Cancer and inflammation 2013

Sample & Assay Technologies

Cancer & Inflammation
Raed Samara, Ph.D.
R&D Scientist


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

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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.
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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

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‘TLR in Inflammation’ webinar



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‘TLR in Inflammation’ webinar



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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


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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


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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

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Injury
Inflammation

Cytokines
ROS, RNI
(Extracellular)

ROS, RNI
(Intracellular)


DNA damage
DNA mutations
Tumor Initiation
(DNA damage)

Epigenetic Changes

Adapted from Grivennikov et al, Cell 2010

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Increased tumor multiplicity in mice experiencing DNA damage
after inflammation

Azoxymethane: colonic carcinogen

Aag+/+
Aag (Alkyladenine DNA glycosylase):
Base excision repair enzyme
Aag-/-


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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.

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Oncogenic mutations are increased in mice lacking base
excision repair enzymes after inflammation

LCM

PCR Amplification

Sequencing

Gene: Ctnnb1 (β Catenin 1)


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Conclusions

Inflammatory conditions

RNOS


Oncogenic mutations

Tumor growth


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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


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Focused Content

http://sabiosciences.com/somaticmutation.php

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Focused Content


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Focused Content


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Complete Workflow

Sample
Isolation &
Preparation

Assay
Set-up &
Analysis
Customer support group:
1-888-503-3187
support@SABiosciences.com

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Inflammation in cancer promotion


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Mdr2-KO mice develop hepatocellular carcinoma on the
background of chronic hepatitis (inflammatory condition)
Arrowheads indicate tumors

H&E staining


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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)


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NSAID treatment decreased inflammation

2.5 month old mice
10-day ibuprofen treatment

MPO staining
Decreased neutrophils


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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


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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


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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


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NFkB is critical for tumor promotion

Inactivation of NFkB in
hepatocytes results in decreased
tumor volume

NFkB activated in hepatocytes


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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


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Conclusions

Inflammatory stimulus (Mdr2 KO)

Inflammatory cytokine (TNFa)

NFkB activation

Modulation of gene expression

Tumor growth

Increased survival
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Cancer
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RT2 Profiler PCR Arrays & Assays
Analyze gene expression by Real-time PCR

genes on one array
(370 genes on one 384
array)

for data normalization,
sample quality, and
reaction performance

Focus on your

http://sabiosciences.com/PCRArrayPlate.php

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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


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Complete Workflow

Sample
Isolation &
Preparation

Assay
Set-up &
Analysis
1-888-503-3187

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Inflammation in cancer progression


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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.


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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


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Growth and size of primary mammary tumors are not altered in
absence of CSF-1

CSF-1
No CSF-1


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Metastasis of mammary tumors is decreased in absence of
CSF-1

CSF-1
No CSF-1


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Progression of mammary tumors is decreased in absence of
CSF-1

CSF-1


No CSF-1

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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)


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Conclusions

Development of primary tumor

Growth of primary tumor

Recruitment of TAMs to primary tumor

Metastasis of primary tumor to distant sites


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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

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ELISArray kits
Analyze Protein expression

analytes from 6
samples on one array

for reaction
performance

Also:
Mix-N-Match
Single-Analyte

Focus on your

http://sabiosciences.com/elisarray.php

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ELISArray kits
Focused Content

http://sabiosciences.com/elisarray.php
1-888-503-3187


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miRNA in cancer & inflammation


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miRNA webinars



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miRNA webinars



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Src activation induces transformation of normal cells

MCF10A cells (immortalized normal mammary epithelial cells)

Tamoxifen treatment

Activation of Src


Transformation

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Src-induced transformation is accompanied by an inflammatory
response mediated by NFkB


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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


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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


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The IL6 signaling pathway, mediated through STAT3, regulates
Src-induced transformation

SRC activation

NFkB activation

Increase LIN28B

Increase IL6

Decrease Let-7a

Transformation

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Conclusions

Oncogene activation (Src)

NFkB activation

Modulation of miRNA expression

Inflammatory cytokine (IL6)

Transformation

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67


miScript miRNA PCR Arrays & Assays
Analyze miRNA expression by Real-time PCR

miRNAs on one array
(370 miRNAs on one
384 array)

for data normalization,
sample quality, and
reaction performance

Focus on your

http://sabiosciences.com/mirna.php


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Focused Content
Screen ENTIRE miRNome
(ALL documented miRNAs)

http://sabiosciences.com/mirna_pcr_array.php

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Complete Workflow

Sample
Isolation &
Preparation

Assay
Set-up &
Analysis
1-888-503-3187


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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
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http://sabiosciences.com/seminarlist.php

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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
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2 Profiler (mRNA): FDK-PAFAS12
oRT
omiScript (miRNA): FDK-MIFAS12
oqBiomarker (Copy Number): FDK-CNVINT12
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Required Reagents (w/ Purchase)
• RT2 First-Strand cDNA Synthesis Kit, OR
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Call 1-888-503-3187 to order
Email: support@SABiosciences.com
(US and Canada only)

Webinar-related question?
QIAWEBINARS@QIAGEN.COM

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