wnt part ii 2013
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Sample & Assay Technologies
Zhong Wu, Ph.D.R&D Scientist
Wnt Signaling: a case study with new research solution
Sample & Assay Technologies
Topics to cover
�Overview of Wnt signaling
� Function of Wnt signaling
�Research tools to study Wnt signaling
Sample & Assay Technologies
Topics to cover
�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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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
Sci. Signal. 1, re9 (2008)
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Canonical Wnt pathway
Nat Rev Cancer. 2008;8:387-98
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Noncanonical Wnt pathway
Nat Rev Neurosci. 2010;11(2):77-86
Wnt/JNK Wnt/calcium
Cell polarization in epithelial sheetPolarized cell movementsLarger overlap with Wnt/PCP
Cell adhesionCardiac differentiationGene expression through NFAT, HDAC
CN
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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
Sci. Signal. 1, re9 (2008)
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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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Wnt signaling in embryonic 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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Wnt signaling in tissue renewal
Nature 434, 843-850 (2005) & Development 134, 479-489 (2007)
� Zebrafish tail fin and heart regeneration
�Maintain stem-cell like fate � Intestinal epithelium� Skin� Hematopoietic cells� Neural stem cells
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Wnt signaling in developmental malformation
Development cell 17,9-26 (2009)
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Wnt signaling in cancer
Eur J Cancer 45,2709-2769 (2009), Nature 434, 843-850 (2005) & Wnt homepage
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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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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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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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Study Wnt pathway activity
� Why Wnt pathway activity� Involving in many physiological and pathological process� Mutations of Wnt components lead to altered pathway activity
� Current methods to study Wnt pathway activity� β-catenin translocation� Reporter assay� Wnt target genes� Western blot of active β-catenin
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Cignal reporter from SABiosciences
� Two modality� Dual luciferase� GFP
� Two systems� Plasmid� Lenti
� Over 40 pathways� Wnt and more
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Reporter assay vs Wnt target genes
� Reporter assay� Relatively simple� High throughput� Rely on TRE� Possible disconnection from endogenous gene expression
� Wnt target genes?� Endogenous genes� Readout based on multiple genes� No quantification as a whole set
Quantitative Wnt target genes? – Develop Wnt gene expression signature with companion scoring
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Proposed work flow for Wnt gene expression signature development
1 or 2 cell linesstimulation
inhibition
~ 20 cell lines
Microarray
Initial list ofResponse genes
Expanded dataset
Real-time PCRInitial gene list
Mathematic model classification
Small subset ofsignature genes
signature genesensitivity
Compare to otherpathway activity readout
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Experiment setup for whole genome gene expression profiling
� Whole genome gene expression profiling after altering Wnt/β-catenin pathway activity� Illumina bead chip 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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SABiosciences service core
� 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
Complete service for genomic and expression analysi s that delivers robust and reproducible results.
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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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ββββ-catenin targeted siRNA from QIAGEN
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Experiment setup for whole genome gene expression profiling
� Whole genome gene expression profiling after altering wnt/β-catenin pathway activity� Illumina bead chip service by SABio Service Core
� Treatment � Stimulation – Wnt3a� Inhibition – siRNA (Qiagen flexible tube 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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What is Wnt signaling target PCR Array?
• 84 genes responsive to Wnt pathway transduction
• 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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Gene content on Wnt signaling target PCR Array
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RT2 PCR Arrays Work Flow
•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
control experimental
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Check pathway activity with active ββββ-catenin and Wnt target gene PCR array
10%
FB
S
active beta-catenin
GAPDH
total beta-catenin
SF
SF
+wnt
10%
FB
S
SF
SF
+wnt
FHC 293H
FHC
293H
siRNASerum free
wnt3a
siRNASerum free
wnt3a
24 36 48h
57 69 72h24
RNA
RNA0
1
2
3
4
5
6
7
8
0 400 0 400
wnt3a (ng/ml)
fold
cha
nge
CTNNB1
c-myc
cyclin D1
fra-1
axin2
sox9
0
1
2
3
4
5
6
7
0 200 0 200
wnt3a (ng/ml)
fold
cha
nge
CTNNB1
c-myc
cyclin D1
fra-1
axin2
CCND2
293H
FHC
sinon siCTNNB1
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Experiment problem and approach
Q: Why the effect of Wnt stimulation on FHC cells w as weaker than that on 293H cells?
