Abstract
Multiple sites of phosphorylation on human estrogen receptor α (ERα) have been identified by a variety of methodologies. Now with the emerging availability of phospho-site-specific antibodies to ERα, the relevance of phosphorylation of ERα in human breast cancer in vivo is being explored. Multiple phosphorylated sites in ERα can be detected in multiple breast tumor biopsy samples, providing evidence of their relevance to human breast cancer in vivo. Published data suggest that the detection in primary breast tumors of phosphorylation at some sites in ERα is associated with a better clinical outcome while phosphorylation at other sites is associated with a poorer clinical outcome most often in patients who have been treated with tamoxifen. This suggests the hypothesis that phospho-profiling of ERα in human breast tumors to establish an ‘ERα phosphorylation code’, may be a more accurate marker of prognosis and/or response to endocrine therapy in human breast cancer.
Introduction
Targeting the estrogen receptor (ER) signaling pathway using the selective ER modulator (SERM), tamoxifen, is efficacious in both treating and preventing breast cancer (Jensen & Jordan 2003, Jordan 2003). Owing to the central role of ER in estrogen signaling, the ER status of breast tumors has long been used to successfully predict response to endocrine therapy (Osborne et al. 1996). There are two known ERs, ERα and ERβ, but the ER status of breast tumors and its clinical correlations are based on the measurement of generally only ERα (Harvey et al. 1999) and current clinical assays measure ERα immunohistochemically (IHC) with specific antibodies. The impact of ERβ remains unclear although roles in human breast cancer have been suggested (Leygue et al. 1998, Murphy & Watson 2006, Skliris et al. 2006, Gruvberger-Saal et al. 2007, Honma et al. 2008).
While ERα expression is the gold-standard biomarker for predicting response to endocrine therapy, it is imperfect, predicting treatment response in ∼50% of ER+ tumors (Osborne 1998, Clarke et al. 2003). Therefore, many ER+ tumors are de novo resistant to tamoxifen without any prior exposure. Furthermore, many of these tumors that initially respond to tamoxifen can acquire resistance during and after tamoxifen therapy. This so-called progression from hormone dependence to independence is an important clinical problem limiting the long-term usefulness of the relatively nontoxic endocrine therapies as well as possibly impacting the use of SERMs as preventative agents.
Most acquired tamoxifen resistance (70–80%) occurs despite continued expression of ERα (Robertson 1996). Newer therapies targeting ER via different mechanisms, such as aromatase inhibitors (AI; Goss et al. 2003) and selective ER downregulators (e.g. ICI182780; Robertson 2002), or potential new therapies, such as electrophilic modulators of ER zinc fingers (Wang et al. 2004), were all developed from basic research into molecular mechanisms of ER action, but development of therapy resistance is likely also to be a problem clinically. Understanding molecular mechanism(s) of ER action still holds promise for identifying complementary and/or alternative approaches targeting other levels of ER signaling to treat ER+ breast cancer. Such knowledge may also identify ways to circumvent resistance, as well as offering new biomarkers beyond ERα for the more precise prediction of therapy responses. Current downstream markers of ERα activity such as progesterone receptor (PR) improve prediction (Bardou et al. 2003), but remain imperfect, supporting the need for other biomarkers to assist in the accurate prediction of treatment response.
Molecular mechanisms of estrogen action and possible mechanisms of tamoxifen resistance
Basic research has significantly increased the knowledge of the molecular mechanisms of ER action (Hall et al. 2001, Nawaz & O'Malley 2004). Multifaceted mechanisms underlying estradiol (E2) action have been identified. These include multiple ERs and variants (Murphy et al. 2003); multiple subcellular localization sites (Murphy et al. 2003); multiple transcription coactivators and corepressors (McKenna et al. 1999); multiple posttranslational modifications (PTMs; Nawaz & O'Malley 2004); multiple levels of cross talk with other signaling pathways (Murphy et al. 2003); and multiple levels of control of ER expression, including proteasomal-mediated degradation (Reid et al. 2003). Alterations at any one of these levels could affect responsiveness to SERMs and/or AIs. There is evidence that multiple mechanisms are involved in altered SERM action during progression from hormone dependence to independence in breast cancer (Clarke et al. 2003, Murphy et al. 2003, Santen et al. 2004). In particular, growth factor receptor signaling pathways are frequently upregulated during tumorigenesis and cancer progression. The resulting increased cross talk with ER signaling is thought to be a mechanism of endocrine therapy resistance (Osborne et al. 2005). In part, this is due to kinases, activated by growth factor signaling, being able to phosphorylate and alter ERα activity in a ligand-independent manner (Kato et al. 1995). Effects on ER coactivator activity are also involved (Font de Mora & Brown 2000). It should also be noted that phosphorylation may influence ER protein levels through modulation of targeting ER for proteasomal degradation. ER can be lost during progression in 25–30% of ER+ tumors and it has been suggested that in some cases intra-tumoral factors such as hypoxia, growth factor, and cytokine signaling may act through phosphorylation of ER to cause reversible depression of ERα (Cooper et al. 2004, Creighton et al. 2006, Lopez-Tarruella & Schiff 2007, West & Watson 2010).
Phosphorylation sites identified in ERα
ERα can undergo multiple PTMs, for example, phosphorylation, acetylation, ubiquitylation, and sumoylation (Lannigan 2003, Weigel & Moore 2007). However, relatively little is known about the function and regulation of any of the PTMs that ERα can potentially undergo (Lannigan 2003, Ward & Weigel 2009) and even less is known about their relevance in vivo. As shown in Fig. 1, ERα can be phosphorylated on multiple amino acid residues throughout the whole protein and within all major structural domains: the N-terminal A/B domain, i.e. serine 46 (S46), serine 47 (S47), tyrosine 52 (Y52), serine 102 (S102), serine 104 (S104), serine 106 (S106), serine 118 (S118), serine 154 (S154), and serine 167 (S167); the DNA-binding or C domain, tyrosine 219 (Y219), serine 236 (S236; Chen et al. 1999); the hinge or D domain, serine 305 (S305; Michalides et al. 2004), and the ligand-binding domain or E domain, threonine 311 (T311; Lee & Bai 2002) and tyrosine 537 (Y537; Arnold et al. 1995b). Recently, novel phosphorylation sites in ERα were identified (Britton et al. 2008, Williams et al. 2009). Table 1 lists the sites of phosphorylation in ERα, which have been identified experimentally, using different methodologies. Detection of phosphorylation at some but not all of these sites has been confirmed in human breast tumor biopsy samples (Table 1).
Multiple phosphorylated sites in ERα have been identified by a variety of approaches as listed in Table 1. These are shown schematically in the figure that depicts different structural (A, B, C, D, E) and functional domains: activation function 1 (AF1), DNA-binding domain (DBD), hinge region, and ligand-binding domain (LBD) of human ERα.
Citation: Endocrine-Related Cancer 18, 1; 10.1677/ERC-10-0070
Phosphorylation sites identified experimentally in human estrogen receptor α
| Site of phosphorylation | Domain | Method of identification | Substrate source | References | Breast tumors in vivo |
|---|---|---|---|---|---|
| Ser46/47 | A/B | 1 | TT/COS1 | Williams et al. (2009) | ND |
| Tyr52 | A/B | 3 | TT/HEK | He et al. (2010) | ND |
| Ser102 | A/B | 2 | MCF7 | Atsriku et al. (2009) | ND |
| Ser104 | A/B | 2 | MCF7 | Atsriku et al. (2009) | Y |
| Ser106 | A/B | 2 | MCF7 | Atsriku et al. (2009) | Y |
| Ser118 | A/B | 2 | MCF7 | Atsriku et al. (2009) | Y |
| Ser154 | A/B | 2 | MCF7 | Atsriku et al. (2009) | ND |
| Ser167 | A/B | 2 | MCF7 | Atsriku et al. (2009) | Y |
| Ser212 | C | 2 | MCF7 | Atsriku et al. (2009) | ND |
| Tyr219 | C | 3 | TT/HEK | He et al. (2010) | ND |
| Ser236 | C | 2 | MCF7 | Atsriku et al. (2009) | ND |
| Ser282 | D | 1 | TT/COS1 | Williams et al. (2009) | Y |
| Ser294 | D | 1, 2 | MCF7 | Atsriku et al. (2009) and Williams et al. (2009) | Y |
| Ser305 | D | 3 | TT/HeLa | Wang et al. (2002) | Y |
| Thr311 | E | 1, 3 | TT/Ishikawa | Lee & Bai (2002) | Y |
| Tyr537 | E | 1 | Sf9rER/MCF7 | Arnold et al. (1995a) | ND |
| Ser554 | F | 2 | MCF7 | Atsriku et al. (2009) | ND |
| Ser559 | F | 1, 2 | MCF7 | Atsriku et al. (2009) and Williams et al. (2009) | Y |
1, [32P]H3PO4 labeling; Edman degradation; phosphoamino acid analysis; phosphopeptide mapping. 2, mass spectroscopy. 3, site-directed mutagenesis; in vitro kinase assay; western blotting. ND, not determined; TT, transient transfection; Y, yes. Adapted from Murphy et al. (2006) with permission.
