Gynecologic Oncology 132 (2014) 351–359
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Gynecologic Oncology journal homepage: www.elsevier.com/locate/ygyno
Prognostic significance of the estrogen receptor beta (ERβ) isoforms ERβ1, ERβ2, and ERβ5 in advanced serous ovarian cancer Alessandra Ciucci a, Gian Franco Zannoni b, Daniele Travaglia a, Marco Petrillo a, Giovanni Scambia a, Daniela Gallo a,⁎ a b
Department of Obstetrics and Gynecology, Catholic University of the Sacred Heart, Rome, Italy Department of Histopathology, Catholic University of the Sacred Heart, Rome, Italy
H I G H L I G H T S • This is the first study analyzing the subcellular expression of ERβ1, ERβ2 and ERβ5 in advanced serous ovarian cancers. • The most striking observation of this study was the strong statistical association of cytoplasmic ERβ2 immunoreactivity with reduced survival. • Cytoplasmic ERβ2 expression was also found to be significantly associated with chemoresistance.
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Article history: Received 28 October 2013 Accepted 18 December 2013 Available online 27 December 2013 Keywords: Ovary Epithelial ovarian carcinomas Survival Estrogen Estrogen receptors Estrogen receptor beta
a b s t r a c t Objective. In the present study we have examined the pattern of expression of the full length estrogen receptor β (ERβ1) and two ERβ splice variant isoforms (ERβ2, ERβ5) in well-characterized advanced serous ovarian cancers. Methods. Immunohistochemistry was performed with ERβ1, ERβ2, and ERβ5 antibodies and results were correlated with pathological and clinical follow-up data. Expression of ERβ isoforms in a panel of ovarian cancer cell lines and human tumor xenografts was also assessed. Results. Immunohistochemical staining revealed cellular compartment-specific distribution for each isoform in malignant ovarian tissues exhibiting both nuclear staining and cytoplasmic staining. Patients with cytoplasmic ERβ2 expression had significantly worse outcome (p = 0.006 at the multivariate analysis), the 5-year survival rate being nearly 28% for patients who did express cytoplasmic ERβ2, and 60% in negative patients. Cytoplasmic ERβ2 expression was also found to be significantly associated with chemoresistance. In concordance with clinical results both nuclear and cytoplasmic expressions were observed for the three isoforms in the cancer cell lines and human tumor xenografts tested. Conclusions. This is the first study to uncover an unfavorable prognostic role of ERβ2 in advanced serous ovarian cancer. If anomalies of ERβ2 cytoplasmic expression could be demonstrated to represent an independent unfavorable prognostic marker and/or a marker predicting chemoresistance in advanced serous ovarian cancer, its immunohistochemical assessment at the time of surgery, could help to recognize candidates for clinical trials of new interventions. © 2013 Elsevier Inc. All rights reserved.
Introduction Of the gynecological malignancies, epithelial ovarian cancer is the leading cause of death in the great majority of developed countries with more than 140,000 women dying from this cancer across the world in 2008 [1]. Although many efforts have been made to clarify the etiology of ovarian carcinogenesis and the molecular mechanisms involved in the proliferation of ovarian carcinoma cells, this disease ⁎ Corresponding author at: Department of Obstetrics and Gynecology, Catholic University of the Sacred Heart, Largo A. Gemelli 8, 00168 Rome, Italy. Fax: + 39 06 3051160. E-mail address: [email protected] (D. Gallo). 0090-8258/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygyno.2013.12.027
remains among the less understood of major human malignancies. Increasing clinical data implicate estrogen in the etiology of ovarian cancer. These data come from studies showing that while long-term use of estrogen replacement therapy is associated with increased risk of ovarian cancer incidence or mortality, the use of oral contraceptives is associated with decreased risk. Actually, the protective effect of oral contraceptive has been interpreted as a support to the “estrogen hypothesis”, as oral contraceptive use decreases ovarian estrogen production, and maintains circulating estrogens around early to midfollicular phase levels; other authors, however, suggest that chronic suppression of ovulation reduces cancer risk by decreasing recurrent ovarian injury [reviewed in 2]. Finally, the hypothesis that a decreased risk of ovarian cancer with oral contraceptive use could also be due to
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the cyclic progestational climate, cannot be ruled out [3]. However, it is worthy to note that ovarian cancer is primarily a disease of postmenopausal women, the median age of diagnosis being 63 years, with 48% of patients 65 years or older [4]. Estrogens exert their action through two estrogen receptors (ERα and ERβ) that are encoded by separate genes. These two receptors are members of the nuclear receptor superfamily of ligand-dependent transcription factors and share both structural and functional homologies. They produce different biological effects, first of all by regulating different genes in response to estradiol (E2) and selective estrogen receptor modulators, but they may also regulate the same genes in opposite directions, in agreement with a yin/yang hypothesis. Overall, ERα is thought to promote the expression of genes involved in cell survival and proliferation, thus determining tumor growth and progression, while the natural function of ERβ is thought to be antiproliferative and proapoptotic, and ERβ is therefore described as a tumor suppressor. Several ERβ isoforms have been reported so far. ERβ1 is the full-length receptor coded by exons 1–8. ERβ2–5 share exons 1–7 with ERβ1, but display unique sequences instead of exon 8. These differences in the C-terminal part of ERβ2–5 determine a truncation of the ligand binding domain and ablation of the ligand-dependent activation function AF-2. Therefore, ERβ1 is the only fully functional isoform that is able to bind ligand, whereas ERβ2, -β4, and -β5 do not form homodimers and have no innate activities of their own, but may modulate estrogen action when dimerized with ERβ1 or ERα [reviewed in [5] and references therein]. Besides, evidence is accumulating that estrogens also exert nongenomic actions including mobilization of intracellular calcium, stimulation of adenylate cyclase activity and cAMP production, activation of the MAPK signaling pathway and of the phosphoinositol (PI) 3-kinase signaling pathway. These non-transcriptional mechanisms mainly involve cytoplasmic or cell membrane-bound ERs, and can be triggered by estrogen (ligand-dependent signaling) or receptor kinases in the absence of estrogen (ligand-independent ER activation) [6]. Nongenomic actions of estrogens may indirectly influence gene expression, through the activation of signal transduction pathways that eventually act on target transcription factors [reviewed in 7]. In the normal ovary, the levels of ERβ are high and predominate over ERα, being ERβ1, -β2, and -β5 the most represented isoforms [8,9]. Besides, literature data suggest that a high percentage of ovarian tumors express ERβ and that a progressive decline of ERβ occurs during development or progression of ovarian cancer [reviewed in [5] and references therein]. However, limited information is available on its prognostic role in the disease. In a previous study aimed at investigating the prognostic significance of ER (ERα, ERβ) and PR expressions in a cohort of uniformly treated patients with advanced serous ovarian cancer, we found that, in this specific setting, cytoplasmic ERβ signaling may be more important for patient survival than its nuclear signaling. Specifically, our results suggested that advanced serous ovarian cancers with high cytoplasmic total ERβ expression may define patients with aggressive biology, and possibly resistant to chemotherapy [10]. To gain insights into the role of estrogen in ovarian cancer and to extend our previous observations on the association between cytoplasmic ERβ signaling and clinical outcome, we have examined in the present study the pattern of expression of the full length ERβ (ERβ1) and two ERβ splice variant isoforms (ERβ2, ERβ5) in a similar series of patients with advanced serous ovarian cancer, and correlated the pattern of expression with clinicopathological parameters and with response to chemotherapy. Expression of ERβ isoforms in a panel of ovarian cancer cell lines and human tumor xenografts was also assessed. Materials and methods Patients The study included 56 patients with advanced serous ovarian cancer admitted to the Gynecologic Oncology Unit, Catholic University of
Rome, between March 2000 and December 2008. In our institution a written informed consent is routinely requested from patients for collection of their clinical data, as well as paraffin embedded sections for research use. Clinicopathological characteristics of the overall series are summarized in Table 1. According to standard guidelines, maximal surgical effort was attempted in all patients resulting in complete resection (residual tumor 0 mm) in 35 (62.5%) cases. All patients received platinum-based chemotherapy (75–100 mg/m2 for cisplatin, AUC = 5 for carboplatin, per cycle). Fifty patients (89.3%) also received paclitaxel (135–175 mg/m2 for each cycle). Recurrence of disease was defined according to GCIG CA125 criteria [11,12] and/or radiological confirmation of tumor progression. To define chemosensitivity, we used the common definition of platinum resistance, defining as “sensitive” patients that relapsed 6 months or more after prior platinum-containing chemotherapy, and as “resistant” patients that relapsed less than 6 months after chemotherapy was stopped, or that progressed while on therapy [13]. Follow-up data were available for all 56 patients (median follow-up, 47 months; range, 9–162 months). During the follow-up period, progression and death of disease were observed in 44 and 34 patients, respectively.
