ENERGY BALANCE JOURNAL CLUB

Zobeida Cruz-Monserrate Ph.D.

Instructor

Cancer Biology Department

A High-Fat Diet Activates Oncogenic Kras and COX2 to Induce Development of

Pancreatic Ductal Adenocarcinoma in MiceBincy Philip, Christina L. Roland, Jaroslaw Daniluk, Yan Liu, Deyali Chatterjee,Sobeyda B. Gomez, Baoan Ji, Haojie Huang, Huamin Wang, Jason B. Fleming,

Craig D. Logsdon, Zobeida Cruz-Monserrate

Source: American Cancer Society, 2013

Men306,920

Women273,430 26% Lung & bronchus

14% Breast

9% Colon & rectum

7% Pancreas

5% Ovary

4% Leukemia

3% Non-Hodgkin lymphoma

3% Uterine corpus

2% Liver & intrahepaticbile duct

2% Brain and other nervous system

Lung & bronchus 28%

Prostate 10%

Colon & rectum 9%

Pancreas 6%

Liver & intrahepatic 5%bile duct

Leukemia 4%

Esophagus 4%

Urinary bladder 4%

Non-Hodgkin 3% lymphoma

Kidney 3%

Pancreatic Cancer: A Deadly Disease 2013 Estimated US Cancer Deaths

• Highest death to incidence ratio (0.99) of all cancers• 5-year survival rate below 6%

• Median survival ~ 6 months• Surgical resection is the only effective treatment

• < 20% of the patients are eligible• Rarely detected at an early stage (small lesions)

– High rate of dissemination• Conventional cancer treatments fail

– Resistance to chemotherapy– Treatment options limited

Pancreatic Ductal Adenocarcinoma (PDAC) is Deadly and Difficult to Diagnose Early

Prevention and Early Detection

Most pancreatic cancers (around 75%)

Develop in the exocrine pancreas

ExocrineEndocrine

Omary, M.B., et. al., 2007. 117: p. 50-9

Pancreas Microanatomy

Normal duct• single cell layer• low cuboidal

PanIN-1A/1B• elongated cells• mucin• papillary growth

PanIN-2• early nuclear abnormalities

PanIN-3• luminal budding• nuclear atypia• mitosis

Carcinoma• invasion• desmoplasia

Hruban, R.H., et al., 2001. 25(5): p. 579-86

PDAC >90% Mutant K-Ras

Multistep Progression Model of PDACPancreatic Intraepithelial Neoplasias (PanINs)

Non-Modifiable PDAC Risk Factors

Modifiable PDAC Risk Factors

Obesity

Obesity is a Risk Factor for Many Cancers Including Pancreatic

Eugenia E. Calle* and Rudolf Kaaks 2004:4:579-591

Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Ras

re

Cell Type Specific Promoter

Duct

Islet

Acini

Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Ras

re

Cell Type Specific Promoter

Elastase-Cre-ErTamoxifen Regulated

Duct

Islet

Acini

Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Ras

re

Duct

Islet

AciniAcinar Cell Specific Cre

Cell Type Specific Promoter

Elastase-Cre-ErTamoxifen Regulated

Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Ras

re

Duct

Islet

AciniAcinar Cell Specific Cre

Cell Type Specific Promoter

XOncogenic K-Ras

“flox-stopped“

Endogenous Promoter “Knock In”

K-RasG12D

loxP loxP

Stop Poly Ap-K-Ras

loxP = locus of recombination

Elastase-Cre-ErTamoxifen Regulated

Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Ras

re

Elastase-Cre-Er

Cell Type Specific Promoter

XOncogenic K-Ras

“flox-stopped“

Endogenous Promoter “Knock In”

K-RasG12D

loxP loxP

Stop Poly Ap-K-Ras

loxP = locus of recombination

Endogenous mutant K-Ras expression in acinar cells

AcinarmK-Ras

K-RasG12D

Cre mediated deletion via Tamoxifen after

birth

LSL/BAC

Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Ras

re

Elastase-Cre-Er

Cell Type Specific Promoter

XOncogenic K-Ras

“flox-stopped“

Endogenous Promoter “Knock In”

K-RasG12D

loxP loxP

Stop Poly Ap-K-Ras

loxP = locus of recombination

AcinarmK-Ras

K-RasG12D

a

Normal Pancreas at early age

Cre mediated deletion via Tamoxifen after

birth

Endogenous mutant K-Ras expression in acinar cells

LSL/BAC

Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Ras

re

Elastase-Cre-Er

Cell Type Specific Promoter

XOncogenic K-Ras

“flox-stopped“

Endogenous Promoter “Knock In”

