Glioblastoma is the most aggressive primary brain tumour with no curative treatment and inevitable relapse. Therapeutic resistance is, at least, related to the presence of cancer stem-like cells in these tumours. Here, we aimed to demonstrate that the GD3 ganglioside was a relevant marker and actionable target for glioblastoma cancer stem-like cells. To this end, we used commercial glioblastoma cell lines, human glioblastoma samples, organotypic culture and xenografted mouse models to study GD3 antigen expression and consequences of its downregulation through a shRNA strategy targeting the ST8SIA1 mRNA which encodes the key enzyme for GD3 synthesis. We performed mono-dimensional Thin Layer Chromatography to analyse ganglioside composition of the glioblastoma samples and RNA-seq analyses to reveal oncogenic pathways and more specifically transcripts affected by ST8SIA1 silencing. Besides, we evaluated GD3 role in stemness of glioblastoma cancer cell, phenotype, microenvironment interaction, and invasion abilities. We showed that GD3 is the main ganglioside in glioblastoma and that patient-derived cancer stem-like cell lines strongly expressed GD3. This GD3 + population decreased significantly after cell differentiation. GD3+ cells sorted from patient samples had stem-like cell properties: they were plastic, clonogenic, and tumorigenic after orthotopic engraftment. Silencing of ST8SIA1/GD3 was associated with a decrease in sphere size, self-renewal and migratory capacities and increased mouse survival. Moreover, increased temozolomide sensitivity was recorded. Finally, data from RNA-seq showed that silencing ST8SIA1/GD3 decreased oncogenic pathways and more specifically the expression of ADAMTS1 and IL33 transcripts. Taken together, our results suggest that GD3 ganglioside is essential for glioblastoma cancer stem-like cell properties, opening promising targeted therapeutic development.

Microtubule Targeting Agents (MTAs) constitute a vital category of tubulin-binding compounds, deployed across anticancer therapies. Despite the array of MTA drugs developed by pharmaceutical entities, the quest for novel efficacious molecules continues unabated. We unveil an innovative in vitro MTA screening methodology employing nano differential scanning fluorimetry (nanoDSF), presenting distinct advantages over known assays. This novel approach not only assesses compound-tubulin binding but also quantitatively analyzes their impact on tubulin polymerization. Proposed nanoDSF assay was rigorously validated using the Prestwick Chemical Library, which encompasses 1,520 approved compounds, successfully identifying all previously known MTAs. Furthermore, this screening has unearthed potential anti-tubulin agents among drugs currently utilized for non-related medical conditions, offering insights into their mechanisms of action in inhibiting cancer cell proliferation and/or inducing cytotoxicity. These discoveries herald new opportunities for drug repositioning involving the newly identified MTAs and substantially streamline the process of screening extensive chemical libraries for MTAs featuring novel chemical structures. ### Competing Interest Statement The authors have declared no competing interest.

