MOLECULAR CARCINOGENESIS

1044-CH

CHEMICAL

VIRUS

RADIATION

Body Surface

Deactivation Excretion

Activation

NORMALCELL

Inhibition

• Defects in Terminal Differentiation• Defects in Growth Control• Resistance to Cytotoxicity• Defects in Programmed Cell Death

GeneticChange

SelectiveClonal

ExpansionGeneticChange

GeneticChange

INITIATEDCELL

PRE-NEOPLASTIC

LESIONMALIGNANT

TUMORCLINICALCANCER

CANCERMETASTASIS

GeneticChange

• Activation of Proto-Oncogenes• Inactivation of Tumor Suppressor Genes• Inactivation of Genomic Stability Genes

Nucleus

AcquiredDisease Tumor Site Risk

Viral

Hepatitis B Liver 88Hepatitis C Liver 3

Bacterial

Helicobacter Pylori Gastric 11PID Ovary 3

Parasitic

S. hematobium Urinary Bladder 2-14S. japonicum Colon 2-6Liver Fluke Liver 14

Chemical/ Physical

Acid reflex Esophagus 50-100

Metabolic Disease Obesity Colon 1.5

CHRONIC INFLAMMATION AND CANCER

InheritedDisease Tumor Site RiskHemochromatosis Liver 219Hereditary Pancreatitis Pancreas 120 Crohn’s Disease Colon 3Ulcerative Colitis Colon 6

“Chronic infection and associated inflammation contribute to about 1/3 of cancers worldwide”

-B.N. Ames, PNAS, 1995“18% of human cancers, i.e., 1.6 million per year, are related toinfection.”

- B. Stewart and P. KleihuesWorld Cancer Report, IARC Press, p. 57, 2003

3000-CH

CANCERS ASSOCIATED WITH OBESITY

• Breast (postmenopausal)• Endometrium• Cervical• Ovarian• Colorectal• Kidney• Liver/ Gall Bladder • Pancreatic• Esophageal• Hematopoietic

• Prostate• Colorectal• Kidney• Liver/Gall Bladder• Pancreatic• Esophageal• Hematopoietic

In MenIn Women

Calle, E et al., NEJM 348:1625-38, 2003

2786*-CH

REACTIVE NITROGEN AND OXYGEN SPECIES DERIVED FROM INFLAMMATORY CELLS

Myeloperoxidase

NO2•

H2O2HOClHOBr

Oxidation & Halogenation

Oxidation & Nitration

Neutrophil

NO2-

+Cl-/Br-

NO•

O2•

N2O3 Deamination

Macrophage

ONOOS

OD

H2O2

ONOOCO2-

Nitrosoperoxycarbonate

Nitrous Anhydride

NO2•

CO3•

OH •iNOS

-

O2

CO2

-Oxidation & Nitration

Of DNA and Proteins

1760-CH

FREE RADIALS AND INFLAMMATION

ROS •OH O2- •(Hydroxyl (Superoxide) radical)

RNS NO • ONOO- N2O3(Nitric Oxide) (Peroxynitrite) (Nitroxyl Radical)

MDA(malondialdehyde)

4HNE(4-hydroxynonenal)

DNA Damageand Mutation

Nitrosamines/Deamination8--oxo-dG8-nitroguanineEtheno AdductsM1G AdductS-nitrosothiolSSB’sDSB’s

Lipid Peroxidation

Arachidonic AcidCascade

Eicosenoids

Cell Proliferation

Protein Damage (DNA Repair Enzymes, Caspases)

CaM

CaM

CaM

Ca+++2

L-ARGININE

NO

NO

CaM

CaM

InactivecNOS

InactivecNOS

ActivecNOS

iNOSAlways active

Billiar

NITRIC OXIDE SYNTHASE

CaM

Citrulline

p53 MODIFICATIONS

p53

P-Ser-15

P-Ser-20

P-Ser-33

P-Ser-46

P-Ser-315

P-Ser-392

K-Lys-382

2016*-CH

NITRIC OXIDE DAMAGES DNA AND ACTIVATES p53 IN MCF-7 CELLS

DNA DAMAGE

SPER/NO

MCF-7 Cells

% Cells in G2/M

Mitotic Index

2122A*-CH

DOWNSTREAM PROTEINS

NO-INDUCED p53 PHOSPHORYLATIONTRANSACTIVATES DOWNSTREAM PROTEINS

AND ENGAGES A G2/M ARREST

INDUCIBLE NITRIC OXIDE SYNTHASE (NOS2)AND CYCLOOXYGENASE-2 (COX2)

