E6 Oncoproteins Research Articles

The Dimeric Form of HPV16 E6 Is Crucial to Drive YAP/TAZ Upregulation through the Targeting of hScrib.

Simple SummaryUnderstanding the mechanisms of action of HPV oncoproteins is pivotal for the rationale development of anti-cancer drugs to treat HPV-related malignancies. The aim of the present study was to explore more in detail the mechanism of action of the HPV16 oncoprotein E6 that directly fosters the YAP/TAZ signaling pathway, a conserved cascade highly active in HPV-related cancers. We confirmed previous evidence about the importance of the PDZ-protein targeting in this process, highlighting here the importance of hScrib degradation, and discovered that the targeting of the Scribble module involves the dimeric form of HPV16 E6. The findings here presented extend our knowledge about the mechanism through which the oncoprotein E6 targets a PDZ-host factor to degradation in cancer cells.Human papillomavirus is the most common viral infectious agent responsible for cancer development in humans. High-risk strains are known to induce cancer through the expression of the viral oncogenes E6 and E7, yet we have only a partial understanding of the precise mechanisms of action of these viral proteins. Here we investigated the molecular mechanism through which the oncoprotein E6 alters the Hippo-YAP/TAZ pathway to trigger YAP/TAZ induction in cancer cells. By employing E6 overexpression systems combined with protein–protein interaction studies and loss-of-function approaches, we discovered that the E6-mediated targeting of hScrib, which supports YAP/TAZ upregulation, intimately requires E6 homodimerization. We show that the self-association of E6, previously reported only in vitro, takes place in the cytoplasm and, as a dimer, E6 targets the fraction of hScrib at the cell cortex for proteasomal degradation. Thus, E6 homodimerization emerges as an important event in the mechanism of E6-mediated hScrib targeting to sustain downstream YAP/TAZ upregulation, unraveling for the first time the key role of E6 homodimerization in the context of its transforming functions and thus paving the way for the possible development of E6 dimerization inhibitors.

Molecular and clinical aspects of oropharyngeal squamous cell carcinoma associated with human papillomavirus

Currently, the role of human papillomavirus (HPV) in carcinogenesis is well known: more than 90 % of HPV-positive oropharyngeal squamous cell carcinomas are caused by HPV type 16 (HPV-16). HPV E6 and E7 oncoproteins play a significant role in the development of this tumor. The E6- mediated degradation of suppressor protein p53 results in G2/M-phase checkpoint dysregulation and inhibition of apoptosis. HPV oncoprotein E7 binds to pRb, promoting its degradation and the release of E2F transcription factor. Diagnostic assays for HPV detection include immunohistochemical staining for p16, polymerase chain reaction, in situ hybridization, and next-generation sequencing. Immunohistochemical examination (determination of p16 protein expression) is an economical and very specific way to detect a viral infection. Patients with HPV-positive oropharyngeal squamous cell carcinoma demonstrate significantly better response to treatment and overall survival rates than those with HPV-negative oropharyngeal squamous cell carcinoma. Despite the fact that five-year overall survival rate in patients with HPV-positive oropharyngeal squamous cell carcinoma after treatment exceeds 80 %, some patients have poor survival. Unfortunately, currently available methods of risk stratification still do not endure their timely identification. Further research is needed to address these problems.

Molecular Landscape and Computational Screening of the Natural inhibitors against HPV16 E6 Oncoprotein.

Background:Human Papillomavirus (HPV) is a small, non-enveloped, icosahedral and double-stranded DNA virus with a genome of 8 kb, belonging to the papillomaviridae family. HPV has been associated with 99.7% cases of cervical squamous cell carcinoma worldwide. The HPV E6 protein is known as a potent oncogene and is closely allied with the events that result in the malignant transformation of virally infected cells. Objective:The present study aims to target plant derived anticancer molecules for HPV driven cancer using a computational approach. Methods: In this study, E6 oncoprotein was targeted by 101 plant-derived nutraceuticals using the molecular docking method. The multiple sequence analysis and phylogenetic analysis of low risk and high risk 28 HPV E6 proteins were performed. Results:Withanolide D, Ginkgetin, Theaflavin, Hesperidin, and Quercetin-3-gluconide were identified as the potential inhibitors of HPV 16 E6 protein. The zinc finger domain was identified on all variants of HPV E6 oncoprotein while high-risk HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV58, HPV68 and HPV73: probable risk HPV53 and low-risk HPV43 and HPV70 contain PDZ domain. Conclusion:The current study using bioinformatics analysis approaches reveals a promising platform for developing anti-cancerous competitive inhibitors targeting HPV.

