FOXO1 mitigates the SMAD3/FOXL2C134W transcriptomic effect in a model of human adult granulosa cell tumor

Paola Benaglio,Martina Belli,Christian Secchi,Dwayne Stupack,Francesca Mulas,Shunichi Shimasaki

doi:10.1186/s12967-021-02754-0

Abstract

BackgroundAdult granulosa cell tumor (aGCT) is a rare type of stromal cell malignant cancer of the ovary characterized by elevated estrogen levels. aGCTs ubiquitously harbor a somatic mutation in FOXL2 gene, Cys134Trp (c.402C < G); however, the general molecular effect of this mutation and its putative pathogenic role in aGCT tumorigenesis is not completely understood. We previously studied the role of FOXL2C134W, its partner SMAD3 and its antagonist FOXO1 in cellular models of aGCT.MethodsIn this work, seeking more comprehensive profiling of FOXL2C134W transcriptomic effects, we performed an RNA-seq analysis comparing the effect of FOXL2WT/SMAD3 and FOXL2C134W/SMAD3 overexpression in an established human GC line (HGrC1), which is not luteinized, and bears normal alleles of FOXL2.ResultsOur data shows that FOXL2C134W/SMAD3 overexpression alters the expression of 717 genes. These genes include known and novel FOXL2 targets (TGFB2, SMARCA4, HSPG2, MKI67, NFKBIA) and are enriched for neoplastic pathways (Proteoglycans in Cancer, Chromatin remodeling, Apoptosis, Tissue Morphogenesis, Tyrosine Kinase Receptors). We additionally expressed the FOXL2 antagonistic Forkhead protein, FOXO1. Surprisingly, overexpression of FOXO1 mitigated 40% of the altered genome-wide effects specifically related to FOXL2C134W, suggesting it can be a new target for aGCT treatment.ConclusionsOur transcriptomic data provide novel insights into potential genes (FOXO1 regulated) that could be used as biomarkers of efficacy in aGCT patients.

Highlights

Adult granulosa cell tumor is a rare type of stromal cell malignant cancer of the ovary characterized by elevated estrogen levels. aGCTs ubiquitously harbor a somatic mutation in Forkhead box L2 (FOXL2) gene, Cys134Trp (c.402C < G); the general molecular effect of this mutation and its putative pathogenic role in aGCT tumorigenesis is not completely understood
Transcriptomics analysis of the human granulosa HGrC1 cell in presence of FOXL2C134W To investigate the transcriptional changes associated with the SMAD3/FOXL2C134W program, and its interaction with Forkhead box O1 (FOXO1), HGrC1 cells transiently expressing SMAD3 and FOXL2WT/FOXL2C134W, with or without FOXO1-3A (FOXO1 hereafter), were profiled using RNA sequencing (Fig. 1a)
To identify genes selectively induced by FOXL2 or FOXL2C134W, we assessed differential expression analysis between FOXL2WT and controls, FOXL2C134W and controls, and F OXL2C134W and F OXL2WT, and identified a total of 452, 939 and 717 differential expressed genes (DEGs, q < 0.05, Additional file 2: Table S1) genes respectively, many of which were shared among the different conditions (Fig. 1c)

Summary

Introduction

Adult granulosa cell tumor (aGCT) is a rare type of stromal cell malignant cancer of the ovary characterized by elevated estrogen levels. aGCTs ubiquitously harbor a somatic mutation in FOXL2 gene, Cys134Trp (c.402C < G); the general molecular effect of this mutation and its putative pathogenic role in aGCT tumorigenesis is not completely understood. In 2011, Benayoun et al demonstrated that FOXL2 plays a key role in the homeostasis of GCs and its failure leads to ovarian aging and tumorigenesis [24] This mutation hyperactivates aromatase and downregulates GC apoptosis pathways [10]. The study revealed low numbers of target genes and the authors noted the SVOG3e cell line may not respond appropriately to F OXL2C134W This may be possibly due to the absence of the needed partner SMAD3. Using FOXL2C134W overexpression and silencing molecular approaches in a non-luteinized GC cell line (HGrC1) treated with TGFβ, they showed that SMAD4 is an important FOXL2C134W partner and the TGFβ/FOXL2C134W molecular events trigger the expression of oncogenic, EMT, and stemness pathways in an aGCT model [37]. It is well accepted that TGFβ has an important role in tumor progression and involves SMAD mediators that control expression of hundreds of genes in different ways in diverse contexts and physiologies [38, 39] additional investigations are needed to find its specific action(s) in aGCTs

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