Abstract 476: A human scFv as a tool to understand the biogenesis of a subset of oncogenic microRNAs

Abstract

Abstract Trans-acting factors such as RNA-binding proteins (RBPs) and microRNAs (miRNA) have recently been recognized as prime regulators of gene expression during tumorigenesis. Nucleolin (NCL) is the most abundant nucleolar RNA-binding protein, controlling critical cellular processes such as chromatin remodeling, ribosome biogenesis and miRNA biogenesis. In the cytoplasm, NCL regulates gene expression post-transcriptionally by controlling mRNA stability and/or translation via ARE- and miRNA-mediated pathways. In contrast, NCL at the cell surface plays pivotal roles in cancer progression and metastasis in many types of tumors. Therefore, surface-NCL is an appealing target for cancer therapy. Earlier our group described 4LB5, a human-derived single chain fragment variable (scFv) that specifically targets surface-NCL exerting cytotoxic effects, both in vitro and in vivo. We have identified the central RNA-binding domains (RBD1-4) of NCL as 4LB5-interacting sites. 4LB5-mediated blocking of NCL-RBDs significantly impairs the biogenesis of a subset of oncogenic miRNAs, such as miR-21, -221 and -222, and their binding to the microprocessor complex. The structural fold of all the 4 RBDs is highly conserved, but there are significant differences in their sequences. RBD1-2 have been well studied in their mechanism and capability to form high-affinity RNA-binding complexes, but RBD 3-4 have not been explored to the same extent. We hypothesize that consecutive or selective combinations of RBDs in NCL might have distinct roles in RNA biology. NCL RBD1-2 domains are well documented in the literature for their high affinity for mRNAs and rRNAs. However, RNA-specificity on NCL-RBDs has yet to be elucidated. In this study, we used protein-antibody and protein-RNA docking analyses to identify potential differential binding affinities between multiple NCL modules (RDB1-2, RBD3-4 and RBD1-4) and either 4LB5 or miRNAs. In silico results were also evaluated by multiple biochemical assays (ELISA, Surface Plasmon Resonance, Pull-Down and Electrophoretic Mobility Shift Assays) using NCL Modules, 4LB5 and RNA molecules. Finally, competition assays were performed to assess ability of our scFv to impair miRNA binding to NCL. Our results indicate the importance of the NCL region targeted by 4LB5 in microRNA maturation. Functional studies will help us to better elucidate the biologic role of NCL RBDs, and their involvement in cancer pathogenesis. Citation Format: Valerio Embrione, Michael Scarpati, Zachary D. Uzzel, Rahimah Ahmad, Fortuna A. De Sury, Tyler J. Sheetz, Vincenzo Coppola, Shaneen M. Singh, Anjana D. Saxena, Carlo M. Croce, Dario Palmieri. A human scFv as a tool to understand the biogenesis of a subset of oncogenic microRNAs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 476.