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

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.

Local rewiring of genome - nuclear lamina interactions by transcription

“…To extend this analysis to a larger number of genes, we switched to a more flexible gene activation system that does not require a custom-made TALE for every promoter of interest. We chose a previously established human RPE-1 cell line that stably expresses the SunCas-CRISPRa system (Tame et al, 2017; Tanenbaum et al, 2014), in which multiple copies of VP64 can be targeted to a promoter of interest by a single sgRNA. We first used this system to activate NLGN1 , a gene of 885 kb that is located in a LAD.…”

“…The RPE-1 cell line stably expressing SunTag-CRISPRa (Tame et al, 2017) was kindly provided by the R. Medema lab (Netherlands Cancer Institute, Amsterdam) and cultured in DMEM-F12 supplemented with 10% FCS. F121-9 mES cells were kindly provided by J. Gribnau (Erasmus Medical Center, Rotterdam, the Netherlands) and cultured in feeder-free 2i medium according to the 4DNucleome protocol (https://data.4dnucleome.org/protocols/cb03c0c6-4ba6-4bbe-9210-c430ee4fdb2c/).…”

Genetics and Expression Profile of the Tubulin Gene Superfamily in Breast Cancer Subtypes and Its Relation to Taxane Resistance

“…Even more problematic, in BC [ 55 ], clear cell ovarian carcinoma [ 56 ] and melanoma [ 57 ], studies have reported that high expression of βIII-tubulin was linked to sensitivity to taxane-based chemotherapy and not resistance. A very recent report indicated that βIII-tubulin overexpression has a negligible effect on the sensitivity to taxol in cultured cell lines [ 58 ]. We have also shown that in MCF-7 cells selected for taxol resistance, the expression level of βIII-tubulin is not elevated but decreased [ 42 ].…”

“…Even more problematic, high expression of βIII-tubulin was found associated with sensitivity to taxane-based chemotherapy and not resistance in BC [ 42 , 55 ], clear cell ovarian carcinoma [ 56 ] and melanoma [ 57 ]. A very recent report showed that βIII-tubulin overexpression has a negligible effect on the sensitivity to taxol in cultured cell lines [ 58 ]. We have also shown that in MCF-7 cells selected for taxol resistance, the expression level of βIII-tubulin is not elevated but decreased and βII- and βIV-tubulin levels are increased [ 42 ].…”

Persistent Increased Frequency of Genomic Instability in Women Diagnosed with Breast Cancer: Before, during, and after Treatments

“…These antimitotic agents stimulate the polymerization of the microtubules. This site-specific binding seems to antagonize the breakdown of this cytoskeletal key protein, with consequent formation of stable and abnormal microtubules, blocking the progression of G2 and M phase in the cell cycle [ 68 – 70 ].…”