High-risk High-risk Human Papillomavirus E6 Research Articles

Ubiquitination of the HPV Oncoprotein E6 Is Critical for E6/E6AP-Mediated p53 Degradation.

High-risk Human papillomaviruses (HPVs) types are associated with more than 90% of premalignant and malignant squamous lesions of the uterine cervix. The E6 oncoprotein of high-risk HPVs is a key determinant in cell transformation because it induces the degradation of the host pro-apoptotic tumor suppressor p53. E6 recruits the intracellular ubiquitin ligase E6AP and subsequently induces proteasome-dependent p53 degradation. Neither E6 nor E6AP alone interact with p53; however, the precise mechanism of the functional regulation of the E6/E6AP/p53 complex is unclear. Here, we showed that the high-risk HPV E6 proteins are ubiquitinated during E6/E6AP/p53 complex assembly and degraded by the proteasome system. Increasing p53 expression enhanced E6/E6AP/p53 assembly and facilitated E6 ubiquitination and degradation. The dominant negative mutant of p53 R175H, which does not efficiently bind E6, decreased E6 ubiquitination and increased stability. Furthermore, we showed that the ubiquitin ligase E6AP is essential for E6 ubiquitination, and downregulation of E6AP expression increased E6 stability. We also showed that p53 R175H inhibited E6-mediated p53 degradation. Consistently, the host deubiquitinating enzyme USP15 removed ubiquitin chains from E6 proteins and inhibited E6-mediated p53 degradation. Crucially, ectopic expression of either p53 R175H or USP15 promoted p53-triggered apoptosis in human cervical cancer cells. Considering the importance of ubiquitinated E6 on p53 degradation, the disruption of E6 ubiquitination represents an attractive pharmacological intervention against HPV-positive human cervical cancer.ImportanceVirtually 100% of cervical cancers are linked to HPV infection. Commercial HPV vaccines are estimated to prevent up to 90% of HPV-associated cancers, while they do not eliminate persistent HPV infections and have no effect on the progression to malignancy. Hence, the development of novel therapeutic interventions against HPV is urgently required. The HPV oncoprotein E6 binds to the intracellular E3 ubiquitin ligase E6AP and p53 resulting in the degradation of p53. In this study, we demonstrate that HPV E6 is ubiquitinated by E6AP in presence of p53. Crucially, ubiquitination of E6 is important for p53 degradation and blockage of E6 ubiquitination negatively interferes with E6-mediated p53 degradation and enhances the apoptotic effects of p53 and the cytotoxicity of DNA damage in HPV-positive cervical cancer cells. Importantly, our data suggest that the HPV oncogene E6 might be an optimal pharmacologic.

Modulation of microRNA-mRNA Target Pairs by Human Papillomavirus 16 Oncoproteins.

ABSTRACTThe E6 and E7 proteins are the major oncogenic drivers encoded by high-risk human papillomaviruses (HPVs). While many aspects of the transforming activities of these proteins have been extensively studied, there are fewer studies that have investigated how HPV E6/E7 expression affects the expression of cellular noncoding RNAs. The goal of our study was to investigate HPV16 E6/E7 modulation of cellular microRNA (miR) levels and to determine the potential consequences for cellular gene expression. We performed deep sequencing of small and large cellular RNAs in primary undifferentiated cultures of human foreskin keratinocytes (HFKs) with stable expression of HPV16 E6/E7 or a control vector. After integration of the two data sets, we identified 51 differentially expressed cellular miRs associated with the modulation of 1,456 potential target mRNAs in HPV16 E6/E7-expressing HFKs. We discovered that the degree of differential miR expression in HFKs expressing HPV16 E6/E7 was not necessarily predictive of the number of corresponding mRNA targets or the potential impact on gene expression. Additional analyses of the identified miR-mRNA pairs suggest modulation of specific biological activities and biochemical pathways. Overall, our study supports the model that perturbation of cellular miR expression by HPV16 E6/E7 importantly contributes to the rewiring of cellular regulatory circuits by the high-risk HPV E6 and E7 proteins that contribute to oncogenic transformation.

