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Background Interferon (IFN) signalling pathways, a key element of the innate immune response, contribute to resistance to conventional chemotherapy, radiotherapy, and immunotherapy, and are often deregulated in cancer. The deubiquitylating enzyme USP18 is a major negative regulator of the IFN signalling cascade and is the predominant human protease that cleaves ISG15, a ubiquitin-like protein tightly regulated in the context of innate immunity, from its modified substrate proteins in vivo. Methods In this study, using advanced proteomic techniques, we have significantly expanded the USP18-dependent ISGylome and proteome in a chronic myeloid leukaemia (CML)-derived cell line. USP18-dependent effects were explored further in CML and colorectal carcinoma cellular models. Results Novel ISGylation targets were characterised that modulate the sensing of innate ligands, antigen presentation and secretion of cytokines. Consequently, CML USP18-deficient cells are more antigenic, driving increased activation of cytotoxic T lymphocytes (CTLs) and are more susceptible to irradiation. Conclusions Our results provide strong evidence for USP18 in regulating antigenicity and radiosensitivity, highlighting its potential as a cancer target.

The deubiquitylating enzyme USP18 is a major negative regulator of the interferon (IFN) signalling cascade. IFN pathways contribute to resistance to conventional chemotherapy, radiotherapy, and immunotherapy and are often deregulated in cancer. USP18 is the predominant human protease that cleaves interferon-stimulated gene ISG15, a ubiquitin-like protein tightly regulated in the context of innate immunity, from its modified substrate proteins in vivo. In this study, using advanced proteomic techniques, we have expanded the USP18-dependent ISGylome and proteome in a chronic myeloid leukaemia (CML)-derived cell line (HAP1) treated with type I IFN. Novel ISGylation targets were characterised that modulate the sensing of innate ligands, antigen presentation and secretion of cytokines. Consequently, CML USP18-deficient cells are more antigenic, driving increased activation of cytotoxic T lymphocytes (CTLs) and are more susceptible to irradiation. Our results suggest USP18 as a pharmacological target in cancer immunotherapy and radiotherapy.

The cyclin-dependent kinase (CDK) inhibitors, p18INK4c and p16INK4a, both have the credentials of tumor suppressors in human cancers and mouse models. For p16INK4a, the underlying rationale is its role in senescence, but the selective force for inactivation of p18INK4c in incipient cancer cells is less clear. Here, we show that in human fibroblasts undergoing replicative or oncogene-induced senescence, there is a marked decline in the levels of p18INK4c protein and RNA, which mirrors the accumulation of p16INK4a. Downregulation of INK4c is not dependent on p16INK4a, and RAS can promote the loss of INK4c without cell-cycle arrest. Downregulation of p18INK4c correlates with reduced expression of menin and E2F1 but is unaffected by acute cell-cycle arrest or inactivation of the retinoblastoma protein (pRb). Collectively, our data question the idea that p18INK4c acts as a backup for loss of p16INK4a and suggest that the apparent activation of p18INK4c in some settings represents delayed senescence rather than increased expression. We propose that the contrasting behavior of the two very similar INK4 proteins could reflect their respective roles in senescence versus differentiation. Cancer Res; 72(1); 165–75. ©2011 AACR.

Both the DNA damage response (DDR) and epigenetic mechanisms play key roles in the implementation of senescent phenotypes, but very little is known about how these two mechanisms are integrated to establish senescence-associated gene expression. Here we show that, in senescent cells, the DDR induces proteasomal degradation of G9a and GLP, major histone H3K9 mono- and dimethyltransferases, through Cdc14B- and p21(Waf1/Cip1)-dependent activation of APC/C(Cdh1) ubiquitin ligase, thereby causing a global decrease in H3K9 dimethylation, an epigenetic mark for euchromatic gene silencing. Interestingly, induction of IL-6 and IL-8, major players of the senescence-associated secretory phenotype (SASP), correlated with a decline of H3K9 dimethylation around the respective gene promoters and knockdown of Cdh1 abolished IL-6/IL-8 expression in senescent cells, suggesting that the APC/C(Cdh1)-G9a/GLP axis plays crucial roles in aspects of senescent phenotype. These findings establish a role for APC/C(Cdh1) and reveal how the DDR integrates with epigenetic processes to induce senescence-associated gene expression.

