Research Project: Dietary Strategies for Cancer Prevention

Location: Jean Mayer Human Nutrition Research Center On Aging

2022 Annual Report

Objectives
Vitamins and Carcinogenesis Lab Objective 1: Define the cellular pathways by which obesity, obesigenic diets, and the intake of the 1-carbon nutrients modulate the risk of developing cancers of the colorectum and other common cancers in both animal models and human samples, and exploit these mechanistic insights in order to devise targeted means of mitigating cancer risk. • Sub-objective 1A: Determine whether the pro-inflammatory/pro-carcinogenic NF'B pathway plays the predominant role in mediating the obesity-promoted increased risk of colorectal carcinogenesis. • Sub-objective 1B: Determine whether supplemental levels of dietary vitamin B6 provide additional suppression of obesity-promoted tumorigenesis and colonic inflammation when combined with curcumin + salsalate, beyond that provided by the two latter agents alone. Objective 2: Examine how modifications in the microbiome alter biochemical and molecular processes that lead to colorectal cancer, and explore how intentional manipulations of the microbiome, or its products, can be exploited for cancer prevention. Objective 3: In both genetic and chemically-induced rodent models of colorectal carcinogenesis examine whether select alternative protein sources (e.g. insect-based foodstuffs) suppress pro-carcinogenic pathways and tumorigenesis compared to soy protein and other dietary sources of protein more common in the American diet. Nutrition and Cancer Biology Lab Objective 1: Investigate mechanistically the anti-inflammatory and anti-carcinogenic effect of phytochemical-rich whole food approaches, and purified phytochemicals as well as their derivatives, in preventing inflammation-promoted (e.g., induced by a high-sugar diet, diabetes, and aging) cancer development. Objective 2: Determine the ability of phytochemical-rich whole foods and dietary phytochemicals to prevent cancer development in liver and colon by targeting multiple signaling pathways (e.g. membrane and nuclear receptors) and inter-organ crosstalk (among liver, pancreas, mesenteric adipose tissue, and gut microbiome).

Approach
Vitamins and Carcinogenesis Lab We will identify novel strategies by which colorectal cancer (CRC), and other cancers that commonly afflict elderly Americans, can be prevented. Our aim is to lessen the risk that accompanies cancer-promoting features typifying the U.S. diet, such as its obesigenic character and emphasis on processed animal meat. Using a combination of in vitro experiments and animal models we identify biochemical and molecular pathways that mediate the effects of specific nutrients or dietary patterns on carcinogenesis. We then identify means of modulating those pathways to mitigate cancer risk. We will examine how the inflammatory state created by obesity and high-fat diets activates procancerous pathways in the colon. We are exploring the use of pharmacologic, nutritional, and microbial agents to block those pathways. The third objective is an exploratory aim, designed to generate preliminary data. We will examine whether substituting protein-rich powder derived from roasted crickets attenuates the enhanced risk of CRC that accompanies the habitual consumption of processed meats which are a prominent source of protein in the American diet. This strategy has the added value of promoting food sustainability. Our research will provide novel avenues for reducing the societal burden of common age-related cancers. Nutrition and Cancer Biology Lab We will conduct animal studies to investigate how one dietary phytochemical, xanthophyll beta-cryptoxanthin (BCX), inhibits metabolic syndrome, nonalcoholic fatty liver disease and liver cancer (hepatocellular carcinoma) development in the liver. Of particular interest is understanding how BCX prevents the development of hepatocellular carcinoma in rodents consuming a diet high in refined carbohydrates (HRCD). We will examine the protective effects of intact BCX, independent of its metabolites, regulating key cell signaling pathways in both young and old animals. We will examine multiple organs (liver, pancreas, adipose tissue, and gut) as well as how these organs communicate, while noting gender differences. Specifically, we will use genetically-altered carotenoid cleavage enzyme (beta-carotene 15,15’-oxygenase and beta-carotene 9’,10’-oxygenase) double knockout mice strains to determine whether HRCD-induced liver metabolic syndrome and tumorigenesis can be prevented by intact BCX itself or sweet red pepper extract (SRPE)-rich in BCX. We will treat mice (male and female) with a single injection of a hepatic carcinogen, diethylnitrosamine (DEN), followed by continued exposure to HRCD with or without BCX (or SRPE) intervention. We will examine the effects of dietary BCX intervention against fatty liver, inflammation, fibrosis, and in livers. We will investigate the protective effects of xanthophyll BCX against HRCD-promoted HCC in both young and old mice respectively. We will determine if the BCX protective action process a common mechnism or pathway, such as intestinal permeability/gap junction/adipose/liver axis, salvage pathway of NAD+ biosynthesis enzyme, and circadian transcription factors, and thereby reducing aging/metabolic syndrome-associated liver cancer development.

