Epothilone B inhibits migration of glioblastoma cells by inducing microtubule catastrophes and affecting EB1 accumulation at microtubule plus ends

Biochemical Pharmacology 84 (2012) 432–443

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Epothilone B inhibits migration of glioblastoma cells by inducing microtubule catastrophes and affecting EB1 accumulation at microtubule plus ends Alessandra Pagano a, Ste´phane Honore´ a,b,*, Renu Mohan c, Raphael Berges a, Anna Akhmanova c, Diane Braguer a,b a b c

INSERM UMR 911, Centre de Recherche en Oncologie Biologique et en Oncopharmacologie, Aix-Marseille Universite´, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France APHM, CHU Timone, 264 Rue Saint-Pierre, 13385 Marseille, France Cell Biology, Faculty of Science, Utrecht University, 3584 CH Utrecht, The Netherlands

A R T I C L E I N F O

A B S T R A C T

Article history: Received 3 April 2012 Accepted 14 May 2012 Available online 23 May 2012

Invasion of normal brain tissue by tumor cells is a major contributing factor to the recurrence of glioblastoma and its resistance to therapy. Here, we have assessed the efficacy of the microtubule (MT) targeting agent Epothilone B (patupilone) on glioblastoma cell migration, a prerequisite for invasive tumor cell behavior. At non-cytotoxic concentrations, patupilone inhibited glioblastoma cell movement, as shown by transwell cell migration, random motility and spheroid assays. This anti-migratory effect was associated with a reduced accumulation of EB1 and other MT plus end tracking proteins at MT ends and with the induction of MT catastrophes, while the MT growth rate and other MT dynamic instability parameters remained unaltered. An increase in MT catastrophes led to the reduction of the number of MTs reaching the leading edge. Analysis of the effect of patupilone on MT dynamics in a reconstituted in vitro system demonstrated that the induction of MT catastrophes and an alteration of EB1 accumulation at MT plus end are intrinsic properties of patupilone activity. We have thus demonstrated that patupilone antagonizes glioblastoma cell migration by a novel mechanism, which is distinct from suppression of MT dynamic instability. Taken together, our results suggest that EB proteins may represent a new potential target for anti-cancer therapy in highly invasive tumors. ß 2012 Elsevier Inc. All rights reserved.

Keywords: Microtubule EB proteins Glioblastoma Cell migration Epothilone B

1. Introduction Microtubules (MTs) are ubiquitous cellular polymers that play a critical role in many cellular activities, such as maintenance of cell structure, migration, protein trafficking, chromosome segregation, and mitosis. Although they constitute a robust part of the cytoskeleton, they are highly dynamic. MT plus ends alternate between periods of growth and shortening, which can be intervened by phases of no detectable length changes, corresponding to a ‘‘paused’’ state. Such a process termed MT dynamic instability can be described by a combination of dynamic parameters including the rates of MT growth and shortening, the duration of MT pauses and frequencies of transition from a growing or paused state to a shortening state (an event referred to as ‘‘catastrophe’’), and from a shortening state to a growing or paused state (termed ‘‘rescue’’) [1,2]. The dynamic instability is

* Corresponding author at: Faculte´ de Pharmacie INSERM 911, CS30064, 27 bd Jean Moulin 13385 Marseille Cedex 05, France. Tel.: +33 491835507. E-mail addresses: [email protected] (A. Pagano), [email protected] (S. Honore´), [email protected] (R. Mohan), [email protected] (R. Berges), [email protected] (A. Akhmanova), [email protected] (D. Braguer). 0006-2952/$ – see front matter ß 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bcp.2012.05.010

crucial for MTs to explore intracellular space. Through this search process, MT plus ends can be captured and stabilized at target destinations such as kinetochores of the mitotic spindle and at the cell cortex during cell migration. The search-and-capture of MTs is important for generating an asymmetrical MT array and maintaining cell shape [3–5]. During cell migration, selective stabilization of MT plus ends at the cell cortex results in polarization of the MT array that facilitates cell migration [6]. MT dynamics and stability are spatially and temporary regulated by several MT-Associated Proteins (MAPs) [7]. In particular, the plus end tracking proteins (+TIPs) accumulate specifically at the plus end of growing MTs in a comet-like manner [8]. +TIPs include the EB proteins such as EB1 and EB3, the CAP-Gly proteins CLIP 170, p150glued and SxIP motif containing plus TIPs such as the CLASPs and APC [9–11]. +TIPs such as EB1, CLIP-170, CLASPs and APC have been shown to play crucial role in cell migration by regulating MT dynamics and interacting with the actin cytoskeleton during the process of adhesion and migration [4,12–14]. EB1 is considered as the master regulator at the MT plus end, since it has been shown to act as a loading factor for other MTinteracting proteins both in vitro and in vivo [9,15–17]. In contrast to most other +TIPs, EB1 and its homologs can track growing MT

