crosstalk between proliferation and migration
TRANSCRIPT
How alternative splicing converts p53 tumour suppressor into an oncogene
Pierre Roux CRBM/ UMR5237 Montpellier, France
Crosstalk between proliferation and migration!
Dynamics of cancer cell invasion
Primary tumor
Invasive tumor
Metastasis
Mode of migration
Mesenchymal Amœboid
!"#$!%#&
EMT(epithelio-mesenchymal transition) - Loss of cell-cell adhesion - Increased motility: Rac1- and metalloprotease dependent EAT (epithelio-amoeboid transition) - Loss of cell-cell and cell-ECM adhesion - Increased motility: RhoA-dependent Independent of Rac1 and metalloproteases
« Guardian of the genome »
•! The most frequent mutated gene in cancer
•! Inactivated function in almost all tumors
•! Activates DNA repair when DNA has sustained damages
•! Arrests growth by holding the cell cycle in G1/S or G2
•! Initiates apoptosis if DNA damage proves to be irreparable
The «!Peto’s paradox!»: no correlation between body size and cancer risk&
•! Larger and longer-lived animals should have a higher risk of cancer than smaller, shorter-lived animals!
•! No link between the size of an animal and its risk of developing cancer. !
•! Elephants: Enhanced DNA-damage response and tumor resistant Why don't elephants get cancer?"
Haupt S., and Haupt, Y. ; F1000Res. 2017 Nov 22;6:2041; Sulak et al.;Elife. 2016 Sep 19;5.
Species Humans Elephants
Average survival ~70 years 60–70 years Number of pairs of p53 copies
1 1
Number of pairs of p53 transgenes
0 19
Cancer incidence 1:4 0
The elephant genome encodes 20 copies of the tumor suppressor gene TP53
Comparison of humans and elephants and parameters of relevance to cancer-free survival
The role of p53 is crucial in cancer
'()&&
"*+,-./012!%'.'+.101!
3*//&454/*&,66*1+!
7+*2&4*//!
!"#$%&'#()#()*+,(%)-#./0-1'2+,2+,(,#3%%4&5#6)7'4,2%%7#
#8"#9%2#6,'7#+,#+#:(%1+4;'4#
<"#=%>#7%#3?<#,'&'@2#2A'#@'&&6&+4#4',3%),'!+7+32'7#B#
NEEDS: '()&08&4,84*69&&
!! #:*&2.1+&;6*<=*8+&2=+,+*>&?*8*&&
!! @8,4AB,+*>&08&,/2.1+&,//&4,84*61&&
7*8*14*84*!C,>0,A.8&+:*6,'5!
3:*2.+:*6,'5!
%>:*10.8D!@8B,10.8D!
"0?6,A.8D!
!!Transcription factor
7+6*11&10?8,/1&
'()&
'()&'()&'()&'()&'()&
'()&'()&'()&'()&'()&'()&'()&'()&'()&'()&'()&'()&
'()&'()&'()& '()&'()& '()&
'()&
'()C!&'()&+,6?*+&?*8*1&
"E"F&
'()&'()&'()&'()&'()&'()&'()&'()&'()&'()&'()&'()&
P Ac Me
'()&6*?=/,A.8&
CBP, p300
SET9
ATM, CHK2
Low p53 level '()&'()&'()&'()&
'()&'()&'()&'()&'()&'()&'()&'()&'()&'()&'()&
'()&'()&'()&
'()&'()& '()&'()& '()&'()&
'()&'()&'()&'()&'()&P
Stabilized p53
Active p53
!!Post-translational regulation
A novel tumor suppressor activity for p53: inhibition of migration and invasion
p53 Apoptosis Cell cycle arrest
p53: The most frequently mutated gene in cancer Inactivated in almost all cancers
Senescence Adhesion Invasion
Migration Rho GTPases
Mutant p53 controls EMT/EAT and promotes invasion
p53 and EMT: Gadea et al., EMBO J., 2002 Gadea et al., J. Cell Science, 2005 Gadea et al., J. Cell Biol, 2007; Vinot et al., Meth. Enzymol., 2008; Roger et al., J. Cell Sci., 2010.
