Anna C. Pfalzer

Diet- and Genetically-Induced Obesity Differentially Affect the Fecal Microbiome and Metabolome in Apc1638N Mice.

Obesity is a risk factor for colorectal cancer (CRC), and alterations in the colonic microbiome and metabolome may be mechanistically involved in this relationship. The relative contribution of diet and obesity per se are unclear. We compared the effect of diet- and genetically-induced obesity on the intestinal microbiome and metabolome in a mouse model of CRC. Apc1638N mice were made obese by either high fat (HF) feeding or the presence of the Leprdb/db (DbDb) mutation. Intestinal tumors were quantified and stool microbiome and metabolome were profiled. Genetic obesity, and to a lesser extent HF feeding, promoted intestinal tumorigenesis. Each induced distinct microbial patterns: taxa enriched in HF were mostly Firmicutes (6 of 8) while those enriched in DbDb were split between Firmicutes (7 of 12) and Proteobacteria (5 of 12). Parabecteroides distasonis was lower in tumor-bearing mice and its abundance was inversely associated with colonic Il1b production (p<0.05). HF and genetic obesity altered the abundance of 49 and 40 fecal metabolites respectively, with 5 in common. Of these 5, adenosine was also lower in obese and in tumor-bearing mice (p<0.05) and its concentration was inversely associated with colonic Il1b and Tnf production (p<0.05). HF and genetic obesity differentially alter the intestinal microbiome and metabolome. A depletion of adenosine and P.distasonis in tumor-bearing mice could play a mechanistic role in tumor formation. Adenosine and P. distasonis have previously been shown to be anti-inflammatory in the colon and we postulate their reduction could promote tumorigenesis by de-repressing inflammation.

Interactions between the colonic transcriptome, metabolome and microbiome in mouse models of obesity-induced intestinal cancer

Obesity is a significant risk factor for colorectal cancer (CRC); however, the relative contribution of high-fat (HF) consumption and excess adiposity remains unclear. It is becoming apparent that obesity perturbs both the intestinal microbiome and metabolome, and each has the potential to induce protumorigenic changes in the epithelial transcriptome. The physiological consequences and the degree to which these different biologic systems interact remain poorly defined. To understand the mechanisms by which obesity drives colonic tumorigenesis, we profiled the colonic epithelial transcriptome of HF-fed and genetically obese (DbDb) mice with a genetic predisposition to intestinal tumorigenesis (Apc(1638N)); 266 and 584 genes were differentially expressed in the colonic mucosa of HF and DbDb mice, respectively. These genes mapped to pathways involved in immune function, and cellular proliferation and cancer. Furthermore, Akt was central within the networks of interacting genes identified in both gene sets. Regression analyses of coexpressed genes with the abundance of bacterial taxa identified three taxa, previously correlated with tumor burden, to be significantly correlated with a gene module enriched for Akt-related genes. Similarly, regression of coexpressed genes with metabolites found that adenosine, which was negatively associated with inflammatory markers and tumor burden, was also correlated with a gene module enriched with Akt regulators. Our findings provide evidence that HF consumption and excess adiposity result in changes in the colonic transcriptome that, although distinct, both appear to converge on Akt signaling. Such changes could be mediated by alterations in the colonic microbiome and metabolome.

Diet- and Genetically-induced Obesity Produces Alterations in the Microbiome, Inflammation and Wnt Pathway in the Intestine of Apc(+/1638N) Mice: Comparisons and Contrasts.

