ANTICANCER RESEARCH 35: 3307-3316 (2015)

Bortezomib Causes ER Stress-related Death of Acute Promyelocytic Leukemia Cells Through Excessive Accumulation of PML–RARA

MARIKO TAKENOKUCHI, KAZUHIDE MIYAMOTO, KATSUYASU SAIGO and TAIZO TANIGUCHI

Faculty of Pharmacological Sciences, Himeji Dokkyo University, Himeji, Hyogo, Japan

Abstract. Background/Aim: The success of proteasome incorporating proteasome inhibitors in the treatment of inhibitors in therapy of multiple myeloma has led to their use diseases expressing aberrant . Furthermore, monitoring for other malignancies. For the proteasome inhibitor of UPS-related enzymes might have use in predicting the bortezomib, combination therapies with histone deacetylase treatment response to proteasome inhibitors and in assessing inhibitors, which up-regulate -proteasome system their therapeutic effects. (UPS)-related enzymes, produce a beneficial effect. However, the mechanisms underlying the effect of bortezomib are not The ubiquitin–proteasome system (UPS) controls normal completely understood. We hypothesized that bortezomib homeostasis in cells, and therefore influences cellular causes excessive accumulation of aberrant proteins, which processes such as intracellular protein processing and augments endoplasmic reticulum (ER) stress, leading to death degradation, apoptosis, inflammation, antigen presentation, of malignant cells. Materials and Methods: The NB4 cell line cell growth and survival, and cell-cycle control (1-3). established from a patient with acute promyelocytic leukemia Ubiquitination is catalyzed by E1 ubiquitin-activating (APL) expressing the promyelocytic leukemia/retinoic acid enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin receptor alpha (PML–RARA) fusion protein was used to assess ligases. E1 enzymes activate ubiquitin ATP-dependently and changes in cell viability and apoptosis caused by bortezomib, transfer ubiquitin to E2 enzymes. Really Interesting New as well as alterations in PML–RARA and UPS-related enzymes (RING), Homologous to the E6-AP Carboxyl Terminus via western blotting and immunoprecipitation assays. Results: (HECT), or U-box domain E3s mediate substrate binding and Bortezomib time- and dose-dependently reduced cell viability recruit E2s, thereby allowing transfer of the E2 ubiquitin and induced apoptosis. Bortezomib significantly increased the cargo to a substrate (4, 5). A poly-ubiquitinated protein is abundance of ubiquitinated-PML–RARA (Ub-PML–RARA), then recognized by the 19S regulatory subunit of the 26S ubiquitin-conjugating human enzyme 8 (UbcH8), and Ub- proteasome and degraded by the 20S core particle (6). UbcH8, indicating that UbcH8 is the E2 ubiquitin-conjugating Several strategies to block UPS components in the enzyme for PML–RARA. Moreover, UbcH8 abundance was treatment of cancer have been devised. Bortezomib (Velcade®, dose-dependently increased in the culture supernatant of PS-341) is a first-in-class proteasome inhibitor that selectively bortezomib-treated cells. Conclusion: UbcH8 may have a and reversibly blocks the 26S chymotrypsin-like activity of the utility as a biomarker of treatment response to bortezomib in proteasome and ultimately leads to modulation of the patients with APL. Furthermore, bortezomib impairs the UPS abundance and functions of many intracellular proteins, which that controls normal protein homeostasis by causing excessive is associated with cytotoxic effects on malignant cells (7). accumulation of PML–RARA augmenting ER stress and Bortezomib has been used worldwide as a treatment for leading to APL cell death. The study provides a rationale for multiple myeloma (MM) since its approval by the United States Food and Drug Administration in 2003 (8). The anti- myeloma effect of bortezomib is at least partially due to inhibition of the transcription factor nuclear factor kappa-light- Correspondence to: Mariko Takenokuchi, Faculty of Pharmacological chain-enhancer of activated B-cells (NFĸB) (7, 9, 10). NFĸB Sciences, Himeji Dokkyo University, 7-2-1 Kamiohno, Himeji, Hyogo is normally sequestered in the cytoplasm as an inactive 670-8524, Japan. Tel/Fax: +81 79 2236853, e-mail: tmariko@himeji- complex via association with its endogenous inhibitor IĸBα. du.ac.jp When cells are stimulated, IĸBα is rapidly phosphorylated by Key Words: Proteasome inhibitor, ubiquitin-proteasome system, IĸB kinase. Phosphorylated IĸBα is specifically recognized by PML–RARA, ER stress. Skp1-Cul1-F-box (SCFβ-TrCP) E3 ligase, ubiquitinated, and