E-cadherin Slug, Zeb1
ROCK RhoA
wt p53 mut p53
Mode of migration
Mesenchymal Amœboid
1: Mutant p53 disrupts adherens junctions in inhibiting E-Cadherin expression through Slug and Zeb1 2: Mutant p53 promotes amoeboid-like mode of migration in 3D. 3: Mutant p53 activates the RhoA/ROCK pathway.
"=+,8+&'()&'6.2.+*1&,2.*-.0>G/0H*&2.>*&.;&20?6,A.8&08&)E&
Epithélium!
Proteases!
!1-integrin!
"*1*84:52,/&
Epithelial Mesenchymal!Transition (EMT)!
E-cadherin!Epithelial Amoeboïd!
Transition (EAT)!
%2.*-.0>&&
!"#$!%#&
3,6408.2,&!"#$!%&#
"!#%7#%7@7&
%>:*10.8&
@8B,10.8!"0?6,A.8!
##>2#3?<# 3?<#7'C@(')2#
"=+,8+&'()&'6.2.+*1&,2.*-.0>G/0H*&2.>*&.;&20?6,A.8&08&)E&
Epithélium!
Proteases!
!1-integrin!
"*1*84:52,/&
Epithelial Mesenchymal!Transition (EMT)!
E-cadherin!Epithelial Amoeboïd!
Transition (EAT)!
%2.*-.0>&&
!"#$!%#&
3,6408.2,&!"#$!%&#
"!#%7#%7@7&
@8B,10.8!"0?6,A.8!
Epithélium
1-integrin
Epithelial AmoeboïdEpithelial AmoeboïdEpithelial AmoeboTransition (EAT)Transition (EAT)Epithelial Mesenchymal
Transition (EMT)
E-cadherin!"#$!%#&!"#$!%#&
3,6408.2, !"#$!%&#
"!#%7#%7@7&
%>:*10.8&
##>2#3?<# 3?<#7'C@(')2#
Wt TP53 generates alternative splice isoforms
Biomarker of metastasis risk? Poorly reliable biomarker
####################
Mutant p53 Alternative Splicing Mutation
IJ7&
EI%&K08>08?&E.2,08&&&&&&&&&&&&&&&L/0?.2*60M,A.8&,81,4AB,A.8&&))N&
'()"&'()!& &JOEO#7POQ!I3& DE#;F#
))N&
"JJEJCR3SPJ@I77& DE#;F#
#TU'()&#TU'()!&#TU'()"&
####################%#V&TU& DG#;F#))N&
%#V&TU& &JOEO#7POQ!I3& D!#;F#
"JJEJCR3SPJ@I77&))N&
%#V&TU& D!#;F#
#N))'()&
#&N))'()!&
#&N))'()"&
%#V&N))&& <?#;F#####################
))N&
####################%#VN))& &JOEO#7POQ!I3& ###8G#;F#####################
))N&
%#VN))& "JJEJCR3SPJ@I77& ###8G#;F#
'()& ####################
W6./08*&>.2,08&
?<#;F#
TP53 gene and its products
! "
#
P1’" P2"
P1"!""!
i9"1 2 3 4 5 6 7 8 9 10 11"
# p53"# 40p53" # 133p53"
Wt TP53
p53 isoforms
1 2 3 4 2 3
1 2 3 4 gene
protein
Alternative splicing pre mRNA
mRNA
Alternative splicing
Isoform A
1 2 3 1 2 3 4
Alternative splicing
1
Isoform B
Transcription
Alternative splicing generates modified proteins
Alternative splicing as an unexplored program in oncology
Major
3 4
DBD
DBD
DBD OD
DBD OD
DQTSFQKENC
DBD DQTSFQKENC
MLLDLRWCYFLINSS
DBD MLLDLRWCYFLINSS
DBD
DBD
DQTSFQKENC
MLLDLRWCYFLINSS
p53α
p53β
p53γ
Δ40p53α
Δ40p53β
Δ40p53γ
Δ133p53α
Δ133p53β
Δ133p53γ
133
p53isoforms
1 3 102 4 5 6 7 8 9 11
P1 P2
TP53 βγ
TAD2 DBD OD TAD1
TAD2TAD1
TAD2TAD1
TAD2
TAD2
TAD240
DBD OD
DBD DQTSFQKENC
MLLDLRWCYFLINSS DBD
Δ160p53α
Δ160p53β
Δ160p53γ
160
!133p53" is associated with bad prognosis in breast cancer (A. Thompson, JC Bourdon)
#133p53ß is: -! associated with reduced disease free survival (DFS) and global survival, independently of p53
mutations -! a clinical biomarker to predict high risk of relapse and metastases in advanced breast cancer.
