Estrogen receptor β is associated with expression of cancer associated genes and survival in ovarian cancer

Ovarian cancer is the leading cause of death from a gynecological malignancy in the developed world [1]. Ovarian cancers are influenced by steroid hormones. Antiestrogenic treatment inhibits growth of ovarian cancer in vitro and in vivo [2‐4]. Progesterone receptor (PR) and ER expression are reported to be associated with improved ovarian cancer survival, independent of clinical prognostic factors, but these associations have not been consistently repeated [5, 6]. In the clinical setting antiestrogens are commonly used in patients with relapsed ovarian cancers after multiple lines of cytostatic therapies that have exhausted further treatment. Available data remains inconclusive, mostly due to heterogeneity of ovarian cancers and inadequate study settings as low number of included patients, missing subgroup analyses and absent evaluation of ER expression. However, use of tamoxifen and aromatase inhibitors have effects in a certain subgroup of patients [7, 8].

Previous studies clearly suggest a tumor suppressive role of ERβ in ovarian cancer as it has been shown for breast or prostate cancer [9‐11]. Our group demonstrated that ERβ reduces proliferation and migration, but activates apoptosis of ovarian cancer cells [12] and that specific ERβ-agonists significantly inhibit growth of different ovarian cancer cell lines [13]. Furthermore, our results from a phenotype-genotype association study suggested that the single nucleotide polymorphism rs3020449 in the promoter region of ESR2 gene might affect progression of ovarian cancer [14].

ERβ is the predominant ER in normal ovarian tissue. In ovarian cancer the expression of ERβ is significantly lower and the ERα/ERβ ratio is significantly higher than in normal ovarian tissue [15‐17]. High expression of ERβ in ovarian cancers is associated with a better progression-free and overall survival [15, 17].

Ovarian cancer marker cancer antigen 125 (CA125) is overexpressed in the majority of ovarian cancers and it has been shown to be involved in the metastatic process [18]. CA125 regulates cell adhesion by interacting with mesothelin, galectin-1, E-cadherin and β-catenin [18, 19]. Moreover, CA125 can promote proliferation and migration [20]. Also for the broad spectrum tumor marker carcinoembryonic antigen (CEA) a regulative role of cell adhesion and thus an influence on metastasis of cancer cells has been suggested [21]. Another antigen selectively expressed in ovarian cancer is CA72–4. As it is highly detectable in all ovarian cancer subtypes it is another effective tumor marker in ovarian cancer [22]. However, its function in carcinogenesis of ovarian cancer is still unknown.

Recently, it has been reported that a shuffle of ERβ between nucleus and cytoplasm plays important roles in regulation of gene transcription, RNA maturation and post-transcriptional control [23]. To further approach the significance of subcellular ERβ localization in ovarian cancer, we examined co-expression of nuclear and cytoplasmic ERβ with various cancer-associated genes and steroid hormone receptors and tested to what extent receptor localization would affect survival of ovarian cancer patients.

Several studies showed a wide range of expression levels of steroid hormone receptors in ovarian cancer [28‐30]. In our study including 171 ovarian cancer cases, 70.35% of all cases were ERα positive. This is in line with the data of Lee et al. who found this receptor to be expressed in 77.3% of all cases [30]. In other studies, ERα was only expressed in 31.4–36% of ovarian cancer specimens [28, 29]. These differences might be due to different applied ERα antibodies detecting a specific set of splice variants. Investigating ERβ expression reveals even more difficulties as specific antibodies have not been available for a long time. Moreover, it is important to distinguish between nuclear and cytoplasmic ERβ expression as their role in carcinogenesis differs [31, 32]. In our study, nuclear ERβ was expressed in 49.62% and cytoplasmic ERβ in only 20.61% of all cases. Other studies showed rates of positive ERβ expression between 48 and 60% [28, 33]. In the study of De Stefano et al. including 58 serous ovarian cancers nuclear ERβ was expressed in 89.66% and cytoplasmic ERβ was expressed in 77.59% [17].

As expression of ERβ declines during tumorigenesis of breast, colon and prostate cancer, this receptor has been proposed to act as a tumor suppressor [9‐11, 34]. ERβ expression is highest in normal ovarian tissue whereas it decreases during dedifferentiation processes [16]. This is in line with our data showing a significantly lower expression of nuclear ERβ in G3 ovarian cancers than in better differentiated tumors. In vitro, we showed reduced proliferation and migration of ovarian cancer cells after overexpression of ERβ as well as increased rates of apoptosis [12]. Moreover, we found a significant growth-inhibition of ovarian cancer cells by treatment with specific ERβ-agonists [13]. Thus, these data support the tumor suppressive role of ERβ in ovarian cancer.

