Trans-lesion synthesis (TLS) is an important DNA-damage tolerance mechanism that permits ongoing DNA synthesis in cells harbouring damaged genomes. The E3 ubiquitin ligase RAD18 activates TLS by ...promoting recruitment of Y-family DNA polymerases to sites of DNA-damage-induced replication fork stalling. Here we identify the cancer/testes antigen melanoma antigen-A4 (MAGE-A4) as a tumour cell-specific RAD18-binding partner and an activator of TLS. MAGE-A4 depletion from MAGE-A4-expressing cancer cells destabilizes RAD18. Conversely, ectopic expression of MAGE-A4 (in cell lines lacking endogenous MAGE-A4) promotes RAD18 stability. DNA-damage-induced mono-ubiquitination of the RAD18 substrate PCNA is attenuated by MAGE-A4 silencing. MAGE-A4-depleted cells fail to resume DNA synthesis normally following ultraviolet irradiation and accumulate γH2AX, thereby recapitulating major hallmarks of TLS deficiency. Taken together, these results demonstrate a mechanism by which reprogramming of ubiquitin signalling in cancer cells can influence DNA damage tolerance and probably contribute to an altered genomic landscape.
The E3 ubiquitin ligase Rad18 mediates tolerance of replication fork-stalling bulky DNA lesions, but whether Rad18 mediates tolerance of bulky DNA lesions acquired outside S-phase is unclear. Using ...synchronized cultures of primary human cells, we defined cell cycle stage-specific contributions of Rad18 to genome maintenance in response to ultraviolet C (UVC) and H(2)O(2)-induced DNA damage. UVC and H(2)O(2) treatments both induced Rad18-mediated proliferating cell nuclear antigen mono-ubiquitination during G(0), G(1) and S-phase. Rad18 was important for repressing H(2)O(2)-induced (but not ultraviolet-induced) double strand break (DSB) accumulation and ATM S1981 phosphorylation only during G(1), indicating a specific role for Rad18 in processing of oxidative DNA lesions outside S-phase. However, H(2)O(2)-induced DSB formation in Rad18-depleted G1 cells was not associated with increased genotoxin sensitivity, indicating that back-up DSB repair mechanisms compensate for Rad18 deficiency. Indeed, in DNA LigIV-deficient cells Rad18-depletion conferred H(2)O(2)-sensitivity, demonstrating functional redundancy between Rad18 and non-homologous end joining for tolerance of oxidative DNA damage acquired during G(1). In contrast with G(1)-synchronized cultures, S-phase cells were H(2)O(2)-sensitive following Rad18-depletion. We conclude that although Rad18 pathway activation by oxidative lesions is not restricted to S-phase, Rad18-mediated trans-lesion synthesis by Polη is dispensable for damage-tolerance in G(1) (because of back-up non-homologous end joining-mediated DSB repair), yet Rad18 is necessary for damage tolerance during S-phase.
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.
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.
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 is a causal factor, the chemoprotective effect was recapitulated in mice without any butyrate-producing bacteria if they were provided a butyrate-fortified diet. 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 colonocytes 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) and functioned as an HDAC inhibitor to increase 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.
Citation Format: Dallas Donohoe, Darcy Holley, Leonard Collins, Stephanie Montgomery, Alan Whitmore, Andrew Hillhouse, Kaitlin Curry, Sarah Renner, Alicia Greenwalt, Elizabeth Ryan, Virginia Godfrey, Mark Heise, Deborah Threadgill, James Swenberg, David Threadgill, Scott Bultman. Dietary fiber protects against colorectal tumorigenesis in a microbiota- and butyrate-dependent manner. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY04-02. doi:10.1158/1538-7445.AM2014-SY04-02
Abstract
It is controversial whether dietary fiber protects against colorectal cancer 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 energentic and epigenetic functions in colonocytes and tumor-suppressive properties in colorectal cancer cell lines. We colonized BALB/c mice with wild-type or mutant strains of a butyrate-producing bacterium in a gnotobiotic facility, provided them with high- or low-fiber diets that were otherwise identical and isocaloric, and used azoxymethane (AOM) to induce colorectal tumors. Analysis of these gnotobiotic mouse models demonstrated that fiber conferred a significant tumor-suppressive effect but in a microbiota- and butyrate-dependent manner. To confirm that butyrate is a causal factor, the anticancer chemoprotective effect was recapitulated in mice without any butyrate-producing bacteria when they were provided a tributyrin-fortified diet. 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 colonocytes utilize butyrate as their primary energy source, whereas cancerous colonocytes rely on glucose because of the Warburg effect. Due to this metabolic difference, butyrate accumulated in tumors and functioned as an HDAC inhibitor to increase histone acetylation levels globally and at pro-apototic (Fas) and cell-cycle (p21 and p27) target genes, which culminated in increased apoptosis and decreased 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 to normal colonic tissues. 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.
Citation Format: Dallas Donohoe, Darcy Holley, Leonard Collins, Stephanie Montgomery, Alan Whitmore, Kaitlin Curry, Sarah Renner, Alicia Greenwalt, Elizabeth Ryan, Virginia Godfrey, Deborah Threadgill, James Swenberg, David Threadgill, Scott Bultman. A gnotobiotic mouse model demonstrates that dietary fiber protects against colorectal tumorigenesis in a microbiota- and butyrate-dependent manner. abstract. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr PL03-01.
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