Cervical cancer is the second largest cause of cancer-related death in women worldwide, and it is caused by persistent infection with a subset of high-risk human papillomaviruses (HPVs) Citation[1,2]. Globally, infection with types HPV16 and 18 accounts for more than 70% of invasive cervical carcinomas Citation[3], with the remaining 30% being caused by HPV31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 Citation[4]. High-risk HPV infections can typically last up to 12 months, but are mostly cleared by the immune system Citation[5]. However, approximately 10% of women fail to clear HPV infections, resulting in long-term persistent infection. In the absence of treatment, individuals can progress or regress from states up to high-grade squamous intraepithelial lesions, and thereafter may progress to cervical cancer. The HPV E6 and E7 oncoproteins play key roles in the development and maintenance of the malignant state. Indeed, removal of either E6 or E7 from cells derived from cervical tumors results in growth arrest and apoptosis, highlighting their relevance as targets for therapeutic intervention.
The mechanisms by which E6 and E7 drive the development of malignancies are varied and complex Citation[6,7]. However, key features are the ability of E7 to override cell-cycle regulation by interactions with the pRb family of tumor suppressors, and the ability of E6 to overcome apoptotic pathways by association with the p53 tumor suppressor, which it targets for proteasome-mediated degradation Citation[8]. However, it is clear that other activities of E6 play critical roles in the development of malignancy, since E6 mutants deficient for degradation of p53 can still immortalize cells, suggesting that interactions with other cellular factors are necessary for cancer development. One class of cellular factors that appear to fit this role are the PSD-95/Dlg/ZO-1 (PDZ) domain-containing substrates of E6. Importantly, transgenic mice encoding E6 proteins defective for binding to PDZ domains do not develop hyperplasia or tumors Citation[9], and mutation of the PDZ binding domain of E6 in the context of complete viral genomes leads to reduced growth rates, loss of viral episomes and frequent integration of viral genomes into host chromosomes, indicating the importance of these interactions for viral pathogenesis Citation[10].
Interestingly, only the so-called high-risk HPV E6 proteins have a C-terminal PDZ binding motif (PBM) designated by the sequence x-S/T-x-V/L, and this is absent in those HPV E6 proteins that are only associated with benign disease. Through this PBM, E6 can interact with a variety of cellular proteins that possess PDZ domains Citation[11]. A number of these E6 targets are found at regions of cell–cell contact and have been implicated in the regulation of cell polarity, including hDlg1 and hDlg4, the human homologs of Dlg Citation[12–14]; hScrib, a homolog of the Drosophila scribble protein Citation[15], MAGI-1, -2 and -3 Citation[16–18]; MUPP1, a multi-PDZ protein Citation[19]; PTPN 3, a membrane-associated tyrosine phosphatase Citation[20,21]; and PATJ Citation[22]. Interestingly, not all of the high-risk HPV E6 proteins recognize their different PDZ domain-containing substrates with equal affinity, and minor alterations in the PBM of E6 greatly alter substrate selection. For example, hScrib is preferentially targeted by HPV16 E6, while hDlg is preferentially recognized by HPV18 E6 Citation[23]. Whether these differences will ultimately be reflected in different disease pathologies associated with the different high-risk virus types remains to be determined; however, this does remain an intriguing possibility and is worthy of further consideration.
Furthermore, it is also worth noting that the interactions between E6 and its PDZ domain-containing substrates are not constitutive. Embedded within the HPV E6 PBM, there is also a consensus recognition site for PKA phosphorylation on either the Thr or the Ser residue at the -3 position. In the case of many ligand–PDZ interactions, such phosphorylation events inhibit the interaction Citation[24,25]. This would also seem to be true for HPV18 E6, where phosphorylation by PKA greatly inhibits E6–PDZ-domain recognition Citation[26]. In addition, post-translational modification of either hDlg or hScrib can potentially affect their respective susceptibilities to E6 targeting Citation[27,28]. Thus, changes in cellular signaling pathways can be expected to profoundly influence the efficiency with which E6–PDZ interactions take place. So what might be the relevance of this with respect to a possible role in cancer development? As noted earlier, loss of E6–PDZ binding capacity in the context of the HPV genome results in loss of viral replicative capacity and frequent integration of the viral DNA into the host genome Citation[10]. One of the hallmarks of cervical cancer progression is the frequent integration of the viral DNA into the host genome, and although there is considerable debate as to the role of this in disease development, the presence of integrated DNA sequences is likely to be of concern due to the concomitant high levels of E6 and E7 gene expression. Therefore, one possibility is that loss of PDZ binding capacity through PKA phosphorylation of E6 might create an environment that is conducive to the loss of viral episomes and to consequent viral integration into the host genome. Obviously, while this is somewhat speculative, it does nonetheless suggest that the levels of PKA activity within the infected cervical epithelium might be one marker that could be assessed as a means of determining whether viral DNA integration was more or less likely, and studies are currently in progress to investigate these aspects further.
Remaining questions relate to how E6–PDZ interactions might contribute to the viral life cycle and disease progression. Certainly, E6 overriding the regulation of cell proliferation by hScrib provides one possible explanation Citation[28]. It is also tempting to speculate that alterations to cell polarity by E6 in HPV-infected basal keratinocytes may perturb segregation of cell fate determinants during basal cell division, and thereby expand the population of replication-competent cells Citation[29]. This would be favorable for viral replication and might also contribute to the early stages of disease development. Finally, there are some intriguing recent studies that suggest PDZ targeting might be related to viral avoidance of the innate immune response. For example, the virulence of influenza A virus has been linked to its capacity to target one or more PDZ domain-containing proteins Citation[30], which in turn has been associated with downregulation of the JAK/STAT signaling pathway Citation[31,32]. Intriguingly, hScrib has been implicated in the regulation of this pathway Citation[33]. Thus, it is possible that E6, through targeting certain PDZ substrates, can impair the innate immune response signaling pathways. Indeed, previous studies have shown that E6 can impair JAK/STAT signaling Citation[34], though whether E6–PDZ domain-containing substrates play a role in this activity remains to be determined.
Thus, while much is known about the requirements for E6 and E7 in the maintenance of tumor growth, a lot remains to be done to understand precisely how these two viral proteins continue to drive the development of malignancy. In addition, there remains a pressing need to be able to differentiate between infected individuals who can be considered to be at ‘high risk‘ of disease progression and those that should hopefully proceed to resolve their infection without the need for surgical intervention. In this article, we have attempted to highlight one particular aspect of E6 function that may help to provide clues to some of the molecular mechanisms that underlie HPV-induced development of cancer, and which in turn might provide us with the identity of markers that can be used to determine the risks of tumor progression.
Financial & competing interests disclosure
Work in L Banks‘ laboratory is supported by research grants from the Associazione Italiana per la Ricerca sul Cancro, the Association for International Cancer Research, Telethon, the Wellcome Trust and the Yoshida (YKK) Scholarships Foundation (to K Nagasaka). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
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