NF-κB2 mutation targets survival, proliferation and differentiation pathways in the pathogenesis of plasma cell tumors
- Brian A McCarthy†1,
- Liqun Yang†1, 4,
- Jane Ding1,
- Mingqiang Ren1,
- William King2,
- Mohammed ElSalanty3,
- Ibrahim Zakhary3,
- Mohamed Sharawy3,
- Hongjuan Cui4Email author and
- Han-Fei Ding1Email author
© McCarthy et al.; licensee BioMed Central Ltd. 2012
Received: 2 December 2011
Accepted: 29 May 2012
Published: 29 May 2012
Abnormal NF-κB2 activation has been implicated in the pathogenesis of multiple myeloma, a cancer of plasma cells. However, a causal role for aberrant NF-κB2 signaling in the development of plasma cell tumors has not been established. Also unclear is the molecular mechanism that drives the tumorigenic process. We investigated these questions by using a transgenic mouse model with lymphocyte-targeted expression of p80HT, a lymphoma-associated NF-κB2 mutant, and human multiple myeloma cell lines.
We conducted a detailed histopathological characterization of lymphomas developed in p80HT transgenic mice and microarray gene expression profiling of p80HT B cells with the goal of identifying genes that drive plasma cell tumor development. We further verified the significance of our findings in human multiple myeloma cell lines.
Approximately 40% of p80HT mice showed elevated levels of monoclonal immunoglobulin (M-protein) in the serum and developed plasma cell tumors. Some of these mice displayed key features of human multiple myeloma with accumulation of plasma cells in the bone marrow, osteolytic bone lesions and/or diffuse osteoporosis. Gene expression profiling of B cells from M-protein-positive p80HT mice revealed aberrant expression of genes known to be important in the pathogenesis of multiple myeloma, including cyclin D1, cyclin D2, Blimp1, survivin, IL-10 and IL-15. In vitro assays demonstrated a critical role of Stat3, a key downstream component of IL-10 signaling, in the survival of human multiple myeloma cells.
These findings provide a mouse model for human multiple myeloma with aberrant NF-κB2 activation and suggest a molecular mechanism for NF-κB2 signaling in the pathogenesis of plasma cell tumors by coordinated regulation of plasma cell generation, proliferation and survival.
NF-κB2 is a member of the NF-κB family of transcription factors that also include NF-κB1 (p105/p50), RelA (p65), RelB, and c-Rel. The full-length NF-κB2 precursor protein p100 contains an amino-terminal Rel homology domain and a carboxyl-terminal region with seven ankyrin repeats. In response to certain cytokines, NF-κB2 is phosphorylated at specific serine residues in its carboxyl-terminal region, leading to partial proteasomal degradation of the carboxyl terminus for the production of p52. The Rel homology domain of p52 then forms active NF-κB dimers with RelB or other Rel proteins, which, once in the nucleus, bind a common DNA sequence motif known as the κB site and regulate the expression of genes crucial to the development and functions of lymphocytes [1, 2].
Constitutive NF-κB2 signaling has been implicated in the pathogenesis of lymphomas. Several mechanisms have been identified wherein activation of NF-κB2 is uncoupled from its normal modes of regulation. Most of these mechanisms target upstream regulators, such as the NF-κB inducing kinase and IκB kinases [3, 4]. Sustained NF-κB2 activation can also be caused by chromosomal translocations and rearrangements at the NF-κB2 locus, which occur in a variety of lymphoid malignancies including T-cell lymphoma, chronic lymphocytic leukemia, multiple myeloma, and B-cell lymphoma . A cardinal feature of these genetic alterations is the generation of C-terminally truncated NF-κB2 mutants that lack various portions of the ankyrin-repeat domain [6–12]. To determine whether NF-κB2 mutation can directly initiate lymphomagenesis, we have generated transgenic mice with targeted expression in lymphocytes of p80HT, a lymphoma-associated NF-κB2 mutant [11, 12]. These transgenic mice develop predominantly B-cell tumors, demonstrating that NF-κB2 mutations can have a causal role in lymphomagenesis .
