NF-kappaB/STAT3/PI3K signaling crosstalk in iMyc E mu B lymphoma - PubMed (original) (raw)
NF-kappaB/STAT3/PI3K signaling crosstalk in iMyc E mu B lymphoma
Seong-Su Han et al. Mol Cancer. 2010.
Abstract
Background: Myc is a well known driver of lymphomagenesis, and Myc-activating chromosomal translocation is the recognized hallmark of Burkitt lymphoma, an aggressive form of non-Hodgkin's lymphoma. We developed a model that mimics this translocation event by inserting a mouse Myc cDNA gene into the immunoglobulin heavy chain locus, just upstream of the intronic Emu enhancer. These mice, designated iMyc E mu, readily develop B-cell lymphoma. To study the mechanism of Myc-induced lymphoma, we analyzed signaling pathways in lymphoblastic B-cell lymphomas (LBLs) from iMyc E mu mice, and an LBL-derived cell line, iMyc E mu-1.
Results: Nuclear factor-kappaB (NF-kappaB) and signal transducer and activator of transcription 3 (STAT3) were constitutively activated in iMyc E mu mice, not only in LBLs but also in the splenic B-lymphocytes of young animals months before tumors developed. Moreover, inhibition of either transcription factor in iMyc E mu-1 cells suppressed growth and caused apoptosis, and the abrogation of NF-kappaB activity reduced DNA binding by both STAT3 and Myc, as well as Myc expression. Inhibition of STAT3 signaling eliminated the activity of both NF-kappaB and Myc, and resulted in a corresponding decrease in the level of Myc. Thus, in iMyc E mu-1 cells NF-kappaB and STAT3 are co-dependent and can both regulate Myc. Consistent with this, NF-kappaB and phosphorylated STAT3 were physically associated with one another. In addition, LBLs and iMyc E mu-1 cells also showed constitutive AKT phosphorylation. Blocking AKT activation by inhibiting PI3K reduced iMyc E mu-1 cell proliferation and caused apoptosis, via downregulation of NF-kappaB and STAT3 activity and a reduction of Myc levels. Co-treatment with NF-kappaB, STAT3 or/and PI3K inhibitors led to additive inhibition of iMyc E mu-1 cell proliferation, suggesting that these signaling pathways converge.
Conclusions: Our findings support the notion that constitutive activation of NF-kappaB and STAT3 depends on upstream signaling through PI3K, and that this activation is important for cell survival and proliferation, as well as for maintaining the level of Myc. Together, these data implicate crosstalk among NF-kappaB, STAT3 and PI3K in the development of iMyc E mu B-cell lymphomas.
Figures
Figure 1
NF-κB and STAT3 are constitutively activated in LBLs and iMycEμ-1 cells. (A and B) EMSA using an NF-κB-specific probe (A) and a STAT3-specific probe (B), respectively, showing constitutive DNA-binding by NF-κB and STAT3 in LBL tumors (left panel), iMycEμ-1 cells (right panel) and control (C) C57BL/6 splenic B cells. (C) EMSA competition assay (left panel) demonstrating specificity of NF-κB probe. Right panel shows a super-shift assay for NF-κB, using antibodies (Abs) specific for the denoted subunits. Asterisks denote non-specific bands that are covered by the super-shifted bands in lanes 2 and 6. (D) Competition (left panel) and super-shift (right panel) assays for STAT3. Competitor is an unlabelled oligonucleotide probe, and mutator is an unlabelled probe with a mutation that abrogates DNA-binding. P-STAT3 super-shift Abs are both specific for phosphorylated STAT3 at Tyr-705; Ab 1 is sc-7993X and Ab 2 is sc-8059X. SP1 and Myc Abs were used as negative controls. Arrowheads denote shifted bands. Images are representative, and image splicing was carried out only for the same experiment, the same gel and the same exposure times.
Figure 2
Constitutive activation of NF-κB and STAT3 in iMycEμ mice occurs early. (A and B) EMSA showing elevated DNA-binding activity of NF-κB (A) and STAT3 (B), respectively, in both premalignant (average spleen weight 80-150 mg) and malignant (average spleen weight ~750 mg) splenic B220-positive B cells from iMycEμ and control BL6 (average spleen weight 80-100 mg) mice. (C) Western blot showing the level of Myc protein in control, premalignant and malignant splenic B cells, defined as above, and in iMycEμ-1 cells. β-actin was used as a loading control. (D and E) Cytokine array (Left panel) and ELISA (Right panel) for IL6 and IL10, showing the levels of IL6 and IL10 protein in splenic B cells (D) and in splenic B220-negative cells (E) from 2-month old control or iMycEμ mice. ImageQuant software was used to quantitate the results of cytokine array, and data are expressed as arbitrary densitometric units (AU) after normalization to positive controls (Pos). "Neg" denotes negative controls.
