Viral hit and run-oncogenesis: Genetic and epigenetic scenarios

Cancer Letters 305 (2011) 200–217

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Cancer Letters journal homepage: www.elsevier.com/locate/canlet

Mini-review

Viral hit and run-oncogenesis: Genetic and epigenetic scenarios Hans Helmut Niller a,*, Hans Wolf a, Janos Minarovits b a b

Institute for Medical Microbiology and Hygiene of the University of Regensburg, Franz-Josef-Strauß-Allee 11, D-93053 Regensburg, Germany Microbiological Research Group of the National Center for Epidemiology, Pihenö ut 1, H-1529 Budapest, Hungary

a r t i c l e

i n f o

Keywords: Epstein–Barr virus Tumor virus Recombination Chromatin DNA-methylation Transformation Tumor suppressor gene

a b s t r a c t It is well documented that viral genomes either inserted into the cellular DNA or coreplicating with it in episomal form can be lost from neoplastic cells. Therefore, ‘‘hit and run”mechanisms have been a topic of longstanding interest in tumor virology. The basic idea is that the transient acquisition of a complete or incomplete viral genome may be sufficient to induce malignant conversion of host cells in vivo, resulting in neoplastic development. After eliciting a heritable change in the gene expression pattern of the host cell (initiation), the genomes of tumor viruses may be completely lost, i.e. in a hit and run-scenario they are not necessary for the maintenance of the malignant state. The expression of viral oncoproteins and RNAs may interfere not only with regulators of cell proliferation, but also with DNA repair mechanisms. DNA recombinogenic activities induced by tumor viruses or activated by other mechanisms may contribute to the secondary loss of viral genomes from neoplastic cells. Viral oncoproteins can also cause epigenetic dysregulation, thereby reprogramming cellular gene expression in a heritable manner. Thus, we expect that epigenetic scenarios of viral hit and run-tumorigenesis may facilitate new, innovative experiments and clinical studies in spite of the fact that the regular presence of a suspected human tumor virus in an early phase of neoplastic development and its subsequent regular loss have not been demonstrated yet. We propose that virus-specific ‘‘epigenetic signatures”, i.e. alterations of the host cell epigenome, especially altered DNA methylation patterns, may help to identify viral hit and run-oncogenic events, even after the complete loss of tumor viruses from neoplastic cells. Ó 2010 Elsevier Ireland Ltd. All rights reserved.

1. Transformation and/or oncogenesis Epstein–Barr virus (EBV), Human herpesvirus 8 (HHV8), also called Kaposi’s sarcoma-associated herpesvirus (KSHV), hepatitis C virus (HCV), human T-cell lymphotropic virus type 1 (HTLV-1), hepatitis B virus (HBV), and the high risk human papillomaviruses (HPV), e.g. HPV types 16 and 18, are clearly recognized as human tumor viruses. For hepatitis B virus, the efficacy of vaccines has been proven for many years. As to the recently developed papillomavirus

* Corresponding author. E-mail addresses: [email protected] (H.H. Niller), [email protected] (H. Wolf), minimicrobi@ hotmail.com (J. Minarovits). 0304-3835/$ - see front matter Ó 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2010.08.007

vaccines, efficacy is assumed without much doubt. The papillomaviruses (genome size: <8.000 base pairs), together with the polyomaviruses (genome size: 4.700– 5.400 bp) and adenoviruses (genome size: 36.000 bp), are assigned to the group of ‘‘small DNA transforming viruses”. Some members of this group are able to transform cells in culture, and to cause tumors in experimental animals including rodents, sometimes in monkeys. The term viral ‘‘transformation” is rather well defined: upon infection, previously naive cells adopt a different phenotype: they lose their contact inhibition, show serum independent growth, form colonies in soft agar and tumors in syngeneic or immune suppressed rodents. Transforming or immortalizing viral functions have been attributed to transforming proteins, like large tumor antigen (T-Ag) of polyomaviruses,

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the E6 and E7 proteins of papillomaviruses, and the E1A and E1B proteins of adenoviruses. The transforming proteins bind to and inactivate the tumor suppressor proteins p53 and/or pRb, respectively, an interaction which turned out to be crucial for viral transformation. The spectacular ability of some viruses to transform the phenotype of their target cells in vitro has only partially translated into their ability to cause human cancer. On the one hand, human adenoviruses and human polyomaviruses (with the exception of Merkel cell carcinoma virus) could not be linked to the development of human neoplasms yet. On the other hand, EBV, a large DNA virus that causally contributes to the development of several human neoplasms, can transform B-lymphocytes both in cell culture and in severely immune suppressed patients. Then again, hepatitis B virus, a small DNA virus that causally contributes to liver cancer, does not readily transform cells in culture. Thus, although both abilities are sometimes combined within the same virus, ‘‘oncogenesis” and ‘‘transformation” are distinct functional entities. Therefore, in this review both terms are not used as synonyms. Due to the obvious imperfect concordance between viral transformation of cultured cells and human oncogenesis, two major research directions have developed. On the one hand, disguised transforming functions are being searched for in acknowledged tumor viruses, that do not transform native cultured cells. On the other hand, hit and run-mechanisms are being searched for in cases where a link between a virus and a neoplasm seems likely on epidemiological grounds, but the viral genome cannot be found in the tumor tissue. In this case, viral genomes or small viral fragments may be looked for in malignant tumors or in tumor precursor stages. Ironically, both transforming functions and hit and run-mechanisms have been looked for in nontransforming viruses that are apparently not linked with human cancer.

2. Hit and run-scenarios 2.1. Hit and run-transformation Rodent cells are easily transformed in culture, much easier than human cells. There have been early reports on hit and run-transformation of rodent cells through DNA fragments of herpes simplex viruses (HSV) type 1 and type 2 [1] (reviewed in [2]). Those reports have been followed up sporadically [3,4]. Transforming capability has also been ascribed to cytomegalovirus [5,6]. Further, papillomavirus type 18 [7], combinations of cytomegalovirus and adenovirus genes [8] or herpesvirus and papillomavirus genes have been reported to hit and run-transform rodent [7–9] or human cells [10]. Reports on rodent cell transformation may be put into perspective by the fact that such cells have been efficiently hit and run-transformed through transient transfection of small plasmid DNAs which contained viral regulatory sequences, but did not contain any obviously transforming reading frames [11]. Further, a transformed cell, even if mammalian, is not necessarily a malignant tumor cell, and the tumorous growth of transformed cells

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in immune suppressed mice may differ from a malignant tumor developing in an immunocompetent organism. 2.2. Hit and run-oncogenesis Hit and run-oncogenesis is principally conceivable in tumor virology. A virus may infect its target tissue, trigger a misdirected immune response, mutagenic activity, or a permanent chromatin reorganization which activates oncogenes or silences tumor suppressor and DNA repair genes. Cells may become malignant and remain so, even if virus-triggered recombinogenic activities lead to the secondary loss of the virus. To conclusively prove hit and run-oncogenesis, a complete chain of evidence from epidemiology, histology and molecular biology is required. According to current causal criteria which have been developed from Koch’s postulates and which are widely accepted in tumor virology [12,13], viral hit and run-oncogenesis remains unproven so far. Generally, epidemiological plausibility, the consistent presence of the viral genome or parts thereof in the tumor cells, and tumor-promoting effects of the viral genes on infected or transfected cells are considered as hints for causality [12,13]. For hit and run-mechanisms one would consider the transient, but regular presence of viral genomes or parts thereof at an early stage of the respective tumor. However, apart from anecdotal reports, a clear-cut proof for hit and run-oncogenesis has not been achieved so far in clinical oncology. This situation may change, because novel high throughput sequencing methods by now allow the identification of viral genome fragments in pre-dysplastic tissue and in early stages of neoplastic development, either by systematic studies aiming to discover tumor-associated viral genomes, or as a by-product of cancer genome projects. In addition, we propose that recent developments in epigenetics, especially the concept of an ‘‘epigenetic signature” left by a tumor virus on the host cell epigenome, may help to identify viral hit and run-oncogenic events, even after the complete loss of tumor virus genomes from neoplastic cells. For an overview of genetic and epigenetic mechanisms potentially involved in viral hit and run-oncogenesis, see Tables 1 and 2, respectively, and Fig. 1. 3. Tumor viruses 3.1. Hepatitis B virus Hepatocellular carcinoma (HCC), one of the most common human cancers, frequently develops on the basis of a long lasting chronic HBV infection or subsequent cirrhosis which may end in HCC. The molecular mechanisms leading to carcinoma are ambiguous. The HBV surface antigen (HBsAg) and X protein (HBx or pX) may contribute to oncogenesis through oxidative stress, transactivation of host genes, inhibition of DNA repair, and liver cell hyperplasia. Further, viral integration into the cellular genome seems to play a crucial role. In woodchuck HCC, viral integration frequently occurs at the c-Myc (MYC) or N-myc (MYCN) oncogene loci. Although not entirely random, viral integration in human HCC does not prefer such a distinguished

