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[Cancer Research 66, 7991-7998, August 15, 2006]
© 2006 American Association for Cancer Research

Cell, Tumor, and Stem Cell Biology

Coregulation of Estrogen Receptor by ERBB4/HER4 Establishes a Growth-Promoting Autocrine Signal in Breast Tumor Cells

Yun Zhu1, Lacey L. Sullivan1, Sujit S. Nair3, Christopher C. Williams1, Arvind K. Pandey1, Luis Marrero2, Ratna K. Vadlamudi3 and Frank E. Jones1

1 Department of Biochemistry, Tulane University Health Sciences Center, Tulane Cancer Center; 2 Louisiana State University Health Sciences Center, Gene Therapy Program, The Morphology and Imaging Core Laboratory, New Orleans, Louisiana; and 3 Department of Obstetrics and Gynecology, University of Texas Health Sciences Center, San Antonio, Texas

Requests for reprints: Frank E. Jones, Department of Biochemistry, SL43, Tulane University Health Sciences Center, Tulane Cancer Center, 1430 Tulane Avenue, New Orleans, LA 70112-2699. Phone: 504-988-6585; Fax: 504-584-2739; E-mail: fjones{at}tulane.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although crosstalk between cell-surface and nuclear receptor signaling pathways has been implicated in the development and progression of endocrine-regulated cancers, evidence of direct coupling of these signaling pathways has remained elusive. Here we show that estrogen promotes an association between extranuclear estrogen receptor {alpha} (ER) and the epidermal growth factor receptor (EGFR) family member ERBB4. Ectopically expressed as well as endogenous ERBB4 interacts with and potentiates ER transactivation, indicating that the ERBB4/ER interaction is functional. Estrogen induces nuclear translocation of the proteolytic processed ERBB4 intracellular domain (4ICD) and nuclear translocation of 4ICD requires functional ligand-bound ER. The nuclear ER/4ICD complex is selectively recruited to estrogen-inducible gene promoters such as progesterone receptor (PgR) and stromal cell–derived factor 1 (SDF-1) but not to trefoil factor 1 precursor (pS2). Consistent with 4ICD-selective promoter binding, suppression of ERBB4 expression by interfering RNA shows that 4ICD coactivates ER transcription at the PgR and SDF-1 but not the pS2 promoter. Significantly, ERBB4 itself is an estrogen-inducible gene and the ERBB4 promoter harbors a consensus estrogen response element (ERE) half-site with overlapping activator protein-1 elements that bind ER and 4ICD in response to estrogen. Using a cell proliferation assay and a small interfering RNA approach, we show that ERBB4 expression is required for the growth-promoting action of estrogen in the T47D breast cancer cell line. Our results indicate that ERBB4 is a unique coregulator of ER, directly coupling extranuclear and nuclear estrogen actions in breast cancer. We propose that the contribution of an autocrine ERBB4/ER signaling pathway to tumor growth and therapeutic response should be considered when managing patients with ER-positive breast cancer. (Cancer Res 2006; 66(16): 7991-8)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Breast cancer is the most commonly diagnosed cancer in North American women and is second to lung cancer as the leading cause of cancer-related deaths in these women (1). The development and progression of breast cancer to a lethal metastatic disease involve, in part, the complex interplay between growth factor and steroid receptors. For example, patients with estrogen receptor {alpha} (ER)–positive tumors have a favorable prognosis and can be effectively treated with a selective estrogen receptor modulator such as tamoxifen. On the other hand, overexpression/amplification of oncogenic members of the epidermal growth factor receptor (EGFR) family, including EGFR, ERBB2, and ERBB3, in breast cancer inversely correlates with ER expression and these patients have poor prognosis (2, 3). When coexpressed with ER in preclinical models of breast cancer, ERBB2 promotes cellular proliferation and tamoxifen resistance (4). Interestingly, expression of the final EGFR family member to be identified, ERBB4, in primary breast tumors strongly correlates with ER expression (5–8). Furthermore, patients with tumors coexpressing ERBB4 and ER have fewer recurrences (9) and improved survival (10) when compared with patients expressing ER alone. These clinical observations imply a unique relationship between ERBB4 and ER signaling in breast cancer. Attempts to decipher the molecular contributions of ERBB4 signaling to the biology of ER(+) breast tumors, however, have led to the identification of several divergent ERBB4-regulated cellular responses. On the one hand, we and others have shown that ectopic ERBB4 expression in breast cancer cell lines induces cellular differentiation (11) and apoptosis (12). In contrast, overwhelming evidence suggests that ERBB4 contributes to estrogen-induced proliferation of ER(+) breast cancer cells (6, 13, 14).

The complexity of ERBB4 signaling in breast cancer may be explained by novel ERBB4 proteolytic processing events that result in the release of an independently signaling ERBB4 intracellular domain (4ICD). Ligand activation of ERBB4 results in tumor necrosis factor-{alpha} (TNF{alpha})–converting enzyme–mediated shedding of the ligand binding extracellular domain (15). The membrane-associated TNF{alpha}-converting enzyme cleavage product (ERBB4 m80) serves as a substrate for presenilin-dependent {gamma}-secretase activity and {gamma}-secretase cleavage of ERBB4 m80 generates an untethered cytosolic 4ICD (16–18). We have recently shown that 4ICD harbors an intrinsic nuclear localization signal and 4ICD functions as a nuclear chaperone for the STAT5A transcription factor (19). Furthermore, subsequent binding of 4ICD/STAT5A complexes to STAT5A target promoters results in transactivation of genes involved in lactation (19). In vivo models have revealed a critical role for coupled ERBB4/4ICD and STAT5A signaling during both mammary epithelial differentiation and lactation (20, 21). Alternatively, 4ICD induces cellular apoptosis by localizing to mitochondria (12, 18) and activating a cell death cascade involving proapoptotic members of the BCL-2 family (12). Significantly, cytosolic immunostaining of 4ICD in primary breast tumors was associated with tumor apoptosis (12). Interestingly, the transcriptional coregulation and apoptotic functions of ERBB4 require {gamma}-secretase processing to release 4ICD (18). Thus, a physiologic function for the membrane-associated ERBB4 holoreceptor remains to be described.

