Research Project: Cancer Prevention via Diet

Location: Jean Mayer Human Nutrition Research Center On Aging

2018 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. 1.4: Investigate the protective effect of beta-cryptoxanthin (BCX, a provitamin A carotenoid) against high sugar diet- induced NAFLD and liver cancer via gut/adipose/liver axis 1.5: Determine the biological functions of intact BCX in absence of carotene cleavage enzymes beta-carotene 15,15'-oxygenase 1 ( BCO1) and beta-carotene 9',10'-oxygenase (BCO2) double knock out (KO) mice. 1.6 Determine the SIRT1 activity as a molecular target for BCX protection against high sugar diet-induced NAFLD and liver cancer.

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
VITAMIN AND CARCINOGENESIS LAB: Over the past year our efforts have largely been focused on identifying dietary, pharmacologic, and microbiologic agents that safely and effectively suppress colonic tumor development in obese laboratory animals, and determining the cellular pathways through which they exert their beneficial effects. One of our publications this past year demonstrated that the combination of curcumin-the most bioactive component of turmeric-in combination with salsalate — an aspirin derivative lacking the gastrotoxicity of aspirin -suppresses obesity-induced colonic inflammation and reduces the pro-cancerous cell signaling that accompanies this inflammation. Further, these anti-tumorigenic effects were far more effective with the combination of the two agents compared to either agent alone. We have now completed a subsequent experiment and prepared a manuscript that demonstrates that this combination regimen suppresses colonic cancer development in obese mice by over 80%, and we once again showed the combination is more effective than either agent alone. Also, in this latter experiment we showed additive effects of this combination in suppressing a pro-cancerous network of cell signaling far more extensive than what we showed in the publication mentioned above. Several years ago we observed that vitamin B6 had anti-inflammatory properties, and we have now completed an experiment in obese mice demonstrating an additive effect in suppressing inflammation and colon cancer development with the combination of curcumin with supplemental levels of B6. Prior experiments by us demonstrated that the normal colonic commensal, Parabacteroides distasonis, has anti-inflammatory properties. We have been also exploring the use of this animal as a cancer preventive. We have completed a series of experiments demonstrating that P. distasonis cell membrane attenuates inflammation in colonic cells and have published a paper demonstrating that freeze-dried organisms are highly effective in blocking colonic tumor development in mice fed a high-fat diet. NUTRITION AND CANCER BIOLOGY LAB: We have carried out three major animal studies: We investigated the effects of tomato powder, a whole food containing substantial amounts of lycopene, on nonalcoholic fatty liver disease development and gut microbiome in the absence of carotenoid cleavage enzymes (BCO1 and BCO2). BCO1-/-/BCO2-/- double knockout mice were fed a high fat diet alone or with tomato powder for 24 weeks. This was associated with increased SIRT1 activity, nicotinamide phosphoribosyl transferase expression and adenosine triphosphate kinase phosphorylation, and it subsequently decreased lipogenesis and hepatic fatty acid uptake, and increased fatty acid ß-oxidation. Tomato powder feeding decreased mRNA expression of pro-inflammatory genes in liver and mesenteric adipose tissue, which were associated with increased mesenteric adipose tissue adiponectin expression, plasma adiponectin and hepatic adiponectin receptor-2. Furthermore, tomato powder feeding increased gut microbial richness and diversity and decreased gut relative abundance of the genus Clostridium. The present study demonstrates that dietary tomato feeding independent of carotenoid cleavage enzymes inhibits high fat diet-induced fatty liver and inflammation with potential modulating gut microbiota. We investigated the effect of ablation of both BCO1/BCO2 in the development of nonalcoholic fatty liver disease and its underlying molecular mechanism(s). Wild type mice and BCO1/BCO2 double knockout mice (BCO1/BCO2 BCO1-/-/BCO2-/- double knockout) from a C57BL/6J genetic background were fed standard rodent chow diet for 24 weeks. There were no significant differences in final body weight, liver weight, mesenteric fat weight, and hepatic vitamin A (retinol and retinyl ester) levels between wild type and BCO1/BCO2 double knockout mice, but carotenoids were accumulated in the livers of BCO1/BCO2 double knockout mice. BCO1/BCO2 double knockout mice developed hepatic steatosis and had higher levels of hepatic and plasma triglyceride and total cholesterol to wild type. Hepatic changes in the BCO1/BCO2 double knockout mice were associated with significant: 1) increases in mRNA and protein levels of lipogenesis markers (SCD1, ACC, CD36), and decreases in the gene expressions of fatty acid ß-oxidation markers (CPT1, PPARa); 2) upregulation of the cholesterol metabolism markers (CYP7A1, CYP8B1, HMG-CoAR); 3) alterations to the microRNAs (miR-34a, miR-33, miR-122) related to triglyceride accumulation and cholesterol metabolism; 4) increases in hepatic oxidative stress markers (HO-1) but decreases in anti-oxidant enzymes (SOD1, SOD2, GPX, catalase); and 5) decreases in nuclear protein levels of farnesoid X receptor, mRNA levels of FXR, small heterodimer partner (SHP), and sirtuin 1 (SIRT1). Taken together, the present study provided novel experimental evidence that the ablation of both BCO1/BCO2 led to the development of NAFLD, indicating that BCO1/BCO2 could play a significant role in maintaining normal hepatic lipid and cholesterol homeostasis, potentially through the regulation of the FXR/miR-34a/SIRT1 pathway. We investigated whether the protective effect of red sweet pepper rich in ß-cryptoxanthin against high refined carbohydrate diet-induced NAFLD was dependent on, or independent of BCO1/BCO2, and whether the upregulation of sirtuin 1 (SIRT1, a highly conserved NAD+-dependent protein deacetylase) was the underlying mechanism of protection of sweet red pepper. BCO1/BCO2 double knock out mice and wild type mice at 6 weeks of age were randomly assigned to either the high refined carbohydrate diet (66.5% of energy as carbohydrate including maltodextrin and sucrose) or the high refined carbohydrate diet with sweet red pepper extract (paprika rich in 11% beta-cryptoxanthin, equivalent to 10mg beta-cryptoxanthin /kg diet which was equivalent to that on daily human consumption of 3-4 ounces of a sweet red pepper) group for 24 weeks. We observed that feeding with sweet red pepper extract rich in beta-cryptoxanthin significantly reduced hepatic steatosis score in both wild type and BCO1/BCO2 double knock out mice compared to their respective high refined carbohydrate diet counterparts (P < 0.05) without significant changes of hepatic vitamin A (retinol and retinyl ester) levels. Moreover, sweet red pepper extract feeding significantly reduced hepatic concentrations of triglyceride and total cholesterol levels in BCO1/BCO2 double knock out mice, compared to the corresponding high refined carbohydrate diet group. These hepatic changes by beta-cryptoxanthin feeding were related to the significant upregulation of mRNA and protein levels of nicotinamide phosphoribosyltransferase and SIRT1, and downregulation of miR-34a levels in the livers. Additionally, sweet red pepper extract feeding decreased mRNA levels of the cholesterol biosynthesis marker (HMG-CoAR) and lipogenesis markers (SREBP-1C, ACC, FAS) and increased in mRNA levels of PPARa. The present study provided compelling experimental evidence that sweet red pepper rich in beta-cryptoxanthin has protective effects against high refined carbohydrate diet-induced nonalcoholic fatty liver disease potentially by regulating microRNA 34a/NAMPT/SIRT1 pathway independent of carotenoid cleavage enzymes.

