Investigation of farnesyltransferase inhibitors mechanism in human tumor cells.

摘要

FTIs were developed based on the idea of blocking Ras malignant transformation. With the increasing investigation on its mechanism, it is clear that FTIs are not highly selective inhibitors of mutant Ras. The actual targets of FTIs in tumor cells remain controversial. Here, I investigate the mechanism of FTIs and examined two potential targets of FTIs, N-Ras and Rheb in human tumor cells. Also, a new proteomics approach TAS was proposed to identify the farnesylated proteins in vivo..;N-Ras is an isoform of H-Ras and is a substrate for both FTase and GGTase. In N-ras mutant cells, I found out that even though the postprenylation was not completely inhibited by FTI, FTI treatments abrogated the membrane association of N-Ras. Accompanied with N-Ras membrane dissociation, FTIs exhibited inhibitory effects on oncogenic N-Ras signaling and reversed the transformed phenotypes of HT 1080, which includes Raf/MEK activity, actin filament organization as well as anchorage-independent growth. In addition, FTIs' treatments effectively inhibit the cell growth; cell cycle progression as well as cell invasion of N-Ras mutated human tumor cells.;The effects of FTIs on Rheb-mediated signaling were examined in Tsc2-null MEFs as well as human tumor cells. I examined the biological function of Rheb in mTOR/p70SK/S6 signaling. The requirement of farnesylation for human Rheb function was verified by using of RhebC181S (prenylation defective Rheb) and RhebM184L (geranylgeranylated Rheb) mutant. By using Tsc2-/- MEF cells as a model cells, we found out that the altered signaling and growth properties caused by elevated Rheb activity can be reversed by treating with FTIs. The involvement of Rheb in tumor cells as FTI target was examined in tumor cell lines including sarcoma cells. Importantly, we observed that FTI treatments result in downregulation of mTOR/p70S6K/S6 signaling in several sarcoma cells.;Due to the limit of current proteomics methods for protein prenylation and identifying targets of FTIs in vivo, we reported a new approach TAS (tagging-via-substrate) to detect farnesylated proteins in vivo. TAS technology involves metabolic incorporation of a synthetic azido-farnesyl analog with farnesylated proteins. This technology enables global profiling of farnesylated proteins by enriching farnesylated proteins and reducing the complexity of farnesylation subproteome.