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  • Needs to be repeated: vit D ; biomarker for K23
  • Needs to be repeated: vit D ; biomarker for K23
  • The pivotal molecular abnormality in BCC carcinogenesis is inappropriate activation of the hedgehog (HH) signaling pathway [17]. Essentially all BCC tumors have an overactive HH signaling pathway, commonly occurring by mutational inactivation of the tumor suppressor Patched1 ( PTCH1 ) gene
  • This developmental pathway is best known for its role in embryonic patterning and hair follicle development [18]. In adult cells, genes responsible for cell proliferation, growth, and invasion are activated when the HH pathway is on. Ptch1 is a transmembrane receptor for the HH ligand, and is the key inhibitor of HH signaling. Ptch1 inhibits HH target genes by repressing Smoothened (Smo) rendering downstream Gli transcription factors inactive. The HH ligand binds to the Ptch receptor, thus relieving inhibition of Smo and activating Gli transcription factors. Most BCC tumors have inactivating mutations in the PTCH1 gene that cause Smo signaling to be constitutively activated [21]. Gli transcription factors are then continuously active and increase the expression of HH target genes. The key biomarker for the HH pathway is Gli mRNA levels . All BCC tumors have increased Gli levels, and molecularly targeted drugs against BCC have focused on decreasing the HH pathway and decreasing Gli mRNA [22]. One such example is cyclopamine, a plant alkaloid that inhibits Smo [23]. Model systems ( in vitro and in vivo ) showed that cyclopamine effectively inhibited BCCs [24], but clinical applications of cyclopamine showed severe side effects that would preclude its use as a chemopreventive agent for BCC
  • Humans with the rare autosomal domin dis BCNS inherit a defective copy of PTCH1. these pts have multiple tens to hundreds of BCCs. And characteristic develop abln include palmar pits, frontal bossing, jaw cysts, abn calcification. Inc risk for extracutan tumors medullobl and rhab. See CPC by Erv 2008 NEJM. 120 mutations identified, majority lead to premature stop codon. BCCs in BCNS pts 60% on sun exposued sites vs 85% of BCCs in nonsyndromic pts. Number of BCCs not related to lifetime sun exposure (Giovanna 1993).
  • Elements of translational research. Necessary steps to developing new drugs/therapeutics. Why BCCs are a great model to study molecularly targeted therapeutics. All you had to do was shut down the HH pathway. Why I was drawn to studying BCCs and the Epstein lab.
  • Needs to be repeated: vit D ; biomarker for K23
  • Ptch1 mice were bred with Cox1 and Cox2 ko mice (developed Smithies lab/Taconic). We compared microscopic BCCs in three groups: Ptch1 mice wt cox 1/cox2, Ptch1 mice deleted for Cox1, and Ptch1 mice deleted for COx2. deletion of cox1 or cox2 decreases number of bccs, signif dec BCC burden. Deletion of Cox2 seemed to sign reduce BCCx compare to cox 1.
  • no dose response; not as dramatic at genetic deletion of cox2 enzy
  • Needs to be repeated: vit D ; biomarker for K23
  • Statins and vit D are not HH pathway specific, act on other pathways. But vit D and statins are relatively safe to take and tolerable used for many decades. Min tox profile, good as chemopreventive agents
  • Elements of translational research. Necessary steps to developing new drugs/therapeutics. Why BCCs are a great model to study molecularly targeted therapeutics. All you had to do was shut down the HH pathway. Why I was drawn to studying BCCs and the Epstein lab.
  • BCC tumors from Ptch1+/- K14-Cre-ER2 p53 fl/fl mice stain blue due to ß-galactosidase activity which is encoded by the lacZ gene that was inserted to replace the wildtype Ptch1 gene in Ptch1+/- mice.
  • Cedric: measure oxysterols, vitamin D3 or 25OH?, total cholesterol in skin and tumor treated samples.
  • Elements of translational research. Necessary steps to developing new drugs/therapeutics. Why BCCs are a great model to study molecularly targeted therapeutics. All you had to do was shut down the HH pathway. Why I was drawn to studying BCCs and the Epstein lab.
  • Needs to be repeated: vit D ; biomarker for K23
  • By screening a small molec library, Curis identified compounds inhibited Smo and decreased Gli levels in reporter assays. One compounds was formulated into a cream and a second compound was form for oral delivery Shown to inhibit microscopic BCCs in an ex vivo assay Phase II trial failed of topical formulation Minimal effect on BCCs in wildtype humans Did not decrease Gli1 mRNA in BCCs Hh Antagonist cream abandoned
  • No irritation on mice with w/0 BCCs
  • Placebo treated BCCs increased by 40% in tumor area while BCCs treated with Cur-61414 decreased in size by 40% (p<0.0001) and this correlated with a significant reduction of Gli1 expression in Cur-61414-treated BCCs. We asked whether this dec in BCC size was due to an inhibition of prolifer or induction of apoptosis in BCCs or something else
  • Treated BCCs with topical Cur 414 for 4 days and looked for markers of prolifer (ki67 staining) and apoposis (CC3) . We saw intense Ki67 staining, showing highly prolifer cells. Cur 414 treated tumors and marked dec in Did not activate apoptosis howev the caveat is that thse mice were deleted for p53 .
