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 p73/p63 death damage D Np73 TAp73 P2 P1 3 ’ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 a 3’ 5’  b g  TA DBD SAM p63 identity 25% 86% 53% 53% p53 identity 23% 63% 35% a OD TA2 SAM TI TA DBD TAp73 C N 641 aa b 516 aa g 448 aa  471 aa  411 aa a D Np73 N C 586 aa b 461 aa  393 aa g 416 aa  356 aa

Intrinsic Extrinsic Intracellular Systemic Inflammation Signaling Immunity Cytokines Infertility p73 Maternal Cell Reproduction Death Aneuploidy Transcriptional Spindle Cancer Assembly Complex Cycle Neuro- Neuro- Arrest degeneration development days weeks seconds hours months

Intrinsic Extrinsic Intracellular Systemic Inflammation Signaling Immunity Infertility Cytokines p73 Cell Death Aneuploidy Transcriptional Spindle Cancer Assembly Free Complex Radicals Cycle Neuro- Neuro- Arrest degeneration development Metabolic Senescence?? Aging?? days weeks seconds hours months

program: p73 in senescence & aging p73 protein is critical for cancer progression HOW is p73 acting at molecular level? PLAN A Rufini (1) TAp73 regulates metabolism in senescence/ M Agostini aging (via Cox4i1) MV Nicklison Chirou I Amelio (2) TAp73 regulates GLS2 & serine biosynthesis B Rotblat F Romeo G Viticchie ’ V Landre’ RA Knight Alex Rufini Max Agostini Ivano Amelio G Melino M R C Leicester, UK Univ. ROMA TW Mak, Toronto Maria Victoria Tor Vergata The Boss Niklison Chirou Richard Knight

Premature Ageing in TAp73-/- mice Low Cox4i1 protein (& mRNA) Oxygen consumption 160 140 O 2 flux (pmol/s*10 6 cells) 120 100 80 60 40 WT KO 20 0 1 in MEF, lung, heart Silencing TAp73 Cox4i1 is a DIRECT TARGET of TAp73 Rufini et al. [Melino, Mak] G&D 2012

TAp73 (1) The End 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) Rufini et al. [Melino, Mak] G&D 2012 Mitochondrial disfunction may contribute to the Univ. Roma aging phenotype triggered by depletion of TAp73 Tor Vergata

Intrinsic Extrinsic Intracellular Systemic Inflammation Signaling Immunity Cytokines Cell Death Infertility p73 Spindle Transcriptional Assembly Aneuploidy Complex Cox4i1 Cancer Cycle Metabolic Arrest targets Neuro- Neuro- development Free degeneration Radicals metabolism Aging Senescence days weeks seconds hours months

The Unified Theory of ageing The TAp73-Cox4i pathway of ageing Gong Nature 1999 Rossi EmboJ 2005 ITCH TAp73 Serine Cox4i (DePinho; Nat Rev MCB 2012) (Melino & Mak 2013)

part 2: p73 regulates GLS & Serine biosynthesis p73 protein is critical for cancer progression HOW is p73 acting at molecular level? PLAN I Amelio (1) TAp73 regulates metabolism in senescence/ M Agostini aging (via Cox4i1) MV Nicklison Chirou B Rotblat (2) TAp73 regulates GLS2 & serine biosynthesis F Romeo G Viticchie ’ V Landre’ RA Knight G Melino Alex Rufini Max Agostini Ivano Amelio M R C Leicester, UK Univ. ROMA TW Mak, Toronto Maria Victoria Tor Vergata The Boss Niklison Chirou Richard Knight

is TAp73 regulating METABOLISM ? is this occuring in CANCER? Positive Enrichment Negative Enrichment Pathway ΔNp73 ΔNp73 TAp73 TAp73 1,73E-006 1,39E-002 Glycolysis not sign not sign Electron transport 5,63E-004 2,91E-002 chain not sign not sign 3.65E-003 0.01970142 not sign not sign SERINE biosynthesis 3,07E-006 1.77E-002 DNA packaging not sign not sign Chromosome 1,36E-005 3,56E-002 not sign not sign segregation

Glycolysis' a Glucose the Serine'biosythesis' SERINE HYDROXYMETIL- p-SERINE PHOPSHO GLYCERATE SERINE TRASFERATSE PHOSPHATASE DEYDROGENASES (SHMT) 3-phospho (PSPH) (PHGDH) Phospho Glycine p-Serine METABOLISM Serine glycerate hydroxupyruvate AMINO NAD + TRASNFERASE NADPH (PSAT1) PEP PYRUVATE α -Ketoglutarate KINASE M2 Glutamate (PKM2) DNA Protein GSH Pyruvate RNA GLUTAMINASE-2 (GLS-2) Glutamatine TCA Lactate Serine Cycle Glutamine'Anaplerosis' Breast cancer ( Van’t Veer et al) Breast cancer (GSE3494) b Lung cancer (GSE311210) Prostate cancer (GSE25136) Survival Probability Survival Probability Time (Years) Time (Months) Time (Years) Time (Years) HIGH Serine Enzymes INTERMEDIATE LOW Serine Enzymes

