Loss of an Estrogen Receptor Isoform (ER{alpha}{Delta}3) in Breast Cancer and the Consequences of Its Reexpression: Interference with Estrogen-Stimulated Properties of Malignant Transformation

I. Erenburg, B. Schachter, R. Mira y Lopez and L. Ossowski

Department of Cell Biology (I.E., B.S., L.O.) Rochelle Belfer Chemotherapy Foundation Laboratory (I.E., R.M.L., L.O) Department of Medicine Department of Obstetrics and Gynecology (B.S.) Mount Sinai School of Medicine New York, New York 10029


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Comparison of mRNA ratios of a non-DNA-binding estrogen receptor (ER{alpha}) isoform, missing exon 3 (ER{alpha}{Delta}3), to the full-length ER{alpha}, in normal breast epithelium to that in primary breast cancers and breast cancer cell lines revealed a 30-fold reduction of this ratio in cancer cells (P < 0.0001). To test what functions may have been affected by the loss of ER{alpha}{Delta}3, stable clones of MCF-7 cells expressing ectopic ER{alpha}{Delta}3 protein, at the range of physiological ER{alpha}, were generated. In vector-transfected controls the ER{alpha}{Delta}3-mRNA and protein were less than 10% while in the ER{alpha}{Delta}3-expressing clones, ER{alpha}{Delta}3-mRNA and protein ranged from 36–76% of the total ER{alpha}. Estrogen (E2) stimulated the expression of pS2-mRNA in pMV7 vector control cells, but the stimulation was reduced by up to 93% in ER{alpha}{Delta}3-expressing clones. In addition, several properties associated with the transformed phenotype were also strongly affected when ER{alpha}{Delta}3 protein was reexpressed. Compared with vector-transfected control cells, the saturation density of the ER{alpha}{Delta}3-expressing clones was reduced by 50–68%, while their exponential growth rate was only slightly (14.5 ± 5%) lower. The in vivo invasiveness of the ER{alpha}{Delta}3-expressing cells was significantly reduced (P = 0.007) by up to 79%. E2 stimulated anchorage-independent growth of the pMV7 vector control cells, but reduced it to below baseline levels in ER{alpha}{Delta}3 clones. The reduction of the pS2 response to E2 in the ER{alpha}{Delta}3-expressing clones and the E2 block of anchorage-independent growth to below baseline were more pronounced than expected from the dominant negative function of ER{alpha}{Delta}3. These observations suggest that E2 may activate an additional ER{alpha}{Delta}3-dependent inhibitory pathway. The drastic reduction of ER{alpha}{Delta}3 to ER{alpha} ratio in breast cancer, and the fact that when present in breast cancer cells this isoform leads to a suppression, rather than enhancement, of the transformed phenotype by E2 suggests that the regulation of ER{alpha}-mRNA splicing may need to be altered for the breast carcinogenesis to proceed.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen (E2), a key regulator of normal breast growth and differentiation, has been shown to promote both cancer cell proliferation and invasion (1, 2). Although the estrogen receptor (ER{alpha}) is the major mediator of estrogen action, the precise mechanism by which ER{alpha} contributes to altered estrogen response in cancer remains unclear.

ER is one of many transcriptional regulatory proteins of the steroid receptor family that act principally as ligand-activated DNA-binding dimers (3). ER has distinct functional domains, including two transcriptional activating regions (the N-amino-terminal AF1 and the C-terminal, ligand-dependent, AF2), vs. internal zinc fingers, DNA-binding domain, dimerization regions, and several nuclear localization sequences (4) (Fig. 1AGo). Like other ligand-activated transcriptional regulators (5), ER is not a single protein, but rather a set of proteins coded by two genes giving rise to ER{alpha} and ERß (6) as well as isoforms generated by alternative splicing (exon skipping) of the single ER{alpha}pre-mRNA. Since alternatively spliced ER{alpha}-mRNAs were first noted in breast tumors and tumor cell lines, before their normal counterparts were thoroughly examined, it was proposed that overexpression of aberrant ER{alpha} isoforms is characteristic of breast cancer (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24). One ER{alpha} isoform (ER{alpha}{Delta}3), missing exon 3, which encodes the second zinc finger of the DNA-binding domain, was shown to be an in-frame deletion that in an in vitro translation reaction yielded a protein of 61.2 kDa (20). This protein was unable to form specific complexes with estrogen response elements (EREs) or transactivate an ERE-reporter plasmid in transient transfection assays (20). At an ER{alpha}{Delta}3 to ER{alpha} ratio of 1:1, ER{alpha} binding to an ERE and transactivation of an ERE-chloramphenicol acetyltransferase (CAT) reporter were inhibited by approximately 30%, suggesting that ER{alpha}{Delta}3 can function as a dominant negative receptor (20). The importance of dominant negative receptors in controlling cellular responses to agonists and antagonists has been underscored by several recent studies of the steroid receptor family (25, 26, 27, 28). Since ER{alpha}{Delta}3 is a naturally occurring form of ER{alpha}, if expressed at high relative levels to full-length ER{alpha}, it may have a profound effect on several estrogen-dependent functions. For example, ER{alpha}{Delta}3 expression in normal breast tissue may provide a means of regulating the magnitude of estrogen responses, and a relative loss of ER{alpha}{Delta}3 expression in breast tumor tissue may lead to unchecked estrogen stimulation. Alternatively, a rise in ER{alpha}{Delta}3 expression during breast carcinogenesis may facilitate the disabling of the normal differentiation-inducing function of estrogen. Finally, the isoform may represent such a minor component that it would not influence estrogen-mediated pathways in either normal or malignant breast tissue.



View larger version (37K):
[in this window]
[in a new window]
 
Figure 1. Analysis of ER{alpha} and ER{alpha}{Delta}3 mRNA Expression in Normal Breast and Breast Cancer Tissue and Cells

A, Diagram of mRNA and protein structures of ER{alpha} and ER{alpha}{Delta}3. B1: Southern blots of four cDNAs, obtained by RT-PCR of mRNA of breast cancers, probed with either an exon 4 probe (to detect both ER{alpha} and ER{alpha}{Delta}3) lanes 1a-4a, 5, and 6, or an exon 3 probe (to detect only full-length ER{alpha}), lanes 1–4. The examples shown represent the entire range of ER{alpha}{Delta}3 to ER{alpha} ratios found in breast cancers; they are 0.25, 0.10, 0.08, and 0.04 for lanes 1a, 2a, 3a, and 4a, respectively. B2: Detection with exon 4 probe only. Southern blot of ER{alpha}-positive breast cancer cell lines: lane 1, BT 474; lane 2, MDAMB175vii; lane 4, MDAMB361; lane 6, MDAMB134vi; lane 7, T47D; lane 8, MCF-7; lane 9, ER{alpha}-positive endometrial cancer cell line, Ishikawa. ER{alpha}-negative breast cancer cell lines: lane 3, MDAMB231; lane 5, MDAMB461. C, Southern blot of epithelial cells from 10 reduction mammoplasties (lanes 1–10) and Hs 578Bst, a normal, immortalized myoepithelial cell line (lane 11) detected with exon 4 probe. (Lanes 1–3 represent purified luminal epithelial cell preparations; lanes 4–10 represent pools of epithelial cells with predominance of basal cells). D, ER{alpha}{Delta}3 to ER{alpha} RNA ratios in breast cancers (group 1), breast cancer cell lines (group 2), normal epithelium (group 3) (the pure luminal epithelium, n = 3, indicated by circled crosses), and fibroblasts (group 4) (isolated from reduction mammoplasty, n = 4, or breast cancer, n = 2). Each point in the scattergram represents the scanned relative intensity of ER{alpha}{Delta}3 and ER{alpha} bands produced by Southern blotting of cDNAs generated by RT-PCR of RNA extracted from individual tissue or cell samples. The median of ER{alpha}{Delta}3 to ER{alpha} ratios was 0.11 for breast cancers, 0.10 for breast cancer cell lines, 3.40 for normal epithelium, and 2.40 for fibroblasts. ANOVA analysis (using SYSTAT program, SYSTAT, Inc., Evanston, IL) of the ER{alpha}{Delta}3 to ER ratio in the three groups, tumors, tumor cell lines, and normal epithelium, showed a significant difference (P < 0.0001). Post hoc analysis showed that the primary breast cancers were not different from the tumor cell lines (P = 0.978), but primary breast cancers and breast cancer cell lines were different from normal epithelium, P < 0.0001 and P = 0.001, respectively.

