100 101 102 103 104
FL2-Height
064
Events
100 101 102 103 104
FL2-Height
064
Events
Infections, Inflammation and Cancer:Infections, Inflammation and Cancer:Oxidative StressOxidative Stress
Prof. Dr. Christoph Gaschewww.meduniwien.ac.at/innere3/gaschelab/
New Cancer Cases in U.S. per year (2000)New Cancer Cases in U.S. per year (2000)
EsophagusStomachPancreasBilarySmall intestineColonRectumLiver
12.30021.50028.300
6.9004.700
93.80036.40015.300
219.200179.400184.200180.400
Digestive SystemRespiratory SystemBreastProstate
130.200
1/10 ♀
1/29 ♂+ ♀
1/10 ♂
GI Location, Inflammatory Lesion and CancerGI Location, Inflammatory Lesion and Cancer
Barrett‘s esophagus
Chronic Gastritis
Chronic Pancreatitis
Cholangitis (PSC)
Celiac Disease
Crohn‘s Disease
Ulcerative Colitis
Hepatitis
Esophagus
Stomach
Pancreas
Bilary
Small intestine
Colon
Rectum
Liver
Inflammation–Driven Carcinogenesis
Infection, Inflammation and Cancer
“Contributes to about 15-20% of cancers world-wide”
From: Hofseth and Ying, BBA-Reviews on Cancer, 2006
Inflammatory Bowel Diseases
• Diarrhea• Abdominal pain• Fever, anemia, thrombocytosis• Chronic bowel inflammation• Extraintestinal manifestations
(joints – skin – eyes)• Associate diseases
(psoriasis, PSC, spondilytis)
Crohn
Colitis
Endoscopy HistologyLocation
Colitis-associated cancer
Familial 10-30%
Sporadic 65-85%
Hereditary nonpolyposis CRC 5%
Familial adenomatous polyposis 5%
Rare CRC syndromes 0.1%
Colitis-related CRC 2%
CRC Incidence in Ulcerative Colitis
Eaden JA et al. Gut 2001;48:526–35.
Duration
Extent Severity
Inflammation
Rutter M et al. Gastroenterology 2004;126:451–9.Ekbom A et al. N Engl J Med 1990;323:1228–33.Gyde SN et al. Gut 1988;29:206–17.
Inflammation-independent
– Family history of CRC– Primary sclerosing cholangitis– Discontinuation of 5-ASA therapy
Screening Reduces MortalityReduces Mortality
Chemoprophylaxis Reduces IncidenceReduces Incidence
CRC Prevention in Colitis Patients
„Bush to have colonoscopy under anesthesia“
WASHINGTON (CNN) -- President Bush will undergo a colonoscopy -- a diagnostic examination to check for early signs of colon cancer.
Bush said: "I do recommend and urge that people get these precautionary tests“
COLONOSCOPY
Anatomy of the Colon
COLONOSCOPY
Colonoscopic Polypectomy
Hochgradige Dysplasie = Carcinoma in situ
Karzinom = Wachstum in Submukosa
BiologyBiology
Adenom – Karzinom SequenzAdenom – Karzinom Sequenz
Argument For Colonoscopy
Transverse Colon dist. Ascending Colon
Ulcerative Colitis - Chromoendoscopy
Kiesslich et al., Gastroenterology 2003
Normal Endoscopy • ~ 30X• No microscopic details
Zoom Endoscopy • ~100X• Crypts visible, but no details
Confocal Endoscopy • ~1000X• Cellular resolution of crypts
Confocal Laser Endomicroscopy
Confocal Laser Endomicroscopy
Kiesslich, Gastro 2004
Cost-Effectiveness Cost/Year Life Saved
• Mandatory motorcycle helmets $2,000• Colorectal cancer screening $25,000• Breast cancer screening $35,000• Dual airbags in cars $120,000
