Oncogene (2001) 20, 7820 ± 7823 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc

Oncogenic DNA

Harald zur Hausen*,1

1Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany

Oncogene (2001) 20, 7820 ± 7823 anogenital papillomaviruses were isolated directly from cervical biopsies (DuÈ rst et al., 1983; Keywords: viruses; cancer; transformation Boshart et al., 1984; Schwarz et al., 1935). The most recent types linked to human are represented by virus (Choo et al., 1989) and Human Herpesvirus type 8 (HHV-8) (Chang et Introduction al., 1994). There exist good reasons to argue that still other virus types are involved in human cancers: The early discoveries of Gross (1951) of viral causation Human immunode®ciency viruses types I and II of murine leukemias, the subsequent demonstration of induce severe immunosuppression and facilitate cancer as the causative factors and the presence of induction by other persisting infections, in particular similar viruses particularly in lymphoproliferative by HHV-8, Epstein-Barr virus and human papilloma- disorders in cattle, cats, and various rodent species, viruses. Thus, these agents contribute indirectly to underlined for decades the justi®cation of attempts to human . It is likely, in addition, that search for similar agents in human cancers, subse- speci®c cutaneous papillomavirus infections contribute quently identi®ed as retroviruses. When Stehelin et al. indirectly to skin carcinogenesis by blocking apoptosis (1976) proved the cellular origin of retroviral onco- in cells exposed to ultraviolet light (Jackson and , it appeared to be almost odd to search for Storey, 2000). agents who possess their own oncogenes and may be At present it is dicult to apply stringent criteria for involved in human cancers. the identi®cation of human tumor viruses. Commonly, Yet, until today, with the exception of the human T- these agents are strictly host-speci®c and several of lymphotropic type I as causative agent of them cannot be maintained under tissue culture adult T- leukemia, endemic in the coastal regions of conditions. The application of Koch's postulates southern Japan, di€erent groups of viruses emerged as therefore turns out to be impossible. Several attempts prime factors even for some common human cancers. have been made to rede®ne criteria to determine the Besides Hepatitis C, they all contain DNA as genetic role of viruses in human carcinogenesis (Evans, 1976; material and belong to very di€erent virus families. zur Hausen, 1994a; Vonka, 2000). Major problems Roughly 15% of the global cancer burden can be arise due to direct and indirect modes of interaction in linked to these infections. An additional 5% can be cell transformation. For these reasons it appears to be attributed to bacterial (Helicobacter pylori) and para- useful to di€erentiate between these two alternatives. If sitic infections (Schistosoma, Opistorchis, Clonorchis). we understand a direct contribution as expression of DNA virus types known to be involved in human speci®c viral oncogenes or the selective insertion of cancers are listed in Table 1. viral into de®ned cellular genes, the following criteria seem to be useful (modi®ed from zur Hausen (1994a)): Identi®cation of human cancer viruses . The regular presence and persistence of the respec- The initial discovery of herpes-type viral particles tive viral DNA in tumor biopsies and cell lines de- (Epstein-Barr virus) later on linked to speci®c human rived from the same tumor type; malignant disease was made by Epstein et al. (1964). . The demonstration of growth-promoting activity of T-lymphotropic retrovirus type I was discovered in speci®c viral genes or of virus-modi®ed host cell 1980 and linked to adult T-cell leukaemia (Poiesz et genes in tissue culture systems or in suitable al., 1980, Hinuma et al., 1981). A ®rm link of systems; Hepatitis to liver cancer became apparent . The demonstration that the malignant phenotype from epidemiological studies published by Beasley et depends on the continuous expression of viral onco- al. (1981). Starting in 1983 and 1984 novel types of genes or on the modi®cation of host cell genes con- taining viral sequences; . Epidemiological evidence that the respective virus *Correspondence: H zur Hausen; infection represents a major risk factor for cancer E-mail: [email protected] development. Oncogenic DNA viruses H zur Hausen 7821 Table 1 DNA viruses linked to human cancers Virus Tumor types Mode of interaction

Epstein-Barr virus (EBV) Burkitt's lymphoma Direct Lymphomas under immunosuppression Hodgkin's disease (*40%) Subset of T-cell lymphomas Human Herpesvirus Type 8 Kaposi's sarcoma Direct Body cavity lymphoma Human papillomaviruses (HPV) HPV 16, 18, 31, 33 and others Anogenital cancers, oral cancers (*20%) Direct HPV 5, 8, 14, 17, 20 and others Cancers in patients with epidermodysplasia Direct? verruciformis Indirect? HPV 20, 23, 38 and others Squamous cell carcinoma of the skin Indirect? virus Direct? Indirect?

