We hypothesized that key antiproliferative target genes for the vitamin D receptor (VDR) were repressed by an epigenetic mechanism in prostate cancer cells resulting in apparent hormonal insensitivity. To explore this possibility, we examined nuclear receptor corepressor expression in a panel of nonmalignant and malignant cell lines and primary cultures, and found frequently elevated SMRT corepressor mRNA expression often associated with reduced sensitivity to 1a,25-dihydroxyvitamin D 3 (1a,25(OH) 2 D 3 ). For example, PC-3 and DU-145 prostate cancer cell lines had 1.8-fold and twofold increases in SMRT mRNA relative to normal PrEC cells (Po0.05). Similarly, 10/15 primary tumour cultures (including three matched to normal cells from the same donors) had elevated SMRT mRNA levels; generally NCoR1 and Alien were not as commonly elevated. Corepressor proteins often have associated histone deacetylases (HDAC) and reflectively the antiproliferative action of 1a,25(OH) 2 D 3 can be 'restored' by cotreatment with low doses of HDAC inhibitors such as trichostatin A (TSA, 15 nM) to induce apoptosis in prostate cancer cell lines. To decipher the transcriptional events that lead to these cellular responses, we undertook gene expression studies in PC-3 cells after cotreatment of 1a,25(OH) 2 D 3 plus TSA after 6 h. Examination of known VDR target genes and cDNA microarray analyses revealed cotreatment of 1a,25(OH) 2 D 3 plus TSA cooperatively upregulated eight (outof1176)genes,includingMAPK-APK2andGADD45a. MRNA and protein time courses and inhibitor studies confirmed these patterns of regulation. Subsequently, we knocked down SMRT levels in PC-3 cells using a small interfering RNA (siRNA) approach and found that GADD45a induction by 1a,25(OH) 2 D 3 alone became very significantly enhanced. The same distortion of gene responsiveness, with repressed induction of GADD45a was found in primary tumour cultures compared and to matched peripheral zone (normal) cultures from the same donor. These data demonstrate that elevated SMRT levels are common in prostate cancer cells, resulting in suppression of target genes associated with antiproliferative action and apparent 1a,25(OH) 2 D 3 -insensitivity. This can be targeted therapeutically by combination treatments with HDAC inhibitors.

The Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all virus-associated tumors where it plays an essential role in the maintenance, replication and transcription of the EBV genome. Transcriptional profiling of EBNA1-expressing carcinoma cells demonstrated that EBNA1 also influences the expression of a range of cellular genes including those involved in translation, transcription and cell signaling. Of particular interest was the ability of EBNA1 to enhance expression of STAT1 and sensitize cells to interferon-induced STAT1 activation with resultant enhancement of major histocompatibility complex expression. A negative effect of EBNA1 on the expression of TGFbeta1-responsive betaig-h3 and PAI-1 genes was confirmed at the protein level in EBV-infected carcinoma cells. This effect resulted from the ability of EBNA1 to repress TGFbeta1-induced transcription via a reduction in the interaction of SMAD2 with SMAD4. More detailed analysis revealed that EBNA1 induces a lower steady-state level of SMAD2 protein as a consequence of increased protein turnover. These data show that EBNA1 can influence cellular gene transcription resulting in effects that may contribute to the development of EBV-associated tumors.

The Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all virus-associated tumours, including nasopharyngeal carcinoma (NPC), where it plays an essential role in EBV genome maintenance, replication and transcription. Previous studies suggest that EBNA1 may have additional effects relevant to oncogenesis, including enhancement of cell survival, raising the possibility that EBNA1 may influence cellular gene expression. We have recently demonstrated by gene expression microarray profiling in an NPC cell model that EBNA1 influences the expression of a range of cellular genes, including those involved in transcription, translation and cell signalling. Here, we report for the first time that EBNA1 enhances activity of the AP-1 transcription factor in NPC cells and demonstrate that this is achieved by EBNA1 binding to the promoters of c-Jun and ATF2, enhancing their expression. In addition, we demonstrate elevated expression of the AP-1 targets interleukin 8, vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1a in response to EBNA1 expression, which enhances microtubule formation in an in vitro angiogenesis assay. Furthermore, we confirm elevation of VEGF and the phosphorylated isoforms of c-Jun and ATF2 in NPC biopsies. These findings implicate EBNA1 in the angiogenic process and suggest that this viral protein might directly contribute to the development and aggressively metastatic nature of NPC. INTRODUCTIONEpstein-Barr virus (EBV) is a ubiquitous human gammaherpesvirus associated with both lymphoid and epithelial tumours (Kieff & Rickinson, 2001). The pattern of EBV latent protein expression differs in these tumours, with the EBV nuclear antigen EBNA1 alone being expressed in Burkitt's lymphoma (BL) while EBNA1 and two membrane proteins (LMP1 and LMP2A/B) are expressed in Hodgkin's lymphoma (HL) and nasopharyngeal carcinoma (NPC) (Kieff & Rickinson, 2001;Young & Rickinson, 2004; RaabTraub, 2002). The consistent expression of EBNA1 in all EBV-related malignancies is a result of the indispensable role that EBNA1 plays in maintenance and replication of the EBV genome via sequence-specific binding to the viral origin of replication, oriP (Raab-Traub, 2002). In addition to the role that EBNA1 plays in viral genome maintenance, it also interacts with viral gene promoters, thereby contributing to the transcriptional regulation of the EBNAs and of LMP1. Recent studies have also demonstrated that EBNA1 can interact with the ubiquitin-specific protease USP7, which has been implicated in the stabilization of p53. Holowaty & Frappier (2004) showed that EBNA1 can bind with a higher affinity to the same region of USP7 as p53 and MDM2 and suggest that, as a consequence, EBNA1 can protect against either UV-or p53-induced apoptosis. A more direct involvement of EBNA1 in carcinogenesis has been suggested by the ability of B-cell-directed EBNA1 expression to produce B-cell lymphomas in transgenic mice (Wilson et al., 1996). However, dominant-negative EBNA1 (dnEBNA1) studies in a lymphoblastoid cell l...