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Table 1 Genetic mechanisms potentially involved in viral hit and run-oncogenesis. Virus

Mechanism

Hepatitis B virus

Insertional mutagenesis Chromosomal alterations induced by the integration of the viral genome into the cellular DNA Induction of genetic instability (loss of heterozygosity) pX blocks DNA repair by binding to XAP-1 (X-associated protein-1)

Hepatitis C virus

Inhibition of mitotic spindle checkpoint functions; Induction of polyploidy by HCV core protein Induction of a mutator phenotype via up-regulation of activation induced deaminase (AID) and inhibition of DNA repair

Polyomaviruses

MCV DNA integration into the host cell genome is an early event during the genesis of Merkel cell carcinoma and may induce, therefore, genetic instability JCV may confer a mutagenic activity via inactivation of p53 and pRb

Human adenovirus – transforming serotypes

E4orf3, E4orf4 increase mutation frequency; E4orf6 interferes with DNA double strand repair

EBV

LMP1 can induce AID in the absence of EBNA2 EBNA1 induces RAG expression

HTLV-1

Tax induces centrosome over duplication and asymmetrical chromosome segregation

Table 2 Epigenetic mechanisms potentially involved in viral hit and run-oncogenesis. Virus

Mechanism

Hepatitis B virus

pX up-regulates DNMT1, DNMT3A1 and DNMT3A2; pX down-regulates DNMT3B; these processes may induce local hypermethylation associated with gene silencing and global hypomethylation at repetitive sequences

Hepatitis C virus

Up-regulation of DNMT1 may result in gene silencing

Polyomaviruses

BK virus Large T antigen up-regulates DNMT1 that may result in gene silencing

Human papillomavirus

E7 stimulates Dnmt1 activity; several epigenetic Alterations of tumor suppressor genes in cervix carcinoma [247]

Human adenovirus (tumorigenic serotypes)

E1A up-regulates DNMT1 and stimulates Dnmt1 activity that may result in gene silencing; E1A binds to TRRAP/Gcn5 of the Tip60 HAT and nucleosome remodeling complex and induces genome wide relocalization of p300/CBP HATs resulting in histone acetylation and promoter activation

EBV

LMP1 up-regulates DNMT1, 3A and 3B resulting in gene silencing

KSHV

LANA up-regulates DNMT1, 3A and 3B; recruitment of Dnmts to cellular promoters results in site specific CpG methylation and histone deacetylation and gene silencing Tax causes dissociation of transcription factors from the Shp1 (PTPN6 gene) promoter that leads to hypermethylation and permanent silencing of the promoter of a putative tumor suppressor gene

HTLV-1

HIT

RUN

Tumor virus infection

Loss of viral genome(s) g ( )

Initiation of transformation induction of genetic instability and/or epigenetic dysregulation

Maintenance of transformation mediated by viral functions and/or altered cellular functions

Neoplastic progression mediated by altered cellular functions

Time ADDITIONAL HITS (non viral) viral), SELECTION Fig. 1. Putative time course of viral hit and run-oncogenesis.

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genetic locus. However, insertional mutagenesis at critical cancer-related gene loci has been found in several cases. Furthermore, deletions, translocations and gross chromosomal abnormalities have been shown to occur in HBV-related HCC. Compared to non-viral HCC, the rate of chromosomal aberrations is significantly increased in HBV-induced HCC. Viral integration occurs at the early steps of hepatocarcinogenesis. By means of chromosomal alterations, HBV insertion may initiate specific oncogenetic pathways (reviewed in [14,15]). There is evidence for the integration into the host cell genome and the subsequent loss of HBV genomes which may contribute to oncogenesis via a hit and run-mechanism in HBV-induced HCC: An extensive analysis on six livers of patients with cirrhosis or HCC was performed. 258 macrocirrhotic nodules and their satellites from 30 liver segments were examined. The integration status of HBV genomes and the genetic stability at 12 highly polymorphic microsatellite loci was examined. In most nodules, HBV maintained its episomal replicative forms without being integrated, while loss of heterozygosity (LOH) at the selected microsatellite loci was marginal. However, a segment from a cirrhotic liver with heterogeneous integration events showed progressive LOH within a group of clonally and spatially related cirrhotic nodules, while the HBV genome was lost with the progress of genetic instability. Thus, viral integration may have induced genetic instability which in turn led to the progress of cirrhosis, and to the subsequent loss of integrated virus [16]. 3.2. Hepatitis C virus Both cytolytic and non-cytolytic immune mechanisms provided by cytotoxic T-cells contribute to the clearance of viral genomes from an infected liver after primary infection [17,18]. However, HCV infection turns chronic in the majority of cases, because clearance of the virus is mostly insufficent. Chronic HCV infection can cause liver cirrhosis and HCC as well. HCV replicates its RNA genome in the cytoplasm, and unlike retroviruses or HBV, it does not integrate into the cellular genome. The core protein and the nonstructural proteins NS3 and NS5A have been linked to hepatic oncogenesis. All three proteins may exert their effects through transactivating cellular genes after translocation of their truncated forms to the nucleus. They interact with the cell cycle or apoptosis-associated proteins p53 and p21/Waf1 (CDKN1A), and the transcription factor NF-jB through the TGF-b signaling pathway (reviewed in [19]). The core protein has been shown to interfere with a multitude of cell cycle and apoptosis proteins, transcription factors, cytokines and growth factor receptors. In hepatocytes or transgenic mouse livers, the HCV core protein inhibits mitotic spindle checkpoint functions by reducing pRb (RB1) transcription, and leads to chromosomal polyploidy [20]. The NS3 protein which carries three enzymatic functions, a serine protease, an RNA helicase, and an ATP-dependent NTPase, was mostly linked to cellular proliferation and apoptosis, and may contribute to liver fibrosis. NS3 interacts with the checkpoint kinase ataxiateleangiectasia-mutated (ATM) which is essential for the

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cellular response to irradiation, thereby rendering the cell more sensitive to DNA damage [21]. NS5A is part of the viral replicase complex and best known for the relative interferon resistance it confers to HCV. It interferes also with the cell cycle and apoptosis [19]. Shortly after the discovery of hepatitis C virus [22], tissues of HCV-induced HCC were examined for the presence of the HCV genome and its replication. In a set of 16 livers, the positive-stranded HCV genome was found in 23% of the cancerous, but in 62% of the non-cancerous surrounding tissues of HCC-tumors by RT-PCR. The negative-stranded replication intermediates of HCV were found in 46% of non-cancerous tissues, but not found in the cancerous tissues. Thus, HCV replication may be suppressed in advanced HCC, and the HCV genome may be lost in a significant fraction of HCC cells [23]. Since HCV-associated liver cancer is persistently and consistently infected, HCV-HCC will certainly not turn out as a hit and run-classic. However, the possibility exists that the virus becomes dispensable for tumor growth in some cases and is cleared, after the cancer is established. The relative risk to incur a B-cell NHL is modestly increased by 2–4-fold in HCV-infected individuals (reviewed in [24]). In immunocytomas of HCV-infected individuals, the hypervariable region of the immunoglobulin (Ig) heavy chain carries frequent somatic mutations and intraclonal diversity. The direct infection of normal B-cells through HCV in vivo has not been described yet. Therefore, in most cases, this activity is probably not due to the direct infection of B-cells, but most likely to the chronic antigenic stimulus [25] or due to the interaction of the viral envelope protein E2 with its putative receptor CD81 on B-cells [26]. But then, HCV is also able to directly infect lymphomatous B-cells and may thereby increase cellular mutational activities [27]. Indeed, an examination of HCV-infected B-cell lines, lymphomas and HCC showed that the mutation frequency was increased at the Ig heavy chain, BCL6, TP53, and b-catenin (CTNNB1) gene loci. Furthermore, activation induced cytidine deaminase (AICDA, AID) as an essential part of the Ig somatic hypermutation (SHM) machinery was found to be strongly induced through the NF-jB-pathway in HCV infected liver cells [28]. Furthermore, AID-expressing transgenic mice developed HCC at a high percentage which resembled human HCC in expressing the a-fetoprotein (AFP) gene and carrying similar TP53 mutations [29]. Thus, HCV induces a mutator phenotype in the liver and in B-cells which may lead to the post-primary phases of tumor development even in the absence of the virus genome [30]. Accordingly, in a series of HCV-associated B-cell lymphomas, the successful antiviral therapy led to a longlasting complete remission of the lymphomas [31–34]. This resembles the regression of gastric lymphomas of the mucosa-associated lymphoid tissue (MALT) which is associated with Helicobacter pylori infection, after the bacteria are eradicated (reviewed in [35]). In contrast to HCV, chronic HBV infection does not lead to an increased lymphoma rate, although it is usually accompanied with a high serum load of viral protein. This may be due to an immunosuppressive function of HBsAg on myeloid dendritic cells [36].

