Chrysosplenetin-induced TMED3 aggregation triggers unfolded protein response in pancreatic cancer

Pancreatic cancer (PC) continues to demand urgent therapeutic innovation given its limited treatment options. Here, through phenotypic screening of a natural product library followed by systematic validation, we identified chrysosplenetin (CHR) as a bioactive compound with anti-PC activity. Transcriptomic profiling and functional analyses demonstrated that CHR induced endoplasmic reticulum (ER) stress, thereby activating the unfolded protein response (UPR) and subsequent apoptosis, while paradoxically triggering a protective autophagy. Genetic or pharmacological inhibition of autophagy potentiated CHR-induced antitumor efficacy. Using an integrated approach including proteomic analysis, bio-layer interferometry, cellular thermal shift assay, and molecular docking, we confirmed TMED3 as a direct target of CHR. Functional studies revealed that disruption of TMED3 expression partially restored ER homeostasis, attenuating CHR-induced UPR activation and apoptosis. Furthermore, CHR combined with standard chemotherapy or autophagy inhibitors exhibited enhanced antitumor activity in preclinical models, providing a basis for future therapeutic exploration of the TMED3–ER stress axis. Together, our findings establish TMED3 as a novel therapeutic target in PC, revealing that disrupting ER proteostasis via TMED3 perturbation represents a potential therapeutic strategy warranting further investigation.