Reduced Response of IRE1α/Xbp-1 Signaling Pathway to Bortezomib Contributes to Drug Resistance in Multiple Myeloma Cells

Abstract

Purpose

Proteasome inhibition with bortezomib eliminates multiple myeloma (MM) cells by partly disrupting unfolded protein response (UPR). However, the development of drug resistance limits its utility and resistance mechanism remains controversial. We aimed to investigate the role of IRE1α/Xbp-1 mediated branch of the UPR in bortezomib resistance.

Methods

The expression level of Xbp-1s was measured in 4 MM cell lines and correlated with sensitivity to bortezomib. LP1 and MY5 cells with different Xbp-1s level were treated with bortezomib; then pivotal UPR regulators were compared by immunoblotting. RPMI 8226 cells were transfected with plasmid pEX4-Xbp-1s and exposed to bortezomib; then apoptosis was determined by immunoblotting and flow cytometry. Bortezomib-resistant myeloma cells JJN3.BR were developed and the effect on UPR signaling pathway was determined.

Results

By analyzing 4 MM cell lines, we found little correlation between Xbp-1s basic level and bortezomib sensitivity. Bortezomib induced endoplasmic reticulum stress-initiated apoptosis via inhibiting IRE1α/Xbp-1 pathway regardless of Xbp-1s basic level. Exogenous Xbp-1s reduced cellular sensitivity to bortezomib, suggesting the change of Xbp-1s expression, not its basic level, is a potential marker of response to bortezomib in MM cells. Furthermore, sustained activation of IRE1α/Xbp-1 signaling pathway in JJN3.BR cells was identified.

Conclusions

Our data indicate that reduced response of IRE1α/Xbp-1 signaling pathway to bortezomib may contribute to drug resistance in myeloma cells.

Introduction

In the last decade, the survival of patients with multiple myeloma (MM) has improved greatly. Much of this progress can be attributed to autologous stem cell transplantation and novel pharmacologic agents including proteasome inhibitors (1). Bortezomib is the first proteasome inhibitor applied extensively in the treatment of MM, and the ideal response rate has been achieved. Despite advances, MM remains an incurable disease, since an emerging challenge is drug resistance (2, 3). About one-third of patients are intrinsically resistant to bortezomib, and the long-term use of bortezomib therapy also results in the development of acquired drug resistance. This has hindered its therapeutic activity. The drug resistance mechanism must be understood in order to improve further efficacy.

The binding target of bortezomib has been characterized as 26S proteasome subunit β5 encoded by the PSMB5 gene. One compelling possibility accounting for bortezomib resistance is the development of mutations in the bortezomib binding site. Indeed, overexpression of PSMB5 or mutations of the PSMB5 gene have been identified in various types of tumor cells, including MM cell lines (4). Nevertheless, these studies were carried out in vitro, by developing myeloma cells adapted to bortezomib exposure. It remains controversial whether these models could be the representation of clinical bortezomib resistance (5). Gene sequencing studies have revealed that PSMB5 mutations are infrequent in patients with MM who are clinically bortezomib-resistant (6). Furthermore, proteasome activity differences have not been found in primary drug-resistant myeloma cells (7). These findings suggest that other mechanisms may be involved in clinical bortezomib resistance.

Induction of endoplasmic reticulum (ER) stress has been identified as the important mechanism for antitumor activity of bortezomib. Multiple myeloma cells are typified by strong activity of secretory immunoglobulin synthesis resulting in ER stress. The ER stress can be adaptively managed by ER-associated protein degradation (ERAD) pathway and unfolded protein response (UPR). However, when the stress becomes prolonged and uncompensated, cell apoptosis occurs due to the terminal UPR. Bortezomib is known to induce the terminal UPR in MM cells. It disturbs ER homeostasis through inhibiting proteasome-associated ERAD, but it disrupts the UPR as well (8, 9). Thus, there is a possibility that dependence on UPR renders bortezomib sensitivity in MM cells.

