Bone marrow stromal cells (BMSCs) and osteoclasts (OCs) confer multiple myeloma (MM) cell survival through elaborating factors. We demonstrate herein that IL-6 and TNF family cytokines, TNFα, BAFF and APRIL, but not IGF-1 cooperatively enhance the expression of the serine/threonine kinase Pim-2 in MM cells. BMSCs and OCs upregulate Pim-2 expression in MM cells largely via the IL-6/STAT3 and NF-κB pathway, respectively. Pim-2 short interfering RNA reduces MM cell viability in cocultures with BMSCs or OCs. Thus, upregulation of Pim-2 appears to be a novel anti-apoptotic mechanism for MM cell survival. Interestingly, the mammalian target of rapamycin inhibitor rapamycin further suppresses the MM cell viability in combination with the Pim-2 silencing. The Pim inhibitor (Z)-5-(4-propoxybenzylidene) thiazolidine-2, 4-dione and the PI3K inhibitor LY294002 cooperatively enhance MM cell death. The Pim inhibitor suppresses 4E-BP1 phosphorylation along with the reduction of Mcl-1 and c-Myc. Pim-2 may therefore become a new target for MM treatment.
Multiple myeloma (MM) cells enhance osteoclast (OC) formation and suppress osteoblast (OB) differentiation of bone marrow stromal cells (BMSCs). Such effects of MM cells not only create destructive bone lesions, but also provide a cellular microenvironment to protect MM cells from various apoptotic insults.1, 2, 3, 4, 5 IL-6 and TNF family cytokines, TNFα, BAFF and APRIL, are among predominant anti-apoptotic factors for MM cells elaborated by the bone marrow microenvironment surrounding MM.1, 6, 7, 8 IL-6 is mainly produced by BMSCs,1, 6 whereas OCs are a major producer of BAFF and APRIL in the MM bone marrow microenvironment.9, 10 The serine/threonine kinase Pim-2 has been demonstrated to be transcriptionally upregulated to promote survival of hematopoietic cells in response to ambient growth factors and cytokines.11 We demonstrate herein that Pim-2 is upregulated in MM cells by BMSCs and OCs, and acts as an important pro-survival mediator.
Materials and methods
The following reagents were purchased from the indicated manufacturers: rh IL-6, BAFF and SDF-1α from PEPROTECH EC (London, UK); rh TNFα, APRIL, IGF-1, VEGF, neutralizing mouse monoclonal antibodies against IL-6 and against IL-6 receptor from R&D Systems (Minneapolis, MN, USA); cucurbitacin I, parthenolide, rapamycin, LY294002 and the Pim inhibitor (Z)-5-(4-propoxybenzylidene)thiazolidine-2,4-dione from Calbiochem (Darmstadt, Germany); mouse monoclonal antibody against human Pim-2 and rabbit polyclonal antibodies against STAT-3 and Mcl-1 from Santa Cruz Biotechnology (Santa Cruz, CA, USA); rabbit polyclonal antibodies against phosphorylated NF-κB p65, NF-κB p65, 4E-BP1, phosphorylated 4E-BP1 (Ser 65), phosphorylated 4E-BP1 (Thr 37/46) and phosphorylated STAT-3, mouse monoclonal antibody against phosphorylated Bad, rabbit monoclonal antibodies against human Pim-2 and c-Myc, horseradish peroxidase-conjugated horse anti-mouse IgG and horseradish peroxidase-conjugated goat anti-rabbit IgG from Cell Signaling Technology (Beverly, MA, USA); mouse monoclonal anti-Bad from BD Transduction Laboratories (Franklin-Lakes, NJ, USA); rabbit anti-β-actin from Sigma (St Louis, MO, USA).
Cells and cultures
Human non-hematopoietic cell lines MG63, MCF7 and Colo205, human myeloid cell lines KU812F, HL-60 and U937, human lymphoid cell lines CEM and IM-9, and human MM cell lines RPMI8226 and U266 were obtained from American Type Culture Collection (Rockville, MD, USA). The MM cell line INA-6 and MM.1S were kindly provided by Dr Renate Burger (University of Kiel, Kiel, Germany) and Dr Steven Rosen (Northwestern University, Chicago, IL, USA), respectively. TSPC-1 and OPC MM cell lines were established in our laboratory.12 Primary MM cells and BMSCs were isolated from fresh bone marrow aspirates from patients with MM and cultured as described previously.11 BMSCs were a homogeneous population of spindle-shaped cells expressing CD44, CD73, CD90 and CD105, but neither CD45 nor factor VIII. OCs were generated from peripheral blood mononuclear cells as reported previously.11 Cells were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM of L-glutamine (Sigma), 100 U/ml of penicillin G and 100 μg/ml of streptomycin (Sigma). All procedures involving human specimens were performed with written informed consent according to the Declaration of Helsinki and using a protocol approved by the Institutional Review Board for human protection.
