Lymphoma

RelA and RelB cross-talk and function in Epstein–Barr virus transformed B cells

Abstract

In this study, we determined the respective roles of RelA and RelB NF-κB subunits in Epstein–Barr virus (EBV)-transformed B cells. Using different EBV-immortalized B-cell models, we showed that only RelA activation increased both survival and cell growth. RelB activity was induced secondarily to RelA activation and repressed RelA DNA binding by trapping the p50 subunit. Reciprocally, RelA activation repressed RelB activity by increasing expression of its inhibitor p100. To search for such reciprocal inhibition at the transcriptional level, we studied gene expression profiles of our RelA and RelB regulatable cellular models. Ten RelA-induced genes and one RelB-regulated gene, ARNTL2, were repressed by RelB and RelA, respectively. Apart from this gene, RelB signature was included in that of RelA Functional groups of RelA-regulated genes were for control of energy metabolism, genetic instability, protection against apoptosis, cell cycle and immune response. Additional functions coregulated by RelA and/or RelB were autophagy and plasma cell differentiation. Altogether, these results demonstrate a cross-inhibition between RelA and RelB and suggest that, in fine, RelB was subordinated to RelA. In the view of future drug development, RelA appeared to be pivotal in both classical and alternative activation pathways, at least in EBV-transformed B cells.

Introduction

Epstein–Barr virus (EBV) is responsible for immunodeficiency-related diffuse large B-cell lymphomas (DLBCLs) of posttransplant or human immunodeficiency virus-infected patients. EBV is also associated with various other cancers, including DLBCLs of the elderly, Burkitt, Hodgkin’s or T-cell lymphomas and nasopharyngeal carcinomas.1 In vitro, EBV infects and transforms primary B cells, leading to the continuous proliferation of lymphoblastoid cell lines. This proliferation program, also called latency III program, is driven by the EBNA2 (Epstein–Barr Nuclear Antigen 2) protein, which regulates expression of the entire set of EBV latent genes, including the BNLF1 gene coding for the main EBV oncogene, the latent membrane protein 1 (LMP1). Other EBV latent proteins are EBNA1, required for episomal maintenance of the EBV genome, the EBNA3 proteins that modulate EBNA2 and regulate cell proliferation and LMP2A that mimics the B-cell receptor. Some EBV-associated B-cell lymphomas such as endemic Burkitt lymphomas or the rare primary effusion and plasmaplastic lymphomas express only the EBNA1 (so-called latency I). However, most EBV-associated tumors express LMP1, even in latency III or II (expression of EBNA1 and LMP proteins). LMP1 is a transmembrane protein acting as a constitutive active CD40 receptor, thereby continuously activating NF-κB.2 Previous results demonstrated that preserved NF-κB activity and protection against apoptosis would be the minimal prerequisite for all LMP1 natural mutated variants isolated from both normal and Reed-Sternberg cells from Hodgkin’s lymphomas.3

Two NF-κB activation pathways have been described, so-called classical (or canonical) and alternative (or non-canonical) (Vallabhapurapu and Karin4 for review). The classical pathway is induced in response to a variety of stimuli, such as CD40-Ligand, TNFα, IL-1, IL-6, bacterial lipopolysaccharide, as well as LMP1 of EBV. It involves RelA- or c-Rel- and p50-containing complexes. In resting cells, NF-κB dimers containing these subunits are retained in the cytoplasm by physical interaction with IκBα, β, ɛ or the p105 precursor of p50. Following activation of the classical NF-κB pathway, the IκBs and p105 are rapidly phosphorylated by the IκB kinase complex (IKK), containing the catalytically active kinases IKKα and IKKβ and the regulatory scaffold protein NEMO (NF-κB Essential Modulator or IKKγ).5 Phosphorylation of IκBs leads to their proteasomal degradation, releasing NF-κB dimers that translocate into the nucleus where they activate transcription of specific target genes.4 With much slower kinetics, the alternative NF-κB activation pathway is induced by a restricted subset of receptors such as the Lymphotoxin β receptor, B-cell-activating factor receptor or CD40, which then leads to NF-κB-inducing kinase activation.6 NF-κB-inducing kinase phosphorylates IKKα, which in turn mediates phosphorylation and proteolysis of p100, the precursor of p52. P100 acts as an IκB molecule specifically trapping RelB-containing complexes (i.e., RelB/p50 and RelB/p52 dimers). P100 proteolysis allows nuclear translocation of these complexes.7

Most reports addressing the question of NF-κB activation by LMP1 used reporter gene assays or studied the involvement of NEMO/IKKα/IKKβ or NF-κB-inducing kinase/IKKα, and most studies were performed in non-B-cell lineages.8, 9, 10, 11 Very few studies have addressed the question of the specific roles of RelA and RelB in EBV-transformed B cells. This question is of importance not only to understand the place of both subunits in B-cell transformation but also in the view of developing new drugs targeting NF-κB. Here, we addressed the question of the function, DNA-binding activity and gene expression signatures of RelA and RelB in EBV-immortalized B-cell line models.

