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Molecular Endocrinology, doi:10.1210/me.2007-0064
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Molecular Endocrinology 21 (7): 1526-1536
Copyright © 2007 by The Endocrine Society

Modulation of Estrogen Receptor {alpha} Protein Level and Survival Function by DBC-1

Amy M. Trauernicht, Se Jin Kim, Nam Hee Kim and Thomas G. Boyer

Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245

Address all correspondence and requests for reprints to: Thomas G. Boyer, Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, Texas 78245-3207. E-mail: boyer{at}uthscsa.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Acquired resistance to endocrine therapy represents a major clinical obstacle to the successful management of estrogen-dependent breast cancers expressing estrogen receptor {alpha} (ER{alpha}). Because a switch from ligand-dependent to ligand-independent activation of ER{alpha}-regulated breast cancer cell growth and survival may define a path to endocrine resistance, enhanced mechanistic insight concerning the ligand-independent fate and function of ER{alpha}, including a more complete inventory of its ligand-independent cofactors, could identify novel markers of endocrine resistance and possible targets for therapeutic intervention in breast cancer. Here, we identify the deleted in breast cancer 1 gene product DBC-1 (KIAA1967) to be a principal determinant of unliganded ER{alpha} expression and survival function in human breast cancer cells. The DBC-1 amino terminus binds directly to the ER{alpha} hormone-binding domain both in vitro and in vivo in a strict ligand-independent manner. Furthermore, like estrogen, the antiestrogens tamoxifen and ICI 182,780 (7{alpha},17ß-[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol) disrupt the DBC-1/ER{alpha} interaction, thus revealing the DBC-1/ER{alpha} interface to be a heretofore-unrecognized target of endocrine compounds commonly used in hormonal therapy. Notably, RNA interference-mediated DBC-1 depletion reduces the steady-state level of unliganded but not liganded ER{alpha} protein, suggesting that DBC-1 may stabilize unliganded ER{alpha} by virtue of their direct association. Finally, DBC-1 depletion promotes hormone-independent apoptosis of ER{alpha}-positive, but not ER{alpha}-negative, breast cancer cells in a manner reversible by endocrine agents that disrupt the DBC-1/ER{alpha} interaction. Collectively, these findings establish a principal biological function for DBC-1 in the modulation of ER{alpha} expression and hormone-independent breast cancer cell survival.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
BREAST CANCER IS the leading cause of death among American women between the ages of 20 and 59 yr (1). Among a variety of established etiological factors linked to breast cancer, the steroid hormone estrogen [17-ß-estradiol (E2)] has long been implicated in disease pathogenesis. Numerous animal studies have revealed that E2 can induce and promote breast cancer, whereas estrogen ablation therapy or the administration of antiestrogens can oppose these effects (2, 3, 4). The physiological effects of E2 in the breast are mediated by cognate receptors that are expressed as two structurally related subtypes, estrogen receptor {alpha} (ER{alpha}) and ß (ERß) (5, 6, 7, 8). ER{alpha} is the predominant receptor isoform expressed in breast cancer cells, and approximately 70% of breast cancer patients score positive for ER{alpha} at diagnosis (9, 10, 11, 12). ER{alpha} is therefore a dominant etiologic and valuable predictive factor with respect to breast cancer development and hormone sensitivity status. Endocrine therapy, which seeks to block ER-mediated mitogenic signaling, has emerged as one of the most important systemic therapies in breast cancer management; however, therapeutic resistance, either inherent (de novo resistance) or acquired during treatment (acquired resistance) remains a significant clinical roadblock to effective disease management (13).

Although de novo resistance to endocrine therapy derives primarily from loss of ER{alpha} expression, the biological mechanism underlying acquired endocrine resistance is incompletely understood and almost certainly multifactorial in nature (14, 15). Nonetheless, the emergence of endocrine resistance is often coincident with a shift from ligand-dependent to ligand-independent control of ER{alpha}-regulated breast cancer cell growth and survival, possibly reflecting bidirectional molecular crosstalk between ER{alpha} and growth factor signaling pathways (14, 16, 17). Because ligand-independent activation of ER{alpha} may therefore define a path to endocrine resistance, enhanced mechanistic insight concerning the ligand-independent function and regulation of ER{alpha}, including a more complete inventory of its ligand-independent cofactors, could identify novel prognostic markers of endocrine resistance and possible targets for therapeutic intervention in breast cancer. Toward this objective, we have undertaken a proteomics-based approach to isolate ligand-independent ER{alpha} protein interaction networks. Herein, we identify the deleted in breast cancer-1 gene product DBC-1 (KIAA1967) to be a direct ligand-independent binding partner of ER{alpha}. Functional analyses further reveal DBC-1 to be a principal determinant of unliganded ER{alpha} protein levels and survival activity in human breast cancer cells.

The gene encoding DBC-1 was originally identified during a genetic search for candidate breast tumor suppressor genes on a human chromosome 8p21 region frequently deleted in breast cancers. However, refined deletion analysis within this region revealed a second gene, deleted in breast cancer 2 (DBC-2), to encode a likely breast tumor suppressor, and further confirmed that DBC-1 expression is not substantially extinguished in cancers from any source (18). In fact, a search of the Oncomine database of published cancer microarray data (www.oncomine.org), which currently permits analysis of gene expression data derived from 132 DNA microarray datasets among 24 different cancer types, reveals DBC-1 to be statistically significantly upregulated in breast carcinoma vs. normal breast tissue as well as breast ductal carcinoma vs. other cancers (19, 20). Furthermore, DBC-1 was found in three independent studies totaling 369 breast tumor samples to be statistically significantly overexpressed in ER-positive vs. ER-negative breast tumors (21, 22, 23).

Little is currently known regarding the molecular and cellular function of DBC-1 in breast or other tissues. Recently, DBC-1 was linked physically to the TNF-{alpha}/nuclear factor {kappa}B (NF-{kappa}B) pathway by proteomic analysis (24), whereas caspase-dependent processing of DBC-1 early in apoptosis induced by diverse stimuli, including TNF-{alpha}, was shown to unmask a proapoptotic function for the DBC-1 carboxyl terminus in the cytosol of moribund cells (25). However, full-length DBC-1 is predominantly localized to the nucleus of healthy cells (25), and its normal biological function therein has heretofore remained unknown. Based on our identification of DBC-1 as a ligand-independent ER{alpha}-interacting protein as well as its provocative expression profile in breast cancers, we therefore undertook to explore the physical basis, biological regulation, and functional consequence of the interaction between DBC-1 and ER{alpha} in human breast cancer cells. Our findings reveal that the DBC-1 amino terminus binds directly to the ER{alpha} hormone-binding domain both in vitro and in vivo in a strict E2-independent manner. Furthermore, like E2, the antiestrogens tamoxifen and ICI 182,780 (7{alpha},17ß-[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol) disrupt the DBC-1/ER{alpha} interaction, thus revealing the DBC-1/ER{alpha} interface to be an unanticipated target of these endocrine compounds. Finally, DBC-1, in a manner dependent on direct interaction with ER{alpha}, suppresses breast cancer cell apoptosis in the absence of hormone. These findings thus establish a principal biological function for DBC-1 in the modulation of ER{alpha} expression and survival activity and further identify DBC-1 as a possible endocrine response determinant and potential therapeutic target in breast cancer.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DBC-1 Interacts with ER{alpha} in Vivo in a Ligand-Independent Manner
During the course of a targeted search for ligand-independent ER{alpha} interaction partners, we identified DBC-1 by mass spectrometric-based peptide sequence analysis of proteins coimmunoprecipitated specifically with unliganded, but not liganded, ER{alpha} (supplemental Fig. 1, which is published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). To validate the ligand-independent interaction between DBC-1 and ER{alpha} in vivo, we used a mammalian two-hybrid interaction analysis. Chimeric proteins consisting of DBC-1 fused to the GAL4 DNA-binding domain and ER{alpha} fused to the VP16 activation domain were expressed with or without one another in HeLa cells and examined for their respective abilities to activate transcription from a reporter template controlled by GAL4 DNA-binding sites in both the absence and presence of E2. In the absence of E2, DBC-1 and ER{alpha} exhibited a robust interaction that was disrupted by addition of E2 to the cell culture medium (Fig. 1AGo). Additional analysis of DBC-1 amino and carboxyl truncation derivatives revealed that the ligand-independent association between DBC-1 and ER{alpha} is mediated entirely by the amino-terminal half of DBC-1 (Fig. 1BGo).