Immunohistochemistry Three-micrometer-thick paraffin sections were mounted on Superfrost coated slides, and dried overnight. The sections were deparaffinized in xylene and rehydrated in graded solutions of ethanol; the endogenous peroxidase was blocked with 3% H2O2 for 5 min. Antigen retrieval procedure was performed by microwave oven heating in citrate buffer (pH = 6) for all ERβ isoforms. Sections were incubated with 20% normal goat serum for 30 min at room temperature to reduce nonspecific binding. Cells expressing ERβ1 (clone PPG5/10, Dako, Glostrup, Denmark, dilution 1:50), ERβ2 (clone 57/3, Serotec Ltd, Oxford, United Kingdom, dilution 1:100), and ERβ5 (clone 5/25 Serotec, dilution 1:100) were identified after overnight incubation at 4 °C. Antibodies used in the present study were previously validated by other authors and widely used in clinical studies for detection in paraffin-embedded tissue sections [14–19]. Sections were incubated with the secondary, anti-mouse EnVision System-HRP (DakoCytomation, Carpinteria, CA, USA) for 30 min, at room temperature. The slides were developed with diaminobenzidine (DAB substrate System, DakoCytomation), counterstained with Mayer's hematoxylin, dehydrated in ethanol and xylene, and finally mounted.
Table 1 Clinicopathological features of the overall series. Characteristics
No. of patients (%)
All cases Median age (range) Grade G1 G2 G3 FIGO stage III IV Residual tumor 0 mm N0 mm Primary chemotherapy Platinum/paclitaxel Platinum-based Chemosensitivity Sensitive Resistant
56 54 (33–79) 1 (1.8) 11 (19.6) 44 (78.6) 54 (96.4) 2 (3.6) 35 (62.5) 21 (37.5) 50 (89.3) 6 (10.7) 40 (71.4) 16 (28.6)
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Expression was evaluated by considering the percentage of cells exhibiting immunoreaction in a minimum of 500 histologically identified neoplastic cells. Scoring of steroid hormone receptors was calculated as previously reported [10,20]. Briefly, the mean percentage of stained cells (both nuclear staining and cytoplasmic staining) was categorized as follows: 0 = 0%, 1 = 1–10%, 2 = 11–33%, 3 = 34–66%, and 4 = 67–100%. The intensity of staining was also evaluated, and graded from 1 to 3, where 1 = weak staining, 2 = moderate staining, and 3 = strong staining. The two values obtained were multiplied to calculate an immunoreactive score (IRS, maximum value 12). For statistical analysis, the samples were grouped into negative (score below or equal to 2) or positive (score higher than 2), as previously suggested [10,21–24]. To further examine ERβ as a predictor of survival, and to assess whether cellular compartment is important, we stratified all patients by the absence versus presence of ERβ isoforms in the cytoplasm (ERβ cyto− vs ERβ cyto+).
8 × 106 cells (A2780, OVCAR-3, and SKOV-3) was injected subcutaneously in the right flank of each animal (0.2 ml per mice). For this study n = 3 mice were studied in each group. In addition, to assess differences in estrogen receptor status following estrogen removal, a further group of female (n = 3) were anesthetized, bilaterally ovariectomized (OVX) and, after a week recovery, inoculated with 8 × 106 A2780 cells. Inoculated animals were observed daily with tumors measured at least twice per week using Vernier calipers. Tumor weight was calculated from two dimensional measurements (mm) [26,27]: Tumor weight = length × width2 / 2. All mice were sacrificed when tumor burden reached approximately 500 mg. All tumors were fixed in 4% paraformaldehyde and stored paraffin embedded. For immunohistochemistry, 3 μm sections from all tumors were used. ERβ1, ERβ2 and ERβ5 were detected using the following antibodies: ERβ1 (clone PPG5/10, Dako, Glostrup, Denmark, dilution 1:50), ERβ2 (clone 57/3, Serotec Ltd, Oxford, United Kingdom, dilution 1:100), and ERβ5 (clone 5/25 Serotec, dilution 1:100) overnight at 4 °C in a humidified chamber. Immunohistochemical analysis was carried out as described above.
Culture of ovarian cancer cell lines
Statistical analysis
To determine whether ERβ isoforms are expressed in human ovarian cancer cells, we analyzed 3 human ovarian cancer cell lines: A2780, OVCAR-3, and SKOV-3. The ovarian carcinoma cell lines A2780 and SKOV-3 were purchased from the European Collection of Cell Cultures (ECACC, Salisbury, UK). NIH:OVCAR-3 was purchased from the CLS Cell Lines Service GmbH (Eppelheim, Germany). OVCAR-3 and A2780 cells were cultured in RPMI 1640 medium (Lonza, Basel, Switzerland), SKOV-3 were grown in Dulbecco's modified Eagle's medium (Lonza). The medium was supplemented with 10% fetal bovine serum (FBS, Lonza), 2 mM glutamine and antibiotics (100 mg/ml streptomycin and 100 IU/ml penicillin) (Lonza). All cultures were maintained at 37 °C under a humidified atmosphere of 5% CO2 and 95% air.
The expression of ERβ isoforms, and their association according to clinicopathological parameters were evaluated using the Fisher's exact test. Disease-free survival and overall survival were calculated from the date of diagnosis to the date of progression/death, or the date last seen. The prognostic effect of the various parameters on clinical outcome (i.e. recurrence or death of disease) was tested by plotting survival curves according to Kaplan–Meier method, and comparing groups using the log rank test, as well as by multivariate analysis using the Cox model. Kaplan–Meier survival estimates were generated from the date of histological diagnosis to the time of the last follow-up or death. In univariate analysis, each parameter was categorized for subsequent statistical analysis. Only variables with p-value b 0.2 in the univariate analysis were included in multivariate model. p values were two-sided, with p ≤ 0.05 considered as significant. All statistical analyses were performed using the GraphPad Prism5 Software (San Diego, CA, USA). Cox analysis was performed using the StatPlus 2009.