Materials and methods

Patients and biopsies

For this study, 11 patients, three with EBV-positive posttransplant DLBCLs (EBV-DLBCL n° 1–3, following kidney transplantation for patients 1 and 2 and curative bone marrow transplantation for T-cell lymphoma for patient 3; patient 1 was under mycophenolate and tacrolimus and patients 2 and 3 were under cyclosporine A regimen. Patient 3 initially received one dose of antilymphocyte globulins), one with EBV positive human immunodeficiency virus-associated DLBCL (EBV-DLBCL n° 4), four with EBV-positive DLBCLs of the elderly (EBV-DLBCL n° 5–8), and three with nontumoral reactive lymph nodes (LN n° 1–3), were enrolled according to institutional regulations and after approval by the IRB of the university hospital of Limoges. EBERs were detected in all tumor cells. Tumor cells expressed CD20 and LMP1 confirmed by immunohistochemical analysis in all cases.

Plasmid constructs

Complementary DNAs for LMP1,12 Luciferase (Promega, Paris, France), IκBαS32,36A,12 RelA, RelB13 and p100/p5213 were cloned into the previously described pRT-1 doxycycline-inducible episomal vectors.14 The RelA insert was obtained by long distance amplification of the corresponding mRNA from an LCL cell line.

Cell lines, CD40-Ligand stimulation and cell transfection

Classical lymphoblastoid cell lines PRI, 1602, TSOC, LCL.4 and LCL.6 have been described in previous reports.3, 15 EREB2–5 cells are a nonclassical LCL cell line with an estradiol-inducible EBV-latency III proliferation program due to an estrogen receptor fused to the EBNA2 viral protein.16 Transfection, hygromycin selection and CD40-Ligand stimulation of EREB2–5 cells were performed as previously described.17, 18

Protein extracts and electrophoretic mobility shift assays (EMSAs)

Methods for cytoplasmic and nuclear extracts are described elsewhere.19 The PRE double-stranded oligonucleotide probe and the EMSA technique are described in Supplementary Materials and Methods.

Immunoprecipitation and western blot

Immunoprecipitation of p50 was performed as described in Supplementary Materials and Methods. Western blots were performed as previously described.20 The antibodies used are detailed in Supplementary Materials and Methods.

Analysis of apoptosis by flow cytometry

Cells were double stained with AnnexinV-FITC (BD Pharmingen, San Diego, CA, USA) and propidium iodide (Sigma-Aldrich, Saint-Louis, MO, USA) in cold PBS-CaCl2-MgCl2 (Invitrogen, Cergy-Pontoise, France) as described20 and analyzed by FACS Calibur cytometer (BD Pharmingen, Paris, France).

Proliferation assays

Transfected EREB2–5 cells were plated in 96-well plates (103/well), in 10% fetal calf serum medium containing or not doxycycline. Over 2 days, proliferation rates were measured using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Metabolism Tetrazolium Salt assay from Promega).

Cell sorting and RNA isolation

EREB2–5 cell line conditions used for gene expression profiling are detailed in Supplementary Materials and Methods. When indicated, NGFRt-expressing EREB2–5 transfected cells were purified with MACS microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) following previously published protocols.12 Isolation of the high-quality total RNA was performed from samples (cell lines and tissues) in the TRIzol Reagent (Invitrogen) using the RNeasy mini kit (Qiagen, Valencia, CA, USA). High quality (integrity and purity) of RNA was verified by the Agilent RNA 6000 Nano LabChip kit and the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

Gene expression profiling

Amplification of RNAs and hybridization onto microarrays were performed on an Affymetrix Gene Atlas system with the Affymetrix Human Genome U219 Array. Data analysis was performed according to both the LIMMA and SAM methods.21, 22, 23 Biological functions of genes were studied using ‘Gene Set Enrichment Analysis’ (www.broadinstitute.org/gsea/index.jsp). Details are in Supplementary Materials and Methods.

Gene quantification with TaqMan low density array

cDNAs were reverse transcribed from total RNA samples using the High Capacity cDNA Archive Kit (Life Technologies, Carlsbad, CA, USA). PCR products were amplified from 200 ng of each cDNA sample using the TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assays preloaded in each reaction well of TaqMan low density arrays (Life Technologies). Thereafter, TaqMan low density arrays were run on the 7900HT system for quantitative real-time PCR analysis. Details are in Supplementary Materials and Methods.

