Background
Deregulated NF-κB activity plays a critical role in the survival and radiation resistance of tumor cells in a variety of human neoplasias including B cell lymphomas (BCLs) [
1‐
5]. NF-κB comprises a family of transcription factors that control genes implicated in B-cell activation, proliferation and resistance to apoptosis [
6]. Five known, structurally conserved members of the NF-κB/Rel family function as dimers in various combinations: p50, p52, p65 (Rel A), Rel B and c-Rel. Classic NF-κB, the p50 and p65 heterodimer, is an activator of gene transcription, whereas the p50/p50 homodimer both represses and activates the transcription of target genes [
7]. NF-κB exists in an inactive form in the cytoplasm because of its interaction with the inhibitory protein, IκBα [
8]. NF-κB activation is controlled by the IκB kinase (IKK) complex; after stimulation by cytokines and/or growth factors, IKK phosphorylates IκB, which results in its subsequent ubiquitination and proteasomal degradation. The degradation of IκB allows NF-κB to translocate to the nucleus, where it can activate or repress target genes [
9]. NF-κB not only plays a role in the survival of neoplastic B cells, but is also critical for the development and survival of normal B cells [
10].
Another family of transcription factors whose members are constitutively activated in many human tumors is the STAT family. These proteins can control various cellular events such as proliferation, differentiation and cell survival [
11]. One member in particular, STAT3, has been shown to be constitutively activated in a number of human tumor cell lines and primary tumors, including several hematological malignancies [
12,
13]. STAT3 can be activated by IL6, interferons, epidermal growth factor or leptin, through the activity of members of the receptor-associated Janus kinase (JAK) family, which comprises JAK1, JAK2, JAK3, or TYK2 [
14‐
16]. JAKs phosphorylate STAT3 at tyrosine (Tyr)-705, leading to its dimerization and subsequent translocation to the nucleus where it activates target genes [
17]. In addition, maximal transcriptional activation of STAT3 requires phosphorylation at serine (Ser)-727 in response to cytokine stimulation [
18‐
20].
Yet another important pathway of signal transduction in B cells and B-cell neoplasms is one involving phosphatidyl inositol-3 kinase (PI3K) and AKT. Aberrant activation of this pathway is a common molecular alteration in human malignancies [
21‐
25]. PI3K becomes activated by receptor tyrosine kinases or other cell-surface receptors, resulting in an elevation in the production of the membrane lipid phospho-inositol (3,4,5)P
3 (PIP
3) from phospho-inositol(4,5)P
2 (PIP
2). The level of PIP
3 is negatively controlled by the phosphatase and tensin homolog (PTEN), which converts PIP
3 back to PIP
2. AKT binds PIP
3 at the plasma membrane, and this leads to phosphorylation of AKT at Ser-473 in its regulatory domain. This activated form of AKT can then phosphorylate, and thereby regulate the function of, many cellular proteins that are involved in cell proliferation and survival, as well as in tumorigenesis and metastasis [
26‐
30].
Although activation of NF-κB, STAT3 and/or the PI3K/AKT pathway in B cell neoplasms has been described [
23‐
25], the mechanism by which these pathways contribute to the development of BCLs remains unclear, as do the circumstances under which this occurs. We recently developed the iMyc
Eμ mouse, an experimental model system for studying Myc-driven neoplastic transformation of B cells. Previous studies have shown that, on a mixed background of segregating C57BL/6 and 129/SvJ alleles, the iMyc transgene causes the development of various B cell-derived lymphomas: lymphoblastic B-cell lymphomas (LBLs) in 50% of the mice; diffuse large B-cell lymphomas (DLBCLs) in 25% of the mice, and plasmacytomas (PCTs) in 20% of the mice [
31]. In the study described here, we investigated the role of NF-κB, STAT3 and PI3K signaling in LBL, the most prevalent tumor type in the iMyc
Eμ mice. We found that constitutive activation of NF-κB and STAT3 begins well before frank tumors develop, with co-activation of NF-κB and STAT3 playing a role in tumor maintenance, and activation of the PI3K/AKT pathway in the neoplastic B cells being responsible, in part, for the constitutive activation of NF-κB and STAT3. Inhibition of any one of these three pathways resulted in Myc downregulation, inhibited growth growth and promoted apoptosis in iMyc
Eμ-LBL-derived cells. We report, for the first time, a physical association of NF-κB with STAT3 in B cells, and provide evidence for the convergence of PI3K, NF-κB and STAT3 signaling in Myc-driven lymphomagenesis.