Materials and methods: EB1 Western blot analysis GBM stem cells were lysed (lysis buffer: Tris 50 mM pH 8.0, NaCl 250 mM, Triton-X100 1%, SDS 0.1% with a cocktail of proteases and phosphatases inhibitors added freshly (all from Sigma-Aldrich). Thirty µg of total protein lysate were loaded onto a 12% SDS-PAGE gel. Nitrocellulose membrane (Bio-Rad laboratories, Marnes la Coquette, France) was blocked with 5% milk (powder) in Phosphate Buffer Saline (PBS) (Life technologies)-Tween (Sigma-Aldrich) pH 7.4 for 1 h and then incubated in PBS-Tween 0.1%-5% milk solution with mouse anti-EB1 antibody (clone 5, BD Biosciences, Le pont de Claix, France) (1/1000) and mouse α-tubulin (clone DM1A, Sigma Aldrich). After washing, membranes were incubated with anti-mouse peroxydase-conjugated secondary antibodies (Jackson Immunoresearch, Baltimore, USA) for 1 h. The bound antibodies were then detected using chemiluminescence detection kit (Millipore, Saint Quentin en Yvelines, France). Signals were recorded with G:BOX (Syngene/Ozyme, Saint Quentin en Yvelines, France ) and quantification was done with Image J software. Immunofluorescence analysis Indirect immunofluorescence was performed as previously described (15). Briefly, cells were fixed for 5 min with cold methanol (-20{degree sign}C), incubated with the anti-EB1 antibody 1/100 for 1 h and anti-mouse antibody Alexa 568 nm (Molecular Probes) 1/200; and FITC-coupled anti-α-tubulin antibody (clone DM1A; Sigma-Aldrich) 1/200 for 1 h at room temperature in the presence of DAPI for nuclear staining. For GFAP staining, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton-X100 for 10 minutes. Primary antibody against GFAP (rabbit IgG, 10€‰Î¼g/ml, Dako) was incubated overnight at 4€‰{degree sign}C. Secondary antibody, Texas-red Goat anti-rabbit IgG (H+L) was purchased from Jackson Immunoresearch (Newmarket, UK), and was incubated for 1 h. Cells were analyzed using a Leica DM-IRBE microscope at a magnification of x100. Real-time quantitative PCR RNA samples were processed using a LightCycler 480 instrument (Roche Applied Science) and a LightCycler 480 SYBR Green I Master Mix (Roche Applied Science). Briefly, total DNA-free RNA (1 μg) was reverse-transcribed into cDNA using 1 μg of random hexamers and Superscript II reverse transcriptase as recommended by the manufacturer (Invitrogen Life Technologies, Cergy Pontoise, France). Measurements were performed in triplicate for each sample, and relative expression ratios of target gene transcripts (GFAP, TUBB3, CNP) and reference gene transcripts (18S, GAPDH, and ACTB) were calculated using qPCR efficiencies and cycle threshold (Ct) deviations of tumor and normal adult brain samples (control: Agilent Technologies) (25). RNA expression levels in GBM CSLCs were subsequently expressed as percentages compared to normal adult human brain samples corresponding to 100% of expression. Forward and reverse primers used for each gene are listed in Supplemental Table 1. Supplementary Table 1: Sequences of primers used in RT-qPCR Supplementary Figure 1: Chemical structure of BAL27862 and BAL101553 Supplementary Figure 2: Modulation of EB1 expression in GBM6 cells using siRNA or shRNA against EB1. (A) Western blot analysis of EB1 in GBM6 shEB1 clones (GBM6 shEB1, GBM6 shEB1#2), in GBM6 GFP shEB1 clone and in control clones (GBM6 wt, GBM6 sh0 and GBM6 GFP sh0). Ratios EB1/tubulin, relative to controls, from at least three independent experiments are presented under the blots. (B) Analysis of EB1 expression by Western blot of GBM6 wt or treated with siRNA against EB1 (GBM6 siEB1) or siRNA control (GBM6 si0). (C) Immunofluorescence staining of tubulin (green) and EB1 (red) in GBM6 wt, GBM6 si0 and GBM6 siEB1. Bar = 10 μm.

Glioblastoma (GBM) contains cancer stem cells (CSC) that are resistant to treatment. GBM CSC expresses glycolipids recognized by the A2B5 antibody. A2B5, induced by the enzyme ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyl transferase 3 (ST8Sia3), plays a crucial role in the proliferation, migration, clonogenicity and tumorigenesis of GBM CSC. Our aim was to characterize the resulting effects of neuraminidase that removes A2B5 in order to target GBM CSC. To this end, we set up a GBM organotypic slice model; quantified A2B5 expression by flow cytometry in U87-MG, U87-ST8Sia3 and GBM CSC lines, treated or not by neuraminidase; performed RNAseq and DNA methylation profiling; and analyzed the ganglioside expression by liquid chromatography–mass spectrometry in these cell lines, treated or not with neuraminidase. Results demonstrated that neuraminidase decreased A2B5 expression, tumor size and regrowth after surgical removal in the organotypic slice model but did not induce a distinct transcriptomic or epigenetic signature in GBM CSC lines. RNAseq analysis revealed that OLIG2, CHI3L1, TIMP3, TNFAIP2, and TNFAIP6 transcripts were significantly overexpressed in U87-ST8Sia3 compared to U87-MG. RT-qPCR confirmed these results and demonstrated that neuraminidase decreased gene expression in GBM CSC lines. Moreover, neuraminidase drastically reduced ganglioside expression in GBM CSC lines. Neuraminidase, by its pleiotropic action, is an attractive local treatment against GBM.

