Abeloff's Clinical Oncology, 4th Edition

Part I – Science of Clinical Oncology

Section B – Genesis of Cancer

Chapter 11 – Viruses and Human Cancer

Paul F. Lambert,Bill Sugden

SUMMARY OF KEY POINTS

  

   

The last 35 years have revealed the existence of human tumor viruses.

  

   

Human tumor viruses contribute to at least 15% of all human cancers.

  

   

The study of tumor viruses led to the discovery of cellular oncogenes and tumor suppressor genes, which are key contributors to cancers regardless of the etiology.

  

   

Virally induced tumors can be eradicated by the development of prophylactic viral vaccines, or other public health measures for preventing transmission.

INTRODUCTION

Viruses cause cancers in people. Today, approximately 15% of all human cancers are thought to have a viral etiology,[1] and this fraction is likely to grow as we investigate additional cancers for a potential viral cause and identify new human viruses. Identifying a human cancer as having a viral etiology has substantive consequences both for its treatment and for its prevention. The known virally caused human cancers often express virally encoded products in the tumor cells. These viral products are potential targets for antiviral, tumor-specific therapies. Viral infections can be prevented by vaccines; therefore, it might be possible to eliminate those human cancers that require viral contributions for their development.

HUMAN TUMOR VIRUSES

The search for human tumor viruses has been propelled by a long appreciation that viruses can cause cancers in birds and rodents. Viruses were isolated as filterable extracts from avian tumors in the first decade of the twentieth century and were shown to induce tumors in susceptible animals. [2] [3] Parallel findings were made in mice in the 1940s.[4] These animal tumor viruses were in the retrovirus family and led researchers to look for retroviruses as human tumor viruses in the 1960s and 1970s. However, most of the human tumor viruses that were subsequently identified are in different virus families and do not conform to some of the expectations that have been derived from the study of highly oncogenic animal retroviruses. For example, highly oncogenic tumor viruses induce tumors in animals rapidly. The inoculation of Rous sarcoma virus into the wing web of newborn chicks can induce fatal sarcomas within 2 weeks in 100% of susceptible animals.[5] In addition, highly oncogenic tumor viruses are oncogenic because they have acquired and express potent derivatives of cellular proto-oncogenes. In fact, many of the known human proto-oncogenes were first identified as homologs of the oncogenes that are transduced by the highly oncogenic animal retroviruses.[6] Known human tumor viruses, however, usually do not induce cancers rapidly; often, 15 to 50 years will elapse between the primary infection and tumor development. Nor do human tumor viruses express cellularly derived oncogenes; rather, some of them have evolved to inhibit cellular tumor suppressor genes. These differences between highly oncogenic animal viruses and human tumor viruses probably have contributed to the reticence in our recognizing that viruses do cause cancers in people.

A second obstacle in our recognizing that viruses can be tumorigenic in their human host is that we lack convincing animal models in which to test these viruses directly. For all practical purposes, all known human tumor viruses infect only people. In addition, we now know that human viruses that are found not to be tumorigenic in people are tumorigenic when experimentally introduced into test animals. For example, human adenovirus 12, which causes only respiratory infections in people, is highly oncogenic when inoculated into newborn hamsters.[7] The lack of an experimentally tractable animal host for human tumor viruses has required multiple lines of evidence to affirm that a given virus can contribute to a given human cancer. In particular, epidemiologic findings have been combined with genetic and molecular analyses in cell culture to identify human tumor viruses. Experiments with mice that are transgenic for viral genes have also supported these identifications.

Here, we shall introduce the six known human tumor viruses, the tumors with which they are associated, data that support these associations, and models to explain the viral contributions to these tumors. These viruses will be presented in the order of their discovery; early findings have often provided insights for analyses of subsequently identified viruses. Finally, we shall outline the kinds of virus-specific therapies that it might be possible to develop and the likelihood of developing vaccines to human tumor viruses in order to limit or eliminate specific human cancers.

Epstein-Barr Virus

Epstein-Barr Virus (EBV) was identified through the insight and advocacy of Dennis Burkitt, who, as a young surgeon, having identified Burkitt's lymphoma as a new disease entity, analyzed the geographic and climatic distribution of this childhood lymphoma, determined that it overlapped with that of malaria, and posited it to have an infectious etiology.[8] At his urging and with his proffered biopsy samples, Tony Epstein and his colleagues identified EBV in Burkitt's lymphoma–derived cells.[9] To do so, they developed the expertise to propagate these cells in culture.[10] Cell lines derived from EBV-positive Burkitt's lymphomas proved to be powerful tools to associate EBV with different human diseases. Different EBV-positive cell lines express different viral antigens and thereby have served as test samples for patients’ expressing antibodies to EBV-encoded antigens.

The analyses of these antibodies by serology led the Henles in Philadelphia to propose EBV as the cause of infectious mononucleosis. [11] [12] A colleague in their laboratory who had lacked antibodies to EBV-encoded antigens developed those antibodies on presenting with infectious mononucleosis. The etiologic role for EBV in this “self-limiting lymphoproliferation” was subsequently established by careful, prospective epidemiologic studies in which serology was used to demonstrate that only immunologically naive people were at risk of developing infectious mononucleosis; on doing so, they would express antibodies first to EBV-encoded antigens of the IgM class and only later to those of the IgG class.[13] Thus, about 85% of infectious mononucleosis cases were shown to arise from a primary infection with EBV. Serologic studies also allowed the Henles to propose that nasopharyngeal carcinoma (NPC) might be caused by EBV because NPC patients were characterized by having atypically high titers to EBV-associated antigens.[14] However, the data that linked EBV causally to Burkitt's’ lymphoma and NPC by the early 1970s was only “guilt by association.” While EBV caused most infectious mononucleosis on primary infection, serology had also demonstrated that children in the parts of Africa in which Burkitt's lymphoma is endemic and adults in the parts of China in which NPC is prevalent had all been infected with EBV, that is, were “EBV-seropositive,” long before they developed these cancers.

The serologic analyses of EBV in the 1960s and 1970s illustrate a conundrum for viruses and human cancers: “How can many people be infected with a given virus, and yet how can that virus contribute to tumor development in only a few infected subjects after long periods of time?” This apparent paradox applies, in fact, to most cancers associated with human tumor viruses and explains a major reluctance to consider viruses as etiologic agents for human cancers. The World Health Organization, without resolving this conundrum, sponsored a prospective epidemiologic survey in Uganda to assay 42,000 youngsters serologically for evidence for or against EBV's contributing causally to Burkitt's lymphoma. The region that was studied had a high incidence of this cancer. Children were bled, their serum was stored, and those who were later identified as developing Burkitt's lymphoma were bled again, and their titers to EBV-antigens were determined. This prospective survey found 14 youngsters who developed Burkitt's lymphoma over the 5 years they were followed, and those who did develop the lymphoma had, prior to tumor development, on average a 3.4-fold higher titer of antibodies to one class of EBV-antigens than did the children who did not develop the lymphoma.[15] That is, for children in the portions of the world in which EBV-associated Burkitt's lymphoma is endemic, a high titer of antibodies to a given set of EBV-encoded antigens represents a 30-fold risk factor for developing Burkitt's lymphoma.[15]

Do these findings prove that EBV causes Burkitt's lymphoma? No, they do not; proof in such cases for which direct experiments are not feasible ultimately comes from the accretion of supporting findings in the absence of confounding data. The World Health Organization study did, however, make it unlikely that EBV is a passenger virus that merely replicates well in tumor cells, because the antibody titers were elevated 7 to 54 months before tumor detection.[15] Similar prospective surveys were carried out in China and identified antibodies of the IgA class to the same set of EBV antigens as a risk factor for developing NPC.[16]

The association of EBV with Burkitt's lymphoma and NPC and the demonstration that EBV causes the bulk of infectious mononucleosis have led researchers to consider other diseases with which EBV might be associated. During the last 20 years, EBV has also been linked to post-transplant lymphoproliferation disease (PTLD),[17] oral hairy leukoplakia,[18] approximately one third to one half of Hodgkin's disease, [19] [20] and one tenth of gastric carcinomas.[21] These linkages have been made not only through serology, but also by molecular genetic analyses that render the linkages more robust. The latter analyses have been made possible by the elucidation of the molecular virology of EBV in cell culture.[22]

EBV is a herpesvirus; it has a double-stranded DNA of 165,000 to 170,000 base pairs[23] and encodes approximately 80 genes[24] ( Fig. 11-1 ). Like other herpesviruses, EBV has two distinct phases to its life cycle. It can infect cells, express a small subset of its genes (see Fig. 11-1 ), and cohabit with the cell without killing it. This is its “latent” phase. EBV can also emerge from its latency, express all or most of its genes, amplify its DNA, assemble progeny virions, and kill its host cell by lysis. This is its “lytic” phase. Unlike neurotropic herpesviruses such as herpes simplex virus type 1 and varicella zoster virus, EBV in its latent phase need not be maintained in a nonproliferating host cell. Rather, it has the capacity to both initiate and maintain proliferation in at least the β-lymphocytes that it infects in cell culture and at early stages of primary infection in vivo. [25] [26] It is EBV's ability to affect proliferation and survival of its infected host cell that likely renders it oncogenic.