 
To distinguish between these possibilities we have compared the relative levels of ER{alpha}{Delta}3 and ER{alpha} expression in primary breast cancers, and breast cancer cell lines to that in luminal and basal epithelium and fibroblasts purified from reduction mammoplasty specimens. This comparison, and the subsequent analysis of breast cancer cells expressing ectopic ER{alpha}{Delta}3, yielded strong support for the hypothesis that ER{alpha}{Delta}3 causes a profound change in cell response to E2 and that the relative loss of ER{alpha}{Delta}3 may be important in carcinogenesis.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Comparison of ER{alpha} and ER{alpha}{Delta}3 Expression in Cancer and Normal Breast Cells
RNA extracted from aliquots of 33 breast cancers was analyzed for ER{alpha} and ER{alpha}{Delta}3 expression using a semiquantitative RT-PCR assay capable of distinguishing between mRNA encoding the full-length and the ER{alpha}{Delta}3 forms of the receptor. The two forms of ER{alpha} were detected by Southern blot analysis of ER{alpha}-cDNA amplified with primers within exons 2 and 4, using internal probes hybridizing either with exon 4, to detect both forms of the receptor, or within exon 3 to detect only the full-length ER{alpha}. The median ratio of ER{alpha}{Delta}3 to ER{alpha} expression in breast tumors was 0.11 (range of 0.03–0.47) (Fig. 1BGo1 and 1D, group 1). Analysis of nine cancer cell lines, (seven of which were ER{alpha} positive) confirmed the low relative levels of ER{alpha}{Delta}3 in pure populations of cancer cells (median ratio of 0.10, range 0.06–0.30, Fig. 1BGo2 and 1D, group 2). A similar test was performed on epithelial cells isolated from 10 reduction mammoplasties, which included seven pools of epithelial cells and three purified populations of luminal cells. [An immortalized, myoepithelial mammary cell line (Hs 578Bst) was also examined]. The median ratio of ER{alpha}{Delta}3 to ER{alpha} in all 10 preparations of epithelial cells was 3.4 (range 0.36 to 9.80) or approximately 30-fold greater than the ratio found in breast cancer (P < 0.0001), (Fig. 1Go, C and D, group 3). The three 90% pure luminal cell populations (purity determined by the K8/K5 ratio) had ER{alpha}{Delta}3 to ER{alpha} ratios of 0.53, 0.36, and 0.42 (Fig. 1DGo, group 3, encircled crosses). Individually, these were 3- to 4-fold greater than the median ratio of ER{alpha}{Delta}3 to ER{alpha} in breast cancers and 7- to 10-fold greater than the median ratio in breast cancers with low ER level (see below). This comparison is important because, like luminal cells, a large proportion of breast cancers expresses cytokeratin K8 (29, 30), suggesting the possibility that breast cancer may arise in luminal cells. Until formally excluded, however, the possibility that breast cancer originates in stem cells found in the basal layers of breast acini must also be considered. We found that in pools of epithelial cells, which due to culture conditions had a preponderance of stem/basal cells (Fig. 1DGo, group 3) (31), the ER{alpha}{Delta}3 to ER{alpha} ratio was even greater (median 4.00, range 0.55–9.80). We also tested six preparations of breast fibroblasts, which under culture conditions became predominantly (~ 80%) myofibroblasts, as determined by their content of smooth muscle {alpha}-actin. Regardless of their origin (reduction mammoplasty, n = 4; or breast cancer, n = 2) (Fig. 1DGo, group 4), they were also found to have high ER{alpha}{Delta}3 to ER{alpha} ratios (median 2.4, range 1.5–4.5).

In an attempt to determine whether the loss of ER{alpha}{Delta}3 was associated with the carcinogenic event per se, or with cancer progression, we analyzed the breast cancer patients data by stratifying them according to their menopausal status (pre- and postmenopausal) or tumor size (<1.5 cm or >1.5 cm) or presence or absence of lymph node involvement. With one exception, these analyses did not identify a subpopulation of patients with a significant difference in the ER{alpha}{Delta}3 to ER{alpha} ratios among the tumors. The exception was a group of tumors (6/33) with ER{alpha} levels lower than 5 fmol/mg protein, deemed ER{alpha} negative by clinical standards and considered more aggressive, in which the median ratio (0.05) was significantly different (P < 0.001) from the median ratio (0.11) of all tumors.

Cumulatively, these results, showing that the ratio of ER{alpha}{Delta}3 to full-length ER{alpha} is substantially reduced in all breast cancer cell lines and in breast cancers, even when tumors are smaller than 1.5 cm and have not spread to the lymph nodes, suggest that a loss of the ER{alpha}{Delta}3 isoform may be associated with an early event in carcinogenesis.

Transfection and Isolation of MCF-7 Cells Expressing ER{alpha}{Delta}3; Characterization of the Native and Transgenic Protein
The findings described above suggested that restoring ER{alpha}{Delta}3 in cancer cells to normal relative levels may result in attenuation of their transformed phenotype. To test this, ER{alpha}{Delta}3-cDNA was subcloned into a pMV7 vector (32). The ER{alpha}{Delta}3/pMV7 construct was first tested by transiently transfecting COS cells, negative for ER{alpha}, an experiment that showed (Fig. 2Go) that transfected cells express and properly localize ER{alpha}{Delta}3 to the nucleus and that the ER{alpha}{Delta}3 protein reacts with a well characterized rat anti-ER{alpha} antibody (H226) (33).



View larger version (59K):
[in this window]
[in a new window]
 
Figure 2. Detection of Transfected ER{alpha}{Delta}3 Protein in COS Cells

COS cells transiently transfected with ER{alpha}{Delta}3/pMV7 vector using Lipofectin (GIBCO) were plated on coverslips 16 h after transfection, allowed to attach, fixed with 3% paraformaldehyde, and incubated overnight at 4 C with H226 antibody (35 µg/ml). Biotin-coupled anti-rat IgG secondary antibody (Sigma) and rhodamine-conjugated strepavidin (Sigma) were used for protein visualization. Left panel, Immunofluorescent detection of ER{alpha}{Delta}3 in nuclei of three COS cells. Right panel, Nomarski optic view of the same field. Magnification x400.

 
Stable clonal lines of MCF-7 cells were then selected from cultures transfected (or infected) with either the ER{alpha}{Delta}3-coding constructs, or the pMV7 vector alone for negative control, and analyzed both for ER{alpha}{Delta}3-mRNA, by RT-PCR (Fig. 3AGo), and protein expression, by immunoprecipitation and Western blotting (Fig. 3BGo). Several pMV7vector control clones were analyzed individually for ER{alpha} and ER{alpha}{Delta}3-mRNA and found to have similar overall levels of ER and invariably very low levels (<10% of total ER{alpha}) of the ER{alpha}{Delta}3-mRNA. Thus, for all further experiments, a pool of approximately 50 pMV7 vector-transfected MCF-7 clones was used as a control. Attempts to generate specific ER{alpha}{Delta}3 antibodies that recognize the exon 2/exon 4 splice junction were unsuccessful. Therefore, the ER{alpha}{Delta}3 protein was identified on the basis of its reactivity with two antibodies recognizing different N-terminal epitopes of ER{alpha}, its faster mobility than ER{alpha} on SDS-PAGE, and the correlation of its expression with that of ER{alpha}{Delta}3-mRNA.



View larger version (42K):
[in this window]
[in a new window]
 
Figure 3. Characterization of RNA and Protein from Clonal Cell Lines Obtained by Stable Transfection or Infection of MCF-7 Cells with ER{alpha}{Delta}3/pMV7 or pMV7 Alone

A, Southern blot with exon 4 probe of RT-PCR-cDNA from ER{alpha}{Delta}3-expressing clones and pMV7 pool vector control. Lanes 1–4 are clone ER{alpha}{Delta}3–1, 2, 3, and 4, respectively; lane 5 is pMV7 pool vector control. B, Western blot of total ER{alpha} immunoprecipitated from 400 µg of protein extract. Left panel, Experiment 1, lanes 1 and 2, clones ER{alpha}{Delta}3–1 and 2, electrophoresis for 8 h. Right panel, Experiment 2, lanes 3 and 4, clones ER{alpha}{Delta}3–3 and 4; lane 5, pMV7-pool vector control, electrophoresis for 10 h. Arrows indicate the 65-kDa ER{alpha} and 61-kDa ER{alpha}{Delta}3. The ER{alpha}{Delta}3-mRNA as a percent of total ER was as follows: clone ER{alpha}{Delta}3–1, 59%; 2, 57%; 3, 64%; 4, 67%. The protein was as follows: ER{alpha}{Delta}3–1, 36%; 2, 40%; 3, 76%; 4, 60%. (A second determination of the ER{alpha}{Delta}3-mRNA and protein fraction in clone 1 yielded 66% and 42%, respectively; in clone 4, 67% in both assays).

