Indole-3-carboxylic acid promotes senescence of colorectal cancer cells by inhibiting the binding of Netrin-1 to UNC5B.

BACKGROUND: Colorectal cancer (CRC) progression is linked to aberrant cellular senescence. Netrin-1/UNC5B signaling regulates tumor cell survival, yet whether indole-3-carboxylic acid (I3C) modulates CRC senescence via this pathway remains unknown. This study aims to investigate I3C's mechanism in CRC senescence and its connection to Netrin-1/UNC5B signaling. METHODS: HCT-116 and HT29 cells were exposed to I3C. Senescence was assessed and the targeted binding relationship and key binding sites between I3C and UNC5B were verified. UNC5B knockdown/mutant cell lines were established; combined with intervention using exogenous recombinant Netrin-1 and Wnt/β-catenin inhibitor XAV939, downstream mechanism of Netrin-1/UNC5B pathway was clarified. A xenograft model was constructed to validate the anti-tumor effect and molecular targets of I3C. RESULTS: I3C induced CRC cell senescence concentration- and time-dependently. I3C specifically bound UNC5B residues (SER604, THR590, GLU718), inhibiting Netrin-1/UNC5B interaction, with SER604 identified as a functionally critical regulatory residue. Notably, loss-of-function of UNC5B attenuated I3C's pro-senescence effect. Netrin-1/UNC5B signaling could activate Wnt/β-catenin pathway (upregulating Axin2, c-Myc, Cyclin D1), counteracting doxorubicin-induced senescence, while XAV939 reversed this activation. Functional assays confirmed that I3C promoted senescence and inhibited proliferation, migration, and epithelial-mesenchymal transition in a wild-type UNC5B-dependent manner, primarily via SER604. Exogenous recombinant Netrin-1 could antagonize the aforementioned effects, whereas UNC5B knockdown abrogated this antagonistic action. In vivo, I3C suppressed tumor growth dependently on Netrin-1/UNC5B axis. CONCLUSIONS: I3C promotes CRC cellular senescence and suppresses tumor progression by blocking Netrin-1/UNC5B binding, likely through modulating Wnt/β-catenin pathway. This reveals a novel anticancer mechanism of I3C and a potential CRC therapeutic target.