The etiology of colorectal cancer (CRC) has been linked to deficiencies in mismatch repair and adenomatous polyposis coli (APC) proteins, diet, inflammatory processes, and gut microbiota. However, the mechanism through which the microbiota synergizes with these etiologic factors to promote CRC is not clear. We report that altering the microbiota composition reduces CRC in APC Min/+ MSH2 −/− mice, and that a diet reduced in carbohydrates phenocopies this effect. Gut microbes did not induce CRC in these mice through an inflammatory response or the production of DNA mutagens but rather by providing carbohydrate-derived metabolites such as butyrate that fuel hyperproliferation of MSH2 −/− colon epithelial cells. Further, we provide evidence that the mismatch repair pathway has a role in regulating β-catenin activity and modulating the differentiation of transit-amplifying cells in the colon. These data thereby provide an explanation for the interaction between microbiota, diet, and mismatch repair deficiency in CRC induction.

Polyp incidence is not reduced in germ-free APCmice (), indicating that gut microbes have little to no role in disease progression in this background. Here we investigated whether gut microbiota and diet have the capacity to affect polyp formation in an APCMSH2mouse model of CRC. We found that altering the microbiota composition with oral antibiotics dramatically reduced CRC specifically in APCMSH2mice. We also demonstrated that a diet reduced in carbohydrates can phenocopy this effect and show that gut microbes stimulated CRC development through the production of carbohydrate-derived metabolites such as butyrate. Furthermore, we show that MSH2 and MLH1 deficiency led to enhanced β-catenin activity and cellular hyperproliferation in colon epithelial cells, and this activity was dependent on gut microbes. These data thereby provide an explanation for the puzzling link between MMR deficiency and CRC and implicate both diet and gut microbiota in disease pathogenesis.

The APC(multiple intestinal neoplasia) mouse is a well-established animal model of human adenomatous polyposis (). These mice spontaneously develop multiple polyps in the small intestine and colon. Combining the APCmutation with MSH2 deficiency results in a large increase in polyp numbers (). Although the enhanced mutation frequency associated with MMR deficiency might lead to mutation in genes involved in regulating the Wnt-β-catenin axis (), mutations in these genes are expected to be rare and might not explain the pronounced synergistic effect of the MSH2 and APCmutation on tumor formation. As the etiology of HNPCC is associated with aggressive and rapid adenoma development, it is critical to understand how major environmental factors such as microbiota and diet interact with the MSH2genotype and whether these affect disease progression.

The intestinal microbiota has been identified as a key contributor to the development of CRC (). Many of these studies highlight the role of inflammation in producing a niche for specific microbes to elicit their oncogenic effects (). However, since the etiology of most CRCs does not have an inflammatory component (), it is unclear whether these recent findings can be extrapolated to the development of other types of CRC. Another etiologic factor that contributes to CRC development is a “western diet” (). Dietary glycemic load and total carbohydrate intake have also been implicated in CRC development in some studies (). Notably, a comprehensive meta-analysis found a positive association of total carbohydrate intake with CRC (), and a recent prospective study showed that carbohydrate intake was associated with an increased risk of recurrence of CRC (). However, it remains unclear how the microbiota and diet affect CRC and how their effects intersect with host genetics and with other known etiologic factors to promote CRC.

Colorectal cancer (CRC) is associated with numerous genetic changes. Some of the most frequently affected genes are the adenomatous polyposis coli (APC) gene () and genes involved in DNA mismatch repair (MMR) (). APC is a tumor suppressor that is involved in diverse physiological roles and regulates Wnt signaling by controlling the levels of β-catenin (). Mutation in or inactivation (via silencing) of MMR genes, such as MutS homolog 2 (MSH2) and MutL homolog 1 (MLH1), are the most common lesions in hereditary nonpolyposis colorectal cancer (HNPCC or Lynch Syndrome) and occur in ∼20% of sporadic CRC (); 56% of CRC tumors that are defective for MMR also harbor APC mutations (). MMR is a multifunctional DNA-repair pathway that corrects errors arising during DNA replication and maintains the fidelity of the genome through other means (). Based on the known roles of the MMR pathway, it is unclear why MMR deficiency predisposes to CRC more so than to other cancers, suggesting a unique interaction between MMR genes within colon epithelial cells and the cell’s environment.

Collectively, these results indicate that the gut microbiota plays a key role in CRC by providing metabolites such as butyrate that “fuel” the transformation of APC Min/+ MSH2 −/− colonic epithelial cells.

To test whether butyrate directly affects polyp formation in APCMSH2mice, antibiotic-treated mice were fed a diet enriched in tributyrin, a stable form of butyrate that breaks down into three butyrate molecules in the gastrointestinal tract (). Tributyrin led to a large increase in butyrate levels in the small intestine ( Figure S7 C). Strikingly, tributyrin stimulated both polyp numbers ( Figure 7 A) and epithelial cell proliferation ( Figures 7 B and S3 B) in the small intestines of antibiotic-treated APCMSH2mice. However, tributyrin had no effect on polyp number or cellular proliferation in the colon ( Figures 7 C, 7D, and S3 C), which was likely due to an insufficient increase of butyrate in the colon in tributyrin-fed mice ( Figure S7 C). Hence, we administered sodium butyrate directly in the colons of antibiotic-cocktail treated mice by rectal instillation. This experiment revealed that 50 μM and 0.5 mM of sodium butyrate, which represent concentrations of butyrate found in the distal part of the colon (), stimulated proliferation of colon epithelial cells in APCMSH2mice but not in controls ( Figures 7 E–7G and S3 D). In contrast, consistent with its action as an HDACi, high concentrations of sodium butyrate (i.e., 10 and 100 mM) did not increase colon epithelial cell proliferation in APCMSH2mice ( Figures 7 E–7G and S3 D). These data show that butyrate facilitates the aberrant hyperproliferation phenotype of colon epithelial cells in APCMSH2mice.

