Meningioma Cultures Research Articles

Stem-like signatures in human meningioma cells are under the control of CXCL11/CXCL12 chemokine activity.

Meningiomas are mainly benign brain tumors, although about 20% of histologically benign cases are clinically aggressive and recur after resection. We hypothesize that meningioma brain invasiveness and recurrence may be related to the presence of cancer stem cells and their high responsiveness to the CXCL12-CXCR4/CXCR7 chemokine axis. The aim of this study was to isolate meningioma stem cells from human samples, characterize them for biological features related to malignant behavior, and to identify the role of CXCR4/CXCR7 in these processes. Meningioma stem cells were isolated from patient-derived primary cultures in stem cell-permissive conditions, and characterized for phenotype, self-renewal, proliferation and migration rates, vasculogenic mimicry (VM), and in vivo tumorigenesis, in comparison with differentiated meningioma cells and stem-like cells isolated from normal meninges. These cell populations were challenged with CXCL12 and CXCL11 and receptor antagonists to define the chemokine role in stem cell-related functions. Stem-like cells isolated from meningioma cultures display higher proliferation and migration rates, and VM, as compared to meningioma non-stem cells or cells isolated from normal meninges and were the only tumorigenic population in vivo. In meningioma cells, these stem-like functions were under the control of the CXCR4/CXCR7 chemokine axis. We report a role for CXCL11 and CXCL12 in the control of malignant features in stem-like cells isolated from human meningioma, providing a possible basis for the aggressive clinical behavior observed in subsets of these tumors. CXCR4/CXCR7 antagonists might represent a useful approach for meningioma at high risk of recurrence and malignant progression.

892 Validation of Docetaxel as a Radiosensitizer in High-Risk Meningiomas Based on DNA Methylation Profiling

INTRODUCTION: Genome-wide methylation profiling has emerged as a transformative prognostic tool in meningiomas, and has identified discrete epigenetic clusters that share distinct clinical and biological features. The association of these signatures with underlying pathological mechanisms has not been well-explored. METHODS: From a collection of over two-hundred well-annotated tumors, we found 981 genes in which methylation signal was predictive of recurrence. Using pathway enrichment analysis, Docetaxel was identified as a candidate therapeutic agent; a medication that is frequently utilized in other common malignancies. This chemotherapy was validated in vitro and in vivo using patient derived meningiomas, and also tested in combination with radiotherapy. RESULTS: Based on 15 patient-derived cultures from our institution and 2 public cell lines, we found that Docetaxel could inhibit meningioma growth at concentrations that were favorable relative to other cancers. This drug triggered apoptosis in meningiomas of all tumor grades and methylation classes, with IC50 values ranging from 0.8 nm to 4.4 nm in vitro. Combining Docetaxel with radiation therapy resulted in synergistic cytotoxic effects on meningioma cultures, associated with elevated markers of cell death and dsDNA breaks. We confirmed our results via orthotopic meningioma models of the cell lines CH157 and IOMM-Lee, which exhibited dose-dependent improvements in mortality and tumor volume in response to Docetaxel and radiation. CONCLUSIONS: Our results leverage DNA methylation patterns to repurpose FDA-approved medications in the treatment of meningiomas, and suggest a clinical trial of Docetaxel with radiotherapy may be efficacious in aggressive lesions.

