Immune escape by Epstein-Barr virus associated malignancies

Persistent Epstein-Barr virus (EBV) infection remains asymptomatic in the majority of virus carriers, despite the potent growth transforming potential of this virus. The increased frequency of EBV associated B cell lymphomas in immune compromised individuals suggests that tumor-free chronic infection with this virus is in part due to immune control. Here we discuss the evidence that loss of selective components of EBV specific immunity might contribute to EBV associated malignancies, like nasopharyngeal carcinoma, Burkitt's and Hodgkin's lymphoma, in otherwise immune competent patients. Furthermore, we discuss how current vaccine approaches against EBV might be able to target these selective deficiencies.     

The putative roles of the ubiquitin/proteasome pathway in resistance to anticancer therapy

The ubiquitin/proteasome (UP) pathway plays a significant role in many important biological functions and alterations in this pathway have been shown to contribute to the pathology of many human diseases, including cancer. Proteasome inhibition has been well established as a rational strategy for the treatment of multiple myeloma and is currently under investigation for the treatment of other haematological malignancies and solid tumours. Recent evidence suggests that proteasome inhibition may also sensitise tumour cells to the actions of both conventional chemotherapy and radiotherapy, suggesting that this pathway may modify clinical response to anticancer therapy. However, conflicting evidence exists as to the roles of the UP pathway in resistance to treatment. This review endeavours to discuss such roles.     

Molecular epidemiology of Epstein-Barr virus (EBV) in EBV-related malignancies

The prevalent strain of Epstein-Barr virus (EBV) in EBV-related malignancies and in healthy adults in Southern Japan was examined by means of polymerase chain reaction (PCR) and/or restriction fragment length polymorphism (RFLP) analysis. In EBV-related gastric cancers, 51/73 cases were subtype A, 4 were subtype B and the EBNA-2 region was not amplified in 18 cases. Sixty-three were wild-type F, and only one was variant “f”. Sixty-one cases had type C and 2 type D. EBNA-2 subtype A was found in 10/12 EBV-related T/NK-cell lymphomas, and 11 samples harbored the wild-type F. Neither subtype B nor the “f” variant was detected. Type C EBV was found in 8 cases and type D in 3 specimens. Two Japanese nasopharyngeal carcinomas (NPC) harbored subtype A with wild-type F and type C. Throat washings from healthy adults harbored wild-type F virus in 60/153 cases, and 25 of these samples were EBNA-2 subtype A. Type C viruses were detected in 92% of cases and type D in 7.4%. Therefore, the prevalent strain in EBV-related malignancies in Southern Japan was the same as in the healthy population in this geographical region. Int. J. Cancer 72:72–76, 1997. © 1997 Wiley-Liss Inc.     

Active-site directed probes to report enzymatic action in the ubiquitin proteasome system

Irreversible covalent inhibitors equipped with reporter groups, also termed activity-based probes, allow the study of target enzymes based on catalytic activity instead of expression level, which does not necessarily indicate protein function and subsequent cellular consequences. Activity-based probes offer advantages over traditional techniques: they can be applied to the cell or tissue of choice and molecular imaging and pharmacology applications are possible. Here the design and use of probes directed at enzymatic activities in the ubiquitin proteasome system are discussed. This system holds promise for the development of new, targeted anticancer therapies and the probes discussed here might aid in fulfilling this promise.     

Specific neutralizing antibodies in Epstein-Barr virus associated diseases

The results of Epstein-Barr virus (EBV) neutralization tests on 650 sera from patients with EBV-associated diseases and controls were compared with the titers of antibodies to EB viral capsid antigens (anti-VCA) and to EBV-induced early antigens (anti-EA). All anti-VCA negative sera failed to neutralize EBV. Among 546 anti-VCA positive sera, only eight yielded negative results in neutralization tests. Four of these came from patients in the early acute phase of infectious mononucleosis who subsequently developed neutralizing antibodies. The ratios between neutralizing and anti-VCA titers varied over a wide range indicating that different antibodies are involved in the two tests. In line with the regular presence of mostly elevated anti-VCA titers, the geometric mean neutralizing titers in Burkitt's lymphoma and nasopharyngeal carcinoma were also higher (5- to 7-fold) than in appropriate controls. In Hodgkin's disease, an over-representation of high neutralizing as well as anti-VCA titers was noted, although some patients had neither of these antibodies. All patients with infectious mononucleosis developed neutralizing antibodies with a geometric mean ultimately matching that of controls whereas in the early acute phase it was lower than in the controls. In contrast, the geometric mean anti-VCA titer in the early acute phase was higher than in controls and then declined in convalescence toward the control value. There was no evident relationship between neutralizing and anti-EA activities whether directed against the D or the R component of the EA complex.     

