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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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Related in: MedlinePlus

mRNAs containing highly structured 5′-UTRs are preferentially translated in DLBCL. (a) The cDNA microarray data were analysed and the average 5′-UTR lengths, and ΔG values were calculated for those mRNAs in the ‘up' and ‘down' lists, and the significance of the difference to the whole array was assessed by Student's t-tests. The data show that the mRNAs in the upregulated list have longer 5′-UTRs (P=0.002; i), have greater −ΔG values (P=0.001; ii) and have more nucleotides incorporated into hairpins (P=0.006; iii); all three criteria would be expected to decrease the translatability of mRNAs in these groups. (b(i)) The cDNAs corresponding to the 5′-UTRs of DAXX, ERCC5 and BCL2 were subcloned into a luciferase reporter vector based on pGL3. (b(ii)) The reporter constructs were transfected into cell lines derived from DLBCL and control B cell, and luciferase activity determined. The data show that there is greater expression of luciferase in the DLBCL-derived cell lines than in the control cell lines for all plasmids, suggesting that the 5′-UTRs are less inhibitory in these cell lines. Significance was assessed by paired Student's t-tests on three independent experiments (*P<0.05, **P<0.01 and ***P<0.001). (c(i)) Unrelated DNA sequences of increasing length (Supplementary Table 2) were subcloned into the 5′-UTR of pGL3 upstream of the luciferase cistron. (c(ii)) These plasmids were transfected into the control cell line (GM01953) and cell line derived from DLBCL (DoHH-2, OCI-LY19 and DB), and luciferase activity determined. (d) Protein extracts derived from cell lines representative of either DLBCL (Val, DoHH-2, SuDHL-6, OCI-LY19 and DB) and two control B-cell lines (GM03201 and GM01953) were separated by SDS-polyacrylamide gel electrophoresis (PAGE). Samples were immunoblotted with eukaryotic initiation factor (eIFs)-specific antibodies. The data show that only eIF4B levels were increased in these cell lines. (e(i)) Extracts were generated from B cells purified from tonsils of tumor-free individuals or tumors from patients with DLBCL, and separated by SDS-PAGE and immunoblotted to examine eIF4B levels. Tubulin was used as a loading control. The data show that eIF4B levels were elevated in the five DLBCL samples tested. (e(ii)) Tissue microarrays that contained tissues from 362 lymphomas, including 196 DLBCL, 76 FL and 52 chronic lymphocytic leukemias (CLLs) were probed by an antibody against eIF4B and scored as described in the experimental procedures section. These data show that there is significantly higher expression of eIF4B in DLBCL compared with that in CLL and FL. Significance between the different categories was assessed by χ2-test (*P<0.05).
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fig3: mRNAs containing highly structured 5′-UTRs are preferentially translated in DLBCL. (a) The cDNA microarray data were analysed and the average 5′-UTR lengths, and ΔG values were calculated for those mRNAs in the ‘up' and ‘down' lists, and the significance of the difference to the whole array was assessed by Student's t-tests. The data show that the mRNAs in the upregulated list have longer 5′-UTRs (P=0.002; i), have greater −ΔG values (P=0.001; ii) and have more nucleotides incorporated into hairpins (P=0.006; iii); all three criteria would be expected to decrease the translatability of mRNAs in these groups. (b(i)) The cDNAs corresponding to the 5′-UTRs of DAXX, ERCC5 and BCL2 were subcloned into a luciferase reporter vector based on pGL3. (b(ii)) The reporter constructs were transfected into cell lines derived from DLBCL and control B cell, and luciferase activity determined. The data show that there is greater expression of luciferase in the DLBCL-derived cell lines than in the control cell lines for all plasmids, suggesting that the 5′-UTRs are less inhibitory in these cell lines. Significance was assessed by paired Student's t-tests on three independent experiments (*P<0.05, **P<0.01 and ***P<0.001). (c(i)) Unrelated DNA sequences of increasing length (Supplementary Table 2) were subcloned into the 5′-UTR of pGL3 upstream of the luciferase cistron. (c(ii)) These plasmids were transfected into the control cell line (GM01953) and cell line derived from DLBCL (DoHH-2, OCI-LY19 and DB), and luciferase activity determined. (d) Protein extracts derived from cell lines representative of either DLBCL (Val, DoHH-2, SuDHL-6, OCI-LY19 and DB) and two control B-cell lines (GM03201 and GM01953) were separated by SDS-polyacrylamide gel electrophoresis (PAGE). Samples were immunoblotted with eukaryotic initiation factor (eIFs)-specific antibodies. The data show that only eIF4B levels were increased in these cell lines. (e(i)) Extracts were generated from B cells purified from tonsils of tumor-free individuals or tumors from patients with DLBCL, and separated by SDS-PAGE and immunoblotted to examine eIF4B levels. Tubulin was used as a loading control. The data show that eIF4B levels were elevated in the five DLBCL samples tested. (e(ii)) Tissue microarrays that contained tissues from 362 lymphomas, including 196 DLBCL, 76 FL and 52 chronic lymphocytic leukemias (CLLs) were probed by an antibody against eIF4B and scored as described in the experimental procedures section. These data show that there is significantly higher expression of eIF4B in DLBCL compared with that in CLL and FL. Significance between the different categories was assessed by χ2-test (*P<0.05).

