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Molecular Endocrinology, doi:10.1210/me.2004-0048
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Molecular Endocrinology 18 (6): 1396-1410
Copyright © 2004 by The Endocrine Society

A Cytoplasmic Substrate of Mitogen-Activated Protein Kinase Is Responsible for Estrogen Receptor-{alpha} Down-Regulation in Breast Cancer Cells: The Role of Nuclear Factor-{kappa}B

Jamie N. Holloway, Shalini Murthy and Dorraya El-Ashry

Lombardi Cancer Center (J.N.H.), Georgetown University, Washington, D.C. 20007; and University of Michigan Comprehensive Cancer Center (S.M., D.E.-A.), Ann Arbor, Michigan 48109-0640

Address all correspondence and requests for reprints to: Dorraya El-Ashry, University of Michigan Comprehensive Cancer Center, 1150 West Medical Center Drive, MSRB III, Room 5220B/5323, Ann Arbor, Michigan 48109-0640. E-mail: elashryd{at}umich.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen receptor {alpha} (ER{alpha}) negative breast tumors often present with enhanced expression and/or activation of growth factor receptors, resulting in increased growth factor signaling and hyperactivation of MAPK (ERK1 and ERK2). We have pre-viously shown that ER{alpha}(+) MCF-7 cells with elevated growth factor signaling lose expression of ER{alpha} without any ligand-independent transcriptional activation, and this is a reversible effect attributable to ERK1/2 hyperactivation. Here, we show that down-regulation of ER{alpha} is not mediated by a specific ERK-1 vs. ERK-2 substrate. Despite up-regulated activator protein-1 activity in response to ERK1/2 activation, and in ER{alpha}(–) and hormone-independent breast cancers, we find that increased activator protein-1 activity is not responsible for ER{alpha} down-regulation. Interestingly, our findings implicate a cytoplasmic substrate of ERK1/2. However, RSK1, the best-characterized cytoplasmic ERK1/2 substrate, does not down-regulate ER{alpha} in our models. On the other hand, inhibition of nuclear factor-{kappa}B (which is linked to chemoresistance in cancer in general and has elevated activity in hormone-independent and ER{alpha}– breast cancer) significantly enhances ER{alpha} activity, suggesting that indirect elevation in nuclear factor-{kappa}B activity (due to hyperactive ERK1/2) is at least partially responsible for ER{alpha} down-regulation in these cell line models.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
BREAST CANCER CAN present as estrogen receptor {alpha} (ER{alpha}) positive (+) or negative (–). The presence or absence of ER{alpha} is a key prognostic feature of this disease: ER{alpha}+ tumors have a better prognosis and respond to hormonal therapy (1), whereas ER{alpha}– tumors have overall a worse prognosis and are resistant to hormonal therapy (2, 3). ER{alpha}+ tumors can progress over time and after antiestrogen therapy to ER{alpha}– tumors. For example, in patients with ER{alpha}+ primary tumors who relapse after adjuvant tamoxifen therapy, 50% of the recurrent tumors lack ER{alpha} expression. About a third of metastatic tumors that initially respond to tamoxifen subsequently develop resistance and lose ER{alpha} expression over this period of time (4, 5). Progression to an ER{alpha}– phenotype typically involves the constitutive overexpression of growth promoting genes that are normally regulated by estrogen, thereby leading to a loss of estrogen dependence, resistance to antiestrogens, and a more aggressive phenotype overall. It is also possible that ER{alpha}– tumors develop de novo. Immunohistochemical studies of normal breast tissue suggested that approximately 6–12% of the ductal epithelial cells are ER{alpha}+ (6, 7), although some studies with more sensitive antibodies indicate that the actual number is higher (8). Interestingly, the proliferating ductal epithelial cells in the normal breast do not express ER{alpha} (6, 8, 9, 10, 11), suggesting that ER{alpha}– cells give rise to ER{alpha}– tumors. Whether the ER{alpha}– phenotype is acquired or de novo, the lack of ER{alpha} expression denotes a more aggressive phenotype as well as resistance to anti-estrogens, and as a result, precludes the use of tamoxifen, relegating patients to more toxic chemotherapies (2, 3).

ER{alpha}– tumors and cell lines often overexpress certain growth factor receptors such as the EGFR [epidermal growth factor (EGF) receptor] and c-erbB-2. EGFR and c-erbB-2 overexpression are also important prognostic indicators in breast cancer, independent of their inverse correlation with ER{alpha} expression (12, 13, 14, 15, 16, 17). This increased growth factor receptor expression and/or activation correlates with increased MAPK activity, both in tumors and in cell lines (12, 18, 19, 20). In addition to overexpression and/or constitutive activation of growth factor receptors (with the resulting hyperactivation of MAPK), many hormone-independent and ER{alpha}– tumors also have elevated activator protein (AP)-1 activity (21, 22). Elevated activity of AP-1 and related family members has also been implicated in breast tumor progression in xenograft models: c-jun overexpression in MCF-7 cells results in a hormone-resistant, tumorigenic phenotype (23).

Another transcription factor that might be linked to hormone-independent breast cancer, as well as elevated growth factor signaling, is nuclear factor-{kappa}B (NF-{kappa}B). NF-{kappa}B activity is elevated in hormone-independent breast cancer (24), and is implicated in enhanced cell survival and chemoresistance in cancer (25, 26, 27). NF-{kappa}B exists in the cytoplasm in the form of a complex with I{kappa}B (inhibitor of {kappa}-B). Cytokines, chemokines, and intracellular stress lead to the phosphorylation of I{kappa}B by IKK (I{kappa}B kinase), releasing NF-{kappa}B, which can then translocate into the nucleus and modulate the transcription of target genes (reviewed in Ref.28). Although MAPK does not directly phosphorylate I{kappa}B or activate IKK, there is evidence to show that hyperactivation of MAPK leads to elevated NF-{kappa}B-mediated transcriptional activity through induction of an autocrine factor, most likely heparin-binding EGF (HB-EGF) (29, 30). These data suggest that our model cell lines (with elevated growth factor signaling pathways) might have elevated NF-{kappa}B activity and that this might be involved in the interactions between up-regulated growth factor signaling and decreased ER{alpha} expression.

