1. Sample & Assay Technologies
Wnt Signaling: New research solutions
Zhong Wu, Ph.D.
R&D Scientist

2. Sample & Assay Technologies
Outline
Overview of Wnt signaling
Function of Wnt signaling
Research tools to study Wnt signaling
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3. Sample & Assay Technologies
Outline
Overview of Wnt signaling
Wnt discovery and family members
Canonical
Noncanonical
Function of Wnt signaling
Research tools to study Wnt signaling
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4. Sample & Assay Technologies
Wnts and receptors
Discovery of Wnt
Murine oncogene Int-1
Homolog to Drosophila segment polarity gene
Wingless
Wnt proteins
19 members
Highly conserved
Secreted glycoproteins
Activate cell surface receptors
Wnt receptors
FZD/LRP receptor complex
10 members of FZD
LRP5 and LRP6 coreceptor
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Different Wnt signaling pathways
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Canonical Wnt pathway
J Biol Chem. 2006;281:22429-33
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Noncanonical Wnt pathway
Wnt/JNK
Wnt/calcium
CN
Cell polarization in epithelial sheet
Polarized cell movements
Larger overlap with Wnt/PCP
Cell adhesion
Cardiac differentiation
Gene expression through NFAT, HDAC
Nat Rev Neurosci. 2010;11(2):77-86
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8. Sample & Assay Technologies
What dictates different Wnt pathways?
Different Wnt proteins
Wnt1,Wnt3A, Wnt8 - canonical
Wnt5A, Wnt11 - noncanonical
Receptor mediated
Wnt/Frizzled/LRP – β-catenin signaling
Frizzled – PCP and Ca2+ signaling
Wnt/Ryk – Src signaling
Wnt/Ror2 – JNK signaling
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9. Sample & Assay Technologies
Topics to cover
Overview of Wnt signaling
Wnt discovery and family members
Canonical
Noncanonical
Function of Wnt signaling
Development
Tissue homeostasis
Pathological condition
Research tools to study Wnt signaling
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10. Sample & Assay Technologies
Wnt signaling in embryonic development
Function during development
Cell fate determination
Proliferation
Migration, motility and polarity specification
Tissue patterning
Organogenesis and organization of the body plan
Almost every development process
Axial formation
Mesoderm specification
Production of presomitic mesoderm
Direct patterning of the nervous system
Suppress chondrogenic potential of joint interzone
cells
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11. Sample & Assay Technologies
Wnt signaling in tissue renewal
Zebrafish tail fin and heart regeneration
Maintain stem-cell like fate
Intestinal epithelium
Skin
Hematopoietic cells
Neural stem cells
Nature 434, 843-850 (2005) & Development 134, 479-489 (2007)
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12. Sample & Assay Technologies
Wnt signaling in developmental malformation
Development cell 17,9-26 (2009)
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13. Sample & Assay Technologies
Wnt signaling in cancer
Eur J Cancer 45,2759-2767 (2009), Nature 434, 843-850 (2005) & Wnt homepage
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14. Sample & Assay Technologies
Wnt signaling in CNS-related diseases
Alzheimer’s disease
Reduced β-catenin signaling
Tau hyperphosphorylation by GSK3β
LRP6 association
Parkinson’s disease
Wnt-β-catenin and Wnt-PCP
Dopaminergic development and
neurogenesis
Schizophrenia
Wnt1 overexpression
SNPs in FZ3
DISC1 interacts with GSK3β
Nat Rev Neurosci. 2010;11(2):77-86
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15. Sample & Assay Technologies
Wnt signaling in other pathological condition
High bone mass and Osteoporosis
LRP5 activating (LRP5G171V)
LRP5 lose of function mutation
Type II diabetes
Wnt5B
TCF7L2 (TCF4)
Pulmonary fibrosis
Activated β-catenin signaling
N Engl J Med 2002;346:1513-1521, Am J Hum Genet, 2004,75:832-43
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16. Sample & Assay Technologies
Topics to cover
Overview of Wnt signaling
Wnt discovery and family members
Canonical
Noncanonical
Function of Wnt signaling
Development
Tissue homeostasis
Pathological condition
Research tools to study Wnt signaling
A real case study about Wnt pathway activity
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17. Sample & Assay Technologies
Where to measure pathway activity?
physiological
pathological
WNT
WNT
Fzd
Fzd
L
R
P
Dsh
Pathway
Activity?
APC Axin
GSK3β
β
P
TCF/
LEF1
Dsh
APC
Axin
GSK3β
β
P
β -catenin
β -catenin
L
R
P
β -catenin
β -catenin
Myc
TCF/
LEF1
CCND1
Myc
CCND1
Downstream effect!
