(2008) 27, 7018–7030 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc REVIEW Human papillomaviruses, cervical and

M Thomas, N Narayan, D Pim, V Tomaic´ , P Massimi, K Nagasaka, C Kranjec, N Gammoh and L Banks

International Centre for Genetic Engineering and Biotechnology, Trieste, Italy

Human papillomaviruses (HPVs) are the causative agents HPV-45 and HPV-58. These cancer-associated of a number of human , of which cervical cancer is types are therefore referred to as high-risk types. In the most important.This occurs following persistent contrast, HPV types such as HPV-6 and HPV-11, infection with a limited number of viral subtypes and is which give rise only to benign lesions of the cervix, are characterized by continued expression of the viral E6 and referred to as low-risk types (reviewed in de Villiers E7 oncoproteins.A unique characteristic of the cancer- et al., 2004). causing HPV types is the presence of a PDZ recognition The HPV life cycle is intimately linked to the motif on the carboxy terminus of the E6 oncoprotein. differentiation programme of its infected, epithelial, Through this motif, E6 directs the proteasome-mediated target tissue. As can be seen from Figure 1, HPVs are degradation of cellular involved in the regulation thought to infect the basal cells of the of cell polarity and in control.These through microtraumas, and as the cells differentiate and include components of the Scrib and Par polarity stratify there is coordinate expression of the viral complexes, as well as a number of other PDZ domain- products, ultimately resulting in the generation of new containing substrates.Thus, PVs are now providing novel infectious particles (Doorbar, 2006). A key feature insights into the functioning of many of these cellular of this life cycle is that the cells in which the virus proteins, and into which of these functions, in particular, replicates are terminally differentiating and have thus are relevant for maintaining normal cellular homeostasis. withdrawn from the . However, HPVs, which In this review, we discuss the biological consequences of have a very small genome (approximately 8kb), do not papillomaviral targeting of these cell polarity regulators, encode any gene products that are capable of replicating both with respect to the viral life cycle and, most their DNA directly; they rely exclusively on the cellular importantly, to the development of HPV-induced malig- DNA replicative machinery to complete their replication nancy. programme. The combined action of two viral proteins, Oncogene (2008) 27, 7018–7030; doi:10.1038/onc.2008.351 E6 and E7, causes an expansion in the population of DNA replication-competent cells, in which the host cell Keywords: HPV; E6; PDZ; cell polarity DNA replication machinery is reactivated, allowing high-level amplification of the viral genome (reviewed in Doorbar, 2006). Normally, the induction of such a replicative cycle in the host cell is highly controlled and efficient, but, for Introduction reasons that are still unclear, the controls sometimes break down and events can occur that lead to host cell Cervical cancer is the second largest cause of cancer- immortalization and, ultimately, to cancer. During this related death in women worldwide, and it occurs process, the viral genome often becomes integrated into following persistent infection, sometimes for decades, the host DNA, with the concomitant loss of expression with a specific subset of human papillomavirus (HPV) of many viral gene products, effectively causing the types (reviewed in zur Hausen, 1999, 2002). At present, ‘death’ of the virus as a replicating entity. However, in well over 100 different HPV types have been described, cases where the E6 and E7 proteins continue to be but only a small proportion of these actually contribute expressed the cells survive and, eventually, can undergo to the development of human malignancies. Of these, malignant transformation (Smotkin and Wettstein, perhaps the most well known are types HPV-16 and 1986; Androphy et al., 1987; Banks et al., 1987). We HPV-18, which, between them, are responsible for the now know that these two proteins alone are responsible development of approximately 80% of the world’s cases for the drive towards malignancy. They are invariably of cervical cancer. Other HPV types that are frequently expressed in tumours and in cell lines derived from found in cervical cancer include HPV-31, HPV-33, them, even many years after the initial transforming events, suggesting that they are required for the Correspondence: Dr L Banks, International Centre for Genetic maintenance of the transformed phenotype. Indeed, Engineering and Biotechnology, Padriciano 99, Trieste I-34012, Italy. this requirement has been confirmed in many studies E-mail: [email protected] using a variety of techniques to ablate the expression Human papillomaviruses, cervical cancer and cell polarity M Thomas et al 7019

Productive 1-2 yearsPersistent Up to 20 years HPV-Induced life–cycle infection malignancy 2-3 weeks Lack of oncogene activation immune clearance +/- integration Infection Virus shedding Packaging amplification Genome E7/E6 E7/E6 S-phase competent

Basal layer

Dermis Basal membrane Dermis

Figure 1 Progression from a productive HPV infection to malignancy. The left-hand panel shows the presumed route of HPV infection of the epithelial mucosa basal cell layer through microtraumas. There is coordinate expression of the different viral gene products, with E6/E7 causing an expansion of S-phase competent cells. This allows viral genome amplification and, ultimately, the synthesis and shedding of new viral particles within a period of 2–3 weeks. In the case of persistent infection, lesions are not resolved and high levels of viral DNA can be detected over extended periods of time. This ultimately pre-disposes the host to the development of a malignancy. This is characterized by a loss of differentiation, no viral replication, and high levels of E6 and E7 oncoprotein expression. HPV, human papillomavirus. Adapted from Doorbar (2006).