In Silico Approaches: A Way to Unveil Novel Therapeutic Drugs for Cervical Cancer Management.

Cervical cancer (CC) is the fourth most common pathology in women worldwide and presents a high impact in developing countries due to limited financial resources as well as difficulties in monitoring and access to health services. Human papillomavirus (HPV) is the leading cause of CC, and despite the approval of prophylactic vaccines, there is no effective treatment for patients with pre-existing infections or HPV-induced carcinomas. High-risk (HR) HPV E6 and E7 oncoproteins are considered biomarkers in CC progression. Since the E6 structure was resolved, it has been one of the most studied targets to develop novel and specific therapeutics to treat/manage CC. Therefore, several small molecules (plant-derived or synthetic compounds) have been reported as blockers/inhibitors of E6 oncoprotein action, and computational-aided methods have been of high relevance in their discovery and development. In silico approaches have become a powerful tool for reducing the time and cost of the drug development process. Thus, this review will depict small molecules that are already being explored as HR HPV E6 protein blockers and in silico approaches to the design of novel therapeutics for managing CC. Besides, future perspectives in CC therapy will be briefly discussed.

In SilicoDesign and Immunological Studies of Two Novel Multiepitope DNA-Based Vaccine Candidates Against High-Risk Human Papillomaviruses.

Human papillomaviruses (HPV)-16 and 18 are the most prevalent types associated with cervical cancer. HPV L1 and L2 capsid proteins and E7 oncoprotein play crucial roles in HPV-related diseases. Hence, these proteins were proposed as target antigens for preventive and therapeutic vaccines. In this study, two multiepitope DNA-based HPV vaccine candidates were designed using in silico analysis including the immunogenic and conserved epitopes of HPV16/18 L1, L2 and E7 proteins (the L1-L2-E7 fusion DNA), and of heat shock protein 70 (HSP70) linked to the L1-L2-E7 DNA construct (the HSP70-L1-L2-E7 fusion DNA). Next, the expression of the L1-L2-E7 and HSP70-L1-L2-E7 multiepitope DNA constructs was evaluated in a mammalian cell line. Finally, immunological responses and antitumor effects of the DNA constructs were investigated in C57BL/6 mice. Our data indicated high expression rates of the designed multiepitope L1-L2-E7 DNA (~ 56.16%) and HSP70-L1-L2-E7 DNA (~ 80.45%) constructs in vitro. The linkage of HSP70 epitopes to the L1-L2-E7 DNA construct significantly increased the gene expression. Moreover, the HSP70-L1-L2-E7 DNA construct could significantly increase immune responses toward Th1 response and CTL activity, and induce stronger antitumor effects in mouse model. Thus, the designed HSP70-L1-L2-E7 DNA construct represents promising results for development of HPV DNA vaccine candidates.

CRISPR/Cas9-based inactivation of human papillomavirus oncogenes E6 or E7 induces senescence in cervical cancer cells

Human papillomaviruses (HPVs) such as HPV16 and HPV18 can cause cancers of the cervix, anogenital and oropharyngeal sites. Continuous expression of the HPV oncoproteins E6 and E7 are essential for transformation and maintenance of cancer cells. Therefore, therapeutic targeting of E6 or E7 genes can potentially treat HPV-related cancers. Here we report that CRISPR/Cas9-based knockout of E6 or E7 can trigger cellular senescence in HPV18 immortalized HeLa cells. Specifically, E6 or E7-inactivated HeLa cells exhibited characteristic senescence markers like enlarged cell surface area, increased β-galactosidase expression and loss of lamin B1. Since E6 and E7 are bicistronic transcripts, inactivation of HPV18 E6 resulted in knockout of both E6 and E7 and increasing levels of p53/p21 and pRb/p21, respectively. Knockout of HPV18 E7 resulted in decreased E6 expression with activation of pRb/p21 pathway. Taken together, our study demonstrates cellular senescence as an alternative outcome of HPV oncogene inactivation by CRISPR/Cas9.

