to altered gene expression in colon carcinogenesis. A recently recognized molecular
alteration is the CpG island methylator phenotype (CIMP)
(85). CpG islands are dense aggregates of cytosine-guanine
dinucleotide sequences that may occur in the promoter region
of genes. Extensive methylation of the cytosine bases is associated with promoter silencing and loss of gene expression.
Many genes involved in cell cycle control, cell adhesion, and
DNA repair can be methylated in colon cancer (75). So-called
type A methylation, i.e., the estrogen receptor, occurs as a
function of age and is found in both normal colon and colon
cancer. Type C methylation, however, is cancer associated,
leading to pathogenic silencing of genes such as hMLH1,
MGMT, p16, and p14.
In general, tumors manifest either the CIN or the MSI
phenotype. However, there can be overlap between the CIMP
and MSI phenotype. For example, hypermethylation of hMLH1
can produce the MSI-H cancer phenotype. By the same token,
methylation of MGMT rather than hMLH1 underlies MSI-L
cancers (48). The process of methylation is an area of intense
investigation, and it is anticipated that this line of research will
help to better define the molecular pathways involved in CRC
in a variety of clinical settings including IBD.
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mucosa of UC patients (45). In colitis-associated neoplasms,
hMLH1 hypermethylation was observed in 6 of 13 (46%)
MSI-H, 1 of 6 (16%) MSI-L, and 4 of 27 (15%) MSS
specimens, implicating this epigenetic change as a cause of
microsatellite instability (31). The cell cycle inhibitor p16INK4a,
the loss of which has been implicated in sporadic CRC, is
commonly hypermethylated in UC neoplasms (42). Approximately 10% of biopsies without dysplasia already demonstrate
p16 promoter hypermethylation, the rate increasing with higher
grades of dysplasia and reaching 100% in cancer specimens.
p14ARF is an indirect regulator of p53, and it resides at the same
locus as p16INK4␣. Loss of p14ARF function by promoter hypermethylation has been reported in 50% of adenocarcinomas,
33% of dysplastic lesions, and even in 60% of mucosal samples without dysplasia from patients with UC (76).
Thus the three main molecular pathways of colon carcinogenesis (the APC/tumor suppressor gene/CIN pathway, the
MSI pathway, and the CIMP pathway) can all be induced early
in the process of CAC, in colitic mucosa before dysplasia
develops (Fig. 1). This strongly suggests that inflammation
plays a causative role. In addition to the above-mentioned
tumor-associated genes that are progressively expressed in the
process of CAC carcinogenesis, in UC patients, several inflammation-associated genes such as cyclooxygenase-2 (COX-2)
(1), nitric oxide (NO) synthase-2 (NOS)-2 (43), and the interferon-inducible gene 1– 8U (38) are also increased in inflamed
mucosa and remain elevated in colonic neoplasms. The central
question is how inflammation results in neoplastic transformation and progression.
For one thing, the colonic mucosa of patients with IBD
demonstrates enhanced epithelial cell turnover. Compared with
normal colonic biopsies taken from patients with sporadic
adenomas, mucosal biopsies from patients with UC demonstrate higher rates of mitosis as well as apoptosis, especially in
areas of active, as opposed to quiescent, inflammation (3).
However, whereas increased epithelial cell turnover likely
contributes to carcinogenesis, it is insufficient to cause cancer.
Rather, in the setting of heightened epithelial cell turnover,
mutagenic assault and sustained DNA damage caused by
factors within an inflammatory cell-rich microenvironment
appear to drive the carcinogenic process. As such, tumors
behave similar to wounds that fail to heal (20).
A leading theory is that the oxidative stress that accompanies
chronic inflammation contributes to neoplastic transformation.
Indeed, IBD is considered one of the main “oxyradical overload” diseases whereby chronic inflammation, be it inherited or
acquired, results in a cancer-prone phenotype (44). Oxidative
stress, with its associated cellular damage, is thought to play a
key role in the pathogenesis of the colitis itself as well as in
colon carcinogenesis (Fig. 2). Colitis is triggered in a genetically susceptible individual by an environmental insult such as
gastrointestinal infection, NSAID use, or other environmental
toxins. The inflammatory cells that contribute to the colitis
generate reactive oxygen and nitrogen species (RONS). Neutrophils and macrophages, which are important in the acute
inflammatory process, generate free radicals and other prooxidant molecules. Inflamed tissues from patients with active UC
Fig. 2. Proposed model of how inflammation associated with colitis promotes
the development of colonic dysplasia and cancer.
