Wilms' Tumor 1 Suppressor Gene Mediates Antiestrogen Resistance Via Down-Regulation of Estrogen Receptor-A Expression in Breas

Wilms’ Tumor 1 Suppressor Gene Mediates Antiestrogen Resistance via Down-Regulation of Estrogen Receptor-A Expression in Breast Cancer Cells

Youqi Han, Lin Yang, Fernando Suarez-Saiz, Serban San-Marina, Jie Cui, and Mark D. Minden

Department of Cellular and Molecular Biology, Ontario Cancer Institute, University Health Network, Toronto, Ontario, Canada

Abstract site sequence–specific manner. Our study clearly The antiestrogen tamoxifen has been used in the implicates WT1 as a mediator of antiestrogen resistance treatment of hormone-responsive breast cancer for in breast cancer through down-regulation of ERA over a decade. The loss of estrogen receptor (ER) expression and supports the development of WT1 expression is the most common mechanism for de novo inhibitors as a potential means of restoring antiestrogen resistance. Wilms’ tumor 1 suppressor antiestrogen responsiveness in breast cancer therapy. gene (WT1) is a clinically useful marker that is (Mol Cancer Res 2008;6(8):1347–55) associated with poor prognosis in breast cancer patients; its high level expression is frequently observed Introduction in cases of breast cancer that are estrogen and The steroid hormone estrogen is a key regulator of growth progesterone receptor negative. The lack of expression and differentiation in normal mammary glands and plays a of these receptors is characteristic of tumor cells that major role in the growth and behavior of breast cancer cells (1). are not responsive to hormonal manipulation. To The effects of 17h-estradiol (E ) are mediated through its determine whether there is a linkage between WT1 2 binding to estrogen receptor (ER)-a and ERh, which function expression and antiestrogen resistance in breast cancer as ligand-activated transcription factors for growth-promoting cells, we studied the effect of WT1 on tamoxifen genes (2). The antiestrogen tamoxifen, a widely used drug in responsiveness in ERA-positive MCF-7 cells. We found the treatment of receptor positive breast cancer, primarily acts that overexpression of WT1 in MCF-7 markedly by competing with estrogen for binding to ER, leading to either abrogated tamoxifen-induced cell apoptosis and cell growth arrest or apoptotic cell death (3, 4). In contrast, 17B-estradiol (E )–mediated cell proliferation. The 2 ER-negative breast cancers are much less responsive to expressions of ERA and its downstream target genes endocrine manipulation (5). were significantly repressed following overexpression of The Wilms’ tumor 1 suppressor gene (WT1) was initially WT1, whereas the down-regulation of WT1 by WT1 identified as a tumor suppressor gene in a subset of pediatric shRNA could enhance ERA expression and the Wilms’ tumors. Despite its original reputation as a tumor sensitivity to tamoxifen treatment in ERA-negative suppressor, a growing body of experimental evidences indicates MDA468 and HCC1954 cells that express high levels of that WT1 functions as an oncogene in leukemias and a variety WT1. Furthermore, we have confirmed that the WT1 of solid tumors including breast cancer (6). The expression of protein can bind to endogenous WT1 consensus sites in WT1 in breast cancer has both prognostic and biological the proximal promoter of ERA and thus inhibit the effects. Several groups have found that high levels of WT1 transcriptional activity of the ERA promoter in a WT1 expression in breast cancer are associated with a worse prognosis (7-9). This is possibly due to a proliferation and survival effect provided by WT1 because down-regulation of WT1 inhibits breast cancer growth (10). Received 11/29/07; revised 4/11/08; accepted 5/12/08. WT1 is a modular transcription factor with an NH2- Grant support: Ontario Cancer Institute, the National Cancer Institute of Canada Terry Fox New Initiative Program, and the Canadian Breast Cancer Research terminal glutamine and proline-rich domain involved in Initiative. self-association, transcriptional repression, and transcriptional The costs of publication of this article were defrayed in part by the payment of activation. The four Cys-Cys-His-His type zinc finger page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. structure in the COOH terminus is involved in DNA and Note: Supplementary data for this article are available at Molecular Cancer RNA binding, nuclear localization, and protein-protein Research Online (http://mcr.aacrjournals.org/). interactions (11-13). Through alternative splicing, there are Requests for reprints: Mark D. Minden, Princess Margaret Hospital/Ontario Cancer Institute, University Health Network, Room 5-217, 610 University four predominant protein isoforms of WT1 that differ by the Avenue, Toronto, Ontario, Canada M5G 2M9. Phone: 416-946-2015; Fax: 416- presence of a 17-amino-acid insert between the activation/ 946-2082. E-mail: [email protected] Copyright D 2008 American Association for Cancer Research. repression and zinc finger domains and a 3-amino-acid insert doi:10.1158/1541-7786.MCR-07-2179 (KTS) that is found between the third and fourth zinc fingers

