Page 1
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
Page 2
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
Page 3
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
Page 4
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
Page 5
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)
Page 6
CaM
CaM
CaM
Ca+++2
L-ARGININE
NO
NO
CaM
CaM
InactivecNOS
InactivecNOS
ActivecNOS
iNOSAlways active
Billiar
NITRIC OXIDE SYNTHASE
CaM
Citrulline
Page 7
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
Page 8
% Cells in G2/M
Mitotic Index
2122A*-CH
DOWNSTREAM PROTEINS
NO-INDUCED p53 PHOSPHORYLATIONTRANSACTIVATES DOWNSTREAM PROTEINS
AND ENGAGES A G2/M ARREST
Page 9
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
Page 11
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)
Page 12
EXAMPLES OF CHRONIC INFLAMMATORY CONDITIONS ASSOCIATED WITH
INCREASED p53 MUTATION LOAD
• ULCERATIVE COLITIS
• HEMOCHROMATOSIS
• WILSON DISEASE
• VIRAL HEPATITIS
Page 13
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
Page 15
509B*-CH
• DNA Repair • Homologous
Recombination• Chromosomal
Segregation
Transcription
Senescence
Programmed Cell Death
p53(1979)
Development
Cell CycleCheckpoints
Page 16
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
Page 17
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
Page 18
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
Page 19
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
Page 20
CELL DEATH
1935-CH
Programmed Non-programmed
Page 21
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%
Page 22
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%
Page 23
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
Page 24
< 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
Page 25
• 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
Page 26
• 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
Page 27
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.
Page 29
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%
Page 30
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