Clinical Cancer Research CR Balducci Advances in Breast Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Clinical Cancer Research 13, 7029, December 1, 2007. doi: 10.1158/1078-0432.CCR-07-0587
© 2007 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bayliss, J.
Right arrow Articles by El-Ashry, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bayliss, J.
Right arrow Articles by El-Ashry, D.
Related Collections
Right arrowCommentary

Imaging, Diagnosis, Prognosis

Reversal of the Estrogen Receptor–Negative Phenotype in Breast Cancer and Restoration of Antiestrogen Response

Jill Bayliss1, Amy Hilger1, Prakash Vishnu3, Kathleen Diehl2 and Dorraya El-Ashry1

Authors' Affiliations: 1 Department of Internal Medicine, Division of Hematology/Oncology, and 2 Department of Surgery, University of Michigan Medical Center, Ann Arbor, Michigan; and 3 Department of Internal Medicine, Detroit Medical Center/Wayne State University, Detroit, Michigan

Requests for reprints: Dorraya El-Ashry, University of Michigan Health System, 1150 West Medical Center Drive, MSRB III, Room 5220B, Ann Arbor, MI 48109-0640. Phone: 734-764-5585; Fax: 734-734-0101; E-mail: elashryd{at}umich.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: In breast cancer, the presence of estrogen receptor {alpha} (ER) denotes a better prognosis and response to antiestrogen therapy. Lack of ER{alpha} correlates with overexpression of epidermal growth factor receptor or c-erbB-2. We have shown that hyperactivation of mitogen-activated protein kinase (MAPK) directly represses ER{alpha} expression in a reversible manner. In this study, we determine if inhibition of MAPK in established ER{alpha}– breast cancer cell lines and tumors results in reexpression of ER{alpha}, and further, if reexpression of ER{alpha} in these ER{alpha}– tumors and cell lines could restore antiestrogen responses.

Experimental Design: Established ER{alpha}– breast cancer cell lines, ER{alpha}– breast tumors, and tumor cell cultures obtained from ER{alpha}– tumors were used in this study. Inhibition of hyperactive MAPK was accomplished via the MAPK/ERK kinase 1/2 inhibitor U0126 or via upstream inhibition with Iressa or Herceptin. Western blotting or reverse transcription-PCR for ER{alpha} was used to assess the reexpression of ER{alpha} in cells treated with U0126. Growth assays with WST-1 were done to assess restoration of antiestrogen sensitivity in these cells.

Results: Inhibition of MAPK activity in ER{alpha}– breast cancer cell lines results in reexpression of ER{alpha}; upstream inhibition via targeting epidermal growth factor receptor or c-erbB-2 is equally effective. Importantly, this reexpressed ER{alpha} can now mediate an antiestrogen response in a subset of these ER{alpha}– breast cancer cell lines. Treatment of ER{alpha}– tumor specimens with MAPK inhibitors results in restoration of ER{alpha} mRNA, and similarly in epithelial cultures from ER{alpha}– tumors, MAPK inhibition restores both ER{alpha} protein and antiestrogen response.

Conclusions: These data show both the possibility of restoring ER{alpha} expression and antiestrogen responses in ER{alpha}– breast cancer and suggest that there exist ER{alpha}– breast cancer patients who would benefit from a combined MAPK inhibition/hormonal therapy.

Breast cancer patients with tumors that do not express estrogen receptors (ER{alpha}) have a very poor prognosis. The presence of ER{alpha} not only confers increased disease-free survival and overall survival but, more importantly, also predicts for response to hormonal therapies such as tamoxifen (1–4). ER{alpha}– breast cancers, which account for approximately one third of all invasive breast cancers, are associated with a more aggressive and metastatic phenotype and overexpression of tyrosine kinase receptors such as the epidermal growth factor receptor (EGFR) or c-erbB-2, with resultant hyperactivation of downstream signaling pathways including p42/44 mitogen-activated protein kinase (MAPK; refs. 5–15). Several questions exist about the biological pathways and alterations involved in determining ER{alpha} expression and its regulation during breast cancer evolution. Current evidence suggests that most ER{alpha}– breast cancers arise from ER{alpha}+ cells that stop expressing the receptor (16). Proposed mechanisms for the origin of ER{alpha}– breast cancers include that of pressures being exerted on ER{alpha}+ cells by hyperactivation of MAPK due to overexpression of EGFR or c-erbB-2 (17), estrogen withdrawal (18, 19), or hypoxia (20, 21), as well as more permanent epigenetic alterations such as methylation of CpG islands in the ER{alpha} gene promoter region (22, 23). Using engineered cell line models of up-regulated growth factor signaling through EGFR, erbB-2, Raf, and MAPK/ERK kinase, we have previously shown that hyperactivation of MAPK directly represses ER{alpha} expression and, importantly, that this repression is reversible via inhibition of MAPK (17, 24). Recently, we have established a MAPK gene profile that accurately distinguishes ER{alpha}– from ER{alpha}+ breast cancer, further showing the link between hyperactive MAPK and the ER{alpha}– phenotype (25).

In this study, we have investigated the likelihood of reversing the ER{alpha}– phenotype and restoring response to antiestrogens in established ER{alpha}– breast cancer lines and in ER{alpha}– tumor specimens. The hypothesis that we explore here is that abrogation of the MAPK pathway by either direct inhibition of hyperactivated MAPK or upstream inhibition of overexpressed growth factor receptor (EGFR and/or erbB-2) signaling will result in reexpression of ER{alpha} and, thus, restoration of estrogen-dependence and antiestrogen sensitivity in a subset of ER{alpha}– breast cancers.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Drugs. U0126 was from Upstate and Herceptin (trastuzumab) was from Genentech. DMSO and 17β-estradiol (E2) were from Sigma. (Z)-4-Hydroxy-tamoxifen was from Calbiochem (EMD Biosciences) and ICI 182,780 was from Tocris Bioscience. Iressa was from AstraZeneca.

Cell lines. Cells were incubated in a 37°C, 5% CO2 forced-air humidified incubator. SUM 149 and SUM 229 cells were grown in F-12 Nutrient Mixture (Ham) with 5% fetal bovine serum, 10 nmol/L insulin, 5 µg/mL gentamicin (all Life Technologies, Inc./Invitrogen), 0.5 µg/mL fungizone (Cambrex), and 1 µg/mL hydrocortisone (Sigma). SUM 190 cells were grown in F-12 Nutrient Mixture (Ham) but without serum and with the following additions: 5 mmol/L ethanolamine, 10 mmol/L HEPES, 0.5 g/L albumin bovine serum fraction V, 10 nmol/L T3 (as 3,3',5-triiodo-L-thyronine sodium salt; all from Sigma), 5 µg/mL transferrin, and 50 µmol/L selenium (Life Technologies). (ca)erbB-2-MCF-7 cells were grown in phenol red–free modified IMEM with L-glutamine, without gentamicin sulfate (Life Technologies), with 10% charcoal-stripped calf serum (Valley Biomedical).