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3.3. Polyomaviruses, JCV At the time of their isolation in 1971, the two polyomaviruses BKV and JCV have been the only genuinely human polyomaviruses. BKV was isolated from the urine of a renal transplant patient, while JCV was isolated from the brain of a patient with progressive multifocal leukencephalopathy (PML). In severely immune suppressed individuals, hemorrhagic cystitis and nephropathy are associated with BKV reactivation, while PML is associated with JCV-reactivation (reviewed in [37,38]). Very recently, two new human polyomaviruses, KIPyV [39] and WUPyV [40], have been discovered in respiratory secretions, with unclear pathogenic significance. Further, Merkel cell carcinoma virus (MCV), another ubiquitous polyoma virus [41], is a novel cancer-associated virus [42,43]. All five specifically human polyomaviruses are reactivated in immunosuppressed individuals [44,45]. MCV is present in approximately 80% of all Merkel cell carcinomas (MCC), a rare malignant neuroendocrine skin tumor [42,46]. The clonal integration of the MCV genome into the host DNA is likely to be an early event in Merkel cell carcinogenesis [42]. MCV-positive carcinomas carry a better overall survival rate than MCV-negative carcinomas [47]. Since patients with MCVnegative tumors were positive for MCV-antibodies, the virus-negative carcinomas might signify advanced tumor stages with a secondary loss of virus genomes [41]. The integration of high risk HPV strains plays a key role in tumorigenesis, too [48]. Contrary, the regular genomic integration of the JCV genome has not been found in human tumors. Nevertheless, BKV and JCV have been suspected to cause several specific types of human tumors. The molecular mechanisms which inactivate p53 and pRb correspond to those that are established for the high risk HPV types 16 and 18. However, clear epidemiological and molecular links of BKV or JCV with human cancers are so far missing, and the data is still controversial (reviewed in [38]). Humans are ubiquitously infected with BKV and JCV. Since JCV is associated with PML, neural oncogenesis has been looked for in the first instance. When injected into the brains of rodents or monkeys, JCV readily causes brain tumors. Even when given intravenously, JCV causes different types of brain tumors in monkeys, with a latency period of about 2 years. These tumors are non-permissively infected with JCV, express the T-Ag, but not the lytic genes, while the viral DNA is often integrated. Mice transgenic for JCV T-Ag under the control of its own neural tissue-specific promoter develop neuroectodermal tumors. These mouse tumors are histologically similar to human tumors of neural crest origin. Viral proteins, mainly T-Ag and agnoprotein, a small late highly basic protein, have been found to interfere with the cell cycle, DNA repair and apoptotic mechanisms (reviewed in [49]). JCV seems to confer a mutagenic activity to the infected cells which is at least in part due to the emergence of DNA damage secondary to the inactivation of p53 and pRb through T-Ag action (reviewed in [50]). Between 60% and 80% of diverse human brain tumors have been found to contain JCV DNA and, at a slightly lower percentage, JCV proteins. This included also non-PML patients. In brain tumors whose JCV DNA has been sequenced,

mutations of the non-coding control region were frequently observed. Besides neural tumors, JCV DNA and proteins have been found in diverse gastrointestinal tumors, at percentages of between 30% and 60%, while normal epithelia were usually positive for JCV DNA, but negative for viral proteins. This may indicate the intestine as the regular locus of viral latency. Further, certain JCV-positive lung cancers have been reported. However, due to conflicting data and due to the virus-negative fraction of suspected tumors, the causal involvement of JCV in tumor development cannot be proven at present (reviewed in [49]). There have been several studies that failed to detect JCV DNA in brain tumor samples at all, or detected JCV only in a low fraction of tumors. This may have been due to technical issues, like the inferior quality of formalin fixed DNA, the low abundance of JCV DNA in such samples, or low PCR sensitivity (reviewed in [49]). However, the use of advanced techniques like immunohistochemical labeling for T-Ag, followed by laser capture microdissection and PCR amplification of JCV DNA from single tumor cells looks quite convincing [51,52]. A similar controversy as for brain tumors continues on the regular presence or absence of JCV DNA in colorectal carcinomas (CRC). CRC are distinguished by their regular aneuploidy and diffuse chromosomal instability (CIN) [53,54]. JCV DNA was found at 10-fold higher levels in tumor tissue than in the surrounding normal epithelia. Furthermore, through immunohistochemistry, viral proteins were only found in tumor tissue, but not the surrounding epithelia which were also regularly JCV-infected. Introduction of T-Ag into diploid cells led to CIN in the short term [55]. Therefore, T-Ag expression may induce CIN in CRC and thereby lead to a progressive loss of heterozygosity (LOH) at tumor suppressor genes. Going along with the increasing LOH, JCV may be lost from the tumor tissue, and thus explain the virus-negative fraction of JCVassociated brain and CRC tumors (reviewed in [50,56]). An analogous discussion including hit and run-mechanisms is carried on about BKV that potentially may cause neural, skin and urinary tract tumors mainly. However, due to the ubiquitous latency of BKV in human populations, a convincing association between infection and tumors has not been established so far (reviewed in [13,57,58]). The genuinely simian polyoma virus SV40 was discovered as a contaminant of polio vaccines which were used between 1955 and 1963. Since the virus kept circulating in human populations long after the use of contaminated vaccines has been discontinued, it became a suspect for causing or modifying several human cancers, mainly pleural mesothelioma. However this issue is still highly controversial and needs further clarification (reviewed in [38]). 3.4. Adenoviruses Adenoviruses usually cause benign, self-limiting infectious diseases of the upper respiratory tract, the gastrointestinal tract, the conjunctiva or the urinary bladder. In the severely immune suppressed, e.g. liver transplant patients, adenoviruses can cause serious systemic disease which may affect all organs. Infectious adenoviruses persist over long time in lymphoid and epithelial tissues

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without causing any symptoms. Their early proteins E1A and E1B are able to inactivate the tumor suppressor proteins pRb and p53, respectively. More recently, Ad5 E4orf3 and E4orf6 early proteins have been shown to enhance cellular transformation together with E1A and E1B, or to replace E1B function in transforming cells (reviewed in [59]). Although specific adenovirus subtypes can transform human and rodent cells and cause malignant tumors upon injection into rodents, adenoviruses are not associated with human cancers. Therefore, all the potential evidence for hit and run-mechanisms in adenoviruses can so far only refer to transformed cells in culture or to tumors in immune suppressed rodents, but not to the genuinely malignant cells of human cancer. Adenovirus type 12 (Ad12) is known to cause multiple tumors in newborn syrian hamsters at the injection site. Usually, multiple copies of Ad12 are integrated into a variable single chromosomal site for each clonal tumor. Several subclones of adenovirus transformed, oncogenic cell lines have been examined by PCR for the loss of viral genomes. Some subclones were found to have shed most of the viral genomes, but all of them retained diverse small DNA fragments mostly from the E4 region. Therefore, Ad12 genomes may be gradually lost from tumor cell lines, while the tumorigenicity of the cells remained preserved [60]. An analysis on possible adenoviral hit and run-mechanisms was conducted through combinatorial transfection of the Ad5 E1A, E1B, E4orf3 and E4orf6 genes into primary baby rat kidney cells. Cells that were transformed by E1A together with E1B regularly retained the viral DNAs and proteins. Contrary, when E4orf3 or E4orf6 replaced the E1B function, viral DNAs and proteins were regularly lost from the cells shortly after transformation, as demonstrated by PCR and immunoblotting or immunoprecipitation. A subset of those consistently virus DNA-negative transformed cell lines even exhibited tumorigenicity in nude mice. Furthermore, in a mutagenicity assay, E4orf3 or E4orf6 together with E1A were able to increase the mutation frequency at the hypoxanthine phosphoribosyltransferase (HPRT1) locus in Chinese hamster ovary (CHO) cells [61]. E4orf6 functions as part of an E3 ubiquitin ligase complex. One mechanism by which the E4 proteins orf3 and orf6 contribute to transformation may be their inhibition of the catalytic subunit of the DNA dependent protein kinase (DNA-PK) which is necessary for the repair of double stranded (ds) DNA breaks. Further, they block apoptosis through E4orf6-mediated proteasomal degradation of p53 (reviewed in [59]). Besides p53, additional DNA repair proteins are inactivated by E4orf6-mediated proteasomal degradation: DNA ligase IV as a central component of the non-homologous end-joining DNA repair system [62], and Mre11 as a member of the Mre11/ Rad50/Nbs1 (MRN) DNA repair complex [63]. Further, the degradation of integrin-a3, a cell surface protein involved in the regulation of cell adhesion and differentiation may contribute to transformation [64]. Through its interference with ds DNA break repair proteins, E4orf6 also inhibits V(D)J recombination and adenoviral DNA concatenation, the latter of which is considered to be a cellular defense mechanism against viral replication. Besides inhibiting ds DNA break repair by targeting repair proteins for proteaso-

