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Translation initiation complex eIF4F is a therapeutic target for dual mTOR kinase inhibitors in non-Hodgkin lymphoma.

Demosthenous C, Han JJ, Stenson MJ, Maurer MJ, Wellik LE, Link B, Hege K, Dogan A, Sotomayor E, Witzig T, Gupta M - Oncotarget (2015)

Bottom Line: Treatment with the active-site dual mTOR inhibitor CC214-1 reduced the level of the eIF4F complex by decreasing the cap bound fraction of eIF4G and increasing the levels of 4E-BP1.Inhibition of eIF4E with shRNA further decreased the CC214-1 induced inhibition of the eIF4F complex, c-Myc, cyclin D3 translation, and colony formation.These studies demonstrate that the eIF4F complex is deregulated in aggressive lymphoma and that dual mTOR therapy has therapeutic potential in these patients.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology, Department of Internal Medicine, Mayo Clinic, Rochester, MN, USA.

ABSTRACT
Deregulated mRNA translation has been implicated in disease development and in part is controlled by a eukaryotic initiation complex eIF4F (composed of eIF4E, eIF4G and eIF4A). We demonstrate here that the cap bound fraction from lymphoma cells was enriched with eIF4G and eIF4E indicating that lymphoma cells exist in an activated translational state. Moreover, 77% (110/142) of diffuse large B cell lymphoma tumors expressed eIF4E and this was associated with an inferior event free survival. Over-expression of wild-type eIF4E (eIF4E(WT)) but not cap-mutant eIF4E (eIF4E(cap mutant)) increased the activation of the eIF4F complex. Treatment with the active-site dual mTOR inhibitor CC214-1 reduced the level of the eIF4F complex by decreasing the cap bound fraction of eIF4G and increasing the levels of 4E-BP1. CC214-1 inhibited both the cap dependent and global protein translation. CC214-1 inhibited c-Myc, and cyclin D3 translation by decreasing polysomal fractions from lymphoma cells. Inhibition of eIF4E with shRNA further decreased the CC214-1 induced inhibition of the eIF4F complex, c-Myc, cyclin D3 translation, and colony formation. These studies demonstrate that the eIF4F complex is deregulated in aggressive lymphoma and that dual mTOR therapy has therapeutic potential in these patients.

No MeSH data available.


Related in: MedlinePlus

Effect of CC214-1 treatment on eIF4F complex and cell growth in the presence of decreased or increased eIF4E(A, left panel)In vitro cap-affinity assay (mGTP) in HEK293 cells stably transfected by eIF4E shRNA and control shRNA plasmids. (A, right panel)In vitro cap-affinity assay was performed in the HEK293 cells transiently transfected by PCDNA3.1 eIF4Ecap mutant and eIF4EWT. The experiments were repeated 3 times. (B) HEK293 cells transiently transfected by PCDNA3.1 eIF4Ecap mutant and eIF4EWT were treated with CC214-1 (1 μM) and thymidine incorporation assay (left panel) and flow cytometry (right panel) were performed. Bars represent mean ± SD from 3 different experiments. (C) Colony forming assay in CC214-1 (1 μM) treated HEK293 cells, stably transfected with eIF4E shRNA and control shRNA. Bars represent mean ± SD from 3 replicates. (D) HEK293 cells after transfection by control and c-Myc siRNAs, were treated with 1 μM CC214-1 and thymidine incorporation was performed. Bars represent mean ± SD from 3 replicates. The experiment was repeated 3 times. The effect of the siRNAs on c-Myc expression was analyzed by western blot.
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Figure 6: Effect of CC214-1 treatment on eIF4F complex and cell growth in the presence of decreased or increased eIF4E(A, left panel)In vitro cap-affinity assay (mGTP) in HEK293 cells stably transfected by eIF4E shRNA and control shRNA plasmids. (A, right panel)In vitro cap-affinity assay was performed in the HEK293 cells transiently transfected by PCDNA3.1 eIF4Ecap mutant and eIF4EWT. The experiments were repeated 3 times. (B) HEK293 cells transiently transfected by PCDNA3.1 eIF4Ecap mutant and eIF4EWT were treated with CC214-1 (1 μM) and thymidine incorporation assay (left panel) and flow cytometry (right panel) were performed. Bars represent mean ± SD from 3 different experiments. (C) Colony forming assay in CC214-1 (1 μM) treated HEK293 cells, stably transfected with eIF4E shRNA and control shRNA. Bars represent mean ± SD from 3 replicates. (D) HEK293 cells after transfection by control and c-Myc siRNAs, were treated with 1 μM CC214-1 and thymidine incorporation was performed. Bars represent mean ± SD from 3 replicates. The experiment was repeated 3 times. The effect of the siRNAs on c-Myc expression was analyzed by western blot.

Mentions: eIF4E levels have been found to be elevated in several types of cancer.[15–18] To elucidate the role of eIF4E availability in the response to CC214-1 on eIF4F complex, eIF4E was knocked down and then the cells were treated with CC214-1. CC214-1 was able to inhibit the binding of eIF4E and eIF4G to cap in both eIF4E shRNA as well as in the control shRNA cells; however, inhibition was slightly more in the eIF4E depleted cells (Figure 6A, left panel). Next, we sought to determine the CC214-1 effect on eIF4F complex integrity while eIF4E is overexpressed. Wild type eIF4E was transiently expressed and assays for cap affinity, proliferation, and apoptosis were performed. CC214-1 suppressed the binding of eIF4G to cap in HEK293cap mutant and HEK293PCDNA3.1 cells. CC214-1 was able to inhibit binding of eIF4G to cap in cells over-expressing wild type-eIF4E but the binding was less inhibited as compared to HEK293PCDNA3.1 and HEK293cap mutant (Figure 6A, right panel).