Selection of Indonesian Medicinal Plant Active Compounds as Inhibitor Candidates of Oncoproteins E6 and E7 Human Papillomavirus Type 16 by Molecular Docking

Cervical cancer cases caused by infection with Human Papillomavirus (HPV), especially HPV 16 (60.5% of cases) continue to increase every year with a high mortality rate. The current anti-cancer drugs were not only specifically targeting cancer cells, but healthy cells and can cause serious side effects. Therefore, it is necessary to find safer alternative therapies, e.g., using active compounds from natural products. The purpose of this study was to find the active compounds of Indonesian medicinal plants potentially as an inhibitor of oncoprotein E6 and E7 HPV 16, the main protein causing cervical cancer by in silico method. In this study, 711 active compounds from 187 medicinal plant species were selected based on molecular weight, solubility, gastrointestinal absorption index, and drug-likeness. Compounds that meet the criteria were tested for their affinity and interaction profile with E6 and E7 proteins through the molecular docking method. The results of this study showed 164 compounds that met the criteria. The molecular docking analysis showed nine of the most potent compounds as E6 inhibitors on the E6AP binding site and six compounds on the p53 binding site. Besides that, there were eleven most potent compounds as E7 inhibitors. The results of this study indicate that there are natural compounds that can inhibit E6 and E7 proteins and have further potential to be used as anti-HPV drugs. However, further research is needed to test these compounds in vitro and in vivo.

Serum HPV16 E7 Oncoprotein Is a Recurrence Marker of Oropharyngeal Squamous Cell Carcinomas

Simple SummaryClassical markers alone, such as HPV DNA, p16 and HPV mRNA expression, are not enough to stratify HPV-positive head and neck squamous cell carcinoma (HNSCC) patients, but when combined with serological markers, the latter are strong indicators of prognosis in oropharyngeal squamous cell carcinoma (OPSCC) patients. Specifically, HPV16 E7 oncoprotein in serum at the time of diagnosis, correlates with disease recurrence and patient overall survival. To our knowledge, this is the first study to investigate HPV E7 oncoprotein in patient serum. The E7 oncoprotein detection in serum at the time of diagnosis may be useful as a non-invasive procedure for HPV-positive OPSCC patient stratification and follow-up, helping to identify patients at risk for tumor recurrence and metastasis during follow-up, and ultimately, providing a tool for clinicians to identify patients for de-escalation treatment or those to be kept under close surveillance.Despite improved prognosis for many HPV-positive head and neck squamous cell carcinomas (HNSCCs), some cases are still marked by recurrence and metastasis. Our study aimed to identify novel biomarkers for patient stratification. Classical HPV markers: HPV-DNA, p16 and HPV mRNA expression were studied in HNSCC (n = 67) and controls (n = 58) by qPCR. Subsequently, ELISA tests were used for HPV16 L1 antibody and HPV16 E7 oncoprotein detection in serum at diagnosis and follow-up. All markers were correlated to relapse-free survival (RFS) and overall survival (OS). HPV-DNA was found in HNSCCs (29.85%), HPV16-DNA in 95% of cases, HPV16 E7 mRNA was revealed in 93.75%. p16 was overexpressed in 75% of HPV-positive HNSCC compared to negative samples and controls (p < 0.001). Classical markers correlated with improved OS (p < 0.05). Serological studies showed similar proportions of HPV16 L1 antibodies in all HNSCCs (p > 0.05). Serum E7 oncoprotein was present in 30% HPV-positive patients at diagnosis (p > 0.05) and correlated to HNSCC HPV16 E7 mRNA (p < 0.01), whereas it was associated to worse RFS and OS, especially for oropharyngeal squamous cell carcinoma (OPSCC) (p < 0.01). Detection of circulating HPV16 E7 oncoprotein at diagnosis may be useful for stratifying and monitoring HPV-positive HNSCC patients for worse prognosis, providing clinicians a tool for selecting patients for treatment de-escalation.