Chemical structure of BAL27862 and BAL101553

Modulation of EB1 expression in GBM6 cells using siRNA or shRNA against EB1

Sequences of primers used in RT-qPCR

ABSTRACTSynergistic drug combinations are an attractive anticancer strategy but prove challenging to identify. Here we present a stepwise approach consisting in revealing core cancer vulnerabilities and exploiting them through drug combination screen to uncover synergistic treatments for glioblastoma patients.MethodsWe established an innovative method, based on high-throughput screening, target deconvolution and functional genomics, to reveal core vulnerabilities in glioblastoma. Combination drug screen targeting these vulnerabilities was then designed to unveil synergistic associations. The therapeutic potential of the top drug combination was validated in two different clinically-relevant models: an organotypicex vivomodel and a syngeneic orthotopic mouse model of glioblastoma.ResultsLarge-scale monotherapy drug screening identified 83 potent anti-glioblastoma compounds. Target deconvolution using public chemoinformatic databases uncovered 1,100 targets and interactors of the hit compounds. Screening of a focused siRNA library targeting the top 292 drug interactors revealed 22 targetable vulnerabilities, 9 of which were confirmed as core glioblastoma vulnerabilities by mining the CRISPR screen cohort data from the online Cancer Dependency Map portal. Six selective inhibitors of the core vulnerabilities were then screened in combination with a custom-made library of 88 compounds and synergies amongst the 528 tested pairwise combinations were predicted. The combinations of CHK1 / MEK and AURKA / BET inhibitors were highlighted and validated in 3D tumor spheroids. Using an organotypicex vivomodel and a syngeneic orthotopic mouse model, we definitively ascertained the efficacy of dual AURKA / BET inhibition in glioblastoma.ConclusionsCollectively, we uncovered that dual inhibition of BET proteins and aurora kinase A is highly synergistic against GBM. Moreover, our study indicates that our approach to exploit drug poly-pharmacology for the rational design of drug combination screens represent a valuable strategy to discover synergistic treatments against refractory cancers.

Glioblastoma (GBM) recurrences appear in most cases around the resection cavity borders and arise from residual GBM cells that cannot be removed by surgery. Here, we propose a novel treatment that combines the advantages of nanomedicine and local drug delivery to target these infiltrating GBM cells. We developed an injectable lipid nanocapsule (LNC)-based formulation loaded with lauroyl-doxorubicin prodrug (DOXC12). Firstly, we demonstrated the efficacy of intratumoral administration of DOXC12 in GL261 GBM-bearing mice, which extended mouse survival. Then, we formulated an injectable hydrogel by mixing the appropriate amount of prodrug with the lipophilic components of LNC. We optimized the hydrogel by incorporating cytidine-C16 (CytC16) to achieve a mechanical stiffness adapted for an application in the brain post-surgery (DOXC12-LNCCL). DOXC12-LNCCL exhibited high DOXC12 encapsulation efficiency (95%) and a size of approximately 60 nm with sustained drug release for over 1 month in vitro. DOXC12-LNCCL exhibited enhanced cytotoxicity compared to free DOXC12 (IC50 of 349 and 86 nM, respectively) on GL261 GBM cells and prevented the growth of GL261 spheroids cultured on organotypic brain slices. In vivo, post-surgical treatment with DOXC12-LNCCL significantly improved the survival of GL261-bearing mice. The combination of this local treatment with the systemic administration of anti-inflammatory drug ibuprofen further delayed the onset of recurrences. In conclusion, our study presents a promising therapeutic approach for the treatment of GBM. By targeting residual GBM cells and reducing the inflammation post-surgery, we present a new strategy to delay the onset of recurrences in the gap period between surgery and standard of care therapy.