Epstein-Barr virus oncogenesis and the ubiquitin-proteasome system

Epstein-Barr virus (EBV), a human herpesvirus associated with lymphoid and epithelial cell tumors, encodes several proteins that exploit the ubiquitin-proteasome system to regulate latency and allow the persistence of infected cells in immunocompetent hosts. Further modifications of ubiquitin-dependent proteolysis by activated cellular oncogenes contribute to malignant transformation. A detailed understanding of these processes may lead to the development of new therapeutic strategies for EBV-associated cancers.     

The ubiquitin–proteasome system is inhibited by p53 protein expression in human ovarian cancer cells

The ubiquitin–proteasome system (UPS) and autophagy provide major cellular pathways for protein degradation. Since the p53 pathway controls autophagy, we investigated whether p53 regulates UPS in ovarian tumour cell lines. A reporter cell line (SKOV3-EGFPu) was established to measure UPS function against a constant genetic background. Transient expression of either wild type or mutant p53 in SKOV3-EGFPu cells reduced UPS activity as compared to vector control. These results, together with those from endogenous p53 expression in seven ovarian cancer cell lines, suggest that expression of both wild-type and mutant p53 protein impairs UPS function. Thus, p53 expression may regulate protein homeostasis by down-regulating UPS function in response to cellular stress.     

Global burden of deaths from Epstein-Barr virus attributable malignancies 1990-2010

Background

Epstein-Barr virus (EBV) is an oncogenic virus implicated in the pathogenesis of a number of human malignancies of both lymphoid and epithelial origin. Thus, a comprehensive and up-to-date analysis focused on the global burden of EBV-attributable malignancies is of significant interest.

Methods

Based on published studies, we estimated the proportion of Burkitt’s lymphoma (BL), Hodgkin’s lymphoma (HL), nasopharyngeal carcinoma NPC), gastric carcinoma (GC) and post-transplant lymphoproliferative disease (PTLD) attributable to EBV, taking into consideration age, sex and geographical variations. This proportion was then imputed into the Global Burden of Disease 2010 dataset to determine the global burden of each EBV-attributable malignancy in males and females in 20 different age groups and 21 world regions from 1990 to 2010.

Results

The analysis showed that the combined global burden of deaths in 2010 from all EBV-attributable malignancies was 142,979, representing 1.8% of all cancer deaths. This burden has increased by 14.6% over a period of 20 years. All 5 EBV-attributable malignancies were more common in males in all geographical regions (ratio of 2.6:1). Gastric cancer and NPC accounted for 92% of all EBV-attributable cancer deaths. Almost 50% of EBV-attributed malignancies occurred in East Asia. This region also had the highest age-standardized death rates for both NPC and GC.

Conclusions

Approximately 143,000 deaths in 2010 were attributed to EBV-associated malignancies. This figure is likely to be an underestimate since some of the less prevalent EBV-associated malignancies have not been included. Moreover, the global increase in population and life-expectancy will further increase the overall burden of EBV-associated cancer deaths. Development of a suitable vaccine could have a substantial impact on reducing this burden.

     

Epstein-Barr virus associated gastric carcinoma: epidemiological and clinicopathological features

In this paper, the roles of Epstein-Barr virus (EBV) in gastric carcinogenesis are discussed, reviewing mainly epidemiological and clinicopathological studies. About 10% of gastric carcinomas harbor clonal EBV. LMP1, an important EBV oncoprotein, is only rarely expressed in EBV-associated gastric carcinoma (EBV-GC) while EBV-encoded small RNA is expressed in almost every EBV-GC cell, suggesting its importance for developing and maintaining this carcinoma. In addition, the hypermethylation-driven suppressor gene downregulation, frequently observed in EBV-GC, appears to give a selective advantage for carcinoma cells. EBV reactivation is suspected to precede EBV-GC development since antibodies against EBV-related antigens, including EBV capsid antigen (VCA), are elevated in prediagnostic sera. Interestingly, the average anti-VCA immunoglobulin G antibody titer in EBV-GC patients was significantly higher among men than among women, whereas EBV-negative GC cases did not show such a sex difference. A higher frequency of human leucocyte antigen-DR11 in EBV-GCs suggests that major histocompatibility complex-restricted EBV nuclear antigen 1 epitope recognition may enhance EBV reactivation. EBV infection of gastric cells by lymphocytes with reactivated EBV is suspected to be the first step of EBV-GC development. Male predominance of EBV-GC suggests the involvement of lifestyles and occupational factors common among men. The predominance of EBV with XhoI+ and BamHI type i polymorphisms in EBV-GC in Latin America suggests a possibility of some EBV oncogene expressions being affected by EBV polymorphism. The lack of such predominance in Asian countries, however, indicates an interaction between EBV polymorphism and the host response. In conclusion, further studies are necessary to examine the interaction between EBV infection, its polymorphisms, environmental factors, and genetic backgrounds. ( Cancer Sci 2008; 99: 195 –201)     