Abstract 1210: NFX1-123: A potential therapeutic target in cancer

Abstract Background: NFX1-123 is the longer splice variant isoform of the NFX1 gene and is highly expressed in cervical cancer. Cervical cancer is caused by high-risk HPV infections, and NFX1-123 is a protein partner with the HPV oncoprotein E6. Together, NFX1-123 and E6 affect cellular growth, longevity, differentiation, and the immune response. The expression status of NFX1-123 in cancers beyond cervical cancer, and its potential as therapeutic target, has not been investigated. Methods: TSVdb of TCGA was used to quantify NFX1-123 expression in 25 primary cancers tissues compared to adjacent normal tissues. The NFX1-123 protein structure was predicted using I-TASSER: Interactive Threading ASSEmbly Refinement tool. The modeled structure was submitted to the MTi-Openscreen Virtual screening web-server using ZINC-Database to retrieve suitable drug molecules, and further screening was evaluated by PyRx interphase and AutoDock Vina. The top four compounds, found to bind in silico to NFX1-123, were tested experimentally to determine their inhibitory effects on NFX1-123-related cellular growth and survival by MTT assay. Results: 44% of cancers (11 of 25) had significant differences in NFX1-123 expression when compared to adjacent normal solid tissues. Nine of 11 cancers (88%) had greater NFX1-123 expression: breast invasive carcinoma, cholangiocarcinoma, colon adenocarcinoma, renal cell carcinoma, renal papillary cell carcinoma, hepatocellular carcinoma, lung adenocarcinoma, sarcoma, and stomach adenocarcinoma. Two of 11 cancers (18%) had reduced NFX1-123 expression: lung squamous cell carcinoma, and pheochromocytoma and paraganglioma. Additionally, HPV+ head and neck cancers had greater expression of NFX1-123 compared to HPV- head and neck cancers. Bioinformatics and proteomic predictive analysis revealed the 3-D structure of the NFX1-123; with this structure, drug libraries were screened for high binding affinity compounds. 17 drugs with binding energies range from -11.3 to -10 Kcal/mol. were found. The top four compounds were used to treat HPV- and HPV+ cervical and head and neck cancer cell lines in culture, and two (R428 and Ketoconazole) were found to reduce NFX1-123 protein levels and inhibit cell growth and survival. R428 was also found to inhibit NFX1-123 and regulate autophagy and autophagy-mediated cell death. Conclusion: Nine out of 25 (36%) cancers expressed high levels of NFX1-123, and drug targeting of NFX1-123 can lead to cell growth inhibition. NFX1-123 may be a potential novel therapeutic target in cancers that highly express NFX1-123. Citation Format: Sreenivasulu Chintala, Anand Anbarasu, Rachel A. Katzenellenbogen. NFX1-123: A potential therapeutic target in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1210.

Human Papillomavirus (HPV)–Specific T-Cells Can be Generated from Unimmunized Donors for Third Party Cell Therapy of HPV-Associated Neoplasms

