Abstract 5269: Farnesyltransferase inhibition induces activation and redistribution of Ras

Abstract

Introduction Osteosarcoma (OS) is the most common bone cancer and often has poor outcomes: despite surgery and chemotherapy, only 30% of patients with metastases at diagnosis survive their disease. This stagnant survival curve generates interest in finding novel therapeutic targets and new treatment options to improve outcomes. Tipifarnib (TF) (ZARNESTRA®, R115777) is a small molecule farnesyltransferase inhibitor (FTI). Inhibition of farnesylation causes relocalization of Ras in responsive OS cells, resulting in alternate activation of downstream effectors of Ras in sensitive vs. resistant lines in response to FTI. Markers for susceptibility and pathway(s) affected by FTI in OS need to be determined to identify patients who might benefit from FTI. Methods For Immunofluorescence (IF), OS cells were treated with 1 µM TF or no drug for 24 hours, fixed and stained for pan Ras, and analyzed by standard IF techniques. OS187 cells were treated with 0, 0.01 or 1 µM TF with or without z-vad (a pan caspase inhibitor) for up to 4 days, and cell yield determined daily by automated counting (ViCell). OS cells were treated with 0, 0.01 or 1 µM TF for 24 hours and lysates assessed by western blot for downstream targets of RAS, including MEK, p38 and ERK. OS187 was treated with SB 203580 (p38 MAPK inhibitor), U0126 (MEK inhibitor) TF, or all possible combinations for 72 hours. Cell yield was determined as above. Results OS cells demonstrate variable growth inhibition from FTI, with a 2-log difference between highly responsive (OS187) and highly resistant OS lines (Saos-2). In OS187, response to FTI was associated with redistribution of Ras from a diffuse, perimembranous pattern in untreated cells to a punctuate and perinuclear pattern with FTI as assessed by IF. Neither z-vad, U0126 or SB 203580 rescued cells from FTI-induced growth inhibition induced. Ras activity increased with FTI. Activation of Ras effectors differs between sensitive and resistant cell lines: p38, ERK, MEK, MKK3/6, ATF2 and MSK-1 are activated in sensitive cells in response to FTI. Conclusion Subcellular relocalization of Ras in response to FTI suggests that altering prenylation changes Ras’ interaction with effector molecules. Though we have shown that FTI induces cell death and sub-G1 DNA content in responding cells (Hall et al., 2010), the fact that z-vad does not rescue OS cells from growth inhibition and death suggests that FTI-induced death in OS is caspase independent. While multiple members of the MAPK cascade (especially p38) are activated in response to FTI, neither a p38 nor a MEK inhibitor rescued OS cells from FTI-induced growth inhibition. This suggests that either multiple MAPK components need to be inhibited to rescue cells, or that growth inhibition and cell death are independent of MAPK pathway activation. Regardless of mechanism, the strong response of some OS cells to low concentrations of TF suggests that some patients may benefit from this class of drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5269.