 
As expected (Fig. 3AGo, lane 5), control pMV7 cells expressed predominantly full-length ER{alpha}-mRNA and a small amount of ER{alpha}{Delta}3, similar to that observed in the parental MCF-7 cell line (Fig. 1BGo2, lane 8). In contrast, all four ER{alpha}{Delta}3 clones had a predominance of ER{alpha}{Delta}3-mRNA (Fig. 3AGo, lanes 1–4; ratios of ER{alpha}{Delta}3 to ER{alpha} ranged from 1.3–2.1 or 57- 67% of total ER{alpha} was ER{alpha}{Delta}3), indicating that the transgene mRNA was efficiently expressed in these cells.

Extracts of the individual clones shown in Fig. 3BGo were subjected to immunoprecipitation with a polyclonal rabbit anti-ER{alpha} antibody followed by Western blotting with the H226 antibody, recognizing the amino terminus of ER{alpha}. In addition to the 65-kDa band, representing the full-length ER{alpha} protein, all ER{alpha}{Delta}3 clones also contained a prominent 61-kDa band, which corresponded to the predicted molecular mass of the ER{alpha}{Delta}3 protein. Expression of the ER{alpha}{Delta}3 protein in these clones ranged from 36–76% of total ER{alpha} (a relative ratio of ER{alpha}{Delta}3 to ER{alpha} of 0.6–3.3), comparable to that observed in the normal mammary epithelium. Although the relative ratios of the RNAs and the proteins did not match perfectly, the two clones (clones 3 and 4, Fig. 3Go, A and B) with the highest proportion of ER{alpha}{Delta}3-mRNA (64% and 67%) also had the highest proportion of ER{alpha}{Delta}3-protein (76% and 60%). In parental MCF-7 extracts, a faint band (~5% of the total ER{alpha}), comigrating with the ER{alpha}{Delta}3 form, could be detected only when excess protein was loaded onto the gel (Fig. 4Go, lane 2). This and the correspondence between the low intensity of the ER{alpha}{Delta}3-mRNA band and the 61-kDa protein band (Fig. 1BGo, lane 8; Fig. 3AGo, lane 5, and 3B, lane 5; Fig. 4Go, lane 2, respectively) suggest that both pMV7-carrying and the parental MCF-7 cells produce small amounts of the ER{alpha}{Delta}3 mRNA and protein. The identity of the 61-kDa band as ER{alpha}{Delta}3, and not as an underphosphorylated form of full-length ER{alpha}, was confirmed in a dephosphorylation experiment. Total immunoprecipitated ER{alpha} from pMV7 or ER{alpha}{Delta}3 cells was mixed with protease inhibitors; one aliquot of each was dephosphorylated by incubation with calf intestinal phosphatase (CIP), and the other was incubated under identical conditions but without CIP. Analysis of products by Western blotting showed that without CIP, ER{alpha} from both pMV7 pool vector control and ER{alpha}{Delta}3 cells produced a comigrating doublet of bands, the upper corresponding to full-length ER{alpha} and the lower to ER{alpha}{Delta}3 protein (compare lanes 2 and 3 of Fig. 4Go). CIP treatment shifted the migration coefficient of both bands in the vector control cells as well as the ER{alpha}{Delta}3 clone to new positions, once more as a comigrating doublet (Fig. 4Go, lanes 1 and 4). No lower bands or smear was detected, indicating that proteolysis during CIP incubation was effectively blocked by the protease-inhibitors cocktail. Comigration of the lower molecular mass protein from pMV7 pool vector control cells with that of the ER{alpha}{Delta}3 protein in the transfected clone, both before and after dephosphorylation, strongly suggests the presence of endogenously produced ER{alpha}{Delta}3 protein. Similar results were obtained using the ER-positive Ishikawa cells, an endometrial carcinoma cell line (data not shown).



View larger version (62K):
[in this window]
[in a new window]
 
Figure 4. Identification of the Native 61-kDa Protein as ER{alpha}{Delta}3 on the Basis of Its Dephosphorylation Pattern

Two equal aliquots of ER{alpha} immunoprecipitated with rabbit polyclonal anti-ER{alpha} antibody (Zymed) from 2 mg of protein extracts of pMV7pool vector control or ER{alpha}{Delta}3–3 clone grown in medium with FBS (to enhance the ER{alpha}{Delta}3 to ER{alpha} ratio) were resuspended in 25 µl phosphatase buffer with protease inhibitors (100 µg/ml leupeptin, 100 µg/ml aprotinin, 20 µg/ml pepstatin) and incubated for 30 min at 30 C with 3 U (or without, controls) of CIP (Boehringer Mannheim). The products were analyzed by Western blotting using H226 antibody. The amount of protein loaded per lane was 2.5 times more than in Figs. 3Go or 6. Lanes 1 and 2, pMV7pool vector control: lane 1, CIP treatment; lane 2, buffer control; lanes 3 and 4, ER{alpha}{Delta}3–3, lane 3, buffer control; lane 4, CIP treatment. The dephosphorylated shifted doublets of ER{alpha} and ER{alpha}{Delta}3 are indicated.

 
ER{alpha}{Delta}3 Suppresses Estrogen-Stimulated Gene Expression
ER{alpha}{Delta}3 has been shown to interfere with ER{alpha} binding to its specific DNA response element in vitro, as well as with E2-induced transcription of an ERE-CAT reporter in transient transfection of COS cells in vivo (20). These studies suggested that ER{alpha}{Delta}3 functions as a dominant negative receptor to inhibit ER{alpha} regulation of gene expression through its cognate DNA response element. To determine whether the ER{alpha}{Delta}3 expressed in MCF-7 cells can interfere with estrogen induction of an endogenous gene, the expression of pS2, a gene with several imperfect EREs in its promoter, was assessed. pMV7 pool vector control cells and three individual ER{alpha}{Delta}3 clones were incubated either with the pure antiestrogen, ICI 164,384 (1 x 10-7 M), to establish the baseline of pS2 expression, or with E2 (1 x 10-8 M). Total RNA was prepared and analyzed by Northern blot to determine pS2 expression. (Ribo-somal RNA, Fig. 5Go, bottom, which served as loading control, shows that similar amounts of RNA were used in each lane). As shown in the blot in Fig. 5Go, in which the number of scanned units is inserted under each lane, the ICI 164,384 treatment reduced pS2 expression to below detection in all but one (ER{alpha}{Delta}3–4) clone. The maximal induction of pS2 by E2 (5.8 scanned units) was observed in the pMV7 pool vector control cells. In clone ER{alpha}{Delta}3–3, with the highest ER{alpha}{Delta}3 protein level, E2 induced only 0.4 scanning units, a 93% reduction compared with pMV7 vector control. In the two additional clones the effect was intermediate (21 and 60% reduction). A longer exposure of the Northern blot allowed the visualization of the pS2-band in all the ICI 164,384-treated cells and the calculation of fold stimulation of pS2 by E2 in each individual clone, compared with pMV7 pool vector control. This comparison showed that the presence of ER{alpha}{Delta}3 blocked the E2-induced stimulation by 83%, 91%, and 95% for clone ER{alpha}{Delta}3–4, 2, and 3, respectively (results not shown). Therefore, at least in the two clones (ER{alpha}{Delta}3–2 and 3) in which base line pS2 expression was similar to that of pMV7 pool vector control, ER{alpha}{Delta}3 prevented E2 stimulation of pS2 more effectively than predicted from its dominant negative function. (The third clone, ER{alpha}{Delta}3–4, fell into the same category if comparison of fold induction of pS2 by E2 was considered). Overall these findings confirm that the ER{alpha}{Delta}3 interferes with the ER{alpha}-regulated gene expression in vivo and suggests that more than a single mechanism may be responsible for this effect.



View larger version (55K):
[in this window]
[in a new window]
 
Figure 5. Effect of ER{alpha}{Delta}3 Expression on Estrogen Regulation of pS2-mRNA

pMV7 pool vector control (1 x 106) and ER{alpha}{Delta}3–3 clonal cells were plated in 100-mm tissue culture dishes in the presence of FBS for 3 days and treated for 48 h either with the pure antiestrogen ICI 164,384 (1 x 10-7 M), to establish the baseline of pS2 expression, or E2 (1 x 10-8 M). pS2 expression was determined by Northern blot analysis of 20 µg total RNA using pS2 cDNA probe. Ethidium bromide-stained ribosomal RNA (lower panel) was used as a loading control. pS2 mRNA in pMV7 pool, and clone ER{alpha}{Delta}3–4, -2, and -3 cells treated either with ICI 164,384 (shown in lanes designated as -) or E2, (shown in lanes designated + to indicate E2 addition). Exposure 2 h. Clone ER{Delta}3–3 expresses the highest level (76% of total) of ER{alpha}{Delta}3 protein (see Fig. 3Go).