FIGURE 1. Abrogation of E2-induced cell proliferation and tamoxifen-mediated apoptosis in WT1-transfected MCF-7 cells. Stable transfectants of control MCF-7 cells (Vec) and MCF-7 cells overexpressing WT1 (WT1B and WT1D) were untreated (Con) and treated with ethanol (ETOH), 10 nmol/L E2,or1Amol/L tamoxifen (TAM)for4d.A. Surviving cell counts were done with trypan blue staining. The viable cell numbers in untreated samples are represented as 100%. Columns, average of four experiments; bars, SD. B. Dot plots show a representative experiment of flow cytometric analysis for staining of Annexin V-phycoerythrin (Annexin V-PE; horizontal axis) and 7-AAD (vertical axis). The percentages of cell populations in the different quadrants are indicated.

(14, 15). The different isoforms are referred to as A, B, C, and are WT1 positive whereas 60% of more advanced ER- D, where A lacks both the 17-amino-acid and KTS inserts; B negative tumors are WT1 positive. In the current publication, contains the 17-amino-acid insert but lacks KTS; C lacks the we have investigated whether there is any relationship 17-amino-acid insert but contains KTS; and D contains both between the level of ER and WT1 in breast cancer cells. the 17-amino-acid and KTS inserts. Using cell lines, we have found that the overexpression of Previous publications suggest an inverse correlation WT1 directly results in the down-regulation of ERa between WT1 expression and ER status in breast cancer. expression and the induction of antiestrogen resistance in Silberstein et al. (16) found that 22% of ER-positive tumors ER-positive cells.

Results were completely abolished in WT1-overexpressing MCF-7 E2-Induced Cell Proliferation and Tamoxifen-Mediated cells (Fig. 1B). Apoptosis Are Abolished by Overexpression of WT1 Protein WT1Down-Regulates Expressions of ER a and Its To determine the effect of WT1 on cell viability, trypan blue Downstream Target Genes exclusion was used to compare cells treated with E2 or The above-noted change in responsiveness to E2 and tamoxifen; results are presented as percent change in cells tamoxifen in an ER-positive cell line might be explained if excluding trypan blue. As can be seen in Fig. 1A, the addition the cells were no longer dependent on ER signaling for of E2 to parental MCF-7 cells enhanced cell viability by f1.75 survival, possibly through the loss of ERa receptors. To test times at 4 days, whereas viability was reduced by 50% in this, we measured the level of ERa protein in WT1-over- tamoxifen-treated cells at the same time point. In contrast, expressing MCF-7 cells. In keeping with our prediction, we MCF-7 cells overexpressing WT1B or WT1D did not show any found that concomitant with the increase in the level of WT1 change in cell viability with the addition of either E2 or protein, there was a sharp reduction in the amount of tamoxifen. To confirm this, we further analyzed cell apoptosis endogenous ERa protein (Fig. 2A). In agreement with this, by flow cytometry. The stably transfected MCF-7 cells were cells overexpressing WT1B and WT1D had reduced levels of simultaneously stained with fluorescent dyes Annexin V and ERa RNA regardless of the growth conditions (Fig. 2B and C). 7-amino-actinomycin D (7-AAD) that serve as indicators for We further sought confirmation of this observation by apoptosis at early and later stages, respectively. As expected, searching published microarray data of gene expression 4 days of treatment with E2 resulted in a decrease of the in breast cancer cell lines (17). As shown in Supplementary subpopulation undergoing apoptosis from 2.9% to 0.9% in Fig. S1, higher level of WT1 expression was associated with control MCF-7 cells. In contrast, and in keeping with the trypan lower levels of ERa expression in the majority of breast cancer blue results, tamoxifen treatment increased the population of cell lines. cells undergoing apoptosis to 15.1%. Significantly, both the Because ERa is a transcription factor, it is predicted that loss E2-enhanced growth and tamoxifen induction of apoptosis of ERa would result in decreased expression of its normal