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plus ends in an autonomous manner and regulate MT dynamic instability in in vitro reconstituted systems [15,18,19]. In cells, EB1 has been described to promote cell migration by stabilizing MT at the cell cortex [12,20]. Moreover, EB1 and EB3 proteins have been described as inhibitors of MT catastrophes promoting persistent MT growth [19,21,22]. MT-targeting agents (MTAs) that can be subdivided into MTstabilizing (e.g. Taxanes) and MT-destabilizing agents (e.g. Vinca alkaloids) are major chemotherapeutic drugs. They inhibit cell proliferation by suppressing the dynamics of spindle MTs, which are essential for proper chromosome separation during cell division. Beside their potent anti-mitotic properties, at very low and non-cytotoxic concentrations MTAs exert anti-migratory and anti-angiogenic activities both in vitro and in vivo, for example, by disturbing endothelial cell migration and differentiation into capillary network [23,24]. We have previously reported that anti-migratory concentrations of MTAs, such as paclitaxel and vinflunine, significantly increase interphase MT dynamic instability in endothelial cells and inhibit MT targeting to peripheral adhesion sites [1,7,25]. Importantly, anti-migratory effects of vinflunine on endothelial cells were associated with the alteration of MT stabilization at the cell cortex and a decrease of EB1 comet length at MT plus ends [1]. The inhibition of cell migration by MTAs has also been described to be associated with suppression in MT dynamic instability; however the precise mechanism of their anti-migratory action remains poorly understood [26]. Glioblastomas are the most frequent primary tumors of the central nervous system. They are very aggressive, highly angiogenic and associated with very bad prognosis. One of the main biological features of glioblastoma is the local invasion of its constituent neoplastic cells into the surrounding brain tissue [27]. This invasive behavior presents a major obstacle to an effective treatment of brain tumors. Moreover, the transient cell cycle arrest in actively migrating glioblastoma cells decreases their sensitivity to the action of cytotoxic drugs. Therefore, it is very important to target glioblastoma cell migration [28]. Although many of the genetic alterations that deregulate the processes of cell growth and death and are involved in tumor initiation have been elucidated in recent years, less progress has been made in understanding cell migration. As a consequence, no specific therapeutic strategies against tumor cell migration are currently available to manage glioblastoma invasion. The Epothilones are a new class of MT stabilizing agents, which bind to the taxane-binding site on the MT and have a strong anticancer activity both in vitro and in vivo. This drug family is active against cancer cells resistant to paclitaxel or to cells developing resistance to taxanes [29–31]. Among the Epothilones, Epothilone B (Patupilone, EPO 906) is able to cross the blood–brain barrier since its activity is fully P-gp independent [32]. Moreover, patupilone is fully active in cells expressing high level of the bIIItubulin isotype [33], which is overexpressed in glioblastomas [34]. For all these reasons, patupilone might be a good candidate for treatment of human gliobastoma, and clinical trials are currently under way. In the present study, we investigated the anti-migratory potential of patupilone and its effects on MT dynamics in glioblastoma cells. At nanomolar, subtoxic concentrations that efficiently inhibited tumor cell motility in several migration assays, patupilone decreased the accumulation of EB1 at MT plus ends as demonstrated by the reduction of both EB1 comet length and fluorescence intensity. Similar effects were also observed for other plus TIPs that concentrate at the plus end in an EB1-dependent manner. We also showed that downregulation of EB1 by RNA interference significantly inhibited cell migration, highlighting the role of EB1 as a master regulator in MT dynamics and cell

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migration. Analysis of MT dynamics revealed that patupiloneinduced alteration of EB1 comets was independent of any effect on MT growth rate when used at non-cytotoxic concentrations. Interestingly, it was associated with an increase in MT catastrophes, which was the only MT dynamic instability parameter that correlated with the inhibition of MT targeting to the cell cortex and inhibition of cell migration. Moreover, patupilone also decreased the accumulation of EB proteins at MT plus ends and induced MT catastrophes in a reconstituted in vitro system demonstrating that the two effects were an intrinsic property of patupilone. Taken together, our results suggest that EB1 family proteins may represent new potential targets for anti-cancer therapy in highly invasive tumors.

2. Materials and methods 2.1. Drugs and cell culture Patupilone (Epothilone B, Sigma–Aldrich, St. Louis, MO) was dissolved in DMSO (Sigma–Aldrich, St. Louis, MO) to obtain a stock solution (10 mM). Paclitaxel was from Novasep Synthesis (Nancy, FR). Human glioblastoma cells (U87MG, ATCC) were routinely maintained at 37 8C and 5% CO2 in EMEM medium, with NEAA (Invitrogen, Carlsbad, CA), containing 10% fetal bovine serum, 2 mmol/L of glutamine, 1% penicillin and streptomycin (Invitrogen, Carlsbad, CA). U87MG cells were used for no more than 15 passages. 2.2. Transwell migration Cells (50,000 per condition) were added to the upper side of a transwell migration chamber (0.8 mm filter, BD Bioscience, San Jose, CA) in serum free EMEM medium (Lonza, Basel, CH) The lower side of the chamber was filled with 10% FCS (Lonza, Basel, CH) in EMEM medium. Cells were allowed to migrate for 5 h; subsequently, the chambers were removed, non-migrating cells on the upper part of the filter were removed with a cotton stick, and the cells on the lower side of the filter were fixed with 1% glutaraldheyde (Sigma–Aldrich, St. Louis, MO) and stained with 1% Crystal-violet (Sigma–Aldrich, St. Louis, MO) solution in 20% MetOH (Sigma–Aldrich, St. Louis, MO). After washing and drying, pictures of the filters were taken with a Nikon TE 2000 microscope (Nikon microscopyU, Melville, NY), equipped with a 10/N.A. 0.45 objective lens, a digital camera (CCD camera coolsnap HQ, Princeton Instruments, Trenton, NJ). Four fields per filter were imaged, and quantification of transmigrated cells was performed using Metamorph software (Universal Imaging Corporation, Downigton, PA). Results were expressed as percent of cells that underwent migration, setting control at 100% (mean  SD). At least four independent experiments were performed. 2.3. Random motility assay Cells were seeded in 24-well culture plates, coated with 10 mg/ ml fibronectin (Sigma–Aldrich, St. Louis, MO). One hour later, the cells were either treated with patupilone (Sigma–Aldrich, St. Louis, MO) at various concentrations or left untreated and subjected to time-lapse video-microscopy. Images were collected every 10 min for 10 h (600 min) with a Nikon TE 2000 microscope (Nikon microscopyU, Melville, NY) equipped with a 10 objective, connected to a digital camera (CCD coolsnap HQ; Princeton Instruments, Trenton, NJ). Random motility parameters were measured as previously described [1]. For each experiment, 30 cells per condition were tracked. Three independent experiments were performed.