Glioblastoma multiform (GBM) is the most frequent primitive brain tumor with a high recurrence and mortality. Histone deacetylase inhibitors (HDACi) have evoked great interest because they are able to change transcriptomic profiles to promote tumor cell death but also induce side effects due to the lack of selectivity. We show in this paper new anticancer properties and mechanisms of action of low concentrations of vorinostat on various GBM cells which acts by affecting microtubule cytoskeleton in a non-histone 3 (H3) manner. Indeed, vorinostat induces tubulin acetylation and detyrosination, affects EB stabilizing cap on microtubule plus ends and suppresses microtubule dynamic instability. We previously identified EB1 overexpression as a marker of bad prognostic in GBM. Interestingly, we show for the first time to our knowledge, a strong decrease of EB1 expression in GBM cells by a drug. Altogether, our results suggest that low dose vorinostat, which is more selective for HDAC6 inhibition, could therefore represent an interesting therapeutic option for GBM especially in patients with EB1 overexpressing tumor with lower expected side effects. A validation of our hypothesis is needed during future clinical trials with this drug in GBM.

Glioblastoma (GBM) are aggressive brain tumors with limited treatment options. Cancer stem-like cells (CSLCs) contribute to GBM invasiveness, representing promising targets. BAL101553, a prodrug of BAL27862, is a novel small molecule tubulin-binding agent, promoting tumor cell death through spindle assembly checkpoint activation, which is currently in Phase 1/2a in advanced solid tumor patients including GBM. This study aimed to evaluate long-term daily oral BAL101553 treatment of mice orthotopically grafted with GBM CSLCs (GBM6) according to EB1 expression-level, and to decipher its mechanism of action on GBM stem cells. Oral treatment with BAL101553 for 100 days provoked a large EB1 expression level-dependent survival benefit, together with a decrease in tumor growth and brain invasion. Formation of vascular structures by the fluorescent GBM6-GFP-sh0 cells, mimicking endothelial vascular networks, was observed in the brains of control grafted mice. Following BAL101553 treatment, vessels were no longer detectable, suggesting inhibition of the endothelial trans-differentiation of GBM stem cells. In vitro, BAL27862 treatment resulted in a switch to the endothelial-like phenotype of GBM6 towards an astrocytic phenotype. Moreover, the drug inhibited secretion of VEGF, thus preventing normal endothelial cell migration activated by CSLCs. The decrease in VEGF secretion was confirmed in a human GBM explant following drug treatment. Altogether, our data first confirm the potential of EB1 expression as a response-predictive biomarker of BAL101553 in GBM we previously published and add new insights in BAL101553 long-term action by counteracting CSLCs mediated tumor angiogenesis. Our results strongly support BAL101553 clinical studies in GBM patients.

A2B5+ cells isolated from human glioblastomas exhibit cancer stem cell properties. The A2B5 epitope belongs to the sialoganglioside family and is synthetized by the ST8 alpha-N-acetyl-neuraminidase α-2,8-sialyltransferase 3 (ST8SIA3) enzyme. Glycolipids represent attractive targets for solid tumors; therefore, the aim of this study was to decipher A2B5 function in glioblastomas. To this end, we developed cell lines expressing various levels of A2B5 either by genetically manipulating ST8SIA3 or by using neuraminidase. The overexpression of ST8SIA3 in low-A2B5-expressing cells resulted in a dramatic increase of A2B5 immunoreactivity. ST8SIA3 overexpression increased cell proliferation, migration, and clonogenicity in vitro and tumor growth when cells were intracranially grafted. Conversely, lentiviral ST8SIA3 inactivation in low-A2B5-expressing cells resulted in reduced proliferation, migration, and clonogenicity in vitro and extended mouse survival. Furthermore, in the shST8SIA3 cells, we found an active apoptotic phenotype. In high-A2B5-expressing cancer stem cells, lentiviral delivery of shST8SIA3 stopped cell growth. Neuraminidase treatment, which modifies the A2B5 epitope, impaired cell survival, proliferation, self-renewal, and migration. Our findings prove the crucial role of the A2B5 epitope in the promotion of proliferation, migration, clonogenicity, and tumorigenesis, pointing at A2B5 as an attractive therapeutic target for glioblastomas.