0250-7005/2015 $2.00+.40 3307 ANTICANCER RESEARCH 35: 3307-3316 (2015) transferred to the proteasome for degradation, while NFĸB is was ubiquitinated in the presence of ubiquitin conjugating translocated to the nucleus and mediates transcription of many human enzyme 8 (UbcH8), UbcH6, UbcH5a, UbcH5b, or (11, 12). Therefore, inhibition of IĸBα degradation via UbcH5c (34, 35), indicating that each of these proteins can proteasomal inhibition prevents transcriptional activation of act as E2-conjugating enzymes for SIAH1. NFĸB target genes (13). In most MM cell lines and primary As with MM, we hypothesize that bortezomib causes MM samples, NFĸB is constitutively activated, contributing to excessive accumulation of aberrant PML–RARA protein in carcinogenesis (14, 15). Because of its well-established role APL cells, augmenting ER stress and leading to cell death. in MM, the NFĸB pathway is thought to be a primary target of The aim of the study was to explore our hypothesis using the bortezomib. However, bortezomib-induced NFĸB inhibition PML–RARA-expressing APL cell line NB4. has not been demonstrated in MM cells from patients (16, 17), and Hideshima et al. reported that bortezomib activated, rather Materials and Methods than inactivated, the canonical NFĸB pathway in MM cells (18), suggesting that bortezomib-induced cytotoxicity was not Materials. The proteasome inhibitor bortezomib (Millennium, fully attributable to NFĸB inhibition. Cambridge, MA, USA) was obtained as a commercial formulation Accumulation of aberrant proteins, such as misfolded or (Velcade®) from Janssen Pharmaceutical K.K. (Tokyo, Japan), unfolded proteins, in the lumen of the endoplasmic reticulum dissolved in dimethyl sulfoxide (1.0 mM stock solution), and stored at −20˚C. For the cell viability assays, trypan blue (Sigma-Aldrich, (ER) induces ER stress. The unfolded protein response St. Louis, MO, USA), and sodium3’-[1-(phenylaminocarbonyl)-3,4- (UPR) is a feedback mechanism that prevents ER stress in tetrazolium]-bis(4-methyoxy-6-nitro) benzene sulfonic acid hydrate response to aberrant protein accumulation. The initial role of (XTT)-based cell proliferation test kits (Roche Diagnostics, Tokyo, the UPR is to restore normal cell function by inducing Japan) were purchased. For apoptosis assays, Sharp DNA Ladder expression of molecular chaperones, but if this process fails, Marker (RBC Bioscience, New Taiwan, Taiwan, ROC), and Muse™ then the ER can remove the aberrant proteins via the Annexin-V & Dead Cell reagent (Merck Millipore, Billerica, MA, proteasome in a process termed ER-associated degradation USA) were purchased. For immunoprecipitation and western blot analysis, primary antibodies to RARA (sc-551), horseradish (ERAD) (19). MM is characterized by the production of peroxidase (HRP)-labeled antibodies to actin (I-19), and anti-rabbit large amounts of monoclonal immunoglobulin (M-protein). (sc-2030) and anti-mouse (sc-2005) secondary antibodies were In MM, misfolded or incompletely assembled subunits of purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). oligomeric immunoglobulins, including M-protein, are UbcH8 (UBE2L6) was purchased from Abgent (San Diego, CA, degraded via the proteasome in ERAD (20), and Meister et USA), UbcH6 (UBE2E1) was purchased from Bioss Antibodies al. (21) and Gu et al. (22) reported that induction of (Woburn, MA, USA), UbcH5 (UBE2D1) was purchased from immunoglobulin production increases the sensitivity of Abcam (Cambridge, UK), and ubiquitin was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Protein A/G Sepharose 4 myeloma cells to bortezomib. These observations led us to Fast Flow was purchased from GE Healthcare (Tokyo, Japan). hypothesize that bortezomib causes excessive accumulation of aberrant proteins, such as M-protein, in MM cells, Cell culture. The NB4 APL cell line was a generous gift from Dr. Eiji augmenting ER stress and ultimately leading to cell death. Tatsumi (Kobe Tokushukai Hospital, Kobe, Japan). NB4 cells were Acute promyelocytic leukemia (APL) is characterized by grown in RPMI-1640 medium supplemented with 10% fetal bovine the presence of a promyelocytic leukemia/retinoic acid serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin receptor alpha (PML–RARA) fusion gene resulting from (Gibco, Tokyo, Japan) at 37˚C with 5% CO2, and the cells were used translocation t(15;17)(q22;q12) (23). The specific for experiments after 3 h of growth in FBS-free RPMI-1640 medium. PML–RARA transcript permits a precise diagnosis and provides a marker by which residual or recurrent disease Cell viability assays. Cell proliferation was measured with an XTT- based cell proliferation test according to the manufacturer’s may be identified (24-26). The PML–RARA oncoprotein instructions. Briefly, NB4 cells (1×104 cells/100 μl) were cultured inhibits granulocytic differentiation at the promyelocytic with or without 10 or 100 nM bortezomib in 96-well plates for 24, stage and promotes malignant transformation of 48, and 72 h at 37˚C. After the incubation, XTT solution was added hematopoietic progenitor cells, mainly by acting as a to the wells and the cells were incubated for 8 h. XTT is metabolized repressor of (27-29). There are some reports by the mitochondria of viable cells into soluble formazan, which was that PML–RARA, which does not occur naturally in normal detected at 450 nm using an ARVO MX micro plate reader (Perkin cells, undergoes proteasomal degradation as an aberrant Elmer, Waltham, MA, USA). The total number of viable cells was determined using the trypan blue dye exclusion test. protein (30), and proteasome inhibitors are known to induce apoptosis in APL cells (31, 32). While the identity of the Apoptosis assay. Analysis of apoptosis was performed by DNA UPS-related enzymes involved in M-protein degradation fragmentation assay (DNA laddering) and annexin-V/7- remain unclear, the E3 ligase seven in absentia homolog 1 aminoactinomycin D (AAD) assay. After the cells (2×106 cells/60 (SIAH1) is known to mediate PML–RARA degradation (33). mm dish) were cultured with or without 10 or 100 nM bortezomib Our group reported that the RING finger domain of SIAH1 for 48 h, the presence of internucleosomal DNA cleavage in NB4

