Search

Supplementary Materials and Methods

CRMP5 depletion does not promote apoptosis.(A): Apoptosis is monitored in GL15 cells transfected with CRMP5-siRNA1 and control-siRNA using caspase 3 antibody (1/400, BD Pharmingen, 559565). 5-bromo-2'-deoxyuridine (BrdU) indicates proliferating cells in both conditions. (B): TUNEL staining (marker of apoptosis) (Promega, G3250) in CRMP5-siRNA1 transfected GL15 cells Scale bar=20µm. (C): TUNEL staining quantification in CRMP5-siRNA transfected GL15 cells after 4 days. The total number of cells counted is indicated by DAPI quantification; TUNEL-stained cells were counted for each condition. The results were obtained from 3 independent experiments.

Clinicopathological characteristics of patients according to CRMP5 status

Collapsin response mediator protein 5 (CRMP5) belongs to a family of five cytosolic proteins that play a major role in nervous system development. This protein was first described in cancer-induced autoimmune processes, causing neurodegenerative disorders (paraneoplastic neurologic syndromes). CRMP5 expression has been reported to serve as a biomarker for high-grade lung neuroendocrine carcinomas; however, its functional roles have not been examined in any setting of cancer pathophysiology. In this study, we report two different CRMP5 expression patterns observed in human glioblastoma (GBM) biopsies that establish connections between CRMP5 expression, Notch receptor signaling, and GBM cell proliferation. We demonstrated that elevated CRMP5 promotes Notch receptor expression and Akt activation in human tumor cell lines, GBM stem cells, and primary tumor biopsies. We have shown that the high CRMP5 and Notch expression in GBM xenograft is related to stem cells. This suggests that high CRMP5 expression pattern in GBM biopsies encompasses a subset of stem cells. Mechanistically, CRMP5 functioned by hijacking Notch receptors from Itch-dependent lysosomal degradation. Our findings suggest that CRMP5 serves as a major mediator of Notch signaling and Akt activation by controlling the degradation of the Notch receptor, with implications for defining a biomarker signature in GBM that correlates with and may predict patient survival. Cancer Res; 75(17); 3519–28. ©2015 AACR.

Transcriptional regulation of proteins involved in neuronal polarity is a key process that underlies the ability of neurons to transfer information in the central nervous system. The Collapsin Response Mediator Protein (CRMP) family is best known for its role in neurite outgrowth regulation conducting to neuronal polarity and axonal guidance, including CRMP5 that drives dendrite differentiation. Although CRMP5 is able to control dendritic development, the regulation of its expression remains poorly understood. Here we identify a Sox5 consensus binding sequence in the putative promoter sequence upstream of the CRMP5 gene. By luciferase assays we show that Sox5 increases CRMP5 promoter activity, but not if the putative Sox5 binding site is mutated. We demonstrate that Sox5 can physically bind to the CRMP5 promoter DNA in gel mobility shift and chromatin immunoprecipitation assays. Using a combination of real-time RT-PCR and quantitative immunocytochemistry, we provide further evidence for a Sox5-dependent upregulation of CRMP5 transcription and protein expression in N1E115 cells: a commonly used cell line model for neuronal differentiation. Furthermore, we report that increasing Sox5 levels in this neuronal cell line inhibits neurite outgrowth. This inhibition requires CRMP5 because CRMP5 knockdown prevents the Sox5-dependent effect. We confirm the physiological relevance of the Sox5-CRMP5 pathway in the regulation of neurite outgrowth using mouse primary hippocampal neurons. These findings identify Sox5 as a critical modulator of neurite outgrowth through the selective activation of CRMP5 expression.