 
ER{alpha}{Delta}3 Expression Alters Saturation Density of MCF-7 Cells
During the initial selection in medium with FBS, the pMV7-ER{alpha}{Delta}3-transfected cells formed smaller colonies than the parental cells or the vector-transfected clones, and fewer number of cells were obtained from confluent cultures. To further evaluate this difference, ER{alpha}{Delta}3 clones and pMV7 pool vector controls were plated at 2 x 105 cells per 60-mm dish in medium with 10% FBS; the medium was changed every third day. Cells were detached and counted over a period of 8 days. Results in Fig. 6Go (mean of three determinations) show that there were only small differences in the rate of cell division between the clones and the control cells during the exponential phase of their growth. The control cells underwent 0.78 divisions per day, while the clones underwent from 0.61 to 0.7 divisions per day (a 14 ± 5% reduction). It was striking, however, that no increase in cell number of the ER{alpha}{Delta}3-expressing clones was observed once 2.1 to 3.4 x 106 cells accumulated per dish. This plateau cell density level was only 32–50% of that found in the pMV7 pool vector control cells (6.9 x 106) (Fig. 6Go) (P < 0.0001), perhaps indicating that cells expressing ER{alpha}{Delta}3 are more sensitive to signals of contact inhibition. Similarly reduced saturation density of ER{alpha}{Delta}3-expressing clones was obtained when these cells were cultured in 10-8 M E2 supplement (results not shown). This was the first suggestion that the expression of ER{alpha}{Delta}3 shifts the transformed phenotype of breast cancer cells toward behavior expected of normal cells.



View larger version (17K):
[in this window]
[in a new window]
 
Figure 6. Effect of ER{alpha}{Delta}3 Expression on Growth Rate and Saturation Density

pMV7pool vector control (0.2 x 106) and ER{alpha}{Delta}3–1, -2, -3, and -4 clonal cells were plated in 60-mm tissue culture dishes in the presence of FBS. Cells were maintained for 8 days and medium was replaced every 3 days. On days 1 and 2 and every second day thereafter, cells in three dishes of each cell type were detached and counted. The results shown are the mean of three determinations. (SEM values were smaller than the symbols and thus are not shown). Comparison of the saturation plateau of each of the four clones and the vector control by ANOVA statistics performed on day 8 showed a significant difference (P < 0.0001).

 
E2 Regulation of ER{alpha}{Delta}3 to ER{alpha} Protein Ratio
Shifting ER{alpha}{Delta}3-expressing cells into estrogen depleted, charcoal-stripped FBS (csFBS) changed the ER{alpha}{Delta}3 to ER{alpha} protein ratio. Western blot detection of ERs immunoprecipitated from an equal amount of protein of clone ER{alpha}{Delta}3–2 cells, grown either in the presence of csFBS or E2-supplemented FBS, showed that there was more overall receptor protein in cells grown in medium with csFBS (Fig. 7Go) and that the gain was predominantly in the full-length receptor, thus increasing the ER{alpha} to ER{alpha}{Delta}3 protein ratio. (Similar results were obtained with clone ER{alpha}{Delta}3–1; data not shown.) To shift the ER{alpha}{Delta}3 to ER{alpha} ratio in favor of the transgenic protein, all further experiments were carried out on cells grown in medium with FBS and estradiol (although for daily cell maintenance the ER{alpha}{Delta}3-expressing clones were kept in medium supplemented with csFBS).



View larger version (63K):
[in this window]
[in a new window]
 
Figure 7. The Effect of E2 on the Relative ER{alpha}{Delta}3 to ER{alpha} Protein Level

Protein (400 µg), extracted from ER{alpha}{Delta}3–2 cells plated at 4 x 106 per 100-mm dish and grown either in csFBS or FBS with 1 x 10-8 M E2 for 72 h, was immunoprecipitated with rabbit anti-ER{alpha} antibodies (Zymed) and analyzed by Western blotting using H226 antibody as described.

 
ER{alpha}{Delta}3 Attenuates the Transformed Phenotype of MCF-7 Cells
An in vitro property of tumor cells that is thought to predict their in vivo tumorigenicity is their ability for anchorage-independent growth. We examined the consequence of ER{alpha}{Delta}3 expression on the anchorage-independent growth of MCF-7 cells (Fig. 8Go). As shown by others, estrogen stimulated the ability of parental MCF-7 cells (and of the pMV7 pool vector control cells) to form colonies in soft agar. In contrast, hormone treatment drastically reduced the ability of ER{alpha}{Delta}3 expressing clones to form colonies in agar, even below the baseline level (Fig. 8Go). Accordingly, these data suggest that, in vivo, ER{alpha}{Delta}3 may reverse the tumorigenic phenotype of breast cancer cells through an as yet undetermined mechanism.



View larger version (14K):
[in this window]
[in a new window]
 
Figure 8. Anchorage-Independent Growth of ER{alpha}{Delta}3 and pMV7pool Cells

Low melt agarose (Seaplaque, 1% in lower and 0.4% in upper layer) was prepared in DMEM with insulin (5 µg/ml) and 10% FBS (±1 x 10-8 M E2). To assess anchorage-independent growth, pMV7pool and ER{alpha}{Delta}3 clones 1, 2, and 3 cells (2 x 103 cells/ml) mixed with agarose (upper layer) were distributed on top of 5 ml of lower DMEM/agarose layer, grown for 2 weeks, and scored for colony formation. Colonies were scored in one fourth to one half of each dish. The results are the mean of duplicate determinations. Stimulation or inhibition by E2 is expressed as percent of colonies in agarose containing medium with FBS alone. The cloning efficiency of the pMV7 pool cells under control conditions (medium with FBS alone) was 6.5%.

 
To assess the effect of ER{alpha}{Delta}3 expression on the ability of MCF-7 cells to invade host tissue, which is linked to protease production known to be under the control of E2 in these cells (34, 35, 36, 37, 38), we inoculated chick embryo chorioallantoic membrane (CAMs), in vivo, with pMV7 pool vector control cells or ER{alpha}{Delta}3-expressing clones 1, 2, 3, and 4 grown in the presence of E2 and metabolically labeled with [125I]UdR for 24 h. Invasion was measured 24 h after inoculation by a previously described method (39). We determined that, compared with the pMV7 pool vector control cells or the parental MCF-7 cells (results not shown), the ability of ER{alpha}{Delta}3-expressing clones to invade the CAM was reduced by 52–79% (Fig. 9Go).



View larger version (17K):
[in this window]
[in a new window]
 
Figure 9. The Effect of ER{alpha}{Delta}3 Expression on in Vivo Invasion

Eight replicate chick embryo chorioallantoic membranes (CAMs) were inoculated with 3 x 105 cells per CAM of pMV7pool vector control or ER{alpha}{Delta}3- clones 1, 2, 3, or 4 cells grown in the presence of 1 x 10-8 M E2 for 72 h and labeled with 0.2 µCi/ml of[125I]UdR for the last 24 h (specific activity 0.1 to 0.2 cpm/cell). Preparation of CAMs for inoculation and quantification of invasion was as described except that CAMS were resealed before inoculation for 22 h. The results are expressed as median percent invasion. Statistical analysis (ANOVA) performed on all pMV7 controls (n = 24) and all ER{alpha}{Delta}3-expressing clones (n = 32) indicated that the groups were significantly different (P = 0.007).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We determined that a non-DNA-binding ER{alpha} isoform (ER{alpha}{Delta}3) is expressed in normal breast epithelial cells with a median ratio of ER{alpha}{Delta}3 to ER{alpha} of 3.40, with a subset of purified luminal epithelial cells having a median ratio of ER{alpha}{Delta}3 to ER{alpha} of 0.40. In contrast, the median ER{alpha}{Delta}3 to ER{alpha} ratio in breast tumors and tumor cell lines is only 0.10, indicating a substantial underrepresentation of ER{alpha}{Delta}3 in cancer cells (P < 0.0001).