Evaluation of immunohistochemical staining
Immunocytochemistry Cells (105 cells/well) were plated into a four-well chamber slide (Nunc® Lab-Tek® Chamber Slide, Nunc, Inc., Naperville, IL). The cells were cultured for 24 h and used for immunostaining. Slides were washed twice with PBS, fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. The endogenous peroxidase was blocked with 3% H2O2 for 5 min. After washing twice with PBS, cells were incubated with a blocking solution containing 20% normal horse serum in PBS for 30 min at room temperature. Excess blocking solution was drained, and samples were incubated with primary antibodies: ERβ1 (clone PPG5/10, Dako, Glostrup, Denmark, dilution 1:100), ERβ2 (clone 57/3, Serotec Ltd, Oxford, United Kingdom, dilution 1:75), and ERβ5 (clone 5/25 Serotec, dilution 1:75) overnight at 4 °C in a humidified chamber. The samples were then rinsed three times with PBS and incubated with secondary antibody, EnVision System-HRP (Dako, Carpinteria, CA), for 30 min at room temperature. The immunoreactivity was detected using the 3,3′-diaminobenzidine substrate (DAB substrate System, Dako). The slides were counterstained with Mayer's hematoxylin, dehydrated in ethanol and xylene, and finally mounted. Experimental animals and xenograft models Female athymic mice [Athymic Nude-nu], 5 weeks old and within a weight range of approximately 18–22 g, were obtained from Charles River (Lecco, Italy) and housed under controlled condition. Procedures and facilities followed the requirements of the Commission Directive 86/609/EEC concerning the protection of animals used for experimental and other scientific purposes. Italian legislation is defined in the Decreto Legislativo No. 116 of 27 January 1992. In addition the UKCCCR guidelines for the welfare of animals in experimental neoplasia were followed [25]. On the day of dosing, cells were trypsinized and a suspension of
Results Estrogen receptor beta (ERβ) isoforms in advanced serous ovarian cancers and their relationship to clinicopathological criteria Fig. 1 shows representative pictures for ERβ1, ERβ2, and ERβ5 immunostaining performed in serial sections from the same patient. All three ERβ isoforms exhibited both nuclear and cytoplasmic distributions. Overall, in our cohort, ERβ1 and ERβ5 were predominantly expressed at the nuclear level, while ERβ2 was mainly localized in the cytoplasm (Fig. 1A). Specifically, of the 56 advanced serous ovarian cancers studied 11 (14.3%) lacked ERβ1 protein expression, the majority of tumors (59%) showing medium-level of ERβ1 nuclear staining (7.4 ± 0.5) without cytoplasmic immunoreactivity; when present, cytoplasmic immunopositivity was of low intensity. Only three out of 56 specimens (5%) lacked ERβ2 expression; medium-level of nuclear staining (8 ± 0.5) was observed in cancers, with about 45% of patients showing an associated cytoplasmic reaction of medium intensity. ERβ5 was expressed by all the primary cancer studied: immunopositivity was primarily nuclear (10 ± 0.3), with about 40% of patients also showing an associated low cytoplasmic reaction (Fig. 1B–D). Table 2 shows ERβ isoform status in advanced serous ovarian cancer patients according to clinicopathological parameters. Cytoplasmic ERβ2 expression (IRS N 2) was more frequent in chemoresistant patients (p = 0.03). Cytoplasmic ERβ1 was also found to be significantly associated with residual tumor extent (p = 0.04). No correlation was demonstrated between nuclear ERβ isoform expression and any of the parameters examined.
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Fig. 1. A) Representative pictures for ERβ1, ERβ2, and ERβ5 immunostaining performed in serial sections from the same patient and showing both nuclear and cytoplasmic protein expressions in advanced serous ovarian cancer. B) Bar chart showing the distribution of negative, nuclear, cytoplasmic, and nuclear/cytoplasmic ERβ isoform staining in our cohort of patients. C) and D) Bar chart showing, for each isoform, the immunoreactive receptor score (IRS) (mean ± SEM) in nuclear and cytoplasmic compartments. *p b 0.05, ***p b 0.0001.
Association between immunohistochemical results and survival data The prognostic role of ERβ isoform expression for both DFS and OS was tested in univariate and multivariate analyses adjusted for clinicopathological parameters. Median DFS for the study population was 15 months. In univariate analysis, cytoplasmic ERβ2 expression was the only molecular marker significantly associated with a shorter DFS (median of 14 months
compared to 23 months in negative patients, p = 0.04) (Fig. 2C, Table 3A). The 5-year recurrence rate was nearly 88% for patients who did express cytoplasmic ERβ2, and 65% in negative patients. The prognostic role of age at diagnosis, histological grade, and residual tumor was also tested in univariate analyses. The presence of any residual tumor at primary surgery was found to be associated with a higher risk of recurrence of disease (Table 3A). In the multivariate Cox regression analysis after controlling for volume of residual tumor, the
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Table 2 Expression of estrogen receptor beta isoforms in the overall series. Characteristics Age ≤54 N54 Grade G1–2 G3 Residual tumor 0 mm N0 mm Chemosensitivity Sensitive Resistant
No. of patients (%)
Nuclear ERβ1+ %
Cytoplasmic ERβ1+ %
29 (51.8) 27 (48.2)
82.7 74.0
27.6 26.0
12 (21.4) 44 (78.6)
83.3 77.3
35 (62.5) 21 (37.5) 40 (71.4) 16 (28.6)
Nuclear ERβ2+ %
Cytoplasmic ERβ2+ %
Nuclear ERβ5+ %
Cytoplasmic ERβ5+ %
93.1 96.3
51.7 37.0
100 100
41.4 37.0
41.7 22.8
100 93.2
41.7 45.5
100 100
33.3 40.9
80.0 76.2
14.3 47.6⁎
91.4 100
37.1 57.1
100 100
31.4 52.3
72.5 93.8
22.5 37.5
95.0 93.8
35.0 68.8⁎
100 100
37.5 43.8
+ = positive cases. ⁎ p b 0.05, Fisher's exact test.
association between cytoplasmic ERβ2 expression and earlier disease recurrence did not achieve statistical significance (HR 1.6; 95% CI 0.9–2.9, p = 0.1, Table 3A). Median OS for the study population was 47 months. In univariate survival analysis, cytoplasmic ERβ2 expression was again the only molecular marker significantly associated with a shorter OS (p = 0.0006). The median OS had not been reached at the time of the analysis in patients with tumors that exhibited no cytoplasmic ERβ2 expression, compared to 33 months in tumors with positive cytoplasmic immunoreaction (Fig. 2D, Table 3B). The 5-year survival rate was nearly 28% for patients who did express cytoplasmic ERβ2, and 60% in negative patients. The prognostic role of age at diagnosis, histological grade, and residual tumor was also tested in univariate analyses: volume of residual tumor was the only variable associated with a shorter OS (p = 0.005, Table 3B). In the multivariate Cox regression analysis after controlling for volume of residual tumor, cytoplasmic ERβ2 expression retained its significance as independent poor prognostic factors (HR 2.8; 95% CI 1.3–5.7, p = 0.006, Table 3B). We were then prompted at analyzing the prognostic relevance of cytoplasmic ERβ2 expression in platinum-sensitive ovarian cancer patients (n = 40), and, interestingly enough, its unfavorable role was maintained in this subset: cases with cytoplasmic ERβ2 expression had a shorter OS (median OS = 53 months) than cases with no cytoplasmic ERβ2 expression (median OS undefined) (p = 0.004). The 5-year survival rate was nearly 43% for patients who did express cytoplasmic ERβ2, and 72% in negative patients. Multivariate Cox analysis in platinum-sensitive patients showed that cytoplasmic ERβ2 maintained its significance as negative prognostic factor, after controlling for volume of residual tumor (HR 2.8; 95% CI 1.0–7.6, p = 0.04).
Assessment of ERβ isoform expression in ovarian cancer cell lines and xenografts To determine whether ERβ isoforms are expressed in human ovarian cancer cells, we analyzed 3 human ovarian cancer cell lines: OVCAR-3, SKOV-3 and A2780. Immunocytochemistry confirmed the expression of ERβ1, ERβ2 and ERβ5 in the tested ovarian cancer cells and, in keeping with clinical data, both nuclear and cytoplasmic expressions were observed (Fig. 3). The retention of this receptor status during in vivo tumor growth was then verified in intact female mice. We found that ovarian cancer xenografts were all positive for the three isoforms, although showing different levels and subcellular distributions. ERβ1 exhibited a prominent cytoplasmic localization, while ERβ2 and ERβ5 were mainly nuclear, being ERβ5 the isoform predominantly expressed (Fig. 3). To verify the possible modulation of hormone receptor expression by endogenous estrogen, we ovariectomized an additional group of female and assessed the pattern of ERβ1, ERβ2 and ERβ5 expression in A2780 grown subcutaneously in these mice.
Notably, we found that when compared to intact female, tumors from OVX mice showed higher levels of cytoplasmic ERβ2 staining (Fig. 3), this suggesting that estrogen removal could induce a shift in the cellular compartment-specific distribution for this isoform. On the other hand, no relevant differences were observed between intact and OVX females in ERβ1 and ERβ5 expression pattern.
