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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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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.
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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.

Mentions: Having observed signaling crosstalk between constitutively activated NF-κB and STAT3, we investigated the role of some other major signaling pathways in LBLs and iMycEμ-1 cells. Given that PI3K, mTOR and MAPK signaling are important for cell survival and proliferation [21-25], we examined activation of these pathways. The PI3K downstream effector AKT was phosphorylated on both Ser-473 and threonine (Thr)-308 in nearly all LBLs and iMycEμ-1 cells, indicating that it was constitutively activated (Figure 6A). In contrast, phosphorylated forms of ERK, p38 and p70S6K were not readily apparent, indicating that the MAPK and mTOR signaling pathways were not activated. In many types of tumors, the loss or mutation of PTEN leads to elevated activity of the PI3K/AKT pathway [47]. Thus, we evaluated the PTEN levels in LBLs and iMycEμ-1 cells by Western blotting and RT-PCR. PTEN protein or mRNA remained unchanged compared to levels in normal splenic B cells (Figure 6B and 6C, respectively). Activation of AKT from these particular tumor samples and quantitation of PTEN mRNA are shown in additional file 3. Sequencing of PTEN showed no mutation in the Pten gene in either LBLs or iMycEμ-1 cells (data not shown). Additionally, because activating mutations of PIK3CA can result in the constitutive phosphorylation and activation of AKT [48], we sequenced the Pik3ca gene. However, we did not find mutations in this gene in any LBLs or iMycEμ-1 cells (data not shown). These results suggest that constitutive activation of the AKT, but not mTOR or MAPK, pathways is involved in the pathogenesis of iMycEμ lymphoma, independent of loss or mutation of either Pten or Pik3ca.


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)

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

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

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.
Mentions: Having observed signaling crosstalk between constitutively activated NF-κB and STAT3, we investigated the role of some other major signaling pathways in LBLs and iMycEμ-1 cells. Given that PI3K, mTOR and MAPK signaling are important for cell survival and proliferation [21-25], we examined activation of these pathways. The PI3K downstream effector AKT was phosphorylated on both Ser-473 and threonine (Thr)-308 in nearly all LBLs and iMycEμ-1 cells, indicating that it was constitutively activated (Figure 6A). In contrast, phosphorylated forms of ERK, p38 and p70S6K were not readily apparent, indicating that the MAPK and mTOR signaling pathways were not activated. In many types of tumors, the loss or mutation of PTEN leads to elevated activity of the PI3K/AKT pathway [47]. Thus, we evaluated the PTEN levels in LBLs and iMycEμ-1 cells by Western blotting and RT-PCR. PTEN protein or mRNA remained unchanged compared to levels in normal splenic B cells (Figure 6B and 6C, respectively). Activation of AKT from these particular tumor samples and quantitation of PTEN mRNA are shown in additional file 3. Sequencing of PTEN showed no mutation in the Pten gene in either LBLs or iMycEμ-1 cells (data not shown). Additionally, because activating mutations of PIK3CA can result in the constitutive phosphorylation and activation of AKT [48], we sequenced the Pik3ca gene. However, we did not find mutations in this gene in any LBLs or iMycEμ-1 cells (data not shown). These results suggest that constitutive activation of the AKT, but not mTOR or MAPK, pathways is involved in the pathogenesis of iMycEμ lymphoma, independent of loss or mutation of either Pten or Pik3ca.

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