The strong association between ERBB4 and ER expression in breast cancer and the multiple functions of 4ICD led us to investigate the molecular mechanisms underlying ERBB4/ER coexpression and the effect of coupled 4ICD/ER signaling on breast cancer cells. Here we present evidence that 4ICD functions as an ER transcriptional coregulator, selectively binding with ER to gene promoters containing estrogen response elements (ERE). Furthermore, we show that ERBB4 is an estrogen-inducible gene thereby completing a functionally important ERBB4/ER autocrine signaling loop in breast cancer that regulates gene expression and promotes tumor cell proliferation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines. The T47D human breast cancer cell line was purchased from the American Type Culture Collection (Manassas, VA) and maintained according to the recommendations of the manufacturer. The MCF-7/B cell line stably overexpressing human BCL-2 and resistant to ERBB4 apoptotic activity (12) has been described elsewhere (22).

Plasmid constructs. The ERE luciferase reporter gene (ERE-luc) was a kind gift of Rakesh Kumar (M.D. Anderson, Houston, TX). The ERBB4, ERBB4-EGFP, ERBB4muNLS-EGFP (19), ERBB4V673I-EGFP (18), and 4ICD-Flag (12) expression vectors have been described elsewhere. The glutathione S-transferase (GST) vectors fused to ER domains A/B, C, D, E, and F (23) have been described elsewhere.

Luciferase reporter assay. MCF-7/B cells were seeded at 2 x 105 per well in a six-well plate and cultured for 24 hours in phenol red–free medium with 5% charcoal-stripped fetal bovine serum (FBS). Transfections and luciferase transcription assays were performed as described elsewhere (19) using 200 ng or 1 µg of ERE-luc as the reporter. At 24 hours posttransfection, indicated samples were incubated with 100 pmol/L 17-ß-estradiol (Sigma, St. Louis, MO) and/or 100 ng/mL ICI 182780 (Tocris, Ellisville, MO) for an additional 16 hours. Each sample was prepared in duplicate and the complete experiment was repeated at least thrice. Significant differences between data sets were determined using paired Student's t test.

Suppression of ERBB4 and ER expression. To suppress expression of endogenous ERBB4 or ER, T47D cells were transfected with erbB-4/HER4 siRNA SMARTpool or ESR1 siRNA SMARTpool, respectively, using siIMPORTER transfection reagent (Upstate Biotechnology, Charlottesville, VA) according to the instructions of the manufacturer. Cells similarly transfected with Nonspecific Negative Control Pool (Upstate Biotechnology) were analyzed as RNA interference (RNAi) controls.

Reverse transcription-PCR. T47D breast cancer cells were cultured in phenol red–free medium supplemented with 5% charcoal-stripped FBS for 48 hours, treated with 100 pmol/L 17-ß-estradiol for 16 hours, and total RNA was extracted using a RNeasy Mini Kit (Qiagen, Valencia, CA) according to the instructions of the manufacturer. First-strand cDNA was synthesized from 5 µg of total RNA in a volume of 40 µL using the Superscript First-Strand Synthesis System for reverse transcription-PCR (RT-PCR; Invitrogen, Carlsbad, CA) and 1 µL was amplified with 35 cycles of PCR using PCR SuperMix (Invitrogen) according to the instructions of the manufacturer. Oligonucleotide primers for progesterone receptor (PgR) PCR were forward 5'-CCATGTGGCAGATCCCACAGGAGTT and reverse 5'-TGGAAATTCAACACTCAGTGCCCGG; for stromal cell–derived factor 1 (SDF-1), forward 5'-GCCAGAGCCAACGTCAAGCATCTC and reverse 5'-GGCAAAGTGTCCAAAACAAAGCCC; for trefoil factor 1 precursor (pS2) PCR, forward 5'-GCGCCCTGGTCCTGGTGTCCAT and reverse 5'-GAAAACCACAATTCTGTCTTTCAC; for ERBB4 PCR, forward 5'-GAGAAGATTCTTGGAAACAGAG and reverse 5'-GGATGATCCATACTTGCCATG; and ß-actin PCR primers were QuantumRNA ß-actin Internal Standards (Ambion, Austin, TX).

Chromatin immunoprecipitation assay. Chromatin immunoprecipitation was done as previously described (19) with the following modifications. Chromatin was prepared from T47D breast cancer cells cultured in phenol red–free medium supplemented with 5% charcoal-stripped FBS for 48 hours and stimulated with 100 pmol/L 17-ß-estradiol for 1 hour. Fragmented chromatin was immunoprecipitated using antibodies directed against ERBB4 (Upstate) or ER Ab-1 (NeoMarkers, Fremont, CA) and amplified with 35 to 45 cycles of PCR using PCR SuperMix (Invitrogen). Oligonucleotide primers for the PgR promoter were forward 5'-TCTGCTGGCTCCGTACTGCGG and reverse 5'-GGCTTTGGGCGGGGCCTCCC; for the SDF-1 promoter region harboring an ERE half-site and associated SP-1 site, forward 5'-GAGCCTGAGAAGGTCAAAGG (nt –216 to –197; ref. 24) and reverse 5'-GCGCTTTAGAGGGGAGAGC (nt –17 to –35; ref. 24); for the pS2 promoter, forward 5'-GTTGTCAGGCCAAGCCTTTT and reverse 5'-GAGCGTTAGATAACATTTGCG; for ERBB4 ERE half-site I, forward 5'-GCTTTTATGGAAGAGAGGTGC and reverse 5'-CGTCTTCATGGAGCCTGTTA; for ERBB4 ERE half-site II, forward 5'-CATCACAGGACAAAGCCATC and reverse 5'-GCTTGAATATTCTCCAGATCC; and for ERBB4 ERE half-site III, forward 5'-GATGAGGAGGAAGATAAGGC and reverse 5'-CCAAACAGCAGCATTCTGTC.

