Interactions Between Papillomavirus Proteins and Tumor Suppressor Gene Products

INTERACTIONS BETWEEN PAPILLOMAVIRUS PROTEINS AND TUMOR SUPPRESSOR GENE PRODUCTS Karen H. Vousden Ludwig Institute for Cancer Research, St. Mary's Hospital Medical School, London W2 1PG, England

I. Introduction 11. Human Papillomaviruses A. Human Tumor Virus

B. Normal Viral Life Cycle C. High-Risk HPV-Encoded Oncoproteins 111. Regulation of Cell Growth A. Control of the Cell Cycle B. Tumor Suppressor Genes IV. ViraVHost Protein Interactions A. Activities of E7 B. Activities of E6 C. Function of HPV-Encoded Oncoproteins in the Normal Viral Life Cycle D. E6 and E7-Targeting a Common Pathway? V. HPV Oncoproteins-Tools and Targets References

I. Introduction

Human papillomaviruses (HPVs) are small DNA viruses that have provided unique insight into the mechanisms that regulate growth and oncogenic progression of human cells. Most members of this large group of common viruses pursue a life cycle that has little serious impact on the host, infecting epithelial tissue and producing self-limiting hyperproliferations, more commonly known as warts (von Knebel Doeberitz, 1992). T h e intense interest in these viruses has resulted from the observation that some of the genital HPV types cause lesions that are not strictly benign; the malignant progression of HPV-infected cells appears to give rise to almost all cervical cancers (zur Hausen, 1991b; Munoz and Bosch, 1992). The importance of this relationship is obvious, since cervical cancer is the second most common cause of cancer death among women worldwide and the most common cancer (without sex adjustment) in parts of the developing world (Parkin et al., 1988). Other viruses 1 ADVANCES IN CANCER RESEARCH, VOL. 64

Copyright 0 1994 hy Academic Press, Inc. All rights of reproduction in any form reserved.

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KAREN H. VOC‘SDEN

such as hepatitis B virus, Epstein-Barr virus, and human T cell leukemia virus type 1 (HTL\’-l) have also been strongly implicated in the development of several human cancers (zur Hausen, 1991a); the longterm goal of preventing these cancers by prophylactic vaccination to eliminate viral infection is further advanced for some of these viruses than for the HPVs. Unlike the situation for these other viruses, however, scientists have an increasingly clear understanding of how the HPVs contribute to oncogenesis at the molecular level and how several virally encoded proteins exhibit activities consistent with a role in malignant progression. These viral proteins therefore provide targets for the development of therapeutic drugs and the potential to treat, as well as prevent, the majority of cervical cancers. II. Human Papillomaviruses A . H U M A N~ r U M O KVIRUS

Epidemiological studies predicted the involvement of a transmissible agent in the etiology of cervical cancer many years before the role of certain genital HPV types was clearly identified (zur Hausen, 1976). Several different HPV types infect the genital tract and only some of these, the so-called high-risk HPV types, are associated with malignancies (De Villiers, 1989). High-risk HPVs, most frequently HPV16 or 18, are evident in over 90% of cervical cancers; these viral types have been intensively studied. T h e second group of genital HPV types, the low-risk viruses, is found almost exclusively in benign lesions. T h e most common of these are HPVG and I 1 and although there is rarely evidence of malignant progression, the appearance of often large genital warts illustrates the ability of these viruses to induce abnormal growth and hyperproliferation. ‘The natural history of infection with these HYV types is slowly being elucidated, although rhc absence of serological tests to determine incidence of infection has somewhat hampered these studies. As might be expected from a sexually transmitted agent, the incidence of genital HPV infection is rather low until early adulthood, when the commencement of sexual activity is paralleled by a rapid rise in infection which apparently declines in subsequent years (Schiffman, 1992). Less than 5 4 of healthy women over 40 years old show any evidence of HPVIG or 18 infection (de Sanjose et al., 1992),in stark contrast to those with cervical cancers, 90% of which are positive for these HPV types (van den Brule et al., 1991). T h e detection of high-risk HPV in many more young women than will be expected to develop cervical cancer indicates that infection with

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these HPV types is not sufficient for full malignant progression. Evidence now suggests that these viruses give rise to cervical intraepithelial neoplasia, previously recognized as premalignant lesions that sometimes, although not always, progress to invasive carcinoma (Koutsky et al., 1992). T h e epidemiology is consistent with the notion that high-risk HPV infection only becomes a problem when viral infection becomes persistent and additional oncogenic events accumulate (zur Hausen, 1991b). HPV infection therefore only contributes to part of the multistep oncogenic process (Vogelstein and Kinder, 1993). The nature of such additional events is not clear, although evidence implicates environmental carcinogens through mechanisms such as mutation of cell genes and immune suppression (Jackson and Campo, 1993). Although a role for HPV infection is clear in most cervical cancers, a small but consistent proportion of these malignancies arises without strong evidence of HPV involvement. It is possible that as yet unidentified HPV types are involved in these tumors or that viral sequences have been lost during progression. T h e epidemiology of these cancers is somewhat different from that of the HPV-positive cancers, however, and it seems more likely that in rare cases these malignancies can arise through HPV-independent mechanisms (Riou et al., 1990; Higgins et al., 1991). B. NORMAL VIRALLIFECYCLE

