Roma 26-27 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
Small G-proteins or small GTPases GTP-protein GDP-protein on off GEFs Regulatory proteins: GAPs
Guanine nucleotide exchange factors (GEFs) GDP -GTP exchange Activation!!!
GTPase activating proteins (GAPs) Increase GTPase activity it Inactivation!!
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
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
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
Onc genes erb-a erb-b b ros ras abl
Comincia l'era della Oncologia Molecolare
The Ras Family
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
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
How is the big killer activated Ras mutations inhibit the GTPase activity blocking the protein ti in its active conformation
The search of achille's heel of Ras
GTP ANALOGUES Direct inhibition only produced molecules with low binding affinity
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
Engagement g of effector proteins: The GTPase cascade RAF
A th t ti i th GTP Are there mutations in the GTPase cascade?
Hereditary neurofibromatosis Caused by NF1 mutations NF1 is a GTPase activating protein (GAP)
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
GTPase activating proteins (GAPs) accelerate the small GTPase hydrolysis INACTIVATION!! Mutated TSC1/TSC2 RHEB ACTIVATION!!!
The AKT/mTOR Pathway Rheb GTPase
Rheb is amplified in prostate cancer
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;67:8089-8094 2007 by American Association for Cancer Research
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
Ras effectors inhibitors (synergistic activity has been seen)
Summary of possible anti-ras approaches