Mitochondries, Hypoxie et Cancer - reseau...
Transcript of Mitochondries, Hypoxie et Cancer - reseau...
CNRS - UMR 6543 Institute of Signaling, Developmental Biology and Cancer Research University of Nice-Sophia Antipolis - FRANCE
Mitochondries, Hypoxie et Cancer
Blood vessel
necrosis
Colon tumor
+ O2 - O2
HIF Blood vessel
+ O2
HIF igfbp1, 2, 3, p21… Cellular proliferation
and Viability
ca9, ca12, mct4 … pH
vegf, vegfR, endo, nos2, ho1, adrenomedullin, krt, mmp2, cathd… Vascular development and
vasomotor tone
vegf, vegfR, endo, nos2, ho1, adrenomedullin, krt, mmp2, cathd…Vascular development and
cerulop, epo, transf, transf R… Erythropoiesis
and iron metabolism
glut-1, ald, pgk-1… Glucose/energy metabolism
pH Bnip3, bnip3L …
Autophagy and Survival
0 1 2 3 4 5
N 24 h 48 h 72 h
Are
a (µ
m2)
H 1%
LS174 PC3 * **
LS174 - H 1% - 72 h
2 µm 2 µm
Higher magnification
Cyto. c
N
PC3 LS174
H 1
%
PC3
LS174
H 1 % (72 h)
- Tet
LS-sh hif-1α
HIF-1α Cyto. c merge +
Tet
HIF-1α Cyto. c
Formation of enlarged mitochondria in hypoxia is HIF-1 dependent
N (72 h)
PC3
DiOC6(3)
FL3-
H
N (72 h) + CLCCP (15 min)
10 0 10 1 10 2 10 3 10 4 10 0
10 1
10 2
10 3
10 4
45.7 % 51 %
10 0 10 1 10 2 10 3 10 4 10 0
10 1
10 2
10 3
10 4
91.5 % 7.5 %
H 1% (72 h)
10 0 10 1 10 2 10 3 10 4 10 0
10 1 1
0 2 10 3
10 4
88.4 % 8.8 %
LS174
FL1-H
FL3-
H
10 0 10 1 10 2 10 3 10 4 10 0
10 1
10 2
10 3
10 4
7.3 %
89.4 %
10 0 10 1 10 2 10 3 10 4 10 0
10 1
10 2
10 3
10 4
9.2 %
10 10 10 10 10 0 1 2 3 4 10 0
10 1
10 2
10 3
10 4
3.4 %
95.2 % 89.1 %
FL1-H
Enlarged mitochondria are functional
0 1 2 3 4
N H N H LS174 PC3
Mfn1 Mfn2 OPA1
Pro
tein
qua
ntifi
catio
n
72 h
Mfn1 96 kDa
Mfn2 96 kDa
OPA1 96 kDa
Tubulin
HIF-1α N H N H
LS174 PC3 72 h
x63
:siMfn1 (40 nM) x63
LS174 H 1 % (72 h)
+ - Mfn1 Tubulin
Mitochondrial fusion leads to enlarged mitochondria in hypoxia
LS174
siC
tl si
Mfn
1 (4
0 nM
)
Cyto. c
siM
fn1
(100
nM
) si
Mfn
1 (1
00 n
M)
Overexpression of Mfn1 mediates excessive mitochondrial fusion giving enlarged mitochondria
LS174
+ - Mfn1
Tubulin
: pMfn1 (10 µg) HeLa
+ -
N (72 h)
LS174 x63
N (72 h)
pEm
pty
Vect
or
pMfn
1
HeLa HeLa LS174 Cyto. c
LS174 - H1% - 72h
siBNIP3 siBNIP3L siCtl
Cyto. c
Cox4
Merge
BNIP3 and BNIP3L are required in the fusion process
Cells with enlarged mitochondria are protected from apoptotic stimuli
0 10 20 30 40 50
Casp
ase
3/
7 (
RLU
) x 1
03 72 h
0 50
100 150 200 250 300 350
N H 1%
24 h
- + + N H 1%
- - + - + : Stau
*
0 10 20 30 40 50 60 70
Casp
ase
3/
7 (
RLU
)x 1
03
: Etop
*
- + + N H 1%
-
72 h
: Stau siCtl! siMfn1!