Despite the frequency with which HPV infection occurs in vivo, these viruses are extremely difficult to culture in experimental systems. Consequently very little is known about their natural replication. Two important points are clear, however. The first is that malignant progression is not part of the normal life cycle of any of the genital HPVs. The loss of normal differentiation associated with malignant progression precludes virion production, which requires terminally differentiated keratinocytes (Dollard et al., 1992; Meyers et al., 1992). In many cases cancer cells contain only incomplete viral genome9 that have become integrated into host chromosomes (Cullen et al., 1991; Das et al., 1992). Since all virally encoded proteins must principally play a role in viral replication, any oncogenic activity exhibited by them should be viewed as an unfortunate manifestation of their normal function. That any viral activity exists only to participate in malignant progression seems extremely unlikely. Related to this first idea is the second point of importance when considering the normal viral life cycle, which is that both high- and lowrisk viruses share the ability to disrupt the normal regulation of cell growth and to give rise to hyperproliferation lesions. These viruses in-

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fect cells that embark on a program of differentiation to form an epithelium consisting of predominantly nondividing cells. T h e small HPV genome cannot encode all the proteins necessary for its own replication; the viral activities that have been identified as potentially oncogenic probably play a principal role in the induction of unscheduled host cell division, thereby allowing the virus to utilize the host replicative machinery. It is therefore of interest to note that both high- and low-risk viruses display some ability to deregulate normal growth control in cultured cells (Storey et al., 1990; Halbert et a[.,1992). All the HPV types are likely t o use similar mechanisms to perturb normal epithelial cell proliferation. The oncogenic potential of the high-risk viruses is paralleled, however, by substantially higher in vitl-o transforming and immortalizing activities. T h e overall difference between the high- and low-risk viruses is likely to reflect the sum of many parameters including viral protein function, control o f - viral gene expression, target cell type, and immune response to infection. T h e consequences of these differences to the normal viral life cycle remain unclear. (;.

HIGH-RISKHPV-ENCODED ONCOPROTEINS

The ability of DNA from the high-risk HPV types to transform and immortalize cells in culture allowed the identification of E6 and E7 as the principal viral oncogenes (Vousden, 199I), although other viral proteins such as E5 ma) also contribute to oncogenesis (Banks and Matlashewski, 1993).Each of these viral genes can function independently in primary or established rodent cells, although E7 generally shows the strongest activity. In primary human genital epithelial cells, a cell type more closely related to the natural target cell of these viruses, cooperation between E6 and E7 is required for efficient immortalization (Hawley-Nelson et al., 1989; Munger el ul., 1989a). This assay is of particular interest since the HPV-immortalized human cells display alterations in growth and differentiation that are ciosely analogous to those seen in cervical intraepithelial neoplasia (McCance el al., 1988; Hudson ef al., 1990) and, like the zn 7~2710 HPV-induced lesions, these cells are not directly tumorigenic (Pirisi et ul., 1988; Kaur and McDougall, 1989). Oncogenic progression can be achieved following the introduction of other oncogenes or exposure to mutagens (DiPaolo et al., 1989; Hurlin et al., 1991; Klingelhutz et al., 1993). This tissue culture system is likely to provide an excellent model in which to identify steps contributing to cervical carcinogenesis. T h e E6 and E7 proteins encoded by the low-risk viruses function only poorly in each o f these immortalization or transformation assays (Schle-

PAPILLOMAVIRUS PROTEINS AND TUMOR SUPPRESSORS

5

gel et al., 1988; Storey et al., 1988; Barbosa et al., 1991) and at least some of the critical differences in the oncogenic activities of the high- and lowrisk viruses lie in the biochemical activities of the E6 and E7 proteins themselves. Much effort has been expended to identify the mechanisms by which E6 and E7 function, with particular emphasis on activities that are absent from or reduced in the low-risk E6 and E7 proteins, to pursue the long-term goal of designing drugs to interfere with the oncogenic activities of the viral proteins. In a series of studies that have drawn heavily on previous analyses of other small DNA tumor viruses such as adenovirus and simian virus 40 (SV40), the mechanism of action of both E6 and E7 was shown to be related to their ability to form complexes with and perturb the normal functions of cell proteins that are involved in regulating cell growth. The intersection of the fields of tumor virology, cell cycle control, and tumor suppression has allowed remarkably rapid progress in our understanding of the significance of these viralhost protein interactions. Ill. Regulation of Cell Growth