Glioblastoma (GBM) is characterized by highly aggressive growth and invasive behavior. Due to the highly lethal nature of GBM, new therapies are urgently needed and repositioning of existing drugs is a promising approach. We have previously shown the activity of Proscillaridin A (ProA), a cardiac glycoside inhibitor of the Na(+)/K(+) ATPase (NKA) pump, against proliferation and migration of GBM cell lines. ProA inhibited tumor growth in vivo and increased mice survival after orthotopic grafting of GBM cells. This study aims to decipher the mechanism of action of ProA in GBM tumor and stem-like cells. ProA displayed cytotoxic activity on tumor and stem-like cells grown in 2D and 3D culture, but not on healthy cells as astrocytes or oligodendrocytes. Even at sub-cytotoxic concentration, ProA impaired cell migration and disturbed EB1 accumulation at microtubule (MT) plus-ends and MT dynamics instability. ProA activates GSK3β downstream of NKA inhibition, leading to EB1 phosphorylation on S155 and T166, EB1 comet length shortening and MT dynamics alteration, and finally inhibition of cell migration and cytotoxicity. Similar results were observed with digoxin. Therefore, we disclosed here a novel pathway by which ProA and digoxin modulate MT-governed functions in GBM tumor and stem-like cells. Altogether, our results support ProA and digoxin as potent candidates for drug repositioning in GBM.

Metabolic reprogramming is a hallmark of cancer development, mediated by genetic and epigenetic alterations that may be pharmacologically targeted. Among oncogenes, the kinase Akt is commonly overexpressed in tumors and favors glycolysis, providing a rationale for using Akt inhibitors. Here, we addressed the question of whether and how inhibiting Akt activity could improve therapy of non-small cell lung cancer (NSCLC) that represents more than 80% of all lung cancer cases. First, we demonstrated that Akt inhibitors interacted synergistically with Microtubule-Targeting Agents (MTAs) and specifically in cancer cell lines, including those resistant to chemotherapy agents and anti-EGFR targeted therapies. In vivo, we further revealed that the chronic administration of low-doses of paclitaxel - i.e. metronomic scheduling - and the anti-Akt perifosine was the most efficient and the best tolerated treatment against NSCLC. Regarding drug mechanism of action, perifosine potentiated the pro-apoptotic effects of paclitaxel, independently of cell cycle arrest, and combining paclitaxel/perifosine resulted in a sustained suppression of glycolytic and mitochondrial metabolism. This study points out that targeting cancer cell bioenergetics may represent a novel therapeutic avenue in NSCLC, and provides a strong foundation for future clinical trials of metronomic MTAs combined with Akt inhibitors.

AbstractGlioblastoma patients have limited treatment options. Cancer stem-like cells (CSLC) contribute to glioblastoma invasiveness and repopulation; hence, they represent promising targets for novel therapies. BAL101553 is a prodrug of BAL27862, a novel microtubule-destabilizing agent inhibiting tumor cell proliferation through activation of the spindle assembly checkpoint, which is currently in phase I/II clinical development. Broad anticancer activity has been demonstrated against human cancer models, including tumors refractory to conventional treatments. We have shown that overexpression of microtubule + end-binding 1-protein (EB1) correlates with glioblastoma progression and poor survival. Here, we show that BAL27862 inhibits the growth of two glioblastoma CSLCs. As EB1 is overexpressed in the CSLC line GBM6, which displays a high tumorigenicity and infiltrative pattern of migration in vivo, we investigated drug activity on GBM6 according to EB1 expression. BAL27862 inhibited migration and colony formation at subcytotoxic concentrations in EB1-expressing control cells (GBM6-sh0) but only at cytotoxic concentrations in EB1-downregulated (GBM-shE1) cells. Three administrations of BAL101553 were sufficient to provoke an EB1-dependent survival benefit in tumor-bearing mice. Patterns of invasion and quantification of tumor cells in brain demonstrated that GBM6-sh0 cells were more invasive than GBM6-shEB1 cells, and that the antiproliferative and anti-invasive effects of BAL101553 were more potent in mice bearing control tumors than in EB1-downregulated tumors. This was associated with inhibition of stem cell properties in the GBM6-sh0 model. Finally, BAL27862 triggered astrocytic differentiation of GBM6 in an EB1-dependent manner. These results support the potential of BAL101553 for glioblastoma treatment, with EB1 expression as a predictive biomarker of response. Mol Cancer Ther; 15(11); 2740–9. ©2016 AACR.

