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In both humans with long-standing ulcerative colitis and mouse models of colitis-associated carcinogenesis (CAC), tumors develop predominantly in the distal part of the large intestine but the biological basis of this intriguing pathology remains unknown. Herein we report intrinsic differences in gene expression between proximal and distal colon in the mouse, which are augmented during dextran sodium sulfate (DSS)/azoxymethane (AOM)-induced CAC. Functional enrichment of differentially expressed genes identified discrete biological pathways operating in proximal vs distal intestine and revealed a cluster of genes involved in lipid metabolism to be associated with the disease-resistant proximal colon. Guided by this finding, we have further interrogated the expression and function of one of these genes, apolipoprotein A-I (ApoA-I), a major component of high-density lipoprotein. We show that ApoA-I is expressed at higher levels in the proximal compared with the distal part of the colon and its ablation in mice results in exaggerated DSS-induced colitis and disruption of epithelial architecture in larger areas of the large intestine. Conversely, treatment with an ApoA-I mimetic peptide ameliorated the phenotypic, histopathological and inflammatory manifestations of the disease. Genetic interference with ApoA-I levels in vivo impacted on the number, size and distribution of AOM/DSS-induced colon tumors. Mechanistically, ApoA-I was found to modulate signal transducer and activator of transcription 3 (STAT3) and nuclear factor-κB activation in response to the bacterial product lipopolysaccharide with concomitant impairment in the production of the pathogenic cytokine interleukin-6. Collectively, these data demonstrate a novel protective role for ApoA-I in colitis and CAC and unravel an unprecedented link between lipid metabolic processes and intestinal pathologies.

Nasopharyngeal carcinoma is closely associated with Epstein-Barr virus (EBV) infection. The EBV-encoded LMP1 has cell transformation property. It suppresses cellular senescence and enhances cell survival in various cell types. Many of the downstream events of LMP1 expression are mediated through its ability to activate NF-kappaB. In this study, we report a novel function of LMP1 to induce Id1 expression in nasopharyngeal epithelial cells (NP69) and human embryonal kidney cells (HEK293). The Id1 is a basic helix-loop-helix (bHLH) protein and a negative transcriptional regulator of p16(INK4a). Expression of Id1 facilitates cellular immortalization and stimulates cell proliferation. With the combination of both specific chemical inhibitors and genetic inhibitors of cell signaling, we showed that induction of Id1 by LMP1 was dependent on its NF-kappaB activation domain at the carboxy-terminal region, CTAR1 and CTAR2. Induction of Id1 by LMP1 may facilitate clonal expansion of premalignant nasopharyngeal epithelial cells infected with EBV and may promote their malignant transformation.

Expression of the oncogenic Epstein-Barr virus (EBV)-encoded Latent Membrane Protein 1 (LMP1) activates signalling on the NF-kappaB axis through two distinct domains in the cytoplasmic C-terminus of the protein, namely CTAR1 (aa 187-231) and CTAR2 (aa 351-386). Whilst this effect is responsible for some of the functional consequences of LMP1 expression, additional LMP1-mediated signalling pathways may exist which contribute to the pleiotropic activities of this protein. In this study we provide evidence of a kinase cascade being activated by LMP1. Thus, we demonstrate that stable or transient expression of the LMP1 prototype from B95.8 in cells of epithelial or B cell origin activates the c-Jun N-terminal kinase (JNK, also known as the stress-activated protein kinase, SAPK) pathway, an effect which was found to be mediated through CTAR2 but not CTAR1. LMP1 from the Cao viral strain or LMP1 homologues from the simian EBV naturally infecting baboons and rhesus monkeys were also able to activate JNK. This phenomenon translates to induction of AP-1, a transcription factor which is readily activated by growth factors and mitogens. Interestingly, an LMP1/ CD40 chimaera comprising of the N-terminus and transmembrane domain of LMP1 and the cytoplasmic tail of CD40 which shares a common TRAF binding motif with CTAR1, effectively induced JNK. As NF-kappaB and JNK are co-activated in LMP1-expressing cells, we investigated whether the two pathways are overlapping or independent. We have found that inhibition of NF-kappaB by metabolic inhibitors or a constitutively active mutated IkappaBalpha does not impair the ability of LMP1 to signal on the JNK axis. Conversely, whilst a dominant negative mutated SEK (JNKK) inhibited LMP1-induced JNK activation, it did not affect NF-kappa-B suggesting that these two LMP1-mediated pathways are divergent.

