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Signal transduction 2013

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  • 1. Sample & Assay Technologies Signal transduction targets: Research solutions for pathway analysis Jennifer Gibbons, Ph.D. R&D Scientist
  • 2. Sample & Assay Technologies Biology-focused solutions for pathway analysis • SABiosciences is now a Company Signal transduction targets, Frederick, MD, 3/15/13 2
  • 3. Sample & Assay Technologies Background: Signal transduction The process of disseminating an extracellular signal, resulting in a functional response Ligand Receptor Downstream Signaling Transcription Factor Target Gene Expression Biological Function Signal transduction targets, Frederick, MD, 3/15/13 3
  • 4. Sample & Assay Technologies Background: Signal transduction Developmental-related pathways TGFβ superfamily WNT Notch Hedgehog Immune/Inflammatory response pathways NFκB JAK/STAT Stress-related pathways p53 Oxidative Stress Hypoxia Signal transduction targets, Frederick, MD, 3/15/13 4
  • 5. Sample & Assay Technologies TGFβ superfamily Four signaling pathways BMP (Bone Morphogenic Protein) Development/differentiation (not just bone) – Stem Cells – CNS TGFβ (Transforming Growth Factor Beta) Proliferation Differentiation Activin Growth & development Reproduction Nodal Development Pathological dysregulation Fibrosis Cancer Signal transduction targets, Frederick, MD, 3/15/13 5
  • 6. Sample & Assay Technologies TGFβ superfamily signaling pathways BMP TGFβ Activin Nodal http://www.genome.jp/kegg/pathway/hsa/hsa04350.html Signal transduction targets, Frederick, MD, 3/15/13 6
  • 7. Sample & Assay Technologies Ligands TGFβ superfamily signaling pathways Receptors Signaling/TFs Target Genes http://www.genome.jp/kegg/pathway/hsa/hsa04350.html Signal transduction targets, Frederick, MD, 3/15/13 7
  • 8. Sample & Assay Technologies WNT signaling Three signaling pathways Canonical (β-catenin signaling) Development Cell cycle/proliferation Planar cell polarity Cellular polarization Cytoskeletal changes Calcium Cell adhesion Pathological dysregulation Cancer Neurodegenerative diseases Pulmonary fibrosis Signal transduction targets, Frederick, MD, 3/15/13 8
  • 9. Sample & Assay Technologies WNT signaling pathways Canonical PCP WNT/Ca2+ http://www.genome.jp/kegg/pathway/hsa/hsa04310.html Signal transduction targets, Frederick, MD, 3/15/13 9
  • 10. Sample & Assay Technologies WNT signaling pathways Ligands Receptors Downstream Signaling TFs Target Genes http://www.genome.jp/kegg/pathway/hsa/hsa04310.html Signal transduction targets, Frederick, MD, 3/15/13 10
  • 11. Sample & Assay Technologies Other signaling pathways Notch signaling Biological processes Development & differentiation Molecular mechanism Receptor processing – γ-secretase complex p53 signaling Biological processes DNA damage Oncogenesis Molecular mechanism Transcription factor (p53) regulation JAK/STAT signaling Biological processes Immune & inflammatory response Molecular mechanism JAK activation of STATs by phosphorylation STAT homo/heterodimerization for gene transcription Signal transduction targets, Frederick, MD, 3/15/13 11
  • 12. Sample & Assay Technologies Notch signaling pathway Ligands/receptors/cofactors TFs Target genes http://www.genome.jp/kegg/pathway/hsa/hsa04330.html Signal transduction targets, Frederick, MD, 3/15/13 12
  • 13. Sample & Assay Technologies p53 signaling pathway http://www.genome.jp/kegg/pathway/hsa/hsa04115.html Signal transduction targets, Frederick, MD, 3/15/13 13
  • 14. Sample & Assay Technologies JAK/STAT signaling pathway Signal transduction targets, Frederick, MD, 3/15/13 14
  • 15. Sample & Assay Technologies Signal transduction experimental design QIAGEN offers a variety of products for signal transduction Gene expression RT-PCR Epigenetics miRNA DNA methylation Histone modifications Functional studies Transcription factor reporter assays siRNA/shRNA Signal transduction targets, Frederick, MD, 3/15/13 15
  • 16. Sample & Assay Technologies Principles of qRT-PCR: Overview Real-time PCR Amplify and simultaneously quantify target DNA Reverse transcription real-time PCR Amplify and simultaneously quantify mRNA Ct values: Threshold cycle Signal transduction targets, Frederick, MD, 3/15/13 16
  • 17. Sample & Assay Technologies Gene expression: Which pathway to choose? Focus your research with the RT2 Profiler Signal Transduction PathwayFinder Hypothesis: An ω-3 diet (unlike the ω-6 Western diet) will suppress prostate tumorigenesis Methods: After confirming phenotypic effect, identify the molecular mechanism using PAMM-014 (RT2 Profiler Signal Transduction PathwayFinder) Signal transduction targets, Frederick, MD, 3/15/13 17
