Pancreatic cancer cells elevate NAD+production in response to nutrient limitation and chemotherapeutic stress

Introduction: Pancreatic cancer (PC) cells are consistently in a state of aneuploidy, nutrient deficiency, and hypoxia-induced stress, which may necessitate increased NAD + levels to maintain critical survival functions such as antioxidant defense, mitochondrial activity, and DNA repair. Therefore, we hypothesized that PC cells adapt to chemotherapy and nutrient deprivation in their microenvironment by elevating NAD+ levels. Methods: We used liquid chromatography-mass spectrometry (LC-MS) to measure NAD + and its precursors in cancer versus non-cancer cells under stress (nutrient deprivation and chemotherapy). Western blot and qPCR were used to assess enzyme expression in NAD+ synthesis pathways following chemotherapy. Athymic nude mice and C57BL/6 mice were injected with MIA PaCa-2, respectively. Once palpable tumors formed, the mice received treatments with either vehicle or oxaliplatin (5 mg/kg, administered twice weekly) for 21 days, and tumors were collected and tested for NAD+ and its precursors. Results: Baseline intracellular NAD + and NAM levels were higher in PC cells (PANC-1, MIA PaCa-2, and BxPC-3) compared to non-cancer cells (HPDE and HPNE) and stayed elevated after 72 hours of treatment with complete media. During increased cellular stress, such as glucose limitation, NAD + and NAM levels rose more significantly in MIA PaCa-2 cells than in HPNE cells, demonstrating pancreatic cancer cells' ability to adapt to the increased NAD + demand required to counteract oxidative and energy stress caused by glucose withdrawal. Intracellular NAD + levels consistently remained higher in PANC-1 and MIA PaCa-2 cells than in HPNE cells during four days of growth. Interestingly, NAM concentrations in the media declined by ~ 40% over 72 hours across all cell lines, regardless of glucose level, indicating strong utilization of external NAD+ precursors. Both Oxaliplatin and 5-fluorouracil (5FU) significantly increased NAD + and NAM levels in MIA PaCa-2 and PANC-1 cells, supporting the model that NAD + is necessary for fueling survival mechanisms under stress. Oxaliplatin treatment increased mRNA levels of NMNAT1 and protein levels of NAPRT, NRK, and NMNAT1, the main enzymes in the Preiss-Handler and NAD + salvage pathways. Although chemotherapy did not change intracellular NAMPT (iNAMPT) mRNA levels, it increased iNAMPT protein levels, whereas extracellular NAMPT (eNAMPT) protein levels remained unchanged. Chemotherapy also increased NAD+ levels in both MIA PaCa-2 and KPC mouse xenograft models in vivo. Conclusion: These findings suggest that PC cells actively prioritize NAD + synthesis as an adaptive mechanism to survive the harsh microenvironment and develop chemoresistance. Therefore, targeting NAD+ synthesis pathways offers a therapeutic vulnerability to enhance chemotherapy susceptibility in pancreatic cancer.