Some potential functions of phosphorylation at different sites in ERα
The exact role of phosphorylation at individual or multiple sites is underexplored although effects on transcription, nuclear localization, dimerization, DNA binding, coactivator recruitment, and ligand binding (Weis et al. 1996, Chen et al. 1999, Endoh et al. 1999, Likhite et al. 2006) have been demonstrated in cell culture models (Murphy et al. 2006). More recently, roles of ERα phosphorylation in RNA splicing (Auboeuf et al. 2002, 2007, Masuhiro et al. 2005) as well as in ER protein stability (Medunjanin et al. 2005, Grisouard et al. 2007) and regulation of other types of PTMs (Cui et al. 2004) have been suggested. A list of experimentally derived important functions of phosphorylation at different sites in ERα is shown in Table 2.
Experimentally identified functional roles of site-specific phosphorylation in estrogen receptor α
Interestingly, within the A/B domain of ERα often only small effects on transcriptional function were observed when any one site, e.g. S104, S106, and S118, was mutated to eliminate phosphorylation. While the effects of mutating all three sites appeared to be additive (Le Goff et al. 1994), giving ∼50% reduction in transcriptional activity. Importantly, lack of phosphorylation of all of these sites does not eliminate estrogen-induced ERα transcriptional activity. Other data were reported showing that combinations of phosphorylation sites affected activity more than individual sites alone (Joel et al. 1998, Medunjanin et al. 2005). Furthermore, other data suggest that it is the combination of phosphorylation sites within ERα rather than any one individual site that may be important for mediating effects of any individual kinase (Rogatsky et al. 1999). The concept that combinations of PTMs of ERα rather than individual sites may be of primary importance in affecting function and response to endocrine therapies is emerging (Barone et al. 2010, Skliris et al. 2010) and supports the hypothesis of a PTM code for ERα, as discussed below.
Phosphorylation, at least at S118, has been suggested to be involved in protein turnover via a proteosome-mediated mechanism (Valley et al. 2005, Grisouard et al. 2007). How other sites of phosphorylation may also affect receptor turnover is not clear and underexplored. However, proteosome-mediated turnover of steroid receptors has been shown to be essential for the dynamic and cyclical nature of receptor occupancy on target gene promoters, which is in turn critically important for transcriptional activity (Reid et al. 2003). Therefore, the further characterization of how phosphorylation at other sites may also affect receptor turnover and stability would be of interest.
An important hypothesis that has developed from laboratory models is that ligand-independent phosphorylation of ERα may cause tamoxifen resistance in vivo. For example, a well-studied p-ERα site (Lannigan 2003) is S118 (Fig. 1). Both E2 and growth factors, e.g. epidermal growth factor (EGF) or insulin-like growth factor 1, stimulate phosphorylation of S118 (Joel et al. 1998, Chen et al. 2002, Lannigan 2003). Mitogen activated protein kinase (MAPK) (ERK1/2), an important enzyme activated by growth factor receptor pathways, can phosphorylate S118 in a ligand-independent manner in vitro (Kato et al. 1995) and in vivo (Joel et al. 1998). Interestingly, estrogen treatment is the most powerful stimulator of phosphorylation at S118 and it is independent of MAPK (ERK1/2; Joel et al. 1998). However, which kinase is responsible for estrogen-induced p-S118 is not clear. CDK7, IKKα, and GSK3β are the possible candidates (Chen et al. 2002, Medunjanin et al. 2005, Park et al. 2005). Since ligand-independent ERα activation may underlie tamoxifen resistance, and EGFR/HER2 upregulation is associated with clinical resistance to tamoxifen in breast cancer (Pietras et al. 1995, Dowsett et al. 2001, Knowlden et al. 2003, Schiff et al. 2004), a role of p-S118-ERα has been suggested. S167 is another site of ERα phosphorylation. AKT (Campbell et al. 2001) and pp90rsk (Joel et al. 1998) can phosphorylate ERα at S167 and increased p-AKT has been associated with poor clinical outcome in breast cancer patients treated with tamoxifen (Kirkegaard et al. 2005). Also, experimental data suggest that ligand-independent phosphorylation of S305 may have a role in tamoxifen resistance in breast cancer cells as well (Michalides et al. 2004, Holm et al. 2009). However, the relevance of p-ERα in breast cancer in vivo is unclear (Lannigan 2003).
Regulation of ERα phosphorylation
Table 3 provides a list of several kinases that have been shown experimentally to have a role in regulation of ERα phosphorylation at various sites but the contributions of specific kinases in vivo are not known. Those studies providing evidence of a potentially direct role of an individual kinase in phosphorylating ERα are also shown in Table 3.
Candidate kinases involved in site-specific estrogen receptor α phosphorylation
Correlation of expression of kinases with individual p-ERα expression in human breast tumor samples (Murphy et al. 2004b, Sarwar et al. 2006, Jiang et al. 2007, Yamashita et al. 2008) is one approach to gain insight into the kinases involved in regulation in vivo. In this regard, even when p-S118 and/or p-S167 are found associated with the parameters of an intact estrogen-dependent signaling pathway and better clinical outcome on tamoxifen, they are also found to be positively associated with several activated kinases, i.e. MAPK/ERK1/2, p90RSK, and/or AKT in primary human breast tumor biopsy samples. This supports the possibility that they may be involved in phosphorylation of ERα in breast tumors in vivo, and/or that an intact estrogen-dependent signaling pathway is involved in regulation of pathways involving activation of MAPK/ERK1/2, p90RSK, and/or AKT (Cheskis et al. 2008, Santen et al. 2009). In addition, when only PAK1-positive tumors, independent of location, were considered, a positive correlation of p-S305 with nuclear PAK1 expression was found (Bostner et al. 2010), suggesting a role of PAK1 in nuclear ERα phosphorylation at S305.
Interestingly, in primary human breast tumor samples there is generally a lack of correlation of overexpression of EGFR or HER2 with p-ERα (Weitsman et al. 2006, Jiang et al. 2007, Murphy et al. 2009). Although some studies found a weak positive association of HER2 expression and p-S118 (Jiang et al. 2007, Yamashita et al. 2008, Zoubir et al. 2008), overall most studies suggest that overexpression of EGFR and HER2 signaling pathways, at least in primary breast tumors, is unlikely to be involved in estrogen independence and tamoxifen resistance de novo.
Studies of p-ERα in human breast cancer biopsy samples
Over the past 5 years or so antibodies to specific phosphorylated sites in ERα have become available, enabling the determination of the relevance of these PTMs in human breast tissues in vivo. Validation of such antibodies for IHC is extremely important and has been reported in some cases (Holm et al. 2009). There are also some reports concerning the effect of breast biopsy collection and processing times on phospho-epitope detection (Skliris et al. 2009), however, such studies are limited in scope (Barnes et al. 2008).
Published studies to date in which phospho-specific sites in ERα have been determined using IHC in human breast tumor biopsy samples are listed in Table 4. The majority of these studies have focused on p-S118, p-S167, and p-S305 although more recently other novel sites have been determined as antibodies become available or have been custom generated (Skliris et al. 2009). However, more effort is required to generate reliable, high-quality antibodies suitable for IHC, western blotting, immunoprecipitation, and chromatin immunoprecipitation not only for phospho-specific sites but also for other posttranslationally modified sites in ERα.
