TP53 Genetic Variations In Human Cancer

1
TP53 Genetic VariationsIn Human Cancer
http//www-p53.iarc.fr

• Pierre Hainaut, Magali Olivier
• Molecular Carcinogenesis and Biomarkers Group
• International Agency for Research on Cancer,
  Lyon, France

2
What Is TP53 ?
3
TP53 Is A Tumor Suppressor Gene
mice with tumor
p53/
1 at 18 months
p53/-
2 at 9 months
p53-/-
75 at 6 months
Donehower et al. 1992
4
TP53 Germline Mutations Are Responsible For
Li-Fraumeni Syndrome
The Li-Fraumeni syndrome (LFS, OMIM151623) is a
rare autosomal disorder characterized by a
familial clustering of early onset tumors (lt45),
with a predominance of sarcomas, breast cancers,
brain tumors and adrenocortical carcinomas.
BRAIN
SOFT TISSUE SARCOMAS
BREAST
ADRENAL GLAND
BONE SARCOMAS
In 1990, Malkin et al. found that this syndrome
may be caused by a germline mutation in the TP53
gene.
5
p53 Is A Transcription Factor
6
p53 is a Sensor of Various Stresses

Genotoxic
stress
UV, x ,
g
, rays
Carcinogens
Cytotoxic
drugs
Stress conditions
non
-
genotoxic


Oncogenic

Stress
Stress
Oxidizing stress
Excess growth signals
Physical stress
Activated
oncogenes
Hypoxia?
(e.g RAS)
p53
p53
Modulation of p53 activity
protein interactions
specific DNA binding regulation of target genes
7
Responses To p53 Activation
8
p53 Activation Breaking p53-MDM2 Association
Genotoxic stress
ATM/ATR
P
Chk2
CK2
p53
P53 ubiquitination and degradation
Proteasome
Target gene
p53
p53
9
A New Model For p53-MDM2 Association
Stommel J and Wahl G 2004
10
Pathways of p53 Activation
Genotoxic stress
Oncogenic stress
ATM/ATR
P
Chk2
CK2
Non-genotoxic stress
b-catenin
c-myc
p53
Rac1
P38 MAPK
E2F
p16
ras
Target gene
11
Post-Translational Modifications
From Olsson et al., 2007
12
The p53 pathway positive and negative feedback
loops
From Harris Levine, 2005
13
TP53 Family MembersSimilar Structure
TP53
TP73
TP63
1983
1997
1998
Family of Transcription Factors
Transactivation domain
DNA-binding domain
Oligomerisation Regulation domain
14
TP53 Family MembersDifferent Functions
TP53 17p13(h) 23 female death by
exencephaly High frequency of tumor development
TP73 1p36(h) hydrocephalus, chronic infections,
inflammation, abnormalities in pheromone sensory
pathways. No increased susceptibility to
spontaneous tumorigenesis.
TP63 3q27-28(h) Defects in the limb,
craniofacial and epithelial development. No
Skin!
15
Sequence Identity Between p53, p63, p73
p53
TA
DNA BINDING
TET
25
65
35
p63
DNA BINDING
TA
TET
SAM
PS
p53
TA
DNA BINDING
TET
25
65
35
TA
TET
SAM
DNA BINDING
PS
p73
p63
TA
TET
SAM
PS
DNA BINDING
40
85
60
50
p73
DNA BINDING
TA
TET
SAM
PS
From Courtois et al. 2004
16
Targeting The Same Genes?
TP53 Ubiquitous expression
TP73 Tissue, cell-type and proliferation status
specific expression
TP63 Tissue, cell-type and proliferation status
specific expression
Pigs (p53 inducible genes)
cell cycle apoptosis degradation/inhibition
differentiation
17
p63 p73 Isoforms Are Generated By Alternative
Splicing And Alternative Promoters
From Yang and McKeon, 2000
18
p63 And p73 Protein Isoforms
TA
a
p63
DN
b
g
TA (mice)
a
b
TA
DN, DN
g
p73
DExon2
d
DExon3
e
DExon2/3
z
19
p53 Isoforms Described In 2005!
From Bourdon et al. 2005
20
2004 25 years of P53
1979 discovery 1983 defined as an
oncogene 1985 cloning of the human
gene 1989 the WT form is defined as a tumor
suppressor 1989 LOH and first mutations
identified in cancer 1990 TP53 is constitutively
mutated in Li-Fraumeni syndrome 1990 p53 is a
transcription factor 1991 participation of p53
in the cellular response to DNA damage 1991 p53
induces apoptosis 1991 selective G to T
mutation of TP53 gene in HCC from
Africa 1992 TP53-/- mice develop tumors
spontaneously 1993 p53 induces G1 arrest via
p21Waf1 1993 TP53 mutation is associated with
poor prognosis in breast cancer 1994 crystal
structure of p53 in complex with DNA 1996 p53 is
induced by hypoxia 1997 role of mdm2 in the
regulation of p53 stability demonstrated in
mice 1997 TP73, first TP53 related gene
discovered 1999 10,000 human mutations
described 1999 p53 plays a role in DNA
repair 2002 constitutive expression of p53
accelerates ageing in mice 2002 discovery of a
N-terminally truncated variant of p53 2003 p53
plays a role in global chromatin
remodelling 2004 targeting wt p53 and mutant p53
for cancer therapy
21
The IARC TP53 DATABASE A LSDB To Study TP53
Gene Variations In Human Cancer
http//www-p53.iarc.fr/
22
Why Study TP53 Mutations?
TP53 somatic mutations are frequent in most types
of sporadic human cancers (frequencies vary from
5 to 70 depending on cancer type and stage).
TP53 mutations may also be inherited in families
with a predisposition to multiple cancers, as in
the Li-Fraumeni syndrome (LFS).
Over 25,000 mutations have been reported in the
literature, with more than 2300 different
missense mutations.
In several cancers, the nature of TP53 mutations
and their distribution along the coding sequence
have allowed the identification of tumor-specific
mutation spectra, revealing clues on the
mechanisms that might have caused the mutation.
The presence of a TP53 mutation may be predictive
of the tumor response to treatment and patient
survival.
23
The IARC DatabaseInformation System For TP53
Mutations
SCIENTIFIC LITERATURE WEB Data Knowledge