We have previously used stable overexpression of various growth factor signaling components in ER{alpha}+ MCF-7 breast cancer cells to study the interaction between ER{alpha} signaling and growth factor receptor signaling in breast cancer (31, 32, 33, 34). Using cell lines that overexpress constitutively active forms of Raf-1, MEK1, or c-erbB-2 and ligand-activatable EGFR, we have shown that the resultant hyperactivation of MAPK (ERK1/2) activity through these signaling pathways leads to the down-regulation of ER{alpha}. We have further shown that this down-regulation is not a consequence of ligand-independent ER{alpha} activation, and that it is reversible in vitro. Abrogation of ERK activity using either pharmacologic inhibitors or dominant-negative ERK (dnERK) constructs reverses the ER{alpha} down-regulation and restores ER{alpha} activity (34). The objective of this study is to further elucidate the mechanisms by which elevated ERK signaling leads to ER{alpha} down-regulation. First, using dnERK1 and dnERK2, we show that the ERK substrate(s) responsible for ER{alpha} down-regulation is a common ERK1/ERK2 substrate and not specific to either ERK isoform. Next, the potential role of two key ERK substrates, AP-1 and RSK1, in ER{alpha} down-regulation was analyzed. Overexpression and increased activation of AP-1, a transcription factor, has been correlated with hormone independence and the ER{alpha}-negative phenotype (21, 35). RSK is a kinase responsible for the activation of various transcription factors (including ER{alpha}) and is also involved directly in chromatin remodeling (reviewed in Ref.36). Although our hyperactive MAPK cell lines have significantly increased AP-1 activity relative to parental MCF-7 cells, inhibition of this AP-1 activity using a dominant-negative jun does not reverse the ER{alpha} down-regulation. Similarly, inhibition of increased RSK activity using a dominant-negative RSK1 (dnRSK1) does not restore ER{alpha} expression. Instead, dnRSK1 reduces ligand-induced ER{alpha} activation in our control cells, further supporting the possibility that phosphorylation of ER{alpha} by RSK enhances ER{alpha} transactivation (37). Interestingly, studies with an ERK2 deletion construct that selectively abrogates nuclear ERK1/2 activities show that the ERK substrate responsible for ER{alpha} down-regulation is located in the cytoplasm. Finally, we show that these cell lines have elevated NF-{kappa}B activity compared with parental MCF-7 cells, and this elevation in NF-{kappa}B activity is attributable to enhanced growth factor signaling through ERK1/2. Inhibiting this NF-{kappa}B activity either pharmacologically, or through the expression of a constitutively active I{kappa}B [(ca)I{kappa}B], partially restores ER{alpha} activity and expression in these cells. Our findings suggest a role for cytoplasmic substrates of MAPK in ER{alpha} down-regulation in breast cancer and further support a role for MAPK-induced NF-{kappa}B activity in this down-regulation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ER{alpha} Down-Regulation Due to Hyperactive MAPK Is Not Specifically Attributable to ERK1 or 2
Previous work from our lab has shown that hyperactivation of MAPK results in the down-regulation of ER{alpha} protein and message and that this down-regulation is reversible through the abrogation of MAPK (ERK 1/2) signaling, either via pharmacologic inhibition or through expression of dnERK1 and dnERK2 (34). Although ERK1 and ERK2 share many substrates, they have some isoform-specific substrates as well (38). Therefore, our first step in the elucidation of a substrate responsible for ER{alpha} down-regulation in breast cancer cells was to determine whether the MAPK-induced down-regulation was a specific effect of the hyperactivation of either ERK1 or ERK2, or whether signaling through either MAPK would down-regulate ER{alpha}. Cell line models with drastically reduced ER{alpha} expression (described previously in Ref.34) were obtained by the stable transfection and overexpression of various signal transduction factors into ER{alpha}(+) MCF-7 breast cancer cells. These signaling factors were a constitutively active c-Raf-1 [yielding Raf14c cells or (ca)Raf cells] (39), a constitutively active MEK-1 construct (40) [yielding MEK15c or (ca)MEK cells], a wild-type EGFR that can be activated by ligand [MCE5 or EGFR(+) cells] (32), or a wild-type c-erbB-2 [a clone with constitutively high levels of autophosphorylation and constitutive downstream signaling, MB3 or erbB2(+) cells] (33). All of these cell lines grow continuously in the absence of estrogen, have very high levels of MAPK activity (consistent with the levels of MAPK activity found in ER{alpha}– breast cancer cell lines) and express extremely low levels of ER{alpha} when compared with control transfected MCF-7 cells (co-MCF7) (34). Each cell line expresses between 4 and 20 fmol of ER{alpha}/mg protein, a significant reduction when compared with the control transfected cell lines (Table 1Go), which exhibit about 120 fmol/mg protein when growing in the continuous presence of estrogen (co-MCF7) or about 400 fmol/mg protein when growing in the continuous absence of estrogen (co-MCF7/s). By selecting single clones of multiple cell lines, all with hyperactive MAPK but generated in different ways, we have substantially reduced the possibility that the reversible down-regulation of ER{alpha} seen in these lines is a chance effect due to clonal variation.


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  		Table 1. ER{alpha} Expression in MCF-7-Derived Cells with Hyperactive MAPK

