Functional characterization of triokinase/FMN cyclase as a ferroptosis regulator and potential direct target of icaritin in hepatocellular carcinoma

BACKGROUND: Triokinase/FMN cyclase (TKFC) is a bifunctional homodimeric enzyme in which the triokinase activity-requiring both the K and L domains from opposing subunits-catalyzes the ATP-dependent phosphorylation of triose sugars, while the L domain alone catalyzes the Mn2 + -dependent cleavage of FAD into cyclic FMN and AMP. Despite its metabolic importance, the role of TKFC in hepatocellular carcinoma (HCC) and regulated cell death remains unclear. METHODS: We employed clinical proteomics, multi-cohort validation, tissue microarray analysis, molecular dynamics simulations, cellular thermal shift assays (CETSA), and in vitro/in vivo models to investigate TKFC expression, prognostic significance, and ferroptosis-regulatory function. RESULTS: TKFC was significantly downregulated in HCC, with expression inversely correlating with tumor grade and positively with patient survival. TKFC overexpression inhibited proliferation, migration, and invasion while inducing ferroptosis through elevated ROS, enhanced lipid peroxidation, and suppression of SLC7A11, GPX4, and FTH1. Molecular docking and 200 ns simulations revealed stable Icaritin (ICA) binding to TKFC, confirmed by CETSA in living cells. TKFC overexpression synergistically potentiated ICA-induced ferroptosis and antitumor efficacy in vitro and in vivo. CONCLUSION: This study establishes TKFC as an endogenous ferroptosis regulator and direct molecular target of Icaritin in HCC, positioning it as both a prognostic biomarker and therapeutic target for precision therapy.