 
 

Figure 11-1  Map of the EBV genome. The genome of the B95-8 strain of EBV in its circular double-stranded DNA form of 165 kbp is depicted as it is found in latently infected B cells. The genome is a linear DNA within the viral particle and is circularized on infection at its terminal repeat (TR) elements found at the ends of the linear molecule. The segments of the circle denoted with letters represent the fragments of the DNA generated by cleavage with the BamHI enzyme and used to map EBV's open reading frames. The EBV genome encodes approximately 100 genes. Shown as boxes are the exons for coding segments of those viral genes that are expressed in B cells infected in vitro, including EBNA1, EBNA2, EBNA3A/B/C, EBNA-LP, LMP1, and LMP2A/B. The boxes denoted EBERS are the two small RNAs that are encoded by EBV. Dashed lines represent primary transcripts originating from viral promoters denoted with a lowercase p. Also shown are the positions of the origins of replication, OriP and OriLyt, that support the latent and lytic replication of the viral genome, respectively. The expanded arcs represent the two regions of EBV DNA found recently to encode miRNAs (miRNAs derived from the BHRF1 and the BART transcripts). Many of these miRNA genes are deleted in the B95-8 strain.

 

 

EBV induces and maintains infected β-lymphocytes to proliferate by maintaining its DNA extrachromosomally and expressing at least five viral genes that regulate expression of both viral and cellular genes and control viral DNA replication.[27] EBV-infected cells that contain intact viral DNA and express two or more viral genes are hallmarks of all EBV-associated diseases. The identification of viral DNA and of viral gene products, our gradual appreciation of the functions of these viral gene products, and the immune responses to them now constitute much of the persuasive evidence linking EBV causally to its associated cancers.

EBV clearly can induce and maintain proliferation of infected B cells. Genetic experiments in which two viral genes, EBNA2 and LMP1 (see Fig. 11-1 ), within the context of the virus are expressed conditionally demonstrate that each gene product when assayed alone needs to function for infected B cells to continue to proliferate. [28] [29] These observations are particularly telling because they help to explain the multistep evolution of Burkitt's lymphoma. Many other genetic analyses have shown that three additional viral genes—EBNA1, EBNA3a, and EBNA3c (see Fig. 11-1 )—contribute to some facet of cell proliferation.[27] EBNA1 has recently been found to be required for survival of infected cells and, at least indirectly, to be required to maintain cell proliferation as well.[30] EBNA3a acts at the stage of initiation of proliferation.[31] All of these viral “transforming” genes except for EBNA1 are recognized by the host's cytotoxic T-cell response.[32] EBNA1 encodes a stretch of gly-gly-ala residues that inhibits its proteolytic degradation and subsequent presentation by class I HLA molecules.[33] The host's cytotoxic response is sufficiently robust that patients recovering from infectious mononucleosis lack B cells that express RNAs encoding these transforming genes. The surviving EBV-infected B cells are in distinct, differentiated states in which they no longer proliferate and detectably express only another viral protein, LMP2 (see Fig. 11-1 ), a viral gene product that is not required for cellular proliferation. [34] [35]

The failure of this robust immune response to EBV's transforming proteins contributes to PTLD. The infected proliferating B cells in these immunosuppressed patients express all five of EBV's transforming proteins.[6] Two kinds of successful treatments for PTLD demonstrate the critical role of the patient's immune response in failing to limit this “iatrogenic” tumor. First, if immunosuppression can be reduced for the patient such that the transplant is still tolerated, PTLD may regress. Second, several groups have amplified the donor's T cells that are cytotoxic for EBV's transforming proteins prior to bone marrow transplantation. Treatment of PTLD patients with these syngenic, specific T-killer cells has cured the disease.[36] These encouraging findings underscore the important role of the immune response in limiting the survival of EBV-infected cells.

Two startling features of tumor cells that have been freshly isolated from Burkitt's lymphoma biopsies help to explain the evolution of this tumor when considered in the context of EBV's transforming genes and the immune response to them. These tumor cells express only EBNA1 among the required viral transforming proteins, yet they proliferate.[37] They also display a chromosomal translocation between one of three human immunoglobulin loci and the c-myc proto-oncogene. [38] [39] The juxtaposition of an immunoglobulin locus to c-myc drives expression of the proto-oncogene in these B cells, as it does in murine plasmacytomas that display similar translocations.[40] These observations can be arranged to provide a satisfying, though necessarily speculative, model for the genesis of Burkitt's lymphoma. First, EBV infects young children living in regions of central Africa in which malaria is endemic.[41] The malaria is a T-cell immunosuppressive that decreases the children's ability to limit proliferation of infected cells.[42] Youngsters with severe infections do have increased antibody titers to viral antigens but have more proliferating B cells and are at increased risk for the chromosomal translocations, fostered by the recombination mechanism, that occur in B cells and use signals at immunoglobulin loci.[43] A rare immunoglobulin/c-myc translocation provides the cell constitutive proliferative signals that can substitute for those provided by EBV's LMP1, EBNA2, EBNA3a, and EBNA3c. A developmental switch occurs in a cell such that these four viral transforming genes are not transcribed, EBNA1 is transcribed from a different promoter, and the cell continues to proliferate but can no longer be recognized by the host's residual cytotoxic T-cell response. The cell proliferates, acquires additional mutations (often mutations inactivating p53), and evolves rapidly into a Burkitt's lymphoma.[44] This model fits well with what we know today. However, each of the cancers with which EBV is causally associated is sufficiently idiosyncratic to make it impractical to extend this model beyond Burkitt's lymphoma.

All of EBV-associated cancers do contain EBV DNA and express EBNA1 and EBERs (see Fig. 11-1 ), which are small viral RNAs, and some also express LMP1.[45] We know less about the genesis of these other tumors, but recent findings with NPC provide evidence for an unexpected contribution of EBV to its etiology. Huang and colleagues have shown that EBV infects cells that already can be distinguished as being “preneoplastic” in the evolution of NPC.[46] This finding might lead one to think that EBV is merely a passenger in this tumor. However, 100% of NPC tumors are infected with EBV, making it likely that the virus contributes some selective advantage to infected, preneoplastic cells such that they are the ones that evolve into tumors. It is not known what this selective advantage is, but it is reasonable to hypothesize that EBV could provide these cells proliferative or survival signals, as it does to Burkitt's lymphoma cells during their evolution.

Two additional cancers associated with EBV differ dramatically from Burkitt's lymphoma, NPC, and PTLD in their viral association. Whereas effectively all cases of Burkitt's lymphoma in Africa, all of NPC, and all of PTLD are EBV-positive, only approximately 30% to 50% of cases of Hodgkin's disease and 10% of cases of gastric carcinoma are EBV-positive. [20] [47] This lack of a general association makes it difficult to demonstrate a causal role for EBV in the fraction of tumors that are virus-positive. However, in the cases that are EBV-positive, it has been claimed that all of the tumor cells have been infected. This finding depends on single-cell assays to detect viral gene products such as the EBERs and the LMP1 protein in the tumor cells. Such assays are not 100% efficient, nor is the identification of tumor cells perfect, so it is accurate to conclude only that the bulk of the tumor cells are infected. Were we to know that all of the tumor cells in a given patient are EBV-positive, then we could conclude that viral infection was an early event in the evolution of that tumor. The retention of this extrachromosomal genome would favor the virus's contributing a selective advantage to the evolving tumor cell such that the rare infected cell outgrew any uninfected, precancerous siblings. Any contributions of EBV to virus-positive Hodgkin's disease and gastric carcinoma are therefore uncertain. The strongest evidence for there being some viral contribution is the recognition that EBV does contribute to the other cancers for which most or all cases are EBV-positive.

The many clinical and basic scientific studies of EBV and its associated cancers can be extracted to yield some lessons for tumor viruses in general. First, the viral genomic nucleic acid remains in tumor cells and expresses one or more viral genes. Second, the virus contributes information to infected cells, which provides them a selective advantage in evolving towards tumor cells. However, this information is not sufficient for tumor formation. Additional, multiple rare events must occur in the infected cells for them to evolve into tumors. These additional essential events explain both why only a fraction of the people who become infected with a given tumor virus develop the associated tumors and why they usually do so only after long delays.

