Background
Breast cancer is the most common female cancer (30.8% of all newly diagnosed cancers in women in Germany, 2012) and also the most common cause of cancer-related death in women [
1]. The development of treatment from conventional to targeted anti-tumor therapies, e. g. human epidermal growth factor receptor 2 (HER2) blockade, has contributed much to improve breast cancer therapy [
2]. However, as these therapies are not suitable for all patients, the search for new, individualized and specific alternatives of treatment is ongoing.
Eicosanoides, which include leukotriens and prostaglandins (PG), are tissue hormones that can act in an autocrine or paracrine manner. They contribute to uncountable physiological and pathological processes: The balance of thromboxane A2 and prostacyclin e. g. is essential for hemostasis, whereas PGE
2 is the most abundant prostaglandin in humans and is known as a key mediator in inflammation. Cyclooxygenase (COX) enzymes are the primary enzymes in the synthesis of eicosanoides and exist in two isoforms: COX-1 is considered to be ubiquitously expressed, whereas COX-2 is normally absent from most human tissues. COX-2 upregulation can be elicited by various cytokines and growth factors and is involved in the regulation of the inflammatory response. Both COX-enzymes catalyze the conversion of arachidonic acid to PGG
2 and subsequently to PGH
2, which itself is the precursor molecule for the synthesis of the different eicosanoids, including PGE
2, prostacyclin or thromboxane A
2 [
3,
4].
Besides its known essential function in physiological processes and inflammation, PGE
2 plays an important role in tumorigenesis: In various cancer samples, elevated PGE
2 levels have been found. Similarly, constitutive COX-2 overexpression - which is thought to be responsible for the increase in PGE
2 levels - has been detected in various tumors [
5]. Both increased PGE
2 and COX-2 overexpression have been associated with tumorigenesis and progression [
3,
6]. Therefore, the role of prostaglandins and their mediators in tumorigenesis and their potential as possible therapeutic targets have come into focus of recent research [
4,
7].
In breast cancer, COX-2 overexpression is found in about 40% of all cases, it is inversely associated with survival rates and is positively associated with various characteristics of aggressive disease [
3,
6]. Studies in murine and human breast cancer models have shown the influence of COX-2 overexpression on breast tumor development, e. g. via the mechanisms of inducing angiogenesis or improving cell mobility and invasiveness [
8,
9]. Experimental blockade of COX-2 in a mouse model has reduced tumor progression [
10]. Similarly, COX-2 antagonist (COXib) treatment in humans has shown an impressive preventive effect with a significant reduction in the risk of breast cancer in a case control study [
11]. These strikingly beneficial effects of inhibiting COX-2 show the potential of the COX-2-PGE
2-axis in breast cancer prevention and therapy. Unfortunately, the use of COXibs in clinical practice is limited due to their strong cardiovascular toxicity (thrombosis, atherogenesis, hypertension). The side effects are thought to be caused by the selective depression of prostacyclin synthesis (via COX-2 inhibition), whereas thromboxane A
2 synthesis via COX-1 is not influenced and in consequence, the balance between pro- and antithrombotic agents is disturbed [
3]. Therefore, elucidating the role of other components involved in the exertion of PGE
2-effects in search for targeted breast cancer therapies with lesser side effects has come into focus of research.
PGE
2 mediates its effects via four G-protein coupled receptors, the EP receptors 1–4 with different intracellular signaling pathways [
4,
12]. EP2 and EP4 receptor are coupled to Gs-protein/protein kinase A/adenylate cyclase and induce intracellular elevation of cyclic adenosine monophosphate (cAMP). EP1 receptor is associated to Gq-protein and mediates elevation of intracellular calcium and phospholipase C activation [
7,
12]. As a remarkable feature, EP3 (Prostaglandin E2 receptor 3) exists in multiple isoforms (generated by alternative mRNA splicing); eight different isoforms are identified in humans and three isoforms in mice signaling via different G-proteins. Most human EP3 isoforms inhibit cAMP generation via Gi-protein (on the contrary to EP2 and EP4 which increase cAMP levels), some isoforms can also increase intracellular calcium like EP1, some might also signal via Gs proteins [
4].
