Blockade of GCH1/BH4 Axis Activates Ferritinophagy to Mitigate the Resistance of Colorectal Cancer to Erastin-Induced Ferroptosis

Abstract

Ferroptosis, a type of cell death triggered by excessive accumulation of iron-dependent lipid peroxidation, possesses an excellent potential in cancer treatment. However, many colorectal cancer (CRC) cell lines are resistant to ferroptosis induced by erastin and RSL3, the classical ferroptotic inducers. Moreover, the underlying mechanism of resistance remains poorly elucidated. This study sought to discover the major factor contributing to ferroptosis resistance in CRC. The study findings will help design strategies for triggering ferroptosis for application in individualized tumor therapy. Here, we show that tetrahydrobiopterin (BH4) determines the sensitivity of CRC cells to ferroptosis induced by erastin. GTP cyclohydrolase-1 (GCH1) is the first rate-limiting enzyme of BH4. Genetic or pharmacological inhibition of GCH1 decreased BH4 and assisted erastin in cell death induction, lipid peroxidation enhancement, and ferrous iron accumulation. BH4 supplementation completely inhibited ferroptotic features resulting from GCH1 knockdown. Unexpectedly, GCH1 knockdown failed to enhance RSL3-induced cell death in CRC. Mechanistically, GCH1 knockdown drastically activated ferritinophagy during erastin treatment rather than RSL3 treatment. Administration of an autophagy inhibitor reversed erastin resistance in GCH1-knockdown cells. GCH1 inhibitor and erastin co-treatment in vivo synergistically inhibited tumor growth in CRC. Overall, our results identified GCH1/BH4 metabolism as a burgeoning ferroptosis defense mechanism in CRC. Inhibiting GCH1/BH4 metabolism promoted erastin-induced ferroptosis by activating ferritinophagy, suggesting that combining GCH1 inhibitors with erastin in the treatment of CRC is a novel therapeutic strategy.

Keywords: GCH1; colorectal cancer; erastin; ferritinophagy; ferroptosis; tetrahydrobiopterin.

FIGURE 1

Association between GCH1/BH4 metabolism and CRC ferroptosis. (A–C) The correlation between BH4 and erastin-induced cell death. (A) Cell viability upon treatment with different doses of erastin for 24 h in CRC cell lines. (B) BH4 abundance across 4 CRC cell lines at baseline. (C) BH4 levels were measured in HCT116 and HT29 cells treated with erastin for 24 h. The error bars represent standard deviation from three replicates (***p < 0.001, ****p < 0.0001, compared between the two groups by unpaired t-test). (D–F) Association between GCH1 and ferroptosis in CRC. (D) GCH1 expression of colon adenocarcinoma (COAD) vs normal adjacent tissues in Gene Expression Profiling Interactive Analysis tool (GEPIA) *p < 0.05. (E) GSEA showing the regulation of oxidative stress-induced cell death and ferrous iron binding pathways with apparent enrichment in low or high expression of GCH1 among patients with CRC. ES, enrichment score. (F) GCH1 protein level was measured in HCT116 and HT29 cells treated with RSL3 or erastin for 24 h. (G‒H), mRNA (G) and protein (H) expression of ferroptosis-related genes in the transfected cells. (G) Red color marked molecules with statistical difference (p < 0.05) between siNC and siGCH1 transfected cells.

FIGURE 2

Genetic deletion of GCH1/BH4 metabolism and induction of erastin synergistically induce ferroptosis in CRC cells. (A) BH4 levels were largely decreased by GCH1 knockdown for 24 h. (B,C) GCH1 knockdown sensitized HCT116 and HT29 cells to erastin. (B) Cell viability in HCT116 and HT29 cells treated with different doses of erastin and co-treated with 2 µM ferrostatin-1 for 24 h (****p < 0.0001, ns, not significant, the total variation between the two groups examined by two-way ANOVA with Tukey’s post hoc test). (C) Representative FACS plots of annexin V-FITC/PI staining in HCT116 cells. (D‒E), GCH1 knockdown promoted erastin-induced lipid peroxidation, measured using the MDA assay kit (D) or BODIPY-C11 staining (E) in HCT116 and HT29 cells (Bar graph showing the ratio of lipid ROS normalized to DMSO-treated siNC cells). (F,G) GCH1 knockdown promoted erastin-induced cellular Fe²⁺ (F) and mitochondrial Fe²⁺ (G) accumulation, detected separately using FerroOrange and MitoferroGreen staining. Scale bar, 20 µM. The error bars represent standard deviation from at least three replicates (##p < 0.01, ###p < 0.001, ####p < 0.0001, compared between siNC and siGCH1 by unpaired t-test) (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared between the two groups by unpaired t-test).