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mal degradation, E4orf6 also leads to a significant cellular radiosensitization [65]. While viral interference with DNA repair mechanisms may explain the transforming potential, the subsequent loss of adenoviral DNA upon transformation may, perhaps, be selected for in order to keep the transformed cells continuously proliferating [61]. Since adenoviruses carry prominent transforming abilities, but have not been found to be associated with human cancer, they might, however unlikely, turn out as the sneakiest hit and run-oncogenic agents of all. On the other hand, they may, more down-to-earth, serve as a suitable probe to highlight and work out the distinction between transformation and oncogenesis. 3.5. Herpesviruses, EBV Herpesviruses are large DNA viruses with a linear ds DNA genome inside a membrane coated icosahedral capsid. EBV, a ubiquitous human herpesvirus that infects more than 90% of the world population is the causative agent of infectious mononucleosis (IM). Being the prototypical tumor virus, it is associated with a wide variety of neoplasms. These include lymphoid and epithelial tumors, Burkitt’s lymphoma (BL), Hodgkin’s lymphoma (HL), extranodal natural killer (NK) T-cell lymphoma, lymphoproliferations in solid organ transplant or bone marrow recipients (PTLD, posttransplant lymphoproliferative disease), AIDS-associated lymphomas, undifferentiated nasopharyngeal carcinoma (NPC), gastric carcinoma, carcinomas of the salivary glands, rare cases of thymic carcinoma, the mesothelial tumor leiomyosarcoma, and possibly breast carcinoma. Some of those tumors, namely NPC, endemic BL, NK/T-cell lymphoma, primary CNS lymphoma or HL of AIDS patients, leiomyosarcoma and the lymphoepithelial type of gastric carcinoma, are associated with a monoclonal EBV-infection of the tumor cells in more than 90% of the cases, approaching 100% in some cases. Other tumors, namely HL, BL of AIDS patients, the adenomatous type of gastric carcinoma, and the extremely rare follicular dendritic cell tumors are associated with EBV-infection at lower proportions between 10% and 50% (reviewed in [66–72]). Of the EBV latency genes, mainly EBNA2 and LMP1 have been linked with B-cell transformation, whereas the multiply spliced BART (also called CST) transcripts and their presumable gene products have been linked with the immortalization of epithelial cells [73]. The small EBV encoded nuclear RNAs (EBERs) exert an anti-apoptotic function [74]. They can be secreted from infected cells and activate innate immunity through Toll-like receptor 3 signaling [75]. Due to their abundant transcription in tumors, EBER in situ hybridization has traditionally served to classify tumor cells as EBV-infected or not. Not only latent, but also lytic viral replication seems to play a role in tumorigenesis. The strong elevation of antibody titers against lytic viral antigens months to years before the onset of BL [76], HL [77], gastric carcinoma [78], and NPC has led to the development of serologic tests as a cancer screening tool in South China [79]. Lytic EBV replication is recombinogenic for the viral genome, and may be recombinogenic for cellular DNA as well. Rearranged and defective viral genomes have been observed in the lytic BL-cell line P3HR1 [80,81]. The

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rearranged ‘‘WZhet DNA” disrupts the latent state, and it does not stably persist in the cells [80]. The recombination points of het DNA have been mapped with nucleotide resolution. They occur regularly in salivary samples from immune suppressed patients and in epithelial samples from patients with oral hairy leukoplakia [82,83]. By contrast, WZhet DNA could not at all be observed in paraffin embedded materials from numerous patients suffering from EBVassociated benign or malignant diseases [84]. Thus, rearranged viral DNA may mostly occur in the absence of an intact immune system, e.g. in cell culture, or it may also regularly occur in vivo in a transient fashion. 3.5.1. Burkitt’s lymphoma In BL tumor cells, multiple circular EBV episomes are attached to the chromosomes through the nuclear matrix attachment region of the viral genome [85–87]. Therefore, the regular maintenance of viral episomes in BL-cells is the rule, episome loss the exception. However, large deletions and rearrangements of the viral genome occur naturally in BL-cell lines [88–90]. Viral integration into host cell chromosomes was also regularly observed [91,92]. Further, in vitro infected BL-cell lines regularly carried integrated EBV genomes, while in lymphomas or PTLDs of immune suppressed patients EBV integration did rather not occur [93]. EBV episomes have been shown to coexist with integrated genomes in several BL-cell lines [94]. The loss of the episomal copies left the integrated copies of the BL60 subline P7 the only ones remaining [95]. But even integrated viral genomes were lost from BL60 cells after fusion with its corresponding EBV-positive lymphoblastoid cell line (LCL) IARC 277. In this case, the episomal copies were the only ones remaining [96]. Virus loss has been explained through the virus-induced generation of genomic fragile sites in lymphoma cells which may lead to the partial or complete loss of viral genomes together with fragments of the cellular genome [97]. The regular loss of the episomal copies from the unusual Akata BL-cells led to a proportion of EBV-negative Akata sublines of about 50% [98]. A further indication for EBV-triggered genomic instability and the loss of EBV genomes from BL-cells was observed in the Mutu BL tumor. An early passage clone that exhibited a class III viral latency pattern contained episomal copies and a large amount of lytic replication accompanied by rearranged WZhet DNA which parallels the induction of the lytic cycle of virus replication. Upon long-term culture, both the episomal copies and rearranged WZhet DNA were lost, while the cells now contained two separately integrated copies. All viral DNA may disappear from BL tumors in vivo as well. The absence of integrated copies or their secondary loss, and the loss of all episomal viral genomes may explain a fraction of EBV-negative BLs [99]. A case in point are sporadic BLs that were originally misclassified as EBV-negative tumors, but turned out to contain integrated fragments of the EBV genome [100]. Regular viral integration and rare excision events, therefore, may signalize the overall genomic instability of BL tumors [100]. Another indication for virus loss was observed in the Oma-BL1 tumor. This tumor contained both EBV-positive and EBV-negative cells which are derived from the same patient and are monoclonal with regard to their cellular

genome. Both EBV-positive and -negative cells could be subcloned from early passage tumor cells, but not from established cell lines anymore [101]. Overall, integration into the cellular genome and subsequent loss of EBV genomes, i.e. events that may form the basis of hit and run-tumorigenesis have in some cases been observed in BL-cells in vitro. But obviously, there is no hit and run in endemic BL tumors. This may be due to the continuous need for apoptosis resistance which is conferred to the tumor cells by the EBV genome. Furthermore, the EBV genome contains binding sites for the multifunctional viral EBNA1 protein, the human oncoprotein c-Myc, and CCCTCbinding factor at its locus control region (LCR) [85–87,102– 107]. This viral LCR provides chromosomal attachment and transcription functions, and may provide enough stickiness for the viral genome to regularly hang onto the nucleus. Hit and run-events might turn out to explain a fraction of the EBV-negative cases of sporadic or AIDS-associated BL. 3.5.2. Hodgkin’s lymphoma Classical HL is associated with EBV-infected HRS cells in 30–50% of cases in industrialized countries, with lower and higher rates depending on the respective HL subtype, while in parts of Latin America, Africa and Asia the EBV-positivity rate is up to 100%. In Europe, the mixed cellularity and lymphocyte depleted HL subtypes contain high rates of about 80% of EBV-positive cases, while the lymphocyte predominant and nodular sclerosis subtype contain low ratios of EBV-positive cases. The presence of EBV-positive tumor cells signifies a better survival in young HL patients, but a worse prognosis in older patients [108]. Strikingly, IM is associated with a 3-fold increased risk for children and young adults to develop EBV-positive HL later [109,110]. This is underscored by the fact that early stage HL involving the cervical lymph nodes which are mostly affected by IM is more frequently EBV-positive than early stage disease in non-cervical lymph nodes. In later disease stages this difference disappears, possibly due to the tumors spreading throughout the body [111]. Further, there are HL cases that have developed directly out of an IM [112]. However, passing through primary EBV-infection in childhood without symptoms does not increase the risk for HL [113]. In contrast, asymptomatic primary infection may even be helpful for developing one’s own immune system [114]. A functional immune system plays a role for the genesis of HL. The high risk of AIDS patients to develop HL is decreased for all HL subtypes when the CD4 cell counts at AIDS onset are strongly decreased. At very low CD4 cell counts between 0 and 49 per ll blood, the nodular sclerosing subtype of HL which is mostly EBV-negative was not at all observed, while the mixed cellularity subtype which is mostly EBVpositive was relatively increased [115]. The same HLA-A1 alleles which are associated with an increased risk of EBV-positive HL are also associated with an increased risk of experiencing IM instead of asymptomatic primary infection [116–118]. Seemingly paradoxical at first, the EBV-positivity rate of HRS cells is lowest for the young adults who are at the highest risk to develop HL after IM. Especially, the nodular sclerosis subtype which occurs most frequently in this age group is even less EBV-positive in young adults than in others [119]. However, large