Breast: 276 tumors
Cum
ulat
ive
Dis
eaes
e-Fr
ee S
urvi
val
1.0
0.0
0.2
0.4
0.6
0.8
0 1000 2000 3000 4000 5000days
6133p53`
(negative)
n=250 e=61
(positive)
n=23 e=11
LOG Rank test, r� = 6.221, p < 0.013
Cum
ulat
ive
Dis
eaes
e-Fr
ee S
urvi
val
1.0
0.0
0.2
0.4
0.6
0.8
0 1000 2000 3000 4000 5000days
6133p53a
(negative)
n=165 e=44
(positive)
n=56 e=14
LOG Rank test, r� = 0.006, p < 0.936
Cum
ulat
ive
Dis
eaes
e-Fr
ee S
urvi
val
1.0
0.0
0.2
0.4
0.6
0.8
0 1000 2000 3000 4000 5000
days
6133p53_
(negative)
n=176 e=44
(positive)
n=97 e=28
LOG Rank test, r� = 0.857, p < 0.354
Development of highly metastatic MDA-MB231-luc cell lines by in vivo selection (breast model)
Implantation into immunodeficient mice
In vitro isolation and expansion
immunodeficient
/'2+,2+,',#H%41+I%)#
W,6*8+,/&X=2,8&-6*,1+&4,6408.2,&4*//&/08*&&"E%G"KF)N9&1/0?:+/5&2*+,1+,A4&
/(@'#+,#+#J@'&&#,%42'4#J#
!,2#4%6)7#
8)7#4%6)7#
K<L9F#
Expression of $133p53" is associated with metastatis index (breast model)
!,2#4%6)7#
<2A#4%6)7#
?2A#4%6)7#
"#!$!%&'()!*+,-./!01234!$!516%%54&23457)65%!
7'.8+,8*.=1&2*+,1+,101&+.&JI&M?#2A#4%6)7N#
"*+,1+,101&>011*208,A.8&
W,11,?*1&8=2-*6& 3*//&/08*1&
W602,65&+=2.6&+,H*&6,+*& C,+*& L6?,81&
E,5&.;&Y61+&>*+*4A.8&
N& O+4')2+&#@'&&#&()'# !PPQ# 8PQ# RS-:4#L9# E,5&ZF&
F& T"D#3%%&# !PPQ# ?PQ# RS-:4#L9#&6)U,#
F+5#D?#
)& KE#&6)U#3%%&## !PPQ# !PPQ# RS-:4#L9# F+5#<?#
T& .<#3%%&# !PPQ# !PPQ# RS-:4#L9# F+5#8V#
(& 3)&JIE& NUU[& NUU[& %\$-6&JI& E,5&FU&
/FR#/T8<!#
/FR#F<=8L9#
K<L9F#
]N))'()^
exp
ress
ion!
C3LND compared to parental MDA-MB231!
