1 / 32

Hedgehog Signaling Regulates Proliferation in Chondrosarcoma: Implications for Novel Therapy

Hedgehog Signaling Regulates Proliferation in Chondrosarcoma: Implications for Novel Therapy. Dung Tiet * Sevan Hopyan Puvi Nadesan Ben Alman Jay Wunder. Mount Sinai Hospital, Toronto, Canada Hospital for Sick Children, Toronto, Canada. Treatment of Chondrosarcoma. Radiation resistant

telma
Download Presentation

Hedgehog Signaling Regulates Proliferation in Chondrosarcoma: Implications for Novel Therapy

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Hedgehog Signaling Regulates Proliferation in Chondrosarcoma:Implications for Novel Therapy Dung Tiet* Sevan Hopyan Puvi Nadesan Ben Alman Jay Wunder Mount Sinai Hospital, Toronto, Canada Hospital for Sick Children, Toronto, Canada

  2. Treatment of Chondrosarcoma • Radiation resistant • Chemotherapy resistant • Requires radical surgery

  3. Chondrosarcoma: Clues to Etiology • Enchondromas are benign precursor lesions • Enchondromas may arise from abnormal regulation of growth plate chondrocytes • Normal growth plate regulation: Indian hedgehog (IHH) and Parathyroid hormone related protein (PTHrP)

  4. Growth Plate Chondrocyte Differentiation Resting zone PTHrP Proliferative zone PTHrP-R Prehypertrophic zone Indian Hedgehog Hypertrophic zone Programmed Cell Death Perichondrium

  5. HedgehogSignaling PKA PKA

  6. PTH PTHrP PTHR-1 NHERF Gq Gs GDP AC GTP PLC cAMP IP3 DAG PKA Ca++ PKC Known pathways in PTHrP Signaling

  7. IHH Proliferation Differentiation PTHrP

  8. Chondrosarcoma: Clues to Etiology • Some cases of enchondromatosis are caused by mutation of the PTHrP protein receptor (PTHR1)  Constitutive activation of Hedgehog signaling • Transgenic mice - mutant PTHR1 - Gli2  Multiple enchondroma-like lesions Hopyan et al, Nature Genetics 30: 306, 2002

  9. Treatment Implications • Hedgehog signaling causes cartilage neoplasia • Hedgehog blockade might be used to treat these tumours

  10. Chondrosarcoma: Study Goals • Expression of IHH/PTHrP pathway genes • Tumor explant culture system to assess the effects of Hedgehog blockade • Tumor xenograft model to examine the effects of Hedgehog blockade

  11. Chondrosarcoma: Methods • Sarcoma tumor bank for frozen specimens • Gene expression by quantitative RT-PCR • Tumor explant culture system • 4 growth plate, 2 enchondroma, 10 chondrosarcoma • agonists: Shh, PTHrP • Hedgehog blockade: cyclopamine • proliferation (thymidine), differentiation (ColX) • Tumor xenograft model • 12 chondrosarcoma • Hedgehog blockade: triparanol • proliferation (BrdU), cellularity, size, apoptosis (TUNEL), Hedgehog target gene expression,

  12. Hedgehog Blocking Agents • Cyclopamine • identified as a teratogen • binds and inactivates SMO • Triparanol • an inhibitor of cholesterol synthesis • increases PTCH repression of SMO

  13. Gene Expression in Chondrosarcoma

  14. Gene Expression in Chondrosarcoma Are IHH/PTHrP pathway members expressed? • IHH, PTCH, GLI1, GLI2, GLI3, PTHrP, PTHR1 • Consistent expression in all ECA and CSA Is Hedgehog signaling active? • Levels of PTCH and GLI1 were higher in ECA and CSA compared to normal articular cartilage or cortical bone

  15. Chondrosarcoma Tumor Explants

  16. Chondrosarcoma Tumor Explants 1 2 3 4 5 6 7 - + - + - + - + - + - + - + -GAPDH -Type X collagen + Cyclopamine

  17. Growth Plate Enchondroma 26 28 26 28 PCR cycles IHH AS IHH Proliferation AS IHH Differentiation PTHrP 0.1%FCS PTHrP Is the IHH/PTHrP Signaling Pathway Functional in Cartilage Tumors?

  18. Constitutively Active Hedgehog Signaling in Cartilaginous Neoplasia IHH Proliferation X Differentiation PTHrP

  19. Tumor Xenograft Model Each tumor cut sample into 10 equal pieces Human chondrosarcoma samples (n=12) NOD-SCID mice Feed mice with triparanol Feed mice with olive oil Place samples in fixative for staining Freeze samples for molecular analysis Collect tumours

  20. Do Mice Uptake Triparanol?Serum Cholesterol Levels * n = 3, p-value < 0.01 using Student t-test

  21. Does Triparanol Block Hedgehog Target Gene Expression in the Bones of Treated Mice? Triparanol Olive Oil GAPDH PTCH n=12, p-value <0.05 using student t-test

  22. Triparanol: Tumor Size * n =12, p-value = 0.01 using Student t-test

  23. Triparanol: Tumor Proliferation n=3, p-value < 0.05 using Student t-test

  24. Triparanol: Cellularity Olive Oil Triparanol n=12, p-value <0.05 using student t-test

  25. Triparanol: Apoptosis n=3, p-value < 0.86 using Student t-test

  26. Conclusion • Hedgehog signaling is active in chondrosarcoma • PTHrP cannot downregulate IHH • Constitutive Hedgehog signaling • Triparanol attenuates the proliferative phenotype of chondrosarcoma • Hedgehog blocking agents may provide a novel therapeutic strategy for chondrosarcoma • Hedgehog signaling and blockade are also functional in other cancers: Breast, GI, Prostate, Basal cell carcinoma, Medulloblastoma

  27. Acknowledgements Nalan Gokgoz Irene Andrulis Bob Bell Peter Ferguson

  28. Histology Size mm3 Carrier Triparanol

  29. Collaborators: Toronto J. Wunder, I. Andrulis, R. Bell, W. Cole, CC. Hui Boston H. Juppner, R. Gensure Funding: CIHR, IHRT, NCIC, Terry Fox Run, Hospital for Sick Children, Canadian Research Chairs program

More Related