Gel electrophoresis and Western blotting. Whole-cell protein lysates were prepared from cells grown to ~80% confluence using Gold Lysis Buffer [20 mmol/L Tris (pH 7.9), 137 mmol/L NaCl, 5 mmol/L EDTA, 10% glycerol, 1% Triton X-100, supplemented with 77 µg/mL aprotinin, 47 µg/mL leupeptin, 250 µg/mL pefabloc SC, 1 µg/mL pepstatin, and 184 µg/mL sodium orthovanadate]. Protein concentrations were obtained using the BCA Protein Assay Kit (Pierce). Protein was denatured in Laemmli sample buffer (Bio-Rad), supplemented with 2-mercaptoethanol, by boiling for 3 min followed by quick chilling on ice. The denatured protein was loaded on 7.5% or 10% Tris-glycine PAGEr Gold Precast gels (Cambrex) and electrophoresed in 1x Tris-glycine-SDS buffer (25 mmol/L Tris, 192 mmol/L glycine, 0.1% SDS, pH 8.3; Bio-Rad). Gels were transferred onto Hybond-P polyvinylidene difluoride membrane (Amersham) in 1x Tris-glycine buffer (25 mmol/L Tris, 192 mmol/L glycine, 20% methanol, pH 8.3; Bio-Rad). Following transfer, the membranes were blocked for 1 h at room temperature in blocking buffer [10 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.1% Tween 20, 5% bovine serum albumin]. Membranes were incubated overnight at 4°C in primary antibody diluted in blocking buffer. The following primary antibodies were used: mouse anti-ER (1D5; Zymed) and phospho-Thr202/Tyr204 p44/p42 MAPK (Cell Signaling Technology). Membranes were washed with TBS-T [10 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.1% Tween 20]. Secondary antibody, diluted in blocking buffer, was added for 1 h at room temperature. The following secondary antibodies were used: enhanced chemiluminescence antimouse IgG horseradish peroxidase–linked whole antibody (from sheep; Amersham) and enhanced chemiluminescence antirabbit IgG horseradish peroxidase–linked whole antibody (from donkey; Amersham). Membranes were washed with TBS-T. Chemiluminescent detection was accomplished using SuperSignal West Pico substrate (Pierce) following the manufacturer's protocol. Following detection, membranes were stripped with Restore Western Blot Stripping buffer (Pierce) for 15 min at room temperature, then washed in TBS-T. The stripped membranes were then probed for actin to verify even loading. Actin (I-19) horseradish peroxidase–conjugated antibody (Santa Cruz Biotechnology) was used following the above protocol, with the exclusion of the secondary antibody step.

Growth assays. Twenty-four hours before adding the treatments, 1,000 to 3,000 cells (depending on cell line) were plated in each well of a 96-well tissue culture plate in 100-µL medium. Treatments were made at 2x concentration in medium and 100 µL were added to the preplated cells. Cells were incubated for 0 (in growth medium only), 2, 4 or 6 days. SUM cell lines were refed and re-treated every 24 to 48 h (depending on cell line and dose of inhibitor). At each time point, 1/10 volume of Cell Proliferation Reagent WST-1 (Roche Applied Science) was added to each well and the plates were incubated at 37°C for 60 to 90 min (consistent time per cell line, but differing times for each cell line). Absorbances were read at 450/630 nm on a microplate reader [Dynex Revelation (Dynex Technologies) or Bio-Rad Benchmark Plus].

Ex vivo tumors, tumor dissociation, and tumor cell cultures. Breast tumors were procured from surgical patients by the Tissue Procurement Core of the University of Michigan Comprehensive Cancer Center, following the Institutional Review Board protocol. The freshly procured tissue was used immediately for experiments or dissociation. For experiments using tissue chunks (ex vivo tumors), tumor was minced into small pieces and randomly divided into treatment groups. Each group was cultured in a 100-mm TC plate containing phenol red–free, modified IMEM with L-glutamine, without gentamicin sulfate (Life Technologies/Invitrogen), with 10% charcoal-stripped calf serum (Valley Biomedical), 50 µg/mL gentamicin, and the appropriate treatment. Treatments consisted of vehicle control (DMSO) or 10 µmol/L U0126. Samples were incubated at 37°C, 5% CO2 for 20 h before harvest for RNA (except for tumor no. 15, which was incubated for 48 h). To create cell cultures, tumors were minced into small pieces with a scalpel and dissociated in serum-free modified IMEM, with L-glutamine, without gentamicin sulfate and phenol red, supplemented with 300 units/mL collagenase type 3, 100 units/mL hyaluronidase (both from Worthington Biochemical), 2% bovine serum albumin fraction V, and 5 µg/mL recombinant human insulin (Sigma), at 37°C, 5% CO2 with gentle agitation for 5 to 16 h until the majority of tissue was digested. The dissociated cells were centrifuged at 100 x g for 5 min to pellet the epithelial cells. The epithelial cell pellet was plated in phenol red–containing modified IMEM with L-glutamine, without gentamicin sulfate (Life Technologies/Invitrogen), with 10% fetal bovine serum (Valley Biomedical), 5 µg/mL recombinant human insulin, and 50 µg/mL gentamicin (Life Technologies/Invitrogen) in tissue culture flask. Cells were continuously passaged with 0.05% trypsin, 0.53 mmol/L EDTA until only the tumor epithelial cell population remained. These stable tumor cell populations were named DT5 [ER–/progesterone receptor (PR)–/H2N– designation by pathology], DT6 (ER+/PR+/H2N– designation by pathology), DT13 (ER–/PR–/H2N+ designation by pathology), and DT16 (ER–/PR–/H2N– designation by pathology). DT5, DT13, and DT16 were also grown in the same media but minus phenol red and insulin and with 10% charcoal-stripped calf serum.

