Targeting the Urotensin II/UT G Protein-Coupled Receptor to Counteract Angiogenesis and Mesenchymal Hypoxia/Necrosis in Glioblastoma

Abstract

Glioblastomas (GBMs) are the most common primary brain tumors characterized by strong invasiveness and angiogenesis. GBM cells and microenvironment secrete angiogenic factors and also express chemoattractant G protein-coupled receptors (GPCRs) to their advantage. We investigated the role of the vasoactive peptide urotensin II (UII) and its receptor UT on GBM angiogenesis and tested potential ligand/therapeutic options based on this system. On glioma patient samples, the expression of UII and UT increased with the grade with marked expression in the vascular and peri-necrotic mesenchymal hypoxic areas being correlated with vascular density. In vitro human UII stimulated human endothelial HUV-EC-C and hCMEC/D3 cell motility and tubulogenesis. In mouse-transplanted Matrigel sponges, mouse (mUII) and human UII markedly stimulated invasion by macrophages, endothelial, and smooth muscle cells. In U87 GBM xenografts expressing UII and UT in the glial and vascular compartments, UII accelerated tumor development, favored hypoxia and necrosis associated with increased proliferation (Ki67), and induced metalloproteinase (MMP)-2 and -9 expression in Nude mice. UII also promoted a "tortuous" vascular collagen-IV expressing network and integrin expression mainly in the vascular compartment. GBM angiogenesis and integrin αvβ3 were confirmed by in vivo ^99mTc-RGD tracer imaging and tumoral capture in the non-necrotic area of U87 xenografts in Nude mice. Peptide analogs of UII and UT antagonist were also tested as potential tumor repressor. Urotensin II-related peptide URP inhibited angiogenesis in vitro and failed to attract vascular and inflammatory components in Matrigel in vivo. Interestingly, the UT antagonist/biased ligand urantide and the non-peptide UT antagonist palosuran prevented UII-induced tubulogenesis in vitro and significantly delayed tumor growth in vivo. Urantide drastically prevented endogenous and UII-induced GBM angiogenesis, MMP, and integrin activations, associated with GBM tumoral growth. These findings show that UII induces GBM aggressiveness with necrosis and angiogenesis through integrin activation, a mesenchymal behavior that can be targeted by UT biased ligands/antagonists.

Keywords: UT receptor; angiogenesis; biased ligand; glioblastoma; necrosis; urotensin II.

FIGURE 1

Characterization of UII and UT expression in non-tumoral and glioma samples. (A) UII and UT co-labeling (brown) with anti-CD34 immunoreactivity (pink) on consecutive sections from different grades of astrocytomas [PA (I), AII, and AIII], oligodendrogliomas (OII and OIII), and oligoastrocytomas (OAII and OAIII) along the histopathological classification showed a more intense staining in AIII and GBM tumors (left panels) compared with O and mixed OA tumors (right panels). (B) UII and UT immunohistochemical co-expression with anti-CD34 structures on consecutive sections of GBM samples revealed staining in vascular cells of hyperplasic vessels (upper panels) and in pseudopalisading cells around areas of necrosis (lower panels). Co-expression of UT (brown) with α-SMA (pink) (upper right panel) in smooth muscle/pericyte vascular components. CA9 staining (brown) highlights hypoxic environment around necrotic areas (lower right panel) exhibiting immunoreactivity for UII and UT. The black star and the dotted line in the lower panels indicate necrosis location. (C) UII and UT expression on consecutive sections of three different non-tumoral tissues showed a strong staining in the gray matter (GM) especially in neurons (upper and middle panels) compared to oligodendrocytes from the GM (peri-neuronal satellitosis) and in the white matter (WM) (upper and lower panels). The dotted line delineates the frontier between GM and WM. (D) Quantification of experiments presented in (A,B) and represented as the mean ± SEM of UT (left panel) and UII (right panel) scores (see section “Materials and Methods”). *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA and multiple comparison test with Tukey’s correction). (E) Vascular density, diameter, and circularity quantified from NT and malignant glioma (AII/OII to GBM) samples based on the CD34-positive structures and histological characteristics from, at least in part, three different images per patient tumor slice. Left, Histograms of mean ± SEM (Kruskal–Wallis and Dunn’s post-test: ***P < 0.001). Right, Scatter plot of the correlation between UII scoring value versus vascular density (left) and UT scoring value versus vascular density (right). Score correlation in glioma showed a significant correlation between UII and UT and vascular density more particular within AIII and GBM samples. (F) Total score correlation in glioma samples showed a significant correlation between UII and its receptor UT. Disease-free survival curves of 20 primary GBM for UII (left panel) and UT (right panel). In (E,F), the correlation is considered significant when P < 0.05, r represents the Pearson correlation coefficient. H/E: Hematoxylin–Eosin staining. Scale bar = 10 μm for (A,B), and (C, middle and lower panels), and 20 μm for (C, upper panel).

