Generacion y caracterizacion de modelos murinos para el estudio del papel del oncogen H-Ras en la tumorogenesis y en el sindrome Costello

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Departamento de Biología Molecular. Fecha de lectura: 19-11-2008To study the role of H-Ras oncogenes in a physiological setting, we have targeted the endogenous H-Ras locus by introducing an oncogenic Gly12Val mutation miscoding mutation along with a floxed STOP transcriptional cassette mutation using homologous recombination in embryonic stem cells. Cre-mediated expression of the H-RasLSLG12Vgeo allele can be monitored by bicistronic expression of a beta-galactosidase gene targeted at the 3’ end of the locus, as previously described for the K-Ras gene (Guerra et al., 2003). The resulting mice, H-RasLSLG12Vgeo were crossed with the RERT strain carrying an inducible CreERT2 recombinase knocked-in in the locus encoding the large subunit of the RNA polymerase II (Guerra et al., 2003). H-RasG12V expression was activated postnatally (P20) upon exposure to a synthetic steroid, 4OH-tamoxifen. In contrast with our previous results with an endogenous K-RasG12V oncogene, expression of the H-RasG12V allele did not result in tumour development after two years even though percentages of activation measured by X-gal staining ranged from 10 to 20% in most tissues. Germ line activation of H-RAS oncogenes is the primary cause of Costello syndrome (CS; Aoki et al., 2005). We have generated a mouse model of CS by crossing the H-Ras+/LSLG12Vgeo with the EIIaCre strain (Lakso et al., 1996) which expresses Cre recombinase at the zygote stage. Mice carrying the H-RasG12V allele were crossed to C57BL/6J mice to eliminate the EIIaCre transgene and subsequently among themselves to generate the homozygous strain. Germ line expression of the endogenous H-RasG12V oncogene results in hyperplasia of the mammary gland. Yet, we only observed tumours in a small percentage of animals. Moreover, long term exposure of these mice to the tumour promoter 12-o-tetradecanoylphorbol-13-acetate (TPA) does not result in the formation of papillomas. These observations indicate that activation of H-Ras oncogenes contributes to tumour progression, but not to tumour initiation. More importantly, H-RasG12V mutant mice closely phenocopy some of the abnormalities observed in CS patients including facial dysmorphia and cardiomyopathies. These mice also show alterations in the homeostasis of the cardiovascular system, including development of systemic hypertension, extensive vascular remodeling and fibrosis in both heart and kidneys. This phenotype is age-dependent and is a consequence of the abnormal upregulation of the renin-angiotensin II system. Treatment with Captopril, an inhibitor of angiotensin II biosynthesis, prevented development of the hypertension condition, vascular remodeling and heart and kidney fibrosis. In addition, it partially alleviated the observed cardiomyopathies. These mice should help in elucidating the etiology of CS symptoms, identifying additional defects and evaluating potential therapeutic strategies. References: Aoki, Y., Niihori, T., Kawame, H., Kurosawa, K., Ohashi, H., Tanaka, Y., Filocamo, M., Kato, K., Suzuki, Y., Kure, S. y Matsubara, Y. 2005. Germline mutations in HRAS proto-oncogene cause Costello syndrome. Nat. Genet. 37:1038-1040. Guerra, C., Mijimolle, N., Dhawahir, A., Dubus, P., Barradas, M., Serrano, M., Campuzano, V., y Barbacid, M. 2003. Tumor induction by an endogenous K-ras oncogene is highly dependent on cellular context. Cancer Cell. 4:111-120. Lakso, M., Pichel, J.G., Gorman, J.R., Sauer, B., Okamoto, Y., Lee, E., Alt, F.W., y Westphal, H. 1996. Efficient in vivo manipulation of mouse genomic sequences at the zygote stage. Proc. Natl. Acad. Sci. U. S. A. 93:5860-5865