E3 ubiquitin ligases and human papillomavirus ...

Review Journal of International Medical Research 2014, Vol. 42(2) 247–260 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0300060513506655 imr.sagepub.com

E3 ubiquitin ligases and human papillomavirusinduced carcinogenesis Zhifeng Lou1,* and Sheng Wang2,*

Abstract Human papillomavirus (HPV) infection is clinically very common. It is usually a major risk factor in the development of cutaneous benign lesions, cervical cancer and a variety of other malignancies. The biological function of ubiquitination as an intracellular proteasomal-mediated form of protein degradation and an important modulator in the regulation of many fundamental cellular processes has been increasingly recognized over the last decade. HPV proteins have been demonstrated to evolve different strategies to utilize the ubiquitin system for their own purposes. The putative roles of E3 ubiquitin ligases in HPV-induced carcinogenesis have become increasingly apparent, although the mechanisms remain unclear. In this review we provide an update on the mechanisms of the involvement of E3 ubiquitin ligases in HPV-induced carcinogenesis, focusing on their interaction with HPV proteins and their roles in several signalling pathways. Targeting the E3 ubiquitin ligases might offer potential therapeutic strategies for HPV-related diseases in future.

Keywords Human papillomavirus, ubiquitin, E3 ubiquitin ligase, carcinogenesis Date received: 27 June 2013; accepted: 15 July 2013

Introduction Human papillomaviruses (HPV) are small double-stranded DNA viruses. More than 130 HPV genotypes have been cloned from a wide variety of clinical lesions.1 HPV have a predilection for either cutaneous or mucosal epithelial surfaces and can be divided into low- and high-risk HPV. Low-risk HPV predominantly generate benign squamous 1

Department of Dentistry, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, People’s Republic of China 2 Department of Stomatology, 118th Hospital of PLA, Wenzhou, People’s Republic of China

epithelial lesions, commonly known as warts, whereas the high-risk types are associated with malignant diseases such as cervical cancer, skin tumours in patients with epidermodysplasia verruciformis, anal and rectal cancers, and head and neck squamous cell carcinoma (HNSCC), including oral *Both authors contributed equally to this work. Corresponding author: Sheng Wang, Department of Stomatology, 118th Hospital of PLA, 15 Jiafusi Road, 325000 Wenzhou, People’s Republic of China. Email: [email protected]; [email protected]

Creative Commons CC-BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 3.0 License (http://www.creativecommons.org/licenses/by-nc/3.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed on the SAGE and Open Access page Downloaded from imr.sagepub.com by guest on Januaryas 9, specified 2016 (http://www.uk.sagepub.com/aboutus/openaccess.htm).

248

Journal of International Medical Research 42(2)

cavity cancer.2–4 The two most common low-risk viruses causing warts on the anogenital epithelium are HPV6 and HPV11; the two major high-risk or oncogenic viruses are HPV16 and HPV18. Although the role of HPV in HNSCC is highly controversial, recent data from meta-analyses and case– control studies indicate that HPV may be an independent risk factor for the development of oropharyngeal and oral carcinoma.5–7 The HPV genome can be divided into three regions: an early region encoding six nonstructural proteins (E1, E2, E4, E5, E6 and E7); a late region encoding two structural proteins (L1 and L2); a noncoding long control region. The E1, E2, E4 and E5 proteins are required for viral DNA replication, the E6 and E7 oncoproteins cooperate to transform and immortalize infected cells, and the L1 and L2 proteins are needed for the production of viral particles.8 Because they infect the basal layer of the squamous epithelium and have no viraemic phase, HPV are isolated from circulating immune cells. The limited innate immune response, the low levels of viral gene expression in the basal epithelium and the lack of cytopathic effects generally lead to a delayed adaptive immune response to initial HPV infection, thus favouring the establishment of persistent viral infection.3 Because of the rather limited coding capacity of viral genomes, many aspects of the life cycle of HPV rely on specific interactions between viral proteins and intracellular regulatory proteins, so as to redirect cellular signalling to the benefit of the virus. Modification of cellular proteins by the reversible covalent attachment of ubiquitin, much like phosphorylation, is involved in the control of most fundamental cellular processes. As one of the most pivotal regulatory mechanisms in biology, the ubiquitin system is extensively used to promote HPV replication, persistent viral infection and HPV-induced carcinogenesis. Good examples are the interactions of HPV E6 and E7

oncoproteins with tumour suppressors p53 and pRb, respectively, leading to their degradation and inactivation, and contributing to persistent viral infection, cellular transformation, immortalization and carcinogenesis.9 In this review we will provide an update on the contributions of E3 ubiquitin ligases to HPV-induced carcinogenesis and the associated mechanisms, focusing on their interaction with HPV proteins and their roles in several signalling pathways.

The ubiquitin system As a highly conserved 76-amino-acid polypeptide, ubiquitin serves as a signal for the target protein to be recognized and degraded in the 26 S proteasome. The ubiquitin–proteasome pathway plays a critical role in various cellular functions including antigen processing, cell-cycle regulation, apoptosis, signal transduction and transcriptional regulation.10 Ubiquitin is covalently attached to one or more lysine residues of cellular proteins via an enzymatic cascade involving three classes of enzyme, termed E1 (ubiquitin-activating enzymes), E2 (ubiquitin-conjugating enzymes) and E3 (ubiquitin–protein ligases). The process of ubiquitination and the ubiquitin–proteasome pathway have been discussed in detail.10,11 E3 ubiquitin ligases can be classified into two groups according to their substrates. One group is characterized by homology to the E6-associated protein carboxyl terminus (HECT) domain.12 The archetype is the E6-associated protein (E6-AP) recruited by HPV E6 protein.13 The second group, the so-called really interesting new gene (RING) finger E3 ubiquitin ligases, can be categorized further into unimolecular and multimolecular E3s. Examples of unimolecular RING E3s are murine double minute 2 (Mdm2)14 and Casitas B-lineage lymphoma (Cbl).15 Prototypes of the multimolecular RING E3s are the Skp1/Cullin1/F-box (SCF) protein family.16

