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CLARA – April 7th 2015
Role of ribosome in translational control
during tumorigenesis
Virginie MARCEL, PhD
CR2 INSERM CSS1
Team “Nuclear domains and pathologies”, Dr JJ Diaz
Team « Nuclear Domains and Pathologies »
Protein
CANCER
mRNA
Ribosome
Human ribosome
• 80 ribosomal proteins
• 4 rRNA (5S, 5.8S, 18S, 28S)
• 2 subunits
Anger et al, Nature 2013; Melnikov et al, Nat Struct Mol Biol 2012
Yeast ribosome
Therizols et al, based on Ben-Shem et al, Sciences 2011
Ribosome: a ribozyme
Human ribosome
• 80 ribosomal proteins
• 4 rRNA (5S, 5.8S, 18S, 28S)
• 2 subunits
Anger et al, Nature 2013; Melnikov et al, Nat Struct Mol Biol 2012
Yeast ribosome
Therizols et al, based on Ben-Shem et al, Sciences 2011
Ribosome: a ribozyme
PTC
Human ribosome
• 80 ribosomal proteins
• 4 rRNA (5S, 5.8S, 18S, 28S)
• 2 subunits
Anger et al, Nature 2013; Melnikov et al, Nat Struct Mol Biol 2012
Ribosome structure
• highly conserved through evolution
• ribozymes
-
decoding activity
proof-reading activity
formation of peptide-bound
Cech Science 2000 ; Melnikov et al, Nat Struct Mol Biol 2012
DC
Yeast ribosome
Therizols et al, based on Ben-Shem et al, Sciences 2011
Ribosome: complexification of structure
Ribosome core
PTC
2005 - Bactérie
Protéine
2011 - Levure
ARN
2013 - Homme
DC
Yeast ribosome
Therizols et al, based on Ben-Shem et al, Sciences 2011
Anger et al., Nature 2013
Ribosome: heterogeneity of composition
Traduction
PTC
Phénotype
Traduction
Phénotype
DC
Yeast ribosome
Therizols et al, based on Ben-Shem et al, Sciences 2011
Ribosome: heterogeneity of composition
Ribosomal proteins
PTC
P
Xue and Barna, Nat Rev Mol Cell Biol 2012
DC
Yeast ribosome
Therizols et al, based on Ben-Shem et al, Sciences 2011
Ribosome: heterogeneity of composition
Ribosomal proteins
PTC
P
Xue and Barna, Nat Rev Mol Cell Biol 2012
ribosomal RNA
DC
Yeast ribosome
Therizols et al, based on Ben-Shem et al, Sciences 2011
205205 combinations
Ribosome: rRNA modifications
C/D box snoRNA
Fibrillarin
PTC
CH3
Ψ
DC
Yeast ribosome
Therizols et al, based on Ben-Shem et al, Sciences 2011
H/ACA box snoRNA
Dyskerin
Ribosome: rRNA modifications
PTC
Fisher et al, Nat Struc Mol Biol 2015
DC
Yeast ribosome
Therizols et al, based on Ben-Shem et al, Sciences 2011
p53 inactivation alters rRNA methylation pattern
Human mammary epithelial cells
initiation
Fold Change of rRNA methylation (HMEshp53/HME)
hTERT
hTERT
shp53
6
5
4
3
2
1
0
Site
rRNA
75
627
5.8S
Marcel et al, Cancer Cell 2013
1326/28
1490
18S-DC
1703
27
1248
18S
1804
4197
4362
4426
4464
28S-PTC
4493
4506
389
1858
28S
2848
3739
28S-H69
FBL is a p53 target gene
Human mammary epithelial cells
initiation
p53
FBL mRNA expression level
• in response to p53 inactivation
2.0
**
1.6
1.2
0.8
0.4
0.0
HME
HMLE
(SV40 T/t antigens)
p53
FBL
β- Actin
Mdm2
FBL gene
hTERT
shp53
• Luciferase assays
p53RE-1
p53
Exon
2
p53RE-2
• A series of 80 breast cancer tumours
Relative FBL mRNA expression level
hTERT
Exon
1
24 P = 0.0006
20
16
12
8
4
0
WTp53
(n=59)
MTp53
(n=21)
p21
Retrospective study, Miller et al, PNAS 2005
P < 10-4 (n=251)
p53 inactivation alters translational initiation of IGF-1R
• Bi-cistronic luciferase assays
• Polysome fractionment
HME-shp53
HME
CAP
CMVp
Renilla
IRES
OD (260nm)
0,400
Firefly
OD (260nm)
0,600
polysomes
0,360
0,320
0,280
polysomes
0,500
0,400
0,300
0,240
0
1.6
1.2
Scramble
*
siRNA-FBL
0.8
0.4
0.0
HME
Fractions
0,200
HME-shp53
10
20
30
12,0
10,0
8,0
6,0
4,0
2,0
c-myc, FGF1, FGF2, VEGF, EMCV
0,0
HME
HME-shp53
• Endogenous expression of IGF-IR
