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CtIP Silencing as a Novel Mechanism of Tamoxifen
Resistance in Breast Cancer
Minhao Wu,1David Ramos Soler,2Martin C. Abba,1Maria I. Nunez,1
Richard Baer,3Christos Hatzis,4Antonio Llombart-Cussac,5
Antonio Llombart-Bosch,2and C. Marcelo Aldaz1
1
Department of Carcinogenesis, The University of Texas M. D. Anderson Cancer Center, Smithville,
Texas;
2
Department of Pathology, Medical School, The University of Valencia, Valencia, Spain;
3
Institute for Cancer Genetics, Columbia University Medical Center, New York, New York;
4
Nuvera Biosciences, Inc., Woburn, Massachusetts; and
5
Department of Oncology,
Fundacio´n Instituto Valenciano de Oncologı´a, Valencia, Spain
Abstract
Acquired resistance to the antiestrogen tamoxifen
constitutes a major clinical challenge in breast cancer
therapy. However, the mechanisms involved are still
poorly understood. Using serial analysis of gene
expression, we identified CtIP, a BRCA1- and CtBP-
interacting protein, as one of the most significantly
down-regulated transcripts in estrogen receptor
A–positive (ER+) MCF-7 tamoxifen-resistant breast
cancer cells. We further confirmed the association of
CtIP down-regulation with tamoxifen resistance in an
additional ER+ breast cancer line (T47D), strengthening
the relevance of the phenomenon observed. In
additional studies, we found CtIP protein expression in a
majority of ER+ breast cancer cell lines that we tested,
but no or very little CtIP expression in ER-negative lines.
Furthermore, CtIP protein expression status correlates
with clinical response to neoadjuvant endocrine therapy,
and patients with progressive disease express
significantly lower CtIP protein in their primary breast
carcinomas than those who respond. Meta-analysis of
seven publicly available gene expression microarray
data sets showed that CtIP expression is significantly
associated with ER, disease-free survival, and breast
cancer metastasis status. Importantly, we found that
silencing endogenous CtIP in tamoxifen-sensitive
breast cancer cells confers tamoxifen resistance. On the
other hand, reexpression of CtIP in tamoxifen-resistant
breast cancer cells restores sensitivity to the inhibitory
growth effects of tamoxifen. Together, our findings
indicate that CtIP silencing might be a novel mechanism
for the development of tamoxifen resistance in
breast cancer, suggesting that CtIP is likely associated
with ER function, and that CtIP gene and protein
expression may be useful biomarkers for breast cancer
prognosis and clinical management. (Mol Cancer Res
2007;5(12):1285 – 95)
Introduction
Estrogen plays a pivotal role in the etiology and progression
of human breast cancer. Therefore, for a long time, treatment of
breast cancer has been directed toward inhibiting the tumor-
promoting effects of estrogen. Tamoxifen, a nonsteroidal
antiestrogen, has been the gold standard for endocrine treatment
of all stages of estrogen receptor a(ER) positive breast cancer
for >25 years and was the first approved drug by the Food and
Drug Administration as a cancer chemopreventive agent for
reducing breast cancer incidence in both premenopausal and
postmenopausal women at high risk of breast cancer develop-
ment (1). As adjuvant therapy, tamoxifen reduces the risk of
recurrence and improves overall survival in early breast cancer
(2). It was also shown to be effective for patients with untreated
metastatic breast cancer (2). Despite the benefits of tamoxifen
in treating breast cancer, unfortunately, many tumors that
initially respond to tamoxifen therapy develop resistance. This
phenomenon has become a serious obstacle in breast cancer
treatment. In the clinic, almost all patients with advanced
metastatic disease and as many as 40% of patients receiving
adjuvant tamoxifen eventually relapse and die from their
disease (3). The mechanisms involved in the development of
tamoxifen resistance are still poorly understood. Numerous
mechanisms have been proposed to contribute to the develop-
ment of tamoxifen resistance, including altered drug metabo-
lism, loss of expression or mutation of ER, lack of expression of
progesterone receptor, increased expression of ERh, posttrans-
lational modifications of ER, altered expression of coregulators
(e.g., increased expression of the coactivator AIB1 or decreased
expression of the corepressor NCoR), and increased growth
factor signaling (e.g., HER2 signaling pathways; reviewed in
refs. 3-9). Interestingly, a recent study showed that DIBA, an
ER zinc finger inhibitor, restores the antagonistic action of
tamoxifen in tamoxifen-resistant breast cancer cells through
targeted disruption of the ER DNA-binding domain and its
interaction with the proximal NH
2
-terminal domain to suppress
ligand-dependent and ligand-independent ER transcription and
Received 3/15/07; revised 7/23/07; accepted 8/15/07.
Grant support: Susan G. Komen Breast Cancer Foundation Award, BC-
TR0402877 (C.M. Aldaz), and Department of Defense Concept Award,
W81XWH-04-1-0622-01 (C.M. Aldaz).