Published studies of the determination of p-estrogen receptor α (ERα) expression in human breast cancer biopsy samples
| p-ERα | Number of cases | References | Biomarker association |
|---|---|---|---|
| p-S104/106 | 301 | Skliris et al. (2009) | Positive with PR |
| p-S118 | 45 | Murphy et al. (2004b) | Negative with grade |
| p-S118 | 113 | Murphy et al. (2004a) | Positive with PR |
| p-S118 | ? | Gee et al. (2005) | Positive with PR |
| p-S118 | 75 | Yamashita et al. (2005) | Positive with PRA |
| p-S118 | 370 | Skliris et al. (2009) | Positive with PR |
| p-S118 | 301 | Sarwar et al. (2006) | Negative with grade |
| p-S118 | 279 | Bergqvist et al. (2006) | Positive with PR |
| p-S118 | 290 | Jiang et al. (2007) | Negative with grade |
| p-S118 | 16 | Yamashita et al. (2009) | Decreased expression with neoadjuvant AI treatment (P<0.0001) |
| p-S118 | 80 | Zoubir et al. (2008) | Decreased expression with neoadjuvant Tam and AI treatment (P=0.0001) |
| p-S167 | 290 | Jiang et al. (2007) | Negative with size |
| p-S167 | 75 | Yamashita et al. (2005) | Positive with PRA |
| p-S167 | 16 | Yamashita et al. (2009) | Decreased expression with neoadjuvant AI treatment (P=0.0005) |
| p-S305 | 377 | Holm et al. (2009) | Positive with grade |
| Positive with MI | |||
| p-S305 | 841 | Bostner et al. (2010) | Positive with small tumor size |
| p-T311 | 406 | Skliris et al. (2009) | Positive with PR |
PR, progesterone receptor (ligand binding or IHC); MI, mitotic index; AI, aromatase inhibitor. Adapted from Murphy et al. (2009a) with permission.
In some of these studies, associations with known histopathological markers were found and these are listed in Table 4. Although contradictory results are sometimes found, possibly due to small numbers of cases and different patient characteristics in the study cohorts, differences in scoring and quantification methods, as well as different definitions of positivity and negativity, common themes have emerged. First, in contrast to what was expected, either p-S118 or p-S167 was found associated with the parameters of less aggressive and more differentiated tumors as well as an intact estrogen-responsive signaling pathway (Murphy et al. 2004b, Jiang et al. 2007).
In addition, recently p-S305 has been a focus; however, in contrast to p-S118 and p-S167, detection of p-S305 is more likely to be associated with features of more aggressive tumors (Holm et al. 2009; Table 4). In apparent contrast to this latter finding, p-S305 has also been found to be associated with smaller size (Bostner et al. 2010). One study also compared the level of p-S118 and p-S167 expression in primary breast tumors compared to secondary tumors from 10 patients after relapse (Yamashita et al. 2005) and found that there was increased levels of both p-S118 and p-S167 in the secondary versus the primary tumors, although this was statistically significant only for p-S118. These observations suggest the possibility that p-ERα may be a useful biomarker in metastatic breast cancer as well.
From the above studies it seems that some phosphorylation sites in ERα such as p-S118 may be associated with better clinical outcome and others such as p-S305 may be associated with poor clinical outcome. Published studies reporting relationships of p-ERα with clinical outcome in breast cancer are listed in Table 5.
Clinical outcome studies of p-estrogen receptor α (ERα) expression in human breast cancer
| p-ERα | Number of cases | References | Type of hormonal therapy | Outcome | Significance |
|---|---|---|---|---|---|
| p-S118 | 113 | Murphy et al. (2004a) | Tamoxifen | Positive longer RFS | P=0.0018 univariate |
| p-S118 | ? | Gee et al. (2005) | Tamoxifen | Positive longer TTP | P<0.009 univariate |
| p-S118 | 75 | Yamashita et al. (2005) | Tamoxifen | No effect | |
| p-S118 | 301 | Sarwar et al. (2006) | Tamoxifen | No effect | |
| p-S118 | 108/279 | Bergqvist et al. (2006) | Tamoxifen | No effect | |
| p-S118 | 290 | Jiang et al. (2007) | Tamoxifen | No effect | |
| p-S118 | 278 | Yamashita et al. (2008) | Low longer RFS | P=0.0003 multivariate | |
| p-S118 | 114 | Generali et al. (2009) | Letrozole | Positive better clinical response | P=0.004 |
| Neoadjuvant 6 months | |||||
| p-S118a | 239 | Kok et al. (2009) | Tamoxifen | Positive better RFS | P=0.037 multivariate |
| p-S118 | 80 | Zoubir et al. (2008) | AI and Tam neoadjuvant | Larger decreased expression associated with better outcome | P=0.017 |
| p-S167 | 290 | Jiang et al. (2007) | Tamoxifen | Positive longer RFS | P=0.006 univariate |
| Positive longer OS | P=0.023 multivariate | ||||
| p-S167 | 75 | Yamashita et al. (2005) | Tamoxifen | Positive longer RFS | P=0.033 univariate |
| p-S167 | 278 | Yamashita et al. (2008) | High longer RFS | P=0.0002 multivariate | |
| p-S305 | 377 | Holm et al. (2009) | Tamoxifen | Negative longer RFS | P=0.01 multivariate |
| p-S305 | 334 | Kok et al. (2010) | Tamoxifen | No effect aloneb | |
| p-S305 | 841 | Bostner et al. (2010) | Tamoxifen | No effect aloneb |
RFS, relapse free survival; TTP, time to progression; OS, overall survival.
Clinical trial material.
Significant when interactions with PKA and/or PAK1 expression considered.
Association of p-ERα with clinical outcome in breast cancer
Several studies have now been published where p-ERα expression has been explored with respect to clinical outcome in breast cancer patients, most often focusing on patients treated with tamoxifen. In contrast to what would have been expected from laboratory model systems, higher expression of either p-S167 and/or p-S118 is most often but not always associated with a better clinical outcome in patients on tamoxifen therapy (Table 5; Murphy et al. 2004a, Yamashita et al. 2005, 2008, Jiang et al. 2007). Most recently, the predictive and prognostic value of p-S118 was assessed in a randomized controlled trial of no systemic treatment versus 2 years of adjuvant tamoxifen therapy (Kok et al. 2009). Improved recurrence-free survival was found in those patients whose tumors expressed high levels of p-S118. This study is consistent with our previously published retrospective analysis (Murphy et al. 2004a), which we have also recently confirmed in a larger cohort of patients representing over 300 cases (Skliris et al. 2010). In addition, there are data to support the view that combinations of p-S118 with known biologically relevant biomarkers such as PR may further improve the prediction of prognosis and response to endocrine therapy (Murphy et al. 2004a). Such data support the combined use of biologically relevant markers for the improved prediction of therapy response.
Interestingly, the results published by Jiang et al. (2007) and Yamashita et al. (2008) show that high levels of p-S167 expression are the better predictor of benefit from tamoxifen and also suggest that both of these phosphorylation sites either alone or in combination in primary breast tumors may be useful biomarkers of endocrine therapy response.
These data strongly support undertaking further studies, potentially using the tissue microarrays generated from the collected tissue samples of large endocrine therapy clinical trials, such as Arimidex, Tamoxifen, Alone or in Combination (ATAC) trial (Forbes et al. 2008), to determine the value of measuring p-S118 and/or p-S167 as more precise biomarkers of endocrine therapy response in human breast cancer. However, standardization of antibodies and methodologies for such analyses should be decided upon and used such that the protocols can be more easily transferred and reproduced in a clinical laboratory environment. Furthermore, there may be other novel sites of phosphorylation in ERα that may be more tightly associated with prognosis and clinical benefit from endocrine therapies. Supporting such speculation are recently published data focusing on some novel phosphorylation sites (Murphy et al. 2009a, Williams et al. 2009, Skliris et al. 2010).
Since the majority of studies so far reported have only determined p-ERα in primary breast tumors and therefore only address associations with de novo/intrinsic endocrine resistance, an important gap in our knowledge is the relationships of phosphorylated forms of ERα with acquired resistance in vivo.
Multiple phosphorylated forms of ERα
Detection in any one tumor of multiple phosphorylated forms of ERα is another emerging theme (Jiang et al. 2007, Yamashita et al. 2008, Skliris et al. 2009, 2010). In some cases, one p-ERα isoform was positively correlated with one or more other p-ERα isoforms (Yamashita et al. 2005, 2008, Jiang et al. 2007, Skliris et al. 2009). Furthermore, mass spectroscopy data (Atsriku et al. 2009) and co-immunoprecipitation data (Murphy et al. 2009) support the idea that there is a population of ERα molecules phosphorylated at multiple sites at least in MCF7 human breast cancer cells, which endogenously express ERα.
