Microtubule-Regulated RNA Translation: Implications for Taxane Chemotherapy

微管调节的 RNA 翻译：对紫杉烷化疗的影响

• 批准号: 8655336
• 负责人: PARASKEVI GIANNAKAKOU
• 金额: $ 36.11万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8655336
• 关键词: Affect; Antimitotic Agents; Binding Sites; Bioinformatics; CPE-binding protein; Cell Nucleus; Cell model; Cells; Chemotherapy-Oncologic Procedure; Clinical; Clinical Sensitivity; Coupled; Cytoskeleton; Data; Drug Targeting; Drug resistance; Epithelial; Event; Genetic Translation; Goals; Individual; Interphase; Lead; Link; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Messenger RNA; MicroRNAs; Microtubule Stabilization; Microtubules; Modification; Molecular; Mutation; Pathway interactions; Patients; Peptide Signal Sequences; Pharmaceutical Preparations; Polyribosomes; Predisposition; Process; Proteins; RNA; Recruitment Activity; Regulation; Research; Resistance; Ribosomes; Role; Sampling; Signal Pathway; Signal Transduction; Site; Solid Neoplasm; Taxane Compound; Therapeutic; Translational Regulation; Translational Repression; Translations; Tubulin; Work; base; cancer cell; cancer therapy; chemotherapy; clinically relevant; hypoxia inducible factor 1; inhibitor/antagonist; knock-down; novel; protein transport; prototype; public health relevance; research study; response; stem; taxane; trafficking; transcriptome sequencing; tumor

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to decipher the microtubule-regulated pathways in interphase and their potential aberrations in cancer in order to better understand the molecular basis of clinical sensitivity and resistance to taxanes and other microtubule inhibitors used in cancer chemotherapy. We have recently identified a novel mechanism by which the microtubule cytoskeleton, and its perturbation by taxanes regulates HIF-1¿ signaling axis and activity. Briefly, we showed that microtubule disruption stalls the translation of HIF- 1¿ by targeting HIF mRNA to cytoplasmic P-bodies which are sites of translational suppression. The objective here, is to identify what other cancer-specific mRNAs are regulated by microtubules as well as the signaling cascade that originates with microtubule damage and culminates with translational suppression. Our central hypothesis is that the clinical activity of taxanes stems primarily from their effects on interphase microtubules and the downstream pathways that become affected following drug-induced microtubule perturbation. In this proposal we focus on the interphase microtubule-dependent pathway of translational regulation. Elucidation of these pathways will not only help us understand the molecular basis of clinical taxane sensitivity and resistance, but it will also lead to the identification of novel targets for cancer therapy, whose therapeutic exploitation can greatly synergize with existing microtubule-based chemotherapy. Specific Aim I: Identify which other mRNAs, in addition to HIF-1 ¿, require dynamic microtubules for their translation and as such represent novel downstream targets of taxane activity. To do so we will: Ia. Perform polysome and ribosome profiling coupled with RNA-Seq in untreated and taxane-treated cells to identify which mRNAs change translational status, and how, following drug treatment. Ib. Perform RNA-Seq of Ago2-associated mRNAs in order to identify which mRNAs are enriched in P-body sites following taxane treatment. Ic. Perform bioinformatic analyses of the microtubule-regulated mRNAs in order to identify the underlying "microtubule-susceptibility-recognition" sequence. Specific Aim II: Identify the signaling pathways that link microtubule damage to P-body-HIF mRNA targeting. IIa. Perform HIF mRNA pull-down coupled with TOF/TOF-Mass Spectrometry in untreated and taxane-treated cells in order to identify the proteins that target HIF-1¿ mRNA to P-bodies following microtubule disruption. IIb. Assess the role of the identified proteins in taxane sensitivity and resistance by pathway perturbation, knock-down and over-expression experiments that will allow modulation of the microtubule-P-body-mRNA axis. IIc. Evaluate the cytoplasmic polyadenylation element binding protein (CPEB) as one candidate protein involved in HIF-1¿ mRNA P-body targeting.

描述（由申请人提供）：拟议研究的长期目标是破译间期微管调节途径及其在癌症中的潜在畸变，以便更好地了解癌症化疗中使用的紫杉烷类和其他微管抑制剂的临床敏感性和耐药性的分子基础。我们最近发现了一种新的机制，通过这种机制，微管细胞骨架及其受紫杉烷类干扰调节HIF-1 <$信号轴和活性。简而言之，我们发现微管破坏通过将HIF mRNA靶向细胞质P体（翻译抑制位点）来阻止HIF- 1？的翻译。本文的目的是确定哪些其他癌症特异性mRNA受微管以及起源于微管损伤并最终导致翻译抑制的信号级联调节。我们的中心假设是，紫杉烷类的临床活性主要源于其对间期微管和下游通路的影响，这些通路在药物诱导的微管扰动后受到影响。在这个建议中，我们专注于间期微管依赖的翻译调控途径。阐明这些途径不仅有助于我们了解临床紫杉烷敏感性和耐药性的分子基础， 涉及鉴定用于癌症治疗的新靶点，其治疗开发可以与现有的基于微管的化学疗法大大协同。具体目标一：确定除了HIF-1 <$以外，哪些其他mRNA需要动态微管进行翻译，因此代表了紫杉烷活性的新下游靶点。为此，我们将：在未处理和紫杉烷处理的细胞中进行多核糖体和核糖体分析，并结合RNA-Seq，以确定药物治疗后哪些mRNA改变了翻译状态以及如何改变。IB.进行Ago 2相关mRNA的RNA-Seq，以鉴定哪些mRNA在紫杉烷处理后富集在P体位点。IC.对微管调节的mRNA进行生物信息学分析，以确定潜在的“微管敏感性识别”序列。具体目标II：确定连接微管损伤与P体-HIF mRNA靶向的信号通路。IIA.在未处理和紫杉烷处理的细胞中进行HIF mRNA下拉结合TOF/TOF-质谱，以鉴定微管破坏后将HIF-1？mRNA靶向P体的蛋白质。IIb.通过通路干扰、敲低和过表达实验来评估所鉴定的蛋白质在紫杉烷敏感性和抗性中的作用，这些实验将允许微管-P-体-mRNA轴的调节。IIc.评价胞质聚腺苷酸化元件结合蛋白（CPEB）作为参与HIF-1 <$mRNA P体靶向的候选蛋白。