Traditional indicators provide insufficient information of the regulatory status of the whole
pathway signaling
Multi-gene expression based methods measure pathway alteration as a function of the
downstream effect of pathway regulation on multiple gene expression changes
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18. Sample & Assay Technologies
Wnt
A simple way to measure downstream effects
Notch
NFκB
κ
TGFβ
β
IL6/STAT3
Signature gene
gene expression change
measure by real-time PCR
Specific gene panel with companion score to monitor individual pathway activity
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19. Sample & Assay Technologies
Proposed work flow for Wnt gene expression signature
development
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20. Sample & Assay Technologies
Experiment setup for whole genome gene expression profiling
Whole genome gene expression profiling after altering Wnt/β-catenin pathway
activity
Illumina bead chip analysis by SABio Service Core
Treatment
Stimulation – Wnt3a
Inhibition – siRNA for β-catenin (Qiagen FlexiTube siRNA)
Wnt Pathway activity readout
Wnt target genes (Wnt signaling target PCR array)
Active β-catenin protein levels
Cell lines
293H
FHC
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21. Sample & Assay Technologies
QIAGEN-SABiosciences service core
Complete service for genomic and expression analysis that
delivers robust and reproducible results
Whole genome
Illumina expression profiling
Illumina genotyping
Pathway/focused panel
Mutation profiling
Methylation
PCR array
miRNA PCR array
Individual gene/Locus
Mutation
Methylation
qRT-PCR SYBR Green
Sample prep
Cell, tissue or biofluids
Fixed tissue
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22. Sample & Assay Technologies
Complete solution for gene silencing from QIAGEN
Predesigned siRNA
FlexiTube siRNA or siRNA Premix
FlexiPlate siRNA
QIAGEN AllStars RNAi controls
Custom siRNA synthesis
HP Custom siRNA – 20 nmol
Plasmid based RNAi
SureSilencing shRNA plasmids
Transfection reagents
HiPerFect transfection reagent
HiPerFect HTS reagent
FlexiTube siRNA Premix
siRNA/DNA cotransfection
Attractene Transfection Reagent
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23. Sample & Assay Technologies
β-catenin targeted siRNA from QIAGEN
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24. Sample & Assay Technologies
Experiment setup for whole genome gene expression profiling
Whole genome gene expression profiling after altering Wnt/ β-catenin pathway
activity
Illumina bead chip analysis by SABio Service Core
Treatment
Stimulation – Wnt3a
Inhibition – siRNA for β-catenin (Qiagen FlexiTube siRNA)
Wnt Pathway activity readout
Wnt target genes (Wnt signaling target PCR array)
Active β-catenin protein levels
Cell lines
293H
FHC
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25. Sample & Assay Technologies
What is Wnt signaling target PCR Array?
• 84 genes responsive to
Wnt signaling pathway
• 5 Housekeeping Genes
• Genomic DNA
Contamination Control
• Reverse Transcription
Controls (RTC) n=3
• Positive PCR Controls
(PPC) n=3
Supported PCR Array Formats
96-Well Plate
384-Well Plate
100-Well Disc
96x96 Chip
Over 100 different pathways available!
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26. Sample & Assay Technologies
control
RT2 PCR Arrays Work Flow
experimental
cDNA Synthesis (C-03 kit)
Genomic DNA Removal Step (5 min.)
Reverse Transcription Step (20 min.)
Load Plates
1 Sample per PCR Array
2 minutes with multi-channel pipet
Run 40 cycle qPCR Program
Standard cycling conditions for all Real
Time PCR Instruments
2 hours
Upload and Analyze Data (FREE)
15 minutes from Raw Ct to
Fold Change Data
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27. Sample & Assay Technologies
Experiment setup for whole genome gene expression profiling
Whole genome gene expression profiling after altering Wnt/ β-catenin pathway
activity
Illumina bead chip analysis by SABio Service Core
Treatment
Stimulation – Wnt3a
Inhibition – siRNA for β-catenin (Qiagen FlexiTube siRNA)
Wnt Pathway activity readout
Wnt target genes (Wnt signaling target PCR array)
Active β-catenin protein levels
Cell lines
293H
FHC
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28. Sample & Assay Technologies
Check pathway activity with active β-catenin and Wnt target
gene PCR array
sinon
Serum free
wnt3a
293H
8
7
RNA
24
36
48h
Serum free
wnt3a
siRNA
FHC
fold change
siRNA
57
293H
CTNNB1
c-myc
6
cyclin D1
5
fra-1
4
axin2
sox9
3
2
RNA
24
siCTNNB1
1
69 72h
0
0
400
0
400
wnt3a (ng/ml)
total beta-catenin
7
SF+wnt
CTNNB1
SF
10% FBS
SF+wnt
293H
6
fold change
active beta-catenin
SF
10% FBS
FHC
5
4
3
FHC
c-myc
cyclin D1
fra-1
axin2
CCND2
2
1
0
GAPDH
0
200
0
200
wnt3a (ng/ml)
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29. Sample & Assay Technologies
Experiment problem and approach
Q: Why the effect of Wnt stimulation on FHC cells was
weaker than that on 293H cells?
