Research Project: Cancer Prevention via Diet

Location: Jean Mayer Human Nutrition Research Center On Aging

2015 Annual Report

Objectives
LAB NAME: Vitamins and Carcinogenesis 1: Define the role that intake, or nutrient status, of each of the one-carbon nutrients plays in determining the risk of common cancers and to examine select environmental and genetic factors that further modify the effects of 1-carbon nutrients. Through the use of cell culture, animal models and human studies, determine the cell-signaling pathways(s) through which the effects of 1-carbon nutrients is exerted, and elucidate genetic and epigenetic mechanisms by which this occurs. In animal models, also examine how parental intake of these nutrients influences cancer risk in offspring. 1.1: Determine the effect of maternal B vitamin intake on tumorigenesis in offspring. 1.2: Determine the effect of paternal B vitamin intake on tumorigenesis in offspring. 2: Define the cellular pathways by which obesity, and related factors, enhance cancer risk and explore means of attenuating that risk. By use of cell culture studies, animal models, and human studies, focus on how the chronic, low-grade inflammation produced by obesity incites molecular processes that lead to cancer. 2.1: Define the effects of obesity-induced elevations of colonic IL-1ß on Wnt and NF'B activation in the colonic mucosa of mice. 2.2: Determine the effect of genetic and immunologic blockade of IL-1ß on obesity-induced tumorigenesis. 2.3: Determine the effects of an orally-available inhibitor of Akt on obesity-promoted tumorigenesis in AOM-treated mice. 2.4: Determine whether obesity in humans raises pro-inflammatory cytokine levels in the colonic mucosa and whether the elevation in cytokines is accompanied by activation of mucosal Wnt and NF'B. LAB NAME: Nutrition and Cancer Biology 1: Determine the ability of carotenoid-enriched food, carotenoids, and apocarotenoids (carotenoid cleavage metabolites by beta-carotene 9’,10’-oxygenase, BCO2) to induce SIRT1 by regulating microRNAs for preventing obesity-related inflammatory responses and cancer development in liver and colon. 1.1: Determine the protective effects of tomato extract, lycopene and apolycopenoids against high-fat diet-induced inflammatory responses and tumorigenesis. 1.2: Investigate the ability of tomato carotenoid and apolycopenoids to modulate SIRT1 and its down-stream effectors as a unique mechanism for preventing inflammation and tumorigenesis. 1.3: Determine whether SIRT1 activity is required for the preventive action of tomato carotenoids and apolycopenoids.

Approach
LAB NAME: Vitamins and Carcinogenesis Alterations in dietary and nutritional habits have an important role to play in cancer prevention. The nutrients involved in 1-carbon metabolism (methionine, choline, and the B-vitamins, folate, B2, B6, and B12), as well as obesity have drawn considerable attention in this regard and are the focus of this laboratory. Our mission is to examine the complex roles that obesity and these 1-carbon nutrients play in modifying cellular pathways that lead to human carcinogenesis and thereby define means by which nutrition can be used to reduce the risk of developing cancer. The program of research emphasizes how dietary intake interacts with the genetic background to modify molecular and signaling pathways which alter the development of cancer, and to examine how other exogenous factors, such as alcohol consumption, also play a role. The laboratory focuses on colorectal and breast cancer, and utilizes cell culture studies, animal models, and human studies to accomplish our research goals. LAB NAME: Nutrition and Cancer Biology We will use both C57BL/6J mice and specific genetically-altered carotenoid cleavage enzyme (beta-carotene 9’,10’-oxygenase, BCO2) knockout mice strains to determine whether high-fat diet-induced liver inflammation and tumorigenesis can be prevented by apo-10’-lycopenoid (lycopene cleavage metabolite by BCO2), lycopene or tomato extract supplementation. We will examine the effects of dietary tomato carotenoids on modulating sirtuin 1 (SIRT1, a key metabolic sensor that directly links environmental nutrient signals to amelioration of inflammation as well as tumor development secondary to high-fat diet-induced obesity) expression/activity and its down-stream effectors, as well as its regulation by microRNAs in liver, intestine, colon and adipose tissues. We will use sirt1y/y homozygous mice that ablates the SIRT1 catalytic activity, as compared with their corresponding wild-type littermates, to determine the contribution of the tomato carotenoids and their metabolites to SIRT1 signaling pathway in liver, colon and adipose tissues, thus leading to the prevention of diet-induced obesity associated inflammation and cancer development.

