Epstein-Barr virus (EBV) associated Burkitt’s lymphoma is characterised by the deregulation of c-Myc expression and a restricted viral gene expression pattern in which the EBV nuclear antigen-1 (EBNA1) is the only viral protein to be consistently expressed. EBNA1 is required for viral genome propagation and segregation during latency. However, it has been much debated whether the protein plays a role in viral-associated tumourigenesis. We show that the lymphomas which arise in EµEBNA1 transgenic mice are unequivocally linked to EBNA1 expression and that both C-Myc and Mdm2 deregulation are central to this process. Tumour cell survival is supported by IL-2 and there is a skew towards CD8 positive T-cells in the tumour environment, while the immune check-point protein PD-L1 is upregulated in the tumours. Additionally, several isoforms of Mdm2 are upregulated in the EµEBNA1 tumours, with increased phosphorylation at ser166, an expression pattern not seen in Eµc-Myc transgenic tumours. Concomitantly, E2F1, Xiap, Mta1, C-Fos and Stat1 are upregulated in the tumours. Using four independent inhibitors of Mdm2 we demonstrate that the EµEBNA1 tumour cells are dependant upon Mdm2 for survival (as they are upon c-Myc) and that Mdm2 inhibition is not accompanied by upregulation of p53, instead cell death is linked to loss of E2F1 expression, providing new insight into the underlying tumourigenic mechanism. This opens a new path to combat EBV-associated disease.

The lymphomagenic action of myc genes in conjunction with Epstein-Barr virus nuclear antigen-1 (EBNA-1) have been examined using transgenic mice in several separate tests. Synergy between Myc and EBNA-1 in lymphomagenesis was revealed in a cross breed study where co-expression of transgenic myc and EBNA-1 led to a tumor latency period reduced significantly in some crosses. In the resulting bitransgenic tumors, expression of the E-myc genes was not af- The virus was discovered in cell cultures from endemic Burkitt's lymphoma (BL) samples 2 and has since been found to have a very high association (Ͼ95%) with this disease. The more rare, sporadic form of BL (occurring worldwide) shows 15-25% association with EBV. Similarly, only a proportion (30 -40%) of the AIDS form of BL is positive for EBV genomes. Nevertheless, in all EBV-associated cases, the viral episomal genome is thought to be clonal, indicating that the virus was present in the tumor progenitor cell. This would argue that the probability of evolving into a tumor is higher for an EBV carrying than an EBV negative B-lymphocyte suggesting that the virus is causal in the genesis of the tumor. 3 Notably, all BL tumors carry a reciprocal chromosomal translocation between chromosome 8, in the proximity of the c-myc gene and one of chromosomes 14, 2, or 22 at the immunoglobulin heavy (IgH) or light (IgL) chain loci. This leads to deregulation of c-myc expression. 4 -6 Several transgenic mouse models to examine myc induced tumorigenesis have been developed. Overexpression of c-myc or N-myc in B cells using the IgH (E) intronic enhancer predisposes transgenic mice to B cell lymphoma. [7][8][9][10][11][12][13] The exact pathology of the c-myc induced tumors and the timing of onset varies according to the precise transgene construct used. Using an IgL locus enhancer instead to drive c-myc overexpression slightly later in B cell development results in a transgenic lymphoma with a pathology startlingly similar to BL. 14 In EBV-associated BL tumors a restricted subset of the viral latent genes is expressed. Of 6 EBV nuclear antigens (EBNA), EBNA-1 is the only one that has been detected consistently. 15,16 In addition the 2 small, polymerase III, untranslated RNAs (EBER-1 and -2) are expressed. LMP2A transcripts and the BamHI-A rightward transcripts have also been detected in BL biopsies. 17 EBV is a potent B cell transforming virus, nevertheless the role it plays in the genesis of BL remains unclear. The theory that EBV continues to contribute to the malignant phenotype of established tumor cells is suggested by the stable retention of the viral genome in cultured BL cell lines and by the loss of tumorigenicity of sub clones of BL cell lines that have lost the viral genome. 18,19 In this manner, the EBERs have been shown to contribute to the transformed phenotype in Akata cells, 20 and EBER expression can substitute for the presence of viral genomes in EBV positive Akata and Mutu I cell lines. This was not the case, however, for all the BL cell lines examined, suggest...