Discussion This is the first study to analyze the subcellular expression of wt ERβ and its different isoforms (ERβ2 and ERβ5) in a well-validated cohort of advanced serous ovarian cancer with complete follow-up data, using specific well-validated antibodies. The immunohistochemical expression of the three ERβs was analyzed in our cohort and staining revealed unique cellular compartment-specific distribution for each isoform in malignant ovarian tissue. Both nuclear staining and cytoplasmic staining were observed for all three isoforms in advanced serous ovarian cancers, being ERβ5 the most represented, a finding also reported by other authors examining both ovarian carcinoma cell lines, and ovarian carcinoma tissues [9]. One of the striking observations of this study was the strong statistical association of cytoplasmic ERβ2 immunoreactivity with reduced survival, a finding also reported in familial and sporadic breast cancer [15,17]. The functional role of cytoplasmic ERβ (and specifically of cytoplasmic ERβ2) is far from being elucidated. There is ample evidence to support nongenomic functions of ERβ in various cell types. Indeed, nongenomic signaling properties has been demonstrated for ERβ in different cancer cell lines, a mechanism implying a cross-communication (in a cooperative fashion) with transmembrane growth factor receptor signaling pathways that ultimately promote downstream signaling for tumor cell proliferation and survival [28,29]. Moreover, a mitochondrial localization of ERβ has been demonstrated in many cell types, by immunocytochemistry, immunohistochemistry, and immunoblots (using a large group of diversified antibodies) and also verified by proteomics [30]. In parallel, mechanistic studies have been carried out to unravel the possible role of mitochondrial ERβ protein. Zhang and colleagues [31] recently reported a ligand-independent cell-survival function of mitochondrial ERβ in lung cancer cells. Specifically they propose that in the absence of ERβ, Bad is able to interact with Bcl-XL (and Bcl-2) to free Bax to oligomerize and initiate apoptosis, whereas in the presence of ERβ Bad is sequestered and prevented from interaction with Bcl-XL, thus inhibiting apoptosis. As well, there has been increasing evidence pointing to the mitochondrial respiratory chain (MRC) as a novel and important target for the ligand-dependent actions of both ERα and ERβ in a number of cell types and tissues, with studies supporting the notion that by up-regulating the MRC biogenesis and functions, E2 and ERs contribute to inhibition of apoptosis, causing the normal balance of cell growth/cell death toward survival and enhanced
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Fig. 2. A) and B) Representative pictures for ERβ2 immunostaining showing nuclear/cytoplasmic (A) and only nuclear (B) expressions in ovarian cancer tissues. C) and D) Kaplan–Meier survival curve for the probability of disease-free survival (C) and overall survival (D) according to expression of cytoplasmic ERβ2 in advanced serous ovarian cancer patients. Any expression of ERβ2 in the cytoplasm is significantly associated with disease-free survival and overall survival disadvantage (p = 0.04 and p = 0.0006, respectively).
Table 3A Univariate and multivariate analyses of factors affecting DFS in advanced ovarian cancer. Variables
Residual tumor 0 mm N0 mm ERβ2 expression (subcellular localization) Only nuclear expression Cytoplasmic expression
Univariate
Multivariate
HR (95% CI)
p
HR (95% CI)
p⁎
2.9 (1.4–5.9)
0.003
2.2 (1.2–4.2)
0.01
1.9 (1.0–3.6)
0.04
1.6 (0.9–2.9)
0.1
DFS = disease-free survival. HR = hazard ratio. CI = confidence interval. Only variables with p-value b 0.2 in the univariate analysis were included in multivariate model. χ2 of the model = 9.8; p value = 0.007. ⁎ ps were derived from the COX proportional hazards model.
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Table 3B Univariate and multivariate analyses of factors affecting OS in advanced ovarian cancer. Variables
Residual tumor 0 mm N0 mm ERβ2 expression (subcellular localization) Only nuclear expression Cytoplasmic expression
Univariate
Multivariate
HR (95% CI)
p
HR (95% CI)
p⁎
2.8 (1.3–5.9)
0.005
2.0 (1.0–4.0)
0.10
3.5 (1.7–7.1)
0.0006
2.8 (1.3–5.7)
0.006
OS = overall survival. HR = hazard ratio. CI = confidence interval. Only variables with p-value b 0.2 in the univariate analysis were included in multivariate model. χ2 of the model = 15.1; p value = 0.0005. ⁎ ps were derived from the COX proportional hazards model.
cell growth [30,32]. Finally, there are data supporting the notion that mitochondrial ERβ functions as a mitochondrial vulnerability factor involved in Ψm maintenance, potentially through a mitochondrial transcription dependent mechanism [33]. Such data also support a potential bi-faceted role of mitochondrial ERβ proteins in apoptosis. In
this context, it is worthy to note that we also found a statistically significant association between cytoplasmic ERβ2 immunoreactivity and chemoresistance. The molecular mechanisms of tumor resistance to chemotherapeutic agents such as cisplatin and paclitaxel involve multiple factors, including reduced drug uptake or increased drug
Fig. 3. A) Expression of ERβ1, ERβ2 and ERβ5 in OVCAR-3, SKOV-3 and A2780 human ovarian cancer cells using immunocytochemical analysis. Pictures show nuclear and cytoplasmic expressions of antibodies (magnification 40×). B) Expression of ERβ1, ERβ2 and ERβ5 in OVCAR-3, SKOV-3 and A2780 subcutaneous xenografts in intact athymic female mice. ERβ1 exhibited a prominent cytoplasmic localization after in vivo growth, while ERβ2 and ERβ5 were mainly expressed in the nucleus. Compared to intact female, A2780 tumor grown in OVX mice showed higher levels of cytoplasmic ERβ2 staining, while no differences were observed for the remaining isoforms (magnification 40×).
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efflux, reduced DNA binding, increased DNA repair, and impaired apoptosis [34,35]. The mitochondrial anti-apoptotic action of ERβ might contribute toward chemotherapy resistance in ovarian cancer. Few studies have examined the prognostic role of ERβ in ovarian cancer [reviewed in 36]. Chan and colleagues [37] reported that a higher level of ERβ mRNA expression was significantly associated with longer DFS and OS, while a recent study suggested that tumor nuclear ERβ1 level did not affect progression-free and cause-specific survival, although influencing overall survival [24]. These previous results are barely comparable to present data since they either refer to mRNA level (that is, a measurement including non-tumor cells and not taking into account translational control or turnover of protein), they assess a specific ERβ isoform (i.e. ERβ1), or do not consider cytoplasmic protein staining. Besides, both studies also considered low-stage disease and thus there may be sub-groups in which the amount of nuclear ERβ protein is prognostic. Notably, we also showed a reasonable concordance in ERβ isoform immunoreactivity between clinical samples, ovarian cancer cell lines and ovarian cancer xenografts. Indeed, we found that in different ovarian cancer cell lines grown in vivo as subcutaneous tumor xenografts the overall expression of ERβ1, ERβ2 and ERβ5, observed both in the nuclear and cytoplasmic compartments, resembled the pattern of expression observed in our series of advanced ovarian cancer. These preliminary pre-clinical experiments also suggest a role for endogenous estrogen in modulating ERβ2 compartmentalization, since we observed that A2780 tumors from ovariectomized mice showed a higher cytoplasmic ERβ2 staining when compared to intact females. Thus, we could speculate that in menopausal women, the abrupt decrease in ovarian estrogen might, through the modulation of ERβ2 status, confer a more aggressive phenotype and ultimately contribute to ovarian cancer development. However, additional preclinical experimental models need to be used to confirm this hypothesis, particularly in the light of very recent data published by Domcke and colleagues [38], relative to the pronounced differences in molecular profiles between commonly used ovarian cancer cell lines and highgrade serous ovarian cancer tumor samples. These future in vivo preclinical studies should also address the possible effect of estrogen modulation on ER status and ovarian cancer growth in models that more accurately mimic the feature behavior of human ovarian cancer, such as the intraperitoneal xenografts. We recognize that our cohort is small (56 patients), however, the possibility of using ERβ2 for advanced serous ovarian cancer prognosis shows great promise. Indeed, if anomalies of ERβ2 cytoplasmic expression could be demonstrated to represent an independent unfavorable prognostic marker and/or a marker predicting chemoresistance in advanced serous ovarian cancer, its immunohistochemical assessment at the time of surgery, could help to recognize candidates for clinical trials of new interventions. Meanwhile, additional investigations are warranted to identify the mechanism responsible for cytosolic localization of endogenous ERβ, and the specific contribution of each ERβ isoform in relation to their differential subcellular distribution within the target cells, a condition that may underlie altered function of ERβ as well as its variants. Definitely, membrane, cytoplasmic and nuclear estrogen receptors are not separate units, but rather the components of a complex mechanism in which they both cooperate with each other. Conflict of interest statement The authors declare no conflict of interest.