Hepatitis B Virus

Hepatitis B virus (HBV) was identified by virtue of its being recognized as an antigen in sera of donors by antibodies in sera of other infected donors.[48] Thoughtful analyses by Blumberg and his colleagues correlated the presence of the antigen with hepatitis, a correlation that was strengthened by the seroconversion of a lab member who contracted hepatitis.[48] By the late 1960s, blood that was donated to blood banks was screened for the antigen, positive samples were removed, and only negative samples were used for transfusions. This early insightful intervention led to a significant reduction in transfusion-associated hepatitis.[49] Blumberg and his colleagues also demonstrated a striking association between HBV, antibodies to its antigens, and hepatocellular carcinoma (HCC).[48] These early findings have been built on to demonstrate that HBV does cause HCC, which is either the fifth or sixth most common cancer in people today. HBV is estimated to cause between 50% and 70% of the approximately 500,000 new cases of HCC in the world each year. [50] [51] Most of the rest of these cases are attributable to hepatitis C virus (HCV), a member of the flavivirus family.

Two kinds of data have established HBV's causal role in HCC. A prospective survey of 22,707 male civil servants in Taiwan was initiated at the end of 1975.[52] Of these subjects, 3454 were found to be positive for HBV's surface antigen (HBsAg), indicating that they were chronically infected with HBV. The whole group of 22,707 members was observed on average for 8.9 years. By the end of 1986, 152 of the 3454 HBsAg-positive men had developed HCC, while only 9 of the 19,253 HBsAg-negative men had developed it. The relative risk of the HBsAg-positive cohort for developing HCC was therefore 100 times greater than that for the negative group.[53] This prospective epidemiologic study provides robust data that the presence of HBV is strongly associated with the development of HCC.

The second kind of data demonstrating that HBV can cause HCC has been derived by removing HBV from a population and determining whether the incidence of HCC declines in the population. Taiwan began vaccinating children in 1984, first with a plasma-derived antigen and eventually with a recombinant antigen. Between 1984 and 1994, the incidence of infection as monitored by the presence of HBsAg dropped from 9.8% to 1.3% among children 12 years or younger.[54]

Similar findings have been made in the Gambia, where children who are vaccinated during their first year develop into only 10% as many chronically infected 9-year-olds as do unvaccinated children.[55]HCC is a cancer that peaks in people between 50 and 60 years of age but does occur rarely in children from 6 to 14 years of age. The incidence of HCC in this latter population in Taiwan dropped from 0.64 per 100,000 per year when averaged from 1981 to 1990 to 0.36 per 100,000 per year when averaged between 1990 and 1994 and is statistically significant (P < 0.01).[56] This decline presumably reflects the eightfold reduction of chronic HBV infection in children who are at risk for developing HCC. The finding that removing a virus from a population decreases an associated cancer in that population is compelling evidence that the virus contributes causally to the cancer. We can expect that a decline of HCC in the vaccinated adult population will be more striking in decades to come.

Although it is clear that HBV causes HCC in people, it is not clear how it does so. Researchers now invoke two distinct contributions, direct or indirect, to explain HBV's oncogenesis. HBV encodes one gene, pX ( Fig. 11-2 ), that can affect viral and cellular transcription and has been proposed to contribute directly to oncogenesis. HCC in general evolves in patients who have marked liver cirrhosis. HBV can contribute to that cirrhosis by providing targets for T-cell killing and thereby could contribute indirectly to oncogenesis. The molecular virology of HBV has illuminated the viral life cycle but has yet to identify its mode of oncogenesis.[57] HBV is a small, enveloped virus with a double-stranded DNA genome, one strand of which is incomplete. The complete viral duplex DNA is 3.2 kbp in length (see Fig. 11-2 ), serves as a template for transcription by RNA polymerase II, and is replicated via reverse transcription of a greater than full-length RNA transcript of approximately 3.4 kbp. All members of the hepadnavirus family preferentially infect hepatocytes. This tropism is apparently mediated by a cellular receptor that is expressed in hepatocytes and by viral transcription being controlled in part by cellular transcription factors principally expressed in hepatocytes. Unlike most DNA viruses, HBV undergoes its complete life cycle to yield progeny virions, which exit from hepatocytes via secretory pathways, without killing the host cell. This anomalous behavior of hepadnaviruses means that in the absence of an exogenous function of the host, an infected hepatocyte could survive, carry out its normal functions, and release large amounts of infectious HBV for long periods of time. Accordingly, some chronically infected people do have large amounts of HBV in their sera.

 
 

Figure 11-2  Map of the HBV genome. The 3200-bp genome of HBV is a circular double-stranded DNA in infected cells (black). The genome in the viral particle is partially double-stranded DNA because of an incomplete extension of the plus-strand by the viral DNA polymerase, P. Shown as colored boxes are the coding segments for the structural (blue) and nonstructural (yellow) viral genes. The arrowheads represent the sites at which translation of the viral proteins initiates. These viral proteins are translated from multiple mRNAs: one for Pre-S1, Pre-S2, and S; one for Pre-S2 and S; one for core and P; and one for x. See Ganem and Schneider[280] for details of HBV's genome and life cycle.

 

 

Mammalian hepadnaviruses encode pX, which is not found in the avian species; only the mammalian members are known to cause HCC in their hosts. This correlation has focused interest on pX as being likely to contribute to the oncogenesis of mammalian hepadnaviruses. It is difficult to gauge pX's potential role in HBV's oncogenesis; however, much information about it is in the literature, yet no ready synthesis of this information explains such a role. Most HCC tumor biopsies retain viral sequences encoding pX,[58] but few express the protein detectably.[59] It has been proposed that pX associates withp53 and inhibits its activation of apoptosis[60]; however, pX is not detected in HCC biopsies,[59] and between 30% and 90% of such biopsies have mutations in p53. [58] [59] The viral protein pX in studies in cell culture can bind one subunit of RNA polymerase II as well as TFIIB. [61] [62] It also associates with Smad4, an integral member of the TGF-β signaling pathway, to foster this pathway's signaling.[63]How these different transcriptional activities of pX might contribute to the evolution of HCC is unclear.

HBV is often integrated in HCC tumors,[64] and it has been proposed that integration of the viral DNA could affect transcription of nearby cellular genes. This suggestion has been strengthened by the recognition that the woodchuck member of the hepadnavirus family does contribute to HCC via insertional mutagenesis.[65] However, HBV has not been found to integrate at sites that can be interpreted to affect its oncogenesis. In addition, HBV DNAs cloned from HCC biopsies have been tested and found not to score as enhancer sequences in hepatoma cells in culture.[66]

The hypothesis that HBV contributes to the development of HCC indirectly by inducing rounds of cirrhosis and subsequent liver regeneration is appealing. HBV infection can be acute or chronic; it appears that acute infection correlates with a robust cytotoxic T-cell response to all viral antigens, while chronic infection correlates with a weak T-cell response.[67] These cytotoxic responses do lead to death of hepatocytes; however, experiments in mice that are transgenic for HBV genes and in infected chimpanzees indicate that there is also a potent noncytotoxic mechanism for limiting viral expression in infected hepatocytes. [68] [69] In these experiments, immune cells release γ-interferon, which by some means inhibits viral gene expression and promotes loss of viral DNA from infected cells. [68] [69] The administration of IL-18 limits viral replication efficiently in a transgenic mouse model by inducing production of both type 1 and type 2 interferons.[70] Chronic, not acute, infection correlates with the eventual development of HCC. How these two modes of eliminating infected cells would be balanced to yield long-term or chronic infection, the resulting cirrhosis, and the concomitant hepatocellular regeneration required for the accumulation of mutations predisposing to HCC is not known.

A role for cytotoxic T cells in the evolution of HCC has been modeled in mice that are transgenic for HBV surface proteins (see Fig. 11-2 ). These mice are tolerant to these viral antigens, but when the mice are reconstituted with syngeneic, nontransgenic bone marrow and subsequently challenged with syngeneic, immune, nontransgenic splenocytes, they develop cirrhosis and maintain cytotoxic T cells that are specific for HBsAg.[71] These animals have long-term liver damage and develop HCC by 18 to 20 months of age.

There are two consequences of the model in which HBV contributes to HCC indirectly. The first is that HBV's oncogenesis should be limited to the liver because it depends on the liver's capacity to regenerate to provide the proliferation required to accumulate mutations that predispose to cancer. HBV does cause only HCC. Second, no function of HBV would be required to maintain proliferation of tumor cells. This latter consequence would mean that therapies targeting functions such as pX's enhancement of transcription would be ineffective.