Progress Report
This report documents research conducted under 2 projects in a Non-Assistance Cooperative Agreement between ARS and TUFTS UNIVERSITY. Additional details for the research are associated with projects 8050-51000-096-01S, Defining Mechanisms by Which Select Nutrients Determine Cancer Risk, and 8050-51000-096-02S, Molecular Targets of Carotenoids and Their Metabolites in Cancer Prevention. LAB:Vitamins and Carcinogenesis - Our major efforts over the past year were devoted to conducting our animal and human studies, developing ideas for new studies, and submitting grant applications. More than ten such grant applications were submitted over the course of the year. We incorporated a Ref-Seq transcriptome analysis into our human colonoscopy study. This study compared the biochemical and molecular milieu of the colon in obese and lean subjects. The laboratory and data analysis portions of that Ref-Seq are now completed, have been combined with our biochemical data, and a manuscript is in preparation. Observations from this study provided preliminary data for several grant applications. These observations are immediately relevant to Objective 2. We also completed the analysis of the cancer cluster study that integrated changes in the microbiome, transcriptome, and metabolome among mice whose intestinal tumorigenesis has been promoted by either diet- or genetically-induced obesity. The data demonstrated that both genetic and diet-induced obesity promote intestinal tumorigenesis, although these two modalities of producing obesity induced very distinct microbial patterns. The intestinal abundance of the bacterium P.distasonis, an organism known to suppress inflammation, was inversely associated with colonic IL-1ß (p<0.05). Fecal adenosine, which is also anti-inflammatory, was depleted in tumor-bearing mice (P<0.05), and its concentration was inversely associated with colonic levels of the pro-inflammatory cytokines, IL-1ß and Tnf alpha (P<0.05). A manuscript has been submitted for publication. Observations from this study provided preliminary data for several grant applications. These observations are also immediately relevant to Objective 2. We completed our transgenerational study in mice that examined whether 1-carbon nutrient intake in the father impacts on the paternal sperm methylome, and offspring transcriptome and tumorigenesis. No impact on offspring tumorigenesis was evident. However, supplementation led to hepatic steatosis in female offspring, and several changes in lipid metabolism in the hepatic transcriptome. The data are analyzed and a manuscript has been submitted for publication. These observations are immediately relevant to Objective 1 (sub-objective 1b). We completed our mouse study that utilized IL-1 beta knockout animals in order to identify the role of that cytokine in mediating the pro-cancerous effect of obesity on colon cancer. We are analyzing all our endpoints at present. These observations are immediately relevant to Objective 2. LAB: Nutrition and Cancer Biology - We have carried out three major animal studies: Utilizing the beta-carotene 9’,10’-oxygenase knockout (BCO2-KO) and wild-type mice, we investigated the potential protective effects of lycopene against high fat diet (HFD)-promoted hepatic tumorigenesis, and elucidated the underlying mechanisms by which lycopene exhibited these chemopreventive effects. We demonstrated that lycopene supplementation for 24 weeks is effective in inhibiting carcinogen-initiated liver tumor incidence and multiplicity in two different strains of mice, the BCO2-KO knockout strain and its respective wild-type. Intriguingly, lycopene chemopreventive effects in wild-type mice were associated with reduced hepatic pro-inflammatory signaling and inflammatory foci. In contrast, the protective effects of lycopene in BCO2-KO were associated with reduced hepatic oxidative stress biomarkers. Lycopene supplementation in BCO2-KO mice also suppressed tumorigenic signals, which was associated with increased hepatic microRNA (miR)-199a/b and miR-214 levels. These results provided novel experimental evidence that dietary lycopene can prevent HFD-promoted liver cancer development. We examined whether or not the biological functions of BCO2 are essential for the protective effects of lycopene and apo-10’-lycopenoic acid (APO10LA, a metabolite of lycopene cleavage by BCO2) against high-fat diet fat (HFD)-induced fatty liver development. We demonstrated that both lycopene or APO10LA supplementation for 12 weeks inhibited the severity of fatty liver in BCO2-/- knockout mice through differential mechanisms. The inhibition of fatty liver development by lycopene was associated with increased fatty acid oxidation and an uptake in mesenteric adipose tissue; APO10LA-mitigated steatosis in males was associated with a reduction in hepatic cholesterol and triglyceride synthesis markers. Our data suggest that lycopene and APO10LA inhibit HFD-induced fatty liver in BCO2-KO male mice through differential mechanisms. Sex disparity of BCO2-KO was observed in the outcomes of HFD-induced liver steatosis and plasma lipids. We have initiated an animal study using sirt1y/y homozygous mice carrying a point mutation (H355Y, sirt1y/y, 129/SvJ background) that ablates the catalytic activity, as well as their wild type littermates. We have initiated the mice with a very low dose of carcinogen at 2 weeks of age, and then exposed to the mice to HFD for 10 months. This study is currently ongoing at the 6-month time point and we expect that HFD exposure with absence of sirt1 activity will significantly promote liver tumor development at 10 months.