Multiple myeloma (MM) is a common, incurable malignant tumor of plasma cells. Although much is known about individual genes and signaling pathways that are activated in MM cells, the interplay and connections between these genes and pathways that drive MM development are not well understood. Many MM cell lines have constitutively nuclear NF-κB activity and are sensitive to inhibitors of NF-κB signalling [14–16]. Recent studies have also shown that approximately 40% of MM cell lines and 17% of primary MM tumors have mutations in genes encoding regulators and effectors of NF-κB signaling, which result primarily in constitutive activation of the NF-κB2 pathway [6, 17]. These findings provide genetic evidence for a critical role of NF-κB2 signaling in the pathogenesis of human MM. However, a causal relationship between aberrant activation of NF-κB2 signaling and the development of MM remains to be established. Also, it is unclear at the molecular and cellular levels how NF-κB2 signaling may drive the tumorigenic process.
In the present study, we conducted a detailed analysis of the tumors developed in p80HT mice. Our analysis revealed that approximately half of the tumors are plasma cell tumors that share some of the key pathological features of human MM. Gene expression profiling suggests that p80HT targets multiple cellular processes, including survival, proliferation and differentiation, to promote the development of plasma cell tumors.
p80HT transgenic mice carry the human p80HT coding sequence  under the control of an H-2Kb promoter and an immunoglobulin μ chain enhancer (pHSE3’ expression vector), which direct the transgene expression specifically in T and B lymphocytes [13, 18]. The p80HT mice were generated and maintained on the C57BL/6 J x SJL/J background . All animal studies were pre-approved by the Institutional Animal Care and Use Committee of Georgia Health Sciences University (GHSU).
Histology and immunohistochemistry
Tumor samples were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 5 μm, and stained with Hematoxylin and Eosin (H&E). All tumor samples were examined microscopically in two independent laboratories. For immunohistochemical staining, tumor sections were deparaffinized, rehydrated, and treated with 10 mM citrate buffer (pH 6.0) at 95 °C for 25 min in water bath for antigen retrieval. After quenching of endogenous peroxidase activity with 3% H2O2 and blocking with normal rabbit serum, sections were incubated sequentially with rat anti-mouse CD138 (clone 281-2, 2.5 μg/ml, BD Pharmingen, San Diego, CA) overnight at 4 °C, biotinylated rabbit anti-rat IgG (Vector Laboratories, Burlingame, CA) 30 min at room temperature, and the ABC reagent kit (Vector Laboratories) 30 min at room temperature. The immunostaining was visualized with 3, 3′-diaminobenzidine (Sigma-Aldrich, St. Louis, MO). Section were then counterstained with Hematoxylin before being examined using a light microscope.
Serum protein electrophoresis
Blood samples were collected into microtubes by tail bleeding and sera obtained by microfuge centrifugation for 5 min at 6,000 xg. Serum protein electrophoresis was conducted using a HYDRAGEL K-20 system according to the manufacturer’s instruction (Sebia, Norcross, GA).
For bone mineral density measurement and 3D morphometric analysis, collected femur and spine samples were scanned in a CT (computed tomography) system (Skyscan 1172, Skyscan, Aartlesaar, Belgium). Scanning was performed at an image pixel size of 36.65 μm. Reconstruction of the scanned images was done using a Skyscan Nrecon program. The reconstructed datasets loaded into Skyscan CT-analyzer software for measurement of bone mineral density and 3D morphometric parameters. Fifteen slices measuring 0.55 mm were chosen in femoral head and the body of a thoracic vertebra, and a standardized round region of interest of 1.13 mm diameter was identified along the those slices. The bone mineral density was measured in each region of interest after calibration with hydroxyl apatite phantoms of known density.
Single-cell suspensions were prepared from the bone marrow of p80HT mice according to standard procedures. Red blood cells were lysed in ACK buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.3) and dead cells removed by passing through Lympholyte-M (Cedarlane Laboratories, Burlington, NC). The remaining cells were then stained with Phycoerythrin (PE)-conjugated rat anti-mouse CD138 (clone 281-2, 2.5 μg/ml, BD Pharmingen), sorted on a FACScan machine (BD Biosciences) and analyzed with FlowJo (Tree Star, Ashland, OR).