Immunofluoresence and deconvolution microscopy. Immunofluorescent detection of ER and ERBB4 was done on T47D breast cancer cells cultured in phenol red–free medium supplemented with 5% charcoal-stripped FBS for 48 hours and stimulated with 100 pmol/L 17-ß-estradiol for 1 hour. Immunofluoresence was done exactly as described elsewhere (19) using rabbit anti-ERBB4 (Santa Cruz Biotechnology, Santa Cruz, CA) primary antibody with Alexafluor 488–conjugated goat anti-rabbit immunoglobulin G (IgG; Molecular Probes, Carlsbad, CA) secondary antibody and mouse anti-ER Ab-1 (NeoMarkers) primary antibody with Alexafluor 568–conjugated goat anti-mouse IgG (Molecular Probes) secondary antibody. Deconvolution microscopy was done on cells fixed in 4% paraformaldehyde exactly as described elsewhere (19).

Isolation of nuclear and cytosolic/membrane subcellular fractions. Nuclear and cytosolic subcellular fractions were isolated from T47D breast cancer cells cultured in phenol red–free medium supplemented with 5% charcoal-stripped FBS for 48 hours and stimulated with 100 pmol/L 17-ß-estradiol for 1 hour exactly as described elsewhere (19).

In vitro transcription/translation and GST pulldown assay. In vitro transcription/translation was done with linearized pBl4ICD-Flag using the TnT Quick Coupled Transcription/Translation System (Promega, Madison, WI) supplemented with 20 µCi of Redivue L-[35S]methionine (Amersham, Piscataway, NJ) exactly as described by the manufacturer. The GST pulldown assay was done using equal amounts of GST alone or GST fused to the independent ER domains A/B, C, D, E, and F as described elsewhere (25). In some experiments, 10 nmol/L 17-ß-estradiol was added to the pulldown assay.

Immunoprecipitation and Western blot analysis. Immunoprecipitations from cell lysates prepared from T47D breast cancer cells cultured in phenol red–free medium supplemented with 5% charcoal-stripped FBS for 48 hours and stimulated with 100 pmol/L 17-ß-estradiol for 1 hour were done using rabbit anti-ERBB4 antibody (Cell Signaling) or control rabbit IgG (Santa Cruz Biotechnology) exactly as described elsewhere (26). Total cell lysates and immunoprecipitates were analyzed by Western blot as described elsewhere (26) with primary antibodies ERBB4 (Santa Cruz Biotechnology), ER Ab-1 (NeoMarkers), {alpha}-tubulin (Upstate), or histone H3 (Santa Cruz Biotechnology). Secondary antibodies were IRDye800 Conjugated Affinity Purified Anti-Rabbit or Anti-Mouse IgG (Rockland Immunochemicals, Gilbertsville, PA) used at a dilution of 1:5,000 and detected using an Odyssey Infrared Imaging System (Licor Biosciences, Lincoln, NE).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell proliferation was measured as a function of metabolism by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma) assay. T47D breast cancer cells were seeded at 6,000 per well in a 96-well plate and cultured in 100 µL of phenol red–free medium supplemented with 5% charcoal-stripped FBS for 24 hours. Indicated wells were transfected with siGenome SMARTpool ERBB4 (Dharmacon) as described above. At 48 hours posttransfection, cells were treated with 100 pmol/L 17-ß-estradiol and incubated for an additional 72 hours. The cells were incubated with 25 µL of medium containing 5 mg/mL of MTT at 37°C for 4 hours, followed by incubation with 75 µL of 20% SDS in 50% dimethylformamide for an additional 6 hours at room temperature. The absorbance of each well at 550 nm was obtained using a microplate reader. Each sample was prepared in triplicate and the data represent the mean and SE of at least three independent experiments. Statistically significant differences between data sets were determined using paired Student's t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ERBB4 functions as an ER coregulator. We have previously shown that ERBB4/4ICD functions as a STAT5A transcriptional coactivator (18, 19, 27) and others have shown that ERBB4 potentiates expression of an ERE reporter gene fusion (6). To determine if ERBB4 contributes to 17-ß-estradiol (estrogen)–stimulated ER transactivation, we cotransfected the ER(+) MCF-7/B breast cancer cell line with an ERBB4 expression vector and an ERE-luciferase (ERE-luc) fusion vector. Luciferase expression in the presence or absence of estrogen was determined. As expected, we observed a robust 10-fold increase in luciferase activity when ERE-luc–transfected cells were stimulated with estrogen (Fig. 1A ). The estrogen-induced expression was abolished in the presence of the pure antiestrogen ICI-182780, indicating that estrogen-stimulated ERE-luc activity in these cells requires a functional ER (Fig. 1A). ERBB4 expression potentiated ERE-luc response to estrogen by 30-fold and this activity was ablated in the presence of ICI-182780 (Fig. 1A). These results suggest that ERBB4 is an estrogen-regulated ER coactivator.


