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

PKA

GDPGs

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

Chondrosarcoma Tumor Explants

1 2 3 4 5 6 7 - + - + - + - + - + - + - +

-GAPDH-Type X collagen

+ Cyclopamine

Growth Plate

Enchondroma ASIHH

ASIHH

0.1%FCS PTHrP

26 28 26 28 PCR cycles

Is the IHH/PTHrP Signaling Pathway Functional in Cartilage Tumors?

IHH

PTHrP

Proliferation

Differentiation

IHH

PTHrP

Proliferation

DifferentiationX

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

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

Oil Triparanol

Se

rum

Ch

ole

ste

rol (

mM

)

* n = 3, p-value < 0.01 using Student t-test

Does Triparanol Block Hedgehog Target Gene Expression in the

Bones of Treated Mice?

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Oil TriparanolPer

cen

tag

e o

f PT

CH

exp

ress

ing

cel

ls

Oli

ve O

il

Tri

para

nol

GAPDHPTCH

n=12, p-value <0.05 using student t-test

* n =12, p-value = 0.01 using Student t-test

Triparanol: Tumor Size

TriparanolOil

-40

-20

0

20

40

60

80

100

Pe

rce

nt

ch

an

ge

in t

um

ou

r v

olu

me

n=3, p-value < 0.05 using Student t-test

0

10

20

30

40

50

60

70

80

90

Oil Triparanol

Pe

rce

nt

Brd

U P

os

itiv

eTriparanol: Tumor Proliferation

Olive Oil

Triparanol

Triparanol: Cellularity

n=12, p-value <0.05 using student t-test

Triparanol: Apoptosis

0

0.5

1

1.5

2

2.5

Oil Triparanol

Pe

rce

nta

ge

of

Ce

lls U

nd

erg

oin

g

Ap

oto

sis

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 GokgozIrene Andrulis

Bob BellPeter Ferguson

0

200

400

600

800

1000

1200

1400

CSA ECA

co

un

ts p

er

min

ute

ethanolcyclopamine

020406080

100120140160180

triparanol carrier

size

Sizemm3

Triparanol

Carrier

Histology

Collaborators:Toronto

J. Wunder, I. Andrulis, R. Bell, W. Cole, CC. HuiBoston

H. Juppner, R. Gensure

Funding: CIHR, IHRT, NCIC, Terry Fox Run, Hospital for Sick Children, Canadian Research Chairs program