TAE226-mediated inhibition of focal adhesion kinase interferes with ...

Invest New Drugs (2010) 28:825–833 DOI 10.1007/s10637-009-9326-5

PRECLINICAL STUDIES

TAE226-mediated inhibition of focal adhesion kinase interferes with tumor angiogenesis and vasculogenesis Alexander Schultze & Sebastian Decker & Jasmin Otten & Andrea Kristina Horst & Gabi Vohwinkel & Gunter Schuch & Carsten Bokemeyer & Sonja Loges & Walter Fiedler

Received: 17 August 2009 / Accepted: 10 September 2009 / Published online: 26 September 2009 # Springer Science + Business Media, LLC 2009

Summary Neoangiogenesis plays an important role in tumor growth and metastasis. Evaluation of new antiangiogenic targets may broaden the armament for future therapeutic concepts. Focal adhesion kinase (FAK), expressed in endothelial and tumor cells, is essential for adhesion and mobility of adherent cells. In the current study we analyzed the anti-angiogenic properties of the FAK inhibitor TAE226 on the proliferation of blood outgrowth endothelial cell (OEC) and differentiation of endothelial progenitor cells (EPC), derived from peripheral blood CD133+ cells, tube formation and on neovascularization in a HT29 xenotransplant model. The effects of TAE226 were compared to those of the rapamycin analogue RAD001. The combination of both drugs was also studied. We showed that HT29 tumor cells and OEC were most sensitive to the action of TAE226 compared to EPC in vitro. In contrast, RAD001 Alexander Schultze and Sebastian Decker contributed equally A. Schultze : S. Decker : J. Otten : G. Vohwinkel : G. Schuch : C. Bokemeyer : S. Loges : W. Fiedler University Medical Center Hamburg-Eppendorf, Hubertus Wald University Cancer Center Hamburg, Dept. of Oncology/Hematology with sections BMT and Pneumology, Hamburg, Germany A. K. Horst Department of Clinical Chemistry, Center of Clinical Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany W. Fiedler (*) University Medical Center Hamburg-Eppendorf, Hubertus Wald University Cancer Center Hamburg, Dept. of Oncology/Hematology with sections BMT and Pneumology, Martinistrasse 52, 20246 Hamburg, Germany e-mail: [email protected]

affected the proliferation of both types of endothelial cells stronger than that of HT29 cells. Furthermore we could show that TAE226 inhibited tube formation in a dose dependent manner. In a HT29 subcutaneous tumor model TAE226 and RAD001 diminished MVD at commonly employed doses to a similar degree. Combination of both compounds did not show synergy in vitro or in vivo. Since TAE226 has been shown to inhibit the PI3 kinase, Akt kinase, mTor pathway, addition of RAD001 may not increase this effect. In conclusion, we have shown that treatment with TAE leads to a reduction of neoangiogenesis in vitro and in a mouse model. The effects are mediated by inhibition of angiogenesis and vasculogenic OEC and EPC. Keywords Vasculogenesis . Angiogenesis . Focal adhesion kinase . TAE226 . OEC . EPC Abbreviations BW Body weight EC Endothelial cell EPC Endothelial progenitor cell FAK Focal adhesion kinase HT29 Colon carcinoma cell line mTOR Mammalian target of rapamycin MVD Microvessel density OEC Outgrowth endothelial cell RAD001 Small molecule drug inhibiting mTOR TAE226 Small molecule drug inhibiting FAK VEGF Vascular endothelial growth factor

Introduction In the last years the contribution of neoangiogenesis to malignant tumor growth has been recognized. As a result of

