Med Oncol (2015)32:239 DOI 10.1007/s12032-015-0671-2

ORIGINAL PAPER

TRAF6 is required for BLyS-mediated NF-jB signaling in multiple myeloma cells Xinfeng Wang1 • Jingjing Wang1 • Hong Liu1 • Ruirong Xu1 • Runsheng Ding1 Shenhua Jiang1 • Xudong Wang2 • Hongming Huang1

•

Received: 9 February 2015 / Accepted: 22 July 2015 Ó Springer Science+Business Media New York 2015

Abstract Tumor necrosis factor receptor-associated factor 6 (TRAF6) transduces signals from members of the IL1R/TLR and TNFR superfamilies to the transcription factors NF-jB and AP1. Elevated expression of the TNF family member B-lymphocyte stimulator (BLyS) in multiple myeloma (MM) has been described recently. However, the precise process by which BLyS signals in myeloma cell remains unknown. Here, we identified increased expression of TRAF6 in MM patient cells and the MM cell lines U266, RPMI8226, and KM3. Furthermore, rhBLyS induced TRAF6 up-regulation in these cells in a dose-dependent manner. Both the classical and alternative NF-jB pathways were activated by rhBLyS treatment. Depletion of TRAF6 by siRNA decreased levels of p-p65 and p-p100, even after stimulation with rhBLyS. Down-regulation of TRAF6 also abrogated rhBLyS-mediated cell viability. These findings suggest that TRAF6 is required for BLyS-mediated NF-jB signaling in myeloma cells and is a potential molecular therapeutic target in MM. Keywords myeloma

TRAF6  BLyS  BAFF  NF-jB  Multiple

Xinfeng Wang and Jingjing Wang are co-first authors. & Xudong Wang [email protected] & Hongming Huang [email protected] 1

Affiliated Hospital of Nantong University, Department of Hematology, Nantong 226001, Jiangsu, China

2

Center of Clinical Laboratory Medicine, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu, China

Introduction Multiple myeloma (MM) is a late-stage B cell malignancy characterized by the clonal proliferation of plasma cells within the bone marrow. Myeloma cells have undergone extensive somatic hyper-mutation, and the disease remains incurable despite available therapies. The nuclear factor kappa B (NF-jB) pathway has been identified as an important signaling pathways dysregulated in MM [1], with inappropriate NF-jB activation prolonging MM cell survival and conferring resistance to chemotherapy [2]. TRAF6 is an adapter molecule that mediates signaling from the IL-1R/TLR family and the TNFR superfamily [3]. Moreover, TRAF6 is an important factor in the regulation of NF-jB activation [4]. As a well-defined key molecule in osteoclasts of myeloma, inhibition of TRAF6 by siRNA also impairs IL-1-induced NF-jB and c-Jun/AP-1 activation of myeloma cells, significantly inhibits myeloma cell proliferation, and enhances their apoptosis in vitro [5]. B-lymphocyte stimulator (BLyS), also known as B cell activating factor (BAFF), is a member of the TNF ligand family [6]. BLyS is expressed in both membrane-bound and soluble forms by various cell types, including monocytes, macrophages, and dendritic cells. Several studies have identified high levels of BLyS in the blood serum and bone marrow of MM patients, and MM cell lines express high levels of BLyS [7–9]. BLyS promotes MM cell proliferation by activating both the classical and alternative NF-jB pathways; however, the precise mechanism by which this activation occurs in myeloma cells remains unknown. To better understand the role of TRAF6 in BLyS-mediated NF-jB activation in myeloma, this study investigates TRAF6 and NF-jB family member expression in the myeloma cell line RPMI8226 following stimulation with

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exogenous BLyS. The effect of siRNA-mediated TRAF6 depletion on NF-jB activation was also assessed.

Materials and methods Human bone marrow myeloma cells and myeloma cell lines

2 h, and washed three times more for 5 min in TBST. Finally, the membrane was subjected to autoradiography and signals quantified by densitometry. Results were analyzed using Quantity One software (Bio-Rad Laboratories, Richmond, CA, USA). The relative expression of the target protein was measured as the gray value ratio of target protein content to b-actin (target protein/b-actin) content. Transfection of RPMI8226 cells with TRAF6 siRNA