• Extract TP53 mutation data from publications
• Organize and annotate data into a format that
  allows easy retrieval and analysis
• Provide a web-based tool to analyse TP53 mutation
  patterns in cancers

• Extraction
• Annotation
• Integration

IARC TP53 DATABASE

• Interfacing

IARC TP53 WEBSITE Public release of structured
data and knowledge
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Data Available

• Somatic mutations in human sporadic cancers
• Mutation spectra
• Mutation prevalence
• Clinical impact of mutations

• TP53 status in cell-lines
• Gene and protein status
• ATCC link

• Germline mutations and Li-Fraumeni syndrome
• Mutation types
• Tumor spectra

• Polymorphisms in human populations
• List of known polymorphisms

• Structure/Function properties of mutant proteins
• List of common mutants that have been
  characterized by functional tests in human or
  yeast cells
• Structure prediction of mutant proteins

• Mouse-models with engineered TP53 gene
• List of existing mouse-models

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Criteria For Inclusion

• TP53 somatic mutations associated with human
  sporadic cancers that have been identified by
  sequencing and published in peer-reviewed
  literature. This includes mutations found in
  normal, pre-neoplastic and neoplastic tissues,
  including metastases, as well as in cell-lines
  derived from such tissues

• Human TP53 germline mutations (identified by
  sequencing and published in peer-reviewed
  literature) in individuals affected or not by a
  cancer

• P53 mutants that have been tested in human cells
  or yeast assay for functional activities such as
  specific DNA-binding, transcriptional activation,
  dominant-negative effects on the wild-type
  protein, gain of function...

• Mouse-models with engineered TP53 gene that are
  included in caMOD database or have been reported
  in peer-reviewed literature.

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Database Structure And Contents
SOMATIC CONTEXT
POLYMORPHISMS

• Tumor type, grade, stage
• Molecular alterations
• Patient demographics, life-style
• Patient follow-up
• Mutation prevalence
• Prognostic value

• Frequency
• SNP databases links

TP53 MUTATION

• Type
• Position
• Effect

GERMLINE CONTEXT
STRUCTURE/FUNCTION

• Li-Fraumeni syndrome
• Tumor type
• Family structure
• Phenotypes

• Structural data
• Functional data in yeast human cells
• Predicted impact

MOUSE MODELS

• Model name,
• Affected tissues
• caMOD link
• PubMed

The database is maintained in SQL server.
Detailed annotations and contents are available
at http//www-p53.iarc.fr/Help.html.
27
Web Analysis Tools
http//www-p53.iarc.fr
Mutation patterns
Functional properties of mutations
Frequencies/Distributions of mutations
Mutation prevalence
28
Mutation Summary
29
Database Figures And Facts