 
To determine whether the ERK substrate responsible for ER{alpha} down-regulation is specific to ERK-1 or ERK-2, cells were transiently cotransfected with either an empty vector, dnERK1 (41), dnERK2 (42), or both, in addition to a luciferase (luc) reporter construct (Fig. 1Go). The estrogen response element (ERE)-luc construct was derived as previously described by the removal of the glucocorticoid response element from the mouse mammary tumor virus (MMTV) promoter. It was replaced with two tandem EREs and then inserted upstream of the luciferase gene. The NON-luc reporter (a critical control that corrects for any nonspecific activation due to the remaining elements of the MMTV promoter) is identical with the ERE-luc, except that the ERE sequences have been scrambled to produce a nonsense sequence. We have previously shown, both by ligand binding assays and by immunohistochemistry, that transfection of a combination of dnERKs 1 and 2 is sufficient to return ER{alpha} expression, and through transient transfection assays with ERE-luc and NON-luc that this reexpressed ER{alpha} is functional. Importantly, we found that receptor expression, as measured by ligand binding assay or by immunohistochemistry, correlated very well with receptor function as measured by the transient transfection assays with ERE-luc (34), confirming that receptor activity mirrors receptor expression. In addition to assessing ER expression by ligand binding assay, many studies were performed using either immunohistochemistry or Western blotting (34). Because all of these latter studies used ER{alpha}-specific antibodies, and ER{alpha} expression measured in this way fully correlated with ER activity by ERE-luc assay, it is presumed that we are looking at ER{alpha}-specific effects. Furthermore, the dnERK constructs do not affect ERE-luc activity in the co-MCF7 cells, either in the absence or presence of estrogen (Fig. 1AGo), again indicating that modulation of ERK activity does not affect the level of activation of preexisting receptor. In all four cell line models with hyperactive MAPK [(ca)Raf in Fig. 1BGo, (ca)MEK in 1C, erbB2(+) in 1D, and ligand-activated EGFR(+) in 1E], expression of either dnERK is sufficient to restore ER{alpha} to levels comparable with the combination of dnERKs 1 and 2 that we previously described as able to return ER{alpha} expression. Although any one dnERK alone was perhaps a little less effective at restoring ER{alpha} function compared with the combination of both dnERKs, both dnERK-1 and dnERK-2 restored ER{alpha} function to levels that would be physiologically relevant. Parallel transfection of the NON-luc does not reveal nonspecific promoter activation, but rather that the ER{alpha} activity measured is a direct result of estrogen-induced ER{alpha} signaling. Because both dnERKs were sufficient to return ER{alpha} to comparable levels, the ERK substrate responsible for ER{alpha} down-regulation is unlikely to be specific to either one.



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  		Fig. 1. Down-Regulation of ER{alpha} Is Not Mediated Exclusively by Either ERK1 or ERK2

A–E, Co-MCF7, (ca)Raf, (ca)MEK, erbB2(+), and EGF-treated EGFR(+) cells were transiently cotransfected with 1.25 µg total dnERK constructs (either dnERK1 alone, or dnERK2 alone, or a 1:1 combination of both dnERKs) and 0.75-µg luciferase reporter constructs and treated with control or estrogen containing media. Fold induction in ERE-luc activity after estrogen treatment is used as a measure of ER{alpha} activity. The NON-luc construct is an identical plasmid, except that the EREs are scrambled to result in a nonsense sequence. Experiments are representative of at least three individual experiments, each performed in triplicate. Error bars represent SEM.

 
Neither AP-1 nor RSK Are Responsible for ER{alpha} Down-Regulation Due to Hyperactive MAPK
AP-1 plays a prominent role in the transcriptional activation of many genes. Furthermore, AP-1 activity is more frequently elevated in ER{alpha}– breast cancer (21, 35, 43, 44), and c-jun overexpression confers a hormone-independent, ER{alpha}–, tumorigenic phenotype on MCF-7 cells in animal models (23). In addition, the expression of AP-1 family members such as fra-1, fra-2, and some jun family members are specifically induced by MAPK (45, 46). Thus, it would be expected that AP-1 activity would be elevated in systems with hyperactive MAPK. Therefore, to determine the role of elevated AP-1 activity in ER{alpha} down-regulation in these MCF-7 models with hyperactive MAPK, a dominant-negative jun construct (TAM67) (47) was used to inhibit AP-1 activity. AP-1 molecules consist of a dimer composed of a jun family member and either another jun family member or a fos family member. TAM67 acts as a dominant negative by forming inactive homo- and heterodimers with jun and fos family members in the cell, thereby eliminating AP-1 mediated transcriptional activity.

The first panel of Fig. 2Go represents the transient cotransfection of TAM67 or its parental vector, pCDNA3, along with the AP-1-luc reporter in our model cell lines, as well as in control transfected MCF-7 cells (co-MCF7). All of these cell lines show elevated AP-1 activity (relative to co-MCF7), which is inhibited 50–75% by the TAM67 construct, reducing the AP-1 activity in these hyperactive MAPK cells to levels comparable to ER{alpha}+ co-MCF7 cells (Fig. 2AGo). If AP-1 were playing a central role in the down-regulation of ER{alpha} in these breast cancer cells, then the expression of TAM67 (and therefore the specific abrogation of AP-1 activity) in the low ER{alpha} model cells should result in the restoration of ER{alpha} activity and expression. However, transfection of TAM67 did not restore ER{alpha} levels to any significant extent in any of the cell lines (Fig. 2Go, B–E). In all of these cell lines, expression of TAM67 did not result in estrogen induction that was significantly different from the vector control, despite AP-1 activity being consistently reduced. Cotransfection of dnERKs, on the other hand, fully restored ER{alpha} function in all these cell lines. These data indicate that elevated AP-1 activity does not play a role, either directly or indirectly, in the down-regulation of ER{alpha} that results from the hyperactivation of MAPK.



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  		Fig. 2. Down-Regulation of ER{alpha} Is Not Mediated by AP-1 Activity

A, Expression of TAM67 inhibits AP-1 activity. Cells were transiently transfected with 1.25 µg TAM67 or vector control DNA and 0.75 µg AP-1-luc reporter construct, and basal AP-1 activity was determined. Activity of CMV-luc transfected into the cells in the same experiment was used for normalization and calculation of relative AP-1 activity. The black bars represent basal AP-1-luc activity, whereas the white bars represent AP-1-luc activity upon cotransfection of the TAM67 plasmid. The first bar shows AP-1 activity in co-MCF7 cells. The next four pairs show AP-1 activity in (ca)Raf, (ca)MEK, erbB2(+), and EGF-treated EGFR(+) cells respectively. B–E, (ca)Raf, (ca)MEK, erbB2(+), and EGF-treated EGFR(+) cells were transiently cotransfected with 1.25 µg TAM67 (dnjun construct) and 0.75 µg ERE-luc or NON-luc reporter constructs and were treated with control or estrogen containing media. Fold induction in ERE-luc activity after estrogen treatment is used as a measure of ER{alpha} activity. Experiments are representative of at least three individual experiments, each performed in triplicate. Error bars represent SEM.