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2.4. Cell migration from spheroids Spheroid formation from hanging drops was performed according to the previously described protocols [35]. Briefly, drops containing U87MG cells at the concentration of 2.25  106 cells/ml were suspended from the lids of culture dishes for 24 h. The resulting cell aggregates were transferred to culture dishes basecoated with agar. After 48 h three-dimensional spheroids were transferred, let to adhere on fibronectin (Sigma–Aldrich, St. Louis, MO) coated wells for 1 h and radial migration of cells from spheroids in the presence or absence of patupilone was evaluated after overnight migration (18–20 h). The extent of migration was measured by Metamorph software (Universal Imaging Corporation, Downigton, PA) as the area of migration of the cells from the spheroid after subtraction of the internal area of the spheroid. 2.5. Cytotoxicity assay Cells were seeded in 96-well plates (5000 cells/well). After 24 h cells were treated with the drug. Growth inhibition of U87MG cells was measured after 72 h of drug treatment by using the MTT (3(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide, Sigma–Aldrich, St. Louis, MO) cell proliferation assay, as previously described [36,37]. 2.6. Indirect immunofluorescence analysis Cells were grown on 8-well Labtek II chamber slides (Labtek, Thermo Scientific,Nunc, Roskilde, DK) precoated with fibronectin (10 mg/ml) and incubated with various Patupilone concentrations for 4 h. For actin staining, cells were fixed with 3.7% formaldehyde, permeabilized with 1% saponin, and incubated with phalloidinTRITC (1/2,000; Sigma–Aldrich, St. Louis, MO) for 30 min at 37 8C. For endogenous a-tubulin, EB1, CLIP170, p150glued, CLASP staining, cells were fixed either for 5 min with cold methanol ( 20 8C) or with MetOH-EGTA 1 mM (Sigma–Aldrich, St. Louis, MO) for 20 min at 20 8C, according to the antibody used and incubated with the following primary antibodies: anti-EB1 antibody (clone 5; BD Biosciences, San Jose, CA), rabbit anti-CLIP-170 (a gift of N. Galjart), anti-CLASP-1 (1A6, a gift of N. Galjart). The secondary antibodies used were TRITC-coupled anti-mouse and anti-rabbit secondary antibodies (Jackson Immunoresearch, West Grove, PA). In some experiments cells were also labeled for a-tubulin using a FITCcoupled anti-a-tubulin antibody (clone DM1A; Sigma–Aldrich, St. Louis, MO). After mounting, cells were observed using either a Leica DM-IRBE microscope (Leica microsystem, Wetzlar, D), equipped with a 100/N.A. 1.30 objective lens, or with a Nikon TE 2000 microscope (Nikon microscopy U, Melville, NY), equipped with a 60/N.A. 1.49 objective lens and coupled to a digital CCD camera (Coolsnap HQ, Princeton Instruments, Trenton, NJ) or by a Leica SP5 confocal microscope system (Leica microsystem,Wetzlar, D), equipped with a 100/N.A. 1.3 objective lens. Quantification of EB1 comet length and maximal fluorescence intensity was performed for at least 90 MT ends with Image J software using images of double fluorescently stained cells for both tubulin and EB1 in which tubulin fluorescence was used as an internal control. Quantification of MT tip distance from cell margin was performed for at least 190 MT by using Image J software on confocal images of cells stained for actin and tubulin. Actin staining was used to visualize the cell margin. 2.7. Western blot analysis Cells were lysed in the lysis buffer (Tris 50 mM pH 8.0, NaCl 250 mM Triton-X100 1%; SDS 0.1%, Sigma–Aldrich, St. Louis, MO). 30 mg of total protein lysate was loaded onto a 12% SDS-PAGE gel.