EXTH-87. DECITABINE POTENTIATES MENINGIOMA IMMUNOTHERAPY TARGETING NY-ESO-1

Abstract INTRODUCTION No immunotherapy exists for meningiomas. NY-ESO-1 is a potential target because in meningiomas it’s the most frequently expressed Cancer-Testis-antigen. Decitabine is a hypomethylating agent known to upregulate CT-antigens. We tested the effects of decitabine on NY-ESO-1 expression in primary and immortalized meningioma cultures, and its impact on the efficacy of NY-ESO-1 T-cell-receptor-transduced T-cells(TCRs), to understand its potential role in meningiomas immunotherapy. METHODS We established primary meningiomas cultures from fresh tumor specimens and quantified NY-ESO-1 expression using Western blot and qRT-PCR. Cultures were pre-treated with 1uM of decitabine for at least 48 hours before assays. In vitro cytolysis was measured using the xCelligence Real-Time Cell Analyzer System in meningioma co-cultures with MHC-Class-I(HLA-A*0201)–restricted NY-ESO-1(157 – 165)-specific TCRs. RESULTS Nine primary and three immortalized meningioma cultures(Grade I – III) were evaluated for baseline NY-ESO-1 expression. High expression was defined as > 100-fold overexpression compared to glioma(U251) cultures. Decitabine treatment significantly increased NY-ESO-1 expression in 6 primary and 2 immortalized(SF1335, grade 1; IOMM-LEE, grade 3) cultures with low baseline expression(median increase over 100 fold; p > 0.001); whereas it slightly decreased NY-ESO-1 expression in 3 primary and 1 immortalized culture(CH157, WHO grade 3) with high baseline expression(median decrease 20%;p< 0.001). Decitabine pre-treatment of meningioma before co-culture with NY-ESO-1 TCRs at a ratio of 1:1, increased tumor cytolysis for SF1335, from 20% to 40%, and for IOMM-LEE, from 0% to 85%, measured at 10 hours. NY-ESO-1 expression increased 5000-fold vs 10-fold for IOMM-LEE vs SF1335, respectively. Decitabine treatment also increased MHC-Class-I expression for IOMM-LEE but not SF1335. Decitabine pre-treatment did not change cytolysis of CH157 at 65%(10 hrs). CONCLUSIONS Decitabine upregulates NY-ESO-1 expression in meningiomas with low baseline expression, and it increases cytolytic effects of NY-ESO-1 TCRs proportional to NY-ESO-1 and MHC-Class-I molecule upregulation. Decitabine may be a clinically feasible adjunct to meningioma immunotherapy.

EXTH-88. DOCETAXEL TARGETS AGGRESSIVE METHYLATION PROFILES AND SERVES AS A RADIOSENSITIZER IN HIGH-RISK MENINGIOMAS

Abstract DNA methylation profiling has emerged as a transformative prognostic tool in meningiomas, identifying distinct epigenetic subgroups that share unique clinical and biological features. Hypothesizing that methylation patterns may also reflect underlying pathogenic mechanisms, we investigated if this modality could be informative for the selection of efficacious chemotherapies in meningiomas, focusing on FDA-approved compounds that are well-tolerated in patients. Using a methylation dataset of 210 tumors, we identified 981 genes in which adjacent CpG sites were predictive of progression-free survival. Pathway enrichment of these genes nominated Docetaxel as a candidate therapeutic; a well-established taxane that is routinely used for breast, prostate, and lung cancers. Using 15 primary patient-derived cultures and 2 cell lines, we found that Docetaxel could inhibit meningioma growth at concentrations that were favorable relative to other cancers. This drug triggered apoptosis in meningiomas of all tumor grades and methylation classes, with IC50 values ranging from 0.8nm to 4.4nm in vitro. Combining Docetaxel with radiation therapy resulted in synergistic cytotoxic effects on meningioma cultures, associated with elevated markers of cell death and dsDNA breaks. We confirmed our results via orthotopic meningioma mouse models of the cell lines CH157 and IOMM-Lee, which exhibited dose-dependent improvements in mortality and tumor volume in response to Docetaxel and radiation. Our results leverage DNA methylation patterns to repurpose FDA-approved medications in the treatment of meningiomas, and suggest a clinical trial of Docetaxel with radiotherapy may be efficacious in higher-grade lesions.

BIOM-52. DIAGNOSTIC POTENTIAL OF EXTRACELLULAR VESICLES IN MENINGIOMA PATIENTS

Abstract Standard monitoring after meningioma resection relies on serial MRI examinations, which are time-consuming, expensive and provide no information on molecular alterations that may indicate progression towards a more aggressive tumor. Extracellular vesicles (EVs) secreted by tumor cells play an important role in cell-cell communication, and tumor-derived EVs circulating in patient blood can serve as biomarkers. We investigated the potential role of plasma EVs in meningioma patients for tumor detection and determined whether EVs secreted by meningioma cells reflect epigenetic, genomic and proteomic alterations of original tumors. EV concentrations were quantified in patient plasma (n = 46). Short-term meningioma cultures were established (n = 26) and secreted EVs were isolated. Methylation and copy number profiling was performed using 850k arrays, and mutations were identified by targeted gene panel sequencing. Differential quantitative mass spectrometry was employed for proteomic analysis. We found that the levels of circulating EVs were significantly elevated in meningioma patients compared to healthy individuals, and that plasma EV concentrations correlated with malignancy grade and extent of peritumoral edema. Postoperatively, EV counts dropped to normal levels, and the magnitude of the postoperative decrease was associated with extent of tumor resection (Simpson grade). Methylation profiling of EV-DNA allowed correct tumor classification as meningioma in all investigated cases, and accurate methylation subclass assignment in nearly all cases. Copy number variations present in tumors, as well as tumor-specific mutations were faithfully reflected in meningioma EV-DNA. Proteomic EV profiling did not permit original tumor identification but revealed tumor-associated proteins such as desmoplakin that could potentially be utilized to enrich meningioma EVs from biofluids. In conclusion, elevated EV levels in meningioma patient plasma may aid in tumor diagnosis and assessment of treatment response. Meningioma EV-DNA mirrors genetic and epigenetic tumor alterations and facilitates molecular tumor classification.

