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

微管 (MT) 干扰剂 (MTA)：作用和耐药机制

• 批准号: 8552754
• 负责人: Antonio Fojo
• 金额: $ 46.23万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8552754
• 关键词: Acetylation; Affect; Aftercare; Antineoplastic Agents; Area; Binding; Biological; Biological Assay; Biological Factors; Biopsy; Cell Death; Cell Line; Cell Nucleus; Cell Survival; Cell physiology; Cells; Chemical Structure; Chemicals; Chemotherapy-Oncologic Procedure; Cisplatin; Clinic; Clinical; Clinical Data; Clinical Oncology; Clinical Trials; Complex; Conduct Clinical Trials; Cytoplasm; Cytoskeleton; DNA Damage; Data; Development; Disease; Dose; Drug Delivery Systems; Drug resistance; Dynein ATPase; Epothilone B Analogue; Epothilones; Eukaryotic Cell; Family; Future; Gene Targeting; Glucocorticoid Receptor; Goals; Growth; Head and Neck Cancer; Hodgkin Disease; Human; Interphase; Interphase Cell; Intracellular Transport; Investigation; Ixabepilone; Kaposi Sarcoma; Kinesin; Knowledge; Laboratories; Lead; Malignant Neoplasms; Malignant neoplasm of cervix uteri; Malignant neoplasm of lung; Malignant neoplasm of ovary; Malignant neoplasm of prostate; Malignant neoplasm of thyroid; Measurement; Mediating; Methodology; Methods; Microtubule Stabilization; Microtubule stabilizing agent; Microtubules; Mitotic spindle; Modeling; Modification; Monitor; Motor; Multiple Myeloma; Mutation; New Agents; Non-Hodgkin' s Lymphoma; Nuclear; Nuclear Translocation; Paclitaxel; Patients; Pharmaceutical Preparations; Pharmacodynamics; Phase II Clinical Trials; Post-Translational Protein Processing; Process; Proteins; Randomized Clinical Trials; Recurrence; Regimen; Renal Cell Carcinoma; Renal carcinoma; Research; Resistance; Role; Sampling; Signal Transduction; System; Therapeutic; Thinking; Transcriptional Regulation; Travel; Tubulin; Vinca Alkaloids; Vincristine; Work; alpha Tubulin; base; cancer cell; cell motility; chemotherapy; drug development; interest; kidney cell; macromolecule; malignant breast neoplasm; malignant stomach neoplasm; neoplastic cell; novel; polymerization; preclinical study; resistance mechanism; response; success; trafficking; tumor

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.A few years ago we conducted 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. In the laboratory we had observed that tubulin acetylation and detyrosination had been correlated with MT stabilization, and so 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 an are currently assessing post-translationally modified tubulin levels as 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. Whether transport or accumulation of differentially post-translationally modified p53 is occurring is also a line of investigation.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 identify targets that associate with MT in interphase as the true ?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 is more important.