Western blot was performed as previously described [38]. We used as primary antibodies an anti-EB1 antibody (clone 5; BD Biosciences, San Jose, CA 1/500) and anti-a-tubulin (clone DM1A, Sigma–Aldrich, St. Louis, MO 1/1000); and anti-mouse IgG-horseradish peroxidase (Jackson Immunoresearch, West Grove, PA 1/2000) as a secondary antibody. 2.8. Transfection of U87MG cells U87MG cells were transfected by nucleofection according to the manufacturer instructions. Briefly, 6  105 cells were resuspended in 100 ml of the specific Nucleofactor buffer T. (Amaxa system, Lonza, Basel, CH) 2.5 to 5 mg of plasmid DNA (pEGFP-a-tubulin, Clontech Laboratories, Mountain View, CA, and pEB1-EGFP, [39]) was added to the cell suspension, which was transferred to a 2.0 mm electroporation cuvette and nucleofected (program number X-001). After transfection, cells were immediately transferred in EMEM complete medium. Twenty-four hours later, cells were treated for 5 h with patupilone at various concentrations and time-lapse microscopy analysis was performed. 2.9. EB1 silencing Twenty-four hours after plating, U87MG cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) with siRNA for EB1 (Hs_MAPRE1-5) and all STAR control (Qiagen France, Courtaboeuf, FR). Cells were analyzed for protein down-regulation 72 h later by Western blot and immunofluorescence experiments. 2.10. Fluorescent time-lapse video microscopy Transfected cells were placed in mounting medium (RPMI w/o phenol red supplemented with 2 mM glutamine, Na pyruvate, NEAA, FCS 1%, Hepes pH 7.4 15 mM, Invitrogen, Carlsbad, CA, Lonza, Basel, CH), supplemented with 0.1 mg/ml ascorbic acid (Aguettant, Lyon, FR) to reduce photodamage, in a double coverslip chamber maintained at 37 8C. Time-lapse acquisitions for MT dynamics experiments (GFP-tubulin, EB1-GFP, transfected cells) were performed with a Nikon TE 2000 microscope (Nikon microscopyU, Melville, NY) equipped with a 60/N.A. 1.49 objective lens. Thirty-one images per cell were acquired at 4-s intervals using a digital camera (CCD camera Coolsnap FX; Princeton Instruments, Trenton, NJ). 2.11. Analysis of MT dynamics Analysis of MT dynamics in GFP-a-tubulin transfected cells was performed as described previously, using the track point function of the Metamorph software (Universal Imaging Corporation, Downigton, PA) [1]. Changes in MT length exceeding 0.5 mm were considered as growth or shortening events. Length changes of less than 0.5 mm were considered as phases of attenuated dynamics or pauses. The rates of growth and shortening were determined by linear regression. Mean and SEM were calculated per event. The time-based catastrophe frequency was calculated by dividing the number of transitions from growth or pause to shortening by the total time of growth and pause for each individual MT. The distance-based catastrophe frequency was calculated by dividing the number of transitions from growth or pause to shortening by the total length grown for each individual MT. The time-based rescue frequency was calculated by dividing the total number of transitions from shortening to pause or growth by the time spent shortening for each individual MT. The distance-based rescue frequency was calculated by dividing the total number of transitions from shortening to pause or growth by the total length shortened for each individual MT. Mean and SEM of transition

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frequencies were calculated per MT (n = 30 MTs, 4–5 cells, for each experimental condition from three independent experiments). Overall dynamicity was calculated as the total length grown and shortened divided by the life span of the MT population. The parameters of growth length, growth rate and catastrophe frequency were similarly calculated by tracking EB1-GFP tracks displacement over time [40]. 2.12. In vitro plus end tracking assay of GFP-EB3 and measurements of MT dynamics The assay was performed by growing MTs in the presence of 15 mM brain tubulin (Cytoskeleton, Denver, CO) from GMPCPPstabilized (Sigma–Aldrich, St. Louis, MO) and paclitaxel-stabilized (Novasep, Nancy, FR) MT seeds, which were immobilized on coverslips using Poly-L-Lysine PEG-biotin (SuSoS, Du¨bendorf, CH)– streptavidin (Invitrogen, Carlsbad, CA, Lonza, Basel, CH) links, as described previously [41]. MT dynamics was measured by automated kymograph analysis described previously [42]. GFPEB3 rather than GFP-EB1 protein was chosen for the assay because the plus end tracking properties of EB3 are not affected by a Nterminal fluorescent tag, while this tagging strategy inhibits plus end tracking behavior of EB1 [41].

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(10 nM), EB1 comets were strongly affected (73% and 54% reduction of the mean comet length and maximum fluorescence intensity, respectively, as compared to control, p < 0.001, Fig. 2C). The decrease in the accumulation of EB1 at MT plus ends was not associated with any change in EB1 expression as shown by Western blot analysis (Fig. 2D). Interestingly, patupilone also affected the MT plus end tracking of the EB1 binding proteins CLIP170, p150glued and CLASP-1 (Fig. 2S). These results suggest that patupilone may exert its anti-migratory effect by affecting +TIP accumulation at MT plus ends. 3.3. EB1 is involved in glioblastoma cell migration We then investigated whether EB1 is a key protein in U87MG cell migration by using RNA silencing. EB1 siRNA strongly reduced EB1 protein expression, as shown by western blot of total cell lysates (Fig. 3A) and EB1 comet labeling at MT plus ends, as shown by immunofluorescence analysis (Fig. 3B). Downregulation of EB1 significantly affected random cell motility (Fig. 3C, mean velocity: 0.37  0.05 mm/min vs 0.54  0.05 mm/min for EB1 siRNA and control siRNA, respectively, p < 0.05; mean distance to origin, 63.8  8.8 mm vs 106.8  14.7 mm for EB1 siRNA and control siRNA, respectively, p < 0.05), confirming the crucial role of EB1 in glioblastoma cell migration.

