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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 9, 2005 as doi:10.1096/fj.04-3178fje. |
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,1
Schools of
* Medicine and
Medical Sciences, College of Life Sciences and Medicine, University of Aberdeen, Medical School, Foresterhill, Aberdeen, UK
1Correspondence: School of Medicine, College of Life Sciences and Medicine, University of Aberdeen, Medical School, Foresterhill, Aberdeen, AB25 2ZD, UK. E-mail: a.schofield{at}abdn.ac.uk
SPECIFIC AIMS
Docetaxel is one of the most active drugs used to treat breast cancer and its cellular target is the microtubule, specifically the ß-tubulin subunit that comprises a series of isotypes and can modulate function. This study has examined the alterations in ß-tubulin isotypes (mRNA and proteins) in vitro and has sequenced the ß-tubulin gene to determine whether there were mutations, both of which may represent important mechanisms of acquired resistance to docetaxel in breast cancer cells.
PRINCIPAL FINDINGS
1. Differential expression of mRNA ß-tubulin isotypes in docetaxel-resistant estrogen receptor-positive and estrogen receptor-negative breast cancer cells
Human breast cancer cell lines MCF-7 (estrogen receptor-positive) and MDA-MB-231 (estrogen receptor-negative) were made resistant to docetaxel by short-term in vitro exposure to docetaxel. MCF-7 docetaxel-resistant sublines (MCF-7 TAX30) demonstrated a 666-fold greater resistance to docetaxel than MCF-7 cells. Furthermore, MDA-MB-231 docetaxel-resistant sublines (MDA-MB-231 TAX30) showed a 1375-fold greater resistance than MDA-MB-231 cells.
To determine whether an altered gene expression level of a specific ß-tubulin isotype is correlated with resistance to docetaxel in breast cancer cells, mRNA levels were identified by semiquantitative RT-PCR analysis. The experiments were repeated in triplicate with RNA isolated from three independent extractions. Flow cytometric analysis was performed to demonstrate cell synchrony and that cells were in the same phase of cell cycle, since ß-tubulin isotypes are differentially expressed during the cell cycle. There were no significant differences between the percentage of cells in G1 phase and G2/M phase in either MCF-7 or MDA-MB-231 breast cancer cells sensitive or resistant to docetaxel.
For the MCF-7 (estrogen receptor-positive) cells, RT-PCR analysis showed increased expression of class I (2.59±0.68-fold), II (1.64±0.15-fold), III (2.21±0.35-fold), IVa (3.55±0.96-fold), and IVb (1.10±0.04) ß-tubulin mRNA levels in MCF-7 TAX30 cells compared with MCF-7 cells sensitive to docetaxel. In contrast, class VI ß-tubulin mRNA expression was decreased in MCF-7 TAX30 cells (4.69±1.59-fold).
In MDA-MB-231 (estrogen receptor-negative) cells, there was an increase in class II (1.54±0.09-fold), III (1.07±0.16-fold), IVb (4.20±0.33-fold), and VI (10.53±3.29-fold) ß-tubulin mRNA expression in the MDA-MB-231 TAX30 docetaxel-resistant sublines. Class I (1.18±0.08-fold) and class IVa (1.78±0.30-fold) ß-tubulin mRNA expression was decreased in MDA-MB-231 TAX30 cells.
2. Differential expression of ß-tubulin isotypes at the protein level in docetaxel-resistant estrogen receptor-positive and estrogen receptor-negative breast cancer cells
Levels of the ß-tubulin protein isotypes were measured by Western analyses. Proteins were extracted from the cell lines and separated by electrophoresis through 12% polyacrylamide gels. Each membrane was incubated with 1:100 dilution (in 5% (w:v) milk/Tris-buffered saline with 10% (v:v) Tween 20) of either mouse monoclonal anti-human class I, II, III, IV ß-tubulin antibodies or 1:5000 ß-actin (internal loading control) for 1 h 45 min at room temperature. It was not possible to examine the protein expression of class VI ß-tubulin because no antibody was available commercially. The experiments were repeated in triplicate with protein isolated from three independent extractions.
In the MCF-7 (estrogen receptor-positive) cells, it was observed that in the docetaxel-resistant sublines ß-tubulin isotype proteins were increased for class I (1.76±0.19-fold), class II (2.10±0.12-fold), class III (2.91±0.76-fold), and class IV (2.81±0.71-fold) isotypes. The increased mRNA expression observed in these docetaxel-resistant cells correlates with a subsequent increased expression of the corresponding proteins.