Real-time PCR. Before RNA extraction, tumor chunks were stored in RNAlater (Ambion). Tissue chunks were homogenized in TRIzol reagent (Life Technologies/Invitrogen) with a glass dounce homogenizer. RNA was extracted according to the TRIzol manufacturer's protocol. RNA from cell lines was also extracted with TRIzol reagent per manufacturer's protocol. RNA was DNase treated using TURBO DNase (RNase-Free; Ambion) following the manufacturer's protocol. The DNase-treated RNA was then subjected to reverse transcription using TaqMan reverse transcription reagents (Applied Biosystems). RNA was denatured at 65°C for 5 min followed by a quick chill on ice before addition to the reverse transcription reaction. Plus reverse transcription reactions were carried out in a final volume of 10 µL with 0.1 µg RNA, 1x reverse transcription buffer, 5.5 mmol/L MgCl2, 500 µmol/L of each deoxynucleotide triphosphate, 2.5 µmol/L random hexamers, 0.4 units/µL RNase inhibitor, and 3.125 units/µL MultiScribe reverse transcriptase. Minus reverse transcription reactions were carried out in the same manner as for plus reverse transcription reactions, but MultiScribe reverse transcriptase was omitted. The plus and minus reverse transcription reactions were incubated at 25°C for 10 min, 37°C for 60 min, 95°C for 5 min, followed by a 4°C hold in a thermocycler. Real-time PCR was carried out to determine relative quantification using the comparative CT method (2–{Delta}{Delta}CT; ref. 26). Plus reverse transcription reactions were run in triplicate per sample per gene. Minus reverse transcription reactions and no template controls were run in duplicate per sample per gene to verify that all amplifications are due to cDNA. Real-time PCR for ER{alpha} and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) on the tumor chunk RNA was done on the iCycler IQ Real-time PCR Detection System (Bio-Rad) in a 25-µL reaction volume of 1x iQ SYBR Green Supermix (Bio-Rad), 5 ng of reverse transcription reaction, and 300 nmol/L of each ER{alpha} primer or 200 nmol/L of each GAPDH primer. Reactions were cycled for 10 min at 95°C, followed by 40 cycles of 95°C for 15 s/60°C for 1 min (during which time data collection and real-time analysis were enabled). The following primer sequences were used: ER{alpha}-F, 5'-CCACCAACCAGTGCACCATT; ER{alpha}-R, 5'-GGTCTTTTCGTATCCCACCTTTC (27); GAPDH-F, 5'-CACCAGGGCTGCTTTTAACTCTGGTA; GAPDH-R, 5'-CCTTGACGGTGCCATGGAATTTGC (GAPDH primer sequences obtained from Bio-Rad Tech Note 2804). Real-time PCR for GREB1 and GAPDH on cell line RNA was done on the Applied Biosystems 7900HT Sequence Detection System in a 25-µL reaction volume of 1x TaqMan Universal PCR Master Mix (Applied Biosystems), 10-ng reverse transcription reaction, and 1x TaqMan Gene Expression Assay. Reactions were cycled at 95°C for 10 min and 40 cycles of 95°C for 15 s/60°C for 1 min. The TaqMan Gene Expression Assays (Applied Biosystems) GREB1 Hs00536409_m1 and GAPDH Hs99999905_m1 were used. Results were calculated using the comparative CT method (2–{Delta}{Delta}CT). Samples were normalized to GAPDH and are relative to vehicle control (DMSO)–treated samples.

Statistical analysis. To determine if differences in growth assays between different hormone treatments were significant, a two-tailed, type 1 Student's t test was done with n = 5 for all cell treatment groups. The type 1 test was chosen because in each growth assay, it was different treatments of the same cells being compared and not different cell types being compared. In each assay, U0126 + tam was compared with U0126 alone and, similarly, U0126 + ICI 182,780 was compared with U0126 alone. For DT16 cells in Fig. 5, the additional comparisons of U0126 + tam + E2 with U0126 + tam and U0126 + ICI 182,780 + E2 with U0126 + ICI 182,780 were done. For coMCF-7 cells (in Fig. 2), tam was compared with control and ICI 182,780 was compared with control. In each case, a P value is indicated in the figure.


Figure 5
View larger version (34K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. MAPK inhibition in DT16 cells restores antiestrogen sensitivity. Proliferation of DT16 cells treated with indicated concentrations of U0126 and/or estradiol, 4-hydroxy-tamoxifen, or ICI 182,780 measured by WST-1 assay at day 6.

 

Figure 2
View larger version (23K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Reexpression of ER{alpha} can restore antiestrogen responses. A, SUM 229 cells treated with 1 µmol/L U0126 or DMSO vehicle (Co) for indicated times and Western blotted for ER{alpha} and actin. B, left, proliferation of coMCF-7 cells treated with 4-hydroxy-tamoxifen (4HT), ICI 182,780 (ICI), or nontreated (Control) at day 6 measured by WST-1 assay; right, proliferation of SUM 229 cells treated daily with U0126 and every other day with estrogen, antiestrogens, or antiestrogen + estrogen at day 6 measured by WST-1 assay. C, proliferation of SUM 149 cells treated as in B, except a higher U0126 dose of 10 µmol/L was used.

 

   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Restoration of ER{alpha} expression in ER{alpha}– breast cancer cell lines via direct MAPK inhibition or via targeted inhibition of EGFR and c-erbB-2. We have previously shown the mechanistic link between hyperactivation of MAPK and down-regulation of ER{alpha} expression using MCF-7 cell lines stably overexpressing constitutively active Raf-1, constitutively active MAPK/ERK kinase (MEK), constitutively active c-erbB-2, or ligand-activatable (i.e., +EGF) EGFR as models of overexpressed growth factor signaling. The four engineered cell lines, referred to as (ca)Raf, (ca)MEK, (ca)erbB-2, and EGFR + EGF, all show hyperactivation of MAPK, estrogen-independent growth, and loss of ER{alpha} expression (17). Importantly, inhibition of this hyperactive MAPK restored not only the expression but also the transcriptional function of ER{alpha} (17, 24).

We have now extended these studies to established ER{alpha}– breast cancer cell lines. Using three ER{alpha}– SUM breast cancer cell lines [SUM 229, which overexpresses EGFR; SUM 190, which overexpresses both EGFR and erbB-2; and SUM 149, which models inflammatory breast cancer and has very high levels of RhoC leading to hyperactivation of nuclear factor {kappa}B (NF-{kappa}B) in addition to EGFR overexpression], we assessed whether inhibition of MAPK activity could result in restoration of ER{alpha} expression. In fact, inhibition of MAPK activity via the pharmacologic MAPK/ERK kinase inhibitor U0126 resulted in significant levels of ER{alpha} expression in each of the three cell lines (Fig. 1A-C ). In Fig. 1A, the relative ER{alpha} expression levels of both coMCF-7 cells (which express ~100-150 fmol/mg protein) and coMCF-7/lt-E2 cells (coMCF-7 grown long-term in estrogen-depleted media; these cells express ~400 fmol/mg protein) are shown for comparison. ER{alpha} reexpression was sustained over the 24-h time period in which U0126 was effective in inhibiting the MAPK activity of each of these cell lines. Upstream inhibition of MAPK via inhibition of the overexpressed EGFR (with Iressa in SUM 190 cells) or erbB-2 (with Herceptin in our constitutively active erbB-2 MCF-7 line) is also effective in restoring ER{alpha} expression (Fig. 1D). We also assessed the effects of MAPK inhibition on the expression of two other factors that could be involved in estrogen responses in breast cancer cells, ERβ and GPR30. None of these cell lines exhibited appreciable expression of ERβ, nor did MAPK inhibition have any effect on this expression. All the cell lines, on the other hand, expressed GPR30, but similar to ERβ, MAPK inhibition in these cells had no effect on the GPR30 expression (data not shown).