or Crohn’s colitis demonstrate increased expression of NOS
and other RONS (40, 43, 50, 66). With the use of GAPDH
enzyme as a molecular marker, McKenzie et al. (55) showed
that oxidation of thiols in the active site of GAPDH, with
subsequent inhibition of enzyme activity, occurred in colonic
epithelial cells from inflamed mucosa of Crohn’s disease and
UC but not from paired samples of unaffected mucosa. Measurements of 8-hydroxydeoxyguanosine (8-OHdG), a mutagen
formed by the action of hydroxyl radical at the C8 position of
deoxyguanosine DNA base, in mucosal biopsies of patients
with UC were reported to be increased in patients with UC
compared with normal controls, with levels even higher in UC
patients who had dysplasia (23). Interestingly, 8-OHdG levels
were increased with longer duration of UC and were lowest in
the rectum, suggesting that the meselamine enemas that most
of these patients were using might have had an antioxidant
effect.
Direct support that RONS participate in colonic inflammation comes from interventional studies in animals. Intrarectal
administration of peroxynitrite causes colonic inflammation in
rats (67). The use of oxygen radical scavengers such as superoxide dismutase, catalase, and NOS inhibitors attenuate colonic inflammation in animal models of chemical-induced
colonic injury (78). Likewise, inducible NOS knockout mice
(iNOS⫺/⫺) manifest an attenuated colitis in response to injury
(41), and in the APCMin/⫹ mouse, which is genetically susceptible to multiple intestinal adenomas, crossing them with
iNOS⫺/⫺ mice or treating them with an iNOS inhibitor significantly reduced the prevalence of adenomas (2). Curiously,
however, treating APCMin/⫹ mice with 5-ASA did not appreciably reduce adenoma size or number, although treatment
with other NSAIDs (piroxicam, sulindac) was effective (70).
Although the APCMin/⫹ mouse is not a model of colon cancers
that arises in the setting of inflammation, these data still offer
insights into mechanisms of colon carcinogenesis. The role
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THE ROLE OF CHRONIC INFLAMMATION IN
COLON CARCINOGENESIS
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It is not yet clear whether chronic inflammation contributes to
this molecular pathway or whether some other factor(s) predisposes. For example, using fluorescent in situ hybridization
with probes specific for chromosomes 8, 11, 17, and 18,
Rabinovitch et al. (65) reported that patients with UC who had
a neoplasm (HGD or cancer) in the colon (so-called progressors) demonstrated CIN both in the neoplastic lesions themselves as well as in nondysplastic rectal mucosa remote from
the neoplastic areas. Although normal mucosa from control
subjects without UC did not display CIN, inflamed but nondysplastic mucosa of UC patients who did not harbor a neoplasm in their colon (nonprogressors) also did not exhibit CIN.
This observation was reinforced by studies in which DNA
fingerprinting demonstrated substantial genomic instability in
both the dysplastic as well as nondysplastic mucosa of UC
patients harboring a neoplasm (16). These findings suggest that
widespread genomic instability occurs in patients with UC who
develop colonic neoplasia but not in patients with UC who
have not yet developed neoplasia despite comparable disease
duration and the presence of inflammation. Thus this type of
genomic instability may be a marker of cancer risk in UC
perhaps apart from the inflammatory process. It has been
observed that a possible mechanism to explain the chromosomal instability associated with UC is telomere shortening
(63). Given the importance of the CIN pathway in CAC, it will
be important to more directly study the role of inflammation in
this process.
Oxidants can also alter DNA methylation patterns (78). It is
not yet known whether the hypermethylation of genes involved
in CAC (45) are affected by oxidant injury.
In addition to damaging DNA and other macromolecules,
oxyradicals can also induce key genes involved in the inflammatory and carcinogenic process. For example, the transcription factor NF-␬B can stimulate iNOS to generate NO and
COX-2 to generate prostanoids that have proinflammatory and
carcinogenic effects (88). Activated NF-kB is found in inflamed mucosal biopsies of patients with IBD (71). Among the
factors that can regulate NF-kB activity, TNF-␣ induces NFkB, whereas peroxisome proliferator-activated receptor-␥
(PPAR␥) attenuates NF-kB. At the present time, it is not clear
whether TNF-␣ itself plays a role in carcinogenesis nor is it
known whether inhibiting TNF-␣, which is so effective in
reducing the inflammation of Crohn’s disease, can abrogate the
carcinogenic process. PPAR␥ ligands have been shown to
inhibit intestinal inflammation (83), and impaired expression of
PPAR␥ has been described in colonic epithelial cells of patients with UC (24). Treatment of APC deficient mice with
PPAR␥ ligands has yielded mixed results, with both increases
and decreases in adenoma growth reported (12, 58). The role of
PPAR␥ in colitis-associated cancer has not been investigated.
