Phosphorylation of estrogen receptor α serine 167 is predictive of response to endocrine therapy and increases postrelapse survival in metastatic breast cancer
© Yamashita et al.; licensee BioMed Central Ltd. 2005
Received: 29 January 2005
Accepted: 28 June 2005
Published: 27 July 2005
Endocrine therapy is the most important treatment option for women with hormone-receptor-positive breast cancer. The potential mechanisms for endocrine resistance involve estrogen receptor (ER)-coregulatory proteins and crosstalk between ER and other growth factor signaling networks. However, the factors and pathways responsible for endocrine resistance are still poorly identified.
Using immunohistochemical techniques, we focused on the expression and phosphorylation of hormone receptors themselves and examined the phosphorylation of ER-α Ser118 and ER-α Ser167 and the expression of ER-α, ER-β1, ER-βcx/β2, progesterone receptor (PR), PRA, and PRB in the primary breast carcinomas of 75 patients with metastatic breast cancer who received first-line treatment with endocrine therapy after relapse.
Phosphorylation of ER-α Ser118, but not Ser167, was positively associated with overexpression of HER2, and HER2-positive tumors showed resistance to endocrine therapy. The present study has shown for the first time that phosphorylation of ER-α Ser167, but not Ser118, and expression of PRA and PRB, as well as ER-α and PR in primary breast tumors are predictive of response to endocrine therapy, whereas expression of ER-β1 and ER-βcx/β2 did not affect response to the therapy. In addition, patients with either high phosphorylation of ER-α Ser167, or high expression of ER-α, PR, PRA, or PRB had a significantly longer survival after relapse.
These data suggest that phosphorylation of ER-α Ser167 is helpful in selecting patients who may benefit from endocrine therapy and is a prognostic marker in metastatic breast cancer.
The development and progression of breast cancer are influenced by steroid hormones, particularly estrogen, via their interaction with specific target receptors. Endocrine therapy has become the most important treatment option for women with estrogen receptor (ER)-positive breast cancer. Nevertheless, many breast cancer patients with tumors expressing high levels of ER are unresponsive to endocrine therapy, and all patients with advanced disease eventually develop resistance to the therapy. The potential mechanisms behind either this intrinsic or acquired endocrine resistance involve ER-coregulatory proteins and crosstalk between the ER pathway and other growth factor signaling networks [1, 2]. An understanding of the molecular mechanisms that modulate the activity of the estrogen signaling network has enabled new ways of overcoming endocrine resistance to be developed.
ER-α is phosphorylated on multiple amino acid residues . Serines 104, 106, 118, and 167 are all located within the activation function (AF)1 region of ER-α, and their phosphorylation provides the important mechanism that regulates AF1 activity [4, 5]. In response to estradiol binding, human ER-α is phosphorylated mainly on Ser118 and to a lesser extent on Ser104 and Ser106 . Although some authors have also reported that Ser167 is a major estradiol-induced phosphorylation site [5, 6], this response to estradiol has not been universally observed [4, 7]. Interestingly, in response to the activation of the mitogen-activated protein kinase (MAPK) pathway, phosphorylation occurs on Ser118 and Ser167 [8, 9]. However, the role of phosphorylation of Ser118 and Ser167 of ER-α in human breast cancer has not been investigated.
ER-β and its splicing isoforms are widely expressed in both normal and malignant breast tissue . Although several groups have reported results regarding the possible function of ER-β, and its potential as a prognostic or predictive factor in breast cancer, the data remain inconclusive and are often contradictory [11, 12]. ER-βcx (also called ER-β2), a splice variant of ER-β, is considered to be a dominant repressor of ER-α; it is identical to ER-β1 (wild-type ER-β) except that the last exon, 8, is replaced by 26 amino acid residues . The role of ER-β and its isoforms, especially with respect to the response of breast cancer to endocrine therapy, has also not been elucidated.
Progesterone receptors (PRs) occur as two isoforms, PRA and PRB, transcribed from two distinct promoters on a single gene. PRA, but not PRB, lacks the 164 amino acid N-terminal residues that contain AF3, and this is the cause of their functional differences . In the mammary gland, the overexpression of PRA relative to PRB results in extensive epithelial cell hyperplasia, excessive ductal branching, and a disorganized basement membrane, all features associated with neoplasia . In contrast, the overexpression of PRB leads to premature arrest of ductal growth and inadequate lobuloalveolar differentiation . However, little is known about the unique roles of the two PR isoforms in breast cancer.
In this study, we focused on the expression and phosphorylation of the hormone receptors themselves and, using immunohistochemistry (IHC), examined the phosphorylation of ER-α Ser118 and Ser167 and the expression of ER-α, ER-β1, ER-βcx/β2, PR, PRA, and PRB in primary breast tumor specimens from 75 patients with metastatic breast cancer who received first-line treatment with endocrine therapy on relapse. Our results show that patients with primary breast tumors in which there is either high phosphorylation of ER-α Ser167 or high expression of ER-α, PR, PRA, or PRB significantly responded to endocrine therapy and had a better survival after relapse.
