Abstract LB-222: Identifying cFLIP as a marker and also a potentially “druggable” target of SAHA+TRAIL (TNF-Related Apoptosis Inducing Ligand), cytotoxicity in malignant pleural mesothelioma (MPM)

Abstract

Abstract Background: Despite expressing adequate levels of receptors for TRAIL significant percentages of cancer cells are resistant to TRAIL-induced apoptosis. We have previously reported that histone deacetylase inhibitor SAHA (vorinostat) + TRAIL combination induces profound supra-additive cytotoxicity in MPM in vitro. We observe that only MPM cells with some TRAIL sensitivity are very susceptible to SAHA+TRAIL cytotoxicity. We hypothesize that TRAIL-initiated death signal at the membrane level determine cellular susceptibility to SAHA+TRAIL. The objective of this study is to identify the molecular marker that predicts TRAIL and thus TRAIL+SAHA sensitivity in MPM cells. Materials and methods: Intrinsic sensitivity of 8 MPM and primary normal endothelial cells to TRAIL and SAHA+TRAIL is determined by cell viability assay; basal expression of cFLIP, caspase 8, DR4/DR5, FADD, RIP, TRADD in these cells are evaluated by western blots. Selective knockdown of FLIP is achieved by siRNA. FLIP mRNA levels were determined by quantitative RT-PCR. Results: 5/8 MPM cells are very suceptible to SAHA+TRAIL cytotoxicity (combination-sensitive cells) while 3 others are classified as combination-resistant. Western blot analysis identifies an inverse relationship between cFLIP as well as procaspase 8 expression and sensitivity to SAHA+TRAIL with only the difference in cFLIP levels as quantified by densitometric analysis being distinctive between two groups: 0.87±0.02 for 5 combination-sensitive MPM cells versus 0.15±0.05 for 3 combination-resistant MPM cells. siRNA-mediated partial cFLIP knockdown restores DISC activity in high cFLIP expressing cells as evidenced by caspase 8 and 3 catalytic processing with stronger caspase activation being noted in combination-treated cells. Quantitative RT-PCR demonstrates high level of cFLIP messenger RNA in high cFLIP expressors indicating that cFLIP is transcriptionally upregulated in these cells. Selective cFLIP downregulation in high cFLIP expressing cells restores susceptibility to TRAIL and strongly sensitizes them to SAHA+TRAIL cytotoxicity. Additionally, selective complete cFLIP knockdown abrogates the need for SAHA to achieve profound TRAIL-mediated cell death in MPM cells regardless of their intrinsic cFLIP epxression. Conclusdion: Our study identifies cFLIP expression as a marker of cellular sensitivity to SAHA+TRAIL in MPM in vitro. More importantly, gene knockdown experiments provide the proof of concept that cFLIP is a pontential “druggable” target and downregulation of which sensitizes resistant cancer cells to TRAIL and SAHA+TRAIL. Ongoing works aim to validate this observation in a larger panel of novel MPM cells and to define treatments strategies to downregulate cFLIP in tumor cells expressing high levels of this antiapoptotic protein. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-222. doi:10.1158/1538-7445.AM2011-LB-222