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A role for eukaryotic initiation factor 4B overexpression in the pathogenesis of diffuse large B-cell lymphoma.

Horvilleur E, Sbarrato T, Hill K, Spriggs RV, Screen M, Goodrem PJ, Sawicka K, Chaplin LC, Touriol C, Packham G, Potter KN, Dirnhofer S, Tzankov A, Dyer MJ, Bushell M, MacFarlane M, Willis AE - Leukemia (2013)

Bottom Line: This process is driven by signaling through mammalian target of rapamycin, resulting in increased synthesis of eukaryotic initiation factor (eIF) 4B complex (eIF4B), a known activator of the RNA helicase eIF4A.Reducing eIF4B expression alone is sufficient to decrease synthesis of proteins associated with enhanced tumor cell survival, namely DAXX, BCL2 and ERCC5.Our work provides new insights into the mechanisms by which the cancer-promoting translational machinery drives lymphomagenesis.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Toxicology Unit, Leicester, UK.

ABSTRACT
Dysregulated expression of factors that control protein synthesis is associated with poor prognosis of many cancers, but the underlying mechanisms are not well defined. Analysis of the diffuse large B-cell lymphoma (DLBCL) translatome revealed selective upregulation of mRNAs encoding anti-apoptotic and DNA repair proteins. We show that enhanced synthesis of these proteins in DLBCL is mediated by the relief of repression that is normally imposed by structure in the 5'-untranslated regions of their corresponding mRNAs. This process is driven by signaling through mammalian target of rapamycin, resulting in increased synthesis of eukaryotic initiation factor (eIF) 4B complex (eIF4B), a known activator of the RNA helicase eIF4A. Reducing eIF4B expression alone is sufficient to decrease synthesis of proteins associated with enhanced tumor cell survival, namely DAXX, BCL2 and ERCC5. Importantly, eIF4B-driven expression of these key survival proteins is directly correlated with patient outcome, and eIF4B, DAXX and ERCC5 are identified as novel prognostic markers for poor survival in DLBCL. Our work provides new insights into the mechanisms by which the cancer-promoting translational machinery drives lymphomagenesis.

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mRNAs identified by the translational profiling show differential protein expression. (a(i) and b(i)) Protein extracts were generated from DLBCL cell lines or from control B cells (GM03201 and GM01953). These were subjected to SDS-polyacrylamide gel electrophoresis (PAGE), immunoblotted and probed with the antibodies indicated to assess differences in expression in the proteins from genes with a role in apoptosis (a) or DNA repair processes (b). (a(ii) and b(ii)) To measure total RNA levels, samples were taken and RNA was extracted. These samples were subjected to northern analysis using probes against DAXX, BCL2, FAS and MYC, or reverse transcriptase-PCR using primers against TRAIP, ERCC5, CCNH, BRCA2, MNAT1, actin and tubulin. Actin/tubulin were used as loading controls. The data show that there is a large increase in the expression of a number of proteins that function in apoptotic and DNA repair pathways with no net change in mRNA levels, in agreement with the array data. (c) B cells were purified from tumors obtained from individuals with DLBCL or from tonsils. Cell extracts were generated and samples separated by SDS-PAGE and immunoblotted with the antibodies shown. The data show that there is increased expression of DAXX, ERCC5 and BCL2 in patient samples in agreement with the cell line and the translational profiling data.
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fig2: mRNAs identified by the translational profiling show differential protein expression. (a(i) and b(i)) Protein extracts were generated from DLBCL cell lines or from control B cells (GM03201 and GM01953). These were subjected to SDS-polyacrylamide gel electrophoresis (PAGE), immunoblotted and probed with the antibodies indicated to assess differences in expression in the proteins from genes with a role in apoptosis (a) or DNA repair processes (b). (a(ii) and b(ii)) To measure total RNA levels, samples were taken and RNA was extracted. These samples were subjected to northern analysis using probes against DAXX, BCL2, FAS and MYC, or reverse transcriptase-PCR using primers against TRAIP, ERCC5, CCNH, BRCA2, MNAT1, actin and tubulin. Actin/tubulin were used as loading controls. The data show that there is a large increase in the expression of a number of proteins that function in apoptotic and DNA repair pathways with no net change in mRNA levels, in agreement with the array data. (c) B cells were purified from tumors obtained from individuals with DLBCL or from tonsils. Cell extracts were generated and samples separated by SDS-PAGE and immunoblotted with the antibodies shown. The data show that there is increased expression of DAXX, ERCC5 and BCL2 in patient samples in agreement with the cell line and the translational profiling data.

