Malignant Meningioma Cell Research Articles

PO-511 High miR-9 levels represent a novel prognostic biomarker to predict development of malignant meningioma

IntroductionMeningioma is the most common primary tumour affecting the central nervous system; it is classified as benign (WHO I,~80%), atypical (WHO II,~15%–20%) and anaplastic/malignant (WHO III,~1%–3%). The 3 year recurrence rate in WHO I meningioma is estimated in about 50% and it is much greater in WHO II and III tumours. MicroRNAs (miRNAs) represent a large class of small RNAs driving regulation of gene expression at post-transcriptional level and playing a role in cell proliferation, differentiation, apoptosis and carcinogenesis. Several studies showed that miRNAs are involved in tumour progression and therefore proposed as diagnostic tools. Here, we evaluated miRNAs signature in meningioma to identify novel biomarkers of tumour progression.Material and methodsMeningioma (MN) specimens were collected from consented patients, according to the ethics. The 96-miRNA profiling was performed using the QuantimirTM Cancer MicroRNA qPCR Array (System Biosciences, UK) following the instructions of the supplier. Validation studies were achieved using TaqMan MicroRNA reagents (Applied Biosystems). Bioinformatic analysis was done using the NormFinder software and in silico studies were performed using the following datasets for putative miRNA targets: TargetScanHuman7.1, DIANATOOLS microT-CDS, mirDIP and the UTRdb tool. Probability (p) values were calculated using the Student’s t-Test, led by the GraphPad Prism 5.01 (p<0.05±SEM).Results and discussionsWe established a new miRNA dataset by identifying six miRNA signatures (p<0.01) differentially regulated in benign versus malignant meningioma cells (miR-9, −10b, −125b, −143,–145 and −199). Validation studies by qPCR confirmed that the miR-9 was upregulated in malignant KT21-MG1 cells (10.71 folds; Log2 scale, p=0.0006) and WHO III tissues (3.75 folds versus WHO I=0, Log2 scale, p=0.044). Finally, highly stringent in silico studies suggested that the miR-9 targets and downregulates the ubiquitin-protein ligase E3C (UBE3C), as confirmed by proteomic analysis in malignant KT21-MG1 and IOMM-Lee cells.ConclusionIn this study, we identified the miR-9 as significantly upregulated in WHO III tumour-derived meningioma cells and tissues when compared to lower grades. Since miR-9 targets the ubiquitin-protein ligase E3C, involved in the ubiquitin-proteasome pathway and therefore regulating protein homeostasis, this protein, together with miR-9, could represent potential novel diagnostic and/or prognostic biomarkers in meningioma.

Primary culture of human malignant meningioma cells and its intracranial orthotopic transplantation in nude mice

To obtain stable primary cultures of human malignant meningioma cells and establish an intracranial in-situ tumor model in nude mice. Ten surgical specimens of highly suspected malignant meningioma were obtained with postoperative pathological confirmation. Primary malignant meningioma cells were cultured from the tissues using a modified method and passaged. After identification with cell immunofluorescence, the cultured cells were inoculated into the right parietal lobe of 6 nude mice using stereotaxic apparatus and also transplanted subcutaneously in another 6 nude mice. The nude mice were executed after 6 weeks, and HE staining and immunohistochmistry were used to detect tumor growth and the invasion of the adjacent brain tissues. The primary malignant meningioma cells were cultured successfully, and postoperative pathology reported anaplastic malignant meningioma. Cell immunofluorescence revealed positivity for vimentin and EMA in the cells, which showed a S-shaped growth curve in culture. Flow cytometry revealed a cell percentage in the Q3 area of (95.99∓2.58)%. Six weeks after transplantation, tumor nodules occurred in the subcutaneous tumor group, and the nude mice bearing the in situ tumor showed obvious body weight loss. The xenografts in both groups contained a mean of (36∓5.35)% cells expressing Ki-67, and the intracranial in situ tumor showed obvious invasion of the adjacent peripheral brain tissues. We obtained stable primary cultures of malignant meningioma cells and successfully established a nude mouse model bearing in situ human malignant meningioma.