Figure 1
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  		Figure 1. ERBB4 is an ER coactivator recruited to estrogen-responsive promoters. A, ERBB4 potentiates estrogen activation of an ERE-luc reporter. MCF-7/B breast cancer cells were incubated in growth medium supplemented with 5% charcoal-stripped FBS for 24 hours and cotransfected with 200 ng of ERE-luc and an ERBB4 expression vector. At 24 hours posttransfection, cells were cultured in the presence or absence of 100 pmol/L 17-ß-estradiol and/or 100 ng/mL ICI-182780 (ICI) and the luciferase reporter gene assay was done after 16 hours. Columns, mean of at least three independent experiments; bars, SE. Asterisk, ERBB4 significantly potentiates estrogen stimulation of ERE-luc. B, expression analysis of estrogen-regulated genes. T47D breast cancer cells were incubated in the presence of control RNAi or ERBB4 RNAi in growth medium supplemented with 5% charcoal-stripped FBS for 48 hours and stimulated with 100 pmol/L 17-ß-estradiol for 16 hours. Total RNA was extracted and PgR, SDF-1, and pS2 expression was analyzed by RT-PCR. ß-Actin RNA was amplified as a control for RNA quantitation. Western blot analysis of treated samples confirmed specific RNAi-induced suppression of ERBB4 expression whereas {alpha}-tubulin expression was unaffected (bottom). C, estrogen stimulates recruitment of ERBB4 and ER to promoter EREs. T47D cells were incubated in growth medium supplemented with 5% charcoal-stripped FBS for 48 hours and incubated in the presence or absence of 100 pmol/L 17-ß-estradiol for 1 hour. Fragmented chromatin was immunoprecipitated with antibodies directed against ERBB4 or ER and amplified by PCR using oligonucleotide primers flanking EREs in the PgR, SDF-1, or pS2 promoter.

 
ERBB4 is a selective ER coregulator recruited to gene promoters harboring EREs. To confirm coactivation of estrogen-regulated gene expression by endogenous ERBB4, we treated T47D breast cancer cells with estrogen and determined the expression levels of the three estrogen-regulated genes PgR, SDF-1, and pS2. The T47D breast cancer cell line expresses both ER and a cleavable form of ERBB4. As expected, estrogen stimulated expression of PgR, SDF-1, and pS2 in this experimental system (Fig. 1B). When ERBB4 expression in the presence of estrogen was suppressed through transfection of an interfering RNA directed against ERBB4 (ERBB4 RNAi), we found that estrogen-stimulated PgR and SDF-1 expression returned to basal levels (Fig. 1B). These results suggest that ERBB4 is required for ER transactivation of PgR and SDF-1 but not the pS2 gene.

We have previously shown that the 4ICD nuclear protein is recruited to gene promoters recognized by STAT5A (19). To determine if ERBB4 is recruited to promoter regions harboring EREs, we did a chromatin immunoprecipitation assay using chromatin prepared from estrogen-stimulated T47D cells and antibodies directed against ERBB4 or ER. Estrogen stimulated recruitment of both ERBB4 and ER to the PgR and SDF-1 promoters (Fig. 1C), genes for which estrogen-regulated expression requires ERBB4 (Fig. 1B). Although estrogen recruited ER to the pS2 promoter, an increase in ERBB4 recruitment was not observed (Fig. 1C). Consistent with this observation, estrogen stimulates pS2 expression independent of ERBB4 (Fig. 1B). Taken together, our results show that ERBB4 is an estrogen-stimulated ER coregulator that selectively binds to and activates gene promoters harboring EREs.

Estrogen stimulates nuclear accumulation of the 4ICD. Our results suggest that ERBB4 regulates ER transactivation, in part, through estrogen-stimulated recruitment of ERBB4 and ER to gene promoters. Because physiologic signals that activate ERBB4 promote proteolytic cleavage of ERBB4 releasing 4ICD, we hypothesized that 4ICD may play a role in coregulating ER transactivation functions. To elucidate the molecular mechanism underlying ERBB4 coregulation of ER at gene promoters harboring EREs, we first determined the effect of estrogen stimulation of T47D cells on 4ICD nuclear translocation. In mock-stimulated T47D cells, some basal nuclear accumulation of both ER and ERBB4/4ICD was observed (Fig. 2A, top ); however, significant levels of both proteins were also detected in the perinuclear region (Fig. 2A, top, asterisks). Following estrogen stimulation, the majority of ER translocated to the nucleus with nearly the entire 4ICD population localizing to the nucleus (Fig. 2A, middle, compare asterisks between top and middle). Nuclear translocation of 4ICD in response to estrogen was abolished in cells where ER expression was suppressed by RNAi (Fig. 2A, bottom), suggesting that ligand-bound ER functions as a 4ICD nuclear chaperone. Western blot analysis of cytosolic/membrane extracts and nuclear extracts shows that estrogen stimulated nuclear accumulation of both ER and 4ICD (Fig. 2B). Consistent with our previous results, the ERBB4 holoreceptor was excluded from the nuclear compartment (ref. 19; Fig. 2B).


