Polyunsaturated fatty acid biosynthesis pathway determines ferroptosis sensitivity in gastric cancer

Abstract

Ferroptosis is an iron-dependent regulated necrosis mediated by lipid peroxidation. Cancer cells survive under metabolic stress conditions by altering lipid metabolism, which may alter their sensitivity to ferroptosis. However, the association between lipid metabolism and ferroptosis is not completely understood. In this study, we found that the expression of elongation of very long-chain fatty acid protein 5 (ELOVL5) and fatty acid desaturase 1 (FADS1) is up-regulated in mesenchymal-type gastric cancer cells (GCs), leading to ferroptosis sensitization. In contrast, these enzymes are silenced by DNA methylation in intestinal-type GCs, rendering cells resistant to ferroptosis. Lipid profiling and isotope tracing analyses revealed that intestinal-type GCs are unable to generate arachidonic acid (AA) and adrenic acid (AdA) from linoleic acid. AA supplementation of intestinal-type GCs restores their sensitivity to ferroptosis. Based on these data, the polyunsaturated fatty acid (PUFA) biosynthesis pathway plays an essential role in ferroptosis; thus, this pathway potentially represents a marker for predicting the efficacy of ferroptosis-mediated cancer therapy.

Keywords: ELOVL5; FADS1; arachidonic acid; ferroptosis; lipid peroxidation.

Fig. 1.

Mesenchymal-type gastric cancer cells (GCs) are sensitive to ferroptotic cell death. (A) Relative viability of GCs treated with RSL3 (0.1 to 5 μM) for 24 h. Data are the means ± SD (n = 3 independent experiments). (B and C) Scatterplots between area under curve (AUC) for RSL3 in A and mesenchymal (B) or stromal (C) scores in GCs. Dots indicate each GC line, and Pearson’s correlation coefficients and P values are shown in graphs. (D) Relative viability of mesenchymal-type (Hs746T and SNU-484) and intestinal-type (NCI-N87 and SNU-719) GCs treated with RSL3 and ML210 (0.01 to 10 μM) for 24 h. Data are the means ± SD (n = 3 independent experiments). (E) Relative viability of Hs746T and SNU-484 cells treated with 1 μM RSL3 in the presence and absence of ferrostatin-1 (Fer-1, 1 μM) or liproxstatin-1 (Lip-1, 200 nM). Data are the means ± SD (n = 3 independent experiments, with ***P < 0.001 according to two-sided Student’s t tests). (F) Relative viability of Hs746T and SNU-484 cells treated with 5 μM ML210 and inhibitors. Data are the means ± SD (n = 3 independent experiments, with **P < 0.01 and ***P < 0.001 according to two-sided Student’s t tests). (G) Relative viability of Hs746T cells treated with zVAD-fmk (zVAD, 10 μM), necrostatin-1 (Nec-1, 30 μM), Fer-1 (1 μM), and/or RSL3 (1 μM). Data are the means ± SD (n = 3 independent experiments, with **P < 0.01 and ***P < 0.001 according to two-sided Student’s t tests).

Fig. 2.

ELOVL5 and FADS1 expressions are up-regulated in mesenchymal-type GCs. (A) Fold changes in the expression of genes associated with lipid and iron metabolism in mesenchymal-type GCs (Hs746T, SNU484, SNU-668, and YCC-16 cells) compared with intestinal-type GCs (MKN-45, NCI-N87, SNU-601, and SNU-719 cells) based on the results of a microarray analysis. Volcano plot showing fold changes and P values of mRNA expression levels. Significantly up-regulated or down-regulated genes (P value <0.001, |fold change| > 3) are shown in red or blue, respectively. (B) Levels of the ELOVL2, ELOVL4, ELOVL5, FADS1, FADS2, and ACSL4 mRNAs in GCs were analyzed using qRT-PCR. Relative expression levels were normalized to the β-actin expression levels. Data are the means ± SD (n = 3 independent experiments). (C) Scheme showing the incorporation of LA into the n-6 PUFA synthesis pathway. LA, linoleic acid; GLA, gamma-linoleic acid; EDA, eicosadienoic acid; DGLA, dihomo-γ-linolenic acid; AA, arachidonic acid; AdA, adrenic acid. (D) Western blots showing the levels of ELOVL5, FADS1, ACSL4, and GPX4 proteins in mesenchymal- and intestinal-type GCs. For the detection of ELOVL5 protein, samples were not boiled. (E) Volcano plot showing fold changes and P values for lipid species in mesenchymal-type GCs and intestinal-type GCs. Free AA, AdA, and PE (18:0/22:4) are highlighted in red. (F) Levels of PUFAs and PE detected in GCs using LC-MS/MS. Intensities were normalized to the total sum of the peak areas. Data are the means ± SD (n = 5 independent experiments), with *P < 0.05 and ***P < 0.001 compared to intestinal-type cells (n = 20) using one-sided Wilcoxon rank-sum test (n.s. denotes not significant). (G) Relative amounts of labeled (m + 18) and unlabeled (m + 0) PUFAs and PE in NCI-N87, SNU-719, Hs746T, and YCC-16 cells cultured in medium containing charcoal-stripped FBS and [U-¹³C₁₈] LA for 5 d.

