Diet and Cancer Prevention: Translational Research in Colon Cancer Matthew R. Young Laboratory of Cancer Prevention, National Cancer Institution, Frederick National Laboratory for Cancer Research

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Diet and Cancer Prevention Translational
Research in Colon Cancer Matthew R.
YoungLaboratory of Cancer Prevention, National
Cancer Institution, Frederick National
Laboratory for Cancer Research
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Division of Cancer Prevention

• Menu
• Translational Cancer Prevention
• Colon Cancer
• Anatomy of Colon Cancer
• Risk factors of Colon Cancer
• Colon Cancer Prevention
• Polyp Prevention Trial (PPT)
• Mouse Models
• Metabolomics
• Cancer Stem Cells
• Nutrition

Laboratory of Cancer Prevention Nancy
Colburn
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Young Kim Chief Shakir Saud
The Prevention Cage Matthew Young
Proprietor NCI-Frederick Frederick National
Lab youngma_at_mail.nih.gov 301 846 6448
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Cancer progression1. Initiation can be a single
mutagenic event.2. Promotion results from
chronic exposure to tumor promoters e.g. TPA,
EGF, UV radiation, TNF? 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.
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TRANSLATING PREVENTIONHow do we approach
Translational Research in Cancer Prevention?
Behavior modification in the general
population Smoking cessation, Weight
reduction Diet modification ExerciseDrugs 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
supplementsVaccines in the general
population HVP vaccine to prevent cervical
cancer. HBV vaccine to prevent liver
cancerAntibiotics in high risk groups Block
H-pylori induce gastric and esophageal cancer
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Colon Cancer is the third most common cause of
cancer-related death
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Risk factors Associated with Colon Cancer
?African-American race..?A
personal history of colorectal cancer or polyps.
? Age. ? Inflammatory intestinal conditions.
? Inherited syndromes that increase colon
cancer risk. ? Family history of colon cancer
and colon polyps. ? Low-fiber, high-fat diet. A
sedentary lifestyle. ? Diabetes.. ? Obesity.
? Smoking.. ? Alcohol.. ? Radiation therapy
for cancer
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Trends in overweight prevalence
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Colon cancer rates and risk. ?5-year
survival is 90 for CRC patients at an
early,localized stage. ?Only 39 of cases are
diagnosed at this stage. Biomarkers for colon
cancer prevention. We need to be able to
detect early stages of CRC. We need more
biomarkers that show a positive affect of life
style changes to prevent colon cancer. ?Diet.
?Exercise. ?Anti- diabetes drugs such as
metformin.
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The Anatomy of the Colon
Differentiated cells
Transit- amplifying cells
Stem cell niche
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Tumor Promotion in the Colon
Activated Myofibroblast HGF
Myofibroblast
Myeloid cells IL-6 TNFa
Normal organization of the intestinal crypt
Accumulation of other genetic lesions, RAS and
PTEN, Progression towards an invasive growing CRC
Loss of wild-type APC or ß-catenin
mutation Transformation of healthy crypts towards
an adenoma
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Stages of colon carcinogenesis 50 of US
population have adenoma(s) by age 70 years
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Relationship of diet and the gut microbiome to
cancer risk. Dietary constituents and energy
availability afft energy balance and gut bacteria
causing inflammation and DNA damage in normal
cells leading to proliferation and failed
apoptosis.
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Mortality Rates are Highest in African American
Men
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The microbiome in high colon cancer risk
population. ?Diet can effect
colonic mucosal health and cancer risk and these
effects are mediated predominantly through the
microbiota. ?The African Americans diet is high
fat, high protein, low complex carbohydrate The
Native Africans diet is the low fat, high complex
carbohydrate ?Colon cancer rates are low in
Native Africans ?Does the diet of African
Americans increase their risk for colon
cancer??The higher risk of colon cancer in
AFRICAN AMERICANS may be linked to a diet that
promotes MICROBES to produce potentially
carcinogenic secondary BILE ACIDS and less
anti-neoplastic SCFAs (short-chain fatty acids).
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The gut microbiota in development and disease

Nicholson et al Science, 2012
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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.
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The Polyp Prevention Trial (PPT) Multicenter
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