Results

RelA and RelB differentially regulate proliferation and survival in EBV-immortalized B cells

To assess functional roles of RelA and RelB, we cloned the corresponding cDNAs and that of their respective inhibitor, IκBαS32,36A super-repressor and p100, into the doxycycline-regulatable pRT-1 vector.14 The same vector coding for Luciferase15 was used as control. After estradiol deprivation to induce arrest of the EBV-latency III program, EREB2–5 B cells were either pre-exposed or not to doxycyline for 24 h and then treated with estradiol for both 24 and 48 h. In this model, estradiol treatment of cells induces the EBV-latency III program with LMP1 expression that induces both NF-κB activation pathways.24

At 48 h, overexpression of each protein was checked by Luciferase assays or by western blot (Figure 1a). As expected, induction of the EBV-latency III program (E24hEBV and E48hEBV) reverted spontaneous apoptosis and induced cell growth in estradiol-starved E0hEBV cells in Luciferase-expressing cells (Figures 1b and c). RelA induction by doxycycline weakly over-increased protection against apoptosis and markedly over-increased cell proliferation when compared with Luciferase or uninduced EBV-latency III proliferating control cells, respectively (Figures 1b and c). Induction of the super-repressor form of IκBα, IκBαS32,36A, which inhibits the classical pathway,19, 25 abolished both EBV-induced protection against apoptosis and growth (Figures 1b and c). Surprisingly, induction of both RelB and p100 decreased EBV-latency III protection against apoptosis (Figure 1b). RelB also repressed EBV-induced proliferation of EREB2–5 cells, whereas p100 had no effect (Figure 1c). These results clearly suggest that RelA activation was associated with both proliferation and protection against apoptosis in EBV-latency III immortalized B cells. The role of RelB on apoptosis and proliferation would be more complex.

Figure 1
figure1

Effect of RelA, IκBαS32,36A, RelB and p100 on apoptosis and proliferation of EBV-latency III program-induced EREB2–5 cells. EREB2–5 cells were stably transfected with the doxycycline-regulatable pRT-1 vector coding for Luciferase, RelA, IκBαS32,36A, RelB or p100. After estradiol starvation for 72 h, cells were treated (+) or not (−) with doxycycline (Dox.) for 24 h. Then, the EBV-latency III program was re-induced or not (E0hEBV) by addition of estradiol for 24 (E24hEBV) or 48 h (E48hEBV). (a) At 48 h, analysis of Luciferase activity (lanes 1–2) and western blots for the expression of RelA (lanes 3–4), IκBαS32,36A (lanes 5–6), RelB (lanes 7–8) p100/p52 (lanes 9–10) and α-tubulin (αTub., lanes 3–10). (b) Annexin-V labeling was assessed by flow cytometry on estradiol-starved doxycycline-treated pRT-1-transfected EREB2–5 cells (E0hEBV) and after 24 (E24hEBV) or 48 h (E48hEBV) estradiol re-induction of the EBV-latency III program in the presence of doxycycline. For each pRT-1-transfected EREB2–5 cells, percentages of Annexin-V-positive cells were normalized to those of E0hEBV cells, corresponding to 100%. Statistically significant differences (one-way ANOVA) between the three time points E0hEBV, E24hEBV and E48hEBV are indicated as follows: *(P<0.05), **(P<0.01), ***(P<0.001) and ****(P<0.0001). ns, non-significant. (c) Proliferation rates were assessed using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Metabolism Tetrazolium Salt assay from Promega). Relative proliferation rate was defined as the ratio of the absorbance at 492 nm between estradiol-starved pRT-1-transfected EREB2–5 cells (E0hEBV) and 24 (E24hEBV) or 48 h (E48hEBV) estradiol re-induction of the EBV-latency III program in the presence or not of doxycycline. T-test significant differences between cells treated or not with doxycycline at the same time point are indicated by *(P<0.05) and **(P<0.01). Each experiment was performed at least three times.

Dynamics of NF-κB complexes in EBV-immortalized B cells: reciprocal inhibition of RelA and RelB

To correlate functional assays with NF-κB DNA binding activities, we performed EMSA. Controls for specificity are given in Supplementary Figure S1. First, nuclear extracts from five EBV-latency III immortalized B cell lines, all with LMP1 expression, were isolated. NF-κB DNA binding patterns systematically corresponded to three bands, BI, BII and BIII (Figure 2a). Supershift experiments indicated that band B-I was likely to correspond to RelA/p50 dimers, band B-II to both c-Rel and RelB complexes and band B-III to p50 homodimers. Kinetics of EBV-latency III program induction in EREB2–5 cells showed that NF-κB activation paralleled LMP1 expression levels and reached maximum rates at 6 h (Figure 2b, lanes 1–5). After LMP1 expression or stimulation of its cell homolog CD40 for 24 h, the same NF-κB complexes with the same bands BI, BII and BIII were identified (Figure 2b, lanes 7–18). At the protein level, NF-κB subunits of the classical pathway, that is, RelA- and c-Rel-containing complexes, were rapidly translocated into the nucleus starting at 1 h after EBV-latency III induction (E1hEBV), whereas RelB complexes were activated later at 24 h (E24hEBV, Figure 2c). These results clearly show a rapid and strong activation of the classical NF-κB pathway and much delayed, slower and weaker activation of the alternative pathway by the EBV-latency III program.