Targeting the ubiquitin–proteasome system to activate wild-type p53 for cancer therapy

Ubiquitination plays a key role in regulating the tumour suppressor p53. It targets p53 for degradation by the 26S proteasome. The ubiquitin pathway also regulates the activity and localisation of p53. Ubiquitination requires ubiquitin-activating and -conjugating enzymes and ubiquitin ligases. In addition, ubiquitination can be reversed by the action of deubiquitinating enzymes. Here we give an overview of the role of components of the ubiquitin–proteasome system in the regulation of p53 and review progress in targeting these proteins to activate wild-type p53 for the treatment of cancer.     

Global and non-random CpG-island methylation in gastric carcinoma associated with Epstein-Barr virus

DNA hypermethylation may play a primary role in the genesis of Epstein-Barr virus (EBV)-associated gastric carcinoma (GC) (EB-VaGC). Methylation-specific PCR targeting CpG-islands demonstrated markedly increased methylation of specific genes, such as p14, p15 and p16 genes, in EBVaGC in vivo . A high frequency of methylation was observed in an EBVaGC strain of severe combined immunodeficiency mice, and the expression of methylated genes in the strain was apparently lower than the expression of the unmethylated genes in EBV-negative GC strains. Although over-expression of DNA methyltransferases ( DNMTs ) is known to be associated with some human cancers, real-time PCR demonstrated that DNMTs expression was suppressed in EBVaGC. The DNA methylation of specific genes, independently of DNMTs expression, may be important in the development of EBVaGC. (Cancer Sci 2003; 94: 76–80)     

Update on the proteasome inhibitor bortezomib in hematologic malignancies

The ubiquitin-proteasome system plays a crucial role in eukaryotic cells in maintaining protein homeostasis. Through the disruption of a variety of pathways and cell cycle checkpoints, proteasome inhibition leads to apoptosis and in experimental models can overcome chemoresistance. Bortezomib is the first of its class of proteasome inhibitors tested in humans that showed promising activity in several tumor types, and especially in hematologic malignancies, in phase I studies. The remarkable results obtained in phase II studies in multiple myeloma (MM) led to its fast-track approval by the US Food and Drug Administration in May 2003 for relapsed MM. More recent observation also revealed promising activity in non-Hodgkin's lymphoma. This review will explore the rationale for the use of bortezomib in hematologic malignancies as well as provide an update on the results of ongoing studies and future directions for the use of this new agent in hematologic malignancies. The mechanism of action of bortezomib and its nonoverlapping toxicity profile make it a very appealing drug for combination with other chemotherapeutic or biologic agents. Bortezomib represents an excellent example of how progress in understanding the biology of cancer cells can impact clinical practice and lead toward a new era of rational therapeutics.     

Biochemistry of latent Epstein-Barr virus infection and associated cell growth transformation

