Fernández-Pisonero, IsabelLorenzo-Martín, L FranciscoDrosten, MattiasSantos, EugenioBarbacid, MarianoAlarcón, BalbinoBustelo, Xosé R2025-06-182025-06-182025-07Oncogene . 2025 Jul;44(24):1905-1921.https://hdl.handle.net/20.500.12105/26763R-RAS2/TC21, a member of the R-RAS subfamily of GTP-binding proteins, shares structural and signaling properties with the RAS subfamily proteins H-, K-, and N-RAS. However, little information is available regarding its role in normal cells and the level of functional redundancy with R-RAS and classical RAS proteins. In this work, we used loss and gain-of-function approaches to assess these issues in mouse embryonic fibroblasts (MEFs). Using primary MEFs from Rras2, Rras or Rras; Rras2 embryos, we show here that endogenous R-RAS2/TC21 is required for activation of the phosphatidylinositol 3 kinase (PI3K)-AKT axis, the proliferation, and the adhesion properties of these cells. Endogenous R-RAS does not influence any of these cell parameters. We also show that the depletion of R-RAS2/TC21 worsens the proliferative and morphological defects elicited by the combined loss of H-, K- and N-RAS proteins in MEFs. Conversely, the ectopic expression of an active version of R-RAS2/TC21, but not of R-RAS, overcomes such defects. This rescue activity involves the inhibition of the tumor suppressor TP53 and is PI3K-, mTORC-, and MEK/ERK-dependent. These results indicate that R-RAS2/TC21, R-RAS, and RAS subfamily GTPases play different roles in MEFs. They also show that R-RAS2 provides subsidiary signals that are essential for the short-term proliferation and long-term viability of MEFs lacking RAS signaling.The XRB's project leading to these results has received funding from the Spanish Association against Cancer (GC16173472GARC), the Castilla-Leon government (CSI018P23), grants funded by MCIN/AEI/10.13039/501100011033/ plus the European Research Development Fund << A way of making Europe >> of the European Union (PID2021-122666OB-I00, PDC2022-133027-I00, PLEC2022-009217), << la Caixa >> Banking Foundation (HR20-00164), the << Programa Excelencia >> of the Fundacion Cientifica AECC 2022 (EPAEC222641CICS), and the << Escalera de Excelencia >> of the Education Ministry of the Castilla y Leon autonomous government plus the European Research Development Fund (CLU-2023-2-01).engVoRhttp://creativecommons.org/licenses/by-nc-nd/4.0/HUMAN R-RASTC21ACTIVATIONTRANSFORMATIONMIGRATIONPROLIFERATIONTC21/R-RAS2EXPRESSIONKINASEAttribution-NonCommercial-NoDerivatives 4.0 International4016487044241905-1921Oncogeneopen access