Novel mechanisms of overcoming apoptotic resistance in Pancreatic Cancer


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Julie Nicoll1,Daniel Longley1,Chris Scott1
1CCRCB, Queen's University Belfast

Abstract

Background

Apoptotic resistance is a barrier that must be overcome in the treatment of all cancers. The extrinsic apoptosis pathway relies on activation of Death Receptor 5 (DR5), receptor crosslinking and subsequent intracellular Death Inducing Signalling Complex (DISC) formation, caspase activation and finally apoptosis. A key inhibitor of this pathway is FLIP, which prevents DISC formation and activation of procaspase-8. Previous studies have shown FLIP to be highly upregulated in Pancreatic Cancer, whereas healthy pancreatic tissue samples were consistently shown to be FLIP negative. Therefore, we hypothesised that inhibition of FLIP, in combination with activation of DR5 could result in cell death in Pancreatic Cancer cells.

Method

Three human Pancreatic Cancer cell lines (MiaPaca2, ASPC-1 and PANC-1) were used for all in vitro experiments. These cell lines were treated with single and co-treatment combinations of Entinostat, FLIPi and iz-TRAIL.  CellTiter-Glo Assays (Promega) were used to evaluate reduction in cell viability. High Content analysis was performed in combination with Annexin V and Propidium Iodide staining to confirm the induction of Apoptosis. 

Results

To assess the role of FLIP as a resistance mechanism in Pancreatic Cancer, FLIP was downregulated via a directly acting novel FLIP inhibitor, reliant on protein-protein interaction, and via the Class I-specific HDACi Entinostat, which downregulates FLIP at a transcriptional level. DR5 activation was achieved via iz-TRAIL treatment, which resulted in reduction in cell viability, even at low ng/ml treatment concentrations. Down-regulation of FLIP via siRNA transfections resulted in increased sensitivity to iz-TRAIL. Subsequent experiments showed co-treatments of Entinostat or FLIPi with iz-TRAIL synergistically reduce cell viability in three Human Pancreatic Cancer Lines. The FLIPi was shown to have greater potency in all lines, compared to Entinostat, which was expected due to their varying mechanisms of action. These treatments were confirmed to induce apoptosis, following Annexin/PI staining and High Content Microscopy analysis. In line with previous results, co-treatment groups were shown to have greater levels of apoptosis than single agent treatment groups.

Conclusion

These findings indicate a potential mechanism of overcoming apoptotic resistance in Pancreatic Cancer via downregulation of FLIP and simultaneous activation of DR5, via co-treatments of Entinostat or FLIPi and iz-TRAIL.