Meningioma Cell Lines Research Articles

Forkhead box M1 (FOXM1) transcription factor is a key oncogenic driver of aggressive human meningioma progression.

Aggressive meningioma remains incurable with neither chemo- nor targeted therapies proven effective, largely due to unidentified genetic alterations and/or aberrant oncogenic pathways driving the disease progression. In this study, we examined the expression and function of Forkhead box M1 (FOXM1) transcription factor during meningioma progression. Human meningioma samples (n=101) were collected, followed by Western blotting, quantitative PCR, immunohistochemical and progression-free survival (PFS) analyses. For in vitro assays, FOXM1 was overexpressed or knocked-down in benign (SF4433 and SF4068) or malignant (SF3061 and IOMM-Lee) human meningioma cell lines respectively. For in vivo studies, siomycin A (a FOXM1 inhibitor)-pretreated or control IOMM-Lee cells were implanted subcutaneously in nude mice. FOXM1 expression was increased in higher grades of meningioma and correlated with the mitotic index in the tumour tissue. Moreover, FOXM1 was increased in recurrent meningioma compared with the matched primary lesions. The patients who had higher FOXM1 expression had shorter PFS. In the subsequent in vitro assays, knockdown of FOXM1 in malignant meningioma cell lines resulted in decreased tumour cell proliferation, angiogenesis and invasion, potentially via regulation of β-catenin, cyclin D1, p21, interleukin-8, vascular endothelial growth factor-A, PLAU, and epithelial-to-mesenchymal transition-related genes, whereas overexpression of FOXM1 in benign meningioma cell lines had the opposite effects. Last, suppression of FOXM1 using a pharmacological inhibitor, siomycin A, decreased tumour growth in an in vivo mouse model. Our data demonstrate that FOXM1 is a key transcription factor regulating oncogenic signalling pathways in meningioma progression, and a promising therapeutic target for aggressive meningioma.

Antitumor Effects of Chrysophanol in Malignant Optic Nerve Meningioma Cell Lines are Mediated via Caspase Activation, Induction of Mitochondrial Mediated Apoptosis, Mitochondrial Membrane Depolarization and Targeting the Mitogen-Activated Protein Kinase Signaling Pathway

Anthroquinones are considered remarkable anticancer agents. Chrysophanol is an important anthroquinone and it has shown to have the potential to inhibit the growth of the range of cancers. However, there are no studies regarding the anticancer effects of chrysophanol against the malignant meningioma of optic nerve. In this review, the potential of chrysophanol in the treatment of malignant -meningioma of optic nerve was explored by evaluating its anticancer activity against the malignant meningioma CH157-MN cells. The 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide assay was used for cell viability determination. The 4',6-diamidino-2-phenylindole (DAPI), acridine orange and ethidium bromide (AO/EB) and annexin V/PI assays were used to determine the induction of apoptosis. The potential of reactive oxygen species and the mitochondrial membrane was estimated by flow cytometry. Western blot analysis was performed to determine the protein expression. The results showed that chrysophanol caused significant decline in the viability of the CH157-MN cells and exhibited an IC50 of 30 µmol/L. Anticancer effects were found to be due to the induction of apoptosis as evident form the DAPI and AO/EB staining. The annexin V/PI staining revealed that the apoptotic cells increased from 1.77% in control to 37.21% at 60 µmol/L concentration of chrysophanol. The Bcl-2/Bax expression ratio was decreased and the caspases-3 and 9 were activated upon chrysophanol treatment of the CH157-MN cells. Chrysophanol also triggered the formation of reactive oxygen species and reduction of the mitochondrial membrane potential in the CH157-MN cells and also blocked the Mitogen-activated protein kinase signaling pathway. The findings of the present study suggest that chrysophanol may prove beneficial in the treatment of malignant meningioma of optic nerve. Key Message: The study revealed the anticancer potential of chrysophanol against the malignant optic nerve meningioma.

Identification of KIF11 As a Novel Target in Meningioma.

