Molecular mechanisms underlying human papillomavirus E6 and E7 oncoprotein-induced cell transformation

Accepted Manuscript Title: Molecular mechanisms underlying human papillomavirus E6 and E7 oncoprotein-induced cell transformation Author: Suruchi Mittal Lawrence Banks PII: DOI: Reference:

S1383-5742(16)30082-5 http://dx.doi.org/doi:10.1016/j.mrrev.2016.08.001 MUTREV 8177

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Mutation Research

Received date: Revised date: Accepted date:

16-5-2016 18-7-2016 2-8-2016

Please cite this article as: Suruchi Mittal, Lawrence Banks, Molecular mechanisms underlying human papillomavirus E6 and E7 oncoproteininduced cell transformation, Mutation Research-Reviews in Mutation Research http://dx.doi.org/10.1016/j.mrrev.2016.08.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Molecular mechanisms underlying human papillomavirus E6 and E7 oncoprotein-induced cell transformation

Suruchi Mittal* and Lawrence Banks

International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34149 Trieste, Italy.

* Author for Correspondence

Abstract

Human papillomaviruses (HPVs) are the causative agents of 5% of all human cancers, with cervical cancer being the most important. Two viral oncoproteins, E6 and E7, are essential for the development and maintenance of malignancy. Both proteins function by targeting critical pathways that are essential for maintaining cellular homeostasis. As a consequence of these activities, this produces an environment that is favourable for the normal viral life cycle, but when perturbed, can result in the initiation of changes to the host cell, which ultimately results in the development of a malignancy. In this review we discuss the role of these different functions of the viral on-

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coproteins during the viral life cycle and carcinogenesis, with an emphasis on how induction of DNA damage by the viral oncoproteins, in conjunction with the stem like nature of the target cells, can ultimately result in the development of cancer.

Introduction

Human papillomaviruses (HPVs) are small, double-stranded DNA viruses that specifically infect epithelia and the HPV family contains approximately 200 virus types. The specific tropism of the virus types differ; whilst most HPV types cause self-limiting benign lesions, a small subset of so-called high-risk types are the causative agents of numerous human malignancies, which include cervical cancer, other anogential cancers and certain head and neck cancers. [1],[2],[3]. This concept of high- and low-risk virus types emanates from observations made more than 30 years ago which showed that certain HPV types were more frequently found in cancers [2],[4],[5],[6]. These observations were later supported by a plethora of studies demonstrating that the “high-risk” virus types are defined by unique biological properties where the viruses have inherent ability to interfere with the control of proliferation and the maintenance of genomic stability within the infected cell. 2

Nearly all cervical cancers (>99%) are linked with high- risk HPV infection. [1],[7],[8] and cervical cancer is the fourth most common cancer in women, with an estimated 528,000 new cases in 2012 globally. [9]. Normally, HPV infections resolve without any intervention within a few months of acquisition, and about 90% of infections are cleared within 2 years, but persistent infection and failure of the immune system to clear the high-risk HPVs can lead to the development of cervical neoplasia. HPV16 and HPV18 are the most regularly found HPV types in cervical cancer and account for approximately 80% of the world’s cases of cervical cancer. The other cancercausing high-risk HPV types are 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 [3].

One of the hallmarks of HPV-induced malignancy is the continual expression of the two crucial viral oncoproteins, E6 and E7, throughout the development of the tumor which appears to sustain the cancer phenotype. Indeed, there is persistent high-level expression of E6 and E7 in cervical cancerderived cell lines [10],[11],[12]. Inhibition of E6 and/or E7 expression or function leads to cancer cell growth arrest and apoptosis, demonstrating the continued importance of E6 and E7 in maintaining the transformed phenotype of the cells many years after the initial transforming events [13],

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[14],[15],[16],[17]. Therefore, these two oncoproteins represent ideal targets for therapeutic intervention in HPV-induced malignancies.

Human papillomavirus life cycle: from infection to malignant progression

The HPV life cycle is tightly linked to the differentiation profile of the host epithelial cells [18]. HPVs infect cells in the basal layer of stratified squamous epithelia, which is exposed as result of small micro-wounds. Their life cycle starts with an infection of these basal cells, which are the only proliferating cells in normal epithelia, as the differentiated cells in the suprabasal layers have normally exited the cell cycle. HPVs establish their genomes as extrachromosomal elements, or episomes, after infection and are solely dependent on the cellular replication proteins to mediate viral DNA synthesis. In the scenario of an HPV infection, the suprabasal cells remain in the cell cycle and, as they differentiate, a subset of cells re-enter S phase thereby allowing replication of HPV genomes in a process known as amplification [19]. This process is followed by capsid protein synthesis, virion assembly and ultimately the release of new infectious virus particles [18] (see Figure 1). The HPV-infected cells maintain their proliferative potential and remain 4

