DNA damage induced by chronic inflammation contributes to colon carcinogenesis in mice (original) (raw)
Tumorigenesis induced by azoxymethane initiation and DSS promotion. It has been proposed that chronic inflammation enhances tumorigenesis via the induction of DNA damage by RONS (24). To test this hypothesis, we examined whether Aag-dependent DNA repair influences chronic inflammation–dependent tumorigenesis. We induced chronic colonic inflammation in Aag+/+ and _Aag_–/– mice by feeding repeated cycles of DSS. DSS cycles were preceded (or not) by a single injection of the colonic carcinogen azoxymethane (AOM). C57BL/6J mice are relatively refractory to spontaneous tumors of epithelial origin (25), and it was previously shown that a 70%–90% colon tumor incidence in C57BL/6J mice requires 12 cycles of DSS (23). We therefore chose a colon carcinogenesis protocol wherein an initial exposure to AOM is followed by fewer cycles of DSS (modified from refs. 1, 7). The treatment scheme is illustrated in Figure 1A.
Increased tumor multiplicity in Aag–/– versus Aag+/+ animals treated with AOM and DSS. (A) Treatment scheme. White blocks represent 16 days of normal water except the last which is 7 days. Black blocks represent 5 days of 2.5% DSS in water except for the last, which was 4 days of 2% DSS. Mice were also treated with only AOM or only DSS. (B) Tumor multiplicity in Aag+/+ animals (white), and Aag–/– animals (black). AOM+DSS Aag+/+, n = 12; AOM+DSS Aag–/–, n = 23; AOM Aag+/+, n = 15; AOM Aag–/–, n = 19; DSS Aag+/+, n = 10; and DSS Aag–/–, n = 10. Data are mean ± SD. (C) Pathology scores for Aag+/+ (circles) and Aag–/– (diamonds) mice. See Supplemental Methods for a complete description of scoring criteria. Lines indicate the median. (D) Photomicrographs depicting, from left to right, normal untreated Aag+/+ colon; AOM+DSS-treated Aag+/+ colon bearing a lesion with an average dysplasia score, a sessile adenoma; normal untreated Aag–/– colon; and AOM+DSS-treated Aag–/– colon bearing a lesion with an average dysplasia score and a higher scoring pedunculated adenoma. Scale bars: 200 μm.
Figure 1B shows the analysis of tumor multiplicity in Aag+/+ and _Aag_–/– animals treated with AOM and DSS, either alone or in combination. For AOM+DSS there was a 3.0-fold increase (P < 0.0001) in tumor multiplicity in _Aag_–/– animals with 22 ± 8.2 tumors per mouse; Aag+/+ animals had only 7.7 ± 6.7 tumors per mouse. AOM+DSS-treated _Aag_–/– animals also scored higher for severity of dysplasia and for the magnitude of the area affected (Figure 1C). All tumors were located in the mid- to distal colon, and included sessile and pedunculated adenomas with high-grade dysplasia and a moderate inflammatory component but no invasion of the muscularis mucosae (Figure 1D).
Treatment with a single dose of AOM did not lead to tumor development in either _Aag_–/– or Aag+/+ mice (Figure 1B). Treatment with 5 cycles of DSS alone led to a similar result for _Aag_–/– and Aag+/+ animals in terms of tumor multiplicity. For _Aag_–/– animals, the multiplicity was 0.3 ± 0.8 tumors per mouse (3 tumors in 10 animals), and for Aag+/+ animals, 0.2 ± 0.6 tumors per mouse (2 tumors in 10 animals). Although there were no significant differences in tumor multiplicity or incidence between Aag+/+ and _Aag_–/– mice treated with 5 cycles of DSS, it was clear that there were major pathological differences between Aag+/+ and _Aag_–/–animals, as shown in Figure 1D and in Figure 2 (see below).
