FUNDC1 drives cholangiocarcinoma progression via RAC1 interaction and ferroptosis suppression.

Cholangiocarcinoma (CCA) is a highly lethal epithelial malignancy that can arise at any site within the biliary tract. Our findings revealed that FUN14 domain-containing protein 1 (FUNDC1) expression was significantly elevated in CCA samples compared to matched peritumoral tissues. Survival analysis indicated that patients with high FUNDC1 expression had shorter overall survival. Mitochondrial membrane potential (MMP) alterations were observed. Specifically, knockdown of FUNDC1 induced MMP disruption in CCA cells. Additionally, we detected damaged mitochondria and increased production of reactive oxygen species (ROS), which were further exacerbated by FUNDC1 knockdown in CCA cells. Further analysis revealed upregulation of Gpx4 and SLC7A11, along with downregulation of NCOA4 in CCA patients. These changes were accompanied by increased glutathione (GSH) levels and decreased malondialdehyde (MDA) and C11-BODIPY levels in CCA cells, effects that were nullified by FUNDC1 knockdown in vivo. We also observed increased expression of Ras-related C3 botulinum toxin substrate 1 (RAC1), a member of the Rho GTPase family, in human CCA samples. Immunoprecipitation analysis demonstrated that an extracellular segment of FUNDC1 (amino acids 96-133) mediated its interaction with RAC1. Furthermore, while FUNDC1 knockdown effectively induces ferroptosis, RAC1 knockdown does not. Notably, this induction is abrogated upon overexpression of FUNDC1. In vivo experiments also confirmed that knockdown of FUNDC1 and RAC1 significantly reduced tumor volume. These findings suggest that RAC1 is involved in FUNDC1-induced malignant transformation in CCA. Overall, our data indicate that FUNDC1 promotes CCA progression through mitochondrial function-dependent ferroptosis, highlighting a promising target for the development of FUNDC1-based therapies for CCA.