A. CONTROL OF

THE

CELLCYCLE

T h e fate of cells within an organism is normally regulated to maintain a balance between cell growth and cell loss. In epithelial tissue, replication is limited to the basal layers. As cells leave these layers they become committed to a pathway of terminal differentiation, resulting ultimately in death. The decision for cell division is regulated by the cellular environment; signals from the extracellular milieu, such as growth factors, are transmitted to the cell nucleus through a complex cascade of signal transduction (Pelech, 1993). Cell proliferation proceeds through a tightly regulated and ordered series of events that constitute the cell cycle. T h e two distinct phases of DNA replication (S phase) and mitotic division (M phase) are separated by the GI and G, periods during which checkpoints and feedback controls operate to monitor the fidelity and completion of the preceding stage and to allow preparation to take place for the next phase of the cell cycle (Murray, 1992). T h e GI period is of particular importance in the interaction between the cell and its environment, since only during this phase is the cell sensitive to inadequacies in nutrient or growth factor levels (Pardee, 1989). Failure to satisfy conditions for progress through the cell cycle at any of the checkpoints results in arrest and possible exit from the cell cycle. Central to this control are the family of cyclin-dependent kinases (cdks), first identified in yeast,

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KAREN H . VOC'SDEN

which play a pivotal role in regulating progress into both S and M phase (Pines and Hunter, 1991). Active forms of the enzyme consist of a catalytic subunit, encoded by the cdk gene family, associated with a regulatory subunit from the family of cyclins. A growing number of cdks and cyclins are being identified with multiple combinations of catalytic and regulatory domains (Xiong and Beach, 1991; Pines, 1993). It is now clear that, in mammalian cells, the activity of the different members of this family of enzymes is specific to certain stages of the cell cycle. Many of the components involved in transmitting the mitogenic signal from growth factors have been identified as potential oncogenes including growth factors themselves, growth factor receptors, and the cytoplasmic kinases responsible for signal transduction (Cantley et al., 1991). Researchers have determined that growth-factor-mediated sigtials can be received by the cell cycle machinery through the G , cyclins, such as the three D-type cyclins (Sherr, 1993) and not surprisingly, alterations in expression of these cyclins have also been identified during cancer development (Hunter and Pines, 1991). B . TUMOR SUPPRESSOR GENES

Perturbations of any of the normal controls of cell proliferation potentially contribute to malignant conversion, either by gain of inappropriate positive signals, as just described, o r by loss of controlling negative signals. Such negative controls are encoded by tumor suppressor genes, which characteristically suffer a loss-of-function mutation in both alleles during cancer development (Marshall, 1991). T h e paradigm for tumor suppressor genes, the Rb-1 gene originally identified in retinoblastomas (Knudson, 197 l), has now been joined by a growing number of genes whose loss of function contributes to cancer development; subsequently many of these genes have been shown to play a role in preventing or delaying progress through the cell cycle (Knudson, 1993). Although the original identification of tumor suppressor genes was through association of hereditary mutation in one allele with predisposition to certain cancers, subsequent studies have shown that somatic mutation within the tumor suppressor loci frequently contributes to the development of sporadic cancers. Indeed, the most common target for mutation identified in human cancers is the tumor suppressor gene p53 (Hollstein et nl., 1991). Several cell-encoded proteins have been detected in association with E6 and E7 and the identification of some of these as the products of tumor suppressor genes has revealed at least some of the mechanisms of viral oncoprotein function.

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IV. Viral/Host Protein Interactions A.

ACTIVITIES OF E7 1 . Interactions of E7 with the pRB Family

The identification of cell proteins that form a complex with the highrisk E7 proteins was greatly aided by the observation that E7 contains a domain with significant sequence similarity to the transforming proteins encoded by two other DNA tumor viruses, adenovirus ElA and SV40 LT (Phelps et al., 1988). This region of each of these proteins is responsible for an interaction with a family of cell proteins that includes the product of the retinoblastoma gene, pRB, and the related p107 and p130 proteins (Munger et al., 198913; Dyson et al., 1992; Davies et al., 1993). Mutational analysis of E7 demonstrated that the ability to bind these cell proteins correlates with transforming and immortalizing activity (Barbosa et al., 1990; Chesters et al., 1990; Phelps et al., 1992), although in the context of the full-length viral genome apparently other viral functions might be substituted for the pRB-binding activity of E7 in the human cell assay (Jewers et al., 1992). The importance of the E7pRB interaction to oncogenic activity is further supported by the reduced affinity of the low-risk E7 proteins for pRB. Substitution of a single amino acid within the pRB-binding region of HPV6 E7 for that found in HPV16 E7 both dramatically increases the affinity of the E7 protein for pRB and also restores transforming activity (Heck et al., 1992; Sang and Barbosa, 1992). Interestingly, in a small sample of cervical carcinoma cell lines, E7 expression could apparently substitute for somatic Rb-1 mutation since these changes were only found in the two HPV-negative cell lines examined (Scheffner et al., 1991). These studies therefore support a role for loss of pRB activity in cervical cancer development and suggest that this loss might be achieved following expression of E7. To test this hypothesis directly, however, an understanding of the normal function of pRB and other related proteins was required. 2 . Normal Functions of the pRB Protein Family pRB, p107, and p130 are related proteins that all play a role in regulating cell growth and progress through the cell cycle. One of the best understood mechanisms by which this family of proteins functions is their interaction with the family of E2F transcription factors (Cobrinik et al., 1993; Zamanian and La Thangue, 1993; Zhu et al., 1993), which in turn participate in the control of expression of several cell genes

