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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
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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 • Chemotherapy resistant • Requires radical surgery
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)
Growth Plate Chondrocyte Differentiation Resting zone PTHrP Proliferative zone PTHrP-R Prehypertrophic zone Indian Hedgehog Hypertrophic zone Programmed Cell Death Perichondrium
HedgehogSignaling PKA PKA
PTH PTHrP PTHR-1 NHERF Gq Gs GDP AC GTP PLC cAMP IP3 DAG PKA Ca++ PKC Known pathways in PTHrP Signaling
IHH Proliferation Differentiation PTHrP
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
Treatment Implications • Hedgehog signaling causes cartilage neoplasia • Hedgehog blockade might be used to treat these tumours
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
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,
Hedgehog Blocking Agents • Cyclopamine • identified as a teratogen • binds and inactivates SMO • Triparanol • an inhibitor of cholesterol synthesis • increases PTCH repression of SMO
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
Chondrosarcoma Tumor Explants 1 2 3 4 5 6 7 - + - + - + - + - + - + - + -GAPDH -Type X collagen + Cyclopamine
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?
Constitutively Active Hedgehog Signaling in Cartilaginous Neoplasia IHH Proliferation X Differentiation PTHrP
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
Do Mice Uptake Triparanol?Serum Cholesterol Levels * n = 3, p-value < 0.01 using Student t-test
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
Triparanol: Tumor Size * n =12, p-value = 0.01 using Student t-test
Triparanol: Tumor Proliferation n=3, p-value < 0.05 using Student t-test
Triparanol: Cellularity Olive Oil Triparanol n=12, p-value <0.05 using student t-test
Triparanol: Apoptosis n=3, p-value < 0.86 using Student t-test
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
Acknowledgements Nalan Gokgoz Irene Andrulis Bob Bell Peter Ferguson
Histology Size mm3 Carrier Triparanol
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