Transcript PPT slides - Johns Hopkins FAMRI

A Cell-Autonomous Requirement for Hedgehog
Signaling in Small Cell Lung Cancer
Craig D. Peacock, Ph.D
Sidney Kimmel Comprehensive Cancer Center
Disclosure
Infinity
Novartis
•Advisory
•Access to novel small molecules
•No Financial Relationship
Cancer Stem Cell: Concept
……that tumors arise from a rare population with stem cell properties
- Durante F. Nesso fisio-pathologico tra la struttura dei nei materni e la
genesi di alcuni tumori maligni. Arch Memor Observ Chir Pract 1874;11:217–26.
- Cohnheim J. Congenitales, quergestreiftes Muskelsarkon der Nireren.
Virchows Arch 1875;65:64
Cancer Stem Cell
Tumor Cell
“heterogeneous”
self-renewal  aberrantly regulated
 differentiation  tumorigenesis
 migration/invasion  metastatic capacity

Cancer Stem Cell: Self-renewal vs Growth
“hierarchical”
Cancer Stem Cell
Clonality
Growth
Apoptosis
Chemosensitivity
Tumor Cell
Hedgehog (Hh) Signaling Pathway
• Evolutionarily conserved
(Drosophila to Homo sapiens)
• One of the key regulators
of animal development
• tissue patterning
• cell division
• differentiation
• Silenced in adults
• stem cell maintenance
(NSC, HSC)
• tissue regeneration
Hedgehog (Hh) Signal Transduction
• Canonical pathway
• normal development
• immortalized embryonic fibroblasts
• relationship to epithelia?
• emphasis on transcriptional readouts
• Gli1, Ptch1, Ptch2, Hhip
Hh Ligands
transcription
Gli1
Ptch1
Ptch2
Hhip
•1o cilium
• membrane-bound organelle, non-cycling cells
• localization of SMO during activation
• described in pancreas cancer.
• other cancers??
Hedgehog and Self-Renewal in Cancer
2007 vol. 104 no. 10 4048-4053
Hedgehog (Hh) Signaling and Cancer
Ligand-independent
Medulloblastoma
Basal Cell Carcinoma
Ligand-dependent
NSCLC
Breast Cancer
Pancreatic Cancer
SCLC
Myeloma
Ovarian Carcinoma
Paracrine
Autocrine/Juxtacrine
Hh signaling and SCLC
Shh
Gli1
Shh
Shh
Gli1
• SCLC tumorigenesis
 persistence of juxtacrine Hh signaling
recapitulates late embryonic airway development
 recapitulates airway repair
Watkins et al. Nature, 2003
Inhibition of Hh signaling in SCLC
NCI-H249
Watkins et al. Nature, 2003
Better Models of SCLC
NOG/SCID
(CL)
HhL pre-E/P
Daniel et al. Cancer Research, 2009
HhL post-E/P
Cancer Stem Cells: Clinical Relevance
Cancer Stem Cell
Chemotherapy
Radiotherapy
Tumor Cell
Conventional Therapies

