p73 & cancer metabolism Gerry Melino Ivano Amelio Massimiliano - - PowerPoint PPT Presentation
p73 & cancer metabolism Gerry Melino Ivano Amelio Massimiliano Agostini Maria Victoria Nicklison-Chirou Alessandro Rufini Tak Mak 22 October 2013 Disclosure Sanct Petersburg Univ. Roma CHANEL Tor Vergata BioUniverSA cell DNA p53
Tor Vergata Disclosure CHANEL BioUniverSA
Maria Victoria Nicklison-Chirou Massimiliano Agostini
Gerry Melino
Ivano Amelio Alessandro Rufini Tak Mak
22 October 2013 Sanct Petersburg
1 2 3 3’ 4 5 6 7 8 9 10 11 12 13 14
P1 P2
TAp73 DNp73 5’ 3’
TA DBD SAM 15
a b g
TAp73
25% 86% 53% 53%
N
TA DBD OD SAM TA2
C
p63 identity 23% 63% 35% p53 identity
a b g
TI
641 aa 516 aa 448 aa 471 aa 411 aa
DNp73
N C a b g
586 aa 461 aa 393 aa 416 aa 356 aa
p53 p73/p63
DNA damage cell death
seconds hours days weeks months Intrinsic Extrinsic
Transcriptional
Cell Death Cycle Arrest Infertility Cancer Spindle Assembly Complex Aneuploidy
Intracellular Systemic
Neuro- degeneration Inflammation Immunity Signaling Cytokines Neuro- development
Maternal Reproduction
seconds hours days weeks months Intrinsic Extrinsic Free Radicals Metabolic Cell Death Cycle Arrest Infertility Cancer Spindle Assembly Complex Aneuploidy
Intracellular Systemic
Senescence?? Aging?? Neuro- degeneration Inflammation Immunity Signaling Cytokines Neuro- development
Transcriptional
(1) TAp73 regulates metabolism in senescence/ aging (via Cox4i1) (2) TAp73 regulates GLS2 & serine biosynthesis
PLAN
TW Mak, Toronto
A Rufini M Agostini MV Nicklison Chirou I Amelio B Rotblat F Romeo G Viticchie’ V Landre’ RA Knight G Melino M R C Leicester, UK
Tor Vergata
program: p73 in senescence & aging
Richard Knight The Boss Max Agostini Maria Victoria Niklison Chirou Alex Rufini Ivano Amelio
p73 protein is critical for cancer progression HOW is p73 acting at molecular level?
Rufini et al. [Melino, Mak] G&D 2012
20 40 60 80 100 120 140 160 1
O2 flux (pmol/s*106cells)
Oxygen consumption
WT KO in MEF, lung, heart
Low Cox4i1 protein
(& mRNA)
Silencing TAp73 Cox4i1 is a DIRECT TARGET of TAp73
TAp73-/- show significant Neural abnormalities, Cancer development TAp73-/- show significant senescence & aging TAp73-/- MEF are more sensitive to oxidative stress and senesce TAp73-/- MEF & organs show reduced Oxygen consumption reduced ATP production reduced GSH levels high ROS production increased anion superoxide production “impaired mitochondrial respiration” TAp73 drives Cox4i1 (increasing O2 consumption, ATP, GSH, low ROS)
TAp73 (1) The End
Tor Vergata
Mitochondrial disfunction may contribute to the aging phenotype triggered by depletion of TAp73
Rufini et al. [Melino, Mak] G&D 2012
seconds hours days weeks months Intrinsic Extrinsic Transcriptional Cell Death Cycle Arrest Cancer Spindle Assembly Complex Aneuploidy
Intracellular Systemic
Neuro- degeneration Inflammation Immunity Signaling Cytokines Neuro- development Infertility
Free Radicals
Metabolic targets
Senescence Aging metabolism
Cox4i1
(DePinho; Nat Rev MCB 2012)
The TAp73-Cox4i pathway of ageing
(Melino & Mak 2013)
TAp73 Cox4i
Gong Nature 1999 ITCH Rossi EmboJ 2005 Serine
The Unified Theory of ageing
(1) TAp73 regulates metabolism in senescence/ aging (via Cox4i1) (2) TAp73 regulates GLS2 & serine biosynthesis
PLAN
TW Mak, Toronto
I Amelio M Agostini MV Nicklison Chirou B Rotblat F Romeo G Viticchie’ V Landre’ RA Knight G Melino M R C Leicester, UK
Tor Vergata
part 2: p73 regulates GLS & Serine biosynthesis
Richard Knight The Boss Max Agostini Maria Victoria Niklison Chirou Alex Rufini Ivano Amelio
p73 protein is critical for cancer progression HOW is p73 acting at molecular level?
is TAp73 regulating METABOLISM ? is this occuring in CANCER?