K-RasG12D

loxP loxP

Stop Poly Ap-K-Ras

loxP = locus of recombination

AcinarmK-Ras

K-RasG12D

Could this mouse model be used to test risk factors like obesity?

a

Normal Pancreas at early age

Endogenous mutant K-Ras expression in acinar cells

Cre mediated deletion via Tamoxifen after

birth

LSL/BAC

High Fat-Induced PDAC Mouse Model

AcinarmK-Ras

K-RasG12D

Endogenous mutant K-Ras expression in acinar cells

LSL/BAC

% kcal of each nutrient Caloric

Breakdown Control Diet High Fat DietProtein 18.3 18.1

Fat 10.2 61.6Carbohydrate 71.5 20.3

High Fat-Induced PDAC Mouse Model

Isocaloric

AcinarmK-Ras

K-RasG12D

Endogenous mutant K-Ras expression in acinar cells

LSL/BAC

% kcal of each nutrient Caloric

Breakdown Control Diet High Fat DietProtein 18.3 18.1

Fat 10.2 61.6Carbohydrate 71.5 20.3

High Fat-Induced PDAC Mouse Model

Isocaloric

AcinarmK-Ras

K-RasG12D

Endogenous mutant K-Ras expression in acinar cells

LSL/BAC

High Fat Diet Increased Total Body Weight and Pancreas Weight Compared to Control Diet

High Fat Diet Increased Inflammation, Fibrosis, and PanIN Lesions on Mice with K-RasG12D Mutation

High Fat Diet Increased Inflammation, Fibrosis, and PanIN Lesions on Mice with K-RasG12D Mutation

High Fat Diet Increased Areas of Collagen Deposition on Mice with K-RasG12D Mutation

High Fat Diet Increased Areas of Activated Stellate Cells on Mice with K-RasG12D Mutation

Stellate Cells

Endogenous Mutant K-Ras Endogenous Mutant K-Ras is not Sufficient is not Sufficient to to Transform Most CellsTransform Most Cells

Developmental promoter EMBRYONIC expression in all cell types

Acinar cell promoterADULT expression in acinar cells

(Unless inflammation was induced)

Multiple PanIN lesions; PDAC- 2/29 (7%) 1 year

No PanIns No tumors – 0/11 (0%) 1 year

Endogenous K-Ras mutations Endogenous K-Ras mutations generates cancer with low efficiencygenerates cancer with low efficiency

Mutant K-Ras at Endogenous Levels Requires Mutant K-Ras at Endogenous Levels Requires an Inflammatory Insult to Transform Acinar an Inflammatory Insult to Transform Acinar CellsCells

Bac-Elastase CreERT100% efficient

100% specific for adult acinar cells

If oncogenic mutant Ras is always “on” then there should be effects on cell function.

Mutant K-Ras at Endogenous Levels Requires Mutant K-Ras at Endogenous Levels Requires an Inflammatory Insult to Transform Acinar an Inflammatory Insult to Transform Acinar CellsCells

mutant Ras NO Stimulant

mutant Ras+ LPS Stimulant

High Fat Diet

Link Between Ras and Obesity?

High Fat Diet Increased Ras Activity in Mice with K-RasG12D Mutation

High Fat Diet Activates of K-Ras Downstream Pathways in Mice with K-RasG12D Mutation

Ras Must Be Active to Initiate Downstream Signaling Resulting in Progression of PDAC

Ras-GDP

GEFsGEFs

GAPsGAPs

Ras-GTP

INACTIVE ACTIVE

PI3KPI3K

RafRaf

RalGDSRalGDS

MAPKMAPK

Receptors (GF, hormones, etc)

Cox2Cox2PGE2

High Fat Diet Increases Cox-2 in Pancreas on Mice with K-RasG12D Mutation

High Fat Diet Promotes Recruitment of Macrophages on Mice with K-RasG12D Mutation

