angiogenin and Glioblastoma

Angiogenin is a multifunctional secreted ribonuclease that is upregulated in human cancers and downregulated or mutationally inactivated in neurodegenerative diseases. A role for angiogenin in glioblastoma was inferred from the inverse correlation of angiogenin expression with patient survival but had not been experimentally investigated.. Angiogenin knockout mice were generated and the effect of angiogenin deficiency on glioblastoma progression was examined. Angiogenin and plexin-B2 genes were knocked down in glioblastoma cells and the changes in cell proliferation, invasion and vascular association were examined. Monoclonal antibodies of angiogenin and small molecules were used to assess the therapeutic activity of the angiogenin-plexin-B2 pathway in both genetic and xenograft animal models.. Deletion of Ang1 gene prolonged survival of PDGF-induced glioblastoma in mice in the Ink4a/Arf. Angiogenin and its receptor, plexin-B2, are a pair of novel regulators that mediate invasion, vascular association and proliferation of glioblastoma cells. Inhibitors of the angiogenin-plexin-B2 axis have therapeutic potential against glioblastoma.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Glioblastoma; Humans; Mice; Nerve Tissue Proteins; Ribonuclease, Pancreatic

Angiogenin is a small protein that exerts potent stimulating effects on angiogenesis. In this study, we aimed to examine the expression of angiogenin in different subtypes of glioblastoma and estimated its independent prognostic value.. The genomic and survival data from The Cancer Genome Atlas-glioblastoma were extracted for a secondary study. Results The expression of angiogenin was upregulated in glioblastoma tissues and varied significantly in different subtypes. Although the proneural subtype had the lowest angiogenin expression, high angiogenin expression was associated with significantly worse overall survival. However, this association was not observed in other subtypes. By performing univariate and multivariate analysis using Cox regression model, we observed that high angiogenin expression was an independent indicator of shorter overall survival in proneural glioblastoma (hazard ratio: 1.669, 95% confidence interval: 1.033-2.696, P = .036), after adjustment of age, gender, isocitrate dehydrogenase 1 mutation, temozolomide chemotherapy and radiation therapy. In addition, we also observed a correlation between elevated angiogenin expression and the hypomethylated status of its DNA. The hypermethylation group had significantly better overall survival.. Angiogenin upregulation might serve as a biomarker for unfavorable overall survival in the proneural subtype of glioblastoma.

Topics: Aged; Biomarkers, Tumor; Brain Neoplasms; Computational Biology; DNA Methylation; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Kaplan-Meier Estimate; Male; Middle Aged; Prognosis; Ribonuclease, Pancreatic; ROC Curve; Up-Regulation

Angiogenin (ANG) is a secreted ribonuclease (RNase) with cell-type- and context-specific roles in growth, survival, and regeneration. Although these functions require receptor-mediated endocytosis and appropriate subcellular localization, the identity of the cell surface receptor remains undefined. Here, we show that plexin-B2 (PLXNB2) is the functional receptor for ANG in endothelial, cancer, neuronal, and normal hematopoietic and leukemic stem and progenitor cells. Mechanistically, PLXNB2 mediates intracellular RNA processing that contribute to cell growth, survival, and regenerative capabilities of ANG. Antibodies generated against the ANG-binding site on PLXNB2 restricts ANG activity in vitro and in vivo, resulting in inhibition of established xenograft tumors, ANG-induced neurogenesis and neuroprotection, levels of pro-self-renewal transcripts in hematopoietic and patient-derived leukemic stem and progenitor cells, and reduced progression of leukemia in vivo. PLXNB2 is therefore required for the physiological and pathological functions of ANG and has significant therapeutic potential in solid and hematopoietic cancers and neurodegenerative diseases.

Topics: Animals; Breast Neoplasms; Cell Proliferation; Female; Glioblastoma; Hematopoietic Stem Cells; Heterografts; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Nerve Tissue Proteins; Neurogenesis; Ribonuclease, Pancreatic

Angiogenin (Ang) is known to induce cell proliferation and inhibit apoptosis by cellular signaling pathways and its direct nuclear functions, but the mechanism of action for Ang in astrocytoma is not yet clear. Astrocytoma is the most frequent one among various neurogliomas, of which a subtype known as glioblastoma multiforme (GBM) is the most malignant brain glioma and seriously influences the life quality of the patients. The expression of Ang and Bcl-xL were detected in 28 cases of various grades of astrocytoma and 6 cases of normal human tissues by quantitative real-time PCR. The results showed that the expression of Ang and Bcl-xL positively correlated with the malignant grades. Cytological experiments indicated that Ang facilitated human glioblastoma U87MG cell proliferation and knock-down of endogenous Ang promoted cell apoptosis. Furthermore, Ang activated NF-κB pathway and entered the U87MG cell nuclei, and blocking NF-κB pathway or inhibiting Ang nuclear translocation partially suppressed Ang-induced cell proliferation. The results suggested that Ang participated in the regulation of evolution process of astrocytoma by interfering NF-κB pathway and its nucleus function. In addition, four and a half LIM domains 3 (FHL3), a novel Ang binding partner, was required for Ang-mediated HeLa cell proliferation in our previous study. We also found that knockdown of FHL3 enhanced IκBα phosphorylation and overexpression of Ang inhibited FHL3 expression in U87MG cells. Together our findings suggested that Ang could activate NF-κB pathway by regulating the expression of FHL3. In conclusion, the present study established a link between Ang and FHL3 proteins and identifies a new pathway for regulating astrocytoma progression.