Human Papillomaviruses

Cervical cancer is caused by human papillomaviruses (HPVs). Over 100 genotypes of HPVs have been identified to date. Most HPV genotypes infect squamous epithelia lining the skin; a subset are mucosotropic and infect stratified, squamous epithelia lining the anogenital tract and oral cavity. A subset of these mucosotropic HPVs, the so-called high-risk HPVs, are associated with more than 99% of human cervical cancers, other anogenital cancers, and a subset of squamous carcinomas of the head and neck, particularly those of the oropharynx. HPV16 and HPV18, the high-risk HPVs that are most common in cancers, are present in over 85% of human cervical carcinomas. The mucosotropic HPVs are thought to be transmitted sexually. The association of specific mucosotropic HPVs with human cancers was first recognized in the 1980s when Harald zur Hausen and associates at the German Cancer Research Institute in Heidelberg detected the presence of then novel HPV genotypes in human cervical cancers and in cell lines derived from such cancers. [72] [73] In these cell lines, which include HeLa cells, the HPV DNA is often integrated into the host genome, and only a subset of viral genes, E6and E7, is expressed. [74] [75] This discovery led to the hypothesis that HPV E6 and E7 genes contribute to cervical cancer, a premise that is now well supported by experimental research.

An association of papillomaviruses with cancer was first demonstrated in the early 1930s with the recognition that a subcellular, transmittable (i.e., infectious) agent causes squamous carcinomas in cottontail rabbits.[76] The agent was later identified to be a virus, cottontail rabbit papillomavirus, that induces warts in the rabbits. A subset of these infected rabbits develops cancers at the original site of cottontail rabbit papillomavirus infection. Other animal papillomaviruses induce frank cancer. Bovine papillomaviruses (BPV), which represent a class of papillomaviruses that induce fibropapillomas, characterized by hyperplasia of both the dermal fibroblasts and epidermal epithelial cells, can induce epithelial tumors of the alimentary canal in cows.[77] Such tumors are thought to arise when animals ingest bracken fern, which contains quercetin, a potent chemical carcinogen. [78] [79] [80] Thus, papillomaviruses and chemical carcinogens act together to induce tumors in cattle.

The study of papillomaviruses in the laboratory began in earnest in the late 1970s when Doug Lowy, Peter Howley, and their colleagues at the National Institutes of Health in Bethesda discovered that BPV-1 infects and transforms a mouse fibroblast cell line, C127, in cell culture. [81] [82] [83] The parental C127 cells, while immortalized, are contact inhibited. Infection by BPV-1 or transfection of C127 cells with a bacterial recombinant plasmid containing the entire BPV-1 genome yields foci of cells that are no longer contact inhibited. Transformed C127 cells harbor the viral genome as a nuclear plasmid and express viral early genes. The viral gene products E5 (E referring to “early,” 5 referring to the fifth largest translational open reading frame), E6, and E7 [84] [85] [86] [87] [88] [89] contribute to this transformation ( Fig. 11-3 ). Thus, by the time HPVs were recognized as potential etiologic agents in human cervical cancers in the mid-1980s, a wealth of information pointing to the transforming potential of animal papillomaviruses in tissue culture had been established.

 
 

Figure 11-3  Map of the HPV genome. Indicated by the circle is the approximately 7900-bp circular double-stranded DNA genome of HPV16 as found in viral particles and infected cells. Shown by the boxes outside the circle are the various translational open reading frames (ORFs) that encode the viral proteins. These include the early (E) and late (L) ORFs. Most early ORFs (those highlighted in yellow) are found expressed throughout the viral life cycle within stratified squamous epithelia, whereas the late ORFs, which encode the capsid proteins (highlighted in blue), and E4 (highlighted in green), are selectively expressed in the productively infected, terminally differentiated epithelial cells. Note, among the early ORFs are E6 and E7, the two ORFs encoding like-named oncoproteins that are commonly found expressed in HPV-associated anogenital and oral cancers. RNA synthesis of papillomaviruses is complex, yielding many potential mRNAs, all of which terminate at polyadenylation sites located at the end of the E5 and L1 open reading frames. The LCR or long control region encodes multiple cis-acting elements that regulate viral transcription and synthesis of viral DNA. See Howley and Lowy[281] for details of the HPV genome and life cycle.

 

 

The seminal studies of zur Hausen and colleagues in the early to mid-1980s, identifying HPV DNA in cell lines derived from cervical cancers, checkmated a long-argued role for herpes simplex virus type II (HSV-II) in human cervical cancer. The posited role for HSV-II in cervical cancer arose from findings that cervical cancer patients often had antibodies to HSV-II, a sexually transmitted agent. However, their tumor cells lack HSV-II DNA, and today it is accepted that HSV-II does not contribute causally to cervical cancer. This early error provides an important lesson to researchers and epidemiologists who are trying to identify biologic agents that contribute to cancer. Proof by today's standards requires a smoking gun (in this case, the gun includes the viral genome and expression of viral genes in cancer cells).

In 1985, both the zur Hausen and Howley laboratories reported that HPV DNAs (see Fig. 11-3 ) were integrated in chromosomal DNAs in cell lines derived from cervical cancers. [74] [75] This finding initially led to speculation that HPVs contribute to cervical cancer by integrating in or nearby to cellular genes that protect against cancer (i.e., inactivating tumor suppressor genes) or activate those cellular genes that can promote cancers (i.e., promoter/enhancer insertion at proto-oncogenes), akin to the mechanism of oncogenesis by certain oncogenic avian and rodent retroviruses. However, a role for HPV as an insertional mutagen in cancer is not consistent with its different sites of integration in different cancers, which have not been found to be near known or suspected tumor suppressor genes or proto-oncogenes. Rather, it is likely that the integration of the HPV genome leads to the selective upregulation of expression of two viral genes, E6 and E7 (see Fig. 11-3 ), which encode gene products that directly contribute to cancer. The mechanism for this upregulation remains poorly understood but might reflect (1) derepression of E6 and E7 expression from the viral promoter resulting from the disruption of a viral transcription factor, E2, that can repress their transcription[90]; (2) an increase in the stability of the E6 and E7 mRNAs resulting from the disruption on integration of an mRNA instability element present in the 3′ end of the E6 and E7 mRNAs[91]; or (3) increased transcriptional initiation from the viral promoter directing expression of E6 and E7 following integration of the viral DNA. The recognition of the increased expression of E6 and E7 in cervical carcinomas, coupled with the knowledge that E6 and E7 contribute to the transforming potential of BPV-1 in mouse fibroblasts, has provided the impetus to examine the tumorigenic activities of these two viral genes.

Evidence for a critical role of increased expression of HPV E6 and E7 in the genesis of cervical cancers comes from multiple studies: (1) Cervical epithelial cells harboring integrated HPV16 DNA have a selective growth advantage over cells harboring normal extrachromosomal viral genomes, and this growth advantage correlates with the increased expression of E6 and E7.[92] (2) E6 and E7 bind and inactivate the tumor suppressor gene products, p53 and pRB, respectively. [93] [94] (3) p53 and pRB are wild-type in cell lines derived from HPV-positive cervical cancers, whereas they are mutated in HPV-negative, cervical cancer-derived cell lines.[95] (4) The expression of the E6 and E7 viral genes is required for survival of cervical cancer-derived cell lines. [96] [97] [98] [99] [100] [101] Together, these observations strongly support the hypothesis that E6 and E7 contribute causally to human cervical cancers at least by blocking the functions of cellular tumor suppressors.

The E6 and E7 genes from the high-risk HPVs are transforming in tissue culture. They act independently or synergistically to immortalize multiple cell types, including human foreskin keratinocytes,cervical epithelial, or mammary epithelial cells. [102] [103] [104] [105] [106] [107] In addition, E7 cooperates with an activated ras to transform baby rat kidney or human cervical epithelial cells. [108] [109] [110] The oncogenic properties of high-risk HPV E6 and E7 in vivo have been validated through the characterization of HPV transgenic mice. [111] [112] [113] [114] [115] [116] [117] [118]

E6 and E7 are best known for their ability to associate with the cellular tumor suppressors, p53 and pRB, respectively. [93] [94] The discovery of these interactions represents a major advance in our understanding of the mechanisms of oncogenesis: The inhibition of tumor suppressors predisposes cells to evolve into tumors. E6 induces degradation of p53, at least in part via recruitment of a ubiquitin ligase, E6-AP. [119] [120] [121] E6 inhibits p53 protein's transcriptional regulatory activities in tissue culture cells. [122] [123] Association of E7 with pRB also promotes the degradation of pRB [124] [125] and disrupts pRB's capacity to bind and functionally inactivate the cellular E2F transcription factors. [110] [126] While these abilities of E6 and E7 to inactivate p53 and pRB, respectively, likely play an important role in their oncogenic potentials, it is important to recognize that E6 and E7 both can bind additional cellular factors, and certain of these interactions likely contribute to HPV-associated carcinogenesis.[127] [128] Which of these many interactions contribute to oncogenic potential largely remains to be determined. In the case of E6, one group of cellular interacting partners implicated in E6-mediated carcinogenesis are the PDZ domain proteins such as Dlg and Scribble, proteins that were originally identified in Drosophila to be tumor suppressors. [128] [129]

E6 proteins from multiple papillomaviruses bind to cellular proteins other than p53 and E6AP. These other interacting partners include p300, [130] [131] paxillin, [132] [133] E6 target protein-1 (E6TP1),[134]interferon regulatory factor-3 (IRF3),[135] E6 binding protein-1 (E6BP1),[136] Bak,[137] protein kinase PKN,[138] myc,[139] the mammalian homolog of Drosophila disk-large tumor suppressor gene product (DLG), [140] [141] Scribble,[142] MAGI-1,[143] and MUPP1[144] In addition, E6 can induce expression of telomerase activity by a yet-to-be-defined activity, [145] [146] [147] [148] a property that correlates with E6's immortalizing potential.