It is curious that the same hormone, estrogen, exerts the tightly controlled effects on growth and differentiation of normal breast cells during puberty and on their cyclical proliferation in an adult nonpregnant female, while also acting as a potent mitogen in breast cancer during its uncontrolled growth and invasion (1, 2). This dichotomy suggests that, during oncogenic transformation, mammary epithelial cells may undergo signaling pathway changes leading to aberrant or inappropriate estrogenic responses. The evidence presented in the current study is the first demonstration that a selective loss of ER{alpha}{Delta}3 may contribute to the phenotypic changes of cancer. The observation showing that small tumors, or tumors that have not spread to the lymph nodes, have ratios of ER{alpha}{Delta}3 to ER{alpha} as low as the more advanced tumors, suggests that the loss of ER{alpha}{Delta}3 may be an early event in carcinogenesis. (However, the finding of significantly lower ratios in tumors with ER{alpha} < 5 fmol/mg, considered more aggressive, hints that a further drop of ER{alpha}{Delta}3 may be associated with disease progression.) It is also worth noting that, in spite of the fact that this latter group of tumors and the normal breast cells have similarly low levels of the receptor mRNA, they have contrasting ratios of ER{alpha}{Delta}3 to ER{alpha}, indicating that the high relative level of ER{alpha}{Delta}3 in normal cells is not the consequence of their overall low receptor level.

The high relative expression of ER{alpha}{Delta}3 in normal breast epithelium and fibroblasts may provide them with a mechanism to regulate and limit the magnitude of responses to estrogen. It can be argued then, that to attain maximum estrogen stimulation of growth and invasive potential during carcinogenesis, breast cells need to be released from the effects of ER{alpha}{Delta}3. Accordingly we demonstrated that a selective loss of this receptor occurs in breast tumors and breast cancer cell lines and that the reintroduction of physiologically relevant levels of ER{alpha}{Delta}3 into breast cancer cells attenuates the mitogenic action of estrogen and reverses several features that distinguish transformed from normal cells.

Most studies of ER{alpha} in normal human mammary tissue have used relatively insensitive immunohistochemical or biochemical techniques. Consequently, only a subset of luminal epithelial cells, and no other cells in normal breast tissue, were considered receptor positive (40). Our study, using RT-PCR, demonstrated ER{alpha} expression both in luminal and basal/myoepithelial cells of the normal breast epithelium (Fig. 1CGo), as well as in stromal fibroblasts. Moreover, mammary fibroblasts, demonstrated to be estrogen responsive (41, 42), were confirmed to express ER{alpha} protein by more sensitive immunofluorescence techniques using a strepavidin amplification of anti-ER{alpha} antibodies (our unpublished results). Thus, several different cell types in the normal adult breast may respond directly to E2. Also, as the high ER{alpha}{Delta}3 ratio (median 2.4) is preserved in breast cancer fibroblasts, it is likely that their presence in cancer tissue may contribute to some degree to the difference in ER{alpha}{Delta}3 to ER{alpha} ratios found in tumors.

The current study has identified the ER{alpha}{Delta}3 protein in cell lines expressing the ER{alpha}{Delta}3 transgene as well as in parental MCF-7 cells and Ishikawa cells; in all cases the ER{alpha}{Delta}3 to ER{alpha} protein ratios were similar to the ER{alpha}{Delta}3 to ER{alpha} RNA ratios (Figs. 3Go and 4Go). The very low abundance of ER{alpha} in normal mammary cells precludes such a direct analysis of ER{alpha} protein in these cells. However, the finding, that in cell lines ER{alpha}-RNA ratios reflect those of the corresponding proteins, makes the likelihood of such correspondence in normal cells highly plausible.

The relevance of our findings is further underscored by the demonstration of an autoregulatory loop in the clones with reexpressed ER{alpha}{Delta}3. In these cells exposure to estrogen can shift the complement of estrogen receptors from mostly ER{alpha} to predominantly ER{alpha}{Delta}3 (Fig. 7Go). This is achieved by a more pronounced down-modulation of ER{alpha} than of ER{alpha}{Delta}3 and, as shown previously (43, 44), may occur via several mechanisms, including mRNA and protein stability. If a similar mechanism of auto-regulation exists in endogenous tissue, it is likely that during periods of peak estrogen availability, a rise in the ER{alpha}{Delta}3 to ER{alpha} protein ratio may protect breast tissue from overstimulation. Thus, oncogenic transformation of breast cancer cells, resulting in a selective reduction in ER{alpha}{Delta}3 expression, would lead to a disruption of this response, promote unchecked estrogen action, and establish permissive conditions for further carcinogenic events.

The reestablishment of a less tumorigenic phenotype in the ER{alpha}{Delta}3-transfected MCF-7 cells deserves further comment because certain of the properties, such as reduced plateau density and reduced invasion, may be the result of dominant negative inhibition by ER{alpha}{Delta}3, while others, such as anchorage-independent growth, may be mediated via additional pathways. Since, as noted, in the ER{alpha}{Delta}3-transfected clones, the relative level of this isoform is highest in the presence of E2, it is interesting that E2 treatment of these clones causes a slight reduction of growth and, more importantly, a much lower saturation density, as is characteristic of a normal phenotype. These effects are specific to the ER{alpha}{Delta}3 isoform, since a similar transfection of full-length ER{alpha} into either MCF-7 or T47D cells (which are also ER{alpha}-positive) did not reduce their proliferative response to hormone (45). Although not yet examined, a testable hypothesis is that a dominant negative receptor interferes with E2 stimulation of genes critical for growth regulation, such as cyclin D1, myc, and the fos/jun family of transcription factors (46, 47). These gene products, in turn, may reduce growth factor receptor expression, resulting in a lower saturation plateau.

The reduced invasiveness of the ER{alpha}{Delta}3-expressing cells may also be mediated via a dominant negative effect. It is known that E2 is necessary for MCF-7 tumor growth and metastasis in nude mice. E2 also stimulates the expression of several proteolytic enzymes (such as plasminogen activators, collagenase IV, cathepsin D), shown to be involved in cancer invasion (34, 35, 36, 37, 38). A testable hypothesis is that the presence of ER{alpha}{Delta}3 will effectively interfere with stimulation of these proteases by E2 to result in reduced invasiveness.

In contrast to the above effects, E2 not only failed to stimulate anchorage-independent growth in ER{alpha}{Delta}3-expressing cells, but inhibited it to below baseline levels. This observation cannot be explained purely on the basis of a dominant negative effect, since even in the two ER{alpha}{Delta}3-expressing clones, in which the isoform protein was only 36–40% of the total ER{alpha}, E2 reduced the growth in agar to below baseline level. Moreover, in a clone with only 40% of ER{alpha}{Delta}3, E2-stimulated pS2 expression was reduced by more than 60%, and in a clone with 75% of ER{alpha}{Delta}3 it was almost completely blocked (Fig. 5Go). Comparing these findings with the published results, showing that a cotransfection of ER{alpha}{Delta}3 and ER{alpha} proteins, at ratios comparable to those present in our clones, produced only a 30% inhibition of estrogen-dependent transactivation (20), suggests the existence of an additional pathway of ER{alpha}{Delta}3 action. Since the total ER{alpha} level in the clones is either equal to or less than that in the parental MCF-7 cells, the observed effect could not be due to the general overexpression of ER{alpha} protein, shown by some to lead to E2 inhibition of growth (48, 49). Although we have not yet investigated the mechanism of the suppressive signal transduction pathway of ER{alpha}{Delta}3, it is likely that this receptor isoform, in addition to its dominant negative action, participates in the nonclassic regulation of gene expression via protein-protein interaction with other transcription factors, which have been shown recently to be 1) independent of ER{alpha} binding to DNA (50), and importantly, 2) independent of the ER{alpha}-DNA-binding domain (51).

Thus we have demonstrated a novel function for a non-DNA-binding ER isoform in breast biology. Relative high expression of this isoform in normal mammary tissue may provide a mechanism for attenuating estrogenic effects, and its reduction in breast cancer may lead to excessive, unregulated mitogenic action of this hormone. Our results indicate that, as with tumor suppressor WT1 (52), carcinogenic events in breast can lead to alteration of splice choice pathways, but unlike suggested for other ER{alpha} isoforms (8, 9, 10, 11, 12, 14, 15, 17, 18, 19, 21), rather than being elevated in cancer, the relative ratio of this isoform is diminished. Further studies of the mechanisms through which ER{alpha}{Delta}3 exerts its effect will clarify its role in controlling E2 responsiveness in mammary cells. Identifying ways to redirect the pathway toward enhanced expression of ER{alpha}{Delta}3, or finding alternative means of increasing its relative ratio, may provide a novel avenue for future breast cancer therapy.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Breast Tissue Specimens
Specimens were provided by the Department of Pathology, Mount Sinai Hospital, either as liquid nitrogen-frozen tissue or in medium for cell separation experiments. The clinical profile of the breast cancer patients (a total of 33) was as follows: median age 63.5, range 32–85 yr; 17% premenopausal; 27% had tumors 1.5 cm or smaller, median 2.0 cm, range 0.8 to 8.0 cm; 36% had no lymph node involvement, 36% were lymph node positive, 28% did not undergo lymph node dissection. Except for two lobular carcinomas, all tumors were infiltrating ductal carcinomas with varying degrees of differentiation. Tumors from six of the 33 patients (18%) had ER levels below 5 fmol/mg. Median ER was 189 fmol/mg (range <5 to 1498 fmol/mg).

