Blocking VEGF Receptor on Brain CSCs Increases Effectiveness of Anticancer Therapy

Researchers find VEGFR2 signaling supports cell viability, self-renewal, and tumorigenicity.

Directly blocking signaling by VEGFR2 expressed on cancer stem cells in glioblastoma may provide an approach to boosting the effectiveness of anti-VEGF therapies such as bevacizumab (Roche’s Avastin), according to Danish researchers. A European team led by investigators at the Danish Cancer Society Research Center and Centre for Genotoxic Stress Research (CGSR) has shown that VEGFR2 is preferentially expressed on the cell surface of the CD133+ human glioma stem-like cells. Binding of tumor-released VEGF to the receptor triggers an autocrine signaling cascade that is key to bolstering cancer cell viability, self-renewal, and tumorigenicity, they claim

Effectively, the team suggests, bevacizumab can’t inhibit the prosurvival effects of this VEGFR2-mediated signaling, whereas glioblastoma stem cell viability can be reduced by inhibiting VEGFR2 tyrosine kinase activity or through shRNA-mediated knockdown of VEGFR2 or the co-receptor NRP1. Reporting their findings in the Journal of Experimental Medicine, Jiri Bartek, M.D., and colleagues suggest 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.” Their studies are detailed in a paper titled “Autocrine VEGF–VEGFR2–Neuropilin-1 signaling promotes glioma stem-like cell viability and tumor growth.”

Glioblastoma multiforme (GBM) is just about invariably fatal, and tumors will recur despite initial treatment with bevacizumabs. The VEGF receptor VEGFR2 is crucial for VEGF-mediated responses in endothelial cells. While it has previously been thought that VEGF receptors are expressed almost exclusively by endothelial cells, more recent studies have suggested that VEGFRs expressed by tumor cells themselves play a role in GBM survival and resistance to VEGF inhibitors.  

Research by the CGSR team has now shown that VEGFR2 is in fact expressed preferentially on the surface of CD133+ human glioma stem-like cells (GSCs) and that VEGF-VEGFR2-Neurophilin-1 (NRP1) signaling plays a key role in the viability, self-renewal, and tumorigenicity of these cells. Neuropilin-1 (NRP1) is a nonsignaling co-receptor of VEGFR2 previously identified in endothelial cells.

The team’s initial studies showed that the receptor was present both on the surface and in the cytosol of CD133+ cells. Moreover, analysis of human GBM biopsies showed that VEGFR2+/CD133+ cell populations were located close to vascular structures.  

The potential involvement of NRP1 in autocrine VEGF signaling was indicated by the finding that the protein colocalized with autophosphorylated and total VEGFR2 on frozen sections from clinical GBM specimens. Co-immunoprecipitation studies demonstrated that formation of the VEGFR2-NRP1 complex could be blocked by treating GBM cells with the humanized antibody bevacizumab but not by the tyrosine kinase inhibitor SU1498. As with other receptor tyrosine kinases such as EGFR and PDGFR, activation of VEGFR2 by phosphorylation rapidly led to receptor (and in this case NRP1) internalization.  shRNA-mediated knockdown of NRP1 led to dramatically decreased VEGFR2 protein levels, “indicating a crucial role of NRP1 in VEGFR2 protein stability,” the authors add.

Importantly, when compared with GBM cells that expressed only low levels of VEGFR2 (VEGFR2^L), those that expressed high levels of the receptor both on the surface and in the cytosol (VEGF2^H cells) exhibited a higher self-renewing potential, as evidence by their capacity to form tumor spheres. This finding correlated with results from other cell viability tests.

To gain further insights into VEGFR2 signaling by GSCs, the team assessed VEGF secretion by VEGFR2^L and VEGFR2^H cells sorted from bulk tumor populations. As expected, highly elevated levels of VEGF were detected in the conditioned media from cultured VEGFR2^H in comparison with media from their VEGFR2^L counterparts. This VEGF secretion was blocked by treating the cells with bevacizumab and significantly decreased following administration of the VEGFR2 tyrosine kinase inhibitor SU1498. Most notably, untreated VEGFR2^H cells more quickly formed tumors when transplanted into experimental mice than the VEGFR2^L cells.  

To assess the contribution of VEGFR2 signaling to GSC self-renewal, survival, and tumor growth, the authors evaluated the effects of shRNA directed against human VEGFR2. VEGFR2^H GBM cells were infected with lentivirus particles expressing shRNAs directed against one of two independent gene regions. This shRNA-mediated knockdown resulted in increased apoptosis and decreased viability of the VEGFR2^H cells. Mice transplanted with the knockdown cells also demonstrated reduced tumor formation and longer survival.  shRNA-mediated knockdown of NRP1 similarly resulted in enhanced apoptosis and decreased viability of GSCs, “thus further supporting the role of VEGFR2–NRP1 interaction in GSCs’ survival,” the team states.

Encouragingly, while treating VEGFR2^H cells with the VEGFR2 kinase inhibitor SU1498 resulted in increased levels of cell death, combining the inhibitor with ionizing radiation enhanced apoptosis even further. In contrast, bevacizumab treatment alone didn’t lead to any significant apoptosis. “These results highlight the differential impact of the biologically effective direct inhibition of VEGFR2 tyrosine kinase activity (by SU1948) versus the apparent inability of bevacizumab to evoke a desirable antitumor effect,” the investigators remark. “We speculate that the latter phenomenon might reflect insufficient neutralization by bevacizumab of the continuously secreted VEGF ligand, thereby allowing for VEGFR2 signaling and, consequently, continued survival and growth of the tumor.”

To add further weight to the observations that VEGFR2 kinase inhibition and antibody-mediated ligand neutralization have different biological effects, the investigators evaluated the viability of VEGFR2^H cells treated using either SU1498 or bevacizumab or left untreated, irradiated, or sham-irradiated. Although bevacizumab treatment alone didn’t impact on the viability of VEGFR2^H cells, exposure to SU1498 led to a significant decrease in cell viability, and this effect was further increased when inhibitor treatment was followed by ionizing radiation (IR).

Bevacizumab in combination with IR did result in a significant decrease in cell viability, but this effect was less pronounced compared with tyrosine kinase inhibition by SU1498. And when either bevacizumab- or SU1498-treated cells were transplanted into immunocompromized mice, it was the tyrosine kinase inhibitor-treated cells that showed impaired tumorigenic capacity.

“In conclusion, the functional abrogation of the VEGFR2-NRP1-VEGF-regulated pathways can undermine GBM survival under various conditions including exposure to IR and reduce tumor formation,” the authors conclude. “These results supported our concept that persistent autocrine VEGFR2 signaling represents a potent regulator of GSC growth under both in vitro and in vivo conditions. Moreover, this signaling apparently not only operates in GSC cell pools but stays active in a larger pool of progeny tumor cells, which can reactivate the autocrine loop by recycling internalized VEGFR2 back to the cell surface … This concept offers new insights into GBM biology and identifies the VEGF-VEGFR2-NRP1 interplay as a novel and attractive target in therapy of malignant gliomas.”