Mentions: The enhanced translation of subsets of mRNAs in DLBCL could be due to unique RNA elements, as the majority of mammalian mRNAs contain regulatory RNA motifs, including upstream open reading frames, internal ribosome entry segments, terminal oligopyrimidine tracts and microRNA-binding sites.31 Therefore, the UTRs of the mRNAs in the ‘up' and ‘down' lists were examined to assess whether there were unique features that could contribute to their up- or downregulation. The proportion of mRNAs containing internal ribosome entry segments, upstream open reading frames, terminal oligopyrimidine tracts or microRNA target sites remained unchanged between the gene lists and the whole array (Supplementary Figure 5A). However, the data show a significant increase in the average 5′-UTR length and in the predicted minimum free energy (ΔG) of folding in the list of genes that were translationally upregulated in DLBCL, compared with the complete array (Figures 3a(i and ii)). Further examination of the 5′-UTR length (Supplementary Figure 5B) reveals that the cumulative distribution corresponding to the translationally upregulated group is shifted towards the longer lengths compared with the downregulated and unchanged groups; the ΔG data shows a similar pattern with a shift towards more negative free energies in the translationally upregulated group (Supplementary Figure 5C(i)). These data suggest that a population of mRNAs with long and/or structured 5′-UTRs is enriched in the translationally upregulated group.


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 containing highly structured 5′-UTRs are preferentially translated in DLBCL. (a) The cDNA microarray data were analysed and the average 5′-UTR lengths, and ΔG values were calculated for those mRNAs in the ‘up' and ‘down' lists, and the significance of the difference to the whole array was assessed by Student's t-tests. The data show that the mRNAs in the upregulated list have longer 5′-UTRs (P=0.002; i), have greater −ΔG values (P=0.001; ii) and have more nucleotides incorporated into hairpins (P=0.006; iii); all three criteria would be expected to decrease the translatability of mRNAs in these groups. (b(i)) The cDNAs corresponding to the 5′-UTRs of DAXX, ERCC5 and BCL2 were subcloned into a luciferase reporter vector based on pGL3. (b(ii)) The reporter constructs were transfected into cell lines derived from DLBCL and control B cell, and luciferase activity determined. The data show that there is greater expression of luciferase in the DLBCL-derived cell lines than in the control cell lines for all plasmids, suggesting that the 5′-UTRs are less inhibitory in these cell lines. Significance was assessed by paired Student's t-tests on three independent experiments (*P<0.05, **P<0.01 and ***P<0.001). (c(i)) Unrelated DNA sequences of increasing length (Supplementary Table 2) were subcloned into the 5′-UTR of pGL3 upstream of the luciferase cistron. (c(ii)) These plasmids were transfected into the control cell line (GM01953) and cell line derived from DLBCL (DoHH-2, OCI-LY19 and DB), and luciferase activity determined. (d) Protein extracts derived from cell lines representative of either DLBCL (Val, DoHH-2, SuDHL-6, OCI-LY19 and DB) and two control B-cell lines (GM03201 and GM01953) were separated by SDS-polyacrylamide gel electrophoresis (PAGE). Samples were immunoblotted with eukaryotic initiation factor (eIFs)-specific antibodies. The data show that only eIF4B levels were increased in these cell lines. (e(i)) Extracts were generated from B cells purified from tonsils of tumor-free individuals or tumors from patients with DLBCL, and separated by SDS-PAGE and immunoblotted to examine eIF4B levels. Tubulin was used as a loading control. The data show that eIF4B levels were elevated in the five DLBCL samples tested. (e(ii)) Tissue microarrays that contained tissues from 362 lymphomas, including 196 DLBCL, 76 FL and 52 chronic lymphocytic leukemias (CLLs) were probed by an antibody against eIF4B and scored as described in the experimental procedures section. These data show that there is significantly higher expression of eIF4B in DLBCL compared with that in CLL and FL. Significance between the different categories was assessed by χ2-test (*P<0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: mRNAs containing highly structured 5′-UTRs are preferentially translated in DLBCL. (a) The cDNA microarray data were analysed and the average 5′-UTR lengths, and ΔG values were calculated for those mRNAs in the ‘up' and ‘down' lists, and the significance of the difference to the whole array was assessed by Student's t-tests. The data show that the mRNAs in the upregulated list have longer 5′-UTRs (P=0.002; i), have greater −ΔG values (P=0.001; ii) and have more nucleotides incorporated into hairpins (P=0.006; iii); all three criteria would be expected to decrease the translatability of mRNAs in these groups. (b(i)) The cDNAs corresponding to the 5′-UTRs of DAXX, ERCC5 and BCL2 were subcloned into a luciferase reporter vector based on pGL3. (b(ii)) The reporter constructs were transfected into cell lines derived from DLBCL and control B cell, and luciferase activity determined. The data show that there is greater expression of luciferase in the DLBCL-derived cell lines than in the control cell lines for all plasmids, suggesting that the 5′-UTRs are less inhibitory in these cell lines. Significance was assessed by paired Student's t-tests on three independent experiments (*P<0.05, **P<0.01 and ***P<0.001). (c(i)) Unrelated DNA sequences of increasing length (Supplementary Table 2) were subcloned into the 5′-UTR of pGL3 upstream of the luciferase cistron. (c(ii)) These plasmids were transfected into the control cell line (GM01953) and cell line derived from DLBCL (DoHH-2, OCI-LY19 and DB), and luciferase activity determined. (d) Protein extracts derived from cell lines representative of either DLBCL (Val, DoHH-2, SuDHL-6, OCI-LY19 and DB) and two control B-cell lines (GM03201 and GM01953) were separated by SDS-polyacrylamide gel electrophoresis (PAGE). Samples were immunoblotted with eukaryotic initiation factor (eIFs)-specific antibodies. The data show that only eIF4B levels were increased in these cell lines. (e(i)) Extracts were generated from B cells purified from tonsils of tumor-free individuals or tumors from patients with DLBCL, and separated by SDS-PAGE and immunoblotted to examine eIF4B levels. Tubulin was used as a loading control. The data show that eIF4B levels were elevated in the five DLBCL samples tested. (e(ii)) Tissue microarrays that contained tissues from 362 lymphomas, including 196 DLBCL, 76 FL and 52 chronic lymphocytic leukemias (CLLs) were probed by an antibody against eIF4B and scored as described in the experimental procedures section. These data show that there is significantly higher expression of eIF4B in DLBCL compared with that in CLL and FL. Significance between the different categories was assessed by χ2-test (*P<0.05).
Mentions: The enhanced translation of subsets of mRNAs in DLBCL could be due to unique RNA elements, as the majority of mammalian mRNAs contain regulatory RNA motifs, including upstream open reading frames, internal ribosome entry segments, terminal oligopyrimidine tracts and microRNA-binding sites.31 Therefore, the UTRs of the mRNAs in the ‘up' and ‘down' lists were examined to assess whether there were unique features that could contribute to their up- or downregulation. The proportion of mRNAs containing internal ribosome entry segments, upstream open reading frames, terminal oligopyrimidine tracts or microRNA target sites remained unchanged between the gene lists and the whole array (Supplementary Figure 5A). However, the data show a significant increase in the average 5′-UTR length and in the predicted minimum free energy (ΔG) of folding in the list of genes that were translationally upregulated in DLBCL, compared with the complete array (Figures 3a(i and ii)). Further examination of the 5′-UTR length (Supplementary Figure 5B) reveals that the cumulative distribution corresponding to the translationally upregulated group is shifted towards the longer lengths compared with the downregulated and unchanged groups; the ΔG data shows a similar pattern with a shift towards more negative free energies in the translationally upregulated group (Supplementary Figure 5C(i)). These data suggest that a population of mRNAs with long and/or structured 5′-UTRs is enriched in the translationally upregulated group.

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