Shikonin triggers apoptosis in lung adenocarcinoma through the FHL2-FOXO1 signaling axis

Abstract Background: FHL2 is an oncogenic scaffold protein overexpressed in lung LUAD, making it a promising therapeutic target. SHK a natural naphthoquinone, exhibits anti-tumor activity, but its molecular mechanism in LUAD, particularly its relationship with FHL2, remains unclear. Aim of the study: The present study aimed to define the effects of SHK on NSCLC and identify the potential molecular mechanisms. Methods: The anti-tumor effects of SHK were evaluated in LUAD cell lines (A549, H1299) and a patient-derived xenograft (PDX) model using CCK-8, colony formation, Transwell, and flow cytometry assays. Integrated proteomics, bioinformatics, molecular docking, and cellular thermal shift assay (CETSA) were employed to identify SHK's direct target. The underlying mechanism was investigated through co-immunoprecipitation (Co-IP), Western blot, qRT-PCR, cycloheximide (CHX) chase assay, and immunofluorescence/immunohistochemistry (IF/IHC). Results: In both in vitro and PDX models, SHK potently inhibited LUAD growth, metastasis, and induced cell death. At the molecular level, FHL2 was identified as a direct target of SHK. Direct binding of SHK to FHL2 promoted its proteasomal degradation, thereby disrupting the FHL2-FOXO1 complex. The dissociation of this complex enhanced FOXO1 acetylation and promoted its nuclear translocation, leading to the subsequent activation of the caspase cascade and apoptosis. Conclusion: Our findings elucidate a novel signaling pathway through which SHK inhibits LUAD: by directly targeting FHL2 for degradation, SHK disrupts the FHL2-FOXO1 complex, which activates FOXO1-mediated transcription and triggers apoptosis. This study not only provides mechanistic insight into SHK's anti-tumor function but also nominates the FHL2-FOXO1 axis as a potential therapeutic target for LUAD.