Paul G. Murray

Epstein–Barr virus and oncogenesis: from latent genes to tumours

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus associated with the development of both lymphoid and epithelial tumours. As a common virus infection, EBV appears to have evolved to exploit the process of B cell development to persist as a life-long asymptomatic infection. However, the virus can contribute to oncogenesis as evidenced by its frequent detection in certain tumours, namely Burkitts lymphoma (BL), post-transplant B cell lymphomas, Hodgkins disease (HD) and nasopharyngeal carcinoma (NPC), and by its unique ability to efficiently transform resting B cells in vitro into permanently growing lymphoblastoid cell lines (LCLs). These transforming effects are associated with the restricted expression of EBV genes such that only a subset of so-called latent virus proteins are expressed in virus infected tumours and in LCLs. Distinct forms of EBV latency are manifest in the different tumours and these appear to be a vestige of the pattern of latent gene expression used by the virus during the establishment of persistent infection within the B cell pool. This review summarises our current knowledge of EBV latent gene function and how this relates to the role of the virus in the aetiology of different tumours.

HIF activation identifies early lesions in VHL kidneys: Evidence for site-specific tumor suppressor function in the nephron

Mutations in the von Hippel-Lindau (VHL) gene are associated with hereditary and sporadic clear cell renal carcinoma. VHL acts in a ubiquitin ligase complex regulating hypoxia-inducible factor-1 (HIF-1), but the link between this function and cancer development is unclear. Here we show that in the kidneys of patients with VHL disease, HIF activation is an early event occurring in morphologically normal single cells within the renal tubules. In comparison, dysplastic lesions, cystic lesions, and tumors showed evidence of additional mechanisms that amplify HIF activation. Detection of cells with constitutive HIF activation identified a large number of previously unrecognized foci of VHL inactivation. In proximal tubules these were almost entirely unicellular, whereas multicellular foci were almost exclusively seen in the distal nephron.

Novel markers for differentiation of lobular and ductal invasive breast carcinomas by laser microdissection and microarray analysis

BackgroundInvasive ductal and lobular carcinomas (IDC and ILC) are the most common histological types of breast cancer. Clinical follow-up data and metastatic patterns suggest that the development and progression of these tumors are different. The aim of our study was to identify gene expression profiles of IDC and ILC in relation to normal breast epithelial cells.MethodsWe examined 30 samples (normal ductal and lobular cells from 10 patients, IDC cells from 5 patients, ILC cells from 5 patients) microdissected from cryosections of ten mastectomy specimens from postmenopausal patients. Fifty nanograms of total RNA were amplified and labeled by PCR and in vitro transcription. Samples were analysed upon Affymetrix U133 Plus 2.0 Arrays. The expression of seven differentially expressed genes (CDH1, EMP1, DDR1, DVL1, KRT5, KRT6, KRT17) was verified by immunohistochemistry on tissue microarrays. Expression of ASPN mRNA was validated by in situ hybridization on frozen sections, and CTHRC1, ASPN and COL3A1 were tested by PCR.ResultsUsing GCOS pairwise comparison algorithm and rank products we have identified 84 named genes common to ILC versus normal cell types, 74 named genes common to IDC versus normal cell types, 78 named genes differentially expressed between normal ductal and lobular cells, and 28 named genes between IDC and ILC. Genes distinguishing between IDC and ILC are involved in epithelial-mesenchymal transition, TGF-beta and Wnt signaling. These changes were present in both tumor types but appeared to be more prominent in ILC. Immunohistochemistry for several novel markers (EMP1, DVL1, DDR1) distinguished large sets of IDC from ILC.ConclusionIDC and ILC can be differentiated both at the gene and protein levels. In this study we report two candidate genes, asporin (ASPN) and collagen triple helix repeat containing 1 (CTHRC1) which might be significant in breast carcinogenesis. Besides E-cadherin, the proteins validated on tissue microarrays (EMP1, DVL1, DDR1) may represent novel immunohistochemical markers helpful in distinguishing between IDC and ILC. Further studies with larger sets of patients are needed to verify the gene expression profiles of various histological types of breast cancer in order to determine molecular subclassifications, prognosis and the optimum treatment strategies.

Functional epigenetics identifies a protocadherin PCDH10 as a candidate tumor suppressor for nasopharyngeal, esophageal and multiple other carcinomas with frequent methylation

