NRF2 interacts with distal enhancer and inhibits nitric oxide synthase 2 expression in KRAS-driven pancreatic cancer cells.

Nitric oxide is a pleiotropic free radical produced by three nitric oxide synthases (NOS1-3), of which inducible NOS2 is involved in tumor initiation and progression. In this study, RNA-seq, ChIP-seq and qRT-PCR experiments combined with bioinformatic analyses showed that NRF2 is a repressor of NOS2 gene by maintaining a distal enhancer located 22 kb downstream of TSS in an inactive state. Deletion of NRF2 leads to activation of the enhancer, which exerts a pioneering function before it is fully activated. Specifically, NRF2 controls the expression of NOS2 in response to intracellular oxidative stress and extracellular oxygen pressure. We found that abrogation of NOS2 expression by siRNAs partially reduced the ability of WT Panc-1 cells to form 3D spheroids, but strongly reduced the formation of 3D spheroids by NRF2-depleted Panc-1 cells. Mechanistically, this effect correlates with the finding that NOS2 and nitric oxide stimulate epithelial-to-mesenchymal transition in NRF2-depleted Panc-1 and MIA PaCa-2 cells. We also found that knockdown of NOS2 leads to blockade of 3D matrigel invasion of NRF2-depleted PDAC cells, demonstrating that a short-circuit in the reciprocal regulation of NOS2 and NRF2 attenuates the malignancy of PDAC cells. In summary, we show for the first time that: (i) NRF2 is a suppressor of NOS2 in pancreatic cancer cells; (ii) NRF2 binds to and inactivates an enhancer located 22 kb downstream of TSS of the NOS2 gene; (iii) activation of NOS2 requires suppression of NRF2; (iv) NOS2 is required for NRF2-depleted Panc-1 cells to maintain their malignancy and invasiveness.

Suppression of the KRAS-NRF2 axis shifts arginine into the phosphocreatine energy system in pancreatic cancer cells.

In pancreatic ductal adenocarcinomas (PDAC), the KRASG12D-NRF2 axis controls cellular functions such as redox homeostasis and metabolism. Disruption of this axis through suppression of NRF2 leads to profound reprogramming of metabolism. Unbiased transcriptome and metabolome analyses showed that PDAC cells with disrupted KRASG12D-NRF2 signaling (NRF2-/- cells) shift from aerobic glycolysis to metabolic pathways fed by amino acids. Metabolome, RNA-seq and qRT-PCR analyses revealed a blockade of the urea cycle, making NRF2-/- cells dependent on exogenous arginine for survival. Arginine is channeled into anabolic pathways, including the synthesis of phosphocreatine, which generates an energy buffer essential for cell growth. A similar switch was observed in tumor clones that had survived FOLFIRINOX therapy or blockade of KRAS signaling. Inhibition of the creatine pathway with cyclocreatine reduced both ATP and invasion rate in 3D spheroids from NRF2-deficient PDAC cells. Our study provides basis for the rational development of combination therapies for pancreatic cancer.