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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)
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Ob/Ob Obese MiceSingle mutation within the Ob
(leptin) geneDevelop obesity, hyperphagia,
hyperinsulinemia, and hyperglycemiaInjected 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
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Navy Beans and their Fractions Decrease Colon
Lesion Incidence in AOM-Induced Obese Mice
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Biomarkers that predict Colon Cancer and Efficacy
of interventions in mice and humans IL-6 a
response biomarker for dietary prevention of
colonCarcinogenesis in Ob mice,Mentor-Marcel
Can Prev Res ,2009
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Interleukin-6 as a potential indicator for
prevention of high-risk adenoma recurrence by
dietary flavonols in the Polyp Prevention Trial.
Intake of flavonols, especially of isorhamnetin,
kaempferol, and quercetin, was inversely
associated with ?serum IL-6 concentrations?high-
risk adenoma recurrence?advanced adenoma
recurrenceA decrease in IL-6 concentration
during the trial was inversely associated with
?high-risk adenoma recurrence?advanced adenoma
recurrence
Bobe et al, CaPR 2010
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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
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MRI is useful for monitoring efficacy of dietary
and/or pharmacological interventions in colon
carcinogenesis
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MRI is useful for monitoring efficacy of dietary
and/or pharmacological interventions in colon
carcinogenesis



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Bean Extract diet inhibits inflammation induced
colon cancer
P 0.09
p0.02
Morbidity (in )
Control Bean Extract N16
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Metabolomics for identificaqtion 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
Group n Description
AC 6 Control diet (AIN93-G), control mice
BC 6 Bean diet, control mice
AT 10 Control diet, AOM/DSS treated mice
BT 10 Bean diet, AOM/DSS treated mice
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Biomarkers associated with bean diet. 431 total
compounds identified in plasma556 total
compounds identified in feces.
ANOVA Contrasts ANOVA Contrasts ANOVA Contrasts ANOVA Contrasts ANOVA Contrasts

Plasma Cont TumorsCont no Tumors Bean TumorBean no T Bean no TCont no T Bean TumorCont Tumor
Total biochemicals p0.05 53 46 53 69
Biochemicals (??) 23 30 838 23 30 2247
Total biochemicals 0.05ltplt0.10 36 26 16 19
Biochemicals (??) 1422 323 97 910

Feces Cont TumorsCont no Tumors Bean TumorBean no T Bean no TCont no T Bean TumorCont Tumor
Total biochemicals p0.05 54 84 117 225
Biochemicals (??) 3915 795 8235 16560
Total biochemicals 0.05ltplt0.10 35 41 46 37
Biochemicals (??) 287 374 2323 2116
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Bean diet decreased every bile acid and BA
metabolite observed in feces (except
lithocholate) intestines. Bean diet decreased
every bile acid (and BA metabolite) observed in
feces when compared to control diet with the
exception of lithocholate. Most of the observed
bile acids are derived from gut microflora
synthesis and/or metabolism of primary bile
acids, Indicates a notable diet-induced
decrease in gut microflora content and/or
metabolism. Alternatively, this may suggest a
dramatic increase in bile acid re-absorption from
the intestines.
BCBean no Tumor ACControl no Tumor BTBean
Tumor ATControl Tumor
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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.
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Metabolomics for identifying biomarkers from the
LIFE study Serum was collected from participants
before and after consumption of bean enriched
weight maintenance diet
Intervention Dry beans 1 and ½ cups/day (250
g/d) Low glycemic index diet - 37 vs 69 Total
fiber from beans 39 vs 17 g/d Cholesterol
142 vs 180 mg/d
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Candidate molecular biomarkers identified from
exfoliated colonocytesTwo- and three-gene
combinations provide robust classifiers with
potential to noninvasively identify
discriminative molecular signatures for
differential diagnostic purposes.
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Potential biomarkers associagted with consumption
of reduced energy legume enriched diets Mean
body weight - 4.4 Plt0.001 BMI -
4.5 Plt0.001 Other markers significantly
reduced (Plt0.001)Total Cholesterol, LDL-C, TG,
C-peptide, fasting glucose, Leptin
weight loss allowed
Zhang et al., Lipids 2010
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Metabolomics for identification of Biomarkers for
Dietary Intervention and Protection 274 named
biochemicals identified 87 biochemicals were
significanly different between pre and post bean
diets
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Metabolomics from Mice fed Bean Extracts
Correlates with Metabolomics from Human (LIFE)
Study N-acetylornithine and pipecolate increase
in both man and mouse after consuming bean diet
N-acetylornithine
Ain93 G Bean Ain93G Bean
AOM/DSS
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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
Young et al unpublished.
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Fecal 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
Young et al unpublished.
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The NCI-Translational Research Working Group
Lifestyle Alteration Developmental Pathway
Lifestyle Alteration High dry beans reduce risk
of advanced adenoma by 70, Lanza et al.,
2006Animal Models Bean-based diets reduced
incidence of colon cancer in obese mice, and
colitis associated colon cancer, Bobe et al.,
2008, Young et al, unpublished Develop
Biomarkers Bean and flavonol-rich diets
decrease serum IL-6 and colon carcinogenesis in
obese mice and in humans , Mentor Marcel et al.,
2009 Bobe et al 2009 Develop Imaging Assays
Colon carcinogenesis in mice can be followed by
MRI, Young et al., 2009
Is there a relevant animal model?
Develop and validate biochemical behavioral
and/or imaging assays to measure effect of
lifestyle alteration
Pilot Study to assess efficacy of lifestyle
alteration
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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