Mentions: To assess the changes in expression of proteins that are associated with tumorigenesis, mRNAs whose protein products are involved in apoptosis and DNA repair were studied. Cell lysates were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted for the proteins shown (Figures 2a and b, Supplementary Table 3). Differences in expression were observed in all cases, in agreement with the profiling data (Figure 1 and Supplementary Figure 2). For proteins that function in apoptosis, the data show that TRADD (tumor necrosis factor receptor type 1-associated death domain protein) and FAS were downregulated, whereas DAXX, BCL2, TRAIP and c-Myc were upregulated, albeit to different extents (Figure 2a(i)). There were no corresponding changes in the total amount of mRNA present (Figure 2a(ii), Supplementary Figure 3). For example, the level of TRADD mRNA was similar in the control cell lines and the DLBCL cell lines, and although FAS mRNA levels were more variable, neither of these correlated with the expression of the protein. The cell lines used in the study carry the t(14:18) translocation that couples the immunoglobulin heavy chain locus to the BCL2 gene resulting in transcriptional upregulation of BCL2 mRNA.30 As expected, the levels of BCL2 mRNA were higher in four out of five of the DLBCL-derived cell lines. However, this change was insufficient to account for the large increase in observed protein levels in some of the lymphoma cell lines. The differential expression of these apoptotic proteins was shown to affect cell sensitivity to FAS-mediated apoptosis (Supplementary Figure 4).


A role for eukaryotic initiation factor 4B overexpression in the pathogenesis of diffuse large B-cell lymphoma.

Horvilleur E, Sbarrato T, Hill K, Spriggs RV, Screen M, Goodrem PJ, Sawicka K, Chaplin LC, Touriol C, Packham G, Potter KN, Dirnhofer S, Tzankov A, Dyer MJ, Bushell M, MacFarlane M, Willis AE - Leukemia (2013)

mRNAs identified by the translational profiling show differential protein expression. (a(i) and b(i)) Protein extracts were generated from DLBCL cell lines or from control B cells (GM03201 and GM01953). These were subjected to SDS-polyacrylamide gel electrophoresis (PAGE), immunoblotted and probed with the antibodies indicated to assess differences in expression in the proteins from genes with a role in apoptosis (a) or DNA repair processes (b). (a(ii) and b(ii)) To measure total RNA levels, samples were taken and RNA was extracted. These samples were subjected to northern analysis using probes against DAXX, BCL2, FAS and MYC, or reverse transcriptase-PCR using primers against TRAIP, ERCC5, CCNH, BRCA2, MNAT1, actin and tubulin. Actin/tubulin were used as loading controls. The data show that there is a large increase in the expression of a number of proteins that function in apoptotic and DNA repair pathways with no net change in mRNA levels, in agreement with the array data. (c) B cells were purified from tumors obtained from individuals with DLBCL or from tonsils. Cell extracts were generated and samples separated by SDS-PAGE and immunoblotted with the antibodies shown. The data show that there is increased expression of DAXX, ERCC5 and BCL2 in patient samples in agreement with the cell line and the translational profiling data.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4017261&req=5