Three ER transmembrane proteins (IRE1α, PERK, ATF6) initiate the UPR, and the IRE1α/Xbp-1 pathway has a prosurvival role. Xbp-1 is an important transcription factor that regulates genes responsible for proteins folding and degrading (10). It was found that myeloma cells whose Xbp-1 gene was silenced became more sensitive to bortezomib (11). However, an in vivo study by Leung-Hagesteijn et al (12) showed that therapy-resistant myeloma cells are Xbp-1s negative plasma cells or plasma blasts. The relationship of Xbp-1 expression and bortezomib sensitivity is controversial. In addition, it has been reported that abnormal activation of UPR in bortezomib-treated MM cells leads to cell death, especially the suppression of IRE1α/Xbp-1 signaling pathway (13). We further hypothesized that response of IRE1α/Xbp-1 signaling pathway may be an important factor to determine sensitivity of MM cells to bortezomib-induced apoptosis. In the present study, we sought to explore the mechanism of bortezomib resistance.

Methods

Cell culture and related reagents

Human MM cell lines JJN3, RPMI 8226, MY5, and LP1 were provided by Professor Jingxuan Pan (Zhongshan Ophthalmic Center, Sun Yat-sen University). All the cell lines were cultured in IMDM (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Biological Industries, Cromwell, CT, USA), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 μM L-glutamine at 37°C in a humidified atmosphere of 95% air and 5% CO₂.

Development of BR cells

Bortezomib (Velcade) was provided by Xian Janssen Pharmaceutical Ltd. (Shaanxi, China). To establish human myeloma cell adaptively resistant to bortezomib, JJN3 cell lines were cultured in gradually increasing concentration of bortezomib continuously. The initial drug concentration was 4 nmol/L; then we increased the concentration to 15 nmol/L in stepwise increments of 0.5 nmol/L, in which MM cells retained stable growth.

Transfection and bortezomib treatment

Spliced Xbp-1 cDNA was cloned based on the sequence of GenBank/NM_001079539. Plasmid pEX4-Xbp-1s was produced by Shanghai GenePharma Co., Ltd., and their successful construction was identified by sequence analysis, without any base pair mutation.

For transfection, RPMI 8226 cells (2 × 10⁵) were seeded into 6-well plates and transfected with plasmid pEX4-Xbp-1s using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. After 48 hours, the cells were exposed to bortezomib and subjected to apoptosis assay by flow cytometry and immunoblotting analysis.

Immunoblotting

Cell lysates were separated by denaturing gel electrophoresis and transferred to nitrocellulose membranes and immunoblotted with primary antibodies: anti- caspase-3, IRE1α, Xbp-1s, PERK, eIF2α, phospho-eIF2α, ATF4, BIP/GRP78, and CHOP (Cell Signaling Technology, Beverly, MA, USA); phopsho-IRE1α (Ser724) (Abcam, Cambridge, UK); PARP (BD Biosciences, East Rutherford, NJ, USA); Actin (Sigma-Aldrich, St. Louis, MO, USA); and active caspase-3 (BD Biosciences). Then, anti-rabbit or anti-mouse fluorescence-labeled secondary antibody (1:10,000) was added. Protein bands were detected by Odyssey Infrared Imaging System (LI-COR, Lincoln, NE, USA), measured by densitometry using ImageJ software, and normalized to β-actin control.

Cell viability assay

Cell viability was evaluated using CEllTiter 96 AQueous One Solution Cell Proliferation Assay Kit (Promega, Madison, WI, USA). We took the average OD values at each concentration to draw up the cell proliferation curve using GraphPad Prism 6.0 software. The IC50 was determined by curve fitting of the dose-response curve and the mean of 3 determinations was calculated.

Apoptosis analysis by flow cytometry

Apoptosis was determined by Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) double staining (Sigma-Aldrich, Shanghai) and analyzed with a FACS C6 flow cytometer.

Statistical analysis

Data were analyzed using SPSS 19.0 statistical software. Data from all the experiments are expressed as means and error bars in the figures depict standard deviation (SD). The statistical analysis was conducted using one-way analysis of variance or t test. A p value of <0.05 was deemed significant.

