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Epstein-Barr virus-encoded EBNA1 inhibits the canonical NF-kappaB pathway in carcinoma cells by inhibiting IKK phosphorylation.

Valentine R, Dawson CW, Hu C, Shah KM, Owen TJ, Date KL, Maia SP, Shao J, Arrand JR, Young LS, O'Neil JD - Mol. Cancer (2010)

Bottom Line: Inhibition of p65 NF-kappaB in murine and human epidermis results in tissue hyperplasia and the development of squamous cell carcinoma.Furthermore, inhibition of NF-kappaB is employed by viruses as an immune evasion strategy which is also closely linked to oncogenesis during persistent viral infection.Our findings therefore further implicate EBNA1 in playing an important role in the pathogenesis of NPC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Research UK Cancer Centre, School of Cancer Sciences, University of Birmingham, Edgbaston, Birmingham, UK.

ABSTRACT

Background: The Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all EBV-associated tumours, including undifferentiated nasopharyngeal carcinoma (NPC), where it is indispensable for viral replication, genome maintenance and viral gene expression. EBNA1's transcription factor-like functions also extend to influencing the expression of cellular genes involved in pathways commonly dysregulated during oncogenesis, including elevation of AP-1 activity in NPC cell lines resulting in enhancement of angiogenesis in vitro. In this study we sought to extend these observations by examining the role of EBNA1 upon another pathway commonly deregulated during carcinogenesis; namely NF-kappaB.

Results: In this report we demonstrate that EBNA1 inhibits the canonical NF-kappaB pathway in carcinoma lines by inhibiting the phosphorylation of IKKalpha/beta. In agreement with this observation we find a reduction in the phosphorylation of IkappaBalpha and reduced phosphorylation and nuclear translocation of p65, resulting in a reduction in the amount of p65 in nuclear NF-kappaB complexes. Similar effects were also found in carcinoma lines infected with recombinant EBV and in the EBV-positive NPC-derived cell line C666-1. Inhibition of NF-kappaB was dependent upon regions of EBNA1 essential for gene transactivation whilst the interaction with the deubiquitinating enzyme, USP7, was entirely dispensable. Furthermore, in agreement with EBNA1 inhibiting p65 NF-kappaB we demonstrate that p65 was exclusively cytoplasmic in 11 out of 11 NPC tumours studied.

Conclusions: Inhibition of p65 NF-kappaB in murine and human epidermis results in tissue hyperplasia and the development of squamous cell carcinoma. In line with this, p65 knockout fibroblasts have a transformed phenotype. Inhibition of p65 NF-kappaB by EBNA1 may therefore contribute to the development of NPC by inducing tissue hyperplasia. Furthermore, inhibition of NF-kappaB is employed by viruses as an immune evasion strategy which is also closely linked to oncogenesis during persistent viral infection. Our findings therefore further implicate EBNA1 in playing an important role in the pathogenesis of NPC.

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(A) Transient transfection of increasing concentrations of the EBNA1 expression plasmid pSG5-EBNA1 into parental Ad/AH cells inhibits NF-κB luciferase reporter activity in a dose-dependent manner. (B) Transient transfection of increasing concentrations of a dominant-negative EBNA1 (dnEBNA1) abrogates the ability of wild-type EBNA1 (wtEBNA1) to inhibit NF-κB luciferase activity in parental Ad/AH cells. Reporter assays were performed in biological and technical triplicate and error bars indicate SD (* = P < 0.05 relative to EBNA1-free controls).
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Figure 2: (A) Transient transfection of increasing concentrations of the EBNA1 expression plasmid pSG5-EBNA1 into parental Ad/AH cells inhibits NF-κB luciferase reporter activity in a dose-dependent manner. (B) Transient transfection of increasing concentrations of a dominant-negative EBNA1 (dnEBNA1) abrogates the ability of wild-type EBNA1 (wtEBNA1) to inhibit NF-κB luciferase activity in parental Ad/AH cells. Reporter assays were performed in biological and technical triplicate and error bars indicate SD (* = P < 0.05 relative to EBNA1-free controls).

