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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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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.
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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.

Mentions: To determine whether constitutive activation of the PI3K/AKT pathway plays a critical role in the proliferation and survival of iMycEμ-1 cells, we cultured them in the presence of the PI3K inhibitor LY294002 (LY). Treatment with LY substantially reduced phosphorylation of AKT (Figure 7A), and resulted in growth suppression (Figure 7B, see additional file 4A) and apoptosis (Figure 7C, see additional file 4B). In keeping with the Western blot results (see Figure 6A), inhibition of ERK by PD98059 (PD), of p38 by SB203580 (SB), of mTOR by rapamycin (Rap), or of JNK by AEG 3482 (AEG) had a marginal to no effect on iMycEμ-1 cell proliferation (Figure 7B, see additional file 4B and 4C). These results show that the PI3K/AKT pathway, but not the MAPK or mTOR pathways, plays an important role in the proliferation and survival of iMycEμ-1 cells. The requirement of PI3K/AKT signaling for constitutive activation of NF-κB, STAT3 and Myc was then examined by EMSA. Inhibition of PI3K significantly reduced NF-κB, STAT3 and Myc activity (Figure 7D) and also led to a reduction of Myc protein (Figure 7E). These effects were identical to those seen following the inhibition of either NF-κB (see Figure 3) or STAT3 (see Figure 4) alone, strongly suggesting crosstalk amongst PI3K/AKT, NF-κB and STAT3.


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)

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

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

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.
Mentions: To determine whether constitutive activation of the PI3K/AKT pathway plays a critical role in the proliferation and survival of iMycEμ-1 cells, we cultured them in the presence of the PI3K inhibitor LY294002 (LY). Treatment with LY substantially reduced phosphorylation of AKT (Figure 7A), and resulted in growth suppression (Figure 7B, see additional file 4A) and apoptosis (Figure 7C, see additional file 4B). In keeping with the Western blot results (see Figure 6A), inhibition of ERK by PD98059 (PD), of p38 by SB203580 (SB), of mTOR by rapamycin (Rap), or of JNK by AEG 3482 (AEG) had a marginal to no effect on iMycEμ-1 cell proliferation (Figure 7B, see additional file 4B and 4C). These results show that the PI3K/AKT pathway, but not the MAPK or mTOR pathways, plays an important role in the proliferation and survival of iMycEμ-1 cells. The requirement of PI3K/AKT signaling for constitutive activation of NF-κB, STAT3 and Myc was then examined by EMSA. Inhibition of PI3K significantly reduced NF-κB, STAT3 and Myc activity (Figure 7D) and also led to a reduction of Myc protein (Figure 7E). These effects were identical to those seen following the inhibition of either NF-κB (see Figure 3) or STAT3 (see Figure 4) alone, strongly suggesting crosstalk amongst PI3K/AKT, NF-κB and STAT3.

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