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 6

Heparin and its conjugate LHbisD4 can target doppel on TECs.

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Heparin and its conjugate LHbisD4 can target doppel on TECs. (A) Structu...
(A) Structure of the LMWH–doxycholic acid conjugate LHbisD4, in which 4 molecules of dimeric deoxycholic acid were conjugated to 1 molecule of LMWH. (B) Surface plasmon resonance (SPR) analysis of PrP-LHbisD4 (left) and doppel-LHbisD4 (right). The KD was calculated from the response curves (3 experiments). (C) Globular domain structure (in light brown) of doppel in complexation with LHbisD4 fragments (upper). Detailed view of the LHbisD4 fragment–binding sites (lower panels). Residues interacting with the LHbisD4 fragments are shown as orange sticks and are labeled. (D) Proposed mechanism of LMWH and LHbisD4 binding with doppel. The basic residues of the flexible N-terminal end of doppel facilitate an interaction with the negatively charged LMWH. The conjugation of deoxycholic acids allows additional hydrophobic binding with the globular α-2a and α-2b helical secondary structure of doppel. The proposed site of direct interaction is near the glycosylation sites; therefore, it may not be as accessible as suggested by the modeling or the studies with recombinant doppel. (E) LHbisD4 binding with TECs, doppel-depleted TECs (TEC–/–dpl), and CD137-knockdown TECs (TEC–/–CD137). Scale bar: 20 μm (n = 3 experiments). (F) Correlation between doppel expression and LHbisD4 binding in isolated TECs of different cancerous cell lines was determined by flow cytometry (n = 3 experiments). LHbisD4 bound with different TECs, depending on doppel expression.