Human Meningiomas Research Articles

The role of MAPK signaling pathway in the Her-2-positive meningiomas

Meningiomas are common types of adult nerve system tumors. Although most cases are considered benign, due to its high rate of recurrence and easy malignant progression to anaplastic meningioma they present a puzzle for the current treatment. The HER-2 oncogene has important value for meningioma cells development and progression. So far, little is known about the effect on the exact underlying signal pathway and molecular mechanisms of HER-2-positive meningioma cells. The goal of the present study was to determine the effects of HER-2 gene and possible involvement of MAPK signal pathway in human malignant meningioma. We applied q-PCR analysis, immunofluorescence (IF) staining, western blot analysis, animal model, MAPK inhibition, MTT assay and cell invasion analysis for the investigation. The results demonstrated that the downregulation of the expression of HER-2 significantly inhibited cell motility and proliferation of human meningioma cells in vivo. Accordingly, in the HER-2-overexpression meningioma cells with the inhibition of ERK1/2, ERK5, JNK, in the cells with the ERK1/2, ERK5 inhibition, protein expression was markedly suppressed as well as the cell proliferation resistance. No difference was observed in the HER-2-overexpression meningioma cells with the inhibition of JNK. These findings suggest that HER-2 gene can affect the proliferation ability of human meningioma cells in vivo and MAPK signal pathway may contribute to the carcinogenesis and development of human meningiomas combinating with HER-2.

RAS Promotes Proliferation and Resistances to Apoptosis in Meningioma.

In this study, we investigated the influence of elevated RAS expression on the growth of meningioma in vivo and in vitro. The IOMM-LEE cells, representing a cell line derived from malignant meningioma, were divided into blank control group (cells without any drug treatment), negative control group (cells treated with an equal volume of normal saline to replace drug), and farnesyl thiosalicylic acid (FTS)-treated group (cells treated with FTS). Methyl-thiazole-tetrazolium bromide (MTT) assay and flow cytometer (with cells after FTS (75μmol/L) treatment for 48h) were utilized to determine the proliferation and apoptosis, respectively, of IOMM-LEE cells after RAS inhibition. Western blot analysis was used for semi-quantitative analysis of p-ERK and p-AKT levels. Animal model of human meningioma was established with sub-renal capsule transplantation, and mice were divided into two groups: experimental group (50mg/kg group, 75mg/kg group, and 100 mg/kg, hypodermic injection with FTS) and control group. Proliferating cell nuclear antigen (PCNA) was detected by immunohistochemistry (IHC). Western blot analysis was used for detecting ERK and AKT signal pathway. The proliferation of IOMM-LEE cells decreased dramatically and apoptosis rate increased significantly in FTS-treated group compared to blank control group and negative control group (all P < 0.05). At FTS concentration of 75μmol/L, the apoptosis rate of IOMM-LEE cells reduced significantly over time (P < 0.05). Cell cycle analysis showed that IOMM-LEE cells exhibited G1-arrest in the FTS-treated group, compared to no cell-cycle arrest in blank control group and the negative control group (P < 0.05). Further, significantly decreased ERK and AKT phosphorylation levels were detected in IOMM-Lee cells after FTS (75μmol/L) treatment for 48h, compared to blank control group and negative control group (P < 0.05). The results in vivo experiments showed that after FTS treatment, tumor volume, PCNA LI, and the levels of p-ERK and p-Akt decreased significantly in 75mg/kg group and 100mg/kg group when compared with the control group and 50mg/kg group (all P < 0.05). Our findings provide strong evidence that RAS protein is highly expressed in meningioma cells, and the RAS activity is inhibited by downregulating ERK and AKT signal pathway, which may further inhibit the growth of meningioma.

Differentially Expressed MicroRNAs in Meningiomas Grades I and II Suggest Shared Biomarkers with Malignant Tumors.

