Page 1
TP53 Somatic Mutations are Frequent in Human Cancer
0 5 10 15 20 25 30 40 45 50
CervixThyroïde
TestisBone
KidneyHematol.Prostate
Soft tissueUterusBreast
LiverBrain
StomachBladder
SkinLung
Head&NeckPancreas
ColorectumOvary
Esophagus
Percent of Tumors with a TP53 Mutation
Page 2
p53 status and DNA damage
p53 wild type
Bax
p21p53AIP1
MDM214-3-3
p63p73 p53
growth arrestapoptosis
p53 null
p63p73
growth arrestapoptosis
X
mutant p53
X
p63
mp53
p73
mp53
growth arrestapoptosis
XX
DNA DAMAGE
Bax
p21p53AIP1
MDM214-3-3
Bax
p21p53AIP1
MDM214-3-3
p53 abnormalities in human cancer
Page 3
mp53
mp53
History of the protein complex of mp53/p73
72R p73
72R p63
Di Como et al., 1999Strano et al., 2000Gaiddon et al., 2001Bensaad et al., 2003
p63
p73
mp53p73
mp53p63
mp53
p73
p63
p53 Baxp53Aip1Mdm2p2114-3-3σ
DBD Domain DBD Dom
ain
mp53
Baxp53Aip1Mdm2p2114-3-3σ
Marin et al., 2000 Bergamaschi et al., 2003Irwin et al., 2003
Lang et al., 2004Olive et al., 2004
Strano et al. 2002
Page 5
NF-Y
CyclinACyclinBCdk1Cdc25c
X
Bax
p21
p63p73
p53AIP1
MDM214-3-3
mp53
CCAATbox
Gain of function activity
mp53mp53
p300
DNA Damage
Protein-protein interaction Transcriptional competent complexes
Page 6
Group 1 – 144 probesetsExpression level increases with CDDP concentration. For CDDP 10, expression levels are lower with Pon.
Page 7
Group 1 – 144 probesetsExpression level increases with CDDP concentration. For CDDP 10, expression levels are lower with Pon.
Page 8
Group 4 – 14 probesetsExpression level increases with CDDP concentration. Expression levels are higher upon Pon addition.
Page 9
Group 4 – 14 probesetsExpression level increases with CDDP concentration. Expression levels are higher upon Pon addition.
Page 10
Pon
Upregulated genes38 probesets
prob
eset
s
5
10
15
20
25
30
35
- - - - -+ + + +
-1.5
-1
-0.5
0
0.5
1
1.5
2
-2
Rela
tive
ID4
mRN
A
PonCDDP (g/ml) 24h
H1299-p53His175 H1299-p53wt H1299-pIND
- + - + - + - + - + - + - + - + - +- - 5 105 10 - - 5 105 10 - - 5 105 10
0,20,4
0,6
0,81,0
1,2
1,4
0
0
10
20
30
40
50
60
1 2 3 4
Pon-Pon+
ID4
ID4
mRN
A
80S polysomes
Pon-
Pon+
ID4 mRNA is specifically induced by mutant p53R175H
Page 11
BRCA1
ID-4
BRCA1 promoterFeedback loop
Anchorage-independent cell growth
ID4 & developmentID-4 –/– mice: Neural proliferation defects and premature differentiation(regulation of neural stem cell proliferation and differentiation)
ID4 & cancerIn mammary and ovarian tumors:
ID4 overexp. in rat mammary carcinoma,seminoma and B-cell leukemia
ID4 promoter hypermethylationIn gastric and colorectal cancer
ID-4
ID proteins are involved in the control of cell proliferation, differentiation, transformation and angiogenesis
Page 12
ID4 promoter is bound by mutant p53R175H
http://www.genomatix.de/cgi-bin/matinspector
TATA
E-boxCCAATNF-kB
Sp1 Sp1 Sp1 CDE CDE
ZF5 ZF5MAZ
A B C D
-850 +300
mp53mp53
mp53
E2F1
mp53
NFKB
SKBR3: p53His175
No
Ab
p53
p65
p53/
No
Ab
p53/
p65
p53/
E2F
1
E2F1
Neg. control
p300
PCAF
p53/
No
Ab
p53/
p30
0p5
3/ P
CAF
ReChIP
A
B
C
DID4
prom
oter
ChIP
p53
Inpu
t
A
B
C
DID4
prom
oter
No
Ab
p21 promoter
p300 PCAFPCAF
Chromatin sheared to 200-400 bp lenght
MCF7: p53wt
Page 13
SKBR3 ChIP
0
2
4
6
8
1 2 3
anti-p53 Ab1N
o Ab
p53
Ab1
AB C D
Rel.