Hypothesis: Defect in Wnt signaling pathway transduction in
FHC cell?
Approach: Expression profiling of 293H and FHC cells on
Wnt pathway genes with Wnt Signaling Pathway PCR
array.
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30. Sample & Assay Technologies
Wnt pathway PCR Array
Signaling
Function
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31. Sample & Assay Technologies
Differential expression of receptors and ligands in 293H vs
FHC cell
293H vs FHC
FZD5
293H
FZD3
Wnt5A
Wnt3
FHC
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32. From microarray to PCR - Custom PCR array
Sample & Assay Technologies
Wnt3a 400 ng/ml
siCTNNB1
sinon
siCTNNB1
8
fold change
7
CTNNB1
293H
c-myc
6
cyclin D1
5
fra-1
4
axin2
sox9
3
2
1
0
0
400
0
wnt3a (ng/ml)
400
–
+
–
–
+
+
+
–
Custom PCR array
TMEM2
MYC
FGFR3
PODXL
SMAD6
CALM1
TMED1
ASB13
LEF1
FEZ2
SKP2
PGM1
MRPL54
DIMT1L
SMAD3
MKX
SCD5
MTSS1
CA2
CCND2
LAPTM4B
NAV2
HSPA12A
SORBS2
C3orf31
MT1A
C5orf13
AKT1
MKKS
MTP18
DPM3
ZAK
ILK
W DR54
DPYSL2
CNTNAP2
CHSY1
ICK
NEFL
RPRML
PIGY
MRFAP1
FAM125B
NUAK1
CYP4V2
HES5
FTSJ1
TMEM98
GARNL4
CCND1
LOC653506
HCFC1R1
FAM44B
CXADR
IGFBP5
TARBP2
EYA2
CERK
PRMT6
SH3D19
PHB
THOC7
REEP5
FAM134B
12 Genes
8 Samples
24 Genes
4 Samples
32 Genes
3 Samples
48 Genes
2 Samples
96 Genes
1 Sample
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33. Sample & Assay Technologies
Proposed work flow for Wnt gene expression signature
development
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34. Identify Wnt signature genes with
Custom Wnt response gene PCR array
Sample & Assay Technologies
CCD1079SK
Wnt3a 400 ng/ml
LiCl 20 uM
–
–
+ –
– +
BJ
–
–
U105MG
Lncap
– +
– –
Custom Wnt response gene
PCR array
– +
– –
+
–
active CTNNB1
GAPDH
U373MG
siCTNNB1
MCF-7
HT29
LnCap
HepG2
– + – + – + – + – +
CTNNB1
GAPDH
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35. Sample & Assay Technologies
Interesting question during experiment
wnt3a
X
Y
LEF1
Q: What’s the mechanism involved in differential Wnt
target gene expression between HepG2 and IMR90 cells?
Hypothesis: Epigenetic silencing of Wnt pathway
transducers in HepG2 cell?
Approach: Examine methylation status of Wnt pathway
transduction genes with Wnt signaling EpiTech Methyl II
qPCR array.