Meningiomas represent the most common primary tumors of the central nervous system, but few microRNA (miRNA) profiling studies have been reported so far. Deep sequencing of small RNA libraries generated from two human meningioma biopsies WHO grades I (benign) and II (atypical) were compared to excess dura controls. Nineteen differentially expressed miRNAs were validated by RT-qPCR using tumor RNA from 15 patients and 5 meninges controls. Tumor suppressor miR-218 and miR-34a were upregulated relative to normal controls, however, miR-143, miR-193b, miR-451 and oncogenic miR-21 were all downregulated. From 10 selected putative mRNA targets tested by RT-qPCR only four were differentially expressed relative to normal controls. PTEN and E-cadherin (CDH1) were upregulated, but RUNX1T1 was downregulated. Proliferation biomarker p63 was upregulated with nuclear localization, but not detected in most normal arachnoid tissues. Immunoreactivity of E-cadherin was detected in the outermost layer of normal arachnoids, but was expressed throughout the tumors. Nuclear Cyclin D1 expression was positive in all studied meningiomas, while its expression in arachnoid was limited to a few trabecular cells. Meningiomas of grades I and II appear to share biomarkers with malignant tumors, but with some additional tumor suppressor biomarkers expression. Validation in more patients is of importance.

Correlation of leptin receptor expression with BMI in differential grades of human meningiomas.

Meningioma is one of the most common primary brain tumor, especially in postmenopausal women. The most important risk factors include radiation, primary head injury or genetic alterations, however it is currently unclear why postmenopausal women are predominantly affected. The aim of the present study was to evaluate leptin receptor (LEPR) expression and body mass index (BMI) in patients with meningiomas of differential grades. Specimens of 158 meningiomas were classified as either G1 (low-grade meningiomas, n=114) or G2/G3 (high-grade meningiomas, n=44). Immunohistochemistry was performed to assess LEPR expression. The mean BMIs of the female and male patient groups were 28.43±5.29 and 23.93±4.66, respectively. Mean BMI was significantly higher in the female group, by ~4.50 kg/m2. Patient age significantly correlated with LEPR expression, with the highly positive (++) and positive (+) groups having mean ages of 62.3±12.07 and 52.3±13.04, respectively. A strong positive correlation (r=0.73) was observed between leptin receptor expression and BMI, with the LEPR (++) group having a mean BMI of 30.11±4.49, compared to 22.12±2.48 for the LEPR (+) group. Furthermore, in the low-grade meningioma group, mean BMI was higher in female patients than male patients (28.13±5.54 and 25.38±4.57, respectively; P=0.01). Additionally, there was strong positive correlation between BMI and leptin receptor expression in the low-grade meningioma group (r=0.69). For the high-grade meningioma group, mean BMI was 29.49±4.26 and 21.76±3.98 in female and male patients, respectively, and LEPR expression strongly correlated with BMI in this group (r=0.80). The present study demonstrates a correlation between patient BMI, age, and LEPR expression status in low- and high-grade meningiomas. Our results indicate that in addition to endogenous hormones, such as estrogen or progesterone, or fatty tissue-associated proinflammatory cytokines, LEPR expression status may be a risk factor for meningioma growth and progression.

Multimodal optical analysis of meningioma and comparison with histopathology.