3308 Takenokuchi et al: Bortezomib Causes ER Stress-related Death of APL Cells cells was investigated using DNA gel electrophoresis. The Sharp Protein A Sepharose 4 Fast Flow and 1 μg of rabbit IgG at 4˚C for DNA Ladder Marker kit was used according to the manufacturer’s 1 h under gentle agitation. After centrifugation at 18,000 ×g at 4˚C instructions. The patterns of DNA ladders were examined by for 20 min, the supernatant (cell lysate) was collected. Next, 100 electrophoresis on a 1.5% agarose gel containing ethidium bromide, μl of each cell lysate was incubated with 2 μg of antibodies to followed by observation under ultraviolet illumination on a RARA, UbcH5, UbcH6, or UbcH8 at 4˚C overnight under gentle Luminescent Image Analyzer (LAS-3000; Fujifilm, Tokyo, Japan). rotation, followed by incubation for 2 h at 4˚C with Protein A After the same treatments, 100 μl of the cell suspension was labeled Sepharose 4 Fast Flow. After centrifugation at 18,000 ×g for 20 for 20 min in the dark with the same volume of the Muse™ min at 4˚C, the precipitates were washed three times in lysis buffer. Annexin-V & Dead Cell reagent. Subsequently, quantitative Afterwards, the precipitates were added to 50 μl of 2× Laemmli detection of annexin-V/7-AAD-positive cells was performed with a sample buffer and boiled for 5 min to denature the protein and Muse™ Cell Analyzer (Merck Millipore). separate it from the protein-A beads, and western blotting was performed as described in the previous section. Western blot. To analyze protein expression in the cells and culture To detect target proteins in the culture supernatants, 15 ml of supernatants, NB4 cells were cultured with or without 10 or each supernatant sample from the bortezomib-treated cells was 100 nM bortezomib for 48 h. For cellular proteins, the cells were precleared with 1 ml of Protein G Sepharose 4 Fast Flow to washed with ice cold PBS and then extracted with lysis buffer remove immunoglobulin. After centrifugation at 25,000 ×g at 4˚C (50 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 0.1% sodium for 20 min, 10 μg of each primary antibody and 1 ml of Sepharose dodecyl sulfate, 0.5% deoxycholic acid, 0.02% sodium azide, 1% 4 Fast Flow were added to each supernatant sample. After NP-40, 2.0 mg/ml aprotinin, 1 mM phenylmethylsulfonylfluoride) incubation for 2 h at 4˚C and centrifugation at 25,000 ×g for 20 supplemented with Complete Protease Inhibitor Cocktail tablets min at 4˚C, the precipitates were subjected to western blotting as (Roche Diagnostics, Tokyo, Japan). After storage on ice for 30 min described above. at 4˚C, the lysates were centrifuged at 20,000 ×g for 30 min at 4˚C and the supernatants (soluble proteins) were collected. The protein Results concentration was measured using Pierce 660 nm Protein Assay (Pierce Biotechnology, Rockford, IL, USA). After each sample was mixed 1:1 with 5× Laemmli sample buffer (Bio-Rad Bortezomib reduces cell viability and induces apoptosis of Laboratories, Hercules, CA, USA) and boiled for 5 min, equivalent NB4 cells. To evaluate the antitumor effect of bortezomib, we amounts of protein were fractionated on 7% (for PML–RARA and used the NB4 APL cell line, which expresses the PML–RARA ubiquitin) or 15% (for UbcH5, UbcH6, UbcH8 and ubiquitin) fusion protein and mimics several