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Introduction
Tumor necrosis factor-related apoptosis−inducing-ligand (TRAIL) holds great promise as an anti−cancer-agent, but 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 and regulates TRAIL-induced-apoptosis through modulating c-Fos/c-FLIP.
Methods
RT−PCR, Western blot and immunofluorescence assays were applied to evaluate protein expression. RNAi, ChIP, EMSA, hydrodynamic−based-transfection were performed to analyze the interaction among FBXL10/c−Fos/NF−kB. Prostate xenografts were carried out for in−vivo analyses.
Results
FBXL10 was suppressed and c−Fos was upregulated in TRAIL-sensitive cancers after treatment with TRAIL. However, in TRAIL-resistant cancers, FBXL10 and c−Fos were not affected. 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 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 down−regulate FBXL10 via inhibiting NF−kB signaling. Using ChIP and EMSA assays, we found that NF−kB−p65 directly binds the FBXL10 promoter, and promotes expression of FBXL10.
Conclusions
Differentiating molecular mechanisms between TRAIL-sensitive and TRAIL-resistant cancer cells will improve the efficacy of apoptotic therapies. In this study, we demonstrate that FBXL10 plays an anti−apoptotic role and indicates a novel NF−kB/FBXL10/c−Fos/c−FLIP signaling pathway in TRAIL−mediated apoptosis.