Western blot analysis
Cells were collected and lysed in lysis buffer (Cell Signaling) supplemented with 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail solution (Sigma). Cell lysates were subjected to western blot analysis as described previously.13
Bone marrow clot sections from MM patients were fixed in neutral-buffered formalin and embedded in paraffin. Paraffin-embedded tissue sections were deparaffinized and hydrated. For antigen retrieval the sections were microwaved in 10 mM sodium citrate buffer (pH 6.6). After a 10-min blocking process, the sections were incubated with rabbit monoclonal anti-human Pim-2 antibody (Cell Signaling) or control rabbit IgG (Vector Laboratories, Burlingame, CA, USA) overnight at 4 °C. Immunoreactivity was detected by biotinylated secondary antibodies and horseradish peroxidase-conjugated streptavidin (DAKO LSAB+ System, horseradish peroxidase; DAKO, Carpinteria, CA, USA) followed by diaminobenzidine substrate (DAKO).
Quantitative real-time PCR
RNA isolation and quantitative real-time PCR were performed as described previously.13Primers used were as follows: human Pim-2, sense 5′-IndexTermAGGGATTGAGGATCAGGGGT-3′ and antisense 5′-IndexTermCACAGGTTCTGGGAGGAAGG-3′. Human GAPDH, used as a housekeeping gene for quantity normalization, sense 5′-IndexTermAATCCCATCACCATCTTCCA-3′, antisense 5′-IndexTermTGGACTCCACGACGTACTCA-3′.
MM cells were transfected with 6-carboxyfluorescein-labeled Pim-2 short interfering RNA (siRNA) (sense, 5′-IndexTermGUGCCAAACUCAUUGAUUUTT-3′ and antisense, 5′-IndexTermAAAUCAAUGAGUUUGGCACTT-3′) and scrambled siRNA (sense, 5′-IndexTermAUCCGCGCGAUAGUACGUATT-3′ and antisense, 5′-IndexTermUACGUACUAUCGCGCGGAUTT-3′) (B-Bridge, Mountain View, CA, USA) using a Human Nucleofector Kit (Amaxa Biosystems, Cologne, Germany). The transfected cells were sorted by a flow cytometer (EPICS ELITE; Beckman Coulter, Brea, CA, USA).
Cell viability assays
Viable cell numbers were counted by trypan blue dye exclusion assay as described previously.11 Cell viability was also determined by Cell Counting Kit-8 assay (DOJINDO, Kumamoto, Japan) according to the manufacturer's instructions. The absorbance of each well was measured at 450 nm with a microtiter plate reader (Model 450 micro plate reader; Bio-Rad Laboratories, Hercules, CA, USA). Apoptosis was evaluated by staining the cells with an annexinV-FITC and propidium iodide labeling kit (MEBCYTO Apoptosis Kit; MBL, Nagano, Japan) according to the manufacturer’s instructions.
Data are expressed as means±s.d. unless otherwise specified. Statistical significance was determined by a one-way analysis of variance (ANOVA) with Scheffe’s post hoc tests. The minimal level of significance was P=0.05.
Results and discussion
BMSCs and OCs enhance Pim-2 expression in MM cells
Pim-2 protein was constitutively expressed at higher levels in MM cell lines and some other hematopoietic cell lines than in non-hematopoietic cell lines and normal peripheral blood mononuclear cells (Figure 1a and Supplementary Figure 1). Pim-2 protein expression was also observed in MM cells in bone marrow specimens of patients with MM (Figure 1b). Interestingly, Pim-2 mRNA levels were upregulated substantially (about 2- to 9-fold) in all MM cell lines and MM cells from all patients with MM when cocultured with BMSCs (Figure 2a upper). Consistently, Pim-2 protein levels were also enhanced by coculturing with BMSCs (Figure 2a lower). Although peripheral blood mononuclear cells-derived OCs also upregulated Pim-2 expression in most of these MM cells (Figure 2b), BMSCs more potently enhanced Pim-2 expression in MM cells than OCs. These results demonstrate that Pim-2 is overexpressed in MM cells and that BMSCs and OCs stimulate Pim-2 expression in MM cells.
