Peroxisome-derived and liver-specific hydroxyacid oxidase 1-mediated hydrogen peroxide promotes ferroptosis by augmenting peroxisomal ROS

Introduction While peroxisome-associated molecules have been implicated in ferroptosis, the mechanistic relationship between peroxisomes and ferroptosis remains poorly understood. Key unresolved questions include whether peroxisomal hydrogen peroxide (H 2 O 2 )-generating enzymes, such as hydroxyacid oxidase 1 (HAO1), contribute to ferroptosis, as well as how they function at the molecular level. Objectives This study was designed to investigate the role of HAO1-mediated peroxisomal H 2 O 2 production in ferroptosis and its impact on liver cancer progression. Methods We evaluated HAO1 expression in liver cancer and its association with patient prognosis using clinical samples and bioinformatic analyses. RNA sequencing was performed to identify HAO1-regulated biological pathways. Cell viability was measured with a CCK-8 assay, and ferroptosis was assessed using a C11-BODIPY 581/591 probe, iron assay, and GSH detection. To delineate how HAO1-induced alterations in peroxisomal H 2 O 2 affect ferroptosis, we employed the H 2 O 2 -specific HyPer probe in conjunction with immunofluorescence and flow cytometry. The role of HAO1 in malignant progression was further examined through in vitro and in vivo functional experiments. Results HAO1 was found to enhance lipid peroxidation in liver cancer cells by boosting reactive oxygen species (ROS) levels within peroxisomes in an enzyme activity-dependent manner. Furthermore, HAO1 downregulated the p-STAT3/SLC7A11/GPX4 pathway, disrupting glutathione synthesis and impeding ROS scavenging, thereby heightening the susceptibility of cancer cells to ferroptosis. Through this dual mechanism, HAO1 synergistically promoted lipid peroxidation, triggering ferroptosis and inhibiting liver cancer cell proliferation and metastasis. Moreover, coenzyme Flavin Mononucleotide (FMN) was shown to augment the tumor-suppressive activity of HAO1. Conclusion Our findings shed light on the key role of HAO1 in the peroxisome-mediated redox regulation of ferroptosis during liver cancer progression, suggesting a potential therapeutic strategy.