Usnic acid inhibits colorectal cancer liver metastasis by targeting CRYAB phosphorylation and inducing ferroptosis

Colorectal cancer liver metastasis (CCLM) presents a significant challenge in oncology, characterized by aggressive progression and poor prognosis. Usnic acid (UA), a secondary metabolite derived from the lichen Usnea, has shown extensive bioactivity, particularly notable anticancer effects. Despite the well-established pro-apoptotic role of UA through reactive oxygen species (ROS) generation, its influence within the hypoxic tumor microenvironment, particularly in CCLM, remains inadequately elucidated. In this study, we employed an integrative approach utilizing multi-omics analyses and molecular dynamics simulations to identify CRYAB (αB-crystallin) as a UA target in CCLM. We assessed the impact of UA on the phosphorylation status of serine 59 (S59) in CRYAB using cellular thermal shift assays. By developing organoid models from both CCLM and primary colorectal cancer tissues, we examined tumor characteristics, pathway enrichment, and the effects of hypoxic conditions on CRYAB dynamics. We utilized mutation analysis to explore the functional implications of S59 phosphorylation. Our findings reveal that CRYAB is upregulated in CCLM tissues with increased S59 phosphorylation. UA binds with high affinity to CRYAB, significantly decreasing S59 phosphorylation in a dose-dependent manner. CCLM organoids exhibited elevated CRYAB and FGFR1 expression, along with activation of pathways involved in epithelial-mesenchymal transition (EMT), hypoxia, and proliferation. Hypoxia further promoted CRYAB phosphorylation and organoid growth; however, UA treatment mitigated these effects, leading to apoptosis and reduced cell migration. Notably, mutations at S59 altered CRYAB localization and modified downstream signaling, highlighting its role in tumor metastasis. This study reveals that CRYAB phosphorylation is a key driver of liver metastasis progression in colorectal cancer and confirms that usnic acid exerts its anti-metastatic effects by targeting this post-translational modification. Mechanistically, beyond its known pro-apoptotic activity, usnic acid induces ferroptosis by inhibiting CRYAB phosphorylation at site S59. These findings establish a mechanistic framework for usnic acid as a potential therapeutic agent against metastatic colorectal cancer, highlighting the therapeutic potential of targeting CRYAB phosphorylation and ferroptosis in managing colorectal cancer liver metastases. • Multi-omics analysis identifies CRYAB as the target of usnic acid in inhibiting colorectal cancer liver metastasis. • CRYAB phosphorylation, linked to the tumor hypoxic microenvironment, is significantly elevated in CCLM samples and correlates with tumor proliferation and migration. • CRYAB phosphorylation promotes its interaction with NEDD4L, preventing GPX4 ubiquitination and inhibiting ferroptosis. • Both usnic acid and phosphorylation site mutation disrupt CRYAB-NEDD4L interaction, leading to GPX4 deactivation and ferroptosis induction.