Human papillomavirus E6 and E7 oncoproteins as risk factors for tumorigenesis

Human papillomavirus (HPV) is small, double-stranded DNA virus that infects mucosal and cutaneous epithelial tissue. HPV is sexually transmitted and the viral DNA replicates extrachromosomally. The virus is non-enveloped and has an icosahedral capsid. There are approximately 118 types of HPV, which are characterized as high-risk or lowrisk types. High-risk HPVs cause malignant transformation while the low-risk ones cause benign warts and lesions. The expression of E6 and E7 is normally controlled during the normal viral life cycle when viral DNA replicates extrachromosomally. HPV E6 and E7 oncoproteins are overexpressed when the viral genome integrates into the host DNA. Deregulated overexpression of E6 and E7 oncoproteins can cause several changes in cellular pathways and functions leading to malignant transformation of cells and tumorigenesis. In this review, we focus on several cellular mechanisms and pathways that are altered in the presence of E6 and E7, the target proteins of E6 and E7 inside the host cell and how they contribute to the development of the transformed phenotype.     

Hallmarks of HPV carcinogenesis: The role of E6, E7 and E5 oncoproteins in cellular malignancy

Human papillomavirus (HPV) is the most common sexually transmitted infectious agent worldwide, being also responsible for 5% of all human cancers. The integration and hypermethylation mechanisms of the HPV viral genome promote the unbalanced expression of the E6, E7 and E5 oncoproteins, which are crucial factors for the carcinogenic cascade in HPV-induced cancers. This review highlights the action of E6, E7 and E5 over key regulatory targets, promoting all known hallmarks of cancer. Both well-characterized and novel targets of these HPV oncoproteins are described, detailing their mechanisms of action. Finally, this review approaches the possibility of targeting E6, E7 and E5 for therapeutic applications in the context of cancer.     

Inhibition of apoptosis in human laryngeal cancer cells by E6 and E7 oncoproteins of human papillomavirus 16

The carcinogenesis of human papillomaviruses type 16 (HPV-16) is mainly due to its two oncoproteins, E6 and E7. Their carcinogenic features in term of their relationship with Bcl-2 family are still unclear. We thus aimed to analyze the expression of Bcl-2 family members, Bcl-2, Bax, and Bak in laryngeal cancer cells transfected with the E6 or E7 and to determine the sensitivity of these cells to apoptotic stimuli. We employed two human laryngeal cancer cell lines, UMSCC12 and UMSCC11A in this study. These two cell lines were stably transfected with HPV16 E6, E7 or empty vector, pcDNA3.1. We found that E6 and E7 inhibited apoptosis induced by TNF-alpha/CHX in both UMSCC11A and UMSCC12 cells, enhanced the stability of Bcl-2 protein and increased the degradation of Bak protein. Furthermore, it was found that HPV-16 E7 statistically enhanced the expression of Bcl-2 in laryngeal cancer. The alteration of Bak by E6 and E7 was not through the influence on the Bak promoter, as the luciferase assay showed that neither E6 nor E7 changed the Bak promoter activity. We conclude that the evasion of apoptosis mediated by HPV-16 E6 and E7 is associated with increased Bcl-2 and decreased Bak in laryngeal carcinogenesis and that the decreased level of Bak by E6 and E7 is not caused by the regulation of the Bak promoter but by reducing its protein stability.     

Regulation of Na(+)-coupled glucose carrier SGLT1 by human papillomavirus 18 E6 protein