Accomplishments
1. Elevations in proinflammatory molecules and alterations in the regulation of the pro-carcinogenic signaling molecules are features of the obese human colon. Mitgating the enhanced risk of colon cancer conveyed by obesity will require an understanding of the underlying mechanism by which obesity produces this effect. By comparing the biochemical and transcriptome features of colonic biopsies from lean and obese humans, ARS researchers at the Jean Mayer USDA Human Nutrition Research Center on Aging in Boston, Massachusetts, demonstrated, for the first time, that obesity is associated with evidence of biochemical inflammation in the colon and that this exists in conjunction with altered regulation of the pro-carcinogenic pathways mediated by signaling molecules PI3 kinase, NFkB, and ERk. This is a necessary step for identifying cellular pathways in the obese colon that can be targeted for inhibition, thereby attenuating the burden of colon cancer in our obese and overweight population.

2. Obesity is associated with diminished fecal concentrations of the anti-inflammatory compound, adenosine, and colons harboring tumors have depletion of P. diastonis, a microorganism that suppresses inflammation. Mitgating the enhanced risk of colon cancer conveyed by obesity will require an understanding of the underlying mechanism by which obesity produces this effect. This study compared the colonic transcriptomes, metabolomes, and microbiomes of lean mice versus those made obese by either dietary or genetic means. ARS researchers at the Jean Mayer USDA Human Nutrition Research Center on Aging in Boston, Massachusetts, observed that obesity is strongly associated with drops in fecal adenosine, which, in turn, was inversely related to inflammatory cytokines in the colon. Further, tumor-bearing animals had a depletion of P. diastonis in the colon. Since several lines of evidence indicate that both adenosine and P. diastonis are ‘anti-inflammatory’, this supports the concept that obesity-induced inflammation is an important means by which obesity enhances the risk of colon cancer. Further, these data point to the importance of gut microbiota in obesity/diet-induced colon cancer.

3. Lycopene as an effective dietary agent against the development of fatty liver disease and liver cancer. Obesity is associated with increased liver cancer risks and mortality. High intake of tomatoes, rich in the carotenoid lycopene, is associated with a decreased risk of chronic disease. ARS-funded researchers at the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University in Boston, Massachusetts, investigated the potential protective effect of lycopene against the development of fatty liver and fatty liver-promoted cancer in animal models. They demonstrated that dietary lycopene is an effective dietary agent for preventing liver cancer or reducing cancer risk for patients with fatty liver disease. Furthermore, the lycopene-mediated chemopreventive effects were associated with reduced hepatic inflammatory responses in wild-type mice, while they were associated with inhibition of oxidative stress markers in carotenoid cleavage enzyme knockout mice. These findings show that lycopene is chemopreventive against hepatic tumorigenesis through two different mechanisms depending on the present or absence of carotenoid cleavage enzyme.