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Cancer and inflammation 2013
 

Cancer and inflammation 2013

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    Cancer and inflammation 2013 Cancer and inflammation 2013 Presentation Transcript

    • Sample & Assay Technologies Cancer & Inflammation Raed Samara, Ph.D. R&D Scientist
    • Sample & Assay Technologies Cancer & Inflammation Webinar Outline Inflammation – brief overview Cancer Inflammation in Cancer Initiation qBiomarker Somatic Mutations System Inflammation in Cancer Promotion RT2 Profiler System Inflammation in Cancer Progression ELISArrays miRNA in Cancer & Inflammation miScript miRNA System Title, Location, Date 2
    • Sample & Assay Technologies QIAGEN webinars Access recorded webinars and slides http://sabiosciences.com/home.php Title, Location, Date 3
    • Sample & Assay Technologies QIAGEN webinars Access recorded webinars and slides Sign up for upcoming webinars Access previous webinars and slides Title, Location, Date 4
    • Sample & Assay Technologies Keep up to date: Follow Pathway focused biology on Facebook Latest information on pathway and disease research, resources and demos.
    • Sample & Assay Technologies Biology-Focused Solutions for Pathway Analysis SABiosciences is now a Company http://sabiosciences.com/home.php Title, Location, Date 6
    • Sample & Assay Technologies Cancer & Inflammation Webinar Outline Inflammation – brief overview Cancer Inflammation in Cancer Initiation qBiomarker Somatic Mutations System Inflammation in Cancer Promotion RT2 Profiler System Inflammation in Cancer Progression ELISArrays miRNA in Cancer & Inflammation miScript miRNA System Promotions Title, Location, Date 7
    • Sample & Assay Technologies Introduction: Inflammation Definition: a protective tissue response to tissue damage or microbes, which serves to destroy, dilute, or wall off both the injurious agent and the injured tissues. ̣ Microbes/Infection Tissue Damage Acute Inflammation Infection Clearance Tissue Homeostasis Epigenetic Changes mRNA Changes Cytokines & Chemokines Signaling Pathways Immune system composition Chronic Inflammation Pre-cancer & Cancer Chronic Inflammatory Diseases Title, Location, Date 8
    • Sample & Assay Technologies ‘TLR in Inflammation’ webinar http://sabiosciences.com/home.php Title, Location, Date 9
    • Sample & Assay Technologies ‘TLR in Inflammation’ webinar Access previous webinars and slides Title, Location, Date 10
    • Sample & Assay Technologies Cancer & Inflammation Webinar Outline Inflammation – brief overview Cancer Inflammation in Cancer Initiation qBiomarker Somatic Mutations System Inflammation in Cancer Promotion RT2 Profiler System Inflammation in Cancer Progression ELISArrays miRNA in Cancer & Inflammation miScript miRNA System Promotions Title, Location, Date 11
    • Sample & Assay Technologies Cancer “Cancer is the result of uncontrolled growth of a given cell type that occurs together with the invasion of surrounding tissue and the spread of malignant cells.” Karin & Greten (2005) Nature Reviews Immunology (5): 749 Three Mechanistic phases Initiation Genomic alterations (DNA Damage) (DNA mutations) Promotion Proliferation & Growth Progression Invasion & Metastasis Inflammation Title, Location, Date 12
    • Sample & Assay Technologies Link between Cancer & Inflammation Bacteria, viruses, chemicals, chronic inflammation Malignancy Inflammatory Stimulus Bladder cancer Schistosomiasis Gastric lymphoma H. pylori-induced gastritis MALT lymphoma H. pylori Hepatocellular carcinoma HBV, HCV Kaposi’s sarcoma HHV8 Bronchial carcinoma Silica, Asbestos Mesothelioma Asbestos Ovarian cancer Endometriosis Colorectal cancer IBD Esophageal cancer Barrett’s metaplasia Papillary thyroid carcinoma Thyroiditis Prostate cancer Prostatitis Title, Location, Date 13
    • Sample & Assay Technologies Cancer & Inflammation Webinar Outline Inflammation – brief overview Cancer Inflammation in Cancer Initiation qBiomarker Somatic Mutations System Inflammation in Cancer Promotion RT2 Profiler System Inflammation in Cancer Progression ELISArrays miRNA in Cancer & Inflammation miScript miRNA System Promotions Title, Location, Date 14