Mentions: To assess whether EBNA1 influenced NF-κB activity we initially performed luciferase reporter assays in a range of carcinoma cell lines using a synthetic NF-κB reporter and found that NF-κB activity in Ad/AH, AGS and Hone1 cells stably expressing levels of EBNA1 comparable to those found in EBV infection was inhibited by 8, 5 and 2.6 fold, respectively (Fig. 1). Transient expression of EBNA1 in Ad/AH cells achieved by transfection using a range of concentrations of EBNA1 plasmid DNA resulted in a dose-dependent decrease in NF-κB reporter activity with a 2-fold reduction seen at the highest concentration of input DNA (Fig. 2A). To assess whether inhibition of NF-κB required a fully functional EBNA1, a dominant-negative EBNA1 (dnEBNA1) was titrated against wild-type EBNA1 in Ad/AH cells. Increasing doses of dnEBNA1, which dimerises with wild-type EBNA1 impairing its function, resulted in almost complete abrogation of the ability of wild-type EBNA1 to inhibit NF-κB activity (Fig. 2B). In addition, the dnEBNA1 alone had no effect on NF-κB reporter activity (Fig. 2B). Next we performed electromobility shift assays (EMSA) using a consensus NF-κB probe to assess whether the reduction in reporter activity was due to a reduction in NF-κB DNA binding. EMSAs performed on Ad/AH cells stably expressing EBNA1 indicated a 2-fold basal reduction in band intensity indicating a reduction in nuclear protein binding to the NF-κB probe, when compared with the neo control cells, which was consistent with our observed reduction in reporter activity (Fig. 3). Furthermore, the ability of two different pro-inflammatory cytokines, TNFα and IL-1β, to enhance NF-κB DNA binding was ablated in those cells stably expressing EBNA1 (Fig. 3 and data not shown). Similar results were observed in Ad/AH cells stably infected with a recombinant EBV (rEBV) carrying the neomycin drug selectable marker (Fig. 4A). In addition, the ability of TNFα, a potent activator of the canonical NF-κB pathway, to enhance binding with the NF-κB probe in C666-1 cells was considerably lower than in Ad/AH parental cells (Fig. 4B). EMSAs performed on Ad/AH cells stimulated with TNFα incubated with both the labeled NF-κB probe and a 100-fold excess of unlabeled probe (cold competition) resulted in complete abrogation of probe binding (Fig. 3, middle panel), thus demonstrating the high degree of specificity in the EMSA assays.


Epstein-Barr virus-encoded EBNA1 inhibits the canonical NF-kappaB pathway in carcinoma cells by inhibiting IKK phosphorylation.

Valentine R, Dawson CW, Hu C, Shah KM, Owen TJ, Date KL, Maia SP, Shao J, Arrand JR, Young LS, O'Neil JD - Mol. Cancer (2010)

(A) Transient transfection of increasing concentrations of the EBNA1 expression plasmid pSG5-EBNA1 into parental Ad/AH cells inhibits NF-κB luciferase reporter activity in a dose-dependent manner. (B) Transient transfection of increasing concentrations of a dominant-negative EBNA1 (dnEBNA1) abrogates the ability of wild-type EBNA1 (wtEBNA1) to inhibit NF-κB luciferase activity in parental Ad/AH cells. Reporter assays were performed in biological and technical triplicate and error bars indicate SD (* = P < 0.05 relative to EBNA1-free controls).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: (A) Transient transfection of increasing concentrations of the EBNA1 expression plasmid pSG5-EBNA1 into parental Ad/AH cells inhibits NF-κB luciferase reporter activity in a dose-dependent manner. (B) Transient transfection of increasing concentrations of a dominant-negative EBNA1 (dnEBNA1) abrogates the ability of wild-type EBNA1 (wtEBNA1) to inhibit NF-κB luciferase activity in parental Ad/AH cells. Reporter assays were performed in biological and technical triplicate and error bars indicate SD (* = P < 0.05 relative to EBNA1-free controls).
Mentions: To assess whether EBNA1 influenced NF-κB activity we initially performed luciferase reporter assays in a range of carcinoma cell lines using a synthetic NF-κB reporter and found that NF-κB activity in Ad/AH, AGS and Hone1 cells stably expressing levels of EBNA1 comparable to those found in EBV infection was inhibited by 8, 5 and 2.6 fold, respectively (Fig. 1). Transient expression of EBNA1 in Ad/AH cells achieved by transfection using a range of concentrations of EBNA1 plasmid DNA resulted in a dose-dependent decrease in NF-κB reporter activity with a 2-fold reduction seen at the highest concentration of input DNA (Fig. 2A). To assess whether inhibition of NF-κB required a fully functional EBNA1, a dominant-negative EBNA1 (dnEBNA1) was titrated against wild-type EBNA1 in Ad/AH cells. Increasing doses of dnEBNA1, which dimerises with wild-type EBNA1 impairing its function, resulted in almost complete abrogation of the ability of wild-type EBNA1 to inhibit NF-κB activity (Fig. 2B). In addition, the dnEBNA1 alone had no effect on NF-κB reporter activity (Fig. 2B). Next we performed electromobility shift assays (EMSA) using a consensus NF-κB probe to assess whether the reduction in reporter activity was due to a reduction in NF-κB DNA binding. EMSAs performed on Ad/AH cells stably expressing EBNA1 indicated a 2-fold basal reduction in band intensity indicating a reduction in nuclear protein binding to the NF-κB probe, when compared with the neo control cells, which was consistent with our observed reduction in reporter activity (Fig. 3). Furthermore, the ability of two different pro-inflammatory cytokines, TNFα and IL-1β, to enhance NF-κB DNA binding was ablated in those cells stably expressing EBNA1 (Fig. 3 and data not shown). Similar results were observed in Ad/AH cells stably infected with a recombinant EBV (rEBV) carrying the neomycin drug selectable marker (Fig. 4A). In addition, the ability of TNFα, a potent activator of the canonical NF-κB pathway, to enhance binding with the NF-κB probe in C666-1 cells was considerably lower than in Ad/AH parental cells (Fig. 4B). EMSAs performed on Ad/AH cells stimulated with TNFα incubated with both the labeled NF-κB probe and a 100-fold excess of unlabeled probe (cold competition) resulted in complete abrogation of probe binding (Fig. 3, middle panel), thus demonstrating the high degree of specificity in the EMSA assays.