Figure 1
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  		Fig. 1. DBC-1 and ER{alpha} Interact In Vivo in a Ligand-Independent Manner

A, B, Mammalian two-hybrid interaction analysis. A, HeLa cells cultured in hormone-free medium for 3 d were transfected with the indicated combinations of mammalian expression plasmids encoding the yeast GAL4 DNA-binding domain (GAL4), the Herpes simplex virus VP16 transactivation domain (VP16), a GAL4-DBC-1 chimera, and a VP16-ER{alpha} chimera. Twenty-four hours after transfection, cells were treated without (–E2) or with (+E2) E2 (10–7 M) for an additional 24 h before cell harvest and assay of transfected whole-cell lysates for luciferase activity produced from a cotransfected GAL4 DNA-binding site driven-reporter template. Luciferase values are expressed relative to the luciferase activity obtained in cells transfected with both the GAL4 and VP16 expression vectors, which was arbitrarily assigned a value of 1. Luciferase activities were first normalized to ß-galactosidase activity obtained by cotransfection of a ß-galactosidase expression vector. Error bars represent the SD from the average of at least three independent transfections performed in duplicate. Note that estrogen abolishes the interaction between GAL4-DBC-1 and VP16-ER{alpha}. B, Top, HeLa cells cultured for 3 d in hormone-free medium (–E2) were transfected with the indicated combinations of mammalian expression plasmids encoding GAL4, VP16, a GAL4-DBC-1 N-terminal chimera (amino acids 1–478), a GAL4-DBC-1 C-terminal chimera (amino acids 479–923), and a VP16-ER{alpha} chimera. Forty-eight hours after transfection, cells were harvested, and transfected whole-cell lysates were assayed for luciferase activity produced from a cotransfected GAL4 DNA-binding site driven-reporter template as described in A. Note that ER{alpha} interacts exclusively with the N terminus of DBC-1. Bottom, Harvested whole-cell lysates were resolved by SDS-12% PAGE and processed by immunoblot analysis with antibodies specific for GAL4-DBD or ER{alpha} as indicated by arrows. Note that differences in the relative expression levels of the GAL4-DBC-1 chimeras cannot explain differences in their respective ER{alpha}-binding capabilities. Results are representative of at least three independent experiments. C, D, Coimmunoprecipitation analysis. C, MCF-7 cells cultured in hormone-free medium for 3 d were treated without (–E2) or with (+E2) E2 (10–7 M) for 1 h before cell harvest and immunoprecipitation (IP) of whole-cell lysates with antibodies specific for ER{alpha} (top) or DBC-1 (bottom). Immunoprecipitates were resolved by SDS-10% PAGE and processed by immunoblot analysis using antibodies specific for DBC-1 or ER{alpha} as indicated by arrows. Note specific immunoprecipitation of DBC-1 by ER{alpha}-specific antibodies and ER{alpha} by DBC-1-specific antibodies only in the absence, but not in the presence, of estrogen. Results are representative of at least three independent experiments. D, T-47D (top) and BG-1 (bottom) cells cultured in hormone-free medium for 3 d were treated without (–E2) or with (+E2) E2 (10–7 M) for 1 h before cell harvest and immunoprecipitation of whole-cell lysates with antibodies specific for ER{alpha}. Immunoprecipitates were resolved by SDS-10% PAGE and processed by immunoblot analysis using antibodies specific for DBC-1 or ER{alpha} as indicated by arrows. Results are representative of at least three independent experiments.

 
To confirm the ligand-independent in vivo association between DBC-1 and ER{alpha} using a more biologically relevant approach, we examined the ability of antibodies specific for ER{alpha} or DBC-1 to coprecipitate one another in MCF-7 human breast cancer cells, which express both ER{alpha} and DBC-1. This analysis revealed that DBC-1 was specifically and reciprocally coimmunoprecipitated along with unliganded, but not liganded, ER{alpha}, demonstrating that the two endogenous proteins interact in a strict ligand-independent manner in human breast cancer cells (Fig. 1CGo). We also confirmed a ligand-independent interaction between endogenous DBC-1 and ER{alpha} in both T-47D human breast and BG-1 human ovarian cancer cell lines, thus revealing the DBC-1/ER{alpha} interaction to be conserved in a variety of ER{alpha}-expressing cell lines (Fig. 1DGo).

Heat shock protein 90 (HSP90) together with additional heat shock family members and immunophilins are known to form a heteromeric chaperone complex that sequesters neosynthesized and unliganded ER{alpha} in an inactive state, primes it for ligand binding, and protects it from proteolytic degradation (26, 27, 28). We initially examined the physical relationship between unliganded ER{alpha} in complex with HSP90-based chaperones and DBC-1 by coimmunoprecipitation analysis using MCF-7 whole-cell lysates. Whereas unliganded ER{alpha} immunoprecipitates included not only DBC-1 but also HSP90 (data not shown), DBC-1 immunoprecipitates included unliganded ER{alpha} but neither HSP90 nor the immunophilin cyclophilin 40 (CYP40) (Fig. 2AGo). Thus, DBC-1 is not a component of the classical HSP90-based molecular chaperone complex. Subsequently, we sought to identify the subcellular pool of unliganded ER{alpha} in specific association with DBC-1 by coimmunoprecipitation analysis using fractionated MCF-7 cell lysates. ER{alpha}/DBC-1 complexes were found exclusively in the nuclear fraction (Fig. 2BGo), thus revealing that unliganded ER{alpha} is distributed among at least two distinct protein complexes in human breast cancer cells: a cytosolic HSP90-based molecular chaperone complex and a nuclear DBC-1-containing protein complex.


Figure 2
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  		Fig. 2. DBC-1 and Unliganded ER{alpha} Associate in the Nucleus Independently of HSP90

A and B, Coimmunoprecipitation analysis. A, MCF-7 cells cultured in hormone-free medium for 3 d were treated without (–E2) or with (+E2) E2 (10–7 M) for 1 h before cell harvest and immunoprecipitation (IP) of whole-cell lysates with antibodies specific for DBC-1. Immunoprecipitates were resolved by SDS-10% PAGE and processed for immunoblot analysis with antibodies specific for DBC-1, HSP90, ER{alpha}, or CYP40 as indicated by arrows. Results are representative of at least three independent experiments. B, MCF-7 cells cultured in hormone-free medium for 3 d were fractionated into cytoplasmic (cyto) and nuclear (nuc) extracts. Equivalent amounts of each extract were immunoprecipitated with antibodies specific for ER{alpha}. Immunoprecipitates were resolved by SDS-10% PAGE and processed for immunoblot analysis with antibodies specific for DBC-1 or ER{alpha} as indicated by arrows. Note that an additional immunoprecipitation containing four times the amount of cytoplasmic extract (4x cyto) failed to yield a detectable amount of DBC-1 in either the input or immunoprecipitate. Results are representative of at least three independent experiments.