516%%54&23457)65%!516%%54&23457)65%!516%%54&
E,5&.;&Y61+&E,5&.;&Y61+&>*+*4A.8&
E,5&ZF&
F+5#D?#
F+5#<?#
F+5#8V#
E,5&FU&
0,00
0,50
1,00
1,50
2,00
K<L9F#/FR#/T8<!#
#133p53 silencing reduces metastasis in highly metastatic MDA-MB231-luc C3LND&&&&
!G#7+5,#!D#7+5,#
"E%G"KF)NG/=4&3)JIE&#
"E%G"KF)NG/=4&3)JIE&_&1:`N))&#
Met
asta
sis
inde
x
`�!"#$%
&'()*+!&,-
6.((&'(`�a�
''
('
/'
''
+0)1," +0(
23&'(_�
23&'(_�
6.((&'(_
0,00E+00
2,00E+06
4,00E+06
6,00E+06
8,00E+06
1,00E+07
1,20E+07
Control
sh #133
#133p53 silencing decreases invasion and EMT markers '()& ####################
'()"&'()!& &JOEO#7POQ!I3&
"JJEJCR3SPJ@I77&
#TU'()&#TU'()!&#TU'()"&
####################
&JOEO#7POQ!I3&"JJEJCR3SPJ@I77&
#N))'()&#&N))'()!&#&N))'()"&
&JOEO#7POQ!I3&"JJEJCR3SPJ@I77&
5’ UTR"
5’ UTR"
5’ UTR"
10N))&
MDA-MB231-D3H2LN
siE7
si133-2
sip53`
siNS
6133p53`
640p53`
51
28
39
39
19
p53`
Ku 80
E-Cadherin
64
97
*
KJC8
Sapu
286133p53` a
Vimentin 39
51
p53 (`��a640p53��_��`��a6133p53_6160p53_
p53(_
Rela
tive m
RN
A E
-Cad
herin
exp
ressio
n
siN
S
si1
33-2
sip
53`
siE
7
0.00
0.01
0.02
0.03
0.10
0.15
0.20* *
*
*
MDA-MB231-D3H2LN
@8B,10.8&
0
20
40
60
80
100
120
Δ133p53 silencing reduces cancer stem cell formation
No
sphe
res/
4000
cel
ls
0
10
20
30
40 *
ctrl
Sh133
10
20
30
No
sp
he
res/4
00
0 c
ells
0
C3LND
MDA-M
B 2
31
D3H2LN
*
MammosphereformaAon
- The CSC phenotype and metastasis development are closely linked
`N))'()&10/*8408?&>*46*,1*1&+:*&'6.'.6A.8&.;&3ETT_$3EFTG&4*//1&"! in promoting mammospheres
formation:
CD44
CD
24
Sh Luc
Sh #133
96.36%
70.76%
!"#$"%&'()#*+%&((,-.,""/0))1
/23)
4,566
7
8
'8
(8
98
:8
588
#133p53ß regulates cancer stem cell formation
"! in promoting mammospheres formation:
"! in inducing regulators of pluripotency (c-myc, Sox2, Oct3/4, Nanog)
Fold
incr
ease
0,00
2,00
4,00
6,00
8,00
10,00
12,00
C-MYC
OCT3-4
NANOG
SOX2
First plating
Re-plating
"3Pa&
!133p53 is associated with bad prognosis in CRC
#133p53 is: -! associated with reduced disease free survival (DFS) and global survivall -! a clinical biomarker to predict high risk of relapse and metastases in advanced CRC
Rectum: 60 tumors
P = 0.027
#133p53
Cum
ulat
ive
Dis
ease
Fr
ee S
urvi
val
Low expression
High expression
YEARS
Primary tumor
Low #133 expression
Low risk: Reduced-intensity chemotheray
#133p53! expression is associated with invasiveness in colon carcinoma
1
2
0
3
4
HCT116
Control siE7 siTAp53
Re
lati
ve
fo
ld i
nv
as
ion
*
*
'()& ####################
'()"&'()!& &JOEO#7POQ!I3&
"JJEJCR3SPJ@I77&
#TU'()&#TU'()!&#TU'()"&
####################
&JOEO#7POQ!I3&"JJEJCR3SPJ@I77&
#N))'()&#&N))'()!&#&N))'()"&
&JOEO#7POQ!I3&"JJEJCR3SPJ@I77&
5’ UTR"
5’ UTR"
5’ UTR"
JOEO#7POQ!I3&
"JJEJCR3SPJ@I77
JOEO#7POQ!I3&
"JJEJCR3SPJ@I77&
JOEO#7POQ!I3&
"JJEJCR3SPJ@I77
5’ UTR
5’ UTR
5’ UTR
10!a&
#133p53! expression is associated with invasiveness in colon carcinoma
1
2
0
3
4
HCT116
Control siE7 siTAp53R
ela
tiv
e f
old
in
va
sio
n
*
*
#N))'()&#&N))'()!&#&N))'()"&
&JOEO#7POQ!I3&"JJEJCR3SPJ@I77&
#TU'()"&
'()& ####################
'()"&'()!& &JOEO#7POQ!I3&
"JJEJCR3SPJ@I77&
#TU'()&#TU'()!&
####################
&JOEO#7POQ!I3&"JJEJCR3SPJ@I77&
5’ UTR"
5’ UTR"
5’ UTR"
10#%&
#133p53! expression is associated with invasiveness in colon carcinoma
0.2
0.6
0.4
0.8
1
1.2
0 R
elat
ive
fold
inva
sion
siNS si133-1 si133-2
SW620
**
'()& ####################
'()"&'()!& &JOEO#7POQ!I3&
"JJEJCR3SPJ@I77&
#TU'()&#TU'()!&#TU'()"&
####################
&JOEO#7POQ!I3&"JJEJCR3SPJ@I77&
#N))'()&#&N))'()!&#&N))'()"&
&JOEO#7POQ!I3&"JJEJCR3SPJ@I77&
5’ UTR"
5’ UTR"
5’ UTR"
10N))&
#133p53! expression promotes invasiveness and amoeboid-like migration in colon carcinoma
VPWG#N))'()!&
#133p53 ! promotes EAT!
