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Cancer Therapy: Preclinical mTORC1 Inhibitors Suppress Meningioma Growth in Mouse Models Doreen Pachow 1 , Nadine Andrae 1 , Nadine Kliese 1 , Frank Angenstein 3 , Oliver Stork 2 , Annette Wilisch-Neumann 1 , Elmar Kirches 1 , and Christian Mawrin 1 Abstract Purpose: To evaluate the mTORC1 (mammalian target of rapamycin complex 1) pathway in menin- giomas and to explore mTORC1 as a therapeutic target in meningioma cell lines and mouse models. Experimental Design: Tissue microarrays (53 meningiomas of all WHO grades) were stained for phosphorylated polypeptides of mTOR, Akt, and the mTORC1 targets 4EBP1 and p70S6K, the latter being the consensus marker for mTORC1 activity. Expression of proteins and mRNAs was assessed by Western blotting and real-time PCR in 25 tumors. Cell lines Ben-Men-1 (benign), IOMM-Lee and KT21 (malignant), and pairs of merlin-positive or -negative meningioma cells were used to assess sensitivity toward mTORC1 inhibitors in methyl-tetrazolium and bromodeoxyuridine (BrdUrd) assays. The effect of temsirolimus (20 mg/kg daily) on tumor weight or MRI-estimated tumor volume was tested by treatment of eight nude mice (vs. 7 controls) carrying subcutaneous IOMM-Lee xenografts, or of eight (5) mice xenotransplanted intracranially with IOMM-Lee (KT21) cells in comparison to eight (5) untreated controls. Results: All components of the mTORC1 pathway were expressed and activated in meningiomas, independent of their WHO grade. A significant dosage-dependent growth inhibition by temsirolimus and everolimus was observed in all cell lines. It was slightly diminished by merlin loss. In the orthotopic and subcutaneous xenograft models, temsirolimus treatment resulted in about 70% growth reduction of tumors (P < 0.01), which was paralleled by reduction of Ki67 mitotic index (P < 0.05) and reduction of mTORC1 activity (p70S6K phosphorylation) within the tumors. Conclusion: mTORC1 inhibitors suppress meningioma growth in mouse models, although the present study did not measure survival. Clin Cancer Res; 19(5); 1180–9. Ó2012 AACR. Introduction Meningiomas are common neoplasms that arise from the meningeal coverings of the brain or spinal cord. The majority represents surgically resectable tumors corre- sponding to World Health Organization (WHO) grade I (1). On the other hand, atypical (WHO grade II) and anaplastic (WHO grade III) meningiomas are associated with increased morbidity and mortality (2). Following resection of such aggressive tumors or partial resection of meningiomas located at difficult sites, patients are treated usually with radiation. While tumor regression is achieved only in a small number of cases, the majority shows disease stabilization following radiation (3). Thus far, chemother- apy, hormonal, and immunotherapy trials for recurrent meningioma deliver only scant and partly successful results (4). This might be based partly on the still limited knowl- edge of the molecular basis of meningioma development and progression. Among the few well-characterized molecular changes in meningiomas, the most frequent alterations are LOH on chromosome 22 with a bi-allelic inactivation of the NF2 (neurofibromatosis type 2) tumor suppressor gene observed in roughly half of sporadic cases (5). The NF2 gene encodes the cytoskeletal protein merlin. Recently, the mTORC1 (mammalian target of rapamy- cin complex 1) pathway has been reported to interact with merlin as a new negative regulator of cell growth control (6). mTOR is a serine/threonine kinase involved in a signaling pathway controlling transcription, actin cytoskeleton organization, translational activation, and metabolism in response to environmental cues (7). The protein exists in 2 distinct multiprotein complexes. The rapamycin-sensitive complex mTORC1 regulates cell growth and proliferation in response to growth factors and metabolic conditions, whereas the rapamycin-insen- sitive mTORC2 regulates locally restricted growth processes within a cell (7) and is involved in cell Authors' Afliations: Departments of 1 Neuropathology and 2 Genetics & Molecular Neurobiology, Institute of Biology, Otto-von-Guericke Universi- ty; and 3 Laboratory for Non-Invasive Imaging, Leibniz Institute for Neuro- biology, Magdeburg, Germany Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corresponding Author: Christian Mawrin, Department of Neuropatholo- gy, Otto-von-Guericke University, Leipziger Str. 44, Magdeburg 39120, Germany. Phone: 49391-6715825; Fax: 49391-6713300; E-mail: [email protected] doi: 10.1158/1078-0432.CCR-12-1904 Ó2012 American Association for Cancer Research. Clinical Cancer Research Clin Cancer Res; 19(5) March 1, 2013 1180 on March 12, 2019. © 2013 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst February 13, 2013; DOI: 10.1158/1078-0432.CCR-12-1904

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Cancer Therapy: Preclinical

mTORC1 Inhibitors Suppress Meningioma Growth in MouseModels

Doreen Pachow1, Nadine Andrae1, Nadine Kliese1, Frank Angenstein3, Oliver Stork2,Annette Wilisch-Neumann1, Elmar Kirches1, and Christian Mawrin1

AbstractPurpose: To evaluate the mTORC1 (mammalian target of rapamycin complex 1) pathway in menin-

giomas and to explore mTORC1 as a therapeutic target in meningioma cell lines and mouse models.

