Ctip Silencing As a Novel Mechanism of Tamoxifen Resistance in Breast Cancer

CtIP Silencing as a Novel Mechanism of Tamoxifen Resistance in Breast Cancer

Minhao Wu,1 David Ramos Soler,2 Martin C. Abba,1 Maria I. Nunez,1 Richard Baer,3 Christos Hatzis,4 Antonio Llombart-Cussac,5 Antonio Llombart-Bosch,2 and C. Marcelo Aldaz1

1Department of Carcinogenesis, The University of Texas M. D. Anderson Cancer Center, Smithville, Texas; 2Department of Pathology, Medical School, The University of Valencia, Valencia, Spain; 3Institute for Cancer Genetics, Columbia University Medical Center, New York, New York; 4Nuvera Biosciences, Inc., Woburn, Massachusetts; and 5Department of Oncology, Fundacio´n Instituto Valenciano de Oncologı´a, Valencia, Spain

Abstract breast cancer, suggesting that CtIP is likely associated Acquired resistance to the antiestrogen tamoxifen with ER function, and that CtIP gene and protein constitutes a major clinical challenge in breast cancer expression may be useful biomarkers for breast cancer therapy. However, the mechanisms involved are still prognosis and clinical management. (Mol Cancer Res poorly understood. Using serial analysis of gene 2007;5(12):1285–95) expression, we identified CtIP, a BRCA1- and CtBP- interacting protein, as one of the most significantly down-regulated transcripts in estrogen receptor Introduction A–positive (ER+) MCF-7 tamoxifen-resistant breast Estrogen plays a pivotal role in the etiology and progression cancer cells. We further confirmed the association of of human breast cancer. Therefore, for a long time, treatment of CtIP down-regulation with tamoxifen resistance in an breast cancer has been directed toward inhibiting the tumor- additional ER+ breast cancer line (T47D), strengthening promoting effects of estrogen. Tamoxifen, a nonsteroidal the relevance of the phenomenon observed. In antiestrogen, has been the gold standard for endocrine treatment a additional studies, we found CtIP protein expression in a of all stages of estrogen receptor (ER) positive breast cancer majority of ER+ breast cancer cell lines that we tested, for >25 years and was the first approved drug by the Food and but no or very little CtIP expression in ER-negative lines. Drug Administration as a cancer chemopreventive agent for Furthermore, CtIP protein expression status correlates reducing breast cancer incidence in both premenopausal and with clinical response to neoadjuvant endocrine therapy, postmenopausal women at high risk of breast cancer develop- and patients with progressive disease express ment (1). As adjuvant therapy, tamoxifen reduces the risk of significantly lower CtIP protein in their primary breast recurrence and improves overall survival in early breast cancer carcinomas than those who respond. Meta-analysis of (2). It was also shown to be effective for patients with untreated seven publicly available gene expression microarray metastatic breast cancer (2). Despite the benefits of tamoxifen data sets showed that CtIP expression is significantly in treating breast cancer, unfortunately, many tumors that associated with ER, disease-free survival, and breast initially respond to tamoxifen therapy develop resistance. This cancer metastasis status. Importantly, we found that phenomenon has become a serious obstacle in breast cancer silencing endogenous CtIP in tamoxifen-sensitive treatment. In the clinic, almost all patients with advanced breast cancer cells confers tamoxifen resistance. On the metastatic disease and as many as 40% of patients receiving other hand, reexpression of CtIP in tamoxifen-resistant adjuvant tamoxifen eventually relapse and die from their breast cancer cells restores sensitivity to the inhibitory disease (3). The mechanisms involved in the development of growth effects of tamoxifen. Together, our findings tamoxifen resistance are still poorly understood. Numerous indicate that CtIP silencing might be a novel mechanism mechanisms have been proposed to contribute to the develop- for the development of tamoxifen resistance in 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- Received 3/15/07; revised 7/23/07; accepted 8/15/07. lational modifications of ER, altered expression of coregulators Grant support: Susan G. Komen Breast Cancer Foundation Award, BC- (e.g., increased expression of the coactivator AIB1 or decreased TR0402877 (C.M. Aldaz), and Department of Defense Concept Award, expression of the corepressor NCoR), and increased growth W81XWH-04-1-0622-01 (C.M. Aldaz). The costs of publication of this article were defrayed in part by the payment of factor signaling (e.g., HER2signaling pathways; reviewed in page charges. This article must therefore be hereby marked advertisement in refs. 3-9). Interestingly, a recent study showed that DIBA, an accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ER zinc finger inhibitor, restores the antagonistic action of Requests for reprints: C. Marcelo Aldaz, Department of Carcinogenesis, The University of Texas M. D. Anderson Cancer Center, Science Park Research tamoxifen in tamoxifen-resistant breast cancer cells through Division, 1808 Park Road 1C, P.O. Box 389, Smithville, TX 78957. Phone: 512- targeted disruption of the ER DNA-binding domain and its 237-2403; Fax: 512-237-2475. E-mail: [email protected] Copyright D 2007 American Association for Cancer Research. interaction with the proximal NH2-terminal domain to suppress doi:10.1158/1541-7786.MCR-07-0126 ligand-dependent and ligand-independent ER transcription and

