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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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TransAM analysis demonstrating the p65 and p50 composition of transcriptionally competent NF-κB dimers present in Ad/AH cells stably expressing EBNA1 or a neomycin control vector (neo), Ad/AH cells stably infected with rEBV and C666-1 cells, under basal conditions and following stimulation with 100 ng/ml TNFα. TransAM analysis was performed in biological and technical triplicate and error bars indicate SD (* = P < 0.05 relative to Ad/AH Neo control).
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Figure 5: TransAM analysis demonstrating the p65 and p50 composition of transcriptionally competent NF-κB dimers present in Ad/AH cells stably expressing EBNA1 or a neomycin control vector (neo), Ad/AH cells stably infected with rEBV and C666-1 cells, under basal conditions and following stimulation with 100 ng/ml TNFα. TransAM analysis was performed in biological and technical triplicate and error bars indicate SD (* = P < 0.05 relative to Ad/AH Neo control).

Mentions: Having demonstrated that general NF-κB activity and NF-κB DNA binding in EBNA1 expressing cells was reduced, we sought to determine the relative abundance of the canonical NF-κB subunits p65 and p50 in active nuclear complexes bound to target DNA. We chose to study p65 and p50 because heterodimers of these NF-κB subunits are the most abundant form of NF-κB and exhibit the most powerful transcriptional activation potential [25]. Furthermore, the canonical NF-κB pathway is most commonly associated with innate immunity in general and with cellular differentiation in epithelial cells, both of which impact upon the pathogenesis of NPC. TransAM analysis in Ad/AH cells demonstrated a 4-fold enrichment of p65-containing dimers in neo control cells stimulated with TNFα (relative to un-stimulated neo cells), whereas the abundance of p50 did not change significantly (Fig. 5). In contrast, there was a 2-fold reduction in the basal amount of p65-containing dimers in Ad/AH cells stably expressing EBNA1, when compared with the un-stimulated neo control, and these cells were refractory to stimulation with TNFα. However, the amount of p50-containing dimers in Ad/AH cells stably expressing EBNA1 both pre and post TNFα stimulation was not significantly different from the levels observed in the neo control cells. Results similar to those found in Ad/AH cells stably expressing EBNA1 were also observed in Ad/AH cells stably infected with rEBV and in C666-1 cells. Interestingly, the amount of p50-containing dimers was marginally increased in Ad/AH cells stably infected with rEBV and in the C666-1 cells and this was unaffected by stimulation with TNFα.


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)

TransAM analysis demonstrating the p65 and p50 composition of transcriptionally competent NF-κB dimers present in Ad/AH cells stably expressing EBNA1 or a neomycin control vector (neo), Ad/AH cells stably infected with rEBV and C666-1 cells, under basal conditions and following stimulation with 100 ng/ml TNFα. TransAM analysis was performed in biological and technical triplicate and error bars indicate SD (* = P < 0.05 relative to Ad/AH Neo control).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: TransAM analysis demonstrating the p65 and p50 composition of transcriptionally competent NF-κB dimers present in Ad/AH cells stably expressing EBNA1 or a neomycin control vector (neo), Ad/AH cells stably infected with rEBV and C666-1 cells, under basal conditions and following stimulation with 100 ng/ml TNFα. TransAM analysis was performed in biological and technical triplicate and error bars indicate SD (* = P < 0.05 relative to Ad/AH Neo control).
Mentions: Having demonstrated that general NF-κB activity and NF-κB DNA binding in EBNA1 expressing cells was reduced, we sought to determine the relative abundance of the canonical NF-κB subunits p65 and p50 in active nuclear complexes bound to target DNA. We chose to study p65 and p50 because heterodimers of these NF-κB subunits are the most abundant form of NF-κB and exhibit the most powerful transcriptional activation potential [25]. Furthermore, the canonical NF-κB pathway is most commonly associated with innate immunity in general and with cellular differentiation in epithelial cells, both of which impact upon the pathogenesis of NPC. TransAM analysis in Ad/AH cells demonstrated a 4-fold enrichment of p65-containing dimers in neo control cells stimulated with TNFα (relative to un-stimulated neo cells), whereas the abundance of p50 did not change significantly (Fig. 5). In contrast, there was a 2-fold reduction in the basal amount of p65-containing dimers in Ad/AH cells stably expressing EBNA1, when compared with the un-stimulated neo control, and these cells were refractory to stimulation with TNFα. However, the amount of p50-containing dimers in Ad/AH cells stably expressing EBNA1 both pre and post TNFα stimulation was not significantly different from the levels observed in the neo control cells. Results similar to those found in Ad/AH cells stably expressing EBNA1 were also observed in Ad/AH cells stably infected with rEBV and in C666-1 cells. Interestingly, the amount of p50-containing dimers was marginally increased in Ad/AH cells stably infected with rEBV and in the C666-1 cells and this was unaffected by stimulation with TNFα.

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