Figure 2
figure2

Activation of NF-κB pathways in EBV-latency III immortalized B cells. (a) NF-κB DNA binding activity from nuclear extracts of five classical EBV-latency III immortalized lymphoblastoid B cell lines. Lane 1 to 5: EMSA with the PRE radiolabeled probe for LCL PRI (lane 1), 1602 (lane 2), TSOC (lane 3), LCL.4 (lane 4) and LCL.6 (lane 5). Corresponding western blots for LMP1 and αTub. are shown below. Lane 6–11: supershift experiments from TSOC nuclear extracts using antibodies against Oct2 (irrelevant control, lane 6), RelA (lane 7), p50 (lane 8), RelB (lane 9), p52 (lane 10) and c-Rel (lane 11). (b and c) Kinetic experiments of NF-κB activation on EREB2–5 cells. Estradiol-starved cells were treated for 0, 1, 3, 6 and 24 h with estradiols E0hEBV, E1hEBV, E3hEBV, E6hEBV and E24hEBV, respectively. Estradiol-starved cells were also treated for 24 h with CD40 Ligand (E24hCD40). (b) Lanes 1–6: EMSA for NF-κB DNA binding activity of nuclear extracts from E0hEBV to E24hEBV and E24hCD40 cells (upper panel) with corresponding western blots for LMP1 (middle panel) and αTub.(lower panel). Lanes 7–18: supershift assay of nuclear extracts from E24hEBV (lanes 7–12) and E24hCD40 cells (lanes 13–18). (c) Lanes 1–6: kinetics of nuclear translocation of NF-κB subunits. Western blots from nuclear and cytosolic extracts for p50, RelA, c-Rel, p52 and RelB proteins. Revelations of αTub. and SAM68 were used for loading controls of cytosolic and nuclear extracts, respectively. For EMSAs, bands B-I to B-III are indicated by arrows and supershifted bands are indicated by * for each lane.

Second, we analyzed NF-κB complexes in EREB2–5 cells transfected with RelA, RelB, IκBαS32,36A super-repressor and p100 after 48 h EBV-latency III program induction. Luciferase expression had no effect on NF-κB binding activity (Figure 3a, lane 2). RelA induction was associated with (i) an increase in band B-I containing the RelA/p50 and RelA/RelA dimers (Figure 3b, lanes 2–5) and (ii) a decrease in band B-II, now completely supershifted with c-Rel antibody (Figure 3b, lanes 2, 3 and 8). Thus, RelA overexpression repressed RelB but not c-Rel DNA binding activity. The expression of IκBαS32,36A led to a global decrease in NF-κB DNA binding activity (Figure 3c, lane 2). Over-expression of RelB deeply modified the NF-κB DNA binding activity, with two major bands: one corresponding to B-II and one new band, called B-IV (Figure 3d, lanes 2 and 3). Both bands B-II and B-IV were completely supershifted with RelB antibodies (Figure 3d, lane 6). As attested by the absence of supershift with RelA and c-Rel antibodies, RelB overexpression repressed NF-κB complexes activated by the classical pathway (Figure 3d, lanes 4 and 8). Moreover, band B-IV was completely supershifted with p50 antibodies, suggesting that p50 was the main partner of RelB (Figure 3d, lane 5). Finally, overexpression of p100 did not affect RelA DNA binding activity and specifically induced the loss of RelB binding activity in band B-II, such a loss was suggested by the complete extinction of band B-II in the presence of c-Rel antibody (Figure 3e, lane 8). This result can be interpreted as a specific inhibition of RelB DNA binding activity by an excess of p100 protein, which would trap RelB in the cytoplasm.

Figure 3
figure3

NF-κB DNA binding activity after RelA, IκBαS32,36A, RelB or p100 induction. EREB2–5 cells were stably transfected with the doxycycline-regulatable pRT-1 vector coding for Luciferase, RelA, IκBαS32,36A, RelB and p100. After estradiol starvation, cells were treated (+) or not (−) with doxycycline (Dox.) for 24 h. Then, the EBV-latency III program was re-induced by addition of estradiol for 48 h. (a to e, lanes 1 and 2) EMSA for NF-κB DNA binding activity of EREB2–5 cells transfected with (a) pRT-1-Luciferase vector, (b) pRT-1-RelA, (c) pRT-1-IκBαS32,36A, (d) pRT-1-RelB and (e) pRT-1-p100. (b, d and e, lanes 3–8) The supershift assay of nuclear extracts from cells treated with both estradiol and doxycycline. Nuclear extracts were incubated with anti-Oct2 (irrelevant control, lane 3), anti-RelA (lane 4), anti-p50 (lane 5), anti-RelB (lane 6), anti-p52 (lane 7) or anti-c-Rel (lane 8) antibodies. Bands B-I to B-IV are indicated by arrows, and supershifted bands are indicated by * for each lane.