Figure 2
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  		Figure 2. Estrogen promotes nuclear cotranslocation of ER and 4ICD. A, immunofluorescent detection of ER and ERBB4. T47D cells were incubated in growth medium supplemented with 5% charcoal-stripped FBS for 48 hours and mock stimulated or stimulated with 100 pmol/L 17-ß-estradiol for 1 hour. In some experiments, ER expression was suppressed by RNAi treatment for 72 hours. Cells were double labeled by immunofluoresence for ER (red) and ERBB4/4ICD (green) and DNA was counterstained with Hoechst dye (blue). Samples were analyzed by deconvolution microscopy. Asterisks, perinuclear regions. B, Western blot detection of nuclear ER and 4ICD. T47D cells were incubated in growth medium supplemented with 5% charcoal-stripped FBS for 48 hours and incubated in the presence or absence of 100 pmol/L 17-ß-estradiol for 1 hour. Cell lysates were separated into cytosolic/membrane (Cyto) and nuclear (Nuc) fractions and 50 µg of each fraction were analyzed by Western blot for ERBB4/4ICD and ER protein. {alpha}-Tubulin and histone H3 were included as cytosolic and nuclear loading controls, respectively.

 
Estrogen-stimulated ERBB4 coregulation of ER and nuclear translocation of 4ICD require ERBB4 proteolytic processing but an intact 4ICD nuclear localization signal is dispensable. We have previously shown that ERBB4 coregulation of STAT5A transactivation required both proteolytic processing of ERBB4 at the cell membrane (18) and an intact 4ICD nuclear localization signal (19). These results identified 4ICD as a nuclear chaperone for the STAT5A transcription factor (19). To determine if a similar mechanism mediates nuclear cotranslocation of 4ICD and ER, we determined the effect of independent ERBB4 mutations that abolish {gamma}-secretase processing (referred to as ERBB4V673I; ref. 18) or abrogate nuclear accumulation of 4ICD (referred to as ERBB4muNLS; ref. 19) on ERBB4 transcriptional coregulation of ER. Interestingly, the ERBB4 nuclear localization signal was dispensable for estrogen-stimulated ERBB4 coactivation of ERE-luc; however, {gamma}-secretase processing of ERBB4 to release a soluble 4ICD was required for ERBB4 coactivation of ERE-luc (Fig. 3A ).


Figure 3
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  		Figure 3. Estrogen promotes 4ICD nuclear translocation independent of an intrinsic ERBB4 nuclear localization signal. A, ERBB4 coactivation of ER requires {gamma}-secretase processing. MCF-7/B cells were incubated in growth medium supplemented with 5% charcoal-stripped FBS for 24 hours and cotransfected with 1 µg of ERE-luc and an expression vector for ERBB4, or ERBB4 with a mutated nuclear localization signal (ERBB4muNLS), or ERBB4 with a mutation abolishing {gamma}-secretase processing (ERBB4V673I). At 24 hours posttransfection, cells were cultured in the presence or absence of 100 pmol/L 17-ß-estradiol and the luciferase reporter gene assay was done after 16 hours. Columns, mean of at least three independent experiments; bars, SE. Asterisks, samples significantly greater than the vector control. B, estrogen promotes 4ICD nuclear translocation independent of an intrinsic ERBB4 nuclear localization signal. MCF-7/B cells were incubated in growth medium supplemented with 5% charcoal-stripped FBS for 24 hours and transfected with an expression vector for ERBB4-EGFP, ERBB4muNLS-EGFP, or ERBB4V673I-EGFP. At 24 hours posttransfection, cells were mock stimulated or stimulated with 100 pmol/L 17-ß-estradiol for 1 hour, fixed in 4% paraformaldehyde, counterstained with Hoechst dye, and analyzed by deconvolution microscopy. Estrogen promoted nuclear translocation of ERBB4 and ERBB4muNLS but not ERBB4V673I.

 
Nuclear accumulation of 4ICD following ligand activation of the ERBB4 holoreceptor requires an intact 4ICD nuclear localization signal (19); however, stimulation of ERE-luc by an ERBB4 receptor with a mutated nuclear localization signal raises the possibility that ligand-bound ER mediates 4ICD nuclear translocation independent of an intrinsic 4ICD nuclear localization signal. To test this possibility, we transfected MCF-7/B cells with ERBB4, ERBB4muNLS, or ERBB4V673I, each fused to enhanced green fluorescent protein (EGFP), and determined the effect of estrogen treatment on nuclear translocation of each receptor. Consistent with our previous results (Fig. 2A and B), estrogen stimulated nuclear accumulation of 4ICD (Fig. 3B, ERBB4/Estrogen). In concordance with our transcriptional coactivation data (Fig. 3A), estrogen stimulated nuclear accumulation of 4ICD harboring a mutated nuclear localization signal (Fig. 3B, ERBB4muNLS/Estrogen) but failed to mobilize the unprocessed ERBB4V673I mutant to the nucleus (Fig. 3B, ERBB4V673I/Estrogen). These results imply that estrogen-stimulated ER facilitates nuclear translocation of 4ICD and, by extension, ER can function as a nuclear chaperone for 4ICD lacking an intact nuclear localization signal.

Estrogen stimulates a direct interaction between 4ICD and ER. Our results raise the possibility of a estrogen-induced cytosolic 4ICD and ER complex that cotranslocates to the nucleus and regulates gene expression. To support this contention, we used an in vitro GST pulldown assay and a coimmunoprecipitation assay with endogenous protein to characterize the putative 4ICD and ER interaction. In an in vitro pulldown assay, each ER functional domain was independently fused to GST (Fig. 4A ), affinity purified, and incubated with 35S-labeled 4ICD (residues 673-1309). Our results indicate that 4ICD directly interacts with ER domain A/B harboring the ligand-independent activation function (AF-1) and ER domain E, which contains the ligand-dependent AF-2 (Fig. 4B). We further show that 4ICD interaction with ER domain E/AF-2 requires estrogen, indicating a ligand-dependent interaction with this region (Fig. 4C).