3. Results 3.1. Patupilone inhibits glioblastoma cell migration at non-cytotoxic concentrations We set out to determine the range of patupilone concentrations that inhibit glioblastoma cell migration without affecting cell proliferation. For this purpose, we first analyzed the cytotoxic effect of patupilone on glioblastoma U87MG cells. As shown by MTT cell proliferation assay (Fig. 1S), after 72 h of treatment the drug efficiently inhibited cell growth with an IC50 of 6 nM, while concentrations 1 nM were not cytotoxic. We then assessed whether patupilone can inhibit U87MG cell migration by using 3 complementary sets of experiments. As shown in Fig. 1A, patupilone significantly inhibited transwell cell migration at the non-cytotoxic concentration of 1 nM, and the effect was more evident at 10 nM. The random motility behavior of U87MG cells on fibronectin (10 mg/ml) was also analyzed by time-lapse videomicroscopy in the presence or absence of the drug during 10 h (Fig. 1B, left and right panel). The average velocity of cell migration was reduced by 42% at 0.1 nM and by 64% at 10 nM (Fig. 1B, p < 0.001). In order to evaluate the effect of the drug on tumor cell migration in a context mimicking in vivo tumor cell migration from primary tumors, we analyzed radial cell migration on fibronectin from U87MG spheroids [35]. As shown in Fig. 1C, the mean area of migration in control cells was 1.11  0.23  106 mm2. The area of migration was reduced by 36% at 1 nM and by 60% at 10 nM. Taken together, our results indicate that patupilone inhibits glioblastoma cell migration independently of its cytotoxic activity. 3.2. Anti-migratory concentrations of patupilone inhibit the accumulation of +TIPs at MT plus ends As shown by indirect immunofluorescence, EB1 which is concentrated at growing MT plus ends [43] appeared as bright comet-like structures (Fig. 2A, control). Interestingly, patupilone affected EB1 comet length and intensity in a concentrationdependent manner (Fig. 2A and B). The mean comet length and maximum fluorescence intensity were reduced by 35% and 19%, respectively, after treatment with 0.1 nM patupilone; by 59% and 38%, respectively, after treatment with 1.0 nM patupilone, as compared to control (p < 0.001, Fig. 2C). At higher concentration

3.4. Reduction of the amount of EB1 at MT plus end by patupilone is not the consequence of an alteration of the MT growth rate but is associated with increased MT catastrophes We then analyzed the effects of patupilone on MT dynamic instability parameters in GFP-a-tubulin transfected U87MG cells. As shown in Table 1, the overall MT dynamicity and the MT growth rate were not significantly affected by the low anti-migratory and non-cytotoxic concentration of patupilone (1 nM) as compared to control conditions. Interestingly, at this patupilone concentration, only the time-based and distance-based MT catastrophe frequencies were significantly increased compared to control (1.97  0.15 min 1 at 1.0 nM vs 1.50  0.15 min 1 in control, p < 0.05; 0.90  0.10 mm 1 at 1.0 nM vs 0.51  0.05 mm 1 in control, p < 0.01). In contrast, the cytotoxic concentration of 10 nM patupilone clearly suppressed MT dynamic instability, as shown by the strong reduction of MT growth rate (6.4  0.4 mm/min at 10 nM vs 10.1  0.9 in control), shortening rate (7.9  0.4 mm/min at 10 nM vs 14.8  1.3 in control) and overall dynamicity (Table 1). In addition, the distance-based catastrophe frequency was further increased (1.83  0.03 mm 1 in 10 nM vs 0.51  0.05 mm 1 in control, p < 0.001) and correlated with a strong decrease in the mean MT growth length (0.86  0.05 mm in 10 nM vs 1.86  0.16 mm in control, p < 0.001). However, the time-based catastrophe frequency was not statistically different from the control condition because MT growth rate was also significantly reduced. To confirm our results with a complementary approach, we also analyzed MT dynamics by using EB1-GFP transfected U87MG cells (Fig. 4). As we showed above for the endogenous EB1, EB1-GFP formed comet-like structures at MT plus ends (Fig. 4A, Control). In patupilone-treated cells, EB1-GFP signal at MT tips was clearly diminished, but it could still be used as a reliable marker of growing MT ends (Fig. 4A; 1 and 10 nM patupilone). At antimigratory and non-cytotoxic concentration (1 nM), MT growth rate was not significantly affected compared to control cells (Fig. 4B, upper left panel). However, the time- and distance-based catastrophe frequencies were significantly increased (Fig. 4B, upper right and lower left panel) (Fig. 4B, lower right panel). After treatment with 10 nM patupilone, distance-based catastrophe frequency was further increased (Fig. 4B, lower left panel), while time-based catastrophe frequency was not significantly different