Diagnostic potential of extracellular vesicles in meningioma patients.

Extracellular vesicles (EVs) play an important role in cell-cell communication, and tumor-derived EVs circulating in patient blood can serve as biomarkers. Here, we investigated the potential role of plasma EVs in meningioma patients for tumor detection and determined whether EVs secreted by meningioma cells reflect epigenetic, genomic, and proteomic alterations of original tumors. EV concentrations were quantified in patient plasma (n = 46). Short-term meningioma cultures were established (n = 26) and secreted EVs were isolated. Methylation and copy number profiling was performed using 850k arrays, and mutations were identified by targeted gene panel sequencing. Differential quantitative mass spectrometry was employed for proteomic analysis. Levels of circulating EVs were elevated in meningioma patients compared to healthy individuals, and the plasma EV concentration correlated with malignancy grade and extent of peritumoral edema. Postoperatively, EV counts dropped to normal levels, and the magnitude of the postoperative decrease was associated with extent of tumor resection. Methylation profiling of EV-DNA allowed correct tumor classification as meningioma in all investigated cases, and accurate methylation subclass assignment in almost all cases. Copy number variations present in tumors, as well as tumor-specific mutations were faithfully reflected in meningioma EV-DNA. Proteomic EV profiling did not permit original tumor identification but revealed tumor-associated proteins that could potentially be utilized to enrich meningioma EVs from biofluids. Elevated EV levels in meningioma patient plasma could aid in tumor diagnosis and assessment of treatment response. Meningioma EV-DNA mirrors genetic and epigenetic tumor alterations and facilitates molecular tumor classification.

Chrysophanol inhibits the osteoglycin/mTOR and activats NF2 signaling pathways to reduce viability and proliferation of malignant meningioma cells

ABSTRACT Chrysophanol shows promising antitumor activity, but how it may work against malignant meningioma is poorly understood. In addition, osteoglycin (OGN) may help mediate the antitumor effects of chrysophanol; thus, this study investigated the potential antitumor mechanism of chrysophanol in malignant meningioma cultures. Meningioma cell line HBL-52 were incubated with varying doses of chrysophanol (0–90 μM) for different time points, and osteoglycin (OGN) was overexpressed or inhibited in some cell cultures to assess its roles. Cell viability was quantified using the CCK8 assay and colony formation assays, while effects on cell cycle distribution and apoptotic rates were examined by flow cytometry and enzyme-linked immunosorbent assays (ELISA) to detect histone DNA levels. Caspase-3 and −9 activities were detected by related commercial kits. Protein expression was assessed using Western blotting. Chrysophanol significantly reduced HBL-52 cell viability, based on reduced colony formation, and proliferation, based on low levels of bromodeoxyuridine incorporation. Annexin V/propidium iodide staining revealed a 30% increase in apoptotic cells at 90 μM chrysophanol (33.7% vs 3.3% in control cultures). Chrysophanol treatment greatly decreased the Bcl-2/Bax expression ratio and increased the expressions of cleaved caspase-3 and −9, and the activities of caspase-3 and −9. Chrysophanol blocked cells in G1 phase and inhibited the OGN/mTOR signaling cascade, but activated neurofibromatosis 2 (NF2) cascade. OGN overexpression activated mTOR, down-regulated NF2, and partially reversed growth inhibition by chrysophanol. Chrysophanol may be useful as a treatment against malignant meningioma by inhibiting OGN/mTOR signaling and activating NF2 signaling.

SSTR-2 as a potential tumour-specific marker for fluorescence-guided meningioma surgery