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 FIGURE 1. CtIP expression in tamoxifen-sensitive and tamoxifen- profiles of two independently derived isogenic MCF-7 breast resistant breast cancer cells. A and B. In vitro characterization of parental cancer cell line variants (TAMR1 and TAMR2) that are 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 resistant to tamoxifen and their parental tamoxifen-sensitive (A) or 10 nmol/L E2 (B). Points, mean of triplicate determinations; bars, SE. MCF-7 line by using SAGE. As shown in Fig. 1A, in contrast C. CtIP expression levels in parental MCF-7, TAMR1, and TAMR2 cells as to their parental MCF-7 cells, both TAMR1 and TAMR2cells determined by SAGE (tag frequencies, normalized to 200,000 tags per library). D. CtIP and ER protein levels were determined by immunoblotting cultured in estrogen-depleted medium proliferated in the in cell lines as indicated. h-Actin was used as a loading control. E. Parental presence of tamoxifen. Additionally, despite continuous 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 exposure to trans-4-hydroxytamoxifen (4-OH-TAM), both determined at various time points as indicated. Points, mean; bars, SE. tamoxifen-resistant variants are still estrogen responsive F. Western blotting for CtIP in parental T-47-D and T-47-D cultured in (Fig. 1B) and express equivalent levels of ER protein as their tamoxifen for 1, 2, and 3 mo. parental MCF-7 cells (Fig. 1D). Therefore, both TAMR1 and TAMR2cells express functional ER, as do their parental MCF-7 cells. The tamoxifen-resistant phenotypes of these cells analysis, we detected CtIP protein expression in the tamoxifen- seem not to be a consequence of changes in ER expression or sensitive parental MCF-7 line, but not in the two tamoxifen- function. These data are also consistent with the clinical resistant lines (Fig. 1D). In fact, both TAMR1 and TAMR2cells findings that the majority of patients with acquired resistance to seem to express little or no CtIP protein. tamoxifen still express functional ERs (11, 12). To determine whether this was a phenomenon exclusive to By SAGE comparative analyses, we identified that the MCF-7 cells, we selected another estrogen-responsive and transcript encoding for CtIP (also known as retinoblastoma tamoxifen-sensitive breast cancer cell line, T-47-D, and deve- binding protein 8, RBBP8) was significantly down-regulated loped a T-47-D/TR variant that is resistant to tamoxifen. To this (15-fold) in both tamoxifen-resistant cell lines when compared end, we followed the same procedure as with MCF-7 cells, with their tamoxifen-sensitive parental MCF-7 counterpart growing parental tamoxifen-sensitive T-47-D cells under chronic (Fig. 1C). Real-time reverse transcription-PCR on the same exposure to 4-OH-TAM (1 Amol/L). After an initial growth arrest RNA samples used for SAGE analysis also confirmed by tamoxifen, T-47-D cells regained exponential growth ability significantly lower CtIP mRNA expression in the tamoxifen- even in the presence of tamoxifen. As seen in Fig. 1E, after resistant cells than in the tamoxifen-sensitive parental MCF-7 being cultured in tamoxifen for over 3 months, T-47-D/TR cells cells (data not shown). Next, we determined whether the proliferated in fetal bovine serum–supplemented growth differential expression of CtIP detected at the mRNA level medium even in the presence of tamoxifen, whereas the growth could also be observed at the protein level. By Western blot of parental cells was significantly inhibited by tamoxifen.