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: C. Marcelo Aldaz, Department of Carcinogenesis, The
University of Texas M. D. Anderson Cancer Center, Science Park Research
Division, 1808 Park Road 1C, P.O. Box 389, Smithville, TX 78957. Phone: 512-
237-2403; Fax: 512-237-2475. E-mail: maaldaz@mdanderson.org
Copyright D2007 American Association for Cancer Research.
doi:10.1158/1541-7786.MCR-07-0126
Mol Cancer Res 2007;5(12). December 2007 1285
influence the recruitment of cofactor to the ER (10). These
findings strengthen the important role of ER in the development
of tamoxifen resistance and suggest a possible new approach
in modifying tamoxifen resistance (10). However, much work is
still needed to learn whether some of the postulated mechanisms
thus far can explain resistance to tamoxifen therapy in a majority
of patients, or simply each of the enumerated possibilities
account for minor portions of resistant cases. Thus, studies
geared at better understanding the most common mechanisms
involved in tamoxifen resistance are of considerable clinical
significance.
With the aim of identifying key genes involved in the
development of tamoxifen resistance, we defined the global
gene expression profiles of recently developed tamoxifen-
resistant MCF-7 breast cancer cell lines and compared them
with their tamoxifen-sensitive parental MCF-7 counterpart by
using serial analysis of gene expression (SAGE). We determined
that the mRNA expression of CtIP, a BRCA1- and CtBP-
interacting protein, was found to be 15-fold down-regulated in
the tamoxifen-resistant cells when compared with their
tamoxifen-sensitive counterparts. In this report, we describe
these observations and show the functional involvement of CtIP
in the development of tamoxifen resistance.
Results
CtIP Expression Is Significantly Down-Regulated in
Tamoxifen-Resistant Breast Cancer Cells
To identify genes implicated in the development of
tamoxifen resistance, we determined global gene expression
profiles of two independently derived isogenic MCF-7 breast
cancer cell line variants (TAMR1 and TAMR2) that are
resistant to tamoxifen and their parental tamoxifen-sensitive
MCF-7 line by using SAGE. As shown in Fig. 1A, in contrast
to their parental MCF-7 cells, both TAMR1 and TAMR2 cells
cultured in estrogen-depleted medium proliferated in the
presence of tamoxifen. Additionally, despite continuous
exposure to trans -4-hydroxytamoxifen (4-OH-TAM), both
tamoxifen-resistant variants are still estrogen responsive
(Fig. 1B) and express equivalent levels of ER protein as their
parental MCF-7 cells (Fig. 1D). Therefore, both TAMR1 and
TAMR2 cells express functional ER, as do their parental
MCF-7 cells. The tamoxifen-resistant phenotypes of these cells
seem not to be a consequence of changes in ER expression or
function. These data are also consistent with the clinical
findings that the majority of patients with acquired resistance to
tamoxifen still express functional ERs (11, 12).
By SAGE comparative analyses, we identified that the
transcript encoding for CtIP (also known as retinoblastoma
binding protein 8, RBBP8) was significantly down-regulated
(15-fold) in both tamoxifen-resistant cell lines when compared
with their tamoxifen-sensitive parental MCF-7 counterpart
(Fig. 1C). Real-time reverse transcription-PCR on the same
RNA samples used for SAGE analysis also confirmed
significantly lower CtIP mRNA expression in the tamoxifen-
resistant cells than in the tamoxifen-sensitive parental MCF-7
cells (data not shown). Next, we determined whether the
differential expression of CtIP detected at the mRNA level
could also be observed at the protein level. By Western blot
analysis, we detected CtIP protein expression in the tamoxifen-
sensitive parental MCF-7 line, but not in the two tamoxifen-
resistant lines (Fig. 1D). In fact, both TAMR1 and TAMR2 cells
seem to express little or no CtIP protein.
To determine whether this was a phenomenon exclusive to
MCF-7 cells, we selected another estrogen-responsive and
tamoxifen-sensitive breast cancer cell line, T-47-D, and deve-
loped a T-47-D/TR variant that is resistant to tamoxifen. To this
end, we followed the same procedure as with MCF-7 cells,
growing parental tamoxifen-sensitive T-47-D cells under chronic
exposure to 4-OH-TAM (1 Amol/L). After an initial growth arrest
by tamoxifen, T-47-D cells regained exponential growth ability
even in the presence of tamoxifen. As seen in Fig. 1E, after
being cultured in tamoxifen for over 3 months, T-47-D/TR cells
proliferated in fetal bovine serum – supplemented growth
medium even in the presence of tamoxifen, whereas the growth
of parental cells was significantly inhibited by tamoxifen.
FIGURE 1. CtIP expression in tamoxifen-sensitive and tamoxifen-
resistant breast cancer cells. Aand B. In vitro characterization of parental
MCF-7, TAMR1, and TAMR2 cells. Growth curves of parental MCF-7,
TAMR1, or TAMR2 cells cultured in the presence of 10 nmol/L 4-OH-TAM
(A) or 10 nmol/L E
2
(B). Points, mean of triplicate determinations; bars, SE.
C. CtIP expression levels in parental MCF-7, TAMR1, and TAMR2 cells as
determined by SAGE (tag frequencies, normalized to 200,000 tags per
library). D. CtIP and ER protein levels were determined by immunoblotting
in cell lines as indicated. h-Actin was used as a loading control. E. Parental
T-47-D and T-47-D tamoxifen-resistant cells (T-47-D/TR) were grown in full
serum medium and treated with 4-OH-TAM (1 Amol/L). Cell numbers were
determined at various time points as indicated. Points, mean; bars, SE.
F. Western blotting for CtIP in parental T-47-D and T-47-D cultured in
tamoxifen for 1, 2, and 3 mo.
Wu et al.
