BACKGROUND: 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 (PPARγ) and the X-linked inhibitor of apoptosis protein (XIAP) as potential specific therapeutic targets. PPARγ is 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-κB signaling pathway. Constitutive NF-κB 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-κB transrepression, and together with PPARγ activation, induces apoptosis. We thus hypothesize that XIAP antagonism sensitizes GCT to pro-apoptotic strategies such as PPARγ activation.
METHODS AND RESULTS: We have used stable isotope labeling with amino acids in cell culture (SILAC), a proteomic approach to identify differentially expressed proteins after combined PPARγ activation (rosiglitazone) and XIAP inhibition (Smac mimetic). Using two GCT-derived cell lines, KGN and COV434, we identified 52 proteins which were differentially expressed (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 PPARγ plays a pivotal role in lipid and glucose metabolism, upregulation of these proteins is a consequence of PPARγ activity being restored. Additionally, we observed downregulation of fascin (-1.65 fold), the main actin-bundling protein associated with cell motility and migration. Overexpression has been observed in metastatic tumors and correlated with clinically aggressive phenotypes, poor prognosis and shorter survival.
We used the xCELLigence RTCA system to monitor invasion of KGN cells through a Matrigel® basement membrane in real-time. KGN cells treated either with Vehicle, RGZ/RA or SM alone or combined RGZ/RA/SM were plated in the upper chamber of a CIM-Plate 16 and cell invasion through the Matrigel® membrane was monitored. When compared to vehicle treated cells, we observed that the RGZ/RA/SM-treated KGN cells were (i) 30% less invasive and (ii) demonstrated a delayed onset of invasion by 17 hours towards serum-containing media with and without drugs in the lower chamber, respectively.
CONCLUSION: We are currently investigating the mechanism involved in XIAP inhibition abrogating transrepression of PPARγ in GCT-derived cells. This study will improve our understanding of the molecular mechanisms in GCT pathophysiology as well as enable identification of new targets for therapeutic strategies. These observations may also translate to ovarian epithelial cell cancers (EOC) as increased apoptosis was observed in serous EOC-derived cell lines expressing both XIAP and PPARγ. 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.
Citation Format: Simon Chu, Dilys TH Leung, Maria Alexiadis and Peter J Fuller. PPARγ AGONISTS AUGMENT ANTICANCER EFFECTS OF XIAP INHIBITION ON HUMAN GRANULOSA CELL TUMOR–DERIVED CELLS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-085.