ER RT-PCR
Total RNA was extracted using RNAzol B reagent (Biotecx Laboratories, Houston, TX), and 1 µg was reverse-transcribed using Superscript reverse transcriptase (GIBCO BRL, Gaithersburg, MD) and a primer specific to exon 4 (5'-GGAGACATGAGAGCTGCCAAC-3') of ER{alpha}. This exon 4 primer and a primer specific to exon 2 (5'-CCGCAAATGCTACGAAGTGG-3') were used to amplify ER{alpha}-cDNA in a 25-cycle reaction of 1 min each at 95 C, 60 C, and 72 C. PCR products were fractionated on a 2% agarose gel, Southern blotted onto Hybond nylon membrane (Amersham, Arlington Heights, IL), and probed using either a 32P end-labeled internal exon 4 probe (5'-GAATGTTGAAACACACAAGCGCC-3'), detecting full-length ER{alpha} and ER{alpha}{Delta}3, or an exon 3-specific probe (5'-CCGCAAATGCTACGAAGTGG-3'), detecting full-length ER{alpha} only. Quantification was performed using the PhosphorImager ImageQuant program (Molecular Dyanamics, Sunnyvale, CA).

Preparation of ER{alpha}{Delta}3 (ER{alpha}{Delta}3/pMV7) Expression Vector
A partial ER{alpha}-cDNA fragment, containing exons 1, 2, and 4, but missing exon 3 (21), (a gift of Dr. R. Miksicek, State University of New York, Stony Brook, NY) was used to replace exon 1–4 in a similarly digested HEGO vector (a gift of Dr. P. Chambon, Strasbourg, France). The resulting ER{alpha}{Delta}3 coding sequence was purified and ligated into a retroviral expression vector pMV7 (32) under the MuLV promoter. This vector also contains the neomycin resistance gene. The "empty" pMV7 plasmid served as a vector control. Both vectors were used to transform DH5{alpha} bacteria and the DNAs purified using Wizard Maxi-Prep kit (Promega, Madison, WI). To prepare ER{alpha}{Delta}3-coding retrovirus for infection, ER{alpha}{Delta}3/pMV7 DNA was transfected into an amphotropic packaging cell line {Psi}-CRIP and selected with G418, and the virus was collected, pooled, and used for infection.

Maintenance of Control and ER{alpha}{Delta}3 Clonal Cells
MCF-7 cells were maintained in RPMI-1640 medium supplemented with insulin (5 µg/ml), penicillin (50 U/ml), streptomycin (50 µg/ml), and 10% FBS (JRH, Lenexa, KS) (final concentration of E2 from serum, ~3 x 10-12 M). All transfected cells were maintained in selection medium with 500 µg/ml G418. For growth of ER{alpha}{Delta}3 clones, 10% csFBS was used, unless otherwise noted.

Separation of Epithelial and Stromal Cells
Normal reduction mammoplasty specimens or breast cancer samples were obtained from the Pathology Department, Mount Sinai Medical Center. Epithelial organoids were separated from stroma by mincing normal breast tissue and incubating it overnight in hyaluronidase/collagenase mixture as described previously (53). The organoids were collected, by filtering the digest through a 400-mesh sieve, and trypsinized into single-cell suspensions. The filtered single cells were plated and enriched for fibroblasts by differential trypsinization. Tumor tissue was minced and incubated in collagenase for 2 h at 37 C. Tumor digest was plated without filtration, and the cultures were enriched for fibroblasts by differential trypsinization. Fibroblasts passaged on plastic differentiated into myofibroblasts so that after two to three passages ~80% stained positively with antibody to smooth muscle {alpha}-actin.

Separation of Basal and Luminal Epithelium
Basal epithelial cells were positively selected from the filtered digest using a monoclonal antibody to the common acute lymphoblastic leukemia (CALLA) antigen (DAKO, Carpinteria, CA) and Dynabeads (Dynal, Norway) coated with goat anti-mouse IgG (a 10:1 bead to cell ratio) essentially as described elsewhere (31). These basal epithelia-enriched cells were cultured in mammary epithelial cell growth medium (MEGM; Clonetics, San Diego CA) with 5 µg/ml transferrin and 10 µM isoproterenol. The CALLA-negative fraction, containing the luminal cells, was densely seeded onto collagen I-coated dishes in MEGM. After a week in culture, RNAs were extracted and cell purity determined by Northern blot analysis of cytokeratin expression (K8-luminal and K5-basal). Cell preparations with K8 to K5 ratios of 10:1 or 1:10, were defined as luminal or basal cells, respectively. Some epithelial cell preparations, not purified further, were used and designated "unselected." RNAs were extracted from these cell types and used in RT-PCR assay for ER{alpha}{Delta}3 and ER{alpha} analysis.

Generation of MCF-7 Cells Expressing ER{alpha}{Delta}3 (Transfection/Infection)
Three micrograms of ER{alpha}{Delta}3/pMV7 or pMV7 DNA were transfected with Lipofectin into MCF-7 cell, as per manufacturer’s recommendation. For retroviral infection (54) 2 ml of growth medium containing the virus and 8 µg/ml of polybrene were added to semiconfluent MCF-7 cells; the cells were rocked for 2 h at 37 C and the inoculum was removed, after which cells were incubated in medium with serum for 48 h and transferred to medium with G418 (selection medium). Infected and transfected cells were maintained in G418-containing medium for 1–2 months before clone isolation.

Immunoprecipitation and Western Blotting
Total cell protein was prepared by four freeze/thaw cycles in a high- salt lysis buffer (0.4 M NaCl, 10% glycerol, 1 mM dithiothreitol, 100 mM Tris, 10 mM EDTA, 50 µg/ml leupeptin, 50 µg/ml aprotinin, 10 µg/ml pepstatin). ER{alpha} and ER{alpha}{Delta}3 were immunoprecipitated with a rabbit anti-ER{alpha} antibody (Zymed, San Francisco, CA) and protein G agarose (Boerhinger Manheim, Indianapolis, IN) from 400 µg total protein diluted with lysis buffer without NaCl for a final NaCl concentration of 0.2 M. Immunoprecipitated material was resuspended in 50 µl of loading buffer and electrophoresed on an 11.5% SDS/PAGE gel for 8–10 h at 200 V. Protein was transferred onto nitrocellulose membrane (Amersham), blocked overnight with 5% nonfat milk, washed in Tris-buffered saline Tween-20/1% nonfat milk, and Western blotted with H226 (0.7 mg/ml) rat anti-ER{alpha} primary antibody (1:100 dilution) overnight at 4 C, and incubated with an horseradish peroxidase-conjugated goat anti-rat secondary antibody (1:10,000 dilution) (Sigma, St. Louis, MO) for 1 h at room temperature. Enhanced chemiluminescence (ECL Kit, Amersham) detected bands were quantitated by densitometry.

Phosphatase Treatment of Protein Extract from pMV7 and ER{alpha}{Delta}3 Clone
Total ER{alpha} was immunoprecipitated from 2 mg of protein from pMV7pool and ER{alpha}{Delta}3 clonal cells, using rabbit polyclonal anti-ER{alpha} antibody (Zymed). Immunoprecipitated material was split into two equal aliquots, resuspended in 25 µl 1x phosphatase buffer (Boerhinger Mannheim, 10x phosphatase buffer: 0.5 M Tris-HCl, pH 8.5, 1 mM EDTA), containing a 2x protease cocktail (100 µg/ml leupeptin, 200 µg/ml bacitracin, 100 µg/ml aprotinin, 20 µg/ml pepstatin). One aliquot each of pMV7pool and ER{alpha}{Delta}3–3 were treated with 3 U of CIP (Boehringer Mannheim) and, along with the mock-treated aliquots, were incubated for 30 min at 30 C. The reaction was terminated by the addition of 25 µl of 2x loading buffer and heating to 95 C for 3 min. Western blot analysis was performed as described above.

Expression of pS2
pMV7pool and ER{alpha}{Delta}3- 2, 3, and 4 clonal cells (1 x 106) were grown for 3 days in 100-mm tissue culture dishes in the presence of FBS and subsequently treated with either ICI 164,384 (1 x 10-7 M) or E2 (1 x 10-8 M) for 2 days. pS2 expression was assessed by Northern blotting 20 µg of total RNA, hybridized with random primed pS2 cDNA probe. Ethidium bromide-stained ribosomal RNA served as loading control. pS2-mRNA level was determined by densitometric scanning.

