Novel microtubule-associated protein complexes for targeted cancer chemotherapy

用于靶向癌症化疗的新型微管相关蛋白复合物

• 批准号: 7678447
• 负责人: Chantal Chanel-Vos
• 金额: $ 5.34万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7678447
• 关键词: Acetylation; Affect; Amino Acid Sequence; Binding; Biological Assay; Cell membrane; Cell physiology; Cells; Chemotherapy-Oncologic Procedure; Client; Clinical Trials; Complex; Coupled; Cytoskeleton; Data; Deacetylase; Deacetylation; Drug usage; Environment; Farnesyl Transferase Inhibitor; Genotype; HDAC6 gene; Human; In Vitro; Individual; Knowledge; Lead; Mass Spectrum Analysis; Measures; Microtubule Stabilization; Microtubule-Associated Proteins; Microtubules; Molecular; Oncogenic; Paclitaxel; Patients; Peptide Sequence Determination; Pharmaceutical Preparations; Physiological; Protein Farnesylation; Proteins; Proteomics; Regulation; Research; Resistance; Role; System; Taxane Compound; Testing; Tubulin; Work; cancer cell; chemotherapy; depolymerization; farnesylation; knock-down; novel; polymerization; protein complex; protein farnesyltransferase; research study; response; taxane; tumor

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：此处提出的研究将剖析Farneylsylansferase抑制剂（FTIS）导致微管蛋白脱乙酰基酶HDAC6失活的机制，并调节微管动力学和紫杉烷活性。我们先前的结果表明，FTIS通过HDAC6失活而与紫杉烷和反向抗紫oi抗抗性。但是，FTI影响HDAC6活性和微管功能的确切机制尚不清楚。我们的初步数据表明，HDAC6，蛋白质Farnesyltransferase（FTase）和微管是同一蛋白质复合物的一部分，该蛋白质复合物受到FTI处理的破坏。为了了解该复合物的生理作用及其在药物反应中的作用，在AIM1中，我们将使用FTase敲除细胞和体外微管蛋白聚合/解聚测定法，确定FTase在微管细胞骨架功能的调节中是否具有直接作用。我们的初步结果表明，FTI处理破坏了FTase和微管蛋白之间的相互作用表明FTase的活性形式与微管结合。鉴于这一结果，在AIM2中，我们将研究FTase与微管网络的关联对于FTase到Farnesylate客户蛋白的能力是否很重要，当时在微管受到破坏时测量FTase的活性。我们还将检验以下假设：FTase是HDAC6底物，而乙酰化/脱乙酰化调节其活性。在我们的初步结果中，我们表明HDAC6不是FTI的直接底物，因此FTI抑制其活性。这一结果与Neiher HDAC6或微管蛋白包含Farnesylation基序的事实表明，在FTase-HDAC6-MT复合物中存在其他蛋白质，从而调节其功能。因此，在AIM3中，我们计划通过纯化和测序与FTase和HDAC6复合物的纯化和测序蛋白来鉴定这些蛋白质。 FTI是一种新型的药物，最初开发用于抑制癌细胞的致癌性RAS转换，目前正在临床试验中进行测试。我们实验室的先前工作表明，除了靶向RAS外，FTIS靶向微管蛋白脱乙酰基酶HDAC6，因此它们会改变细胞的细胞骨架并增强群群的活性，这些群是在各种人类肿瘤中使用的有效化学疗法药物。但是，FTI作用的确切机制仍然未知。我们的建议有望更好地了解FTI在分子水平上的工作方式，并确定潜在的癌症化学疗法的新靶标。这些知识将有助于识别患者群体，即根据其各个肿瘤的基因型，更有可能受益于这种类药物的治疗。