In addition to binding pRB, E7 can bind to other cellular proteins, including p107 and p130, which are related to pRB protein,[149] and can interact with different members of the E2F family of transcription factors. [150] [151] E7 also is argued to complex with cyclins [149] [152] [153] [154] and to inactivate cyclin-associated kinase inhibitors, p21 and p27. [125] [155] Thus, E7 can associate with and/or alter the activities of multiple cellular factors that themselves interact normally and thereby contribute to the normal regulation of the cell cycle. Still other interactions have been identified between E7 and cellular factors, including S4 subunit of the 26 S proteasome;[156] Mi2-beta, a component of the NURD histone deacetylase complex;[157] the fork-head domain transcription factor MPP2;[158] the transcription factor AP-1;[159] insulin-like growth factor-binding protein 3;[160] TATA box-binding protein (TBP); [161] [162] TBP-associated factor-110;[163] and a novel human DnaJ protein, hTid-1.[164] It remains unclear whether any of these additional interactions contribute to E7's oncogenic potential.

There is a growing appreciation of HPV's role not only in anogenital cancers such as cervical cancer, but also in head and neck cancers of the oral cavity and in skin cancers. [165] [166] [167] [168] The multiple interactions of E6 and E7 with cellular proteins that have regulatory functions is consistent with E6 and E7 contributing to cancers through multiple mechanisms. Studies in tissue culture strongly support the hypothesis that continued expression of E6 and E7 is required for the continued growth of cervical cancer cells [96] [97] [98] [99] [100] and, perhaps, with other cancers to which HPVs contribute causally.

Cervical cancers take decades to arise in most patients after initial infection with high-risk HPVs. During this time, the HPV must persist in the patient. Strategies that can interfere with viral persistence could prove effective in preventing the development of cancer. E6 and E7 are important to the replicative phase of the HPV life cycle and therefore for viral persistence, indicating that they are also appropriate targets for antiviral and antitumor drug development. Recent studies have used organotypic tissue culturing to recapitulate the life cycle of HPVs in fully differentiating, stratified squamous epithelial cells. These studies have implicated E7 in reprogramming cells within the terminally differentiating compartment of the epithelia to support the amplification of the viral DNA genome, likely through its inactivation of pRB.[169] E6's inactivation of p53 might be necessary for viral replication by inhibiting cellular stress responses elicited by E7's inactivation of pRB.[170] At least two more HPV proteins, E1 and E2 (see Fig. 11-3 ), contribute to the replication of the viral genome.[171] E1 and E2 bind to an origin-of-DNA replication on the viral genome.[172] E1 is a DNA helicase that unwinds the viral double-stranded DNA genome at its origin and, together with E2, recruits cellular DNA replication proteins that then synthesize the viral DNA. [172] [173] [174] Thus, E1 and E2 both represent potentially useful targets for intervening in the viral life cycle.

One desirable, long-term public health strategy for dealing with this and other human tumor viruses is generation of an effective, prophylactic vaccine for prevention of initial infection. Such a vaccine has recently received FDA approval in the case of the mucosotropic HPVs that are implicated in cervical cancer, although several issues complicate the potential success of this strategy. This vaccine is discussed later. Another approach that is being pursued is the generation of microbicides that could be used to prevent infection by sexually transmitted HPVs.

Human T-Cell Leukemia Virus I

Adult T-cell leukemia/lymphoma (ATLL), a tumor of CD4+ T cells, is caused by human T-cell leukemia virus type I (HTLV-I), the only retrovirus that is now accepted as being oncogenic in people. HTLV-I is found worldwide, with approximately 10 million to 20 million people estimated to be infected today. This number is likely an overestimate because of a failure to distinguish serologically between HTLV-I and HTLV-II. [175] [176] HTLV-I is particularly prevalent in restricted sites, including southern Japan, the Caribbean, and West Central Africa. ATLL is prevalent in those areas in which HTLV-I is common, and it is estimated that as many as 5% of HTLV-I-positive carriers will develop ATLL in their lifetime.[177] HTLV-I also causes a progressive, paralytic myelopathy termed HTLV-I-associated myelopathy or tropical, spastic paraparesis. This neurologic disorder appears to arise preferentially in patients whose HLA haplotypes fail to limit viral load.[178]

The data that link HTLV-I causally to ATLL are varied. ATLL can occur in familial clusters. It is characterized by an average age at onset of 56 years and proves rapidly fatal, 50% of patients dying within 6 months of diagnosis.[177] Patients generally have antibodies to HTLV-I-encoded proteins,[179] and in 88 of 88 primary biopsies of ATLL examined, all had single copies of integrated HTLV-I proviruses.[180] The presence of the provirus of HTLV-I in all of the tumors makes it likely that infection with the virus is an early, contributing event in the evolution of the tumor. The presence of ATLL in familial clusters is consistent with the routes of transmission of HTLV-I. HTLV-I is passed from male to female via semen and from mother to child by breast milk. The latter route has been demonstrated prospectively. Encouraging carrier mothers to refrain from breast-feeding has decreased transmission of HTLV-I to their children by 80%.[181] It appears highly likely that this form of public health intervention will lead to a corresponding decrease in ATLL in Japan in the future. Such a decrease would constitute formal proof of the oncogenic role of HTLV-I in ATLL.

HTLV-I clearly differs from the highly oncogenic animal retroviruses that encode oncogenes derived from cellular proto-oncogenes. HTLV-I is a complex retrovirus that encodes multiple open reading frames in addition to the gag, pol, and env genes that are common to simple retroviruses ( Fig. 11-4 ). However, none of these additional viral genes is obviously related to known proto-oncogenes, and all are thought to affect the viral life cycle either directly or indirectly by affecting the host cell. [182] [183] It is accepted that one viral protein Tax (see Fig. 11-4 ), which regulates both viral and cellular gene expression, is a major contribution of HTLV-I to leukemogenesis. It is also evident that viral infection precedes onset of ATLL by 50 years or more, that many cells are infected, and that only a minority of infected people develop this clonal tumor. These combined observations indicate that multiple rare events in an HTLV-I-infected CD4+ T cell must occur for that cell to evolve into ATLL.

 
 

Figure 11-4  Map of the HTLV-I genome. Indicated by the line drawing is the approximately 9000-bp DNA proviral form of the HTLV-I genome as it is found integrated into the host genome. The genome as present in the viral particle consists of two copies of a single-stranded positive-strand, RNA. Shown are the long terminal repeats (LTRs, green) flanking the unique region that contains the translational open reading frames (boxes) for structural (blue) and nonstructural (yellow) viral proteins. The LTRs contain cis-acting elements that are required for transcription and replication of the viral genome. The structural genes are transcribed late in the viral life cycle from the 5′ LTR while the nonstructural proteins are transcribed early from the 5′ LTR from different, spliced transcripts. See Green and Chen[282] for details of the genome and life cycle of HTLV-I.

 

 

Multiple approaches demonstrate that Tax can transform cells in culture and be oncogenic in animal models. The introduction of a vector that expresses Tax into established, adherent rodent cells can transform them to grow in an anchorage-independent fashion.[184] Strains of rodent cells can be transformed with Tax in combination with the ras oncogene to yield cells that are tumorigenic in nude mice.[185] Tax has also been recombined into Herpesvirus samiri and has been introduced into resting human T cells. These infected cells can proliferate and yield infected, immortalized progeny, whereas the Tax-negative parental virus cannot do so.[186] In accord with these data, variants of HTLV-I from which the Tax gene has been deleted no longer can immortalize human T cells in culture.[187] These results in culture are paralleled and bolstered by others in transgenic animal models. Expression of Tax from the HTLV-I long terminal repeat, LTR (the viral promoter), in transgenic mice leads to mesenchymal tumors.[188] When its expression is directed to lymphoid cells, the transgenic animals develop leukemias.[189] However, the exact means by which Tax transforms cells in culture or is oncogenic in animal models is not obvious.