(F and G) The fraction of cells in the distal colon expressing Ki-67 as a function of the total length of each crypt from APCMSH2(F) or APCMSH2mice (G). Each symbol represents the mean fraction of Ki-67 expression for multiple crypts for each individual mouse. See also Figure S3 D.

(E) Mice with indicated genotypes were treated with cocktail antibiotics for 3 weeks, then rectally administered PBS or sodium butyrate for 24 hr, and Ki-67 expression was measured in colon epithelial cells by immunohistochemistry.

(B and D) Ki-67 expression in the small intestines and colons of tributyrin-fed mice (see also Figures S3 B and S3C).

(A and C) Polyp numbers in the small intestines and colons of tributyrin-fed APCMSH2mice compared to the polyps in the untreated and antibiotic-treated mice (from Figures 3 B and 3C).

Butyrate is produced by specific members of Firmicutes (), and it was notable that metronidazole and the low-carbohydrate diet reduced the phylum Firmicutes without impacting total bacterial abundance ( Figures 1 A and S1 D). Specifically, these treatments led to the reduction of three families within Firmicutes, namely Clostridiaceae, Lachnospiraceae, and Ruminococcaceae ( Figure 6 C), that are known to produce butyrate (). Representatives of genus Coprococcus and Roseburia, which belong to Lachnospiraceae and are members of the Clostridium cluster XIVa (), and Anaerotruncus, which belong to Ruminococcaceae and cluster IV (), were also reduced by these treatments as measured by proportion of sequencing reads ( Figure 6 C) and by quantitative PCR (qPCR) ( Figure 6 D). In addition, the gene copy number for butyryl-CoA transferase, a microbial gene responsible for butyrate production, was also reduced by these treatments ( Figure 6 E).

The fact that reducing either gut microbiota or dietary carbohydrates resulted in the reduction in both Ki-67 and β-catenin expression in APCMSH2mice led us to hypothesize that bacterial metabolites might fuel the aberrant hyperproliferation of colon epithelial cells in these mice. We analyzed the colon contents from mice subjected to either a low-carbohydrate diet or metronidazole treatment because these treatments did not impact total bacterial load ( Figure 1 A) but had an impact on polyp numbers ( Figures 1 D and 2 C). Using two analytical platforms (i.e., NMR and direct flow injection/liquid chromatography-tandem mass spectrometry [DI/LC-MS/MS]), we found that mice under these treatments exhibited a reduction in numerous metabolites ( Figure S6 ). Using other methodologies, we also measured the levels of lactate and short-chain fatty acids (SCFAs), which are major carbohydrate-derived metabolites that are produced by microbial fermentation. We found that cocktail antibiotics or a low-carbohydrate diet reduced the levels of mucosal lactate in these mice ( Figure 6 A); however, metronidazole did not ( Figure S6 ). On the other hand, the only SCFA that was statistically reduced by all antibiotic treatments and by the low-carbohydrate diet was butyrate ( Figures 6 B and S7 B). Although butyrate functions as a histone deacetylase inhibitor (HDACi) at high concentrations (), the concentration of butyrate that is produced by gut bacteria in the distal colon, the part of the colon that exhibits the hyperproliferation phenotype and amasses polyps in APCMSH2mice, promotes epithelial cell proliferation (). In addition, butyrate modulates canonical Wnt signaling () and has been shown in some studies to promote CRC (). Hence, we focused our efforts on the microbial-derived metabolite butyrate in subsequent experiments.

(B) Figures showing the distribution of the indicated SCFAs in the distinct parts of the gastrointestinal tract under the various treatments: upper (U), middle (M), and lower (L) small intestine, and proximal (P) and distal colon (D). The concentrations of each SCFA were determined from the ratio of each peak height to the internal standard, with reference to bracketing standard curves. The antibiotic treatments reduced the levels of all indicated SCFAs in the P and D sections, whereas the 7% low-carbohydrate had minimal effect.

(A) A schematic representation of the parts of the small intestine and colon where the concentration of SCFA was measured.

Measurements of the Concentration of Essential SCFAs in the Intestines of Mice under Different Treatments, Related to Figure 6

(E) The abundance of butyryl CoA-transferase gene relative to RNA polymerase B gene under indicated treatments was measured by qPCR, n ≥ 3.

(D) The abundance of clostridium cluster IV and XIVa under indicated treatments was measured by qPCR using 16S rRNA gene primers specific for each species, n ≥ 2.

(C) Comparison of the abundance of specific bacteria in the colon of indicated treatments shown as proportion of reads. Data are derived from the 16S rRNA gene-sequencing analysis, n = 7.

(B) The levels of butyrate (and other SCFAs; see Figure S7 A) were measured in the small intestines and colons from mice under indicated treatments.