uncoupled from differentiation through the inactivation of key cell cycle regulators, including members of the pRb family of proteins, pRb, p107 and p130 [20],[21]. The HPV E7 proteins interact with these essential cell cycle regulators [22],[23], leading to the release and activation of E2F transcription factors that regulate S phase genes [24],[25],[26]. One consequence of this activity of E7 can lead to the activation of the p53 tumour suppressor pathway, potentially resulting in either cell growth arrest or apoptosis [27],[28],[29]. To evade this inhibition, the high-risk E6 proteins have evolved to target p53 for proteasomal-mediated degradation [30],[31],[32]. This cooperative action of the high-risk E6 and E7 oncoproteins abrogates multiple cell cycle checkpoints, thereby allowing genome amplification whilst ensuring the continued survival of the infected cell [8],[33]. As can be imagined, cells that are persistently infected with HPV and undergoing repeated rounds of DNA synthesis with concomitant compromised DNA damage surveillance pathways, are highly prone to mutation [1]. This can range from point mutations to large chromosomal abnormalities [34],[35],[36],[37],[38],[39],[40], which in some cases appears to be linked to the ability of E6 and E7 to perturb the normal centrosome cycle [41],[42], [43],[44],[45]. Normal productive cervical infections can lead to mild as well as to moderate cellular abnormalities, and histologically such condi-

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tions are manifested as cervical intraepithelial neoplasia (CIN 1 and 2). These lesions usually regress within 1-2 years, accompanied by viral clearance as a result of immune responses to E2, E6 and E7 [46],[47],[48],[49] . However the virus has several mechanisms available for overcoming the host immune response, and these include suppression of innate immunity, suppression of T cell effector function, and frequent loss of human leukocyte antigen (HLA) expression, sometimes resulting from genetic events, all of which can lead to immune evasion [50], [51]. Failure of the immune system to clear such persistent HPV infections can result in the development of cervical cancer after several decades [2]. HPVs are maintained as episomes in precancerous lesions whereas, in many high-grade lesions, genomes are found integrated into the host chromosome. However no hotspots for such integration have been identified, but some fragile sites with genomic instability have been proposed [52],[53]. Indeed, recent studies have shown that HPV genomes replicate at fragile sites in the host genome, and while at the same time hijacking the host DNA damage response, this can also increase the chance of viral integration at these sites [54], in turn promoting further rearrangements within this locus [55]. Thus, whilst there is considerable debate about the precise role of viral integration in the development of HPV induced malignancy, it seems likely that the re-

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sultant loss of control of E6/E7 expression which often occurs during viral integration most likely also contributes towards these later stages of disease development.

HPV-associated cancers are initiated by expression of E6 and E7 oncoproteins: evidence of direct oncogenicity

A plethora of studies have demonstrated the role of E6 and E7 in the transforming activity of HPVs. The first direct evidence that established the intrinsic transforming activity of E6 and E7 came from the seminal studies in NIH3T3 cells, where it was shown that the genomic DNA containing HPV16 from cervical cancer could induce malignant transformation, as could the cloned HPV16 genome [56],[57]. Thus, while using immortalized NIH3T3 cells in such assays are not very relevant with respect to the natural target cells of the virus, it nonetheless showed that the HPV16 genome could confer a fully malignant phenotype in such a cellular context. Other studies showed that the major transforming activity in these assays was encoded by the E7 gene [58],[59], since the HPV16 and HPV18 E6 proteins can induce the transformation of established rodent cells, but the efficiency with which this occurs is much lower than that with E7 [60],[61].

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Experiments performed on established cells yielded important information about the transforming ability of the different viral oncoproteins, but the studies on primary rodent cells were deemed to be more relevant assays to decipher the true potential transforming capability of the virus. These studies were done on primary baby rat kidney cells (BRK), primary mouse kidney cells (BMK) and rat embryo fibroblasts (REF). Although the results obtained were consistent within each cell type, BRK and BMK are more relevant owing to their epithelial origin. The first study showing HPV16 could contribute to the immortalization and transformation of primary BRKs was dependent on the co-transfection of EJ-ras [62]. This study demonstrated that the transforming capability was localized to the genomic region encoding E6 and E7 proteins. In the majority of the studies the choice of oncogene was EJ-ras [63], however cooperation with v-fos could also transform primary BMKs. Similar to the results in established rodent cells the E7 oncoprotein was found to be the more potent of the two viral oncoproteins in bringing about cell transformation. Furthermore, these early assays also highlighted critical differences between the oncoproteins derived from the low-risk and high-risk virus types, where only high-risk E7 was found to be able to induce primary cell transformation in cooperation with an activated cellular oncogene [64],[65].