Bone marrow from four MM patients and three healthy donors (HDs) was collected with individual informed consent (Table 1), purified with CD138 microbeads according to the instruction of the manufacturer (Miltenyi Biotech, Bergisch Gladbach, Germany). RPMI8266, KM3, and U266 cells (CRC/PUMC, Beijing, China) were cultured in RPMI-1640 (Gibco, Rockville, MD, USA) supplemented with 10 % fetal bovine serum and penicillin– streptomycin–glutamine in a humidified atmosphere containing 5 % CO2 at 37 °C. Western blotting RPMI8226 cells were lysed with Laemmli buffer [50 mM Tris buffer, pH 6.8, 2 % sodium dodecyl sulfate, and 10 % glycerol containing protease inhibitors (1 lg/mL leupeptin, 1 lg/mL aprotinin and 2 mM phenylmethylsulfonyl fluoride; Sigma-Aldrich, St Louis, MO, USA)]. Forty micrograms of total protein was separated by SDS-PAGE on a 12 % gel and transferred to a PVDF membrane (Millipore, Bedford, MA, USA). The membrane was then blocked by incubation with 5 % bovine serum albumin in tris-buffered saline (TBS; 10 mM Tris–HCl, pH 7.5, 150 mM NaCl) containing 0.1 % Tween-20 (TBST) for 2 h at room temperature (RT), probed with antibodies against TRAF6 (Abcam, Cambridge, UK) or p-p65, p-p100, p100/p52, P50(all Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight, washed three times for 5 min in TBST, probed with a corresponding secondary antibody at RT for Table 1 Clinical features of MM patients and healthy volunteers Parameter

Myeloma patients

Healthy volunteers

Number

4

3

Median age (range), Y

54 (37–70)

38 (30–45)

Male/female

3/1

3/0

Type

IgG, 2; IgA, 1; jFC, 1

Stage (DS)

II B 1; III A 2; III B 1

Organ involved

Anemia, 3; osteolysis, 4

Average number of plasma cells in BM at the diagnosis (%)

25.6 (9–42)

Y years, FC free chain, DS Durie–Salmon, BM bone marrow

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Before transfection, 6 9 105 cells were seeded in each well of six-well plates using PBS with 10 % FBS. The next day, 25–100 nM of TRAF6 siRNA (Shanghai GenePharma Co, Ltd., Shanghai, China) were added to each well with EntransterTM-R (Engreen, Beijing, China) and cultured for a further 48 h. Ribonucleic acid interference oligomers were complementary to TRAF6 mRNA (sense 50 -GCAGA UGGGGCAUUCAUATT-30 , anti-sense 50 -UAUGAAUG CCCCAUCUGCTT-30 ). The negative control siRNAs were sense 50 -UUCUCCGAACGUGUCACGUTT-30 and antisense 50 -ACGUGACACGUUCGGAGAATT-30 . CCK-8 assay of cell proliferation Cell viability was measured using a modified CCK-8 assay. Approximately 4 9 103 cells/well were plated in 200 lL RPMI-1640 with 10 % fetal bovine serum and/or rhBLyS (Peprotech, Rocky Hill, NJ, USA) in a 96-well plate, and incubated at 37 °C in a 5 % CO2 atmosphere. Next, 20 lL CCK-8 solution was added to the culture medium at 24, 48, or 72 h, followed by another 3-h incubation. The optical density (OD) at 450 nm of samples was measured using a Benchmark microplate reader (BIO-TEK Elx800). Experiments were performed independently three times, with each condition in triplicate. Statistical analysis SPSS v19.0 (SPSS, Chicago, IL, USA) was used to perform the Student’s t test or one-way analysis of variance. A p value \.05 was considered statistically significant.

Results TRAF6 is over-expressed in MM cells TRAF6 expression was measured in the MM cell lines KM3, U266, and RPMI8226, and in MM cells from patients via western blotting. TRAF6 protein expression was significantly higher in all cell lines compared with normal control cells (Fig. 1). Further more, comparison of CD138? cells from MM patients with U266, KM3, and

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Fig. 1 TRAF6 protein levels are increased in patient MM cells and MM cell lines. a TRAF6 protein levels in whole cell lysates of CD138? bone marrow mononuclear cells (BMMCs) from four MM patients and three HDs and U266, KM3, and RPMI8226 cell lines

were measured by western blotting. CD138? cells from normal BMMCs served as the control. b Representative flow cytometric profile of purified CD138? primary myeloma cells. All cells were [90 % pure

RPMI8226 cells showed that CD138? cells had higher TRAF6 protein expression than the three myeloma cell lines (Fig. 1).

various concentrations, and the family members of NF-jB (p65, p100, p52) in cell lysates were detected at 48 h. Although a graphical representation of the 50 ng/mL rhBLyS-induced P100 is shown elevated in Fig. 3a, no apparent difference was observed in p52 and p65 when comparing untreated cells. BLyS-induced phosphorylation of NF-jB was also detected, in which RPMI8226 cells were co-cultured with 200 ng/mL rhBLyS for 0, 15, 30, 60, or 120 min. Total p65, p100, and p52 levels were unchanged, while phosphorylated p65 and p100 levels were significantly elevated 15–60 min, which did maximally at 30 min following BLyS treatment (Fig. 3b). All above findings suggests that rhBLyS can induce both the classical and alternative phosphorylation of NF-jB pathways in RPMI8226 cells.