Figure 1
View larger version (24K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. ER{alpha} reexpression on MAPK inhibition. A to C, ER{alpha}– SUM 229, SUM 190, and SUM 149 breast cancer cell lines treated with 10 µmol/L U0126 or DMSO (Co) for the indicated times and Western blotted for ER{alpha} and actin as a loading control. D, left, SUM 190 cells, which overexpress EGFR and c-erbB-2, treated with 0.5 µmol/L Iressa or DMSO for 4 or 24 h and Western blotted for ER{alpha} and actin; right, (ca)erbB-2-MCF-7 cells treated with 5 µg/mL Herceptin or 1.1% benzyl alcohol vehicle (Co) for 1 or 16 h and Western blotted for ER{alpha} or actin. coMCF-7 (~100-150 fmol ER{alpha}/mg protein) and coMCF-7/lt-E2 (~400 fmol ER{alpha}/mg protein) were included in some blots for comparison.

 
ER{alpha} reexpression restores responses to the antiestrogens tamoxifen and Faslodex. To assess the ability of this reexpressed ER{alpha} to restore antiestrogen responsiveness, SUM 229 cells were analyzed in 6-day WST-1 growth assays. The protein results shown in Fig. 1A were achieved with 10 µmol/L U0126; however, this dose used every 24 h was extremely growth inhibitory in SUM 229 cells. Therefore, we carried out a dose response assay to determine a dose that would not, on its own, inhibit growth so that we could observe a tamoxifen or Faslodex response if there was one. U0126 at 1 µmol/L was the maximum tolerated daily dose for these cells, so this dose was then assessed for its ability to restore ER{alpha} expression. Whereas 1 µmol/L U0126 did not result in the same large increase in ER{alpha} expression that 10 µmol/L did, it was still effective in restoring some ER{alpha} expression in SUM 229 cells (Fig. 2A ). Growth assays were then carried out where the effects of 4-hydroxy-tamoxifen and the pure antiestrogen ICI 182,780 (Faslodex) at 10–7 mol/L alone, 1 µmol/L U0126 alone, or the combination of 4-hydroxy-tamoxifen or ICI 182,780 and U0126 on cell proliferation were assessed by a WST-1 assay at 6 days. For comparison, the growth-inhibitory effects of both 4-hydroxy-tamoxifen and ICI 182,780 at 6 days on ER{alpha}+ coMCF-7 is shown in Fig. 2B (left). As can be seen in Fig. 2B (right), 1 µmol/L U016, although not having growth-inhibitory effects on its own in SUM 229 cells, restored the growth-inhibitory effects of both 4-hydroxy-tamoxifen and ICI 182,780. Estrogen addition to ICI 182,780 and U0126 partially reversed the antiestrogen inhibition, indicating that this is a specific ER-mediated effect; however, estrogen by itself did not further induce growth presumably because these cells were growing at their maximum rate and this dose of U0126 was not inhibiting their growth. When a slightly higher dose of U0126 was used to induce a modest growth suppression, then an estrogen dependence effect on growth can also be observed (data not shown). These data suggest that reexpressed ER{alpha}, on inhibition of MAPK activity, is capable of mediating the growth-inhibitory effects of antiestrogens in at least some ER{alpha}– breast cancers. SUM 149 cells, on the other hand, were extremely resistant to the growth-inhibitory effects of U0126 although MAPK activity was inhibited. In this cell line in which 10 µmol/L U0126 resulted in a significant increase in ER{alpha} expression (Fig. 1A), this reexpressed ER{alpha} could not restore the growth-inhibitory effects of antiestrogens (Fig. 2C). This could be due to the hyperactivation of NF-{kappa}B exhibited by these cells, a well-established inducer of antiestrogen resistance (28–30). These two cell lines thus serve as examples of two different subsets of ER{alpha}– breast cancers: those in which EGFR/erbB-2–driven MAPK plays a role in cell proliferation, and thus the concomitant inhibition of MAPK and restoration of ER{alpha} expression restores ER{alpha}-driven growth pathways and antiestrogen sensitivity, and those in which pathways/factors other than growth factor receptor/MAPK drive growth, and thus while inhibition of MAPK restores ER{alpha} expression, these other pathways/factors bypass ER{alpha} and maintain the antiestrogen resistant phenotype.

Breast cancer cell lines exhibiting a basal phenotype do not exhibit MAPK-dependent reexpression of ER{alpha}. We hypothesized that a third subset of ER{alpha}– breast cancers would exist: those in which inhibition of MAPK would not result in reexpression of ER{alpha}. It is well established that a subset of ER{alpha}– breast cancers exhibit hypermethylation of the ER{alpha} promoter resulting in the permanent repression of ER{alpha} (23, 31, 32), and thus MAPK inhibition alone would not be expected to restore ER{alpha} expression in this case. More recently, breast tumors have been defined as having luminal cell properties or basal cell properties, with the basal cell phenotype correlating with lack of ER{alpha} expression, in some cases with BRCA mutation and in many cases with EGFR overexpression (33–35). We therefore examined two ER{alpha}– breast cancer cell lines (SUM 102 and SUM 159) that have been shown by microarray analyses to display the basal phenotype (36) to further analyze the ability of MAPK inhibition to restore ER{alpha} expression. Whereas both cell lines exhibit hyperactive MAPK and U0126 is able to effectively inhibit this MAPK activity, no restoration of ER{alpha} expression could be observed in these cells (Fig. 3 ). These two cell lines, in fact, turn out to exhibit hypermethylation of the ER{alpha} promoter (data not shown). These data suggest that an additional mechanism, hypermethylation of the ER{alpha} promoter, operates to repress ER{alpha} expression in at least two cell lines exhibiting a basal phenotype such that MAPK inhibition alone is not sufficient to restore ER{alpha} expression.