Saturation Density
pMV7pool and ER{alpha}{Delta}3 clonal cells (0.2 x 106) were plated in 60-mm tissue culture dishes (three dishes per cell type per time point) in the presence of FBS. Medium was changed every 3 days, and the cells were detached with trypsin and counted on day 1, 2, 4, 6, and 8. The ER{alpha}{Delta}3-expressing clones were deemed to be in saturation plateau when 2 days of growth did not produce a further increase in cell number. The five groups of cells (four clones and pMV7 pool vector control) were analyzed by ANOVA statistics.

Growth in Soft Agar
A two-layer low melt agarose (Seaplaque; FMC Corporation, Rockland, ME) system was used to assess anchorage-independent growth of pMV7pool and ER{alpha}{Delta}3 clonal cells. A 1% lower layer and an 0.4% upper layer of agarose, prepared in DMEM medium supplemented with insulin (5 µg/ml), penicillin (50 U/ml), streptomycin (50 µg/ml), and 10% FBS (±E2 1 x 10-8 M) inoculated into 60-mm gridded plates. Cells (2 x 103 cells/ml), distributed in the upper layer, were allowed to grow for 2 weeks and colony formation in the two conditions was scored. The effect of E2 was determined by comparison to cloning efficiency in FBS.

Chorioallantoic Membrane Invasion
Invasion was assayed as previously described (39). ER{alpha}{Delta}3 clones or pMV7pool cells were grown in the presence of selection medium supplemented with 10% FBS and estradiol (1 x 10-8 M) for 48 h. Cells were trypsinized, counted, allowed to attach overnight in the same medium (4 x 106 cells per 100-mm dish), and labeled with 0.2 µCi/ml of [125]UdR for 24 h (specific activity of 0.1–0.2 cpm/cell). An artificial air chamber above the CAM of a 10-day-old embryo was created, and the CAM was allowed to reseal for 22 h and the labeled cells (3 x 105 per CAM) were inoculated onto the CAM. After a 24-h incubation, CAMs were washed with PBS, excised, incubated for 20 min in trypsin-EDTA (0.05% trypsin, 1 mM EDTA) to remove surface-attached tumor cells, and rinsed with PBS. The radioactivity remaining in CAMs after the trypsin-EDTA incubation and the PBS wash, expressed as percent of total radioactivity (associated with CAMs before trypsinization and present in washes), represent the proportion of cells that invaded. The ANOVA test was used for the statistical analysis.


    ACKNOWLEDGMENTS
 
We thank Y-K. Jing for help with the characterization of normal epithelial cells, C. A. Chaparro for expert technical assistance, Drs. G. Greene for the gift of the H226 antibodies, P. Chambon for HEGO vector, R. Miksicek for the partial ER{alpha}{Delta}3 clone, S. Lehrer for tumor samples, P. Fedi for breast cancer cell lines, and S. Waxman for helpful discussions and support.


    FOOTNOTES
 
Address requests for reprints to: L. Ossowski, Department of Medicine, Box 1178, 1 Gustave Levy Place, Mount Sinai Medical Center, New York, New York 10029.

This work was supported by a predoctoral fellowship from The Department of Defense, DAMD17–94-J-4140 (to I.E); NIH Grants HD-27557 and ES-90228 (to B.S); NIH Grant CA-54273 (to R.M.L.); NIH Grant CA-40758 (to L.O.); and funds from the Rochelle Belfer Chemotherapy Foundation and Samuel Waxman Cancer Research Foundation.