High Fat Diet Increases Cox-2 in Pancreas on Mice with K-RasG12D Mutation

High Fat Diet Promotes PDAC

AcinarmK-Ras

K-RasG12D

Normal Pancreas at early age

Endogenous mutant K-Ras expression in acinar cells

LSL/BAC

High Fat Diet Promotes PDAC

AcinarmK-Ras

K-RasG12D

Normal Pancreas at early age

Endogenous mutant K-Ras expression in acinar cells

High Fat Diet

PanINs Cancer

LSL/BAC

Cox2Cox2

Cox-2 Deletion in Acinar Cells with “Knock-in” of Oncogenic K-Ras

Cox-2 KO“flox-stopped“

X

AcinarmK-Ras

K-RasG12D

LSL/BAC

Endogenous mutant K-Ras expression in acinar cells

Cox-2 Deletion in Acinar Cells with “Knock-in” of Oncogenic K-Ras

Cox-2 KO“flox-stopped“

X

AcinarmK-Ras

K-RasG12D

AcinarmK-Ras

K-RasG12D

COXKO/LSL/BAC

LSL/BAC

Endogenous mutant K-Ras expression in acinar cells but

NO Cox-2 Expression

Endogenous mutant K-Ras expression in acinar cells

COX-2 is Required in High Fat-Induced PDAC Mouse Model

AcinarmK-Ras

K-RasG12D

COXKO/LSL/BAC

% kcal of each nutrient Caloric

Breakdown Control Diet High Fat DietProtein 18.3 18.1

Fat 10.2 61.6Carbohydrate 71.5 20.3

IsocaloricEndogenous

mutant K-Ras expression in acinar cells but NO Cox-2 Expression

Conditional Knockout of Cox-2 in the Acinar Cells Blocked the Effects of High Fat Diet

Conditional Knockout of Cox-2 in the Acinar Cells Blocked the Effects of High Fat Diet

Conditional Knockout of Cox-2 in the Acinar Cells Blocked the Effects of High Fat Diet

Systemic Cox-2 inhibition Decreases the Effects of High Fat Diet

Systemic Cox-2 inhibition Decreases the Effects of High Fat Diet

High Fat Diet Decreases Survival of Mice Susceptible to PDAC

30 days

High Fat Diet Decreases Survival of Mice Susceptible to PDAC

Control Diet

High Fat Diet

160Days

30 days

High Fat Diet Decreases Survival of Mice Susceptible to PDAC

Control Diet High Fat Diet

205Days

Pancreas Pancreas

Pancreas

30 days

High Fat Diet Decreases Survival of Mice Susceptible to PDAC

Control Diet High Fat Diet

205Days

Pancreas Pancreas

Pancreas

30 days

Pancreas

High Fat Diet

Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Ras

Duct

Islet

Acini

Elastase-Cre-ErTamoxifen Regulated

re

Cell Type Specific Promoter

Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Ras

re

PDX-1 Cre

Cell Type Specific Promoter

Activates Cre during development

Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Ras

re

PDX-1 Cre

Cell Type Specific Promoter

Activates Cre during development

X

Oncogenic K-Ras“flox-stopped“

Endogenous Promoter “Knock In”

K-RasG12D

loxP loxP

Stop Poly Ap-K-Ras

loxP = locus of recombination

Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Ras

re

PDX-1 Cre

Cell Type Specific Promoter

Activates Cre during embryogenesis

X

Oncogenic K-Ras“flox-stopped“

Endogenous Promoter “Knock In”

K-RasG12D

loxP loxP

Stop Poly Ap-K-Ras

loxP = locus of recombination

Cre mediated deletion during development

Endogenous mutant K-Ras expression in every cell of

the pancreas

mK-RasAll cells

K-RasG12D

LSL/PDX-1

High Fat Diet Decreases Survival of Mice Susceptible to PDAC

SomeMice in control diet are

still alive

Cre mediated deletion via Tamoxifen after

birth

Cre mediated deletion during development

High Fat Diet Accelerated PanIN-2 and PanIN-3 Development Compared to Control Diet Using and Embryonic Promoter

Control Diet

High Fat Diet

90Days

137Days

Cre mediated deletion during development

High Fat Diet Accelerated PanIN-2 and PanIN-3 Development Compared to Control Diet Using and Embryonic Promoter

Control Diet

High Fat Diet

90Days

137Days

Cre mediated deletion during development

Summary:

Obesity is a Risk Factor for PDAC in Humans

High Fat Diet Accelerates PanIN formation andCancer Development in PDAC Mouse Models

Ras Activity and Cox-2 are essential for High Fat Diet induced PDAC

HOW DOES FAT LEAD TO RAS ACTIVATION?Mechanisms?

Future Directions with High Fat Induced PDAC Model

Test Prevention Agents

LS

L/B

AC

Acknowledgments Dr. Logsdon’s Laboratory Collaborators

MDACCHuamin Wang, PathologyJason Fleming, Surgical OncologyJoya Chandra, PediatricsDavid McConkey, UrologyKathleen M. Schmeler MD, Ob/GYNRalph B. Arlinghaus, Translational Molecular PathologyAdel El-Naggar, Pathology

The Methodist Hospital Research InstituteChing-Hsuan Tung Ph.D.Wael R. Abd-Elgaliel Ph.D.Rita Serta Ph.D.

City of Hope National Medical CenterAnn David Ph.D.

University of Rhode IslandOleg AndreevYana Reshetnyak

Bincy Philips, MS

Funding SourcesNIDDK Minority Supplement

Phi Beta Psi Sorority