features of clinically sodium dodecyl sulfate polyacrylamide gels. The proteins were isolated APL blast cells (36). NB4 cells were cultured with or transferred to polyvinylidene fluoride membranes (ATTO, Tokyo, Japan) by electroblotting, blocked with Tris(hydroxymethyl) without 10 or 100 nM bortezomib for 24, 48, or 72 h, and cell aminomethane-buffered saline (pH 7.4) with 0.05% Tween 20 viability was determined using the trypan blue dye exclusion (TBST), and supplemented with 0.3% non-fat dry milk for 1 h at test and the XTT assay. Bortezomib reduced NB4 cell viability room temperature (RT). The membranes were next incubated in a time- and dose-dependent manner (Figure 1a, b). After the overnight at 4˚C with primary antibodies against RARA (1:1000 cells were cultured with or without 10 or 100 nM bortezomib dilution in TBST), ubiquitin (1:1000 dilution in TBST), UbcH5 for 48 h, the occurrence of apoptosis was determined by (1:500 dilution in TBST), UbcH6 (1:500 dilution in TBST), or assessment of nucleosomal laddering on a 1.5% agarose gel. UbcH8 (1:500 dilution in TBST). After extensive washing with TBST, the membranes were incubated for 1 h at RT with As shown in Figure 1c, typical DNA fragmentation was dose- peroxidase-conjugated anti-mouse or anti-rabbit secondary dependently induced by bortezomib. Moreover, apoptotic and antibodies (1:100,000 dilution in TBST). β-Actin was used as the necrotic cells were detected by annexin V and 7-AAD internal control. The signals were visualized using an LAS-3000 staining, respectively. Treatment with 10 and 100 nM Image Analyzer (Fujifilm) after the membranes were incubated bortezomib for 48 h resulted in apoptosis rates of 9.82 and with ECL Prime Western Blotting Detection reagent (GE 34.59%, respectively, while vehicle-treated cells had an Healthcare) for 5 min. apoptosis rate of 2.34% (Figure 1d). These results demonstrate that bortezomib produces time- and dose-dependent anti-tumor Immunoprecipitation. To detect ubiquitinated and non-ubiquitinated PML–RARA, UbcH5, UbcH6, and UbcH8, 2×107 NB4 cells were effects on NB4 cells by inducing apoptosis. cultured with or without 10 or 100 nM bortezomib for 48 h and separated into cell pellets and culture supernatants by Bortezomib causes accumulation of ubiquitinated proteins in centrifugation. The cell pellets were washed with ice-cold PBS and NB4 cells. Accumulation of ubiquitinated proteins is a suspended with 1 ml of ice cold lysis buffer [20 mM Tris HCl (pH characteristic feature of proteasomal inhibition. To 8), 137 mM NaCl, 10% glycerol, 1% NP-40, 2 mM EDTA] with investigate the effect of bortezomib on protein ubiquitination, Complete Protease Inhibitor Cocktail tablets. After storage on ice NB4 cells were cultured with bortezomib (10 or 100 nM) for for 30 min at 4˚C, the lysates were centrifuged at 20,000 × g for 20 min at 4˚C and the soluble fractions were collected. After the 48 h, harvested, and subjected to western blot analysis with protein concentration was measured using Pierce 660 nm Protein antibodies for ubiquitinated proteins. Bortezomib treatment Assay, all lysates were diluted to the same concentration. Next, 0.5 caused dose-dependent accumulation of ubiquitinated ml of lysate from each sample was precleared with 100 μl of proteins in NB4 cells (Figure 2).

