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Spliced XBP1 Levels Determine Sensitivity of Multiple Myeloma Cells to Proteasome Inhibitor Bortezomib Independent of the Unfolded Protein Response Mediator GRP78

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Spliced XBP1 Levels Determine Sensitivity of Multiple Myeloma Cells to Proteasome Inhibitor Bortezomib Independent of the Unfolded Protein Response Mediator GRP78

Bojana Borjan et al. Front Oncol.

Abstract

Background: Mechanisms mediating resistance against the proteasome inhibition by bortezomib (BTZ) in multiple myeloma (MM) cells are still unclear. We analyzed the activation of the unfolded protein response (UPR), induction of prosurvival, and apoptotic pathways after proteasome inhibition in BTZ-sensitive and -resistant cells. Thereafter, these findings from tissue culture were proofed on MM cells of BTZ-sensitive and BTZ-refractory patients. Methods: Proteasomal and ABC transporter activities were measured in sensitive and resistant cell lines by the use of the respective substrates. TP53 gene loss and mutations were determined by cytogenetics and targeted NGS. UPR pathways, proteasome subunit levels and protein secretion were studied by Western Blot analysis, and apoptosis was determined by flow cytometry. MM cell lines were stably transfected with inducible GRP78 expression to study unfolded protein expression. Transient knock-down of GRP78 was done by RNA interference. Splicing of XBP1 and expression of GRP78 was studied by real-time PCR in CD138-enriched MM primary cells of BTZ-refractory and -sensitive patients. Results: BTZ-sensitive cells displayed lower basal proteasomal activities. Similar activities of all three major ABC transporter proteins were detected in BTZ-sensitive and resistant cells. Sensitive cells showed deficiencies in triggering canonical prosurvival UPR provoked by endoplasmic reticulum (ER) stress induction. BTZ treatment did not increase unfolded protein levels or induced GRP78-mediated UPR. BTZ-resistant cells and BTZ-refractory patients exhibited lower sXBP1 levels. Apoptosis of BTZ-sensitive cells was correlating with induction of p53 and NOXA. Tumor cytogenetics and NGS analysis revealed more frequent TP53 deletions and mutations in BTZ-refractory MM patients. Conclusions: We identified low sXBP1 levels and TP53 abnormalities as factors correlating with bortezomib resistance in MM. Therefore, determination of sXBP1 levels and TP53 status prior to BTZ treatment in MM may be beneficial to predict BTZ resistance.

Keywords: UPR; bortezomib; myeloma; p53; resistance.

