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Annals of Oncology Advance Access originally published online on April 23, 2008
Annals of Oncology 2008 19(8):1402-1406; doi:10.1093/annonc/mdn151
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© The Author 2008. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

breast cancer

Modulation of ER phosphorylation on serine 118 by endocrine therapy: a new surrogate marker for efficacy

M. Zoubir1,2,{dagger}, M. C. Mathieu3,{dagger}, C. Mazouni4, C. Liedtke5,6, L. Corley7, S. Geha3, J. Bouaziz1, M. Spielmann2, F. Drusche3, W. F. Symmans8, S. Delaloge1,2,{ddagger} and F. Andre1,2,{ddagger},*

1 Translational Research Unit, UPRES EA035, Université Paris XI, Institut Gustave Roussy, Villejuif
2 Breast Cancer Unit, Department of Medicine, Institut Gustave Roussy, Villejuif
3 Department of Pathology, Institut Gustave Roussy, Villejuif
4 Laboratory of translational research, Marseille University, Marseille, France
5 Department of Breast Medical Oncology, The University of Texas, MD Anderson Cancer Center, Houston, USA
6 Department of Gynecology and Obstetrics, University of Muenster, Muenster, Germany
7 Department of Pathology Research, The University of Texas, MD Anderson Cancer Center, Houston
8 Department of Pathology, The University of Texas, MD Anderson Cancer Center, Houston, USA

* Correspondence to: Dr F. Andre, Translational Research Unit, Comite 050, Institut Gustave Roussy, 39 rue C. Desmoulins, 94805 Villejuif, France. Tel: +33-142114371; Fax: +33-142115274; E-mail: fabrice.andre{at}igr.fr


   Abstract
Top
 Abstract
introduction
 patients and methods
 results
 discussion
 funding
 Acknowledgements
 References
 
Background: Phosphorylation of serine 118 (ser118) has been reported to be involved in the activation of estrogen receptor (ER). In the present study, we evaluated whether endocrine therapy modulated ER phosphorylation on ser118.

Patients and methods: We carried out a tissue microarray that included 80 primary breast tumors obtained before the administration of endocrine therapy. A second tissue microarray included 52 tumors obtained after endocrine therapy from the same patients. Immunostainings were carried out for ER, Pser118ER, Her2, insulin growth factor receptor (IGFR), p21-activated kinase 1 (PAK1), pMAPK, bcl2 and progesterone receptor.

Results: Pser118ER staining was higher in Her2- (P = 0.06), IGFR- (P = 0.0002) and pMAPK-expressing tumors (P = 0.001). The level of ER phosphorylation was not different according to the occurrence of clinical tumor response (P = 0.16). Pser118ER expression was significantly reduced by endocrine therapy. The mean Pser118ER score was 163 [standard deviation (SD) 81] before endocrine therapy and 80 (SD 90) after endocrine therapy (P = 0.0001, paired t-test). The magnitude of Pser118ER decrease was higher in tumors that responded to endocrine therapy (mean decrease 128, SD 86) as compared with refractory tumors (mean decrease 38, SD 130) (P = 0.017, t-test).

Conclusion: These findings suggest that endocrine therapy modulates ER on ser118. Pser118ER immunostaining could be used as surrogate marker to monitor treatment efficacy.

Key words: aromatase inhibitor, breast cancer, estrogen receptor, predictive biomarker, tamoxifen


   introduction
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 Abstract
 introduction
patients and methods
 results
 discussion
 funding
 Acknowledgements
 References
 
Endocrine therapy with tamoxifen has been shown to decrease the risk of relapse and death in patients with hormonal receptor-positive disease [1]. More recently, in postmenopausal women, aromatases inhibitors have been shown to reduce the risk of metastatic relapse as compared with tamoxifen [2]. Nevertheless, although treated with optimal endocrine therapy, a significant proportion of patients with estrogen receptor (ER)-positive breast cancer will present a metastatic relapse. In addition to be only partially effective, endocrine therapy is limited by toxic effects. These toxic effects include hot flushes and thromboembolic disease for tamoxifen, bone density loss and arthralgia for aromatases inhibitors [2]. On the basis of these considerations has emerged the need to develop biological parameters to identify tumors that are refractory to endocrine therapies.