IL-6 and TNF family cytokines cooperatively enhance Pim-2 expression in MM cells
To determine the factors responsible for Pim-2 upregulation in MM cells, we examined Pim-2 expression in MM cells under stimulation with anti-apoptotic cytokines overproduced in the MM bone marrow microenvironment. IL-6 and TNF family cytokines, TNFα, BAFF and APRIL, upregulated Pim-2 expression in MM cells (Figure 3a). Interestingly, IL-6 and these TNF family cytokines cooperatively enhanced Pim-2 expression in MM cells (Figure 3a). However, another important anti-apoptotic cytokine, IGF-I, did not increase but rather suppressed Pim-2 expression (Supplementary Figure 2). SDF-1 and VEGF showed no significant effect. These results suggest that IL-6 and these TNF family cytokines are among the predominant factors responsible for Pim-2 upregulation in MM cells in the bone marrow microenvironment in MM.
IL-6 induced STAT3 phosphorylation followed by Pim-2 upregulation in MM cells (Supplementary Figure 3). Addition of cucurbitacin I, a STAT3 inhibitor, suppressed the Pim-2 upregulation by IL-6 (Figure 3b). TNFα induced Pim-2 upregulation following phosphorylation of NF-κB in MM cells (Supplementary Figure 3). The Pim-2 upregulation by TNFα was suppressed by an IKKα/β inhibitor, parthenolide (Figure 3c). Thus, STAT3 and NF-κB activation mediates Pim-2 upregulation in MM cells. These results are consistent with the notion that IL-6 and these TNF family cytokines act together to upregulate Pim-2 expression in MM cells in the bone marrow microenvironment. However, basal expression levels of Pim-2 were associated with neither the levels of phosphorylation of STAT3 nor NF-κB activation in MM cell lines without external stimuli (Supplementary Figure 1). Other signaling pathways may also be involved in endogenous upregulation of basal Pim-2 levels in MM cells.
Inhibition of IL-6/STAT3 and NF-κB pathways abolishes Pim-2 upregulation in MM cells by BMSCs and OCs, respectively
Because BMSCs produce a large amount of IL-6 and are regarded as a major source of IL-6 in the bone marrow microenvironment in MM,1, 6 and because IL-6 potently upregulates Pim-2 expression in MM cells, we next explored the role of IL-6 in Pim-2 upregulation in MM cells by BMSCs. The upregulation of Pim-2 mRNA expression in INA-6 and RPMI8226 cells by BMSCs was almost completely suppressed by a combination of antibodies against IL-6 and IL-6 receptor (Figure 3d). The inhibition of STAT3 by cucurbitacin I also abolished the Pim-2 upregulation in MM cells by BMSCs (Figure 3e left). The inhibition by LY294002 of a PI3K/Akt pathway, a major downstream signaling pathway of IGF-1, did not affect Pim-2 expression in MM cells upregulated by BMSCs (Figure 3e right), which is consistent with the observation of no induction of Pim-2 expression in MM cells by IGF-1 (Supplementary Figure 2). Thus, IL-6 appears to be a predominant BMSC-derived factor responsible for Pim-2 upregulation in MM cells. The IKKα/β inhibitor parthenolide abolished Pim-2 upregulation in MM cells cocultured with OCs (Figure 3f), suggesting a major role of the NF-κB pathway in Pim-2 upregulation in MM cells by OCs.
