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Investigating the Roles and Mechanisms of Action of Estrogens via Estrogen Receptor ß in Granulosa Cell Tumours of the Ovary

Final Report Summary - ESTROVARYTUMOUR (Investigating the Roles and Mechanisms of Action of Estrogens via Estrogen Receptor ß in Granulosa Cell Tumours of the Ovary)

Summary description of project context and objectives

Granulosa cell tumours (hereafter named as GCT) which account for about 5% of ovarian tumours, are a rare form of malignancy affecting women of all ages, with two distinct clinical presentations, the adult and juvenile forms (1). Most juvenile cases are diagnosed early and their prognosis is generally good, though recurrences and metastases have been reported. However, in the adult cases of GCT, 80% of patients with advanced disease die of the consequences of their tumour (1)). The particularity of this disease is its high rate of recurrence, even when the diagnosis is established at an early stage. Besides, these tumours have a tendency to late recurrence, with latency after primary tumour treatment of up to 37 years. Chemotherapy has limited success, and surgery remains the main therapeutic approach.
Despite the importance and insidiousness of GCT, very little is known of its molecular etiology. Abnormal uterine bleeding and pain attributable to the large size of GCT are the most common presenting symptoms. Endometrial hyperplasia and adenocarcinoma are also frequent. The hormones secreted by GCT that are responsible for most of these clinical signs are the sex steroids estrogens, among which 17β-estradiol (E2) that is found elevated in ~70% of patients. Estrogens, which are mainly produced by the ovary upon conversion of androgens, are known to mediate important physiological responses by binding to nuclear estrogen receptors (ER), ERα and ERß. Estrogens play a key role in the regulation of ovarian function by stimulating granulosa cell proliferation, survival and maturation, thereby supporting the growth and differentiation of ovarian follicles (2). In addition to producing estrogens, GCT express ERs (3). Hence, one could hypothesize that these hormones could regulate granulosa cell tumour growth and /or progression.
However, the possible role of estrogens in the growth and metastatic potential of GCT has not been evaluated. Besides, how estrogens would mediate their molecular actions in GCT remained to be investigated.
Given the paucity of information about GCT pathogenesis, the aims of this project were 1) to investigate the action of estrogens in GCT growth and progression (i.e. the metastatic potential), 2) to uncover the signaling pathways of estrogens in these tumours, and 3) to identify the estrogen receptor involved. These major goals were achieved using in vitro culture systems and were published this year in Carcinogenesis (4). The last part of the project, which is still ongoing, consists in developing mouse models of GCT and to test on these models the effect of estrogens on GCT growth and progression to confirm our in vitro findings.

Description of the work performed since the beginning of the project and main results

To address this issues, cell-based and molecular studies were performed using two human ER-positive GCT-derived cell lines, i.e. COV434 and KGN cells established from a primary tumour and from a metastatic recurrence, respectively. Because these cells are cultured in charcoal-stripped serum medium that is depleted of steroids, cell-based studies to assess the effect of E2 can be performed in vitro by treating cells with this hormone.
Our cell-based studies revealed that estradiol does not alter GCT cell growth, but that it inhibits metastatic GCT cell migration and invasion in vitro ((4), please see Figure 1 of the attached article). This is in marked contrast with what is usually found in other gynaecological diseases wherein a tumour-promoting role of estrogens has been documented. Besides, we made the remarkable discovery that estrogens signal via non-genomic actions in GCT cells to rapidly and durably repress ERK1/2 activity in metastatic GCT cells (Figure 2 of the article). No alteration in MAP ERK kinase (MEK1/2) or other MAPK pathways is observed. Overall, our studies indicate that estrogens would specifically repress ERK1/2 pathway via non-genomic signaling to inhibit GCT metastasis spreading (Figures 2 and 3). Unexpectedly, by studying the possible ER involved in estrogen-induced ERK1/2 repression, we found that it involves none of the classical nuclear ERs, but a member of the seven-transmembrane G-protein coupled receptor (GPCR) family, GPR30 also named GPER1 (for G-protein coupled estrogen receptor) (Figures 4 and 5). Importantly, through collaboration with a clinical network, we found that about 90% of human GCTs express GPER, at early or advanced stage of progression (Figure 6). These findings are of particular interest as they suggest that estrogens would have a protective role on this form of ovarian cancer by acting through a novel non-genomic mode of action that has never been reported to date.
For the next step of the project, we are currently completing the development of murine models of GCTs in order to test in vivo the potential benefit of a GPER agonist. We have successfully obtained xenografts of the human metastatic cells used for our in vitro studies. We are now optimizing this system to allow in vivo imaging in order to follow the effect of the treatment on the spreading of metastatic cells. The other animal model consists in a transgenic mouse line developing GCT and metastases in adulthood (5). We are further implementing this mouse model by analysing the ontogenesis of GCT development and the disruption of E2 levels in the course of the disease.

Expected final results and their potential impact and use

Research on GCT is under-represented worldwide. Therefore, little information is available regarding the molecular etiology of this cancer. Patients and clinicians have to face with the lack of efficient curative treatment beyond surgery, and recurrence yields poor prognosis. This project addressed for the first time the role and mechanisms of action of estrogens in this rare but severe form of ovarian cancer.
Because a role for E2 in GCT pathogenesis had long been suspected but never investigated, this work brings important understanding regarding the impact of this hormone in this pathology. In the near future with the completion of in vivo studies, our project should bring valuable information that, we hope, could improve the clinical management of metastatic GCT.
Importantly, in contrast with other gynaecological cancers, in this particular form of ovarian cancer, E2 may well prevent metastasis spreading. These novel findings were unexpected because the carcinogenic effect of excessive estrogen stimulation in females has been largely documented. Demonstrating the protective role of estrogens in this disease is a major breakthrough discovery for cancer research that would possibly improve the clinical management of this disease, since a number of estradiol agonists already safely used on animals are available. One may thus consider the possibility of using GPER selective agonists for the chronic treatment of patients as soon as the disease is diagnosed to prevent relapse in subsequent years. This strategy appears relevant since the usual deleterious side-effects of estrogen therapies on E2-target tissues that express ERalpha and/or ERbeta would be avoided.

References
1. Pectasides D, Pectasides E, Psyrri A. Granulosa cell tumor of the ovary. Cancer Treat. Rev. 2008;34(1):1–12.
2. Couse JF, Yates MM, Deroo BJ, Korach KS. Estrogen receptor-beta is critical to granulosa cell differentiation and the ovulatory response to gonadotropins. Endocrinology 2005;146(8):3247–3262.
3. Chu S, Nishi Y, Yanase T, Nawata H, Fuller PJ. Transrepression of estrogen receptor beta signaling by nuclear factor-kappab in ovarian granulosa cells. Mol. Endocrinol. Baltim. Md 2004;18(8):1919–1928.
4. François CM, Wargnier R, Petit F, Goulvent T, Rimokh R, Treilleux I, Ray-Coquard I, Zazzu V, Cohen-Tannoudji J, Guigon CJ. 17β-estradiol inhibits spreading of metastatic cells from granulosa cell tumors through a non-genomic mechanism involving GPER1. Carcinogenesis 2015;36(5):564–573.
5. Dutertre M, Gouédard L, Xavier F, Long WQ, di Clemente N, Picard JY, Rey R. Ovarian granulosa cell tumors express a functional membrane receptor for anti-Müllerian hormone in transgenic mice. Endocrinology 2001;142(9):4040–4046.

Project website: http://cvscience.aviesan.fr/cv/1203/celine-guigon