• Database content
• gt25000 somatic mutations
• gt390 germline mutations
• gt2300 mutants with functional properties
• gt150 studies on TP53 mutation and clinical
  outcome
• largest dataset of mutations in a single gene.
• Database use
• 5000 visits per month
• 500 downloads per month
• gt2100 citations in the scientific literature
• Links from SwissProt, Entrez, EBI,

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IARC TP53 DATABASE A Resource For Various
Disciplines
Molecular Epidemiology
Molecular Pathology
Medical Genetics
Structure/Function Research
TP53 MUTATION DATABASE
Molecular data
Functional data
Structural data
Clinical data
Recommendations and Guidelines
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Database Credits
Molecular Carcinogenesis and Biomarkers Group P.
Hainaut M. Olivier A. Petitjean
Collaborators C.C. Harris M. Khan, NIH (US) M.
Hollstein, DKFZ (Germany) R. Eeles, ICR (UK) M.
Resnick, NIH/NIEHS (US)
32
TP53 Mutations In Human CancersLessons From
The IARC TP53 Database
33
Types Of Genetic Alterations In Cancer

• Somatic Acquired during development and present
  only in cells undergoing clonal expansion
• Inherited present in the germline and detectable
  in both healthy and cancer cells

34
TP53 Somatic Mutations are Frequent in Human
Cancer
35
TP53 Germline Mutations Predispose To Several
Types of Cancers
Tumor spectrum in TP53 mutation carriers
28.9
Breast
16
Soft tissues
gt80
15.4
Brain
12.8
Bones
6.8
Adrenal gland
3.4
Lung
3.2
Leuk/Lymph.
2.5
Stomach
1.7
Colorectum
1.5
Skin
1.5
Ovary
5.9
Other
0
5
10
15
20
25
30
Olivier et al. Cancer Res. 2003
36
p53 Protein is Targeted by Viruses
37
The Majority Of TP53 Mutations Are Missense
Mutations
Somatic (R11, n23581)
Germline (R11, n376)
38
Missense Mutations are Clustered in the
DNA-binding Domain
N-
-C
Transactivation (1-42 43-62)
Regulation (363-393)
DNA binding (102-292)
Oligomerisation (323-356)
Proline-rich (65-97)
1
2.3
80
3.4
0. 3
Mut. frequency
50.8
45.4
82.1
36.4
72.7
Missense mut.
39
Post-Translational Modification Sites Are Rarely
Mutated In Cancer
Number of missense mutations reported in sporadic
cancers (IARC TP53 database, R12).
1
0
1
0
0
1
2
5
0
2
16
13
75
8
2
0
0
5
0
0
0
0
0
8
16
1
40
Most Frequent Mutations Are In The Loops That
Make Contact With DNA
0
90
180
175
248
273
282
245
249
176
220
Codon
gt
gt
gt
gt
gt
gt
gt
41
Effects Of The Most Frequent TP53 Mutations
The following mutations represent about 20 of
all mutations
Codon
Residue
Mutant
Effects on protein structure
175
Arg
His
Breaks crucial H-bond bridging loops L2 and L3
248
Arg
Gln
Breaks main contact with DNA in minor groove
273
Arg
His
Breaks main contact with DNA in major groove
248
Arg
Trp
Breaks main contact with DNA in minor groove
273
Arg
Cys
Breaks main contact with DNA in major groove
Destabilizes H2 helix and DNA binding in the
major groove and breaks contacts on the ß-
hairpin
282
Arg
Trp
42
Biological Activities Of TP53 Mutants
WT p53
DNA- Binding, Transactivation, Repression
Growth suppression
43
How to Interpret Mutation Patterns?
metabolization and intracellular processing
Type of mutation Position and sequence
context Strand bias/asymmetry
ADAPTATION
Cellular strategies to cope with the lesion DNA
repair
Effects of the mutation on cell behavior
44
Sequence context CpG mutations
CgtT mutations occur frequently at CpG sites by
endogenous mechanisms
Hydrolytic deamination
Enzymatic deamination
From Yang et al., 1995
45
Strand Bias
There is a strand bias (or strand asymmetry) when
a mutation event occurs preferentially on one
strand of DNA
Coding strand or non-transcribed strand
A G T G T C
5
3
DNA
3
T C A C A G
5
3
A G U G U C
5
RNA
Non-coding strand or transcribed strand
46
Mutations As Carcinogen Fingerprints
A mutation is a carcinogen fingerprint if there
is

• Evidence that a characteristic mutation pattern
  is found in exposed compared with non-exposed
  individuals. This pattern should be distinct by
  at least one of the following criterion
• type of mutations, site of mutations, strand
  bias.
• Evidence that the suspected carcinogen induces
  similar mutations in experimental model systems.
• Evidence that mutations occur early in tumor
  development.