Since estrogen treatment has been shown to increase phosphorylation at several sites in ERα it is possible that all of these sites similarly represent a functional ligand-dependent pathway in human breast tumors (Lannigan 2003, Murphy et al. 2006, Weitsman et al. 2006, Williams et al. 2007, 2009). Further support for this conclusion is the observation that both p-S118 and p-S167 are decreased by neoadjuvant treatment with AIs (Zoubir et al. 2008, Yamashita et al. 2009) and that several p-ERα are correlated with the PR status (Murphy et al. 2004a, Yamashita et al. 2005, Bergqvist et al. 2006, Sarwar et al. 2006). Another possibility is that phosphorylation or other PTMs (Cui et al. 2004) at one site increases the possibility of phosphorylation at another site (Yang et al. 2007).
It is interesting that the phospho-epitopes predicting for good clinical outcome are clustered in the N-terminus of ERα and that the one p-ERα consistently associated with a poor clinical outcome in vivo, p-S305, is more C-terminal. Our recent studies have identified two other sites, p-T311 and p-S559 (Murphy et al. 2009a), that seem to be associated with a poorer clinical outcome (Skliris et al. 2010), interestingly, however, T311 and S559 are also located more C-terminally in the ERα protein. The significance of this is unclear at the moment, however, taking into consideration these latter data together with the p-S305 published data, it would seem that not all types of p-ERα necessarily predict good prognosis or outcome to endocrine therapies.
The presence of multiple phosphorylation sites in ERα (Britton et al. 2008, Murphy et al. 2009a) that may have differential effects on activity suggests that it may be necessary to consider the balance of multiple phosphorylation sites in vivo in terms of predicting clinical outcome with respect to endocrine treatment responsiveness. Recently, data have been published where up to seven different phosphorylation sites in ERα in any individual tumor were taken into account by developing a mathematical model that balances the presence of phosphorylation sites associated with good clinical benefit and those associated with poor clinical benefit. The resulting score generated from this analysis (called the P7-score) was found using multivariate analysis to be independently associated with overall survival and relapse-free survival in patients treated with tamoxifen (Skliris et al. 2010), raising the possibility that phospho-profiling of ERα may provide more precise prediction of prognosis and potentially treatment response to endocrine therapies. These interesting results require replication in other cohorts. Furthermore, since large clinical sample numbers are required to achieve the statistical power needed when multiple sites of phosphorylation are to be determined, the development and use of tissue microarray methods will facilitate this process.
Most recently the detection of ERβ phosphorylated on S105 in human breast tumor samples was reported (Hamilton-Burke et al. 2010), and high levels of nuclear staining for p-S105-ERβ were found associated with good prognosis in breast cancer patients. Similar to ERα, there are likely to be more sites in ERβ which can be phosphorylated (Sanchez et al. 2010) and can affect function. These data establish the relevance of p-ERβ in breast cancer in vivo and lead to the speculation that a phosphorylation and/or PTM code for ERβ in breast cancer exists. Therefore, phosphorylation profiling of both ERs may be more informative than either alone (Murphy & Watson 2006). This possibility remains to be explored when more tools, e.g. phospho-site-specific antibodies, become more widely available.
The concept of PTM codes or profiles is best studied and functionally relevant for histones (Sims & Reinberg 2008). But recently, the relevance of a PTM code for nonhistone proteins, of significance to steroid receptors, has been underscored using a knockin allele of an SRC3/AIB1/NCOA3 gene mutated in four conserved phosphorylation sites that resulted in marked changes in systemic function, which were distinct from overexpressing or knocking out the whole gene (York et al. 2010).
Summary and speculation
Retrospective clinical outcome studies and, more recently, a randomized clinical trial (Kok et al. 2009) strongly support a positive association of p-S118 and/or p-S167-ERα with better clinical outcome to tamoxifen. Therefore, these two phosphorylation sites, in contrast to what was predicted from laboratory-based models, are unlikely to be a mechanism of de novo resistance to tamoxifen. Furthermore, data have been published suggesting that p-S118 may also predict response to AIs (Generali et al. 2009). Their role in acquired resistance remains to be determined. In contrast, the results, where p-S305 has been determined in human breast cancer biopsy samples, support its association with lack of benefit from tamoxifen treatment. These data, together with the detection of multiple different phosphorylation sites in any one human breast tumor, support the hypothesis that phospho-profiling of ERα in human breast tumors, to establish an ERα phosphorylation code, may be a more accurate biomarker of prognosis and/or response to endocrine therapy.
Furthermore, since other PTMs such as acetylation, can occur in ERα (Faus & Haendler 2006) by analogy with the ‘histone code’ (Fischle et al. 2003), an ERα PTM code may exist, which more accurately reflects the balance of ligand-mediated and cross talk signal transduction (ligand-independent) pathways affecting the breast tumor cells. ERα is pivotal in breast cancer biology, and it is likely to be an important site, where integration of diverse signals occurs, to regulate breast cancer cell growth and survival. This, we suggest, will be reflected in a PTM code.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the review reported.
Funding
This work was supported by the Canadian Institutes of Health Research (CIHR), the CancerCare Manitoba Foundation (CCMF), and the Canadian Breast Cancer Foundation (CBCF). This work was also supported by the Manitoba Breast Tumor Bank, a member of the Canadian Tumor Repository Network and was funded in part by CCMF and CIHR.
References
Acconcia F, Manavathi B, Mascarenhas J, Talukder AH, Mills G & Kumar R 2006 An inherent role of integrin-linked kinase-estrogen receptor alpha interaction in cell migration. Cancer Research 66 11030–11038 doi:10.1158/0008-5472.CAN-06-2676.
Adeyinka A, Niu Y, Cherlet T, Snell L, Watson P & Murphy L 2002 Activated mitogen-activated protein kinase expression during human breast tumorigenesis and breast cancer progression. Clinical Cancer Research 8 1747–1753.
Arnold S, Obourn J, Jaffe H & Notides A 1994 Serine 167 is the major estradiol-induced phosphorylation site on the human estrogen receptor. Molecular Endocrinology 8 1208–1214 doi:10.1210/me.8.9.1208.
Arnold S, Obourn J, Jaffe H & Notides A 1995a Phosphorylation of the human estrogen receptor on tyrosine 537 in vivo and by Src family tyrosines in vitro. Molecular Endocrinology 9 24–33 doi:10.1210/me.9.1.24.
Arnold S, Vorojeikina D & Notides A 1995b Phosphorylation of tyrosine 537 on the human estrogen receptor is required for binding to an estrogen response element. Journal of Biological Chemistry 270 30205–30212 doi:10.1074/jbc.270.4.1850.
Arnold SF, Melamed M, Vorojeikina DP, Notides AC & Sasson S 1997 Estradiol-binding mechanism and binding capacity of the human estrogen receptor is regulated by tyrosine phosphorylation. Molecular Endocrinology 11 48–53 doi:10.1210/me.11.1.48.
Assender JW, Gee JM, Lewis I, Ellis IO, Robertson JF & Nicholson RI 2007 Protein kinase C isoform expression as a predictor of disease outcome on endocrine therapy in breast cancer. Journal of Clinical Pathology 60 1216–1221 doi:10.1136/jcp.2006.041616.
Atsriku C, Britton DJ, Held JM, Schilling B, Scott GK, Gibson BW, Benz CC & Baldwin MA 2009 Systematic mapping of posttranslational modifications in human estrogen receptor-α with emphasis on novel phosphorylation sites. Molecular and Cellular Proteomics 8 467–480 doi:10.1074/mcp.M800282-MCP200.
Auboeuf D, Hönig A, Berget SM & O'Malley BW 2002 Coordinate regulation of transcription and splicing by steroid receptor coregulators. Science 298 416–419 doi:10.1126/science.1073734.
Auboeuf D, Batsché E, Dutertre M, Muchardt C & O'Malley BW 2007 Coregulators: transducing signal from transcription to alternative splicing. Trends in Endocrinology and Metabolism 18 122–129 doi:10.1016/j.tem.2007.02.003.
Bardou V, Arpino G, Elledge R, Osborne C & Clark G 2003 Progesterone receptor status significantly improves outcome prediction over estrogen receptor status alone for adjuvant endocrine therapy in two large breast cancer databases. Journal of Clinical Oncology 21 1973–1979 doi:10.1200/JCO.2003.09.099.
