CNN3 promotes angiogenesis in osteosarcoma, associated with upregulating VEGF-A and enhancing endothelial cell activity

Background: Angiogenesis plays a pivotal role in driving tumour progression, including tumour growth, local invasion, and distant metastasis. Calponin 3 (CNN3) belongs to the actin-binding protein family and has been shown to be aberrantly expressed in osteosarcoma specimens, but its exact role in the regulation of angiogenesis remains unknown. This study aimed to investigate the function and underlying mechanism of CNN3 in osteosarcoma angiogenesis. Methods: CNN3 and CD31 expression in osteosarcoma specimens was detected by immunohistochemistry. CNN3 in MG-63 and Saos-2 cells was silenced or overexpressed by transfection with small interfering RNA or overexpression plasmids, respectively. Vascular endothelial growth factor-A (VEGF-A) levels were determined by enzyme-linked immunosorbent assay. Human umbilical vein endothelial cells (HUVECs) were co-cultured with CNN3-silenced or -overexpressing osteosarcoma cells using a Transwell chamber. The function of HUVECs were assessed using cell counting, scratch wound healing, Transwell Matrigel invasion, and Matrigel vascular mimicry assays. The protein levels of endothelial-to-mesenchymal transition (EndMT) markers and phosphorylated Akt in HUVECs was determined using western blotting. Results: Immunohistochemical analysis demonstrated a significant positive correlation between CNN3 expression and CD31 (microvascular density marker) levels in osteosarcoma tissues (r=0.7264, P=0.0003). HUVECs co-cultured with CNN3-overexpressing cells exhibited enhanced proliferative, migratory, invasive, and tube-forming capacities along with promoted EndMT, whereas CNN3 silencing suppressed these effects. CNN3 silencing reduced, while its overexpression elevated, VEGF-A secretion in both MG-63 and Saos-2 cells. Furthermore, silencing CNN3 in MG-63 and Saos-2 cells decreased Akt phosphorylation in co-cultured HUVECs, whereas CNN3 overexpression concomitantly increased it. Conclusions: CNN3 promotes osteosarcoma angiogenesis, possibly through upregulating VEGF-A secretion and enhancing endothelial cell functions, suggesting its potential as the therapeutic target for anti-angiogenic strategies.