Proteasome inhibitors disrupt the unfolded protein response in myeloma cells

Abstract

Novel agents that target the proteasome, a proteolytic complex responsible for the degradation of ubiquitinated proteins, have demonstrated remarkable therapeutic efficacy in multiple myeloma, a plasma cell malignancy. However, the mechanism by which these compounds act remains unknown. A signaling pathway called the unfolded protein response (UPR) allows cells to handle the proper folding of proteins. The transcription factor XBP-1, a regulator of the UPR, is also required for plasma cell differentiation, suggesting a link between the UPR and plasma cell differentiation. Here we show that proteasome inhibitors target XBP-1 and the UPR in myeloma cells. Proteasome inhibitors suppress the activity of the translumenal endoplasmic reticulum endoribonuclease/kinase, IRE1 alpha, to impair the generation of the active, spliced XBP-1 species and simultaneously stabilize the unspliced species that acts as a dominant negative. Myeloma cells rendered functionally deficient in XBP-1 undergo increased apoptosis in response to endoplasmic reticulum stress. Identification of compounds that target the activity of IRE1 alpha/XBP-1 may yield novel therapies for the treatment of multiple myeloma and other malignancies that rely on an intact UPR.

Fig. 1.

PIs induce ER stress and caspase-12 activation, but suppress the UPR. (A) BiP and CHOP mRNA induction in NIH 3T3 or J558 myeloma cells treated with MG-132 (20 μM), Tm (10 μg/ml), or both. Cells were pretreated with MG-132 for 1 h and then further treated with Tm for 4 h. Ethidium bromide staining of the gel is shown at the bottom. (B) Alteration in the ratio of XBP-1 protein species in J558 cells treated with increasing amounts of MG-132 for 16 h. Cells undergoing apoptosis were counted by annexin V staining. (C) Inhibition of caspase-12 processing by PIs. Processing of full-length caspase-12 was examined by Western blotting in J558 myeloma cells treated with thap-sigargin (1 μM) or PIs (20 μM) during the indicated time periods. (D) Time course of the XBP-1s to -1u shift. Cells were treated with MG-132 (1 μM) for the indicated times, and XBP-1u and -1s protein levels and cell death were determined. (E) Alteration in the ratio of XBP-1 protein species in the MM.1s human myeloma cell line. Cells were treated with PS-341 (8 nM) in a time course analysis, and XBP-1 protein species were quantified.

Fig. 2.

Effect of PIs on IRE1α-mediated XBP-1 mRNA splicing. (A) XBP-1 mRNA levels in ER-stressed J558 cells treated with Tm for4hinthe absence or presence of MG-132. Cells were pretreated with MG-132 for 1 h before adding Tm. (B) The ratio of XBP-1u to -1s mRNA as revealed by RT-PCR analysis with a probe set spanning the spliced-out region as demonstrated (22). (C) Effect of a panel of PIs on XBP-1 splicing. Cells were treated with Tm for 4 h in the absence or presence of MG-132 (10 μM), PS-341 (10 μM), lactacystin (10 μM), ZL₃VS (50 μM), or AdaAhxL₃VS (50 μM). (D) IRE1α phosphorylation in NIH 3T3 cells treated with Tm as indicated after 2 h of pretreatment with MG-132 (10 μM).

Fig. 3.

PIs stabilize XBP-1u protein to act as a dominant-negative inhibitor of XBP-1s activity. (A) Ubiquitination of XBP-1 in HeLa cells cotransfected with XBP-1u and His-tagged ubiquitin expression plasmids. (B) Degradation rates of XBP-1u and -1s proteins as determined by pulse-labeling J558 cells with [³⁵S]Met/[³⁵S]Cys for 1 h and chasing for the indicated times. (C) Generation and expression of lysine to arginine XBP-1u mutants. Two or three lysine residues in the C terminus of XBP-1u were replaced by arginine to generate XBP-1uKK (235, 252) and XBP-1uKKK (146, 235, 252) by site-directed mutagenesis. dn-XBP contains the N-terminal 188 aa of XBP-1u. Western blot analysis was performed with NIH 3T3 extracts transfected with the indicated plasmids. (D) Effect of XBP-1u on XBP-1s-dependent UPR element (UPRE) activation in PI-treated NIH 3T3 cells with 8-fold excess of XBP-1u plasmids. Transfected cells were treated with MG-132 for 16 h before harvesting for luciferase assays. Values represent fold induction of activity compared with the reporter alone after normalizing to Renilla. (E) Inhibition of XBP-1s-dependent activation of the UPRE reporter in NIH 3T3 cells by XBP-1u lysine to arginine mutants.

Fig. 4.

Cells with an impaired UPR are more sensitive to ER stress-induced apoptosis. (A) Synergistic effect of Tm and MG-132 on apoptosis. Annexin V-positive cells were counted after treating J558 cells for 18 h with suboptimal concentrations of Tm and MG-132 as indicated. (B) Generation of J558-iXBP cells by retroviral transduction of J558 cells with the U6 promoter-based XBP-1 RNAi vector. (C) XBP-1-dependent gene expression in J558 cells that express control GFP, dn-XBP, or iXBP-1 treated with Tm. Generation of dn-XBP-1 J558 cells by infection with a retrovirus containing dn-XBP cDNA inserted into the GFP-RV vector (22). ERdj4, p58^IPK, and BiP gene expression was examined by Northern blot analysis. (D) Increased apoptosis in iXBP-1- and dn-XBP-1-expressing J558 cells. Cells were treated with the indicated amounts of Tm for 48 h, and dead cells were counted after annexin V staining.