Materials and methods
Tissues and cell lines
Primary LBL tumors from iMyc
Eμ mice [
31] and the LBL-derived cell line, iMyc
Eμ-1 [
32], were used in this study. WEHI 231, RAW 8.1, and NFS-1.0 C-1 cell lines were purchased from ATCC (Rockville, MD). All cell lines were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 200 mM L-glutamine, 50 mM 2-mercaptoethanol and antibiotics, 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco-BRL, Rockville, MD), at 37°C in a humidified 5% CO
2 incubator. Highly enriched (>95% pure) splenic B cells were isolated from C57BL/6 (BL6) or iMyc
Eμ mice using CD45R (B220) microbeads and MACS
® separation columns (Miltenyi Biotec, Auburn, CA) according to the manufacturer's protocol. Control cultures were treated with phosphate-buffered saline (PBS) or DMSO where appropriate, and the final concentration never exceeded 0.3%.
Preparation of nuclear and cytosolic extracts
Pellets of 107 cells or powdered-frozen LBL samples were lysed with 400 μl of 10 mM KCl, 0.2 mM EDTA, 1.5 mM MgCl2, 0.5 mM DTT, and 0.2 mM PMSF at 4°C for 10 minutes. The lysate was centrifuged for 5 minutes at 14,000 × g and supernatants were stored as cytosolic extract, at -70°C. The resulting pellet was re-suspended in 100 μl of ice-cold 20 mM HEPES (pH 7.9), 420 mM NaCl, 1.5 mM MgCl2, 20% (v/v) glycerol, 0.2 mM EDTA, 0.5 mM DTT, and 0.2 mM PMSF. After incubation at 4°C for 20 minutes, the lysate was centrifuged for 6 minutes at 14,000 × g, and the supernatant was stored as a nuclear extract, at -70°C. The concentration of cytosolic and nuclear extract was determined using a BCA kit (Bio-Rad, Richmond, CA).
Electrophoretic mobility shift assay (EMSA) and super-shift assay
The DNA-protein binding detection kit (Gibco-BRL) was used with modifications. In brief, DNA-binding reactions were carried out in a final volume of 25 μl of buffer containing 10 mM Tris (pH 7.5), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 4% (w/v) glycerol, 0.1 mg/ml sonicated salmon sperm DNA, 10 μg of nuclear extract, and oligonucleotides. Oligonucleotides containing consensus NF-κB (Promega, Madison, WI), STAT3 (Santa Cruz Biotechnology, Santa Cruz, CA), or Myc-Max binding sites (Santa Cruz Biotechnology) were end-labeled to a specific activity of 105 CPM with γ-[32P]-ATP and T4-polynucleotide kinase, followed by purification on a Nick column (GE Healthcare, Piscataway, NJ). Reaction mixtures with radio-labeled oligonucleotides were incubated at room temperature for 20 minutes, and resolved on 6% non-denaturing polyacrylamide gels after addition of 2 μl bromophenol blue (0.1%). Gels were dried and subjected to autoradiography. For competition assays, 30-fold excess unlabeled oligonucleotides containing consensus or mutated NF-κB, STAT3 or Myc-Max binding sites, respectively, were added for 20 minutes at room temperature, after incubation with the radio-labeled oligonucleotides. For super-shift assays, 2 μg of antibody (Ab) was added for 20 minutes at room temperature after the initial incubation. Abs specific for p50 (sc-114X), p52 (sc-298X), p65 (sc-109X), RelB (sc-226X), c-Rel (sc-70X), Myc (sc-764X), SP-1 (sc-59X), STAT3 (sc-483X) or P-STAT3 (sc-8059X or sc-7993X) were purchased from Santa Cruz Biotechnology.