Photodynamic therapy using talaporfin sodium induces heme oxygenase-1 expression in rat malignant meningioma KMY-J cells.

Photodynamic therapy (PDT) using talaporfin sodium (TS) is tumor cell-selective less invasive therapy for the treatment of malignant glioma. We previously demonstrated that PDT using TS (TS-PDT) treatment exhibits anti-tumor activity against not only glioblastoma cells but also malignant meningioma cells. In general, various stress response proteins have been reported to affect the sensitivity determination for anticancer agents against tumor cells. However, the relationship between the therapeutic effect of TS-PDT and stress response systems in tumor cells is not adequately investigated. In this study, we investigated the gene expression of stress response proteins, including Sod1, Cat1, Gstp1, Gpx1, Nqo1, and Hmox1, in rat malignant meningioma KMY-J cells after treatment of TS-PDT. TS-PDT treatment significantly decreased the cell viability when compared with the no laser irradiation group. In morphological observation, TS at 25.6 µM treatment exhibited a significant cytotoxic effect after 12 hr of laser irradiation to KMY-J cells. After 3 and 6 hr of TS-PDT treatment, mRNA expression of heme oxygenase-1 (HO-1, encoded by Hmox1) was significantly increased by TS-PDT treatment. We also demonstrated that zinc protoporphyrin IX (ZnPPIX), a HO-1 inhibitor, significantly augmented the cytotoxic effect of TS-PDT treatment. These data suggest that HO-1 induction may contribute to a protective response against TS-PDT treatment in the malignant meningioma cells and may attenuate the therapeutic effect for TS-PDT treatment.

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.

High incidence of TERT mutation in brain tumor cell lines.

TERT promoter gene mutations are highly recurrent in malignant glioma. However, little information exists regarding their presence in experimental brain tumor models. To better characterize systems in which TERT mutation studies could be appropriately modeled experimentally, the TERT promoter was examined by conventional sequencing in primary brain tumor initiating cells (BTIC), two matched recurrent BTIC lines, a panel of established malignant glioma cell lines, and two meningioma cell lines. Telomerase gene expression was examined by quantitative PCR. We found that all glioblastoma BTIC lines harbored a TERT mutation, which was retained in two patient-matched recurrent BTIC. The TERT C228T or C250T mutation was found in 33/35 (94%) of established malignant glioma cell lines and both meningioma cell lines examined. Brain tumor cell lines expressed variably high telomerase levels. Thus, a high percentage of glioma cell lines, as well as two meningioma cell lines, harbors TERT mutations. These data characterize tractable, accessible models with which to further explore telomerase biology in these tumor types.

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.

NDRG4 is a novel oncogenic protein and p53 associated regulator of apoptosis in malignant meningioma cells.

Aggressive meningiomas exhibit high levels of recurrence, morbidity and mortality. When surgical and radiation options are exhausted, there is need for novel molecularly-targeted therapies. We have recently identified NDRG4 overexpression in aggressive meningiomas. NDRG4 is a member of the N-Myc Downstream Regulated Gene (NDRG) family of the alpha/beta hydrolase superfamily. We have demonstrated that NDRG4 downregulation results in decreased cell proliferation, migration and invasion. In follow up to our prior studies; here we demonstrate that the predominant form of cell death following NDRG4 silencing is apoptosis, utilizing Annexin-V flow cytometry assay. We show that apoptosis caused by p53 upregulation, phosphorylation at Ser15, BAX activation, Bcl-2 and BcL-xL downregulation, mitochondrial cytochrome c release and execution of caspases following NDRG4 depletion. Sub-cellular distribution of BAX and cytochrome c indicated mitochondrial-mediated apoptosis. In addition, we carried out the fluorescence cytochemical analysis to confirm mitochondrial-mediated apoptosis by changes in mitochondrial membrane potential (Ψm), using JC-1 dye. Immunoprecipitation and immunofluorescence confirmed binding of NDRG4 to p53. In addition, we demonstrate that apoptosis is mitochondrial and p53 dependent. The proapoptotic effect of p53 was verified by the results in which a small molecule compound PFT-α, an inhibitor of p53 phosphorylation, is greatly protected against targeting NDRG4 induced apoptosis. These findings bring novel insight to the roles of NDRG4 in meningioma progression. A better understanding of this pathway and its role in meningioma carcinogenesis and cell biology is promising for the development of novel therapeutic targets for the management of aggressive meningiomas.

