Caracterización de la activación del oncogén K-ras V12 endógeno in vivo mediante una aproximación genética. Validación de Farnesiltransferasa en la terapia antitumoral in vivo mediante una aproximación genética

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 26-04-2005We have targeted a K-ras allele in mouse embryonic stem (ES) cells to express a KRasVl2 oncoprotein along with a marker protein (p-geo) from a single bicistronic transcript. Expression of this oncogenic allele requires removal of a knocked-in STOP transcriptional cassette by Cre recombinase. This mouse model will establish the experimental bases to study the wnsequences of K-ras activation within a physiological context. In vivo, most K-rasV12 oncogene expressing cells remain normal for long periods of time (up to eight months). Only, lung bmnchiolo-alveolar cells acquire hyper-proliferative propetties and progress into malignant adenocarcinomas. Cdk4 R24C mutation, which makes insensible to the 1NK4 inhibitor family members, cooperates with K-rasV12 mutation leading to the development of PanlN 1A metaplasias in pancreatic ductal cells and hyperplasias in pituitary cells. The K-rasVl2 driven adenocarcinomas acquire a spreading phenotype and thus develop metastads in the adjacent lymphoid nodules when wmbined with a p53 null background. In primary mouse embryonic fibroblasls (MEFs) activation of this K-rasVI2 onwgene at physiological level does not induce senescence due to oncogenic stress. Wñat is more. MEFs expressing this K-rasV12 allele proliferate as immortal cells and escape replicative senescence. Farnesyltransferase (FTase) is a heterodimeric enzyme responsible for post-translational modification of proteins carrying a carboxy-terminal CAAX motif. Farnesylation allows substrates, such as the Ras proteins involved in a significant fraction of human cancers, to interact with membranous structures and other protein targets. Using gene-targeted mice, we repott that FTase is essential for early embryonic development, but dispensable for adult homeostasis including responses to stress challenges. Older FTase mice display delayed wound healing and maturation defects in erythroid cells. FTase deficient embryonic fibroblasts have a distinct flat morphology and reduced motili, plating efficiency and proliferation rates. Ablation of FTase has no effect on tumor development induced by an endogenous K-ras oncogene. In a carcinogen-induced skin tumor model, loss of FTase results in decreased tumor progression, but has no effect on the activation of H-ras oncogenes. These results indicate that protein famesylation is not essential for the transformlng activtly of ras oncogenes

Identification of cancer initiating cells in K-Ras driven lung adenocarcinoma

Ubiquitous expression of a resident K-RasG12V oncogene in adult mice revealed that most tissues are resistant to K-Ras oncogenic signals. Indeed, K-RasG12V expression only induced overt tumors in lungs. To identify these transformation-permissive cells, we induced K-RasG12V expression in a very limited number of adult lung cells (0.2%) and monitored their fate by X-Gal staining, a surrogate marker coexpressed with the K-Ras G12V oncoprotein. Four weeks later, 30% of these cells had proliferated to form small clusters. However, only SPC+ alveolar type II (ATII) cells were able to form hyperplastic lesions, some of which progressed to adenomas and adenocarcinomas. In contrast, induction of K-Ras G12V expression in lung cells by intratracheal infection with adenoviral-Cre particles generated hyperplasias in all regions except the proximal airways. Bronchiolar and bronchioalveolar duct junction hyperplasias were primarily made of CC10+ Clara cells. Some of them progressed to form benign adenomas. However, only alveolar hyperplasias, exclusively made up of SPC+ ATII cells, progressed to yield malignant adenocarcinomas. Adenoviral infection induced inflammatory infiltrates primarily made of T and B cells. This inflammatory response was essential for the development of K-RasG12V-driven bronchiolar hyperplasias and adenomas, but not for the generation of SPC+ ATII lesions. Finally, activation of K-RasG12V during embryonic development under the control of a Sca1 promoter yielded CC10+, but not SPC+, hyperplasias, and adenomas. These results, taken together, illustrate that different types of lung cells can generate benign lesions in response to K-Ras oncogenic signals. However, in adult mice, only SPC+ ATII cells were able to yield malignant adenocarcinomas.Work was supported by European Research Council Grant ERC-AG/250297-RAS AHEAD; EU-Framework Programme Grants LSHG-CT-2007-037665/CHEMORES, HEALTH-F2-2010-259770/LUNGTARGET, and HEALTH-010-260791/EUROCANPLATFORM; Spanish Ministry of Economy and Competitiveness Grant SAF2011-30173; Autonomous Community of Madrid S2011/BDM-2470/ONCOCYCLE (to M.B.); National Institutes of Health Grant R01 CA109335-04A1; Spanish Ministry of Economy and Competitivenes Grant SAF2012-32810; and EU-Framework Programme Grant FP7-ENV-2011/ARIMMORA (to I.S.-G.). S.M. was supported by a predoctoral fellowship from Fundación La Caixa.Peer Reviewe

Tumor induction by an endogenous K-ras oncogene is highly dependent on cellular context