An important role for B cells and immunoglobulin deposition in the inflammatory tumor cell environment has been recognized in several cancers, and this is recapitulated in our murine model of inflammation-associated carcinogenesis: transgenic mice expressing the Epstein-Barr virus oncogene LMP1 in epithelia. Similarly in several autoimmune disorders, immunoglobulin deposition represents a key underlying event in the disease process. However, the autoantigens in most cases are not known. In other studies, overexpression of the enzymatically inactive mammalian chitinase-like proteins (CLPs) has been observed in a number of autoimmune disorders and numerous cancers, with expression correlated with poor prognosis, although the function of these proteins is largely unknown. We have now linked these observations demonstrating that overexpression of the CLPs renders them the targets for autoantigenicity during carcinogenic progression. We show that the CLPs, Chi3L1, Chi3L3 /YM1, and Chi3L4/YM2, are abundantly overexpressed in the transgenic epidermis at an early, preneoplastic stage and secreted into the serum. Immunoglobulin G reactive to the CLPs is detected in the serum and deposited in the hyperplastic tissue, which goes on to become inflamed and progressively displastic. The CLPs are also upregulated in chemical carcinogen-promoted lesions in both transgenic and wild-type mice. Expression of the related, active chitinases, Chit1 and AMCase, increases following infiltration of inflammatory cells. In this model, the 3 CLPs are autoantigens for the tissue-deposited immunoglobulin, which we propose plays a causative role in promoting the inflammation-associated carcinogenesis. This may reflect their normal, benign function to promote tissue remodeling and to amplify immune responses. Their induction during carcinogenesis and consequent autoantigenicity provides a missing link between the oncogenic event and subsequent inflammation. This study identifies the CLPs as important and novel therapeutic targets to limit inflammation in cancer and potentially also autoimmune disorders.

Nasopharyngeal carcinoma (NPC) is the most tightly Epstein-Barr virus (EBV)-associated tumour. The EBV oncoprotein latent membrane protein 1 (LMP1) is frequently expressed in NPC tumours and may play a role in the genesis of the disease. NPC tumours often exhibit loss of expression (by deletion or methylation) of the INK4a locus, which encodes the tumour suppressor genes p16INK4a and p14ARF. To investigate the contribution of LMP1 and INK4a loss to tumourigenesis, skin chemical carcinogenesis was conducted using PyLMP1 and INK4a null mice. Surprisingly, INK4a null mice developed significantly fewer papillomas than wild-type mice, nevertheless, the papillomas that did develop grew faster and converted more rapidly to carcinoma than controls. This indicates that while loss of the INK4a locus plays an important role in the later stages of tumourigenesis, initially its loss inhibits papilloma formation. Conversely, LMP1 promoted papilloma formation but paradoxically inhibited papilloma growth. Using cross-breeds, it was found that LMP1 cooperates with loss of the INK4a locus during epithelial tumourigenesis. The expression of LMP1 overcame the inhibition of papilloma formation observed in INK4a null mice, whilst the loss of the INK4a locus counteracted the inhibition of papilloma growth rate found in PyLMP1 mice. This suggests that LMP1 mediates the inhibition of papilloma growth via one or both of the INK4a locus products. Intriguingly, mice heterozygous for INK4a loss showed lesion growth rates intermediate between wild-type and null, demonstrative of haploinsufficiency. We propose that LMP1 acts at the early stages in carcinogenesis to promote the development of benign tumours and that early reduction of INK4a locus expression allows these lesions to expand in size. In addition, loss of the INK4a locus accelerates the development of a more aggressive lesion. Conversely, complete loss of the INK4a locus in an otherwise normal cell might inhibit lesion formation.