Mol Cancer Res 2007;5(12). December 2007
1286
Furthermore, we determined CtIP protein levels in these T-47-D
cells cultured in tamoxifen for 1, 2, and 3 months and compared
them with parental T-47-D cells. We observed that consistent
with the findings obtained from MCF-7 cells, T-47-D cells also
have significantly decreased CtIP protein expression as they
became tamoxifen resistant (Fig. 1F).
Together, the above data confirm that the expression of CtIP
is significantly decreased in tamoxifen-resistant cells at both
mRNA and protein levels and raise the possibility that CtIP
silencing could be a novel mechanism for the development of
tamoxifen resistance.
CtIP Expression in Various Human Breast Cancer Cell
Lines
To perform a comparative analysis of CtIP expression levels,
we next determined CtIP protein expression by Western blot
analysis in 10 human breast cancer lines. As can be observed in
Fig. 2, three of six ER-positive (ER+) breast cancer lines
express abundant CtIP (MCF-7, T-47-D, and ZR-75-1) and one
of these lines (BT-483) expresses some CtIP; that is, in total,
four of six (67%) ER+ cell lines express detectable CtIP. In
contrast, four of four (100%) ER-negative (ER) breast cancer
lines either do not express CtIP at all or very little (UAC 812,
MDA-MB-231, MDA-MB-435, SKBR3).
Poor Clinical Response to Endocrine Therapy Is Associ-
ated with CtIP Deficiency in Breast Cancer Patients
To determine whether there is a relationship between
CtIP status and endocrine therapy response in vivo, we evaluated
CtIP expression by immunohistochemistry in 59 ER+, non-
operable primary breast carcinomas from patients who received
endocrine therapy as single neoadjuvant therapy. Immunohisto-
chemistry was done of samples from tumors before initiation of
antiestrogen therapy. Based on the clinical response to the
therapy after 4 months of follow-up, patients were classified into
four groups: complete response, 4 cases (7%); partial response,
32 cases (54%); stable disease, 17 cases (29%), and progressive
disease, 6 cases (10%). These cases are representative of a larger
cohort previously reported in which complete response was
reported to be between 4% and 10% (13). Immunoreactive
scores (IRS) for CtIP were used to semiquantify immunohisto-
chemical staining intensity and percentage of positive cells
(14, 15). IRS ranging from 0 to 12 represents CtIP protein
staining from undetectable to the highest expression level,
respectively (Fig. 3A). One-way ANOVA analysis of CtIP IRS
revealed significantly different CtIP expression in the four
response groups (P= 0.01). Remarkably, we observed that
patients who had the worst response to endocrine therapy
(defined as progressive disease) had significantly lower CtIP
expression than those who had the best response to endocrine
therapy (defined as complete response; P= 0.006; Fig. 3B).
Moreover, Pearson’s correlation analysis showed a significant
correlation between CtIP status and clinical response to
endocrine therapy (P= 0.004). In this limited small study, data
seem to indicate that poor response to endocrine therapy is
associated with CtIP deficiency in breast cancer patients.
However, these observations have to be confirmed in larger
cohorts.
FIGURE 2. CtIP protein expression in breast cancer cell lines as
determined by Western blot analysis. ER expression status is also
indicated as positive (+) or negative (). As can be observed, all ER
breast cancer lines (100%) tested do not express CtIP protein, whereas
four of six ER+ lines (67%) express this protein.
FIGURE 3. Poor clinical response to endocrine therapy is significantly
associated with CtIP deficiency in primary breast carcinomas. A. CtIP
expression patterns in human breast carcinomas. CtIP expression was
evaluated by immunohistochemistry and semiquantified using IRS in 59
inoperable hormone receptor – positive primary breast carcinomas (before
treatment) from patients who received endocrine therapy as single
neoadjuvant therapy. Representative tumor CtIP immunoreactivity (IRS
scores 0, 4, 8, and 12). B. CtIP status strongly correlates with clinical
response to endocrine therapy (Pearson correlation, P= 0.004), and
patients with progressive disease have significantly lower CtIP IRS than
those who completely respond to endocrine therapy. **, P= 0.006 by
ANOVA and Tukey’s post tests. CR, complete response; PR, partial
response; SD, stable disease; and PD, progressive disease.
CtIP and Tamoxifen Resistance
Mol Cancer Res 2007;5(12). December 2007
1287
CtIP Expression Is Associated with ER, Disease-Free
Survival, and Breast Cancer Metastasis Status
To further explore the clinical relevance of CtIP expression
in breast cancer, we evaluated information of seven publicly
available breast cancer gene expression microarray data sets
(16-22) through the use of the web-based Oncomine cancer
microarray database (23).
6
Clinicopathologic and gene expres-
sion data from a total of 828 breast carcinomas was obtained
using this publicly available resource. Because ER plays a
critical role in the clinical management of breast cancer
patients, we first analyzed levels of CtIP mRNA expression
in the mentioned microarray sets according to ER status of the
tumors (Fig. 4A). By using a meta-analysis approach, we
directly compared CtIP expression profiles between 590 ER+
and 238 ERbreast carcinomas by combining all seven
microarray data sets. We found a significant association
between high CtIP expression and ER (+) status in breast
carcinomas (P< 0.0001; Fig. 4A). Next, we analyzed CtIP
expression profiles versus disease-free survival in two micro-
array data sets that had at least 5 years of follow-up clinical
information available. Analysis from the study of van de Vijver
et al. (19) showed a statistically significant association between
loss of CtIP and disease relapse (P= 0.019; Fig. 4B, left ). A
trend was also found in the Sorlie et al. study (20) but did not
reach statistical significance (P= 0.069), possibly due to the
low number of samples (Fig. 4B, right). However, by using the
meta-analysis approach and pooling both studies together, we
found a highly significant association between decreased
CtIP expression and disease relapse (P= 0.004; Fig. 4B).
