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.

Figure 1. Expression of doppel in clinical and preclinical cancer tissues.

Representative images of lung (A) and colon (B) tissues showing doppel (red), blood vessels (green; CD34), and nuclei (blue). Doppel colocalized with the blood vessels of cancer tissues, but not in normal tissues. Scale bars: 10 μm (merge images) and 20 μm (magnified images). n = 3–5 tissues per group. (C) Relative mRNA expression levels of doppel in mouse NECs derived from brain tissue and TECs derived from SCC7 tumor (data represent 3 experiments). ***P < 0.001 versus NECs, Student’s t test. (D) Whole-mount staining of SCC7 tumor section showing doppel expression (green) in tumor vessels (CD31). Note that incubation of control IgG failed to detect doppel in blood vessels of the tumor, confirming the specificity of the Ab and the accuracy of doppel detection. Scale bars: 50 μm. n = 5 tumors.

Figure 2. Increased doppel expression increases tumoral angiogenesis and EC function.

(A) Total volume of blood vessels in squamous, lung, breast, and colon tumor. An aliquot of tumor (1 g) was dissected to make single cells from the site where the CD31-positive area was calculated (n = 3 sections from each tumor). (B) Doppel expression in individual TECs as determined by flow cytometric analysis (3 experiments). (C) Experimental procedure for evaluation of the gain-of-function effect of doppel in ECs. Luciferase-expressing HUVECs (Hu^+luc) and doppel-transfected Hu^+luc (Hu^+luc+dpl) spheroids were implanted s.c. in a Matrigel-fibrin matrix into female SCID mice. Three weeks after transplantation, the vascularization was analyzed. (D) Noninvasive monitoring of vascularization by bioluminescence imaging (n = 4 mice). (E) Ex vivo bioluminescence counts. ***P < 0.001 versus Hu^+luc, Student’s t test. (F) Hemoglobin content within Hu^+luc and Hu^+luc+dpl plugs was quantified. ***P < 0.001 versus Hu^+luc, Student’s t test. (G) 3D structure of the vascular network formed by Hu^+luc and Hu^+luc+dpl cells, as assessed by confocal microscopy using IF whole-mount staining for hCD34. Scale bar: 50 μm. (H) Immunoperoxidase detection of hCD34-positive blood vessels in Hu^+luc and Hu^+luc+dpl plugs. Scale bar: 20 μm. (I) Characterization and images of vascular network by staining for doppel (red), hCD34 (green), and nuclei (blue) in Hu^+luc and Hu^+luc+dpl plugs. Scale bar: 20 μm. (J) Quantification of hCD34-positive and doppel-positive mean vessel density (MVD) in Hu^+luc and Hu^+luc+dpl plugs. Doppel-positive vessels were not detected in Hu^+luc plugs. ***P < 0.001 versus Hu^+luc, Student’s t test. n = 4 plugs per experiment.

Figure 3. Doppel plays a role in VEGFR2 signaling.

(A) Phosphorylated RTK (p-RTK) signaling array of Hu.dpl exposed to fasting media, complete media, and α-doppel (30 minutes, 10 μg/ml) in the presence of complete media. (B) Quantification of pixel density of p-Tie2, p-VEGFR2, p-AKT, p-ERK1/2, p-RpS6, and p-Src. See also Supplemental Figure 10. (C) Immunoblots of p-VEGFR2, p-AKT, p-ERK1/2, p-Src, and total VEGFR2, doppel, and actin in HUVECs and Hu.dpl cells treated with different concentrations of α-doppel in the presence of VEGF₁₆₅ (100 ng/ml). Cells were pretreated with α-doppel for 30 minutes, and then VEGF₁₆₅ was added for 5 minutes. (D) Immunoblots of p-VEGFR2, total VEGFR2, total doppel, and actin in TECs treated with different concentrations of α-doppel in the presence of mVEGF (100 ng/ml). Dose-dependent inhibition (E) and total number of TEC sprouts (F) by α-doppel stimulated with either 10% FBS or mVEGF (100 ng/ml). Scale bar: 100 μm. Each experiment was repeated 3 times.