Figure 3
View larger version (11K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Failure of MAPK inhibition to restore ER{alpha} expression in ER{alpha}– breast cancer cells with a basal phenotype. SUM 102 and SUM 159 treated with 10 µmol/L U0126 or DMSO vehicle (Co) for indicated times and analyzed by Western blotting for p-MAPK, ER{alpha}, and actin expression.

 
MAPK inhibition of ER{alpha}– breast tumors ex vivo results in reexpression of ER{alpha} mRNA. To extend these data to more clinically relevant models, we have been obtaining specimens of ER{alpha}– tumors from the tissue procurement core. We have assessed specimens from 10 ER{alpha}– tumors. On receipt, tumors were minced and divided randomly into tissue culture dishes containing estrogen-depleted medium supplemented with 10 µmol/L U0126 or vehicle control (DMSO). The minced tumor specimens in medium plus or minus U0126 were incubated at 37°C, 5% CO2 for 20 h. RNA was prepared from all treatment groups and analyzed for ER{alpha} mRNA level by real-time PCR. Of the 10 tumors, 6 displayed increased ER{alpha} mRNA levels after treatment with U0126 (Fig. 4A ).


Figure 4
View larger version (20K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Inhibition of MAPK in ER{alpha}– breast tumors restores both ER{alpha} mRNA and protein expression. A, ER{alpha} mRNA in ex vivo tumors after incubation with 10 µmol/L U0126 or vehicle (DMSO) for 20 h (except for tumor no. 15, which was incubated for 48 h) by reverse transcription-PCR. B, ER{alpha}, p-MAPK, and actin loading control in DT5 (passage 7) and DT6 (passage 6) with MCF-7 cell lysate as a positive control. C, p-MAPK, ER{alpha}, and actin loading control in ER{alpha}– DT5 (left) and DT16 (right) treated with 10 µmol/L U0126 for indicated times. D, p-MAPK, ER{alpha}, and actin loading control in erbB-2–overexpressing DT13 treated with 500 ng/mL Herceptin or the vehicle 1.1% benzyl alcohol (Co) for indicated times. coMCF-7 (~100-150 fmol ER{alpha}/mg protein) or coMCF-7/lt-E2 (~400 fmol ER{alpha}/mg protein) were included in some blots for comparison. E, GREB1 relative mRNA expression in DT13 and DT16 cells treated for 24 h with DMSO (vehicle control), 10 µmol/L U0126, or 10 µmol/L U0126 + 10–8 mol/L E2 (for final 20 h of treatment) by real-time PCR.

 
ER{alpha} reexpression upon MAPK inhibition of dissociated tumor cells from ER{alpha}– breast tumors. To ascertain that the reexpressed ER{alpha} mRNA exhibited by ER{alpha}– tumors ex vivo corresponded to reexpression of ER{alpha} protein in tumor cells, we have established cultures from breast tumors acquired from the tissue procurement core. These cultures have been propagated over many passages, and the cell culture established from an ER{alpha}+ tumor displays high expression of ER{alpha} whereas the cell culture from an ER{alpha}– tumor does not (Fig. 4B). Similarly, the cell culture from the ER{alpha}– tumor exhibits significant hyperactivation of MAPK whereas the cell culture from the ER{alpha}+ tumor has relatively low basal MAPK activation, similar to control MCF-7 cells.

Treatment of three of these ER{alpha}– cell cultures with 10 µmol/L U0126 does result in inhibition of MAPK activity: for DT5, this occurs at 1 and 4 h, but by 8 h, MAPK activity is returning and is almost back to basal levels by 24 h (Fig. 4C); for DT13 and DT16, this also occurs by 1 h and is sustained through 8 h with modest return of MAPK activity occurring by 24 h (Fig. 4C, DT13; data not shown). In all three ER{alpha}– cell cultures, this inhibition of MAPK activity (even the relatively short inhibition in DT5) is sufficient to restore ER{alpha} expression in these cells (Fig. 4C). In DT13 and DT16, in which the MAPK inhibition is mostly sustained through 24 h, ER{alpha} levels are also sustained through 24 h, but in DT5, ER{alpha} levels increase through 8 h and then have dropped back down by 24 h when the MAPK activity is almost fully restored. Re-treatment of these cells with U0126 every 8 h for a 24-h period results in maintenance of the ER{alpha} levels observed with 8 h of MAPK inhibition, showing that ER{alpha} expression can be maintained for the duration of MAPK inhibition (data not shown). In DT13, which overexpresses ErbB-2, Herceptin is also effective in restoring ER{alpha} expression (Fig. 4D) although the inhibition of MAPK by Herceptin occurs early and does not last much beyond 4 h. Similar to the SUM breast cancer cell lines, these DTs exhibited no significant expression of ERβ but did express GPR30, and MAPK inhibition had no effect on these expression profiles (data not shown).

We next assessed whether this reexpressed ER{alpha} was transcriptionally active by assessing the ability of U0126-mediated restoration of ER{alpha} to result in increased expression of the estrogen inducible gene GREB1. DT13 and DT16 were treated with U0126 for 24 h or for 24 h in the presence of 10 nmol/L E2 for the last 20 h of U0126 treatment (Fig. 4E). In both cell cultures, the return of ER{alpha} expression at 24 h of U0126 treatment results in slight increases in GREB1 expression, whereas estrogen treatment along with U0126 is able to further induce the expression of GREB 1.

ER{alpha} reexpression in tumor cell cultures restores responses to estrogen and the antiestrogens tamoxifen and Faslodex. Finally, the ability of restored ER{alpha} to mediate an antiestrogen response in these ER{alpha}– cell cultures from tumors was assessed in DT16 cells in a 6-day growth assay in which cells were treated with 10 µmol/L U0126 every 48 h. As seen with the established ER{alpha}– cell lines, in these dissociated ER{alpha}– tumor cells, reexpression of ER{alpha} on inhibition of MAPK does restore responses to antiestrogens (Fig. 5 ). These antiestrogen responses are specific because estrogen at 10–8 mol/L E2 is able to partially reverse the 4-hydroxy-tamoxifen and ICI 182,780 effects.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have shown the ability to reexpress ER{alpha} in ER{alpha}– breast cancer cells via the inhibition of hyperactive MAPK resulting from overexpression of EGFR or erbB-2 in both established ER{alpha}– breast cancer cell lines and ER{alpha}– tumors. This reexpression of ER{alpha} can be achieved via either direct inhibition of MAPK or inhibition of the upstream growth factor receptor (EGFR or erbB-2) that is driving its hyperactivation. Furthermore, we have established, for the first time, that the restoration of ER{alpha} expression is sufficient to induce antiestrogen responses in a subset of these ER{alpha}– breast cancer cells.