Figure 3
NF-κB inhibition suppresses growth, causes apoptosis and downregulates STAT3 and Myc activity in iMycEμ-1 cells. (A) MTS/PMS cell proliferation assay, after culture with the NF-kB inhibitor lactacystin (LC) at various concentrations as indicated. Data were normalized to vehicle control, and error bars represent the standard deviation from a representative experiment performed in triplicate. (B) Agarose gel showing DNA fragmentation in sample treated with LC, but not in PBS control. (C) EMSA showing reduced NF-κB DNA-binding after LC treatment. (D) Stabilization of IκB protein after treatment with LC, as determined by Western blotting. (E) Reduced STAT3 DNA-binding activity after NF-κB inhibition, as observed by EMSA. (F) EMSA showing that Myc DNA-binding activity is reduced after LC treatment. (G) Western blot showing that Myc protein levels are reduced after NF-κB inhibition. β-actin was used as a loading control for Western blots. All LC incubations were for 24 hours.
Figure 4
STAT3 inhibition reduces growth, leads to apoptosis and downregulates NF-κB and Myc activity in iMycEμ-1 cells. (A) Dose-dependent suppression of proliferation (MTS/PMS) after culture with the STAT3 inhibitor WHI P-131 (WHI) at various concentrations, as indicated. Data were normalized to DMSO treatment controls, and error bars represent the standard deviation for a representative experiment performed in triplicate. (B) Agarose gel showing DNA fragmentation after treatment with WHI. (C, D and E) EMSA revealing reduced STAT3 (C), NF-κB (D) and Myc (E) DNA-binding, respectively, after WHI treatment. (F) Western blot showing a decrease in the level of Myc protein after STAT3 inhibition. β-actin was used as a loading control. All WHI incubations were for 24 hours.
Figure 5
NF-κB and STAT3 associate with one another physically in iMycEμ-1 cells. (A and B) EMSA super-shift assays performed with STAT3-specific probes and NF-κB-specific Abs (A), or NF-κB-specific probes and STAT3-specific Abs (B). Abs were specific for NF-κB subunits, Tyr-705 phosphorylated STAT3 (P-STAT3) and total STAT3, as indicated. Abs against SP1 and Myc were used as negative controls. (C) Co-IP and Western blot showing co-immunoprecipitation of NF-κB p50 and P-STAT3. Abs used for immunoprecipitations (IP) and Western blotting (WB) are designated. Images are representative, and image splicing was only carried out only for the same experiment, the same gel and the same exposure times.
Figure 6
AKT is constitutively phosphorylated, in a PTEN independent-manner, in a majority of LBLs and iMycEμ-1 cells. (A) Western blot analysis of the activating-phosphorylation status of key proteins of the PI3K (AKT, P-AKT S473 and T308), MAPK (ERK 1/2, P-ERK1/2, total p 38, P-p 38) and mTOR (p70S6K, P-p70S6K) signaling pathways. Positive controls for P-ERK1/2, P-p38 and P-p70S6K were from extracts of UV-treated HeLa cells, NIH 3T3 cells and insulin-treated MCF-7 cells, respectively. (B and C) Levels of PTEN protein (B) and mRNA (C) in LBLs and iMycEμ-1. α-tubulin and β-actin served as loading controls, respectively. "C" denotes control BL6 splenic B cells.
Figure 7
PI3K inhibition diminishes NF-κB, STAT3 and Myc activity in iMycEμ-1 cells, and reduces their proliferation and survival. (A) Western blot showing the levels of total AKT, PTEN and phosphorylated AKT (S473 and T308) after treatment with LY294002 (LY). α-tubulin was used as a loading control. (B) MTS/PMS assay after treatment with vehicle control, LY, PD98059 (PD), SB203580 (SB), rapamycin (Rap), or AEG 3482 (AEG) at the indicated concentrations. Data were normalized to DMSO-treatment controls, and error bars represent the standard deviation from a representative experiment performed in triplicate. (C) Representative FACS analyses on LY- (open grey histogram) or DMSO- (filled black histogram) treated cells showing an increase in sub-G0/G1 DNA, as assessed by propidium idodide (PI) staining (left panel), and apoptosis as assessed by increases in both Annexin V (middle panel) and activated caspase 3 (right panel) staining. (D) EMSA showing reduced DNA-binding activity of NF-κB, STAT3 and Myc after treatment with LY, but not PD, SB, AEG or Rap. (E) Western blot demonstrating reduced Myc protein levles after inhibition of PI3K; α-tubulin served as a loading control. (F) NF-κB, STAT3 and Myc DNA-binding activity is reduced in a time-dependent manner after PI3K is inhibited with LY. The incubation time with small-molecule inhibitors was 24 hours unless otherwise indicated.
Figure 8
Co-treatment with small-molecule inhibitors of NF-κB, STAT3 and/or PI3K additively inhibits the proliferation of iMycEμ-1 cells. MTS/PMS cell proliferation assay after 24-hour treatment with low doses of LY, LC and WHI in isolation or in various combinations, as indicated. Box at the bottom gives the average percent (%) growth inhibition. Data were normalized to DMSO-treatment controls, and error bars represent the standard deviation from a representative experiment performed in triplicate.
References
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