Dialogue calcique et Sénescence

Dialogue calcique et Sénescence
David Bernard
« Senescence Escape Mechanisms » lab Tumoral Escape Department
What is cellular senescence ?
-A stable form of cell cycle arrest
(Hayflick et al, Exp Cell Res, 1965)
young
-Morphological alterations
(Hayflick et al, Exp Cell Res,1965)
-β
β galactosidase activity at pH6 (SA b Gal)
(Dimri et al, PNAS, 1995)
-Secretory phenotype (SASP)
(Coppe et al, PLos Biol, 2008)
n
s
senescence
Senescence: a stable proliferation arrest
-where it occurs : in normal (replicative and premature) but
also in cancer cells (premature)
-how is it induced: by various stresses (short telomeres,
oncogenes,oxidative stress, genotoxic drugs…)
-how is it recognized : SAbGal activity, strongly released
cytokines (SASP), may display nuclear heterochromatin dots
(SAHF) and others markers have been proposed
Physiopathological functions of cellular senescence
Senescence
Timely:
-tumor suppressive
-embryonic development
-wound healing
…
Chronic:
-tumor promoter
-progeria/aging
-aging related deseases
…
Physiopathological functions of cellular senescence
Senescence
Timely:
-tumor suppressive
-embryonic development
-wound healing
…
Chronic:
-tumor promoter
-progeria/aging
-aging related deseases
…
Tissue and organism homeostasis
Proliferative disorder
Cancer
Degenerative disorder
Aging
Senescence
Cellular senescence promotes aging:
From old concept to recent (partial) demonstration
proliferating cells
normal human cell
in culture
non proliferating cells
Replicative senescence = replicative limit
1) Cells derived from old human have a decreased replicative limit
when compared to young ones
Cellular senescence promotes aging:
From old concept to recent (partial) demonstration
Skin
young (38) donor
old (73) donor
From Dimri et al, PNAS, 1995
2) Old tissues display more senescent cells in vivo
(allowed by the discovery of the first senescence marker
(SA-b-Gal activity))
Cellular senescence promotes aging:
From old concept to recent (partial) demonstration
From Baker et al, Nature, 2011
3) Eliminating senescent cells improve progeroid syndromes in mouse
Cellular senescence promotes aging:
From old concept to recent (partial) demonstration
Senescent cells
Limit renewal capacity of organs
(proliferation arrest)
Alter the tissue organization
and function by its secretome
(inflammatory mediators, MMP…)
Cellular senescence inhibits tumorigenesis:
From old concept to demonstration
stress
senescence
1) Cancer being a proliferative disease if cells stop to grow
they cannot form tumors
Cellular senescence inhibits tumorigenesis:
From old concept to demonstration
From Serrano et al, Cell, 1997
2) Oncogenes, which are the engine of the tumorigenesis process,
induce normal human cell senescence
Cellular senescence inhibits tumorigenesis:
From old concept to demonstration
From Collado et al, Nature, 2005
3) Senescence escape occurs in malignant tumors
Cellular senescence inhibits tumorigenesis:
From old concept to demonstration
RasV12
From Xue et al, Nature, 2007
4) Senescence induction blocks tumorigenesis process
Cellular senescence inhibits tumorigenesis:
From old concept to demonstration
Senescent cells
the proliferation is blocked
Secreted factors will activate
the immune system
Understand the mechanisms regulating cellular
senescence should allow to better understand aging
and cancer processes and might give rise to new tools
to improve healthy aging as well as to fight cancer.
Functional genetic screen to identify new
regulators of senescence
1) Infection with the whole genome
shRNA library lentivirus
3) Identification of the shRNA
2) Induction of senescence
Model of senescence
Immortalized mammary human epithelial cells (HMEC) upon
oncogenic stress
HMEC-Tert-MEK:ER (or RAF:ER)
120
4-OHT
P-ERK
PS10-H3
-
+
100
80
60
40
20
ACTB
Relative DcR2 mRNA level
Cyclin A
7
6
5
4
3
2
1
0
0
-
4-OHT
SA-β
β-Gal
Relative Dec1 mRNA level
+
+
-
+
-
+
3.5
3
Relative IL8 mRNA level
-
Relative Sprouty-2 mRNA level
4-OHT
senescence
marker
2.5
3
2.5
2
1.5
1
0.5
1
0.5
0
0
4-OHT
2
1.5
-
+
Identified Senescence Regulators
-KCNA1 (Lallet-Daher et al, Cancer Research, 2013)
-ABCC3 (Wiel et al, Oncogene, in press)
-CASP2 (Gitenay et al, Oncotarget, 2014)
-ITPR2 and MCU (Wiel et al, Nature Com, 2014)
mito
ER
-Is calcium flow an important regulators of senescence ?
-Is there a calcic dialog between ER and mitochondria
during senescence ?
-How mtCa2+ participate to senescence ?
ITPR2 knockdown allows OIS escape
ctrl
ctrl
shITPR2_2
shITPR2_3
ER
ITPR2
100
80
60
40
20
0
+
+ 4OHT
+
+
IL8 mRNA relative expression
% of SA-β
β -Gal positive cells
Senescence markers
4OHT -
25
20
15
10
5
0
+ 4OHT
MCU knockdown allows OIS escape
+
+
60
50
40
30
20
10
0
+4OHT
+
+4OHT
4
3
2
1
0
mito
MCU
+
IL8 mRNA relative epxression
-
pRS/shMCU_1pRS/shMCU_2pRS/shMCU_3
% of SA-β
β -Gal positive cells
pRS
Senescence markers
4OHT
pRS
Calcium accumulates in the mitochondria during OIS
Mitochondrial calcium genetic reporter
ITPR2
MCU
proliferating
senescent
Calcium accumulation in the mitochondria during OIS
is inhibited by ITPR2 or MCU knockdown
ITPR2
MCU
0.4
0.6
Fluorescence/area
0.5
- 4OHT
0.4
0.3
0.3
+ 4OHT
0.2
0.2
0.1
0.1
0
0.0
red
?
MCU
JC-1 Red/Green ratio
green
3.5
2.5
3.0
2.0
2.5
2.0
1.5
1.5
1.0
1.0
0.5
0.5
0.0
0.0
merge
Ratiometric JC1 probe
mCa2+ accumulation during OIS leads to mitochondrial
depolarization
ITPR2
proliferating senescent
+ 4OHT
+ 4OHT
mCa2+ accumulation during OIS leads to ROS production
ITPR2
?
100
MCU
11
80
pRS
count
pRS + 4-OHT
18
60
17
pRS/shITPR2_2 + 4OHT
40
pRS/shITPR2_3 + 4OHT
33
20
0
100
101
102
103
Fluorescence intensity
(ROS)
104
Model
ITPR2
∆ψ(M)
∆ψ
senescence
MCU ROS
Wiel et al, Nature Com, 2014
Ca2+ in the mitochondria
Normal level of
Ca2+
High level of
Ca2+
Required for
mitochondrial
activity
Induce cell
death or
senescence
To know more about the role of mitochondria in
cellular senescence
Senescence Escape Mechanism lab
Nadine Martin (CR INSERM)
David Vindrieux (MCF)
Audrey Griveau (Postdoc)
Marine Warnier (Postdoc)
Xingjie Ma (PhD student)
Mylène Ferrand (AI)
Past members:
Hélène Simonnet (MCF)
Clotilde Wiel (PhD student)
Arnaud Augert (PhD student)
Hélène Lallet-Daher (Postdoc)
Delphine Gitenay (Postdoc)
Benjamin Le Calvé (Postdoc)
collaborator
N Prevarskaya (Lille)