EP1–4 receptor expression has been shown in a variety of cancers, including breast cancer [
13‐
15]. Exploiting the role of EP receptors in breast cancer tumorigenesis, it has been found that in COX-2-induced murine mammary tumors, EP1, 2 and 4 are strongly induced compared to normal mammary gland tissue, whereas EP3 receptor expression is rather downregulated, but still detectable [
8]. However, concerning the prognostic relevance of EP receptor expression and the effects of receptor blockade or stimulation on breast cancer development and the course of clinical disease, studies partly show different effects (reviewed in [
4,
7] and [
16]). Most data is available concerning EP2 and EP4, whose elevated expression in mammary tumor cells is mostly associated with enhanced metastasis, tumor cell proliferation and tumor invasiveness [
13,
14,
17]. Limited data is available concerning EP1 in breast cancer: It has been associated with tumor development [
7,
18], but another study associated EP1 expression with suppression of metastasis while it had no effect on the primary tumor and EP1 positive patients had improved survival [
19]. Therefore, EP1 might have a pro-tumorigenic effect on the primary tumor but might also have anti-metastatic effects in breast cancer [
19]. Of the EP receptors, EP3 is the least well-understood in breast cancer with both tumor-promoting and suppressive effects having been published [
4].
As EP3 seems to have a different role than the other EP receptors – EP3 is downregulated in tumor cells and shows rather inhibitory signaling mechanisms – it is suggesting that EP3 might have a protective role in mammary tumor development and its expression on cancer cells might be associated with a more favorable course of the disease. This makes EP3 an interesting target to analyze and might also open the possibility to target EP3 in future specific cancer therapy.
To our knowledge, no sufficient data exists concerning prognostic relevance of EP3 in sporadic breast cancer. Therefore, we performed this study to evaluate the expression of EP3 receptor in sporadic breast cancer and its association with clinicopathological parameters (tumor size, lymph node status, focality, grading, hormone receptors, HER2-amplification, age), progression and survival.
Discussion
Data from literature show COX-2 overexpression in breast cancer resulting in elevated PGE
2 synthesis which is thought to contribute to disease progression. Recent studies have evaluated the mechanisms through which PGE
2 exerts its effects in tumorigenesis and have shown that the expression of PGE
2 receptors EP1–4 is modified in different kinds of cancer. EP2 and EP4 expression is rather associated with an unfavorable outcome, whereas data regarding the role of the other EP receptors EP1 and EP3, especially in breast cancer, is still sparse [
4]. However, as EP3 has the unique feature that it mainly signals via an inhibitory pathway (EP2 and EP4 on the contrary activate a stimulatory pathway), its role in breast cancer and its eligibility as a possible therapeutic target should not be neglected.
This study was performed to analyze the prognostic relevance of EP3 receptor expression in sporadic breast cancer and its association with clinicopathological tumor characteristics (e. g. tumor size, lymph node status, hormone receptors, histology).
We have confirmed that in the majority of sporadic breast cancer cases, EP3 receptor was expressed like it was shown e. g. for different inflammatory breast cancer cell lines [
21]. EP3 expression occurred in all histological subtypes of breast cancer and the expression did not differ between the histological subtypes. Therefore, targeting EP3 diagnostically or therapeutically seems generally possible and could be applied to any histological breast cancer subtype. However, EP3 expression was not compared between healthy tissue and tumor – published studies have shown a downregulation of EP3 in breast cancer compared to healthy breast tissue [
8], in colon cancer [
22] and in prostate cancer [
23].
EP3 receptor expression was independent of other clinicopathological factors (named in Table
1) which partly have known prognostic relevance (like e.g. ER-positivity). COX-2 overexpression in breast cancer on the contrary is mostly associated with clinicopathological factors characteristic for an aggressive phenotype, like large tumor size, negative hormone receptor status or high proliferation [
6,
24]. Other studies, however, did not show an association of COX-2 overexpression with clinicopathological parameters [
25,
26].
