Farnesyl transferase inhibitors affect microtubule stability and function

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Farnesyl transferase inhibitors (FTIs) are a novel class of anti-neoplastic agents that have high anti-tumor activity and are currently in phase II and III clinical trials. These agents work by inhibiting the farnesyl transferase (FT) enzyme, whose function is to post-translationally modify proteins via the addition of a farnesyl group. Although FTI was developed as a specific inhibitor of Ras farnesylation and activity it soon became clear that FTI activity is independent of Ras status. Moreover, the combination of FTIs with taxanes was shown to be synergistic, both in preclinical and clinical models; however, the molecular mechanism underlying this synergistic interaction is largely unknown. To investigate the molecular basis of synergy between FTIs and taxanes we examined the effects of FTI treatment on interphase microtubules. Here we show that FTI treatment affected interphase microtubules in a dose-dependent manner in various human cancer cell lines. Specifically, FTI treated cells displayed bundle-like microtubules and increased levels of acetylated α-tubulin (a marker of microtubule stability) as assessed by confocal microscopy. These data were confirmed by quantitating levels of acetylated α-tubulin by flow cytometry. Moreover, FTI treatment increased tubulin polymerization as assessed by cell fractionation followed by western blotting. These data were extended and confirmed by transmission electron microscopy, showing characteristic microtubule bundles in cells treated with FTI, similar to taxane-induced bundles. These effects were seen using lonafarnib (SCH 66336) concentrations of 10-25 μM. These levels are higher than the Cmax achieved in patients dosed twice daily with 200 mg of lonafarnib. In vitro experiments using purified bovine brain tubulin are underway to determine if FTI binds microtubules directly or exerts its effects on cellular microtubules via another pathway. It remains to be determined to what extent the observed antitubulin effects of FTIs contribute to the antitumor activity of this new class of compounds. Nonetheless, these data provide significant insight into the mechanism of synergy between FTIs and taxanes.