Microtubule-associated protein tau: A marker
of paclitaxel sensitivity in breast cancer
Roman Rouziera,b, Radhika Rajanc, Peter Wagnera, Kenneth R. Hessd, David L. Goldd, James Stece,f,g, Mark Ayerse,f,h,
Jeffrey S. Rosse,f,i, Peter Zhangc, Thomas A. Buchholzj, Henry Kuererk, Marjorie Greena, Banu Aruna,
Gabriel N. Hortobagyia, W. Fraser Symmansc, and Lajos Pusztaia,l
Departments ofaBreast Medical Oncology,cPathology,dBiostatistics and Applied Mathematics,jRadiation Oncology, andkSurgical Oncology, University of
Texas M. D. Anderson Cancer Center, Houston, TX 77030;eMillennium Pharmaceuticals, Cambridge, MA 02139;iAlbany Medical College, Albany, NY 12208;
andbDepartment of Gynecological Oncology, Institut Gustave-Roussy, 94805 Villejuif, France
Edited by Lawrence H. Einhorn, Indiana University, Indianapolis, IN, and approved March 15, 2005 (received for review December 2, 2004)
Breast cancers show variable sensitivity to paclitaxel. There is no
used U133A chips to identify genes that are associated with
pathologic complete response (pCR) to preoperative paclitaxel-
containing chemotherapy in stage I–III breast cancer (n ? 82). Tau
was the most differentially expressed gene. Tumors with pCR had
significantly lower (P < 0.3 ? 10?5) mRNA expression. Tissue arrays
from 122 independent but similarly treated patients were used for
validation by immunohistochemistry. Seventy-four percent of pCR
cases were tau protein negative; the odds ratio for pCR was 3.7
(95% confidence interval, 1.6–8.6; P ? 0.0013). In multivariate
analysis, nuclear grade (P < 0.01), age <50 (P ? 0.03), and tau-
negative status (P ? 0.04) were independent predictors of pCR.
Small interfering RNA experiments were performed to examine
whether down-regulation of tau increases sensitivity to chemo-
therapy in vitro. Down-regulation of tau increased sensitivity of
breast cancer cells to paclitaxel but not to epirubicin. Tubulin
polymerization assay was used to assess whether tau modulates
binding of paclitaxel to tubulin. Preincubation of tubulin with tau
resulted in decreased paclitaxel binding and reduced paclitaxel-
induced microtubule polymerization. These data suggest that low
tau expression renders microtubules more vulnerable to paclitaxel
and makes breast cancer cells hypersensitive to this drug. Low tau
expression may be used as a marker to select patients for paclitaxel
therapy. Inhibition of tau function might be exploited as a thera-
peutic strategy to increase sensitivity to paclitaxel.
adjuvant therapy ? drug resistance
breast cancers can improve survival rates (1, 2). There are several
commonly used combination chemotherapy regimens that are
considered acceptable standard adjuvant or neoadjuvant treat-
ments (3, 4). More recently, it has been demonstrated that incor-
poration of paclitaxel into anthracycline-containing chemotherapy
regimens can improve disease-free survival (5). However, the
that only a minority of patients may benefit from inclusion of this
drug. Because inclusion of paclitaxel increases the length, cost, and
potential toxicity of therapy, it would be clinically helpful if physi-
cians could identify patients at the time of diagnosis who are most
likely to benefit from this drug. Currently, no such predictive
Administration of chemotherapy before surgery provides an
opportunity to directly measure tumor response and identify mo-
lecular predictors. Several large retrospective studies have demon-
strated that complete eradication of all invasive cancer from the
breast and axillary lymph nodes after preoperative chemotherapy,
pathologic complete response (pCR), is associated with excellent
cancer-free survival (6, 7). Therefore, molecular predictors of pCR
could help identify individuals who are most likely to benefit from
a particular therapy.
hemotherapy administered either before surgery (neoadju-
vant) or after surgery (adjuvant) for patients with stage I–III
We initiated a pharmacogenomic study to identify genes that
could predict pCR to preoperative sequential weekly paclitaxel
followed by 5-fluorouracil, doxorubicin, and cyclophosphamide
reported in ref. 8. In the current analysis, we used gene expression
that are differentially expressed between cases with pCR and those
gene was microtubule-associated protein tau. Tau mRNA expres-
sion was low in cases with pCR. Next, we performed immunohis-
tochemistry (IHC) on tissue arrays from 122 independent patients
who received similar preoperative chemotherapy to validate this
negative correlation between tau expression and pCR.
