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Molecular Endocrinology 17 (3): 356-365
Copyright © 2003 by The Endocrine Society

The NEDD8 Pathway Is Required for Proteasome-Mediated Degradation of Human Estrogen Receptor (ER)-{alpha} and Essential for the Antiproliferative Activity of ICI 182,780 in ER{alpha}-Positive Breast Cancer Cells

Meiyun Fan, Robert M. Bigsby and Kenneth P. Nephew

Medical Sciences (M.F., K.P.N.), Indiana University School of Medicine, Bloomington, Indiana 47405; and Departments of Obstetrics & Gynecology (R.M.B., K.P.N.) and of Cellular and Integrative Physiology (R.M.B., K.P.N.), Indiana University Cancer Center (R.M.B., K.P.N.), Indiana University School of Medicine, Indianapolis, Indiana 46202

Address all correspondence and requests for reprints to: Kenneth P. Nephew, Ph.D., Medical Sciences, Indiana University School of Medicine, 302 Jordan Hall, 1001 East Third Street, Bloomington, Indiana 47405-4401. E-mail: knephew{at}indiana.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroid hormone receptors, including estrogen receptor-{alpha} (ER{alpha}), are ligand-activated transcription factors, and hormone binding leads to depletion of receptor levels via preteasome-mediated degradation. NEDD8 (neural precursor cell-expressed developmentally down-regulated) is an ubiquitin-like protein essential for protein processing and cell cycle progression. We recently demonstrated that ubiquitin-activating enzyme (Uba)3, the catalytic subunit of the NEDD8-activating enzyme, inhibits ER{alpha} transcriptional activity. Here we report that Uba3-mediated inhibition of ER{alpha} transactivation function is due to increased receptor protein turnover. Coexpression of Uba3 with ER{alpha} increased receptor degradation by the 26S proteasome. Inhibition of NEDD8 activation and conjugation diminished polyubiquitination of ER{alpha} and blocked proteasome-mediated degradation of receptor protein. The antiestrogen ICI 182,780 is known to induce ER degradation. In human MCF7 breast cancer cells modified to contain a disrupted NEDD8 pathway, ICI 182,780 degradation of ER{alpha} was impaired, and the antiestrogen was ineffective at inhibiting cell proliferation. This study provides the first evidence linking nuclear receptor degradation with the NEDD8 pathway and the ubiquitin-proteasome system, suggesting that the two pathways can act together to modulate ER{alpha} turnover and cellular responses to estrogens. Based on our observation that an intact NEDD8 pathway is essential for the antiproliferation activity of the ICI 182,780 in ER{alpha} positive breast cancer cells, we propose that disruptions in the NEDD8 pathway provide a mechanism by which breast cancer cells acquire antiestrogen resistance while retaining expression of ER{alpha}.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ESTROGEN REGULATES DIVERSE biological processes through estrogen receptors (ER{alpha} and ERß) (1). Receptor levels and dynamics have a profound influence on target tissue responsiveness and sensitivity to estrogen (2). ER{alpha} is a short-lived protein with a half-life of about 4 h, which is reduced to 3 h by 17ß-estradiol (estradiol), and to less than 1 h by the steroidal antiestrogens, ICI 182,780 and ICI 164,384 (3, 4). Receptor turnover rates provide estrogen target cells with the capacity for rapid regulation of receptor levels and thus dynamic hormone responses. An attenuated transcriptional response has been associated with down-regulation of ER{alpha}, and receptor up-regulation has been shown to enhance the cellular response to estrogen (2). Nonetheless, mechanisms governing ER{alpha} protein levels remain poorly understood.

It has recently been shown that degradation of ER{alpha} and other members of the nuclear receptor superfamily occurs through the ubiquitin-proteasome pathway (5). Ubiquitination is a multistep process involving the action of a ubiquitin-activating enzyme (E1 or Uba), a ubiquitin conjugation enzyme (E2 or Ubc), and a ubiquitin ligase (E3) (6). Because the high specificity for target proteins is primarily conferred by E3, regulation of E3 activity may play a crucial role in governing protein degradation in vivo. A large number of E3s are cullin-based ubiquitin ligases (7), including SCF (Skp1/Cul1/F-box/ROC1) and VCB (von Hippel-Lindau-Cul2/elongin B/elongin C) complexes. One important level of regulation of these cullin-based ubiquitin ligases involves modification of the cullin subunit with NEDD8, an ubiquitin-like protein (7).

