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Aberrant ovarian granulosa cell proliferation and apoptosis may lead to granulosa cell tumors (GCT), the pathogenesis of which involves transcription factors GATA4, FOXL2, and SMAD3. FOXL2 gene harbors a point mutation (C134W) in a vast majority of GCTs. GATA4 is abundantly expressed in GCTs and its expression correlates with poor prognosis. The TGF-β mediator SMAD3 promotes GCT cell survival through NF-κB activation, and interacts with FOXL2. Here, we find that the expression patterns of these factors overlap in the normal human ovary and 90 GCTs, and positively correlate with each other and with their mutual target gene CCND2, which is a key factor for granulosa cell proliferation. We have explored the molecular interactions of FOXL2, GATA4, and SMAD3 and their roles in the regulation of CCND2 using co-immunoprecipitation, promoter transactivation, and cell viability assays in human GCT cells. We found that not only SMAD3, but also GATA4 physically interact with both wild type and C134W-mutated FOXL2. GATA4 and SMAD3 synergistically induce a 8-fold increase in CCND2 promoter transactivation, which is 50% reduced by both FOXL2 types. We confirmed that wild type FOXL2 significantly decreases cell viability. Interestingly, GATA4 and SMAD3 caused a marked reduction of GCT cell apoptosis induced by wild type FOXL2. Thus, the effects of GATA4 and SMAD3 on both cell viability and apoptosis are distinct from those of wild type FOXL2; a perturbation of this balance due to the oncogenic FOXL2 mutation is likely to contribute to GCT pathogenesis.

Context: Ovarian granulosa cell tumors (GCT) are highly vascularized and express vascular endothelial growth factor A (VEGF) and its functional receptor VEGF receptor 2 (VEGFR-2). Angiogenesis inhibitors have been used in the treatment of ovarian carcinomas, whereas their roles in GCT remain unknown., Objective: The aim was to assess serum levels of VEGF and endostatin, an endogenous angiogenesis inhibitor, in GCT patients and to study the effect of bevacizumab (BVZ), a VEGF-binding monoclonal antibody, on human GCT cells in vitro., Design: Using ELISA, we measured soluble VEGF and endostatin in the sera of 54 GCT patients and in conditioned media from cultures of 14 primary GCT and an established GCT cell line (KGN). The expression of activated VEGFR-2 was analyzed in GCT tissues using immunohistochemistry. GCT cells were treated with BVZ and analyzed for cell number and apoptosis., Results: Serum VEGF was elevated in GCT patients, and the levels significantly decreased after tumor removal (P < 0.05), whereas serum endostatin levels changed conversely. Human GCT expressed activated VEGFR-2 protein, and the level of expression was associated with tumor VEGF and vascularization. In addition, the cultured GCT cells produced significant amounts of VEGF but not endostatin. Treatment of KGN cells with BVZ significantly reduced the number of viable cells by 41% and induced a 3.3-fold increase in apoptosis. Furthermore, BVZ induced a mean 2.6-fold increase in apoptosis in six primary GCT cell cultures studied., Conclusions: These data suggest an autocrine role for VEGF in GCT and encourage clinical studies on anti-VEGF treatments in this disease.

Ovarian granulosa cell tumors (GCTs) are sex cord stromal tumors that constitute 3-5% of all ovarian cancers. GCTs usually present with an indolent course but there is a high risk of recurrence, which associates with increased mortality, and targeted treatments would be desirable. Anti-Müllerian hormone (AMH), a key factor regulating sexual differentiation of the reproductive organs, has been implicated as a growth inhibitor in ovarian cancer. GCTs and normal granulosa cells produce AMH, but its expression in large GCTs is usually downregulated. Further, as the lack of specific AMH-signaling pathway components leads to GCT development in mice, we hypothesized that AMH inhibits growth of GCTs. Utilizing a large panel of human GCT tissue samples, we found that AMH type I receptors (ALK2, ALK3 and ALK6) and type II receptor (AMHRII), as well as their downstream effectors Smad1/5, are expressed and active in GCTs. AMHRII expression was detected in the vast majority (96%) of GCTs and correlated with AMH mRNA and protein expression. AMH mRNA level was low in large GCTs, confirming previous findings on low-AMH protein expression in large human as well as mouse GCTs. To study the functional role of AMH in this peculiar ovarian cancer, we utilized a human GCT cell line (KGN) and 10 primary GCT cell cultures. We found that the AMH-Smad1/5-signaling pathway was active in these cells, and that exogenous AMH further activated Smad1/5 in KGN cells. Furthermore, AMH treatment reduced the number of KGN cells and primary GCT cells, with increasing amounts of AMH leading to augmented activation of caspase-3 and subsequent apoptosis. All in all, these data support the premise that AMH is a growth inhibitor of GCTs.

Epidermal growth factor receptor (EGFR) is implicated in the progression of many human cancers, but its significance in ovarian granulosa cell tumor (GCT) pathobiology remains poorly understood. We assessed the EGFR gene copy number, surveyed themRNAand protein expression patterns of EGFR in 90 adult GCTs, and assessed the in vitro sensitivity of GCTcells to EGFR inhibition. Low-level amplification of EGFR gene was observed in five GCTs and highlevel amplification in one sample. EGFR mRNA was robustly expressed in GCTs. Most tumors expressed both unphosphorylated and phosphorylated EGFR protein, but the protein expression did not correlate with clinical parameters, including the risk of recurrence. Smallmolecule EGFR inhibitors reduced the EGF-induced activation of EGFR and its downstream signaling molecules at nanomolar doses, but cell viability was reduced, and caspase-3/7 was activated in GCTcells only at micromolar doses. Based on the present results, EGFR is active and abundantly expressed in the majority of GCTs, but probably has only minor contribution to GCTcell growth. Given the high doses of EGFR inhibitors required to reduce GCTcell viability in vitro, they are not likely to be effective for GCT treatment as single agents; they should rather be tested as part of combination therapies for these malignancies [ABSTRACT FROM AUTHOR]