Functional Exploration of the Adult Ovarian Granulosa Cell Tumor-Associated Somatic FOXL2 Mutation p.Cys134Trp (c.402C>G)

Abstract

BackgroundThe somatic mutation in the FOXL2 gene c.402C>G (p.Cys134Trp) has recently been identified in the vast majority of adult ovarian granulosa cell tumors (OGCTs) studied. In addition, this mutation seems to be specific to adult OGCTs and is likely to be a driver of malignant transformation. However, its pathogenic mechanisms remain elusive.Methodology/Principal FindingsWe have sequenced the FOXL2 open reading frame in a panel of tumor cell lines (NCI-60, colorectal carcinoma cell lines, JEG-3, and KGN cells). We found the FOXL2 c.402C>G mutation in the adult OGCT-derived KGN cell line. All other cell lines analyzed were negative for the mutation. In order to gain insights into the pathogenic mechanism of the p.Cys134Trp mutation, the subcellular localization and mobility of the mutant protein were studied and found to be no different from those of the wild type (WT). Furthermore, its transactivation ability was in most cases similar to that of the WT protein, including in conditions of oxidative stress. A notable exception was an artificial promoter known to be coregulated by FOXL2 and Smad3, suggesting a potential modification of their interaction. We generated a 3D structural model of the p.Cys134Trp variant and our analysis suggests that homodimer formation might also be disturbed by the mutation.Conclusions/SignificanceHere, we confirm the specificity of the FOXL2 c.402C>G mutation in adult OGCTs and begin the exploration of its molecular significance. This is the first study demonstrating that the p.Cys134Trp mutant does not have a strong impact on FOXL2 localization, solubility, and transactivation abilities on a panel of proven target promoters, behaving neither as a dominant-negative nor as a loss-of-function mutation. Further studies are required to understand the specific molecular effects of this outstanding FOXL2 mutation.

The FOXL2 C134W mutation is characteristic of adult granulosa cell tumors of the ovary

“…The presence of the mutation in the KGN cell line, which was derived from an 81-year-old woman with recurrent metastatic disease, 25 is consistent with previous analyses 9,10 and suggests that it may be a useful tool to study adult granulosa cell tumors. We found that the COV434 cell line, established from a metastatic granulosa cell tumor removed from a 27-year-old female, 26,27 lacked the FOXL2 mutation and did not express the FOXL2 gene.…”

“…8 The mutation was found in only one juvenile granulosa cell tumor 8 and not found in a wide range of other tumors, both gonadal and non-gonadal. 9,10 Curiously, diminished and/or loss of FOXL2 expression has been reported in advanced stage/aggressive juvenile granulosa cell tumors. 11 In this study, we sought to confirm the presence of this mutation in two geographically independent cohorts of granulosa cell tumors and to examine the expression pattern of the FOXL2 gene in these tumors.…”

“…8 The mutant might be postulated to be either an activating mutation or a gain-of-function mutation. In an initial in vitro assay, Benayoun et al 10 found limited evidence to support activation. Given the high homology with other members of the FOX family, several of which also have fundamental roles in ovarian function, such as FOXO3a, 35 a gain of function is a distinct possibility.…”

Mouse forkhead L2 maintains repression of FSH-dependent genes in the granulosa cell

“…6), it is possible that FOXL2 and SF-1 compete for this site, leading to loss of transcriptional regulation. In addition to SF-1, FOXL2 transcriptional regulation may be altered by other transcription factors known to bind to FOXL2 , Blount et al 2009, Kim et al 2009, Lamba et al 2009), as well as FOXL2 binding to itself (Lamba et al 2009, Benayoun et al 2010, Fleming et al 2010. Further, posttranslational modifications such as sumoylation (Marongiu et al 2010, phosphorylation (Benayoun et al 2008a, and acetylation (Benayoun et al 2008a) may all act to modify FOXL2 transcriptional activity under the influence of FSH and the various signaling cascades that have been identified in granulosa cell proliferation and differentiation.…”

Differential apoptotic activities of wild-type FOXL2 and the adult-type granulosa cell tumor-associated mutant FOXL2 (C134W)

“…The FOXL2 mutant is defective in eliciting the cell death response induced by some apoptotic stimulants On the basis of the fact that the majority of FOXL2 mutations found in GCTs were heterozygotes of the 402C FOXL2 and 402G FOXL2 alleles (Shah et al, 2009), we attempted to test the possibility that the mutant may behave as a dominant-negative mutation. As KGN cells are also heterozygous for FOXL2 (Benayoun et al, 2010), FOXL2 was first knocked down using siRNA targeting of the 3 0 untranslated region to avoid any compounding effects of being heterozygotic, and its efficient depletion was confirmed by immunoblot analysis using an anti-FOXL2 antibody (Figure 5c). Subsequently, the knocked-down cells were transfected with increasing amounts (1, 2 and 3 mg) of either WT-or mutated-FOXL2-coding plasmids, and changes in cellular apoptosis were assessed.…”