Exposure of the developing brain to toxins, drugs, or deleterious endogenous compounds during the perinatal period can trigger alterations in cell division, migration, differentiation, and synaptogenesis, leading to lifelong neurological impairment. The brain is protected by cellular barriers acting through multiple mechanisms, some of which are still poorly explored. We used a combination of enzymatic assays, live tissue fluorescence microscopy, and anin vitrocellular model of the blood–CSF barrier to investigate an enzymatic detoxification pathway in the developing male and female rat brain. We show that during the early postnatal period the choroid plexus epithelium forming the blood–CSF barrier and the ependymal cell layer bordering the ventricles harbor a high detoxifying capacity that involves glutathioneS-transferases. Using a functional knock-down rat model for choroidal glutathione conjugation, we demonstrate that already in neonates, this metabolic pathway efficiently prevents the penetration of blood-borne reactive compounds into CSF. The versatility of the protective mechanism results from the multiplicity of the glutathioneS-transferase isoenzymes, which are differently expressed between the choroidal epithelium and the ependyma. The various isoenzymes display differential substrate specificities, which greatly widen the spectrum of molecules that can be inactivated by this pathway. In conclusion, the blood–CSF barrier and the ependyma are identified as key cellular structures in the CNS to protect the brain fluid environment from different chemical classes of potentially toxic compounds during the postnatal period. This metabolic neuroprotective function of brain interfaces ought to compensate for the liver postnatal immaturity.SIGNIFICANCE STATEMENTBrain homeostasis requires a stable and controlled internal environment. Defective brain protection during the perinatal period can lead to lifelong neurological impairment. We demonstrate that the choroid plexus forming the blood–CSF barrier is a key player in the protection of the developing brain. Glutathione-dependent enzymatic metabolism in the choroidal epithelium inactivates a broad spectrum of noxious compounds, efficiently preventing their penetration into the CSF. A second line of detoxification is located in the ependyma separating the CSF from brain tissue. Our study reveals a novel facet of the mechanisms by which the brain is protected at a period of high vulnerability, at a time when the astrocytic network is still immature and liver xenobiotic metabolism is limited.

In the hematopoietic system, tyrosine kinases of Syk family are essential components of immunoreceptor ITAM-based signaling. While an increasing number of data involved immunoreceptors in neural functions, the contribution of Syk kinases remains obscure. In previous studies we depicted phosphorylated forms of Syk kinases in specialized populations of migrating neurons or projecting axons. Moreover, we identified ephrin/Eph as guidance molecules utilizing the ITAM-bearing molecule CD3zeta and associated Syk kinases for growth cone collapsing response induced in vitro. From here, we show that in the developing spinal cord, Syk is phosphorylated in navigating commissural axons. By analyzing axon trajectories in open book preparations of Syk−/− ; ZAP-70−/− double KO embryos, we found that Syk kinases are dispensable for attraction towards the midline but confer growth cone responsiveness to repulsive signals required to expel commissural axons from the midline. Known to serve repulsive function at midline, ephrinB3/EphB2 consist in obvious candidates in driving the Syk-dependent repulsive response. Indeed, Syk kinases were found as required for ephrinB3-induced growth cone collapse in cultured commissural neurons. Besides, in fragments of commissural neuron-enriched tissues, Syk is present under a constitutively phosphorylated state and ephrinB3 decreases its level of phosphorylation. Furthermore, directly altering Syk kinase activity through pharmacological inhibition was sufficient to induce growth cone collapse, suggesting that Syk inhibition is a general requirement for growth cone collapse. In conclusion, Syk kinases act as a molecular switch of growth cone adhesive and repulsive responses.

EphA4, an Ephrins tyrosine kinase receptor, behaves as a dependence receptor (DR) by triggering cell apoptosis in the absence of its ligand Ephrin-B3. DRs act as conditional tumor suppressors, engaging cell death based on ligand availability; this mechanism is bypassed by overexpression of DRs ligands in some aggressive cancers. The pair EphA4/Ephrin-B3 favors survival of neuronal progenitors of the brain subventricular zone, an area where glioblastoma multiform (GBM) are thought to originate. Here, we report that Ephrin-B3 is highly expressed in human biopsies and that it inhibits EphA4 pro-apoptotic activity in tumor cells. Angiogenesis is directly correlated with GBM aggressiveness and we demonstrate that Ephrin-B3 also supports the survival of endothelial cells in vitro and in vivo. Lastly, silencing of Ephrin-B3 decreases tumor vascularization and growth in a xenograft mice model. Interference with EphA4/Ephrin-B3 interaction could then be envisaged as a relevant strategy to slow GBM growth by enhancing EphA4-induced cell death.

