Folic Acid Supplementation Promotes Hypomethylation in Both the Inflamed Colonic Mucosa and Colitis-Associated Dysplasia.

PURPOSE: The purpose of this study was to assess the effect of folic acid (FA) supplementation on colitis-associated colorectal cancer (CRC) using the azoxymethane/dextran sulfate sodium (AOM/DSS) model. METHODS: Mice were fed a chow containing 2 mg/kg FA at baseline and randomized after the first DSS treatment to receive 0, 2, or 8 mg/kg FA chow for 16 weeks. Colon tissue was collected for histopathological evaluation, genome-wide methylation analyses (Digital Restriction Enzyme Assay of Methylation), and gene expression profiling (RNA-Seq). RESULTS: A dose-dependent increase in the multiplicity of colonic dysplasias was observed, with the multiplicity of total and polypoid dysplasias higher (64% and 225%, respectively) in the 8 mg FA vs. the 0 mg FA group (p < 0.001). Polypoid dysplasias were hypomethylated, as compared to the non-neoplastic colonic mucosa (p < 0.05), irrespective of FA treatment. The colonic mucosa of the 8 mg FA group was markedly hypomethylated as compared to the 0 mg FA group. Differential methylation of genes involved in Wnt/ß-catenin and MAPK signaling resulted in corresponding alterations in gene expression within the colonic mucosa. CONCLUSIONS: High-dose FA created an altered epigenetic field effect within the non-neoplastic colonic mucosa. The observed decrease in site-specific DNA methylation altered oncogenic pathways and promoted colitis-associated CRC.

High Variability in Cellular Proliferation, Gene Expression, and Cytokine Production in the Nonneoplastic Colonic Epithelium of Young Apc+/Min-FCCC Mice.

An urgent need exists to identify efficacious therapeutic preventive interventions for individuals who are at high risk of developing colorectal cancer. To maximize the benefits of preventive intervention, it is vital to identify the time interval during which the initiation of a preventive intervention will lead to an optimal outcome. The goal of the present study was to determine if oncogenic events can be detected in the nonneoplastic colonic mucosa of Apc+/Min-FCCC mice prior to formation of the first adenoma, thus defining an earlier point of intervention along the cancer continuum. Tissues taken at three potential points of intervention were characterized: prior to Apc mutation (wild type Apc+/+-FCCC mice); after initiation but prior to colon adenoma formation (tumor-free Apc+/Min-FCCC mice); and after formation of the first colon adenoma (tumor-bearing Apc+/Min-FCCC mice). Experimentation focused on molecular processes that are dysregulated in early colon lesions: 1) cellular proliferation (proliferative index and size of the proliferative zone); 2) cellular stemness (expression of Ascl2, Grem1, Lgr5 and Muc2); 3) EGFR signaling (expression of Ereg); and 4) inflammation (expression of Mmp9, Ptsg2, and Reg4, as well as secretion of 18 cytokines involved in immune activation and response). Interestingly, the nonneoplastic colonic mucosa of wild type, tumor-free Apc+/Min-FCCC , and tumor-bearing Apc+/Min-FCCC mice did not display significant differences in average epithelial cell proliferation (fold change 0.8-1.3, p≥0.11), mucosal gene expression (fold change 0.8-1.4, p≥0.22), or secretion of specific cytokines from colonic mucosa (fold change 0.2-1.5, p≥0.06). However, the level of cytokine secretion was highly variable, with many (22% of wild type, 31% of tumor-free Apc+/Min-FCCC , and 31% of tumor-bearing Apc+/Min-FCCC ) mice categorized as outliers (> 1.5 x interquartile ranges below the first quartile or above the third quartile) due to elevated expression of at least one cytokine. In summary, no differences were observed in proliferation, stemness, and EGFR signaling in the colonic mucosa of wild type vs Apc+/Min-FCCC mice, with low baseline cytokine expression, prior to the formation of the first colon adenoma. The results of this study provide valuable baseline data to inform the design of future cancer prevention studies.

The Loss of TBK1 Kinase Activity in Motor Neurons or in All Cell Types Differentially Impacts ALS Disease Progression in SOD1 Mice.

DNA sequence variants in the TBK1 gene associate with or cause sporadic or familial amyotrophic lateral sclerosis (ALS). Here we show that mice bearing human ALS-associated TBK1 missense loss-of-function mutations, or mice in which the Tbk1 gene is selectively deleted in motor neurons, do not display a neurodegenerative disease phenotype. However, loss of TBK1 function in motor neurons of the SOD1G93A mouse model of ALS impairs autophagy, increases SOD1 aggregation, and accelerates early disease onset without affecting lifespan. By contrast, point mutations that decrease TBK1 kinase activity in all cells also accelerate disease onset but extend the lifespan of SOD1 mice. This difference correlates with the failure to activate high levels of expression of interferon-inducible genes in glia. We conclude that loss of TBK1 kinase activity impacts ALS disease progression through distinct pathways in different spinal cord cell types and further implicate the importance of glia in neurodegeneration.

Self-antigen MASH2 combined with the AS15 immunostimulant induces tumor protection in colorectal cancer mouse models.

Human achaete scute homolog 2 (HASH2) and its murine ortholog MASH2 are potential targets for colorectal cancer immunotherapy. We assessed immunogenicity and antitumor potential of recombinant MASH2 protein combined with AS15 immunostimulant (recMASH2+AS15) in CB6F1 and Apc+/Min-FCCC mice. CB6F1 mice received 4 injections of recMASH2+AS15 or AS15 alone before challenge with TC1-MASH2 tumor cells (Tumor Challenge). Apc+/Min-FCCC mice received 9 injections of recMASH2+AS15 or vehicle (phosphate buffer saline [PBS] or AS15 alone), before (two independent Prophylactic Studies) or after (Immunotherapy) colon adenomas were detectable by colonoscopy. CB6F1 mice immunized with recMASH2+AS15 had a significantly smaller mean tumor size and improved survival rate compared to controls (104 mm2 vs. 197 mm2 [p = 0.009] and 67% vs. 7% [p = 0.001], respectively). In Prophylactic Study 1, the mean number of colon adenomas was significantly lower in Apc+/Min-FCCC mice receiving recMASH2+AS15 compared to PBS (1.8 [95% confidence interval 1.0-3.3] vs. 5.2 [3.7-7.4], p = 0.003). Fewer microadenomas were observed in recMASH2+AS15 groups compared to PBS in both Prophylactic Studies (Study 1: mean 0.4 [0.2-1.0] vs. 1.5 [0.9-2.4], p = 0.009; Study 2: 0.4 [0.2-0.6] vs. 1.1 [0.8-1.5], p = 0.001). In the Immunotherapy Study, fewer colon adenomas tended to be observed in recMASH2+AS15-treated mice (4.1 [2.9-6.0]) compared to controls (AS15 4.7 [3.3-6.6]; PBS 4.9 [3.5-6.9]; no significant difference). recMASH2+AS15 induced MASH2-specific antibody and CD4+ responses in both mouse models. recMASH2+AS15 partially protected mice against MASH2-expressing tumors and reduced spontaneous colorectal adenomas in Apc+/Min-FCCC mice, indicating that MASH2/HASH2 antigens are targets for colorectal cancer immunotherapy.