Tax can be considered a paradigm for viral proteins that affect host cells in that it has multiple, distinct functions that are not found in any one cellular protein. Apparently, HTLV-I during its evolution has assimilated multiple cellular activities in this one gene product. Tax can be viewed as having at least three kinds of activities: It activates transcription via NF-κB and CBP, the CREB (cyclic AMP response element-binding protein)-binding protein; it inhibits transcription, perhaps through binding histone deacetylase-1, HDAC-1; and it inhibits several tumor suppressor gene products. [182] [190] [191] [192] [193] [194] Tax potently activates transcription from HTLV-I's own LTR[195] and activates the promoters for IL-2, IL-2 receptor a chain, and c-fos. [196] [197] [198] This transcriptional activation could obviously contribute to proliferation of an infected T cell. It also can activate the promoter for Bcl-x and thereby help to inhibit apoptosis.[199] Tax can positively regulate transcription by binding CREB and CBP as well as some members of the NF-κB family. [200] [201] [202] Tax not only binds members of the NF-κB family directly, but also can activate NF-κB's homing to the nucleus by binding to IκBa and promoting its degradation. [203] [204] Some of Tax's protein : protein associations have been functionally validated through chromatin immunoprecipitations that have documented the binding of Tax, CREB, and CBP to HTLV-I's LTR in intact, HTLV-I-transformed T cells.[205]

Tax can also inhibit transcription. It has been shown to inhibit both expression of the β-DNA polymerase gene[190] and some promoters that are regulated by CBP/p300.[206] It has been proposed that the latter inhibition occurs by Tax's binding to CBP and effectively sequestering CBP such that it is unavailable to bind other DNA-binding transcriptional factors. It has also been shown that Tax binds HDAC-1 as measured by coimmunoprecipitations.[191] Were Tax to tether HDAC-1 to a promoter, the resulting localized histone deacetylation would presumably lead to its decreased support of transcription.[207]

A third activity of Tax is its inhibition of some cellular tumor suppressors. Tax has been shown to bind to and inhibit the function of p16INK4A.[192] p16INK4A binds cyclin-dependent kinase 4 (CDK4), a kinase that, when activated, can phosphorylate pRb, yielding release of E2F transcription factors and promotion of the G1 to S transition of the cell cycle. On binding p16INK4A, Tax can increase the kinase activity of CDK4 kinase.[192] p16INK4A is often found to be mutationally inactivated in tumors. Consistent with Tax's functionally inactivating p16INK4A, p16INK4A was shown to be wild-type in sequence in two HTLV-I-infected T-cell lines in which Tax is expressed but deleted in four uninfected T-cell lines.[192] Tax also appears to bind cyclin D3 and might thereby foster, by a second means, the activity of CDK4 and CDK6.[193] Tax's binding to p16INK4A and cyclin D3 would inhibit control of the cell cycle and promote cell proliferation. Tax can also inhibit the transcriptional activity of p53 by an NF-κB-dependent mechanism that culminates in the phosphorylation of p53 as certain residues. [194] [208] Such an inhibition of p53 is likely to limit its induction of apoptosis and increase the rate of survival of HTLV-I-infected, proliferating T cells.

The multiple activities of Tax likely contribute to HTLV-I's associated leukemogenesis but must be insufficient for the development of ATLL. Multiple T cells are initially infected by HTLV-I, but over the course of the 50 to 60 years of its development, only one infected cell and its progeny give rise to the tumor. The additional genetic and epigenetic events that are necessary for this evolution are not known. It is also clear that the expression of viral genes in infected cells is surprisingly low; measurements of RNAs encoding Tax indicate that between 0.1% and 10% of freshly harvested, infected T cells express any Tax message in vivo.[209] These findings indicate that the regulation of HTLV-I expression, be it at the level of T-cell development or immune response to viral antigens, is likely also to be critical to the development of ATLL.

Human Hepatitis C Virus

Human HCV is accepted as an etiologic agent for hepatocellular carcinoma (HCC) along with HBV. Approximately half of the cases in the U.S. are ascribed to HCV.[210] Infection with HCV constitutes a 20-fold risk for men in Taiwan to develop HCC.[211] HCV represents the most common chronic viral infection among blood-borne pathogens in the United States, where the rate of infection during the period from 1988 to 1994 was estimated to be 1.8%.[212] The World Health Organization estimates the worldwide infection rate to be 3%, yielding over 170 million infected individuals.[213] The rates of infection vary widely, with rates as low as 0.01% to 0.1% in Scandinavia and the United Kingdom and as high as 17% to 26% in Egypt.[212] Infection is thought to arise from contaminated blood, use of shared needles among intravenous drug users, organ transplantation, hemodialysis, sexual transmission, and vertical transmission. The establishment of sensitive tests for identifying contaminated blood and blood products fortunately has now greatly reduced the rate of infection in the general population. As with people who are chronically infected with HBV, HCC in HCV-positive individuals correlates with chronic hepatitis and cirrhosis. Di Bisceglie has estimated that 20% of people who are chronically infected with HCV develop cirrhosis per decade, and by two decades, 2% to 7% of chronically infected people develop HCC.[214] It is argued that the hyperproliferative state that is induced by chronic hepatitis and cirrhosis leads to an accumulation of genetic changes and contributes to the onset of liver cancer. What specifically HCV contributes to this scheme is not known.

HCV is a flavivirus, indicating that it is unique among the human tumor viruses for having RNA as its only genetic material ( Fig. 11-5 ). It was identified in 1989 during a search for the causal agent of non-A, non-β hepatitis.[215] This flavivirus contains a 9.6-kbp, positive-stranded RNA as its genome, which encodes a single translation product of approximately 3000 amino acids (see Fig. 11-5 ). This polyprotein is cleaved by both cellularly and virally encoded proteases to yield at least 10 proteins.[216] The study of HCV has been daunting for at least two reasons. Its sequence varies in infected people such that there are six recognized genotypes with multiple subtypes among patients and a spectrum of quasispecies within any one infected individual.[217] Members of these quasispecies within one patient vary by 1% to 2% in their sequence.[218] In addition, there is no available cell culture for HCV. Researchers have made heroic efforts to overcome this latter hurdle and have met with partial success.

 
 

Figure 11-5  Map of the HCV genome. The 9500-nucleotide-long positive-strand RNA of HCV is shown in black. It encodes a single 3000-amino-acid polyprotein that is proteolytically cleaved into mature proteins (indicated on the map by labeled boxes with alternative names indicated above the boxes) by virally encoded proteases (NS3/4A and possibly 2B). At the 5′ and 3′ ends are short noncoding regions that contain signals for replication of the viral genome. The 5′ NCR also contains an internal ribosome entry site. Structural proteins are shown in blue. Nonstructural proteins, including proteases, helicases and RNA polymerase, are shown in yellow. See Majo and colleagues[283] for details of HCV's genome and life cycle.

 

 

In 1999, Lohmann and colleagues described the replication of a subgenomic derivative of HCV in a cell line derived from a human hepatocellular carcinoma.[219] These subgenomic replicons were difficult to establish, but once established, they exhibited bona fide characteristics of flaviviral nucleic acid replication. One thousand to five thousand molecules of plus-strand RNA were present in each cell; minus-strand RNA was present at 10% to 20% of the level of the plus-strand; and this viral RNA replication was insensitive to treatment of cells with actinomycin D.[219] This mode of nucleic acid replication in cells will allow the elucidation of the cis and trans elements it requires but with an unexpected twist. The efficient replication of the subgenomic derivatives of HCV requires mutations in at least two viral genes that enhance replication in cells but abrogate infectivity of intact HCV RNA.[220] Chimpanzees are the only nonhuman host for HCV. The parental strain of HCV that is used to derive the subgenomic replicons is infectious in this primate host, while a derivative of it, containing the mutations required for the efficient replication of the subgenomic replicons, is not.[220] This finding must limit the conclusions to be drawn from the analyses of replication of these mutated subgenomic replicons in cells. They also indicate that the generation of the many sequence variants that constitute the quasispecies within any one infected patient might contribute to the successful replication of HCV.

The lack of a cell culture host for infection with HCV and/or a practicable animal host has led researchers to seek chimeric animal models of infection. Researchers formerly developed a chimeric animal model for HBV, which has been adapted to the study of HCV. Mice that are transgenic for the plasminogen activator urokinase, expressed in the liver (uPA), have hepatocytes with a selective disadvantage such that transplanted, nontransgenic hepatocytes repopulate the liver.[221] When these animals are crossed with nu/nu mice, they not only can tolerate rat hepatocytes, but also support rat hepatocytes reconstituting their livers.[222] These findings paved the way for Petersen and colleagues to cross mice that are transgenic for uPA with mice that are null for Rag-2, which eliminates their β- and T-cell responses, and to repopulate their livers with woodchuck hepatocytes.[223] These chimeric animals now supported infection with woodchuck HBV and became chronically infected with 106 to 1011 virions per milliliter of serum.[223] Human hepatocytes can also repopulate the livers of uPA-transgenic animals as long as they cannot reject the xenograft. SKID mice that are homozygously transgenic for uPA accept human hepatocytes such that 50% or more of their hepatocytes are of human origin.[224] These animals can be infected with HCV such that 75% of them become persistently infected with 104 to 106 viral RNA molecules per milliliter of serum, and the infections can be passaged serially in them.[224] These reconstituted animals are obviously difficult to generate but should provide some insights into the life cycle of HCV and could serve as models in which to test inhibitors of HCV infection.

