Glycogen synthase kinase-3, DDX3 and cIAP-1 associate with TRAIL-R2 to form an anti-apoptotic complex

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Resistance of tumor cells to death receptor-mediated apoptosis is a major concern in the development of death receptor agonists as anti-cancer therapies. However, the mechanisms by which tumor cells can develop resistance to death receptor-mediated apoptosis are unclear. Thus, the identification of agents that sensitize cells to death receptor agonists and that can counteract resistance is important both to clarify the resistant mechanism and to overcome the resistance. A few reports have indicated that glycogen synthase kinase-3 (GSK3) (Hoefflich et al 2000; Song et al 2004), DDX3 (Li et al 2006), and cIAP-1 (Wang et al 1998) have anti-apoptotic actions in death receptor-mediated apoptosis, so might contribute to resistance. In the present study, we investigated the anti-apoptotic mechanism of GSK3 in blocking apoptosis induced by activation of tumor necrosis factor-related apoptosis-inducing ligand receptor-2 (TRAIL-R2) to test if GSK3 inhibitors might improve cancer therapies, especially for resistant cancer cells. Our study showed that inhibition of GSK3 dramatically increased TRAIL-R2-induced tumor cell death. This potentiation of apoptosis by GSK3 inhibitors was also evident in a cell line derived from MDA-231 breast cancer cells that was developed for high resistance to TRAIL-R2-induced apoptosis, showing that these agents overcome resistance to death receptor-induced apoptosis. We found that prior to activation, TRAIL-R2 is capped by an anti-apoptotic complex containing GSK3, DDX3, and cIAP-1, and that GSK3 is required for DDX3 and cIAP-1 binding to TRAIL-R2. This anti-apoptotic complex inhibits formation of the death-inducing signaling complex (DISC) at TRAIL-R2, thereby restraining apoptotic signaling. Stimulation of death receptors can overcome this anti-apoptotic complex by disabling these proteins, causing inactivation of GSK3 and cleavage of DDX3 and cIAP-1, to enable progression of the apoptotic signaling cascade. In cells resistant to TRAIL-R2-induced apoptosis, GSK3 was not inactivated by death receptor stimulation, so the anti-apoptotic complex maintained a blockade of apoptotic signaling, and inhibition of GSK3 overcame this block, allowing death receptor stimulation to cause apoptosis even in resistant cells. Thus, the anti-apoptotic complex of GSK3, DDX3 and cIAP-1 associated with TRAIL-R2 counterbalances apoptotic signaling and may contribute to cancer cell resistance to death receptor-induced apoptosis.