Transcript TITLE

MOLECULAR CARCINOGENESIS
• Defects in Terminal Differentiation
• Defects in Growth Control
• Resistance to Cytotoxicity
• Defects in Programmed Cell Death
CHEMICAL
Body Surface
R
A Deactivation Excretion
D
Genetic
Change
I
Activation
A
T
Nucleus
I
O
N
Inhibition
NORMAL
CELL
VIRUS
Selective
Clonal
Expansion
Genetic
Change
Genetic
Change
Genetic
Change
PREINITIATED NEOPLASTIC MALIGNANT
CELL
TUMOR
LESION
CLINICAL
CANCER
• Activation of Proto-Oncogenes
• Inactivation of Tumor Suppressor Genes
• Inactivation of Genomic Stability Genes
CANCER
METASTASIS
1044-CH
CHRONIC INFLAMMATION AND CANCER
Inherited
Disease
Acquired
Tumor Site Risk
Hemochromatosis
Liver
219
Hereditary Pancreatitis Pancreas 120
Crohn’s Disease
Colon
3
Ulcerative 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 to
infection.”
- B. Stewart and P. Kleihues
World Cancer Report, IARC
Press, p. 57, 2003
Disease
Tumor Site
Risk
Viral
Hepatitis B
Hepatitis C
Liver
Liver
88
3
Bacterial
Helicobacter Pylori Gastric
PID
Ovary
11
3
Parasitic
S. hematobium
S. japonicum
Liver Fluke
Urinary Bladder 2-14
Colon
2-6
Liver
14
Chemical/ Physical
Acid reflex
Esophagus
Metabolic Disease
Obesity
Colon
50-100
1.5
CANCERS ASSOCIATED WITH
OBESITY
In Women
• Breast (postmenopausal)
• Endometrium
• Cervical
• Ovarian
• Colorectal
• Kidney
• Liver/ Gall Bladder
• Pancreatic
• Esophageal
• Hematopoietic
Calle, E et al., NEJM 348:1625-38, 2003
In Men
• Prostate
• Colorectal
• Kidney
• Liver/Gall Bladder
• Pancreatic
• Esophageal
• Hematopoietic
3000-CH
REACTIVE NITROGEN AND OXYGEN SPECIES
DERIVED FROM INFLAMMATORY CELLS
Myeloperoxidase
NO2-
O2
N2O3
NO•
Nitrous
Anhydride
Neutrophil
O2 •
Macrophage
H2O2+Cl-/Br-
H2O2
ONOO -
CO2
ONOOCO2-
Oxidation &
HOCl
Halogenation
HOBr
NO2• Oxidation & Nitration
Deamination
OH •
NO2• Oxidation & Nitration
CO3•
Nitrosoperoxycarbonate
Of DNA and Proteins
2786*-CH
FREE RADIALS AND INFLAMMATION
ROS
•OH
O2- •
(Hydroxyl (Superoxide)
radical)
NO •
RNS
ONOO-
(Nitric Oxide) (Peroxynitrite) (Nitroxyl Radical)
Protein Damage
Lipid
Peroxidation
(DNA Repair Enzymes, Caspases)
DNA Damage
and Mutation
Nitrosamines/Deamination
8--oxo-dG
8-nitroguanine
Etheno Adducts
M1G Adduct
S-nitrosothiol
SSB’s
DSB’s
N2O3
MDA
(malondialdehyde)
4HNE
(4-hydroxynonenal)
Arachidonic Acid
Cascade
Eicosenoids
Cell
Proliferation
1760-CH
NITRIC OXIDE SYNTHASE
Inactive
cNOS
Inactive
cNOS
Active
cNOS
CaM
Ca+++2
CaM
CaM
CaM
NO
Citrulline
L-ARGININE
NO
CaM
iNOS
CaM
Always active
Billiar
NITRIC OXIDE DAMAGES DNA AND
ACTIVATES p53 IN MCF-7 CELLS
DNA DAMAGE
p53 MODIFICATIONS
SPER/NO
p53
MCF-7 Cells