 
RSK is a cytoplasmic substrate of MAPK that translocates to the nucleus upon activation, activates several transcription factors, and regulates chromatin structure through the phosphorylation of histones. Thus, increased activation of RSK1 could lead to induction of a factor that represses ER{alpha} expression. On the other hand, RSK1 has also been reported to phosphorylate ER{alpha} directly on serine 167, leading to ligand-independent activation of ER{alpha} (37). These data indicate that RSK1 might play a role in enhancing ER{alpha} activity, either in a ligand-dependent or ligand-independent fashion. In our model cell lines, RSK1 expression is unchanged in response to elevated MAPK activity, but its activation correlates with MAPK activity, and therefore is high in these model cell lines (Fig. 3AGo).



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  		Fig. 3. Down-Regulation of ER{alpha} Is Not Mediated by RSK1

A, RSK1 activity correlates with MAPK activity in breast cancer cells. Whole cell lysates were prepared from cells in normal culture conditions grown to approximately 80% confluence. EGFR(+) cells were treated or not with 10 ng/ml EGF for 10 min. Western blots were performed on 5 µg of total protein for phospho-RSK1 and phospho-MAPK. A Western blot for RSK1 is shown as a loading control. B–F, co-MCF7, (ca)Raf, (ca)MEK, erbB2(+), and EGF-treated EGFR(+) cells were transiently cotransfected with 1.25 µg dnRSK1 construct and 0.75 µg luciferase reporter constructs and were treated with control or estrogen containing media. Fold induction in ERE-luc activity after estrogen treatment is used as a measure of ER{alpha} activity. Experiments are representative of at least three individual experiments, each performed in triplicate. Error bars represent SEM.

 
To determine the effects of elevated RSK1 activity on ER{alpha} in these model cell lines, a dnRSK1 construct was obtained (48). Because RSK1 had been reported to activate ER{alpha}, the effects of dnRSK1 on ER{alpha} activity in co-MCF7 cells was first examined. Indeed, cotransfection of dnRSK1 into ER{alpha}+ co-MCF7 cells results in reduced estrogen-induced transcriptional activation (Fig. 3BGo), indicating the dnRSK1 has inhibitory activity against RSK1. If RSK1 hyperactivation were responsible for the down-regulation of ER{alpha}, then expression of dnRSK1 in the low ER{alpha} model cell lines would result in the restoration of ER{alpha} activity to significantly higher levels (comparable to those seen with dnERKs). However, transient transfection of dnRSK1 into our model cells was not able to restore ER{alpha} to levels comparable with dnERK transfection, indicating that RSK1 is not responsible for the MAPK-induced down-regulation of ER{alpha} (Fig. 3Go, C–F). In fact, in co-MCF-7 cells, the estrogen-inducible ER{alpha} activity even decreases slightly. These data collectively indicate that hyperactivation of RSK1 is not mediating the down-regulation of ER{alpha} induced by hyperactive MAPK.

A Cytoplasmic Substrate of MAPK Is Responsible for ER{alpha} Down-Regulation
MEK acts as a cytoplasmic anchor for MAPK, but upon MEK activation by Raf, MAPK is activated and released into the cytoplasm, where it can activate cytoplasmic substrates such as RSK1. In addition, active MAPK translocates to the nucleus and phosphorylates nuclear substrates such as elk (reviewed in Ref.49). A mutant ERK2 construct lacking the region that associates with MEK (ERK2{Delta}19–25) (50) was used to determine the role of cytoplasmic vs. nuclear MAPK activity in ER{alpha} down-regulation. Deletion of the MEK association region causes the construct to be constitutively localized in the nucleus, where it cannot be activated by MEK. As a result, this construct prevents any MAPK activity in the nucleus by binding to (and therefore blocking docking sites on) nuclear MAPK substrates. However, endogenous cellular MAPK can still be activated in the cytoplasm and can continue to activate cytoplasmic substrates. Essentially, this construct functions as a dominant negative in the nucleus without affecting cytoplasmic MAPK signaling (50). As would be expected, nuclear MAPK activity (measured by Elk activation) is elevated relative to co-MCF-7 in our model cell lines with hyperactive MAPK (Fig. 4AGo). The ERK2{Delta}19–25 construct greatly reduces this elevated elk activation to levels comparable to co-MCF-7, i.e. it greatly reduces nuclear MAPK activity. If a nuclear substrate were responsible for the down-regulation of ER{alpha}, then expression of the ERK2{Delta}19–25 construct in these ER{alpha}– models would be expected to block the activation of that substrate by MAPK, and thus lead to the restoration of ER{alpha} activity and expression. However, expression of the ERK2{Delta}19–25 construct did not lead to the restoration of ER{alpha} in any of the cell lines (Fig. 4Go, B–E); in fact, it was unable to increase ER{alpha} activity to above basal levels in all cases and was not able to return ER{alpha} activity to the level obtained with dnERK transfection. This indicates that a cytoplasmic substrate of MAPK is responsible for the down-regulation of ER{alpha} in these breast cancer cell lines.



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  		Fig. 4. Down-Regulation of ER{alpha} Is Mediated by a Cytoplasmic Substrate of MAPK

A, ERK2{Delta}19–25 represses activity of a nuclear MAPK substrate. Cells were transiently transfected with 0.625µg ERK2{Delta}19–25 or vector control DNA, 0.625 µg pFAelk, and 0.75 µg pFA-luc reporter construct, and basal Elk activity was determined. Activity of CMV-luc transfected into the cells in the same experiment was used for normalization and calculation of relative Elk activity. The black bars represent basal Elk activity, whereas the white bars represent Elk activity upon cotransfection of the ERK2{Delta}19–25 plasmid. The first bar shows Elk activity in co-MCF-7 cells; the next four pairs show Elk activity in in (ca)Raf, (ca)MEK, erbB2(+), and EGF-treated EGFR(+) cells respectively. B–E, (ca)Raf, (ca)MEK, erbB2(+), and EGF-treated EGFR(+) cells were transiently cotransfected with 1.25 µg ERK2{Delta}19–25 construct and 0.75 µg luciferase reporter constructs and were treated with control or estrogen-containing media. Fold induction in ERE-luc activity after estrogen treatment is used as a measure of ER{alpha} activity. Experiments are representative of at least three individual experiments, each done in triplicate. Error bars represent SEM.

