Human Papillomavirus E6 Proteins Research Articles

DARPin-induced reactivation of p53 in HPV-positive cells.

Infection of cells with high-risk strains of the human papillomavirus (HPV) causes cancer in various types of epithelial tissue. HPV infections are responsible for ~4.5% of all cancers worldwide. Tumorigenesis is based on the inactivation of key cellular control mechanisms by the viral proteins E6 and E7. The HPV E6 protein interacts with the cellular E3 ligase E6AP, and this complex binds to the p53 DNA-binding domain, which results in degradation of p53. Inhibition of this interaction has the potential to reactivate p53, thus preventing oncogenic transformation. Here we describe the characterization of a designed ankyrin repeat protein that binds to the same site as the HPV E6 protein, thereby displacing the E3 ligase and stabilizing p53. Interaction with the designed ankyrin repeat protein does not affect p53 DNA binding or the crucial MDM2 negative feedback loop but reactivates a p53-dependent transcriptional program in HeLa (HPV18-positive) and SiHa (HPV16-positive) cells, suggesting a potential therapeutic use.

Amino acid substitution patterns in the <i>E6</i> and <i>E7</i> Proteins of HPV type 16: Phylogeography and Evolution

The E6 and E7 proteins of human papillomavirus (HPV) play a key role in the oncogenesis of papillomavirus infection. Data on the variability of these proteins are limited, and the factors affecting their variability are poorly understood. We analyzed the variability of the currently known sequences of HPV type 16 (HPV16) E6 and E7 proteins, taking into account their geographic origin and year of sample collection, as well as the direction of their evolution in major geographic regions of the world. All sequences belonging to HPV16 genome fragments encoding E6 and E7 oncoproteins were downloaded from the NCBI GenBank database on October 6, 2022. Samples were filtered according to the following parameters: the sequence includes at least one of the two whole open reading frames, the collection date and the country of origin are known. A total of 3,651 full-genome nucleotide sequences encoding the E6 protein and 4,578 full-genome nucleotide sequences encoding the E7 protein were sampled. The nucleotide sequences obtained after sampling and alignment were converted to amino acid sequences and analyzed using MEGA11, R, RStudio, Jmodeltest 2.1.20, BEAST v1.10.4, Fastcov, and Biostrings software. The highest variability in E6 protein structure was recorded at positions 17, 21, 32, 85, and 90, while in E7, positions 28, 29, 51, and 77 were the most variable. The samples were divided geographically into 5 heterogeneous groups: African, European, American, Southwest and South Asia and Southeast Asia. Unique amino acid substitutions (AA-substitutions) in the E6/E7 proteins of HPV16, presumably characteristic of certain ethnic groups, were identified for a number of countries. They are mainly localized in sites of known B- and T-cell epitopes and relatively rarely in structural and functional domains. The revealed differences in AA-substitutions in different ethnic groups and their colocalization with clusters of B- and T-cell epitopes suggest their possible relationship with the geographical distribution of alleles and haplotypes of the major histocompatibility complex (HLA). This may lead to the recognition of a different set of B- and T-cell epitopes of the virus, resulting in regional differences in the direction of epitope drift. Phylogenetic analysis of the nucleotide sequences encoding the E6 protein of HPV16 revealed a common ancestor, confirmed regional clustering of the E6 protein gene sequences by the set of the most common AA-substitutions, and identified cases of reversion of individual AA-substitutions when the virus distribution region changed. For the E7 protein, a similar analysis was not possible due to high sequence homology. Covariance analysis of the pooled sample revealed that there was no relationship between amino acid residues in the E6 protein, in the E7 protein, and between E6 and E7. Data obtained are important for the development of therapeutic vaccines against HPV of high carcinogenic risk.

What are the essential determinants of human papillomavirus carcinogenesis?

Human papillomavirus (HPV) infection is the leading viral cause of cancer. Over the past several decades, research on HPVs has provided remarkable insight into human cell biology and into the pathology of viral and non-viral cancers. The HPV E6 and E7 proteins engage host cellular proteins to establish an environment in infected cells that is conducive to virus replication. They rewire host cell signaling pathways to promote proliferation, inhibit differentiation, and limit cell death. The activity of the "high-risk" HPV E6 and E7 proteins is so potent that their dysregulated expression is sufficient to drive the initiation and maintenance of HPV-associated cancers. Consequently, intensive research efforts have aimed to identify the host cell targets of E6 and E7, in part with the idea that some or all of the virus-host interactions would be essential cancer drivers. These efforts have identified a large number of potential binding partners of each oncoprotein. However, over the same time period, parallel research has revealed that a relatively small number of genetic mutations drive carcinogenesis in most non-viral cancers. We therefore propose that a high-priority goal is to identify which of the many targets of E6 and E7 are critical drivers of HPV carcinogenesis. By identifying the cancer-driving targets of E6 and E7, it should be possible to better understand the distinct roles of other targets, perhaps in the viral life cycle, and to focus efforts to develop anti-cancer therapies on the subset of virus-host interactions for which therapeutic intervention would have the greatest impact.