Figures

Figure 1
Figure 1
MM cells are sensitive to inhibition of proteasome and disturbance of endoplasmic calcium homeostasis. To examine the apoptosis-inducing potential of bortezomib (BTZ) and thapsigargin (TG), cells were treated with increasing doses of the drug for 24 h. Flow cytometric analysis of cell death in multiple myeloma cell lines (OPM-2, NCI-H929, MM1.S, and U266), solid tumor cell lines (MDA-MB-231, HRT-18, PC-3) and primary fibroblasts (PFF). Apoptosis was determined using Annexin V-PerCP eFluor 710 staining of externalized phosphatidylserine. The cells were incubated with increasing concentrations of the (A) BTZ, reversible proteasome inhibitor or (B) thapsigargin, a drug disturbing Ca homeostasis of the endoplasmic reticulum. *p < 0.05, i.e., first concentration with significant cell death. Statistical analyses were performed with the GraphPad Prism™ software for Windows. Student's T-test 2-tailed, two-way ANOVA, and Mann–Whitney U-Tests were used to study differences between groups.
Figure 2
Figure 2
Analysis of proteasome levels, activity, and ubiquitinated proteins in MM and solid tumor cell lines. Proteasome core subunit expression beta 5i (A) and beta 3 (B) was analyzed by Western Blot (105 cells per lane). Experiments were repeated three times to calculate relative expression normalized to cell number and compared with reference control PFF (mean expression was set to 1). In comparison to BTZ-resistant solid tumor cell lines and fibroblasts, MM cells displayed lower proteasomal subunit expression when correlated to equal cell numbers. (C) Equal cell numbers of MM and solid tumor cell lines were exposed to BTZ (10 nM) for 6 h. Thereafter, chymotrypsin-like (CT-L) activity was detected by an assay with viable cells monitoring cleavage of substrate peptide after uptake. CT-L activity is displayed as relative luminescence unit (RLU). BTZ-resistant cells display significant higher residual CT-L activity after BTZ-treatment. (D) Dot blot analysis of the accumulation of ubiquitinated proteins in MM and solid tumor cells after treatment with the proteasome inhibitor. Equal cell numbers of MM and solid tumor cell lines were exposed for 6 h to BTZ (10 nM). Thereafter, proteins were analyzed by an anti-Ubiquitin Lys-48 specific antibody in a Dot Blot analysis and subsequently quantified. Mean of three experiments ± SD is shown. *p < 0.05.
Figure 3
Figure 3
Proteasome inhibition does not lead to higher amounts of unfolded proteins in MM cell line. (A) OPM-2TetRGRP78−FLAG were stimulated with 10 μg/mL tetracycline for 48 h to achieve a robust expression of GRP78-FLAG. Confocal immunofluorescence shows inducible GRP78-FLAG expression stained by anti-FLAG primary antibody. The nuclei are counterstained with DAPI (blue). Scale bar represents 10 μm. (B) After 48 h of tetracycline induction, OPM-2TetRGRP78−FLAG were treated with 10 nM BTZ for additional 6 h. Cell lysates were immunoprecipitated (IP) with anti-FLAG antibody to enrich co-precipitated unfolded proteins like the λ light chains, which subsequently were detected by Western Blot analysis. (C) Western Blot analysis of OMP-2WT cells displaying total intracellular λ light chain protein levels in a time course up to 12 h after treatment with 10 nM BTZ. A representative result of three independent experiments is shown.
Figure 4
Figure 4
MM cells lack full ability to respond by canonical UPR prosurvival signaling after disturbance of ER homeostasis. MM, PC-3 and PFF cells were treated with TG (5 nM), and activation of UPR was analyzed in a time course of 3–24 h. (A) Proteins specific to the three main branches of UPR (i.e., upregulation of GRP78 and IRE1α, and PERK phosphorylation) were detected by Western blot. The data represents at least three repeated experiments. The red square indicates significant upregulation of protein or phosphorylation-induced mobility shift (p-shift; PERK). Numbers indicate mean upregulation as determined by densitometry of chemoluminescence signals of two experiments. (B) Moreover, real-time PCR analysis was performed to monitor changes in HSPA5 (GRP78) and ERN1 (IRE1α) gene expression after BTZ-treatment. Mean ± SD of three independent experiments. (C) Splicing of the XBP1 primary transcript was analyzed in MM and solid tumor cell lines after induction of ER stress. Mean ± SD of three independent experiments. *p < 0.05.
Figure 5
Figure 5
MM cells sensitive to BTZ display high IRE1α and sXBP1 levels. (A) Western Blot analysis of IRE1α and GRP78 protein expression in sensitive and resistant cells. (B) Western Blot analysis of IRE1α and tubulin protein expression in MM plasma B cells and peripheral blood mononuclear cells (PBMCs) and CD-19 enriched B-cells. (C) Spliced XBP1 transcript levels were analyzed in sensitive and resistant cells by real-time PCR. RNA levels in cell lines were normalized to 18S and expression values of PFF (upper panel). RNA levels in cell lines were normalized to 18S and expression values of PBMCs (lower panel). Mean ± SD of three independent experiments. *p < 0.05.
Figure 6
Figure 6
MM cells of patients sensitive to BTZ treatment have high sXBP1 levels. Relative mRNA levels of sXBP1 (A) and HSPA5 gene expression (B) were analyzed in CD138± enriched cells isolated from bone marrow aspirates of BTZ-responsive (n = 10) and resistant (n = 10) MM patients by real-time PCR. RNA levels in cell lines were normalized to ACTB, *p < 0.05.
Figure 7
Figure 7
BTZ-sensitive MM cell lines have intact p53/NOXA responses for induction of apoptosis. (A) Western Blot analysis of p53 and NOXA protein expression after treatment with BTZ (10 nM, 8 h). GAPDH was used as a loading control. The data represents at least three independent experiments. Red squares indicate significant upregulation and mean value after densitometric quantification of chemoluminescence. (B) Real time PCR analysis of PMAIP1 (NOXA) and BBC3 (Puma) gene expression after treatment with BTZ (10 nM, 8 h). The data is expressed as mean ± SD of three independent experiments. *p < 0.05. (C) Frequency of TP53 mutation and deletions across CD138± cells isolated from bone marrow aspirates from BTZ-responsive (n = 10) and refractory (n = 10) MM patients. NGS was performed on all patients showing no 17p deletion in cytogenetics. One BTZ-refractory patient (MM#18) with no 17p deletion displayed a M246V TP53 mutation. All NGS results of TP53 mutational status can be found in Table S2.

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