Splenic B cells were isolated from 1-year-old p80HT mice that were positive for serum M-protein and from their wild-type littermates (n = 3 per genotype) using rat anti-mouse B220 (RA3-6B2, BD Pharmingen) and magnetic beads (EasySep, Stemcell Technologies, Vancouver, BC, Canada), followed by total RNA extraction using Trizol reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). RNA samples were quantified with a spectrophotometer (NanoDrop Technology, Wilmington, DE) and their quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA). RNA samples were further purified with an Ambion WT Expression kit (Life Technologies, Carlsbad, CA) and then run on the Mouse Gene 1.0ST microarray chip (Affymetrix, Santa Clara, CA). Chip hybridization, washing, and imaging were performed in the GHSU Cancer Center Genomics Core using Affymetrix protocols and software with .cel and .txt files generated. Data were normalized, significance determined by ANOVA and fold change was calculated with the Partek Genomics Suite (Partek Inc., St. Louis, MO). Preliminary microarray analysis was performed by the Bioinformatics Resource in the GHSU Cancer Center Genomics Core. Further analysis was done with Microsoft Excel (Redmond, Washington), DAVID , and Ingenuity Pathway Analysis (IPA Ingenuity® Systems http://www.ingenuity.com), with significance of ± 1.5 fold, and P < 0.01. Depending on the software application, filters were applied to duplicates, ESTs, probes with only family similarity and hypothetical proteins. The microarray data in this manuscript are in compliance to MIAME guidelines and have been deposited in the Gene Expression Omnibus at the National Center for Biotechnology Information and is accessible through GEO Series accession number GSE30080 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE30080).
Quantitative reverse-transcription PCR (qRT-PCR)
Relative expression of mRNA was determined on the IQ5 Detection System (Bio-Rad, Hercules, CA) using either a SYBR Green (SA Biosystems, Frederick, MD) or a TaqMan gene expression assay kit (Life Technologies), according to the manufacturer’s instructions. Independent samples (n = 3-5) were assayed twice with each assay being conducted in triplicate. TaqMan assays were performed using the following specific primers after extraction by the RNeasy Mini Kit (Qiagen, Valencia, CA): CD27 Mm01185212_g1, Ccnd1 Mm00432359_m1, Ccnd2 Mm00438071_m1, IL-15 Mm00434210_m1 and Gapdh Mm03302249_g1. The following SYBR Green primers were used after Trizol extraction: survivin forward 5′-GCGGAGGCTGGCTTCA-3′, reverse 5′-CCTGGCTCTCTGTCTGTCCA-3′; IL-10 forward 5′-TGCTATGCTGCCTGCTCTTA-3′, reverse 5′-TCATTTCCGATAAGGCTTGG-3′. Normalized mRNA expression was calculated in comparison to control samples relative to Gapdh. Significance was determined by two-tailed Student t test (P < 0.05).
Human MM cell lines RPMI-8226 (CCL-155, ATCC, Manassas, VA) and EJM were cultured in RPMI1640 with L-glutamine, 10% FBS and 25 μg/ml gentamicin, and H929 in RPMI1640 supplemented with 10% FBS, 1% penicillin/streptomycin and 50 μM β-mercaptoethanol. All cell lines were cultured in humidified air at 37 °C and 5% CO2. For blocking Stat3 activity, S3I-201 (BioVision, Milpitas, CA) was dissolved in DMSO and 100 mM stock solution was prepared. The MM cell lines were treated with S3I-201 at the final concentrations of 50, 100, or 200 μM. DMSO (0.05-0.2%) was used as negative control (untreated). Trypan blue exclusion assays were performed to quantify the numbers of viable and dead cells at various time points following S3I-201 treatment.