FIGURE 2

Gene expression of UTS2, UTS2R, and UTS2D and patient survival in gliomas. (A) UTS2R, UTS2, and UTS2D are expressed in all histological OII/III, OAII/III, AII/III, and GBM classes and according to the glioma tumor grade, n = 66 tumors of the TCGA database. Histograms show gene expression from RNA-seq analysis. The data are from the following cohorts: OII/III, OAII/III, AII/III, and GBM. Of all glioma tissues, UTS2 expression exhibits significant higher levels in GBM compared to others (P < 0.0001). (B,C) Based on mRNA information on the subgroup classification of GBM from Verhaak et al. (2010) available in TCGA, neural, mesenchymal, and pro-neural groups expressed higher levels of UTS2R, UTS2, and UTS2D, respectively. (D) Kaplan–Meier statistical analysis of the prognostic significance of UTS2R, UTS2, and UTS2D. The overall survival period of glioma patients with UTS2R and UTS2 high expression was shorter than that of GBM patients with low expression. Green line, low expression; red line, high expression. N indicates the number of samples in each low or high expression group. *P < 0.05; **P < 0.01; ***P < 0.001.

FIGURE 3

Gene expression of UTS2, UTS2R, and UTS2D in glioma and hCMEC/D3 cell lines. (A) UTS2R, UTS2, and UTS2D gene expression were analyzed by qPCR in gliomas. UTS2R, UTS2, and UTS2D mRNA expressions are presented as ΔΔCt, related to the GAPDH gene expression in the anaplastic astrocytoma cell line SW1088 and in U87, U251, 8MG, and 42MG GBM cell lines. Data were expressed as mean ± SEM of three independent cultures and normalized to mRNA first to the housekeeping gene GAPDH and then to levels of gene of interest in SW1088. (B) Effect of UII on UTS2 and UTS2R gene expression in U87, U251, 8MG, and 42MG GBM cell lines and the endothelial hCMEC/D3 cell line. Cells were treated by UII (10^–9 M, 24 h) in the absence of FBS. UTS2R and UTS2 mRNA expressions were expressed as mean ± SEM of ΔΔCt from three independent cultures normalized to the housekeeping gene UBC. Statistical significance of treatments vs. control condition was assessed with Mann–Whitney test. *P < 0.05; **P < 0.01. (C) Representative example of the effect of UII on prepro-UII and UT protein expression detected by Western blot of U87, U251, 8MG, and 42MG or hCMEC/D3 cells exposed to UII (10^–9 M, 24 h) in the absence of FBS. For UT, a doublet of bands (∼42–53 kDa) and a higher molecular mass (∼70 kDa) correspond to human UT as previously shown (Lecointre et al., 2015, Supplementary Material). β-tubulin antibody was used as protein loading control.

FIGURE 4

In vitro effect of urotensinergic ligands on human glioma and endothelial cell migration and tubulogenesis. (A) Microphotographs of UT and UII staining on the GBM cell line U87 and EC hCMEC/D3 from human origins. Scale bar, 20 μm. (B) Representative fields (Top panel) of migrating U87 and hCMEC/D3 cells quantified from the Boyden chamber assay and quantification of U87 (Left) and hCMEC/D3 (Right) cell number in the absence or the presence of FBS 10% or UII (10^–12, 10^–11, 10^–10, 10^–9, or 10^–8 M). (C) Microphotographs of the tubulogenesis assay illustrating in vitro properties of UT agonists (UII, URP, and UII_4–11), biased ligand (urantide), or antagonist (palosuran) on angiogenic features of hCMEC/D3 and HUV-EC-C cells. Scale bars, 400 μm. (D) Data are presented as the% of control ± SEM from six independent experiments, and branch length, major junctions, and segment number were quantified and compared to control conditions. Statistical significance of treatments vs. control condition was assessed with one-way ANOVA with Dunnett post hoc test. ns, non-significant; *P < 0.05; **P < 0.01; ***P < 0.001. Statistical significance of co-treatments (UII + urantide; UII + palosuran) vs. UII alone was conducted with Student t-test. ^##P < 0.01; ^###P < 0.001.