 
The Role of NF-{kappa}B in ER{alpha} Down-Regulation by Hyperactive MAPK
NF-{kappa}B activity is elevated in hormone-independent and ER-negative breast tumors (24), and hyperactivation of MAPK leads to enhanced NF-{kappa}B activity through induction of autocrine factors such as HB-EGF (29, 30, 51, 52). We therefore asked whether NF-{kappa}B activity is elevated in MCF-7 breast cancer cells with elevated MAPK activity. As shown in Fig. 5AGo, NF-{kappa}B activity is about 5-fold higher than parental MCF-7 in all of our model cell lines. This elevated NF-{kappa}B activity is attributable to hyperactivation of MAPK because NF-{kappa}B activity is returned to normal levels (basal levels in co-MCF7 cells) by dnERKs 1 and 2 (Fig. 5AGo). The elevated NF-{kappa}B activity can also be inhibited by Parthenolide [a specific pharmacological inhibitor of NF-{kappa}B (53)] and through cotransfection of (ca)I{kappa}B (which acts as a dominant negative for NF-{kappa}B, obtained from Upstate Biologicals, Charlottesville, VA). Both (ca)I{kappa}B and Parthenolide, at the usual dose of 2 µM, result in decreased NF-{kappa}B activity to levels observed in co-MCF7 cells. Neither Parthenolide nor I{kappa}B alter MAPK activity (data not shown); nor does inhibition of NF-{kappa}B activity alter ERE-luc activity in the co-MCF7 cells (Fig. 5BGo). Interestingly, inhibition of elevated NF-{kappa}B activity in the hyperactive MAPK cell lines with either Parthenolide or (ca)I{kappa}B is about 40–50% as effective in restoring ER{alpha} activity as are the dnERKs (Fig. 5Go, C–F). Increasing doses of Parthenolide do result in further inhibition of NF-{kappa}B activity in the hyperactive MAPK cell lines (Fig. 6AGo); however, this further decrease in NF-{kappa}B activity does not result in further increases in ER{alpha} activity (Fig. 6BGo), suggesting that the 40–50% effectiveness in restoration of ER{alpha} is the maximal effect. Collectively, these data demonstrate that the indirect but specific elevation of NF-{kappa}B activity by hyperactive MAPK plays a role in ER{alpha} down-regulation in our cell lines, but it is not the only player. Overall, our data suggest that an additional cytoplasmic MAPK substrate(s), along with NF-{kappa}B, leads to ER{alpha} down-regulation in MCF-7 cells with hyperactive MAPK.



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  		Fig. 5. Elevated NF-{kappa}B Activity Is Partially Responsible for ER{alpha} Down-Regulation

A, Expression of dnERKs inhibits NF-{kappa}B activity. Cells were transiently transfected with 1.25 µg dnERks, (ca) I{kappa}B or vector control DNA and 0.75 µg NF-{kappa}B luciferase reporter construct, and basal NF-{kappa}B activity was determined. Activity of CMV-luc transfected into the cells in the same experiment was used for normalization and calculation of relative NF-{kappa}B activity. The white bars represent basal NF-{kappa}B-luc activity, the black bars represent NF-{kappa}B-luc activity in the presence of 2 µM Parthenolide, the light gray bars represent NF-{kappa}B-luc activity in the presence of dnERKs, and the dark gray bars represent NF-{kappa}B activity in the presence of (ca)I{kappa}B. B–F, co-MCF7, (ca)Raf, (ca)MEK, erbB2(+), and EGF-treated EGFR(+) cells were transiently cotransfected with 1.25 µg vector DNA or (ca)I{kappa}B, and 0.75 µg ERE-luc or NON-luc reporter constructs and were treated with control or estrogen-containing media. One set of plates was treated with 2 µM Parthenolide (DMSO at 1:10,000 was used as vehicle control). Fold induction in ERE-luc activity after estrogen treatment is used as a measure of ER{alpha} activity. Experiments are representative of at least three individual experiments, each performed in triplicate. Error bars represent SEM.

 


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  		Fig. 6. Elevated NF-{kappa}B Activity Has a Threshold Effect on ER{alpha}

A, Higher doses of Parthenolide inhibit NF-{kappa}B activity to levels below basal MCF-7. (ca)Raf cells were transfected with the NF-{kappa}B-luc reporter and luciferase activity was measure in the presence of increasing doses of Parthenolide (2–20 µM). NF-{kappa}B activity in co-MCF-7 cells was used for comparison. B, Higher doses of Parthenolide have no further effect on NF-{kappa}B activity in cells with hyperactive MAPK. (ca)Raf cells were transiently cotransfected with 1.25µg vector DNA and 0.75 µg ERE-luc or NON-luc reporter constructs and were treated with control or estrogen-containing media. Cells were treated with the indicated doses of Parthenolide (DMSO at 1:10,000 was used as vehicle control). Fold induction in ERE-luc activity after estrogen treatment is used as a measure of ER{alpha} activity. Experiments are representative of at least three individual experiments, each done in triplicate. Error bars represent SEM. Similar experiments with the (ca)MEK, erbB-2(+), and EGFR(+) cells yielded similar results (data not shown).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The inverse correlation between ER{alpha} and EGFR or c-erbB-2 overexpression/ hyperactivation in breast cancer is well established (54, 55, 56, 57). Because ER{alpha}– tumors display a more aggressive phenotype, have a poor prognosis, and do not respond to antiestrogen therapy, therapeutic targets enabling the restoration of ER{alpha} expression may provide beneficial treatment strategies. Elucidation of the mechanisms involved in the generation of the ER{alpha}– phenotype is crucial for the development of such therapies. We have previously demonstrated that hyperactivation of MAPK induced by enhanced expression and/or activation of EGFR or c-erbB-2 results in down-regulation of ER{alpha} expression, suggesting that elevated mitogenic signaling induced by overexpression/ hyperactivation of these growth factor receptors is directly responsible for generating the ER{alpha}– phenotype (34). Importantly, abrogation of the hyperactive MAPK restores ER{alpha} expression and activity in our model cell lines. To identify the MAPK substrate(s) responsible for down-regulation of ER{alpha} expression, the specificity of this response to ERK1 vs. ERK2 was first examined. It has been reported that although ERK1 and 2 share common substrates, they also display some differential substrate specificity (38). In our previous study, we used pharmacologic inhibitors or dnERK1 and dnERK2 combined to inhibit MAPK activity. To determine whether the inhibition of either ERK alone would restore ER{alpha} expression/activity, experiments were performed comparing the ability to restore ER{alpha} activity by dnERK1 alone or dnERK2 alone relative to the combination of dnERK1 and 2 (Fig. 1Go). In these experiments, it did not appear that there was a differential ERK substrate involved in down-regulating ER{alpha}. However, it is possible that these constructs may not be completely specific, i.e. that dnERK1 may inhibit ERK2 to some extent, and vice versa. Therefore, to completely rule out a differential ERK specificity, it would be necessary to knock out expression of each ERK separately using small inhibitory RNA or antisense technology.