Mechanisms of RelA and RelB reciprocal inhibition

As p50 was the main partner of RelA and RelB by EMSA, we looked for the p50 partners by immunoprecipitation. P50 was indeed associated with RelA, RelB and p52 in Luciferase control cells (Figure 4, lane1). Induction of RelA was associated with a marked increased in immunoprecipitated RelA/p50 heterodimers and a slight increase in immunoprecipitated RelB/p50 complexes (Figure 4, lane 2). Furthermore, p50 was additionally associated with p100 in these RelA-overexpressing cells, which correlates with an increase in p100 expression in the input (Figure 4, lanes 1 and 5). These results led to the hypothesis that RelA overexpression could indirectly repress DNA binding of RelB/p50 complexes by increasing p100 expression, which in turn would trap RelB-containing complexes in the cytoplasm.

Figure 4
figure4

p50 Immunopreicipitation after induction of RelA and RelB. EREB2–5 cells were stably transfected with the doxycycline-regulatable pRT-1 vector coding for Luciferase, RelA or RelB. After estradiol starvation, cells were treated with doxycycline for 24 h and then the EBV-latency III program was re-induced by addition of estradiol for 48 h. Western blot detection of RelA, p105/p50, RelB and p100/p52 from immunoprecipitated p50 and input extracts of EREB2–5 overexpressing Luciferase (lanes 1 and 4), RelA (lanes 2 and 5) and RelB (lanes 3 and 6). Input control by western blot for αTub. are shown (lanes 4–6).

When RelB was overexpressed, RelB/p50 dimers were strongly increased and, concomitantly, RelA/p50 complexes were decreased when compared with Luciferase control cells (Figure 4, lane 3). In this condition, p100 expression and association with p50 were also increased (Figure 4, lanes 3 and 6). This p100 induction by RelB was likely to exert some inhibitory negative feedback on RelB but was certainly not sufficient to inhibit binding of all RelB/p50 heterodimers to DNA, as attested by the EMSA experiments presented in Figure 3.

Altogether, results presented in Figure 3 and 4 demonstrated a cross-talk between RelA and RelB so that RelA was likely to inhibit RelB DNA binding by overexpression of p100 and RelB repressed RelA DNA binding by competing for their association with p50.

RelA and RelB transciptomic signatures

To evidence cross-talk between RelA and RelB at the transcriptional level, that is, their cross-inhibition, we searched traces of such interplay using high-throughput gene expression profiling.

We first established the list of genes deregulated in EBV-transformed B cells. To avoid gene deregulation due to in vitro cell line artifacts, we also addressed genes truly deregulated in EBV-associated DLBCLs with the expression of LMP1 (three cases of EBV-related posttransplant DLBCL, one case with EBV-positive human immunodeficiency virus-associated DLBCL and four with EBV-positive DLBCLs of the elderly). Such variants of EBV-associated DLBCLs would also allow to prevent any bias due to a particular subtype of EBV-associated B-cell lymphomas.

On one hand, the different B cell lines were divided in two groups, those in which the EBV-latency III program was on or off. Cells expressing LMP1 alone were also included (Supplementary Figure S2). Selected genes yielded a cell line list of 1550 genes upregulated by the EBV-latency III program (Supplementary Table S1). On the other hand, EBV-DLBCL tumors were compared with three non-tumoral lymph nodes with benign follicular hyperplasia. This led to an EBV-related tumor list of 3415 genes (Supplementary Table S2). A total of 726 genes were in common between both lists, being thus bona fide EBV-induced genes in EBV-transformed B cells both in vivo and in vitro (Supplementary Figure S3 and Supplementary Table S3). Among these 726 genes, 612 (84%) were regulated by LMP1 alone and numerous genes were known targets of c-Myc, E2F1, Jun or NF-κB (Supplementary Figure S3 and Supplementary Table S3).

Then, EREB2–5 cells overexpressing Luciferase, RelA, RelB, IκBαS32,36A super-repressor and p100 were sorted after doxycycline induction (i.e., E.Luc, E.RelA, E.RelB, E.IκBαSS32,36A and E.p100 cells, respectively), and their gene expression profiles were analyzed to identify RelA and RelB target genes among the 726 EBV upregulated genes.