Expression of the Epstein-Barr virus (EBV) transforming LMP1 in B cells activates the transcription factor NF-kappaB and induces phenotypic changes through two distinct domains in the cytoplasmic C-terminus of the protein. The aa 187-231 domain of LMP1, which is important for growth transformation, binds tumour necrosis factor (TNF) receptor associated factor (TRAF) 1 and TRAF3 and this interaction mediates subsequent signalling events. The TRAFs also associate with CD40, a member of the TNFR family, which upon ligation activates NF-kappaB and induces phenotypic changes similar to those mediated by LMP1. This study demonstrates that LMP1 expression in carcinoma cell lines and SV40-transformed keratinocytes results in induction of the pleiotropic cytokine interleukin 6 (IL6), an effect which is also observed upon CD40 ligation. The mechanism by which either LMP1 expression or CD40 ligation induces IL6 production was found to be NF-kappaB-dependent. Mutational analysis identified domains in the C-terminus of LMP1 which are important for NF-kappaB activation and IL6 secretion. LMP1 and CD40 share a common PxQxT core TRAF binding motif and mutations in or adjacent to this sequence impaired the ability of LMP1 or CD40 to induce NF-kappaB activation and IL6 secretion. The importance of TRAF interactions in mediating these effects was confirmed using dominant negative TRAF2 and TRAF3 mutants which also identified differences in the signalling events mediated by the two NF-kappaB activating domains of LMP1. A20, an anti-apoptotic protein which interacts with TRAF2 and blocks CD40-mediated NF-kappaB activity, also blocked NF-kappaB and IL6 secretion in LMP1-transfected epithelial cells. These results suggest that LMP1 regulates IL6 production in epithelial cells in a manner similar to CD40 ligation and implicate TRAFs as common mediators in the transduction of signals generated via the CD40 and LMP1 pathways. As a role for IL6 in regulating epithelial cell growth has previously been suggested, the control of IL6 secretion via the CD40 and LMP1 pathways may have implications for the growth of both normal and transformed epithelial cells.

CD40, a tumour necrosis factor (TNF) receptor family member, is expressed in a variety of cell types, including B lymphocytes, macrophages, fibroblasts, endothelial and epithelial cells, and this widespread expression is likely to account for its central role in normal physiology and disease pathogenesis. In this study, we provide evidence to support a role for constitutive CD40 signalling in cell transformation. We show that the ligand for CD40 (CD40L/CD154) is expressed in CD40-positive human breast tumour biopsies, suggesting that the constitutive activation of the CD40 receptor in vivo may contribute to the oncogenic process. Coexpression of CD40 and CD40L confers oncogenic effects on immortalized human epithelial cells in vitro, increasing their proliferation, motility and invasion. Expression of LMP:CD40, a hybrid molecule comprising the N-terminus and transmembrane domains of the Epstein-Barr virus-encoded latent membrane protein-1 (LMP1) fused to the cytoplasmic tail of CD40, mimics a constitutively active CD40 receptor and promotes the transformation of immortalized rodent fibroblasts in vitro and their oncogenicity in vivo. The observed effects of aberrant CD40 activation on cell transformation are largely diminished upon suppression of the oncogenic NF-kappaB signalling pathway. Taken together, our results suggest a role for the constitutive engagement of the CD40L/CD40/NF-kappaB activation pathway in cell transformation and neoplastic growth. Strategies that neutralize this pathway may therefore be useful in cancer treatment and prevention.

The oncogenic Epstein-Barr virus (EBV)-encoded latent infection membrane protein 1 (LMP1) constitutively activates the 'canonical' NF-kappaB pathway that involves the phosphorylation and degradation of IkappaBalpha downstream of the IkappaB kinases (IKKs). In this study, we show that LMP1 also promotes the proteasome-mediated proteolysis of p100 NF-kappaB2 resulting in the generation of active p52, which translocates to the nucleus in complex with the p65 and RelB NF-kappaB subunits. LMP1-induced NF-kappaB transactivation is reduced in nf-kb2(-/-) mouse embryo fibroblasts, suggesting that p100 processing contributes to LMP1-mediated NF-kappaB transcriptional effects. This pathway is likely to operate in vivo, as the expression of LMP1 in primary EBV-positive Hodgkin's lymphoma and nasopharyngeal carcinoma biopsies correlates with the nuclear accumulation of p52. Interestingly, while the ability of LMP1 to activate the canonical NF-kappaB pathway is impaired in cells lacking IKKgamma/NEMO, the regulatory subunit of the IKK complex, p100 processing remains unaffected. As a result, nuclear translocation of p52, but not p65, occurs in the absence of IKKgamma. These data point to the existence of a novel signalling pathway that regulates NF-kappaB in LMP1-expressing cells, and may thereby play a role in both oncogenic transformation and the establishment of persistent EBV infection.

The Epstein-Barr virus (EBV) encoded Latent Membrane Protein-1 (LMP1) mimics a constitutively active receptor molecule, and has been shown to activate NF-kappaB and the MAPK and JNK pathways. Two regions within the cytosolic domain of LMP1 have been found to effect cell signalling. One of these, the carboxy-terminal activation region-1 (CTAR1), binds members of the TRAF family of proteins, and the other (CTAR2) binds TRADD, suggesting that LMP1 transduces signals similarly to the Tumour Necrosis Factor Receptor family of receptors. The ability to bind TRAFs, to activate NF-kappaB and the JNK pathway, to upregulate cellular genes such as CD54 (ICAM-1 adhesion molecule), and to affect cell growth and apoptosis has led to the suggestion that LMP1 signalling is similar to, or even identical to CD40. However, we now show that while ligand-induced CD40 signalling is impaired in the Jurkat T cell line, LMP1 was fully functional; therefore demonstrating that LMP1 and CD40 signalling differ. Mutated LMP1 genes, in which one or other of the CTAR1 and CTAR2 domains was non-functional, behaved more like CD40 in being unable to upregulate the CD54 cell surface marker in Jurkat cells. However, the CTAR1 domain of LMP1, which shared a TRAF-binding sequence motif with CD40, differed from CD40 in being unable to activate NF-kappaB in Jurkat. Cotransfection experiments with LMP1 mutants demonstrated that CTAR1 can cooperative with CTAR2 on separate LMP1 molecules, provided that they exist within the same oligomeric complex.