▪HPV is a known cause of oropharyngeal cancer, cervical cancer, and other genital neoplasms. Although treatments exist for HPV-associated malignancies, patients unresponsive to these therapies have a poor prognosis. Recent findings from vaccine studies suggest that T cell immunity is essential for disease control. As “off the shelf” third party EBV-specific T-cells have been successful in treating patients with EBV-associated tumors, we hypothesized that the development of a manufacturing platform for HPV-specific T cells from healthy donors could be used in a third party setting to treat patients with HPV-associated cancers. Most protocols for generating virus-specific T-cells require prior exposure of the donor to the targeted virus. Since the seroprevalence of high-risk HPV types varies greatly by age and ethnicity, manufacturing of donor-derived HPV-specific T cells is challenging. Hence, we implemented systematic changes to GMP-compliant protocols to improve antigen presentation, priming, and expansion for the manufacture of HPV-specific T cells.By optimizing dendritic cell maturation and function using lipopolysaccharide (LPS) and IFNg, and adding IL-21 for priming, we achieved reliable expansion of naïve T-cells specific for oncoproteins E6 and E7 of the HPV16 serotype to clinically relevant numbers (mean 578-fold expansion, n=10). Ex vivo expanded, HPV-specific T cells secreted IFNγ (137±148 IFNγ spot forming cells, SFC/1x105 cells and 68±145 IFNγ SFC/1x105cells against HPV E6 and E7 respectively, compared to 7±8 IFNγ SFC/1x105 cells against an irrelevant antigen, n=10) and other cytokines (in five evaluated products,T cells secreted high levels of IL6 (3792±966 pg/mL), IL7 (18000±0 pg/mL), IL8 (4766 ±2271 pg/mL), IP10 (234 ±220pg/mL), MIP1b (4915.848±2050.065 pg/mL) and TNFa (1945±823pg/mL). In contrast to T cells directed against other viral antigens, most T cells expanded against HPV antigens were CD4+ with a mean of 68.6±23.3% vs 18.2±20.9% CD8+ T cells (n=10). Expanded populations primarily recognized antigens through MHC class II confirmed by a decrease in responses to HPV antigen in the presence of HLA class II blocking antibodiesIn summary, we successfully generated HPV16 specific T cells from healthy donors, and established the conditions for a robust and reproducible. GMP-compliant protocol using this product as an off the shelf T cell immunotherapeutic for patients with HPV-positive malignancies. Other groups have previously developed T cell therapies for HPV targets, but most efforts have focused on generating autologous products for patients with HPV malignancies. Other studies using TCR gene modified T cells have been developed, but the strategy limits the HLA types that can be targeted. Since immune dysfunction frequently accompanies patients with HPV malignancies, the development of an “off the shelf” third-party HPV-specific T-cell product potentially represents a readily available effective cell therapeutic for patients with HPV-associated cancers. DisclosuresBollard:Torque: Membership on an entity's Board of Directors or advisory committees; Cellectis: Membership on an entity's Board of Directors or advisory committees; Neximmune: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Vitamin A deficiency in K14E7HPV expressing transgenic mice facilitates the formation of malignant cervical lesions.

Infection with high-risk human papillomavirus (HR-HPV) is the main cause of cervical cancer (CC), but viral infection alone does not guarantee the development of this malignancy. Indeed, deficiencies of dietary micronutrients could favor cervical cancer development in individuals that harbor HR-HPV infections. The status of retinoid levels, natural and synthetic derivatives of vitamin A, is important in maintaining cellular differentiation of the cervical epithelium. Moreover, many studies show a link between deficient intake of retinoids or alteration of the retinoid receptors and CC development. In spite of this, the effect of vitamin A deficiency (VAD) in presence of HR-HPV oncoproteins on cervical carcinogenesis in vivo has not been reported. Transgenic mice expressing E6 or E7 oncoproteins (K14E6 or K14E7 mice, respectively) were used to evaluate the possible role of VAD in the development of malignant cervical lesions. The survival of the mice in VAD condition was studied, and histopathological analysis and immunohistochemical detection of molecular cancer markers such as the tumor suppressor retinoic acid receptor beta (RARβ), proliferating cell nuclear antigen (PCNA), cleaved caspase 3, and the tumor suppressor protein p16INK4A (inhibitor of CDK4) were performed. Our results show that K14E6/VAD mice showed moderate cervical dysplasia; notably, K14E7/VAD mice developed severe cervical dysplasia and cervical in situ carcinoma at an early age. VAD synergizes with HPV16E7 oncoprotein expression favoring cervical carcinogenesis in vivo.

Elucidation of rutin's role in inducing caspase-dependent apoptosis via HPV-E6 and E7 down-regulation in cervical cancer HeLa cells.

Over the recent few years rutin has gained wider attention in exhibiting inhibitory potential against several oncotargets for inducing apoptotic and antiproliferative activity in several human cancer cells. Several deregulated signaling pathways are implicated in cancer pathogenesis. Therefore we have inclined our research towards exploring the anticancerous efficacy of a very potent phytocompound for modulating the incontinent expression of these two crucial E6 and E7 oncogenes. Further, inhibitory efficacy of rutin against human papillomavirus (HPV)-E6 and E7 oncoproteins in cervical cancer has not been elucidated yet. This research addresses the growth inhibitory efficacy of rutin against E6 and E7 oncoproteins in HeLa cells, which is known to inactivate several tumor suppressor proteins such as p53 and pRB. Rutin treatment exhibited reduced cell viability with increased cell accumulation in G0/G1 phase of cell cycle in HeLa cell lines. Additionally, rutin treatment has also led to down-regulation of E6 and E7 expression associated with an increased expression of p53 and pRB levels. This has further resulted in enhanced Bax expression and decreased Bcl-2 expression releasing cytochrome c into cytosol followed by caspase cascade activation with cleavage of caspase-3, caspase-8 and caspase-9. Further, in silico studies have also supported our in vitro findings by exhibiting significant binding energy against selected target oncoproteins. Therefore, our research findings might recommend rutin as one of the potent drug candidate in cervical cancer management via targeting two crucial oncoproteins associated with viral progression.