FIGURE 2. Alteration of expressions of ERa and its target genes in WT1-transfected MCF-7 cells. A. Western blots show the abundance of WT1, ERa, and h-actin (right)in stable transfectants of control MCF-7 cells and MCF-7 cells overexpressing WT1 [WT1B (B) and WT1D (D)]. B to E. The stable transfectants of MCF-7 cells overexpressing vector, WT1B, or WT1D were untreated and treated with ethanol, 10 nmol/L E2,or1Amol/L tamoxifen for 2 d. Quantitative real- time PCRs were done with primers against mWT1 (B), ERa (C), PR (D), and PS2 (E). The expressions in empty vector – transfected cells untreated are represented as 1.0. Columns, average of three experiments.

FIGURE 3. Enhanced expressions of ERa and its target genes in MDA468 and HCC1954 cells transfected with WT1 shRNA. A. Western blots show the abundance of WT1 and h-actin (middle) in the transiently transfected MDA468 and HCC1954 cells with control pSuper vector lacking insert (pS), control nonsilencing shRNA (pSCN), or WT1 shRNAs [pSWT1a (a) and pSWT1b (b)] for 1 d. B to D. MDA468 and HCC1954 cells were transfected with either control nonsilencing shRNA or WT1 shRNAs (pSWT1a and pSWT1b) for 1 d, and then treated with either ethanol or 10 nmol/L E2 for 1 d. Quantitative real- time PCRs were done with primers against hWT1 (B), ERa (C), and PS2 (D). The expressions in pSCN-transfected cells treated with ethanol are represented as 1.0. Columns, average of three experiments; bars, SD.

targets in WT1-overexpressing cells. To test this, we assessed WT1 Gene Silencing Enhances the Expression and the levels of two ERa-regulated genes, progesterone receptor Activity of ERa (PR) and PS2 (18-21). As can be seen in Fig. 2D and E, the If WT1 inhibits the expression of ERa, then it is predicted basal level of PR is reduced in WT1-expressing cells. The that reducing the level of WT1 should result in increased ERa addition of E2 resulted in increased expression of PR and expression. MDA468 and HCC1954 cells are ERa-negative PS2in both parental and WT1-expressing MCF-7 cells. breast cancer cell lines with high level of endogenous WT1 However, the degree of response was substantially reduced in (Supplementary Fig. S1). To test if knockdown of WT1 WT1-overexpressing cells. This is consistent with the reduced expression would alter endogenous ERa abundance, we levels of ERa in those cells. transfected transiently WT1 shRNAs into MDA468 and