BackgroundMeningiomas are the most frequently occurring primary intracranial tumours in adults. Surgical removal can only be curative by complete resection; however surgical access can be challenging due to anatomical localization and local invasion of bone and soft tissues. Several intraoperative techniques have been tried to improve surgical resection, including intraoperative fluorescence guided imaging; however, no meningioma-specific (fluorescent) targeting has been developed yet. Here, we aimed to identify the most promising biomarkers for targeted intra-operative fluorescence guided meningioma surgery.MethodsOne hundred forty-eight meningioma specimens representing all meningioma grades were analysed using immunohistochemistry (IHC) on tissue microarrays (TMAs) to determine expression patterns of meningioma biomarkers epithelial membrane antigen (EMA), platelet-derived growth factor β (PDGF-β), vascular endothelial growth factor α (VEGF-α), and somatostatin receptor type 2 (SSTR-2). Subsequently, the most promising biomarker was selected based on TArget Selection Criteria (TASC). Marker expression was examined by IHC in 3D cell culture models generated from freshly resected tumour material.ResultsTMA-IHC showed strongest staining for SSTR-2. All cases were positive, with 51.4% strong/diffuse, 30.4% moderate/diffuse and only 18.2% focal/weak staining patterns. All tested biomarkers showed at least weak positivity in all meningiomas, regardless of WHO grade. TASC analysis showed that SSTR-2 was the most promising target for fluorescence guided imaging, with a total score of 21 (out of 22). SSTR-2 expression was determined on original patient tumours and 3D cultures of three established cultures.ConclusionsSSTR-2 expression was highly sensitive and specific in all 148 meningiomas, regardless of WHO grade. According to TASC analysis, SSTR-2 is the most promising receptor for meningioma targeting. After establishing in vitro meningioma models, SSTR-2 cell membrane expression was confirmed in two of three meningioma cultures as well. This indicates that specific fluorescence in an experimental setting can be performed for the further development of targeted fluorescence guided meningioma surgery and near-infrared fluorescent tracers targeting SSTR-2.

Altered expression of E-Cadherin-related transcription factors indicates partial epithelial-mesenchymal transition in aggressive meningiomas

E-Cadherin has been suggested to be involved in meningioma progression but is also known as a key player of epithelial to mesenchymal transition (EMT). We wondered whether the adherens junction protein E-Cadherin, the tight junction protein Zo-1, and transcription factors suppressing E-Cadherin expression (Slug, Snail, Twist, Zeb-1) are differentially expressed between histopathological subtypes of meningioma, and if the expression of these factors is related to biological features of meningiomas. Analyzing 85 meningiomas of various histopathological subtypes and grades of malignancy by immunohistochemistry and 50 of them in addition by real-Time-PCR, we observed significantly reduced expression of Zeb-1, Twist and Slug, together with slightly increased expression levels for E-Cadherin and Zo- 1 in fibroblastic WHO-grade I tumors compared to meningothelial WHO grade I tumors, contradicting the hypothesis of EMT in the fibroblastic meningiomas characterized by mesenchymal appearance. However, comparing aggressive WHO grade II or III meningiomas with WHO-grade I tumors, we observed altered expression levels (loss of E-Cadherin and Zo-1, increased expression of Zeb-1 and Slug) indicating molecular features of EMT in aggressive meningiomas. This was supported by reduced E-Cadherin and increased Slug levels in recurrent compared to non-recurrent meningiomas. The expression levels of E-cadherin and Zo-1 were positively correlated with expression of NF2 mRNA. In primary meningioma cultures and IOMM-Lee meningioma cells, EMT induction by TGF-ß resulted in altered morphology and increased expression of EMT associated transcription factors. Meningioma cells with allelic losses of NF2 showed generally higher levels of various EMT relevant proteins, but were unresponsive to TGF-ß treatment. Our data indicate that aggressive meningiomas of WHO grade II/III are characterized by molecular alterations indicating partial EMT. This might contribute to the aggressive biology of these tumors.

P38MAPK activation and DUSP10 expression in meningiomas

The mitogen activated protein kinase (MAPK) p38MAPK has been implicated in regulation of cell proliferation and apoptosis. However, expression, activation and regulation has not been studied in meningiomas, to our knowledge. p38MAPK is regulated, in part, by dual specificity phosphatases (DUSP) that inactivate signaling by dephosphorylation. DUSP10 is also a likely participant in regulating meningioma proliferation. Five fetal and an adult human leptomeninges and 37 meningioma cultures (MC) were evaluated for DUSP10 as well as phosphorylation of its substrates p38MAPK and p44/42MAPK by western blot and DUSP10 expression by polymerase chain reaction. Platelet derived growth factor-BB (PDGF-BB), transforming growth factor B1 (TGFB1) and cerebrospinal fluid effects on DUSP10 and signaling were also studied in vitro. DUSP10 and phospho-p38MAPK and phospho-p44/42MAPK were detected in all six leptomeninges. DUSP10 was detected in 13 of 17 World Health Organization grade I, 11 of 14 grade II and four of six grade III meningiomas. Phospho-p38MAPK was detected in nine of 17 grade I, two of six grade II, and four of six grade III meningiomas. In the majority of meningiomas DUSP10 expression correlated inversely with phosphorylation of p38MAPK. PDGF-BB increased DUSP10 in MC2 and MC4 and weakly in MC3. TGFB1 increased phosphorylation of p38MAPK and caspase 3 activation. Thus p38MAPK and DUSP10 likely participate in the pathogenesis of meningiomas.