Downloaded from imr.sagepub.com by guest on January 9, 2016

Lou and Wang

249

It is now clear that polyubiquitin chains linked through lysine at position 48 of ubiquitin (Lys48) target substrate proteins for proteasome-mediated degradation, whereas polyubiquitin chains of alternative linkages (such as Lys63) carry out signalling functions independently of proteolysis.17 Mono-ubiquitination of some proteins (such as histones, calmodulin and actin) serves as a signal for endocytosis and histone-mediated transcriptional regulation without targeting for degradation.18 The removal of ubiquitin residues from substrates is catalysed by deubiquitinating enzymes. The deubiquitinating enzymes are of great importance in numerous critical cellular functions such as cell growth, gene silencing, oncogenesis, endocytosis and DNA replication.19

HPV oncoproteins that assemble E3 ubiquitin ligases The E6-associated protein is a member of the HECT-domain-containing E3 ubiquitin ligase family, with a HECT domain that is involved in the ubiquitination of bound substrates and divergent N-termini that mediate substrate specificity.13,20 Isolated and identified in 1993, E6-AP is believed to form a complex with both E6 and the substrate protein, causing ubiquitination and subsequent proteasome-mediated degradation of the substrate protein.21 E6AP-mediated proteolysis of the substrate proteins plays an important role in many cellular processes associated with HPVinduced carcinogenesis. Substrates of the E6/E6-AP complex, extensively reviewed elsewhere, include p53,13 Bak (Bcl-2 antagonist killer),22 c-myc (cellular myelocytomatosis oncogene),23 E6TP1 (E6 targeting protein 1),24 MCM7 (minichromosome maintenance protein 7),25 MGMT (O6methylguanine DNA methyl transferase),26 NFX1 (nuclear transcription factor, X-box binding 1),27 hDlg (human homologue of

the Drosophila discs large tumour suppressor protein),28 hScrib (human homologue of the Drosophila Scribble tumour suppressor protein)29 and PTPN3 (protein tyrosine phosphatase, nonreceptor type 3)30 (Table 1). E6 was reported to direct the degradation of hDlg in an E6-AP-null in vitro system, providing evidence for the possible existence of a second E6-associated ubiquitin ligase.31 Annexin A1 and human alteration/ deficiency in activation protein 3 (hADA3) have been shown to be novel substrates for E6-AP-mediated degradation (Table 1). Annexin A1 is thought to be a novel substrate for E6-AP-mediated ubiquitination and subsequent degradation,32 indicating that E6-AP plays a role in controlling the diverse functions of annexin A1, such as inhibition of cell proliferation, regulation of cell differentiation, apoptosis and processes involved in carcinogenesis. These results may explain research showing that, in comparison with normal cervical epithelium, the expression of annexin A1 is significantly reduced during the progression of cervical neoplasia.33 hADA3 appears to be required for p53-mediated transcriptional activation of various target promoters and p53mediated G1 cell-cycle arrest.34 A study reported significantly increased hADA3 expression, decreased cell proliferation and increased apoptotic rate in HPV-16-positive SiHa cells treated with either E6 siRNA or E6-AP siRNA.35 These data suggest that HPV16 E6 might initiate cellular transformation as a consequence of hADA3 inhibition, induced by E6-AP-dependent ubiquitination and proteasomal degradation. The ability of high-risk HPV to target cellular proteins for proteasome-mediated degradation is not restricted to E6. It has been reported that HPV E7 proteins bind pRb through the conserved LXCXE motif, resulting in disruption of the pRb/E2F repressor complex and uncontrolled G1


250


Table 1. Examples of interactions between human papilloma virus (HPV) proteins and E3 ubiquitin ligases. HPV protein

E3 ubiquitin ligase

Speculated or confirmed effects

References

E6

E6-AP

Degradation of p53, Bak and c-myc Degradation of E6TP1 Degradation of MCM7 and MGMT Degradation of NFX1 Degradation of hDlg, hScrib and PTPN3 Degradation of annexin A1 Degradation of hADA3 Degradation of TIP-2/GIPC Ubiquitination of E6 Ubiquitination of RIP1 Regulation of E6/E6-AP complex Degradation of pRb Ubiquitination and degradation of E7 Ubiquitination and degradation of E7 Ubiquitination of RIP1 Degradation of IGFBP3 Inhibition of effect of c-Cbl on EGFR Inhibition of APC Increase in E2 transcriptional activity Ubiquitination and degradation of E2

13, 22, 23 24 25, 26 27 28–30 32 35 65 55 60 88 36–42 50 51 60 73 46 48 80 56

E7

E5 E2

Unknown cIAP, TRAF EDD Cullin-containing E3 SCF SOCS1-containing E3 cIAP, TRAF Unknown c-Cbl APC Mdm2 SCF

See text for details. APC, anaphase-promoting complex; Bak, Bcl-2 antagonist killer; c-Cbl, cellular Casitas B-lineage lymphoma; cIAP, cellular inhibitor of apoptosis: c-myc, cellular myelocytomatosis oncogene; E6-AP, E6-associated protein; E6TP1, E6 targeting protein 1; EGFR, epidermal growth factor receptor; hADA3, human alteration/deficiency in activation protein 3; hDlg, human homologue of Drosophila discs large tumour suppressor protein; hScrib, human homologue of the Drosophila Scribble tumour suppressor protein; IGFBP3, insulin-like growth factor binding protein 3; MCM7, minichromosome maintenance protein 7: Mdm2 murine double minute 2; MGMT, O6-methylguanine DNA methyl transferase; NFX1, nuclear transcription factor, X-box binding 1; PTPN3, protein tyrosine phosphatase, nonreceptor tyrosinase 3; RIP1, receptor-interacting protein 1; SCF, Skp1/Cullin1/F-box; SOCS1, suppressor of cytokine signalling 1; TIP-2/GIPC, Tax interacting protein, clone 2/GAIP interacting protein, C terminus; TRAF, tumour necrosis factor receptor-associated factor; EDD, E3 identified by differential display.