Aag–/– animals are more susceptible than Aag+/+ animals to DSS-induced colitis. (A) Change in colon length. (B) Spleen weight as a percentage of body weight. AOM+DSS Aag+/+, n = 14; AOM+DSS Aag–/–, n = 31; AOM Aag+/+, n = 15; AOM Aag–/–, n = 19; DSS Aag+/+, n = 10; DSS Aag–/–, n = 10. Data are mean ± SD. (C) Histopathology scores for severity of inflammation, epithelial defects, and crypt atrophy. A description of the histopathological endpoints for inflammation examined can be found in Supplemental Methods. Epithelial defect scores were based on increased gland dilation and surface epithelial attenuation. (D) Photomicrographs showing a DSS-treated Aag+/+ colon bearing a lesion with an average epithelial defect score (left) and a DSS-treated Aag–/– colon bearing a lesion with an average epithelial defect score (right). Scale bars: 100 μm.
DSS-induced general pathologies are more severe in Aag–/– mice. We assessed the following colitis-related disease markers: decreased colon length due to healing ulcers and fistulas (Figure 2A) and increased spleen weight (Figure 2B). As shown in Figure 2A, colons from Aag–/– mice were significantly shorter than those of Aag+/+ mice treated with AOM+DSS or treated with DSS alone. Changes in spleen weight also indicate that DSS-induced colitis affects Aag–/– more severely than Aag+/+ mice. Colitis-associated splenomegaly, as determined by spleen weight and histopathology, was attributable to extramedullary hematopoiesis, presumably to replenish blood lost through stools due to tumors or ulcerations and to supply neutrophils to inflamed areas. Figure 2B shows a significantly greater increase in spleen weight in _Aag_–/– versus Aag+/+ animals for all treatment groups (P < 0.01). Note that the Aag+/+ spleen weight only increased when DSS treatment was preceded by AOM treatment, yet DSS treatment alone led to a significant increase in the _Aag_–/– spleen weight compared with untreated Aag–/– animals (P = 0.002) and compared to similarly treated Aag+/+ animals (P = 0.002). Taken together, these results show that Aag–/– animals respond more profoundly to, and manifest slower recovery from, DSS-induced chronic inflammation compared with Aag+/+ animals.
Intestinal changes at the cellular level were determined by histopathological analysis. H&E-stained colon sections were scored blindly for inflammation, epithelial defects, and crypt atrophy. Scoring criteria are described briefly in Methods and in full in Supplemental Methods (supplemental material available online with this article; doi:10.1172/JCI35073DS1). Five cycles of DSS induced similar levels of inflammation in Aag+/+ and Aag–/– animals, as judged histologically by the degree of leukocyte infiltration, aggregation, and follicle formation (see Supplemental Methods for description of scoring criteria). However, significant differences in epithelial defects were seen for Aag–/– versus Aag+/+ mice (Figure 2, C and D). Even though there is no histological difference in the level of chronic inflammation, the cellular and tissue defects are more severe in Aag–/– animals. These results prompted us to evaluate the effects of longer DSS administration in the Aag mutant model.
Aag deficiency enhances tumor development with extensive cycles of DSS. Given 7 cycles of DSS in drinking water (Figure 3A), Aag–/– animals developed more tumors than Aag+/+ mice and were much more severely affected for most pathophysiological criteria measured. With regard to tumor development, 7 of the 18 Aag–/– animals (39%) developed tumors, while none of the similarly treated WT mice (n = 7) developed any tumors (P = 0.07). All 7 affected Aag–/– animals had 1 colonic polyp displaying significant dysplasia (score of 2.5–3; Figure 3D).
Increased severity of disease in Aag–/– versus Aag+/+ animals treated with 7 cycles of DSS. (A) Treatment scheme. (B) Decrease in colon length for Aag–/– animals (n = 18) compared with Aag+/+ animals (n = 7). (C) Increase in spleen weight as a percentage of body weight for Aag+/+ (n = 7) and Aag–/– animals (n = 18). Data are mean ± SD. (D) Pathology scores for Aag+/+ (circles) and Aag–/– (diamonds) mice. (E) Histopathology of colonic disease induced by 7 cycles of DSS. From left to right: Aag+/+ colon exhibited moderate inflammation and glandular epithelial hyperplasia; Aag+/+ colon exhibited mild dysplasia characterized by epithelial cell pleomorphism and mild branching, with hyperplasia and inflammation; Aag–/– colon exhibited moderate to severe inflammation, crypt atrophy, mucosal collapse, and segmental epithelial cell loss; Aag–/– colon exhibited a portion of an intraepithelial neoplasia with mucosal dysplasia characterized by loss of columnar orientation, elongation, branching and infolding, glandular ectasia, inflammation, and crypt abscesses. Scale bars: 100 μm.
