Inducible nitric oxide synthase is repressed by Nuclear Factor Erythroid 2-Related Factor 2 in KRAS-driven pancreatic cancer cells

Nitric oxide is a pleiotropic free radical produced by three nitric oxide synthases (NOS1-3), of which the inducible NOS2 is involved in carcinogenesis. In this study, RNA-seq, ChIP-seq, and qRT-PCR experiments combined with bioinformatic analysis showed that NRF2 is a repressor of the NOS2 gene by maintaining a distal enhancer located 22 Kb downstream of the transcription start site (TSS) in a poised state. NRF2 depletion primed the activation of this enhancer, exerting a pioneering activity before achieving full activation. Specifically, NRF2 controls the inducible expression of NOS2 in response to intracellular oxidative stress and extracellular oxygen partial pressure. Abrogation of NOS2 expression using esiRNAs partially reduced or severely impaired the ability to form 3D spheroids respectively of WT and NRF2-depleted Panc-1 cells. Mechanistically, NOS2 and nitric oxide release are required to sustain the epithelial-to- mesenchymal transition (EMT) process. Importantly, silencing of NOS2 blocked 3D Matrigel invasion of NRF2-depleted Panc-1 cells, demonstrating that a short circuit in the reciprocal regulation of NOS2 and NRF2 attenuates PDAC cell malignancy. Overall, our data demonstrate for the first time that: i) NRF2 is a repressor of the NOS2 gene in pancreatic cancer cells; ii) identify a poised enhancer whose priming is triggered in conditions that simulate or stimulate NRF2 depletion; iii) NOS2 activity is required for NRF2-depleted Panc-1 cells to maintain their malignancy and invasiveness. Overall design: H3K27ac chipped samples from Panc-1 wt, NRF2KO and their input: cells were grown for 7 days in 3D matrix generating 3D spehorids. Cells were fixed with 1% formaldeyde for 15 min at room temperature and then quenched with 5 ml of PBS/GLYCINE for 5 min at room temperature. Matrigel was melted and Cells were lysed in 0.1 ml ChIP lysis buffer (50 mM Hepes-KOH, pH 7.5, 140 mM NaCl, 1% Triton X-100, and 0.1% deoxycholate with 0.1 mM PMSF and the EDTA-free Protease Inhibitor Cocktail). Samples were incubated on ice for 10 min and then centrifuged at 3,000 rpm for 3 min at 4°C. The pellet was resuspended in 500 µl lysis buffer 2 (10 mM Tris, pH 8.0, 200 mM NaCl with 0.1 mM PMSF and the EDTA-free Protease Inhibitor Cocktail) and incubated at room temperature for 10 min. Samples were centrifuged at 3,000 rpm for 5 min at 4°C. Next, the pellet was resuspended in 300 µl lysis buffer 3 (10 mM Tris, pH 8.0, 100 mM NaCl, 0.5% deoxycholate, and 0.1% SDS with 0.1 mM PMSF and the EDTA-free Protease Inhibitor Cocktail). Cells were sonicated using a Biorupter (Diagenode) for 15 min (30 s on, 1 min off). Next, 30 µl of 10% Triton X-100 was added to each tube, and then samples were centrifuged at max speed for 15 min at 4°C. The supernatant was transferred to new tubes, and the DNA concentration was quantified. Samples were precleared for 1 h at 4°C on a rotator using 8 µl protein A Dynabeads (Zymo) in ChIP lysis buffer. Samples were centrifuged at max speed for 15 min at 4°C, after which the supernatant was transferred to a new tube. 1/100 input was collected. 3 µg Antibodies were added O/N. 8 µl of protein A Dynabeads were added and chromatin was immunoprecipitated for 2h at 4°C. The following washes were performed for 15 min each by rotating for 15 min at 4°C: ChIP lysis buffer (twice), ChIP lysis buffer + 0.65 M NaCl, wash buffer (10 mM Tris-HCl, pH 8.0, 250 mM LiCl, 0.5% NP-30, 0.5% DOC, and 1 mM EDTA, pH 8.0), and TE (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA, pH 8.0). Beads were incubated with RNASEA solution (100ul TE/2ul Rnasi A (10mg/ml). DNA was eluted by adding to the beads 100ul of TES (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, pH 8.0, and 1% SDS) for 15 min at 65°C with brief vortexing every 2 min. Reversal of cross-linking was performed by incubating samples overnight at 65°C: transfer into 0.5ml tubes and place in termocycler. Proteins were digested using 150ug of proteinase K (0.5ul proteinase K) by incubating at 37°C for 5h. Finally, the DNA was purified using the Zymo Research Kit and eluted 2 times in 40ul H20.