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KAREN H. VOUSDEN

essential for cell cycle progression (Fig. 1; Wiman, 1993). Binding of pRB to E2F negatively regulates the activity of the transcription factor (Heldin et al., 1993), rendering it either inactive or able to function as an inhibitor of transcription from E2F-responsive promoters. For pRB, this activity has been shown to be limited to the under- or hypophosphorylated form of the protein that is present in cells during G , (Nevins, 1992); cumulative phosphorylation of pRB as the cells traverse S and G, correlates with the dissociation of most (although not all) of these complexes. Interestingly, the phosphorylation of pRB can be carried out by the G I specific cdks; overexpression of the cyclin component of these enzymes overcomes the cell cycle arrest induced by pRB (Hinds et al., 1992; Sherr, 1993). The direct association between pRB and cyclins D2 and D3 appears to reflect the ability of the cyclins to participate in the regulation of pRB activity (Ewen et al., 1993; Kato et al., 1993) and evidence exists that both p107 and p130 also interact with the D-type cyclins, suggesting that they may be similarly regulated by phosphorylation (Hannon et al., 1993; Li et al., 1993). p107 and p130 also form complexes with cyclins A and E through a region of similarity in these two proteins that is not shared by pRB (Faha et al., 1992; Lees et al., 1992; Hannon et al., 1993; Li et al., 1993), although rather intriguingly neither cyclin A nor E expression can abrogate the growth-inhibitory effect of p107 (Zhu et al., 1993). Despite the functional similarity shared by the pRB family of proteins, there are also obvious differences with respect to the form of E2F with which they interact and the exact mechanisms by which they regulate the activity of the transcription factor (Cress et al., 1993; Dyson et al., 1993; Zhu et al., 1993). This diversity of function probably reflects the complexity of EPF-dependent transcriptional control and the participation of each of the pRB-related proteins in the regulation of different stages of cell cycle progression. Interactions with E2F factors only represent a facet of the potential pRB activities in regulating cell growth, however. pRB has been shown to complex with several other cell proteins including other transcription factors (Rustgi et al., 1991; Kim et d., 1992; Hageemeier et al., 1993; Wang et al., 1993), the cytoplasmic tyrosine kinase c-Abl (Welch and Wang, 1993), and members of the cyclin D family (Dowdy et al., 1993; Ewen et al., 1993). Although some of these interactions, such as those with cyclins D2 and D3, may reflect control of pRB function, in many cases the converse is likely to be true, that is, pRB is responsible for regulating the activity of the bound protein. Of particular interest in this respect is the possibility that pRB regulates the growth-promoting activity of cyclin D1 (Dowdy et al., 1993).

PAPILLOMAVIRUS PROTEINS AND TUMOR SUPPRESSORS

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3 . Consequences of the E7IpRB Interaction

Identification of functions for the pRB protein family was rapidly followed by analyses that revealed that E7, which also preferentially binds to underphosphorylated pRB (Imai et al., 1991),could target negatively regulating proteins complexing with E2F and induce expression of E2F-responsive genes (Fig. 1; Phelps et al., 1991; Chellappan et al. 1992; Morris et al., 1993; Lam et al., 1994). Several genes that appear to play important roles in allowing entry into S phase are regulated by E2F and mutational analysis has indicated that the induction of inappropriate E2F-dependent transcription is likely to account for the ability of E7 to induce DNA synthesis in quiescent cells (Banks et al., 1990). This result is consistent with those of studies showing that expression of E2F

A Cell cycle progression Inactivation

of pR6 by

phorphorylation

Transcription

I

G2

No transcription

B

Inactivation of unphosphorylated pR6

FIG. 1 . Participation of pRB in the control of the E2F transcription factors, which consist of heterodimeric combinations of DP1 and various E2F proteins. (A) Regulation during the G , phase of a normal cell and (B) the consequent disruption of this activity in E7-expressing cells.