FIGURE 5

In vivo chemoattractant effects of the urotensinergic ligands toward pro-angiogenic cells in Matrigel plugs. (A,B) Macrophotographs of resected Matrigel plugs (200 μl) containing vehicle (PBS), EGF (500 ng), EG-VEGF (500 ng) (A) or UII, mUII, URP, UII_4–11 (50 ng, each), urantide (50 ng), or palosuran (50 ng) (B) 3 weeks after implantation in C57B/l6 mice. Matrigel invasion was detected by immunohistochemical analysis of macrophages (F4/80, red), EC matrix collagen-IV (COLL-IV, red), and smooth muscle cells (α-SMA, green). Scale bars, 40 μm. Quantification of macrophage number or smooth muscle cell and EC structure areas. Data are presented as percentage of control ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 from at least four independent experiments. Statistical significance is given by one-way ANOVA with Dunnett post hoc test vs. control.

FIGURE 6

Effects of the urotensinergic system on glioma development in vivo. (A) Tumor growth (left) and survival (right) of Swiss Nude mice transplanted with U87 cells. When tumor reached 100 mm³, intratumoral injections of vehicle (saline) or UII (0.1 μg) were performed daily and tumor growth or median survival of treated mice was measured. **P < 0.01; ***P < 0.001. Median survival: vehicle (n = 7), 33 days; UII (n = 7), 22 days (P = 0.0126). (B) Microphotographs of UT (green) and UII (red) expression in vehicle (PBS)- and UII-treated U87 xenografts. UII and UT were expressed in tumor parenchyma, in perinecrotic (N) areas and in vascular structures. (C) Tumor growth (Left) and mice survival (Right) during treatments with vehicle, URP, or UII_4–11 (0.1 μg, each) of xenografted U87. Median survival: vehicle (n = 4), 22 days; URP (n = 4), 28 days (P = 0.5875); UII_4–11 (n = 4), 26 days (P = 0.8931). (D) Top panel, typical gallery of representative example of U87 xenografts treated with vehicle, UII (0.1 μg), palosuran (1 μg), UII + palosuran (0.1/1 μg), urantide (1 μg), and UII + urantide (0.1/1 μg). Middle panel, tumor growth during the different treatments. *P < 0.05; **P < 0.01; ***P < 0.001. Bottom panel, mice survival during the different treatments. Median survival: vehicle (n = 10), 23 days; UII (n = 10), 16 days (P = 0.0005); palosuran (n = 10), 23 days (P = 0.2922), UII + palosuran (n = 9), 23 days (P = 0.86), urantide (n = 10), 43 days (P = 0.0067), UII + urantide (n = 10), 27 days (P = 0.0645). (E) Immunolabeling of UT (green) and UII (red), and tumor distribution in vehicle, UII, urantide, and UII + urantide-treated U87 xenografts. Data presented as mean ± SEM. Animal survival was analyzed with Kaplan–Meier method, using the log-rank test for comparison. Statistical significance for tumor growth in (A,C,D) was given by using two-way ANOVA with Bonferroni post hoc test comparison with vehicle. Cell nuclei stained with DAPI (blue). Scale bars: 20 μm.

FIGURE 7

Effects of the urotensinergic system on cell proliferation, hypoxia, and necrosis in U87 GBM. (A) Representative microscopic fields (Top panel) of U87 xenografts immunostained with an antibody directed against the proliferation marker Ki67 (red). Bottom left, quantification of the number of proliferative cells (Ki67⁺) after 15 days of daily intratumoral injections of the UT ligands; bottom right, linear regression of tumor volume vs. proliferation index. Statistical significance of vehicle versus UII, palosuran, urantide, or co-treatments at day 15. **P < 0.01; ***P < 0.001. Statistical significance of UII versus co-treatments at day 15. ###P < 0.001. A significant positive correlation was given by the Pearson coefficient correlation r = 0.6579 (P < 0.0001). Scale bars: 40 μm. (B) Top, microscopic fields of U87 xenografts of tumoral hypoxia revealed by pimonidazole labeling (green) after 15 days of daily intratumoral injections of vehicle, UII (0.1 μg), or urantide (1 μg). Bottom, quantification of pimonidazole stained area (Left) and intensity (Right). Scale bars: 40 μm. n = 4 in each treatment groups. ***P < 0.001. (C) Top, H&E necrotic area coloration after 15 days of daily intratumoral injections of vehicle, UII (0.1 μg), or urantide (1 μg). Bottom, quantification of the necrotic area after 15 days (left) or after tumors reached 1000 mm³ (Right). Scale bars: 2 mm. n = 6 in each treatment groups. **P < 0.01; ***P < 0.001.