Reverse transcription polymerase chain reaction (RT-PCR)
Semi-quantitative RT-PCR was performed by extracting total RNA using TRIzol (Sigma-Aldrich, St. Louis, MO), and this was followed by double-stranded cDNA synthesis from 1 μg of total RNA, using the AMV reverse transcriptase kit (Roche, Indianapolis, IN). Thermal cycling conditions were as follows: 95°C for 5 minutes followed by 20, 25, 30, 35, or 40 cycles (depending on the target gene) of amplification at 57°C, 72°C, and 95°C, for 1 minute each. PCR products were resolved by electrophoresis on 1% agarose gels containing ethidium bromide. Primer sequences are as follows:
PTEN, forward 5'-GGCGGTGTCATAATGTCTCTCA-3'
reverse 5'-CCCATTTTCCACTTTTTCTGAGG-3'
β-actin, forward 5'-ATGGCATTGTTACCAACTGGGACG-3'
reverse 5'-CTCTTTGATGTCACGCACGATTTC-3'.
Whole-cell extracts and Western blotting
Whole-cell lysates were obtained by re-suspending pellets of 107 cells or powdered-frozen LBL samples in RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 ng/ml PMSF, 0.03% aprotinin, 1 μM sodium orthovanadate) at 4°C for 30 minutes. Lysates were centrifuged for 6 minutes at 14 000 × g, and supernatants were stored at -70°C as a whole-cell extract. Total protein concentrations were determined by BCA (Bio-Rad). Western blotting was performed with 40 μg of total protein resolved by SDS-PAGE and transferred to PVDF membranes. Membranes were probed with Abs against c-Myc (sc-764), PTEN (sc-7974), or IκBα (sc-847) from Santa Cruz Biotechnology, ERK1/2 (9102), P-ERK1/2 (4377), p38 (9212), P-p38 (9211), AKT (9272), P-AKT (9271), P-AKT (9275), p70S6K (9202), or P-p70S6K (9205) from Cell Signaling (Danvers, MA), α-tubulin (T6074) or β-actin (A5316) from Sigma-Aldrich. Proteins were visualized using horseradish peroxidase-conjugated secondary Ab (1:5000) and the ECL detection kit from Amersham (GE Healthcare). To confirm equal loading, membranes were stripped and re-probed using an Ab specific for α-tubulin or β-actin. Total cell extracts from UV-treated HeLa and NIH 3T3 cells were used as positive controls for P-ERK1/2 (sc-2221) and P-p38 (sc-2210), respectively (Santa Cruz Biotechnology). Total cell extract from insulin-treated MCF-7 cells was used as a positive control for P-p70S6K (9203) (Cell Signaling).
Proliferation assay
Proliferation was determined using the Cell Titer 96® MTS/PMS assay (Promega). Briefly, 3 × 104 cells were re-suspended in 100 μl growth medium and plated into 96-well plates (Costar, Cambridge, MA). After 20 hours at 37°C and 5% CO2, 20 μl of MTS/PMS solution was added to each well and cells were incubated for another 4 hours before the absorbance at 490 nm was measured on a Multiskan Spectrum (Thermo Scientific, Hudson, NH).
Apoptosis assay
Apoptosis was evaluated using the DNA fragmentation assay and fluorescence activated cell sorting (FACS)-based analysis of propidium iodide (PI), annexin V, and Caspase-3 reactivity. For DNA fragmentation, DNA was extracted using the Puregene kit (Gentra Systems, Minneapolis, MN) and resolved by electrophoresis on 1.0% agarose gels containing ethidium bromide. For the identification of cells with sub-G0/G1 DNA content, cells were resuspended in PI/Rnase buffer (BD Pharmingen, San Diego, CA) for 20 minutes at 37°C in the dark before FACS analysis. Annexin-V reactivity was determined by applying a phycoerythrin (PE)-labeled Ab (BD Pharmingen) to cells co-stained with 7-amino-actinomycin D (7-AAD). Activated caspase-3 was detected using a FITC- or PE-conjugated Ab (BD Pharmingen).