In Silico Drug Designing for the Treatment of Cervical Cancer

Cervical cancer is a significant global health issue, especially in developing countries where limited resources and access to health services pose challenges. Human papillomavirus (HPV) is the primary cause of cervical cancer and plays a crucial role in its development. Despite the availability of prophylactic vaccines, effective treatment options for patients with pre-existing HPV infections or HPV-induced carcinomas remain elusive. High-risk (HR) HPV E6 and E7 oncoproteins serve as biomarkers in cervical cancer progression. Research has focused on these proteins as potential targets for novel therapeutics and to find a novel compound that can overcome the problem of drug ineffectiveness in cancerous cells as they become multi-drug resistant to the existing drugs and treatments. Computational-aided methods, including in silico approaches, have been valuable in discovering and developing small molecules that can block or inhibit the action of the E6 oncoproteins. This review discusses existing small molecules explored as HPV E6 protein blockers and outlines future perspectives in cervical cancer therapy. In molecular docking studies, it is identified that four compounds exhibit the properties of a lead molecule, possess more negative binding energy, and form a highly stable ligand-receptor complex. These four compounds will help in future research and development. Advancing these compounds through further studies will help develop potential and effective drugs for treating cervical cancer.

No genetic causal association between human papillomavirus and lung cancer risk: a bidirectional two-sample Mendelian randomization analysis

IntroductionSeveral observational or retrospective studies have previously been conducted to explore the possible association between lung cancer and human papillomavirus (HPV) infection. However, there may be inconsistencies in the data and conclusions due to differences in study design and HPV testing methods. There are currently no studies that provide conclusive evidence to support the involvement of HPV in the occurrence and development of lung cancer. Therefore, the relationship between HPV and lung cancer remains controversial and uncertain. This study aimed to explore whether HPV infection is causally related to lung cancer risk by systematically performing a two-way Two-Sample Mendelian Randomization (TSMR) analysis.MethodsIn the International Lung Cancer Consortium (ILCCO) genome-wide association study dataset, we included 11,348 lung cancer (LUCA) cases, including 3275 squamous cell carcinoma (LUSC) cases, 3442 adenocarcinoma (LUAD) cases, and 15,861 cases of control. Using genetic variants associated with the HPV E7 protein as instrumental variables, we summarized statistics associated with HPV infection in the MRC IEU OpenGWAS database, which included the HPV-16 E7 protein and the HPV-18 E7 protein. Two-sample Mendelian randomization (MR) results are expressed as odds ratios (OR) and 95% confidence intervals (CI).ResultsBased on a comprehensive analysis of genome-wide association study (GWAS) data from public databases, we mainly used inverse-variance weighted (IVW) to estimate causal relationships, while using MR-Egger, weighted median, simple mode, and weighted mode, and other four methods as supplements. Two-sample MR Analysis revealed no causal relationship between exposure factors (HPV-16 E7 protein and HPV-18 E7 protein) and outcome factors (lung cancer (LUCA) and its subtypes squamous cell carcinoma (LUSC) and adenocarcinoma (LUAD)) in forward MR Analysis using the IVW approach.HPV-16 E7 protein and LUCA and its subtypes LUSC and LUAD by IVW method results: [OR] = 1.002; 95% [CI]: 0.961 − 1.045; p = 0.920; [OR] = 1.023; 95% [CI]: 0.966 − 1.084; p = 0.438; [OR] = 0.994; 95% [CI]: 0.927 − 1.066; p = 0.872); HPV-18 E7 protein and LUCA and its subtypes LUSC and LUAD by IVW method results: [OR] = 0.965; 95% [CI]: 0.914 − 1.019; p = 0.197; [OR] = 0.933; 95% [CI]: 0.834 − 1.043; p = 0.222; [OR] = 1.028; 95% [CI]: 0.945 − 1.118; p = 0.524. It was observed through reverse MR that LUCA and its subtypes LUSC and LUAD were used as exposure factors, and HPV infection (HPV-16 E7 protein and HPV-18 E7 protein) was used as the outcome factors, the results of the IVW method are also invalid.LUCA and HPV-16 E7 protein and HPV-18 E7 protein by IVW method results: [OR] = 1.036; 95% [CI]: 0.761 − 1.411; p = 0.82; [OR] = 1.318; 95% [CI]: 0.949 − 1.830; p = 0.099; LUSC and HPV-16 E7 protein and HPV-18 E7 protein by IVW method results: [OR] = 1.123; 95% [CI]0.847 − 1.489; p = 0.421; [OR] = 0.931; 95% [CI]: 0.660 − 1.313; p = 0.682; LUAD and HPV-16 E7 protein and HPV-18 E7 protein by IVW method results: [OR] = 1.182; 95% [CI] 0.983 − 1.421; p = 0.075; [OR] = 1.017; 95% [CI]: 0.817 − 1.267; p = 0.877.Our results indicate that there is no causal relationship between genetically predicted HPV infection and LUCA and its subtypes LUSC and LUAD. In addition, in the reverse MR analysis, we did not observe a significant causal relationship between LUCA and its subtypes LUSC and LUAD on HPV infection.ConclusionsOur findings do not support a genetic association between HPV infection and lung cancer.