Estradiol induces the activation of its receptor through conformation change [3] and phosphorylation in ser118 site [4]. Following activation by estradiol, ER induces transcription of genes that present ERE or AP1 elements in their promoter [3, 5]. Recent data indicate that not only B-estradiol but also activation of kinase pathways are able to phosphorylate ER on ser118 site. Both PAK1 [6] and MAPK [7, 8] have been shown to mediate this phenomenon. Overall, these findings indicate that the detection of Pser118ER in breast cancer samples could be a surrogate marker of an activated form of ER.

There are only a few reports to date that focused on the clinical significance of ER phosphorylation in breast cancer. Sarwar et al. [9] have reported that Pser118 form of ER is detected mainly in well-differentiated tumors and that this phosphorylation could be increased in relapsing tumors. Murphy et al. [10] have reported that the expression of Pser118ER predicted better outcome in a series of patients treated with tamoxifen. Finally, the same team [11] reported that Pser118ER expression was associated with MAPK activation and a lower grade.

Altogether, these data indicate that phosphorylation of ER on ser118 could be a biomarker of ER activation. In the present study, we analyzed whether endocrine therapy modulates ER phosphorylation on ser118 and whether this modulation correlates with treatment efficacy.


   patients and methods
Top
 Abstract
 introduction
 patients and methods
results
 discussion
 funding
 Acknowledgements
 References
 
patients
The present study includes 80 postmenopausal patients treated with preoperative endocrine therapy for a breast adenocarcinoma between 1995 and 2005. Patients were retrospectively selected from the clinical database of Institut Gustave Roussy. An overall 112 patients were initially identified to have received a preoperative endocrine therapy during this period in the same institute. Tumor samples at diagnosis could be retrieved and processed in 80 of these 112 patients (71%). In the same 80 patients, the surgical specimen obtained after endocrine therapy was found and processed for 52 cases. Clinicopathological characteristics were extracted from medical files. All patients presented an ER-positive tumor at diagnosis. A modified Scarff–Bloom–Richardson grade was used to determine tumor grade. Patients were treated either with tamoxifen or aromatase inhibitor. A clinical response was defined as a 50% or more reduction in the longest diameter of the breast tumor.

tissue arrays and immunostainings
A tissue array that includes breast tumor sample at diagnosis from the 80 patients was constructed at the Department of Pathology, MD Anderson Cancer Center. This tissue array contains two spots per sample. A second tissue array includes surgical specimen of the same breast tumors obtained after endocrine therapy. This second tissue array was constructed at Department of pathology, Institut Gustave Roussy. Fifty-two tumors were included in this ‘post-treatment’ tissue-array. Immunostainings were carried out for ER (clone E6F11, Novocastra, Newcastle, UK, 1 : 40 dilution), progesterone receptor (PR) (clone PGR636, Dako Cytomation, Glostrub, Denmark 1 : 150 dilution), Her2 (DA485, Dako, 1 : 800 dilution), IGF-1R (Cell Signaling, Rabbit antibody, ref 3027, 1 : 70 dilution), p-MAPK (clone 4376, Cell Signaling, 1 : 50 dilution), PAK1 (clone 2602, Cell Signaling, Danvers, USA, 1 : 50), bcl2 (clone 124, Dako Cytomation, 1 : 25 dilution) and Pser118ER (SAB, Anti-pER rabbit, ref 110072, 1 : 300 dilution). ER, PR, phospho-ER, PAK1 and pMAPK expressions were evaluated by a composite score that consisted in the product of intensity and percentage of tumor cells stained. For Her2, samples were positive if >10% tumor cells were intensively stained. For bcl2, a tumor was considered positive if >10% of tumor cells were stained.

statistical analysis
Spearman's correlation was used to correlate Pser118ER staining with pMAPK, PAK1, ER and PR expressions. t-test was used to correlate Pser118ER staining with Her2, IGFR1 and bcl2 expressions. t-test was used to correlate the evolution of Pser118ER score with treatment efficacy. Paired t-test was used to compare the Pser118ER score before and after endocrine therapy.


   results
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 Abstract
 introduction
 patients and methods
 results
discussion
 funding
 Acknowledgements
 References
 
patient characteristics
Eighty patients were included in the present study. This represents 71% of the patients retrospectively identified to have been treated with preoperative endocrine therapy between 1995 and 2005 at the Institut Gustave Roussy. Patient characteristics are summarized in Table 1. Briefly, the median age was 79, the median clinical tumor size was 42 mm and 12% of the patients presented a high-grade tumor. Thirty-two (40%) and 48 (60%) patients were treated with tamoxifen or aromatase inhibitor, respectively. Forty-four (55%) patients presented an objective clinical response. No patient presented a complete pathologic response. Forty-seven per cent and 60% of patients treated with tamoxifen or aromatase inhibitors presented a clinical response, respectively (P = 0.23).