Materials and methods
Cell culture and transfections
COS-7 cells (ATCC American Type Culture Collection, Manassas, VA, USA) were grown in DMEM containing 10% fetal calf serum, 2 mM L-glutamine, and penicillin–streptomycin (50 IU/ml and 50 mg/ml, respectively) at 37°C with 5% CO2 as described previously . T47D cells (ATCC) were grown in RPMI-1640 supplemented with 10% fetal calf serum, 2 mM L-glutamine, and penicillin–streptomycin (50 IU/ml and 50 mg/ml, respectively), at 37°C with 5% CO2. Six microliters of FuGENE6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN, USA), 3 μg of an expression vector for human ER-α cDNA (pSG5/puromycine hERα, full length, kindly provided by Pierre Chambon, Strasbourg, France) were used for transfection into COS-7 cells as described previously . After transfection, cells were starved in serum-free DMEM without phenol red for 20 hours.
Cells were treated in the absence or presence of 17β-estradiol (E2) (10 nM, Sigma-Aldrich Co, St Louis, MO, USA) and/or epidermal growth factor (EGF) (100 ng/ml, human recombinant EGF, Sigma-Aldrich) for 30 min, pelleted by centrifugation and solubilized in lysis buffer containing 10 mM Tris-HCl, pH 7.6, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 1% Triton X-100, 1 mM phenylmethylsulfonylfluoride (PMSF), 5 μg/ml aprotinin, 1 μg/ml pepstatin A, and 2 μg/ml leupeptin, as described previously . Equal amounts of total protein from whole-cell lysates were prepared and used for SDS–PAGE. Immunoblotting was performed as described previously  using polyvinylidene difluoride (PVDF) membranes (Invitrogen, Carlsbad, CA, USA; Catalogue no. LC2002), and polyclonal antibody against ER-α (H-184, Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1:200 dilution), polyclonal rabbit antiphospho-ER-α (Ser118) antibody (Cell Signaling Technology, Beverly, MA, USA) (1:500 dilution), polyclonal rabbit antiphospho-ER-α (Ser167) antibody (Cell Signaling) (1:500 dilution) as primary antibodies and horseradish-peroxidase-conjugated goat antibodies to rabbit IgG as secondary antibodies in conjunction with enhanced chemiluminescence substrate mixture (SuperSignal WestPico Chemiluminescent Substrate, Pierce, Rockford, IL, USA) in accordance with the manufacturer's instructions. Phospho-ER-α Ser118 antibody detects ER-α only when the receptor is phosphorylated at Ser118, and not at Ser106 or Ser167; and phospho-ER-α Ser167 antibody detects ER-α only when the receptor is phosphorylated at Ser167, and not at Ser106 or Ser118, by immunoblotting, as described by Chen and colleagues .
Generation of specific antibodies for ER-β proteins
To detect specific ER-β1 and ER-βcx/β2 proteins, rabbit polyclonal antibodies were generated against synthesized peptides of the C-terminal region of ER-β1 (CSPAEDSKSKEGSQNPQSQ) and ER-βcx/β2 (MKMETLLPEATMEQ), in accordance with the method of Ogawa and colleagues  and purified on affinity columns bound with each synthetic peptide as described previously . To confirm the specificity of these polyclonal antibodies, immunoblot analysis was performed using COS-7 cells transfected with either expression plasmid encoding ER-β1 or ER-βcx/β2 (kindly donated by Masami Muramatsu, Saitama, Japan) as previously described . Immunoblotting with specific anti-ER-β antibodies showed that the polyclonal antibody for ER-β1 detected a specific band at 60 kDa only in the lysates of COS-7 cells transfected with an ER-β1 expression plasmid, and not in those transfected with an ER-βcx/β2 expression plasmid, as described previously by Ogawa and colleagues . Conversely, the polyclonal antibody for ER-βcx/β2 detected a specific band at 55 and 51 kDa only in the lysates of COS-7 cells transfected with an ER-βcx/β2 expression plasmid, and not in those transfected with an ER-β1 expression plasmid, as described previously by Ogawa and colleagues .
Patients and breast cancer tissues
Clinicopathological characteristics of patients with metastatic breast cancer, their primary tumors, and treatment
Number of patients
Total number of patients
Age at diagnosis (years)
29 to 77
Tumor size (cm)
Number of positive lymph nodes
Disease-free interval (months)
Mean ± standard deviation
39.9 ± 26.4
1 to 123
First-line endocrine therapy
LH-RH agonist + tamoxifen
First-line endocrine therapy for metastatic breast cancer and response criteria
When the patients relapsed and were diagnosed with metastatic breast cancer, they started endocrine therapy (Table 1). Patients were assessed monthly for clinical response, which was defined according to World Health Organization criteria as complete response, partial response, no change, or progressive disease. The presence of progressive disease indicated treatment failure; all other clinical responses were considered to show efficacy of treatment.
