Role of the VEGFR3/VEGFD Receptor Axis in TGFβ1 Activation of Primary Prostate Cell Lines

BACKGROUND. Reports indicate that vascular endothelial growth factor receptor type 3 (VEGFR3) regulates cellular functions such as invasion, proliferation, and chemo-resistance. However, the exact function of the VEGFR3 signaling axis in prostate epithelial cells is poorly characterized. METHODS. The goal of this study was to evaluate whether TGF beta 1 in combination with VEGFD can promote pre-malignant invasive activities of intermediate basal cells (IBC-10a) isolated from human prostate cancer (Gleason score 6). RESULTS. hTERT immortalized IBC-10a cells normally grew as confluent cobblestoned monolayers, but treatment with TGF beta 1 (10 ng/ml for 2-6 hr) dissociated the cell-cell junctions and induced VEGFR3 translocation to the cell surface. This event was not inhibited by 10 mu M cycloheximide or puromycin, indicating transcription and protein synthesis were not required. We further discovered that TGF beta 1 in combination with VEGFD induced a significant increase in the invasive activity of IBC-10a cells (> 26% and 53% after 24 and 48 hr, respectively) in modified Boyden Chamber assays. TGF beta RII receptor antibodies specifically blocked TGF beta 1 induction of VEGFR3 translocation to the cell surface and blocked VEGFD-induced invasion. Zymograms revealed that TGF beta 1 (and not VEGFR3) stimulated the secretion of MMP-2 and MMP-9, presumably to promote cell invasion. The cell invasion assays confirmed that antibodies specific for TGF beta II receptor, MMP-2 and MMP-9 and VEGFR3, independently blocked TGF beta 1-induced invasion. CONCLUSIONS. For the first time, we have demonstrated the mechanism by which TGF beta 1 stimulates VEGFD/VEGFR3 receptor axis activation leading to increased cell migration and invasion by primary intermediate basal cell cultures. Prostate 69: 982-990, 2009. (C) 2009 Wiley-Liss, Inc.