Kinesins play an important role in many physiological functions including intracellular vesicle transport and mitosis. The emerging role of kinesins in different cancers led us to investigate the expression and functional role of kinesins in meningioma. Therefore, we re-analyzed our previous microarray dataset of benign, atypical, and anaplastic meningiomas (n = 62) and got evidence for differential expression of five kinesins (KIFC1, KIF4A, KIF11, KIF14 and KIF20A). Further validation in an extended study sample (n = 208) revealed a significant upregulation of these genes in WHO°I to °III meningiomas (WHO°I n = 61, WHO°II n = 88, and WHO°III n = 59), which was most pronounced in clinically more aggressive tumors of the same WHO grade. Immunohistochemical staining confirmed a WHO grade-associated upregulated protein expression in meningioma tissues. Furthermore, high mRNA expression levels of KIFC1, KIF11, KIF14 and KIF20A were associated with shorter progression-free survival. On a functional level, knockdown of kinesins in Ben-Men-1 cells and in the newly established anaplastic meningioma cell line NCH93 resulted in a significantly inhibited tumor cell proliferation upon siRNA-mediated downregulation of KIF11 in both cell lines by up to 95% and 71%, respectively. Taken together, in this study we were able to identify the prognostic and functional role of several kinesin family members of which KIF11 exhibits the most promising properties as a novel prognostic marker and therapeutic target, which may offer new treatment options for aggressive meningiomas.

LINC00702/miR-4652-3p/ZEB1 axis promotes the progression of malignant meningioma through activating Wnt/β-catenin pathway

Long noncoding RNAs (lncRNAs) have been acknowledged as significant regulators in the progression of various cancers, including malignant meningioma. Being a newly identified lncRNA, long intergenic non-protein coding RNA 702 (LINC00702) has not been comprehensively studied in malignant meningioma. In this study, the role of LINC00702 was identified and explored in malignant meningioma. The LINC00702 expression was determined in malignant meningioma tissues and cell lines by qRT-PCR. Then the association between LINC00702 expression and the prognosis of malignant meningioma patients was analyzed by Kaplan-Meier analysis. The functional role of LINC00702 in malignant meningioma cell proliferation and migration was analyzed by loss-of function assays. Subcellular fractionation assay and FISH assay revealed the cytoplasmic localization of LINC00702. Bioinformatics analysis and mechanism experiments demonstrated that LINC00702 acted as the molecular sponge of miR-4652-3p to upregulate ZEB1 in malignant meningioma. Furthermore, high level of LINC00702 was demonstrated to be associated with the activity of Wnt/β-catenin signaling. Moreover, miR-4652-3p and ZEB1 involved in LINC00702-mediated Wnt/β-catenin signaling. At last, rescue assays revealed that miR-4652-3p and ZEB1 attenuated LINC00702-mediated cell proliferation and migration.

Immunohistochemical Characterization of Procaspase-3 Overexpression as a Druggable Target With PAC-1, a Procaspase-3 Activator, in Canine and Human Brain Cancers.

Gliomas and meningiomas are the most common brain neoplasms affecting both humans and canines, and identifying druggable targets conserved across multiple brain cancer histologies and comparative species could broadly improve treatment outcomes. While satisfactory cure rates for low grade, non-invasive brain cancers are achievable with conventional therapies including surgery and radiation, the management of non-resectable or recurrent brain tumors remains problematic and necessitates the discovery of novel therapies that could be accelerated through a comparative approach, such as the inclusion of pet dogs with naturally-occurring brain cancers. Evidence supports procaspase-3 as a druggable brain cancer target with PAC-1, a pro-apoptotic, small molecule activator of procaspase-3 that crosses the blood-brain barrier. Procaspase-3 is frequently overexpressed in malignantly transformed tissues and provides a preferential target for inducing cancer cell apoptosis. While preliminary evidence supports procaspase-3 as a viable target in preclinical models, with PAC-1 demonstrating activity in rodent models and dogs with spontaneous brain tumors, the broader applicability of procaspase-3 as a target in human brain cancers, as well as the comparability of procaspase-3 expressions between differing species, requires further investigation. As such, a large-scale validation of procaspase-3 as a druggable target was undertaken across 651 human and canine brain tumors. Relative to normal brain tissues, procaspase-3 was overexpressed in histologically diverse cancerous brain tissues, supporting procaspase-3 as a broad and conserved therapeutic target. Additionally, procaspase-3 expressing glioma and meningioma cell lines were sensitive to the apoptotic effects of PAC-1 at biologically relevant exposures achievable in cancer patients. Importantly, the clinical relevance of procaspase-3 as a potential prognostic variable was demonstrated in human astrocytomas of variable histologic grades and associated clinical outcomes, whereby tumoral procaspase-3 expression was negatively correlated with survival; findings which suggest that PAC-1 might provide the greatest benefit for patients with the most guarded prognoses.

Telomerase activity, TERT expression, hTERT promoter alterations, and alternative lengthening of the telomeres (ALT) in meningiomas - a systematic review.

