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Research Project: Cancer Prevention via Diet

Location: Jean Mayer Human Nutrition Research Center On Aging

2016 Annual Report

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.

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
LAB NAME: Vitamins and Carcinogenesis Our major efforts over the past year have been devoted to conducting our animal and human studies, developing ideas for new studies, as well as submitting grant applications. Eight grant applications were submitted over the course of the year. Three new grants were funded over the past year and reviews of two others are presently pending. We completed final analysis of a human translational study in which we compared the biochemical and molecular environments of the colons of obese and lean subjects. This study is the first to demonstrate that mediators of inflammation are elevated in the colons of obese individuals and are accompanied by activation of several pro-cancerous molecular pathways. Observations from this study provided preliminary data for multiple grant applications and a manuscript has been submitted for publication. These observations are immediately relevant to Objective 2. We completed analyzing and writing up the results of a 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. Two original research publications arising from this study were accepted for publication this past year. Observations from this study also provided preliminary data for multiple grant applications. These observations are immediately relevant to Objective 2. We completed analyzing data from our transgenerational study in mice that examined whether 1-carbon nutrient intake in the father impacts on the paternal sperm methylome, and offspring transcriptome, tumorigenesis and metabolic phenotype. Two original research papers were published with these data and some of the observations were used for preliminary data for a pending grant application. These observations are immediately relevant to Objective 1. We completed analyzing our mouse study that utilized IL-1 beta knockout animals in order to identify the role of that cytokine in mediating the pro-cancerous effects of obesity on colon cancer, and a manuscript is being developed. These observations are immediately relevant to Objective 2. LAB NAME: Nutrition and Cancer Biology We investigated the effects of whole tomato (tomato powder, TP) supplementation on high fat diet (HFD)-induced hepatic steatosis and inflammation in the absence of carotenoid cleavage enzyme (beta-carotene 15,15’-oxygenase (BCO1) and beta-carotene 9’,10’-oxygenase (BCO2) in mice (BCO1-/-BCO2-/- double knockout KO) for 24 weeks. We found that dietary TP reduced significantly HFD-induced hepatic steatosis and triglyceride content in the liver (27% reduction), which was associated with significant increase in SIRT1 deacetylase activity, higher expression of nicotinamide phosphoribosyltransferase, and increased AMPK phosphorylation. TP supplementation down-regulated lipogenesis marker acetyl-CoA carboxylase and induced peroxisome proliferator-activated receptor-a related genes in the liver that down-regulates fatty acid uptake receptor gene (Cd36) and the key enzyme of triglyceride synthesis (diglyceride acyltransferaseand-1) and, increases fatty acid ß-oxidation markers (carnitine palmitoyltransferase I and acyl-CoA oxidase 1). In addition, TP supplementation decreased pro-inflammatory genes [tumor necrosis factor-a, interleukin (IL)-1ß, and IL-6] and increased anti-inflammatory genes expression (IL-10) in liver and visceral adipose tissue. Furthermore, there was a significant increase of mRNA expression of adiponectin in visceral adipose tissue and elevation of plasma levels of adiponectin in the HFD+TP group. The present study indicates that dietary TP supplementation inhibits HFD-induced hepatic steatosis and inflammatory responses independent of carotenoid cleavage enzymes, potentially through elevation of adiponectin and SIRT1 activity. We have conducted 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 the mice to HFD for 10 months. We observed that HFD exposure with absence of SIRT1 activity significantly increased hepatic triglyceride levels which was correlated with increased hepatic steatosis score at 10 months. There was a significant decrease mRNA expression of adiponectin in adipose tissue of SIRT1 mutation mice, which