Viral nuclear oncoproteins EBNA3A and EBNA3C are essential for the efficient immortalization of B cells by Epstein–Barr virus (EBV) in vitro and it is assumed that they play an essential role in viral persistence in the human host. In order to identify cellular genes regulated by EBNA3A expression, cDNA encoding EBNA3A was incorporated into a recombinant adenoviral vector. Microarray analysis of human diploid fibroblasts infected with either adenovirus EBNA3A or an empty control adenovirus consistently showed an EBNA3A-specific induction of mRNA corresponding to the chaperones Hsp70 and Hsp70B/B′ and co-chaperones Bag3 and DNAJA1/Hsp40. Analysis of infected fibroblasts by real-time quantitative RT-PCR and Western blotting confirmed that EBNA3A, but not EBNA3C, induced expression of Hsp70, Hsp70B/B′, Bag3 and DNAJA1/Hsp40. This was also confirmed in a stable, inducible expression system. EBNA3A activated transcription from the Hsp70B promoter, but not multimerized heat-shock elements in transient transfection assays, consistent with specific chaperone and co-chaperone upregulation. Co-immunoprecipitation experiments suggest that EBNA3A can form a complex with the chaperone/co-chaperone proteins in both adenovirus-infected cells and EBV-immortalized lymphoblastoid cell lines. Consistent with this, induction of EBNA3A resulted in redistribution of Hsp70 from the cytoplasm to the nucleus. EBNA3A therefore specifically induces (and then interacts with) all of the factors necessary for an active Hsp70 chaperone complex.

Epstein-Barr virus (EBV) contributes to the development of several human cancers including the endemic form of Burkitt's lymphoma (BL). In culture, EBV induces the continuous proliferation of primary B cells as lymphoblastoid cell lines (LCLs) and if EBV-negative BL-derived cells are infected with EBV, latency-associated viral factors confer resistance to various inducers of apoptosis. Nuclear proteins EBNA3A and EBNA3C (but not EBNA3B) are necessary to establish LCLs and their expression may be involved in the resistance of BL cells to cytotoxic agents. We have therefore created recombinant EBVs from which each of the EBNA3 genes has been independently deleted, and revertant viruses in which the genes have been re-introduced into the viral genome. Infection of EBV-negative BL cells with this panel of EBVs and challenge with various cytotoxic drugs showed that EBNA3A and EBNA3C cooperate as the main determinants of both drug resistance and the downregulation of the proapoptotic Bcl-2-family member Bcl-2-interacting mediator of cell death (Bim). The regulation of Bim is predominantly at the level of RNA, with little evidence of post-translational Bim stabilization by EBV. In the absence of Bim, EBNA3A and EBNA3C appear to provide no survival advantage. The level of Bim is a critical regulator of B cell survival and reduced expression is a major determinant of lymphoproliferative disease in mice and humans; moreover, Bim is uniquely important in the pathogenesis of BL. By targeting this tumour-suppressor for repression, EBV significantly increases the likelihood of B lymphomagenesis in general, and BL in particular. Our results may also explain the selection pressure that gives rise to a subset of BL that retain expression of the EBNA3 proteins.

The INK4a/ARF tumor suppressor locus, a key executor of cellular senescence, is regulated by members of the Polycomb group (PcG) of transcriptional repressors. Here we show that signaling from oncogenic RAS overrides PcG-mediated repression of INK4a by activating the H3K27 demethylase JMJD3 and down-regulating the methyltransferase EZH2. In human fibroblasts, JMJD3 activates INK4a, but not ARF, and causes p16INK4a-dependent arrest. In mouse embryo fibroblasts, Jmjd3 activates both Ink4a and Arf and elicits a p53-dependent arrest, echoing the effects of RAS in this system. Our findings directly implicate JMJD3 in the regulation of INK4a/ARF during oncogene-induced senescence and suggest that JMJD3 has the capacity to act as a tumor suppressor.