Pathway
Positive Enrichment Negative Enrichment TAp73 ΔNp73 TAp73 ΔNp73
Glycolysis
not sign
1,73E-006 1,39E-002
not sign
Electron transport chain
not sign
5,63E-004 2,91E-002
not sign
SERINE biosynthesis
not sign
3.65E-003 0.01970142
not sign
DNA packaging
not sign
3,07E-006 1.77E-002
not sign
Chromosome segregation
not sign
1,36E-005 3,56E-002
not sign
the SERINE METABOLISM
Survival Probability
Time (Years) Time (Years) Time (Years)
Lung cancer (GSE311210) Breast cancer (Van’t Veer et al)
b
Breast cancer (GSE3494)
Time (Months) Survival Probability
Prostate cancer (GSE25136)
HIGH Serine Enzymes LOW Serine Enzymes INTERMEDIATE
a
Glucose 3-phospho glycerate Lactate Phospho hydroxupyruvate p-Serine Serine Glutamate α-Ketoglutarate NAD+ NADPH
PHOPSHO GLYCERATE DEYDROGENASES (PHGDH) AMINO TRASNFERASE (PSAT1) p-SERINE PHOSPHATASE (PSPH)
PEP Pyruvate
PYRUVATE KINASE M2 (PKM2)
Glutamatine TCA Cycle
Serine'biosythesis' Glycolysis' Glutamine'Anaplerosis'
Serine
Glycine
DNA RNA GSH Protein
SERINE HYDROXYMETIL- TRASFERATSE (SHMT)
GLUTAMINASE-2 (GLS-2)
TAp73 induces SERINE METABOLISM
Serine level Glycine level Glutammate level α-Ketoglutarate level Succynil-CoA level GSH level
0 8 (h) Doxy 0 8 (h) Doxy 0 8 (h) Doxy 0 8 (h) Doxy 0 8 (h) Doxy 0 8 (h) Doxy
p<0,05 p<0,05 p<0,05 0,05>p>0,01 0,05>p>0,01
0.0 0.5 1.0 1.5 2.0 2.5
mRNA Levels
PHGDH PSAT-1 PSPH
0 4 8 12 24 0 4 8 12 24 0 4 8 12 24 (h) Doxy
b
PHGDH PSAT-1 PSPH 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
Luciferase Activity pcDNA TAp73 DNp73
c
a
Human PSPH Promoter
Score 0.7
β Δ α α
b
Human PSAT-1 Promoter
a
Score 0.8
β α
c
Δ Δ
c
α β Human PHGDH Promoter
Score 0.9
+1 +1 +1
a
SaOs Tet-On TAp73α
TAp73 induces SERINE METABOLISM
0.0 0.2 0.4 0.6 0.8 1.0 1.2
ATF-4 mRNA level siCTR siTAp73
d
ATF-4
not via PHGDH, PSAT-1, PSPH
TAp73 PHGDH PSAT PSPH 0.0 0.2 0.4 0.6 0.8 1.0 1.2
mRNA levels siCTR siTAp73
e
p 0.01 p 0.01 ns
Human GLS-2 Promoter
Score 0.92
a
CAAGT
2 4 6 8
GLS-2 mRNA level pcDNA TAp73
H1299
p 0.001
b
1 2 3 4
GLS-2 mRNA level
p 0.001
24 (h) Doxy
SaOs Tet-On TAp73α
c
pcDNA TAp73alfa 0.0 0.5 1.0 1.5 2.0 2.5 3.0
Luciferase Activity 1 2 3 4 5 6
Promoter Binding Enrichment
d
p 0.001
p 0.001
e
p 0.001 GLS-2 Promoter MDM2 Promoter
Anti IgG Anti HA Input
TAp73 induces SERINE METABOLISM via GLS2
Complete
10 20 30 40 50
BrdU Incorporation (%) siCTR siTAp73
a
p 0.01 p 0.01
siCTR siTAp73 siCTR siTAp73 20 40 60 80 100 120
Cell Cycle Profile (%) G1 S G2
Complete
b
20 40 60 80 100 120 140 20 40 60 80 100 120
Time Percent survival Lung cancer (GSE311210)
p73/GLS-2 Interaction p73/GLS-2 NO interaction
2 4 6 8 6 8 10 12 14
TAp73 GLS-2
R=0.4
c d
p73/GLS-2 Interaction p73/GLS-2 NO Interaction
TAp73 induces SERINE METABOLISM via GLS2
glucose
TAp73
glycerate-3
phospho-enol
pyruvate lactate PSPH PKM2 PHGDH PSAT-1 3-phospho
phospho
SERINE
glycine nucleic acids, aminoacids lipids, ATP, (PROLIFERATION) Gln-, Ser - (aminoacid deprivation) ATF4 GCN2 GLS2 α-ketoglutarate TCA cycle ROS glutamate GSH glutamine
TAp73 induces SERINE METABOLISM via GLS2
TP7 3
TP53 TP63
TAp73 DNp73
ROLE OF 14 isoforms? Interfamily INTERACTIONS? OPEN QUESTIONS
(with Tak Mak) Tomasini G&D 2008 Tomasini PNAS 2009 Wilhelm G&D 2010 Agostini PNAS 2011 Agostini PNAS 2011 b Melino CDD 2011 Levine Nat Rev MCB 2011 Rivetti PNAS 2012 Rufini G&D 2012 Terrinoni PNAS 2013
Overall CONCLUSION
p73-deficient mice show metabolic defects These are at least in part mediated by:
..… contributing to senescence & aging The End
Thanks You !! for your kind attention
Wittgenstein