Protocadherins constitute the largest subgroup in the cadherin superfamily of cell adhesion molecules. Their major functions are poorly understood, although some are implicated in nervous system development. As tumor-specific promoter methylation is a marker for tumor suppressor genes (TSG), we searched for epigenetically inactivated TSGs using methylation-subtraction combined with pharmacologic demethylation, and identified the PCDH10 CpG island as a methylated sequence in nasopharyngeal carcinoma (NPC). PCDH10 is broadly expressed in all normal adult and fetal tissues including the epithelia, though at different levels. It resides at 4q28.3 – a region with hemizygous deletion detected by array-CGH in NPC cell lines; however, PCDH10 itself is not located within the deletion. In contrast, its transcriptional silencing and promoter methylation were frequently detected in multiple carcinoma cell lines in a biallelic way, including 12/12 nasopharyngeal, 13/16 esophageal, 3/4 breast, 5/5 colorectal, 3/4 cervical, 2/5 lung and 2/8 hepatocellular carcinoma cell lines, but not in any immortalized normal epithelial cell line. Aberrant methylation was further frequently detected in multiple primary carcinomas (82% in NPC, 42–51% for other carcinomas), but not normal tissues. The transcriptional silencing of PCDH10 could be reversed by pharmacologic demethylation with 5-aza-2′-deoxycytidine or genetic demethylation with double knockout of DNMT1 and DNMT3B, indicating a direct epigenetic mechanism. Ectopic expression of PCDH10 strongly suppressed tumor cell growth, migration, invasion and colony formation. Although the epigenetic and genetic disruptions of several classical cadherins as TSGs have been well documented in tumors, this is the first report that a widely expressed protocadherin can also function as a TSG that is frequently inactivated epigenetically in multiple carcinomas.

The stress-responsive gene GADD45G is a functional tumor suppressor, with its response to environmental stresses frequently disrupted epigenetically in multiple tumors

The CpG island of GADD45G was identified as a target sequence during the identification of hypermethylated genes using methylation-sensitive representational difference analysis combined with 5-aza-2′-deoxycytidine demethylation. Located at the commonly deleted region 9q22, GADD45G is a member of the DNA damage-inducible gene family. In response to stress shock, GADD45G inhibits cell growth and induces apoptosis. Same as other GADD45 members, GADD45G is ubiquitously expressed in all normal adult and fetal tissues. However, its transcriptional silencing or down-regulation and promoter hypermethylation were frequently detected in tumor cell lines, including 11 of 13 (85%) non-Hodgkins lymphoma, 3 of 6 (50%) Hodgkins lymphoma, 8 of 11 (73%) nasopharyngeal carcinoma, 2 of 4 (50%) cervical carcinoma, 5 of 17 (29%) esophageal carcinoma, and 2 of 5 (40%) lung carcinoma and other cell lines but not in any immortalized normal epithelial cell line, normal tissue, or peripheral blood mononuclear cells. The silencing of GADD45G could be reversed by 5-aza-2′-deoxycytidine or genetic double knockout of DNMT1 and DNMT3B, indicating a direct epigenetic mechanism. Aberrant methylation was further frequently detected in primary lymphomas although less frequently in primary carcinomas. Only one single sequence change in the coding region was detected in 1 of 25 cell lines examined, indicating that genetic inactivation of GADD45G is very rare. GADD45G could be induced by heat shock or UV irradiation in unmethylated cell lines; however, this stress response was abolished when its promoter becomes hypermethylated. Ectopic expression of GADD45G strongly suppressed tumor cell growth and colony formation in silenced cell lines. These results show that GADD45G can act as a functional new-age tumor suppressor but being frequently inactivated epigenetically in multiple tumors.

Target cells of Epstein-Barr-virus (EBV)-positive post-transplant lymphoproliferative disease: similarities to EBV-positive Hodgkin's lymphoma.

BACKGROUND Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disease (PTLD) encompasses a histologically broad range of lesions, arising from the expanded pool of EBV-infected B cells in the immunocompromised host. Identification of the precise cellular origin of these tumours could clarify their pathogenesis. METHODS Of 13 cases of EBV-positive cases of PTLD characterised by histological analysis, pattern of EBV gene expression, and clinical course, 11 had monoclonal or biclonal lesions in which we determined the progenitor B cell by immunoglobulin heavy chain (IgH) genotyping. RESULTS Two tumours had a naive B cell genotype and two showed patterns of IgH somatic mutation typical of antigen-selected (post-germinal-centre) memory cells. All four arose early post-transplant and expressed the markers of EBV transformation--Epstein-Barr nuclear antigen (EBNA) 2 and latent membrane protein (LMP) 1. However, seven tumours, either of early or late onset and including some with downregulated EBNA 2 and LMP 1, arose from post-germinal cells with randomly mutated or sterile IgH genotypes usually incompatible with B-cell survival in vivo. INTERPRETATION PTLD can arise from a broad range of target B cells and not only from the pool of antigen-selected memory cells that EBV generally colonises in immunocompetent individuals. Tumour development seems frequently associated with the EBV-induced rescue and expansion of B cells that have failed the physiological process of germinal centre selection into memory. This finding shows an unexpected connection between pathogenesis of PTLD and that of EBV-positive Hodgkins lymphoma, another B-cell malignancy of atypical post-germinal-centre cell origin.

The Epstein-Barr virus and its association with human cancers.

