NICD3 mediates pro-angiogenic effects through SMAD3/TGFBI axis in colorectal cancer.

The consensus molecular subtype 4 (CMS4) of colorectal cancer (CRC) represents an aggressive, mesenchymal phenotype associated with the poorest clinical prognosis. Although angiogenesis is fundamental to CRC progression, current anti-VEGF therapies are frequently compromised by primary or acquired resistance, creating an urgent need to identify alternative vascular drivers. NOTCH3 is significantly enriched in CMS4 CRC, yet its direct role in modulating the tumor vasculature remains elusive. Here, integrating multi-center transcriptomic datasets with clinical validation, we identified NOTCH3 as a critical engine of CRC angiogenesis. In Stage IV clinical samples, NOTCH3 expression correlated significantly with microvessel density (MVD). Functionally, overexpression of the NOTCH3 intracellular domain (NICD3) dramatically enhanced endothelial tube formation and migration in vitro, while driving robust tumor vascularization in xenograft models. Through transcriptomic profiling, we identified the secreted factor transforming growth factor-beta-induced protein (TGFBI) as the essential downstream effector. Mechanistically, we demonstrate that NICD3 physically interacts with and upregulates SMAD3, thereby facilitating the direct transcriptional activation of TGFBI. Crucially, to translate these mechanistic insights, we utilized molecular docking and drug screening to identify the flavonoid Hesperidin (HES) as a potent binder of TGFBI. HES treatment effectively abolished NICD3-driven angiogenesis and suppressed tumor progression in vivo. Collectively, our findings characterize a novel NICD3/SMAD3/TGFBI signaling axis as a key vulnerability in CRC and propose HES as a promising precision therapeutic strategy for CRC patients with features of CMS4.