CRC is a Genetic Disease with different Phenotypes
Inflammatory Cell
Angiogenesis
NO, oxidative stress(DNA damage)
Metastasis
Cytokine production(survival and growth stimulus)
Arguments for a Mutator Phenotype
• Too many mutations in cancer
• Mutations in random DNA regions
• Presence of Clonal and Random Mutations
Biology“Mutator Phenotypes” in CRC
• CHROMOSOMAL INSTABILITY(CIN)– 80 – 85%
– Aneuploid
– Loss of heterozygosity of tumor supressor genes
– Familial Adenomatous Polyposis (FAP)
• MICROSATELLITE INSTABILITY(MIN)– 15 – 20%
– Diploid
– Frameshift mutations of tumor supressor genes
– Hereditary Non-Polyposis Colorectal Cancer (HNPCC) Lynch Syndrome
Prototype:Prototype:
DNA:DNA:
Mutations:Mutations:
Microsatellite Instability: “MSI”
Fingerprint of DNA mismatch repair deficiency Hereditary Non-Polyposis Colorectal Cancer (HNPCC)
Diploid tumors with characteristic clinical features
MSI-H (high) found in 12-15% of CRC complete loss of MMR activity
(germline mutation or promoter hypermethylation)
MSI-L (low) found in another 15-20% of CRC not due to loss of DNA MMR genes
found in chronically inflamed UC mucosa
Defective DNA Mismatch Repair System
Permits polymerase errors (during DNA replication) Permits polymerase errors (during DNA replication)
to persist and to be passed on to the next generationto persist and to be passed on to the next generation
- Insertion/ deletion loops at repetitive DNA - Insertion/ deletion loops at repetitive DNA
sequences (microsatellites) sequences (microsatellites)
- single base mismatches- single base mismatches
MICROSATELLITE INSTABILITY (MSI)MICROSATELLITE INSTABILITY (MSI)
hMSH2hMSH2
hMSH6hMSH3
hMLH1hMLH1
hPMS2hPMS2
hMutS
GATCCGAT
CTAGGTTA
CACACACA
GTGTGTGT
CA
GATCCGAT
CTAGGCTA CACACACA
GTGTGTGT
hMSH6
hMSH2
hMSH6
hMSH2hMSH2
hMSH3
hMutS hMutS
OR
Single base mispairs Insertion or deletion loops (IDLs)
hMSH2
hMSH6
hMLH1
hPMS2
OR
hMutL
hMutL
Marker Location Repeat FunctionTGFRII 3p22 (A)10 Differentiation, growth control
IGFIIR 6q26-27 (G)8 Differentiation, growth control
BAX 19q13.3-4 (G)8 Apoptosis, p53 pathway
hMSH3 5q11-12 (A)8 MMR-gene (MutS-complex)
hMSH6 2p15-16 (C)8 MMR-gene(MutS-complex)
E2F4 16q22.1 (CAG)13 Transcription Factor, Apoptosis
MLH3 14q14.3 (A)9 MMR-gene
MSI Target Genes
Multiple Pathways to Colorectal CancerMultiple Pathways to Colorectal Cancer
CIMPCIMP
CancerCancer
CINCIN
MINMIN
Adenoma
- APC
hMLH1Methylation
Mut. in MMR geneshMLH1, hMSH2
K-ras, p53, DICLOH at 17p & 18q
TGFbR2, IGF2R, BAX
Meth. in p16,PTEN, TIMP3 etc
Mechanism unknown
Mechanism unknown
Normal
Mut. in Target genes:Primary Pathway
SPORADIC COLORECTAL CANCER
COLITIS-ASSOCIATED COLORECTAL CANCER
Differences between sporadic and colitis-associated colorectal cancer
APC = adenomatous polyposis of the colon; CIN = chromosomal instability; COX2 = cyclooxygenase 2; MSI = microsatellite instability
Dysplasia-Carcinoma: Endoscopist’s ViewDysplasia-Carcinoma: Endoscopist’s View
