Cancer Prevention: Translational Research in Colon CancerMatthew R. YoungGene Regulation Section Laboratory of Cancer PreventionCCR, NCI

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. Cancer prevention prolongs the natural lifetime due to reduced death from cancer

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 PREVENTIONHow do we approach Translational Research in Cancer Prevention? Behavior modification in the general population:

Smoking cessation, Weight reduction Diet modificationExercise

Drugs in high risk groups: Tamoxifen: to prevent breast cancerDFMO + Sulindac: to prevent colon cancerAspirin: breast and colonNSAIDS (Celecoxib) Adenoma Prevention TrailDiet 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..Sedentary Life styleAgeDiabetesA personal history of colorectal cancer or polyps. SmokingObesityInflammatory intestinal conditions. RadiationAlcoholInherited syndromes that increase colon cancer risk.Family history of colon cancer and colon polyps.

Low-fiber, high-fat diet.

Trends in overweight prevalence

Stem cell niche

Transit- amplifying

cells

Differentiated cells

The Anatomy of the Colon

Normal organization of the intestinal crypt

Loss of wild-type APC or β-catenin mutationTransformation of healthy crypts towards an adenoma

Accumulation of other genetic lesions, RAS and PTEN,Progression towards an invasive growing CRC

Myofibroblast

Activated Myofibroblast

HGF

Myeloid cellsIL-6 TNFa

ProgressionInitiation Promotion

Tumor Promotion in the Colon

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

CancerPrevention

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.

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

monthscomplete 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

P-trend: 0.001A

dvan

ced

Ade

nom

a R

ecur

renc

e O

R (

95%

CI)

Q2 Q3 Q43.4 12.0 41.5

∆ Dry Bean Intake (T(1,2,3)-T0; in g/d)

Dry Bean Intake Inversely Associatedwith Advanced Adenoma Recurrence

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 diet3) Bean Residue fraction diet 4) Bean ethanol Extract fraction diet

Navy Beans and their Fractions Decrease Colon Lesion Incidence* in AOM-Induced Obese Mice

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

Decrease in serum levels of IL-6 anindicator of efficacious response to bean diet

0.05.0

10.015.020.025.030.035.040.045.050.055.060.065.070.075.080.0

IL-6

Ser

um

IL

-6 (

% d

etec

tab

le)

No Bean

WH Bean

RES Bean

EXT Beanp=.151

p=.0009

p=.018

p=.0035

0.00

5.00

10.00

15.00

20.00

25.00

30.00

Fo

ld c

ha

ng

e r

ela

tiv

e t

o N

o A

OM

No

Be

an

s

IL-6 Tnfrsf8 Stat 4 Sftpd

AOMAOM + Bean extract

**

* *

Bean diet attenuates colon gene expression changes induced by AOM

in ob/ob mice

Human Relevance of IL-6 as a Biomarker of Response to Dietary Intervention

Interleukin-6 as a Potential Indicator for Dietary PreventionOf High Risk Adenoma Recurrence in the Polyp Prevention Trial, Bobe G et al, Cancer Prevention Research, 2010

Colon Carcinogenesis stages in the mouse

Day 0 7 14 21 28 35 42 49 56 70

1-2% DSS in dist. water

AOM 10 mg/kg BWMice 6 wks of age

First tumors appearedStart Diet

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

PolypsDetected

Initial polypsEnlarge

Tumor burdenUnhealthy

Normal

Untreated 29 50 60Days after AOM injection

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

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.

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.

Feces (F)

American Diet American Diet

Legume DietLegume Diet

Blood (B) B B B B B

F F F

Legume DietAd libitum

Weight change

Legume DietAd Libitum

Weight change

weeks

Legume Inflammation Feeding ExperimentElaine Lanza, Cytonix; Terry Hartman, PSU; Robb

Chapkin Tx A & M

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.

D Legume P-value D Healthy American P-valuesTNFR1 - 3.7% 0.005 - 4.4% 0.001CRP -20.2% 0.018 -18.3% 0.007C-peptide - 2.8% 0.407 - 0.1 0.605

American Diet American Diet

Legume Diet Legume Diet

Potential biomarkers associated with consumption of Legume Enriched and Healthy American diets

American Diet American Diet

Legume Diet Legume Diet Legume Diet

Ad libitumWeight change

Potential biomarkers associated with consumption of reduced energy legume enriched diets. Mean body weight - 4.4 P<0.001BMI -4.5% P<0.001

Other markers significantly reduced (P<0.001)

Total Cholesterol, LDL-C, TG, C-peptide, fasting glucose, Leptin

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

American Diet

American DietLegume Diet

Legume Diet

B B

B B

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 complianceDiscovery of biomarkers of efficacy

Metabolite Name

Sca

led

In

ten

sity

Treatment Group

01

23

45

pipecolate

1 2 3 4 1 2 3 4

Baseline End

Median ValueExtreme Mean Value

Data Points

Upper QuartileLower Quartile

“Max” of distribution“Min” of distribution

___

Box and Whiskers Legend

Metabolomics for identifying biomarkers from the LIFE study274 named biochemicals identified; 87 biochemicals were significanly different between pre and post bean dietsPipecolate increased more than 6-fold in post bean diet.

Diet-derived Metabolites Diet-derived metabolites showed significantly different

plasma levels in pre-diet and post-diet samples.0

.00

.51

.01

.5

1,5-anhydroglucitol (1,5-AG)

1 2 3 4 1 2 3 4

Baseline End

01

23

45

trigonelline (N-methylnicotinate)

1 2 3 4 1 2 3 4

Baseline End

Trigonelline (N-methylnicotinate)

Gut Bacterial Metabolites. Several metabolites generated by gut bacterial metabolism showed significantly

different plasma levels in pre-diet and post-diet samples

01

23

45

pipecolate

1 2 3 4 1 2 3 4

Baseline End

01

23

4

indolepropionate

1 2 3 4 1 2 3 4

Baseline End

Potential Markers of Dietary CompliancePotential 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

01

23

45

pipecolate

1 2 3 4 1 2 3 4

Baseline End

01

23

4

indolepropionate

1 2 3 4 1 2 3 4

Baseline End

0.0

0.5

1.0

1.5

2.0

2.5

N-acetylornithine

1 2 3 4 1 2 3 4

Baseline End

0.0

0.5

1.0

1.5

1,5-anhydroglucitol (1,5-AG)

1 2 3 4 1 2 3 4

Baseline End

AOM 2% DSS Diet serum/feces

0 7 12 14 21 28 35 42 49 56 63 70 Days

Metabolomics for identifying biomarkers from the AOM/DSS induced CRC in mice

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) StudyDecreased 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 ZhangRobb 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

Pdcd4Hsin-Sheng Yang, Joan Cmarik, Aaron Jansen, Halina ZakowiczArti 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

LCP 2009Laboratory of Cancer Prevention