Abstract 1809: Marizomib (NPI-0052) activity as a single agent in malignant glioma

Abstract Background: Glioblastoma multiforme (GBM) is a highly aggressive brain tumor, which displays innate resistance to multiple treatment modalities. Previous studies have shown that the proteasome plays a vital role in the physiology of GBM, and that proteasome inhibition can be used as a strategy for treating malignant gliomas. Marizomib (NPI-0052) is a second generation irreversible proteasome inhibitor, which has a more lipophilic structure and has a broader and more prolonged inhibition profile for 20S proteasome activities compared to bortezomib and carfilzomib, two proteasome inhibitors approved by FDA for treatment of multiple myeloma. While bortezomib and carfilzomib have only modest activity as a treatment of malignant gliomas, marizomib might potentially be a novel therapeutic strategy for primary brain tumors. Unfortunately, to date, the number of studies that have analyzed the effect of marizomib on glioma is limited. Methods: In these studies, we investigated the in vitro activities of marizomib in primary cell cultures derived from a multitude of human brain tumors (high-grade and low-grade gliomas and meningiomas), normal neural stem/progenitor cells (NSC) and as well as in the established human malignant glioma lines U251-MG and D54-MG. The effect of marizomib on cell proliferation, proteasome activity, motility, apoptosis and Reactive Oxygen Species (ROS) were evaluated in glioma cell lines. The inhibition of marizomib by the ROS quenching agent, N-acetyl cysteine (NAC) was also tested. Results: The sensitivities varied in function of the pathology of the tumor, with the malignant glioma stem-like cells being the most severely affected, in contrast with the low-grade glioma, meningioma and NSC-derived cultures. Marizomib inhibited the proliferation of U251-MG and D54-MG cell lines with a half maximal effective concentration (EC50) of 52nM and 20nM respectively, along with a significant decrease in cell migration and invasion. Treatment with marizomib at a concentration of 60nM for 4 hours inhibited proteasome chymotrypsin-like (CT-L, β5) activity by 85% in U251-MG and D54-MG cells. Marizomib treatment of human glioma cells was associated with increased free radical production and apoptosis, along with activation of caspase-3 and cleavage of PARP. Those effects of marizomib can be suppressed by exposure to the ROS quenching agent N-acetyl cysteine (NAC). Conclusion: These preclinical studies demonstrate a significant anti-tumor effect of marizomib in malignant glioma cells. Marizomib has relatively little effect on neural stem/progenitor cells suggesting minimal neurotoxicity, while severely affecting both malignant glioma stem cells and glioma cell lines. But importantly, unlike bortezomib and carfilzomib, marizomib can cross the blood brain barrier. Additional research into the use of marizomib as a potential treatment for malignant gliomas as a single agent or in combination with SOC therapies for glioma is warranted. Citation Format: Kaijun Di, Xing Gong, Dana M. Curticiu, Michael A. Palladino, Daniela A. Bota. Marizomib (NPI-0052) activity as a single agent in malignant glioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1809. doi:10.1158/1538-7445.AM2014-1809

Therapeutic Potential of HSP90 Inhibition for Neurofibromatosis Type 2

The growth and survival of neurofibromatosis type 2 (NF2)-deficient cells are enhanced by the activation of multiple signaling pathways including ErbBs/IGF-1R/Met, PI3K/Akt, and Ras/Raf/Mek/Erk1/2. The chaperone protein HSP90 is essential for the stabilization of these signaling molecules. The aim of the study was to characterize the effect of HSP90 inhibition in various NF2-deficient models. We tested efficacy of the small-molecule NXD30001, which has been shown to be a potent HSP90 inhibitor. The antiproliferative activity of NXD30001 was tested in NF2-deficient cell lines and in human primary schwannoma and meningioma cultures in vitro. The antitumor efficacy of HSP90 inhibition in vivo was verified in two allograft models and in one NF2 transgenic model. The underlying molecular alteration was further characterized by a global transcriptome approach. NXD30001 induced degradation of client proteins in and suppressed proliferation of NF2-deficient cells. Differential expression analysis identified subsets of genes implicated in cell proliferation, cell survival, vascularization, and Schwann cell differentiation whose expression was altered by NXD30001 treatment. The results showed that NXD30001 in NF2-deficient schwannoma suppressed multiple pathways necessary for tumorigenesis. HSP90 inhibition showing significant antitumor activity against NF2-related tumor cells in vitro and in vivo represents a promising option for novel NF2 therapies.