COX-2 activity plays an important role in sporadic carcinogenesis where it has been shown that normal colonic mucosa
does not express COX-2, but with the adenoma-carcinoma
sequence, this enzyme is induced. Among the many effects of
cyclooxygenases, they can activate procarcinogens, indirectly
produce free radicals, and promote angiogenesis (81). In animal models, COX-2 inhibition can dramatically reduce the
development of colon cancers, just as in humans, use of COX-2
inhibitors and other NSAIDs is protective against colon cancer
(34). In UC, COX-2 expression is somewhat enhanced in
inflamed mucosa, but it is further induced in dysplastic and
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played by the NO/iNOS system in both colonic inflammation
and tumor development is quite complex and does not permit
simple conclusions or extrapolation to the issue of carcinogenesis in UC at the present time (6, 22). The fact that mice
deficient in glutathione peroxidase enzymes develop inflammation and cancer of the small and large intestine further
supports the notion that antioxidant pathways are important for
preventing the inflammation-neoplasia sequence (17).
How then might oxidative stress contribute to colorectal
carcinogenesis? Free radicals have the potential to affect a
large array of metabolic processes, because their targets include DNA, RNA, proteins, and lipids (44, 53). If key genes or
proteins responsible for colonocyte homeostasis are targeted,
dysplasia and subsequent carcinoma arise. The p53 tumor
suppressor gene is one important target. Hussain et al. (43)
examined the mutation spectrum of p53 at codons 247 and 248
in biopsies taken from inflamed colonic mucosa of UC patients
compared with normal, non-UC controls. They noted that more
than half of the UC cases exhibited a higher frequency of
G-to-A transitions at the CpG site of codon 248 and C-to-T
transitions at the third base of codon 247 compared with
controls. Moreover, in paired biopsies from UC patients, this
pattern of mutation was only seen in inflamed rather than
noninflamed mucosa. Increased NOS-2 activity was associated
with these p53 mutations, suggesting that oxidative stress
was playing a role. These investigators also observed that
increases in posttranslational modifications of p53 were
associated with increased iNOS activity in inflamed tissues
from UC patients, further implicating activated p53 in
response to oxidant injury (40).
More direct evidence for the role of RONS in colon carcinogenesis comes from the work of Gasche et al. (33), who used
model colon cancer cell lines to observe that hydrogen peroxide (H2O2) produced frameshift mutations in a reporter gene. In
their system, cells that were genetically deficient in DNA
MMR activity were particularly susceptible to frameshift mutations, but even at higher concentrations of H2O2, cells with
normal MMR activity displayed frameshift mutations. In subsequent studies, these investigators found that H2O2 inactivated
the DNA MMR system, apparently by damaging the protein
complexes responsible for DNA base mismatch repair (15).
Conceivably therefore, even in the absence of mutations of the
relevant genes, oxidative stress may put enough “pressure” on
the DNA MMR system to create microsatellite instability, and
this presumably contributes to the MSI phenotype seen both in
nonneoplastic and in neoplastic mucosa of UC patients. A
recent discovery suggests that increased activity of enzymes
responsible for the process of base excision-repair might also
contribute to MSI in UC tissues (39). MSI can be detected in
chronically inflamed mucosa from UC patients, even in those
with rather short disease duration before the risk of neoplasia
ostensibly rises (9). This lends credence to the concept that
inflammation alone can cause MSI. Curiously, however, MSI
is not found in normal colonic mucosa from healthy controls or
from patients with other types of benign inflammatory colitis
including Crohn’s colitis (9, 59). It is tempting to speculate that
certain RONS may cause MSI and that the spectrum of RONS
differs in the local tissue microenvironment depending on the
inflammatory background of the underlying disease.
As mentioned above, chromosomal instability represents the
major pathway by which cancers seem to arise in IBD patients.
