Caveolin-1 promotes tumor cell proliferation and vasculogenic mimicry formation in human glioma

Abstract

Vasculogenic mimicry (VM) plays an important role in human glioma progression and resistance to antiangiogenic therapy as a compensatory neovascularization mechanism in malignant tumors. Caveolin-1 (Cav-1) has been found to contribute to VM formation. However, it remains largely unknown whether Cav-1 expression correlates with VM in glioma. In this study, we examined CAV-1 expression levels and VM in human glioma cell lines and in 94 human gliomas with different grades of malignancy, and present Cox proportional hazards regression. The molecular role of Cav-1 in glioma cells was investigated using quantitative polymerase chain reaction (qRT-PCR) assays, western blotting, CCK-8 assays, and tubule formation assays. Cav-1 expression and VM formation were positively correlated with each other and both were closely associated with glioma development and progression as evidenced by the presence of cystic tumor, shortened survival time, and advanced-stage glioma in glioma patients with Cav-1 overexpression/increased VM formation. Cav-1 promoted U251 glioma cell proliferation and VM formation in a Matrigel-based 3D culture model. VM-associated factors including hypoxia-inducible factor 1α (HIF-1α) and p-Akt was significantly elevated by Cav-1 overexpression but suppressed by siCav-1 in U251 cells. Collectively, our study identified Cav-1 as an important regulator of glioma cell proliferation and VM formation, contributing to glioma development and progression.

Figure 1. Correlation of Cav-1 expression and/or vasculogenic mimicry (VM) formation with human glioma grades and overall survival of glioma patients. A, Immunohistochemical staining for GFAP (brown, yellow arrow), Cav-1 (brown, green arrow), and CD31/PAS co-staining for VM channels (light purple, red arrow) in glioma specimens. Magnification 40× (scale bar 100 μm) and 400× (scale bar 20 μm). B, The mean number of VM channels (left) and Cav-1-positive tumor cells (right) in low-grade (LGG) (n=39) and high-grade gliomas (HGG) (n=55). Data are reported as means±SE. ****P<0.0001. C, Kaplan-Meier analyses of overall survival in all glioma patients with differential Cav-1 expression and/or VM formation.

Figure 2. Immunolocalization and correlation between vasculogenic mimicry (VM) formation and Cav-1 expression in glioma (n=94). A, Cav-1 (red) and CD31 (green) expression in the blood vessels of normal tissues and low-grade gliomas and abundant Cav-1-positive (red) and CD31-negative cells around the VM-like structures (blue arrow heads) in high-grade glioma tissues (scale bar 20 μm). B, ROC analysis shows cause-effect relationship between factors and survival. Diagonal segments are produced by ties. C, Correlation between VM formation and Cav-1 expression.

Figure 3. Cav-1 promotes U251 glioma cell proliferation in vitro. U251 cells were transfected with empty vector pEGFP-C3 or the vectors expressing Cav-1, siCav-1, or negative control siRNA (siNC). The transfection efficiency was confirmed by qPCR (A) and western blot analysis (B). C, Cell proliferation was measured at time points as indicated using the CCK-8 assay. D, Human umbilical vein endothelial cells (HUVECs) and U251 glioma cells were transfected with pEGFP-C3, pEGFP-C3-Cav-1, siCav-1, or siNC and verified by enhanced chemiluminescence assay. Images were captured at magnification 40 (scale bar 500 μm) and 100× (scale bar 100 μm). Representative images are shown. E, Branch points in 3 randomly selected fields were counted using ImageJ software. Data are reported as means±SE. **P<0.01, ***P<0.001, ****P<0.0001 vs other groups (transfected with pEGFP-C3, siCav-1, or siNC; ANOVA) for n=3. NC: negative control.

Figure 4. Cav-1 regulates p-Akt and HIF-1α expression. U251 cells were transfected with pEGFP-C3, pEGFP-C3-Cav-1, siCav-1, or siNC. The mRNA expressions of Akt and HIF-1α were determined by qPCR (A) and protein levels of HIF-1α (B), and Akt and p-Akt (C) were assessed by western blot analysis. ****P<0.0001 vs other groups (transfected with pEGFP-C3, siCav-1, or siNC; ANOVA) for n=3. NC: negative control.