    • Sample & Assay Technologies Inflammation in cancer initiation Injury Inflammation Cytokines ROS, RNI (Extracellular) ROS, RNI (Intracellular) Base excision repair DNA damage DNA mutations Tumor Initiation (DNA damage) Epigenetic Changes Adapted from Grivennikov et al, Cell 2010 Title, Location, Date 15
    • Sample & Assay Technologies Inflammation in cancer initiation Injury Inflammation Cytokines ROS, RNI (Extracellular) ROS, RNI (Intracellular) Base excision repair DNA damage DNA mutations Tumor Initiation (DNA damage) Epigenetic Changes Adapted from Grivennikov et al, Cell 2010 Title, Location, Date 16
    • Sample & Assay Technologies Inflammation in cancer initiation Title, Location, Date 17
    • Sample & Assay Technologies Inflammation in cancer initiation Increased tumor multiplicity in mice experiencing DNA damage after inflammation Azoxymethane: colonic carcinogen Aag+/+ Aag (Alkyladenine DNA glycosylase): Base excision repair enzyme Aag-/- Title, Location, Date 18
    • Sample & Assay Technologies Inflammation in cancer initiation Accumulation of Nitrotyrosine in both strains Nitrotyrosine has been identified as an indicator of cell damage and inflammation, as well as an indicator of exposure to NO. Repeated cycles of DSS treatment leads to positive nitrotyrosine staining in colons of treated animals. Similar staining intensity is detected for both Aag+/+ and Aag-/- colons. Title, Location, Date 19
    • Sample & Assay Technologies Inflammation in cancer initiation Oncogenic mutations are increased in mice lacking base excision repair enzymes after inflammation LCM PCR Amplification Sequencing Gene: Ctnnb1 (β Catenin 1) Title, Location, Date 20
    • Sample & Assay Technologies Inflammation in cancer initiation Conclusions Inflammatory conditions RNOS Base excision repair Oncogenic mutations Tumor growth Title, Location, Date 21
    • Sample & Assay Technologies Cancer & Inflammation Webinar Outline Inflammation – brief overview Cancer Inflammation in Cancer Initiation qBiomarker Somatic Mutations System Inflammation in Cancer Promotion RT2 Profiler System Inflammation in Cancer Progression ELISArrays miRNA in Cancer & Inflammation miScript miRNA System Promotions Title, Location, Date 22
    • Sample & Assay Technologies QIAGEN tools for cancer & inflammation qBiomarker Somatic Mutation PCR Arrays & Assays Analyze Somatic Mutations by Real-time PCR Profile 85 different mutations on one array (372 mutations on one 384 array) Appropriate controls for data normalization and reaction performance Focus on your questions and papers http://sabiosciences.com/somaticmutation.php Title, Location, Date 23
    • Sample & Assay Technologies QIAGEN tools for cancer & inflammation qBiomarker Somatic Mutation PCR Arrays & Assays Focused Content http://sabiosciences.com/somaticmutation.php Title, Location, Date 24
    • Sample & Assay Technologies QIAGEN tools for cancer & inflammation qBiomarker Somatic Mutation PCR Arrays & Assays Focused Content Title, Location, Date 25
    • Sample & Assay Technologies QIAGEN tools for cancer & inflammation qBiomarker Somatic Mutation PCR Arrays & Assays Focused Content Title, Location, Date 26
    • Sample & Assay Technologies QIAGEN tools for cancer & inflammation qBiomarker Somatic Mutation PCR Arrays & Assays Complete Workflow Sample Isolation & Preparation Assay Set-up & Analysis Customer support group: 1-888-503-3187 support@SABiosciences.com Title, Location, Date 27
    • Sample & Assay Technologies Cancer & Inflammation Webinar Outline Inflammation – brief overview Cancer Inflammation in Cancer Initiation qBiomarker Somatic Mutations System Inflammation in Cancer Promotion RT2 Profiler System Inflammation in Cancer Progression ELISArrays miRNA in Cancer & Inflammation miScript miRNA System Promotions Title, Location, Date 28
    • Sample & Assay Technologies Inflammation in cancer promotion Title, Location, Date 29
    • Sample & Assay Technologies Inflammation in cancer promotion Mdr2-KO mice develop hepatocellular carcinoma on the background of chronic hepatitis (inflammatory condition) Arrowheads indicate tumors H&E staining Title, Location, Date 30
