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