Ras e la Cascata delle Piccole GTPasi
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1 Roma Giugno 2015 Multidisciplinarietà e Biologia Molecolare applicate alla pratica Clinica i Oncologica: un'opportunità ed un vantaggio per tuttitti Ras e la Cascata delle Piccole GTPasi Alvaro Leone Anatomia patologica Ospedale San Camillo-Forlanini Roma Direttore: Dr.ssa Lucia Grillo
2 Small G-proteins or small GTPases GTP-protein GDP-protein on off GEFs Regulatory proteins: GAPs
3 Guanine nucleotide exchange factors (GEFs) GDP -GTP exchange Activation!!!
4 GTPase activating proteins (GAPs) Increase GTPase activity it Inactivation!!
5 Post-Translational modification (prenylation) 1. Farnesyltransferase (Ras): the covalent addition of a C15 farnesyl isoprenoid (F) lipid to the cysteine residue of the CAAX motif 2. Geranylgeranyltransferase I (Rho): the covalent addition of a C-20 geranylgeranyl isoprenoid (GG) lipid to the cysteine residue of the CAAX motif 3. Geranylgeranyltransferase II (Rab): the covalent addition of a C-20 geranylgeranyl isoprenoid (GG) lipid to the cysteine residue of the CC, CXC, CCX, CCXXX
6 GTPase functions Ras: regulators of cell proliferation and differentiation Rab, Arf, Sar: regulators of membrane trafficking (the secretory and endocytic pathways) Ran: regulators of nucleocytoplasmic transport Rho: regulators of actin cytoskeletal l organization, i which h influences cell morphology and movement Ral: vescicle trafficking and cell survival Gem/Rad: Tissue differentiation
7 RAS the big Killer Most frequent mutated oncogenes in human tumors Attempts to inhibit function failed!! In 2014 the National Cancer Institute of Bethesda (USA) launched an Anti-RAS project with a 10 million dollars yearly budget
8
9 Onc genes erb-a erb-b b ros ras abl
10 Comincia l'era della Oncologia Molecolare
11 The Ras Family
12 Not all Ras genes are created equal Differences among the ras genes in human cancer K-Ras can confer stem-cell like properties to certain cell lines Mice that lack K-ras dye during embryogenesis while the ones Lacking H-Ras or N-Ras are viable K-Ras has a stronger oncogenic activity So there may be not one single anti-ras therapy that fits all Ras-mutant cancers
13
14 Differences among K-Ras mutations G12C is a hallmark of exposure to tobacco smoke and activates the RAL-GDS pathway as the g12v mutation G12D mutation prefers the RAF/MAPK and PI3K pathway G12C and G12V mutations have a worse outcome than G12D Possibly because they engage different downstream effectors
15 How is the big killer activated Ras mutations inhibit the GTPase activity blocking the protein ti in its active conformation
16 The search of achille's heel of Ras
17 GTP ANALOGUES Direct inhibition only produced molecules with low binding affinity
18 Farnesyl-Transferase inhibitors (FTI) Post-Translational modifications that regulate Ras membrane association K-Ras and N-RAS genes bypass FTI by Geranylgeranyl transferase activation!! All the experiments had been performed All the experiments had been performed using H-Ras that does not show GGTase activity
19 Engagement g of effector proteins: The GTPase cascade RAF
20 A th t ti i th GTP Are there mutations in the GTPase cascade?
21 Hereditary neurofibromatosis Caused by NF1 mutations NF1 is a GTPase activating protein (GAP)
22 Tuberous sclerosis TSC is characterized by benign tumors (hamartomas) in different organs It's caused by TSC1 and TSC2 inactivating mutations The TSC1/TSC2 complexis a GTPase activating Protein) Inhibits RHEB small GTPase Mutations cause the loss of TSC1/TSC2 GTPase activity Therefore inducing i Rheb constitutive activation
23 GTPase activating proteins (GAPs) accelerate the small GTPase hydrolysis INACTIVATION!! Mutated TSC1/TSC2 RHEB ACTIVATION!!!
24 The AKT/mTOR Pathway Rheb GTPase
25 Rheb is amplified in prostate cancer
26 Rac1 is necessary for K-Ras induced lung transformation Purple Kras G12D plus Rac1 Light blue Kras G12D Rac1 loss of function. Kissil J L et al. Cancer Res 2007;67: by American Association for Cancer Research
27 RalA inhibition slows down tumor growth Ras-G12D Transformed IN VIVO IN VITRO Suggests inhibition of Metastatic potential Or Ras-G12D/RalA-siRNA in mice
28 Ras effectors inhibitors (synergistic activity has been seen)
29
30
31 Summary of possible anti-ras approaches
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