  • 18. Sample & Assay Technologies Mouse Signal Transduction PathwayFinder Functional gene groupings identify what pathways may be affected in your experimental system Signal transduction targets, Frederick, MD, 3/15/13 18
  • 19. Sample & Assay Technologies Western blots confirm PCR array results Signal transduction targets, Frederick, MD, 3/15/13 19
  • 20. Sample & Assay Technologies Maybe WNT signaling involved? Signal transduction targets, Frederick, MD, 3/15/13 20
  • 21. Sample & Assay Technologies Gene expression: Which pathway to choose? Focus your research with the RT2 Profiler Signal Transduction PathwayFinder Hypothesis: An ω-3 diet (unlike the ω-6 Western diet) will suppress prostate tumorigenesis Methods: After confirming phenotypic effect, identify the molecular mechanism using PAMM-014 (RT2 Profiler Signal Transduction PathwayFinder) Conclusions: NFκB signaling is increased during prostate cancer tumorigenesis, whereas apoptosis is decreased. These effects are reversed during the ω-3 diet, inhibiting tumorigenesis. Signal transduction targets, Frederick, MD, 3/15/13 21
  • 22. Sample & Assay Technologies Gene expression analysis: JAK/STAT signaling Background: Human parvovirus B19 can cause inflammatory cardiomyopathy The viral protein NS1 may play a role in this pathophysiological process Hypothesis: JAK/STAT signaling is important for immune and inflammatory processes, and may be involved in B19 infection. Methods: Infect endothelial cells (HMEC-1) with NS1. Examine STAT activation and target gene expression Signal transduction targets, Frederick, MD, 3/15/13 22
  • 23. Sample & Assay Technologies Gene expression analysis: JAK/STAT signaling Conclusions: Upregulation of STAT3 signaling by NS1 may play a role in the mechanism for viral evasion of the immune response. Signal transduction targets, Frederick, MD, 3/15/13 23
  • 24. Sample & Assay Technologies Signal transduction experimental design QIAGEN offers a variety of products for signal transduction Gene expression RT-PCR Epigenetics miRNA DNA methylation Histone modifications Functional studies Transcription factor reporter assays siRNA/shRNA Signal transduction targets, Frederick, MD, 3/15/13 24
  • 25. Epigenetics overview Sample & Assay Technologies Activated transcription factors miRNA shRNA siRNA Protein “A” NFκB + p53 Transcription initiation complex mRNA ”A” – Histones p53 BS Me Me Me Me Me NFκB BS DNA methylation Histone-DNA interactions Ac Structural gene Me Me Me DNA methylation Signal transduction targets, Frederick, MD, 3/15/13 25
  • 26. Sample & Assay Technologies miRNA products from QIAGEN miRNeasy Isolation Target identification miRNA studies Expression miScript miRNA PCR Array & Assays 3’ UTR reporters miRNA Targets Arrays Function miRNA mimics & inhibitors Signal transduction targets, Frederick, MD, 3/15/13 26
  • 27. Sample & Assay Technologies miRNA: WNT signaling Background: What is the molecular mechanism for TIMP-1 regulation in mesenchymal stem cells? Methods: Knockdown TIMP-1 and identify differentially-expressed miRNAs via miFinder miScript PCR Array. Confirm WNT signaling genes are targets. Results: WNT signaling is identified as a part of TIMP-1 regulation. Hsa-let-7f is shown as a link between TIMP-1 and WNT signaling. Signal transduction targets, Frederick, MD, 3/15/13 27
  • 28. Sample & Assay Technologies miRNA: WNT signaling miFinder 3’-UTR reporter Signal transduction targets, Frederick, MD, 3/15/13 28
  • 29. Epigenetics overview Sample & Assay Technologies Activated transcription factors miRNA shRNA siRNA Protein “A” NFκB + p53 Transcription initiation complex mRNA ”A” – Histones p53 BS Me Me Me Me Me NFκB BS DNA methylation Histone-DNA interactions Ac Structural gene Me Me Me DNA methylation Signal transduction targets, Frederick, MD, 3/15/13 29
  • 30. Sample & Assay Technologies Pyrosequencing Signal transduction targets, Frederick, MD, 3/15/13 30
  • 31. Sample & Assay Technologies DNA methylation: WNT signaling Hypothesis: SFRP1 expression is commonly lost during renal cancer due to hypermethylation. Can SFRP1 methylation correlate with renal cancer risk? Methods: SFRP1 methylation analysis of age-matched renal cancer samples via pyrosequencing. Signal transduction targets, Frederick, MD, 3/15/13 31
  • 32. Sample & Assay Technologies DNA methylation: WNT signaling Signal transduction targets, Frederick, MD, 3/15/13 32
  • 33. Sample & Assay Technologies DNA methylation: WNT signaling Hypothesis: SFRP1 expression is commonly lost during renal cancer due to hypermethylation. Can SFRP1 methylation correlate with renal cancer risk? Methods: SFRP1 methylation analysis of age-matched renal cancer samples via pyrosequencing. Conclusion: SFRP1 hypermethylation increases with age and correlates with cancer risk. Signal transduction targets, Frederick, MD, 3/15/13 33