High Expression of Vascular Endothelial Growth Factor, Ki-67, and Protein 53 are Risk Factors for Poor Response of Paclitaxel-Carboplatin Neoadjuvant Chemotherapy in Stadium IB3, IIA2, and IIB Cervical Cancer.

The therapeutic strategy for stage IB3, IIA2, and IIB cervical cancer is still controversial. The modalities are chemoradiation, radical hysterectomy surgery, or administration of neoadjuvant chemotherapy followed by radical hysterectomy. Response to chemotherapy is determined by tumor vascularization or angiogenesis, proliferative activity, and genetic instability of cervical cancer. The marker of tumor cell proliferation is the Ki-67 protein. In cervical cancer, the p53 gene is suppressed by human papillomavirus (HPV). The HPV E6 protein promotes the degradation of p53 thereby inhibiting stabilization and activation of p53. This study aimed to prove that high expression of VEGF, Ki-67, and p53 are risk factors for a poor response to neoadjuvant chemotherapy. This was a case-control study that was conducted at the Department of Obstetrics and Gynecology in one tertiary hospital in Denpasar from October 2021 to April 2022. There were 56 samples included in this study, which were divided into two equal groups, namely good response and poor response to neoadjuvant chemotherapy. Data were analyzed using the software SPSS-24 including the Kolmogorov-Smirnov normality test, Chi-square, and multiple regression logistics. Data were presented in tables and described narratively. It was found that the risk of a poor response to chemotherapy on the expression of VEGF VEGF, Ki-67, and p53 were 11.5, 15.0, and 8.33 times, respectively. We obtained a formula for calculating chemotherapy response, y = -7.3+ 1.6 VEGF + 1.6 Ki-67 + 1.8 p53. High VEGF, Ki-67, and p53 expressions were scored 1, and low expressions were scored 2. The limit value used is 0.05. The result y < 0.05 means the risk of poor response to chemotherapy and the value of y > 0.05 means good response. This formulation can be used as a parameter to assess the risk of poor response to neoadjuvant chemotherapy in stage IB3, IIA2, and IIB cervical cancer which can be applied in clinical practice in the treatment of cervical cancer.

Characterization of novel compounds that directly bind to and inactivate the HPV E6 protein for treatment of HPV-associated cervical and oropharyngeal cancers.

e15122 Background: The human papillomavirus (HPV) E6 protein is necessary for viral genome replication and is always expressed in HPV-induced malignancies. We performed in silico screening of chemical libraries for molecules that bind to a pocket on the cancer associated HPV-16 E6 protein. We focused on the protein-protein interaction of E6 with one of its cellular binding partners, the ubiquitin ligase E6AP. In a trimolecular complex with E6, E6AP ubiquitinates p53, leading to its destruction by the proteasome. We designed, synthesized, and characterized &gt;300 compounds that 1) bind to the E6AP interaction surface of the HPV-16 E6 protein; 2) are armed with a ‘warhead’ to make an irreversible bond with a specific cysteine in this HPV-16 E6 binding pocket; 3) and restore levels of p53 and selectively decrease viability of HPV-16 expressing tumor derived cells. Methods: E6 inhibitor optimization was guided by in silico docking strategies, co-crystallization, and medicinal chemistry, followed by biochemical and cellular assays to iteratively design and synthesize molecules with increased E6 inhibitory activities. Results: We used whole protein mass spectrometry to interrogate covalent bonding to E6, showing irreversible binding to the cysteine in the targeted E6 binding pocket without the other 13 cysteines in E6, with Ki in the range of seconds. Biolayer interferometry and fluorescence polarimetry measured blockade of E6 with E6AP, with IC50 in the range of 400-600 nM. To establish in cell inactivation of E6, human cervical cancer derived SiHa cells that express HPV-16 E6 were incubated with compounds, showing dose dependent increase of p53 levels up to 8-fold after 24 hrs, an effect that was not observed in the HPV-16- cell line RPE-1 expressing wild-type p53. Viability of two cervical (SiHa, CaSki) and two oropharyngeal HPV-16+ cancer cell lines (UM-SC-47, UM-SCC-104) was reduced with an IC50 of ~2-3 µM, with an IC50&gt;30µM in RPE-1 cells and telomerase immortalized human keratinocytes. These in vitro and cell-based experiments revealed dependence on the cysteine residue in the E6AP binding pocket of E6. For in vivo proof-of-concept studies, we developed mouse xenograft models, performed by engrafting SiHa, UM-SC-47 and UM-SCC-104 cancer derived cells subcutaneously into the flank of immunodeficient mice. Lead compounds were administered by intraperitoneal injection and demonstrated nearly complete inhibition (&gt; 70% tumor reduction) in HPV-16 tumor growth compared to vehicle. Anti-tumor activity was not observed with the HPV-negative tumor cell line C33A. Conclusions: HPV-16 is identified in about 50% of all cervical cancers and the majority of head and neck malignancies. Inactivation of the HPV E6 protein represents the opportunity for a novel molecular targeted approach for medical treatment of HPV-16 expressing cancers of the cervix and oropharynx.