One 4-μm section of each submitted paraffin block was stained first with hematoxylin and eosin to verify that an adequate number of invasive carcinoma cells were present and that the fixation quality was adequate for immunohistochemical (IHC) analysis. Serial sections (4 μm) were prepared from selected blocks and float-mounted on adhesive-coated glass slides, for ER-α, ER-β, and PR staining as described previously . Primary antibodies included monoclonal mouse antihuman ER-α antibody (1D5, DAKO, Glostrup, Denmark) at 1:100 dilution for ER-α; polyclonal rabbit antiphospho-ER-α (Ser118) antibody (Cell Signaling) at 1:25 dilution for phosphorylated ER-α Ser118; polyclonal rabbit antiphospho-ER-α (Ser167) antibody (Cell Signaling) at 1:50 dilution for phosphorylated ER-α Ser167; polyclonal rabbit anti-ER-β1 antibody at 1:10000 dilution for ER-β1; polyclonal rabbit anti-ER-βcx/β2 antibody at 1:2000 dilution for ER-βcx/β2; monoclonal mouse antihuman PR antibody (636, DAKO) at 1:100 dilution for PR; monoclonal mouse antihuman PR antibody (Ab-7, Neo Markers, Fremont, CA) at 1:100 dilution for PRA; and monoclonal mouse antihuman PR antibody (Ab-2, Neo Markers) at 1:100 dilution for PRB. With respect to the PRA and PRB antibodies, it has been reported that whereas AB-7 can recognize high-PRA and low-PRB forms, this antibody recognizes PRA only in 10% formalin-fixed and paraffin-embedded tissue sections, and AB-2 recognizes exclusively PRB in these same media . The DAKO Envision system (DAKO EnVision labelled polymer, peroxidase) was used as the detection system as described previously . HER2 immunostaining was done and evaluated using a method similar to the HercepTest (DAKO) .
Immunostained slides were scored after the entire slide had been evaluated by light microscopy. The expression and phosphorylation of hormone receptors were scored by assigning proportion and intensity scores, in accorance with the procedure of Allred and colleagues . In brief, a proportion score represented the estimated proportion of tumor cells staining positive, as follows: 0 (none); 1 (<1/100); 2 (1/100 to 1/10); 3 (>1/10 to 1/3); 4 (>1/3 to 2/3); and 5 (>2/3). Any brown nuclear staining in invasive breast epithelium counted towards the proportion score. An intensity score represented the average intensity of the positive cells, as follows: 0 (none); 1 (weak); 2 (intermediate); and 3 (strong). The proportion and intensity scores were then added to obtain a total score, which could range from 0 to 8.
The Mann–Whitney U test or the Kruskal–Wallis test was used to compare the IHC scores of hormone receptors with clinicopathological characteristics. The Mann–Whitney U test and the unpaired t-test were used to compare the IHC scores of hormone receptors with response to endocrine therapy. The Spearman rank correlation test was used to study relations between expression and phosphorylation of hormone receptors and disease-free interval. To examine the change of expression and phosphorylation status between the primary and recurrent tumors, the one-sample Wilcoxon signed rank test was used. Estimation of overall survival was performed using the Kaplan–Meier method, and differences between survival curves were assessed with the log-rank test. Cox's proportional hazards model was used for univariate and multivariate analyses of prognostic values.
Phosphorylation of ER-α Ser118 and ER-α Ser167 is induced in response to EGF
To further validate the ability of site-specific antibodies for phospho-ER-α Ser118 and Ser167 in ER+ and PR+ breast cancer cells, phosphorylation of ER-α Ser118 and Ser167 was analyzed in T47D cells. Cells were grown in serum- and estrogen-deprived conditions and treated with vehicle (medium) (Fig. 1b, lane 1), E2 for 10 min (lane 2) and 30 min (lane 3), EGF for 10 min (lane 4) and 30 min (lane 5), or E2 and EGF for 10 min (lane 6) and 30 min (lane 7). ER-α was inducibly phosphorylated on Ser167 in response to EGF (Fig. 1b, lanes 1, 4, 5, 6, and 7, second panel). Phosphorylation of Ser167 was increased with EGF treatment for 30 min compared with that for 10 min (lane 5 vs 4, lane 7 vs 6). On the other hand, ER-α was not phosphorylated on Ser167 in response to E2 (Fig. 1b, lanes 2 and 3, second panel). In addition, ER-α was not phosphorylated on Ser118 in response to either E2 or EGF (Fig. 1b, lanes 2 to 7, top panel) in T47D cells. Expression of ER-α was observed equally under both conditions (Fig. 1b, bottom panel). We concluded from immunoblotting that, in response to EGF in COS-7 and T47D cells, ER-α Ser167 was inducibly rather than constitutively phosphorylated, and that the phosphorylation of ER-α Ser118 was constitutive and further induced by EGF in COS-7 cells, but that Ser118 phosphorylation was not observed after the stimulation of T47D cells by either E2 or EGF.