Abstract A26: Importance of long noncoding RNAs in human papillomavirus-related cancers

Abstract The objective of this study is to identify the importance of long non-coding RNAs (lncRNAs) in the induction of carcinogenesis by high-risk human papillomavirus (HPV)-16 oncoprotein E6 in HPV-related cancers. Dysregulation of long non-coding RNAs (lncRNAs) occurs in most human cancers, however, there are only a few studies showing dysregulation of specific lncRNAs in human papillomavirus (HPV)-related cancers. High-risk HPV infection (e.g. HPV-16 and HPV-18) is one the most common causes of cervical cancer, as well as a subset of head and neck squamous cell carcinoma (HNSCC). It is well known that one of the main factors contributing to HPV-related carcinogenesis is the expression of high-risk HPV E6 viral oncoprotein and its interaction with several human proteins, such as the tumor suppressor p53 and the anti-apoptotic protein Bak. It is hypothesized that HPV-16 E6 changes the expression of host lncRNAs to regulate downstream processes important in the induction of carcinogenesis. To test this hypothesis, we stably express high-risk HPV-16 E6 in primary human keratinocytes and conducted high-throughput RNA sequencing analysis to identify HPV-16 E6-mediated changes in lncRNAs expression. Our preliminary data shows over 500 lncRNAs up- or down- regulated by greater than 2-fold with the expression of HPV-16 E6 compared to control. We validated the expression changes of many individual lncRNAs after the expression of HPV-16E6, via q-RT-PCR and Northern blots. In addition we observe similar expression patterns when comparing normal cells to HPV+ cervical cancer cell lines. Currently, we are characterizing specific lncRNAs of interest that are altered with expression of HPV-16 E6 by using 5' and 3' rapid amplification of cDNA ends (RACE) experiments as well as determining certain lncRNA's mechanism of action contributing to cancer by conducting knockdown and rescue experiments followed by behavior assays (e.g. invasion, angiogenesis, and proliferation). To aid in the determination of the function of the lncRNAs, we plan to use Fluorescence in situ Hybridization (FISH) assays to identify their localization. We believe this project will be significant in that it will identify and show the importance of lncRNAs in HPV-related cancers as well as give us new information about a novel mechanism by which high-risk HPV infection contributes to cervical and HNSCC carcinogenesis. Citation Format: Jamie A. Barr, Karen E. Hayes, Ivan Martinez. Importance of long noncoding RNAs in human papillomavirus-related cancers. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A26.

Structural Insights into a Wildtype Domain of the Oncoprotein E6 and Its Interaction with a PDZ Domain

The high-risk human papilloma virus (HPV) oncoproteins E6 and E7 interact with key cellular regulators and are etiological agents for tumorigenesis and tumor maintenance in cervical cancer and other malignant conditions. E6 induces degradation of the tumor suppressor p53, activates telomerase and deregulates cell polarity. Analysis of E6 derived from a number of high risk HPV finally yielded the first structure of a wild-type HPV E6 domain (PDB 2M3L) representing the second zinc-binding domain of HPV 51 E6 (termed 51Z2) determined by NMR spectroscopy. The 51Z2 structure provides clues about HPV-type specific structural differences between E6 proteins. The observed temperature sensitivity of the well-folded wild-type E6 domain implies a significant malleability of the oncoprotein in vivo. Hence, the structural differences between individual E6 and their malleability appear, together with HPV type-specific surface exposed side-chains, to provide the structural basis for the different interaction networks reported for individual E6 proteins. Furthermore, the interaction of 51Z2 with a PDZ domain of hDlg was analyzed. Human Dlg constitutes a prototypic representative of the large family of PDZ proteins regulating cell polarity, which are common targets of high-risk HPV E6. Nine C-terminal residues of 51Z2 interact with the second PDZ domain of hDlg2. Surface plasmon resonance in conjunction with the NMR spectroscopy derived complex structure (PDB 2M3M) indicate that E6 residues N-terminal to the canonical PDZ-BM of E6 significantly contribute to this interaction and increase affinity. The structure of the complex reveals how residues outside of the classical PDZ-BM enhance the affinity of E6 towards PDZ domains. Such mechanism facilitates successful competition of E6 with cellular PDZ-binding proteins and may apply to PDZ-binding proteins of other viruses as well.