and/or activities of these proteins, including ribozymes transforming activity is still under investigation; how- (Alvarez-Salas et al., 1998), anti-sense RNA (Steele ever, it seems very likely that the ability of E6 to target et al., 1992; von Knebel Do¨ beritz et al., 1992), RNA cell polarity regulators will be an important factor. In interference (Butz et al., 2003; Yoshinouchi et al., 2003) direct support of this is the exciting realization that E6 and blocking peptides (Butz et al., 2000). In all cases, the proteins from the HPV types linked to cancer develop- cells cease to grow and enter either apoptotic or ment (the high-risk types) all share the ability to target senescent states. Thus, both of these viral gene products certain cellular proteins that are involved in the represent ideal therapeutic targets for intervention in regulation of cell polarity, through a unique carboxy- HPV-induced malignancy. terminal recognition motif. The molecular bases of how E6 and E7 bring about cell transformation are still the subjects of very active research. However, it is clear that the two proteins have complementary activities, with E7 being largely respon- A molecular signature for malignant potential sible for driving cell proliferation and E6 for enhancing cell survival. This is exemplified by the cooperative As we noted above, a small subset of HPV types is linked activity of the two proteins in bringing about immorta- to the development of malignancy, whereas many HPV lization of keratinocytes, the natural target cells of the types cause only benign lesions. Analysis of the sequences virus in vivo. Primary human keratinocytes have quite a of E6 proteins derived from the cancer-causing mucosal short lifespan in tissue culture before they cease to HPV types reveals a remarkable conservation of amino- proliferate and senesce. However, the combination of E6 acid sequences at the extreme carboxy termini of the and E7 is a very potent inducer of keratinocyte proteins. As can be seen in Figure 2, all of these E6 immortalization, whilst either protein alone exhibits proteins have a class I PDZ (PSD95/Dlg/ZO-1)-binding only weak activity in such assays (Barbosa and Schlegel, motif (x-T/S-x-L/V (Songyang et al., 1997)) at their 1989; Hawley-Nelson et al., 1989). Likewise, in the skin carboxy termini. None of the E6 proteins from low-risk of transgenic mice, E7 is a potent inducer of benign cell HPV types have this motif. Of all the protein interaction proliferation, while the addition of E6 results in much domains of E6, it is the only one that is strictly high-risk- more aggressively transformed lesions (Song et al., specific, and so it can be considered to be a marker of 2000). The precise mechanism by which E6 exerts its malignant potential in mucosal HPVs.

Oncogene Human papillomaviruses, cervical cancer and cell polarity M Thomas et al 7020 The HPV-16 and HPV-18E6 oncoproteins have been shown to interact, through this motif, with a large number of PDZ domain-containing cellular substrates. As can be seen from Table 1, many of these proteins are CC CC CC CC implicated in processes that regulate epithelial cell polarity, underlining the importance of this pathway HPV-16 : C MS C C------RSSRTRRETQL both for viral replication and for virus-induced malig- HPV-33 : C AA C W------RS--RRRETAL nancy. In the following discussion, we shall attempt to HPV-31 : C IV C W------RRPRTETQV highlight those interactions that, in our opinion, have been the most extensively verified, and for which there is HPV-35 : C MS C W------KPTRRETEV a clear role in the regulation of cell polarity. First, HPV-58 : C AV C W------RPRRRQTQV however, we shall consider the biological implications of High-risk HPV-52 : C SE C W------RPRPVTQV E6 E6’s PDZ-binding capacity. HPV-18 : C HS C CNRARQE--RLQRRRETQV HPV-45 : C NT C CDQARQE--RLRRRRETQV HPV-39 : C RR C WTTKRED-RRLT-RRETQV Biological consequences of HPV E6 interaction HPV-59 : C RG C RTRARHLRQQRQARSETLV with PDZ-containing targets HPV-56 : C LG C W------RQTSREPRESTV HPV-66 : C LQ C W------RHTSRQATESTV In mice Low-risk HPV- 6 : C LH C WTTCMED------MLP Many important insights into the biological effect of E6 HPV-11 : C LH C WTTCMED------LLP HPV E6’s ability to bind PDZ-containing targets have been provided by a series of studies in transgenic mice, High-risk in which the HPV-16 E6 and/or E7 oncoproteins were RhPV-1 : C PS C ------ASRV E7 expressed from the keratin 14 promoter. This promoter Figure 2 The high-risk HPV E6 oncoproteins contain a PDZ is activated in basal epithelium, thus ensuring that the recognition motif. Schematic diagram showing the HPV E6 E6 and E7 proteins are expressed in a physiologically oncoprotein comprising two zinc fingers, with a highly conserved relevant tissue (Arbeit et al., 1994; Herber et al., 1996; carboxy terminal tail. High-risk types are those frequently found in Song et al., 1999). Mice expressing both oncoproteins cervical cancers whereas the low-risk types are predominantly exhibited a high frequency of epithelial neoplasias found only in benign lesions. For comparison, RhPV-1 E7 oncoprotein is also shown. HPV, human papillomavirus; RhPV-1, and also developed cervical cancer when treated rhesus papillomavirus type 1. with oestrogen. Individual expression of either E6 or E7 also resulted in skin tumours, but with interesting

Table 1 PDZ-containing targets of papillomavirus oncoproteins

PDZ-containing Targets of Papillomavirus Oncoproteins

PROTEIN FUNCTION EFFECT ON TARGET PROTEIN REFERENCE

Dlg Cell polarity/tumour suppressor ubiquitination and proteasomal degradation Gardiol et al. (1999)

Scribble Cell polarity/tumour suppressor ubiquitination and proteasomal degradation Nakagawa and Huibregtse (2000)

MAGI-1 Cell polarity/tumour suppressor Glaunsinger et al. (2000) MAGI-2 PTEN localisation to membrane ubiquitination and proteasomal degradation MAGI-3 Thomas et al. (2002)

