Analysis of molecular mechanisms underlying FGF2 cell cycle inhibition in mouse Y1 adrenocortical and tumor cells dependent of amplified WT-Kras

• Grant number: 17/19545-8
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: November 01, 2017
• End date: February 29, 2020
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Hugo Aguirre Armelin
• Grantee: Edmilson Ozorio dos Santos
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Associated research grant: 13/07467-1 - CeTICS - Center of Toxins, Immune-Response and Cell Signaling, AP.CEPID

Abstract

We previously reported that Kras-driven Y1 tumor cells exhibit an unlikely association of high basal levels of K-Ras-GTP with cell cycle arrest by Fibroblast Growth Factor 2 (FGF2). Furthermore, in Y1 cells, experimental ectopic expression of an inducible dominant negative mutant, Ras-N17, reduced K-Ras-GTP basal levels and rescued FGF2 mitogenic activity (Costa et al. 2008; Salotti et al, 2011).More recently, computer simulations with our K-Ras dynamical model predicted that the Y1 cells should express at least two GEFs to catalyze the transition KRas-GDP à KRas-GTP, keeping high basal levels of [K-Ras-GTP]. Experimental testing validated this prediction: first, qRT-PCR analysis demonstrated that Y1 cells express two main GEFs, SOS and RasGRP4. Second, Y1 clonal sublines selected for resistance to FGF2 (Y1FRs), showed: i) Y1 normal pattern of FGFR expression and dependence on FGF2 for proliferation; ii) very low levels of K-Ras expression; iii) only negligible levels of the RasGRP4-GEF (M. Reis et al, 2016). Third, Y1 sublines knocked out for the Kras gene, by the CRISPR-Cas9 technique, displayed a normal-like phenotype in culture and were not tumorigenic in Nude mice (M. Dias, C. Fonseca & H. Armelin, unpublished results).Altogether, our results with mouse Y1 cells showed that the KRas oncogenic activity depends on the KRas oncoprotein concentration and the interplay between Ras-GEFs, such as SOS and GRP4. In addition, results from our initial tests suggested that these conclusions with mouse cells translate to human cell lines experimentally transformed by oncogenic KRas and HRas (M. Dias, J. Zeidler, J. Galvão & H. Armelin, unpublished results).The purpose of this project is to advance our knowledge of the molecular mechanisms underlying FGF2 toxic effects in Y1 cells.