NEDD8 conjugation (neddylation) resembles ubiquitination and involves the action of amyloid precursor protein-binding protein (APP-BP1)/Uba3, a heterodimeric E1-like enzyme, and Ubc12, an E2-like enzyme (8). Whether a ligase is required for neddylation is unknown. To date, the only known substrates of NEDD8 are cullin family members (9, 10). Cullin neddylation is conserved and plays an important regulatory role for cullin-based E3 activity in yeast, plant, and mammalian cells (7, 11, 12, 13). Interrupting NEDD8 modification of cullins in mammalian cells has been shown to block ubiquitination of certain proteins involved in different cellular functions, including p27, I{kappa}B{alpha}, HIF{alpha}, and NF{kappa}B precursor p105 (14, 15, 16, 17, 18, 19). Recent studies have revealed that cullin neddylation is a tightly controlled dynamic process (20, 21, 22, 23, 24), and the effect of neddylation on protein polyubiquitination appears to be specific (17, 18).

We recently identified the NEDD8 activating enzyme, Uba3 as an ER-interacting protein and inhibitor of transactivation by steroid nuclear receptors (25). We further demonstrated that an intact neddylation pathway is required for Uba3-mediated inhibition of ER transcriptional activity (25). Taken together with recent reports linking the ubiquitin and NEDD8 pathways (7), our findings raise the intriguing possibility for a role of neddylation in ER{alpha} ubiquitination and degradation. Here we show that Uba3 enhances ER{alpha} degradation by the 26S proteasome, and expression of dominant-negative mutants of Uba3 or Ubc12 impaired ER{alpha} ubiquitination and ligand-induced ER{alpha} degradation. Blocking the neddylation pathway with the dominant-negative Ubc in ER{alpha}-positive human breast cancer cells inhibited both receptor degradation and the growth inhibitory effect of the antiestrogen ICI 182,780 (known clinically as Faslodex or Fulvestrant). Collectively, these data show that the NEDD8 pathway plays an essential role in ubiquitination and proteasomal degradation of ER{alpha} and indicate that disruptions in the pathway may contribute to the development of antiestrogen resistance in human breast cancer.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Uba3 Enhances Proteasomal Degradation of ER{alpha}
To test the hypothesis that the neddylation pathway restricts ER{alpha} activity by modulating receptor degradation, we transfected HeLa cells with ER{alpha}, alone or in combination with an expression vector for Uba3, APP-BP1, or Ubc12, or with an empty vector (pcDNA3.1, Invitrogen, Carlsbad, CA); a green fluorescence protein (GFP) expression vector was cotransfected to serve as a means of normalizing transfection efficiency and sample preparations. Steady-state levels of ER{alpha} protein were determined by Western blot analysis. Coexpression of Uba3 decreased ER{alpha} protein level but had no effect on GFP expression (Fig. 1AGo). Treatment of the transfected HeLa cells with MG132, a specific proteasome inhibitor, blocked Uba3-stimulated down-regulation of ER{alpha} (Fig. 1BGo), confirming that the Uba3-induced ER{alpha} degradation is through the 26S proteasome. Overexpression of APP-BP1 or Ubc12 had no significant effect on ER{alpha} protein levels (data not shown), a result consistent with our previous observation that Uba3 is the limiting factor in neddylation-associated inhibition of ER{alpha} transcriptional activity (25).



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  		Figure 1. Uba3 Enhances Proteasomal Degradation of ER{alpha}

A, Coexpression of Uba3 decreases ER{alpha} protein level in transfected HeLa cells. HeLa cells were transfected with pSG5-ER and pcDNA-Uba3 or pcDNA vector. Whole cell extracts were prepared 24 h post transfection and analyzed by Western blotting to determine ER{alpha} protein level. B, Proteasome inhibitor MG132 restores expression level of ER{alpha} in cells transfected with Uba3. Transfected HeLa cells (same as in A) were treated with 20 µM MG132 for 6 h before protein extracts and ER{alpha} level analysis. GFP was used as an internal control to correct for transfection efficiency and SDS-PAGE loading. Representative results of three independent experiments are shown.

