Therapeutic potential of pentamidine for glioma-initiating cells and glioma cells through multimodal antitumor effects.

Glioma-initiating cells, which comprise a heterogeneous population of glioblastomas, contribute to resistance against aggressive chemoradiotherapy. Using drug reposition, we investigated a therapeutic drug for glioma-initiating cells. Drug screening was undertaken to select candidate agents that inhibit proliferation of two different glioma-initiating cells lines. The alteration of proliferation and stemness of the two glioma-initiating cell lines, and proliferation, migration, cell cycle, and survival of these two differentiated glioma-initiating cell lines and three different glioblastoma cell lines treated with the candidate agent were evaluated. We also used a xenograft glioma mouse model to evaluate anticancer effects of treated glioma cell lines. Among the 1301 agents, pentamidine-an antibiotic for Pneumocystis jirovecii-emerged as a successful antiglioma agent. Pentamidine treatment suppressed proliferation and stemness in glioma-initiating cell lines. Proliferation and migration were inhibited in all differentiated glioma-initiating cells and glioblastoma cell lines, with cell cycle arrest and caspase-dependent apoptosis induction. The in vivo study reproduced the same findings as the in vitro studies. Pentamidine showed a stronger antiproliferative effect on glioma-initiating cells than on differentiated cells. Western blot analysis revealed pentamidine inhibited phosphorylation of signal transducer and activator of transcription 3 in all cell lines, whereas Akt expression was suppressed in glioma-initiating cells but not in differentiated lines. In the present study, we identified pentamidine as a potential therapeutic drug for glioma. Pentamidine could be promising for the treatment of glioblastomas by targeting both glioma-initiating cells and differentiated cells through its multifaceted antiglioma effects.

Glioma-derived extracellular vesicles promote tumor progression by conveying WT1.

Glioma persists as one of the most aggressive primary tumors of the central nervous system. Glioma cells are known to communicate with tumor-associated macrophages/microglia via various cytokines to establish the tumor microenvironment. However, how extracellular vesicles (EVs), emerging regulators of cell-cell communication networks, function in this process is still elusive. We report here that glioma-derived EVs promote tumor progression by affecting microglial gene expression in an intracranial implantation glioma model mouse. The gene expression of thrombospondin-1 (Thbs1), a negative regulator of angiogenesis, was commonly downregulated in microglia after the addition of EVs isolated from different glioma cell lines, which endogenously expressed Wilms tumor-1 (WT1). Conversely, WT1-deficiency in the glioma-derived EVs significantly attenuated the Thbs1 downregulation and suppressed the tumor progression. WT1 was highly expressed in EVs obtained from the cerebrospinal fluid of human patients with malignant glioma. Our findings establish a novel model of tumor progression via EV-mediated WT1-Thbs1 intercellular regulatory pathway, which may be a future diagnostic or therapeutic target.

RBPJ contributes to the malignancy of glioblastoma and induction of proneural-mesenchymal transition via IL-6-STAT3 pathway.

Notch signaling plays a pivotal role in many cancers, including glioblastoma (GBM). Recombination signal binding protein for immunoglobulin kappa J region (RBPJ) is a key transcription factor of the Notch signaling pathway. Here, we interrogated the function of RBPJ in GBM. Firstly, RBPJ expression of GBM samples was examined. Then, we knocked down RBPJ expression in 2 GBM cell lines (U251 and T98) and 4 glioblastoma (GBM) stem-like cell lines derived from surgical samples of GBM (KGS01, KGS07, KGS10 and KGS15) to investigate the effect on cell proliferation, invasion, stemness, and tumor formation ability. Expression of possible downstream targets of RBPJ was also assessed. RBPJ was overexpressed in the GBM samples, downregulation of RBPJ reduced cell proliferation and the invasion ability of U251 and T98 cells and cell proliferation ability and stemness of glioblastoma stem-like cells (GSC) lines. These were accompanied by reduced IL-6 expression, reduced activation of STAT3, and inhibited proneural-mesenchymal transition (PMT). Tumor formation and PMT were also impaired by RBPJ knockdown in vivo. In conclusion, RBPJ promotes cell proliferation, invasion, stemness, and tumor initiation ability in GBM cells through enhanced activation of IL-6-STAT3 pathway and PMT, inhibition of RBPJ may constitute a prospective treatment for GBM.