(A) Lactate imaging in mouse colon samples. Left panel shows the hematoxylin and eosin (H&E)-stained samples (above) and the bioluminescence imaging of lactate (bottom). Right panel depicts the relative lactate contents measured in the colon samples from treated APC Min/+ MSH2 −/− mice.

Figures showing the fecal concentration of different metabolites, measured by DI/LC-MS/MS or NMR spectroscopy, in mice untreated (Untreat) or treated with either a 7% low-carbohydrate diet (7% LC) or metronidazole (Metro). ND: nondetected. LysoPC a: lysoPhosphatidylcholine acyl; SM (OH): hydroxysphingomyeline; SM: sphingomyeline; PC aa: Phosphatidylcholine diacyl; PC ae: Phosphatidylcholine acly-alkyl. Each molecule’s name is followed by the number of carbon atoms that it possesses. The levels of 84 other metabolites were either not different between any of the samples, or were not consistently reduced or increased in either of the treated (i.e., 7% LC and Metro) samples (data not shown). Please contact the corresponding author for the entire metabolomic analysis data set.

Metabolomic Analysis of the Colon Contents of Mice Treated with Metronidazole or Fed the 7% Low-Carbohydrate Diet, Related to Figure 6

In addition to repairing mismatched base pairs, the MMR system also induces apoptosis through the DNA damage response (DDR) and inhibits recombination between nonidentical sequences (). However, it is unclear which of these functions are responsible for suppressing polyp formation, hyperproliferation, and aberrant β-catenin activity in colon epithelial cells. MSH2mice are deficient in all functions of MMR; however, MSH2mice are active for DDR but cannot carry out other functions of MMR (). We also assessed whether MLH1mice, which are defective in both DNA repair and DDR and are mutated for a gene (MLH1) that is frequently inactivated in CRC, are similar to MSH2mice with respect to the phenotypes that are reported in this study. Indeed, increased polyp numbers were observed in MLH1(as previously reported in) and MSH2mice on the APCbackground ( Figures S5 A and S5B). We found that colon epithelial cells from MLH1and MSH2mice displayed a hyperproliferative phenotype ( Figures S5 C and S5D). Further, we observed increased nuclear β-catenin in MLH1and MSH2mice relative to controls ( Figure S5 E) and found increased expression of downstream targets of β-catenin in MLH1mice ( Figure S5 F), indicative of increased β-catenin activity. These data indicate that deregulated β-catenin and hyperproliferation are general properties in MMR-mutated colon epithelial cells and that MMR’s role in mismatch repair and/or homologous recombination sensitizes colon epithelial cells to transformation by microbial-derived metabolites.

Data were normalized to HPRT cDNA levels. The data in (A) and (B) were analyzed by the Mann-Whitney test.

(E) Immunohistochemistry analysis for β-catenin expression of colons from mice of the indicated genotypes. Magnification is 100×.

(C and D) The fraction of cells in the distal colon of indicated mice expressing Ki-67. Each symbol represents the mean fraction of Ki-67 expression for multiple crypts for each individual mouse.

(A and B) Polyp count in 6-week-old APC Min/+ MLH1 +/× (A +/− Mlh1 +/− ), APC Min/+ MLH1 −/− (A +/− Mlh1 −/− ), APC Min/+ MSH2 +/G674D (A +/− M +/GD ), and APC Min/+ MSH2 G674D/G674D (A +/− M GD/GD ) in the small intestine and colon, respectively.

Mice Deficient in MLH1 or in the ATPase Activity of MSH2 Have Enhanced β-Catenin Activity and Hyperproliferation of Colon Epithelial Cells

To further investigate the nature of the hyperproliferation phenotype observed in MSH2-deficient colonic epithelial cells, we assessed the status of the Wnt/β-catenin signaling pathway, which regulates the proliferation and differentiation of intestinal epithelial cells within the crypts (). In APCMSH2mice, β-catenin was present in a normal membrane pattern and localized to the nucleus of the cells at the crypt base ( Figures 5 A, 5B, and S4 A). In contrast, accumulation of nuclear β-catenin was observed in colonic epithelial cells that were located above the first third of the crypt of APCMSH2colons ( Figures 5 A, 5B, and S4 A). This defect was also observed in MSH2mice, indicating that the APCmutation does not lead to aberrant β-catenin localization ( Figure S4 B). Antibiotic treatment and the low-carbohydrate diet restored the nuclear β-catenin expression in APCMSH2mice to that seen in APCMSH2mice ( Figures 5 A and S4 A). β-catenin transcript levels were also elevated in MSH2colonic epithelial cells compared to controls ( Figure 5 C). These results correlate well with the expression of Ki-67 ( Figure 4 ), suggesting that enhanced β-catenin activity drives hyperproliferation of MSH2colonic epithelial cells. Furthermore, we found that the expression of c-MYC protein, which is a downstream target of β-catenin, was elevated beyond the normal proliferative zone in APCMSH2mice ( Figure S4 C). Other downstream targets of β-catenin, such as stem cell markers Lgr5, CD44, EphB2, and EphB3 (), were also elevated at the transcript level in MSH2colonic epithelial cells compared to controls ( Figure 5 C). Hence, the enhanced β-catenin activity defines these cells as actively proliferating and undifferentiated. Indeed, the APCMSH2colonic crypts were characterized by reduction in expression levels of p21, a protein that governs cell-cycle arrest and differentiation ( Figure S4 D). By comparison, p21expression was lost in an adenomatous polyp ( Figure S4 E). Therefore, the abnormal β-catenin activity in MSH2and APCMSH2colonic crypts probably leads to expansion of the proliferating/undifferentiated compartment likely consisting of transit-amplifying cells. Strikingly, gut microbes had the capacity to modulate β-catenin activity specifically in the colon of MSH2hosts, and reduction of gut microbiota or dietary carbohydrates normalized the proliferation and differentiation phenotype to levels observed in controls.