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Studies performed in primary human keratinocytes more closely resemble the natural sequence of immortalization event seen in vivo, and represents a more relevant transformation system for assessing the transforming potential of different HPV types and their associated oncoproteins. These are also the natural host cells for HPV and are significantly harder to transform than rodent cells. The early immortalization studies were performed using keratinocytes from a range of anatomical sites, including genital tract and oral mucosa, all of which provided comparable results. Primary human keratinocytes have a short life-span in tissue culture before they cease to proliferate and senesce. However, the introduction of HPV-16 or HPV-18 genomes into foreskin, cervical and oral keratinocytes resulted in the extended lifespan of these cells in tissue culture. In all cases this required the combined expression of E6 and E7, which highlights the cooperative activities of the viral oncogenes in driving cell proliferation and establishment of immortalisation [66],[67],[68],[69],[70],[71],[72],[73]. Furthermore, this also emphasizes the important role played by acquisition of additional oncogenic mutations in order to achieve a fully transformed phenotype. As with the primary rodent cell assays, the low-risk HPV E6 and E7 oncoproteins were again not effective in inducing immortalisation of primary human keratinocytes [70],[71],

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again demonstrating a striking correlation between in vitro transforming potential and oncogenic potential in vivo.

Numerous studies have been carried out using HPV transgenic mouse models and these have provided a wealth of information regarding the in vivo biological properties of HPV oncogenes, in particular HPV16 E6 and E7. The majority of these studies have been based on expressing the viral oncogenes from the karatin 14 promoter, thus ensuring expression of E6 and E7 in the basal keratinocytes. The first studies using an entire HPV16 genome demonstrated a potent propensity for the viral oncogenes to induce cervical malignancy in these transgenic mice, a process which was dramatically increased following the addition of exogenous oestrogen [74]. Further studies also began to dissect the role of E6 and E7 in the formation of these tumours, both at the cervix, and at other anatomical sites. Interestingly, E7 would appear to be the more potent of the two oncoproteins in the cervix and in head and neck tissues, although in both cases E6 appears to increase tumour size and frequency. In contrast, at cutaneous sites, E6 would appear to be the more potent of the two viral oncoproteins. [75],[76],[77],[78].

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Maintenance of the transformed phenotype requires continual expression of E6 and E7: targets for therapeutic intervention There is an absolute requirement for the stable and continued expression of HPV E6 and E7 oncogenes to drive the continuous growth of cell lines derived from cervical cancers. This is based on evidence obtained from various studies using different approaches to block the expression or functions of E6 and E7. These include using blocking peptides wherein it was shown that peptide aptamers against E6 resulted in the apoptotic elimination of only HPV-16 positive cells [79],[80]. A study that used antisense RNA against HPV E6/E7 in HPV positive cells showed that the RNA expressing cells have slower growth rates and reduced ability to form colonies in soft agar [81]. Similar results were also obtained using BPV-1 E2 to suppress the expression of HPV 18 E6 and E7 in HeLa cells, where suppression of E7 resulted in sensescence, whilst suppression of E6 resulted in both sensescence and apoptosis [82]. Recently siRNA approaches have been used to suppress E6 and E7 expression, again with similar effects on the continued proliferation of cervical tumour derived cell lines [15],[16],[83],[84],[85]. More recently the CRISPR/Cas 9 system which is a programmable RNAguided endonuclease system has emerged as a powerful genome editing tool

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in many organisms, including prokaryotes, C. elegans, and zebrafish [86], [87],[88]. A promising approach would be to deploy this technique to permanently inactivate the viral E6 and/or E7 gene by targeted mutagenesis, which is predicted to result in the death of HPV-transformed cells. A recent report utilized bacterial Cas9 RNA-guided endonuclease, together with single guide RNAs (sgRNAs) specific for E6 or E7. This resulted in the introduction of inactivating deletion and insertion mutations into the E6 or E7 gene [89]. The authors also show the induction of p53 or Rb respectively, leading to cell cycle arrest and eventual cell death. Both HPV16 and HPV18 transformed cells were found to be responsive to targeted HPV genomespecific DNA cleavage. Similarly another study has also shown the disruption of HPV16 E7 at specific sites through the single-guide RNA (sgRNA)guided CRISPR/Cas 9 system [90]. They report the induction of apoptosis and growth inhibition in HPV-positive SiHa and Caski cells, but not in HPV-negative C33A and HEK293 cells. This disruption of E7 DNA was followed by E7 protein downregulation, and subsequent upregulation of pRb [90]. Taken together these studies provide compelling evidence for the continued requirement for E6 and E7 in tumour maintenance and highlights their relevance as therapeutic targets in HPV-associated malignancies.