Experimental Design: Tissue microarrays (53 meningiomas of all WHO grades) were stained for

phosphorylated polypeptides of mTOR, Akt, and the mTORC1 targets 4EBP1 and p70S6K, the latter being

the consensus marker for mTORC1 activity. Expression of proteins and mRNAs was assessed by Western

blotting and real-time PCR in 25 tumors. Cell lines Ben-Men-1 (benign), IOMM-Lee and KT21 (malignant),

and pairs of merlin-positive or -negative meningioma cells were used to assess sensitivity toward mTORC1

inhibitors in methyl-tetrazolium and bromodeoxyuridine (BrdUrd) assays. The effect of temsirolimus (20

mg/kg daily) on tumor weight or MRI-estimated tumor volume was tested by treatment of eight nude mice

(vs. 7 controls) carrying subcutaneous IOMM-Lee xenografts, or of eight (5) mice xenotransplanted

intracranially with IOMM-Lee (KT21) cells in comparison to eight (5) untreated controls.

Results: All components of the mTORC1 pathway were expressed and activated in meningiomas,

independent of their WHO grade. A significant dosage-dependent growth inhibition by temsirolimus and

everolimus was observed in all cell lines. It was slightly diminished by merlin loss. In the orthotopic and

subcutaneous xenograftmodels, temsirolimus treatment resulted in about 70%growth reduction of tumors

(P < 0.01), which was paralleled by reduction of Ki67 mitotic index (P < 0.05) and reduction of mTORC1

activity (p70S6K phosphorylation) within the tumors.

Conclusion:mTORC1 inhibitors suppress meningioma growth in mouse models, although the present

study did not measure survival. Clin Cancer Res; 19(5); 1180–9. �2012 AACR.

IntroductionMeningiomas are common neoplasms that arise from

the meningeal coverings of the brain or spinal cord. Themajority represents surgically resectable tumors corre-sponding to World Health Organization (WHO) grade I(1). On the other hand, atypical (WHO grade II) andanaplastic (WHO grade III) meningiomas are associatedwith increased morbidity and mortality (2). Followingresection of such aggressive tumors or partial resection ofmeningiomas located at difficult sites, patients are treatedusually with radiation. While tumor regression is achievedonly in a small number of cases, the majority shows disease

stabilization following radiation (3). Thus far, chemother-apy, hormonal, and immunotherapy trials for recurrentmeningioma deliver only scant and partly successful results(4). This might be based partly on the still limited knowl-edge of the molecular basis of meningioma developmentand progression.

Among the few well-characterized molecular changesin meningiomas, the most frequent alterations are LOH onchromosome 22 with a bi-allelic inactivation of theNF2 (neurofibromatosis type 2) tumor suppressor geneobserved in roughly half of sporadic cases (5). The NF2gene encodes the cytoskeletal protein merlin.

Recently, the mTORC1 (mammalian target of rapamy-cin complex 1) pathway has been reported to interactwith merlin as a new negative regulator of cell growthcontrol (6). mTOR is a serine/threonine kinase involvedin a signaling pathway controlling transcription, actincytoskeleton organization, translational activation, andmetabolism in response to environmental cues (7). Theprotein exists in 2 distinct multiprotein complexes. Therapamycin-sensitive complex mTORC1 regulates cellgrowth and proliferation in response to growth factorsand metabolic conditions, whereas the rapamycin-insen-sitive mTORC2 regulates locally restricted growthprocesses within a cell (7) and is involved in cell

Authors' Affiliations: Departments of 1Neuropathology and 2Genetics &Molecular Neurobiology, Institute of Biology, Otto-von-Guericke Universi-ty; and 3Laboratory for Non-Invasive Imaging, Leibniz Institute for Neuro-biology, Magdeburg, Germany

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Christian Mawrin, Department of Neuropatholo-gy, Otto-von-Guericke University, Leipziger Str. 44, Magdeburg 39120,Germany. Phone: 49391-6715825; Fax: 49391-6713300; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-12-1904

�2012 American Association for Cancer Research.

