Microtubule-Regulated RNA Translation: Implications for Taxane Chemotherapy

描述(申请人提供)：这项拟议研究的长期目标是破译间期微管调节通路及其在癌症中的潜在异常，以便更好地了解癌症化疗中临床对紫杉烷和其他微管抑制剂的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，通过微管细胞骨架及其被紫杉烷的干扰来调节HIF-1的信号轴和活性。简而言之，我们发现，微管破裂通过将HIF mRNA靶向翻译抑制部位的细胞质P小体来阻止HIF-1的翻译。这里的目的是确定哪些其他癌症特异的mRNAs受微管以及起源于微管损伤并最终以翻译抑制为终点的信号级联的调节。我们的中心假设是，紫杉烷的临床活性主要源于它们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，我们关注的是翻译调控的间期微管依赖途径。阐明这些途径不仅有助于我们了解临床紫杉类药物敏感性和耐药性的分子基础，而且还将导致 为癌症治疗寻找新的靶点，其治疗开发可以与现有的基于微管的化疗极大地协同。具体目标一：确定除了HIF-1之外，哪些其他mRNAs需要动态微管进行翻译，因此是紫杉烷活性的新下游靶点。要做到这一点，我们将：IA。在未经处理和紫杉烷处理的细胞中，结合RNA-Seq进行多聚体和核糖体分析，以确定哪些mRNAs在药物治疗后改变了翻译状态，以及如何改变。IB。对Ago2相关的mRNAs进行RNA序列分析，以确定哪些mRNAs在紫杉烷处理后富含在P-Body部位。IC.对微管调节的mRNAs进行生物信息学分析，以确定潜在的“微管-敏感性-识别”序列。特定目的II：确定将微管损伤与P-Body-HIF mRNA靶向联系起来的信号通路。亚拉。在未经处理和紫杉烷处理的细胞中进行HIF mRNA下拉结合TOF/TOF-MS分析，以确定在微管破坏后将HIF-1 mRNA靶向P小体的蛋白质。IIB。通过调节微管-P-体-信使核糖核酸轴的途径扰动、敲除和过度表达实验，评估已识别的蛋白质在紫杉烷敏感性和耐药性中的作用。IIC。评价胞浆多聚腺苷酸化元件结合蛋白(CPEB)作为HIF-1mRNA P体靶向候选蛋白的作用。

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.