Immunohistochemical staining for phosphorylation of ER-α Ser118 and ER-α Ser167, and expression of ER-β 1, ER-βcx/β2, PRA, and PRB in human breast cancer
Correlation between expression and phosphorylation of ER-α, ER-β, and PR and clinicopathological factors in primary breast tumors
We examined the phosphorylation of ER-α Ser118 and Ser167, and expression of ER-α, ER-β1, ER-βcx/β2, PR, PRA, and PRB in 75 primary invasive breast carcinomas by IHC. The IHC scores for ER-α, ER-β, and PR were compared among patient subgroups, according to clinicopathological factors. Phosphorylation of ER-α Ser118, but not of ER-α Ser167, was positively correlated with expression levels of HER2 (P = 0.038), whereas expression of ER-α (1D5) tended to be inversely correlated with HER2 overexpression. PR (636) expression was significantly associated with age (P = 0.0018). There was no difference between the expression and phosphorylation of hormone receptors and other clinicopathological factors.
Correlation between expression and phosphorylation of ER-α, ER-β, and PR in primary breast tumors
Correlations between immunohistochemistry scores for expression and phosphorylation of hormone receptors in primary breast tumors
Phosphorylation of ER-α Ser167, but not Ser118, and expression of PRA and PRB in primary breast tumors are predictive of response to endocrine therapy in metastatic breast cancer
Correlation between immunohistochemistry scores for hormone receptors and response to endocrine therapy in breast cancer
Responders (n = 35)a
Nonresponders (n = 40)a
5.8 ± 2.3 (7; 0–8)
4.1 ± 2.9 (5; 0–8)
4.3 ± 2.6 (5; 0–8)
4.2 ± 2.4 (4; 0–8)
2.5 ± 2.0 (2; 0–6)
1.6 ± 1.7 (2; 0–5)
4.2 ± 2.2 (4; 0–8)
4.5 ± 2.3 (5; 0–8)
3.1 ± 2.4 (3; 0–8)
3.0 ± 2.5 (2; 0–8)
5.5 ± 2.5 (6; 0–8)
3.6 ± 2.7 (4; 0–8)
4.6 ± 2.0 (5; 0–8)
2.4 ± 2.5 (2; 0–8)
4.0 ± 2.1 (4; 0–8)
2.7 ± 2.4 (3; 0–8)
Correlation between immunohistochemistry scores for hormone receptors and disease-free interval
Spearman correlation coefficient
Spearman rank correlation test (P)
Correlation between expression and phosphorylation of ER-α, ER-β, and PR in primary breast tumors and disease-free interval
We then examined whether the expression and phosphorylation levels of hormone receptors in primary breast tumors affected disease-free interval in relapsing breast cancer patients. Spearman correlation coefficients between the IHC scores of ER-α, ER-β, and PR and disease-free interval are shown in Table 4. The time to relapse after primary surgery was significantly longer in patients with primary breast tumors with high phosphorylation levels of ER-α Ser167 or with high expression levels of ER-α (1D5), PR (636), PRA, or PRB (P = 0.0076, P = 0.035, P = 0.018, P = 0.0061, and P = 0.023, respectively). On the other hand, no significant relation was observed between either phosphorylation of ER-α Ser118 or expression of ER-β1 or ER-βcx/β2 and disease-free interval.
Comparison of IHC scores of ER-α, ER-β, and PR in primary and secondary tumors
Comparison of immunohistochemistry scores for hormone receptors in primary and secondary tumors
4.9 ± 2.7
3.1 ± 3.2
4.9 ± 2.8
7.3 ± 1.6
3.7 ± 1.6
5.5 ± 2.2
5.5 ± 2.2
7.0 ± 2.0
4.1 ± 2.6
6.1 ± 2.6
4.1 ± 3.2
2.9 ± 3.5
3.5 ± 2.6
6.1 ± 2.6
3.7 ± 2.4
5.7 ± 2.6
Patients with high phosphorylation of ER-α Ser167 and high expression of PRA and PRB in primary breast tumors had a significantly longer survival after relapse
Univariate and multivariate analysis of factors predicting postrelapse survival
Lymph node status
Using IHC techniques, we investigated the phosphorylation of ER-α Ser118 and ER-α Ser167, and expression of ER-α, ER-β1, ER-βcx/β2, PR, PRA, and PRB, in primary breast tumor specimens from 75 patients with metastatic breast cancer who, on relapse, received endocrine therapy as first-line treatment. Our results indicate that patients with primary breast tumors with high phosphorylation of ER-α Ser167, or high expression of ER-α, PR, PRA, or PRB, significantly respond to endocrine therapy and had a better survival after relapse.