We next focused on the role of AP-1 in the MAPK-induced down-regulation of ER{alpha} expression. Because ERKs induce the expression of various AP-1 family members (45, 58) and result in elevated AP-1 activity, all of our model cell lines displayed increased AP-1 activity. And although AP-1 has been identified as part of a complex that binds to an enhancer element in the ER{alpha} promoter (59), up-regulation of AP-1 activity is seen in ER{alpha}– and hormone-independent breast cancers (21, 35, 43, 60). In addition, stable overexpression of c-jun in MCF-7 cells leads to the formation of hormone-independent, ER{alpha}– tumors in nude mice (23). Collectively, these data seem to implicate elevated AP-1 activity in ER{alpha} down-regulation. However, the data presented here (Fig. 2Go) represent the first mechanistic study to investigate this correlation, analyzing whether inhibition of elevated AP-1 activity consequent to MAPK hyperactivation can restore ER{alpha} expression/activity. Results from these experiments show that inhibition of the elevated AP-1 activity in our cell line models is not enough to restore ER{alpha} expression, suggesting that MAPK-induced elevation in AP-1 activity does not, on its own, result in the loss of ER{alpha}. This is in apparent contradiction to what might be expected based on the report by Smith et al. (23) that overexpression of c-jun leads to the loss of ER{alpha} activity and expression in MCF-7 cells. The authors in that report suggest that this might occur either due to squelching of common cofactors required for both ER{alpha}-mediated and AP-1-mediated transcriptional activity (similar to that described in Refs.60, 61) or maybe through AP-1 activity negatively affecting ER{alpha} transcription, although the one AP-1 element identified so far on the ER promoter is a positive regulatory element (59). Because AP-1 molecules can be composed of both homo- and heterodimers, and different dimers have differential affinities for the consensus AP-1 sites, the exact composition of the AP-1 molecule(s) in each instance is likely to influence the final outcome on target genes. It is therefore possible that elevated AP-1 activity due to hyperactive MAPK in our MCF-7-derived cell lines may be due to dimers that markedly differ from those that result from c-jun overexpression, thereby accounting for the very different results reported earlier (23). In addition, AP-1 activity in our hyperactive MAPK cells is three to four times higher than in co-MCF7. Because we don’t know the degree to which AP-1 activity is enhanced by c-jun overexpression, it is possible that a threshold level of AP-1 activity is required to alter ER{alpha} expression and that this threshold level might be higher than what is seen in our cell lines. Our data here suggests that the negative effects of hyperactive MAPK on ER{alpha} transcription are not attributable to MAPK-induced AP-1 but might be due to some other ERK substrate(s), and that inhibiting AP-1 activity is not enough to relieve this effect of hyperactive MAPK.

Data obtained using the ERK2{Delta}19–25 construct (Fig. 4Go) confirm the fact that AP-1 does not play a role in MAPK-induced ER{alpha} down-regulation, as they indicate that nuclear substrates of MAPK, such as the AP-1 family members, are not responsible for ER{alpha} down-regulation. In fact, these data point to a cytoplasmic substrate of MAPK as responsible for ER{alpha} down-regulation in our model cell lines. Upon activation, this substrate may itself translocate to the nucleus to affect ER{alpha} transcription, or it may activate another molecule, either cytoplasmic or nuclear, which results in the loss of ER{alpha}.

We have preliminary data indicating that the down-regulation of ER{alpha} occurs via both transcriptional repression and induction of protein degradation (our unpublished data). Although these experiments clearly show that the MAPK effect on ER{alpha} is not a direct one in the nucleus, that is, interacting with the ER{alpha} promoter to result in transcriptional repression, they do not address what role, if any, MAPK may play in any proteasomal degradation of ER{alpha} protein that may be occurring. Several reports indicate that MAPK can directly phosphorylate ER{alpha} on serine residue 118 (62, 63), and if the phosphorylation of this residue leads to ubiquitination and degradation, then the abrogation of nuclear MAPK activity could also eliminate this method of down-regulation. However, this effect cannot be identified in a system with transcriptional repression of ER{alpha} smaller posttranslational effects on ER{alpha} levels would be masked unless the strong transcriptional repression is relieved. Therefore, to elucidate any role of MAPK hyperactivation in ER{alpha} degradation, it would be necessary to use tagged ER{alpha} constructs under the control of a heterologous promoter.