(E) Expression of β-catenin and p21 (CIP1/WAF1) in an adenomatous polyp. The magnification for β-catenin and p21 (CIP1 / WAF1) is 40×, for c-MYC is 20×.

(D) Expression of p21 (CIP1/WAF1) in the colon of mice of the indicated genotype and treatment was measured using p21 (F-5) Alexa Fluor 488 antibody purchased from Santa Cruz Biotechnology, Inc. The staining was carried out on frozen sections of Swiss-rolled colons.

(C) The expression level of c-MYC was analyzed using anti-c-MYC (c-19) (Santa Cruz Biotechnology, Inc) by immunofluorescence (left panels) and expressed as fraction of cells (right panel) in the distal colon expressing the proteins as a function of the total length of each crypt (as measured from the bottom of the crypt to the top of the mucosal surface).

(A) Representative immunohistochemistry analyses for β-catenin expression in APC Min/+ MSH2 +/− and APC Min/+ MSH2 −/− mice under various conditions. Each panel represents a different mouse.

(C) cDNA levels were quantified by qPCR for β-catenin, and downstream targets of β-catenin (Lgr5, CD44, EphB2, EphB3). Data are normalized to HPRT cDNA levels. cDNA levels for another house-keeping gene, Tata-box binding protein (TBP), are also shown.

(B) Same as (A) except that β-catenin was visualized by immunofluorescence. Right panels are representative confocal images of colonic crypts (magnification 63×). Boxes within the left panels are magnified and shown on the right panels.

(A) Immunohistochemistry analysis for β-catenin expression of colons from mice of the indicated treatments and genotypes. Representative figures are depicted, with figures from other mice shown in Figure S4 A. Arrows identify cells with nuclear β-catenin expression. Magnification is 100×.

We next assessed whether microbial-derived metabolites influence colon epithelial proliferation particularly in APCMSH2mice. We used the comet assay because it detects both ssDNA and dsDNA breaks and can be used to detect replication-derived breaks (). Figures S2 D–S2F show typical experimental results of the comet assays that were used to calculate the fraction of cells with DNA breaks ( Figure 4 A). Such results revealed that colon epithelial cells harvested from APCMSH2mice had approximately two times the number of cells with DNA breaks as cells harvested from APCMSH2mice. Interestingly, antibiotic treatment or a low-carbohydrate diet reduced the number of cells with DNA breaks in APCMSH2mice ( Figure 4 A). These DNA breaks are likely ssDNA breaks given that our previous analysis did not show elevated levels of dsDNA breaks (i.e., γH2AX) in APCMSH2mice ( Figure S2 C). Furthermore, we found that ∼12% of colon epithelial cells in APCMSH2mice were in the S-G-M phases of the cell cycle compared to ∼8% in APCMSH2mice ( Figure 4 B). We also measured expression of the nuclear protein Ki-67, which does not stain cells in G, in colon epithelial cells. Strikingly, Ki-67 expression was elevated in the distal colons of APCMSH2mice relative to controls, and antibiotics or a low-carbohydrate diet reduced Ki-67 expression in these mice ( Figures 4 C, 4D, and S3 A). Furthermore, the increased Ki-67 expression in APCMSH2mice occurred specifically in the distal region of the colons in APCMSH2mice ( Figure 4 E), which is noteworthy because >90% of polyps in the colon of APCMSH2mice occur in the distal region of the colon. Ki-67-positive cells were also increased in MSH2mice compared to their wild-type (WT) controls ( Figure 4 D), indicating that MSH2 deficiency alone is associated with abnormal cellular proliferation. We also observed an increase in apoptosis in colon epithelial cells from APCMSH2mice compared to controls, but not in antibiotic-treated APCMSH2mice ( Figure 4 F). Taken together, these data show that APCMSH2colonic epithelial cells hyperproliferate relative to controls, which is dependent on gut microbiota and dietary carbohydrates.

(D) Same as in (A) except that Ki-67 expression is reported in the colon for APC Min/+ MSH2 +/− and APC Min/+ MSH2 −/− mice rectally administered various concentrations of sodium butyrate.

(C) Same as (A) except that Ki-67 expression is reported in the colon for APC Min/+ MSH2 −/− mice with the indicated treatments.

(B) Same as (A) except that Ki-67 expression in the small intestine is reported for APC Min/+ MSH2 −/− mice with the indicated treatments.

(A) Ki-67 expression of tissue from the distal part of the colon was detected by immunohistochemistry with anti-Ki-67 antibody. Ki-67 staining was carried out on frozen sections of Swiss-rolled colons. Staining was performed with rabbit primary polyclonal anti-Ki-67 antibody (Abcam) followed by secondary goat anti-rabbit antibody conjugated with horse radish peroxidase. Slides were selected with well orientated crypts and the total length of each crypt was measured and compared to the length of the crypt containing Ki-67 positive cells. Each symbol represents the fraction of Ki-67 expression relative to the crypt length for one crypt (as measured from the bottom of the crypt to the top of the mucosal surface). Each column represents the values obtained for at least five crypts per mouse for the indicated genotype and treatments.