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KAREN H. VOCSDEN

itself is sufficient for entry into S phase (.Johnson et al., 1993). Point mutational analysis of E7 has revealed slightly different sequence requirements for interaction with pRB and p107 (Davies el al., 1993) and E7 mutants that retain plO7-binding activity but fail to interact with pRB have been identified. Such mutants have been useful in analyzing the contribution of pRB and p107 to transcriptional regulation of genes such as B-tnyb, whose expression is repressed during the Go and early G , stages of the cell cycle by E2F/pl07-containing complexes. Activation of the B-myb promoter by E7 correlates with the ability of the viral protein to complex p107 rather than pRB, supporting evidence that pRB is not involved in the regulation of this promoter in mouse cells (Lam et al., 1994). Although E7 clearly disrupts the interaction between pRB and EZF, it can become part of a pl07-E2F-containing complex (Arroyo et ul., 1993; Lam et nl., 1994), further emphasizing the differences among these viral-host protein interactions. The consequence of the presence of E7 in the p 107-E2F complex remains unknown, but such interactions are likely to account for the ability of E7 to associate with a cyclin A-dependent kinase that can phosphorylate p107 and p130 (Davies et at., 1993; Tommasino et al., 1993). Interestingly, the interaction between E7 and the kinase occurs later in the cell cycle, during G,, and may reflect an activity of E7 in overcoming G 2 , as well as G I , blocks in cell growth (Vousden and Jat, 1989). Although analyses of E7 activity have been mostly limited to the effect on E2F regulation, the consequences of the E7 interaction with the pRB family are likely to be manifold. Several of the interactions between pRB and other cell proteins have been shown to be disrupted by E7 in uitro, pointing to extensive pleiotropy of E7 function. Such interactions may result in loss of normal transcriptional control through disruption of pKB complexes with transcription factors and the potential of E7 to release cyclin D 1 from negative regulation by pRB suggests an additional means by which the E7-pRB interaction may affect cell proliferation. Although the consequences of the E7 interaction for many of the pRB complexes have not yet been fully explored, it is already clear that they will vary. Evidence suggests that E7 and E2F d o not bind to identical sites o n pRB (Huang rt al., 1993; Wu et al., 1993a) and it may be possible to disrupt the E7-pKB interaction without significantly affecting normal pRB-E2F complex formation. This will not be true, however, for all the cellular interactions involving pRB. The interaction of pRB with Myc, for example, can be successfully competed using a peptide of E7 containing only the pRB-binding domain (Rustgi et af., 1991); the cyclin D family also appears to complex pRB through the sequence motif found in E7 (Dowdy et al., 1993). Other activities of pRB, for example the

PAPILLOMAVIRUS PROTEINS AND TUMOR SUPPRESSORS

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recently described regulation of the cytoplasmic tyrosine kinase c-Abl, cannot be perturbed by E7; presumably these functions of pRB remain unaffected in E7-expressing cells (Welch and Wang, 1993). 4 . PRB-Independent Activities of E7

Despite the obvious attraction of assigning many of the biological activities of E7 to the known interactions, mutations in E7 that do not affect any of the known protein interactions can nevertheless also render the E7 protein transformation and immortalization incompetent (Banks et al., 1990; Phelps et al., 1992). Although these additional activities of E7 have not been identified, their importance is graphically demonstrated by the observation that, in the context of the complete viral genome, the pRB-binding activity of E7 is not necessary for keratinocyte immortalization or wart formation (Jewers et al., 1992; Defeo-Jones et al., 1993). Complementation studies have indicated that these additional E7 activities can substitute for pRB-independent activities of E 1A (Davies and Vousden, 1992), although their mechanisms of action do not appear to be identical. B. ACTIVITIES OF E6 1 . Interactions of HPV E 6 with p53

Although there is no strong structural similarity between E6 and oncoproteins encoded by other DNA tumor viruses, a functional parallel exists in the ability of E6, adenovirus ElB, and SV40 LT to target p53, the product of another important tumor suppressor gene (Werness et al., 1990). T h e interaction between the high-risk E6 proteins and p53 results in the rapid degradation of the p53 proteins through ubiquitin-targeted proteolysis (Scheffner et al., 1990). This activity of E6 depends on the interaction of E6 and another cell protein, E6-AP (Huibregtse et al., 1991); the E61E6-AP complex functions as a ubiquitin-protein ligase (Scheffner et al., 1993). Although p53 is normally the target of ubiquitindependent degradation (Ciechanover et al., 199l),whether E6 enhances the normal mechanism of p53 turnover or whether a novel pathway is utilized is not known. T h e ability of E6 to target p53 proteolysis suggests that, like E7, the consequence of the viral-host protein interaction is the inactivation of the cell protein. Advances in our understanding of p53 function have allowed the verification of this hypothesis and there is good evidence that the E6-p53 interaction is an important component of the oncogenic activity of the high-risk HPV types.

