Abstract 4099: F-box protein 10, an NF-κB-dependent anti-apoptotic protein, regulates TRAIL-induced apoptosis through modulating c-Fos/c-FLIP pathway

Background: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) holds great promise as a potential anti-cancer agent, but some tumors develop resistance to TRAIL. AP-1 family member, c-Fos, is an important modulator of apoptosis. Although F-box protein 10 (FBXL10) has been implicated to regulate an AP-1 family protein, significance and mechanism of FBXL10 in apoptosis has not been investigated. Methods: In this study, different types of TRAIL-sensitive cells (A498, PC3, MBA-MD-231) and TRAIL-resistant cells (LNCaP, PC3TR) were used, and a quantitative analysis of apoptosis was performed by using MTS and caspase-3, PARP activity assay and flow cytometry assay of annexin V- and propidium iodide-stained cells. RNAi, Chromatin immunoprecipitation (ChIP), EMSA, hydrodynamic-based transfection, oligonucleotide decoy technologies were performed to analyze the molecular interaction between FBXL10, c-Fos and NF-κB. The anti-apoptotic role of FBXL10 in vivo was analyzed by Lexatumumab (an agonistic human monoclonal antibody against TRAIL-R2) intravenous injection into mice carrying xenograft prostate cancers. All statistical tests were two-sided. Results: We found that FBXL10 was suppressed and c-Fos was upregulated in TRAIL sensitive cancer cells and xenografts after treatment with TRAIL. However, in TRAIL resistant cancer cells and xenografts, FBXL10 and c-Fos were not affected. Expression of FBXL11, a FBXL10 homologous protein and c-Jun, was not affected by TRAIL. Silencing of FBXL10 sensitized resistant cells to TRAIL. Conversely, over-expression of FBXL10 repressed TRAIL-induced apoptosis. To behave as an anti-apoptotic molecule, we found that FBXL10 directly binds and represses c-Fos promoter in vitro and in vivo. In addition, FBXL10 regulates c-FLIP, another anti−apoptotic molecule, by a c-Fos dependent pathway. We also found that expression of FBXL10 is NF-κB – dependent, and TRAIL or proteasome inhibitors down-regulate FBXL10 via inhibiting NF-κB signaling. Using ChIP, EMSA assays and oligonucleotide decoy technologies, we found that NF-κB -p65 directly binds the FBXL10 promoter in vivo and in vitro, and promotes expression of FBXL10. Conclusions: Differentiating molecular mechanisms between TRAIL sensitive and TRAIL resistant cancer cells will improve the efficacy of apoptotic therapies for cancer patients. In this study, we demonstrate a novel functional role for FBXL10 as an anti-apoptotic molecule, and describe a new apoptotic mediated pathway that involves NF-κB/FBXL10/c-Fos/c-FLIP. Therefore, silencing FBXL10 can help overcome resistant cancer cells for pro-apoptotic therapies. 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 4099. doi:10.1158/1538-7445.AM2011-4099