Designing a Novel Multiepitope Vaccine from the Human Papilloma Virus E1 and E2 Proteins for Indonesia with Immunoinformatics and Molecular Dynamics Approaches.

One of the deadliest malignant cancer in women globally is cervical cancer. Specifically, cervical cancer is the second most common type of cancer in Indonesia. The main infectious agent of cervical cancer is the human papilloma virus (HPV). Although licensed prophylactic vaccines are available, cervical cancer cases are on the rise. Therapy using multiepitope-based vaccines is a very promising therapy for cervical cancer. This study aimed to develop a multiepitope vaccine based on the E1 and E2 proteins of HPV 16, 18, 45, and 52 using in silico. In this study, we develop a novel multiepitope vaccine candidate using an immunoinformatic approach. We predicted the epitopes of the cytotoxic T lymphocyte (CTL) and helper T lymphocyte (HTL) and evaluated their immunogenic properties. Population coverage analysis of qualified epitopes was conducted to determine the successful use of the vaccine worldwide. The epitopes were constructed into a multiepitope vaccine by using AAY linkers between the CTL epitopes and GPGPG linkers between the HTL epitopes. The tertiary structure of the multiepitope vaccine was modeled with AlphaFold and was evaluated by Prosa-web. The results of vaccine construction were analyzed for B-cell epitope prediction, molecular docking with Toll like receptor-4 (TLR4), and molecular dynamics simulation. The results of epitope prediction obtained 4 CTL epitopes and 7 HTL epitopes that are eligible for construction of multiepitope vaccines. Prediction of the physicochemical properties of multiepitope vaccines obtained good results for recombinant protein production. The interaction showed that the interaction of the multiepitope vaccine-TLR4 complex is stable based on the binding free energy value -106.5 kcal/mol. The results of the immune response simulation show that multiepitope vaccine candidates could activate the adaptive and humoral immune systems and generate long-term B-cell memory. According to these results, the development of a multiepitope vaccine with a reverse vaccinology approach is a breakthrough to develop potential cervical cancer therapeutic vaccines.

Abstract 2471: State-of-the-art Specific T cell Activating Modulator on ePENDY platform (STAM.eP) activates specific CD8+ T cell by stimulating TCR signal

