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)

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

Hedgehog Signaling PKA

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

IHH PTHrP Proliferation Differentiation

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 Gene Expression in Chondrosarcoma

Chondrosarcoma Tumor Explants

GAPDH -Type X collagen + Cyclopamine

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

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

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

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? Olive OilTriparanol GAPDH PTCH n=12, p-value <0.05 using student t-test

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

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

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

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

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 Conclusion

Acknowledgements Nalan Gokgoz Irene Andrulis Bob Bell Peter Ferguson

Size mm 3 Triparanol Carrier Histology

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