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Contents lists available at ScienceDirect
Gynecologic Oncology journal homepage: www.elsevier.com/locate/ygyno
Prognostic significance of the estrogen receptor beta (ERβ) isoforms ERβ1, ERβ2, and ERβ5 in advanced serous ovarian cancer Alessandra Ciucci a, Gian Franco Zannoni b, Daniele Travaglia a, Marco Petrillo a, Giovanni Scambia a, Daniela Gallo a,⁎ a b
Department of Obstetrics and Gynecology, Catholic University of the Sacred Heart, Rome, Italy Department of Histopathology, Catholic University of the Sacred Heart, Rome, Italy
H I G H L I G H T S • This is the first study analyzing the subcellular expression of ERβ1, ERβ2 and ERβ5 in advanced serous ovarian cancers. • The most striking observation of this study was the strong statistical association of cytoplasmic ERβ2 immunoreactivity with reduced survival. • Cytoplasmic ERβ2 expression was also found to be significantly associated with chemoresistance.
a r t i c l e
i n f o
Article history: Received 28 October 2013 Accepted 18 December 2013 Available online 27 December 2013 Keywords: Ovary Epithelial ovarian carcinomas Survival Estrogen Estrogen receptors Estrogen receptor beta
a b s t r a c t Objective. In the present study we have examined the pattern of expression of the full length estrogen receptor β (ERβ1) and two ERβ splice variant isoforms (ERβ2, ERβ5) in well-characterized advanced serous ovarian cancers. Methods. Immunohistochemistry was performed with ERβ1, ERβ2, and ERβ5 antibodies and results were correlated with pathological and clinical follow-up data. Expression of ERβ isoforms in a panel of ovarian cancer cell lines and human tumor xenografts was also assessed. Results. Immunohistochemical staining revealed cellular compartment-specific distribution for each isoform in malignant ovarian tissues exhibiting both nuclear staining and cytoplasmic staining. Patients with cytoplasmic ERβ2 expression had significantly worse outcome (p = 0.006 at the multivariate analysis), the 5-year survival rate being nearly 28% for patients who did express cytoplasmic ERβ2, and 60% in negative patients. Cytoplasmic ERβ2 expression was also found to be significantly associated with chemoresistance. In concordance with clinical results both nuclear and cytoplasmic expressions were observed for the three isoforms in the cancer cell lines and human tumor xenografts tested. Conclusions. This is the first study to uncover an unfavorable prognostic role of ERβ2 in advanced serous ovarian cancer. If anomalies of ERβ2 cytoplasmic expression could be demonstrated to represent an independent unfavorable prognostic marker and/or a marker predicting chemoresistance in advanced serous ovarian cancer, its immunohistochemical assessment at the time of surgery, could help to recognize candidates for clinical trials of new interventions. © 2013 Elsevier Inc. All rights reserved.
Introduction Of the gynecological malignancies, epithelial ovarian cancer is the leading cause of death in the great majority of developed countries with more than 140,000 women dying from this cancer across the world in 2008 [1]. Although many efforts have been made to clarify the etiology of ovarian carcinogenesis and the molecular mechanisms involved in the proliferation of ovarian carcinoma cells, this disease ⁎ Corresponding author at: Department of Obstetrics and Gynecology, Catholic University of the Sacred Heart, Largo A. Gemelli 8, 00168 Rome, Italy. Fax: + 39 06 3051160. E-mail address: [email protected] (D. Gallo). 0090-8258/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygyno.2013.12.027
remains among the less understood of major human malignancies. Increasing clinical data implicate estrogen in the etiology of ovarian cancer. These data come from studies showing that while long-term use of estrogen replacement therapy is associated with increased risk of ovarian cancer incidence or mortality, the use of oral contraceptives is associated with decreased risk. Actually, the protective effect of oral contraceptive has been interpreted as a support to the “estrogen hypothesis”, as oral contraceptive use decreases ovarian estrogen production, and maintains circulating estrogens around early to midfollicular phase levels; other authors, however, suggest that chronic suppression of ovulation reduces cancer risk by decreasing recurrent ovarian injury [reviewed in 2]. Finally, the hypothesis that a decreased risk of ovarian cancer with oral contraceptive use could also be due to
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the cyclic progestational climate, cannot be ruled out [3]. However, it is worthy to note that ovarian cancer is primarily a disease of postmenopausal women, the median age of diagnosis being 63 years, with 48% of patients 65 years or older [4]. Estrogens exert their action through two estrogen receptors (ERα and ERβ) that are encoded by separate genes. These two receptors are members of the nuclear receptor superfamily of ligand-dependent transcription factors and share both structural and functional homologies. They produce different biological effects, first of all by regulating different genes in response to estradiol (E2) and selective estrogen receptor modulators, but they may also regulate the same genes in opposite directions, in agreement with a yin/yang hypothesis. Overall, ERα is thought to promote the expression of genes involved in cell survival and proliferation, thus determining tumor growth and progression, while the natural function of ERβ is thought to be antiproliferative and proapoptotic, and ERβ is therefore described as a tumor suppressor. Several ERβ isoforms have been reported so far. ERβ1 is the full-length receptor coded by exons 1–8. ERβ2–5 share exons 1–7 with ERβ1, but display unique sequences instead of exon 8. These differences in the C-terminal part of ERβ2–5 determine a truncation of the ligand binding domain and ablation of the ligand-dependent activation function AF-2. Therefore, ERβ1 is the only fully functional isoform that is able to bind ligand, whereas ERβ2, -β4, and -β5 do not form homodimers and have no innate activities of their own, but may modulate estrogen action when dimerized with ERβ1 or ERα [reviewed in [5] and references therein]. Besides, evidence is accumulating that estrogens also exert nongenomic actions including mobilization of intracellular calcium, stimulation of adenylate cyclase activity and cAMP production, activation of the MAPK signaling pathway and of the phosphoinositol (PI) 3-kinase signaling pathway. These non-transcriptional mechanisms mainly involve cytoplasmic or cell membrane-bound ERs, and can be triggered by estrogen (ligand-dependent signaling) or receptor kinases in the absence of estrogen (ligand-independent ER activation) [6]. Nongenomic actions of estrogens may indirectly influence gene expression, through the activation of signal transduction pathways that eventually act on target transcription factors [reviewed in 7]. In the normal ovary, the levels of ERβ are high and predominate over ERα, being ERβ1, -β2, and -β5 the most represented isoforms [8,9]. Besides, literature data suggest that a high percentage of ovarian tumors express ERβ and that a progressive decline of ERβ occurs during development or progression of ovarian cancer [reviewed in [5] and references therein]. However, limited information is available on its prognostic role in the disease. In a previous study aimed at investigating the prognostic significance of ER (ERα, ERβ) and PR expressions in a cohort of uniformly treated patients with advanced serous ovarian cancer, we found that, in this specific setting, cytoplasmic ERβ signaling may be more important for patient survival than its nuclear signaling. Specifically, our results suggested that advanced serous ovarian cancers with high cytoplasmic total ERβ expression may define patients with aggressive biology, and possibly resistant to chemotherapy [10]. To gain insights into the role of estrogen in ovarian cancer and to extend our previous observations on the association between cytoplasmic ERβ signaling and clinical outcome, we have examined in the present study the pattern of expression of the full length ERβ (ERβ1) and two ERβ splice variant isoforms (ERβ2, ERβ5) in a similar series of patients with advanced serous ovarian cancer, and correlated the pattern of expression with clinicopathological parameters and with response to chemotherapy. Expression of ERβ isoforms in a panel of ovarian cancer cell lines and human tumor xenografts was also assessed. Materials and methods Patients The study included 56 patients with advanced serous ovarian cancer admitted to the Gynecologic Oncology Unit, Catholic University of
Rome, between March 2000 and December 2008. In our institution a written informed consent is routinely requested from patients for collection of their clinical data, as well as paraffin embedded sections for research use. Clinicopathological characteristics of the overall series are summarized in Table 1. According to standard guidelines, maximal surgical effort was attempted in all patients resulting in complete resection (residual tumor 0 mm) in 35 (62.5%) cases. All patients received platinum-based chemotherapy (75–100 mg/m2 for cisplatin, AUC = 5 for carboplatin, per cycle). Fifty patients (89.3%) also received paclitaxel (135–175 mg/m2 for each cycle). Recurrence of disease was defined according to GCIG CA125 criteria [11,12] and/or radiological confirmation of tumor progression. To define chemosensitivity, we used the common definition of platinum resistance, defining as “sensitive” patients that relapsed 6 months or more after prior platinum-containing chemotherapy, and as “resistant” patients that relapsed less than 6 months after chemotherapy was stopped, or that progressed while on therapy [13]. Follow-up data were available for all 56 patients (median follow-up, 47 months; range, 9–162 months). During the follow-up period, progression and death of disease were observed in 44 and 34 patients, respectively.