Fig. 3.

The down-regulation of ELOVL5 and FADS1 expression alleviates ferroptosis. (A and B) Relative viability and lipid peroxidation levels in WT, ELOVL5-, and FADS1-KO YCC-16 cells treated with RSL3. Data are the means ± SD (n = 3 independent experiments, with ***P < 0.001 according to two-sided Student’s t tests). (C) Relative amounts of labeled (m + 18) and unlabeled (m + 0) PUFAs and PE in WT, ELOVL5-, and FADS1-KO YCC-16 cells cultured in medium containing charcoal-stripped FBS and [U-¹³C₁₈] LA. (D) Bar plots showing the ratios of AdA (C22:4) to AA (C20:4) and AA (C20:4) to DGLA (C20:3) in ELOVL5- and FADS1-KO YCC-16 cells. Levels of PUFAs and PE in ELOVL5- and FADS1-KO YCC-16 cells determined using LC-MS/MS. Intensities were normalized to the cellular protein level. Data are the means ± SD (n = 5 independent experiments), with *P < 0.05, **P < 0.01 and ***P < 0.001 according to a two-sided Student’s test (n.s. denotes not significant).

Fig. 4.

Inhibition of desaturase activity by SC-26196 or CP-24879 ameliorates ferroptosis. (A and B) Relative cell viability and LDH levels in Hs746T cells pretreated with 5 μM FADS2 inhibitor (SC-26196) or FADS1/2 inhibitor (CP-24879) for 4 h and treated with RSL3 for 24 h. Data are the means ± SD (n = 3 independent experiments, with **P < 0.01 according to two-sided Student’s t tests). (C) Lipid peroxidation levels in Hs746T cells pretreated with 5 μM FADS2 inhibitor (SC-26196) or FADS1/2 inhibitor (CP-24879) for 4 h and treated with RSL3 for 1 h. Data are the means ± SD (n = 3 independent experiments, with ***P < 0.001 according to two-sided Student’s t tests). (D) Cell death determined by LDH release from ELOVL5- or FADS1-depleted Hs746T cells cultured in cysteine/methionine-deficient medium for 24 h. Data are the means ± SD (n = 3 independent experiments, with *P < 0.05, **P < 0.01 and ***P < 0.001 according to two-sided Student’s t tests). (E) Cell death measured by LDH release from Hs746T cells pretreated with FADS inhibitors for 4 h, followed by an incubation with cysteine/methionine-deficient medium for 24 h. Data are the means ± SD (n = 3 independent experiments, with **P < 0.01 and ***P < 0.001 according to two-sided Student’s t tests).

Fig. 5.

Exogenous AA supplementation restores the sensitivity of intestinal-type GCs to ferroptosis. (A) Relative viability of GCs pretreated with 2.5 μM of AA for 16 h and treated with RSL3 for 24 h. Data are the means ± SD (n = 3 independent experiments). (B and C) Levels of the indicated lipids in NCI-N87 and SNU-719 cells treated with 2.5 μM AA for 3 h determined using LC-MS/MS. Intensities were normalized to cell numbers. Data are the means ± SD (n = 4 independent experiments), with *P < 0.05, and **P < 0.01 according to two-sided Student’s tests (n.s. denotes not significant). (D) Lipid peroxidation levels in NCI-N87 cells pretreated with 2.5 μM PUFAs for 16 h and treated with RSL3 for 1 h. Data are the means ± SD (n = 3 independent experiments), with ***P < 0.001 according to two-sided Student’s tests. (E and F) Cell viability and cell death as measured by LDH release from NCI-N87 cells pretreated with LA and AA for 16 h and treated with RSL3 for 24 h. Data are the means ± SD (n = 3 independent experiments), with **P < 0.01 and ***P < 0.001 according to two-sided Student’s tests. (G and H) Cell viability of and LDH release from NCI-N87 cells cultured with cysteine/methionine-deficient medium in the presence and absence of AA and Fer-1. Data are the means ± SD (n = 3 independent experiments, n.s. denotes not significant, ***P < 0.001 according to two-sided Student’s t tests).

Fig. 6.

ELOVL5 and FADS1 expression is down-regulated through DNA hypermethylation. (A and B) Manhattan plot of the methylation levels and statistical significance of methylation at each CpG site in the promoter regions of ELOVL5 (A) and FADS1/2 (B) in mesenchymal-type (Hs746T, SNU484, SNU-668, and YCC-16) and intestinal-type (NCI-N87, SNU-719, SNU-601, and MKN-45) GCs. The putative enhancer/promoter region of ELOVL5 (chr6: 53,211,316 to 53,214,820) is highlighted in orange. The putative regions of the FADS1 promoter (chr11: 61,584,650 to 61,586,300), FADS2 promoter (chr11: 61,594,300 to 61,595,600) and putative enhancer (chr11: 61,587,300 to 61,589,000) are colored in green, blue, and orange, respectively (36).