HCC1954 cells and examined the effect on ERa expression. WT1Protein Is Specifically Bound to a WT1Site in the As shown in Fig. 3A, both WT1 shRNAs markedly decreased Endogenous ERa Promoter the level of endogenous WT1 protein in these cell lines, To determine if the observed repression of ERa requires whereas control nonsilencing shRNA showed no significant direct binding of WT1 to the ERa promoter, we conducted effect on the level of WT1 protein. The WT1 gene silencing chromatin immunoprecipitation assays followed by PCR with by WT1 shRNAs was further confirmed by quantitative real- DNA from MCF-7 cells or MCF-7 cells engineered to time PCR with primers specific for human WT1 (Fig. 3B). overexpress His-tagged WT1B or WT1D (Fig. 4A). The Coincident with the knockdown of WT1 RNA expression, primers bracketed a potential WT1 binding site in the ERa ERa RNA expression was significantly up-regulated, a promoter (nucleotides À254 to À116; ref. 22). MCF-7 cells process irrespective of E2 treatment (Fig. 3C). Moreover, have low but detectable levels of endogenous WT1 and the expression of PS2,anERa target gene, was obviously express ERa, whereas the WT1-overexpressing cells have a increasedonE2 stimulation in WT1 knockdown cells reduced ER . Antibody against acetylated histone H4 was (Fig. 3D). The above results further indicate that WT1 is included as a positive control. In the control MCF-7 cell line, indeed involved in down-regulating the expression and the PCR-amplifiable fragment was immunoprecipitated with activity of ERa. antibody against WT1 but not with anti-His antibody. In cells expressing His-tagged WT1 constructs, the fragment was precipitated with both antibodies, indicating that exogenous as well as the endogenous WT1 proteins were recruited to the proximal ERa promoter. Conversely, when WT1 was knocked down in MDA468 cells, there was a marked reduction in the amount of ERa promoter that could be precipitated with the WT1 antibody (Fig. 4B). Taken together, our observations indicate that the WT1 protein specifically binds to DNA containing a WT1 site in the proximal ERa promoter, and that this is important in abrogating the transcriptional activation of the promoter in breast cancer cells.

WT1Regulates the Transcriptional Activity of the ER a Promoter In an effort to further elucidate the effect of WT1 on ERa transcriptional activity and to directly show sequence specificity requirement of the WT1 binding site, we used luciferase reporters driven by the ERa promoter that harbored a single WT1 binding site. As shown in Fig. 5, reporter expression was obviously enhanced when the WT binding site mutant was transfected into MCF-7 or MDA468 cells, as compared with the native form of the promoter. Cotransfection of WT1B or D with the wild-type promoter into MCF-7 cells further decreased wild-type reporter expression. In contrast, cotransfection of the wild-type promoter along with WT1 shRNA into MDA468 cells significantly increased the reporter expression as compared with control pSuper vector lacking insert. These effects were abolished with the reporter construct containing WT1 site mutant, thus further confirming the regulation of the ERa promoter by binding of WT1 to the ERa WT1 element in breast cancer cell lines.

Knockdown of WT1Increases Cell Sensitivity to Tamox- ifen Treatment Because the above results show that down-regulation of ERa by WT1 results in antiestrogen resistance in breast cancer FIGURE 4. Specific recruitment of WT1 protein to WT1 consensus motifs in the ERa promoter. A. Chromatin immunoprecipitation analysis cells, we sought to see if sensitivity of cells to tamoxifen is for the cross-linked sheared chromatin from control MCF-7 and MCF-7 restored following the down-regulation of WT1 in ERa- cells expressing His-tagged WT1 (WT1B and WT1D) as indicated on the left. The input and immunoprecipitation with preimmune serum (PI), anti- negative cells. To test this, we transfected MDA468 and WT1, anti-His, or anti – acetyl-histone H4 antibody are indicated on top. HCC1954 cells with WT1 shRNA and treated the cells with The 1-kb DNA ladder is shown on the left lane as a marker. B. Chromatin E2 or tamoxifen for 4 days. As expected, the down-regulation immunoprecipitation analysis of chromatin from MDA468 cells transfected with either control nonsilencing shRNA or WT1 shRNAs (pSWT1a and of WT1 resulted in a reduction of cell viability (Fig. 6). pSWT1b) for 1 d. Tamoxifen treatment further decreased viability by f20%,

there was a significant overexpression of WT1 in ERa-negative as compared with ERa-positive samples (P < 0.05).