3309 ANTICANCER RESEARCH 35: 3307-3316 (2015)

Figure 1. Bortezomib reduces cell viability and induces apoptosis of NB4 cells. NB4 cells were cultured with bortezomib (BTZ) (10 or 100 nM) or vehicle for 0, 24, 48, or 72 h. Cell viability was determined using the trypan blue dye exclusion test (a) and the XTT assay (b). The data are presented as the mean±S.D. of the results of three independent experiments in triplicate. After 48 h of BTZ treatment at the indicated concentrations, the occurrence of apoptosis was determined by assessment of nucleosomal laddering (DNA fragmentation) on a 1.5% agarose gel electrophoresis (c) and quantitative detection of annexin- V/7- aminoactinomycin D (AAD) -positive cells using MuseTM Cell Analyzer (d). Data are representative of three independent experiments (c, d).

3310 Takenokuchi et al: Bortezomib Causes ER Stress-related Death of APL Cells

Bortezomib increases the abundance of ubiquitinated PML–RARA in NB4 cells and cell culture supernatant. To investigate the effect of bortezomib on the abundance of the PML–RARA fusion protein, NB4 cells were cultured with bortezomib (10 or 100 nM) or vehicle for 48 h and the culture supernatant was collected by centrifugation. Total protein was extracted from cells and subjected to western blotting with anti-RARA antibodies. Two bands were detected at approximately 110 kDa, which corresponded to the molecular weight of PML–RARA. The density of the lower band was reduced by bortezomib, whereas the density of the upper band was increased (Figure 3a). Immunoprecipitation with antibodies against RARA confirmed that the upper band was ubiquitinated PML–RARA (Ub-PML–RARA) (Figure 3b). Next, we examined whether PML–RARA was released into the culture supernatant. The supernatants were subjected to immunoprecipitation with antibodies to RARA, followed by western blotting with anti-RARA or anti-ubiquitin antibodies. In the supernatant, PML–RARA was detected at a very low level in comparison to that of the cell pellet Figure 2. Bortezomib causes accumulation of ubiquitinated proteins in NB4 (Figure 3c, left), and was not detected with the anti-ubiquitin cells. NB4 cells were cultured with bortezomib (BTZ) (10 or 100 nM) or antibodies (Figure 3c, right). These results demonstrate that vehicle for 48 h. Whole-cell lysates were extracted and western blot analysis bortezomib increased the abundance of Ub-PML–RARA, was performed with anti-ubiquitin antibodies. β-Actin was used as the and this effect was correlated with reduced cell viability and loading control. Data are representative of three independent experiments. induction of apoptosis (Figure 1).

