Epstein-Barr Virus LMP2A Transforms Epithelial Cells, Inhibits Cell Differentiation, and Activates Akt

Abstract

The Epstein-Barr virus LMP2A protein was expressed in a human keratinocyte cell line, HaCaT, and effects on epithelial cell growth were detected in organotypic raft cultures and in vivo in nude mice. Raft cultures derived from LMP2A-expressing cells were hyperproliferative, and epithelial differentiation was inhibited. The LMP2A-expressing HaCaT cells were able to grow anchorage independently and formed colonies in soft agar. HaCaT cells expressing LMP2A were highly tumorigenic and formed aggressive tumors in nude mice. The LMP2A tumors were poorly differentiated and highly proliferative, in contrast to occasional tumors that arose from parental HaCaT cells and vector control cells, which grew slowly and remained highly differentiated. Animals injected with LMP2A-expressing cells developed frequent metastases, which predominantly involved lymphoid organs. Involucrin, a marker of epithelial differentiation, and E-cadherin, involved in the maintenance of intercellular contact, were downregulated in LMP2A tumors. Whereas activation of the mitogen-activated protein kinase pathway was not observed, phosphatidylinositol-3-kinase (PI3-kinase)-dependent activation of the serine-threonine kinase Akt was detected in LMP2A-expressing cells and LMP2A tumors. Inhibition of this pathway blocked growth in soft agar. These data indicate that LMP2A greatly affects cell growth and differentiation pathways in epithelial cells, in part through activation of the PI3-kinase-Akt pathway. Epstein-Barr virus (EBV) is a ubiquitous herpesvirus of the familyGammaherpesviridae. Primary infection occurs in the oropharyngeal epithelium, which is permissive for virus replication. Trafficking B lymphocytes are subsequently infected by progeny virions that go on to establish a lifelong latent infection in the B-cell compartment with periodic reactivation and reinfection of the oropharyngeal epithelium, virus release, and transmission of virus via the salivary route (27). EBV is associated with a number of human malignancies, such as Burkitt's lymphoma, Hodgkin's lymphoma, posttransplant lymphoproliferative disease, and the epithelial cell malignancy nasopharyngeal carcinoma (NPC) (27,42,(44)(45)(46)56). In EBV latency, only a subset of genes is expressed. Different types of latency can be distinguished according to the latent gene products expressed. In type I latency, characteristic of Burkitt's lymphoma, only the EBV nuclear antigen 1 (EBNA1), transcripts from the BamHI-A region of the genome (10), and the EBERs, small, nonpolyadenylated RNAs with an unknown function, are expressed. In B-cell lines immortalized by EBV in vitro, all latent genes are expressed, which include EBNA1 through -6, BamHI-A transcripts, EBERs, and three latent membrane proteins, LMP1, LMP2A, and LMP2B (latency III). The pattern of gene expression is more restricted in NPC, with expression of EBNA1, EBERs, BamHI-A transcripts, and the latent membrane proteins (latency II) (27).LMP2A and -2B arise from two different promoters, and transcription proceeds across the fused ...

Mechanisms of cell immortalization mediated by EB viral activation of telomerase in nasopharyngeal carcinoma

“…The exact role of LMP2A inhibition of telomerase activity is still unknown in EBV-associated cell immortalization. The inhibitory effect is consistent with the current view that LMP2A is dispensable in EBVinduced cell immortalization in B cells, but seems contradictory against the notion that LMP2 promotes survival and inhibits apoptosis of Burkitt's lymphoma cells and in gastric carcinoma HSC-39 cell line by a mechanism of inducing Akt phosphorylation and activation [78,79]. It has been speculated that LMP2A inhibits telomerase activity to suppress B-cell activation in order to maintain viral latency [17].…”

The Shb signalling scaffold binds to and regulates constitutive signals from the Epstein–Barr virus LMP2A membrane protein

“…LMP2A and bovine leukemia virus gp 30 were first shown to issue ITAM-dependent signals in T cells (Beaufils et al, 1993). Some functions of the ITAM-containing viral proteins may relate to tumorigenicity, such as survival signalling by activation of the PI3K-Akt pathway by LMP2A and KSHV K1 (Scholle et al, 2000;Swart et al, 2000;Tomlinson and Damania, 2004) and stimulation of cellular migration and invasiveness by LMP2A and MMTV env (Katz et al, 2005;Lu et al, 2006).…”

“…Tyrosine residues involved in Shb binding to LMP2A control constitutive Akt activation The LMP2A ITAM tyrosines Y74 and Y85 were previously shown to bind the Syk tyrosine kinase (Longnecker et al, 1991) and mutations of these tyrosines were shown to influence Syk binding to LMP2A as well as LMP2A-mediated Akt activation and survival signalling (Scholle et al, 2000;Swart et al, 2000). Here, we confirm the negative effect of the Y74F and Y85F mutations on the ability of LMP2A to activate Akt (Figure 6, lanes 11 and 12) and extend this observation by showing that the Y64F mutation suppresses Akt activation by LMP2A, either alone (lanes 5 and 6) or in combination with the Y74F mutation (lanes 9 and 10).…”

“…In B cells, it can deliver signals that permit survival of B-cell receptor (BCR)-negative cells in the peripheral circulation (Caldwell et al, 1998;Merchant et al, 2000). These signals were shown to depend on a constitutive activation of the PI3K (phosphoinositide 3-kinase)-Akt signalling pathway (Scholle et al, 2000, Swart et al, 2000 mediated by the LMP2A ITAM motif. Depending on its expression level, LMP2A can direct B-cell differentiation either to the B1 or the follicular (B2) and marginal zone B cells (Casola et al, 2004).…”

“…Although Syk can mediate PI3K activation in B cells, it is not clear whether the association between Syk and LMP2A leads to both PI3K activation and Syk ubiquitination or whether LMP2A inactivates Syk and another interacting protein mediates PI3K activation. If so, then such a protein must utilize the LMP2A ITAM motif, which was shown to be required for LMP2A-mediated Akt-activation (Scholle et al, 2000;Swart et al, 2000;Morrison and Raab-Traub, 2005). One such regulatory protein is the signalling scaffold Shb, which is recruited to the ITAM SH2-binding motifs of the CD3z upon T-cell receptor (TCR)-stimulation (Welsh et al, 1998).…”