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36. Sample & Assay Technologies
EpiTech Methyl II qPCR Array Protocol Overview
Innovative technology enabling fast and accurate detection of
CpG island DNA methylation of individual genes, and disease or
pathway focused gene panels
(0.5 – 4 ug of Genomic DNA)
Raw Ct
Values
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37. Sample & Assay Technologies
Differentially methylated genes identified by EpiTech Methyl
II qPCR Wnt signaling array
100
90
Unmethylated
80
HepG2
% Total input DNA
Methylated
70
60
50
40
30
20
10
0
FZD1
FZD2
FZD9
SOX17
WIF1
100
90
Unmethylated
IMR90
% Total input DNA
80
Methylated
70
60
50
40
30
20
10
0
FZD1
FZD2
FZD9
SOX17
WIF1
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38. Sample & Assay Technologies
Sixteen genes were identified as Wnt signature genes
Positive treatment
Negative treatment
Distinct signature gene expression levels between Wnt pathway activity stimulated
samples and inhibited samples
19 out of 20 treatment samples were correctly classified by 16 unfolded protein response
gene expression signature
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39. Sample & Assay Technologies
Sensitivity of Wnt signature genes
HCT116 (CCT036477, 6h)
0
1.25
2.5
5
10
293H (CHIR99021, 24h)
20 µM
0
1.25
2.5
5
10
20 µM
Active-β-catenin
β
GAPDH
Pathway activity based
on Wnt gene signature
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Notch
+
-
Specificity of Wnt signature genes
+
Wnt
-
+
IL6
-
NFκB
+
-
+
TGFβ
-
Pathway activity score
1.2
0.9
0.6
0.3
0
-0.3
-0.6
-0.9
-1.2
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41. Sample & Assay Technologies
Wnt Signaling Pathway RT2 Profile PCR Array Plus
Content
Experimentally derived signature
genes (16, wells F9-G12)
Pathway relevant genes (68)
HK gene (5)
GDC (1)
RTC (3)
PPC (3)
Free companion algorithm
Convert signature gene expression
change to pathway activity score
Data analysis
Gene expression profiling
Pathway activity readout
Two biological readouts in a single realtime PCR experiment
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42. Sample & Assay Technologies
Wnt Signaling Pathway RT2 Predictor PCR Array
Content
Experimentally derived signature gene
(16)
HK gene (5)
GDC (1)
RTC (1)
PPC (1)
Free companion algorithm
Convert signature gene expression
change to pathway activity score
Data analysis
Pathway activity readout
Processing multiple samples within
one plate
96-well plate – 4 samples
384-well plate – 16 samples
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43. Sample & Assay Technologies
Pathway activity score
Summary of Pathway Activity Scores
Sample
Pathway activity score
P value
value*
A
-0.925142
0.01
B
0.904624
0.01
C
0.146092
0.82
Significant call of pathway activity change
P < 0.05
Score >0.3 or <-0.3
Available Plus and Predictor arrays
Wnt
NFκB
IL6/STAT3
Notch
TGFβ
Oxidative stress
ER stress
Mitochondrial
Hypoxia
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Application example: cancer stem cell analysis with Wnt
Pathway Plus Array
Wnt pathway activity confers chemoresistance to cancer
stem-like cells in a neuroblastoma cell line
CD133-
Tumour Biol. 2012 Aug 11. [Epub ahead of print]
Wnt Signaling RT2 PCR Array Plus uncovered:
Differential Wnt pathway gene expression profiles
associated with CD133+ (stem like) and CD133neuroblastoma cells
CD133+
Activation of Wnt pathway signaling in CD133+ cells
(sample 1) while repression of Wnt signaling in
CD133- cells (sample 3) upon Dox treatment.
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45. Sample & Assay Technologies
Application example: neural stem cell analysis with RT2
Predictor PCR Arrays
NSC (undifferentiated)
NSC (differentiated 10d)
Notch
Wnt
IL6/STAT3
NFkB
TGFβ
RT2 Predictor array indicted activation of Notch and TGFβ pathway activity while
repression of Wnt signaling during neural stem cell differentiation process
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46. Sample & Assay Technologies
Available Plus and Predictor arrays
RT2 Profiler Plus PCR Array
Human WNT Signaling Pathway Plus
Human IL6/STAT3 Signaling Pathway Plus
Human TGFb/BMP Signaling Pathway Plus
Human Notch Signaling Pathway Plus
Human NFkB Signaling Pathway Plus
Human Oxidative Stress Plus
Human Hypoxia Signaling Pathway Plus
Human Unfolded Protein Response Plus
Human Mitochondrial Energy Metabolism Plus
RT2 Predictor PCR Array
Human WNT Signaling Pathway Activity
Human IL6/STAT3 Signaling Pathway Activity
Human TGFb Signaling Pathway Activity
Human Notch Signaling Pathway Activity
Human NFkB Signaling Pathway Activity
Human Oxidative Stress Toxicity
Human Hypoxia Signaling Pathway Activity
Human Unfolded Protein Response Toxicity
Human Mitochondrial Energy Metabolism Toxicity
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47. Sample & Assay Technologies
Summary
Overview of Wnt signaling
Wnt discovery and family members
Canonical - β-catenin
Nonncanonical – Wnt/JNK & Wnt/Ca2+
Function of Wnt signaling
Development – many aspects
Tissue homeostasis – maintain stem cell like feature
Pathological condition – cancer, bone, nervous
Wnt gene signature development with various tools
– A gene expression signature to measure Wnt pathway activity
RT2 Profile PCR array – study gene expression changes related to Wnt
signaling
siRNA – gene silencing for functional Wnt pathway study
EpiTech Methyl array – explore mechanism of gene expression changes
RT2 Predictor PCR array – assess pathway activity with real-time PCR
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48. Sample & Assay Technologies
Additional Resources
Questions?
Contact Technical Support
9 AM – 6 PM Eastern M – F
Contact: 1-888-503-3187 OR
support@SABiosciences.com
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