Evidence for a direct role of HCV in HCC has come from studies of the HCV Core protein. The HCV Core protein can cooperate with an activated form of the ras oncogene to transform primary rodent cells in tissue culture.[225] Mice that are transgenic for the HCV Core protein develop HCC.[226] Several potential mechanisms have been invoked to explain the transforming potential of HCV Core protein. It has been found to enhance cell proliferation through the stimulation of the mitogen-activated protein kinase [227] [228] In addition, HCV Core protein can inactivate a transcription factor, lZIP, and this inactivation correlates with transformation in rodent cells.[229]

More recently, the Core protein has been found to activate STAT3, and this activation may contribute to its transforming potential.[230] Whether the HCV Core protein contributes directly to human HCC is uncertain. Neither replication of subgenomic replicons in cell culture nor infection of chimeric mice populated with human hepatocytes will allow ready testing of the possible role of the Core protein in HCV's oncogenesis.

Current treatments for HCV are unsatisfactory. The combination of interferon α plus ribovirin, a general antiviral nucleotide analog, has supported a clearance of detectable HCV in 40% of a treated group over 1 year. Those who responded had higher titers of the virus.[231] These observations may indicate that treatments with multiple, independently acting drugs are the most likely route to successful treatments for HCV.[232] In the United States, approximately 10,000 deaths are attributed to HCV each year, and today infection with HCV is the leading indication for liver transplantation.[212] Clearly, this mode of treatment is not practical for most infected people. The lack of satisfactory treatments for chronic HCV infection and its associated HCC make it important to continue to develop tractable means for studying HCV's life cycle and to elucidate its mode of oncogenesis.

Kaposi's Sarcoma Herpes Virus

The identification of Kaposi's sarcoma herpesvirus (KSHV) represents the culmination of much scientific detective work. Kaposi's sarcomas (KS) were known before the AIDS epidemic [233] [234] but increased markedly among HIV-positive people. Researchers therefore looked for molecular evidence of an infectious agent present in KS lesions. In 1994, Chang and Moore and their colleagues used a newly developed enrichment procedure based on PCR to identify DNA sequences that are present in KS but absent in normal cells. They identified a new herpesvirus, KSHV (also termed human herpesvirus 8, or HHV-8), which is related to EBV and Herpesvirus saimiri.[235] Retrospective studies indicate that KSHV was prevalent in different parts of the world prior to the spread of HIV and that in Africa, for example, the prevalence of KSHV has not changed. What has changed there is the incidence of KS; in regions where it was formerly infrequent, the incidence of KS rose threefold between 1988 and 1996.[236] Similarly, the frequency of infection with KSHV of certain cohorts in the United States has not altered with the advent of HIV,[237] but the incidence of KS has.[238] KSHV has also been detected in one class of lymphomas termed body-cavity-based lymphomas or primary effusion lymphomas (PEL)[239] and in an atypical lymphoproliferative disorder termed multicentric Castleman's disease.[240]

KSHV is now accepted as contributing causally to KS and PEL. Each of these malignancies displays intriguing features that are likely to reflect their viral etiology. Single-cell assays of early KS lesions have detected KSHV in a minority of cells that surround their vascular spaces, while in the more advanced, nodular lesions, more than 90% of the spindle cells characteristic of these lesions are KSHV-antigen-positive.[241] The viral antigen-positive cells also stain with antibodies against the VEGF receptor-3, indicating that they are lymphatic or proliferating endothelial cells.[241] That only a subset of the cells that are characteristic of early KS lesions appear to be infected with KSHV likely indicates that infected cells affect the development of their neighbors. This possibility is supported by the multiple cellular homologs of cytokines and receptors encoded by KSHV that may allow infected cells to interact with adjacent, uninfected cells. [242] [243]

PEL cells are B cells in origin and, in six of seven cases examined, have nongermline immunoglobulin mRNAs, indicating that they are likely derived from B cells that have encountered antigen.[244] They often, however, fail to express β-cell activation antigens.[245] PEL cells usually, but not always, are coinfected with EBV. [239] [245] The role of EBV in the etiology of this lymphoma has not yet been clearly determined. However, because EBV is often retained in these cells as they are propagated in vitro and is maintained in them in vivo, EBV is likely to provide the tumor cells some selective advantage.

KSHV encodes many genes with clear homologies to cellular genes, some of which are candidates for contributing to viral oncogenesis ( Fig. 11-6 ). Three categories of these cellular homologs are particularly likely to be important for tumor development: cyclins, inhibitors of apoptosis, and cytokines and receptors. KSHV encodes its own cyclin. Cellular cyclins bind specific, dependent kinases (CDK) whose activities promote progression through the cell cycle and are controlled by cellular inhibitors, including p16INK4A, p21CIP1, and p27Kip1. The KSHV-encoded cyclin can promote cellular proliferation in part by overcoming these cellular inhibitors. It does so apparently by extending the substrates that are phosphorylated by CDK6 to include p27Kip1. [246] [247] Phosphorylation of p27Kip1 at position 187 leads to its downregulation and promotes passage through the G1 phase of the cell cycle. One measure of the activity of the KSHV cyclin CDK6 complex is that it can foster progression of nuclei isolated from cells in G1 to undergo DNA synthesis.[248] The KSHV cyclin can also induce cells to undergo apoptosis.[249] It is striking that in cells lacking p53, the expression of the KSHV cyclin promotes cells both to become aneuploid and to survive so that they continue to proliferate. In fact, mice that are transgenic for KSHV cyclin and are p53-null develop T- and β-cell lymphomas with a mean latency of 3 months, which is shorter than the latency for the p53-null mice alone.[250]

 
 

Figure 11-6  Map of the KSHV genome. The genome of KSHV consists of approximately 160 kbp of linear double-stranded DNA in the viral particle. The viral genome becomes circularized on infection via the 20 to 40 copies of terminal repeats found at the ends of the linear genome. The terminal repeats also contain the origin of plasmid replication used during the latent phase of the viral life cycle. Shown by the boxes are positions of those viral genes that are expressed during latency and/or are thought to contribute to oncogenesis by KSHV. The arrows represent the positions from which the RNAs encoding the labeled proteins are expressed. Several of these primary transcripts are polycistronic. Shown on the inside of the circle are the positions of the recently identified miRNAs that are encoded by KSHV. These also are derived from a polycistronic message.

 

 

KSHV also encodes its own inhibitors of apoptosis: viral Bcl-2 and viral FLIP (an inhibitor of cellular FLICE, a protein that mediates Fas ligand–induced cell death) (see Fig. 11-6 ). The viral Bcl-2 shares its limited sequence homology with cellular Bcl-2 in those regions that are critical for inhibiting apoptosis and fails to dimerize with cellular Bcl-2 and Bcl-XL, thus avoiding regulation by potential cell-binding partners.[251] It can inhibit the apoptosis that is induced by efficient expression of KSHV's cyclin.[252] Viral FLIP can inhibit apoptosis mediated by the Fas pathway and induced by cytotoxic T-lymphocytes.[253] Finally, KSHV encodes homologs of cytokines and cytokine receptors (see Fig. 11-6 ). Its viral IL-6 may promote proliferation of PEL cells, although they appear to be dependent on cellular IL-6 and not viral IL-6 for their continued growth.[254] KSHV also encodes a G protein-coupled receptor, viral GPCR, which is likely to be pivotal for the development of KS. The expression of viral GPCR in endothelial cells in mice leads to KS-like tumors, while the individual expression of KSHV cyclin, KSHV Bcl-2, or KSHV FLIP does not.[255] What is particularly exciting is that cells that are inoculated into mice that express viral GPCR promote tumor formation by coinoculated cells that individually express viral cyclin, viral FLIP, or both.[255] These findings support a model in which viral GPCR contributes to the development of KS by affecting neighboring cells not infected by KSHV. The model is also supported by the findings that viral GPCR induces expression of the cellular VEGF receptor-2 in endothelial cells that proliferate in the presence of VEGF and that viral GPCR induces expression of VEGF. [256] [257] Clearly, these observations define an autostimulatory loop in which KSHV GPCR alone can maintain proliferation of endothelial cells. These collected observations help to explain the viral contributions to KS and perhaps PEL. These explanations are not yet complete, however, because several of these putative viral oncogenes, including viral IL-6, viral Bcl-2, and viral GPCR, are expressed during the lytic phase of KSHV's life cycle. The lytic phase of a herpesvirus is traditionally thought to lead to death of the host cell. How these putative viral oncogenes could affect oncogenesis and be expressed only in cells that are destined to die soon is an enigma that has yet to be resolved.