Abstract After decades of research, therapeutic cancer vaccine approach now sheds light on developing efficient cancer therapy treating patients who have resistance to other cancer immunotherapy. However, cancer vaccine approach using conventional modalities such as protein, peptide, and mRNA is considered a powerful cancer treatment only when the patients have healthy immune systems. In addition, dendritic cell (DC) cancer vaccine can be a great alternative therapy, but manufacture of DC vaccines in vitro is a labor-intensive, time- and money-consuming procedure. Here, we developed Specific T cell Activating Modulator (STAM) on our engineered IgM backbone, ePENDY (engineered PENtamer boDY), with high avidity based on multivalent binding sites to overcome the disadvantages of other therapeutic cancer vaccines. STAM on ePENDY (STAM.eP) consists of ten peptide-loaded-HLAs linked to ePENDY to simulate priming signal of CD8+ T cell by TCR cluster and a costimulatory signal molecule is linked to J chain of ePENDY to prevent CD8+ T cell from becoming anergic status. With these features, STAM.eP mimics cognately licensed DC that can present tumor peptide-loaded MHC class I molecule to CD8+ T cell and promote its priming. Thus, one of the greatest advantages of STAM.eP is to be a potential treatment for immunosuppressive cancer patients with DCs that do not respond appropriately. Another advantage is that any tumor specific antigen (TSA), including from intracellular proteins, can be applied to STAM.eP concept to activate specific CD8+ T cells for targeting cancer cells. In this study, we used Human Papillomavirus (HPV) E7-derived peptide which is an oncoprotein associated with cervical and head/neck cancers. We observed that HPV E7-derived peptide loaded STAM.eP, named IMB-401, increases antigen-specific CD8+ T cell population and shows cytotoxicity activity against cancer cells expressing HPV E7 protein in vitro test model. In vivo test model, it was also observed that population of specific CD8+ T cells and memory T cells were increased in the blood and spleens of transgenic mice treated with IMB-401. Furthermore, we confirmed that IMB-401 shows anti-tumor efficacy due to an increase of antigen-specific CD8+ T cell in vivo humanized mouse model. These results suggest that IMB-401 can be a new therapeutic cancer vaccine technology that replaces DC function and is expected to have robust anti-tumor activity in combination with immune checkpoint inhibitors. Citation Format: Kyung-gi Hyun, Sungmuk Kang, Yongjun Kang, Jihye Koo, Suho Park, Sunghyun Byun, Sunghyun Yoon, Jaebeom Lee, Hong Jai Lee, Hong Kyu Lee, Eunyoung Cho, Chungmin Lee, Kyung-Chul Choi, Gyongsik Ha. State-of-the-art Specific T cell Activating Modulator on ePENDY platform (STAM.eP) activates specific CD8+ T cell by stimulating TCR signal [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2471.

Amino Acid Substitution Patterns in the E6 and E7 Proteins of HPV Type 16: Phylogeography and Evolution

The E6 and E7 proteins of the high risk human papillomaviruses (HR HPVs) play a key role in the oncogenesis associated with papillomavirus infection. Data on the variability of these proteins are limited, and the factors affecting their variability are still poorly understood. We analyzed the variability of the currently known sequences of the HPV type 16 (HPV16) E6 and E7 proteins, taking into account their geographic origin and year of sample collection, as well as the direction of their evolution in the major geographic regions of the world. All sequences belonging to HPV16 genome fragments encoding the E6 and E7 oncoproteins were downloaded from the NCBI GenBank database on October 6, 2022. Samples were filtered according to the following parameters: the sequence has to include at least one of the two whole open reading frames, and given date of collection, and the country of origin. A total of 3651 full-genome nucleotide sequences encoding the E6 protein and 4578 full-genome nucleotide sequences encoding the E7 protein were sampled. The nucleotide sequences obtained after sampling and alignment were converted to amino acid sequences and analyzed using the MEGA11, R, RStudio, Jmodeltest 2.1.20, BEAST v1.10.4, Fastcov, and Biostrings software. The highest variability in the E6 protein was recorded for amino acid (AA) residues in the positions 17, 21, 32, 85, and 90. The most variable in E7 were aa positions 28, 29, 51, and 77. The samples were divided geographically into five heterogeneous groups as derived from Africa, Europe, America, South-West and South Asia, and South-East Asia. Unique amino acid substitutions (AA-substitutions) in the E6/E7 proteins of HPV16, presumably characteristic to certain ethnic groups, were identified for a number of countries. They weare mainly localized in the sites of known B- and T-cell epitopes and relatively rarely the domains critical for in structure and protein function. The revealed differences in AA-substitutions in different ethnic groups and their colocalization with the clusters of B- and T-cell epitopes suggested their possible relation to the geographical distribution of alleles and haplotypes of the major histocompatibility complex (HLA). This may lead to the recognition of a different set of B- and T-cell epitopes of the virus in different geographic areas, resulting in the regional differences in the direction of epitopic drift. Phylogenetic analysis of the nucleotide sequences encoding the E6 protein of HPV16 revealed a common ancestor, confirmed regional clustering of the E6 protein sequences sharing common AA-substitutions, and identified cases of reversion of individual AA-substitutions when the change of geographical localization. For the E7 protein, such analysis was not possible due to the high sequence homology. Covariance analysis of the pooled of E6 and E7 sequences revealed that there was no associations between amino acid residues in any aa position within E6 or E7 as well as aa positions of E6 and E7 proteins. The data presented here are important for the development of universal therapeutic vaccines against HPV of high carcinogenic risk.

