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NF-kappaB/STAT3/PI3K signaling crosstalk in iMyc E mu B lymphoma.

Han SS, Yun H, Son DJ, Tompkins VS, Peng L, Chung ST, Kim JS, Park ES, Janz S - Mol. Cancer (2010)

Bottom Line: Inhibition of STAT3 signaling eliminated the activity of both NF-kappaB and Myc, and resulted in a corresponding decrease in the level of Myc.Consistent with this, NF-kappaB and phosphorylated STAT3 were physically associated with one another.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Iowa Carver College of Medicine, Department of Pathology, Iowa City, IA, USA.

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.

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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.
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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.

Mentions: Both NF-κB and STAT3 are important for the proliferation and survival of normal B cells and several types of non-Hodgkin's lymphoma (NHL) [33-37]. We used EMSA to examine NF-κB and STAT3 activity in both iMycEμ-derived LBLs and the iMycEμ-1 cell line. All nine LBLs and the iMycEμ-1 cells showed abnormal activation of both NF-κB (Figure 1A) and STAT3 (Figure 1B) when compared to isolated splenic B cells from control C57BL/6 (BL6) mice.


NF-kappaB/STAT3/PI3K signaling crosstalk in iMyc E mu B lymphoma.

Han SS, Yun H, Son DJ, Tompkins VS, Peng L, Chung ST, Kim JS, Park ES, Janz S - Mol. Cancer (2010)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2876994&req=5

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.
Mentions: Both NF-κB and STAT3 are important for the proliferation and survival of normal B cells and several types of non-Hodgkin's lymphoma (NHL) [33-37]. We used EMSA to examine NF-κB and STAT3 activity in both iMycEμ-derived LBLs and the iMycEμ-1 cell line. All nine LBLs and the iMycEμ-1 cells showed abnormal activation of both NF-κB (Figure 1A) and STAT3 (Figure 1B) when compared to isolated splenic B cells from control C57BL/6 (BL6) mice.

Bottom Line: Inhibition of STAT3 signaling eliminated the activity of both NF-kappaB and Myc, and resulted in a corresponding decrease in the level of Myc.Consistent with this, NF-kappaB and phosphorylated STAT3 were physically associated with one another.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Iowa Carver College of Medicine, Department of Pathology, Iowa City, IA, USA.

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.

Show MeSH
Related in: MedlinePlus