Telomerase activity and (human) Telomerase Reverse Transcriptase (hTERT) expression are considered hallmarks in oncogenesis of neoplasms and are upregulated by alterations of the hTERT promoter. In meningiomas, numerous studies investigated hTERT expression, telomerase activity, promoter mutations, and methylations. Moreover, reports about hTERT-targeted chemotherapy in meningiomas have recently been published. We provide a systematic review of the literature about the role of hTERT in meningiomas. TERT expression and telomerase activity is found in benign and high-grade meningiomas and increase with WHO grade. Remarkably, rates of TERT expression/telomerase activity usually exceed mutation frequency and both telomerase activity and TERT expression have also been found in hTERT promoter wildtype meningiomas, indicating further mechanisms of TERT upregulation. Although hTERT promoter methylation has been reported in the vast majority of meningiomas, correlation with TERT expression remains controversial. Rates of promoter mutations, and methylation were shown to increase with rising WHO grade. Moreover, promoter methylation and mutations strongly correlate with prognosis. Although mutations predicted malignant progression, de novo mutations in high-grade recurrences of former benign lesions were also observed. Retroviral transduction of the TERT gene enabled immortalization in several grade I-III meningioma cell lines. In vitro analyses revealed significant effects on viability in hTERT-mutated meningioma cells after targeted treatment. Alternative mechanisms of telomere lengthening are usually absent in meningiomas. TERT and hTERT promoter alterations play a major role during oncogenesis of meningiomas with implications for prognosis and potentially treatment.

Mebendazole and radiation in combination increase survival through anticancer mechanisms in an intracranial rodent model of malignant meningioma

Meningiomas are a frequent tumor of the central nervous system. Although mostly benign, approximately 5% present as atypical or malignant tumors. Treatments for atypical meningiomas include gross total resection and radiotherapy, but about 33% of patients have recurrent tumors, sometimes as a higher grade. Recently, the brain penetrant anthelmintic drug, mebendazole, has shown promise as an anticancer agent in rodent models of glioblastoma and medulloblastoma. The half maximal inhibitory concentration (IC50) effect on colony formation, cell proliferation, and caspase-3/7 markers of apoptosis of mebendazole with and without radiation was measured in vitro. Mice intracranially implanted with KT21MG1 human meningioma were administered mebendazole alone or in combination with radiation. Survival benefit was evaluated, while tumors were investigated by immunohistochemical staining for apoptosis, cell proliferation, and vascular density. In vitro experiments on meningioma cell lines showed the IC50 for mebendazole in the range of 0.26-0.42µM. Mebendazole alone induced cytotoxicity, however the combination had a greater reduction in colony formation and resulted in higher levels of cleaved caspase-3. The in vivo study showed both, mebendazole alone and the combination, to have a survival benefit with an increase in apoptosis, and decreases in tumor cell and vascular proliferation. These preclinical findings indicate that mebendazole alone or in combination with radiation can be considered for the treatment of malignant meningioma. The mechanism of action for this combination may include an increase in apoptosis, a reduction in proliferation and angiogenesis, or a combination of these effects.

Photodynamic therapy with talaporfin sodium induces dose- and time-dependent apoptotic cell death in malignant meningioma HKBMM cells.

To investigate the effect of photodynamic therapy (PDT) with the talaporfin sodium (mono-L-asparthyl chlorine e6: NPe-6) on human malignant meningioma cell line HKBMM cells in vitro. After incubation with NPe6 for 4 h, cells underwent PDT (diode laser irradiation: 3.4 mW/cm2 and 1 J/cm2. Cell viability was determined in 2 malignant meningioma cell lines (human origin; HKBMM cells and rat origin; KMY-J cells) and human malignant glioma U251 cells with Cell Counting Kit-8 assay. The HKBMM cells were examined for caspase-3 activity, annexin V or propidium iodide (PI) staining, and lactate dehydrogenase leakage. Morphological change was also investigated with phase-contrast microscopy. In human malignant meningioma HKBMM cells, viability showed a dose- and time-dependent decrease. After 24 h of laser irradiation, NPe6 at 20 μg/ml or more induced a significant decrease in cell viability in both HKBMM cells and KMY-J cells, although they more resistance than the malignant glioma cell line U251 cells. Two kinds of morphological change were also observed in the HKBMM cells, shrinkage of the cell body, indicating apoptosis, and swelling of the cell body, indicating necrosis. In addition, both caspase-3 activity and DNA fragmentation, biochemical markers indicative of apoptosis, showed a dose-dependent increase. The percentage of necrotic cells showing positive staining for annexin V or PI was greater than that of apoptotic cells at a high concentration of NPe6. Lactate dehydrogenase leakage, a biochemical marker of necrosis, also showed a marked increase at a high concentration of NPe6. Photodynamic therapy with NPe6 induced dose- and time-dependent apoptosis in human malignant meningioma HKBMM cells. At a high concentration of NPe6, however, it induced necrosis.

