Licochalcone B inhibits Neuritin 1-driven perineural invasion and osteosarcoma progression via the Hedgehog signaling pathway.

BACKGROUND: Osteosarcoma (OS), the most common primary malignant bone tumor, remains an aggressive malignancy with limited targeted therapeutic options. Neuritin 1 (NRN1) has been implicated in neuronal development and tumor progression. Licochalcone B (LCB), a bioactive chalcone derived from Glycyrrhiza uralensis Fisch, exhibits antitumor effects in various cancers. However, the precise role of NRN1 and the therapeutic efficacy of LCB via regulating NRN1 in OS remain unclear. PURPOSE: To elucidate the oncogenic properties of NRN1 and evaluate LCB as a therapeutic agent targeting NRN1 in OS. METHODS: Bulk and single-cell RNA-seq data from multiple clinical cohorts were integrated to profile the transcriptional characteristics, clinical significance, and immune landscape of NRN1 in OS. Molecular docking was employed to predict the binding affinity between LCB and NRN1. Cellular thermal shift assay and drug affinity responsive target stability assay were performed to verify the direct binding of LCB to NRN1. A series of in vitro and in vivo experiments, including dorsal root ganglion-OS co-culture, sciatic nerve invasion models, multiphoton microscopy, and orthotopic OS xenograft models, were performed to investigate the pro-tumorigenic effects of NRN1 and the therapeutic potential of LCB. RESULTS: Elevated NRN1 expression in malignant OS cells presented an independent prognostic value for predicting poor patient survival and perineural invasion. NRN1 overexpression accelerated cell proliferation, migration, invasion, and tumor growth, which were attenuated by LCB treatment. Mechanistically, NRN1 activated the Hedgehog signaling pathway (smoothened/GLI family zinc finger 2) to drive malignant progression. Notably, NRN1 established a bidirectional tumor-neurotropic interaction that facilitated perineural invasion and metastasis, whereas LCB disrupted this crosstalk by directly binding and suppressing NRN1, thereby preserving nerve structural integrity and reducing lung metastasis. Additionally, NRN1 expression shaped an immunosuppressive microenvironment in OS via transforming growth factor beta 1-mediated natural killer cell exhaustion, which was effectively reversed by LCB through downregulating the NRN1-transforming growth factor beta 1 axis. CONCLUSION: LCB exerts comprehensive antitumor effects in OS by targeting NRN1, concomitantly suppressing Hedgehog signaling, attenuating perineural invasion, and restoring natural killer cell-mediated antitumor immunity, which may serve as a promising therapeutic strategy for improving patient outcomes.