Tau protein promotes tubulin polymerization and stabilizes
microtubules (9). We hypothesized that loss of tau expression may
sensitize breast cancer cells to paclitaxel by rendering microtubules
more vulnerable to this drug. To test this hypothesis, we performed
small interfering RNA (siRNA) experiments to knock down tau
expression in breast cancer cells and examined the sensitivity of
these cells to paclitaxel. We also performed tubulin polymerization
assays in the presence or absence of tau and paclitaxel to examine
whether paclitaxel binding is modulated by tau.
Patients and Samples. Fine-needleaspirationsofbreastcancerwere
collected during a prospectively designed pharmacogenomic
marker discovery program at the Nellie B. Connally Breast Center
of University of Texas M. D. Anderson Cancer Center (10). The
program includes marker discovery (n ? 85 patients in single arm
study) and validation (n ? 220 patients in a randomized study)
phases to develop a gene signature-based predictor of pCR to
sequential paclitaxel and T?FAC preoperative chemotherapy for
stage I–III breast cancer. The discovery phase of this program has
been completed. Results on the single most predictive gene that
emerged from the microarray analysis are reported here. Table 1
presents the clinical characteristics of the 82 informative patients
included in the marker discovery analysis. Thirty-three of the 82
cases were also included in our previous report (8). All patients
underwent breast surgery after completion of chemotherapy,
This paper was submitted directly (Track II) to the PNAS office.
ER, estrogen receptor.
fM.A. and J.S. are former employees and J.S.R. is a current employee of Millennium
Pharmaceuticals, which provided partial funding for this study.
gPresent address: Praecis Pharmaceuticals, 830 Winter Street, Waltham, MA 02451-1420.
hPresent address: Clinical Discovery, Bristol-Myers Squibb, P.O. Box 4000, Princeton,
lTo whom correspondence should be addressed at: Department of Breast Medical Oncol-
ogy, University of Texas M. D. Anderson Cancer Center, Unit 424, 1515 Holcombe Boule-
vard, Houston, TX 77030-4009. E-mail: firstname.lastname@example.org.
© 2005 by The National Academy of Sciences of the USA
June 7, 2005 ?
vol. 102 ?
no. 23 ?
and the extent of residual cancer was measured in the surgical
specimen. Grossly visible residual tumor was identified, and rep-
resentative sections were submitted for histopathologic study. In
the absence of grossly visible residual cancer, all slices of the
specimen were radiographed, and areas of radiologically and?or
architecturally abnormal tissue were submitted for histopathologic
study. This study was approved by the institutional review board of
For immunohistochemical validation on independent cases, a
tissue microarray was used that was previously constructed from
formaldehyde-fixed, paraffin-embedded tissues of pretreatment
core needle biopsies left over after diagnosis. All of these patients
with stage I–III breast cancer received preoperative T?FAC che-
motherapy on a previous randomized clinical trial. One hundred
twenty-two patients had sufficient pretreatment tissue available for
tissue array analysis of tau expression. IHC and data analysis were
conducted in accordance with a laboratory protocol approved by
the institutional review board of the University of Texas M. D.
Anderson Cancer Center.
Twelve human breast tumor cell lines (T47D, BT20, ZR75.1,
MCF7, MDA-MB-231, MDA-MB-361, MDA-MB 435, MDA-453,
MDA-468, BT 549, BT 474, and SKBR3) were obtained from the
American Type Culture Collection. All culture media components
were purchased from the M. D. Anderson Tissue Culture Core
Microarray Data Analysis.RNAwasextractedfromFNAsamplesby
using the RNeasy Kit (Qiagen, Valencia, CA). The RNA yield and
11. cRNA was generated by using standard T7 amplification
protocol without second-round amplification. Fragmented and
biotin-labeled cRNA was hybridized to Affymetrix U133A gene
chips overnight at 42°C as described in the Affymetrix technical
DCHIP V1.3 software (http:??dchip.org) was used to generate
probe level intensities and quality measures, including median
intensity, percentage of probe set outliers, and percentage of single
probe outliers for each chip. Three chips failed the quality-control
process, and subsequent analysis was performed on 82 samples.
