Based on the role of Tau in microtubule stabilization, it has been hypothesized that Tau competes with taxanes for microtubule binding sites. In this model, elevated Tau expression leads to decreased taxane binding to microtubules and in turn, decreased taxane efficacy and suboptimal clinical response to taxane-based chemotherapy regimens. Previously published studies have reported that Tau is one of the genes that discriminate between breast cancer cases with pathologic complete response (pCR) and those with residual disease following paclitaxel-based chemotherapy [10
]. However, while Tau-mediated modulation of taxane response was an intriguing hypothesis, it remained unclear whether Tau overexpression represented a true mechanism of taxane resistance or was a marker of another biological phenomenon. In addition, the role of particular Tau isoforms was poorly understood. In this study, we sought to address these questions by studying the functional role of Tau in modulating taxane response by both gene silencing and overexpression, and characterizing the expression of the various Tau isoforms in a panel of taxane resistant breast cancer cells.
Tau is encoded by a single-copy gene which produces three transcripts of 2, 6 and 9 kb. These transcripts are differentially expressed and localized depending on the cell type and stage of maturation, and produce multiple alternatively spliced isoforms. Taking into account all of the splice sites, Tau can hypothetically produce at least 30 variants by splicing alone, excluding further post-translational modifications of each isoform [19
]. The probe sets used in gene expression studies that originally identified Tau as a potential modulator of taxane response targeted Tau domains shared by all isoforms. We sought to characterize the expression of each of the six known major Tau splice variants in our taxane resistant breast cancer variants (). Our results demonstrate that T-47D and MDA-MB-231 taxane resistant variants express higher levels of all Tau isoforms compared to parental cells both at the protein level () as well as the mRNA level ().
To further characterize the expression of Tau isoforms, we sought to determine whether the breast cancer variants express exons 4A, 6 and 8, which are typically absent in the human brain. Interestingly, we showed that exon 6, but not exons 4A and 8, is expressed in 0N, 1N and 2N Tau isoforms in breast cancer cells (). Exon 6 is an alternatively spliced cassette whose expression profile differs from that of other regulated exons, implying the existence of distinct regulatory factors. Inclusion of the proline rich exon 6 results in a Tau protein containing a more rigid and extended hinge region, which might alter microtubule spacing [32
]. Exon 6 may be involved in modulating the dynamicity or extent of the microtubule network. However, it is not clear whether its inclusion in the breast cancer variants has a functional significance. The lack of expression of exon 8 (data not shown) is consistent with the finding that no human tissue has yet been demonstrated to express exon 8. Exon 4A is the longest exon and is exclusively present in the 9kb Tau transcript. We have shown that exon 4A is expressed in skeletal muscle and heart but not other types of tissues, including our breast cancer variants (data not shown).
Because Tau overexpression was found in four of our breast cancer taxane resistant variants, we sought to ascertain the functional role of Tau via knockdown and overexpression experiments. It should be noted that two of these variants also expressed P-gp, and that taxane resistance in the P-gp positive variants was completely reversed by PSC. Since the MCF-7 parental cell line expressed relatively high baseline levels of Tau, we used it for the stable shRNA mediated Tau silencing experiments. Our results demonstrated that the depletion of Tau did not alter cellular response to taxanes (). We also knocked down Tau in the human ovarian carcinoma OVCAR-3 cell line, which expresses moderate baseline levels of Tau. We again showed that although Tau was successfully depleted (), the sensitivity to taxanes was not affected. To confirm our findings, we used eight independent siRNAs including two siRNAs previously published by Rouzier et al
]. Although we observed >90% Tau silencing, taxane sensitivity was not altered relative to wild-type and non-targeting controls in both the MCF-7 and ZR-75-1 breast cancer cells (). This is in contrast to other reports, that down regulation of Tau by transient siRNA transfection conferred sensitivity to taxanes in these two cell lines [10
There are important differences between our set of experiments and those reported by Rouzier et al.
] and Wagner et al
]. First, we used both an shRNA approach via lentiviral delivery, which allowed us to select cells with stable Tau knockdown, as well as a transient siRNA transfection method. Second, our drug exposure was for 72 hr for the SRB cell proliferation assays rather than 48 hr, allowing cells to undergo at least 3 cell divisions in the presence of drug. Third, we confirmed our results with a clonogenic assay to assess cell survival after drug exposure rather than the less reliable tetrazolium based assay or CellTiter-Glo luminescent cell viability assays used in the prior studies.
Tau belongs to the same family of proteins as MAP4; therefore, we hypothesized that perhaps down regulation of Tau is compensated by changes in expression of other microtubule associated proteins or tubulin proteins. We examined the expression of MAP4, stathmin-1, and tubulin isoforms by Western blot and did not show any significant changes in expression (). We also measured the kinetics of taxane-driven tubulin polymerization in MCF-7 cells knocked down in Tau, demonstrating no difference in comparison to parental or luciferase shRNA controls (data not shown). Altogether, these results show that depletion of Tau does not confer taxane sensitivity, and suggest that Tau overexpression in taxane resistant breast cancer clinical specimens may be an epiphenomenon associated with another taxane resistance mechanism(s).
To further evaluate a possible causal relationship between Tau overexpression and taxane resistance, we developed three T-47D clones that stably express Tau-3R, three clones stably expressing Tau-4R, and three clones stably expressing vector alone. We hypothesized that if Tau indeed competes with taxanes for binding to microtubules, then cell lines overexpressing Tau should show resistance to taxanes. Our results show that the overexpression of Tau did not result in resistance to taxanes (). In fact, some of the clones overexpressing Tau showed slightly more sensitivity to taxanes compared to vector alone.
Tau expression is reported to be regulated by estrogen. The Tau gene contains an imperfect ER response element upstream of its promoter, and Tau expression has been induced in an estrogen-dependent fashion [33
]. Tau levels were substantially higher in our T-47D/T×TP50 and MDA-31/T×TP50 variants, as measured by quantitative real time PCR, while estrogen receptor levels did not significantly change from parental cells (data not shown). It was previously suggested that high levels of Tau may be a potential marker of sensitivity to anti-estrogen therapy whereas low levels of Tau may be a potential marker of sensitivity to paclitaxel therapy [35
]. Our findings indicate that upregulation of Tau in taxane resistant variants is not dependent upon upregulation of estrogen receptor.
A recently published, extensive analysis of Tau expression in the NSABP-B28 study confirmed that high Tau expression was associated with estrogen receptor (ER) expression in breast cancers. However, there was no association of Tau expression and clinical benefit from paclitaxel chemotherapy in either ER positive or negative patients (36). Thus, Tau expression does not appear to be a useful biomarker for clinical sensitivity to taxanes in breast cancers.
In summary, in this study we show that modulation of Tau expression (knockdown and overexpression) does not result in altered cellular responses to taxanes in several breast cancer cell lines and one ovarian cancer cell line. These findings indicate that Tau expression is not a mechanism of resistance to taxanes.