Accomplishments
1. The combination of curcumin and salsalate suppresses colon cancer development in obese mice. Obesity and overweight are prominent risk factors for colon cancer, and this increased risk appears to be mediated, in large part, by colonic inflammation that is induced by a high-fat diet or excess body fat. ARS-funded researchers in Boston, Massachusetts combined a highly bioactive component of turmeric, curcumin, with salsalate, a derivative of aspirin that lacks toxicity, and examined whether the combination of these two agents has an additive effect in suppressing colonic inflammation, pro-cancerous cell signaling and tumor formation in obese mice fed a high-fat diet. The lab successfully showed these suppressive effects and that the combination had more robust effects than either agent alone. These results provide the rationale and justification for trials in humans that may similarly demonstrate cancer-preventive effects in humans.

2. Increased consumption of tomatoes may prevent fatty liver disease and cancer. Nonalcoholic fatty liver disease affects 30% of men, 20% of women, and 3-10% of children in the United States while the incidence of and death rate from liver cancer continues to increase. ARS-funded researchers in Boston, Massachusetts have found strong experimental evidence using mice that tomato powder, representing a whole food containing a substantial amount of lycopene, effectively prevents risk of high fat diet-induced fatty liver, inflammation, and liver cancer. Furthermore, the tomato powder promoted healthy gut microbial richness and diversity and decreased some harmful gut bacteria. These findings may lead to dietary recommendations to increase tomato consumption to reduce the risk of fatty liver disease and liver cancer and maintain a healthy gut.