INTERACTIONS IN HUMAN CARCINOGENESIS

Hypoxia

1079A*-CH

Cytokines

NOS2

NO

Genomicinstability

Apoptosis

Mutantp53

• Selective Clonal Expansion• DNA damage

Lipid Peroxidation

p53

•

HIF1

e.g., IL-1TNF-

COX2 Prostaglandins (e.g., PGE2)

p53NFB

Hypoxia

K-ras

1909-CH

2978-CH

VENN DIAGRAM OF 1396 “p53-DEPENDENT” GENES MODULATED BY

CELLULAR STRESS

NO H2O2

HU Hypoxia

140 genes

139 genes

666 genes

225 genes7

5

29

40 14

4

1433

11

Hypoxia

35

HU

34

(T-test at p<0.001 for each treatment and time point)

EXAMPLES OF CHRONIC INFLAMMATORY CONDITIONS ASSOCIATED WITH

INCREASED p53 MUTATION LOAD

• ULCERATIVE COLITIS

• HEMOCHROMATOSIS

• WILSON DISEASE

• VIRAL HEPATITIS

p53 MUTATION LOAD IS INCREASED IN ULCERATIVE COLITIS

054-PH

UC vs. Non-UC (p < 0.001)

UC vs. Non-UC ( p < 0.001)

ULCERATIVE COLITIS

NORMALCONTROL

G TO A (CpG SITE OF CODON 248 )

C TO T (CODON 247)

0

10

20

30

Ab

solu

te M

uta

tio

n f

req

uen

cy x

10

-7

1908-CH

509B*-CH

• DNA Repair • Homologous

Recombination• Chromosomal

Segregation

Transcription

Senescence

Programmed Cell Death

p53(1979)

Development

Cell CycleCheckpoints

p21WA F1

14-3-3 Gadd45

GADD45, p48, p53R2APE1, Pol

PUMA, NOXA, BAX, Apaf1,

XPB, XPD, WRN, BLM

p21WAF1

Others

NOS2

ATM, ATR, CHK2 p14ARF

mdm2

E2F

1306I*-CH

DNA RepairDNA Repair

p53

ApoptosisApoptosis

DNA Damage Oncogene ActivationHypoxia

p53 IS AT THE CROSSROADS OF CELLULAR STRESS RESPONSE PATHWAYS

Cell CycleCell CycleCheckpointsCheckpoints SenescenceSenescence

EXAMPLES OF p53 NEGATIVE FEEDBACK LOOPS

• Posttranslational Modification and Proteolytic Cleavage

Oliver et al., Nature 362: 857, 1993Wu et al., Genes Dev. 7: 1126, 1993

• ATM-Dependent DNA Damage Pathway

Matsui et al., J. Biol. Chem. in press, 2004

• Nitric Oxide Pathway

2900-CH

Ubiquitination

Inducible Nitric Oxide Synthase DNA Damage

p53 MDM2

CHK2 Kinase p53 PhosphorylationTransrepression

p53Transrepression

Forrester et al., PNAS 93: 2442, 1996Ambs et al., PNAS 95: 8823, 1998

Cytochrome C+

ProCaspase 9+

APAF1

APOPTOSOME

EXECUTIONERCASPASES

Lipid Peroxidation

Fe2+

1996A*-CH

p53

MitochondrialDepolarization

PUMA, NOXA, BAX, p53AIP1PIG3Ferredoxin reductase

O2 H2O2 H2O + O2

MnSOD GPX1

CAT•

MODEL OF CELLULAR STRESS INDUCED p53 ACTIVATION AND APOPTOSIS

Caspase 3

APOPTOSIS

PUMA, NOXA, p53AIP1, Bax

Mitochondrial depolarization

Cytochrome C

1917*-CH

Apoptotic stimuli

RE

p53

Apaf-1Pro-caspase 9Apoptosome

APAF-1 IS A TRANSCRIPTIONAL TARGET OF p53 IN DNA DAMAGE-INDUCED APOPTOSIS

CELL DEATH

1935-CH

Programmed Non-programmed

MUTATIONAL SPECTRA OF THE p53, APC, ATM AND BRCA-1 GENES IN ALL HUMAN CANCERS

797B-CH

Missense 75%

Frameshift 9%

p53 (n=15,122)

ATM (n=617)

Nonsense 7%

Splice site 2%

Frameshift56%

Nonsense 14%

Missense 28%

In Frame Del/Ins. 2%Silent 5%

APC (n=1,451)

Nonsense 32%

Missense 4%

Splice site 4%

Nonsense 11%

Missense 30%

Splice site 5%

BRCA-1 (n=3,703)