RSK1 is the best-characterized cytoplasmic substrate of MAPK. However, it is not the cytoplasmic effector that results in ER{alpha} down-regulation (Fig. 3Go). RSK1 is a kinase that is responsible for the activation of many substrates; among them are multiple transcription factors and several histones (reviewed in Ref.36). RSK1 was a good candidate as the MAPK substrate responsible for the down-regulation of ER{alpha} because we had previously established that a major component of ER{alpha} down-regulation in our model cells was transcriptional (our unpublished data). The fact that RSK1 itself alters chromatin structure through the phosphorylation of histones, and that many of its substrates are also involved in transcriptional regulation, led us to investigate it initially. In addition, the data provided by the ERK2{Delta}19–25 construct further encouraged us to pursue RSK1 because it is a cytoplasmic substrate of MAPK. Also of interest to us was the fact that RSK1 has been reported to phosphorylate ER{alpha} directly, on serine reside 167 (37), although in this case it resulted in ligand-independent activation of ER. Although we show that the activation of RSK1 through MAPK does not lead to ligand-independent activation of ER{alpha}, as the phosphorylation and results from (37) might suggest, it appears that RSK1 may play a role in the ligand-dependent activation of ER{alpha}. Abrogation of signaling through RSK1 did not restore estrogen-induced ER activity, and in fact, it seemed to decrease the ability of estrogen to induce ER{alpha} activity, at least in the control (MCF-7) cells. This implicates the phosphorylation of ER{alpha} on S167 as potentially important in traditional estrogen-dependent signaling. This result also confirms that the dnRSK1 construct is functional and has an effect on cell signaling. Therefore, a cytoplasmic substrate of MAPK other than RSK1 must be responsible for the down-regulation of ER{alpha} induced by hyperactivation of MAPK in these breast cancer cells.

It has been known for some time that NF-{kappa}B activity is elevated in hormone-independent and ER{alpha}– breast cancers (24). However, the correlation between NF-{kappa}B activity and MAPK was less clear—it is now evident that, whereas MAPK does not directly activate NF-{kappa}B (either through I{kappa}B phosphorylation, or through activation of IKK), hyperactive MAPK enhances NF-{kappa}B activity through induction of an autocrine factor, likely HB-EGF (30, 51, 52). Thus, hyperactivation of MAPK can indirectly lead to elevated NF-{kappa}B activity. We have shown here that our MCF-7-derived model cell lines all have high basal levels of NF-{kappa}B activity as a consequence of ERK1/2 hyperactivation (Fig. 5Go). Interestingly, inhibiting this level of NF-{kappa}B activity (bringing it down to levels normally seen in MCF-7 cells) partially restores ER{alpha} activity in these cell lines, indicating that loss of ER{alpha} is attributable to elevated NF-{kappa}B activity, along with (other) cytoplasmic substrate/s of ERK1/2. It is important to note that, under our experimental conditions, ERE-luc activity mirrors ER{alpha} expression [as we have shown earlier (34)], indicating that increase in ER{alpha} activity after NF-{kappa}B inhibition is attributable to restoration of ER{alpha} expression. This is further reinforced by the fact that NF-{kappa}B inhibitors have no effect on ERE-luc activity in co-MCF7 cells. Therefore, the enhanced ERE-luc activity in our hyperactive MAPK cell lines after NF-{kappa}B inhibition is due to increased ER{alpha} expression, rather than due to increased activation of preexisting receptor through modulation of coactivators and/or corepressors. We also find that further reducing NF-{kappa}B activity beyond the basal level inherent to MCF-7s does not increase ER{alpha} activity any further (Fig. 6Go), suggesting that down-regulation of ER{alpha} due to deregulation of NF-{kappa}B activity is a threshold effect. The basal level of activity seen in MCF-7 cells has no adverse effect on ER{alpha}; rather, it is the elevation in NF-{kappa}B activity beyond this limiting level, which contributes to down-regulating ER{alpha} expression. This threshold effect mimics that observed for hyperactivation of MAPK; that is, we had previously observed that neither the basal MAPK activity levels in MCF-7 cells nor the modest elevation of MAPK that occurs in estrogen-independent MCF-7 cells (those long-term adapted for growth in the absence of estrogen such as our co-MCF-7/s cells) has a detrimental effect on ER{alpha} expression levels; only hyperactivation to a very high level as seen in ER{alpha}– breast cancer cells resulted in down-regulation of ER{alpha} expression (34).

This and future studies further elucidating the interaction/s between hyperactive growth factor signaling and ER{alpha} expression in breast cancer could have substantial clinical impact in patients. Methylation of the ER{alpha} promoter is present at the time of diagnosis of breast cancer in about 25% of ER{alpha}– breast cancer cases (64), and in these tumors abrogation of MAPK activity would not be expected to return ER{alpha} expression (because demethylation of the ER{alpha} promoter would have to occur first). However, in patients with ER{alpha}– tumors without any hypermethylation of the promoter, a therapeutic regimen that combines a signal transduction inhibitor/a monoclonal antibody against a key signaling molecule, along with an antiestrogen, could prove beneficial. Iressa, which inhibits the kinase activity of EGFR, and Herceptin, a monoclonal antibody raised against c-erbB-2, can both be expected to reduce MAPK activity, and would be good candidates for such combination therapies. Although further studies are essential to show that these inhibitors of mitogenic signaling can restore ER{alpha} expression/function in breast tumors, coupling signal transduction inhibitors or monoclonal antibodies with antiestrogens could result in therapies that are better tolerated than standard chemotherapy for the treatment of ER{alpha}– breast cancer. In addition, once the MAPK substrate/s responsible for ER{alpha} down-regulation is identified, that could provide an additional drug target. The implication of elevated NF-{kappa}B activity in development of the ER{alpha}– phenotype also has clinical potential. High NF-{kappa}B activity is thought to contribute to resistance to chemotherapy and to radiation-induced apoptosis in cancers generally (25, 26, 27), as well as to influence tumor growth and metastasis through promoting cell survival and cell cycle entry (27, 65). Interestingly, it has been reported that cotreatment with NF-{kappa}B inhibitors enhances the paclitaxel sensitivity of MDA-MB-231 breast cancer cells (which have very high basal NF-{kappa}B activity) (66). Therefore, combinations of NF-{kappa}B inhibitors with standard chemotherapy/radiation might prove generally beneficial in breast and other cancers. Added to this, our data (establishing a role for NF-{kappa}B in the loss of ER{alpha} expression) make NF-{kappa}B a very attractive candidate for combination therapies in breast cancer.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture and Reagents
All cells with hyperactive MAPK were maintained in phenol red-free improved MEM (IMEM) (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% charcoal-stripped calf serum (CCS). Control transfected MCF-7 cells were maintained in IMEM supplemented with 10% fetal bovine serum. All cell lines used in this study have been described earlier (34); the (ca)MEK cells were generated by transfection of MCF-7 cells as described before (34, 39) using a constitutively active MEK construct [a generous gift from Dr. Natalie Ahn (40)]. After transfection, cells were grown in PRF-IMEM/10% CCS supplemented with 10 µg/ml gentamicin (Invitrogen Life Technologies). Hormone treatments after transfections were performed by supplementing the gentamicin containing CCS media with 17ß-estradiol (Sigma, St. Louis, MO), at a final concentration of 10–9 M; in the case of EGF-treated EGFR(+) cells, the medium was also supplemented with 10 ng/ml EGF (Upstate Biologicals). Parthenolide was obtained from Sigma, and stored as a 10,000x stock in dimethylsulfoxide (DMSO) at –20 C. Lipofectamine and PLUS reagent were obtained from Invitrogen. Cells were seeded in 75-cm2 T-flasks (Costar, Cambridge, MA) and grown in a forced air humidified incubator at an atmosphere of 5% CO2 and 37 C.

