ABSTRACT: Pancreatic cancer (PC) is a highly aggressive malignancy with a dismal prognosis. Approximately 95% of cases are pancreatic ductal adenocarcinoma (PDAC), which represents the most prevalent histological subtype and is currently the third leading cause of cancer-related mortality in the United States. The limited efficacy of existing therapies highlights an urgent need for novel therapeutic strategies. Metabolic reprogramming is a hallmark of cancer that enables tumor cells to sustain their energetic and biosynthetic demands, thereby supporting uncontrolled proliferation and survival. Targeting these metabolic adaptations represents a promising therapeutic avenue for PDAC. Mitochondrial oxidative phosphorylation (OxPhos) and glycolysis are the two principal energy-producing pathways in mammalian cells, including PDAC cells. Although numerous metabolic inhibitors have been developed for pancreatic and other cancers, clinical trials employing single-agent metabolic therapies have shown limited success, in part due to the pronounced metabolic heterogeneity and plasticity of PDAC. Cancer cells can dynamically shift between glycolysis and OxPhos in response to changes in nutrient and oxygen availability within the tumor microenvironment, thereby evading metabolic stress. We hypothesized that simultaneous inhibition of both glycolysis and OxPhos would overcome this metabolic plasticity and produce enhanced antitumor efficacy. In this study, we evaluated the in vitro effects of two small-molecule metabolic inhibitors, SR4 and lonidamine (LND), targeting OxPhos and glycolysis, respectively, in PDAC models. SR4 is a novel mitochondrial uncoupler that functions as a fatty acid–activated proton transporter, whereas LND inhibits aerobic glycolysis primarily through suppression of mitochondrial-associated hexokinase II, which catalyzes the first committed step of glycolysis. Combined treatment with SR4 and LND produced highly synergistic cytotoxic effects and significantly suppressed proliferation, clonogenic survival, migration, and invasion across PDAC cell lines with distinct metabolic phenotypes. Mechanistically, the dual therapy markedly inhibited glycolysis, mitochondrial OxPhos, and overall cellular bioenergetic capacity, independent of baseline metabolic dependency. The combination treatment also increased reactive oxygen species (ROS) levels and induced mitochondrial membrane depolarization. Furthermore, flow cytometry, protein expression and RNA transcriptomic analyses revealed that dual inhibition more effectively triggered cell cycle arrest, cellular senescence, autophagy/mitophagy, and apoptosis compared with either agent alone. Collectively, these findings demonstrate that concurrent targeting of mitochondrial uncoupling and glycolytic inhibition disrupts cellular bioenergetics and adaptive metabolic responses in PDAC, supporting this dual-metabolic strategy as a promising therapeutic approach for PC.