exit and S-phase entry.36,37 High-risk HPV E7 proteins destabilize pRb via a ubiquitin/ proteasome-dependent mechanism.38,39 40 Huh et al. proposed that the HPV16 E7associated cullin 2/ubiquitin ligase complex contributes to aberrant degradation of pRb in HPV16 E7-expressing cells (Table 1). Low-risk HPV E7 proteins bind pRb with lower affinity than the high-risk HPV E7 proteins.37 Recent reports show that expression of the low-risk HPV6 E7 protein causes destabilization of the pRb family member p130 by ubiquitination of phospho-p130

through the complex.41,42

cullin

1/ubiquitin

ligase

HPV proteins that interfere with E3 ubiquitin ligases The third oncoprotein in HPV, HPV E5, has been shown to induce transformation and anchorage-independent growth,43 to co-operate with E7 to stimulate proliferation of primary cells in vivo44 and to enhance immortalization by E6/E7.45 HPV E5 delays degradation of activated


Lou and Wang

251

epidermal growth factor receptor (EGFR) and upregulates EGFR-mediated signal transduction. This may be a consequence of interaction of E5 with the EGFR, which disrupts binding of EGFR to cellular Cbl (cCbl) (Table 1),46 an E3 ubiquitin ligase that ubiquitinates phosphorylated tyrosine kinase receptors.15 Expression of the HPV E2 protein leads to G2/M arrest.47 Retardation of the G2/M transition is believed to be the result of interference with the activity of anaphase-promoting complex (APC), a large multiprotein E3 ubiquitin ligase that is crucial for progression into and out of M-phase. Furthermore, high-risk, but not low-risk, HPV E2 proteins induce genomic instability as a consequence of perturbation of APC-dependent proteolysis48 (Table 1).

HPV proteins degraded by E3 ubiquitin ligases Human papillomavirus E7 is a short-lived protein ubiquitinated on the N-terminal residue and degraded by the ubiquitin–proteasome pathway.49 Two independent pathways for E7 ubiquitination have been clarified (Table 1): one involves the SCF ubiquitin ligase complex;50 the second involves the interferon-g-inducible suppressor of cytokine signalling-1 (SOCS1), a cytokine signalling suppressor functioning as an antioncogene against various haematopoietic oncogenic proteins. SOCS1 induces ubiquitination and degradation of E7 in a SOCS-box-dependent manner, causing a concomitant increase in pRb levels.51 These two E7 degradation pathways seem to be complementary, as the first appears to be predominant in the nucleus50 whereas the second appears to be predominant in the cytoplasm.51 Interestingly, the stability of E7 is also regulated by its interaction with the deubiquitinating enzyme ubiquitin-specific peptidase 11 (USP11). E7 binds to USP11, which reduces the ubiquitination and

therefore enhances the stability of E7.52 Thus, the stability of E7 depends on the net result of two opposing activities controlling the rate of ubiquitination. In addition to E7, HPV E2 and E6 have both been reported to be ubiquitinated and degraded by the proteasome.53–55 In the case of E2, the aminoterminal transactivation domain is required,53 whereas with E6 its degradation is independent of E6/E6-AP binding capability.55 The major ubiquitin ligase involved in E2 degradation was reported to be SCF.56

E3 ubiquitin ligases in signalling pathways associated with HPV-induced carcinogenesis E3 ubiquitin ligases in NF-B signalling Activation of nuclear factor-kB (NF-kB) has been implicated in a variety of cellular processes related to transformation and oncogenesis. NF-kB-dependent proliferation and protection from apoptosis are likely to be important in HPV-induced carcinogenesis. HPV E6- and E7-positive cells have been shown to be sensitized to interleukin-1b-mediated NF-kB activation and exhibit elevated NF-kB components.57 In addition, microarray data suggest that NF-kB-responsive genes are upregulated by E6 protein in cervical keratinocytes.58 Data on NF-kB expression and activation in relation to HPV in oral cancer have also been reported.59 Although the exact mechanisms are still being debated, further studies have revealed that HPV16 E6 and E7 proteins inhibit tumour necrosis factormediated apoptosis by upregulating the expression of cellular inhibitor of apoptosis-2 (cIAP-2), a potent and critical antiapoptotic protein, leading to activation of the NF-kB signalling pathway.60,61 The RING E3 ubiquitin ligases tumour necrosis factor receptor-associated factor (TRAF) and cIAP are involved in NF-kB activation by HPV oncoproteins (Table 1).


252


E3 ubiquitin ligases in TGF- /Smad signalling One of the most widely studied roles of transforming growth factor-b (TGF-b) is its function as a tumour suppressor during the early stage of tumourigenesis. TGF-b binds to its receptors and transduces the antiproliferative signal by the intracellular proteins termed signalling mother against decapentaplegic peptides (Smads).62 Alterations of the TGF-b/Smad signalling pathway, which is tightly regulated by E3 ubiquitin ligases,63 are related to a range of human cancers, including cervical carcinoma.64 FavreBonvin et al.65 reported that HPV18 E6 protein interacts with a protein containing the PDZ domain, TIP-2/GIPC (Tax interacting protein, clone 2/GAIP interacting protein, C terminus) (Table 1), triggering its degradation through the ubiquitin–proteasome pathway. As this PDZ protein has been found to be involved in TGF-b signalling by favouring expression of the TGF-b type III receptor at the cell membrane, the depletion of TIP-2/GIPC in HeLa cells hampers TGF-b signalling and also diminishes the antiproliferative effect of TGF-b. Like the degradation of other PDZ proteins by E6-AP, the interaction between TIP-2/GIPC and HPV18 E6 is possibly mediated through E6-AP, though this point remains to be further defined. Moreover, HPV16 E7 sequesters Smads in the nucleus and inhibits the ability of Smads to bind to their target DNA sequence,66 indicating that suppression of Smadmediated signalling by HPV E7 oncoprotein may contribute to HPV-induced carcinogenesis. However, whether ubiquitination plays a role in the interaction of HPV E7 with Smad proteins needs further investigation.

