Fig. 5: FASN and the Lands cycle limit PUFA content of phospholipids of KMLC.

Quantification of total (a) and newly synthetized (b) arachidonic acid (FA 20:4) in the PL fraction of the indicated cell lines (n = 3 biologically independent samples). Tracer incorporation was measured after 7-h incubation with ethyl acetate-1,2 ¹³C₂. c, d Incorporation of arachidonic acid (AA) alkyne in the indicated cell lines treated as indicated, and its quantification. n = 20–42 cells over 2 biologically independent samples. e Venn Diagram of the “metabolism of lipids and lipoproteins” (R-HSA-556833) genes upregulated in KM (H460, A549) and KRAS-WT (H661, H522) LC cells treated with FASNi. f, g Gene expression volcano plot and top KEGG pathways specifically upregulated in KMLC cells upon FASNi treatment. h, i Cell viability after siRNA-mediated knockdown of the indicated genes (n = 2 biologically independent experiments). Venn diagram summarizes lethal genes specific for KMLC cells (H460 and A549, Dunnett’s multiple comparison test with cutoff adj p < 0.05). j, k Quantification of newly synthetized- palmitate (FA 16:0) and total arachidonic acid (FA 20:4) in the PL fraction of indicated cell lines after shRNA-mediated knockdown of lysophosphatidylcholine acyltransferase 3 (LPCAT3) and phospholipase A2 group IV C (PLA2G4C) (n = 4 and n = 3 biological independent samples). l, m Working model explaining the role of FASN in the regulation of the Lands cycle in KMLC. FASN is active: KM induces ROS that oxidize the PUFA acyl chain on PC. PLA2 removes the oxidized fatty acid (FA) on PC synthetizing a LysoPC. FASN and SCD1 produce saturated FA (SFA) and MUFA, respectively. SFA/MUFA are transferred to CoA by ACSL3. These acyl-CoAs are used by LPCAT3 to re-acylate the LysoPC forming again PC. Inhibition of FASN (m) causes the depletion of SFA/MUFA and uptake of exogenous PUFA for the re-acylation of LysoPC. This process increases the amount of PUFA-PC and PUFA-LysoPC, which are oxidized under oxidative stress (oxPUFA-PC and oxPUFA-LysoPC). Accumulation of these lipid species leads to cell death via ferroptosis. Bars represent mean ± SD. In a, b, d, j, k Statistical analyses were done using two-tailed unpaired Student’s t-test. In f p-values were generated using one-way ANOVA and adjusted for multiple comparisons using a Benjamini–Hochberg correction (FDR). In h statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test.