Microtubule (MT) Interfering Agents (MTAs): Mechanisms of Action and Resistance

真核细胞的细胞骨架参与多种细胞功能，如运动、分泌、信号传导和增殖。微管（MT）是细胞骨架的一个组成部分。在抗癌药物中，针对微管蛋白或 MT 的药物即使不是最有效，也是最有效的一类药物。与微管蛋白或 MT 结合的化合物种类繁多，并且还在不断扩大。绝大多数是天然产物，它们的化学结构非常多样化。长春花生物碱于 20 世纪 50 年代推出，尽管它们可用于治疗多种恶性肿瘤，但开发针对 MT 的新药物的兴趣逐渐下降，直到紫杉醇的推出。紫杉醇可以说是自顺铂以来最有效的药物，其卓越的活性激发了人们对微管蛋白和 MT 作为化疗靶点的兴趣。紫杉醇的临床成功带来了丰富的新科学知识，强化了微管蛋白/MT系统作为癌症化疗靶标的重要性，并刺激了识别新型微管蛋白活性药物的努力。在MT靶向剂（MTA）领域，我们目前的研究目标是（1）增加我们对MTA如何与微管蛋白相互作用并导致细胞死亡的理解； (2)了解MTAs的耐药机制； (3) 开发检测方法来监测 MTA 在患者中的药效学。在临床上，我们继续进行检查 MTA 的试验。鉴于我们成功识别了紫杉醇和埃坡霉素耐药细胞的突变，并受到所积累的信息和吸取的教训的鼓舞，我们开始选择 HTI-286，一种当时由 Wyeth-Ayerst 开发的合成半甾林。在研究半斯特林耐药细胞系的同时，我们还在肾细胞癌患者中开展了 BMS-247550（伊沙匹隆）的 II 期临床试验。 BMS-247550 是埃坡霉素 B 类似物和 MT 稳定剂。作为这项正在进行的试验的一部分，我们试图在治疗前和第五剂 BMS-247550 后获得肿瘤活检。最初的目标是通过定量给药前后微管蛋白聚合程度来检查 BMS-247550 的药效学。该测量将使我们能够确定 BMS-247550 是否在肿瘤细胞中具有稳定的 MT。然而，我们也考虑了证明 MT 稳定性的替代方法。我们的半甾醇抗性细胞中的微管蛋白乙酰化和去酪氨酸化与 MT 稳定性相关，受到这些结果的鼓舞，我们选择研究患者样本中的这些化学修饰。作为第一步，我们证明去酪氨酸（谷氨酸末端）和乙酰化 α-微管蛋白的水平与 BMS-247550 在培养的肾癌细胞和卵巢癌细胞中诱导的 MT 稳定性良好相关，表明这些修饰可用于监测 MT 稳定性。更重要的是，在检查患者样本时，我们发现用 BMS-247550 治疗后，8 个连续肿瘤活检中有 8 个的谷氨酸封端和/或乙酰化 α-微管蛋白水平增加了 2 至 100 倍。这些数据表明 BMS-247550 到达肿瘤并接合 MT 靶点，导致 MT 稳定，与其避免 Pgp 并达到其靶点的能力一致。我们得出的结论是，谷氨酸末端和/或乙酰化 α-微管蛋白水平是 BMS-247550 药效作用的简单而可靠的标志物。我们相信，评估翻译后修饰的微管蛋白水平可以为其他 MTA 的药效学效应提供简单而可靠的测定。我们反复观察到我们的紫杉醇和埃博霉素耐药细胞系在 p53 中获得了突变，这让我们感到困惑，我们着手确定 p53 是否可以以有意义的方式与微管蛋白相互作用。我们发现 wt 和 mt p53 都与 MT 相关，并且在使用 MT 解聚药物治疗后这种相互作用消失。此外，我们还发现，只有在具有功能性 MT 网络的细胞中，DNA 损伤后 p53 才会在细胞核中积累。用长春新碱或紫杉醇进行预处理可减少 p53 的核积累，表明 p53 的核转位需要功能性 MT 网络。在大多数细胞中，MT 的组织方式是“负端”靠近细胞核，“正端”靠近细胞外围。基于 MT 的细胞内运输是通过驱动蛋白（正端定向 MT 马达）和动力蛋白（负端定向 MT 马达）介导的。 MT 驱动蛋白的两个家族都需要 ATP 来沿着 MT 及其货物移动。我们已经证明动力蛋白家族介导 p53 向细胞核的转运，最近我们已经能够证明 p53 在与动力蛋白结合之前寡聚化，然后这种结合发生在细胞质中。只有这样，p53-动力蛋白复合物才会与微管结合并进入细胞核。 p53中参与此的残基已被鉴定为在p53寡聚化中重要的残基，因此这些残基的突变不仅损害p53寡聚化及其反式激活其靶基因的能力，而且还干扰p53向细胞核的运输——实际上是双重打击，损害了靶基因的反式激活。 p53 与细胞 MT 的关联可能在几个方面很重要。首先，这可能为调节p53亚细胞定位提供机制基础。其次，我们的研究结果表明 p53 是 MT 活性药物的间接靶标。在这方面，我们的研究结果可以解释 MT 活性药物可能影响 p53 水平并激活 p53 依赖性检查点的证据。第三，通过结合 MT，p53 与其他细胞蛋白更加接近。 此外，MT 可以为 p53 提供储存库。这一假设与大量的p53与MT结合以及MT存储p53的大容量是一致的。 DNA 损伤后，MT 能够结合较高水平的 mt p53 以及增加的 wt p53 水平就证明了这一点。最重要的是，我们的数据表明 p53/MT 关联对于 p53 核积累很重要。由于 p53 通过转录调控发挥其许多作用，因此易位至核靶标对于生物反应至关重要。我们的数据显示，在 DNA 损伤之前功能性 MT 网络的破坏会导致 p53 靶基因的反式激活受损，这进一步支持了 MT 在 p53 细胞内运输中的作用。随着我们的前进，我们计划重点关注这项工作的几个方面。我们计划进一步检查翻译后修饰作为 MT 稳定性的替代物。虽然 MTA 已成功开发，但没有药物与 MT 结合的临床证据，但我们相信，一种简单、灵敏且可靠的检测方法来监测这些药物的药效作用将对其未来的开发有价值。此外，我们的工作表明，MT 促进细胞内运输和几种蛋白质的核积累，并且正在阐明它们如何与微管相互作用。最终目标是确定间期微管是否是有效的药物靶点。目前的观点倾向于干扰有丝分裂纺锤体作为 MTA 的主要作用，然而，我们认为干扰间期纺锤体也可能非常重要。在临床上，我们参与了新型埃博霉素B的临床试验[摘要截断为7800个字符]

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 an [summary truncated at 7800 characters]