MUPP1 Signalling complex scaffold ubiquitination and proteasomal degradation Lee et al. (2000)

PATJ TJ formation and integrity ubiquitination and proteasomal degradation Latorre et al. (2005) plus E6*-directed degradation Storrs and Silverstein (2007)

PSD95 Signalling complex scaffold ubiquitination and proteasomal degradation Handa et al. (2007)

PTPH1/ Protein tyrosine phosphatase ubiquitination and proteasomal degradation Jing et al. (2007) PTPN3 Töpffer et al. (2007)

PTPN13 Non- phosphatase ? Spanos et al. (2008b)

TIP-1 Down regulator of PDZ interactions ? Hampson et al. (2004)

TIP-2/GIPC TGFβ signalling ubiquitination and proteasomal degradation Favre-Bonvin et al. (2005)

CAL Intracellular trafficking ubiquitination and proteasomal degradation Jeong et al. (2007)

PAR3 Cell polarity/tumour suppressor ubiquitination and proteasomal degradation Tomaic et al. (2008) plus relocalisation

Oncogene Human papillomaviruses, cervical cancer and cell polarity M Thomas et al 7021 differences. Expression of E7 alone gave rise to a high the HPV-16 E6 PDZ-binding motif was required for the frequency of mainly benign, differentiated tumours. In induction of anchorage-independent growth in coopera- contrast, expression of E6 alone resulted in significantly tion with an activated ras oncoprotein in both murine fewer tumours, which were, however, far more aggres- and human tonsillar keratinocyte cells (Spanos et al., sive and prone to progression into metastatic cancer 2008a, b). This is particularly interesting in the light of (Song et al., 2000). Co-carcinogen studies with chemical recent studies showing that the loss of certain PDZ carcinogens or with oestrogen in these mice indicated domain-containing substrates of E6 (Dow et al., 2008 that whereas E7 promotes immortalization, E6 pro- and reviewed in Humbert et al. (2008), this issue) also motes progression into full malignancy (Song et al., confers cooperativity with an activated ras oncoprotein. 2000; Riley et al., 2003; Brake and Lambert, 2005). In As noted above, one of the major activities of the the reproductive tract, E6 has significantly weaker HPV E6 oncoprotein is its ability to inhibit , activity than E7 (Riley et al., 2003); however, after which is largely thought to occur through its interaction prolonged oestrogen treatment, cervical tumours did with the pro-apoptotic regulators p53, Bak (reviewed in result, although this activity did not seem to require Thomas et al., 1999) and pro-caspase 8(Filippova et al., PDZ-binding capacity (Shai et al., 2007). Most im- 2007). However, it has also been shown that HPV-16 E6 portantly, however, the ability of E6 to cooperate with can inhibit apoptosis in human airway epithelial cells in E7 in increasing tumour size and frequency, both in the a PDZ-dependent manner (James et al., 2006). When cervix and at other tumour sites, was dependent upon one considers that one of the principal activities of E6 is the ability of E6 to bind to its PDZ domain-containing inhibition of the cell apoptotic response to viral substrates (Nguyen et al., 2003; Shai et al., 2007, 2008). infection, the demonstration that E6 could block Obviously, the prime candidates for these activities of tumour necrosis factor (TNF)-induced apoptosis E6 are those substrates implicated in the regulation of through a nuclear factor-kB (NF-kB), PDZ-dependent cell polarity (see below), but the dissection of which mechanism is particularly intriguing, although the PDZ target(s) is involved will require further dissection of domain-containing targets of E6 that are required for E6’s PDZ-binding capacity, as well as the use of this activity remain to be determined (James et al., conditional knockout mice in some of the polarity 2006). Interestingly, the potential link between PDZ candidates. domain targeting and NF-kB signalling has also been highlighted by studies with HTLV-1 Tax (Higuchi et al., 2007; reviewed in Javier (2008), this issue). In tissue culture Induction of an epithelial-to-mesenchymal transition As in transgenic mice, the transforming activity of E6 (EMT) is a hallmark of cancer development (reviewed in in vitro is not as strong as that seen with the E7 Etienne-Manneville (2008), this issue; Moreno-Bueno oncoprotein. However, a number of different in vitro et al., 2008, this issue). Not surprisingly, both HPV-16 assays are available for assessing the ability of E6 to and HPV-18E6 can induce EMT-like changes in human induce cell transformation, and for dissecting the keratinocytes (Watson et al., 2003; Spanos et al., 2008a). requirement for different domains of the protein in these However, this activity is largely dependent upon an assays. The first demonstration that HPV-16 E6 had intact PDZ-binding motif and, as we will see in the PDZ-binding potential also showed that an intact PDZ- following discussion, the nature of the targets with binding motif was important for the ability of HPV-16 which E6 interacts through this motif provides a E6 to induce morphological transformation in rat 3Y1 rational explanation for this observation. cells (Kiyono et al., 1997). This study demonstrated that the motif was also required for the ability of HPV-16 E6 to confer tumorigenicity upon a mouse fibroblast cell In the viral life cycle line. An intriguing parallel with these studies was the The best method available to recapitulate the normal finding that a similar conserved PDZ-binding motif is polarity of epithelial tissue is the organotypic raft also present on the adenovirus type 9 E4-ORF1 gene culture. Since the papillomavirus life cycle is absolutely product and the human T-cell leukaemia virus type 1 dependent on epithelial differentiation, it is also the only (HTLV-1) Tax oncoprotein, which, in both cases, is tissue culture system in which studies of the virus life required for optimal transforming activity (Kiyono et al., cycle can be made. Work done in the context of 1997; Lee et al., 1997; Weiss and Javier, 1997; reviewed the whole virus, in this case HPV-31, showed that the in Javier (2008), this issue), thereby demonstrating a thickening of the basal layer of epithelium and the fascinating conservation of function between widely presence of nuclei throughout the suprabasal layers that different families of tumour . are normally seen in HPV infections are lost in the In human keratinocytes, the most relevant system for presence of PDZ-binding defective mutants of E6 (Lee assessing the role of E6 in cell transformation in vitro, and Laimins, 2004). In addition, the growth rate of the the role of the PDZ domain is less clear. In the case of mutant E6 virus-infected cells is reduced in comparison HPV-31, the E6 PDZ-binding site does not appear to be with wild-type virus-infected cells and, interestingly, required for the immortalizing activity of the virus, but there is a decrease in episomal copy number and an it is required for increasing cell proliferation (Lee and increase in the frequency of viral genome integration Laimins, 2004). However, in a different assay of events (Lee and Laimins, 2004). Taken together, these keratinocyte-transforming potential, it was found that findings suggest that the PDZ-binding activities of high-