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.

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.

A role for matrix remodelling proteins in invasive and malignant meningiomas.

Meningiomas are one of the most common brain tumours in adults. Invasive and malignant meningiomas present a significant therapeutic challenge due to high recurrence rates and invasion into surrounding bone, brain, neural and soft tissues. Understanding the molecular mechanism of invasion could help in designing novel therapeutic approaches in order to prevent the need for repeat surgery, decrease morbidity and improve patient survival. The aim of this study was to identify the key factors and underlying mechanisms which govern invasive properties of meningiomas. Formalin-fixed paraffin-embedded (FFPE) as well as frozen tumour tissues from bone-invasive, non-invasive and malignant meningiomas were used for RNA microarray, quantitative real-time PCR or Western blot analyses. Malignant meningioma cell lines (F5) were subject to MMP16 downregulation or overexpression and used for in vitro and in vivo functional assays. Subdural xenograft meningioma tumours were generated to study the invasion of tumour cells into brain parenchyma using cell lines with altered MMP16 expression. We establish that the expression level of MMP16 was significantly elevated in both bone-invasive and brain invasive meningiomas. Gain- and loss-of-function experiments indicated a role for MMP16 in meningioma cell movement, invasion and tumour cell growth. Furthermore, MMP16 was shown to positively regulate MMP2, suggesting this mechanism may modulate meningioma invasion in invasive meningiomas. Overall, the results support a role for MMP16 in promoting invasive properties of the meningioma tumours. Further studies to explore the potential value for clinical use of matrix metalloproteinases inhibitors are warranted.

MS-29 * SORAFENIB AND REGORAFENIB INHIBIT GROWTH AND MIGRATION OF MENINGIOMA CELLS

Sorafenib and Regorafenib (Fluoro-Sorafenib) are closely related multikinase inhibitors, which possess activity in the treatment of some human cancers. The potential role of both drugs in meningiomas have not been elucidated so far. Therefore, we have studied their anti-tumor activity in various in vitro meningioma models. A moderate antiproliferative activity of both drugs applied for 48 h (p < 0.001) to malignant IOMM-Lee meningioma cells was found in microtiter-tetrazolium (MTT) assays with an IC50 of 15 µM for Regorafenib and an IC50 between 7.5 and 15 µM for Sorafenib. MTT assays just reflect the number of viable cells and may thus indicate solely an inhibition of proliferation of the rapidly growing IOMM-Lee cells. However, some degree of cell death induction must have been occured, because a strong induction of nucleosome liberation (p < 0.05) was observed for both drugs by ELISA. Both drugs consistently had lower effects (MTT assay) in two syngenous derivatives of the benign SF4068 meningioma line exhibiting a massive NF2 loss (shRNA mediated) as compared to two syngenous SF4068 derivatives with NF2 wildtype. This suggests a modifying role of the NF2 status on the effects of Regorafenib and Sorafenib. Moreover, both drugs (15 µM) dramatically inhibited cell migration (gap assay, p < 0.05) and invasion (matrigel transwell assay, p < 0.01). Therefore, Sorafenib and Regorafenib seem to target preferentially meningioma cell motility and brain invasion.

