FOXO1 Mitigation of FOXL2C143W/SMAD3 Transcriptomic Landscape in a Model of Granulosa Cell Tumor

Background: Adult granulosa cell tumor (aGCT) is a rare type of stromal cell malignant cancer of the ovary. Postmenopausal genital bleeding is the main aGCT clinical sign which is attributed to estrogen excess driven by CYP19 upregulation. Typically, aGCTs that are diagnosed at an initial stage can be treated with surgery. However, recurrences are mostly fatal(1). Current studies are focused on finding new molecular markers and targets that aim to treat the aGCTs recurrence. Between 95-97% of aGCTs harbor a somatic mutation in the FOXL2 gene, Cys134Trp (c.402C<G)(2). A TGF-β pathway protein, SMAD3, was identified as an essential partner in FOXL2(C134W) transcriptional activity driving CYP19 upregulation(3). Recently, the antitumoral FOXO1 gene has been recognized as a potential target for suppressing the FOXL2(C134W) pathogenic action(4). Aim: The objective of this study was to examine whether FOXO1 upregulation affects the FOXL2(C143W)/SMAD3 transcriptomic landscape. Methods: RNA-seq analysis was performed comparing the effect of FOXL2(WT)/SMAD3 and FOXL2(C143W)/SMAD3 overexpression in presence of FOXO1 by transfection of an established human GC line (HGrC1). RNA-seq libraries were prepared using the illumina TrueSeq and sequenced using an illumina HiSeq Platform4000. To quantify transcript abundance for each sample we used salmon (1.1.0) with default parameters, using indexes from hg38. Data was subsequently imported in R using the tximport package and processed with the DESeq2 package. Results: RNA-seq data show that FOXL2(C143W)/SMAD3 significantly drives 717 genes compared with the WT and enabled us to identify targets (TGFB2, SMARCA4, HSPG2, MKI67, NFKBIA) and neoplastic pathways directly associated with the mutant. To provide evidence that the differences in gene expression were attributed to a direct consequence of FOXL2 binding, we annotated gene promoters with previously published FOXL2 ChIP-seq analysis. The majority (73-40%) of the differential expressed genes (DEGs) between FOXL2(C134W) and FOXL2(WT) had a FOXL2 binding site at their promoters, which was a significantly higher proportion than in non-DEGs (Fisher’s exact test, murine: p= 7.9x10(-157); human, p= 9.9x10(-39)). Surprisingly, the number of DEGs between FOXL2(C134W) + FOXO1 and FOXL2(WT) was much lower (230) with respect to the number of DEGs between FOXL2(C134W) and FOXL2(WT) (717, of which 130 in common; linear regression slope ß = 0 .58), suggesting that the effect of FOXL2(C134W) compared with FOXL2(WT) is moderated by the addition of FOXO1. Conclusions: Our transcriptomic study provides the first evidence that FOXO1 can efficiently mitigate 40% of the altered genome-wide effect specifically related to FOXL2(C134W) in a model of human aGCT.1 Farkkila, A. et al. Ann Med (2017). 2 Jamieson, S. & Fuller, P. J. Endocr Rev (2012). 3 Belli, M. et al. Endocrinology (2018). 4 Belli, M et al. J Endocr Soc (2019).