DCHIP software was used for normalization; this program normal-
izes all arrays to one standard array that represents a chip with
median overall intensity. Normalized gene expression values were
transformed to the log scale (base 10) for analysis. To identify
those with residual disease (n ? 61), genes were ranked by P values
obtained with two-sample, unequal-variance t tests.
were selected for coring, and 5-?m sections (0.6-mm diameter)
were placed in a tissue array slide. Antigen retrieval was performed
with boiling in citrate buffer (pH 6.0) for 10 min in a microwave
oven after deparaffinization. The slides were incubated with anti-
tau monoclonal antibody that recognizes all isoforms of human tau
protein, irrespective of phosphorylation status (1:50 dilution, clone
T1029, United States Biological, Swampscott, MA) overnight at
4°C. Anti-mouse horseradish peroxidase-labeled secondary anti-
body (DAKO Envision TM? System) and diaminobenzidine sub-
strate were used to generate signal. Normal breast epithelium
served as an internal positive control. Omission of the primary
antibody served as a negative control. The specificity of the
antibody was demonstrated with Western blot. Tau staining of
staining than normal epithelium (Fig. 1b); 2?, similar to normal
epithelium (Fig. 1c); 3?, uniform staining more intense than
normal cells (Fig. 1d). Cases with 0 or 1? staining intensity were
considered tau negative, and tumors with 2??3? staining were
determined by using staining intensity of normal epithelial cells as
a reference and without knowledge of the clinical outcome. Slides
were scored without knowledge of the clinical outcome. Correla-
tion with complete response was assessed in univariate analysis (?2
test) and in multivariate analysis (logistic regression) including
patient age, tumor size, histological type and grade, and estrogen
receptor (ER), progesterone receptor, HER2, and tau status. ER
and HER2 status was determined by the routine clinical pathology
laboratory of University of Texas M. D. Anderson Cancer Center.
was assessed by Western blot using two different monoclonal
anti-tau antibodies (clone T14 from Zymed and clone T1029 from
United States Biological). Results were concordant with both
antibodies. To transiently knock down tau expression, cells were
transfected with a control siRNA (directed against lamin) or two
distinct anti-tau siRNAs (5?-AATCACACCCAACGTGCA-
GAA-3? and 5?-AACTGGCAGTTCTGGAGCAAA-3?) follow-
ing the manufacturer’s instructions (Qiagen).
Twenty-four hours after siRNA transfection, the medium was
changed, and cells were treated with various concentrations of
paclitaxel and epirubicin for a further 48 h. Proliferation rates were
determined with the CellTiter-Glo Luminescent Cell Viability
Assay (Promega). Chemosensitivity was determined from three
separate experiments, each performed in triplicate.
The effect of tau suppression on cellular uptake of paclitaxel was
examined by using fluorescent-conjugated paclitaxel (Oregon
Green 488, Molecular Probes). Spontaneously fluorescent epiru-
bicin was used to examine epirubicine uptake (12, 13). Forty-eight
hours after siRNA transfection, 3 ? 105cells were trypsinized and
resuspended in 1 ml of medium containing 1 ?M Oregon Green
paclitaxel or 16 ?M epirubicin and incubated for 20, 50, or 80 min
Table 1. Clinical information and demographics of the patients
included in the DNA and tissue microarray studies
Median age (range)
Invasive lobular and mixed
Tumor node metastasis
Black’s modified nuclear grade
Weekly T (80 mg?m2) ? 12 ? FAC ? 4
3-weekly T (225 mg?m CI) ? 4 ? FAC ? 4
52 years (29–79)
51 years (29–72)
T, paclitaxel; FAC, 5-flurouracil, doxorubicin, and cyclophosphamide.
*Cases where ?10% of tumor cells stained positive for ER with IHC were
†Cases that showed either 3? IHC staining or had gene copy number ?2.0
were considered HER2 positive.
www.pnas.org?cgi?doi?10.1073?pnas.0408974102Rouzier et al.
at 37°C. Subsequently, cells were analyzed by FACS using
CELLQUEST software (BD Biosciences, Franklin Lakes, NJ). The
amount of fluorescence per cell (arbitrary fluorescence units) was
taken as the measure of drug uptake. Results were displayed as
histograms of mean fluorescence and standard deviation of three
independent experiments. The percentage of fluorescent cells and
nonfluorescent cells was also determined.