    • Sample & Assay Technologies Inflammation in cancer promotion NFkB activation is involved in Mdr2-KO hepatocarcinogenesis NFkB Nuclear staining TNFa leads to NFkB activation (red = Kupffer cells; yellow = hepatocytes) NFkB activation is seen predominantly adjacent to inflamed portal tracts, NOT in all neoplastic hepatocytes, suggesting an inflamed phenomenon. (blue = bile epithelium; yellow = hepatocytes) Title, Location, Date 31
    • Sample & Assay Technologies Inflammation in cancer promotion NSAID treatment decreased inflammation 2.5 month old mice 10-day ibuprofen treatment MPO staining Decreased neutrophils Title, Location, Date 32
    • Sample & Assay Technologies Inflammation in cancer promotion TNFa drives NFkB activation Treatment with anti-TNFa decreased NFkB nuclear staining (activation) Source of TNFa is the non-hepatocyte fraction of the liver H: hepatocyte fraction R: non-hepatocyte fraction Title, Location, Date 33
    • Sample & Assay Technologies Inflammation in cancer promotion NFkB is critical for tumor promotion Generate DKO mice to directly assess the role of NFkB in hepatocarcinogenesis ∆N-IκBhep Mdr2 KO X NFkB inactivated in hepatocytes (Dox-activated NFkB) Double mutant (DM) Mdr2-/- ∆N-IκBhep Title, Location, Date 34
    • Sample & Assay Technologies Inflammation in cancer promotion NFkB is dispensable for early stages of HCC development 4 month old mice No change in inflammation 7 month old mice No change in dysplasia Title, Location, Date 35
    • Sample & Assay Technologies Inflammation in cancer promotion NFkB is critical for tumor promotion Inactivation of NFkB in hepatocytes results in decreased tumor volume NFkB activated in hepatocytes Title, Location, Date 36
    • Sample & Assay Technologies Inflammation in cancer promotion NFkB inhibition results in suppression of anti-apoptotic factors Increased hepatocyte apoptosis in DM mice (DM mice & KO + anti-TNF = inactivation of NFkB in hepatocytes) Liver protein extracts from KO mice Inactivation of NFkB decreased the expression of anti-apoptotic genes Title, Location, Date 37
    • Sample & Assay Technologies Inflammation in cancer promotion Conclusions Inflammatory stimulus (Mdr2 KO) Inflammatory cytokine (TNFa) NFkB activation Modulation of gene expression Tumor growth Title, Location, Date Increased survival 38
    • Sample & Assay Technologies Cancer & Inflammation Webinar Outline Inflammation – brief overview Cancer Inflammation in Cancer Initiation qBiomarker Somatic Mutations System Inflammation in Cancer Promotion RT2 Profiler System Inflammation in Cancer Progression ELISArrays miRNA in Cancer & Inflammation miScript miRNA System Promotions Title, Location, Date 39
    • Sample & Assay Technologies QIAGEN tools for cancer & inflammation RT2 Profiler PCR Arrays & Assays Analyze gene expression by Real-time PCR Profile 84 different genes on one array (370 genes on one 384 array) Appropriate controls for data normalization, sample quality, and reaction performance Focus on your questions and papers http://sabiosciences.com/PCRArrayPlate.php Title, Location, Date 40
    • Sample & Assay Technologies QIAGEN tools for cancer & inflammation RT2 Profiler PCR Arrays & Assays Focused Content Array Species NFkB Pathway Human/Mouse/Rat NFkB Targets Human/Mouse/Rat Apoptosis Human/Mouse/Rat TNF Pathway Human/Mouse/Rat Others (>140) Varies Array Species IL6/STAT3 Pathway Human/Mouse/Rat JAK/STAT Pathway Human/Mouse/Rat Inflammatory responses Human/Mouse/Rat Title, Location, Date 41
    • Sample & Assay Technologies QIAGEN tools for cancer & inflammation RT2 Profiler PCR Arrays & Assays Complete Workflow Sample Isolation & Preparation Assay Set-up & Analysis Customer support group: 1-888-503-3187 support@SABiosciences.com Title, Location, Date 42
    • Sample & Assay Technologies Cancer & Inflammation Webinar Outline Inflammation – brief overview Cancer Inflammation in Cancer Initiation qBiomarker Somatic Mutations System Inflammation in Cancer Promotion RT2 Profiler System Inflammation in Cancer Progression ELISArrays miRNA in Cancer & Inflammation miScript miRNA System Promotions Title, Location, Date 43