Meningioma is the most frequent primary central nervous system tumor. The risk of recurrence and the prognosis are correlated with the extent of the resection that ideally encompasses the infiltrated dura mater and, if required, the infiltrated bone. No device can deliver real-time intraoperative histopathological information on the tumor environment to help the neurosurgeon to achieve a gross total removal. This study assessed the abilities of nonlinear microscopy to provide relevant and real-time data to help resection of meningiomas. Nine human meningioma samples (four World Health Organization Grade I, five Grade II) were analyzed using different optical modalities: spectral analysis and imaging, lifetime measurements, fluorescence lifetime imaging microscopy, fluorescence emitted under one- and two-photon excitation and the second-harmonic generation signal imaging using a multimodal setup. Nonlinear microscopy produced images close to histopathology as a gold standard. The second-harmonic generation signal delineated the collagen background and two-photon fluorescence underlined cell cytoplasm. The matching between fluorescence images and Hematoxylin and Eosin staining was possible in all cases. Grade I meningioma emitted less autofluorescence than Grade II meningioma and Grade II meningioma exhibited a distinct lifetime value. Autofluorescence was correlated with the proliferation rates and seemed to explain the observed differences between Grade I and II meningiomas. This preliminary multimodal study focused on human meningioma samples confirms the potential of tissue autofluorescence analysis and nonlinear microscopy in helping intraoperatively neurosurgeons to reach the actual boundaries of the tumor infiltration. Correspondence between H&E staining (top pictures) and the two-photon fluorescence imaging (bottom pictures).

Aryl hydrocarbon receptor (AHR) signalling pathway is activated and involved in tumor progression of human meningioma

Aryl hydrocarbon receptor (AHR) signalling pathway is activated and involved in tumor progression of human meningioma

MNGO-08MENINGIOMA GROWTH INHIBITION AND RADIOSENSITIZATION BY THE SMALL MOLECULE YAP INHIBITOR VERTEPORFIN

Meningiomas frequently harbor inactivating mutations in the tumor suppressor protein Merlin (NF2), an upstream activator of the Hippo signaling pathway. This aberration drives tumor growth and aggressiveness through inactivation of this pathway and unchecked activation of its downstream effector, Yes-associated protein (YAP). The transcriptional coactivator YAP has been previously implicated in driving meningioma growth and malignant transformation of non-neoplastic arachnoid cells. YAP exerts its effect by direct binding to the TEAD family of transcription factors and transactivation of growth-related genes. However, the efficacy of pharmacologic modulation of this pathway in meningioma has yet to be explored. Verteporfin is a small molecule inhibitor of the porphyrin family, recently implicated as a direct inhibitor of YAP. Here, we report that Verteporfin modulates growth and radiation resistance in NF2 driven meningiomas through inhibition of the YAP-TEAD interaction. In particular, our data demonstrates that Verteporfin treatment in the NF2 mutant KT21MG1 human malignant meningioma cell line, results in a decreased activity of oncogenic YAP, resulting in decreased mRNA expression of YAP downstream targets (CTGF, Mcl-1 and ANKRD1) (p ≤ 0.05). Moreover, Verteporfin inhibits meningioma cell growth and proliferation in a dose dependent manner (MTT reduction and viable cell number) (p ≤ 0.05). Interestingly, pretreatment with Verteporfin results in increased sensitivity to irradiation in vitro (p ≤ 0.05). In addition, we explore the in vivo efficacy of Verteporfin treatment in a murine skull base xenograft model of human meningioma. Taken together, our results show that Verteporfin is a potent inhibitor of meningioma cell growth and radioresistance, making it an attractive drug candidate for treatment of this disease. Hence, this targeted therapy towards the NF2-YAP axis holds promising alternatives to current standard of care.

PDGF activation in PGDS-positive arachnoid cells induces meningioma formation in mice promoting tumor progression in combination with Nf2 and Cdkn2ab loss.

The role of PDGF-B and its receptor in meningeal tumorigenesis is not clear. We investigated the role of PDGF-B in mouse meningioma development by generating autocrine stimulation of the arachnoid through the platelet-derived growth factor receptor (PDGFR) using the RCAStv-a system. To specifically target arachnoid cells, the cells of origin of meningioma, we generated the PGDStv-a mouse (Prostaglandin D synthase). Forced expression of PDGF-B in arachnoid cells in vivo induced the formation of Grade I meningiomas in 27% of mice by 8 months of age. In vitro, PDGF-B overexpression in PGDS-positive arachnoid cells lead to increased proliferation.We found a correlation of PDGFR-B expression and NF2 inactivation in a cohort of human meningiomas, and we showed that, in mice, Nf2 loss and PDGF over-expression in arachnoid cells induced meningioma malignant transformation, with 40% of Grade II meningiomas. In these mice, additional loss of Cdkn2ab resulted in a higher incidence of malignant meningiomas with 60% of Grade II and 30% of Grade III meningiomas. These data suggest that chronic autocrine PDGF signaling can promote proliferation of arachnoid cells and is potentially sufficient to induce meningiomagenesis. Loss of Nf2 and Cdkn2ab have synergistic effects with PDGF-B overexpression promoting meningioma malignant transformation.

Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1

BackgroundThere is a paucity of effective therapies for recurrent/aggressive meningiomas. Establishment of improved in vitro and in vivo meningioma models will facilitate development and testing of novel therapeutic approaches.MethodsA primary meningioma cell line was generated from a patient with an olfactory groove meningioma. The cell line was extensively characterized by performing analysis of growth kinetics, immunocytochemistry, telomerase activity, karyotype, and comparative genomic hybridization. Xenograft models using immunocompromised SCID mice were also developed.ResultsHistopathology of the patient tumor was consistent with a WHO grade I typical meningioma composed of meningothelial cells, whorls, and occasional psammoma bodies. The original tumor and the early passage primary cells shared the standard immunohistochemical profile consistent with low-grade, good prognosis meningioma. Low passage KCI-MENG1 cells were composed of two cell types with spindle and round morphologies, showed linear growth curve, had very low telomerase activity, and were composed of two distinct unrelated clones on cytogenetic analysis. In contrast, high passage cells were homogeneously round, rapidly growing, had high telomerase activity, and were composed of a single clone with a near triploid karyotype containing 64–66 chromosomes with numerous aberrations. Following subcutaneous and orthotopic transplantation of low passage cells into SCID mice, firm tumors positive for vimentin and progesterone receptor (PR) formed, while subcutaneous implant of high passage cells yielded vimentin-positive, PR-negative tumors, concordant with a high-grade meningioma.ConclusionsAlthough derived from a benign meningioma specimen, the newly-established spontaneously immortal KCI-MENG1 meningioma cell line can be utilized to generate xenograft tumor models with either low- or high-grade features, dependent on the cell passage number (likely due to the relative abundance of the round, near-triploid cells). These human meningioma mouse xenograft models will provide biologically relevant platforms from which to investigate differences in low- vs. high-grade meningioma tumor biology and disease progression as well as to develop novel therapies to improve treatment options for poor prognosis or recurrent meningiomas.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0596-8) contains supplementary material, which is available to authorized users.

Canine intracranial meningiomas: Immunohistochemical evaluation of tissue factor, fibrin/fibrinogen and D-dimers

The haemostatic system influences angiogenesis, cell growth and metastasis in solid tumours. The aim of this study was to investigate tissue factor (TF) expression, fibrin/fibrinogen and D-dimer deposition, as well as the occurrence of intravascular thrombosis (IVT) in canine intracranial meningiomas using immunohistochemistry. All but three (26/29) meningiomas expressed TF. TF immunolabelling was significantly higher in high-grade (grades II and III) than in low-grade (grade I) meningiomas. Fibrin/fibrinogen and D-dimer deposits were detected in all meningiomas and staining scores were statistically different between different meningioma grades. IVT was detected in 19/29 specimens, but no statistical differences were observed between different malignancy grades. In conclusion, the haemostatic system may be involved in meningioma pathobiology and may be a potential therapeutic target for canine meningiomas, as also suggested for human meningiomas.

Activation of Yes-Associated Protein in Low-Grade Meningiomas Is Regulated by Merlin, Cell Density, and Extracellular Matrix Stiffness.