Bortezomib increases the abundance of ubiquitin-containing Bortezomib increases the abundance of ubiquitin- E2-conjugating enzyme UbcH8 in NB4 cells. PML–RARA containing E2-conjugating enzyme UbcH8 in the culture turnover is critically dependent on the E3 ligase SIAH1 (33, supernatant from NB4 cells. Next, we measured UbcH8 and 37). We previously reported that the RING finger domain of Ub-UbcH8 in the culture supernatants of bortezomib- SIAH1 was mono-ubiquitinated in the presence of UbcH5a, cultured NB4 cells to determine whether these proteins are UbcH5b, UbcH5c, UbcH6, or UbcH8 (34, 35). Based on released into the medium. Western blotting of the culture these reports, we searched for the E2-conjugating enzyme supernatant after immunoprecipitation with each E2 for PML–RARA. Western blotting after immuno- antibody showed that abundance of Ub-UbcH8, but not precipitation with antibodies to UbcH5, UbcH6, or UbcH8 UbcH8, was increased in a dose-dependent manner by showed that UbcH5 and UbcH6 were abundant in NB4 cells bortezomib (Figure 5a, right). UbcH5 was not detected before treatment, and their expression levels remained (data not shown), while Ub-UbcH6 was detected at a low unchanged after bortezomib treatment (Figure 4a, left and level (Figure 5a, left). Western blotting with antibodies middle, lower bands). Moreover, Ub-UbcH5 and Ub-UbcH6 against ubiquitin showed that the abundance of Ub-UbcH8 were detected at low levels only after treatment with 100 was increased in a dose-dependent manner by bortezomib nM bortezomib (Figure 4a, left and middle, upper bands). (Figure 5b, right), whereas Ub-UbcH6 and Ub-UbcH5 were In contrast, levels of UbcH8 and Ub-UbcH8 were not detected after bortezomib treatment (Figure 5b, left). dramatically increased from their initial low levels after These data indicate that accumulated Ub-UbcH8 in NB4 bortezomib treatment (Figure 4a, right, upper and lower cells treated with bortezomib may be released from the band). Ub-UbcH8 expression was increased by bortezomib cells into the culture medium, and this process likely occurs treatment in a dose-dependent manner (Figure 4b, right), during cell death. and Ub-UbcH6 expression was slightly increased by bortezomib treatment (Figure 4b, left), whereas Ub-UbcH5 Discussion was not detected (data not shown). These results indicate that UbcH8 may act as the E2-conjugating enzyme for Due to the success of proteasome inhibitors in treating MM, PML–RARA. Moreover, Ub-UbcH8 and UbcH8 are more they are now being translated to other hematological responsive to bortezomib than PML–RARA. malignancies, including acute leukemia. Bortezomib showed

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Figure 3. Bortezomib increases the abundance of ubiquitinated promyelocytic leukemia/retinoic acid receptor alpha (PML–RARA) in NB4 cells and the cell culture supernatants. NB4 cells were cultured with bortezomib (BTZ) (10 or 100 nM) or vehicle for 48 h, and the cells and their culture supernatants were separated by centrifugation. Total protein was extracted and western blot analysis with antibodies against RARA was performed (a). The cells were immunoprecipitated with anti-RARA, followed by western blotting with anti-ubiquitin antibodies (b). The culture supernatants were immunoprecipitated with antibodies to RARA , followed by western blotting with anti-RARA (left) or anti-ubiquitin (right) antibodies (c). β-Actin was used as the loading control. The white triangle indicates 110-kDa PML–RARA (P/R). The black triangles indicate 118-kDa mono-ubiquitinated- PML–RARA (Ub-P/R). IP: Immunoprecipitation; asterisk: IgG heavy chain. Data are representative of three independent experiments.

modest clinical activity as a single-agent in patients with MM PML–RARA expressed in APL is a misfolded fusion and leukemia, but combination therapies with conventional oncoprotein that is linked to APL transformation (39, 40). chemotherapeutics, including other proteasome inhibitors Mutated proteins and proteins accidentally generated by such as carfilzomib and histone deacetylase inhibitors various mechanisms, such as chromosomal rearrangement, (HDACi), were clearly superior (38). However, the are recognized as misfolded proteins and degraded by the mechanisms underlying the effects of bortezomib are not UPS (41). Although the mechanism for degradation of completely understood. Although there are some reports that aberrant PML–RARA proteins has been investigated, the bortezomib produces antitumor effects indirectly by inhibiting degradation mechanism of M-protein is not well-understood. NFĸB, we believe that bortezomib produces antitumor effects In this study, using the PML–RARA-expressing APL cell on MM cells by acting directly on M-protein, an aberrant line NB4, we tested the hypothesis that bortezomib impairs protein. After bortezomib treatment, accumulation of aberrant protein quality control mechanisms by causing excessive proteins may cause ER stress-related cell death. To test this accumulation of aberrant proteins such as PML–RARA, hypothesis, we studied APL as a substitute for MM. leading to ER stress-related cell death.

3312 Takenokuchi et al: Bortezomib Causes ER Stress-related Death of APL Cells

Figure 4. Bortezomib increases the abundance of ubiquitin-containing E2-conjugating enzyme UbcH8 in NB4 cells. NB4 cells were cultured with bortezomib (BTZ) (10 or 100 nM) or vehicle for 48 h. The cells were immunoprecipitated with antibodies to UbcH5, UbcH6, or UbcH8, and western blotting was subsequently performed with each antibody (a) or with anti-ubiquitin antibodies (b). The white triangles indicate 17-kDa UbcH5 (U5), 24-kDa UbcH6 (U6), and 17-kDa UbcH8 (U8). The black triangles indicate 25-kDa ubiquitin-containing-UbcH5 (Ub-U5), 32-kDa ubiquitin- containing-UbcH6 (Ub-U6), and 25-kDa ubiquitin-containing-UbcH8 (Ub-U8). IP: Immunoprecipitation. Data are representative of three independent experiments.