Tubulin Polymerization Assays. Bovine brain tubulin polymerization
Polymerization Assay Kit (Cytoskeleton, Denver). Purified tau
protein was purchased from Cytoskeleton (ref. no. TA01). Fluo-
rescent BODIPY-paclitaxel (BODIPY 564?570) was purchased
time. Fluorescence emission was read at excitation?emission wave-
(PerSeptive Biosystems, Framingham, MA). Binding of paclitaxel
to microtubules was also assessed with
(Moravek Biochemicals, Brea, CA).
Gene Expression Analysis Reveals Tau mRNA as the Best Single Gene
Discriminator of pCR to Preoperative Chemotherapy with Paclitaxel,
5-Fluorouracil, Doxorubicin, and Cyclophosphamide. We used gene
expression profiling to discover genes that are associated with
extreme chemotherapy sensitivity. The most significantly differen-
tially expressed gene between cases with pCR and residual cancer
was microtubule-associated protein tau. Tau mRNA expression
measured by all four Affymetrix probe sets that were directed
against this molecule was significantly lower (unequal-variance t
test, P ? 0.3 ? 10?5) in tumors that achieved pCR (Fig. 5, which
is published as supporting information on the PNAS web site). All
cases with pCR had low tau expression; however, some cases with
residual cancer also had low tau expression. There was no differ-
ential expression of any other microtubule-associated proteins or
tubulin subtypes in our gene expression data.
Low Tau Protein Expression Assessed by IHC Predicts Higher pCR Rate.
We examined tau protein expression in 122 independent cases by
and anthracycline-containing chemotherapy. None of these pa-
tients was included in the microarray study. Thirty-eight patients
had pCR (31%). Cytoplasmic expression of tau protein was seen in
all normal breast epithelium (2? IHC score) and blood vessels but
not in fibroblasts or adipocytes (Fig. 1). Normal epithelium pro-
vided a convenient internal control for scoring and represent cells
that are resistant to therapeutic doses of paclitaxel. Sixty-four
(2??3?). Forty-four percent of tau-negative tumors (28?64) had
pCR, compared with 17% of tau-positive tumors (10?58) (P ?
28?38; Fig. 1e). The odds ratio for pCR in tau-negative tumors was
receptor expression identified high nuclear grade (P ? 0.05) and
ER-negative status (P ? 0.06) as independent factors associated
with low tau expression. Similar multiple logistic regression includ-
grade, and ER, progesterone receptor, and HER2 receptor expres-
sion as covariates identified nuclear grade 3 histology (P ? 0.01),
age ?50 (P ? 0.03), and tau-negative status (P ? 0.04) as
independent predictors of pCR (Table 2). Tau was more powerful
likely to achieve pCR.
These results confirmed the microarray data that low tau ex-
pression is associated with significantly higher rate of pCR com-
pared with normal tau expression. However, only half of the tau
protein-negative cases had pCR, which suggests that tau expression
of response than tau protein expression assessed by IHC.
Down-Regulation of Tau Expression in Breast Cancer Cells Increases
Sensitivity to Paclitaxel. We hypothesized that lower than normal
tau expression may increase sensitivity to paclitaxel due to its effect
on microtubules. We assessed tau protein expression in breast
cancer cell lines with Western blot by using the same antibody that
was used for IHC. Four cell lines (ZR75.1, T47D, MCF7, and
(Fig. 2a). ZR75.1 and MCF-7 cells were used for further in vitro
studies. These cells are known to be relatively resistant to paclitaxel
(14). T47D and MDA-MB 435 cells could not be successfully
transfected with anti-tau siRNA.
siRNA transfection efficiency was estimated to be 30–40% (see
epithelium (a) and invasive breast cancer with 1? (b), 2? (c), and 3? (d)
staining (magnification ?40). (e) The proportion of patients with pCR and
residual disease as a function of tau IHC scores (n ? 122).
Tau protein expression by IHC. (a–d) Tau expression in normal breast
Rouzier et al.