Results

Sensitivity of myeloma cell lines to bortezomib is little associated with basic XBP-1s level

Four human MM cell lines were examined for bortezomib sensitivity, and their basic Xbp-1s expression levels were determined by immunoblotting. As is known, the IRE1α/Xbp-1 signaling pathway is differently activated in various stress states. Xbp-1s is a stress response molecule whose level can vary with cell states and culture conditions. That is the reason why we detected Xbp-1s protein level with the same aliquot of cells used for analysis of myeloma sensitivity to bortezomib. Four MM cell lines with different basic Xbp-1s level were used in the current study. As shown by immunoblotting, weak expression of Xbp-1s was observed in MY5 and JJN3 myeloma cells, but higher expression level in RPMI 8226 and LP1 cell lines (Fig. 1A). Besides, each cell line was treated with increasing concentrations of bortezomib for 72 hours. We found that JJN3 cells were the least sensitive to bortezomib, with an IC50 value of 21.98 nmol/L that was much more than the other 3 cell lines (p<0.01; Fig. 1B). These data demonstrate that there was no significant association between Xbp-1s protein level and sensitivity to bortezomib. In addition, another interesting finding was that inhibition effect of bortezomib on JJN3 cells was not enhanced significantly even when the concentration exceeded 50 nmol/L. We suppose that secondary drug resistance could be developed in JJN3 cell lines.

Fig. 1

The basic level of Xbp-1s is little associated with sensibility to bortezomib in multiple myeloma (MM) cells. (A) Xbp-1s expression was accessed by immunoblotting analysis in 4 MM cell lines. (B) Growth inhibitory effects of bortezomib on 4 MM cell lines with high or low expression of XBP-1s were evaluated. Cell viability (percentage relative to control) was determined by MTS assay (left). The data represent mean ± SD of 3 independent experiments. **p<0.01 (right).

Deregulation of UPR contributes to bortezomib-induced apoptosis regardless of Xbp-1s basic level

It seemed that bortezomib might similarly induce ER stress-initiated apoptosis of MM cells with different Xbp-1s basic level. To further investigate this possibility, we treated LP1 and MY5 cells with increasing concentration of bortezomib for 24 hours, then analyzed the relevance of UPR to bortezomib-induced apoptosis. The induction of BIP (a chaperone protein), CHOP (a transcriptional factor associated with ER stress-induced apoptosis), and Xbp-1s is a certain marker for UPR, and the cleavage of PARP and activation of caspase-3 are considered as hallmarks of apoptosis. Consequently, bortezomib induced substantial apoptosis as determined by Annexin V/PI double staining (Fig. 2, A and B). Activation of caspase-3 and cleavage of PARP were observed in bortezomib-treated myeloma cells, accompanied by moderate upregulation of CHOP and BIP at the protein level. Mildly elevated expression of Xbp-1s was detected at low concentration of bortezomib. All of these changes were indicative of ER stress. However, with the increase of drug concentration, the expression level of Xbp-1s was dramatically decreased, and the phosphorylation of IRE1α showed no significant change (Fig. 2C). These results confirm that bortezomib led to ER stress-induced apoptosis in MM cells regardless of Xbp-1s basic level, and the suppression of IRE1α/Xbp-1 signaling pathway could be one of the main mechanisms.

Fig. 2

Bortezomib induced apoptosis by disrupting unfolded protein response (UPR) in multiple myeloma (MM) cells regardless of Xbp-1s basic level. LP1 and MY5 cells were treated with increasing concentration of bortezomib for 24 hours; flow cytometry analysis was done after Annexin V-FITC/PI double staining. (A) Representative histograms are shown. (B) Results from 3 independent experiments are shown. *p<0.05, **p<0.01. (C) Dose-course Western blotting analysis of PARP, Caspase-3, and the key UPR markers are shown.

