Wnt part i 2013

522 views

Published on

0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total views
522
On SlideShare
0
From Embeds
0
Number of Embeds
5
Actions
Shares
0
Downloads
34
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide

Wnt part i 2013

  1. 1. Sample & Assay Technologies Wnt Signaling: New research solutions Zhong Wu, Ph.D. R&D Scientist
  2. 2. Sample & Assay Technologies Outline Overview of Wnt signaling Function of Wnt signaling Research tools to study Wnt signaling 2
  3. 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 3
  4. 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 4
  5. 5. Sample & Assay Technologies Different Wnt signaling pathways 5
  6. 6. Sample & Assay Technologies Canonical Wnt pathway J Biol Chem. 2006;281:22429-33 6
  7. 7. Sample & Assay Technologies 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 7
  8. 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 8
  9. 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 9
  10. 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 10
  11. 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) 11
  12. 12. Sample & Assay Technologies Wnt signaling in developmental malformation Development cell 17,9-26 (2009) 12
  13. 13. Sample & Assay Technologies Wnt signaling in cancer Eur J Cancer 45,2759-2767 (2009), Nature 434, 843-850 (2005) & Wnt homepage 13
  14. 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 14
  15. 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 15
  16. 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 16
  17. 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 17
  18. 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 18
  19. 19. Sample & Assay Technologies Proposed work flow for Wnt gene expression signature development 19
  20. 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 20
  21. 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 21
  22. 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 22
  23. 23. Sample & Assay Technologies β-catenin targeted siRNA from QIAGEN 23
  24. 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 24
  25. 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! 25
  26. 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 26
  27. 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 27
  28. 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) 28
  29. 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. 29
  30. 30. Sample & Assay Technologies Wnt pathway PCR Array Signaling Function 30
  31. 31. Sample & Assay Technologies Differential expression of receptors and ligands in 293H vs FHC cell 293H vs FHC FZD5 293H FZD3 Wnt5A Wnt3 FHC 31
  32. 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 32
  33. 33. Sample & Assay Technologies Proposed work flow for Wnt gene expression signature development 33
  34. 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 34
  35. 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. 35
  36. 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 36
  37. 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 37
  38. 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 38
  39. 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 39
  40. 40. Sample & Assay Technologies 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 40
  41. 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 41
  42. 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 42
  43. 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 43
  44. 44. Sample & Assay Technologies 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. 44
  45. 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 45
  46. 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 46
  47. 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 47
  48. 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 QIAWEBINARS@QIAGEN.COM E-Learning Center: Recorded Seminars 48

×