 
The Neddylation Pathway Is Required for Ligand-Mediated Degradation of ER{alpha}
Estradiol stimulates ER{alpha} degradation through the ubiquitin-proteasome pathway (26, 27, 28, 29, 30). Having established a role for Uba3 in this process, it was important to assess whether neddylation pathway is required for ligand-induced degradation of ER{alpha}. To address this issue, we used a dominant-negative mutant of Ubc12 (Ubc12C111S). Due to a single Cys-to-Ser substitution at the active Cys residue, Ubc12C111S forms a stable complex with NEDD8, resulting in sequestration of NEDD8 and inhibition of subsequent NEDD8 conjugation (31, 32). Dominant-negative inhibition of NEDD8 conjugation by Ubc12C111S has been shown to impair efficient ubiquitination and protein degradation (14, 15, 17, 18). Treatment of ER{alpha}-transfected HeLa cells with estradiol resulted in a time-dependent decrease in ER{alpha} protein levels; receptor levels were reduced by 80% at 6–8 h. (Fig. 2AGo). In contrast, the effects of estradiol on receptor levels were less dramatic in cells expressing Ubc12C111S, producing a reduction of only 40% by 6–8 h (Fig. 2AGo). Consistent with this observation, Uba3C216S, a dominant-negative mutant of Uba3 (31, 32), also inhibited estradiol-induced ER{alpha} down-regulation (Fig. 2BGo). Addition of the proteasome inhibitor MG132 before estradiol treatment completely abolished ligand-induced down-regulation of ER{alpha} (Fig. 2BGo), confirming that exogenous ER{alpha} in HeLa cells undergoes proteasome-dependent degradation in response to estradiol. Collectively, these results demonstrate that a functional NEDD8 pathway is required for efficient, ligand-induced, proteasome-mediated degradation of ER{alpha}.



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  		Figure 2. Expression of Ubc12C111S or Uba3C216S Inhibits Ligand-Induced ER{alpha} Degradation

A, HeLa cells were transfected with pSG5-ER and pcDNA vector (upper panel) or pcDNA-Ubc12C111S (lower panel). Twenty-four hours after transfection, cells were treated with 100 nM estradiol for the indicated times and analyzed for ER{alpha} protein level using Western blotting. Relative ER{alpha} levels in cells cotransfected with vector (gray) or Ubc12C111S (black) from two independent experiments are shown in corresponding histogram. B, HeLa cells were transfected with pSG5-ER and pcDNA vector (upper panel) or pcDNA-Uba3C216S (lower panel). Twenty-four hours after transfection, cells were treated with vehicle or 20 µM MG132 for 1 h followed by incubation with vehicle or 100 nM estradiol for 6 h, as indicated. ER{alpha} protein levels were analyzed by immunoblotting. Relative ER{alpha} levels in cells cotransfected with vector (gray) or Uba3C216S (black) from three independent experiments are shown in corresponding histogram. GAPDH was used as an internal control to correct SDS-PAGE loading.

 
The NEDD8 Pathway Is Required for Efficient Ubiquitination of ER{alpha}
Having established a role for Uba3 and Ubc12 in ER{alpha} down-regulation, it was important to examine the effect of NEDD8 on receptor ubiquitination. HeLa cells were cotransfected with ER{alpha} and hemagglutinin (HA)-tagged ubiquitin, along with wild-type Ubc12 or Uba3 or the corresponding mutant forms of these neddylation enzymes (Ubc12C111S or Uba3C216S). At 24 h post transfection, cells were treated with MG132 or vehicle, followed by estradiol treatment. Immunoprecipitation assays using an anti-ER{alpha} antibody were performed and the levels of ubiquitinated ER{alpha} in the precipitated immunocomplex were assessed by Western blotting with an anti-HA antibody. The polyubiquitinated ER{alpha} exhibited a ladder of higher molecular weight species on the blot membrane (Fig. 3Go). Expression of dominant-negative Ubc12C111S or Uba3C216S markedly decreased ER{alpha} ubiquitination in either the absence (Fig. 3Go, left panel) or presence of estradiol and MG132 (Fig. 3Go, right panel), compared with cells transfected with control vector or wild-type Ubc12 or Uba3. These results suggest that a functional neddylation pathway is required for the efficient ubiquitination of ER{alpha}.



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  		Figure 3. An Intact NEDD8 Pathway Is Required for Efficient ER{alpha} Ubiquitination

HeLa cells were transfected with pSG5-ER, and pcDNA-HA-Ubiquitin, alone with indicated construct. Twenty-four hours after transfection, cells were either untreated (left panel) or treated with 20 µM MG132 for 1 h followed by 100 nM estradiol exposure for 3 h (right panel). Protein extracts were prepared and subjected to immunoprecipitation using anti-ER{alpha} antibody. Polyubiquitinated ER{alpha} was detected by Western blotting using anti-HA antibody, and was visualized as a ladder of higher molecular weight species on the blot. The blot was striped and reprobed by anti-ER{alpha} antibody to assess the amount of precipitated ER{alpha} (lower panels). The heavy chain of the anti-ER{alpha} IgG used for immunoprecipitation exhibits a 57-kDa band in the ER{alpha} blot. Representative results of three independent experiments are shown.