ClinicalCancer

Research

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migration. Merlin was shown to enhance the kinaseactivity of mTORC2 (8).The development of effective therapeutic strategies in

meningiomas has been hampered by incomplete under-standing of the signals that influence tumor cell growth.Recent studies showed that activation of the phosphoinosi-tide 3-kinase (PI3K)/Akt/mTORC1 pathway seems to be alucid feature of meningiomas (9–11). Hyperactivation ofAkt is associated with elevated mTORC1 signaling. UsingmTORC1 inhibitors as anti-cancer agents became popularwith the development of derivatives, such as temsirolimusand everolimus that have a more favorable pharmacoki-netic profile (12). In preclinicalmodels, the rapamycin estertemsirolimus has been shown to be effective in variouscancers (13–15), suggesting that temsirolimus is a promis-ing anti-cancer therapeutic (16). In the present study, wedeliver evidence from human meningioma samples, cellculture, and animal studies that the mTORC1 signalingpathway is frequently activated in human meningiomasand that mTORC1 targeting decelerates meningiomagrowth in mouse models.

Materials and MethodsTumor material and immunohistochemical studiesSnap-frozen or paraffin-embeddedmeningioma samples

were retrieved from the files of the Department of Neuro-pathology at the University of Magdeburg (Magdeburg,Germany). Use of the tumor material was approved by thelocal ethical board. ForWestern blotting and real-time PCR,25 meningiomas of different WHO grades were analyzed.Immunohistochemistry was conducted using a tissuemicroarray (TMA) as previously described (17). The TMAcomprised 53meningioma samples: 30 tumorswere grade I(10 meningothelial, 7 fibroblastic, 7 transitional, 3 angio-matous, 1 microcystic, 2 secretory subtypes), 14 were atyp-ical grade II, and 9 were anaplastic grade III meningiomasaccording to the recent WHO classification (1). Thirty TMAtumors were derived from females.The following antibodies were used for immunohis-

tochemistry: phospho-p70S6K (Thr389; Epitomics,1:100), phospho-Akt (Ser473; Santa Cruz; 1: 400), phos-pho-mTOR (Ser2448; Cell Signaling, 1:100), phospho-4EBP (Thr37/46; Cell Signaling, 1:200), Ki-67/Mib-1

(Dako, 1:500), mTOR (Cell Signaling, 1:100), 4EBP1 (CellSignaling, 1:100), Akt (Cell Signaling, 1:400), and p70S6K(LifeSpan Biosciences, 1:100).

Cell cultureThe benign meningioma cell line Ben-Men-1 (18) was

acquired fromWerner Paulus (Neuropathology, Universityof Muenster, Muenster, Germany) and the malignant lineIOMM-Lee from David H. Gutmann (Department of Neu-rology, Washington University School of Medicine, St.Louis, MO). Men and NF2-deficient Men-shNF2 cellsderived from an atypical meningioma, immortalized ACand AC-shNF2 arachnoidal cells, as well as the malignantKT21 meningioma cell line were obtained from Anita Lal(Brain TumorResearchCenter,University ofCalifornia, SanFrancisco, CA; ref, 19). All cells were cultured in high-glucose Dulbecco’s Modified Eagle’s Media (DMEM), sup-plemented with 10% fetal calf serum (FCS) and penicillin/streptomycin. The identity of the cell lines was analyzedusing the AmpFSTR kit and the software GeneMapper IDv3.2 from Applied Biosystems (ABI). While IOMM-Lee andBen-Men-1 exhibit similar and strong expression of merlin,as shown in our laboratory by Western blotting, KT21 cellsdo not express merlin (19).

MTT and BrdUrd assaysCells in microtiter plates (2000/well) were treated for 24

hours with the indicated concentrations of temsirolimus(Torisel, Pfizer) or everolimus (RAD001, NOVARTIS)before standard MTT or bromodeoxyuridine (BrdUrd)assays, as described previously (20). In some experiments,treated cells were in addition irradiated with the indicateddosage of X-rays in aGulmayD3225machine (Gulmay Inc)and analyzed by an MTT assay 24 hours thereafter.

Western blottingAbout 30 mg of human or mouse tumor tissue were

homogenized in 300 mL radioimmunoprecipitation assay(RIPA) buffer (10 mmol/L Tris-HCl, pH 7.4; 150 mmol/LNaCl; 50 mmol/L NaF; 1 mmol/L EDTA; 1% Triton-X-100;0.1% SDS; 0.5% Deoxycholat), supplemented with dithio-threitol (DTT), sodium vanadate, and protease inhibitors.The suspension was centrifuged for 10 minutes at 14,000rpm at 4�C and supernatant used for Western blotting. Toextract proteins from PBS-washed cell cultures, cells wereharvested by scraping in RIPA buffer. Forty micrograms ofprotein per lane was separated by SDS-PAGE and blottedonto a nitrocellulose membrane. Primary antibodies (4�Covernight, dilution 1:1,000) included phospho-p70S6K1(Thr389), p70S6K1, mTOR, phospho-mTOR (Ser2448),4EBP, phospho-4EBP (Thr37/46) and Akt (all from CellSignaling), as well as phospho-Akt (Ser473) (1:500, SantaCruz) and b-actin (1:1000, Sigma Aldrich). Blots weredeveloped with enhanced chemiluminescence (Millipore).