Alternatively spliced variants of the regucalcin gene in various human normal and tumor tissues.

Regucalcin plays a pivotal role as a suppressor protein in signal transduction in various cell types. The regucalcin gene, which is localized on the X chromosome, consists of 7 exons and 6 introns. Decreased liver regucalcin gene expression has been suggested to play a suppressive role in the development of hepatocellular carcinogenesis in animal models. This study was undertaken to determine the changes in regucalcin gene expression in various human normal and tumor tissues, including liver, kidney, brain and lung tissues. The full-length and alternatively spliced variants of regucalcin mRNA were found to be expressed in various human tissues. This expression was suppressed in tumor tissues of hepatocellular carcinoma, kidney transitional cell carcinoma, brain malignant meningioma and lung non-small cell carcinoma. The full-length regucalcin protein was found to be highly expressed in normal human liver and kidney tissues; its expression was suppressed, however, in the liver and kidney tumor tissues. The spliced variant proteins were found to be expressed in the normal liver and kidney tissues, and decreased in the tumor tissues. Such alternative variants were not observed in the liver and kidneys of rats and mice. The alternatively spliced variants of the regucalcin gene were found to be expressed in various human normal and tumor tissues.

Enhancing the effect of 5-aminolevulinic acid based photodynamic therapy in human meningioma cells

The interest in photodynamic therapy (PDT) in cancer treatment has continuously increased since its' advent in the 1960s. In neurosurgery, 5-aminolevulinic acid based photodynamic therapy (5-ALA PDT) is used for malignant glioma in vitro and in vivo. Data about PDT in meningioma, which is the second most frequent primary brain tumor, is sparse. Preliminary work indicated that meningiomas are more resistant to PDT than gliomas. In the present study we evaluated whether the effect of 5-ALA PDT on malignant meningioma cells can be increased by ciprofloxacin and changes in incubation time, factors known to influence the effectiveness of PDT in other tumor entities. An in vitro-PDT model of a malignant human meningioma cell line (KT21-MG) was chosen as screening method. Cultured cells were incubated with varying 5-ALA concentrations for 4h and exposed to PDT (control group). Two experimental groups, one with 5-ALA and ciprofloxacin (group I) and one with 24-h ALA incubation time (group II), were studied, respectively. Cell viability was assessed by WST-1 assay. Ciprofloxacin and longer ALA incubation time significantly increased the lethal effect of 5-ALA PDT on meningioma cells. Efficacy of 5-ALA PDT could be increased by adjunction of ciprofloxacin in conventional clinical dosing and by prolongation of ALA incubation time. As ciprofloxacin is a common antibiotic with good tissue penetration, low toxicity and a favorable risk profile it represents a clinically interesting method for future PDT trials in meningiomas.

The expression of disabled-2 is common reduced in meningiomas

Disabled-2 (Dab2) is frequently down-regulated in several types of cancers. We examined the expression level of Dab2 in human meningiomas and meningioma cells, aimed to investigate its role in the oncogenesis and development of meningiomas. Western blot analysis was employed to detect Dab2 expression in 90 fresh tissues of meningiomas, 10 leptomeninges and two kinds of human malignant meningioma cell lines. Independent samples t-test, analysis of variance, Pearson Chi-square test and likelihood ratio test were used to analyze the expression level of Dab2 and its relations to clinic-pathological characteristics of meningiomas. Dab2 was significantly down-regulated in classic meningiomas than the atypical or anaplastic meningiomas. The reduced or loss of expression of Dab2 were significantly correlated with the lower classification of meningiomas and negatively correlated with the invasive ability of adjacent tissues. Furthermore, it was reduced or lost in malignant meningioma cell lines (IOMM-Lee and KT21-MG1). The lower classification of meningiomas correlated with previous comorbidities; not with the gender, age of patients and smoking. Dab2 is expressed at variable level in meningiomas with different grade of malignancy and probably plays a pivotal role in the early stage of oncogenesis of malignant meningiomas.

