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Tumor suppression and inflammation:Controlling the senescence secretory phenotype
Adam Freund1,2,3 , Christopher K. Patil2,3, Shruti Waghray1, Judith Campisi2,3
1. Department of Molecular and Cell Biology, University of California, Berkeley, CA2. Buck Institute for Age Research, Novato CA
3. Lawrence Berkeley National Laboratory, Berkeley CA
Tumor Suppressor mechanisms falls into two general classes
Caretakers
Stabilize the genome by preventing or repairing mutations
e.g. DNA repair proteins
Gatekeepers
Prevent the growth of potential cancer cells
e.g. apoptosis and senescence (p53, p16, etc)
Strong/mitogenicstress signals
Oncogenes/ oncogenicmutations
Chromatin instability
Non-telomeric DNA damage
Short/ dysfunctional
telomeres (ReplicativeSenescence) Irreversible
cell cyclearrest
Cellular senescence is caused by potentially oncogenic stimuli
Cellular senescence: irreversible cell cycle arrest
Senescence occurs in vivo in response to oncogenic mutations
Michaloglou, C, et al. (2005). BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature 436, 720-724
Senescence markers
IrreversibleGrowth Arrest
The two sides of senescence
InflammatorySecretory
Profile
-Cell autonomous tumor suppression -Alteration of the microenvironment
-Stimulation of malignant phenotypes
-Global tissue degeneration
The Senescence-Associated Secretory Phenotype (SASP) is activated by genotoxic stress
DNA damage Hyper-replication ROS
DNA damage response
ATM/NBS1/Chk2
SASP
But this is an extremely rapid response (<1 hour)
And this takes days to develop
What other molecular events need to happen?
?
p38MAPK activation is associated with DNA damage-induced senescence
0
1
2
3
4
5
6
7
0 5 10
po
pu
lati
on
do
ub
lings
days after irradiation
PRE
SEN(XRA)
Growth Curve
0
5
10
15
20
- 2 4 6 8 10
PRE SEN(XRA)
Fold
Ch
ange
fro
m P
RE IL-6
8-10 days
PRE
SEN(XRA)
Ionizing radiation (XRA)
Hsp27-P
Hsp27
Tubulin
p38-P
p38
Days after irradiation: PRE 2 4 6 8 10
p38MAPK is a mitogen activated protein kinase and part of a well-known stress-response pathway
p38MAPK activity is required for markers of the SASP
PRE
PRE+p38 inhibitor
IL-6 DAPI
SEN(XRA)
SEN(XRA)+p38 inhibitor
0
1
2
3
4
5
6
7
8
9
- - p38 inhibitor
PRE SEN(XRA)
Fold
ch
ange
fro
m P
RE
Secreted protein levels - ELISA
IL-6
IL-8
GM-CSF
p38MAPK activity is required for expression of most SASP factors
0
50
100
150
200
250
300
350
400
1 2 1 2
shGFP shp38α shGFP shp38α
Avg
nu
mb
er o
f ce
lls p
er f
ield
Invasion of MDA-MB-231 cells
0123456789
1 2 1 2
shGFP shp38α shGFP shp38α
PRE SEN(XRA)
Fold
ch
ange
fro
m P
RE
shG
FP
Secreted IL-6 levels - ELISA
SB = SB203580 = p38 inhibitor
p38MAPK activity is also required for the Ras-induced SASP
EmptyVector
SEN(RAS)
p38-P
p38
Hsp27-P
Hsp27
p16
Ras
Tubulin
0
1
2
3
4
5
6
7
8
9
10
0 2 4 6 8 10
po
pu
lati
on
do
ub
lings
days after infection
Growth Curve
Empty Vector
SEN(RAS)
8-10 days
PRE
SEN(RAS)
Constitutive expression of RasV12
SB = SB203580 = p38 inhibitor
p38MAPK activity is sufficient to induce a SASP
SB = SB203580 = p38 inhibitor
MKK6