Furthermore, CtIP mRNA expression levels were significantly
lower in invasive breast carcinomas that had distant metastasis
when compared with breast cancer counterparts that did not
metastasize (P= 0.029; Fig. 4C).
Silencing Endogenous CtIP in Tamoxifen-Sensitive
MCF-7 Cells Confers Tamoxifen Resistance In vitro
To further explore the putative role of CtIP in the
development of tamoxifen resistance, we first examined
whether silencing the expression of endogenous CtIP in
tamoxifen-sensitive cells can induce a tamoxifen-resistant
phenotype. Knockdown of CtIP protein levels in tamoxifen-
sensitive parental MCF-7 cells was achieved using RNA
interference techniques. As shown in Fig. 5A, in the resulting
clone [MCF-7/CtIP; small interfering (siRNA)] stably trans-
fected with a vector expressing siRNA targeting CtIP mRNA,
the silencing of CtIP protein levels reproduces quite closely
the expression difference observed between the tamoxifen-
sensitive (parental MCF-7) and tamoxifen-resistant (TAMR1
and TAMR2) cells. The negative control siRNA clone
[MCF-7/() control siRNA] showed unchanged CtIP protein
levels when compared with parental MCF-7 cells (Fig. 5A).
In addition, we observed an equal level of ER expression in
all three clones, indicating that ER protein expression was
not affected by the siRNA intervention (Fig. 5A).
To test whether silenced CtIP expression in parental MCF-7
cells leads to the tamoxifen-sensitive to tamoxifen-resistant
transition, we next compared cell proliferation between MCF-7/
CtIP siRNA and MCF-7/() control siRNA cells under various
stimuli. Reduced CtIP expression increased MCF-7/CtIP
siRNA cell proliferation under estrogen-deprived conditions
compared with control siRNA cells (Fig. 5B). When hormone-
starved experimental and control siRNA cells were treated with
4-OH-TAM, growth of the control cells was inhibited by
exposure to tamoxifen, whereas growth of MCF-7/CtIP siRNA
cells was not inhibited by tamoxifen, indicating acquired
resistance to tamoxifen (P< 0.05; Fig. 5C). Furthermore, CtIP-
silenced MCF-7/CtIP siRNA cells grew significantly faster than
control cells in the presence of both estrogen plus tamoxifen
(P< 0.01), indicating that unlike in control cells, tamoxifen
indeed fails to inhibit the stimulatory effect of estrogen in CtIP-
silenced MCF-7 cells (Fig. 5D). Interestingly, the MCF-7/CtIP
siRNA cells still retain response to estrogen-stimulated cell
proliferation when exposed to estradiol (Fig. 5E), indicating
that ER is still functional and capable of regulating cell growth.
In fact, after 10 days in culture in the presence of estrogen,
the cell number of both control and MCF-7/CtIP siRNA cells
increased f145-fold (Fig. 5E), which is five to eight times
more than that of the MCF-7/CtIP siRNA cells cultured in the
presence of tamoxifen (Fig. 5C) or in the absence of estrogen
(Fig. 5B), respectively (over the same time period), suggesting
that estrogen still seems to stimulate cell proliferation in MCF-7
cells regardless of CtIP status. Taken together, these data
indicated that CtIP silencing leads to resistance to the inhibitory
growth effects of tamoxifen in vitro.
Reexpression of CtIP in Tamoxifen-Resistant Cells
Restores Sensitivity to the Inhibitory Growth Effects of
Tamoxifen
Next, we addressed the reciprocal question of whether
reexpression of CtIP in tamoxifen-resistant cells abrogates
resistance to tamoxifen. Because both tamoxifen-resistant lines
have similar proliferation profiles in the presence of tamoxifen,
we selected the TAMR1 cell line for further functional studies.
To this end, a Tet-off – inducible gene expression system was
used to reexpress CtIP in the tamoxifen-resistant TAMR1 cells.
TAMR1 cells were transiently cotransfected with Tet-off–
inducible vectors containing NH
2
-terminal Flag-tagged full-
length human CtIP cDNA. After transfection, cells were
immediately divided equally into two batches. The first batch
was treated with 4-OH-TAM or vehicle, and cultured in the
presence of doxycycline. The second batch was treated with
4-OH-TAM or vehicle, but cultured in the absence of
doxycycline. Expression of FLAG-CtIP was analyzed by
immunoblotting after 72 h of transfection. FLAG-CtIP was only
detected in TAMR1 cells cultured without doxycycline (Fig. 6A,
left), indicating the restoration of CtIP is tightly controlled by
doxycycline. Cell proliferation was determined in TAMR1 cells
with or without CtIP reexpression. This experiment showed
that cells with restored CtIP protein expression displayed a
significant growth inhibition by tamoxifen in comparison with
control cells having no CtIP restoration (Fig. 6A, middle ).
Without tamoxifen treatment (vehicle control), transient CtIP
restoration had no significant effect on TAMR1 cell proliferation
(Fig. 6A, right ). Therefore, restoration of CtIP abrogates
resistance to tamoxifen in tamoxifen-resistant cells.
6
http://www.oncomine.org
Wu et al.