Frameshift 54%

Silent 9%

Frameshift51%

In Frame Del/Ins. 2%

249

157

Sequence-Specific DNA Binding Domain

N C

EVOLUTIONARILY CONSERVED17-29 97 292 324 352

Missense

Transactivation Domain

Oligomerization andNuclear Localizationand Export Domains

0

567L-CH

Tobacco SmokingLung ,Codon 157

G:C to T:A 78%

EXAMPLES OF p53 MUTATION HOTSPOTS ASSOCIATED WITH CARCINOGEN EXPOSURE

Aflatoxin B1 and HBVLiver, Codon 249

G:C to T:A 98%

281

SunlightSkin, Codon 281

CC to TT 100%

400

200

100

300

HemochromatosisLiver, Codon 220

220

A:T to G:C 100%

HYPOTHESIS:

• p53 mutation hotspots in clonally derived human cancers reflect the preferential:• sites of carcinogen-DNA adduct formation in

the gene• sites of slow repair of DNA damage• mutagenic potential of certain carcinogen-DNA

adducts• pathobiological effects of the p53 mutant

leading to a selective clonal expansion advantage, including “gain of function” or an increase in genomic instability

1156-CH

< 3.3

< 5.6

< 9.0

< 15.0

< 98.9

North AmericaN=15 Western Europe

N=82

AfricaN=28

JapanN=242

ChinaN=171

TaiwanN=113

G:C to C:G

G:C to T:A

G:C to A:TCpG

G:C to A:T Non-CpG

A:T to T:A

A:T to G:C

A:T to C:G

Del + ins.p53 MUTATION DIAGRAM

Incidence of HCC per 100,000

2115-CH

WORLDWIDE p53 MUTATIONAL SPECTRA IN HCC FROM DIFFERENT GEOGRAPHICAL AREAS

• STRENGTH OF ASSOCIATION• Consistency

• Positive correlation in 3 different ethnic populations on 3 continents

• Temporality• 249ser p53 mutant cells observed in non-tumorous liver

in high HCC incidence geographic areas• Specificity

• 249ser p53 mutations are uncommon in other cancer types

• 249ser p53 mutation in serum DNA is a biomarker of liver cancer risk

ASSESSMENT OF CAUSATION BY THE BRADFORD-HILL CRITERIA

From: Hussain and Harris, Cancer Res. 58: 4023-37, 1998 926C-CH

HYPOTHESIS: Dietary aflatoxin B1 exposure can produce 249ser (AGG->AGT) p53 mutations during human liver carcinogenesis

• BIOLOGIC PLAUSIBILITY

• AFB1 is a potent carcinogen in rodents, monkeys and humans

• AFB1 is enzymatically activated by human hepatocytes to 8,9-AFB1 oxide that binds to DNA, including the 3rd base (G) at codon 249

• AFB1 exposure to human liver cells in vitro produces codon 249ser p53 mutations

• 249ser p53 expression inhibits apoptosis and p53-mediated transcription and enhances liver cell growth rates in vitro

ASSESSMENT OF CAUSATION BY THE BRADFORD-HILL CRITERIA

From: Hussain and Harris, Cancer Res. 58: 4023-37, 1998 926D-CH

HYPOTHESIS: Dietary aflatoxin B1 exposure can produce 249ser (AGG->AGT) p53 mutations during human liver carcinogenesis

p53 CODON 249ser MUTANT IN SERUM DNA AND SERUM HBVSAg ARE

BIOMARKERS OF LIVER CANCER RISK• HBSAg/249p53 mutant RR(95%CI)

minus/minus 1

plus/minus 10(5-20)

minus/plus 13(5-35)

plus/plus 399(49-3272)Kirk, GD et al., Proc. 11th Int. Symposium on Viral Hepatitis and

Liver Diseases, Sydney, 2003.

FORTY PERCENT OF LIVER CANCER IN QIDONG, PRC IS ATTRIBUTABLE TO AFLATOXIN DIETARY EXPOSURE

Ming, l et al., Hepatology 36: 1214-20, 2002.

COLORECTAL CARCINOGENESIS

• SPORADIC:

Normal Adenomatous Mucosa Polyps Carcinoma

• ULCERATIVE COLITIS ASSOCIATED:

Ulcerative Colitis Dysplasia Carcinoma

885-CH

Mutation

K-ras APC -catenin p53~45% 85% ~10% ~55%

~15% 6% ?% ~55%

EXAMPLES OF GENETIC LESIONSIN BRONCHIAL DYSPLASIA,

CARCINOMA-IN-SITU AND LUNG CARCINOMALesion Dysplasia CIS Carcinoma• LOH• 3p12, 14, 21• 9p21• 17p13

• p53 (p17p13)• p16 (9p21)• Telomerase• Ki-ras• FHIT (3p14)• Rb

1049-CH