The reexpression of ER{alpha} in established ER{alpha}– breast cancer cell lines has only been previously shown via inhibition of DNA methylation or histone deacetylation in those cell lines in which the ER{alpha} promoter has been shown to be methylated (23, 31, 37). The methylation of ER{alpha} promoter is presumably a means of permanent repression secondary to some other down-regulating event. The down-regulation of ER{alpha} expression by hyperactive MAPK is a more direct mechanism and is dynamic and reversible (i.e., the down-regulation is reversed by the inhibition of MAPK activity and occurs again shortly after return of MAPK activity). And as we found with the two cell lines in which the ER{alpha} promoter turned out to be hypermethylated, despite the very high levels of MAPK exhibited by these cells and the effectiveness of U0126 in inhibiting MAPK, ER{alpha} expression could not be restored. Our data indicate that in addition to hypermethylation of the ER{alpha} promoter, hyperactivation of MAPK resulting from overexpression of EGFR or erbB-2 can also be directly responsible for the lack of ER{alpha} expression in ER{alpha}– tumors. Importantly, this MAPK-meditated down-regulation of ER{alpha} expression can be targeted to result in reexpression of ER{alpha}. In fact, it has recently been shown that in a small study of 10 ER{alpha}–/erbB-2+ patients treated for various lengths of time with Herceptin, 3 patients reexpressed ER{alpha} (38). A more recent study by Massarweh et al. suggests that this mechanism can also be exploited in ER{alpha}+/erbB-2+ tumors that lose ER{alpha} expression during treatment. They found that resistance to estrogen deprivation/fulvestrant in an ER{alpha}+/erbB-2+ MCF-7 xenograft model was accompanied by up-regulation of MAPK activity and loss of ER{alpha} expression, and subsequent cotreatment with Iressa resulted in inhibition of MAPK activity and increased ER{alpha} expression (39).

Regardless of the different potential mechanisms for down-regulating/restoring ER{alpha} expression, the reexpressed ER{alpha} must not only be functional on reexpression (i.e., induce the regulation of estrogen-responsive genes) but must also be able to regulate growth in response to estrogen/antiestrogens to be clinically relevant. In studies wherein demethylation of the ER{alpha} promoter or use of histone deacetylase inhibitors restored ER{alpha} expression, this ER{alpha} was functional in that it could regulate ERE-luciferase activity as well as the expression of specific estrogen-regulated genes such as the progesterone receptor (37, 40, 41). In addition, in both our previous studies in our hyperactive MAPK cell line models, wherein reexpression of ER{alpha} on inhibition of MAPK also restored ER{alpha} transcriptional activity (17, 24), and our current study, wherein the reexpressed ER{alpha} in both DT13 and DT16 was able to induce the expression of GREB1 (Fig. 4E), an estrogen-induced gene, we show the restoration of ER{alpha} function. Importantly, for clinical applicability, the ability of the reexpressed ER{alpha} on MAPK inhibition to mediate the growth inhibition of antiestrogens in both established ER{alpha}– breast cancer cell lines and dissociated tumor cell cultures shows clearly for the first time the potential for a novel therapeutic strategy for ER{alpha}– breast cancer. In these studies, two different cell line types were observed. In the SUM 229 cell line, which was quite sensitive to MAPK inhibition in terms of growth inhibition, restoration of ER{alpha} expression correlated with restoration of response to both 4-hydroxy-tamoxifen and ICI 182,780 (fulvestrant, Faslodex); however, in the SUM 149 cell line, which also exhibits hyperactivation of NF-{kappa}B and RhoC overexpression (42–44), the reexpressed ER{alpha} was not able to restore responses to either antiestrogen. This is most likely due to the hyperactivation of NF-{kappa}B, which is known to result in antiestrogen resistance in breast cancer cells (28–30, 45). Thus, although the MAPK repression of ER{alpha} mechanism is operative in these cells and can thus be targeted to allow for reexpression of ER{alpha}, the cells have additional cell signaling alterations that allow them to bypass ER{alpha} and remain antiestrogen resistant although now ER{alpha}+. Indeed, these cells were very resistant to growth inhibition induced by MAPK inhibition whereas even modest inhibition of NF-{kappa}B significantly affected their proliferation (data not shown). Not surprisingly, the level of reexpressed ER{alpha} necessary to restore antiestrogen effects does not need to be as high as that observed in MCF-7 cells, ~100 to 150 fmol/mg protein (Figs. 2 and 4). There are several established ER{alpha}+ breast cancer cell lines with varying ER{alpha} levels, all lower than that of MCF-7, which are estrogen dependent and exhibit full estrogen responsiveness, such as T47D, ZR75.1, and BT474.

Together, these data are suggestive of a number of important possibilities for the treatment of ER{alpha}– breast cancer (Fig. 6 ). First, it is clear that in the large majority of ER{alpha}– breast tumors, hyperactivation of MAPK by upstream overexpressed/hyperactive EGFR or c-erbB-2 represses ER{alpha} expression, and thus can be targeted to allow for reexpression of ER{alpha}. This targeting can be at the level of MAPK activity itself or via upstream inhibition of EGFR/erbB-2 signaling. In the subset of ER{alpha}– tumors exhibiting hypermethylation of the ER{alpha} promoter, such targeting alone is not successful in restoring ER{alpha} expression but would most likely be necessary to maintain ER{alpha} expression after demethylation of the promoter because these tumors also exhibit high MAPK activity. Importantly, restoration of ER{alpha} expression simultaneously restores estrogen/antiestrogen responses in those ER{alpha}– tumors in which MAPK signaling seems to be the predominant mediator of proliferation. However, where alternative signaling pathways, such as NF-{kappa}B, seem to be the predominant proliferation mediators, concomitant inhibition of the alternate signaling pathway would be necessary to allow the restored ER{alpha} to mediate antiestrogen responses. Furthermore, in those tumors exhibiting hypermethylation of the ER{alpha} promoter, in which it has recently been shown that inhibitors of histone deacetylases are equally effective in relieving the repression of ER{alpha} transcription (37), a combination of a histone deacetylase inhibitor and MAPK inhibition may be an effective means of restoring antiestrogen responses. Finally, these data indicate that ER{alpha} status, rather than being solely positive or negative, is a dynamic process strongly affected by the signaling environment of breast cancer cells.


Figure 6
View larger version (13K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 6. Model of ER{alpha}– breast cancer subsets with potential mechanisms and therapies for each.