 
ER{alpha} Protein Levels in MCF7 Breast Cancer Cell Lines Stably Expressing Dominant-Negative Ubc12C111S
MCF7 human breast cancer cells express high levels of ER{alpha} and proliferate in response to estrogen treatment (33, 34), providing a model to study endogenous ER{alpha} function. To further investigate the role of neddylation in ER{alpha} function under physiological relevant conditions, we transfected Ubc12C111S into MCF7 cells and established the stable cell line MCF7/C111S. As a control, MCF7/Vec (MCF7 cells stably transfected with empty vector) was also established. Expression of the Ubc12C11S mutant protein in MCF7/C111S cells was confirmed by Western blotting and, consistent with a previous report (31), the mutant was detected as 26- and 31-kDa proteins (Fig. 4Go, lanes 3–8). In the regular growth medium containing phenol red and 10% fetal bovine serum (FBS), the level of ER{alpha} in MCF7/Vec cells was very low; after 3 d of culture in hormone-free medium containing 3% dextran-coated charcoal-stripped FBS (csFBS) and no phenol red, ER{alpha} expression was dramatically increased (Fig. 4Go, lanes 1 and 2). The culture medium (regular growth medium vs. hormone-free medium) showed no effect on the expression level of Ubc12C111S. In three MCF7/C111S clones, receptor levels varied among the clones and, when cultured in growth medium, detectable ER{alpha} was seen in two of the three clones (Fig. 4Go, lanes 5 and 7). When cultured in estrogen-free medium, however, ER{alpha} levels were high in all three clones (Fig. 4Go, lanes 4, 6, 8).



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  		Figure 4. The Expression of Ubc12C111S and ER{alpha} in Three Independent MCF7/C111S Clones

MCF7/C111S cells stably expressing mutant Ubc12C111S were maintained in growth medium (lanes 1, 3, 5, and 7) or hormone-free medium for 3 d (lanes 2, 4, 6, and 8) and analyzed by immunoblotting using anti-HA (upper panel) or anti-ER{alpha} (lower panel) antibodies, respectively. GAPDH was used as an internal control to correct for SDS-PAGE loading.

 
Ubc12C111S Inhibits ICI 182,780-Induced Down-Regulation of ER{alpha}
In contrast to estradiol, which down-regulates ER{alpha} in target tissues through both transcriptional and posttranslational mechanism (35, 36), the pure antiestrogen ICI 182,780 causes ER{alpha} protein degradation without affecting ER{alpha} mRNA levels (3, 36). Based on our observations that the NEDD8 pathway is essential for ER{alpha} degradation in transfected HeLa cells (Fig. 2Go), it was of interest to examine the effect of the antiestrogen on ER{alpha} degradation in MCF7/C111S cells. Cells were cultured in hormone-free medium for 3 d before ICI 182,780 treatment. Under this condition, comparable amounts of ER{alpha} were observed in MCF7/C111S and MCF7/Vec cells (compare 0-h lanes in Fig. 5AGo). Treatment with ICI 182,780 rapidly (by 1 h) decreased ER{alpha} levels in the MCF7/Vec cells; by 4 h post treatment, the levels of ER{alpha} were reduced by 95% (Fig. 5AGo). In the MCF7/C111S cells, the effects of ICI 182,780 on ER{alpha} levels were much less dramatic (Fig. 5AGo). Thus, although ER degradation was not completely inhibited by expression of the dominant-negative Ubc12C111S, these results confirm our observations using transient transfection in HeLa cells and further suggest that the NEDD8 pathway is required for efficient degradation of endogenous ER{alpha}. To examine the effect of another antiestrogen on ER{alpha} degradation in this system, cells were cultured in the presence of various doses of 4-hydroxytamoxifen (4-OHT) and ER{alpha} levels were examined. In both MCF7/Vec and MCF7/C111S cells, ER{alpha} levels remained unchanged or were slightly increased after treatment with 4-OHT (Fig. 5BGo). Stabilization of ER{alpha} by tamoxifen has been reported by others (30), perhaps due to inhibition of the basal rate of ER degradation by the antiestrogen.