Lopinavir inhibits meningioma cell proliferation by Akt independent mechanism

Recent studies suggest that HIV-1 protease inhibitors may have anti-neoplastic effects on some malignancies. The anti-neoplastic effects of lopinavir have not been established or studied in brain tumors. Primary cultures of three fetal leptomeninges and 18 meningiomas were treated with lopinavir alone or with PDGF-BB. DNA synthesis was assessed by CyQUANT. Lopinavir effects on basal and PDGF-stimulated phosphorylation of the Akt-mTOR, MEK1/2-MAPK and STAT3 pathways, phosphorylation of Rb, Caspase 3 activation and reductions in survivin were assessed by Western blots. Lopinavir produced a significant reduction in PDGF-BB stimulation of DNA synthesis in a leptomeningeal culture (P = 0.0013) and 1 of 6 WHO grade I and 1 of 4 grade II meningiomas at 24 h and in 3 of 6 WHO grade I, 4 of 4 grade II and 1 of 1 grade III cell cultures (P = 0.0001) at 72 h. Lopinavir reduced PDGF-BB stimulation of phosphorylation/activation of MAPK in the 22 week fetal leptomeningeal cell cultures and in cells from 1 grade I meningioma at 24 h, but in none of 4 grade I and 5 grade II meningiomas at 6 h. Lopinavir had no notable effect on basal or PDGF-stimulated p-mTOR, p-MEK1/2, or p-STAT3, activation of Caspase 3 or survivin levels. Lopinavir treatment for 24 h had no effect on basal Rb phosphorylation but reduced Rb phosphorylation in all four meningioma cultures. These studies suggest that lopinavir may inhibit meningioma growth, and does so in part by cell cycle arrest. Additional evaluation of lopinavir as a potential adjunct chemotherapy is warranted.

Bone Morphogenetic Protein 4 and Its Receptors Are Expressed in the Leptomeninges and Meningiomas and Signal via the Smad Pathway

The roles of bone morphogenetic proteins (BMPs) and their receptors (BMPRs) in meningioma biology are not known. In this study, frozen tissues from 26 World Health Organization Grades I to III meningiomas were analyzed by Western blot for BMP-2/4, BMPR IA, and BMPR II, and activation of downstream p-Smad1, p38 mitogen-activated protein kinase (MAPK), and p44/42 MAPK signaling molecules. Sections from 20 normal leptomeninges, 2 arachnoid cysts, and 51 meningiomas were analyzed for BMP-4 and p44/42 MAPK by immunohistochemistry. Primary meningioma cultures from 11 meningiomas were treated with BMP-4 and evaluated for cell proliferation and signaling pathway activation. Conditioned media from 7 cultures were analyzed for BMP-4 by ELISA. Bone morphogenetic protein 4 was variably detected in adult leptomeninges but was detected in 89% or 84% of Grade I meningiomas and in 60% of Grade II meningiomas by Western blot and immunohistochemistry, respectively. Bone morphogenetic protein receptors IA and II were detected in leptomeninges and in all meningiomas studied, and activated Smad1 was detected in all meningiomas studied. Bone morphogenetic protein 4 stimulated meningioma cell proliferation and phosphorylation/activation of Smad1 but not p38 MAPK or p44/42 MAPK in vitro, and it was detected in conditioned media from 4 of 7 cultures. These findings suggest that BMP-4 and BMPRs may play autocrine/paracrine roles and interact with other transforming growth factor-beta superfamily members in regulating meningioma growth and differentiation.

Evidence of ubiquitous in vivo and in vitro expression of pro-apoptotic Smac/DIABLO protein in meningioma cell lines

Although meningiomas are one of the most common tumors in the central nervous system, the adjuvant treatment for recurrent or malignant meningiomas is not satisfactory. An intense interest in evaluating new molecular markers that may serve as potential therapeutic targets exists. Changes in apoptosis mechanisms play important roles in tumor pathogenesis. One pro-apoptotic protein is Smac/DIABLO, which neutralizes the inhibitors of apoptosis (IAPs). As Smac/DIABLO has not been previously analyzed in meningiomas, We investigated the expression of Smac/DIABLO and survivin in primary meningioma cultures in vivo and in vitro. Expression of Smac/DIABLO, survivin and single-stranded (ss)DNA in vivo were determined immunohistochemically in 100 meningioma surgical specimens, dura and normal human cortex. The expression of the apoptotic enzymes in vitro was analyzed after RNA and protein isolation of all meningiomas via Western blotting and PCR. All examined meningiomas and normal cerebral cortex displayed intense positive cytoplasmic Smac/DIABLO immunoreactivity. Survivin and ssDNA were expressed in all surgical specimens and showed weak staining overall. Examination of Smac/DIABLO protein via Western blotting showed distinct signals in the cytoplasmic extracts. PCR analysis displayed no changes of Smac/DIABLO and survivin expression in different meningioma grades, normal human cortical cortex or dura. Constant high-level Smac/DIABLO respectively low-level survivin expression in meningiomas and normal brain demonstrate similar apoptotic behavior of meningiomas compared to normal brain tissue. These findings indicate no pathological overexpression of survivin in meningiomas as is evident in several other cancer types impeding apoptosis.