 
The DBC-1 N Terminus Interacts Directly with the ER{alpha} Hormone-Binding Domain In Vitro
To determine whether DBC-1 interacts directly with unliganded ER{alpha} and to map the reciprocal binding domains on each protein, we tested the ability of glutathione S-transferase (GST)-ER{alpha} derivatives to bind to full-length DBC-1 or DBC-1 truncation fragments produced by in vitro translation. DBC-1 bound most efficiently to GST-ER{alpha} derivatives 1–595 (full-length ER{alpha}) and 302–595 (ER{alpha} hormone-binding domain), although DBC-1 also exhibited weak binding to GST-ER{alpha} derivative 251–301 (ER{alpha} hinge region) (Fig. 3AGo). Reciprocally, GST-ER{alpha} 1–595 (full-length ER{alpha}) bound to the extreme amino terminus of DBC-1 (amino acids 1–150) (Fig. 3BGo). Thus, in the absence of ligand, the ER{alpha} hormone-binding domain can accommodate the DBC-1 amino terminus.


Figure 3
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  		Fig. 3. The DBC-1 N Terminus Binds to the ER{alpha} Hormone-Binding Domain in Vitro

A and B, GST pull-down assays were performed using full-length in vitro translated DBC-1 and GST-ER{alpha} fragments (A) or in vitro translated DBC-1 fragments and GST-full-length ER{alpha} (B) as indicated. Numbers refer to amino acid coordinates. 35S-labeled in vitro translated proteins were incubated with glutathione-Sepharose-immobilized GST derivatives, and bound proteins were resolved by SDS-12% PAGE before detection by PhosphorImager analysis. Input represents 10% of the 35S-labeled in vitro translated proteins used in binding reactions. The amount of each DBC-1 derivative retained by GST-ER{alpha} (percentage bound) was quantified and expressed as a percentage of the total input. % bound refers to the average and SD of at least three independent experiments. *, P < 0.05, statistically significant binding values relative to GST alone. Note that DBC-1 binds primarily to GST-ER{alpha} derivatives 1–595 (full-length ER{alpha}) and 302–595 (ER{alpha} hormone-binding domain), whereas GST-ER{alpha} binds primarily to DBC-1 derivative 1–150 (N terminus). Schematic diagrams of ER{alpha} and DBC-1 indicate fragments used in binding reactions. AF-1, Activation function 1; DBD, DNA-binding domain; AF-2/HBD, activation function 2/hormone-binding domain; NLS, putative nuclear localization sequence; LZip, putative leucine zipper.

 
The DBC-1/ER{alpha} Interface Is a Novel Target of Antiestrogens
Antiestrogens are currently the most widely administered endocrine agents for the management of ER{alpha}-expressing breast cancers (29, 30). Mechanistically, antiestrogens competitively displace E2 from the ER{alpha} hormone-binding domain and either block ER{alpha} function or induce destabilization and degradation of ER{alpha}. Tamoxifen, a prototype of the former class, is a selective ER modulator with antiestrogenic properties in breast and the most widely administered antiestrogen in breast cancer therapy (29, 30). Among the latter class of antiestrogens, ICI 182,780 (Faslodex; fulvestrant) is a selective ER down-regulator and an effective second-line therapeutic agent used to treat breast cancers that have progressed on previous tamoxifen therapy (29, 30, 31). Because these compounds bind directly to the ER{alpha} hormone-binding domain, we examined the influence of each agent on the DBC-1/ER{alpha} interaction. To this end, we tested the ability of ER{alpha}-specific antibodies to coimmunoprecipitate endogenous DBC-1 present in MCF-7 and BG-1 cells cultured in the absence or presence of E2, tamoxifen, or ICI 182,780. Strikingly, we observed that, like E2, both tamoxifen and ICI 182,780 disrupted the DBC-1/ER{alpha} interaction, thus revealing the DBC-1/ER{alpha} interface to be a heretofore unrecognized target of these endocrine compounds (Fig. 4Go, A and B).


Figure 4
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  		Fig. 4. Tamoxifen and ICI 182,780 Disrupt the Interaction between DBC-1 and ER{alpha}

A and B, MCF-7 (A) or BG-1 (B) cells cultured in hormone-free medium for 3 d were treated with vehicle (–E2), E2 (10–7 M; +E2), 4-hydroxytamoxifen (10–6 M; 4-OHT), or ICI 182,780 (10–7 M; ICI) for 1 h before cell harvest and immunoprecipitation (IP) of whole-cell lysates with antibodies specific for ER{alpha}. Immunoprecipitates were resolved by SDS-7.5% PAGE and processed for immunoblot analysis with antibodies specific for DBC-1 or ER{alpha} as indicated by arrows. Results are representative of at least three independent experiments.

 
DBC-1 Is an ER{alpha}-Dependent Prosurvival Factor in Breast Cancer Cells
To examine the biological consequence of the DBC-1/ER{alpha} interaction in human breast cancer cells, we first established conditions for RNA interference (RNAi)-mediated DBC-1 depletion in MCF-7 cells. Strikingly, we observed that RNAi-mediated DBC-1 knockdown was accompanied by a significant reduction in the steady-state level of ER{alpha} protein but not ER{alpha} mRNA, suggesting that DBC-1 modulates ER{alpha} protein synthesis or stability (Fig. 5Go). Notably, DBC-1 knockdown preferentially reduced the steady-state level of unliganded, but not liganded, ER{alpha} protein, consistent with the possibility that DBC-1 may stabilize unliganded ER{alpha} by virtue of their direct physical association (Fig. 5Go).


Figure 5
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  		Fig. 5. RNAi-Mediated DBC-1 Suppression Is Accompanied by Reduced Steady-State Levels of Unliganded ER{alpha}

MCF-7 cells cultured in hormone-free medium for 3 d were electroporated with control (siCNTL) or DBC-1-specific (siDBC-1) siRNA (21 nM) as indicated. Electroporated cells were cultured without (–E2) or with (+E2) E2 (10–7 M) for an additional 3 d before cell harvest. A, Harvested whole-cell lysates were resolved by SDS-10% PAGE and processed by immunoblot analysis with antibodies specific for DBC-1, ER{alpha}, or TFIIEß as indicated by arrows. Results are representative of at least three independent experiments. B, Top, Immune signals were quantified using a Kodak ImageStation 2000R. ER{alpha} protein levels were normalized to TFIIEß and plotted relative to the ER{alpha} protein level in control siRNA cells cultured in the absence of E2, which was arbitrarily assigned a value of 1. Error bars represent the SD from the average of at least three independent experiments. Bottom, RNA was processed by quantitative RT-PCR analysis for the levels of DBC-1, ER{alpha}, and GAPDH mRNAs. ER{alpha} RNA levels were normalized to GAPDH levels and expressed relative to the level of ER{alpha} RNA in control siRNA cells cultured in the absence of E2, which was arbitrarily assigned a value of 1. Error bars represent the SD from the average of at least three independent experiments performed in duplicate.