Abstract 4398: In vitro Tumor Treating Fields (TTFields) alter proliferation and morphology of patient-derived high-grade meningioma cell lines

Abstract Introduction: Meningiomas are the most common neoplasm of the central nervous system (CNS), accounting for 37% of primary CNS tumors. Though derived from the arachnoid cap cells within the meninges, high-grade meningiomas (WHO grades II and III) invade into the normal brain tissue, and have a high rate of recurrence. As there are no efficacious chemotherapies for these high-grade meningiomas, standard of care is currently limited to maximal surgical resection followed by radiotherapy, with a median overall survival of only 2.5-3 years. Clinical trials with TTFields therapy are under way for patients with high-grade meningiomas. In this study, in vitro experiments comparing TTFields to untreated controls were performed on two patient-derived anaplastic (WHO grade III) meningioma cell lines to determine the effects of TTFields on cell proliferation and clonogenicity. Methods: Studies utilizing patient tumor specimens were approved by the Wayne State University Institutional Review Board and written informed consent was obtained from participants. Meningioma tumor specimens were collected immediately following microsurgical resection. Single-cell suspensions from the tumor tissues were prepared by enzymatic and mechanical disruption. Cells were plated on plastic coverslips (10,000 each) in DMEM/F12 media supplemented with 10% fetal bovine serum. TTFields were applied at ~1.6 V/cm at 100, 200, 300, or 400 kHz (groups n=4-5) with one control group. Culture media were replaced every day. After 12 days of treatment, cell proliferation was assessed with the XTT assay and cells were harvested and replated for clonogenic assays (500 cells/well), which were stained with crystal violet and counted with an automated colony counter. Harvested cells were also fixed and immunostained with the diagnostic meningioma cytoplasmic marker vimentin to observe the cell morphology in control vs. TTFields-treated cells. Control vs. TTFields-treated groups were compared by one- or two-tailed t-tests. Results: The 12-day TTFields treatment significantly reduced cell proliferation, with the greatest decrease found in the 200 kHz group (0.508±0.21; mean±SEM) compared to the control (1.515±0.08; two-tailed t test p=0.0019). Similar results were found in the clonogenic assay, with control cell counts of 4219±793 compared to the 200kHz treated cells 1793±444 (one-tailed t test p=0.0279). In the vimentin-stained cells, the control cells displayed a typical meningioma spindle morphology, while the TTFields-treated cells demonstrated pleomorphic and irregular morphologies. Conclusions: Application of TTFields in vitro reduced proliferation and clonogenicity and altered morphology of patient-derived anaplastic meningioma cell lines. This is the first report on the in vitro effects of TTFields on meningioma. Citation Format: Sharon K. Michelhaugh, Sam Kiousis, Sam A. Michelhaugh, Neil V. Klinger, Sandeep Mittal. In vitro Tumor Treating Fields (TTFields) alter proliferation and morphology of patient-derived high-grade meningioma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4398.

Investigation of the aryl hydrocarbon receptor and the intrinsic tumoral component of the kynurenine pathway of tryptophan metabolism in primary brain tumors.

There is mounting evidence supporting the role of tryptophan metabolism via the kynurenine pathway (KP) in the pathogenesis of primary brain tumors. Under normal physiological conditions, the KP is the major catabolic pathway for the essential amino acid tryptophan. However, in cancer cells, the KP becomes dysregulated, depletes local tryptophan, and contributes to an immunosuppressive tumor microenvironment. We examined the protein expression levels (in 73 gliomas and 48 meningiomas) of the KP rate-limiting enzymes indoleamine 2,3-dioxygenase (IDO) 1, IDO2, and tryptophan 2,3-dioxygenase (TDO2), as well as, the aryl hydrocarbon receptor (AhR), a carcinogenic transcription factor activated by KP metabolites. In addition, we utilized commercially available small-molecules to pharmacologically modulate IDO1, IDO2, TDO2, and AhR in patient-derived glioma and meningioma cell lines (n = 9 each). We observed a positive trend between the grade of the tumor and the average immunohistochemical staining score for IDO1, IDO2, and TDO2, with TDO2 displaying the strongest immunostaining. AhR immunostaining was present in all grades of gliomas and meningiomas, with the greatest staining intensity noted in glioblastomas. Immunocytochemical staining showed a positive trend between nuclear localization of AhR and histologic grade in both gliomas and meningiomas, suggesting increased AhR activation with higher tumor grade. Unlike enzyme inhibition, AhR antagonism markedly diminished patient-derived tumor cell viability, regardless of tumor type or grade, following in vitro drug treatments. Collectively, these results suggest that AhR may offer a novel and robust therapeutic target for a patient population with highly limited treatment options.