E3 ubiquitin ligases in IGF signalling Insulin-like growth factor (IGF)-I is a broad-spectrum growth factor that has

mitogenic and antiapoptotic activities: high levels of IGF-I in the circulation are reported to be positively associated with heightened risk of many epithelial and glandular tumours.67,68 Data suggest a possible role of IGF-I receptor in primary and metastatic undifferentiated carcinoma of the head and neck.69 IGF binding protein 3 (IGFBP3) is a major component of IGF binding proteins in the circulation and is an important regulator of biologically active IGFs. Functioning independently of IGF-I, IGFBP3 contributes to both p53-dependent and -independent apoptosis.70 It has been shown that the serum concentration of IGFBP3 is inversely associated with the incident detection of oncogenic HPV and the incidence of oncogenic HPV-positive cervical neoplasia.71 HPV16 E7 protein binds to and inactivates IGFBP3,72 which may augment the serum level of active IGFI, contributing to the progression of cervical disease. Further studies revealed that HPV16 E7 oncoprotein induces polyubiquitination and proteasome-dependent proteolysis of nuclear IGFBP3 in cervical cancer cells,73 suggesting that the inactivation of IGFBP3 and the inhibition of apoptosis are important for the oncogenic activity of E7. The E3 ubiquitin ligase mediating the degradation of IGFBP3 has not yet been identified.

Other E3 ubiquitin ligases Mdm2 Research data suggest that Mdm2 might be related to HPV-induced diseases. Overexpression of Mdm2 protein was found to contribute to the pathogenesis of oral carcinoma and oral verrucous hyperplasia.74 In addition, the risk of oral squamous cell carcinoma associated with HPV16 L1 seropositivity was modified by Mdm2 promoter polymorphisms.75 Identified as a p53 E3 ubiquitin ligase, Mdm2 was considered to be the regulator of normal


Lou and Wang

253

physiological p53 degradation.14 Mdm2 may be an alternative mechanism causing p53 protein dysfunction in oral squamous cell carcinoma.76 Furthermore, p53 was shown to drive Mdm2 expression in a negative-feedback loop.77 The role of Mdm2 in degrading p53 was best demonstrated by murine studies in which inactivation of p53 was able to entirely rescue the embryonic lethality of loss of Mdm2 function.78 The effects of Mdm2 on p53 are concentration-dependent: low Mdm2 induces p53 monoubiquitination and nuclear export; high Mdm2 promotes polyubiquitination and nuclear degradation.79 Interestingly, research showed that Mdm2 enhances transcriptional activity of HPV E2 protein, which is critical for the regulation of the viral life cycle.80 (Table 1).

Nedd4 Gap junction biogenesis requires the oligomerization of six connexin proteins into a hexameric connexon and their subsequent signalling to the plasma membrane.81 Among the gap junction proteins encoded by the 21 connexin genes identified in humans, connexin 43 (Cx43) is the most widely expressed and best studied.82 Certain HPV-associated cancers, especially those of the cervix, have previously demonstrated downregulation of Cx43. In addition, loss of Cx43 expression was believed to be an early event in cervical carcinogenesis, and reexpression of this gene in cervical carcinoma cells was associated with decreased neoplastic potential.83 Tomakidi et al.84 found an almost complete disappearance of Cx43 when HaCaT cells were grown in an organotypic raft culture system to induce differentiation. However, the mechanisms behind these data remain unexplained. Leykauf et al.85 revealed that Cx43 binds, both in vitro and in vivo, the HECT E3 ubiquitin ligase Nedd4 (neuronal precursor cell-expressed developmentally down-regulated 4).

All three WW domains of rat Nedd4 interact with rat Cx43, and this interaction is modulated by the phosphorylation state of Cx43. The authors further showed that the Nedd4 domain WW2 binds to the canonical PY motif at the C-terminus of Cx43, triggering the internalization of Cx43 and posterior degradation of the gap junction proteins. Thus, the disruption of Cx43 by the Nedd4-mediated pathway may contribute to the development of HPV-associated cervical cancer.

EDD E3 identified by differential display (EDD), which was originally isolated as a progestininduced gene, encodes a 350-kDa E3 ubiquitin ligase containing a HECT domain.86 Although alterations in EDD function have been linked to cancer development,87 little is known about the biochemical activities of this protein. EDD was identified as a new HPV18 E6 binding partner.88 Moreover, the authors claimed that changes in the levels of EDD expression may have a significant impact on the ability of HPV E6/E6-AP ubiquitin ligase complex to direct the degradation of several cellular substrates, p53 in particular, and thereby directly influence the development of HPV-induced malignancy.88

Therapeutic intervention with E3 ubiquitin ligases for HPV-related disease Only a small fraction of the genes involved in the ubiquitin system have been identified, and no comprehensive evaluation has been made of the genetic alterations of these components in human diseases. Defining the genetic and genomic targets of new drugs will therefore be important in finding therapeutic strategies. Selective inhibition of a deubiquitinating enzyme may allow more rapid degradation of given substrate(s). Bortezomib, a highly selective proteasomal


254


inhibitor that has been approved by the US Food and Drug Administration, is the product of rational drug design directed towards selective inhibition of the proteolysis of a group of proteasomal substrates.89 Similarly, specific targeting of an E3 ligase would diminish the ubiquitination of its substrate(s). The E3 ligases will probably provide exceptional specificity in the therapeutic modulation of the ubiquitin system because the system is hierarchical, with the greatest diversity at the E3 ligase level.90 As described above, Mdm2 is an oncogenic RING finger E3 for p53. Theoretically, inhibition of the Mdm2–p53 interaction or inhibition of the conjugation of ubiquitin to p53 might lead to stimulation of the tumoursuppressor activity of p53. Furthermore, inactivating Mdm2 might prove beneficial in the treatment of tumours carrying wildtype p53, in that Mdm2 might work as a ubiquitin ligase for other antioncogenic proteins.91 Chemical library screens for Mdm2 inhibitors have identified Nutlins (cis-imidazoline derivatives) that occupy the p53 binding pocket and thus prolong p53 action, including p53-dependent cell-cycle arrest and apoptosis.92 Importantly, Nutlins are effective in vivo upon oral administration to halt the growth of nude mouse tumour xenografts without noticeable toxicity to healthy tissues. 93 Recently Nutlin-3 has been shown to be effective against many cancer cell lines, including HNSCC.93 Moreover, another p53-stabilizing small molecule, 2,5-bis(5-hydroxymethyl-2-thienyl)furan (RITA), isolated from a screen at the National Cancer Institute,94 binds the N-terminus of p53 and seems to prevent the recognition of p53 by Mdm2 or to stabilize the N-terminal a-helix domain of p53 in the Mdm2 groove, or both.95 The RITA-stabilized p53 is transcriptionally active as it can facilitate expression of endogenous p53target genes.95 Most excitingly, RITA slows the growth of mice tumour xenografts in a concentration-dependent manner,96

