Cancer Prevention: Translational Research in Colon Cancer Matthew R Cancer Prevention: Translational Research in Colon Cancer Matthew R. Young Gene Regulation Section Laboratory of Cancer Prevention CCR, NCI

Translational Cancer Prevention: Translational Cancer Prevention: How do we approach Translational Research in Cancer Prevention? Colon Cancer: Anatomy of Colon Cancer Risk factors for Colon Carcinogenesis Colon Cancer Prevention: Polyp Prevention Trial (PPT) Mouse Metabolomics Cancer Stem Cells Nutrition

Molecular Targeted Carcinogenesis Prevention: Benefits at any stage Molecular Targeted Carcinogenesis Prevention: Benefits at any stage. Cancer prevention prolongs the natural lifetime due to reduced death from cancer

Cancer progression: 1. Initiation can be a single mutagenic event. 2 Cancer progression: 1. Initiation can be a single mutagenic event. 2. Promotion results from chronic exposure to tumor promoters e.r. TPA, EGF, UV radiation, TNFά or stress lead to benign tumors. 3. Progression results when benign tumors progress to carcinoma. Receptors activation increases protein kinase activity, resulting in and increase in transcription factors. Translation factors lead to mis-regulation of target proteins.

TRANSLATING PREVENTION How do we approach Translational Research in Cancer Prevention? Behavior modification in the general population: Smoking cessation, Weight reduction Diet modification Exercise Drugs in high risk groups: Tamoxifen: to prevent breast cancer DFMO + Sulindac: to prevent colon cancer Aspirin: breast and colon NSAIDS (Celecoxib) Adenoma Prevention Trail Diet supplements Vaccines in the general population: HVP vaccine: to prevent cervical cancer. HBV vaccine: to prevent liver cancer Antibiotics in high risk groups: Block H-pylori induce gastric and esophageal cancer

Colon Cancer is the third most common cause of cancer-related death

Risk factors Associated with Colon Cancer African-American race Risk factors Associated with Colon Cancer African-American race.. Sedentary Life style Age Diabetes A personal history of colorectal cancer or polyps. Smoking Obesity Inflammatory intestinal conditions. Radiation Alcohol Inherited syndromes that increase colon cancer risk. Family history of colon cancer and colon polyps. Low-fiber, high-fat diet.

Trends in overweight prevalence

The Anatomy of the Colon Differentiated cells The intestinal epithelial layer consists of several differentiated cell types and is lined with mesenchymal cells14 (see Box 1 Figure). The bottom of the small intestinal crypt contains Paneth cells and ISCs, whereas the remainder of the crypt is largely occupied by transit-amplifying cells, which are estimated to divide twice a day and are key to the rapid renewal of the epithelium14. At the top of the crypt, proliferation halts, and cells differentiate into either secretory (goblet, Paneth and enteroendocrine) cells or enterocytes. Although the colon lacks villi, the organization is roughly the same, except that Paneth cells are not present and differentiated cells occupy a large part of the crypt. Both the colon crypt (left) and the small intestinal crypt (right) contain a stem-cell compartment at the crypt bottom. CBCCs and the +4 stem cell have been indicated to be present between and just above the Paneth cells, respectively. Of note, Paneth cells are not detected in the colon, yet a Paneth-like cell has been suggested to be present at the crypt bottom. All four lineages (three in the colon) — enterocytes, Paneth cells, goblet cells and enteroendocrine cells — appear in different, but set ratios. Paneth cells move down to the bottom and are long-lived, whereas other lineages move up and are shed (a few days later) into the lumen while undergoing apoptosis. Rare cell types reported to exist in crypts, such as tuft cells, are not shown. Transit- amplifying cells Stem cell niche

Tumor Promotion in the Colon Progression Initiation Promotion Activated Myofibroblast HGF Myofibroblast Myeloid cells IL-6 TNFa Normal organization of the intestinal crypt. b, After the loss of wild-type APC or β-catenin mutation, the transformation of healthy crypts towards an adenoma starts. This is accompanied by several changes in crypt appearance and behaviour. First, cells show a more immature phenotype and a higher proliferative index (more red and pink cells with irregular shape). Second, crypt fission, a physiological process in which a crypt splits, is observed and results in expansion of the pre-malignant clone. Third, the attraction of myeloid cells (round light blue), producing factors such as interleukin-6 and tumour-necrosis factor-α (light blue dots) is detected and promotes carcinogenesis. Although normal myofibroblasts (blue) are still present, factors produced by premalignant and infiltrating cells activate myofibroblasts (orange). The myofibroblasts produce, among other factors, large amounts of hepatocyte growth factor (HGF), which promotes dedifferentiation (orange dots). c, The accumulation of other genetic lesions, in RAS and PTEN, for example, induces progression towards an invasive growing CRC. At this stage, stromal cells become an even more pronounced part of the tumour through the production of factors that further promote tumour progression (orange dots). Note the continued presence of several types of differentiated (tumour) cell throughout the whole sequence. Normal organization of the intestinal crypt Loss of wild-type APC or β-catenin mutation Transformation of healthy crypts towards an adenoma Accumulation of other genetic lesions, RAS and PTEN, Progression towards an invasive growing CRC

