Background: Granulosa cell tumours (GCT) are uncommon ovarian cancers characterised by an indolent clinical course and significant late recurrence rates. Aside from invasive surgery, there are limited therapeutic options, highlighting the need for targeted therapies. The Hudson institute laboratory has previously shown that targeting X-linked Inhibitor of Apoptosis Protein (XlAP) using small molecule inhibitors known as Smac-Mimetics (SM) in combination with other compounds, is a compelling therapeutic strategy in GCT. XlAP inhibition sensitises cancer cells to anti-cancer therapies through the regulation of key pro-survival pathways, namely NF-κB (Nuclear factor kappa-light-chain-enhancer of activated B cells)
Methods: High-throughput screening (HTS) using established drug libraries was performed in a GCT-derived cell line KGN (steroidogenic human granulosa like cell line) and a transformed non-luteinized granulosa cell line (hGrC1), following pre-treatment with SM - Compound A (CmpdA: 500nM). Drug combination hits were defined as >80% reduction in viability compared to CmpdA monotherapy alone. Subsequent validation studies were conducted to measure the effects on cell proliferation, apoptosis and NF-κB transactivation. The impact of the combination therapy was assessed by RNA-seq analysis to identify differentially expressed genes, significant pathways and functional enrichment.
Results: Here we report the use of a histone deacetylase inhibitor (HDACi), panobinostat, in combination with CmpdA, as a potential treatment for GCT. Using cell proliferation and viability assays, we demonstrated that 100nM panobinostat acts synergistically with 500nM CmpdA to significantly decrease cell proliferation and increase apoptosis, as demonstrated by increased caspase 3/7 activity. Apoptotic pathways were further assessed using flow cytometry. A potential mechanism of action for the compounds was tested using real-time PCR and luciferase reporter assays. In KGN cells, panobinostat demonstrated increased NF-kB activity and TNFa expression, which decreased when combined with CmpdA, suggesting that there is enhanced activation of the extrinsic pathway of apoptosis. Transcriptomic analysis showed the top significantly enriched pathways related to mechanisms of cancer pathogenesis. In particular, we observed significant downregulation of SIRT1, a non-classical Class III HDAC that is potentially important in GCT tumorogenesis through its interactions with the FOXL2 transcription factor, affecting cancer pathways such as cell senescence, proliferation, and apoptosis.
Conclusion: We present a promising combination therapeutic strategy for GCT that reduced cell proliferation, increased apoptosis, and reduced inflammatory gene expression. Further studies are now needed to confirm and translate these findings, and to identify the links between SIRT1 gene, and the pathognomonic FOXL2C134W mutation of GCT.