Prostate-Specific Membrane Antigen (PSMA) Promotes Angiogenesis of Glioblastoma Through Interacting With ITGB4 and Regulating NF-κB Signaling Pathway

Abstract

Background: Glioblastoma multiforme (GBM) is the most common primary malignant tumor in the central nervous system (CNS), causing the extremely poor prognosis. Combining the role of angiogenesis in tumor progression and the role of prostate-specific membrane antigen (PSMA) in angiogenesis, this study aims to explore the functions of PSMA in GBM.

Methods: Clinical GBM specimens were collected from 60 patients who accepted surgical treatment in Fudan University Shanghai Cancer Center between January 2018 and June 2019. Immunohistochemical staining was used to detect PSMA and CD31 expression in GBM tissues. Prognostic significance of PSMA was evaluated by bioinformatics. Human umbilical vein endothelial cells (HUVECs) transfected with PSMA overexpression plasmids or cultured with conditioned medium collected based on GBM cells, were used for CCK8, Transwell and tube formation assays. High-throughput sequencing and immunoprecipitation were used to explore the underlying mechanism. Furthermore, the in vivo experiment had been also conducted.

Results: We demonstrated that PSMA was abundantly expressed in endothelium of vessels of GBM tissues but not in vessels of normal tissues, which was significantly correlated with poor prognosis. Overexpression of PSMA could promotes proliferation, invasion and tube formation ability of human umbilical vein endothelial cells (HUVECs). Moreover, U87 or U251 conditioned medium could upregulated PSMA expression and induce similar effects on phenotypes of HUVECs, all of which could be partially attenuated by 2-PMPA treatment. The mechanistic study revealed that PSMA might promote angiogenesis of GBM through interacting with Integrin β4 (ITGB4) and activating NF-κB signaling pathway. The in vivo growth of GBM could be alleviated by the treatment of 2-PMPA.

Conclusion: This study identified PSMA as a critical regulator in angiogenesis and progression of GBM, which might be a promising therapeutic target for GBM treatment.

Keywords: GBM; HUVEC; PSMA; angiogenesis; tumor progression.

FIGURE 1

PSMA is abundantly expressed in neurovascular structure of GBM and predicts poor prognosis. (A) IHC was performed to detect PSMA expression in normal and GBM tissues and representative images of GBM tissues with faint, moderate and maximum PSMA expression were shown (Scale bar = 100 μm). (B) The Immunofluorescence colocalization detection of CD31 and PSMA in GBM tissues (Scale bar = 100 μm). (C) Kaplan-Meier survival analysis was performed to reveal the significance of PSMA expression in prognosis of GBM patients.

FIGURE 2

Overexpression of PSMA promotes proliferation, invasion and tube formation of HUVECs. (A) qPCR was used to detect PSMA expression in HUVECs with or without PSMA overexpression. (B) CCK8 assay was performed to examine the effects of PSMA overexpression on proliferation of HUVECs. (C) Transwell assay was used to evaluate the effects of PSMA overexpression on invasion ability of HUVECs (Scale bar = 100 μm). (D) Tube forming assay was carried up to assess the effects of PSMA overexpression on tube formation ability of HUVECs (Scale bar = 50 μm). Data were presented as mean with SD. **P < 0.01.

FIGURE 3

PSMA plays critical role in angiogenesis of GBM. (A) qPCR was used to detect PSMA expression in HUVECs cultured with normal medium or U87/U251 conditioned medium, and treated with vehicle or 2-PMPA. (B) CCK8 assay was performed to examine the effects of U87/U251 conditioned medium culturing and inhibitor 2-PMPA on proliferation of HUVECs. (C) Transwell assay was used to evaluate the effects of U87/U251 conditioned medium culturing and inhibitor 2-PMPA on invasion ability of HUVECs (Scale bar = 100 μm). (D) Tube forming assay was carried up to assess the effects of U87/U251 conditioned medium culturing and inhibitor 2-PMPA on tube formation ability of HUVECs (Scale bar = 50 μm). Data were presented as mean with SD. *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 4

PSMA promotes GBM angiogenesis through regulating NF-κB signaling pathway. (A) The enrichment analysis of DEGs identified by RNA sequencing was performed based on KEGG. (B) The heat-map showed the expression of DEGs of TNF signaling pathway in HUVECs with or without PSMA overexpression. (C) GSEA analysis indicated that DEGs in TNF signaling pathway were significantly enriched in NF-κB signaling pathway. (D) Five key factors in TNF or NF-κB signaling pathways, including IL-6, TNF-α, CXCL1, EDN1 and ICAM1 were subjected to qPCR for detecting their expression in HUVECs with or without PSMA overexpression. Data were presented as mean with SD. *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 5

PSMA promotes GBM angiogenesis through interacting with ITGB4. (A) HUVECs with or without PSMA overexpression were lysed and immunoprecipitated with anti-PSMA antibody. (B) The expression of PSMA and ITGB4 was detected in input complex, and the expression of PSMA was detected in IP complex. (C) The expression of ITGB4 in GBM tissues (left, n = 163) and normal tissues (right, n = 207) was analyzed through GEPIA online tools. (D) The correlation between ITGB4 expression and GBM patients’ prognosis was analyzed through GEPIA online tools. *P < 0.05.

FIGURE 6

PSMA promotes GBM development in vivo. (A) Bioluminescence imaging was performed to evaluate the growth of xenografts, (B) and the intensity of which was scanned as a representation of tumor growth. (C,D) The expression of PSMA, ITGB4, and phosphorylation of NF-κB p65 were also detected after using inhibitor 2-PMPA in vivo. Data were presented as mean with SD. *P < 0.05.