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  		Table 1. Patient characteristics and biomarker expression

 
correlation between Pser118ER immunostaining and IGFR1/MAP kinase pathway
Illustrative immunostainings are reported in Figure 1. The distribution of biomarker expression is summarized in Table 1. As shown in Table 2, Pser118ER immunostaining highly correlated with phospho-MAPK (P = 0.001, Spearman's correlation) and IGFR1 expressions (P = 0.0002, t-test). In addition, a trend was observed for a correlation between Her2 and Pser118ER expression (P = 0.06, t-test). No significant correlation was found between Pser118ER and ER (P = 0.60, Spearman's correlation), PR (P = 0.29, Spearman's correlation), PAK1 (P = 0.20, Spearman's correlation) or bcl2 (P = 0.86, t-test).


Figure 1
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  		Figure 1. Illustrative immunostainings for Her2 (Figure 1A), IGFR1 (Figure 1B), phospho-MAPK (Figure 1C), PAK1 (Figure 1D), ER (Figure 1E), Pser118ER (Figure 1F), PR (Figure 1G) and bcl2 (Figure 1H).

 

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  		Table 2. Correlation between Pser118ER and other biomarkers

 
Pser118ER immunostaining and efficacy of endocrine therapy
As reported in Table 3, Pser118ER score was not different between sensitive and refractory tumors (mean score 179 versus 150, P = 0.15). When the analysis focused on patients treated with aromatase inhibitors (n = 40), a trend was observed for a higher Pser118ER staining in sensitive tumors (mean score 182 versus 139, P = 0.09). At the opposite, although the number of patients was small (n = 26), no difference of Pser118ER staining was observed according to tamoxifen efficacy (mean values 170 versus 162, P = 0.72).


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  		Table 3. Expression of biomarkers according to treatment efficacy

 
Since ER phosphorylation has been reported as a potential biomarker for ER activation, we evaluated whether estrogen inhibitors would decrease the Pser118ER immunostaining. In order to achieve this goal, we compared the Pser118ER score before and after endocrine therapy in 38 paired tumor samples. These paired tumor samples were obtained at diagnosis (before endocrine therapy) and during surgery (after preoperative endocrine therapy) in the same patients. As reported in Figure 2, the P118serER immunostaining decreased after endocrine therapy. The mean Pser118 score was 163 [standard deviation (SD) 81] before endocrine therapy and 80 (SD 90) after endocrine therapy (P = 0.0001, paired t-test). At the opposite, ER expression was not affected by endocrine therapy. The ER score was 163 (SD 96) before endocrine therapy and 195 (SD 89) after endocrine therapy (P = 0.18, paired t-test). The mean Pser118ER decrease was 71 (SD 145) for patients treated with tamoxifen (n = 14) and 90 (SD 101) for those treated with aromatase inhibitors (n = 24).


Figure 2
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  		Figure 2. Evolution of Pser118ER and ER during endocrine therapy. Evolutions of Pser118ER (Figure 2A) and ER (Figure 2B) are reported in 38 paired samples obtained before and after endocrine therapy. Each tumor is represented by a plot and connected to its paired sample. Black squares represent mean scores. Scores are compared by t-test.

 
Since the decrease of Pser118ER score observed in the present study could indicate that endocrine therapy abrogates ER activation, we evaluated whether the magnitude of Pser118ER decrease correlated with efficacy of endocrine therapy. As reported in Figure 3, the magnitude of Pser118ER decrease was higher in tumor sensitive to endocrine therapy as compared with refractory tumors. The mean decrease in Pser118ER score was 128 (SD 86) in patients who presented a clinical response to endocrine therapy. At the opposite, the mean decrease in Pser118ER score was 38 (SD 130) in patients who presented a refractory disease (P = 0.017, t-test). Only one of eight patients (12%) who presented an increase in Pser118ER score during endocrine therapy presented a clinical response. At the opposite, 18 of 30 (60%) patients who presented a decrease in Pser118ER score during endocrine therapy presented a clinical response (P = 0.01, {chi}2 test).