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  		Figure 5. ER{alpha} Degradation Is Impaired in MCF7/C111S Cells

A, ICI 182,780-induced ER{alpha} degradation is impaired in MCF7/C111S cells. MCF7/Vec (upper panel) and MCF7/C111S cells (lower panel) were cultured in hormone-free medium for 3 d and treated with 1 nM ICI 182,780 for the indicated times. B, 4-OHT does not cause ER{alpha} degradation in MCF7 cells. MCF7/Vec (upper panel) and MCF7/C111S cells (lower panel) were cultured in hormone-free medium for 3 d and treated with indicated doses of 4-OHT for 6 h. ER{alpha} protein levels were determined by Western blotting with anti-ER{alpha} antibody. The histogram shows the relative ER{alpha} levels after ICI 182,780 or 4-OHT treatment. Relative ER{alpha} levels in MCF7/vec (gray) from three independent experiments or MCF7/C111S (black) from three independent MCF7/C111S clones are shown in corresponding histogram. GAPDH was used as an internal control to correct SDS-PAGE loading.

 
Disrupting the NEDD8 Pathway Confers Antiestrogen Resistance in Breast Cancer Cells
Estradiol is mitogenic in MCF7 cells and stimulates cell proliferation through activation of ER{alpha} (37). The pure antiestrogen ICI 182,780, on the other hand, blocks ER{alpha}-mediated transactivation and induces ER{alpha} protein degradation, resulting in growth inhibition of breast cancer cells (38). Because expression of Ubc12C111S inhibited ICI 182,780-induced ER{alpha} down-regulation (Fig. 5AGo), we examined the growth inhibitory effect of ICI 182,780 in MCF7/C111S cells. No significant difference was observed in basal cell proliferation rates between MCF7/C111S and MCF7/Vec cells in hormone-free medium (data not shown). Treatment with the antiestrogen (1 nM) inhibited the basal cell growth of MCF7 and MCF7/Vec cells (Fig. 6AGo). In contrast, MCF7/C111S cells were partially resistant to ICI 182,780. Specifically, over an 8-d period, the antiestrogen inhibited the growth of control cells by 50% compared with 20–25% growth inhibition of the MCF7/C111S cells (Fig. 6AGo, left panel). Dose-response analysis showed that MCF7/C111S cells were resistant to a broad range (0.01–10 nM) of ICI 182,780 concentrations (Fig. 6AGo, right panel). On the other hand, estradiol-induced proliferation of MCF7/C11S and control cells was similar (2-fold increase in cell number over a 6-d treatment period; data not shown). The effect of 4-OHT on MCF7/C111S and MCF7/Vec cell proliferation was examined in a time- and dose-response analysis. The response of the cell lines to 4-OHT was similar (Fig. 6BGo), suggesting that Ubc12C111S expression did not confer cells resistance to growth inhibitory effect of antiestrogens in general. These results suggest that the expression of Ubc12C111S conferred resistance of MCF7 cells to the growth inhibitory effects of ICI 182,780, but disrupting the NEDD8 pathway had no effect on the mitogenic response of MCF7 breast cancer cells to estradiol or the growth inhibitory effects of 4-OHT.



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  		Figure 6. Interruption of the NEDD8 Pathway Confers Resistance to ICI 182,780 in Human Breast Cancer Cells

A, Time- and dose-dependent growth inhibition of ICI 182,780. For time-response analysis, cells were treated with 1 nM ICI 182,780 and cell numbers were determined 0, 2, 4, 6, and 8 d after drug exposure. For dose-response assay, cells were treated with indicated doses of ICI 182,780 and cell numbers were determined on d 7. B, Time- and dose-dependent antiproliferative effect of 4-OHT. For time-response analysis, cells were treated with 10 nM 4-OHT and cell numbers were determined 0, 3, 6, and 9 d later. For the dose-response assay, cells were treated with indicated doses of 4-OHT and cell numbers were determined on d 7. For all assays, cells were cultured in hormone-free medium for 3 d before treatment and cell numbers were determined by MTT assay. Relative proliferation rate was expressed as percentage of cells grown in hormone-free medium. Each experiment was repeated three times in quadruplicate.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
ER{alpha} is a short-lived protein whose degradation is primarily mediated by the ubiquitin-proteasome pathway (26, 27, 28, 29, 30). The recently described ubiquitin-like pathways, including the NEDD8 and SUMO (small ubiquitin-like modifier) conjugation systems (39), have been implicated in nuclear receptor regulation (40, 41, 42, 43, 44) and the NEDD8 pathway has been shown to enhance protein polyubiquitination (12, 14, 15, 16, 17, 18, 19, 45, 46, 47). Our previous investigation into the role of the NEDD8 pathway in nuclear hormone receptor regulation showed that Uba3, the catalytic subunit of the NEDD8 activating enzyme complex, interacts with ER{alpha} and inhibits receptor function (25). Here we report that Uba3-mediated inhibition of ER{alpha} transactivation is due to increased receptor turnover and that an intact neddylation pathway is essential for ER{alpha} ubiquitination and degradation. By impairing the NEDD8 pathway in human MCF7 breast cancer cells, we demonstrated that the cells became resistant to the growth inhibitory effects of ICI 182,780. Thus, our data suggest that neddylation plays an important role in ER{alpha} degradation and we speculate that alterations in the NEDD8 pathway may provide a mechanism by which tumors can acquire antiestrogen resistance.