Barlund M, Monni O, Kononen J, Cornelison R, Torhorst J, Sauter G, Kallioniemi O-P & Kallioniemi A 2000 Multiple genes at 17q23 undergo amplification and overexpression in breast cancer. Cancer Research 60 5340–5344.
Barnes RO, Parisien M, Murphy LC & Watson PH 2008 Influence of evolution in tumor biobanking on the interpretation of translational research. Cancer Epidemiology, Biomarkers and Prevention 17 3344–3350 doi:10.1158/1055-9965.EPI-08-0622.
Barone I, Iacopetta D, Covington KR, Cui Y, Tsimelzon A, Beyer A, Ando S & Fuqua SA 2010 Phosphorylation of the mutant K303R estrogen receptor α at serine 305 affects aromatase inhibitor sensitivity. Oncogene 29 2404–2414 doi:10.1038/onc.2009.520.
Bergqvist J, Elmberger G, Ohd J, Linderholm B, Bjohle J, Hellborg H, Nordgren H, Borg A, Skoog L & Bergh J 2006 Activated ERK1/2 and phosphorylated oestrogen receptor α are associated with improved breast cancer survival in women treated with tamoxifen. European Journal of Cancer 42 1104–1112 doi:10.1016/j.ejca.2006.01.028.
Bostner J, Skoog L, Fornander T, Nordenskjold B & Stal O 2010 Estrogen receptor-α phosphorylation at serine 305, nuclear p21-activated kinase 1 expression, and response to tamoxifen in postmenopausal breast cancer. Clinical Cancer Research 16 1624–1633 doi:10.1158/1078-0432.CCR-09-1733.
Britton D, Scott G, Schilling B, Atsriku C, Held J, Gibson B, Benz C & Baldwin M 2008 A novel serine phosphorylation site detected in the N-terminal domain of estrogen receptor isolated from human breast cancer cells. Journal of the American Society for Mass Spectrometry 19 729–740 doi:10.1016/j.jasms.2008.02.008.
Campbell R, Bhat-Nakshatri P, Patel N, Constantinidou D, Ali S & Nakshatri H 2001 Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance. Journal of Biological Chemistry 276 9817–9824 doi:10.1074/jbc.M010840200.
Castana E, Vorojeikina D & Notides A 1997 Phosphorylation of serine-167 on the human oestrogen receptor is important for oestrogen response element binding and transcriptional activation. Biochemical Journal 326 149–157.
Chen D, Pace P, Coombes R & Ali S 1999 Phosphorylation of human estrogen receptor α by protein kinase A regulates dimerization. Molecular and Cellular Biology 19 1002–1015.
Chen D, Washbrook E, Sarwar N, Bates G, Pace P, Thirunuvakkarasu V, Taylor J, Epstein R, Fuller-Pace F & Egly J et al. 2002 Phosphorylation of human estrogen receptor alpha at serine 118 by two distinct signal transduction pathways revealed by phosphorylation-specific antisera. Oncogene 21 4921–4931 doi:10.1038/sj.onc.1205420.
Cheskis BJ, Greger J, Cooch N, McNally C, McLarney S, Lam HS, Rutledge S, Mekonnen B, Hauze D & Nagpal S et al. 2008 MNAR plays an important role in ERa activation of Src/MAPK and PI3K/Akt signaling pathways. Steroids 73 901–905 doi:10.1016/j.steroids.2007.12.028.
Clarke R, Liu M, Bouker K, Gu Z, Lee R, Zhu Y, Skaar T, Gomez B, O'Brien K & Wang Y et al. 2003 Antiestrogen resistance in breast cancer and the role of estrogen receptor signaling. Oncogene 22 7316–7339 doi:10.1038/sj.onc.1206937.
Cooper C, Liu G, Niu Y, Santos S, Murphy L & Watson P 2004 Intermittent hypoxia induces proteasome-dependent down-regulation of estrogen receptor α in human breast carcinoma. Clinical Cancer Research 10 8720–8727 doi:10.1158/1078-0432.CCR-04-1235.
Creighton CJ, Hilger AM, Murthy S, Rae JM, Chinnaiyan AM & El-Ashry D 2006 Activation of mitogen-activated protein kinase in estrogen receptor α-positive breast cancer cells in vitro induces an in vivo molecular phenotype of estrogen receptor α-negative human breast tumors. Cancer Research 66 3903–3911 doi:10.1158/0008-5472.CAN-05-4363.
Cui Y, Zhang M, Pestell R, Curran EM, Welshons WV & Fuqua SA 2004 Phosphorylation of estrogen receptor α blocks its acetylation and regulates estrogen sensitivity. Cancer Research 64 9199–9208 doi:10.1158/0008-5472.CAN-04-2126.
Dowsett M, Harper-Wynne C, Boeddinghaus I, Salter J, Hills M, Dixon M, Ebbs S, Gui G, Sacks N & Smith I 2001 HER-2 amplification impedes the antiproliferative effects of hormone therapy in estrogen receptor-positive primary breast cancer. Cancer Research 61 8452–8458.
Dutertre M & Smith C 2003 Ligand-independent interactions of p160/steroid receptor coactivators and CREB-binding protein (CBP) with estrogen receptor-alpha: regulation by phosphorylation sites in the A/B region depends on other receptor domains. Molecular Endocrinology 17 1296–1314 doi:10.1210/me.2001-0316.
Elsberger B, Tan BA, Mitchell TJ, Brown SB, Mallon EA, Tovey SM, Cooke TG, Brunton VG & Edwards J 2009 Is expression or activation of Src kinase associated with cancer-specific survival in ER-, PR- and HER2-negative breast cancer patients? American Journal of Pathology 175 1389–1397 doi:10.2353/ajpath.2009.090273.
Endoh H, Maruyama K, Masuhiro Y, Kobayashi Y, Goto M, Tai H, Yanagisawa J, Metzger D, Hashimoto S & Kato S 1999 Purification and identification of p68 RNA helicase acting as a transcriptional coactivator specific for the activation function 1 of human estrogen receptor α. Molecular and Cellular Biology 19 5363–5372.
Faus H & Haendler B 2006 Post-translational modifications of steroid receptors. Biomedicine and Pharmacotherapy 60 520–528 doi:10.1016/j.biopha.2006.07.082.
Fischle W, Wang Y & Allis CD 2003 Binary switches and modification cassettes in histone biology and beyond. Nature 425 475–479 doi:10.1038/nature02017.
Font de Mora J & Brown M 2000 AIB1 is a conduit for kinase-mediated growth factor signaling to the estrogen receptor. Molecular and Cellular Biology 20 5041–5047 doi:10.1128/MCB.20.14.5041-5047.2000.
Forbes JF, Cuzick J, Buzdar A, Howell A, Tobias JS & Baum M 2008 Effect of anastrozole and tamoxifen as adjuvant treatment for early-stage breast cancer: 100-month analysis of the ATAC trial. Lancet Oncology 9 45–53 doi:10.1016/S1470-2045(07)70385-6.
Gee J, Robertson J, Gutteridge E, Ellis I, Pinder S, Rubini M & Nicholson R 2005 Epidermal growth factor receptor/HER2/insulin-like growth factor receptor signalling and oestrogen receptor activity in clinical breast cancer. Endocrine-Related Cancer 12 (Suppl 1) S99–S111 doi:10.1677/erc.1.01005.
Generali D, Buffa FM, Berruti A, Brizzi MP, Campo L, Bonardi S, Bersiga A, Allevi G, Milani M & Aguggini S et al. 2009 Phosphorylated ERα, HIF-1α, and MAPK signaling as predictors of primary endocrine treatment response and resistance in patients with breast cancer. Journal of Clinical Oncology 27 227–234 doi:10.1200/JCO.2007.13.7083.
Goss P, Ingle J, Martino S, Robert N, Muss H, Piccart M, Castiglione M, Tu D, Shepherd L & Pritchard K et al. 2003 A randomized trial of letrozole in postmenopausal women after five years of tamoxifen therapy for early-stage breast cancer. New England Journal of Medicine 349 1793–1802 doi:10.1056/NEJMoa032312.
Grisouard J, Medunjanin S, Hermani A, Shukla A & Mayer D 2007 Glycogen synthase kinase-3 protects estrogen receptor α from proteasomal degradation and is required for full transcriptional activity of the receptor. Molecular Endocrinology 21 2427–2439 doi:10.1210/me.2007-0129.