Discussion There are a number of key descriptors of breast cancer with biological and therapeutic relevance, one of the more important being the status of expression of ERa (25). Whereas ER-positive tumors tend to be localized and respond well to hormonal manipulation (26), ER-negative tumors are more aggressive, disseminate widely, and do not respond to hormone therapy (5). A number of mechanisms for the development of ERa negativity have been identified (27-32). This includes hypermethylation of the ERa promoter in f60% of cases (33) and repression by transcription factors such as pRB2/p130 and LMO4 (34, 35). In the present study, we provide evidence that WT1 can interfere with both the growth-enhancing effect of estrogen and the antiestrogen effect of tamoxifen in MCF-7 cells. Shown herein are the results with WT1B and WT1D isoforms; similar results were obtained with WT1A and WT1C (data not shown). This effect was found to be due to reduced levels of ERa in the WT1-overexpressing cells. In keeping with the reduction in ERa protein, there was reduced expression of two bona fide ERa target genes, PR and PS2 (18-21). Conversely, the reduction of WT1 in the ERa-negative cell line MDA468 restored ERa expression. Using chromatin immunoprecipitation, we confirm the binding of WT1 to an upstream region of the ERa promoter that contains a WT1 binding site. Furthermore, we found that the effect of WT1 on ERa- promoter-luciferase constructs was dependent on an intact WT1 binding site. Together, these results are in keeping with the suggestion that the effect of WT1 is through direct binding to a canonical WT1 site in the ERa promoter DNA. A potential interaction between WT1 and ERa has previously been reported in another setting in breast cancer cells. In those studies, it was shown that the ERa and WT1 FIGURE 5. Regulation of the transcriptional activity of the ERa proteins could physically interact in breast cancer cells and that promoter by WT1. A. MCF-7 cells were transiently cotransfected with this association reduced or blocked ERa-mediated induction of luciferase reporter constructs (control pGL2 vector, ERPWT/Luc, or ERPmut/Luc) and expression plasmids of WT1 (horizontal axis; control insulin-like growth factor I receptor (IGF-IR; ref. 36). These pcDNA3 vector, WT1B, or WT1D) as indicated. Luciferase activity was studies do not contradict the work presented in this article, but shown as fold induction as compared with luciferase vector control (vertical axis) on the left. Columns, average of three independent rather highlight the variability of WT1 function dependent on experiments. B. MDA468 cells were transiently cotransfected with the cellular context within which it is expressed. It is interesting luciferase reporter constructs (control pGL2 vector, ERPWT/Luc, or to note in the above-mentioned study that a variant of MCF-7 ERPmut/Luc) and shRNA plasmids (horizontal axis; control pSuper vector, control nonsilencing shRNA, WT1 shRNAa, or WT1 shRNAb), cells was isolated by prolonged growth in the absence of respectively, as indicated. estrogen. These MCF-7 C4 cells had markedly reduced levels of ERa, reduced levels of IGF-IR, and increased levels of WT1. This phenotype that is ERa negative, IGF-IR low/negative, and whereas E2 treatment resulted in a 21% to 35% enhancement of WT1 positive is similar to that found in some cases of cell viability in these cells. metastatic breast cancer. We postulate that MCF-7 C4 cells mimic this form of metastatic breast cancer. Furthermore, we Clinical Association between WT1and ERa in Primary predict that the inhibition of WT1 in these cells by shRNA Breast Cancer would lead to reexpression of ERa. The results presented above suggest that breast cancer WT1 is a zinc finger transcription factor initially identified samples having high level of WT1 expression should have low as the gene mutated in a minority of cases of Wilms’ tumor. levels of ERa (i.e., ER-negative disease). To see if this is the Further research has shown that WT1 in its native form is case, we used Oncomine to assess the expression of WT1 in highly expressed in a variety of tumors and is associated with ER-positive and ER-negative cases of breast cancer. For this an adverse prognosis (7, 9). In the present study, we have found study, we assessed microarray data provided by Perou et al. (23) that WT1 negatively regulates the expression of ERa.Inthe and van de Vijver et al. (24). In keeping with the prediction, same cells, we have found that WT1 can enhance the

