Giovanni Blandino: geni e tumori

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  • 1. TP53 Somatic Mutations are Frequent in Human Cancer 0 5 10 15 20 25 30 40 45 50 Cervix Thyro ï de Testis Bone Kidney Hematol. Prostate Soft tissue Uterus Breast Liver Brain Stomach Bladder Skin Lung Head&Neck Pancreas Colorectum Ovary Esophagus Percent of Tumors with a TP53 Mutation
  • 2. p53 status and DNA damage p53 abnormalities in human cancer p53 wild type Bax p21 p53AIP1 MDM2 14-3-3  p63 p73 p53 growth arrest apoptosis p53 null p63 p73 growth arrest apoptosis X mutant p53 X p63 mp53 p73 mp53 growth arrest apoptosis X X DNA DAMAGE Bax p21 p53AIP1 MDM2 14-3-3  Bax p21 p53AIP1 MDM2 14-3-3 
  • 3. mp53 History of the protein complex of mp53/p73 72R p73 72R p63 Di Como et al., 1999 Strano et al., 2000 Gaiddon et al., 2001 Bensaad et al., 2003 p63 p73 p73 mp53 p63 mp53 p73 p63 p53 Bax p53Aip1 Mdm2 p21 14-3-3 σ DBD Domain DBD Domain mp53 Bax p53Aip1 Mdm2 p21 14-3-3 σ Marin et al., 2000 Bergamaschi et al., 2003 Irwin et al., 2003 Lang et al., 2004 Olive et al., 2004 Strano et al. 2002 mp53 mp53
  • 4.  
  • 5. NF-Y CyclinA CyclinB Cdk1 Cdc25c X Bax p21 p63 p73 p53AIP1 MDM2 14-3-3  mp53 Gain of function activity mp53 p300 DNA Damage Protein-protein interaction Transcriptional competent complexes CCAATbox mp53
  • 6. Group 1 – 144 probesets Expression level increases with CDDP concentration. For CDDP 10, expression levels are lower with Pon.
  • 7. Group 1 – 144 probesets Expression level increases with CDDP concentration. For CDDP 10, expression levels are lower with Pon.
  • 8. Group 4 – 14 probesets Expression level increases with CDDP concentration. Expression levels are higher upon Pon addition.
  • 9. Group 4 – 14 probesets Expression level increases with CDDP concentration. Expression levels are higher upon Pon addition.
  • 10. ID4 mRNA is specifically induced by mutant p53R175H Pon Upregulated genes 38 probesets probesets 5 10 15 20 25 30 35 - - - - - + + + + -1.5 -1 -0.5 0 0.5 1 1.5 2 -2 Relative ID4 mRNA Pon CDDP (  g/ml) 24h H1299-p53His175 H1299-p53wt H1299-pIND - + - + - + - + - + - + - + - + - + - - 5 10 5 10 - - 5 10 5 10 - - 5 10 5 10 0,2 0,4 0,6 0,8 1,0 1,2 1,4 0 ID4 ID4 mRNA 80S polysomes Pon- Pon+
  • 11. BRCA1 ID-4 BRCA1 promoter Feedback loop Anchorage-independent cell growth ID4 & development ID-4 –/– mice: Neural proliferation defects and premature differentiation (regulation of neural stem cell proliferation and differentiation ) ID4 & cancer In mammary and ovarian tumors: ID4 overexp. in rat mammary carcinoma, seminoma and B-cell leukemia ID4 promoter hypermethylation In gastric and colorectal cancer ID-4 ID proteins are involved in the control of cell proliferation, differentiation, transformation and angiogenesis
  • 12. ID4 promoter is bound by mutant p53R175H http://www.genomatix.de/cgi-bin/matinspector TATA E-box CCAAT NF-kB Sp1 Sp1 Sp1 CDE CDE ZF5 ZF5 MAZ A B C D -850 +300 mp53 mp53 SKBR3: p53His175 p300 PCAF PCAF Chromatin sheared to 200-400 bp lenght MCF7: p53wt mp53 E2F1 mp53 NFKB No Ab p53 p65 p53/ No Ab p53 / p65 p53 / E2F1 E2F1 Neg. control p300 PCAF p53 / No Ab p53 / p300 p53 / PCAF ReChIP A B C D ID4 promoter ChIP p53 Input A B C D ID4 promoter No Ab p21 promoter
  • 13. SKBR3 ChIP anti-p53 Ab1 No Ab p53 Ab1 A B C D Rel. Enrich. No Ab Ab E2F Same results in MDA MB 231 cells anti-p53 Ab2 B C D Rel. Enrich. No Ab p53 Ab2 A p65 No Ab A B C D Rel. Enrich. anti-p65 anti-p300 p300 No Ab A B C D Rel. Enrich. anti-E2F1 E2F1 No Ab A B C D Rel. Enrich. anti-E2F1 Rel. Enrich. B C D A p53/E2F1 p53/No Ab anti-p300 Rel. Enrich. B C D A p53/p300 p53/No Ab anti-p53/No Ab anti-p53/Ab anti-p65 Rel. Enrich. B C D A p53/p65 p53/No Ab SKBR3 re-ChIP p65 mp53 E2F-1 mp53 p300 E2F-1 mp53 p300
  • 14. Endothelial Cell Migration: Chemoattractant C.M. H1299#41 (Pon-/+ 48h) +361 Mutant p53 induces neoangiogenesis Pon- Pon+ neg Endothelial cell migration Pon - Pon + +143 +121 Pon - + - + - + VgRXR 36h Cl.41 36h Cl.41 24h Endothelial Cell Prol. Conditioned medium from clone 41 Endothelial cell proliferation Matrigel experiments 0.000 0.600 1.200 1.800 2.400 3.000 C- C- (pon+) Pon- Pon + 0,000 0,200 0,400 0,600 0,800 0,0 0,6 1,2 1,8 Hb content (OD/g of matrigel) Pos Neg Pon- Pon+
  • 15. Ribonucleoprotein Immunoprecipitation (RIP) Crosslinking of Cells Immunoprecipitation of RNA-binding proteins RIP-RNA Input-RNA Reverse-crosslinking RNA extraction RT-PCR  -p53 Input  -MyoD  -ID4  -p53  -MyoD  -ID4  -p53  -MyoD  -ID4 IL8 GRO-  MDA-MB-231 MDA-MB-468 SKBR3 MDA-MB-231 MDA-MB-468 SKBR3 RPL19
  • 16. Ribonucleoprotein Immunoprecipitation (RIP) Crosslinking of Cells Immunoprecipitation of RNA-binding proteins RIP-RNA Input-RNA Reverse-crosslinking RNA extraction RT-PCR  -p53 Input  -MyoD  -ID4  -p53  -MyoD  -ID4  -p53  -MyoD  -ID4 IL8 GRO-  MDA-MB-231 MDA-MB-468 SKBR3 MDA-MB-231 MDA-MB-468 SKBR3 RPL19
  • 17. ID-4 PCAF E2F1 mp53 PCAF E2F1 mp53 p300 mp53 ID-4 PCAF ID4 3’UTR 3’UTR 3’UTR mRNA binding mRNA pro-inflammatory/pro-angiogenic cytokines Stabilization mp53 ID-4 Pro-angiogenic factors release