Colon lengths decreased and spleen weights increased more in _Aag_–/– versus Aag+/+ animals (Figure 3, B and C). Moreover, histopathological scores for inflammation, epithelial defects, and crypt atrophy were significantly higher in the _Aag_–/– cohort versus the Aag+/+ mice (Figure 3D). While the extent and severity of dysplasia were generally worse in _Aag_–/– versus Aag+/+ mice, the differences in their histopathological scores did not reach significance (Figure 3D). Figure 3E shows examples of the severity of colonic disease induced by 7 cycles of DSS in Aag+/+ and _Aag_–/– animals. Lesions in Aag+/+ mice included moderate mucosal and submucosal inflammation plus glandular epithelial hyperplasia (Figure 3E, WT untreated) and moderate dysplasia (Figure 3E, WT+DSS), whereas lesions in _Aag_–/– mice were worse, with moderate to severe mucosal and submucosal inflammation, crypt atrophy, and mucosal collapse (Figure 3E, Aag–/– untreated) and with highly dysplastic adenomas also present in some animals (Figure 3E, Aag–/–+DSS). Taken together, these data show that even in the absence of an initiating dose of AOM, _Aag_–/– animals are more susceptible than Aag+/+ to the detrimental effects of chronic inflammation induced by repeated DSS cycles.
Aag suppresses the severity of gastric lesions following infection with H. pylori. To determine whether Aag influences disease progression in another model of chronic inflammation, we infected Aag+/+ and Aag–/– mice with the pathogen H. pylori and analyzed gastric pathology at 32 weeks after infection. Although no gastric cancer developed during this relatively short experimental time frame, Aag–/– mice were clearly more susceptible to developing histopathologic lesions that were markers of increased predisposition to, or were precursors to, gastric cancer (26–28) (Figure 4A). Aag–/– mice had significantly more oxyntic atrophy (P = 0.002), foveolar hyperplasia (P = 0.04), and mucosal metaplasia (P < 0.001) (Figure 4B). Importantly, the innate and adaptive immune responses to the bacteria were unaffected by the Aag genotype: Aag–/– and Aag+/+ mice had similar histopathological scores for inflammation and serum levels of the _H. pylori–_specific IgG1 and IgG2c antibodies (Figure 4C). Also, the colonization of the gastric mucosa by H. pylori, as measured by quantitative PCR, was unaffected by Aag genotype (Figure 4D). In summary, the inflammatory response to H. pylori in Aag+/+ and Aag–/– mice is the same, but Aag–/– mice develop significantly more severe gastric lesions that are precursors to gastric cancer.
Increased severity of stomach pathology in Aag–/– versus Aag+/+ animals infected with H. pylori. (A) Histopathology of gastric disease and mucosal metaplasia induced by H. pylori infection in the stomach mucosa of Aag+/+ or Aag–/– mice. Uninfected Aag+/+ mouse stomach showed normal microscopic architecture (H&E) and a thin surface lining of gastric-type neutral mucins (red; AB/PAS), and anti-TFF2 stained intermittent mucous neck cells within normal oxyntic mucosa. Uninfected Aag–/– mice were indistinguishable from the uninfected Aag+/+, so only the Aag+/+ is shown. For infected Aag–/– mice, moderate gastritis with marked mucous metaplasia, hyperplasia, and oxyntic atrophy (loss of parietal and chief cells) was observed (H&E). Hyperplastic mucous neck cell population replaced resident zymogenic cells, secreting a mixture of gastric (red) and intestinal-type (acidic, blue) mucins (AB/PAS). Mucous metaplasia associated with expanded mucous neck cell population highlighted by TFF2 immunoreactivity. For infected Aag+/+ mice, gastritis but minimal oxyntic alterations were observed (H&E). Mucous neck cells were significantly reduced in number in the Aag+/+ _H. pylori_–infected mouse (AB/PAS). Fewer TFF2-positive mucous neck cells were observed in the infected Aag+/+ mouse. Scale bars: 160 μm. (B) Pathology scores for infected Aag+/+ (circles) and infected Aag–/– (diamonds) mice. (C) Serum IgG1 and IgG2c responses to H. pylori in Aag–/– and Aag+/+ mice 32 weeks after infection. (D) H. pylori colonization levels in the stomach. Data presented as mean ± SD of log-transformed CFU/μg of genomic DNA.