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this research effort, the anti—VEGF antibody bevacizumab and two small molecule VEGF receptor inhibitors have approved for clinical use. To further improve anti-angiogenic therapy, new targets on endothelial cells are being identified. Inhibition of these targets may lead to new therapeutic options either as monotherapy or in combination with established antiangiogenic drugs. For the formation of new blood vessels, endothelial cell mobility and adhesion is required. In this regard, Focal Adhesion Kinase (FAK) plays a central role. FAK is a nonreceptor tyrosine kinase that localizes to cellular focal adhesions and is involved in cell contacts with extracellular matrix [1]. FAK plays an important role in tumor cell survival, migration, extravasation, homing and metastasis [2]. Besides its effects on tumor cells, the participation of (FAK) for endothelial tube formation has been demonstrated in vitro [3–5]. Therefore, FAK inhibition may exert its anti-tumor activity by direct action on tumor cells and by affecting neoangiogenesis. Inhibition of FAK function is therefore a new, attractive therapeutic option in oncology. TAE226 is a novel small molecule ATP competitive inhibitor of FAK. It has demonstrated anti-tumor activity in experimental models in ovarian cancer, gliomas, esophageal and breast cancer [6–9]. In the current study, we investigated the ability of TAE226 to reduce endothelial cell proliferation and viability of blood outgrowth endothelial cells (OEC) and the in vitro differentiation capacity of EPC as well as its effect on revascularization in a HT29 xenotransplant model. The effects of TAE226 were compared to those of RAD001, a m-Tor inhibitor. The parent compound rapamycin had already been shown to possess anti-angiogenic properties [10]. In addition the combination of both agents was studied.

Materials and methods Isolation and culture of primary cells and cell lines For isolation of endothelial progenitor cells (EPC), aliquots of leukapheresis products from G-CSF primed healthy donors or cancer patients were obtained from the Institute of Transfusion Medicine, University-Hospital HamburgEppendorf, after informed consent of the donors and after approval of the local ethics committee. EPC derived endothelial cells (EC) were differentiated from CD133+ cells of leukapheresis products as previously described [11]. Peripheral blood outgrowth endothelial cells (OEC) were isolated from peripheral blood samples from random, healthy donors which were also obtained in an anonymous fashion from the Institute of Transfusion Medicine, University Hospital Hamburg-Eppendorf, and cultured as

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described [12]. Ten OEC clones were analyzed for expression of CD45 and endothelial markers (CD144, CD146 and CD31) by flow cytometry. All clones were negative for CD45 (80%) as already shown [13]. Human Fibroblasts were a kind gift of J. Brandner (Department of Dermatology, University Hospital HamburgEppendorf) and cultured as adherent cells in Dulbecos Modified Eagles Medium supplemented with 20% Fetal Bovine Serum (both Gibco BRL, Karlsruhe, Germany). The human colon cancer cell line, HT29, was bought from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany, # DSM ACC 299). HT29 cells were grown in Roswell Park Memorial Institute 1640 medium (Gibco) supplemented with 20% Fetal Bovine Serum. Immortalized Human Microvascular Endothelial Cells-1 (HMEC-1) were provided within a material transfer agreement by the Centre for Disease Control and Prevention (CDC, Atlanta, GA, USA) to A.K.H. and were kept in Maintainance of Endothelial Cell Growth Medium, MV2 medium (Promocell, Heidelberg, Germany), including endothelial growth supplements. All cells were cultured under standard conditions (37°C, 5% CO2 in a humidified atmosphere). Reagents The drugs, TAE226 and RAD001, were generously provided by Novartis Pharma AG (Basel, Switzerland). For in vitro use TAE226 and RAD001 were dissolved in DMSO (Sigma Aldrich, Steinheim, Germany) to a concentration of 30 and 20 mmol/L respectively and were stored at −20°C. Further dilution was done with Dimethyl sulfoxide (DMSO) and culture medium of the respective cells resulting in a final DMSO concentration of 0.1% (v/v). Medium containing 0.1% (v/v) DMSO served as mock control. For in vivo use reagents were diluted with 0.5% methyl cellulose in water to a final volume of 100 µl. DMSO content did not exceed 0.1% (v/v). Expression analysis with PCR Expression of the targets of TAE226 (Focal Adhesion Kinase) and of RAD001 (mTOR) were evaluated by RTPCR for all primary cells and the colon cancer cell line HT29 using the following primer: FAK forward 5′ - GTGA GGCGTGGGAGGAAG - 3′ and FAK reverse 5′ - AGTTG GGGTCAAGGTAAGCA -3′ to detect both isoforms of FAK and mTOR forward 5′ - TAGCAATGTGAGCG TCCTG - 3′ and mTOR reverse 5′ - TCAGTTGGTCATAG AAGCGAGT - 3′ to prove expression of mTOR. Therefore