FIGURE 8

Effects of the urotensinergic system on angiogenesis in U87 GBM. (A) UII (Top) and UT (Bottom) (red, each) immunostainings co-localized with VSMC and EC detected with anti-αSMA and anti-CD31 (green), respectively, in U87 xenografts. (B) COLL-IV (red) labeling in U87 tumors after 15 days of different treatments (left panel) or after tumors reach 1000 mm³ (right panel). Quantification of intratumoral angiogenesis (vascular area) after 15 days of treatments or after tumors reach 1000 mm³ (vascular area, number of branches and vessel diameter) as percentage of control ± SEM. **P < 0.01; ***P < 0.001 vehicle vs. treatments; ^##P < 0.05; ^###P < 0.001; UII versus UII + palosuran or UII + urantide, from at least four different tumors in each group. Statistical significance was given by one-way ANOVA followed by Dunnett post hoc test. UC, uncountable.

FIGURE 9

Effects of the urotensinergic system on metalloprotease activation in U87 GBM. (A,B) MMP-2 (green) and quantification of labeled intensity and surface (A) or MMP-9 staining (green) and quantification (B) of labeled intensity and surface. Statistical significance was given by one-way ANOVA followed by Dunnett post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001 vehicle versus treatments. ^#P < 0.05; ^##P < 0.01; ^###P < 0.001 UII vs treatments. Cell nuclei stained with DAPI (blue). Scale bars: 20 μm.

FIGURE 10

In vivo imaging of the urotensinergic system-associated integrin expression and angiogenesis. (A) Left, U87 xenografts neo-vessels stained with antibodies directed against the endothelial precursor marker CD34 (green) and α_V integrins (red) after 15 days when daily treated with vehicle (top panel) or α_V integrins (green) in vehicle-, UII (0.1 μg)-, or urantide (1 μg)-treated xenograft after 15 days treatment. Right, Quantification of α_V staining in tumor xenografts after 15 days of different treatments in tumor parenchyma and vascular compartment. *P < 0.05; **P < 0.01; ***P < 0.001; vehicle vs. treatments. Cell nuclei stained with DAPI (blue). Scale bars: 20 μm. (B) Representative MicroSPECT imaging of the ^99mTc-RGD integrin ligand (red shades) binding in living tumors (circled by white dots), in antero-posterior (A-P, top), or dorso-ventral (V-D, bottom) positions and quantification of tracer incorporation in tumors (right) after 15 days of treatment. (C) Quantification of tumoral volume/weight and ^99mTc-RGD γ ray emission in resected tumors, after 15 days of treatment (left, n = 6 in each group of treatment). ID, injected dose. (D) Left, representative fields of necrotic staining with H&E (top), ^99mTc-RGD tracer binding (middle), and merged pictures (bottom) from consecutive slices of tumors acquired by β imaging. Scale bar: 2 mm. Right, Quantification of ^99mTc-RGD in entire histological tumor sections (tumor, T) or when discriminating necrotic tumor areas (NT) in UII-treated tumors (non-necrotic tumor, NNT). n = 6 in each treatment groups. *P < 0.05; ***P < 0.001. Statistical significance in (A–D) experiments was given by one-way ANOVA with Dunnett post hoc test comparison with vehicle.

FIGURE 11

Schematic model illustrating the pleiotrope functions of the urotensinergic system during GBM malignancy. The UT receptor when expressed at both the tumoral and vascular compartments, and activated by UII through endogenous release by tumor cells, relayed accelerated tumor growth and proliferation, hypoxia, and necrosis, leading to exacerbation of the abnormal and tortuous vascularization. These processes are accompanied by metalloprotease (MMPs) release such as MMP-9 by the endothelial compartment likely degrading extracellular matrix, and by increased expression of αv(β3) integrins at least in part by GBM cells. The hypothesis of a contributing mechanism of a UII-induced macrophage tumor invasion can also be proposed, as potential cell partners in necrosis and angiogenesis. UT receptor antagonist palosuran or the biased ligand urantide would constitute a new original strategy to prevent glioma malignancy. Here, the biased ligand urantide exhibits a better multicellular anti-UT activity than palosuran both repressing angiogenesis and tumor growth, suggesting a new avenue for GBM treatment targeting the urotensinergic system.