Differential expression of tubulin tyrosine ligase-like 7 in human epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic cancer (1). We performed discovery of genes associated with epithelial ovarian cancer and of the high-grade serous ovarian cancer (HGSC) subtype, using published and public microarray data (2, 3) to compare global gene expression profiles of normal ovary or fallopian tube with that of primary tumors from women diagnosed with epithelial ovarian cancer or HGSC. We identified the gene encoding tubulin tyrosine ligase-like 7, TTLL7, as among the genes whose expression was most different in epithelial ovarian cancer as compared to the normal fallopian tube. TTLL7 expression was significantly lower in high-grade serous ovarian tumors relative to normal fallopian tube. TTLL7 expression correlated with overall survival in patients with ovarian cancer. These data indicate that expression of TTLL7 is perturbed in epithelial ovarian cancers broadly and in ovarian cancers of the HGSC subtype. TTLL7 may be relevant to pathways underlying ovarian cancer initiation (transformation) or progression.

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

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 (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. Conclusions These 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.

α-Tubulin detyrosination impairs mitotic error correction by suppressing MCAK centromeric activity

Incorrect kinetochore–microtubule attachments during mitosis can lead to chromosomal instability, a hallmark of human cancers. Mitotic error correction relies on the kinesin-13 MCAK, a microtubule depolymerase whose activity in vitro is suppressed by α-tubulin detyrosination—a posttranslational modification enriched on long-lived microtubules. However, whether and how MCAK activity required for mitotic error correction is regulated by α-tubulin detyrosination remains unknown. Here we found that detyrosinated α-tubulin accumulates on correct, more stable, kinetochore–microtubule attachments. Experimental manipulation of tubulin tyrosine ligase (TTL) or carboxypeptidase (Vasohibins-SVBP) activities to constitutively increase α-tubulin detyrosination near kinetochores compromised efficient error correction, without affecting overall kinetochore microtubule stability. Rescue experiments indicate that MCAK centromeric activity was required and sufficient to correct the mitotic errors caused by excessive α-tubulin detyrosination independently of its global impact on microtubule dynamics. Thus, microtubules are not just passive elements during mitotic error correction, and the extent of α-tubulin detyrosination allows centromeric MCAK to discriminate correct vs. incorrect kinetochore–microtubule attachments, thereby promoting mitotic fidelity.

The Structure of MT189-Tubulin Complex Provides Insights into Drug Design

Background: Drugs that interfere with microtubule dynamics are used widely in cancer chemotherapy. Microtubules are composed of αβ-tubulin heterodimers, and the colchicine binding site of tubulin is an important pocket for designing tubulin polymerization inhibitors. We have previously designed and synthesized a series of colchicine binding site inhibitors (CBSIs). However, these compounds showed no anticancer activity in vivo. Then, we have used a deconstruction approach to obtain a new derivative MT189, which showed in vivo anticancer activity. Methods: We crystallized a protein complex including two tubulins, one stathmin-like domain of RB3 and one tubulin tyrosine ligase, and soaked MT189 into the crystals. We collected the diffraction data and determined the tubulin-MT189 structure to 2.8 Å. Results: Here, we report the crystal structure of tubulin complexed with MT189, elucidate how the small-molecular agent binds to tubulin and inhibits microtubule assembly, and explain previous results of the structure-activity-relationship studies. Conclusion: The tubulin-MT189 complex structure reveals the interactions between this agent and tubulin and provides insights into the design of new derivatives targeting the colchicine binding site.