ER-α is phosphorylated on multiple amino acid residues . In general, phosphorylation of serine residues in the AF1 domain of ER-α appears to influence the recruitment of coactivators, resulting in enhanced ER-mediated transcription. In this study, we measured the phosphorylation of ER-α Ser118 and Ser167 as well as the expression of ER-α in breast cancer by IHC using site-specific anti-ER-α-phosphoserine antibodies. Our results showed that phosphorylation of ER-α Ser118, but not of ER-α Ser167, was significantly correlated with expression levels of HER2. It has been reported that ER-α was significantly phosphorylated on Ser118 in response to either estradiol binding or the activation of the mitogen-activated protein kinase (MAPK) pathway, while Ser167 is phosphorylated by AKT, p90 ribosomal S6 kinase (RSK), and casein kinase II as well as MAPK [5, 7, 9, 24]. Murphy and colleagues recently reported that in 45 human breast tumor biopsies phosphorylation of ER-α Ser118 correlated with active MAPK . Because MAPK is located downstream of HER2, it is possible that phosphorylation of ER-α Ser118 is in part caused by HER2-MAPK signaling in breast cancer. On the other hand, phosphorylation of ER-α Ser167 seems to be led by different mechanisms.
Our results also showed that while phosphorylation of both ER-α Ser118 and Ser167 was strongly and positively correlated with expression of ER-β 1 and ER-βcx/β2, there was no observed association between expression of ER-α and ER-β proteins. Both antibodies for ER-β1 and ER-βcx/β2, generated in this study, were specific against their respective C-terminal amino acid residues, and positive nuclear staining was observed in normal breast epithelial cells, noninvasive ductal carcinoma, and invasive carcinoma. Saunders and colleagues also found that there was no quantitative relation between IHC scores for ER-α and ER-β . However, using IHC, it was reported that ER-β expression was positively correlated with ER-α and PR . Specific detection of ER-β1 from other isoforms also indicated a positive correlation between expression of ER-β1 and ER-α . However, no studies have been reported concerning the relation between phosphorylation of ER-α and expression of ER-β proteins.
In our analysis, ER-α expression was positively correlated with PRA but not with PRB. In addition, phosphorylation of both ER-α Ser118 and Ser167 was strongly and positively associated with expression of PRA but not with PRB. This suggests that PRA is preferentially induced following the transcription of ER-α after the phosphorylation of Ser118 and/or Ser167. Two previous studies have reported investigations into the expression of PRA and PRB in breast cancer. The first, an analysis of 202 PR-positive breast cancers by immunoblotting, showed that while there was no significant difference in levels of PRA and PRB in most of the PR-positive tumors, nevertheless expression levels of PRA were higher in 59% of tumors and at least four times as high in 25% . In the second study, of 32 PR-positive breast cancers, it was reported that excess PRB correlated with the absence of HER2, thereby indicating a good prognosis, whereas excess PRA correlated with a poorly differentiated phenotype and higher tumor grade . The normally equal expression of PRA and PRB is disrupted early in carcinogenesis. PRA is usually the predominant isoform in tumors of the breast, and it appears, therefore, that disrupted progesterone signaling may play a role in the development or progression of these cancers [14, 29, 31].
The most important results to come out of this study concern the correlation between clinical response and the phosphorylation and expression of the receptors. We identified that patients with primary breast tumors with high phosphorylation of ER-α Ser167, or high expression of PRA or PRB, significantly responded to endocrine therapy, whereas phosphorylation of ER-α Ser118 and expression of ER-β1 and ER-βcx/β2 did not influence response. Phosphorylation of both ER-α Ser118 and ER-α Ser167 occurs in response to either estradiol binding or activation of growth factor signaling pathways. It is well established that peptide growth factor signaling pathways can activate ER-α, in the absence of its ligand, through phosphorylation of ER-α by MAPK [8, 32]. In addition, the induction of ER-α by MAPK also enhances ER signaling and promotes tumor growth in the presence of estradiol, and such tumors have been shown to be responsive still to antiestrogen therapy . Furthermore, Clark and colleagues reported that, independently of MAPK, p90 ribosomal S6 kinase 2 (Rsk2) specifically activates ER-α by phosphorylation of Ser167 and by docking to the hormone-binding domain of ER-α, and that the antiestrogen 4-hydroxytamoxifen blocks Rsk2-mediated activation of ER-α . Since our results showed that phosphorylation of ER-α Ser167, but not ER-α Ser118, was predictive of response to endocrine therapy, they suggest that, in breast cancer, phosphorylation of ER-α Ser118 occurs frequently without estradiol, whereas phosphorylation of ER-α Ser167 may occur frequently in response to estradiol binding.
It has been reported that HER2-induced MAPK and ER-α activation leads to tamoxifen resistance . Data from these clinical trials demonstrated that the antiproliferative response to endocrine therapy was impeded in ER-α-positive/HER2-positive primary breast cancers . In contrast, a Southwest Oncology Group study reported that overexpression of HER2 was not associated with tamoxifen unresponsiveness or a more aggressive phenotype of ER-α-positive metastatic breast cancer . In our analysis, HER2-positive tumors showed high phosphorylation levels of ER-α Ser118 and were resistant to endocrine therapy.
