Gastroenterology
Volume 132, Issue 2 , Pages 797-801, February 2007

Sporadic Colorectal Cancer: An Infectious Disease?

  • Frank A. Sinicrope

      Affiliations

    • Corresponding Author InformationAddress requests for reprints to: Frank A. Sinicrope, MD, Divisions of Gastroenterology and Hepatology, and Oncology, Mayo Clinic and Mayo College of Medicine, 200 First Street, SW, Rochester, Minnesota 55905. fax: (507) 284-9111.

Divisions of Gastroenterology and Hepatology, and Oncology, Mayo Clinic and Mayo College of Medicine, Rochester, Minnesota

published online 03 February 2007.

Article Outline

 

See “Extracellular superoxide production by Enterococcus faecalis promotes chromosomal instability in mammalian cells” by Wang X and Huycke MM on page 551.

The link between noninfectious chronic inflammation due to inflammatory bowel disease (IBD), and colon carcinogenesis in humans is well established.1 Studies indicate that colon cancer risk increases with the duration, extent, and histologic severity of colitis and inflammation.1 Although a bacterial etiology of colorectal carcinogenesis has been postulated but never established in humans, the microbial flora of the colon are thought to produce a state of continual low-grade inflammation that may contribute to colon carcinogenesis. In this regard, studies in germ-free mice have shown that enteric bacteria are required for colon cancer in some model systems.2, 3 Infection with Helicobacter hepaticus, a murine enteric pathogen, led to IBD with associated colon cancer in immune-dysregulated mice.3, 4 Another enteric pathogen in mice, Citrobacter rodentium, increased epithelial cell proliferation and enhanced intestinal polyp formation in both APCMin5 and carcinogen-treated mice.5 Furthermore, the enteric bacterial flora have been implicated in the pathogenesis of enterocolitis and colon cancer in the C57BL/6 interleukin-10 knockout (IL-10 KO) mouse model. Mice lacking the inflammatory cytokine IL-10 are predisposed to developing enterocolitis in the presence of enteric bacterial flora in contrast to wild-type mice, but remain disease free when maintained under germ-free conditions.6 In this same model, the intestinal commensal bacterium Enterococcus faecalis was shown to produce severe distal colitis7 and was associated with development of rectal dysplasia and adenocarcinoma.8 The importance of host immunity in the IL-10 KO model was shown in a recent study where transfer of regulatory lymphocytes expressing CD4 and CD25 prevents the innate inflammatory events that lead to colon cancer in these mice.9

Although the mechanisms underlying infection-related colonic tumorigenesis remain largely unknown, inflammation-related oxidative stress and DNA damage are believed to be important contributors to colorectal neoplastic development in humans.10 Studies have shown increased DNA damage in colorectal epithelia from ulcerative colitis patients, especially in cases with prolonged disease duration or dysplasia.11 A role for oxidative stress in intestinal tumorigenesis is supported by studies in mice with targeted disruption of glutathione peroxidase isoenzymes that reduce hydroperoxides in intestinal epithelium. In these mice, colonization with commensal microflora led to development of ileocolitis and ileal or colonic tumors in some compared with an absence of pathology in germ-free mice.12 One hypothesis proposes that oxidative stress from free radicals produced by intraluminal bacteria causes chromosomal instability (CIN) and subsequent colorectal carcinogenesis.13 This hypothesis was based initially on ex vivo observations of hydroxyl radical production by human stool specimens.13, 14 A candidate bacterium is E faecalis that has been shown produces extracellular superoxide (O2−) and derivative reactive oxygen species (ROS) in the colonic contents of rats colonized with this organism,15 suggesting that E faecalis is a source of oxidative stress in colonic epithelium. Furthermore, prior studies from the laboratory of Huycke15 have shown that E faecalis-derived ROS, including H2O2 and hydroxyl radical, can damage colonic epithelial cell DNA both in vitro and in vivo.16 These results suggest that ROS generation near the oxygenated luminal surface of colonocytes is a potential source of DNA damage leading to genomic instability. In this month’s Gastroenterology, Wang and Huycke17 show evidence to support this hypothesis in mammalian cells.