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cancerous lesions (1). Whether COX-2 inhibition would be
chemopreventive against CRC in the setting of UC is not
known. Concern has been raised by a study in the IL-10deficient mouse model of chronic colitis, where COX-2 inhibitors paradoxically enhanced the frequency of colonic dysplasia (see below) (35).
among mice receiving only one or two cycles of DSS was 22
and 40%, respectively. Likewise, DSS induced a very high
frequency of dysplasia that arose in areas of healed mucosa as
well as those of acute and chronic inflammation. Animals with
higher inflammation scores had significantly more dysplastic
lesions. Thus colitis markedly accelerates the development of
neoplasia in APC mutant mice.
LESSONS FROM ANIMAL MODELS
DSS COLITIS IN HEALTHY RODENTS
Dysplasia and cancer can be induced in healthy mice or rats
when colitis is induced by repeated cycles (19, 52, 61) or rarely
after a single cycle (19) of DSS. In this model, longer disease
duration (3– 6 mo) was associated with an increased rate of
neoplasia, even in mice given the identical initial colitis insult
and in the setting of clinical remission (19). The localization of
dysplasia and/or cancer is ⬃20, 44, and 36% for the proximal,
middle, and distal colon, respectively (19). Animals that developed neoplasia demonstrated more severe degrees of inflammation, especially in the distal colon (19, 61). The histology of dysplastic and cancerous lesions resembles that of IBD
neoplasms, but in contrast to the human situation, none of the
cancers and only 7% of dysplasias induced by DSS manifested
nuclear p53 immunostaining (19). Parenthetically, in the DSS
model, treatment with the antioxidant N-acetylcysteine reduced
both the inflammation and tumor incidence (77). In general,
these observations support the concept that prolonged and
repetitive inflammation promotes colon cancer.
DSS COLITIS IN CANCER-PRONE MICE
The induction of chronic inflammation by DSS results in
more frequent development of cancer and dysplasia in mice
prone to MSI (51). For example, 28/30 MSH2⫺/⫺ mice developed dysplasia or cancer in response to DSS, and the frequency
of HGD was 47, 23, and 8% for MSH2⫺/⫺, MSH2⫹/⫺, and
MSH2⫹/⫹, respectively. Perhaps not surprisingly, 89% of colonic dysplasias or cancers from MSH2⫺/⫺ mice were MSI
positive, whereas the majority of neoplasms arising in DSStreated heterozygotes or wild-type mice developed independently of the mismatch repair system, because very little MSI
was found in these groups. The inflamed and uninflamed
colonic mucosa remote from dysplastic lesions in the
MSH2⫺/⫺ mice also demonstrated MSI, implying that, similar
to humans, MSI is induced by colonic inflammation and can
precede dysplasia.
The APCMin/⫹ mouse has also been studied in the context of
DSS-induced colitis (18). No cancers arose in APCMin/⫹ mice
that did not receive DSS. In contrast, the frequency of cancers
CANCER AND DYSPLASIA IN IBD-PRONE MICE
Of greater relevance to human CAC, the genetically engineered immunodeficient mice have provided new insights into
aspects of the inflammatory response that may be responsible
for instigating neoplastic progression (Table 1).
IL-2-Deficient, IL-2, and ␤2-Microglobulin Double
Knockout Mice
IL-2 is an essential regulatory cytokine of the immune
system. Among IL-2-deficient mice, 50% die within 9 wk with
splenomegaly, lymphadenopathy, and severe autoimmune hemolytic anemia (37). The rest develop chronic colitis resembling UC and a systemic wasting disease resulting in death
within 6 mo. Within the limited lifespan, dysplasia (37) but not
cancers (80) has been observed. The IL-2null and ␤2-Mnull
double knockout mouse develops pancolitis similar to IL-2
knockout mice, but these mice have milder overall disease and
are able to survive ⬎6 mo. This offers an opportunity to study
colon carcinogenesis. With prolonged observation (6 –12 mo),
32% of these mice develop adenocarcinoma in the proximal
colon. In addition, LGD and HGD are also noted in these
animals (80). Molecular characterization of these cancers revealed that all of the cancers harbored APC gene mutations,
more than one-half had p53 gene mutations, and most exhibited MSI (80).
IL-10 KNOCKOUT MICE
When the IL-10 gene is disrupted, mice develop spontaneous generalized enterocolitis under conventional conditions.