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population-based case-control studies concluded that the risk for EBV-negative HL among young adults clearly is not increased after IM, while EBV-positive HL cases among young adults had a strong correlation with delayed EBVinfection and IM [110,113]. This leaves the EBV-negative cases among young adults for another explanation than previous IM. Therefore, HL may consist of different disease entities that are only in part caused by delayed EBV-infection, in part caused by other factors that are associated with the same lifestyle and behaviour which also predisposes to delayed EBV-infection [120]. A fraction of EBV-negative HL may be explained by hit and run-mechanisms. Some EBV-positive cases of HL have been reported which relapsed in an EBV-negative form. One of those cases was a nodular sclerosing HL in a 5 year old boy whose tumor relapsed 1 year after remission. Both tumors were analyzed by in situ hybridization, and PCR on microdissected HRS tumor cells. The relapsed tumor showed similar clinical, histological, and immunohistochemical features as the initial tumor. Therefore, it was assumed that the virus was required for the initiation, but not for the maintenance of the malignant state, and had been lost [121]. The loss of EBV upon relapse of a HL is unusual, however, since in most cases initial tumor and relapsed tumor exhibit the same EBV-status [122]. Another series of onset and relapse tumors of 23 classical HL cases was analyzed by EBER in situ hybridization and LMP1 immunohistochemistry. 14 cases remained positive, eight cases remained negative, but one nodular-sclerosing tumor changed from EBV-positive at onset to EBV-negative at relapse [123]. In order to exclude the misclassification of tumors, due to the partial loss of EBV genomes or due to the epigenetic silencing of the EBER genes, in situ hybridization with cosmid clones spanning the entire viral genome was applied to HRS cells. Among eight young adult HL cases, the HRS cells of three biopsies were EBV-positive by both LMP1 immunohistochemistry and hybridization of all genomic fragments, while the remaining five were EBVnegative in all tests [124]. Another survey of 72 HRS EBV-negative young adult cases found no correlation of classical HL with EBV-positive serostatus. Patients with EBV-negative HRS samples were more frequently seronegative than a matched control group, a trend that tended to be stronger towards the younger. Furthermore, a PCR survey on genomic DNA from 30 HRS EBV-positive and -negative tumor samples covering seven separate regions of the virus genome did not yield hints for partially deleted or rearranged EBV genomes. Since some young adult patients with HRS EBV-negative HL have never had contact with EBV, the virus cannot be responsible for all classical HL cases [125]. However, the loss of viral genomes from initially EBV-positive HRS cells may be caused by the pressure of a more functional adult immune system. This is underscored by the fact that in severely immune suppressed individuals the HRS EBV-positivity rate is higher [126]. Therefore, a set of 56 classical HL cases was analyzed for the presence of defective viral WZhet DNA. The HRS cells of 32 cases tested EBV-positive by EBER in situ hybridization, while 24 of the cases were EBV-negative. About one-third of both groups contained WZhet DNA. A smaller subset of 42 tumor samples was analyzed by both standard

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PCR and in situ PCR for WZhet DNA with largely congruent results. Another smaller subset of six EBER-negative, WZhet genome-positive tumors was examined for the presence of unrearranged or undeleted EBV genomes. Four tumors contained unrearranged genomes in addition, while two did not. In contrast, all EBER-positive samples contained unrearranged genomes, independent of their WZhet status. Thus, the absence of unrearranged EBV genomes in EBV-infected HL is principally possible, and in some cases the complete loss either of EBV genomes or the eradication of initially EBV-infected HRS cells in the course of tumor development is conceivable on the basis of this study [127]. 3.5.3. Breast cancer Throughout female populations worldwide, breast cancer risk is inversely associated with the risk of contracting so called ‘‘microbial cancers” (i.e. hepatitis B virus-hepatic cancer, H. pylori-gastric cancer, human papillomavirus-cervical cancer) in a strikingly hyperbolic way [128]. The delayed exposure to a common infectious agent has been linked with breast cancer [129,130], and cells resembling HRS cells have been found in breast tumors, too [131]. Consequently, delayed primary EBV-infection was shown to not only correlate with subsequent HL, but also with breast cancer susceptibility [132]. However, a different situation as for other EBV-associated tumors has been observed in breast cancer, and its molecular association with EBVinfection is still disputed [133]. Breast cancer was found to be associated with EBV-infected cells in less than 50% of cases [134]. In EBV-positive tumors, low or variable copy numbers of the viral genome were detected in a minority of tumor cells [135]. Lytic EBV-infection contributed to the detection of viral genomes in carcinoma tissue [136]. The estrogen receptor (ER) -positive subtype which represents the dominant majority of cases in high risk female populations, carries a much lower EBV-positivity rate than the more malignant and advanced ER-negative subtype [132,137]. Therefore, EBV-infection of breast carcinoma cells is generally considered a late event in tumor progression or a disease modifier that may make tumors more aggressive and metastasis-prone [13]. In some cases, hypothetical yet, where EBV-infection may be an early event, the specificity, strength or duration of the immune response may be an important risk factor for breast cancer, besides the intracellular alterations triggered by the EBV genome itself. Accordingly, breast cancer occurs less frequently in severely immune suppressed individuals [138]. 3.5.4. Nasopharyngeal carcinoma Undifferentiated NPC, a highly endemic lymphoepithelioma in southeast Asia, is virtually always EBV-infected [139,140]. Although a hallmark genetic lesion like the translocation of the MYC gene of BL is missing, NPC tumors are characterized by a high load of somatic mutations and epigenetic alterations. Further, food carcinogens and a long-lasting childhood viremia play a major role in the generation of NPC [71,141–143]. Monoclonal viral infection has been found in high grade dysplastic lesions and carcinoma in situ, but not in low grade lesions and in healthy epithelia. However, EBV-infected high grade NPC precursor stages are

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only rarely observed [144–148]. Therefore, EBV-infection has been suggested to take place as a critical second event in tumorigenesis, after genetic alterations of nasopharyngeal cells have already taken place [143,149]. On the other hand, the virus may catch on first, or enter nasopharyngeal cells more than once in a ‘‘multiple hit”-fashion. A more stable viral infection may then be established on the basis of epigenetic and genetic cellular alterations which may be induced by sequential EBV-infections [71]. Besides, viral microRNAs that are transferred from EBV-infected cells to non-infected other cell types may contribute to target cell conditioning [150] and may pave the way for viral infection of non-B cells [151]. As all EBV episomes are rapidly lost from NPC explant cultures within few passages, C666-1 is the only NPC cell line so far that has retained its viral genomes under prolonged culture [152,153]. Upon NPC explantation, individual tumor clones were able to retain viral episomes for prolonged passage. However, along with successive small deletions and viral rearrangements, all subclones eventually turned EBV-negative [153]. NPC cells that have completely lost their EBV genomes stayed tumorigenic in immune suppressed mice. However, infection of EBV-negative NPC cell lines increased their tumorigenicity [154]. Like in BL, EBV episomes coexist with integrated viral genomes in s NPC tumors [155]. The BamHI A-fragment which codes for the BART transcripts, and the putative oncogene BARF1, is strongly transcribed in NPC cells [156,157]. Upon transfection of a rearranged viral 40 kb fragment containing one viral W-repeat and the BamHI fragment A among other fragments, diverse epithelial monkey cell types were immortalized [158]. Also human mammary epithelial cells were growth stimulated through transfection with this cosmid clone [159]. Upon subsequent culture of subclones, the integration of viral into cellular DNA, the amplification of the W-repeat, and the loss of viral DNA from the chromosomes were observed [158,160]. On continued passaging, the viral DNA reappeared in an increasing number of cells within extrachromosomal bodies resembling double minute (DM) chromosomes. The immortalization process took place without a crisis event, in contrast to EBV-mediated B lymphocyte transformation [161]. It was concluded that the DM bodies may have provided a function in preventing telomere shortening [160]. These observations suggest that EBV-induced recombinogenic activities may be able to contribute to hit and run-cancerogenesis in epithelial cells. However, immortalization of simian epithelial cells and EBV genome loss from such cells in culture has so far not been reproduced by other research groups. 3.5.5. Mutagenic events in EBV-infected cells The viral life cycle is deeply intertwined with the B-cells and their specific functions. Accordingly, there are numerous points of contact and parallels between EBV and the Bcell. Upon primary infection, EBV remains latent for life in memory B-cells [162,163]. Remarkably, the left part of the EBV genome appears to be structurally colinear with the rearranged Ig gene loci, suggesting that both the EBV genome and the Ig locus may have descended from a common precursor [164]. The EBV-encoded latent membrane proteins LMP2A and LMP1, functional homologues of two

