Abstract 2912: F-box protein 10: A novel NF-κB–dependent antiapoptotic protein regulates TRAIL-induced apoptosis

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction and Objective Tumor necrosis factor-related apoptosis—inducing ligand (TRAIL) induces selective apoptotic death of human cancer cells while sparing normal human cells. Although TRAIL holds great promise as a potential anti—cancer agent, some tumors develop resistance to TRAIL. Previously, we have shown that the AP—1 family member, c—Fos, is an important modulator of apoptosis. Although FBXL10 has been implicated to regulate an AP—1 family protein, c—Jun, its role in mediating apoptotic pathways has not been previously investigated. Here, we report that FBXL10 is a novel NF—kB dependent anti—apoptotic molecule, which directly binds and represses c—Fos, another AP—1 family member, rather than c—Jun. Repression of c—Fos leads to resistance of cancer cells to TRAIL—induced apoptosis. Methods RT—PCR, Western blot and immunofluorescence assays were applied to evaluate protein expression level. RNAi, Chromatin immunoprecipitation (ChIP), EMSA, hydrodynamic—based transfection, oligonucleotide decoy technologies, plasmid reconstruction and site—directed mutation were performed to analyze the interaction between FBXL10, c—Fos and NF—kB. Orthotopic prostate xenografts were carried out for in—vivo analyses. Results 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—kB—dependent, and TRAIL or proteasome inhibitors down—regulate FBXL10 via inhibiting NF—kB signaling. Using ChIP and EMSA assays, we found that NF—kB—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 demonstrates for the first time, that FBXL10 plays an anti—apoptotic role and indicates a novel NF—kB/FBXL10/c—Fos/c—FLIP signaling pathway in TRAIL—mediated apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2912.