Specific pathogen-free (SPF) housing conditions reduce the
severity and limit the disease to the proximal colon (7). The
development of colitis in these mice is dependent on IL-12 and
requires the presence of enteric bacteria (57). Under SPF
conditions, 100% of the mice develop colitis after 3 mo of age.
After 3 and 6 mo, 25 and 60% of the mice, respectively,
develop adenocarcinoma. Cancers arise mainly in the proximal
colon, and they often express COX-2 in regions containing
inflammatory cells and myofibroblasts (79). Ironically, these
cancers do not seem to manifest alterations of APC, p53, K-ras,
or Msh2 genes, and although circulating TGF-␤ levels are
elevated, there does not appear to be any downregulation or
truncation of the TGF-␤ receptor type II (82). Thus the molecular mechanism of these tumors remains elusive. IL-10
treatment was shown to ameliorate the colitis in all animals and
decreased the cancer occurrence by half even after the colitis
had been established (7). Likewise, in a small study (62),
probiotics also reduced the prevalence of colon cancer and
mucosal inflammatory activity. On the other hand, pure cultures of Enterococcus faecalis induced both IBD and, after 20
wk, dysplasia and cancer of the rectum in IL-10⫺/⫺ mice (5).
Paradoxically, COX-2 inhibitors have been shown to exacerbate, rather than ameliorate, the severity of colitis and the
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To help better understand the role of inflammation in vivo,
investigators have turned to animal models, and indeed, the
existing data suggest that inflammation predisposes to colorectal cancer. Three main approaches have been used: 1) inducing
inflammation with injurious agents such as dextran sulfate
sodium (DSS) in otherwise healthy rodents and monitoring for
the development of neoplasia; 2) inducing inflammation in
mice with a genetic predisposition to colon cancer; and 3)
studying mice with a genetic predisposition to inflammatory
bowel disease for the development of neoplasia. Of the three
approaches, the latter is the most physiologically relevant to
human disease.
Theme
G13
ROLE OF INFLAMMATION IN COLITIS-ASSOCIATED COLORECTAL CANCER
Table 1. Summary of colorectal neoplasia in genetically engineered IBD animal models
Knockout Mouse (Ref)
Background Strain
Inflammation
Tumor
Location
Tumor Histology
C57BL/6 ⫻ 129
UC-like
Carcinoma (32%)
IL-10 KO (7)
C57BL/6 ⫻ 129
Colitis with some
features of Crohn’s,
duodenitis
Carcinoma (60%)
Rag2 KO (28,29)
129/SvEv
Induced by infecting with
Helicobacter hepaticus
dysplasia, tubular
adenoma, carcinoma
in situ,
adenocarcinoma
(100%)
Cecum
Colon
Rag2/Tgf␤1 DKO (27)
129S6 ⫻ CF1
Colitis
Cecum
Colon
TCR␤/p53 DKO (49)
C57BL/6jjcl
UC-like
Gpx1/Gpx2 DKO (17)
Mixed C57BL/6J
and 129Sv/J or
129S3
Ileocolitis
N-cadherin dominant
negative (36)
129/Sv3B6
Crohn’s-like
Dysplasia,
adenocarcinoma
(100%)
Dysplasia,
adenocarcinoma
(70%)
Dysplasia,
adenocarcinoma
(28%); signet ring
cell carcinoma
Adenomas, not cancer
G␣i2 KO (73)
129/Sv
UC-like
Carcinoma (31%)
Rectum
Colon
Colon
Rectum
Ileocecum
Cecum
Ileum
Colon
Small
bowel
Colon
Spontaneous development of
colitis and carcinoma
Colitis induced by normal
flora; colitis and cancer
caused by Enterococcus
faecalis
1. Both colitis and neoplasm
are induced by H.
hepaticus.
2. CD4⫹ CD45RBlo (IL-10)
prevents colitis and
cancer.
Require H. hepaticus to
induce inflammation and
neoplasms
Normal flora is required to
produce inflammation and
tumors
Require bacterial flora,
including Helicobacter
species
Chimeric mouse; adenomas
form in inflamed and noninflamed areas.
Colitis and cancer occur
even in SPF barrier
facility.
KO, knockout; DKO, double knockout; IBD, inflammatory bowel disease; UC, ulcerative colitis; GPx, glutathione peroxidase; TCR, T cell receptor; SPF,
specific-pathogen free.
frequency of dysplasia in IL-10⫺/⫺ mice (35). The effect of
IL-10 in IBD has also been studied in other colitis models (57).