In MDA-MB-231 (estrogen receptor-negative) cells a different pattern emerged. In the docetaxel-resistant sublines there was a decrease in levels of the ß-tubulin isotypes for class I (1.28±0.27-fold), class II (1.30±0.11-fold), and class III (1.54±0.47-fold). However, there was an increased level of the class IV ß-tubulin isotype (2.26±0.24-fold) compared with the docetaxel-sensitive cells. When comparing this with the expression of mRNA, there was not a correlation when ß-tubulin isotypes class II and III were examined. Although class II and class III ß-tubulin isotypes were increased at the mRNA level, there was not a resultant increase in translation into proteins, as detected by Western analysis.
3. Mutations in the ß-tubulin gene are not associated with acquired resistance to docetaxel in breast cancer cells
We have demonstrated that alterations in the expression of ß-tubulin isotypes (both at the mRNA and the protein level) occur in association with acquired resistance to docetaxel. This is important because there is evidence from other studies to suggest that this may be an important mechanism of resistance by modulating microtubular components and dynamics. An alternative mechanism by which microtubular function can be disrupted, and hence sensitivity to docetaxel modulated, is through mutation in the ß-tubulin gene. This is important because mutations in the ß-tubulin gene have been shown to be associated with resistance in patients receiving the taxane paclitaxel and may represent a potential mechanism of resistance in these studies.
Genomic DNA was isolated from the cell lines and the coding region of the ß-tubulin gene was examined using automated fluorescent sequencing. There was no evidence of alteration to the DNA sequence of the ß-tubulin gene in the docetaxel-resistant sublines when compared with their parental docetaxel-sensitive cells.
CONCLUSIONS AND SIGNIFICANCE
An important advance in the treatment of breast cancer has been the use of the taxane, docetaxel. Although up to 50% of patients treated with docetaxel demonstrate a clinical response, tumors of these patients may have either an inherent resistance to docetaxel or develop resistance subsequently, having been initially sensitive to docetaxel. The mechanisms of action of docetaxel remain to be fully elucidated, but of particular importance is its interaction with the microtubular system, which are heterodimers of 
-tubulin and ß-tubulin subunits. Electron crystallographic data has demonstrated docetaxel binds to ß-tubulin subunits, which stabilizes and prevents microtubules depolymerizing. This disruption of mitotic spindle formation results in inhibition of cell division and leads to cell death.
It is apparent that the ß-tubulin gene and its resultant proteins, which exists as six different isotypes in mammalian cells (class I, II, III, IVa, IVb, and VI), may be important in resistance to docetaxel. These different isotypes can be variably and differentially expressed in malignant cells. Preliminary studies have shown a differential expression of ß-tubulin isotypes to affect the ability of chemotherapeutic agents binding to the microtubular system. Increased expression of class III ß-tubulin isotype can affect the stability of the microtubular system and impair the stabilizing effects of taxanes, thus preventing their anti-tumor effects.
The role of differences in ß-tubulin isotype expression in resistance to docetaxel in breast cancer is unclear. A preliminary study in patients with breast cancer suggested that increased class III ß-tubulin isotype mRNA expression may indicate patients who were unlikely to respond to docetaxel. However, the expressions of the other isotypes and their corresponding protein expression was not evaluated nor was the gene for ß-tubulin sequenced to determine whether or not there were mutations present which may be of important significance.
Our study has taken forward previous studies and is the first to demonstrate that an altered expression of ß-tubulin isotypes is associated with acquired resistance to docetaxel in human breast cancer cells. Our study also showed differences in the profile of these changes between estrogen receptor-positive cells and estrogen receptor-negative cells that were resistant to docetaxel. In MCF-7 docetaxel-resistant cells there was an increase protein expression of classes I, II, III, and IV ß-tubulin. MDA-MB-231 docetaxel-resistant cells predominantly exhibited decreased protein expression of classes I, II, and III ß-tubulin isotypes, but with an increase in class IV ß-tubulin isotype.
The existence of isotypes of ß-tubulin, with tissue-specific expression, suggests that different isotypes may have functional significance. There is aberrant expression of class III ß-tubulin (neuron-specific), in tumors of non-neuronal origin and in breast tumors there is predominance of class II ß-tubulin, but functional significance of these changes is unclear. It is possible, that relative compositions of components of ß-tubulin dictate, or predict, tumor cell behavior and responses to chemotherapy acting through microtubules.
To support this concept, it has been confirmed that differences in ß-tubulin compositions influence regulation of the dynamics of the microtubule system. Microtubules composed of class III ß-tubulin dimers are more dynamic (they are less stable and have a spontaneous tendency toward depolymerization) than those composed of other ß-tubulin isotypes and alter the sensitivity of cells to chemotherapeutic agents.