In our study PR expression was significantly higher in early stage ovarian cancers than in cancers with FIGO stages III and IV (p = 0.0027). Moreover, we showed a significantly higher expression of PR in tumors without invasion of lymphatic vessels compared to cancers that did not invade lymphatic vessels. In line with data published earlier, progression-free survival of patients with PR-expressing tumors was significantly longer compared to those with tumors not expressing PR [5, 28, 35]. This could be explained by an induction of apoptosis by PR activation in ovarian cancer [5]. Multiple in vitro studies have shown that increased PR expression could promote the progesterone-induced inhibition of DNA synthesis, cell division and proliferation in ovarian cancer cells [35, 36]. Moreover, PR is transactivated by ERα and PR expression may be a biomarker of improved prognosis because it indicates a functionally intact ER pathway and less aggressive tumor behavior [5].

With regard to overall survival and progression-free survival, data from literature remain inconclusive. In our study, patients with tumors expressing cytoplasmic ERβ had a significant benefit in overall survival compared to those with tumors not expressing this form of ERβ. A recent study revealed that ERβ-positive nuclear staining was associated with decreased progression-free survival (hazard ratio (HR) 1.69; 95% CI 0.91–3.15; p = 0.096) and decreased overall survival (HR 1.91; 95% CI 0.94–3.89; p = 0.075) [37]. In the study by De Stefano et al. expression of cytoplasmic ERβ predicted poor clinical outcome in serous ovarian cancer. However, the study was smaller with 58 included ovarian cancer cases and only serous cases were included [17].

CA125 is overexpressed in the majority of serous ovarian cancers, the most common histological subtype [18]. We found a high correlation between the expression of ERα and the expression of CA125, which is in line with the findings of Sylvia et al. [38]. CA125 interacts with cell-adhesion regulators mesothelin, galectin-1 and E-cadherin and β-catenin [18, 19] and is involved in promotion of proliferation and migration [20]. Park et al. found an ERα-dependent, estrogen-induced suppression of expression and promoter activity of E-cadherin in ovarian cancer cells, whereas epithelial-mesenchymal transition-associated transcription factors, Snail and Slug, were significantly up-regulated [39]. Thus, this possible interaction between CA125 and ERα in regulation of cell adhesion should be elucidated in future studies. Also CEA has a regulative function in cell adhesion and thus an influence on metastasis of cancer cells has been suggested [21]. Another antigen selectively expressed in ovarian cancer is CA72–4. As it is highly detectable in all ovarian cancer subtypes it is another effective tumor marker in ovarian cancer [22]. However, its function in carcinogenesis of ovarian cancer is still unknown. We were able to show a high correlation between the expression of nuclear ERβ and CEA as well as CA72–4. Moreover, we observed a significant correlation between expression of ERα and PR (Additional file 2), which is in line with studies published earlier [28, 40].

As discussed above, the role of ERβ as a tumor suppressor in ovarian cancers is still controversial. Our data and those published by others clearly suggest a contradictory role of nuclear and cytoplasmic ERβ. Our results demonstrating an improvement of overall survival for patients with ovarian cancers expressing cytoplasmic ERβ corroborate a tumor suppressive role of ERβ. In contrast, nuclear expression of this receptor previously has been reported to shorten progression-free survival [37]. This is in accordance with our finding that expression of nuclear ERβ is associated with tumor markers CEA and CA72–4. Different binding partners depending on the subcellular location of ERβ might explain these effects [23]. Our data suggest that future studies should further assess the different roles of nuclear and cytoplasmic ERβ in ovarian cancer as previous controversial results might be due to a distinct function of ERβ depending on its location.

To our knowledge, we are the first to show a correlation between nuclear ERβ and CEA as well as CA72–4 expression in ovarian cancer. Moreover, our data support the role of ERβ as a tumor suppressor in ovarian cancer, as our survival analyses show a significant benefit in overall survival of patients with tumors expressing cytoplasmic ERβ. Further studies will be necessary to examine the relevance of our data in the clinical setting.