72 h
H 1%
- + 0
100 200 300 400
- +
Casp
ase
3/
7 (R
LU
) x 1
03
*
siBNIP3!- +
*
This protection is reversible
H 1 % (72 h) + Stau. Stau. + 24 h reoxy.
Stau. + 48 h reoxy. Stau. + 72 h reoxy.
0 5 10 15 20
H 1 % (72 h) + 24 h + 48 h + 72 h
Apopto
tic
nucl
ei (
%)
reoxygenation
0% 20% 40% 60% 80%
100%
Dead cells Low DiOC6 (3) High DiOC6 (3)
- - + + N H 1% - 72 h
: Stau.
N - 72 h + Stau. H 1% - 72 h + Stau.
cyto. c
Cytochrome C is not released
: Stau 0
10 20 30 40 50 60
Cas
pas
e 3/7
(R
LU)
x 10
3 A549
- + - + N H 1%
SKMel
- + - + N H 1%
CCL39 786-0 HeLa LS174 A549
MCF7 CAL33 BE PC3 SKMel
SKMel A549 72 h
N
H 1%
J. Chiche et al. J. Cell. Physiol. 2009
miRNA Profiling of Non Small Cell Lung Cancer
miR-210
Dr B. Mari - CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, UMR6097, F-06560 Sophia Antipolis, France miR-210 is overexpressed in late stages of lung cancer and mediates mitochondrial alterations associated with modulation of HIF-1 activity
Marie-Pierre Puisségur1,2!, Nathalie M. Mazure2,3, Thomas Bertero1,2, Ludivine Pradelli2,4, Sébastien Grosso1,2, Karine Robbe-Sermesant1,2, Thomas Maurin1,2!, Kevin Lebrigand1,2, Bruno Cardinaud1,2!, Véronique Hofman2,4, Sandra Fourre1,2, Virginie Magnone1,2, Jean Ehrland Ricci2,5, Jacques Pouysségur2,3, Pierre Gounon2,6, Paul Hofman2,4, Pascal Barbry1,2 and Bernard Mari1,2
Soumis Cell Death and Differentiation
miRNA-210 is induced by hypoxia in Lung Cancer
In vivo: correlation between miR-210 expression and hypoxic markers in lung tumors
0
1
2
3
4
24h 48h 72h
Normoxia
In vitro: A549 cells in 1% O2 Taqman PCR
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miR-210 induces a phenotypic and functional modification of mitochondria
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Mitochondrial Membrane
Potential: JC-1 Dye
3 days 4 days
Citric Acid Cycle and Oxydative Phosphorylation:
The Electron Transport Chain (ETC)
o NDUFA4 : NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4, 9kDa. Mammalian complex I of mitochondrial respiratory chain is composed of 45 different subunits. This protein has NADH dehydrogenase activity and oxidoreductase activity. It transfers electrons from NADH to the respiratory chain. The immediate electron acceptor for the enzyme is believed to be ubiquinone.
o SDHD : Complex II of the respiratory chain, which is specifically involved in the oxidation of succinate, carries electrons from FADH to CoQ. The complex is composed of four nuclear-encoded subunits and is localized in the mitochondrial inner membrane. The subunit D protein is one of two integral membrane proteins anchoring the complex to the matrix side of the membrane. Mutations in SDHD have been linked to hereditary paraganglioma.
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Silencing SDHD mimicks miR-210 effect on mitochondria shape
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miR-210 inhibition reduces HIF activity in hypoxic A549 cells
: LNA-210 - + Normoxia Hypoxia (1 % O2)
48 h
72 h
- - - + - -
- + - - - + - -
: LNA-159s : miR-210 : miR-Neg
A
HIF-1α
Tubulin
HIF-1α
Tubulin
- + - - - + - -
- + - - - + - -
C