June 7, 2005 ?
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below), and tau down-regulation lasted for at least 72 h after
transfection (Fig. 2b). Decreased tau expression significantly in-
creased the sensitivity of ZR75.1 cells to paclitaxel compared with
control cells transfected with lamin siRNA or no siRNA (Fig. 2c).
The IC50of paclitaxel was reduced from ?10 ?M to 20 nM in the
tau knock-down cell pool. However, cell kill did not reach 100%,
which is consistent with partial transfection efficacy and limited
(48-h) exposure to paclitaxel. This finding also suggests that other
mechanisms of resistance may exist in these cells. Tau down-
regulation did not result in increased sensitivity to epirubicin (Fig.
2d). Identical results were obtained with MCF7 cells (data not
shown). These data demonstrate that tau protein partially protects
cells from the cytotoxic effects of paclitaxel. Suppression of tau
expression renders cells more sensitive to paclitaxel, but not to
Microtubules Formed in the Presence of Tau Bind Less Paclitaxel and
Show Decreased Paclitaxel-Induced Stabilization in Vitro. Pharmaco-
(15, 16). We hypothesized that the opposite may also occur;
physiological stabilization of microtubules by tau may reduce
paclitaxel binding to tubulin. To examine this hypothesis, we first
measured the uptake of fluorescent paclitaxel in tau knock-down
ZR75.1 cells and lamin siRNA-treated control cells. Intracellular
paclitaxel is mostly bound to tubulin; therefore, cells with low tau
expression are expected to take up more paclitaxel. Forty-four
Green paclitaxel for 20–80 min and then analyzed by FACS.
Control cells showed unimodal distribution of fluorescence inten-
bimodal distribution indicating a fraction of highly fluorescent cells
(mean ? 100 units) (Fig. 3 a and b). We assume that the highly
fluorescent subpopulation represents the successfully transfected
cells. Twenty-seven percent (?6.3%) of cells showed ?10 fluores-
cence units in the tau knock-down pool compared with 7.2%
conducted with spontaneously fluorescent epirubicin. The distri-
butions were unimodal and similar in both the control and tau
knocked-down cells (Fig. 3 d and e). These results suggest that cells
with lowered tau protein expression accumulate more paclitaxel,
but not epirubicin.
Next, we examined whether tau could reduce paclitaxel-induced
tubulin polymerization in vitro. The rate of tubulin polymerization
can be monitored by measuring optical absorbance of the tubulin
solution at 340 nm (17, 18). When paclitaxel and tau were added
simultaneously to the tubulin solution, their combined effect was
partially additive (Fig. 4a). However, when tubulin was preincu-
bated with tau before adding paclitaxel, which may represent a
more physiological condition, the ability of paclitaxel to induce
maximal tubulin polymerization was reduced in a dose-dependent
of substrate because tubulin already polymerized by tau cannot be
recruited by paclitaxel or (ii) tau directly competing with paclitaxel
binding to tubulin.
To examine whether binding of paclitaxel to tubulin is affected
by tau, we used fluorescent BODIPY-paclitaxel. When BODIPY-
Table 2. Multivariate analysis of predictive factors of pCR
1 or 2
2.6 [0.09–7.2] 0.08
cell lines (a) and in ZR75.1 cells 36–72 h after tau siRNA transfection (b). (c and
in parental, lamin siRNA-transfected, and tau siRNA-transfected ZR75.1
cells indicated increased sensitivity to paclitaxel but not to epirubicin in tau
knock-down cells. Error bars indicate 95% confidence intervals of triplicate
Tau down-regulation sensitizes ZR75.1 breast cancer cells to pacli-
www.pnas.org?cgi?doi?10.1073?pnas.0408974102Rouzier et al.
paclitaxel binds to microtubules, it displays enhanced fluorescence
(19). BODIPY-paclitaxel (5 ?M) was added to tubulin solution
after 30 min of preincubation with (i) tau (15 ?M), (ii) regular,
nonfluorescent paclitaxel (10 and 20 ?M), or (iii) control buffer
(Fig. 4 c and d). Fluorescence was measured 30 min later. Prein-
cubation with regular paclitaxel reduced the binding of BODIPY-
paclitaxel to tubulin in a dose-dependent manner due to direct
10 ?M regular paclitaxel (Fig. 4d). These experiments were re-
peated with tritium-labeled paclitaxel, and similar results were
esis that tau-stabilized microtubules bind less paclitaxel.
