TUBB3 overexpression has a negligible effect on the sensitivity to taxol in cultured cell lines.

Mihoko A Tame,Anna G Manjón,Jonne A Raaijmakers,René H Medema,Daria Belokhvostova

doi:10.18632/oncotarget.17740

Mihoko A Tame, Anna G Manjón + Show 3 more

Open Access

https://doi.org/10.18632/oncotarget.17740

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Abstract

Microtubules are cellular targets for a variety of anticancer therapies because of their critical function in mitosis. Taxol belongs to a class of microtubule targeting agents that suppresses microtubule dynamics and interferes with the functioning of the mitotic spindle, thereby effectively blocking cell cycle progression of rapidly proliferating tumor cells. Despite its antitumor activity, drug resistance remains a common obstacle in improving its overall clinical efficacy. Previous studies have shown that the expression of a specific β-tubulin isotype, βIII-tubulin/TUBB3, is dysregulated in drug-refractory tumors. However, whether enhanced TUBB3 expression is directly involved in promoting taxol resistance remains a subject of debate. Here, we have used several approaches to assess the functional relation of TUBB3 overexpression and taxol resistance. First, we generated a number of taxol-resistant cell lines, to find that TUBB3 expression was elevated in a resistant cell line (RPE-20) derived from untransformed retinal pigment epithelial (RPE) cells, but the abundance of TUBB3 remained unchanged in four other cell lines after taxol treatment. However, although RPE-20 cells displayed enhanced TUBB3 levels, we find that simultaneous up-regulation of the P-glycoprotein (P-gP) drug-efflux pump is responsible for the resistance to taxol. Indeed, we could show that TUBB3 levels were dynamically regulated upon taxol exposure and withdrawal, unrelated to the resistance phenotype. Next, we generated cell lines in which we could induce robust overexpression of TUBB3 from its endogenous locus employing the CRISPRa system. We demonstrate that solely enhancing TUBB3 expression results in a very minor decrease in the sensitivity to taxol. This was further substantiated by selective depletion of TUBB3 in a series of breast cancer cell lines expressing high levels of TUBB3. We find that TUBB3 depletion had a minimal effect on the sensitivity to taxol in one of these cell lines, but had no effect in all of the others. Based on these findings we propose that TUBB3 overexpression can only marginally affect the sensitivity to taxol in cultured cell lines.

Highlights

Microtubules, polymers of α/β heterodimers, are dynamic cytoskeletal structures that are essential for many cellular functions, including cell movement, intracellular transport and cell division
This suggests that P-gP is an important driver of taxol resistance in retinal pigment epithelial (RPE) cells and their proliferative capacity is severely compromised when forced to adapt to taxol through alternative mechanisms
These results suggest that while the induction of P-gP activity provides the major mechanism of taxol-resistance in RPE cells, they may have adapted their MT dynamics to the stabilizing effect of taxol, albeit that the contribution of the altered MT dynamics to the overall sensitivity to taxol appears to be very minor

Summary

Introduction

Microtubules, polymers of α/β heterodimers, are dynamic cytoskeletal structures that are essential for many cellular functions, including cell movement, intracellular transport and cell division. Since uncontrolled cycles of cell divisions and chronic cell proliferation is a hallmark of many cancers [2], microtubules (MTs) have been exploited www.impactjournals.com/oncotarget as therapeutic targets to curb proliferation of transformed cells using a variety of microtubule-targeting agents (MTAs), known as anti-mitotics [3]. Paclitaxel (hereafter referred to as taxol) is an MTA that suppresses microtubule dynamics and thereby disrupts mitotic progression. This mode of action is thought to be responsible for the potent ability of taxol to prevent cell proliferation in tumors [4, 5]. Taxol is used for the treatment of a variety of solid tumors, such as ovarian, breast and lung cancers [6]. Determining molecular mechanisms of taxol resistance is of great clinical value for the design of treatment plans

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