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cDNA of 2×106 cells was prepared using the RNeasy MiniKit (Qiagen, Hilden, Germany) and Ready-To-Go You-Prime First-Strand-Beads (GE Healthcare, Buckinghamshire, UK) according to the manufacturer´s instructions using random hexanucleotides (Invitrogen, Karlsbad). In vitro treatment of cells with TAE226 and RAD001 To determine the effect of Focal Adhesion Kinase Inhibitor TAE226 and mTOR Inhibitor RAD001 (Novartis, Basel, Switzerland) on proliferation of OEC, HT29 cells and fibroblasts, cells were incubated for 1 week with different concentrations of one or both drugs. After 28 days of expansion CD133+ EPC were differentiated for 1 week in absence or presence of TAE226, RAD001 or the combination to determine effect on differentiation of EPC to EC. Adherent EC were counted at the end of the differentiation period (1 week). For all experiments, 17,500 cells were seeded into each well of 8-well chamber slides in 400 µl of culture medium 1 day before incubation with inhibitors. Medium with inhibitor was replaced daily. Data represent 3 independent experiments, each performed in duplicate. After 8 days of treatment cells were trypsinized and viable cells, identified by trypan blue exclusion, were counted in a Neubauer chamber. Tube formation assay HMEC-1 grown to 80% confluence were harvested following trypsinization and counted. 10 000 cells/well were seeded onto Matrigel (BD Biosciences, Heidelberg, Germany) in 96 well plates. Twenty four hours after addition of MV2 medium supplemented with final concentrations of TAE226 ranging from 0,01 to 10 μM, cells were fixed by addition of 4% paraformaldehyde (PFA) and tube formation was documented by photography using a digital camera (CANON EOS) mounted on a ZEISS Axioplan inverse microscope. Data represent 3 independent experiments, each performed in duplicate. In vivo treatment with TAE226 and RAD001 All animal experiments were carried out consistently with the institutional guidelines for the welfare of animals in experimental neoplasia and were approved by the local licensing authority (Behörde für Soziales, Gesundheit, Familie, Verbraucherschutz; Amt für Gesundheit und Verbraucherschutz, Hamburg, Germany, project no. 66/07). The experiment was supervised by the institutional animal welfare officer. Male, 6 to 8 weeks old and pathogen free severe combined immunodefiency mice were purchased from

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Taconic (Lille Skensved, Denmark). Animals were kept under aseptic conditions in individually ventilated cages and were provided with sterile water and food ad libitum. Tumor inoculation and gavage of drugs were carried out using a sterile laminar flow hood. On day 1, 1×106 HT29 colon cancer tumor cells were injected s.c. on the flanks of the animals. For s.c. injection mice were anaesthesized with a 80% carbon dioxide / 20% oxygen medical gas mixture. Treatment with drugs began 1 day after inoculation of tumor cells. Schedules for treatment by gavage were 5 times a week with 2 days off for TAE226 (dose range per gavage: 12.5–100 mg/kg body weight (BW)). Between 0.625–5 mg RAD001/kg BW were given twice weekly. Dosage of drugs was as previously described to have an anti-tumor effect in vivo [14–16]. One group of mice (n=6) received both drugs at a dosis of 100 mg TAE226/kg BW (5 times a week) and 5 mg RAD001/kg BW (twice a week) to determine a synergistic or additive effect in vivo. After three weeks of treatment, mice were anaesthesized with a 80% carbon dioxide / 20% oxygen medical gas mixture and sacrificed by cervical dislocation. Tumors were resected and shock frozen in liquid nitrogen. Samples were stored at −80°C until further processing. Effect on neovascularization in vivo Tumor samples were embedded in TissueTec and stored at −80°C. To determine the effect on neovascularization, 7 μm cryosections were fixed with ice cold methanol and airdryed. Specimens were stained with hematoxylin and eosin for histological diagnosis and immunohistochemically for CD31 using a purified rat anti-mouse CD31 antibody and antiRat Ig HRP Detection Kit (both from BD Biosciences, Heidelberg, Germany). Purified Rat IgG 2ak Isotype Antibodies (BD Biosciences, Heidelberg, Germany) served as control. For quantification of microvessel densitiy (MVD) hotspots of angiogenesis within the tumor tissue were identified and vessels were counted by a blinded investigator (S.D.) in four 100 times fields (area per field = 3.142 mm2). MVD was expressed as mean vessel count / mm2. Statistical analysis Descriptive statistics was used to present data as mean +/− standard error. Analysis of statistical significance was done by ANOVA using SPSS 15.0 software (SPPS Inc., Chicago, Illinois). A p-value of 5% or lower was considered to be statistically significant. IC50 values were calculated using CalcuSyn 2.0 software (Biosoft, Cambridge, UK).