In this study, we used transcriptional profiling as a screening tool to
identify genes whose expression is associated with pCR to T?FAC
preoperative chemotherapy for stage I–III breast cancer. Tumors
that show pCR to preoperative chemotherapy represent extremely
chemotherapy-sensitive cancers. The single best gene to discrimi-
current study was also included in a separate analysis when RNA
cDNA result-based predictor; however, it was among the top
differentially expressed genes (20). Low tau mRNA expression was
significantly more common among cases with pCR compared with
those with residual disease. In the current study, no cases with high
tau mRNA expression had pCR. This inverse correlation with
pathologic response was confirmed with IHC in an independent
used as a marker to identify breast cancers that are particularly
sensitive to paclitaxel-containing chemotherapy. We also demon-
strated that reducing tau expression in breast cancer cells renders
these cells more sensitive to paclitaxel but not to epirubicin in vitro.
to paclitaxel, which could explain how low tau expression leads to
some residual cancer, suggesting that there are other mechanisms
of resistance that can protect cells from paclitaxel even if the
microtubules are hypervulnerable because of low tau expression.
The observation that tau expression modulates response to
paclitaxel is unique but consistent with previously reported data.
Expression of tau outside the central nervous system, including in
epithelial cells, is well documented (21–23). Tau contains an
imperfect estrogen response element upstream of its promoter and
is an estrogen-induced protein in cultured neurons and in MCF-7
cells (24–26). Indeed, three of the four cell lines that expressed tau
were ER positive (MCF7, ZR75, and T47D), and we also observed
an association between low tau expression and ER-negative status
ER-negative breast cancers are more sensitive to paclitaxel che-
motherapy. Furthermore, induction of tau expression with retinoic
acid in neuroblastoma cells increased their resistance to paclitaxel,
suggesting a protective effect (27). Tau is implicated in the pathol-
ogy of Alzheimer’s disease, and its microtubule binding character-
istics have been studied extensively. Tau is able to bind to both the
outer and inner surfaces of microtubules, and it may bind to the
same inner-surface pocket as paclitaxel (28). Some investigators
reported that under some conditions, tau may enhance binding of
paclitaxel to microtubules (17, 19). In these reports, paclitaxel
exposure either preceded tau exposure or was concomitant to it.
When tau is added to paclitaxel-stabilized microtubules, tau binds
further enhances paclitaxel-induced polymerization (29, 30). We
also observed this additive effect when the two molecules were
added to tubulin concomitantly (Fig. 4a). Kinetic studies also
showed that tau binds to microtubules differently depending on
of paclitaxel show moderate binding affinity and rapid dissociation
kinetics. In contrast, when microtubules are assembled in the
presence of tau without paclitaxel, tau shows strong binding with
slow dissociation. Our experiments are complementary to these
presence or absence of tau. Our findings suggest that microtubules
assembled in the presence of tau are less susceptible to paclitaxel
binding and pharmacological hyperpolymerization.
Several important questions remain to be examined. There are
numerous alternative splice variants of tau, and each contains
multiple phosphorylation sites that can affect interactions with
antibody that we used were directed against shared domains of the
tau confers partial protection to other microtubule-binding che-
motherapy drugs, including docetaxel, epothilons, or vinca alka-
loids, also remains to be investigated.
cells. (a and b) FACS analysis of cells transfected with lamin siRNA (a) and tau
siRNA (b) after exposure to Oregon Green fluorescent paclitaxel. (c) The
units 20, 50, and 80 min after incubation with 1 ?M Oregon Green paclitaxel.
(d and e) FACS analysis after exposure (80 min) to spontaneously fluorescent
epirubicin in lamin (d) and tau (e) knocked-down cells.
Fluorescent paclitaxel uptake is increased in tau knock-down ZR75.1
Rouzier et al.
June 7, 2005 ?
vol. 102 ?
no. 23 ?
