Wnt part i 2013

Sample & Assay Technologies

Zhong Wu, Ph.D.R&D Scientist

Wnt Signaling: New research solutions

Sample & Assay Technologies Outline

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� Overview of Wnt signaling

� Function of Wnt signaling

� Research tools to study Wnt signaling

Sample & Assay Technologies Outline

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� Overview of Wnt signaling� Wnt discovery and family members� Canonical� Noncanonical

� Function of 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


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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

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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� Function of Wnt signaling� Development� Tissue homeostasis� Pathological condition


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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� 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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� Whole genome gene expression profiling after altering Wnt/ β-catenin pathway activity� Illumina bead chip analysis by SABio Service Core




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


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� Whole genome gene expression profiling after altering Wnt/ β-catenin pathway activity� Illumina bead chip analysis by SABio Service Core




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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E-Learning Center:Recorded Seminars

Questions?Contact Technical Support 9 AM – 6 PM Eastern M – FContact: 1-888-503-3187 OR [email protected]

[email protected]

Wnt part i 2013

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