Autophagy Assassins: Exosome-Mediated Inhibition of Calcineurin-TFEB Signaling

Overview

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers, with a survival rate of approximately 13%. 90% of PDAC tumors possess KRAS mutations, which drive tumor hyperproliferation, promoting reliance on autophagy for tumor survival. The transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, is tightly related to autophagy. More specifically, TFEB regulates the CLEAR gene network, controlling lysosome biogenesis and autophagy. In low-nutrient environments, TFEB localization is regulated by the phosphatase calcineurin, which dephosphorylates TFEB and causes its nuclear localization, upregulating the CLEAR network. Disruption of this dephosphorylation and localization of TFEB is a promising target to disrupt autophagy and subsequent tumor survival in PDAC. To disrupt calcineurin-TFEB signaling, this study investigated whether exosomal delivery of an NFATc1-derived peptide containing the calcineurin-binding LxVP motif would competitively bind calcineurin, prevent TFEB nuclear localization, and disrupt autophagy signaling in PDAC cells. In-silico docking simulations using CABS-dock were performed to confirm peptide binding to the calcineurin LxVP pocket. Exosomes were loaded with NFATc1-YLAVP peptide using saponin-mediated permeabilization, and encapsulation was validated using a protease protection assay with Proteinase K ± saponin through fluorescence signals, giving a 40.3% loading efficiency. PANC-1 cells were seeded, treated with varying peptide concentrations loaded into the exosomes, and starved with HBSS to induce autophagy. Immunofluorescence microscopy was used to quantify TFEB nuclear localization, and data analysis yielded a 49.7% reduction. These results demonstrate a successful exosome-mediated peptide delivery strategy capable of inhibiting calcineurin-TFEB signaling and reducing autophagy activity in PDAC cells.

Competition history

• CSEF 2026 Medicine & Physiology (Senior Division) · Entry S-15-34

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