TAp73 induces SERINE METABOLISM Serine level Glycine level Glutammate level p<0,05 p<0,05 p<0,05 0 8 (h) Doxy 0 8 (h) Doxy 0 8 (h) Doxy 0,05>p>0,01 0,05>p>0,01 0 8 (h) Doxy 0 8 (h) Doxy 0 8 (h) Doxy α -Ketoglutarate level Succynil-CoA level GSH level

+1 Human PHGDH Promoter a Score 0.9 TAp73 induces +1 SERINE a Human PSAT-1 Promoter c METABOLISM Score 0.8 -1288 -596 -592 +1 a b c not via PHGDH, PSAT-1, PSPH Human PSPH Promoter α Score 0.7 pcDNA -1119 1.4 -1123 b 2.5 TAp73 SaOs 1.2 Luciferase Activity PHGDH c Tet- On TAp73α DNp73 α 2.0 1.0 PSAT-1 mRNA Levels PSPH 0.8 1.5 α β 0.6 1.0 Δ 0.4 β 0.2 0.5 0.0 Δ 0.0 β PHGDH PSAT-1 PSPH 0 4 8 12 24 0 4 8 12 24 0 4 8 12 24 (h) Doxy 1.2 e ns siCTR 1.2 Δ 1.0 d siTAp73 mRNA levels siCTR 1.0 ATF-4 mRNA level 0.8 siTAp73 p 0.01 0.8 0.6 p 0.01 0.6 0.4 ATF-4 0.4 0.2 0.2 0.0 TAp73 PHGDH PSAT PSPH 0.0 α

TAp73 induces a Human GLS-2 Promoter SERINE Score 0.92 -1240 METABOLISM -792 CAAGT -787 via GLS2 c d SaOs Tet-On b H1299 4 GLS-2 Promoter 8 TAp73α p 0.001 6 e MDM2 Promoter p 0.001 p 0.001 3.0 GLS-2 mRNA level GLS-2 mRNA level Promoter Binding 5 3 6 Luciferase Activity p 0.001 2.5 Enrichment p 0.001 4 2.0 2 4 3 1.5 1 2 1.0 2 0.5 1 0 0 0.0 24 pcDNA TAp73 0 0 pcDNA TAp73alfa (h) Doxy Anti IgG Anti HA Input

TAp73 induces b a 120 SERINE p 0.01 50 G2 p 0.01 Cell Cycle Profile (%) 100 BrdU Incorporation (%) siCTR S METABOLISM 40 siTAp73 80 G1 30 via GLS2 60 20 40 20 10 0 0 siCTR siTAp73 siCTR siTAp73 Complete - Ser & Gly Complete - Ser & Gly p73/GLS-2 Interaction d c Lung cancer (GSE311210) 14 120 100 12 Percent survival GLS-2 80 10 60 8 40 p73/GLS-2 Interaction R=0.4 p73/GLS-2 NO Interaction 20 p73/GLS-2 NO interaction 6 0 -2 0 2 4 6 8 0 20 40 60 80 100 120 140 TAp73 Time

TAp73 induces SERINE Gln - , Ser - METABOLISM ( aminoacid deprivation ) via GLS2 GCN2 glucose ATF4 PHGDH PSAT-1 PSPH glycerate-3 phospho 3-phospho glycine SERINE -phosphate -serine -hydroxypyruvate α -ketoglutarate glutamate GSH GLS2 phospho-enol nucleic acids, -pyruvate aminoacids glutamine lipids, ATP, PKM2 ( PROLIFERATION ) TCA pyruvate cycle TAp73 lactate ROS

Overall CONCLUSION The End D Np73 TAp73 OPEN TP63 TP53 TP7 QUESTIONS 3 Interfamily ROLE OF INTERACTIONS? 14 isoforms? p73-deficient mice show metabolic defects These are at least in part mediated by: (with Tak Mak) Tomasini G&D 2008 - Cox4i1 Tomasini PNAS 2009 - GLS2 Wilhelm G&D 2010 Agostini PNAS 2011 Agostini PNAS 2011 b - (Serine pathway) Melino CDD 2011 Levine Nat Rev MCB 2011 Rivetti PNAS 2012 ..… contributing to senescence & aging Rufini G&D 2012 Terrinoni PNAS 2013

Thanks You !! for your kind attention on things we are not able to speak, we should be silent Wittgenstein