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. 2012 Mar 12;209(3):507-20.
doi: 10.1084/jem.20111424. Epub 2012 Mar 5.

Autocrine VEGF-VEGFR2-Neuropilin-1 signaling promotes glioma stem-like cell viability and tumor growth

Petra Hamerlik  1 , Justin D Lathia, Rikke Rasmussen, Qiulian Wu, Jirina Bartkova, MyungHee Lee, Pavel Moudry, Jiri Bartek Jr, Walter Fischer, Jiri Lukas, Jeremy N Rich, Jiri Bartek
Affiliations

Autocrine VEGF-VEGFR2-Neuropilin-1 signaling promotes glioma stem-like cell viability and tumor growth

Petra Hamerlik et al. J Exp Med. .

Abstract

Although vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) is traditionally regarded as an endothelial cell protein, evidence suggests that VEGFRs may be expressed by cancer cells. Glioblastoma multiforme (GBM) is a lethal cancer characterized by florid vascularization and aberrantly elevated VEGF. Antiangiogenic therapy with the humanized VEGF antibody bevacizumab reduces GBM tumor growth; however, the clinical benefits are transient and invariably followed by tumor recurrence. In this study, we show that VEGFR2 is preferentially expressed on the cell surface of the CD133(+) human glioma stem-like cells (GSCs), whose viability, self-renewal, and tumorigenicity rely, at least in part, on signaling through the VEGF-VEGFR2-Neuropilin-1 (NRP1) axis. We find that the limited impact of bevacizumab-mediated VEGF blockage may reflect ongoing autocrine signaling through VEGF-VEGFR2-NRP1, which is associated with VEGFR2-NRP1 recycling and a pool of active VEGFR2 within a cytosolic compartment of a subset of human GBM cells. Whereas bevacizumab failed to inhibit prosurvival effects of VEGFR2-mediated signaling, GSC viability under unperturbed or radiation-evoked stress conditions was attenuated by direct inhibition of VEGFR2 tyrosine kinase activity and/or shRNA-mediated knockdown of VEGFR2 or NRP1. We propose that direct inhibition of VEGFR2 kinase may block the highly dynamic VEGF-VEGFR2-NRP1 pathway and inspire a GBM treatment strategy to complement the currently prevalent ligand neutralization approach.

