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