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Sample & Assay Technologies
Zhong Wu, Ph.D.R&D Scientist
Wnt Signaling: New research solutions
Sample & Assay Technologies Outline
2
� Overview of Wnt signaling
� Function of Wnt signaling
� Research tools to study Wnt signaling
Sample & Assay Technologies Outline
3
� Overview of Wnt signaling� Wnt discovery and family members� Canonical� Noncanonical
� Function of Wnt signaling
� Research tools to study Wnt signaling
Sample & Assay Technologies Wnts and receptors
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� 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
Sample & Assay Technologies
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Different Wnt signaling pathways
Sample & Assay Technologies
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Canonical Wnt pathway
J Biol Chem. 2006;281:22429-33
Sample & Assay Technologies
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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
Sample & Assay Technologies What dictates different Wnt pathways?
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� 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
Sample & Assay Technologies Topics to cover
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� 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
Sample & Assay Technologies Wnt signaling in embryonic development
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� 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
Sample & Assay Technologies Wnt signaling in tissue renewal
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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
Sample & Assay Technologies Wnt signaling in developmental malformation
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Development cell 17,9-26 (2009)
Sample & Assay Technologies Wnt signaling in cancer
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Eur J Cancer 45,2759-2767 (2009), Nature 434, 843-850 (2005) & Wnt homepage
Sample & Assay Technologies Wnt signaling in CNS-related diseases
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� 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
Sample & Assay Technologies Wnt signaling in other pathological condition
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� 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
Sample & Assay Technologies Topics to cover
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� 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
Sample & Assay Technologies Where to measure pathway activity?
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LRP
FzdWNT
Dsh
GSK3ββββ
APC Axin
ββββ-catenin
TCF/LEF1
ββββ-catenin
P
Myc
CCND1
physiological
LRP
WNT
Dsh
GSK3ββββ
Axin
TCF/LEF1
Pββββ-catenin
ββββ-catenin
APC
MycCCND1
Fzd
pathological
PathwayActivity?
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
Sample & Assay Technologies A simple way to measure downstream effects
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gene expression change
measure by real-time PCR
Sig
natu
re g
ene
Wnt Notch NFκκκκB TGFββββ IL6/STAT3
Specific gene panel with companion score to monitor individual pathway activity
Sample & Assay TechnologiesProposed work flow for Wnt gene expression signature development
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Sample & Assay Technologies Experiment setup for whole genome gene expression profiling
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� 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
Sample & Assay Technologies QIAGEN-SABiosciences service core
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� 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 analysis that delivers robust and reproducible results
Sample & Assay Technologies Complete solution for gene silencing from QIAGEN
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� 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
Sample & Assay Technologies β-catenin targeted siRNA from QIAGEN
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Sample & Assay Technologies Experiment setup for whole genome gene expression profiling
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� 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
Sample & Assay Technologies What is Wnt signaling target PCR Array?
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Supported PCR Array Formats 96-Well Plate 384-Well Plate 100-Well Disc 96x96 Chip
Over 100 different pathways available!
• 84 genes responsive toWnt signaling pathway
• 5 Housekeeping Genes
• Genomic DNA Contamination Control
• Reverse Transcription Controls (RTC) n=3
• Positive PCR Controls (PPC) n=3
Sample & Assay Technologies RT2 PCR Arrays Work Flow
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control 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
Sample & Assay Technologies Experiment setup for whole genome gene expression profiling
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� 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
Sample & Assay TechnologiesCheck pathway activity with active β-catenin and Wnt target gene PCR array
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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
Sample & Assay Technologies Experiment problem and approach
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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?
Sample & Assay Technologies Wnt pathway PCR Array
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Signaling
Function
Sample & Assay TechnologiesDifferential expression of receptors and ligands in 293H vs FHC cell
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293H vs FHC
293H
FHC
FZD5FZD3
Wnt3Wnt5A
Sample & Assay Technologies From microarray to PCR - Custom PCR array
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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 TechnologiesProposed work flow for Wnt gene expression signature development
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Sample & Assay TechnologiesIdentify Wnt signature genes with Custom Wnt response gene PCR array
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– +
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
Sample & Assay Technologies Interesting question during experiment
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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 II qPCR array.
wnt3a
LEF1
YX
Hypothesis: Epigenetic silencing of Wnt pathway transducers in HepG2 cell?
Sample & Assay Technologies EpiTech Methyl II qPCR Array Protocol Overview
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(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
Sample & Assay TechnologiesDifferentially methylated genes identified by EpiTech Methyl II qPCR Wnt signaling array
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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
Sample & Assay Technologies Sixteen genes were identified as Wnt signature genes
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� 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
Positive treatmentNegative treatment
Sample & Assay Technologies Sensitivity of Wnt signature genes
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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
Sample & Assay Technologies Specificity of Wnt signature genes
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+ - + - + - + - + -Notch Wnt IL6 NFκB TGFβ
-1.2
-0.9
-0.6
-0.3
0
0.3
0.6
0.9
1.2
Pat
hway
act
ivity
sco
re
Sample & Assay Technologies Wnt Signaling Pathway RT2 Profile PCR Array Plus
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� 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 real-time PCR experiment
Sample & Assay Technologies Wnt Signaling Pathway RT2 Predictor PCR Array
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� 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
Sample & Assay Technologies Pathway activity score
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Summary of Pathway Activity Scores
0.82C
0.010.904624B
-0.925142A
P valuePathway activity score Sample
0.01
C
0.010.904624B
A
P value*Sample
0.146092
� 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
Sample & Assay TechnologiesApplication example: cancer stem cell analysis with Wnt Pathway Plus Array
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CD133+
CD
133-
Wnt pathway activity confers chemoresistance to cancer stem-like cells in a neuroblastoma cell line
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 CD133-neuroblastoma cells
� Activation of Wnt pathway signaling in CD133+ cells (sample 1) while repression of Wnt signaling in CD133- cells (sample 3) upon Dox treatment.
Sample & Assay TechnologiesApplication example: neural stem cell analysis with RT2
Predictor PCR Arrays
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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
Sample & Assay Technologies Available Plus and Predictor arrays
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RT2 Profiler Plus PCR Array
RT2 Predictor PCR ArrayHuman 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
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
Sample & Assay Technologies Summary
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� 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
Sample & Assay Technologies Additional Resources
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