Gelsolin inhibits malignant phenotype of glioblastoma and is regulated by miR-654-5p and miR-450b-5p.

We have previously shown that gelsolin (GSN) levels are significantly lower in the blood of patients with glioblastoma (GBM) than in healthy controls. Here, we analyzed the function of GSN in GBM and examined its clinical significance. Furthermore, microRNAs involved in GSN expression were also identified. The expression of GSN was determined using western blot analysis and found to be significantly lower in GBM samples than normal ones. Gelsolin was mainly localized in normal astrocytes, shown using immunohistochemistry and immunofluorescence. Higher expression of GSN was correlated with more prolonged progression-free survival and overall survival. Gelsolin knockdown using siRNA and shRNA markedly accelerated cell proliferation and invasion in GBM in vitro and in vivo. The inactive form of glycogen synthase kinase-3ß was dephosphorylated by GSN knockdown. In GBM tissues, the expression of GSN and microRNA (miR)-654-5p and miR-450b-5p showed an inverse correlation. The miR-654-5p and miR-450b-5p inhibitors enhanced GSN expression, resulting in reduced proliferation and invasion. In conclusion, GSN, which inhibits cell proliferation and invasion, is suppressed by miR-654-5p and miR-450b-5p in GBM, suggesting that these miRNAs can be targets for treating GBM.

Fibulin-7 is overexpressed in glioblastomas and modulates glioblastoma neovascularization through interaction with angiopoietin-1.

Glioblastoma (GBM) is pathologically characterized by highly malignant neoplastic cells, focal necrosis and aberrant blood vessels composed of disorganized endothelial cells and pericytes. The recent cancer microarray database revealed upregulation of fibulin-7 (Fbln7), a member of the fibulin family, but provided no information on the tissue localization or biological function. In the present study, we demonstrated that Fbln7 is markedly overexpressed by the GBM tissue among astrocytic tumors, and immunolocalized mainly to endothelial cells and pericytes of the glomeruloid and hypertrophied microvessels. The production of Fbln7 by endothelial cells and pericytes was confirmed in cultured human umbilical vein endothelial cells (HUVEC) and human brain vascular pericytes (HBVP) and vascular endothelial growth factor (VEGF) stimulated the Fbln7 expression in HUVEC. Fbln7 bound to angiopoietin-1, but not angiopoietin-2 or Tie2 receptor, through interaction between the N-terminal portions of Fbln7 and angiopoietin-1, and it blocked phosphorylation of Tie2 receptor in HUVEC. In a coculture assay using HUVEC and HBVP, multilayered and irregular-shaped tube-like structures of HUVEC were induced by treatment with a high concentration of VEGF. This was accompanied by Fbln7 overproduction by HUVEC and angiopoietin-1 expression by HBVP. The production of aberrant VEGF-induced tube-like structures was attenuated by treatment with antibody or synthetic peptides specific to the Fbln7 N-terminal domain or knockdown of Fbln7. These data demonstrate that Fbln7 is overexpressed by endothelial cells and pericytes of the abnormal microvessels in GBM, and suggest that Fbln7 may contribute to the aberrant vessel formation by modulation of the angiopoietin-1/angiopoietin-2-Tie2 axis.

Nucleoporin TPR (translocated promoter region, nuclear basket protein) upregulation alters MTOR-HSF1 trails and suppresses autophagy induction in ependymoma.