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Figures

Figure 1.
Figure 1.
VEGFR2 is enriched on the surface of human GSCs. (A) Graph of cell surface VEGFR2+ cell fractions among CD133+ versus CD133− GBM cells based upon FACS analysis of 17 freshly dissociated human glioma specimens (**, P = 0.0017). (B) Immunohistochemical detection of VEGFR2 in clinical GBM specimens (left and middle image) of both surface (arrowhead) and intracellular (arrow; cytosolic) VEGFR2; normal brain (NB; right) shows VEGFR2 in endothelial cells/vessels (*). Bar, 50 µm. (C) Graph of surface and cytosolic VEGFR2 in 4 GBM (two independent experiments). (D) Representative plots of FACS-analyzed surface (mem) versus cytosolic (cyto) VEGFR2 in CD133+ and CD133− GBM cells (specimens T4121 and T3691).
Figure 2.
Figure 2.
VEGFR2H GBM cells localize in the perivascular niche in vivo. (A) Immunofluorescence on frozen sections of human GBM detects VEGFR2H/CD133+CD31− cells next to tumor vessel. (B) Immunofluorescence on sections of human GFP-marked GBM VEGFR2H/CD31− (T556) cell xenografts after 15 d of tumor growth in mouse brain. Bars, 50 µm.
Figure 3.
Figure 3.
Total and activated VEGFR2 localize to the cytosol of GBM cells and undergo endocytosis and recycling. (A) Indirect immunofluorescence staining for VEGFR2, phosphorylated VEGFR2, and NRP1 on frozen sections of human gliomas. DAPI shown in blue. (B) Immunoprecipitation (IP)/Western blotting (WB) of VEGFR2 and NRP1 in GBM cells, lysed after overnight recovery/starvation of freshly dissociated specimen T556, pretreated or not with SU1498 (10 µM) or bevacizumab (Bev; 0.5 mg/ml) for 2 h, and then exposed (15 min) or not to recombinant VEGF165 (100 µg/ml). pVEGFR2, Tyr1054-phosphorylated VEGFR2. Data are representative of three independent experiments. (C) Effect of shRNA knock-down of NRP1 in T556 GSCs on phosphorylated and total VEGFR2. Data are representative of two independent experiments. (D) Analysis of VEGFR, pVEGFR2 and NRP1 in membrane (biotin-labeled) and cytosolic (biotin-free) fractions of GBM T556 cells. ERK kinase: control cytosolic protein. Representative example from three experiments. (E) VEGFR2, NRP1, and TfR (positive control) examined during endocytosis and recycling. Biotinylated surface proteins (+BIO, high level); biotin label stripped immediately (Strip); proteins allowed to internalize for 10 min (Inter, partly protected from strip) and recycle for 20 min (Recy, de-protected by resurfacing again) before stripping, respectively. Recy-Con was performed along with Recy-sample, but the final stripping was omitted. Experiment is representative of three. (F) Confocal microscopy imaging in sorted CD133+ GBM cells stained for VEGFR2, NRP1, and EEA1 (a marker of early endosomes). DAPI is shown in blue. Representative images of three independent staining experiments are shown. Bars: (A and F) 50 µm.
Figure 4.
Figure 4.
VEGFR2 expression accounts for enhanced self-renewal, VEGF secretion, and viability of GSCs. Matched numbers of VEGFR2L and VEGFR2H cells from xenografted GBM specimens (T556 and T4121) were assayed for tumorsphere formation (A) and (T556 and T1966) viability (B). (A) Data are means ± SD of two independent experiments (n = 3; P < 0.001). (B) Viability of GSCs. Data are means ± SD of two independent experiments (n = 3; P < 0.001). (C) VEGF secretion by VEGFR2H versus VEGFR2L cells (GBM T556, 24 h) detected by ELISA (**, P < 0.01). (D) ELISA-detected VEGF secreted by VEGFR2H GBM cells after neutralization by bevacizumab (0.5 mg/ml, 24 h), and inhibition of VEGFR2 kinase (SU1498, 5 µM), compared with nontreated controls (Con). Mean ± SD from two experiments in duplicate (***, P < 0.001).
Figure 5.
Figure 5.
VEGFR2–NRP1 expression in GSCs accounts for preferential growth and survival in vitro and in vivo. (A) Kaplan-Meier survival curves for mice brain transplanted with VEGFR2H versus VEGFR2L human T556 GBM cells (4 mice per group; **, P = 0.0067, log-rank analysis). (B) Kaplan-Meier survival curves for mice after brain transplantation of T556 GSCs with lentivirus-mediated knockdown of VEGFR2 via two shRNAs (shVEGFR2-1 and shVEGFR2-2 targeting human VEGFR2), compared with a nontargeting shRNA (shNT). 5 mice per group; P = 0.0754 for shVEGFR2-1; **, P = 0.0054 for shVEGFR2-2 by log-rank analysis. (C) GSC death (T556; caspase 3/7 activity, left) and viability (right) after VEGFR2 knockdown (shVEGFR2-1 and shVEGFR2-2) compared with a nontargeting shRNA (shNT). n = 3; P = 0.0061 for shVEGFR2-1; P = 0.0024 for shVEGFR2-2 for caspase 3/7; P < 0.001 for shVEGFR2-1 and -2 for viability. (D) GSC death (T556; caspase 3/7 activity, left) and viability (right) after NRP1 knockdown (shNRP1-1 and shNRP1-2) compared with control shNT. n = 3; P = 0.0001 for both NRP1 shRNAs in either assay. (C and D) Data are means ± SD from three independent experiments.
Figure 6.
Figure 6.
Chemical abrogation of VEGFR2 signaling decreases survival and enhances apoptosis in T556 GSCs. (A) Annexin V FACS analysis of dissociated xenograft VEGFR2H cells pretreated with bevacizumab (Bev, 0.5 mg/ml) or SU1498 (SU, 5 µM) or untreated for 2 h, and then irradiated (IR, 8 Gy) or sham-irradiated, and stained 24 h after IR for Annexin V. Mean ± SD (n = 3); one of two experiments with similar results. (B) Effect of SU1498 or bevacizumab alone or combined with IR on cell growth of VEGFR2H cells (P = 0.0047). Mean ± SD (n = 3). Representative of three experiments is shown. (C) Survival effect of in vitro pretreatment of VEGFR2H cells with SU1498 (10 µM; 24 h) or bevacizumab (0.5 mg/ml; 4 d) on their tumorigenicity in vivo (n = 6; **, P = 0.0078 by log-rank analysis of survival curves).
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