Interestingly, EP3 receptor expression was not associated with an unfavorable course of the disease, like it is known e. g. from EP2 and EP4 [
13,
14,
17,
27]. On the contrary, instead of a negative influence of EP3, an association of EP3 with improved survival and improved progression-free survival could be shown. EP3 was even a significant prognostic factor when other factors with known prognostic influence were accounted for (Table
3). To our knowledge, this is the first report of EP3 as a prognosticator for survival or progression-free survival in breast cancer.
Data concerning EP3 in breast cancer is sparse. EP3 is described as irrelevant in one study, where treatment of breast cancer cells with EP3 antagonists had no effect on metastasis [
15]. Another study in inflammatory breast cancer, however, showed a beneficial effect of EP3, as treatment of inflammatory breast cancer cells with the EP3 agonist sulprostone reduced the ability of the tumor cells to undergo vasculogenic mimicry, a characteristic of very aggressive tumor types [
21].
Regarding the role of EP3 in other cancer types, both pro- and anti-tumorigenic effects have been described.
Some data suggest a pro-tumorigenic effect of EP3 receptor expression, as EP3 has been associated to angiogenesis and lymphangiogenesis: When Lewis lung carcinoma cells were injected in mice, tumor-associated angiogenesis, metastasis and tumor growth were reduced in EP3 knockout mice compared to wild type mice. Also, levels of vascular endothelial growth factor, matrix-metalloproteinase-9 and of podoplanin, a marker for lymphatic endothelial cells, were reduced in EP3 knockout mice and induced by EP3 agonists [
28‐
30].
Other studies, on the contrary, suggest an anti-tumorigenic effect of EP3 receptor expression. EP3 knockout e.g. enhanced azoxymethane-induced colon carcinogenesis [
22] and contributed to squamous cell carcinoma development [
31]. Similarly, EP3 knockdown in prostate cancer cells or treatment of prostate cancer cells with EP3 antagonists accelerated tumor cell growth [
32]. Consistent with this data, EP3 overexpression in prostate cancer cells impaired tumor growth in vitro and stimulation of cells overexpressing EP3 with the EP3 agonist sulprostone further enhanced the inhibitory effect [
23]. Therefore, the authors of these studies hypothesize that the reduction of EP3 expression during tumorigenesis might be consistent with tumor-suppressive properties of EP3.
Differences between the above named studies might be partly due to the different EP3 isoforms, as EP3 isoforms mainly signal via Gi proteins but partly also via the Ca
2+/phospholipase cascade or via Gs proteins [
4]. Therefore, different effects in studies concerning EP3 might be to some extent due to different expression patterns of the isoforms. In prostate cancer e.g., EP3 II, an EP3 isoform coupled to Gi protein, was the major isoform found and EP3-expression also showed inhibitory effects on tumor cell growth in this study [
23]. In mice, three EP3 isoforms exist and overexpression of all three variants has been associated with reduced tumor cell growth [
33]. For future studies in breast cancer it is therefore necessary to clarify which isoforms are expressed in which manner to better understand the effects and to facilitate comparing different studies.
In summary, our study has shown that in breast cancer, EP3 was a significant prognosticator for improved progression-free and overall survival without association of its expression to known clinicopathological parameters. This is contrary to part of the above named studies, where EP3 has shown negative effects and is also contrary to the negative effects of the other EP receptors (EP2, EP4) in breast cancer [
4]. The positive prognostic influence of EP3 in breast cancer is surprising insofar, as both COX-2 overexpression and PGE
2 elevation have been shown to have pro-tumorigenic effects in breast cancer, and EP3 is part of the signaling pathway of PGE
2 and is therefore mediating PGE
2 effects. As EP3 seems to have a beneficial effect in breast cancer, it is likely, that one or more of the functional effects of PGE
2 in tumorigenesis (proliferation, invasiveness, metastasis, anti-apoptotic effect) might be inhibited by EP3. Our future work will concentrate on identification of the factor that is regulated by EP3. By improving the understanding of the functional aspects of EP3 and its regulated factors, we aim to evaluate its eligibility as a possible future target in breast cancer prevention and treatment.