Forty-six genes were very likely to be upregulated by RelA and/or RelB in EBV-DLBCL-transformed B cells (Supplementary Materials and Methods for details and Figure 5). Of note, 44/46 genes were upregulated by LMP1 with a fold change ranging from 1.6–85 (Supplementary Table S4). As an independent experiment, we looked at the deregulation of these genes in the series published by Basso et al.26 (GEO Accession Number: GSE2350), which includes purified resting B cells from cord blood, lymphoblastoid cell lines and immunoblastic DLBCLs from both AIDS-related immunodeficient and immunocompetent patients. Indeed, these DLBCL variants are frequently associated with constitutive NF-κB activation.27, 28 Of the 46 RelA and/or RelB target genes, 34 could be retrieved from the gene list of Basso et al., and among them 31 were indeed deregulated either in lymphoblastoid cell lines or in immunoblastic DLBCLs (Supplementary Table S5). Deregulation of these genes was confirmed by quantitative PCR on cDNA from EBV-DLBCL tumors and LCL cells (Supplementary Figures S4 and S5).

Figure 5
figure5

RelA and RelB traces among EBV-latency III-deregulated genes in EBV-transformed B cells. The different cell line conditions are indicated at the top of the panels: E0hEBV−120h+Dox-Luc, EcontEBV+Dox-Luc, EcontEBV+Dox-RelA, EcontEBV+Dox-IκBαS32,36A, EcontEBV+Dox-RelB and EcontEBV+Dox-p100. Genes were arrayed in a descending order according to their expression level in EcontEBV+Dox-Luc. Gene symbols are indicated on the right. RelA- or RelB-regulated genes were those that were overexpressed in E.RelA or E.RelB when compared with E.IκBαS32,36 A or E.p100 cells, respectively, and that were downregulated in E.IκBαSS32,36A or E.p100 cells, respectively when compared with E.Luc cells. Expression profiles of (a) 11 RelA and RelB co-dominant target genes, corresponding to intersection between the RelA and RelB lists, (b) 24 RelA dominant target genes, corresponding to the RelA but no to the RelB list, and (c) 11 RelA or RelB target genes only, corresponding to genes whose expression was repressed by RelB or RelA, respectively. (d) List of RelA (n=45) and RelB target genes (n=12) among the 726 upregulated genes by EBV in LCLs and EBV-DLBCLs. Log2-value color codes are shown below each panel. EREB2–5 line conditions are detailed in Supplementary Materials and Methods. Note the induction of NFKBIA/IKB mRNA in the EBVcontEBV+Dox-IκBαS32,36A condition (IκBαS32,36A overexpression).

Among the RelA and/or RelB target genes selected by our analysis, 45/46 were induced by RelA (Figure 5). Eleven of these 45 genes were also induced by RelB, that is, RelA- and RelB-co-regulated genes (Figure 5a). Twenty four of these 45 genes were apparently not regulated by RelB (so-called RelA-dominant genes) (Figure 5b). This can be interpreted as a simultaneous and overlapping RelA repression by RelB and RelB weak gene upregulation. The remaining 11 RelA-regulated genes were repressed by RelB, so-called RelA-only genes (Figure 5c). RelB induced the expression of one gene, ARNTL2 (Aryl hydrocarbon receptor nuclear translocator-like protein 2), whose expression was repressed by RelA (Figure 5c).

These results clearly suggest that RelB may repress transcription of some RelA target genes and vice versa. Additionally, it appeared that RelB signature was almost entirely included in the one of RelA (Figure 5d).

Functions of RelA- and/or RelB-regulated genes are shown in Supplementary Figure S6 and Supplementary Table S6. Genes dominantly regulated by RelA were associated with metabolism, genetic instability, protection against apoptosis, cell cycle and immune response. Additional functions of genes codominantly regulated by RelA and RelB were autophagy, transcription factors regulating cell cycle and plasma cell differentiation and NF-κB regulators. The only RelB-specific gene (i.e., repressed by RelA) was ARNTL2, a gene involved in the regulation of circadian rhythm and protection against hypoxia.

Discussion

NF-κB pathways have been implicated in Hodgkin’s lymphomas, multiple myelomas and both EBV-negative and EBV-associated DLBCLs.29, 30, 31, 32, 33 Recently, it has been demonstrated in a mouse transgenic model that LMP1-expressing B cells are under permanent control of both T-cell-acquired and NK cell innate immune responses.34 We indeed demonstrated that LMP1 is able to promote autologous T cell targeting of EBV-infected B cells in humans.12 Despite this permanent immune control, LMP1 remains the main oncogenic agent of EBV, as demonstrated both in human EBV-related tumors and different mice models, including the model developed by Zhang et al.34 Indeed, almost all LMP1-positive EBV-associated B-cell lymphomas are associated with a more or less profound immunodeficiency status. The oncogenic potential of EBV is directly linked to continuous activation of NF-κB by LMP1.35 We thus worked on LMP1-expressing EBV-transformed B cells, raising the question of the respective roles of RelA and RelB. The model that emerged from our results is that RelA and RelB cross-talked together so that RelB was subordinated to RelA and would help RelA in reinforcing the regulation of some genes like those involved in autophagy or metabolism.