We confirmed that bortezomib time- and dose-dependently previously reported that the RING finger domain of SIAH-1 induced death of NB4 cells, and demonstrated that some cell was mono-ubiquitinated in the presence of UbcH8, UbcH6, death was due to apoptosis. We also showed that bortezomib UbcH5a, UbcH5b, or UbcH5c (34, 35). Among these E2- caused accumulation of Ub-PML–RARA in a dose- conjugating enzymes, UbcH8 was detected at a very low level, dependent manner. When the amount of misfolded and whereas UbcH6 was detected at a moderate level and UbcH5 unfolded proteins exceeds the repair capacity of the ER, the was detected at a high level, indicating that UbcH5 and UPR is activated to re-establish ER homeostasis and avoid UbcH6 were endogenously abundant in NB4 cells. High levels cell damage. The UPR involves ERAD, in which aberrant of UbcH5 and UbcH6 were confirmed in an NB4-transplanted ER proteins are retrotranslocated to the cytosol, where they mouse APL model (data not shown). The levels of both are ubiquitinated and degraded by the proteasome (19). UbcH8 and Ub-UbcH8 were dramatically increased after Therefore, accumulation of PML–RARA caused by bortezomib treatment, while those of UbcH5/6 and Ub- proteasomal inhibition may exacerbate ER stress and UbcH5/6 showed little change. These data raise the possibility subsequently cause the UPR to switch from pro-survival that UbcH8 is the E2-conjugating enzyme for PML–RARA. mode to anti-survival mode. Our results suggest that It has been reported that modulating the abundance of accumulation of Ub-PML–RARA accounts, at least in part, enzymes catalyzing ubiquitination can affect target protein for cell death caused by bortezomib treatment. turnover (37). Kramer et al. reported that HDACi exposure To understand the process of PML–RARA degradation in increased expression of the E2-conjugating enzyme UbcH8, the proteasome, its ubiquitin-related enzymes were identified. triggering proteasomal degradation of PML–RARA (44) , and SIAH1 has been identified as the E3 ligase mediating these results provide further support for the notion that UbcH8 PML–RARA degradation (33). However, we detected SIAH1 is the E2-conjugating enzyme for PML–RARA. only after loading large amounts of protein or immuno- MM is characterized by the clonal expansion of plasma precipitation, corroborating reports of difficulties in detecting cells, the presence of a M-protein in the serum/urine, lytic SIAH1 due to its rapid turnover (42, 43). Therefore, we bone lesions, and organ damage. Because detection of serum focused on the E2-conjugating enzyme for PML–RARA. We M-protein is used for both disease diagnosis and follow-up

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Figure 5. Bortezomib increases the abundance of ubiquitin-containing E2-conjugating enzyme UbcH8 in the culture supernatant from NB4 cells. NB4 cells were cultured with bortezomib (BTZ) (10 or 100 nM) or vehicle for 48 h, and cells and supernatants were separated by centrifugation. Each supernatant was immunoprecipitated with anti-UbcH5, anti-UbcH6, or anti-UbcH8 and western blotting was performed with each antibody (a) or with anti-ubiquitin antibodies (b). The white triangles indicate 24-kDa UbcH6 (U6) and 17-kDa UbcH8 (U8). The black triangles indicate 32-kDa ubiquitin-containing- UbcH6 (Ub-U6) and 25-kDa ubiquitin-containing-UbcH8 (Ub-U8). IP: Immunoprecipitation. Data are representative of three independent experiments.