The HPV16 E6 Oncoprotein Causes Prolonged Receptor Protein Tyrosine Kinase Signaling and Enhances Internalization of Phosphorylated Receptor Species

The high-risk human papillomavirus (HPV) E6 proteins are consistently expressed in HPV-associated lesions and cancers. HPV16 E6 sustains the activity of the mTORC1 and mTORC2 signaling cascades under conditions of growth factor deprivation. Here we report that HPV16 E6 activated mTORC1 by enhanced signaling through receptor protein tyrosine kinases, including epidermal growth factor receptor and insulin receptor and insulin-like growth factor receptors. This is evidenced by sustained signaling through these receptors for several hours after growth factor withdrawal. HPV16 E6 increased the internalization of activated receptor species, and the signaling adaptor protein GRB2 was shown to be critical for HPV16 E6 mediated enhanced EGFR internalization and mTORC1 activation. As a consequence of receptor protein kinase mediated mTORC1 activation, HPV16 E6 expression increased cellular migration of primary human epithelial cells. This study identifies a previously unappreciated mechanism by which HPV E6 proteins perturb host-signaling pathways presumably to sustain protein synthesis during the viral life cycle that may also contribute to cellular transforming activities of high-risk HPV E6 proteins.

Abstract 2043: Inhibition of cervical carcinoma cells proliferation by miR-34c

Abstract MicroRNAs (miRNAs) play central roles in controlling cell proliferation, differentiation, and apoptosis. Aberrant miRNA expression is recognized as an important molecular mechanism for human carcinogenesis. Infection with high-risk human papillomaviruses (HPVs) is causally associated with the onset of cervical dysplasia including cancer. Although high-risk HPV infection leads to hyperproliferation and immortalization, it is insufficient to produce a fully transformed phenotype. High-risk HPV E6 proteins (i.e. HPV-16 and 18) inactivate and destroy P53 protein leading to a p53-null phenotype and the subsequent repression of the p53 effector network including pro-apoptotic proteins and cell cycle inhibitors. Microarray analyses in normal and tumor cervical cells have linked only a few miRNAs to cervical cancer. However, no miRNAs have been established in the malignant process that may follow a high-risk HPV infection. Here, we analyze the function of miR-34c (a p53 effector) in HPV-positive cells. Steady-state miR-34c levels are very low in both immortal and tumor HPV-positive cells. Over-expression of miR-34c in cervical carcinoma cells caused inhibition of cell proliferation and anchorage independent growth resulting in G2/M arrest and significant apoptosis. Interestingly, no effect was noticed in HPV-negative tumor cells and HPV-immortalized keratinocytes suggesting a differential regulatory pathway of mi-34c in HPV-positive tumor cells. Analyses of miR-34c potential targets showed no significant effect on BCL-2 levels although it produced MAP-2 down-regulation. These results suggest that activation of miR-34c in the absence of functional p53 may play an important role in proliferation and apoptosis of HPV-positive cervical tumor cells that could be used as therapeutic or diagnostic/prognostic tool for cervical cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2043.

Correlation between high-risk HPV E6 and telomerase in cervical cancer

High-risk human papillomavirus (HPV) infection is a major cause of cervical cancer. The high telomerase activity of cervical cancer cells is closely related with high-risk HPV infection. E6 is the main oncogene of high-risk HPV, which can activate telomerase, and degradate and inactivate P53, furthermore E6 can cause a number of other tumor suppressor genes abnormality, and oncogenes activation. Cells immortalization resulted from telomerase activation caused by high-risk HPV E6 is a key point for tumorigenesis in cervix. Key words: Human papillomavirus ; Oncogene proteins ; Tolemerase ; Cervix neoplasms ;

A Magnetic Immunochromatographic Strip Test for Detection of Human Papillomavirus 16 E6

Cervical cancer kills 230 000 women annually. Low-resource regions of the world are disproportionately burdened with 80% of the cases. Efficient screening methods are the key to decreasing the death toll from this disease. High-risk human papillomavirus (HPV) types have been identified as the etiological agent for >99% of cervical cancers. Infection with HPV is ubiquitous and is often resolved by the host. High-risk HPV infections leading to cervical cancer require the production of both HPV E6 and E7 oncoproteins (1)(2)(3)(4)(5). Thus, an assay capable of detecting high-risk HPV E6 from cervical swab samples may have a high positive predictive value and may help to accurately identify women at increased risk of progression to cancer. Internal studies have shown that cervical cancer cell lines produce ∼1 ng of E6 per 1 000 000 cells (data not shown). Work is continuing to quantify E6 amounts in cervical cell samples from clients with cancer, high-grade lesions (cervical intraepithelial neoplasia 2/3), and low-grade lesions (cervical intraepithelial neoplasia 1). Protein determinations performed on provider-collected cervical swabs indicate that the swabs collect 500 000 …

