Abstract 3856: Loss fo N or K-Ras B does not mimic the effects of farnesy transferase inhibited osteosarcoma or 293T cells

Abstract

Ras proteins are small GTPases that regulate growth, differentiation, survival, and cell death. Prenylation, the addition of either a farnesyl (15 carbon chain lipid) or geranylgeranyl (20 carbon chain lipid) group, allows Ras and other small GTPases to anchor into the plasma membrane, a necessary step for Ras to activate multiple signaling pathways, including the MAPK cascades. The specific CAAX motif at the C terminus of a protein determines whether it can be farnesylated, geranylgeranylated, or both. There are four highly homologous Ras proteins: K-Ras A, K-Ras B, N-Ras, and H-Ras, each containing a CAAX motif that allows farnesylation. K-Ras B and N-Ras can be alternately geranylgeranylated (GGd) if farnesylation is blocked by a specific small molecule (FTI). Oncogenic Ras mutations are found commonly in cancer, and Ras also can be a major mediator of signals initiated by activated receptor tyrosine kinases (RTKs). Since RTK signaling plays a major role in the biology of sarcomas, we were interested in the mechanisms by which FTIs might affect osteosarcoma cell growth and survival. Osteosarcoma cell lines have variable responses to FTI treatment, including reduced proliferation and programmed cell death at a range of concentrations from 1 nM to 100 nM (Hall et al. 2010). Further, 293T fibroblast cells also have decreased cell viability and increased subG1 and G2/M cell cycle arrest with FTI treatment. 293T cells respond similarly to OS187 our most sensitive osteosarcoma cell line. To assess Ras activity in response to FTI, we co-precipitated activated Ras with the Ras target protein Raf, then probed by western for Ras. Surprisingly, Ras activity was increased in response to FTI in both osteosarcoma cell lines and 293T cells. We also found that the activity of downstream effectors, ERK and p38 MAPK, also increased with FTI (Hall et al, 2010). It was unclear whether the changes in cell growth and survival were due to decrease in farnesylated Ras or the increase in geranylgeranylated Ras. To determine if loss of N-Ras or K-Ras B activity could account for reduced cell yield following FTI, we knocked down each of these proteins in our most responsive osteosarcoma cell line, OS187, and in 293T cells. There was no significant difference in cell yield or cell cycle when either of these proteins was knocked down. To determine if alternate prenylation of N or K-Ras B are responsible for decreased cell yield and/or cell cycle arrest, mutant forms of these proteins will need to be transduced into OS187 and 293T cells for further analysis. In conclusion, FTI caused growth inhibition and cell death for both osteosarcoma cells and 293T cells. FTIs increased Ras activation and the activity of downstream effectors. Since knocking out N or K-Ras B did not reproduce any of these effects, the effects of FTI are not due to the loss of farnesylated Ras. These observations suggest that the effects may be due to increased geranylgeranylated N Ras or K-Ras B. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3856. doi:10.1158/1538-7445.AM2011-3856