Critical Role of Novel O-GlcNAcylation of S550 and S551 on the p65 Subunit of NF-B in Pancreatic Cancer

Simple Summary: NF-kappa B is an inflammatory protein that contributes to the low rate of survival observed in pancreatic cancer patients. The activation of NF-kappa B causes high expression of proteins that drive and sustain pancreatic cancer growth. Thus, it is important to understand how NF-kappa B is regulated to help with the diagnosis and treatment of pancreatic cancer. In this study, we discovered that the typical NF-kappa B subunit p65 is modified by O-GlcNAc at serines (S)550 and S551. To characterize the role of O-GlcNAcylated p65 at S550 and S551, we overexpressed p65 serine-toalanine (S-A) mutants, such as S550A, S551A, and S550A/S551A, in pancreatic cancer cells. Using this model, we show that the p65 mutants reduce NF-kappa B transcriptional activity, nuclear translocation, p65 phosphorylation, and target gene expression. We also observed that the p65 mutants blocked pancreatic cancer cell growth and migration. This suggests the contribution of p65 O-GlcNAcylation at S550 and S551 to pancreatic cancer phenotypes. Taken together, our study uncovers a novel aspect of NF-kappa B regulation, which could aid future therapeutic development by targeting O-GlcNAc transferase (OGT) in pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, with a mere 5-year survival of similar to 10%. This highlights the urgent need for innovative treatment options for PDAC patients. The nuclear factor kappa B (NF-kappa B) is a crucial transcription factor that is constitutively activated in PDAC. It mediates the transcription of oncogenic and inflammatory genes that facilitate multiple PDAC phenotypes. Thus, a better understanding of the mechanistic underpinnings of NF-kappa B activation holds great promise for PDAC diagnosis and effective therapeutics. Here, we report a novel finding that the p65 subunit of NF-kappa B is O-GlcNAcylated at serine 550 and 551 upon NF-kappa B activation. Importantly, the overexpression of either serine-to-alanine (S-A) single mutant (S550A or S551A) or double mutant (S550A/S551A) of p65 in PDAC cells impaired NF-kappa B nuclear translocation, p65 phosphorylation, and transcriptional activity, independent of I kappa B alpha degradation. Moreover, the p65 mutants downregulate a category of NF-kappa B-target genes, which play a role in perpetuating major cancer hallmarks. We further show that overexpression of the p65 mutants inhibited cellular proliferation, migration, and anchorage-independent growth of PDAC cells compared to WT-p65. Collectively, we discovered novel serine sites of p65 O-GlcNAcylation that drive NF-kappa B activation and PDAC phenotypes, thus opening new avenues by inhibiting the NF-kappa B O-GlcNAcylation enzyme, O-GlcNAc transferase (OGT), for PDAC treatment in the future.