Finally, our results showed that expression of ER-β1 and ER-βcx/β2 does correlate with response to endocrine therapy. No significant differences in the expression of ER-β1, ER-β2, and ER-β5 mRNAs between tamoxifen-sensitive and -resistant groups, has been reported . Taken together, these data suggest that the expression of ER-β proteins is not predictive of response to endocrine therapy in breast cancer. However, a significant correlation between a PR-negative phenotype and the presence of ER-βcx/β2 in ER-α-rich tumor foci and expression of ER-βcx/β2 with low PR expression has been shown to correlate with a poor response to tamoxifen .
In our analysis, the expression of PRA and PRB as well as PR was strongly predictive of response to endocrine therapy. In contrast, it has been reported, in a study of T47D human breast tumor xenografts, that tamoxifen treatment preferentially inhibited the growth of PRA tumors, whereas PRB tumors were unaffected . Another study reported that, although estrogen induced PR expression in all breast cancer cell lines studied, the expression ratio of PRA/PRB induced by estrogen was dependent on the cell line, and that these results suggested that the PRA and PRB promoters were differentially regulated by estrogen in different breast cancer cells . Further studies are obviously needed to resolve these apparent discrepancies and in order to identify the functional importance of altered PR isoform expression and how this might affect the response of breast tumors to endocrine therapy.
The present study has shown for the first time that patients with primary breast tumors with either high phosphorylation of ER-α Ser167 or high expression of PRA or PRB respond significantly to endocrine therapy and have a better survival after relapse. Our data suggest that phosphorylation of ER-α Ser167 is helpful in selecting patients who may benefit from endocrine therapy and is a prognostic marker in metastatic breast cancer.
Dulbecco's modified essential medium
epidermal growth factor
mitogen-activated protein kinase
This work was supported by Grants-in Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture in Japan 16591267.
- Johnston SR, Head J, Pancholi S, Detre S, Martin LA, Smith IE, Dowsett M: Integration of signal transduction inhibitors with endocrine therapy: an approach to overcoming hormone resistance in breast cancer. Clin Cancer Res. 2003, 9: 524S-532S.PubMedGoogle Scholar
- Schiff R, Massarweh S, Shou J, Osborne CK: Breast cancer endocrine resistance: how growth factor signaling and estrogen receptor coregulators modulate response. Clin Cancer Res. 2003, 9: 447S-4454S.PubMedGoogle Scholar
- Lannigan DA: Estrogen receptor phosphorylation. Steroids. 2003, 68: 1-9. 10.1016/S0039-128X(02)00110-1.PubMedView ArticleGoogle Scholar
- Le Goff P, Montano MM, Schodin DJ, Katzenellenbogen BS: Phosphorylation of the human estrogen receptor. Identification of hormone-regulated sites and examination of their influence on transcriptional activity. J Biol Chem. 1994, 269: 4458-4466.PubMedGoogle Scholar
- Arnold SF, Obourn JD, Jaffe H, Notides AC: Serine 167 is the major estradiol-induced phosphorylation site on the human estrogen receptor. Mol Endocrinol. 1994, 8: 1208-1214. 10.1210/me.8.9.1208.PubMedGoogle Scholar
- Castano E, Vorojeikina DP, Notides AC: Phosphorylation of serine-167 on the human oestrogen receptor is important for oestrogen response element binding and transcriptional activation. Biochem J. 1997, 326: 149-157.PubMedPubMed CentralView ArticleGoogle Scholar
- Clark DE, Poteet-Smith CE, Smith JA, Lannigan DA: Rsk2 allosterically activates estrogen receptor alpha by docking to the hormone-binding domain. EMBO J. 2001, 20: 3484-3494. 10.1093/emboj/20.13.3484.PubMedPubMed CentralView ArticleGoogle Scholar
- Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, Masushige S, Gotoh Y, Nishida E, Kawashima H, et al: Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science. 1995, 270: 1491-1494.PubMedView ArticleGoogle Scholar
