Abstract 1650: Characterization of nucleolar biological functions via Auxin inducible degron-mediated acute depletion in triple negative breast cancer cells

Abstract

Abstract Nucleoli are large nuclear sub-compartments where vital cellular processes, such as rRNA production and ribosome assembly, take place. Aggressive tumor types, such as Triple Negative Breast Cancer (TNBC), require increased ribosome biogenesis to support their enhanced proliferation. Therefore, it has been suggested that the nucleolus could be a valid anti-neoplastic target. However, we still have a limited understanding of the cancer-specific functions of the nucleolar components, preventing the effective development of therapeutic agents. The characterization of nucleolar proteins is technically challenging. Several nucleolar proteins are essential, and their complete abrogation cannot be achieved through conventional RNAi or CRISPR-KO approaches. Additionally, many nucleolar proteins display peculiar biophysical properties, and their functions are connected to their abundance. Therefore, artificial overexpression systems might not fully recapitulate their endogenous biological functions. To overcome these challenges, we implemented an Auxin Inducible Degron (AID) system to acutely abrogate endogenous nucleolin, one of the most abundant nucleolar proteins, and asses its impact on the proliferation of TNBC cell lines. We used CRISPR/Cas9 editing in TNBC cell lines to modify the endogenous NCL gene, fusing it with an AID tag. Then, we stably transduced the cells with a modified Oryza Sativa TIR1 (OsTIR1-F74G) E3 ubiquitin ligase. In response to the synthetic phytohormone phenyl-auxin, OsTIR1 leads to the proteasomal degradation of AID-containing endogenous nucleolin in less than 6h, sparing any other human protein. RNA sequencing analysis showed that nucleolin degradation significantly alters the expression of genes involved in cell cycle progression. Accordingly, nucleolin degradation reduces cancer cell proliferation in vitro, associated with increased propidium iodide staining, suggestive of a G2/M cell cycle arrest. However, immunofluorescence analyses revealed that nucleolin abrogation leads to the accumulation of bi-nucleated cells with actin patches. These findings are indicative of potential cytokinesis defects due to altered spindle tension during mitosis. Notably, nucleolin abrogation increased the efficacy of chemical inhibitors of the Anaphase Promoting Complex on suppression of cell proliferation. To the best of our knowledge, this study is the first reporting the acute abrogation of an endogenous, highly abundant nucleolar protein, using the AID system. This approach allowed the characterization of nucleolin biological functions with unprecedented temporal resolution, shedding light on new biological functions in the regulation of cell division. Finally, our results suggest that nucleolin inhibition could enhance the therapeutic activities of drugs affecting cell cycle progression in TNBC. Citation Format: Nastaran Samadi Rad, Joseph Mills, Anna Tessari, Damu Sunil Kumar, Vollter Anastas, Saranya Lamba, Ashley B Reers, Ilaria Cosentini, Zirui Zhu, Thomas J Magliery, Emanuele Cocucci, Lara Rizzotto, Dario Palmieri. Characterization of nucleolar biological functions via Auxin inducible degron-mediated acute depletion in triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1650.