A role for genomic instability in cancer development was postulated nearly a century ago with the observation that cancer cells have an abnormal chromosome number and are, thus, aneuploid. This postulate was proven to be correct and in the case of human colorectal cancers (CRCs), at least 80% show CIN characterized by loss or gains of whole chromosomes or large parts of chromosomes during cell division.18 CIN causes aneuploidy and an increased rate of loss of heterozygosity, an important mechanism of inactivating tumor-suppressor genes.18 An unresolved question is whether CIN is an early event and thus a driving force of colorectal tumorigenesis. Furthermore, the mechanisms that induce and influence CIN are only partially understood. Microsatellite instability (MIN) is another distinct form of genomic instability that is caused by defective DNA mismatch repair (MMR).19 MIN tumor cells show an accelerated accumulation of mutations in repetitive nucleotide sequences located throughout the genome.18 MSI occurs in approximately 15% of CRCs and results from either germline MMR mutations giving rise to HNPCC, or hypermethylation of the hMLH1 promoter in sporadics.20, 21 MIN tumors have normal or nearly normal chromosome numbers.

In this issue of Gastroenterology, Wang and Huycke17 demonstrate that the E faecalis can promote CIN in mammalian cells by mechanisms that require ROS and involve cyclooxygenase-2 (COX-2) induction and activity. The authors measured the ability of E faecalis to promote CIN using human–hamster hybrid cells (ALN) that contain 1 copy of chromosome 11. Pretreatment of ALN cells with E faecalis produced increased mutant fractions compared with controls, with the number of mutant clones equivalent to that produced by treatment of the cells with 2 Gy γ irradiation. Deletions in chromosome 11 were found in 80% of ALN cells with mutant clones indicating CIN. As acknowledged by the authors, ALN cells are transformed cells that display CIN, so it remains unknown whether E faecalis can induce CIN in chromosomally stable cells. Protection from CIN was afforded by treatment with antioxidants and a selective COX-2 inhibitor. An increase in mutant fractions in ALN cells was also promoted by exogenous ω6 polyunsaturated fatty acids, but was inhibited by antioxidants. Polyunsaturated fats are known to react rapidly with oxygen (lipid peroxidation) to generate highly reactive intermediates that can induce single- and double-strand breaks in DNA leading to genetic instability.13, 14 Of note, antioxidants serve as chain terminators of lipid peroxidation. Wang and Huycke17 found that macrophages pretreated with E faecalis showed increased mutant fractions in contrast with cells treated with Escherichia coli that produce negligible superoxide and did not promote CIN. Furthermore, the authors found that COX-2 was up-regulated in macrophages by E faecalis-derived superoxide and, moreover, made the novel observation that mutant fractions in ALN cells were decreased when COX-2 was silenced using siRNA. Based on these results, the authors concluded that macrophage COX-2 induction by superoxide from E faecalis can promote CIN in mammalian cells. Additionally, these observations were interpreted to indicate that a COX-2–related mechanism can link the oxidative physiology of E faecalis to the propagation of genomic instability through a bystander effect.

Overall, the manuscript by Wang and Huycke17 provides important mechanistic insights into bacteria-mediated colon carcinogenesis, and suggests that infection-induced CIN in vitro can presumably be an initiating event in tumorigenesis. The authors correctly point out that other factors, commensal bacteria, and inflammatory mediators may also induce COX-2 in addition to ROS.22 Furthermore and, although provocative, the data presented do not elucidate the specific mechanism(s) underlying the effect of COX-2 on mutagenesis. Moreover, no in vivo data are provided and such studies are needed to confirm the study findings in a relevant in vivo model. These limitations notwithstanding, the data do indeed support a potential role for E faecalis in the etiology of sporadic CRC. Importantly, Wang and Huycke17 found that the COX-2 inhibitor, celecoxib, can attenuate E faecalis-induced mutant clones, suggesting an additional mechanism by which COX-2 inhibition may prevent colorectal tumorigenesis. Protection from ROS-induced mutagenesis by COX-2 inhibition is consistent with other studies where the selective COX-2 inhibitor, nimesulide, was shown to block mutagenic oxidative damage in the colonic mucosal DNA of rats induced by dextran sodium sulphate (DSS).23 Furthermore, nimesulide also suppressed the increase in superoxide induced by nongenotoxic DSS in the colonic mucosa.