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Molecular targets of E6 HPV E6 proteins have a plethora of interacting partners, and this is reflected in the large number of different biological functions that have been assigned to the E6 oncoprotein. Some of these are shown in Figure 2. Obviously a major activity of the E6 oncoproteins derived from the mucosal high-risk types is their propensity to interact with the p53 tumour suppressor [91] and target it for proteasome mediated degradation. This activity of E6 requires the recruitment of the cellular ubiquitin ligase, E6-AP [32],[92],[30] and the subsequent tri-partite complex of E6/E6-AP/p53 [93] results in polyubiquitination of p53 and subsequent degradation. Clearly the ability of E6 to degrade p53 has major implications for overcoming p53 tumour suppressor pathways. Interestingly however, E6 seems to have acquired multiple ways to ablate p53 function. For example, whilst E6 proteins from the low risk viruses are unable to target p53 for degradation, they can nonetheless still act as potent inhibitors of p53 transcriptional activity, via the ability of E6 to interact with the coactivator p300 [94],[95],[96], [97],[98],[99]. In transgenic animals it is clear that simple abrogation of p53 function is not sufficient for all of E6’s transforming activity [100],[101] although the ability to associate with E6-AP does seem to play a pivotal role

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[77],[102]. This most likely in part reflects the important role E6-AP has in helping to maintain E6 steady state levels, where it is thought that the interaction with E6-AP does not simply help in the degradation of E6 substrates, but also helps stabilize E6 itself [103]. Several E6 target proteins such as paxillin, IRF3, E6BP1 and E6TP1 all share a common α-helical structural motif which has been demonstrated to mediate their binding to E6 and which was originally described for E6-AP [104],[105],[106],[107] . Not surprisingly, mutations in E6 that disrupt its ability to bind α-helical partners also causes defects in multiple biological properties [108]. However as with all multi-functional proteins, elucidating which particular cellular partner is relevant for which biological activity of E6 is extremely difficult. It is also interesting to note that the ability of E6 proteins derived from high risk virus types to interact with p53 and E6-AP is also shared with some of the E6 proteins from the low risk mucosal types [109],[110]. Thus whilst there are clear differences in how the different virus types modulate p53 function and interact with E6-AP, it is also clear that many aspects of this are also highly conserved, and are most likely more relevant for aspects of the virus life cycles rather than malignant progression.

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A class of cellular targets that are only bound by the high risk virus types are those proteins which possess PDZ (PSD/Dlg/ZO) domains [111],[112], [113]. The molecular basis for this is simple: the high risk HPV E6 proteins have a short stretch of amino acids at their extreme carboxy terminus which confers interaction with PDZ domain containing substrates. This PDZ binding motif (PBM) is absent from the majority of low risk virus types and can be considered as a marker of oncogenic potential.. Many of the cellular targets that are bound by E6 through this motif are important for the regulation of cell adhesion, cell contact and cell polarity. To date at least 12 different PDZ domain containing substrates of E6 have been described, and include the tumour suppressors Discs large, Scribble and MAGI-1[114]. These interactions appear highly complex and subject to multiple levels of regulation including post translational control of E6’s PBM, resulting in E6 substrateswitching to 14-3-3 proteins [115]. Most importantly, these interactions appear to play major roles in the ability of E6 to maintain the transformed phenotype. For example, restoration of MAGI-1expression in HPV transformed cells results in apoptosis [116]. In addition, HPV E6 also appears to usurp the normal activities of many of these proteins. In the case of Dlg, E6 appears to use the interaction to promote increased cell invasion [117], whilst

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in the case of hScrib, this appears to be required for maintaining high levels of E6 protein expression [118].

Molecular targets of E7 Like E6, the E7 oncoprotein is also highly multifunctional with a large number of cellular interacting partners and a correspondingly broad range of biological activities. These are summarized in Figure 3 but one of the most important targets of the high-risk HPV E7 proteins is the cellular tumor suppressor, pRB [119]. Furthermore, the association of high risk HPV E7 with pRB and the recruitment of the cullin 2 ubiquitin ligase promotes the proteasome mediated degradation of pRB [120],[121],[122],[123]. As a result, this disrupts the interaction of pRB with E2F transcription factors, thereby promoting the G1/S phase cell cycle transition [24],[25]. HPV E7 also interacts with the other pocket proteins, p107 and p130, via the same Leu-X-CysX-Glu (LXCXE) motif by which E7 binds pRB [20],[21],[23]. The aberrant pRB degradation by high-risk HPV E7 proteins and the resulting activation of E2F-mediated transcription represents an important strategy by which these viruses achieve and maintain S-phase competence in differentiated epithelial cells. HPV16 E7 can also directly bind to E2F1 and enhance E2F1-