P-Ser-15
P-Ser-20
P-Ser-33
P-Ser-46
P-Ser-315
P-Ser-392
K-Lys-382
2016*-CH
NO-INDUCED p53 PHOSPHORYLATION
TRANSACTIVATES DOWNSTREAM PROTEINS
AND ENGAGES A G2/M ARREST
DOWNSTREAM PROTEINS
% Cells in G2/M
Mitotic Index
2122A*-CH
INDUCIBLE NITRIC OXIDE SYNTHASE (NOS2)
AND CYCLOOXYGENASE-2 (COX2)
INTERACTIONS IN HUMAN CARCINOGENESIS
Hypoxia
HIF1a
NOS2
•
Cytokines
e.g., IL-1b
TNF-a
Hypoxia
Genomic
instability
NO
•
•
p53
Selective Clonal Expansion
DNA damage
Mutant
p53
Lipid Peroxidation
COX2
NFB
K-ras
p53
Prostaglandins
(e.g., PGE2)
Apoptosis
1079A*-CH
1909-CH
VENN DIAGRAM OF 1396
“p53-DEPENDENT” GENES MODULATED BY
CELLULAR STRESS
NO
Hypoxia
H 2 O2
29
666
genes
HU
139
genes
34
35
14
33
40
5
14
11
140
genes
HU
4
7
225
genes
Hypoxia
(T-test at p<0.001 for each treatment and time point)
2978-CH
EXAMPLES OF CHRONIC INFLAMMATORY
CONDITIONS ASSOCIATED WITH
INCREASED p53 MUTATION LOAD
• ULCERATIVE COLITIS
• HEMOCHROMATOSIS
• WILSON DISEASE
• VIRAL HEPATITIS
Absolute Mutation frequency x 10
-7
p53 MUTATION LOAD IS INCREASED
IN ULCERATIVE COLITIS
30
G TO A (CpG SITE OF CODON 248 )
UC vs. Non-UC (p < 0.001)
C TO T (CODON 247)
20
UC vs. Non-UC ( p < 0.001)
10
0
ULCERATIVE NORMAL
COLITIS CONTROL
054-PH
1908-CH
Transcription
Senescence
p53
Cell Cycle
Checkpoints
(1979)
Programmed
Development
Cell Death
• DNA Repair
• Homologous
Recombination
• Chromosomal
Segregation
509B*-CH
p53 IS AT THE CROSSROADS OF CELLULAR
STRESS RESPONSE PATHWAYS
DNA Damage
Hypoxia
NOS2
Oncogene Activation
E2F
p14ARF
ATM, ATR, CHK2
p53
mdm2
Cell Cycle
Checkpoints DNA Repair Apoptosis Senescence
p21WA F1
14-3-3s
Gadd45
GADD45, p48, p53R2
APE1, Pol b
PUMA, NOXA, BAX,
p21WAF1
Apaf1,
XPB, XPD, WRN, BLM
Others
1306I*-CH
EXAMPLES OF p53 NEGATIVE FEEDBACK LOOPS
• Posttranslational Modification and Proteolytic Cleavage
p53
MDM2
Ubiquitination
Oliver et al., Nature 362: 857, 1993
Wu et al., Genes Dev. 7: 1126, 1993
• ATM-Dependent DNA Damage Pathway
CHK2 Kinase
p53 Phosphorylation
Transrepression
Matsui et al., J. Biol. Chem. in press, 2004
• Nitric Oxide Pathway
Inducible Nitric Oxide Synthase
DNA Damage
Transrepression
Forrester et al., PNAS 93: 2442, 1996
Ambs et al., PNAS 95: 8823, 1998
p53
2900-CH
MODEL OF CELLULAR STRESS
INDUCED p53 ACTIVATION AND APOPTOSIS
Mitochondrial
Depolarization
p53
PUMA,
NOXA,
BAX,
p53AIP1
PIG3
Ferredoxin
reductase
Cytochrome C
+
ProCaspase 9
+
APAF1
MnSOD
O•2
GPX1
H 2 O + O2
H 2 O2
2+
CAT
Fe
Lipid Peroxidation
EXECUTIONER
CASPASES
APOPTOSOME
1996A*-CH
APAF-1 IS A TRANSCRIPTIONAL TARGET OF p53 IN
DNA DAMAGE-INDUCED APOPTOSIS
p53
Apoptotic stimuli
RE