It is more dicult to develop stringent criteria for transformations mediated primarily by the Epstein ± agents acting as indirect carcinogens. Here an evalua- Barr virus nuclear antigen 2 (EBNA-2) and related tion depends on epidemiological data, on experimental proteins, EBNA-2 transactivates genes in the Notch- results explaining possible modes of interaction (e.g. pathway by interacting with the cellular protein RBP-J prevention of apoptosis, ampli®cation of persisting kappa (Hsieh et al., 1996; Strobl et al., 1997). The nucleic acids of other potential tumor viruses, or regulation of viral expression by cellular factors immunosuppression by the respective agent), and on has been less intensively studied in comparison to clinical observations. The identi®cation here is usually human papillomaviruses. Yet, it is an interesting more a question of plausibility than of a stringent observation that overexpression of the cellular myc experimental deduction. gene results in a Burkitt-like phenotype of the respective cells, suppressing the expression most EBNA genes, but permitting the expression of EBNA 1 (Polack et al., 1996). Mechanisms by which viruses contribute to cancer The contribution of to the development of hepatocellular carcinoma is presently Our understanding of virus-induced oncogenesis has poorly understood. It is still not possible to assign a substantially increased during the past two decades. direct or indirect role of this virus to human The emerging picture reveals a remarkable variation in carcinogenesis. Although a large number of liver cancer mechanistic contributions. The two oncogenes of high biopsies in Hepatitis B-endemic regions contain risk anogenital papillomaviruses, the E6 and E7 genes fragments of the viral , no consistent pattern of HPV types 16 and 18, modify a large set of cellular has been observed up to now (see review Arbuthnot genes, either by changing their expression pattern, by and Kew, 2001). As an alternative explanation, the functionally inactivating them or by targeting them for virus may act as an indirect carcinogen by mediating a degradation (see reviews by Mantovani and Banks and chronic in¯ammation of the a€ected liver inducing an MuÈ nger et al. (this issue). In addition, however, their abundant production of reactive oxygen radicals. own expression is tightly regulated by cellular factors Three other modes of mechanistic contributions of and depends on the state of di€erentiation of infected viruses to carcinogenesis deserve some discussion: the keratinocytes. The interruption of this cellular control immunosuppression induced by human immunode®- in infected basal layer cells emerges as a prime factor in ciency viruses has been mentioned before. As a cell immortalization, but also in malignant conversion consequence of the AIDS epidemic, secondary virus- (reviewed in zur Hausen, 1994b, 1996). Whereas events linked tumors emerge as increasingly important linked to immortalization seem to result from mod- contributions to the spectrum of tumors caused by i®cations of intracellular signaling cascades, in malig- infections. nant conversion intercellular regulatory mechanisms The recent demonstration of prevention of Bak- are a€ected, rendering the malignant cells non- induced apoptosis by the E6 protein of several responsive to TNF-a mediated changes in the hetero- cutaneous papillomavirus types (Thomas and Banks, dimerization of the AP-1 complex (Soto et al., 1993, 1998; Jackson and Storey, 2000; Mantovani and 2000) and reducing their ability to induce endogenous Banks, this issue) may represent another most -b synthesis (Bachmann et al., 2001). interesting example for an indirect contribution of Infections by Epstein ± Barr virus and human virus infections to carcinogenesis. A high percentage of Herpesvirus type 8 similarly result in the induction of squamous cell carcinomas of the skin contains the viral oncogenes that a€ect a large number of host cell same HPV types (Shamanin et al., 1996; Astori et al., proteins. The transforming mechanism, however, 1998; de Villiers, 1998) although their copy number is di€ers since other signaling cascades are modi®ed. In far below one copy per tumor cell. This would be