- Joel PB, Smith J, Sturgill TW, Fisher TL, Blenis J, Lannigan DA: pp90rsk1 regulates estrogen receptor-mediated transcription through phosphorylation of Ser-167. Mol Cell Biol. 1998, 18: 1978-1984.PubMedPubMed CentralView ArticleGoogle Scholar
- Leygue E, Dotzlaw H, Watson PH, Murphy LC: Expression of estrogen receptor beta1, beta2, and beta5 messenger RNAs in human breast tissue. Cancer Res. 1999, 59: 1175-1179.PubMedGoogle Scholar
- Palmieri C, Cheng GJ, Saji S, Zelada-Hedman M, Warri A, Weihua Z, Van Noorden S, Wahlstrom T, Coombes RC, Warner M, et al: Estrogen receptor beta in breast cancer. Endocr Relat Cancer. 2002, 9: 1-13. 10.1677/erc.0.0090001.PubMedView ArticleGoogle Scholar
- Speirs V: Oestrogen receptor beta in breast cancer: good, bad or still too early to tell?. J Pathol. 2002, 197: 143-147. 10.1002/path.1072.PubMedView ArticleGoogle Scholar
- Ogawa S, Inoue S, Watanabe T, Orimo A, Hosoi T, Ouchi Y, Muramatsu M: Molecular cloning and characterization of human estrogen receptor betacx: a potential inhibitor of estrogen action in human. Nucleic Acids Res. 1998, 26: 3505-3512. 10.1093/nar/26.15.3505.PubMedPubMed CentralView ArticleGoogle Scholar
- Graham JD, Clarke CL: Expression and transcriptional activity of progesterone receptor A and progesterone receptor B in mammalian cells. Breast Cancer Res. 2002, 4: 187-190. 10.1186/bcr450.PubMedPubMed CentralView ArticleGoogle Scholar
- Shyamala G, Yang X, Silberstein G, Barcellos-Hoff MH, Dale E: Transgenic mice carrying an imbalance in the native ratio of A to B forms of progesterone receptor exhibit developmental abnormalities in mammary glands. Proc Natl Acad Sci USA. 1998, 95: 696-701. 10.1073/pnas.95.2.696.PubMedPubMed CentralView ArticleGoogle Scholar
- Shyamala G, Yang X, Cardiff RD, Dale E: Impact of progesterone receptor on cell-fate decisions during mammary gland development. Proc Natl Acad Sci USA. 2000, 97: 3044-3049. 10.1073/pnas.97.7.3044.PubMedPubMed CentralView ArticleGoogle Scholar
- Yamashita H, Xu J, Erwin RA, Farrar WL, Kirken RA, Rui H: Differential control of the phosphorylation state of proline-juxtaposed serine residues Ser725 of Stat5a and Ser730 of Stat5b in prolactin-sensitive cells. J Biol Chem. 1998, 273: 30218-30224. 10.1074/jbc.273.46.30218.PubMedView ArticleGoogle Scholar
- Yamashita H, Iwase H, Toyama T, Fujii Y: Naturally occurring dominant-negative Stat5 suppresses transcriptional activity of estrogen receptors and induces apoptosis in T47D breast cancer cells. Oncogene. 2003, 22: 1638-1652. 10.1038/sj.onc.1206277.PubMedView ArticleGoogle Scholar
- Yamashita H, Nevalainen MT, Xu J, LeBaron MJ, Wagner KU, Erwin RA, Harmon JM, Hennighausen L, Kirken RA, Rui H: Role of serine phosphorylation of Stat5a in prolactin-stimulated beta-casein gene expression. Mol Cell Endocrinol. 2001, 183: 151-163. 10.1016/S0303-7207(01)00546-9.PubMedView ArticleGoogle Scholar
- Chen D, Washbrook E, Sarwar N, Bates GJ, Pace PE, Thirunuvakkarasu V, Taylor J, Epstein RJ, Fuller-Pace FV, Egly JM, et al: Phosphorylation of human estrogen receptor alpha at serine 118 by two distinct signal transduction pathways revealed by phosphorylation-specific antisera. Oncogene. 2002, 21: 4921-4931. 10.1038/sj.onc.1205420.PubMedView ArticleGoogle Scholar
- Yamashita H, Nishio M, Toyama T, Sugiura H, Zhang Z, Kobayashi S, Iwase H: Coexistence of HER2 over-expression and p53 protein accumulation is a strong prognostic molecular marker in breast cancer. Breast Cancer Res. 2004, 6: R24-R30. 10.1186/bcr738.PubMedPubMed CentralView ArticleGoogle Scholar
- Clarke CL, Zaino RJ, Feil PD, Miller JV, Steck ME, Ohlsson-Wilhelm BM, Satyaswaroop PG: Monoclonal antibodies to human progesterone receptor: characterization by biochemical and immunohistochemical techniques. Endocrinology. 1987, 121: 1123-1132.PubMedView ArticleGoogle Scholar
- Allred DC, Harvey JM, Berardo M, Clark GM: Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Mod Pathol. 1998, 11: 155-168.PubMedGoogle Scholar
- 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: 9817-9824. 10.1074/jbc.M010840200.PubMedView ArticleGoogle Scholar
- Murphy L, Cherlet T, Adeyinka A, Niu Y, Snell L, Watson P: Phospho-serine-118 estrogen receptor-alpha detection in human breast tumors in vivo. Clin Cancer Res. 2004, 10: 1354-1359.PubMedView ArticleGoogle Scholar