Pim-2 inhibition and rapamycin cooperatively suppress MM cell survival
To clarify the roles of Pim-2 in MM cell survival, we next examined the effect of Pim-2 silencing in MM cells. Pim-2 silencing by Pim-2 siRNA reduced Pim-2 expression in RPMI8226 cells to about 62% and 29% in the absence and presence of IL-6, respectively, compared with that by control siRNA (Supplementary Figure 4a). IL-6, BMSCs or OCs are able to enhance the survival of the IL-6-dependent INA-6 cell line, which otherwise undergoes apoptosis. The viability of INA-6 cells was partially but significantly reduced by Pim-2 silencing in the presence of IL-6, BMSCs or OCs (Figure 4a). Because Pim-2 upregulation is largely independent of the PI3K/Akt pathway (Figure 3e), and because the PI3K/Akt pathway is an important survival pathway in MM cells,14, 15 we examined the effects of inhibition of both Pim-2 and PI3K/Akt pathways on MM cell viability in the presence of BMSCs. Rapamycin is an inhibitor of mammalian target of rapamycin, a downstream signaling molecule of the PI3K/Akt pathway. Pim-2 silencing or addition of rapamycin alone partially reduced the viability of INA-6 cells supported by BMSCs (Figure 4b left), whereas the two treatments in combination further reduced INA-6 cell viability. The combinatory treatment also potently suppressed the survival of RPMI8226 cells (Figure 4b right) that can grow in the absence of BMSCs. These results suggest a cooperative role of Pim-2 and mammalian target of rapamycin pathways in MM cell survival.
The Pim inhibitor (Z)-5-(4-propoxybenzylidene)thiazolidine-2,4-dione suppresses MM cell viability
Because Pim-2 knockdown by Pim-2 siRNA was partial in MM cells due to low transfection efficiency in MM cells, we extended the experiments with the Pim inhibitor (Z)-5-(4-propoxybenzylidene)thiazolidine-2,4-dione, which preferentially suppress Pim-2 rather than Pim-1.16, 17 The Pim inhibitor dose-dependently suppressed the viability of MM cell lines including INA-6, RPMI8226, MM.1S, TSPC-1 and OPC (Figure 4c). Addition of the Pim inhibitor increased the percentage distribution of annexinV-positive cells in INA-6 cells in the absence or presence of BMSCs (Figure 4d), suggesting that anti-apoptotic activity was mediated by the Pim pathway. The Pim inhibitor in combination with the PI3K inhibitor LY294002 cooperatively reduced numbers of viable INA-6 cells both in the absence and in the presence of BMSCs (Figure 4e).
We further examined the effects of the Pim inhibitor on phosphorylation of 4E-BP1 and expression of growth and survival mediators, because the Pim pathway has been demonstrated to be responsible for the phosphorylation of 4E-BP1 to trigger protein translation for cell growth and survival.11, 18 Treatment with the Pim inhibitor markedly suppressed the phosphorylation of 4E-BP1 along with the reduction of Mcl-1 and c-Myc protein levels in INA-6 and RPMI8226 cells (Figure 4f). However, phosphorylation of Bad and the levels of other apoptosis-related factors including Bcl-xL, Bcl-2 and Bim showed no appreciable change in RPMI8226 cells upon treatment with the Pim inhibitor (Supplementary Figure 4b). Thus, the induction of apoptosis by the Pim inhibitor is suggested to be associated at least in part with the reduction of phosphorylated 4E-BP1, Mcl-1 and c-Myc.
The present observations demonstrate that Pim-2 is a novel pro-survival mediator for MM cells, and suggest that the MM bone marrow microenvironment upregulates Pim-2 expression in MM cells through activation of the JAK2/STAT3 pathway for IL-6 and the NF-κB pathway for TNF family cytokines to promote MM cell survival. Therefore, Pim-2 overexpressed in MM cells in the MM bone marrow microenvironment appears to be an important therapeutic target. IGF-1 is another critical microenvironment-derived survival factor for MM cells,14, 15 and inhibition of the IGF-1/PI3K/Akt pathway by Akt or mammalian target of rapamycin inhibitors has drawn considerable attention as a new therapeutic modality against MM.19, 20 Because Pim-2 upregulation is largely independent of the PI3K/Akt pathway (Figure 3e), and because inhibition of Pim-2 and PI3K/Akt pathways cooperatively reduce MM cell survival (Figures 4b and e), Pim-2 should be targeted to improve anti-MM efficacy together with PI3K/Akt pathway inhibitors. A therapeutic impact of Pim-2 inhibition on MM survival would be further warranted when potent specific inhibitors for Pim-2 become available.
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This work was supported in part by Grants-in-aid for Scientific Research (A) to TM and (C) to MA, and for the 21st Century Center of Excellence Program from the Ministry of Education, Culture, Science and Sports of Japan, and a Grant-in-aid for Cancer Research (17–16) to MA from the Ministry of Health, Labor and Welfare of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Leukemia website
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