Pharmacological inhibitors
The following small-molecule inhibitors were used: LY294002 (LY) (Promega); Lactacystin (LC), PD98059 (PD), SB203580 (SB), and rapamycin (Rap) (Biomol, Plymouth Meeting, PA); WHI P-131 (WHI) and AG 490 (Biosource, Carlsbad, CA); and AEG 3482 (AEG) (Tocris, Ellisville, MO).
Co-Immunoprecipitation (Co-IP)
Co-IP was performed using the Universal Magnetic Co-IP kit according to the manufacturer's protocol (Active Motif, Carlsbad, CA). Briefly, 500 μg of nuclear extract was incubated with 5 μg of phosphorylated STAT3 (P-STAT3) Ab (sc-7993), NF-κB p50 Ab (sc-114), or Rabbit IgG control (sc-2345) for 3 hour at 4°C. 25 μl of Protein G Magnetic Beads were added to each tube and then incubated for 1 hour at 4°C. Immunoprecipitates were washed 4 times each with 500 μl wash buffer using a magnetic stand, after which the pellets were resuspended with 2 × reducing loading buffer. Western blot analysis was performed using a primary NF-κB p50 Ab (sc-114) or P-STAT3 Ab (sc-7993), respectively.
IL6 and IL10 assays
IL6 or IL10 expression was assesssed using the RayBio Mouse Cytokine Antibody Array III kit (RayBiotech, Norcross, GA) or the Mouse IL-6 or IL-10 Enzyme-Linked Immunosorbent Assay (ELISA) kit (eBioscience, San Diego, CA), according to the manufacturer's protocol. Samples tested were 107 splenic B or B220-negative cells from 2-month-old BL6 or iMycEμ mice, separated by CD45R (B220) microbeads and MACS® separation columns (Miltenyi Biotec).
Discussion
An enhanced understanding of the signal transduction pathways underlying the development of B-cell neoplasms is an important step towards identifying novel targets for tumor therapy and prevention. Although previous studies have demonstrated that NF-κB, STAT3 and/or PI3K play critical roles in growth control, survival, and chemotherapy resistance of B-cell and plasma-cell neoplasms [
50‐
52], the precise function of NF-κB, STAT3 and/or PI3K in the development of these tumors is not completely understood. In this study, we used the iMyc
Eμ LBL model to uncover signaling crosstalk between NF-κB, STAT3 and PI3K signaling. To our knowledge, this is the first report of crosstalk amongst these pathways in B lymphoma cells. We found that constitutive activation of the PI3K/AKT, but not the mTOR or MAPK pathways, was found to be at least partially responsible for aberrant NF-κB and STAT3 activity. Inhibition of NF-κB, STAT3 or PI3K signaling in iMyc
Eμ B cells, respectively, led to growth suppression, apoptosis and downregulation of Myc. Combined inhibition had an additive effect on proliferation, suggesting that NF-κB and STAT3 converge downstream of PI3K. Our finding that NF-κB and STAT3 are physically associated in iMyc
Eμ-1 B cells supports this interpretation. Signaling crosstalk of NF-κB, STAT3 and PI3K may play an important role in Myc-induced B-cell lymphoma in mice.