X3#NNb: G&:=2,8&4./.8&4,6408.2,&4*//1&
G&c+&'()&&G&6*+,081&*'0+:*/0,/&4:,6,4+*601A4&
####################
E-Cadherin
p53 status SW480 CoLo205 HCT116 LoVo SKCo1 SW620
wt mutant mutant wt mutant mutant
Meta Meta Meta Meta
Re
lati
ve
fo
ld e
xp
re
ss
ion
Subclasses of p53 isoforms
*
*
*
*
*
*
**
*
HCT 1
16
SW
480
Lovo
SW
620
Colo
205
0
5
10
15
20
25
All isoformsalphabetagamma 6133
TAp53
1
32
4
5
0
Rel
ativ
e fo
ld in
vasi
on
VPWG#N))'()
31!
The %133p53ß isoform promotes RhoA/ROCK rounded blebbing invasion&=K0!!E#W#X!<<3?<Y"Z[O#
\]E8P#W#$^K_#()A(:(2%4#
Gilles Gadea et al.!T`0#=^]B#
#122p53/#122p53
1" 3" 10"2" 4" 5 "6 "7 "8 "9" 11"
P2
'()*# β"γ"
DBD" OD "$122p53! 122"
DBD OD $122p53!122122ATG"
•! Mice modelling $133p53 :
•! are highly tumour-prone,
•! display chronic inflammation and autoimmunity.
•! display elevated serum IL-6
Δ122p53 (a model of Δ133p53) mice show decreased survival and a very aggressive tumor
spectrum"
Antony Braithwaite, University of Otago, NZ
!133p53 isoform promotes tumour invasion and metastasis via interleukin-6 activation of JAK-STAT and RhoA-ROCK signalling
IL6
ROCK RhoA
#133p53
Mode of migration
Mesenchymal Amœboid
JAK
STAT
P!
Tumour cell invasion Immune cell migration
Campbell et al.,"Nat. Commun; 2018 Jan 17;9(1):254. !
MYPT
MLC LIM
#133p53! reprograms epithelial cells to pro-metastatic cells
#N))'()!&
E-cadherin Slug, Zeb1
ROCK RhoA
wt p53
!133p53ß is a transcript produced by the WT TP53 gene.
-! Promotes invasiveness in WT p53 cells -! Induces amoeboid movement -! Promotes cancer stem cell formation
Mode of migration
Mesenchymal Amœboid
To conclude
•! p53 isoforms expression is a more important mechanism than TP53 mutations to predict patients outcome
•! This explains why patients with WT TP53 can have metastasis and reciprocally why patients with mutant TP53 gene do not systematically have metastasis and relapse
•! The %133p53s isoforms are reliable targets to reduce metastasis
1. How %133p53 isoforms interfer with p53 tumor suppressor expression and activity ?
2. How %133p53 isoforms interact with oncogenic mutant p53 activity ?
1! 2!
!Cell cycle arrest and senescence"
!
p53"
p21"
Mdm2"
p53"
Mdm2"
p21"
!No cell cycle arrest "
and bypass senescence "!