PUMA, NOXA, p53AIP1, Bax
Mitochondrial
depolarization
Apoptosome Pro-caspase 9
Apaf-1
Cytochrome C
Caspase 3
APOPTOSIS
1917*-CH
CELL DEATH
Programmed
Non-programmed
1935-CH
MUTATIONAL SPECTRA OF THE p53, APC, ATM
AND BRCA-1 GENES IN ALL HUMAN CANCERS
p53
Frameshift 9%
Splice site 2%
In Frame Del/Ins. 2%
Silent 5%
(n=15,122)
APC
Nonsense
7%
Nonsense
32%
Frameshift
51%
Missense
75%
ATM
Frameshift
56%
(n=1,451)
Missense 4%
Silent 9%
Splice site 4%
BRCA-1
(n=617)
Nonsense
14%
Missense
28%
In Frame
Del/Ins. 2%
(n=3,703)
Nonsense 11%
Frameshift
54%
Missense
30%
Splice site
5%
797B-CH
EXAMPLES OF p53 MUTATION HOTSPOTS ASSOCIATED
WITH CARCINOGEN EXPOSURE
Tobacco Smoking
Lung ,Codon 157
400
Hemochromatosis
Liver, Codon 220
Aflatoxin B1 and HBV
Liver, Codon 249
249
300
G:C to T:A
98%
200
G:C to T:A
78%
A:T to G:C 100%
157
100 Missense
Sunlight
Skin, Codon 281
281
220
CC to TT 100%
0
17-29
97
EVOLUTIONARILY CONSERVED
N
292
324
352
C
Transactivation
Domain
Sequence-Specific
DNA Binding Domain
Oligomerization and
Nuclear Localization
and Export Domains
567L-CH
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
WORLDWIDE p53 MUTATIONAL SPECTRA IN HCC FROM
DIFFERENT GEOGRAPHICAL AREAS
North America
N=15
Western Europe
N=82
Japan
N=242
China
N=171
Taiwan
N=113
Africa
N=28
Incidence of HCC
per 100,000
< 3.3
< 9.0
p53 MUTATION DIAGRAM
Del + ins. G:C to C:G
A:T to C:G
G:C to T:A
< 15.0
A:T to G:C
< 5.6
< 98.9
A:T to T:A
G:C to A:T
CpG
G:C to A:T
Non-CpG
2115-CH
ASSESSMENT OF CAUSATION BY
THE BRADFORD-HILL CRITERIA
HYPOTHESIS: Dietary aflatoxin B1 exposure can produce 249ser
(AGG->AGT) p53 mutations during human liver carcinogenesis
• 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
From: Hussain and Harris, Cancer Res. 58: 4023-37, 1998
926C-CH
ASSESSMENT OF CAUSATION BY
THE BRADFORD-HILL CRITERIA
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
From: Hussain and Harris, Cancer Res. 58: 4023-37, 1998
926D-CH
p53 CODON 249ser MUTANT IN SERUM
DNA AND SERUM HBVSAg ARE
BIOMARKERS OF LIVER CANCER RISK
•
HBSAg/249p53 mutant
minus/minus
plus/minus
minus/plus
plus/plus
RR(95%CI)
1
10(5-20)
13(5-35)
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
Mucosa
Mutation
K-ras APC b-catenin p53
Adenomatous
~55%
Polyps
Carcinoma ~45% 85% ~10%
• ULCERATIVE COLITIS ASSOCIATED:
Ulcerative
Colitis
Dysplasia
Carcinoma ~15% 6%
?%
~55%
885-CH
EXAMPLES OF GENETIC LESIONS
IN BRONCHIAL DYSPLASIA,
CARCINOMA-IN-SITU AND LUNG CARCINOMA
Lesion
• LOH
• 3p12, 14, 21
• 9p21
• 17p13
• p53 (p17p13)
• p16 (9p21)
• Telomerase
• Ki-ras
• FHIT (3p14)
• Rb
Dysplasia
CIS
Carcinoma
1049-CH