Tuesday, March 21, 2023
3/21/2023
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 (CBFß) 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 CBFß. Yet, this protein is upregulated in OS, demonstrates increased expression in metastatic lesions, and is associated with reduced disease free and overall survival. CBFß appears to control RUNX2 expression through post-transcriptional mechanisms independent of RUNX2 protein stability or proteasomal degredation. Recently, CBFß 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 CBFß 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 CBFß knockout cells transfected with either wild-type CBFß or asite-directed mutant of CBFß 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 CBFß and 2) Identify the contribution of CBFß to cap-dependent protein translation in osteosarcoma. De novo protein synthesis assays, investigation of CBFß interactions with translational machinery proteins, and the ability CBFß to influence RNA-binding and cap-binding protein interactions with RUNX2 mRNA will be used to uncover potential mechanisms by which CBFß exerts post-transcriptional control of RUNX2. Ribosome footprinting, or Ribo-seq, combined with RNA-seq will identify the role of CBFß on translational efficiency of RUNX2 and RUNX2-target gene products. Ribo-seq data will also provide a global view of the influence of CBFß 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.
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