The benzofuran derivative DOP binds CTGF and suppresses angiogenesis and tumor growth in preclinical models of colorectal cancer

Objective:

First-line anti-angiogenic therapies for colorectal cancer (CRC), which predominantly target the VEGF axis, are frequently hampered by acquired resistance driven by single-pathway blockade and compensatory pro-angiogenic pathway activation. Connective tissue growth factor (CTGF) is a key pro-angiogenic driver in tumors, yet its therapeutic targeting remains underinvestigated. Here, we investigated the antiangiogenic activity and underlying mechanisms of disodium 6-methoxy-2-methyl-3-[1-(3,4,5-trimethoxyphenyl)ethenyl]benzofuran-7-dibenzyl phosphate, a benzofuran derivative designated DOP, together with its antitumor effects in colorectal cancer.

Methods:

We assessed the effects of DOP on endothelial cell proliferation, tube formation, and migration using human umbilical vein endothelial cells (HUVECs), and evaluated in vivo angiogenesis via Matrigel plug and zebrafish models. DARTS and CETSA were used to assess DOP-associated changes in CTGF stability, while MST assays assessed DOP-CTGF binding. Molecular docking and molecular dynamics simulations were used to explore a potential binding mode. Proposed binding residues were investigated by site-directed mutagenesis, and the functional involvement of CTGF was assessed using knockdown and rescue experiments. Proteomic profiling was used to identify candidate pathways, followed by western blot analysis of selected signaling proteins. Antitumor efficacy was evaluated in an HCT116 xenograft mouse model, while body weight and histopathological changes in major organs were assessed.

Results:

DOP inhibited HUVEC proliferation, tube formation, and migration and suppressed angiogenesis in Matrigel plug and zebrafish models. Purified-protein MST supported binding of DOP to CTGF, while CTGF knockdown-rescue experiments supported a contribution of CTGF to the effects of DOP on endothelial migration and tube formation. Proteomic profiling guided further investigation of VEGF/VEGFR2 and Notch signaling, and western blotting demonstrated DOP-associated changes in these pathways. In the HCT116 xenograft model, DOP inhibited tumor growth, reduced intratumoral microvessel density, and increased tumor necrosis. Under the tested dosing regimen, no obvious body-weight loss or overt histopathological abnormalities were observed in the examined major organs during the experimental period.

Conclusion:

DOP exhibited antiangiogenic activity and inhibited tumor growth in an HCT116 xenograft model. The findings support CTGF as a binding partner of DOP and a contributor to its endothelial effects. These findings support further investigation of DOP as an antiangiogenic candidate for CRC.

Graphical abstract: https://links.lww.com/AHM/A244