E6/E7 Variants of Human Papillomavirus 16 Associated with Cervical Carcinoma in Women in Southern Mexico.

Persistent infection with the human papillomavirus 16 (HPV 16) is the cause of half of all cervical carcinomas (CC) cases. Moreover, mutations in the oncoproteins E6 and E7 are associated with CC development. In this study, E6/E7 variants circulating in southern Mexico and their association with CC and its precursor lesions were evaluated. In total, 190 DNA samples were obtained from scrapes and cervical biopsies of women with HPV 16 out of which 61 are from patients with CC, 6 from patients with high-grade squamous intraepithelial lesions (HSIL), 68 from patients with low-grade squamous intraepithelial lesions (LSIL), and 55 from patients without intraepithelial lesions. For all E7 variants found, the E7-C732/C789/G795 variant (with three silent mutations) was associated with the highest risk of CC (odd ratio (OR) = 3.79, 95% confidence interval (CI) = 1.46–9.85). The analysis of E6/E7 bicistron conferred to AA-a*E7-C732/C789/G795 variants revealed the greatest increased risk of CC (OR = 110, 95% CI = 6.04–2001.3), followed by AA-c*E7-C732/C789/G795 and A176/G350*E7-p. These results highlight the importance of analyzing the combinations of E6/E7 variants in HPV 16 infection and suggest that AA-a*E7-C732/C789/G795, AA-c*E7-C732/C789/G795, and A176/G350*E7-p can be useful markers for predicting CC development.

Expression of the E5 Oncoprotein of HPV16 Impacts on the Molecular Profiles of EMT-Related and Differentiation Genes in Ectocervical Low-Grade Lesions.

Infection with human papillomavirus type 16 (HPV16) is one of the major risk factors for the development of cervical cancer. Our previous studies have demonstrated the involvement of the early oncoprotein E5 of HPV16 (16E5) in the altered isoform switch of fibroblast growth factor receptor 2 (FGFR2) and the consequent expression in human keratinocytes of the mesenchymal FGFR2c isoform, whose aberrant signaling leads to EMT, invasiveness, and dysregulated differentiation. Here, we aimed to establish the possible direct link between these pathological features or the appearance of FGFR2c and the expression of 16E5 in low-grade squamous intraepithelial lesions (LSILs). Molecular analysis showed that the FGFR2c expression displayed a statistically significant positive correlation with that of the viral oncoprotein, whereas the expression values of the epithelial FGR2b variant, as well as those of the differentiation markers keratin 10 (K10), loricrin (LOR) and involucrin (INV), were inversely linked to the 16E5 expression. In contrast, the expression of EMT-related transcription factors Snail1 and ZEB1 overlapped with that of 16E5, becoming a statistically significant positive correlation in the case of Snail2. Parallel analysis performed in human cervical LSIL-derived W12 cells, containing episomal HPV16, revealed that the depletion of 16E5 by siRNA was able to counteract these molecular events, proving to represent an effective strategy to identify the specific role of this viral oncoprotein in determining LSIL oncogenic and more aggressive profiles. Overall, coupling in vitro approaches to the molecular transcript analysis in ectocervical early lesions could significantly contribute to the characterization of specific gene expression profiles prognostic for those LSILs with a greater probability of direct neoplastic progression.

Interpretations on the Interaction between Protein Tyrosine Phosphatase and E7 Oncoproteins of High and Low-Risk HPV: A Computational Perception.