Translation initiation complex eIF4F is a therapeutic target for dual mTOR kinase inhibitors in non-Hodgkin lymphoma.

Demosthenous C, Han JJ, Stenson MJ, Maurer MJ, Wellik LE, Link B, Hege K, Dogan A, Sotomayor E, Witzig T, Gupta M - Oncotarget (2015)

Effect of CC214-1 treatment on eIF4F complex and cell growth in the presence of decreased or increased eIF4E(A, left panel)In vitro cap-affinity assay (mGTP) in HEK293 cells stably transfected by eIF4E shRNA and control shRNA plasmids. (A, right panel)In vitro cap-affinity assay was performed in the HEK293 cells transiently transfected by PCDNA3.1 eIF4Ecap mutant and eIF4EWT. The experiments were repeated 3 times. (B) HEK293 cells transiently transfected by PCDNA3.1 eIF4Ecap mutant and eIF4EWT were treated with CC214-1 (1 μM) and thymidine incorporation assay (left panel) and flow cytometry (right panel) were performed. Bars represent mean ± SD from 3 different experiments. (C) Colony forming assay in CC214-1 (1 μM) treated HEK293 cells, stably transfected with eIF4E shRNA and control shRNA. Bars represent mean ± SD from 3 replicates. (D) HEK293 cells after transfection by control and c-Myc siRNAs, were treated with 1 μM CC214-1 and thymidine incorporation was performed. Bars represent mean ± SD from 3 replicates. The experiment was repeated 3 times. The effect of the siRNAs on c-Myc expression was analyzed by western blot.
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Figure 6: Effect of CC214-1 treatment on eIF4F complex and cell growth in the presence of decreased or increased eIF4E(A, left panel)In vitro cap-affinity assay (mGTP) in HEK293 cells stably transfected by eIF4E shRNA and control shRNA plasmids. (A, right panel)In vitro cap-affinity assay was performed in the HEK293 cells transiently transfected by PCDNA3.1 eIF4Ecap mutant and eIF4EWT. The experiments were repeated 3 times. (B) HEK293 cells transiently transfected by PCDNA3.1 eIF4Ecap mutant and eIF4EWT were treated with CC214-1 (1 μM) and thymidine incorporation assay (left panel) and flow cytometry (right panel) were performed. Bars represent mean ± SD from 3 different experiments. (C) Colony forming assay in CC214-1 (1 μM) treated HEK293 cells, stably transfected with eIF4E shRNA and control shRNA. Bars represent mean ± SD from 3 replicates. (D) HEK293 cells after transfection by control and c-Myc siRNAs, were treated with 1 μM CC214-1 and thymidine incorporation was performed. Bars represent mean ± SD from 3 replicates. The experiment was repeated 3 times. The effect of the siRNAs on c-Myc expression was analyzed by western blot.
Mentions: eIF4E levels have been found to be elevated in several types of cancer.[15–18] To elucidate the role of eIF4E availability in the response to CC214-1 on eIF4F complex, eIF4E was knocked down and then the cells were treated with CC214-1. CC214-1 was able to inhibit the binding of eIF4E and eIF4G to cap in both eIF4E shRNA as well as in the control shRNA cells; however, inhibition was slightly more in the eIF4E depleted cells (Figure 6A, left panel). Next, we sought to determine the CC214-1 effect on eIF4F complex integrity while eIF4E is overexpressed. Wild type eIF4E was transiently expressed and assays for cap affinity, proliferation, and apoptosis were performed. CC214-1 suppressed the binding of eIF4G to cap in HEK293cap mutant and HEK293PCDNA3.1 cells. CC214-1 was able to inhibit binding of eIF4G to cap in cells over-expressing wild type-eIF4E but the binding was less inhibited as compared to HEK293PCDNA3.1 and HEK293cap mutant (Figure 6A, right panel).

Bottom Line: Treatment with the active-site dual mTOR inhibitor CC214-1 reduced the level of the eIF4F complex by decreasing the cap bound fraction of eIF4G and increasing the levels of 4E-BP1.Inhibition of eIF4E with shRNA further decreased the CC214-1 induced inhibition of the eIF4F complex, c-Myc, cyclin D3 translation, and colony formation.These studies demonstrate that the eIF4F complex is deregulated in aggressive lymphoma and that dual mTOR therapy has therapeutic potential in these patients.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology, Department of Internal Medicine, Mayo Clinic, Rochester, MN, USA.

ABSTRACT
Deregulated mRNA translation has been implicated in disease development and in part is controlled by a eukaryotic initiation complex eIF4F (composed of eIF4E, eIF4G and eIF4A). We demonstrate here that the cap bound fraction from lymphoma cells was enriched with eIF4G and eIF4E indicating that lymphoma cells exist in an activated translational state. Moreover, 77% (110/142) of diffuse large B cell lymphoma tumors expressed eIF4E and this was associated with an inferior event free survival. Over-expression of wild-type eIF4E (eIF4E(WT)) but not cap-mutant eIF4E (eIF4E(cap mutant)) increased the activation of the eIF4F complex. Treatment with the active-site dual mTOR inhibitor CC214-1 reduced the level of the eIF4F complex by decreasing the cap bound fraction of eIF4G and increasing the levels of 4E-BP1. CC214-1 inhibited both the cap dependent and global protein translation. CC214-1 inhibited c-Myc, and cyclin D3 translation by decreasing polysomal fractions from lymphoma cells. Inhibition of eIF4E with shRNA further decreased the CC214-1 induced inhibition of the eIF4F complex, c-Myc, cyclin D3 translation, and colony formation. These studies demonstrate that the eIF4F complex is deregulated in aggressive lymphoma and that dual mTOR therapy has therapeutic potential in these patients.

No MeSH data available.


Related in: MedlinePlus