though its oral bioavailability was not tested in this study. One question that has not been answered is whether RITA and Nutlins might show synergistic antitumour activity in the mouse tumour xenograft model, as they attack different aspects of the Mdm2–p53 recognition process.92,95 It is also necessary to understand why RITA causes cell-cycle perturbations and a slight increase in apoptosis in p53-negative cells.95 Does this mean that RITA targets other cellular proteins, such as other p53-related proteins p63 or p73? Finally, possible sideeffects of long-term Nutlin/RITA administration should be further addressed in animal models. So although it is clear that certain small molecules have the desired effect of activating p53 by suppressing its degradation, further studies are required to determine whether these molecules will be useful in the treatment of human cancers, including cervical cancer and oral cavity carcinoma. Although the exact nature of the contribution of E6-AP to HPV-induced disease is not fully understood,, E6-AP and HPV itself are considered to be pharmacological targets for cervical cancer. It has been shown that knocking down E6-AP expression by in vitro-selected ribozymes in HeLa cells induces apoptosis on exposure to genotoxic compounds.96 Additionally, pharmacological targeting of HPV E6 with zincejecting compounds inactivates E6 by disrupting its functionally crucial zinc fingers and thus silences aberrant E6-AP activity.97 Such small-molecule zinc ejectors could potentially be applicable for clinical use in the topical treatment of cervical papillomas and cervical cancer, if their in vivo safety and bioavailability can be more thoroughly demonstrated. In principle, inhibitors of the HECT domain of E6-AP might be useful in the therapy of cervical cancer. Such HECT domain inhibitors might take advantage of the extra step involved in HECT-dependent protein ubiquitination


Lou and Wang

255

that is not required for other classes of E3s. Because this step requires a large conformational change in the HECT domain,98 it might be possible to find molecules that block this transition or stabilize the HECT domain in a nonfunctional conformation.

events biochemically as they happen during HPV infection in vivo. Advanced mechanistic understanding of ubiquitin signalling would be extremely valuable in the future development of possible treatments for HPV-associated diseases, including cervical cancer and HNSCC.

Conclusions and future prospects Ubiquitin modification is a mechanism for controlling the function and availability of the regulatory proteins in cells; it may also provide a platform for HPV to achieve successful infection and carcinogenesis. In some cases, HPV proteins have evolved so that they structurally resemble the cellular E3 ubiquitin ligases, to permit degradation of target proteins and thereby enhance the efficiency of replication. In other cases, HPV proteins are capable of inhibiting targets of these E3 ubiquitin ligases that might retard viral replication. In addition, some HPV proteins are themselves regulated by degradation. Other E3 ubiquitin ligases also participate in cytokine signalling during HPV infection. It is likely that further studies of HPV will reveal additional examples of such processes, and, as in the past, these examples may shed light on important cellular pathways. In order to further understand the significance of E3 ubiquitin ligases in the immunopathogenesis of HPV infection and HPVinduced carcinogenesis, future studies should focus more on the identification of new substrates of ubiquitination and the identification of more ubiquitin-binding domains. Fortunately, technological advances (such as the use of mass spectrometry) should assist the discovery of new substrates of ubiquitination. The discovery of nearly 20 types of ubiquitin-binding domains in recent years will provide a boost to the identification of ubiquitin receptors. If we know both the ubiquitination targets and the ubiquitin receptors, it may be possible to reconstruct ubiquitin signalling

Declaration of conflicting interest The authors declare that there are no conflicts of interest.

Funding This work was supported by grants from the Education Bureau of Zhejiang Province (Y201121544) and the Health Bureau of Zhejiang Province (2012RCB025).

Acknowledgements The authors thank Dr Qiang Zhou for his critical review of this article.

References 1. de Villiers EM, Fauquet C, Broker TR, et al. Classification of papillomaviruses. Virology 2004; 324: 17–27. 2. Scheffner M and Whitaker NJ. Human papillomavirus-induced carcinogenesis and the ubiquitin-proteasome system. Semin Cancer Biol 2003; 13: 59–67. 3. Einstein MH, Schiller JT, Viscidi RP, et al. Clinician’s guide to human papillomavirus immunology: knowns and unknowns. Lancet Infect Dis 2009; 9: 347–356. 4. Chaturvedi AK, Engels EA, Anderson WF, et al. Incidence trends for human papillomavirus-related and -unrelated oral squamous cell carcinomas in the United States. J Clin Oncol 2008; 26: 612–619. 5. Dayyani F, Etzel CJ, Liu M, et al. Metaanalysis of the impact of human papillomavirus (HPV) on cancer risk and overall survival in head and neck squamous cell carcinomas (HNSCC). Head Neck Oncol 2010; 2: 15.