The finding that NF-κB, STAT3 and PI3K are constitutively activated in LBLs and iMyc
Eμ-1 cells is in keeping with the aberrant activity of these pathways observed in various types of B cell neoplasms. Constitutive activation of NF-κB has frequently been observed in follicular lymphoma [
53,
54], DLBCL [
55], mucosa-associated lymphoid tissue (MALT) lymphoma [
56], multiple myeloma (MM) [
57], and mantle-cell lymphoma (MCL), as well as MCL cell lines, in which inhibition of this constitutive activation induces growth arrest and apoptosis [
58‐
60]. Aberrant STAT3 activation has been documented in MM [
61], Hodgkin's disease [
62], anaplastic lymphoma kinase-positive (ALK) DLBCL [
63], and activated B-cell (ABC) DLBCL, in which JAK2/STAT3 inhibitors trigger arrest and apoptosis [
40]. Activation of the PI3K pathway is one of the most common defects in human malignancies, including Burkitt's lymphoma, MCL, and Hodgkin's lymphoma [
21‐
25]. The repeated discovery of the involvement of NF-κB, STAT3 and PI3K in distinct forms of B-cell neoplasias underscores the importance of these signaling pathways in B-cell transformation.
Several findings support crosstalk among NF-κB, STAT3 and PI3K signaling in the iMyc
Eμ system. Inhibition of NF-κB abrogated constitutive STAT3 activity, inhibition of STAT3 reciprocally reduced constitutive NF-κB activity, and inhibition of PI3K suppressed activation of both NF-κB and STAT3 in iMyc
Eμ-1 cells. When inhibitor combinations affecting NF-κB and STAT3 or either and PI3K were applied, additive suppression of proliferation was observed, indicating that the NF-κB and STAT3 pathways converge. The physical association between the active forms of NF-κB and STAT3 in iMyc
Eμ-1 cells provides direct evidence for such crosstalk and convergence. Partial characterization of this complex revealed interactions between the NF-κB subunits p50, p65, and/or c-Rel, either directly or indirectly, with phosphorylated STAT3. The exact compositions of the complexes, and the ultimate functions of these interactions, are not yet defined. Although crosstalk among transcription factors is a common mode of gene regulation, and several studies have already reported physical and functional interactions between NF-κB and STAT3 in various cell types [
42‐
46,
64‐
66], to our knowledge, this is the first description of a physical association between NF-κB and STAT3 in neoplastic B cells. A recent study showed that constitutive STAT3 activity can maintain constitutive NF-κB activity in solid tumors [
46], and our finding supports the possibility of a reciprocal activity of NF-κB and STAT3 in the maintenance of hematopoietic tumors.
We have explored the potential involvement of other pathways in the proliferation and survival of iMyc
Eμ -1 cells and on NF-κB and STAT3 signaling, but found that only the PI3K pathway was involved. It is very interesting that the Eμ-myc model of B-cell lymphoma, one of the earliest transgenic mice ever developed to still be widely used today [
67], also showed a requirement for PI3K, but not mTOR or ERK, activity in mitogen-induced B-cell growth [
68]. This suggests that the PI3K pathway may be a key modulator of Myc-driven B cell lymphomagenesis. Moreover, inhibition of PI3K abrogated STAT3 and NF-κB activity, and simultaneous inhibition of PI3K with NF-κB or STAT3 resulted in an additive growth inhibition, implying that PI3K functions upstream of NF-κB and STAT3 in iMyc
Eμ B cells. To follow up on how PI3K might be constitutively activated, we assessed the known causes of aberrant PI3K activity - loss or mutation of
Pten[
69‐
71] or mutation of
Pi3kca[
47,
48,
72,
73] - but did not find these alterations in either LBLs or iMyc
Eμ-1 cells. This finding is consistent with other studies indicating that neither PTEN nor
PI3KCA is involved in B-cell malignancies [
74,
75]. The reason for constitutive activation of PI3K remains to be determined.