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Fig. 1. Patupilone inhibits cell migration of human glioblastoma cells. (A) Percentage of transwell migration of U87MG cells in the presence of patupilone as compared to vehicle alone. Values are expressed as mean  SD of four different experiments (**p  0.01, ***p  0.001 vs control, Student’s t-test). Panels on the right show representative images of transwell bottom sides stained with 0.1% Crystal-violet solution and observed by phase contrast microscopy. (B) Mean velocity of cells incubated with patupilone at various concentrations or with vehicle alone (mean  SEM of 30 cells; one representative experiment is shown; ***p  0.001 vs control, Student’s t-test). Panels on the right show representative tracks of three cells for each condition. (C) Area of cell migration from spheroids in the presence of patupilone or vehicle alone. Values are expressed as mean  SD of four different experiments; *p  0.05, **p  0.01 (vs control, Student’s t-test). Panels on the right show representative images for each condition.

from control due to the suppression of the MT growth rate (Fig. 4B, upper right and left panels). Therefore the results obtained with EB1-GFP transfected cells confirmed those obtained using GFP-atubulin transfected cells, and demonstrated that the distancebased catastrophe frequency and MT net growth are reliable parameters associated with anti-migratory activity of patupilone.

Importantly, such an effect was not restricted to U87MG cells, since we observed a similar effect on HeLa cells transiently transfected with EB3-GFP. Indeed, patupilone affected EB3-GFP comets in a concentration dependent manner from 1 to 10 nM (Fig. 3S-A). Importantly, as visualized on kymographs presented in Fig. 3S-B, patupilone at 1 and 5 nM altered EB3-GFP comets without

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Fig. 2. Patupilone reduces EB1 comet length. (A) Cells were either untreated or treated for 5 h with patupilone at the indicated concentrations, fixed with cold methanol and immunofluorescence detection of EB1 was performed; size bars = 5 mm. (B) Cells were either untreated or treated with patupilone at the indicated concentration, fixed with cold methanol and double immunofluorescence detection of EB1 (red) and a-tubulin was (green) performed. Panels on the right show the intensity of EB1 and tubulin fluorescence and comet length, quantified for representative MT indicated in the images on the left by white arrows; size bars = 5 mm. (C) Quantification of mean comet fluorescence intensity (SEM, circle) and length (SEM, triangle) as a function of patupilone concentrations (n = 100 comets). (D) Cells were treated with patupilone at the indicated concentrations for 5 h. EB1 protein was detected in total lysates by Western blot (30 mg of protein), a-tubulin was used as loading control.

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Fig. 3. Downregulation of EB1 reduces glioblastoma cell migration. (A) Cells were transfected either with siRNA control or EB1 siRNA, and after 72 h total cell lysates (30 mg) were tested for the expression of EB1 and EB3 protein. a-Tubulin was used as loading control. (B) Cells were transfected with siRNA control or EB1 siRNA. After 72 h cells were fixed with cold methanol and immunofluorescence detection of EB1 and EB3 was performed; size bars = 5 mm. (C). Histograms represent the mean velocity (left) and distance to origin (right) of cells transfected with control siRNA or EB1 siRNA (mean  SEM of 30 cells; one representative experiment is shown; *p  0.01 vs control, Student’s t-test).

affecting MT growth rate, since the slope of the EB3-GFP trajectory was unchanged in these conditions. Interestingly, the MT net growth length was strongly reduced indicating an increased catastrophe frequency. 3.5. Patupilone-induced MT catastrophes are associated with a reduced MT density at the cell cortex An increase in MT catastrophe frequency at the cell periphery should be responsible for an inhibition of MT targeting and capture at the cell cortex. Therefore, we analyzed MT organization at the cell cortex in patupilone-treated cells by indirect immunofluorescence analysis of MT and actin cytoskeleton using confocal microscopy. As expected, in control cells MT projected radially toward the cell periphery and MT plus ends reached the cell edge. The mean distance from the MT tips to the cell margin was 2.6 mm, in contrast to patupilone-treated cells, where such distance was increased to 4 and 7.5 mm for 1 and 10 nM patupilone, respectively (Fig. 5A and B). Indeed, 60% of the MT tips in control cells were localized at a distance of less than 2 mm from the cell margin (Fig. 5C). Only 38% and 22% the MTs were able to reach this zone at 1 and 10 nM patupilone, respectively (Fig. 5C). Taken together, these results suggest that the induction of MT catastrophes by patupilone inhibits the ability of MTs to reach the cell cortex,

which might be a likely cause of suppressed cell migration in the presence of the drug. 3.6. Alteration of EB accumulation at MT plus ends and induction of MT catastrophes are intrinsic properties of MT stabilizing agents In order to decipher whether alteration of EBs comet-like accumulation at MT plus end is an intrinsic property of patupilone or whether it is a consequence of intracellular signaling, we analyzed the effects of patupilone in a reconstituted MT tip tracking system in vitro using 75 nM GFP-EB3 (Fig. 6). We have analyzed MT dynamic instability parameters and the mean fluorescence intensity of GFP-EB3 comets at MT plus ends during the growth phases. Patupilone decreased the mean fluorescence intensity of GFP-EB3 comets in a concentration-dependent manner, confirming that patupilone affects EB tip tracking, as observed in cells (Fig. 6A and B). Furthermore, we also analyzed the effect of paclitaxel, the leader of MT stabilizing drugs binding to the taxane-binding site, for which we observed the same effect on EB3 tip tracking (Fig. 6A and B). However, the two drugs differently affected MT dynamic instability parameters (Fig. 6C). Indeed, patupilone slightly suppressed the MT growth rate and increased the MT shortening rate a low concentrations (1 and 10 nM). At 100 nM patupilone, the MT shortening rate was no more affected