The Epstein-Barr virus (EBV) has been linked to the development of a variety of human malignancies, including Burkitts lymphoma, Hodgkins disease, nasopharyngeal carcinoma, some T cell lymphomas, post-transplant lymphoproliferative disease, and more recently, certain cancers of the stomach and smooth muscle. This review summarizes these associations and in particular the role of the viral latent genes in the transformation process.

Constitutive activation of phosphatidyl-inositide 3 kinase contributes to the survival of Hodgkin's lymphoma cells through a mechanism involving Akt kinase and mTOR

The molecular mechanisms underlying the pathogenesis of the malignant Hodgkins/Reed–Sternberg (HRS) cells of Hodgkins lymphoma (HL) are largely unknown. This study investigates the contribution of phosphatidyl‐inositide 3 kinase (PI3‐kinase) and demonstrates that Akt, a substrate of PI3‐kinase, is constitutively activated in HL‐derived cell lines. Several downstream effectors of Akt signalling, including glycogen synthase kinase 3 (GSK‐3) α and β and mTOR substrates 4E‐BP1 and p70 S6 kinase, were also phosphorylated in HL cells. The mTOR inhibitor, rapamycin, inhibited phosphorylation of these proteins. Furthermore, LY294002 inhibited phosphorylation of p70 S6 kinase and 4E‐BP1, suggesting that the phosphorylation of p70 S6 kinase and 4E‐BP1 in HL cells is PI3‐kinase dependent. Importantly, HRS cells of primary tumour samples not only expressed high levels of activated Akt but also displayed phosphorylation of downstream targets of Akt activation including GSK‐3, 4E‐BP1, and p70 S6 Kinase. Inhibition of PI3‐kinase and mTOR showed only modest effects on cell survival at the lower serum concentrations. However, rapamycin and doxorubicin acted synergistically to reduce HL cell survival. A combination of rapamycin and chemotherapy should be investigated in the treatment of HL. Copyright

Hypoxia-regulated carbonic anhydrase IX expression is associated with poor survival in patients with invasive breast cancer

Tumour hypoxia is a microenvironmental factor related to poor response to radiation, chemotherapy, genetic instability, selection for resistance to apoptosis, and increased risk of invasion and metastasis. Hypoxia-regulated carbonic anhydrase IX (CA IX) has been studied in various tumour sites and its expression has been correlated with the clinical outcome. The purpose of this study was to investigate the correlation of CA IX expression with outcome in patients with invasive breast cancer. We conducted a retrospective study examining the effects of carbonic anhydrase IX (CA IX) on survival in patients with breast cancer. To facilitate the screening of multiple tissue blocks from each patient, tissue microarrays were prepared containing between two and five representative samples of tumour per patient. Immunohistochemistry was used to examine expression of CA IX in patients with breast cancer. The study includes a cohort of 144 unselected patients with early invasive breast cancer who underwent surgery, and had CA IX expression and follow-up data available for analysis. At the time of analysis, there were 28 deaths and median follow-up of 48 months with 96% of patients having at least 2 years of follow-up. CA IX was negative for 107 patients (17 deaths) and positive for 37 patients (11 deaths). Kaplan–Meier survival curves show that survival was superior in the CA IX-negative group with a 2-year survival of 97% for negatives and 83% for positives (log-rank test P=0.01). Allowing for potential prognostic variables in a Cox regression analysis, CA IX remained a significant independent predictor of survival (P=0.035). This study showed in both univariate and multivariate analysis that survival is significantly inferior in patients with tumour expressing CA IX. Prospective studies are underway to investigate this correlation in clinical trial setting.

Expression of the Epstein-Barr Virus-Encoded Epstein-Barr Virus Nuclear Antigen 1 in Hodgkin's Lymphoma Cells Mediates Up-Regulation of CCL20 and the Migration of Regulatory T Cells

In approximately 50% of patients with Hodgkins lymphoma (HL), the Epstein-Barr virus (EBV), an oncogenic herpesvirus, is present in tumor cells. After microarray profiling of both HL tumors and cell lines, we found that EBV infection increased the expression of the chemokine CCL20 in both primary Hodgkin and Reed-Sternberg cells and Hodgkin and Reed-Sternberg cell-derived cell lines. Additionally, this up-regulation could be mediated by the EBV nuclear antigen 1 protein. The higher levels of CCL20 in the supernatants of EBV-infected HL cell lines increased the migration of CD4(+) lymphocytes that expressed FOXP3, a marker of regulatory T cells (Tregs), which are specialized CD4(+) T cells that inhibit effector CD4(+) and CD8(+) T cells. In HL, an increased number of Tregs is associated with the loss of EBV-specific immunity. Our results identify a mechanism by which EBV can recruit Tregs to the microenvironment of HL by inducing the expression of CCL20 and, by doing so, prevent immune responses against the virus-infected tumor population. Further investigation of how EBV recruits and modifies Tregs will contribute not only to our understanding of the pathogenesis of virus-associated tumors but also to the development of therapeutic strategies designed to manipulate Treg activity.