Irinotecan: a potential new chemotherapeutic agent for atypical or malignant meningiomas

There is currently no effective chemotherapy for meningiomas. Although most meningiomas are treated surgically, atypical or malignant meningiomas and surgically inaccessible meningiomas may not be removed completely. The authors have investigated the effects of the topoisomerase I inhibitor irinotecan (CPT-11) on primary meningioma cultures and a malignant meningioma cell line in vitro and in vivo. The effects of irinotecan on cellular proliferation in primary meningioma cultures and the IOMM-Lee malignant meningioma cell line were measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay and flow cytometry. Apoptosis following drug treatment was evaluated by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and the DNA laddering assays. The effects of irinotecan in vivo on a meningioma model were determined with a subcutaneous murine tumor model using the IOMM-Lee cell line. Irinotecan induced a dose-dependent antiproliferative effect with subsequent apoptosis in the primary meningioma cultures (at doses up to 100 microM) as well as in the IOMM-Lee human malignant meningioma cell line (at doses up to 20 microM) irinotecan. In the animal model, irinotecan treatment led to a statistically significant decrease in tumor growth that was accompanied by a decrease in Bcl-2 and survivin levels and an increase in apoptotic cell death. Irinotecan demonstrated growth-inhibitory effects in meningiomas both in vitro and in vivo. Irinotecan was much more effective against the malignant meningioma cell line than against primary meningioma cultures. Therefore, this drug may have an important therapeutic role in the treatment of atypical or malignant meningiomas and should be evaluated further for this purpose.

Induction of Apoptosis in Primary Meningioma Cultures by Fenretinide

Fenretinide, a synthetic retinoid that induces apoptosis in tumor cells in vitro, is being evaluated in clinical trials as a chemotherapeutic agent against several malignancies. Due to its ease of administration, long-term tolerability, and low incidence of long-term side effects, we explored its potential as a therapeutic agent against meningiomas by examining its efficacy in vitro against such cells in primary culture. Cells, cultured from freshly resected benign, atypical, or malignant meningiomas, were exposed to fenretinide (10 mumol/L). Treatment effects were assessed using flow cytometry, Western blot analysis, semiquantitative reverse transcription-PCR for retinoid receptor expression, and changes in insulin-like growth factor-I (IGF-I)-induced proliferation. Fenretinide induced apoptosis in the three grades of meningioma primary cells tested, as shown by the appearance of a sub-G(1) fraction in flow cytometric analysis and by the detection of poly-adenosyl ribonucleotidyl phosphorylase cleavage indicating caspase activation. Fenretinide treatment also increased levels of the death receptor DR5 and caused mitochondrial membrane depolarization. The levels of the retinoid receptors, retinoic acid receptor alpha and retinoid X receptor gamma, were up-regulated in response to fenretinide, suggestive of ligand-induced receptor up-regulation. IGF-I-induced proliferation in the meningioma cells was abolished by fenretinide. We conclude that fenretinide induces apoptosis in all three histologic subtypes of meningioma and exerts diverse cellular effects, including DR5 up-regulation, modulation of retinoid receptor levels, and inhibition of IGF-I-induced proliferation. These results provide preliminary evidence that fenretinide has activity against meningiomas and suggest that further studies are warranted to explore its potential as a therapeutic agent against meningiomas.