The NF2 gene product Merlin is a protein containing ezrin, radixin, and moesin domains; it is a member of the 4.1 protein superfamily associated with the membrane cytoskeleton and also interacts with cell surface molecules. The mammalian Hippo cascade, a downstream signaling cascade of merlin, inactivates the Yes-associated protein (YAP). Yes-associated protein is activated by loss of the NF2 gene and functions as an oncogene in meningioma cells; however, the factors controlling YAP expression, phosphorylation, and subcellular localization in meningiomas have not been fully elucidated. Here, we demonstrate that merlin expression is heterogeneous in 1 NF2 gene-negative and 3 NF2 gene-positive World Health Organization grade I meningiomas. In the NF2 gene-positive meningiomas, regions with low levels of merlin (tumor rims) had greater numbers of cells with nuclear YAP versus regions with high merlin levels (tumor cores). Merlin expression and YAP phosphorylation were also affected by cell density in the IOMM-Lee and HKBMM human meningioma cell lines; nuclear localization of YAP was regulated by cell density and extracellular matrix (ECM) stiffness in IOMM-Lee cells. These results suggest that cell density and ECM stiffness may contribute to the heterogeneous loss of merlin and increased nuclear YAP expression in human meningiomas.

Minimal interleukin expression in canine intracranial meningiomas

MENINGIOMAS are a common CNS neoplasm in both humans and dogs. The propagation of meningiomas has been linked with several inflammatory interleukins; human meningioma cells have been identified to synthesise...

Molecular imaging correlates of tryptophan metabolism via the kynurenine pathway in human meningiomas.

Increased tryptophan metabolism via the kynurenine pathway (KP) is a key mechanism of tumoral immune suppression in gliomas. However, details of tryptophan metabolism in meningiomas have not been elucidated. In this study, we evaluated in vivo tryptophan metabolism in meningiomas and compared it with gliomas using α-[(11)C]-methyl-L-tryptophan (AMT)-PET. We also explored expression patterns of KP enzymes in resected meningiomas. Forty-seven patients with MRI-detected meningioma (n = 16) and glioma (n = 31) underwent presurgical AMT-PET scanning. Tumoral AMT uptake and tracer kinetic parameters (including K and k3' evaluating unidirectional uptake and trapping, respectively) were measured, correlated with meningioma grade, and compared between meningiomas and gliomas. Patterns of KP enzyme expression were assessed by immunohistochemistry in all meningiomas. Meningioma grade showed a positive correlation with AMT k3' tumor/cortex ratio (r = 0.75, P = .003), and this PET parameter distinguished grade I from grade II/III meningiomas with 92% accuracy. Kinetic AMT parameters could differentiate meningiomas from both low-grade gliomas (97% accuracy by k3' ratios) and high-grade gliomas (83% accuracy by K ratios). Among 3 initial KP enzymes (indoleamine 2,3-dioxygenase 1/2, and tryptophan 2,3-dioxygenase 2 [TDO2]), TDO2 showed the strongest immunostaining, particularly in grade I meningiomas. TDO2 also showed a strong negative correlation with AMT k3' ratios (P = .001). PET imaging of tryptophan metabolism can provide quantitative imaging markers for differentiating grade I from grade II/III meningiomas. TDO2 may be an important driver of in vivo tryptophan metabolism in these tumors. These results can have implications for pharmacological targeting of the KP in meningiomas.

Effects of human epidermalgrowth factor receptor-2/neu small interfering RNA-mediated gene silencing on cell proliferative activity in human meningiomas