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Results Expression of targets Expression of the targets of TAE226 (Focal Adhesion Kinase, 2 isoforms) and the target of RAD001 (mammalian Target of Rapamycin) were examined by RT-PCR. OEC, EPC, HT29 cells and fibroblasts expressed both targets (data not shown). Proliferation and differentiation assay Outgrowth Endothelial cells (OEC) and EPC were treated for 1 week with TAE226, RAD001 or the combination to determine effect of these drugs on proliferation and differentiation. Fibroblasts and tumor cells served as controls. Data represent 3 independent experiments, each performed in duplicate. The numbers of OEC, EPC derived EC, fibroblasts and tumor cells were reduced compared to control by both drugs in a dose dependent manner (Figs. 1 and 2). Sensitivity to TAE226 at 100 nM was highest for HT29 tumor cells and OEC with a fraction of viable cells of 38± 9% and 64±22%, respectively. IC50 values were calculated to be 45 nM and 324 nM, respectively. This reflects the higher sensitivity of OEC at low concentrations of TAE226 compared to the EPC (IC50=943 nM). Differentiation of EPC to EC was less inhibited. Treatment of EPC with 1 mM TAE226 resulted in a reduction of differentiated EC to 23±17 (p=0,003). To study the effect of TAE226 on proliferation of fibroblasts served as a control for nontumor and non-endothelial cell tissues. Fibroblasts were the least sensitive to TAE226 (Fig. 1 and 2) indicating a specific action of TAE226. Treatment with RAD001 resulted in potent inhibition of OEC and EPC compared to fibroblasts and HT29 cells (n=2 and n=3, respectively). EPC were the most sensitive cell type (IC50=0,28 nM) followed by OEC with an IC50 value of 27 nM. HT29 tumor cells were less sensitive (IC50= 123 nM). There was no significant inhibition of proliferation of fibroblasts at 200 nM RAD001. Incubation with 20 nM RAD001 reduced the percentage of viable OEC and EPC derived EC after 1 week of treatment to 48±31% and 36± 34%, respectively. In contrast 100 nM of the drug was needed to achieve a similar inhibition of HT29 cells (49± 25%). Maximal inhibition of HT29 cells was only reached at 40±23% at 1,000 nM. Remarkably, already 200 nM RAD001 were sufficient to inhibit differentiation of EPC to EC potently with only 13±10% of viable cells. Treatment with both drugs did not show a synergistic or additive effect. Simultaneous treatment with 300 nM TAE226 and 150 nM RAD001 decreased proliferation of OEC only by 25%. HT29 tumor cells treated with

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1,000 nM of each drug showed no significant reduction of proliferation (93% viable cells compared to control). IC50 value for combination experiments with fibroblasts was 300 nM of each drug. Effect of TAE226 on tube formation in vitro Tube formation of HMEC1 cells was investigated using increasing concentrations of TAE226 for 48 h. As shown in Fig. 3, treatment with TAE226 resulted in a dose dependent decrease in tube formation. Clear effects on tube formation were seen with 100 nM TAE226 and a total inhibition of tube formation could be achieved with 1,000 nM TAE226. No impact on the viability of the cells could be detected. Effect on revascularization in vivo To determine effect of TAE226 and RAD001 on tumor vessels in vivo we used a human colon cancer model in SCID mice and analyzed microvessel density (MVD) of animals treated with TAE226, RAD001, the combination or placebo. One day after inoculation of HT29 tumor cells we started oral treatment with 12,5, 25, 50 and 100 mg/kg BW TAE226 (5 times a week, n=3), 0,625, 1,25 2,5 and 5 mg/kg BW RAD001 (twice a week, n=3), the combination or placebo (n=3). After three weeks of treatment, mice were sacrificed. Tumors were removed and analyzed. Cryosections of the tumor samples were stained for CD31 to determine MVD (Fig. 4). There was a significant decrease in MVD in the group receiving 5 mg RAD001/kg BW (to 56±15%) and in the group treated with 100 mg TAE226/kg BW (to 72±13%) compared to placebo (p=0,003 and p=0,027 respectively). In a combination experiment mice received 5 mg RAD001/kg BW and 100 mg TAE226/kg BW (same schedules as single agents). There was no significant reduction of microvessel density (86±24% compared to control).

