RNA-mediated gene silencing of VEGF-C inhibits the growth of human pancreatic cancer in vivo

Abstract

Purpose: Vascular endothelial growth factor-C (VEGF-C), and its receptors, VEGF receptor-2 and VEGF receptor-3, are central to angiogenesis and lymphangiogenesis. Pancreatic cancer VEGF-C expression is associated with poorer prognosis for patients with this disease. We sought to determine the role of VEGF-C on growth of pancreatic cancer in vitro and in vivo. Methods: Expression of VEGF-C was determined in 3 human pancreatic cancer (PaCa) cell lines (HPAF-II, MIA PaCa-2, PANC-1) by RT-PCR, Western blot, and ELISA. Expression of the preferential VEGF-C receptor, VEGFR3, was determined by RT-PCR and Western blot. Small interfering RNA (siRNA) was used to suppress VEGF-C expression. Specificity of the VEGF-C siRNA was confirmed by Western blot in VEGF-C transfected NIH3T3 cells. Efficiency of RNA interference was confirmed using VEGF-C ELISA on MIA PaCa-2 cell culture supernatants. Cell proliferation following exogenous human VEGF-C, VEGF-C siRNA or control siRNA treatment was assessed in vitro by MTT and [3H]-thymidine. Nude mice (n 4/group) were subcutaneously xenografted with 2 106 MIA PaCa-2 cells, which overexpress VEGF-C. Tumor growth and VEGF-C expression were compared in mice administered either vehicle, 150 g/kg VEGF-C specific siRNA, or 150 g/kg control siRNA twice weekly for 4 weeks by tail vein injection. Statistical comparison was made using t-test for paired observations and ANOVA for multiple comparisons. Results: All PaCa cell lines expressed VEGF-C and VEGFR3 receptor. VEGF-C was expressed at significantly higher levels in the poorly differentiated MIA PaCa-2 cells. VEGF-C siRNA was specific and effectively downregulated expression in vitro. Treatment of PaCa cell lines with VEGF-C siRNA in vitro did not affect proliferation, nor did treatment with exogenous VEGF-C. Treatment with VEGF-C siRNA in vivo suppressed tumor growth at 4 weeks when compared to controls: Vehicle 250 4 mm3; Control siRNA 230 26 mm3 (p 0.2); VEGF-C siRNA 87 25 mm3 (p 0.01). VEGF-C ELISA of tumor homogenates confirmed RNA interference: Vehicle 496 7 pg/mg; Control siRNA 362 95 pg/mg (p 0.2); VEGF-C siRNA 115 55 pg/mg (p 0.01). Conclusions: These data demonstrated that VEGF-C plays an important role in PaCa growth in vivo. VEGF-C does not appear to promote PaCa cell growth in vitro. Identifying strategies that target VEGF-C may serve as potential therapeutic interventions in patients with pancreatic adenocarcinoma.