DysplasiaDysplasiaColitisColitis CancerCancer
AdenomaAdenomaNormalNormal CancerCancer
Cancer
NO, oxidativestress
MSI, CIN,CIMP
Accumulation
of mutations, clonal selection
Inflammation as Mutator PhenotypeInflammation as Mutator Phenotype
Dysplasia
O6 meG Mispairing
C C
C
C
C
C
CC
N
N
NN
NC
H
CH3 O
NH
HO
O N
O6 MethylguanineThymine
DeoxyriboseDeoxyribose
6
12 3
4
5
H
78
91 23
45
6
CH3
H
Why is human DNA so stable?Multiple DNA repair systems
DNA mismatch repair (MMR)
- defective in HNPCC + 12% sporadic CRCA
Base excision repair (BER)
- several, targeted systems
- excises and replaces damaged bases Nucleotide excision repair (NER)
- defective in Xeroderma Pigmentosa (XP)
Cell cycle checkpoints (G1/S and G2/M) prevent replication of damage genome
MSI: Defect in DNA MMR
•Inherited (HNPCC):• germline mutation in DNA MMR gene• acquired loss of function of wildtype allele • MSI-H
•Hypermethylation•Promoter hypermethylation on both MLH1 alleles •MSI-H
•Oxidative stress induced MMR inactivation
•Affecting mostly “CA repeats”•MSI-L
Activated Neutrophils Cause an hMLH1-Independent and hMSH2-Dependent G2/M
Arrest in Colon Epithelial Cells
www.meduniwien.ac.at/innere3/gaschelab
S-phase
M-phaseG1-phase
G2-phase
STOP
STOP
STOP
STOPSTOP
Sense DNA damage and ensure the integrity of the genome and fidelity of replication
Cell Cycle Checkpoints
Model & Experimental Setup
Day 0
Target Cells(e.g. HCT116)
Day 2-4
Harvesting
Target Cells
Day 1Co-culture with
Effector CellsHL60 or PMNs
Analysisof Target Cells
Cell cycleApoptosisImmunoblotting
differentiated HL60(6 days in 1.3% DMSO)
Neutrophils (PMNs)primary
Effector Cells
Semipermeable membrane
1h PMA(phorbol 12-myristate
13-acetate)
Activation
Target Cells in the Co-Culture System
MMR deficient
HCT116
Lovo
MLH1-/-
MSH2-/-
MMR proficient
HCT116+chr3
Lovo+chr2
MLH1wt/-
MSH2wt/-
102
85
kDaHCT116
MSH2
HCT116+chr3
Lovo
Lovo +chr2
MLH1
Koi et. al. Cancer Research 1994Watanabe et. al. Mol Carcinog 2000
G2/M arrest in HCT116 Following 24h Co-culture with Activated PMNs
18.8%
47.0%
34.2%
1 : 5
17.2%
45.2%
37.6%
1 : 101 : 0 1 : 1HCT116 : PMN ratio
22.3%
57.0%
20.7% 17.1%
53.3%
29.6%
HCT116+chr3
1 : 10
36.1%
26.1%37.8%
53.1%
22.5%24.5%
1 : 0G1-Phase
S-Phase
G2/M
5
10
15
20
25
30
HCT116 HCT116+chr3
G2/
M a
rres
t (%
)
51015202530354045
Lovo Lovo+chr2
G2/
M a
rres
t (%
)
5
10
15
20
25
30
HEC59 HEC59+chr2
G2/
M a
rres
t (%
)
hMSH2-dependent G2/M Arrest
w/o PMN
PMN (w/o PMA)
PMN (+ PMA)
hMSH6 recruitment to the chromatin
H3-
RPA-
PCNA-
hPMS2-
hMSH6---
hMSH2- hMLH1
0:1
8 h
0:1
24h
20:1
8 h
20:1
24
h
HU
8 h
HU
24
h
S-phase
M-phaseG1-phase
G2-phase
STOP
STOP
STOP
STOPSTOP
G2/M Cell Cycle Checkpoint
Chk1pSer317-
p53pSer15-
p21waf1/cip1-
α-tubulin-
cleaved caspase 7-
HCT116+chr3
Genotoxic stress
Sensor proteins
Transducer proteins
Signal proteins
Effector proteins
ResponsesG1/S G2/M
Inflammation causedDNA damage
ATM / ATR
Chk1 Chk2
cdc25
p53
p21
MMR
? MSHa
Inflammation-driven Checkpoint Activation
P P
?