 
Because DBC-1 is a direct binding partner and key determinant of steady-state ER{alpha} protein levels, we examined its role in ER{alpha}-dependent breast cancer cell proliferation and survival. RNAi-mediated DBC-1 depletion significantly reduced E2-independent, but not E2-dependent, MCF-7 cell proliferation, an observation concordant with the fact that DBC-1 preferentially binds to and modulates the levels of unliganded ER{alpha} (Fig. 6Go). Because transient DBC-1 knockdown cells experienced an initial (~2-fold) reduction in cell number on d 3 after small interfering RNA (siRNA) delivery followed by growth kinetics similar to control siRNA knockdown cells, we hypothesized that the influence of DBC-1 silencing on ligand-independent cell proliferation may derive, at least in part, from an increase in apoptotic cell death. To address this question, we examined the influence of DBC-1 knockdown on the apoptotic fate of MCF-7 cells cultured in the absence of E2. Under these conditions, DBC-1 depletion increased the percentage of apoptotic cells from 6.2 to 12.8%, thus revealing an antiapoptotic function for DBC-1 in the absence of hormone (Fig. 7AGo). To determine whether DBC-1 promotes hormone-independent cell survival through its direct interaction with ER{alpha}, we also monitored the influence of DBC-1 knockdown on the apoptotic fate of MCF-7 cells cultured in the presence of E2, which disrupts the DBC-1/ER{alpha} interaction, or ICI 182,780, which not only disrupts the DBC-1/ER{alpha} interaction but also drastically depletes ER{alpha} protein levels. Notably, DBC-1 depletion had no effect on MCF-7 cell apoptosis in the presence of either E2 or ICI 182,780 (Fig. 7AGo). Furthermore, DBC-1 depletion did not enhance apoptosis of ER{alpha}-negative MDA-MB-231 breast cancer cells cultured in the absence of E2 (Fig. 7BGo). Together, these observations suggest that DBC-1 functions to promote E2-independent breast cancer cell survival in an ER{alpha}-dependent manner. We note that we have also attempted to examine the influence of DBC-1 overexpression on breast cancer cell proliferation and survival; however, we have not been able to achieve overexpression of DBC-1 protein in ER{alpha}-expressing breast cancer cells, suggesting that DBC-1 expression levels are tightly regulated in this context.


Figure 6
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  		Fig. 6. RNAi-Mediated DBC-1 Depletion Inhibits Estrogen-Independent Proliferation in Human Breast Cancer Cells

MCF-7 cells cultured in hormone-free medium for 3 d were electroporated with control or DBC-1-specific siRNA (21 nM) as indicated and cultured without (–E2) or with (+E2) E2 (10–7 M). Culture medium was replaced every 2 d. Cell proliferation was monitored by counting with trypan blue exclusion for 7 d after electroporation. P values are compared with controls. Error bars represent the SD from the average of at least three independent experiments performed in triplicate.

 

Figure 7
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  		Fig. 7. DBC-1 Is an ER{alpha}-Dependent Prosurvival Factor in Human Breast Cancer Cells

A and B, MCF-7 (A) or MDA-MB-231 (B) cells cultured in hormone-free medium for 3 d were electroporated with control or DBC-1-specific siRNA (21 nM) as indicated. Forty-eight hours after electroporation, cells were treated with vehicle (–E2), E2 (10–7 M; +E2), ICI 182,780 (10–7 M; ICI), or a combination of E2 and ICI 182,780 (E2+ICI) for an additional 24 h before cell harvest. Top, Harvested cells were stained with Annexin V-FITC and propidium iodide before quantification of apoptosis by flow cytometric analyses. P values are compared with controls. Error bars represent the SD from the average of at least three independent experiments performed in triplicate. Bottom, Cell lysates from representative apoptosis assays in A and B were resolved by SDS-10% PAGE and processed by immunoblot analysis with the indicated antibodies specific for DBC-1 or TFIIEß as a loading control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Here we describe for the first time a biological function for DBC-1 in the modulation of ER{alpha} expression and survival activity in human breast cancer cells. Our identification of DBC-1 as a heretofore unrecognized determinant of steady-state ER{alpha} protein levels is a novel finding with implications for the regulation and function of ER{alpha} in normal and malignant breast epithelial cells. A compelling body of experimental, clinical, and epidemiological evidence suggests that dysregulation of ER{alpha} expression is a driving force in the initiation and progression of estrogen-sensitive breast tumors. ER{alpha} is the predominant receptor isoform expressed in breast cancer cells, and increased numbers of ER{alpha}-expressing cells as well as increased individual cell ER{alpha} content can be observed at the earliest stages of breast tumorigenesis (32, 33). It is thus likely that alterations in pathways leading to ER{alpha} synthesis and/or degradation underlie the dysregulation of ER{alpha} and its consequent manifestations, including enhanced proliferation in breast tumors. Therefore, revelation of the mechanism by which DBC-1 modulates ER{alpha} expression should yield important insight concerning the physiological regulation and, possibly, the pathological dysregulation of ER{alpha} in normal and malignant breast epithelial cells, respectively.

In this regard, previous work has revealed that unliganded ER{alpha} is sequestered by an HSP90-based molecular chaperone complex that protects the neosynthesized receptor from proteolytic degradation and primes it for ligand binding (26, 27, 34, 35). Our observation that HSP90 and CYP40 cannot be coprecipitated along with unliganded ER{alpha} by DBC-1-specific antibodies coupled with our finding that DBC-1 and unliganded ER{alpha} interact in the nucleus suggests that the cellular reserve of unliganded ER{alpha} is partitioned among at least two pools: one comprising cytosolic HSP90-based molecular chaperones and the other nuclear DBC-1. Notably, we observed that DBC-1 depletion preferentially reduced the steady-state level of unliganded ER{alpha} protein, suggesting the possibility that unliganded ER{alpha} is stabilized by its direct physical association with DBC-1. Thus, DBC-1 could function as a chaperone of ER{alpha} in the nucleus in a manner analogous to that of HSP90 toward ER{alpha} in the cytosol. Additional studies will be required to elucidate the mechanism by which DBC-1 modulates ER{alpha} steady-state protein levels.

Several observations herein suggest a novel antiapoptotic function for the population of unliganded ER{alpha} bound by DBC-1. First, apoptosis triggered by DBC-1 depletion in the absence of hormone was not observed in MCF-7 cells codepleted of ER{alpha} with ICI 182,780, nor in ER{alpha}-negative MDA-MB-231 breast cancer cells. These findings thus reveal an apparent DBC-1-dependent ER{alpha} requirement for suppression of apoptosis in the absence of hormone. Second, E2-mediated disruption of the interaction between DBC-1 and unliganded ER{alpha} abrogated the increase in MCF-7 cell apoptosis observed to accompany DBC-1 knockdown, suggesting that DBC-1-bound ER{alpha} functions to suppress hormone-independent apoptosis. We therefore speculate that a specific pool of unliganded ER{alpha} bound by DBC-1 may promote breast cancer cell growth and survival in the absence of hormone.

The underlying mechanism by which DBC-1 and ER{alpha} collaborate to promote hormone-independent breast cancer cell growth and survival remains to be established. As discussed above, DBC-1 could directly stabilize a pool of unliganded ER{alpha} dedicated to these functions. Whether or not DBC-1 additionally directly participates in ER{alpha}-regulated cell growth and survival processes is presently unknown. An intriguing possibility is that DBC-1 might function to mediate crosstalk between ER{alpha} and the NF-{kappa}B survival pathway. Emerging evidence indicates that bidirectional molecular crosstalk between the ER{alpha} and NF-{kappa}B pathway contributes to hormone-independent breast cancer cell growth and the development of antiestrogen resistance (36, 37, 38, 39, 40). Previously, DBC-1 has been linked physically to the NF-{kappa}B pathway through a demonstrated interaction with I{kappa}B kinase ß (24), although our findings herein link DBC-1 physically and functionally to ER{alpha}. Possibly, DBC-1 could thus serve to stabilize and channel ER{alpha} toward functional interactions with the NF-{kappa}B pathway. Future studies will be required to establish whether and how DBC-1-mediated crosstalk between the ER{alpha} and NF-{kappa}B signaling pathways might contribute to hormone-independent breast cancer cell growth and survival.

