of Oncogenic K-Ras - MD Anderson Cancer Center
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ENERGY BALANCE JOURNAL CLUB A High-Fat Diet Activates Oncogenic Kras and COX2 to Induce Development of Pancreatic Ductal Adenocarcinoma in Mice Bincy 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 Zobeida Cruz-Monserrate Ph.D. Instructor Cancer Biology Department Pancreatic Cancer: A Deadly Disease 2013 Estimated US Cancer Deaths Lung & bronchus 28% Prostate 10% Men 306,920 Women 273,430 26% Lung & bronchus 14% Breast Colon & rectum 9% 9% Colon & rectum Pancreas 6% 7% Pancreas Liver & intrahepatic 5% bile duct 5% Ovary 4% Leukemia 3% Non-Hodgkin lymphoma Leukemia 4% Esophagus 4% Urinary bladder 4% 3% Uterine corpus Non-Hodgkin lymphoma 3% 2% Liver & intrahepatic bile duct Kidney 3% 2% Brain and other nervous system Source: American Cancer Society, 2013 Pancreatic Ductal Adenocarcinoma (PDAC) is Deadly and Difficult to Diagnose Early • 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 Prevention and Early Detection Pancreas Microanatomy Endocrine Exocrine Most pancreatic cancers (around 75%) Develop in the exocrine pancreas Omary, M.B., et. al., 2007. 117: p. 50-9 Multistep Progression Model of PDAC Pancreatic Intraepithelial Neoplasias (PanINs) Normal duct PanIN-1A/1B PanIN-2 PanIN-3 Carcinoma • single cell layer • low cuboidal • elongated • early • luminal budding • nuclear atypia • mitosis • invasion • desmoplasia cells • mucin • papillary growth nuclear abnormalities PDAC >90% Mutant K-Ras Hruban, R.H., et al., 2001. 25(5): p. 579-86 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 Duct Islet Acini Elastase-Cre-Er Tamoxifen Regulated Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Ras re Cell Type Specific Promoter Duct Islet Acini Acinar Cell Specific Cre Elastase-Cre-Er Tamoxifen Regulated Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Ras re Oncogenic K-Ras “flox-stopped“ X Cell Type Specific Promoter Endogenous Promoter “Knock In” Stop p-K-Ras Duct loxP loxP loxP = locus of recombination Islet Acini Acinar Cell Specific Cre Elastase-Cre-Er Tamoxifen Regulated K-RasG12D Poly A Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Ras re Oncogenic K-Ras “flox-stopped“ X Endogenous Promoter “Knock In” Cell Type Specific Promoter Elastase-Cre-Er Stop p-K-Ras loxP K-RasG12D loxP loxP = locus of recombination Cre mediated deletion via Tamoxifen after birth LSL/BAC Acinar mK-Ras K-RasG12D Endogenous mutant K-Ras expression in acinar cells Poly A Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Ras re Oncogenic K-Ras “flox-stopped“ X Endogenous Promoter “Knock In” Cell Type Specific Promoter Elastase-Cre-Er Stop p-K-Ras K-RasG12D loxP loxP loxP = locus of recombination Cre mediated deletion via Tamoxifen after birth a LSL/BAC Acinar mK-Ras K-RasG12D Endogenous mutant K-Ras expression in acinar cells Normal Pancreas at early age Poly A Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Ras re Oncogenic K-Ras “flox-stopped“ X Could this mouse model be used to test risk factors like obesity? Endogenous Promoter “Knock In” Cell Type Specific Promoter Elastase-Cre-Er Stop p-K-Ras K-RasG12D loxP loxP loxP = locus of recombination Cre mediated deletion via Tamoxifen after birth a LSL/BAC Acinar mK-Ras K-RasG12D Endogenous mutant K-Ras expression in acinar cells Normal Pancreas at early age Poly A High Fat-Induced PDAC Mouse Model LSL/BAC Acinar mK-Ras K-RasG12D Endogenous mutant K-Ras expression in acinar cells High Fat-Induced PDAC Mouse Model LSL/BAC Acinar mK-Ras K-RasG12D Endogenous mutant K-Ras expression in acinar cells % kcal of each nutrient Caloric Breakdown Protein Fat Carbohydrate Control Diet 18.3 10.2 71.5 High Fat Diet 18.1 61.6 20.3 Isocaloric High Fat-Induced PDAC Mouse Model LSL/BAC % kcal of each nutrient Caloric Breakdown Protein Fat Carbohydrate Acinar mK-Ras K-RasG12D Endogenous mutant K-Ras expression in acinar cells Control Diet 18.3 10.2 71.5 High Fat Diet 18.1 61.6 20.3 Isocaloric CONTROL DIET HIGH FAT DIET BAC (n=10) BAC (n=10) LSL/BAC (n=10) LSL/BAC (n=10) 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 is not Sufficient to Transform Most Cells Developmental promoter EMBRYONIC expression in all cell types Multiple PanIN lesions; PDAC- 2/29 (7%) 1 year Endogenous K-Ras mutations generates cancer with low efficiency Acinar cell promoter ADULT expression in acinar cells No PanIns No tumors – 0/11 (0%) 1 year (Unless inflammation was induced) Mutant K-Ras at Endogenous Levels Requires an Inflammatory Insult to Transform Acinar Cells Bac-Elastase CreERT 100% 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 an Inflammatory Insult to Transform Acinar Cells 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 PGE2 Cox2 Receptors (GF, hormones, etc) PI3K GEFs RasGDP RasGTP Raf GAPs INACTIVE ACTIVE RalGDS MAPK 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 LSL/BAC Acinar mK-Ras K-RasG12D Endogenous mutant K-Ras expression in acinar cells Normal Pancreas at early age High Fat Diet Promotes PDAC LSL/BAC Acinar mK-Ras High Fat Diet K-RasG12D Endogenous mutant K-Ras expression in acinar cells Normal Pancreas at early age Cox2 PanINs Cancer Cox-2 Deletion in Acinar Cells with “Knock-in” of Oncogenic K-Ras LSL/BAC Cox-2 KO “flox-stopped“ Acinar mK-Ras K-RasG12D Endogenous mutant K-Ras expression in acinar cells X Cox-2 Deletion in Acinar Cells with “Knock-in” of Oncogenic K-Ras LSL/BAC Cox-2 KO “flox-stopped“ Acinar mK-Ras K-RasG12D X Endogenous mutant K-Ras expression in acinar cells COXKO/LSL/BAC Acinar mK-Ras K-RasG12D Endogenous mutant K-Ras expression in acinar cells but NO Cox-2 Expression COX-2 is Required in High Fat-Induced PDAC Mouse Model COXKO/LSL/BAC % kcal of each nutrient Acinar mK-Ras K-RasG12D Endogenous mutant K-Ras expression in acinar cells but NO Cox-2 Expression Caloric Breakdown Protein Fat Carbohydrate Control Diet 18.3 10.2 71.5 High Fat Diet 18.1 61.6 20.3 Isocaloric CONTROL DIET HIGH FAT DIET COXKO/BAC (n=10) COXKO/BAC (n=10) COXKO/LSL/BAC (n=10) COXKO/LSL/BAC (n=10) 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 30 days 160 Days High Fat Diet High Fat Diet Decreases Survival of Mice Susceptible to PDAC Control Diet High Fat Diet 30 days 205 Days Pancreas Pancreas Pancreas High Fat Diet Decreases Survival of Mice Susceptible to PDAC Control Diet High Fat Diet 30 days 205 Days Pancreas High Fat Diet Pancreas Pancreas Pancreas Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Ras re Cell Type Specific Promoter Duct Islet Acini Elastase-Cre-Er Tamoxifen Regulated Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Ras re Cell Type Specific Promoter PDX-1 Cre Activates Cre during development Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Ras re Cell Type Specific Promoter Oncogenic K-Ras “flox-stopped“ X Endogenous Promoter “Knock In” PDX-1 Cre Activates Cre during development Stop p-K-Ras loxP K-RasG12D loxP loxP = locus of recombination Poly A Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Ras re Oncogenic K-Ras “flox-stopped“ X Cell Type Specific Promoter Endogenous Promoter “Knock In” PDX-1 Cre Stop p-K-Ras Activates Cre during embryogenesis loxP K-RasG12D loxP loxP = locus of recombination Cre mediated deletion during development LSL/PDX-1 mK-Ras All cells K-RasG12D Endogenous mutant K-Ras expression in every cell of the pancreas Poly A High Fat Diet Decreases Survival of Mice Susceptible to PDAC Some Mice 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 137 Days High Fat Diet Cre mediated deletion during development 90 Days High Fat Diet Accelerated PanIN-2 and PanIN-3 Development Compared to Control Diet Using and Embryonic Promoter Control Diet 137 Days High Fat Diet Cre mediated deletion during development 90 Days Summary: Obesity is a Risk Factor for PDAC in Humans High Fat Diet Accelerates PanIN formation and Cancer 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 LSL/BAC Test Prevention Agents Acknowledgments Dr. Logsdon’s Laboratory Collaborators MDACC Huamin Wang, Pathology Jason Fleming, Surgical Oncology Joya Chandra, Pediatrics David McConkey, Urology Kathleen M. Schmeler MD, Ob/GYN Ralph B. Arlinghaus, Translational Molecular Pathology Adel El-Naggar, Pathology The Methodist Hospital Research Institute Ching-Hsuan Tung Ph.D. Wael R. Abd-Elgaliel Ph.D. Rita Serta Ph.D. Bincy Philips, MS Funding Sources NIDDK Minority Supplement Phi Beta Psi Sorority City of Hope National Medical Center Ann David Ph.D. University of Rhode Island Oleg Andreev Yana Reshetnyak