  • 34. Epigenetics overview Sample & Assay Technologies Activated transcription factors miRNA shRNA siRNA Protein “A” NFκB + p53 Transcription initiation complex mRNA ”A” – Histones p53 BS Me Me Me Me Me NFκB BS DNA methylation Histone-DNA interactions Ac Structural gene Me Me Me DNA methylation Signal transduction targets, Frederick, MD, 3/15/13 34
  • 35. Sample & Assay Technologies Histone ChIP: TGFβ signaling Background: What are the epigenetic alterations during Ras-induced cellular senescence? Methods: Genome-wide gene expression by microarray. Histone modifications analyzed by genome-wide ChIP-seq and correlated with gene expression. Active: H3K4me3 Repressed: H3K27me3 Signal transduction targets, Frederick, MD, 3/15/13 35
  • 36. Sample & Assay Technologies Histone ChIP: TGFβ signaling Active Repressed Signal transduction targets, Frederick, MD, 3/15/13 36
  • 37. Sample & Assay Technologies Histone ChIP: TGFβ signaling Background: What are the epigenetic alterations during Ras-induced cellular senescence? Methods: Genome-wide gene expression by microarray. Histone modifications analyzed by genome-wide ChIP-seq. Conclusions: TGFβ signaling is involved in Ras-induced senescence Signal transduction targets, Frederick, MD, 3/15/13 37
  • 38. Sample & Assay Technologies Signal transduction experimental design QIAGEN offers a variety of products for signal transduction Gene expression RT-PCR Epigenetics miRNA DNA methylation Histone modifications Functional studies Transcription factor reporter assays siRNA/shRNA Signal transduction targets, Frederick, MD, 3/15/13 38
  • 39. Sample & Assay Technologies Reporter assay overview Signal transduction targets, Frederick, MD, 3/15/13 39
  • 40. Epigenetics overview Sample & Assay Technologies Activated transcription factors miRNA shRNA siRNA Protein “A” NFκB + p53 Transcription initiation complex mRNA ”A” – Histones p53 BS Me Me Me Me Me NFκB BS DNA methylation Histone-DNA interactions Ac Structural gene Me Me Me DNA methylation Signal transduction targets, Frederick, MD, 3/15/13 40
  • 41. Sample & Assay Technologies Reporter assay & shRNA: Notch signaling Background: Luminal breast cancer (ER+, PR+) is common and has a favorable prognosis Antiestrogen therapy is a common treatment Relapses often include “basal” breast cancer (ER-, PR-), which has a poor prognosis Hypothesis: Luminal breast cancers often include a subset of basal breast cancer cells. This subset grows rapidly during antiestrogen treatment Methods: Develop breast cancer cell lines with progressive luminal to basal characteristics Microarray gene expression analysis Signal transduction targets, Frederick, MD, 3/15/13 41
  • 42. Sample & Assay Technologies Reporter assay & shRNA: Notch signaling Results: Notch signaling is commonly dysregulated in basal breast cancer cells Activated during anti-estrogen therapy Inhibited by γ-secretase inhibitors Signal transduction targets, Frederick, MD, 3/15/13 42
  • 43. Sample & Assay Technologies Reporter assay & shRNA: Notch signaling Reporter assay Luminal Luminobasal Signal transduction targets, Frederick, MD, 3/15/13 43
  • 44. Sample & Assay Technologies Reporter assay & shRNA: Notch signaling Hypothesis: Luminal breast cancers often include a subset of basal breast cancer cells. This subset grows rapidly during antiestrogen treatment Methods: Develop breast cancer cell lines with progressive luminal to basal characteristics Microarray gene expression analysis Conclusions: Luminal breast cancer should be treated with anti-estrogens plus γ-secretase inhibitors Signal transduction targets, Frederick, MD, 3/15/13 44
  • 45. Sample & Assay Technologies Conclusions Many signal transduction pathways Typical receptor to target gene TGFβ WNT Transcription factor-focused JAK/STAT p53 QIAGEN offers many methods to study these cellular processes Gene expression RT2 Profiler PCR Arrays & Assays Epigenetics miScript miRNA PCR System EpiTect Methyl II PCR Arrays & Assays EpiTect ChIP qPCR Arrays & Assays Functional studies Cignal Reporter Assays SureSilencing shRNA Plasmid Flexitube/Flexiplate siRNA, GeneSolution, Premix, AllStar Control Signal transduction targets, Frederick, MD, 3/15/13 45
  • 46. Sample & Assay Technologies Thank you for attending! PCR Array Starter Pack- Promotion Code FDK-PAFAS22 PCR Arrays of any Pathway (FREE) 2 96-well/100-well (2 samples) OR 1 384-well (4 samples) Required Reagents (w/ Purchase) RT2 First-Strand cDNA Synthesis Kit RT2 SYBR Green Mastermix (2-Pack) Email: Support@SABiosciences.com OR QIAWEBINARS@QIAGEN.COM Call 1-888-503-3187 to order (US and Canada only) Signal transduction targets, Frederick, MD, 3/15/13 46

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