VEGF: Autocrine-paracrine Function in Oral Squamous Cell Carcinoma (OSCC)

Studies from ours and other laboratories have confirmed that OSCC cells and tumors release high levels of vascular endothelial growth factor (VEGF) and also retain VEGF's primary target receptor, KDR. To date, the primary focus on VEGF–OSCC interactions has been on VEGF's role in tumor-associated angiogenesis. OBJECTIVE: This study investigated the hypothesis that VEGF plays a biphasic role in OSCC development due to direct VEGF-OSCC cell interactions. METHODS: Western blot confirmation of KDR expression in two OSCC cell lines set precedent for further evaluation of VEGF's effect on OSCC cell: 1) mitogenesis [MTT proliferation assay], 2) intracellular signaling [fluorogenic probes to detect reactive oxygen species (ROS)], 3) invasion of a synthetic basement membrane (InnoCyte™ cell invasion assay). RESULTS: High endogenous VEGF production rendered OSCC cells refractory to exogenous VEGF. siRNA was therefore used to inhibit endogenous VEGF production for up to 96h (n=10, p<0.01, Tukey-Kramer multiple comparisons test). Addition of exogenous _hrVEGF165 (30ng/ml and 50ng/ml) to siRNA-silenced cells resulted in dose-dependent increases in cell proliferation (n=12, p<0.05, Yates corrected Chi-square test). In contrast, exogenous VEGF did not affect proliferation in non-siRNA treated OSCC cells. _hrVEGF165 initiated ROS-mediated intracellular signaling, resulting in fluorescent intensities similar to the direct probe activator, H₂O₂ (0.2mM). Invasion assay results show: 1) VEGF (100ng/ml) is comparable to fetal bovine serum (FBS, 10%) as an OSCC chemoattractant, 2) 24h _hrVEGF165 pretreatment (100ng/ml with 100ng/ml VEGF as chemoattractant) increased OSCC invasion relative to all other groups [10%FBS pretreatment + 10%FBS chemoattractant, VEGF(100mg/ml) chemoattractant only, 10%FBS chemoattractant only, negative control] (n=6, p<0.001, Tukey-Kramer multiple comparisons test). CONCLUSIONS: Our results demonstrate, for the first time, that OSCC cells are both targets and effectors for VEGF. This new evidence introduces the prospect that anti-VEGF therapy could fulfill both anti-angiogenic and anti-tumorigenic functions. [Supported by: NIHR01CA95901]