expression of c-myc (37). It is not known what mechanisms are transfer to overexpress WT1 were cultured in selective medium at play that allow the same protein to have such opposite roles containing 0.5 mg/mL G418. For the treatment of cells with E2 in the same cell. However, the ability of WT1 to increase the or tamoxifen, cells were seeded in triplicate onto 10-cm plates expression of a gene associated with proliferation and to reduce (2 Â 105 per plate) and allowed to reach logarithmic growth the expression of a gene whose loss is associated with phase in culture medium supplemented with 10% fetal bovine resistance to hormonal manipulation helps in our understanding serum. At 30% confluence, cells were washed with PBS and of how WT1 is associated with an inferior prognosis in patients cultured for 48 h in phenol-red–free medium supplemented with breast cancer. This observation argues for further research with 10% dextran-charcoal–stripped serum (Hyclone). Cells aimed at blocking WT1 activity in breast cancer. were then treated with 10 nmol/L E2 (Sigma) or 1 Amol/L tamoxifen (Sigma) for the indicated times. Materials and Methods Cell Culture Plasmids The human breast cancer lines MCF-7 (ATCC HTB22) and The expression plasmids for mouse WT1B and WT1D MDA 468 (ATCC HTB132) were routinely subcultured in isoforms were constructed by cloning the corresponding coding MEM supplemented with 10% heat-inactivated fetal bovine region respectively into pcDNA3 or pcDNA3.1/His vectors serum and antibiotics (penicillin/streptomycin/fungizone) in a (Invitrogen) under a cytomegalovirus promoter. To generate plasmid expressing WT1 shRNA, double-stranded oligonucleo- humidified atmosphere of 5% CO2 at 37jC. The third human breast cancer line, HCC1954, was cultured in RPMI 1640 tides were cloned into the HindIII/BglII sites in pSuper vector containing 10% fetal bovine serum, 10 mmol/L HEPES, 1.5 g/L (Oligoengine, Inc.). The sequences of WT1 shRNAa and bicarbonate, 4.5 g/L glucose, 1 mmol/L pyruvate, and shRNAb used are gatccccTCAGGGTTACAGCACGGTCttcaa- antibiotics. The breast cancer cells engineered through gene gagaGACCGTGCTGTAACCCTGAtttttggaa and gatccccTGA- CATCCCAGCTTGAATGttcaagagaCATTCAAGCTGG- GATGTCAtttttggaa, respectively. The uppercase letters represent WT1 specific sequence and lowercase letters represent hairpin sequences. The control nonsilencing shRNA sequence (Qiagen) used is gatccccTTCTCCGAACGTGT- CACGTttcaagagaACGTGACACGTTCGGAGAAtttttggaaa. The reporter ERPWT/Luc was generated by amplifying the human ERa promoter (from nucleotides À287 to +202 relative to the predominant transcription start site) with PCR amplification and cloning into the KpnI/BglII sites immediately upstream of luciferase in pGL2enhancer vector (Promega; ref. 38). Mutagenesis in situ of the ERPWT/Luc plasmid (ERPmut/ Luc) was done using Transformer Site-Directed Mutagenesis Kit (Clontech) in accordance with the manufacturer’s instruc- tions. WT1 site CGCCCCCGC was changed to CGCAGCTGC in ERPmut/Luc. All constructs were verified with restriction enzyme digests and direct nucleotide sequencing.

