Wnt part ii 2013

Sample & Assay Technologies

Zhong Wu, Ph.D.R&D Scientist

Wnt Signaling: a case study with new research solution


Topics to cover

�Overview of Wnt signaling

� Function of Wnt signaling

�Research tools to study Wnt signaling


Topics to cover

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

� Function of Wnt signaling



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


Different Wnt signaling pathways

Sci. Signal. 1, re9 (2008)


Canonical Wnt pathway

Nat Rev Cancer. 2008;8:387-98


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


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)


Topics to cover

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

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



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


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


Wnt signaling in developmental malformation

Development cell 17,9-26 (2009)


Wnt signaling in cancer

Eur J Cancer 45,2709-2769 (2009), Nature 434, 843-850 (2005) & Wnt homepage


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


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


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


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


Cignal reporter from SABiosciences

� Two modality� Dual luciferase� GFP

� Two systems� Plasmid� Lenti

� Over 40 pathways� Wnt and more


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


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


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


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.


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


ββββ-catenin targeted siRNA from QIAGEN


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


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!


Gene content on Wnt signaling target PCR Array


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


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


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?


Wnt pathway PCR Array

Signaling

Function


Differential expression of receptors and ligands in 293H vs FHC cell

293H vs FHC29

3H

FHC

FZD5FZD3

Wnt3Wnt5A


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


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


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?


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


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


Sixteen genes were identified as Wnt signature genes

Pro

babi

lity

of p

athw

ay a

ctiv

atio

n


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


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


Additional Resources

E-Learning Center:Recorded Seminars

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Documents

Wnt part ii 2013