Real-time PCRRNA isolation from tissue samples was conducted with

TRIzol reagent according to the manufacturer’s instructions

Translational RelevanceMeningiomas are frequent intracranial tumors with-

out a clear effective pharmaceutical treatment optionthus far.We show that ahighpercentageofmeningiomashas an activated mTOR signaling pathway. In an ortho-topic meningioma xenograft mouse model, we providefurther evidence that tumor growth responds unequiv-ocally to early systemic treatment with an mTOR inhib-itor, asmonitoredbyMRI.Ourdatapresent thefirst hintsthat mTOR inhibition may be a suitable additional toolto control meningioma growth in humans after surgery.

mTOR in Meningiomas

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(Invitrogen) and transcribed into cDNA by random prim-ing. SYBR-Green real-time PCR was carried out using anSDS7000 (Applied Biosystems). The difference of Ct valuesbetween gene of interest (GOI) and reference gene glycer-aldehyde-3-phosphate dehydrogenase [GAPDH (DCt)] wastranslated into a relative expression (Er) ofGOI according tothe formula: Er ¼ 1/2DCt. Primers and annealing tempera-tures are listed in the Supplementary Table S1.

Temsirolimus treatment of tumor-bearing nude miceAll experiments were done in accordance with the regula-

tions of animal protection. Fifteen 8- to 10-week-old nudemice (Swiss Nude, Charles River) were injected subcutane-ously on both sides with 3� 106 IOMM-Lee cells in 100 mLPBS/Matrigel (1:1). Two days later, 8 mice were intraperi-toneally (i.p.) injected daily (5 d/wk) with 20 mg/kg tem-sirolimus for 3 weeks. Seven untreated tumor-bearing miceserved as a control group. The tumor volume (V) wasestimated weekly by measurement (caliper rule) of 2 per-pendicular axes according to the formula V ¼ p/6 � a � b2

(a > b). After killing themice (day 21), tumorswere resected,weighed, and either stored for Western blotting (�80�C) orembedded in paraffin for immunohistochemistry.

Sixteen 8- to 10-week-old nude mice were used forsubarachnoidal tumor inoculation with IOMM-Lee cells.The animals were anesthetized i.p. (Rompun/Ketamin) andstabilized in a stereotactic head frame. Two holes weredrilled 2 mm anterior of the bregma and 1.5 mm left andright from the sagittal suture, just deep enough to penetratebone and underlying meninges with minimal alteration ofthe neocortex. Approximately 2.5 � 105 cells in 5 mL PBSwere slowly (1minute) injected per hole to a depth of 1mmwith aHamilton syringe. After 2 days, 8mice received adailydose of 20 mg/kg temsirolimus i.p., whereas the remaining8 received only the diluent PBS (control group). In a secondexperiment with subarachnoidal xenografts, 10 mice wereinjected with the same amount of KT21 cells. Five micewere temsirolimus-treated according to the same schemewhereas 5 animals received PBS.

Meningioma growth was monitored in isoflurane-anes-thetized mice by magnetic resonance imaging at days 2and 9 after inoculation (IOMM-Lee cells) or at days 10 and17 (KT21 cells) using a Bruker Biospec 47/20 scanner (4.7 T,Bruker BioSpin GmbH) equipped with a BGA09 (400 mT/m) gradient system. A 25-mm Litzcage system (DotyScien-tific Inc., Columbus, SC, USA) was used for RF excitationand signal reception. T2-weighted imageswere acquiredby arapid acquisition relaxation enhanced (RARE) sequencewith the following parameters: TR 4000 ms, TE 15 ms, slicethickness 800 mm, field of view 25.6 � 25.6 mm2, matrix256 � 256 (i.e., nominal in plane resolution 100 � 100mm), RARE factor 8, 6 averages, total scanning time for onedirection 12min 48 s. The public domain Java-based imageprocessing and analysis program ImageJ (http://rsb.info.nih.gov/ij/) was used to calculate the tumor volume at days9 (IOMM-Lee) and 17 (KT21). For this volumetric analysis,the tumor was segmented manually in each section and thedetermined areamultiplied by the slice thickness (0.8mm).

Analysis of LOH in the NF2 geneThree DNA markers, one within the NF2 gene and 2

flanking markers, were analyzed by PCR using 6-FAM–labeled fluorescent primers and 60�C anneal temperature(21). Primer sequences are listed in Supplementary TableS1. The PCR products were separated on an ABI310Ccapillary sequencer (Applied Biosystems) and alleles weredetermined by automatic comparison to ROX-labeledstandards.

Statistical analysisComparisons of multiple drug concentrations in vitro

were conducted by ANOVA, followed by Tukey post hoctest. The Mann–Whitney U test was used to analyze differ-ences of final tumor weight, final tumor volume, and Ki67index between treated and untreated animals to analyzeMRI-estimated tumor volumes and real-time PCR datawhile Pearson correlation coefficient was calculated toquantify correlations between mRNA levels. Significancewas assumed for P � 0.05. All calculations were conductedusing SPSS, Version 16.0.