真核细胞的细胞骨架参与各种细胞功能，例如运动，分泌，信号传导和增殖。微管（MTS）是细胞骨架的组成部分。在抗癌药物中，针对微管蛋白或MT的药物是最有效的药物最有效类别的药物之一。与微管蛋白或MTS结合的化合物列表很大，并且继续扩展。绝大多数是天然产品，它们的化学结构非常多样化。 VINCA生物碱是在1950年代引入的，尽管它们在广泛的恶性肿瘤中很有用，但对开发针对MTS的新代理商的兴趣逐渐下降，直到引入紫杉醇。可以说是自顺铂以来最有效的药物，紫杉醇的显着活性刺激了小管蛋白和MT作为化学疗法的靶标。紫杉醇的临床成功导致了大量新的科学知识，增强了微管蛋白/MT系统作为癌症化疗的目标，并刺激了识别新型微管蛋白活性剂的努力。在MT靶向剂（MTA）的领域，我们当前的研究目标是（1）提高我们对MTA与微管蛋白相互作用的理解并导致细胞死亡； （2）了解对MTA的抗性机制； （3）开发测定方法以监测患者MTA的药效学。在诊所中，我们继续进行检查MTA的试验。几年前，我们对肾细胞癌患者进行了BMS-247550（ixabepilone）的II期临床试验。 BMS-247550是雌酮B模拟和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之后获得肿瘤活检。最初的目标是通过定量药物给药之前和之后的微管蛋白聚合度的程度来检查BMS-247550的药效学。这种测量将使我们能够确定BMS-247550是否在肿瘤细胞中稳定了MT。但是，我们还考虑了证明MT稳定的替代方法。在实验室中，我们观察到小管蛋白乙酰化和驱异染料与MT稳定相关，因此我们选择研究患者样品中的这些化学修饰。作为第一步，我们证明了驱异触及的（GLU终止）和乙酰化的α-微管蛋白与BMS-247550在培养的肾脏和卵巢癌细胞中诱导的MT稳定良好相关，这表明这些修饰可用于监测MT稳定。更重要的是，在检查患者样品时，我们发现在BMS-247550治疗后，在8个连续肿瘤活检中，有8个中，GLU终止和/或乙酰化α-微管蛋白的水平增加了2至100倍。这些数据表明BMS-247550达到了肿瘤并参与了MT靶标，导致MT稳定与避免PGP并达到目标的能力一致。 We conclude that glu-terminated and/or acetylated alpha-tubulin levels are simple and reliable markers for the pharmacodynamic effects of BMS-247550 an are currently assessing post-translationally modified tubulin levels as 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在p53中的突变，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MTS相关，并且在用MT-脱聚合药物治疗后，这种相互作用消失了。 此外，我们表明p53仅在具有功能性MT网络的细胞中DNA损伤后在细胞核中积聚。用vincristine或紫杉醇的预处理减少了p53的核积累，表明p53的核易位需要功能性MT网络。在大多数细胞中，MT与其“减去末端”的核及其“加上”的“末端”对细胞周围的态度进行了组织。基于MT的细胞内转运是通过驱动蛋白，加端的定向MT电动机和Dyneins，减去端电动机的介导的。 MT运动蛋白的两个家族都要求ATP与货物一起沿MT沿MT移动。我们已经证明了Dynein家族介导p53向细胞核的转运，最近我们能够证明p53寡聚在与动力蛋白的关联之前进行了寡聚，然后在细胞质中发生这种关联。只有这样，p53-dynein复合物才会与微管相关并传播到核。 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在几种方面可能很重要。首先，这可能提供了调节p53亚细胞定位的机械基础。其次，我们的发现表明p53是MT活性药物的间接目标。在这方面，我们发现可以通过我们的发现来解释MT活性药物可能影响p53水平并激活依赖p53检查点的证明。第三，通过结合MT，p53与其他细胞蛋白近距离接近。此外，MTS可以为P53提供储层。该假设与与MTS结合的大量p53和p53存储的MT大量容量一致。在DNA损伤之后，MT结合MT较高水平的MT p53的能力以及WT p53的水平增加来证明这一点。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通过转录调节发挥许多作用，因此转移到核靶标对于生物学反应至关重要。我们的数据表明，在DNA损伤之前的功能MT网络破坏会导致p53靶基因的反式激活损害进一步支持MT在p53细胞内运输中的作用。转运后修改后的p53的运输还是积累也是一项调查。随着我们的前进，我们计划专注于这项工作的几个方面。我们计划进一步研究翻译后修饰作为MT稳定性的替代物。尽管MTA已成功开发而没有药物参与MT参与的临床证据，但我们认为一种简单，敏感和可靠的测定法以监测这些药物的药效效应在其未来的发展中具有价值。此外，我们的工作表明，MTS促进了几种蛋白质的细胞内运输和核积累，并且正在阐明它们与微管的相互作用。最终目标是确定与MT相间的MT相关的目标是对吗？药物目标？当前的思维有利于对有丝分裂纺锤体的干扰作为MTA的主要作用，但是，我们认为干扰相间纺锤体更为重要。