Objective To investigate the effects of human epidermal growth factor receptor-2 (Her-2)/neu gene on cell proliferative activity in human meningiomas. Methods The surgicall resected fresh samples from 5 cases of human meningiomas with Her-2 overexpression were selected,and cells were primarily cultured.The expression of Her-2 mRNA and protein was detected by reverse transcriptase -polymerase chain reaction(RT-PCR)and Western blotting respectively.Methyl thiazol tetrazolium (MTT)assay was employed to examined the cell viability.The cell cycle distribution was measured by flow cytometry. Results At 72nd h after transfection, as compared with the silencing group and the negative control group, the Her-2 m RNA and protein expression was reduced by 56. 3% and 52. 7% respectively(P< 0. 05). At 72nd h after transfection, the proliferation inhibition rate was 50. 08%.The proportion of cells in G0/G1 phase was increased by 18. 11%, and that in S phase decreased by 39. 34%(P< 0. 05). Conclusion Her-2 small interfering RNA(siRNA)-mediated gene may inhibit the proliferative ablity of human meningiomas. Key words: Meningioma; Human epidermalgrowth factor receptor-2; Small interfering RNA; Proliferation

Combined treatment by octreotide and everolimus: Octreotide enhances inhibitory effect of everolimus in aggressive meningiomas.

Treatment for recurrent and aggressive meningiomas remains an unmet medical need in neuro-oncology, and chemotherapy exhibits limited clinical activity, if any. Merlin expression, encoded by the NF2 gene, is lost in a majority of meningiomas, and merlin is a negative regulator of mTORC1. The sst2 somatostatin receptor, targeted by octreotide, is highly expressed in meningiomas. To investigate new therapeutic strategies, we evaluated the activity of everolimus (mTOR inhibitor), BKM-120 and BEZ-235 (new Pi3K/Akt/mTOR inhibitors), octreotide and a combined treatment (octreotide plus everolimus), on cell proliferation, signaling pathways, and cell cycle proteins, respectively. The in vitro study was conducted on human meningioma primary cells extracted from fresh tumors, allowing the assessment of somatostatin analogs at the concentration levels used in patients. The results were correlated to WHO grades. Further, everolimus decreased cell viability of human meningiomas, but concomitantly, induced Akt activation, reducing the antiproliferative effect of the drug. The new Pi3K inhibitors were not more active than everolimus alone, limiting their clinical relevance. In contrast, a clear cooperative inhibitory effect of octreotide and everolimus was observed on cell proliferation in all tested meningiomas, including WHO grades II-III. Octreotide not only reversed everolimus-induced Akt phosphorylation but also displayed additive and complementary effects with everolimus on downstream proteins involved in translation (4EB-P1), and controlling cell cycle (p27Kip1 and cyclin D1). We have demonstrated a co-operative action between everolimus and octreotide on cell proliferation in human meningiomas, including aggressive ones, establishing the basis for a clinical trial.

Role of HER-2 activity in the regulation of malignant meningioma cell proliferation and motility.

Meningiomas are common types of intracranial tumor. Invasive and malignant meningiomas present a significant therapeutic challenge due to high rates of recurrence and invasion. Understanding the molecular mechanism of invasion may assist in designing novel therapeutic approaches and improving patient survival rates. The HER‑2 gene has been demonstrated to be a useful predictor of tumor aggression, which promotes the survival and growth of cancer cells through the mitogen‑activated protein kinase and/or phosphatidylinositol 3‑kinase (PI3K)/AKT pathway. Until now, few studies have investigated the associateion between meningiomas and the expression of HER‑2, and the significance of HER‑2 in meningiomas remains to be elucidated. The present study aimed to investigate the effects of the HER‑2 gene on the biological behaviors of human malignant meningioma cells. The results demonstrated that downregulation of the expression of HER‑2 by small interfering RNA in human meningioma cells significantly inhibited cell motility and proliferation, led to cell cycle arrest at the G0/G1‑phase and increased early apoptosis. By contrast, the overexpression of HER‑2 group resulted in meningioma cell invasion, migration and proliferation being significantly enhanced, cell cycle was promoted at the G1/S‑phase and early apoptosis was decreased. Accordingly, the inhibition of HER‑2 also prevented the protein expression of PI3K and phosphorylated AKT. The results demonstrated that regulation of the HER‑2 gene can affect the proliferation, apoptosis, invasion and metastasis abilities of human meningioma cells in vitro. Furthermore, PI3K/AKT may contribute to the carcinogenesis and development of human meningiomas in combination with HER-2.