Cells were suspended in standard SDS sample buffer and protein concentrations were determined using a protein assay kit (Bio-Rad, Hercules, CA) with bovine serum albumin as reference. Proteins (50 μg) were separated on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed with the following primary antibodies: mouse anti-cyclin D1 (sc-20044, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-cyclin D2 (#2924, 1:1000, Cell Signaling, Danvers, MA), rabbit anti-Stat3 (sc-482, 1:1000, Santa Cruz Biotechnology), rabbit anti-phospho-Stat3-Tyr705 (clone D3A7, #9145, 1:2000, Cell Signaling), mouse anti-survivin (clone D8, sc-17779, 1:100, Santa Cruz Biotechnology), and mouse anti-α-tubulin (B-5-1-2, 1:5000; Sigma-Aldrich). Horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit IgG (Santa Cruz Biotechnology) were used as secondary antibodies. Proteins were visualized using a SuperSignal West Pico chemiluminescence kit (Pierce, Rockford, IL).
p80HT mice develop B-cell and plasma cell tumors
Gene expression profiling of B cells from p80HT mice
Top 20 up and downregulated genes in p80HT B cells
Gene ontology/biological function
Neuropeptide signaling pathway
Positive regulation of cell proliferation
Lymph node development
G-protein coupled receptor protein signaling pathway
Immune response/regulation of gene expression
Positive regulation of transcription, DNA-dependent
Positive regulation of peptidyl-tyrosine phosphorylation
Rab GTPase binding
Wnt receptor signaling pathway
Cell adhesion/signal transduction
Cytokine-mediated signaling pathway
Regulation of transcription
AMP catabolic process
Response to virus
Regulation of cell cycle
Pre-B cell receptor (BCR) signals
Negative regulation of transcription
Regulation of protein amino acid phosphorylation
Zinc ion binding
Positive regulation of cell-matrix adhesion
Protein amino acid phosphorylation
T cell activation
Negative regulation of transcription
Carboxylic acid metabolic process
Negative regulation of cell proliferation
Integrin-mediated signaling pathway
Fatty acid metabolic process
Regulation of transcription, DNA-dependent
Phosphatidylinositol metabolic process
Negative regulation of transcription
Regulation of cell proliferation
IL-10 (+4.02 fold, P = 0.007) promotes the proliferation and survival of MM cells , and cooperates with CD27 (+3.23 fold, P = 0.038) in driving the differentiation of B-cells to plasma cells . IL-15 (+4.57-fold, P = 0.001) has been shown to increase proliferation and inhibit apoptosis in both MM cell lines and primary MM cells . CD80 (+3.5 fold, P < 0.001) interacts with its ligand CD28 (+1.9 fold, P < 0.001) to promote the survival and proliferation of MM cells [24, 25]. Blimp1/Prdm1 (+1.7 fold, P = 0.007) is a transcriptional repressor required for the formation and maintenance of mature plasma cells . Upregulation of cyclin D is a major oncogenic event in MM pathogenesis , and in p80HT B cells, the expression of both cyclin D1 and cyclin D2 was significantly increased (cyclin D1, +2.47 fold, P = 0.038; cyclin D2, +2.58 fold, P = 0.009).
Focus list of differentially expressed TNF genes in P80HT B cells
Gene ontology/biological function
Positive regulation of B cell proliferation/Ig secretion
Regulation of cell proliferation
Regulation of apoptosis
Cell surface receptor linked signal transduction
We confirmed the upregulation of CD27, cyclin D1, cyclin D2, IL-10, IL-15 and survivin in p80HT B cells by qRT-PCR (Figure 3C). Survivin, also called baculoviral IAP repeat-containing protein 5 (BIRC5), is a member of the inhibitor of apoptosis proteins (IAP) and is critical for the survival of human MM cells . Importantly, plasma cell tumor samples from p80HT mice (n = 5) showed even higher levels of cyclin D1, IL-10 and survivin than p80HT B cells (Figure 3C), suggesting that plasma cells or their precursor B cells with high-level expression of these genes were preferentially selected in the development of plasma cell tumors in p80HT mice.
Together, our gene expression profiling and pathway analysis suggest that p80HT promotes the development of plasma cell tumors by coordinated regulation of the generation, proliferation and survival of plasma cells.