Mol Cancer Res 2007;5(12). December 2007
1288
To further confirm the observations derived from the
transient transfection experiments, we developed double stably
transfected TAMR1 Tet-off FLAG-CtIP cells. Among the
various stably transfected clones obtained, clone 32 was
selected for further study. This clone showed no CtIP expression
in the presence of doxycycline but similar CtIP protein levels to
those produced in parental MCF-7 cells upon doxycycline
withdrawal, as determined by Western blot analysis (Fig. 6B).
FIGURE 4. CtIP expression is associated with ER status and prognosis in breast cancer. CtIP gene expression profiles and clinicopathologic data of 828
breast carcinomas were obtained from seven published and publicly available breast cancer microarray data sets as described in Materials and Methods.
Data were collected and visualized using the Oncomine cancer microarray resource. A. Oncomine database output and meta-analysis showing CtIP
expression patterns relative to ER status across seven breast cancer microarray studies. Columns, CtIP transcripts represented as normalized expression
units; bars, SE (95% CI). B. Oncomine database output of CtIP expression patterns relative to disease-free survival in two of the seven data sets that have
5 y follow-up information available. The van de Vijver et al. data set (left ) shows a significant association between loss of CtIP expression and relapse
(P= 0.019). The Sorlie et al. study (right ) shows a trend that does not reach statistical significance (P= 0.069). Meta-analysis (pooling both studies together)
shows an excellent statistical inverse correlation between decreased CtIP expression and breast cancer relapse (P= 0.004). C. Oncomine database output
of CtIP expression patterns related to metastasis status (left, the van de Vijver et al. study; right, the Sorlie et al. study). The analysis shows a statistically
significant decrease of CtIP expression in association with metastatic breast carcinomas (P= 0.029).
CtIP and Tamoxifen Resistance
Mol Cancer Res 2007;5(12). December 2007
1289
Next, we measured the growth of the double-stably transfected
TAMR1 Tet-off FLAG-CtIP cells under conditions in which
CtIP restoration was either induced or repressed. Cells from
clone 32 were cultured in two different conditions. Half of the
cells were grown in hormone-free medium containing doxycy-
cline whereas the other half was cultured in the same medium
but devoid of doxycycline. After 3 days of incubation, cells
were treated with either 17-h-estradiol (E
2
), 4-OH-TAM, or
vehicle control. As shown in Fig. 6C, the growth of TAMR1
Tet-off FLAG-CtIP cells was significantly inhibited by
tamoxifen when doxycycline was removed from the medium
compared with cells from the same clone but cultured in the
presence of doxycycline (P< 0.05). Cells still responded well to
estrogen regardless of doxycycline status (Fig. 6D). Taken
together, these results show that sensitivity to the inhibitory
growth effects of tamoxifen in previously tamoxifen-resistant
cells is restored, at least partially by CtIP reexpression.
Discussion
SAGE studies identified CtIP as one of the most
significantly down-regulated transcripts in two independently
developed tamoxifen-resistant breast cancer cell lines when
compared with their tamoxifen-sensitive parental MCF-7 line.
Immunoblotting analyses not only validated the SAGE
observations but also showed a dramatic difference in CtIP
FIGURE 5. Silencing of
CtIP protein expression in ta-
moxifen-sensitive MCF-7 cells
confers tamoxifen resistance
in vitro.A. Western blot anal-
ysis of CtIP and ER proteins in
parental MCF-7 cells, MCF-7/
() control siRNA, and MCF-7/
CtIP siRNA stable clones.
Growth curves of control and
MCF-7/CtIP siRNA cells
treated with vehicle control (B)
or 4-OH-TAM (C) for 2 wk. D.
Growth curves of MCF-7/CtIP
siRNA and () control cells
grown in hormone-free medium
and treated with E
2
(10 nmol/L)
and 4-OH-TAM (1 Amol/L) for 6
d. E. Growth curves of control
and MCF-7/CtIP siRNA cells
exposed to E
2
for 10 d. All
experiments were done in trip-
licate. Points, mean; bars, SE. *
and **, P<0.05andP< 0.01 by
ttest, respectively. Note that
Y-axis scale for cell number in
Dis different from that in B,
C, and E.
Wu et al.
Mol Cancer Res 2007;5(12). December 2007
1290
protein expression levels, with high levels of protein
expression in parental ER+ MCF-7 breast cancer cells but
very little or no CtIP protein product in the ER+ tamoxifen-
resistant derivative isogenic cell lines. In addition to the
observations in MCF-7 cells, we also found significantly
reduced CtIP protein expression in ER+ T-47-D breast cancer
cells when they became tamoxifen resistant. Furthermore,
consistent with the in vitro observations, we found significant
association of CtIP deficiency in breast cancers from patients
with poor clinical response to endocrine therapy. By meta-
analysis of gene expression data sets, we also found an
association of CtIP expression levels with poor outcome.
