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