Stat3 inhibition induces growth arrest and cell death in human MM cell lines
Despite much effort, the development of mouse models for human MM remains a challenge . A major advance in this area of research has been provided recently by the generation of a transgenic mouse line with spontaneous MYC activation driven by Activation-Induced Deaminase (AID) . These transgenic mice developed bone marrow plasma cell tumors that recapitulate many features of human MM. However, rearrangements of the MYC gene are present in only 15% of MM patients , calling for the development of additional mouse models that target distinct signaling pathways important in MM pathogenesis. Chromosomal translocations and rearrangements at the NF-κB2 locus have been shown to occur in primary human MM . The human MM cell lines JK6L and CAG also carry genetic mutations in the NF-κB2 gene . These mutations lead to the generation of C-terminally truncated NF-κB2 proteins similar to the tumor-derived NF-κB2 mutant p80HT . Currently, no mouse models are available for human MM with aberrant activation of NF-κB2 signaling. We have recently reported that transgenic mice with targeted expression of p80HT in lymphocytes developed predominantly B-lineage lymphomas with the tumor incidence of 79% by 70 weeks . In this investigation, we conducted detailed histological and immunohistochemistry examination of 12 tumor samples from the previous study , which revealed that half of them (6/12) were plasma cell tumors. To corroborate this finding, we generated additional p80HT mice with a focus on their development of plasma cell tumors. Approximately 40% of the newly generated p80HT mice produced M-protein by 1 year of age. Most of these M-protein-positive p80HT mice developed plasma cell tumors with diffuse osteoporosis. Some of them also had osteolytic bone lesions and/or significant accumulation of plasma cells in the bone marrow. These findings provide the first direct evidence for a causal role of NF-κB2 mutation in the pathogenesis of plasma cell tumors that share some key histopathological and clinical features of human MM.
The transgenic mice express p80HT in both T and B cells, and our previous study suggests that p80HT promotes tumor development primarily by enhancing the survival of T and B cells . Also, the lymphoma development in p80HT mice is characterized by a prolonged latent period , suggesting that additional genetic and/or epigenetic alterations are required for the malignant transformation of p80HT lymphocytes and their clonal expansion. We speculate that this might be the major reason why p80HT mice develop a wide spectrum of B cell lymphomas including plasma cell tumors, as well as T cell lymphomas, which depend on the type and developmental stage of the lymphocytes that have acquired secondary genetic and/or epigenetic alterations.
We have previously identified TRAF1 as a target gene critical for the oncogenic activity of p80HT . TRAF1 deficiency reestablished B cell homeostasis and significantly delayed the tumor development in p80HT mice  (unpublished data). However, constitutive overexpression of TRAF1 in lymphocytes is not tumorigenic in mice  suggesting that additional target genes must be critical for the development of plasma cell tumors in p80HT mice. We performed gene expression profiling of B cells from M-protein-positive p80HT mice for the reason that genes activated in these plasma cell precursors are anticipated to drive the development of plasma cell tumors. It is important to note that the three M-protein-positive p80HT mice used for the microarray assay might not develop plasma cell tumors in the end. Nonetheless, the gene expression profiling revealed the activation of many genes, in addition to TRAF1, in p80HT B cells that are known to promote the proliferation and survival of MM cells, as well as the differentiation of B cells to plasma cells.
IL-15 is the second most upregulated gene in p80HT B cells. IL-15 is important to the proliferation of MM cells and their ability to evade apoptosis . An autocrine loop between IL-15 and its receptor has been identified as a mechanism for tumor cell expansion in MM . The upregulation of both IL-15 and its receptor IL-15Ra (+1.7 fold, P = 0.045) in the B cells of p80HT mice suggest that this pathway is important in the pathogenesis of plasma cell tumors in our mouse model.
Another top upregulated gene is IL-10. Using the transcription factor database search tool DECODE (SaBiosciences) and data from the University of California Santa Cruz Genome Browser, we identified potential two κB-binding sites in the IL-10 promoter (unpublished data), suggesting that p80HT may directly upregulate IL-10 expression. IL-10 exerts its biological functions primarily through Stat3 [30, 31]. It has long been recognized that IL-10 promotes the differentiation of B cells to plasma cells [22, 40–43]. This action of IL-10 is likely mediated, at least in part, by Blimp1, a known target gene of Stat3 . Consistent with the notion, Blimp1 expression was significantly upregulated (+1.7 fold, P = 0.007) in p80HT B cells. Blimp1 is a transcriptional repressor required for the formation and maintenance of mature plasma cells . Blimp1 is also required for the formation of plasmacytoma in a mouse model . Other Stat3 target genes include cyclin D1, cyclin D2, and survivin [32, 33], and they were all markedly upregulated in p80HT B cells.