key B-cell surface receptors, are important for establishing viral latency in B-cells. One of the cytokines up-regulated by LMP1 is interleukin-10 (IL10) which is involved in the class switch recombination (CSR) of the Ig genes. In addition, EBV contains its own functional IL10 homologue encoded in the BCRF1 reading frame [165]. Through BCRF1, EBV may interfere with the antiviral immune response during the acute phase of infection in order to establish viral latency as a hiding-place from the NK-cells.

3.5.5.1. Germinal Center reaction. Mutagenic key events in the life of a B-cell are somatic hypermutation (SHM) and CSR of the Ig genes within the germinal centers (GC) of lymphoid organs. A physiological GC reaction yields B-cell clones of several hundreds to thousands of cells. For a Bcell to successfully undergo both SHM and CSR, it must stay in contact with an antigen-presenting dendritic cell through the B-cell receptor, and with a T-helper cell through the CD40 receptor. If the contacts are lost, the Bcell normally undergoes apoptosis. LMP2A and LMP1 are able to interfere with or replace the functions of the B-cell receptor and CD40, respectively [166,167]. Through LMP2A and LMP1, EBV may rescue a B-cell which would be apoptosis-bound, due to a failed GC reaction, from imminent apoptosis. Apparently, the c-myc translocation of endemic BL, and several other oncogene translocations in NHLs mostly occur as molecular accidents of the GC reaction [168]. Thus, the EBV-infection of a B-cell which suffers an oncogene translocation during the GC reaction may increase the likelihood that such a dangerously mutated B-cell survives the strong apoptotic forces of the GC [104]. The W-repeat units, also called the major internal repeat 1 of EBV, contain CSR-related sequences which are able to bind an identical regulatory protein complex as Ig switch recombination sequences do. During primary infection of B-cells, the W-repeats are instrumental in the recombination of EBV genomes [169–171]. The EBNA2 deletions in the BL-cell lines P3HR1 and Daudi are likely due to the recombinogenic activity of the W-repeat [89,172,173]. Also the rearranged viral genomes observed in tissues from HL, sporadic BL, thymic carcinoma, and in the epithelial lesions from oral hairy leukoplakia patients may have been generated by similar recombinogenic activities in EBV-infected cells [83,100,127,174]. EBV-infection has been shown to support a high CSR activity in B-cell lines [171]. LMP1 induces AID, the key enzyme involved in SHM and CSR. Thus, LMP1 can induce CSR independent of surface CD40 expression. Through the dysregulation of CSR, LMP1 may contribute to accidental mutagenesis. Infection of B-cells in vitro induced the expression of AID and error-prone DNA polymerase-g (POLH), another key enzyme involved in SHM and CSR. The expression of both enzymes coincided with an increased number of mutations in the cellular protooncogenes, BCL6, TP53, and the b-globin (HBB) gene in EBV-positive lymphoma cell lines [175]. Further, EBV-infected cell lines continued hypermutating their IgH genes [176–178]. An increased genetic instability of B-cells [179] and epithelial cells [180] was observed after EBV-infection. Burkitt-like translocations between the MYC and Ig genes

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were enhanced through the induction of AID expression in B-cells [181]. However, contrary to He et al. who reported that AID was induced in LCLs through LMP1 signaling [182], Tobollik et al. reported that AID could be induced by LMP1 only in specific settings when EBNA2 was not expressed [183]. They found that AID was not regularly expressed in latency class III cells. The EBV growth program even silenced AID expression through its master transcription factor EBNA2. Further, in GC cells in vivo, the coexpression of AID and the EBER, LMP1 or EBNA2 genes of EBV was only rarely observed [183]. This is consistent with the observation that EBV-infected cells, although physically located in GCs, did usually not participate in GC reactions [184,185]. Only under conditions of immune hyperstimulation, clonal expansions of EBV-infected B-cells in GC reactions were observed [184]. Recently, it was confirmed that EBV-infected cells, even when expressing GC-markers, did not proliferate under physiological circumstances in the GC reaction [186,187]. Most likely, LMP1-expression in the physiological GC mostly permits only an abortive GC reaction [188]. 3.5.5.2. VDJ-recombination. Another combinatorial key event in the life of a B-cell is the VDJ-recombination of the Ig genes with the help of the recombination activating gene (RAG) proteins 1 and 2. The two RAG proteins, together forming a DDE-type recombinase (D: aspartate, E: glutamate), are transiently expressed in the bone marrow and thymocytes. They recognize specific recombination signal sequences (RSS) flanking the V, D and J segments which are instrumental in the recombination of the B- and T-cell receptor genes. The RAG proteins were induced by EBNA1 in EBV-negative BL-cells [189]. Transgenic mice expressing EBNA1 in B-cells under the control of the immunoglobulin enhancer showed an increased level of the RAG mRNAs in preneoplastic B-cells, but not in the frequently resulting lymphomas of the transgenic mouse lines [190]. Some EBV-infected B-cell lines expressed high levels of the RAG proteins [191]. Further, in the peripheral blood of IM patients, RAG expression was regularly found in PBMCs [192]. The viral DNA-binding protein BALF2, a processivity factor for lytic replication, exhibits a structural homology with the RAG proteins and other herpesviral DDE-type recombinases, and is most likely a functional recombinase [193]. Furthermore, BALF2 and the RAG genes are coregulated through similar cis-acting transcriptional elements [194]. However, the RAG genes are not expressed in most established BL, HL and follicular lymphoma cell lines in general. Further, there was no RAG expression in EBV-infected lymphoma samples [195]. In GCs they are not expressed either. However, in extrafollicular bystander lymphocytes, RAG expression was observed [196]. Apparently, the RAG proteins are not directly involved in the oncogene translocations of NHLs [168]. Then again, in EBV-associated lymphomas or epithelial tumors they may be involved in mutagenic activities other than classical oncogene translocations. The RAGs may be expressed transiently and may be regularly switched off after accomplishing their recombinogenic feat. Like the internal W-repeats, also the terminal repeats (TR) of EBV are GC-rich recombinogenic sequences whose

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number varies between viral strains. They are the sites of circularization and linearization of the viral genome during lytic replication [197,198]. The TRs contain the same sequence motifs and protein binding sites that are found at the VDJ-RSS [194]. Viral linearization and circularization is not mediated by simple cleavage and ligation, but by homologous recombination which facilitates genome rearrangement and ds DNA break repair. Ds DNA breaks of the viral genome have been shown to occur in replication compartments. Therefore, replication protein A (RPA), Rad51, Rad52, and the MRN complex which are involved in homologous recombinational repair (HRR), are loaded onto the replicating EBV genomes in an ATM checkpoint-dependent manner, together with the viral replication factors BALF2 and BMRF1 [199]. Thus, during lytic replication, HRR proteins, the RAG proteins or BALF2 are probably involved in rearranging the viral genomes. They may also be responsible for further rearrangements of viral and cellular DNA. In addition, they may accomplish the traceless elimination of EBV genomes from an infected tumor cell, in this way leaving a formerly EBV-infected tumor cell virus-free [200]. 3.5.5.3. Immunoglobulin-related gene expression in non-B cells. Proteins that are involved in immunoglobulin gene mutation and recombination may play a broader oncogenic role, not only for B-cell lymphomas, but also for T-cell lymphomas and carcinomas. Indeed, there has been an early report of immunoglobulin gene transcripts in cancer cells which was detected with a highly sensitive RT-PCR [201]. Since every tissue-specific gene is illegitimately transcribed in any cell type at very low abundance, the significance of this finding was ambiguous [202]. In addition to immunoglobulin and RAG transcripts, however, the expression of cytoplasmic and secreted immunoglobulins was found by laser capture microdissection and immunohistochemistry in breast, colon, liver, and lung cancer tissue, and even normal lung epithelia. Secreted immunoglobulins acted as tumor growth factor in nude mice. Furthermore, several epithelial cancer cell lines exhibited RAG and immunoglobulin transcription [203]. RAG mediated recombination may further be responsible for deletions at the CDKN2A (p16INK4a) tumor suppressor locus in T-cell acute lymphatic leukemia (T-ALL) cases [204]. The expression of AID, RAG and immunoglobulin transcripts and immunoglobulin proteins was recently reported in a set of diverse tumor cell lines and 66 different epithelial tissues of increasing malignancy [205]. Accordingly, AID transcripts were found in all of six different breast cancer cell lines, while the immunoglobulin heavy chain variable (VH) genes were VDJ-rearranged in four lines, somatically mutated in five and switched in two cases. EBV-infection was not found in any of the cell lines [206]. The finding of immunoglobulin-related gene expression in epithelial cancers and the induction of RAG expression in preneoplastic lymphatic samples of EBNA1-transgenic [190] mice may lead the way to a more systematic examination of the expression profiles of immunoglobulin, RAG, and AID genes also in EBV-infected epithelial cancers. Actually, IgG-j was found to be induced by LMP1 through NF-jB and AP1 signaling in the NPC cell line HNE2. The