Immunohistochemistry Three-micrometer-thick paraffin sections were mounted on Superfrost coated slides, and dried overnight. The sections were deparaffinized in xylene and rehydrated in graded solutions of ethanol; the endogenous peroxidase was blocked with 3% H2O2 for 5 min. Antigen retrieval procedure was performed by microwave oven heating in citrate buffer (pH = 6) for all ERβ isoforms. Sections were incubated with 20% normal goat serum for 30 min at room temperature to reduce nonspecific binding. Cells expressing ERβ1 (clone PPG5/10, Dako, Glostrup, Denmark, dilution 1:50), ERβ2 (clone 57/3, Serotec Ltd, Oxford, United Kingdom, dilution 1:100), and ERβ5 (clone 5/25 Serotec, dilution 1:100) were identified after overnight incubation at 4 °C. Antibodies used in the present study were previously validated by other authors and widely used in clinical studies for detection in paraffin-embedded tissue sections [14–19]. Sections were incubated with the secondary, anti-mouse EnVision System-HRP (DakoCytomation, Carpinteria, CA, USA) for 30 min, at room temperature. The slides were developed with diaminobenzidine (DAB substrate System, DakoCytomation), counterstained with Mayer's hematoxylin, dehydrated in ethanol and xylene, and finally mounted.
Table 1 Clinicopathological features of the overall series. Characteristics
No. of patients (%)
All cases Median age (range) Grade G1 G2 G3 FIGO stage III IV Residual tumor 0 mm N0 mm Primary chemotherapy Platinum/paclitaxel Platinum-based Chemosensitivity Sensitive Resistant
56 54 (33–79) 1 (1.8) 11 (19.6) 44 (78.6) 54 (96.4) 2 (3.6) 35 (62.5) 21 (37.5) 50 (89.3) 6 (10.7) 40 (71.4) 16 (28.6)
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Expression was evaluated by considering the percentage of cells exhibiting immunoreaction in a minimum of 500 histologically identified neoplastic cells. Scoring of steroid hormone receptors was calculated as previously reported [10,20]. Briefly, the mean percentage of stained cells (both nuclear staining and cytoplasmic staining) was categorized as follows: 0 = 0%, 1 = 1–10%, 2 = 11–33%, 3 = 34–66%, and 4 = 67–100%. The intensity of staining was also evaluated, and graded from 1 to 3, where 1 = weak staining, 2 = moderate staining, and 3 = strong staining. The two values obtained were multiplied to calculate an immunoreactive score (IRS, maximum value 12). For statistical analysis, the samples were grouped into negative (score below or equal to 2) or positive (score higher than 2), as previously suggested [10,21–24]. To further examine ERβ as a predictor of survival, and to assess whether cellular compartment is important, we stratified all patients by the absence versus presence of ERβ isoforms in the cytoplasm (ERβ cyto− vs ERβ cyto+).
8 × 106 cells (A2780, OVCAR-3, and SKOV-3) was injected subcutaneously in the right flank of each animal (0.2 ml per mice). For this study n = 3 mice were studied in each group. In addition, to assess differences in estrogen receptor status following estrogen removal, a further group of female (n = 3) were anesthetized, bilaterally ovariectomized (OVX) and, after a week recovery, inoculated with 8 × 106 A2780 cells. Inoculated animals were observed daily with tumors measured at least twice per week using Vernier calipers. Tumor weight was calculated from two dimensional measurements (mm) [26,27]: Tumor weight = length × width2 / 2. All mice were sacrificed when tumor burden reached approximately 500 mg. All tumors were fixed in 4% paraformaldehyde and stored paraffin embedded. For immunohistochemistry, 3 μm sections from all tumors were used. ERβ1, ERβ2 and ERβ5 were detected using the following antibodies: ERβ1 (clone PPG5/10, Dako, Glostrup, Denmark, dilution 1:50), ERβ2 (clone 57/3, Serotec Ltd, Oxford, United Kingdom, dilution 1:100), and ERβ5 (clone 5/25 Serotec, dilution 1:100) overnight at 4 °C in a humidified chamber. Immunohistochemical analysis was carried out as described above.
Culture of ovarian cancer cell lines
Statistical analysis
To determine whether ERβ isoforms are expressed in human ovarian cancer cells, we analyzed 3 human ovarian cancer cell lines: A2780, OVCAR-3, and SKOV-3. The ovarian carcinoma cell lines A2780 and SKOV-3 were purchased from the European Collection of Cell Cultures (ECACC, Salisbury, UK). NIH:OVCAR-3 was purchased from the CLS Cell Lines Service GmbH (Eppelheim, Germany). OVCAR-3 and A2780 cells were cultured in RPMI 1640 medium (Lonza, Basel, Switzerland), SKOV-3 were grown in Dulbecco's modified Eagle's medium (Lonza). The medium was supplemented with 10% fetal bovine serum (FBS, Lonza), 2 mM glutamine and antibiotics (100 mg/ml streptomycin and 100 IU/ml penicillin) (Lonza). All cultures were maintained at 37 °C under a humidified atmosphere of 5% CO2 and 95% air.
The expression of ERβ isoforms, and their association according to clinicopathological parameters were evaluated using the Fisher's exact test. Disease-free survival and overall survival were calculated from the date of diagnosis to the date of progression/death, or the date last seen. The prognostic effect of the various parameters on clinical outcome (i.e. recurrence or death of disease) was tested by plotting survival curves according to Kaplan–Meier method, and comparing groups using the log rank test, as well as by multivariate analysis using the Cox model. Kaplan–Meier survival estimates were generated from the date of histological diagnosis to the time of the last follow-up or death. In univariate analysis, each parameter was categorized for subsequent statistical analysis. Only variables with p-value b 0.2 in the univariate analysis were included in multivariate model. p values were two-sided, with p ≤ 0.05 considered as significant. All statistical analyses were performed using the GraphPad Prism5 Software (San Diego, CA, USA). Cox analysis was performed using the StatPlus 2009.