Survivin is a member of the inhibitor of apoptosis protein family with dual roles in regulation of cell cycle progression and apoptosis . Survivin expression is increased during MM progression, and knockdown of survivin induces cell death in human MM cells . It has been shown previously that the human survivin gene promoter region contains κB-binding sites , and our sequence examination revealed 4 potential κB-binding sites within the mouse survivin promoter region (unpublished data). These observations suggest that p80HT may transcriptionally upregulate survivin expression either directly or indirectly through the IL-10-Stat3 signaling pathway.
Cyclins D1, D2 and D3 (encoded by CCND1, D2 and D3) interact with and activate cyclin-dependent kinase 4 (Cdk4) or Cdk6 to facilitate the G1/S cell-cycle transition . Upregulation of cyclin D expression occurs in the vast majority of MM tumors and has been considered a crucial and early oncogenic event in MM pathogenesis . Approximately 20% of MM tumors show elevated levels of cyclin D1 or D3 as the result of chromosomal translocations that juxtapose potent immunoglobulin (Ig) gene enhancers next to CCND1 (11q13) or CCND3 (6p21) , and ~7% of MM tumors have Ig translocations involving c-MAF (16q23) or MAFB (20q11), which encode transcription factors that target CCND2 [27, 48]. In the rest of MM tumors, the transcription factors responsible for upregulating the expression of cyclin D genes remain to be identified. Cyclin D1 is a known target gene of the NF-κB1 signaling pathway [49, 50]. However, to the best of our knowledge, a role for NF-κB2 in regulation of cyclin D expression has not been previously described. Thus, our findings that cyclin D1 and cyclin D2 were significantly upregulated in p80HT B cells suggest a novel mechanism for early activation of cyclin D genes in the development of MM.
To determine the clinical relevance of our findings, we assessed the response of human MM cell lines to the inhibition of Stat3, a key downstream component of the IL-10 signaling pathway. Our investigation revealed a correlation between the activity of Stat3 and the sensitivity to Stat3 inhibition in MM cells. Also consistent with our model, Stat3 signaling was found to be essential for high-level expression of cyclin D1 and survivin in MM cells, providing a molecular mechanism for the critical role of Stat3 signaling in the proliferation and survival of MM cells. Thus, targeting the Stat3 signaling pathway may represent a therapeutic strategy for human MM.
Our findings provide the first direct evidence for a causal role of NF-κB2 mutation in the pathogenesis of mouse plasma cell tumors that share some key histopathological and clinical features of human MM. The NF-κB2 mutant p80HT promotes the development of plasma cell tumors by transcriptional activation of genes critical for the generation, proliferation and survival of plasma cells, including cyclin D1, cyclin D2, Blimp1, survivin, IL-10 and IL-15. We further present evidence that targeting the IL-10-Stat3 signaling pathway may represent a therapeutic strategy for human MM.
We are indebted to W.M. Kuehl of the National Cancer Institute for his insightful comments on the manuscript. We thank H.C. Morse of the National Institute of Allergy and Infectious Diseases, W. Gunning of the University of Toledo College of Medicine, and T. Nagy of the University of Georgia for histological examination of tumor samples. We also thank W.M. Kuehl and D. Sullivan of the Moffit Cancer Center for providing the MM cell lines EJM and H929, respectively. This work was supported by a grant from the National Cancer Institute (R01 CA106550) and Georgia Cancer Coalition Distinguished Scholar Award to HFD, and grants from and the National Basic Research Program of China (No. 2012cb114603) and the National Natural Science Foundation of China (No. 31172268) to HC. BAM is a Multiple Myeloma Research Foundation Research Fellow.
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