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Sample & Assay Technologies Signal transduction targets: Research solutions for pathway analysis Jennifer Gibbons, Ph.D. R&D Scientist

Signal transduction 2013

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Page 1: Signal transduction 2013

Sample & Assay Technologies

Signal transduction targets: Research solutions for pathway analysisJennifer Gibbons, Ph.D.R&D Scientist

Page 2: Signal transduction 2013

Sample & Assay Technologies Biology-focused solutions for pathway analysis

Signal transduction targets, Frederick, MD, 3/15/13 2

• SABiosciences is now a Company

Page 3: Signal transduction 2013

Sample & Assay Technologies Background: Signal transduction

Signal transduction targets, Frederick, MD, 3/15/13 3

The process of disseminating an extracellular signal, resulting in a functional response

Ligand ReceptorDownstream

Signaling

Transcription Factor

Target Gene Expression

Biological Function

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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

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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

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Sample & Assay Technologies TGFβ superfamily signaling pathways

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http://www.genome.jp/kegg/pathway/hsa/hsa04350.html

BMP

TGFβ

Activin

Nodal

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Sample & Assay Technologies TGFβ superfamily signaling pathways

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http://www.genome.jp/kegg/pathway/hsa/hsa04350.html

Ligands Receptors Signaling/TFs Target Genes

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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

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Sample & Assay Technologies WNT signaling pathways

Signal transduction targets, Frederick, MD, 3/15/13 9

http://www.genome.jp/kegg/pathway/hsa/hsa04310.html

Canonical

PCP

WNT/Ca2+

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Sample & Assay Technologies WNT signaling pathways

Signal transduction targets, Frederick, MD, 3/15/13 10

http://www.genome.jp/kegg/pathway/hsa/hsa04310.html

TFsTarget Genes

DownstreamSignalingReceptorsLigands

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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

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Sample & Assay Technologies Notch signaling pathway

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http://www.genome.jp/kegg/pathway/hsa/hsa04330.html

Ligands/receptors/cofactors TFsTargetgenes

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Sample & Assay Technologies p53 signaling pathway

Signal transduction targets, Frederick, MD, 3/15/13 13

http://www.genome.jp/kegg/pathway/hsa/hsa04115.html

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Sample & Assay Technologies JAK/STAT signaling pathway

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Sample & Assay Technologies Signal transduction experimental design

Gene expression� RT-PCR

Epigenetics� miRNA� DNA methylation� Histone modifications

Functional studies� Transcription factor reporter assays� siRNA/shRNA

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QIAGEN offers a variety of products for signal transduction

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Sample & Assay Technologies Principles of qRT-PCR: Overview

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Ct values: Threshold cycle

Real-time PCR� Amplify and simultaneously quantify target DNAReverse transcription real-time PCR� Amplify and simultaneously quantify mRNA

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Sample & Assay Technologies Gene expression: Which pathway to choose?

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)

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Focus your research with the RT2 Profiler Signal Transduction PathwayFinder

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Sample & Assay Technologies Mouse Signal Transduction PathwayFinder

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Functional gene groupings identify what pathways may be affected in your experimental system

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Sample & Assay Technologies Western blots confirm PCR array results

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Sample & Assay Technologies Maybe WNT signaling involved?

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Sample & Assay Technologies Gene expression: Which pathway to choose?

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

Focus your research with the RT2 Profiler Signal Transduction PathwayFinder

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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

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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.

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Sample & Assay Technologies Signal transduction experimental design

Gene expression� RT-PCR

Epigenetics� miRNA� DNA methylation� Histone modifications

Functional studies� Transcription factor reporter assays� siRNA/shRNA

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QIAGEN offers a variety of products for signal transduction

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Sample & Assay Technologies Epigenetics overview

Signal transduction targets, Frederick, MD, 3/15/13 25

Structural gene NFκB BS

Activated transcription factors

p53 BS

p53

NFκB

Protein “A”

mRNA ”A”

MeMeMe

Histones

Histone-DNA interactions

DNA methylation

Me AcMe Me MeMe

DNA methylation

miRNAshRNAsiRNA

Transcription initiation complex

+

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Sample & Assay Technologies miRNA products from QIAGEN

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3’ UTR reportersmiRNA Targets Arrays

Target identification Expression

Isolation

Function

miRNA mimics & inhibitors

miScript miRNA PCR Array & Assays

miRNeasy

miRNAstudies

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Sample & Assay Technologies miRNA: WNT signaling

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Background: What is the molecular mechanism for TIMP-1 regulation in mesenchymal stem cells?

Methods: Knockdown TIMP-1 and identify differentially-expressed miRNAs via miFindermiScript 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.

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Sample & Assay Technologies miRNA: WNT signaling

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miFinder

3’-UTR reporter

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Sample & Assay Technologies Epigenetics overview

Signal transduction targets, Frederick, MD, 3/15/13 29

Structural gene NFκB BS

Activated transcription factors

p53 BS

p53

NFκB

Protein “A”

mRNA ”A”

MeMeMe

Histones

Histone-DNA interactions

DNA methylation

Me AcMe Me MeMe

DNA methylation

miRNAshRNAsiRNA

Transcription initiation complex

+

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Sample & Assay Technologies Pyrosequencing

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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.

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Sample & Assay Technologies DNA methylation: WNT signaling

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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.

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Sample & Assay Technologies Epigenetics overview

Signal transduction targets, Frederick, MD, 3/15/13 34

Structural gene NFκB BS

Activated transcription factors

p53 BS

p53

NFκB

Protein “A”

mRNA ”A”

MeMeMe

Histones

Histone-DNA interactions

DNA methylation

Me AcMe Me MeMe

DNA methylation

miRNAshRNAsiRNA

Transcription initiation complex

+

Page 35: Signal transduction 2013

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

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Sample & Assay Technologies Histone ChIP: TGFβ signaling

Signal transduction targets, Frederick, MD, 3/15/13 36

Active

Repressed

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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

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Sample & Assay Technologies Signal transduction experimental design

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

QIAGEN offers a variety of products for signal transduction

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Sample & Assay Technologies Reporter assay overview

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Sample & Assay Technologies Epigenetics overview

Signal transduction targets, Frederick, MD, 3/15/13 40

Structural gene NFκB BS

Activated transcription factors

p53 BS

p53

NFκB

Protein “A”

mRNA ”A”

MeMeMe

Histones

Histone-DNA interactions

DNA methylation

Me AcMe Me MeMe

DNA methylation

miRNAshRNAsiRNA

Transcription initiation complex

+

Page 41: Signal transduction 2013

Sample & Assay Technologies Reporter assay & shRNA: Notch signaling

Signal transduction targets, Frederick, MD, 3/15/13 41

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

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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

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Sample & Assay Technologies Reporter assay & shRNA: Notch signaling

Signal transduction targets, Frederick, MD, 3/15/13 43

Reporter assay

Luminal

Luminobasal

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Sample & Assay Technologies Reporter assay & shRNA: Notch signaling

Signal transduction targets, Frederick, MD, 3/15/13 44

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-secretaseinhibitors

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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

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Sample & Assay Technologies Thank you for attending!

Signal transduction targets, Frederick, MD, 3/15/13 46

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