Stages of colon carcinogenesis ~50% of US population have adenoma(s) by age 70 years

TRANSLATING PREVENTION TRANSLATING PREVENTION. Basic research uses molecular processes, molecular target identification and targeted drug discovery. Preclinical research uses target validation and target discovery as well as response biomarkers and molecular targets as endpoints. Clinical research used drug-based and dietary interventions as well as response biomarfer and molecular target identification. Cancer Prevention 12

The Polyp Prevention Trial (PPT) Multicenter randomized controlled trial examining the effect of a low-fat (20% of total energy intake), high-fiber (18 g/1000 kcal), high-vegetable and -fruit (5-8 daily servings) dietary pattern on the recurrence of adenomatous polyps of the large bowel, Eligibility one or more adenomas removed within 6 months complete nonsurgical polyp removal complete colonic examination age 35 years or older; no history of colorectal cancer, inflammatory bowel disease, or large bowel resection; satisfactory completion of a food frequency questionnaire and 4-day food record DIETARY COOKED NAVY BEANS AND THEIR FRACTIONS ATTENUATE COLON CARCINOGENESIS IN AZOXYMETHANE-INDUCED OB/OB OBESE MICE

Dry Bean Intake Inversely Associated with Advanced Adenoma Recurrence Advanced Adenoma Recurrence OR (95% CI) P-trend: 0.001 Q2 Q3 Q4 3.4 12.0 41.5 ∆ Dry Bean Intake (T(1,2,3)-T0; in g/d)

Ob/Ob Obese Mice Single mutation within the Ob (leptin) gene Develop obesity, hyperphagia, hyperinsulinemia, and hyperglycemia Injected with colon carcinogen azoxymethane (AOM) to induce colon cancer Placed on diets after final AOM( injection for 40 weeks 1) Control diet (modified AIN-93G) 2) Cooked Whole navy bean diet 3) Bean Residue fraction diet 4) Bean ethanol Extract fraction diet

Navy Beans and their Fractions Decrease Colon Lesion Incidence Navy Beans and their Fractions Decrease Colon Lesion Incidence* in AOM-Induced Obese Mice Colon lesions (Includes focal hyperplasia (centralized), dysplasia, adenoma, and adenocarcinoma. ---Early stage (best stage to target interventions) 16

Biomarkers that predict Colon Cancer and Efficacy of interventions in mice and humans IL-6 a response biomarker for dietary prevention of colon Carcinogenesis in Ob mice, Mentor-Marcel Can Prev Res ,2009

Decrease in serum levels of IL-6 an indicator of efficacious response to bean diet 1/2/08---- Figure 1: Bean diets produced changes in serum levels of inflammation-associated proteins in AOM-induced ob/ob mice: a. Serum levels of IL-6 decreased in mice on residue (p=.009), on bean extract (p=.018), and bean overall (p<.0035). IL-6 values are depicted as percent (%) detectable ± SEM; b. Serum levels of MCP-1 were increased in mice on whole bean (p=.004), residue (p=.02), bean extract (p=.015) and bean overall (p=.002). MCP-1 values are depicted as geometric means (pg/ml) ± 95% CI; c. Serum levels of leptin were decreased in mice on whole bean (p=.032), residue (p=.0001), bean extract (p=.027), and bean overall (p=.001). Leptin values are depicted as geometric means (pg/ml) ± 95% CI. For all figures, p-values spanning bar graphs represent overall bean diets (i.e. whole bean, residue and extract groups combined). All comparisons are to mice on control (no bean) diet. Total mice per group: Control (n= 37), whole bean (n=32), residue (n=33), and extract (n=35). 10/30/07----values flank detection limit so use % detectable 11/9/07-------ADD p-val for ALL Bean ADD SE to graph: IL-6 SE (Ctl)= .077 IL-6 SE (WH)= .088 IL-6 SE (RES)= .078 IL-6 SE (EXT)= .082 18

