Abstract 2025 Investigating the role of the microtubule severing enzyme, katanin, in EMT and EMT

Cancer metastasis and cell division are interconnected and highly dynamic processes. The underlying characterizations of cytoskeletal rearrangements, altered cell-to-cell contacts, adapted transcriptional regulation, and dynamic signaling events are assumed to be similar if not the same for both processes. Metastasis is explained through cancer cells undergoing transitions between a highly proliferative epithelial state to a migratory mesenchymal state, and vice-versa. These transitions are referred to as epithelial-to-mesenchymal (EMT) and mesenchymal-to-epithelial (MET) transitions. Previous studies emphasized the underlying highly coordinated rearrangement of the cytoskeletal network within EMT and MET, with a large focus in actin and intermediate filament cytoskeletons. However, the microtubule network has been understudied comparatively. This cytoskeleton component undergoes dynamic rearrangements during the same processes overlapping cell division and EMT/MET. Microtubule stability is in part coordinated by severing enzymes altering their biochemical and biophysical properties. For example, katanin, is a microtubule severing enzyme that is targeted to branch points and defects within the cytoskeleton lattice to alter microtubule stability. Furthermore, katanin is also known to be targeted by DYRK2 and LAPSER1. DYRK2 and LAPSER1 modulate the activity of katanin, and these proteins are known coordinators of cell cycle and signaling within EMT and MET. This research aims to investigate the microtubule cytoskeleton dynamics in EMT and MET through the microtubule severing enzyme katanin and the proteins that regulate katanin activity. This will be focused on lung cancer, using the A549 non-small cell lung cancer adenocarcinoma cell line. To investigate the influence of katanin in EMT/MET. To investigate the effects of katanin perturbation on EMT/MET signaling dual fluorescent reporter, inducible CRISPR-Cas9, and overexpression cell lines will be generated. Using these cell lines, proliferation, migration, and invasion assays will be performed to assess the dynamics of EMT/MET following katanin perturbation. Additionally, proteomic mass spectrometry experiments will provide details on interactions, protein levels, and modifications of katanin in EMT and MET processes. We have established a model for studying EMT and MET within A549 cells following perturbation of katanin levels. Preliminary experiments show increase in katanin protein levels during mesenchymal phenotypes. Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number T32GM145388. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.