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 (ERÀ) 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+, nonoperable primary breast carcinomas from patients who received endocrine therapy as single neoadjuvant therapy. Immunohisto- FIGURE 3. Poor clinical response to endocrine therapy is significantly chemistry was done of samples from tumors before initiation of associated with CtIP deficiency in primary breast carcinomas. A. CtIP expression patterns in human breast carcinomas. CtIP expression was antiestrogen therapy. Based on the clinical response to the evaluated by immunohistochemistry and semiquantified using IRS in 59 therapy after 4 months of follow-up, patients were classified into inoperable hormone receptor – positive primary breast carcinomas (before four groups: complete response, 4 cases (7%); partial response, treatment) from patients who received endocrine therapy as single neoadjuvant therapy. Representative tumor CtIP immunoreactivity (IRS 32cases (54%); stable disease, 17 cases (29%),and progressive scores 0, 4, 8, and 12). B. CtIP status strongly correlates with clinical disease, 6 cases (10%). These cases are representative of a larger response to endocrine therapy (Pearson correlation, P = 0.004), and cohort previously reported in which complete response was patients with progressive disease have significantly lower CtIP IRS than those who completely respond to endocrine therapy. **, P = 0.006 by reported to be between 4% and 10% (13). Immunoreactive ANOVA and Tukey’s post tests. CR, complete response; PR, partial scores (IRS) for CtIP were used to semiquantify immunohisto- response; SD, stable disease; and PD, progressive disease. chemical staining intensity and percentage of positive cells

(14, 15). IRS ranging from 0 to 12represents 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 FIGURE 2. CtIP protein expression in breast cancer cell lines as seem to indicate that poor response to endocrine therapy is determined by Western blot analysis. ER expression status is also associated with CtIP deficiency in breast cancer patients. indicated as positive (+) or negative (À). As can be observed, all ERÀ breast cancer lines (100%) tested do not express CtIP protein, whereas However, these observations have to be confirmed in larger four of six ER+ lines (67%) express this protein. cohorts.

