Two Antagonistic Microtubule Targeting Drugs Act Synergistically to Kill Cancer Cells.

Paclitaxel is a microtubule stabilizing agent and a successful drug for cancer chemotherapy inducing, however, adverse effects. To reduce the effective dose of paclitaxel, we searched for pharmaceutics which could potentiate its therapeutic effect. We screened a chemical library and selected Carba1, a carbazole, which exerts synergistic cytotoxic effects on tumor cells grown in vitro, when co-administrated with a low dose of paclitaxel. Carba1 targets the colchicine binding-site of tubulin and is a microtubule-destabilizing agent. Catastrophe induction by Carba1 promotes paclitaxel binding to microtubule ends, providing a mechanistic explanation of the observed synergy. The synergistic effect of Carba1 with paclitaxel on tumor cell viability was also observed in vivo in xenografted mice. Thus, a new mechanism favoring paclitaxel binding to dynamic microtubules can be transposed to in vivo mouse cancer treatments, paving the way for new therapeutic strategies combining low doses of microtubule targeting agents with opposite mechanisms of action.

A New Quantitative Cell-Based Assay Reveals Unexpected Microtubule Stabilizing Activity of Certain Kinase Inhibitors, Clinically Approved or in the Process of Approval.

Agents able to modify microtubule dynamics are important anticancer drugs. The absence of microtubules resulting from drug-induced depolymerization is easy to detect. However the detection of a stabilized microtubule network needs specific assays since there is not a significant visual difference between normal and stabilized microtubule networks. Here, we describe a quantitative cell-based assay, suitable for automation, which allows the detection of stabilized microtubules without the need of microscopic examination. The rationale of this assay is based on the drug-induced resistance of the microtubule network to the depolymerizing agent combretastatin A4 and the subsequent detection of the residual microtubules by immunoluminescence. Using this assay to screen a kinase inhibitor library allowed the selection of seven known kinase inhibitors: selonsertib, masatinib, intedanib, PF0477736, SNS-314 mesylate, MPI0479605, and ponatinib. The yet undescribed ability of these inhibitors to stabilize cellular microtubules was confirmed using additional markers of stable microtubules and time-lapse video-microscopy to track individual microtubules in living cells. None of the compounds interacted, however, directly with tubulin. By employing other inhibitors of the same kinases, which have structurally unrelated scaffolds, we determined if the microtubule stabilizing effect was due to the inhibition of the targeted kinase, or to an off-target effect. Many of these inhibitors are clinically approved or currently assayed in phase 2 or phase 3 clinical trials. Their microtubule-stabilizing effect may account for their therapeutic effect as well as for some of their adverse side effects. These results indicate also a possible repurposing of some of these drugs.

[On the road to deciphering the tubulin code: focus on acetylation and detyrosination]. / Déchiffrage du code tubuline - Le voile se lève sur le rôle de l'acétylation et de la détyrosination.

Microtubules are cytoskeletal fibers formed by the assembly of α- and ß-tubulin heterodimers. They contribute to cell morphology, mobility and polarity, as well as to cellular transport processes and cell division. The microtubular network constantly adapts to cellular needs and may be composed of very dynamic or more stable microtubules. To regulate their diverse functions in a spatio-temporal manner, microtubules are subjected to numerous reversible post-translational modifications, which generate the "tubulin code". This review focuses on two modifications characteristic of stable microtubules - acetylation and detyrosination of α-tubulin - and their deregulation in certain pathologies.

Tau regulates the localization and function of End-binding proteins 1 and 3 in developing neuronal cells.

The axonal microtubule-associated protein tau is a well-known regulator of microtubule stability in neurons. However, the putative interplay between tau and End-binding proteins 1 and 3 (EB1/3), the core microtubule plus-end tracking proteins, has not been elucidated yet. Here, we show that a cross-talk between tau and EB1/3 exists in developing neuronal cells. Tau and EBs partially colocalize at extending neurites of N1E-115 neuroblastoma cells and axons of primary hippocampal neurons, as shown by confocal immunofluorescence analyses. Tau down-regulation leads to a reduction of EB1/3 comet length, as observed in shRNA-stably depleted neuroblastoma cells and TAU-/- neurons. EB1/3 localization depends on the expression levels and localization of tau protein. Over-expression of tau at high levels induces EBs relocalization to microtubule bundles at extending neurites of N1E-115 cells. In differentiating primary neurons, tau is required for the proper accumulation of EBs at stretches of microtubule bundles at the medial and distal regions of the axon. Tau interacts with EB proteins, as shown by immunoprecipitation in different non-neuronal and neuronal cells and in whole brain lysates. A tau/EB1 direct interaction was corroborated by in vitro pull-down assays. Fluorescence recovery after photobleaching assays performed in neuroblastoma cells confirmed that tau modulates EB3 cellular mobility. In summary, we provide evidence of a new function of tau as a direct regulator of EB proteins in developing neuronal cells. This cross-talk between a classical microtubule-associated protein and a core microtubule plus-end tracking protein may contribute to the fine-tuned regulation of microtubule dynamics and stability during neuronal differentiation. We describe here a novel function for tau as a direct regulator of End binding (EB) proteins in differentiating neuronal cells. EB1/3 cellular mobility and localization in extending neurites and axons is modulated by tau levels and localization. We provide new evidence of the interplay between classical microtubule-associated proteins (MAPs) and "core" microtubule plus-end tracking proteins (+TIPs) during neuronal development.

Regulation of brain-type creatine kinase by AMP-activated protein kinase: interaction, phosphorylation and ER localization.

AMP-activated protein kinase (AMPK) and cytosolic brain-type creatine kinase (BCK) cooperate under energy stress to compensate for loss of adenosine triphosphate (ATP) by either stimulating ATP-generating and inhibiting ATP-consuming pathways, or by direct ATP regeneration from phosphocreatine, respectively. Here we report on AMPK-dependent phosphorylation of BCK from different species identified by in vitro screening for AMPK substrates in mouse brain. Mass spectrometry, protein sequencing, and site-directed mutagenesis identified Ser6 as a relevant residue with one site phosphorylated per BCK dimer. Yeast two-hybrid analysis revealed interaction of active AMPK specifically with non-phosphorylated BCK. Pharmacological activation of AMPK mimicking energy stress led to BCK phosphorylation in astrocytes and fibroblasts, as evidenced with a highly specific phospho-Ser6 antibody. BCK phosphorylation at Ser6 did not affect its enzymatic activity, but led to the appearance of the phosphorylated enzyme at the endoplasmic reticulum (ER), close to the ER calcium pump, a location known for muscle-type cytosolic creatine kinase (CK) to support Ca²âº-pumping.