These in vivo data suggest that CtIP gene and protein
expression may be useful biomarkers for breast cancer
prognosis and clinical management, although it would require
further studies in larger patient cohorts. In addition, we found
a significant reverse association of CtIP expression with ER
status, suggesting that CtIP may function in a pathway or
pathways associated with ER signaling and perhaps regulate
ER-mediated cell proliferation. All ERbreast cancer cells
FIGURE 6. Tamoxifen-resistant cells regain sensitivity to the inhibitory growth effects of tamoxifen upon restoration of CtIP protein expression. A. Left,
expression of FLAG-CtIP protein in TAMR1 cells transiently cotransfected with inducible FLAG-CtIP expression vectors and cultured in the presence or
absence of doxycycline (DOX) for 3 d. Middle and right, transient CtIP restoration partially abrogates resistance to tamoxifen in TAMR1 cells. TAMR1 cells
were transiently cotransfected with inducible FLAG-CtIP expression vectors and treated with 4-OH-TAM (middle ) or vehicle control (right ) in the presence or
absence of doxycycline for 3 d. Cell proliferation was determined as shown. Percent cell number (middle) represents cell numbers relative to vehicle control –
treated cells. Columns, mean of triplicate determinations; bars, SE. B. Total CtIP protein expression in the double-stably transfected TAMR1 Tet-off FLAG-
CtIP clone 32 cells in the presence or absence of doxycycline. CtIP expression in parental MCF-7 cells is shown for comparative purposes. C. CtIP
reexpression upon doxycycline withdrawal restores sensitivity to the inhibitory growth effects of tamoxifen in TAMR1 Tet-off FLAG-CtIP clone 32 cells. The
proliferation of clone 32 cells under the treatment of 4-OH-TAM was determined in the presence (black columns ) or absence (white columns) of doxycycline.
*, P< 0.05 by ttest. D. Effect of E
2
on the proliferation of clone 32 cells cultured with or without doxycycline. Note that Yaxis scales for cell number in Cand
Dare different from each other.
CtIP and Tamoxifen Resistance
Mol Cancer Res 2007;5(12). December 2007
1291
tested (intrinsically resistant to tamoxifen) express either none or
very little CtIP protein. Interestingly, BT-474, an ER+ but
tamoxifen-resistant breast cancer cell line as reported by Wang
et al. (10), also expresses nearly undetectable CtIP protein when
compared with ER (+) and tamoxifen-sensitive lines such as
MCF-7 and T-47-D, which further supports the important role of
CtIP in the development of tamoxifen resistance. Finally, based
on RNA interference and reciprocal reexpression studies, we
were able to reproduce the tamoxifen-resistant phenotype
simply by shutting down the expression of one gene
(i.e., CtIP). These data show that CtIP silencing is critical for
the development of tamoxifen resistance in breast cancer in vitro
models and suggest that CtIP silencing may be a novel
mechanism by which cells can circumvent the inhibitory effects
of tamoxifen to resume proliferation and ultimately acquire
resistance to the antiestrogen tamoxifen.
The human CtIP (also known as RBBP8) encodes an 897-
amino-acid nuclear protein that is widely expressed in various
human tissues (24-27). It was initially identified as a cofactor of
transcriptional corepressor CtBP (24). CtIP is also known to
interact with tumor suppressors, Rb family proteins (Rb and
p130; refs. 25, 28) and BRCA1 (26, 29-31), as well as the
transcriptional repressors such as LIM-only protein LMO4 (30)
and Ikaros family members (32). Recent studies suggest that
CtIP plays an important role in cell cycle regulation and DNA
damage response (33-37). Emerging evidence also suggests that
CtIP may itself be a tumor susceptibility gene. Analysis of CtIP
cDNA from 89 human tumor cell lines revealed 5 missense and
11 silent mutations (26). In a more recent screening study of
109 colon cancers, CtIP was found to be a frequent target for
microsatellite instability (38). More importantly, it has been
shown that inactivation of CtIP in mice leads to early
embryonic lethality, and the life span of Ctip
+/
heterozygotes,
which have Ctip haploid insufficiency, was shortened due to
the development of multiple types of tumors. These findings
show that CtIP is a critical protein in early embryogenesis and
implicates an important role of CtIP in tumorigenesis (39). In
addition, CtIP interacts with the BRCT domains of BRCA1
where most mutations occur in BRCA1 breast cancer patients,
and such protein-protein interaction is abolished by tumor-
associated mutations in the BRCT domains (26, 29, 31),
suggesting that interaction between CtIP and BRCA1 is
of functional relevance in the breast cancer suppressor activity.
It has been shown that amino acid residues 299 to 345 of
CtIP mediate its interaction with the BRCT domains of BRCA1
(26, 27, 37). Recently, it was also reported that phosphorylation
at Ser
327
in CtIP seemed to be critical for its interaction with
BRAC1 BRCT domains (37, 40).
Available evidence suggests that CtIP is involved in
transcriptional repression (reviewed in refs. 41, 42). Signifi-
cantly, recent studies showed that BRCA1, CtIP, and ZBRK1
form a repressor complex at a recognition site of ZBRK1 in
ANG1 promoter and a defect of this complex formation
derepresses ANG1 transcription, promoting endothelial cell
survival and vascular enlargement (43). Interestingly, other
studies showed that BRCA1 physically interacts with ER and
inhibits transcriptional activity of the receptor (44, 45). In this
study, we found that high CtIP expression is significantly
associated with ER+ status. Thus, it is possible that CtIP may
functionally be linked with ER signaling via its interaction with
BRCA1. Moreover, CtIP was also shown to form a complex
with BRCA1 and the transcriptional corepressor CtBP, which is
important for the repression of p21 promoter activity (29).
Together, it raises the possibility that in physiologic conditions,
CtIP could bridge BRCA1 and CtBP to form a transcriptional
repressor complex, which in turn may modulate ER signaling
pathways through the interaction between BRCA1 and ER.