Finally, our finding that ER{alpha} and DBC-1 collaborate to suppress apoptosis and promote hormone-independent breast cancer cell growth could have implications for breast cancer prognosis and/or treatment. It is generally believed that a balance between proliferation and apoptosis influences the response of breast tumors to hormonal therapy, and dysregulation of apoptotic signaling pathways has been suggested as a possible basis for treatment failure (29, 30, 41, 42). Accordingly, alterations in DBC-1 expression and/or activity could tip the balance between breast cancer cell growth and death; if so, DBC-1 could represent a novel biomarker of breast tumor response to endocrine therapy. In this regard, no published data currently exists concerning the relationship between DBC-1 and clinical response of breast tumors to endocrine therapy. Nonetheless, it would be useful to know whether overexpression or amplification of DBC-1 is linked to treatment failure. Furthermore, although DBC-1 is not deleted in most breast cancers, it would be of interest to know the hormone receptor and endocrine response status of the relatively small percentage of breast cancers that do harbor DBC-1 deletions. For example, might ER-positive patients carrying DBC-1 deletions be underrepresented among the patient pool refractory to endocrine therapy? Answers to these and related questions should help to clarify the possible role of DBC-1 as a predictor of breast tumor response to endocrine therapy. From a possible therapeutic perspective, disruption of the DBC-1/ER{alpha} interface might provide a targeted means to reduce in breast tumors the number of hormone-refractory cells that arise through selection in response to prolonged endocrine treatment. Future experiments will be required to validate this hypothesis and further investigate the full spectrum of ER{alpha}-dependent and ER{alpha}-independent biological activities linked to DBC-1.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Expression Plasmids
pCS2+-ER{alpha} was constructed by subcloning a BamHI-BamHI fragment carrying the full-length coding region of ER{alpha} cDNA from pG/ER(G) (provided by Dr. Dider Picard, University of Geneva, Geneva, Switzerland) (43) into pCS2+ (44). pACT-ER{alpha} was constructed by subcloning a BamHI-BamHI fragment carrying the full-length coding region of ER{alpha} cDNA from pCS2+-ER{alpha} into the pACT VP16 fusion vector (Promega, Madison, WI). GST-ER{alpha} (1–184), GST-ER{alpha} (185–250), GST-ER{alpha} (251–301), and GST-ER{alpha} (302–595) were gifts from Dr. Yi-Jun Zhu (Northwestern University, Evanston, IL) (45). GST-ER{alpha} (1–595) was generated by amplifying ER{alpha} by PCR and inserting it into the EcoRI site of pGEX-4T-3 vector (GE Healthcare, Little Chalfont, UK).

pSport1-DBC-1 was a clone obtained from RZPD Deutsches Ressourcenzentrum für Genomforschung (www.rzpd.de) (RZPD clone DKFZp761O0817Q; KIAA1967). pCS2+DBC-1 was constructed by first amplifying the amino-terminal half of DBC-1 by PCR and inserting it into the ClaI/EcoRI site of pCS2+.His6.FLAG, which yielded pCS2+.His6.FLAG-5'DBC-1.SphI. The carboxyl-terminal half of DBC-1 was amplified by PCR and then inserted into the SphI/EcoRI site of pCS2+.His6.FLAG-5'DBC-1.SphI to yield pCS2+DBC-1, which contains a STOP codon between the DBC-1 coding sequence and the His6.FLAG fusion. This construct was confirmed by sequencing. pCS2+.His6.FLAG-DBC-1 was generated by amplifying the carboxyl-terminal half of DBC-1 by PCR and then inserting it into the SphI/EcoRI site of pCS2+.His6.FLAG-5'DBC-1.SphI to create a version of DBC-1 fused to C-terminal 6XHis and FLAG tags. pCS2+-DBC-1 amino-terminal fragments (1–478, 1–300, 1–230, 1–200, 1–150, and 150–478) were generated by amplifying fragments by PCR and inserting them into the EcoRI/XhoI site of pCS2+. pCS2+-DBC-1 (479–923) was generated by amplifying the carboxyl-terminal half of DBC-1 by PCR and inserting it into the EcoRI/XhoI site of pCS2+. pBIND-DBC1 (1–478) was constructed by amplifying the amino-terminal half of DBC-1 by PCR and inserting it into the SalI/XbaI site of the pBIND GAL4 fusion vector (Promega). pBIND-DBC1 (479–923) was constructed by amplifying the carboxyl-terminal half of DBC-1 by PCR and inserting it into the XbaI/NotI site of pBIND. pBIND-DBC-1 was constructed by subcloning an XbaI/NotI carboxyl-terminal fragment of DBC-1 from pBIND-DBC1 (479–923) into pBIND-DBC1 (1–478).

Reporter Plasmids
pG5luc, carrying five GAL4 DNA-binding sites upstream of the major late promoter of adenovirus driving expression of the firefly luciferase gene, was purchased from Promega.

Cell Lines and Culture Conditions
The HeLa (American Type Culture Collection, Manassas, VA), T-47D (American Type Culture Collection), MCF-7 (American Type Culture Collection), AmphoPack 293 (Clontech, Mountain View, CA), and MDA-MB-231 (American Type Culture Collection) cells were routinely cultured in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT) and penicillin-streptomycin-L-glutamine (Invitrogen). BG-1 cells, from Dr. Kenneth S. Korach (National Institute of Environmental Health Sciences, Research Triangle Park, NC) (46), were routinely cultured in DMEM/F12 (Invitrogen) supplemented as listed above. All cell lines except BG-1 and MDA-MB-231 cells were cultured at 37 C in a 10% CO2 humidified chamber; BG-1 and MDA-MB-231 cells were cultured at 5% CO2.

GST Pull-Down Assays
GST and GST fusion proteins were expressed in and purified from BL21-CodonPlus(DE3)-RIPL Escherichia coli (Stratagene, La Jolla, CA). Cells were grown at 37 C to A600 of 1.0, and then isopropyl-1-thio-ß-D-galactopyranoside was added to a final concentration of 0.5 mM. For GST, GST-ER{alpha} (1–184), GST-ER{alpha} (185–250), and GST-ER{alpha} (251–301), the cells were grown at 30 C for another 5 h. For GST-ER{alpha} (302–595), the cells were grown at 20 C for another 5 h. For GST-ER{alpha} (1–595), the cells were grown at 16 C for another 5 h. Cells were pelleted, washed once with PBS, and resuspended in lysis 250 buffer [50 mM Tris-HCl, 250 mM NaCl, 5 mM EDTA, and 0.1% Nonidet P-40 (NP-40)] supplemented with protease inhibitors (20 µM antipain, 2 µM pepstatin, 20 µM leupeptin, and 2 µg/ml aprotinin). Resuspended cells were subjected to one round of freeze-thaw, followed by sonication and clarification by centrifugation at 35,000 x g for 30 min at 4 C.

