MON-326 The impact of FOXL2C134W on gene expression in immortalized non-luteinized granulosa cell lines

Abstract Disclosure: T.S. Hegarty: None. P.J. Fuller: None. S. Chu: None. Background: Adult granulosa cell tumors (aGCT) are rare endocrine-related ovarian cancers, comprising approximately 5% of all ovarian cancers. These tumors harbor a pathognomonic FOXL2 gene mutation, involving a cysteine-to-tryptophan substitution at amino acid 134 (C134W). FOXL2 is a key transcriptional regulator of granulosa cell identity and function, and the FOXL2C134W mutant has been shown to alter its DNA binding specificity and transcriptional activity, inducing genes associated with oncogenesis, stemness and epithelial-to-mesenchymal transition (EMT). This study investigates the transcriptional impact of the FOXL2C134W mutation in an immortalized non-luteinized granulosa (hGrC1) cell line model. Methods: CRISPR-Cas9 gene editing introduced a homozygous FOXL2C134W mutation into hGrC1 cells. Cell proliferation was assessed using the xCELLigence Real-Time Cell Analysis SP system. RNA sequencing (RNA-seq) was performed to identify differentially expressed genes (DEGs) in FOXL2C134W hGrC1 cells compared to parental controls, with DEGs defined by log2FC≥1 and Q value of <0.05. Published wildtype FOXL2 ChIP-sequencing data (GSE236785) and FOXL2C134W ChIP-sequencing data (GSE126171) were analyzed to delineate target genes. Gene set enrichment analysis (GSEA) was used to assess pathway-level changes in gene expression. Results: FOXL2C134W mutation did not alter cell proliferation compared to parental hGrC1 cells. RNA-seq identified 355 DEGs in FOXL2C134W hGrC1 cells with 101 upregulated and 254 downregulated genes. Among these, 47 DEGs overlapped with FOXL2WT target genes, and 12 DEGs with FOXL2C134W target genes. GSEA revealed significant enrichment (p<0.01) of EMT-associated genes in FOXL2C134W hGrC1 cells. Notably, Microfibril associated protein 5 (MFAP5), an EMT-associated gene implicated in tumor progression and metastasis, exhibited a marked upregulation (log2FC = 10) in FOXL2C134W hGrC1 cells. Discussion: Our findings demonstrate that the FOXL2C134W mutation drives transcriptional reprogramming in granulosa cells, specifically upregulating EMT-associated pathways. The overexpression of MFAP5, a gene correlated with advanced aGCT stages and cancer invasiveness, underscores its potential as a biomarker and therapeutic target. These insights advance our understanding of the molecular mechanisms underlying aGCT pathogenesis and paves the way for targeted therapeutic strategies for this rare and challenging cancer. Presentation: Monday, July 14, 2025