Received for publication May 21, 1997. Revision received September 12, 1997. Accepted for publication September 15, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Topper YJ, Freeman CS 1980 Multiple hormone interactions in the developmental biology of the mammary gland. Physiol Rev 60:1049–1106[Free Full Text]
  2. Lippman ME, Dickson RB 1990 Regulation of normal and malignant breast epithelial proliferation. In: Growth Regulation of Cancer II. Alan R. Liss, Washington, DC, vol. 115:127–172
  3. Truss M, Chelepakis G, Pina B, Barettino D, Bruggemeier U, Kalff M, Slater EP, Beato M 1992 Transcriptional control by steroid hormones. J Steroid Biochem Mol Biol 41:241–248[CrossRef][Medline]
  4. Kumar V, Green S, Stack G, Berry M, Jin JR, Chambon P 1987 Functional domains of the human estrogen receptor. Cell 51:941–951[Medline]
  5. Koenig RJ, Lazar MA, Hodin RA, Brent GA, Larsen PR, Chin WW, Moore DD 1989 Inhibition of thyroid hormone action by a non-hormone binding c-erbA protein generated by alternative RNA splicing. Nature 337:659–661[CrossRef][Medline]
  6. Mosselman S, Polman J, Dijkema R 1996 ERß: identification and characterization of a novel human estrogen receptor. FEBS Lett 392:49–53[CrossRef][Medline]
  7. Leygue ER, Watson PH, Murphy LC 1996 Estrogen receptor variants in normal human mammary tissue. J Natl Cancer Inst 88:284–290[Abstract/Free Full Text]
  8. Fuqua SAW, Fitzgerald SD, Allred DC, Elledge RM, Nawaz Z, McDonnell DP, O’Malley BW, Greene GL, McGuire WL 1992 Inhibition of estrogen receptor action by a naturally occurring variant in human breast tumors. Cancer Res 52:483–486[Abstract]
  9. Fuqua SAW, Fitzgerald SD, Chamness GC, Tandon AK, McDonnell DP, Nawaz Z, O’Malley BW, McGuire WL 1991 Variant human breast tumor estrogen receptor with constitutive transcriptional activity. Cancer Res 51:105–109[Abstract]
  10. Castles CG, Fuqua SAW, Klotz DM, Hill SM 1993 Expression of a constitutively active estrogen receptor variant in the estrogen receptor negative BT-20 human breast cancer cell line. Cancer Res 53:5934–5939[Abstract]
  11. Zhang QX, Borg A, Fuqua SAW 1993 An exon 5 deletion variant of the estrogen receptor frequently co-expressed with wild-type estrogen receptor in human breast cancers. Cancer Res 53:5882–5884[Abstract]
  12. Parker MG, Rea D 1996 Effects of an exon 5 variant of the estrogen receptor in MCF7 breast cancer cells. Cancer Res 56:1556–1563[Abstract]
  13. Daffada AAI, Dowsett M 1995 Tissue-dependent expression of a novel splice variant of the human oestrogen receptor. J Steroid Biochem Mol Biol 55:413–421[CrossRef][Medline]
  14. Villa E, Camellini L, Dugani A, Zucchi F, Grottola A, Merighi A, Buttafoco P, Losi L, Manenti F 1995 Variant estrogen receptor messenger RNA species detected in human primary hepatocellular carcinoma. Cancer Res 55:498–500[Abstract]
  15. Koehorst SGA, Jacobs HM, Thijssen JHH, Blankenstein MA 1993 Wild type and alternatively spliced estrogen receptor messenger RNA in human meningioma tissue and MCF7 breast cancer cells. J Steroid Biochem Mol Biol 45:227–233[CrossRef][Medline]
  16. Daffada AAI, Johnston SR, Smith IE, Detre S, King N, Dowsett M 1995 Exon 5 deletion variant receptor messenger RNA expression in relation to tamoxifen resistance and progesterone receptor/pS2 status in human breast cancer. Cancer Res 55:288–293[Abstract]
  17. Daffada AAI, Johnston SRD, Nicholls J, Dowsett M 1994 Detection of wild type and exon 5 deleted splice variant oestrogen receptor (ER) mRNA in ER-positive and negative breast cancer cell lines by reverse transcription/polymerase chain reaction. J Mol Endocrinol 13:265–273[Abstract]
  18. Dotzlaw H, Alkhalaf M, Murphy LC 1992 Characterization of estrogen receptor variant mRNAs from human breast cancers. Mol Endocrinol 6:773–785[Abstract]
  19. Pfeffer U, Fecaroota N, Catagnetta L, Vidali G 1993 Estrogen receptor variant messenger RNA lacking exon 4 in estrogen responsive human breast cancer cell lines. Cancer Res 53:741–743[Abstract]
  20. Wang Y, Miksicek RJ 1991 Identification of a dominant negative form of the human oestrogen receptor. Mol Endocrinol 5:1707–1715[Abstract]
  21. Miksicek RJ, Lei Y, Wang Y 1993 Exon skipping gives rise to alternatively spliced forms of the estrogen receptor in breast tumor cells. Breast Cancer Res Treat 26:163–174[Medline]
  22. Friend KE, Ang LW, Shupnik MA 1995 Estrogen regulates the expression of several different estrogen receptor mRNA isoforms in rat pituitary. Proc Natl Acad Sci USA 92:4367–4371[Abstract]
  23. Madsen MW, Reiter BE, Larsen SS, Bieand P, Lykkesfeldt AE 1997 Estrogen receptor messenger RNA splice variants are not involved in antiestrogen resistance in sublines of MCF-7 human breast cancer cells. Cancer Res 57:585–589[Abstract]
  24. Pfeffer U, Fecarotta E, Vidali G 1995 Coexpression of multiple estrogen receptor variant messenger RNAs in normal and neoplastic breast tissues and in MCF-7 cells. Cancer Res 55:2158–2165[Abstract]
  25. Tung L, Mohamed MK, Hoeffler JP, Takimoto GS, Horwitz KB 1993 Antagonist-occupied human progesterone B-receptors activate transcription without binding to progesterone response elements and are dominantly inhibited by A-receptors. Mol Endocrinol 7:1256–1265[Abstract]
  26. Ince BA, Zhuang Y, Wrenn CK, Shapiro DJ, Katzenellenbogen BS 1993 Powerful dominant negative mutants of the human estrogen receptor. J Biol Chem 268:14026–14032[Abstract/Free Full Text]
  27. Schodin DJ, Zhuang Y, Shapiro DJ, Katzenellenbogen BS 1995 Analysis of mechanisms that determine dominant negative estrogen receptor effectiveness. J Biol Chem 270:31163–31171[Abstract/Free Full Text]
  28. Ince BA, Schodin DJ, Shapiro DJ, Katzenellenbogen BS 1995 Repression of endogenous estrogen receptor activity in MCF-7 human breast cancer cells by dominant negative estrogen receptors. Endocrinology 136:31194–3199
  29. Taylor-Papadimitriou J, D’Souza B, Burchell J, Kyprianou N, Berdichevsky F 1993 The role of tumor-associated antigen in the biology and immunotherapy of breast cancer. Ann NY Acad Sci 698:31–47[Medline]
  30. Bartek J, Bartkova J, Kyprianou N, Lalani E-N, Staskova Z, Shearer M, Chang S, Taylor-Papadimitriou J 1991 Efficient immortalization of luminal epithelial cells from human mammary glands by introduction of simian virus 40 large tumor antigen with a recombinant retrovirus. Proc Natl Acad Sci USA 88:3520–3524[Abstract]
  31. Stampfer M, Hallowes RC, Hackett AJ 1980 Growth of normal human mammary cells in culture. In Vitro 16:415–425[Medline]
  32. Kirschmeier PT, Housey GM, Johnson MD, Perkins AS, Weinstein IB 1988 Construction and characterization of a retroviral vector demonstrating efficient expression of cloned cDNA sequences. DNA 7:219–225[Medline]
  33. Greene GL, Press MF 1986 Structure and dynamics of the estrogen receptor. J Steroid Biochem 24:1–7[CrossRef][Medline]
  34. Mira-Y-Lopez R, Osborne MP, DePalo AJ, Ossowski L 1991 Estradiol modulation of plasminogen activator production in organ cultures of human breast carcinomas: correlation with clinical outcome of anti-estrogen therapy. Int J Cancer 47:827–832[Medline]
  35. Burg B, Groot RP, Isbrucker L, Kruijer W, Laat SW 1990 Stimulation of tPA-responsive element activity by a cooperative action of insulin and estrogen in human breast cancer cells. Mol Endocrinol 4:1720–1726[Abstract]
  36. Touitou I, Cavailles V, Garcia M, Defrenne A, Rocheford H 1989 Differential regulation of cathepsin D by sex steroids in mammary cancer and uterine cells. Mol Cell Endocrinol 66:231–238[CrossRef][Medline]
  37. Davis MD, Butler WB, Brooks SC 1995 Induction of tissue plasminogen activator mRNA and activity by structurally altered estrogens. J Steroid Biochem Mol Biol 52:421–430[CrossRef][Medline]
  38. Thompson EW, Reich R, Shima TB, Albini A, Graf J, Martin GR, Dickson RB, Lippman ME 1988 Differential regulation of growth and invasiveness of MCF7 breast cancer cells by anti-estrogen. Cancer Res 48:6764–6768[Abstract]
  39. Ossowski L 1988 In vivo invasion of modified chorioallantoic membrane by tumor cells: the role of cell surface bound urokinase. J Cell Biol 107:2437–2445[Abstract]
  40. Peterson OW, Hoyer PE, Deurs B 1987 Frequency and distribution of estrogen receptor positive cells in normal, non-lactating human breast tissue. Cancer Res 47:5748–5751[Abstract]
  41. Cullen KJ, Lippman ME 1991 Stromal-epithelial interactions in breast cancer. In: Dickson RB, Lippman ME (eds) Genes, Oncogenes, and Hormones: Advances in Cellular and Molecular Biology of Breast Cancer, Chapter 20. Kluwer Academic Publishers, pp 413–431
  42. Haslam SZ 1991 Stromal-epithelial interactions in normal and neoplastic mammary gland. In: Dickson RB, Lippman ME (eds) Regulatory Mechanisms in Breast Cancer: Advances in Cellular and Molecular Biology of Breast Cancer, Chapter 19. Kluwer Academic Publishers pp 401–420
  43. Borras M, Hardy L, Lempereur F, el-Khissiin AH, Legros N, Gol-Winkler R, Leclercq G 1994 Estradiol-induced down regulation of estrogen receptor. Effects of various modulators of protein synthesis and expression. J Steroid Biochem Mol Biol 48:325–336[CrossRef][Medline]
  44. Kaneko KJ, Furlow JD, Gorski J 1993 Involvement of the coding sequence for the estrogen receptor gene in autologous ligand-dependent down regulation. Mol Endocrinol 7:879–888[Abstract]
  45. Zajchowski DA, Sager R, Webster L 1993 Estrogen inhibits the growth of estrogen receptor negative, but not estrogen receptor positive cells expressing a recombinant estrogen receptor. Cancer Res 53:5004–5011[Abstract]
  46. Umayahara Y, Kawamori R, Watada H, Jmano E, Iwama N, Morishima T, Yamasaki Y, Kajimoto Y, Kamada T 1994 Estrogen regulation of the insulin-like growth factor I gene transcription involves an AP-1 enhancer. J Biol Chem 269:16433–16442[Abstract/Free Full Text]
  47. Watts CKW, Sweeney KJE, Warlters A, Musgrove A, Sutherland RL 1994 Antiestrogen regulation of cell cycle progression cyclin D1 gene expression in MCF7 human breast cancer cells. Breast Cancer Res Treat 31:95–105[Medline]
  48. Maminta ML, Molteni A, Rosen ST 1991 Stable expression of the human estrogen receptor in HeLa cells by infection: effect of estrogen on cell proliferation and c-myc expression. Mol Cell Endocrinol 78:61–69[CrossRef][Medline]
  49. Touitou I, Mathieu M, Rochefort H 1990 Stable transfection of the estrogen receptor cDNA into Hela cells induces estrogen respoonsiveness of endogenous cathepsin D gene but not of cell growth. Biochem Biophys Res Commun 169:109–115[Medline]
  50. Kraus WL, Weis KE, Katzenellenbogen BS 1995 Inhibitory cross-talk between steroid hormone receptors: differential targeting of estrogen receptor in the repression of its transcriptional activity by agonist and antagonist occupied progestin receptors. Mol Cell Biol 15:1847–1857[Abstract]
  51. Webb P, Lopez GN, Uht RM, Kushner PJ 1995 Tamoxifen activation of the estrogen receptor/AP1 pathway: potential origin for the cell-specific estrogen like effects of antiestrogens. Mol Endocrinol 9:443–456[Abstract]
  52. Haber DA, Park S, Maheswaran S, Englert C, Re GG, Hazen-Martin DJ, Sens DA, Garvin AJ 1993 WT1 mediated growth suppression of Wilms’ tumor cells expressing a WT1 splicing variant. Science 262:2057–2059[Medline]
  53. Dairkee S, Heid HW 1993 Cytokeratin profile of immunomagnetically separated epithelial subsets of the human mammary gland. In Vitro Cell Dev Biol 29A:427–432
  54. Danos O, Mulligan RC 1988 Safe and efficient genera-tion of recombinant retroviruses with amphotropic and ecotropic host ranges. Proc Natl Acad Sci USA 85:6460–6464[Abstract]