Background Glioblastoma (GBM) is the most frequent and aggressive type of adult brain tumor. Most GBMs express telomerase; a high level of intra-tumoral telomerase activity (TA) is predictive of poor prognosis. Thus, telomerase inhibitors are promising options to treat GBM. These inhibitors increase the response to radiotherapy (RT), in vitro as well as in vivo. Since typical treatments for GBM include RT, our objective was to evaluate the efficiency of Imetelstat (TA inhibitor) combined with RT. Findings We used a murine orthotopic model of human GBM (N = 8 to11 mice per group) and μMRI imaging to evaluate the efficacy of Imetelstat (delivered by intra-peritoneal injection) alone and combined with RT. Using a clinically established protocol, we demonstrated that Imetelstat significantly: (i) inhibited the TA in the very center of the tumor, (ii) reduced tumor volume as a proportion of TA inhibition, and (iii) increased the response to RT, in terms of tumor volume regression and survival increase. Conclusions Imetelstat is currently evaluated in refractory brain tumors in young patients (without RT). Our results support its clinical evaluation combined with RT to treat GBM. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0376-3) contains supplementary material, which is available to authorized users.

International audience; The collapsin response-mediator proteins (CRMPs) are multifunctional proteins highly expressed during brain development but down-regulated in the adult brain. They are involved in axon guidance and neurite outgrowth signalling. Among these, the intensively studied CRMP2 has been identified as an important actor in axon outgrowth, this activity being correlated with the reorganisation of cytoskeletal proteins via the phosphorylation state of CRMP2. Another member, CRMP5, restricts the growth-promotional effects of CRMP2 by inhibiting dendrite outgrowth at early developmental stages. This inhibition occurs when CRMP5 binds to tubulin and the microtubule-associated protein MAP2, but the role of CRMP5 phosphorylation is still unknown. Here, we have studied the role of CRMP5 phosphorylation by mutational analysis. Using non-phosphorylatable truncated constructs of CRMP5 we have demonstrated that, among the four previously identified CRMP5 phosphorylation sites (T509, T514, T516 and S534), only the phosphorylation at T516 residue was needed for neurite outgrowth inhibition in PC12 cells and in cultured C57BL/6J mouse hippocampal neurons. Indeed, the expression of the CRMP5 non-phosphorylated form induced a loss of function of CRMP5 and the mutant mimicking the phosphorylated form induced the growth inhibition function seen in wildtype CRMP5. The T516 phosphorylation was achieved by the glycogen synthase kinase-3β (GSK-3β), which can phosphorylate the wildtype protein but not the non-phosphorylatable mutant. Furthermore, we have shown that T516 phosphorylation is essential for the tubulin-binding property of CRMP5. Therefore, CRMP5-induced growth inhibition is dependent on T516 phosphorylation through the GSK-3β pathway. The findings provide new insights into the mechanisms underlying neurite outgrowth.

International audience; Glioblastomas are the most frequent and most aggressive primary brain tumors in adults. The median overall survival is limited to a few months despite surgery, radiotherapy, and chemotherapy. It is now clearly established that hyperactivity of cyclin-dependent kinases (CDKs) is one of the processes underlying hyperproliferation and tumoral growth. The marine natural products meridianins and variolins, characterized as CDK inhibitors, display a kinase-inhibitory activity associated with cytotoxic effects. In order to improve selectivity and efficiency of these CDK inhibitors, a series of hybrid compounds called meriolins have been synthesized. The potential antitumoral activity of meriolins was investigated in vitro on glioma cell lines (SW1088 and U87), native neural cells, and a human endothelial cell line (HUV-EC-C). The impact of intraperitoneal or intratumoral administrations of meriolin 15 was evaluated in vivo on 2 different nude mice-xenografted glioma models. Meriolins 3, 5, and 15 exhibited antiproliferative properties with nanomolar IC50 and induced cell-cycle arrest and CDK inhibition associated with apoptotic events in human glioma cell lines. These meriolins blocked the proliferation rate of HUV-EC-C through cell cycle arrest and apoptosis. In vivo, meriolin 15 provoked a robust reduction in tumor volume in spite of toxicity for highest doses, associated with inhibition of cell division, activation of caspase 3, reduction of CD133 cells, and modifications of the vascular architecture. Meriolins, and meriolin 15 in particular, exhibit antiproliferative and proapoptotic activities on both glioma and intratumoral endothelial cells, constituting key promising therapeutic lead compounds for the treatment of glioblastoma.