Several recent studies have focused on the role of the ubiquitin-proteasome pathway in nuclear receptor down-regulation (26, 27, 28, 29, 30). Enhancement of ER{alpha} ubiquitination by estradiol was first reported by Nirmala and Thampan (48), and Nawaz et al. (27) showed that a functional ubiquitin-proteasome system is required for ER{alpha} degradation. Both basal and ligand-induced ER{alpha} ubiquitination occurs at the nuclear matrix (49), but how ER{alpha} is targeted for ubiquitination has not been fully established. Previously, we had shown that Uba3 interacts directly with ER and that this interaction is augmented by estradiol (25). Here, we show that overexpression of Uba3 enhanced degradation of ER{alpha} and that disruption of Uba3 activity reduces estradiol-induced receptor degradation. Taken together, these data support a role for Uba3 in the regulation of basal as well as ligand-induced ER{alpha} turnover.

The present study is the first to link the NEDD8 pathway to ubiquitination of ER{alpha}. The exact mechanism connecting the two pathways, however, remains unclear. The only known substrates for direct neddylation are members of the cullin family (10). Some of the cullins have been identified as core subunits of specific ubiquitin ligase complexes (7). Mechanistically, conjugation of NEDD8 to cullins may up-regulate ubiquitin ligase activity of specific E3s by facilitating the formation of an ubiquitin E2-E3 complex (45). In this regard, the interaction between Uba3 and ER{alpha} could result in the functional recruitment and activation of a cullin-based ubiquitin-protein ligase, which, in turn, targets ER{alpha} for degradation by the ubiquitin-proteasome system. The hypothetical model depicting the role of neddylation pathway in proteasome-mediated degradation of ER{alpha} is shown in Fig. 7Go. Together with our previously reported data (25), these observations indicate that such targeted degradation of ER{alpha} leads to reduced hormonal responsiveness.



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  		Figure 7. Hypothetical Model Depicting the Role of Neddylation Pathway in Proteasome-Mediated Degradation of ER{alpha}

The physical interaction between Uba3 and ER{alpha} promotes the functional recruitment and activation of a cullin-based ubiquitin-protein ligase to augment receptor polyubiquitination. Uba3 and APP-BP1, the heterodimeric activating enzyme for NEDD8, and Ubc12, the NEDD8 conjugating enzyme, promote cullin NEDD8 modification of specific ubiquitin E3 ligases. Neddylated cullins enhance the formation and activity of the ubiquitin E2-E3 complex. The potency of ER{alpha}-Uba3 interaction appears to correlate with ER{alpha} turnover rate. In the absence of ligand, ER{alpha} interacts weakly with Uba3, resulting in basal ubiquitination and degradation of ER{alpha}; however, estradiol augments the ER{alpha}-Uba3 interaction to enhance ER{alpha} ubiquitination. On the other hand, 4-OHT interrupts the ER{alpha}-Uba3 interaction and stabilizes ER{alpha}, and MG132 blocks ER{alpha} degradation by inhibiting proteasome activity. APP-BP1, Amyloid precursor protein-binding protein; E2, ubiquitin conjugation enzyme; E3, ubiquitin protein ligase; estradiol, 17ß-estradiol; Nd, neural precursor cell-expressed developmentally down-regulated (NEDD8); {downarrow} and {perp}, Stimulation and inhibition, respectively.

