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 TGFb1 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 TGFb1 (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 mM cycloheximide or puromycin, indicating transcription and protein synthesis were not required. We further discovered that TGFb1 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. TGFbRII receptor antibodies specifically blocked TGFb1 induction of VEGFR3 translocation to the cell surface and blocked VEGFD-induced invasion. Zymograms revealed that TGFb1 (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 TGFbII receptor, MMP-2 and MMP-9 and VEGFR3, independently blocked TGFb1-induced invasion. CONCLUSIONS. For the first time, we have demonstrated the mechanism by which TGFb1 stimulates VEGFD/VEGFR3 receptor axis activation leading to increased cell migration and invasion by primary intermediate basal cell cultures. Prostate 69: 982–990, 2009. # 2009 Wiley-Liss, Inc.