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. 2014 Dec;4(12):1387-97.
doi: 10.1158/2159-8290.CD-14-0501. Epub 2014 Sep 29.

A gnotobiotic mouse model demonstrates that dietary fiber protects against colorectal tumorigenesis in a microbiota- and butyrate-dependent manner

Dallas R Donohoe  1 , Darcy Holley  1 , Leonard B Collins  2 , Stephanie A Montgomery  3 , Alan C Whitmore  4 , Andrew Hillhouse  5 , Kaitlin P Curry  1 , Sarah W Renner  1 , Alicia Greenwalt  1 , Elizabeth P Ryan  6 , Virginia Godfrey  7 , Mark T Heise  4 , Deborah S Threadgill  8 , Anna Han  9 , James A Swenberg  2 , David W Threadgill  10 , Scott J Bultman  11
Affiliations

A gnotobiotic mouse model demonstrates that dietary fiber protects against colorectal tumorigenesis in a microbiota- and butyrate-dependent manner

Dallas R Donohoe et al. Cancer Discov. 2014 Dec.

Abstract

Whether dietary fiber protects against colorectal cancer is controversial because of conflicting results from human epidemiologic studies. However, these studies and mouse models of colorectal cancer have not controlled the composition of gut microbiota, which ferment fiber into short-chain fatty acids such as butyrate. Butyrate is noteworthy because it has energetic and epigenetic functions in colonocytes and tumor-suppressive properties in colorectal cancer cell lines. We used gnotobiotic mouse models colonized with wild-type or mutant strains of a butyrate-producing bacterium to demonstrate that fiber does have a potent tumor-suppressive effect but in a microbiota- and butyrate-dependent manner. Furthermore, due to the Warburg effect, butyrate was metabolized less in tumors where it accumulated and functioned as a histone deacetylase (HDAC) inhibitor to stimulate histone acetylation and affect apoptosis and cell proliferation. To support the relevance of this mechanism in human cancer, we demonstrate that butyrate and histone-acetylation levels are elevated in colorectal adenocarcinomas compared with normal colonic tissues.

Significance: These results, which link diet and microbiota to a tumor-suppressive metabolite, provide insight into conflicting epidemiologic findings and suggest that probiotic/prebiotic strategies can modulate an endogenous HDAC inhibitor for anticancer chemoprevention without the adverse effects associated with synthetic HDAC inhibitors used in chemotherapy.

