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

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

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

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

Group 1 – 144 probesetsExpression level increases with CDDP concentration. For CDDP 10, expression levels are lower with Pon.

Group 1 – 144 probesetsExpression level increases with CDDP concentration. For CDDP 10, expression levels are lower with Pon.

Group 4 – 14 probesetsExpression level increases with CDDP concentration. Expression levels are higher upon Pon addition.

Group 4 – 14 probesetsExpression level increases with CDDP concentration. Expression levels are higher upon Pon addition.

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

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

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

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

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

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

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

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