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

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days after irradiation

PRE

SEN(XRA)

Growth Curve

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PRE SEN(XRA)

Fold

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m P

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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

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- - p38 inhibitor

PRE SEN(XRA)

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Secreted protein levels - ELISA

IL-6

IL-8

GM-CSF

p38MAPK activity is required for expression of most SASP factors

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1 2 1 2

shGFP shp38α shGFP shp38α

Avg

nu

mb

er o

f ce

lls p

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ield

Invasion of MDA-MB-231 cells

0123456789

1 2 1 2

shGFP shp38α shGFP shp38α

PRE SEN(XRA)

Fold

ch

ange

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m P

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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

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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

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Vector MKK6EE SEN(RAS) SEN(XRA) SEN(REP)

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f ce

lls w

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≥3

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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

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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

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IL6 IL8 GMCSFFo

ld c

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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

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PRE 2 hr 4 hr 8 hr 24 hr 48 hr 6 day 8 day 10 day

Fold

ch

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m P

RE

NF-kB DNA binding activity

Time after XRA:

RelA = most common NF-kB subunit

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- 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

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- +SB - +SB

PRE SEN(XRA)

Fold

ch

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m P

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NF-κB transcriptional activity

ATM

p38-P

p38

Tubulin

PRE SEN(XRA)shATM: - -+ +

Genotoxic stress

p38 ATM

NF-κB

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shGFP shATM 11 shATM 12Fold

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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

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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