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mediated transcription [124]. E2F1 is responsible for mediating the transcriptional control of the E2F6 gene, which is upregulated at the G1/S-phase transition to exert an opposing effect on the activities of E2F-responsive promoters, thereby directing appropriate cell cycle exit and differentiation [125]. However, HPV E7 also associates with E2F6 and abrogates its ability to function as a transcriptional repressor [126]. By this interaction HPV E7 is able to counterbalance the up-regulation of E2F6 that occurs as a consequence of the activation of E2F1 by E7, thus ensuring that the cells remain in an S-phase-competent state which is essential for the viral life cycle. It is interesting to note that E2F6 has a role in maintaining quiescence [127], and its deregulation by HPV E7 protein is an important strategy in allowing HPVs to bypass negative growth signals, thus allowing cells to exit from G0. Another important feature of high risk E7 protein is the potential to bind various chromatin modifiers such as histone deacetylases (HDACs) through sequences distinct from those with which they bind to RB and to target HDACs to repress transcription as well as facilitating their removal from other promoters to activate transcription [128],[129],[130], [131]. The E7RB-HDAC interaction is necessary for the episomal maintenance of the viral genome as well as for the productive viral replication in the supra basal layers [128],[131].

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High-risk HPV E7 proteins also contribute to cell cycle dysregulation by regulating cyclin dependent kinases (cdks). Cdks are the engines that drive the cell division cycle and are regulated by cyclin-dependent kinase inhibitors (CKIs). Expression of cyclins E and A , which are the regulatory subunits of cdk2 are under E2F control and they are both expressed at higher levels in E7 expressing cells [132]. The CKIs p21 and p27 regulate growth arrest during epithelial differentiation, and p21 is thought to act as a tumour suppressor in cervical carcinogenesis [133]. cdk2 is the major target of p21 and p27 in human keratinocytes [134]; high risk E7 proteins regulate cdk2 activity by binding to p21 and p27 via their carboxy termini thereby efficiently neutralizing the inhibitory effects on cyclin E- and cyclin Aassociated kinase activities [135],[136]. This leads to increased cdk2 activity in E7-expressing cells despite the high levels of p21 [135],[137]. Low-risk E7 proteins can also bind p21 but with a much reduced efficiency and a decreased ability to abrogate the inhibitory effects of p21. Both high-risk and low- risk HPV E7 can bind indirectly to cyclin E and cyclin A–cdk2 complexes through RB, p107 or p130, as well as directly to cdk2 and/or cyclin subunits thereby allowing sustained cdk2 activity [138],[139]. High-risk E7 has also been shown to increase the levels of the CDC25A phosphatase [140],[141] which can induce tyrosine dephosphorylation of cdk2 thereby

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promoting its activation. Interestingly both p21 and p27 have been implicated in TGF-β mediated growth inhibition [142],[143],[144], therefore HPV16 E7's ability to inactivate these CKIs may contribute to its ability to abrogate TGF-β mediated growth inhibition. Anoikis, is a form of programmed cell death that is triggered when cells attempt to enter S-phase despite a lack of matrix attachment [145]. The hallmark feature of cellular transformation is the resistance to anoikis and anchorage independent growth. Both high and low risk, as well as BPV1 E7 associate with p600 [146],[147]. p600 has been identified as a novel microtubule associated protein (MAP) which is developmentally regulated in neurons [148]. RNAi knockdown of p600 in mammalian cells have implicated p600 in anoikis [149],[147], suggesting that the interaction between E7 and p600 might deregulate anoikis and protect detached cells from apoptosis [149],[147]. Interestingly HPV 16E7 associates with p600 via the CR1 domain, which is essential for the transforming capacity of HPV 16 E7 [150],[151],[152], indicating that the interaction between E7 and p600 is a critical element for the life cycle many PVs, but which might also play an important role in the transforming activity of the cancer-causing types.

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Genomic instability, DNA damage response and epigenetic control by E6 and E7 HPV E6 and E7 are essential for maintenance of the transformed phenotype, however they are not sufficient to directly transform cells. Subsidiary events such as genomic instability are necessary for malignant progression of the cells. A distinctive feature of cells that express E6 and E7 is the presence of genomic instability, which has been observed in multiple epithelial cell types [34],[35],[36],[37],[38],[39],[40]. Many abnormalities such as monosomies, trisomies, chromatid gaps and breaks, double minutes and aberrant chromosomes have been associated with cells expressing E6 and E7. This induction of genetic instability is proposed to be an early event in HPV-induced cancers, occurring before integration of the virus into host chromosomes [43],[44]. Interestingly aneuploidy can be detected in pre -malignant HPVassociated cervical lesions which is in accordance with this concept [153],[154]. It is important to note that such activities are restricted to highrisk E6 and E7 proteins as no such activities are seen in cells expressing their low-risk counterparts [155]. Expression of HPV E6 and E7 leads to induction of numerous mitotic defects including multipolar mitoses, anaphase bridges and aneuploidy [156]. Abnormal multipolar mitoses are a characteristic feature of most high-risk HPV lesions accompanied by abnormal cen20