 
In addition to its effect on ER{alpha}, Uba3 inhibits the transactivation function of other steroid receptors, ERß, androgen receptor (AR) and progesterone receptor (PR) (25). Others have reported that NEDD8 interacts with aryl hydrocarbon receptor and the interaction affects the transcriptional activity and stability of the receptor protein (40). Furthermore, the NEDD8 protein has been found to colocalize with AR (50). Together with the observations that turnover of ER, AR, PR, and aryl hydrocarbon receptor occurs via degradation by the 26S proteasome (28, 51, 52, 53), these results provide compelling evidence for integration of the neddylation and ubiquitin-proteasome pathways in steroid hormone action. Because receptor levels can have a profound influence on target tissue responsiveness to hormone, NEDD8 and ubiquitin pathways, by modulating receptor protein turnover, could play important roles in determining and perhaps limiting cellular responses to steroid hormones and antihormones.

The antiestrogen ICI 182,780 is a 7{alpha}-alkylsulfinyl analog of estradiol lacking agonist activity (54). The drug is used as a second-line endocrine agent in patients who have developed tamoxifen-resistant breast cancer (38). Although the drug clearly displays complex pharmacology, rapid degradation of ER{alpha} protein has been associated with the antiproliferative effects of ICI 182,780 on breast cancer cells (38, 54). Despite its potent antitumor effects, the drug does not circumvent the development of antiestrogen resistance (55, 56, 57, 58). Moreover, the fact that most tumors acquiring ICI 182,780 resistance do so while retaining expression of ER{alpha} and estrogen responsiveness (55, 59) suggests that administration of the antiestrogen may possibly lead to the selection of tumor cells defective in ER{alpha} down-regulation pathway(s), which in turn may confer a proliferative advantage in either the presence or absence of estrogens. Mechanism underlying persistent expression of ER{alpha} in tumors with acquired resistance may thus present an important therapeutic target for future drug intervention. In this context, the loss of NEDD8 expression during malignant transformation of prostate cancer was recently reported (60). Because our results show an intact NEDD8 pathway is essential for ER{alpha} ubiquitination and degradation, we speculate that disruptions in the NEDD8 pathway may provide a mechanism by which breast cancer cells acquire ICI 182,780 resistance while retaining expression of ER{alpha}.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
The following antibodies and reagents were used in this study: anti-ER (HC20; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-HA (3F10; Roche Molecular Biochemicals, Indianapolis, IN); anti-GFP (GFP01, NeoMarkers, Inc., Fremont, CA); anti-GAPDH (glyceraldehyde phosphate dehydrogenase; Chemicon International, Inc., Temecula, CA); antirabbit IgG and protein G-agarose beads (Oncogene Research Products, San Diego, CA); SuperSignal West Pico Chemiluminescent Substrate (Pierce Chemical Co., Rockford, IL); protease inhibitor cocktail set III (Calbiochem-Novabiochem Corp., San Diego, CA); Bio-Rad Laboratories, Inc. (Hercules, CA) protein assay kit; FBS and csFBS (HyClone Laboratories, Inc., Logan, UT); LipofectAMINE Plus Reagent, geneticin, and other cell culture reagents were from Life Technologies, Inc. (Rockville, MD). Estradiol, 4-OHT, MG132, and 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from Sigma (St. Louis, MO). ICI 182,780 was purchased from Tocris Cookson Ltd. (Ellisville, MO).

Plasmid Construction
The construction of pSG5-ER(HEGO), pcDNA-Uba3, pcDNA-HA-Uba3C216S, pcDNA-HA-Ubc12, and pcDNA-HA-Ubc12C111S was described previously (25). The pcDNA-HA-ubiquitin was kindly provided by Y. Xiong (61). The pCMV (cytomegalovirus)-GFP was purchased (Promega Corp., Madison, WI).

Cell Lines
The human cervical carcinoma cell line, HeLa, and the breast cancer cell line, MCF-7 were purchased from ATCC (Manassas, VA). HeLa cells were maintained in MEM with 2 mM L-glutamine, 1.5 g/liter sodium bicarbonate, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, 50 U/ml penicillin, 50 µg/ml streptomycin, and 10% FBS. MCF7 cells were maintained in MEM with 2 mM L-glutamine, 0.1 mM nonessential amino acids, 50 U/ml penicillin, 50 µg/ml streptomycin, 6 ng/ml insulin, and 10% FBS. Before experiments involving in transient transfection and hormone treatment, cells were cultured in hormone-free medium (phenol red-free MEM with 3% csFBS) for 3 d.

Transient Transfection Assays
HeLa cells were cultured in hormone-free medium for 3 d and transfected with equal amount of total plasmid DNA (adjusted by corresponding empty vectors) by using LipofectAMINE Plus Reagent according to the manufacturer’s guidelines. Five hours later, the DNA/LipofectAMINE mixture was removed and cells were cultured in hormone-free medium. All cells were also cotransfected with pCMV-GFP as internal control to correct for transfection efficiency and SDS-PAGE loading.

