Abstract SY08-03: Metaboloepigenetic effects of microbial-produced butyrate in cancer prevention.
Authors: Donohoe Dallas , Montgomery Stephanie , Collins Leonard , Holley Darcy , Godfrey Virgina , Swenberg James , Bultman Scott 
Published: August 2014

Abstract
It is controversial whether dietary fiber protects against colorectal cancer because of conflicting results from human epidemiologic studies. These studies have been complicated by the participants' genetic heterogeneity and differences in the composition of microbiota within their gastrointestinal tracts. To eliminate these confounding variables, we utilized a gnotobiotic mouse model of colorectal cancer. Our experiments were designed to investigate the function of butyrate because it is a short-chain fatty acid produced by bacterial fermentation of fiber in the colon at high (mM) levels and has potent energetic and epigenetic properties in host colonocytes. Here, we report that fiber did, in fact, have a chemoprotective effect but in a microbiota- and butyrate-dependent manner. The incidence, number, size, and histopathologic progression of AOM/DSS-induced colorectal tumors were significantly diminished when BALB/c mice were provided a high-fiber diet only if they were colonized with defined microbiota that included a butyrate-producing bacteria. This chemoprotective effect was attenuated when mice were colonized with the same microbiota except that the wild-type butyrate producer was replaced by a mutant strain with a 0.8-kb deletion in the butyryl-CoA synthesis operon. To confirm that butyrate was a causal factor, the chemoprotective effect was also observed in mice without any butyrate-producing bacteria if their diet was fortified with a butyrate derivative. Our data support a general mechanism that includes microbial fermentation of fiber rather than fiber exclusively speeding colonic transit to minimize the exposure of colonocytes to ingested carcinogens. Our data also support a molecular mechanism that is metaboloepigenetic. Normal coloncytes utilize butyrate as their preferred energy source, whereas cancerous colonocytes rely on glucose because of the Warburg effect. Due to this metabolic difference, butyrate accumulated in tumors (as measured by LC-MS/MS) and functioned as an HDAC inhibitor to increase global histone acetylation levels and apoptosis. To support the applicability of this model to human cancer, we demonstrate that butyrate also accumulates at higher levels in human colorectal tumors than in normal colonic tissue, and this is associated with higher levels of histone acetylation in tumors. These results link diet and microbiota to a common metabolite that influences epigenetics and cancer predisposition.
To investigate the metaboloepigenetic mechanism in more detail, we evaluated the effect of butyrate in colorectal cancer cell lines in the presence of the Warburg effect and when it was prevented from occurring by growing the tumor cells in low glucose or depleting lactate dehydrogenase levels (siLDHA). Low doses of butyrate (0.5-1 mM) inhibited cell proliferation in the presence of the Warburg effect by acting as an epigenetic factor (by inducing histone acetylation) but stimulated proliferation in the absence of the Warburg effect by acting as an energy source. Low doses of butyrate also stimulated the proliferation of non-cancerous colonocytes, which do not undergo the Warburg effect without any experimental manipulation. Higher doses of butyrate (2-5 mM), which exceed the metabolic capacity of the cell to oxidize butyrate (but are still physiologically relevant), induced histone acetylation and apoptosis regardless of the Warburg effect. At the lower doses, where butyrate was metabolized, it was converted to acetyl-CoA, and this was important not only for energetics but also for epigenetics because it served as a HAT co-factor to stimulate histone acetylation. Although the acetyl-CoA/HAT and HDAC inhibition mechanisms both stimulate histone acetylation, they were differentially utilized and upregulated different target genes. The acetyl-Co-A/HAT mechanism was predominant in normal cells and at low butyrate doses regardless of the Warbug effect and upregulated cell proliferation genes, whereas the HDAC inhibition mechanism was predominant in cancerous colonocytes and at high butyrate doses regardless of the Warburg effect and upregulated pro-apoptotic genes. These data have important implications in vivo. Because mucus produced by goblet cells within the crypts flows upward into the lumen, an endogenous butyrate gradient is believed to exist with lower concentrations at the base of crypts (&lt;1 mM) than in the lumen (2-5 mM). Therefore, butyrate may contribute to normal colonic homeostasis by promoting the proliferation of stem cells and transit amplifying cells near the base while inducing apoptosis in cells exfoliating into the lumen. And due to the Warburg effect in tumor cells, butyrate is not readily metabolized, accumulates, and functions primarily as an HDAC inhibitor regardless of position within the epithelium. These results indicate that metabolic transformation (i.e., the Warburg effect) can drive aberrant epigenetic (histone acetylation) and transcriptome profiles in tumor cells compared to their cell of origin.
Citation Format: Dallas Donohoe, Stephanie Montgomery, Leonard Collins, Darcy Holley, Virgina Godfrey, James Swenberg, Scott Bultman. Metaboloepigenetic effects of microbial-produced butyrate in cancer prevention. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY08-03. doi:10.1158/1538-7445.AM2013-SY08-03