Approach: Expression profiling of 293H and FHC cell s on Wnt pathway genes with Wnt Signaling Pathway PCR array.
Hypothesis: Defect in Wnt signaling pathway transdu ction in FHC cell?
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Wnt pathway PCR Array
Signaling
Function
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Differential expression of receptors and ligands in 293H vs FHC cell
293H vs FHC29
3H
FHC
FZD5FZD3
Wnt3Wnt5A
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From microarray to PCR - Custom PCR array
TMEM2MYCFGFR3PODXLSMAD6CALM1TMED1ASB13LEF1FEZ2SKP2PGM1MRPL54DIMT1LSMAD3MKXSCD5MTSS1CA2CCND2LAPTM4BNAV2HSPA12ASORBS2C3orf31MT1AC5orf13AKT1MKKSMTP18DPM3ZAKILKWDR54DPYSL2CNTNAP2CHSY1ICKNEFLRPRMLPIGYMRFAP1FAM125BNUAK1CYP4V2HES5FTSJ1TMEM98GARNL4CCND1LOC653506HCFC1R1FAM44BCXADRIGFBP5TARBP2EYA2CERKPRMT6SH3D19PHBTHOC7REEP5FAM134B
+ – + –– – + +Wnt3a 400 ng/ml
siCTNNB1Custom PCR array
12 Genes8 Samples
24 Genes4 Samples
32 Genes3 Samples
48 Genes2 Samples
96 Genes1 Sample
0
1
2
3
4
5
6
7
8
0 400 0 400
wnt3a (ng/ml)
fold
cha
nge
CTNNB1
c-myc
cyclin D1
fra-1
axin2
sox9
293H
sinon siCTNNB1
Sample & Assay Technologies
Identify Wnt signature genes with Custom Wnt response gene PCR array
– +
U373MG
– +
MCF-7
– +
HT29
– +
LnCap
– +
HepG2
siCTNNB1
active CTNNB1
LiCl 20 uM
– + –CCD1079SK BJ U105MG
– +–– +– –
– +– –
Wnt3a 400 ng/ml
Lncap
– +– –
GAPDH
CTNNB1
GAPDH
Custom Wnt response gene PCR array
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Interesting question during experiment
Q: What’s the mechanism involved in differential Wnt target gene expression between HepG2 and IMR90 cells?
Approach: Examine methylation status of Wnt pathway transduction genes with Wnt signaling EpiTech Methyl qPCR array.
wnt3a
LEF1
YX
Hypothesis: Epigenetic silencing of Wnt pathway transducers in HepG2 cell?
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EpiTech Methyl qPCR Array Protocol Overview
(0.5 – 4 ug of Genomic DNA)
Raw CtValues
Innovative technology enabling fast and accurate de tection of CpG island DNA methylation of individual genes, and disease or pathway focused gene panels
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Differentially methylated genes identified by EpiTech Methyl qPCR Wnt signaling array
HepG2
IMR90
Unmethylated
Methylated
Unmethylated
Methylated
0
10
20
30
40
50
60
70
80
90
100
FZD1 FZD2 FZD9 SOX17 WIF1
% T
otal
inpu
t DN
A
0
10
20
30
40
50
60
70
80
90
100
FZD1 FZD2 FZD9 SOX17 WIF1
% T
otal
inpu
t DN
A
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Sixteen genes were identified as Wnt signature genes
Pro
babi
lity
of p
athw
ay a
ctiv
atio
n
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Sensitivity of Wnt signature genes
Active- ββββ-catenin
GAPDH
0 1.25 2.5 5 10
HCT116 (CCT036477, 6h)
20 µµµµM 0 1.25 2.5 5 10
293H (CHIR99021, 24h)
20 µµµµM
Pathway activity based on Wnt gene signature
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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� 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� Cignal Reporter assay – assess pathway activity of Wnt signaling
Sample & Assay Technologies
Additional Resources
E-Learning Center:Recorded Seminars
Questions?Contact Technical Support 9 AM – 6 PM Eastern M – FContact: 1-888-503-3187 OR support@SABiosciences.com
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