Inactive accumulated form
"133p53!"133p53!
1. How %133p53 isoforms interfer with p53 tumor suppressor expression and activity ?
Normal cell: induced expression
Cancer cells: constitutive expression
Véronique Gire, Fanny Tomas
2. How %133p53s isoforms interact with mutant p53 ?
HUMAN TP53 GENE
!"#$%&'! ()$%&'
!"#$%&'!()$%&'
p53 mutants p53 mutants
Tumor suppressor p53
???!
Mutant p53
Dominant negative effect!
Nikola Arsic
What make differences between WT $133p53 isoforms and mutant p53?
•! Mutant p53 and %133p53 isoforms share:
#! Similar pro-oncogenic functions opposite to p53 tumour suppression
#! Identical cellular localisation, both present in cytoplasm and nucleus, contrary to WT p53, which is exclusively detected in the nucleus.
#! Unfolded protein conformation
#! Specific proteins partners (Pontin, HSP70 and p63 family members), which do not bind to WT p53
%'.'+.101&6*101+,84*&! 3,84*6&7+*2&
&4*//!
3:*2.+:*6,'5&6*101+,84*&!@8B,10.8& "0?6,A.8!
`N))'()1&"=+,8+&'()&
!"#!
Mutant p53 and %133p53s isoforms promote cancer aggressiveness
From: Gain of function of mutant p53 by coaggregation with multiple tumor suppressors, Xu et al, 2011; Nature Chemical Biology
Pro-oncogenic functions of mutant p53 is implemented by coaggregation of wild-type p53 that promotes
dominant-negative effect
p53R273H"p53R175H"
WT p53"
Only p53 structural mutant p53R175H creates protein aggregates
p53"amyloid aggregates"
Hoechst "
Control"
3D reconstitution"H1299 cells"
Δ133p53βR175H"
Control" WT Δ133p53β"
$133p53!R280K
3D reconstitution"
$133p53" creates protein aggregates independently of the mutation status
H1299 cells"
Δ133p53β"
amyloid aggregates"Hoechst "
Nikola Arsic
!"#`#$%&'(#
!"#)*+**
**
+*
,*
%-(&.
-/
0()1&
62++)*+`#
3&#
62++)*+_#3
&#
%-(&.
-/
62++)*+`#
3&#
62++)*+_#3
&#
45678
!"#)*+**
+*
,*
0()1&
%-(&.
-/
62++)*+`#
9,:;<
#
62++)*+_#9
,8+=
#
62++)*+_62++)*+`
62++)*+`#
9,8+=
#
6/$>
%-(&.
-/
62++)*+`#
9,:;<
#
62++)*+_#9
,8+=
#
62++)*+`#
9,8+=
#
6/$>
!"#`#$%&'(#
0?7_#6/$>0?7_#6/$>
4@A74"7,+2
62++)*+_62++)*+`
6/$>6/$>
**
+*
,*
B<7"97+
%-(&.
-/
62++)*+_#3
&#
62++)*+_#9
,8+=
#
62++)*+`#
3&#
62++)*+`#
9,8+=
#
62++)*+`#
928*=
#
0()1&
6/$>6/$>
0?7_#6/$>
62++)*+_62++)*+`
!"#)*+
!"#`#$%&'(#**
%-(&.
-/
62++)*+_#3
&#
62++)*+_#9
,8+=
#
62++)*+`#
3&#
62++)*+`#
9,8+=
#
62++)*+`#
928*=
#
!"#6/$>#!"#6/$>#
!"#6/$>#
$133p53" does not interact with WT p53
Nikola Arsic
Δ133p53βR175H"
Control" WT Δ133p53β"
$133p53!R280K
3D reconstitution"
$133p53" creates protein aggregates independently of the mutation status
H1299 cells"
Δ133p53β"
amyloid aggregates"Hoechst "
Nikola Arsic
Interaction with protein partner p63 reduces aggregation state of $133p53"
!"
#$""
%$$%!$&"$
""
!"
#$""
%$$%!$&"$
'&( )"(
6*+,!
_!
6*+,!
_6%!!+"!
`-./0123!
456.751
6%!!+"!
`-./0123
6*+,!
_!
6*+,!