Anti-tumor activity of Organometallic Compounds

Modern medicine has been focusing on proving new and alternative chemotherapeutic compounds with high tumor cytotoxicity and reduced side effects to combat drug resistance and improve drug efficacy in cancer treatment. Organometallic compounds have unique physio-chemical properties like structural diversity, ligand exchange, redox and catalytic properties which are now being used to develop potential candidates for cancer therapy. The impressive clinical effectiveness of cisplatin is limited by the significant side effects and emergence of drug resistance. One of the metals showing promise is ruthenium. Ruthenium compounds often have lower cytotoxicity compared to cisplatin and are better tolerated in vivo. RuIII complexes maintain their metallic oxidation state till they reach the tumor, wherein a hypoxic condition is reduced to RuII. Moreover, in many cancer types, iron uptake is upregulated. Ruthenium, being in the same group as iron is transported into the cells by the transferrin receptor which is overexpressed in many tumors. Therefore, the properties of tumors enhance both Ru uptake and toxicity relative to normal tissues. DNA cleavage will lead to cell cycle arrest until the damage is repaired, or if it is too extensive to repair, it will undergo senescence and death that may be either dependent or independent of p53 tumor suppressor gene function. This project is focused on the synthesis, purification, and characterization of monometallic and bimetallic ruthenium compounds and evaluate its effects on glioblastoma, meningioma and pancreatic cell lines to analyze their effect on the proliferation of the tumor cells and their capacity to induce apoptosis.This project was supported by NIH Grants 5P20RR016477 and P20GM103434 to the West Virginia IDeA Network for BioMedical Research Funding.

EPH receptor signaling as a novel therapeutic target in NF2-deficient meningioma.

Meningiomas are the most common primary brain tumor in adults, and somatic loss of the neurofibromatosis 2 (NF2) tumor suppressor gene is a frequent genetic event. There is no effective treatment for tumors that recur or continue to grow despite surgery and/or radiation. Therefore, targeted therapies that either delay tumor progression or cause tumor shrinkage are much needed. Our earlier work established mammalian target of rapamycin complex mTORC1/mTORC2 activation in NF2-deficient meningiomas. High-throughput kinome analyses were performed in NF2-null human arachnoidal and meningioma cell lines to identify functional kinome changes upon NF2 loss. Immunoblotting confirmed the activation of kinases and demonstrated effectiveness of drugs to block the activation. Drugs, singly and in combination, were screened in cells for their growth inhibitory activity. Antitumor drug efficacy was tested in an orthotopic meningioma model. Erythropoietin-producing hepatocellular receptor tyrosine kinases (EPH RTKs), c-KIT, and Src family kinase (SFK) members, which are biological targets of dasatinib, were among the top candidates activated in NF2-null cells. Dasatinib significantly inhibited phospho-EPH receptor A2 (pEPHA2), pEPHB1, c-KIT, and Src/SFK in NF2-null cells, showing no cross-talk with mTORC1/2 signaling. Posttreatment kinome analyses showed minimal adaptive changes. While dasatinib treatment showed some activity, dual mTORC1/2 inhibitor and its combination with dasatinib elicited stronger growth inhibition in meningiomas. Co-targeting mTORC1/2 and EPH RTK/SFK pathways could be a novel effective treatment strategy for NF2-deficient meningiomas.

The expression of the MSC-marker CD73 and of NF2/Merlin are correlated in meningiomas.

