Insights into the role of tubulin ligase-like family member 4 (TTLL4) in selected breast cancer cells -

Abstract

Tubulin Tyrosine Ligase-Like family member 4 (TTLL4) is one of a family of proteins which share a Tubulin Tyrosine Ligase (TTL) domain. TTLL4 functions in polyglutamylating numerous substrates, mainly β-tubulin. It is located in different cell compartments including the cytoskeleton where it is able to interact with tubulin permitting its branching and elongation. TTLL4 was shown to be overexpressed in various types of cancer; however, its expression and putative role in breast cancer has not been fully inspected. After looking into the Cancer Cell Line Encyclopedia (CCLE) database, TTLL4 mRNA expression levels vary across a panel of 58 breast cancer cell lines, but are consistently higher than TTLL4 expression in normal tissue. TTLL4 promotes the initiation of β-tubulin polyglutamylation. Therefore, Paclitaxel (Taxol®), the β-tubulin targeting chemotherapeutic drug, may have an effect on TTLL4 or vice versa. As such we hypothesize TTLL4 expression is upregulated in breast cancer than in normal tissue and that its expression levels impact cellular sensitivity to the tubulin-targeting chemotherapy drug Paclitaxel. It is also possible that TTLL4 expression and-or function are altered in response to treatment with Paclitaxel. For the purpose of this study, we examined the change of TTLL4 expression at the transcript and protein levels under baseline conditions and after Paclitaxel treatment. We assessed the transcript expression of TTLL4 by Real-time PCR, where RNA was extracted from MCF-7 and MDA-MB-231 cells under baseline conditions. This expression was also determined post treatment with Paclitaxel for 8hrs, 2hrs and 30 mins at the following concentrations: 0nM, 14nM, 55nM and 220nM (IC50) for MCF-7 cells, and 0nM, 20nM, 80nM and 320nM (IC50) for MDA-MB-231 cells. Under baseline conditions, MCF-7 cells showed higher TTLL4 expression than MDA-MB-231 cells. Upon treatment with Paclitaxel, both cell lines showed no significant change in TTLL4expression. To examine the protein expression of