(F) Colonic epithelial cells were stained with annexin V and PI, and the % apoptosis was estimated by flow cytometry (n = 6 per group).

(E) Ki-67-positive cells per crypt in the proximal and distal colon for the indicated mice (n ≥ 3) under various treatments.

(D) The fraction of cells in the distal colon expressing Ki-67 as a function of the total length of each crypt. Each symbol represents the mean fraction of Ki-67 expression for multiple crypts for each individual mouse (derived from Figure S3 A).

(C) Ki-67 expression in the distal colon for the indicated mice. Magnification is 20×.

(B) The % of colon epithelial cells from indicated genotypes/treatments showing a greater than 2N DNA, as measured by propidium iodide (PI) staining.

(A) Colonic epithelial cells derived from mice with the indicated treatments were collected, and the % DNA breaks were measured by the alkaline comet assay. Figures S2 D–S2F show typical data for identifying breaks. Each symbol represents an individual mouse.

Our results show that polyp development in the colons of APCMSH2mice is dependent on gut microbiota particularly between weeks 3 and 6 after birth ( Figure 1 D), corresponding to the time when mice are weaned. Owing to the enormous metabolic potential of the gut microbiota, we hypothesized that gut microbes might contribute to CRC development by providing diet-derived metabolites that facilitate tumor initiation and/or progression. Given that carbohydrates have been implicated in CRC, we tested whether carbohydrate-derived metabolites were associated with CRC. Three-week-old APCMSH2mice were given a normal or low-carbohydrate diet ( Table S1 ). Approximately 58% of the calories provided with the normal diet derived from carbohydrates, compared to 7% for the low-carbohydrate diet. Weight gain was normal with the low-carbohydrate diet ( Figure 3 A), indicating that mice on this diet were not under caloric restriction. The low-carbohydrate diet did not alter the total bacterial abundance ( Figure 1 A) but led to substantial changes in the microbial communities ( Figures 1 B and S1 D). Strikingly, the low-carbohydrate diet reduced polyp numbers in the small intestines and colons of APCMSH2mice ( Figures 3 B and 3C). By comparison, treating mice with antibiotics from weeks 3 to 6 produced a similar decrease in polyp number ( Figures 3 B and 3C). Furthermore, there was no additive effect on polyp number by combining the low-carbohydrate diet and the antibiotic treatment ( Figures 3 B and 3C) suggesting that both treatments function by the same mechanism. Taken together, these data indicate that carbohydrate-derived metabolites produced by gut microbes drive CRC development in APCMSH2mice.

(B and C) Polyp count was measured for the small intestines and colons of mice treated with an antibiotic cocktail (Abx: ampicillin, metronidazole, and neomycin) and/or 7% LC diets from weeks 3 to 6.

(A) The body weights of A +/− M −/− mice (8 per group) were fed normal diets (ND) with or without tributyrin (TriB) or 7% low-carbohydrate diet (7% LC) from week 3 to week 6.

MSH2 is a central component of MMR (). Inasmuch as our results showed that reduction of the gut microbiota decreased CRC in APCMSH2hosts, we hypothesized that antibiotics reduced the occurrence of mutations by mitigating the mutagenic effects of the microbiota (). Thus, we used the lacI-containing transgenic mouse mutation detection system to measure the mutation frequency in colonic epithelial cells. We found that there was an increase in the mutation frequency in colon epithelial cells from MSH2mice compared to MSH2mice ( Figure S2 A), as expected (). However, the mutation frequencies were similar between colon epithelial cells from untreated or antibiotic-treated MSH2mice ( Figure S2 A). Furthermore, antibiotic treatment did not reduce the mutation frequency in adenomas isolated from APCMSH2mice ( Figure S2 B). Because some DNA mutagens cause DNA breaks in the absence of point mutations, we assessed whether γH2AX levels, a cellular marker for double-stranded DNA (dsDNA) breaks, were affected by antibiotics or the genotype. However, we found that γH2AX levels did not vary between genotypes and treatments and were >10-fold lower than in irradiated colon epithelial cells ( Figure S2 C), suggesting that microbiota did not induce dsDNA breaks in APCMSH2mice. Taken together, these data suggest that gut microbiota induce CRC through a mechanism that is independent of both inflammation and DNA damage.

(F) Tail moments were measured for colonic epithelial cells harvested from individual mice under various treatments. The tail moment of each individual cell was calculated by multiplying the percentage DNA in the tail by the tail length and divided by 100. At least 75 comets per mouse were analyzed. For the purposes of this study, cells with tail moments greater than 4.5 were considered to be cells with DNA breaks.

(E) To calibrate the comet assay, colonic epithelial cells derived from an A +/− M +/− mice were incubated with 0, 50, and 100μM H 2 O 2 for 10 min to induce DNA breaks.

(D) DNA breaks were measured by the “comet assay.” This assay was carried out under alkaline conditions which allow detection of both ssDNA and dsDNA breaks. Colonic epithelial cells were prepared from minced colons in PBS containing 3 mM EDTA, 0.5 mM DTT. The cells were dissociated by incubation at 37°C for 20 min in presence of 70U of collagenase (Sigma). Alkaline comet assay and silver staining of the comets was carried out according to the manufacturer’s instructions (Trevigen). Images showing the DNA migration in cells derived from indicated mice.