    • Sample & Assay Technologies Inflammation in cancer progression Title, Location, Date 44
    • Sample & Assay Technologies Inflammation in cancer progression TAMs and CSF-1 Tumor-associated macrophages (TAMs) populate most solid tumors. TAMs are associated with poor prognosis. Colony-stimulating factor (CSF-1) is a regulator of the proliferation, differentiation, and survival of macrophages. CSF-1 expression is prevalent in invasive tumor cells. Title, Location, Date 45
    • Sample & Assay Technologies Inflammation in cancer progression Generate DM mice to directly assess the role of CSF-1 in cancer progression CSF1op MMTV-PyMT X CSF1 null recessive mutation (CSF-1 not expressed) Double mutant (DM) PyMT-CSF1op Title, Location, Date 46
    • Sample & Assay Technologies Inflammation in cancer progression Growth and size of primary mammary tumors are not altered in absence of CSF-1 CSF-1 No CSF-1 Title, Location, Date 47
    • Sample & Assay Technologies Inflammation in cancer progression Metastasis of mammary tumors is decreased in absence of CSF-1 CSF-1 No CSF-1 Title, Location, Date 48
    • Sample & Assay Technologies Inflammation in cancer progression Progression of mammary tumors is decreased in absence of CSF-1 CSF-1 Title, Location, Date No CSF-1 49
    • Sample & Assay Technologies Inflammation in cancer progression Presence of TAMs is decreased in absence of CSF-1 CSF-1 No CSF-1 +/op op/op 7-wk mice (F4/80 staining) 19-20-wk mice (F4/80 staining) Title, Location, Date 50
    • Sample & Assay Technologies Inflammation in cancer progression Conclusions Development of primary tumor Growth of primary tumor Recruitment of TAMs to primary tumor Metastasis of primary tumor to distant sites Title, Location, Date 51
    • Sample & Assay Technologies Inflammation in cancer progression There is a correlation between TAM abundance and poor prognosis. However, it is not only the number of TAMs but also the cytokine-expression profile of TAMs that might link inflammation with tumor progression Balkwell et al. Cancer Cell 7; Mar 2005; 211 Title, Location, Date 52
    • Sample & Assay Technologies Cancer & Inflammation Webinar Outline Inflammation – brief overview Cancer Inflammation in Cancer Initiation qBiomarker Somatic Mutations System Inflammation in Cancer Promotion RT2 Profiler System Inflammation in Cancer Progression ELISArrays miRNA in Cancer & Inflammation miScript miRNA System Promotions Title, Location, Date 53
    • Sample & Assay Technologies QIAGEN tools for cancer & inflammation ELISArray kits Analyze Protein expression Profile 12 different analytes from 6 samples on one array Appropriate controls for reaction performance Also: Mix-N-Match Single-Analyte Focus on your questions and papers http://sabiosciences.com/elisarray.php Title, Location, Date 54
    • Sample & Assay Technologies QIAGEN tools for cancer & inflammation ELISArray kits Focused Content http://sabiosciences.com/elisarray.php Customer support group: 1-888-503-3187 support@SABiosciences.com Title, Location, Date 55
    • Sample & Assay Technologies Cancer & Inflammation Webinar Outline Inflammation – brief overview Cancer Inflammation in Cancer Initiation qBiomarker Somatic Mutations System Inflammation in Cancer Promotion RT2 Profiler System Inflammation in Cancer Progression ELISArrays miRNA in Cancer & Inflammation miScript miRNA System Promotions Title, Location, Date 56
    • Sample & Assay Technologies miRNA in cancer & inflammation Title, Location, Date 57
    • Sample & Assay Technologies miRNA webinars http://sabiosciences.com/home.php Title, Location, Date 58
    • Sample & Assay Technologies miRNA webinars Access previous webinars and slides Title, Location, Date 59
    • Sample & Assay Technologies Title, Location, Date 60
    • Sample & Assay Technologies miRNA in cancer & inflammation Src activation induces transformation of normal cells MCF10A cells (immortalized normal mammary epithelial cells) Tamoxifen treatment Activation of Src Title, Location, Date Transformation 61
