Microtubule-Regulated RNA Translation: Implications for Taxane Chemotherapy

描述（由申请人提供）：拟议研究的长期目标是解读癌症间期微管调控通路及其潜在畸变，以便更好地了解紫杉烷和其他用于癌症化疗的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，通过微管细胞骨架及其紫杉烷的扰动调节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靶向胞质p体（翻译抑制位点）来阻止HIF- 1¿的翻译。这里的目的是确定其他癌症特异性mrna是由微管调控的，以及信号级联反应，该信号级联反应起源于微管损伤，最终以翻译抑制结束。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的影响，以及在药物诱导的微管扰动后受到影响的下游途径。在这一建议中，我们重点关注翻译调控的间期微管依赖途径。这些途径的阐明不仅有助于我们了解临床紫杉烷敏感性和耐药的分子基础，而且还将引导临床研究

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.