ACADS-dependent fatty acid oxidation drive mitochondrial remodeling in pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer projected to become the second leading cause of cancer-related deaths by 2030. While PDAC cells rely primarily on glycolysis, emerging evidence indicates that mitochondrial metabolism also contributes to tumor survival. The metabolic impact of stromal fibroblasts on pancreatic cancer mitochondria, particularly through fatty acid oxidation (FAO), remains poorly understood. Here, we investigated whether fibroblast exposure alters mitochondrial remodeling in PDAC cells and if this remodeling depends on mitochondrial fatty Acyl-CoA Dehydrogenase (ACADS) mediated FAO. Mitochondrial changes were assessed in Panc1 cells cultured alone and co-cultured with 3T3-L1 fibroblasts. MitoTracker Red staining and confocal microscopy were used to visualize mitochondrial content. Mitochondrial and metabolic proteins were quantified with Western blotting. Carnitine Palmitoyltransferase 1 (CPT1) was inhibited using Etomoxir (5-100 μM, 24 hrs). Compared with fibroblasts grown separately, Panc1 cells showed lower baseline mitochondrial mass. However, when direct co-cultured, Panc1 cells exhibited increased mitochondrial content and elevated expression of ACADS and Mitochondrially Encoded Cytochrome c Oxidase (MTCO1), suggesting mitochondrial adaptation in response to fibroblast interaction. To test whether this remodeling required downstream short-chain FAO, we inhibited CPT1, the rate-limiting enzyme for mitochondrial fatty-acid import. Although CPT1 does not directly regulate ACADS, Etomoxir-mediated CPT1 inhibition significantly decreased ACADS levels, consistent with reduced FAO flux. CPT1 blockade also triggered metabolic stress, evidenced by increased phosphorylated AMP-activated protein kinase alpha (p-AMPKalpha), and resulted in a dose-dependent increase in Dynamin-Related Protein 1 (DRP1), indicating enhanced mitochondrial fission and dysfunction. Collectively, our preliminary data suggests that PDAC cells depend on intact fatty-acid oxidation to maintain mitochondrial integrity.