Lessons Learned in T1 Research: mouse to human

1. Lessons Learned in T1 Research: mouse to human Jean Y. Tang MD PhD Assistant Professor Department of Dermatology

2. Mechanism of disease In vitro experiments Animal studies Human clinical trials New drugs Epidemiological studies

3. Lessons learned: challenges • Jack of all trades, Master of none • Journals • Conferences • Students and trainees • Not that many role models – find a true believer and the experts Slower time to publication Grants: enthusiasm from NIH

4. KL2/K23 Mentors: • Ervin Epstein, Children’s Hospital Oakland Research Institute • Mary-Margaret Chren, Dept of Dermatology, UCSF • Charles McCulloch, Steve Cummings, Dept of Epidemiology and Biostatistics, UCSF

5. Translational Research in BCC Screen for drugs in cell lines and Ptch1+/- mice Clinical trials in PTCH1 +/- BCNS patients Observational studies at Kaiser/UCSF

6. Summary of chemopreventive agents against BCCs

8. Epidemiology of BCC • 1 million BCC cases per yr in US • Estimated annual incidence of 0.1% to 0.5% • Rare risk of metastasis: < 0.5% • 5th most costly cancer for Medicare • The age-adjusted incidence per 100,000 white individuals: 475 cases in men, 250 cases in women • The estimated lifetime risk of BCC in the white population is 33-39% in men and 23-28% in women. • Risk of second BCC: 44% in 3 yr

9. BCC basic science: • Almost all BCCs have mutations in PTCH1 tumor suppressor gene • All BCCs have increased Hedgehog signaling

10. HH HH HH Ptch Ptch Ptch Ptch Ptch Ptch Mutant Mutant Mutant Ptch Ptch Ptch CPN Smo Smo Smo Smo Smo Smo Smo Smo Smo Smo Smo Gli Gli Gli off off off Gli Gli Gli on on on Gli Gli Gli on on on Gli Gli off Hedgehog signaling pathway regulates cell proliferation and growth

11. Basal cell nevus syndrome Basal cell nevus syndrome are PTCH1+/-

12. (A) Photo of multiple circled BCCs on the back of a patient with Basal Cell Nevus Syndrome. (B) Photo of Ptch1+/- K14-Cre-ER2 p53 fl/fl mice with multiple BCCs Ptch1+/- mice mimic BCNS phenotype: develop BCC tumors after IR or UV treatmentGoodrich and Scott, Science 1997Aszterbaum, Oro, Scott, Epstein Nature Medicine 1999

13. Roadmap for finding new therapeutics Mechanism of disease Hedgehog pathway BCC cell lines In vitro experiments Ptch1 +/- mice Animal models PTCH1+/- Basal cell nevus syndrome patients Human clinical trials Patients with sporadic BCCs Epidemiological studies

14. Summary of chemopreventive agents against BCCs

15. Genetic deletion of Cox1 or Cox2 decreases microscopic BCCs in Ptch1+/- mice IR-treated Ptch1+/- mice wild type (n=24), deleted for Cox1 (n=12) or for Cox2 (n=6). Mean and SEM. p<0.05 Cox1 and Cox 2 KO: Smithies Cell 1995 * *

16. Celecoxib decreases microscropic BCC burden in Ptch1+/- mice (p<0.05)

17. Study design: Phase II randomized, double-blinded, placebo controlled trial • Subjects: 60 patients with Basal Cell Nevus Syndrome (BCNS) • Treatment: oral Celecoxib at 200mg BID versus placebo for 24 months followed by 12 months of observation • Primary endpoint: change in BCC numbers during study periods

18. Baseline characteristics of study participants are similar in two groups (mean and SD)

19. BCNS subject with low number of BCCs at baseline (<15 tumors)

20. BCNS subject with high number of BCCs at baseline (>15 tumors)

21. How to analyze BCC development • Regession technique: Linear mixed models • Calcuates a slope or rate (number of BCCs/yr) for each patient • Compare percent change in rate of BCCs in placebo and celecoxib groups • Accounts for drop-outs • Adjust for age, gender, BCC at baseline