Clarified GST lysates were bound to glutathione-Sepharose beads (GE Healthcare) for 45 min at 25 C, followed by washing four times for 5 min each with lysis 250 buffer containing 0.2% BSA and protease inhibitors. DBC-1 or fragments of DBC-1 were labeled with [35S]methionine (TNT SP6 quick-coupled transcription/translation system; Promega) and incubated with immobilized GST proteins in PD buffer (50 mM Tris-HCl, 200 mM KCl, 5 mM MgCl2, 5 mM EDTA, and 0.05% NP-40) for 2 h at 4 C. Binding reactions were washed with PD buffer three times for 5 min each at 4 C and subsequently boiled in 20 µl of 1x Laemmli’s sample buffer. Eluates were resolved by SDS-12% PAGE and visualized by PhosphorImager analysis (GE Healthcare).

Mammalian Two-Hybrid Interaction Analysis
HeLa cells grown under hormone-free conditions for 2 d were plated at 1 x 105 cells per well in 12-well plates (Dow Corning, Corning, NY). After 24 h, the cells were transfected using FuGENE 6 (Roche, Indianapolis, IN) according to the recommendations of the manufacturer. In defining the ER{alpha}-DBC-1 interaction, transfection mixtures consisted of pCH110 (47), an internal control plasmid, expressing ß-galactosidase under control of the simian virus 40 promoter (167 ng), pG5luc reporter (167 ng), pACT-ER{alpha} (334 ng), and the various pBIND-DBC-1 constructs (334 ng), including pBIND-DBC-1, pBIND-DBC-1 (1–478), and pBIND-DBC-1 (479–923). pBIND empty vector was used as an appropriate control for interaction with pACT-ER{alpha}. pACT empty vector was used as an appropriate control for interaction with the various pBIND-DBC-1 constructs. After 48 h, cells were harvested and assayed for luciferase activity according to the guidelines of the manufacturer (Promega). Luciferase activity was corrected for the corresponding ß-galactosidase activity to give relative activity. ß-Galactosidase activity was assayed according to the instructions of the manufacturer (Tropix, Bedford, MA). Transfections were repeated a minimum of three times in duplicate. For experiments with ligand treatment, E2 (Sigma, St. Louis, MO) was added to cells at 10–7 M for 24 h before harvest.

For Western blot analysis, 48 h after transfection, whole-cell lysates were prepared in radioimmunoprecipitation assay buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% deoxycholate, 1% NP-40, and 0.1% SDS) supplemented with protease inhibitors and clarified by centrifugation. Equivalent amounts of lysates were boiled in Laemmli’s sample buffer and resolved by SDS-10% PAGE. Proteins were analyzed by immunoblot using antibodies against GAL4-DBD (RK5C1; Santa Cruz Biotechnology, Santa Cruz, CA) and ER{alpha} (HC-20; Santa Cruz Biotechnology).

Coimmunoprecipitations
T-47D, MCF-7, or BG-1 cells were grown under hormone-free conditions for 3 d and treated without or with E2 (10–7 M), 4-hydroxytamoxifen (10–6 M; Sigma), or ICI 182,780 (10–7 M; Tocris, Ellisville, MO) for 1 h before cell harvest and coimmunoprecipitation. Whole-cell lysates were prepared in 0.5% NP-40 lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, and 0.5% NP-40) supplemented with protease inhibitors and clarified by centrifugation. Nuclear and cytoplasmic extracts were prepared as described previously (48). Lysates were adjusted to binding buffer (50 mM Tris-HCl, 175 mM NaCl, 5 mM EDTA, 0.2% NP-40, and 10% glycerol, supplemented with protease inhibitors) concentration. Lysates were then subjected to immunoprecipitation with rabbit polyclonal anti-ER{alpha} (HC-20; Santa Cruz Biotechnology) antibody or mouse polyclonal anti-DBC-1 antibody [produced in our laboratory against recombinant DBC-1 (amino acids 475–923)] and protein A-Sepharose beads. Immune complexes were washed three times with binding buffer, boiled in Laemmli’s sample buffer, and resolved by SDS-10% PAGE. Proteins were transferred to nitrocellulose membranes and visualized by using antibodies against DBC-1, ER{alpha}, HSP90 (rabbit polyclonal; Genetex, San Antonio, TX), CYP40 (rabbit polyclonal; Abcam, Cambridge, MA), appropriate peroxidase-conjugated secondary antibodies (Bio-Rad, Hercules, CA), and enhanced chemiluminescence detection (GE Healthcare).

DBC-1 Silencing by siRNA
To selectively knock down the expression of endogenous DBC-1 protein, an siRNA pool consisting for four different target sequences was used (catalog no. 010427; Dharmacon, Chicago, IL). These RNA duplexes (3 µg per 2 x 106 cells), as well as a negative control duplex that does not pair with any human mRNA (Dharmacon), were electroporated in MCF-7 or MDA-MB-231 cells using the cell line Nucleofector kit V (Amaxa, Gaithersburg, MD). Immediately after control or DBC-1 siRNA electroporation, cells were seeded at a concentration of 1 x 106 per 60 mm plate. In all experiments, cells were allowed to grow for 3 d in phenol-red-free medium supplemented with 10% charcoal/dextran-treated FBS and without or with indicated chemical treatments. Cells were harvested 3 d after electroporation.

Western Blot Analysis
Three days after electroporation, whole-cell lysates were prepared in radioimmunoprecipitation assay buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% deoxycholate, 1% NP-40, and 0.1% SDS) supplemented with protease inhibitors and clarified by centrifugation. Lysates were boiled in Laemmli’s sample buffer and resolved by SDS-10% PAGE. Proteins were analyzed by immunoblot using antibodies against DBC-1 (produced in our laboratory), ER{alpha} (HC-20; Santa Cruz Biotechnology), and TFIIEß (C-21; Santa Cruz Biotechnology) as described previously. Quantification of Western blots was performed using the Kodak ImageStation 2000R (Eastman Kodak, Rochester, NY).

Quantitative Real-Time RT-PCR
Three days after electroporation, RNA was isolated from cells using TRIzol reagent (Invitrogen). RNA was reverse transcribed using random hexamers and Superscript III (Invitrogen) following the instructions of the manufacturer. Quantitative RT-PCR was performed using ABsolute SYBR Green ROX Mix (ABgene, Rochester, NY) on an ABI PRISM 7900HT Fast real-time PCR system (Applied Biosystems, Foster City, CA). The gene-specific primers used were as follows: DBC-1, 5'-ATG TCC CAG TTT AAG CGC CAG-3' and 5'-CAA CCC CAA AGT AGT CAT GCA A-3'; ER{alpha}, 5'-CCA CCA ACC AGT GCA CCA TT-3' and 5'-GGT CTT TTC GTA TCC CAC CTT TC-3'; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-CCT GTT CGA CAG TCA GCC G-3' and 5'-CGA CCA AAT CCG TTG ACT CC-3'.

Proliferation Assays
Three days before electroporation, cells were grown in phenol-red-free medium supplemented with 10% charcoal/dextran-treated FBS. Immediately after control or DBC-1 siRNA electroporation, cells were seeded at a concentration of 10 x 104 per well in six-well plates. In all experiments, triplicates of cells were allowed to grow for 7 d in phenol-red-free medium supplemented with 10% charcoal/dextran-treated FBS and without or with E2 (10–7 M) at 37 C and 10% CO2. Cell viability was determined using the trypan blue exclusion assay, and viable cells were counted with the use of a hemacytometer. Proliferation assays were repeated a minimum of three times.