As a side conclusion of our work, we found that p50 was the main partner of RelA and RelB in EBV-immortalized B cells. Overexpressed RelB trapped most, if not all, available p50 subunits in the cell, inhibiting therefore DNA binding of both RelA/p50 and c-Rel/p50 complexes. Thus, in EBV-infected B cells, RelA and RelB were in competition for p50, explaining mechanistically how RelB exerted an inhibitory activity on RelA. Although poorly represented in our cell models, RelB/p52 complexes were also increased under RelB overexpression. Although processing of p105 to p50 is constitutive, p100 degradation or processing to p52 results from the activation of alternative signaling, allowing nuclear translocation of RelB-containing dimers associated with p50 or p52, respectively.36 The nfkb2/p100−/−-deficient mice model showed a functional overlap between p50 and p52 when bound to RelB.37 As RelB blocks RelA DNA binding by trapping its main partner, the p50 subunit, it can be put forward that the inhibitory effect of RelB overexpression on both proliferation arrest and increased apoptosis corresponds to its inhibitory effect on RelA. It is only because p100 overexpression was associated with both increased apoptosis and specific lack of RelB DNA binding activity that we can infer that RelB is likely to have a role in protection against apoptosis.

Several recent studies provided data that interconnections between the alternative and classical NF-κB pathways exist through expression control of NF-κB subunits.38 Regulatory regions of relb and nfkb2/p100 genes contain κB binding sites, and the expression of these genes is dependent on RelA.39, 40 In addition, experiments on rela−/− murine embryonic fibroblasts suggested that RelA-dependent expression of RelB is the main determinant of alternative pathway responses through Lymphotoxin β receptor stimulation.41 Consistently, as the overexpression of RelA increased p100 and RelB expression and RelB/p50 association together with decreased RelB DNA binding activity, we interpreted these results as the effect of RelB trapping by p100. However, another alternative would be that RelA and RelB association would directly inhibit RelB DNA binding activity, as reported in the murine embryonic fibroblast model.42 In any case, DNA binding of RelB was always weak in EBV-latency III immortalized B cells when compared with that of RelA or c-Rel or with RelB activity induced by CD40 stimulation. Kinetic experiments on induction of the EBV-latency III program demonstrated that RelA complexes were primarily translocated into the nucleus and paralleled LMP1 expression. Upon EBV stimulation, RelB activation was observed later at 24 h and was concomitant with TRAF3 degradation and p100 cleavage to p52 (not shown). Thus, not only RelB is secondarily induced by RelA but also it is negatively retro-controlled by RelA.

To further search for functional traces of RelA and RelB interactions in EBV-related DLBCLs, we established gene expression profiles of both our B-cell line models and EBV-associated DLBCL tumors. A total of 726 six genes were upregulated in both tumors and cell lines, most of them being regulated by the LMP1 oncogene of EBV. On the basis of RelA and RelB inhibition or activation experiments, 46 genes were found to be regulated either by RelA or RelB. The number of RelA- and/or RelB-regulated genes seemed to be low, when compared with the number of genes induced by LMP1 alone, and probably not all NF-κB target genes were selected following our methodology. However, LMP1 is a very strong activator, and overexpression of IκBαS32,36A or p100 could have not completely abolished NF-κB DNA binding activity in EBV-immortalized B cells. Moreover, as tissue control for EBV-related DLBCLs, we took lymph nodes with benign follicular hyperplasia, that is, lymph nodes with current intense secondary B-cell immune response. Thus, it is not excluded that some NF-κB activation would exist in these control tissues. We can hypothesize that we detected the most NF-κB-sensitive genes deregulated in DLBCLs when compared with benign lymph nodes submitted to an intense immune response.

The expression of ARNTL2 gene was induced by RelB and repressed by RelA overactivation (so-called RelB only). Reciprocally, 10 RelA-regulated genes were repressed by RelB overexpression (so-called RelA only). These results are a strong indication that RelA and RelB reciprocal inhibition may occur at the transcriptional level in transformed B cells. Genome-wide gene expression analysis derived from Lymphotoxin β receptor-stimulated murine embryonic fibroblasts revealed that the majority of the induced genes require both RelA- and RelB-containing dimers.43 Here, RelB gene expression profile was almost included in that of RelA. As RelB activity was secondarily induced by RelA in EBV-transformed B cells and genes repressed by RelB in our experimental in vitro conditions were in fact overexpressed both in vitro and in EBV-related DLBCLs, it is very likely that the optimal equilibrium between RelA and RelB would exist in EBV-transformed B cells so that RelA-specific functions remained and RelA/RelB co-regulated functions were secondarily reinforced.