Discussion Neoangiogenesis is required for growth of tumor or metastatic lesions. Two processes involved in vessel formation can be separated: angiogenesis and vasculogenesis. In angiogenesis preexisting vessels contribute to the emerging vascular network. In vasculogenesis, circulating endothelial progenitor cells take part in vessel development in situ. Two types of endothelial progenitor cells have been described: Outgrowth endothelial cells (OEC) and CD133+ EPC [11, 12]. Both cell types are mobilized during the process of neoangiogesis. While CD133+ EPC are derived from the bone marrow, the

Invest New Drugs (2010) 28:825–833 Fig. 1 Proliferation and differentiation of cells treated with TAE226 in vitro. Cells were seeded into 8-well chamber slides (17,500 per well) and were treated for 1 week with different concentrations of TAE226. After 1 week viable adherent cells were counted (by trypan exclusion using a Neubauer chamber) to determine the effect on proliferation of OEC (a), Fibroblasts (d) and HT29 tumor cells (c) as well as the effect on differentiation of EPC to adherent EC (b) through treatment with drugs. Bars indicate percentage of mean cell counts compared to mock control (data represent 3 independent experiments, each performed in duplicate). Indicated p-values (*) are given for the lowest concentration showing a statistically significant difference between treated cells and mock control

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830 Fig. 2 Proliferation and differentiation of cells treated with RAD001 in vitro. Cells were seeded into 8-well chamber slides (17,500 per well) and were treated for 1 week with different concentrations of RAD001. After 1 week viable adherent cells were counted (by trypan exclusion using a Neubauer chamber) to determine the effect on proliferation of OEC (a), Fibroblasts (d) and HT29 tumor cells (c) as well as the effect on differentiation of EPC to adherent EC (b) through treatment with drugs. Bars indicate percentage of mean cell counts compared to mock control (data represent 3 independent experiments, each performed in duplicate). Indicated p-values (*) are given for the lowest concentration showing a statistically significant difference between treated cells and mock control

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Fig. 3 Tube formation assay with HMEC1 cells. 104 HMEC cells per well were seeded into a 96 well plate onto Matrigel and were covered with MV2 medium containing TAE226 at concentrations from 0 to 10,000 nM (10 μM). After 24 h of incubation cells were fixed with 4% PFA and tube formation was assessed by light microscopy (×100 field), bar 10 μm

origin of OEC remains unclear. Since OEC from patients with myeloproliferative diseases such as CML or polycythemia vera do not share the typical genetic features seen in blood and bone marrow cells such as the Philadelphia chromosome or JAK2 mutations, their hematologic origin has been questioned [13, 17]. Our group has developed in vitro assays to analyse anti-angiogenic properties of new targeted compounds [18, 19]. For these assays human OEC are cultured from buffy coat cells from healthy donors and CD133+ EPC are purified from leukapheresis products from G-CSF primed stem cell donors. Expanded EPC are differentiated in vitro to endothelial cells (EC). The effect of targeted drugs on this differentiation process can be evaluated in vitro. Focal adhesion kinase is a non-receptor tyrosine kinase receptor which is expressed by various tumor types including colon-, esophageal- and ovarian cancer. FAK is activated by the beta-subunit of integrins [20]. Genetic inactivation of beta1 integrin or FAK causes early embryonic lethal phenotypes by reducing cellular proliferation and

mobility [21]. After FAK activation intracellular pathways such as the phosphatidylinositol 3′-kinase-AKT pathway become operational and can lead to p53 inhibition [22]. TAE226 is a novel small molecule ATP competitive, bisanilino pyrimidine inhibitor of FAK and IGF-IR signaling [7, 14]. Since activated endothelial cells express integrins such as αVβ3 and FAK, we reasoned that TAE226 may impede angiogenesis in in vitro and in vivo models. We therefore analyzed the effect of TAE226 towards human OEC and EPC in vitro, its effects on tube formation and its impact on neovascularization in a HT29 xenotransplant model in vivo. Effects were compared to the antiangiogenic effects of the rapamycin analog RAD001. Rapamycin has shown its anti-angiogenic potency in vivo [10] and also in one of our in vitro models of vasculogenesis [18]. The combination of TAE226 and RAD001 was also studied. In our experiments we could show that TAE226 has dose dependent activity on the proliferation of OEC and HT29 tumor cells whereas EPC were less sensitive. Inhibition of