Tumor cells utilize preferably glucose for energy production. They accomplish cellular glucose uptake in part through Na⁺-coupled glucose transport mediated by SGLT1 (SLC5A1). This study explored the possibility that the human papillomavirus 18 E6 protein HPV18 E6 (E6) participates in the stimulation of SGLT1 activity. E6 is one of the two major oncoproteins of high-risk human papillomaviruses, which are the causative agent for cervical carcinoma. According to Western blotting, SGLT1 is expressed in the HPV18-positive cervical carcinoma cell line HeLa. To explore whether E6 affects SGLT1 activity, SGLT1 was expressed in Xenopus oocytes with and without E6 and electrogenic glucose transport determined by dual electrode voltage clamp. In SGLT1-expressing oocytes, but not in oocytes injected with water or expressing E6 alone, glucose triggered a current (I_g). I_g was significantly increased by coexpression of E6 but not by coexpression of E2. According to chemiluminescence and confocal microscopy, coexpression of E6 significantly increased the SGLT1 protein abundance in the cell membrane. The decay of I_g following inhibition of carrier insertion by Brefeldine A (5 μM) was not significantly affected E6 coexpression. Accrodingly, E6 was not effective by increasing carrier protein stability in the membrane. In conclusion, HPV18 E6 oncoprotein participates in the upregulation of SGLT1.     

Interaction of oncogenic papillomavirus E6 proteins with fibulin-1

Human papillomavirus (HPV) infection is the primary risk factor for the development of cervical cancer. The papillomavirus E6 gene is essential for virus-induced cellular transformation and the viral life cycle. Important insight into the mechanism of E6 function came from the discovery that cancer-related HPV E6 proteins promote the degradation of the tumor suppressor p53. However, mounting evidence indicates that interaction with p53 does not mediate all E6 activities. To explore the p53-independent functions of E6, we performed a yeast two-hybrid screen and identified fibulin-1 as an E6 binding protein. Fibulin-1 is a calcium-binding plasma and extracellular matrix protein that has been implicated in cellular transformation and tumor invasion. The interaction between E6 and fibulin-1 was demonstrated by both in vitro and in vivo assays. Fibulin-1 is associated specifically with cancer-related HPV E6s and the transforming bovine papillomavirus type 1 E6. Significantly, overexpression of fibulin-1 specifically inhibited E6-mediated transformation. These results suggest that fibulin-1 plays an important role in the biological activities of E6.     

Mechanisms of tumor suppressor protein inactivation by the human papillomavirus E6 and E7 oncoproteins

There are now several examples where experimental and epidemiologic data have implied a causative role for viruses in human cancer. Human papillomavirus (HPV) DNA is found in approximately 90% of cervical cancers. Only a subset of the HPV types that infect the anogential tissues, however, are associated with cancer. Interestingly, only the cloned DNA of this subset is capable of immortalizing human primary genital keratinocytes in culture. The oncoproteins of the HPVs are encoded by the E6 and E7 genes. Analogous to the oncoproteins of certain other DNA tumor viruses, the E6 and E7 proteins have been shown to functionally inactivate the tumor suppressor proteins p53 and pRB, respectively. We will review what is known of the mechanisms by which the E6 and E7 proteins inactivate these tumor suppressors and the evidence that these activities are related to the transforming capabilities of the HPVs associated with cancer.     

Oncogenic comparison of human papillomavirus type 58 E7 variants

Human papillomavirus 58 (HPV58) ranks the second or third in East Asian cervical cancers. Current studies on HPV58 are scarce and focus on the prototype. Previously, we identified the three most common circulating HPV58 E7 strains contained amino acid alterations: G41R/G63D (51%), T20I/G63S (22%) and T74A/D76E (14%) respectively. Among them, the T20I/G63S variant (V1) had a stronger epidemiological association with cervical cancer. We therefore suggested that V1 possessed stronger oncogenicity than the other two variants. Here, we performed phenotypic assays to characterize and compare their oncogenicities with HPV58 E7 prototype. Our results showed that overexpression of V1 conferred a higher colony‐forming ability to primary murine epithelial cells than prototype (P < 0.05) and other variants, implicating its higher immortalising potential. Further experiments showed that both V1 and prototype enhanced the anchorage‐independent growth of NIH/3T3 cells (P < 0.001), implicating their stronger transforming power than the two other variants. Moreover, they possessed an increased ability to degrade pRb (P < 0.001), which is a major effector pathway of E7‐driven oncogenesis. Our work represents the first study to compare the oncogenicities of HPV58 E7 prototype and variants. These findings deepened our understanding of HPV58 and might inform clinical screening and follow‐up strategy.     