International audience; Despite a standard of care combining surgery, radiotherapy (RT), and temozolomide chemotherapy, the average overall survival (OS) of glioblastoma patients is only 15 months, and even far lower when the patient cannot benefit from this combination. Therefore, there is a strong need for new treatments, such as new irradiation techniques. Against this background, carbon ion hadrontherapy, a new kind of irradiation, leads to a greater biological response of the tumor, while minimizing adverse effects on healthy tissues in comparison with RT. As carbon ion hadrontherapy is restricted to RT-resistant patients, photon irradiation resistance biomarkers are needed. Long telomeres and high telomerase activity have been widely associated with photon radioresistance in other cancers. Moreover, telomere protection, telomere function, and telomere length (TL) also depend on the shelterin protein complex (TRF1, TRF2, TPP1, POT1, TIN2, and hRAP1). We thus decided to evaluate an enlarged telomeric status (TL, telomerase catalytic subunit, and the shelterin component expression level) as a potential radioresistance biomarker in vitro using cellular models and ex vivo using patient tumor biopsies. In addition, nothing was known about the role of telomeres in carbon ion response. We thus evaluated telomeric status after both types of irradiation. We report here a significant correlation between TL and the basal POT1 expression level and photon radioresistance, in vitro, and a significant increase in the OS of patients with long telomeres or a high POT1 level, in vivo. POT1 expression was predictive of patient response irrespective of the TL. Strikingly, these correlations were lost, in vitro, when considering carbon irradiation. We thus propose (1) a model of the implications of telomeric damage in the cell response to both types of irradiation and (2) assessment of the POT1 expression level and TL using patient tumor biopsies to identify radioresistant patients who could benefit from carbon hadrontherapy.

International audience; Collapsin Response Mediator Protein 5 (CRMP5) belongs to a family of five cytosolic proteins highly expressed in the developing nervous system but downregulated in the adult brain. When expressed at the adult stage, CRMP5 is involved in neurological disorders. Indeed, CRMP5 is found expressed in cancer cells of some brain tumors, such as glioblastoma, or in small cell lung cancer causing paraneoplastic neurological syndromes as a result of cancer-induced auto-immune processes. Nevertheless, its role in cancer pathology is still obscure. Here, we show a new short isoform, derived from C-terminal processing of CRMP5, presenting a nuclear localization both in human glioblastoma, and in cancer cell lines (H69, GL15). By mutational analysis, we demonstrate that nuclear translocation occurs via nuclear localization signal (NLS), where the essential residue for nuclear location is K391. Direct CRMP5/ tubulin interaction, previously shown during brain development, does not occur for cytosolic CRMP5 in pathological conditions, leading to the suggestion that in cancer cells CRMP5 is not sequestered in the cytosol; therefore it may undergo C-terminal truncation allowing the exposure of the NLS for active translocation. Moreover, we show that the function associated with the CRMP5 nuclear targeting is an increase of cell proliferation activity.

Mucopolysaccharidosis IIIB is a lysosomal disease characterized by a severe neurological deterioration, the pathophysiological mechanisms of which are poorly understood. Recently FGF pathway was shown to be altered leading us to explore a downstream target involved in brain development: the collapsin response mediator protein-1 (CRMP-1). CRMP-1 transcript level was normal but a cleavage of CRMP-1 was observed with an abnormal expression of the truncated form until adult age. This truncated CRMP-1 protein could play a role in post-natal cortex maturation and be involved in neuronal alterations occurring in lysosomal diseases.