(C) Representative western blot (of a total of three) for γH2AX and β-actin of colon epithelial cells from the indicated mice and treatments. Control colon epithelial cells were exposed to 50 μM H 2 O 2 or 4 and 8 grays of ionizing radiation. Colonic epithelial cells were isolated by scraping off the mucus layer from colons, and protein extracts were prepared by lysing cells with 1% SDS solution. Membranes were blotted with anti-phospho-histone H2AX (Millipore) and anti-β-actin (Sigma) followed by horse radish peroxidase-conjugated anti-rabbit IgG antibody and chemiluminescence detection. In a separate experiment, we sequentially diluted the 8 gray-irradiated sample to achieve a similar levels of γH2AX signal to unirradiated samples. This analysis showed that the 8 gray-irradiated sample had >10-fold more γH2AX levels than unirradiated samples (data not shown). The data were analyzed by the Mann-Whitney test.

(B) The frequency of mutations at the lacI gene in normal colon epithelial tissue (colon: n = 2), and pooled adenomas obtained from untreated (adenoma: n = 2) or antibiotic-treated (adenoma Abx: n = 2) MSH2 −/− mice. Error bars represent SD.

(A) The mutant frequency at the lacI gene in MSH2 +/− (n = 3) and MSH2 −/− untreated (n = 4) or antibiotic-treated (n = 3) mice was measured by the Big Blue mutagenesis screen. Error bars represent SD.

Recent reports highlight the role of inflammatory cells, including specific lymphocyte subsets, and the gut microbiota in promoting CRC (). However, the influence of gut microbes on CRC development is likely not through an inflammatory response in this APCMSH2mouse model. First, the composition of the inflammatory cell component of the lamina propria was within normal limits in all genotypes and treatments ( Figure 2 A). Second, neutrophils and plasma cell numbers did not vary between the two genotypes ( Figure 2 B), including IFNγ-positive Th1 cells ( Figure 2 C). Third, the numbers of Treg and Th17 cells, which influence colitis-associated CRC (), were similar between the different genotypes and treatments ( Figures 2 D and 2E). Fourth, polyp numbers were not reduced in APCMSH2mice that were RAG1 deficient ( Figure 2 F), indicating that lymphocytes do not enhance CRC in APCMSH2mice. Fifth, ablating either the inflammasome through caspase-1 deletion or NOD1/2 signaling through RIP2 deletion does not alter polyp numbers in this mouse model ( Figures 2 G and 2H) unlike in colitis-associated models of CRC (). Hence, these genetic manipulations could not recapitulate the 6-fold reduction of colonic polyps that was achieved with antibiotic treatment.

(F–H) Polyp counts in the small intestines and colons of 6-week-old APC Min/+ MSH2 −/− RAG1 +/− (A +/− M −/− R1 +/− ) and APC Min/+ MSH2 −/− RAG1 −/− (A +/− M −/− R1 −/− ) mice (F), APC Min/+ MSH2 −/− Caspase-1 +/− (A +/− M −/− C +/− ) and APC Min/+ MSH2 −/− Caspase-1 −/− (A +/− M −/− C −/− ) mice (G), and APC Min/+ MSH2 −/− RIP2 +/− (A +/− M −/− Rp +/− ) and APC Min/+ MSH2 −/− RIP2 −/− (A +/− M −/− Rp −/− ) mice (H).

(C) Quantification by flow cytometry of lamina propria IFNγ Th1 cells within colonic tissue of the indicated mice.

(B) Quantification of polymorphonuclear leukocytes (PMN) and plasma cells (PC) per high-powered field (HPF) in histological sections of colonic tissue of the indicated mice, n ≥ 7.

We graded the polyps according to established histopathological criteria ( Figure S1 G). This analysis showed that compared to untreated mice, antibiotic-cocktail-treated APCMSH2mice had fewer grade 1 to ≥ 3 polyps in both the small intestine and colon ( Figures 1 E and 1F). Metronidazole and ampicillin also reduced grade 1 to ≥ 3 polyps but only in the colon ( Figure 1 F and data not shown). The fact that antibiotic treatment led to a reduction in grade 1 polyps, which include aberrant crypt foci, argues that gut microbiota in APCMSH2mice act at an early stage in the formation of CRC, perhaps even as a tumor initiator. Moreover, that metronidazole treatment reduced polyp numbers without affecting total bacterial abundance indicates that not all members of the gut microbiota contribute equally to CRC development in this animal model.

