Sharmila , Velapasamy (2018) Transforming growth factor-beta signalling in the regulation of epstein-barr virus infection in nasopharyngeal epithelial cells / Sharmila Velapasamy. PhD thesis, University of Malaya.
Abstract
Undifferentiated nasopharyngeal carcinoma (NPC) is consistently associated with Epstein-Barr virus (EBV) infection. However, the molecular events that regulate the establishment of stable EBV infection in nasopharyngeal epithelial (NPE) cells remain largely undefined. It is now recognised that persistent EBV latent infection in NPE cells is dependent on specific pre-existing genetic changes. There is evidence to show that Transforming growth factor-beta (TGF-β) signalling is de-regulated in NPC. TGF-β signalling regulates a variety of cellular processes and functions as a tumour suppressor in the early stage of epithelial carcinogenesis by inhibiting cell proliferation and promoting differentiation, apoptosis and senescence. Our preliminary data showed that epithelial cells that are no longer responsive to TGF-β1-induced growth inhibition are more amenable to stable EBV infection, while epithelial cells harbouring an intact TGF-β signalling pathway are not able to sustain EBV genomes, suggesting that de-regulation of TGF-β signalling is a prerequisite for the establishment of stable EBV infection. To test this hypothesis, two different approaches were used to disrupt TGF-β signalling in three immortalised NPE cell lines (NP361hTert, NP460hTert and NP550hTert): (1) inhibiting the type 1 TGF-β receptor (TGFR-1) kinase with a chemical inhibitor, SB431542 and (2) overexpression of a kinase-deficient form (dominant negative) of the type 2 TGF-β receptor (DnTGFBR2). Functional disruption of the TGF-β signalling pathway was confirmed by a reduced growth inhibitory response to TGF-β1, inhibition of TGF-β1-induced phosphorylation of Smad2 and a loss of Smad2/3-dependent transcription activity following TGF-β1 stimulation. These cells were then infected with a GFP-tagged recombinant EBV (Akata strain) and FACS used to determine the percentages of GFP-positive cells over a period of 21 days post-infection. Compared to the respective controls, both TGFR-1 kinase inhibition and overexpression of DnTGFBR2 resulted in higher numbers of cells carrying the EBV genomes in all three cell lines. Given that EBV infection is associated with growth inhibition and senescence, as well as entry into the lytic cycle in a manner that is linked to epithelial cell differentiation, the influence of TGF-β signalling disruption on cellular differentiation, EBV-induced senescence and induction of the EBV lytic cycle, was investigated. The results showed that both the inhibition of TGFR-1 kinase and overexpression of DnTGFBR2 suppressed the differentiation of NPE cells in response to serum and calcium as well as TGF-β1, as shown by reduced involucrin expression. Following EBV infection of these cells, the expression of SIRT1 was readily detected while p16 and p21 levels were significantly decreased, indicating that the cells were resistant to EBV-induced senescence and growth inhibition. Further, the expression of EBV-encoded BZLF1 was reduced, suggesting that disruption of TGF-β signalling suppressed EBV lytic cycle induction. Lastly, to conclusively demonstrate an essential role for TGF-β signalling in sustaining EBV infection in NPE cells, TGFBR2 was knocked out using CRISPR/Cas9. Similarly, knockout of TGFBR2 resulted in a more persistent EBV infection in NP460hTert, and these effects were reversed following expression of a wild-type TGFBR2. Collectively, the results of this study demonstrate that disruption of TGF-β signalling supports stable EBV infection in NPE cells, possibly by suppressing cellular differentiation, EBV-induced senescence and EBV lytic cycle induction.
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