Figure 4. Doppel interacts with VEGFR2 on TECs.

Representative images of the PLA of doppel (A), VEGFR2 (B), and doppel-VEGFR2 interactions (C) identified on TECs. PLA signals are shown with red dots, cytoskeletal staining (FITC-phalloidin) is shown in green, and nuclear staining (DAPI) is shown in blue. Scale bars: 5 μm (A–C). (D) Quantification of doppel, VEGFR2, and doppel-VEGFR2 heterodimer PLA signals in TECs. Co-IP followed by immunoblotting (IB) of VEGFR2 and doppel in Hu.dpl cells and TECs (E), immunoblot of VEGFR1 and doppel (F), and immunoblot of VEGFR3 and doppel (G) in Hu.dpl cells. Input: whole-cell lysates. Single asterisk indicates a light or heavy chain. Each experiment was repeated 3–5 times.

Figure 5. Doppel inhibition spatially regulates the VEGFR2 internalization process.

(A) Flow cytometric analysis of VEGFR2 in permeabilized versus nonpermeabilized TECs following incubation with α-doppel and α-VEGFR2. VEGFR2 was internalized as a result of doppel blocking. (B) VEGFR2 and doppel internalization kinetics rate following incubation with α-doppel (upper panel; 10 μg/ml) and α-VEGFR2 (lower panel; 10 μg/ml). (C) Biochemical detection of a membrane and intracellular pool of VEGFR2 in unstimulated TECs and VEGF- (5 minutes; 100 ng/ml), α-doppel– (30 min; 10 μg/ml), and control IgG–stimulated (30 minutes; 10 μg/ml) TECs. Pan-cadherin and RSP20 were used for membrane and cytoplasmic markers, respectively. (D) IF staining of TECs for VEGFR2 (red) or EEA1 (green) and VEGFR2 (green) or LAMP1 (red) following incubation with VEGF (5 min; 100 ng/ml), α-doppel (30 min; 10 μg/ml), and control IgG (30 min; 10 μg/ml) and (E) quantification of the colocalized fraction of fluorescence signal. Nuclei were stained with DAPI (blue). Scale bar: 10 μm. Panels on the right are magnified images of the outlined portion of each image (scale bar: 5 μm). **P < 0.01, **P < 0.01, and ***P < 0.001 versus nontreated cells, Student’s t test. (F) Total VEGFR2 in TECs by Western blot analysis following incubation with α-doppel (10 μg/ml) and control IgG (10 μg/ml) at different time points. **P < 0.01 and ***P < 0.001 versus initial (zero), Student’s t test. (G) VEGFR2 degradation rate following incubation with α-doppel (12 hours, 10 μg/ml) in the absence or presence of different endocytosis and protein translation inhibitors. ***P < 0.001 versus no inhibitor, Mann-Whitney U test. (H) Immunoblot showing that VEGF₁₆₅ (100 ng/ml) stimulated the phosphorylation of AKT, ERK1/2, Src, and total GAPDH in cells when treated with α-doppel (10 μg/ml) or control IgG (10 μg/ml) in the presence or absence of the endocytosis inhibitor dynasore. Each experiment was repeated 3 times. CHX, cycloheximide.

Figure 6. Heparin and its conjugate LHbisD4 can target doppel on TECs.

(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 K_D 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.

Figure 7. LHbisD4 inhibits angiogenic signaling in TECs.

(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.

Figure 8. In vivo TEC homing ability of LHbisD4 following oral delivery.