ResultsThe mTOR pathway is activated in meningiomasirrespective of the grade of malignancy

Analyzing a TMA containing 53 meningioma samplesas well as tumor-free meningeal tissue, we found thatthe majority of tumors were immunopositive for phos-pho-mTOR and for the phosphorylated forms of themTORC1 substrates p70S6K and 4EBP (Fig. 1A, Table1). Phosphorylation of Akt, a kinase upstream ofmTORC1, was frequently seen. The RNA expression levelsof mTOR, p70S6K, and 4EBP were found not to bechanged between grade I and II/III meningiomas (Fig.1B). Western blot analyses using meningioma lysatesconfirmed the general activation of mTORC1 signalingin meningiomas (Fig. 1C).

Inhibition of meningioma cell proliferation bymTORC-1 inhibitors

As shown for temsirolimus-treated IOMM-Lee cells in Fig.2A, the inhibitor reduced cell density in a dose-dependentmanner. MTT assays revealed significantly reduced cellviability in both IOMM-Lee and Ben-Men-1 cells (Fig. 2B;comparable data for everolimus are not shown). Meningi-oma cell proliferation measured by BrdUrd could be sig-nificantly suppressedbyboth temsirolimus and everolimus,with stronger effects in IOMM-Lee than in Ben-Men-1 cells(Fig. 2C). Finally, on the basis of regularly applied radiationtherapy following resection of aggressive or invasivemenin-giomas,wewonderedwhether irradiation of the cells wouldsynergistically act with mTORC1 inhibition. As shownin Fig. 2D, the percentage of cells surviving a combinedtreatment of temsirolimus and irradiation (5 Gy) wasusually lower than the percentage of surviving cells undertemsirolimus alone, if data were normalized to the corre-sponding drug-free control. Although all treatments exhib-ited a significant decline of viable cells, a statistically

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significant interaction between drug and irradiation couldnot be proven.

Meningioma growth inhibition by temsirolimus in vivoFollowing the confirmation of a functionally active

mTORC1pathway inmeningioma cells, we used 3 differentxenograft models to evaluate meningioma tumor growthduring intraperitoneal temsirolimus treatment. As shownin Fig. 3A and in Supplementary Fig. S1, subcutaneouslyimplanted IOMM-Lee cells showed a clearly reduced growth

in the treatment group (8 mice) compared with untreatedanimals (7 mice) and a significantly reduced weightof explanted tumors from sacrificed mice. Histologic anal-yses of tumor explants showed reduced cell density andlower mitotic activity in the temsirolimus group (Fig. 3B).Assessing the proliferation activity using Ki67 immunohis-tochemistry, a significant reduction of proliferation wasevident (Fig. 3C). Phosphorylation of p70S6K (the consen-sus parameter for mTORC1 activity) was reduced (Fig. 3D),confirming the systemic effect of i.p. temsirolimus

Figure 1. Expression and activationof mTORC1 signaling pathway inmeningiomas. A, analyzing a TMAcontaining 53 human meningiomasas well as normal meninges,immunohistochemistry revealsphosphorylation of mTOR, p70S6K,4-EBP, and Akt in meningiomas.Note that nontumorous meninges (�)are always devoid of staining. B,expression analysis of mTOR,p70S6K, and 4-EBP mRNA inmeningiomas of different grade ofmalignancy. Mean � SEM are given.The comparison of WHO grade I withmeningiomas of higher grades (Utest) for any of these transcripts didnot reveal a significant differencebetween both groups. The group ofhigh-grade meningiomas withsufficient RNA quality could not befurther split into grades II and III dueto low sample numbers. C, Westernblot analyses show overall strongexpression and activation of mTORsignaling molecules in humanmeningiomas of grade I and II.

C

B

A Grade I Grade II Grade IIIMeninges

P-mTOR

P-p70S6K

P-4EBP

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P-Akt

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(N = 10)

WHO grade

II/III (N = 10)

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WHO grade I

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WHO grade

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NA

exp

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0

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0.001

0.0015

0.002

WHO grade

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WHO grade

II/III (N = 10)

Rela

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P m

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A e

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nmTOR p70S6K 4EBP

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β-Actin

Grade I Grade II

mTOR in Meningiomas

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treatment.We also observed increased Akt phosphorylationin the treatment group, an adverse effect already describedafter mTORC1 inhibition (22).

