Establishing the role of cytoplasmic Core Binding Factor beta in the regulation of osteosarcoma protein translation

项目总结/摘要 骨肉瘤是人类最常见的原发性骨肿瘤， 影响青少年和年轻人。高达30%的确诊患者将无法存活5年， 多模式治疗，包括手术、化疗，在某些情况下还包括放射治疗。没有有意义 过去40年来，生存时间有所改善，说明人们对小说的迫切需求 治疗的形式。 核心结合因子β（CBF β）蛋白是异源二聚体转录因子复合物的一个亚基 与Runt相关转录因子2（RUNX2）结合以协调有组织的骨骼发育。两 在某些情况下，这些组分在OS中过表达，并且它们的正常活性失调。没有固有的DNA结合 也没有其自身的转录活性，对细胞质CBF β的活性知之甚少。然而，这种蛋白质 在OS中上调，在转移性病变中表达增加， 无病生存和总生存。CBF β似乎通过转录后调控RUNX2的表达， 与RUNX2蛋白稳定性或蛋白酶体降解无关的机制。最近，CBF β已被 参与调节乳腺癌细胞中蛋白质翻译的起始。该项目旨在确定和 描述了CBF β在启动OS中帽依赖性蛋白翻译中的非经典调节作用， 恶性细胞被认为是对之上瘾的。该项目的目标将通过以下方式实现： 用野生型CBF β或CBF β的asite定向突变体转染的CBF β敲除细胞的用途， 与RUNX2的结合位点。将在另外的OS细胞系和正常成骨细胞中使用抑制肽 细胞本项目的具体目标是：1）确定RUNX2的转录后调控机制 CBF β的蛋白表达和2）确定CBF β对CAP依赖性蛋白翻译的贡献， 骨肉瘤 从头蛋白质合成测定，CBF β与翻译机制相互作用的研究 蛋白质，以及CBF β影响RNA结合和帽结合蛋白与RUNX2 mRNA相互作用的能力 将用于揭示CBF β发挥RUNX2转录后调控的潜在机制。 核糖体足迹法或Ribo-seq与RNA-seq相结合将确定CBF β在翻译中的作用。 RUNX2和RUNX2靶基因产物的效率。Ribo-seq数据还将提供一个全球性的观点， CBF β对蛋白质翻译的影响，以及随后的途径分析将提供有关 潜在的新目标。 这些研究与目前的K01 SERCA研究相结合，将推动研究 申请人的独立性，并将用于开发和完善研究假设，包括在一个 未来的R01应用。

Project Summary/Abstract Osteosarcoma (OS) is the most common primary bone neoplasms in people, with the majority of cases affecting adolescents and young adults. Up to 30% of those diagnosed will not survive 5 years with current, multimodal therapy which includes surgery, chemotherapy and, in some cases, radiation therapy. No meaningful improvements in survival times have been made in the past 40 years, illustrating the desperate need for novel forms of therapy. The core binding factor beta (CBFβ) protein is one subunit of a heterodimeric transcription factor complex that binds to Runt-related transcription factor 2 (RUNX2) to coordinate organized skeletal development. Both components are overexpressed in OS and their normal activity is dysregulated. With no inherent DNA binding nor transcriptional activity of its own, very little is known about the activity of cytoplasmic CBFβ. Yet, this protein is upregulated in OS, demonstrates increased expression in metastatic lesions, and is associated with reduced disease free and overall survival. CBFβ appears to control RUNX2 expression through post-transcriptional mechanisms independent of RUNX2 protein stability or proteasomal degredation. Recently, CBFβ has been implicated in regulating the initiation of protein translation in breast cancer cells. This project aims to identify and describe a noncanonical, regulatory role of CBFβ in initiating cap-dependent protein translation in OS, a process to which malignant cells are thought to be addicted. The goals of this project will be accomplished through the use of a CBFβ knockout cells transfected with either wild-type CBFβ or asite-directed mutant of CBFβ that targets the binding site with RUNX2. An inhibitory peptide will be used in additional OS cell lines and normal osteoblast cells. The Specific Aims of this project will 1) determine the mechanism of post-transcriptional control of RUNX2 protein expression by CBFβ and 2) Identify the contribution of CBFβ to cap-dependent protein translation in osteosarcoma. De novo protein synthesis assays, investigation of CBFβ interactions with translational machinery proteins, and the ability CBFβ to influence RNA-binding and cap-binding protein interactions with RUNX2 mRNA will be used to uncover potential mechanisms by which CBFβ exerts post-transcriptional control of RUNX2. Ribosome footprinting, or Ribo-seq, combined with RNA-seq will identify the role of CBFβ on translational efficiency of RUNX2 and RUNX2-target gene products. Ribo-seq data will also provide a global view of the influence of CBFβ on protein translation, and subsequent pathway analysis will provide information about potential novel targets in OS. These studies, when combined with those of the current K01 SERCA, will advance research independence of the applicant and will be used to develop and refine research hypotheses for inclusion in a future R01 application.