 

   Acknowledgments
 
We thank Drs. James Rae and Michael Johnson for critical review of the manuscript, Drs. Marc Lippman and Dan Hayes for thoughtful discussions, and Dr. Steve Ethier, Deputy Director, Barbara Ann Karmanos Cancer Institute, Detroit, MI, for providing the SUM breast cancer cell lines.


   Footnotes
 
Grant support: NIH grant 1 R01 CA113674-01A2 (D. El-Ashry), American Cancer Society grant RSG-01-0330-01 (D. El-Ashry), Susan G. Komen Foundation grant BCTR0600890 (D. El-Ashry), NIH-University of Michigan Cancer Center support grant 5 P30 CA46592, and in part by the Fashion Footwear Association of New York/QVC Presents/Shoes on Sale.

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.

Received 3/12/07; revised 7/ 6/07; accepted 7/18/07.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Knight WA, Livingston RB, Gregory EJ, McGuire WL. Estrogen receptor as an independent prognostic factor for early recurrence in breast cancer. Cancer Res 1977;37:4669–71.[Abstract/Free Full Text]
  2. De Sombre ER, Thorpe SM, Rose C, et al. Prognostic usefulness of estrogen receptor immunocytochemical assays for human breast cancer. Cancer Res 1986;46:4256–64s.
  3. Clark GM, McGuire WL. Steroid receptors and other prognostic factors in primary breast cancer. Semin Oncol 1988;15:20–5.[Medline]
  4. McGuire WL, Tandon AK, Allred DC, Chamness GC, Clark GM. How to use prognostic factors in axillary node-negative breast cancer patients [see comments]. J Natl Cancer Inst 1990;82:1006–15.[Free Full Text]
  5. Nicholson S, Halcrow P, Sainsbury JR, et al. Epidermal growth factor receptor (EGFR) status associated with failure of primary endocrine therapy in elderly postmenopausal patients with breast cancer. Br J Cancer 1988;58:810–4.[Medline]
  6. Nicholson S, Sainsbury JR, Halcrow P, Chambers P, Farndon JR, Harris AL. Expression of epidermal growth factor receptors associated with lack of response to endocrine therapy in recurrent breast cancer. Lancet 1989;1:182–5.[Medline]
  7. Nicholson S, Richard J, Sainsbury C, et al. Epidermal growth factor receptor (EGFR); results of a 6 year follow-up study in operable breast cancer with emphasis on the node negative subgroup. Br J Cancer 1991;63:146–50.[Medline]
  8. Sainsbury JR, Farndon JR, Sherbet GV, Harris AL. Epidermal-growth-factor receptors and oestrogen receptors in human breast cancer. Lancet 1985;1:364–6.[CrossRef][Medline]
  9. Sainsbury JR, Farndon JR, Needham GK, Malcolm AJ, Harris AL. Epidermal-growth-factor receptor status as predictor of early recurrence of and death from breast cancer. Lancet 1987;1:1398–402.[Medline]
  10. Toi M, Osaki A, Yamada H, Toge T. Epidermal growth factor receptor expression as a prognostic indicator in breast cancer. Eur J Cancer 1991;27:977–80.[Medline]
  11. Gusterson BA, Gelber RD, Goldhirsch A, et al. Prognostic importance of c-erbB-2 expression in breast cancer. International (Ludwig) Breast Cancer Study Group. J Clin Oncol 1992;10:1049–56.[Abstract]
  12. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987;235:177–82.[Abstract/Free Full Text]
  13. Perren TJ. cv-erbB-2 oncogene as a prognostic marker in breast cancer [editorial]. Br J Cancer 1991;63:328–32.[Medline]
  14. Sivaraman VS, Wang H, Nuovo GJ, Malbon CC. Hyperexpression of mitogen-activated protein kinase in human breast cancer [see comments]. J Clin Invest 1997;99:1478–83.
  15. Salh B, Marotta A, Matthewson C, Flint J, Owen D, Pelech S. Investigation of the Mek-MAP kinase-Rsk pathway in human breast cancer. Anticancer Res 1999;19:731–40.[Medline]
  16. Allred DC, Mohsin SK, Fuqua SA. Histological and biological evolution of human premalignant breast disease. Endocr Relat Cancer 2001;8:47–61.[Abstract]
  17. Oh AS, Lorant LA, Holloway JN, Miller DL, Kern FG, El-Ashry D. Hyperactivation of mapk induces loss of er{alpha} expression in breast cancer cells. Mol Endocrinol 2001;15:1344–59.[Abstract/Free Full Text]
  18. Jeng MH, Yue W, Eischeid A, Wang JP, Santen RJ. Role of MAP kinase in the enhanced cell proliferation of long-term estrogen deprived human breast cancer cells. Breast Cancer Res Treat 2000;62:167–75.[CrossRef][Medline]
  19. Santen RJ, Song RX, McPherson R, et al. The role of mitogen-activated protein (MAP) kinase in breast cancer. J Steroid Biochem Mol Biol 2002;80:239–56.[CrossRef][Medline]
  20. Stoner M, Saville B, Wormke M, Dean D, Burghardt R, Safe S. Hypoxia induces proteasome-dependent degradation of estrogen receptor {alpha} in ZR-75 breast cancer cells. Mol Endocrinol 2002;16:2231–42.[Abstract/Free Full Text]
  21. Kronblad A, Hedenfalk I, Nilsson E, Pahlman S, Landberg G. ERK1/2 inhibition increases antiestrogen treatment efficacy by interfering with hypoxia-induced down-regulation of ER{alpha}: a combination therapy potentially targeting hypoxic and dormant tumor cells. Oncogene 2005;24:6835–41.[CrossRef][Medline]
  22. Lapidus RG, Ferguson AT, Ottaviano YL, et al. Methylation of estrogen and progesterone receptor genes 5' CpG islands correlates with ER and PR gene expression in breast tumors. Clin Cancer Res 1996;2:805–10.[Abstract]
  23. Ottaviano YL, Issa JP, Parl FF, Smith HS, Baylin SB, Davidson NE. Methylation of the estrogen receptor gene CpG island marks loss of estrogen receptor expression in human breast cancer cells. Cancer Res 1994;54:2552–5.[Abstract/Free Full Text]
  24. Holloway JN, Murthy S, El-Ashry D. A cytoplasmic substrate of mitogen-activated protein kinase is responsible for estrogen receptor-{alpha} down-regulation in breast cancer cells: the role of nuclear factor-{kappa}B. Mol Endocrinol 2004;18:1396–410.[Abstract/Free Full Text]
  25. Creighton CJ, Hilger AM, Murthy S, Rae JM, Chinnaiyan AM, El-Ashry D. Activation of mitogen-activated protein kinase in estrogen receptor {alpha}-positive breast cancer cells in vitro induces an in vivo molecular phenotype of estrogen receptor {alpha}-negative human breast tumors. Cancer Res 2006;66:3903–11.[Abstract/Free Full Text]
  26. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(–{Delta}{Delta}C(T)) method. Methods 2001;25:402–8.[CrossRef][Medline]
  27. Bieche I, Parfait B, Laurendeau I, Girault I, Vidaud M, Lidereau R. Quantification of estrogen receptor {alpha} and β expression in sporadic breast cancer. Oncogene 2001;20:8109–15.[CrossRef][Medline]
  28. Nakshatri H, Bhat-Nakshatri P, Martin DA, Goulet RJ, Jr., Sledge GW, Jr. Constitutive activation of NF-{kappa}B during progression of breast cancer to hormone-independent growth. Mol Cell Biol 1997;17:3629–39.[Abstract]
  29. Degraffenried LA, Chandrasekar B, Friedrichs WE, et al. NF-{kappa}B inhibition markedly enhances sensitivity of resistant breast cancer tumor cells to tamoxifen. Ann Oncol 2004;15:885–90.[Abstract/Free Full Text]
  30. Riggins RB, Zwart A, Nehra R, Clarke R. The nuclear factor kappa B inhibitor parthenolide restores ICI 182,780 (Faslodex; fulvestrant)-induced apoptosis in antiestrogen-resistant breast cancer cells. Mol Cancer Ther 2005;4:33–41.[Abstract/Free Full Text]
  31. Ferguson AT, Lapidus RG, Baylin SB, Davidson NE. Demethylation of the estrogen receptor gene in estrogen receptor-negative breast cancer cells can reactivate estrogen receptor gene expression. Cancer Res 1995;55:2279–83.[Abstract/Free Full Text]
  32. Lapidus RG, Nass SJ, Butash KA, et al. Mapping of ER gene CpG island methylation-specific polymerase chain reaction. Cancer Res 1998;58:2515–9.[Abstract/Free Full Text]
  33. Perou CM, Sorlie T, Eisen MB, et al. Molecular portraits of human breast tumours. Nature 2000;406:747–52.[CrossRef][Medline]
  34. Sorlie T, Tibshirani R, Parker J, et al. Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci U S A 2003;100:8418–23.[Abstract/Free Full Text]
  35. Sorlie T, Perou CM, Tibshirani R, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A 2001;98:10869–74.[Abstract/Free Full Text]
  36. Neve RM, Chin K, Fridlyand J, et al. A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell 2006;10:515–27.[CrossRef][Medline]
  37. Zhou Q, Atadja P, Davidson NE. Histone deacetylase inhibitor LBH589 reactivates silenced estrogen receptor {alpha} (ER) gene expression without loss of DNA hypermethylation. Cancer Biol Ther 2007;6:64–9.[Medline]
  38. Munzone E, Curigliano G, Rocca A, et al. Reverting estrogen-receptor-negative phenotype in HER-2-overexpressing advanced breast cancer patients exposed to trastuzumab plus chemotherapy. Breast Cancer Res 2006;8:R4.[CrossRef][Medline]
  39. Massarweh S, Osborne CK, Jiang S, et al. Mechanisms of tumor regression and resistance to estrogen deprivation and fulvestrant in a model of estrogen receptor-positive, HER-2/neu-positive breast cancer. Cancer Res 2006;66:8266–73.[Abstract/Free Full Text]
  40. Yang X, Ferguson AT, Nass SJ, et al. Transcriptional activation of estrogen receptor {alpha} in human breast cancer cells by histone deacetylase inhibition. Cancer Res 2000;60:6890–4.[Abstract/Free Full Text]
  41. Yang X, Phillips DL, Ferguson AT, Nelson WG, Herman JG, Davidson NE. Synergistic activation of functional estrogen receptor (ER)-{alpha} by DNA methyltransferase and histone deacetylase inhibition in human ER-{alpha}-negative breast cancer cells. Cancer Res 2001;61:7025–9.[Abstract/Free Full Text]
  42. Pan Q, Kleer CG, van Golen KL, et al. Copper deficiency induced by tetrathiomolybdate suppresses tumor growth and angiogenesis. Cancer Res 2002;62:4854–9.[Abstract/Free Full Text]
  43. van Golen KL, Wu ZF, Qiao XT, Bao L, Merajver SD. RhoC GTPase overexpression modulates induction of angiogenic factors in breast cells. Neoplasia 2000;2:418–25.[CrossRef][Medline]
  44. van Golen KL, Wu ZF, Qiao XT, Bao LW, Merajver SD. RhoC GTPase, a novel transforming oncogene for human mammary epithelial cells that partially recapitulates the inflammatory breast cancer phenotype. Cancer Res 2000;60:5832–8.[Abstract/Free Full Text]
  45. Campbell RA, Bhat-Nakshatri P, Patel NM, Constantinidou D, Ali S, Nakshatri H. Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor {alpha}: a new model for anti-estrogen resistance. J Biol Chem 2001;276:9817–24.[Abstract/Free Full Text]