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KAREN H . VOUSDEK

2. Normal Functions of p53 Although loss of wild-type p53 function appears to play a role in most malignancies (Hollstein et al., 1991), there is no clear function for p53 in the proliferation of most cells; mice that lack functional p53 alleles develop normally (Donehower et al., 1992). Inability to express p53 is associated with a high cancer rate in these animals, however, and cells lacking in wild-type p53 display an increased frequency of genetic instability (Livingstone et al., 1992; Yin et al., 1992). Convincing evidence is now available that under some circumstances p53 functions as a checkpoint in the event of DNA damage, after exposure to agents such as ionizing radiation or certain cytotoxic drugs (Lane, 1992). This response is accompanied by a rapid increase in p53 levels due to stabilization of the protein, which results in either arrest at the G, stage of the cell cycle or induction of progranimed cell death. Under normal circumstances, therefore, cells sustaining D N A damage cease proliferation, either temporarily uniil DNA repair has been effected o r permanently through apoptosis. Loss of p53 function can be associated with an inability to undergo G, arrest in the presence of damaged DNA (Kuerbitz et ul., 1992) and the consequent potential to acquire oncogenic genetic lesions is predicted t o contribute to tuniorigenesis. Of course, the cellular responses to DNL4damage are more complex than this and the role of p53 is also less straightforward. This is illustrated by studies indicating that the extremely efficient induction of p53 activity in response to UV daniage is not accompanied by a clear G, arrest, although this response may be related to the activation of proteins that suppress p53 function (Lu and Lane, 1993; Perry et d., 1993). The exact mechanisms by which pi53 functions are not yet clear, although several potentially important activities of p53 have been described and a growing number of inreresting p53-binding proteins have been isolated (Pietenpol and Vogelstein, 1993).Current evidence suggests that the ability of p53 to specifically trans-activate transcription from promoters containing p53-binding sites plays an important role in mediating cell cycle arrest (Fig. 2; Vogelstein and Kinzler, 1992);cell genes that may be transcriptionally regulated by p53 are now being identified (KaStan et nl., 1992; El-Deiry et al., 1993; Wu ei al., 1993b; see Section IV,D). Interestingly, one of these p53-regulated genes, mdm-2, encodes a protein that in turn associates with and negatively regulates the transcriptional activity of p53 in an autoregulatory feedback loop (Momand et al., 1992; Oliner et al., 1993; Wu et al., 1993b). p53 also shows a more general ability to repress transcription of many other cell genes (Ginsberg et al., 1991), particularly those with TATA-containing promoters (Mack et d.,

PAPILLOMAVIRUS PROTEINS AND TUMOR SUPPRESSORS

A DNA damage

L!22&& p53 p53

p53 Stabilization

Transcriptional activation

13

(_> Cell cycle arrest

B DNA damage

p53 Degradation

No transcriptional activation

Cell cycle progression

FIG. 2. (A) Induction of p53 activity following DNA damage in normal cells resulting in the transcriptional activation of cell genes and cell cycle arrest. (B) Targeted degradation of p53 by E6 abrogates the block on cell cycle progression.

1993). This activity may also contribute to the inhibition of cell cycle progression. Evidence for a third activity of p53 in directly interfering with DNA synthesis by binding replication proteins such as RPA (Dutta et al., 1993; He et al., 1993; Li and Botchan, 1993) underscores the important point that regulation of cell proliferation by p53 is likely to be a complex and multifaceted process.

3 . Consequences of the E6lp53 Interaction Initial studies revealed that, although E6 expression in cells results in a reduction of the half-life of the endogenous p53 protein, this effect is not necessarily reflected by a reduction in the total p53 content of E6-expressing cells compared with normal cells (Hubbert et al., 1992;

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KAREN H. VOUSDEN

Lechner et al., 1992). This result suggests that E6 preferentially targets nascent p53 and implies the existence of a stable pool of p53 within the cell that is not sensitive to E6 although the implications of these observations are not yet understood. T h e identification of the damage-response functions of p53 led to the realization that the E6-p53 interaction may not play an important role in normal cycling cells, which are clearly not growth inhibited by p53. Analyses of the effects of E6 under conditions in which p53 would be expected to induce growth arrest, following DNA damage, have shown that E6-expressing cells do not accumulate p53 protein and subsequently fail to undergo the G, arrest (Fig. 2; Kessis et al., 1993). E6 therefore seems to fulfill its predicted role of inhibiting the growthsuppressing activity of p53. Not surprisingly, expression of E6 can abrogate both the truns-activating and the trans-repressing transcriptional activities of p53 (Lechner et al., 1992; Mietz et al., 1992). Evidence suggests that simply the interaction between E6 and p53 is sufficient for a reduction of p53 activity (Lechner et al., 1992; Crook et al., 1994). E6 has been shown to inhibit p53 DNA binding (M. S. Lechner and L. A. Laimins, personal communication), suggesting at least one mechanism for the abrogation of the transcriptional truns-activation. The transcriptional activity of p53 appears to be mediated or modulated through complex formation with several cell proteins such as TBP, CBF, or WT1 (Seto et al., 1992; Agoff et ul., 1993; Maheswaran et ul., 1993) although the ability of E6 to perturb these interactions remains to be determined. 4 . 156 in Oncogenesis

The significance of the E6-p53 interaction in cancer development is supported by the observation that, unlike many other epithelial tumors, HPV-positive cervical cancers very rarely show evidence of somatic p53 mutations (Crook P t nf., 1991c, 1992; Scheffner et al., 1991; Fujita et al., 1992; Choo and Chong, 1993). The straightforward interpretation of these observations is that expression of E6 in HPV-positive cancers abrogates the tumor suppressor activity of p53 and thus eliminates selection for somatic mutation within the p53 gene itself, a notion supported by the ability of mutant p53 to substitute for E6 in the immortalization of human keratinocytes (Sedman et al., 1992). p53 mutations have been detected in the much rarer HPV-negative cancers (Crook et al., 1991c, 1992; Scheffner et al., 1991), although a significant proportion of these also present without evidence for alterations in the p53 gene (Park et al., 1994). Loss of p53 function through indirect mechanisms is also seen in other types of tumor that display a low incidence of p53 mutation. Sarcomas, for example, frequently demonstrate amplification of the mdm-2 gene (Oliner et a/., 1992); presumably inactivation of p53 in these cancers

