Targeting Wnt3a and Loxl2 Synergistically Induces Ferroptosis in Liver Cancer Stem Cells and Suppresses Tumorigenesis

Hepatocellular carcinoma (HCC) has a poor prognosis and high mortality. Ferroptosis, an iron-dependent regulated cell death process, is implicated in cancer development and treatment. Wnt signaling and lysyl oxidase (Lox) family members are associated with ferroptosis. This study investigates how Wnt3a and/or Loxl2 knockdown affects liver cancer stem cells (LCSCs) and orthotopic tumor growth in mice, and explores the role of ferroptosis-related genes. Bioinformatics identified ferroptosis- and HCC-associated differentially expressed genes (DEGs) correlated with Wnt3a/Loxl2. LCSCs sorted from Hep3B were transduced with lentivirus for gene knockdown. Ferroptosis markers and DEG expression were analyzed. Wnt3a/Loxl2 knockout mice were generated using CRISPR-Cas9, and orthotopic tumor models were established. Tumor inhibition rates, ferroptosis-related indicators, and DEG expression were assessed. 199 ferroptosis-related DEGs were identified in HCC; ZEB1 was selected as a key gene via PPI analysis. Wnt3a/Loxl2 knockdown increased Fe²⁺ and MDA, and decreased GSH, most evidently in double-knockdown cells. In vivo, single- and double-knockout groups showed suppressed tumor growth, with inhibition rates of 51%, 71%, and 93%, respectively. Tumor tissues exhibited similar ferroptosis marker changes. ZEB1 was upregulated in both cellular and animal knockout models. Wnt3a/Loxl2 knockdown promotes ferroptosis in LCSCs and inhibits orthotopic tumor growth, with the strongest effect following dual-gene knockout. ZEB1 may be an important regulatory factor in this process.