In SCID mice, transfer of CD4⫹CD45RBhi T cells from normal donors into CB-17 SCID mice resulted in severe colitis.
IL-10 has been shown to prevent colitis in this setting. Oral
administration of Lactococcus lactis-secreting IL-10 reduced
DSS-induced colitis. Taken together, it appears that IL-10
plays an inhibitory/regulatory role in these animal models and
that lacking IL-10 resulted in colitis. The chronic inflammation
further contributed to the formation of adenocarcinoma.
RAG2-DEFICIENT MICE
Rag2-deficient mice lack functional T and B lymphocytes
because of an inability to properly rearrange antigen receptors.
This mouse has been a valuable model to study the function of
various subsets of lymphocytes by transferring specific lymphocytes from immunocompetent mice. Colitis can be induced
in this mouse by adoptive transfer of CD4⫹CD45RBhi T cells.
This colitis is preventable by cotransfer of CD4⫹CD25⫹ or
CD4⫹CD45RBlo regulatory T cells. The latter subset of T cells
is believed to harbor regulatory cells that inhibit intestinal
inflammation. In this animal, significant colitis may also be
induced by infection with a widespread enteric mouse bacterial
pathogen, Helicobacter hepaticus, in the absence of T cells
(28). Interestingly, after the infection, these mice not only
contract colitis, but also inevitably develop colonic tumors,
including sessile tubular adenoma, dysplasia, carcinoma in
situ, and adenocarcinoma (100%) (28). However, they do not
produce pedunculated or villous adenomas. In contrast, if they
are maintained in a H. hepaticus-free environment, these ani-
mals do not have colon tumors or colitis. Adoptive transfer of
CD4⫹CD45RBlo regulatory T cells to these mice significantly
suppressed colitis. After the regulatory T cell transfer, the
majority of animals had minimal or no colitis and did not
develop neoplasia; neoplasia was found in those animals that
developed moderate to severe local colitis. It is believed that
IL-10 produced by these regulatory T cell is pivotal in inhibiting inflammation and interrupting carcinogenesis, because
transferring IL-10-deficient CD4⫹CD45RBlo regulatory cells
did not protect these animals from colitis and carcinoma (29).
This model provides further evidence supporting IL-10 as a
protective agent against colitis and that there is a link between
chronic colitis and the formation of colonic neoplasms. Alternatively, IL-10 may be able to inhibit or arrest carcinogenesis
via a T cell-independent mechanism, because administration of
IL-10 has been shown to suppress cancers in SCID mice (57).
TGF-␤1 AND RAG2 DOUBLE KNOCKOUT MICE
Tgfb1⫺/⫺ mice develop an autoimmune disease with inflammatory lesions in multiple organs. Crossing these mice with
Rag2 knockout mice rescues them from the autoimmune phenotype and permits them to live as long as 8 mo. They develop
hyperplasia of the cecum and colon, with a primarily granulocytic inflammation of the submucosa. This model shares similarity with Rag2-deficient mice, including the location of
colitis, the universal development of both dysplasia and adenocarcinoma, and the requirement of H. hepaticus for the
induction of colitis and colon tumors (26 –29). The most
significant difference, perhaps, is the genetic background of
these two models. In this model, colitis is required but is not
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IL-2/␤2M DKO (80)
Comments
Theme
G14
ROLE OF INFLAMMATION IN COLITIS-ASSOCIATED COLORECTAL CANCER
sufficient for cancer formation; a genetic predisposition to
cancer appears to contribute. Whether the enteric bacteria
directly affect carcinogenesis or induce inflammation that results in neoplastic events is unclear. The cancers in this model
do not demonstrate MSI.
T CELL RECEPTOR-␤ KNOCKOUT MICE
GLUTATHIONE PEROXIDASE-1/-2 DOUBLE KNOCKOUT MICE
Glutathione peroxidases (GPX) are a family of intracellular
antioxidant enzymes that reduce H2O2 and organic hydroperoxides by oxidizing glutathione. Mice with targeted disruption
of both Gpx1 and Gpx2 develop ileocolitis between 2 and 7 wk
of age. After 4 mo of age, ⬃40% of animals develop tumors
(28% adenocarcinomas) that are often nonpolypoid and primarily located in the distal ileum (17). Higher tumor rates were
correlated with higher inflammation scores in the ileum but not
the colon, suggesting that inflammation is necessary but not
sufficient to cause tumors. Tumor incidence was highest in
colonies that were raised in non-SPF conditions. Essentially no
tumors developed in germ-free or SPF colonies nor were there
any tumors noted in animals that had at least one wild-type
Gpx1 or Gpx2 allele. This model is instructive, because these
mice have otherwise intact immune systems and mucosal
barrier function, and it highlights the importance of the antioxidant system in protecting against inflammation and neoplasia in the setting of bacterial infection.