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Table 1 Parameters of MT dynamic instability in patupilone-treated cells. Parameters Rate (mm/min  SEM) Duration (min  SEM)

Length (mm  SEM) %Time spent

Catastrophe Rescue Catastrophe Rescue Overall dynamicity (mm/min)

Growth Shortening Growth Shortening Pause Growth Shortening Growth Shortening Pause min 1 min 1 mm 1 mm 1

Control

1.0 nM Patupilone

10.0 nM Patupilone

10.1  0.9 14.8  1.3 0.21  0.01 0.14  0.01 0.33  0.03 1.85  0.16 2.06  0.16 32.41 14.24 53.35 1.50  0.15 8.02  0.60 0.51  0.05 0.75  0.08 5.34

9.0  0.4 15.6  1.2 0.18  0.01* 0.14  0.01 0.31  0.02 1.55  0.13* 2.11  0.16 27.16 16.86 55.97 1.97  0.15* 8.26  0.7 0.90  0.10** 0.79  0.10 5.23

6.4  0.4** 7.9  0.4** 0.15  0.01* 0.14  0.01 0.48  0.03*** 0.86  0.05*** 0.97  0.07*** 16.10 13.33 70.57 1.55  0.13 7.82  0.7 1.83  0.3*** 1.25  0.08*** 2.26

Analysis of MT dynamics was performed for more than 30 MTs per condition by time-lapse videomicroscopy of U87MG cells transfected with GFP-a-tubulin. * p  0.05 each condition vs control, Student’s t-test. ** p  0.01 each condition vs control, Student’s t-test. *** p  0.001 each condition vs control, Student’s t-test.

as compared to control. In contrast, paclitaxel increased MT dynamic instability by increasing both MT growth and shortening rates. Importantly, patupilone decreased the MT growth rate while paclitaxel increased the MT growth rate (Fig. 6C). Moreover, both

drugs were able to increase the time and/or the distance-based catastrophe frequencies. However, at 100 nM, the distance-based catastrophe frequency was increased for patupilone but not for paclitaxel (Fig. 6C).

Fig. 4. Analysis of MT plus end dynamics. (A) Representative images from time-lapse videomicroscopy of U87MG cells transfected with EB1-GFP cells, either untreated or treated with patupilone at the indicated concentrations. EB1 comets were tracked over time to measure MT dynamics; size bars = 5 mm. (B) Parameters of EB1-GFP dynamics. All values are expressed as mean  SEM of 30–35 comet tracks analyzed (*p  0.05, **p  0.01, ***p  0.001 each condition vs control, Student’s t-test).

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Fig. 5. Patupilone affects MT targeting to the cell cortex. (A) U87MG cells were either untreated or treated with patupilone at the indicated concentrations and stained for MTs (green) and actin (red) and analyzed by confocal microscopy (100/N.A. 1.30 objective lens); size bars = 5 mm. (B) Quantification of the mean MT tip distance from the cell margin (mm  SEM, n > 190 MTs, 5–10 cells; ***p  0.001 each condition vs control, Student’s t-test). (C) Distribution of MT numbers according to the distance of their tips to the cell margin. One representative experiment is shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

These results show that the alteration of EB accumulation at MT tips by MT stabilizing drugs patupilone and paclitaxel is independent of the effects on the MT growth rate as observed for patupilone in glioblastoma cells. This experiment also indicates that induction of MT distance-based catastrophes is an intrinsic property of patupilone. 4. Discussion In this study, we investigated the mechanism of the antimigratory effect of patupilone on glioblastoma cells. At a very low concentration, patupilone reduced the accumulation of EB1 protein at MT plus ends and increased MT catastrophes, without affecting MT growth rate. Interestingly, the antimigratory effect was observed at concentrations of the drug that did not inhibit cell proliferation (from 0.1 to 1 nM), suggesting that the mechanism underlying the anti-migratory effect differs from that of the

cytotoxic action of the drug. Indeed, it is well known for all MTAs that the MT-related mechanism of cytotoxicity involves suppression of MT dynamic instability [2]. Suppression of MT dynamics by drugs has been also associated with inhibition of migration in CHO cells [26]. In the present work, we showed that the suppression of MT growth and shortening rates, and the resulting changes in the overall dynamicity are not involved in the anti-migratory activity of patupilone in glioblastoma cells. Importantly, we found that among the dynamic instability parameters, only the distancebased catastrophe frequency was related to the anti-migratory effect of patupilone. The increase in catastrophe frequency by a MT stabilizing agent is counterintuitive but it has already been observed previously with paclitaxel in MCF7 breast cancer cells. However, no assigned functional consequences were revealed [44]. Patupilone-mediated induction of catastrophes was associated with the reduction of the amount of EB1 at MT plus ends in a concentration dependent manner, without affecting the EB1