Production of recombinant HPV11/16 E6/E7-MBP-His6 fusion proteins and their potential to induce cytokine secretion by immune cells in peripheral blood

Human papillomavirus (HPV) infection poses a significant threat to public health worldwide. Targeting the function of HPV E6 and E7 proteins and activating the host immune response against these proteins represent promising therapeutic strategies for combating HPV-related diseases. Consequently, the efficient production of soluble, high-purity E6 and E7 proteins is crucial for function and host immune response studies. In this context, we selected the pMCSG19 protein expression vector for Escherichia coli to produce soluble MBP-His6 tagged HPV11/16 E6/E7 proteins, achieving relatively high purity and yield. Notably, these proteins exhibited low toxicity to peripheral blood mononuclear cells (PBMCs) and did not compromise their viability. Additionally, the recombinant proteins were capable of inducing the secretion of multiple cytokines by immune cells in peripheral blood, indicating their potential to elicit immune responses. In conclusion, our study offers a novel approach for the production of HPV11/16 E6/E7 fusion proteins with relatively high purity and yield. The fusing HPV11/16 E6/E7 proteins to MBP-His6 tag may serve as a valuable method for large-scale protein production in future research endeavors.

Human papillomavirus E7 protein induces homologous recombination defects and PARPi sensitivity

PurposeCervical cancer is a common gynecological malignancy, pathologically associated with persistent infection of high-risk types of human papillomavirus (HPV). Previous studies revealed that HPV-positive cervical cancer displays genomic instability; however, the underlying mechanism is not fully understood.MethodsTo investigate if DNA damage responses are aggravated in precancerous lesions of HPV-positive cervical epithelium, cervical tissues were biopsied and cryosectioned, and subjected to immunofluorescent staining. Cloned HA-tagged E6 and E7 genes of HPV16 subtype were transfected into HEK293T or C33A cells, and indirect immunofluorescent staining was applied to reveal the competency of double strand break (DSB) repair. To test the synthetic lethality of E7-indued HRD and PARP inhibitor (PARPi), we expressed E7 in C33A cells in the presence or absence of olaparib, and evaluated cell viability by colony formation.ResultsIn precancerous lesions, endogenous DNA lesions were elevated along with the severity of CIN grade. Expressing high-risk viral factor (E7) in HPV-negative cervical cells did not impair checkpoint activation upon genotoxic insults, but affected the potential of DSB repair, leading to homologous recombination deficiency (HRD). Based on this HPV-induced genomic instability, the viability of E7-expressing cells was reduced upon exposure to PARPi in comparison with control cells.ConclusionIn aggregate, our findings demonstrate that HPV-E7 is a potential driver for genome instability and provides a new angle to understand its role in cancer development. The viral HRD could be employed to target HPV-positive cervical cancer via synthetic lethality.

HPV16 E7 protein antagonizes TNF-α-induced apoptosis of cervical cancer cells via Daxx/JNK pathway

Human papillomavirus (HPV) E7 protein as an important viral factor was involved in the progression of cervical cancer by mediating the cellular signaling pathways. Daxx (Death domain-associated protein) can interact with a variety of proteins to affect the viral infection process. However, the interaction and its related function between HPV16 E7 and Daxx have not been adequately investigated. Here, it was found that HPV16 E7 can interact with Daxx in HeLa or C33A cells by co-immunoprecipitation. HPV16 E7 protein treatment can up-regulate Daxx protein expression, while the interference in Daxx expression and the agonists for JNK can both reduce the antagonistic effects of HPV16 E7 on TNF-α-induced apoptosis, suggesting that Daxx/JNK pathway may be involved in the anti-apoptotic activity of HPV16 E7.

Human papillomaviruses:Knowns, mysteries, and unchartered territories.

There has been an explosion in the number of papillomaviruses that have been identified and fully sequenced. Yet only a minute fraction of these has been studied in any detail. Most of our molecular research efforts have focused on the E6 and E7 proteins of "high-risk,"cancer-associated human papillomaviruses (HPVs). Interactions of the high-risk HPV E6 and E7 proteins with their respective cellular targets, the p53 and the retinoblastoma tumor suppressors, have been investigated in minute detail. Some have thus questioned if research on papillomaviruses remains an exciting and worthwhile area of investigation. However, fundamentally new insights on the biological activities and cellular targets of the high-risk HPV E6 and E7 proteins have been discovered and previously unstudied HPVs have been newly associated with human diseases. HPV infections continue to be an important cause of human morbidity and mortality and since there are no antivirals to combat HPV infections, research on HPVs should remain attractive to new investigators and biomedical funding agencies, alike.

