ERBB3 influences the ferroptosis pathway via modulation of lipid peroxidation and GSH synthesis in gastric cancer

Abstract

Gastric cancer remains one of the most lethal malignancies worldwide, with high relapse rates and limited survival for patients with advanced disease. Despite advances in targeted therapies and immune checkpoint inhibition, intrinsic tumor heterogeneity poses challenges for effective treatment. The HER3 receptor (ERBB3) has emerged as an important player in cancer progression, contributing to aggressive tumor behavior and poor prognosis. Recent evidence indicates that activating ferroptosis-an iron-dependent, non-apoptotic form of cell death-offers a promising strategy to inhibit cancer growth. In gastric cancer, ferroptosis plays a crucial role, and promoting this process may open new avenues for therapeutic intervention. Ferroptosis is characterized by iron-mediated lipid peroxidation of cell membranes and is critically regulated by the cystine/glutamate antiporter system (SLC7A11) and glutathione peroxidase 4 (GPX4). Our study aimed to investigate the relationship between ERBB3 and ferroptosis in gastric cancer. We found that high ERBB3 expression correlated with resistance to ferroptosis-inducing agents, including GPX4 and SLC7A11 inhibitors, across multiple cell lines. Vice versa, ERBB3 inhibition with TX1-85-1 induced lipid peroxidation in gastric cancer cells, with effects most pronounced in cell lines expressing higher SLC7A11 levels. Knockdown of ERBB3 reproduced these effects, suggesting SLC7A11 as a predictive marker. Importantly, combined inhibition of ERBB3 and GPX4 significantly enhanced lipid peroxidation and cytotoxicity, while ERBB3 activation by co-treatment with the ERBB3 ligand heregulin reduced lipid peroxidation in cells with lower baseline SLC7A11 expression. Analysis of glutathione levels and SLC7A11 expression further supported the role of ERBB3 in modulating ferroptosis sensitivity. These findings identify ERBB3 as a critical regulator of ferroptosis and a promising target for enhancing ferroptosis-mediated cell death. Its inhibition in combination with ferroptosis inducers may thus represent a particularly promising and efficacious therapeutic strategy in gastric cancer.

Fig. 1. ERBB3 expression is associated with ferroptosis resistance.

A Correlation analysis of ERBB3 expression with sensitivities to 481 compounds from the CTRP database v2. Z-scores: ML210 (8.11), ML162 (8.12), RSL3 (7.07), and Erastin (5.82). B mRNA expression analysis of ERBB3, SLC7A11, and GPX4 normalized to RPLP0 in five gastric cancer cell lines. C Correlation between Erastin area under the drug response curve (AUC) values from the CTRP database v2 and ERBB3 expression from the DepMap Expression 24Q2 dataset across 756 cell lines. Pearson coefficient: 0.319; p = 2.59E-19. D Correlation between RSL3 AUC values (CTRP database v2) and ERBB3 expression in 772 cell lines (DepMap Expression 24Q2 dataset). Pearson coefficient: 0.362; p = 2.86E-25. E Correlation between ACSL4 expression and ERBB3 expression in 1517 cell lines from the DepMap Expression 24Q2 dataset. Pearson coefficient: –0.354; p = 5.33E-46. F Co-expression analysis of ACSL4 and ERBB3 (RNA-Seq V2 RSEM) in 413 samples from the TCGA Firehose Legacy cohort. Pearson coefficient: –0.26; p = 1.46E-7.

Fig. 2. Lipid peroxidation induction upon ERBB3 inhibition or siRNA-mediated knockdown.

A FACS analysis of lipid peroxidation via BODIPY™ 581/591 C11 staining. The upper panel summarizes data from ≥3 independent experiments, while the lower panel displays representative histograms. Cells were incubated for 48 h with TX1-85-1 (5 µM for Hs746T, MKN7, MKN45, and NCI-N87 cells; 3 µM for MKN74 cells). B Lipid peroxidation following siRNA-mediated knockdown of ERBB3 (20 nM siRNA). Cells were incubated for 72 h prior to analysis. C FACS analysis of lipid peroxidation via BODIPY™ 581/591 C11 staining from n = 3 independent experiments. Cells were incubated for 48 hours with 5 µM TX1-85-1, 1 µM Ferrostation-1, 100 µM α-Tocopherol, or the combination.

Fig. 3. Combinatory effects of ERBB3 inhibition plus SLC7A11- or GPX4 inhibition on lipid peroxidation and cell viability.

A FACS analysis of lipid peroxidation via BODIPY™ 581/591 C11 staining. The upper panel summarizes data from ≥3 independent experiments, and the lower panel presents representative histograms. Cells were treated for 48 h with RSL3 (4 µM), Erastin (10 µM), and TX1-85-1 (1 µM for MKN74, 5 µM for MKN7 and MKN45). B Cell death staining using propidium iodide from n = 3 independent experiments. Cells were treated for 48 h with RSL3 (4 µM), Erastin (10 µM), and TX1-85-1 (1 µM for MKN74, 5 µM for MKN7 and MKN45 cells). C IncuCyte^® proliferation assay depicting normalized confluency over 6 days. Cells were seeded in 96-well plates, and confluency was measured every 6 h. Treatment conditions: Erastin (1 µM), RSL3 (0.5 µM), and TX1-85-1 (2 µM for MKN45, 1 µM for MKN7, and 0.5 µM for MKN74).

Fig. 4. Rescue effects of ERBB3 stimulation with heregulin, diminishing lipid peroxidation.

FACS analysis of lipid peroxidation via BODIPY™ 581/591 C11 staining. A Quantitation of data from ≥3 independent experiments, B representative histograms. Cells were treated for 48 h with RSL3 (4 µM for MKN7, MKN45, MKN74; 1 µM for Hs746T), Erastin (10 µM for all cell lines), and HRG (30 ng/ml).

Fig. 5. Alterations of SLC7A11 expression and glutathione levels upon ERBB3 stimulation.

A Total glutathione (GSH) levels detected colorimetrically in cells treated with HRG (30 ng/ml) for 48 h. B mRNA expression levels of SLC7A11 normalized to the housekeeping gene RPLP0 (set to 100%) following treatment with HRG (30 ng/ml) for 48 h. C Western blot of SLC7A11 upon HRG stimulation for 48 h in MKN7 cells. Representative image of n = 3 experiments. D GSH/GSSG ratios in MKN7 and MKN45 cells treated with Erastin (10 µM) and HRG (30 ng/ml) for 48 h.