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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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EBNA1 inhibits p65 phosphorylation in carcinoma cell lines. Western blot analyses of total and phosphorylated (ser 536) p65 in (A) Ad/AH and (B) Hone1 cells stably expressing EBNA1 or a neomycin control vector (neo) under basal conditions or following stimulation with TNFα. Western blotting for EBNA1 and β-actin serve as EBNA1 expression and protein loading controls, respectively.
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Figure 6: EBNA1 inhibits p65 phosphorylation in carcinoma cell lines. Western blot analyses of total and phosphorylated (ser 536) p65 in (A) Ad/AH and (B) Hone1 cells stably expressing EBNA1 or a neomycin control vector (neo) under basal conditions or following stimulation with TNFα. Western blotting for EBNA1 and β-actin serve as EBNA1 expression and protein loading controls, respectively.

Mentions: We next sought to determine whether the EBNA1-induced reduction in NF-κB activity and reduced levels of p65 in active NF-κB dimers was as a result of alterations in the expression or phosphorylation status of p65. In its transcriptionally inactive form unphosphorylated p65 NF-κB is retained in the cytoplasm via interactions with specific inhibitors, the IκBs. Upon stimulation of the canonical NF-κB pathway the inhibitory IκBα protein becomes phosphorylated, ubiquitinated and degraded by the 26S proteasome. NF-κB dimers subsequently translocate to the nucleus where phosphorylated p65-containing dimers can modulate the expression of target genes [26]. Specifically, phosphorylation of p65 at Ser 536 by IKKα and IKKβ has been implicated in p65 nuclear translocation and transcriptional activity [26]. Immunoblot analysis confirmed enhanced serine 536 phosphorylation of p65 following TNFα stimulation of Ad/AH and Hone1 neo control cells as expected (Fig. 6A and 6B, respectively). In contrast, there was almost complete inhibition of p65 phosphorylation in stable EBNA1-expressing Ad/AH and Hone1 cells basally and following TNFα stimulation. Furthermore, the reduction in p65 phosphorylation in EBNA1-expressing cells was not due to a reduction in total levels of p65 protein (Fig. 6A and 6B, respectively). EBNA1-expressing Hone1 cells were also refractory to stimulation with another potent activator of the canonical NF-κB pathway, IL-1β, exhibiting reduced p65 phosphorylation in EBNA1-expressing cells which was in contrast to enhanced levels of phospho-p65 observed in the neo control cells (Fig. 7A). Immunofluorescence staining in Ad/AH cells also revealed that the ability of TNFα to stimulate translocation of p65 from the cytoplasm to the nucleus, as seen in the neo control cells, was almost completely inhibited in cells stably expressing EBNA1 (Fig. 7B). These observations were, therefore, in agreement with the above reporter assays, EMSA and TransAM data.


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)

EBNA1 inhibits p65 phosphorylation in carcinoma cell lines. Western blot analyses of total and phosphorylated (ser 536) p65 in (A) Ad/AH and (B) Hone1 cells stably expressing EBNA1 or a neomycin control vector (neo) under basal conditions or following stimulation with TNFα. Western blotting for EBNA1 and β-actin serve as EBNA1 expression and protein loading controls, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2818691&req=5

Figure 6: EBNA1 inhibits p65 phosphorylation in carcinoma cell lines. Western blot analyses of total and phosphorylated (ser 536) p65 in (A) Ad/AH and (B) Hone1 cells stably expressing EBNA1 or a neomycin control vector (neo) under basal conditions or following stimulation with TNFα. Western blotting for EBNA1 and β-actin serve as EBNA1 expression and protein loading controls, respectively.
Mentions: We next sought to determine whether the EBNA1-induced reduction in NF-κB activity and reduced levels of p65 in active NF-κB dimers was as a result of alterations in the expression or phosphorylation status of p65. In its transcriptionally inactive form unphosphorylated p65 NF-κB is retained in the cytoplasm via interactions with specific inhibitors, the IκBs. Upon stimulation of the canonical NF-κB pathway the inhibitory IκBα protein becomes phosphorylated, ubiquitinated and degraded by the 26S proteasome. NF-κB dimers subsequently translocate to the nucleus where phosphorylated p65-containing dimers can modulate the expression of target genes [26]. Specifically, phosphorylation of p65 at Ser 536 by IKKα and IKKβ has been implicated in p65 nuclear translocation and transcriptional activity [26]. Immunoblot analysis confirmed enhanced serine 536 phosphorylation of p65 following TNFα stimulation of Ad/AH and Hone1 neo control cells as expected (Fig. 6A and 6B, respectively). In contrast, there was almost complete inhibition of p65 phosphorylation in stable EBNA1-expressing Ad/AH and Hone1 cells basally and following TNFα stimulation. Furthermore, the reduction in p65 phosphorylation in EBNA1-expressing cells was not due to a reduction in total levels of p65 protein (Fig. 6A and 6B, respectively). EBNA1-expressing Hone1 cells were also refractory to stimulation with another potent activator of the canonical NF-κB pathway, IL-1β, exhibiting reduced p65 phosphorylation in EBNA1-expressing cells which was in contrast to enhanced levels of phospho-p65 observed in the neo control cells (Fig. 7A). Immunofluorescence staining in Ad/AH cells also revealed that the ability of TNFα to stimulate translocation of p65 from the cytoplasm to the nucleus, as seen in the neo control cells, was almost completely inhibited in cells stably expressing EBNA1 (Fig. 7B). These observations were, therefore, in agreement with the above reporter assays, EMSA and TransAM data.

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