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Journal of Experimental & Clinical Cancer Research

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Open Access 01-12-2020 | Metastasis | Research

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

Authors: Julia Arnold, Juliana Schattschneider, Christine Blechner, Christoph Krisp, Hartmut Schlüter, Michaela Schweizer, Marcus Nalaskowski, Leticia Oliveira-Ferrer, Sabine Windhorst

Published in: Journal of Experimental & Clinical Cancer Research | Issue 1/2020

Abstract

Background

The 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.

Methods

Screening 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.

Results

Among 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 (TTLL4^plus) in MDA-MB231 and MDA-MB468 breast cancer cells (TTLL4^plus cells) significantly increased polyglutamylation of β-tubulin. Moreover, trafficking of secretory vesicles and MVBs was increased in TTLL4^plus cells. EVs derived from TTLL4^plus cells promote adhesion of MDA-MB231 and MDA-MB468 cells to hCMEC/D3 cells and increase permeability of hCMEC/D3 cell layer.

Conclusions

These data suggest that TTLL4-mediated microtubule polyglutamylation alters exosome homeostasis by regulating trafficking of MVBs. The TTLL4^plus-derived EVs may provide a pre-metastatic niche for breast cancer cells by manipulating endothelial cells of the BBB.

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Title: Tubulin Tyrosine Ligase Like 4 (TTLL4) overexpression in breast cancer cells is associated with brain metastasis and alters exosome biogenesis
Authors: Julia Arnold
Juliana Schattschneider
Christine Blechner
Christoph Krisp
Hartmut Schlüter
Michaela Schweizer
Marcus Nalaskowski
Leticia Oliveira-Ferrer
Sabine Windhorst
Publication date: 01-12-2020
Publisher: BioMed Central
Keywords: Metastasis
Breast Cancer
Breast Cancer
Published in: Journal of Experimental & Clinical Cancer Research / Issue 1/2020
Electronic ISSN: 1756-9966
DOI: https://doi.org/10.1186/s13046-020-01712-w

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