Inflammatory Cell
Cytokine production(survival and growth stimulus)
NO, oxidative stress(DNA damage)
MetastasisAngiogenesis
Greten F, Karin M Cell 2004
Reduction of Tumor Numbers in DSS-Colitis Through Target Deletion of IKK
NFkB as Tumor Promotion Gene
NFkB activation inhibits apoptosis – “cultivates” the tumor
Blockade of IL-6R signaling reduces colon tumorigenesis in mice
Bec
ker,
Neu
rath
, Im
mun
ity
2004
Inflammatory Cell
Cytokine production(survival and growth stimulus)
NO, oxidative stress(DNA damage)
MetastasisAngiogenesis
Neo-Angiogenesis: Normal versus Tumor Tissue
Coussens LM, Werb Z, Nature 2002
Inflammatory Cell
Cytokine production(survival and growth stimulus)
NO, oxidative stress(DNA damage)
MetastasisAngiogenesis
differentiated de-differentiated differentiated epithelial “mesenchymal“ epithelial
adhesion dissemination adhesion
primary tumor invasive front metastasis
MET
EMT
Dynamic Tumor Progression Model
Epithelial Mesenchymal Transformation
(Brabletz et al., Path Res Pract, 1998)
primary tumor metastasiscenter invasive front center
E-cadherin
-catenin
(Brabletz et al., PNAS, 98, 2001)
differentiated de-differentiated differentiated
Inflammatory Cell
Cytokine production(survival and growth stimulus)
NO, oxidative stress(DNA damage)
MetastasisAngiogenesis
www.meduniwien.ac.at/innere3/gaschelab
(Chemo) Prophylaxis(Chemo) Prophylaxis
• Exercise• Low-fat, low protein, rich in fruits and vegetables• Fiber?• Chemoprophylaxis (for high risk groups)
– NSAIDs, COX-2 Inhibitors (Aspirin, Celebrex)– Mesalazine, Urso-deoxycholic acid – Folate – Calcium/Vitamin D– Estrogen
Aspirin - MesalazineAspirin - MesalazineStructural – Biological SimilaritiesStructural – Biological Similarities
OH
COOH
Salicylic Acid
O-CO-CH3
COOH
Aspirin
OH
COOH
5-ASA
NH2
• Anti-inflammatory• Prevention of sporadic
colorectal cancer
• Anti-inflammatory in colon• Prevention of IBD-related
colorectal cancer
Sulfasalazine - Mesalazine
• Treatment of RA • Treatment of UC• Mesalazine active moiety
Sulfasalazine (salicyl-azo-sulfapyridine)
Mesalazine Sulfapyridine
N Swartz, Acta Med Scand 1942Azad Kahn, Truelove. Lancet 1977
CRC in IBDCRC in IBDChemoprevention with 5-ASA
Year Dose % Reduction (OR)
Pinczowski Gastroenterology 1994 SAP 1.5–3g 60% (0.3 - 0.7)
Moody Eur J Gastr Hepat 1996 SAP 1.5–3g 90% (0.02 - 0.3)
Eaden Alim Pharm Ther 2000 5-ASA >1.2g 91% (0.03 – 0.28)
SAP>2g 59% (0.18 – 0.92)
Lindberg Dis Colon Rectum 2001 SAP n.s. (0.3 - 1.7)
Bernstein Am J Gastroenterol 2003 5-ASA n.s. (0.58 – 3.73)
Van Staa Gut 2005 5-ASA/SAP 40% (0.4-1.0)
Rubin Clin Gastroenterol Hepatol 2006 5-ASA >1.2g 72% (0.09 – 0.85)
5-ASA for ChemopreventionMeta-Analysis
Velayos, Am J Gastroenterol 2005
Intermediate Use
5-Year cancer survival by prior use of 5-ASA
Years after colorectal cancer diagnosis
Pe
rce
nt s
urvi
val
0
20
40
60
80
100
0 1 2 3 4 5
Log rank p-value <0.0001
High Use
Low Use
Velayos et al, DDW 2006
QuestionQuestion
• What are the molecular mechanisms of 5-ASA?What are the molecular mechanisms of 5-ASA?