Transfection and Luciferase Assays The cells were transiently cotransfected using Lipofectamine (Invitrogen) into a 12-well plate with 0.5 Ag of luciferase reporter (pGL2vectors as control), 0.05 Agofh-galactosidase internal control vector, and 1 Ag of plasmid expressing WT1 (pcDNA3 containing no insert as control) or plasmid expressing human WT1 shRNA (pSuper containing no insert as control) in each well according to the manufacturer’s protocol. After 48 h, cells were washed twice with PBS and harvested for the assay of luciferase and h-Gal activities according to standard methods (Promega). Luciferase activity was determined by normalizing each plate to h-galactosidase activity. Luciferase induction was calculated by dividing the luciferase activity from cells transfected with luciferase reporter by that from cells FIGURE 6. Enhanced sensitivity to tamoxifen treatment in WT1 down- transfected with pGL2control. regulated cells. MDA468 (A) and HCC1954 (B) cells were transfected with either control nonsilencing shRNA or WT1 shRNAs (pSWT1a and pSWT1b) for 1 d, and then treated with ethanol, 20 nmol/L E2,or2Amol/L Quantitative Real-time PCR tamoxifen for 4 d. Surviving cell counts were done with trypan blue The total RNA was extracted using RNeasy Mini Kits staining. The viable cell numbers in pSCN-transfected cells treated with ethanol are represented as 100%. Columns, average of three experi- (Qiagen) and converted into cDNA with reverse transcriptase ments; bars, SD. (Life Technologies, Inc.) at 42jC for 45 min. The PCR

reactions containing SYBR Green Master Mix (PE Applied GATC-3¶ (forward) and 5¶-CTCCAGGCACAACTCGATTTG- Biosystems), 100 nmol/L primer, and template cDNA in a 3¶ (reverse). The DNA specificity of the primers was shown by 25-AL final volume were done in an ABI PRISM 7799 sequence the nucleotide sequence of single correctly sized band detector. The SYBR Green PCR program consisted of an initial visualized by agarose gel electrophoresis. denaturation at 95jC for 10 min, followed by 40 PCR cycles: 95jC for 15 s, 60jC for 60 s with the continual measurement of Apoptotic Assay fluorescence. The primer sequences for gene amplification were The cells were stained with Annexin V-phycoerythrin and as follows: hWT1,5¶-GAGAGCCAGCCCGCTATTC-3¶ (for- 7-AAD (BD PharMingen) according to the manufacturer’s ward) and 5¶-CATGGGATCCTCATGCTTG-3¶ (reverse); recommendation. Appropriate gates based on individual con- mWT1,5¶-TCAAGGACTGCGAGAGAAGG-3¶ (forward) and trols, defining the Annexin V-phycoerythrin and 7-AAD stained 5¶-TGGTGTGGGTCTTCAGATGG-3¶ (reverse); ERa,5¶-GAC- cells, were used to determine the percentages of cell population AGGGAGCTGGTTCACATG-3¶ (forward) and 5¶-ACGAGAC- at earlier stage (Annexin V positive, 7-AAD negative) and later CAATCATCAGGATCTC-3¶ (reverse); PR,5¶-GAGCCCACA- stage (Annexin V positive, 7-AAD positive) of apoptosis. ATACAGCTTCG-3¶ (forward) and 5¶-TCCAGTGCTCTCA- ¶ ¶ CAACTCTGAC-3 (reverse); PS2, 5 -CCATGGAGAACA- Data Mining ¶ ¶ AGGTGATCTG-3 (forward) and 5 -CGAACGGTGTCGTC- The Oncomine database was queried for the expression of ¶ ¶ GAAAC-3 (reverse); and GAPDH,5-GAAGGTGA- WT1 in breast cancer specimens using the differential ¶ ¶ AGGTCGGAGTC-3 (forward) and 5 -GAAGATGGTGATGG- expression tool (39). GATTTC-3¶ (reverse). 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Mol Cancer Res 2008;6(8). August 2008 Downloaded from mcr.aacrjournals.org on September 25, 2021. © 2008 American Association for Cancer Research. Wilms' Tumor 1 Suppressor Gene Mediates Antiestrogen Resistance via Down-Regulation of Estrogen Receptor- α Expression in Breast Cancer Cells

Youqi Han, Lin Yang, Fernando Suarez-Saiz, et al.

Mol Cancer Res 2008;6:1347-1355.

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