_6%!!+"!
`-./0123!
456.751
6%!!+"!
`-./0123
89/0123/�6%!!+"!`
89/+,!
89!`�24.:6;<+=762.26.
6%!!+"!`89/0123
3D reconstitution"
H1299 c"
Control" WT Δ133p53β"
ΔNp63α" WT Δ133p53β+ΔNp63α"
Δ133p53β"ΔNp63α"Hoechst"
Current view: p53 and p63 are independent EMT regulators
metastasis
Primary tumor
!"#&
p63 p53
- p53 and p63 do not interact
metastasis
!"#&
Mode of migration
Mesenchymal Amœboid
Δ133p53β and p63 cooperate in disrupting adherens junctions and in promoting invasion
Δ133p
53βE
GFP
ΔNp6
3α
ΔNp6
3γ
ΔNp6
3γ
ΔNp6
3α
control
E-Cadherin
TubulinN-Cadherin
Adhesion
Inva
sion
inde
x
sh6N
p63
shC
ontr
ol
0
2
4
6
8
*
6133p53`
sh6
Np6
3
shC
ontr
ol
*10
Inva
sion
inde
x
613
3p53
`
6N
p63a
6N
p63_
6N
p63a
6N
p63_
6133p53`
Con
trol
0
20
40
60
80
*
MCF7:WTp53
WT ∆133p53ß synergizes with ∆Np63 in promoting adherent junctions disruption and enhanced invasion
"Np63!"133p53ß!
"133p53ß Storage of inactive %133p53ß in aggregates
"133p53ß!
"Np63!
CCT! Invasion
Release
Active complexes CCT!
Oncoprotein reversible aggregation mechanism
Nikola Arsic
-! Stored in aggregates in an inactive form -! Aggregation state of %133p53ß inversely correlates to its activity level
-! %133p53ß aggregates are reversible depending on presence or absence of interacting partners
"Np63!"133p53ß!Storage of inactive
%133p53ß in aggregates "133p53ß!
"Np63!
CCT!Invasion
Release
Active complexes CCT!
mutant p53 and WT $133p53" aggregates are distinct
Nikola Arsic
Mutant p53
WT p53
Inhibition of WT p53 function in aggregates
Invasion
%133p53ß
To conclude
•! %133p53 isoform: oncoprotein reversible aggregation mechanism
•! %133p53 oncogenic activity mechanism differ from this of mutant p53
•! p53 and p63 (p73?) isoforms activities are coordinated
p63 and p53 signaling cooperate in regulating EMT
metastasis
Primary tumor
!"#&p63 isoforms
p53 isoforms
- p63 and p53 isoforms interact each other
p53!p53!
- This extends the number of combinaison of transcriptional heterocomplex complex
3?<Y#
X!<<3?<Y#
3?<#
X!<<3?<#
X93E<a#
0R3E<#
TAp63!TAp63TAp63!TAp63
"Np63!
TAp63
Np63"133p53ß!
p53!p53!p53
p53!
p53
"133p53ß!
3?<b#
X!<<3?<b#
X93E<c#
0R3E<c#
TAp63!TAp63TAp63!TAp63
"Np63!
TAp63
Np63"133p53ß!
p53
p53!
p53
"133p53ß!
X93E<b#
0R3E<b#
Etc ………
How can p53 integrate so many signals at once?
-pH-Glucose-Nutrients
Neighboring cells - cellcontact
- secreAonofcytokines
H2O2
.OH
.O2
-
Protein oxidation - Meta-tyrosine - Amino group - Thiol group
Nucleic Acid oxidation Damages to:
• Genomic DNA • Mitochondrial DNA • Ribosome (rRNA)
• tRNA • mRNA
• microRNA, snoRNA…
Carbohydrate oxidation Extracellular signals
Lipid Peroxidation - Cytoplasmic mb (Na+/K+ )
- Endoplasmic Reticulum mb (Ca2+ ) - Mitochondrial mb (ATP )
- Lysosomal mb (release protease) - Nuclear mb (nuclear structure)
mitochondria
UV
Carcinogenes
proliferation Energy
metabolism
Cell cycle arrest G1/G2 Cell motility,
angiogenesis
Senescence Differentiation Inflammation
Organogenesis
Cell death : -extrinsic apoptosis -intrinsic apoptosis
-necroptosis - Autophagic cell death
Cell Stemness Cell repair
How do p53 choose the target genes to induce in order to trigger a coordinated and defined cellular response adapted to the damages and the tissue type?