Mesenchymal stem cells (MSC) have been found in various cancers and were discussed to influence tumor biology. Cells fulfilling the complete MSC criteria, including surface marker expression (CD73, CD90, CD105) and tri-lineage differentiation, have been isolated solely from a low percentage of high-grade meningiomas. In contrast, pure co-expression of the surface-markers was relatively frequent, raising the question for an additional role of these membrane proteins in meningiomas. Therefore, here we analyzed the expression of CD73, CD90 and CD105 in a series of meningiomas of all grades. Although no significant association of any marker with meningeal tumor growth per se or with tumor-grade was observed, we detected a positive Pearson correlation (r = 0.55, p ≤ 0.05) in low-grade tumors between CD73 and the most relevant tumor suppressor NF2/Merlin, supported by a tendency of lower CD73 expression in cases with allelic losses at the NF2-locus, which express significantly lower NF2/Merlin-mRNA (p ≤ 0.05). In two pairs of syngenous meningeal or meningioma cell lines with or without shRNA-mediated knockdown of NF2/Merlin a nearly complete loss of CD73 mRNA expression was observed after the knockdown (p ≤ 0.001). This suggested that the correlation observed in tumors may result from a direct functional link between Merlin and CD73. Since CD73 is a 5'-exonucleotidase (termed NT5E), we discuss a potential role of NT5E-mediated purinergic signaling to modulate actin-cytoskeleton and cell contacts, which may be a functional link to NF2/Merlin.

Effect of TAGLN2 in the regulation of meningioma tumorigenesis and development.

To explore the role of transgelin-2 TAGLN2 in the development and progression of meningioma and the potential regulatory. TAGLN2 knockdown expression and overexpression in vitro models were constructed using lentivirus in meningioma cell line CH157; their corresponding transfection efficiencies were verified by qRT-PCR and Western Blot. Actions of TAGLN2 on the proliferation of meningioma cells were explored by CCK8 and colony formation assays. The effect of TAGLN2 on invasion of meningioma cells was analyzed by transwell cell invasion assay. Biological function of TAGLN2 on apoptosis of meningioma cells was determined by flow cytometry. Finally, Western Blot was used to investigate the detailed mechanism of TAGLN2 on regulating the biological functions of meningioma cells. After down-regulating the expression of TAGLN2, there were significantly decreased capacities of cells proliferation and colony formation of meningioma cells, meanwhile, cell invasion was significantly decreased but the apoptosis rate was increased. On the contrary, up-regulation of TAGLN2 expression, the proliferation, colony formation ability were significantly increased as well as the invasion capacity, whilst apoptosis rate was decreased. Western Blot showed that expressions of p-PI3K and p-AKT were inhibited after knockdown of TAGLN2, which were significantly increased after TAGLN2 was overexpressed. TAGLN2 can affect the proliferation, invasion and apoptosis of meningioma cells and may participate in the development of meningioma through regulating the PI3K/AKT signaling pathway.

LB-100, a novel Protein Phosphatase 2A (PP2A) inhibitor, sensitizes malignant meningioma cells to the therapeutic effects of radiation

Atypical and anaplastic meningiomas (AAM) represent 20% of all meningiomas. They are associated with poor outcomes due to their tendency to recur. While surgery and radiation (RT) are first line therapy, no effective systemic medical treatment has been identified. Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase involved in cell cycle regulation and DNA repair. Here, we examined radiosensitizing effects of LB-100, a novel inhibitor of PP2A against AAM as a novel treatment strategy. Three human-derived immortalized meningioma cell lines, IOMM-LEE, GAR, and CH-157, were used to investigate the radio-sensitizing potential of LB-100 in AAM. Survival fraction by clonogenic assay, immunofluorescence, cell cycle analysis and protein expression were evaluated in vitro. The antitumor effects of combining LB-100 with RT were verified in vivo by using intracranial orthotopic xenograft mouse model. Pharmacologic PP2A inhibition with LB-100 prior to RT enhanced the radiosensitivity of meningioma cells and reduced survival fraction in clonogenic assays. LB-100 increased DNA double-strand breakage (measured by γ-H2AX), mitotic catastrophe cell death, and G2/M cell cycle arrest in irradiated meningioma cells. Also, LB-100 decreased activation of STAT3 and expression of its downstream proteins. In vivo, LB-100 and RT combined treatment prolonged the survival of mice with xenografts compared to RT alone. Taken together, these results provide convincing preclinical data to support the use of LB-100 as a radiosensitizing agent for treatment of malignant meningioma. Its potential for clinical application deserves further investigation.

Let-7d suppresses proliferation and invasion and promotes apoptosis of meningioma by targeting AEG-1.

