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