Lipid Peroxidation Regulators GPX4 and FSP1 as Prognostic Markers and Therapeutic Targets in Advanced Gastric Cancer

Abstract

Gastric cancer is one of the most common cancers worldwide, and new therapeutic strategies are urgently needed. Ferroptosis is an intracellular iron-dependent cell death induced by the accumulation of lipid peroxidation, a mechanism different from conventional apoptosis and necrosis. Therefore, induction of ferroptosis is expected to be a new therapeutic strategy. Glutathione peroxidase 4 (GPX4) and ferroptosis suppressor protein 1 (FSP1) have been identified as the major inhibitors of ferroptosis. Herein, we performed immunohistochemistry for GPX4, FSP1, and 4-HNE using tissues from patients with gastric cancer and investigated the relationship between these factors and prognosis. Patients with high GPX4 expression or high GPX4 expression and low 4-HNE accumulation tended to have a poor prognosis (p = 0.036, 0.023), whereas those with low FSP1 expression and high 4-HNE accumulation had a good prognosis (p = 0.033). The synergistic induction of cell death by inhibiting GPX4 and FSP1 in vitro was also observed, indicating that the cell death was non-apoptotic. Our results indicate that the expression and accumulation of lipid peroxidation-related factors play an important role in the clinicopathological significance of gastric cancer and that novel therapeutic strategies targeting GPX4 and FSP1 may be effective in treating patients with gastric cancer who have poor prognosis.

Keywords: chemotherapy; ferroptosis; gastric cancer; lipid peroxidation.

Figure 1

Immunostaining of gastric cancer tissue for GPX4 (a,b), FSP1 (c,d), and 4-HNE (e,f). (a,b) Representative weak (a) and strong (b) staining in GPX4 immunostaining. (c,d) Representative weak (c) and strong (d) staining in FSP1 immunostaining. (e,f) Representative weak (e) and strong (f) staining in 4-HNE immunostaining. The magnification is 200×, and the scale bar is 50 μm.

Figure 2

Kaplan–Meier analysis of gastric cancer, stratified by result of immunohistochemistry. (a) High expression of GPX4 correlated with poor prognosis (p = 0.036). (b) No significant difference was observed between FSP1 high and low groups (p = 0.677). (c) No significant difference was observed between 4-HNE high and low groups (p = 0.245). (d) Stratification based on GPX4 and 4-HNE. (e) The prognosis of high GPX4 expression and low 4-HNE accumulation was worse than that of the others (p = 0.023). (f) Stratification based on FSP1 and 4-HNE. (g) The prognosis of low FSP1 expression and high 4-HNE accumulation was better than that of the others (p = 0.033).

Figure 3

Effect of GPX4 on the proliferation and sensitivity to apoptosis-inducing drugs. (a,b) Cell proliferation assay under treatment with RSL3, a GPX4 inhibitor. Cell proliferation was inhibited in both MKN-45 (a) and KATO III (b) cells (MKN-45, p = 0.010 at 1.25 μM RSL3, p = 0.002 at 2.5 μM RSL3, 48 h after treatment; KATO III, p = 0.028 at 0.25 μM RSL3, p = 0.006 at 1 μM RSL3, 72 h after treatment). (c,d) Histogram of flow cytometry performed with propidium iodide 48 h after administration of RSL3, a GPX4 inhibitor, and cisplatin, an apoptosis inducer. (e,f) Cell death rate measured by flow cytometry. In both MKN-45 and KATO III cells, RSL3 treatment significantly increased cell death with cisplatin treatment (MKN-45, p = 0.006; KATO III, p = 0.043).

Figure 4

Synergistic effects of GPX4 and FSP1 inhibition on cell death induction. (a–c) Cell death rates with administration of RSL3, an inhibitor of GPX4, and iFSP1, an inhibitor of FSP1. In MKN-45 (a) and KATO III (b) cells, the synergistic effects on cell death induction were observed; in AGS (c) cells, combined treatment did not dramatically increase the cell death rate. (d,e) Investigation of type of cell death by cell death inhibitors. Cell death was fully inhibited by ferrostatin-1, vitamin E (vit E), liproxstatin, ferroptosis inhibitors, and necrostatin, necroptosis inhibitor. Cell death was partially inhibited by deferoxamine, an iron chelator.