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expression of immunoglobulin proteins was weaker than in B-cell lines, but strong enough for visualizing them in western blots [207].

4. Epigenetic mechanisms 4.1. Epigenetic changes during oncogenesis In multicellular organisms the establishment of cell identity and cell-type specific gene expression patterns is regulated by epigenetic mechanisms. This means that coregulated activation or silencing of certain gene-sets is achieved by epigenetic systems that leave mitotically and/or meiotically heritable marks on DNA or chromatin. In mammals the major epigenetic regulatory systems include DNA methylation, the Polycomb–trithorax group of protein complexes and histone modifications (reviewed in [208–212]). Epigenetic regulatory systems can achieve permanent, heritable changes in gene function without affecting the nucleotide sequence. Therefore, epigenetic marks constitute a molecular memory for longer-lasting signaling stimuli during the history of a neoplastic cell clone. This corresponds to the establishment of an epigenetic field for cancerization, in which epigenetic dysregulation coincides with the development of malignancy. In an epigenetic cancer field, the degree of epigenetic dysregulation decreases with the anatomical distance from the center of a tumor [213]. A distinct feature of cancer cell genomes is global hypomethylation associated with local hypermethylation [214]. The latter frequently affects the regulatory regions of tumor suppressor genes. Therefore, epigenetic and especially DNA methylation changes became promising biomarkers for monitoring the conversion of a cell towards a malignant cell clone (reviewed in [215]). As an experimental example for the epigenetic memory of signaling, the durable knock-down of the estrogen receptor (ER) in the breast carcinoma cell line MCF-7 led to a progressive methylation of more than 70 gene loci as a consequence. Correspondingly, ER-expressing and ER-nonexpressing breast cancer cells carried distinct methylation patterns [216]. Another example for specific methylation patterns corresponding with specific tumor types is gastric carcinoma. Different types of gastric carcinoma are distinguished by their different methylation patterns (reviewed in [71]). The p16INK4a and p14ARF (CDKN2A) promoters were more frequently methylated in EBV-positive than in EBV-negative gastric carcinomas. However, if methylation was present in both types of gastric carcinoma, the methylation patterns at specific CpG-dinucleotides in both promoters was different in dependence of the EBV-status of the tumor [217]. Furthermore, a highly specific association of methylation at the TP73 [218] and the HOXA10 [219] gene promoters was found with EBV-positive gastric carcinoma cases. Thus, specific epigenome profiles may generally correspond to specific tumor types. We propose that hit and run-oncogenesis may also leave permanent traces through epigenetic dysregulation. This means that tumor viruses or other carcinogenic agents may exert a short hit or a longer-lasting stimulus that results in the epigenetic reprogramming of the target cell.

Subsequently, the presence of the modifying agent may become dispensable for the continuation of multistep oncogenesis, while the epigenetic alterations may remain. An example for a transcriptional hit and run-scenario can be observed in transcription factor-induced chromatin remodeling. In the presence of its ligand dexamethasone, glucocorticoid receptor (GR) binds to its cognate binding sites. GR directs the binding of Switch/Sucrose Non-fermentable (SWI/SNF) to closed promoter chromatin which is remodeled into open chromatin in the presence of ATP. GR in turn is lost from the promoter during in the remodeling process [220,221]. Another example is the proteasomal deubiquitinating enzyme Uch37 which is part of the Ino80 chromatin remodeling complex. Through its transient contact with other proteasomal proteins, Uch37 becomes activated, thereby activating the Ino80 chromatin remodeling complex in turn [222]. Chromatin remodeling complexes may offer contact points for viruses to hit onto transcriptional regulation, DNA repair, and permanent epigenetic alterations in the infected cell. 4.2. Epigenetic reprogramming mediated by viral oncoproteins It was demonstrated recently, that the HBV-encoded pX, a pleiotropic regulator, is capable to elicit such paradoxical epigenetic changes in liver cells by affecting the expression of DNA methyltransferases. First of all, pX up-regulated cyclin D1 and activated DNMT1 expression via the cyclin D1Cdk4/6-pRb-E2F1 pathway, that resulted in a DNA methylation-mediated decrease of the tumor suppressor p16INK4a level in HepG2 cells [223]. Independently of Dnmt1, two variants of Dnmt3a, termed Dnmt3a1 and Dnmt3a2 which are translated from differentially spliced trancripts of the DNMT3A gene, were also found in increased amounts in liver cells transfected with a pX-expressing vector [224]. This resulted in regional hypermethylation of specific tumor suppressor genes. In parallel, pX downregulated Dnmt3b in the very same cells. Because Dnmt3b is involved in the methylation of satellite 2 repeat sequences, pX induced a global hypomethylation of these repeats [224]. Interestingly, a CpG-rich sequence hypomethylated in hepatocellular carcinomas which carried integrated HBV genomes was found in the peri-centromeric region of human acrocentric chromosomes, although this sequence, despite its similar localization, differed from the typical a-satellite sequence [225]. Similarly to HBV, HCV also activates DNA methyltransferases. Activation of DNA methyltransferase 1 and 3b by the core protein of HCV may switch off the E-cadherin (CDH1) promoter via hypermethylation [226]. A similar mechanism may silence the promoters of interferon-stimulated genes [227]. It is noteworthy that the promoters of several tumor suppressor genes were inactivated in both HCV and HBV-associated hepatocellular carcinomas [228–230]. It was demonstrated that oncoproteins of papilloma and polyomaviruses and human adenoviruses inactivate members of the retinoblastoma pocket protein family and thereby release activating E2F transcription factors that control the coordinated transcription of genes in-

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volved in DNA replication and cell cycle progression (reviewed in [231,232]). One of the targets of E2F is the DNMT1 gene [233]. Accordingly, both BKV T-Ag, and the human adenovirus E1A protein could stimulate E2F activity and thereby activate the transcription of the DNMT1 gene [234]. In addition, E1A and the E7 oncoprotein of HPV type 16 could directly associate with Dnmt1 and stimulate its activity in vitro [235]. In addition, the E1A protein also interacts with the TRRAP and Gcn5 subunits of the Tip60 histone acetyl transferase (HAT) and nucleosome remodeling complex. Further, E1A led to the genome wide relocalization of p300/CBP HATs in order to achieve a gene expression profile that is suitable for adenovirus replication (reviewed in [236]). Interestingly, the methylation pattern of cellular genes changed in Ad12-transformed rodent cells, while the increased methylation of these cellular sequences was maintained, even after the complete loss of integrated adenovirus genomes [237]. Chromosomal insertion of foreign (Ad12, plasmid, or bacteriophage lambda) DNA was associated with enhanced methylation of cellular DNA segments [237] (reviewed in [238]). This observation suggests that the epigenetic alterations elicited by a tumor virus may prevail even in the absence of the viral genome. Hit and run-transformation was simulated in vivo in a model experiment where the large T-Ag of the BKV relative SV40 was expressed transiently in the salivary glands of transgenic mice. The expression of T-Ag was under the control of a tetracyclin-dependent promoter and, therefore, could be conditionally switched on for selected time-periods. Ductal hyperplasia with polyploid cells developed increasingly in the submandibular glands. When T-Ag expression was silenced after 4 months, the hyperplasia regressed. However, when T-Ag expression was switched off after 7 months only, the ductal hyperplasia was irreversible. Independent of further T-Ag expression, the ductal cells remained transformed [239,240]. Obviously, interference of a transforming viral protein with the p53 and pRb tumor suppressor pathways makes the cellular genome vulnerable for additional molecular accidents that may lead the way to a full-blown malignancy later on. SV40, however, also affects epigenetic regulation. SV40-induced immortalization of fibroblasts resulted in a stabilization of Dnmt levels [241]. In addition, in a lung cancer model, the expression of DNMT3B was essential for the oncogenic transformation induced by SV40 large T-Ag [242]. De novo methylation of selected cellular genes was associated both with immortalization and oncogenic transformation in both experiments. Latent infection by oncogenic herpesviruses is regularly associated with epigenetic dysregulation of the target cell genomes (reviewed in [71,243]). Expression of the EBVencoded oncoprotein LMP1 up-regulated the expression and activity of cellular DNMTs 1, 3a and 3b in vitro, and resulted in the hypermethylation of the E-cadherin (CDH1) promoter [244]. LMP1 activated DNMT1 via the c-jun NH(2)-terminal kinase/activator protein-1 (JNK-AP1) signaling pathway [245]. KSHV is the causative agent of Kaposi’s sarcoma, primary effusion lymphoma and multicentric Castleman’s disease. The latency associated nuclear antigen (LANA)