However, subcutaneous meningioma growth might notbe representative for the situation in human patients.Therefore, we used an orthotopic model with IOMM-Leecell implantation in the subarachnoid space of nude mice(21). MRI was suitable to monitor tumor growth (Fig. 3E)and it was clearly evident that systemic temsirolimusapplication reduced intracranial tumor growth signifi-cantly (8 treated vs. 8 untreated mice) if assessed byvolumetric MRI imaging analysis (Fig. 3F). The tumorsshowed growth characteristics highly reminiscent of thesituation in human patients, with tumor spreading in thesubarachnoidal space seen in histologically processedxenografts (Fig. 3G, top). Moreover, hematoxylin andeosin (H&E) sections clearly showed reduced mitoticactivity in temsirolimus-treated orthotopic meningiomacell grafts (Fig. 3G, bottom). Finally, we could confirmeven for the orthotopic model that explanted tumors inthe treatment group had strongly reduced phosphoryla-tion of p70S6K and mTOR, supporting specific mTORC1pathway inhibition in meningioma cells following sys-temic temsirolimus treatment (Fig. 3H). We repeated theintracranial xenograft model with the in vivo slowly grow-

ing malignant meningioma cell line KT21, resulting in amean tumor volume of only 1.26 � 0.25 mm3 in thecontrol group at day 17 (five mice) as estimated by MRI(Fig. 3J). However, this volume exceeded significantly (P< 0.01) the volume in 5 temsirolimus-treated mice (0.36� 0.037 mm3). The dose-dependent sensitivity of KT21cells toward 1, 2, or 10 mmol/L temsirolimus (Fig. 3I) invitro (MTT assay) was significant (P < 0.01) and similar tothat of IOMM-Lee and Ben-Men-1 cells.

mTORC1 inhibition in meningioma cells depends onthe NF2 function

More than 50% of human sporadic meningiomas harboralterations in theNF2 tumor suppressor gene (5). Thus, wewondered whether mTOR inhibition by temsirolimus oreverolimus might be modulated by NF2/merlin. We usedcells with short hairpin RNA (shRNA)-mediated NF2knockdown which have been characterized extensively(19). As shown in Fig. 4A, both immortalized arachnoidal(AC) andmeningioma (Men) cells stably transformed withNF2-shRNA constructs had reducedmerlin levels indicativeof effective NF2 downregulation. Merlin-deficient menin-gioma cells showed a distinctmorphology (shown forMen/Men-shNF2 in Fig. 4B) and responded differently to temsir-olimus treatment. Assessing the proliferation activitysuggested that meningioma cells lacking merlin are lesssensitive to temsirolimus or everolimus treatment, respec-tively (Fig. 4C). Comparable data were found for AC/AC-shNF2 cells (data not shown). This effect was at least partlydependent on cell density, as shown for everolimus in Fig.4D. Driven by these observations, we analyzed by Westernblotting whether a differential activation of the mTORpathway may occur in MEN and MEN-shNF2 cells. How-ever, no difference of mTOR or p70S6K phosphorylationstatus could be observed between the 2 lines, suggestingthat a moderately decreased sensitivity of merlin-deficientmeningioma cells toward temsirolimus does not rely uponmTOR signaling in this model. We further analyzed humanmeningioma samples for NF2 mRNA expression and forthe presence of NF2 LOH. There was indeed a moderatepositive correlation (r ¼ 0.69, P < 0.01) between theexpression ofNF2 and p70S6KmRNA, although the samplesize analyzed was small. We also re-evaluated the TMA forphospho-p70S6K immunoexpression in tumor sampleswith knownNF2 status. Among these 22 tumors, the groupwithNF2wild-type did not differ from theNF2 LOH groupwith respect to phospho-mTOR and phospho-4EBP expres-sion (Table 2). In contrast, the percentage of phospho-p70S6K immunopositive tumors (the consensus indicatorof mTORC1 activity) was higher within the NF2 LOHgroup, supporting a relation between NF2/merlin andp70S6K as recently suggested (6). However, this issue needsdeeper investigation in following studies.

DiscussionIn the present study, we found that mTORC1 is activated

in themajority ofmeningiomas and that systemicmTORC1inhibition can impairmeningioma tumor formation in vivo.

Table 1. Summary of immunoexpressionpatterns of mTOR and related proteins in 52meningiomas

WHO grade I(n ¼ 30)

WHO gradeII (n ¼ 13)

WHO gradeIII (n ¼ 9)

Phospho-p70S6K� 13 (43) 4 (31) 2 (22)þ 15 (50) 9 (69) 7 (78)þþ 2 (7) 0 (0) 0 (0)þþþ 0 (0) 0 (0) 0 (0)

Phospho-mTOR� 10 (33) 7 (54) 2 (22)þ 5 (17) 3 (23) 5 (56)þþ 7 (23) 3 (23) 2 (22)þþþ 8 (27) 0 (0) 0 (0)

Phospho-4EBP� 17 (57) 6 (46) 2 (22)þ 10 (33) 6 (46) 3 (33)þþ 3 (10) 1 (8) 1 (11)þþþ 0 (0) 0 (0) 3 (33)

Phospho-Akt� 3 (10) 0 (0) 0 (0)þ 7 (23) 0 (0) 3 (33)þþ 5 (17) 3 (23) 1 (11)þþþ 17 (57) 10 (77) 5 (56)

NOTE:�, no expression;þ, weak expression;þþ, moderateexpression;þþþ, strong expression. Percentages are givenin brackets.