(A) Absorption of LMWH and LHbisD4 in rats after oral delivery at a dose of 10 mg/kg (n = 4–6 rats). (B) Experimental procedure to evaluate the ability of LHbisD4 to target doppel in vivo. Cy5.5-labeled LHbisD4 (10 mg/kg) was administered orally to female BALB/c nude mice that were s.c. implanted with SCC7-derived TECs and doppel-depleted TEC (TEC^–/–dpl) spheroids. A perfused vascular network formed within 21 days of implantation. In vivo distribution (C) and ex vivo image (D) of Cy5.5-labeled LHbisD4 in TECs and TEC^–/–dpl plugs 4 hours after oral delivery (n = 3 mice). (E) Total fluorescent photon counts for LMWH and LHbisD4 in TECs and TEC^–/–dpl plugs. LMWH (2.5 mg/kg) was injected i.v., and LHbisD4 (10 mg/kg) was administered orally. All values were normalized to the hemoglobin content of each plug. *P < 0.05 and ***P < 0.001 for TECs versus TEC^–/–dpl, Student’s t test.

Figure 9. LHbisD4 targets doppel-expressing vasculatures with broad tumor specificity.

Whole-body distribution (A) and organ accumulation (B) of Cy5.5-labeled LHbisD4 in SCC7 tumor–bearing mice 8 hours after oral administration at a dose of 10 mg/kg (n = 3 mice). See also Supplemental Figure 5, A and B. (C) Localization of LHbisD4 in the SCC7 tumor (n = 3 tumors). LHbisD4 was mainly localized in the doppel-expressing blood vessels of tumor sections. Scale bars: 20 μm. (D) LHbisD4 localization in vivo was assessed by IF staining of various organs following oral administration of Cy5.5-labeled LHbisD4 (10 mg/kg) to SCC7 tumor–bearing mice (n = 3 tumors). LHbisD4 is stained in green, blood vessels in red, and nuclei in blue. Scale bars: 50 μm (tumor sections) and 100 μm (other organs). (E) Amount of LHbisD4 in the plasma and in SCC7 tumor at different time points after oral administration at a dose of 10 mg/kg (n = 4 mice). (F and G) Cy5.5-labeled LHbisD4 distribution in 6 different tumor models of different cancer types (breast, head and neck, colorectal, brain, and lung cancers) in mice 8 hours after oral administration at a dose of 10 mg/kg and (H) total photon counts in the tumors at different time points.

Figure 10. Therapeutic efficacy of LHbisD4.

(A) Orally administered LHbisD4, at a dose of 10 mg/kg daily, inhibited SCC7 tumor growth (n = 11–12 mice). **P < 0.01 versus control, Student’s t test. (B) Images of isolated tumors after termination of the experiment. Scale bar: 1 mm. (C) Tumors were excised after the study to calculate the final tumor weight. ***P < 0.001 versus control. (D and E) Representative images of SCC7 tumor–bearing mouse tumor sections stained for PCNA (proliferating cells) and CD31 (blood vessels) and their staining score (n = 11 mice). Scale bars: 50 μm. *P < 0.05 versus control; ***P < 0.001 versus control, Student’s t test. (F) Total volume of isolated TECs after termination of the experiment (n = 11 tumor sections). *P < 0.01 versus control, Student’s t test. (G) Tumor growth inhibition study of orally administered LHbisD4 in MDAMB-231 tumor at doses of 2.5, 5, and 10 mg/kg once daily or 5 and 10 mg/kg twice daily (n = 5–7 mice). ***P < 0.001 between each of the groups and the control group. **P < 0.01 between the 10 mg/kg once daily and the 10 mg/kg twice daily groups, Mann-Whitney U test. (H) Tumors were excised at the end of the study to calculate the final tumor weight. *P < 0.05, Mann-Whitney U test. (I) Tumor sections from MDAMB-231 tumor–bearing mice were stained for CD31 (blood vessels) after LHbisD4 treatment at different doses (n = 5–7 mice). Scale bar: 50 μm. (J) Dual staining of doppel (green) and PCNA (red) in a section of control and LHbisD4-treated samples. Scale bar: 50 μm.

Figure 11. Strategy for targeting doppel-expressing angiogenic tumors.

GAG-based therapeutic material binds with the tumor endothelial marker doppel, which constitutively interacts with surface VEGFR2 in TECs, but not NECs. This subsequently triggers induced internalization of the doppel-VEGFR2 complex and inhibits VEGF signaling and angiogenesis in tumors.