RNA interference of human papillomavirus type 18 E6 and E7 induces senescence in HeLa cells

The human papillomavirus oncoproteins E6 and E7 promote cell proliferation and contribute to carcinogenesis by interfering with the activities of cellular tumor suppressors. We used a small interfering RNA molecule targeting the E7 region of the bicistronic E6 and E7 mRNA to induce RNA interference, thereby reducing expression of E6 and E7 in HeLa cells. RNA interference of E6 and E7 also inhibited cellular DNA synthesis and induced morphological and biochemical changes characteristic of cellular senescence. These results demonstrate that reducing E6 and E7 expression is sufficient to cause HeLa cells to become senescent.     

Targetting of the N‐terminal domain of the human papillomavirus type 16 E6 oncoprotein with monomeric scFvs blocks the E6‐mediated degradation of cellular p53

The E6 protein of cancer‐associated human papillomavirus type 16 (HPV16) binds to cellular p53 and promotes its degradation through the ubiquitin pathway. In an attempt to identify the regions of E6 that could be targetted for functional inhibition, we generated monoclonal antibodies to the HPV16 E6 oncoprotein (16E6) and analysed their effect on E6‐mediated p53 in vitro degradation. The isolated antibodies recognize the 16E6 oncoprotein expressed in the CaSki carcinoma cell line and strongly inhibit the proteolysis of p53 in vitro by binding specifically to a region of 10 residues located at the N‐terminal end of 16E6. The variable regions of these antibodies were cloned and expressed in E. coli as single chain Fvs (scFvs). Purified scFvs were present in monomeric form and totally abolished 16E6‐mediated p53 degradation by preventing the formation of E6/p53 protein complexes. Our results demonstrate that monovalent binding of scFvs to the N‐terminal end of 16E6 abrogates the biological mechanisms leading to the degradation of p53, and they suggest that this region of 16E6 may be a useful in vivo target for blocking the oncogenic activity of HPV16 E6 protein. Copyright © 1999 John Wiley & Sons, Ltd.     

Down-regulation of p21 contributes to apoptosis induced by HPV E6 in human mammary epithelial cells

Infection with human papillomaviruses (HPV) is strongly associated with the development of cervical cancer. The HPV E6 gene is essential for the oncogenic potential of HPV. E6 induces cell proliferation and apoptosis in cervical cancer precursor lesions and in cultured cells. Although induction of telomerase and inactivation of the tumor suppressor p53 play important roles for E6 to promote cell growth, the molecular basis of E6-induced apoptosis is poorly understood. While it is expected that inactivation of p53 by E6 should lead to a reduction in cellular apoptosis, numerous studies demonstrated that E6 could in fact sensitize cells to apoptosis. Understanding the mechanism of p53-independent apoptosis is of clinical significance. In the present study, we investigated the mechanism of apoptosis during E6-mediated immortalization of primary human mammary epithelial cell (HMEC). E6 by itself is sufficient to immortalize HMECs and is believed to do so at least in part by activation of telomerase. During the process of E6-mediated HMEC immortalization, an increased apoptosis was observed. Mutational analysis demonstrated that E6-induced apoptosis was distinct from its ability to promote cell proliferation, activate telomerase, or degrade p53. While the known pro-apoptotic E6 target proteins such as Bak or c-Myc did not appear to play an important role, down-regulation of the cyclin-dependent kinase inhibitor p21^Waf1/Cip1 (p21) by E6 correlated with its ability to induce apoptosis. Ectopic expression of p21 inhibited E6-induced apoptosis. Moreover, a p53 degradation defective E6 mutant was competent for p21 down-regulation and apoptosis induction. The anti-apoptotic function of p21 may not simply be the result of p21-induced growth arrest. These studies demonstrate an E6 activity to down-regulate p21 that is important for induction of apoptosis.     