MRNA-LNP vaccination-based immunotherapy augments CD8+ T cell responses against HPV-positive oropharyngeal cancer

Although mRNA vaccines are known as potent activators of antigen-specific immune responses against infectious diseases, limited understanding of how they drive the functional commitment of CD8+ T cells in tumor microenvironment (TME) and secondary lymphoid organs hinders their broader application in cancer immunotherapy. Here, we systematically evaluated the immunological effects of a lipid nanoparticle (LNP)-encapsulated mRNA vaccine that encodes human papillomavirus E7 protein (HPV mRNA-LNP), a tumor-specific antigen of HPV-positive oropharyngeal squamous cell carcinoma (OPSCC). HPV mRNA-LNP vaccination activated overall and HPV-specific CD8+ T cells, as well as differentially drove the functional commitment of CD8+ T cells through distinct IFN-response and exhaustion trajectories in the spleen and TME, respectively. Combination therapies of HPV mRNA-LNP vaccination with immune checkpoint blockades boosted HPV-specific CD8+ T cells while maintaining their anti-tumor function, thus further promoting tumor regression. Our results showed that the HPV mRNA-LNP vaccination combined with immune checkpoint blockade is a promising approach for immunotherapy of HPV-positive OPSCC.

HPV E6 inhibits E6AP to regulate epithelial homeostasis by modulating keratinocyte differentiation commitment and YAP1 activation.

Human papillomaviruses (HPV) cause persistent infections by modulating epithelial homeostasis in cells of the infected basal layer. Using FUCCI and cell-cell competition assays, we have identifed regulatory roles for E6AP and NHERF1, which are the primary HPV11 E6 cellular targets, as well as being targets of the high-risk E6 proteins, in processes governing epithelial homeostasis (i.e. cell density, cell cycle entry, commitment to differentiation and basal layer delamination). Depletion of E6AP, or expression of HPV11 or 16E6 increased keratinocyte cell density and cell cycle activity, and delayed the onset of differentiation; phenotypes which were conspicuously present in HPV11 and 16 infected patient tissue. In line with proposed E6 functions, in HPV11 condyloma tissue, E6AP and NHERF1 were significantly reduced when compared to uninfected epithelium. In experimental systems, loss of HPV11 E6/E6AP binding abolished 11E6's homeostasis regulatory functions, while loss of E6/NHERF1 binding reduced the cell density threshold at which differentiation was triggered. By contrast, a NHERF1-binding mutant of 16E6 was not compromised in its homeostasis functions, while E6AP appeared essential. RNA sequencing revealed similar transcriptional profiles in both 11 and 16E6-expressing cells and E6AP-/- cells, with YAP target genes induced, and keratinocyte differentiation genes being downregulated. HPV11 E6-mediated Yap activation was observed in 2D and 3D (organotypic raft) cell culture systems and HPV-infected lesions, with both NHERF1, which is a regulator of the Hippo and Wnt pathways, and E6AP, playing an important role. As the conserved binding partner of Alpha group HPV E6 proteins, the precise role of E6AP in modulating keratinocyte phenotype and associated signalling pathways has not previously been defined. Our study suggests a model in which the preserved functions of the low and high-risk Alpha E6 proteins modulate epithelial homeostasis via E6AP activity, and lead to alteration of multiple downstream pathways, including those involving NHERF1 and YAP.

Current Status of Human Papillomavirus–Targeted Therapies Development in Head and Neck Cancer

PURPOSE While the incidence of smoking-related head and neck squamous cell carcinoma (HNSCC) has been declining, that of human papillomavirus (HPV)–mediated HNSCC has rapidly increased in the past decades worldwide. Despite rapid advances in therapeutics for solid tumors with novel immunotherapy and targeted therapeutic agents, no breakthroughs have yet been made in the treatment of advanced HPV+ HNSCCs. This review aims to summarize the concepts and designs, early trial results, and future directions of various HPV-targeted experimental therapeutics for HPV+ HNSCC. MATERIALS AND METHODS Guided by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement, a systemic literature search of PubMed was conducted for HPV-targeted therapeutics using the following search terms: HPV, head and neck squamous cell carcinoma, and therapy. For clinical trial data, publications, major oncology conference abstracts, and the National Institutes of Health Clinical Trials Registry (ClinicalTrials.gov) information were reviewed. This review focused on the ones that are in the clinical stage and currently in active ongoing evaluation. The therapeutics not actively evaluated in HNSCC, in the preclinical stage, or terminated for further development were excluded. RESULTS Diverse approaches are being actively explored to target HPV+ HNSCC, including therapeutic vaccines of various modalities, HPV-specific immune cell–activating agents, and adaptive cellular therapeutics. All these novel agents use immune-based mechanisms and target constitutively expressed oncogenic HPV E6 and/or E7 viral proteins. Most therapeutics demonstrated excellent safety but single-agent activities are only modest. Many are being tested in combination with immune checkpoint inhibitors. CONCLUSION Our review summarized various novel HPV-targeted therapeutics that are in the clinical phase for HPV+ HNSCC. Early-phase trial data suggest the feasibility and promising efficacy. Further strategies, including selecting the optimal combination and understanding and overcoming resistant mechanisms, are warranted for successful development.