- Saunders PT, Millar MR, Williams K, Macpherson S, Bayne C, O'Sullivan C, Anderson TJ, Groome NP, Miller WR: Expression of oestrogen receptor beta (ERbeta1) protein in human breast cancer biopsies. Br J Cancer. 2002, 86: 250-256. 10.1038/sj.bjc.6600035.PubMedPubMed CentralView ArticleGoogle Scholar
- Fuqua SA, Schiff R, Parra I, Moore JT, Mohsin SK, Osborne CK, Clark GM, Allred DC: Estrogen receptor beta protein in human breast cancer: correlation with clinical tumor parameters. Cancer Res. 2003, 63: 2434-2439.PubMedPubMed CentralGoogle Scholar
- Omoto Y, Inoue S, Ogawa S, Toyama T, Yamashita H, Muramatsu M, Kobayashi S, Iwase H: Clinical value of the wild-type estrogen receptor beta expression in breast cancer. Cancer Lett. 2001, 163: 207-212. 10.1016/S0304-3835(00)00680-7.PubMedView ArticleGoogle Scholar
- Graham JD, Yeates C, Balleine RL, Harvey SS, Milliken JS, Bilous AM, Clarke CL: Characterization of progesterone receptor A and B expression in human breast cancer. Cancer Res. 1995, 55: 5063-5068.PubMedGoogle Scholar
- Bamberger AM, Milde-Langosch K, Schulte HM, Loning T: Progesterone receptor isoforms, PR-B and PR-A, in breast cancer: correlations with clinicopathologic tumor parameters and expression of AP-1 factors. Horm Res. 2000, 54: 32-37. 10.1159/000063434.PubMedView ArticleGoogle Scholar
- Mote PA, Bartow S, Tran N, Clarke CL: Loss of co-ordinate expression of progesterone receptors A and B is an early event in breast carcinogenesis. Breast Cancer Res Treat. 2002, 72: 163-172. 10.1023/A:1014820500738.PubMedView ArticleGoogle Scholar
- Bunone G, Briand PA, Miksicek RJ, Picard D: Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation. EMBO J. 1996, 15: 2174-2183.PubMedPubMed CentralGoogle Scholar
- Atanaskova N, Keshamouni VG, Krueger JS, Schwartz JA, Miller F, Reddy KB: MAP kinase/estrogen receptor cross-talk enhances estrogen-mediated signaling and tumor growth but does not confer tamoxifen resistance. Oncogene. 2002, 21: 4000-4008. 10.1038/sj.onc.1205506.PubMedView ArticleGoogle Scholar
- Kurokawa H, Lenferink AE, Simpson JF, Pisacane PI, Sliwkowski MX, Forbes JT, Arteaga CL: Inhibition of HER2/neu (erbB-2) and mitogen-activated protein kinases enhances tamoxifen action against HER2-overexpressing, tamoxifen-resistant breast cancer cells. Cancer Res. 2000, 60: 5887-5894.PubMedGoogle Scholar
- Dowsett M, Harper-Wynne C, Boeddinghaus I, Salter J, Hills M, Dixon M, Ebbs S, Gui G, Sacks N, Smith I: HER-2 amplification impedes the antiproliferative effects of hormone therapy in estrogen receptor-positive primary breast cancer. Cancer Res. 2001, 61: 8452-8458.PubMedGoogle Scholar
- Elledge RM, Green S, Ciocca D, Pugh R, Allred DC, Clark GM, Hill J, Ravdin P, O'Sullivan J, Martino S, et al: HER-2 expression and response to tamoxifen in estrogen receptor-positive breast cancer: a Southwest Oncology Group Study. Clin Cancer Res. 1998, 4: 7-12.PubMedGoogle Scholar
- Murphy LC, Leygue E, Niu Y, Snell L, Ho SM, Watson PH: Relationship of coregulator and oestrogen receptor isoform expression to de novo tamoxifen resistance in human breast cancer. Br J Cancer. 2002, 87: 1411-1416. 10.1038/sj.bjc.6600654.PubMedPubMed CentralView ArticleGoogle Scholar
- Saji S, Omoto Y, Shimizu C, Warner M, Hayashi Y, Horiguchi S, Watanabe T, Hayashi S, Gustafsson JA, Toi M: Expression of estrogen receptor (ER) (beta)cx protein in ER(alpha)-positive breast cancer: specific correlation with progesterone receptor. Cancer Res. 2002, 62: 4849-4853.PubMedGoogle Scholar
- Sartorius CA, Shen T, Horwitz KB: Progesterone receptors A and B differentially affect the growth of estrogen-dependent human breast tumor xenografts. Breast Cancer Res Treat. 2003, 79: 287-299. 10.1023/A:1024031731269.PubMedView ArticleGoogle Scholar
- Vienonen A, Syvala H, Miettinen S, Tuohimaa P, Ylikomi T: Expression of progesterone receptor isoforms A and B is differentially regulated by estrogen in different breast cancer cell lines. J Steroid Biochem Mol Biol. 2002, 80: 307-313. 10.1016/S0960-0760(02)00027-4.PubMedView ArticleGoogle Scholar