Bean diet attenuates colon gene expression changes induced by AOM in ob/ob mice AOM + Bean extract * * * * IL-6 Tnfrsf8 Stat 4 Sftpd

Human Relevance of IL-6 as a Biomarker of Response to Dietary Intervention Interleukin-6 as a Potential Indicator for Dietary Prevention Of High Risk Adenoma Recurrence in the Polyp Prevention Trial, Bobe G et al, Cancer Prevention Research, 2010

Colon Carcinogenesis stages in the mouse

Two-Stage Colon Carcinogenesis Model AOM/DSS Mice develop ACFs, dysplastic lesions, adenomas and adenocarcinomas. Lesions have elevated b-catenin, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) activity Day 0 7 14 21 28 35 42 49 56 70 1-2% DSS in dist. water AOM 10 mg/kg BW Mice 6 wks of age First tumors appeared Start Diet

MRI is useful for monitoring efficacy of dietary and/or pharmacological interventions in colon carcinogenesis Polyps Detected Initial polyps Enlarge Tumor burden Unhealthy Normal Untreated 29 50 60 Days after AOM injection

MRI is useful for monitoring efficacy of dietary and/or pharmacological interventions in colon carcinogenesis

Bean Extracts and Isorhamnetin diets inhibit inflammation induced colon cancer. Isorhamnetin, Kaempferol and Bean Extracts decreased tumor burden Isorhamnetin and the Bean Extracts decreased morbidity associated with AOM/DSS treatment

Pilot study to assess efficacy of lifestyle alteration Pilot study to assess efficacy of lifestyle alteration. Legume Inflammation Feeding Experiment (LIFE). The effects of a high legume (dry bean) diet on markers of insulin resistance (IR) and inflammation in patients at high risk for CRC.

Legume Inflammation Feeding Experiment Elaine Lanza, Cytonix; Terry Hartman, PSU; Robb Chapkin Tx A & M Feces (F) American Diet Legume Diet Blood (B) B F Ad libitum Weight change Ad Libitum weeks

Candidate molecular biomarkers identified from exfoliated colonocytes Candidate molecular biomarkers identified from exfoliated colonocytes. Two- and three-gene combinations provide robust classifiers with potential to noninvasively identify discriminative molecular signatures for differential diagnostic purposes.

Potential biomarkers associated with consumption of Legume Enriched and Healthy American diets D Legume P-value D Healthy American P-value sTNFR1 - 3.7% 0.005 - 4.4% 0.001 CRP -20.2% 0.018 -18.3% 0.007 C-peptide - 2.8% 0.407 - 0.1 0.605 American Diet Legume Diet

Potential biomarkers associated with consumption of reduced energy legume enriched diets. Mean body weight - 4.4 P<0.001 BMI -4.5% P<0.001 Other markers significantly reduced (P<0.001) Total Cholesterol, LDL-C, TG, C-peptide, fasting glucose, Leptin American Diet Legume Diet Ad libitum Weight change

Metabolomics for identification of Biomarkers for Dietary Intervention and Protection. Metabolomics: The systematic study of all metabolites in an organism and how they change in relation to a biological perturbation such as diet, disease or intervention

Metabolomics for identifying biomarkers from the LIFE study Metabolomics for identifying biomarkers from the LIFE study. Serum was collected from participants before and after consumption of bean enriched weight maintenance diet. Anticipated results: Identification of biomarkers for compliance Discovery of biomarkers of efficacy American Diet Legume Diet B

Box and Whiskers Legend Metabolomics for identifying biomarkers from the LIFE study 274 named biochemicals identified; 87 biochemicals were significanly different between pre and post bean diets Pipecolate increased more than 6-fold in post bean diet. Median Value Extreme Mean Value Data Points Upper Quartile Lower Quartile “Max” of distribution “Min” of distribution ___  Metabolite Name Box and Whiskers Legend Scaled Intensity Treatment Group

Trigonelline (N-methylnicotinate) Diet-derived Metabolites Diet-derived metabolites showed significantly different plasma levels in pre-diet and post-diet samples. Trigonelline (N-methylnicotinate)

Gut Bacterial Metabolites Gut Bacterial Metabolites. Several metabolites generated by gut bacterial metabolism showed significantly different plasma levels in pre-diet and post-diet samples

Potential Markers of Dietary Compliance Potential candidates for markers of compliance to bean diet include the following. Gut bacterial metabolites pipecolate and indole proprionate. Diet derived 1,5-anhydroglucitol (1,5-AG) Modified amino acid N-acetylornithine

Metabolomics for identifying biomarkers from the AOM/DSS induced CRC in mice AOM 2% DSS Diet serum/feces 0 7 12 14 21 28 35 42 49 56 63 70 Days

Metabolomics from Mice fed Bean Extracts Correlates with Metabolomics from Human (LIFE) Study Pipecolate and N-acetyl-ornithine, proposed biomarkers of bean diet compliance identified elevated in both bean diet plasma groups. Also a similar subtle yet significant decrease in 1,5-anhydroglucitol was observed in both animal groups on bean diet.