Therefore, we hypothesize that in tamoxifen-sensitive cells,
BRCA1, CtIP, and CtBP form a transcriptional repressor
complex that leads to inhibition of a full ER+ transcriptional
response, accounting for the inhibitory growth effects of
tamoxifen. To circumvent the transcriptional inhibitory effects
of tamoxifen, tamoxifen-resistant cells silence CtIP expression,
which, in turn, disrupts the repressor complex and allows breast
cancer cells to resume proliferation. Further characterization of
the CtIP signaling pathways may provide insights into how CtIP
silencing leads to the development of tamoxifen resistance.
Materials and Methods
Cell Lines and Chemicals
Parental MCF-7 cells used in this study have been described
previously (46). The tamoxifen-resistant MCF-7 isogenic cell
line variants (termed TAMR1 and TAMR2) were generated
in our laboratory by culturing MCF-7 cells under continuous
4-OH-TAM (the active metabolite of tamoxifen; 1 Amol/L)
exposure for f2 years. These cells were maintained in phenol
red-free IMEM medium containing 5% fetal bovine serum
(TAMR1) or 5% charcoal-stripped fetal bovine serum
(TAMR2), glutamine (2 mmol/L), gentamicin (50 Ag/mL)
and 4-OH-TAM (1 Amol/L). The characterization of parental
MCF-7 and TAMR1 cells has been previously described
(47, 48). Tamoxifen-resistant T-47-D cells (T-47-D/TR) were
generated by growing regular ER+ T-47-D cells in the presence
of 4-OH-TAM for over 3 months. These cells were maintained
in DMEM (Cambrex Bio Science) medium containing 10%
fetal bovine serum and 4-OH-TAM (1 Amol/L). Other breast
cancer cell lines used, including SUM-44-PE, ZR-75-1, MDA-
MB-231, MDA-MB-435, SKBR3, and BT-474, were main-
tained in DMEM (Cambrex Bio Science) supplemented with
10% fetal bovine serum. The BT-483 cell line was maintained
in RPMI (Cambrex Bio Science) supplemented with 10% fetal
bovine serum. The UACC-812 breast cancer cell line was
grown in L-15 medium (Invitrogen) supplemented with 10%
fetal bovine serum. We purchased 4-OH-TAM and E
2
from
Sigma-Aldrich.
Serial Analysis of Gene Expression
SAGE was done on tamoxifen-sensitive parental MCF-7 line
and tamoxifen-resistant TAMR1 and TAMR2 lines. SAGE
library generation, sequencing, and tag extraction were done as
previously described (46, 49, 50). SAGE data were analyzed
using an ANOVA-based multivariate approach called multiple
linear contrast analysis. Two contrasts were defined to identify
differentially expressed genes in the tamoxifen-resistant cells,
one comparing the average expression level in the two resistant
cell lines to that of the parental strain and the second one
comparing the expression levels between the two resistant
Wu et al.
Mol Cancer Res 2007;5(12). December 2007
1292
strains. The null hypothesis for first contrast tests for lack of
differential expression between resistant and parental strains
and the second tests for consistent expression between the two
resistant cell lines. Significance of the null hypotheses for these
tests was set at the 95% level after a Bonferroni-type adjustment
for the multiplicity of comparisons.
Western Blot Analysis
Cells were washed twice with ice-cold PBS and then lysed
with radioimmunoprecipitation assay buffer [10 mmol/L Tris,
5 mmol/L EDTA, 150 mmol/L NaCl, 0.1% SDS, 1% Triton
X-100, 1% deoxycholate, (pH 7.2), 1protease inhibitor
cocktail (Roche)]. Cell lysates were flushed 10 times through
21-gauge needle and microcentrifuged at 21,000 gfor
10 min at 4jC. Supernatants were collected and protein
concentration was measured with a Pierce Protein Assay Kit,
according to the manufacturer’s instruction. Equal amount
(30– 50 Ag) of protein from each sample was separated on 6%
to 10% SDS-PAGE and transferred to polyvinylidene difluoride
membranes by electroblotting. Blots were first treated with
blocking buffer [5% milk in 1TBS containing 0.1% Tween
20 (TBS-T)] for 1 h and then incubated with primary antibodies
for 1 to 2 h. After washing thrice in TBS-T, blots were
incubated with horseradish peroxidase–labeled secondary
antibodies for 1 h. Labeled proteins were detected using KPL
Protein Detector chemiluminescence detection reagents and
exposed to X-ray films. All procedures described were carried
out at room temperature. Antibodies used were as follows:
CtIP (14-1; ref. 27; T-16, Santa Cruz Biotechnology; 19E8,
GeneTex), ER (HC-20, Santa Cruz Biotechnology), anti-Flag
(M2, Sigma), and h-actin (AC-15, Sigma).
Human Samples
Primary breast cancer formalin-fixed, paraffin-embedded
tissue samples were collected retrospectively from 59 postmen-
opausal patients with stage II to III ER (+) breast carcinomas
(median age 78 years; range 60 to 92 years). The 59 patients
were treated at a single institution (Instituto Valenciano
Oncologı´a, Valencia, Spain) between 1999 and 2002 with 4
months neoadjuvant endocrine therapy consisting of tamoxifen
(23 patients) or letrozole (36 patients) for large nonoperable or
locally advanced ER+ breast cancers. These patients were part of
a larger study published elsewhere (13). All patients gave written
informed consent before the submission of tumor samples for
CtIP analyses, and the local ethic committee approved the study
protocol and informed consent form. Determination of response
to the referred endocrine adjuvant therapy was made after
4 months of patient follow-up. We used one-way ANOVA
followed by Tukey’s test post hoc comparisons, and Pearson’s
correlation test to assess the association between CtIP protein
status (raw IRS scores) and clinical response to endocrine
therapy. All statistical analyses were two side, and P< 0.05 was
considered as statistically significant. Analyses were conducted
using SPSS version 11.5 software (SPSS, Inc.).