fig2: mRNAs identified by the translational profiling show differential protein expression. (a(i) and b(i)) Protein extracts were generated from DLBCL cell lines or from control B cells (GM03201 and GM01953). These were subjected to SDS-polyacrylamide gel electrophoresis (PAGE), immunoblotted and probed with the antibodies indicated to assess differences in expression in the proteins from genes with a role in apoptosis (a) or DNA repair processes (b). (a(ii) and b(ii)) To measure total RNA levels, samples were taken and RNA was extracted. These samples were subjected to northern analysis using probes against DAXX, BCL2, FAS and MYC, or reverse transcriptase-PCR using primers against TRAIP, ERCC5, CCNH, BRCA2, MNAT1, actin and tubulin. Actin/tubulin were used as loading controls. The data show that there is a large increase in the expression of a number of proteins that function in apoptotic and DNA repair pathways with no net change in mRNA levels, in agreement with the array data. (c) B cells were purified from tumors obtained from individuals with DLBCL or from tonsils. Cell extracts were generated and samples separated by SDS-PAGE and immunoblotted with the antibodies shown. The data show that there is increased expression of DAXX, ERCC5 and BCL2 in patient samples in agreement with the cell line and the translational profiling data.
Mentions: To assess the changes in expression of proteins that are associated with tumorigenesis, mRNAs whose protein products are involved in apoptosis and DNA repair were studied. Cell lysates were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted for the proteins shown (Figures 2a and b, Supplementary Table 3). Differences in expression were observed in all cases, in agreement with the profiling data (Figure 1 and Supplementary Figure 2). For proteins that function in apoptosis, the data show that TRADD (tumor necrosis factor receptor type 1-associated death domain protein) and FAS were downregulated, whereas DAXX, BCL2, TRAIP and c-Myc were upregulated, albeit to different extents (Figure 2a(i)). There were no corresponding changes in the total amount of mRNA present (Figure 2a(ii), Supplementary Figure 3). For example, the level of TRADD mRNA was similar in the control cell lines and the DLBCL cell lines, and although FAS mRNA levels were more variable, neither of these correlated with the expression of the protein. The cell lines used in the study carry the t(14:18) translocation that couples the immunoglobulin heavy chain locus to the BCL2 gene resulting in transcriptional upregulation of BCL2 mRNA.30 As expected, the levels of BCL2 mRNA were higher in four out of five of the DLBCL-derived cell lines. However, this change was insufficient to account for the large increase in observed protein levels in some of the lymphoma cell lines. The differential expression of these apoptotic proteins was shown to affect cell sensitivity to FAS-mediated apoptosis (Supplementary Figure 4).

Bottom Line: This process is driven by signaling through mammalian target of rapamycin, resulting in increased synthesis of eukaryotic initiation factor (eIF) 4B complex (eIF4B), a known activator of the RNA helicase eIF4A.Reducing eIF4B expression alone is sufficient to decrease synthesis of proteins associated with enhanced tumor cell survival, namely DAXX, BCL2 and ERCC5.Our work provides new insights into the mechanisms by which the cancer-promoting translational machinery drives lymphomagenesis.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Toxicology Unit, Leicester, UK.

ABSTRACT
Dysregulated expression of factors that control protein synthesis is associated with poor prognosis of many cancers, but the underlying mechanisms are not well defined. Analysis of the diffuse large B-cell lymphoma (DLBCL) translatome revealed selective upregulation of mRNAs encoding anti-apoptotic and DNA repair proteins. We show that enhanced synthesis of these proteins in DLBCL is mediated by the relief of repression that is normally imposed by structure in the 5'-untranslated regions of their corresponding mRNAs. This process is driven by signaling through mammalian target of rapamycin, resulting in increased synthesis of eukaryotic initiation factor (eIF) 4B complex (eIF4B), a known activator of the RNA helicase eIF4A. Reducing eIF4B expression alone is sufficient to decrease synthesis of proteins associated with enhanced tumor cell survival, namely DAXX, BCL2 and ERCC5. Importantly, eIF4B-driven expression of these key survival proteins is directly correlated with patient outcome, and eIF4B, DAXX and ERCC5 are identified as novel prognostic markers for poor survival in DLBCL. Our work provides new insights into the mechanisms by which the cancer-promoting translational machinery drives lymphomagenesis.

Show MeSH
Related in: MedlinePlus