BLyS-induced TRAF6 up-regulation In previous studies, our colleagues had confirmed myeloma cells (KM-3 and CZ-1) bind BLyS at high levels, and BLyS contributes to MM cell survival and proliferation in vitro (7). To investigate a potential relationship between BLyS and TRAF6 in myeloma cells, the three cell lines (U266, KM3, and RPMI8226) were treated with 0, 50, 100, or 200 ng/mL rhBLyS and harvested at 24 and 48 h respectively. TRAF6 proteins levels were elevated in a dosedependent manner at both 24 and 48 h in both RPMI8226 and U226 cell lines upon rhBLyS treatment. TRAF6 expression was also gradually increased at KM3 cells, even the growth trended to be slow from 100 to 200 ng/mL rhBLyS stimulation Fig. 2). BLyS-induced NF-jB pathway activation To determine whether BLyS activated NF-jB signaling in MM cells, RPMI8226 cells were treated by rhBLyS with

Fig. 2 TRAF6 expression in myeloma cell lines (RPMI8266, KM3, and U266 cells) after stimulation with rhBLyS. The cells were cultured with increasing concentrations of rhBLyS for 24 or 48 h

Effects of silencing TRAF6 on myeloma cell proliferation The transfection efficiency of 50 nmol/L siTRAF6C was determined by many methods in RPMI8226 cells. It showed 87 % (Fig. 4a) transfected cells using fluorescent microscopy after 48-h transfection. TRAF6 mRNA expression after 24-h incubation (Fig. 4b) and TRAF6

followed by western blotting to detect TRAF6 levels. a RPMI8226 cells, b U266 cells, c KM3 cells

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Fig. 3 BLyS activates NF-jB in RPMI8226 cells. a RPMI8226 cells were treated by rhBLyS with various concentrations and the family members of NF-jB (p65, p100, p52) in cell lysates were detected at 48 h by western blotting. b RPMI8226 cells were cultured with rhBLyS (200 ng/mL) for indicated times followed by western blotting to determine levels of NF-jB pathway components

Fig. 4 Effects of siTRAF6C transfection on RPMI8226 cells. a Transfection efficiency of SiTRAF6C (50 nM) by fluorescence microscopy after 48 h. b TRAF6 mRNA expression after 24-h

incubation with siTRAF6C. c Western blot analysis of TRAF6 protein levels following transfection with siTRAF6C. Rnc, non-targeting control siRNA; R0, no treatment; Rsi, TRAF6C siRNA

protein levels after 48-h incubation (Fig. 4c) both decreased, suggesting that siTRAF6C transfected myeloma cells.

Depletion of TRAF6 reduced RPMI8226 cell viability

TRAF6 depletion impaired BLyS-induced NF-jB pathway activation To investigate whether TRAF6 regulated NF-jB signaling in MM cells, the levels of p65, p-p65, p52, p100, and p-p100 in RPMI8226 cells were measured by western blotting following transfection with 50 nM TRAF6C siRNA at 48 h. Levels of p-p65, p65, p-p100, and p100 were found decreased in the silencing groups, especially the decrease in p-p65 and p-p100 was more significantly (Fig. 5a). To determine whether TRAF6 played a role in BLySmediated NF-jB induction, RPMI8226 cells were exposed to 200 ng/mL rhBLyS for 30 min following transfection with 0, 25, 50, or 100 nM of TRAF6C siRNA 48 h prior. Levels of p-p65 and p-p100 were decreased following the use of 50 and 100 nM TRAF6C siRNA. Moreover, the activation of p65 and p100 previously observed following treatment with rhBLyS (Fig. 2) did not occur (Fig. 5b).

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To determine the influence of BLyS and TRAF6 on myeloma cell growth, RPMI8226 cells were subjected to CCK8 assay following exposure to 200 ng/mL rhBLyS or transfection with 50 nM TRAF6C siRNA. Stimulation with rhBLyS promoted RPMI8226 cell growth, while TRAF6depleted cell viability was significantly decreased (Fig. 6a). Last, to determine the role of the BLyS-TRAF6 relationship in myeloma cell growth, TRAF6-depleted RPMI8226 cells were exposed to 200 ng/mL rhBLyS. Cell growth within the control group increased significantly following the addition of rhBLyS, while cell growth in the TRAF6-depleted group was not significant (Fig. 6b).