Oncogene Human papillomaviruses, cervical cancer and cell polarity M Thomas et al 7022 risk E6 proteins are important in the viral life cycle and PDZ3 and the PDZ1 domain of MAGI-1 and MAGI-3) have an unexpected role in maintaining viral episomes, has shown that the selection is also determined something which might be related to potential altera- and modulated by the sequences of the PDZ domains tions in cell polarity during asymmetric cell division themselves. It should also be noted that, although many (reviewed in Januschke and Gonzalez (2008), this issue) of the PDZ-containing targets of HPV E6 proteins of the basal epithelial cells. contain multiple PDZ domains, so far only one PDZ A final point worth emphasizing is that studies in the domain per protein has been identified as the E6 target role of the PDZ-binding motif on the viral life cycle are (Lee et al., 2000; Gardiol et al., 2002; Thomas et al., still ongoing. This is important for HPV-16 and HPV- 2001, 2002, 2005), again emphasizing the specificity of 18, for which mutants of the E6 proteins in the context these interactions. Thus, it is highly likely that PV types of a whole viral genome are being generated by several other than HPV-16 and HPV-18may target a different laboratories. This is also particularly relevant when one spectrum of PDZ domain-containing cellular proteins; considers the potential differences in the PDZ domain- exciting evidence that strongly supports this possibility containing substrates of E6 (see below) between will be discussed later. different HPV types. Clearly, understanding the role of Finally, it is worth noting that within the HPV E6 these interactions in the viral life cycle will, by necessity, PDZ-binding motif, there is also embedded a consensus have to be studied on a case-by-case basis. recognition site for protein kinase A (PKA) phosphor- ylation on either the Thr or Ser residues at the À3 position. In the case of many ligand–PDZ interactions, Specificity of PDZ binding such events inhibit the interaction (Chetkovich et al., 2002; Sonoda et al., 2006). This A number of PDZ-containing proteins have been would also seem to be true for HPV-18E6, where described as targets of HPV E6 proteins and these are phosphorylation by PKA greatly inhibits E6-PDZ summarised in Table 1. This is almost certainly an domain recognition (Kuhne et al., 2000). At present, incomplete record since the majority of studies have there is no information on how heavily E6 is phos- considered only the E6 proteins of HPV-16 and phorylated by PKA, either during malignancy or during HPV-18. As was seen in Figure 2, the PDZ domains the course of the viral life cycle, but it is clear that this of other HPV E6 proteins are quite diverse in their offers an extra degree of regulation to the whole set of sequences, and this may be reflected in their target interactions and should be borne in mind when one proteins. In support of this hypothesis, there is considers different experimental settings. substantial evidence to show that HPV-18E6 binds more strongly than HPV-16 E6 to the Dlg1 and MAGI- 1 proteins, and is more efficient in inducing their HPV and the Scrib polarity complex degradation (Pim et al., 2000; Thomas et al., 2001). Exchanging the last amino-acid residue of each E6 A large number of PDZ domain-containing substrates protein from the HPV-18ETQV to the HPV-16 ETQL with roles in the regulation of cell polarity have been (or vice versa) reverses this (Thomas et al., 2001). found to be targets of the HPV E6 oncoprotein (Table 1). Conversely, hScrib is bound more strongly by HPV-16 Several of these targets are proteins found at regions of E6 than by HPV-18E6, again dependent upon the cell–cell contact, including the MAGI-1, -2 and -3 carboxy terminal amino-acid residue, and, equally, it is proteins (Glaunsinger et al., 2000; Thomas et al., 2002) degraded more effectively in the presence of HPV-16 E6 and MUPP1 (Lee et al., 2000), which are found at (Thomas et al., 2005), thus showing that the exact subapical tight junctions (Ide et al., 1999; Hamazaki sequence of the PDZ-binding motif is instrumental in et al., 2001), and the Dlg1 and Scribble proteins substrate selection. An extra dimension was added to (Gardiol et al., 1999; Nakagawa and Huibregtse, 2000; this with the description of the crystal structures of the Thomas et al., 2005), found at lateral adherens junctions MAGI-1 PDZ1 and the Dlg1 PDZ2 and PDZ3 (Bilder and Perrimon, 2000; Firestein and Rongo, 2001). domains, each bound to a peptide homologous to the This apparent level of redundancy is certainly a concern, HPV-18E6 C terminus (Zhang et al., 2007b). The Dlg but some of this is most likely a reflection of differences PDZ3 is not an in vivo target of HPV-18E6, and between different E6 proteins: targeting the same analysis showed that it made a minimum of binding pathway but through different substrates. The classic contacts with the peptide; in contrast, the MAGI-1 example of this was noted above where HPV-16 E6 and PDZ1 and Dlg PDZ2 domains, both of which are in vivo HPV-18E6 each preferentially targets Scrib and Dlg1, targets of HPV-18E6, each made several additional respectively (Thomas et al., 2005). The molecular basis contacts with the peptide, including those with amino- for this is now apparent, but it does emphasize the need acid residues outside the canonical PDZ-binding motif. for caution when comparing different HPV types. It was also found that the exact sequence, not only of Certainly, Scrib and Dlg have all the appearances of the PDZ-binding motif, but also of the upstream and being bona fide targets of the respective E6 oncoproteins, non-canonical amino-acid residues, contributes to the with many reports detailing their binding with E6 and specificity of substrate selection (Thomas et al., 2008). In their subsequent proteolytic degradation in a variety of parallel, comparison of different but very closely related experimental settings (Gardiol et al., 1999; Nakagawa PDZ domain-containing substrates (the Dlg PDZ2 and and Huibregtse, 2000). Perhaps one of the most striking

