The research proposed here will dissect the mechanism by which farnesyltransferase inhibitors (FTIs) lead to the inactivation of the tubulin deacetylase HDAC6, and regulate microtubule dynamics and taxane activity. Our previous results showed that FTIs synergize with the taxanes and reverse taxol-resistance via the inactivation of HDAC6. However, the exact mechanism by which FTIs affect HDAC6 activity and microtubule function is not known. Our preliminary data show that HDAC6, the protein farnesyltransferase (FTase) and microtubules are part of the same protein complex, which is disrupted by FTI treatment. To understand the physiological role of this complex and its effects in drug response, in Aim1, we will determine whether FTase has a direct role in the regulation of microtubule cytoskeleton functions, using FTase knock- down cells and in vitro tubulin polymerization/depolymerization assays. Our preliminary results showing that FTI treatment disrupts the interaction between FTase and tubulin suggests that the active form of FTase is bound to microtubules. In view of this result, in Aim2, we will investigate whether the association of FTase with the microtubule network is important for the ability of FTase to farnesylate client proteins, by measuring FTase's activity when microtubules are disrupted. We will also test the hypothesis that FTase is an HDAC6 substrate and that acetylation/deacetylation regulates its activity. In our preliminary results we showed that HDAC6 is not a direct substrate for FTIs and hence FTIs inhibit its activity. This result together with the fact that neiher HDAC6 nor tubulin contains a farnesylation motif, suggests that additional proteins are present in the FTase-HDAC6-MT complex regulating its function. Thus, in Aim3, we plan to identify these proteins by purifying and sequencing proteins that are specifically bound to the FTase and HDAC6 complexes. FTIs, a novel class of agents, originally developed to inhibit oncogenic Ras-transformation of cancer cells, are currently being tested in clinical trials. Previous work from our lab showed that FTIs target the tubulin deacetylase HDAC6, in addition to targeting Ras, and as such they modify the cells'cytoskeleton and enhance the activity of the taxanes, which are potent chemotherapy drugs used in a variety of human tumors. However, the exact mechanism of FTI action is still unknown. Our proposal promises to provide a better understanding of how FTIs work at the molecular level, as well identify potential novel targets for cancer chemotherapy. This knowledge will help in turn identify groups of patients, that according to their individual tumors genotypes, are more likely to benefit from treatment with this class of agents.
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