Oncogene Oncogenic DNA viruses H zur Hausen 7822 compatible with the speculation that these viruses are tumorigenic for other animal species if inoculated not required for the maintenance of the transformed under the same conditions. Bovine papillomaviruses phenotype but contribute indirectly by permitting the types 1 and 2 even induce tumors (sarcoids) in horses survival of UV-damaged cells that otherwise would be and donkeys under natural conditions. With the prone to apoptosis. exception of the rhesus monkey virus SV40, very few virus infections have been shown to additional studies have been conducted to test human e€ectively induce persisting of polyoma- and cancers for a possible involvement of animal viruses. papillomaviruses, even under conditions of abortive These agents may not be able to replicate in human infection (Schlehofer et al., 1983; Schmitt et al., 1989; tissues, but still could express early cellular growth- Heilbronn and zur Hausen, 1989). Similarly, human stimulating functions. A careful study, excluding some (CMV) e€ectively promotes the re- of these agents as direct or indirect carcinogens for plication of the human polyomavirus JC in human humans is still missing (zur Hausen, 2001). ®broblasts, although these cells are non-permissive for The epidemiological pattern of some human tumors JC in the absence of CMV (Heilbronn et al., 1993). would be compatible with an infectious etiology. This These modes of interaction could represent an accounts for instance for slightly more than 50% of additional indirect contribution of viruses to carcino- Hodgkin's disease, speci®cally the EBV-negative tumors. genesis. Besides these experimental ®ndings, however, Other lymphomas and childhood leukemias may repre- there exists at this stage no epidemiological or clinical sent additional candidates. We should, however, bear in evidence supporting this notion. mind that for a number of tumor types presently linked In view of the fact that RNA-containing tumor- to infections, epidemiological clues were missing prior to viruses still reveal di€erent mechanistic contributions the isolation of the responsible agents. This accounts for to oncogenesis, we are confronted with a broad example for gastric cancers, hepatocellular carcinomas spectrum of various interventions by infections to linked to infection and for HPV-linked redirect the cellular growth pattern. oral cancers. Based on these considerations at this stage no human cancer can be excluded from a potential etiological involvement of infectious agents, unless other Do we know all human tumors linked to virus infections etiological factors have been ®rmly documented. and do we know all human cancer viruses? During the past decade the discovery of novel types of Perspectives viruses, potentially involved in the stimulation of cell growth increased substantially. Most of these isolates The identi®cation of infectious agents as causative were hitherto unknown papillomavirus types, most factors for presently close to 20% of the global cancer frequently derived from the analysis of squamous cell burden can be considered as one of the major carcinomas of the skin (SCC), from normal skin achievements in cancer research of the past two or biopsies or from mucosal surfaces (Boxman et al., three decades. Besides our increasing understanding of 1997; Astori et al., 1998; de Villiers, 1998). It appears the mechanism underlying cancer development, prac- that many of these viruses do not cause overt disease tical consequences of these results are presently and probably produce at best microlesions barely emerging: they are only visible for therapy of the detectable without magnifying lenses. Besides their respective tumors to a limited degree, more so for possible involvement in SCC by an indirect (anti- diagnostics and the identi®cation of persons at risk, apoptotic) function, their possible relationship to other most remarkably however for cancer prevention. The tumors remains unknown. Two virus types belonging preventive value of early postnatal Hepatitis B to di€erent virus families were identi®ed in 1989 and vaccination already became recognizable in regions 1994; Hepatitis C virus and HHV-8. The possible with a high prevalence of Hepatitis B virus persistence. involvement of the two much longer known human Clinical trials with vaccines against high risk papillo- polyomaviruses BK and JC in some human cancers is maviruses are well on their way and reveal a still under discussion, although the data remain remarkable immunogenicity of the vaccine prepara- controversial (see Imperiale, this issue). tions used. Eradication of Helicobacter pylori infec- A number of human pathogenic viruses have been tions by antibiotic treatment may turn out to prevent shown in the past to be tumorigenic upon inoculation gastric cancer. Thus, the patient search for infections as into newborn rodents (reviewed in zur Hausen, 2001). cancer-causing factors is presently paying o€. This will Similarly, a number of animal viruses turn out to be be even more visible in forthcoming years.

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