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encoded by KSHV was reported to be associated with Dnmt1, Dnmt3a and Dnmt3b [246]. This resulted in de novo methylation of the H-cadherin (CDH13) promoter. In addition, LANA relocalized Dnmt3a and to some extent also Dnmt3b from the nuclear matrix to the chromatin. Thus, LANA may induce promoter-specific de novo DNA methylation by recruiting a de novo DNA methyltransferase activity [246]. In addition, LANA silenced the promoter of the transforming growth factor-b type II receptor (TGFbR2) in primary effusion lymphoma cells by site-specific induction of CpG methylation and deacetylation of proximal histones [247]. The retrovirus HTLV-1 infects CD4-positive human Tcells, and can cause adult T-cell leukemia (ATL) or neurological diseases, especially tropical spastic paraparesis, also called HTLV-1-associated myelopathy (TSP/HAM), in a small proportion of infected individuals. The proviral genome of HTLV-1 encodes the transactivator protein tax which has transforming capabilities. Tax interacts with numerous transcriptional activators, like CREB/ATF, AP1, NF-jB which drive T-cell proliferation and inhibit apoptosis, and with chromatin modifying enzymes, like histone acetyl and methyl transferases (HMT), and the SWI/SNF complex (reviewed in [248]). Tax expression causes multipolar mitoses by inducing centrosome overduplication [249], and asymmetrical chromosome segregation [250]. Expression of the viral tax oncoprotein is frequently lost in T-cell lymphoma and leukemic cells due to deletions of the proviral genome or to epigenetic silencing by CpG methylation (reviewed in [251]). The resulting epigenetic dysregulation could prevail, even in the absence of tax, during the further steps of oncogenesis and tumor progression. Tax may act as a hit and run-oncoprotein, because it can induce promoter silencing by inducing dissociation of transcription factors from the promoter of the Src homology-2-containing protein tyrosine-phosphatase 1 (Shp1, PTPN6) gene, followed by hypermethylation of the promoter [252]. Because Shp1 is a tumor suppressor protein, tax-induced epigenetic reprogramming of target cells could be an important early step for leukemogenesis.

5. Concluding remarks At present it is difficult to judge the relevance of viral hit and run-mechanisms which have been observed in cell culture or on selected tumor samples for clinical oncology. It cannot be excluded that such a mechanism or possible epigenetic variants thereof, like ‘‘hit and hide” or ‘‘hit and hang around”-oncogenesis may contribute to the generation of several human neoplasms. In the variant model, the viral genome would not be lost, but silenced by epigenetic mechanisms, e.g. the HTLV-1 tax gene in ATLs. Actually, anecdotal evidence has continuously increased in favour of such mechanisms. However, a negative case is hard to prove. Novel experimental methods may be required to fill in the gaps of viral hit and run-oncogenesis. Ideally, the regular presence of a suspected tumor virus in pre-malignant tissue and its regular subsequent loss which accompanies the development from dysplasia to malignancy should be observed in a series of tissue sec-

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tions. Secondly, the infection of EBV-negative tumor cells with EBV, regularly followed by an induced episome loss or the induced episome loss from naturally EBV-infected tumor cell lines could make hit and run-models more plausible. Thirdly, genome-wide epigenetic analyses may establish specific epigenetic changes that are regularly conferred to cellular genomes by specific tumor viruses. Such epigenetic signatures may be permanently retained even after a traceless elimination of the respective viral genome from an established cancer cell and may be recognized as a footprint of past viral infection. Conflicts of interest None declared. Acknowledgment We thank Dr David H Dreyfus, Yale School of Medicine and Keren Pharmaceutical, for helpful discussions. References [1] G.R. Skinner, Transformation of primary hamster embryo fibroblasts by type 2 simplex virus: evidence for a ‘‘hit and run” mechanism, Br. J. Exp. Pathol. 57 (1976) 361–376. [2] D.A. Galloway, J.K. McDougall, The oncogenic potential of herpes simplex viruses: evidence for a ‘hit-and-run’ mechanism, Nature 302 (1983) 21–24. [3] D.A. Galloway, J.K. McDougall, Alterations in the cellular phenotype induced by herpes simplex viruses, J. Med. Virol. 31 (1990) 36–42. [4] G. Bauer, S. Kahl, I.S. Sawhney, P. Hofler, R. Gerspach, B. Matz, Transformation of rodent fibroblasts by herpes simplex virus: presence of morphological transforming region 1 (MTR 1) is not required for the maintenance of the transformed state, Int. J. Cancer 51 (1992) 754–760. [5] J.A. Nelson, B. Fleckenstein, D.A. Galloway, J.K. McDougall, Transformation of NIH 3T3 cells with cloned fragments of human cytomegalovirus strain AD169, J. Virol. 43 (1982) 83–91. [6] D.J. Clanton, R.J. Jariwalla, C. Kress, L.J. Rosenthal, Neoplastic transformation by a cloned human cytomegalovirus DNA fragment uniquely homologous to one of the transforming regions of herpes simplex virus type 2, Proc. Natl. Acad. Sci. USA 80 (1983) 3826–3830. [7] T. Iwasaka, Y. Hayashi, M. Yokoyama, K. Hara, N. Matsuo, H. Sugimori, ‘Hit and run’ oncogenesis by human papillomavirus type 18 DNA, Acta Obstet. Gynecol. Scand. 71 (1992) 219–223. [8] Y. Shen, H. Zhu, T. Shenk, Human cytomagalovirus IE1 and IE2 proteins are mutagenic and mediate ‘‘hit-and-run” oncogenic transformation in cooperation with the adenovirus E1A proteins, Proc. Natl. Acad. Sci. USA 94 (1997) 3341–3345. [9] T. Iwasaka, M. Yokoyama, Y. Hayashi, H. Sugimori, Combined herpes simplex virus type 2 and human papillomavirus type 16 or 18 deoxyribonucleic acid leads to oncogenic transformation, Am. J. Obstet. Gynecol. 159 (1988) 1251–1255. [10] J.A. DiPaolo, C.D. Woodworth, N.C. Popescu, D.L. Koval, J.V. Lopez, J. Doniger, HSV-2-induced tumorigenicity in HPV16-immortalized human genital keratinocytes, Virology 177 (1990) 777–779. [11] C.C. Lau, I.K. Gadi, S. Kalvonjian, A. Anisowicz, R. Sager, Plasmidinduced ‘‘hit-and-run” tumorigenesis in Chinese hamster embryo fibroblast (CHEF) cells, Proc. Natl. Acad. Sci. USA 82 (1985) 2839– 2843. [12] H. zur Hausen, Viruses in human cancers, Eur. J. Cancer. 35 (1999) 1174–1181. [13] J.S. Pagano, M. Blaser, M.A. Buendia, B. Damania, K. Khalili, N. RaabTraub, B. Roizman, Infectious agents and cancer: criteria for a causal relation, Semin. Cancer Biol. 14 (2004) 453–471. [14] D. Cougot, C. Neuveut, M.A. Buendia, HBV induced carcinogenesis, J. Clin. Virol. 34 (Suppl. 1) (2005) S75–S78. [15] I. Chemin, F. Zoulim, Hepatitis B virus induced hepatocellular carcinoma, Cancer Lett. 286 (2009) 52–59.

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