Oncogene Human papillomaviruses, cervical cancer and cell polarity M Thomas et al 7023 confirmations is through the use of proteomic techno- forms of Dlg have now been extensively described logy. If E6 proteins are overexpressed in cells, and (McLaughlin et al., 2002; Garcia-Mata et al., 2007; protein complexes from these cells are then analysed by Roberts et al., 2007), as has the regulation of Dlg by immunoprecipitation and mass spectroscopy, then the multiple phosphorylation events (Hanada et al., 1997; preferentially bound PDZ domain-containing substrates Koh et al., 1999; Sabio et al., 2005; Massimi et al., 2006). of HPV-16 and HPV-18E6 are indeed seen to be hScrib Recent studies have begun to combine these observa- and hDlg (Bohl et al., 2007; V Tomaic´ , personal tions and it is becoming clear that nuclear accumulation observations). of Dlg is, in part, cell cycle-regulated, being phospho- A salient characteristic of the high-risk HPV E6 rylated by cyclin-dependent kinases 1 and 2, thus oncoproteins is their ability to target substrates for providing a link between cell cycle regulation and Dlg proteolytic degradation through the protea- function (Narayan et al., 2008). Intriguingly, these some pathway. This is an extremely efficient way for the phospho-acceptor sites, plus several others, are also viral oncoproteins to overcome the often massive phosphorylated by mitogen-activated protein kinase stoichiometric imbalance that they frequently encoun- (MAPK) following exposure to osmotic stress (Sabio ter, with the cellular target proteins often being present et al., 2005). Once again, certain of these phosphory- in much higher quantities. In the cases of Dlg and Scrib, lated forms of Dlg are preferentially degraded by the E6 both are indeed targeted for proteasome-mediated oncoprotein (Massimi et al., 2006). Most intriguingly, degradation. However, it is clear that E6 does not however, some of these phosphorylation events result in induce degradation of the entire cellular complement of a redistribution of Dlg from nuclear/membrane-bound Scrib and Dlg, as they are both still readily detectable in pools to an accumulation in insoluble complexes at sites HPV-containing cell lines. Careful analysis in the case of of cell contact (Massimi et al., 2008). A striking feature HPV-18E6 and Dlg1 reveals that the virus actually only of this relocalization upon exposure to osmotic stress is induces degradation of certain cellular pools of the that the other two components of the Scrib polarity protein. Intriguingly, this appears to involve differential complex, Scrib and Hugl1, behave in exactly the same phosphorylation events and nuclear localization way, demonstrating that the whole complex is intimately (Massimi et al., 2004, 2006). linked to the MAPK signalling cascade (Figure 3). It is Viral targeting of specific nuclear pools of Dlg also worth highlighting that such signalling cascades are provides very strong evidence for Dlg having multiple active during epithelial cell differentiation (Eckert et al., functions within the cell and not solely being utilized as 2002; Efimova et al., 2003; Ivanova et al., 2006) and a scaffolding protein at sites of cell contact. Nuclear during which a redistribution and accumulation of the

PATJ Crb3 P Pals1 Cdc42 GTP

Subapical Par6 aPKC HPV HPV E6 region Par3 E6* Pip RhPV PTEN E7 MAGI-1 Tight HPV junction E6 Cdk1/2?

P P P MA P Dlg PK HPV Diffe P rentia P 18E6 Hugl-1 tion P M AP Scrib D K Adherens iffer Dlg P ent P M iati D A on junction i P ff K Hugl-1 er en ti at io HPV n 16E6 Scrib

Figure 3 Oncogenic papillomaviruses exert a coordinated attack on cell polarity regulators. A schematic diagram showing the different components of the cell polarity ‘supercomplex’ that are susceptible to the papillomavirus oncoproteins. HPV-16 E6 preferentially targets Scribble, whereas HPV-18E6 preferentially targets Dlg. MAGI-1 is also highly susceptible to HPV E6 degradation. PATJ is a substrate for full-length E6 and for the alternatively spliced isoform, E6*. Meanwhile, RhPV-1 E7 targets Par3. Also shown are some of the post-translational modifications that are known to result in enhanced phosphorylation and relocalization, with MAPK driving an accumulation of the Scrib complex at sites of cell–cell contact. HPV, human papillomavirus; MAPK, mitogen-activated protein kinase; RhPV-1, rhesus papillomavirus type 1.