Bottom Line: Inhibition of p65 NF-kappaB in murine and human epidermis results in tissue hyperplasia and the development of squamous cell carcinoma.Furthermore, inhibition of NF-kappaB is employed by viruses as an immune evasion strategy which is also closely linked to oncogenesis during persistent viral infection.Our findings therefore further implicate EBNA1 in playing an important role in the pathogenesis of NPC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Research UK Cancer Centre, School of Cancer Sciences, University of Birmingham, Edgbaston, Birmingham, UK.

ABSTRACT

Background: The Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all EBV-associated tumours, including undifferentiated nasopharyngeal carcinoma (NPC), where it is indispensable for viral replication, genome maintenance and viral gene expression. EBNA1's transcription factor-like functions also extend to influencing the expression of cellular genes involved in pathways commonly dysregulated during oncogenesis, including elevation of AP-1 activity in NPC cell lines resulting in enhancement of angiogenesis in vitro. In this study we sought to extend these observations by examining the role of EBNA1 upon another pathway commonly deregulated during carcinogenesis; namely NF-kappaB.

Results: In this report we demonstrate that EBNA1 inhibits the canonical NF-kappaB pathway in carcinoma lines by inhibiting the phosphorylation of IKKalpha/beta. In agreement with this observation we find a reduction in the phosphorylation of IkappaBalpha and reduced phosphorylation and nuclear translocation of p65, resulting in a reduction in the amount of p65 in nuclear NF-kappaB complexes. Similar effects were also found in carcinoma lines infected with recombinant EBV and in the EBV-positive NPC-derived cell line C666-1. Inhibition of NF-kappaB was dependent upon regions of EBNA1 essential for gene transactivation whilst the interaction with the deubiquitinating enzyme, USP7, was entirely dispensable. Furthermore, in agreement with EBNA1 inhibiting p65 NF-kappaB we demonstrate that p65 was exclusively cytoplasmic in 11 out of 11 NPC tumours studied.

Conclusions: Inhibition of p65 NF-kappaB in murine and human epidermis results in tissue hyperplasia and the development of squamous cell carcinoma. In line with this, p65 knockout fibroblasts have a transformed phenotype. Inhibition of p65 NF-kappaB by EBNA1 may therefore contribute to the development of NPC by inducing tissue hyperplasia. Furthermore, inhibition of NF-kappaB is employed by viruses as an immune evasion strategy which is also closely linked to oncogenesis during persistent viral infection. Our findings therefore further implicate EBNA1 in playing an important role in the pathogenesis of NPC.

Show MeSH
Related in: MedlinePlus