Gruvberger-Saal S, Bendahl P, Saal L, Laakso M, Hegardt C, Edén P, Peterson C, Malmström P, Isola J & Borg A et al. 2007 Estrogen receptor β expression is associated with tamoxifen response in ERα-negative breast carcinoma. Clinical Cancer Research 13 1987–1994 doi:10.1158/1078-0432.CCR-06-1823.
Guo JP, Shu SK, Esposito NN, Coppola D, Koomen JM & Cheng JQ 2010 IKKepsilon phosphorylation of estrogen receptor alpha Ser-167 and contribution to tamoxifen resistance in breast cancer. Journal of Biological Chemistry 285 3676–3684 doi:10.1074/jbc.M109.078212.
Gutierrez MC, Detre S, Johnston S, Mohsin SK, Shou J, Allred DC, Schiff R, Osborne CK & Dowsett M 2005 Molecular changes in tamoxifen-resistant breast cancer: relationship between estrogen receptor, HER-2, and p38 mitogen-activated protein kinase. Journal of Clinical Oncology 23 2469–2476 doi:10.1200/JCO.2005.01.172.
Hall J, Couse J & Korach K 2001 The multifaceted mechanisms of estradiol and estrogen receptor signaling. Journal of Biological Chemistry 276 36869–36872 doi:10.1074/jbc.R100029200.
Hamilton-Burke W, Coleman L, Cummings M, Green CA, Holliday DL, Horgan K, Maraqa L, Peter MB, Pollock S & Shaaban AM et al. 2010 Phosphorylation of estrogen receptor β at serine 105 is associated with good prognosis in breast cancer. American Journal of Pathology 177 1079–1086 doi:10.2353/ajpath.2010.090886.
Harvey J, Clark G, Osborne C & Allred D 1999 Estrogen receptor status by immunohistochemistry is superior to the ligand binding assay for predicting response to adjuvant endocrine therapy in breast cancer. Journal of Clinical Oncology 17 1474–1481.
He X, Zheng Z, Song T, Wei C, Ma H, Ma Q, Zhang Y, Xu Y, Shi W & Ye Q et al. 2010 c-Abl regulates estrogen receptor alpha transcription activity through its stabilization by phosphorylation. Oncogene 29 2238–2251 doi:10.1038/onc.2009.513.
Holm C, Kok M, Michalides R, Fles R, Koornstra RH, Wesseling J, Hauptmann M, Neefjes J, Peterse JL & Stal O et al. 2009 Phosphorylation of the oestrogen receptor α at serine 305 and prediction of tamoxifen resistance in breast cancer. Journal of Pathology 217 372–379 doi:10.1002/path.2455.
Honma N, Horii R, Iwase T, Saji S, Younes M, Takubo K, Matsuura M, Ito Y, Akiyama F & Sakamoto G 2008 Clinical importance of estrogen receptor-β evaluation in breast cancer patients treated with adjuvant tamoxifen therapy. Journal of Clinical Oncology 26 3727–3734 doi:10.1200/JCO.2007.14.2968.
Jensen E & Jordan V 2003 The estrogen receptor: a model for molecular medicine. Clinical Cancer Research 9 1980–1989.
Jiang J, Sarwar N, Peston D, Kulinskaya E, Shousha S, Coombes R & Ali S 2007 Phosphorylation of estrogen receptor-α at Ser167 is indicative of longer disease-free and overall survival in breast cancer patients. Clinical Cancer Research 13 5769–5776 doi:10.1158/1078-0432.CCR-07-0822.
Joel P, Smith J, Sturgill T, Fisher T, Blenis J & Lannigan DA 1998 pp90rsk1 regulates estrogen receptor-mediated transcription through phosphorylation of Ser-167. Molecular and Cellular Biology 18 1978–1984.
Jordan V 2003 Is tamoxifen the Rosetta stone for breast cancer? Journal of the National Cancer Institute 95 338–340 doi:10.1093/jnci/95.5.338.
Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, Masushige S, Gotoh Y, Nishida E & Kawashima H et al. 1995 Activation of the estrogen receptor through phosphorylation by mitogen activated protein kinase. Science 270 1491–1494 doi:10.1126/science.270.5241.1491.
Kirkegaard T, Witton C, McGlynn L, Tovey S, Dunne B, Lyon A & Bartlett J 2005 AKT activation predicts outcome in breast cancer patients treated with tamoxifen. Journal of Pathology 207 139–146 doi:10.1002/path.1829.
Knowlden J, Hutcheson I, Jones H, Madden T, Gee J, Harper M, Barrow D, Wakeling A & Nicholson R 2003 Elevated levels of epidermal growth factor receptor/c-erbB2 heterodimers mediate an autocrine growth regulatory pathway in tamoxifen-resistant MCF-7 cells. Endocrinology 144 1032–1044 doi:10.1210/en.2002-220620.
Kok M, Holm-Wigerup C, Hauptmann M, Michalides R, Stal O, Linn S & Landberg G 2009 Estrogen receptor-α phosphorylation at serine-118 and tamoxifen response in breast cancer. Journal of the National Cancer Institute 101 1725–1729 doi:10.1093/jnci/djp412.
Kok M, Zwart W, Holm C, Fles R, Hauptmann M, Van't Veer LJ, Wessels LF, Neefjes J, Stal O & Linn SC et al. 2010 PKA-induced phosphorylation of ERalpha at serine 305 and high PAK1 levels is associated with sensitivity to tamoxifen in ER-positive breast cancer. Breast Cancer Research and Treatment 125 1–12 doi:10.1007/s10549-010-0798-y.
Lahn M, Kohler G, Sundell K, Su C, Li S, Paterson BM & Bumol TF 2004 Protein kinase C alpha expression in breast and ovarian cancer. Oncology 67 1–10 doi:10.1159/000080279.
Landesman-Bollag E, Romieu-Mourez R, Song DH, Sonenshein GE, Cardiff RD & Seldin DC 2001 Protein kinase CK2 in mammary gland tumorigenesis. Oncogene 20 3247–3257 doi:10.1038/sj.onc.1204411.
Lannigan D 2003 Estrogen receptor phosphorylation. Steroids 68 1–9 doi:10.1016/S0039-128X(02)00110-1.
Lee H & Bai W 2002 Regulation of estrogen receptor nuclear export by ligand-induced and p38-mediated receptor phosphorylation. Molecular and Cellular Biology 22 5835–5845 doi:10.1128/MCB.22.16.5835-5845.2002.
Le Goff P, Montano MM, Schodin DJ & Katzenellenbogen BS 1994 Phosphorylation of the human estrogen receptor. Identification of hormone-regulated sites and examination of their influence on transcriptional activity. Journal of Biological Chemistry 269 4458–4466.
Leygue E, Dotzlaw H, Watson P & Murphy L 1998 Altered estrogen receptor α and β mRNA expression during human breast tumorigenesis. Cancer Research 58 3197–3201.
Likhite VS, Stossi F, Kim K, Katzenellenbogen BS & Katzenellenbogen JA 2006 Kinase-specific phosphorylation of the estrogen receptor changes receptor interactions with ligand, deoxyribonucleic acid, and coregulators associated with alterations in estrogen and tamoxifen activity. Molecular Endocrinology 20 3120–3132 doi:10.1210/me.2006-0068.
Lopez-Tarruella S & Schiff R 2007 The dynamics of estrogen receptor status in breast cancer: re-shaping the paradigm. Clinical Cancer Research 13 6921–6925 doi:10.1158/1078-0432.CCR-07-1399.
Masuhiro Y, Mezaki Y, Sakari M, Takeyama K, Yoshida T, Inoue K, Yanagisawa J, Hanazawa S, O'Malley B & Kato S 2005 Splicing potentiation by growth factor signals via estrogen receptor phosphorylation. PNAS 102 8126–8131 doi:10.1073/pnas.0503197102.
McKenna N, Lanz R & O'Malley B 1999 Nuclear receptor coregulators: cellular and molecular biology. Endocrine Reviews 20 321–344 doi:10.1210/er.20.3.321.
Medunjanin S, Hermani A, Servi BD, Grisouard J, Rincke G & Mayer D 2005 Glycogen synthase kinase-3 interacts with and phosphorylates estrogen receptor α and is involved in the regulation of receptor activity. Journal of Biological Chemistry 280 33006–33014 doi:10.1074/jbc.M506758200.