Children with ependymoma have high mortality rates because ependymoma is resistant to conventional therapy. Genomic and transcriptomic studies have identified potential targets as significantly altered genes in ependymoma patients. Although several candidate oncogenes in ependymoma were recently reported, the detailed mechanisms for the roles of these candidate oncogenes in ependymoma progression remain unclear. Here, we report an oncogenic role of the nucleoporin TPR (translocated promoter region, nuclear basket protein) in regulating HSF1 (heat shock transcription factor 1) mRNA trafficking, maintaining MTORC1 activity to phosphorylate ULK1, and preventing macroautophagy/autophagy induction in ependymoma. High expression of TPR were associated with increased HSF1 and HSPA/HSP70 expression in ependymoma patients. In an ependymoma mouse xenograft model, MTOR inhibition by rapamycin therapeutically suppressed TPR expression and reduced tumor size in vivo. Together, these results suggest that TPR may act as a biomarker for ependymoma, and pharmacological interventions targeting TPR-HSF1-MTOR may have therapeutic potential for ependymoma treatment.Abbreviations: ATG: autophagy related; BECN1: beclin 1; BSA: bovine serum albumin; CQ: chloroquine; DMSO: dimethyl sulfoxide; GEO: gene expression omnibus; GFP: green fluorescence protein; HSF1: heat shock transcription factor 1; HSPA/HSP70: heat shock protein family A (Hsp70); LMNB1: lamin B1; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAPK: mitogen-activated protein kinase; MAPK8/JNK: mitogen-activated protein kinase 8; MTORC1: mechanistic target of rapamycin kinase complex 1; NPC: nuclear pore complex; NUP: nucleoporin; PBS: phosphate-buffered saline; q-PCR: quantitative real time PCR; SDS: sodium dodecyl sulfate; SQSTM1: sequestosome 1; STED: stimulated emission depletion microscopy; STX17: syntaxin 17; TCGA: the cancer genome atlas; TPR: translocated promoter region, nuclear basket protein; ULK1: unc-51 like autophagy activating kinase 1.

Identification of 2-Fluoropalmitic Acid as a Potential Therapeutic Agent Against Glioblastoma.

BACKGROUND: Glioblastomas (GBMs) are aggressive malignant brain tumors. Although chemotherapy with temozolomide (TMZ) can extend patient survival, most patients eventually demonstrate resistance. Therefore, novel therapeutic agents that overcome TMZ chemoresistance are required to improve patient outcomes. PURPOSE: Drug screening is an efficient method to find new therapeutic agents from existing drugs. In this study, we explored a novel anti-glioma agent by drug screening and analyzed its function with respect to GBM treatment for future clinical applications. METHODS: Drug libraries containing 1,301 diverse chemical compounds were screened against two glioma stem cell (GSC) lines for drug candidate selection. The effect of selected agents on GSCs and glioma was estimated through viability, proliferation, sphere formation, and invasion assays. Combination therapy was performed to assess its ability to enhance TMZ cytotoxicity against GBM. To clarify the mechanism of action, we performed methylation-specific polymerase chain reaction, gelatin zymography, and western blot analysis. RESULTS: The acyl-CoA synthetase inhibitor 2-fluoropalmitic acid (2-FPA) was selected as a candidate anti-glioma agent. 2-FPA suppressed the viability and stem-like phenotype of GSCs. It also inhibited proliferation and invasion of glioma cell lines. Combination therapy of 2-FPA with TMZ synergistically enhanced the efficacy of TMZ. 2-FPA suppressed the expression of phosphor-ERK, CD133, and SOX-2; reduced MMP-2 activity; and increased methylation of the MGMT promoter. CONCLUSION: 2-FPA was identified as a potential therapeutic agent against GBM. To extend these findings, physiological studies are required to examine the efficacy of 2-FPA against GBM in vivo.

Identification of GSK3ß inhibitor kenpaullone as a temozolomide enhancer against glioblastoma.

Cancer stem cells are associated with chemoresistance and rapid recurrence of malignant tumors, including glioblastoma (GBM). Although temozolomide (TMZ) is the most effective drug treatment for GBM, GBM cells acquire resistance and become refractory to TMZ during treatment. Therefore, glioma stem cell (GSC)-targeted therapy and TMZ-enhancing therapy may be effective approaches to improve GBM prognosis. Many drugs that suppress the signaling pathways that maintain GSC or enhance the effects of TMZ have been reported. However, there are no established therapies beyond TMZ treatment currently in use. In this study, we screened drug libraries composed of 1,301 existing drugs using cell viability assays to evaluate effects on GSCs, which led to selection of kenpaullone, a kinase inhibitor, as a TMZ enhancer targeting GSCs. Kenpaullone efficiently suppressed activity of glycogen synthase kinase (GSK) 3ß. Combination therapy with kenpaullone and TMZ suppressed stem cell phenotype and viability of both GSCs and glioma cell lines. Combination therapy in mouse models significantly prolonged survival time compared with TMZ monotherapy. Taken together, kenpaullone is a promising drug for treatment of GBM by targeting GSCs and overcoming chemoresistance to TMZ.