Figure 3
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  		Figure 3. Evolution of Pser118ER immunostaining according to treatment efficacy. The value of each plot is calculated as the difference between Pser118ER score after treatment and the Pser118 score before treatment. A decrease in ER phosphorylation is therefore represented by a negative value. Each plot represents a value for a single tumor. Squares represent the means of the Pser118ER evolution. Values are compared between responders and nonresponders by a t-test.

 

   discussion
Top
 Abstract
 introduction
 patients and methods
 results
 discussion
funding
 Acknowledgements
 References
 
In the present study, we have reported that (i) ER phosphorylation on ser118 correlates with activation of MAPK pathway, (ii) ER phosphorylation on ser118 decreases under endocrine therapy and (iii) the level of Pser118ER decrease is higher in tumor sensitive to endocrine therapy.

The ER phosphorylation on ser118 leads to ER activation [4, 12]. This phenomenon occurs after B-estradiol/ER binding [4] and/or after kinase activation. Among the kinases, MAPK, PAK1 and AKT have been reported to mediate in vitro the phosphorylation of ER on ser118 in tumor cell lines. There are only a few studies carried out on tumor samples that addressed this issue. Murphy et al. [11] have reported that phospho-MAPK immunostaining correlates with the phosphorylation of ER. Our results corroborate these data by reporting a high level of correlation between Pser118ER immunostaining and the expression of phospho-MAPK (P = 0.001, t-test). Unfortunately, we were not able to correlate PAK1 expression with Pser118ER immunostaining. This might be due to the fact that we did not assess the activated form of PAK1 in our study. Interestingly, Pser118ER also correlated with IGF-1R expression and trend to correlate with Her2 expression. These data indicate that activation of IGF-1R and Her2 could lead to ER phosphorylation. IGF-1R [13] and Her2 inhibitors [14] could reverse resistance to endocrine therapy in patients with IGF1R and/or Her2-overexpressing tumors who present persistence of ER phosphorylation during endocrine therapy.

The main finding of the present study was the observation that endocrine therapy decreases ER phosphorylation at ser118 and that such decrease is predictive for treatment efficacy. The present finding that aromatase inhibitors decrease ER phosphorylation well corroborates with the previously reported data that 17B-estradiol induces ER phosphorylation on ser118 in vitro [4]. At the opposite, the finding that ER phosphorylation was decreased by tamoxifen was unexpected. Several reports indeed suggested that OH-tamoxifen was more likely to induce ER phosphorylation in vitro. Nevertheless, it must be pointed out that ER changes induced by tamoxifen are highly related to coactivators and -repressors expressed by tumor cells. An ongoing study aims at evaluating whether tamoxifen could have differential effect on ER phosphorylation status according to AIB1 expression (Zoubir, personal communication).

The present data suggest that the modulation of ER phosphorylation by endocrine therapy could be used as surrogate marker to evaluate both the bioactivity and efficacy of endocrine therapy. Several other surrogate markers, including Ki67, are being investigated [15]. Further studies will aim at determining whether Pser118ER could be complementary to Ki67 to monitor bioactivity of endocrine therapy and whether the assessment of Phospho-ER expressed by normal tissues could be informative in patients treated in adjuvant setting. Although the Pser118ER expression at diagnosis did not correlate with the efficacy of endocrine therapy (P = 0.15), there was a trend toward a higher Pser118ER score in tumors sensitive to aromatase inhibitors as compared with refractory tumors (P = 0.09, t-test). At the opposite, there was no difference according to treatment efficacy in patients treated with tamoxifen (P = 0.72). Although sample sizes were too small to address an interaction between Pser118 and the drug, this observation would deserve further investigations to determine whether the Pser118ER expression at diagnosis could predict efficacy of aromatase inhibitors over tamoxifen.

Overall, our study suggests that Pser118ER immunostaining could be used as surrogate marker to monitor the efficacy of endocrine therapy. Further studies will determine whether its determination either in tumor (preoperative setting) or in normal tissues (adjuvant setting) could help to monitor and adjust endocrine therapy.


   funding
Top
 Abstract
 introduction
 patients and methods
 results
 discussion
 funding
Acknowledgements
 References
 
Career development award of the American Society of Clinical Oncology to F.A.; Deutsche Forschungsgemeinschaft to C.L.