Overexpression of RLIP76 Required for Proliferation in Meningioma Is Associated with Recurrence.

The GTPase-activating protein RLIP76 is overexpressed in and correlates with the pathological grade of many malignant tumor cells. But the potential correlation between RLIP76 and clinical outcomes in patients with meningioma remains unknown. In this study, we examined the expression of RLIP76 in meningioma and correlated the RLIP76 expression to the patient outcome. RLIP76 expression in tumor tissues was examined with immunohistochemistry, quantitative reverse-transcription polymerase chain reaction(RT-PCR) and Western-blot. Immunohistochemistry showed an increased RLIP76 immunostaining score in anaplastic and atypical meningiomas versus classical meningiomas. Statistical analyses revealed that RLIP76 immunostaining positively correlated with immunostaining for Ki-67, a nuclear protein highly expressed in proliferating cells(r=0.29, p=0.034 by Spearman's correlation coefficient). Clinicopathological evaluation suggested that RLIP76 expression be associated with tumor grade and recurrence(P<0.05). Univariate and Cox analysis indicated that RLIP76 was an independent prognostic factor for tumor recurrence. Furthermore, the human malignant meningioma cell lines IOMM-Lee and CH157-MN stably transfected with short hairpin RNA (siRNA) targeting RLIP76 were then examined by in vitro growth assays, and apoptosis assays. RLIP76 knockdown in IOMM-Lee and CH157-MN cells inhibited cell proliferation and induced apoptosis. Western blot analysis revealed that cells underexpressing RLIP76 exhibited decreased B-cell lymphoma-2(Bcl-2) expression but increased apoptosis effector caspase-3 expression. These findings demonstrate that high RLIP76 expression is associated with a poor outcome of meningioma and may provide a new gene therapy approach for patients with malignant meningiomas.

Aminolevulinic acid-mediated photodynamic therapy of human meningioma: an in vitro study on primary cell lines.

Objective: Five-aminolevulinic acid (5-ALA)-induced porphyrins in malignant gliomas are potent photosensitizers. Promising results of ALA-PDT (photodynamic therapy) in recurrent glioblastomas have been published. Recently, 5-ALA-induced fluorescence was studied in meningioma surgery. Here, we present an experimental study of ALA-PDT in an in vitro model of primary meningioma cell lines. Methods: We processed native tumor material obtained intra-operatively within 24 h for cell culture. Epithelial membrane antigen (EMA) immunohistochemistry was performed after the first passage to confirm that cells were meningioma cells. For 5-ALA-PDT treatment, about 5000 cells per well were seeded in 20 wells of a blank 96-well plate. Each block of 4 wells was inoculated with 150 µL of 0, 25, 50 and 100 µg/mL 5-ALA solutions; one block was used as negative control without 5-ALA and without PDT. Following incubation for 3 h PDT was performed using a laser (635 nm, 18.75 J/cm2). The therapeutic response was analyzed by the water soluble tetrazolium salt (WST-1) cell viability assay 90 min after PDT. Results: 5-ALA-PDT was performed in 14 primary meningioma cell lines. EMA expression was verified in 10 primary cell cultures. The remaining 4 were EMA negative and PDT was without any effect in these cultures. All 10 EMA-positive cell lines showed a significant and dose-dependent decrease in viability rate (p < 0.001). Cell survival at 5-ALA concentrations of 12.5, 25, 50 and 100 μg/mL was 96.5% ± 7.6%, 67.9% ± 29.9%, 24.0% ± 16.7% and 13.8% ± 7.5%, respectively. For the negative controls (no 5-ALA/PDT and ALA/no PDT), the viability rates were 101.72% ± 3.5% and 100.17% ± 3.6%, respectively. The LD50 for 5-ALA was estimated between 25 and 50 µg/mL. Conclusion: This study reveals dose-dependent cytotoxic effects of 5-ALA-PDT on primary cell lines of meningiomas. Either 5-ALA or PDT alone did not affect cell survival. Further efforts are necessary to study the potential therapeutic effects of 5-ALA-PDT in vivo.