Stable Transfection
MCF7 cells were transfected with pcDNA-HA-Ubc12C111S or empty vector by using LipofectAMINE Plus Reagent and selected in growth medium containing 0.5 mg/ml geneticin for 3 wk. Drug-resistant colonies were chosen and expanded in growth medium containing 0.3 mg/ml geneticin. The expression of HA-Ubc12C111S in the stable cell lines (MCF7/C111S) was detected by Western blotting with anti-HA antibody. Geneticin-resistant clones from vector transfectants (MCF7/Vec) were pooled, maintained in growth medium containing 0.3 mg/ml geneticin, and used as control cells.

Preparation of Cell Extracts and Immunoblotting
Whole cell extracts were prepared by suspending cells (~2 x 106) in 0.1 ml of ice-cold lysis buffer (25 mM HEPES, pH 7.5; 0.3 M NaCl; 0.2% sodium dodecyl sulfate; 0.5% sodium deoxycholate; 0.2 mM EDTA; 0.5 mM dithiothreitol; 0.1% Triton X-100; 10 µl protease inhibitor cocktail set III). After 15 min on ice, extracts were sonicated (3 x 10 sec), insoluble material was removed by centrifugation (15 min at 12,000 x g), and protein concentration in the supernatant was determined using the Bio-Rad Laboratories, Inc. protein assay kit. The protein extracts were mixed with 1/4 vol of 5x electrophoresis sample buffer and boiled for 5 min at 90 C. Protein extract (50 µg per lane) was then fractionated by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and probed with antibodies. Primary antibody was detected by horseradish peroxidase-conjugated second antibody and visualized using enhanced SuperSignal West Pico Chemiluminescent Substrate. The band density of exposed films was evaluated with ImageJ software (http://rsb.info.nih.gov/ij/).

Immunoprecipitation
For immunoprecipitation, 500 µg whole cell extract was diluted to protein concentration of 1 µg/µl using PBS containing protease inhibitor cocktail and incubated with 5 µl antirabbit IgG and 20 µl protein G-agarose beads for 1 h at 4 C. After centrifugation at 12,000 x g for 15 sec, the precleared supernatants were incubated with 5 µl anti-ER antibody overnight at 4 C, followed by another 1-h incubation with 30 µl protein G-agarose beads. The beads were then pelleted by brief centrifugation, washed three times with PBS and once with PBS containing 0.4 M NaCl, and resuspended in 30 µl SDS-PAGE loading buffer for SDS-PAGE and Western blotting.

Cell Proliferation Assays
To assess the effects of estradiol, ICI 182,780, or 4-OHT on cell proliferation, cells (1000/well) were plated in 96-well dishes in hormone-free medium for 3 d before drug exposure. For time-response analysis, cell numbers were determined by MTT assay (62) at indicated times after drug treatment; and for dose-response analysis, cell number was determined by MTT assay at d 7.


   FOOTNOTES
 
The authors gratefully acknowledge the following agencies for supporting this work: NIH Grants CA-74748 (to K.P.N.) and HD-37025 (to R.M.B.); the U.S. Army Medical Research Acquisition Activity, Award Numbers DAMD 17-02-1-0418 and DAMD17-02-1-0419 (to K.P.N.); American Cancer Society Research Grant TBE-104125 (to K.P.N.), the Walther Cancer Institute (to M.F.); and Hoosiers Outrun Cancer/Bloomington Hospital Foundation (to K.P.N.).

Abbreviations: APP-BP1, Amyloid precursor protein-binding protein; AR, androgen receptor; csFBS, charcoal-stripped FBS; E2, ubiquitin conjugation enzyme; E3, ubiquitin ligase; ER, estrogen receptor; estradiol, 17ß-estradiol; FBS, fetal bovine serum; HA, hemagglutinin; GAPDH, glyceraldehyde phosphate dehydrogenase; GFP, green fluorescent protein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NEDD8, neural precursor cell-expressed developmentally down-regulated; 4-OHT, 4hydroxytamoxifen; PR, progesterone receptor; Uba, ubiquitin-activating enzyme; Ubc, ubiquitin-conjugation enzyme.

Received for publication September 13, 2002. Accepted for publication December 11, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   ERα
Coregulators:   Uba3
Ligands:   17β-Estradiol  |  4-Hydroxytamoxifen



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