Pilot Study to assess efficacy of lifestyle
alteration
Develop and validate biochemical behavioral
and/or imaging assays to measure effect of
lifestyle alteration
Study of efficacy in larger, more diverse
population
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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
Study of efficacy in larger, more diverse
population
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Conclusions1. Diets supplemented with Navy
Bean Extracts Prevent (reduced) colitis
associated carcinogenesis in mice as predicted
from the Polyp Prevention Trial. 2. Krebs cycle
intermediates are increased in AOM/DSS treated
mice after consumption of the Bean Diet3.
Carnitine and Markers of FA Oxidation decrease in
mice on the bean diet indicating an increase in
FA metabolism4. 84 plasma metabolites and 230
fecal metabolites are candidate markers of
compliance.5. 29 plasma and 38 fecal
metabolites are candidate markers of efficacious
intervention with BE6. Pipecolate and
n-acetylornithine, increase and 1,5-AG decreases
in men and mice fed a bean enriched diet are
candidates for markers of compliance in clinical
dietary intervention studies.
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Resveratrol is an anti-oxidant,
anti-inflammatory, anti-cancer and perhaps
anti-microbial
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Resveratrol Resveratrol inhibits
Colitis-induced tumor formationResveratrol
decreased arachidonate and reduced Cox-2 levels,
suggesting an anti inflammatory
activityResveratrol inhibits polyamine
synthesis and decreases ODC expression
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Other intervention1. Diets supplemented with
Isorhamnetin (a flavonol that is high in beans)
prevented colitis associated carcinogenesis in
mice2. Diets supplemented with Diallyl
disulfide (from garlic) prevented colitis
associated carcinogenesis in mice3. Berberine
(a Traditional Chinese Medicine) prevented
colitis associated colon carcinogenesis4.
Berberine activated AMPK
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Can we identify a diet that produces a healthy
microbiome that will ultimately lead to a reduce
risk of disease? The Human Gut Microbiota and
UndernutritionTackling the scourge of
undernutritionJeffrey I. Gordon, et al., Sci
Transl Med 6 June 2012Human gut microbiome
viewed across age and geographyTanya Yatsunenko,
et. al.,Nature, 14 June 2012

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BIOMARKERS AND MOLECULAR TARGETS OF NON-TOXIC
DIETARY INTERVENTIONS FOR CANCER PREVENTION
Laboratory of Cancer Prevention Nancy
Colburn Shakir Saud Weidong LI Noriko Yoshikawa,
Alyson Baker Qiou Wei, Glenn Hegamyer, Chris
Dextras 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 CIP Amiran Dzutsev
Division of Cancer Prevention Young Kim John
Milner Gerd Bobe, Prevention Fellow Roycelynn
Mentor-Marcel, Prevention Fellow
Small Animal Imaging Program Pete
Choyke Marcelino Bernardo Lilia Ileva Joe
Kalen Lisa RIffle