Mesalazine – anti-inflammatory actions• Modulation of inflammatory cytokine production• COX inhibition • iNOS inhibition • NFB inhibition• PPAR- activation• Antimicrobial
Kennedy M, et al. Int J Mol Med 1999Kaiser GC, et al. Gastroenterology 1999
Yan F, et al. J Biol Chem 1999Rousseaux C, et al. J Exp Med 2005
Swidsinski A, et al. J Clin Microbiol 2005
Mesalazine – cancer-preventive actions
• Oxygen scavenger• Inhibition of -catenin • Inhibition of EGF-R signaling• CpG demethylation• Cell cycle checkpoint activation• Increase in replication fidelity
Joshi R, et al. Free Radic Res 2005Bos KL, et al. Carcinogenesis 2006
Monteleone G, et al. Cell Death Differ 2006Goel A, et al. Presented at UEGW 2006
Reinacher-Schick A, et al. Carcinogenesis 2003Luciani G, et al. Gastroenterology 2007
Gasche C, et al. Cancer Res 2005
• 5x106 PMA-activated neutrophils• ROS-production tested by lucigenin• Measuring light emission (luminometer)
C. Campregher, DDW 2006
5-ASA
01.23.45.6.7
0 30 60 90 120[min]
RL
U
-PMA+PMA2.5mM 5-ASA5.0mM 5-ASA10mM 5-ASA
Aspirin
-PMA+PMA2.5mM ASA5.0mM ASA10mM ASA
01.23.45.6.7
0 30 60 90 120[min]
RL
U
ROS - Scavanging ActivityAspirin vs. Mesalazine
Growth inhibition in HT29
Reinacher-Schick A, et al. Carcinogenesis 2003
Mesalazine –effect on cell cycle
35mM
2.5
2
1.5
1
0.5
0
co
20mM
25mM30mM
40mM
****
***
***
***extin
ctio
n
Mesalazine
extin
ctio
n
2.5
2
1.5
1
0.5
0
co
40M
80M
120M
160M
Sulindac
G2/M block?
Mesalazine – cell cycle and checkpoints
celldivision
chromosomereplication
chromosomesegregation
S-
M- G1-
phase
G2-
ensure integrity of the genome
block entry into mitosis until DNA
replication is complete and
replication errors are repaired
block chromosome segregation
until chromosomes are correctly
aligned on the mitotic spindle
STOP
STOP
STOP
STOP
Mesalazine – cell proliferation
0.00
0.20
0.40
0.60
0.80
1.00
1.20
0 0.31 0.62 1.25 2.5 5 10 20
Mesalazine concentration
RU
HCT116
HT29
LoVo
Luciani et al. Gastroenterology 2007
20 mM
20 mM
10 mM
10 mM
10 mM
5 mM
HCT1160 mM
0 mM
LoVo
HT290 mMG1
G2/M
S
Mesalazine – cell cycle changes
Luciani et al. Gastroenterology 2007
Mesalazine –increase in S-phase population
HCT116
51
32
17G2
S
G1
0mM
HCT116 p53-/-
48
37
45G2
S
G1
HCT116+ch3
47
39
44G2
S
G1
1868
14G2
S
G1
10mM
28
58 14G2
S
G1
2361
16G2
S
G1
20 40 20 40 20 400mM Ap
h. 6
0µM
2hr 4hr 8hr
- p53pSer15
- p21
- Chk1pSer345
- Rad17 pSer645
0 20 40
HT29
0 20 40
- Chk1pSer317
HCT116
mM
Mesalazine – activation of replication checkpoint
Luciani et al. Gastroenterology 2007
A C
B
Luciani et al. Gastroenterology 2007
M1 - dim GFP
M2 - strong GFP
Quantitation of Frameshift Mutations Quantitation of Frameshift Mutations in in vitrovitro
Positive Negative Mixed
Gasche C, et al. Proc Natl Acad Sci 2003
M1 - dim GFP
M2 - strong GFP
M1 - dim GFP
M2 - strong GFP
Mesalazine – improves replication fidelity
Gasche C, et al. Cancer Res 2005GFP = green fluorescent protein
Mutation Rate
Treatment Method of the mean
HCT116-A1.3 HCT116-A2.1
None 8.9 ± 1.8 (100%) 6.0 ± 1.1 (100%)
Mesalazine 6.7 ± 1.8 (75%) 4.9 ± 1.4 (82%)
Aspirin 9.6 ± 2.3 (108%) 7.8 ± 1.6 (130%)
5-ASA
Intra S-phase checkpoint
p53
Cdc25 C
G2/M arrest
Replication stallingSlowing down of replication rate
Prevention of Mitotic Entry
ATR Claspin
Chk1Rad17
ModelModel