mRNA
HME
β- Actin
HMLE
IGF-IR expression level
Precursor protein
IGF-IR precursor
0
***
14,0
(polysomal fraction/total
cytosolic fraction)
**
**
IGF1R mRNA distribution
IGF-IR IRES activity (Fluc/Rluc)
0,200
2.0
*
ns
HME
HMLE
Fractions
10
20
30
Clinical significance in breast cancer
• In a series of 80 primary breast tumours
• Relapse-free survival
• Breast cancer-specific survival
Low FBL
High FBL
Breast cancer-specific survival
Relapse-free survival
Low FBL
High FBL
Log Rank Test, P = 0.034
Log-Rank Test, P = 0.008
Time from surgery (days)
• Multivariate analyses: HR=4.35; CI95%[1.16-16.32]
Time from surgery (days)
Identification of “cancer specific ribosomes”
Cytoplasm
Nucleus
Nucleolus
p53
STOP
rDNA
Transcription
Fidelity
RNA pol I
FBL
rRNA
Maturation
mRNA
Translation
AAAA
CAP
AAAA
CAP
Initiation
IRES
40S
AAAA
60S
Subset of pro-tumor proteins
(IGF-1R, c-myc, VEGFA, VEGFB, FGF1, FGF2)
Marcel et al, Cancer Cell 2013
Marcel et al, Oncotarget 2013
Marcel et al, Med Sci 2014
Marcel et al, Mol Cell Biol 2015
Marcel et al, Oncogene 2015
Evolution of ribosome concepts
0
2000
1774 : Discovery of the Nucleolus
1896 : 1st publication on Nucleolus & Cancer
1960
Ribosome
"Translation nano-machine"
Ribosome
“Ribozyme”
Yoon – Science. 2006
Whitaker - Cancer Cell. 2010
Kondrashov - Cell 2011
Xue - Nat. Rev. Mol. Cell. Biol. 2012
Xue – Nature. 2014
The Regulatory
ribosome
Diaz – Nature 1996
Catez – Mol Cell Biol 2001
Belin - PLoS One 2009
Marcel- Cancer Cell 2013
1980
2000
2009 Nobel - Structure
Yonath A.
Steitz T.
Ramakrishnan V.
2009 : Ribosome profiling – Ingolia N. - Science 2009
2015
Ribosomes
(PubMed 1960 - 2015)
The Therapeutic Target
ribosome
Bywater - Cancer Cell. 2012
Peltonen - Cancer Cell. 2014
Marcel – Oncotarget. 2013
Future directions
Signaling
Repertoire
Functional
Consequences
Clinical significance
snoRNA
mRNA
AAAA
Fibrillarin
Ribosome
Translation
Cancer
Biogenesis
 Validation of ribosome as targets for anti-cancer therapies
Acknowledgements
“Nuclear domains and Pathologies” Team
Fundings
Jean-Jacques DIAZ
Jean-Christophe SAURIN
Frédéric CATEZ
Hichem MERTANI
Jenny ERALES
Zeina BASH IMAM
Florian LAFORETS
Nathalie PION
Former members
Sandra GHAYAD
Stéphane BELIN
Gabriel THERIZOLS
Yasmine TAFER
Sabine HACOT
Marie-Alexandra ALBARET
Collaborators
Alain Puisieux, Anne-Pierre Morel, Charles Dumontet, Julie Vendrell (CRCL, Lyon)
Anne-Catherine Prats, Eduardo Solano-Gonzàlez (IMMC, Toulouse)
Philippe Bouvet, Rong Cong (ENS, Lyon)
Jean-Christophe Bourdon, Lee Jordan, Alastair Thompson (University of Dundee, UK)
SAVE THE DATE
CALL FOR ABSTRACTS
TRANSLATIONAL CONTROL
From Basics…
…to Cancer
April 23-24th 2015, Montpellier
SCIENTIFIC SESSIONS
1 - The Translation Machinery: Biogenesis, Structure…
2 - Multifaceted Regulation of Gene Expression
3 - The Cancerous Translation Apparatus
SPEAKERS
Davide Ruggero
UCSF, CA
Edouard Bertrand, IGMM, Montpellier
Eric Chevet, Inserm U1053, Bordeaux
Marat Yusupov
Michel Cohen-Tannoudji,
IGBMC, Illkirch
Robin Fahraeus, UMR940, Paris
Matthias Hentze
EMBL, Heidelberg
Pasteur Institute, Paris
Sébastien Fribourg, IECB, Bordeaux
Pierre-Emmanuel Gleizes, LBME, Toulouse
Olivier Namy, IGM, Orsay
Shu-Bing Qian
Martin Teichman, IECB, Bordeaux
Cornell U., NY
Stephan Vagner, Curie Institute, Paris
ORGANIZERS
Alexandre David, IGF, Montpellier
Venue
Jean-Jacques Diaz, CRCL, Lyon
Stéphane Pyronnet, CRCT, Toulouse
CALL FOR ABSTRACTS (ORAL AND POSTERS) : DEADLINE FEBRUARY 8, 2015
Registration - free but mandatory, limited seats- at http://www.cancerogso-colloques.org/Translation