Ovarian granulosa cell tumours (GCT) are hormonally active cancers characterised by indolent growth and late, invasive relapse. Aside from surgery, the therapeutic options are very limited. We previously reported (ESA 2014) the nuclear receptor, peroxisome proliferator-activated receptor-gamma (PPARγ), and the X-linked inhibitor of apoptosis protein (XIAP) to be potential specific therapeutic targets for the treatment of GCT. Inhibiting XIAP releases PPARγ transrepression by the constitutive NF-κB activity, and together with PPARγ activation, induces apoptosis in two GCT-derived cell lines, KGN and COV434. Our aims are to: 1) identify differentially expressed proteins after combined PPARg and XIAP treatment; and 2) investigate the mechanism by which XIAP inhibition abrogated NF-κB transrepression of PPARγ in GCT-derived cells.
We have used stable isotope labelling with amino acids in cell culture (SILAC), a proteomic approach to identify differentially expressed proteins after combined PPARg activation (rosiglitazone) and XIAP inhibition (Smac mimetic) for 24 hours. The role of upstream regulator of NF-κB, TGF-β-activated kinase (TAK) 1, which is activated via XIAP, was investigated using transactivation assays and a specific TAK1 inhibitor, 5Z-7-oxozeaenol.
In KGN and COV434, we identified a total of 407 proteins, 31 of which were upregulated by ≥1.5 fold with the combined treatment. These included several proteins involved in metabolic processes such as acyl-CoA desaturase (4.50-fold), phosphoglycerate kinase 1 (2.87-fold) and α-enolase (1.75-fold). We demonstrated that the inhibition of XIAP and TAK1 significantly reduced NF-κB-mediated transactivation in GCT in vitro.
As PPARγ plays a pivotal role in lipid and glucose metabolism, upregulation of proteins associated with metabolic processes is consistent with the restoration of PPARγ activity. Constitutive NF-κB signalling is potentially a consequence of a positive feed-forward loop involving its effector protein XIAP. Findings suggested that TAK1 may also be involved in constitutive NF-κB signalling in GCT in vitro.