Background: BAL101553 is the prodrug of the novel small molecule BAL27862, undergoing clinical evaluation in advanced cancer patients as an i.v. (phase 2a) and oral (phase 1) formulation. BAL27862 binds the colchicine site of tubulin with distinct effects on microtubule organization, resulting in activation of the ‘spindle assembly checkpoint’ and tumor cell death. BAL27862 has broad activity against diverse cancer models, including tumors refractory to conventional treatments. The drug efficiently distributes to tumor and to brain, with cytotoxic effects in glioblastoma (GBM) lines. We have shown that End-binding 1-protein (EB1) overexpression correlates with GBM progression and sensitizes GBM tumors to Vinca alkaloids. Moreover, EB1 is overexpressed in the GBM stem-like cells, GBM6, that display a high tumorigenicity with an infiltrative pattern of migration in vivo. Here, we investigate the activity of BAL27862/BAL101553 on GBM6 stem-like cells according to EB1 expression level in vitro and in orthotopically transplanted nude mice. Material and methods: Effects of BAL27862 on wild-type and EB1-downregulated GBM6 cells were analyzed using sulforhodamin B survival, clonogenic and transwell migration assays. Nude mice were orthotopically grafted with GBM6GFPSh0 (EB1+) and GBM6GFPShEB1 (EB1-) cells. BAL101553 (25mg/kg) or vehicle were administrated i.v. at D30, D33 and D36. Stem cell phenotype characterization, tumor volume and brain invasion were analyzed at D45, D75 and D105. Overall survivals were analyzed. Stem-like cell differentiation was analyzed by flow cytometry and real-time PCR by analyzing stem cell markers (A2B5, CD133), markers of differentiation (GFAP, β-III-tubulin and CNPase) and self-renewal assay. Results: BAL27862 inhibited GBM6 survival after 72h (half maximal effective concentration [EC50] = 20nM) with EB1-downregulation resulting in a ∼2-fold increase in EC50 (40nM). BAL27862 inhibited clonogenicity and cell migration in GBM6 cells, even at a low, non-cytotoxic concentration (6 nM). However, these effects were only detectable with cytotoxic concentrations (≥ 20 nM) in EB1 downregulated GBM6 cells. Just three BAL101553 administrations over a week provoked a mice survival increase of 69 and 32 days (vs. vehicle controls) after GBM6GFPSh0 and GBM6GFPShEB1 tumor grafting, respectively. Analysis of the pattern of invasion, and quantification of fluorescent tumor cells and A2B5+cells in brain, demonstrated that control cells were more invasive than EB1-downregulated cells, and that anti-invasive effects of BAL101553 were more potent in control cells than in EB1-downregulated cells. BAL27862 also acted on stem-like cells by inducing apoptosis and differentiation, as shown by the decrease in self renewal and stem cell markers. Conclusion: BAL27862 is the first tubulin-binding agent to show a strong inhibitory activity against stem-like cell proliferation and invasion. These results support further investigation of BAL101553 for the treatment of GBM patients, with EB1 expression a potential predictive biomarker for drug responders. Financial supports: (SIRIC label) INCa-DGOS-Inserm 6038, A*MIDEX project “Investissements d9Avenir” (No. ANR-11-IDEX-0001-02), the ITMO Cancer AVIESAN as part of the Cancer Plan and Basilea Pharmaceutica. Citation Format: Raphael Berges, Aurelie Tchoghandjian, Stephane Honore, Dominique Figarella-Branger, Felix Bachmann, Heidi Lane, Diane Braguer. The novel tubulin-binding ‘tumor checkpoint controller’ BAL101553 exerts EB1 expression-dependent antitumor effects on glioblastoma stem-like cells in vitro and in vivo. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A183.