trosome numbers [42],[157]. Development of aneuploidy is the result of the cooperative actions of E6 and E7, which induces centrosome abnormalities leading to chromosomal missegregation. Aberrant centrosome numbers are accompanied by nuclear atypia in the cells expressing high levels of E6, which become evident only after prolonged passaging. However in the cells that express high levels of E7 show rapid centrosome amplification, which correlates with cell division errors and occurs before the detection of genomic instability [43]. These studies indicate that abnormal centrosome duplication is an early event that might drive chromosomal instability. Multiple rounds of centrosome synthesis are induced by HPV E7 in a single S phase via the formation of multiple immature centrioles from a single maternal centriole [158]. The E7 mediated centrosome duplication is dependent on the high levels of CDK2 activity [159] indicating the importance of pRB degradation by HPV E7. It is important to note that the E7 LXCXE pRb-binding motif is essential for aberrant centrosome synthesis, but HPV E7 can also induce centrosome abnormalities in mouse embryonic fibroblasts deficient in Rb family members, albeit at a much reduced level [160]. This activity could be the result of binding of E7 to γ-tubulin, a centrosome regulator, via sequences that also overlap with the pRb-binding domain [161].

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Under normal situations cells with aberrant mitoses are targeted for cell death, however the synergistic actions of E6 and E7 allow cells with abnormal centrosomes to accumulate, possibly by relaxing the G2–M checkpoint response that is normally regulated by p53 [162] as well as by inhibition of apoptotis [163]. E6 and E7 have also been shown to independently bypass mitotic checkpoints in addition to inducing centrosome abnormalities [164], [165]. This leads to the accumulation of polyploid cells resulting in aneuploidy. Interference with these check points are of critical importance for viral replication but can also lead to genomic instability in HPV-immortalized cells. Since the malignant progression takes many years, it is likely that these mitotic defects occur infrequently and do not often lead to viable progeny. However this accumulation of chromosomal alterations might ultimately confer a growth advantage to a sub clonal population of HPV positive cells, leading to the outgrowth of a cellular population that contributes to viral persistence and, ultimately, malignant progression. HPV E6 and E7 have been shown to induce abrogation of the normal DNA damage response [166],[167],[168],which is impeded by the activity of E6 and E7 via inhibition of p53 mediated cell cycle arrest. This is carried out in part by the ability of E6 to bind and inactivate p53 [169] and E7's functional disruption of the cell-cycle regulator pRb and inactivation of p21 [137], the 22

cyclin-dependent kinase inhibitor that is induced when p53 is activated in response to DNA damage [170]. Both E6 and E7 have been shown to independently induce DNA damage [171] thereby increasing the frequency of foreign DNA integration into the host genome [172]. E6 and E7 can lead to activation of the ATM–ATR pathway (ataxia telangiectasia mutated–ATM and RAD3-related DNA damage repair pathway). The ATM pathway has been shown to be activated in differentiated keratinocytes by high risk E7 [173], which appears to be important for differentiation-dependent viral genome amplification but not for the stable maintenance of episomes in undifferentiated cells [173]. The integrated copies of HPV genomes after replication can also activate ATM and ATR which can lead amplification of integrated HPV sequences and the flanking cellular sequences [55]. The activation of DNA damage response in cells containing episomal as well as integrated copies of the viral genome could result in chromosomal alterations and induction of genomic instability, which is an important event in the progression towards malignancy. The ATM–ATR DNA damage response leads to the induction of cell cycle checkpoints at S or G2–M, but importantly E7 can abrogate these checkpoints thereby promoting mitotic activity. E7 can also lead to the degradation of claspin106 which is a key regulator of the ATR–CHK1 DNA damage-signalling pathway, that is activated in response 23

to replication stress. Proteolysis of claspin is an essential event for the DNA damage checkpoint recovery [174]. This accelerated degradation of claspin in G2-M is an important strategy used by E7 to deceive cells into initiating checkpoint recovery, allowing aberrant mitotic entry in the presence of DNA damage. This act leads to genomic instability through defective DNA repair. Thus the abrogation of the DNA-damage responses by E6 and E7 is expected to contribute to the accumulation of genetic alterations in HPVpositive cells, which in turn leads to the malignant nature of these cells. Both E6 and E7 can influence cellular and viral gene expression by modulating the methylation machinery. HPV-16 E6 can induce upregulation of the DNA methyltransferase DNMT1 via suppression of p53 [175]. It has also been shown that HPV-16 E7 can directly bind to and activate DNMT1 [176]. Interestingly both DNMT1 and DNMT3B were shown to be upregulated in CIN3 lesions and cervical carcinomas [177],[178],[179], supporting the concept of interaction of E6 and E7 with the methylation machinery of the cells. It has also been suggested that viral DNA methylation represents a generic phenomenon of de novo methylation of foreign DNA, serving as a host defense mechanism to silence viral replication and transcription [180],[181].

