Abstract 1318: 17β-estradiol inhibits metastatic granulosa cell migration by repressing ERK1/2 pathway via an extra-nuclear mechanism of action.

Abstract

Abstract Granulosa cell tumor (GCT) is a rare but severe form of ovarian tumor that is characterized by its tendency to recur years after surgical ablation. Because there is no efficient curative treatment beyond surgery, ∼20% of adult patients die of the consequences of their tumor. However, very little is known of the molecular etiology of this pathology. Although GCTs express the estrogen receptors (ER) ERα and ERβ, the effect of 17β-estradiol (E2) in disease progression remains elusive. The purpose of our study was to evaluate the potential role of E2 in GCT pathogenesis and to determine its underlying mechanisms of actions. To address this issue, molecular studies were performed using two human ER-positive GCT-derived cell lines, i.e., COV434 and KGN cells established from a primary tumor and from a metastatic recurrence, respectively. In both cell lines, E2 treatment (1 to 100 nM) up to 72 hours did not affect cell growth, as assessed by MTT assay and automatic cell counting. Importantly, horizontal migration assay showed that a 24-hour E2 treatment (10 nM) significantly inhibited KGN cell migration by ∼25%, in terms of migration distance and migrating cells. In contrast, no effect was observed in the primary tumor-derived COV434 cell line. Western blot studies revealed that in KGN, but not in COV434 cells, E2 treatment (10 nM) led to a rapid (as early as 5 minutes) and durable (up to 24 hours) decrease by ∼30% in the phosphorylation levels of ERK1/2, while it had no effect on ERK1/2 abundance. This was accompanied by the down-regulation of the expression of myosin light chain-2 (MLC-2), which is involved in stress fiber contraction during cell migration. ERK1/2 inhibition by the MEK1/2 inhibitor U0126 mimicked E2 actions, suggesting that the effect of E2 in KGN cells mostly results from the alteration of ERK1/2 pathway. To identify the ER involved in E2 action in KGN cells, a pharmacological approach with ER agonists was used. As none of the ER agonists could mimic E2 action, we investigated the possibility that the G protein coupled estrogen receptor (GPER or GPR30), which belongs to the G protein coupled receptor family, was involved. GPER expression was confirmed by RT-PCR and Western blot assays. Treatment of KGN cells with the GPER agonist G-1 (50 nM) rapidly and durably inhibited ERK1/2 pathway and cell migration. Pre-treatment with the GPER antagonist G15 (200 nM) completely prevented the E2-induced repression of ERK1/2 activity and inhibition of cell migration. Altogether, our study reveals that E2 would prevent metastasis spreading by inhibiting ERK1/2 activity to down-regulate MLC2 expression through a GPER-dependent mechanism. It, thus, brings evidence that extra-nuclear actions of E2 can be inhibitory to cancer pathways. Further, our observations may have translational implications, as they suggest the potential benefit of selective GPER agonists for patients with recurrent GCT. Citation Format: Charlotte M. François, Richard Wargnier, Joëlle Cohen-Tannoudji, Celine J. Guigon. 17β-estradiol inhibits metastatic granulosa cell migration by repressing ERK1/2 pathway via an extra-nuclear mechanism of action. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1318. doi:10.1158/1538-7445.AM2013-1318