Progress Report
Vitamins and Carcinogenesis: Over the past year, the investigators defined two mechanistic pathways by which the combination of curcumin and B6 interact in an additive fashion to produce a potent anti-tumor effect. In studies conducted in human colonic organoids derived from obese human subjects, they demonstrated that the B6/curcumin combination suppresses cytokine release, underscoring relevance to human cancer biology. These observations are immediately relevant to Objective #1. The investigators previously found that gut bacterium P. distasonis (Pd) suppresses the formation of obesity-promoted colonic neoplasms in mice and reduces inflammatory cytokines and increased gut barrier integrity in vitro and in vivo. Subsequently, the investigators have generated and published additional data demonstrating: • That freeze-dried extracts of Pd reduce colonic tumorigenesis in non-obese mice, complementing prior observations in obese animals. • That Pd treatment increased intestinal protein expression of the tight junction proteins Zonula occludens-1 and Occludin, thereby suggesting that the enhanced gut barrier function and anti-tumor effect might be mediated by these proteins. • Cell and molecular studies are ongoing to identify anti-inflammatory proteins from Pd. An NIH R01 grant was secured to expand the scope of mechanistic questions (the PI of this R01, who was a member of this project at the time the R01 was awarded, has since left the institution). These studies are immediately relevant to Objective #2. The investigators successfully designed and configured highly unconventional rodent diets to determine whether a diet whose protein is sourced from cricket powder is less tumorigenic than one whose protein is sourced from processed pork, soy flour, or milk solids. Macro- and micronutrient compositions, including total protein, total fat, saturated fat, fiber, calcium, folate and vitamin B6 were analyzed to ensure that diet components that were potential confounding effect modifiers were normalized. A mouse study is now underway to test the hypothesis and to examine mechanistic questions on biochemical, molecular, microbiologic and metabolomic bases. The investigators examined the quantitative and qualitative nature of cobalamins in cricket powder, which is relevant since the availability of several 1-carbon nutrients act in combination to determine colon cancer risk. They determined that >90% of the cobalamins that have previously been regarded as sources of vitamin B12 are actually corrinoids that cannot be absorbed and/or utilized by the human. This work is immediately relevant to Objective #3. Nutrition and Cancer Biology: In support of Objective 1 and Objective 2, we carried out the following studies: Study 1) To determine whether the protective effects of Beta-Cryptoxanthin (BCX) against a high refined carbohydrate diet (HRCD)-promoted hepatocellular carcinoma (HCC) development associated with metabolomic and gut microbial profiles in wild type (WT) mice; Study 2) To determine if mice with systemic ablation of beta-carotene 15,15’-oxygenase (BCO1-/-)) /beta-carotene 9’.10’-oxygenase (BCO2-/- ) double knockout (DKO) display distinctive phenotypes in response to HRCD-induced non-alcoholic fatty liver disease (NAFLD) development compared to WT mice; and Study 3) To determine the sexually dimorphic mechanisms in NAFLD development in response to a HRCD in male and female mice possessing a heterozygous H355Y mutation that ablates SIRT1 deacetylase activity (SIRT1y/+) and WT mice. In Study 1, we performed 16S rDNA sequencing, targeted and untargeted metabolomics, high performance liquid chromatography (HPLC) analysis for determination of hepatic vitamin A status, and a series of colorimetric assays to systemically characterize the protective effects of BCX against diethyl-nitrosamines (DEN)-initiated, HRCD-promoted HCC progression in wild type mice at 8 months. Mice fed BCX displayed significantly lower tumor burden and steatosis, higher hepatic vitamin A status, and lower plasma levels of alanine transaminase (ALT) and lactate dehydrogenase (LDH). Moderate negative correlations were also identified amongst hepatic vitamin A levels and primary outcomes, such as HCC tumor number, steatosis score, and relevant biomarkers (ALT, LDH, sphingosine-1-phosphate (S1P), and Adiponectin). Our metabolomics data showed significant modulations in plasma metabolites with BCX treatment from phosphatidylcholine, sphingomyelin, ceramide, bile acid, and other phospholipid classes. Overall, these data support a potential chemopreventive role for BCX supplementation against the progression of HRCD-promoted HCC development in a mouse model. In the Study 2, we performed 16S rDNA sequencing, targeted and untargeted metabolomics, and a series of colorimetric assays to further characterize the collective contributions of BCO1 and BCO2 to HRCD-induced NAFLD development using male, BCO1-/-/BCO2-/- DKO and WT mice at 8 months. Although NAFLD severity was similar between the groups, DKO mice displayed significantly lower plasma adiponectin levels and higher hepatic levels of malondialdehyde (MDA). Furthermore, BCO1-/-/BCO2-/- DKO mice possessed significantly reduced alpha diversity (observed Operational Taxonomic Units (OTUs), Shannon Index, and Simpson Index) and significant compositional differences based on Bray Curtis dissimilarity measures. Our metabolomics data revealed that chow-fed BCO1-/-/BCO2-/- DKO mice displayed significant differences in ceramide, cholesterol ester, triglyceride, and phospholipid classes compared to WT mice. When fed a HRCD, BCO1-/-/BCO2-/- DKO mice had significantly higher levels of select metabolites from various phospholipid classes. Collectively, these data indicate that the carotenoid cleavage enzymes, BCO1 and BCO2, affect the regulation of diverse lipid classes and the gut microbiome in a mouse model. In the Study 3, we fed male and female WT and heterozygous SIRT1y/+ mice a liquid HRCD for 5 weeks and 9 weeks to assess early pathological changes in NAFLD development through evaluating relevant biomarkers, hepatic expression of inflammatory markers and TLR signaling, metabolic endotoxemia [Plasma lipopolysaccharide-binding protein (LBP), CD14, and liposaccharide (LPS)], and the gut microbiome. Although SIRT1y/+ mice did not display a distinctive phenotype compared to WT mice, male mice possessed significantly higher levels of hepatic triglycerides, hepatic malondialdehyde (MDA), plasma alanine aminotransferase (ALT), plasma CD14, and plasma (LPS) compared to female mice. In accordance with these data, male mice also had significantly higher hepatic genetic expression of a variety of inflammatory markers and toll-like receptor (TLR) signaling. On the contrary, female mice had significantly higher levels of plasma adiponectin at each timepoint. After 9 weeks of HRCD, male and female mice displayed significant compositional differences based on Bray Curtis dissimilarity measures. Furthermore, through differential abundance analyses using DESeq2, male mice were shown to have significantly higher levels of Helicobacter Hepaticus, a bacterial species previously tied to hepatic inflammation and tumorigenesis. Interestingly, the abundance of H. Hepaticus correlated with hepatic triglycerides, hepatic MDA, plasma ALT, plasma LBP, and plasma CD14. Overall, these data indicate that NAFLD pathogenesis in response to a liquid HRCD is sexually dimorphic and is more severe in male mice compared to female mice, potentially through differences in adiponectin, metabolic endotoxemia, and the gut microbiome.