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Different signaling pathways have been studied in menin-giomas in the last few years. PI3K/Akt activation has beenreportedby several groups (10, 23–25). Akt iswell known to

be an upstream element ofmTORC1 (7) and to be activatedin meningioma cells by platelet-derived growth factor(PDGF; ref. 9). PDGF also induces phosphorylation of

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Figure 2. mTORC1 inhibition substantially affects meningioma cell survival and proliferation in vitro. IOMM-Lee meningioma cells show marked morphologicchanges after application of 1, 2, or 10 mmol/L temsirolimus (A). Temsirolimus significantly reduces cell viability in IOMM-Lee and Ben-Men-1 cells in adose-dependent fashion (���, P < 0.001; B). Assessment of proliferation activity by BrdUrd assay shows significant inhibition by everolimus (left) ortemsirolimus (right; ���,P<0.001)with stronger effects onmalignant IOMM-Lee cells thanonBen-Men-1 cells (C). Concomitant irradiationwith 5Gy enhancesthe significant dosage-dependent effects of temsirolimus (���, P < 0.001) at concentrations of 1 and 5 mmol/L in both IOMM-Lee and Ben-Men-1cells (D). Asterisks (���) in all parts of the figure refer to the highly significant differences between any analyzed treatment and the corresponding untreatedcontrol, as seen in ANOVA followed by Tukey post hoc test.

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Figure 3. Temsirolimus significantly reduces meningioma tumor growth in vivo. Nude mice were intraperitoneally treated with temsirolimus as described inMaterials and Methods or left untreated either following subcutaneous (A–D) or orthotopic intracranial (E–H) implantation of 3� 106 (subcutaneous model) orbilaterally 2.5 � 105 (intracranial model) IOMM-Lee meningioma cells. For verification of growth inhibition with another cell line, intracranial xenografts ofKT21cellswere used (J),which hadshownsimilar sensitivity toward temsirolimus in vitro (I). In theorthotopicmodel, the control group received i.p. injections ofthe solvent PBS precisely following the time schedule of temsirolimus injections. Subcutaneously implanted meningiomas have reduced tumor size (A)following temsirolimus treatment, as determined at day 21 by U test. The treated tumors, prepared from sacrificed animals at day 21, show reduced mitoticactivity (B) and reduced proliferation rate as assessed byKi67 staining (U test; C). The specificmTOR inhibition is shownbyWestern blotting showing reducedphosphorylation of mTOR and p70S6K in tumors from treated animals (D). In an orthotopic model with meningioma cells grown in the subarachnoidalspace, MRI shows reduced tumor extent and tumor volume at day 9 (U test) in systemically treated animals (E and F). The tumors show histologically reducedmitotic activity (G) and reduced phosphorylation of mTOR and p70S6K by Western blotting (H), confirming the specificity of mTORC1 inhibition bytemsirolimus in this model [arrow in G (b) indicates tumor growth and arrows in G (c) indicate mitotic figures]. I, the significant in vitro response of KT21meningioma cells towards temsirolimus (ANOVA and Tukey post hoc test), whereas J shows a significant MRI-estimated growth suppression of this secondmeningioma line in the intracranial model at day 17 (U test).

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p70S6K, the expression of which was reported to beincreased in malignant meningiomas (26, 27). p70S6Ktransfection of malignant IOMM-Lee cells resulted inincreased tumor size in mice (27). However, the impact ofthe mTORC1 pathway for potential treatment of meningi-omas remained unclear.We observed an activation of this pathway in menin-

giomas of all WHO grades. This suggests that not only therare group of anaplastic meningiomas but also the com-mon WHO grade I tumors might be a meaningful targetfor mTORC1 inhibitors. Their successful use in slowlygrowing subependymal giant cell astrocytoma (SEGA) inpatients with Tuberous’ sclerosis implies that mTORC1targeting might not be restricted to rapidly growingcancers (28).

On the basis of our finding of frequent mTORC1 activa-tion in meningiomas, we analyzed the biologic effects ofeverolimus and temsirolimus onmeningioma cell viability.We could clearly show that both inhibitors were effective inreducingmeningioma cell viability andproliferation.More-over, evidences were found that the NF2 gene status mayaffect the response to both inhibitors. This result could notbe explained by differentially activated mTOR pathways in2 isogenic meningioma cell lines with and without merlinexpression, although a functional interaction betweenthe NF2 product merlin and mTORC1 had recently beenshown (6).