Commentary

The Dynamics of Estrogen Receptor Status in Breast Cancer: Re-shaping the Paradigm
Sara Lopez-Tarruella and Rachel Schiff
Clin. Cancer Res. 2007 13: 6921-6925. [Full Text] [PDF]



This article has been cited by other articles:


Home page
 Clin. Cancer Res.Home page
C. L. Arteaga, A. O'Neill, S. L. Moulder, M. Pins, J. A. Sparano, G. W. Sledge, and N. E. Davidson
A Phase I-II Study of Combined Blockade of the ErbB Receptor Network with Trastuzumab and Gefitinib in Patients with HER2 (ErbB2)-Overexpressing Metastatic Breast Cancer
Clin. Cancer Res., October 1, 2008; 14(19): 6277 - 6283.
[Abstract] [Full Text] [PDF]

Home page
 JNCI J Natl Cancer InstHome page
L. Lusa, B. Peissel, S. Manoukian, E. Marchesi, P. Radice, M. A. Pierotti, and M. Gariboldi
Re: Molecular Basis for Estrogen Receptor {alpha} Deficiency in BRCA1-Linked Breast Cancer
J Natl Cancer Inst, May 21, 2008; 100(10): 752 - 753.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bayliss, J.
Right arrow Articles by El-Ashry, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bayliss, J.
Right arrow Articles by El-Ashry, D.
Related Collections
Right arrowCommentary

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online