was associated with decreased hepatic PGC-1a and ACOX1, two of down-stream genes of adiponectin in livers. This ongoing study suggests that SIRT1 activity ablation down-regulates adiponectin in adipose tissue, and SIRT1 may be a molecular target in adipose tissue for tomato carotenoid intervention against obesity-related complications. Recent epidemiological studies have suggested that consumption of high-refined carbohydrates/sugar diet can induce non-alcohol fatty liver disease. To investigate the different effects of isocaloric high-fat diet (HFD) and high-refined carbohydrate diet (HRCD) on hepatic steatosis and the underlying mechanisms, especially the role of microRNA-34a/SIRT1 deacetylase axis, C57BJ/6L mice were isocaloric pair-fed with Lieber-DeCarli liquid diet containing either HFD or HRCD for 16 weeks. We demonstrated that despite the similar final body weights, HRCD feeding: 1) induced more severe hepatic steatosis; 2) up-regulated hepatic expression of miR-34a accompanied with significant decrease of SIRT1 protein level and activity, and phosphorylation of AMPK; 3) up-regulated de novo lipogenesis (DNL) related proteins expression (ACC, SCD1), and down-regulated expressions of miR-122, miR-370 and miR-33; 4) decreased mRNA expressions of Cpt1, PPARa and Pgc1a genes related to fatty acid oxidation; 5) increased hepatic total cholesterol concentration and decreased mRNA expression of cholesterol metabolism related genes; and 6) induced higher hepatic inflammatory response accompanied with significant increased mRNA expressions of IL1ß, TNFa and MCP1. Thus, isocaloric high refined carbohydrate diet (HRCD) feeding induced greater severity in hepatic steatosis and inflammatory response than HFD feeding, potentially through miR-34a/SIRT1 axis mediated promotion of DNL, inhibition of fatty acid oxidation and cholesterol metabolism. To further understand mechanism(s) of inducing hepatic inflammatory response by HRCD, we used a mouse model lacking tumor progression locus 2 (TPL2), a serine-threonine kinase, functions as a critical regulator of inflammatory pathways and mediates oncogenic events. We observed that TPL2 knockout mice fed with HRCD for 24 weeks had significantly decreased hepatic steatosis and expression of de novo lipogenesis related markers, as well as lower incidences of liver tumor and developed hepatocellular adenoma only, which is contrast to wild-type mice where they all developed hepatocellular carcinoma. TPL2 knockout mice had significantly down-regulated phosphorylation of JNK and ERK, and levels of mRNA expression of pro-inflammatory cytokines (IL-1ß, IL-18, MCP-1 and NALP3), which correlated with the reduced incidence and number of hepatic inflammatory foci. Furthermore, TPL2 ablation resulted in decreased (ACC, SCD1, SREBP1C and AKT phosphorylation), as well as reduction of endoplasmic reticulum stress biomarkers PERK and eIF-2a. The study suggested that TPL2 could be a molecular target for preventing high refined carbohydrate diet-associated fatty liver and liver cancer development. Early epidemiologic studies have reported that tobacco smoking is an independent risk factor for non-alcoholic fatty liver diseases. We investigated whether the tobacco carcinogen 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces lesions in both lungs and livers of ferrets with or without lycopene intervention for 26 weeks. We demonstrate that NNK exposure results in higher incidences of lung tumors, liver tumors, and nonalcoholic steatohepatitis (NASH, which is characterized by severe inflammatory cell infiltration with concurrent fat accumulation in liver, hepatocellular ballooning degeneration and increased inflammatory cytokine expressions), as well as elevations in bilirubin and AST levels in ferrets. Lycopene supplementation at two doses prevented NNK-induced expressions of a7 nicotinic acetylcholine receptor in the lung and cytochrome P450 enzyme (CYP2E1) in the liver and attenuated the NNK-induced mortality and pathological lesions in both the lungs and livers of ferrets. The present study provided strong experimental evidence that the tobacco carcinogen NNK can induce both hepatocellular carcinoma and NASH in the ferrets which can be a useful model for studying tobacco carcinogen-associated inflammation and liver cancer. Furthermore, lycopene could provide potential benefits against smoke carcinogen-induced pulmonary and hepatic injury.