To gain insight into the mechanism of CRC development, we manipulated four known components—Apc and Msh2 mutations, the microbiota, and diet—separately and in combination and measured their effects on polyp numbers in mice. We used mice bearing the APC) and MSH2mutations. To measure the contribution of the microbiota, we altered gut bacterial community structure with antibiotics (ampicillin metronidazole, neomycin, and vancomycin), which were provided to the mice through their drinking water continuously in utero to 6 weeks post-birth. Mice on the antibiotic cocktail exhibited an ∼10,000-fold reduction of colonic bacteria ( Figure 1 A and Figure S1 A available online) and had enlarged cecums ( Figure S1 B), but their body weights were normal ( Figure S1 C). Ampicillin alone reduced bacterial load by ∼1,000-fold, whereas metronidazole did not ( Figure 1 A). The composition of the microbiota was strongly affected in both the metronidazole- and ampicillin-treated mice ( Figures 1 A, 1B, and S1 D). Antibiotic-cocktail treatment had little to no effect on polyp numbers in APCMSH2mice at 3, 6 ( Figures 1 C and 1D), and 12 weeks of age ( Figures S1 E and S1F), supporting a previous report (). Combining the APC mutation with the MSH2mutation led to a large increase in the number of polyps in the small intestine and colon ( Figures 1 C and 1D) as previously observed (). A 16S rRNA gene-based microbiota sequencing analysis showed that the microbial composition was similar between the APCMSH2and APCMSH2genotypes ( Figures 1 B and S1 D), indicating that the increased polyp count in APCMSH2mice was not due to differences in microbial composition. Antibiotic-cocktail treatment only had a marginal effect on polyp numbers in APCMSH2mice at 3 weeks of age ( Figures 1 C and 1D). However, at 6 weeks of age, antibiotic treatment led to an ∼2 and an ∼6-fold reduction in polyp numbers in the small intestines and colons of APCMSH2mice, respectively ( Figures 1 C and 1D). Treatment with single antibiotics (ampicillin or metronidazole) led to reduced polyp numbers in the colons of APCMSH2mice but not in the small intestines ( Figures 1 C and 1D).

(G) Histological grading of polyps in antibiotic-treated mice. Photomicrographs depicting polyps of different histological grades in the colon of A +/− M −/− mice: Grade 1 adenoma represents aberrant crypt foci; Grade 2 adenoma up to moderate dysplasia; Grade 3 adenoma with high-grade dysplasia; Grade 3.5 intramucosal carcinoma. Magnification is 100×.

(E and F) Polyp Counts in APC Min/+ MSH2 +/− mice at 12 weeks of age in the small intestine (E) and colon (F).

(D) Bar chart depicting the relative proportions of the bacterial phyla present in the indicated mice and treatments (each bar represents a single mouse) derived from the 16S rRNA gene sequencing. Although the supplementary plot showing bacterial abundances depicts the phylum level, the principal components analysis was done at the family level.

(C) Body weight of the A +/− M +/− and A +/− M −/− mice untreated or cocktail antibiotic-treated at 6 weeks of age, n ≥ 12.

(A) Stool samples from the most distal part of the small intestine (SI) and the distal portion of the colon of APC Min/+ MSH2 +/− mice were collected under sterile conditions. The mass of each sample was measured, and the stools were resuspended in PBS. Serial dilutions of the resulting supernatant were plated on MacConkey agar plates, and the bacterial colonies were counted after 18 hr incubation at 37°C. Each bar shows the number of bacterial colonies/g stool weight. The errors represent SD from two independent plating experiments for different mice (n = 2).

Effect of Antibiotic Treatment on the Microbiota within the Small Intestine and Colon, Related to Figure 1

(E and F) Pathology scores of 6-week-old mice for polyps observed in the small intestine and colon, respectively.

(C and D) Polyp count in 3- and 6-week-old APC Min/+ MSH2 +/− (A +/− M +/− ) and APC Min/+ MSH2 −/− (A +/− M −/− ) mice untreated and treated with the indicated oral antibiotics in the small intestine and colon, respectively.

(B) Principal components analysis of 16S rRNA gene-sequencing analysis of gut microbes obtained from the indicated mice and treatments. PC1 and PC2 explain 24.14% and 18.24% of variation, respectively.

(A) Total bacterial content was estimated by measuring the abundance of 16S rRNA gene copies/g sample, collected from the colons of APC Min/+ MSH2 +/− mice that were either untreated (Untreat) or treated with the antibiotic cocktail (Abx), ampicillin (Amp), metronidazole (Metro), and 7% low-carbohydrate diet (7% LC).

Discussion

Roy et al., 2006 Roy C.C.

Kien C.L.

Bouthillier L.

Levy E. Short-chain fatty acids: ready for prime time?. Franceschi et al., 2001 Franceschi S.

Dal Maso L.

Augustin L.

Negri E.

Parpinel M.

Boyle P.

Jenkins D.J.

La Vecchia C. Dietary glycemic load and colorectal cancer risk. Gnagnarella et al., 2008 Gnagnarella P.

Gandini S.

La Vecchia C.

Maisonneuve P. Glycemic index, glycemic load, and cancer risk: a meta-analysis. Meyerhardt et al., 2012 Meyerhardt J.A.

Sato K.

Niedzwiecki D.

Ye C.

Saltz L.B.

Mayer R.J.

Mowat R.B.

Whittom R.

Hantel A.

Benson A.

et al. Dietary glycemic load and cancer recurrence and survival in patients with stage III colon cancer: findings from CALGB 89803. Min/+MSH2−/− mice. Our analysis revealed that gut microbes stimulated polyp formation by providing metabolites that stimulated hyperproliferation and transformation of colon epithelial cells in these mice. Hence, in a genetic model of CRC, gut microbiota stimulate polyp formation through a mechanism that is distinct from other models of CRC. Carbohydrates account for about 50% of the daily intake of adults receiving a western-style diet, with starch and sucrose as the largest components (). Previous studies have linked carbohydrate-rich diets to CRC in humans (), although a mechanistic insight into this relationship is lacking. Our study supports the carbohydrate-cancer link by showing that a diet reduced in carbohydrates resulted in reduced polyp formation in APCMSH2mice. Our analysis revealed that gut microbes stimulated polyp formation by providing metabolites that stimulated hyperproliferation and transformation of colon epithelial cells in these mice. Hence, in a genetic model of CRC, gut microbiota stimulate polyp formation through a mechanism that is distinct from other models of CRC.

