Tubulin Tyrosine Ligase Like 4 (TTLL4) overexpression in breast cancer cells is associated with brain metastasis and alters exosome biogenesis

Journal of Experimental & Clinical Cancer Research

Michaela Schweizer¹  Juliana Schattschneider²  Marcus Nalaskowski²  Sabine Windhorst²  Julia Arnold²  Christine Blechner²  Leticia Oliveira-Ferrer³  Hartmut Schlüter⁴  Christoph Krisp⁴ 

[1] Core Facility Morphology und Electron Microscopy, Center for Molecular Neurobiology Hamburg, ZMNH, University Medical Center Hamburg-Eppendorf, Falkenried 94, 20251, Hamburg, Germany;Department of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246, Hamburg, Germany;Department of Gynecology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246, Hamburg, Germany;Institute of Clinical Chemistry and Laboratory Medicine, Mass Spectrometric Proteomics, University Medical Center Hamburg-Eppendorf, 20246, Hamburg, Germany;

关键词: Breast cancer;    Metastasis;    Exosome;    Microtubule;    Polyglutamylation;   

DOI  :  10.1186/s13046-020-01712-w

来源: Springer

【 摘 要 】

BackgroundThe survival rate is poor in breast cancer patients with brain metastases. Thus, new concepts for therapeutic approaches are required. During metastasis, the cytoskeleton of cancer cells is highly dynamic and therefore cytoskeleton-associated proteins are interesting targets for tumour therapy.MethodsScreening for genes showing a significant correlation with brain metastasis formation was performed based on microarray data from breast cancer patients with long-term follow up information. Validation of the most interesting target was performed by MTT-, Scratch- and Transwell-assay. In addition, intracellular trafficking was analyzed by live-cell imaging for secretory vesicles, early endosomes and multiple vesicular bodies (MVB) generating extracellular vesicles (EVs). EVs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), Western blotting, mass spectrometry, and ingenuity pathway analysis (IPA). Effect of EVs on the blood-brain-barrier (BBB) was examined by incubating endothelial cells of the BBB (hCMEC/D3) with EVs, and permeability as well as adhesion of breast cancer cells were analyzed. Clinical data of a breast cancer cohort was evaluated by χ2-tests, Kaplan-Meier-Analysis, and log-rank tests while for experimental data Student’s T-test was performed.ResultsAmong those genes exhibiting a significant association with cerebral metastasis development, the only gene coding for a cytoskeleton-associated protein was Tubulin Tyrosine Ligase Like 4 (TTLL4). Overexpression of TTLL4 (TTLL4plus) in MDA-MB231 and MDA-MB468 breast cancer cells (TTLL4plus cells) significantly increased polyglutamylation of β-tubulin. Moreover, trafficking of secretory vesicles and MVBs was increased in TTLL4plus cells. EVs derived from TTLL4plus cells promote adhesion of MDA-MB231 and MDA-MB468 cells to hCMEC/D3 cells and increase permeability of hCMEC/D3 cell layer.ConclusionsThese data suggest that TTLL4-mediated microtubule polyglutamylation alters exosome homeostasis by regulating trafficking of MVBs. The TTLL4plus-derived EVs may provide a pre-metastatic niche for breast cancer cells by manipulating endothelial cells of the BBB.

【 授权许可】

CC BY