23. Celecoxib reduces BCC development in subjects with less severe disease (< 15 BCCs)

24. Lessons learned: importance of finding a reliable mouse model • Ptch1+/- mice are a reliable model for testing new anti-BCC agents in humans • Moderate effect of celecoxib on BCCs in mice and in BCNS patients • Greater effect on tumor size rather than number in both mice and BCNS patients

25. Lessons learned: statistics • Statistics – linear mixed models for determining slope of BCCs in RCT • Regression models for tumors in mice • Go to class • Building a database (and managing) • Go to class

26. Lessons learned: • Long time to publication • Journals and co-authors disagree on whether to present data from mice and clinical trial study together • Cancer Prevention Research

27. Summary of chemopreventive agents against BCCs

28. Vitamin D3, Statins New and relatively safe agents that decrease Hedgehog signaling Cyclopamine Smo Gli Corcoran and Scott, Proceedings of the Natl Acad Sci 2006 Bijlsma and Peppelenbosch, PLOS Biology 2007

29. Mechanism of disease Statins blocks Hedgehog pathway BCC cell lines In vitro experiments Ptch1 +/- mice Animal models Human clinical trials Statin therapy and risk of subsequent BCCs in Kaiser cohort Epidemiological studies

31. Lessons learned • Have a good biomarker or target gene (Gli1 mRNA) • Have a good way to measure bioavailability (24OHase) • Have a reliable cell line

32. Vitamin D3 decreases Gli mRNA in BCC cells

33. Vitamin D3 and 1,25(OH)2 D activate the Vitamin D receptor

34. Ptch1+/- K14-Cre-ER2 p53 fl/fl mice treated with vehicle control (Left) versus topical vitamin D3 (right) at 7 months of age. • Example of large BCC tumor on the dorsal skin these transgenic mice • Histological confirmation of BCC. Topical Vitamin D3 decrease BCC development by 50% (p<0.05)

35. Topical vitamin D3 decreases BCC development in mice *Adjusted for gender and coat color of mouse

36. Lessons learned • Have a good biomarker or target gene (Gli1 mRNA) • Have a good way to measure bioavailability (24OHase) • Pilot trial of topical and oral vitamin D on human BCC

37. Mechanism of disease Vit D3 blocks Hedgehog pathway BCC cell lines In vitro experiments Ptch1 +/- mice Animal models PTCH1+/- Basal cell nevus syndrome patients Human clinical trials Vit D3 levels in BCC pts Epidemiological studies

38. Lesson Learned • Translating from mouse studies to epidemiologic studies (skip the human clinical trial/pilot) • Mouse to Epi (Ralph Gonzalez)

39. The association of serum vitamin D with skin cancer risk in elderly men Jean Y. Tang1,3, Neeta Parimi2, Angela Wu1,3, John Boscardin1, Meg Chren1,Steven R. Cummings1,2, Ervin Epstein3, and Douglas C. Bauer1,2 1 University of California San Francisco, 2 San Francisco Coordinating Center, California Pacific Medical Center Research Institute, 3 Children’s Hospital Oakland Research Institute

41. Table 3 Association of increasing serum 25(OH)D levels with non-        melanoma skin cancer   * Adjusted for age (continuous variable), BMI (continuous variable), season of blood draw, and clinic site † Adjusted for age, BMI, season of blood draw, clinic site, outdoor walking activity (continuous variable), and cigarette smoking (yes/no)

43. Summary of chemopreventive agents against BCCs

44. Topical Cur-414 cream Oral Hh antag Cyclopamine Smo Gli Williams and Wang , Proceedings of the Natl Acad Sci 2003

45. Placebo cream does not reduce BCC tumors Day 0 Day 20 Day 35

46. Untreated BCC

47. Topical Hh Antagonist BCCs decreases BCC tumors Day 0 Day 20 Day 35

49. Hh Antagonist cream decreases Gli1 mRNA in BCC tumors S Tu Tu Tu S Tu S Tu Re S Re Re Tu S Tu Tu Hh Antagonist cream Placebo Lesson: benefits of collaborator

50. Lesson: Benefits of UCSF Cancer Center core labs