Proteasome inhibitors induce a terminal unfolded protein response in multiple myeloma cells

Abstract

Multiple myeloma (MM) is an incurable plasma cell malignancy. The 26S proteasome inhibitor, bortezomib, selectively induces apoptosis in MM cells; however, the nature of its selectivity remains unknown. Here we demonstrate that 5 different MM cell lines display similar patterns of sensitivity to 3 proteasome inhibitors (PIs) but respond differently to specific NF-kappaB inhibition. We further show that PIs initiate the unfolded protein response (UPR), a signaling pathway activated by the accumulation of misfolded proteins within the endoplasmic reticulum (ER). Consistent with reports that prosurvival/physiologic UPR components are required for B-cell differentiation into antibody-secreting cells, we found that MM cells inherently expressed the ER chaperones GRP78/Bip and GRP94/gp96. However, bortezomib rapidly induced components of the proapoptotic/terminal UPR, including PERK, the ER stress-specific eIF-2alpha kinase; ATF4, an ER stress-induced transcription factor; and its proapoptotic target, CHOP/GADD153. Consistent with our hypothesis that PIs induce the accumulation of misfolded ER-processed proteins, we found that the amount of immunoglobulin subunits retained within MM cells correlated with their sensitivity to PIs. These findings suggest that MM cells have a lower threshold for PI-induced UPR induction and ER stress-induced apoptosis because they constitutively express ER stress survival factors to function as secretory cells.

Figure 1.

Different multiple myeloma cell lines have different levels of sensitivity to proteasome inhibitors. Five different human multiple myeloma cell lines were cultured for 12 (A) and 24 (B) hours in increasing concentrations of the proteasome inhibitors epoxomycin (EPOX), PSI, and Bortezomib (BZ). Cell viability was determined by flow cytometry following Annexin V–FITC and propidium iodide staining. The data are presented as the mean ± SD from at least 3 independent experiments.

Figure 2.

Multiple myeloma cell lines respond differently to direct inhibition of NF-κB than they do to direct inhibition of the 26S proteasome. (A) Whole-cell lysates were prepared from untreated multiple myeloma cell lines, cells treated with 5 μM BAY 11-7082 or cells treated with 100 nM (8226/S and U266 cells), 25 nM (KMS-11, KMS-18), or 5 nM (MM.1S) bortezomib and incubated with a double-stranded NF-κB oligonucleotide probe. Samples were run on a native polyacrylamide gel, and NF-κB binding was visualized by autoradiography. (B) Five different human multiple myeloma cell lines were cultured for 24 hours in increasing concentrations of the NF-κB inhibitor BAY 11-7082. Cell viability, as assessed by Annexin V–FITC and propidium iodide staining, is presented as the mean ± SD from at least 3 independent experiments.

Figure 3.

Multiple myeloma cell lines constitutively express high levels of physiologic UPR components. Five different multiple myeloma cell lines were treated for 12 and 24 hours with 50 μM tunicamycin (TM), 1 μM brefeldin A (BfA), 30 μM melphalan (Mel), 250 nM staurosporine (STS), or the indicated concentration of the proteasome inhibitors epoxomycin (EPOX), PSI, or bortezomib (BZ). Whole-cell lysates were isolated, subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes. The membranes were sequentially probed for GRP94, GRP78, and actin. The relative induction (Rel. Ind.) is the amount of protein present in treated samples relative to untreated cells after normalizing protein loading to actin using densitometry. Cell viability was assessed from an aliquot of the treated cells by Annexin V–FITC and propidium iodide staining. Representative blots from at least 3 independent experiments are shown.

Figure 4.

eIF-2α is phosphorylated in multiple myeloma cells treated with a variety of cytotoxic agents. The indicated multiple myeloma cell lines were cultured in 50 μM tunicamycin (TM), 1 μM brefeldin A (BfA), 30 μM melphalan (Mel), 250 nM staurosporine (STS), or the indicated concentration of the proteasome inhibitors epoxomycin (EPOX), PSI, or bortezomib (BZ). After 12 and 24 hours of treatment, cell viability was determined by Annexin V–FITC and propidium iodide staining, and whole cell lysates were isolated to sequentially evaluate the amount of phosphorylated and total eIF-2α present by Western blot analysis. The data are representative of at least 3 different experiments.

Figure 5.

Proteasome inhibitors specifically induce the expression of the transcription factor ATF4. The indicated multiple myeloma cell lines were treated for up to 12 hours with 50 μM tunicamycin (TM), 30 μM melphalan (Mel), or the indicated concentration of bortezomib (BZ). Whole-cell lysates were prepared, and Western blots for ATF4 were performed. The blots were then stripped and reprobed for actin to confirm equal protein loading. Representative blots from 3 independent experiments are shown.

Figure 6.

Proteasome inhibitors rapidly up-regulate a protein specifically involved in ER stress–induced apoptosis. (A) The 8226/S, U266, and MM.1S human multiple myeloma cell lines were cultured for up to 24 hours in 50 μM tunicamycin (TM), 1 μM brefeldin A (BfA), or the indicated concentration of epoxomycin (EPOX) or bortezomib (BZ). At the indicated time points, an aliquot of cells was taken to assess cell viability by Annexin V–FITC and propidium iodide staining, and the remainder of the cells was used to prepare whole-cell lysates. Induction of GADD153 and actin expression were determined by Western blot analysis. Blots representative of at least 3 independent experiments are shown. (B) Five different human multiple myeloma cell lines were treated for 12 and 24 hours with 50 μM tunicamycin (TM), 1 μM brefeldin A (BfA), 30 μM melphalan (Mel), 250 nM staurosporine (STS), or the indicated concentration of the proteasome inhibitors epoxomycin (EPOX), PSI, or bortezomib (BZ). Western blot and cell viability analyses were performed as described above. The data are representative of at least 3 independent experiments.