Obesity is an established risk factor for colorectal cancer (CRC). Our previous study indicated that obesity increases activity of the pro-tumorigenic Wnt-signaling. Presently, we sought to further advance our understanding of the mechanisms by which obesity promotes CRC by examining associations between microbiome, inflammation and Wnt-signaling in Apc+/1638N mice whose obesity was induced by one of two modalities, diet- or genetically-induced obesity. Three groups were employed: Apc+/1638NLepr+/+ fed a low fat diet (10% fat), Apc+/1638NLepr+/+ fed a high fat diet (60% fat, diet-induced obesity), and Apc+/1638NLeprdb/db fed a low fat diet (genetically-induced obesity). All animals received diets for 16 weeks from 8 to 24 weeks of age. The abundance of 19 bowel cancer-associated bacterial taxa were examined by real-time PCR. The abundance of Turicibacter and Desulfovibrio decreased, but F. prausnitizii increased, in diet-induced obese mice (p < 0.05). In contrast, in genetically-induced obese mice, Bifidobacterium, A. muciniphila and E. rectale decreased, but Peptostrptococcus, and E. coli increased (p < 0.05). Both diet- and genetically-induced obesity altered the expression of genes involved in bacterial recognition (MyD88) and increased inflammation as indicated by elevated levels of cytokines (IFNγ and TNF-α for genetically-induced obesity, and IL-6 for diet-induced obesity). The elevated inflammation was associated with altered expression of genes that are integral components of the Wnt-signaling cascade in a fashion indicating its activation. These findings demonstrate that the composition of the small intestinal microbiome is affected differently in diet- and genetically-induced obesity, but both are associated with elevated intestinal inflammation and alterations of the Wnt pathway towards enhancing tumorigenesis.

Paternal B Vitamin Intake Is a Determinant of Growth, Hepatic Lipid Metabolism and Intestinal Tumor Volume in Female Apc1638N Mouse Offspring

Background The importance of maternal nutrition to offspring health and risk of disease is well established. Emerging evidence suggests paternal diet may affect offspring health as well. Objective In the current study we sought to determine whether modulating pre-conception paternal B vitamin intake alters intestinal tumor formation in offspring. Additionally, we sought to identify potential mechanisms for the observed weight differential among offspring by profiling hepatic gene expression and lipid content. Methods Male Apc1638N mice (prone to intestinal tumor formation) were fed diets containing replete (control, CTRL), mildly deficient (DEF), or supplemental (SUPP) quantities of vitamins B2, B6, B12, and folate for 8 weeks before mating with control-fed wild type females. Wild type offspring were euthanized at weaning and hepatic gene expression profiled. Apc1638N offspring were fed a replete diet and euthanized at 28 weeks of age to assess tumor burden. Results No differences in intestinal tumor incidence or burden were found between male Apc1638N offspring of different paternal diet groups. Although in female Apc1638N offspring there were no differences in tumor incidence or multiplicity, a stepwise increase in tumor volume with increasing paternal B vitamin intake was observed. Interestingly, female offspring of SUPP and DEF fathers had a significantly lower body weight than those of CTRL fed fathers. Moreover, hepatic trigylcerides and cholesterol were elevated 3-fold in adult female offspring of SUPP fathers. Weanling offspring of the same fathers displayed altered expression of several key lipid-metabolism genes. Hundreds of differentially methylated regions were identified in the paternal sperm in response to DEF and SUPP diets. Aside from a few genes including Igf2, there was a striking lack of overlap between these genes differentially methylated in sperm and differentially expressed in offspring. Conclusions In this animal model, modulation of paternal B vitamin intake prior to mating alters offspring weight gain, lipid metabolism and tumor growth in a sex-specific fashion. These results highlight the need to better define how paternal nutrition affects the health of offspring.

The influence of tumor necrosis factor-α on the tumorigenic Wnt -signaling pathway in human mammary tissue from obese women

Epidemiological studies have convincingly suggested that obesity is an important risk factor for postmenopausal breast cancer, but the mechanisms responsible for this relationship are still not fully understood. We hypothesize that obesity creates a low-grade inflammatory microenvironment, which stimulates Wnt-signaling and thereby promotes the development of breast cancer. To test this hypothesis, we evaluated the correlations between expression of multiple inflammatory cytokines and Wnt pathway downstream genes in mammary tissues from women (age ≥ 50) undergoing reduction mammoplasty. Moreover, we specifically examined the role of tumor necrosis factor-α (TNF-α), an important proinflammatory cytokine associated with obesity and a possible modulator of the Wnt pathway. The regulatory effects of TNF-α on Wnt pathway targets were measured in an ex vivo culture of breast tissue treated with anti-TNF-α antibody or TNF-α recombinant protein. We found that BMI was positively associated with the secretion of inflammatory cytokines IL-1β, IL-6 and TNF-α, all of which were negatively correlated with the expression of SFRP1. The transcriptional expression of Wnt-signaling targets, AXIN2 and CYCLIN D1, were higher in mammary tissue from women with BMI ≥ 30 compared to those with BMI < 30. Our ex vivo work confirmed that TNF-α is causally linked to the up-regulation of active β-CATENIN, a key component in the Wnt pathway, and several Wnt-signaling target genes (i.e. CYCLIN D1, AXIN2, P53 and COX-2). Collectively, these findings indicate that obesity-driven inflammation elevates Wnt-signaling in mammary tissue and thereby creates a microenvironment conducive to the development of breast cancer.