Figure 4
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  		Figure 4. Estrogen stimulates an interaction between 4ICD and ER. A, schematic of ER functional domains fused to GST with residues indicated. B, mapping of 4ICD interaction domains in ER. Independent ER domain GST fusions were incubated with 35S-labeled 4ICD (ERBB4 residues 673-1309) and 4ICD binding was analyzed by GST pulldown assay. Input of each GST fusion is shown at the bottom. Interaction between 4ICD and ER domains A/B and E harboring AF-1 and AF-2, respectively, was observed. C, 4ICD interaction with AF-2 is ligand dependent. Binding of 4ICD to ER domain E harboring AF-2 was analyzed by GST pulldown assay in the presence or absence of 10 nmol/L 17-ß-estradiol. D, estrogen stimulates endogenous ER and ERBB4/4ICD complex formation. T47D cells were incubated in growth medium supplemented with 5% charcoal-stripped FBS for 48 hours and incubated in the presence or absence of 100 pmol/L 17-ß-estradiol for 1 hour. Control rabbit IgG and ERBB4 immunoprecipitations were done on cell lysates and analyzed by Western blot for ERBB4/4ICD and ER protein.

 
To support our in vitro assay, we stimulated T47D cells with estrogen and determined if ER exists in a complex with ERBB4/4ICD. Indeed, estrogen stimulation of T47D cells resulted in the recruitment of ER to an ERBB4 immunoprecipitated complex (Fig. 4D). Taken together, our results suggest a novel signaling mechanism with estrogen stimulating an association between ER and 4ICD. Subsequent nuclear cotranslocation of the complex results in selective ER/4ICD recruitment to gene promoters containing EREs and activation of gene expression.

ERBB4 is an estrogen-inducible gene recruited with ER to the ERBB4 promoter. Clinically, ERBB4 expression in breast cancer is significantly associated with ER expression (5–8), raising the possibility that ERBB4 is an estrogen-inducible gene. We therefore determined the effect of estrogen treatment of T47D cells on ERBB4 expression by RT-PCR. Our results show that estrogen stimulation of T47D cells for 16 hours induced an increase in ERBB4 mRNA expression (Fig. 5A ), showing for the first time that ERBB4 is an estrogen-inducible gene. To further evaluate estrogen regulation of ERBB4 expression, we examined the ERBB4 promoter for the presence of EREs using Genomatix/MatInspector software. Three potential ERE half-sites were identified within the ERBB4 promoter, positioned at –2097, –4150, and –4487 relative to the ERBB4 mRNA translational start codon (Fig. 5B). To determine if estrogen promotes recruitment of ER and/or 4ICD to ERE half-sites within the ERBB4 promoter, we did chromatin immunoprecipitation analysis of chromatin isolated from estrogen-stimulated T47D cells using antibodies directed against ER or ERBB4. Immunoprecipitated chromatin was amplified using three PCR primer pairs, each designed to span one ERBB4 promoter ERE half-site. The results indicate that estrogen stimulated a dramatic increase in the association of both ER and 4ICD at ERE I positioned at –2097. Recruitment of ER and 4ICD to ERE III positioned at –4487 was at the lower detection limits of this assay and ERE II positioned at –4150 failed to recruit ER or 4ICD (Fig. 5C). These results suggest that recruitment of ER and 4ICD to ERE I of the ERBB4 promoter potentiates estrogen-induced expression of ERBB4. Significantly, our observations provide evidence that ERBB4 may coactivate its own estrogen-stimulated expression in breast cancer cells, thereby establishing an estrogen-regulated ER/ERBB4 autocrine signaling loop in these cells.


Figure 5
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  		Figure 5. ERBB4 is an estrogen-regulated gene contributing to the estrogen response of T47D breast cancer cells. A, estrogen stimulates ERBB4 expression. T47D cells were incubated in growth medium supplemented with 5% charcoal-stripped FBS for 48 hours and incubated in the presence or absence of 100 pmol/L 17-ß-estradiol for 16 hours. Total RNA was extracted and analyzed for ERBB4 expression by RT-PCR. ß-Actin RNA was amplified as a control for RNA quantitation. B, schematic of ERBB4 promoter. Gray boxes, three potential ERE half-sites identified by Genomatix/MatInspector software; double arrows, regions of chromatin amplified by oligonucleotide primers flanking each ERE half-site. C, estrogen stimulates recruitment of ERBB4 and ER to the ERBB4 promoter. T47D cells were incubated in growth medium supplemented with 5% charcoal-stripped FBS for 48 hours and incubated in the presence or absence of 100 pmol/L 17-ß-estradiol for 1 hour. Fragmented chromatin was immunoprecipitated with antibodies directed against ERBB4 or ER and amplified by PCR using oligonucleotide primers flanking ERBB4 promoter ERE half-site I, II, or III. D, ERBB4 contributes to estrogen-induced proliferation of T47D cells. T47D cells were incubated in the presence or absence of ERBB4 RNAi in growth medium supplemented with 5% charcoal-stripped FBS for 48 hours and stimulated with 100 pmol/L 17-ß-estradiol for an additional 72 hours. An MTT assay was done. Columns, mean of at least three independent experiments; bars, SE. Asterisks, estrogen alone was significantly greater than control or estrogen with ERBB4 RNAi.

