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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 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.
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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.

Mentions: Recent studies have shown that NF-κB and STAT3 physically associate with one another in several cell types [42-46]. Our findings indicate that constitutively activated NF-κB and STAT3 may cooperatively regulate each other. Thus, we investigated whether STAT3 and NF-κB are physically associated in iMycEμ-1 cells. Super-shift assays were performed with a STAT3-specific oligonucleotide probe and antibodies specific for p 50, p 65, or c-Rel NF-κB subunits. As shown in Figure 5A, our results showed a clear shift in DNA-bound STAT3 when a p 50 Ab was added (lane 2). Addition of a p 65 Ab (lane 3) or c-Rel Ab (lane 4) led to a slight decrease in band intensity (~1/3 of control). This suggests that p65 and c-Rel may be involved in the complex, consistent with our previous observation of shifts in NF-κB DNA-binding with these subunits (see Figure 1C). In the reciprocal experiment, only the addition of an anti-STAT3 Ab (Figure 5B, lane 2) or a P-STAT3 (Tyr 705) Ab (lane 3) affected DNA-binding of NF-κB. These super-shift results indicate that NF-κB and P-STAT3 are physically associated. For further verification, we performed Co-IP and Western blotting for P-STAT3 or the p50 subunit of NF-κB. In keeping with the super-shift results, NF-κB and P-STAT3 were co-immunoprecipitated (Figure 5C). Thus, NF-κB and STAT3 reside in the same complex in iMycEμ-1 cells.


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

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

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.
Mentions: Recent studies have shown that NF-κB and STAT3 physically associate with one another in several cell types [42-46]. Our findings indicate that constitutively activated NF-κB and STAT3 may cooperatively regulate each other. Thus, we investigated whether STAT3 and NF-κB are physically associated in iMycEμ-1 cells. Super-shift assays were performed with a STAT3-specific oligonucleotide probe and antibodies specific for p 50, p 65, or c-Rel NF-κB subunits. As shown in Figure 5A, our results showed a clear shift in DNA-bound STAT3 when a p 50 Ab was added (lane 2). Addition of a p 65 Ab (lane 3) or c-Rel Ab (lane 4) led to a slight decrease in band intensity (~1/3 of control). This suggests that p65 and c-Rel may be involved in the complex, consistent with our previous observation of shifts in NF-κB DNA-binding with these subunits (see Figure 1C). In the reciprocal experiment, only the addition of an anti-STAT3 Ab (Figure 5B, lane 2) or a P-STAT3 (Tyr 705) Ab (lane 3) affected DNA-binding of NF-κB. These super-shift results indicate that NF-κB and P-STAT3 are physically associated. For further verification, we performed Co-IP and Western blotting for P-STAT3 or the p50 subunit of NF-κB. In keeping with the super-shift results, NF-κB and P-STAT3 were co-immunoprecipitated (Figure 5C). Thus, NF-κB and STAT3 reside in the same complex in iMycEμ-1 cells.

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