  • Statins and vit D are not HH pathway specific, act on other pathways. But vit D and statins are relatively safe to take and tolerable used for many decades. Min tox profile, good as chemopreventive agents
  • Elements of translational research. Necessary steps to developing new drugs/therapeutics. Why BCCs are a great model to study molecularly targeted therapeutics. All you had to do was shut down the HH pathway. Why I was drawn to studying BCCs and the Epstein lab.
  • Needs to be repeated: vit D ; biomarker for K23

Transcript

  • 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 Epidemiological studies New drugs
  • 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. Clinical trials in PTCH1 +/- BCNS patients Observational studies at Kaiser/UCSF Screen for drugs in cell lines and Ptch1+/- mice Translational Research in BCC
  • 6. Summary of chemopreventive agents against BCCs In vitro Mice Observational studies Clinical Trials Celecoxib (oral) ↓ ↓ mixed ↓ in subset Statin No No No N/A Vitamin D3 ↓↓ ↓↓ ↓ Biomarker study Hh Antagonist (Genentech) ↓↓↓ ↓↓↓↓ none Enrolling RCT Itraconazole (antifungal) ↓↓ ↓↓ none Biomarker study
  • 7. Challenge Lesson Learned Celecoxib (oral) Combining mouse and RCT Statistics, finding the experts Statins Difficulty in combining mouse and epidemiologic results (Kaiser) OK to disagree Vitamin D3 Difficulty of doing it all yourself: lab, mouse, pilot clinical trial
    • 1°Mentor and his lab
    • SFCC, EpiBio mentors
    • -Public databases
    Hh Antagonist Collaborations with Genentech -Good to have lab skills -RCT design Itraconazole Collaborations with basic lab FDA approved drug -Nice to collaborate -Time to start the pilot trial
  • 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%
    • 5 th 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. Hedgehog signaling pathway regulates cell proliferation and growth Ptch Smo Gli off HH Ptch Smo Gli on Mutant Ptch Smo Gli on CPN Smo Gli off Ptch Smo Gli off Ptch Smo Gli off HH Ptch Smo Gli on HH Ptch Smo Gli on Mutant Ptch Smo Gli on Mutant Ptch Smo Gli on Smo Gli
  • 11. Basal cell nevus syndrome Basal cell nevus syndrome are PTCH1+/-
  • 12. Ptch1+/- mice mimic BCNS phenotype: develop BCC tumors after IR or UV treatment Goodrich and Scott, Science 1997 Aszterbaum, Oro, Scott, Epstein Nature Medicine 1999 (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
  • 13. Mechanism of disease In vitro experiments Animal models Human clinical trials Hedgehog pathway BCC cell lines Ptch1 +/- mice PTCH1+/- Basal cell nevus syndrome patients Roadmap for finding new therapeutics Epidemiological studies Patients with sporadic BCCs
  • 14. Summary of chemopreventive agents against BCCs In vitro Mice Observational studies Clinical Trials Celecoxib (oral) ↓ ↓ mixed ↓ in subset Statin No No No N/A Vitamin D3 ↓↓ ↓↓ ↓ Biomarker study Hh Antagonist (Genentech) ↓↓↓ ↓↓↓↓ none Enrolling RCT Itraconazole (antifungal) ↓↓ ↓↓ none Biomarker study
  • 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) Placebo (N=27) Celecoxib (N=33) Age 42 ± 12 47 ± 12 Number of BCC tumors greater than 3mm 24 ± 27 45 ± 76 Body Mass Index 31 ± 1.3 30 ± 1.1 % Male 48% 58% % Caucasian 96% 100% % With >15 BCCs at baseline 41% 39% % Seen at California site 59% 55%
  • 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
  • 22.  
  • 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 In vitro Mice Observational studies Clinical Trials Celecoxib (oral) ↓ ↓ mixed ↓ in subset Statin No No No N/A Vitamin D3 ↓↓ ↓↓ ↓ Biomarker study Hh Antagonist (Genentech) ↓↓↓ ↓↓↓↓ none Enrolling RCT Itraconazole (antifungal) ↓↓ ↓↓ none Biomarker study
  • 28. 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 Vitamin D3, Statins
  • 29. Mechanism of disease In vitro experiments Animal models Human clinical trials Statins blocks Hedgehog pathway BCC cell lines Ptch1 +/- mice Epidemiological studies Statin therapy and risk of subsequent BCCs in Kaiser cohort
  • 30.  
  • 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. Topical Vitamin D3 decrease BCC development by 50% (p<0.05)
    • 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.
