ENERGY BALANCE JOURNAL CLUB

1. A High-Fat Diet Activates Oncogenic Kras and COX2 to Induce Development of Pancreatic Ductal Adenocarcinoma in Mice ENERGY BALANCE JOURNAL CLUB 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

2. Pancreatic Cancer: A Deadly Disease 2013 Estimated US Cancer Deaths Men306,920 Women273,430 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% 26% Lung & bronchus 14% Breast 9% Colon & rectum 7% Pancreas 5% Ovary 4% Leukemia 3% Non-Hodgkin lymphoma 3% Uterine corpus 2% Liver & intrahepatic bile duct 2% Brain and other nervous system Source: American Cancer Society, 2013

3. 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

4. Pancreas Microanatomy Endocrine Exocrine Most pancreatic cancers (around 75%) Develop in the exocrine pancreas Omary, M.B., et. al., 2007. 117: p. 50-9

5. Multistep Progression Model of PDAC Pancreatic Intraepithelial Neoplasias (PanINs) 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 PDAC >90% Mutant K-Ras Hruban, R.H., et al., 2001. 25(5): p. 579-86

6. Non-Modifiable PDAC Risk Factors

7. Modifiable PDAC Risk Factors Obesity

8. Obesity is a Risk Factor for Many Cancers Including Pancreatic Eugenia E. Calle* and Rudolf Kaaks 2004:4:579-591

9. Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Ras re Cell Type Specific Promoter Duct Islet Acini

10. Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Ras re Cell Type Specific Promoter Duct Islet Acini Elastase-Cre-Er Tamoxifen Regulated

11. 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

12. 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 K-RasG12D Poly A p-K-Ras Duct loxP loxP loxP = locus of recombination Islet Acini Acinar Cell Specific Cre Elastase-Cre-Er Tamoxifen Regulated

13. K-RasG12D 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” Elastase-Cre-Er Stop K-RasG12D Poly A p-K-Ras loxP loxP Cre mediated deletion via Tamoxifen after birth loxP = locus of recombination LSL/BAC Acinar mK-Ras Endogenous mutant K-Ras expression in acinar cells

14. K-RasG12D 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” Elastase-Cre-Er Stop K-RasG12D Poly A p-K-Ras loxP loxP Cre mediated deletion via Tamoxifen after birth loxP = locus of recombination LSL/BAC Acinar mK-Ras Endogenous mutant K-Ras expression in acinar cells Normal Pancreas at early age

15. K-RasG12D 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? Cell Type Specific Promoter Endogenous Promoter “Knock In” Elastase-Cre-Er Stop K-RasG12D Poly A p-K-Ras loxP loxP Cre mediated deletion via Tamoxifen after birth loxP = locus of recombination LSL/BAC Acinar mK-Ras Endogenous mutant K-Ras expression in acinar cells Normal Pancreas at early age

16. K-RasG12D High Fat-Induced PDAC Mouse Model LSL/BAC Acinar mK-Ras Endogenous mutant K-Ras expression in acinar cells

17. K-RasG12D High Fat-Induced PDAC Mouse Model LSL/BAC Acinar mK-Ras Endogenous mutant K-Ras expression in acinar cells Isocaloric

18. K-RasG12D High Fat-Induced PDAC Mouse Model LSL/BAC Acinar mK-Ras Endogenous mutant K-Ras expression in acinar cells Isocaloric

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

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

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

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

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

24. 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)

25. 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.

26. Mutant K-Ras at Endogenous Levels Requires an Inflammatory Insult to Transform Acinar Cells mutant Ras NO Stimulant mutant Ras + LPS Stimulant

27. High Fat Diet Link Between Ras and Obesity?

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

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

30. Ras Must Be Active to Initiate Downstream Signaling Resulting in Progression of PDAC Cox2 PGE2 Receptors (GF, hormones, etc) PI3K Ras-GTP GEFs Raf Ras-GDP GAPs RalGDS INACTIVE ACTIVE MAPK

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

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

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

34. K-RasG12D High Fat Diet Promotes PDAC LSL/BAC Acinar mK-Ras Endogenous mutant K-Ras expression in acinar cells Normal Pancreas at early age

35. K-RasG12D High Fat Diet Promotes PDAC LSL/BAC Acinar mK-Ras High Fat Diet Endogenous mutant K-Ras expression in acinar cells Normal Pancreas at early age Cox2 PanINs Cancer

36. K-RasG12D Cox-2 Deletion in Acinar Cells with “Knock-in” of Oncogenic K-Ras LSL/BAC Cox-2 KO “flox-stopped“ Acinar mK-Ras X Endogenous mutant K-Ras expression in acinar cells

37. K-RasG12D K-RasG12D Cox-2 Deletion in Acinar Cells with “Knock-in” of Oncogenic K-Ras LSL/BAC Cox-2 KO “flox-stopped“ Acinar mK-Ras X Endogenous mutant K-Ras expression in acinar cells COXKO/LSL/BAC Acinar mK-Ras Endogenous mutant K-Ras expression in acinar cells but NO Cox-2 Expression

38. K-RasG12D COX-2 is Required in High Fat-Induced PDAC Mouse Model COXKO/LSL/BAC Acinar mK-Ras Endogenous mutant K-Ras expression in acinar cells but NO Cox-2 Expression Isocaloric

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

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

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

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

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

44. High Fat Diet Decreases Survival of Mice Susceptible to PDAC 30 days

45. High Fat Diet Decreases Survival of Mice Susceptible to PDAC Control Diet 30 days 160 Days High Fat Diet

46. High Fat Diet Decreases Survival of Mice Susceptible to PDAC Control Diet High Fat Diet 30 days 205 Days Pancreas Pancreas Pancreas

47. High Fat Diet Decreases Survival of Mice Susceptible to PDAC Control Diet High Fat Diet 30 days 205 Days Pancreas Pancreas High Fat Diet Pancreas Pancreas

48. Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Ras re Cell Type Specific Promoter Duct Islet Acini Elastase-Cre-Er Tamoxifen Regulated

49. Mouse Models Allow Conditional Specific “Knock-in” of Oncogenic K-Ras re Cell Type Specific Promoter PDX-1 Cre Activates Cre during development

50. 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 K-RasG12D Poly A p-K-Ras Activates Cre during development loxP loxP loxP = locus of recombination