 
ERBB4 contributes to the estrogen response of breast cancer cells. We next determined the effect of ERBB4 expression on estrogen-stimulated proliferation of the T47D breast cancer cell line. T47D cells were stimulated with estrogen in the presence or absence of RNAi directed against ERBB4. Cell proliferation was determined by MTT assay after 72 hours of estrogen treatment. As expected, estrogen stimulated a significant increase in cellular MTT conversion, an indication of cellular proliferation (Fig. 5D). A significant decrease in MTT conversion was observed, however, when ERBB4 expression was suppressed in estrogen-treated T47D cells (Fig. 5D). These results indicate that the potent estrogen-stimulated proliferative response in T47D breast cancer cells requires ERBB4 expression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Crosstalk between cell-surface and nuclear receptors and their contribution to the development and progression of endocrine regulated cancers have been areas of intense investigation. In breast cancer, these studies have identified signaling crosstalk between the EGFR-family and ER involving activation of parallel or overlapping signaling pathways (28, 29). Here we show for the first time direct coupling of transmembrane and nuclear receptor signaling, forming an autocrine feedback loop that regulates breast cancer cell gene expression and contributes to tumor cell proliferation. Our results support a model of ERBB4/ER coupled signaling in breast cancer cells, which involves an estrogen-stimulated interaction between extranuclear ER and ERBB4/4ICD. Subsequent proteolytic processing of ERBB4 by TNF{alpha}-converting enzyme followed by {gamma}-secretase results in release of an independently signaling 4ICD. Estrogen promotes nuclear cotranslocation of ER and 4ICD and ultimately recruitment of ER and selective recruitment of 4ICD to estrogen responsive gene promoters. Nuclear 4ICD functions as an ER coactivator when bound with ER to gene promoters including PgR and SDF-1, and possibly ERBB4 itself. Estrogen activation of ERBB4 expression establishes a novel transmembrane and nuclear receptor autocrine signaling loop that selectively potentiates expression of estrogen-regulated genes and affects breast tumor cell proliferation (Fig. 6 ).


Figure 6
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  		Figure 6. Estrogen stimulates a growth-promoting ER and ERBB4 autocrine signal in breast cancer cells. Our results show that estrogen stimulates ERBB4/4ICD complex formation with extranuclear ER. Following proteolytic processing of ERBB4 by TNF{alpha}-converting enzyme (TACE) and {gamma}-secretase, the ER/4ICD complex translocates to the nucleus where 4ICD is selectively recruited with ER at the PgR, SDF-1, and ERBB4 promoters. Estrogen-stimulated expression of ERBB4 completes a breast cancer cell autocrine loop that promotes tumor cell proliferation. See text for additional details.

 
Our results support a novel ER signaling pathway that directly integrates the extranuclear and genomic actions of estrogen. Classic or genomic estrogen signaling involves binding of estrogen to the ER followed by activation or repression of gene expression regulated by direct association of the estrogen/ER complex with gene promoters harboring EREs. Extranuclear actions of estrogen are thought to be mediated through a membrane-associated ER, and estrogen activation of this nonclassic receptor pathway results in lateral stimulation of receptor tyrosine kinases and G-protein coupled receptors (30) or in activation of secondary messengers including intracellular calcium (31) and cyclic AMP (32, 33). Here we show that extranuclear ER associates with ERBB4 in response to estrogen stimulation and estrogen promotes nuclear cotranslocation of ER and the ERBB4 proteolytic product, 4ICD. This ER/4ICD complex directly regulates estrogen genomic activity by binding to and activating expression of estrogen-regulated genes. Our results support this model over a model where the 4ICD nuclear protein independently translocates and interacts with nuclear ER residing at target promoters. Indeed, we show that 4ICD lacking a functional nuclear localization signal translocates to the nucleus in response to estrogen, implying that extranuclear ER functions as a nuclear chaperone for 4ICD. Furthermore, estrogen-induced nuclear translocation of 4ICD is abolished when ER expression is suppressed by RNAi, further supporting a critical role for ER during estrogen-induced 4ICD nuclear translocation. Although estrogen stimulates association between extranuclear ER and other cell-surface receptors, including the receptor tyrosine kinases ERBB2 (34) and insulin-like growth factor (35), crosstalk between ER and these receptors results in activation of nongenomic estrogen signaling pathways, including mitogen-activated protein kinase and phosphatidylinositol 3-kinase (28, 35), which may indirectly affect ER transactivation (30). In this context, the ERBB4/ER signaling axis is a unique cell-surface and nuclear receptor signaling pathway that directly couples extranuclear and nuclear estrogen actions.

The exact molecular mechanisms regulating the transcriptional coactivator function of 4ICD remain to be determined. Similar to the EGFR-family members EGFR (36) and ERBB2 (37), 4ICD harbors independent transcriptional activity (17, 38). However, artificial transcription assays and an independently expressed 4ICD protein fusion were employed in these studies, thus limiting the significance of these observations. A physiologically activated and proteolytically processed ERBB4 holoreceptor with independent transactivation activity remains to be confirmed. Alternatively, ERBB4 may regulate transcription indirectly by facilitating transcription factor association with target promoters. For example, we have previously shown that the STAT5A transcription factor interacts with 4ICD, and 4ICD mediates nuclear translocation and subsequent DNA binding of STAT5A at STAT5A target promoters (19). Similarly, estrogen stimulates 4ICD/ER complex binding to the PgR, SDF-1, and ERBB4 promoters, and transcriptional activation of the PgR and SDF-1 genes, and possibly ERBB4 itself, requires ERBB4 expression. In contrast, 4ICD fails to associate with ER at the pS2 promoter. One interpretation of these observations is that 4ICD sequesters and enhances recruitment of transcription factors, including ER, to selectively activate a subset of ER target promoters. In the absence of ERBB4/4ICD, ER and other unbound transcriptional complexes may now be recruited to transactivate gene promoters indirectly modulated by 4ICD. We are currently doing a global analysis of transcription factors recruited with 4ICD to estrogen-regulated genes to substantiate this molecular model of 4ICD transcriptional coregulation.