Accomplishments

Review Publications
Tanprasertsuk, J., Scott, T., Johnson, M., Poon, L., Nelson, P., Davey, A., Woodard, J., Vishwanathan, R., Barbey, A.K., Barger, K., Wang, X., Johnson, E.J. 2021. Brain a-tocopherol concentration is inversely associated with neurofibrillary tangle counts in brain regions affected in earlier BRAAK stages: a cross-sectional finding in the oldest OLD. Journal of Aging Research & Lifestyle. 10:8-16. https://doi.org/10.14283/jarlife.2021.2.
Tanprasertsuk, J., Ferland, G., Johnson, M., Poon, L., Scott, T.M., Barbey, A.K., Barger, K.K., Wang, X., Johnson, E. 2019. Concentrations of circulating phylloquinone, but not cerebral menaquinone-4, are positively correlated with a wide range of cognitive measures: exploratory findings in centenarians. Journal of Nutrition. 150(1): 82-90. https://doi.org/10.1093/jn/nxz200.
Maruvada, P., Stover, P.J., Mason, J.B., Bailey, R.L., Davis, C.D., Field, M., Finnell, R.H., Garza, C., Green, R., Gueant, J., Jacques, P.F., Klurfeld, D.M., Lamers, Y., MacFarlane, A.J., Miller, J.W., Molloy, A.M., O'Connor, D.L., Pfeiffer, C.M., Potischman, N.A., Rodricks, J.V., Rosenberg, I.H., Ross, S.A., Shane, B., Selhub, J., Stabler, S.P., Trasler, J., Yamini, S., Zappala, G. 2020. Knowledge gaps in understanding the metabolic and clinical effects of excess folates/folic acid: a summary, and perspectives, from an NIH workshop. American Journal of Clinical Nutrition. 112(5):1390-1403. https://doi.org/10.1093/ajcn/nqaa259.
Liu, C., Rafacho, B.M., Wang, X. 2020. Xanthophyll beta-cryptoxanthin treatment inhibits hepatic steatosis without altering vitamin A status in beta-carotene 9'10'-oxygenase knockout mice. Hepatobiliary Surgery and Nutrition. https://doi.org/10.21037/hbsn-20-404.
Xia, B., Zhu, R., Zhang, H., Chen, B., Liu, Y., Dai, X., Ye, Z., Zhao, D., Mo, F., Gao, S., Wang, X., Bromme, D., Wang, L., Wang, X., Zhang, D. 2022. Lycopene improves bone quality and regulates AGEs/RAGE/NF-kB signaling pathway in high-fat diet-induced obese mice. Oxidative Medicine and Cellular Longevity. https://doi.org/10.1155/2022/3697067.