NeuroD1-GPX4 signaling leads to ferroptosis resistance in hepatocellular carcinoma

Cell death resistance is a hallmark of tumor cells that drives tumorigenesis and drug resistance. Targeting cell death resistance-related genes to sensitize tumor cells and decrease their cell death threshold has attracted attention as a potential antitumor therapeutic strategy. However, the underlying mechanism is not fully understood. Recent studies have reported that NeuroD1, first discovered as a neurodifferentiation factor, is upregulated in various tumor cells and plays a crucial role in tumorigenesis. However, its involvement in tumor cell death resistance remains unknown. Here, we found that NeuroD1 was highly expressed in hepatocellular carcinoma (HCC) cells and was associated with tumor cell death resistance. We revealed that NeuroD1 enhanced HCC cell resistance to ferroptosis, a type of cell death caused by aberrant redox homeostasis that induces lipid peroxide accumulation, leading to increased HCC cell viability. NeuroD1 binds to the promoter of glutathione peroxidase 4 (GPX4), a key reductant that suppresses ferroptosis by reducing lipid peroxide, and activates its transcriptional activity, resulting in decreased lipid peroxide and ferroptosis. Subsequently, we showed that NeuroD1/GPX4-mediated ferroptosis resistance was crucial for HCC cell tumorigenic potential. These findings not only identify NeuroD1 as a regulator of tumor cell ferroptosis resistance but also reveal a novel molecular mechanism underlying the oncogenic function of NeuroD1. Furthermore, our findings suggest the potential of targeting NeuroD1 in antitumor therapy. Aberrant expression of cell death resistance-related genes is common in tumor cells and contributes to their resistance to cell death and tumorigenesis. Transcription factor NeuroD1 has been found to be highly expressed in various tumors and could promote tumorigenesis. However, whether it is involved in tumor cell death resistance remains unclear. In this study, we demonstrate that NeuroD1 enhances the resistance of hepatocellular carcinoma (HCC) cells to ferroptosis. Knocking down NeuroD1 leads to the increase of reactive oxygen species (ROS), toxic lipid peroxides, as well as DNA and mitochondrial damages. These subsequently trigger ferroptosis. We further revealed that NeuroD1 acts as a transcriptional activator of glutathione peroxidase 4 (GPX4), a key molecule that reduces lipid peroxide and thus protects cells against ferroptosis, thereby upregulates the expression level of GPX4 and suppresses HCC cells ferroptosis. Our results also confirmed the critical role of NeuroD1/GPX4 in tumorigenesis in vivo. Together, our findings reveal NeuroD1 as a crucial ferroptosis suppressor gene, thereby unraveling a novel mechanism of tumor cells ferroptosis resistance. Furthermore, our results suggest that targeting NeuroD1 might be a potential antitumor therapeutic strategy.