Whereas tau is a promising single gene marker of sensitivity to Download full-text
paclitaxel-containing chemotherapy, it is also clear that many
tumors, despite low tau expression, are not fully sensitive to
treatment, suggesting additional pathways of resistance. This ob-
servation is consistent with the commonly held belief that response
to chemotherapy is a multifactorial process and that no single
marker will be informative in all cases. Indeed, tubulin mutations,
variable expression of tubulin isoforms, overexpression of multi-
drug resistance transporters, or bcl-2 may all contribute to resis-
tance to paclitaxel in tau-negative tumors (32–35). Multigene
predictors that use information from several distinct molecular
pathways of resistance will likely be more powerful than any single
gene. However, low tau expression represents a unique molecular
mechanism of hypersensitivity to paclitaxel. Inhibition of tau func-
tion could be explored as a potential therapeutic strategy to
increase the anticancer activity of this drug.
This work was supported in part by the Nellie B. Connally Breast Cancer
Research Fund, grants from Millennium Pharmaceuticals and The Dee
Simmons Fund, the University of Texas M. D. Anderson Cancer Center
Aventis Drug Development Award (to L.P.), and Susan G. Komen
Breast Cancer Foundation Grant LF2002-044HM (to W.F.S.) R.
Rouzier was supported by the Association pour la Recherche sur le
1. Early Breast Cancer Trialists Collaborative Group (1998) Lancet 352, 930–942.
2. Fisher, B., Bryant, J., Wolmark, N., Mamounas, E., Brown, A., Fisher, E. R., Wickerham,
D. L., Begovic, M., De Cillis, A., Robidoux, A., et al. (1998) J. Clin. Oncol. 16, 2672–2685.
3. Carlson, R. W., Anderson, B. O., Bensinger, W., Cox, C. E., Davidson, N. E., Edge, S. B.,
Farrar, W. B., Goldstein, L. J., Gradishar, W. J., Lichter, A. S., et al. (2000) Oncology
(Huntington, NY) 14, 33–49.
4. Goldhirsch, A., Wood, W. C., Gelber, R. D., Coates, A. S., Thurlimann, B. & Senn, H. J.
(2003) J. Clin. Oncol. 21, 3357–3365.
5. Henderson, I. C., Berry, D. A., Demetri, G. D., Cirrincione, C. T., Goldstein, L. J., Martino,
S., Ingle, J. N., Cooper, M. R., Hayes, D. F., Tkaczuk, K. H., et al. (2003) J. Clin. Oncol. 21,
6. Rouzier, R., Extra, J. M., Klijanienko, J., Falcou, M. C., Asselain, B., Vincent-Salomon, A.,
Vielh, P. & Bourstyn, E. (2002) J. Clin. Oncol. 20, 1304–1310.
R. L., Singh, G., Binkley, S. M., Sneige, N., et al. (1999) J. Clin. Oncol. 17, 460–469.
8. Ayers, M., Symmans, W. F., Stec, J., Damokosh, A. I., Clark, E., Hess, K., Lecocke, M.,
Metivier, J., Booser, D., Ibrahim, N., et al. (2004) J. Clin. Oncol. 22, 2267–2269.
9. Drechsel, D. N., Hyman, A. A., Cobb, M. H. & Kirschner, M. W. (1992) Mol. Biol. Cell 3,
10. Pusztai, L., Symmans, W. F. & Hortobagyi, G. N. (2005) Breast Cancer 12, 73–85.
11. Symmans, W. F., Ayers, M., Clark, E. A., Stec, J., Hess, K. R., Sneige, N., Buchholz, T. A.,
Krishnamurthy, S., Ibrahim, N. K., Buzdar, A. U., et al. (2003) Cancer 97, 2960–2971.
12. Kimchi-Sarfaty, C., Gribar, J. J. & Gottesman, M. M. (2002) Mol. Pharmacol. 62, 1–6.
13. Harris, N. M., Anderson, W. R., Lwaleed, B. A., Cooper, A. J., Birch, B. R. & Solomon, L. Z.
(2003) Cancer 97, 71–78.
14. Dougherty, M. K., Schumaker, L. M., Jordan, V. C., Welshons, W. V., Curran, E. M., Ellis,
M. J. & El-Ashry, D. (2004) Cancer Biol. Ther. 3, 460–467.
15. Hong, M., Zhukareva, V., Vogelsberg-Ragaglia, V., Wszolek, Z., Reed, L., Miller, B. I.,
Geschwind, D. H., Bird, T. D., McKeel, D., Goate, A., et al. (1998) Science 282, 1914–1917.
