FOXO1/3 and PTEN Depletion in Granulosa Cells Promotes Ovarian Granulosa Cell Tumor Development

Abstract

The forkhead box (FOX), FOXO1 and FOXO3, transcription factors regulate multiple functions in mammalian cells. Selective inactivation of the Foxo1 and Foxo3 genes in murine ovarian granulosa cells severely impairs follicular development and apoptosis causing infertility, and as shown here, granulosa cell tumor (GCT) formation. Coordinate depletion of the tumor suppressor Pten gene in the Foxo1/3 strain enhanced the penetrance and onset of GCT formation. Immunostaining and Western blot analyses confirmed FOXO1 and phosphatase and tensin homolog (PTEN) depletion, maintenance of globin transcription factor (GATA) 4 and nuclear localization of FOXL2 and phosphorylated small mothers against decapentaplegic (SMAD) 2/3 in the tumor cells, recapitulating results we observed in human adult GCTs. Microarray and quantitative PCR analyses of mouse GCTs further confirmed expression of specific genes (Foxl2, Gata4, and Wnt4) controlling granulosa cell fate specification and proliferation, whereas others (Emx2, Nr0b1, Rspo1, and Wt1) were suppressed. Key genes (Amh, Bmp2, and Fshr) controlling follicle growth, apoptosis, and differentiation were also suppressed. Inhbb and Grem1 were selectively elevated, whereas reduction of Inha provided additional evidence that activin signaling and small mothers against decapentaplegic (SMAD) 2/3 phosphorylation impact GCT formation. Unexpectedly, markers of Sertoli/epithelial cells (SRY [sex determining region Y]-box 9/keratin 8) and alternatively activated macrophages (chitinase 3-like 3) were elevated in discrete subpopulations within the mouse GCTs, indicating that Foxo1/3/Pten depletion not only leads to GCTs but also to altered granulosa cell fate decisions and immune responses. Thus, analyses of the Foxo1/3/Pten mouse GCTs and human adult GCTs provide strong evidence that impaired functions of the FOXO1/3/PTEN pathways lead to dramatic changes in the molecular program within granulosa cells, chronic activin signaling in the presence of FOXL2 and GATA4, and tumor formation.

Constitutive Activation of PI3K in Oocyte Induces Ovarian Granulosa Cell Tumors

“…Development of GCTs has been observed in genetic mouse models: null mice for Inhα (21), Esr2 (47), mice with GC-specific activating mutation in Ctnnb1 (48), and GC-specific null mutation in Smad1/Smad5 (25), Foxo1/Foxo3 and Foxo1/Foxo3/Pten (12). Although these mutations have not been detected in human GCTs, these mice are excellent tools to understand the signaling pathways that are potentially involved in the pathogenesis of human GCTs.…”

“…Recent studies detected recurrent in-frame duplications in exon 3 of AKT1 gene in JGCTs (10,11). In mice, loss of negative regulators of the PI3K-AKT pathway, PTEN and FOXO1/3 in GCs, resulted in GCTs (12), suggesting the crucial role of the PI3K-AKT pathway in the pathogenesis of JGCTs.…”

SMAD3 Activation: A Converging Point of Dysregulated TGF-Beta Superfamily Signaling and Genetic Aberrations in Granulosa Cell Tumor Development?

“…Development of sex cord-stromal tumors has been documented in a number of genetically engineered mouse models [4, 10, 11, 26–32]. INHA is known as a tumor suppressor from the creation of mice lacking INHA [4].…”

“…New evidence suggests that GCTs resulting from genetic modifications of some non-TGFB superfamily genes may also harbor TGFB/activin signaling activation [31, 32]. A recent elegant study by the Richards laboratory reveals that triple deletion of forkhead box protein O1 ( Foxo1 ), Foxo3, and phosphatase and tensin homolog ( Pten ) in the mouse ovary causes GCT formation.…”

“…This mouse model mimics human GCTs in hormone profiles (e.g., increased levels of estradiol and inhibins). Tumor tissues from these mice are positive for phospho-SMAD2/3, suggesting an activation of TGFB/activin signaling [31]. Another mouse model with constitutive activation of phosphoinositide 3-kinase (termed PIK3CA) in the oocyte demonstrates abnormal follicle growth, ovulatory defects, and formation of bilateral GCTs [32, 47].…”