Discussion Roles for TRAF6 in numerous physiological and pathological processes have been described. In cancer, overexpression of TRAF6 can activate the NF-jB pathway and facilitate tumorigenesis, metastasis, and invasion [10–12]. In myeloma, inhibition of TRAF6 by siRNA or decoy

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Fig. 5 TRAF6 is required for BLyS-induced NF-jB activation. a RPMI8226 cells were transfected with 50 nM TRAF6C siRNA for 48 h and lysates subjected to western blot to determine levels of p65, p-p65, p100, and p-p100 protein. b RPMI8226 cells were pre-treated

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with indicated concentrations of 50 nM TRAF6 siRNA prior to 30 min stimulation with 200 ng/mL rhBLyS. Western blotting measured levels of p65, p-p65, p100/p52, and p-p100 in RPMI8226 cells

Fig. 6 TRAF6 is required for RPMI8226 cell viability. a CCK-8 assay following transfection of RPMI8226 cells with 50 nM TRAF6C siRNA. b CCK-8 assay following treatment of TRAF6-depleted cells with 200 ng/mL BLyS (compared with control: *p \ .05; **p \ .01)

peptides decreases tumor cell proliferation, increases tumor cell apoptosis [5, 13], and reduces osteoclast-mediated bone resorption [14]. Therefore, TRAF6 has been proposed as a potential therapeutic target for the treatment of MM [15]. However, the mechanism by which TRAF6 functions in myeloma cells is incompletely understood. We identified increased expression of TRAF6 in both MM patient cells and MM cell lines. Furthermore, TRAF6 expression in MM patient cells seemed even higher than that in MM cell lines. This suggests that as an adapter protein, TRAF6 may be up-regulated by additional factors in myeloma patients. This is supported by the identification of a dysregulated microenvironment [16], which includes abnormal cytokine expression, as a major feature of MM. Many receptors can bind TRAF6, including CD40, TLR4, IL-1, IL-17, BLyS, and RANK [17–19]. Elevated expression of BLyS has been recently identified in MM patients and MM cell lines, where it promotes the survival and proliferation of myeloma cells [20]. Therefore, we investigated whether BLyS could signal via TRAF6 in myeloma cells. Stimulation with rhBLyS increased TRAF6

expression in MM cells. This finding is consistent with a previous study that found BAFF-R-dependent activation of B cell lymphoma cells required TRAF6 [21]. This suggests that the development of myeloma and B cell lymphoma may share common mechanisms. Previous studies have shown that BLyS can activate both the classical and alternative NF-jB pathways [7, 22]. To determine whether TRAF6 is required for BLyS-mediated NF-jB expression in MM cells, we co-cultured RPMI8226 cells with rhBLyS for multiple time points and assessed NF-jB activation. Both p65 and p100 were maximally phosphorylated 30 min following exposure to BLyS, supporting previous findings that signaling by BLyS was necessary and sufficient to activate NF-jB1 and NFjB2 pathways in myeloma cells [22]. Transfection of RPMI8226 cells with siRNA-targeting TRAF6 significantly decreased activation of both the classical and alternative NF-jB pathways, supporting previous findings from Chen et al. [5]. Additionally, even though TRAF6 signal level increased by the incentive of BLyS, depletion of TRAF6 by siRNA disrupted BLyS-induced activation of

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both the classical and alternative NF-jB pathways. These findings demonstrate that TRAF6 is essential for mediating activation of NF-jB by BLyS. NF-jB-positive MM cell lines are sensitive to growth inhibition by blockers of NF-jB signaling [23, 24]. Our current study demonstrated that down-regulation of TRAF6 inhibits NF-jB activation and reduces RPMI8226 cell viability even after stimulation of BAFF-R by BLyS. These findings suggest a novel role for TRAF6 in mediating the viability of MM cells, possibly in response to signaling by BAFF-R. Taken together, our findings suggest that targeting the BLyS-TRAF6-NF-jB pathway should be investigated as a strategy to treat MM. Our study demonstrates that TRAF6 is over-expressed in myeloma cells where it plays a regulatory role in BLySmediated NF-jB signaling. This is the first study to identify a TRAF6-dependent signaling pathway associated with BLyS in myeloma. These findings therefore enhance our understanding of the role played by TRAF6 in myeloma cell growth. Acknowledgments Thanks for technical help from Ju Shaoqing, Ph.D., of Surgical Comprehensive Laboratory, Affiliated Hospital of Nantong University. Funding source This work was supported by the National Youth Science Foundation (81201857) and the Natural Science Foundation of Jiangsu Province, China (BK2011388). Compliance with ethical standards Conflict of interest interest to declare.

All the authors do not have any conflict of

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