p38-P
p38
p16
Tubulin
MKK6
p38
MKK6EE = constitutively active form of MKK6 = constitutively active endogenous p38
p38MAPK activity induces a SASP and is downstream of genotoxic stress
SEN(XRA)Factor MKK6EE-induced
SEN(RAS)Factor MKK6EE-induced
GRO + GM-CSF +IL-6 + IL-6 +IL-8 + GRO +
MCP-2 + MIP-1alpha +
MCP-3 + IL-1beta +
GCP-2 + ENA-78 +MCP-1 GRO-alpha +GM-CSF + RANTES
IGFBP-2 IL-8 +IL-10 MCP-3 +
PRE SEN53BP1 foci
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Vector MKK6EE SEN(RAS) SEN(XRA) SEN(REP)
% o
f ce
lls w
ith
≥3
53
BP
1 f
oci
53BP1 foci
p38MAPK inhibition does not affect the DNA damage response
2 4 8 24 48 72Hours after irradiation:
ATM-P
ATM
Chk2-P
Chk2
p53-P (Ser15)
p53 total
p21
Tubulin
2 4 8 24 48 72
p38 inhibitor: - - - - - - - + + + + + + +
0%10%20%30%40%50%60%70%80%90%
100%
1h 1d 2d 4d 6d 8d
% o
f ce
lls w
ith
≥3
53
BP
1 f
oci
time after irradiation
53BP1 foci
PRE
PRE +p38 inhibitor
SEN(XRA)
SEN(XRA) +p38 inhibitor
p38MAPK inhibition modulates the SASP at the mRNA level
Quantitative RT-PCR
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
SEN(XRA) +SB SEN(XRA) +SB SEN(XRA) +SB
IL6 IL8 GMCSFFo
ld c
han
ge f
rom
SEN
(XR
A)
mRNA
Protein
Protein versus mRNA level
SB = SB203580 = p38 inhibitor
2 main ways to modulate mRNA levels:1. Stability2. Transcription
NF-κB binding sites are overrepresented in the promoters of p38MAPK-induced genes
The NF-κB Pathway
NF-κB is activated at senescence
RelA DAPI
PRE
SEN(XRA)
SEN(XRA)+p38 inhibitor
0
1
2
3
4
5
6
PRE 2 hr 4 hr 8 hr 24 hr 48 hr 6 day 8 day 10 day
Fold
ch
ange
fro
m P
RE
NF-kB DNA binding activity
Time after XRA:
RelA = most common NF-kB subunit
0
1
2
3
4
5
6
- MKK6 REP RAS XRA XRA + p38
inhibitor
SEN
Fold
ch
ange
fro
m P
RE
NF-kB DNA binding activity
PRE
NF-κB transcriptional activity is p38MAPK-dependent
0
2
4
6
8
10
12
- +SB - +SB
PRE SEN(XRA)
Fold
ch
ange
fro
m P
RE
NF-κB transcriptional activity
ATM
p38-P
p38
Tubulin
PRE SEN(XRA)shATM: - -+ +
Genotoxic stress
p38 ATM
NF-κB
0.0
0.2
0.4
0.6
0.8
1.0
1.2
shGFP shATM 11 shATM 12Fold
ch
ange
fro
m S
EN(X
RA
) sh
GFP
NF-κB transcriptional activity
RelA is required for the majority of the SASP
o3
RelA
RelB
C-Rel
Tubulin 0
20
40
60
80
100
120
shGFP shRelA #1
shRelA #2
shGFP shRelA #1
shRelA #2
Empty Vector SEN(RAS)Fo
ld c
han
ge f
rom
SEN
(RA
S) s
hG
FP (
x10
0)
RelA is required for SEN(RAS) SASP markers
IL-6
IL-8
GM-CSF
The p38MAPK and RelA-dependent SASP factors overlap
The top 10 SASP factors, and their dependence on RelA, p38MAPK, and ATM
p38 inhibition
RelA knockdown
SEN(XRA)SASP
Factor RelA p38MAPK ATM
GRO + + +
IL-6 + + +
IL-8 + + +
MCP-2 + +
MCP-3 + +
GCP-2 + +
MCP-1 + +
GM-CSF + +
IGFBP-2
IL-10 +
Model: The SASP pathways
Senescence-inducing stimulus (DNA damage, oncogene expression)
p38 ATM
NF-κB
Transcription of SASP factors
Judy Campisi
Present lab membersPierre DesprezAlbert DavalosArt OrjaloChris PatilRemi-Martin LabergeChris WileyBridget GenglerMichael VelardeYing Zou
Past lab membersFrancis Rodier Jean Philipppe Coppe
Acknowledgments
Buck Institute for Age Research
Lawrence Berkeley National Laboratory