Notch signaling governs colorectal cancer metastasis via transcriptional control of TGF-β effectors SMAD2/SMAD3.

[BACKGROUND] The molecular interplay between Notch and TGF-β signaling in colorectal cancer (CRC) metastasis remains poorly understood.

[METHODS] Genetic ablation of RBP-J, the central transcriptional mediator of Notch signaling, was performed in CRC cells. In vitro functional assays assessed migration, invasion, and transendothelial migration. Metastatic colonization was evaluated in vivo using orthotopic, intrasplenic, and intravenous murine models. Whole-transcriptome analysis, chromatin immunoprecipitation sequencing (ChIP-seq), and luciferase reporter assays were used to analyze RBP-J-mediated transcriptional regulation of SMAD2/SMAD3. Rescue experiments reconstituted SMAD2/SMAD3 in RBP-J knockout (KO) cells to verify functional necessity.

[RESULTS] RBP-J knockout profoundly impaired CRC cell migration, invasion, and transendothelial migration in vitro, and suppressed metastatic colonization across multiple in vivo models. Transcriptomic and ChIP-seq analyses revealed RBP-J directly binds to SMAD2 and SMAD3 promoters and activates their transcription. Reconstitution of SMAD2/SMAD3 restored the migratory and metastatic capacities of RBP-J KO cells. Mechanistically, Notch signaling primes TGF-β responsiveness by maintaining SMAD2/SMAD3 expression and phosphorylation, establishing a feedforward loop essential for metastasis.

[CONCLUSION] Notch signaling orchestrates TGF-β-driven metastasis through direct transcriptional control of SMAD2/SMAD3, defining a hierarchical regulatory axis. This offers novel therapeutic targets for metastatic CRC.