Exogenous Xbp-1s decreases the sensitivity of myeloma cells to bortezomib

Exogenous Xbp-1s directly plays biological effects independently on endoribonuclease activity of IRE1α. To examine the role of IRE1α/Xbp-1 signaling pathway in bortezomib resistance, RPMI 8226 cells transfected with pEX4-Xbp-1s plasmid or empty vector were treated with bortezomib or culture medium for 24 hours. We next determined the effect of exogenous Xbp-1s on bortezomib-induced apoptosis in MM cells. A massive apoptotic cell death was found in RPMI 8226 cells transfected with empty vector, whereas Xbp-1s-transfected cells display attenuated apoptosis, as indicated by activation of caspase-3, cleavage of PARP (Fig. 3A), and Annexin V/PI double staining (Fig. 3, B and C). These data demonstrate that overexpression of Xbp-1s potentiated the capacity of MM cells to develop bortezomib resistance.

Fig. 3

Overexpression of Xbp-1s induced survival in multiple myeloma (MM) cells. The RPMI 8226 cells were transfected with plasmid (empty vector or Xbp-1s). Twenty-four hours after transfection, the cells were treated with or without bortezomib and subjected to the apoptosis analysis (flow cytometry, B and C) or immunoblotting with PARP, Caspase-3, and Xbp-1s (A). Data shown are 1 representative result (B) and 3 independent experiments (C). ***p<0.0001.

Induction of bortezomib resistance in the MM cell line

As shown in previous studies, myeloma cells are typically sensitive to bortezomib-induced apoptosis. To establish bortezomib-resistant myeloma cells, we exposed JJN3 parental cells initially to low bortezomib dose (4 nmol/L), and then slowly increased the concentration to 15 nmol/L. The BR subline displayed a significant decrease in its sensitivity to the antiproliferation effect of bortezomib. Drug-naive JJN3 cells were sensitive to bortezomib with an IC50 value of 21.98 nmol/L. Whereas resistant subline JJN3.BR exhibited an approximately 50% decrease of viability, even it was exposed to 50 nmol/L bortezomib, indicating a nearly 2-fold increase in resistance (Fig. 4A).

Fig. 4

Reduced response of IRE1 α/Xbp-1 signaling mediates resistance to bortezomib. (A) JJN3 and JJN3.BR cells were incubated in escalating concentration of bortezomib for 72 hours followed by MTS assay. (B) Sustained activation of IRE1 α/Xbp-1 pathway was observed in JJN3. BR cells were exposed to bortezomib. After incubating with bortezomib for 24 hours, cell lysates were harvested and immunoblotting result (left) and the gray value analysis by ImageJ software from 3 independent experiments are shown. **p<0.01 (right). (C) Effects of bortezomib on PERK signaling and BIP were not identical in JJN3 and JJN3.BR cells. The cells were treated with bortezomib for 24 hours and subjected to immunoblotting with indicated antibodies.

Downregulation of XBP-1s and reduced response of IRE1α/Xbp-1 signaling to bortezomib in resistant myeloma cells

Bortezomib-resistant subline JJN3.BR was generated through the above method. To further investigate if attenuated response of IRE1α/Xbp-1 signaling to bortezomib occurs and plays an important role in bortezomib resistance, we next made a comparison of the changes in IRE1α/Xbp-1 signaling pathway proteins and other UPR markers between parental cell line and bortezomib-resistant subline. First, protein levels of Xbp-1s were found to be decreased substantially at baseline in JJN3.BR cells compared with JJN3 cells. Bortezomib led to the cleavage of PARP in both drug-naive and BR cells, but it was more enhanced in JJN3 cells. As shown by Western blotting, unlike JJN3 cells, JJN3.BR cells failed to inhibit the expression level of Xbp-1s in response to bortezomib, whereas the phosphorylation of IRE1α was enhanced (Fig. 4B). Immunoblotting profiling also confirmed that persistent activation of PERK signaling pathway was found in both cell lines. However, the JJN3.BR subline exhibited significantly less CHOP level but more BIP protein expression, which helped stabilize the ER homeostasis and support cellular survival (Fig. 4C). These results revealed that response of IRE1α/Xbp-1 signaling pathway may be an important factor that affects the sensitivity of myeloma cells to bortezomib.

