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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 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.
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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.

Mentions: To investigate the role of NF-κB in proliferation and survival, we cultured iMycEμ-1 cells in the presence of the NF-κB inhibitor, Lactacystin (LC). LC treatment for 24 hours inhibited growth of iMycEμ-1 cells in dose-dependent fashion, as measured by MTS (Figure 3A). DNA laddering indicated that LC also induced apoptosis (Figure 3D). By EMSA, we confirmed that 5 μM LC inhibited NF-κB activity (Figure 3C) by stabilizing IκB (Figure 3D). Notably, other NF-κB inhibitors, BAY-11 7085 or Helenin, which function by blocking IκB phosphorylation or preventing DNA-binding by NF-κB, respectively, had similar inhibitory effects on the proliferation of iMycEμ-1 cells (data not shown). We then examined whether inhibiting NF-κB altered STAT3 or Myc activity. As shown in Figure 3E and 3F, treatment with LC dramatically reduced the activity of both STAT3 and Myc. The reduction in Myc activity corresponded to a remarkable decrease in the level of Myc protein (Figure 3G). EMSA competition and super-shift assays were done as before, to demonstrate the specificity of Myc DNA-binding (see additional file 2). These data imply that NF-κB is necessary for the proliferation and survival of iMycEμ-1 cells, and to link NF-κB to the activities of STAT3 and Myc.


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

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

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.
Mentions: To investigate the role of NF-κB in proliferation and survival, we cultured iMycEμ-1 cells in the presence of the NF-κB inhibitor, Lactacystin (LC). LC treatment for 24 hours inhibited growth of iMycEμ-1 cells in dose-dependent fashion, as measured by MTS (Figure 3A). DNA laddering indicated that LC also induced apoptosis (Figure 3D). By EMSA, we confirmed that 5 μM LC inhibited NF-κB activity (Figure 3C) by stabilizing IκB (Figure 3D). Notably, other NF-κB inhibitors, BAY-11 7085 or Helenin, which function by blocking IκB phosphorylation or preventing DNA-binding by NF-κB, respectively, had similar inhibitory effects on the proliferation of iMycEμ-1 cells (data not shown). We then examined whether inhibiting NF-κB altered STAT3 or Myc activity. As shown in Figure 3E and 3F, treatment with LC dramatically reduced the activity of both STAT3 and Myc. The reduction in Myc activity corresponded to a remarkable decrease in the level of Myc protein (Figure 3G). EMSA competition and super-shift assays were done as before, to demonstrate the specificity of Myc DNA-binding (see additional file 2). These data imply that NF-κB is necessary for the proliferation and survival of iMycEμ-1 cells, and to link NF-κB to the activities of STAT3 and Myc.

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