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Figures

Figure 1
Figure 1
A high-fiber diet protects against colorectal tumors in a microbiota- and butyrate-dependent manner. (A, B) Scatter plots showing tumor multiplicity for mice in each treatment group following low (A) and high (B) carcinogen exposures. Means are shown, and symbols denote groups with statistically-significant differences: * versus #, p < 0.01; * versus +, p = 0.05; # versus +, p < 0.05. (C) Tumor size presented as mean ± SE with significant differences indicated (* versus #, p < 0.01). (D) Percentage of low-grade versus high-grade tumors for each treatment group based on H&E characteristics of dysplasia within tumors as described in panel E. (E) H&E-stained sections representative of normal colonic epithelium, low-grade tumors, and high-grade tumors. Compared to normal colon, low-grade tumors exhibit hyperplasia (circled region) and are less differentiated with fewer goblet cells that are identified by mucous vacuoles (black dot). High-grade tumors exhibit these features plus a loss of polarity (dotted circled region) and have crypt abscesses with apoptotic material (star). A subset of high-grade tumors display signs of potential invasion based on tumor cells within the muscularis (inset).
Figure 2
Figure 2
Colorectal tumors exhibit characteristics of the Warburg effect, accumulate butyrate, and have increased H3ac levels. (A) Representative western blot of LDHA in normal colonic tissue and a colorectal tumor. β-actin serves as a loading control. (B) Quantification of LDHA levels normalized to β-actin in normal colonic tissues and tumors based on western blot data. Each histogram shows the mean ± SE based on 3 biological replicates (i.e. colonic tissue and tumors from 3 separate mice) with significant differences indicated (*, p < 0.05). (C) Lactate levels in normal colonic tissue and colorectal tumors based on LC-MS measurements. Results are normalized to μg of protein, and each histogram shows the mean ± SE of 4-5 biological replicates (i.e. colonic tissue and tumors from 4-5 separate mice) with significant differences indicated (*, p < 0.05). (D) Representative LC-MS chromatograms showing butyrate levels in normal colonic tissue and a colorectal tumor. (E) Butyrate levels in normal colonic tissues and tumors from mice in control and experimental treatment groups. Values are based on LC-MS measurements, and each histogram shows the mean ± SE of 5 biological replicates (i.e. colonic tissue and tumors from 5 separate mice) with significant differences indicated (*, p < 0.05; **, p < 0.01). (F) Representative IHC image showing H3ac staining intensity in tumor cells and adjacent normal colonic epithelial cells. (G) Western blot analysis of H3ac and total H3 levels in tumors from mice of the different treatment groups. (H) Quantification of western blot data with H3ac levels normalized to total H3 in tumors from mice of each treatment group. LFD, low-fiber diet; HFD, high-fiber diet. Each histogram shows the mean ± SE based on 5 biological replicates for each treatment group with significant differences indicated (*, p < 0.05; **, p < 0.01). (I) HDAC activity levels normalized to protein levels in normal colonic tissue and tumors from control and experimental treatment groups. Each histogram shows the mean ± SE for 5 independent experiments with significant differences indicated (*, p < 0.05; **, p < 0.01).
Figure 3
Figure 3
Tumors from mice provided a high-fiber diet and colonized with B. fibrisolvens (experimental treatment group) have increased H3ac and expression levels of target genes and undergo increased apoptosis and decreased cell proliferation. (A,C,E) Quantitative ChIP assays showing H3ac enrichment at the promoters of the Fas, p21, and p27 genes in normal colonic tissues and tumors as indicated. qPCR results for each ChIP were normalized to input, and the values of normal colonic tissues from control mice were set at 1.0. Each histogram shows the mean ± SE for 3-4 independent experiments with significant differences indicated (*, p < 0.05; **, p < 0.01). (B,D,F) RT-qPCR analysis of Fas, p21, and p27 mRNA levels normalized to Gapdh levels in normal colonic tissues and tumors. The values of normal colonic tissues from control mice were set at 1.0. Each histogram shows the mean ± SE for 5 independent experiments with significant differences indicated (*, p < 0.05; **, p < 0.01). (G) Quantification of cleaved caspase 3 positive cells in normal colonic epithelial cells and tumors from the control and experimental treatment groups. Each histogram shows the mean ± SE based on 10 biological replicates for each group with significant differences indicated (*, p < 0.05; **, p < 0.01). (H) Quantification of Ki-67 levels in normal colonic epithelial cells and tumors from the control and experimental treatment groups. The values of normal colonic tissues from control mice were set at 1.0. Each histogram shows the mean ± SE based on 10 biological replicates for each group with significant differences indicated (*, p < 0.05).
Figure 4
Figure 4
A model depicting the mechanism of the fiber-microbiota-butyrate axis in tumor suppression and evidence to support the relevance of this model to human cancer prevention. (A- D) Relevance of the model to human cancer. (A) Butyrate levels in macroscopically normal colonic tissue and colorectal adenocarcinomas from humans based on LC-MS measurements. Each histogram shows the mean ± SE from 11 biological replicates of each with significant differences indicated (*, p < 0.05). (B) A scatter plot of the samples from panel A. The samples were obtained from the University of North Carolina at Chapel Hill (white, left) and Colorado State University (black, right) tissue procurement facilities. (C) Western blot analysis of H3ac and total H3 levels in macroscopically normal colonic tissue and colorectal adenocarcinomas from 2 pairs of human samples. (D) Quantification of western blot data with H3ac levels normalized to total H3 in macroscopically normal colonic tissue and colorectal adenocarcinomas. Each histogram shows the mean ± SE from 5 biological replicates of each with significant differences indicated (*, p < 0.05). (E) Final working model.Dietary fiber is fermented by microbiota into butyrate, which is a short-chain fatty acid that is abundant (mM levels) in the lumen of the colon. In normal colonocytes, butyrate undergoes oxidative metabolism in the mitochondria to promote homeostasis. In the cancerous colonocyte, due to the Warburg effect, butyrate is metabolized to a lesser extent and accumulates in the nucleus where it functions as an HDAC inhibitor to induce apoptosis and inhibit cell proliferation. The importance of the Warburg effect in this model draws upon previous work (22). In addition to these cell-autonomous effects within the cancer cell, some of the tumor suppression could be mediated by butyrate promoting Treg cell differentiation and having anti-inflammatory effects in the presence of a complex gut microbiota.
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