Adoptive transfer of genetically modified Wilms' tumor 1–specific T cells in a novel malignant skull base meningioma model

Meningiomas are the most commonly diagnosed primary intracranial neoplasms. Despite significant advances in modern therapies, the management of malignant meningioma and skull base meningioma remains a challenge. Thus, the development of new treatment modalities is urgently needed for these difficult-to-treat meningiomas. The goal of this study was to investigate the potential of build-in short interfering RNA-based Wilms' tumor protein (WT1)-targeted adoptive immunotherapy in a reproducible mouse model of malignant skull base meningioma that we recently established. We compared WT1 mRNA expression in human meningioma tissues and gliomas by quantitative real-time reverse-transcription polymerase chain reaction. Human malignant meningioma cells (IOMM-Lee cells) were labeled with green fluorescent protein (GFP) and implanted at the skull base of immunodeficient mice by using the postglenoid foramen injection (PGFi) technique. The animals were sacrificed at specific time points for analysis of tumor formation. Two groups of animals received adoptive immunotherapy with control peripheral blood mononuclear cells (PBMCs) or WT1-targeted PBMCs. High levels of WT1 mRNA expression were observed in many meningioma tissues and all meningioma cell lines. IOMM-Lee-GFP cells were successfully implanted using the PGFi technique, and malignant skull base meningiomas were induced in all mice. The systemically delivered WT1-targeted PBMCs infiltrated skull base meningiomas and significantly delayed tumor growth and increased survival time. We have established a reproducible mouse model of malignant skull base meningioma. WT1-targeted adoptive immunotherapy appears to be a promising approach for the treatment of difficult-to-treat meningiomas.

UPAR and cathepsin B shRNA impedes TGF-β1-driven proliferation and invasion of meningioma cells in a XIAP-dependent pathway

Overexpression of transforming growth factor β1 (TGF-β1) has been linked to immune suppression, tumor angiogenesis, tumor cell migration, tumor cell survival, and tumor cell invasion in many cancers. In the present study, we found abundant expression of TGF-β1 in the microenvironment of four different pathological types of meningioma tumors. TGF-β1 induced invasion in malignant meningioma cells with an associated upregulation of urokinase-type plasminogen activator (uPA), uPAR, cathepsin B, and MMP-9, and this increase in proliferation was coupled with the expression of anti-apoptotic and pro-survival signaling molecules. In addition to the intense immunoreactivity of meningioma tumors to X-linked inhibitor to apoptosis (XIAP), its knockdown abolished the TGF-β1-induced proliferation of these cells. The stimulation of XIAP expression and the activation of pSMAD-2 is mediated by phosphatidylinositol 3-kinase (PI3K)- and MEK-dependent pathways, and the addition of anti-TGF-β1 antibodies prevented their expression with a consequent decrease in invasion. Bicistronic shRNA constructs targeting uPAR and cathepsin B (pUC) quenched TGF-β1-driven invasion and survival of meningioma cells by downregulation of XIAP and pSMAD-2 expression. Animal models with intracranial tumors showed elevated levels of TGF-β1, XIAP and pSMAD-2, and pUC treatment prevented this increased expression. Thus, targeted silencing of TGF-β1-induced signaling by pUC in meningioma would provide new treatment approaches for management of meningioma.