Bordonaro et al., 2008 Bordonaro M.

Lazarova D.L.

Sartorelli A.C. Hyperinduction of Wnt activity: a new paradigm for the treatment of colorectal cancer?. Tarapore et al., 2012 Tarapore R.S.

Siddiqui I.A.

Mukhtar H. Modulation of Wnt/β-catenin signaling pathway by bioactive food components. Lazarova et al., 2004 Lazarova D.L.

Bordonaro M.

Carbone R.

Sartorelli A.C. Linear relationship between Wnt activity levels and apoptosis in colorectal carcinoma cells exposed to butyrate. Roediger, 1980 Roediger W.E. Role of anaerobic bacteria in the metabolic welfare of the colonic mucosa in man. The expression and activity of β-catenin are precisely regulated to create a gradient that defines the fate of intestinal epithelial cells (). Several dietary factors can modify β-catenin activity (). Between them, butyrate has been shown to modulate canonical Wnt signaling, and depending on the status of β-catenin activity, colon epithelial cells respond differently to butyrate (). In normal colonic epithelium, butyrate is the major source of energy () and synergizes with low levels of Wnt signaling to facilitate normal growth and proliferation. However, MSH2-deficient colon epithelial cells have deregulated β-catenin activity and likely have a different response to microbial-derived butyrate that might result in increased proliferation and apoptosis.

Jiricny, 2006 Jiricny J. The multifaceted mismatch-repair system. −/− cells given that β-catenin regulates colon epithelial cell proliferation ( Gregorieff et al., 2005 Gregorieff A.

Pinto D.

Begthel H.

Destrée O.

Kielman M.

Clevers H. Expression pattern of Wnt signaling components in the adult intestine. Liu et al., 2000 Liu W.

Dong X.

Mai M.

Seelan R.S.

Taniguchi K.

Krishnadath K.K.

Halling K.C.

Cunningham J.M.

Boardman L.A.

Qian C.

et al. Mutations in AXIN2 cause colorectal cancer with defective mismatch repair by activating beta-catenin/TCF signalling. (Cip1/WAF1), which induces cell-cycle inhibition and differentiation of colonic epithelium ( van de Wetering et al., 2002 van de Wetering M.

Sancho E.

Verweij C.

de Lau W.

Oving I.

Hurlstone A.

van der Horn K.

Batlle E.

Coudreuse D.

Haramis A.P.

et al. The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Heijmans et al., 2013 Heijmans J.

van Lidth de Jeude J.F.

Koo B.K.

Rosekrans S.L.

Wielenga M.C.

van de Wetering M.

Ferrante M.

Lee A.S.

Onderwater J.J.

Paton J.C.

et al. ER stress causes rapid loss of intestinal epithelial stemness through activation of the unfolded protein response. Van der Flier et al., 2007 Van der Flier L.G.

Sabates-Bellver J.

Oving I.

Haegebarth A.

De Palo M.

Anti M.

Van Gijn M.E.

Suijkerbuijk S.

Van de Wetering M.

Marra G.

Clevers H. The Intestinal Wnt/TCF Signature. In this report, we found that MSH2 and MLH1 deficiency leads to a hyperproliferative phenotype of colon epithelial cells. Although components of the MMR system can induce cell-cycle arrest in response to extensive DNA damage (), we did not find evidence that the microbiota produce genotoxic agents in this animal model ( Figure S2 ). Indeed, the DDR function of MSH2 is not involved in the hyperproliferative property of colon epithelial cells ( Figure S2 ). Instead, this defect is likely due to the aberrant activation of β-catenin signaling in MSH2cells given that β-catenin regulates colon epithelial cell proliferation (). Notably, overexpression of both β-catenin and c-MYC occurred in all crypts in MSH2- and MLH1-deficient mice in an APC-independent manner, suggesting that MMR proteins have a direct role in modulating β-catenin signaling rather than by, for example, suppressing mutations in this pathway (), which would be expected to lead to sporadic overexpression of β-catenin in some but not all crypts. On the other hand, the increased proliferation of colon epithelial cells in MMR mutant mice could be due to a defect in differentiation from transit-amplifying (proliferating) cells to terminally differentiated (nonproliferating) cells. Indeed, the expanded proliferating cells in MMR mutant colons were positive for β-catenin and c-MYC expression (pro-proliferative) and negative for p21, which induces cell-cycle inhibition and differentiation of colonic epithelium (), supporting the notion that MMR deficiency leads to an expanded transit-amplifying cell population. In fact, endoplasmic reticulum stress has been identified as a differentiation signal for stem cells (), and it is conceivable that genetic stress that is sensed by MMR during the rapidly proliferating transit-amplifying cell stage might have a similar effect. Regardless of the mechanism, because deregulated β-catenin signaling is a marker of early neoplastic changes of intestinal epithelium (), our results suggest that MSH2-deficient colonic epithelial cells are highly predisposed to transformation.