The hScrib/Dlg apico-basal control complex is differentially targeted by HPV-16 and HPV-18 E6 proteins

The E6 proteins of the high-risk Human papillomaviruses (HPV) types have a well-documented ability to target certain cellular proteins for ubiquitin-mediated degradation via the proteasome. Previous studies have shown that E6 proteins interact differently with different target proteins, and that the viral proteins, depending upon the target, may recruit diverse cellular ubiquitin-protein ligases. In this study, we have examined the abilities of E6 proteins from HPV-16 and HPV-18 to interact with and induce the degradation of two PDZ domain-containing targets, Dlg and hScrib. We have also mapped the binding site of E6 on hScrib and shown that the interaction of E6 with hScrib is distinct from its interactions with other PDZ domain-containing targets. This is reflected in the efficiency with which the two viral E6 proteins can inhibit hScrib's suppression of cell transformation.Dlg and hScrib have complementary activities in the control of epithelial cell polarity and the fact that both are targeted by high-risk HPV E6 proteins underlines their importance. Our finding that they are each targeted differently by HPV-16 and HPV-18 E 6 s suggests that the two viruses are subjected to somewhat different constraints and provides a possible explanation for the apparent redundancy in targeting both parts of this important control mechanism.

Centrosomes, genomic instability, and cervical carcinogenesis.

High-risk human papillomavirus (HPV)-associated carcinogenesis of the uterine cervix is a particularly useful model to study basic mechanisms of genomic instability in cancer. Cervical carcinogenesis is associated with the expression of two high-risk HPV-encoded oncoproteins, E6 and E7. Aneuploidy, the most frequent form of genomic instability in human carcinomas, develops as early as in nonmalignant cervical precursor lesions. In addition, cervical neoplasia is frequently associated with abnormal multipolar mitotic figures, suggesting disturbances of the cell-division process as a mechanism for chromosome segregation defects. Spindle poles are formed by centrosomes, and the high-risk HPV E6 and E7 oncoproteins can each induce abnormal centrosome numbers. These two HPV oncoproteins, however, induce centrosome abnormalities through fundamentally different mechanisms and, presumably, with different functional consequences. High-risk HPV E7, which targets the pRB tumor suppressor pathway, can provoke abnormal centrosome duplication in phenotypically normal cells. On the contrary, cells expressing the HPV E6 oncoprotein, which inactivates p53, accumulate abnormal numbers of centrosomes in parallel with multinucleation and nuclear atypia. These two pathways are not mutually exclusive, since co-expression of HPV E6 and E7 has synergistic effects on centrosome abnormalities and chromosomal instability. Taken together, these findings support the general model in which chromosomal instability arises as a direct consequence of oncogenic insults and can develop at early stages of tumor progression.

Oncogenic human papillomavirus E6 proteins target the MAGI-2 and MAGI-3 proteins for degradation.

The E6 proteins from the high-risk human papillomavirus (HPV) types have previously been shown to target a number of PDZ domain-containing proteins for proteasome-mediated degradation. These include the hDlg tumour suppressor and the MAGI-1 protein. In this study we show that high-risk HPV E6 proteins also target the related MAGI-2 and MAGI-3 proteins for degradation. Moreover, we show that the interaction is specific to one PDZ domain, and that co-expression of this domain can protect each of the full-length MAGI proteins from E6-mediated degradation. These data provide clear indicators for the potential design of compounds that could specifically inhibit the interaction of oncogenic HPV E6 proteins with an important class of target proteins.

Interactions of the PDZ-protein MAGI-1 with adenovirus E4-ORF1 and high-risk papillomavirus E6 oncoproteins.