    • Sample & Assay Technologies miRNA in cancer & inflammation Src-induced transformation is accompanied by an inflammatory response mediated by NFkB Title, Location, Date 62
    • Sample & Assay Technologies miRNA in cancer & inflammation Src-induced transformation is accompanied by increased LIN28B and decreased Let-7a expression Real-time PCR Extract mRNA SRC activation Reverse transcribe to cDNA NFkB activation Screen 365 miRNAs Increase LIN28B Decrease Let-7a Title, Location, Date 63
    • Sample & Assay Technologies miRNA in cancer & inflammation Let-7a and LIN28B regulate IL6 expression and activity during Src-induced transformation Increasing Let-7a levels decreases IL6 levels SRC activation NFkB activation Decreasing LIN28B levels decreases IL6 levels Increase LIN28B Increase IL6 Decrease Let-7a Title, Location, Date 64
    • Sample & Assay Technologies miRNA in cancer & inflammation The IL6 signaling pathway, mediated through STAT3, regulates Src-induced transformation SRC activation NFkB activation Increase LIN28B Increase IL6 Decrease Let-7a Transformation Title, Location, Date 65
    • Sample & Assay Technologies miRNA in cancer & inflammation Conclusions Oncogene activation (Src) NFkB activation Modulation of miRNA expression Inflammatory cytokine (IL6) Transformation Title, Location, Date 66
    • Sample & Assay Technologies Cancer & Inflammation Webinar Outline Inflammation – brief overview Cancer Inflammation in Cancer Initiation qBiomarker Somatic Mutations System Inflammation in Cancer Promotion RT2 Profiler System Inflammation in Cancer Progression ELISArrays miRNA in Cancer & Inflammation miScript miRNA System Promotions Title, Location, Date 67
    • Sample & Assay Technologies QIAGEN tools for cancer & inflammation miScript miRNA PCR Arrays & Assays Analyze miRNA expression by Real-time PCR Profile 84 different miRNAs on one array (370 miRNAs on one 384 array) Appropriate controls for data normalization, sample quality, and reaction performance Focus on your questions and papers http://sabiosciences.com/mirna.php Title, Location, Date 68
    • Sample & Assay Technologies QIAGEN tools for cancer & inflammation miScript miRNA PCR Arrays & Assays Focused Content Screen ENTIRE miRNome (ALL documented miRNAs) http://sabiosciences.com/mirna_pcr_array.php Title, Location, Date 69
    • Sample & Assay Technologies QIAGEN tools for cancer & inflammation miScript miRNA PCR Arrays & Assays Complete Workflow Sample Isolation & Preparation Assay Set-up & Analysis Customer support group: 1-888-503-3187 support@SABiosciences.com Title, Location, Date 70
    • Sample & Assay Technologies QIAGEN tools for cancer & inflammation Gene Expression RT-PCR Somatic mutations Copy Number Variations & Alterations Epigenetics miRNA DNA methylation Histone modifications Functional Studies Reporter assays siRNA/shRNA Protein Expression Cytokine & Chemokine levels ̣ ̣ ̣ ̣ http://sabiosciences.com/home.php http://sabiosciences.com/seminarlist.php Title, Location, Date 71
    • Sample & Assay Technologies Keep up to date: Follow Pathway focused biology on Facebook Latest information on pathway and disease research, resources and demos.
    • Sample & Assay Technologies Cancer & Inflammation Webinar Outline Inflammation – brief overview Cancer Inflammation in Cancer Initiation qBiomarker Somatic Mutations System Inflammation in Cancer Promotion RT2 Profiler System Inflammation in Cancer Progression ELISArrays miRNA in Cancer & Inflammation miScript miRNA System Promotions Title, Location, Date 73
    • Sample & Assay Technologies Thank you for attending Would you like to try a Cancer & Inflammation-related PCR Array? PCR Array Starter Pack Promotion PCR Arrays of any Pathway (FREE) • 2 96-well/100-well (2 samples) OR • 1 384-well (4 samples) 2 Profiler (mRNA): FDK-PAFAS12 oRT omiScript (miRNA): FDK-MIFAS12 oqBiomarker (Copy Number): FDK-CNVINT12 oEpiTect ChIP (Chromatin modifications): FDK-CPFAS12 Required Reagents (w/ Purchase) • RT2 First-Strand cDNA Synthesis Kit, OR • RT2 SYBR Green Mastermix (2-Pack) Call 1-888-503-3187 to order Email: support@SABiosciences.com (US and Canada only) Webinar-related question? QIAWEBINARS@QIAGEN.COM Title, Location, Date 74