The expression of ‘steroid superfamily’ receptors in meningiomas

The meningioma is a tumour that arises from the cells that form a fibrous membrane covering the brain. They account for around 15% of intracranial tumours and about 7% of all people dying from brain tumours. These tumours occur more frequently in women than in men and this has lead to the suggestion that oestrogens have a role in their development and growth. Studies a decade ago with antioestrogens have proved inconclusive and therefore a medical therapy based on anti-endocrine treatment has not been developed. Radiotherapy or surgery is still the only means of treatment. With the latter in mind, we have re-visited the ‘endocrine hypothesis’ by performing the most extensive screening study so far performed for members of the ‘superfamily’ of steroid receptors. RNA was extracted from around 140 tumours and reverse transcription-PCR was performed. This analysis revealed that the ERα, ERβ, AR, PR, GRα, GRβ, PPARα, PPARβ, PPARγ was expressed in, 6%, 0%, 56%, 26% 83%, 0%, 46%, 91%, and 13% of the tumour samples, respectively. Our recent studies, as well as those of some other groups, have indicated that glucocorticoids reduce apoptotic cell death in response to chemotherapeutic agents. Given that glucocorticoid receptors are expressed in so may tumours and the fact that glucocorticoids are given to patients prior to radiotherapy, we have tested whether these steroid hormones modulate cell death. When added alone, the glucocorticoid, dexamethasone (100nM), reduced cellular metabolic activity by 25%, as measured by the redox sensitive dye, WST-1. The glucocorticoid receptor antagonist RU486, at a concentration of 10µM, was able to reverse this effect. Classical apoptosis was observed to be induced in meningioma cultures by the addition of the HMG-CoA enzyme inhibitor, Fluvastatin (10µM). Prior treatment of meningioma cultures with dexamethasone for 24h before exposure to Fluvastatin for 72h reduced the extent of apoptotic cell death observed. As these experiments with glucocorticoids are preliminary, extrapolation to the treatment scenario is not yet justified. We are therefore currently extending these studies by combining glucocorticoid pre-treatment of meningioma cultures with subsequent gamma irradiation.

Growth hormone receptor expression and function in meningiomas: effect of a specific receptor antagonist.

This study was undertaken to explore the effects of growth hormone (GH) and the GH-stimulated peptide insulin-like growth factor-1 (IGF-1) on the growth rate of meningiomas. Polymerase chain reaction and ribonuclease protection assays were used to demonstrate that GH receptor messenger RNA was present in all 14 meningioma specimens studied, regardless of tumor grade. Both wild type (GHRwt) and a previously described exon 3 deletion isoform (GHRd3) of the GH receptor were identified in individual tumor specimens. The importance of the GH receptor was assessed using a GH receptor antagonist (B2036). Blockade of the GH receptor with B2036 reduced serum-induced DNA synthesis, as measured by thymidine incorporation, by 8 to 33% (mean 20%) in primary meningioma cultures. Tumors that expressed the GHRwt and GHRd3 isoforms, or a combination of the two, were all responsive to antagonist treatment. The importance of IGF- in stimulating meningioma cell growth was also assessed. It was found that IGF-1 increased thymidine incorporation in primary meningioma cultures in a dose-dependent manner: 1 ng/ml, 5 ng/ml, and 10 ng/ml resulted in increases in thymidine incorporation of 21%, 43%, and 176%, respectively, over baseline values. In these studies the authors demonstrate that activation of the GH/IGF-1 axis significantly increases the growth rate of meningiomas. Blockade of the GH receptor on tumor cells inhibited tumor growth. If these findings are confirmed in animal studies, agents that downregulate the GH/IGF-1 axis might represent a potential adjuvant therapy in the management of patients with meningioma.

Progesterone Receptor Synthesis in Human Meningiomas: Relation to the Estrogen-Induced Proteins pS2 and Cathepsin-D and Influence of Epidermal Growth Factor, Forskolin and Phorbol Ester in vitro

Autonomous expression of progesterone receptors (PR) in human meningiomas is well established. To evaluate whether, similar to progesterone receptors, other estrogen-inducible proteins are also autonomously expressed in meningiomas, concentrations of pS2 and cathepsin-D (Cath-D) were measured in 52 meningiomas. No pS2 protein was detectable in 52/52 tested meningiomas. The Cath-D protein was measurable in all 52 meningiomas, but the mean concentration of Cath-D in meningioma cytosols was 2.4-fold lower than that of a group of 54 breast tumors (p < 0.001). These results indicate that autonomous expression is a PR-related rather than an estrogen receptor-related phenomenon and, consequently, that estradiol is probably not responsible for PR synthesis in human meningiomas. To evaluate the role of other, non-estradiol-dependent signalling pathways in PR synthesis, the effects of EGF, Forskolin and phorbol ester on PR synthesis were tested in vitro. No PR was detectable after the addition of EGF to six different primary cultures. Forskolin and TPA addition caused a morphological change in meningioma cells, but did not induce PR or pS2 synthesis in two different primary meningioma cultures. We conclude that PR synthesis in human meningiomas cannot be triggered by switching on the signalling pathways activated by these growth factors.