Proteasomal targeting of a viral oncogene abrogates oncogenic phenotype and enhances immunogenicity

Department of Molecular and Cellular Pathology, University of Queensland, Brisbane, Queensland, Australia
Blood (Impact Factor: 10.45). 01/2004; 102(13):4535-40. DOI: 10.1182/blood-2003-03-0870
Source: PubMed


The ability of viral or mutated cellular oncogenes to initiate neoplastic events and their poor immunogenicity have considerably undermined their potential use as immunotherapeutic tools for the treatment of human cancers. Using an Epstein-Barr virus-encoded oncogene, latent membrane protein 1 (LMP1), as a model, we report a novel strategy that both deactivates cellular signaling pathways associated with the oncogenic phenotype and reverses poor immunogenicity. We show that cotranslational ubiquitination combined with N-end rule targeting of LMP1 enhanced the intracellular degradation of LMP1 and total blockade of LMP1-mediated nuclear factor-kappaB (NF-kappaB) and signal transducer and activator of transcription (STAT) activation in human cells. In addition, although murine cells expressing LMP1 were uniformly tumorigenic, this oncogenicity was completely abrogated by covalent linkage of LMP1 with ubiquitin, while an enhanced CD8+ T cell response to a model epitope fused to the C-terminus of LMP1 was observed following immunization with ubiquitinated LMP1. These observations suggest that proteasomal targeting of tumor-associated oncogenes could be exploited therapeutically by either gene therapy or vaccination.

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Available from: Jaikumar Duraiswamy
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    • "Stable in frame fusion of Ub to the N-terminus of proteins has been shown to augment their proteasomal degradation and thus enhances their MHC-I antigen presentation [4], [5], [6], [7], [8], [9], [10]. The associated proteolytic pathway has been termed Ub fusion degradation pathway (UFD; [5], [6]). "
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    ABSTRACT: N-terminal stable in frame fusion of ubiquitin (Ub) has been shown to target the fusion protein for proteasomal degradation. This pathway, called the Ub fusion degradation (UFD), might also elevate MHC class I (MHC-I) antigen presentation of specific antigens. The UFD, mainly studied on cytosolic proteins, has been described to be mediated by polyubiquitination of specific lysine residues within the fused Ub moiety. Using the well characterized melanoma-specific antigen MelanA as a model protein, we analyzed the requirements of the UFD for ubiquitination and proteasomal degradation of a transmembrane protein. Here we show that fusion of the non-cleavable Ub(G76V) variant to the N-terminus of MelanA results in rapid proteasomal degradation via the endoplasmic reticulum-associated degradation (ERAD) pathway and, consequently, leads to an increased MHC-I antigen presentation. While lysine residues within Ub are dispensable for these effects, the presence of one single lysine residue, irrespectively of its location along the fusion protein, is sufficient to induce degradation of MelanA. These results show that the ubiquitination, ER to cytosol relocation and proteasomal degradation of a transmembrane protein can be increased by N-terminal fusion of Ub at the presence of at least one, position independent lysine residue. These findings are in contrast to the conventional wisdom concerning the UFD and indicate a new concept to target a protein into the ubiquitin-proteasome system (UPS) and thus for enhanced MHC-I antigen presentation, and might open up new possibilities in the development of tumor vaccines.
    Full-text · Article · Feb 2013 · PLoS ONE
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    • "All LMP1 expression vectors showed normal to high levels of LMP1 expression which was quite comparable to the levels seen in EBV-infected B cells. Furthermore, data presented in figure 2 clearly shows that expression vectors encoding LMP1 protein fused to GFP at the C-terminus are fully capable of activating NF-κB and STAT3 which is comparable to that seen with LMP1 protein without GFP [18]. It is interesting to note that LMP1 sequences displayed some differences with respect to their ability to activate NF-κB and STAT3, although this variation not particularly associated with any specific disease setting from which the LMP1 sequence was isolated. "
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    ABSTRACT: Previous studies have indicated that Epstein-Barr virus (EBV) can modulate the Wnt pathway in virus-infected cells and this effect is mediated by EBV-encoded oncogene latent membrane protein 1 (LMP1). Here we have reassessed the role of LMP1 in regulating the expression of various mediators of the canonical Wnt cascade. Contradicting the previous finding, we found that the levels of E-cadherin, beta-catenin, Glycogen Synthase Kinase 3ss (GSK3beta), axin and alpha-catenin were not affected by the expression of LMP1 sequences from normal B cells or nasopharyngeal carcinoma. Moreover, we also show that LMP1 expression had no detectable effect on the E-cadherin and beta-catenin interaction and did not induce transcriptional activation of beta-catenin. Taken together these studies demonstrate that EBV-mediated activation of Wnt pathway is not dependent on the expression of LMP1.
    Preview · Article · Feb 2008 · PLoS ONE
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    • "This is associated with decreased activation of NFjB and STAT in human cells and a subsequent increase in sensitivity to apoptosis of ubiquitinated-LMP1-expressing fibroblasts. There was abrogation of the oncogenic phenotype in nude mice, whilst in BALB/c mice an enhanced CD8 + T cell response to a model epitope fused to the C-terminus of LMP1 was observed following immunization with ubiquitinated LMP1 (Tellam et al, 2003). It is important to stress that a model epitope was used in this study to assess the immunogenicity of ubiquitinated LMP1, and formal validation of this approach would require further studies based on an LMP1- derived epitope. "
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    ABSTRACT: Survivors of Hodgkin's lymphoma (HL) frequently have many years to experience the long-term toxicities of combined modality therapies. Also, a significant proportion of HL patients will relapse or have refractory disease, and less than half of these patients will respond to current salvage strategies. 30-50% of HL cases are Epstein-Barr virus associated (EBV-positive HL). The virus is localized to the malignant cells and is clonal. EBV-positive HL is more frequent in childhood, in older adults (>45 years) and in mixed cellularity cases. The survival of EBV-positive HL in the elderly and the immunosuppressed is particularly poor. Despite improvements in our understanding of EBV-positive HL, the true contribution of EBV to the pathogenesis of HL remains unknown. Increased knowledge of the virus' role in the basic biology of HL may generate novel therapeutic strategies for EBV-positive HL and the presence of EBV-latent antigens in the malignant HL cells may represent a target for cellular immunotherapy.
    Preview · Article · Jun 2004 · British Journal of Haematology
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