The oncoproteins of small DNA tumor viruses promote tumorigenesis by complexing with cellular factors intimately involved in the control of cell proliferation. The major oncogenic determinants for human adenovirus type 9 (Ad9) and high-risk human papillomaviruses (HPV) are the E4-ORF1 and E6 proteins, respectively. These seemingly unrelated viral oncoproteins are similar in that their transforming activities in cells depend, in part, on a carboxyl-terminal PDZ domain-binding motif which mediates interactions with the cellular PDZ-protein DLG. Here we demonstrated that both Ad9 E4-ORF1 and high-risk HPV E6 proteins also bind to the DLG-related PDZ-protein MAGI-1. These interactions resulted in MAGI-1 being aberrantly sequestered in the cytoplasm by the Ad9 E4-ORF1 protein or being targeted for degradation by high-risk HPV E6 proteins. Transformation-defective mutant viral proteins, however, were deficient for these activities. Our findings indicate that MAGI-1 is a member of a select group of cellular PDZ proteins targeted by both adenovirus E4-ORF1 and high-risk HPV E6 proteins and, in addition, suggest that the tumorigenic potentials of these viral oncoproteins depend, in part, on an ability to inhibit the function of MAGI-1 in cells.

Human scribble (Vartul) is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6AP ubiquitin-protein ligase.

The high-risk human papillomavirus (HPV) E6 proteins stimulate the ubiquitination and degradation of p53, dependent on the E6AP ubiquitin-protein ligase. Other proteins have also been shown to be targeted for degradation by E6, including hDlg, the human homolog of the Drosophila melanogaster Discs large (Dlg) tumor suppressor. We show here that the human homolog of the Drosophila Scribble (Vartul) (hScrib) tumor suppressor protein is also targeted for ubiquitination by the E6-E6AP complex in vitro and that expression of E6 induces degradation of hScrib in vivo. Characterization of the E6AP-E6-hScrib complex indicated that hScrib binds directly to E6 and that the binding is mediated by the PDZ domains of hScrib and a carboxyl-terminal epitope conserved among the high-risk HPV E6 proteins. Green fluorescent protein-hScrib was localized to the periphery of MDCK cells, where it colocalized with ZO-1, a component of tight junctions. E6 expression resulted in loss of integrity of tight junctions, as measured by ZO-1 localization, and this effect was dependent on the PDZ binding epitope of E6. Thus, the high-risk HPV E6 proteins induce the degradation of the human homologs of two Drosophila PDZ domain-containing tumor suppressor proteins, hDlg and hScrib, both of which are associated with cell junction complexes. The fact that Scrib/Vart and Dlg appear to cooperate in a pathway that controls Drosophila epithelial cell growth suggests that the combined targeting of hScrib and hDlg is an important component of the biologic activity of high-risk HPV E6 proteins.

The E6 oncoproteins of high-risk papillomaviruses bind to a novel putative GAP protein, E6TP1, and target it for degradation.

The high-risk human papillomaviruses (HPVs) are associated with carcinomas of the cervix and other genital tumors. Previous studies have identified two viral oncoproteins, E6 and E7, which are expressed in the majority of HPV-associated carcinomas. The ability of high-risk HPV E6 protein to immortalize human mammary epithelial cells (MECs) has provided a single-gene model to study the mechanisms of E6-induced oncogenic transformation. In this system, the E6 protein targets the p53 tumor suppressor protein for degradation, and mutational analyses have shown that E6-induced degradation of p53 protein is required for MEC immortalization. However, the inability of most dominant-negative p53 mutants to induce efficient immortalization of MECs suggests the existence of additional targets of the HPV E6 oncoprotein. Using the yeast two-hybrid system, we have isolated a novel E6-binding protein. This polypeptide, designated E6TP1 (E6-targeted protein 1), exhibits high homology to GTPase-activating proteins for Rap, including SPA-1, tuberin, and Rap1GAP. The mRNA for E6TP1 is widely expressed in tissues and in vitro-cultured cell lines. The gene for E6TP1 localizes to chromosome 14q23.2-14q24.3 within a locus that has been shown to undergo loss of heterozygosity in malignant meningiomas. Importantly, E6TP1 is targeted for degradation by the high-risk but not the low-risk HPV E6 proteins both in vitro and in vivo. Furthermore, the immortalization-competent but not the immortalization-incompetent HPV16 E6 mutants target the E6TP1 protein for degradation. Our results identify a novel target for the E6 oncoprotein and provide a potential link between HPV E6 oncogenesis and alteration of a small G protein signaling pathway.