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Several reports have documented the association of HPV E6 and E7 with histone modifying enzymes as well as their transcriptional cofactors [94],[95],[129],[182]. HPV E7 collaborates with class I histone deacetylases (HDACs) [129],[183], which in turn is connected with increased levels of E2F2-mediated transcription in differentiating cells [183]. In addition, E7 can also associate with histone acetyl transferases (HATs), including p300, pCAF, and SRC1 [182],[184],[185],[186]. Although the biological relevance of some of these interactions require further clarification, it has been shown that HPV 16 E7 interaction is responsible for cytoplasmic relocalization and functional inactivation of steroid coactivator-1 (SRC1) [185]. The histone methyl transferase EZH2 has been recognized as a transcriptional target of HPV E7 proteins [187]. Taken together, these studies indicate that the HPV oncoproteins have the potential to alter the epigenetic program of the infected host cell.

HPV carcinogenesis: transformation zone, squamo-columanar junction and cervical cancer stem cells It is clear from the above discussion that the high-risk HPV E6 and E7 oncoproteins can impact upon many cell regulatory pathways, and as a result

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have the capacity to perturb cellular homeostasis in multiple ways. However an additional element which requires consideration, is the time frame over which these activities of E6 and E7 are exerted, and also the precise nature of the target cell in which this is believed to occur. For example, whilst the complete viral life-cycle can be completed in a relatively short period of 2-3 weeks, it is clear that infections can persist for long periods of time. Indeed, the acquisition of a malignant phenotype is always preceded by longstanding HPV infections [2],[188], thereby providing ample time for acquisition of additional genetic alterations in the target cell which are believed to initiate malignant progression. A key question is the identity of the target cell for HPV infection and transformation and whether these cells have any stem cell characteristics. Although several studies have proposed multiple target cells for HPV infection, there is an increasing acceptance for the hypothesis that stem cells or “reserve cells” of the transformation zone are the primary sites of persistent HPV infection [189]. This is based on the anatomical observation that many cervical cancers arise in the TZ. The long latency periods between infection with HPV and development of cervical dysplasia can be explained only if such reserve cells are infected as they can serve as vehicles for long-term viral latency in the cervix. However, a recent report has shown a discrete population of cells with a unique gene expression sig-

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nature, at the squamo-columnar junction (SCJ) of the cervix, that could be responsible for the majority of HPV-associated cervical carcinomas [190]. These junctional, single layered cuboidal epithelial cells of embryonic origin, has a gene expression profile which is different from that of the adjacent endocervical and ectocervical/TZ epithelium (see Figure 4). These SCJ specific genes include keratin 7 (KRT7), anterior gradient 2(AGR2), matrix metalloproteinase 7 (MMP7) and guanine deaminase (GDA) [190], all of which are found to be expressed in CIN lesions and cervical cancers. Most importantly, HPV E6 and E7 do not induce these proteins in vitro in squamous epithelial cells, and their expression is lost if the SCJ is removed by cone biopsy or loop electrical excision. This indicates that the SCJ-specific expression profile in CIN lesions and cervical cancers is not acquired during the process of transformation, and instead represents the embryonal origin of the cells. Interestingly, of all the SCC and adenocarcinoma analysed, most CIN2/CIN3 lesions and one-third of CIN1 lesions were positive for an SCJ expression profile [190],[191]. The proximity of these cells to the SC junction reinforces the historically supported assumption that cervical cancer and its precursors originate in this site [192],[193]. These results suggest that this distinctive population of cells at the SC junction forms the core group that can give rise to multiple tumor phenotypes. Interestingly, HPV infection of

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the squamous epithelium at sites that do not harbour these SC junction cells, including the ectocervix, vagina and vulva, seems to be less efficient in generating malignancy [194]. These studies also raise the possibility that the productive viral infections might arise exclusively from infection of the basal cells of squamous epithelium lining the ectocervix or adjacent transformation zone [195]. Indeed a recent report suggests that squamocolumnar junction (SCJ) excision appears to reduce the risk of new CIN 2/3 lesions, suggesting that SCJ cryoablation could be effective in preventing cervical cancer. [196]. Several studies have established which phenotypic and functional markers can be used to facilitate the identification and characterization of the cervical cancer stem cells derived from the primary tumors [197],[198], as well as from cervical cancer cell lines [189], with CD44 being particularly useful [198]. Notably the role of Notch in the progression of cervical squamous cell carcinomas has been shown by examining its expression and function in the context of CD66+cells, a known mediator of metastasis [199]. The authors studied the CD66 and Notch in clinical specimens and explants of human cervical cancer, including specimens grown in a pathophysiologically relevant murine model. Their results of gene expression profiling CD66+ cells from primary tumors showed enhanced features of Notch signaling, metasta28