Binding sequence-dependent regulation of the human proliferating cell nuclear antigen promoter by p53

Exposure of a lung epithelial cell line to ionizing radiation (IR) arrests cell cycle progression through 48 h post-exposure. Coincidentally, IR differentially activates expression of the cell cycle inhibitor, p21/WAF1, and the DNA replication protein, proliferating cell nuclear antigen (PCNA). p21/WAF1 mRNA levels remain elevated through 48 h post-exposure to IR, while PCNA mRNA levels increase transiently at early times. Since p21/WAF1 inhibits DNA replication by directly binding PCNA, the relative levels of the two proteins can determine cell cycle progression. The PCNA p53-binding site displayed reduced p53 binding affinity in vitro relative to the distal p21/WAF1 p53-binding site. Substitution of the p21/WAF1 site for the resident p53-binding site in the PCNA promoter altered the responses to increasing amounts of p53 or IR in transient expression assays. The p21/WAF1 p53-binding site sustained activation of the chimeric PCNA promoter under conditions (high p53 levels or high dose IR) that the PCNA p53-binding site did not. Binding site-specific regulation by wild-type p53 was not observed with mutant p53 harboring a serine to alanine change at amino acid 46. Limited activation of the PCNA promoter by p53 and its modified forms would restrict the amount of PCNA made available for DNA repair.     

Regulation of the human papillomavirus type 18 E6/E6AP ubiquitin ligase complex by the HECT domain-containing protein EDD

Human papillomavirus (HPV) E6 oncoproteins target many cellular proteins for ubiquitin-mediated proteasomal degradation. In the case of p53, this is mediated principally by the E6AP ubiquitin ligase. Several studies have reported that E6 can target certain of its substrates in an apparently E6AP-independent fashion and that several of these substrates vary in the degree to which they are degraded by E6 at different stages of malignancy. To more fully understand the regulation of the E6AP/E6 proteolytic targeting complex, we performed a mass spectroscopic analysis of HPV type 18 (HPV-18) E6 protein complexes and identified the HECT domain-containing ubiquitin ligase EDD as a new HPV-18 E6 binding partner. We show that EDD can interact independently with both E6 and E6AP. Furthermore, EDD appears to regulate E6AP expression levels independently of E6, and loss of EDD stimulates the proteolytic activity of the E6/E6AP complex. Conversely, higher levels of EDD expression protect a number of substrates from E6-induced degradation, partly as a consequence of lower levels of E6 and E6AP expression. Intriguingly, reduction in EDD expression levels in HPV-18-positive HeLa cells enhances cell resistance to apoptotic and growth arrest stimuli. These studies suggest that changes in the levels of EDD expression during different stages of the viral life cycle or during malignancy could have a profound effect upon the ability of E6 to target various substrates for proteolytic degradation and thereby directly influence the development of HPV-induced malignancy.     

Bacterial expression and purification of human papillomavirus type 18 L1

The human papillomavirus (HPV) 18 L1 gene, which encodes the L1 major capsid protein, was isolated from a female patient in Pusan, Korea Republic and was cloned into pGEX-4T-1 vector. The HPV-18L1 gene was expressed in Escherichia coli as a fusion protein with a glutathione-S-transferase (GST) tag. The soluble recombinant fusion protein, GST-18 L1 fusion, was isolated to high purity. HPV-18 L1 was purified from the GST-18 L1 fusant after biotinylated thrombin cleavage, and then the treated thrombin was removed serially using streptavidin conjugated resin. The purified HPV-18 L1 was confirmed by western blotting using a rabbit anti-denatured papillomavirus polyclonal antibody. The virus-like particles (VLP) from the purified full-length 18 L1 protein without any extra amino acid sequences was observed through the analysis of the electron microscope. This is the first study to report the expression and purification of HPV-18 L1 in E. coli. This expression and purification system offers a simple method of expressing and purifying HPV L1 protein, and could potentially be an effective route for the development and manufacturing of highly purified HPV-18 L1-based cervical cancer vaccines.