PAPILLOMAVIRUS PROTEINS AND TUMOR SUPPRESSORS

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occurs through interaction with enhanced levels of the mdm2 protein. Preliminary analyses have indicated that mdm-2 is not frequently amplified in HPV-positive cervical cancers (A. Farthing and K. H. Vousden, unpublished observations), consistent with the notion that expression of E6 is sufficient to inactivate p53 and to allow malignant progression. Although E6 expression clearly interferes with the wild-type activity of p53, the p53 gene remains a target for oncogenic mutations even in HPV-positive cells. Evidence exists that some p53 point mutations can induce both loss of wild-type growth-suppressing function and gain of a positive transforming activity (Shaulsky et al., 1991; Sun et al., 1993; Dittmer et al., 1993). The interaction with E6 would be predicted to prevent only the normal function of p53 and expression of mutant p53 might play a role during HPV-associated tumorigenesis, although possibly at a later stage of the oncogenic process (Crook and Vousden, 1992). Importantly, many p53 mutations render the protein insensitive to E6directed degradation (Crook and Vousden, 1992; Scheffner et al., 1992b), thus allowing the expression of a positive transforming function in E6-containing cells. Although the mechanism by which these mutant p53 proteins contribute to malignant progression of HPV-positive cancers is not known, it may be germane to note that in rodent cells strong synergy exists between E7 and mutant forms of p53 in transformation (Peacock et al., 1990; Crook et al., 1991a).

5. $153-IndependentActivities of E 6 Although much emphasis has been placed on the E6-p53 interaction, evidence suggests that some activities of E6, such as the transformation of rodent cells (Sedman et al., 1992), are not dependent on this interaction and that other important functions of E6 remain to be identified. Of particular interest is the observation that the ability of E6 to target proteins for ubiquitination and degradation is not limited to p53 (Scheffner et al., 1992a; Scheffner et al., 1993), raising the possibility that other important regulators of cell growth are also targets of E6-directed degradation. C. FUNCTION OF HPV-ENCODED ONCOPROTEINS IN THE NORMAL VIRALLIFECYCLE

Although the activities of E6 and E7 in abrogating the activities of tumor suppressor gene products can easily be understood in terms of a contribution to tumorigenesis, the importance of these functions to the virus is more likely to be in maintaining cell replication during infection. In the case of E7, perturbation of the control of E2F activity might play a

16

K A R E N H . VOUSDEN

role in maintaining DN'A synthesis is a cell that has embarked on a program of epithelial differentiation and would normally stop dividing. The ability of E7 to interact with p107 is shared by both high- and lowrisk viruses (K.Davies and K. H. \lousden, unpublished observations); the low-risk E7 proteins also retain the ability to activate transcription of EPF-dependent promoters (Storey et al., 1990; Munger et al., 1991). The association o f E7 with pRB, on the other hand, correlates well with the oncogenic activities of the protein in experimental models, suggesting that this interaction does contribute to the malignant potential of the virus. 'l'he low-risk E7 proteins show a much lower affinity for pKB, although they d o retain some binding activity; the relevance of these differences to normal viral replication are not clear. The normal function of E6 may also be in the prevention of cell growth arrest, either following a stress response to viral infection o r during the normal course of epithelial cell differentiation and death. T h e E6 proteins encoded b y the low-risk HPV types interact with p53 much less efficiently than the high-risk proteins (Werness et ul., 1990; Crook et al., 1991b) and, in in zdro assays, are unable to target p53 for degradation (Scheffner ef al., 1990; Crook et ul., 1Wlb), although indirect ejidence suggests that these proteins also retain some degradation activity (Scheffner et al.. 1992a; Band et al., 1993). Corisisterit with these observations is the modest ability of the low-risk E6 proteins to abrogate p53 transcriptional control (Lechner Pt al., 1992; Mietz et al., 1992; Hoppe-Seyler and Butz, 1993; Crook el al., 1994). The weak interactions of the low-risk E6 and E7 proteins with p.53 and pKB may be sufficient to contribute to the replication of these viruses. However, the clearly enhanced efficiency displayed by the high-risk HPV oncoproteins in targeting proteins with an established tumor suppressor activity may be a crucial coniponent contributing to the overall enhanced oncogenic potential displayed by these virus types.

D. E6

AXD

E'~-TARCETING A COMMON PATHWAY?