sis and stemness. Overall their findings suggest that CD66+ cells have a tumor-propagating subset in cervical cancers [199]. Another paper from the same group investigated the existence and differentiation state, as well as the neoplastic potential of CD66+ cells in a pre-cancer cell line harboring HPV31b episomes [200]. By using this precancerous cell line, they demonstrate that CD66High cells have an intermediate differentiation state with a cellular milieu connected with both viral replication and neoplastic potential. HPV16 has been shown to have a statistical positive correlation with stem cell-associated genes PSCA, PIWIL1, and TBX2 in cervical squamous cell carcinoma, which indicate that HPV16 viral oncoproteins might be involved in up-regulating the expression of these genes thereby suggesting a complex molecular mechanism for the role of HPV16 infection during cervical carcinogenesis [201]. A recent report has shown the isolation and enrichment of cervical cancer stem-like cells (CaCxSLCs) from cervical primary tumors and cancer cell lines by novel sequential gating using a set of functional and phenotypic markers (ABCG2, CD49f, CD71, CD133). The authors used defined conditioned media for assessing sphere formation and expression of self renewal and stemness markers by FACS, confocal microscopy and qRTPCR [202]. Importantly they show that these stem like cells have spheroid forming ability and also express self-renewal/stemness markers such as

29

Oct4, Sox2, Nanog, Lrig1 and CD133. They also demonstrate overexpression of E6 and HES1 transcripts in both cervical primary tumors and cancer cell lines. The enrichment of CaCxSLCs display high tumorigenic capacity and lead to recapitulated primary tumor histology in nude mice. The siRNA silencing of HPVE6 or Hes1 abolished sphere formation, and downregulated AP-1-STAT3 signaling with the induction of re-differentiation [202]. Similarly in the case of head and neck squamous cell carcinoma (HNSCC), the strongest association with HPV has been found in oropharyngeal sites, and there are early investigations to suggest that cancer stem cells (CSC) may play a significant role in carcinogenesis. In a small study of six oropharyngeal HNSCC tumor specimens, HPV positive tumors had a higher proportion of CSCs compared to HPV negative tumors [203]. Another study of four HPV negative oral and oropharyngeal HNSCC cell lines infected with the HPV genome resulted in tumors with increased growth and self-renewal capacity [204]. Although these findings are on small sample sizes and are not definitive, they may suggest a relationship between HPV and CSC formation which needs further exploration. Although restricted, the evidence that supports the existence of “cancer stem cell” heterogeneity in cervical tumors is relatively convincing. These find-

30

ings serve as a foundation to further examine the functional role of “cancer stem cell " / “reserve cell” / “squamocolumnar junction cells” and their contribution to the development and progression of cervical cancer. Cellular heterogeneity represents one of the greatest challenges in cancer therapeutics, therefore there remains a need to identify hallmark characteristics of these cells with the therapeutic potential to target them for the better treatment options in the future. Also these naive cells represent the cells capable of tumor recurrence and metastasis, thus they are potentially the “more appropriate” target cells for therapeutic advancements in the future. Concluding perspectives It has become clear over the last decade that cancer is a complex group of diseases resulting from multiple genetic alterations [205]. Emerging “omics” technology and personalized medicine has provided new insights into the design of novel therapeutic strategies based on genome, epigenome and transcriptome levels. However the complexity and large number of mutations in human solid tumors undermines the promise of “personalized medicine” due to the difficulties in pin pointing the “driver” mutations as opposed to the “passenger” mutations that do not directly contribute to the cancer process.

31

HPV-associated cancers are initiated by uniform oncogenic hits and thus represent distinctive experimental systems to tackle some of these issues. In HPV-associated cervical cancers E6 and E7 oncogene expression drives cancer initiation and progression. The high risk HPV E6 and E7 oncoproteins target two hallmark tumor suppressor pathways p53 and pRB. These pathways are deregulated at some level in the majority of human solid tumors and thus represent ubiquitous “driver mutations”. Another attribute of the high-risk HPVs is that they can frequently exist in an infected host cell at a low copy number for decades, often without causing clinically overt lesions. This formulates the foundation for the hypothesis that high risk HPVs might have evolved with the propensity to infect “stem like” cells in order to establish a persistent infection. In addition, the ability of stem cells to differentiate is also important for the production of viral particles [206] [207]. Interestingly the virus can program the infected stem cells to undergo symmetric division instead of asymmetric division [208], thereby aiding to maximize the proliferation and production of viral particles. Taken together, HPVs represent a wonderful model system for identifying the key cellular players in the development of cancer, and would appear to be also valuable in shedding light on the role of stem cells in this process.

32

Furthermore, understanding the molecular basis behind the mechanism of action of E6 and E7 wil be important in designing novel therapeutic strategies for targeting not just HPV –associated cancer, but cancers in general.

Acknowledgements. We are most grateful to Miranda Thomas for valuable comments on the manuscript. The laboratory is supported by a research grant from the Associazione Italiana per la Ricerca sul Cancro. SM is the recipient of an Arturo Falaschi ICGEB Fellowship.

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