Evaluation of immunohistochemical staining
Immunocytochemistry Cells (105 cells/well) were plated into a four-well chamber slide (Nunc® Lab-Tek® Chamber Slide, Nunc, Inc., Naperville, IL). The cells were cultured for 24 h and used for immunostaining. Slides were washed twice with PBS, fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. The endogenous peroxidase was blocked with 3% H2O2 for 5 min. After washing twice with PBS, cells were incubated with a blocking solution containing 20% normal horse serum in PBS for 30 min at room temperature. Excess blocking solution was drained, and samples were incubated with primary antibodies: ERβ1 (clone PPG5/10, Dako, Glostrup, Denmark, dilution 1:100), ERβ2 (clone 57/3, Serotec Ltd, Oxford, United Kingdom, dilution 1:75), and ERβ5 (clone 5/25 Serotec, dilution 1:75) overnight at 4 °C in a humidified chamber. The samples were then rinsed three times with PBS and incubated with secondary antibody, EnVision System-HRP (Dako, Carpinteria, CA), for 30 min at room temperature. The immunoreactivity was detected using the 3,3′-diaminobenzidine substrate (DAB substrate System, Dako). The slides were counterstained with Mayer's hematoxylin, dehydrated in ethanol and xylene, and finally mounted. Experimental animals and xenograft models Female athymic mice [Athymic Nude-nu], 5 weeks old and within a weight range of approximately 18–22 g, were obtained from Charles River (Lecco, Italy) and housed under controlled condition. Procedures and facilities followed the requirements of the Commission Directive 86/609/EEC concerning the protection of animals used for experimental and other scientific purposes. Italian legislation is defined in the Decreto Legislativo No. 116 of 27 January 1992. In addition the UKCCCR guidelines for the welfare of animals in experimental neoplasia were followed [25]. On the day of dosing, cells were trypsinized and a suspension of
Results Estrogen receptor beta (ERβ) isoforms in advanced serous ovarian cancers and their relationship to clinicopathological criteria Fig. 1 shows representative pictures for ERβ1, ERβ2, and ERβ5 immunostaining performed in serial sections from the same patient. All three ERβ isoforms exhibited both nuclear and cytoplasmic distributions. Overall, in our cohort, ERβ1 and ERβ5 were predominantly expressed at the nuclear level, while ERβ2 was mainly localized in the cytoplasm (Fig. 1A). Specifically, of the 56 advanced serous ovarian cancers studied 11 (14.3%) lacked ERβ1 protein expression, the majority of tumors (59%) showing medium-level of ERβ1 nuclear staining (7.4 ± 0.5) without cytoplasmic immunoreactivity; when present, cytoplasmic immunopositivity was of low intensity. Only three out of 56 specimens (5%) lacked ERβ2 expression; medium-level of nuclear staining (8 ± 0.5) was observed in cancers, with about 45% of patients showing an associated cytoplasmic reaction of medium intensity. ERβ5 was expressed by all the primary cancer studied: immunopositivity was primarily nuclear (10 ± 0.3), with about 40% of patients also showing an associated low cytoplasmic reaction (Fig. 1B–D). Table 2 shows ERβ isoform status in advanced serous ovarian cancer patients according to clinicopathological parameters. Cytoplasmic ERβ2 expression (IRS N 2) was more frequent in chemoresistant patients (p = 0.03). Cytoplasmic ERβ1 was also found to be significantly associated with residual tumor extent (p = 0.04). No correlation was demonstrated between nuclear ERβ isoform expression and any of the parameters examined.
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Fig. 1. A) Representative pictures for ERβ1, ERβ2, and ERβ5 immunostaining performed in serial sections from the same patient and showing both nuclear and cytoplasmic protein expressions in advanced serous ovarian cancer. B) Bar chart showing the distribution of negative, nuclear, cytoplasmic, and nuclear/cytoplasmic ERβ isoform staining in our cohort of patients. C) and D) Bar chart showing, for each isoform, the immunoreactive receptor score (IRS) (mean ± SEM) in nuclear and cytoplasmic compartments. *p b 0.05, ***p b 0.0001.
Association between immunohistochemical results and survival data The prognostic role of ERβ isoform expression for both DFS and OS was tested in univariate and multivariate analyses adjusted for clinicopathological parameters. Median DFS for the study population was 15 months. In univariate analysis, cytoplasmic ERβ2 expression was the only molecular marker significantly associated with a shorter DFS (median of 14 months
compared to 23 months in negative patients, p = 0.04) (Fig. 2C, Table 3A). The 5-year recurrence rate was nearly 88% for patients who did express cytoplasmic ERβ2, and 65% in negative patients. The prognostic role of age at diagnosis, histological grade, and residual tumor was also tested in univariate analyses. The presence of any residual tumor at primary surgery was found to be associated with a higher risk of recurrence of disease (Table 3A). In the multivariate Cox regression analysis after controlling for volume of residual tumor, the
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Table 2 Expression of estrogen receptor beta isoforms in the overall series. Characteristics Age ≤54 N54 Grade G1–2 G3 Residual tumor 0 mm N0 mm Chemosensitivity Sensitive Resistant
No. of patients (%)
Nuclear ERβ1+ %
Cytoplasmic ERβ1+ %
29 (51.8) 27 (48.2)
82.7 74.0
27.6 26.0
12 (21.4) 44 (78.6)
83.3 77.3
35 (62.5) 21 (37.5) 40 (71.4) 16 (28.6)
Nuclear ERβ2+ %
Cytoplasmic ERβ2+ %
Nuclear ERβ5+ %
Cytoplasmic ERβ5+ %
93.1 96.3
51.7 37.0
100 100
41.4 37.0
41.7 22.8
100 93.2
41.7 45.5
100 100
33.3 40.9
80.0 76.2
14.3 47.6⁎
91.4 100
37.1 57.1
100 100
31.4 52.3
72.5 93.8
22.5 37.5
95.0 93.8
35.0 68.8⁎
100 100
37.5 43.8
+ = positive cases. ⁎ p b 0.05, Fisher's exact test.
association between cytoplasmic ERβ2 expression and earlier disease recurrence did not achieve statistical significance (HR 1.6; 95% CI 0.9–2.9, p = 0.1, Table 3A). Median OS for the study population was 47 months. In univariate survival analysis, cytoplasmic ERβ2 expression was again the only molecular marker significantly associated with a shorter OS (p = 0.0006). The median OS had not been reached at the time of the analysis in patients with tumors that exhibited no cytoplasmic ERβ2 expression, compared to 33 months in tumors with positive cytoplasmic immunoreaction (Fig. 2D, Table 3B). The 5-year survival rate was nearly 28% for patients who did express cytoplasmic ERβ2, and 60% in negative patients. The prognostic role of age at diagnosis, histological grade, and residual tumor was also tested in univariate analyses: volume of residual tumor was the only variable associated with a shorter OS (p = 0.005, Table 3B). In the multivariate Cox regression analysis after controlling for volume of residual tumor, cytoplasmic ERβ2 expression retained its significance as independent poor prognostic factors (HR 2.8; 95% CI 1.3–5.7, p = 0.006, Table 3B). We were then prompted at analyzing the prognostic relevance of cytoplasmic ERβ2 expression in platinum-sensitive ovarian cancer patients (n = 40), and, interestingly enough, its unfavorable role was maintained in this subset: cases with cytoplasmic ERβ2 expression had a shorter OS (median OS = 53 months) than cases with no cytoplasmic ERβ2 expression (median OS undefined) (p = 0.004). The 5-year survival rate was nearly 43% for patients who did express cytoplasmic ERβ2, and 72% in negative patients. Multivariate Cox analysis in platinum-sensitive patients showed that cytoplasmic ERβ2 maintained its significance as negative prognostic factor, after controlling for volume of residual tumor (HR 2.8; 95% CI 1.0–7.6, p = 0.04).
Assessment of ERβ isoform expression in ovarian cancer cell lines and xenografts To determine whether ERβ isoforms are expressed in human ovarian cancer cells, we analyzed 3 human ovarian cancer cell lines: OVCAR-3, SKOV-3 and A2780. Immunocytochemistry confirmed the expression of ERβ1, ERβ2 and ERβ5 in the tested ovarian cancer cells and, in keeping with clinical data, both nuclear and cytoplasmic expressions were observed (Fig. 3). The retention of this receptor status during in vivo tumor growth was then verified in intact female mice. We found that ovarian cancer xenografts were all positive for the three isoforms, although showing different levels and subcellular distributions. ERβ1 exhibited a prominent cytoplasmic localization, while ERβ2 and ERβ5 were mainly nuclear, being ERβ5 the isoform predominantly expressed (Fig. 3). To verify the possible modulation of hormone receptor expression by endogenous estrogen, we ovariectomized an additional group of female and assessed the pattern of ERβ1, ERβ2 and ERβ5 expression in A2780 grown subcutaneously in these mice.
Notably, we found that when compared to intact female, tumors from OVX mice showed higher levels of cytoplasmic ERβ2 staining (Fig. 3), this suggesting that estrogen removal could induce a shift in the cellular compartment-specific distribution for this isoform. On the other hand, no relevant differences were observed between intact and OVX females in ERβ1 and ERβ5 expression pattern.