Plasmids
Luciferase reporter plasmids (pGLB-mERE and pGLB-mNON) were obtained by the insertion of an altered MMTV promoter containing a tandem repeat of a consensus ERE or a scrambled version of the same sequence instead of the glucocorticoid response element into the HindIII site of the pGLB basic plasmid (Promega Corp., Madison, WI) (67). The dnERK1 and dnERK2 constructs and parental vector pCep4L were kindly provided by Dr. Melanie Cobb (University of Texas Southwestern, Dallas, TX). The TAM67 construct was a gift of Dr. Powel Brown (Baylor College of Medicine Breast Center, Houston, TX). The ERK2{Delta}19–25 construct was a gift of Dr. Michael Weber (University of Virginia, Charlottesville, VA), and Dr. John Blenis (Harvard Medical School, Boston, MA) kindly provided the dnRSK1 and pRK7 (parental vector) constructs. pCep4L (dnERK parental vector), pCDNA3 (TAM67 and ERK2{Delta}19–25 parental vector), and pRK7 (dnRSK1 parental vector) were used as empty vector controls in transient transfections. The Stratagene PathDetect kit was the source of the AP-1-luc, NF-{kappa}B-luc, pFAelk, and pFRluciferase plasmids. The constitutively active I{kappa}B plasmid [(ca)I{kappa}B or I{kappa}B (S32A/S36A) in pUSE] was obtained from Upstate Biologicals.

Transfections
Cells were plated at approximately 60–70% confluence in Falcon six-well plates and allowed to attach overnight. They were then transfected using the LipoPlus method. Briefly, 100 µl serum-free IMEM per well was mixed with 4 µl Plus reagent and 2 µg total plasmid DNA [0.75 µg of each luciferase containing reporter plasmid, and 1.25 µg total of the effector plasmid(s)]. This mixture was incubated at room temperature for 15 min, combined with a mixture containing 100 µl serum-free IMEM and 4 µl Lipofectamine, and then incubated for an additional 15 min. This mixture was then added to the cells, which were incubated for 3 h at 5% CO2 and 37 C. After transfection, the cells were washed with PBS and incubated for 48 h in appropriate treatment media. Control transfection with a cytomegalovirus (CMV)-luc plasmid was performed in all experiments as a measure of transfection efficiency. All transfections in each experiment were performed in triplicate.

Luciferase Assays
Cells were washed twice with PBS and lysed in 200 µl of passive lysis buffer (Promega) with gentle agitation for 15 min at room temperature. Ten to 25 µl of lysate (actual volumes determined based on the amount needed to get CMV-luc readings in the middle of the linear range of the instrument) were used to measure luciferase activity using Promega’s Luciferase Assay system. The luciferase values (relative light units) were normalized for protein concentration. Values were corrected by the subtraction of relative light units per microgram of protein for the mock-transfected cells. Triplicates were averaged, and fold activation by estrogen was plotted; the error bars represent SEM. Results shown are representative of at least three experiments with similar results, each done in triplicate.

Ligand Binding Assays
Whole cell extracts were prepared from cells grown to 75–80% confluence as described before (34). Extracts were diluted to a concentration of 2 mg/ml, and incubated with 10 nM [3H]-17ß-estradiol in the presence and absence of x100 unlabeled estradiol for 16 h at 4 C. Dextran-coated charcoal was then used to adsorb free hormone and pelleted by centrifugation. Aliquots of the supernatant were counted in 10 ml of liquid scintillation fluid using a Beckman Coulter Inc. (Fullerton, CA) liquid scintillation counter. Receptor expression values were determined as described in Ref.68 and are expressed as femtomoles per milligram of protein. Assays were performed a minimum of three times on each cell line over time in culture.


   ACKNOWLEDGMENTS
 
We acknowledge Drs. Sofia Merajver, Francis Kern, and James Rae for critical review of this manuscript and thoughtful and helpful discussions.


   FOOTNOTES
 
This work was supported by Department of Defense Grant DAMD17-01-0247 (to J.N.H.); American Cancer Society Grant RSG-01-0330-01 (to D.E.A.); Susan G. Komen Foundation Grant BCTR2000565 (to D.E.A.); and National Institutes of Health-University of Michigan Cancer Center Support Grant 5 P30 CA46592.

J.N.H. and S.M. made equal contributions.

Abbreviations: AP, Activator protein; CCS, charcoal-stripped calf serum; CMV, cytomegalovirus; co-MCF-7, control transfected MCF-7; DMSO, dimethylsulfoxide; dnERK, dominant-negative ERK; dnSK1, dominant-negative RSK1; EGF, epidermal growth factor; EGFR, EGF receptor; ER{alpha}, estrogen receptor-{alpha}; ERE, estrogen response element; HB-EGF, heparin-binding EGF; I{kappa}B, inhibitor of {kappa}-B; IKK, I{kappa}B kinase; IMEM, improved MEM; luc, luciferase; MMTV, mouse mammary tumor virus; NF-{kappa}B, nuclear factor-{kappa}B.

Received for publication February 5, 2004. Accepted for publication March 22, 2004.


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

Nuclear Receptors:   ERα
Ligands:   17β-Estradiol



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