EMT
Responses p53
-pH-Glucose-Nutrients
Neighboring cells - cellcontact
- secreAonofcytokines
H2O2
.OH
.O2
-
Protein oxidation - Meta-tyrosine - Amino group - Thiol group
Nucleic Acid oxidation Damages to:
• Genomic DNA • Mitochondrial DNA • Ribosome (rRNA)
• tRNA • mRNA
• microRNA, snoRNA…
Carbohydrate oxidation Extracellular signals
Lipid Peroxidation - Cytoplasmic mb (Na+/K+ )
- Endoplasmic Reticulum mb (Ca2+ ) - Mitochondrial mb (ATP )
- Lysosomal mb (release protease) - Nuclear mb (nuclear structure)
mitochondria
UV
Carcinogenes
proliferation Energy
metabolism
Cell cycle arrest G1/G2 Cell motility,
angiogenesis
Senescence Differentiation Inflammation
Organogenesis
Cell death : -extrinsic apoptosis -intrinsic apoptosis
-necroptosis - Autophagic cell death
Cell Stemness Cell repair
How do p53 choose the target genes to induce in order to trigger a coordinated and defined cellular response adapted to the damages and the tissue type?
EMT
Responses p53/p63/p73familyisoforms
How can p53 integrate so many signals at once?
Conventional view: biological response specificity is driven by p53 levels
proliferation Energy
metabolism
Cell cycle arrest G1/G2 Cell motility,
angiogenesis
Senescence Differentiation Inflammation
Organogenesis
Cell death : -extrinsic apoptosis -intrinsic apoptosis
-necroptosis - Autophagic cell death
Cell Stemness Cell repair
Levelofp53
Target genes: > 8000/10000 genes
p53
- transcripAon- post-translaAonalmodificaAons- degradaAon
proliferation Energy
metabolism
Cell cycle arrest G1/G2 EMT
Senescence Differentiation Inflammation
Organogenesis
Cell death : -extrinsic apoptosis -intrinsic apoptosis
-necroptosis - Autophagic cell death
Cell Stemness Cell repair
'()$'b)$'a)&@1.;.621&4.2'/*\*1&
Target genes: > 8000/10000 genes
Model: biological response specificity is driven by composition of p53 family members complexes
X!<<3?<#
3?<Y#
X!<<3?<Y#
3?<#
X!<<3?<#
X93E<#
0R3E<#
Invasion Migration
Angiogenesis
TAp63!TAp63!
Target genes: "Np63!
TAp63
Np63 "133p53ß!
p53!p53!
p53!
p53
"133p53ß!
3CK"9&I0H./,&%6104&W0*66*&C.=\&de6.80<=*&V06*&W:0/0''*&P.6+&3/,06*&#./M,&&V0//*1&V,>*,&3:601+*//*&%8?=0//*&&J,=6*8+&f=//0*8&7+e':,80*&d08.+&J,=6e/08*&C.?*6&W*??5&C,58,=>&!22,8=*/&d0?8,/&7,2*6&%->,//,:&P,885&#.2,1&&
7'/04.19&E0>0*6&74:*66*6&g3!L$37Lh&3,61+*8&K6.4H&I,+:,/0*&3,:=M,4&3e40/*&%'./0+&",5/01&36*8&&Pi&",:=+*,=&g3=60*&@81A+=+*h&Ci&I,j2,8&&
3.//,-.6,A.819&f3&K.=6>.8&gE=8>**h&fi&#,M0&g".8+'*//0*6h&Vi&K,6/.B,+M&g!B65h&Vi&P,B6*&g#.=/.=1*h&%i! Q,5,B,&g3CK"h&&&
3.//,-.6,A.81&g4/0804h9&%i! #:.2'1.8gE=8>**h&Ki! "i&E*/&60.&g".8+'*//0*6h&!i&36,'*M&g".8+'*//0*6h&fW&E*/.6>&g#.=/.=1*h&