Backgroundlet-7d has been indicated to act as a tumor suppressor in various cancers. However, the function and molecular mechanism of let-7d in meningioma progression have not been elucidated.Materials and methodsQuantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression levels of let-7d and AEG-1 mRNA in meningioma tissues and cell lines. The protein level of AEG-1 was measured by Western blot analysis. MTT assay, Transwell invasion assay and flow cytometry analysis were carried out to determine the proliferation, invasion and apoptosis of IOMM-Lee and CH-157MN cells, respectively. Target gene of let-7d was verified by luciferase reporter analysis.Resultslet-7d expression was downregulated, and AEG-1 expression was upregulated in meningioma tumor tissues. let-7d overexpression suppressed proliferation and invasion and induced apoptosis in IOMM-Lee and CH-157MN cells. Moreover, AEG-1 was a direct target of let-7d. Restoration of AEG-1 expression reversed let-7d-mediated suppression of the proliferation and invasion and let-7d-induced apoptosis in IOMM-Lee and CH-157MN cells.Conclusionlet-7d repressed proliferation and invasion and promoted apoptosis of meningioma cells by targeting AEG-1. The present study provided a better understanding of the meningioma pathogenesis and a promising therapeutic target for meningioma patients.

Osteoglycin promotes meningioma development through downregulation of NF2 and activation of mTOR signaling

BackgroundMeningiomas are the most common primary intracranial tumors in adults. While a majority of meningiomas are slow growing neoplasms that may cured by surgical resection, a subset demonstrates more aggressive behavior and insidiously recurs despite surgery and radiation, without effective alternative treatment options. Elucidation of critical mitogenic pathways in meningioma oncogenesis may offer new therapeutic strategies. We performed an integrated genomic and molecular analysis to characterize the expression and function of osteoglycin (OGN) in meningiomas and explored possible therapeutic approaches for OGN-expressing meningiomas.MethodsOGN mRNA expression in human meningiomas was assessed by RNA microarray and RNAscope. The impact of OGN on cell proliferation, colony formation, and mitogenic signaling cascades was assessed in a human meningioma cell line (IOMM-Lee) with stable overexpression of OGN. Furthermore, the functional consequences of introducing an AKT inhibitor in OGN-overexpressing meningioma cells were assessed.ResultsOGN mRNA expression was dramatically increased in meningiomas compared to a spectrum of other brain tumors and normal brain. OGN-overexpressing meningioma cells demonstrated an elevated rate of cell proliferation, cell cycle activation, and colony formation as compared with cells transfected with control vector. In addition, NF2 mRNA and protein expression were both attenuated in OGN-overexpressing cells. Conversely, mTOR pathway and AKT activation increased in OGN-overexpressing cells compared to control cells. Lastly, introduction of an AKT inhibitor reduced OGN expression in meningioma cells and resulted in increased cell death and autophagy, suggestive of a reciprocal relationship between OGN and AKT.ConclusionWe identify OGN as a novel oncogene in meningioma proliferation. AKT inhibition reduces OGN protein levels in meningioma cells, with a concomitant increase in cell death, which provides a promising treatment option for meningiomas with OGN overexpression.

370 Hypoxic Conditions and Hypoxia-Inducible Factor 1-Alpha are Critical in the Radioresistance of Meningioma

Abstract INTRODUCTION Meningiomas are common benign intracranial neoplasms treated with a combination of surgical resection, radiotherapy, and chemotherapy. Hypoxia-inducible factor 1 alpha (HIF1A) can predict disease aggressiveness, response to oxygen tension and regulate meningioma pathogenesis. The role of HIF1A and hypoxia was explored in the radiation resistance of meningioma, hypothesizing that HIF1A confers resistance to radiotherapy in meningiomas and that oxygen tension is key to radiation responsiveness. METHODS Various primary meningioma cell lines were studied (IOMM-LE, JEN, GAR, SAM). GAR primary cell lines were utilized in addition to a shRNA-HIF1A knockdown (GAR1589). Cell proliferation, viability, and apoptosis were evaluated in response to radiation in both normoxia (21% O2) and hypoxia (1% O2). RESULTS >Doubling times of the various meningioma cell lines ranged from 19.5 to 213.2 hours. Cell density impacted responsiveness to radiation, with confluent cells showing greater dose tolerance before reduced proliferation. In normoxic environments, HIF1A knockout resulted in increased susceptibility to single radiation fractions of 2 Gy (P = 0.05) and 10 Gy (P = 0.02); however, both cells had strong susceptibility at 20 Gy. Hypoxic environments reduced the viability differences between GAR and GAR1589 cells and slightly increased GAR cell viability at 2 Gy (P < 0.05). Fractionated radiotherapy (2 Gy) daily showed reduced GAR and GAR1589 cell viability in normoxia and hypoxia. Cell viability for GAR1589 at 3 fractions of 2-Gy radiation was reduced, but this effect was eliminated in hypoxic environments. Apoptosis was increased in GAR1589 cells at baseline and with increased fractions of radiation; however, this effect was reduced in hypoxic conditions. CONCLUSION These results support important findings for meningioma response to radiation, including 1) importance of cell density, 2) dependence on HIF1A mutation, and 3) a critical role for oxygen tension. These results may be helpful in understanding the mechanisms of meningioma resistance to radiation and serve as a model to trial novel adjuvant therapies and treatment types.