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Fig. 6. GFP-EB3 tip tracking on dynamic MTs in vitro. (A) Kymographs of dynamic MTs grown in the presence of 75 nM GFP-EB3 in the presence or absence of patupilone and paclitaxel. Two kymographs of 2 different MTs per condition are shown. (B) Mean fluorescence intensity of GFP-EB3 comets at MT plus ends during the growth phases. (C) Parameters of MT dynamic instability of the MT plus end in vitro.

expression level. The ability of MTAs including paclitaxel to modify EB1 comet pattern has been shown in different cell types, i.e. cancer, neuronal and endothelial cells [1,45,46]. On the other side, EB1 down-regulation has been recently shown to induce MT catastrophes in cells [21]. In addition, patupilone also affected CLIP170 or p150glued comet pattern. However, the formation of MT plus end comets with these CAP-Gly proteins depends on the presence of EB1 [47]. Thus, the disruption of the plus TIPs network at MT plus end could be the consequence of the altered accumulation of EB1 at MT plus end. It is known that the length of EB1 comets at MT plus end is influenced by the MT growth rate. Indeed, during MT polymerization, new high affinity binding sites for EB1 are generated at MT plus end. These high affinity binding sites exist for a period of time and then progressively disappear from the MT lattice, making the

binding of EB1 resembling to a comet. The time of existence of EB1 binding sites at MT plus end, termed ‘‘decoration time’’ have been evaluated to approximately 9 s in vitro [41]. The fact that the length and fluorescence intensity EB1 comets were affected independently of the MT growth rate suggests that patupilone directly affects the time of existence of EB1 binding sites and/or the number of binding sites at MT plus end. Moreover, patupilone affected EB comets and increased the catastrophe frequency in an in vitro reconstituted system, indicating that these two effects are intrinsic properties of patupilone. The effect of patupilone on EB proteins may be the consequence of the alteration of at least one of the three potential mechanisms for EB1 binding to MTs, i.e. the recognition of (1) a specific structure of the sheet at MT plus end, (2) the a-lattice, and (3) the nucleotide state of tubulin (GTP cap) [10]. By using a monoclonal antibody

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against GTPgS-tubulin [48] we found that the GTP-bound tubulin distribution on MTs was not affected by patupilone at any of the concentrations tested, suggesting that the effect of patupilone is not the consequence of an alteration of the nucleotide state of tubulin (data not shown). Moreover, it has been recently proposed that EB1 may recognize a region between tubulin subunits, close to the guanine nucleotide binding site of b-tubulin [49], which is expected to undergo a conformational change upon GTP hydrolysis [50]. Interestingly, it has been shown that paclitaxel gives rise to long-range allosteric changes in the b-tubulin monomer which affects such hypothesized EB1 binding site [51]. As patupilone also binds to the taxane binding site on tubulin and has the same effect on EB3 tip tracking in vitro, we can hypothesize that the inhibition of EBs accumulation at MT plus end is the consequence of an allosteric conformational change that affects the EB1 binding site. However, we cannot exclude complementary intracellular mechanisms involved in EBs comet disruption by patupilone such as oxidative signaling [52,53]. EB1 is overexpressed in several cancers and associated with poor prognosis and tumor progression [54,55]. Moreover, EB1 has been described as an oncogene and has been demonstrated to be essential in cell migration [12,20,54,56]. We confirmed that the downregulation of EB1 in glioblastoma cells, using specific siRNA, impairs cell migration. In addition, we showed that patupiloneinduced alteration of EB1 comets and induction of MT catastrophes was associated with the disruption of MT targeting to the cell cortex and consequent inhibition of cell migration. These results expand our previous work showing a similar effect on endothelial cells with vinflunine, a MT depolymerizing agent [1,25]. Local invasion in glioblastoma requires the migration of tumor cells away from the original tumor site. This infiltration of malignant gliomas into the surrounding brain regions compromises neurologic function and is a major cause of morbidity. Because patupilone was antimigratory in glioblastoma cells at very low concentrations in vitro, we can propose that a metronomic scheduling with low doses and prolonged drug exposure times, may be appropriate to affect glioblastoma migration in vivo. The metronomic scheduling of patupilone has already been shown to exert a strong anti-angiogenic effect [57]. The metronomic chemotherapy has now grown beyond the anticipated scope of anti-angiogenic chemotherapy, with accumulating evidence demonstrating that such treatments may also act by stimulating an antitumor immune response and could ultimately lead to reinduction of tumor dormancy [58]. Taken together, our results suggest that MTA inhibit cell migration without affecting MT growth and shortening rates, potentially through an EB1-dependent mechanism and that plusend protein-directed therapeutic strategies could be beneficial in glioblastoma tumors. Acknowledgements This work was partly supported by the Canceropole PACA and INCa (RS019), by an EMBO Long Term Fellowship and a MarieCurie fellowship to RM and by the Netherlands Organization for Scientific Research (NOW) ALW VICI grant to AA. AP was supported in part by grants from FUI Provence-Alpes Cote d’Azur council, ‘‘Conseil Ge´ne´ral 13’’, and Association pour la Recherche sur les Tumeurs Ce´re´brales (ARTC). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bcp.2012.05.010.

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