Immunohistochemistry Analysis
Immunohistochemical staining was done as previously
described (51) with a rabbit polyclonal CtIP antibody (H-300,
1:100 dilution, Santa Cruz Biotechnology). CtIP expression
levels was scored blindly by two independent pathologists
(D.R.S. and M.I.N.) using the IRS method as previously
described (14, 15). In brief, the IRS was calculated by
multiplying the percentage of CtIP-positive cells (scored 0 to
4: 0, 0%; 1, 0-25%; 2, 26-50%; 3, 51-75%; 4, >75%) with the
CtIP staining intensity (scored 0 to 3: 0, none; 1, weak; 2,
moderate; 3, strong).
Meta-analysis of Breast Cancer Microarray Data Sets
CtIP gene expression profiles and clinicopathologic data of
828 breast carcinomas were obtained from seven published and
publicly available breast cancer microarray data sets (16-22).
The Oncomine cancer microarray database was used for data
collection, processing, and visualization (23).
6
CtIP gene
expression was log-transformed, median centered for each
gene expression data set, and SD was normalized to one per
array. The gene module application was used for differential
expression analysis (two-sided ttest). We used a meta-analysis
approach to determine and summarize the CtIP mRNA
expression pattern from the seven independent studies. We
computed summary estimates (effect sizes) of CtIP expression
changes by the standardized mean difference method using the
exact tvalues and sample size for each groups. To calculate the
pooled effects of CtIP profile, each study was weighed by the
inverse of the individual and between-study variance according
to a random-effects model. Meta-analysis was carried out using
comprehensive meta-analysis software (Biostat, Inc.). All effect
sizes were presented with 95% confidence interval –based on
the estimated variances.
In vitro Cell Proliferation Assays
Cells were cultured in estrogen-free medium for 48 h. On
day 0, 1 10
4
cells in estrogen-free medium were plated
in triplicate in 12-well plates. E
2
(10 nmol/L), 4-OH-TAM
(10 nmol/L), or ethanol (vehicle control, 1 AL/mL) was added
directly into the medium at the same time. Fresh medium
with the adequate treatment was changed every 2 days. Cell
counts were done at various time points as indicated in the
figures.
RNA Interference
siRNA expression cassette (SEC) encoding siRNA targeting
CtIP mRNA (from 2,492 to 2,512, NM_002894; AATGA-
TAGCTTGGAAGATATG) was generated using the Silencer
Express siRNA Expression Cassette Kits (Ambion). A negative
control SEC expressing siRNA with no significant homology to
human, mouse, or rat genome sequences was also generated
by the same method. The SECs were then cloned into the
mammalian expression pSEC-puro vector (Ambion). To obtain
cell clones that stably expressed siRNAs targeting CtIP, MCF-7
cells were transfected with either pSEC-CtIP-puro or pSEC-
Control-puro by electroporation. One day after, puromycin
(600 ng/mL) was added into the culture medium. After 3 weeks
of selection, puromycin-resistant clones were picked up,
expanded, and analyzed for CtIP expression levels by
immunoblotting. The stable clone displaying the lowest level
of CtIP protein expression was chosen for further studies.
CtIP and Tamoxifen Resistance
Mol Cancer Res 2007;5(12). December 2007
1293
Generation of Double-Stably Transfected Tet-off TAMR1
Cells with Doxycycline-Inducible Restoration of CtIP
The Tet-off gene expression system was purchased from BD
Biosciences Clontech. Full-length human CtIP cDNA with
three NH
2
-terminal Flag epitope tags was cloned into
pTRE2hyg response vector (pTRE2hyg-FLAG-CtIP). To
generate double-stable Tet-off TAMR1 cell clones, cells were
cotransfected with pTet-Off and pTRE2hyg-FLAG-CtIP vec-
tors. After electroporation, cells were plated in 10-cm dishes
and allowed to grow in regular medium containing 4-OH-TAM
(1 Amol/L) plus doxycycline (1 Ag/mL) for 48 h. Cells were
then selected for resistance to G418 (800 Ag/mL) and
hygromycin B (200 Ag/mL). Fresh doxycycline (1 Ag/mL)
was added to tamoxifen-containing medium (regular culture
medium for TAMR1 cells) every 2 days to maintain a constant
suppression of CtIP expression during the selection process.
Hygromycin and G418 double-resistant colonies began to
appear after 3 to 4 weeks of selection. Thirty-seven large and
healthy colonies were isolated using cloning cylinders and
transferred to individual wells for expansion. Each clone was
first screened by immunoblotting using anti-Flag M2 antibody
for doxycycline-responsive CtIP expression in the presence or
absence of 1 Ag/mL doxycycline. We then used anti-CtIP (14-1)
antibody to assess the total CtIP protein in the positive clones.
Clone 32, in which the level of total CtIP protein expressed
upon the withdrawal of doxycycline was similar to that
produced in the parental MCF-7 cells, was chosen for further
studies.
Acknowledgments
We thank Dr. John H. Ludes-Meyers for helpful discussions.
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