Long-term Incubation with Proteasome Inhibitors (PIs) Induces IκBα Degradation via the Lysosomal Pathway in an IκB Kinase (IKK)-dependent and IKK-independent Manner

Background: IB, a cytoplasmic inhibitor of NF-B, is degraded via the proteasome. Results: Paradoxically, proteasome inhibitors (PIs) induce IB degradation via the lysosome in an IKK-dependent and IKK-independent manner. Conclusion: PI-induced IB degradation results in NF-B activation that confers resistance to PI-induced cancer cell death. Significance: This provides a molecular mechanism to enhance the anti-cancer efficacy of PIs. Proteasome inhibitors (PIs) have been reported to induce apoptosis in many types of tumor. Their apoptotic activities have been suggested to be associated with the up-regulation of molecules implicated in pro-apoptotic cascades such as p53, p21(Waf1), and p27(Kip1). Moreover, the blocking of NF-B nuclear translocation via the stabilization of IB is an important mechanism of PI-induced apoptosis. However, we found that long-term incubation with PIs (PS-341 or MG132) increased NF-B-regulated gene expression such as COX-2, cIAP2, XIAP, and IL-8 in a dose- and time-dependent manner, which was mediated by phosphorylation of IB and its subsequent degradation via the alternative route, lysosome. Overexpression of the IB superrepressor (IB-SR) blocked PI-induced NF-B activation. Treatment with lysosomal inhibitors (ammonium chloride or chloroquine) or inhibitors of cathepsins (Z-FF-FMK or Z-FA-FMK) or knock-down of LC3B expression by siRNAs suppressed PI-induced IB degradation. Furthermore, we found that both IKK-dependent and IKK-independent pathways were required for PI-induced IB degradation. Pretreatment with IKK specific inhibitor, SC-514, partially suppressed IB degradation and IL-8 production by PIs. Blockade of IKK activity using insolubilization by heat shock (HS) and knock-down by siRNAs for IKK only delayed IB degradation up to 8 h after treatment with PIs. In addition, PIs induced Akt-dependent inactivation of GSK-3. Inactive GSK-3 accelerated PI-induced IB degradation. Overexpression of active GSK-3 (S9A) or knock-down of GSK-3 delayed PI-induced IB degradation. Collectively, our data demonstrate that long-term incubation with PIs activates NF-B, which is mediated by IB degradation via the lysosome in an IKK-dependent and IKK-independent manner.