CtIP Expression Is Associated with ER, Disease-Free transition, we next compared cell proliferation between MCF-7/ Survival, and Breast Cancer Metastasis Status CtIP siRNA and MCF-7/(À) control siRNA cells under various To further explore the clinical relevance of CtIP expression stimuli. Reduced CtIP expression increased MCF-7/CtIP in breast cancer, we evaluated information of seven publicly siRNA cell proliferation under estrogen-deprived conditions available breast cancer gene expression microarray data sets compared with control siRNA cells (Fig. 5B). When hormone- (16-22) through the use of the web-based Oncomine cancer starved experimental and control siRNA cells were treated with microarray database (23).6 Clinicopathologic and gene expres- 4-OH-TAM, growth of the control cells was inhibited by sion data from a total of 828 breast carcinomas was obtained exposure to tamoxifen, whereas growth of MCF-7/CtIP siRNA using this publicly available resource. Because ER plays a cells was not inhibited by tamoxifen, indicating acquired critical role in the clinical management of breast cancer resistance to tamoxifen (P < 0.05; Fig. 5C). Furthermore, CtIP- patients, we first analyzed levels of CtIP mRNA expression silenced MCF-7/CtIP siRNA cells grew significantly faster than in the mentioned microarray sets according to ER status of the control cells in the presence of both estrogen plus tamoxifen tumors (Fig. 4A). By using a meta-analysis approach, we (P < 0.01), indicating that unlike in control cells, tamoxifen directly compared CtIP expression profiles between 590 ER+ indeed fails to inhibit the stimulatory effect of estrogen in CtIP- and 238 ERÀ breast carcinomas by combining all seven silenced MCF-7 cells (Fig. 5D). Interestingly, the MCF-7/CtIP microarray data sets. We found a significant association siRNA cells still retain response to estrogen-stimulated cell between high CtIP expression and ER (+) status in breast proliferation when exposed to estradiol (Fig. 5E), indicating carcinomas (P < 0.0001; Fig. 4A). Next, we analyzed CtIP that ER is still functional and capable of regulating cell growth. expression profiles versus disease-free survival in two micro- In fact, after 10 days in culture in the presence of estrogen, array data sets that had at least 5 years of follow-up clinical the cell number of both control and MCF-7/CtIP siRNA cells information available. Analysis from the study of van de Vijver increased f145-fold (Fig. 5E), which is five to eight times et al. (19) showed a statistically significant association between more than that of the MCF-7/CtIP siRNA cells cultured in the loss of CtIP and disease relapse (P = 0.019; Fig. 4B, left). A presence of tamoxifen (Fig. 5C) or in the absence of estrogen trend was also found in the Sorlie et al. study (20) but did not (Fig. 5B), respectively (over the same time period), suggesting reach statistical significance (P = 0.069), possibly due to the that estrogen still seems to stimulate cell proliferation in MCF-7 low number of samples (Fig. 4B, right). However, by using the cells regardless of CtIP status. Taken together, these data meta-analysis approach and pooling both studies together, we indicated that CtIP silencing leads to resistance to the inhibitory found a highly significant association between decreased