Ligand-dependent EphB4 activation serves as an anchoring signal in glioma cells.

Of the erythropoietin-producing human hepatocellular receptors (Ephs), EphB4 has recently emerged as a potential target in several cancers due to its roles in modified cell migration and invasion. As little is known about the roles of EphB4 in glioma, we sought to investigate its function in glioma by in vitro cell migration and invasion assays, immunoblotting and immunostaining. EphB4 was expressed in glioma cell lines and stem-like cell lines. The stimulation of glioma cells with ephrin-B2, the sole ligand of EphB4, conducted EphB4 phosphorylation and suppressed migration and invasion that downregulation of EphB4 using small interfering RNA abrogated. The stimulation also suppressed the phosphorylation of Akt. We confirmed by immunostaining that EphB4-positive cells existing only in the tumor core, whereas ephrin-B2-positive cells widespread in both the tumor core and the invasive area signifying that EphB4-ephrin-B2 reaction occurred only at the tumor core. Taken together, our data suggest that ephrin-B2-dependent EphB4 phosphorylation acts as an anchoring signal to reduce the malignancy by inhibiting Akt phosphorylation in the glioma core, whereas the scarcity of signaling in the tumor periphery promotes invasion into the surrounding brain.

Potential Strategies Overcoming the Temozolomide Resistance for Glioblastoma.

Glioblastoma (GBM) is a highly malignant type of primary brain tumor with a high mortality rate. Although the current standard therapy consists of surgery followed by radiation and temozolomide (TMZ), chemotherapy can extend patient's post-operative survival but most cases eventually demonstrate resistance to TMZ. O6-methylguanine-DNA methyltransferase (MGMT) repairs the main cytotoxic lesion, as O6-methylguanine, generated by TMZ, can be the main mechanism of the drug resistance. In addition, mismatch repair and BER also contribute to TMZ resistance. TMZ treatment can induce self-protective autophagy, a mechanism by which tumor cells resist TMZ treatment. Emerging evidence also demonstrated that a small population of cells expressing stem cell markers, also identified as GBM stem cells (GSCs), contributes to drug resistance and tumor recurrence owing to their ability for self-renewal and invasion into neighboring tissue. Some molecules maintain stem cell properties. Other molecules or signaling pathways regulate stemness and influence MGMT activity, making these GCSs attractive therapeutic targets. Treatments targeting these molecules and pathways result in suppression of GSCs stemness and, in highly resistant cases, a decrease in MGMT activity. Recently, some novel therapeutic strategies, targeted molecules, immunotherapies, and microRNAs have provided new potential treatments for highly resistant GBM cases. In this review, we summarize the current knowledge of different resistance mechanisms, novel strategies for enhancing the effect of TMZ, and emerging therapeutic approaches to eliminate GSCs, all with the aim to produce a successful GBM treatment and discuss future directions for basic and clinical research to achieve this end.

Identification of antipsychotic drug fluspirilene as a potential anti-glioma stem cell drug.

Glioma stem cell (GSC)-targeted therapy is expected to be one of the most innovative approaches to treat patients with glioblastoma (GBM). A number of the drugs that restrain the signaling pathway essential for GSC maintenance have been under clinical trials. Here, we identified fluspirilene, a traditional antipsychotic drug, as a GSC-targeting agent, selected from thousands of existing drugs, and investigated its therapeutic effects against GBM with the purpose of drug repositioning. To develop novel therapeutics targeting GSCs, we initially screened drug libraries for small-molecule compounds showing a greater efficacy, compared to that of controls, in inhibiting the proliferation and survival of different GSC lines using cell proliferation assay. Drugs already reported to show therapeutic effects against GBM or those under clinical trials were excluded from subsequent screening. Finally, we found three drugs showing remarkable antiproliferative effects on GSCs at low concentrations and investigated their therapeutic effects on GSCs, glioma cell lines, and in a GBM mouse model. Of the three compounds, fluspirilene demonstrated a significant inhibitory effect on the proliferation and invasion of glioma cells as well as in the model mice treated with the drug. These effects were associated with the inactivation of the signal transducer and activator of transcription 3 (STAT3). Redeveloping of fluspirilene is a promising approach for the treatment of GBM.