16. Rao, S., He, L., Chakravarty, S., Ojima, I., Orr, G. A. & Horwitz, S. B. (1999) J. Biol. Chem.
17. Diaz, J. F., Barasoain, I. & Andreu, J. M. (2003) J. Biol. Chem. 278, 8407–8419.
18. Lu, Q. & Wood, J. G. (1993) J. Neurosci. 13, 508–515.
19. Ross, J. L., Santangelo, C. D., Makrides, V. & Fygenson, D. K. (2004) Proc. Natl. Acad. Sci.
USA 101, 12910–12915.
20. Stec, J., Wang, J., Coombes, K., Ayers, M. & Hoersch, S. (2005) J. Mol. Diagn., in press.
21. Bernard-Marty, C., Treilleux, I., Dumontet, C., Cardoso, F., Fellous, A., Gancberg, D.,
Bissery, M. C., Paesmans, M., Larsimont, D., Piccart, M. J., et al. (2002) Clin. Breast Cancer
22. Gu, Y., Oyama, F. & Ihara, Y. (1996) J. Neurochem. 67, 1235–1244.
23. Hattori, H., Matsumoto, M., Iwai, K., Tsuchiya, H., Miyauchi, E., Takasaki, M., Kamino, K.,
Munehira, J., Kimura, Y., Kawanishi, K., et al. (2002) J. Gerontol. A Biol. Sci. Med. Sci. 57,
24. West, M., Blanchette, C., Dressman, H., Huang, E., Ishida, S., Spang, R., Zuzan, H., Olson,
J. A., Jr., Marks, J. R. & Nevins, J. R. (2001) Proc. Natl. Acad. Sci. USA 98, 11462–11467.
25. Matsuno, A., Takekoshi, S., Sanno, N., Utsunomiya, H., Ohsugi, Y., Saito, N., Kanemitsu,
H., Tamura, A., Nagashima, T., Osamura, R. Y., et al. (1997) J. Histochem. Cytochem. 45,
26. Ferreira, A. & Caceres, A. (1991) J. Neurosci. 11, 392–400.
27. Guise, S., Braguer, D., Remacle-Bonnet, M., Pommier, G. & Briand, C. (1999) Apoptosis 4,
28. Kar, S., Fan, J., Smith, M. J., Goedert, M. & Amos, L. A. (2003) EMBO J. 22, 70–77.
29. Chau, M. F., Radeke, M. J., de Ines, C., Barasoain, I., Kohlstaedt, L. A. & Feinstein, S. C.
(1998) Biochemistry 37, 17692–17703.
30. Al-Bassam, J., Ozer, R. S., Safer, D., Halpain, S. & Milligan, R. A. (2002) J. Cell Biol. 157,
31. Makrides, V., Massie, M. R., Feinstein, S. C. & Lew, J. (2004) Proc. Natl. Acad. Sci. USA
32. Zhang, C. C., Yang, J. M., Bash-Babula, J., White, E., Murphy, M., Levine, A. J. & Hait,
W. N. (1999) Cancer Res. 59, 3663–3670.
33. Verdier-Pinard, P., Wang, F., Martello, L., Burd, B., Orr, G. A. & Horwitz, S. B. (2003)
Biochemistry 42, 5349–5357.
34. Horwitz, S. B., Cohen, D., Rao, S., Ringel, I., Shen, H. J. & Yang, C. P. (1993) J. Natl. Cancer
Inst. Monogr., 55–61.
35. Orr,G.A.,Verdier-Pinard,P.,McDaid,H.&Horwitz,S.B.(2003) Oncogene 22,7280–7295.
with modest additive effect when combined. (b) Preincubation of tubulin with tau for 30 min before adding paclitaxel (20 mM) decreased the maximum
paclitaxel-induced polymerization in a dose-dependent manner. (c) Fluorescence emission of BODIPY-paclitaxel is enhanced when it binds to increasing
(5 mM) was added, the increase in fluorescence was reduced, which suggests that tau inhibits BODIPY-paclitaxel binding to microtubules.
www.pnas.org?cgi?doi?10.1073?pnas.0408974102Rouzier et al.