Discussion

Multiple myeloma is known to be extremely sensitive to the proteasome inhibitor bortezomib. This mainly relates to the fact that those malignant plasma cells are characterized by massive protein synthesis, and untimely degradation of these proteins would provoke activation of ERAD pathway and the UPR (14-15-16). That likely explains the efficacy of bortezomib, which interferes with these key processes (8, 9). The UPR signaling pathway concerning IRE1α/Xbp-1 has been linked to bortezomib sensitivity (11, 17, 18).

Xbp-1 is an important transcript factor for regulating protein folding and degradation, and Xbp-1s is the active isoform (19). In the current study exploring the link between bortezomib sensitivity and Xbp-1s expression, we found that bortezomib sensitivity is little associated with basic Xbp-1s level in tested myeloma cell lines. Our results seemed to be inconsistent with previous studies. Several lines of evidence reveal that mature MM cells with high Xbp-1s level are more sensitive to bortezomib, and high Xbp-1 is an indicator of better outcome in MM patients treated with bortezomib. Though Xbp-1s level is reported to be associated with the differentiated status of MM cells, it also changes with cell states and culture conditions. Yang et al (11) showed that Xbp-1 gene silencing significantly enhanced the proapoptotic activity of bortezomib in MM cells. Hence, we suppose that Xbp-1s level does not determine sensitivity to bortezomib directly, but its response to bortezomib may be a marker of drug sensitivity.

Bortezomib causes the accumulation of more abnormal proteins through inhibiting ERAD, which exacerbates ER stress and activates the UPR signaling pathways. However, bortezomib induces the activation of ATF6 and PERK signaling pathways in response to ER stress, whereas Xbp-1s activity is minimally enhanced or even suppressed (8, 9). In our study, the response of IRE1α/Xbp-1 signaling pathway to bortezomib in the 2 tested myeloma cell lines was similar, and ER stress-induced apoptosis occurred following bortezomib treatment regardless of Xbp-1s level. Besides, the decline in sensitivity to bortezomib that followed exogenous Xbp-1s was in line with our assumption. Our results suggest that the specific inhibition on IRE1α/Xbp-1 pathway is an important antimyeloma mechanism of bortezomib, and the inhibition level may affect drug sensitivity.

Exploring the link between bortezomib resistance and response of IRE1α/Xbp-1 signaling pathway led us to develop a bortezomib-resistant myeloma cell subline. Analysis of the bortezomib-resistant myeloma cells revealed that downregulation of Xbp-1s level was accompanied by low level of CHOP and BIP. This points to the possibility that cellular adaptability gradually improves during the bortezomib-resistance induction process. We next found that UPR was significantly activated when cells were exposed to higher dose of bortezomib, but the consequences were different between the 2 cell lines. Notably, IRE1α/Xbp-1 signaling pathway was almost totally inhibited in parental cells, whereas there were no remarkable changes in the bortezomib-resistant subline. In parental cells, prolonged ER stress with the failure of protective mechanism by UPR predominantly induced proapoptotic gene CHOP expression, leading to cell death. However, CHOP gene was slightly activated in resistant cells and the expression of UPR-protective component BIP was more enhanced. This is in accord with the strong transcriptional activity of Xbp-1s, which can upregulate the expression of chaperone molecules and promote myeloma cell survival (18). These results strongly implicate deregulation of the IRE1α/Xbp-1 signaling pathway in acquired bortezomib resistance.

In conclusion, our study demonstrates the relationship between Xbp-1s basic level and bortezomib sensitivity is weak. In the context of myeloma, Xbp-1s basic level cannot fully reflect bortezomib response, but the change of Xbp-1s level followed by bortezomib treatment may be a potential response marker. The current findings suggest that reduced response of IRE1α/Xbp-1 signaling pathway is associated with bortezomib resistance, and inhibition of IRE1α/Xbp-1 signaling pathway may reverse bortezomib resistance in MM, though further research is needed to confirm these findings.

Footnotes

Financial support: Funded by the Natural Science Foundation of Guangdong Province, China (S20130110016838), Science and Technology Planning Project of Guangdong Province, China (2014A020212061), National High-tech R&D Program of China (863 program, 2013AA020104), and National Natural Science Foundation of China (81400170).

Conflict of interest: None of the authors has conflict of interest with this submission.

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