  • 35. Topical vitamin D3 decreases BCC development in mice *Adjusted for gender and coat color of mouse Unadjusted p value Adjusted* p value Vitamin D3 -62% .008 -53% .042 1,25(OH)2D -36% .39 -33% .43 Statin** -58% .10 -40% .30
  • 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 In vitro experiments Animal models Human clinical trials Vit D3 blocks Hedgehog pathway BCC cell lines Ptch1 +/- mice PTCH1+/- Basal cell nevus syndrome patients Epidemiological studies Vit D3 levels in BCC pts
  • 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. Tang 1,3 , Neeta Parimi 2 , Angela Wu 1,3 , John Boscardin 1 , Meg Chren 1 ,Steven R. Cummings 1,2 , Ervin Epstein 3 , and Douglas C. Bauer 1,2
    • 1 University of California San Francisco, 2 San Francisco Coordinating Center, California Pacific Medical Center Research Institute, 3 Children’s Hospital Oakland Research Institute
  • 40.  
  • 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)
  • 42.  
  • 43. Summary of chemopreventive agents against BCCs In vitro Mice Observational studies Clinical Trials Celecoxib (oral) ↓ ↓ mixed ↓ in subset Statin No No No N/A Vitamin D3 ↓↓ ↓↓ ↓ Biomarker study Hh Antagonist (Genentech) ↓↓↓ ↓↓↓↓ none Enrolling RCT Itraconazole (antifungal) ↓↓ ↓↓ none Biomarker study
  • 44. Cyclopamine Smo Gli Williams and Wang , Proceedings of the Natl Acad Sci 2003 Topical Cur-414 cream Oral Hh antag
  • 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 35 Day 20
  • 48.  
  • 49. S Tu Re Re Re Tu Tu S S Tu S Tu S Tu Tu Hh Antagonist cream Placebo Tu Hh Antagonist cream decreases Gli1 mRNA in BCC tumors Lesson: benefits of collaborator
  • 50. Lesson: Benefits of UCSF Cancer Center core labs
  • 51. Topical Cur-414 treated BCC
  • 52.  
  • 53. Lessons learned: mechanism of disease
    • Topical and oral HH antagonists significantly reduce murine BCCs by decreasing tumor proliferation and/or inducing follicular differentiation.
    • We know how to collect tumors, do these assays, get to mechanism of disease in future trials
  • 54.  
  • 55. Investigator sponsored trial in BCC prevention
    • Genentech Hh antagonist GDC 0449: 150mg daily
    • Phase II, placebo controlled RCT in 41 Basal Cell Nevus Syndrome subjects
    • Primary endpoint: change in BCCs at 12 mo and 18 mo
  • 56. New and relatively safe agents that decrease Hedgehog signaling Cyclopamine Smo Gli J Kim and P Beachy, Stanford Itraconazole
  • 57. Mechanism of disease In vitro experiments Animal models Human clinical trials Small molecule library screen for inhibitors of Hedgehog pathway Cell based assays Ptch1 +/- mice Patients with sporadic BCCs
  • 58. Day 0, BCC A: 10mm Day 4 – irritation and necrosis, 11 mm Day 11 – residual BCC, 3mm Itraconazole #178
  • 59. Cyclodextrin #5460 Day 0 – BCC B (11 mm), BCC D (10 mm), BCC G (9mm) Day 7 – BCC B (11 mm), BCC D (10 mm), BCC G (9mm)
  • 60. Lessons learned:
    • Collaboration with another basic lab
      • Post-doc fellows who are experts at specific assays (5 years) vs training someone new
    • Focus on getting the first pilot clinical trial of itraconazole on human BCCs
      • Measure Gli1mRNA in BCCs
      • Measure Ki67
      • Paired t-test (at biopsy and at excision)
    • New opportunities/markets
  • 61. Lessons learned:
    • Easier to translate FDA approved drug or a drug manufactured and tested by Pharma - already have paid the $$$
    • New agent (vitamin D) – investigator pays
      • Efficacy
      • Stability
      • GMP grade for human
      • IND
  • 62. Challenge Lesson Learned Celecoxib (oral) Combining mouse and RCT Statistics, finding the experts Statins Difficulty in combining mouse and epidemiologic results (Kaiser) OK to disagree Vitamin D3 Difficulty of doing it all yourself: lab, mouse, pilot clinical trial
    • 1°Mentor and his lab
    • SFCC, EpiBio mentors
    • -Public databases
    Hh Antagonist Collaborations with Genentech -Good to have lab skills -RCT design Itraconazole Collaborations with basic lab FDA approved drug -Nice to collaborate -Time to start the pilot trial
  • 63.
    • Children’s Hospital Oakland
    • Ervin Epstein
    • Po Lin So
    • Tony Zheng Xiao
    • Elana Shpall
    • Angela Wu
    • Kris Chang
    • Yefim Khaimsky
    • UCSF EpiBiostat
    • Charles McCulloch
    • Ralph Gonzalez
    • Steve Hulley
    • Steve Cummings
    • Doug Bauer
    • Neeta Parimi
    • John Boscardin
    • Michael Kohn
    • Kaiser Division of Research
    • Maryam Asgari
    • Genentech
    • Fred de Sauvage
    • Tracy Tang
    • Chris Callahan
    • UCSF Derm
    • Meg Chren
    • Dan Bikle
    • Loretta Chan
    • Funding Sources
    • NRCC – CTSA KL2
    • NIAMS – K23
    • Prevent Cancer Foundation
    • American Skin Association