The cytoskeleton of eukaryotic cells participates in various cellular functions such as motility, secretion, signaling and proliferation. Microtubules (MTs) are an integral part of the cytoskeleton. Among anti-cancer agents, drugs targeting tubulin or MTs are among the most, if not the most, effective class of agents. The list of compounds that bind to tubulin or the MTs is large and continues to expand. The overwhelming majority are natural products, and their chemical structures are remarkably diverse. The vinca alkaloids were introduced in the 1950's, and although they were useful in a wide range of malignancies, interest in developing new agents targeting MTs gradually declined, until the introduction of Taxol. Arguably the most effective agent since cisplatin, the remarkable activity of Taxol stimulated interest in tubulin and MTs as targets for chemotherapy. The clinical success of Taxol has led to a wealth of new scientific knowledge, reinforced the importance of the tubulin/MT system as a target for cancer chemotherapy and spurred efforts to identify novel tubulin-active agents. In the field of MT targeting agents (MTAs), our current research goals are to (1) to increase our understanding of how MTAs interact with tubulin and lead to cell death;(2) understand the mechanisms of resistance to MTAs;and (3) develop assays to monitor the pharmacodynamics of MTAs in patients. In the clinic, we continue to conduct trials examining MTAs. Given our success in identifying mutations in paclitaxel- and epothilone-resistant cells, and encouraged by the information accumulated and the lessons learned, we began selections with HTI-286 a synthetic hemiasterlin in development at that time by Wyeth-Ayerst. As we were investigating the hemiasterlin resistant cell lines, we were also conducting a phase II clinical trial with BMS-247550 (ixabepilone) in patients with renal cell carcinoma. BMS-247550 is an epothilone B analogue and MT-stabilizing agent. As a part of this ongoing trial, we were attempting to obtain tumor biopsies before therapy and after the fifth dose of BMS-247550. The original goal had been to examine the pharmacodynamics of BMS-247550 by quantitating the degree of tubulin polymerization before and after drug administration. This measurement would allow us to establish whether BMS-247550 had stabilized MTs in the tumor cells. However, we also considered alternate methods to demonstrate MT stabilization. Encouraged by the results in our hemiasterlin resistant cells where tubulin acetylation and detyrosination had been correlated with MT stabilization, we chose to investigate these chemical modifications in the patient samples. As a first step we demonstrated that the levels of detyrosinated (glu-terminated) and acetylated alpha-tubulin correlated well with MT stabilization induced by BMS-247550 in cultured renal and ovarian cancer cells, suggesting these modifications could be used to monitor MT-stabilization. More importantly, in examining the patient samples we found that after treatment with BMS-247550, the levels of glu-terminated and/or acetylated alpha-tubulin increased 2- to 100-fold in 8 out of 8 serial tumor biopsies. These data indicate BMS-247550 reached the tumors and engaged the MT target, leading to MT stabilization consistent with its ability to avert Pgp and reach its target. We conclude that glu-terminated and/or acetylated alpha-tubulin levels are simple and reliable markers for the pharmacodynamic effects of BMS-247550. We believe that assessing post-translationally modified tubulin levels may provide a simple and reliable assay of the pharmacodynamic effects of other MTAs. Puzzled by the recurrent observation that our paclitaxel and epothilone resistant cell lines had acquired mutations in p53, we set out to determine if p53 could interact with tubulin in a meaningful way. We found that both wt and mt p53 associate with MTs and this interaction is lost following treatment with MT-depolymerizing drugs. Furthermore we showed that p53 accumulates in the nucleus following DNA damage only in cells with a functional MT network. Pre-treatment with either vincristine or paclitaxel reduced nuclear accumulation of p53, indicating nuclear translocation of p53 requires a functional MT network. In most cells, MTs are organized with their 'minus ends'near the nucleus and their 'plus ends'towards the cell periphery. MT-based intracellular transport is mediated via the kinesins, plus-end directed MT motors, and the dyneins, minus-end directed MT motors. Both families of MT-motor proteins require ATP to move along MTs with their cargoes. We have demonstrated the dynein family mediates transport of p53 to the nucleus and more recently we have been able to show that p53 oligomerizes prior to association with dynein and that this association then occurs in the cytoplasm. Only then does the p53-dynein complex associate with microtubules and travel to the nucleus. The residues in p53 involved in this have been identified as the residues important in the oligomerization of p53, so that mutations at these residues not only impairs p53 oligomerization and hence its ability to trans-activate its target genes, but also interferes with the trafficking of p53 to the nucleus - in effect a double hit impairing the trans-activation of target genes. The association of p53 with cellular MTs may be important in several ways. First, this may provide a mechanistic basis to regulate p53 subcellular localization. Second, our findings suggest p53 is an indirect target for MT-active agents. In this regard, the demonstration that MT active drugs may affect p53 levels and activate p53 dependent checkpoints could be explained by our findings. Third, by binding MTs, p53 is brought in close proximity to other cellular proteins. Moreover, MTs could provide a reservoir for p53. This hypothesis is consistent with the substantial amount of p53 bound to MTs and the large capacity of MTs for p53 storage. This is evidenced by the ability of MTs to bind the higher levels of mt p53, and the increased levels of wt p53, following DNA damage. Most importantly, our data indicate the p53/MT association is important for p53 nuclear accumulation. As p53 exerts many of its effects by transcriptional regulation, translocation to nuclear targets is critical for biological responses. Our data showing that disruption of a functional MT-network prior to DNA damage results in impaired trans-activation of p53-target genes further supports a role of MTs in p53 intracellular trafficking. As we go forward we plan to focus on several aspects of this work. We plan to further examine post-translational modifications as surrogates for MT stability. While MTAs have been successfully developed without clinical evidence of MT engagement by drug, we believe a simple, sensitive, and reliable assay to monitor the pharmacodynamic effect of these agents would be of value in their future development. In addition, our work has revealed that MTs facilitate intracellular trafficking and nuclear accumulation of several proteins and are in the process of clarifying how they interact with microtubules. The ultimate goal is to determine whether interphase microtubules are effective drug targets. Current thinking favors interference with the mitotic spindle as the principal effect of MTAs, however, we believe that interfering with the interphase spindle may also be very important. In the clinic we have been involved in the conduct of clinical trials with a novel epothilone B analog, ixabepilone (BMS-247550), and have conducted numerous translational studies in conjunction with these phase I and phase II trials. These studies will continue.