Medunjanin S, Weinert S, Schmeisser A, Mayer D & Braun-Dullaeus RC 2010 Interaction of the double-strand break repair kinase DNA-PK and estrogen receptor-α. Molecular Biology of the Cell 21 1620–1628 doi:10.1091/mbc.E09-08-0724.
Michalides R, Griekspoor A, Balkenende A, Verwoerd D, Janssen L, Jalink K, Floore A, Velds A, van't Veer L & Neefjes J 2004 Tamoxifen resistance by a conformational arrest of the estrogen receptor α after PKA activation in breast cancer. Cancer Cell 5 597–605 doi:10.1016/j.ccr.2004.05.016.
Miller WR, Watson DM, Jack W, Chetty U & Elton RA 1993 Tumour cyclic AMP binding proteins: an independent prognostic factor for disease recurrence and survival in breast cancer. Breast Cancer Research and Treatment 26 89–94 doi:10.1007/BF00682703.
Morena AM, Oshima CT, Gebrim LH, Egami MI, Silva MR, Segreto RA, Giannotti Filho O, Teixeira VP & Segreto HR 2004 Early nuclear alterations and immunohistochemical expression of Ki-67, Erb-B2, vascular endothelial growth factor (VEGF), transforming growth factor (TGF-beta1) and integrine-linked kinase (ILK) two days after tamoxifen in breast carcinoma. Neoplasma 51 481–486.
Murphy L & Watson P 2006 Is oestrogen receptor-β a predictor of endocrine therapy responsiveness in human breast cancer? Endocrine-Related Cancer 13 327–334 doi:10.1677/erc.1.01141.
Murphy L, Cherlet T, Lewis A, Banu Y & Watson P 2003 New insights into estogen receptor function in human breast cancer. Annals of Medicine 35 614–631 doi:10.1080/07853890310014579.
Murphy L, Niu Y, Snell L & Watson P 2004a Phospho-Serine-118 estrogen receptor-α expression in primary human breast tumors in vivo is associated with better disease outcome in women treated with tamoxifen. Clinical Cancer Research 10 5902–5906 doi:10.1158/1078-0432.CCR-04-0191.
Murphy LC, Cherlet T, Adeyinka A, Niu Y, Snell L & Watson P 2004b Phospho-Serine-118 estrogen receptor-α detection in human breast tumors in vivo. Clinical Cancer Research 10 1354–1359 doi:10.1158/1078-0432.CCR-03-0112.
Murphy L, Weitsman G, Skliris G, Teh E, Li L, Peng B, Davie J, Ung K, Niu Y & Troup S et al. 2006 Potential role of estrogen receptor α phosphorylation at serine 118 in human breast cancer in vivo. Journal of Steroid Biochemistry and Molecular Biology 102 139–146 doi:10.1016/j.jsbmb.2006.09.021.
Murphy L, Skliris G, Rowan B, Al-Dhaheri M, Williams C, Penner C, Troup S, Begic S, Parisien M & Watson P 2009 The relevance of phosphorylated forms of estrogen receptor in human breast cancer in vivo. Journal of Steroid Biochemistry and Molecular Biology 114 90–95 doi:10.1016/j.jsbmb.2009.01.017.
Nawaz Z & O'Malley B 2004 Urban renewal in the nucleus: is protein turnover by proteasomes absolutely required for nuclear receptor regulated transcription? Molecular Endocrinology 18 493–499 doi:10.1210/me.2003-0388.
Osborne C 1998 Steroid hormone receptors in breast cancer management. Breast Cancer Research and Treatment 51 227–238 doi:10.1023/A:1006132427948.
Osborne C, Elledge R & Fuqua S 1996 Estrogen receptors in breast cancer therapy. Science and Medicine 3 32–41.
Osborne C, Shou J, Massarweh S & Schiff R 2005 Crosstalk between estrogen receptor and growth factor receptor pathways as a cause for endocrine therapy resistance in breast cancer. Clinical Cancer Research 11 865s–870s.
Park K, Krishnan V, O'Malley B, Yamamoto Y & Gaynor R 2005 Formation of an IKKalpha-dependent transcription complex is required for estrogen receptor-mediated gene activation. Molecular Cell 18 71–82 doi:10.1016/j.molcel.2005.03.006.
Pietras R, Arboleda J, Reese D, Wongvipat N, Pegram M, Ramos L, Gorman C, Parker M, Sliwkowski M & Slamon D 1995 HER-2 tyrosine kinase pathway targets estrogen receptor and promotes hormone independent growth in human breast cancer cells. Oncogene 10 2435–2446.
Plaza-Menacho I, Morandi A, Robertson D, Pancholi S, Drury S, Dowsett M, Martin LA & Isacke CM 2010 Targeting the receptor tyrosine kinase RET sensitizes breast cancer cells to tamoxifen treatment and reveals a role for RET in endocrine resistance. Oncogene 29 4648–4657 doi:10.1038/onc.2010.209.
Plotnikov A, Li Y, Tran TH, Tang W, Palazzo JP, Rui H & Fuchs SY 2008 Oncogene-mediated inhibition of glycogen synthase kinase 3 beta impairs degradation of prolactin receptor. Cancer Research 68 1354–1361 doi:10.1158/0008-5472.CAN-07-6094.
Rayala SK, Talukder AH, Balasenthil S, Tharakan R, Barnes CJ, Wang RA, Aldaz CM, Khan S & Kumar R 2006 P21-activated kinase 1 regulation of estrogen receptor-alpha activation involves serine 305 activation linked with serine 118 phosphorylation. Cancer Research 66 1694–1701 doi:10.1158/0008-5472.CAN-05-2922.
Reid G, Hubner M, Metivier R, Brand H, Denger S, Manu D, Beaudouin J, Ellenberg J & Gannon F 2003 Cyclic, proteasome-mediated turnover of unliganded and liganded ERα on responsive promoters is an integral feature of estrogen signaling. Molecular Cell 11 695–707 doi:10.1016/S1097-2765(03)00090-X.
Robertson J 1996 Oestrogen receptor: a stable phenotype in breast cancer. British Journal of Cancer 73 5–12.
Robertson J 2002 Estrogen receptor downregulators: new antihormonal therapy for advanced breast cancer. Clinical Therapeutics 24 (Suppl A) A17–A30 doi:10.1016/S0149-2918(02)85032-9.
Rogatsky I, Trowbridge J & Garabedian M 1999 Potentiation of human estrogen receptor α transcriptional activation through phosphorylation of serines 104 and 106 by the cyclin A–CDK2 complex. Journal of Biological Chemistry 274 22296–22302 doi:10.1074/jbc.274.32.22296.
Sanchez M, Picard N, Sauve K & Tremblay A 2010 Challenging estrogen receptor β with phosphorylation. Trends in Endocrinology and Metabolism 21 104–110 doi:10.1016/j.tem.2009.09.007.
Santen R, Song R, Zhang Z, Yue W & Kumar R 2004 Adaptive hypersensitivity to estrogen: mechanism for sequential responses to hormonal therapy in breast cancer. Clinical Cancer Research 10 337S–345S doi:10.1158/1078-0432.CCR-031207.
Santen RJ, Fan P, Zhang Z, Bao Y, Song RX & Yue W 2009 Estrogen signals via an extra-nuclear pathway involving IGF-1R and EGFR in tamoxifen-sensitive and -resistant breast cancer cells. Steroids 74 586–594 doi:10.1016/j.steroids.2008.11.020.
Sarwar N, Kim J, Jiang J, Peston D, Sinnett H, Madden P, Gee J, Nicholson R, Lykkesfeldt A & Shousha S et al. 2006 Phosphorylation of ERα at serine 118 in primary breast cancer and in tamoxifen-resistant tumours is indicative of a complex role for ERα phosphorylation in breast cancer progression. Endocrine-Related Cancer 13 851–861 doi:10.1677/erc.1.01123.
Schiff R, Massarweh S, Shou J, Bharwani L, Mohsin S & Osborne C 2004 Cross-talk between estrogen receptor and growth factor pathways as a molecular target for overcoming endocrine resistance. Clinical Cancer Research 10 331S–336S doi:10.1158/1078-0432.CCR-031212.