   Acknowledgements
Top
 Abstract
 introduction
 patients and methods
 results
 discussion
 funding
 Acknowledgements
References
 
We thank Martin Schlumberger and Jean Charles Soria for their support. M.Z. was a fellow of Diplome Universitaire Européen de Recherche Clinique en Cancérologie (DUERCC). We thank the Automated Image Acquisition and Tissue MicroArray Core Lab Facility at MD Anderson Cancer Center.


   Footnotes
 
{dagger} Both authors equally contributed to this work. Back

{ddagger} Both authors equally contributed to this work as senior scientist. Back

Received for publication January 21, 2007. Accepted for publication March 6, 2008.


   References
Top
 Abstract
 introduction
 patients and methods
 results
 discussion
 funding
 Acknowledgements
 References
 
1. Early Breast Cancer Trialists’ Collaborative Group (EBCTCG). Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials. Lancet (2005) 365:1687–1717.[CrossRef][Web of Science][Medline]

2. Coates AS, Keshaviah A, Thurlimann B, et al. Five years of letrozole compared with tamoxifen as initial adjuvant therapy for postmenopausal women with endocrine-responsive early breast cancer: update of study BIG 1-98. J Clin Oncol (2007) 25:486–492.[Abstract/Free Full Text]

3. Osborne CK, Schiff R. Estrogen-receptor biology: continuing progress and therapeutic implications. J Clin Oncol (2005) 23:1616–1622.[Free Full Text]

4. Cheng J, Zhang C, Shapiro DJ. A functional serine 118 phosphorylation site in estrogen receptor-alpha is required for down-regulation of gene expression by 17beta-estradiol and 4-hydroxytamoxifen. Endocrinology (2007) 148:4634–4641.[Abstract/Free Full Text]

5. Piccart M, Parker LM, Pritchard KI. Oestrogen receptor downregulation: an opportunity for extending the window of endocrine therapy in advanced breast cancer. Ann Oncol (2003) 14:1017–1025.[Abstract/Free Full Text]

6. Rayala SK, Talukder AH, Balasenthil S, et al. P21-activated kinase 1 regulation of estrogen receptor-alpha activation involves serine 305 activation linked with serine 118 phosphorylation. Cancer Res (2006) 66:1694–1701.[Abstract/Free Full Text]

7. Chen D, Washbrook E, Sarwar N, 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.[CrossRef][Web of Science][Medline]

8. Kato S, Endoh H, Masuhiro Y, et al. Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science (1995) 270:1491–1504.[Abstract/Free Full Text]

9. Sarwar N, Kim JS, Jiang J, et al. Phosphorylation of ERalpha at serine 118 in primary breast cancer and in tamoxifen-resistant tumours is indicative of a complex role for ERalpha phosphorylation in breast cancer progression. Endocr Relat Cancer (2006) 13:851–861.[Abstract/Free Full Text]

10. Murphy LC, Niu Y, Snell L, Watson P. Phospho-serine-118 estrogen receptor-alpha expression is associated with better disease outcome in women treated with tamoxifen. Clin Cancer Res (2004) 10:5902–5906.[Abstract/Free Full Text]

11. Murphy L, Cherlet T, Adeyinka A, et al. Phospho-serine-118 estrogen receptor-alpha detection in human breast tumors in vivo. Clin Cancer Res (2004) 10:1354–1359.[Abstract/Free Full Text]

12. 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.[Abstract/Free Full Text]

13. Duran I, Salazar R, Casanovas O, et al. New drug development in digestive neuroendocrine tumors. Ann Oncol (2007) 18:1307–1313.[Abstract/Free Full Text]

14. Jones A. Combining trastuzumab (Herceptin) with hormonal therapy in breast cancer: what can be expected and why? Ann Oncol (2003) 14:1697–1704.[Abstract/Free Full Text]

15. Dowsett M, Smith IE, Ebbs SR, et al. Prognostic value of Ki67 expression after short-term presurgical endocrine therapy for primary breast cancer. J Natl Cancer Inst (2007) 99:167–170.[Abstract/Free Full Text]


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