examination in patients with MM, we measured levels of There are reports that simultaneous treatment with an PML–RARA, Ub-PML–RARA, UbcH8 and Ub-UbcH8 in HDACi and a proteasome inhibitor results in accumulation the culture supernatant to investigate extracellular leakage of of aberrant proteins, leading to ER stress-related lethality accumulated proteins. As expected, Ub-UbcH8 abundance in (47). In addition, co-administration of bortezomib and the the culture supernatant was increased in a dose-dependent HDACi belinostat induced apoptosis of cultured and primary manner by bortezomib, whereas Ub-PML–RARA was APL cells (48). Taken together with these reports, our results detected at a very low level. When the UPR and ERAD fail, indicate that UbcH8 expression is directly associated with ER stress can trigger the lysosome-mediated autophagic PML–RARA degradation in the proteasome, under which protein degradation pathway to preserve cell survival (45). condition blocking the proteasome can induce further PML–RARA is known to be prone to aggregation, a feature accumulation of Ub-PML–RARA. Consequently, increased that makes it a good substrate for autophagic degradation ER stress may cause the UPR to switch to anti-survival (46). However, aggregate proteins are often less likely to mode. Therefore, up-regulation of UbcH8 caused by bind with antibodies. Autophagic degradation or low binding proteasome inhibitor and HDACi co-treatment makes APL affinity for the antibody could be the reason why cells more susceptible to cell death. Furthermore, the level PML–RARA was detected only at a very low level in the of UbcH8 before treatment might predict the response to culture supernatant. protease inhibitors. These observations raise the possibility

3314 Takenokuchi et al: Bortezomib Causes ER Stress-related Death of APL Cells that quantitative determination of serum UbcH8 or Ub- 9 Sartore-Bianchi A, Gasparri F, Galvani A, Nici L, Darnowski UbcH8 in patients with APL can be a predictive indicator for JW, Barbone D, Fennell DA, Gaudino G, Porta C and Mutti L: the response to bortezomib. Bortezomib inhibits nuclear factor-kappaB dependent survival and has potent in vivo activity in mesothelioma. Clinical cancer In the present study, we report that the proteasome inhibitor research: an official journal of the American Association for bortezomib, through its potent inhibition of protein quality Cancer Research 13: 5942-5951, 2007. control mechanisms, augments ER stress by causing excessive 10 Hideshima T, Chauhan D, Richardson P, Mitsiades C, Mitsiades accumulation of aberrant proteins, induces failure of the ER N, Hayashi T, Munshi N, Dang L, Castro A, Palombella V, stress-adaptive UPR, and leads to cell death. This study provides Adams J, and Anderson KC: NF-kappa B as a therapeutic target a rationale for incorporating proteasome inhibitors in the in multiple myeloma. The Journal of biological chemistry 277: treatment of patients with some diseases involving expression 16639-16647, 2002. of aberrant proteins. We also propose that monitoring UPS- 11 Biswas DK and Iglehart JD: Linkage between EGFR family receptors and nuclear factor kappaB (NF-kappaB) signaling in related enzymes, including E1 ubiquitin-activating enzymes, E2 breast cancer. Journal of cellular physiology 209: 645-652, ubiquitin-conjugating enzymes, and E3 ubiquitin ligases, may 2006. be a method of predicting the treatment response to proteasome 12 Solt LA and May MJ: The IkappaB kinase complex: master inhibitors and assessing their therapeutic effects. regulator of NF-kappaB signaling. Immunologic research 42: 3- 18, 2008. Conflicts of Interest 13 Hayden MS and Ghosh S: Signaling to NF-kappaB. Genes & development 18: 2195-2224, 2004. The Authors declare that they have no conflicts of interest with 14 Annunziata CM, Davis RE, Demchenko Y, Bellamy W, Gabrea regard to this study. A, Zhan F, Lenz G, Hanamura I, Wright G, Xiao W, Dave S, Hurt EM, Tan B, Zhao H, Stephens O, Santra M, Williams DR, Dang L, Barlogie B, Shaughnessy JD Jr., Kuehl WM, and Staudt Acknowledgements LM: Frequent engagement of the classical and alternative NF- kappaB pathways by diverse genetic abnormalities in multiple We are grateful to our colleagues at the Faculty of Pharmacological myeloma. Cancer cell 12: 115-130, 2007. Sciences of Himeji Dokkyo University for their secretarial and 15 Keats JJ, Fonseca R, Chesi M, Schop R, Baker A, Chng WJ, Van technical assistance. We are also grateful to Dr. Eiji Tatsumi (Kobe Wier S, Tiedemann R, Shi CX, Sebag M, Braggio E, Henry T, Tokushukai Hospital, Kobe, Japan) for the generous gift of NB4 Zhu YX, Fogle H, Price-Troska T, Ahmann G, Mancini C, Brents cells. 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