indiscriminately kills dividing cells

evaluated by rapid tumor size reduction

spares stem cell compartment
 extensive capacity for self-renewal
Recurrence
 quiescence
 active telomerase expression
 activation of anti-apoptotic pathways
 increased membrane transporter
activity (SP)
 metastatic capacity
Tumor Volume (mm3)
Hh Antagonist and SCLC Regeneration
End E/P
Time (Days)
Chemo
Hh Antagonist
Smo antagonist
 does not effect bulk tumor
 delays SCLC regrowth ← chemoresistant fraction
Travaglione et al. AACR Proceedings, 2008
Hh Antagonist and SCLC Regeneration
* p<0.001
Smo antagonist
 does not effect bulk tumor
 delays SCLC regrowth ← chemoresistant fraction
Hh Inhibition and Traditional Readouts
Inhibition of Hh Pathway in Stromal but not Tumor Cells
GLI1 - Tumor
1.00
Vehicle
I926
0.10
D1
D4
D7
D10
Post-Chx (days)
10.00
Relative Gene
Expression
Relative Gene
Expression
10.00
Gli1 - Stroma
1.00
0.10
Vehicle
I926
0.01
D1
D4
D7
Post-Chx (days)
D10
Hedgehog Signaling and Cancer
Nature, 2009.
SCLC
Mouse
Stroma
O’Toole et al. Expert Opin. Ther. Targets, 2009
transcription
Gli1
Ptch1
Ptch2
Hhip
Modulation of Hh Signaling in SCLC
Effects on Clonogenic Growth – LX22CL Bulk Tumor
Modulation of Hh Signaling in SCLC
Effects on Clonogenic Growth – Innately Chemoresistant Fraction
E/P
Modulation of Hh Signaling in SCLC
Effects on Traditional Readouts of Pathway Activity
Relative Transcript Levels
5
E/P
IPI-926 (50nM) : Vehicle
4
ShhN (1ug/ml) : Vehicle
3
2
1
0
GLI1
2.0
AdSmoM2 : AdGFP
40
30
20
10
0
Relative Transcript Levels
Relative Transcript Levels
50
PTCH1
shSMO : shGFP
1.5
1.0
0.5
0.0
SMO
GLI1
PTCH1
SMO
GLI1
PTCH1
Conclusions
Clonal capacity ≠ growth
Hh pathway regulates clonal capacity in SCLC
• Shh ligand
• Ligand specific (5E1)
• Smo specific (IPI926, shRNA, AdSmoM2)
• Transient pathway modulation produces long term effects
• Regulates tumor regeneration in vivo
No consistent change in PTCH1 and GLI1 mRNA expression
SMO Localization in the Primary Cilia of
Chemoresistant LX22CL Cells in vitro
Fliegauf et al. NatRevMCB.,2007
AcT
SMO
Merge
Watkins Lab
Analysis of Hh Signaling in a Mouse
Model of SCLC
Detection of Hh signaling in mSCLC
8
6
4
2
0
300
Rb/p53
Rb/p53
SmoM2
*
200
100
0
Rb/p53 Rb/p53
SmoM2
tumor area/mouse (mm2)
*
10
# CC3+ cells/mm2
# PH3+ cells/mm2
# of tumors/mouse
Increased Hh Signaling Augments mSCLC
25
*
20
15
10
5
0
Rb/p53
400
Rb/p53
SmoM2
ns
300
200
100
0
Rb/p53
Rb/p53
SmoM2
*, p<0.05
Decreased Hh Signaling Moderates mSCLC
Smo
*, p<0.05
Modulation of Hh Signaling in mSCLC
Effects on Clonogenic Growth (self-renewal)
10 µm
lacZ (-) cells
mSCLC lines
1-1 1-2 2-1 2-2
Parental
Shh
Ptc1
X-gal
Smo
Clone 1
Cone 2
Gli1
Cgrp
Gapdh
Cgrp/DAPI
Chemonaive mSCLC
2.00
LDE225(100nM) : Vehicle
1.50
1.00
0.50
Ptch1
Gli1
Hhip
Possibilities
 Off target
• Smo antagonists @ 2 x IC50
 Abnormal regulation of Ptch1/Gli1 transcription in SCLC
• Normal tissues
• Mesenchymal cell lines
 Alternative Gli-dependent transcriptional targets
 Gli1-independent transcriptional targets
•Gli1-/- mice viable and normal
 Non-canonical Hh signaling
•Non-transcriptional Smo signaling
Non-transcriptional Smo signaling
AA metabolism/5-lipoxygenase
• cytoskeletal rearrangement
• migratory response to Shh
Gli-independent
Smo  Src family kinase
• Commissural axon guidance
 Shh-induced
 rapid, Gli-independent
SmoGi Rac1RhoA
• HhL-induced endothelial tubulogenesis
• HhL-induced fibroblast migration
 rapid, Gli-independent
Bijlsma et al. Cell. Signal., 2007
Yam et al. Cell, 2009
Chincilla et al. Cell Cycle, 2010
Polizio et al. JBC, 2011
Multiple Pathways Converge on GLI
GANT61
HPI-1
ATO
Lauth & Toftgard. Cell Cycle, 2007
CANONICAL HEDGEHOG PATHWAY
rhShhN
5E1
LIGANDS
shPTCH1
PTCH1
SAG
SMO
SANT-1
shSMO
pLenti6-mSmoM2*RFP
1o cilium
shSUFU
shGLI3
pCMV6-AC-Gli1*GFP
GLIA
Inhibition
Activation
pCMV6-XL5-Shh
shKIF3A
SUFU
?
GLIR
Self-renewal
Migration/Invasion
Proliferation/Apoptosis
Chemoresistance
GANT61/HPI-1
pcDNA6.2-Gli3R*GFP
Cancer Stem Cell: Self-renewal vs Growth
SCLC Stem Cell
Tumor Bulk
Clonality
Growth
Apoptosis
Chemosensitivity
Innately
Chemoresistant
Chemonaive
SMO
GLIA
CANONICAL HEDGEHOG PATHWAY
rhShhN
5E1
LIGANDS
shPTCH1
PTCH1
SAG
SMO
SANT-1
shSMO
pLenti6-mSmoM2*RFP
1o cilium
pCMV6-AC-Gli1*GFP
shKIF3A
SUFU
shSUFU
shGLI3
Inhibition
Activation
pCMV6-XL5-Shh
GLIA
?
GLIR
GLI1, PTCH1, HHIP
Other reported targets
Novel Biomarkers
(microarray)
GANT61/HPI-1
pcDNA6.2-Gli3R*GFP
CANONICAL HEDGEHOG PATHWAY
rhShhN
5E1
LIGANDS
shPTCH1
PTCH1
SAG
SMO
1o cilium
shGLI3
pCMV6-AC-Gli1*GFP
GLIA
SANT-1
shSMO
pLenti6-mSmoM2*RFP
shSUFU
Inhibition
Activation
pCMV6-XL5-Shh
shKIF3A
SUFU
?
GLIR
Non-Canonical Pathways
(pathway reporter array)
GANT61/HPI-1
pcDNA6.2-Gli3R*GFP
HEPM: A Canonical Pathway Control
Validation of Hh pathway modifiers  Biomarker identification
 Validation of Novel Hh Antagonists
100.00
SHH
SHH+SANT
10.00
1.00
0.10
HHIP
PTCH1
PTCH2
SMO
GLI1
GLI2
GLI3
Small Cell Lung Cancer
Peukert & Moslin. ChemMedChem, 2010
15-20% of lung cancers
aggressive/metastatic
>200,000 deaths/year
no effective treatment
Clinical Markers?
Hedgehog Pathway History
Nusslein-Volhard & Weichaus. Nature, 1980
Taipale et al. Nature, 2000
Hedgehog Mutants in Popular Culture
Leela
Mike
Wenlock & Mandeville
B.O.B
Acknowledgements
Ana Markovic
Wendy Devereux
Jonathan Rhodes
Bill Matsui
Steve Baylin
MIMR
Neil Watkins
Luciano Martelotto
Julien Sage
Kwon-Sik Park
FAMRI
NHMRC
Damon Runyon