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. 2009 Jul 30;114(5):1046-52.
doi: 10.1182/blood-2009-01-199604. Epub 2009 May 12.

Bortezomib induces canonical nuclear factor-kappaB activation in multiple myeloma cells

Teru Hideshima  1 , Hiroshi Ikeda, Dharminder Chauhan, Yutaka Okawa, Noopur Raje, Klaus Podar, Constantine Mitsiades, Nikhil C Munshi, Paul G Richardson, Ruben D Carrasco, Kenneth C Anderson
Affiliations

Bortezomib induces canonical nuclear factor-kappaB activation in multiple myeloma cells

Teru Hideshima et al. Blood. .

Abstract

Bortezomib is a proteasome inhibitor with remarkable preclinical and clinical antitumor activity in multiple myeloma (MM) patients. The initial rationale for its use in MM was inhibition of nuclear factor (NF)-kappaB activity by blocking proteasomal degradation of inhibitor of kappaBalpha (IkappaBalpha). Bortezomib inhibits inducible NF-kappaB activity; however, its impact on constitutive NF-kappaB activity in MM cells has not yet been defined. In this study, we demonstrate that bortezomib significantly down-regulated IkappaBalpha expression and triggered NF-kappaB activation in MM cell lines and primary tumor cells from MM patients. Importantly, no inhibition of p65 (RelA) nuclear translocation was recognized after bortezomib treatment in a murine xenograft model bearing human MM cells. Bortezomib-induced NF-kappaB activation was mediated via the canonical pathway. Moreover, other classes of proteasome inhibitors also induced IkappaBalpha down-regulation associated with NF-kappaB activation. Molecular mechanisms whereby bortezomib induced IkappaBalpha down-regulation were further examined. Bortezomib triggered phosphorylation of IkappaB kinase (IKKbeta) and its upstream receptor-interacting protein 2, whereas IKKbeta inhibitor MLN120B blocked bortezomib-induced IkappaBalpha down-regulation and NF-kappaB activation, indicating receptor-interacting protein 2/IKKbeta signaling plays crucial role in bortezomib-induced NF-kappaB activation. Moreover, IKKbeta inhibitors enhanced bortezomib-induced cytotoxicity. Our studies therefore suggest that bortezomib-induced cytotoxicity cannot be fully attributed to inhibition of canonical NF-kappaB activity in MM cells.

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Figures

Figure 1
Figure 1
Bortezomib down-regulates IκBα expression. (A) MM.1S, RPMI 8226, and U266 cells were cultured with bortezomib (10 nM and 20 nM) for 8 hours. (B) RPMI 8226 cells were cultured with bortezomib (20 nM) for the indicated time periods. (C) Primary tumor cells from MM patients were cultured with bortezomib (20 nM) for 8 hours. Whole-cell lysates (A-C) were immunoblotted with indicated Abs.
Figure 2
Figure 2
Bortezomib activates NF-κB in MM cell lines. (A) RPMI 8226 cells were cultured with increasing doses (10-40 nM) of bortezomib for 8 hours. (B) MM.1S, U266, OPM1, INA6, RPMI 8226, and OPM2 cells were treated with bortezomib (20 nM) for 8 hours. (C) MM.1S, U266, and RPMI 8226 cells were treated with bortezomib (10 nM) for indicated time periods. (D) RPMI 8226 cells were treated with bortezomib (20 nM) for 8 hours. Nuclear extracts were subjected to EMSA (A-C) or supershift assay (D) using anti-p65, (65) p50, (50) p52, (52) RelB (B), and cRel (cR) Abs. Oct-1 served as a loading control for EMSA. Exposure time of autoradiography varied for each cell line. (E) Alteration of gene expression after bortezomib treatment (20 nM, 6 hours) in RPMI 8226 cells. (F) RPMI8226 cells were cultured with DMSO control medium (□) or bortezomib (20 nM) for 1 hour (formula image), 3 hours (formula image) and 6 hours (■). Gene expression of Bcl-xL, CD44, IRF4, and ICAM1 was analyzed by DNA microarray.
Figure 3
Figure 3
Bortezomib induces DNA synthesis. (A) RPMI 8226 cells were treated with bortezomib (10 and 20 nM) for 8 hours. Cells were labeled with BrdU for an additional 2 hours and assessed for growth by DNA synthesis. (B) RPMI 8226 cells were incubated with bortezomib (20 nM) for 8 hours. Cells were washed with media to remove bortezomib, and then cultured for another 24 hours or 48 hours. Cell survival was determined by MTT assay. (C) Immunohistochemical analysis for p65 expression was performed on tumor tissue from RPMI 8226 xenograft mice, untreated and treated with bortezomib. *P < .01.
Figure 4
Figure 4
Neither caspase nor c-Jun N-terminal kinase inhibitors block bortezomib-induced IκBα down-regulation. (A) RPMI 8226 cells were cultured with bortezomib (20 nM for 8 hours and 12 hours) in the presence of control media (lane 2), Z-VAD-FMK (lane 3), or Z-DEVD-FMK (lane 4). Whole-cell lysates were subjected to immunoblotting with indicated Abs (A), and nuclear extracts were subjected to EMSA (B).
Figure 5
Figure 5
Bortezomib triggers IKKβ phosphorylation; conversely, inhibition of IKKβ blocks bortezomib-induced IκBα down-regulation and NF-κB activation. (A) RPMI 8226 cells were cultured with bortezomib (20 nM) or MG132 (MG, 0.5 μM). TNFα (TNF, 5 ng/mL) served as a positive control for p-RIP2 and p-IKKβ. (B) MM.1S, U266, and OPM1 MM cell lines, as well as primary tumor cells from MM patients, were cultured with bortezomib (20 nM for 8 hours). (C) MM.1S, RPMI 8226, and U266 cells were cultured with bortezomib (10 nM and 20 nM) for 8 hours. (D) RPMI 8226 cells were cultured with bortezomib (20 nM) for 8 hours in the presence (5 and 10 μM) or absence of MLN120B (MLN). Whole-cell lysates and nuclear extracts were immunoblotted with indicated Abs and EMSA, respectively. RPMI 8226 cells (E) and primary tumor cells (F) from MM patients (no. 1 and 2) were cultured with bortezomib (10 nM) for 24 hours, in the presence (5 μM, formula image; 10 μM, ■) or absence (□) of MLN120B, and cell growth was assessed by MTT assay. * P < .01.
Figure 6
Figure 6
Bortezomib activates NF-κB in normal healthy donor-derived PBMCs. (A) PBMCs were cultured with bortezomib (10 nM and 20 nM) for 8 hours. Whole-cell lysates were immunoblotted with indicated Abs. (B) PBMCs and BMSCs from MM patient were cultured with bortezomib (10 nM and 20 nM) for 8 hours. (C) PBMCs were separated into adherent and nonadherent subsets, followed by bortezomib (20 nM) treatment for 8 hours. Nuclear extracts were subjected to EMSA. (D) PBMCs were cultured with or without bortezomib (20 nM for 8 hours). Nuclear extracts were subjected to supershift assays using anti-p65 (65), p50 (50), p52 (52), RelB (B), and cRel (cR) Abs.
Figure 7
Figure 7
Possible mechanism whereby bortezomib triggers canonical NF-κB activation. Bortezomib either directly or indirectly (via RIP2) activates IKKβ, which subsequently phosphorylates IκBα, an inhibitor of p50/p65. After nonproteasomal degradation of IκBα, p50/p65 translocates to nucleus. IKKβ inhibitors block down-regulation of IκBα and NF-κB activity, as well as enhance bortezomib-induced cytotoxicity.
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