1. LAB NAME: VITAMINS AND CARCENOGENESIS: The patterns of altered gene expression in the colon due to diet- and genetically-induced obesity are highly discrepant, but the observed changes in both forms of obesity converge on Akt activation. Mitigating 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 spectrum of gene expression in the colons of lean mice versus those made obese by either dietary or genetic means. Tufts University researchers at USDA in Boston, Massachusetts, observed that although obesity produced by these two modalities changed the profile of colonic gene expression very differently, with each type of obesity one of the most prominent changes was an apparent activation of the pro-inflammatory (and pro-cancerous) Akt cell signaling pathway. These observations indicate that obesity from any cause upregulates a common cellular pathway that likely contributes to the increased risk of colon cancer and that targeting this pathway for inhibition is a candidate means of reducing the risk of obesity-associated colon cancer.

2. LAB NAME: VITAMINS AND CARCENOGENESIS: Blocking the actions of pro-inflammatory molecule, IL-1ß alone reduces several of the pro-cancerous effects that obesity produces in the colon. Mitigating 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 in obese mice incorporated a genetically-modified animal whose tissues were incapable of responding to the effects of one of the major protein mediators of inflammation, called IL-1ß. Tufts University researchers at USDA in Boston, Massachusetts, observed that obese mice lacking the ability to respond to this protein did not develop biochemical inflammation in the colon, had less activation of the pro-cancerous Wnt cellular pathway, and displayed lower levels of cellular proliferation, all of which are indications of a lesser proclivity towards cancer. These observations identify a candidate molecule that could be targeted for inhibition in order to reduce the risk of obesity-associated colon cancer.

3. LAB NAME: NUTRITION AND CANCER BIOLOGY: High refined carbohydrate diet induces greater severity in fatty liver disease than high fat diet. Consumption of high-fat diet has been proposed to be one risk factor for increased prevalence of nonalcoholic fatty liver disease, a major form of chronic liver disease in adults and children. Tufts University researchers at USDA in Boston, Massachusetts, provided strong experimental evidence that under the same energy intake and body weight gain in mice, feeding of high refined carbohydrate diet induced greater severity in fatty liver disease and inflammatory response than a high fat diet feeding. Furthermore, they demonstrated that the lack of tumor progression locus 2 enzyme which mediates inflammatory pathways, inhibited high refined carbohydrate diet-associated fatty liver, inflammation and liver cancer development. Thus, reducing the intake of refined carbohydrate/sugars may be an effective strategy to alleviate the prevalence of nonalcoholic fatty liver disease and reduce risk for liver cancer.

4. LAB NAME: NUTRITION AND CANCER BIOLOGY: Lycopene is a protective agent against smoking-related lesions in lung and liver. High intake of tomatoes, rich in carotenoid lycopene, is associated with a decreased risk of smoking-related chronic disease. Using ferret as a model-mimicking humans, Tufts University researchers at USDA in Boston, Massachusetts, provided strong experimental evidence that a tobacco carcinogen induces not only lung cancer but also non-alcoholic fatty liver, steatohepatitis, and liver cancer. Furthermore, dietary lycopene attenuated tobacco carcinogen-induced pathological lesions in both the lungs and livers of ferrets. These findings show that the ferret can be a useful non-rodent model for studying inflammation and cancer development, and dietary lycopene is capable of reducing fatty liver disease and cancer risk among cigarette smokers.

Review Publications
Stice, C.P., Hussain, S., Liu, C., Ausman, L., Wang, X., Greenberg, A. 2016. Deletion of tumor progression locus 2 attenuates alcohol induced hepatic inflammation. Hepatobiliary Surgery and Nutrition. 5(1):29-37.
Pfalzer, A.C., Nesbeth, P.C., Parnell, L.D., Iyer, L.K., Liu, Z., Kane, A.V., Chen, C., Tai, A.K., Bowman, T.A., Obin, M.S., Mason, J.B., Greenberg, A., Choi, S., Selhub, J., Paul Pottenplackel, L., Crott, J.W. 2015. Diet- and genetically-induced obesity differentially affect the fecal microbiome and metabolome in Apc1638N mice. PLoS One. 10(8):e0135758. doi:10.1371/journal.pone.0135758.
Li, X., Liu, C., Ip, B.C., Hu, K., Smith, D.E., Greenberg, A., Wang, X. 2015. Tumor progression locus 2 ablation suppressed hepatocellular carcinoma development by inhibiting hepatic inflammation and steatosis in mice. Journal of Experimental and Clinical Cancer Research. 34:138. doi: 10.1186/s13046-015-0254-2.
Li, X., Lian, F., Liu, C., Hu, K., Wang, X. 2015. Isocaloric pair-fed high-carbohydrate diet induced more hepatic steatosis and inflammation than high-fat diet mediated by miR- 34a/SIRT1 axis in mice. Scientific Reports. 5:16774. doi: 10.1038/srep16774.
Aizawa, K., Liu, C., Tang, S., Veeramachaneni, S., Hu, K., Smith, D.E., Wang, X. 2016. Tobacco carcinogen (NNK) induces both lung cancer and non-alcoholic steatohepatitis and hepatocellular carcinomas in ferrets which can be attenuated by lycopene supplementation. International Journal of Cancer. doi: 10.1002/ijc.3016.