Vascular endothelial growth factor-C protects prostate cancer cells from oxidative stress by the activation of mammalian target of rapamycin complex-2 and AKT-1

Abstract

Recurrence and subsequent metastatic transformation of cancer develops from a subset of malignant cells, which show the ability to resist stress and to adopt to a changing microenvironment. These tumor cells have distinctly different growth factor pathways and antiapoptotic responses compared with the vast majority of cancer cells. Long-term therapeutic success can only be achieved by identifying and targeting factors and signaling cascades that help these cells survive during stress. Both microarray and immunohistochemical analysis on human prostate cancer tissue samples have shown an increased expression of vascular endothelial growth factor-C (VEGF-C) in metastatic prostate cancer. We have discovered that VEGF-C acts directly on prostate cancer cells to protect them against oxidative stress. VEGF-C increased the survival of prostate cancer cells during hydrogen peroxide stress by the activation of AKT-1/protein kinase Balpha. This activation was mediated by mammalian target of rapamycin complex-2 and was not observed in the absence of oxidative stress. Finally, the transmembrane nontyrosine kinase receptor neuropilin-2 was found to be essential for the VEGF-C-mediated AKT-1 activation. Indeed, our findings suggest a novel and distinct function of VEGF-C in protecting cancer cells from stress-induced cell death, thereby facilitating cancer recurrence and metastasis. This is distinctly different from the known function of VEGF-C in inducing lymphangiogenesis.

Figure 1

(A) Western Blot of LNCaP, LNCaP C4-2 and PC3 whole cell lysates for the protein expression of Neuropilin-2 and VEGF-R3. (B and C) Apoptosis Assays with H₂O₂ (3 mM) treated LNCaP C4-2 cells for 5 hour with prior addition of increasing concentrations of VEGF-C. Cells were serum starved overnight before the addition of VEGF-C and H₂O₂. (B) Graphical representation of the apoptotic experiment after evaluation of three independent results. Dead cells were counted in 10 randomly selected high-power (40X) fields for each experimental condition. We calculated the average of all the fields for each experimental condition. (C) Cell death was measured by PI (red) and YOPRO (green). Hoechst staining was used to visualize the nucleus.

Figure 2

(A) Western Blots for pAKT-1 (S473), pAKT-1 (T308), AKT-1, pGSK3β (S9), pFoxO1 (T24) in whole cell lysates after H₂O₂ and VEGF-C treatment. Densitometry quantizations of the western blot results are presented below of each figure. The data represented as ratio of intensities of the bands with respect to “no VEGF-C control”. (B) Stable clones of VEGF-C (ΔNΔC) overexpressing LNCaP C4-2 as well as mock transfected cells were treated with increasing doses of H₂O₂. pAKT-1 (S473) levels in the clones were determined by western blots. (C) Western blots for pS6 and p4EBP1 after VEGF-C and H₂O₂ treatment of LNCaP C4-2. Densitometry quantization of the western blot results are presented below of each figure. The data represented as ratio of intensities of the bands with respect to “no VEGF-C control”.

Figure 3

(A) Whole cell lysates of VEGF-C and H₂O₂ treated LNCaP C4-2 cells were immunoprecipitated with mTOR antibody. The precipitant was then blotted for rictor and mTOR. Lower two panels represent the total protein levels of rictor and mTOR in LNCaP C4-2 cells after H₂O₂ and VEGF-C treatment. (B) Rictor knocked-down LNCaP C4-2 cells were treated with VEGF-C (150 ng/ml) and H₂O₂ (3 mM). Whole cell lysates were subjected to an immunoblot. (C) RNAi for Neuropilin-2 was performed in LNCaP C4-2 cells with increasing doses of siRNA. The whole cell lysates were subjected to an immunoblot for Neuropilin-2. (D) LNCaP C4-2 cells were treated with Neuropillin-2 siRNA for 72 hour. After 63 hour of siRNA transfection, VEGF-C (150 ng/ml) was added to the serum-starved cells (serum-starvation started after 48 hour of siRNA transfection) for the rest 9 hour. These cells were challenged with H₂O₂ (3 mM) for the last 5 hours of the experiment. Whole cell lysates were subjected to an immunoblot.

Figure 4

(A) Left panel: Increasing concentration of VEGF-C was added to H₂O₂ (1 mM) treated LNCaP cells. The whole cell lysate was subjected to an immunoblots for pAKT-1 (S473), total AKT-1, phospho-GSK-3β, total GSK-3β and GDI (as a loading control). Right panel: Apoptosis Assay for H₂O₂ (1 mM) treated LNCaP cells for 5 hour with prior addition of increasing concentrations of VEGF-C. Cells were serum starved overnight before the addition of VEGF-C and H₂O₂. Cell death was measured by PI (red) and YOPRO (green). Hoechst staining was used to visualize the nucleus (details in supplement figure 5). Comparisons of the apoptotic LNCaP cells at each treatment are graphically represented after evaluating three independent results. (B) PC3 cell were treated with siRNA specific for VEGF-C or scrambled siRNA and then challenged with increasing concentrations of H₂O₂ for 5h. Left panel: The immunoblots for pAKT-1 (S473) in the scrambled and VEGF-C siRNA treated PC-3 cells. Right panel: Endogenous VEGF-C level in PC-3 cells were knocked down by VEGF-C specific siRNA. Apoptosis Assay of control and VEGF-C siRNA treated PC-3 cells after 5-hour incubation with H₂O₂ (1 mM and 5mM) was performed as described previously. Comparisons of the apoptotic PC-3 cells at each treatment are graphically represented after evaluating three independent results. (C) Western blot results of phospho-FOXO-1 and GSK-3β in H₂O₂ treated PC-3 cells where the endogenous levels of VEGF-C were knocked down. (D) Schematic representation of the signaling pathways for VEGF-C mediated mTORC-2 and AKT-1 activation in prostate cancer cells under oxidative stress. The ROS mediated dissociation of mTORC2 can be reversed by VEGF-C binding to Neuropilin-2. This restores the activity status of AKT-1, which in turn induces the phosphorylation of GSK3β and FoxO1.