'l'he gradual expansion of our understanding of various aspects of the regulation of growth control has recently allowed several pieces of the puzzle to be brought together in a pathway involving p53, pRB, and the cdks (Fig. 3 ) . A gene identified as transcriptionally activated in response to p53, called WAFI (El-Deiry et al., 1993), was independently isolated as C I P I , encoding a cdk-interacting protein (Harper et al., 1993); S D I l , a gene active in senescent cells (Noda et al., 1994); and p21, a component of the cyclin-cdk complexes in normal but not transformed cells (Xiong et ul., 1993). The product of this gene, subsequently

PAPILLOMAVIRUS PROTEINS AND TUMOR SUPPRESSORS

17

DNA damage

t

i

I

- A

p53 Stabilization

t

Point of E6 function

Transcriptionalactivation

Kinase inactive

I

CS~Icycle arrest

Point of E7 function

FIG. 3. Model depicting possible functions of E6 and E7 in a common pathway. In a normal cell, activation of p53 following DNA damage results in increased expression of Picl, which inhibits pRB phosphorylation and prevents release of the pRB-mediated block to cell cycle progression. E6 interferes with this pathway by targeting p53 for degradation, thus preventing Picl expression and allowing phosphorylation of pR3. E7 relieves this block by interfering directly with pRB function and may not be expected to prevent the activation of p53 or consequent inactivation of the pRB kinase.

renamed P I C I , negatively regulates the activity of the G,-specific cdks and consequently inhibits entry into DNA synthesis, thus establishing a direct link between p53 activity and regulators of cell cycle progression. A further step can be taken along this pathway, since the GI cdks inhibited by Picl are capable of phosphorylating and inactivating pRB. Transcriptional activation of PIC1 by p53 would therefore be predicted to result in an inability to escape from the pRB-mediated GI arrest of growth. This model is clearly an oversimplification and p53 almost certainly does not function exclusively through Picl. With this caveat in mind, however, it is of interest to consider the potential roles of the HPV oncoproteins in such a pathway. A straightforward corollary of the model is that proteins that inactivate pRB might function downstream of p53 and be capable of overcoming a p53-mediated growth arrest. Identification of SV40 LT as an antagonist of Picl function is complicated by the ability of the viral protein to abrogate the activity of both p53 and pRB, but at least some support for the model is provided by the observation that, in rat cells, expression of either E7 or adenovirus E1A (both pR3binding proteins) can efficiently overcome the growth-inhibitory effects of wild-type p53 (Vousden et al., 1993). It is possible that both E6 and E7 may function independently to overcome DNA-damage-induced cell cycle arrest, E6 functioning by directly inhibiting p53 function and E7

18

KAREN H . VOUSDEN

acting downstream to release the pRB-induced block. A prediction of this model is that expression of E7 alone would not prevent, and may even induce, an efficient, albeit futile, p53 response. It is therefore intriguing to note that human cells expressing E7, but not E6, contain elevated levels of wild-type p53 protein (Demers et nl., 1994). Clearly, p53 and pRB exhibit other important activities; the fact that each of the small DNA tumor viruses has developed mechanisms to interfere with both cell proteins strongly indicates that many of their functions are not equivalent. T h e identification of a pathway potentially linking the activities of these proteins, however, has allowed the first steps toward untangling the complex webs through which positive and negative regulators of growth function. V. HPV Oncoproteins-Tools

and Targets

T h e identification of- the mechanisms by which E6 and E7 function has presented a panoply of potential uses for these viral proteins both in probing the normal regulation of cell growth and in the design of therapeutic drugs to treat cervical disease. The abilities of E6 and E7 to inactivate at least two tumor suppressor gene products have enormous value as tools to investigat.e the normal function of these cell proteins. Differential abilities of E7 mutants to interact with pRB or p107, for example, have also been used to study the independent activities of these cell proteins in regulating transcription. These studies have contributed to the accumulation of evidence that p107 and pRB display distinct, if related, activities. Identification of additional cell proteins that interact with E6 o r E7 will alniost certainly reveal other factors wit.h a role in the regulation of cell growth. Possibly the most exciting consequence of the rapid advance in our understanding of the functions of E6 and E7 at the molecular level, however, is the identilication of viral-host protein interactions as targets for the action of chemotherapeutic drugs. T h e observation that E6 and E7 expression is generally maintained in cervical cancers and cancer cell lines, combined with evidence that continued expression is necessary for tumor cell growth (von Knebel Doeberitz et nl., 1988; Steele et al., 1992; IIwarig et nl., 1993), provides the additional incentive that anti-E6 or -E7 therapies might also be useful for the treatment of advanced stage disease. Small peptides that interfere with the interactions between E7 and cell proteins such as pRB and p107 have been described (Jones et nl., 1990; Davies et nl., 1993), although a biological effect of these peptides on the growth of E7-transformed cells has not yet been identified. T h e

PAPILLOMAVIRUS PROTEINS AND TUMOR SUPPRESSORS

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possibility remains that they will function as agonists rather than antagonists of E7 function. Despite the obvious problems and caveats, the development of small molecules that target E7 or E6 function holds much promise. The highrisk genital HPVs are the most convincing examples of human tumor viruses, playing a role in the development of the second most common female cancer worldwide. Viral oncoproteins have been identified, and the enormous advances in unraveling their mechanism of action have participated in the convergence of many different areas of research. T h e application of our understanding of the interactions between viral and host proteins directly to treating such a common human disease may be a fitting culmination to these studies.

ACKNOWLEDGMENTS I am extremely grateful to Rachel Davies, Xin Lu, and Roger Watson for their helpful comments and to Lou Laimins for sharing unpublished data. I also apologize to the authors of the many excellent papers that I have been unable to cite.

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