growth effects of tamoxifen in vitro. CtIP expression and disease relapse (P = 0.004; Fig. 4B). Furthermore, CtIP mRNA expression levels were significantly Reexpression of CtIP in Tamoxifen-Resistant Cells lower in invasive breast carcinomas that had distant metastasis Restores Sensitivity to the Inhibitory Growth Effects of when compared with breast cancer counterparts that did not Tamoxifen metastasize (P = 0.029; Fig. 4C). Next, we addressed the reciprocal question of whether reexpression of CtIP in tamoxifen-resistant cells abrogates Silencing Endogenous CtIP in Tamoxifen-Sensitive resistance to tamoxifen. Because both tamoxifen-resistant lines MCF-7 Cells Confers Tamoxifen Resistance In vitro have similar proliferation profiles in the presence of tamoxifen, To further explore the putative role of CtIP in the we selected the TAMR1 cell line for further functional studies. development of tamoxifen resistance, we first examined To this end, a Tet-off–inducible gene expression system was whether silencing the expression of endogenous CtIP in used to reexpress CtIP in the tamoxifen-resistant TAMR1 cells. tamoxifen-sensitive cells can induce a tamoxifen-resistant TAMR1 cells were transiently cotransfected with Tet-off– phenotype. Knockdown of CtIP protein levels in tamoxifen- inducible vectors containing NH2-terminal Flag-tagged full- sensitive parental MCF-7 cells was achieved using RNA length human CtIP cDNA. After transfection, cells were interference techniques. As shown in Fig. 5A, in the resulting immediately divided equally into two batches. The first batch clone [MCF-7/CtIP; small interfering (siRNA)] stably trans- was treated with 4-OH-TAM or vehicle, and cultured in the fected with a vector expressing siRNA targeting CtIP mRNA, presence of doxycycline. The second batch was treated with the silencing of CtIP protein levels reproduces quite closely 4-OH-TAM or vehicle, but cultured in the absence of the expression difference observed between the tamoxifen- doxycycline. Expression of FLAG-CtIP was analyzed by sensitive (parental MCF-7) and tamoxifen-resistant (TAMR1 immunoblotting after 72h of transfection. FLAG-CtIP was only and TAMR2) cells. The negative control siRNA clone detected in TAMR1 cells cultured without doxycycline (Fig. 6A, [MCF-7/(À) control siRNA] showed unchanged CtIP protein left), indicating the restoration of CtIP is tightly controlled by levels when compared with parental MCF-7 cells (Fig. 5A). doxycycline. Cell proliferation was determined in TAMR1 cells In addition, we observed an equal level of ER expression in with or without CtIP reexpression. This experiment showed all three clones, indicating that ER protein expression was that cells with restored CtIP protein expression displayed a not affected by the siRNA intervention (Fig. 5A). significant growth inhibition by tamoxifen in comparison with To test whether silenced CtIP expression in parental MCF-7 control cells having no CtIP restoration (Fig. 6A, middle). cells leads to the tamoxifen-sensitive to tamoxifen-resistant Without tamoxifen treatment (vehicle control), transient CtIP restoration had no significant effect on TAMR1 cell proliferation (Fig. 6A, right). Therefore, restoration of CtIP abrogates 6 http://www.oncomine.org resistance to tamoxifen in tamoxifen-resistant cells.