47
Examples Of Cancer Specific Mutations
Source
Mutagen
Adduct
TP53 pattern
CC to TT Various codons Skin cancer 7 Other
cancers 0
UV radiations
G to T Codon 249 Liver cancer gt50 Other
cancers lt2
Aflatoxins
G to T Codons 157, 158, 248, 273 Lung cancer
30 Other cancers lt10
Tobacco smoke
48
A Specific Mutation Pattern In Lung Cancer from
Smokers
G to T
9
12
30
Smokers (N419)
Non-tobacco related, (N4516)
Non-Smokers (N153)
Pfeifer et al. Oncogene (2002)
49
Mutagenesis And Selection in Lung Cancer
GgtT mutations at hotspot residues
100
All Lung cancers
80
60
40
20
of all mutations
0
157
158
245
248
273
100
Breast, Brain, CRC
80
60
40
20
0
157
158
245
248
273
50
TP53 Mutations In Skin CancerEffect Of UV
Exposure
Repair defects
UV exposure
Sporadic Skin SCC
Skin SCC in XP patients
All sporadic cancers
CC gt TT
UV-induced mutations
Gene-environment interaction evidence
51
TP53 Mutations In Breast CancerEffect Of
Genetic Background
All Breast
24 AT
2
4
3
12
15
5
10
7
21
21
ATgtCG
ATgtGC
ATgtTA
GCgtAT
GCgtAT at CpG
GCgtCG
GCgtTA
del
ins
other
52
Functional Selection Of Mutations
The majority of missense mutations found in human
cancers are deficient for trans-activation
trans-activation classes based on experimental
data in yeast from Kato et al. (2003). See
http//p53-test.iarc.fr/Help.htmlTA_class for
details.
53
Clinical Applications Of TP53 Mutations
54
From Early Detection To Gene Therapy

• Cancer diagnosis
• Early detection of cancerous lesion
• Identification of cancer type
• Marker of clonality
• Cancer etiology
• Identification of cause/exposure
• Patient outcome
• Predict patient survival
• Surveillance of recurrence
• Cancer treatment
• Prediction of treatment response, treatment
  selection
• Gene therapy
• Re-activating p53 in mutated cells

55
Prognostic Value Of TP53 Mutations
Number of studies reporting that TP53 mutations
are
TUMOR SITE
Related to bad prognosis
Related to good prognosis
Not related to prognosis
Bladder
4
-
5
Brain
6
2
6
Breast
22
-
6
Colorectum
11
-
10
Corpus uteri
3
-
-
Esophagus
4
1
3
Head Neck
7
-
3
Hematol.
13
-
1
Lung
8
-
6
Ovary
8
1
5
Pancreas
1
1
-
Sarcomas
3
-
1
Stomach
1
1
3
56
TP53 Mutations Are Associated With Shorter
Survival in Breast Cancer
Tumor grade lt3, tumor size lt5 cm, node negative
and ER or PR positive cases
(n204)
No mutation
40 decrease in survival at 5 years
Mutation
Olivier et al., Clin Cancer Res, 2006
57
TP53 Missense Mutations Within The DNA-binding
Loops And Non-Missense Mutations Are Associated
With The Worst Prognosis In Breast Cancer
No Mutation
Missense in DBM
Other missense
Non-missense mutations
Olivier et al., Clin Cancer Res, 2006
58
TP53 Gene Therapy

• ONYX-015
• Is an attenuated chimeric human group C
  adenovirus, that has been developed to
  preferentially replicates in and lyses tumor
  cells that are p53 negative (McCormick, Cancer
  Biol Ther. 2003).
• Advexin (Ad5CMV-p53)
• Is a non-replicating, non-integrating adenoviral
  vector that carries the TP53 gene (Gabrilovich,
  Expert Opin Biol Ther., 2006).

59
Targeting p53 With Small Molecules
Lane Fisher (2004)

• Inhibition of mdm2/p53 interaction (Lane
  Fisher, 2004)
• Kill cancer cells with WT p53
• Reactivation of mutant p53 (Bykov et al., 2002)
• Kill cancer cells with MUT p53

60
Further Information
See list of references at http//www-p53.iarc.fr/
References.html And p53 story at http//p53.free
.fr/ p53 Knowledgebase at http//p53.bii.a-star.
edu.sg/index.php