The most prevalent and common sexually transmitted infection is caused by human papillomavirus (HPV) among sexually active women. Numerous genotypes of HPV are available, among which the major oncoproteins E6 and E7 lead to the progression of cervical cancer. The E7 oncoprotein interacts with cytoplasmic tumor suppressor protein PTPN14, which is the key regulator of cellular growth control pathways effecting the reduction of steady-state level. Disrupting the interaction between the tumor suppressor and the oncoprotein is vital to cease the development of cancer. Hence, the mechanism of interaction between E7 and tumor suppressor is explored through protein–protein and protein–ligand binding along with the conformational stability studies. The obtained results state that the LXCXE domain of HPV E7 of high and low risks binds with the tumor suppressor protein. Also, the small molecules bind in the interface of E7–PTPN14 that disrupts the interaction between the tumor suppressor and oncoprotein. These results were further supported by the dynamics simulation stating the stability over the bounded complex and the energy maintained during postdocking as well as postdynamics calculations. These observations possess an avenue in the drug discovery that leads to further validation and also proposes a potent drug candidate to treat cervical cancer caused by HPV.

Combo Approaches for HPV16+ Cancers.

Beyond anti-PD-1 therapy as a mainstay, additional strategies are being developed for HPV-associated cancers. Emerging data from two ongoing trials have shown some early efficacy with vaccines directed at HPV16-specific E6 and E7 viral oncoproteins, as well as with other therapies targeting different facets of antitumor immunity.

Design of a multi-epitope vaccine against cervical cancer using immunoinformatics approaches

Cervical cancer, caused by human papillomavirus (HPV), is the fourth most common type of cancer among women worldwide. While HPV prophylactic vaccines are available, they have no therapeutic effects and do not clear up existing infections. This study aims to design a therapeutic vaccine against cervical cancer using reverse vaccinology. In this study, the E6 and E7 oncoproteins from HPV16 were chosen as the target antigens for epitope prediction. Cytotoxic T lymphocytes (CTL) and helper T lymphocytes (HTL) epitopes were predicted, and the best epitopes were selected based on antigenicity, allergenicity, and toxicity. The final vaccine construct was composed of the selected epitopes, along with the appropriate adjuvant and linkers. The multi-epitope vaccine was evaluated in terms of physicochemical properties, antigenicity, and allergenicity. The tertiary structure of the vaccine construct was predicted. Furthermore, several analyses were also carried out, including molecular docking, molecular dynamics (MD) simulation, and in silico cloning of the vaccine construct. The results showed that the final proposed vaccine could be considered an effective therapeutic vaccine for HPV; however, in vitro and in vivo experiments are required to validate the efficacy of this vaccine candidate.

Bovine Delta Papillomavirus E5 Oncoprotein Interacts With TRIM25 and Hampers Antiviral Innate Immune Response Mediated by RIG-I-Like Receptors.

Persistent infection and tumourigenesis by papillomaviruses (PVs) require viral manipulation of various of cellular processes, including those involved in innate immune responses. Herein, we showed that bovine PV (BPV) E5 oncoprotein interacts with a tripartite motif-containing 25 (TRIM25) but not with Riplet in spontaneous BPV infection of urothelial cells of cattle. Statistically significant reduced protein levels of TRIM25, retinoic acid-inducible gene I (RIG-I), and melanoma differentiation-associated gene 5 (MDA5) were detected by Western blot analysis. Real-time quantitative PCR revealed marked transcriptional downregulation of RIG-I and MDA5 in E5-expressing cells compared with healthy urothelial cells. Mitochondrial antiviral signalling (MAVS) protein expression did not vary significantly between diseased and healthy cells. Co-immunoprecipitation studies showed that MAVS interacted with a protein network composed of Sec13, which is a positive regulator of MAVS-mediated RLR antiviral signalling, phosphorylated TANK binding kinase 1 (TBK1), and phosphorylated interferon regulatory factor 3 (IRF3). Immunoblotting revealed significantly low expression levels of Sec13 in BPV-infected cells. Low levels of Sec13 resulted in a weaker host antiviral immune response, as it attenuates MAVS-mediated IRF3 activation. Furthermore, western blot analysis revealed significantly reduced expression levels of pTBK1, which plays an essential role in the activation and phosphorylation of IRF3, a prerequisite for the latter to enter the nucleus to activate type 1 IFN genes. Our results suggested that the innate immune signalling pathway mediated by RIG-I-like receptors (RLRs) was impaired in cells infected with BPVs. Therefore, an effective immune response is not elicited against these viruses, which facilitates persistent viral infection.