N-CADHERIN DOMINANT-NEGATIVE MICE
These mice are immunologically intact but have a defect in
their intestinal mucosal barrier. Cadherins are essential for
mediating cell adhesion and normal development. Expression
of a dominant-negative form of N-cadherin lacking extracellular domain (NCAD⌬) resulted in loss of endogenous Ecadherin from the cell surface. Because complete disruption of
cadherin production results in embryonic lethality in mice, a
chimeric mouse model was created that expressed dominantnegative N-cadherin in the small intestine (36). This was
generated by introducing genetically manipulated embryonic
stem cells from 129/Sv strain (NCAD⌬) into normal B6
blastocytes. The resultant 129/Sv3 B6 chimeric mouse intestine contained patches of 129/Sv (NCAD⌬)-derived cryptvillus units and patches of crypt-villus units of B6 background
that were easily distinguishable from 129/Sv by their lectinbinding properties. All chimeric mice developed IBD similar to
Crohn’s disease, presumably because disruption of the epithelial barrier results in inflammation. Inflammation is present
only in 129/Sv (NCAD⌬) patches but not in B6 epithelium,
and consequently, foci of dysplasia and adenoma were only
found in 129/Sv (NCAD⌬) areas. No adenocarcinomas were
observed during the 19 mo of the experiment. This model
supports the association between inflammation and tumorogen-
G␣i2ⴚ/ⴚ MICE
Mice deficient in the G protein subunit ␣i2 develop profound
alterations in thymocyte maturation and function (73). From 13
wk of age onward, these mice develop severely active colitis,
especially in the distal colon, and the intensity and extent of
colitis progress with age. By 15–36 wk of age, 31% of mice
developed cancer that was not polypoid or metastatic. Large
pools of mucin were found in some cancers.
FUTURE DIRECTIONS
Chemoprevention of CRC is an area of great interest and
intense investigation. Just as aspirin and NSAIDs appear to
prevent CRC in the general population, evidence is accumulating that 5-aminosalicylic acid (5-ASA) compounds may
prevent colorectal neoplasia in patients with UC (21). Whereas
the mechanism of this is not known, it has been shown
experimentally that 5-ASA is able to prevent damage to
GAPDH caused by exposure to hypochlorite, the most potent
oxidant produced by neutrophils (56). This ability of 5-ASA to
scavenge RONS was not shared by other medications used to
treat IBD such as methylprednisolone, 6-mercaptopurine, or
metronidazaole (56). Rectal administration of 5-ASA in patients without IBD has been shown to induce apoptosis selectively in tumor cells but not in nearby normal epithelial cells
(14). The chemopreventive role of 5-ASA in IBD deserves
further study in humans and in animal models. In addition,
given the importance of prooxidants in causing molecular
damage to colonic epithelium, further investigating the potential role of antioxidants as chemopreventive agents is also
worthwhile. Moreover, more work is needed to identify the
molecular underpinnings of how inflammation might contribute to chromosomal instability and abnormal methylation of
genes. Another area of interest is the role of bacteria in causing
cancer. Although an infectious origin to human CRC is not
usually considered, recent data suggest that polyoma virus
infection can cause CIN in human colonic epithelial cells (69).
Whether bacteria can also cause CRC in humans, analogous to
H. pylori causing gastric cancer, is not known. Because several
of the animal models of CRC in IBD implicate bacteria in the
pathogenesis of both the inflammation and cancer, insights
from these studies, in addition to asking why some colitis
models do not get CRC, may provide further food for thought.
Finally, to date, researchers in the field of CAC have typically
drawn insights from the work performed on SCC. Curiously, a
recent study (30) showing elevated C-reactive protein levels in
individuals who were prone to sporadic CRC suggests that
inflammation may play more of a role in SCC than previously
appreciated. So, just as understanding the uncommon conditions FAP and HNPCC have taught us about the pathogenesis
of SCC, lessons learned from IBD as a model of colonic
inflammation might also help us further our knowledge about
how inflammation might cause CRC in general.
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