Pleomorphism and drug resistant cancer stem cells are characteristic of aggressive primary meningioma cell lines

BackgroundMeningioma tumors arise in arachnoid membranes, and are the most reported central nervous system (CNS) tumors worldwide. Up to 20% of grade I meningioma tumors reoccur and currently predictive cancer stem cells (CSCs) markers for aggressive and drug resistant meningiomas are scarce.MethodsMeningioma tissues and primary cell lines were investigated using whole transcriptome microarray analysis, immunofluorescence staining of CSCs markers (including CD133, Sox2, Nestin, and Frizzled 9), and drug treatment with cisplatin or etoposide.ResultsUnsupervised hierarchical clustering of six meningioma samples separated tissues into two groups. Analysis identified stem cells related pathways to be differential between the two groups and indicated the de-regulation of the stem cell associated genes Reelin (RELN), Calbindin 1 (CALB1) and Anterior Gradient 2 Homolog (AGR2). Immunofluorescence staining for four tissues confirmed stemness variation in situ. Biological characterization of fifteen meningioma primary cell lines concordantly separated cells into two functionally distinct sub-groups. Pleomorphic cell lines (NG type) grew significantly faster than monomorphic cell lines (G type), had a higher number of cells that express Ki67, and were able to migrate aggressively in vitro. In addition, NG type cell lines had a lower expression of nuclear Caspase-3, and had a significantly higher number of CSCs co-positive for CD133+ Sox2+ or AGR2+ BMI1+. Importantly, these cells were more tolerant to cisplatin and etoposide treatment, showed a lower level of nuclear Caspase-3 in treated cells and harbored drug resistant CSCs.ConclusionCollectively, analyses of tissues and primary cell lines revealed stem cell associated genes as potential targets for aggressive and drug resistant meningiomas.

Cytoplasmic overexpression of RNA-binding protein HuR is a marker of poor prognosis in meningioma, and HuR knockdown decreases meningioma cell growth and resistance to hypoxia.

HuR regulates cytoplasmic mRNA stability and translatability, and the HuR expression level has been shown to correlate with poor disease outcome in several cancer types; however, the prognostic value and potential pro-oncogenic properties of HuR in meningioma remain unclear. Thus, in the present study, we analysed 85 meningioma tissue samples to establish the relationship between HuR expression, tumour cell proliferation, and/or patient survival. In addition, we examined the anti-proliferative effects of HuR knockdown in two meningioma cell lines (IOMM-Lee and Ben-Men-1) and conducted transcriptome-wide analyses (IOMM-Lee cells) to elucidate the molecular consequences of HuR knockdown. The results of the present study showed HuR cytoplasmic expression to correlate positively with tumour grade (p = 1.2 × 10-8 ) and negatively with progression-free and overall survival (p = 0.01) time in human meningioma tissues. In vitro, siHuR-induced HuR knockdown was shown to reduce the growth of both Ben-Men-1 (p = 2 × 10-8 ) and IOMM-Lee (p = 4 × 10-9 ) cells. Transcriptome analyses revealed HuR knockdown in IOMM-Lee cells to deregulate the HIF1A signalling pathway (p = 1.5 × 10-6 ) and to up-regulate the expression of genes essential for the assembly of the cytoplasmic mRNA processing body, global genome nucleotide-excision repair, poly(A)-specific ribonuclease activity, the positive regulation of apoptosis and of cell cycle arrest, and the negative regulation of RNA splicing [p(FDR) < 0.001]. Interestingly, HuR knockdown under hypoxic culture conditions further potentiated the effects of HuR knockdown on cell growth, apoptosis, and HIF1A expression. We thus conclude that cytoplasmic HuR expression is a marker of poor prognosis in meningioma and that HuR is a promising potential therapeutic target for use in tumours refractory to standard therapies. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.