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Fig. 4 Effect of Treatment with RAD001 and TAE226 on neovascularization in vivo. Severe combined immunodefiency (SCID) mice, inoculated subcutaneously with human colon cancer tumor cells (HT29) were treated for 3 weeks with different concentrations of RAD001 and TAE226. Treatment started 1 day after inoculation of the tumor cells. Schedule for RAD001 was twice a week and five times a week for TAE226. Dose ranges were from 0.625 to 5 mg RAD001/kg

BW and 12.5–100 mg TAE226/kg BW, respectively. a After sacrification of mice tumors were immunohistochemically stained for CD31 to determine effect on neovascularization within treatment groups compared to placebo (×50 field), bar 20 μm. b Neovascularization was quantified as mean vessel count / mm2 ±SE, determined from four 100 times fields within hot spots of angiogenesis

OEC and HT29 tumor cells was achieved at concentrations comparable to those described for various tumor cell lines [23]. This is in contrast to the action of RAD001 which inhibits proliferation and viability of both types of endothelial progenitors more efficiently than that of HT29 cells. Both compounds may therefore differ in their mode of action by inhibiting different types of endothelial progenitors to a different extent. Since OEC and EPC have a different origin they may have complementary roles in vasculogenesis. Although the contribution of each cell type to the formation of neovessels remains to be determined, it might be important for the future development of antiangiogenic drugs to have more insight into the cellular target distribution of new compounds. The anti-angiogenic effect of TAE226 is further supported by our experiments which show dose dependent inhibition of tube formation in vitro. These results are in line with experiments describing reduced tube formation after knock-down of FAK in HUVEC [4, 5]. Administration of TAE226 to tumor bearing mice led to a reduction of neovascularization (quantified as microvessel density) in a HT29 xenotransplant model. The impact of

RAD001 on MVD was of slightly higher magnitude than that of TAE226 (46±13% and 60±11%, p=0,035) at doses which are commonly used in mouse models [14]. The difference in efficacy on tumor vessels could possibly be explained with the different sensitivity of OEC and EPC to the drugs. RAD001 inhibited potently both endothelial progenitors (OEC and EPC) in vitro whereas the effect of TAE226 in vitro was stronger against OEC but less potent against EPC. The targeting of both vasculogenic mechanisms (OEC and EPC) could be more effective in reducing MVD than targeting only one vasculogenic cell type. In agreement with our observations, reduction of MVD and endothelial cell apoptosis has already been described after treatment with TAE226 and docetaxel in an ovarian cancer xenotransplant model [6]. In our experiments we did not detect synergy between treatment with TAE226 and RAD001 neither in vitro on endothelial cell proliferation and viability nor on MVD in vivo. One possible explanation might be that both agents target the same pathway. Activation of FAK leads to stimulation of the PI3 kinase, Akt, mTor pathway [22]. Inhibition of FAK should therefore inhibit this pathway. In esophageal cancer cells TAE226 has been shown to reduce

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expression of mTor and Akt and to inhibit phosphorylation of mTor [9]. Consequently, insufficient quantities of activated mTor may be present intracellular to allow effective engagement by RAD001. In summary, we have shown that the anti-neoplastic action of TAE226 is two sided. On one hand it has a direct anti-proliferative action on tumor cells such as HT29 and secondly it impedes angiogenesis and vasculogenesis by interfering with proliferation of OEC, differentiation of EPC, blocking tube formation in vitro and reducing neovascularization in a mouse model. Potent inhibition of both known vasculogenic cell types (OEC and EPC) by RAD001 showed a stronger inhibition of neovascularization than by TAE226 in vivo which could not be explained with a direct effect on tumor cells.

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14. Acknowledgements This research was supported by Novartis Pharma AG, Basel, Switzerland. A.S. receives a bursary of the Werner Otto Foundation for Researchers in Medical Science at the University of Hamburg.

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