Coordinated action of human papillomavirus type 16 E6 and E7 oncoproteins on competitive endogenous RNA (ceRNA) network members in primary human keratinocytes

BackgroundmiRNAs and lncRNAs can regulate cellular biological processes both under physiological and pathological conditions including tumour initiation and progression. Interactions between differentially expressed diverse RNA species, as a part of a complex intracellular regulatory network (ceRNA network), may contribute also to the pathogenesis of HPV-associated cancer. The purpose of this study was to investigate the global expression changes of miRNAs, lncRNAs and mRNAs driven by the E6 and E7 oncoproteins of HPV16, and construct a corresponding ceRNA regulatory network of coding and non-coding genes to suggest a regulatory network associated with high-risk HPV16 infections. Furthermore, additional GO and KEGG analyses were performed to understand the consequences of mRNA expression alterations on biological processes.MethodsSmall and large RNA deep sequencing were performed to detect expression changes of miRNAs, lncRNAs and mRNAs in primary human keratinocytes expressing HPV16 E6, E7 or both oncoproteins. The relationships between lncRNAs, miRNAs and mRNAs were predicted by using StarBase v2.0, DianaTools-LncBase v.2 and miRTarBase. The lncRNA-miRNA-mRNA regulatory network was visualized with Cytoscape v3.4.0. GO and KEEG pathway enrichment analysis was performed using DAVID v6.8.ResultsWe revealed that 85 miRNAs in 21 genomic clusters and 41 lncRNAs were abnormally expressed in HPV E6/E7 expressing cells compared with controls. We constructed a ceRNA network with members of 15 lncRNAs – 43 miRNAs – 358 mRNAs with significantly altered expressions. GO and KEGG functional enrichment analyses identified numerous cancer related genes, furthermore we recognized common miRNAs as key regulatory elements in biological pathways associated with tumorigenesis driven by HPV16.ConclusionsThe multiple molecular changes driven by E6 and E7 oncoproteins resulting in the malignant transformation of HPV16 host cells occur, at least in part, due to the abnormal alteration in expression and function of non-coding RNA molecules through their intracellular competing network.

Proteasomal degradation of p130 facilitate cell cycle deregulation and impairment of cellular differentiation in high-risk Human Papillomavirus 16 and 18 E7 transfected cells.

The High-Risk Human Papillomaviruses (HR-HPVs) 16 and 18 are known to cause cervical cancer, which is primarily attributed to E6 and E7 oncoproteins. In addition, recent studies have focused on the vital role of the p130 pocket protein as an oncosuppressor to limit the expression of E2F transcription factors required for cell cycle progression. In view of this, the current study was conducted to investigate the mechanism by which transfection with HPV16/18 E7 leads to the deregulation of the host cell cycle, altering the localisation of p130, and expression of differentiation genes in Human Keratinocytes (HaCaT) cells. Co-immunoprecipitation, Western blot analysis, immunofluorescence microscopy, flow cytometry, quantitative-Polymerase Chain Reaction (qPCR), and the inhibition of p130 by MG132 inhibitor were employed to investigate the loss of p130 and its disruption in HPV 16/18 E7-transfected HaCaT cells. The HPV16- and HPV18-transformed cells, known as CaSki and HeLa, respectively, were also used to complement the ectopic expressions of E7 in HaCaT cells. Normal keratinocytes displayed higher level of p130 expression than HPV-transformed cells. In addition, the immunofluorescence analysis revealed that both HPV 16/18 E7-transfected HaCaT and HPV-transformed cells exhibited higher level of cytoplasmic p130 compared to nuclear p130. A significant increase in the number of S/G2 phase cells in HPV-transformed cells was also recorded since E7 has been shown to stimulate proliferation through the deactivation of Retinoblastoma Protein (pRB)-dependent G1/S checkpoint. Furthermore, the findings recorded the down-regulation of keratinocyte differentiation markers, namely p130, keratin10, and involucrin. The proteasomal degradation of the exported p130 confirmed the cellular localisation pattern of p130, which was commonly observed in cancerous cells. The findings provide strong evidence that the localisation of nuclear p130 nuclear was disrupted by HPV16/18 E7 led to the deregulation of the cell cycle and the impairment of cellular differentiation ultimately lead to cellular transformation.