Taking both RelB and RelA together, one of the main NF-κB signatures was that of metabolism with regulation of genes involved, for example, in cholesterol synthesis, fatty acid degradation or the pentose phosphate pathway. Recent publications have identified NF-κB as a major regulator of energy homeostasis via the regulation of metabolic functions.44 Here, ACSL1 (acyl-CoA synthetase long chain family, member 1) codes for an enzyme that produces acetyl-CoA, the requisite building block for lipid biosynthesis, and also contributes to energy production through the β-oxidation into mitochondria.45, 46 Tumor cells reactivate de novo lipid synthesis such as that of fatty acids, which are important substrates for energy production, essential components of all biological lipid membranes and participate in several signaling pathways that regulate in turn metabolism, proliferation, survival and invasive properties.47

Consistently with functional results, the RelA signature indicated that it is very likely to participate in strong proliferation of EBV-transformed B cells. Indeed, other RelA-regulated genes revealed that RelA was in the center of key functions associated with cell transformation, including regulation of genes involved in cell cycle progression such as BATF (basic leucine zipper transcription factor, ATF-like) and CCNE1 (cyclin E1). The B-cell-specific transcription factor, BATF, promotes latent EBV infection with transforming potential and contributes to in vivo progression of EBV-associated lymphomagenesis.48, 49, 50 G1/S checkpoint alteration by cyclin E1 (CCNE1) overexpression is associated with high cell proliferation and chromosomal instability in many human tumors and related to a poor clinical outcome in DLBCLs.51, 52 RelA could also participate in genetic instability through upregulation of APOBEC3.53 RelA-regulated genes could also modulate the tumor microenvironment through regulatory molecules with pro- or anti-tumor effects such as CD58/LFA3 or IL1R2, respectively.54, 55

In addition to RelA, RelB could contribute to increased energy metabolism by targeting genes directly involved in cholesterol or sorbitol pathways, as well as by increasing the expression of DRAM1 (damage-regulated autophagy modulator) autophagy regulator. Autophagy may promote the survival of tumor cells within the tumor microenvironment by providing an alternate route of nutriment acquisition when tumor cells are deprived of blood flow. In addition, both RelA and RelB regulated i) the well-known NF-κB tumor markers of EBV-DLBCLs such as NFKBIA/IKBA, TNFRSF8/CD30 and TRAF132 and ii) ZBTB32 (zinc-finger and BTB domain-containing protein 32), involved in plasma cell differentiation.56 ZBTB32 is a transcriptional partner of Blimp-1 (B-lymphocyte-induced maturation protein-1), a tumor marker related to apoptosis protection of tumor cells engaged in terminal B-cell differentiation such as in EBV-DLBCL tumors.48, 56

ARNTL2 was found to be regulated by RelB only. With CLOCK, ARNTL1-2 regulates the circadian rhythm of cells and is a cancer risk factor.57 ARNTL2 expression is increased in B leukemia and colorectal cancer, which is related to tumor invasiveness and aggressiveness.58, 59 ARNTL2 is also a partner of HIF1α (Hypoxia-Inducible Factors 1 alpha) suggesting a potential physiological link between circadian rhythm and cellular oxygen status.60

In conclusion, our results demonstrated a cross-talk between RelA and RelB so that RelB appeared to be subordinated to RelA and modulated RelA activity. In that context, NF-κB dependent transformation of B cells could occur in two steps. The first step would be induction of the entire set of RelA target genes by RelA alone, including RelB and its inhibitor p100. The second step would be attenuation of some RelA-specific genes by RelB and long-term reinforced expression of RelA and RelB co-regulated genes. RelA alone would act directly on tumor development and progression through direct regulation of genes associated with energy metabolism, genetic instability, survival and cell cycle progression but also anti-tumor immunity. The presence of RelB would favor long-term maintenance of EBV-associated tumors, reinforcing the energy metabolism subversion, protection against apoptosis and promoting resistance to hypoxic stress. RelA would be the right NF-κB subunit to target in the view of new drug development, at least for EBV-transformed B cells.

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Acknowledgements

This work was supported by Institut National du Cancer, Cancéropôle Grand-Sud-Ouest, Comité Orientation Recherche Cancer du Limousin, Conseil Régional du Limousin, Comités Limousin de la Ligue contre le Cancer and Association pour la Recherche sur le Cancer. We thank the tumor banks of the University hospitals of Bordeaux and Limoges for providing biopsies and Véronique Pantesco (Microarray Core Facility of the Institute for Research in Biotherapy, Montpellier, France) for Affymetrix analyses. We thank Dr Jeanne Cook-Moreau, CNRS-UMR-7276 Limoges, for English corrections and Mr Lionel Forestier platform GENOLIM, University of Limoges, France. N Faumont was supported by Comité Orientation Recherche Cancer du Limousin, A Chanut and S Durand-Panteix by Association pour la Recherche sur le Cancer. A Chanut was also supported by Société Française d’Hématologie.

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Chanut, A., Duguet, F., Marfak, A. et al. RelA and RelB cross-talk and function in Epstein–Barr virus transformed B cells. Leukemia 28, 871–879 (2014). https://doi.org/10.1038/leu.2013.274

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Keywords

  • EBV
  • NF-κB
  • cell cycle
  • metabolism
  • B-cell lymphoma.

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