Oncogene Human papillomaviruses, cervical cancer and cell polarity M Thomas et al 7024 whole Scrib complex at sites of cell contact is also important feature in the development of diverse cancers, observed (Watson et al., 2002; Cavatorta et al., 2004; but the precise mechanism by which it occurs can differ Schimanski et al., 2005; Gardiol et al., 2006). depending on whether there is HPV involvement or not, All of the above implies that, in the context of the with evidence to suggest that transcriptional downregula- viral life cycle and the induction of malignancy, nuclear tion also plays a role (Cavatorta et al., 2008; reviewed in hyperphosphorylated forms of Dlg are particularly Moreno-Bueno et al. (2008), this issue). susceptible to E6 degradation. Obviously, understand- ing the functions of these different forms of Dlg is now a major research aim, but the evidence would point to A lesson from monkeys their having novel roles in cell cycle regulation, stress signalling and differentiation. Recent studies would also Rhesus macaques, Maccaca mulatta, are the only suggest that hDlg can interact with key elements of the species, other than human, in which infection with the transcriptional machinery, implying a potential function natural PV, in this case rhesus papillomavirus type 1 for Dlg in regulating (P Massimi, (RhPV-1), which is closely related to HPV-16, is both personal observations), thereby providing a potential sexually transmitted and gives rise to cervical neoplasia link between the regulation of cell polarity and (Ostrow et al., 1991; de Villiers et al., 2004; Wood et al., transcriptional control. This once again highlights the 2007). Thus, it is a high-risk PV type, but examination of unique insights that DNA tumour viruses can provide in the E6 protein sequence showed no sign of a PDZ- identifying hitherto unknown functions and activities of binding motif. However, a type 1 PDZ-binding motif is their cellular substrates. found on the carboxy terminal of the E7 protein The splicing arrangement of the early transcripts of (Figure 1), and its sequence (A-S-R-V) is, within the high-risk, mucosal HPVs leads to one or more mRNA constraints of a type 1 motif, quite different from that of species that encode truncated versions of the E6 protein, the motifs found on HPV-16 or HPV-18E6 (Tomaic ´ commonly known as E6*. Whilst early studies of the et al., 2008). As we have seen that small differences in properties of these proteins demonstrated their effects on PDZ recognition motifs can have large effects upon the the functions of E6 (Pim et al., 1997, 1999), more recent preferred PDZ domain target, it should be clear that the studies have shown that they may, by themselves, have spectrum of RhPV-1 E7 targets might be very different functions that relate to certain PDZ domain-containing from that of the HPV E6 proteins. And thus it proved: substrates, with E6* being shown to degrade PATJ in RhPV-1 E7 binds only weakly to hScrib and hDlg, the absence of a PDZ recognition motif (Storrs and although its interaction with the pRb tumour suppressor Silverstein, 2007). Interestingly, phosphorylation events was equivalent to that of the HPV-16 E7, showing that that drive proteins such as Dlg into cytoskeletally- the protein itself was in no way defective. A mass- associated complexes may render these substrates more spectroscopic analysis of RhPV-1 E7 binding partners susceptible to E6*-dependent degradation than they are to then revealed that the preferred PDZ-containing sub- that induced by full-length E6, although the precise strate of RhPV-1 E7 is Par3, which it can target for mechanism is still under investigation (Pim et al., proteasome-mediated degradation (Tomaic´ et al., 2008). submitted for publication). This appears to be a biologically relevant substrate of The biochemical data supporting the identification of RhPV-1 E7 as mutants of E7 that cannot bind Par3 hDlg and hScrib as targets of HPV E6 are very strong, but are defective in their ability to transform primary what about the situation in vivo? Certainly, during the rodent cells. development of cervical cancer there is a marked Par3, also known as Bazooka in Drosophila, is, progressive loss of both hScrib and hDlg1 protein together with Par6 and aPKC, a member of the PAR expression, consistent with their being targets of the complex, which with the Crumbs and Scrib complexes HPV E6 oncoprotein (Watson et al., 2002; Cavatorta controls cellular apico-basal polarity (Bilder, 2004; et al., 2004; Nakagawa et al., 2004). Indeed, by the time Margolis and Borg, 2005; Humbert et al., 2006; for the tumour is fully invasive, the hScrib and hDlg proteins reviews see Aranda et al., 2008; Humbert et al., 2008). are essentially undetectable in cervical cancers. However, So it now appears that while at least some of the HPVs it is important to note that both proteins, as well as target members of the Scrib complex through their E6 Hugl-1, are also lost at the later stages of many other protein, the RhPV-1 targets a member of the PAR epithelial cancers (Fuja et al., 2004; Navarro et al., 2005; complex through its E7 protein (Figure 3). Thus, the Schimanski et al., 2005; Gardiol et al., 2006), making it viruses target the same pathway by the same mechan- difficult to determine which changes are E6-specific and ism, albeit with different cellular target proteins and which are the results of other factors. Perhaps this is best different viral effectors. This degree of functional exemplified by the analysis of hDlg in C33I cells. These are conservation indicates that a very strong evolutionary derived from a cervical cancer, but are one of the very few stimulus is at work here. such cell lines that lack HPV DNA sequences. In this case, the hDlg levels are also very low, but interestingly, this is not due to enhanced proteolytic degradation, but is rather PAR and Scribble complexes due to suppression of hDlg transcription (Mantovani et al., 2001). Taken together, these reports suggest that the The PAR and Scrib complexes are mutually dependent loss of components of the Scrib polarity complex is an for their correct cellular localization (reviewed in