Apoptosis Assays
Three days before electroporation, cells were grown in phenol-red-free medium supplemented with 10% charcoal/dextran-treated FBS. Immediately after control or DBC-1 siRNA electroporation, cells were seeded at a concentration of 1 x 106 per 60 mm plate. In all experiments, cells were allowed to grow for 3 d in phenol-red-free medium supplemented with 10% charcoal/dextran-treated FBS and without or with E2 (10–7 M), ICI 182,780 (10–7 M; Tocris), or a combination of the two at 37 C and 10% CO2. Seventy-two hours after electroporation, trypsinized cells (1 x 105) were stained with Annexin V-fluorescein isothiocyanate (FITC) (BD Pharmingen, San Diego, CA) and propidium iodide (Becton Dickinson, Franklin Lakes, NJ) according to the instructions of the manufacturer. Flow-cytometric analyses to quantify apoptosis were done in an FACSCalibur (Becton Dickinson). All Annexin V-FITC-positive cells were considered apoptotic. Apoptosis assays were repeated a minimum of three times.

Data Analysis
Statistical significance was assessed by comparing mean ± SD values with Student’s t test for independent groups. P ≤ 0.05 was considered statistically significant.


   ACKNOWLEDGMENTS
 
We are grateful for the various plasmids received from D. Picard, Y. J. Zhu, and Deutsches Ressourcenzentrum für Genomforschung. We thank W. S. Lane and the Harvard Microchemistry Facility for performing the peptide sequencing by LC/MS/MS. We also thank K. Korach for providing BG-1 cells. We thank E. White for providing DBC-1 rabbit polyclonal antibody used in the initial stage of these studies. We are also grateful to P. Garza for DBC-1 antibody production. We thank P. Garza, N. Ding, W. Tan, S. Kim, H. Zhou, L. N. Ngo, P. R. Yew, R. E. Cendaña, X. N. Zhu, H. Li, V. B. Holcomb, T. Marple, and other colleagues for assistance, advice, discussion, and comments.


   FOOTNOTES
 
This work was supported by National Institutes of Health Predoctoral Fellowship 5T32CA086800-04, National Institutes of Health Grant CA098301-01, and United States Army Department of Defense Grant DAMD17-02-1-0584.

Disclosure Summary: The authors have nothing to disclose.

First Published Online May 1, 2007

Abbreviations: CYP40, Cyclophilin 40; E2, 17-ß-estradiol; ER, estrogen receptor; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S-transferase; HSP90, heat shock protein 90; NF-{kappa}B, nuclear factor {kappa}B; NP-40, Nonidet P-40; RNAi, RNA interference; siRNA, small interfering RNA.

