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Introduction: Ovarian granulosa cell tumors (GCT) are hormonally active cancers characterized by indolent growth and late, invasive relapse. GCT are unusual in that they have an unexplained propensity for late recurrence. ~80% of patients with aggressive or recurrent tumors die from their disease. Aside from surgery the therapeutic options are very limited. We have identified the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) and the X-linked inhibitor of apoptosis protein (XIAP) as potential specific therapeutic targets. PPARgamma, a transcription factor that impedes proliferation and promotes terminal differentiation in granulosa cells (GC), is highly expressed in GCT. Its activity however is transrepressed by constitutive activity of the critical pro-survival NF-kappaB signaling pathway. Constitutive NF-kappaB signaling is potentially a consequence of a positive feed-forward loop involving XIAP, which is also highly expressed in GCT. As a potent inhibitor of apoptosis, XIAP is an attractive therapeutic target. We have shown in vitro that inhibiting XIAP releases NF-kappaB transrepression, and together with PPARgamma activation, induces apoptosis. We thus hypothesize that XIAP antagonism sensitizes GCT to pro-apoptotic strategies such as PPARgamma activation. Methods and Results: Using two GCT-derived cell lines, KGN and COV434 cells, we investigated the anti-tumor effects of combined XIAP inhibition using Smac-mimetics and PPARgamma activation using thiazolidinediones (TZD). Transactivation assays revealed that NF-kappaB transrepression of PPARgamma can be relieved by NF-kappaB or XIAP inhibition. Combined Smac-mimetic and TZD treatment significantly induced apoptosis, reduced cell viability and proliferation in the GCT-derived cells in monolayer and 3D spheroid culture, as well as in a primary GCT explant model. We used the xCELLigence RTCA system to monitor cell invasion and showed that the Smac-mimetic and TZD co-treatment caused a significant delay in cell invasion. Cellular bioenergetic profiling of the KGN cells was assessed using the Seahorse XFp system, and we demonstrate that the combined treatment compromises cell metabolism in KGN cells. We utilized stable isotope labeling with amino acids in cell culture (SILAC) to identify differentially expressed proteins in the KGN cells following TZD and Smac-mimetic co-treatment. We identified a total of 32 differentially expressed proteins, 22 of which were upregulated by ≥1.5 fold with the combined treatment. Upregulated proteins included several involved in metabolic processes including acyl-CoA desaturase (4.50-fold), phosphoglycerate kinase 1 (2.87-fold) and α-enolase (1.75-fold). As PPARgamma plays a pivotal role in lipid and glucose metabolism, upregulation of these proteins is a consequence of PPARgamma activity being restored. Conclusion: This study provides evidence that PPARgamma and XIAP co-treatment has anti-neoplastic effects in GCT. As therapeutics that target these proteins are already in clinical or pre-clinical use, expedient translation to the clinic is possible. We propose that a combination therapy involving the abrogation of XIAP may be of greater efficacy for the treatment of GCT and potentially other ovarian cancer subtypes.
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