Curcumin and Salsalate Suppresses Colonic Inflammation and Procarcinogenic Signaling in High-Fat-Fed, Azoxymethane-Treated Mice

High-fat diets (HFDs) and excess adiposity increase proinflammatory cytokines in the colon, altering gene expression in a manner that promotes the development of colorectal cancer (CRC). Thus, compounds that reduce this biochemical inflammation are potential chemopreventive agents. Curcumin (CUR), a dietary polyphenol, and salsalate (SAL), a non-steroidal anti-inflammatory drug, are both anti-inflammatories. We investigated the inhibitory effects of CUR with or without SAL on inflammatory cytokines and procarcinogenic signaling in azoxymethane (AOM)-treated A/J mice. A sub-tumorigenic AOM dose was chosen to produce a biochemical and molecular procarcinogenic colonic environment without tumors. Mice were fed either a HFD (60% of kilocalories) or low-fat diet (LFD) (10% of kilocalories). One HFD treatment group received 0.2% CUR in the diet; one received 0.2% CUR + 0.15% SAL; and one received 0.4% CUR + 0.3% SAL. The HFD mice developed 30% greater fat mass than the LFD mice (p < 0.05). The colonic concentrations of interleukin-1β (IL-1β) and interleukin-6 (IL-6) in the HFD mice were decreased by 50-69% by the high-dose combination regimen (p < 0.015). Only the combination regimens significantly suppressed phosphorylation of protein kinase B (Akt) and nuclear factor-κB (NF-κB) p65 (p < 0.044). The combination of CUR and SAL reduces the concentration of proinflammatory cytokines and diminishes activation of Akt and NF-κB more effectively than CUR alone, providing a scientific basis for examining whether this combination mitigates the risk of CRC in obese individuals.

Interleukin-1 Signaling Mediates Obesity-Promoted Elevations in Inflammatory Cytokines, Wnt Activation, and Epithelial Proliferation in the Mouse Colon

Obesity is a prominent risk factor for colorectal cancer (CRC). One mechanism by which obesity promotes the development of CRC is by generating a chronic, low-grade state of colonic inflammation. Interleukin-1β (IL-1β), a proinflammatory cytokine often elevated in obesity, is known to activate several procarcinogenic signaling pathways that are implicated in colonic carcinogenesis. We therefore sought to define the role of IL-1β in mediating some of the early biochemical and molecular events leading up to obesity-promoted CRC. Twenty-five wild-type (WT) C57BL/6J mice and 24 lacking a functional IL-1 receptor (IL1R-/-) were each randomized to either low-fat or high-fat diets, resulting in lean and obese mice. Compared to WT lean controls, WT obese mice displayed 30%-80% greater concentrations of IL-1β and tumor necrosis factor-α (TNF-α) in the colonic mucosa (IL-1β: P = 0.04; TNF-α: P < 0.05), activation of the Wnt signaling cascade [evidenced by a 2-fold increase in colonic crypt cells displaying intranuclear β-catenin (P < 0.03)], and a significant expansion of the proliferation zone of the colonic crypt (P < 0.04). These obesity-induced alterations in colonic cytokines, Wnt signaling, and proliferation were absent in the obese IL1R-/- mice. In the absence of IL-1 signaling, obesity-induced elevations of colonic IL-1β, TNF-α, Wnt activation, and enhanced epithelial proliferation no longer occur. These observations underscore the important mechanistic roles that IL-1 signaling appears to play in mediating the procancerous effects of obesity in the colon, thereby identifying a potential target for future strategies aimed at chemoprevention.

Fathers Sperm MeDIP raw data

Raw data of methyl-DNA immunoprecipitation analysis of fathers sperm with Nimblegen arrays. Sample number in the top row.