To further confirm the observations derived from the selected for further study. This clone showed no CtIP expression transient transfection experiments, we developed double stably in the presence of doxycycline but similar CtIP protein levels to transfected TAMR1 Tet-off FLAG-CtIP cells. Among the those produced in parental MCF-7 cells upon doxycycline various stably transfected clones obtained, clone 32was 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).

FIGURE 5. Silencing of CtIP protein expression in tamoxifen-sensitive MCF-7 cells confers tamoxifen resistance in vitro. A. Western blot analysis of CtIP and ER proteins in parental MCF-7 cells, MCF-7/ (À) 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 (À) control cells grown in hormone-free medium and treated with E2 (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 E2 for 10 d. All experiments were done in triplicate. Points, mean; bars, SE. * and **, P <0.05andP < 0.01 by t test, respectively. Note that Y-axis scale for cell number in D is different from that in B, C, and E.

Next, we measured the growth of the double-stably transfected estrogen regardless of doxycycline status (Fig. 6D). Taken TAMR1 Tet-off FLAG-CtIP cells under conditions in which together, these results show that sensitivity to the inhibitory CtIP restoration was either induced or repressed. Cells from growth effects of tamoxifen in previously tamoxifen-resistant clone 32were cultured in two different conditions. Half of the cells is restored, at least partially by CtIP reexpression. cells were grown in hormone-free medium containing doxycycline whereas the other half was cultured in the same medium but devoid of doxycycline. After 3 days of incubation, cells Discussion were treated with either 17-h-estradiol (E2), 4-OH-TAM, or SAGE studies identified CtIP as one of the most vehicle control. As shown in Fig. 6C, the growth of TAMR1 significantly down-regulated transcripts in two independently Tet-off FLAG-CtIP cells was significantly inhibited by developed tamoxifen-resistant breast cancer cell lines when tamoxifen when doxycycline was removed from the medium compared with their tamoxifen-sensitive parental MCF-7 line. compared with cells from the same clone but cultured in the Immunoblotting analyses not only validated the SAGE presence of doxycycline (P < 0.05). Cells still responded well to observations but also showed a dramatic difference in CtIP

protein expression levels, with high levels of protein analysis of gene expression data sets, we also found an expression in parental ER+ MCF-7 breast cancer cells but association of CtIP expression levels with poor outcome. very little or no CtIP protein product in the ER+ tamoxifen- These in vivo data suggest that CtIP gene and protein resistant derivative isogenic cell lines. In addition to the expression may be useful biomarkers for breast cancer observations in MCF-7 cells, we also found significantly prognosis and clinical management, although it would require reduced CtIP protein expression in ER+ T-47-D breast cancer further studies in larger patient cohorts. In addition, we found cells when they became tamoxifen resistant. Furthermore, a significant reverse association of CtIP expression with ER consistent with the in vitro observations, we found significant status, suggesting that CtIP may function in a pathway or association of CtIP deficiency in breast cancers from patients pathways associated with ER signaling and perhaps regulate with poor clinical response to endocrine therapy. By meta- ER-mediated cell proliferation. All ERÀ breast 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 t test. D. Effect of E2 on the proliferation of clone 32 cells cultured with or without doxycycline. Note that Y axis scales for cell number in C and D are different from each other.