Aag does not suppress tumorigenesis in the absence of chronic inflammation. It was possible that Aag might influence tumor development independent of chronic inflammation. However, this did not appear to be the case. When crossed with ApcMin mice, a robust genetic model for identifying modifiers of intestinal tumorigenesis that does not involve chronic inflammation, Aag+/+ and Aag–/– mice had nearly identical tumor responses (Supplemental Figure 1). Aag did not affect tumor multiplicity in either the small intestine (Supplemental Figure 1A) or the colon (Supplemental Figure 1B) or the distribution of tumors throughout the entire intestine (Supplemental Figure 1C). Furthermore, tumor size was unaffected by Aag (Supplemental Figure 1D). In summary, in the absence of inflammation, we observed no effect of Aag on colon tumorigenesis.
It was also possible that Aag–/– mice were differentially sensitive to the single dose of AOM used to initiate tumorigenesis. C57BL/6J animals form appreciable numbers of aberrant crypt foci (ACF) in response to AOM (29), but in the absence of treatment with exogenous tumor promoters, neoplasia is infrequent. To determine whether Aag modulates this initiation response, colons from animals treated with AOM alone were examined for ACF formation. Aag deficiency had no effect on ACF formation (Supplemental Figure 2). Similarly, Aag deficiency had no significant effect on tumor responses following 5 weekly treatments with AOM, as 2 of 8 Aag+/+ animals and 1 of 12 Aag–/– animals each developed a single tumor. This result is consistent with the low overall tumor response of C57BL/6J animals to AOM in the absence of subsequent chronic inflammation. This confirms that the heightened susceptibility of Aag–/– mice to tumor formation in the 2-stage carcinogenesis model is not due to a differential response to the initiating carcinogen, AOM, but rather to the chronic inflammation induced by DSS exposure.
Finally, it was possible that Aag–/– animals respond aberrantly to episodic bouts of colitis because of an altered innate inflammatory response. To address this issue, we used Citrobacter rodentium to evaluate the effects of acute inflammation in Aag–/– versus Aag+/+ animals; such infection induces acute hyperplastic colitis (30). No clinical disease was observed in Aag+/+ or Aag–/– mice infected with C. rodentium (data not shown). Fecal bacterial shedding was detected at 4 days after infection and was indistinguishable between Aag+/+ and Aag–/– mice throughout 30 days of the experiment (Supplemental Figure 3). Similarly, there were no differences between Aag+/+ and Aag–/– mice with regard to colonic lesions throughout the course of the infection, and these lesions were largely resolved by 30 days after infection. Thus, we observed no effect of Aag on mounting an acute inflammatory reaction.
Modified bases following DSS treatment. Given the results presented above, our working hypothesis was that DSS-induced colitis results in RONS-induced DNA damage that is poorly repaired in the absence of Aag and that the unrepaired DNA lesions enhance tumorigenesis. In support of this hypothesis, we demonstrated that reactive nitrogen species are indeed formed after DSS treatment, by positive staining for nitrotyrosine, a known marker for NO-induced peroxynitrite formation, in colons of both WT and Aag–/– mice treated with AOM+DSS (Supplemental Figure 4). In addition, we used a recently developed and extremely sensitive LC/MS method to measure endogenous levels of a selection of DNA lesions known to be induced by RONS (31). We quantified the modified DNA bases εA, εC, 8oxoG, and Hx in colonic epithelial DNA from untreated and DSS-treated mice. Levels for all modified bases in the untreated groups were not appreciably different between Aag-proficient and Aag-deficient animals, suggesting that Aag does not affect the accumulation of these lesions in the colons of relatively young animals that have not experienced colitis (Figure 5). After a single cycle of DSS, levels of εA and εC increased significantly relative to the untreated groups (P < 0.0002) and increased with time in both groups of animals after DSS was terminated (P < 0.04) (Figure 5, A and B). These increases were more dramatic in the Aag-deficient versus Aag-proficient animals. At 1 and 7 days after treatment, the εA increase was 1.5- and 1.8-fold higher in Aag-deficient than that in Aag-proficient animals, respectively (Figure 5A), and εC was 1.4- and 2.0-fold higher (Figure 5B), respectively. These differences represent significant effects of Aag on the levels of both ε-lesions in response to DSS (P = 0.01 for both) treatment. Levels of εA in the Aag-deficient mice showed the greatest difference of any lesion relative to untreated mice. Seven days after DSS treatment was terminated, Aag-deficient mice had 14-fold higher levels of εA compared with the mean levels from all untreated mice.