256


6. Gillison ML, D’Souza G, Westra W, et al. Distinct risk factor profiles for human papillomavirus type 16-positive and human papillomavirus type 16-negative head and neck cancers. J Natl Cancer Inst 2008; 100: 407–420. 7. Syrja¨nen S. The role of human papillomavirus infection in head and neck cancers. Ann Oncol 2010; 21(Suppl. 7): vii243–vii245. 8. Zhou Q, Zhu K and Cheng H. Toll-like receptors in human papillomavirus infection. Arch Immunol Ther Exp (Warsz) 2013; 61: 203–215. 9. Mammas IN, Sourvinos G, Giannoudis A, et al. Human papilloma virus (HPV) and host cellular interactions. Pathol Oncol Res 2008; 14: 345–354. 10. Glickman MH and Ciechanover A. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev 2002; 82: 373–428. 11. Ciechanover A. The ubiquitin-proteasome proteolytic pathway. Cell 1994; 79: 13–21. 12. Ingham RJ, Gish G and Pawson T. The Nedd4 family of E3 ubiquitin ligases: functional diversity within a common modular architecture. Oncogene 2004; 23: 1972–1984. 13. Scheffner M, Huibregtse JM, Vierstra RD, et al. The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell 1993; 75: 495–505. 14. Fang S, Jensen JP, Ludwig RL, et al. Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53. J Biol Chem 2000; 275: 8945–8951. 15. Joazeiro CA, Wing SS, Huang H, et al. The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase. Science 1999; 286: 309–312. 16. Ang XL and Wade Harper J. SCF-mediated protein degradation and cell cycle control. Oncogene 2005; 24: 2860–2870. 17. Chen ZJ. Ubiquitin signalling in the NFkappaB pathway. Nat Cell Biol 2005; 7: 758–765. 18. Johnson ES. Ubiquitin branches out. Nat Cell Biol 2002; 4: E295–E298. 19. Wing SS. Deubiquitinating enzymes–the importance of driving in reverse along the

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

ubiquitin-proteasome pathway. Int J Biochem Cell Biol 2003; 35: 590–605. Schwarz SE, Rosa JL and Scheffner M. Characterization of human hect domain family members and their interaction with UbcH5 and UbcH7. J Biol Chem 1998; 273: 12148–12154. Huibregtse JM, Scheffner M and Howley PM. Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53. Mol Cell Biol 1993; 13: 775–784. Thomas M and Banks L. Human papillomavirus (HPV) E6 interactions with Bak are conserved amongst E6 proteins from high and low risk HPV types. J Gen Virol 1999; 80: 1513–1517. Gross-Mesilaty S, Reinstein E, Bercovich B, et al. Basal and human papillomavirus E6 oncoprotein-induced degradation of Myc proteins by the ubiquitin pathway. Proc Natl Acad Sci U S A 1998; 95: 8058–8063. Gao Q, Kumar A, Singh L, et al. Human papillomavirus E6-induced degradation of E6TP1 is mediated by E6AP ubiquitin ligase. Cancer Res 2002; 62: 3315–3321. Ku¨hne C and Banks L. E3-ubiquitin ligase/ E6-AP links multicopy maintenance protein 7 to the ubiquitination pathway by a novel motif, the L2G box. J Biol Chem 1998; 273: 34302–34309. Srivenugopal KS and Ali-Osman F. The DNA repair protein, O(6)-methylguanineDNA methyltransferase is a proteolytic target for the E6 human papillomavirus oncoprotein. Oncogene 2002; 21: 5940–5945. Xu M, Luo W, Elzi DJ, et al. NFX1 interacts with mSin3A/histone deacetylase to repress hTERT transcription in keratinocytes. Mol Cell Biol 2008; 28: 4819–4828. Kuballa P, Matentzoglu K and Scheffner M. The role of the ubiquitin ligase E6-AP in human papillomavirus E6-mediated degradation of PDZ domain-containing proteins. J Biol Chem 2007; 282: 65–71. Nakagawa S and Huibregtse JM. Human scribble (Vartul) is targeted for ubiquitinmediated degradation by the high-risk papillomavirus E6 proteins and the E6AP


Lou and Wang

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

257

ubiquitin-protein ligase. Mol Cell Biol 2000; 20: 8244–8253. Jing M, Bohl J, Brimer N, et al. Degradation of tyrosine phosphatase PTPN3 (PTPH1) by association with oncogenic human papillomavirus E6 proteins. J Virol 2007; 81: 2231–2239. Grm HS and Banks L. Degradation of hDlg and MAGIs by human papillomavirus E6 is E6-AP-independent. J Gen Virol 2004; 85: 2815–2819. Shimoji T, Murakami K, Sugiyama Y, et al. Identification of annexin A1 as a novel substrate for E6AP-mediated ubiquitylation. J Cell Biochem 2009; 106: 1123–1135. Wang LD, Yang YH, Liu Y, et al. Decreased expression of annexin A1 during the progression of cervical neoplasia. J Int Med Res 2008; 36: 665–672. Nag A, Germaniuk-Kurowska A, Dimri M, et al. An essential role of human Ada3 in p53 acetylation. J Biol Chem 2007; 282: 8812–8820. Hu Y, Ye F, Lu W, et al. HPV16 E6-induced and E6AP-dependent inhibition of the transcriptional coactivator hADA3 in human cervical carcinoma cells. Cancer Invest 2009; 27: 298–306. Dyson N, Guida P, Mu¨nger K, et al. Homologous sequences in adenovirus E1A and human papillomavirus E7 proteins mediate interaction with the same set of cellular proteins. J Virol 1992; 66: 6893–6902. Mu¨nger K, Werness BA, Dyson N, et al. Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product. EMBO J 1989; 8: 4099–4105. Boyer SN, Wazer DE and Band V. E7 protein of human papilloma virus-16 induces degradation of retinoblastoma protein through the ubiquitin-proteasome pathway. Cancer Res 1996; 56: 4620–4624. Jones DL, Thompson DA and Mu¨nger K. Destabilization of the RB tumor suppressor protein and stabilization of p53 contribute to HPV type 16 E7-induced apoptosis. Virology 1997; 239: 97–107. Huh K, Zhou X, Hayakawa H, et al. Human papillomavirus type 16 E7 oncoprotein

41.

42.

43.

44.

45.

46.

47.

48.

