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

Blood vessel

necrosis

Colon tumor

+ O2 - O2

HIF

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

PC3 cells!H 1% - 24h!

1 µm!G. Bellot et al. MCB 2009

PC3

H 1% - 72 h

5 µm

PC3 Hyp. 1% - 24h

5 µm

PC3 Hyp. 1% - 48h

5 µm

5 µm

LS174 Hyp. 1% - 24h

5 µm

LS174 Hyp. 1% - 48h

5 µm

LS174 Hyp. 1% - 72h

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

2 µm

2 µm

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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A positive regulatory loop between HIF and miR-210

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

Mitochondria project : MC Brahimi-Horn J Chiche N Mazure

J Pouysségur

M Rouleau (FACs analysis)

P Gounon (Electron Microscopy)

Dr J. C. Martinou (Fusion - Suisse)

miR-210 project : MP Puisségur T Bertero S Grosso B Mari