Review Publications
Wang, X.D. Caudill, M.A. 2018. Vitamin A. IN: Stipanuk, M.H., Caudill, M.A., editors. Biochemical, Physiological, and Molecular Aspects of Human Nutrition. 4th edition. Philadelphia, PA: Saunders. p. 721-741.
Li, C., Liu, C., Fu, M., Hu, K., Aizawa, K., Takahashi, S., Hiroyuki, S., Cheng, J., Von Lintig, J., Wang, X. 2018. Tomato powder inhibits hepatic steatosis and inflammation potentially through restoring SIRT1 activity and adiponectin function independent of carotenoid cleavage enzymes in mice. Molecular Nutrition and Food Research. 62(8):e1700738. https://doi.org/10.1002/mnfr.201700738.
Cheng, J., Miao, B., Hu, K., Fu, X., Wang, X. 2018. Apo-10'-lycopenoic acid inhibits cancer cell migration and angiogenesis and induces peroxisome proliferator-activated receptor gamma. Journal of Nutritional Biochemistry. 56:26-34. https://doi.org/10.1016/j.jnutbio.2018.01.003.
Romano, F.R., Heinze, C.R., Barber, L.G., Mason, J.B., Freeman, L.M. 2016. Association between body condition score and cancer prognosis in dogs with lymphoma and osteosarcoma. Journal of Veterinary Internal Medicine. 30(4):1179-1186. https://doi.org/10.1111/jvim.13965.
Bird, J.K., Ronnenberg, A.G., Choi, S., Du, F., Mason, J.B., Liu, Z. 2014. Obesity is associated with increased red blood cell folate despite lower dietary intakes and serum concentrations. Journal of Nutrition. 145(1):79-86. https://doi.org/10.3945/jn.114.199117.
Sabet, J.A., Park, L.K., Iyer, L.K., Tai, A.K., Koh, G., Pfalzer, A., Parnell, L.D., Mason, J.B., Liu, Z., Byun, A.J., Crott, J.W. 2016. Paternal B vitamin intake is a determinant of growth, hepatic lipid metabolism and intestinal tumor volume in female Apc1638N mouse offspring. PLoS One. 11(3):e0151579. https://doi.org/10.1371/journal.pone.0151579.
Wu, X., Pfalzer, A.C., Koh, G., Tang, S., Crott, J.W., Thomas, M.J., Meydani, M., Mason, J.B. 2017. Curcumin and salsalate suppresses colonic inflammation and procarcinogenic signaling in high-fat-fed, azoxymethane-treated mice. Journal of Agricultural and Food Chemistry. 65(33):7200-7209. https://doi.org/10.1021/acs.jafc.7b02648.
Song, M., Brasky, T.M., Catalin, M., Weng, D.Y., Taslim, C., Llanos, A.A., Dumitrescu, R.G., Liu, Z., Mason, J.B., Spear, S.L., Kallakury, B.V., Freudenheim, J.L., Shields, P.G. 2016. Genetic variation in one-carbon metabolism in relation to genome-wide DNA methylation in breast tissue from healthy women. Carcinogenesis. 37(5):471-480. https://doi.org/10.1093/carcin/bgw030.
Mason, J.B., Black, R., Booth, S.L., Brentano, A., Broadbent, B., Connolly, P., Finley, J.W., Goldin, J., Griffin, T., Hagen, K., Lesnik, J., Lewis, G., Pan, Z., Morales Ramos, J.A., Ranalli, M., Rojas, M.G., Shockley, M., Stull, V., Swietlik, D.M. 2018. Fostering strategies to expand the consumption of edible insects: the value of a tripartite coalition between academia, industry, and government. Current Developments in Nutrition. https://doi.org/10.1038/s41598-017-10675-6.
Koh, G., Kane, A., Lee, K., Xu, Q., Wu, X., Roper, J., Mason, J.B., Crott, J.W. 2018. Parabacteroides distasonis attenuates toll-like receptor 4 signaling and Akt activation and blocks colon tumor formation in high-fat diet-fed azoxymethane-treated mice. International Journal of Cancer. https://doi.org/10.1002/ijc.31559.