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

确定细胞质核心结合因子β在骨肉瘤蛋白翻译调节中的作用

• 批准号: 10570684
• 负责人: Luke A. Wittenburg
• 金额: $ 11.14万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-15 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10570684
• 关键词: Address; Adolescent and Young Adult; Affect; Binding; Binding Sites; Biological Assay; Biology; Bone neoplasms; Breast Cancer Cell; Cell Line; Cell Survival; Cells; Combined Modality Therapy; Complex; Core-Binding Factor; Cytoplasm; DNA Binding; Data; Development; Diagnosis; Disease; Family; Future; Genetic Transcription; Global Change; Goals; Grant; Human; Investigation; Knock-out; Knowledge; Measures; Messenger RNA; Molecular Target; Mutation; Neoplasm Metastasis; Nuclear; Oncogenic; Oncoproteins; Operative Surgical Procedures; Osteoblasts; Pathogenesis; Pathway Analysis; Pathway interactions; Patients; Peptides; Persons; Post-Transcriptional Regulation; Primary Neoplasm; Process; Proteasome Inhibition; Protein Biosynthesis; Proteins; RNA Binding; RNA Cap-Binding Proteins; Radiation therapy; Regulation; Reporting; Research; Ribosomes; Role; Signal Pathway; Signal Transduction; Skeletal Development; System; Therapeutic; Time; Transfection; Translation Initiation; Translations; cancer cell; chemotherapy; driver mutation; gene product; improved; in vivo; insight; knowledge base; member; molecular targeted therapies; mutant; neoplastic cell; new therapeutic target; novel; osteoblast differentiation; osteosarcoma; overexpression; posttranscriptional; primary bone cancer; protein expression; ribosome profiling; targeted agent; therapeutically effective; transcription factor; transcriptome sequencing; treatment strategy; tumor heterogeneity

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.

项目摘要/摘要 骨肉瘤(OS)是人类最常见的原发骨肿瘤，占大多数病例 影响青少年和青壮年。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。没有意义 在过去的40年里，生存时间得到了改善，这说明了对小说的迫切需求 各种形式的治疗。 核心结合因子β(cbfβ)蛋白是异源二聚体转录因子复合体的一个亚基 它与Run-Related转录因子2(RUNX2)结合，以协调有组织的骨骼发育。两者都有 各组分在OS中过度表达，其正常活动受到失调。没有固有的DNA结合 也不是其自身的转录活性，对胞质CBFβ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，并与减少相关 无疾病和总存活率。CBFβ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变性无关的机制。最近，cbfβ一直在 参与调控乳腺癌细胞中蛋白质翻译的启动。该项目旨在确定和 描述CBFβ在OS中启动帽子依赖的蛋白质翻译过程中的非规范调节作用 恶性细胞被认为对这种物质上瘾。该项目的目标将通过 靶向野生型CBFβ或Asite定向突变体的CBFβ基因敲除细胞的应用 与RUNX2的结合部位。一种抑制肽将用于其他OS细胞系和正常成骨细胞 细胞。该项目的具体目标将1)确定RUNX2转录后调控的机制 CBFβ的蛋白表达和2)确定CBFβ在大鼠帽依赖蛋白翻译中的作用 骨肉瘤。 蛋白质合成实验、脑血流量β与翻译机制相互作用的研究 蛋白质，以及CBFRNA影响β结合和帽结合蛋白与RUNX2mRNA相互作用的能力 将被用来揭示CBFβ对RUNX2进行转录后控制的潜在机制。 核糖体足迹或ribo-seq与rna-seq相结合将确定cbfβ在翻译中的作用。 RUNX2和RUNX2靶基因产物的效率。Ribo-seq数据还将提供全球视角 CBFβ对蛋白质翻译的影响，后续的途径分析将提供以下信息 操作系统中潜在的新目标。 这些研究与目前K01 SERCA的研究相结合，将推动研究 申请人的独立性，并将被用来发展和完善研究假设，以纳入 未来的R01应用。