Discussion This is the first study to analyze the subcellular expression of wt ERβ and its different isoforms (ERβ2 and ERβ5) in a well-validated cohort of advanced serous ovarian cancer with complete follow-up data, using specific well-validated antibodies. The immunohistochemical expression of the three ERβs was analyzed in our cohort and staining revealed unique cellular compartment-specific distribution for each isoform in malignant ovarian tissue. Both nuclear staining and cytoplasmic staining were observed for all three isoforms in advanced serous ovarian cancers, being ERβ5 the most represented, a finding also reported by other authors examining both ovarian carcinoma cell lines, and ovarian carcinoma tissues [9]. One of the striking observations of this study was the strong statistical association of cytoplasmic ERβ2 immunoreactivity with reduced survival, a finding also reported in familial and sporadic breast cancer [15,17]. The functional role of cytoplasmic ERβ (and specifically of cytoplasmic ERβ2) is far from being elucidated. There is ample evidence to support nongenomic functions of ERβ in various cell types. Indeed, nongenomic signaling properties has been demonstrated for ERβ in different cancer cell lines, a mechanism implying a cross-communication (in a cooperative fashion) with transmembrane growth factor receptor signaling pathways that ultimately promote downstream signaling for tumor cell proliferation and survival [28,29]. Moreover, a mitochondrial localization of ERβ has been demonstrated in many cell types, by immunocytochemistry, immunohistochemistry, and immunoblots (using a large group of diversified antibodies) and also verified by proteomics [30]. In parallel, mechanistic studies have been carried out to unravel the possible role of mitochondrial ERβ protein. Zhang and colleagues [31] recently reported a ligand-independent cell-survival function of mitochondrial ERβ in lung cancer cells. Specifically they propose that in the absence of ERβ, Bad is able to interact with Bcl-XL (and Bcl-2) to free Bax to oligomerize and initiate apoptosis, whereas in the presence of ERβ Bad is sequestered and prevented from interaction with Bcl-XL, thus inhibiting apoptosis. As well, there has been increasing evidence pointing to the mitochondrial respiratory chain (MRC) as a novel and important target for the ligand-dependent actions of both ERα and ERβ in a number of cell types and tissues, with studies supporting the notion that by up-regulating the MRC biogenesis and functions, E2 and ERs contribute to inhibition of apoptosis, causing the normal balance of cell growth/cell death toward survival and enhanced
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Fig. 2. A) and B) Representative pictures for ERβ2 immunostaining showing nuclear/cytoplasmic (A) and only nuclear (B) expressions in ovarian cancer tissues. C) and D) Kaplan–Meier survival curve for the probability of disease-free survival (C) and overall survival (D) according to expression of cytoplasmic ERβ2 in advanced serous ovarian cancer patients. Any expression of ERβ2 in the cytoplasm is significantly associated with disease-free survival and overall survival disadvantage (p = 0.04 and p = 0.0006, respectively).
Table 3A Univariate and multivariate analyses of factors affecting DFS in advanced ovarian cancer. Variables
Residual tumor 0 mm N0 mm ERβ2 expression (subcellular localization) Only nuclear expression Cytoplasmic expression
Univariate
Multivariate
HR (95% CI)
p
HR (95% CI)
p⁎
2.9 (1.4–5.9)
0.003
2.2 (1.2–4.2)
0.01
1.9 (1.0–3.6)
0.04
1.6 (0.9–2.9)
0.1
DFS = disease-free survival. HR = hazard ratio. CI = confidence interval. Only variables with p-value b 0.2 in the univariate analysis were included in multivariate model. χ2 of the model = 9.8; p value = 0.007. ⁎ ps were derived from the COX proportional hazards model.
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Table 3B Univariate and multivariate analyses of factors affecting OS in advanced ovarian cancer. Variables
Residual tumor 0 mm N0 mm ERβ2 expression (subcellular localization) Only nuclear expression Cytoplasmic expression
Univariate
Multivariate
HR (95% CI)
p
HR (95% CI)
p⁎
2.8 (1.3–5.9)
0.005
2.0 (1.0–4.0)
0.10
3.5 (1.7–7.1)
0.0006
2.8 (1.3–5.7)
0.006
OS = overall survival. HR = hazard ratio. CI = confidence interval. Only variables with p-value b 0.2 in the univariate analysis were included in multivariate model. χ2 of the model = 15.1; p value = 0.0005. ⁎ ps were derived from the COX proportional hazards model.
cell growth [30,32]. Finally, there are data supporting the notion that mitochondrial ERβ functions as a mitochondrial vulnerability factor involved in Ψm maintenance, potentially through a mitochondrial transcription dependent mechanism [33]. Such data also support a potential bi-faceted role of mitochondrial ERβ proteins in apoptosis. In
this context, it is worthy to note that we also found a statistically significant association between cytoplasmic ERβ2 immunoreactivity and chemoresistance. The molecular mechanisms of tumor resistance to chemotherapeutic agents such as cisplatin and paclitaxel involve multiple factors, including reduced drug uptake or increased drug
Fig. 3. A) Expression of ERβ1, ERβ2 and ERβ5 in OVCAR-3, SKOV-3 and A2780 human ovarian cancer cells using immunocytochemical analysis. Pictures show nuclear and cytoplasmic expressions of antibodies (magnification 40×). B) Expression of ERβ1, ERβ2 and ERβ5 in OVCAR-3, SKOV-3 and A2780 subcutaneous xenografts in intact athymic female mice. ERβ1 exhibited a prominent cytoplasmic localization after in vivo growth, while ERβ2 and ERβ5 were mainly expressed in the nucleus. Compared to intact female, A2780 tumor grown in OVX mice showed higher levels of cytoplasmic ERβ2 staining, while no differences were observed for the remaining isoforms (magnification 40×).
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efflux, reduced DNA binding, increased DNA repair, and impaired apoptosis [34,35]. The mitochondrial anti-apoptotic action of ERβ might contribute toward chemotherapy resistance in ovarian cancer. Few studies have examined the prognostic role of ERβ in ovarian cancer [reviewed in 36]. Chan and colleagues [37] reported that a higher level of ERβ mRNA expression was significantly associated with longer DFS and OS, while a recent study suggested that tumor nuclear ERβ1 level did not affect progression-free and cause-specific survival, although influencing overall survival [24]. These previous results are barely comparable to present data since they either refer to mRNA level (that is, a measurement including non-tumor cells and not taking into account translational control or turnover of protein), they assess a specific ERβ isoform (i.e. ERβ1), or do not consider cytoplasmic protein staining. Besides, both studies also considered low-stage disease and thus there may be sub-groups in which the amount of nuclear ERβ protein is prognostic. Notably, we also showed a reasonable concordance in ERβ isoform immunoreactivity between clinical samples, ovarian cancer cell lines and ovarian cancer xenografts. Indeed, we found that in different ovarian cancer cell lines grown in vivo as subcutaneous tumor xenografts the overall expression of ERβ1, ERβ2 and ERβ5, observed both in the nuclear and cytoplasmic compartments, resembled the pattern of expression observed in our series of advanced ovarian cancer. These preliminary pre-clinical experiments also suggest a role for endogenous estrogen in modulating ERβ2 compartmentalization, since we observed that A2780 tumors from ovariectomized mice showed a higher cytoplasmic ERβ2 staining when compared to intact females. Thus, we could speculate that in menopausal women, the abrupt decrease in ovarian estrogen might, through the modulation of ERβ2 status, confer a more aggressive phenotype and ultimately contribute to ovarian cancer development. However, additional preclinical experimental models need to be used to confirm this hypothesis, particularly in the light of very recent data published by Domcke and colleagues [38], relative to the pronounced differences in molecular profiles between commonly used ovarian cancer cell lines and highgrade serous ovarian cancer tumor samples. These future in vivo preclinical studies should also address the possible effect of estrogen modulation on ER status and ovarian cancer growth in models that more accurately mimic the feature behavior of human ovarian cancer, such as the intraperitoneal xenografts. We recognize that our cohort is small (56 patients), however, the possibility of using ERβ2 for advanced serous ovarian cancer prognosis shows great promise. Indeed, if anomalies of ERβ2 cytoplasmic expression could be demonstrated to represent an independent unfavorable prognostic marker and/or a marker predicting chemoresistance in advanced serous ovarian cancer, its immunohistochemical assessment at the time of surgery, could help to recognize candidates for clinical trials of new interventions. Meanwhile, additional investigations are warranted to identify the mechanism responsible for cytosolic localization of endogenous ERβ, and the specific contribution of each ERβ isoform in relation to their differential subcellular distribution within the target cells, a condition that may underlie altered function of ERβ as well as its variants. Definitely, membrane, cytoplasmic and nuclear estrogen receptors are not separate units, but rather the components of a complex mechanism in which they both cooperate with each other. Conflict of interest statement The authors declare no conflict of interest.
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