Targeting prion-like protein doppel selectively suppresses tumor angiogenesis
Taslim A. Al-Hilal, Seung Woo Chung, Jeong Uk Choi, Farzana Alam, Jooho Park, Seong Who Kim, Sang Yoon Kim, Fakhrul Ahsan, In-San Kim, Youngro Byun
Taslim A. Al-Hilal, Seung Woo Chung, Jeong Uk Choi, Farzana Alam, Jooho Park, Seong Who Kim, Sang Yoon Kim, Fakhrul Ahsan, In-San Kim, Youngro Byun
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Research Article Oncology

Targeting prion-like protein doppel selectively suppresses tumor angiogenesis

Abstract

Controlled and site-specific regulation of growth factor signaling remains a major challenge for current antiangiogenic therapies, as these antiangiogenic agents target normal vasculature as well tumor vasculature. In this article, we identified the prion-like protein doppel as a potential therapeutic target for tumor angiogenesis. We investigated the interactions between doppel and VEGFR2 and evaluated whether blocking the doppel/VEGFR2 axis suppresses the process of angiogenesis. We discovered that tumor endothelial cells (TECs), but not normal ECs, express doppel; tumors from patients and mouse xenografts expressed doppel in their vasculatures. Induced doppel overexpression in ECs enhanced vascularization, whereas doppel constitutively colocalized and complexed with VEGFR2 in TECs. Doppel inhibition depleted VEGFR2 from the cell membrane, subsequently inducing the internalization and degradation of VEGFR2 and thereby attenuating VEGFR2 signaling. We also synthesized an orally active glycosaminoglycan (LHbisD4) that specifically binds with doppel. We determined that LHbisD4 concentrates over the tumor site and that genetic loss of doppel in TECs decreases LHbisD4 binding and targeting both in vitro and in vivo. Moreover, LHbisD4 eliminated VEGFR2 from the cell membrane, prevented VEGF binding in TECs, and suppressed tumor growth. Together, our results demonstrate that blocking doppel can control VEGF signaling in TECs and selectively inhibit tumor angiogenesis.

Authors

Taslim A. Al-Hilal, Seung Woo Chung, Jeong Uk Choi, Farzana Alam, Jooho Park, Seong Who Kim, Sang Yoon Kim, Fakhrul Ahsan, In-San Kim, Youngro Byun

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Figure 7

LHbisD4 inhibits angiogenic signaling in TECs.

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LHbisD4 inhibits angiogenic signaling in TECs. (A) Flow cytometric analy...
(A) Flow cytometric analysis of VEGFR2 in nonpermeabilized TECs following incubation with LHbisD4 (10 μg/ml) at different time points. (B) Immunoblot of mVEGF-stimulated (100 ng/ml) phosphorylation of VEGFR2, total VEGFR2, total doppel, and actin in NECs derived from brain and in TECs following incubation with different concentrations of LHbisD4 in the presence or absence of the endocytosis inhibitor dynasore. Dynasore was pretreated for 2 hours prior to the incubation of LHbisD4. Cells were then treated with LHbisD4 for 30 minutes and stimulated with mVEGF for 5 minutes. (C) Densitometric measurement of the p-VEGFR2 signal (normalized to VEGFR2 and actin bands) from each experiment. Results are expressed as percentages relative to the mVEGF-treated group. **P < 0.01 and ***P < 0.001 versus mVEGF treatment alone, Mann-Whitney U test. (D) Representative images of TECs with staining for VEGFR2 (red) or EEA1 (green) and VEGFR2 (green) or LAMP1 (red) following incubation with LHbisD4 (30 min; 10 μg/ml). Nuclei were stained with DAPI (blue). Scale bars: 10 μm. Panels on the right are magnified images of the outlined portion of each image (scale bars: 5 μm). (E) Colocalized fraction of fluorescence signal between VEGFR2 and EEA1 or LAMP1. ***P < 0.001 versus control, Student’s t test. (F) Total VEGFR2 in TECs by Western blotting following incubation with LHbisD4 (10 μg/ml) at different time points. **P < 0.01 and ***P < 0.001 versus initial (zero), Student’s t test. (G) TEC-sprouting assay following incubation with different concentrations of LHbisD4 in the presence or absence of mVEGF. Scale bar: 100 μm. **P < 0.01 and ***P < 0.001 versus mVEGF treatment alone, Student’s t test. Each experiment was performed 3 times.