Abstract B041: MEK/ERK-dependent regulation of TFEB in response to chemotherapeutic stress

Abstract

Our previous studies identified Transcription factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis, as highly expressed in pancreatic cancer (PDAC) cells, where it exhibits prominent nuclear localization even under basal conditions. Functional studies demonstrated that TFEB depletion in PDAC cells impairs clonogenic growth in vitro and limits tumor growth in orthotopic mouse models, supporting a pro-tumorigenic role for TFEB in PDAC. More recently, we demonstrated that the chemotherapeutic agent gemcitabine and the nucleolar stressor CX-5461 drive TFEB nuclear accumulation. Interfering with TFEB sensitized PDAC cells to gemcitabine and CX-5461, implicating TFEB as a mediator of chemoresistance. Still, the signaling mechanisms coupling chemotherapeutic stress to TFEB regulation remain unknown. Therefore, our study aimed to identify signaling events triggered by chemotherapeutic drugs that impact TFEB. Experiments were performed using pancreatic cancer cells (MIA PaCa-2, PANC1) and PDAC patient-derived organoids PDM-41. We found that gemcitabine and CX-5461 activate MEK/ERK signaling. Consistent with a protective role for this pathway, co-treatment with the MEK inhibitor trametinib enhanced the growth-inhibitory effects of gemcitabine in PDM-41 organoids. In parallel, as previously reported, we observed that gemcitabine and CX-5461 induced an electrophoretic mobility shift of TFEB, indicative of changes in TFEB phosphorylation, although less pronounced than that induced by the mTOR inhibitor Torin1. Despite these differences, both chemotherapeutics and Torin1 promoted TFEB nuclear accumulation. Still, whereas Torin1 reduced phosphorylation of TFEB at serine 211 (S211), a phospho-site classically associated with TFEB cytoplasmic retention, gemcitabine failed to reduce S211 phosphorylation and instead modestly increased TFEB S211 phosphorylation. Notably, trametinib prevented the gemcitabine-induced increase in TFEB S211 phosphorylation, implicating MEK/ERK signaling in this regulatory event. Collectively, these findings reveal that chemotherapeutic agents and mTOR inhibition converge on TFEB nuclear localization through distinct phosphorylation-dependent mechanisms. Furthermore, our results challenge the current paradigm that S211 phosphorylation is associated with cytoplasmic TFEB retention and suggest that S211-phosphorylated TFEB can accumulate in the nucleus under specific stress conditions. These observations identify a previously unrecognized MEK/ERK-TFEB signaling axis and provide a mechanistic framework for understanding TFEB-dependent chemoresistance in PDAC. Microsoft Copilot was used for language editing and refinement of the abstract to improve clarity and readability; the scientific content and conclusions were developed and reviewed by the authors.