Topics: Cell Line, Tumor; Cell Proliferation; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Intracellular Signaling Peptides and Proteins; LIM Domain Proteins; Male; Neoplasm Proteins; NF-kappa B; Ribonuclease, Pancreatic; Signal Transduction

In our earlier reports, we showed that downregulation of uPA and uPAR inhibited glioma tumor angiogenesis in SNB19 cells, and intraperitoneal injection of a hairpin shRNA expressing plasmid targeting uPA and uPAR inhibited angiogenesis in nude mice. The exact mechanism by which inhibition of angiogenesis takes place is not clearly understood.. In the present study, we have attempted to investigate the mechanism by which uPA/uPAR downregulation by shRNA inhibits angiogenesis in endothelial and glioblastoma cell lines. uPA/uPAR downregulation by shRNA in U87 MG and U87 SPARC co-cultures with endothelial cells inhibited angiogenesis as assessed by in vitro angiogenesis assay and in vivo dorsal skin-fold chamber model in nude mice. Protein antibody array analysis of co-cultures of U87 and U87 SPARC cells with endothelial cells treated with pU2 (shRNA against uPA and uPAR) showed decreased angiogenin secretion and angiopoietin-1 as well as several other pro-angiogenic molecules. Therefore, we investigated the role of angiogenin and found that nuclear translocation, ribonucleolytic and 45S rRNA synthesis, which are all critical for angiogenic function of angiogenin, were significantly inhibited in endothelial cells transfected with uPA, uPAR and uPA/uPAR when compared with controls. Moreover, uPA and uPAR downregulation significantly inhibited the phosphorylation of Tie-2 receptor and also down regulated FKHR activation in the nucleus of endothelial cells via the GRB2/AKT/BAD pathway. Treatment of endothelial cells with ruPA increased angiogenin secretion and angiogenin expression as determined by ELISA and western blotting in a dose-dependent manner. The amino terminal fragment of uPA down regulated ruPA-induced angiogenin in endothelial cells, thereby suggesting that uPA plays a critical role in positively regulating angiogenin in glioblastoma cells.. Taken together, our results suggest that uPA/uPAR downregulation suppresses angiogenesis in endothelial cells induced by glioblastoma cell lines partially by downregulation of angiogenin and by inhibition of the angiopoietin-1/AKT/FKHR pathway.

Topics: Activating Transcription Factors; Active Transport, Cell Nucleus; Angiopoietin-1; Angiopoietin-2; Animals; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Coculture Techniques; Dose-Response Relationship, Drug; Down-Regulation; Endothelial Cells; Gene Knockout Techniques; Glioblastoma; Humans; Mice; Neovascularization, Pathologic; Phosphorylation; Receptor, TIE-2; Receptors, Urokinase Plasminogen Activator; Ribonuclease, Pancreatic; RNA, Ribosomal; RNA, Small Interfering; Signal Transduction; Transcription, Genetic; Urokinase-Type Plasminogen Activator

Vascular endothelial growth factor (VEGF) has been identified as a critical regulator of angiogenesis. Currently, several different strategies are being used to target the VEGF-VEGF receptor signal transduction pathway in glioblastoma. Although anti-VEGF therapy seems be effective in normalizing abnormal tumor vasculature, leading to an enhanced response to radiation and chemotherapy, tumors eventually become resistant to the therapy and adopt a highly infiltrative and invasive phenotype.. In the present study, we evaluated the effects of anti-VEGF therapy (bevacizumab) on glioblastoma invasion both in vitro and in vivo and evaluated the angiogenesis- and invasion-related mediators of developed resistance to this therapy.. We found that glioblastoma tumors escaped from antiangiogenic treatment by (a) reactivating angiogenesis through up-regulation of other proangiogenic factors and (b) invading normal brain areas, which was seen in association with up-regulation of matrix metalloproteinase (MMP)-2, MMP-9, and MMP-12; secreted protein, acidic, cysteine-rich; and tissue inhibitor of metalloproteinase 1. In addition to the paracrine effects of VEGF on endothelial cells, autocrine VEGF signaling seemed to regulate glioblastoma invasion because anti-VEGF therapy increased tumor invasiveness in vitro.. Collectively, these findings reinforce the importance of VEGF in regulating tumor invasion and identify potential mediators of resistance to targeted VEGF therapy. These results will be important for developing novel combination therapies to overcome this resistance phenotype.

Topics: Angiogenesis Inhibitors; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Bevacizumab; Blotting, Western; Cell Line, Tumor; Cell Movement; Chemokine CXCL9; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Kaplan-Meier Estimate; Matrix Metalloproteinase 12; Mice; Mice, Nude; Neoplasm Invasiveness; Reverse Transcriptase Polymerase Chain Reaction; Ribonuclease, Pancreatic; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-2; Xenograft Model Antitumor Assays