Metabolomics from Mice fed Bean Extracts Correlates with Metabolomics from Human (LIFE) Study Decreased lysophospholipids Decreased medium- chain fatty acids Decrease in carnitine/acylcarnitines, No notable change in long-chain FA; Collectively indicating increased FA metabolism for energy in bean diet-fed animals

Fetal metabolomics from mice fed bean extract diet Increase in Alcohol sugars, Krebs cycle intermediates (citrate, alpha-ketoglutarate, fumarate and malate) were also significantly elevated in feces of bean extract fed mice. Fecal nucleotide breakdown products including nitrogenous bases, ribose and 2-deoxyribose, as well as phosphate were substantially increased in bean extract fed mice

The NCI-Translational Research Working Group: Lifestyle Alteration Developmental Pathway LIFE: Short term feeding study to measure the effects of a bean diet on markers of insulin resistance (IR) and inflammation in patients at high risk for CRC. Develop Biomarkers-Metabolomics: Metabolic biomarkers of compliance identified in human serum Develop Biomarkers-Metabolomics: Metabolic biomarkers of compliance identified in human serum also detected in mouse serum and feces. Young et al., unpublished

The NCI-Translational Research Working Group: Lifestyle Alteration Developmental Pathway LIFE: Short term feeding study to measure the effects of a bean diet on markers of insulin resistance (IR) and inflammation in patients at high risk for CRC. Develop Biomarkers-Metabolomics: Metabolic biomarkers of compliance identified in human serum Develop Biomarkers-Metabolomics: Metabolic biomarkers of compliance identified in human serum also detected in mouse serum and feces. Young et al., unpublished

Metabolomic analysis from the Polyp Prevention Trial: Identification of metabolic biomarkers associated with reduced adenoma recurrence. 3 groups of 125 participants 2 time points, baseline and after 3 years Control: Participants with no change in tumors Intervention, bean consumption: participants who consumed high bean diet and showed a reduced recurrence of adenomas Tumor Positive: Participants with increased recurrence of adenoma after 3 yr

BIOMARKERS AND MOLECULAR TARGETS OF NON-TOXIC DIETARY INTERVENTIONS FOR CANCER PREVENTION Laboratory of Cancer Prevention Nancy Colburn, Noriko Yoshikawa, Alyson Baker, Qiou Wei, Glenn Hegamyer, Shakir Saud, Elaine Lanza LIFE Study, Terryl J. Hartman, Pennsylvania State, Zhiying Zhang Robb Chapkin, Texas A & M Obese mice, Marcie Bennink, Michigan State University, Kati Barrett Division of Cancer Prevention, John Milner, Young Kim, Gerd Bobe, Prevention Fellow, Roycelynn Mentor-Marcel, Prevention Fellow Statistician Paul Albert, NCI Small Animal Imaging Program, Pete Choyke, Marcelino Bernardo, Lilia Ileva, Joe Kalen, Lisa RIffle

AP-1 and NF-kB Matthew Young, Arindam Dhar, Jing Hu, Connie Matthews, Moon-IL Kang, Brett Hollingshead, Qiou Wei, Gerd Bobe,Roycelynn Mentor-Marcel Jim McMahon, MTDP NCI; Curt Henrich, MTDP, Powel Brown, Baylor Univ; Peter Choyke and SAIP, NCI; Elaine Lanza, Cytonix; Terry Hartman, PSU; Rob Chapkin, TX A&M; Gary Stoner, OHU; Michel Toledano, IBITECS, France Pdcd4 Hsin-Sheng Yang, Joan Cmarik, Aaron Jansen, Halina Zakowicz Arti Santhanam, Tobias Schmid, Brett Hollingshead, Noriko Yoshikawa, Nahum Sonenberg, McGill Univ.; Myung Cho, Seoul Nat Univ; Alex Wlodawer, Nicole LaRonde, NCI; Michele Pagano, NYU; Heike Allgayer, Klinikum Mannheim; Bruce Shapiro, NCI 45

Laboratory of Cancer Prevention LCP 2009