FIGURE 3

Supplementation of BH4 protects CRC cells from ferroptosis promoted by GCH1 knockdown. (A) Supplementation of 50 µM BH4 restored the BH4 levels in siGCH1 transfected cells. (B-F) BH4 supplemented in (A) reversed cell death, lipid peroxidation, and ferrous iron accumulation caused by GCH1 knockdown during erastin treatment. (B) Cell viability was assessed by CCK-8. Lipid peroxidation was detected using the MDA assay kit (C) or BODIPY-C11 staining (D) (The ratio of lipid ROS normalized to erastin-treated siNC cells). The cellular Fe²⁺ (E) and mitochondrial Fe²⁺ (F) were detected using FerroOrange and MitoferroGreen staining, separately. Scale bar, 20 µM. The error bars represent standard deviation from at least three replicates (#p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001, ns, not significant, compared between siNC and siGCH1 by unpaired t-test) (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant, compared between the two groups by unpaired t-test).

FIGURE 4

Blockade of GCH1/BH4 sensitizes erastin-induced ferroptosis by activating ferritinophagy. (A) Western blot analysis of GCH1, xCT, GPX4, NCOA4, FTH1, LC3B, and GAPDH in HCT116 and HT29 cells treated with 40 µM erastin for 24 h. The numbers below the LC3 lane indicate the ratio of LC3B-II/LC3B-I. (B) Autophagic flux was determined by the accumulation of LC3B-II in a 4-h treatment period with 25 nM bafilomycin A1 (BafA1). (C) Cell viability after pretreatment with 3-methyladenine (3-MA) for 24 h and treatment with erastin for 24 h in HCT116 and HT29 cells. (D) Western blot analysis of GCH1, FTH1, GPX4, and GAPDH in HCT116 and HT29 cells treated with RSL3 for 24 h. The error bars represent standard deviation from at least three replicates (**p < 0.01, ****p < 0.0001, ns, not significant, compared between the two groups by unpaired t-test).

FIGURE 5

Co-treatment of DAHP and erastin suppresses tumor growth in vivo. (A,B) The tumor volume (A) and body weight (B) of HCT116 xenograft tumors with the indicated treatment (n = 8 mice/group, two-way ANOVA). (C) Weight of isolated tumors at day 11 (n = 8 mice/group, unpaired t-test). (D,E) In parallel, total pterin, BH4 (D), and MDA levels (E) in the isolated tumors were assayed (n = 5 mice/group, unpaired t-test). (F,G) Representative western blot images (F) and quantitative analysis results (G) of indicated proteins in isolated tumors (n = 6 mice/group, unpaired t-test). (H) Representative H&E and IHC staining graphs of isolated tumors with indicated treatment. Scale bars, 50 μm. GCH1, Ki67, Act-caspase 3 and 4-HNE (I) positive cells shown in (H) were quantified using ImageJ (n = 5 mice/group, unpaired t-test). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6

Schematic description of GCH1/BH4 metabolism suppression of erastin-induced ferroptosis by reducing lipid peroxidation and silencing NCOA4-mediated ferritinophagy in CRC. DAHP, an inhibitor of GCH1; TFR1, transferrin receptor 1; TF, transferrin; DMT1, divalent metal transporter 1; GSH, Glutathione; GPX4, Glutathione peroxidase 4; GCH1, GTP cyclohydrolase-1; BH4, tetrahydrobiopterin; PTPS, 6-pyruvoyl tetrahydropterin synthase; SR, sepiapterin reductase; Mito, mitochondria.