We next analyzed the effects of temsirolimus in threedifferent nude mouse models. A strong reduction ofsubcutaneous tumor growth and a clear growth

Figure 4. mTOR inhibition inmeningioma cells depends on theNF2 status. A, arachnoidal (AC) ormeningioma cells (Men) with shRNA-mediated downregulation of the NF2gene product merlin were used. Theygrow differentially and show markedmorphologic changes aftertemsirolimus application (B). Cellularsurvival following temsirolimus oreverolimus is significantly reducedfor all concentrations tested(���, P < 0.001), but merlin-deficientcells are clearly less responsivecompared to wild-type cells (C).Asterisks in (C) indicate a statisticallysignificant difference to untreatedcontrols, as seen in ANOVA followedby Tukey post hoc test. Differencesbetween NF2-positive or -negativecells treated with identicaleverolimus concentrations becameevident, if survival data of MEN orMEN-shNF2 cells seeded at identicaldensity and treated with 3 differentdrug concentrations were analyzedby ANOVA and Tukey post hoc test.Among these 6 columns shown perseeding density, the indicateddifferences between the cell typesoccurred, besides effects of theeverolimus dosage (not indicated inthe Figure; D).

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inhibition in the subarachnoidal space using the ortho-topic model were observed, although it must be statedthat a meaningful estimation of tumor volumes in theorthotopic model was only possible at a single timeinterval after inoculation, whereas no tumor had beendetectable after the second (shorter) time interval chosen.The determination of a precise growth kinetics was there-fore not possible. Nevertheless, together with the mini-mal adverse effects of temsirolimus in humans, theseresults are first hints, which suggest that systemicmTORC1 inhibition may be a suitable strategy to treatpatients with meningioma. However, it should be empha-sized that the sole intention of the present study was tomeasure growth inhibition by early application of a hightemsirolimus dosage, whereas survival times of mice werenot measured. This fact limits the conclusions to bedrawn from the study about clinical applications.

The specific inhibition of mTORC1 kinase activity insubcutaneous and orthotopic tumors was confirmed byWestern blotting. Interestingly, the decrease of p70S6K-phosphorylation was accompanied by an elevated phos-phorylation of Akt, suggesting enhanced activity of PI3K.It has been observed that mTORC1 is able to attenuatereceptor tyrosine kinase (RTK) signaling via PI3K and Akt,thereby creating a negative feedback loop. For insulinreceptors and insulin-like growth factor I (IGF-I) recep-tors, this goal is achieved by mTORC1-dependent down-regulation of insulin responsive substrate 1 (IRS-1), anessential coupling protein connecting these 2 types ofRTKs with intracellular signaling cascades. This negativefeedback loop may be hampered by targeting mTORC1,thus favoring the activity of the PI3K/Akt system (22).Because of its well-known function as a PI3K antagonist,the tumor suppressor PTEN may also influence mTORC1activity via Akt, but PTEN is rarely mutated or deleted inmeningiomas.

Taken together, our data implicate that mTORC1 inhi-bition might be a reasonable strategy to treat patients with

meningioma in need of concomitant chemotherapy tocontrol residual meningioma growth after surgery. More-over, our orthotopic model shows that systemically appliedtemsirolimus has specific effects in tumors growing in thesubarachnoidal space.

Disclosure of Potential Conflicts of InterestC. Mawrin received a commercial research grant for funding for consum-

ables from Pfizer. No potential conflicts of interest were disclosed by theother authors.

Authors' ContributionsConception and design: D. Pachow, E. Kirches, C. MawrinDevelopment of methodology:D. Pachow, N. Andrae, N. Kliese, O. Stork,E. Kirches, C. MawrinAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): D. Pachow, N. Kliese, F. Angenstein, A. Wilisch-Neumann, C. MawrinAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): D. Pachow, E. Kirches, C. MawrinWriting, review, and/or revision of the manuscript: D. Pachow, E.Kirches, C. MawrinAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): N. Andrae, O. Stork

AcknowledgmentsThe technical expertise of Ines Schellhase and Sandra Hartmann and

experimental support through Dr. Jorge R. Bergado-Acosta is highly appre-ciated. NOVARTIS kindly provided everolimus. The authors thank CorneliusCalkhoven (FLI Jena, Germany) for helpful discussion and Werner Paulus(Muenster), David Gutmann (St. Louis), and Anita Lal (San Francisco) forproviding cell lines.

Grant SupportThe work was supported by the Deutsche Krebshilfe (grant no. 108987)

and theGermanResearch Foundation (SFB854TP10 toO. Stork). Additionalsupport was given by a research grant from Pfizer Inc.. The meningiomaresearch of C. Mawrin is further supported by the Wilhelm Sander Stiftung(grant no. 2010.017.1).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received June 13, 2012; revised November 20, 2012; accepted December5, 2012; published OnlineFirst February 13, 2013.

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Phospho-p70S6K Phospho-mTOR Phospho-4EBP

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NF2 LOH (n ¼ 12) Immunopositive 10 (83%) 8 (67%) 5 (50%)Immunonegative 2 (17%) 4 (33%) 5 (50%)

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