Detection of high-risk Human Papillomavirus in prostate cancer from a UK based population

Human papillomavirus (HPV) infection is one of the sexually transmitted diseases which have been implicated in the etiology of multiple cancers. To date, several studies have been conducted to evaluate the incidence of high-risk (HR) HPV in prostate cancer (PCa) which have generated widely conflicting data. Hence, this leaves a lack of awareness on the causal role of persistent HPV infection in the development of PCa. Although this has been investigated in a handful of countries, to the best of our knowledge, no prior studies have been conducted in the UK. In this study, polymerase chain reaction (PCR) and Sanger sequencing were implemented to analyze a total of 49 fresh prostate specimens (35 benign and 14 malignant specimens) for the presence of viral DNA of 12 HR-HPV types. Data obtained confirmed the presence of HR-HPV in 32.7% of analyzed benign and malignant prostate tissues with HPV 35 being identified as the most frequent type. Moreover, HR-HPV positivity rate was found to be higher in abnormal prostate tissues (adenocarcinoma and benign with prostatitis) compared those with normal prostate condition. Using immunohistochemistry, we have confirmed the expression of HPV E7 protein in prostate tissues positive for HPV DNA. This observation, the first reported from a UK population, suggests that the presence of HPV in prostate tissue is likely to be a related factor in the progression of certain cases of prostate cancer.

Fragment-based design and MD simulations of human papilloma virus-16 E6 protein inhibitors

The main objective of this study is to screen potential small molecule inhibitors against HPV (Human Papilloma Virus)-16 E6 protein (HPV16 E6P) using a fragment-based approach. Twenty-six natural HPV inhibitors were selected based on the review of the literature. Among them, Luteolin was selected as the reference compound. These 26 compounds were used to generate novel inhibitors against HPV16 E6P. Fragment script and BREED of Schrodinger software were used to build novel inhibitor molecules. The result in 817 novel molecules was docked into the active binding site of HPV E6 protein and the top ten compounds were screened based on binding affinity compared to Luteolin for further study. Compounds Cpd5, Cpd7, and Cpd10 were the most potent inhibitors of HPV16 E6P and these were non-toxic and showed high Gastrointestinal (GI) absorption and positive drug-likeness score. Complexes of these compounds were stable in the 200 ns Molecular Dynamics (MD) simulation. These 3 HPV16 E6P inhibitors could be the lead molecules as new drugs for HPV-related diseases. Communicated by Ramaswamy H. Sarma

MmuPV1 E7's interaction with PTPN14 delays Epithelial differentiation and contributes to virus-induced skin disease.

Human papillomaviruses (HPVs) contribute to approximately 5% of all human cancers. Species-specific barriers limit the ability to study HPV pathogenesis in animal models. Murine papillomavirus (MmuPV1) provides a powerful tool to study the roles of papillomavirus genes in pathogenesis arising from a natural infection. We previously identified Protein Tyrosine Phosphatase Non-Receptor Type 14 (PTPN14), a tumor suppressor targeted by HPV E7 proteins, as a putative cellular target of MmuPV1 E7. Here, we confirmed the MmuPV1 E7-PTPN14 interaction. Based on the published structure of the HPV18 E7/PTPN14 complex, we generated a MmuPV1 E7 mutant, E7K81S, that was defective for binding PTPN14. Wild-type (WT) and E7K81S mutant viral genomes replicated as extrachromosomal circular DNAs to comparable levels in mouse keratinocytes. E7K81S mutant virus (E7K81S MmuPV1) was generated and used to infect FoxN/Nude mice. E7K81S MmuPV1 caused neoplastic lesions at a frequency similar to that of WT MmuPV1, but the lesions arose later and were smaller than WT-induced lesions. The E7K81S MmuPV1-induced lesions also had a trend towards a less severe grade of neoplastic disease. In the lesions, E7K81S MmuPV1 supported the late (productive) stage of the viral life cycle and promoted E2F activity and cellular DNA synthesis in suprabasal epithelial cells to similar degrees as WT MmuPV1. There was a similar frequency of lateral spread of infections among mice infected with E7K81S or WT MmuPV1. Compared to WT MmuPV1-induced lesions, E7K81S MmuPV1-induced lesions had a significant expansion of cells expressing differentiation markers, Keratin 10 and Involucrin. We conclude that an intact PTPN14 binding site is necessary for MmuPV1 E7's ability to contribute to papillomavirus-induced pathogenesis and this correlates with MmuPV1 E7 causing a delay in epithelial differentiation, which is a hallmark of papillomavirus-induced neoplasia.