Recent studies of cell lines derived from PEL and human endothelial cells are providing the foundation for understanding infections by KSHV in vivo. PEL cells maintain KSHV DNA extrachromosomally and consistently express the viral protein LANA-1 (see Fig. 11-6 ), which is required for viral plasmid replication. [258] [259] In general, these cells support the latent phase of KSHV's life cycle and also express FLIP, the viral cyclin and the viral inhibitor of cellular FLICE, but few other viral proteins. Some PEL cell lines support an inefficient spontaneous conversion to the lytic phase of the viral cycle, which can be further induced by treatment of cells with tetradecanoyl phorbol acetate or introduction of a plasmid that includes ORF50 (see Fig. 11-6 ), a viral inducer of the lytic cycle.[260] The released virus is infectious on human dermal microvascular endothelial cells. [261] [262] These dermal microvascular endothelial cell cultures can be passaged to yield populations in which all cells are infected, express LANA-1, assume a spindle-cell morphology, and can proliferate indefinitely.[262] The spindle shape is characteristic of cells in KS lesions in vivo. Staining of these infected dermal microvascular endothelial cell-derived spindle cells indicates that 5% to 10% of them spontaneously support early stages of KSHV's lytic cycle and 1% to 2% express genes diagnostic of the late stages of the viral life cycle.[262] If endothelial cells that are infected in vivo by KSHV display a similar distribution of cells supporting the latent, early, and late lytic phases of the viral life cycle, then the enigma of the oncogenic contribution of viral genes that are expressed only during the lytic cycle may be resolved. A sustained, spontaneous conversion of 5% to 10% of KSHV-infected cells to support the viral lytic cycle might allow enough infected cells to express enough KSHV GPCR to promote the bystander-dependent tumor evolution proposed by Bais and colleagues.[257]

TREATMENT AND PREVENTION OF VIRAL TUMORS

Tumors that are associated causally with viruses are treated variously: Surgical resections, when appropriate, are used in combination with chemotherapy and/or radiation therapy. These treatments are disappointing in that overall survival rates are often low and so far have not been able to capitalize on any specific antiviral therapies. Current survival rates after different therapies obviously vary with the malignancy, its stage, and the setting in which it is treated. For example, youngsters with Burkitt's lymphoma have a 4-year overall survival rate of 65%.[263] Approximately 45% of NPC patients remain disease-free after treatment for 10 years, but this value is highly dependent on the stage at which the NPC was diagnosed.[141] In the United States, 26% of HBV-positive, surgically resected HCC patients have a 5-year, local disease-free survival compared with 38% for the analogous HCV-positive group.[264] A retrospective study of German patients with a mix of HBV- and HCV-associated HCC found a median survival of 7 years.[265] The 5-year median survival for cervical carcinoma patients ranges from 65%[266] to 75% to 90%[267] in different studies. The median survival for patients once they have been diagnosed with ATLL is only 10 months.[268] The overall survival rates of KS patients who are HIV-positive have changed with the introduction of highly active antiretroviral therapies. Prior to this therapy, median overall survival was 13 months; with highly active antiretroviral therapies, it surpasses 28 months.[269] The median survival for PEL patients has been described as “dismal,” but there are case reports of some combination therapies being helpful.[270] These actuarial findings indicate that developing antiviral therapies directed at those viral gene products that maintain tumor phenotypes is a highly desirable goal.

Early detection of premalignant disease has been an effective means for reducing the incidence of cervical cancer. The introduction of routine cytological (Pap smear) screening of women in the United States and other countries with well-developed health care systems has led to an approximate threefold decline in cervical cancers over the last 40 years. Pap smears represent a first-level screening tool for the clinician. Women with positive Pap smears are routinely subjected to further examination with histopathologic examination and, if necessary, removal of lesions. More recently, HPV DNA testing has emerged as an alternative means for identifying women who are at risk of developing cervical cancers, although the utility of this testing appears to be restricted to women 40 years of age or older. This restriction reflects the fact that many younger women who are sexually active will have nascent infections that will be detected by using the HPV DNA test but are not necessarily going to lead to cancer because many HPV infections will spontaneously resolve. The detection of high-risk HPVs in older women, on the other hand, is more likely to indicate the presence of persistent HPV infections, which are highly correlative with progressive cervical disease leading to cancer. Another use of HPV DNA testing is as an adjunct screen for women with positive Pap smears, that is, to determine whether they are infected with high-risk HPVs associated with cervical cancer.

The ultimate goal, the development of effective vaccines that can prevent initial infection or induce the elimination of viral persistence, is being pursued for all human tumor viruses. Success or the glimmer of success has been achieved for two of them: HBV and HPV.

Hepatitis B Virus Vaccine

Although there are four serotypes of HBV, highly effective vaccines for HBV have been developed that consist only of its surface antigen, HBsAg. Today, this subunit vaccine is usually synthesized with a recombinant DNA that is expressed in yeast. Vaccines for HBV began to be used in the early 1980s, and since 2001, 129 countries throughout the world routinely vaccinate infants and/or adolescents against HBV.[271] These vaccinations are effective. The fraction of children who are infected with HBV has been reported to have declined between 7.5- and 10-fold in Taiwan and the Gambia, respectively. [54] [55]This decline has been paralleled by a detectable drop in the frequency of HCC in children in Taiwan, an age group for which this viral tumor is rare.[56]

The current HBV vaccines apparently are free of unwanted side effects but can select for variants of HBV that are resistant to the neutralizing antibodies they elicit.[271] One domain of the HBsAg in particular elicits neutralizing antibodies efficiently, and mutations in it can allow for viral escape. The frequency of these escape mutants in populations of vaccinated children has risen significantly.[272]One means to overcome the selection for such escape mutants would be to generate vaccines for HBV that include more than the HBsAg as an immunogen. Such vaccines are now being developed.

Human Papillomavirus Vaccine

The second family of human tumor viruses for which an effective prophylactic vaccine has been developed are those human papillomaviruses that are associated with cervical cancer, other anogenital cancers, and certain head and neck cancers, in particular, HPV16 and HPV18. These two viruses account for approximately 85% of cervical cancers worldwide and, in a recent study, 16% of new infections.[273] Several HPV prophylactic vaccines, designed to prevent or eliminate acute infections by HPV16 and HPV18, have recently gone through clinical trials.[274] These vaccines are based on the production of viruslike particles (VLPs) composed of the major capsid protein of HPV16 and HPV18, L1, which self-assembles into icosahedrons (see Fig. 11-3 ). These L1-based VLPs induce neutralizing antibodies in vaccinated individuals. Preclinical studies with cottontail rabbit papillomavirus and canine oral papillomavirus demonstrated the effectiveness of VLP-based vaccines in protecting animals from cottontail rabbit papillomavirus and canine oral papillomavirus infection. [275] [276] Clinical trials likewise demonstrated the effectiveness of HPV16 VLP-based vaccines in inducing neutralizing antibodies that are specific for HVP16 and preventing HPV16 infections and in reducing the incidence of premalignant disease. [277] [278] One of these HPV vaccines, Gardosil, which is a product of Merck, received FDA approval in 2006 for administration to young women in the United States. It has also been approved for use in many other countries. A second vaccine, which is being developed by GSK, is expected to gain FDA approval in 2008. One issue that is of relevance to the effectiveness of these vaccines is the specificity of immune responses invoked by VLP-based vaccines for specific HPV genotypes. Approximately two dozen HPV genotypes are associated with anogenital cancers. VLP-based vaccines induce protective immunity that is highly specific for the genotype from which the VLP is generated. This specificity has led to the development of polyvalent vaccines composed of a mixture of VLPs generated with L1 proteins from multiple mucosotropic HPV genotypes. For example, Gardosil is a polyvalent vaccine that is composed of HPV16, HPV18, HPV6, and HPV11 VLPs. HPV6 and HPV11 are low-risk mucosotropic HPVs that induce genital warts but do not contribute to cervical cancer. It remains to be seen whether, once a population is protected from HPV-16- and HPV-18-induced cancers, other HPV genotypes arise to induce a greater incidence of cervical cancers than they currently cause. Studies now in progress are designed to increase the cross-genotype neutralizing capacity of the VLP-based vaccines. Incorporation of the minor capsid protein, L2, might contribute to this property, because neutralizing antibodies induced against L2 tend to be more generally effective at neutralizing multiple genotypes.[279] There is a concern whether the HPVs, particularly HPV16 and HPV18, will evolve to become resistant to VLP-based vaccines. Assuming a high effectiveness of the vaccines that are currently in development and their worldwide distribution and given the long latency of cervical cancer, it is estimated that prophylactic HPV vaccines will lead to a reduction of deaths due to cervical cancer no earlier than 2040.

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