Oncogene Human papillomaviruses, cervical cancer and cell polarity M Thomas et al 7025 Humbert et al., 2006; Aranda et al., 2008, this issue; (2008), this issue), and are targeted by the HPV-16 and Humbert et al., 2008, this issue): the Scrib complex HPV-18E6 proteins for proteasome-mediated degrada- restricts the PAR complex to the apical membrane, tion (Glaunsinger et al., 2000; Thomas et al., 2001, whereas the PAR and Crumbs complexes together 2002). Indeed, biochemically they appear to be the most exclude the Scrib complex from the apical membrane avidly bound PDZ targets of E6 (Zhang et al., 2007b) (Bilder et al., 2003; Tanentzapf and Tepass, 2003). and the most susceptible to degradation (M Thomas, Together with the basolateral targeting of Scribble personal observation). endowed by its LAP domain, this restricts the Scrib The functions of the MAGI proteins are not yet complex to basolateral cellular membranes, below the entirely clear and they may vary depending upon the zonula adherens (Legouis et al., 2003). Meanwhile, the cell type studied. Their known binding partners include Hugl-1 protein suppresses the formation of PAR b-catenin (Dobrosotskaya and James, 2000) and complex in the basal domain, while its phosphorylation mNET-1 (Dobrosotskaya, 2001). MAGI-3 has been by aPKC excludes it from the apical region, driving it to reported to bind the lysophosphatidic activated receptor the lateral domain ( et al., 2003; Yamanaka et al., LPA(2) to enhance Erk and RhoA activation (Zhang 2003, 2006). These protein complexes are thought to et al., 2007a), with potential downstream effects on cell coordinate the transmission of intra- and extracellular survival signalling. Most interestingly, perhaps, they, polarity-related signalling to cellular effectors such as like Dlg, have been reported to interact with the PTEN the cytoskeletal network and the membrane, RNA tumour suppressor (Adey et al., 2000; Wu et al., and protein-trafficking pathways (Humbert et al., 2000a, b; Kotelevets et al., 2005). PTEN is a lipid 2006). As we mentioned earlier, HPV-16 and HPV-18 phosphatase that regulates phosphoinositide (PIP) turn- E6 have also been reported to induce degradation of over, antagonizing PI3 kinase activity and thus down- PATJ, a component of the Crumbs complex (Latorre regulating Akt activity. It exhibits a dynamic membrane et al., 2005; Storrs and Silverstein, 2007), suggesting a association (Vazquez et al., 2006) and is required for sustained viral assault upon these polarity-controlling the generation of the apical plasma membrane, complexes. thus contributing to apico-basal polarity (Comer and It is clear, however, that even slightly altering the Parent, 2007; Martin-Belmonte et al., 2007). Given its balances between the proteins of the Scrib, PAR and function and location, it is perhaps not surprising Crumbs complexes can have quite serious effects on the that it contributes to aPKC regulation (Comer and correct functioning, not only of each complex, but also Parent, 2007) and it binds directly to Par3 (von Stein of the ‘polarity supercomplex’ that comprises them all. et al., 2005) through the Par3 PDZ domain 3, whereas From the discussions here it can be seen that in fact the PDZ2 is the site of PIP binding, providing a focus on the oncogenic papillomaviruses do not solely target the apical membrane for the interaction of these important polarity-related functions of the Dlg, Scrib, Par3 and components of membrane polarity (Wu et al., 2007; PATJ proteins. Thus, at certain stages of the viral life Feng et al., 2008). It is interesting that the RhPV-1 E7 cycle, de-regulation of polarity could be of advantage to impinges on this pathway through Par3 and the HPV E6 the virus, and this can certainly be envisaged in the through the MAGI and Dlg proteins, and it underlines asymmetric division and differentiation of the infected yet again the functional conservation of effect between basal keratinocytes or stem cells that are believed to be the different virus types, though using different cellular the primary targets of HPV infection. However, other targets and viral effector proteins. functions of the polarity complex, related perhaps to It is relevant to note here that PTEN appears to have signalling, differentiation and proliferation control, are a role in the maintenance of stem cell phenotypes most likely to be targeted at other stages of the viral life (reviewed in Tamguney and Stokoe, 2007). In haema- cycle. Clearly, during malignant progression, the dis- topoetic cells, the conditional knockdown of PTEN ruption of the whole polarity complex is, in fact, one of results in an expansion of the population and differ- the later downstream effects, most likely enhancing entiation, at the expense of the stem cell phenotype invasiveness and . (Yilmaz et al., 2006; Zhang et al., 2006). Similarly, in basal stem cells of the prostate, deletion of PTEN results in a marked proliferation and differentiation, MAGI and PTEN often followed by intraepithelial neoplasia (Wang et al., 2006). Considering that papillomaviruses are The MAGI-1, MAGI-2 and MAGI-3 proteins are thought to infect epithelial stem cells and their life cycle MAGUK proteins with inverted structure, having requires an expansion of the infected cell population, guanylate kinase domains at the amino followed by differentiation, it might be speculated terminal end of the polypeptide. In addition, each has that the ability to disrupt PTEN activity gives an WW domains and at least five PDZ domains (Dobro- evolutionary advantage to the virus. Unfortunately, in sotskaya et al., 1997; Wu et al., 2000a, b), giving the those cases where infection is persistent or the viral potential to interact simultaneously with several other genome integrates, the loss of the nuclear activities of proteins. They have been shown to be targets for virus PTEN in preventing chromosomal abnormalities could oncoproteins, such as adenovirus type 9 E4ORF1 contribute to the malignant transformation of the cell protein and the HTLV-1 Tax protein (Glaunsinger (Shen et al., 2007; Trotman et al., 2007; Wang et al., et al., 2000; Ohashi et al., 2004; reviewed in Javier 2007).