In keeping with our results, crosstalk among NF-κB, STAT3 and PI3K signaling is supported in the literature. Notable examples include AKT-mediated phosphorylation of IKK to activate NF-κB [
76,
77], IL-2-mediated induction of PI3K upstream of STAT3 activation in primary human T cells [
78], and the physical interaction between the PI3K p85 subunit and STAT3 during STAT3 activation [
79]. Furthermore, AKT, NF-κB and STAT3 signaling are required for the growth of lymphomas driven by the expression of Epstein-Barr Virus latent membrane protein 1 (EBV-LMP1) [
50], and also for the survival of chronic lymphocytic leukemia (CLL) B cells [
51]. Intriguingly, several recent reports describe a role for p300, an acetyltransferase, as a potential mediator of signaling crosstalk of NF-κB, STAT3 and PI3K/AKT. AKT-mediated phosphorylation of p300 dramatically increases its acetyltransferase activity and can increase acetylation and full transcriptional activation of p65 [
80,
81]. For STAT3, leukemia inhibitory factor (LIF)- or IL6-mediated activation of AKT can lead to phosphorylation of p300, and to subsequent acetylation and activation of STAT3 in 293T and Hep3B cells [
65,
82,
83]. Also, acetylation of p65 by p300 is facilitated by STAT3 and can lead to enhanced nuclear localization of p65 [
46]. Although proof the involvement of p300 in iMyc
Eμ B-cell neoplasia has not yet been demonstrated, p300 is a prime candidate to link the crosstalk of PI3K, NF-κB, and STAT3 signaling, and is of considerable interest for future studies.
To demonstrate that our results are not unique to iMyc
Eμ-1 cells, we investigated whether similar signal transduction pathways were important for tumor maintenance in other mouse B-lymphoma lines (see additional files
5,
6, &
7). Strikingly similar inhibitor sensitivity was seen in WEHI 231 and iMyc
Eμ-1 cells. In fact, the sort of PI3K/NF-κB/STAT3 signaling crosstalk seen in iMyc
Eμ-1 cells was also observed in WEHI 231 cells when we repeated many of the same experiments (see additional files
6 and
7). These findings argue that our results are not a peculiarity of iMyc
Eμ-1 cells, and also make a strong case for the specificity of the small-molecule inhibitors used in our studies.
Premalignant B cells are difficult to obtain from humans, but mouse models, such as iMyc
Eμ are a ready source of these cells and can be used to elucidate the temporal regulation of molecular events in the course of lymphoma development. We found that NF-κB and STAT3 were already constitutively activated in splenic B cells of iMyc
Eμ mice months before overt tumors developed. The literature would suggest that this early activation of NF-kB and STAT3 is caused by an increase in IL6 and/or IL10 [
4,
44,
49]. Our data are novel because they exclude the possibility of elevated IL6 or IL10 from either autocrine or paracrine sources in a pre-tumorigenic state. The reason for constitutive NF-kB and STAT3 activation remains unknown. Intriguingly, NF-κB and STAT3 are known to target Myc [
34,
38‐
41], but Myc protein was only slightly elevated during the premalignant stage in iMyc
Eμ mice. Some of our other results, however, are consistent with Myc as a target downstream of PI3K/NF-κB/STAT3 in tumors of the iMyc
Eμ system. Myc protein was highly elevated during malignancy, and inhibition of any one of the tested effectors of Myc transcription (PI3K, NF-κB or STAT3) resulted in a reduction of Myc protein. Moreover, a loss of Myc activity trailed the reduction of NF-κB and STAT3 activity after PI3K was inhibited in iMyc
Eμ-1 cells. If Myc is upregulated by NF-κB and STAT3, perhaps this occurs at some point between the premalignant and malignant state in iMyc
Eμ B cells. Elucidating the nature of this apparent tumor progression event is ongoing in our laboratory, and will be the subject of a future manuscript.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SSH designed the study, performed most experiments, and wrote the article. DJS conducted proliferation assays, Co-IPs and the cytokine array experiments. HY performed Western blot analysis. VST contributed critical insights and edited the article. LP conducted FACS analysis. STC cultured cells and conducted proliferation assays. JK performed Western blot analysis for c-Myc. ESP contributed critical insights. SJ designed the study, evaluated results, and wrote and approved the article. All authors have read and approved the final manuscript.