End-binding 1 protein (EB1) is a key player in the regulation of microtubule (MT) dynamics. Here, we investigated the role of EB1 in glioblastoma (GBM) tumor progression and its potential predictive role for response to Vinca-alkaloid chemotherapy. Immunohistological analysis of the 109 human GBM cases revealed that EB1 overexpression correlated with poor outcome including progression-free survival and overall survival. Downregulation of EB1 by shRNA inhibited cell migration and proliferation in vitro. Conversely, EB1 overexpression promoted them and accelerated tumor growth in orthotopically-transplanted nude mice. Furthermore, EB1 was largely overexpressed in stem-like GBM6 that display in vivo a higher tumorigenicity with a more infiltrative pattern of migration than stem-like GBM9. GBM6 showed strong and EB1-dependent migratory potential. The predictive role of EB1 in the response of GBM cells to chemotherapy was investigated. Vinflunine and vincristine increased survival of EB1-overexpressing U87 bearing mice and were more effective to inhibit cell migration and proliferation in EB1-overexpressing clones than in controls. Vinca inhibited the increase of MT growth rate and growth length induced by EB1 overexpression. Altogether, our results show that EB1 expression level has a prognostic value in GBM, and that Vinca-alkaloid chemotherapy could improve the treatment of GBM patients with EB1-overexpressing tumor.

We previously showed that vinflunine, a microtubule-targeting drug of the Vinca-alkaloid family exerted its anti-angiogenic/anti-migratory activities through an increase in microtubule dynamics and an inhibition of microtubule targeting to adhesion sites. Such effect was associated with a reduction of EB1 comet length at microtubule (+) ends. In this work we first showed that the pro-angiogenic vascular endothelial growth factor VEGF suppressed microtubule dynamics in living Human Umbilical Vein Endothelial Cells (HUVECs), increased EB1 comet length by 40%, and induced EB1 to bind all along the microtubules, without modifying its expression level. Such microtubule (+) end stabilization occurred close to the plasma membrane in the vicinity of focal adhesion as shown by TIRF microscopy experiments. Vinflunine completely abolished the effect of VEGF on EB1 comets. Interestingly, we found a correlation between the reduction of EB1 comet length by vinflunine and the inhibition of cell migration. By using 2D gel electrophoresis we demonstrated for the first time that EB1 underwent several post-translational modifications in endothelial and tumor cells. Particularly, the C-terminal EEY sequence was poorly detectable in control and VEGF-treated HUVECs suggesting the existence of a non-tyrosinated form of EB1. By using specific antibodies that specifically recognized and discriminated the native tyrosinated form of EB1 and a putative C-terminal detyrosinated form, we showed that a detyrosinated form of EB1 exists in HUVECs and tumor cells. Interestingly, vinflunine decreased the level of the detyrosinated form and increased the native tyrosinated form of EB1. Using 3-L-Nitrotyrosine incorporation experiments, we concluded that the EB1 C-terminal modifications result from a detyrosination/retyrosination cycle as described for tubulin. Altogether, our results show that vinflunine inhibits endothelial cell migration through an alteration of EB1 comet length and EB1 detyrosination/retyrosination cycle.

Bcl-2 is commonly overexpressed in tumors, where it is often associated with unfavorable outcome. However, it has also been linked to a favorable sensitivity to microtubule-targeting agents (MTAs). We show that Bcl-2-overexpressing lung and breast cancer cells were more sensitive to both paclitaxel and vinorelbine. Bcl-2 overexpression also significantly potentiated in vivo efficacy of paclitaxel, in terms of tumor volume decrease and survival benefits, in models of nude mice bearing lung cancer xenografts. To further investigate this favorable effect of Bcl-2, a genomic approach was taken. It revealed that Bcl-2 overexpression induced up-regulation of the proapoptotic protein Bim in lung cancer cells and that, conversely, Bcl-2 silencing decreased Bim expression level. A gene regulation study implicated the transcription factor Forkhead box-containing protein, class O3a in Bim up-regulation. Lastly, we show that Bim was responsible for MTA-triggered lung cancer cell death through a dynamin-related protein 1-mediated mitochondrial fragmentation. The Bcl-2-governed Bim induction evidence offers for the first time an explanation for the favorable higher sensitivity to treatment shown by Bcl-2-overexpressing cells. We suggest that Bim could be a powerful predictive factor for tumor response to MTA chemotherapy. Our data also give new insight into some failures in the efficacy of therapies targeted against Bcl-2.