tested (intrinsically resistant to tamoxifen) express either none or functionally be linked with ER signaling via its interaction with very little CtIP protein. Interestingly, BT-474, an ER+ but BRCA1. Moreover, CtIP was also shown to form a complex tamoxifen-resistant breast cancer cell line as reported by Wang with BRCA1 and the transcriptional corepressor CtBP, which is et al. (10), also expresses nearly undetectable CtIP protein when important for the repression of p21 promoter activity (29). compared with ER (+) and tamoxifen-sensitive lines such as Together, it raises the possibility that in physiologic conditions, MCF-7 and T-47-D, which further supports the important role of CtIP could bridge BRCA1 and CtBP to form a transcriptional CtIP in the development of tamoxifen resistance. Finally, based repressor complex, which in turn may modulate ER signaling on RNA interference and reciprocal reexpression studies, we pathways through the interaction between BRCA1 and ER. were able to reproduce the tamoxifen-resistant phenotype Therefore, we hypothesize that in tamoxifen-sensitive cells, simply by shutting down the expression of one gene BRCA1, CtIP, and CtBP form a transcriptional repressor (i.e., CtIP). These data show that CtIP silencing is critical for complex that leads to inhibition of a full ER+ transcriptional the development of tamoxifen resistance in breast cancer in vitro response, accounting for the inhibitory growth effects of models and suggest that CtIP silencing may be a novel tamoxifen. To circumvent the transcriptional inhibitory effects mechanism by which cells can circumvent the inhibitory effects of tamoxifen, tamoxifen-resistant cells silence CtIP expression, of tamoxifen to resume proliferation and ultimately acquire which, in turn, disrupts the repressor complex and allows breast resistance to the antiestrogen tamoxifen. cancer cells to resume proliferation. Further characterization of The human CtIP (also known as RBBP8) encodes an 897- the CtIP signaling pathways may provide insights into how CtIP amino-acid nuclear protein that is widely expressed in various silencing leads to the development of tamoxifen resistance. 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 Materials and Methods p130; refs. 25, 28) and BRCA1 (26, 29-31), as well as the Cell Lines and Chemicals transcriptional repressors such as LIM-only protein LMO4 (30) Parental MCF-7 cells used in this study have been described and Ikaros family members (32). Recent studies suggest that previously (46). The tamoxifen-resistant MCF-7 isogenic cell CtIP plays an important role in cell cycle regulation and DNA line variants (termed TAMR1 and TAMR2) were generated damage response (33-37). Emerging evidence also suggests that in our laboratory by culturing MCF-7 cells under continuous A CtIP may itself be a tumor susceptibility gene. Analysis of CtIP 4-OH-TAM (the active metabolite of tamoxifen; 1 mol/L) f cDNA from 89 human tumor cell lines revealed 5 missense and exposure for 2years. These cells were maintained in phenol 11 silent mutations (26). In a more recent screening study of red-free IMEM medium containing 5% fetal bovine serum 109 colon cancers, CtIP was found to be a frequent target for (TAMR1) or 5% charcoal-stripped fetal bovine serum A microsatellite instability (38). More importantly, it has been (TAMR2), glutamine (2 mmol/L), gentamicin (50 g/mL) A shown that inactivation of CtIP in mice leads to early and 4-OH-TAM (1 mol/L). The characterization of parental embryonic lethality, and the life span of Ctip+/À heterozygotes, MCF-7 and TAMR1 cells has been previously described which have Ctip haploid insufficiency, was shortened due to (47, 48). Tamoxifen-resistant T-47-D cells (T-47-D/TR) were the development of multiple types of tumors. These findings generated by growing regular ER+ T-47-D cells in the presence show that CtIP is a critical protein in early embryogenesis and of 4-OH-TAM for over 3 months. These cells were maintained implicates an important role of CtIP in tumorigenesis (39). In in DMEM (Cambrex Bio Science) medium containing 10% A addition, CtIP interacts with the BRCT domains of BRCA1 fetal bovine serum and 4-OH-TAM (1 mol/L). Other breast where most mutations occur in BRCA1 breast cancer patients, cancer cell lines used, including SUM-44-PE, ZR-75-1, MDA- and such protein-protein interaction is abolished by tumor- MB-231, MDA-MB-435, SKBR3, and BT-474, were main- associated mutations in the BRCT domains (26, 29, 31), tained in DMEM (Cambrex Bio Science) supplemented with suggesting that interaction between CtIP and BRCA1 is 10% fetal bovine serum. The BT-483 cell line was maintained of functional relevance in the breast cancer suppressor activity. in RPMI (Cambrex Bio Science) supplemented with 10% fetal It has been shown that amino acid residues 299 to 345 of bovine serum. The UACC-812breast cancer cell line was CtIP mediate its interaction with the BRCT domains of BRCA1 grown in L-15 medium (Invitrogen) supplemented with 10% (26, 27, 37). Recently, it was also reported that phosphorylation fetal bovine serum. We purchased 4-OH-TAM and E2 from at Ser327 in CtIP seemed to be critical for its interaction with Sigma-Aldrich. BRAC1 BRCT domains (37, 40). Available evidence suggests that CtIP is involved in Serial Analysis of Gene Expression transcriptional repression (reviewed in refs. 41, 42). Signifi- SAGE was done on tamoxifen-sensitive parental MCF-7 line cantly, recent studies showed that BRCA1, CtIP, and ZBRK1 and tamoxifen-resistant TAMR1 and TAMR2lines. SAGE form a repressor complex at a recognition site of ZBRK1 in library generation, sequencing, and tag extraction were done as ANG1 promoter and a defect of this complex formation previously described (46, 49, 50). SAGE data were analyzed derepresses ANG1 transcription, promoting endothelial cell using an ANOVA-based multivariate approach called multiple survival and vascular enlargement (43). Interestingly, other linear contrast analysis. Two contrasts were defined to identify studies showed that BRCA1 physically interacts with ER and differentially expressed genes in the tamoxifen-resistant cells, inhibits transcriptional activity of the receptor (44, 45). In this one comparing the average expression level in the two resistant study, we found that high CtIP expression is significantly cell lines to that of the parental strain and the second one associated with ER+ status. Thus, it is possible that CtIP may comparing the expression levels between the two resistant