COX-2 is an inducible intracellular enzyme that catalyzes the conversion of arachidonic acid into prostaglandins (PGs) and related eicosanoids.22 COX-2 is up-regulated at sites of inflammation and is frequently overexpressed in colorectal adenomas and carcinomas compared with its absence in normal mucosa.24 In colorectal neoplasms, COX-2 is expressed predominantly by epithelial cells but is also up-regulated in macrophages and myofibroblasts.24, 25 Tumor-associated macrophages express COX-2 when activated, and may play an important role in the stromal–epithelial interaction during carcinogenesis. Localization of COX-2 to macrophages implies a paracrine effect of COX-2 function on neoplastic, as well as histologically normal, epithelia. Although abundant evidence supports a role for COX-2 in epithelial carcinogenesis,22 the most compelling data were shown by the mating of COX-2 knockouts with APCΔ716 mice, where a marked reduction in intestinal polyps was observed in double knockout animals.26 However, the exact mechanism(s) underlying the role of COX-2 in colonic tumorigenesis remains obscure. Most studies have focused on the signaling effects of the eicosanoid products of COX-2 activity, related to cell proliferation, apoptosis resistance, angiogenesis, and invasion (reviewed in Sinicrope22). These biological activities are believed to result primarily through formation of PGs such as PGE2. Although ROS generation has previously been shown to induce COX-2,27 limited data exist to indicate a role for COX-2 in producing DNA damage and in generating CIN. COX-2 possesses peroxidase activity, and formation of DNA adducts as a result of COX-2–mediated lipid peroxidation28 may contribute to COX-2–mediated tumorigenesis. Lipid hydroperoxides are formed enzymatically through the action of COX and lipoxygenase on ω3 and ω6 polyunsaturated fatty acids.29 These findings suggest that inhibition of COX-2–mediated lipid hydroperoxide formation may potentially interrupt colon carcinogenesis. It has also been shown that DNA and/or nucleosides can be oxidized when incubated in vitro with COX-2 and arachidonic acid, and this effect was prevented by COX-2 inhibitors, as well as by antioxidants.30 These results suggest a potential mechanism by which COX-2 may regulate DNA oxidation and the induction of mutations, which may be relevant mechanistically to the data of Wang and Huycke.17 Another potential mechanism for the role of COX-2 in regulating CIN in human colon cancer cells concerns malondialdehyde (MDA), a mutagen produced by spontaneous and enzymatic breakdown of PGH2.31 COX-2 results in the production of PGE2 via the unstable intermediate endoperoxide PGH2. MDA reacts with DNA to form adducts, predominantly the pyrimidopurinone adduct of deoxyguanosine.31 Although not examined in the study by Wang and Huycke,17 MDA could be a potential contributor to CIN as can other products of COX-2 or its downstream effectors.

In an attempt to demonstrate the relevance of E faecalis colonization and colorectal neoplasia risk in humans, this same group32 studied the relationship of colonization with enterococci and the risk of colorectal adenomas and carcinomas. In a prospective case-cohort study, they found that 40% of human stool samples from adults consecutively presenting for colonoscopy contained superoxide-producing enterococci. No association, however, was found between colonization with these bacteria and prevalence of colorectal neoplasms. Repeat cultures of stool 1 year later showed some variability in colonization with colonic enterococci. This negative study has several limitations, including the fact that chronic infection and mutagenicity in the colon are likely to occur over several decades and, thus, may contribute to the age-related incidence of CRC.

In addition to direct mutagenic effects, ROS from commensal colonic flora may also indirectly promote genomic instability by forming carcinogens from dietary procarcinogens.33 In this regard, an oxidative mechanism for CIN can potentially link the association of certain dietary factors with colorectal neoplasia. Despite the abundant epidemiologic evidence, a satisfactory explanation for the effects of high- or low-risk diets on risk of colorectal neoplasia remains elusive. High-fat diets could promote free radical formation via lipid peroxidation, whereas diets with abundant fruits, vegetables, and fiber contain antioxidants that may scavenge ROS.34 The relationship of diet to colonic bacterial microflora and colonization with E faecalis in humans warrants further study. The experimental findings by Wang and Huycke17 are relevant to and have potential implications for the primary prevention of CRC. At present, probiotics and prebiotics are being studied for the chemoprevention of CRC. These agents act to alter the intestinal microflora by increasing concentrations of beneficial bacteria such as lactobacillus and bifidobacteria, and reducing the levels of pathogenic microorganisms.35 Modification of enteric flora in IL-10 KO mice by probiotic lactobacilli was associated with a significant reduction in fecal coliform and enterococci levels in probiotic-fed animals compared with controls.36 These data suggest the potential of probiotics as a chemopreventive strategy against microbially related colonic tumorigenesis. In summary, the data by Wang and Huycke17 provide further evidence for the importance of colonic microflora in colon carcinogenesis. Studies to replicate these provocative data in an in vivo model are eagerly awaited and have the potential to further our understanding of colon carcinogenesis and also, to influence strategies for CRC prevention.

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PII: S0016-5085(07)00014-5

doi:10.1053/j.gastro.2007.01.012

Refers to article:

  • Extracellular Superoxide Production by Enterococcus faecalis Promotes Chromosomal Instability in Mammalian Cells , 06 December 2006

    Xingmin Wang, Mark M. Huycke
    Gastroenterology February 2007 (Vol. 132, Issue 2, Pages 551-561)

Gastroenterology
Volume 132, Issue 2 , Pages 797-801, February 2007

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