There is sufficient knowledge of the biochemistry of Epstein-Barr virus (EBV) persistence and gene expression in latent growth-transforming infection and of the persistence and expression of other oncogenic viruses to permit interesting and possibly useful comparisons. Most smaller oncogenic viral genomes usually persist solely as integrated DNAs despite their ability to circularize. Papilloma and hepatitis viruses may persist as episomes, and parts of their genomes may integrate. Usually, only the oncogenic fragment of adenovirus DNA is integrated into cell DNA. In contrast, the entire EBV genome persists in cells as an episome or as integrated DNA. Thus, EBV may have novel mechanisms to maintain its complete genome as an episome or as a complete integrated virus DNA. Three viral genes are expressed in latently EBV-infected growth-transformed cells, each of which encodes one RNA and one protein. Two of the proteins are probably nuclear DNA-binding proteins; the third is probably a membrane protein. Thus, the repertoire of genes expressed is similar in complexity and intracellular distribution to that expressed by papova and adenoviruses in cellular transformation. The papova and adenovirus-transforming genes are partially analogous to retrovirus oncogenes. This similarity cannot as yet be extended to EBV. There is no homology at the DNA-sequence or protein-sequence level between EBV and other viral or cell oncogenes. Thus, it remains important to pursue analysis of the EBV-transforming genes. Identification of these genes is a first step in discerning their function in latent growth-transforming cell infection. Parts of each of these genes are being made in bacteria. The bacterial products enable us to make antisera that are specific for each of the viral proteins. These antisera can also be used to identify the viral proteins within latently infected growth-transformed cells or within cells stably expressing transfected virus genes. The antisera can also be used to study the association of Epstein-Barr nuclear antigen (EBNA) 1 and 2 with DNA and of the lymphocyte-determined membrane antigen (LYDMA) with the cell membrane. The three genes must be introduced into nontransformed cells to determine whether, alone or in combination, they are sufficient to accomplish cell growth transformation.     

Antiviral agent cidofovir decreases Epstein-Barr virus (EBV) oncoproteins and enhances the radiosensitivity in EBV-related malignancies

The Epstein-Barr virus (EBV) is involved in the carcinogenesis of several human cancers such as nasopharyngeal carcinoma (NPC) and Burkitt lymphoma (BL). Given the consistent role of EBV in transformation and maintenance of malignant phenotype, antiviral strategies provide an attractive approach to target EBV-expressing cells. In that aim, we have tested the Cidofovir, which is an acyclic nucleoside phosphonate analog known to exert an antiproliferative activity in some human virus-related tumors. Here, we show that Cidofovir induces a downregulation of the EBV oncoprotein LMP1 associated with a decrease of the antiapoptotic Bcl-2 and an increase of the proapoptotic Bax protein in Raji (BL) and C15 (NPC) cells. Using BL cell line BL2 B95-8 (BL2 infected with the B95.8 strain of EBV), we addressed the relation between EBV genome expression and modulation of viral oncoproteins by Cidofovir and/or ionizing radiation (IR). Cidofovir was able to significantly reduce LMP1 and EBNA2 mRNA and protein expression. This effect was associated with inhibition of proliferation, stimulation of apoptosis, and decrease of Bcl-2 expression in BL2 B95.8 cells. In addition, Cidofovir enhanced the radiation-induced apoptosis and the radiosensitivity through the proteolytic cleavage of death effectors caspase-9 and -3, which was specifically induced by combined treatment in EBV-positive cells compared to their negative counterparts. Furthermore, the combined treatment in nude mice led to a complete tumor remission without increasing toxicity in two human EBV-related cancer xenografts (Raji and C15). These results provide the basis for a novel anticancer strategy to enhance the therapeutic ratio of IR in EBV-related cancers.     

Induction of the Epstein-Barr virus thymidine kinase gene with concomitant nucleoside antivirals as a therapeutic strategy for Epstein-Barr virus-associated malignancies

Lymphoproliferative diseases (LPDs) associated with the Epstein-Barr virus (EBV) include non-Hodgkin lymphomas, which occur in the setting of immunosuppression, including that induced by human immunodeficiency virus, and posttransplant lymphoproliferative disorders. These LPDs are characterized by actively proliferating, latently infected EBV-positive B lymphocytes and often follow a rapidly progressive fatal clinical course. Pharmacologic treatment for herpesvirus infections has targeted the virus-specific enzyme, thymidine kinase (TK), with nucleoside analogs. The lack of viral TK expression in EBV-positive tumors, caused by viral latency, however, makes antiviral therapy alone ineffective as an antineoplastic therapy. Arginine butyrate selectively activates the EBV TK gene in latently infected EBV-positive tumor cells. We have developed a strategy for treatment of EBV-associated lymphomas using pharmacologic induction of the latent viral TK gene and enzyme in tumor cells using arginine butyrate, followed by treatment with ganciclovir. A phase I/II trial, using an intrapatient dose escalation of arginine butyrate combined with ganciclovir, is underway. This combination therapy has produced complete clinical responses in 5 of 10 previously refractory patients, with partial responses occurring in 2 additional patients. This virus-targeted antitumor strategy may provide a new therapeutic approach to EBV-associated neoplasms.