49.

associates with the cullin 2 ubiquitin ligase complex, which contributes to degradation of the retinoblastoma tumor suppressor. J Virol 2007; 81: 9737–9747. Zhang B, Chen W and Roman A. The E7 proteins of low- and high-risk human papillomaviruses share the ability to target the pRB family member p130 for degradation. Proc Natl Acad Sci U S A 2006; 103: 437–442. Tedesco D, Lukas J and Reed SI. The pRbrelated protein p130 is regulated by phosphorylation-dependent proteolysis via the protein-ubiquitin ligase SCF (Skp2). Genes Dev 2002; 16: 2946–2957. Leechanachai P, Banks L, Moreau F, et al. The E5 gene from human papillomavirus type 16 is an oncogene which enhances growth factor-mediated signal transduction to the nucleus. Oncogene 1992; 7: 19–25. Bouvard V, Matlashewski G, Gu ZM, et al. The human papillomavirus type 16 E5 gene cooperates with the E7 gene to stimulate proliferation of primary cells and increases viral gene expression. Virology 1994; 203: 73–80. Sto¨ppler MC, Straight SW, Tsao G, et al. The E5 gene of HPV-16 enhances keratinocyte immortalization by full-length DNA. Virology 1996; 223: 251–254. Zhang B, Srirangam A, Potter DA, et al. HPV16 E5 protein disrupts the c-Cbl-EGFR interaction and EGFR ubiquitination in human foreskin keratinocytes. Oncogene 2005; 24: 2585–2588. Fournier N, Raj K, Saudan P, et al. Expression of human papillomavirus 16 E2 protein in Schizosaccharomyces pombe delays the initiation of mitosis. Oncogene 1999; 18: 4015–4021. Bellanger S, Blachon S, Mechali F, et al. High-risk but not low-risk HPV E2 proteins bind to the APC activators Cdh1 and Cdc20 and cause genomic instability. Cell Cycle 2005; 4: 1608–1615. Reinstein E, Scheffner M, Oren M, et al. Degradation of the E7 human papillomavirus oncoprotein by the ubiquitin-proteasome system: targeting via ubiquitination of the N-terminal residue. Oncogene 2000; 19: 5944–5950.


258


50. Oh KJ, Kalinina A, Wang J, et al. The papillomavirus E7 oncoprotein is ubiquitinated by UbcH7 and Cullin 1- and Skp2containing E3 ligase. J Virol 2004; 78: 5338–5346. 51. Kamio M, Yoshida T, Ogata H, et al. SOCS1 [corrected] inhibits HPV-E7-mediated transformation by inducing degradation of E7 protein. Oncogene 2004; 23: 3107–3115. 52. Lin CH, Chang HS and Yu WC. USP11 stabilizes HPV-16E7 and further modulates the E7 biological activity. J Biol Chem 2008; 283: 15681–15688. 53. Bellanger S, Demeret C, Goyat S, et al. Stability of the human papillomavirus type 18 E2 protein is regulated by a proteasome degradation pathway through its aminoterminal transactivation domain. J Virol 2001; 75: 7244–7251. 54. Stewart D, Kazemi S, Li S, et al. Ubiquitination and proteasome degradation of the E6 proteins of human papillomavirus types 11 and 18. J Gen Virol 2004; 85: 1419–1426. 55. Kehmeier E, Ru¨hl H, Voland B, et al. Cellular steady-state levels of ‘‘high risk’’ but not ‘‘low risk’’ human papillomavirus (HPV) E6 proteins are increased by inhibition of proteasome-dependent degradation independent of their p53- and E6AP-binding capabilities. Virology 2002; 299: 72–87. 56. Bellanger S, Tan CL, Nei W, et al. The human papillomavirus type 18 E2 protein is a cell cycle-dependent target of the SCFSkp2 ubiquitin ligase. J Virol 2010; 84: 437–444. 57. Havard L, Delvenne P, Frare´ P, et al. Differential production of cytokines and activation of NF-kappaB in HPV-transformed keratinocytes. Virology 2002; 298: 271–285. 58. Nees M, Geoghegan JM, Hyman T, et al. Papillomavirus type 16 oncogenes downregulate expression of interferon-responsive genes and upregulate proliferation-associated and NF-kappaB-responsive genes in cervical keratinocytes. J Virol 2001; 75: 4283–4296. 59. Mishra A, Bharti AC, Varghese P, et al. Differential expression and activation of NF-kappaB family proteins during oral carcinogenesis: role of high risk human

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

papillomavirus infection. Int J Cancer 2006; 119: 2840–2850. Yuan H, Fu F, Zhuo J, et al. Human papillomavirus type 16 E6 and E7 oncoproteins upregulate c-IAP2 gene expression and confer resistance to apoptosis. Oncogene 2005; 24: 5069–5078. Zhou Q, Zhu K and Cheng H. Ubiquitination in host immune response to human papillomavirus infection. Arch Dermatol Res 2011; 303: 217–230. ten Dijke P and Hill CS. New insights into TGF-beta-Smad signalling. Trends Biochem Sci 2004; 29: 265–273. Inoue Y and Imamura T. Regulation of TGF-beta family signaling by E3 ubiquitin ligases. Cancer Sci 2008; 99: 2107–2112. Ki KD, Tong SY, Huh CY, et al. Expression and mutational analysis of TGF-beta/Smads signaling in human cervical cancers. J Gynecol Oncol 2009; 20: 117–121. Favre-Bonvin A, Reynaud C, Kretz-Remy C, et al. Human papillomavirus type 18 E6 protein binds the cellular PDZ protein TIP2/GIPC, which is involved in transforming growth factor beta signaling and triggers its degradation by the proteasome. J Virol 2005; 79: 4229–4237. Lee DK, Kim BC, Kim IY, et al. The human papilloma virus E7 oncoprotein inhibits transforming growth factor-beta signaling by blocking binding of the Smad complex to its target sequence. J Biol Chem 2002; 277: 38557–38564. Renehan AG, Zwahlen M, Minder C, et al. Insulin-like growth factor (IGF)-I, IGF binding protein-3, and cancer risk: systematic review and meta-regression analysis. Lancet 2004; 363: 1346–1353. Shen MR, Hsu YM, Hsu KF, et al. Insulinlike growth factor 1 is a potent stimulator of cervical cancer cell invasiveness and proliferation that is modulated by alphavbeta3 integrin signaling. Carcinogenesis 2006; 27: 962–971. Friedrich RE, Hagel C and Bartel-Friedrich S. Insulin-like growth factor-1 receptor (IGF-1R) in primary and metastatic undifferentiated carcinoma of the head and neck: a possible target of immunotherapy. Anticancer Res 2010; 30: 1641–1643.