TR-13 * EFFECTS OF EVEROLIMUS ON MENINGIOMA GROWTH IN PATIENTS WITH NEUROFIBROMATOSIS TYPE 2

BACKGROUND: Vestibular schwannomas (VS) and meningiomas are associated with biallelic loss of NF2, and may arise sporadically or in the context of the tumor predisposition syndrome neurofibromatosis type 2. Loss of NF2 activates the mammalian target of rapamycin (mTOR) pathway, and published in vitro and in vivo data indicate that mTOR inhibition may be effective in the treatment of NF2-deficient VS and meningiomas. Everolimus is an oral inhibitor of mTOR complex 1 (mTORC1) with anti-tumor activity in a variety of tumors. METHODS: We conducted a retrospective review of patients with NF2 and progressive vestibular schwannomas treated with everolimus on our prospective phase II clinical trial (ClinicalTrials.gov NCT01419639). Everolimus was administered at a daily dose of 10 mg in continuous 28-day courses for up to 12 courses. In this retrospective study, we included patients with at least one volumetrically measurable meningioma (>0.5 cc) who received at least six 28-day courses of therapy. Tumor response was assessed with brain MRI every three months using three-dimensional volumetric analysis. RESULTS: Three patients met criteria and had 10 evaluable meningiomas, with a total combined volume of 11.84 cc at baseline (median 1.20 cc, range 0.53–3.00 cc). Median time on therapy was 9 months (range 6–12 months). Total combined meningioma volume remained stable during treatment (-1.8%). Correspondingly, none of the individual tumors analyzed met criteria for volumetric response or progression during the treatment period (range -10% to +9.4%). CONCLUSIONS: Neither objective response nor progression was appreciated in meningiomas patients during a median treatment duration of 6 months. Further clinical studies will be required to determine whether everolimus affects meningioma growth velocity in patients with NF2. To better understand pharmacologic mTOR signaling pathway modulation in human VS and meningiomas in vivo, we are currently conducting a multi-center pharmacodynamic/pharmacokinetic (“phase 0”) study with everolimus (NCT01880749).

The transducible TAT-RIZ1-PR protein exerts histone methyltransferase activity and tumor-suppressive functions in human malignant meningiomas

Malignant meningiomas are a rare meningioma subtype and tend to have post-surgical recurrence. Significant endeavors have been taken to identify functional therapeutic targets to halt the growth of this aggressive cancer. We have recently discovered that RIZ1 is downregulated in high-grade meningiomas, and RIZ1 overexpression inhibits proliferation while promoting cell apoptosis of the IOMM-Lee malignant meningioma cell line. In this report, we show that the N-terminal PR domain of RIZ1 alone possessed growth-inhibitory activity and anticancer activity in primary human meningioma cells. Interestingly, the effects seem to be dependent on differential RIZ1 protein levels. Transducible TAT-RIZ1-PR protein could also inhibit meningioma tumor growth in nude mice models. We further demonstrate that PR protein exerts histone methyltransferase activity. A microarray analysis of TAT-RIZ1-PR-treated human malignant meningioma cells reveals 969 differentially expressed genes and 848 alternative splicing exons. Moreover, c-Myc and TXNIP, two putative downstream targets of H3K9 methylation, may be involved in regulating RIZ1 tumor-suppressive effects. The reciprocal relationship between RIZ1 and c-Myc was then validated in primary meningioma cells and human tumor samples. These findings provide insights into RIZ1 tumor suppression mechanisms and suggest that TAT-RIZ1-PR protein is a potential new epigenetic therapeutic agent for advanced meningiomas.