We have targeted a K-ras allele in mouse embryonic stem (ES) cells to express a K-Ras(V12) oncoprotein along with a marker protein (beta-geo) from a single bicistronic transcript. Expression of this oncogenic allele requires removal of a knocked in STOP transcriptional cassette by Cre recombinase. Primary mouse embryonic fibroblasts expressing this K-ras(V12) allele do not undergo proliferative senescence and proliferate as immortal cells. In mice, expression of K-ras(V12) throughout the body fails to induce unscheduled proliferation or other growth abnormalities for up to eight months. Only a percentage of K-ras(V12)-expressing lung bronchiolo-alveolar cells undergo malignant transformation leading to the formation of multiple adenomas and adenocarcinomas. These results indicate that neoplastic growth induced by an endogenous K-ras oncogene depends upon cellular contextThis work was supported by grants from the V Framework Programme of the EU (to M.B. and M.S.), Ministerio de Ciencia y Tecnologı́a (to M.B.), Ministerio de Sanidad y Consumo and Comunidad Autónoma de Madrid (to C.G.), and Ligue contre le Cancer (Comité de Dordogne) and INSERM (to P.D.). N.M. was supported by a Beca de Formación para la Investigación (BEFI, Fondo de Investigación Sanitaria) and A.D. by a fellowship from the Programa de Formación de Personal Investigador (FPI, Ministerio de Ciencia y Tecnología

Tumor induction by an endogenous K-ras oncogene is highly dependent on cellular context

We have targeted a K-ras allele in mouse embryonic stem (ES) cells to express a K-Ras(V12) oncoprotein along with a marker protein (beta-geo) from a single bicistronic transcript. Expression of this oncogenic allele requires removal of a knocked in STOP transcriptional cassette by Cre recombinase. Primary mouse embryonic fibroblasts expressing this K-ras(V12) allele do not undergo proliferative senescence and proliferate as immortal cells. In mice, expression of K-ras(V12) throughout the body fails to induce unscheduled proliferation or other growth abnormalities for up to eight months. Only a percentage of K-ras(V12)-expressing lung bronchiolo-alveolar cells undergo malignant transformation leading to the formation of multiple adenomas and adenocarcinomas. These results indicate that neoplastic growth induced by an endogenous K-ras oncogene depends upon cellular contextThis work was supported by grants from the V Framework Programme of the EU (to M.B. and M.S.), Ministerio de Ciencia y Tecnologı́a (to M.B.), Ministerio de Sanidad y Consumo and Comunidad Autónoma de Madrid (to C.G.), and Ligue contre le Cancer (Comité de Dordogne) and INSERM (to P.D.). N.M. was supported by a Beca de Formación para la Investigación (BEFI, Fondo de Investigación Sanitaria) and A.D. by a fellowship from the Programa de Formación de Personal Investigador (FPI, Ministerio de Ciencia y Tecnología

Protein farnesyltransferase in embryogenesis, adult homeostasis, and tumor development

Protein farnesyltransferase (FTase) is an enzyme responsible for posttranslational modification of proteins carrying a carboxy-terminal CaaX motif. Farnesylation allows substrates to interact with membranes and protein targets. Using gene-targeted mice, we report that FTase is essential for embryonic development, but dispensable for adult homeostasis. Six-month-old FTase-deficient mice display delayed wound healing and maturation defects in erythroid cells. Embryonic fibroblasts lacking FTase have a flat morphology and reduced motility and proliferation rates. Ablation of FTase in two ras oncogene-dependent tumor models has no significant consequences for tumor initiation. However, elimination of FTase during tumor progression had a limited but significant inhibitory effect. These results should help to better understand the role of protein farnesylation in normal tissues and in tumor development.This work was supported by grants from the V Framework Programme of the European Union (QLK3-1999-00875) to M.B. and from the Ministerio de Ciencia y Tecnologia (SAF2001-0058) and Fondo de Investigación Sanitaria (00/0109) to J.V. P.D. was supported by the Association pour la Recherche contre le Cancer (ARC). P.J.C. was supported by NIH grant GM46372. N.M. was supported by a BEFI Fellowship from the Fondo de Investigación Sanitaria. The CNIO is partially supported by the RTICCC (Red de Centros de Cáncer; FIS C03/10

Protein farnesyltransferase in embryogenesis, adult homeostasis, and tumor development

Protein farnesyltransferase (FTase) is an enzyme responsible for posttranslational modification of proteins carrying a carboxy-terminal CaaX motif. Farnesylation allows substrates to interact with membranes and protein targets. Using gene-targeted mice, we report that FTase is essential for embryonic development, but dispensable for adult homeostasis. Six-month-old FTase-deficient mice display delayed wound healing and maturation defects in erythroid cells. Embryonic fibroblasts lacking FTase have a flat morphology and reduced motility and proliferation rates. Ablation of FTase in two ras oncogene-dependent tumor models has no significant consequences for tumor initiation. However, elimination of FTase during tumor progression had a limited but significant inhibitory effect. These results should help to better understand the role of protein farnesylation in normal tissues and in tumor development.This work was supported by grants from the V Framework Programme of the European Union (QLK3-1999-00875) to M.B. and from the Ministerio de Ciencia y Tecnologia (SAF2001-0058) and Fondo de Investigación Sanitaria (00/0109) to J.V. P.D. was supported by the Association pour la Recherche contre le Cancer (ARC). P.J.C. was supported by NIH grant GM46372. N.M. was supported by a BEFI Fellowship from the Fondo de Investigación Sanitaria. The CNIO is partially supported by the RTICCC (Red de Centros de Cáncer; FIS C03/10