Enric
h.No Ab
Ab
0
2
4
6
1 2 3
anti-p53 Ab2
B C D
Rel.
Enric
h.
No
Ab
p53
Ab2
A
0
2
4
6
1 2 3
p65
No
Ab
A B C D
Rel.
Enric
h.
anti-p65
0
5
10
15
20
1 2 3
anti-p300
p300
No
Ab
A B C D
Rel.
Enric
h.
0
5
10
15
1 2 3
anti-E2F1
E2F1
No
Ab
A B C D
Rel.
Enric
h.
0
1
2
3
4
5
6
7
1 2 3
anti-E2F1
Rel.
Enric
h.
B C DA
p53/
E2F1
p53/
No
Ab
0
1
2
3
4
5
6
1 2 3
anti-p300
Rel.
Enric
h.
B C DA
p53/
p300
p53/
No
Ab
anti-p53/No Abanti-p53/Ab
0
2
4
6
1 2 3
anti-p65
Rel.
Enric
h.
B C DA
p53/
p65
p53/
No
AbSKBR3 re-ChIP
E2F
p65mp53
E2F-1mp53
p300E2F-1
mp53p300
Same results in MDA MB 231 cells
Page 14
Endothelial Cell Migration: Chemoattractant C.M. H1299#41 (Pon-/+ 48h)+361
Mutant p53 induces neoangiogenesis
Pon- Pon+neg
Endo
thel
ial c
ell m
igra
tion
Pon -
Pon +
0.000
0.600
1.200
1.800
2.400
3.000
C- C- (pon+) Pon- Pon +
+143
0,000
0,200
0,400
0,600
0,800
+121
Pon - + - + - +VgRXR36h
Cl.4136h
Cl.4124h
Endo
thel
ial C
ell P
rol.
0,0
0,6
1,2
1,8
Hb
cont
ent
(OD
/g o
f mat
rigel
)
Pos Neg Pon- Pon+
Conditioned medium from clone 41 Endothelial cell proliferation Matrigel experiments
Page 15
Ribonucleoprotein Immunoprecipitation (RIP)
Crosslinking of Cells Immunoprecipitation of RNA-binding proteins
RIP-RNA
Input-RNA
Reverse-crosslinkingRNA extractionRT-PCR
-p
53
Inpu
t
-M
yoD
-ID
4
-p
53
-M
yoD
-ID
4
-p
53
-M
yoD
-ID
4
IL8
GRO-
MDA-MB-231 MDA-MB-468 SKBR3
MDA-MB-231 MDA-MB-468 SKBR3
RPL19
Page 16
Ribonucleoprotein Immunoprecipitation (RIP)
Crosslinking of Cells Immunoprecipitation of RNA-binding proteins
RIP-RNA
Input-RNA
Reverse-crosslinkingRNA extractionRT-PCR
-p
53
Inpu
t
-M
yoD
-ID
4
-p
53
-M
yoD
-ID
4
-p
53
-M
yoD
-ID
4
IL8
GRO-
MDA-MB-231 MDA-MB-468 SKBR3
MDA-MB-231 MDA-MB-468 SKBR3
RPL19
Page 17
ID-4PCAF
E2F1
mp53
PCAFE2F1
mp53p300
ID4
3’UTR
3’UTR3’UTR
mRNA binding
mp53
ID-4
mRNA pro-inflammatory/pro-angiogenic cytokines Stabilization
mp53
ID-4
Pro-angiogenicfactors release
PCAF