Induction of εA, εC, and 8oxoG lesions by chronic inflammation and Ctnnb1 mutations in colon tumors. Six- to 8-week-old male mice were untreated or were administered 2.5% DSS for 5 days and euthanized 1 or 7 days after treatment. Levels of the modified bases εA (A), εC (B), and 8oxoG (C) were measured from colonic mucosal DNA and are shown for Aag-proficient (+) and Aag-deficient (–) mice. Within treatment groups, we observed no differences between Aag+/+ and Aag–/– mice, and thus these genotypes were combined in the analysis. Data are mean ± SD. (D–F) Point mutations in Ctnnb1. (D and E) Chromatogram showing an example of the most common mutation observed, a G:C to A:T transition in codon 41 of Ctnnb1. The arrow indicates the peaks for the mutant and WT sequences. (E and F) Distribution of mutations in Ctnnb1. Base substitutions are indicated below the sequence, and their frequencies are shown for the different experimental groups. (F) Deletion mutants in Ctnnb1. Chromatograms of the 2 deletion mutants are shown with an arrow below the sequence indicating the deletion junction. Also shown is the deleted sequence (lowercase) with red highlighting of regions of microhomology between the deleted portion and the adjacent retained sequence.
Levels of 8oxoG also increased with DSS treatment (P = 0.05) and, like εA and εC, it increased to higher levels in Aag-deficient versus Aag-proficient animals. At 1 and 7 days after DSS was stopped, the increases in 8oxoG levels were 1.7- and 1.8-fold higher in Aag-deficient mice versus those in Aag-proficient mice (P = 0.01) (Figure 5C). Although there was a trend for increasing levels of Hx in the colonic mucosae of repair deficient mice, the overall effect of inflammation on levels of this lesion were not significant (P = 0.2) (Supplemental Figure 5).
Oncogenic mutations. Microdissected tumors were analyzed to determine whether damage that resulted from RONS affected the mutation spectra in the candidate oncogenes Ctnnb1 and Kras2, which are commonly mutated in mouse colon tumors (Figure 5 and Supplemental Table 1). We sequenced a portion of exon 2 from Ctnnb1 (homologous to exon 3 in human CTNNB1) corresponding to the coding sequence for the glycogen synthase kinase-3β phosphorylation domain. In addition, we sequenced portions of exons 1 and 2 from Kras2, corresponding to the coding sequences for the codon 12 and codon 61 regions, respectively. Oncogenic mutations were detected in Ctnnb1 in most tumors analyzed, while only 1 tumor among all groups had a mutation in Kras2 (a G:C to A:T transition in the second base of codon 13 from a tumor in an Aag–/– mouse treated with AOM+DSS) (Supplemental Table 1). Figure 5E shows an example of the most common mutation, observed at codon 41 in Ctnnb1, and Figure 5F shows the positions for all identified base substitutions. When tumors were initiated with AOM, all but 1 of the mutations were G:C to A:T transitions, reflecting the mutational specificity of this carcinogen. Aag–/– animals that were treated with DSS alone showed a similar fraction of tumors with mutations in Ctnnb1 (5 of 6). However, in this group only 2 of the 5 mutations were G:C to A:T transitions, while the others included an A:T to G:C transition and 2 in-frame deletion mutations (Supplemental Table 1 and Figure 5F). While the sample size is small, the fraction of deletion mutants (2 of 5) represents a significant difference (P = 0.02) from that of the combined tumors from Aag–/– and Aag+/+ mice treated with AOM+DSS (0 of 26). These results suggest that oncogenic deletion mutants are generated by unrepaired RONS-induced lesions in DNA.