Lou and Wang

259

70. Rajah R, Valentinis B and Cohen P. Insulinlike growth factor (IGF)-binding protein-3 induces apoptosis and mediates the effects of transforming growth factor-beta1 on programmed cell death through a p53- and IGFindependent mechanism. J Biol Chem 1997; 272: 12181–12188. 71. Harris TG, Burk RD, Yu H, et al. Insulinlike growth factor axis and oncogenic human papillomavirus natural history. Cancer Epidemiol Biomarkers Prev 2008; 17: 245–248. 72. Mannhardt B, Weinzimer SA, Wagner M, et al. Human papillomavirus type 16 E7 oncoprotein binds and inactivates growthinhibitory insulin-like growth factor binding protein 3. Mol Cell Biol 2000; 20: 6483–6495. 73. Santer FR, Moser B, Spoden GA, et al. Human papillomavirus type 16 E7 oncoprotein inhibits apoptosis mediated by nuclear insulin-like growth factor-binding protein-3 by enhancing its ubiquitin/proteasome-dependent degradation. Carcinogenesis 2007; 28: 2511–2520. 74. Lin HP, Wang YP and Chiang CP. Expression of p53, MDM2, p21, heat shock protein 70, and HPV 16/18 E6 proteins in oral verrucous carcinoma and oral verrucous hyperplasia. Head Neck 2011; 33: 334–340. 75. Chen X, Sturgis EM, Lei D, et al. Human papillomavirus seropositivity synergizes with MDM2 variants to increase the risk of oral squamous cell carcinoma. Cancer Res 2010; 70: 7199–7208. 76. Matsumura T, Yoshihama Y, Kimura T, et al. p53 and MDM2 expression in oral squamous cell carcinoma. Oncology 1996; 53: 308–312. 77. Wu X, Bayle JH, Olson D, et al. The p53mdm-2 autoregulatory feedback loop. Genes Dev 1993; 7: 1126–1132. 78. de Rozieres S, Maya R, Oren M, et al. The loss of mdm2 induces p53-mediated apoptosis. Oncogene 2000; 19: 1691–1697. 79. Li M, Brooks CL, Wu-Baer F, et al. Monoversus polyubiquitination: differential control of p53 fate by Mdm2. Science 2003; 302: 1972–1975. 80. Gammoh N, Gardiol D, Massimi P, et al. The Mdm2 ubiquitin ligase enhances

81.

82.

83.

84.

85.

86.

87.

88.

89.

90.

transcriptional activity of human papillomavirus E2. J Virol 2009; 83: 1538–1543. Bruzzone R, White TW and Paul DL. Connections with connexins: the molecular basis of direct intercellular signaling. Eur J Biochem 1996; 238: 1–27. So¨hl G and Willecke K. Gap junctions and the connexin protein family. Cardiovasc Res 2004; 62: 228–232. King TJ, Fukushima LH, Hieber AD, et al. Reduced levels of connexin43 in cervical dysplasia: inducible expression in a cervical carcinoma cell line decreases neoplastic potential with implications for tumor progression. Carcinogenesis 2000; 21: 1097–1109. Tomakidi P, Cheng H, Kohl A, et al. Connexin 43 expression is downregulated in raft cultures of human keratinocytes expressing the human papillomavirus type 16 E5 protein. Cell Tissue Res 2000; 301: 323–327. Leykauf K, Salek M, Bomke J, et al. Ubiquitin protein ligase Nedd4 binds to connexin43 by a phosphorylation-modulated process. J Cell Sci 2006; 119: 3634–3642. Callaghan MJ, Russell AJ, Woollatt E, et al. Identification of a human HECT family protein with homology to the Drosophila tumor suppressor gene hyperplastic discs. Oncogene 1998; 17: 3479–3491. Clancy JL, Henderson MJ, Russell AJ, et al. EDD, the human orthologue of the hyperplastic discs tumour suppressor gene, is amplified and overexpressed in cancer. Oncogene 2003; 22: 5070–5081. Tomaic V, Pim D, Thomas M, et al. Regulation of the human papillomavirus type 18 E6/E6AP ubiquitin ligase complex by the HECT domain-containing protein EDD. J Virol 2011; 85: 3120–3127. Richardson PG, Barlogie B, Berenson J, et al. Extended follow-up of a phase II trial in relapsed, refractory multiple myeloma: final time-to-event results from the SUMMIT trial. Cancer 2006; 106: 1316–1319. Nalepa G, Rolfe M and Harper JW. Drug discovery in the ubiquitin-proteasome system. Nat Rev Drug Discov 2006; 5: 596–613.


260


91. Zhang Z, Wang H, Li M, et al. MDM2 is a negative regulator of p21WAF1/CIP1, independent of p53. J Biol Chem 2004; 279: 16000–16006. 92. Vassilev LT, Vu BT, Graves B, et al. In vivo activation of the p53 pathway by smallmolecule antagonists of MDM2. Science 2004; 303: 844–848. 93. Roh JL, Kang SK, Minn I, et al. p53Reactivating small molecules induce apoptosis and enhance chemotherapeutic cytotoxicity in head and neck squamous cell carcinoma. Oral Oncol 2011; 47: 8–15. 94. Rubinstein LV, Shoemaker RH, Paull KD, et al. Comparison of in vitro anticancerdrug-screening data generated with a tetrazolium assay versus a protein assay against a diverse panel of human tumor cell lines. J Natl Cancer Inst 1990; 82: 1113–1118.

95. Issaeva N, Bozko P, Enge M, et al. Small molecule RITA binds to p53, blocks p53HDM-2 interaction and activates p53 function in tumors. Nat Med 2004; 10: 1321–1328. 96. Kim Y, Cairns MJ, Marouga R, et al. E6AP gene suppression and characterization with in vitro selected hammerhead ribozymes. Cancer Gene Ther 2003; 10: 707–716. 97. Foster SA and Phelps WC. Zn(2þ) fingers and cervical cancer. J Natl Cancer Inst 1999; 91: 1180–1181. 98. Verdecia MA, Joazeiro CA, Wells NJ, et al. Conformational flexibility underlies ubiquitin ligation mediated by the WWP1 HECT domain E3 ligase. Mol Cell 2003; 11: 249–259.