One unexpected finding from these studies was that ERBB4 itself is an estrogen-inducible gene with both ER and 4ICD recruited to the ERBB4 promoter in response to estrogen. Estrogen stimulated binding of ER and 4ICD to a region of the ERBB4 promoter between –2275 and –1989 nucleotides upstream of the translational start. This region contained a consensus ERE half-site (GGTCA) at –2097 in tandem with an imperfect half-site (CGTCA) at –2086. Although ER dimers bind inefficiently to ERE half-sites (39–41), binding is enhanced by juxtaposed SP-1 or activator protein 1 (AP-1) sequence elements (42–46). Interestingly, the bottom strand of each ERBB4 ERE overlaps with sequences homologous to the AP-1 core binding site (TGAC). A similar combination of an ERE half-site with overlapping AP-1 sites regulates estrogen response of the c-fos promoter (46). Although the exact mechanism of ER recruitment to ERE half-sites remains unresolved, one prevailing model suggests that dimeric ER binding to an ERE half-site is stabilized by interactions with AP-1 and SP-1 transcriptional complexes recruited to juxtaposed response elements (40, 42, 47, 48). Currently, the exact molecular mechanism regulating the selective recruitment of 4ICD to EREs remains to be determined. It is interesting, however, that 4ICD binds to the PgR, SDF-1, and ERBB4 promoters harboring ERE half-sites with associated SP-1/AP-1 sequence elements, but not to the pS2 promoter with its near consensus ERE. These observations require confirmation by examining 4ICD recruitment to other estrogen-regulated genes, but one intriguing possibility is that sequences surrounding target ERE sites determine 4ICD recruitment to estrogen-regulated promoters.

Our findings have led to the molecular characterization of a novel ER/ERBB4 autocrine signaling loop in breast cancer cells; however, does this unique signaling pathway affect cell growth? Similar to earlier reports (13, 14), we show that suppression of ERBB4 expression in the T47D cell line results in a significant decrease in estrogen-induced cell proliferation. In concordance with these findings, others have shown that ectopic overexpression of ERBB4 enhances estrogen-stimulated growth of the ER(+) MCF-7 breast cancer cell line (6). Our results suggest that the 4ICD transcriptional coactivator may contribute to estrogen-induced cellular responses by promoting selective expression of estrogen-regulated genes. Thus, 4ICD may coactivate growth-promoting genes while suppressing expression of growth inhibitory genes. In support of this contention, we show that 4ICD coactivates expression of PgR, which acts in combination with ER to promote breast epithelial proliferation (49–51). Likewise, SDF-1, another 4ICD transcriptional target, contributes to estrogen-induced breast tumor cell proliferation (52) and is associated with poor prognosis in breast cancer patients (53). Identification of additional candidate tumor-promoting estrogen response genes directly regulated by 4ICD is an area of ongoing research in our laboratory.

In concordance with our experimental findings, clinical evidence supports estrogen stimulation of a functional ER/ERBB4 autocrine signaling loop regulating PgR expression in breast cancer. Indeed, ERBB4 expression in breast cancer is significantly associated with ER expression (5–8). Furthermore, loss of PgR expression, an ER/ERBB4 coregulated gene, correlates with the loss of ERBB4 expression in ER(+) tumors (5, 6, 8). ERBB4 regulation of PgR in vivo is supported by the fact that overlapping defects during pregnancy induced mammary gland proliferation and differentiation observed in mice lacking functional ERBB4 or PgR receptors (20, 21, 26, 54–56).

Interestingly, ER and ERBB4 coexpression in breast cancer predicts significantly improved patient disease-free and overall survival when compared to patients with tumors expressing ER alone (9, 10, 57). These clinical observations contradict preclinical models reported here and by others which predict that ER/ERBB4 coexpression contributes to a tumor-promoting phenotype (6, 13, 14). One possible explanation for this apparent paradox is provided by our most recent findings showing that 4ICD is a multifunctional protein in breast cancer cells. Here we show that nuclear 4ICD functions as a growth-promoting transcriptional coregulator; however, we have also recently shown that 4ICD harbors a proapoptotic BH3-domain and ligand-induced mitochondrial localization of 4ICD induces tumor cell killing (12, 18). Furthermore, cytosolic accumulation of 4ICD in primary breast tumors is associated with tumor apoptosis (12). By recruiting 4ICD to the nuclear compartment, ER may indirectly suppress 4ICD apoptotic activity while commandeering the growth-promoting action of a nuclear 4ICD/ER complex. We therefore predict that disruption of an ER and nuclear 4ICD association by endocrine therapy would result in cytosolic accumulation of proapoptotic 4ICD and ultimately tumor cell death. Although speculative, this model would account for the improved clinical response to endocrine therapy observed in patients coexpressing ER and ERBB4 when compared to patients with tumors expressing ER alone (10). Nevertheless, clinical observations and our recent descriptions of multiple 4ICD activities imply that the ER and 4ICD signaling axis has a significant effect on estrogen action and therapeutic response in breast cancer patients. Therefore, the potentially complex influence of ER and ERBB4 autocrine signaling should be considered when interpreting therapeutic responses of patients with ER-positive breast tumors.


   Acknowledgments
 
Grant support: National Cancer Institute/NIH grants RO1CA95783 and RO1CA96717 (F.E. Jones).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Laura McDonald for excellent laboratory management and Nicolette Solano for outstanding administrative assistance (we wish them both luck in their post-Katrina lives), and Rakesh Kumar and members of the Jones lab for helpful discussions during this project.

This work is dedicated to June Allison, caring mother, wife, and friend, who after 14 disease-free years must stage another battle against breast cancer.


   Footnotes
 
Note: Y. Zhu and L.L. Sullivan contributed equally to this work.

Received 12/12/05. Revised 6/14/06. Accepted 6/21/06.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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