Shah Y & Rowan B 2005 The Src kinase pathway promotes tamoxifen agonist action in Ishikawa endometrial cells through phosphorylation-dependent stabilization of estrogen receptor (alpha) promoter interaction and elevated steroid receptor coactivator 1 activity. Molecular Endocrinology 19 732–748 doi:10.1210/me.2004-0298.
Sims RJ III & Reinberg D 2008 Is there a code embedded in proteins that is based on post-translational modifications? Nature Reviews. Molecular Cell Biology 9 815–820 doi:10.1038/nrm2502.
Skliris G, Leygue E, Curtis-Snell L, Watson P & Murphy L 2006 Expression of oestrogen receptor-β in oestrogen receptor-α negative human breast tumours. British Journal of Cancer 95 616–626 doi:10.1038/sj.bjc.6603295.
Skliris GP, Rowan BG, Al-Dhaheri M, Williams C, Troup S, Begic S, Parisien M, Watson PH & Murphy LC 2009 Immunohistochemical validation of multiple phospho-specific epitopes for estrogen receptor α (ERα) in tissue microarrays of ERα positive human breast carcinomas. Breast Cancer Research and Treatment 118 443–453 doi:10.1007/s10549-008-0267-z.
Skliris GP, Nugent ZJ, Rowan BG, Penner CR, Watson PH & Murphy LC 2010 A phosphorylation code for oestrogen receptor-α predicts clinical outcome to endocrine therapy in breast cancer. Endocrine-Related Cancer 17 589–597 doi:10.1677/ERC-10-0030.
Someya M, Sakata K, Matsumoto Y, Satoh M, Narimatsu H & Hareyama M 2007 Immunohistochemical analysis of Ku70/86 expression of breast cancer tissues. Oncology Reports 18 1483–1487.
Tharakan R, Lepont P, Singleton D, Kumar R & Khan S 2008 Phosphorylation of estrogen receptor alpha, serine residue 305 enhances activity. Molecular and Cellular Endocrinology 295 70–78 doi:10.1016/j.mce.2008.07.018.
Thomas RS, Sarwar N, Phoenix F, Coombes RC & Ali S 2008 Phosphorylation at serines 104 and 106 by Erk1/2 MAPK is important for estrogen receptor-alpha activity. Journal of Molecular Endocrinology 40 173–184 doi:10.1677/JME-07-0165.
Valley C, Metivier R, Solodin N, Fowler A, Mashek M, Hill L & Alarid E 2005 Differential regulation of estrogen-inducible proteolysis and transcription by the estrogen receptor α N terminus. Molecular and Cellular Biology 25 5417–5428 doi:10.1128/MCB.25.13.5417-5428.2005.
Wakasugi E, Kobayashi T, Tamaki Y, Nakano Y, Ito Y, Miyashiro I, Komoike Y, Miyazaki M, Takeda T & Monden T et al. 1997 Analysis of phosphorylation of pRB and its regulatory proteins in breast cancer. Journal of Clinical Pathology 50 407–412 doi:10.1136/jcp.50.5.407.
Wang RA, Mazumdar A, Vadlamudi RK & Kumar R 2002 P21-activated kinase-1 phosphorylates and transactivates estrogen receptor-alpha and promotes hyperplasia in mammary epithelium. EMBO Journal 21 5437–5447 doi:10.1093/emboj/cdf543.
Wang L, Yang X, Zhang X, Mihalic K, Fan Y, Xiao W, Howard O, Appella E, Maynard A & Farrar W 2004 Suppression of breast cancer by chemical modulation of vulnerable zinc fingers in estrogen receptor. Nature Medicine 10 40–47 doi:10.1038/nm969.
Ward RD & Weigel NL 2009 Steroid receptor phosphorylation: assigning function to site-specific phosphorylation. Biofactors 35 528–536 doi:10.1002/biof.66.
Weigel N & Moore N 2007 Steroid receptor phosphorylation: a key modulator of multiple receptor functions. Molecular Endocrinology 21 2311–2319 doi:10.1210/me.2007-0101.
Weis K, Ekena K, Thomas J, Lazennec G & Katzenellenbogen B 1996 Constitutively active human estrogen receptors containing amino acid substitutions for tyrosine 537 in the receptor protein. Molecular Endocrinology 10 1388–1398 doi:10.1210/me.10.11.1388.
Weitsman G, Li L, Skliris G, Davie J, Ung K, Curtis-Snell L, Tomes L, Watson P & Murphy L 2006 Estrogen receptor-α phosphorylated at Serine 118 is present at the promoters of estrogen-regulated genes and is not altered due to Her2 over-expression. Cancer Research 66 10162–10170 doi:10.1158/0008-5472.CAN-05-4111.
West NR & Watson PH 2010 S100A7 (psoriasin) is induced by the proinflammatory cytokines oncostatin-M and interleukin-6 in human breast cancer. Oncogene 29 2083–2092 doi:10.1038/onc.2009.488.
Williams C, Smith CL & Rowan BG 2007 Identification of four novel phosphorylation sites in estrogen receptor α: impact on receptor-dependent gene expression and phosphorylation by protein kinase CK2. In The Endocrine Society's 89th Annual Meeting, pp P2–P258. Toronto, Canada.
Williams CC, Basu A, El-Gharbawy A, Carrier LM, Smith CL & Rowan BG 2009 Identification of four novel phosphorylation sites in estrogen receptor α: impact on receptor-dependent gene expression and phosphorylation by protein kinase CK2. BMC Biochemistry 10 36 doi:10.1186/1471-2091-10-36.
Yamashita H, Nishio M, Kobayashi S, Ando Y, Sugiura H, Zhang Z, Hamaguchi M, Mita K, Fujii Y & Iwase H 2005 Phosphorylation of estrogen receptor α serine 167 is predictive of response to endocrine therapy and increases postrelapse survival in metastatic breast cancer. Breast Cancer Research 7 R753–R764 doi:10.1186/bcr1285.
Yamashita H, Nishio M, Toyama T, Sugiura H, Kondo N, Kobayashi S, Fuji Y & Iwase H 2008 Low phosphorylation of estrogen receptor α (ERα) serine 118 and high phosphorylation of ERα serine 167 improve survival in ER-positive breast cancer. Endocrine-Related Cancer 15 755–763 doi:10.1677/ERC-08-0078.
Yamashita H, Takahashi S, Ito Y, Yamashita T, Ando Y, Toyama T, Sugiura H, Yoshimoto N, Kobayashi S & Fujii Y et al. 2009 Predictors of response to exemestane as primary endocrine therapy in estrogen receptor-positive breast cancer. Cancer Science 100 2028–2033 doi:10.1111/j.1349-7006.2009.01274.x.
Yamnik RL, Digilova A, Davis DC, Brodt ZN, Murphy CJ & Holz MK 2009 S6 kinase 1 regulates estrogen receptor alpha in control of breast cancer cell proliferation. Journal of Biological Chemistry 284 6361–6369 doi:10.1074/jbc.M807532200.
Yang C, Xin H, Kelley J, Spencer A, Brautigan D & Paschal B 2007 Ligand binding to the androgen receptor induces conformational changes that regulate phosphatase interactions. Molecular and Cellular Biology 27 3390–3404 doi:10.1128/MCB.02411-06.
York B, Yu C, Sagen JV, Liu Z, Nikolai BC, Wu RC, Finegold M, Xu J & O'Malley BW 2010 Reprogramming the posttranslational code of SRC-3 confers a switch in mammalian systems biology. PNAS 107 11122–11127 doi:10.1073/pnas.1005262107.
Yudt M, Vorojeikina D, Zhong L, Kafar D, Sasson S, Gasiewicz T & Notides A 1999 Function of estrogen receptor tyrosine 537 in hormone binding, DNA binding, and transactivation. Biochemistry 38 14146–14156 doi:10.1021/bi9911132.
Zhao H, Ou-Yang F, Chen IF, Hou MF, Yuan SS, Chang HL, Lee YC, Plattner R, Waltz SE & Ho SM et al. 2010 Enhanced resistance to tamoxifen by the c-ABL proto-oncogene in breast cancer. Neoplasia 12 214–223 doi:10.1593/neo.91576.
Zoubir M, Mathieu M, Mazouni C, Liedtke C, Corley L, Geha S, Bouaziz J, Spielmann M, Drusche F & Symmans W et al. 2008 Modulation of ER phosphorylation on serine 118 by endocrine therapy: a new surrogate marker for efficacy. Annals of Oncology 19 1402–1406 doi:10.1093/annonc/mdn151.
