Neurofilaments (NF) are the most abundant cytoskeletal component of large myelinated axons from adult central and peripheral nervous system. Here, we provide an overview of the complementary approaches, including biochemistry, cell biology and transgenic technology that were used to investigate the assembly, axonal transport and functions of NF in normal and pathological situations. Following their synthesis and assembly in the cell body, NFs are transported along the axon. This process is finely regulated via phosphorylation of the carboxy-terminal part of the two high-molecular-weight subunits of NF. The correct formation of an axonal network of NF is crucial for the establishment and maintenance of axonal calibre and consequently for the optimisation of conduction velocity. The frequent disorganisation of NF network observed in several neuropathologies support their contribution. However, despite the presence of NF mutations found in some patients, the exact relations between these mutations, the abnormal NF organisation and the pathological process remain a challenging field of investigation.

No abstract

Despite aggressive treatment regimes, glioma remains a largely fatal disease. Current treatment limitations are attributed to the precarious locations within the brain where such tumors grow, their highly infiltrative nature precluding complete resection and lack of specificity among agents capable of attenuating their growth. Here, we show that in vitro, glioma cells of diverse origins internalize a peptide encompassing a tubulin-binding site (TBS) on the neurofilament light protein. The internalized peptide disrupts the microtubule network, inhibits migration and proliferation, and leads to apoptosis. Using an intracerebral transplant model, we show that most, if not all, of these responses to peptide exposure also occur in vivo. Notably, a single intratumor injection significantly attenuates tumor growth, while neither peptide uptake nor downstream consequences are observed elsewhere in the host nervous system. Such preferential uptake suggests that the peptide may have potential as a primary or supplementary glioblastoma treatment modality by exploiting its autonomous microtubule-disrupting activity or engaging its capacity to selectively target glioma cells with other cell-disrupting cargos.

No abstract

We previously described a neurofilament derived cell-penetrating peptide, NFL-TBS.40-63, that specifically enters in glioblastoma cells where it disturbs the microtubule network both in vitro and in vivo. The aim of this study is to test whether this peptide can increase the targeted uptake by glioblastoma cells of lipid nanocapsules filled with Paclitaxel, and thus can increase their anti-proliferation in vitro and in vivo. Here, using the drop tensiometry we show that approximately 60 NFL-TBS.40-63 peptides can bind to one 50 nm lipid nanocapsule. When nanocapsules are filled with a far-red fluorochrome (DiD) and Paclitaxel, the presence of the NFL-TBS.40-63 peptide increases their uptake by glioblastoma cells in culture as evaluated by FACS analysis, and thus reduces their proliferation. Finally, when such nanocapsules were injected in mice bearing a glioma tumour, they are preferentially targeted to the tumour and reduce its progression. These results show that nanocapsules functionalized with the NFL-TBS.40-63 peptide represent a powerful drug-carrier system for glioma targeted treatment.

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.

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 over-expression 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.

Glioblastoma 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.