Oncogene Human papillomaviruses, cervical cancer and cell polarity M Thomas et al 7026 Other PDZ-containing targets of E6 CAL (Jeong et al., 2007), the cystic fibrosis trans- membrane regulator-associated ligand, has been shown In addition to those interactions that have been to be subject to degradation in the presence of HPV-16 described above, HPV E6 has been reported to bind to E6. It is involved in the vesicle trafficking of plasma a number of other PDZ-containing targets about which membrane proteins and, as such, may be involved in less is known, and these are summarized in Table 1. cell–cell communication and planar cell polarity (re- MUPP1 has been identified as a common target viewed in Tanos and Rodriguez-Boulan (2008), this of two viral oncoproteins, Ad9 E4ORF1 and HPV E6 issue). For CAL, as for all the more recently described (Lee et al., 2000). It is a multiple PDZ-containing PDZ-containing targets of HPV E6 proteins, further protein, with 13 PDZ domains (Ullmer et al., 1998), work is required to determine the effects and purposes and it is thought to be involved in the selective targeting of these interactions. and assembly of signalling complexes (Becamel et al., 2000). In polarized epithelial cells, it localizes to tight junctions through the Coxsackie and adenovirus Conclusion receptor (Coyne et al., 2004), where it interacts with the members of the and JAM (junctional It is clear that interaction with PDZ domain-containing adhesion molecule) families and may be involved in members of the Scrib, PAR and Crumbs polarity cell polarity signalling, barrier function complexes is a feature of the high-risk mucosotropic and osmotic stress responses (Hamazaki et al., 2001; papillomaviruses, both human and simian, as it is of Kimber et al., 2002; Jeansonne et al., 2003; Lanaspa certain other oncogenic viruses, such as adenovirus and et al., 2007). HTLV-1. However, as we have mentioned above, the TIP-1 and TIP2/GIPC were originally defined as Tax- proteins that are targeted are multifunctional, as are the interacting proteins, partners of the HTLV-1 Tax complexes that they form. It is also clear that only oncoprotein (reviewed in Javier (2008), this issue), and certain subsets of these cellular proteins are targeted by each has been found by two-hybrid assays to bind an E6 the viral proteins, depending upon such things as the protein: TIP-1 with HPV-16 (Hampson et al., 2004) and post-translational modifications, cellular localization or TIP2/GIPC with HPV-18(Favre-Bonvin et al., 2005). potentially even the differential splicing of the viral or TIP-1 is a potential RhoA effector (Reynaud et al., cellular proteins in question. The contribution of these 2000) and TIP-2 is involved in transforming growth interactions to PV infection and, clinically perhaps more factor-b (TGF-b) signalling (Blobe et al., 2001). In each important, to PV-induced malignancy, is still the subject case, the E6 protein was shown to degrade the target, of intense research and furious debate. Nonetheless, one but it is not yet clear at what point in virus replication or feature of all this research is clear: studying the transformation these interactions might occur. interactions of the tumour viruses with their cellular The other reported PDZ targets of E6 have not been target proteins has contributed greatly to our under- described as targets of other oncogenic viruses, and it is standing of the complexity of those proteins and their thus difficult to assess their relevance to transformation functions. A virus that has evolved over millennia to in general. Like MUPP1, PSD95 (Dlg4, another Dlg manipulate the balance of cellular growth and polarity family member) (Handa et al., 2007) is thought to be a controls for its own survival is a good indicator scaffold for signalling complexes, but has so far mainly of the pivotally important points in the network of been described at neuromuscular synapses. The PTPH1/ interactions governing those controls. By subtly altering PTPN3 protein tyrosine kinase (Jing et al., 2007; the delicate equilibria between the polarity complexes, To¨ pffer et al., 2007) and the PTPN13 non-receptor the virus ‘puts a thumb on the balance’ and thus pushes kinase (Spanos et al., 2008b) are also described as HPV the host cell in a favourable direction. By analysing the E6 targets. Tyrosine kinase phosphorylation is involved when, the where and the how modified of favourite in signalling and is commonly altered in transformed tumour virus target proteins, we can gain a clearer cells and cancers. Because such kinases are involved in understanding of what the function of that protein is in the cross-talk of multiple pathways, it could be the healthy cell and why targeting it can lead to speculated that tyrosine phosphorylation/de-phospho- transformation. rylation might play a role in the integrity and function of This again demonstrates the strength of the DNA polarity complexes and the proteins whose functions tumour virus as a research tool for studying cellular impinge on these. Interestingly, shRNA-induced processes. After all, their survival depends on getting it PTPN13 loss in tonsillar epithelial cells will induce right; by studying them, we could improve our own anchorage-independent growth, and PTPN13 loss can survival too! also cooperate with activated ras to induce invasive . Equivalent phenotypes are induced by wild- Acknowledgements type, but not PDZ binding-defective, HPV-16 E6 (Spanos et al., 2008b). This provides compelling We gratefully acknowledge research support provided by the evidence that the PTPN13 interaction plays a role in ICGEB Fellowship Programme, the Associazione Italiana per la HPV-induced malignancy, and it will be interesting to Ricerca sul Cancro and the Association for International Cancer determine how this pathway might impact upon the Research. K Nagasaka is supported by the YKK Scholarship control of cell polarity. Foundation and the Kanzawa Medical Research Foundation.

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