Epstein-Barr virus (EBV) is associated with a number of human cancers, and latent EBV gene expression has been reported to interfere with cell cycle checkpoints and cell death pathways. Here we show that latent EBV can compromise the mitotic spindle assembly checkpoint and rescue Burkitt's lymphoma (BL)-derived cells from caspase-dependent cell death initiated in aberrant mitosis. This leads to unscheduled mitotic progression, resulting in polyploidy and multi- and/or micronucleation. The EBV latent genes responsible for this phenotype are expressed from the P3HR1 strain of virus and several viruses with similar genomic deletions that remove the EBNA2 gene. Although EBNA2 and the latent membrane proteins are not expressed, the EBNA3 proteins are present in these BL cells. Survival of the EBV-positive cells is not consistently associated with EBV lytic gene expression or with the genes that are expressed in EBV latency I BL cells (i.e., EBNA1, EBERs, and BARTs) but correlates with reduced expression of the cellular proapoptotic BH3-only protein Bim. These data suggest that a subset of latent EBV gene products may increase the likelihood of damaged DNA being inherited because of the impaired checkpoint and enhanced survival capacity. This could lead to greater genetic diversity in progeny cells and contribute to tumorigenesis. Furthermore, since it appears that this restricted latent EBV expression interferes with the responses of Burkitt's lymphoma-derived cells to cytotoxic drugs, the results of this study may have important therapeutic implications in the treatment of some BL.

NSP4-encoding genes of 78 human rotavirus strains of common or reassortant genotypes were characterized by reverse transcription-PCR followed by sequencing and phylogenetic analysis. It was found that all the human strains characterized clustered into only two of the five known NSP4 genotypes. Linkage between NSP4 genotypes and VP6 subgroups was 100%, NSP4 genotype A being linked to VP6 of subgroup I (SGI) and NSP4 of genotype B being linked to VP6 of SGII. The diversity among the NSP4- and VP6-encoding genes was significantly less than that among the VP7 and VP4 genes in cocirculating human rotavirus strains. Whereas G and P types appear to be shared among different animal species and humans, the NSP4- and VP6-encoding genes appear to segregate according to their host of origin, suggesting that these two proteins may be host restriction determinants. The NSP4-VP6 association may be structurally determined during rotavirus replication (morphogenesis).

Background: Polycomb group proteins form multicomponent complexes that are important for establishing lineage-specific patterns of gene expression. Mammalian cells encode multiple permutations of the prototypic Polycomb repressive complex 1 (PRC1) with little evidence for functional specialization. An aim of this study is to determine whether the multiple orthologs that are co-expressed in human fibroblasts act on different target genes and whether their genomic location changes during cellular senescence. Results: Deep sequencing of chromatin immunoprecipitated with antibodies against CBX6, CBX7, CBX8, RING1 and RING2 reveals that the orthologs co-localize at multiple sites. PCR-based validation at representative loci suggests that a further six PRC1 proteins have similar binding patterns. Importantly, sequential chromatin immunoprecipitation with antibodies against different orthologs implies that multiple variants of PRC1 associate with the same DNA. At many loci, the binding profiles have a distinctive architecture that is preserved in two different types of fibroblast. Conversely, there are several hundred loci at which PRC1 binding is cell type-specific and, contrary to expectations, the presence of PRC1 does not necessarily equate with transcriptional silencing. Interestingly, the PRC1 binding profiles are preserved in senescent cells despite changes in gene expression. Conclusions: The multiple permutations of PRC1 in human fibroblasts congregate at common rather than specific sites in the genome and with overlapping but distinctive binding profiles in different fibroblasts. The data imply that the effects of PRC1 complexes on gene expression are more subtle than simply repressing the loci at which they bind.

Latent Epstein-Barr virus (EBV) has been shown to protect Burkitt's lymphoma-derived B cells from apoptosis induced by agents that cause damage to DNA, in the context of mutant p53. This protection requires expression of the latency-associated nuclear proteins EBNA3A and EBNA3C and correlates with their ability to cooperate in the repression of the gene encoding the pro-apoptotic, BH3-only protein BIM. Here we confirm that latent EBV in B cells also inhibits apoptosis induced by two other agents – ionomycin and staurosporine – and show that these act by a distinct pathway that involves a p53-independent increase in expression of another pro-apoptotic, BH3-only protein, NOXA. Analyses employing a variety of B cells infected with naturally occurring EBV or B95.8 EBV-BAC recombinant mutants indicated that the block to NOXA induction does not depend on the well-characterized viral latency-associated genes (EBNAs 1, 2, 3A, 3B, 3C, the LMPs or the EBERs) or expression of BIM. Regulation of NOXA was shown to be at least partly at the level of mRNA and the requirement for NOXA to induce cell death in this context was demonstrated by NOXA-specific shRNA-mediated depletion experiments. Although recombinant EBV with a deletion removing the BHRF1 locus – that encodes the BCL2-homologue BHRF1 and three microRNAs – partially abrogates protection against ionomycin and staurosporine, the deletion has no effect on the EBV-mediated block to NOXA accumulation.