Although previous studies have not examined the relationship between the expression of ß-tubulin isotypes and resistance to breast cancer cells in vitro, there is evidence to support possible involvement in, or association with, resistance to other chemotherapeutic agents and tumor types. In cancer cells resistant to paclitaxel, there was an increased expression of mRNA for classes I, II, III, IVa ß-tubulin isotypes. A small study has evaluated ß-tubulin isotypes in vivo in 11 ovarian tumor specimens from patients who had chemotherapy and found an increase in mRNA for classes I, III, and IVa ß-tubulin isotypes in chemotherapy-resistant tumors. There was no evaluation in these studies, however, as to whether these effects at the mRNA level were translated to changes in the different ß-tubulin proteins themselves and this clearly is the key point.
In our study, where the ß-tubulin protein was actually examined, we noted an increased expression of class I, II, III, and IV ß-tubulins in MCF-7 docetaxel-resistant breast cancer cells. While there are some differences in the relative increases in expressions of mRNA and protein for individual isotypes, they do correspond and are similar. This suggests that the changes in mRNA are being translated to the protein level.
However, there was a different pattern in MDA-MB-231 docetaxel-resistant cells, where only the class IV ß-tubulin protein was increased but class I, II and III ß-tubulin protein expressions were decreased. In particular, however, the major change was the increased expression of class IV protein (by a factor of 2.26), the antibody for which is detecting the combined protein product of the mRNA isotypes IVa (decreased by a factor of 1.78) and class IVb (increased by a factor of 4.20). The variability between mRNA expression and protein expression of other ß-tubulin isotypes indicates the complicated possible post-transcriptional changes that are well recognized. This, therefore, illustrates the importance of assessing protein expression with mRNA expression when trying to elucidate these molecular mechanisms.
Our data confirm and extend those of previous studies with regard to differences in ß-tubulin mRNA expression and is the first to demonstrate this in breast cancer cells resistant to docetaxel. Clearly, there are differences between the two cell lines in terms of patterns of expression of mRNAs and proteins. However, one common finding was that there was an increased expression at the protein level of the class IV ß-tubulin isotype. As proteins are the key effector molecules at the cellular level, this may represent a common mechanism of resistance to docetaxel in breast cancer cells. These differences, however, may also be specific to tumor and cell type, but further studies are required to define these relationships more fully.
One of the limitations of the present study may be whether an in vitro model of docetaxel resistance reflects changes that occur in vivo. Nevertheless, an important study has shown that the patterns of gene expression in breast cancer cells in vitro, exposed to chemotherapy (doxorubicin and 5-fluorouracil), was similar to those observed in patients with breast tumors treated with these drugs and mitomycin C. Another point to consider is that it is unclear whether the altered expression of specific ß-tubulin isotypes is a consequence of the exposure of cells to docetaxel or is causative of the resistant phenotype. The changes in ß-tubulin expression may arise as a direct action of docetaxel or via a secondary mechanism, which remains to be clarified. However, while other mechanisms may be important in resistance to docetaxel (for example, P-glycoprotein overexpression), in the case of these cells we have shown that inhibition of P-glycoprotein function does not fully restore sensitivity to docetaxel. However, the evidence as discussed above would suggest a mechanism whereby different compositions of tubulin may result in differential effects, and hence variable anti-tumor activities of docetaxel (Fig. 1
).
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Our results now require evaluation in the clinical setting. It will be important to determine whether these ß-tubulin isotype profiles of tumor biopsies could be used to indicate which patients receiving docetaxel would be most likely to be resistant to it before treatment commenced. This concept of molecular profiling of tumors before treatment starts is important, and in our preliminary clinical studies we showed that patients whose tumors are negative for estrogen receptor and Bcl-2 have a higher likelihood of responding to chemotherapy. Furthermore, Chang and colleagues have used a 92 gene expression profile to determine which tumors would be most likely to respond to docetaxel. However, this did not include an assessment of protein expression.
In conclusion, we have shown for the first time that in acquired resistance there is altered expression of ß-tubulin isotypes, not just at the mRNA level but at the protein level, in docetaxel-resistant breast cancer cells. A specific ß-tubulin expression profile may prove to be important in determining exactly how docetaxel will interact with the microtubular system and thus determine the effectiveness in terms of anti-tumor actions. This requires further study, as it may be fundamentally important in understanding the molecular mechanisms of resistance to docetaxel.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3178fje;
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