Received for publication January 31, 2007. Accepted for publication April 27, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Jemal A, Siegel R, Ward E, Murray T, Xu J, Smigal C, Thun MJ 2006 Cancer statistics, 2006. CA Cancer J Clin 56:106–130[Abstract/Free Full Text]
  2. Hilakivi-Clarke L, Cho E, Cabanes A, DeAssis S, Olivo S, Helferich W, Lippman ME, Clarke R 2002 Dietary modulation of pregnancy estrogen levels and breast cancer risk among female rat offspring. Clin Cancer Res 8:3601–3610[Abstract/Free Full Text]
  3. Koibuchi Y, Sugamata N, Iino Y, Yokoe T, Andoh T, Maemura M, Takei H, Horiguchi J, Matsumoto H, Morishita Y 1999 The mechanisms of antitumor effects of luteinizing hormone-releasing hormone agonist (buserelin) in 7, 12-dimethylbenz(a)anthracene-induced rat mammary cancer. Int J Mol Med 4:145–148[Medline]
  4. Liao DZ, Pantazis CG, Hou X, Li SA 1998 Promotion of estrogen-induced mammary gland carcinogenesis by androgen in the male Noble rat: probable mediation by steroid receptors. Carcinogenesis 19:2173–2180[Abstract/Free Full Text]
  5. Couse JF, Korach KS 1999 Estrogen receptor null mice: what have we learned and where will they lead us? Endocr Rev 20:358–417[Abstract/Free Full Text]
  6. McDonnell DP, Norris JD 2002 Connections and regulation of the human estrogen receptor. Science 296:1642–1644[Abstract/Free Full Text]
  7. Nilsson S, Makela S, Treuter E, Tujague M, Thomsen J, Andersson G, Enmark E, Pettersson K, Warner M, Gustafsson JA 2001 Mechanisms of estrogen action. Physiol Rev 81:1535–1565[Abstract/Free Full Text]
  8. Osborne CK, Schiff R 2005 Estrogen-receptor biology: continuing progress and therapeutic implications. J Clin Oncol 23:1616–1622[Free Full Text]
  9. Clarke RB, Anderson E, Howell A 2004 Steroid receptors in human breast cancer. Trends Endocrinol Metab 15:316–323[CrossRef][Medline]
  10. Ali S, Coombes RC 2000 Estrogen receptor alpha in human breast cancer: occurrence and significance. J Mammary Gland Biol Neoplasia 5:271–281[CrossRef][Medline]
  11. Colditz GA, Rosner BA, Chen WY, Holmes MD, Hankinson SE 2004 Risk factors for breast cancer according to estrogen and progesterone receptor status. J Natl Cancer Inst 96:218–228[Abstract/Free Full Text]
  12. Key TJ, Verkasalo PK, Banks E 2001 Epidemiology of breast cancer. Lancet Oncol 2:133–140[CrossRef][Medline]
  13. Zhou Y, Eppenberger-Castori S, Eppenberger U, Benz CC 2005 The NF{kappa}B pathway and endocrine-resistant breast cancer. Endocr Relat Cancer 12(Suppl 1):S37–S46
  14. Normanno N, Di Maio M, De Maio E, De Luca A, de Matteis A, Giordano A, Perrone F 2005 Mechanisms of endocrine resistance and novel therapeutic strategies in breast cancer. Endocr Relat Cancer 12:721–747[Abstract/Free Full Text]
  15. Huang J, Li X, Hilf R, Bambara RA, Muyan M 2005 Molecular basis of therapeutic strategies for breast cancer. Curr Drug Targets Immune Endocr Metabol Disord 5:379–396[CrossRef][Medline]
  16. Rau KM, Kang HY, Cha TL, Miller SA, Hung MC 2005 The mechanisms and managements of hormone-therapy resistance in breast and prostate cancers. Endocr Relat Cancer 12:511–532[Abstract/Free Full Text]
  17. Schiff R, Massarweh SA, Shou J, Bharwani L, Mohsin SK, Osborne CK 2004 Cross-talk between estrogen receptor and growth factor pathways as a molecular target for overcoming endocrine resistance. Clin Cancer Res 10:331S–336S[CrossRef]
  18. Hamaguchi M, Meth JL, von Klitzing C, Wei W, Esposito D, Rodgers L, Walsh T, Welcsh P, King MC, Wigler MH 2002 DBC2, a candidate for a tumor suppressor gene involved in breast cancer. Proc Natl Acad Sci USA 99:13647–13652[Abstract/Free Full Text]
  19. Radvanyi L, Singh-Sandhu D, Gallichan S, Lovitt C, Pedyczak A, Mallo G, Gish K, Kwok K, Hanna W, Zubovits J, Armes J, Venter D, Hakimi J, Shortreed J, Donovan M, Parrington M, Dunn P, Oomen R, Tartaglia J, Berinstein NL 2005 The gene associated with trichorhinophalangeal syndrome in humans is overexpressed in breast cancer. Proc Natl Acad Sci USA 102:11005–11010[Abstract/Free Full Text]
  20. Bittner M 2005 A window on the dynamics of biological switches. Nat Biotechnol 23:183–184[CrossRef][Medline]
  21. van de Vijver MJ, He YD, van’t Veer LJ, Dai H, Hart AA, Voskuil DW, Schreiber GJ, Peterse JL, Roberts C, Marton MJ, Parrish M, Atsma D, Witteveen A, Glas A, Delahaye L, van der Velde T, Bartelink H, Rodenhuis S, Rutgers ET, Friend SH, Bernards R 2002 A gene-expression signature as a predictor of survival in breast cancer. N Engl J Med 347:1999–2009[Abstract/Free Full Text]
  22. Zhao H, Langerod A, Ji Y, Nowels KW, Nesland JM, Tibshirani R, Bukholm IK, Karesen R, Botstein D, Borresen-Dale AL, Jeffrey SS 2004 Different gene expression patterns in invasive lobular and ductal carcinomas of the breast. Mol Biol Cell 15:2523–2536[Abstract/Free Full Text]
  23. Richardson AL, Wang ZC, De Nicolo A, Lu X, Brown M, Miron A, Liao X, Iglehart JD, Livingston DM, Ganesan S 2006 X chromosomal abnormalities in basal-like human breast cancer. Cancer Cell 9:121–132[CrossRef][Medline]
  24. Bouwmeester T, Bauch A, Ruffner H, Angrand PO, Bergamini G, Croughton K, Cruciat C, Eberhard D, Gagneur J, Ghidelli S, Hopf C, Huhse B, Mangano R, Michon AM, Schirle M, Schlegl J, Schwab M, Stein MA, Bauer A, Casari G, Drewes G, Gavin AC, Jackson DB, Joberty G, Neubauer G, Rick J, Kuster B, Superti-Furga G 2004 A physical and functional map of the human TNF-{alpha}/NF-{kappa}B signal transduction pathway. Nat Cell Biol 6:97–105[CrossRef][Medline]
  25. Sundararajan R, Chen G, Mukherjee C, White E 2005 Caspase-dependent processing activates the proapoptotic activity of deleted in breast cancer-1 during tumor necrosis factor-{alpha}-mediated death signaling. Oncogene 24:4908–4920[CrossRef][Medline]
  26. Bagatell R, Khan O, Paine-Murrieta G, Taylor CW, Akinaga S, Whitesell L 2001 Destabilization of steroid receptors by heat shock protein 90-binding drugs: a ligand-independent approach to hormonal therapy of breast cancer. Clin Cancer Res 7:2076–2084[Abstract/Free Full Text]
  27. Fliss AE, Benzeno S, Rao J, Caplan AJ 2000 Control of estrogen receptor ligand binding by Hsp90. J Steroid Biochem Mol Biol 72:223–230[CrossRef][Medline]
  28. Reid G, Hubner MR, Metivier R, Brand H, Denger S, Manu D, Beaudouin J, Ellenberg J, Gannon F 2003 Cyclic, proteasome-mediated turnover of unliganded and liganded ER{alpha} on responsive promoters is an integral feature of estrogen signaling. Mol Cell 11:695–707[CrossRef][Medline]
  29. Jordan VC 2004 Selective estrogen receptor modulation: concept and consequences in cancer. Cancer Cell 5:207–213[CrossRef][Medline]
  30. Riggins RB, Bouton AH, Liu MC, Clarke R 2005 Antiestrogens, aromatase inhibitors, and apoptosis in breast cancer. Vitam Horm 71:201–237[CrossRef][Medline]
  31. Howell A 2006 Pure oestrogen antagonists for the treatment of advanced breast cancer. Endocr Relat Cancer 13:689–706[Abstract/Free Full Text]
  32. Fanelli MA, Vargas-Roig LM, Gago FE, Tello O, Lucero De Angelis R, Ciocca DR 1996 Estrogen receptors, progesterone receptors, and cell proliferation in human breast cancer. Breast Cancer Res Treat 37:217–228[CrossRef][Medline]
  33. Sommer S, Fuqua SA 2001 Estrogen receptor and breast cancer. Semin Cancer Biol 11:339–352[CrossRef][Medline]
  34. Lee MO, Kim EO, Kwon HJ, Kim YM, Kang HJ, Kang H, Lee JE 2002 Radicicol represses the transcriptional function of the estrogen receptor by suppressing the stabilization of the receptor by heat shock protein 90. Mol Cell Endocrinol 188:47–54[CrossRef][Medline]
  35. Pratt WB, Toft DO 1997 Steroid receptor interactions with heat shock protein and immunophilin chaperones. Endocr Rev 18:306–360[Abstract/Free Full Text]
  36. Zhou Y, Eppenberger-Castori S, Marx C, Yau C, Scott GK, Eppenberger U, Benz CC 2005 Activation of nuclear factor-{kappa}B (NF{kappa}B) identifies a high-risk subset of hormone-dependent breast cancers. Int J Biochem Cell Biol 37:1130–1144[CrossRef][Medline]
  37. Bhat-Nakshatri P, Sweeney CJ, Nakshatri H 2002 Identification of signal transduction pathways involved in constitutive NF-{kappa}B activation in breast cancer cells. Oncogene 21:2066–2078[CrossRef][Medline]
  38. deGraffenried LA, Chandrasekar B, Friedrichs WE, Donzis E, Silva J, Hidalgo M, Freeman JW, Weiss GR 2004 NF-{kappa}B inhibition markedly enhances sensitivity of resistant breast cancer tumor cells to tamoxifen. Ann Oncol 15:885–890[Abstract/Free Full Text]
  39. Pratt MA, Bishop TE, White D, Yasvinski G, Menard M, Niu MY, Clarke R 2003 Estrogen withdrawal-induced NF-{kappa}B activity and bcl-3 expression in breast cancer cells: roles in growth and hormone independence. Mol Cell Biol 23:6887–6900[Abstract/Free Full Text]
  40. Nakshatri H, Bhat-Nakshatri P, Martin DA, Goulet Jr RJ, Sledge Jr GW 1997 Constitutive activation of NF-{kappa}B during progression of breast cancer to hormone-independent growth. Mol Cell Biol 17:3629–3639[Abstract]
  41. Mandlekar S, Kong AN 2001 Mechanisms of tamoxifen-induced apoptosis. Apoptosis 6:469–477[CrossRef][Medline]
  42. Dowsett M, Archer C, Assersohn L, Gregory RK, Ellis PA, Salter J, Chang J, Mainwaring P, Boeddinghaus I, Johnston SR, Powles TJ, Smith IE 1999 Clinical studies of apoptosis and proliferation in breast cancer. Endocr Relat Cancer 6:25–28[Abstract]
  43. Liu JW, Picard D 1998 Bioactive steroids as contaminants of the common carbon source galactose. FEMS Microbiol Lett 159:167–171[CrossRef][Medline]
  44. Turner DL, Weintraub H 1994 Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate. Genes Dev 8:1434–1447[Abstract/Free Full Text]
  45. Qi C, Chang J, Zhu Y, Yeldandi AV, Rao SM, Zhu YJ 2002 Identification of protein arginine methyltransferase 2 as a coactivator for estrogen receptor {alpha}. J Biol Chem 277:28624–28630[Abstract/Free Full Text]
  46. Ignar-Trowbridge DM, Teng CT, Ross KA, Parker MG, Korach KS, McLachlan JA 1993 Peptide growth factors elicit estrogen receptor-dependent transcriptional activation of an estrogen-responsive element. Mol Endocrinol 7:992–998[Abstract]
  47. Zheng L, Annab LA, Afshari CA, Lee WH, Boyer TG 2001 BRCA1 mediates ligand-independent transcriptional repression of the estrogen receptor. Proc Natl Acad Sci USA 98:9587–9592[Abstract/Free Full Text]
  48. Dignam JD, Lebovitz RM, Roeder RG 1983 Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11:1475–1489[Abstract/Free Full Text]

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