strains. The null hypothesis for first contrast tests for lack of 1:100 dilution, Santa Cruz Biotechnology). CtIP expression differential expression between resistant and parental strains levels was scored blindly by two independent pathologists and the second tests for consistent expression between the two (D.R.S. and M.I.N.) using the IRS method as previously resistant cell lines. Significance of the null hypotheses for these described (14, 15). In brief, the IRS was calculated by tests was set at the 95% level after a Bonferroni-type adjustment multiplying the percentage of CtIP-positive cells (scored 0 to for the multiplicity of comparisons. 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, Western Blot Analysis moderate; 3, strong). Cells were washed twice with ice-cold PBS and then lysed with radioimmunoprecipitation assay buffer [10 mmol/L Tris, Meta-analysis of Breast Cancer Microarray Data Sets 5 mmol/L EDTA, 150 mmol/L NaCl, 0.1% SDS, 1% Triton CtIP gene expression profiles and clinicopathologic data of X-100, 1% deoxycholate, (pH 7.2), 1Â protease inhibitor 828 breast carcinomas were obtained from seven published and cocktail (Roche)]. Cell lysates were flushed 10 times through publicly available breast cancer microarray data sets (16-22). 21-gauge needle and microcentrifuged at 21,000 Â g for The Oncomine cancer microarray database was used for data 10 min at 4jC. Supernatants were collected and protein collection, processing, and visualization (23).6 CtIP gene concentration was measured with a Pierce Protein Assay Kit, expression was log-transformed, median centered for each according to the manufacturer’s instruction. Equal amount gene expression data set, and SD was normalized to one per (30–50 Ag) of protein from each sample was separated on 6% array. The gene module application was used for differential to 10% SDS-PAGE and transferred to polyvinylidene difluoride expression analysis (two-sided t test). We used a meta-analysis membranes by electroblotting. Blots were first treated with approach to determine and summarize the CtIP mRNA blocking buffer [5% milk in 1Â TBS containing 0.1% Tween expression pattern from the seven independent studies. We 20 (TBS-T)] for 1 h and then incubated with primary antibodies computed summary estimates (effect sizes) of CtIP expression for 1 to 2h. After washing thrice in TBS-T, blots were changes by the standardized mean difference method using the incubated with horseradish peroxidase–labeled secondary exact t values and sample size for each groups. To calculate the antibodies for 1 h. Labeled proteins were detected using KPL pooled effects of CtIP profile, each study was weighed by the Protein Detector chemiluminescence detection reagents and inverse of the individual and between-study variance according exposed to X-ray films. All procedures described were carried to a random-effects model. Meta-analysis was carried out using out at room temperature. Antibodies used were as follows: comprehensive meta-analysis software (Biostat, Inc.). All effect CtIP (14-1; ref. 27; T-16, Santa Cruz Biotechnology; 19E8, sizes were presented with 95% confidence interval–based on GeneTex), ER (HC-20, Santa Cruz Biotechnology), anti-Flag the estimated variances. (M2, Sigma), and h-actin (AC-15, Sigma). In vitro Cell Proliferation Assays Human Samples Cells were cultured in estrogen-free medium for 48 h. On Primary breast cancer formalin-fixed, paraffin-embedded day 0, 1 Â 104 cells in estrogen-free medium were plated tissue samples were collected retrospectively from 59 postmen- in triplicate in 12-well plates. E2 (10 nmol/L), 4-OH-TAM opausal patients with stage II to III ER (+) breast carcinomas (10 nmol/L), or ethanol (vehicle control, 1 AL/mL) was added (median age 78 years; range 60 to 92years). The 59 patients directly into the medium at the same time. Fresh medium were treated at a single institution (Instituto Valenciano with the adequate treatment was changed every 2days. Cell Oncologı´a, Valencia, Spain) between 1999 and 2002 with 4 counts were done at various time points as indicated in the months neoadjuvant endocrine therapy consisting of tamoxifen figures. (23 patients) or letrozole (36 patients) for large nonoperable or locally advanced ER+ breast cancers. These patients were part of RNA Interference a larger study published elsewhere (13). All patients gave written siRNA expression cassette (SEC) encoding siRNA targeting informed consent before the submission of tumor samples for CtIP mRNA (from 2,492 to 2,512, NM_002894; AATGA- CtIP analyses, and the local ethic committee approved the study TAGCTTGGAAGATATG) was generated using the Silencer protocol and informed consent form. Determination of response Express siRNA Expression Cassette Kits (Ambion). A negative to the referred endocrine adjuvant therapy was made after control SEC expressing siRNA with no significant homology to 4 months of patient follow-up. We used one-way ANOVA human, mouse, or rat genome sequences was also generated followed by Tukey’s test post hoc comparisons, and Pearson’s by the same method. The SECs were then cloned into the correlation test to assess the association between CtIP protein mammalian expression pSEC-puro vector (Ambion). To obtain status (raw IRS scores) and clinical response to endocrine cell clones that stably expressed siRNAs targeting CtIP, MCF-7 therapy. All statistical analyses were two side, and P < 0.05 was cells were transfected with either pSEC-CtIP-puro or pSEC- considered as statistically significant. Analyses were conducted Control-puro by electroporation. One day after, puromycin using SPSS version 11.5 software (SPSS, Inc.). (600 ng/mL) was added into the culture medium. After 3 weeks of selection, puromycin-resistant clones were picked up, Immunohistochemistry Analysis expanded, and analyzed for CtIP expression levels by Immunohistochemical staining was done as previously immunoblotting. The stable clone displaying the lowest level described (51) with a rabbit polyclonal CtIP antibody (H-300, of CtIP protein expression was chosen for further studies.

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Mol Cancer Res 2007;5(12). December 2007 Downloaded from mcr.aacrjournals.org on September 24, 2021. © 2007 American Association for Cancer Research. CtIP Silencing as a Novel Mechanism of Tamoxifen Resistance in Breast Cancer

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