420PD - Systemic Temsirolimus Administration Reduces Tumor Growth in an Orthotopic Mouse Meningioma Model

ABSTRACT Disclosure C. Mawrin: Work was supported in part by Pfizer Inc. and the Deutsche Krebshilfe. D. Pachow: work was supported in part by Pfizer Inc. and the Deutsche Krebshilfe. E. Kirches: work was supported in part by Pfizer Inc. and the Deutsche Krebshilfe Meningiomas represent the most frequent intracranial tumors. While most cases correspond to WHO grade I, about 5% belong to the aggressive atypical subtype, and 1% are anaplastic meningiomas with poor clinical outcome. However, even the treatment of benign subtypes might be challenging in recurrent tumors and if complete resection is impossible. So far, no chemotherapy regime has been proven to be effective; radiotherapy is used to treat aggressive meningiomas. We have previously established that human meningiomas have activated mTORC-1 signalling, and that meningioma cells are responsive to mTORC-1 inhibitors. In the present study, we used an orthotopic mouse meningioma model established in our lab to monitore the effect of systemic temsirolimus treatment on meningioma cells grown in the subarachnoidal space. Following orthotopic injection of malignant IOMM-Lee meningioma cells, mice (n = 8) were treated daily with 20mg/kg temsirolimus intraperitoneally for nine days, beginning at day 3. Control animals (n = 8) were treated with diluent only. Tumor growth was monitored by magnetic resonance imaging (MRI). We observed that at day 9, mice treated with temsirolimus had a significantly (p

Gefitinib enhances the efficacy of photodynamic therapy using 5-aminolevulinic acid in malignant brain tumor cells

Since the ATP-binding cassette transporter ABCG2 plays a physiologically significant role of porphyrin efflux from living cells, the expression of ABCG2 may influences the efficacy of photodynamic therapy (PDT). In this study, we evaluated the effect of gefitinib, a potent ABCG2 inhibitor, on 5-aminolevulinic acid (5-ALA)-PDT in brain tumor cell lines in vitro. Four human glioma cell lines (U87MG, U118MG, A172, and T98G) and a malignant meningioma cell line (IOMM-Lee) were incubated with gefitinib (0.01-1.0μM) before incubation with 5-ALA (1mM). The effects gefitinib on intracellular protoporphyrin IX (PpIX), mRNA and protein expression of ABCG2, and PDT were evaluated, in vitro. At concentrations of 0.1μM or higher, gefitinib enhanced intracellular levels of PpIX in a dose-dependent manner in all those cell lines. Gefitinib decreased mRNA and plasma membrane protein expression of ABCG2, and efficiently enhanced the effect of 5-ALA-PDT in malignant brain tumor cells. Gefitinib can inhibit ABCG2-mediated PpIX efflux from malignant brain tumor cells to increase the intracellular PpIX and thereby enhance the PDT effect.

Retinoblastoma protein-interacting zinc-finger gene 1 (RIZ1) dysregulation in human malignant meningiomas

Retinoblastoma protein-interacting zinc-finger gene 1 (RIZ1) expression is often silenced in many types of human tumors. However, the relationship between RIZ1 expression and malignant meningiomas remains unclear. Here we have found for the first time that the expression of RIZ1 genes are associated with meningiomas progression through extensive analyses of Affymetrix GeneChip microarray data. Further validation methods for gene expression included quantitative PCR (qPCR), western blot and immunohistochemistry analysis, and these methods confirmed that RIZ1 is significantly downregulated in malignant meningioma tissues, as compared with benign meningiomas. In addition, malignant meningioma cells were stably transfected with ectogenic RIZ1 using Lentivirus-mediated transfection, and the transfections were followed by an in vitro 5-bromo-2-deoxyuridin incorporation assay, colony formation assay, cell cycle analysis, invasive analysis, apoptotic assay and western blot analysis. Our results demonstrate that the forced expression of RIZ1 in a malignant meningioma cell line inhibited cellular proliferation and arrested the cells in the G2/M phase of the cell cycle. We also confirmed that overexpression of RIZ1 may induce apoptosis of malignant meningioma cells. Furthermore, RIZ1 overexpression in malignant meningioma cells was associated with the downregulation of c-myc expression. These results from our study indicate that RIZ1 expression is significantly downregulated as the formation of meningiomas progressed, and suggest that RIZ1 may represent a promising candidate tumor suppressor gene that contributes to malignant meningiomas.